nj
THE
ACTINOMYCETES
VOLUME III
Crystals of Candidix
THE ACTINOMYCETES
Vol. Ill
ANTIBIOTICS OF ACTINOMYCETES
hy
Selman A. Waksman
and
Hubert A. Lechevalier
BALTIMORE
THE WILLIAMS & W I L K I N S COMPANY
1962
THE ACTINOMYCETES
Vol. III. Antibiotics of Actinomycetes
Copyright ©, 1962
The William.s & Wilkins Company
Made in the United States of America
Library of Congress
Catalog Card Number
59-9962
Composed and printed at the
WAVERLY PRESS, INC.
Baltimore 2, :Md., U. S. A.
PREFACE
In 1045, tho senior author published a book entitled "Alierobial Antagonisms
and Antibiotic Substances," which presented a comprehensive review of the
knowledge of this subject at that time. Of the antibiotics listed, only six had been
isolated from cultures of actinomycetes. Only one of these, namely streptomycin,
had fovnid at that time practical application in the treatment of human and ani-
mal diseases. A second edition of the book appeared in 1947. The number of anti-
biotic preparations obtained from cultures of actinomycetes did not increase ap-
preciably during this brief period.
In 1953, in a volume entitled "Guide to the Identification and Classification of
the Actinomycetes and their Antibiotics," we described more than a hundred
chemical compounds and preparations isolated from cultures of actinomycetes.
About ten of these had found practical application in chemotherapy.
The publication of the present \'()lume, only 8 years later, emphasizes the tre-
mendous progress made in this field. Alore than 400 substances and preparations
have been isolated. More than 30 have found practical application in chemo-
therapy.
No attempt has been made in this treatise to cover the very extensive litera-
ture on the isolation, identification, and utilization of antibiotics of actinomy-
cetes. Only the more pertinent references to the develf)pments of the subject are
presented in this volume. For a more detailed analysis of the background of our
knowledge of the antagonistic relationships among microorganisms and the forma-
tion of antibiotic substances, especially by species of actinomycetes, the reader
is referred to the 1945 and 1947 volumes. Detailed lit(>rature references are best
found in the special treatises dealing with individual antibiotics.
The authors wish to express their sincere appreciation to Mrs. Mary P. Leche-
valier for her imselfish and painstaking efforts in the assimilation of most of the
literature for Part B of this \'ohmie. They are indebted to Dr. Maxwell Finland
of Boston City Hospital for re\-iewing Chapter 8; to Dr. Vernon Bryson of the
Institute of ]\Iicrobiology for reviewing Chapter 10; and to Dr. 0. K. Saz of the
National Institutes of Health for reviewing and suggesting various corrections
and changes in Chapter 9. We also wish to thank Dr. E. Borowski and Dr. N.
Gerber for many suggestions helpful in preparing Chapter 6. The editorial as-
sistance of Mr. R. A. Day and Mrs. H. B. Kitchen and the secretarial assistance
of Mrs. R. Nehlig are gratefully acknowledged. Some of the necessary transla-
tions were kindly supplied by Dr. Nobuo Sakurai, Dr. Tadashi Arai, and jNIiss
Sandra Czaplicki.
Selman A. Waksman
Hubert A. Lechevalier
^
J^,
TABLE OF CONTENTS
The Actinoiiiycetes
Part A
NATURE, FORAIATION, AND ACTIVITIES OF ANTIBIOTICS
PRODUCED BY ACTINOMYCETES
Preface v
Introductory 1
1. Microbial Antagonisms and Production of Antibiotics 7
2. How Antibiotics Came to be Recognized 12
3. The Search for Antibiotics: Screening Programs 20
4. Production of Antibiotics 31
5. Isolation and Identification of Antibiotics of Actinomycetes 40
6. Chemical Nature of the Antibiotics of Actinomycetes 48
7. Biogenesis of Antibiotics 64
8. Antimicrobial Activities of Antibiotics 77
9. Modes of Action of Antibiotics 90
10. Development of Resistance 104
11. Utilization of Antibiotics in Clinical and in \'eterinarv Medicine. Other
Applications 1 14
An Outlook 119
References to Part A 122
Part B
DESCRIPTIONS OF THE VARIOUS ANTIBIOTICS PRODUCED
BY ACTINOMYCETES
Introductory 139
Keys to the Antibiotics 141
Antibiotics Which Are Active Mainly Against Gram-positive Bacte-
ria " 141
Antibiotics Which Are Active Against Gram-positi\'e and Ciram-
negati\'e Bacteria 146
Antibiotics Which Have Antifungal Activity 150
Substances Which Are Not Active Against Bacteria and 'or Fungi. . 154
List of Antibiotics with Antiprotozoal Activity 155
List of Antibiotics with Antiviral Activitv 156
Vll
iQ8i
CONTENTS
List of Antitumor Substances 1^^>6
List of Amino Acids Present in Hydrolysates of Antibiotics 157
Descriptions of Antibiotics 162
Appendix ^1^
Additional Antibiotics 410
General Index 41'^
Index of Organisms 423
INTRODUCTORY
Detailed studies of the microbiological
population of the soil have revealed not only
the presence of numerous actinomycetes but
also the fact that the growth of certain of
these organisms exerts a depressive effect
upon the growth of other microorganisms,
notably l)acteria and fungi. Casual o))ser\'a-
tions of cultures of actinomycetes isolated by
Gasperini from 1892 to 1895, Aliiller in 1908,
Greig-Smith from 1911 to 1917, Lieske in
1921, Gratia and Dath from 1924 to 1927,
and Rosenthal in 1925 further demonstrated
that these organisms have the capacity to
produce chemical substances, now known
as antibiotics, which inhibit the growth of
other organisms. That these observations
were not isolated instances but were char-
acteristic of a large number of the actino-
mycetes was also established in the ^'arious
sur^'eys carried out by a group of Russian
investigators from 1935 to 19o9.
Systematic investigations of the effect of
actinomycetes upon other soil organisms,
carried out in our own laboratories since
1935 (Waksman, 1937, 1941, 1947), resuUed
in 1940 in the isolation, in crystalline form,
of a pigmented antibiotic which was named
actinomycin. This was followed by the iso-
lation in our laboratories of streptothricin
in 1942, micromonosporin and streptomycin
in 1943, grisein in 1946, neom_ycin in 1948,
and later of a number of other antibiotics,
notably streptocin, ehrlichin, fradicin, can-
dicidin, and candidin. Some of these antibi-
otics, especially streptomycin and neomycin,
have found extensive pi'actical application
in the control of numerous human, animal,
and plant diseases; more recently actinomy-
cin was shown to possess activity against
certain forms of cancer; candicidin and can-
didin give promise as antifungal agents.
Nearly all of them are of scientific interest.
Numerous other antibiotics soon were iso-
lated in various other laboratories through-
out the world.
In recent years, the field of antibiotics has
undergone spectacular developments. The
ever growing imi)ortance of these comjxuuids
in the control of human and animal diseases
as well as of certain plant diseases, in animal
nutrition, in food pi'eser\^ation, in the preser-
vation of biological materials, and in other
fields of human endeavor has revolutionized
medical practice and many (jf the habits of
modern life. The antibiotics have added un-
told wealth to our economy and have re-
sulted directly in the prolongation of mil-
lions of human lives. They have also intro-
duced a new concept in our understanding
of microbial life in natural environments and
have added greatly to our understanding of
certain chemical reactions in biological sys-
tems.
These de\'elopments in the field of medical
science and the important practical applica-
tions in agriculture are due primarily to
specific biological and chemical properties of
the antibiotics, particularly their antimi-
crobial activities. Among these, their selec-
tWe destructive action against various
microbial pathogens and their relative
harmlessness to the hosts attacked by the
pathogens are of particular significance.
Antibiotics affect various microbes at dif-
ferent rates. They are not generalized anti-
septics and disinfectants. Each antibiotic is
characterized by a selective antimicrobial
spectrum, or the ability to inhibit the growth
of or destroy certain microbes but not others.
The various microbes differ, moreover, in
the degree of their sensitivity to each anti-
biotic. On prolonged contact with a given
NATURE, FORMATION, AND ACTIVITIES
antibiotic, microbes usually become resistant
to this antibiotic as a result of the selection,
by the antibiotic, of resistant mutants.
The potentiality of a particular antibiotic
for important therapeutic usefulness in th(>
treatment of one or more infectious diseases
depends largel^^ upon its action on the causa-
tive agents of the disease and its lack of
toxicity to the affected animals or plants.
There are very few infectious diseases now
known, aside from those caused by viruses
and certain protozoa and some fungi that
are not completely or partly controlled by
the use of antibiotics. Nearly all the dis-
eases caused by bacteria, and some of the
diseases caused by fungi, rickettsiae, and
the psittacosis-lymphogranuloma group of
organisms, certain amoebae, and trichomo-
nads, lend themselves readily to antibiotic
therapy. Such diseases include not only those
that afflict man, but also those that attack
animals and plants.
Some antibiotics also possess a marked
growth-promoting effect upon animals and
have thus found practical application in the
nutrition of these animals. This is true es-
pecially of nonruminant animals, such as
swine and poultry. Ruminant animals are
usually excluded, since antibiotics may
affect adversely the bacterial population of
the rumen which assists such animals in the
digestion of cellulosic food materials; but
even some of the ruminant animals may
benefit at a certain stage of their develop-
ment from the use of antibiotics. Antibiotics
at low concentrations may exert a growth-
promoting effect also upon certain microbes.
This effect becomes growtli-inhibiting and
even destructive at higher concentrations;
hence, the concentration in which antibi-
otics are used is of prime importance.
Because of their specific selective action
upon microbial cells, antibiotics are ideal
agents for the preservation of semen, A'irus
preparations, vaccines, and similar biological
materials. Hecently, antibiotics have been
utilized foi- the j^reservation of foods, es-
pecially poultry and certain vegetables.
Since a single antibiotic will not inhibit all
forms of mici'obial life, more than one anti-
biotic may be recjuired. Before the food is
eaten the antibiotics must be inactivated,
as by boiling, since their constant consump-
tion in the food might exert dangerous effects
upon the human body.
The potentialities in the utilization of
antibiotics in the life of modern man are
still far from exhausted. Wherever man has
had to combat microbes — be they injurious
to his own health or to that of his herds and
crops or be they destructive to his industrial
products or to his foodstuffs — he has found
and will continue to find in the antibiotics
a source of great assistance.
Howe\'er, as with every other great dis-
covery that has revolutionized human life,
new problems have arisen as a result of the
usage of antibiotics. The prevalence of cer-
tain microbes resistant to specific antil)iotics
is now a major problem of chemotherapy.
As resistant forms appear, new antibiotics,
or new forms of known antibiotics, have to
be found to eradicate them. This seems to
he an endless process. The problem might be
minimized by a rotation program in the
usage of antibiotics. The disturbance in the
microbiological e(iuilibrium existing in na-
ture by the extensive use of chemical agents
that tend to eliminate certain members of
the microbial population and not others may
not only stimulate the de\'elopment of re-
sistant strains but may also lead to the
appearance of undesirable mutants of mi-
crobes. One should consider, further, the
dangerous potentialities of reduced natural
resistance in the human and animal body as
a result of the elimination of infectious or-
ganisms before the body has had a chance to
react immunologically. And finally, we must
not forget that antibiotics have contributed
many problems to geriatrics })y greatly in-
creasing the average life span of man.
INTRODUCTORY
3
111 tracing the history of antiliiotics, which
brought about a revohition in medical sci-
ence and cHnical practice, the futiu'e his-
torian will no dotibt designate the years
1939 to 1940 as a turning point in the history
of medicine and of microbiology as well. In
those years began a period which has already
been designated, medically speaking, as the
Age of Antibiotics. The actinomycetes have
played a dominant part in this development.
With the single exception of penicillin, they
ha\'e yielded the most important antibiotics
now used in medicine, in \'eterinary science,
and in animal nutrition. Annual production
of antibiotics, mostly produced by actino-
mycetes, has reached the colossal figure of
2..") million pounds in the United States
alone.
In addition to streptomycin and neomy-
cin, the actinomycetes liave contributed
chloramphenicol, the tetracyclines, the
erythromycins, the novobiocins, and the
polyenes, to name only a few. Numerous
industrial plants in this country and abi-oad
are concerned with the manufacture of anti-
biotics produced by actinomycetes. Hun-
dreds of laboratories throughout the world
are engaged in the search for new antibiotics
active upon diseases not subject to control
at the present time; in this search, actino-
mycetes and their antibiotics play a dom-
inant part.
The story is still far from complete. In
presenting this summary, the authors hope
to coordinate information that has been
accumulating so rapidly that even the ex-
perts have had difficulty in absorbing and
assimilating it.
Part A
Nature, Formation, and Activities of Antibiotics
Produced by Actinoniycetes
Chapter 1
Microbial Antagonisms and Production
of Antibiotics
Living organisms are not found in nature
in separated pigeonholes but are in constant
association with one another. During these
contacts various types of reactions can be
noted. Even though these interactions are
often difficult to classify, the following types
can be recognized: (1) symbiosis, (2) com-
petition, (3) predatoriness, (4) parasitism,
and (5) antagonism. In order to simplify the
following discussion, these interactions are
considered as if they occurn^d only between
two individual organisms or groups of or-
ganisms.
Symbiosis
The word .sijmbiosis is used to designate
the harmonious relationship between two
organisms which is beneficial to both part-
ners. In the world of microorganisms there
are many examples of symbiosis.
The lichens represent a symbiotic associ-
ation between an alga and a fungus so har-
monious that it forms physiological and
morphological types which are different frf)m
either fungi or algae. Either one or the
other may be the dominant partner, that
which encircles the weaker partner and is
mainly responsible for the shape of the
lichen. In most cases the fungus is the dom-
inant partner. The fungal mycelium pene-
trates the substratum on which the lichen
grows, l)e it rock, l)ark, wood, or soil, and
secretes acids and enzymes that dissolve and
break down the substratum, often with a
resulting beneficial nutritional effect. The
entrapped alga furnishes the products of its
photosynthesis.
Mycorrhizas are structures formed by
interaction of the mycelium of certain fungi
and the roots of certain higher plants. The
nature of the interrelationship between the
roots and the fungi is still the subject of
numerous studies, but it seems obvious that
the association is beneficial to the plants.
The fungus probably helps the plant by
absorbing water and nutrients from the soil
and benefits from the association by receiv-
ing food from the plant. In some instances
the association is so successful that the plant
produces no chlorophyll and depends on the
fungus as a universal pro\-ider. This is true
of the Ericaceae, Monotropa (Indian pipe),
and the orchid Corallorrhiza. In nature, all
orchids depend on fungi for their very life
even though most of them ha\'e chlorophyll.
In the laboratory, orchid seeds can l)e ger-
minated free of mycorrhizal fungi if pro-
^'ided with sucrose which has been auto-
claved at an acid pll. It is a fair assumption,
then, that fungi fui'uish orchid seeds with a
mixture of sugars and sugar degradation
products.
Symbiosis between animals and micro-
organisms is represented by certain scale
insects and a fungus belonging to the genus
Septohasidium. The insect feeds by sucking
on a plant, the fungus sending hyphae in-
side the insect and growing luxuriant h^ on
NATURE, FORMATION, AND ACTIVITIES
the outside, forming a protective mat. This
fungal mass is inhabited both by mycelium
infected scale insects, the only function of
which consists in feeding the protecting fun-
gus, and by noninfected scale insects which
take care of the reproduction. In this case
protection is traded for food. Further in-
formation on the phenomena of symbiosis in
nature is found in the work of \'uillemin
(1889), Waksman (1937), C'hristensen
(1951), and Gaullery (19r)2).
Competition
Different living organisms may feed on
the same substances, with a resulting con-
flict. Obviously, in nature, microorganisms
capable of utilizing the same food will com-
pete for whatever concentrations of that food
are available. If two organisms can utilize
the same nutrient with the same ease, other
factors will regulate which one of the two
will have the supremacy. For example, at
high temperature, thermophilic organisms
will be favored; in the absence of free oxy-
gen, anaerobic organisms will be favored.
Predatorines8
Any living organism that consumes an-
other living organism is a predator. Ex-
amples of such an association are most com-
mon among animals but are not restricted
to the animal kingdom. Carni\'orous plants
with specialized leaves capture insects. The
leaves of Sarracenia and Nepenthes are
shaped like an urn and are filled with a
licjuid, diluted by rain water. If an insect
is trapped in the urn, the motion of the in-
sect starts the secretion of proteolytic en-
zymes, the pH of the licjuid becoming acid.
Bacterial action is not essential for the de-
composition of the insect, since the sterile
liciuid has definite proteolytic i^roperties.
In other plants, such as Droscra, the insect
is trapped by mucilaginous tentacles which
can also produce proteolytic enzymes. In
plants of the genus Dionea, the leaf folds
along its central nervation and traps the
insect in a forest of hard bristles, proteolytic
enzymes being produced.
Predatoriness is common among protozoa,
but even some of the fungi are predators.
Some of these organisms specialize in catch-
ing nematodes; others can trap protozoa.
This process has been studied extensively b}^
Drechsler (see Duddington, 1957). Certain
fungal species have developed different types
of nematode traps. These may be mycelial
loops strong enough to hold a nematode if
the worm sticks its head through one of
them. The mycelium will then invade the
body of the nematode and digest it. Possibly
the fungi secrete a substance which attracts
nematodes and incites them to stick their
necks in the loops. It is interesting to note
that Pi-amer (1959) has shown that the
nematode-trapping fungi form traps only
under the stimulation of a substance found
in many animal tissues including those of
nematodes.
The biological significance of predatori-
ness is clear in the case of a fox catching a
chicken but harder to explain logically in
the case of higher plants and fungi. The
insect-catching plants are indeed able to
carry out photosynthesis and they rarely
live in such poor soil that no nitrogen would
be a\^ailable to them. The nematode-trap-
ping fungi are able to utilize the organic and
inorganic nutrients of the soil. Still they are
performing what seems to us the nonessen-
tial function of predators. It may be of in-
terest to note here that Vuillemin first ap-
plied, in 1889, the designation "antibiosis"
to phenomena of predatoriness, standing
between strict saprophitism and parasitism,
as a "snake devouring its prey."
Parasitism
Predatoriness differs from parasitism in
that a predator destroys its prey outright,
whereas a parasite usually feeds on the living
host. It is common practice to differentiate
between facultatiN'e and obligator}^ parasites.
The term obligatory parasite may be only an
MICROBIAL ANTAGONISMS AND PRODUCTION OF ANTIBIOTICS
9
expression of our luck oi knowledge of the
nutrition of the organisms that we so label
(Gaullery, 1902). The pathogenic actino-
mycetes all fall in the group of facultative
parasites, as amply illustrated in Volume I,
Chapters 17 and 18, and in \\)lume II,
Chapters 2 and 3.
Antagonism
Antagonism is the phenomenon hy which
one living organism inhibits the growth of
another one by creating an unfavorable set
of conditions such as the production of toxic
chemical substances. In the case of micro-
organisms, antimicrobial sul)stances may be
of two types: (1) chemical compounds toxic
in high concentrations, such as certain acids
(nitric, sulfuric, acetic, butyric, lactic, fu-
maric) and alcohols (ethyl, butyl); (2)
chemical substances toxic in xcry dilute
solutions. These are called antibiotics and
are usually selective in their antimicrobial
action, being much moi-e active against
certain microorganisms than against others.
The phenomena of antagonism among
microorganisms, notably among actinomy-
cetes and bacteria, ha\-e been examined in
detail by Greig-Smith (1917), Millard and
Taylor (1927), Alexopoulos and Ilerrick
(1942), Waksman (1937, 1945, 1947), Florey,
Chain et al. (1949, 1952), and Rehacek ct al.
(1960). The ecological aspects of microbial
antagonisms have been reviewed by Brian
(1957) and Waksman (19(U). In discussing
the phenomena of symbiosis and antagonism,
one cannot overlook the phenomena of
adaptation (Stanier, 1953).
Definition of an Antibiotic
The word antibiotic is now an integral
part of the vocabulary of the layman as well
as of the scientist and the medical man. Like
any term that is employed widely, however,
the word is often used loosely.
As pointed out previously, the word
antibiosis was used in 1889 by Vuillemin to
describe a type of association in which one
living creature was destroying another in
order to sustain its own life. This broad con-
cept changed in time, and Papacostas and
Gate (1928) limited the meaning of the word
in their re\'iew of the problem of bacterial
associations. According to them, when one
organism was exerting an injurious effect
upon another in vitro, the type of association
should be called "antibiosis"; when the
same phenomenon occurred in vivo the
association should be called "antagonism."
The noun antibiotic was introduced by
Waksman in 1942 (1947) to designate a
chemical substance of microbial origin which
had the property to inhil^t the growth of
microorganisms. Waksman in 1947 published
the following definition of the word: "An
antibiotic is a chemical substance, produced
by microorganisms, which has the capacitj'
to inhil)it the growth and even to destroy
bacteria and other microorganisms." Bene-
dict and Langlykke, later in 1947, modified
this definition to comprise substances which
act upon certain organisms at least, in very
dilute solutions. This ([ualification avoided
the inclusion among antibiotics of such
products of microbial metabolism as acetic
acid and ethyl alcohol. Waksman recognized
the \alidity of this argument and corrected
his original definition in 1951.
Se\'eral workers suggested that the word
antibiotic should not be limited to substances
produced by microorganisms. They felt that
the use of a specific term for the product of
microorganisms would seem to imply that
microorganisms have a special property that
other organisms do not have. Since higher
plants and animals are known to produce
substances similar to and in certain cases
identical with antibiotics, why not apply
the term to all substances of biological origin
which have the aforementioned properties?
Mascherpa (1954) proposed the following
definition: "Antibiotics are substances spon-
taneously produced by living organisms (or
synthetically obtained, l)ut with analogous
structui'e to that of natural products) en-
10
NATURE, FORMATIOX. AXl) ACTIVITIES
dowed with .selective antibacterial action
through antimetabolic mechanism." I'me-
zawa (1956) suggested the inclusion among
antibiotics not only of substances of mi-
ci'ol)ial origin but also those produced l)y
higher forms of life; their action should not
be limited to only microbes, but should also
include tumors. The definition thus becomes:
"Antibiotics are chemical substances that
are produced by living organisms and that
have the capacity to inhibit the growth of
microorganisms or other living cells."
Words are but a means of conveying ideas
from the mind of one person to another. The
chief requisite is that the meaning of the
word be clear to everyone.
We feel that the word antibiotic should be
used in its original meaning. An antibiotic
is not, howe\'er, a uniciue type of substance;
it can l)e an antibiotic and something else
at the same time. For instance, chloram-
phenicol is an antibiotic and also a synthetic
chemotherapeutic agent, since it can be
synthesized chemically. Citrinin is an anti-
biotic from PeniciUinm citrUinm and also an
antibiotic-like substance produced by a
plant.
The word antibiotic does not imply any
specific type of action of the substance so
long as the effect is produced by minute
concentrations. Certain antibiotics act in an
indirect fashion. For instance, penatin, pro-
duced by certain penicillia, is toxic to micro-
organisms because of its enzymatic liberation
of hydi-()gen peroxide.
Antibiotic Production in Soil
Although antagonism caused by the pro-
duction of antibiotics is easily demonstrated
under the artificial conditions of laboi'atory
culture, it is rather difhcult to demonstrate
that production of antibiotics by soil micro-
organisms in natural soil does occur. This is
complicated by the fact that most of the
antibiotics are readily destroyed by some
of the microorganisms present in the soil.
One method used to study this phenome-
non consists in inoculating soil with known
antibiotic-producers and, after a period of
incubation, attempting to detect the particu-
lai- antibiotic. Four different types of experi-
ments have thus been performed. The antag-
onist is inoculated: (1) into sterile soil
supplemented with various nutrients, such
as sugars, proteins, and peptones; (2) into
sterile unsupplemented soil; (8) into un-
sterilized soil with supplemented materials;
and (4) into unsterilized unsupplemented
soil.
Demonstration of the production of an
antibiotic in sterile soil supplemented with
organic nutrients is easy, since the environ-
ment is highly artificial and not very differ-
ent from that of pure culture studies in
laboratory media (Siminoff and Gottlieb,
1952). In sterile unsupplemented soils, few
antibiotics have ever been produced.
In the presence of a normal soil microflora,
demonstration of the production of a given
antibiotic in soil becomes most difficult be-
cause members of the microflora compete for
food with the artificially added antagonist
and, moreover, if the antil)iotic is formed, it
is commonly microbiologicall.v degraded.
In\'estigators have surmounted these diffi-
culties by adding massive inocula of the
antagonist imder study to nutritionalh"
favored loci in the soil. The production of
known antil)iotics in normal soil, in loci rich
in organic matter, was demonstrated by in-
oculating organic materials such as straw
and seeds of higher plants with the known
antibiotic-producers and burying them in
soil. After a period of incul)ation, proper
extraction techniques revealed in certain
cases the production of the antibiotic under
investigation. This method was successful
mainly in the study of fungal antibiotics
(Jeffreys et at., 1953; Wright, 1956).
From these experiments we can conclude
that the production of antibiotics, as we
visualize it in the laboratory, does not occur
MICROBIAL ANTAGONISMS AND PRODUCTION OF ANTIBIOTICS
II
ill nature. The useful aiitil)i(>tics, which are
mainly the product of the metabolism of
actinomycetes, fungi, and bacteria, are not
natural products in the same sense that
(juinine is. It is possible to collect l)ark from
Cinchona trees in naturally occurring stands
and to extract from this bark commerciall.y
useful (luinine. It is not possible to extract
soil and isolate from the extract chloram-
phenicol or any of the other important anti-
biotics. Antil)iotics as we know them are
laboratory products obtained by growing
pure cultures of microorganisms under nu-
tritionally rich and well aerated conditions
not to be found in the soil.
The difference between the ease of ex-
tracting ciuinine from a tree grown under
conditions unaltered by man and the im-
possibility of extracting chloramphenicol
from Streptomyces venezuelae naturally oc-
curring in a normal soil may be only a re-
flection of the differences in the physical
size of the tree, the actinomycete, and man.
The actinomycete in a natural soil sample
is indeed a needle in a haystack.
The possibility then remains that small
amounts of antibiotics may play a role in
the microcosm that surrounds the hyphae
of the antibiotic-producing organism. These
antagonistic interactions would probably be
more pronounced in the vicinity of accumu-
lated organic matter. At present, as can be
seen from the excellent discussion of Brian
(IQoT), too little solidly grounded informa-
tion is available to warrant much more than
speculation about the ecological significance
of antibiotic production.
Production of Antibiotics by Actino-
mycetes
Be that as it may, the actinomycetes are
prolific antibiotic-producers in . the labora-
tory and the factory. The compilation of
antibiotics of actinomycetes in Part B of this
book includes the listing and description of
some 400 chemical substances and prepara-
tions. Alost of these were found in the cul-
ture broths of various organisms and a few
in the mycelium. These substances and
preparations vary greatly in their physical
and chemical properties, antimicrobial ac-
tivities, toxicity to animals, and chemo-
therapeutic potentialities.
About 80 antibiotics produced by actino-
mycetes have already found extensive ap-
plication ill the treatment of various human
and animal diseases. These include strepto-
mycin and its derivative dihydrostrepto-
mycin, chloramphenicol, the tetracyclines,
^'iomycin, neomycin, cycloserine, erythro-
mycin, no\T)biocin, oleandomycin, kanamy-
cin, A-ancomycin, cycloheximide, nystatin,
amphotericin B, trichomycin, and paromo-
mycin. They are used in the treatment of a
great variety of infections caused by gram-
positive and gram-negative bacteria, myco-
bacteria, rickettsiae, the members of the
psittacosis-lymphograiiuloma group of intra-
cellular parasites, trichomonads, amoelxie
and other protozoa, monilia and other fungi.
Some antibiotics, such as streptomycin and
cycloheximide, have found application in the
treatment of plant diseases. Some are used in
animal feeding. Some, like the tetracylcines,
have found application in the preservation of
poultry and certain other foodstuffs. This
wide range of usefulness makes the actino-
mycetes the most important of all the anti-
biotic-producing microorganisms. Only four
fungal products (penicillin, fumagillin, vari-
otin, and griseofulvin) and three bacterial
products (bacitracin, polymyxin, and tyro-
thricin) are used in medicine.
Chapter 2
How Antibiotics Came to be
Recognized
Anyone attempting to analyze the his-
torical background of our present day knowl-
edge of antibiotics must take into considera-
tion not only scientific concepts, but also
popular observations and beliefs. Moreover,
there has always been the danger of reading
into observations the results of subsequent
experiments. With our present knowledge,
we are now able to analyze complicated
observations of the past in simple terms,
but we may forget that the pioneer observer
was not thinking along the same lines that
we now are. It is a further problem to decide
whether one observation truly exerted an
influence upon a subsequent line of scientific
development, since this is largely a matter
of interpretation. Was it merely a name
proposed at the right time, or was it the
persistence of one or another investigator
that turned a particular obser\^ation into an
important scientific or practical contrilui-
tion?
Even though we have hindsight at our
disposal, we cannot always analyze faith-
fully the events of the past, but we can try.
1. One may first consider the origin of
our knowledge of penicillin. The French
bacteriologist Duchesne wrote a thesis,
published in 1897, on the antagonisms be-
tween fungi and bacteria. He described ex-
periments in which injection of large
amounts of a culture of PcmciUium
glaucum permitted the sur\nval of guinea
pigs that had received lethal doses of gram-
negative bacteria. He concluded: "One
might thus hope that by pursuing the study
of biological competition between molds and
microbes, one might be led to the discovery
of other facts which would be directly use-
ful and applicable to prophylactic hygiene
and to therapy." Had he lived long enough
or had others appreciated the significance
of his work, the discovery of penicillin
might have come at a much earlier date.
Accurate observations on the production of
l)acteriolytic agents by "a strain of Peni-
cillium glaucum'" were made in 1925
by the Belgian bacteriologist Gratia (1930).
Had he used a specific name ("The trouble
with you, my boy," said the famous im-
munologist Bordet of Brussels to his former
student Gratia of Li'^ge, "is that you do not
christen your babies.") for the metabolic
product of his PcniciUium culture that
exerted an inhibiting effect upon bacteria,
the now famous antibiotic might have been
known under a totally ditferent label.
Finally, Chain and Florey (1940) of Oxford
University, England, after ha\'ing studied
various natural products, including the
lysozyme of Fleming (1922), turned their
attention in 1938-1939 to the antimicrobial
substances produced by microorganisms.
The.y isolated from a culture of PcniciUium
notatum the active antibacterial substance
observed by Fleming in 1928 (1929) and
12
RECOGNITION OF ANTIBIOTICS
i;^
designated later by him as "penicillin," and
demonstrated its effectiveness in the treat-
ment of human diseases. Thus was cul-
minated the historical background of the
most useful of the antibiotics. With hind-
sight we can say that, as was true of the
sulfa drugs, penicillin could have been dis-
covered sooner.
2. An even older antibiotic, pyocyanase,
offers another illustration. This antibiotic
was extensively studied for nearly half a
century. It failed, however, tf) achieve
practical use because it was a, highly com-
plex and somewhat toxic compound. Had
another organism producing a more de-
sirable substance been used, the field of
antibiotics might have been opened much
earlier.
3. The production of antibacterial sub-
stances by aerobic spore-forming bacteria
has been known for many years. It was in-
vestigated hi detail by Xicolle in 11)07, by
Pringsheim in 1920, and by Much and
Sartorius in 1924-192."), to name only a few.
All this work failed to receix'c the attention
it deserved. It took some years before this
problem was taken up again in a more
systematic manner by Dubos (1939), who
successfully isolated in 1938 a group of
active substances known as tyrothricin,
thus opening the field of the antil)acterial
antibiotics.
4. The l''rench clinician N'audremer re-
ported in 1913 that the mold Aspergillus
fiimigatus exerts a marked antitubercular
effect. The extracts of this mokl were used
in the treatment of 200 tubercular patients
with varying degrees of success. Had the
potentialities of such a method of treat-
ment been more clearly visualized, screening
methods might have been established, the
study of chemotherapy of tuberculosis
might have been initiated, and perhaps the
problem might have been solved much
earlier; the world would not have had to
wait nearly three decades longer before
streptomycin was isolated and its anti-
tubercular properties were established.
o. Lieske (1921) in Germany, Krassilni-
kov and Koreniako (1939) and Kriss (1940)
hi Russia, Gratia and Dath (1924) in
Belgium, and Rosenthal (1925) in France,
among others, recognized that cultures of
the various actinomycetes possess marked
antibacterial properties. The agents studied
by these investigators were often bacterio-
lytic. The concept of a nonlytic antibiotic
was not very clear. Gratia and his collab-
orators were able not only to experiment on
animals with the therapeutic powers of
preparations obtained from an actinomycete
(Streptothnx albus), but even to treat with
success human patients suffering from
staphylococcal infections. The methods were
ciuasi-immunological. The lytic properties of
the actinomycetes have been associated with
the phages of d'Herelle and with the lyso-
zyme of Fleming, and to this day the litera-
ture on the actinomycetic agent of Gratia,
later named aclinomucetin by Welsch (1937,
1942), tends to be rather confusing.
(). Whereas most of the above observa-
tions were largely concerned with the anti-
bacterial properties of actinomycetes,
Aliillcr (1908) and, more recently, Alex-
opoulos and Herrick (1942) demonstrated
that as many as 38.8 per cent of such cultures
were also effective against fungi.
The opening of the field of the antibiotics
of actinomycetes was delayed until 1940,
when actinomycin, a substance simpler than
actinomycetin, was isolated in our labora-
tories. Here again, a lack of foresight on our
part should be noted. We understood the
concept of antiliiosis, but we did not foresee
the potentialities of actinomycin as an anti-
tumor agent. This discovery had to wait for
the investigations of Stock (1950), Hack-
mann (1952), and numerous others.
Despite this fumbling, antibiotics be-
came a part of our li\-es, following the route
that we now will trace.
14
NATURE, FORMATION, AND ACTIVITIES
Early Observations
The earliest ob,ser^■ations on the effect of
microbial products upon disease came be-
fore microbes were recognized and their
metabolic processes known. They were
made long before the etiology of human and
animal diseases was established.
The Bible and postbiblical writings, such
as the Apocrypha, contain numerous ref-
erences to the effect of the soil upon the
destruction of disease-producing organisms:
And when thou goest out of thy camp
Take a .spade with you
And cover uj) wliat comes out of nou.
There is actutd reference to the presence
of medicines in pi'oducts of the soil:
The Lord created medicines out of the earth,
And he that is wise shall not abhor them.
Those who gave such adxice may have
had an inkling of many modern ideas which
were the result of subsequent observations.
Since a great many antibiotic-producing
organisms inhabit the soil under our feet,
the above statement may have some justi-
fication. The introduction of pathogenic
microbes into the soil is known to result in
their destruction and for many years was
even thought to induce the development of
organisms that are able to produce anti-
biotics. This theory has not been supported
by more critical experimental evidence.
Folklore abounds in prescriptions con-
sisting of the application of moldy cheese,
rotting meat, and other moldy products to
wounds to treat infections or prevent their
development. Jules Brunei, in an article
published in 1944, quoted the following
statement from a Canadian biochemist: "It
was dvu'ing a \'isit through central Europe
in 1908 that I came across the fact that
amost every farm house followed the practice
of keeping a moldy rye loaf on one of the
beams in the kitchen. When I asked the
reason for this I was told that this was an
old custom and that when any member of
the family received an injury, such as a cut
or a bruise, a thin slice from the outside of
the loaf was cut off, mixed into a paste with
water and applied to the wound with a
bandage. I was assured that no infection
would then result from such a cut."
The North American Indians were also
users of moldy products. In the eighteenth
century, they were reported to have applied
rotten wood to wounds to pi'ex'ent suppura-
tion.
In Europe, bakers' yeast was applied to
abscesses, probably with the intention of
making the abscesses c(jme to a head more
rapidly. This practice was followed by most
of the reputal)le medical authorities, includ-
ing Lieutaud, physician to Louis XVI. The
yeast was also taken orally. Perhaps this
was early vitamin therapy rather than early
antibiotic therapy. We might even consider
this as a precursor of the combined anti-
biotic-vitamin therapy which is now so
popular !
First Experimental Observations
With the growth of scientific medicine
during the nineteenth century, certain
events took place that were to be the basis
of modern antibiotic research. These can be
briefly summarized as follows:
1 . There was a growing knowledge of
mixed infections and the replacement of
parasites by saprophytes when the latter
were introduced into infected animals.
2. Soil microbiology was born, bringing
into focus the complex microbial population
of the soil and the interrelations among
different microorganisms in natural sub-
strates.
3. The investigation of the effect of green
manures upon the control of the potato scab
organism and the effect of organic manures
upon certain root rots of plants led to de-
velopments in the field of plant pathology
which contributed to our undei-standing of
RECOGNITION OF ANTIBIOTICS
15
the interrelations })et\veen disease-produe-
ing and sapropl^vtic microlies in plant
diseases.
4. Direct observations were made on the
effect of metabolic products of saprophytic
organisms upon disease-pi'oducing organisms
and upon infections.
One of the early observations, made by
Roberts in 1874, is of particular interest hi
this connection. Certain liquid extracts in
which a green mold had been growing
luxuriantly became infected with bacteria
only with great difficulty. The possibility
was suggested that the mold held in check
the growth of bacteria. On the other hand,
liquids full of bacteria did not favor good
growth of the mold {PeniciUium glaucum).
An antagonism was also observed between
the growth of different races of bacteria.
Roberts concluded, "There is probably in
such a case a struggle for existence and a
survival of the fittest."
In 1876, the British physicist Tyndall also
reported on the growth of wild cultures of
l)acteria and fungi in organic infusions; he
spoke of "the struggle for existence between
the bacteria and the penicillium. In some
tubes the former were triumphant; in other
tubes of the same infusion the latter was
triumphant." He concluded that ''the
bacteria which manufactiu'e a green pigment
appear to be uniformly victorious in their
fight with the peniciUiiim.'"
These early reports heralded the develop-
ment of penicillin and pyocyanase.
Soon afterward, in 1877, Pasteur and
Joubert noted that "... one can infect
abundantly an animal with anthrax with-
out the animal becoming diseased; it is
sufficient that the fluid contain in sus-
pension simultaneously the anthrax or-
ganism and a common or harmless bac-
terium." They then added prophetically,
"These facts perhaps justify the highest hope
for therapeutics."
Soon numerous other observations were
recorded concerning the effect of saprophytic
microbes upon disease-producing organisms.
Cantani, for example, wrote in 1885: "The
known fact that certain bacteria can destroy
the cultures of other microbes, even those
that are pathogenic, if they come into con-
tact with them in any way, gave me the idea
of exploring this procedure for the treat-
ment of various infectious diseases." He
treated pulmonary tuberculosis by intro-
ducing by nebulization the saprophyte
Bacterium termo into the lungs of a patient.
Even though he reported clinical improve-
ment, the method was never used on a large
scale.
The very same year, C'ornil and Babes
wrote: "The study of the reciprocal action
that l)acteria have one upon another,
persisted in, and enlarged in scope, might
lead to therapeutical results." These authors
are also credited with introducing the agar
cross-streak test. This test was further de-
veloped by Garre, who showed in 1887 that
strains of Pseudomonas were producing a
specific diff'usible substance which was able
to inhibit the growth of staphylococci and
other pathogens.
Two to three years later a number of
papers were published \\hich focused atten-
tion on the great potentialities of Pseudo-
monas aeruginosa. Bouchard, Charrin and
Guignard, Kitasato, Woodhead and Wood,
and Blagovestchensky all demonstrated the
antibiotic powers of this l)acterium against
the anthrax bacillus and other pathogens.
Honl and Bukovsky, in 1899, treated
with an ill-defined extract of Pseudomonas
aeruginosa more than 100 patients who had
infected wounds. The results were good. In
the same year, Emmerich and Low reported
their work on pyocyanase, also an ill-defined
extract of the same bacterium. Pyocyanase
was bactericidal and bacteriolytic (references
to above papers are found in a paper by
Lagodsky, 1951).
Before the turn of the century, attention
16
NATURE, FORMATION, AND ACTIVITIES
was attracted to the antibiotic potentialities
of actinomycetes. Gasperini, in 1890, re-
ported that the organism Strcptothri.r Foer-
steri, isolated as a contaminant, was able to
lyse bacteria and fungi. It was also before the
turn of the century that the first antibiotic
was isolated in a crystalline form. From a
strain of PeniciUium, Gosio, in 189(>, isolated
and crystallized the antibiotic which was
later called mycophenolic acid.
Laying the Foundation for Antibiotic
Research
The first four decades of the present
century were characterized by marked ad-
vances in chemotherap3^ First came the
arsenicals that were shown to have activity
against trypanosomes. Ehrlich and his col-
laborators made a great number of chemical
derivatives of the first compound, atoxyl, in
the hope of finding a substance that would
retain the antimicrobial activity of atoxyl
but be less toxic to the animal body. In 1909,
the 606th compound was found to possess
the desired properties and was named
salvarsan.
Starting from the fact that certain azo
dyes were kn^own to have bactericidal
effects in vitro, a group of chemists from the
Bayer laboratories in Germany synthesized
a large number of these compounds. These
were tested by Gerhard Domagk in mice
infected with hemolytic streptococci. In
1932, one of the dyes, prontosil, was found
to be effective iii vivo. Strangely enough, it
was active only in vivo. This was explained
in 1935 by Trefouel and his collaborators at
the Pasteur Institute. They showed that the
dye was split in the body, that sulfanilamide,
one of the products formed, was the active
portion of the molecule anfl that it was ac-
tive both in vitro and in vivo. Hundreds of
derivatives of sulfanilamide were sub-
sequently synthesized and tested in animals
(references to the above papers are given by
Albert, 1951).
The extensive testing of arsenicals and
sulfa drugs led to the development of micro-
biological, pharmacological, and pharma-
ceutical methods necessary for the progress
of antibiotic research.
During the first four decades of the
twentieth century, while spectacular work
was being done in the field of chemotherapy
with arsenicals and sulfa drugs, numerous
observations, similar to those made during
the nineteenth century, were published in
the field of microbial antagonism. Only the
most important developments will be dis-
cussed here.
The pyocyanase of Iilmmerich and Low
(1899) was studied in experimental animals
and in human patients. P'or a short time
commercial preparations of pyocyanaise
were available in Germany and were used
mainly topically. The theory that pyo-
cyanase was an enzyme did not last long,
since lipoidal, heat-stable fractions of the
crude extract were found to have anti-
bacterial action.
The fungal antibiotic isolated in pure
form by Gosio in 1896 was studied again in
1913 by Alsberg and Black, and later by
Clutterbuck et al. (1932, 1933). This anti-
biotic, the first to be isolated in a pure form,
is now virtually unknown.
In any scientific climate, the minds of
men are molded by the prevailing ideas of
the time. During the first four decades of
the twentieth century, emphasis on en-
zymes and lytic phenomena was to hinder
the development of antibiotics. This is true
of the discovery of the Twort-d'Herelle
phenomenon in 1915-1916. As mentioned
previously, Gratia and his collaborators in
Belgium had studied the bacteriolytic
properties of actinomycetes. It should l)e
stressed that these authors were prol)at)ly
the first to use the product of an actino-
mycete in human therapy. The treatment
seems to ha\-e been used mainly for staphylo-
coccal infections and consisted of injecting
RECOGNITION OF ANTIBIOTICS
17
bacteriophages in eoml)iiuition with what
was then called "mycolysates of staphylo-
cocci." These mycolysates were the products
of the lytic action of an actinomycete on the
pathogen. This actinomycete was identified
as Streptothrix alhus and the antibacterial-
bacteriolytic complex formed by this or-
ganism was named actinomycetin by Welsch
in 1937. In a paper published in l!)oO,
Gratia said: "We ha\'e used this association
of the bacteriophage and of mycolysates of
staphylococci in a very great number of
cases and we believe that we may say that
it is today the most effective treatment of
staphylococcal infections. . . ."
Fleming, working at St. Mary's Hospital
of London, had been interested first in the
cellular aspect and then in the humoral
aspect of immunity. He studied the bac-
teriolytic properties of nasal and ocular
secretions. In 1922, he gave the name lyso-
zyme to a bacteriolytic enzyme al)iuidantly
distributed in nature. Lysozyme was later
found to be a mucopolysaccharidase which
now plays an important role in studies of
bacterial biochemistry. In 1929, Fleming
pul)lished a paper on the antibacterial ac-
tion of a strain of Penicillium. Since the
culture filti'ate of the Penicillium was
mainly active against gram-positive bac-
teria, Fleming considered that it could be
used for the making of selective media for
the isolation of gram-negative bacteria. He
named this active filtrate penicillin. A few
more papers were pulilished on this crude
substance, but they attracted little atten-
tion. In 1982, Clutterbuck, Lovell, and
Raistrick published a paper on the metabolic
products of the PcnicilHion of Fleming,
which was properl^^ identified by Charles
Thom as P. notatum. They found a chem-
ically defined medium on which it grew and
produced the antibacterial substance, which
proved to be markedly unstable. Their con-
cluding sentence, "The investigation of the
isolation and chemical nature of penicillin is
being continued," was almost the epitaph of
Fleming's penicillin !
The Discovery of Antibiotics
The year 1939 was a milestone in the
history of the world. The Second World War
had started, and as the guns and the bombs
began to settle human differences, scientists
and physicians were faced with the problem
of salvaging what they could of the mangled
flesh.
An impetus was thus gi\'en to the search
for antimicrol)ial agents, but this effect was
not to be felt immediately in 1939. This date
is nevertheless a turning point in the history
of chemotherapy. It was about then that the
work in Dubos' laboratory at the Rocke-
feller Institute yielded tyrothricin, and re-
search in the Xew Jersey Agricultural Ex-
periment Station yielded actinomycin. It
was also at that time that Chain aiul Morey
at Oxford I'ni^•ersity carried out their now
famous evaluation of penicillin.
The new study of penicillin showed the
great potentialities of this antibiotic as a
chemotherapeutic drug. With the war in
process, the British investigators needed help
to carry out the titanic jjroblem of producing
penicillin and elucidating its chemical
structure. In 1941, Florey and Heat ley came
to the United States. A vast research pro-
gram was set up which involved the co-
operation of government laboratories, such
as the Northern Regional Research Labora-
tory in Peoria, of universities, especially the
University of Wisconsin, and of many
industries. Better media were developed;
more acti\'e strains of penicillia were iso-
lated; better methods (deep-tank) of culture
were introduced on a large scale. Penicillin
was found to be therapeutically effective
antl could now be produced cheaply. It
opened the eyes of the world to a new type
of chemotherapeutic agent, of low toxicity
and of high activity, against a variety of
infectious diseases caused }),v gram-positive
18
NATURE, FORMATION, AND ACTIVITIES
bacteria, cocci, spirochetes, and other
organisms.
In 19ol, Dubos and Avery incorporated
polysaccharides from the capsule of type III
pneumococci in soil. They isolated from the
soil a bacillus which, when grown in a
synthetic medium containing the poly-
saccharide as the source of carbon, elabo-
rated an enzyme that specifically attacked
the organism. This enzyme could protect
mice from a fatal dose of type III pneumo-
coccus. Dubos continued the soil enrichment
study by introducing living l)acteria into the
soil, rather than the capsular material alone.
From these enriched soils Dubos in 1938
isolated a strain of Bacillus brevis which
produced an antibiotic that he called tyro-
thricin. In 1940, Hotchkiss and Dubos
showed that tyrothricin was composed of
two active antibiotics, tyrocidine and grami-
cidin. Tyrothricin is active chiefly against
gram-positive bacteria and is used topically
in medicine. The tyrothricin complex
opened the field for bacterial polypeptides,
to be followed soon by bacitracin, polymyxin,
subtilin, and a variety of others. The prac-
tical importance of some of these is still not
fully recognized. Even though it is doul)tful
that the soil enrichment method was a
necessary step in the isolation of the active
strain of B. brevis, the work of Dubos showed
the value of systematic screening programs
in the search for antibiotics.
Another blow to the chance isolation pro-
cedure f^f obtaining antibiotic-producing
strains was dealt at about the same time by
what could be called the Rutgers group of
investigators. Ever since 1936, the senior
author of this treatise had been interested in
antagonistic relationships among soil micro-
organisms. Actinomycetes often showed
themseh'es to have outstanding antimicro-
bial activity. In 1940, Waksman and Wood-
ruff reported the isolation of actinomycin,
the first actinomycete-produced antibiotic
to be obtained in a crvstalline form. After
this discovery, the attention of the Rutgers
group turned to products of fungi, and
cku-acin, fumigacin, and chaetomin were
isolated. Actinomycetes were not forgotten,
however, and systematic screening programs
yielded, among others, streptothricin (1942),
and most important of all, streptomycin
(1944). Streptothricin was an interesting
substance. It was basic, stable, and water-
soluble; it was active against gram-negative
and gram-positi\'e bacteria, m^ycobacteria,
and fungi in vitro and in vivo; however, it
had a delayed toxicity that limited its use-
fulness. Streptomycin had the same general
chemical properties as streptothricin but
was less toxic; infections caused by gram-
negative and gram-positive bacteria and
mycobacteria responded to treatment with
this new drug. The chemotherapy of tuber-
culosis was finally made possible.
The Modern Period
The success of the screening methods of
the Rutgers group was partly responsible
for the scrutiny of the actinomycetes in
screening programs throughout the world.
The results were rewarding. Antibiotics were
isolated which are active not only against
gram-positive and gram-negative bacteria,
but also against rickettsiae and the psitta-
cosis-lymphogranuloma group of organisms.
These antibiotics include chloramphenicol
(1947), chlortetracycline (1948), oxytetra-
cycline (19o0), and tetracycline (1953).
Others were to come later, namely, the
macrolides erythromycin (1952), carbomy-
cin (1952), spiramycin (1954), and oleando-
mycin (1954). Still others, similar to strepto-
mycin in certain respects, have found a
place in chemotherapy; these included
neomycin (1949), viomycin (1951), and
kanamycin (1957).
Antibiotics were also found which are
primarily antifungal, such as cycloheximide
(194(5), nystathi (1951), candicidin (1952),
RECOGNITION OF ANTIBIOTICS
19
trichomycin (1952), candidin (1954), and
amphotericin B (1955). Others have been
found which ha^'e pronounced antitumor
action; these include, apart from the actino-
mycins, azaserine (1954), .sarkomycin (1953),
carzinophilin (1954), 6-diazo-5-oxo-L-norleu-
cine (DON) (1956), and mitomycin (1956).
New antibiotics are still being discovered;
most of them are being isolated from cultures
of actinomycetes. Some are still being
introduced into medical practice. As yet,
however, no useful anti\'iral antibiotics have
been isolated.
The uses of antibiotics have been extended
far beyond their original chemotherapeutic
provinces, and e\'en into the fields of animal
feeding and food preservation. Some anti-
biotics are used by the geneticist to select
mutants of bacteria; here streptomycin oc-
cupies a place of choice. Others are used by
the biochemist as specific inhibitors of
metabolic reactions, such as chloramphenicol
(inhibition of protein synthesis) and anti-
mycin A (inhibition of cj'tochrome oxidase).
The marvelous potentialities of the anti-
biotics have not vet been exhausted.
Chapter 3
The Search for Antibiotics:
Screening Programs
The detection of antagonists capable of
producing antibiotics was at first a matter
of chance. It was by chance that one of the
bacterial culture plates of Fleming was con-
taminated by the now famous Penicillimn.
Since then, investigators have tried in many
ways to be more systematic in their methods
of detecting antibiotic-producers.
Basic Screening Procedures
We have already discussed Dubos' en-
richment of soil samples with pathogens in
an attempt to increase the chances of de-
tecting antibiotic-producers. This method
would be logical if antibiotics were lytic
enzymes which would ha^'e the property of
making the pathogen available as food for
the antagonist. Since this is not the case, the
soil enrichment method has remained one of
historical interest with no practical im-
portance, as demonstrated in 1946 b}^ Waks-
man and Schatz.
A second approach comprised efforts to
induce antagonistic properties in a non-
antagonist. Basically the method consisted
in confronthig a pathogen with another mi-
croorganism in a medium poor in nutrients,
in the hope that the microorganism would
become antagonistic toward the pathogen.
Successful results have been reported by
Schiller (1952) in Russia and by Davide
(1949) in Sweden. ]\Iany other investigators,
including us, have tried this method without
success.
By far the most successful method in the
search for antibiotics has consisted in test-
ing the antagonistic properties of large num-
bers of microorganisms in vitro. The general
procedure can be modified in a number of
ways. Briefly, the method comprises the fol-
lowing steps: (1) The substrate to be studied
is plated out on media which permit the
growth of actinomycetes. (2) The various
actinomycetes are isolated in pure cultures,
usually on slants of media favorable for
abundant growth of these organisms. (3)
Each actinomycete culture is inoculated in
Petri dishes containing agar media consid-
ered favorable for the production of antibi-
otics; the inoculation is usually made as a
broad streak so that incubation yields a rib-
bon of growth of even width. (4) After
growth of the actinomycete, at what is con-
sidered a favorable temperature (25-30°C)
for a favorable length of time (.3 to 7 days),
test organisms, against which antagonists are
sought, are streaked at a right angle to the
actinomycetic ribbon. (5) After incubation
of the test organisms under optimal condi-
tions for their growth, the antagonistic po-
tentialities of the actinomycetes are esti-
mated by the width of the inhibition zone.
Such cross-streak tests are illustrated in
Chapter 15 of Volume L
There is, of course, no ideal medium which
permits the plating out of a natural sub-
strate with the resulting growth of all the
actinomycetes pi'esent in the substrate and
20
SEARCH FOR ANTIBIOTICS: SCREENING PROGRAMS
21
which inhibits the growth of all other micro-
organisms. Porter et al. (19(i0) advocate the
use of an arginine-glycerol agar.
The addition of selective inhibitors per-
mits reduction of the number of fungi or
true bacteria and helps in the isolation of
actinomycetes in pure cultures. Corke and
Chase (19o6) have used with success the
antifungal antibiotic cycloheximide to elim-
inate fungal growth. Lawrence (1956) re-
duced the number of contaminating bacteria
and fungi by pretreating the samples to be
plated out for 10 minutes with a 1:140
dilution of phenol. The successful use of
centrifugation of suspensions of substrates
to be plated out, as a means of separating
actinomycetes from other microorganisms,
was reported l)y Rehacek in 19")G. The
suspensions were centrifuged for 20 minutes
in tubes which were subjected to 904 X g
on the surface and 1609 X g on the bottom.
Under such conditions most actinomycetes
were not sedimented, whereas most other
microorganisms were.
Another method consists in increasing the
number of actinomycetes present in a soil
sample before plating out. Tsao ct al. (1960)
dried soil samples and then incubated them,
buffered with calcium carbonate, in a moist
atmosphere. This resulted in an increase in
the percentage of viable actinonwcetes in
these soil samples.
For the isolation of actinomycetes in pure
culture, there is, of course, no universal
medium. The authors consider yeast extract-
glucose agar the best general medium for
this purpose. In certain countries where
standard preparations of yeast extract are
not available, a bakers' yeast-salt medium
is a good substitute (Lechevalier and
Tikhonienko, 1960).
It is always desirable to rememljer that
the use of various other media may permit
the growth of organisms which might not
grow, or which might grow poorly, on yeast-
glucose agar.
The media used for the actual cross-streak
Table 1
Cross-sireak test of 269 actinomvcetes freshly
isolated from soil and from manure
Comparison between results obtained on nu-
trient agar and on yeast-glucose agar.
Mycobacterium smeg-
matis 607
.1/. smegmatis 105. . .
-1/. smegmatis 7992. .
.1/. phlei 23
Percentage of total number tested
On nutrient agar,
width of inhibi-
tion zone
1-10
mm
11-20
mm
10
11
10
27
>20
mm
On yeast-glucose
agar, width of
inhibition zone
1-10
mm
11-20
mm
>20
mm
3
2
19
test must l)e favorable for growth of both
the actinomycete and the test organisms.
The ideal medium for such tests should also
be free from chemicals that might inhibit
the action of the antibiotics produced by the
actinomycete. The selection of such a me-
dium must, of course, be highly empirical.
The influence of the medium used in the
cross-streak test and the results obtained are
illustrated in Table 1. A group of 269 freshly
isolated strains of actinomycetes were tested
for activity against four strains of fast
growing, nonpathogenic mycobacteria on
two different media, nutrient agar and yeast-
glucose agar. The same amount of agar (15
ml) was used in all plates to permit com-
parison of the width of inhibition zones.
One will note that approximately the same
percentage of strains formed narrow and
broad zones of inhibition on both media, but
that the percentages of medium width zones
were higher on nutrient agar than on yeast-
glucose agar. This suggests that certain
types of antibiotics responsible for the
medium width zones were not formed or
were inacti\'ated by yeast-glucose agar.
Demonstration that an antagonist can
produce a diffusible substance efYective upon
the test organisms chosen in a given screen-
ing program must be followed bj^ demon-
22
naturp:, formation, and activities
stration that this substance can also be
produced in liquid media. This is of prime
importance, since antibiotics must be ob-
tained in liquid media for large-scale produc-
tion.
Since the production in liquid media is the
chief concern of an industrial microbiologist,
many large-scale screening programs bypass
completely the cross-streak test and inocu-
late their isolates directly into liquid media,
which are usually incubated on shaking
machines to furnish the aeration necessary
for the growth of most actinomycetes.
The following scheme outlines the basic
screening procedures discussed above :
spraying the test organism (Stansly, 1947)
on the surface of the plate or by flooding this
surface with a water-agar suspension of the
test organism.
One of the disadvantages of these methods
was that they did not permit the testing of
activity against more than a single test
organism. Another difftculty encountered in
the screening of actinomycetes is that media
selective for the growth of these organisms
are often not good for antibiotic production.
An adaptation of Lederberg and Leder-
berg's replica plating method was described
in IQoS by Lechevalier and Corke. The
substrate was plated out with a dilute sus-
I. Selection of natural substrates as a potential sovirce of actinomycetes
II. Isolation from substrate of actinomycetes in pure cultures —
i
Selection ot test organisms ttj r-i j- + <-• <• i- ^ i
. u- u 4^-1 ■ <-• ill- Confrontation ot actinomycetes and
against which antibiotics —* ^ ^ ■ i- i ■ t-
are desired
test organisms on solid media
I
IV. Growth of actinomycetes in li(iuid media *—
i_ "
V. Determination of aiitil)iotic activity in liquid media
Various modifications of this general
scheme ha^^e been made, and a number of
special methods have been devised.
Special Screening Techniques
The crowded-plate technique, used in the
early studies on the isolation of antibiotic-
producing organisms, consisted in plating
out hea\'y suspensions of natural substrates.
Organisms growing on such plates, sur-
rounded by a zone of inhibition, were
selected for further work. This method did
not permit the development of slow-growing
actinomycetes which were submerged by
bacterial growth before they had a chance to
elaborate any antiliiotic.
Attempts have been made to difterentiate
at an early stage between antibiotic-produc-
ing organisms and nonproducers bv inoculat-
ing a test organism directly on plates in
which the substrate had been plated out.
This inoculation was accomplished either In'
pension of the natural stil)strate to obtain
well-isolated colonies. These were trans-
ferred with a sterile velveteen stamp to a
series of plates containing media thought to
be favorable for the production of anti-
biotics. After incubation, the plates were un-
molded and turned upside down. The back
of each plate was inoculated with a test
organism. Colonies ha\'ing the desired
spectrvmi of activity could then be isolated
from the original plate from \A'hi('h all
transfers had been made.
New techniciues have been used to detect
types of biological activities undetectable by
pre\'ious procedures (Gause, 19.58). Such
was the method of Alurat cf al. (1959) which
was designed to detect antil)iotics active
against intracellular bacteria. The method
consists in allowing brucellae to be phago-
cytized by guinea pig monocytes. The re-
maining extracellular brucellae cells are
killed with streptomycin. The living brucella-
SEARCH FOR ANTIBIOTICS: SCREENIXCi PROGRAMS
23
containing monocytes are confronted with
the antibiotic or the culture filtrate to be
studied. If the antibiotic is active intra-
cellularly, the brucellae are killed; if not,
their life is detected by the formation of
bacterial colonies when the monocytes are
flooded with agar.
Among the special techniques, it is suf-
ficient to mention those devised for the
study of antagonists active against plant
pathogens (Cooper and Chilton, 1949; Stessel
(•t al., 1953). The actinomycetes attracted
particular attention because of the early
observations by KenKnight (1941) and
Alexopoulos (1941), among others, that they
were active upon the potato scab organism
and other plant pathogens.
Screening Surveys
Numerous surveys Xvcwe shown that
actinomycetes capable of producing anti-
biotics in vitro are widely distributed in
nature. They are the most common anti-
biotic-producers that can be isolated from
soil (Waksman and Lechevalier, 1901).
.1 ntimicrobial Surreys
Among the previous surveys, in addition
to those already listed, it is sufficient to m(Mi-
tion those of Borodulina (1935), Burkholder
(194(3), Waksman et al. (1946), Landerkin
and Lochhead (1950), Harris and Ruger
(1953), and Craven et al. (1960). A sum-
mary of some of l^oth older and more recent
surveys is shown in Table 2. As can be seen,
Table 2
Surveys of antagonistic activity of actinomycetes
Date of
survey
Total
cultures
tested
Percentages of cultures active against
Bacteria
Myco-
bacteria
Fungi
I
Gram +
Gram —
59
43
46
36
49
39
44
52
-2 *
46
25
47
39
33
a
63
0.6
7
4
8
10
11
a g
0.5
9
8
■a.
19
* 2
21
'5,
:«■
65
51
a
a
-o
"a
7
30
2 ^
o c
'Z 3
^^
56*
24t
48
28
o S
'C be
m
28
20
Nakhimovskaia. . . .
Alexopoulos
Waksman et al
Welsch
Waksman et al
Burkholder
Jones and Schatz . .
Cooper et al
Landerkin et al. . . .
Groupe et al
Rouatt et al
Lechevalier
Waksman et al
Asheshov et al
Aleshina and Ma-
karovskaia
Vanek et al
1937
1941
1942
1942
194G
1946
1947
1949
1950
1951
1951
1951
1952
1954
1955
1958
80
80
244
164
187
7369
176
2452
660
113
544
302
197
1000
1117
739
10
* Antagonistic to Colletotrichum gloeosporioides.
t Antagonistic to Pythinm arrhenomanes.
24
NATURE, FORMATION, AND ACTIVITIES
gi'uiii-positive bacteria and mycobacteria
are inhibited by a larger percentage of
actinomycetes than are gram-negative bac-
teria. Fungi are also inhibited b}^ a large
number of actinomycetes. ^lany inhibitors
of phages can be detected in the culture
filtrates of actinomycetes, but their anti-
phage action does not show correlation with
true antiviral action. As has already been
mentioned, no useful anti\'iral antibiotic has
yet been discovered.
Routien and Finlay (1952) screened
thousands of soil samples obtained from
widely scattered geographical areas. Certain
organisms producing streptomycin, strepto-
thricin, chloramphenicol, actinomycin, and
xanthomycin-like antil)iotics were extremely
common; in fact, they were of worldwide
distribution. Tetracycline-producing cul-
tures were isolated only a few times. One
antibiotic was obtained from only one
culture isolated from one particular soil.
Certain antibiotics were produced by or-
ganisms common in soils in certain localized
areas. Other antibiotics came from soils
collected within a restricted area. I'mezawa
et al. (1949), using the technique of Waks-
man and Schatz (194()) of adding a particu-
lar antibiotic (chloramphenicol) to the
medium before plating out soil samples, were
able to demonstrate the presence of a rather
large number of chloramphenicol-producing
strains, thus suggesting a wide distribution
of these organisms.
Janot d al. (1954) isolated 7941 cultures
of Streptomyces; 351 of these produced anti-
biotics. Streptothricin-protlucers were most
common, followed by streptomycin-, then
tetracycline-producers. Various cultures ca-
pable of forming framycetin (neomycin) were
also fovmd, followed by a group of non-
identified antibiotics.
Hirabayashi (1959) examined oKi cul-
tures of streptomycetes for antiamebic ac-
tivities. Nine of the cultures were found to
have direct antiamebic effects. An acti\'e
substance was isolated from one culture of
Streptomyces and was named protomycin. It
had no antibacterial activities. Protomycin
showed inhibitory activity upon the growth
of Endamoeba histolytica at a concentration
of 25 /xg per ml after 24 hours of incubation
and at a concentration of 1 .(> ng per ml after
48 hours of incubation. Woodruff' and
AIcDaniel (1958) emphasized that, on the
a\'erage, 25 per cent of cultures of strepto-
mycetes isolated were able to produce some
kind of an antibiotic. About 90 per cent of
all the antil)iotic-producing cultures were
found to form streptothricin or closely re-
lated compounds. At least half of the re-
mainder produced streptomycin. Of the
others, one third formed tetracyclines, and
most of the rest could be identified with one
of the hundreds of antibiotics which have
been descril)ed from species of Streptomyces.
In a study of 10,000 cultures isolated, about
2,500 showed antibiotic activity. All but 250
gaA'e streptothricin-like antibiotics; about
125 formed streptomycin; 40 produced
tetracyclines; and 55 yielded other pre-
viously described antibiotics. Only 30
cultures seemed to form new antibiotics.
The above calculations are based upon the
examination of antibacterial antibiotics.
Had a similar study included antifungal
agents as well, especially the polyene type
of compounds, the latter would have been
found to play an important part in the
distribution of antibiotic-producing prop-
erties among actinomycetes. Vanek and co-
workers (1958) found that 16.3 per cent of
739 freshly isolated strains of actinomycetes
produced polyenes. Heptaenes were the
most commonly produced polyenes, as re-
ported in 1956 by Pledger and Leche\-alier.
Plotho (1947) tried to correlate pigment
production with antibiotic activity. A
collection of 291 cultures were classified into
four groups on the basis of pigments pro-
duced either in the mycelium or in the
m(Hlium. Of 61 cultures (21 per cent) show-
.SEARCH FOR ANTIBIOTICS: SCREEXlXCi PROORA.MS
25
iug activity against M ycohactcrium cos, 21
were classified as a colorless group, 20 reel-
yellow, 12 red-blue, and 8 red-brown. It was
suggested that use of media in which pig-
ments could be detected readily might re-
^■eal a correlation l)et\veen antibiotic ac-
tivity and some other property.
Antitumor Survctjs
Considerable attention has recently l)een
focused upon the products of actinomycetes
that possess antitumor activities. A number
of different substances active against neo-
plastic cells ha\'e been isolated and de-
scribed (Table '.]). None of them can be said
to have become cures for cancers, although
some appear to offer definite promise. These
substance^s are not comparable to antil)iotics
in their activities, although many appear to
be definitely antibiotics, since they are also
active upon bacteria or fungi. They are in-
cluded in this treatise because the methods
of approach to their isolation are similar to
those concerned with antibiotics and also
because of their selective action against
different cells, 'iliese substances include
actinomycin (Waksman and Woodruff,
1940), azaserine (Bartz ct a/., lUo:!; I':hrlich
Table 3
Groii pitKj of adi iinin ),retc (lutiliiunir pnidurls
(Umezavvil, 195())
Pigmented sulistancps
Actinomyfin l'lur;iiii\ cin
Cancidin .\c-tinnH()cin
Ractinomycin ( Iriseolutein
Chromomx'cin Actiiuilpukin
Mitomycin
.\cidic .sub.staiicf's c
Sarkom\'cin
Table 4
Results of srrceuing of actinomycetes for antitmnor
substances (Umezawa, 1956)
()\\ molecular wt'iglit
fi Diazo -5-<).\(!-L-norleu-
ciiic
Carzinophiliu
Aza.serine
High molecular weight suh.stances
Carcinomycin Carzinocidin
1 'urine antimetaf)olite
Purcjmycin
Antifungal substances
Tovocamycin Hygroscopin
Mouse
Ehrlich
sarcoma
carcinoma,
180,
ascitic form
ascitic
form
Xo. actinonnoetes tested
705
98
X'o. strains prochicing anti-
tumoi- substances in first
test
130
17
Xo. strains ])ositive in re-
jaeated test
()9
Table 5
Results of scfceninti acti noui ycetes for untilu nior
substances, using HeLa cells (Umezav\a, 195())
Number
Toxic to
chick
embryo
fibroblast
Nonto.xic
to chick
embryo
fibroblast
Strains tested
Active straiTis
522
109
68
14
19
35
9
14
20
Strains active in re-
peated tests
Active against Elhrlich
carcinoma, ascitic
form
Toxic t o mice
5
5
Xo effect on l^hiiich
carcinoma, ascitic
form
15
et al., 19.")4), ()-diazo-")-oxo-L-norleucine
(DOX) (Clarke (/ a/., U)5(i), carcinomycin
(Hasoya, H).").")), carzinocidin (Harada et al.,
n)")()), carzinophiliu (Hata d a!., 19o4), gan-
cidin (Also ct al., H)r)4), melanomycin (Suga-
wara d (iL, HloT), mitontycin (Sugawara and
Hata, 19o()), pluramycin (Alaeda ct al.,
19")()), puromycin (Troy ct al., 1958), sarko-
mycin (Cmezawa et al., 1954; Hooper et al.,
1955), teomycic acid (Oda, 19()0), and many
others.
The screening methods devised to isolate
antibiotics have been modified for the isola-
tion of antitiuiior agents (Tables 4 and 5).
One such method consists of isolating actino-
26
NATURE, FORMATION, AND ACTIVITIES
mycetes from f^oil, growing- them in li(iuid
media, and testing the fihrates for activity
against experimental tumors in mice. The
test systems can be soUd tumors, ascitic
forms, or blood tumors. The results can be
evaluated in terms of reduction of tumor
sizes, weights of animals, number of tumor
cells in the ascitic fluid, or even the pro-
longation of the life of the treated animals
in comparison with the respective conditions
of the untreated controls.
To save time and animals, efforts have
been made to elaborate preliminary screen-
ing programs in n'tro. \'ari()us lines of reason-
ing have been followed:
1. Cancer cells are rapidly dividing cells.
In the search for antimitotic agents, the in-
hibition of mitosis in onion root tips has been
investigated. H<)we^'er, this method gives no
information about the selective action of the
antimitotic agents found.
2. Cancer cells ha\'e active dehydrogenase
systems. One can put such cells in ritro in
presence of an oxidation-reduction indicator
such as methylene blue. If the cells are killed
in vitro they will be unable to reduce meth-
ylene l)lue to the leuco form and they will
be stained blue. Here again, the method is
not selective, but it can be most useful for
assay purposes.
8. Cancer cells ha\-e an impaired respira-
tion mechanism. One can thus select for
further studies substances which would ha\'e
a selective antifermentative action or sub-
stances that would be selectively active
against microbial mutants which would also
ha^'e an impaired respiration. These micro-
bial mutants can be considered as the
e(|uivalent of cancer cells in the microbial
work, (lood correlation has been shown be-
tween antitumor activity and activity
against such mutants. This subject was re-
viewed Iw Clause in 1959 (Udintzev ct a/.,
1959). Specific studies on different actino-
mycins were reported by Hackmann and
Schmidt-Kastner (1957).
The screening procedui'c imoh'ing tests
for antitumor activity can be carried out by
the cylinder plate method with carcinoma
cells and a dye such as 2,()-dichlorophenol-
indophenol. This method showed, for ex-
ample, that Ehrlich carcinoma cells were
about 10 times more sensitive to sarkomycin
than were cells of Yoshida sarcoma, upon
which sarkomycin was almost inactive.
Testing of the toxicity on HeLa cells in
tissue culture was also found to be useful for
the screening and extraction of the active
agent. There were substances which were
destructive to HeLa cells but not to Ehrlich
carcinoma and chicken em})ryo cells, as
shown in Tables 3 to 5 (Umezawa, 1956).
Perlman et al. (1959) made a study of
effects of antibiotics on multiplication of L
cells of mouse fibroblasts in suspension
culture. They reported positive inhibition of
multiplication with less than 1 /xg per ml of
actinomycins B, By , or Diy ; of cyclo-
heximide, gliotoxin, hygromycin, and mito-
mycin C; of clavacin, thiostrepton, and
xanthomycin. Actinomycin By was most
active, with 1.5 Mg PPI" n^l causing a 50
per cent reduction in growth of culture. It
was suggested that this method can be used
in determining the presence of cytotoxic
microbial products.
Once an organism with desirable prop-
erties, as shown by the screening test used,
has been isolated, a series of investigations
must be initiated to e\'aluate the true po-
tentialities of the antitumor substance.
Only a few compounds have found practical
application. The others either were never
described or were described purely because
of their academic interest. Some compounds,
such as mitomycin, off'ered great promise
but were found to be of little practical value
because of their high toxicity. Others, like
actinomycin, seemed to be stillborn but were
only ahead of their time. Still others, like
puromvcin, sarkomycin, carzinophilin, aza-
serine, and DON, proxcd to l)e of limited
value.
In discussing the problem of anticancer
SEARCH FOR ANTIBIOTICS: SCRP:ENIXC; PROCRAMS
27
agents, Chain (1!).")8) made the following
comment: "In the absence of any line of
rational approach to the problem of tumour
therapy, any new substance with any sort
of biological activity is tested by someone
with iTgard to its possible inhibitory effect
on tumours; there is no prima facie scientific
case at all why antibiotics in particular could
be expected to act against tumours. Bac-
terial infections and malignant tumours ha\'e
nothing in common except that in some very
superficial resemblance phenomena of growth
are involved in both diseases."
Antiviral and Antiphagc Surreus
In a recent review of antibiotics, Al)ra-
ham (1959), one of the pioneers in the field,
stated: "In some laboratories they [screen-
ing programs] also aimed at the discovery of
substances that will selectively inhibit the
growth of tumor cells or viruses, though
there is so far little evidence to indicate that
they will have much success in this field."
In a summary of an early sur\'ey dealing
with the problem of antiviral agents, Jones
and associates (194.")) reported that they
tested loO organisms, comprising bacteria,
fungi, and actinomycetes, isolated from
composts, manures, soils, drainage ma-
terial, as well as from soil enriched with
A'irus concentrates, for their ability to in-
activate viruses. Only three of these or-
ganisms gave any indication of possible
inactivation of fowl ])ox \irus, and in one
case, of the laryngotracheitis virus. An ac-
tive principle was isolated from a culture of
one of these organisms; the substance pro\XKl
to be actinomycin. The other two virus-
inactivating organisms were less extensively
studied.
A number of substances capable of modify-
ing or controlling viral infections have been
described in recent years. A l)eneficial effect
was found to be exerted on pre\-iously in-
fected hosts, such as chick embryos. These
compounds included certain bacterial poly-
saccharides, hexamidine, pectin, tannic acid,
tea extracts, crude penicillin, and viscosin. A
preparation obtained from a strain of
Streptomyces lavendulae and designated as
ehrlichin also had an injurious effect upon
influenza virus (Groupe et ai, ]9ol ).
Unfortunately, none of the above ob-
servations, or others of a similar nature, led
to any practical developments as potential
therapeutic agents in viral diseases. Actino-
mycin recei\'ed no further consideration be-
cause of its highly toxic effect upon animals.
Different investigators, constrained by the
difficulties involved in the use of animal and
plant viruses for this type of investigation,
devoted their attention to the bacterial
\-iruses or l)acteriophages (Schatz and Jones,
1947; Asheshov et a!., 1949, 1952; Hamada,
1957; Smejkal, 1960), on the assumption that
if one were successful in obtaining antiphage
agents, he would be dealing with mechanisms
similar to those that might be destructive to
the ultramicroscopic intracellular agents
affecting higher forms of life. Several prep-
arations were actually found to be effective
against phages, l)ut they were totally in-
active for animal viruses.
In an effort to find drugs that would
possess antix'iral properties, Dickinson (1953)
suggested the following po.'^sibilities: (1)
direct inactivation of the extracellular virus;
(2) prevention of adsorption of virus on the
cell or of its penetration into the cell; (3)
inhibition of multiplication of intracellular
virus; (4) prevention of release of the virus
from infected host cells.
Hermann and Rosselet (19()()) proposed a
procedure bv which paper chromatograms
of antiviral agents are de^'eloped by apply-
ing them to virus-infected, agar-overlay,
chick embryo cell tissue cultures. The
plaque-free zones produced indicate the
location of the active material on the paper
chromatogram. The biological activity of
several antiviral agents could thus be
demonstrated. As little as 1 jug of an anti-
biotic (W 122) produced large placiue-free
zones when a paper chromatogram was ap-
28
NATURE, FORMATION, AND ACTIVITIES
plied to the overlay culture 1 hour after
virus infection; large plaque-free areas re-
sulted when 100 yug were applied 46 hours
after \'irus infection.
Certain glycoproteins in the sap of various
plants were found capable of reducing the
infecti\nty of several plant viruses. On
dilution, however, noninfective mixtures re-
gained infectivity, thus indicating a lack of
combining ratio between virus and inhibitor
necessary to cause a loss of infectivity
(Kassanis and Kleczkowski, 1948). It has
been suggested, howe\'er, that the evidence
points not to a combination effect of virus
and proteins, but rather to the effect of the
latter upon the host, or to an antagonistic
behavior between the virus and inhibitor.
Among the inhibiting factors, the action of
ribonuclease on ribonucleic acid viruses is of
particular interest. This enzyme hydrolyzes
the nucleic acid deri\'ed from these viruses,
but seems to have no effect upon the intact
viruses. When mixed with the ^'irus, how-
ever, the enzyme combines with it and in-
hibits its infectivity re\'ersibly in a manner
similar to protein (Bawden and Pirie, 1957).
Other substances capable of inhibiting the
infectivity of plant viruses include certain
yeast polysaccharides (Kleczkowski, 1946).
Table (5
Effect of a nocardia antibiotic on tobacco mosaic
virus multiplication under different light in-
tensities (Schlegel and Rawlins, 1954)
Preparation
Light
Concentra-
tion of
antibiotic
Percentage
inhibition
of virus
multiplica-
tion
Average
per cent
deviation
from mean
Unpurified
antibiotic
fool candles
300
300
25
mg/lOO ml
1
1
1
69
73
90
0.8
5.5
7.7
I'uiified
antibiotic
1100
1100
1100
1100
0.001
0.001
0.005
C.OOl
0.0001
73
74
80
80
23
8.3
4.5
1.9
0.3
5.9
Some of these substances, like primycin,
exert not only antiA'iral but also antibac-
terial effects; some were foimd able to cause
marked destruction of worms. These sub-
stances vary in s()lu!)ility, stability, activity,
and toxicity.
Schlegel and Rawlins (1904) isolated from
a Nocardia a substance found to be efTective
in inhibiting multiplication of tobacco
mosaic virus in floating leaf discs. Its action
appeared to be relatively independent of
changes in host composition produced by
light. It was concluded that it inhibits virus
increase by acting directly upon virus
multiplication rather than on host metab-
olism (Table 6). Other antiviral agents
produced by Nocardia have been studied by
Harris and Woodruff (1953). Kuroya et al.
(1957) examined 418 culture filtrates of
freshly isolated streptomycetes for their
activity against influenza virus cultivated
in vitro. Of the filtrates 21.5 per cent showed
some activity. There was no correlation be-
tween activity and any other biological
properties of the organism.
Unfortunately, none of these numerous
preparations can be said to have a suf-
ficiently suppressive effect upon viral
diseases to warrant use in chemotherapy of
such diseases. In analyzing the failure to
obtain antiviral substances of practical
significance, Waksman (19()0) concluded
that since viruses do not grow and do not
metabolize, the whole approach to their
suppression must be different from that of
antibiotics which act upon the living systems
of bacteria, fungi, and other microorganisms.
Further observations on anti\'iral agents
and reasons for failure to obtain good
preparations ha^'e been examined by Leva-
diti (1952), Kuroya ct al. (1957), Krassilni-
kov d al. (1960), and Waksman (1960).
.1 ntitrichomonal A gents
Yamaguchi and Sabiu'i (1955) found that
of 1244 cultures of actinomycetes, 172 pos-
sessed antitrichomonal properties; a few of
SEARCH FOR ANTIBIOTICS: SCREENING PROGRAMS
29
Table 7
Inhibition of different actinomycetes by their respective antibiotics (Wak-sman et al., 1946)
Antibiotic
Actinomycin .
Streptothricin
Streptomycin
Organism producing
antibiotic
S. antibioticut
S. lavenclulae
S. griseiis
* S. lutea units; crystalline material.
t E. coli units; crude preparation.
Activity of prepara-
tion, per mg
100,000*
loot
125t
Inhibition of
S. antibioticus
10
1
1
6'. lavendulae
mg/ml
0.2
2500
10
6'. griseus
10
100
830
these lost their activity on repeated trans-
fers in synthetic media. Among the sub-
stances so far isolated that possess anti-
trichomonal properties it is sufficient to
mention borrelidin and anisorhycin.
Use of Antibiotics for Isolation of
Specific Antibiotic-producers
The resistance of different organisms to
their own antibiotics and to closely related
forms can be utilized for isolating new strahis
of organisms producing specific antibiotics
and for obtaining even more potent strains
of such organisms. This phenomenon has
long been recognized (Table 7). Waksman,
Reilly, and Johnstone (1946) were the first
to suggest utilization of this property for the
isolation of organisms producing specific
antibiotics. This is further illustrated in
Table 8.
In a study of a number of strains belong-
ing to the same species, or at least the same
series, of *S. lavendulae, Okami demon-
strated (Table 9) the high selectivity in the
antagonistic effects of one upon another.
The type culture of this species (No. 3330),
isolated in 191"), exerted no antibiotic action
upon any of the other strains; it was sensi-
tive, however, to three other cultures be-
longing to this group. Another culture, Xo.
oooo, likewise produced no antibiotic but
was sensitive to several other members of
the group. The original streptothricin-
producing culture, Xo. 3440-8, was active
Table 8
Inhibition of S. frcirliae 3554 by different antibiotics
(Teillon, 1952)
Antibiotic used fimpregnated disc)
Inhibition zone
Streptonncin, 0.5%
mm
14
Streptothricin, 1.0%. .
16
17
17
Neomycin, 0.25%
Oxytetracycline, 0.5%
Chlortetracycline, 0.25%
Chloramphenicol*.
•^Water saturated solution.
Table 9
Reciprocal antagonism tjctween various S.
lavendulae strains (Okami*)
S. lavendulae
strain streaked
3330...
3440-8 .
3516-W
3530...
3531 ..
3532
3542.
3543.
3568.
3465
3544.
3555.
Strain cross-streaked and inhibition
zone (mm)
* Unpublished.
30
NATURE, FORMATION, AND ACTIVITIES
against several strains of S. lavendiilae, but
inactive against others. This was true of
some other forms. In no case was one strain
autoinhibited.
This should not be considered, however,
as justifying the broad generalization, much
emphasized by Krassilnikov (1950, 1951,
1958), that because some antibiotic-produc-
ing organisms are resistant to their own
antibiotics the general conclusion may be
reached that antibiotics form a kind of
defense mechanism for different micro-
organisms living in mixed populations. It
must rather be looked upon as an isolated
instance in a complex group of relationships
among organisms li\ing in mixed populations
(Waksman, 1956; see also Okami et ai, 1960).
Alusilek (1957), for example, demon-
strated that the growth of *S. aurcofaciens
on an agar medium was not inhil)ited l)y the
antibiotics of the tetracycline group, but
was inhibited by other antibiotics. This was
true also of an actinomycin-producing
actinomycete that was not inhil)ited by its
own antibiotic. On the other hand, S.
griseus, S. rimosus, and erythromycin-
producing actinomycetes were not inhibited
by the antibiotics produced by these or-
ganisms or by similar antibiotics; at the
same time, they were not inhibited by
\'arious antibiotics produced by different
other organisms. The conclusion was reached
that the finding of resistance of an unknown
actinomycete to a certain antibiotic cannot
in itself serve as a method for identifying
actinomycetes producing known antibiotics.
Other Antimicrobial Systems
Certain actinomycetes were found capal)le
of producing substances that possess marked
bacteriolytic properties. These were quite
distinct from phages, although some of them
exerted an autolytic effect; they were known
long before the true nature of the antibiotics
became recognized. This is true of the
"bacteriolytic enzyme of Sti'eptothrix"
studied by Gratia (Gratia and Dath, 1924-
1927) and designated by Welsch (1937) as
actinomycetin. It is also true of the "actino-
myces lysozyme," recognized by Kriss
(1939) and by Krassilnikov and Koreniako
(1940). Welsch et at. (1955) later isolated
frcjm the actinomycetin preparation several
substances, one of which was designated as
actinozyme; another was designated as
actinolysin, and was active against both
dead and living bacterial cells. Whether
these substances should be considered as
enzyme systems, and more specifically,
autolytic or generally lytic enzymes, or
whether they should be considered as true
antibiotics is a subject for further study.
Here one approaches the borderline of
biological systems that may be considered
either antibiotic or enzyme, or even vitamin,
since some of the antibiotics have, in certain
concentrations, a definite growth-promoting
effect upon lower and higher forms of life.
Numerous other preparations possessing
antil)iotic properties have been isolated from
cultures of actinomycetes. These range from
well defined compounds such as the phena-
zines ("^'agishita, 1960) to crude preparations.
In many cases the information is so limited
that it is difficult to place the preparation in
one or another of the above groups. Further
study of many of these preparations may
have been abandoned, since no practical
significance could be attached lo them. These
substances are listed in Part B of this
volume.
Chapter 4
Production of Antibiotics
The problem oi the possible formation of
antibiotics in nature has already been dis-
cussed in Chapter 1. In the present chapter
the methods used for the empirical produc-
tion of antibiotics by actinomycetes in vitro
are discussed. What is known of the bio-
genesis and the mechanism of biosynthesis of
antibiotics by these organisms will be con-
sidered in Chapter 7.
The ability to form antibiotics is one of
the numerous reactions characteristic of
various biological systems. The micro-
biologist has learned to recognize and favor
such systems. The chemist has developed
methods for the isolation of antibiotics.
Techniques for their rapid production on a
large scale have been developed by the
engineer. The pharmacologist and the clini-
cian have suggested procedures for their
practical evaluation. By combined effort,
these investigators have succeeded in ob-
taining powerful drugs for combatting a
variety of infectious diseases. To appreciate
the problems invoh'ed, one must consider
the following facts:
1. Xot all kinds of micn)l)es and not even
all strains of a particular group of microbes
are able to form antibiotics. This phenome-
non is not so widely distributed among the
microbes inhabiting our planet as to warrant
any generalization in regard to their role in
microbial life or in the survival of the or-
ganisms capable of forming such antibiotics.
2. Different strains of a microbe capable
of producing a given antibiotic vary greatly,
both (iualitati\'ely and (luantitatively, hi this
capacity, l^roduction of an antibiotic is not a
fixed property of the organism and is evi-
denced only when the organism is grown in
special media and in selected environments.
3. The ability of a particular microbe to
form antibiotics may easily be lost under
certain conditions of culture or as a result of
mutation.
4. The concentration of a j^articuiar anti-
biotic produced by a given microbe can be
greatly increased by strain selection, by
de^'elopment of special media, and by im-
pro\'ement of conditions of cultix'ation.
5. The sam(^ oi'ganism may be able to form
different anlibiotics in different media and
under different conditions of culti\-ation.
Each antibiotic is characterized by a given
chemical structure and by its antibiotic
spectrum.
6. The same culture may have the capac-
ity of forming, in the same medium and at
the same time, several antibiotics or various
chemical modifications of the same anti-
biotic. As many as five to seven different,
although chemically related, antil)iotics have
been found in the same culture of the same
organism.
7. The same antibiotic, or closely related
chemical modifications, can be formed by
different organisms.
Among all groups of mici'oorganisms, the
actinomycetes ha\'e proved to be the richest
source of antibiotics. Alost of these anti-
biotics are produced by members of the
genus Streptomyces; only a few of them are
produced by strains of Xocardio and M icro-
monospora.
31
32
NATURE, FORMATION, AND ACTIVITIES
Production of the Same Antibiotic by
Different Species
111 \'iew of the fact that antibiotic-produc-
ing organisms are biological systems, one
may expect considerable variation, not only
of a (luantitative but also of a qualitative
nature, among the species and strains pro-
ducing the same type of antibiotic. Once a
given antibiotic has been recognized, it is a
common practice to try to increase the yield
by strain selection, impro\'ement in composi-
tion of media, and changes in environmental
conditions such as aeration and temperature.
The production of the same antibiotic or
closely related forms by difTerent organisms
is recognized for such therapeutically im-
portant compounds as streptomycin, tetra-
cycline, neomycin, actinomycin, and various
others, notably the polyenes.
The ability of species other than S. griseus
to produce streptomycin was first demon-
strated by Johnstone and Waksman (1948).
It has since been established that strepto-
mycin and the streptomycin group of anti-
liiotics are produced by a ^-ariety of organ-
isms found among the different series of the
genus Streptomyces (Volume II, Chapter ()).
These include (Okami ct al., 1959): (1) tuft-
pi'oducing organisms with .straight aerial
mycelium: ^>. griseus, S. bikiniensis, S.
rameus; (2) spiral-producing organisms: S.
humidus; (3) verticil-producing organisms:
S. reticuli, S. griseocorneus, S. mashuensis.
S. griseus produces streptomycin and man-
Table 10
Classijicdlio'ii of acliii(niiycin-pr<i(hui>uj organ isms
(Corbaz et al., 1957)
Organism
Chromogenicity
Actinomycin
type*
S antibiotic us
+
+
X
S. michiganensis
S. parvulliis
S. chrysoniallus
S. parvus ...
X
I, X
C, I
X
* Brockmann system of nomenclature used.
nosidostrcptomycin; S. hunii<lus forms dihy-
drostreptomycin (Kavanagh et al., 1960);
and S. griseocarneus forms hydroxystrepto-
myciii.
Neomycin and the neomycin group of
antibiotics are produced by numerous strains
of »S'. fradiae, S. roseojiavus, S. albogriseolus,
S. kanamyeeticus, and by a variety of other
organisms (Waksman et at., 1958). This
group comprises neomycins B and C,
neamine, catenulin, kanamycin, paromo-
mycin, and a number of other compounds,
the exact chemical nature of which is still
undetermined.
The tetracyclines comprise a variety of
compounds, including chlortetracycline, oxy-
tetracycline, tetracycline, demethylchlorte-
tracycline, and others. These are produced by
different organisms, including S. aureo-
faciens, S. rimosus, certain members of the
»S. fiavus group, and others.
A number of organisms are now known to
1)6 capable of producing actinomycins; they
include S. antihioticus, S. flavus, S. flaveolus,
S. parvus, S. chrysomaUus, S. parvuUus, and
S. ceUulosae. Some of the polyenes are pro-
duced by a large num})er of species and
varieties (Vajima, 1955). Blinov (1958)
demonstrated that the candicidin-type poly-
ene is produced l)y at least 11 different
organisms.
Further information on the chemical rela-
tionships of the various groups of antibiotics
is given in Chapter 6.
A very interesting relationship between
micr()l)ial specificity and chemical nature of
antibiotics produced is found among the
actinomycins (Waksman, Geiger, and Rey-
nolds, 194()). Corbaz et al. (1957) demon-
strated (Table 10) that although different
species of Streptomyccs are capable of produc-
ing actinomycin, the chemical composition
of the antibiotic differs. In addition to these
organisms, other actinomycetes as well are
capable of producing actinomycin, as shown
for Micromonospora sp. (Fisher et al., 1951).
PRODUCTION OF ANTIBIOTICS
33
Protluclion of DifTerent Antibiotics by
the Same Organism
Not only may the antibiotic produced by
an organism vary in chemical composition,
but its very nature may differ (Trussel ct al.,
1947). This can easily be demonstrated by
the fact that freciuently the same organism
may produce both antibacterial and anti-
t'lmgal antibiotics. Thus, S. griseus was
found to produce streptomycin, candicidin,
and cycloheximide; S. Jradiac produces
neomycin and fradicin; S. rimosus forms
oxy tetracycline and rimocidin. Frecjuently,
different strains of the same organism pro-
duce different antibiotics. This is true, for
example, of different strains of S. lavcndidac,
which produce several forms of strepto-
thricin and also a \'ariety of such compounds
as ehrlichin and polyenes. Often a change in
composition of medium and in conditions of
growth results in a change in the nature of
the antibiotics produced by the same organ-
ism. Certain organisms are capable of form-
ing in the same medium as many as three
different antibiotics (Despois et al., lOotJ).
S. alhirHiculi, for example, produces euro-
cidin, enteromycin, and cai'bomycin (Osato
etal, 1955).
Production of Antibiotics in Li<{uid and
Solid Media
As was shown in Chapter 3, the fact that
an actinomycete has antagonistic properties
sometimes may not be discovered until after
it has been grown both on solid media and
in licjuid media. For instance, the culture
may form zones of inhiliition agahist test
organisms on solid media, but may refuse
stubbornly to produce antibiotics in li(iuid
media. This may l)e due to differences be-
tween the cultural conditions of some actino-
mycetes on the two different types of media.
In certain cases, for example, this phenome-
non can be explained by the formation of
ammonia by the actinomycete. It can easily
be demonstrated that some actinomycetes
can produce a volatile substance which is
toxic to fungi. Inhilntion of the fungi can be
noted not only on the layer of agar on which
the organism is growing, but also on a layer
that is on top of it and separated by an air
space. The pH of this upper layer of agar
increases during the growth of the actino-
mycete on the layer below, and fungi seeded
on this upper layer will not grow, whereas
some bacteria will. Substitution of a dilute
solution of ammonia for the growing actino-
mycete produces a similar inhibitory effect
on fimgi. These experiments do not rule out
the possibility that actinomycetes form
\'olatile antibiotics different from ammonia,
but the production of this compound can
probably explain certain discrepancies be-
tween inhibitory effects observed when
actinomycetes are grown on solid and in
li(iui(l media.
In general, once an antagonistic actino-
mycete has been selected in a screening
pi'ogram, the antibiotic or antibiotics pro-
duced tjy this organism must l)e obtained in
large enough (juantities to permit their
exti'action, purification, characterization,
and if necessary, the study of their toxicity,
pharmacology, and activity in vivo. This is
accomplished by growing the antagonist in
liciuid cultures which are incubated on shak-
ing machines or in fermentors of various
volumes.
The motion applied to flasks by shaking
machines is either circular (rotary shakers)
or linear (reciprocal shakers). The purpose
of the shaking is to furnish the culture with
the aeration necessary for maximal growth
and the formation of antiV)iotics. The mo-
tion may also play a role in keeping the
organism well dispersed in the liciuid me-
dium, permitting good contact between the
cell surfaces and the nutrients.
Assay Methods
Since antibiotics are chemical substances
capable of inhibiting the growth of micro-
84
NATURE, FORMATION, AND ACTIVITIP^S
organisms, they can be assayed chemically
and/or l)iologically (Grove and Randall.
1955).
Chemical Methods
Chemical methods can be used only after
the chemical properties of a given antibiotic
are known. Examples of these chemical
assays are: (1) use of the blue-colored nin-
hydrin reaction to assay antibiotics with
free amino groups, such as neomycin; (2)
the use of maltol determination to assay
streptomycin and mannosidostreptomycin ;
and (3) the anthrone test to titer mannosido-
streptomycin by determining the amount of
mannose present. Stable antibiotics with
strong and typical light-absorption spectra
can be assayed directly by spectrophoto-
metric methods. Chemical methods of assay
must be used with caution, since contaminat-
ing substances may give false positive values.
For example, contaminating amino acids
would give a positive ninhydrin reaction and
interfere with the chemical assay of neo-
mycin by this reaction.
Biological Alethods
Biological methods of assay of antibac-
terial and antifungal antibiotics are of two
general types: (1) dilution and (2) diffusion
methods. Dilution assays can be made with
or without a standard. It is virtually impossi-
ble to make diffusion assays without a
standard .
Dilution assays can be made in both liquid
and solid media. The use of solid media
permits the assay of a given antibiotic
against a number of test organisms with
minimal labor. The dilution assay on solid
media, one of which was described by Waks-
man and Reilly (1945), is a good procedure
for use without a standard. Activity is ex-
pressed in terms of the minimal amount of
antibiotic that will prevent growth of a test
organism. The measure can be expressed in
micrograms per milliliter or in dilution units.
Dilution assays in li(iuid media are well
suited to statistical analysis; a standard is
usually re(|uired. In such an assay, the
turbidity caused by the growth of a bac-
terium or a yeast is measured and the value
of unknown preparations is determined by
estimating the amount of unknown neces-
sary to permit the same growth as that per-
mitted by a known amount of the standard.
Factors influencing the results of dilution
assays are: (1) the composition of the me-
dium (especially its pH and salt content),
and (2) the age and number of cells of the
test organisms used to inoculate the medium.
Diffusion assays are carried out by placing
cylinders or filter paper discs containing
various amounts of an antibiotic solution on
the surface of an agar layer seeded with a
test organism. Comparison of the sizes of
sterile zones produced by solutions of un-
known concentration with those produced by
known solutions of a standard permits
estimation of the potency of unknown prepa-
rations.
Factors influencing diffusion assays are:
(1) the thickness and evenness of the agar
layer, (2) the rate of evaporation of water
from the agar layer, (3) the composition of
the agar medium (especially its pH and salt
content), (4) the nature of the solvent con-
taining the antibiotic samples (especially
pH and salt content), and (5) the method of
seeding the agar layer with the test organ-
ism, sharper zones of inhibition being formed
if a thin agar seed layer is superimposed on
a thicker unseeded layer of agar.
A comparative study was recently made
of the relationship between the minimal
inhibiting concentration of an antibiotic by
the serial dilution method and the diameter
of the zone of inhibition by the single-disc
method. It was found to correspond to a
straight line, with the exception of spiramy-
cin for which the values fit a second order
equation (Ericsson et al., 19()0). The meas-
urement of antibiotic concentrations in
PRODUCTION OF ANTIBIOTICS
35
clinical medicine must tak(> into considera-
tion the phenomena of absorption and
distribution in different body fluids (I^rics-
son, 19(i0).
Increasing Commercial Yields
For the chemical characterization of an
antibiotic, broths of low titer of activity
may be satisfactory. Howe\'er, for its com-
mercial production, broths of the highest
possible titers are desired. Yields can be in-
ci'eased by two general approaches, which
are usually utilized together. One can either
(1) improve the strain forming the antibiotic
by selection of more and more active mu-
tants, or (2) improve the media and cultural
conditions used for the growth of the anti-
biotic-producing organism.
Strain Selection
The ability of different strains of the same
organism to form different concentrations of
the same antibiotic is illustrated in Table 1 1.
At least four distinct procedures are
utilized for obtaining more active strains
capable of producing higher concentrations
of a particular antibiotic:
1. Selections from a natural population
of a culture and evaluation of antibiotic
potency of different strains of the particular
culture.
2. Selection of strains from a given culture
grown on a medium enriched with the anti-
l)iotic produced l)y the particular culture.
;'). Preliminary treatment of a culture
with ultraviolet and other radiations.
4. Treatment of a culture with various
chemicals, such as ethylene amine, nitrogen
mustard, or colchicine.
Natural selection is accomplished by plat-
ing out actmomycetes so as to get a large
number of isolated colonies, each one origi-
nating, if possible, from a single spore or
from small segments of mycelium. The level
of antibiotic production of these substrains
Table 11
Antibiotic potency of different strains of S. fradiae,
(/roup I (Waksman et al., 1958)
Medium: N-Z-amine-glucose. Potency ex-
pressed in neomycin units per ml of broth.
Incubation period
3 days
4 days
6 days
3535
270
285
324
3554
325
230
195
355(ia
202
241
375
355()b
279
239
297
3572
67
77
78
3594
394
321
687
3719
200
264
230
can be tested in a numbei' of ways. The sul)-
strains can be tested directly in lic[uid or in
solid media. In this case replication methods
are often used as a time-sa^'ing device, but
in final analysis an increase in antil^iotic
formation in lic[uid media is the aim.
Actinomycetes, as a rule, are sensitive to
strictly antibacterial antibiotics and resist-
ant to strictly antifungal antil)iotics. These
properties can be utilized to isolate strains of
actinomycetes which pi-oduce antibacterial
antibiotics of a given type. Active strains
will be more resistant to the action of the
antibacterial antibiotics they produce than
will other actinomycetes, and the more
antibiotic a given strain will accumulate, the
more resistant the sti'ain will be. That this
phenomenon is not universal, however, is
evident from the fact that strains of Strepto-
myces rimosus forming oxytetracycline ai-e
resistant to streptomycin.
Waksman et al. (194()) first suggested the
use of a streptomycin-enriched medium for
the selection of streptomycin-producing
cultures. Although the inactive variants were
thus eliminated, more active cultures were
not obtained. This method was later utilized
by AIcDani(4 and Hodges (1950) with con-
siderable success and resulted in the produc-
o()
NATUHK, FORMATION, AND ACTIVITIES
tioii of highly active cuhures. When tliis
method is combined with other procedures,
such as radiation treatments, highly active
substrains can be obtained. Strain selection
of streptomycin-producing organisms has
been studied further l)y Savage (1949), I)vi-
laney et al. (1949), Dulaney (1953), and
Pittenger and McCoy (19o;-5).
Exposui'e of antagonistic actinomycetes
to various mutagenic agents has been the
most successful method of obtaining better
antibiotic-forming strains. In this area, ultra-
violet light has played an important role, as
shown by the following procedure of Darken
d al. (1960), who discussed the methods used
to improve tetracycline-forming organisms.
An isolate was selected from among the
better produc(n-s. It gave an average of 125
^g per ml of antibiotic. A spore suspension
of the culture was irradiated with ultraviolet
light at an exposure sufficient to kill 99.99
per cent of the spores, and 200 colonies were
transplanted to agar tubes. A total of 19 iso-
lates, or 9.5 per cent, showed various types
of gross morphological variation. When
X-ray treatment was used, an exposure of
100,000 roentgen units gave 99.5 per cent
kill. A total of 340 colonies was picked and,
of these, 149 (44 per cent) were gross mor-
phological variants. (3f 45 isolates obtained
by natural selection and 135 by X-ray treat-
ment, none showed a significant increase in
antibiotic production. Treatment with ultra-
violet irradiation, however, resulted in three
superior isolates.
A culture giving a 20 per cent higher
tetracycline yield than the original isolate, or
an average of 150 /xg per ml, was selected for
medium development studies; 1250 deriva-
tives of this culture were examined. Of these,
160 (23 per cent) of 700 ultraviolet-treated
isolates and 190 (38 per cent) of 500 X-raj^-
treated isolates were morphological variants.
None of the 50 natural selections, however,
gave evidence of gross morphological change
(see also Katagiri, 1954). The variability of
naturally occurring oxytetracycline-produc-
ing strains has been studied by Boi-enstain
and Wolf (1956).
There was a greater numl)er of superior
antibiotic-producers as well as a higher
incidence of morphological types in the X-
ray-treated isolates than in either the ultra-
violet-treated or the untreated isolates. Of
640 ultraviolet-treated cultures, only three
gave significant increases in broth potency
over the parent. Of these, a culture was
selected as a control for the testing of 390
derivatives from the X-ray treatment of one
culture (TVS). The yields obtained with
these selected isolates from this culture
grown in a synthetic medium ranged from an
average of 1955 to 2150 ^g pei" nil.
Goldat (1958) reported that the treatment
of spores of >S. aureofaciens with various
doses of ultra\'iolet rays causes an increase
in the mutation rate. A high dose of ultra-
\'iolet rays and a four-fold photoreactivation
gave the largest number of morphological
mutations; an exposure to smaller doses was
found to l)e more effective for increasing the
variation of activity. A single photoreactiva-
tion with daylight reduced both the lethal
and the mutational effects, whereas a re-
peated reactivation increased the latter.
Alikhanian (1959b) discussed the results
obtained by various investigators in improv-
ing strains of S. griseus, S. rimosus, and >S.
aureofaciens in order to increase production
of streptomycin, oxytetracycline, and chlor-
tetracycline. liable 12 shows the predomi-
nant role played Ijy ultraviolet light and
X-rays.
Some chemical mutagenic agents such as
nitrogen mustard have also found a place in
the searc'h for more active mvitants. Among
other mutagenic agents, actinophage may be
mentioned (Ilina and Alikhanian, 1957).
Further studies on the genetic interactions
among streptomycetes and the selection of
active strains is found in the work of Bradley
PRODUCTION OF AXTIinoTICS
37
Table 12
Strain sclccHoti hi/ raridus methuds of trealiiient ( Alikhaniaii, 19591))
Year
Author
Treatment
Activity
Organism and antibiotic
Old
strain
New
strain
units/ml
S. f//'/.sp(/S-sf l-(^J)t0111,\'cill
1947
Stanley
Ultraviolet rays
400
900
1949
Savage
Ultraviolet rays and X-
180
700
1953
Pittenger and McCoy
ra\'
Ultraviolet rays
180
800-900
1949
Dvilaney cl al .
Ull ra violet rays
250
1000
1953
Dulaney
X-ray
1000
2000
1957,
Alikhanian
Ultraviolet rays and vis-
3200
4000
1959
ii)le light
»S'. /•/H;o.s(/.s--(j.\ytetr:i-
195r.
Boi'pnstain and Wolf
Ultraviolet rays and ni-
500
1500
cvc'line
trogen mustard gas
1958
Mindlin and Alik-
hanian
Ultraviolet rays
1828
3040
tS. aiircofacie)iH-i-\\\ov~
1954
Katagiri
Ultraviolet rays and ni-
1200-1300
tetracycliiip
trogen mustard gas
1958
Goldat
Ultraviolet lays
tiOO
1000
1959
Goldat
X-ray
1000
2200
1959
Lin Wang
X-ray
400
1000
('/ al. (H).')!)), Alikhaniaii d al. (U).")9), and
Mindlin and Alikhaniaii (l'.)58).
Improvemcnl of Media and
Cidlural Conditions
During the de\elopment of a process for
the production of a given antibiotic, the
work on strain selection and the study of
media and cultural conditions go hand in
hand.
In improving media, the following nu-
trients must be investigated: (1) sources of
carbon, (2) sources of nitrogen, (.3) mineral
sources, and (4) growth-promoting sub-
stances.
Several important factors are known to
influence the growth of the antibiotic-pro-
ducing organism: (1) temperature, (2) initial
pH and control of pH during growth, (3)
aeration, and (4) agitation.
The medium used to grow the inoculum,
the age of the inoculum, and its size will
markedly influence the jjroduction of anti-
biotics.
Kffeel of Composition of Medium on
Antibiotic Production
The composition of the medium has a
highly important eflect upon the growth and
metabolic activities of actinomycetes, and
upon the production of antibiotics. Both the
(luantitative yield of speciflc antibiotics and
their chemical structure are greatly influ-
enced by the nature of the nutrients (carbon
and nitrogen sources), their concentrations,
and the presence of specific salts.
When streptomycin was first produced by
freshly isolated strains of »S'. griseus (Schatz,
Bugie, and Waksman, 1944), the maximal
yield of the antil^iotic was 100 to 200 /xg per
ml. In recent years, this yield has been in-
creased to between 5 and 10 mg per ml. Al-
though the proper strain development had a
great deal to do with this phenomenal in-
crease, the selection of suitable media and
optimal conditions of growth w(n-e also
la rgely responsi l)le .
The addition of basic amino acids (argi-
38
NATURE, FORMATION, AND ACTIVITIES
Table 13
Composition of Jour media found very suitable for
production of antibiotics by actinomycetes
(Warren et al., 1955)
Media components
Soyl)ean meal FF grits,
ediV:ile grade
Peptone, technical grade.
Corn steep (liquid)
Glucose, technical grade.
Molasses, Brer Rabbit
(green label)
Dextrin
Curbay, B. G
Glycerol
NaCl
CaCOg
K0HPO4
Media numbers and compo-
sition
A-4 A-4h A-9 A-12
g"i/
iter
10.0
15.0
5.0
10.0
15.0
5.0
2.5
10.0
20.0
5.0
5.0
1.0
1.0
10.0
20.0
10.0
5.0
2.0
2.0
nine, histidine, and leucine) to an autoclaved
aqueous extract of soybeans increased con-
siderably the yield of streptomycin. The
addition of monoamino acids (glutamic,
aspartic) was found to be unfavorable for
both growth and streptomycin production
(Kazanskaia and Andreeva, 1959).
The various constituents of production
media are selected on the basis not only of
their value in permitting the elaboration of
the antibiotics, but also of their costs. Soya
bean and corn products have l)een important
in this respect.
Warren and coworkers (1955) showed that
the four media listed in Table 13 permitted
the production of a large variety of anti-
biotics by actinomycetes. They concluded
that the production of most antibiotics of
actinomycetes should occur at a detectable
level in at least one of them.
Table 14
Influence of nitrogen source on composition of actinomycin complex
(Wak.sman et al., 1958)
Nitrogen source
Relative percentage of components
Organism
I
II
III
IV
V
VI
VII
S antibioticus
L-Glutamic acid
L-Threonine
L-Glutamic acid -|- sarcosine
KN0,3
Glycine
6.2
7.1
7.1
2.1
2.4
24.6
2.8
2.9
35.6
80.4
29.2
27.2
10.0
7.0
8.4
58.4
5.5
50.0
43.2
S antibioticus
,S. chrysomaUus
S. chrysomaUus
40.0
49.8
Table 15
Influence of amino acids on actinomycin synthesis
(Katz and Goss, 1958)
Concentration of
amino acid
added
Relative percentage of actinomycin components
Medium
I
II
III
IV
V
*
0.0
0.25
0.05
0.25
0.25
6.4
31.0
9.1
8.1
4.7
2.3
3.8
25.6
2.0
2.8
3.2
7.1
35.0
3.2
5.8
(58.3
25.3
24.4
32.7
17.2
17.2
30.0
5.9
51.1
30.6
2.6
Glutamic acid + hydroxy-L-proline
Glutamic acid -|- sarcosine ■ .
3.0
0.0
Glutamic acid -f- N-acetylglycine. . .
Glutamic acid -|- L-isoleucine
3.0
38.9
* Denotes an unidentified component which moves faster than Component V in circular i^aper chro-
matography.
PRODUCTION OF ANTIBIOTICS
39
The effect of the medium upon tlie nature
of the antibiotic can best be ilhistrated by
an analysis of the production of different
actinomycins. Schmidt-Kastner first demon-
strated (195()) that by increasing the amino
acid content of the medium or by the addi-
tion of special amino acids, notably sarcosine,
new actinomycins can be produced. These
results were fully confirmed in subsecjuent
studies carried out in our laboratories, as
shown in Tables 14 and 15.
An examination of the chemical nature of
the actinomycins produced by different
organisms revealed certain pertinent differ-
ences in the concentrations of the various
components of the actinomycin molecule. »S.
antibioticus (the original producer of actino-
mycin A) gives, in certain media, the follow-
ing actinomycins: I, 10 per cent; IV, 60 per
cent; V, 30 per cent. Streptomyces 3723 (the
original producer of actinomycin B) gives, in
certain media: I, 10 per cent; IV, 30 per
cent; V, 60 per cent. S. chrysomaUus (the
original producer of actinomycin C) gives:
IV, 10 per cent; V, 50 per cent; VII, 40 per
cent. S. parvuUus (the original producer of
actinomycin D) gives: IV, nearly 100 per
cent.
Katz and Goss (1958) have demonstrated
that it is possible to change to a limited
extent the proportion of any particular com-
ponent in a natural mixture of actinomycins.
Certain components normally produced in
small amounts may be increased until they
represent the major constituents of an
actinomycin complex. Actinomycin \, for
example, was the major component in the
actinomycin mixture produced by *S. anti-
bioticus when L-threonine was the sole
nitrogen source, whereas actinomycin IV
represented the main constituent when L-
glutamic acid was used. Schmidt-Kastner
(1960) demonstrated that actinomycin IV
increased from 10 to 83 per cent of the ac-
tinomycin mixture formed by S. chrysomaUus
when 0.5 per cent i)L-valine was added to a
glycerol-nitrate medium.
Various amino acids, notably hydroxy-L-
proline, sarcosine, N-acetylglycine, and l-
isoleucine, bring about marked (juantitative
changes in the actinomycin complex pro-
duced. Components normally formed in
small amounts were found, in certain specific
conditions, to represent major constituents
of such mixtures.
Among the many other factors influencing
the (luantitative yield of an antibiotic, the
mineral constituents occupy a prominent
place. In this connection, the importance of
the effect of phosphorus starvation upon the
yield of streptomycin must be emphasized.
The presence of iron in the mediiun will favor
the accumulation of mannosidostrepto-
mycin, whereas the addition of calcimn will
lead to the transformation of the latter to
streptomycin. Detailed studies of the role of
trace elements in the profluction of strepto-
mycin have been made by Chesters and
Rolinson (1951) and numerous others.
Chapter 5
Isolation and Identification of
Antibiotics of Actinoniycetes
Principles of Isolation
The isolation of antibiotics of actino-
mycetes requires the use of methods which
have found general application in the isola-
tion of natural products.
As will be discussed in detail in Chapter 6,
antibiotics of actinomycetes have extremely
di\Trse chemical structures. We find among
them acids, bases, amphoteric compounds,
neutral compounds, polypeptides, amino-
sugar complexes, compounds with huge
lactone rings, nitro compounds, guanido
compounds, polyenic compounds, and ace-
tylenic compounds. One cannot help being
amazed by the synthesizing capacities of
this group of organisms.
Most antibiotics are released by actino-
mycetes into the culture medium; a few of
them are formed in the mycelium. Clearly
then, no single procedure will isolate all these
compounds.
When one is dealing with an unknown sub-
stance, it is always advisable to have an idea
of the stability of the substance at various
temperatures and at \'arious pH \-alues
before attempting to devise a method of
extraction. A reliable assay is also necessary
to follow the fate of the antibiotic during
those various attempts.
Since antibiotics are rarely formed alone
by the producing organism, but are often
elaborated as mixtures of complexes of re-
lated substances or along with totally unre-
lated antibiotics, assay methods should give
not only quantitative data but also informa-
tion of a cjualitative nature. Qualitative data
permit one to follow the fate of the various
components of the complexes as they are
resolved by the extraction procedures.
Some crude antibiotics are formed of
components which have a synergistic action.
Without proper bioassays, the activity would
seem to disappear during purification. This is
true of the antibiotic complexes E 129 and
PA 114.
Basically there are two types of methods
for separating antibiotics from accompany-
ing impurities: (1) those which take advan-
tage of differences in solubility between com-
pounds and (2) those which take advantage
of differences in the affinity of such com-
pounds for the surfaces of adsorbents or ion
exchangers. Both methods liave endless
variations and are often used together in the
isolation of a given substance.
The reader will find in Part B of this f)ook
outlines of the extraction and purification
procedures used for each one of the anti-
biotics described. He will note that for many
antibiotics, various methods of extraction
have been used. The (luality of the results
obtained with a given method will often
depend on the medium used for growing the
producing organism. Certain nutrients will
interfere with a given method of extraction,
iust as metabolites formed on a given me-
40
ISOLATION AND IDENTIFICATION OF ANTIBIOTICS
41
clium may also affect the ease of puritication.
Thus, it is wise to have close cooperation
between workers invoh'ed in media cle\'elop-
ment and those who carry out the extraction
of an antibiotic.
Methods of Characterization
The characterization of an antibiotic in-
volves the determination of its properties:
physical, chemical, and biological. Plants,
animals, and other li\'ing systems camiot be
easily classified on the basis of a single char-
acteristic which would be different for each
]i\-ing form. Similarly, ouv cannot usuall}'
differentiate between the products of li\'ing
systems on the basis of one single criterion;
this is true particularly of the \-arious anti-
biotics. A number of properties should be
studied, and an^^ comparison between anti-
biotics must be based on a number of char-
acteristics.
It is impossible to make a list of "classical"
criteria which would always permit one to
differentiate between two antiliiotics and
which would always be adeciuate for the full
characterization of an antibiotic. A list
satisfactory at one time will be inade(iuate a
few years or even a few months later. Scien-
tific methods are forever changing. The
investigator must be adaptable and refrain
from taking a dogmatic stand.
We will discuss various criteria which ha^'e
been used in the characterization of anti-
biotics. In the keys found in Part H of this
book we will try to use these characteristics
to single out each antibiotic which had been
described by the time that the compilation
was completed. This will not always be
possible, since some substances have not
been described in sufhcient detail. In such
cases we simply group similai' substances
together.
Nature of Antibiotic-producing Organisms
It has been observed that certain types of
cultures tend to form certain types of anti-
biotics. For example, members of the Strcpto-
myces griseus group ha\'e been found to
produce streptomycin, cycloheximide, gri-
sein, and candicidin, to name only a few.
Similarly, strains of S. fradiae tend to pro-
duce antibiotics of the neomycin tN'pe and
fradicin. However, the ability of a cultiu'e to
produce a particulai' antibiotic is hardly a
criterion for the identification of the organ-
ism or of the antibiotic. This is due partly to
the confused state of the systematics of this
group of organisms. Further, it has been
shown that the same antibiotic can be pro-
duced by a \-ariety of actinom^'cete species
or e\'en by \'arious genera. In this respect
the paper of Fishei- and coworkers (19ol) is
of interest. These authors isolated and
chemically characterized actinomycin from
a strain of Micromonospora. This antil)iotic
is usually j^roduced l)y streptomycetes and
is known to be a mixture of closely related
substances. It is then conceivable that the
actinomycin elaborated by the Micro-
monospora might ha\'e been different from
the actinomycins of the streptomycetes. But
any difference must ha\'e been minor indeed,
since the amino acid content of the Micro-
monospora actinomycin was not unusual. It
is also unlikely that a Micromonospora would
be confused with a Strcptomyces. In this light,
one should consider with reserve the state-
ment of Krassilnikov (1960) that authors
who claim that the same antibiotic can be
produced by different species "are mistaken
either in speci(>s identification or in the
accuracy of detei'mining the nature of the
antibiotic."
Teillon (1*.)."):)) suggested a scheme for the
separation of antibiotic-producing organ-
isms on the basis of their sensiti\'ity to
known antibiotics (Table l(i).
A ntim icrobial Spectra
The range of antimicrobial activity of any
antibiotic should be determined against
aerobic l)acteria, both gram-positive and
42
NATURE, FORMATION, AND AC'TIVITIlvS
Table 1(3
Dirholoiiiir kei/J'nr iileiitijicalion of Slreptonu/rcfi antibiotics (Teilloii, 10.53)
+ = jiositivp inhibition; = no inhil)ition.
Streptomycin
St rp|)toniycin culture
Oxytetracj-cliue culture
( )xvtetracvline
+
Oxytetracyline culture Streptomvcin culture
Biotype .S. rimosKS Biotype »S. griseiis
+
Chloramphenicol culture
Chlortetracycline culture
Streptothricin culture
Neomycin culture
Antibiotic X culture
-Chlortetracycline h
- Chloramphenicol
Chlortetracycline
culture
i
Biotype S. aureo-
faciens
+
Chloramphenicol
culture
Streptothricin
culture
Neomycin culture
Antibiotic X
culture
Chloramphenicol
culture
i
Biotype S. venezuelae
-Streptothricin 1 +
Streptothricin culture Antibiotic X culture
i
Biotype S. lavendulae
gram-negative, anaerobic baoteria, myco-
bacteria, actinomycetes, and fungi, l)oth
filamentous and yeast forms. In such deter-
mination of activity, dilution methods are
preferable to diffusion methods, since some
antibiotics which are very active on a weight
basis do not diffuse readily in agar media. If
possible, such an antibacterial-antifungal
spectrum should l)e complemented with
studies of antiprotozoal, antialgal, antirick-
ettsial, antiviral, and antitumor activity.
Two different antibiotics may have very
similar antimicrobial spectra. This is true
of the antifungal polyenes candidin and
Streptothricin cultvire
Neomycin culture
Antibiotic X culture
Neomvcin-
Neomvcin culture
i
Biotype <S. fradiae
+
Streptothricin culture
Antibiotic X culture
amphotericin B, which have been differ-
entiated biologically only with great care
(Lechevalier, 1900).
Mutants of microorganisms resistant to
known antibiotics are usually included
among the test organisms used for the deter-
mination of antimicrobial spectra. If there
is no cross-resistance between two otherwise
closely related antibiotics it can be assumed
that the antibiotics are different. However,
there usually is cross-resistance between
closely related compounds and this may even
occur between very different substances, as
was demonstrated by Pledger and Lecheva-
ISOLATION AND IDENTIFICATION OF ANTIBIOTICS
43
Her (1956), who found cross-resistance be-
tween certain antibiotics, such as neomycin,
and a mild silver protein.
In the case of streptomycin, tlependent
nuitants are available which will not grow
except in the presence of streptomycin or
certain of its derivatives.
Development of Resistance
If a large number of cells of a given
microbe are subjected to an antibiotic, most
of the (^ells will be inhibited, but a few will
grow. These cells will be resistant to either
low or high concentrations of the antibiotic.
A population of Escherichia coli, for example,
will contain a few cells that are very resistant
to streptomycin. Only a few cells in the
particular population of E. coli are slightly
more resistant to neomycin than the bulk
of the cells; the slightly more resistant popu-
lation can again be put in contact with
neomycin and still more resistant mutants
will be selected. The ease with which mu-
tants of a given organism resistant to an
antil)iotic can be obtained is a useful cri-
terion for the characterization of the sub-
stance. However, one must keep in mind
that development of resistance to one given
antibiotic may be fast for one microorganism
and slow for another. Polyenic antifungal
antibiotics are noteworthy examples of sub-
stances to which resistance develops slowly
and never achieves high levels. The practical
problems in chemotherapy arising from the
development of resistance are discussed in
Chapter 10.
Microbio static and Microbicidal Action
If a microbial population is subjected
under identical conditions to concentrations
of antibiotics high enough to prevent growth,
and if after a period of contact the anti-
biotics are removed and the cells placed in a
suitable nutrient medium, one can easily
demonstrate that some antibiotics will have
killed the test organism whereas others will
simply have prevented its growth. Leche-
valier (19()0) has shown differences in the
fungicidal action of polyenes which could
be useful in the characterization of these
polyenes.
Use of Inhibitors
The antimicrobial action of certain anti-
biotics is inhibited by specific substances.
For example, the action of streptomycin is
inhibited by cysteine, hydroxylamine, and
nucleic acids; that of neomycin, only by
nucleic acids. The action of chlortetracycline
on E. coli has been found to be competitively
inhibited by riboflavin. The action of oxy-
tetracycline against both gram-positive and
gram-negati\'e bacteria is re\'ersed by mag-
nesium ions, whereas in the case of novobio-
cin, magnesium sulfate reverses the inhibi-
tion of gram-negati\'e bacteria but not that
of gram-positive bacteria (Brock, U)")!)).
Mode of Action
Each antibiotic inhibits one metal)olic
reaction or a series of such reactions in the
microbial cell. These reactions can be used
as criteria for the characterization of the
antibiotic. The mode of action of antibiotics
in general is poorly understood (see Chapter
9), but some antibiotics have become bio-
chemical tools for the study of microbial
metabolism. Vor example, chloramphenicol
is an inhibitor of protein synthesis; nystatin
inhibits endogenous respiration and gly-
colysis in fungal cells; and antimycin A is an
inhibitor of electron transport in the cyto-
chrome C system.
The effect of the antibiotic on the micro-
bial cell may at times alter the morphology
of the sensitive organisms. For example, low
concentrations of fradicin induce the cells of
Candida albicans to l)ecome filamentous,
whereas other antifungal antibiotics, such as
nystatin and candicidin, do not induce any
change in the form of the yeast.
44
NATURE, FORMATION, AND ACTIVITIf:S
Physical Properties of Antibiotics
SOLUBILITY
A survey of the solulMlity of an iintil)iotic
ill x'arious organic solvents and in water, at
various pH values, is a very useful method
of characterization. This method also gives
information aV)Out changes in the substance
being investigated.
An acidic substance will be least solulile
in water at an acid pH and a basic substance
will be least soluble at an alkaline pH. An
amphoteric substance will be least soluble
at the pH of its isoelectric point. This in-
formation can be supplemented with pf)-
tentiometric titrations.
One must remember that the solubility of
a crude preparation can be \'ery different
from that of the pure antil)iotic. Thus, crude
fradicin is soluble in ethanol, and crystalline
fradicin is soluble only in the glycols. As a
rule, the purer the substance, the less soluble
it is. Candicidin is a good example: crude,
it is partly soluble even in water; pure, it is
one of the least soluble antibiotics.
STABILITY
As we have already discussed, knowledge
of the stability of an antibiotic at \'arious
pH values and at various temperatures is an
essential prelude to the study of purification
procedures. Stable antibiotics such as chlor-
amphenicol and neomycin will not lose
activity if autoelaved at a neutral reaction.
Other antibiotics, such as mycomycin, are
highly unstal)le. The half-life of mycomycin
is only o hours at 27°C. In this respect,
certain antibiotics are most unusual; for
example, upon standing, rifomycin B in-
creases in activity as a result of molecular
rearrangement and subsequent transforma-
tion into a more potent antibiotic.
COLOR REACTIONS
Chemists have de^•ised a number of color
reactions which are characteristic of certain
molecular structures, such as the ninhydrin
reaction for primary amino groupings, the
Sakaguchi reaction for guanido groups, and
the I'^ehling test for reducing sugars. The ap-
plication of such tests to antibiotics and
their degradation products is very useful in
characterizing the antibiotic.
In this area, it should be mentioned that
certain antibiotics fluore.sce when excited
by ultraviolet light. The bright yellow
fluorescence of oxytetracycline is one ex-
ample.
LIGHT ABSORPTION
The light absorption of a substance can be
measured in the ultraviolet, the visible, and
the infrared range. To be of any significance,
the infrared spectrum of a substance must
be measured on the pure compound. In
certain cases, such as that of the neomy-
cins, the infrared spectrum gives little infor-
mation. In other cases, it will give the
chemist consitlerable information on the
various functional groupings which are
present in the molecule of the antibiotic. In
contrast, certain antibiotics have such char-
acteristic and intense visible or ultraviolet
light spectra that they can be detected in
the crudest extracts. This is true of the
polyenic antifungal antibiotics. If two sub-
stances have similar absorption spectra, this
indicates the presence in the two molecules
of similar groupings but does not mean
necessarily that the two substances are
identical. Vov example, trichomycin and
candicidin are two different antibiotics with
the same light-absorption spectrum.
Chromatography
Ohromatography is a method which can
be used not only to purify antibiotics, l)ut
also to characterize them. In this respect,
paper chromatography has been of great
value. It can be carried out in either the
vertical (ascending and descending) or the
horizontal plane (circular and centrifugal).
ISOLATION AXl) IDENTIFICATION OF ANTIBIOTICS
45
It can also be carried out moi'e than once on
the same sheet of paper (two-dimensional
chromatography) . I'aper chromatography
can be used to compare the Ivf vahie of one
antibiotic with that of anothei- or to compare
the decomposition products or the deriva-
tives of one antibiotic with those of another.
The antibiotic can be located on the paper
chromatogram biologically (bioautogram)
or by chemical color reactions. Laboratories
actively engaged in the searcii for new anti-
biotics have developed a number of solvent
systems which permit separation of most of
the known antibiotics into small subgroups.
Despite the fact that such paper chroma-
tographic systems play an important role in
the characterization of antibiotics, it should
not be taken for granted that two substances
are identical if they have identical Rf values
in a lumiber of solvent systems. Neomycin B
and neomycin C, for example, were resolved
by paper chromatography only after being
acetylated (Pan and Dutcher, 11).")()).
Electrophoresis
Similar antibiotics may, in certain cases,
be separated from one another by submission
to a difference in electric potential in a
buffered environment. For the isolation and
characterization of antibiotics, electropho-
resis is carried out either in agar or on paper.
Co un tercurre n t Distrib 1 1 Hon
Countercurrent distribution is another
method that can be used for both purification
and characterization of antibiotics. Similar
in principle to partition chromatography,
this method permits separation of substances
as the result of differences in partition coeffi-
cients betw^eeii two immiscible solvent sys-
tems. More laborious than chromatography,
the method recjuires costly instruments.
Countercurrent distril)ution is often used as
a criterion of purity, especially for anti-
l)iotics that cannot be crystallized.
Elementary A ncilysis
The elementary analysis of a pure prepara-
tion of antibiotic is, of course, necessary for
the determination of its empirical formula.
An elementary analysis of crude prepara-
tions of antibiotics can also be most useful for
characterization. Its usefulness is limited,
however, primarily to the negative results
obtained: such analyses can show, for
example, that a substance crjutains no
halogens, no nitrogen, or no sulfur.
Ehijsical ( 'onstants
Once a new or unknown antil)i()tic has
been obtained in a pure form it can then be
characterized on the basis of its physical
constants — such as density, refractive index,
molecular weight, melting point, and optical
rotation — and the physical constants of its
salts and derivatives.
Eventually, the substance is fully charac-
terized when its exact chemical structiu'e is
known in all its spatial intricacies.
Classification of Antibiotics of Actino-
niycetes
The antibiotics of actinomycetes represent
a great variety of chemical compounds. They
vary greatly in their physical properties,
including stability to heat and to oxidation,
and in their solubility in water and organic
solvents. They vary also in their chemical
composition, in their antimicrobial activities,
and in their toxicity to animals. These differ-
ences are frec^uently not only c[ualitative but
also (luantitative in nature. Hence, it is
difhcult to classify them. However, various
systems for the classification of antibiotics
have been proposed.
Several criteria ha\'e been used in estab-
lishing such systems. These are based upon
(1) the nature of the organisms that form
the antibiotics, (2) the specific antimicrobial
spectra or selective action of the antibiotics
upon different groups of microorganisms, (3)
46
NATURE, FORMATION, AND ACTIVITIES
the practical utilization of antibiotics in the
control of human, animal, or plant diseases,
and (4) the chemical nature of the anti-
biotics. Each of these systems has certain
distinct advantages and disadvantages.
BIOLOCilCAL SYSTEM
The biological system offers the advantage
of a knowledge of the specific nature of the
organisms producing the antibiotics. It has
even been argued that the ability of a certain
organism to form a particular antibiotic is
more characteristic of the species than such
properties as pigment production, sugar
utilization, or enzyme formation. The fact,
however, that the same antibiotic may be
produced by different organisms and, fur-
ther, that the same organism, or even strain,
may yield more than one antibiotic, reduces
the usefulness of this system of classification
designed primarily for characterization
piu"poses. The use of this system is further
complicated by the great variability of the
antibiotic-producing organisms, including
ordinary morphological, cultural, and bio-
chemical \'ariations and hereditary mutant
formations; the latter may involve either a
gain or a loss of the ability to produce a
certain antibiotic. Differences in chemical
composition or physical state (such as semi-
solid ve7-sus liquid) of media used for the
cultivation of antibiotic-producing organ-
isms, in the degree of aeration of the culture,
and in the length of the incubation period
may all result in chemical differences in the
antibiotics produced. The sensitivity or
resistance of the organisms to their own
antibiotics and the ability of certain organ-
isms to decompose the antibiotics after they
have been formed are other limiting factors
of such a system. Too little is yet known of
the mechanism of formation of the anti-
biotics in the mycelium and their liberation
into the medium to permit use of this reac-
tion for classification purposes.
ANTIMICROBIAL ACTIVITIES OR ANTIBIOTIC
SPECTRA
The antimicrobial activities or antibiotic
spectra offer another system for classifying
antibiotics. A knowledge of the range of
antimicrobial activities of an unknown anti-
biotic may permit its ready identification
with known substances. Naturally sensitive
and resistant organisms, as well as organisms
made resistant to known antibiotics, can be
used in the classification of antibiotics in
general. Unfortunately, different strains of
the same organism may \'ary considerably in
their sensitivity to a given antibiotic. Fur-
ther, the ease with which many organisms
may produce mutants resistant to an anti-
biotic reduces the usefulness of such a sys-
tem. Incidentally, this system has been
greatly abused for commercial purposes by
the designation of various antibiotics as
members of the "wade spectrum," "broad
spectrum," "narrow spectrum," and "stub-
born spectrum" groups.
PRACTICAL UTILIZATION
The practical utilization of antibiotics also
has much to disc^ualify it as a basis for
classification, since relatively few antibiotics
produced by actinomycetes, among the
many so far disco^^ered, have found practical
application. The fa\'or which a particular
antibiotic finds in the hands of the clinician
or the veterinarian has little to do with its
chemical nature and its })iological origin.
CHEMICAL SYSTEM
The chemical system appears to offer the
most logical basis for the classification of
antibiotics of actinomycetes. Unfortunately,
this system does not take into consideration
the range of antimicrobial activities of the
antibiotics; it does not explain the sensi-
tivity and resistance of different microorgan-
isms to what appear to be chemically closely
related antibiotics; and it does not indicate
ISOLATION AND IDENTIFICATION OF ANTIBIOTICS
47
the practical potentialities of freshly isolated
antibiotics.
In view of the rapidl}' accumulating in-
formation concerning the large number of
actinomycetes capable of producing anti-
biotics under certain conditions, and in view
of the numerous new antibiotics constantly
being isolated in various laboratories
throughout the world, no final system of
classification can be proposed at the present
time. The chemical nature of the substances,
supplemented by a knowledge of the range
of their antimicrobial activities, offers a good
basis for a tentative system of classification,
as outlined in detail in Fart B.
Control and Standardization of Anti-
biotics
Those antibiotics that have found prac-
tical application in disease control, in animal
feeding, or in the preservation of food mate-
rials are carefully controlled by official
government agencies in different countries
(Dony and Guisset, 19G0). Official standards
are usually established for such antibiotics,
their sale for medicinal purposes being
limited primarily to prescriptions. The wide
and often indiscriminate use of antibiotics,
by many physicians and especially in certain
countries, has resulted in the rapid develop-
ment of resistance to antibiotics in general
and to some antibiotics, such as penicillin,
in particular. This has led to recommenda-
tions that a careful study be made of the
sensitivity of the causative agents of infec-
tion to various antibiotics, so that only a
specific antibiotic would always be em-
ployed. Finally, the World Health Organiza-
tion, recognizing the growing importance of
antibiotics in the world at large, has taken
steps to coordinate research in certain im-
portant problems pertaining to the wide use
of antibiotics. Its Expert Committee on
Antibiotics has recently (H)()f) published a
report on standardization uK^hods for assay-
ins; antibiotics.
Chapter 6
Chemical Nature of the Antibiotics
of Actinoiiiycetes
The known antibiotics prodiicctl by
actinomycetes range in complexity from
very simple compounds, such as nitro-
propionic acid (bovinocidin), to very
complex proteins, such as the lytic enzymes
of actinomycetin. Certain chemical types
are linked with a specific range of anti-
microbial activity. For example, the polyenes
are mainly antifungal in nature, and the ex-
ceptions reported in the literature may very
well be due to antibacterial impurities. Also
the streptothricins are active against both
gram-positive and gram-negative bacteria
and even fungi, whereas the nonpolyenic
macrolides are, as a whole, active only
against gram-positive l)acteria. In spite of
these general correlations between types of
structure and activity, it is not yet possible
to predict in all cases the range of anti-
microbial activit}' of an antibiotic purely on
the basis of its chemical structure. Likewise,
it is not yet possible to synthesize com-
pounds, the biological properties of which
would be known beforehand.
In this chapter, the antibiotics are grouped
according to their chemical similarities.
More detailed information and references
will be found in Part B of this book and in
the reviews written on this subject recently
by Abraham and Xewton (1958), Chain
(1958), Van Tamelen (1958), Harman
(1959), and also in the Merck Index (1960).
From a chemical point of vi(^\v, antibiotics
can Ije grouped according to various criteria:
(1) the elements contained in their mole-
cules, (2) the most important groupings in
these molecules, and (3) the structure of
their molecular skeleton.
An examination of the empirical formulas
of the antibiotics of actinomycetes listed in
Part B of this Ixjok reveals that most of
these substances contain carbon, hydrogen,
oxygen, and nitrogen (153 compounds).
Next in frequency are substances containing
only carbon, hydrogen, and oxygen (31
compounds), followed by substances con-
taining carbon, hydrogen, oxygen, nitrogen,
and sulfur (18 compounds). This survey
takes into account only those substances
for which an empirical formula was at least
proposed; howe\'er, their number is probably
high enough to be representative of the
whole group. A few substances have unusual
elementary compositions: chloramphenicol,
chlortetracycline, demethylchlortetracycline,
and exfoliatin contain nonionic chlorine.
Bromine can l)e substituted for this chlorine
in chlortetracycline to form bromtetra-
cycline, and possibly a similar substitution
can be made in the other substances.
Exfoliatin contains only carbon, hj^drogen,
oxygen, and chlorine, whereas the other
antibiotics of this group contain, in addition,
nitrogen. Two antibiotics have unique
elementary composition: grisein contains
sulfui' and iron, and phleomycin contains
48
CHEMICAL NATURE OF ANTIBIOTICS
49
copper in addition to the four most common
elements. One will note that there are no
known antibiotics of actinomycetes with a
molecule composed of only carbon and hy-
drogen.
In the following review of the chemical
structure of the antibiotics of actinomycetes,
an effort has been made to group together
the antibiotics with similar structure. In
some cases the antibiotics are grouped to-
gether because they have an outstanding
functional group in common. For example,
chloramphenicol is discussed with bovinoci-
din because they both have a nitro group.
In the chemical formulas used throughout
this book no pretention is made to show the
spacial arrangement of the atoms.
Nitro Compounds
BovinocidiN (/5-nitropropionic acid) has the
following structure:
NOo
I
CH2— CHo— COOH
This substance, which is also produced by
higher plants and fungi, has weak anti-
my cobacterial activity .
Somewhat more complicated chemically is
chloramphenicol,
NO2
> CHoOH
I
CH(OH)— CH— XHCOCHCI,
Its molecule, which contains nitrobenzene
and two atoms of nonionic chlorine, is very
active biologically. Chloramphenicol is ac-
tive against both gram-positive and gram-
negative bacteria, rickettsiae, and members
of the lymphogranuloma group of intracellu-
lar parasites. It can he synthesized chem-
ically in commercial (luantities.
Azomi/cin, which is discussed under
miscellaneous monocyclic compounds, is also
a nitro compound.
Straight -chained Diazo Compounds
Azascrinc and DON are diazo derivatives
of amino acids. DON is G-diazo-o-oxo-L-
norleucine,
N.— CH— CO— (CH,).,— CH— COOH
NH2
and azaserine is o-diazoacetyl-L-serine,
N2— CH— CO— ()— CHo— CH— COOH
i
NHo
The antitumor activities of these two
similar compounds have aroused interest.
Both compounds also have modest anti-
microbial activity. Three compounds of
undetermined structure, diazomijcins A, B,
and C, are also aliphatic diazo compounds
with a similar type of biological activity.
Alazopcptin, a more complex substance, is a
peptide containing a-alanine and a diazo-
ketoamino acid. It has modest antitumor
activity.
Elaiom>jcin is one of the few natural
products with an alipiialic azoxy linkage:
CH0OCH3
T I
CH;,(CH,),,CH=CH— X=X— CH— CHOH— CH:,
It is actix'c almost exclusively against myco-
bacteria.
Miscclhmeous Monocyclic Compounds
Qticstiomtjcin B is o-aminophenol,
OH
XHo
It has some antimycobacterial activity and
is believed to be the })uilding stone of another
antibiotic, questiomyciu A (O-aminophen-
oxazone), which will l)e discussed with the
actinomycins.
Sarkomijcin is a simple cyclopentane
derivative,
H2C=| ,— COOH
o=L J
50
NATURE, FORMATION, AND ACTIVITIES
which has activity against tumor colls and a
few gram-positive bacteria.
Acetomycin is a neutral saturated acetoxy
lactone,
CH3 CH3
-COCH,
/
CH3CO— o
o
\
o
which is moderately active against myco-
l)acteria and protozoa.
Azomycin is 2-nitro-imidazole,
,1 N
NO2
This antibiotic, which contains an ecjual
number of molecules of nitrogen and carbon,
is active mainly against bacteria.
Cycloseriyie is a ketoaminoisoxazole, which
is active mainly against mycobacteria:
HsNr
-,=0
.N
O
The chemical structure of cycloserine was
identified as D-4-amino-3-isoxazolidone. On
acid hydrolysis, it gives hydroxylamine and
serine. Its biogenetic relationship to alanine
has also been suggested (Abraham and
Newton, 1958). Its synthesis has been
brought about, as summarized by Van
Tamelen (1958).
Another antibiotic with activity only
against mycobacteria is actithiazic acid, a
keto thiazole with an aliphatic acid chain in
position 2:
0=
-N
-(CH.2)5COOH
It is 4-thiazolidone-2-caproic acid. It is
soluble in organic solvents and its sodium
salt is soluble in water. It has been syn-
thesized, the racemate having about half
the activity of the natural levo form. The
toxicity is low, but the antibiotic is not
active in vivo l)ecause of the presence of
biotin, which interferes with its activity
(Grundy r/ a/., 1952).
Cyclohexi 111 ides
This is a group of antibiotics containing a
glutarimide moiety. Of these, the antifungal
agent cydohcximide has been known for the
longest time:
CH,
CH3
=0
CHOHCH.
O
/
NH
\
O
Its molecule has four centers of asymmetry.
Some of the isomers of cycloheximide have
been studied; they have been foimd to have
less biological activity than the mother com-
pound. One of the eight possible dehydro-
cycloheximides, inactone, has no biological
activity. Four substances which have been
studied mainly because of their antitumor
activity, strcptovitacins A, B, and (' and
antitumor .substance E 73, have the same
molecular skeleton as cycloheximide. Their
structural formulas will be found in Part B
of this book.
Stre-ptimidone is an antifungal-antipro-
tozoal antibiotic which also has a glutarimide
moiety to which an aliphatic chain is at-
tached:
CH.CHCCH.CHCHCHs
O
COCH.CHOHCH/ NH
_/
\
O
CHEMICAL NATURE OF ANTIBIOTICS
51
Aureothricin-type Coiiipoiinds
Three antibacterial and antifungal anti-
biotics have the same molecular skeleton,
composed of two rings formed of five atoms
of carbon, two of sulfur, and one of nitrogen.
The nitrogen-containing ring is of the
pyrrolidine type:
,S R
S
/
\-
Aureothricin
Thiolutiii :
Ri
R = NHCOCH.CH,
R, = CU,
R = NHCOCH:,
]{, = CH;,
Holomyciii: R = NHCOCH3
R, = H
Aurcothricin and thioiutin differ only in one
C'Ho group in the side chain R. Holomycin
is des-X-methyl thioiutin. Acid hydrolysis
of aurcothricin and thioiutin yields a weak
amine, pyrrothine (CeHeXoOS^). The hy-
drolysis of holomycin yields des-N-methyl-
pyrrothine, which is called holothin and
which has some antibiotic activity of its
own.
Other sulfur-containing compounds of dif-
ferent types were isolated from cultures of
actinomycetes. They include lavendvilin
(C49H6:i6i8Ni3S:0, suifactin {C^rjUio^HOS-.d,
thiostrepton (7.4 per cent S), and sulfocidin.
Thioaurin (Ci4Hi2X404vS4) is an orange-
yellow substance active against gram-posi-
tive and gram-negative bacteria but not
against fungi.
Compounds Containing a Purine or a
Pyriniidine Base
A few products of actinomycetes contain a
purine nucleus. They include puromycin,
nucleocidin, psicofuranin(% and the angust-
mycins. Thi'ee of these substances, angust-
mijcui (\ antibiotic U 9586, and psicofura-
nine, are presum(>d to have the same struc-
ture:
NH.,
N
-N
N N
y^
OHCHo
>— CH.CJH
OH OH
Their molecule contains two moieties,
adenine (()-aminoj)urine) and the keto-
hexose, D-psicose. The special configuration
of the atoms in the molecule of these three
compounds must vary from one to the other,
since their biological activity differs. Angust-
mycin C has no known biological activity.
Antibiotic U OoSl) has some activity against
bacteria and tumors, and psicofuranine has
some activity against tumors and liacteria
in nro. In vitro antil)acterial activity was
demonstrated when a special medium con-
taining liver extract was used.
Anduxtmt/cin A differs from the previous
compounds in \hv sugar attached to the 6-
amin()i)urine, which is called angustose:
O
^— CH:,
J— OH
OH
OH
Angustmycin A is active mainly against
mycobacteria.
Tubercidin and toi/ocami/cin are similar
compounds with an adenine and a D-ribose
moiety.
Pnromi/cin has a more complicated mole-
cule. It is composed of three moieties: (1)
G-dimethylamino purine, (2) D-8-amino-
ribose, and {'^) o-methyl-L-tyrosine:
52
NATURE, FORMATION. AND ACTIVITIES
CHs CH,
\ /
N
N
"^N^^N-"
OH-
-CH,OH
NH— CO— CH(NH.O— CH,
I
OCH3
Puromycin is active against bacteria (mainly
gram-positive), protozoa, and tumors. Re-
moval of the methyltyrosine moiety from
the molecule caused a loss in antibacterial
activity. The resulting aminonucleoside was,
however, acti\-e against protozoa and
tumors.
Nudeocidin is another meml^er of this
group of purine-containing compounds. It is
active against bacteria and protozoa. It con-
tains a G-aminopurine moiety, a carbohy-
drate moiety, and sulfur.
Three antibiotics, amicetin, amicetin B,
and bamicetin, are formed of four cyclic
moieties, one of which is a pyrimidine: (1)
p-aminobenzoic acid, (2) cytosine, (3) a
six-carbon sugar-like unit in the furan form,
and (4) a dimethylamino sugar, amosamine
(C8Hn04N):
CH.-i CH3
N
OH
OHCH,
'CH,
nw ^O' \ / \
^'^^ N />— NHCO
/ \
O
/
-N^'
In the molecules of both amicetin and
bamicetin,
CH,
!
R = XHCOC— CH.OH
I
NHo
The two antibiotics differ in that l)amicetin
has one less — CHo group in the glycosidic
moiety than does amicetin. In amicetin B,
R = XH2 . These three antibiotics are ac-
tive mainly against gram-positive bacteria
and mycobacteria. It is interesting to note
that bamicetin is more active against gram-
negative bacteria than are the amicetins.
It has been suggested that amicetin B might
be a precursor of amicetin.
Tetracyclines
The tetracyclines ha\'e in common a naph-
thacene nucleus; their range of biological
activity approximates that of chlor-
amphenicol; they are active against gram-
positive and gram-negative bacteria, ricket-
tsiae, and the psittacosis-lymphogranuloma
group of organisms. The three commonly
used antibiotics of this group are tetracycline,
chlortetracycline, and o.vytetracycline. They
have the following formulas:
OH OH
TetnifvcliiK': R, = R, = H
Oxytetiacvfline: Ri = H; R,. = OH
Chlortetracycline: Ri = CI; Ri = H
When chlortetracycline-producing organ-
isms are grown in chlorine deficient media
that contain bromine, bromine is substituted
for chlorine and bromtetracycline is formed.
A slight drop in biological activity follows
this substitution.
CHEMICAL NATURE OF ANTIBIOTICS
53
Mutants of S. aureofacicKS, in chlorine-
containing media, can form another useful
\-ariation of the chlortetracycline molecule
t)y producing demethylchlortetracycline, in
which there is no metlwl group in position (i.
Anhydrochlortetracycline is obtained l)y
acid degradation of chlortetracycline. It
differs from chlortetracycline in the structure
of rings B and C :
CH
OH ()
This compound has a rather specific activity
against actinomycetes (Goodman d al.,
I'.l.'i.V).
Antibicjtic X 340 is a tetracycline, the
complete structure of which is not yet
known. It differs in biological activity from
the other tetracyclines in that it is active
mainly against gram-posit i^'e bacteria.
Acetylenic Compounds
Two antibiotics, cellocidin and myco-
mycin, have acetylenic bonds. Cellocidin is
acetylenedicarboxamide,
H,N— CO— C=C— CO— NHo
It is active mainly against mycobact(>ria,
but also has a moderate action against l)ac-
teria.
Mycomijcin has a wider range of anti-
microbial activity, being active against bac-
teria, mycobacteria, and fungi. It is an un-
saturated carb(«ylic acid with l)oth ethylenic
and acetylenic linkages:
H=C— C=CCH=C=CHCH=
CH— CH=CHCH,C( )()H
This highly unstable substance explodes at
7o°C. A more stable and also biologically ac-
tive isomer is obtained by treatment of
mycomycin with alkali. It is called isomyco-
mycin:
CHsC^C- C=C— C=C— CH=
CH— CH=CH— CH .— C( )OH
Polyenic Compounds
These unsaturated compounds have only
ethylenic bonds. They form a large group of
antibiotics which are mainly antifungal.
These polyenes can be grouped together on
the basis of the number of conjugated carbon
to carbon (loul)le l:)onds present in th(^
molecule. Most of them have four, fi\e, six,
and seven such unsaturated bonds in their
chromophores which are responsible for \'ery
typical three-peaked light-absorption spec-
tra. One antibacterial antibiotic, antibiotic
PA 147, has a diene chromophore and could
be considered as a small polyene :
f. ^OH
H,C=CH
O
Antibiotic PA 147 has a lactone ring, like
the larger polyenes, but the polyenic portion
of the molecule is not all enclosed in the
lactone ring.
The diene, antibiotic PA 147, has on(>
single peak of light absorption at 272 niju.
The polyenes with four conjugated double
bonds, the tetraenes, have three main peaks
of light absorption; the position of the
central peak is at 300 to 30o m/x. The posi-
tion of the central peak is 330 to 340 mn
for the pentaenes, 3oo to 3o9 m^i for the
hexaenes, and 377 to 388 ni/u for the hepta-
enes. To date, no triene or octaene has been
discovered among the products of actino-
mycetes.
The complete structures of one tetraene,
pimaricin, and two pentaenes, filipin and
lagosin (Dhar et al., lUliO), have been re-
ported. Some headway has also been made
in the elucidation of the structures of the
tetraene nj^statin and of the heptaenes
54
NATURE, FORMATION, AND ACTIVITIES
candidin, amphotericin B, caiidicidiii, tri-
chomycin, and perimycin.
Pimaricin has the empirical formula
C34H49NO14 and the following sti-ucture:
heptaenes. Of the five hexaenes reported in
the literature, four are said to ha\'e some
kind of antil)acterial activity.
The chemistry of the heptaenes has been
H2X
-OH
The nitrogen is present in the form of an studied chiefly by Borowski and coworkers
amino group in a five-carbon sugar moiety, (1960). Amphotericin B and candidin are
mycosamine. A large lactone ring of the xevy closely related substances, each of
type found in the bacterial macrolides, such which contains one atom of nitrogen located
as erythromycin, is formed in part by the in the amino sugar, mycosamine. In contrast,
tetraenic chromophore. A carl)oxyl and an candicidin, trichomycin, and perimycin have
epoxide group are attached to the large twoaminogroupsper molecule. In candicidin
lactone ring. Alycosamine is also present in and trichomycin, one of these amino groups
the molecule of another tetraene, nystatin, is located in a mycosamine moiety and the
and in the molecule of some of the hep- other in a p-aminoacetophenone moiety:
taenes. Nystatin has a larger molecule than
pimaricin; it has the empirical formula
Lagosin and filipin are two pentaenes
which do not contain nitrogen:
CH3(CH2)4CHOH
CHa— CO— r ^— NH2
Perimycin dilTers in that one of the amino
groups is located in an amino sugar moiety
OH
Lagosin: R = OH
The large lactone ring of each is formed of
27 carbons and includes the pentaenic
chromophore.
Other pentaenes, such as antibiotic PA
1").'^, contain nitrogen.
The hexaenes are the least known poly-
enes. They have not been isolated as often
as the tetraenes, the pentaenes, and the
OH
Filii)iii :
of unknown structure and the other is in a
p-aminophenylacetone moiety :
CH,— CO— CH,,-
-NH2
Perimycin, unlike other heptaenes, does not
have a carboxyl group.
With the help of the keys, sunnnaries of
CHEMICAL NATURE OF ANTIBIOTICS
55
the data pul)lished on the various polyenes
can he found in Part 1^ of this book. One
should keep in mind that many of the poly-
enes described may be synonyms. This will
be clarified l)y further studies in tlie chem-
istry of these interesting compounds.
Macrolides
Certain antibiotics, which are active
mainly against gram-positive Ixicteria, are
called macrolides because each has a large
macrocychc lactone ring. One will note that
the polyenic macrocyclic lactones also ha\'e
this structtiral feature. Since the nonpolyenic
macrocyclic lactones were described first, the
Table 17
List of niacrolide antibiotics
Antibiotic
Amaromycin
Angolamycin
Antibiotic PA 108. .
Antibiotic PA 133A
Antibiotic PA 133B.
Antibiotic PA 148..
Carbomvcin
Carbomycin B
Erythromycin
Erythromycin B . . .
Erythromycin C . . .
Foromacidin 1)
(rri.seomycin
Leucomycin
Leucomycin B
Methymycin
Miamycin
Narbomycin
Neomethym\cin. . . .
Oleandomycin
IMcromycin
Proactinomycin A..
Proactinomycin B..
Proactinomycin C.
Spiramycin I
Spiramycin II
Spiramycin III
Tylosin
Tertiomycin A
Tertiomycin B
Tenichiomvcin
Empirical formula
C25H39O7N
C4 9-6irl>j7-ill' )l8-^
CssHesOu-^
C2 5H4,,06N
C.,5H4 50,„X
CssHeoOisN
C42H670l6^^
C41-42H67-69O15-I6N
C:i7H670i:iN
C37H67O1.N
C36H6.5O1.-iN
C28H48O8N
C33-38H54-66O11-13N
C41H69O16N
C20H4..O7N
C.:8H4707N
C25H4.3O7N
C35H6I-63O12N
C25H43O7N
C2-H47O8N
C2sH4a08N
C24H4,06N
C45H78O10N2
C47H8oO,6N2
C48H82O16N2
C45-46H75-79O17-I8N
C42H69O16N
C43H71O17N
C28H43O16N
name "macrolide" must be reserved for this
group of sul)stances. Howe\'er, the polyenic
macrocyclic lactones can be called "polyenic
macrolides."
Picromycin was the first compound defi-
nitely known to belong to the macrolides,
but the first antibiotics of this group to be
isolated were the proactinomycins reported
by CJardner and Chain in l'.)42 (see also
Marston, 1949).
The macrolides are soluble in most organic
solvents and, because of their amino sugar
moiety, they are basic. Because of unsatura-
tion in the molecule, most of these com-
pounds absorb ultraviolet light. Table 17
indicates the variation in the size of the
molecule from a carbon skeleton composed
of 24 carbons atoms (proactinomycin C) to
one composed of some 50 carbon atoms
(angolamycin). The spiramycins are uni(iue
in that they contain two atoms of nitrogen
per molecule.
Enjthromiicin is the best known of the
macrolides. It has the following structure:
()H
CH3CH:
CH3O
It contains two sugar moieties — a netitral
sugar, cladinose, and an amino sugar, de-
sosamine. Erythromycin B has a similar
structure except that one of the hydroxyl
groups (marked above with a star) is re-
placed by a hydrogen atom, and erythro-
mycin V difters in that the cladinose moiety
56
NATURE. FORMATION. AND ACTIVITIES
is replaced by a C7Hi:i():{ fragment of un-
known striu'ture.
Oleandomycin is similar to erythromycin
in that it contains desosamine, but its neu-
tral sugar is oleandrose, which is closely
related to cladinose. The structure of this
antibiotic has recently been elucidated by
Hochstein (1960).
CH.,()
OH— ,'
CH.,-i
CH,
CH.,0
-OH
OH— r
CHs- 1
J— OH
■()"
O
cation and characterization of these anti-
biotics.
Methymycin and picromycin do not con-
tain a neutral sugar. They both have the
same lactone ring, to which desosamine is
attached. They differ only in the point of
attachment of desosamine to the lactone
ring. Methymycin has the following struc-
ture, the star indicating the point of attach-
ment of desosamine in the picromycin mole-
cule :
CH3 CH3
Carhomycin contains a large lactone ring
which is not fully saturated and which fea-
tures an epoxide function adjacent to the
unsaturated carbonyl function. Attached to
this ring is the amino sugar mycaminose, to
which is attached the neutral sugar my-
carose :
OH CHa
CHs CH3
ororHoC'HfC'H,)^
CH,
CH,
Table 18 lists some properties of tetra- Neomethymycin differs from merhymycin
phenylboron derivatives of some macrolides. only in the position of a hydroxyl group.
These derivatives can be used in the purifi- The exact structure of the spiramycins
T.^BLE 18
Tetraphetiylborou derivatives of antibiotics (Zief et al., 1957)
Molecular weight
Nitrogen
Sodium tetraplienylboron derivative
Antibiotic
m.p.
Nitrogen
Tlieory
Found
Ervthromvciii
733
830
640-744
689
%
1.91
1.69
1.89-2.17
2.03
183-186
148-153
138-144
157-161
%
1.33 ' 1.40
Carbomycin
1 . 22 : 1 . 20
Leucomvcin
1.32-1.46
1.4
1.32
Oleandomycin
1.79
CHEMICAL NATURE OF ANTIBIOTICS
57
has not been elucidated, but they are known
to he formed of a large macrolide ring to
which is attached mycaminose and my-
caro.se, already found in the carbomycin
molecule, and which in addition contains
another amino sugar moiety of the following
structure:
CH3
N-
CH3
CH3
-OH
Longisporin is an antibacterial antibiotic,
the complete structure of which is not
known. Preliminary data indicate the exist-
ence of a large multilactone ring (three
lactone groups) of 36 carbons.
A compound which is somewhat similar
to the antibacterial macrocyclic lactones is
nocardamine. It has an odd molecular struc-
ture with a nine-membered ring with an ad-
jacent three-membered ring. It has one
weakly acidic and no basic center:
OH
It is active only against mycobacteria.
Streptomycins
Streptomycin is active against gram-posi-
tive and gram-negative bacteria and myco-
bacteria. Its molecule is composed of three
moieties — .streptidine, streptose, and X-
methyl-L-glucosamine :
C
CHO
Streptidine is a diguanido derivative of
inositol. The hydrogenation of streptomycin
under pressure, in presence of platinum oxide
or palladium black, results in the reduction
of the aldehyde group in the pentose,
streptose. The resulting compound, which
is biologically active and is chemically more
stable than streptomycin, is called dihydro-
streptomycin and can also be produced di-
rectly by certain Strcptomyces. Another
chemical transformation is the removal of
an oxygen at the same site, with the forma-
tion of dihydrodcso.rystreptomycin:
CHO— C— OH
I
Streptomycin
I
CH2OH— C— OH
1 )ihy(ho8treptoniycin
CH.OH— CH
Dihydrodeso.xystreptomycin
Streptomycin-producing strains of S.
griscus form not only streptomycin but
also a D-mannoside of streptomycin whicii
was at first called streptomycin B and is
now called manno.sidostreptomycin. The
mannose moiety is attached, in the pyranose
form, to the 4 position of the X-methyl-L-
glucosamine nucleus. Mannosidostreptomy-
cin is only about one third as active as
streptomycin.
OH
Methylglucosamine
Streptose
OH Oh
Streptidine
58
NATURE, FORMATION, AND ACTIVrriES
H ijdro.ri/streptomijci)! , which is pi'ochicod
by certain Streptomijccs, cHft'ers from strepto-
mycin in that it has a hych'oxymethyl group
instead of a methyl group in the streptose
moietv:
CHO
CH3
OH'
Streptomycin
CH,OH
CHO
OH
Hydroxystrcptomycin
This antibiotic seems to offer nf) advantage
over streptomycin.
Pseudostreptomycin is composed of two
molecules of streptomycin linked together
through their aldehyde groups by condensa-
tion with ammonia. The formula is thus:
,S.l/— CHOH— XH— CHOH—SAI
where Si\I stands for streptomycin minus
the — CHO group. Pseudostreptomycin has
little biological activity and is very toxic.
It is converted to streptomycin in acjueous
acidic sohition.
neomycins B and C were later isolated. An
antibiotically active degradation product,
neomycin A or neamine, produced in the
culture or by chemical hydrolysis, w^as also
obtained. The ninhydrin and other color
tests, as well as specific test bacteria, can
be utilized for the separation of the various
neomycins.
Neomycin B is composed of fovn- cyclic
moieties, three of which have a carbon
skeleton composed of six carbons and one of
five carbons. One diaminohexose moiety is
linked to the pentose, D-ribose, to form a
Cii fragment called neobiosamine, and an-
other molecule of a diaminohexose is linked
to a diaminotrihydroxycyclohexane (2-
desoxystreptamine) to form a C12 moiety
called neamine. Neamine has some antibiotic
activity of its own. Neobiosamine and ne-
amine are linked together to form the C23
molecule of neomycin B, which has thus a
total of six amino groups. Neomycin C is an
isomer of neomycin B which is somewhat
less active biologicallv. The difference be-
XH,
C,H,O3(NH0a— 1-0
I
Diaminohexose !
OH OH
d-Ribose
I I
I I
I I
"C«H802(XH2)2
Diaminohexose
— O
NH2
Neobiosamine
Neomvcins
The neomycins are very similar to the
streptomycins, but they do not contain
guanido groups. They are formed of cyclic
aminated moieties. These basic, stable,
water-soluble substances are active against
gram-positive and gram-negative bacteria
and often have antiprotozoal activity.
Neomycin was first isolated in 1948 by
Waksman and Lechevalier from a culture of
;S. fradiae. It w-as soon recognized that this
antibiotic was made up of se\'eral chemical
entities, the mixture being designated as the
"neomycin complex." From this mixture,
OH OH
2-Desoxystreptamine
Neamine
tween the two isomers is in the diamino
hexose which is part of neobiosamine.
Kanamycin A is formed of three aminated
cyclic moieties. These are 2-desoxystrept-
amine, which is also found in neomycin, and
two aminohexoses:
NH.
OH
OHCH,,-
NH.CH.-
OH-
-OH
-O^
OH-
.0 .
OH
-OH
NH..
NH2
CHEMICAL NATURE OF ANTIBIOTICS
59
( )tli('r ant ihioties of the "ncomyciii "iroup"
inchule catcnulin, paromomycin, ht/droxy-
myviii, and amminosidin.
iMisceilaneous Sugar-containing Coni-
poiuids
The simplest of the antibiotics of this
group is trchalosaminc. This antibiotic has
the basic skeleton of the disaccharide treha-
lose, but in one of the glucose moieties a
hydroxyl gi'oup is replaced by an amino
group. Its biological activity is slight.
Novobiocin is an acidic antibiotic, the
molecule of which consists of three moieties:
(1) a sugar, (2) a substituted coumarin, and
(3) a substituted benzoic acid :
Chartreusin is a \veakl3- acidic, glucosidic,
nonnitrogenous substance which is active
against gram-positive bacteria and the exact
structure of which is not yet known. Its
molecule is formed of an aromatic micleus
containing three benzene rings linked to a
disaccharide chain composed of D-fucose
and D-digitalose.
Cclcsticctin is an amphoteric substance
also active against gram-positive bacteria.
Its acid hydrolysate yields a sulfur-contain-
ing base (desalicetin), salicylic acid, L-hygric
acid, and a reducing amino sugar (celestose).
The ristocetins are mixtures of at least
two compounds which are active mainly
against gram-positive bacteria. Acid hy-
CH3O
NHCO
CH2— CH=C(CH3)2
The sugar, which is called noviose, includes
a methoxyl and a carbamate group. In dilute
alkali reaction, the carbamate group is
shifted to the carbon indicated l)y a star to
form an isomer of novobiocin, isonovol)io-
cin, which is biologically inactive.
Hygromycin is a weakly acidic sul)stance
which is active against bacteria and is also
toxic to worms. Its molecule is formed of
three cyclic moieties: (1) an aminated inosi-
tol moiety which is believed to have a meth-
ylenedioxy group attached, (2) a hydrox-
ylated and methylated cinnamic acid moiety,
and (8) a hexose (5-keto-()-desoxyarabo-
hexose) :
OH
CH3— CO-
O
-/ N-
drolysis of the mixture yields D-araljinose,
glucose, mannose, rhamnose, and a ninhy-
drin-positive sul)stance.
Strcptothricins
Strcptothricin was the first basic, water-
solui)le antibiotic with activity against
gram-positive and gram-negative bacteria
to be isolated. Because of its toxicity it was
never used clinically.
As explained in greater detail in Part B
of this book, a large number of substances
closely related to strcptothricin have been
isolated. All the "streptothricin type" anti-
biotics yield on hydrolysis a mixtiu'c of
OH
CH3
-0-
.CH=C— CO— NH/
OH
OH OH
OH
O— CH.
60
NATURE, FORMATION, AND ACTIVITIES
amino sugars and amino acids. Typical
products of the hydrolysis of a strepto-
thricin-type compound include (1) a di-
aminocaproic acid (L-|8-lysine), (2) an
imidazole derivative, streptolidine, and (3)
the amino sugar a-D-gulosamine.
The streptothricins are active not only
against bacteria but also against fungi. The
most important streptothricin-type anti-
biotics are lavendulin, actinorubin, antibiotic
136, roseothricin, geomycin, pleocidin, and
miicothricin. The antimycobacterial anti-
biotic viomijcin also yields |3-lysine upon
hydrolysis.
Miscellaneous Pigmented Antibiotics
which Act as pH Indicators
A number of antibiotics are colored sub-
stances which act as pH indicators, the
color changing with the pH of their solution.
The structure of one of these substances,
actinorhodin, is partially known. It has a
dinaphthazarin nucleus. The structure of
naphthazarin is:
OH O
OH O
Two such units are thus hooked together
and are variously substituted.
Another pH indicator, granaticin, is a
tricyclic tetrahydroxyciuinonedicarboxylic
acid (C22H20O10).
Litmocidin, also a pH indicator, is be-
lieved to be related to the anthocyanin pig-
ments. As such, it is believed to have a
molecular nucleus of the following type:
Nothing is known about the chemical struc-
ture of other pH indicators, such as coeli-
colorin, mitomycin C, nocardorvbin, rhodo-
mycetin, and ruhidin.
The rhodomycins are a group of pH in-
dicator antibiotics active against gram-posi-
tive bacteria. They are formed of a ciuinoid
chromophore called rhodomycinon, to which
is attached a moiety which contains rhodos-
amine, an isomer of desosamine.
Compounds Containing Phenazine or
Quinoxaline Nuclei
One antifungal antibiotic, 1 ,6-dih]jdr<)X]j-
phenazine, and two antibacterial antibiotics,
griseolutein A and B, have a phenazine
nucleus which is substituted as follows:
The hydrolysates of another such indica-
tor, mycorhodin, include reducing sugars.
1 ,G-Dihydroxyj)hen:iziiie: Ri = Rn = H;
R = II, = OH
Griseolutein A: R = OCH3 ;
R, = CH2— O— CO— CH4)H;
R, = H; R3 = COOH
Griseolutein B: R = OCH3 ;
R, = CH.— O— CHOH— CH.OH;
R: = H; R, = COOH
1 ,6-Dihydroxyphenazine is closely re-
lated to the bacterial antibiotic pigment
iodinin, from which it can be obtained by
reduction. Iodinin is the .") , 10-dioxide of
1 , 6-dihydroxyphenazine and is active
against bacteria. In contrast, 1 , 6-dihydroxy-
phenazine has no antibacterial activity.
Both griseolutein A and B are acti\'e against
gram-positive and gram-negative bacteria.
The only difference between the two mole-
cules is two hydrogen atoms in the Ki chain.
Despite this small difference, only slight
cross-resistance was observed between these
two antibiotics.
CHEMICAL NATURE OF ANTIBIOTICS
61
Echinomijcin is an antibacterial antibiotic
containing two fjuinoxaline moieties:
N
These are attached to a polypeptide moiety.
Phenoxaziiies: The Actinom veins
The actinom ijcins are red-colored anti-
biotics, the molecules of which consist of a
phenoxazine nucleus to which are attached
two polypeptide chains. These antibiotics
are active against gram-positive bacteria
and tumors. Actinomycins are usually pro-
duced by actinomycetes as mixtures of
closely related compounds which all have
the same phenoxazine chromophore but
which differ in the amino acid composition
of the polypeptide chains. Qnestiomycin A
((3-aminophenoxazone) is active mainly
against mycobacteria :
NHo
This rather nontoxic compound is closely
related to the chromophore of the actino-
mycins. It is believed to be formed by the
condensation of two molecules of (juestio-
mycin B (o-aminophenol).
The chromophore of the actinomycins
is 3-amino-l ,8-dimethylphenoxazone-(2)-di-
carboxylic acid- (4, 5):
CO OH CO OH
N
NH,
CHs
CH3
In the molecules of the actinomycins, two
cyclic five-membered polypeptides are linked
to the carboxyl groups. Their composition
varies from one actinomycin to the other,
as explained in Part B of this book.
The opening by alkali treatment of the
polypeptide rings produces actinomycinic
acids which have no antibacterial activity
but some antitumor activities.
Some antibiotics other than the actino-
mycins are also polypeptides with an aro-
matic chromophore. They include Icvomycin,
mikamycin A , actinoleukin, and as previously
ment ioned , cchin om ycin .
Oligojieptides
A group of closely related substances, the
antimycins and blastmycin, have antifungal
activity and inhibit electron transport in
cytochrome systems. Their molecule is
composed of two large moieties, antimycic
acid and a neutral fragment. Antimycic
acid itself contains two moieties: (1) L-thre-
onine, and (2) a substituted salicylic acid
fragment. The neutral fragment is composed
of a variously substituted furan ring. The
following structure has been suggested for
these compounds:
I O,
NH
CHO
— CO-
— OH
-NH-
CO— O-
I
-CH
I
CHOH
I
CH3
-R
R.
Aiitimvcic acid
O
CO
I
R2
Neutral
fragment
Blastmycin: R = CH,, ; R, = (CH.JsCHs ;
R2 = CH2— CH(CH3).>
Antimvcin.s: R = C7H16 ; Ri = H;
R2 = CHCH-CH3
I
CH:,
The various antimycins (there are at least
four) probal)ly differ in the structiu'e of the
C7H15 fragment.
A recent paper (Strong et al., 1960) has
modified the concept of the structure of the
antimycins. The substituted salicylic acid
fragment, present in antimycic acid, is
attached to a dilactonic ring.
62
NATURE, FORMATIOX, AND ACTIVITIES
'Valine ■
a-hydroxyisovaleric acid
valine
I
a-hydroxyisovaleric acid
a-hydroxyisovaleric acid
valine
a-hydroxyisovaleric acid
Netropsin is active against both fungi and ^^'hcYeils anu'domtjcin (C40H68O12N4) is formed
bacteria. Its nitrogen content is very high of the following residues:
(CisHoeOaXio), and it contains two pyrrole
rings, a guanido group, two peptide grovips,
and a primary amino group as shown on the
facing page.
A prodigiosin-likc substance which is not
so rich in nitrogen (C25H35()N3) also con-
tains two pyrrole rings.
Eidicin is a basic antifungal antil)iotic
which contains two guanido groups and a
peptide linkage:
NH OH
valine -
The only difference between the two anti-
l)iotics is thus four carbons and eight hy-
drogen atoms. Valinomycin was reported to
have some activity against bacteria, and
NH.,— C— NH(CH2)8— CH— CH(CH2)3NH>
NH ! CO(CH.,)sNHC— NH.
1 II
NH
Acid hydrolysis of the base cleaves it, as amidomycin to be antifungal in nature,
indicated by the dotted line, and 9-guani- Taber (personal communication) reported,
dinononanoic acid and a base, eulicinine, however, that valinomycin also has anti-
are formed. ' fungal activity.
Etamycin is a macrocyclic peptide lactone
Polypeptides which is active against gram-positive bac-
We have already seen that some antibi- teria. Opening of the lactone ring results in
otics have molecules which are partly poly- the loss of antibiotic activity. In the follow-
peptidic in nature, such as the streptothri- ing formula, the amino acids marked with a
cins and the actinomycins. A large number of star had not been found previously in na-
antibiotics of actinomycetes are polypep- ture:
[3 -hydroxy picolinic acid]*
[threonine
O
c=o
. I
[a-phenylsurccsine]* — [L -alanine] — [/3, N -dimethyl leucine
[D -leucine] — [D-allohydroxy proline]
[sarccsine]
tides strido sensu. The exact struct vu-e of
only a few of them is known.
Two rather simple polypeptides, amido-
mycin and valinomycin, have very similar
molecules. Valinomycin is a cyclic polypep-
tide (C36H60O12X4) formed of the following
units :
, valine^
lactic acid
I
valine
I
a-hvdroxvisovaleric acid
a-hydroxyisovaleric acid
valine
lactic acid
' valine
Staphylomycin S and mikaiyiycin B are
very similar to etamycin.
3-Hydroxypicolinic acid is also found in
the molecule of pyridomycin, a polypeptide
antibiotic which is active mainly against
mycobacteria.
The complete structures of other poly-
peptide antibiotics of actinomycetes are not
known. In the case of some of them the
products of hydrolysis are known; of others,
nothing is known except that they are poly-
peptides.
CHEMICAL NATURE OF ANTIBIOTICS
63
NHa— C— NHCH.— C— NH
NH NH
N'
I
CH3
netrop.sin
i'— CONH-
^N
CH,
-CONHCH.CH.,
I
CO
I
NH2
Two of these substances stand apart ciii, neocide, matamycin, amphomycin,
because their molecules contain a metal, cinnamycin, bryamycin, and thiostrepton.
These are the iron-containing grisein and In Part B of this book, a table lists the
the copper-containing phleomycin. amino acids identified in the hydrolysates
Grisein and albomycin are mixtures of of antibiotics of actinomycetes and the
components. Hydrolysates of crude grisein antibiotic or antibiotics in which the acids
and albomycin contain many amino acids.
Pvu'er preparations of grisein contain only
glutamic acid and an unidentified amino
acid, whereas a purified preparation of one
have been fovuid.
Proteins
The most chemically complex antibiotics
of the albomycins contains only ornithine are protein or i)rotein-like sul)stances, such
and serine. Removal of iron from Ili(> grisein
molecule results in a loss of the yellow-
orange color of the compoimd and in a re-
duclion in its l)iol()gical activity against
gram-positive and gram-negative bacteria.
I'lilcomi/cin contains copper and is prob-
as actinomi/aiin, A'arious hactcriolijlic factors,
ccphalomi/cin, and micromonosporin.
As this brief survey of what is known of
the chemical structure of antibiotics of
actinomycetes has shown, these filamentous
l)acteria have the abifity to produce some
at)ly formed of two moieties, a polypeptide intriguing compounds. The elucidation of
and a cart)ohydrate. This cobalt-blue com- the exact structure of an antibiotic, in com-
pound, which is active against liacteria and plete stereochemical detail, is a type of work
mycol)acteria, loses color upon remox'al of the which her(>tofor(> has been mainly of aca-
metal, with a concomitant slight I'edudion demic interest. However, eventually a firmer
in antimycobacterial activity. link will l)e made between structure and
Numerous other polypc^ptides are pro- mode of action, and chemotherapy will be-
duced by actinomycetes, such as phalaniy- come a completely rational discipline.
Chapter 7
Biogenesis of Antibiotics
Only scattered attention has been paid
hei-etofoi'e to the mechanism of formation of
antil)iotics and to their role in the metabo-
lism of the organisms prodiu.-ing these sub-
stances.
In this area, a number of (juestions can be
asked which remain, for the most part, un-
answered. Are antibiotics essential cell con-
stituents? Are they storage products? Are
they waste products of microbial metabo-
lism? Are they a result of "abnormal" or
"shunted" biosynthesis which may be con-
trolled by certain special constituents of the
medium? What role do they play in the life
of the organisms producing them? What
effect do they exert upon these organisms?
Does the fact that they exert an inhibiting
effect upon the growth of other microbes
suggest that they play a role in the sur\'ival
of the organisms producing them?
It has long been recogniz(Kl that environ-
ment exerts a marked effect upon the com-
position of the microbiological population
living in a natural or an artificial substrate.
Important en\'ir()iun('ntal factors include
humidity, aeration, temperature, and pH
as well as the nature and concentration of
available nutrients. These factors also exert
an important influence upon the metabolic
processes and even upon the chemical com-
position, morphology, and life cycle of the
organisms involved. In addition to factors
of nutrition and environment, the nature of
the microbial p()i)ulati()u also depends upon
the presence of other organisms and upon
their metabolic products. These exert a
variety of associative or antagonistic effects
upon the various members of the popu-
lation. We have already discussed briefly
the role of antibiotics in nature, and have
concluded that it is doubtful that antibiotics
play an important role in microbial ecology
(Chapter 1). We will discuss here the part
played by antibiotics directly on the cells
of the producing organisms rather than in-
directly, through their action on other micro-
organisms.
Role of Antibiotics in the Biology of the
Producing Organisms
Let us begin this discussion })y an exami-
nation of the effect of various factors upon
the concentration of antibiotics produced by
certain cultures of actinomycetes. Whether
the active substance is excreted into the
medium, whether it is liberated from the
mycelium when the latter undergoes lysis, or
whether it is extracted from the mycelium
by means of special organic solvents during
the isolation of the antibiotic are other
important (luestions that must be con-
sidered.
In the i)i-oduction of actinomycin, for
example, every change in tlie composition
of the medium and in the cultural conditions
brings about not only a change in the yield
of the antibiotic, but also in the nature and
ratios of its amino acid make-up. The syn-
thesis of the mycelium and the yield of
actinomycin show the same course. Both
reach a maximum at a time of complete
consumption of the carbon source; when
64
BIOGENESIS OF ANTIBIOTICS
65
mycelial growth ceases, actiiiomycin no
longer forms. Although the amount of
actinomycin produced is small as compared
to the mycelium synthesized, it is believed
that the organism synthesizes, in addition
to actinomycin itself, a large number of
closely related compounds. It has been con-
cluded that actinomycin is neither a degrada-
tion nor an autolytic product of the cell
protein, but that its formation is a result of a
side reaction of the assimilating process of
metabolism (Martin and J^ampus, 19")()).
I'^rommer (lOo?) found that phenylthiourea
produces no depression in the respiration of
organisms that produce actinomycin. The
formation of this antibiotic in certain culture
media is inhibited without growth inhibition.
Some of the antibiotics, such as strepto-
mycin, neomycin, and the tetracyclines, are
produced in concentrations of 5 to 10 gm per
liter of culture medium. Although the media
used for the production of these antibiotics
vary greatly in chemical composition, one
may assume that they contain about 80 to
80 gm per liter of nutrients, mostly carbo-
hydrates, lipids, amino acids, proteins, and
certain minerals. One may further assume
that the organism metabolizes most of those
nutrients, thereby converting about 80 per
cent of the consumed suljstrate into cell sub-
stance. This would give a maximal synthesis
of 10 to 2") gm of cell material per liter of
medium. One can thus calculate that the
antibiotic compounds make up approxi-
mately 38 to 40 per cent of the total cell
material synthesized. In some cases, the
transformation is undoubtedly nuich lower,
as in the case of Strcplomijces antihioticns
growing in a glutamic acid-glycerol medium.
Of the lo gm of nutrients per liter, 10 gm ai'e
consumed in 10 to 12 days, giving ].") gm of
cell material and 150 mg of actinomycin. The
cell synthesis comprises only 10 per cent and
antibiotic synthesis only 1 per cent of the
nutrients added to the medium, or 1.5 per
cent of the nutrients consumed.
Assuming the correctness of the above
calculations, one is led to the inevitable con-
clusion that the antibiotic substance is not a
mere metabolic intermediate or an ordinary
\vaste product of the microbial cell, but that
it is a constituent of the living cell proto-
plasm, a storage product, or a special by-
product. If it were a storage product, the
organism should have the capacity to utilize
it for its own metabolism, which is not neces-
sarily the case, although apparently mecha-
nisms exist which bring about the destruc-
tion of at least some of the antibiotics if
allowed to remain in contact with the living-
cells of the organisms producing them.
These calculations place such antibiotics
as streptomycin beyond the scope of an
intermediate or a waste product of metabo-
lism. Thus the biogenesis of the antibiotic is
closely allied to its role in the metabolism of
the organism. Since so many different anti-
biotics are produced by different organisms,
one must postulate a great variety of meta-
bolic reactions. Further, since each organism
frequently produces more than one antibiotic
or several closely related chemical com-
pounds, the (juestion arises as to the extent
and variety of metabolic reactions involved
in the growth of actinomycetes.
Bu'lock (1900) emphasized the fact that
although a single species or a restricted range
of i-elated species of microbes is able to
produce a great variety of chemical com-
poinids, often of great structural complexity,
the fundamental chemistry of these organ-
isms is based upon a limited gi'oup of reac-
tions and compounds and "is i'emarkal)ly
uniform throughout most living things."
We have thus, on the one hand, a limited
variety of basic metabolic pathways; on the
other, a seemingly endless variety of second-
ary metabolites, of which the antil^iotics
represent merely an arbitrary selection. As a
result of recent studies on the l)iosynthesis
of antibiotics and other natural products, it
is possible to demonstrate that certain pre-
66
NATURE, FORMATION, AND ACTIVITIES
cursors (like the acetate or propionate types)
of man}' well known secondary metabolites
are also intermediates of primary metabo-
lism. According to this concept, antibiotics
are to be looked upon as "waste" products
of the general metabolic reactions of the
organisms.
Location of Antibiotics in the Producing
Organism
The cells of bacteria and actinomycetes
may be considered as being made up of three
groups of constituents: cell walls, cell proto-
plasm, and slimy excretion products adher-
ing to the cells, such as capsules. The proto-
plasm comprises: (1) functioning elements,
notably nuclear material, enzymes and co-
enzymes, and other constituents that take
part in the growth and multiplication of the
cell; (2) metabolic constituents, including
sugars, amino acids, fatty acids, vitamins,
which are required for cell synthesis; (3)
waste products of metabolism; and (4)
storage or reserve materials (Holdsworth,
1952; Cummins, 1956; Cummins and Harris,
1956; Ikawa and Snell, 1956; Salton, 1956).
The cjuestion under consideration is:
where do the antibiotics accumulate? The
cell wall is certainly a logical place for them.
Still, they may be considered as storage
materials and even as metabolites. It is true
that the organisms producing actinomycin
or streptomycin are no longer able to utilize
these antibiotics as nutrients. This may be
due, however, to a change in the chemical
structure of the compound after it has been
isolated from the medium, since in the cul-
ture itself the substances tend to l)e de-
stroyed after they have accumulated (see
also Wiebull, 1956; Work, 1957).
As already mentioned, some antibiotics
such as streptomycin are mainly found extra-
cellularly in the culture medium. Others such
as perimycin are found only in the myceliimi
of the producing organism. Still others such
as candicidin are found in lioth locations.
These facts raise a number of questions
which have not yet been successfully an-
swered .
In our early studies on the formation of
streptothricin, it was observed that when a
culture broth was allowed to stand for
several days at the end of the fermentation
period, it tended to increase gradually in
antibiotic potency. This suggested that
streptothricin may have been originally
bound to some chemical constituent in the
cells of the organism; gradually this union
was freed from its association with another
compound by some enzyme system in the
culture.
Eiser and AIcFarlane (1948) found that
the presence of NaCl increased the permea-
bility of the hyphae of Strepfomyces griseus
and allowed the liberation of streptomycin
at a greater rate than in the absence of the
salt. Only lysis of the mycelium served a
similar purpose, since after prolonged incu-
bation (6 days), the streptomycin in the
medium without the salt had risen to the
level of the medium with the salt.
Perlman and Langlykke (1950) found that
large amounts of streptomycin occur bound
in the mycelium of the organism producing
the antibiotic. They suggested that the anti-
biotic may be a part of the cell wall of the
actinomycete. The bound streptomycin
could be released by treatment of the cells
with acid, alkali, or ionizable salts, as well as
by sonic vibrations. Perlman (1953) reported
that various other antibiotics, notably neo-
mycin and chloramphenicol, were also bound
to the mycelium of the organisms producing
them and could be released by various treat-
ments. The binding of the antibiotic was not
a simple ion exchange phenomenon, since
addition of streptomycin to the mycelium
did not result in adsorption of the antibiotic.
Although in our early studies NaCl was ob-
served to have a fa^'orable effect upon the
"production" of streptomycin, this effect was
explained as the result of the freeing of the
BIOGENESIS OF ANTIBIOTICS
67
"bound" antil)i()tic. Actiiall.v, higher produc-
tion of streptomycin may be obtained in the
absence of .saU.
Gwatkin (1954) found that large ([uan-
tities of neomycin, sufficient to account for
nearly all the antibiotic which later appeared
in the medium, had been present in the
mycelivnn of the organism pi'oducing this
antibiotic (N. fradiae). At a proper pH, the
neomycin is released by salt from its com-
bination, which was considered to be a neo-
mycin-micleic acid complex. This complex
was found to be present in the disintegrated
cell material. Neomycin foi'med insoluble
compounds with ribonucleic and deoxy-
I'ibomicleic acids and protamine nucleinate,
but not with nucleotides or hydrolyzed
nucleic acid. Xeamine, a hydrolytic product
of neomycin, did not form any such com-
plexes.
Surikova and Rudakova (1958) compared
\'ari()us methods of extraction of strepto-
mycin from the mycelium of S. griseus. The
most effective method consisted in the
acidification of the mycelium with a mineral
acid (pH 2.o) and subsequent heating. As
much as lo per cent of the streptomycin
could thus be extracted, as compared to the
amount obtained from the broth. In the case
of a soya bean medium, it was possible to ex-
tract about 1 ") per cent of the streptomycin.
The amount of the antibiotic bound with
the mycelium of the organism, as calculated
pel' unit weight of mycelium, did not change
significantly in the process of fermentation.
Legator and Gottlieb (19o.3) found that
chloramphenicol production reached its peak
well after maximal growth had been at-
tained. This was particularly true of organic
media, a rise in the concentration of the
antil)iotic being correlated with an increase
in pH and in ammonium ions, and finall,v
with a lysis of the mycelium. These investi-
gators believed that the antibiotic is not
stored within the cells of the organism to any
large extent. It is either immediatelv secreted
into the medium upon production, or is
formed from a degradation product. When
high concentrations of the antibiotic were
added to the medium, no more chloram-
phenicol was formed, the concentration
needed to arrest further production being
e(lui^•alent to the amount normally formed
by the cell when no antibiotic was added.
Chloramphenicol added at any time during
the growth phase of the organism exerts its
limiting action on further production, al-
though nol on the synthesizin.g al)ility of the
cells.
These data indicate that most antibiotics
are formed intracellularly and are i-eleased
in the culture UK^dium. It is doubtful that
any of the well known antil)iotics are formed
extracelhilarly.
Mechanism of Biosynthesis of Anti-
hioties
On the l)asis of what is known of the
chemical structure of antibiotics of actino-
mycetes and of the mechanism of their bio-
synthesis, Al)raham and Xewton (1960)
grouped antil)iotics into three main classes:
those dei'iA'al)le from sugars, from amino
acids, and fi'om acetate. In the following
discussion we have grouped the antibiotics
according to this general outline.
Atitibiotics Derivable from Sugars
STREPTOMYCIN
It has been definitely established that
some of the nutrients in the medium are
important largely for cell growth and others
for antibiotic synthesis. This has been stud-
ied extensively for penicillin, streptomycin,
and the teti'acy<'lines. Agatov and Kazan-
skaya (19.')8) hiixe shown that in the growth
of streptomycin-producing »S'. griseus, during
the first 2 days, histidine and arginine are
rapidly utilized and lysine is utilized more
slowly; on the third day, rapid utilization of
lysine takes place. This is accompaniexl b.v
68
NATURE, FORMATION, AND ACTIVITIES
liberation of micleic acids and monoamine
acids into the medium. When alanine is
present in the medium, basic amino acids and
nucleic acid metabolites appear in the first 2
days; on the following day the concentra-
tion of histidine and nucleic acid metabolic
products diminishes. Arginine and lysine
remain on the same level. Inositol and sub-
stances containing a guanidine grouping
exert a marked effect upon the biosynthesis
of streptomycin (Egorov, 1959).
Shaposhnikov et al. (1959) made a study
of the formation of streptomycin in synthetic
media containing proline, or histidine with
lysine or proline, succinimide, or succina-
mide. The yield of antibiotics on these media
w^as 74 to 84 per cent of that on soybean
meal medium. Oxyproline was found to be a
unique source of nitrogen, contributing to
the growth of the actinomycete, while only
weakly stimulating the formation of strepto-
mycin (Table 19). When this amino acid was
added to a medium already containing the
basic amino acids, growth was favored but
the antibiotic yield was lowered (Table 20).
Schaiberger (1959) examined in detail the
mechanism leading to the biosynthesis of
Table 19
Maximal mycelial weights and quantities of strep-
tomycin on media with pyrrole compounds
(Shaposhnikov et al., 1959)
Table 20
Maximal mycelial weights and quantity of strepto-
mycin on media with histidine, lysine, and pyr-
role compounds (Shaposhnikov et al., 1959)
Mycelial weight
Amount
of streptomycin
Medium
% of
% of
/xg/ml
control
I
control
II
mg/lOO ml
Control I*
1200
100
1070
Control II
300
112
9
100
Pyrrolidine
440
524
44
408
Proline
738
738
1312
328
109
27
1171
Oxyproline
293
Succinimide ...
327
200
17
184
Succinamide. . . .
280
190
10
170
Mycelial
weight
Amount of streptomycin
Medium
/ig/ml
% of
control
Amount
of strep-
tomycin
in con-
trol
Basic medium* . . .
Pyrrolidine
a-Methylpyrroli-
dine
mg/100 ml
040
089
709
592
725
800
797
999
910
1103
1575
1524
744
1310
53
08
74
84
81
48
75
liS/ml
1809
1340
1568
Succinimide
Succiiuimide
Oxyproline
Proline
1870
1874
1550
1743
* Control I contains an inorganic nitrogen
source. Control II contains an organic nitrogen
source. Both controls have sovbean meal added.
* Basic medium: 2% glucose, 0.39( (NH4)2S04 ,
0.25% NaCl, 0.05% KH.,P04 , 0.3% CaCO^ , distilled
water. Soybean meal, amino acids, or other pyr-
role compounds added on basis of 112 mg of ni-
trogen per 100 ml of medium.
streptomycin. He used a high streptomycin-
producing culture of *S. griseus (S-f-) and a
mutant without streptomycin (S — ) derived
from it. The S— culture was asporogenous
and lacked the ability to synthesize strepto-
mycin; however, its rates of growth and of
sugar utilization were double those of the
S4- strain. The nutritional reciuirements for
growth and streptomycin synthesis by the
S+ culture included glucose, a suitable
inorganic nitrogen source (ammonia), and
six mineral salts (MgS04 , FeS04 , ZnCl,. ,
CaCOa, K2HPO4, and NaCl). These nu-
trients also satisfied the growth require-
ments of the S— strain. Resting mycelial
suspensions confirmed the essentiality of the
salts for streptomycin synthesis, with the
exception of KiHP04 . Exogenous addition
of phosphate inhibited streptomycin syn-
thesis by resting cells. When used as the sole
nitrogen source in a mineral salts-glucose
medium, proline and the amino acids closely
related metabolically (asparagine, histidine.
BlOdKXESIS OF ANTIBIOTICS
69
and glutamic acid) permitted the highest
level (1000 to 3000 fig per ml) of strepto-
mycin synthesis by the 8+ culture. Xor-
leucine and isoleucine strongly inhibited
streptomycin synthesis in experiments with
growing cells as well as with resting cells;
leucine stimulated synthesis. Resting cell
data also suggested that acetate is an inter-
mediate in streptomycin biosynthesis.
Silverman and Rieder (19(30) utilized the
distribution of radioactivity among the
individual carbon atoms to elucidate the
mechanism of the formation of X-methyl-L-
glucosamine in the streptomycin molecule
from D-glucose. Both D-glucose-l-C'' and
D-glucose-6-C^^ were employed. A method
for the isolation of the methyl glucosamine
was described, as was a procedure for the
degradation of this amino sugar. The conclu-
sion was reached that the major portion of
the radioactivity incorporated into the
carbon chain of the amino sugar was in
carbon 1 when D-glucose- l-C'"* was used and
in carbon 6 when D-glucose-6-C^'* was em-
ployed.
These investigators suggested that a pos-
sible mechanism for the inversion of all the
asymmetric carbons of D-glucosc is one of
multiple epimerizations. Other possibilities
were also suggested, such as an extensive
series of oxidations and reductions with
subseciuent inversion of configuration upon
reduction, or a mechanism of multiple isom-
erizations.
The changes occurring during strepto-
mycin production are shown in Table 21.
Little progress has so far been made in the
use of precursors for streptomycin biosyn-
thesis. By using C'^^ compounds, it was shown
(Hunter et al., 1904) that the guanidine
carbon is derived largely, if not entirely,
from CO2 , and that compounds such as
arginine may act as precursors. The strep-
tamine and streptose portions of the strep-
tomycin mf)lecule appear to be formed from
glucose (Hunter and Hockenhull, 1955).
Streptamine itself does not act as a pre-
cursor, but X-methyl-L-glucosamine does.
According to Egorov (1957), a combination
of a guanidine compound (L-arginine,
creatine, guanidine) and inositol favors the
biosynthesis of streptomycin. Asparagine
favors growth l)Ut not streptomycin synthe-
sis. Hydrolysates oi casein and soybean
Table 21
Changes occurring during feriucntation of glucose -meal exlracl-peplone medium
(Dulaney and Perlman, 1947)
Medium: Glucose 1 per cent, meat extract 0.5 per cent, peptone 0.5 per cent, sodium chloride 0.5
per cent.
Mj'celium (mg/ml)
Streptomycin (mg/liter) . ,
Glucose (mg/ml)
Soluble C (mg/ml)
Lactic acid (mg/liter) . . . .
Oxygen demand (Qoo/ml)
Soluble N (mg/ml)
Mycelial N (mg/ml)
Inorganic P (mg/ml)
Ammonia N (mg/liter) . . .
pH
Duration of fermentation (days)
9.0
10.2
292
1.48
118
66
7.35
0.4
8.8
8.6
328
19
1.30
0.04
108
70
7.30
5.1
37
8.0
7.0
114
81
1.10
0.44
34
75
7.55
5.8
194
2.4
5.1
13
82
0.67
0.62
1
63
7.50
5.7
198
1.2
5.0
10
53
0.70
0.57
5
103
7.75
4.8
231
0.6
4.4
16
25
0.73
0.49
2
115
8.25
4.6
270
4.6
12
5
0.90
0.40
19
179
8.55
4.2
186
4.5
6
0.88
0.38
24
232
8.65
3.8
267
4.6
15
1.14
0.29
34
265
8.90
70
NATURE, FORMATION, AND ACTIVITIES
Tabi.e 22
Changes characterizing the three phases of streptomycin production (Hockenhull, 1960)
Phase 1: "growth"
Phase 2: "maturation"
Phase 3:"senescence"
Strejjtoniyciii
Slight production
Maximal rate of pro-
Streptomycin level
duction
ceases to rise or falls
pH
Steady rise
Very slow fall
Rise
Mycelium
Rapid growth
Mycelial weight fairly
constant
Mycelial disintegration
Glucose
Used slowly
Used steadily through-
out
Usually absent
Ammonia
Released into medium
Utilized
Released
Inorganic phosijluite
Released
Utilized
Released
Qo.
High
Moderate
Low
Total oxygen demand
High
High
Low
meal are favorable for the production of the
antil^iotic.
A detailed review of the biogenesis of
streptomycin has recently been published
by Hockenhull (H)()0). Three distinct phases
were recognized in the growth of the organ-
ism and formation of the antibiotic (Table
22). Although the surface growth of the
streptomycin-producing *S. griseus tended at
first to gwe higher yields than the growing
of the organisms under submerged condi-
tions (Thornberry and Anderson, 1948), the
latter method gradually became generally
employed. The first medium, recommended
by Waksman and Schatz (1945), consisted,
in grams per liter, of glucose (10), peptone
(5), meat extract (5), and sodium chloride
(5). Yeast extract, soybean meal, and dried
whole yeast were later used to replace the
meat extract. Glucose was usually the sugar
of choice, in amounts of 10 to more than 25
gm per liter. Certain strains were found to
be able to utilize, for streptf)mycin i)r(Kluc-
tion, fats, oils, or certain fatt.y acids in
place of glucose (Perlman and Wagman,
1952).
Of the various nitrogen sources, L-proline
was found (Table 23) to be the most efTec-
ti\'e for streptomycin synthesis, although it
is only slowly utilized for the growth of the
organism.
Streptomycin is basically a trisaccharide
with various substituent nitrogen groups.
By the use of isotopic carbon (C^), Hunter
and Hockenhull (1955) demonstrated that
the carbon of the glucose was distributed
evenly among the streptamine, streptose,
and the X-methyl-L-glucosamine portions
of the streptomycin molecule. The carbon
of the guanidine group was poorer in radio-
activity, thus indicating that this carbon
came from CO2 , as shown in Table 24 (see
also Xumerof et al., 1954).
The effect of phosphate concentration on
streptomycin production is illustrated in
Table 25. In synthetic media, phosphate
exerted an effect on glucose breakdown and
on streptomycin biosynthesis. An excess of
phosphate caused an increased glucose con-
siunption. Increasing concentrations of phos-
phate first showed an increase, then a de-
crease in streptomycin synthesis. Arsenate
had a similar effect (Hockenhull et al., 1954).
Hockenhull (1960) concluded that the
following factors favored streptomycin bio-
genesis: (1) high oxygen supply, (2) low
inorganic phosphate, (3) adec^uate glucose
concentration, and (4) nitrogen levels that
would not lead to high protein synthesis. It
was suggested that once the enzymes re-
(luired for streptomycin production have
Ijeen formed, further biosynthesis will take
BIOGENESIS OF ANTIBIOTICS
71
Table 23
Effect of organic nitroyen compounds on
streptomycin production (Dulaney,
1948)
Compound added fO.1%)
I )L- Alanine
( Hyc'ine
L-Arginine-HCl
L-Aspartic acid
L-Cysteine HCl
L-C^'stine
Creatine hydrate
L -Glutamic acid
L-Histidine HCl
Hydroxy-L-proline
I )L-Isoleucine
L-Leucine
1)L-Lysine HCl
l)L-Methionine
DL-Norleucine
DL-Phenylalanine
L-Proline
DL-Serine
OL-Threonine
I )L-Tryptophan
L-Tyrosine
I)L-Valine
L'rea
Guanidine nitrate
0.1% Corn steep solids .
0.1% Casein digest
1 )iammonium hydrogen
phosphate
None
Streptomycin produced
Glucose
diammonium
hydrogen phos-
phate medium
No other
nitrogen
source
lig/ml
234
151
101
12
4
112
1-2
39
5
800
3
5
3
3
166
{)laco without the formation of new cells.
HockenhuU (1960) concluded that strepto-
mycin "does not constitute a portion of the
wall polymers and certainly was not a major
constituent."
Streptomycin is a glycoside containing the
monosaccharides streptose and X-methyl-
glucosamine, whereas mannosidostreptomy-
cin contains streptose, X-methylglucos-
amine, and mannose. Both antil)iotics are
pi'csent in the hi'oths of N. griscus. The enzy-
matic conversion of mannosidostreptomycin
to streptomycin, which in\-olves hydrolytic
removal of the mannose unit, is an impor-
tant step in the production of streptonwcin.
Glucose preferentially enters the strepto-
mycin molecule. A threefold increase in
specific radioactivity has been observed in
the streptomycin produced in a medium
containing labelled glucose as compared with
the specific radioactivity of the carbon in the
nutrients supplied (Karow ef ciL, 19.")2). The
supplementation of »S'. griseus broth with
mannose has resulted in an increase in the
proportion of mannosidostreptomycin. Man-
nose, therefore, meets a second criterion for
T.\BLE 24
Conversion of ('^''-glucose to streptomycin
(Hunter and HockenhuU, 1955)
Medium: soya bean meal-distillers' solubles-
glucose. Glucose added 60 hours after inoculation.
Substance
Gluco.se
Streptomycin
Streptidine
Streptamine
BaCO.-i (from guani-
dine groups)
N-Methyl-L-glucosamine
Streptose (by difference)
Specific activity
lic/m
-mole
9
1
29
9
9
8
8
55
10
1
9
2
lic/mg carbon
12.6 X 10-2
11.6 X 10^
10.4 X 10-^
12.3 X 10-2
4.5 X 10-2
12.2 X 10-2
12.8 X 10-2
Table 25
Streptomycin production in a proline medium,
with amount of diarnmonium hydrogen phosphate
as variable (Woodruff and Ruger, 1948)
(XHjIiHPOj concentration
Streptomycin (on 8th day)
mg/ml
nig/liler
5.7
225
2.0
170
().()()
570
0.20
675
0.06
530
0.02
390
0.006
200
0.002
130
Nil
25
72
NATURE, FORMATION, AND ACTIVITIES
controlled biosynthesis in that it modifit^s
fermentation to result in the formation of an
antibiotic which contains mannose as an
integral unit. Mannose is known, however,
to inhibit the action of the enzyme mannosi-
dase, which converts mannosidostreptomy-
cin to streptomycin (HockenhuU et al., 1954).
The formation of mannosidostreptomycin
in a medium supplemented with mannose
is the result of controlled biosynthesis; the
organism has been induced to synthesize a
biologically undesirable compound rather
than an active antibiotic. The controlled bio-
synthesis techniciue may thus be employed
strategically to design new molecules with
desired chemical properties.
The biosynthesis and the degradation of
mannosidostreptomycin, especially the for-
mation and action of the enzyme a-man-
nosidase, were also studied by Abalo and
Varela (1960).
Further information on biosynthesis of
streptomycin is found in the work of Hunter
(1956).
NEOMYCIN
Sebek (1955) observed that when glucose
labelled uniformly with C" was added to a
growing culture, in a medium containing 9
gm of glucose per liter, 19.5 per cent of the
carbon of the sugar was incorporated in the
neomycin. The rest was distributed in the
CO2 , in the filtrate, and in the mycelium.
The fact that the antibiotic was also readily
produced in the presence of other sugars,
including mono-, di-, and polysaccharides,
pentoses, hexoses, and sugar acids, suggested
the operation of a general basic mechanism
of sugar breakdown and antibiotic synthesis.
Antibiotics Dcrirabic from Amino Acids
ACTIXOMYCINS
An actinomycin-producing organism gen-
erally synthesizes a mixture of different
actinomycins. S. antibioticus, for example,
gives a mixture of actinomycins I to V;
occasionally, trace amounts of a sixth com-
ponent are also formed. S. chrysomallus
produces actinomycins IV, VI, and VII.
The quantitative and qualitative nature of
the mixture synthesized can be modified
to a considerable degree by modifying the
medium in which the organism is growing.
The nitrogen source in particular was found
to influence the composition of a given mix-
ture of actinomycins. Actinomycin IV in-
creased from 10 to 83 per cent of the com-
plex produced by S. chrysomallus when
DL-\'aline was added to the medium; when
DL-isoleucine or sarcosine was added, new
actinomycins were formed (Schmidt-Kast-
ner, 1956). Hydroxy-L-proline brought
about an increase in the synthesis of actino-
mycin I from 6 to 7 to 31 per cent of the
complex produced by S. antibioticus (Katz
and Goss, 1958).
Katz (1960) made a detailed study of the
effect of addition of sarcosine upon the syn-
thesis of actinomycins II and III by S.
ayitibioticus. The formation of these two
actinomycins was found to depend, in part,
on the concentration of sarcosine and on the
time and the number of additions of this
amino acid. The effect of sarcosine was
highly specific, compounds structurally and
biochemicall}^ related to it being ineffective.
The actinomycins were found in both the
mycelium and the medium. The addition of
L-proline reversed the effect of a given
concentration of sarcosine; larger amounts
of sarcosine nullified the effect of proline.
Incorporation of DL-pipecohc acid, a pro-
line analog, into the medium resulted in
synthesis of several new actinomycins. When
washed suspensions of *S. antibioticus were
incubated in the presence of 1 mil/ sarcosine,
there was a five fold increase in the synthesis
of actinomycin III but no change in that of
actinomycin II.
Schmidt-Kastner (1956) suggested that
sarcosine interferes with the incorporation
BIOGENESIS OF ANTIBIOTICS
73
Table 26
Neutralization by DL-threonine of DL-isoIeucine
inhibition of aciinotnycin production (Kawamata
et al.
, 1960)
Actinomycin production
Incubation
Control
DL-Isoleucine
lOO/ig. ml
DL-Isoleucine
100yu?'ml +
DL-threonine
100 ng/m\
days
2
4
100
200
6
400
400
7
2000
800
8
4000
1000
9
4000
4000
of proline into certain actinomycin peptides.
The results obtained support the view that
sarcosine competes with and replaces pro-
line in the peptide of certain actinomycins
(Katz and Goss, UJoS).
Kawamata et al. (1960) found that addi-
tion of DL-isoleucine to the medium (starch-
glutamate-salts) represses the formation of
actinomycin, whereas growth of the organ-
ism is not adversely affected and is exen
stimulated. The addition of 1 mg per ml of
isoleucine l)rought about complete inhibition
of antibiotic production. The addition of
DL-threonine to the mediiun completely
reversed this repressive effect, as shown in
Table 26.
Antibiotics Dcrirahh from Acetate
ERYTHUOMYCIX
Erythromycin has two sugar-like groups
attached to a large lactone nucleus. The
lactone nucleus is made up of seven pro-
pionic acid units (Woodward, 11)")7). Acetic,
propionic, and valeric acids may be incor-
porated into the lactone ring. This is true
especially of propionic acid, a fact which
suggests that this acid is an important pre-
cursor, and that the other two acids are
converted into it before incorporation.
Gerzon et al. (1956) suggested that the
macrolide ring arises, at least in the erythro-
mycins, by a process analogous to that by
which many long-chain molecules are built
from acetate, but utilizing propionate in-
stead. The long carbon chains of such anti-
biotics as the methymycins, picromycin, and
narbomycin would be deri\'ed in essentially
the same way except for incorporation of one
acetate moiety.
Alusilek and Sevcik (1958a) demonstrated
that the addition of sodium arsenite (final
concentration of 4 X 10~^ M) to the fermen-
tation medium reduced the biosynthesis of
erythromycin by S. erijthreus bj- 87 per cent,
whereas it increased the accumulation of
pyruvic acid (Figs. 1 and 2). The addition
Eryfhro- .
mycin
4 00-
300-
200-
100-
C)^"^~0 Conrrol
(§)- © AceTaTe
0— —0 As"
(g) (g) As^acerare
13
Days
FuiCRE 1. Inhil)itioii l)y arsenite of erythro-
mycin biosynthesis in submerged cultivation of
S. erythreus. (Reproduced from Mu.silek, V. and
Sevcik, V. Folia Biol. 4: 319-327. 1958b.)
74
NATURE, FORMATION, AND ACTIVITIES
Pyruvate
O o ^""^^"^ © © ^'^^^°^^
'^Iml
0— —% As" (g) (^ As^aceWe
7-
A,
6-
5-
^-
J-
^' \
2-
1-
/^"%^
1 1 III
Days
13
Figure 2. Effect of arseiiite on the accumula-
tion of pyruvate in a submerged culture of S.
erythreus. (Reproduced from Musilek, V. and
Sevcik, V. Symposium on antil)i(itics, Prague
Al)str., p. 32, 1959.)
of O.o per cent sodium acetate or propionate
markedly reduced the inhibitory effect of
arsenite on the biosynthesis of erythromycin.
Other salts of organic acids, as well as gly-
cine, failed to nullify the effect of arsenite.
The latter completely inhibited the oxidative
decarboxylation of pyruvate and oxidation
of acetate by washed suspensions of *S'.
erythreus. Oxidation of glucose was only
partially inhibited. It was therefore con-
cluded that the biosynthesis of erythromycin
is dependent on the oxidative decarboxyla-
tion of pyruvic acid to acetic acid. The par-
ticipation of acetic acid as the initial sub-
strate for the biosynthesis of propionic acid,
presumed to be the precursor of the lactone
nucleus of the erythromycin molecule, was
suggested (Musilek and Sevcik, lOoSb).
According to .Musilek et al. (]9o8), the
})iosynthesis of acetylmethylcarbinol de-
pends largely on the composition of the
medium and on the presence of arsenite,
which regulates the metabolism of pyruvic
acid. In the case of S. aureofacicns, the
stimulating effect of phosphate on the pro-
duction of acetylmethylcarbinol suggested a
partial explanation of the negative effect of
phosphate on the biosynthesis of chlortetra-
cycline (see also Biffi et al., 19o4). The addi-
tion of arsenite to a submerged culture of
S. erythreus reduced erythromycin produc-
tion by nearly 90 per cent, but vitamin B]2
production and growth were reduced by only
20 per cent. No direct relationship could
thus be demonstrated between the produc-
tion of erythromycin and of B12 (Alusilek,
1959).
The addition to submerged culture of
arsenite, which inhibits the second step
of oxidative decarboxylation of pjTUvic acid
to acetic acid {i.e., the oxidation of acetal-
dehyde), allows the accumulation of pyruvic
acid and acetaldehyde at the beginning, but
the anoxidative process of biosynthesis of
acetylmethylcarbinol from these substrates
is intensified. The inhibitory effect of arse-
nite on erythromycin biosynthesis is sup-
pressed to a large degree not only by the
addition of sodium acetate and propionate
but also by sodium formate. This suggested
that formic acid or its metabolically active
equivalent {e.g., formaldehyde) is closely
connected with the biosynthesis of erythro-
mycin. When washed mycelium was used,
the anoxidative biosynthesis of acetyl-
methylcarbinol from free acetaldehyde took
place. Tnlike the oxidation of acetaldehyde,
the oxidation of succinate was not inhibited
by arsenite. These results pointed further to
the essentiality of acetic acid as an inter-
mediate of erythromycin biosynthesis (Mu-
silek and 8e\'cik, 19")9).
Chain (19r)8) postulated the mechanism
of the biosvnthesis of ervthromvcin as fol-
BIOGENESIS OF ANTIBIOTICS
75
lows. According to the st met are of its
molecule, a propionyl unit is found to repeat
itself regularly. The structure of other macro-
lides suggests that both acetyl and pro-
pionyl residues take part in the condensation
process. The macrolides were said to be
built up 1)3' a mechanism similar to that by
which the long-chain fatty acids are syn-
thesized, i.e., by condensation of coenzyme-
activated acetyl residues or propionyl resi-
dues to jjolyketo acids, which ar(^ then re-
duced.
TETRACYCLINES
*S. aureofaciens was found (AlcCormick
ct a/., 19o9) to produce 2 gm of chlortetra-
cycline per liter of media of simple chemical
composition. When washed rvW inoculum
was previously grown in a corn steep me-
dium, the yield increased to 4 gm. With
glycerol as a source of carbon and ammonium
ion as a source of nitrogen, the other ele-
ments required were inorganic, notably sul-
fur, phosphorus, and chlorine.
Chain (19")8) suggested that the position
of the oxygen atoms hi the tetracycline
molecules on alternate carbon atoms indi-
cates some acetate condensation mechanism.
Acetate labelled in positions 1 and 2 with
radioactive carbon was found to be incor-
porated readily into the tctracycliries in
positions consistent with this type of mecha-
nism of biosynthesis. Chain further believed
that the long-chain unsaturated antibiotics
of polyene and polyacetylene nature are also
built up by acetate condensation. The oc-
currence of odd-numbered long fatty acid
chains may be due to clecaiboxylation of a
dicarboxylic acid.
Darken et al. (llKiO) ha\-e shown that in
the production of tetracyclines the phos-
phate-citrate-carbonate ratio is highly criti-
cal. As a result of careful study of these in-
terrelationships, yields in shaker flasks were
raised from an average of loO jug pei" n"il to
1350 /xg per ml. The high calcium carbonate-
citric acid medium permits the calcium ion
to act as a seciuestering agent for tetracy-
cline, ^'ields were raised in tank fermenta-
tions from ^)~) fjLg per ml to 525 ;ug per ml
in the high citric acid-ammonia medium,
which permits fermentation in the preferred
pH range. The seciuestering effect of calcium
carbonate becomes apparent as the yields
are further raised with this culture to an
average of 705 /xg per ml.
Each molecule of chlortetracycline con-
tains one chlorine atom. Antibiotic activity
can also be produced in a chloride-deficient
medium, or one in which chloride utilization
is blocked by the presence of low levels of
bromide, leading to the production of a new
antil)iotic, tetracycline (Gourevitch et al.,
1955). Controlled biosynthesis is a deter-
mining factor in the ])r()duction of tetracy-
cline, chloi-tetracycline, or bromtetracycline,
depending on the presence or absence of
chlorine or !)romine in a biologically a\'ailable
form as a precursor. Tetracycline itself is
luiilt up by one-carbon transfers fi'om simple
starting materials. So far no carbon com-
pound precursors have been found. The
biosynthesis of the various chlor- and brom-
tet]-acyclines has lieen studied further by
Doerschuk r( al. (]!)5(i).
M isceUaucoK.s A ntihiotic.'^
CHLORAMPHENICOL
This antibiotic is made up of two chemical
units, p-nitrophenylserinol antl dichloroace-
tic acid. I^y use of a synthetic glycerol
lactate medium, it was found that various
amino acids and p-nitrophenylserinol stimu-
lated chloramphenicol biosynthesis; dichloro-
acetic acid did not. Both growing and rest-
ing cells of S. venezuelae are capable of
acetylation of p-nitrophenylsei'inol to give
X-acetyl-p-nitrophenylserinol. Howe\'er, the
latter does not act as a precursor of chlor-
amphenicol, but being itself an antibiotic,
it increases the antibiotic potency of the
broth ((iottlieb et at., 1956).
7()
NATURE, FORMATION, AND ACTIVITIES
XOVOBIOCIN
This antibiotic consists of three distinct
moieties: a sugar, substituted coumarin,
and substituted benzoic acid {Yau Tam-
elen, 1958). The addition of the last to the
medium i-esulted in an increase in the yield
of novobiocin, a fact which suggests the
precursory nature of the acid (Jones, 1958).
Conclusions
Examination of the chemical structure of
a large number of antibiotic complexes led
Abraham and Xewton (1958) to conclude
that many antibiotics are built up from
substances that play an essential metalxjlic
or structural role in the life of microor-
ganisms. Certain types of structure, such as
the large lactone or peptide ring, frequently
recur. This was believed to be a reflexion of
specific types of organization in microbial
cells. These workers emphasized that differ-
ent members of the same type of antibiotic
may be produced by such processes as N-,
0-, and C-methylation, X-acylation, and
the introduction of hydroxyl groups, or by
the substitution of one structural unit for
another of a similar type. The yield of a
given antibiotic can vary enormously with
changes in the concentrations of intermedi-
ates which accompany changes in cultural
conditions; modifications of the enzymatic
systems which are a consequence of muta-
tions were also believed to play a role.
Abraham (1959) made a detailed analysis
of the phenomenon of biogenesis of anti-
biotics. According to him, "What we now
know, and can reasonably surmise, about
the origin of the antibiotics points to the
economy with which their novel and some-
times intricate structures are made. Amino
acids and acetate, which feature as common
structural units, have widespread functions
in cellular metabolism. The condensation of
acetate, as acetyl coenzyme A, to yield a
/3-keto acid, and of activated amino acids
to form peptide chains, are familiar proc-
esses. So, too, is the transfer of a methyl
group from S-adenosyl-methionine to nitro-
gen, oxygen, or carbon in another molecule.
D-amino acids are commonl}' present in the
cell walls of bacteria and actinomycetes,
though they have not been detected in other
forms of life. Large peptide and lactone rings,
of a type previously unknown in natural
products, have now been identified so often
in the structures of antibiotics that their
formatiori would seem to have a broad
significance." He concluded that the bio-
synthesis and mode of action of antibiotics
might "be expected to illustrate the diversity
rather than the unity of nature." He added,
however, that this is only partly true.
Chapter 8
Antimicrobial and Antitumor
Activities of Antibiotics
Antibiotics vary greatly in their biological
activities, especially in their antimicrobial
properties, toxicity to animals, and mode of
action upon sensitive organisms. Their prac-
tical potentialities, therefore, notably their
therapeutic effects in the treatment of human
and animal diseases and their growth-
promoting action, also vary greatly. Un-
fortunately, the specific chemical structure of
a compound alone is not necessarily indica-
tive of its biological behavior.
It was at first believed that screening
methods in vitro were not satisfactory for
finding new chemotherapeutic substances.
Fortunately, this concept was found later
to be completely unjustified. Chain and
Flore}^ (1944) emphasized that those anti-
biotics which pass the biological activity and
toxicity tests can be expected to be effecti\'e
as general therapeutic agents. Further stud-
ies confirmed these observations, namely,
that biological activities of antibiotics in
vivo are in general parallel to their activities
in vitro. Usually the only factors controlling
their actual practical potentialities are tox-
icity, selective activity upon various disease-
producing organisms, and the extent of such
activity.
Antimicrobial Spectra of Antibiotics
Antibiotics are characterized primarily by
their selective action upon different micro-
organisms, or their antil)iotic spectra. It was
once believed that this property was so
characteristic of each substance as actually
to identify the antibiotic. However, the ease
with which many antibiotics select resistant
mutants in bacterial populations, and the
frequent variations in the degree of sensi-
tivity among the different strains of the
same organism tend to diminish the signifi-
cance of this property for either the identifi-
cation of the antibiotic or its proper char-
acterization.
The following types of antimicrobial
spectra can be recognized:
1. Antibiotics active mainl}' against my-
cobacteria; examples: actithiazic acid and
nocardamin.
2. Antibiotics active mainly against gram-
positive bacteria; examples: the macrolides
such as erythromycin and other antibiotics
such as thiostrepton.
3. Antibiotics acti\'e against both gram-
positive and gram-negative bacteria; ex-
amples: streptomycin, neomycin, and the
tetracyclines.
4. Antibiotics active against bacteria and
fungi; examples: mycothricin and aureo-
thricin.
o. Antibiotics active mainl^y against fungi;
examples: most of the polyenes, such as
amphotericin B, and other sul)stances such
as cycloheximide.
G. Antibiotics active against inti'acellular
parasites of the lymphogranuloma-psitta-
NATURE, FORMATION, AND ACTIVITIES
oofsis group. Such antibiotics can be active
against other microorganisms; examples: the
macroHdes and the tetraeycHnes.
7. Antibiotics active against protozoa.
Such antil)i()tics usually have other types of
antimicrobial activity also. They include
paromomycin, which is also active against
bacteria, and trichomycin, which is also
active against fungi.
Substances produced by actinomycetes
which are not always strictly antibiotics
have other types of biological activity: (1)
antiviral substances such as ehrlichin, (2)
antitumor substances; these may be anti-
biotics, such as aetinomycir., or have no
known antibiotic activity, such as carcino-
mycin.
The keys and lists in Pai't B of this book
show in more detail the antibiotics belong-
ing to these various groups.
The antibacterial properties of a group of
antibiotics produced by actinomycetes are
shown in Table 27, in which the sensitivities
of different bacteria are summarized accord-
ing to Turpin and Velu (ll)r)7). Streptomy-
cin and neomycin show the greatest activity
against the mycobacteria and various gram-
negative bacteria, especially the Escherichia,
Saimonclla, Proteus, and Pseudomonas
groups; they show little or no activity
against the anaerobic Clostridia, and Km-
ited activity against the neisseriae and
(•(M'tain streptococci. Erythromycins are
highly active upon the various cocci and
certain gram-positi\e bacteria, but show
only limited action upon gram-negative or-
Table 27
Comparative sensitivity of variouf^ bacteria to several antibiotics cif actinomycetes CTiirpin and Velu, 1957)
Re.sults given as ng per ml.
Organism
Streptomycin
Chloram-
phenicol
Chlortetra-
cycline
O.xytetracycline
Neomycin
Erythromycin
Diplococcus pnennioniae .
0.5-50
0.00-12.5
0.03-5
<0.2-<0.4
0.5-150
0.003-0.19
Neisseria gonorrhoeae . . .
1-40
0.078-0.3
0.150-5
0.31-1
R*
0.04-0.50
N . intracellidaris
1-40
0.78-0.25
0.20-5
0.20-3.12
4->300
0.19-3.12
Staphylococcus aureus . .
0.03->256
1-50
0.9-125
0.31-2.5
0.1->300
0.01-02.5
Streptococcus hcuiolyti-
cus
( lioiip A
0,2-100
0.3-0
0.8-5
0.15()-1.5
0.5-150
0.007-0.02
Ciroup D
0.2->100
1-15
0.2-25
0.3-3
1(^500
0.02-3.12
Streptococcus viridaris
3-12
0.(^-2.5
0.062-25
<0.03
1-250
0.02-3.12
Bacillus subtilis
0.050-128
1-5
0.00-5
0.15-42
o.i-o.(;
0.078
Corynebacterium diph -
theriae
0.4-200
0.5-3
0.07
0.15-0.30
0.3-25
0.31-3.12
Mycobacterium tubercu-
losis
0.1-12.5
0-50
0.1-8
Various Clostridia
R
1.50->500
0.1-5
0.2-10
30- > 150
0.78
Brucella uielitensis
0.5-128
1.5(H). 25
0.2-5
2.8
Escherichia coli
0.015->1000
3-50
0.2-25
. 78-5
0.0(>->150
12.5-100
Hemophilus influenzae .
0.5-50
0.2-3.5
0.15-5
0.31-2.5
4->300
0.02-3.12
Klebsiella pneumoniae .
0. 05- > 1000
0.5-25
0.2-50
0.78-3.12
1.5-25
3.12-200
Proteus vulgaris
1->1000
0.12->250
0.2-400
0.25->250
0.25->150
250
Pseudoynonas aeruginosa
0.1->1000
8-> 1000
10-400
5-25
1 . 0-05
125->200
Salmonella diverses
0.004-32
0.25-0
0.2-25
2.5-5.5
0.8-83
02. 5- > 200
Sal. typhosa
0.004-32
0.75-5
0.2-5
<0.3-3.12
0.3-10
02.5
Shigella diverses
0.25-10
0.5-10
0.078-25
0.039-3.12
0.5-10
32
R = resistant.
ANTIMICROBIAL AND ANTITUMOR ACTIVITIES
79
ganisms. The tetracyclines and chloram-
phenicol are active against both gram-posi-
tive and gram-negative bacteria, including
the Clostridia and the brucellae. The last
two groups of antibiotics are also active
upon the rickettsiae and psittacosis-lymph-
ogranuloma group of intracellular parasitic
organisms, Init the first group is not.
The phenomena of cross-resistance and
strain variation further characterize the
above three groups of antibiotics, as will be
discussed later (Chapter 10). The literature
on the various antibiotics is so extensive that
no attempt could be made to cover it in
even a limited way. Only a few pertinent
references can be presented here on the
antimicrobial activities of some of the anti-
biotics produced by actinomycetes, espe-
cially those that have been well recognized
and are of established therapeutic value.
Finland and Haight (1953) isolated 500
strains of hemolytic, coagulase-positive cul-
tures of Staph, aureus from clinical material
and tested them for sensitivity to various
antibiotics (Table 28). They observed that
with an increase in the use of various anti-
biotics in clinical practice, there was a sig-
nificant increase in the proportion of bac-
terial strains resistant to the antibiotics,
notably to chlortetracycline and oxytetra-
cycline. Of the total strains tested, about
three fourths were resistant to penicillin,
one fourth to chlortetracycline, and one
third to oxy tetracycline.
Potee et al. (1954) made a study of 119
strains of freshly isolated cultures of Pro-
teus. They comprised 86 strains of /-'/•.
mirahilis, 12 of Pr. vulgaris, 15 of Pr. m<>r-
ganii, and 6 of Pr. rettgeri. The sensitivity of
these strains to 10 antibiotics varied greatly,
depending on the species, as shown in
Tables 29 and 30. They were all found to
be invariably resistant to erythromycin, al-
though a few showed some sensitivity. Most
of the strains were resistant to streptomycin,
especially after the previous use of this
Table 28
Distribution of 500 strains of Staph, aureus accord-
ing to grade of resistance to five antibiotics which
have been in common use (Finland and Haight,
1953)
Antibiotic
Grade of
resistance*
No. of
strains
% of
strains
Penicillin
s
124
24.8
I
11
2.2
R
365
73.0
Chlortetracy-
s
337
67.4
cline
I
45
9.0
R
118
23 . 6
Oxytetracycline
s
266
53.2
I
73
14.6
R
161
32.2
Chlorami)heni-
s
(j
1.2
col
I
482
96.4
R
12
2.4
Streptomycin
S
99
19.8
I
351
70.2
R
50
10.0
*S = sensitive; I = intermediate; R = re-
sistant.
antibiotic. Many strains were relatively
sensitive to neomycin, which had not been
used previously in these particular cases. The
sensitivity to chloramphenicol and the tetra-
cyclines varied greatly among the species
and strains. In general, neomycin and chlor-
amphenicol were most active; streptomycin
came next; erythromycin and penicillin were
least active. Bacitracin and polymyxin
showed no activity. Almost all strains of all
species were inhibited by neomycin in con-
centrations of 25 to 100 Mg per ml. This
antibiotic was slightly more active in vitro
against most of the strains than were strep-
tomycin and chloramphenicol (Tables 29 and
30). When the above results were compared
to the findings obtained in 1949, no definite
increase in resistance of Proteus to penicillin
or to streptomycin was found; there was a
slight increase in resistance to chlorampheni-
80
NATURE, FORMATION, AND ACTIVITIES
Table 29
Susceptibility of 33 strains of Proteus to different antibiotics (Potee et al., 1954)
Previous antibiotic
Species and
varieties of
Proteus
Minimal inhib
iting concentration, /igm
therapy*
Chlortetra-
cycline
Oxytetra-
cycline
Tetra-
cycline
Chloram-
phenicol
Strepto-
mycin
Neomycin
Erythro-
mycin
T
vulgaris
12.5
12.5
25
25
50
50
400
p
12.5
6.3
12.5
200
>400
50
400
p
6.3
25
12.5
200
>400
25
>400
P, Ct
6.3
12.5
6.3
200
>400
50
>400
P, s
3.1
3.1
6.3
12.5
50
50
400
None
6.3
3.1
12.5
200
>400
25
>400
P
6.3
12.5
6.3
200
>400
25
>400
P
12.5
6.3
3.1
200
50
50
400
None
3.1
3.1
3.1
200
>400
50
400
P
3.1
3.1
3.1
100
>400
50
400
None
12.5
12.5
6.3
25
50
25
200
P
12.5
6.3
12.5
100
>400
50
>400
None
morgan ii
6.3
3.1
6.3
100
50
25
>400
P, Ct
>400
>400
>400
200
>400
12.5
>400
Ot
3.1
3.1
6.3
12.5
50
25
>400
T
>400
>400
>400
>400
>400
25
400
P, T, Cm
>400
>400
>400
>400
>400
25
>400
None
12.5
3.1
12.5
12.5
>400
25
>400
None
6.3
6.3
6.3
25
25
12.5
400
P
>400
>400
>400
>400
>400
12.5
>400
P
12.5
12.5
12.5
50
12.5
25
>400
P
12.5
12.5
12.5
25
12.5
25
400
Ot, T, Cm
50
12.5
12.5
200
>4G0
50
>400
P
100
25
12.5
50
12.5
25
>400
P
25
25
12.5
12.5
12.5
25
>400
P
25
12.5
12.5
25
>400
50
>400
B, Pm
>400
>400
200
100
>400
100
50
P
rettgeri
>400
>400
>400
>400
>400
400
50
P, S
>400
>400
>400
400
>40()
50
400
P, 8
>400
>400
>400
> 400
>400
25
400
P, s
12.5
6.3
6.3
100
>400
25
400
P
12.5
6.3
12.5
100
>400
50
>400
P, S, Ct
>400
>400
400
12.5
>400
25
>400
*T = tetracycline; P = penicillin; Ct = chlortetracycline; Ot = oxytetracycline; Cm
phenicol; S = streptomycin; B = bacitracin; Pm = polymyxin.
chloram-
col and a definite increa.sc in the proportion
of strains resistant to chlortetracycline.
Treatment of patients with penicillin did
not appear to influence the susceptibility to
this antibiotic of strains of Proteus subse-
flLiently isolated from such patients. Strep-
tomycin-resistant strains were more fre-
(|uent among patients previously treated
with this antil)iotic than among those who
had iKit received streptomycin. Cultures iso-
lated from patients treated with tetracy-
clines showed a larger proportion resistant
to these three antibiotics.
The susceptibility of Pseudomonas cul-
ANTIMICROBIAL AND ANTITUMOR ACTIVITIES
81
Table 30
Comparison of antibiotic susceptibilities of 86 strains of Pr. niirabilis with those of
33 strains of other species of Proteus (Potee et al., 1954)
Antibiotic
Species and strains
of Proteus
No.
of strains inhibited by f^g/ml)
3.1
6.3
12.5
25
50
100
200
■iOO
>400
Chlortetracycline
mirabilis
Others*
4
(i
10
2
1
1
2
4
80
9
Oxj-tetracycline
mirabilis
Others
7
6
8
3
2
13
71
9
Tetracycline
mirabilis
Others
3
8
12
1
4
17
43
1
15
1
7
7
Chloramphenicol
mirabilis
Others
1
2
5
13
5
30
2
33
6
1
9
1
5
Streptomycin
mirabilis
Others
3
3
4
13
1
38
17
1
11
22
Neomycin
mirabilis
Others
3
12
16
57
12
17
1
1
Penicillin
mirabilis
Others
1
20
25
8
10
11
1
4
7
32
Erythrom3'cin
mirabilis
Others
2
1
5
1
10
13
17
Bacitracin
mirabilis
Others
86
33
Polymyxin
mirabilis
Others
86
33
Pr. vulgaris, 12 strains; Pr. morganii , 15 strains; and Pr. rettgeri, 6 strains.
tures to various antibiotics is shown in
Table 31. Polymyxin B was by far the most
active agent, nearly all strains being inhib-
ited by 6.3 Mg pel" ml or less. The tetracy-
clines and neomycin were next in activity.
A few strains were sensitive to streptomycin;
most of them were moderately sensitive to
other antibiotics. Typical strains of Ps.
aeruginosa were moderately or highly re-
sistant to erythromycin and chlorampheni-
col, and all strains were resistant to the
highest concentrations of bacitracin and
penicillin used, namely, 400 Mg pei" ml- The
resistance of the strains to any of the anti-
biotics could not be correlated with the
pre\'ious history of treatment of the patient
with homologous or other antimicrobial
agents.
The sensitivity of different pathogenic
organisms to streptomycin is illustrated in
Table 32.
The sensitivity of different bacteria to
different neomycins is shown in Table 33.
Neomycin B was most active, whereas
! NATURE, FORMATION, AND ACTIVITIES
Table 31
Comparison of antibiotic sensitivity of three series of strains of Pseudonionas (Wright et al., 1954)
Antibiotic
Series*
No. of
strains
Per cent of strains inhibited by (pg/in
1)
1.6
3.1
6.3
12.5
25
50
100
200
400
>400
Polymyxin
A
B
C
25
32
110
3
4
72
38
35
24
59
60
4
1
Streptomycin
A
185
1
2
5
1()
28
27
6
2
14
B
32
3
9
44
19
3
9
13
C
110
1
1
2
9
20
18
3
3
44
Chloramphenicol
A
B
27
32
33
59
IG
4
84
C
110
1
1
1
1
8
88
Chlortetracycline
A
B
115
32
1
2
2
9
20
9
49
38
17
Ki
1
38
C
110
1
1
3
5
15
70
Oxytetracycline
B
32
3
()
78
13
C
110
1
1
5
35
41
17
Tetracycline
B
32
3
75
22
C
110
2
5
18
50
24
2
Neomycin
B
32
()
41
28
19
6
C
110
3
3
3
13
13
37
2(>
3
Erythromycin
B
32
22
72
6
C
110
1
2
1
10
50
31
5
* Series A = All strains isolated in 1949 and earlier. Series B = L>()])hilize(l strains isolated in 1949,
studied in 1954. Series C = Current strains, isolated December 1953 to May 1954.
neamine was least active. There was much
variation among the different species and
strains.
The increased use of antibiotics in clinical
practice leads to an increase in resistance of
bacteria. This is well illustrated in Table 34.
There is a difference, however, between
different organisms. Klebsiella, for example,
develops greater resistance than Escherichia.
The correlation between antibiotic sensi-
tivity tests and clinical results was examined
by Abboud and Waisbren (1959).
Antibiotics have been grouped on the
basis of their antistaphylococcal properties
by Waisbren and St relit zer (1960) (Table
35). A detailed study of the behavior of new
antistaphylococcal agents has been made by
Garrod and Waterworth (195(3).
Considerable work has been done and a
most extensive literature has accumulated on
the sensitivity of the tuberculosis organism
Alycohacferium tuberculosis, to various anti-
biotics and other antimycobacterial agents.
Waksman and Lechevalier (1953) have
shown that the human and bovine forms of
M. tuberculosis are highly sensitive to iso-
niazid, the avian form is less sensitive, the
nonpathogenic mycobacteria are still less
sensitive, and the Nocardia and Streptomy-
ces species are fairly resistant. Pollak (1956)
reported that atypical acid-fast mycobac-
teria are sensitive to streptomycin and par-
tially or totally resistant to the synthetic
chemotherapeutic agents p-aminosalicylic
acid and isoniazid.
IVIicrobiostatic versus Microbicidal Ac-
tivity of Antibiotics
Antibiotics can kill microbial cells, or they
may simply prevent their multiplication.
ANTIMICROBIAL AND ANTITUMOR ACTIVITIES
83
Table 32
Sensitivity of pathogenic microorganisms to
streptomycin in vitro (Youmans and
Usher, 1949)
Actinomycetes
Bacillns anthracis
Brucella abortus
Br. meliiensis
Br. .s»/s
Donovania gran ulumuli.'i
Erysipelothrix rhusio-
pathiae
Hemophilus influenzae
H. pertussis
Klebsiella pneumoniae
Leptospira icterohaem-
orrhagiae
Sensitive
.1/.
tuberculosis var.
bovis
A'eisseria gonorrhoeae
N^ . meningitidis (A'. ///-
traccllularis)
Pasteurella multocida
P. pestis
P. tularensis
Salmonella species
Sal. typhosa (Eberthella
ti/phosa)
Shigella dysenteriae
Sh. paradysenteriae
Listeria monocytogenes Streptobacillus monili-
(Lislerella monocyto- form is
genes) Veillonclla gazogenes
Mycobacterium tubercu-
losis var. hominis
Moderately sensitive
Aerobacter aerogenes
Alcaligenes faecalis
Corynebacterium diph -
theriae
Coxiella burnetii (Rick-
ettsia burneti)
Diplococcus pneumoniae
Escherichia coli
Hemophilus ducreyi
Malleomyces mallei
Staphylococcus albus
Staph, aureus
Nocardia asteroides
Proteus morganii
Insensitive
Pr. vulgaris
Pseudomonas aerugi-
nosa (Bacillus pyo-
cyaneus)
Rickettsia akari
R. proivazekii
R. typhi (R . uiooseri)
Streptococcus, a hemo-
lytic
Streptococcus. /3 hemo-
lytic
Streptococcus faecalis
Vibrio cholerae (V.
comma)
Bacteroides fragilis
Bade r (rides fundilifor-
mis
Clostridium species
Malleomyces pseudo-
uiatlei
Rickettsia tsutsugamu-
shi (Rickettsia orien-
talis)
Feline pneumonitis or-
ganism
Virus of himian influ-
enza
Psittacosis organism
Blastomyces dermatidi-
iis
Candida albicans
( 'occidioides im m it is
Cryptococcus neofor-
utans (Torula histo-
lytica)
Geotrichum species
Histoplasma capsulatuui
Endauioeba histolytica
Trichomonas vaginalis
Trypauosoma species
Table 32 — Continued
Lymphogranu-
loma venereum
Meningopneu-
monitis
Virus of mumps
Table 33
Responses of bacteria to neomycin B, neomycin C,
and neamine (Sel)ek, 1958)
Organism
Serratia marcescens
Micrococcus pyogenes var.
albus
Bacillus brevis
Corynebacterium diphthc-
roides
Neisseria catarrhalis
Aerobacter aerogenes
Klebsiella pneumoniae . . .
Escherichia coli
Micrococcus pyogenes var.
aureus
Shigella dysenteriae
Salmonella paratyphi ....
Proteus vulgaris
Salmonella pullorum
Shigella parody sen teriae
var. sonnei
Salmonella schdttnniclleri
Sal. hirschfeldii
Pseudomonas aeruginosa .
Streptococcus pyogenes. . .
Streptocoi-cus viridans. . . .
Diplococcus pneumoniae .
Alcaligenes faecalis
Micrococcus lysodeiklicus
Corynebacterium sp
Sarcina lutea
Maximal tolerated concentra-
tion
Neomy-
cin B
Neomy-
cin C
Neamine
lig/ml
0.8
12.5
1.5
0.8
1.5
6.0
0.8
6.0
6.0
1.5
6.0
6.0
1.5
6.0
6.0
1.5
3.0
12.5
3.0
6.0
12.5
3.0
12.5
12.5
3.0
12.5
25.0
6.0
25.0
25.0
6.0
25.0
25.0
6.0
50.0
25.0
6.0
25.0
50.0
12.5
25.0
25.0
12.5
50.0
>100.0
12.5
100.0
>100.0
12.5
>100.0
>100.0
25.0
25.0
12.5
25.0
25.0
12.5
25.0
50.0
50.0
>100.0
> 100.0
> 100.0
1.5
50.0
>100.0
1.0
250.0
250.0
1.0
250.0
250.0
In the latter case, growth \voiild take place
in the presence of nutrients, after the anti-
biotic had been washed off the cells.
The microbicidal action of antibiotics
84
NATURE, FORMATION, AND ACTIVITIES
Table 34
A^iDiiber and percentage of cuUform bacilli resistant to Jour antibiotics
isolated in two periods (Vaccaro et al., 1956)
Escherichia
Klebsiella
Antibiotic
1951-
-1952
1955-
-1956
1951-
-1952
1955-
-1956
No.
%
No.
%
No.
%
No.
%
St i"eptom\'cin
125
40
118
106
45.4
15.5
42.5
41.7
100
28
69
77
64.1
17.9
44.2
49.3
49
22
33
36
50
22.4
33.0
36.7
72
36
60
63
85.7
Chlorami)henicol
Chlortetracvcline
45.2
71 4
Oxytetracvcline
75
Total strains studied
253
156
98
84
depends on the eoncentration of the aiiti-
})iotic.s which are put in contact with micro-
l)ial cells and on the amount and nature of
nutrients and salts present. Environmental
conditions, such as pH, also play an im-
portant role in the killing process.
Many antibiotics have strong bactericidal
action. The addition of O.o mg of actino-
mycin to a 10-ml suspension of E. coli re-
duced the number of viable cells from 0,400,-
000 to 493,000; 1 mg of the antibiotic
brought about a i-eduction to 4,800; and 2
mg resulted in complete destruction of all
the living cells.
The microbiostatic action of very similar
antibiotics can differ. For example, the
bacteriostatic action of both mannosido-
streptomycin and dihydromannosidostrepto-
mycin is significantly less than that of
streptomycin and dihydrostreptomycin for
all organisms except Salmonella typhosa and
Salmonclki schuttmuUeri. These results are
similar to those pre\'iously reported (Rake
et al., 1947).
Similarly, Lechevalier (1960) repoited
differences in the fungicidal properties of
polyenic antifungal antibiotics.
A strong lytic action, similar in some cases
to the action of enzymes, has also been
indicated for some of the antibiotics. This
effect may be a result of the lysis of the
cells. Autol.ysis is usually defined as the
destruction of some of the cell constituents
by enz^anes originating within the cell. The
lytic effect is bi-ought about, however, by
but few of the antibiotics and does not affect
most of the bacteria. The greatest bacteri-
cidal action of penicillin, for example, occurs
during maximal cell division, when the cells
capable of producing lytic agents undergo
Ij^sis rapidly. It must therefore be concluded
that lysis of the cells follows the killing ef-
fect of penicillin.
Interaction among Antibiotics
It has been noted that when antibiotics
are used in mixtures, one antibiotic will
sometimes repress or boost the antimicrobial
action of another.
Jawetz, in a chapter of a monograph on
neomycin (Waksman et al., 1958), has
pointed out that antibiotics can be divided
into two groups on the basis of their be-
havior when used in mixtures. Group 1
comprises penicillin, streptom^'cin, bacitra-
cin, and neomj^cin, and Group 2 is composed
of the tetracyclines, chloramphenicol, and
erythromycin. Members of Group 1 are
never antagonistic to one another but not
infre(iuently are synergistic. Members of
ANTIMICROBIAL AND ANTITUMOR ACTIVITIES
85
(Iroup 2 are a.s a rule neither antagonistic
nor synergistic to one another. When a
member of Group 1 is added to a member
of Group 2, the effect is unpredictable and
depends on the microorganism.
^'arious methods of determining syner-
gism and antagonism among antibiotics
luu'e been proposed and were reviewed
briefly by Ghal)bert and Patte (1960), in a
paper in which they described a method
permitting the study of the bactericidal
synergistic effect of mixtures of antibiotics.
Actinomycetes have been found to pro-
duce mixtures of antibiotics which are syn-
ergistic. For example, antibiotics PA 114A
and B are moi'e active in combination than
when used alone.
Geminimycin is the j^erfect example of
such synei-gistic antibiotic pairs (Rao e( al.,
1960). It is formed of two compounds, A
and B, which are antibiotically inactive.
The mixture A + B is active against gram-
positive bacteria.
Antifungal Activities of Antibiotics
It has been pointed out elsewhere that
actinomjTete antibiotics active upon fungi,
beginning with cycloheximide and ending
with many of the polyenes, have either no
activity at all or only very limited activity
upon bacteria. These anti})iotics are fre-
(juentl}' spoken of as antimycotics. Different
fungi, often strahis of the same species, differ
greath' in their sensiti\'ity to these agents.
Trichophijion mcntagrophytes and Candida
albicans are commonly used as test organ-
isms, with the agar-cup method. Bergman
(1955) suggested use of conidia only, a
conidial "bank" being recommended for this
purpose. Alcohol is usualh^ used as the
solvent, since most of the agents are not
soluble in water. Sabouraud's agar is com-
monly employed.
A comparative study of the effect of
nystatin, amphotericin B, and candidin on
Table 35
Grouping of antibiotics on the basis of (heir activity
against staphylococci (Waisbren and
Strelitzer, 1960)
Drug
Neomycin
Kanamycin
Paromomycin . .
Novobiocin
Vancomycin
Ristocetin
Nitrofurantoin. . .
Oleandomycin.
Erythromycin. .
Streptomycin . .
Tetracycline
Oxytetracycline. .
Penicillin
Polymyxin B
Bacitracin
Chlortetracycline
Chloramphenicol .
Group"
Minima] inlii-
bitory con-
centration
(interpolated
from means
of inhibitory
tube)
fig/ml
0.35
0.36
0.41
0.51
0.86
1.40
5.79
5.40
5.43
7.62
7.20
8.40
11.46
11.10
11.70
14.52
21.60
Per cent of
strains sus-
ceptible to 6
lig/ml or less
of the agent
(Abboud and
Waisbren,
1959)
100
100
100
96
100
100
84
73
59
59
43 1
40
47t
40
35
35
15
* Significant difTerence.s among members of the
groups were determined by analyzing the differ-
ence of the means of the number of tube dilutions
necessary for inhibition of the 75 strains of stai)h-
ylococci. The following formula was used:
T =
D
VNiSi^ + N.,So2
VNiN2(Ni + Na - 2)
N, + N,
Antibiotics were considered to be significantly
different in activity if the P value of the difference
of means was <0.01; i.e., if there was less than
one chance in 100 that the means of the number
of tubes necessary for inhibition with each anti-
liiotic belonged in the same distribution and were
not representatives of different distributions.
The tubes were numbered as follows: > minimal
inhibitory concentration OlOO) = 11; 100 = 10;
50 = 9; 25 = 8; 12 = 7; 6 = 6; 3 = 5; 1.5 = 4;
0.75 = 3; 0.38 = 2; <0.38 = 1.
t This means that a slightly greater percentage
of strains may be clinically susceptible to peni-
cillin, but that over-all tetracycline is moi-e ac-
tive on a weight for weight basis.
86
NATURE, FORMATION, AND ACTIVITIES
experimental moniliasis has recently been
carried out by Kosunen ( 1 959) .
Antitumor Activities of Antibiotics
The ability of various antibiotics to sup-
press the development of neoplasms resulted
in the isolation of a large number of com-
pounds from cultures of actinomycetes that
possess such a property (Reilly, 1953). This
was brought out in Chapter 3. Sevcik (1959)
divided these compounds into four catego-
ries on the basis of their antibiotic and anti-
tumor spectra:
1. Substances active upon tumors, as well
as upon bacteria. These include actinoxan-
thine, azaserine, 6-diazo-5-oxo-L-no-leucine
(DON), cellocidin, alazopeptin, netropsin,
carzinophilin, aburamycin, actinomycin, rac-
tinomycin, pluramycin, amicetin, gancidin,
actinoleukin, griseolutein, levomycin, sulfo-
cidin, puromycin, desertomycin, mitomycin,
and others.
2. Substances active upon tumors and
upon only one group of bacteria, namely,
M. tuberculosis. These include toyocamycin
and tubercidin.
3. Substances active upon tumors and
fungi, but not upon bacteria. Ihese include
cycloheximide, hygroscopin, and polyenes.
4. Substances active only upon tumors.
These include melanomycin, carzinocidin,
carcinomycin, and sarkomycin.
The above groups were further subdivided
on the basis of their solubility in water.
Sevcik found the third group, comprising
the polyene compounds, to lie the most
widely distributed in nature, although he
doubted their practical usefulness. The nu-
cleotide antibiotics (puromycin, amicetin,
and carzinophilin A) appeared to be most
promising because of their relatively low
toxicity. The cjuinone-type antibiotics (ac-
tinomycins, pluramycin, ractinomycin, ac-
tinoleukin, and levomycin) were highly toxic.
Oda (1960) also summarized our i-ecent
knowledge of antitumor antibiotics. Differ-
ent experimental tumors in animals are used
for sci^eening purposes, such as Yoshida
sarcoma, Ehrlich carcinoma, mouse leu-
kemia, and others in Japan; sarcoma 180,
carcinoma 755, and mouse leukemia in the
United States. Oda emphasized that "the
present situation of antitumor antibiotic
research seems to l^e in the night l:)efore the
discovery of streptomycin and the author
wishes to hei'e introduce an outline of I'e-
search of antitumor antibiotics." As many
as 2 per cent of all cultures of actinomycetes
isolated from soil possess antitumor actiA'ity.
Considerable information has acciunu-
lated concerning the mode of action of some
of these antibiotics, especially actinomycin,
upon the tumor cells. Robineaux et at. (1958),
for example, have shown that in tissue cul-
ture, antimitotic activity of actinomycin C
is completely repressed by glutathione; cyto-
static activity is not affected, however.
They suggested that actinomycin possesses
at least two mechanisms: antimitotic and
cytocidal.
In speaking of the effect of actinomycin U
upon transplantable animal tumors, Sugiura
(1960) stated, "The effectiveness, at least
temporary, of this antibiotic against human
neoplasia (Wilms's tumor, neuroblastoma,
rhabdomyosarcoma, lymphosarcoma, Sw-
ing's tumor, and melanoma) affords some
hope in the attainment of our goal, the cure
of cancer in man."
Various methods for the determination of
cytotoxic metabolites formed by microor-
ganisms ha\'e been suggested. Perlman el al.
(1959) tested a number of antibiotics for
inhibition of multiplication of an L cell line
of mouse fibroblasts and showed that various
actinomycins (Fig. 3) were remarkably ac-
tive.
Antitoxin Activity of Antibiotics
A'arious antibiotics possess remarkable
antitoxin properties. Hinton and Orr (1960),
for example, have shown that inhibition of
ANTIMICROBIAL AND ANTITUMOR ACTIVITIES
87
GLIOTOXm
8 12 16 20 24
CONCENTRATION OF ANTIBIOTIC, M/iG/ML
32
Figure 3. Eft'oct of ;intil)iotics on the multiplication of L cells of mouse tihrohlasts. (Reproducec
from Perlman ct <d. Proc. Soc. Exptl. Biol. Med. 102: 290-292, 1959.)
toxin production in Staph, aureus l)y clilor-
aniphenieol, the tetracyclines, and oleando-
mycin i.^ directly proportional to inhibition
of growth. Streptomycin and bacitracin in-
hilMt toxin production to a degree out of
proportion to growth inhibition.
Chemical Structure and Antibiotic
Activities
The marked variations in the antimicro-
l)ial activities of the antibiotics, or their
antimicrobial spectra, present a type of
problem that defies any attempt at logical
speculation on the possible relation between
chemical structure and biological activity.
Some antibiotics possess a wide or l)road
spectrum of antimicrobial activity; others
are characterized by a very narrow spectrum
of activity.
The following questions logically present
themselves in this connection:
Why do some antibiotics act upon bac-
teria alone, others on fungi alone, and still
others upon both luicteria and fmigi? Why
do some affect the rickettsiae anfl the other
intracellular parasites and not th(^ true
viruses?
Why ai-e the gram-positive bacteria far
more sensiti\'e to the great majority of
antibiotics produced by a<'t inomycetes than
are the gram-negative bacteria?
Why do some antibiotics have a marked
acti\-ity u])on acid-fast l)acteria, whereas
others, e\en though active upon various
gram-positive bacteria, have no effect?
Why do certain closely related groups of
bacteria, such as those found among the
aerobic spore-formers (B. subtilis versus B.
mijcoides) or among the gram-negati\-e bac-
teria {E. coli versus A. aerogenes), often differ
greatly in their sensitivity to certain anti-
biotics?
Why are some antibiotics active only
upon gram-positi\'e bacteria and not upon
gram-negati\'e forms, whereas some are ac-
tive on both gram-posit i\'e and gram-nega-
tive bacteria?
Whv do some l)acteria and not others
NATURE, FORMATION, AND ACTIVITIES
develop i-apid resistance to some anti-
biotics and not to others, as in the case of
staphylococci versus streptococci to peni-
cillin? Why do the rates of development of
resistance differ for different antibiotics, as
in the mechanism of development of resist-
ance among sensitive bacteria to neomycin
versus streptomycin?
Why do some bacteria produce strains
that become nutritionally dependent upon
certain antibiotics, such as streptomycin?
Were answers found to these cjuestions,
one could go a long way in establishing the
correlation between chemical structure and
biological activity of the various antibiotics.
Even if they cannot be answered at pres-
ent, advantage is taken of some of the
known properties of the antibiotics in clas-
sifying them, utihzing them, and suggesting
an interpretation of their possible mode of
action. These can be briefly listed as follows:
1. The phenomena of resistance and of
sensitivity of microorganisms to various
antibiotics permit the recognition of certain
close relationships among such antibiotics,
if not in their chemical structure, at least
in their biological activity. Thus, it is pos-
sible to recognize the similarity among anti-
biotic preparations long before their chemi-
cal nature has been established.
2. Although it is now fully recognized that
the modes of action of various antibiotics
differ, too little is still known about this
phase of antibiotic behavior to warrant
speculation upon any possible relationships
between structure and activity.
3. The toxicity of antibiotics to animal
tissues is known to differ great l.y. This phe-
nomenon is of importance in any effort
to evaluate the practical potentialities of
antibiotics in disease control. The reasons
for it, however, remain obscure. Neomycin,
for example, was shown to have an effect in
the treatment of tuberculosis, but it has so
far not taken a significant place in the arma-
mentarium of phthisiologists, largel}^ be-
cause of its injurious nephrotoxic and oto-
toxic effects when administered parenterally.
Different modes of administration or the
supplementation of certain nutritional fac-
tors may be the answer for the practical
utilization of this antibiotic.
4. Although it has been assumed that
activities of antibiotics in vitro and in vivo
are parallel, there are certain instances in
which they are not. This is true, for exam-
ple, of cycloserine, an antibiotic found to be
more active against the tubercle bacillus
in vivo than in vitro.
,'-). Finally, attention must be directed to
the facts that actinomycetes also produce
growth-promoting substances (B12), and that
some antibiotics, in limited concentrations,
may also exert a growth-promoting effect
upon ^'arious forms of life — a property of
certain antibiotics that has been taken ad-
vantage of in the feeding of poultry, swine,
and other animals. These properties tend to
complicate further our concept of the chemi-
cal structure and biological activity of anti-
biotics.
The following illustrations will sufhce to
emphasize that certain changes in the chemi-
cal structure of the antibiotic molecule may
result in marked changes in its biological
activity:
1. When streptomycin is changed chemi-
cally to dihydrostreptomycin, whereby the
carbonyl group in the central hexose unit is
reduced, the characteristic antibacterial
properties of the drug are retained, although
there is a change in the nature of its poten-
tial toxicity. On the other hand, the treat-
ment of streptomycin by such carbonyl
reagents as hydroxylamine brings about in-
activation of the drug. The replacement of
the CH:j group in the central hexose unit
(streptose) by CHoOH, to give hydroxy-
streptomycin, seems to increase the toxicity
ANTIMICROBIAL AND ANTITUMOR ACTIVITIES
89
of the drug without apparently interfering
with its activity.
2. Alodification of the aromatic aryl, the
(lichloroacetyl, and the C'HoOH groups in
the chloramphenicol molecule results in the
complete destruction of its activity.
3. The degradation of chlortetracycline to
tetracycline, whereby the chlorine atom in
the first ring is replaced by a hydrogen atom,
results in a decrease in the antimicrobial
activities of the molecule, and is believed to
give a less toxic and more stable compound.
Dechlorination of chlortetracycline in-
creases its stability markedly.
4. The activitv of actithiazic acid agauist
the tuberculosis organism has been related
to its thiazolidone structure.
These facts are too limited to justify any
broad generalization concerning specific
structure and activity of antibiotics. One
point is clear, however. The antibiotics of
actinomycetes represent such a wide variety
of chemical structures and biological ac-
tivities that we must conclude that we are
dealing here with a new field of natural
products, \-arying greatly in chemical com-
position, in antimicrobial activities, and in
othei- biological properties that render them
potentially of great importance to human
health and human economy.
Chapter 9
Modes of Action of Antibiotics
Ehilich defined chemotherapeutics as
substances which are bound directly to
pathogenic microbial cells, damaging the
micro])e without affecting the host. At first
this concept was not generally accepted. The
prevailing idea was that the action of chemo-
therapeutic agents consisted in stimulating
the body defenses against the invading mi-
crobes. Later this idea appeared to be sup-
ported by the demonstration that prontosil
was effective in vivo but not in vitro. Ehr-
lich's concept was fully substantiated only
when it was established that the acti\'e sub-
stance in prontosil was sulfanilamide, which
w^as split off in the body, and that this
substance was active both in vitro and in
vivo. The action of antibiotics upon patho-
genic organisms served to support this idea
further, thus contributing materially to the
rapid progress in the utilization of antibiot-
ics as chemotherapeutic agents. Some in-
vestigators went even further in suggesting
that, since the activity of therapeutic agents
upon microbes consists largely in their effect
upon microbial metabolism, all substances
which have such an effect should be con-
sidered as antibiotics (Ericsson and Svartz-
Malmberg, 1959). Such a concept is scarcely
justified, however, since all sorts of com-
plexes, both of natural origin and syntheti-
cally produced, have therapeutic poten-
tialities.
We still know relatively little concerning
the mechanisms involved in the selective
action of antibiotics upon different bacteria
and other microorganisms. This phenome-
non cannot be correlated with either the
morphological or staining properties of the
sensitive organisms. True, some antibiotics
are active largely upon bacteria and others
upon fungi or animal forms, such as amoebae
and trypanosomes, luit the differences in
their action are so marked that no broad
generalizations can yet be made.
Alost investigators have emphasized the
modes of action of clinically useful anti-
biotics. Some (Lardy ci a/., 1908) described
the action of antibiotics which for various
reasons are not useful therapeutically, espe-
cially when considered as potential anti-
tumor agents.
Numerous theories ha^•e been proposed to
explain the modes of action of antibiotics.
This action has been attributed to the fol-
lowing phenomena :
1. The antibiotic interferes with microbial
cell division, thus preventing further growth
of the organism. The cell, unable to divide,
gradually dies.
2. The antibiotic interferes with the meta-
bolic processes of the microbial cells by
substituting for one of the essential nutrients.
A specific inhibitory effect may be exerted
by those substances that are structuralh-
related to normal cell metabolites. Such
substances are taken up by the cell in com-
petition with normal nutrients. Since they
are useless to the cell for further reactions,
they block the process of growth.
3. The antibiotic interferes with various
enzymatic systems, such as the respiratory
mechanism of the microbial cell, especially
90
MODES OF ACTION OF ANTIBIOTICS
91
the hydrogenase system and the phosphate
uptake by the bacteria accompanying glu-
cose oxidation.
4. The antibiotic inhibits celhilar oxida-
tions involving nitrogenous compounds.
5. The antibiotic interferes with the pro-
duction and utilization of a growth factor
essential to the cell.
6. The antibiotic combines with the sub-
strate or with one of its constituents, which
is thereby rendered inactive for liacterial
utilization.
7. The antibiotic favors certain lytic
mechanisms in the cell, resulting in destruc-
tion of the cell.
8. The antibiotic affects the surface ten-
sion of the sensitive organisms, acting as a
detergent.
9. It has also been suggested that the
activity and specificity of an antibiotic are
functions of several factors, such as ditfusi-
bility of the antibiotic into the microl;)ial
cell, adsorption by various enzyme systems,
its reaction with sulfhychyl groups of the
enzymes or with other sulfhydryl-containing
substances adsorbed by the enzymes.
10. The concentration of the antibiotic
and the composition of the mediiun ai'e
highly important in modifying the activity
of the antibiotic. Some antibiotics lose con-
siderable bacteriostatic activity when incu-
bated with sterile l)roth.
The majority of antibiotics exert not only
a marked bacteriostatic effect, but also a bac-
tericidal action. This effect is accelerated by
an increase in temperature from 4 to 42°C,
but is impaired by an increase in acidity of
medium between pH 7.0 and o.O. The rajiid
drop in the number of bacteria within the
first 15 minutes after the application of
penicillin was interpreted as indicative of its
bactericidal action. Young cells are particu-
larly susceptible, whereas mature cells are
neither lysed nor readily killed. The bacterio-
lytic action of penicillin upon sensitive or-
ganisms is greatest at the maximal rate of
multiplication.
Host defenses play a significant role in the
clinical efficacy of antibiotics. Here too,
however, the precise mechanisms still await
clarification. It has been reported, for ex-
ample, that prior treatment of bacteria with
penicillin or with streptomycin sensitizes the
organisms to phagocytosis (Linz, 1953). The
fact that chlortetracycline, oxytetracycline,
and chloramphenicol are therapeutically
effective at bacteriostatic rather than bac-
tericidal concentrations implicates the host
defense mechanisms in clinical medicine.
These observations were sunmiarized by
Eagle and Saz (1955).
Chain and Florey (1944) divided all the
antibiotics into two groups: (1) those that
react with the protoplasm of the cell, thus
killing l)otli microbial and animal cells,
comparable to the action of chemical anti-
septics; and (2) those that I'eact with sul)-
stances ha^•ing a specific significance in the
growth of the bacterial cell. Some of the anti-
})iotics were found to be largely growth in-
hibiting and have therefore been designated
as "bacteriostatics." With the broadening
knowledge of antibiotics, this classification
became too limited in scope.
It has also been suggested that the chemo-
therapeutic potentialities of antibiotics may
be measured by their effects on bacterial
respiration. If the latter is stopped by the
addition of an antibiotic in dilution of 1:
1000, the organisms may be said to have
been killed; such an antibiotic would there-
fore be toxic to animal tissues. If, however,
the antibiotic produces little or no effect on
the respii'ation of bacteria, the probability
was suggested that the antibiotic might pos-
sess chemotherapeutic possibilities.
The antibiotics produced by actinomy-
cetes were shown to affect the growth of
certain bacteria, such as B. inycoides, in the
following manner: Cell division is delayed;
the cells become elongated, reaching enor-
92
NATURE, FORMATION, AND ACTIVITIES
mous size and assuming most peculiar forms ;
spore formation is repressed; delayed non-
spore-forming variants are produced with a
modified type of growth on nutrient media.
The cells of bacteria subject to the action of
streptothricin are greatly enlarged as a re-
sult of incomplete fission.
Action of Specific Antibiotics
Streptomycin
The mode of action of streptomycin upon
bacteria has received much consideration.
Some of the observations appear to be
unrelated. Macheboeuf (1948) reported inhi-
bition by the antibiotics of dephosphoryla-
tion of mononucleotides and depolymeriza-
tion of nucleic acids. Wight and Burk (1951)
reported inhibition of oxygen consumption
of resting cells of E. coli on various substrates
and an inhibitory effect of dihydrostrepto-
mycin upon pyruvate fermentation by E.
coli (Fig. 4). On the other hand, stimulation
by streptomycin of oxygen consumption of
E. coli with some of the same substrates has
200
TIME-HOURS
Figure 4. Effect of streptomycin on the oxygen consumption involving oxalacetic acid and pyruvic
acid. (Reproduced from Wight, K. and Burk, D. Antibiotics & Chemotherapy 1: 380-38G, 1951.)
MODES OF ACTION OF ANTIBIOTICS
93
also l)eeii suggested (Wasserman, 1053).
Zeller (1953) observed an inhibition of
diamine oxidase in mycobacteria and as-
cribed this to the digiianidine residue of the
antibiotic.
According to Geiger (1947), the increased
ability of E. coli cells, first permitted to act
on fumarate or other carbon compounds,
to oxidize amino acids is nullified by strepto-
mycin. Yoshida and Sevag (1958) suggested
that streptomycin interferes with the incor-
poration of phosphate by E. coli cells. The
mechanism of action of streptomycin on
bacteria was described by Linz (1948) as
consisting first of the absorption of strepto-
mycin by the bacterial cells, later followed
by the action of the antibiotic on the essen-
tial SH groups concentrated in enzymatic
complexes.
Lightbown (1954) reported that Pseudo-
monas aeruginosa produced a highly acti\-e
antagonist of streptomycin and dihydro-
streptomj^cin activity. The antagonist has
been identified as a mixture of 4-hydroxy-
quinoline-N-oxides with alkyl chains of
seven, eight, and nine carbons. It inhibits
respiration via the cytochrome system
(Lightbown and Jackson, 1956). Hancock
(1960b) measured the uptake of radioactive
streptomycin by cells of various bacteria.
When growth is inhibited, the radioactivity
taken corresponds to 0.06 ng per mg of
cells for B. megaterium, 1.5 mS for Staph,
aureus, and 1.6 Mg for B. subtilis. The uptake
by the first corresponds to about 5 X 10^
molecules of streptomycin per cell. In the
case of streptomycin-resistant strains, the
uptake is then 1 per cent of this amount.
When the growth-inhibitory effect of strep-
tomycin (25 ij.g per ml) on B. subtilis is
antagonized b}' 2-heptyl-4-hydroxy(iuino-
line-X-oxide (0.4 fig per ml), the uptake of
radioactivity is only about 25 per cent of
that in the absence of the antagonist. Ac-
cording to Hancock, "The uptake of strep-
tomycin into the cell is associated with
aerobic respiratory processes and is reduced
when these are depressed."
Umbreit (1949) and Oginsky (1953) sug-
gested that streptomycin inhibits terminal
oxidation in sensitive organisms by inhibit-
ing the condensation of pyruvate and
oxalacetate to 2-phospho-4-carboxyadipic
acid. This concept has been questioned by
Paine and Clark (1954), who reported that
a strain of Staph, aureus, which under anaero-
bic conditions simply reduced pyruvate to
lactate, was readily killed by the antibiotic.
It has also been observed that the antibiotic
may mhibit, stimulate, or have no effect on
oxygen uptake, depending on the carbon
source on which the organism was grown.
The killing action of streptomycin was cor-
related with the metabolic activity of the
organism but not with its respiration. The
role of streptomycin in the inhibition of
synthesis of enzymes responsible for con-
tinued oxidation in the cells was also sug-
gested.
Katagiri et al. (1960b) could not demon-
strate any inhibiting effect of dihydro-
streptomycin on the oxalacetate-pyruvate
condensation reaction in E. coli, nor was the
anaerobic production of lactate from glucose
inhibited. Anaerobic fermentation of P3^ru-
vate by cells grown in a casein hydrolysate
medium w^as stronglj^ inhibited by dihydro-
streptomycin; the inhibiting efTect of dihy-
drostreptomycin on the phosphoroclastic
split of pyruvate into acetate and formate
could also be demonstrated.
However, although the oxidation of pyru-
vate, acetate, and dicarboxylates by washed
cells of E. coli was affected only slightly by
dihydrostreptomycin or chloramphenicol,
the oxidation of these carbon compounds
was very sensitive to oxytetracj^cline. Dihy-
drostreptomycin showed an accelerating
efTect on aerobic decomposition of a-keto-
glutarate in the presence of appropriate
carbon and nitrogen sources by washed,
dried, or dry ice-treated cells of E. coli or
94
NATURE, FORMATION, AND ACTIVITIES
Ps. flnorescens; no accelerating effect of
chloramphenicol was observed (Katagiri
daL,19G0a).
Evidence has recently been presented
(Anand and Davis, 1960) which indicates
the possibility that streptomycin is lethal to
sensitive cells because of interference with
cell membrane synthesis. This results in
drastic changes in the selective permeability
of the membrane, with attendant leakage of
important constituents such as nucleotides
from the cell to the outside. Inward permea-
bility is also affected. Here again, however,
one must be absolutely certain that the
effects observed are primary effects of the
antibiotic and not due to defects associated
with dead and dying cells. Hurwitz and
Rosano (1960), basing their results upon the
observation that chloramphenicol inhibits
killing by streptomycin when l)oth drugs
are added simultaneously, suggested that a
streptomycin-induced specific protein syn-
thesis precedes killing of the cells by strep-
tomycin. The reverse of this is that cells
lacking the potential for this induced protein
synthesis cannot be killed by this antibiotic.
The latter type cells presumably would in-
clude both host cells and streptomycin-
resistant bacterial mutants.
By using radioactive amino acids for the
aerobic growth of M. tuberculosis (BCG),
Stachiewicz and Quastel (1959) demon-
strated that glycine and serine showed dis-
tribution of radioactivity in a large number
of amino acids of the microbial protein; on
the other hand, the use of radioactive alanine
and valine resulted in a protein in which
only these two amino acids were radioactive.
The effect of dihydrostreptomycin, in con-
centrations at which it exerts inhibitive
effects, upon the aerobic growth of M.
tuberculosis (BCG) consists in the inhibition
of protein synthesis. Xo effect was obtained
on labelled amino acid incorporation in E.
coli, but there was an effect in M. phlei
(see also Erdos and Ullmann, 1959).
According to Erdos et al. (1960), strepto-
mycin inhibits the incorporation of tyrosine
into proteins of a sensitive strain of a sapro-
phytic Mycobacterium; on the other hand,
the incorporation of tyrosine into resistant
strains, as well as in a somewhat dependent
strain, w^as increased by the antibiotic. RNA
synthesis, either in resistant or sensitive
strains, was not influenced by streptomycin.
The antibiotic was said to inhibit transport
of amino acids from RXA to proteins.
According to Shaw et al. (1960), manno-
sidostreptomycinase activity was inhibited
by the addition of Fe++, Ni++, Zn++, or
Cr''+ at the beginning of the fermentation.
The possible inhibition of mannosidostrep-
tomycinase synthesis by Fe++ was suggested.
Cii'^^ added after 144 hours to fermentation
l)roths containing 50 to 60 ppm of Fe+''"
rex'ersed the inhibition of enzyme activity
because of the presence of the ferrous ion.
Streptomycin production was reduced mark-
edly by Ni+"^ or Cu+"'", but was reduced only
slightly in the presence of both Xi++ and
Fe++. Further information on streptomy-
cinase is given by Sakakibara (1951) and on
the microbial degradation of streptomycin,
by Pramer and Starkey (1951) and Klein
and Pramer (1960).
Henry and Hobby (1949) reported that
streptomycin activity varies directly with
concentration of the antibiotic, and inversely
with hydrogen ion concentration. Strepto-
mycin is both bacteriostatic and bactericidal,
depending on various factors. The bacterio-
static action of streptomycin appears after a
certain lag period. This action is antagonized
by most inorganic and organic salts and by
many sulfhydryl compounds. Streptomycin
was believed to inhibit the metabolism of
carbohydrates, ribonucleic acid, benzoic
acid, and amino acids. It was suggested that
the process of cell division or the synthesis
of protoplasm is blocked by interference by
streptomycin wdth one or more enzyme
systems essential to these functions. Han-
MODES OF ACTION OF ANTIBIOTIC'S
95
cock (l!t()Oa) examined in detail the factors
invol\'ed in bactericidal action of strepto-
mycin upon Staph, aureus.
The ability of streptomycin to destroy
chloroplasts was first demonstrated by von
Eiiler (1947). ProvasoU et al. (1951) have
shown that bleached races of the flagellate
Eiiglena gracilis can thus be obtained. In
the absence of an added energy supply,
streptomycin also acts as an inhibitor of
biosynthesis by protoplast lysates and ghosts
of E. coli (Reiner et al., 1958).
The antibacterial activity of streptomycin
can be largely or completely neutralized or
antagonized by an anaerobic environment
and by various chemical agents. These in-
clude glucose and certain other sugars, cer-
tain sulfhydryl compounds, and ketone
reagents. The effect of cysteine, of cevitamic
acid, and of ketone reagents in inhibiting
streptomycin activity may l)e associated
with the blocking of the active grouping in
the molecule of streptomycin.
Streptomychi is adsorbed on the bacterial
surface, resulting in a reduction of th(^ net
negative charge, a change which affects the
electrophoretic mobility of some of the cells
(McQuillen, 1951). The effect of streptomy-
cin on the intermediary carbohydrate metab-
olism, especial!}^ to the acetate or pyruvate
stage, of the bacteria and on amino acid
utilization has been variously postulated
(UiMarco, 1958). The bacterial cell tries to
escape the antibacterial action of the anti-
biotic, which was believed to be due to
interference with the synthesis of cell-wall
material, by the synthesis of a different kind
of cell-wall substance. Streptomycin-re-
sistant mutants of bacteria may show new
deficient characters (Kohiyama and Ikeda,
19G0).
Slreptothricin
Streptothricin and streptomycin are both
active against gram-posit i\'e and gram-
negative bacteria, ])ut they differ in their
antibiotic spectra and in their toxicity to
animals, the first exerting a delayed toxic
action. They are soluble in water but insolu-
ble in alcohol and other organic solvents.
Both have an optimal reaction at pH 8.0,
and both are repressed by glucose and by
acid salts. They are both stable compounds
and are highly resistant to the action of
microorganisms. However, the two sub-
stances can be differentiated in their rela-
tion to cysteine. Streptomycin becomes in-
activated by the addition of 3 to 5 mg of this
compound to 100 jug of the antibiotic,
whereas streptothricin is not affected by
cysteine. Streptothricin is also active upon
fungi, but streptomycin is not.
Chloramphenicol
Chloramphenicol is structurally related to
p-nitrophenylserinol. Numerous attempts
were therefore made to re^•erse the bacterio-
static activity of this antibiotic by aromatic
amino acids. Woolley (1950) reported that
the growth-inhibitory activity of chloram-
phenicol at a concentration of 1 Mg per ml on
E. coli was completely reversed by the addi-
tion of phenylalanine to the medium at a
concentration of 500 ^g per ml. Xo reversal
of acti^'ity was obtained, however, when the
chloramphenicol concentration was greater
than 2 ^g Pf'i" n^l- Similar I'esults were ob-
tained with tyrosine and tryptophan. With
Lactobacillus casei, only phenylalanine was
effective in reversal and, as with E. coli,
reversal occurred only within a narrow range
of concentrations of the antibiotic. Mentzer
et al. (1950) reported the antagonistic ef-
fects of glycine upon the inhibition of E.
coli by chloramphenicol; aspartic acid and
serine also showed some antagonistic poten-
tialities; tryptophan was without effect.
Truhaut et al. (1951) reported that chlor-
amphenicol inhibited both synthesis and
breakdown of tryptophan or its precursors
(anthranilic acid, indol) by Salmonella ty-
phosa.
90
NATURE, FORMATION, AND ACTIVITIES
The most dramatic effects of chloi-amphen-
icol have been reported on protein syn-
thesis and in both bacterial and mammalian
tissues. Earlier work (Hahn and Wisseman,
1951; Saz and Marmur, 1953) had shown
that the antibiotic inhibited the synthesis of
inchiced enzymes mediating the oxidation of
lactose and the phosphorylation of gluconic
acid in E. coli. No effect of the antibiotic
was observed in cells previously adapted to
lactose and gluconate oxidation. Gale and
Folkes (1953) and Wisseman et al. (1954)
found that chloramphenicol, at growth-
inhibitory concentrations, specifically and
immediately inhibited protein synthesis in
bacterial cells, whereas formation of ribo-
nucleic acid (RNA), deoxyribonucleic acid
(DXA), and polysaccharide was inhibited,
if at all, only at much higher levels of the
antibiotic.
Since these original ol)servations were
made, numerous confirmatory reports have
followed. There seems little doubt that, upon
addition to bacterial cultures, chlorampheni-
col abruptly inhibits protein synthesis and
that presumably this inhibition accounts for
its antibiotic activity. Hopkins (1959), for
example, demonstrated that chloramphenicol
inhibits the incorporation of amino acids into
protein of calf thymus nuclei but has no
effect on the uptake of leucine by nuclear
RXA (Fig. 5). It must be noted, however,
that the precise locus of chloramphenicol in-
hibition of protein synthesis remains to be
delineated.
According to Brock (1901), chlorampheni-
col antagonizes the action of antibiotics
which act on growing cells, such as penicillin
and streptomycin; however, it exerts an ad-
ditive effect upon antibiotics which also in-
hibit protein synthesis, such as the tetra-
cyclines and erythromycin. Chloramphenicol
inhibits the incorporation of radioactive
amino acids into protein. It does not inhibit
the activation of amino acids or transfer of
amino acids to soluble RXA, but it prevents
some step in their transfer from soluble RNA
to protein. A similar behavior of the tetra-
1500
1000
500
250
/^
RNA(0067M choromphenicol)
>,
Protein (0067M choramphenicol)
Time (minutes)
Figure 5. Effect of chloramphenicol upon the uptake of leucine-1-C'^ (5.3 ^c per /xmole) into ribo-
nucleic acid and protein of calf 1h>nius nuclei. (Reproduced from Hojjkins, J. W. Proc. Natl. Acad.
Sci.U.8. 45:1461-1470,1959.)
MODES OF ACTION OF ANTIBIOTICS
97
cyclines, erythromycin, and puromyciu ex-
plains the above additive effect.
According to Korotajev (1959), chloram-
phenicol does not influence the aerobic oxi-
dation of pyruvic acid by resting cells of
E. coli but inhibits anaerobic pyruvate
metabolism. Pyruvate consumption by rest-
ing cells oi Shigella flexneri is inhibited under
aerobic conditions and under conditions of
limited oxygen supply. Schneierson et al.
(1960) emphasized that chloramphenicol, in
concentrations that fail to inhibit growth, is
capable of depressing pigment synthesis by
Pseudomonas aeruginosa. The antibiotic
exerts its action by interfering with biosyn-
thesis of the pigment by the organism, no
reducing effect upon pigment already formed
being demonstrated. A single exposure to
chloramphenicol resulted in a complete and
permanent loss of the ability to produce the
pigment in three of the four strains tested.
Tetracyclines
It is generally assumed that chlortetra-
cycline, oxytetracycline, and tetracycline,
because of their close chemical i-elation-
ship, have similar, if not identical, modes of
action (Hahn, 1958). Tetracycline is the
parent compound, chlortetracycline has a
chlorine atom in the unsaturated (D) ring,
and oxytetracycline instead of a hydrogen
has a hydroxyl group in the B ring. The
antibiotic spectra of all three are similar
(Love et al., 1954), and mutual cross-resist-
ance has been found (Wright and I'^inland,
1954). There are, however, reports of ([uali-
tative as well as (luantitative differences in
their inhil)itory effects, and therefore the
possibility must be entertained that differ-
ences exist in their modes of action. Ciuil-
laume and Osteux (1959) have shown that
chlortetracycline inhibits two different enzy-
matic systems in Proteus mirahilis, that ot"
oxidation of glucose, pyruvic acid, and ace-
tate, and that of the citric acid cycle.
The tetracyclines affect oxidation and fer-
mentation in susceptible bacteria; inhibi-
tion of protein synthesis has been reported;
and finally, by virtue of the strong chelating
properties of all three antibiotics, interfer-
ence with \-arious cellular and enzymatic
processes has been suggested as a possible
mechanism of inhibition.
The inhibitory activities of the tetracy-
clines on various oxidative properties of
whole bacteria have been noted. In the in-
terpretation of these data, the difficulty of
distinguishing between primary action and
secondary effects on dead or dying cells
must again be considered. For example, it
has been reported (McCullough and Beal,
1952) that chlortetracycline at concentra-
tions of 250 to 500 ng per ml inhibits oxida-
tion of glucose, pyruvate, fructose, xylose,
and trehalose by Brucella (Fig. 6). Oxidation
of tricarboxylic acid cycle intermediates by
E. coli, Ps. aeruginosa, and Pr. vulgaris was
similarly inhibited by high concentrations
of chlortetracycline. Oxytetracycline in
analogous concentrations was also found to
inhibit these oxidations (Wong et al., 1953).
The tetracyclines were also found to cause
serious derangement of cellular processes
leading to protein synthesis and nucleic acid
formation.
According to Bernheim and De Turk
(1952), chloramphenicol, streptomycin, and
the tetracyclines inhibit the oxidation and
to a lesser extent the deamination of phen-
ylalanine, tyrosine, and phenylserine by a
strain of Ps. aeruginosa. They also inhi})it
the oxidation of succinate and certain other
dicarboxylic acids. It was suggested that
these antibiotics interfere with the formation
of compounds which may be necessary for
the assimilation of ammonia or, in the case
of phenylalanine, for its oxidation. Bern-
heim (1954) has further shown that Ps.
aeruginosa on contact with succinate pro-
duces a cell constituent which can be utilized
for the synthesis of an enzyme that oxidizes
benzoic acid. The above antibiotics inhibit
98
NATURE, FORMATION, AND ACTIVITIES
Q800
UJ
Z)
z
(J 6001-
>
X400
O
o
^200
Ld
CHLORTETR. (10 pg)
o •
/ ^A ___^D DIHYDROSTREPT. (1000 /ug)
oy/'
/ x°
/5/
/7
nt_L
_0— • CHLORTETRA. (10 /jg)
PLUS DIHYDROSTREPT.
(lOOO^g)
i — I 1 — i — I 1 I I I I I
^0 2 4 6 8 10 12
TIME IN HOURS
Figure G. Influence of time on the effect of oxygen uptake by Br. melitensis by chlortetracy-
cline, dihydrostrei^tomycin, and chlortetracycline plus dihydrostreptomycin. Glucose added at zero
hour. (Antil)iotics in presence of cells for 1 hour at room temperature and 35 minutes at 37°C be-
fore addition of glucose.) Endogenous respiration values subtracted. (Reproduced from McCullough,
N. B. and Beal, G. A. J. Infectious Diseases 90: 196-204, 1952.)
the formation and utilization of the particu-
lar constituent.
In Staph, aureus, protein synthesis, as
measured by the incorporation of isotopi-
cally labelled glutamate, was inhibited by
concentrations (0.2 to 0.4 /xg per ml) of
chlortetracycline and oxytetracycline which
were lower than the growth inhibitory levels
(O.o to 1 .0 /ig per ml). A concentration of oO
to 500 iJLg per ml was refiuired for inhibition
of nucleic acid synthesis, free glutamate
incorporation, or glucose fermentation (Gale
and Folkes, 1958). Glutathione synthesis by
suspensions of Proteus was iminhibited even
by high concentrations of oxytetracycline
and chlortetracycline (Samuels, 1953).
Chlortetracylcine in low concentrations
inhibited the synthesis of RNA and DNA by
Lactobacillus casei, and folic acid and vitamin
Bi2 were reported to prevent this inhibition
(Rege and Sreenivasan, 1954).
Reports have indicated that the tetracy-
clines inhibit Aarious enzymatic reactions
and growth of sensitive organisms by inter-
fering with inorganic ion metabolism.
A highly purified nitroreductase isolated
from a chlortetracycline-sensitive E. coli
was found to be markedly sensitive to pre-
cisely those concentrations of the antibiotic
which were growth inhibitor^^ Resolution of
the enzyme complex indicated that the re-
ductase was a manganoflavoprotein and
that chlortetracycline inhibited the nitro-
reductase, presumably as a result of its ca-
MODES OF ACTION OF ANTIBIOTICS
99
pacity to chelate or otherwise bind the
functional Mn. The locus of inhibition was
shown to be at the level of the reoxidation of
reduced flavin mononucleotide. It was fur-
ther observed that the nitroreductase was
indeed a portion of the electron transport
system of the cell. Thus, inhibition of this
aspect of the metabolism of the cell could
conceivably account for the antibiotic prop-
erties of chlortetracycline. It should be noted
that the system described was 100-fold more
sensitive to chlortetracycline than to oxy-
tetracycline and tetracycline. It is of interest
that a similar system isolated from a chlor-
tetracycline-resistant mutant derived by
serial passage from the parent sensitive
strain was resistant to chlortetracycline.
The presence of firmly bound metal was
postulated, and thus the resistant extract
successfully competed with the chlortetra-
cycline for essential cation (Saz et al., 1956;
Saz and Martinez, 1958, 1960).
Weinberg (1954) investigated the effects
of cations on oxytetracycline inhibition of
Ps. aeruginosa and other cells. The various
cations had profound effects on the toxicity
of the antibiotic for the cells. Some metals
were antagonistic to oxytetracycline, while
others potentiated the effects. Rokos ct al.
(1958) have shown that the inhibiting effect
of sodium citrate on the action of chlortetra-
cycline on lipase and D-amylase can be ex-
plained by the removal of the calcium ion
from the system.
The additive effects of chloramphenicol
and tetracyclines were discussed l)y (iale
and Folkes (1953) and Ciak and Hahn
(1958).
Erythromycin
Erythromycin is active largely upon gram-
positive cocci and upon rickettsial organisms.
It is either bacteriostatic or bactericidal,
depending on the sensitivity of the organism
and concentration of the antibiotic. It is
active upon multiplying cells but not upon
fully grown ones (Haight and Finland, 1952).
Neomycin, Kanamycin, and Other Memhers
of the Neomycin Complex
Gale (1952) made a comparative study
of the effect of neomycin and various other
antibiotics on amino acid assimilation by
Staph, aureus. The accumulation of lysine
and glutamic acid within the cells was not
affected. Protein synthesis was interfered
with, however, but not nucleic acid synthe-
sis. Ihe action of neomycin was similar, in
this system, to that of chloramphenicol.
According to Tsukamura (I960), kana-
mycin inhil)its the incorporation of P^^
into the nucleic acid and protein tractions
and the incorpoi'ation of S^^ into the tri-
chloroacetic acid-soluble and protein (tri-
chloroacetic acid-insoluble) fractions of the
parent sensitive strain of Mycohacterium
avium, but not of the kanamycin-resistant
strain. The ratio of RNA to DXA is higher
in the kanamycin-resistant strain than in the
parent sensitive strain.
Novoljiocin
The effect of novobiocin on the turbidity
and viable cells of a growing culture of E. coli
is shown in Fig. 7. In the case of Staph, au-
reus, it causes the accumulation of N-acetyl-
amino sugar. The fact that the L-isomer
exhibits a totally different kind of antibacte-
rial activity, showing a different antibiotic
spectrum and a different dose response
correlation, was interpreted by Hahn (1958)
as illustrating the problematic nature of
structure-activity relationship.
Puromycin
Puromycin was found (Creaser, 1955) to
be an inhibitor of induced enzyme (i3-galac-
tosidase) synthesis in Staph, aureus. Chemi-
cally, this antibiotic consists of an amino-
nucleoside linked to an amino acid.
100
NATURE, FORMATION, AND ACTIVITIES
12 5
HOURS
Figure 7. Effect of novol)iocin (1 mg ])er ml) on turbidity and vial)le count of growing cells of E.
coli; = viable count; = turbidity. (Reproduced from Brock, T. D. and Brock, M. L. Arch.
Biochem. Biophys. 85: 176-185, 1959.)
Actinomyciyi
Actinomycin is a bacteriostatic agent,
active primarily upon gram-positive bac-
teria and to a lesser degree upon gram-nega-
tive bacteria. It is also active upon certain
neoplasms. It is extremely toxic to animals,
a factor which limits its utilization in the
therapy of infectious diseases and certain
forms of cancer. One milligram of actinomy-
cin given to mice, rats, or rabl:)its intra-
venously, intraperitoneally, subcutaneously,
or orally is lethal for 1 kg weight of the
animals. Doses as small as 50 /xg per kg
injected intraperitoneally daily for 6 days
cause death accompanied by severe gross
pathological changes, notabl.y a marked
shrinkage of the spleen. Actinomycin is
rapidly excreted from the body.
Kawamata and Imanishi (1960) suggested
that the carcinogenic effect of actinomycin
may be due to its interaction with deoxyri-
bonucleic acid.
Foley (1955) found that the action of ac-
tinomycin D upon bacteria consists in inter-
ference with pantothenate utilization. This
phenomenon could not be confirmed by
Slotnick (1957) for B. subtilis. Slotnick
(1960) demonstrated that actinomycin D
suppresses the assimilation of ammonia by
B. suhtilis and inhibits completely the for-
mation of certain inducible enzymes. He
concluded that this antibiotic interferes in
some reactions leading to protein synthesis.
Kirk (1960) studied the metabolic reac-
tion between actinomycin D and DXA and
found that the antibiotic has no significant
inhibitory effect on the polynucleotide phos-
phorylase of Staph, aureus, that it inhibits
the incorporation of radioactivity into the
HC104-insolul)le fraction when P^-.j^Q^y-
adenosine triphosphate is incubated with a
crude preparation of the DNA "polymer-
ase" enzyme isolated from Escherichia coli,
and that it inhibits the transformation of
MODES OF ACTION OF ANTIBIOTICS
101
H. injiucnzac from streptomycin sensitivity
to streptomycin resistance when it is added
to high concentrations of transformhig DXA
from a resistant strain.
Kersten (1961) recently reported on the
inhibition of the growth inhibiting effect
of actinomycin upon Ncurospora crassa
and Streptococcus faecalis l)y DNA, RNA,
and some of their degradation products
(purine moieties). The binding of actino-
mycin and nucleic acids was demonstrated
l)y changes in the ahsorption of spectra of
actinomycin.
The effect of actinomycin upon the ana-
erobic carbohydrate metabolism of Candida
albicans has been studied l)y l^rave (1959).
Azasenne and DON
Azaserine (o-diazoacetyl-L-serine) and
DON (6-diazo-5-oxo-L-norleucine) inhibit
the growth of E. coli in a synthetic medium.
They interfere with the incorporation of gly-
cine and formate, y)ut not of adenine, into nu-
cleic acids. According to Levenberg et al.
(1957), they interfere with the biosynthesis
of inosinic acid, behaving as competitive in-
hibitors of glutamine. The action of DON
upon the growth of E. coli, unlike that of aza-
serine, is not antagonized by adenine, gua-
nine, hypoxanthine, and the corresponding
nucleosides (Aiaxwell and Nickel, 1957).
Chain (1958) also demonstrated that aza-
serine interferes with the synthesis of the
purine ring system, thus affecting the forma-
tion of nucleotide. In the synthesis of ino-
sinic acid by cell-free pigeon liver extracts,
azaserine inhibits the formation of formyl-
glycinamide-ribotide and glutamine (in the
presence of adenosine triphosphate), appar-
ently acting as a specific antimetabolite to
glutamine, to which it is structurally related.
The amoebicidal action of azaserine was
investigated by Nakamura (1956).
Polijenes
Among the antifungal agents, nystatin, a
polyene antibiotic, has received the greatest
attention. This is a tetraene compound with
a diene unit, a carbonyl, and a primary
amino group. It inhibits the growth of most
fungi, in concentration of 1 to 10 /ig per ml,
but has no effect upon actinomycetes, bac-
teria, and rickettsiae. It is more effective
upon the mycelium than upon the spores of
the fungi. It is primarily fungicidal and its
action is irreversible. According to Lampen
et ol. (1957), nystatin inhil)its the endoge-
nous respiration and the aerobic and anaero-
bic utilization of glucose and certain other
carbon sources by yeasts and other fungi.
Low levels of the antibiotic show increased
oxygen consumption; high levels show an
initial stimulation, followed by abrupt cessa-
tion of metabolism, when the cells are no
longer viable. Other polyenes (amphotericin)
inliil)it glycolysis. Nonpolyene antifungal
agents (cycloheximide) have no such effect.
The conclusion was reached that nystatin
blocks a reaction of general metabolic signifi-
cance to fungi. I'urther information on the
mode of action of nystatin is found in the
work of Sutton et al. (19()0), Marini et al.
(1960), Tape et al. (1960), and Horvath and
Szentirmai (19()0).
Drouhet el al. (19C)0) studied the effect of
amphotericin B on the growing phase of
yeasts. It produces an inhibition of the
synthesis of proteins, ribonucleic acid,
carbohydrate, and polyphosphate reserves.
The disturbance of phosphorus metabolism
is related to the stimulation of endogenous
or exogenous oxidations. The action of other
polyenes on respiration is distinct from that
of other antifungal antibiotics such as
cycloheximide. Amphotericin B produces an
increase in Oo uptake by resting or growing
cells of C. albicans in the presence or the
absence of carbon substrate. This effect is no
longer observed on yeasts which have been
washed after contact with the antibiotic. It
was suggested that the washing eliminates
the products responsible for the increase in
O2 uptake, products released by alteration
of cell permeability. The mode of action of
102
NATURE, FORMATION, AND ACTIVITIES
other polyenes was studied b}- Henis and
Grossowicz (19G0).
Other Antibiotics
According to Aizawa (lOoo), both the
respiration and the adaptive oxidation of
mannose and galactose by Candida albicans
are inhibited by aureothricin, candimycin,
eurocidin, and trichomycin; they are not af-
fected, however, by penicillin G, chlortetra-
cycline, oxytetracycline, chloramphenicol, or
dihydrostreptomycin.
Abraham (1959) defined some of the main
features of bacterial metabolism that are
open to selective attack by antibiotics. The
formation of the cell walls in liacteria and
actinomycetes differs from that of other
kinds of cells, although the precise nature
of the reactions that are inhibited has yet
to be elucidated. The same is true of certain
highly specific properties of the bacterial cell
which make them sensitive or resistant to a
given antibiotic.
Dependence of JVIicroorganisnis upon
Specific Antibiotics
Among the phenomena related to the
antibacterial properties of streptf)mycin,
the development of resistance and of depend-
ence is of particular interest. The problems
related to resistance will be considered in
detail in Chapter 10. The problem of de-
pendence was first observed by Miller and
Bohnhoff (1950). Each of 18 strains of menin-
gococci yielded two variants. One variant,
designated as A, grew in large yellowish
colonies on streptomycin-free and strepto-
mycin-containing media; it retained the
original virulence for mice. The other, E,
appeared in greatest numbers in concentra-
tions of 100 and 400 mg per ml of strepto-
mycin. Its colonies varied in size and color,
depending upon the concentration of the
antibiotic in which they were developed,
and were dependent on the pi'esence of
streptom3^cin for multiplication. This de-
pendence was demonstrable not only in
vitro but also in vivo, since the organism
exhibited no \'irulence for mice unless strep-
tomycin was administered to the animals
after infection. Both variants retained the
characteristic sugar fermentations and type
specificity of the parent strain.
The production of streptomjTun-depend-
ent strains has also been reported for a
number of other bacteria, including E. coli,
Ps. aeruginosa, B. subtihs, Staph, aureus,
and xl/. tuberculosis.
In a study on the distribution of depend-
ent cells of E. coli in a broth culture of this
organism, Iverson and Waksman (1948)
found that one dependent cell was present
among each 1.5 billion normal sensitive cells.
Streptomycin, and not any accompanying
impurity, was reciuircd for growth of the
dependent organisms. IMannosidostrepto-
mycin and dihydrostreptomycin were also
effective in favoring growth of dependent
strains; but streptomycin that had been
inacti\'ated by cysteine and hydroxylamine
was ineffective, as were streptidine and
streptamine.
Xewcombe and Xyholm (1950) have
shown that streptom3Tin-dependent fcms
of E. coli differ among themselves not only
in the degree of their dependence but also
with regard to other compounds that have
the capacity to replace streptomycin. De-
pendent strains give double mutants, arising
from a second mutation at the original locus,
thus forming a continuous series with respect
to degree of resistance.
Szybalski and Cocito-\andermeulen
(1958) identified among the streptomycin-
dependent mutants of E. coli four nutritional
groups: (1) Growth is supported solely by
streptomycin, or by its dihydro, hj^droxy,
and desoxydihydro derivatives. (2) Catenu-
lin and neamine, the neomycin-related
antibiotics, can substitute for streptomycin.
(3) Streptobiosamine, a streptomycin deg-
MODES OF ACTION OF ANTIBIOTICS
103
radation product, forms a substitute for
streptomycin. (4) A group similar in proper-
ties to the first, but showing restricted
growth on appropriate mixtures of catenuhn
and streptidine or streptobiosamine; it pro-
duced frequent mutants which belonged to
the second class. Other substances related to
streptomycin or neomycin were either inac-
tive (streptamine, streptiu'ea, methylneo-
biosaminide) or inhibitor}^ (neomycin B and
C, kanamycin, streptidine) to all the classes
of streptomycin-dependent mutants. Strep-
tomycin and catenulin, when present alone
in concentrations of 5 to 100 yug per ml, gave
good growth of the second and third classes
of dependent mutants. A mixture of the two
antibiotics, however, in any proportion so
long as the concentrations surpassed 2 /ug per
ml, prevented growth of the organisms. The
dependent cells grown in the presence of one
antibiotic behaved as sensitive cells in
respect to the other, indicating diverse
mechanisms of growth-promoting action for
streptomycin and catenulin. Xeamine ex-
hibited similar nutritional incompatibility
with various streptomycins.
Hashimoto (1955) obtained a partially
streptomycin-dependent strain from the
parent strain of pneumococcus I by succes-
sive cultivation in media containing increas-
ing amounts of streptomycin. This strain
gave the same amount of bacterial growth on
media containing no streptomycin and on
media with 1 mg per ml of streptomycin,
but the growth was faster in the latter.
Crude deoxynucleic acid prepared from the
partially streptomycin-dependent strain
could bring about transformation of the
streptomycin-indifferent strain in similar
ratio. The deoxynucleic acid was believed
to have a factor that controlled the depend-
ence on 50 to 100 /xg of streptomycin and
that was imparted to the streptomycin-
sensitive strains.
Streptomycin-dependent cultures also
show back mutations. Yegian and Budd
(1951) obtained from such strains of Myco-
hacteriirm ranae either parent sensitive cul-
tures or streptomycin-resistant variants,
with no discernible change in colony mor-
phology.
Thind (1958) reported that extracts of
certain streptomycin-sensitive cultures of E.
coll produce a substance which can replace
streptomycin in supporting the growth of
streptomycin-dependent organisms.
Chapter 10
Development of Resistance
Natural and Acquired Resistance
The problem of increased bacterial resist-
ance to chemotherapeutic agents has been
the focus of special attention in recent years,
with increasing utilization of antibiotics for
the control of a variety of bacterial infec-
tions. The first two important antibiotics,
penicillin and streptomycin, have proved to
be of particular interest. They are similar
in some respects and different in others. Both
act primarily upon bacteria, have little
effect upon intracellular parasites (rickett-
siae), and act not at all or to only a very
limited extent upon fungi. They differ, how-
ever, in their respective antibacterial spectra.
Penicillin is active largely against cocci,
gram-positive aerobes and' anaerobes, and
spirochetes, but has only a limited effect
upon gram-negative rods and acid-fast bac-
teria unless used at high concentrations.
Streptomycin, on the other hand, is effective
against both gram-negati\'e and gram-posi-
tive bacteria, including the acid-fast organ-
isms, and is relatively less active against the
cocci and spirochetes than is penicillin.
Bacteria develop resistance to streptomycin
much more rapidly than to penicillin and
may lose that resistance much more slowly
(Finland, 1956).
Different strains of the same species ex-
hibit considerable variation in their sensi-
tivity to a given antibiotic. Staphylococci
show wide ranges of sensitivity to penicillin.
The sensitivity of Mycobacterium tubercu-
losis to streptomycin ranges from 0.1 to 12.5
/xg per ml, or 1 to 125. This natural variation
in sensitivity of a given organism is of great
practical importance from a chemotherapeu-
tic point of view, since it influences the
selection of the particular antibiotic for the
treatment of a given infection, and the con-
centrations to be used.
In addition to the natural variation in
sensitivity, a population of organisms origi-
nally sensitive becomes gradually more
resistant or "fast" to a given antibiotic on
continued contact with it, either in the test
tube or in the body of the host. This phe-
nomenon is not new in either bacteriology
or chemotherapy. It has long been observed,
for example, that upon repeated administra-
tion of a drug, the infecting organism be-
comes less susceptible to it. This decrease
in sensitivity has been assumed to be of two
kinds: (1) a reduction of the sensitive strains
with a selection of the naturally resistant
forms; (2) a change of the sensitive strains
into resistant ones.
Ehrlich and others reported, for example,
that the resistance of trypanosomes to atoxyl
and to dyes could l)e raised by gradut^lly
increasing the doses of the drug. A microbial
strain resistant to one type of compound
could still be sensitive to other agents. The
same organism could be made to develop
resistance against several substances, by a
series of treatments or adaptations. The
ability of various bacteria to become resist-
ant to sulfa drugs has also been well demon-
strated. When the bacteria are removed
from contact with the drug they become
sensitive again; the rate of loss of resistance
104
DEVELOPMENT OF RESISTANCE
105
varies with the organism, some losing their
resistance rapidly and others only very
slowly. Certain strains of bacteria ma_y not
become resistant to the drug at all. An
organism made resistant to one sulfa com-
pound was found to become resistant to
others.
With the introduction of antibiotics for
chemotherapeutic purposes, it was soon
evident that the problem of bacterial resist-
ance would eventually become of paramount
importance. It is sufficient to list here a few
of the recent contributions to this highly
important phase of chemotherapy. Further
information is given in the work of Ramsey
and Padron (1954), Katsunuma and Xaka-
sato (1954), Knight and Collins (1955), Jones
et al. (1956), Finland (1958), Chernomordik
and Kobeleva (1959), and numerous others.
The Oxford group of investigators (Chain
et al., 1940), in their first report on the use of
penicillin for disease control, noted a marked
increase in resistance of Staphylococcus
aureus to penicillin upon continued use. This
observation was soon confirmed by many
other investigators and was found to hold
true also for certain other organisms natu-
rally susceptible to penicillin. The occurrence
of the natural \'ariation in resistance of bac-
teria to penicillin was also soon recognized.
Bacteria acciuire resistance to penicillin when
cultivated in a medium containing gradually
increasing concentrations of the drug, pro-
vided these are kept below the level inhibit-
ing bacterial growth. The tolerance of a
strain of gonococcus was increased 350 times
and of meningococcus 130 times the concen-
tration originally permitting growth. The
increase in resistance of meningococci to
penicillin could also be brought about by
passage of the culture through penicillin-
treated mice. This increase in resistance was
not accompanied by the production of the
enzyme penicillinase, which has the capacity
of destroying penicillin.
Considerable variation has been reported
for the de\'elopment of resistance by bacteria
to streptomycin. Of particular importance
in this connection is the increased resistance
of M. tuberculosis isolated from a host that
has been treated with considerable ciuantities
of this antibiotic. Variation in sensitivity of
E. coli to streptomycin was found to range
from 0.3 to 3.0 ^ig per ml, with an a\'erage of
1 Mg pel' nil for nine strains. In the case of
seven strains of Proteus vulgaris, the varia-
tion was from 0.3 to 2.5 fxg per ml. Similar
variations were obtained for other bacteria.
A strain of Pr. vulgaris made resistant to
streptomycin showed only a slight increase
in resistance to streptothricin, a closely
related antibiotic.
Bacteria develop resistance to strepto-
mycin very rapidly. Among bacteria isolated
from the urinary tract, it was found that
only three to seven transfers were retiuired
to make strains of Pseudomonas resistant to
1000 /xg of streptomycin per ml, and between
four and se\^en transfers for Streptococcus
faecalis. Two strains of E. coli re([uired 7 to
12 transfers. .1. cwrogenes and colon-aeroge-
nes intermediate strains re(iuired 2 to 17
transfers. Proteus strains were sensitive to
3.1 to 0.2 /ug of streptomycin per ml, and
from 7 to 11 transfers were recjuired to make
them resistant to 1000 ^ug per ml. When
Proteus was grown in urine, 12 to 24 trans-
fers were reciuired to make the l)acteria
resistant to 1000 /ig of streptomycin per ml
of urine. Proteus splits urea, thus increasing
the alkalinity of the medium to more than
pH 8, which increases the activity of strepto-
mycin.
Streptomycin-resistant strains showed no
change in susceptibility to either penicillin
or to sulfonamides. This led to the suggestion
that a combination of streptomycin with one
of these drugs might prove effective. It was
possible to increase the resistance of gram-
negative bacteria to more than 50,000 Mg pf^i'
ml of streptomycin by passage through in-
creasing concentrations of this antibiotic. In
106
NATURE, FORMATION, AND ACTIVITIES
most iiLstances this resistance was increased
gradually. However, in some cases, there was
a sudden increase in resistance of isolated
colonies from relatively low values to more
than 50,000 Mg per ml. When a culture of
organisms was made resistant by exposure
to streptomycin in broth, some of the cells
showed marked pleomorphism and in some
instances underwent changes in biochemical
reactions. However, when resistant organ-
isms were obtained from a patient during or
after treatment, no morphological or cultural
differences were observed, as compared with
the sensitive strains isolated from the same
patient.
The development of resistance of fungi to
polyenic antifungal antibiotics has been
studied by Stout and Pagano (1955) and by
Pledger (1957). These authors showed that
by repeated transfer in the presence of poly-
enes the resistance of naturally sensitive
fimgi was only slightly increased. Pledger,
for instance, reported that after 15 to 38
transfers in the presence of candicidin, can-
didin, filipin, and nystatin, the resistance of
strains of ('. albicans and Sacch. ccrevisiae
increased only 1.5- to 8.8-fold.
Mechanism of Development of Resist-
ance
Various explanations have been suggested
for the development of resistance. These can
be summarized as follows: (1) Induced
resistance is due to the killing of sensitive
cells in a given bacterial population, which
permits the more resistant cells to grow
selectively. (2) The phenomenon of resist-
ance is due to the acquisition of new enzyme
systems or to new metabolic activities which
permit the organism to survive in spite of
the presence of the particular inhibiting
agent. (3) Certain treatments tend to reverse
the effect of resistance or to prevent its
occurrence altogether.
Davies, Hinshelwood, and Pryce (1944)
expressed the above concepts of the develop-
ment of adaptation of an organism to an
antibacterial agent as follows: (1) Adapta-
tion occurs by natural selection from an
initially heterogeneous population. (2) Adap-
tation occurs by the modification of the
individual cells, as a result of the establish-
ment in the cells of a mechanism alternative
to that normally in use, or to a quantitative
modification of the existing mechanism. (3)
Adaptation is a change in some center of
organization of the cell. When variations or
adaptive changes occur, there is an actual
modification of the character of the individ-
ual cells, although selection may be super-
imposed on this when modified and unmodi-
fied cells exist together. Postgate and
Hinshelwood (1946) found that most con-
sideration should be given to the hypothesis
that ciualitative and cjuantitative changes
occur in the cell enzymes in response to the
changed medium.
The development of resistance among bac-
teria sensitive to particular antibiotics, on
contact with such antibiotics, is sometimes
highly specific; those bacteria that become
resistant to one antil)iotic may still remain
sensitive to another. Staphylococci which
develop resistance to penicillin are not
affected in their sensitivity to streptomycin
or to various other antibiotics.
When an organism develops resistance to
one antibiotic and yet remains sensitive to
another, the modes of action of these two
compounds are assumed to be different.
According to Chain and Florey (1944), the
formation by a given organism of strains
resistant to dift'erent antibiotics serves to
emphasize "the great variety of ways hi
which the organization of the cell is open to
attack by chemical substances."
Bryson and Demerec (1955) analyzed the
phenomenon of the de^'elopment of resist-
ance to drugs; this was considered to be an
important aspect of the continuing process
of microbial evolution. The use of anti-
biotics resulted in the selection of new types
devp:lopment of resistance
107
of organisms by the principle of survival of
the fittest. These investigators considered
the changes leading to the development of
resistance not as primarily drug-induced but
rather as a result of spontaneously occurring
mutations, leading to modified biochemical
processes in the bacterial cell and thereby
yielding resistant strains. Two types of
resistance were recognized: natural and
acquired. The first occurs in a natural popu-
lation, among species or sti'ains that have
had no previous contact with a given anti-
biotic (Table 36). The second takes place in
a bacterial population that has been in con-
tact with an antibiotic; resistant cells emerge
from an originally sensitive population.
The emergence of antibiotic resistance was
regarded as essentially a phenomenon of
adaptation, of which two categories were
recognized: (1) genetic adaptation, in which
resistant mutants overgrow the population
under the selective effect of the antil)iotic;
(2) physiological, or phenotypic, adaptation.
in which cytoplasmic alterations (adaptive
enzyme formation) are induced by the anti-
biotic, so as to render some of the cells more
resistant without affecting the genetic ap-
paratus (see also Garrod, IQ.IO).
Some organisms, including resistant
species and naturally resistant strains, were
belie\'ed to possess cytochemical systems
that are not vulnerable to specific anti-
biotics. Degrees of resistance and sensitivity
were considered as relative. Two resistance
patterns were recognized: (1) the penicillin
or obligatory multistep pattern; (2) the
streptomycin or facultative one-step pattern.
The nature of the pattern permits prediction
of the pro})al)ility that resistance will develop
rapidly. Mutations to antibiotic I'esistance
were beliex'ed to include a wide variety of
types. Gain or loss of resistance, once muta-
tions to resistance have occurred, depends
primarily on selection, the presence or
absence of antibiotics playing a major part
(Bryson and Demerec, 1955).
Table 36
Approximate concentration required to produce detectable inhibition of cells in streaks of representative
bacterial species on gradient plates (Brj'son and Demerec, 1955)
Escherichia coti
Staph, aureus
Mycobacterium ranae
Bacillus megaterium
Drug
A'g/ml
Drug
Mg/ml
Drug
Mg/ml
Drug
Mg/ml
Bacitracin
700
Viomycin
80
Chlorampheni-
20
Viomycin
10
Viomycin
50
Licheniformin
50
col
Streptothricin
8
Netropsin
8
B
Streptothricin
15
Mycomycetin
3
Catenulin
6
Bacitracin
20
Thiolutin
15
Tetracycline
1.5
Penicillin
5
Netropsin
10
Isoniazid
5
Carbomycin
0.8
Streptothricin
4
My corny ce tin
10
Netropsin
4
O.xytetracycline
0.8
Neomycin
2
Streptothricin
4
Viomycin
2
Streptomycin
0.7
Streptomycin
2
Chlorampheni-
4
Catenulin
2
Erythromycin
0.2
Chlortetracy-
1
col
Streptom^'cin
0.6
Neomycin
0.04
cline
Streptomycin
3
Neomycin
0.5
0.xytetracycline
1
Thiolutin
3
Oxytetracy-
0.4
Chlorampheni-
1
Catenulin
1
cline
col
Neomycin
0.8
Mycomycetin
0.2
Oxytetracy-
0.5
Chlortetracy-
0.05
cline
cline
Chlortetracy-
0.3
cline
Penicillin
0.02
108
NATURE, FORMATION, AND ACTIVITIES
A growing culture may acquire resistance
to an antibiotic by selection of cells initially
less sensitive than the average, or l\y a
sudden modification of the properties of
indi\-idual cells, namely, by mutation. Ac-
cording to Demerec (1950), resistance by
Staph, aureus to streptomycin originates by
gene mutation. Differences in the de\-elop-
meiit of resistance to penicillin and to strep-
tomycin were observed by plating cultures of
various organisms in the presence of increas-
ing concentrations of either antibiotic. The
penicillin-resistant strains produced in the
first step were fairly uniform in their degree
of resistance; on the other hand, strepto-
mycin-resistant strains showed great varia-
bility, some highly resistant forms being
obtained in one step. It was suggested that
penicillin mutations may be a result of a
number of genes, whereas in streptomycin
mutations some genes are more effective
than others.
In the development of our knowledge of
resistance of microorganisms to antibiotics,
several phenomena have been recognized:
1. Microorganisms develop resistance to
antibiotics by different mechanisms; such
development of resistance takes place in
diJertnt ways, as in the case of penicillin
versus streptomycin type of resistance.
2. Some antibiotics show cross-resistance,
as in the case of tetracyclines and chlor-
amphenicol, and among members of the
neomycin group; others do not, as in the
case of penicillin and streptomycin, where
an organism developing resistance to one
antibiotic remains sensitive to another.
3. After continued use of a certain anti-
biotic, there is a gradual development of
resistance among the bacteria that formerly
were highly sensitive to this antibiotic. The
occurrence of staphylococci resistant to
penicillin has reached more than (30 per cent
in some hospitals. This is also true of other
bacteria and other antibiotics, as shown in
Table 37.
Table 37
Propensity of microorganisms to develop antibiotic-resistant
strains (Dowling et al., 1955)
Group
Microorganism
Rapidity of response to proper
antibiotic treatment
Frequency of appearance of resistant
strains in patients during treatment
1
Pneumococci
Meningococci
jS-Hemolytic streptococci
(excluding group D strep-
tococci)
Gonococci
Shigella
Hemophilus influenzae
Rapid
Seldom, if ever
2
Staphylococcus
Str. viridans
Enterococci
Proteus
Pseudomonas group
Coliform bacteria
Mycobacterium tuberculosis
Occasionall}^ rapid ; usually
slow or incomplete
Often
3
Brucella
Salmonella typhosa
Rickettsiae
Rapid; frequently followed
by relapse
Seldom, if ever
DEVELOPMENT OF RESISTANCE
109
4. l^y conilHiiing" two antibiotics, such as
streptomycin with penicilhn, or an antibiotic
and a chemical agent, such as streptomycin
with p-aminosahcyhc acid or isoniazid,
resistance to any one agent can be delayed
if not prevented.
5. Little is known concerning the mecha-
nism of de\'elopment of resistance. The fact
that treatment of sensitive cells with deoxy-
ribonucleic acid isolated from resistant cells
renders the sensitive cells resistant, and
similar observations, may suggest proper
approaches in o\'ercoming the development
of resistance.
The background of the problem of devel-
opment of resistance of microorganisms to
drugs, comprising both natural and acquired
resistance, has been further analyzed in
detail by Abi'aham (11)53), Schnitzer and
Grunberg (1957), DiAIarco (1958), and
others. Abraham (1959) considered the
al)ility of bacteria to acquire resistance to
antibiotics as one of the man}' examples of
the adaptability of microorganisms, involv-
ing the basic problems of protein and nucleic
acid synthesis. The procedure of Lederberg
and Lederl)erg (1952), whereby samples of a
mass of bacteria are first transferred from
the surface of a nutrient agar plate to a
velvet pad and then printed on other plates
to give replicas of the first, suggested that
bacteria highly resistant to streptom3^cin
can be formed without anj^ contact with the
drug. The fact that successive prints from a
normal plate of E. coli to plates containing
streptomycin show a lack of multiplication
of most of the organisms, but give growth of
a small numl^er of colonies of resistant cells
suggests that resistant cells, formed bj^ rare
mutations, already exist on the normal plate
and are later selected in the presence of the
drug. Abraham added, however, that this
does not prove that random mutation repre-
sents the only mechanism by which resist-
ance to antibiotics develops.
Lederberg in 1959, said:
In some favorable instances the spontaneous
origin of drug-resistant mutants can be verified
unambiguouslj^ by contriving to isolate them
without their ever being exposed to the drug.
One method entails indirect selection. To illus-
trate its application, consider a culture of Esch-
erichia coli containing 10^ bacteria per milliliter.
By plating samples on agar containing strepto-
mycin, we infer that one per million bacteria or
10' per milliliter produce resistant clones. But to
count these clones they were selected in the pres-
ence of streptomycin which hypothetically might
have induced the resistance. We may, however,
dilute the original bacteria in jjlain broth to give
samples containing 10° per milliliter. Since lO""*
of the bacteria are resistant, each sample has a
mathematical expectation of 0.1 of including a
resistant bacterium. The individual bacteria being
indivisible by dilution, nine samples in ten will
include no resistants; the tenth will have one, but
now augmented to 10^^ Which one this is can be
readily determined by retrospective assay on the
incubated samples. The procedure can be reiter-
ated to enrich for the resistant organisms until
they are obtained in pure culture. The same result
is reached more conveniently if we spread the
original culture out on a nutrient agar plate rather
than distribute samples into separate test tubes.
Replica plating, transposing a pattern of surface
growth from plate to plate with a sheet of velvet,
takes the place of assaying inocula distributed in
tubes. Dilution sampling and replica plating are
then alternative methods of indirect selection
whereby the test line is spared direct contact with
the drug.
According to Leidy ef at. (1956), strepto-
mycin resistance of a high degree can be
induced in sensitive populations of Hemophi-
lus influenzae and H. parainfluenzae by
deoxyribonucleic acids (DNA) derived
from streptomycin-resistant cells of at least
one heterologous species of Hemophilus.
Comparison of the activity of heterologous
and homologous DNA showed differences
within species and degrees of differences
among species not brought out by other
available methods. According to these re-
sults, H. influenzae is more closely related to
H. parainfluenzae than to H. suis, the rela-
tionship between the last two being remote.
The low proportion of cells in H. influenzae
no
NATURE, FORMATION, AND ACTIVITIES
populations which are made streptomyc-in-
resistant by DXA deri\'ed from strepto-
mycin-resistant H. parainfluenzae and vice
versa has been increased 4- to b")-t'old l)y the
replication of the heterologous species
streptomycin-resistant DNA in the heterolo-
gous species. An alteration of the heterolo-
gous DNA by the host was suggested.
Lightl)Own (1957) made a study of the
development of resistance to streptomycin
by Ps. pyocyaneus. Resistance was found not
to be enzymatic, but to be due to the produc-
tion of alkyl-substituted quinoline-X-oxides,
which are potent inhibitors of bacterial cyto-
chrome electron transport at concentrations
that give increased resistance to strepto-
mycin for organisms such as B. subtilis and
Staph, aureus. Resistance to streptomycin
was believed to depend on the de\'elopment
and choice of alternati^'e pathways of metab-
olism; the effects of the ([uinoline-X-oxides
may be due to the inhibition of the Pasteur
effect, allowing more active glycolysis to
occur aerobically. Obligate aerobes were
believed to possess an alternative to the
cytochrome terminal pathway. This ma}^
be, in part, a flavoprotein oxidase-peroxidase
path. Certain elements, notably Mg, accel-
erate the development of resistance to
streptomycin; others, hke Co, have an
inhibitory effect (Chernomordik and Kobe-
leva, 1959).
Pollak (195()) studied antibiotic resistance
of M. kansasii, an atypical acid-fast organ-
ism previously known as the "yellow bacil-
lus." It was as susceptible to streptomycin as
M. tuberculosis, but about 5 to 10 times as
resistant to /^-aminosalicylic acid and isonia-
zid.
Sebek (1958) investigated growth-inhibi-
tory effects of neomycin B, neomycin C, and
neamine on 24 bacterial species (Table 33).
Particularly striking differences were ob-
tained in Corynebacierium sp. and Sarcina
lutea. The growth of the first was inhibited
by about 250 /xg per ml of neamine or neo-
mycin C but by only 0.4 to 1.6 /xg per ml of
neomycin B. When neomycin C and neamine
were added in \-arying ratios and in different
combinations to neomycin B, the inhibitory
concentrations of neomycin B remained
virtually unchanged. The organism readily
acciuired resistance and cross-resistance to a
high degree to neamine and neomycin C but
only slightly to neomycin B. It was suggested
that the diaminohexose portion of neomycin
B is responsible for the specific growth-
inhibitory effect of this antibiotic on these
two bacteria. Cross-resistance between strep-
tomycin and neomycin has been studied by
Sidi et al. (1958).
Several cross-resistant groups are now
recognized among the antibiotics of actino-
mycetes: (1) streptomycin, streptothricin,
viomycin, and neomycin, as well as kana-
mycin, catenulin, and paromomycin; (2) the
tetracyclines, chloramphenicol, and possibly
penicillin; (3) erythromycin, carbom^'cin,
celesticetin, oleandomycin, and spiramycin.
Kunin et al. (1958) observed that kana-
mycin, paromomycin, and neomycin had
essentially the same activity against strains
of Staph, aureus and of various Enterobac-
teriaceae. Bacteria made resistant to any
one of these three antibiotics by subcultures
on that antibiotic also exhibited complete
cross-resistance to the other two. Freshly
isolated cultures did not show significant
cross-resistance between streptomycin and
these three antibiotics; however, sti'ains
made resistant to any one of the three also
showed increases in resistance to strepto-
mycin; strains made resistant to the latter
exhibited only minor increases in resistance
to the others. Cultures of staphylococci
comprising i)oth parent and resistant (to all
four antibiotics) variants were of the same
phage type; resistant variants of Kl. pneu-
moniae and E. coli, however, retained their
serological specificity. During oral treatment
with paromomycin or kanamycin, fecal
organisms were resistant to the antibiotic
DEVELOPMENT OF RESISTANCE
111
administered and also showed moderate to
marked resistance to the other one and to
neomycin.
Finland (1958) also found cross-resistance
between kanamycin, paromomycin, and
neomycin, but little or none between these
and streptomycin. The fact that they all
show 8th cranial nerve toxicity and renal
toxicity was believed to indicate further the
relationship between chemical structure and
pharmacological properties.
Welsch (1957), who examined the prol)-
lems of resistance facing the clinician, em-
phasized that the antibiotics should be used
with the utmost care. He concluded that
bacterial resistance takes place at three
different biological levels — the species, the
strain, and the individual cell — with different
clinical implications in each instance:
1. Variations of resistance at the level of
the species account for the occurrence of
superinfections as the result of a selective
destruction of some organisms of the normal
flora. The clinician must thus make a first
approximation of the antibiotic spectrum of
the causative organism for emergency
therapeutics.
2. Differences of resistance between natu-
ral strains within a given species are very
marked in staphylococci. The heterogeneity
of the species accounts for the results ob-
tained from the widespread use of an anti-
biotic, in the selection of resistant strains,
and the occurrence of resistant cross-infec-
tions in a treated individual.
3. The occurrence of spontaneous mutants
accounts for variation of resistance among
individuals of a pure bacterial population. It
was suggested that environmental factors
might influence the phenotypic expression of
individual resistant mutants derived from
the same clone. This hypothesis is based on
the fact that the degree of resistance ob-
served is not necessarily quantitatively re-
lated to the concentration of the drug to
which the bacteria were submitted, ^'aria-
tion of resistance at the level of the individ-
ual cell accounts for the progressive in-
effectiveness of chemotherapy sometimes
observed during treatment. The practical
importance of this type of resistance was said
to be limited largely to streptomycin therapy
and to treatment of tuberculosis. The use of
an association of chemotherapeutic agents is
known to minimize the emergence of resist-
ant organisms.
Welsch further emphasized that organ-
isms surviving the bactericidal action of an
antibiotic need not necessarily be resistant
to the drug, as in the case of penicillin and
streptomycin. Further, exposure of l)acteria
to an association of antibiotics does not
necessarily prevent the emergence of resist-
ant individuals.
Bacterial cultures with resistance acquired
in vitro show certain altered biological char-
acteristics, such as partial or complete
dependence on the antibiotic, decreased via-
bility, retarded growth rate, slower meta-
bolic reactions, decreased or altered nutri-
tional re(iuirements, certain pleomorphic
changes, changed cultural properties,
changed staining reactions, and frequently a
reduction in virulence.
Bacterial cells made resistant to anti-
biotics grow at a lower rate and produce less
growth in simple and complex media than
do the original sensitive strains. When the
resistant strains are grow^n in the presence
of the sensitive parent strains but in the
absence of the corresponding antibiotic, the
former are suppressed, especially in simple
media. This was also recently established by
Blackwell and McVeigh (1960) hi their
study of the effect of dihydrostreptomycin
upon E. coll.
Sevag and Rosanoff' (1952) demonstrated
that the synthesis of phenylalanine and as-
partic acid by sensitive cells of Staph, aureus
is blocked by streptomycin. These amino
acids are synthesized by both sensitive and
resistant cells in the absence of streptomycin.
112
NATURE, FORMATION, AND ACTIVITIES
Practical Aspects
The recognition of potentialities of in-
creasing resistance of bacteria to a given
antibiotic, thus resulting in a decrease in the
thei-apeutic value of the antibiotic, led to
rather alarming generalizations. This was
brought out by JMolitor (1946), who said,
"Whether the high incidence of drug-fast-
ness is due to the liberal and indiscriminate
use of these new, practically non-toxic agents,
or whether these drugs are particularly likely
to produce resistant strains, is not known.
Regardless of the cause, however, there is the
prospect that in the relatively near future
penicillin and streptomycin maj^ to a con-
sideral)le degree lose their usefulness in the
therapy of some of the most prevalent infec-
tions unless some means can be devised to
restore the original susceptibility of either
host or pathogenic agent."
In an effort to overcome the development
of drug resistance, it has been suggested that
the selected dose of the antibiotic be large
enough to eliminate the pathogenic organ-
isms from the body rapidly. Penicillin and
streptomycin, because of their low toxicity,
make possible the administration of doses
greater than those re(iuired to stop bacterial
growth. It is necessary to determine the
resistance of the pathogen prior to the treat-
ment. High initial concentrations of the
antibiotic insure the maintenance of high
blood concentrations. It has been empha-
sized that use of the antil)iotic in such prepa-
rations as salves, lozenges, chewing gum, and
sprays is likely to produce adequate concen-
trations only at the site of application and
would tend to create a hazard unless special
forms can be developed which will assure a
completely adequate drug concentration in
blood and body tissue.
The possibility of developing new anti-
biotics offers further promise of overcoming
the resistance of an organism to a given
antibiotic. This makes possible the combined
use of two substances, which would tend to
repress the few resistant cells. It has been
shown, for example, that use of penicilHn
and bacteriophages in combination produces
a synergistic effect. Malignant infections due
to staphylococci, colon bacilli, and certain
streptococci showed good response to such a
combination.
Combinations of Antibiotics
Among the procedures recommended for
overcoming the problem of bacterial resist-
ance, combined therapy of two or more drugs
has been given particular consideration.
Such combined activity may be synergistic,
additive, indifferent, or antagonistic, de-
pending upon the nature of the antibiotics
and upon the bacterial species or strain.
Cavalli-Sforza and Lederberg (1953) ana-
lyzed in detail the phenomenon of combina-
tion of antibiotics. Thej^ considered true
synergism (the joint effect being greater
than expected on simple addition of effects),
simple addition, indifferent effect, and
antagonism. Physiological synergism was
believed to be negatively correlated with
genetic synergism (Klein and Schorr, 1953).
Additive interaction could be expected be-
tween chloramphenicol and tetracycline,
which show cross-resistance and therefore
genetic antagonism. Tetracycline and peni-
cillin show genetic synergism and physiologi-
cal antagonism. This was believed to be due
to the fact that penicillin has a bactericidal
effect only on growing cells, and a bacterio-
static agent would therefore greatly reduce
its action. The association of chlorampheni-
col and sulfa drugs and the association of
isoniazid and streptomycin in the therapy of
tuberculosis were considered as involving
both genetic and physiological synergism.
Jawetz and Gunnison (1953) made a
detailed study of the synergistic or additive
properties of such combinations. Unfortu-
nately, there was freciuently a lack of correla-
tion between the activities of the combina-
DEVELOPMENT OF RESISTANCE
113
tions ill vitro and in vivo. Jtiwetz (1958)
emphasized that the basic mechanisms
underlying combined antibiotic action are
still unknown. He agreed, however, that
certain combinations may give good results
in clinical practice. This conclusion is based
upon laboratory data and clinical judgment.
He outlined the rational use of two anti-
microbial drugs, instead of one, as follows:
1. In mixed infections it is possible that
two drugs, each acting on a separate portion
of the complex microbial flora, might be more
effective than one drug. This applies occa-
sionally to infections of skin, wounds, or
body cavities, particularly when nonabsorb-
able drugs of limited antibacterial spectrum
are used topically {e.g., polymyxin, baci-
tracin, or neonwcin).
2. Toxic side effects may sometimes be
reduced by employing simultaneously two
drugs which have a similar antibacterial ac-
tion but distinct toxic effects. A combination
of such drugs could obtain a given antibac-
terial effect together with a lower toxicity
than would be feasi])le with either of the
components of the mixture used alone.
Streptomycin-dihydrostreptomycin mixtures
may serve as an example.
3. In some clinical situations the rapid
emergence of bacteria resistant to one drug
may impair the chances for cure. The addi-
tion of a second drug sometimes delays the
emergence of resistance. This effect has been
demonstrated uneciuivocally in tuberculosis.
In some other chronic infections the evidence
for its occurrence is questionable. In serious
systemic staphylococcal infections, strepto-
mycin, erythromjTin, novobiocin, or related
drugs should not be used singly, as a rule,
because resistance to each is likely to emerge
rapidly.
4. In certain desperately ill patients with
suspected infection of unknown etiology it
may be desirable to administer several anti-
microbial drugs after all steps have been
taken to establish an etiologic diagnosis.
These drugs are aimed at the organisms
most likely to cause the clinical picture en-
countered and are usually continued o\\\y
until the discovery of an etiologic agent
permits specific therap.v. The initial treat-
ment of meningitis in a small child might
be an example in this category.
"). In some infections the simultaneous
use of two drugs gives an effect not obtain-
able by either drug alone. Perhaps the best
estabhshed example is endocarditis due to
S. faecalis, for which the combined effect of
penicillin and sti'eptomycin is essential for
cure. The "synergistic" drug effects and the
known dynamics of combined drug action
are sunnnai-ized later (Chapter 11).
Chapter 11
Utilization of Antibiotics in Clinical
Medicine and Other Applications
Before the advent of inotlei'ii chemo-
therapy, the treatment of disease in general
and of infectious diseases in particular by
means of chemical agents was arbitrary and
fragmentary in nature. In most instances,
justification for the particular method of
treatment was specula ti\'e rather than
scientific.
The use of plant products offers an inter-
esting illustration. Since ancient times or
long before the role of microbes in the causa-
tion of infectious diseases was recognized,
certain plants have been used for the treat-
ment of \'arious infections. Reference to this
is found in the Herbals of the Chinese, in the
p]bers papyrus of Egypt (the use of onions),
in the Old Testament (Isaiah ad^'ised the
use of figs for the ti'eatment of a l:)oil of
Hezekiah), in the writings of ancient Greece
(Theophrastus) and Rome (Dioscorides), as
well as in folk medicine in South Africa and
in Central and South America. Cinchona
bark has been used in the treatment of
malaria. Chaulmoogra oil for leprosy and the
use of certain alkaloids may be cited as other
classic examples of plant products that have
found, even up to recent times, extensive
application in the treatment of various
diseases. Mention may also be made of the
use of cepharantine in Japan in the treatment
of tuberculosis. Among the other plant
products that possess marked antimicrobial
substances, the phenolic compounds occupy
a prominent place.
We now know that various plants produce
chemical sulostances which possess antibac-
terial properties. However, it is neither these
nor the enzyme-rich plants (such as figs) that
have found extensive application in the
treatment of infections caused by microbes,
but rather the products of microbes them-
selves, namely, the antibiotics.
At first, to combat infections, came the
use of certain preparations, in the form of
vaccines, serums, and antitoxins, obtained
from the causative microbes. These prepara-
tions were used, and many still continue to
be used, either as prophylactics or as thera-
peutics. They range from common cold and
smallpox vaccines to Calmette-Guerin bacil-
lus (BCG) and polio vaccines, from diph-
theria and tetanus antitoxins to antistaphyl-
ococcal serums. Strictly speaking, however,
none of these come under the category of
chemotherapy. They are to be considered as
immunotherapeutic methods rather than as
chemotherapeutic procedures. This is true
despite the fact that some of these prepara-
tions, such as the diphtheria nncl tetanus
antitoxins, have been isolated in a purified
chemical state.
The field of modern chemotherapy is based
largely upon the disco^'ery of four types of
compounds: (1) salvarsan and other arseni-
cals; (2) sulfanilamide and other sulfa drugs,
including the sulfones; (3) other synthetic
chemical compounds, notably 7>-aminosali-
cylic acid and isoniazid; (4) the antibiotics.
114
UTILIZATION OF ANTIBIOTICS
115
It is the last group with which we are con-
cerned here.
It has been said that much greater
progress has been made in the treatment of
infectious diseases during the last 15 or 20
years than in all previous medical history.
The first real progress in combatting infec-
tious diseases was made during the latter
part of the last century with the discovery of
immune sera and antitoxins. This was fol-
lowed by the work of Paul Ehrlich and others
on the great potentialities of the arsenicals
as chemotherapeutic agents, resulting in the
discovery of salvarsan in 1910. A quarter of
a century later came the sulfonamides,
broader in scope and of much wider appHca-
tion. They were soon followed by discovery
of the antibiotics, of which penicillin and
streptomycin were the first two most strik-
ing examples.
The mere listing of the various uses of
antibiotics is sufficient to emphasize their
broad application.
I. Treatment of infectious diseases.
A. Infectious diseases of man.
1. Diseases caused by gram-posi-
tive bacteria; penicillin, chlor-
amphenicol, tetracyclines, eryth-
romycin, and \'ari()us others are
used.
2. Diseases caused by gram-nega-
tive bacteria (gastrointestinal in-
fections, tularemia, plague, chol-
era, and numerous others);
streptomycin, neomycin, chlor-
amphenicol, tetracyclines, and
certain others are effective.
3. Various forms of tuberculosis;
streptomycin and certain other
antibiotics have found particular
application.
4. Diseases caused by rickettsiae
and the psittacosis-lymphogran-
uloma group of organisms; the
tetracyclines, chloramphenicol,
and erythromycin are used.
5. Diseases that can be treated ef-
fectively by combinations of
antibiotics with synthetic com-
pounds.
B. Numerous animal diseases that af-
flict poultry, swine, dogs, cats, and
other domesticated animals.
C. Plant diseases caused by bacteria
and certain fungi.
1. Fireblights and other bacterial
diseases of trees.
2. Bacterial diseases of vegetables,
such as string beans and peppers.
3. Bacterial diseases of flowers, such
as carnations.
4. Diseases caused by fungi, such as
the blue mold of tobacco.
II. Treatment of diseases, noninfectious in
nature, notably neoplasms.
III. Preservation of valual)le biological
products.
A. Virus preparations.
1. Poliovirus.
2. Poultry vaccines
B. Human and animal semen.
C. Foodstuffs, notably fish and poul-
try.
I\\ Animal feeding.
More than 30 antibiotics are now avail-
able for the treatment of various infectious
diseases caused by bacteria, fungi, protozoa
(amoebae, trichomonads, etc.), rickettsiae,
the psittacosis-lymphogranuloma group, and
certain worms. Some are more effective than
others. Some are best used orally, others by
injection, still others by surface application.
The development of resistance against one
necessitates the use of others, l^ndesirable
reactions caused by a particular antibiotic
through one method of administration, as in
the case of neomycin used parenterally, sug-
gest other methods of administration, as oral
or topical for neomycin.
Animal experiments cannot always be
considered as true representations of the
chemotherapeutic potentialities of a given
116
NATURE, FORMATION, AND ACTIVITIES
antibiotic in man. This has been emphasized,
for example, for cycloserine (Alulinos, 1955).
A partial list of antituberculosis agents
produced by microorganisms would indicate
among the bacterial products nisin, licheni-
formin, and viscosin. These were soon dis-
carded, however, in favor of others. Among
the fungal products, only clitocibin appeared
at first to offer promise, but it was discarded
as well. The actinomycetes eventually
.yielded the most valuable products. The
most important of these is streptomycin,
followed by viomycin, cycloserine, neomycin,
and kanamycin (Scowen, 1960).
The development of resistance among the
staphylococci to penicillin, especially under
hospital conditions, has led to a concentra-
tion of attention on this growing problem in
the use of antibiotics (Lowbury, 1960;
Pollock, 1960). Some pessimists have gone
so far as to predict that within 10 years the
antibiotics will no longer be clinically useful
because all bacteria will ha\^e developed
resistance to these drugs. Such predictions
are the height of absurdity and ignorance.
Even the "hospital staphylococci" would
not have developed resistance so rapidly if
the principles of cleanliness had been ad-
hered to and if the antibiotics had not been
expected to take the place of cleanliness; if
self-healing by many patients had been
strictly controlled; and if excess use of anti-
biotics had been avoided. Perhaps the warn-
ing came in good time. Fortunately, other
antibiotics have come to supplement or take
the place of those to which bacteria have
developed resistance.
Treatment of Infectious Diseases
The introduction of antil)i()tics in the
treatment of infectious diseases completely
revolutionized medical science and medical
practice. Diseases like cholera, plague, dysen-
tery, typhoid, and typhus fever, which
formerly at frecjuent intervals decimated the
human race, and the numerous diseases of
childhf)od have now been brought under
practical control. Diseases that onl}^ two
decades ago were believed to be beyond the
control of man are now being successfully
treated. Many of them have been almost
completely eradicated. Tuberculosis, for-
merly known as consumption or as the Great
White l^lague, has receded in a rather short
period of time from first to tenth place as a
killer of mankind. First to be found effective
in its treatment was streptomycin. This was
later supplemented by certain synthetic com-
pounds (p-aminosalicylic acid and isoniazid)
and other antibiotics (viomycin, cycloserine)
to increase the effectiveness of this agent
(Crofton, 1960). As a result of the tremen-
dous developments due largely to antibiotics,
the attitude of the average man to disease,
especially to infectious disease, has changed
in one generation from fear to understand-
ing.
In deciding upon a particular antil)iotic
for the treatment of a given infection, the
close collaboration of the clinic and labora-
tory is most essential (Gould, 1960). The use
of antibiotics in chemotherapy is based upon
the assumption that the drugs act upon the
infectious agents without injuring the host
and without affecting the natural defenses
against the infectious agent. It is further
based upon the close correlation of the effect
of antibiotics upon sensitive organisms in
vitro as opposed to their effect in vivo. Sensi-
tivity tests must therefore be very accurate.
A recent analysis of the problems involved
was made by Ericsson and Svartz-JMalmberg
(1959).
Two important factors must be consid-
ered: (1) variation in sensitivity of individ-
ual strains of the same organism, and (2)
development of resistance. On the basis of
the clinical I'esponse to antibiotic therapy
and rapidity of de\'elopment of resistance,
Dowling et al. (1955) classified disease-pro-
ducing organisms in three groups, as shown
in Table 37.
Group 1 comprises organisms that usually
respond promptly to antibiotics. Resistant
UTILIZATION OF ANTIBIOTICS
117
forms are produced with difficulty, and even
more rarely in clinical use.
Group 2 includes organisms that often
respond only slowly to therapy with anti-
biotics. In some cases, such as pulmonary
tuberculosis or staphylococcal osteomyelitis,
patients may impro\^e and yet retain the
organisms and infection for years, never
becoming entirely well. Resistant forms are
also often encountered.
Group 3 includes organisms causing bru-
cellosis, t^^phoid, and some rickettsial in-
fections. The organism isolated during re-
currence remains sensitive to the antibiotic
originally used, and the infection will re-
spond to another course of therapy.
Antibiotic resistance is particularly serious
in the case of staphylococci, tubercle bacilli,
and certain gram-negati\'e forms, such as
Proteus, Psoidomonas, and the coliform
organisms. Staph^dococcal infections due to
resistant strains are said to constitute the
most serious clinical prol)lem of antil)iotic
resistance. The solution of the problem of the
emergence of antibiotic-resistant staphylo-
cocci has received much attention (Dunlop,
1960). Combination therapy with two anti-
biotics has been recommended, provided the
organism is sensitive to each antibiotic
alone. Unfortunately, there is no evidence
that the incidence of resistant strains
is always reduced by combined therapy.
Lepper et al. (1956) used novobiocin plus
spiramycin in a routine manner for 6
months. A gradual increase in the incidence
of staphylococcus resistance to each anti-
biotic occurred despite this combination
therapy (see also Kass, 1955, and Velu,
1958). The preventive use of antibiotics must
also be mentioned (By waters, 1960; Taylor,
1960).
Animal Diseases
On a par with the revolution that took
place in the treatment of infectious diseases
in man as a result of the introduction of
antibiotics, one must consider the remark-
able role played by these agents in the treat-
ment of infectious diseases in animals.
Brucellosis and mastitis in cattle and numer-
ous infectious diseases of dogs, cats, poultry,
and other domesticated animals can now be
controlled by antibiotics.
Animal Nutrition
In addition to their use for the control of
infectious diseases, antibiotics have also
been employed extensively in the mitrition
of nonherbivorous animals, such as poultry
and swine. This effect is particularly marked
in animals deprived of iS-carotene and vita-
min A (Guerrant, 1960). For further details,
see Jukes (1955) and Goldberg (1959).
Among the uses of antibiotics that invf)lve
both animal feeding and disease control, one
might mention the control of silkworm dis-
eases by the soaking of mulberry leaves in
antibiotic solutions. The use of streptomycin
and tetracyclines resulted not only in the
almost complete suppression of bacterial in-
fections in silkworms, but also resulted in
a higher finality silk (Afrikian, 1960).
Food Pre-servation
In recent years, certain antibiotics, nota-
bly the tetracyclines, which are readily de-
stroyed on boiling, have been used exten-
sively in the preservation of fish and poultry
products (Carey, 1958).
Laboratory Uses
Numerous laboratory processes have bene-
fited greatly from the utilization of anti-
biotics. One might mention their use in the
preparation of selective culture media for
the isolation of (1) gram-negative bacteria,
(2) anaerobic bacteria, (3) fungi, (4) proto-
zoa, and (5) viruses. They are used in genet-
ics for the isolation of special nongrowing
variants which depend upon specific growth
factors. They are used for preservation of
cattle semen and virus preparations. Finally,
their use in tissue cultures and in the produc-
118
NATURE, FORMATION, AND ACTIVITIES
tion of virus vaccines has made possible cer-
tain special forms of prophylactic therapy.
Problems Arising from Use of Anti-
biotics
The tremendous benefits of antibiotics to
the human race were soon fully recognized.
They were lauded as the "miracle drugs."
The medical profession was ready to replace
the concept of "chemotherapy" with that of
"antibiotic therapy." On the other hand,
many persons went to the other extreme.
If no rapid cures for cancer or for the ^'arious
viral diseases were forthcoming, they tended
to blame the antibiotics. When of the hun-
dreds of thousands of cultures tested and
the numerous antibiotics isolated, only 20
or 30 found a place in practical therapy,
many persons felt cheated and asked for
more. Further, the de^'elopment of liacterial
resistance to some antibiotics and the unde-
sirable reactions occasionally produced in
certain patients were given undue pul)licity,
with the resulting designation of the anti-
biotics as "toxins" and "poisons" (Gale,
1960).
Ciradually, howe\'ei', the undesirable char-
acteristics of the antil)iotics are being elimi-
nated through a better understanding of
their use. Combinations of two antibiotics,
such as penicillin and streptomycin, or of
two forms of the same antibiotic, such as
streptomycin and dihydrostreptomycin, or
of an antibiotic with a synthetic chemical
agent, as streptomycin with p-aminosalicylic
acid or with isoniazid in the treatment of
tuberculosis, have tended to increase the
effectiveness of antibiotics and decrease
their limitations (Lacey, 1960). New anti-
biotics are constantly being introduced to
supplement those now in use. Both the
physician and the patient have come to
recognize that every new discovery carries
wdth it an obligation and that no sure cure
can be expected, even from the most nearly
ideal drugs, unless their mode of action,
their specific role in therapy, and especially
their limitations are thoroughly understood.
Treatment of Plant Diseases
The antagonistic effects of microbial
saprophytes upon plant pathogens were
recognized long before the advent of anti-
biotic therapy. Various bacteria and fungi
were utilized for the purpose of controlling
plant diseases. The results were not alwaj^s
convincing, however, and could never be
properly duplicated. On the other hand,
some of the antibiotics, such as streptomy-
cin, have proved to be markedly effective
in the treatment of \'arious bacterial dis-
eases of plants; and actidione, antimycin,
and streptomycin have found a place in the
ti'eatment of certain fungus diseases. A de-
tailed re\iew of this suljject was recently
published by P. Miiller (1959).
Important Therapeutic Applications
A list of the anti})iotics of actinomycetes
that ha\'e found important therapeutic ap-
plications is given here in order of their
disco\'ery :
Actinomycins C and D, used in the treat-
ment of certain forms of cancer, such as
rhabdosarcoma and Wilms' tumor.
Streptomycin and dihydrostreptomycin,
used in the treatment of tuberculosis and of
various infections caused by gram-negative
and gram-positive bacteria; also of certain
plant diseases caused by bacteria and a few
fungi.
Chlortetracycline, oxytetracycline, and
tetracycline, used in the treatment of dis-
eases caused by various gram-posit Ia'c and
gram-negative bacteria, rickettsiae, and the
psittacosis-lymphogranuloma group of or-
ganisms.
Chloramphenicol for the treatment of
diseases caused by gram-posit i\'e and gram-
negative bacteria, rickettsiae, and the psitta-
cosis-lymphogranuloma group of organisms.
UTILIZATION OF ANTIBIOTICS
119
Staphjdococcal infections and typhoid are
very susceptible.
\^iomycin, which has found a Hmited use
in the treatment of forms of tulx'rculosis.
Erythromycin, carbomycin, spiramycin,
and oleandomycin, active mainly upon
gram-positive organisms, particularly peni-
cillin-resistant staphylococci.
Xeomycin (framycetin), active upon
gram-positive and gram-negative bacteria;
largely for topical and oral use, for intestinal
sterilization, and for bacterial diarrheas.
Cycloserine for the treatment of resistant
forms of tuberculosis and of certain gram-
negative urinary tract infections.
Novobiocin, active upon diseases caused
by gram-positive organisms, particularly
penicillin-resistant staphylococci.
Amphomycin, active upon certain gram-
positive organisms; for topical use onl3^
Ristocetin, active upon gram-positive
organisms, especially penicillin-resistant
staphylococci; used intravenously only.
Thiostrepton, active upon gram-positive
organisms; used orally for intestinal sterili-
zation, usually combined with other drugs.
Hygromycin, used in animal feeds only,
for large round worms, nodular worms, whip-
worms.
Cycloheximide (actidione), used in the
treatment of certain plant diseases.
Nystatin, antifungal agent.
Candicidin, trichomycin, used in treat-
ment of diseases caused by fungi.
Amphotericin, active upon fungi causing
systemic mycosis.
Vancomycin, active upon gram-positive
organisms, particularly penicillin-resistant
staphylococci; administered by intravenous
injection only.
Kanamycin, actix'e upon gram-positive
bacteria, particularly penicillin-resistant
staphylococci, and various gram-negative
organisms.
A detailed review of the clinical applica-
tion of most of these antibiotics has been
made recently by Florey (1960).
An Outlook
In view of the lai'ge number of actino-
myeete cultures already isolated and tested,
the numerous antil)iotics and antibiotic prep-
arations obtained, the great variety of chem-
ical compounds now recognized, and their
potential biological activities, one might be
inclined to think that the limit to our
knowledge of antibiotics of actinomycetes
may already have been approached. To
those of us, however, who have devoted
several decades to the study of the actino-
mycetes, who have searched for them in
numerous soils throughout the world, in
peat bogs and in composts, in lakes and in
the sea, all our present knowledge appears as
a mere beginning. Since a single gram of
soil may yield, on proper plating on suitable
media, a million or more colonies of actino-
mycetes, representing numerous species with
many metaboHc potentialities, one can w'ell
appreciate the various biochemical mecha-
nisms invoh'ed. JNIany more species are still
to be discovered.
Aside from their importance in the treat-
ment of infectious diseases in man, animals,
and plants, antibiotics have contributed
materially to \'arious fields of science. In the
hands of qualified investigators, the anti-
biotics have become powerful tools for fur-
ther scientific research. This is true particu-
larly in the fields of chemistry and biology,
especially in their application to agriculture,
medicine, and public health. A few illus-
trations will suthce to indicate the extent of
their contribution.
1. The knowledge of antibiotics has con-
tributed greatly to genetics, especially mi-
crol)ial genetics. The development of bac-
terial resistance to streptomycin and other
antibiotics proved to be an important genetic
marker for studies on the sexual recombina-
tions among bacteria and actinomycetes.
120
NATURE, FORMATION, AND ACTIVITIES
Crossings of parental strains with rearrange-
ment of genetic materials are usually per-
formed with nutritionally deficient mutants
of sti'ains that normally can grow in syn-
thetic media with a sugar as the sole carbon
source. Penicillin is usually employed to
concentrate and recover the induced mu-
tants.
2. A better understanding of biological
synthesis, especially of large molecules (nota-
bly proteins, nucleic acids, and cell-\vall
material), can be attributed to the introduc-
tion of antibiotics. The effect of chloram-
phenicol on the building of the protein
molecule and on amino acid incorporation
established the fact that this antibiotic
uncouples the synthesis of nucleic acid fi'om
that of protein (Lacks and Gros, lUoD). This
is also true of the effect of penicillin upon
protoplast formation in Escherichia coli.
3. The widespread use of antibiotics has
stimulated organic chemical I'esearch, and
several new or very rare compounds \\i\\e
been discovered. Streptose, the first branched-
chain sugar to be identified in a microbiologi-
cal product, and streptidine, a base re-
lated to inositol, were found in streptomycin.
Only three naturally occurring polyacetyl-
enes were known before 1950, but study of
the polyene antibiotics has increased the
number to at least 2o. Dichloroacetic acid
and nitrobenzene, although well known to
the organic chemist, were found in a natural
product for the first time in chloramphenicol.
4. The very fact that there is relatively
little cross-resistance among the various
antibiotics suggests the prol)ability that
different mechanisms of antimicrobial ac-
tivity are involved. This effect of antibiotics
upon microbes causing infectious diseases
offers the clinician a number of possibilities
in the selection of chemotherapeutic agents,
alone or in combination with other anti-
biotics or chemical substances. The whole
principle involved in the preparation of
polio vaccine is based on the preservation
of the polioviruses, by means of antibiotics,
against destruction through bacterial con-
tamination.
5. The gi'owth-promoting efl'ects of anti-
biotics on higher animals appear to be dis-
tinctly different from those exerted by es-
sential growth factors or \'itamins. They are
not so specific as are true vitamins. They
involve processes heretofore scarcely recog-
nized in nature. This stimulating effect of
antibiotics has been ascribed to a disturb-
ance of the intestinal microbial populations
of the animals. A direct effect upon animal
growth has also been postulated. Since anti-
l:)iotics may affect adversely the bacterial
population of the rumen which assists the
animal in the digestion of its cellulosic food
materials, care must be exercised to use
antil)iotics only at a certain stage of the de-
velopment of the animal.
6. Human and animal semen can be pre-
served from bacterial attack by means of
antibiotics. The same is true of the preserva-
tion of foods, especially poultry and certain
vegetal)les. Since antibiotics do not inhibit
all forms of microbial life, more than one
antibiotic may be recjuired for proper preser-
vation. Before the food is eaten, however,
the antibiotics must be destroyed by boiling,
for their constant con.sumption in the food
would tend to ha^•e certain dangerous effects
upon the human body.
7. Antibiotics ha\'e introduced a new con-
cept of microbial life in natural environ-
ments. They have added greatly to oiu*
understanding of various aspects of biology,
under natural conditions. The actinomycetes
ha\'e pro\'ed to be the richest source of anti-
biotic producers. In 1989, on the eve of the
advent of antibiotics, these organisms were
considered as a rather insignificant group of
microloes, largely inhalnting soils, composts,
and lakes. Today, because of their ability to
form antibiotics, their biochemical activities
and their role in natiu-e have become prob-
lems of paramount significance.
UTILIZATION OF ANTIBIOTICS
121
8. Light has Ijcen thrown on nunierou.s
other .scientific prol)lems through the chs-
covery of the potent iahte.s of antibiotics.
Such advances inchide the extensive use of
tissue cultures in biology, a l)ottei' under-
standing of the structure of the bacterial
cell, a clearer picture of superinfections and
of other problems in the field of medical
research.
It is belie^'ed by some that the de\'elop-
ment of resistance to antil)iotics suggests
their i-educed usefulness and gradual elimi-
nation. Xew antibiotics are introduced, onl}^
to be followed by the development of I'esist-
ance to these as well. The pessimistic prophet
tends to see in this the end of the antiljiotic
era. The optimist, however, is greatly
heartened by the progress already made.
He foresees the complete elimination of
tuliei'culosis as the great enemy of man. He
looks forward to the complete control of
children's diseases. Such infections as un-
dulant fever, typhoid, dysentery, cholera,
plague, and even leprosy no longer hold for
him the threat that they did before the
advent of the antilnotics. He even looks
forward to the ultimate control of such dis-
eases as cancer, those caused by A^iruses, and
possiljly others. How soon this may come
about, onlv the futui-e will fell.
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Part B
Descriptions of the Various Antibiotics
Produced by Actinoniycetes
Introductory
Section B contains a compilation of the
basic information published on the anti-
biotics, as well as the antiviral and the
antitumor substances produced by actino-
mycetes.
In presenting this information, emphasis
is laid upon the organism producing the
antibiotic; the method of extraction of the
antibiotic; its physical, chemical, and bio-
logical properties; its acti\'ity in riro;
toxicity; and practical utilization. A few of
the most pertinent references are gi\-en for
each antibiotic.
The authors have tried to make a thorough
review of the literature up to July 1, H)()0.
After this date the coverage of the literature
has been more fragmentary. Specifically,
Section B contains:
1. A compilation of the information avail-
able on the various antil)iotics. The sul)-
stances are arranged alphabetically. Anti-
biotics with no names, only numbers, appear
in the following order: first, those with no
letters before the numbers. The first digit
before which there is a break of some kind
gives the order of listing, for example,
antibiotic l-81d-ls will be foinid before
antibiotic 136. Second, the antibiotics
indicated by a letter with a number, such as
antibiotic F 416, are listed in alphabetical
and numerical order. Some antibiotics,
especially those which have been reported
very recently, will be listed at the end vmder
the heading "List of Additional Antibiotics."
Enough will be said in that section to suggest
the general nature of these substances. These
substances will be indexed but will not be
included in the keys and in the lists.
2. Keys which might help those who try to
identify freshlv isolated antibiotics. It is to
be understood that these keys are not classi-
fication systems, sensu strictu, but they are
presented here for the sole purpose of help-
ing the investigator who is interested in
characterizing antibiotic preparations.
Antibiotics are first separated on the basis
of l)iological activity, then on the basis of
chemical properties. Special attention is
placed on the light-absorption spectra. Dif-
ferences in what one author might con-
sider a maximum of absorption and another
a shoulder could lead to confusion. The keys
lack uniformity because all aiitil)iotics have
not been described with equal care and ac-
cording to the same criteria. The same sub-
stance may, therefore, be listed in the keys
in more than one section. In spite of their
limitations, the keys have been useful to the
authors, and they hope that others will be
al)le to use them.
o. A list of amino acids reported to be
present in hydrolysates of antibiotics.
4. Lists of antibiotics active against proto-
zoa and viruses will be found after the keys.
A list of antitumor substances will also l)e
found there.
One should note that antitumorsubstances,
with no known activity against microorgan-
isms, are not antibiotics as defined by the
senior author. For the sake of completeness,
we have tried to include such substances in
this book.
The usefulness of this compilation is
extended by the index found at the end of
the A'olume, in which synonyms for the
antibiotics described are listed.
In view of the rapidly accumulating in-
formation on antibiotics produced by ac-
tinomvcetes, and in view of the fact that the
139
140 DESCRIPTIONS OF ANTIBIOTICS
same or only slightly different antibiotics This is true particularly of those antibiotic
are often isolated in different laboratories preparations that have not been fully de-
throughout the world, there will be found a scribed and of those that represent mixtures
certain amount of overlapping among some of closely related compounds rather than
of the antibiotics described in this treatise, single chemical entities.
Keys to Antibiotics
KEY NO. 1
Antibiotics Which Are Active Mainly against Gram-positive Bacteria
I. Antibiotics active mainly against mycobacteria and/or actinomycetes
A. Antibiotics for which one maximum of light asborption has been reported
(Each number indicates the maximum, expressed in m/x.)
1. 208 in 0.1 A^ HCl; 218 in 0.1 N NaOH
2. 226 in water at pH G.O
3. 235 in ethanol
4. 237. o in methanol
5. 260
6. 260 in methanol over a wide pH range
7. 268 in 0.1 A^ HCl; 280 to 282.5 in 0.1 N XaOH
all)()vei'ticillin
cycloserine
hygroscopin A
elaiomycin
mycomycetin
august my cin A
phthiomycin
viomycin
bo\iii()cidin
pyridomycin
B
8. 270 at pH 8.4
9. 303 in alcohol
10. 306 in 50 per cent aqueous ethanol; 31() in
0.1 A^ HCl
Antibiotics for which more than one maximum of light absorption has been re-
ported
1. 227 and 270 in acidic solutions
2. 230 and 285, with end-absorption, in water
3. 240, 410, and 425 in cyclohexane
4. 244 and 295 to 300; ninhydrin-positive
5. 251 and 364 in methanol
6. 255 and 320 in water
7. 256 and 365 in methanol
8. 260 and 270, with weak maxima at 290, 307,
327, and 348
C. Antibiotics with no light absorption or for which light al)sorption has not been re-
ported
1. Neutral, white antibiotics
amicetin
tubercidin
(|uestiomycin B
(juestiomycin A
phleomycin (base)
tubermycin B
grisaminc
tubermycin A
isomycomycin
2. Acidic, colorless substance, essentially insol-
uble in water and chloroform
3. Heat-stable substances, probably basic and
water-soluble
4. Reddish substance
5. Lytic agent
acetomycni
longisporin
actithiazic acid
antiphlei antibiotics
antismegmatis antibiotic
nocardin
nocardamin
actinolysin
141
142
THE ACTINOMYCETES, VOL. Ill
II. Antibiotics active against gram-positive bacteria in general
A. Antibiotics for which one maximum of hght absorption has been reported
1. 203 bottromycin
2. 206 gancidin W
3. 208 in 0.1 N HCl alboverticillin
4. 215 in ethanol antibiotic E 129 (patent com-
ponent B)
5. 216 in methanol
6. 217
7. 220
8. 223
9. 225
10. 226 in methanol
11. 226 in water at pH ().0
12. 227.5 in ethanol
13. 230
14. 230-232
15. 230-235 in methanol
16. 233 in ethanol
17. 235 in ethanol
18. 238
19. 240
20.
252 to 253
21.
256 in isopropyl ale
22.
260
23.
268 in 0.1 N HGl
24.
268 to 272 in water
25.
270 in methanol
26.
273 in water
27.
275
28
278 to 280
staphylomycin factor ]\1
megacidin
amaromycin
antibiotic PA 133B
streptogramin
picromycin
mikamycin A
cycloserine
neomethymycin
miamycin
melanosporin
sarkomycin
leucomycin
leiicomycin-like complex
leucomycin B
spiramycin
antibiotic E 300
tertiomycin A
hygroscopin A
carliomycin
antibiotic PA 148
angolamycin
hygrostatin
proactinomycin A
azalomycin B
elaiophylin
borrelidin
mycomycetin
phthiomycin
thermoviridin
xanthicin
speciomycin
flavocidin
antibiotic F 256
carbomycin B
erythromycin
antibiotic PA 108
ristocetins
KEYS TO ANTIBIOTICS
143
29. 280 to 282 in acid
30. 281 in hexane
31. 285 in methanol
32. 289
33. 290 to 295
34. 292
35. 304 in othanol
36. 30(3 in 50 per cent a(iueous ethanol
37. 310 in i\ N H2SO4
38. 350
39. 405 to 430 in dilute HCl
40. 440 in methanol
B. Antibiotics for which two maxima of light absorpt
1 . 207 and 304 in methanol
2. 208 and 245 in ethanol
3. 215 and 257 to 258 in methanol
4. 220 to 2.30 and 275 in methanol
5. 222 and 293
6. 223 to 225 and 322 in ethanol
7. 225 and 280
8. 226 and 275
9. 230 to 240 and 334 to 340 in 0.1 .¥ HCl
10. 230 to 231 and 279 to 285 in ethanol
11. 230 to 231 and 278 to 285 in water
12. 231 and 276
13. 235 and 280 in 0.01 .V XaOH
14. 240 and 295
15. 240 and 267 to 268 in ethanol or methanol
16. 240 and 310 in 0.01 .¥ alcoholic sulfuric acid
17. 240 and 440 to 450
18. 243 and 320 to 325 in afjueous methanol
19. 243 and 312
20. 244 and 284
21. 245 and 255
22. 245 and 270 to 275
23. 245 and 440 to 450
24. 248 and 308 in 70 per cent ethanol at pH 7.5
25. 249 and 317 in isopropyl alcohol
26. 249 and 321 in p?l 7.0 phosphate buffer
27. 250 and 400 to 450
28. 255 and 320 in water
vancomj'cm
valinomycin
matamycin
erythromycin B
oleandomycin
erythromycin C
seligocidin
amicetin
bryamycin
etamycin
pluramycin A
nocardianin
ion have been reported
staphylomycin factor S
puromycin A
mycospocidin
antibiotic PA 114 A
picromycin
methymycin
narbomycin
antibiotic PA 133A
abikox'iromycin
tertiomycin B
leucomycins
antibiotic 446
(juestiomycin B
actinojiocin
aureolic acid (Mg salt)
proactinomycin C
musarin
azalomycin F
celesticetin
actincnnycins
antibiotic F 43
actinoleukin
antibiotic l-81d-ls
aniibiotic SKCC 1377 (pic
rat(\)
proactinomycin B
diazomycins
ractinomycin A
novobiocin
antibiotic X-537-A
amicetin B
ractinomycin B
jirisamine
144
THE ACTINOMYCETES, VOL. Ill
C.
D,
29. 258 to 261 and 355 to 85(i in methanol
30. 260 and 305 in methanol
31. 260 to 280 and 340
32. 275 and 316
33. 280 and 420 to 440 in 0.1 .V HCi
34. 280.5 and 430
35. 330 and 420 in methanol
36. 523 and 560 in dioxane
37. 535 and 575
38. 536 and 567 in methanol
39. 546 and 584 in 2 iV NaOH
Antibiotics for which three maxima of light asborpt
1. 206, 265, and 340 in 0.1 N HCI
2. 209
3. 215
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
218
218
223
227
228
230
234
234
240
240
240
247
258
317
260, and 305 in methanol
255 to 257, and 430 at acid pH
250, and 283 in ethanol
289, and 370 in ethanol
304, and 425, in pH 7.3 phosphate buffer
278, and 400 in ethanol
258, and 425
276, and 410 in 95 per cent ethanol
357.5, and 370 in 0.01 A^ ethanolic RSOi
540, and 580
280, and 305 in methanol
410, and 425 in cyclohexane
413 to 430, and 440 to 450
287, and 390 to 400
420, and 471 in 95 per cent ethanol
333, and 350 in organic solvents, pentaenes
18. 320, 415, and 490 in n-butanol
19. 335 to 341, 355 to 358, and 373 to 380 in or
ganic solvents, hexaenes
20. 460 to 480, 510 to 530, and 560 to 570
Antibiotics for which more than three maxima of
ported
1. 218, 273, 281, and 288
2. 223, 286, 532, and 576 in ethanol
3. 230, 276, 320, 330, 350, and 410 in ethanol
4. 230, 260, 290, and 500 in methanol
5. 235, 305, 525, 551, 563, and 610 in methanol
6. 236 to 240, 266 to 270, 334 to 340, 380, 400 to
401, and 424 to 430
teomycic acid
antibiotic PA 114B
telomycin
pulvomyf'iii
luteomycin
antibiotic L. A. 7017
taitomycin
actinorhodin
collinomycin
rhodomycin complex
rubromycin
ion have been reported
gancidin A
mikamycin B
antitumor antibiotic 289
antibiotic SAX 10
carzinophilin A
antibiotic X 340
rifomycin B
aburamycin isomer
quinocyclines
aburamycin
streptolydigin
rhodomycetin
thiostrepton
questiomycin A
actinomycins
chrysomycin
mycorhodin
pentaene antifungal antibi-
otics I and II
rubidin
flavacid
mediocidin
cryptocidin
litmocidin
light absorption have been re-
antibiotic PA 155A
granaticin
aburamycin
rhodomycins A and B
isorhodomycin A
chartreusin
coerulomycin
KEYS TO ANTIBIOTICS 145
7. 240, 305, 385, and 401 in methanol rifomyeins
8. 269 to 270, 290, 820, 840, and 460 heliomyein
resistomycin
9. 294, 331, 471, and 529 in alkaline ethanol prodigiosin-like antibiotic
10. 504, 535, 542, and 575 in l)enzene rhodomycins
E. Antibiotic with a pr(),<!;rcssi\(' drop in light absorption tVom 210 to 450
("unphom^ycin
F. Antibiotics ha^•ing no typical light-absorption spectra or for which light absorption
data were not reported
1. Antibiotic active only against Sarcina lutea sarcidin
2. Antibiotic actiA'C mainly against Coryncbacter- thermomycin
ium diphtheriae
3. Antibiotic active only against respiratory-de- mntonrycin
ficient staphylococci
4. Antibiotic active mainly against Streptococcus totomycin
hemolytic us
5. Antibiotics presumably active against ^'ari<)us
gram-positive bacteria
a. Water-soluble substances
(1) Acidic substances streptocin
duramycin
(2) Neutral, yellowish green substance actinomycelin
(3) Basic substances
(a) Positive Sakaguchi and ninhydrin antibiotic AX 18
reactions
(b) Positive Sakaguchi reaction, nega- antibiotic A 116
tive ninhydrin reaction cinnamycin
(c) Negative Sakaguchi and ninhydi'in albomycetin
reaction amphomycin
(d) Negative Sakaguchi reaction, posi- trehalosamine
tWe ninhydrin reaction
(e) Unknown Sakaguchi and ninhydrin actinoidin, HCl salt
reactions
(4) Polypeptide or proteinaceous substances actinoxanthine
amphomycin
cinnamycin
crystallomycin
duramycin
micromonosporin
phytostreptin
(5) Red-amber substance, brilliant yellow antibiotic 721
solution at alkaline pH
(6) Red-brown substance. Yellow in acidic antibiotic SKCC 1377
solutions, purple at alkaline pH
b. Substances solul)le in water only at alkaline
reaction
146
THE ACTINOMYCETES, VOL. Ill
(1) pH indicator; blue at alkaline pH and
red at acid pH
(2) Indicator; yellow at acid pH and violet
at alkaline pH
(3) Ba.sic polypeptide
c. Essentially water-insoluble substances
(1) Violet substances
(2) Reddish substances
(3) Yellow substances
(4) Colorless or white substances
(a) Acidic substances
(b) Neutral substance
(c) Basic substance
(d) Polypeptides
(e) Miscellaneous compounds, soluble
in chloroform
(f) Miscellaneous compounds, insoluble
in chloroform
(g) Solubility in chloroform not known
(5) Color unknown
(a) Basic compoiuid
(b) Acidic compound
(c) Antibiotic soluble in chloroform
d. Miscellaneous compounds
(1) Mixture of lytic enzymes
(2) Toxic pigment
(3) Bright yellow pigment
coelicolorin
vinacetin
actinoidin
mycetin
antibiotic 1212
microcins
pluramycin B
actiduins
antibiotic K 125a
vinacetin
zaomycin
antibiotic X 206
antibiotic X 464
aspartocin
nigericin
longisporin
mesenterin
aspartocin
phytoactin
antibiotic F 416
antibiotic J 4
exfoliatin
griseomycin
lustericin
sulfactins
griseoflavin
primycin
antibiotic E 129 (component
O)
monamycin
cardicin
phalamycin
actinomycctin
bacteriolytic factors
antivirubin
chromomycin A3
KEY NO. 2
Antibiolics Which Are Active against Gram-positive and Gram-negative Bacteria
A. Antibiotics for which one maximum of light absorption has been reported
1. 218.5 antibiotic PA 132
KEYS TO ANTIBIOTICS
147
B.
2.
220.5
griseoviridin
3.
226 in water at pH 6.0
cycloserine
4.
228 (very weak maxima at
380
, 394, and 413)
streptozotocin
5.
235
caerulomycin
6.
243
echinomycin
7.
244.5 in ethanol
streptovaricins
8.
252 in water
antibiotic 10 CM
9.
256 in water at pH 7.0
niicleocidin
10.
259 in acid
psicofuranine
antibiotic U 9586
11.
264 in 0.1 M phosphate l)uffer
at pH 7.0
actinobolin
12.
265 in water
cellostatin
13.
265 to 275 at pH 1 to 6
xanthomycin C
14.
267.5 in 0.1 .V HCl
puromycins
If).
268 in 0.1 .V HCl
viomycin
10.
270 to 272 in water
homomycin
hygromycin
17.
272
antibiotic PA 147
18.
275 in 0.1 .V HCl
blasticidin S
19.
275 to 280
snlfocidin
20.
278 in watei-
chloramphenicol
21.
280 to 282 in acid
285 in methanol
\-a neomycin
matamyciii
22.
293 in water
violacetin
23.
299 in 0.1 .V XaOH
cellocidin
24.
300 to 320 in ethanol, more
active against gram-
cnteromycin
negative bacteria than ag
ainst gram-positive
bacteria
25.
306 in 50 per cent ethanol
bamicetin
2G.
313 to 314 in ethanol
azomycin
27.
365
streptocardin
28.
370 in methanol
thiomycin
29.
388 in ethanol
thiohitin
30.
500 to 530
rhodocidin
Antibiotics for which two maxima
of light absorption have been reported
1.
215 and 315
mitomycin fraction R
2.
223 to 225 and 322 in ethanol
methymycin
3.
227 and 303 in 0.1 N HCl
pyridomycin
4.
230 to 240 and 334 to 340
in 0.1 .V HCl
abikoviromycin
5.
232 and 370 in methanol
thioaurin
6.
235 and 300 to 305 in 0.03
A^XaOH
althiomyc-in
7.
238 and 295
netropsin
8.
243 and 318
levomycin
9.
244 and 274
DON
10.
244.5 and 2()3 in ethanol
streptovaricins
11.
244 and 295 to 300
phleomycin
12.
245 and 275
diazomvcins
148
THE ACTINOMYCETES, VOL. Ill
13. 245 and 380 in methanol
14. 248 and 308 in 70 per cent ethanol at pH 7.5
15. 250 and 320
16. 257.5 and 390 to 402 in pH 0.0 phosphate buffer
17. 262 and 364
18. 265 and 362 in methanol
19. 265 and 420
20. 267 and 281
21. 270 and 330 to 340
22. 270 and 370
23. 281 to 283 and 342 to 344 in methanol
24. 288 and 460 in ethanol
25. 290 and 330 at pH 1 or ()
26. 498 and 532 in n-bntanol
streptonigrin
novobiocin
nitrosporin
xanthothricin
rnticin
griseolutein A
grisein
mycomycin
pleomycin
oxytetracycline
griseolutein B
xanthomycin A
xanthomycin B
\'iolarin
C. Antibiotics for which three maxima of light absorption ha\'e been reported
mitomycin A and B
mitomycin C
antibiotic X 340
bromtetracycline
antibiotic 7, 080 R. ;
chlortetracycline
fervenulin
1. 215, 316 to 318, and 530 in water
2. 216, 360, and 560
3. 218, 289, and 370 in ethanol
4. 227, 260, and 370 in 0.1 .V HCl
5. 227, 271, and 304
6. 230, 275, and 367.5 in water
7. 239, 270 to 280, and 340 in pH 7.8 phosphate
buffer
8. 240, 278, and 384 in 0.1 .¥ HCl cyanomycin
9. 248, 312, and 388 aureothricin
10. 250, 303, and 390 in ethanol holomycin
11. 250, 311, and 388 in ethanol thiolutin
12. 288, 301, and 316, tetraene tennecetin
13. 292, 308, and 320, tetraene endomycin A
14. 320, 415, and 490 in n-butanol rubidin
15. 338, 359, and 380, hexaene endomycin B
D. Antibiotics for which four maxima of light absorption ha\'e been reported
1. 207, 237, 286, and 343 mitomycin fraction Y
2. 222, 266, 286, and 369 in methanol nybomycin
3. 228, 258, 288, and 427 aklavin
4. 235, 269, 298.5, and 365 in methanol tetracycline
5. 240, 255, 305, and 375 albofungin
E. Antibiotics which exhibit either only end-absorption of ultraviolet light, or no
typical light absorption, or for which no light-absorption data have been reported
1. Red substances
2. Indicator; red in alkaline solution, yellow in
acid solution
3. Yellow substances
a. Acidic
b. Basic, water-insoluble
antibiotic of Chandrasekhar
cladomycin
nocardorubin
antibiotic of Rolland
raisnomycin
KEYS TO ANTIBIOTICS
149
4. Essentially white substances, basic, water-
soluble
a. Positive Sakaguchi reaction; negative nin-
hydrin reaction
streptomycin group
streptomycin
pseudostreptom^Tin
mannosidostreptomycin
b. Positive Sakaguchi reaction, positive nin-
hNTlrin reaction
c. Positive Sakaguchi reaction; ninhydrin re-
action not reported
d. Negative Sakaguchi and ninhvdrin reactions
e. Negative Sakaguchi reaction; positix'e nin-
hydrin reaction
(1) Substance yielding at least one amino
acid upon hydrolysis
streptothricin group
actinorubin
antibiotic 186
antibiotic 156
antibiotic EI 5
evericin
geomycins
grasseriomycin
grizin
lavendulin
(2) Substances yielding no amino acids upon
hydrolysis; amino sugar compounds
neomycins
kanamycins
catenulin
hydroxymj^cin
f. Unknown ninhydrin and Sakaguchi reactions
antibiotic GB/229?
antibiotic of Alukherjee
hygromycin B
Polypeptides or proteinaceous substances
actinin
antibiotic of Sackmann
echinomycin
neocide
hydroxystreptomycin
dihydrostreptomycin
dihydrodesoxystreptomycin
antibiotic 1943
eurimycin
antibiotic A 6
antibiotic A 20
antibiotic 587/ 13
flaveolin
fuscomycin
roseocitrin B
roseomycin
luridin
mycothricin
pleocidins
racemomycins
roseothricin
streptin
streptolins
streptothricin
desertomycin?
antibiotic GB/229?
monomycni
paromomycin
novomycin?
trehalosamine
neonocardin
virusin 1609
cephalomycin
actinomycetin
bacteriolvtic factors
150
THE ACTINOMYCETES, VOL. Ill
6. Basic substance soluble in water at acid pH, phagolessin
insoluble in chloroform
7. Neutral or weakly basic compound, soluble in phagostatin
chloroform
8. Benzenoid-type compound containing no ni- ramnacin
trogen
9. Contains nitrogen, water-soluble substance desertomycin
totomycin
10. Xo data to permit classification miramycin
KEY NO. 3
Aiilihiotics Wliicli Have Anlifiiiifial Activity'
I. Antibiotics active not only against fungi but also against gram-positive bacteria
A. Active against il/. tuberculosis var. hominis but not against other bacteria
1. Weakly basic substance, light-absorption max- toyocamycin
ima in water at 230 and 277, and in 0.1 to unamycin B
0.05 X HCl at 235 to 236 and 273
2. Oil, light-absorption maximum at 2:55 in hygroscopin A
ethanol
B. Acti^'e against strains of Nocardia asteroides, but not against other bacteria or
streptomycetes
1. Light-absorption maxima at 203 and 268 solu- flavofungin
l)le with difhculty in water
2. Basic, water-soluble substance eulicin
C. Active against strains of Nocardia and Sfreptomijccs, but not against bacteria
1. Light-absorption maximum at 302 in meth- eum3^cetin
anol
D. Active against gram-positive bacteria sfricto scnsu
1. Substances for which one maximum of light al)-
sorption has been reported, as follows:
a. 230 in methanol
b. 240, with shoulder at 255 to 270; yellow,
water-insoluble substance
c. 270 in methanol; yellow substance
d. 275 in methanol
e. 304 in ethanol
f. 310, with strong end-absorption below 250 br,vamycin
in 6 N H2SO4 ; sulfur-containing polypep-
tide
2. Substances for which two maxima of light ab-
sorption have l)cen reported
a. 215 and 257 to 258 in methanol mycospocidin
b. 230 and 285 in water (luestiomycin B
c. 240 and 267 to 268 in ethanol or methanol azalomycin F
musarin
melanosporin
lu'grostatin
xanthicin
antibiotic F 256
seligocidin
In this key, actinomycetes, including mycobacteria, are considered as gram-positive bacteria.
KEYS TO ANTIBIOTICS
151
d. 248 and o20 to o2r) in methanol
e. 244 and 284 in methanol
f. 245 and 27:)
g. 24.-) to 2:)0 and 400 to 4:)0
Substances for which three maxima of light al)
sorption have been reported
a. Pentaenes; about 317, 333, and 3.")0
b. Hexaenes; about 338, 3o7, and 378
antibiotic ¥ 43
antibiotic l-81d-ls
diazomycins
ractinomycins
pentaene antifungal antibi-
otics I and II
cryptocidin
flavacid
mediocidin
questiomycin A
c. 240, 410, and 42.") in cyclohexane
4. Substances which have been reported to have antibiotic F 416
only end-absorption in ultraviolet light lustericin
5. Substances giving a red color at alkaline reac-
tion
a. Heating with XaOH produces red color and camphomycin
a basic gas
b. Greenish yf>llow; acti\'e mainly against
phages; gives inactixc red solutions at alka-
line reaction
6. ^liscellaneous substances
a. Yellowish red to reddish ^■iolet substances
antil)iotic l-81d-ls
chrvsomvcin
t). Xonsulfur-containing polypeptide
c. Acidic substances
ractmomycms
microcins
phytoactin
phytostreptin
nigericin
cardicin
duramvcin
d. Acidic polypeptide
II. Antibiotics active not only against fungi, but also against gram-positi\-e and gram-
negati\'e bacteria
A. Substances for which one light-absorption maximum has been reported
1. 235, with shoulders at 285 and 295 caerulomycin
2. 244, with a shoulder at 2()3 and a plateau at streptovaricins
316; bright orange materials; yellow in acid
solution, red-amber in alkaline solution
3. 2()5 in water cellostatin
4. 275 in 0. 1 N HCl or at 266 to 270 in 0. 1 .V Xa( )H blasticidin S
5. 275 to 280 in ethanol; contains sulfur sulfocidin
6. 293 in water violacetin
7. 388 in ethanol thiolutin
B. Substances for which two light-absorption maxima have been reported
1. 232 and 370 in methanol; contains sulfur thioaurin
2. 244 and 274 DON
3. 266 and 285 in ethanol; water-insoluble base nybomycin
4. 267 and 280, with an inflection at 256; very un- mycomycin
stable acid
152 THE ACTIXOMYCETES, VOL. Ill
C. Substances for which three Ught-absorption maxima have been reported
1. 239, 270 to 280, and 840 in pH 7.8 phosphate fervenuhn
buffer
2. 248 to 2o0, 311 to 312, and 388 in etlianol aureothricin
thiohitin
3. 288 to 292, 300 to 308, and 315 to 320; tetraenes endomycin A
tennecetin
4. 338, 359, and 380; hexaene endomycin B
D. Substances for which more than three hght-absorption maxima have been reported
1. 228, 258, 288, and 427; reddish purple color aklavin
with H2SO4
2. 240, 255, 305, and 375; yellow, water-insoluble albofungin
substance
E. Water-soluble substances with no specific light-absorption spectra, but often with
end-absorption of ultraviolet light
actinorubin grizin
antibiotics 136, A 20, GB/229, hygromycin B
and 587/13 mycothricins
cephalomycin streptothricin
grasseriomycin virusin 1609
F. Miscellaneous substances
1. Basic substance, soluble in water, yellow in flaveolin
acid solution, reddish in alkaline solution
2. Water-insoluble substance; can be autoclavcd ramnacin
from pH 2 to 10
III. Antibiotics active mainly against fungi, and with little or no activity against bacteria
A. Substances for which \'isible and or ultraviolet light al)sorption has been reported
1. Tetraenes: light-absorption maxima at about 290 to 292, 300 to 305, and 317
to 320
amphotericin A nystatin
antibiotic PA 86 pimaricin
antibiotic PA 166 protocidin
antifungal antibiotic 7071 R. P. rimoeidin
antifungal antibiotic J 4B sistomycosin
antimycoin tetraene antifungal antibiotic
chromin tetrin
endomycin A unamycin
etruscomycin
2. Pentaenes: light-absorption maxima at about 317 to 324, 330 to 340, and 349
to 358
aliomycin fungichromatin
antibiotic PA 153 lagosin
cabicidin moldcidin A
eurocidin pentaene antifungal antibi-
filipin otics I and II
fungichromin pentamycin
KEYS T(3 ANTIBIOTICS
153
3. Hexaenos: light-absorption maxima at
about 335 to 338, 355 to 359, and
to 380
ciyptocidin
mediocidin
eiidomyein B
mycelin-IMO
flavacid
4. Heptaones: hght-absorptioii maxima at about
358 to 3()6, 377 to 388,
399 to 410
amphotericin B
ascosin
antibiotic 20/ 1
aureofacin
antibiotic AYF
ayfactin
antibiotic PA 150
candicidin
antifungal antibiotic 757
candidin
antifungal antibiotic A 228
candimycin
antifungal antibiotic of Rao
grubilin
and Uma
perimycin
antifungal heptaenc F 17C'
trichomycin
5. Xonpolyenic substances for which one 1
ght -ab-
sorption maximiun has been reported,
as fol-
lo^^'S :
a. 216
blasticidin A
b. 235 in ethanol
hygroscopin A
c. 237.5 in methanol
elaiomycin
d. 250.5 in pH 7.0 phosphate butter
azaserine
e. 251 in methanol
flavensomycin
f. 258
niger factor
g. 287
cycloheximide
h. 290
fermicidin
i. 302 in methanol
eumycetin
J. 320
moldin
). Xonpolyenic substances for which tw
light -
absorption maxima have been reported
a. 212 and 260
antifungal antibiotic J 4A
b. 225 and 232 in ethanol
oligomycins
c. about 225 to 228 and 320 to 330
antimycins
blastmycins
d. 227 and 337
vulgarin
e. 230 and 280 in water
monilin
f. 232 and 291 in methanol
streptimidone
g. 233 to 236 and 272 to 273 in 0.05 X
HCl
unamycin B
vengicide
h. 242 and 292 in methanol
fradicin
i. 245 and 285
humidin
j. 254 to 255 and 345 in methanol
mycolutein
antibiotic 2814K
k. 263 and 268
flavofungin
1. 270 and 350 in methanol
alomvcin
154
THE ACTIXOMYCETES, VOL. Ill
7. Xoiipolyenic substances for which three or
more light-absorption maxima ha\'e l)een re-
ported
a. 210, 263, and 363
b. 224, 277, and 283 in ethanol
c. 243, 294, 335, 355, and 373 in methanol
d. 272, 372, and 440 to 445 in methanol
8. Substances reported to ha^'e only end-absorp-
tion in ultraviolet light
9. Substances reported to have no absorption in
ultraviolet light
amidomycin
carzinocidin
10. Substances for which hght absorption has not
been reported
a. Soluble in water
bulging factor
antibiotic A 67
b. Insoluble in water
(1) Soluble in chloroform
antibiotic 30-10
cacaomvcetin
mj'coticm
anisomycin
fradicin
1 , 6-dihych'oxyphenazine
niromycins
streptovitacins
cerevioccidin
niromycins
eulicin
mycelin
phaeofacin
(2) Insoluble in chloroform
actmone
rotaventin
KEY NO. 4
Substances, Produced by Actinoniycetes, Which Have Been Reported to Absorb Ultraviolet
and/or Visible Light, and Which Have INot Been Reported to be Active against Bactei'ia
and/or Fungi
I. Substances for which one maximum of light absorption has been reported
A. 206
B. 216
C. 217 in methanol
D. 233 in ethanol
E. 259 in 0.01 N acid or 2()1 in 0.01 N base
F. 260 to 270
G. 260 in water
H. 264
I. 274 in 0.1 N HCl
J. 330 in ethanol
II. Substances for which more than one maximvnn of light absorption has been reported
A. 225 and 337.5 in alkaline aciueous solution antitumor substance 1418A1
B. 230 to 240 and 334 to 340 in 0.1 A^ HCl and 244 to abikoviromycin
246 and 280 to 291 in 0.1 N NaOH
gancidin W
lenamj^cin
antitumor substance 1418A1
hygroscopin B
psicofuranine
melanomycin
angustmycin C
nonactin
isokojic acid
inactone
KEYS TO ANTIBIOTICS
155
C. 235 and 300 in methanol
D. 238 and 335 in acidic water
E. 240 and 285 in pH (i.O phosphate buffer
¥. 242 and 274 in pH 7.0 phosphate buffer
G. 244 and 274
H. 245 and 285
I. 285 and 320
phagocidin
virocidin
carimbose
alazopeptin
DON
antibiotic PA 128
antibiotic 6270
LIST NO. 1
Antibiotics W liicli IIa>e Been Reported to Have Aiitipiotozoaii Activity
I. Substances active against only protozoa
II. Substances active against protozoa and fungi
alioniycin
anisomycin
antifungal antil)iotic A 228
azalomycin ¥
candimvcin
endomycins
etruscomycin
eurocidin
fervenulin
fermicidin
filipin
III. Sul)Stances active against protozoa and gi'am-negative
abikoviromycin
actinobolin
antibiotic 7,080 R. l\
antil)iotic PA 132
azomj^cin
cycloserine
echinomycin
endomycins
fervenulin
hygromycin
geomycins
antibiotic PA 128
protomycin
fradicin
humidin
moldcidin A
nystatin
pimaricin
ractinomycins
rimocidin
rotaventin
streptimidone
strepto\'itacins
trichomycin
and gram-positive bacteria
griseolutein B
neomycin
netropsin
nucleociclin
oxytetracycline
paromomycin
phagolessin
puromycins
sarkomycin
tetracycline
xanthomycins
1\. Substances active against protozoa and gram-positive l)acteria
aburamycin isomer
acetomycin
amphomycin
angolamycin
aureolic acid
azalomycin F
borrelidin
carl>omycins
erythromycins
et am vein
156 THE ACTINOMYCETES, VOL. Ill
granaticin spiramycin
oleandomycin streptocin
prodigiosin-like antibiotic valinomycin
ractinomycins vancomycin
LIST NO. 2
Antibiotic^^ Which Have Been Kepoitetl to Have Antiviral Activity
I. Antil)iotics active only against \'iruses
achromoviromycin notormicni
ehrlichin phagocidin
hygroscopin B phagomycin
myxo\'iromycin virocidm
II. Antibiotics active against virnses and fungi
antimycins i^iger factor
fermicidin niromycins
hygroscopin A
III. Antibiotics active against viruses, fungi, and gram-positive and gram-negative bacteria
aklavin nybomycin
cephalomycin violacetin
grasseriomycin virusin 1609
IV. Antibiotics active against viruses, fungi, and gram-positive bacteria
antibiotic l-81d-ls cardicin
antibiotic F 43 chrysomycin
antibiotic F 256 toyocamycin
antibiotic F 416
V. Antibiotics active against viruses and gram-positive and gram-negative bacteria
bromtetracycline luridin
chloramphenicol mitomycin C
chlortetracycline oxytetracycline
echinomycin phagolessin
enteromycin phagostatin
griseoviridin tetracycline
hygromycin violarm
VI. Antibiotics active against viruses and gram-positive bacteria
actinomycetin erythromycins
antibiotic 1212 flavocidin
antibiotic E 300 heliomycin
antivirubin leucomycin
carbomycins oleandomycin
chartreusin primycin
chromomycins spiramycin
coerulomycin thiostrepton
KEYS TO ANTIBIOTICS
LIST NO. 3
Substances Which Have Been Reported to Have Activity against Tumor!
I. Substances acti\T mainly against tumors
alazopeptin
antibiotic 6270
antitumor substance l-il8Al
antitumor antibiotic E 73
carcinomycin
157
caryomycm
DON
gancidin W
hygroscopin B
lenamycin
melanomycin
psicofuranine
raromycin
II. Substances active against tiunors and t'lmgi
aliomycin tilipin
antimycins hygroscopin A
aureothricin peutamycin
azaserine rimocidin
candimycin str(>ptovitacins
carzinocidin sulfocidin
cellostatin to.yocamycin
diazomycins
III. Substances active against tumors and gram-positive and gram-negative bacteria
actinin griseolutein B
actinobolin mitomycins
aureothricin neocide
antibiotic U 9r)8() netropsin
azomycin psicofiu'anine
cellostatin puromycins
desertomycin sulfocidin
echinomycin streptonigrin
lY. Sul)stances active against tumors and gram-positive bacteria
aburamycin and isomer diazomycins
actinofiocin etamycin
actinoleukin gancidin
actinomycins hygroscopin A
actinoxanthine mutomycin
amicetin netropsin
antitumor antibiotic 289 pluramycins
borrelidin ractinomycins
carzinophilin A sarkomycin
cellocidin toyocamycin
chromomycins t ubercid i 1 1
158
THE ACTINOMYCETES, VOL. Ill
LIST NO. 4
Amino Acids Identified (or Suspected to Be
for Which Abstracts Have Been Written
Alanine
alazopeptin
antibiotic 6270
bryamycin
cephalomycin
geomycin
matamyoin
melanomycin
X neooide
phalamycin
phytoactin
phytostreptin
telomyc'in
thiostrepton
vinaotins A and B
L- Alanine
echinomycin
etamyein
Alanine (X-methyl)
ac'tinomyeins
Alanine (,S-(2-thiazole)-/3-)
bottromycin
a-Aminobntyrie acid
staphylomycin
a-Amino-/3-phenylbutyric acid
bottromycin
Arginine
cephalomycin
cinnamycin
matamycin
melanomycin
neocide
phytoactin
phytostreptin
Aspartic acid
amphomycin
aspartocin
Present; in the Ilych-olysates of Antibiotics
Aspartic Acid — continued
cephalomycin
cinnamycin
duramycin
geomycin
neocide
telomycin
thiostrepton
vancomycin
vinactins A and B
Cysteine
bryamycin
griseoviridin
matamycin
neocide
Cystine
bryamycin
carzinocidin
neocide
thiostrepton
a ,;5-Diaminopropionic acid
viomycin
Glutamic acid
alazopeptin
carzinocidin
cephalomycin
duramycin
geomycin
grisein
melanomycin
neocide
noformicin
thiostrepton
vinactins A and B
lycine
amphomycin
antimycins
aspartocin
KEYS TO ANTIBIOTICS
159
Glycine — contin ued
bottromycin
bryamycin
carzinocidin
cephalomycin
cinnamycin
duramycin
geomycin
matamycin
melanomycin
mikamycin
mycospocidin
neocide
phalamycin
phytoactin
phytostreptin
telomycin
thiostrepton
vinactins A, B, and C
Histidine
melanomyein
Isoleucine
bryamycin
phalamycin
phytoactin
phytostreptin
thiostrepton
Isoleiicine (D-allo-)
actinomycins
Isoleucine (methyl-)
actinomycins
duramycin
Lanthionine
duramycin
Lanthionine (meso-)
cinnamycin
Leucine
antibiotic 156
cephalomycin
Leucine — contin ued
melanomyein
phytoactin
phytostreptin
thiostrepton
D-Leucine
etamycin
Leucine (dimethyl-)
antibiotic 6270
etamycin (L-/5,X-)
Lysine
antibiotic 156
carzinocidin
neocide
thiostrepton
vinactins A and B
^-Lysine
geomycin
mycothricin
racemomycin
roseothi'icin
streptolins
streptothricin
viomycin
Xorvaline
staphylomycin
Ornithine
duramycin
grisein
Phenylalanine
cephalomycin
cinnamycin
duramycin
melanomyein
staphylom_vcin
Phenylalanine (3'-desoxy S'-p-methox}^-)
piu'omycin
Picolinic acid (3 hydroxy-)
etamycin
160
THE ACTINOMYCETES, VOL. Ill
Proline
amphomycin
antibiotic 156
cinnamycin
duramycin
melanomyc'in
neocide
phytoactin
phytostreptin
staphylomycin
thiostrepton
L-Proline
aetinomycins
aspartocin
mikamycin
D-Proline (allohydroxy-)
ctamycin
Proline (4-hydroxy-)
aetinomycins
Proline (4-keto-)
aetinomycins
Sarcosine
aetinomycins
etamycin
vinactin C
Sarcosine (L-a-phenyl-)
etamycin
Serine
antibiotic 156
antibiotic 6270
geomycin
grisein
griseoviridin
matamycin
mycothricin
neocide
phytoactin
Serine — continueed
phytostreptin
vinactins A, B, and C
D-Serine
echinomycin
L-Serine
\'iomycin
Streptolidine
geomycin
racemomycin
roseothricin
streptolins
streptothricin
Threonine
bryamycin
cephalomycin
geomycin
staphylomycin
telomycin
thiostrepton
L-Threonine
aetinomycins
etamycin
pyridomycin
Tryptophan
thiostrepton
Tyrosine
cephalomycin
Valine
amphomycin
antibiotic 156
aspartocin
bottromycin
cephalomycin
cinnamycin
duramycin
KEYS TO ANTIBIOTICS 161
Valine — continued D-Valine — continued
phytoactin valinomyein
phytostreptin L- Valine
thiostrepton valinomyein
D-Valine L-Valine (N-methyl-)
actinomyeins aetinomycins
amidomyein echinomycin
Descriptions of Antibiotics
A b i k o V i ro in vein
Produced by: Strepiuiiiyces abikoensian (1 , 2), also
known as S. abikoensis (3). This culture also
produces a heptaene antifungal substance and
viomycin (7). S. rubescens (1, 2); S. reticidi var.
[atumcidicus (5), said to resemble <S'. abikocusnin
(6); Slreptomyces sp. (4).
Synonyms: Latumcidin (6), virocidin (?).
Method of extraction: I. Broth-filtrate treated
with ethyl acetate at neutrality. Extract con-
centrated and chromatographed on alumina after
addition of petroleum ether; column developed
with petroleum ether, then with ethyl acetate.
Exceedingly unstable; glucose must be added
before the solvent can be removed in vacuo (1, 2).
II. All procedures are carried out in the cold
luider N-i. Broth is adjusted to pH 8, and extracted
with methyl isobutyl ketone. Back-extraction into
water at pH 2.0. Extraction from water with ether
at pH 8, and re-extraction into water at pH 2.0.
Extraction of aqueous layer with methyl isobutyl
ketone at pH 8, followed by addition of concen-
trated H)S04 to precipitate the active substance.
Re-crystallized from methanol or ether (5).
Chemical and physical properties: Unstable
basic substance (5). Sublimes during freeze-drying
(1, 2). The sulfate forms white needles, m.p.
U0-14O.5°C (decomposition) (5) or 120-125°C
(decomposition) (6). Soluble in water and meth-
anol. Slightly soluble in ethanol, butanol, and
acetone. Insoluble in other organic solvents (5).
Ultraviolet absorption spectrum maxima at 230
to 240 niM and 334 to 340 m^ in 0.1 N HCl; or at
244 to 246 niM and 280 to 291 m^ in 0.1 N NaOH
(6). Infrared spectra given in ref. 6. [a]p = +148.9°
(c = 0.1 per cent in 0.1 A'' NaOH). Positive diazo,
Bayer, and bromine tests (5). Conflicting reports
on Molisch and ToUen silver mirror tests. Nega-
tive FeCl.? , Fehling, Ehrlich, Sakaguchi, nin-
hydrin, biuret, and xanthoproteic tests, and tests
for primary, secondary, and amyl amines. Turns
red after decomposition (1, 2, 5). Most unstable at
pH 4 to 7 (5) and less stable in water and ethanol
than in other solvents. Readily auto-oxidizetl (1, 2).
CnHiaO-iNHeSOi- C = 46.6%; H = 5.13%; () =
31.18%; N = 4.99%; S = 12.10% (5).
Biological activity: Active on western and
eastern equine encephalomyelitis viruses in con-
tact tests, but not on Venezuela equine encephalo-
myelitis or on Japanese B encephalitis viruses
(1, 2, 6). Weakly active on bacteria and myco-
bacteria. Conflicting reports on antifungal ac-
tivity (1, 2, 5). Active on Tetrahymena at 5 )ug
per ml (5).
Toxicity: LDoo (niice) 8 mg per kg (1, 2) or 17.3
mg per kg (5) intravenouslj', and 100 mg per kg
subcutaneously (1, 2).
References :
1. Umezawa, H. et al. Japan. Med. J. 4:
331-346, 1951.
2. Umezawa, H. et al. J. Antibiotics (Japan)
5: 469-476, 1952.
3. Okami, Y. J. Antibiotics (Jai)aii) o: 477-
480, 1952.
4. Umezawa, H. et al. Jajjan J. Med. Sci. &
Biol. 6: 261-268, 1953.
5. Sakagami, V. et al. J. Antibiotics (Japan)
11 A: 6-13, 1958.
(). Sakagami, Y. et al. J. Antibiotics (Japan)
11 A: 231-232, 1958.
7. Arai, T. et al. Antibiotics & Chemotherapy
7: 435-442, 1957.
Aburaniycin
Produced by: Slreptomyces aburaviensis.
Synonym: Similar to aureolic acid, but differs in
chemical tests.
Method of extraction: Broth adjusted to pH 9.5
and filtered. Filtrate extracted at pH 2.0 with
ethyl acetate. Ethjd acetate back-extracted with
water at pH 9.0. Chromatography on alumina
and development with 80 per cent methanol.
Active fractions concentrated in vacuo. Addition
of water to concentrate precipitated aburamycin.
Re-crystallization from aqueous ethanol.
Chemical and physical properties: Weakly acidic
substance. Yellow crystals; m.p. 163-165°C. Very
soluble in ethanol, acetone, ethyl acetate, butyl
acetate, butanol, and chloroform. Insoluble in
water, ether, benzene, ligroin, and petroleum
ether. C = 55.57%; H = 7.54%; O = 36.89%.
No N, S, or halogen. Positive Molisch, Fehling,
Benedict, Tollen, Seliwanoff, anthrone, and orcin
tests. Negative biuret, xanthoproteic, ninhydrin,
Tollen phloroglucine, FeCls , Folin, and tyrosine
reactions. [a]o = +24.56° (c = 1 per cent in
methanol). Ultraviolet absorption spectrum
maxima at 230, 276, and 410 m/i with weak maxima
at 320, 330, and 350 m/i (95 per cent ethanol); or
229, 276, and 410 ran with weak maxima at 316 and
330 niM (0.01 N HCl); or 234, 278, 316, and 410 m^
(0.01 N NaOH). Infrared spectrum given in
reference 1. More stable at alkaline than acid pH.
Biological activity: Active on gram-positive
162
DESCRIPTIONS OF ANTIBIOTICS
163
bacteria and M. phlei. Not active on other myco-
bacteria, gram-negative bacteria, or C. albicans.
Is 64 times more active on Staph, aureus at pH 6.0
than at pH 8.5. Some antitmiior activity in mice
against the ascitic form of Ehrlich carcinoma and
the solid form of Crocker sarcoma 180.
Toxicity: LD50 (mice) 2 mgper kg intravenously,
and 2.5 mg per kg subcutaneously. Not well
absorbed from intestinal tract.
Reference: 1. Nishimura, H. et al. J. Antilnotics
(Japan) 1(»A: 205-212, 1957.
Abiiraniycin Isomer
Produced by: Streptomyces sjj. differing from
S. aburaviensis .
Synonym: Antibiotic M5-18903. Both aburamy-
cin and its isomer have similarities with aureolic
acid.
Method oj extraction : I. Broth-filtrate extracted
with chloroform at pH 7.2 to 7.5. Precipitated from
extract by addition of petroleum ether. Chromato-
graphed on alumina with chloroform as solvent
and developer. Elated with 5 per cent ethanol in
chloroform. Active fractions concentrated under
reduced pressure and precipitated with petroleum
ether. Crystallized from chloroform on addition
of petroleum ether. II. Same as that for aburamy-
cin.
Chemical and physical properties: Weakly acidic
substance. Yellow crystals; m.p. 169-171°C.
Soluble in chloroform, acetone, pyridine, ethyl
acetate, dimethylformamide, ethanol, and meth-
anol. Insoluble in water and petroleum ether.
Ultraviolet absorption spectrum maxima at 227
n\^l (E/e'm 200 ± 5), 278 m^ (£'i'cm 400 ± 10), and 400
mix. Weak maxima at 304, 317 (E'l'L 60 ± 2), and
330 viXix (ethanol). Infrared spectrum given in
reference 1. [aj" = —29° (c = 0.5 per cent in
methanol). pKl = 7.1. C = 53.32%; H = 7.44%;
= 35.24%. No N, S, or halogen. Molecular
weight, 1295. Paper chromatographic l)ehavior is
indistinguishable from aburamycin. Can be hydro-
genated to yield a biologically active compound
which has similar ultraviolet spectra in alkaline
and acid solution to the hydrogenated product
from aureolic acid. Acetylation of the hydro-
genated product destroys the biological activity.
The acetyl derivative has m.p. at 205-207°C.
Biological activity: Active on gram-i)ositive
bacteria, including mycobacteria. Not active on
gram-negative bacteria. Not active on bacteria
in vivo. Some activity on Endamoeba histolytica in
vivo. Reduces the worm burden in mice infected
with Syphacia obvelata. Some protection against
leukemia P-1534 in mice, but no activity on sar-
coma 180.
Toxicity: LD50 (mice) 2 mg per kg intravenously.
Mice tolerate a single oral dose of 80 mg per kg.
The antibiotic was shown to be unabsorbed from
the injection site.
Reference: 1. Gale, R. M. et al. Antibiotics Ann.
489-492, 1958-1959.
Acetoniycin
Produced by: Streptomyces ramiilosis (1).
Method of extraction: Broth filtered with a filter-
aid, and filtrate extracted with ethyl acetate.
Extract concentrated in vacuo. Addition of petro-
leum ether to the residue precipitated acetomycin.
Crystallized in the cold from hot methanol (1).
Chemical and physical properties: Neutral,
saturated, ketoacetoxy lactone, with three
methyl groups. Colorless, rough rod-like crystals.
Sublimes at 70°C; m.p. 115-116°C. [a|„ = -167°
(c = 1.47 per cent in ethanol). CuiHuOs:
C = 55.96%; H = 6.65%; O = 37.31%. No ultra-
violet absorption. Infrared spectrum given in
reference 1. Forms a mono-2,4-nitrophenylhy-
drazone: dark yellow crystalline leaves; m.p. 205-
208°C. Positive iodoform reaction. Negative FeCls
test. Structural formula given in Chapter 6.
Bi-jlogical activity: Weak activity on gram-
positive and gram-negative bacteria (100 ^g per
ml); moderateh' active on M. tuberculosis H37Rv
(10 fig per ml), Trichomonas foetus (25 /ig per ml),
and E. histolytica (70 /ug per ml) (1).
Toxicity: LD50 (mice) 100 mg per kg subcutane-
ously (1).
References:
1. Ettlinger, L. et al. Helv. Chim. Acta 41:
216-219, 1958.
2. Keller-Schierlein, W. ei al. Helv. Chim.
Acta 41: 220-228, 1958.
Achroinoviromycin
Produced by: Streptomyces achromogenes.
Remarks: The same strain produces sarcidin.
Method of extraction: Extraction with ethvl ace-
tate at pH 2.0, concentration in vacuo at 40°C.
Freeze drying after addition of 2.0 i)er cent
NaHCO:, to pH 7.5
Chemical and physical properties: Negative
Molisch, Tollen, ninhydrin, Sakaguchi, Millon,
Pauly, and Fed., tests.
Biological activity: Active in vitro and in vivo
against Japanese B encephalitis virus. Not active
against western equine encephalitis virus.
Toxicity: 20 mg per mouse of the crude sul)stance
164
DESCRIPTIONS OF ANTIBIOTICS
did not induce any toxic signs when injecletl sul)-
cutaneously.
Reference: 1. Umezawa, H. et al . Japan. J. Med.
Sci. tt Biol. 6: 261-268, 1953.
Actidiiins
Produced by: Streptomyces sp.
Method of extraction: Isolated from broth-
filtrates and mycelial extracts by acid, or complex
precipitation, solvent extraction, or absorption
techniques. Purified by solvent precipitation,
chromatography, and countercurrent distribution.
Chemical and physical properties: Complex com-
posed of six components, very closely related.
Yellow to reddish substances. Darken at 280°C,
but do not melt up to 350°C. Insoluble in water and
slightly soluble in butanol, ethanol, pyridine, ace-
tic acid, cyclohexane, and dimethylformamide.
Certain variation exists among the components as
to solubility in acetone, methanol, and chloroform.
Soluble in strong mineral acids, and are precipi-
tated on dilution. Most stable at acid and neutral
pH; unstable to alkali. Not readily diffusible.
Reineckates: Soluble in acetone. Rf (butanol-ace-
tic acid-water, 62:12:26) 0.95 (tail). Infrared data
given on components II, III, and VI in reference 1.
All give a yellowish fluorescence in organic sol-
vents; is less intense in aqueous solvents. Contain
C, H, N, and S, and in one case, CI. Acid or alka-
line hydrolysates contain a fluorescent acidic
product and a ninhydrin-positive material com-
posed of a neutral and an acidic fraction.
Biological activity: Active on gram -positive
bacteria. Not active on gram-negative bacteria or
mycobacteria.
Reference: 1. Burton, H. S. Chem. & Ind.
442-443, 1955.
Acliniii
Produced by: Streptothrix {Streptoutyces) felis.
Synonym: Mycetin.
Remarks: Organism isolated from a gramdomat-
ous lesion of a cat; pathogenic for various animals.
Method of extraction: No data.
Chemical and physical properties: Basic poly-
peptide.
Biological activity: Active (in mice) on Ehrlich
adenocarcinoma when administered as the cidture
fluid, destroying the tumor completely. DeAngelis
(1, 2) claims to have first demonstrated, in 1936,
the cancerolytic properties of this culture, and he
considers it to be the first antitumor antibiotic.
Active on gram-positive and gram-negative
bacteria.
Toxicity: Said to be nontoxic when given in doses
which destroy tumors.
I 'lilization: Is said to have caused complete
regression of certain types of cancer in man.
References:
1. DeAngelis, (i. Oncologia 2: 43-62, 1949.
2. DeAngelis, G. Ateneo parmense 28: 248-
260, 1957.
Vfliiioholiii
Produced by: Streptomyces sp.
Method of extraction: Broth-filtrate adsorbed on
Darco G-60 at pH 4.0. Eluted with 30 to 40 per cent
aqueous acetone. Eluates adjusted to pH 3.5 and
concentrated in vacuo. Chromatographed on
Decalso (at pH 6.0) and eluted with 5 per cent
aqueous acetic acid containing 10 per cent ethanol.
Active fractions adsorbed on a Darco G-60-Celite
545 mixture (1:1) and eluted with 20 pei cent
aqueous acetone. Active fraction adjusted to pH
3.5, concentrated in vacuo, and mixed with 0.02 M
cupferron (C6H5N(NO)ONH4) in 50:50 n-butanol-
chloroform to remove iron. Aqueous layer washed
with chloroform, concentrated in vacuo at pH 3.2,
then freeze dried. Purification by salt conversion.
Sulfate crystallized from vvater-ethanol (25:45).
Occasional difficulties were encountered because
of the tendency of actinobolin to form complexes
with aluminum (and other Group III elements).
Aluminum was removed by oxine precipitation or
preferential adsorption on Dowex 50 columns (2).
Chemical and physical properties: Amphoteric
substance. Base is amorphous, white, flufl'y pow-
der. Hydrophilic; verj^ soluble in water. Could not
be extracted from aqueous solution with any com-
mon water-immiscible organic solvent. Basic:
1)K;, = 7.5 and a weakly acidic (enolic) pKa = 8.8.
Gives a purple color with ninhydrin, red-orange
with the Pauly diazo reagent, a red color with
FeChi . Positive KMn04 , Fehling, Folin-Cio-
calteu, and iodoform tests. Negative Moiisch,
I'^hrlich (dimethylaminobenzaldehyde), and Elson-
Morgan tests. No reactive carbonyl. Could not be
hydrogenated. One N atom per molecule was lib-
erated by the Van Slyke procedure; the other N
was nonbasic. CnH.o-o.oNaOe-iiHoO: C = 50.31%;
H = 6.88%; N = 9.17%. Sulfate: colorless crystals;
solul)ilities the same as the base. Ultraviolet ab-
sorption spectrum maxima at 263 niyu (a = 26.6) in
0.1 N HCl; at 264 m/x (a = 25.3) in 0.1 M phosphate
butter at pH 7.0; and at 288 m/x (a = 40.6) in 0.1 N
NaOH. Infrared spectrum given in reference 2.
Most stable at pH 3.0; unstable at pH 7.0 and
above, [a]^ = +54.5° (c = 1 per cent in water).
Acetate: white needles, m.p. 263-266° C. Softens
DESCRIPTIONS OF ANTIBIOTICS
165
at 130°C and resolidifies at 145°C. Sohible in water
and soluble in lower alcohols and acetone while
warm. Slightly soluble in ethyl acetate, [a]^ =
+58° (c = 1 per cent in water). Acetylation of the
acetate or free base with acetic anhydride gives a
l)iologically inactive N-acetyl derivative: white
needles; m.p. 254-255°C; plvl = 8.4 (2).
Biological activity: Moderately active on gram-
positive and gram-negative bacteria. Resistance
develops easily. No cross-resistance with other
common antibiotics (1). Protects mice from in-
fections with Staph, aureus and K. pneumoniae at
high doses. No effect on infections with D. pneu-
uLoniae, Pr. vulgaris, Sal. typhimurium, or M.
tuberculosis. Very slightly active against Plas-
modium lophurae, (chicks) but not Sckistosomci
uiansoni (mice), helminths (rat), or protozoa (1).
Protects X-ray irradiated rats against the trans-
plantable human neoplasms H.S. No. 1 sarcoma
and H. ep. No. 3 carcinoma when given 24 hours
after tumor implantation. Ineffective against
these tumors after they were established (4 days
post-implantation) (4). Not effective against H.ep.
No. 3 in mice, hamsters, or eggs (3). Active on
sarcoma 180 (ascites) , Ehrlich carcinoma (ascites) ,
carcinoma 1025, glioma 2(5, Walter carcinosarcoma
256, and slightly active on adenocarcinoma E()771
(5). Active on a large number of transplanted
mouse leukemias, including lines resistant toother
antitumor agents (6).
Toxicity: LD50 (mice) 800 ± 27 mg per kg and
1550 ± 26 mg per kg (rat) intravenously (1).
References:
1. Pittillo, R. F. ('/ at. Antil)iotics Ann. 497-
504, 1958-1959.
2. Haskell, T. H. and Bartz, Q. R. Antibiotics
Ann. 505-509, 1958-1959.
3. Merker, P. C. and WooUey, (i. W. Anti-
biotics Ann. 515-517, 1958-1959.
4. Teller, M. N. et at. Antibiotics Ann. 518-
521, 1958-1959.
5. Sugiura, K. and Reilly, H. C. Antibiotics
Ann. 522-527, 1958-1959.
6. Burchenal, J. H. et al. Antibiotics Ann.
528-532, 1958-1959.
Actinoflocin
Produced by: Streptouiyces sp. resembling <S.
albus (3). This cvilture also produces si.x other
antibiotics (3, 4).
Synonym: Kikuchi's third substance (1).
Method of extraction: I. Broth extracted with
chloroform at acid pH. Chromatography of con-
centrated extract on celhdose powder. Washed
with 20 per cent NH4CI and eluted with water (3,
4). II. Broth adsorbed on IRC-50 (H+ phase) at
pH 7.8 and eluted with 80 per cent acetone. Eluate
extracted with chloroform at pH 5.0 (4).
Chemical and physical properties: Soluble in most
organic solvents. Sparingly soluble in water.
Ultraviolet absorption spectrum maxima at 231
and 276 m/x. Stable in solution from pH 2.0 to 8.0.
Thermostable (2).
Biological activity: Active on Streptococcus
hemolyticus at 1.56 )ug per ml (2). Slightly active on
B. subtilis. Not active on Sarcina lutea, C. albicans
or A. niger (4). Slight activity on ascitic and solid
forms of Ehrlich carcinoma and sarcoma 180 (2).
Toxicity: LD50 (mice) 3 mg per kg intraperi-
toneally (2).
References:
1. Kikuchi, K. J. Antibiotics (Japan) 8A:145-
147, 1955.
2. Katagiri, K. et al. Chemotherapy (Tokyo)
4: 143, 1956.
3. Sato, K. and Katagiri, K. Chemotherapy
(Tokyo) 5: 182-183, 1957.
4. Katagiri, K. el al. Shionogi Kenkyusho
Nempo 7: 715-723, 1957.
Actiiioidiii
Produced by: Nocardia (Proactinouiyces) acti-
noides.
Method of extraction: Atlsorption on activated
carbon. Elution with 80 jjer cent aqueous acetone
at pH 3.0. Purification by chromatography on
"Permutit" (alumina-sodium silicate). Washed
with distilled water and eluted with 5 per cent
NaCl solution. Salt conversion: picrate to hydro-
chloride. Reprecipitation from methanol with ace-
tone. Also forms a reineckate. Carboxylic cation
exchange resins (H+ form) can also be used to
purify actinoidin.
Chemical and physical properties: Basic polypep-
tide. HCl salt: water-soluble, white amorphous
powder. Free base: Little solubility in water except
at pH 8.5. Slightly soluble in methanol and ethanol.
Insoluble in butanol, acetone, ether, and other
nonpolar solvents. Most stable at acid or neutral
pH. N = 7 per cent (Kjeldahl); amino N = 2 per
cent (Van Slyke). Positive Pauly, Molisch, and
biuret reactions. Product of diazotization reacts
with a-naphthol to give a brownish green color.
Negative orcin, FeCls , Fehling, Seliwanoff tests,
and negative test for amino sugars. Fehling reac-
tion becomes positive after 3 to 5 minutes of heat-
ing in 5 per cent HCl. Mild acid hydrolysis yields
two fractions: 1 = 5 per cent HCl -insoluble; II =
5 per cent HCl-soluble. Fraction I contains many
166
DESCRIPTIONS OF ANTIBIOTICS
components giving positive Pauly reactions.
Fraction II is a complex peptide.
Biological activity: Active on gram-positive V:)ut
not gram-negative bacteria. No cross-resistance
with penicillin, streptomycin, albomycin, erythro-
mycin, or chlortetracycline. Resistance develops
slowly. Active in vivo (mice) on pneumococcal
infections; somewhat less active on staphylococcal
or streptococcal infections. Activity considered
equal to chlortetracycline and better than peni-
cillin.
Toxicity: Mice tolerate 2 gm per kg intra-
venously. Not irritating when instilled as a 1 per
cent solution into the eyes of rabbits. Strong in-
flammatory reaction at the site of a subcutaneous
or intramuscular injection. Causes overgrowth of
gram-negative bacteria in the intestine when
given orally. Poorly absorbed from the intestinal
tract .
Utilization: Limited by painful reactions at
injection site.
Reference: 1. Shorin, V. A. et al . Antibiotiki
2(5): 44-49, 1957.
Acliiioleiikin
Produced by: Streptonit/ces aureus and Strepto-
myces sp. (1, 3).
»Sy«.onj/wi; Related to levomycin, antil)iotic F4.3,
and echinomycin.
Method of extraction: Broth-filtrate extracted
with ethyl or butyl acetate. Extract decolorized
with nitric acid-treated alumina. Effluent evap-
orated in vacuo. Can be purified by (a) chromatog-
raphy on alumina with ethyl acetate or developer.
Active fractions concentrated to dryness in vacuo,
washed with petroleum ether, and crystallized
from ethanol. (b) Countercurrent distribution
(80 per cent aqueous methanol-benzene-carbon
tetrachloride, 2:1 : 1) ; active fractions recrys-
tallized from methanol-chloroform (7:3) (I, 3).
Chemical and physical properties : White platelets
or needles; m.p. 213°C (decomposition). Soluble in
chloroform and pyridine. Sparingly soluble in
acetone, warm ethanol, methanol, ethyl and butyl
acetates. Slightly soluble in carbon tetrachloride
and benzene. Insoluble in water. Ultraviolet ab-
sorption spectrum maxima at 243 and 312 ni/u
(solvent not given). Infrared absorption spectrum
in references, [a],?^ = -302° (c = 0.01 per cent in
ethyl acetate). Negative FeCls , ninhydrin,
Molisch, Sakaguchi, biuret, Tollen, and Fehling
tests. Positive pine-splint test. Ehrlich test gives
a yellow color. C29-3nH4o-42N607-8S: C = 56.41%;
H = 6.74%; N = 13.06%; S = 5.01%. Molecu-
lar weight, 648 (Rast) (1, 3).
Bioloi/ical activity: Active on gram-positive
bacteria. Little or no activity against gram-
negative bacteria (1). Kills HeLa cells at 0.06 jug
per ml (2). Slight activity' on Ehrlich ascites
carcinoma (1, 3).
Toxicity: LD^o (mice) 1.5 mg per kg intra-
peritoneally (1).
References:
1. Ueda, M. et al. J. Antibiotics (Japan) 7 A:
125-126, 1954.
2. Umezawa, H. Giorn. niicrobiol. 2: 160-
193, 1956.
3. Ishihara, S. et al. J. Antibiotics (Japan)
llA: 160-161, 1958.
Acliii«»l> sill
Produced by: Streptomyces albicans.
Method of extraction: Nutrient broth inoculated
with a culture of the organism and incubated at
30° C for 4 to 5 days. The lysed cultvire is filtered.
Cheuiical and physical properties: Unstable,
partly destroyed at 80°C and completely destroyed
at 100°C. Can be separated into two constituents
by means of dialysis; neither of these is active by
itself. In contrast to actinophage, the agent is
active upon dead cells as well as upon living.
Biological activity: Active oidy upon the culture
from which it is isolated.
Utilization: Exerts a favorable effect in the
treatment of actinomycotic infections.
Reference: 1 . Dimitriev, S. and Suteev, G. Med.
Parasitol. Parasitic Diseases (U. S. S. R.) No.
4: 1947.
Actinonijceliii
Produced by: Streptaniyces antibioticus.
Method of extraction: Broth-filtrate extracted
with butanol. Extract dried in vacuo, washed with
organic solvents, such as benzene, dissolved in
ethanol, and filtered. Dried in vacuo. Purified by
chromatography on alumina from butanol and
developed with methanol and methyl acetate (3:1)
Chemical and physical properties: Neutral,
yellowish green sul)stance. Solulile in water at pH
7.0 and in ethanol (with intense fluorescence).
Scarcely soluble in chloroform and am.vl acetate;
insoluble in ether and benzene. Treatment with
concentrated HCl gives a red or gray color; pre-
cipitate forms on boiling. Positive Fehling and
FeCls tests. No reaction with alcoholic NaOH,
picric acid, KCN, or sodium bisulfite. Decolorizes
KMn04 . More stable at pH 7.0 than at alkaline or
acid pH. Thermolabile.
Biological activity: Active on gram-positive
DESCRIPTIONS OF ANTIBIOTICS
1(17
bacteria. Not active on gram negative bacteria,
mycobacteria, or fungi.
Toxicity: 25 mg per kg lethal to rats.
Reference: 1. Cercos, A. P. Rev. invest, agr.
2: 147-156, 1948.
ActiiioiiiN celin
Produced by: Sireploniyces albiis and Strepfo-
inyces sp. (1 , 2).
Nenidiks: Antibacterial factor may be similar to
mycomycin.
In discussing Feist mantel's (5) work on the
close genetic relationship of the tuberculosis or-
ganism and the actinomycetes, based upon the
reactions given to tuberculin and by preparations
of Nocardia farcinica (the culture being desig-
nated as Streptothrix farcinica and the preparation
as "farcin poison") in guinea pigs, Mathieson et al.
(6) used in 1935 the term "actinomycetin" for the
extract of the culture designated by them as
Actinomyces farcinica. In 1937, Welsch (7) used
the designation "actinomycetin" for the sterile
filtrates of an actinomycete which possessed bac-
teriolytic properties.
Method of extraction: I. Broth-filtrate at i)H
3.0 saturated to 75 per cent with ammonium sul-
fate. Precipitate taken up in water and reprecipi-
tated by addition of alcohol or acetone at low-
temperatures. The lipoid antibacterial substance
is extracted from the ammonium sulfate precipi-
tate with ether. Ether concentrated to dryness.
Residue taken up in ether, washed with 5 per cent
HCl, then extracted into 5 per cent NaOH. Re-
extracted into ether at acid pH, then into water at
alkaline pH several times (2). The two peptidases
(Fi and Fi) of the staphylolytic principle (see
below) can be separated by chromatography (3).
II. Culture-filtrate concentrated in vacuo at 35 °C
while Nj is bubbled through the solution. Con-
centrate treated with ammonium sulfate at 0°C
and at pH 8.0. Precipitate which forms taken up in
0.033 M K_.HP()4, centrifuged, and dialyzed against
distilled water at low temperature (4).
Chemical and ph\;sical properties: Actinomycetin
is a general term for all the products, having activ-
ity on microorganisms, which are produced by S.
albus and other Streptoniyces spp. It includes the
following: (a) A colilytic principle, which is a
protein and an enzyme and is not identical to the
other proteases or enzymes present in the mixture
(1). (b) A staphylolytic principle containing two
peptidases, Fi and Fo , which are weakly active
alone, but synergistically active when combined.
Fi : White powder or "pic unique" crystals;
soluble in presence of electrolytes at >pH 5.0.
Basic protein having no protease-like activity.
r2 : Brownish powder, not completely purified;
soluble in the presence of electrolytes at >pH 5.0.
No protease-like activity (3). (c) Lytic system
active on livimj streptococci , in part distinct from
the staphylolytic principle, (d) Activity on heat-
killed or living pncumococci represented by four
lytic agents, at least two of which differ from the
staphylolytic, colilytic, and streptolytic prin-
ciples, (e) Activity (lytic) on heat-killed but not
living gram-negative rods, occurring at 60-65°C
more rapidly than thermal sterilization (1). (f)
Other enzymes capable of digesting a variety of
substrates, such as casein, keratin, fibrin, fibrino-
gen, and mucin (3). (g) Antibacterial substance:
yellowish oily liquid. Acidic, probably unsatu-
rated fatty acids. Soluble in dilute alkali, petro-
leum ether, ether, acetone, chloroform, and carbon
tetrachloride. Less soluble in ethanol. Insoluble in
water and dilute acids. Loses activity on exposure
to air (2). (h) Antiviral substance: differs from the
other agents present in the complex. Thermolabile
(4).
Biological activity: The antil)acterial substance
(g) is active on certain gram-positive bacteria
including M. phlei. Not active on gram-negative
bacteria except Flavobacterium sp. Very active on
certain protozoa, such as Paramecium, Glaucoma,
and Colpidium sp. Biological activity inhibited by
complex organic substances of a protein nature (2).
The colilytic principle (a) is active on heat-killed
E. coli (1). The staphylolytic principle (b) is active
on the cell walls of living gram-positive bacteria
(3). The antiviral substance (h) destroys the abil-
ity of red cells to be agglutinated by influenza
MF 1 (A) and Lee (B), but not influenza PR 8 (A)
or Newcastle disease virus; it destroys the hemag-
glutinin of influenza A (MF 1 and PR 8), influenza
B (Lee), and influenza C, l)ut not the Newcastle
disease virus; it destroys the infectivity of in-
fluenza A (PR 8) in contact tests in eggs and mice.
It is not active in ovo. It is active in protecting
mice from influenza infections, if given by the
intravenous route (4).
Toxicity: 1 gm given intraperitoneally kills mice
in 18 hours or less (4). Antibacterial substance was
toxic to tomato plant roots at 5 /ug per ml (2).
Utilization: Study of the structure of bacterial
cells (3).
References :
1. Welsch, M. et al. Proc. Intern. Congr. Bio-
chem., 3rd Congr., Brussels, 1955, p. 413-
415.
2. Welsch, M. Phenomenes d'antibiose chez les
168
DESCRIPTIONS OF ANTIBIOTICS
actinomycetes. J. Duclot, Gembloux,
France, 1957.
3. Salton, M. R. J. and Ghuy.sen, J. M. Bio-
chim. et Biophys. Acta 24: 160-173, 1957.
4. Malchair, R. Giorn. microbiol. 5: 137-157,
1958.
5. Feistinantel, Dr. Centr. Bakteriol Parasi-
tenk. Abt. 1, Orig. 36: 282-290, 406-415,
1904.
6. Mathieson, D. R. et al. Am. J. Hyg. 21 : 405-
431, 1935.
7. Welsch, M. Compt. rend. Soc. Biol. 122: 244-
246, 1937.
.\ctinoinyciiis
Intruduction: Actinomycin A, discovered by
Waksman and Woodruff in 1940 (1), was relegated
to a decade of oblivion because of its high toxicity
(3). Following the fine work on actinomycin C
(Brockmann and coworkers) and actinomycin B
(by workers in England), and the report that
actinomycin C was effective clinically against
certain neoplastic diseases (21), interest in the
actinomycins was vigorously renewed.
Altogether, actinomycins A, B, C, D, E, F, I, J,
K, M, X, and Z, as well as others, have been re-
ported. All are to.xic, red substances that have
similar biological properties. They can be ex-
tracted and purified in similar ways, V)ut they
differ in certain physical and chemical properties.
The designations actinomycin A, actinomycin B,
etc., do not refer to single substances but to
complexes. The components of the actinomycin
complexes are very closely related. Two different
complexes may contain the same components, but
in different proportions. One component may be
found in one complex but not in another.
Brockmann and his coworkers refer to the
various components by the letter of the complex,
with a subordinate arable number; i.e., Xo , Xi ,
X2 , etc. As the numbers become larger, the Rt
values become larger within a single complex,
regardless of the solvent system used. Roussos and
Vining (62), on the other hand, introduced another
system, using subordinate Roman numerals. These
numerals referred to a particular Rf value in a
specific system of paper chromatography and ap-
plied to a component with that Rf, regardless of its
complex. The numeral was used in conjunction
with the letter of the complex to indicate the
source of the component; e.g., Ai , Bn , Div , etc.
Waksman et al. (77) proposed to clarify this
confusion by terming all actinomycin complexes
"mixtures." Each different component of these
mixtures, when purified and characterized, would
be given a roman numeral, with no reference being
made to the complex which was the source of the
component.
Ai , Bi , Xfi(j = Actinomycin I
All , Bii = Actinomycin II
Am , Bill = Actinomycin III
Aiv , Biv , Div , Ci , Ii , Xi = Actinomycin IV
Ay , By , X2 = Actinomycin V
C> = Actinomycin VI
C3 = Actinomycin VII
Thus, what has been called the actinomycin A
complex becomes a mi.xture containing actino-
mycins I to V. All others, such as the new biosyn-
thetic actinomycins {e.g., E, F) would become
"mixtures" until their components were more
fully characterized. This new system will be used
insofar as possible in the following discussion, al-
though previously used terminology will be em-
ployed parenthetically for historical clarity.
It is evident from this, as previously mentioned,
that the same components can appear in different
complexes. Roussos and Vining (62) found by'
circular paper chromatography that four "actino-
mycin mixtures" (A, B, D, and X) contained
actinomycins I, II, III, IV, and V, but in different
proportions (Table 38). This was further verified
when actinomycins I, IV, and V isolated from the
different mixtures \yere shown to have almost
completelj' identical physical and chemical
properties.
In general, actinomycins have a m.p. of about
215-255 °C. They are soluble in benzene, ethanol,
and acetone, slightlj' soluble in water and ether;
insoluble in aqueous dilute alkali and petroleum
ether. All have peak absorption at about 440 to 450
niju and about 240 injj. in ultraviolet light ; many
also show a peak at about 415 to 430 m^u. All re-
ported thus far are levorotatory. Actinomycins
give a transient purple color in ethanolic NaOH,
and a dark red color in concentrated HCl. They
are most stable at pH 6 to 7, relatively stable at
acid pH, and destroyed at alkaline pH.
Actinomycins are polypeptides linked to a
chromophoric ciuinoid moiet}-. This chromophore
has been shown to be 3-amino-l ,8-dimethyl-
phenoxazone-(2)-dicarboxylic acid (4, 5) for
actinomycins I (Xo^), IV(Div , Ci , Ii), V(X2,
By), VK'c-), and VII (C.,), and is believed to be the
same for all actinomycins (see formula in Chapter 6
and references 56, 57, 65, 98). This belief is based
on the fact that the main barium hydroxide hy-
drolysis degradation product of these components
is the same (see references 25, 39, 45, 47, 65). The
structure of despeptido actinomycin is shown hero:
DESCRIPTIONS OF ANTIBIOTICS
169
Table 38
Percentage of actinomycins contained in various mixtures (62, 77)"
Mixture
Actinomycin
I
II
Ill
IV
V
VI''
VII
A type
6.6
2.9
tr.^
66.7
23.8
B type
9.6
tr.
tr.
28.1
59.3
C'' type
18.1
45.7
34.4
D type
tr.
tr.
tr.
100.0
tr.
X'' type
5.1'
5.1
88.6
V
86.5
" See references to "Actinomycins," Part A.
* Roussos and Vining (62) described a VI component in the "B type" mixture, but this is not synony
lous with the VI of Waksman et al. (77).
' tr. = trace.
■^ Contain.s other minor components not listed in the table.
' Probably represents X^ + Xoa + another less well defined fraction.
■'■ Contains other major and minor components not listed in the table.
CH,
The polypeptide portions of the various actino-
mycins contain a variety of amino acids differing
in their identity, arrangement, and number.
Physical and chemical variations observed be-
tween various components appear to be caused by
these differences. To date, all actinomycins de-
scribed contain L-threonine and sarcosine (Table
39).
The complete structures of actinomycins I to
VII are shown below, and some of their properties
are given in Table 40 (44, 57, 63, 65, 95, 101).
— L-valine-N — CH3 N — CHrL- valine — ,
sarcosine
sarcosine
A
B
C
D
-L-threonine L-threonine-
CO
CH3 ^ CH,
Basic structure of actinomycins
Variations from one actinonivcin to the other
occur at the locations marked .4, B, ('. and D as
follows:
Actinomycin I: .1 = L-proline, B = hydroxy-
proline, C = D = D-valine (44).
Actinomycin II: A = B = sarcosine, C = D = D
valine (101).
Actinomycin III: . I = proline, B = sarcosine,
(' = JJ = D-valine (101).
Actinomycin IV: ,1 = B = L-proline, (' — D = D-
valine (65).
Actinomycin V: .1 = proline,/? = 4-ketoproline,
(• = JJ = D-valine (95).
Actinomycin VI: C = D-valine, D = D-alloiso-
leucine,.! = B = L-proline (57).
Actinomycin VII: r = D = D-alloisoleucine,
A = /i = L-proline (57).
Actinomycins which have been characterized
biologically are active on gram-positive bacteria,
less active on mycobacteria, and inactive on fungi
and gram-negative bacteria, although their activ-
ity varies quantitatively. One report of antiviral
activity exists (80). Many are active on tumors in
animals, and certain actinomycins have been re-
ported to have a temporary activity on neoplasms
in man (21, 27, 41-43, 49, 76). All are very toxic
substances. Many cause splenic atrophy in animals
after multiple doses (CO).
Actinomycin-producers thus far reported are all
members of the family Streptomycetaceae. Some
actinomycin-producing cultures have been found
under various conditions to form the compo-
nents of their "mixtures" in proportions differing
from what was considered the norm, or even to
vield new actinomycins altogether. A certain
170
DESCRIPTIONS OF ANTIBIOTICS
Table 39
Amino acid composition of various (ictinoiii ijnns
Data given as moles of amino acid per mole of actiiiomyein (molecular weight, 1200).
Amino acids
L-Thieonine
D-Valine
L-Proline
Sarcosine
N-CH3-L-valine. . .
D-AUoisoleucine. . .
4-Hydroxyproline. .
4-Ketoproline
N-Methylisoleiicine
N-Methylalanine . .
Actinomycins
IV
VI
VII
El
F=
F4
+
+
+
+
+
2104L
" Designates presence of amino acid.
Streptoniyces sp. was shown to produce, in earlj^
stages of growth, an actinomyci" similar in the
proportions of its components to the "B mixture,"
whereas at later stages of growth, the proportion
of components was changed to that of an "A mix-
ture." This was demonstrated by circular paper
chromatography in 5 per cent Na-o-cresotinate
and ethyl acetate-n-butyl ether (2:1). Under dif-
ferent nutritional conditions, this culture could
produce only A-, or only B-tj'pe "mixtures" (61,
66). A "C-mixture'"-producing culture formed
VII (C3) as the major component actinomycin in
early growth stages, whereas later, VI (C2) became
the major component (28). Addition of sarcosine
to an L-glutamic acid-containing synthetic
medium in which an "A mixture "-producer was
grown changed the proportions of the actinomy-
cins normally formad. Amounts of actinomycins
IV and V were decreased, and II and III increased
(100).
It has also been possible to produce new actin-
omycins when different amino acids or amino acid
derivatives (such as oligopeptides) were added to
the culture media in which "C" or "A mixture"-
producers were grown (58, 86). These new actin-
omycins are referred to below as "biosynthetic"
actinomycin mixtures.
In the descriptions which follow, no data have
been included from reports in which the complex
was tested as though it were a single entity instead
of a mixture; only references to such publications
will be given. The heterogeneity of the complexes
makes such data invalid.
Actinomycin Mixtures (A, B, D, J, X) Containiny
Actinomycins I, II , III , /T', and V
Produced by: Streptoniyces antibiuticus (2, 82)
(A, B mixtures) ; S. parvus and others (8, 31, 35, 80)
(A mixture); S. kitasawaensis (103) (A mixture);
Streptoniyces sp. (10, 13) (B mixture); S. parvullus
(31) (D mixture); S.flavus (6, 17) (J mixture); S.
flaveolus (17) (J mixture); <S. griseus (S. parvus)
(71) (X mixture); *S. michiganensis (72, 99) (X
mixture) ; S. galbiis (99) (X mixture) ; S galbus var.
achromogen.es (99) (X mixture); S. murinus (99)
(X mixture); S. lanatus (99) (X mixture); and
Streptoniyces sp. (18, 22, 23, 28).
Synonyms: Antibiotic X 45 (B mixture) (13).
Actinoflavin (J mixture) (9).
Method of extraction: I. Broth and/or mycelium
extracted with ether, n-butanol, benzene, or butyl
acetate. Extract concentrated to dryness. Residue
treated with petroleum ether and extracted into
acetone, benzene, or cold chloroform, filtered and
evaporated to dryness. Recrystallized from cold
ethanol on addition of successive aliquots of
petroleum ether, or from acetone, ether, warm
ethanol, or other solvents. Purified by chromatog-
raph}' on alumiiui from benzene and elution with
30 per cent acetone in benzene or with ethyl ace-
tate (4, 10, 23, 32). Actinomycins I, II, III, IV,
and V are partially separated by chromatography
of the crude mixture on acid-washed alumina from
benzene-chloroform (3:1) and elvition with the
same mixture, gradually changing the proportion
to 1:3. Particular fractions are further separated
by rechromatographing as before, but gradually
<
^3 CO CO
t^
06 --C' im'
II II II
ffi ^
CO
00 00
GO d CO
II II II
w ^
C = 58.4
H = 7.2
N = 13.1
000
00 Ci t-.
00 d c<i
II II II
ffi ^
++
1
1
1
c£-c
06
^ i
d
00
d
1
1
1
d
00
od
^
ugS
" ^ CO t^
«u 10 CO CO
fcD ...
"^ Tft "^
CO 00 --H
-* CO '^i
5!- SI- ^
1
1
1
1
t^ Ci t^
CO C<l -f
Oi ~r ^
000
^ CO >0
<M -* -r
5:2
1
^ 1
~ -r CO -r
-*"
C^l 1
-f
CO
d »o
05 -r
00
CO
^' CO
00
CO
(>J "O CO
c
.0
in
0.
C/2
C
o3
s
CO ,,0
I ^
II -'
?;= II
U
X
II ~
2 . II
X
c
I "^J
II ^
3Q II
^3
[a];;' = -261 to 268°
(f = . 25% in 95%, ethanol )
or [a]-" = -349° (±10°)
(c = 0.26%; in methanol)
[a]f = -320 to 323°
(c = 0.25% in 95%, ethanol)
[a]" = -325° (±10°)
(c = 0.24% in methanol)
[aln = -328° (±10°)
(c = 0.24% in methanol)
Melting pointf
CO V
00
CO
-co
?5 5
C2
1
?5
10
?5
S
03
0)
c
02
i
Prisms, hexagonal
bipyramids, or
needles
03
.Si ^
^ "a
ai
£
OJ
>
03
3
en C
3.2
n
■0
&
<
>
4^"
d
d
•a
"
'-'
-
^
1
>
>■
>
o — <
c ^
<f
^,
--„
"
XII
^
g
c
^*^
rt*
a;
.^
3::
,li
W
<
171
172
DESCRIPTIONS OF ANTIBIOTICS
proceeding to a 2:3 ratio in the eluent. Complete
separation is made by preparative paper chroma-
tography (N-dibutyl ether-tetrachloroethane-10
per cent sodium o-cresotinate) (62, 101).
Chemical and physical properties: These mixtures
contain actinomycins I(Ai , Bi , Xufi), II(Aii ,
Bii), III(Ain , Bin), IV(Aiv , Biv , Div , X,), and
V (Av , Bv , Xo) in varying proportions (See
Table 38). "J mixture" has been identified with
"X mixture" (83), but precise data have not been
published. The A, B, and X mixtures also contain
a number of other actinomycins, not yet char-
acterized, in trace amounts. Physical and chemical
properties of the major actinomycins I to V are
given in Tal)le 40. The infrared spectrum of IV is
given in reference 36, and infrared data on II and
III are given in reference 101. Amino acid content
of the peptide moieties of actinomycins I to V is
given in Table 39 (24, 77). For information on the
various mixtures see references 1 , 4 (A mixture);
13, 19, 62, 82 (B) ; 32 (D) ; 6, 9, 12, 17 (J) ; 25, 28, 37,
44, 46, 62 (X).
Biological activity: Actinomycins I to V are
active on gram-positive bacteria, less active on
mycobacteria, and inactive on fungi (102). Some
typical activities of components I, IV, and V are
given in Table 41. Actinomycins II and III were
about half as active as IV in vitro (86). Tested in
mice against Gardner lymphosarcoma (6C3HED)
(ascitic form), actinomycins I and V had much less
activity than IV, and II and III had equal or
better activity than IV (60, 86). Actinomycin IV
has been reported active on the following tumors:
Crocker sarcoma 180 (50), malignant melanoma
S91, mammary adenocarcinoma, myeloid and
lymphoid leukemia (53), Ehrlich carcinoma (as-
citic form), Krebs 2 ascitic carcinoma (54), Ridge-
way osteogenic sarcoma, carcinoma 1025 (90), ME
1 melanoma (human amelanotic), MFSl myxo-
fibrosarcoma, KB (Eagle human carcinoma),
P1534 mouse lymphatic leukemia (88), RC mam-
mary carcinoma (91), mammary adenocarcinomas
C 755 and C 3HBA, thymoma (solid), and the
ascites form of an ovarian carcinoma (70). Infor-
mation on the various mixtures is given in refer-
ences 26 and 55. Treatment of actinomycins with
methanolic sodium hydroxide opens the lactone
rings in the polypeptide chains. The resulting
compounds are called actinomycinic acids. The
actinomycinic acid of actinomycin S3 has no anti-
bacterial activity but some antitumor activity
(108).
Toxicity: LDjo (mice) subcutaneously (60):
actinomycin I, > 8 mg per kg; actinomycin IV, 1.1
Table 41
Biological activity of actinomycin components I,
IV, and V against four test organisms (60)
Minimal inhibitory concentration
Actinomycin
Staph,
aureus
B. subtilis
B. cereus
Sarcina
lute a
lig/ml
I
0.4
0.48
0.5
0.175
IV
0.14
0.068
0.07
0.066
V
0.07
0.042
0.052
0.035
mg per kg; actinomycin V, 0.35 mg per kg. LD50
(mice) intraperitoneally (86): actinomycin II, 6
mg per kg; actinomycin III, 1.5 mg per kg. Actin-
omycin I had much less toxicity than IV and V, as
measured by splenic atrophy (60). Actinomycin
IV (D) had a teratogenic effect on the rat foetus
at 10 jug per mother rat (4). It was also intensely
irritating to skin and subcutaneous tissue. In
human beings, side effects included depletion of
bone marrow, stomatitis, pigmentation of the skin,
and gastrointestinal disturbances (93). Other
toxicity studies with actinomycin IV in animals
(70) and normal and neoplastic tissue cell cultures
(68) have been reported.
Utilization: Used experimentally in the treat-
ment of certain neoplastic diseases (69, 92, 93).
Actinomycin mixtures {C and AA-A(') Containing
Acfinotnycins IV, VI, and VII
Produced by: Strcptomyces chrysomallus (20),
<S. griseus (84), S. chrysomallus var. fumigatus
(99), and Slreptomyccs sp. (5, 23, 28, 64, 85, 87).
Synonym: Antibiotic HBF 386 ("C mixture").
Method of extraction: Ground dried mycelium
extracted with benzene. Broth extracted with
butjd acetate. Broth-extract concentrated in
vacuo, residue dissolved in benzene-extract from
mycelium, and the whole chromatographed on
alumina. Eluted with ethyl acetate; eluate con-
centrated. Addition of carbon disulfide to con-
centrate gives i)recipitate of crude ' C mixture."
Washed with ethyl acetate-carbon disulfide
(1:2) and carbon disulfide. Recrystallized from
ethyl acetate, ether, methanol, or ethanol. Sepa-
ration of major components by countercurrent
distribution (5 per cent Na p-xylol sulfonate-
mothyl l)utyl ether-n-dil)utyl ether) (16, 23) or
by chromatography on alumina (33).
Chemical and physical properties: "C mixture"
contains three major components, actinomycins
IV(Ci), VI(C2), and VII(C3), and a number of
DESCRIPTIONS OF ANTIBIOTICS
173
trace components including Co, Cna, Cia, Csa,
Csa , and C4 (36, 37, 87). These components have
the following Rcj values (Rf value relative to
actinomycin VI(Co)) (87):Cu = 0.2; VICC.) = 1.0;
C3a = 1.56; IV (CO = 0.69; C-a = 1.15; C4 = 1.7 ;
Cia = 0.8; VII (Ca) = 1.39. System: n-Dibutyl
ether, n-butanol, and 5 per cent aqueous /3-naph-
thalene sulfonic acid. Other components have
been reported formed when a "C mixture"-pro-
ducing streptomycete was grown in the presence
of various amino acids (64) (see actinomycin
mixtures E and F). The infrared spectrum of
actinomycin IV (Ci) is given in reference 36 and
that of actinomycin VII (C3) in reference 37. The
other physical and chemical properties of actino-
mycins IV (Ci), VKC.), and VII(C3) are given in
Table 40. Their amino acid content is given in
Table 39 (24, 77). On mild acid hydrolysis, actino-
mycin VII (Cs) splits off one molecule of ammonia
andformsa "desamino actinomycin" (C64H89O17N11 ;
brick-red rhomboid crystals; m.p. 239°C). The
chromophore of this degradation product (3 hy-
droxy! , 8 dimethylphenoxazone- (2) -dicarboxylic
acid- (4, 5) (40) is shown below:
COOH
OH
"AA-AC mixture", contains two actinomycins
in major amounts, IV (AA) and VII(AC), and a
minor component with an amino acid content
like that of VII, but not as yet completely char-
acterized (30, 33, 83). References giving data
concerning the "mixtures" include 16, 29, 36, 37,
40 ("C mixture"); and 14, 30, and 33 ("AA-AC
mixture") .
Biological activity: The minimal inhibitory con-
centrations of some of the component actinomycins
of the "C mixture" against/^, suhtilisare as follows
(in ;ug per ml): actinomycin IV (Ci), 0.2; VI (C.),
0.14; VII (C3), 0.2; C«, 19; Cja, 50; and C<>3, 28.
The activity against mammary carcinoma TM
8013 and lymphosarcoma T 24 179 of the "C mix-
ture" is accounted for by actinomycins IV (Ci),
VI(C2), and VII(C3); VII (C3) being the least
active (67). "Desaminoactinomycin" has less than
Koo the activity of the parent compound on
Staph, aureus (40). References giving data on the
biological activity of the "C mixture" include 11,
27, 34, 89, and 91; for the "AA-AC mixture," ref-
erence 5.
Toxicity: lA)^ (mice, no route given, mg per
kg): IV (Ci), 1.8; VI (C), 0.9; VII (C3), 1.6; C„ ,
8; Coa, 24; and Co(3, 41 (67). References giving data
on the toxicity of the "mixtures" include 5 and 27.
Utilization: Same as previous "mixtures." See
references 41-43 and 49. Some anti-allergy activ-
ity has been rei)orted (,42).
Actinoinyciti H or ./■>
A preparation designated as actinomycin J2
is a mixture of actinomycin Ji and duodecyl ester
of 5-ketostearic acid. The last compound has no
antibiotic activity. Actinomycin J 2 is very prob-
ably the equivalent of Waksman and Woodruff's
actinomycin B. Later Waksman, CJeiger, and
Reynolds proposed dropping the term actinomycin
B to designate this fraction, since obviously it is
nothing else than a mixture of actinomycin A
with inert material. They proposed calling ac-
tinomycin A, actinomycin (1, 105, 106).
Acli noiiiyriti Mixture "7"
Produced by: Strcptonryccs parvullus (71, 72),
S. antibioticus (99), and Streptomyces sp. (38).
Synonym: This mixture may contain the same
actinomycins as the "C mixture," but in different
proportions (77).
Method of extraction: Xo data. Prol)al)ly the
same as for the other "mixtures" described.
Chemical and physical properties: Contains one
major com])onent, actinomycin IV (Ii), and four
le.ss well described comj^onents, lo , loa , I2 , and I3
(37, 77). Chemical and physical data on actino-
mycin IV(Ii) are given in Table 40. The amino
acid content of IV (Ii) and lo is given in Table 39.
In : Hexagonal bipyramids of needles; m.p. 242 to
243°C. Specific extinction at 446 m/x = 20.5 (cyclo-
hexane) (37).
The actinomycins described below have been
less well characterized. Whether they contain any
of the known actinomycins (I to VII) is a matter
for further research. They fall into three cate-
gories: Category 1 : Actinomycins which are known
to be "mixtures" for which a certain amount of
data on the component actinomycins is available.
Category 2: "Mixtures" for which no data on the
component actinomycins are available. Oidy
references to the original papers will be given.
Category 3: "Actinomycins" which may be com-
posed of one, or more probably, of a mixture of
actinomycins. All important data have been in-
cluded here, even though subsequent work may
show the substance to be a "mixture." In all cases,
the original author's terminology has been used.
174
DESCRIPTIONS OF ANTIBIOTICS
CATEGORY 1 :
Mixtures of actinomycins for which some com-
ponent data have been pul)lished.
Biosynthetic Acfinoiuycin Mixtures E and F
Produrecl by: Streptomyces sp.
Remarks: This organism, whicli normally jjro-
duced an actinomycin mixture of the "C type," pro-
duced another mixture containing entirely new
actinomycins ("E type") when DL-isoleucine was
added to the medium. It produced still another
mixture ("F type"), along with actinomycins
VI(C2) and X'llfCg) when sarcosine was added
(58, 59).
Method of extraction: Actinomycins of the "F
mixture" were separated by chromatography on
alumina (59).
Chemical and physical properties: Mixture "E"
contains two major component actinomycins,
E, and E.>. E, : C = 58.89%; H = 7.21%; N = 12.28%.
Specific extinction at 444 nxfx is 18.2 (ethanol).
E.2 : C = 52.21%; H = 7.20%; N = 11.9()%. Specific
extinction at 444 m/x is 19.0 (ethanol). Infrared
spectra of Ei and E2 are given in reference 59.
Amino acid content is given in Table 39. The
n-methyl isoleucine probably replaces the N-
methyl valine of actinomycin VII (Ca).
"Mixture F" contains six component actino-
mycins, Fo to P\>, . All have the same ultraviolet
and infrared spectra. Specific extinctions at 444
niM (ethanol): Fi = 19.0; F. = 19.4; F:, = 18.5;
F4 = 19.0; F., = 17.8 (59). See Table 39 for the amino
acid content of the F actinomycins.
Biological activity: Ei has 90 per cent of the
activity of actinomycin VI (C2) against B. siibtilis;
E2 , 82 per cent ; Fo , 74 per cent; Fi , 59 per cent;
F2 , 71 per cent; F.'j , 47 per cent; F4 , 55 per cent;
and F5 , 72 per cent (59). Against Walker carcinoma
(rat). El had slight activity; E2, none; Fo was
active at 5 X W) jug per kg; Fi , F2 , F3 , and F4 were
active at high repeated doses (200 to 1000 /ug per
kg); Fo was active at 100 /ig per kg (single dose).
Against Jensen sarcoma (rats), Fi (the only one
tested) was active. With Yoshida sarcoma (rats)
as the test tumor, Fo , Fi , F2 , and F4 were inactive
at 6 X 00 Mg per kg (highest level tested). Against
Rous sarcoma (chickens), Ei , Ej , Fi , F4 , and F5
were active (75).
Toxicity: Ei , E2, and F, were as toxic as ac-
tinomycin IV (D); Fi was 3^ to ^\o as toxic as IV;
F2 , H as toxic; F3 and F4 , ^5 as toxic (75).
Utilization: See reference 75.
Actinomycin Mixture "A'"
Produced by: Streptomyces melanochronKxjcnes
(73).
Method of extraction: Extracted fioni brotli
with benzene, and from mycelium with acetone-
benzene. Purified by chromatography on alumina
(73).
Chemical and physical properties: Contains three
components. Acid hydrolysates of the "mixture"
contain threonine, sarcosine, proline, valine, and
isoleucine (73).
Biological activity: Typical of the actinomycins
(73).
Toxicity: No valid data (73).
Actinomycin Mixture "M"
Produced by: Streptomyces sp. Has an antimitotic
effect (()2a).
Synonym: "(Jiolitti actinomycin" (63). Prob-
ably a mixture of the "B" or "X" type (48, 83).
Method of extraction: Similar to that of the other
actinomycins (63).
Chemical and physical properties: Has three
components with Rf values of 0.2 (trace), 1.0, and
1.86, and a fourth variably present (Rf = 2.6) as
demonstrated by circular paper chromatography
(ethyl acetate-n-butyl ether-2 per cent aqueous
naphthalene-2-sulfonic acid, 1:1:2) (63).
Biological activity: Same as the other actino-
mycins (63). For data on the mixtvire see reference
74.
Toxicity: No valid data ((J3).
Actin4)))iycin P-i
Synonym: .\ntil)iotic PA I26-P2.
Toxicity: Said to be less toxic to animals than
actinomycins C and D (107).
Actinomycin Mixture "Z"
Produced by: Streptomyces fradiae (83).
Method of extraction: Culture-filtrate extracted
with ethyl acetate. Ivxtract concentrated, then
evaporated in vacuo to an oily residue. Addition
of petroleum ether precipitates Z. Chromato-
graphed on aluminum oxide from benzene and
developed with l)enzene-absolute CCI4 (65:70),
then chloroform-methanol (49:1), followed by the
same mixture (19:1). Active fractions concen-
trated in vacuo. Residue taken up in acetone.
Addition of ether gives "Z mixture." Components
separated by chromatography on alumina with
benzene as solvent. Developed with chloroform-
methanol (99:1) to give Z2, Z3, Z4, and Z.^. Zi fol-
lows further elution with same developer, Zn is
eluted with CHCU-methanol (97:3). Zo-and Z,-
containing fractions are each rechromatographed
on alumina. Zi crystallized from acetone-ether.
14ie mixure of Z2 , Z3 , and Z4 is never separated.
Z5 is also crystallized from acetone-ether (83).
DESCRIPTIONS OF ANTIBIOTICS
175
Chemical (ind phj/sicnl properties: Contains six
components, three of which were not separated.
Amino acid content of Zn , Zi, and Z5 is shown in
Table 39. Z,,: Amorphous orange-brown powder.
Decomposes with darkening at about 250°C.
Ultraviolet absorption spectrum maxima at 236
and 437 mfx (log E\^n = 2.17 or 2.44). Z, : Orange-
red crj^stals; m.p. 25G-260°C (decomposition);
[a]D = -362" (c = 0.185 per cent in CHCI3).
Ultraviolet absorption spectrum maxima at 240
mM (log .elcn, = 2.25), 427 m/x (£1"°,, 2.00), and
442 m/x (log E\lm 2.01). Infrared spectrum identi-
cal to actinomycin "X mixture." C = 53.97%;
H = 6.97%; N = 12.3%; N-CH., = 7.48%. Z, :
Short red rods, in clusters; m.p. 261-267°C (de-
composition). [a\D = —284° (c = 0.244 per cent
in CHCI3). Ultraviolet absorption maxima at
240 m/x (log E'Jc'n. 2.40), 428 ni/x (log E\"L = 2.21),
and 443 m/x (log E\L = 2.24). C = 55.71% H =
6.44%; and N = 12.25% (83).
ArtinoDii/cin Mixture 1048a
Produced by: Slreptoinyces sp. (78).
Method of extraction: Acetone-extract of my-
celium evaporated under reduced pressure, and
residue taken up in ethyl acetate; precipitated on
addition of petroleum ether. Recrystallized from
ethyl acetate. Purified by countercurrent distri-
bution (aqueous 1.7 per cent Na /3-naphthalene-
sulfonate and methyl butyl ether), and chromato-
graphy on alumina with acetone as eluent (78).
Chemical and physical properties: Contains three
major components, I, II, and III, and a minor
component, IV (author's designations). See
Table 39 for the amino acid content of I, II, and
III. /: m.p. 240-244 °C (decomposition). Ultraviolet
absorption s])ectrum maxima at 238 m/x (Eie'm
243) and 440 m/x (E^L 160). // : m.p. 238-240°C
(decomposition). Ultraviolet absorption spectrum
maxima at 240 m/x (Elcm 281) and 444 m/x (E'lJm
191). ///: m.p. 240-242°C (decomposition); ultra-
violet absorption spectrum maxima at 240 m/x
(.ElL 308) and 440 m/x (^Icm 209). IV: m.p.
239-243°C (decomposition) ; ultraviolet absorption
spectrum maxima at 240 (EIL 233) and 442 {E
l^m 162) (78).
Biological activity: Order of activity against
Staph, aureus and B. subtilis: I = II > III > IV.
In antitumor activity (Ehrlich ascites carcinoma),
all are equal (78).
Toxicity: I and II: LD50 (mice) 0.49 mg per kg
(no route given). ///: LD50 (mice) 0.89 mg per kg
(no route given) (78).
Actinomycin Mixture 2IO4L
Produced by: Streptomyces sp. (79).
Method of extraction: See actinomycin 1048A.
Chemical and physical properties: Contains two
major components, I and II, and two minor ones,
III and IV (author's designations, not related to
the usual I, II, III, and IV). /: m.p. 237-240°C.
Ultraviolet absorption spectrum maxima at 240
m/x {E\lr, 296) and 444 m/x (E\1,, 203). [a]" =
— 349.2°. Contains threonine, sarcosine, proline,
n-methyl valine, and alloisoleucine. II: m.p.
230-232°C. Ultraviolet absorption spectrum max-
ima at 240 m/x (i^lL 270) and 440 ni/x (E'lL 180).
[ale = —284.0°. Contains threonine, sarcosine,
proline, valine, N-methyl valine, and alloisoleucine
(79).
Biological activity: Typical of the actinomycins
(79).
Toxicity: Mice tolerate at least 50 /xg per kg for
5 days, intraperitoneally (79).
CATEGORY 2:
Substances known to be "mixtures," but for
which no component data is available
(1) Actinomycin 4A-2 (51).
(2) Actinomcyin Mixture — ? (96, 97).
CATEGORY 3 :
It is not known whether these substances are
single actinomycins or "mixtures"
Actinomycin 1
Produced by: Micromonospora sp. resembling
M. globosa (15).
Method of extraction: Broth extracted with ether,
and extract concentrated. Addition of petroleum
to concentrate gives amorphous powder. Crystal-
lization from aqueous ethanol and recrystalliza-
tion from same solvent or a benzene-ether mixture
(15).
Chemical and physical properties: m.p. 251-252°C.
Ultraviolet absorption maxima at 239 m/x {Eicm
272) and 440 m/x {Eu'n, 192). Acid hydrolysis prod-
ucts include threonine, proline, valine, N-methyl
valine, and sarco.sine. C = 60.06%; H = 6.99%;
N = 12.62% (15).
Biological activity: Active on gram-positive
organisms, mycobacteria, but not Sacch. cerevisiae
or .1 . niger at 50 /xg per ml (15).
Actinomycin 2
Produced by: Streptomyces sp. (7).
Method of extraction: Same as for A-type mix-
ture (7).
176
DESCRIPTIONS OF ANTIBIOTICS
Chemical and physical properties: Red crystals,
m.p. 248-250°C (decomposition). Soluble in tli-
ethyl ether; insoluble in petroleum ether. Ultra-
violet absorption maxima at 446 m/j (Eic'm 160
to 170), and 236 or 242 m/x (£:IL 265 or 230).
[a]" = -260 to -268° (7).
Biological aclivity: Typical of the actinomycins
(7).
Toxicili/: 10 /xg per 20 gm mouse is lethal, intra
venously (7).
Actinomycin 3
Produced by: Streptomyces chri/sonidlhis (81).
Method of extraction: Present in cult ure-l)rot h
and mycelium (81).
Chemical and physical properties: Red substance ;
m.p. 254 °C (decomposition). Soluble in acetone
and chloroform; moderately soluble in benzene,
ethanol, and ethyl acetate; less soluble in ether;
very sparingly soluble in carbon tetrachloride
and water; insoluble in petroleum ether. Yellow-
green fluorescence in ultraviolet light. Ultraviolet
absorption spectrum maximum at 450 ran (acetone
and alcohol). Contains six amino acids, including
L-valine, L-proline, and L-threonine. Forms a
biologically active, said to be less toxic than other
actinomycins, water-soluble degradation product
(81).
Biological activity:Typica\ of actinomycins (81).
Toxicity: 3.3 mg per kg is toxic to mice, in-
travenously or intraperitoneally (81).
Acti noniycni J/.
Produced by: Streptomyces sji. Tliis culture also
produces a eurocidin-like substance (52).
Method of extraction: Essentially similar to that
for the C-type "mixture" (52).
Chemical and physical properties: Red hexagonal
plates; m.p. 252-254°C. Ultraviolet absorption
maxima at about 242 niju {E\'om 40) and 440 to
450 ni;u. Infrared spectrum given in reference 1;
said to l)c similar to that of actinomycin C. C =
57.23%; H = 6.32%; N = 12.55% (52).
Biological activity: Typical of the actinomycins.
Active on Ehrlich (ascites) carcinoma in mice
(52).
Toxicity: 150 yug per kg is lethal to mice in 7
days, but 75 yug per kg is tolerated (52).
.1 u rant in
Produced by: A Streptomyces of the uuruntiacus
group (104).
Synonym: Aurantin is an actinomycin similar to
actinomycin C.
Chemical and physical properties: Dark red
hexagonal bipyramidal crystals; m.p. 251-253°C.
Biological activity: Active against gram-positive
bacteria. Streptococci and B. mycoides completely
inhibited by 0.04 ng per ml; staphylococci and
B. subtilis by 0.4 jug per ml. Active in mice against
Ehrlich carcinoma, sarcoma 180, and lymphoma
L 10. Active in rats against sarcoma M 1, sarcoma
4 J, and (Juerin carcinoma.
Toxicity: A single injection of 900 to 1000 jug
per kg intraperitoneally kills more than 50 per
cent of the mice. A single dose of 500 /xg per kg is
tolerated by the animals. Rats are more easily
killed than mice by aiu'antin.
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London 179: 1306-1307, 1957.
75. Hackmann, C. and Schmidt-Kastner, (i.
Z. Krebsforsch. 61: 607-615, 1957.
178
DESCRIPTIONS OF ANTIBIOTICS
76. Moore, G. E. et al. Caiifer 11: 1204-1214,
1958.
77. Waksman, S. A. et al. Proc. Natl. Acad.
Sci. U. S. 44: 602-612, 1958.
78. Kawamata, J. and Fujita, H. Med. J. Osaka
Univ. 8: 737-742, 1958.
79. Kawamata, J. and Fujita, H. Med. J. Osaka
Univ. 743-751, 1958.
80. Miyakawa, T. et al. Japan. J. Microhiol.
2: 53-62, 1958.
81. Thirumalachar, M. J. and Chosh, I). Indian
Phytopathol. 11: 23-24, 1958.
82. Sevcik, V. et al. Folia Biol. 4: 328-333, 1958.
83. Bossi, R. et al. Helv. Chim. Acta 41: 1645-
1652, 1958.
84. l%ttlinger, L. e/ a/. Arch. Mikrol^iol. (in i)ress
according to reference 83 j.
85. Tsai, J. S. cl al. Med. Dosvviadczalna i
Mikrobiol. 10: 105-125, 1958.
86. Katz,E.e/a/. Abstr. 7th Intern. Congr. Micro-
biol., Stockholm 387-388, 1958.
87. Kepf, K. Experientia 14:207-208,1958.
88. Handler, A. H. Ann. N. Y. Acad. Sci. 76:
775-778, 1958.
89. Loustalot, P. et al. Ann. N. Y. Acad. Sci.
76: 838-848, 1958.
90. Sugiura, K. et al. Cancer Research 18: 66-
77, 1958.
91. Tarnowski, (!. S. and Stock, C. C. Cancer
Research (Suppl.). 24-25, 1958.
92. Moore, G. E. et al. Proc. Am. Assoc. Cancer
Research 2: 328, 1958.
93. Farber, S. In Amino acids and peptides with
antimetabolite activity. Ciba Foundation
Symposium. Little, Brown and Comjjany,
Boston, 1958, pp. 138-145.
94. Tuchmann-Duplessis, H. and Mercier-Parot,
L. Compt. rend. 247:2200 2203,1958.
95. Brockmann, H. and Manegold, J. H. Natur-
wissenschaften 45: 310-311, 1958.
96. Ahmad, N. et al. Ann. Biochem. and Exptl.
Med. 18: 17-20, 1958.
97. Ahmad, N. et al. Ann. Biochem. and Ivxptl.
Med. 18: 21-26, 1958.
98. Brockmann, H. and Muxfeldt, H. Chem.
Ber. 91: 1242-1265, 1958.
99. Frommer, W. Arch. Mikrobiol. 32:187-206,
1959.
100. Katz, E. and Goss, W. A. Biochem. J. 73:
458-465, 1959.
101. Johnson, A. W. and Mauger, A. B. Biochem.
J. 73: 535-538, 1959.
102. Katz,E. Personal communication, 1960.
103. Harada, Y. et al. Japanese Patents 789,
790 and 791, February 18, 1959, and 898
of February 21, 1959 (Chem. Abstr. 5.3:
12595a, 1959).
104. Maevskii, M. M. et al. Antiljiotiki 4(4):
43-45, 1959.
105. Waksman, S. A. et al. Proc. Natl. Acad. Sci.
U. S. 32: 117-120, 1946.
106. Hirato, Y. and Nakanishi, K. Bull. Chem.
Soc. Japan 22: 121-127, 1949.
107. Colsky, I. el al. Cancer Chemothera])y
Repts. 8: 27-32, 1960.
108. Kawata, J. et al. J. Antibiotics (Japan)
13A: 415, 1960.
Actinoiie
Produced by: Streptomijces sp. resembling S.
antibioticiis.
Method of extraction: Broth-filtrate stirred with
activated carbon. Elution with 95 per cent metha-
nol. Eluate concentrated in vacuo. Acetone added
to precipitate impurities. Filtrate freeze-dried.
Purified by precipitating impurities from an ab-
solute methanol solution with cold ether, evapo-
ration in vacuo, and extraction with ether. Ether-
insoluble fraction is lyophilized to give actinone.
Chemical and physical -properties: Contains <1
per cent nitrogen. Insoluble in chloroform. Solu-
ble in ether, butyl acetate, benzene, and petro-
leum ether.
Biological activity: Active against Saccharo-
niyces, W'illia, and Trichophyton species. Not
active on bacteria or other fungi.
Toxicity: Mice tolerate 1 gm per kg intraven-
ously and 500 mg per kg subcutaneously.
Reference: 1. Ikeda, Y. et al. J. Antibiotics
(Japan) 3: 726-729, 1950.
Actiiiorliodin
Pioduced by: Streptomyces coelicolor (2, 3). This
organism produces other red pigments besides
actinorhodin (1).
Method of extraction: Mycelium treated with N
HCl for 15 minutes, filtered, exhaustively washed
with methanol, then dried, ground in a mill with
sand, and extracted with ether. Treatment with
A'^ NaOH gives a deep blue solution of the anti-
biotic (I). It is filtered; then acid is added to
precipitate the antibiotic (II), which is extracted
with acetone in a Soxhlet apparatus, then with
dioxane under COi (2).
Chemical and physical properties: Dinaphtha-
zarin derivative (4). Probable formula (CieHu-
Oi)-2 : C = 60.22%; H = 4.54%; O = 35.48%. De-
composes without melting at about 270°C. Thin,
red needles (2) or microscopically fine prisms (3).
Solul)le in pyridine, piperidine, phenol, and al-
DESCRIPTIONS OF ANTIBIOTICS
179
kali. Slightly soluble in alcohol, acetic acid, ace-
tone, dioxane, tetrahydrofuran, and NaHCOs
solution. Insoluble in petroleum ether, ether,
carbon disulfide, carbon tetrachloride, and water.
Ultraviolet absorption spectrum maxima at 523
and 560 ni/n (dioxane), 573 and (Ul ni/x (concen-
trated H2S()4), and 588 and 641 niM (NaOH). Red
in acetic anhydride, becoming violet-blue with
red fluorescence on addition of boric acid with
warming. Red in dioxane. Deep blue in concen-
trated H2SO4 , becoming red-violet on addition
of boric acid. Bright blue in NaOH (1, 2). Forms
a yellow crystalline triacetate (clustered needles) ,
which gives no absorption of visible light and de-
composes without melting at 260°C (2). In the
mycelium, it exists mainly as protoactinorhodin
(III). Ill is extracted from the dried mycelium
with acetone, and precipitates as pink, micro-
scopic prisms which turn red at 270°C and de-
compose at about 335°C (3). Partial structural
formula of actinorhodin (4) :
rated NaCl. Eluate evaporated to dryness in
vacuo. Residue extracted with boiling methanol.
Cooling of extract precipitates crude actinorul)in.
Purified by chromatography on H2S04-treated
alumina, with 85 per cent methanol as solvent
and developer. Precipitates from active fractions
as helianthate (2).
Chemical and physical properties: Basic sub-
stance: CeHuNsOo or C,H22Nr,04 . HCl salt: white
powder. Dihelianthate: reddish orange needle
clusters; m. p. 206-214°C. C = 51.40%; H = 5.93%;
N = 17.36%; S = 7.34 or 8.87%. Soluble in water
and methanol. Insoluble in ether. Positive biuret,
Fehling (on boiling), and KMn04 tests. Negative
Sakaguchi and Molisch tests. Dialyzable. Stable
to Ijoiling in aqueous solution at pH 6 to 7 for
15 minutes (2). Major component present in crude
preparation indistinguishable by paper chroma-
tography from pure streptothricin (wet butanol-
/3-toluenesulfonic acid) (4).
W
R2
-H O
Ri , R2 , R3 , R4 •
2— COOH, 2— CH:, , CsHieO;
Biological activity: Weakly active on Staph.
aureus (2).
References:
1. Brockmann, H. and Pini, H. Naturwissen-
schaften 34: 190, 1947.
2. Brockmann, H. et al. Chem. Ber. }{.'}: 161-
167, 1950.
3. Brockmann, H. and Loescheke, V. Chem.
Ber. 88: 778-788, 1955.
4. Brockmann, H. and Hieronymus, E. Chem.
Ber. 88: 1.379-1390, 1955.
Acli
iioriinin
Produced by: A Strcptoiuyces sp. that produces
a red mycelium (1).
Synonijm: Streptothricin-like substance.
Method of extraction: Adsorbed from broth-fil-
trate on Decalso at pH 7.0 and elute<l with satu-
Biological activity: Active on gram-positive and
gram-negative bacteria and mycobacteria.
Limited activity on B. cereus-niycoides group and
streptococci. Moderately active on Trichophyton
interdigiiale. Cross-resistance with lavendulin
and streptothricin (1). Active /// vivo (mice) on
K. pneumoniae infections (3).
Toxicity: LDioo (mice) 73.9 mg per kg intra-
peritoneally (3).
References:
1. Kelner, A. and Morton, H. E. J. Bacteriol.
53: 695-704, 1947.
2. Junowicz-Kocholaty, R. and Kocholatj-,
W. J. Biol. Chem. 168: 757-764, 1944.
3. Morton, H. E. Proc. Soc. E.xptl. Biol.
Med. 64: 327-331, 1947.
4. Benedict, R. G. Botan. Rev. 19: 229-320,
1953.
180
DESCRIPTIONS OF ANTIBIOTICS
Ac tin oxaii thine
Produced by: Sireptomyces globisporus (3).
Method of extraction: Antibiotic salted out of
broth-filtrate with (NH4)jS()4 . Precipitate dis-
solved in water and reprecipitated successively
with (NH4)2S04 . Chromatographed on AI2O3 ■
Elution with water, acidification of active frac-
tions to precipitate impurities, concentration,
treatment with ion exchange resins, and precipi-
tation of excess CO3" with BaS()4 . Solution con-
centrated to dryness (1, 2).
Chemical and physical properties: Water-solu-
ble, proteinaceous or peptide-like substance (1).
Inactivated by air in dry form; more stalile in
ar[ueous solution (2).
Biological activity: Very active on gram-positive
bacteria and tumors, including Ehrlich adeno-
carcinoma. No cross-resistance with penicillin,
streptomycin, or chlortetracycline (1, 2). Active
on cotton gummosis (4).
Toxicity: Maximal tolerated doses in mice
(single administration): intraperitoneally, 0.025
mg per kg; intravenously, 0.023 mg per kg; sul)-
cutaneously and intramuscularly, 1.2 mg per kg;
orally, 19.5 mg per kg (5).
References:
1. Buyanovskaya, I. vS. e/ o/. Antibiotiki 2(1):
17-20, 1957.
2. Vikhroba, N.M.e^a/. Antibiotiki 2(1): 21-
25, 1957.
3. Solovieva, N. K. and Delova, I. D. J. Mi-
crobiol. Epidemiol. Immunobiol. 29: 399-
404, 1958.
4. Starygina, L. P. et al. quoted in Rautensh-
tein, Y. I. Mikrobiologiya 28: 146-151,
1959.
5. Vieis, R. A. Antiljiotiki 3(1): 22-27, 1958.
Actithiazic Acid
Produced by: Streptotnyces sp. re.sembling S.
lavendulae (1), *S. virginiae (2), S. acidomyceticiis
(7), S. cinnamonensis (9), S. lydiciis (13), and S.
roseochromogenes (14) .
Synonyms: Antibiotic PA 95 (15), mycobacidin
(1), acidomycin (7), cinnamonin (9), thiazolidone
antiliiotic (12).
Method of extraction: I. Broth-filtrate ex-
tracted with n-butanol or butyl acetate at pH
2.0. Back-extracted into Na^COs solution. Ex-
tract adjusted to pH 4.5, then repeatedly ex-
tracted with butyl acetate. Butyl acetate evapo-
rated, residue taken up in hot ethylene dichloride,
treated with activated carbon, and filtered.
Crystals form on cooling. Recrystallization from
hot water, warm acetone, or methanol (4, 7, 14).
II. Adsorbed from broth-filtrate on carbon.
Eluted with ethanol -methanol (95:5) or 50 per
cent aqueous acetone at pH 7.0, 50°C. Eluate ad-
justed to pH 8.0, filtered, and concentrated under
reduced pressure. Concentrate extracted with
butanol at pH 3.0, or with ethyl acetate at pH
1.0 to 2.0. Purified by chromatography on (a)
silica gel with butanol as solvent and developer;
active fractions washed with water at pH 2.0,
concentrated and cooled to give a precipitate
(5); or (b) alumina and developed with ethyl
acetate, acetone, 1 per cent Ba(OH)2 , and 4 per
cent NaOH, successively. Recrystallized from
ethanol (10) IRA-400 and eluted with 10 to 20
per cent ammonium chloride. Eluate adjusted to
pH 4.5 and freeze dried. Crystallized from chloro-
form with hexane. A hot ethylene dichloride solu-
tion of precipitate filtered through charcoal and
cooled to give crystals. Recrystallized from
methanol, and then water or ethvlene dichloride
(14).
Chemical and physical properties: Monobasic
acid. Colorless needles; m.p. 137.5-141 °C (1, 4, 5,
7, 10). Soluble in dilute NaOH, lower alcohols,
ethyl acetate, benzyl alcohol, and hot water.
Less solul)le in acetone and distilled water. Very
slightly soluble or insoluble in ether, ethylene
dichloride, benzene, chloroform, petroleum ether,
and water (5, 10). [a]'^ = —60.5° (c = 1 i)er cent
in absolute ethanol). End-absorption in ultra-
violet light (5). Blue fluorescence in ultraviolet
light (7). Infrared spectrum given in reference
5. pKa' = 5.8. Negative 2,4-dinitrophenylhydra-
zine, Tollen, ninhydrin, and FeCls tests (10).
Positive bromine and KMn04 tests. A strongly
alkaline solution heated with lead acetate dark-
ens, with evolution of NH3 . Forms a white pre-
cipitate with HgClo . Stable (4). C9Hi50.,NS (5,
10). C = 49.86%; H = 6.86%; N = 6.46%,; S =
14.4%. No C — CH3 . Actithiazic acid is 4-thiazoli-
done-2-caproic acid (5, 6). Structural formula is
given in Chapter 6. Forms biologically active
esters. Methyl actithiazate: Colorless needles;
m.p. 53-54°C. [a]l = -50.9 (c = 1 per cent in
methanol) (6). Oxidation with H2O2 yields pimelic
acid (5); with HNO3 , adipic acid (7).
Biological activity: Active principally on myco-
bacteria and nocardiae; not active on other bac-
teria (2). Not active on tuberculosis infections in
mice (3, 7). Activity reversed by biotin. Inter-
feres with biotin synthesis in mycobacteria (2,
11). A number of esters formed by actithiazic
acid are about twice as active as the parent com-
pound on mycobacteria (6). The Na salt, methyl
and ethyl esters, the hydrazide, and amide and
guanidine derivatives of the antibiotic are active
in vitro on mycobacteria (8).
DESCRIPTIONS OF ANTIBIOTICS
181
Toxicitij: LDjo (mice) 3.5 gm per kg intraven-
ously and 20 gm per kg subcutaneously (7).
References:
1. Tejera, E. et al. Antibiotics & Chemo-
therapy 2: 333, 1952.
2. Grundy, W. E. et al. Antiliiotics & Chemo-
therapy 2: 399-408, 1952.
3. Hwang, K. Antibiotics tt Chemotherapy
2:453-459, 1952.
4. Sobin, B. A. J. Am. Chem. Soc. 74:
2947-2948, 1952.
5. Schenck, J. R. and DeRose, A. F. Arch.
Biochem. Biophys. 40: 263-2(>9, 1952.
6. McLamore, W. M. et al. J. Am. Chem.
Soc. 75: 105-115, 1953.
7. Miyake, A. et al. Pharm. Bull. (Tokyo)
1: 84-88, 1953.
8. Miyake, A. Pharm. Bull. (Tokyo) I:
89-93, 1953.
9. Okami, Y. et al . Japan. J. Aled. Sci. &
Biol. 6: 87-90, 1953.
10. Maeda, K. Japan. J. Med. Sci. & Biol.
6: 143-149, 1953.
11. Umezavva, H. et al. Japan. J. Met!. Sci.
& Biol. 6: 395-403, 1953.
12. Hamada, Y. et al. J. Antibiotics (Japan)
6A: 159-162, 1953.
13. DeBoer, C. et al. Antil)iotics Ann. 88(5-
892, 1955-1956.
14. British Patent 729,208, May 4, 1955.
15. Personal communication, Chas. Pfizer and
Company, January, 1960.
Akla> ill
Produced by: Streptomyces sp.
Method of extraction: I. Culture Huid and my-
celium adjusted to pH 2.0, heated at 80°C for 10
minutes, cooled, and filtered. Mycelium retreated
with HCl at pH 2.0 with heating. Combined ex-
tracts adjusted to pH 5.0 and extracted with
chloroform. Chloroform extracts back-extracted
with water at pH 2.0. II. Aqueou.s extract ad-
justed with NaOH to pH 9.0 under a stream of
nitrogen and extracted with benzene. Benzene
reextracted with water at pH 2.0. Process re-
peated once. Lyophilization of the final aqueous
acidic extract yields crude aklavin hydrochloride.
Chemical and physical properties: Crude aklavin
hydrochloride is very soluble in water, ethanol,
chloroform, dioxane, and pyridine; soluble in
acetone; and moderately soluble in ethyl acetate.
Concentrated HCl solution is yellow; concen-
trated H2SO4 solution intensely reddish purple.
A purplish pink color in alkaline solution is re-
versibly changed to yellow by Na2S204 . Addition
of NH4OH to an ethanol solution of the purple-
blue magnesium acetate-aklavin complex pro-
duces a stable magenta-colored precipitate.
Ultraviolet absorption maxima at 228, 258, and
288 mix, and a visible light peak at 427 m/u. The
crude antibiotic appears to contain several frac-
tions, as shown by covmtercurrent distribution
(ethyl acetate and 0.2 M phosphate buffer,
pH 6.3). Melting points of crystalline salts: 168
or 1S8°C (picrate); 197°C (helianthate) ; 168 or
170°C (hydrochloride). Activity lost in alkaline
solution on exposure to air; diff'usibility in agar
media doubled by heating at 80°C for 10 minutes.
C36H40O18N4 (picrate): C = 52.80%; H = 4.97%;
O = 35.22%.; N = 6.85%. Formula of base: CsoH.-,,-
(JuN or a multiple. Acid hydrolysis indicates that
the antibiotic is made up of two moieties: a non-
l:)asic, water-insoluble moiety, and a colorless,
water-soluble basic structure.
Biological activity: Active on a variety of bac-
teriophages; stimulates others. Some phages
stimulated by aklavin in phage-host system
are inhibited when free. Other phages that are
completely inhibited in phage-host system are
more resistant when free. Higher aklavin concen-
trations usually required for inactivation of free
phage than i)hage in phage-host system. Active
on some gram-negative and gram-positive bac-
teria, mycobacteria, fungi, and viru.ses. Mod-
erately virucidal for eastern equine encephalo-
myelitis virus (EEE), slightly for PR 8 influenza,
and not virucidal for MM virus or the Lansing
strain. Inhibits Y-SK poliomyelitis virus in
tissue culture. Active on EEE inoculated into
mice when a mixture of equal volumes of virus
(0.1 per cent) and antibiotic (0.01 mg ])er ml)
stood for 1 hour before inoculation.
Toxicity: LDo (mice) 150 mg per kg intrave-
nously; 100 mg per kg given intraperitoneally is
not tolerated.
Reference: 1. Strelitz, F. et al. J. Bacteriol.
72: 90-94, 1956.
Alazopeptiii
Produced by: Streptomyces griseoplanus (2).
Synontjm: Distantly related to azaserine and
DON, which are diazo derivatives of amino acids.
Alazopeptin contains diazo groups in a peptide
(!)•
Method of extraction: Broth stirred with acti-
vated carbon. Antibiotic eluted with 50 per cent
acetone. Eluates concentrated in vacuo, residual
solution adjusted to pH 9.0 and extracted with
butanol containing 2 per cent Arquad 2C. Back-
extracted into water at pH 2.0 to 2.5. Aqueous
182
DESCRIPTIONS OF ANTIBIOTICS
extract neutralized to pH 7.0, concentrated in
vacuo, and freeze dried. Purification from a metha-
nol solution on alumina. Developed with metha-
nol and 90 per cent methanol. Further purifica-
tion by partition chromatography on Celite 545
with development with a lower phase of CC14-90
per cent aqueous phenol-water, 82:18:25. Peak
fractions are pooled, diluted with carbon tetra-
chloride, and extracted with water. Aqueous ex-
tract adjusted to pH 7.0 and extracted with ether.
Extract concentrated in vacuo and freeze dried.
Recrystallization from 95 per cent ethanol in the
cold, 90 per cent aqueous methanol, and 70 per
cent aqueous acetone (1).
Chemical and physical properties: Ci6H2iN706-
H2O: C = 43.59%; H = 5.96%; N = 23.64% (Du-
mas); N (diazo) = 13.72%,; N (nondiazo) =
10.4%; H2O = 2.85%. No specific melting point.
Decomposes over a wide temperature range.
Very soluble in water; somewhat soluble in acetic
acid, formamide, dimethyl sulfoxide, and aque-
ous solutions of methanol, ethanol, and acetone.
Insoluble in absolute alcohols, acetone, ethyl
acetate, and ether. [a]f, = +9.5° (c = 4.7 per
cent in water). Stable at alkaline pH and neu-
trality, but not at acid pH. Ultraviolet absorp-
tion maxima (pH 7.0, phosphate buffer) at 242
niM (E]^m 321) and 274 mfx (El^ 549), typical of
aliphatic diazoketones. Infrared spectrum given
in reference 1 . Liberates nitrogen on acidification
with strong acids, with loss of biological activity
and ultraviolet absorption characteristics. Posi-
tive ninhydrin reaction. Positive biuret reaction
after acidification or treatment with l)romine
water. Untreated alazopeptin gives a yellow pre-
cipitate with the biuret reagents. Acid hydrolysis
products include a-alanine, representing 21.4 per
cent of the antibiotic. Oxidation with periodic
acid yields a peptide, which on acid hydrolysis
yields glutamic acid and a-alanine, indicating
that the antibiotic is a peptide containing a-ala-
nine and a Ce diazoketoamino acid (1).
Biological activity: Slightly active in mice on
sarcoma 180 (ascitic and solid) and Ehrlich car-
cinoma (solid form) (4).
Toxicity: LD50 (rat) 150 mg per kg intraperi-
toneally. Toxic to fetuses in utero at one thirtieth
of the adult LD50 (3).
References:
1. De Voe, S. E. et ul. Antibiotics Ann. 730-
735, 1956-1957.
2. Backus, E. J. et al. Antibiotics & Chemo-
therapy 7: 532-541, 1957.
3. Thiersch, J. B. Proc. Soc. Exptl. Biol.
Med. 97: 888-889, 1958.
4. Sugiura, K. Ann. N. Y. Acad. Sci. 76:
575-585, 1958.
Albofiingiii
Produced by: Strcptomyces alhus (2) or S. albiis
var. fungatus (3).
Method of extraction: Mycelium washed with
acetone, then extracted with A^ HCl in acetone.
Extract neutralized with CaCOs ; precipitate
forms on dilution with water. Reprecipitated
from acidic acetone with petroleum ether. Chro-
matographed on alumina from acetone (2).
Chemical and physical properties: Complex, con-
taining .several closely related components. Yel-
low powder. Decomposes without melting at
190°C. Soluble in chloroform, acetone, and di-
methylformamide. Less soluble in alcohols. In-
solul)le in water and ether. Ultraviolet absorption
spectrum maxima at 240, 255, 305, and 375 mn.
Contains C, H, O, and N (2).
Biological activity: Active on yeasts, filamen-
tous fungi, gram-positive bacteria (1), and cer-
tain gram-negative bacteria (3).
References:
1. Caykovskaya, S. M. and Tyebyakina, A. E.
Abstr. Communs. Symposium on Anti-
biotics. Prague, 1959, pp. 142-143.
2. Chochlov, A. S. and Liberman. (1. S. Abstr.
Communs. Symposium on Antibiotics.
Prague, 1959, pp. 154-155.
3. Solovyeva, N. J. et al. Abstr. Communs.
Symposium on Antibiotics. Prague, 1959,
pp. 189-191.
A 1 bom y eel ill
Produced by: Strcptomyces albiis (1) iind a Strcp-
tomyces sp. resembling S. albus (3).
Method of extraction: I. Broth-filtrate ex-
tracted with ethyl acetate, benzene, chloroform,
or butanol at pH 8.0. Back-extracted into acidic
water (pH 1.0). The aciueous extract can be: (a)
concentrated and adjusted to alkaline pH to pre-
cipitate the antibiotic; or (b) extracted into ethyl
acetate at pH 8.0 and chromatographed on AI2O3
with ethyl acetate as developer. Colorless frac-
tion dried in vacuo, taken up in benzene, and
again dried in vacuo. Recrystallized from acetone-
ether (1:9) (1, 3). II. Adsorbed from broth-fil-
trate on a cation exchange resin or activated
charcoal. Eluted with 80 per cent acidic acetone.
Eluate concentrated in vacuo, then treated as in
1(b) (1, 3).
Chemical and physical properties: Basic sub-
stance. Colorless hexagonal crystals; m.p. 166-
167°C (1) or l(i4-l()6°C (3). Soluble in xylene,
DESCRIPTIONS OF ANTIBIOTICS
183
l)enzene, ethylene dichloride, methanol, ethanol,
l)enzyl alcohol, and water at acid pH. Soluble at
liigh temperatures in acetone, ethyl acetate,
dioxane, butanol, and Tetralin. Scarcely soluble
in ether, carbon bisulfide, distilled water, and
petroleum ether. No characteristic ultraviolet
absorption in ethyl ether or ethanol. Infrared
data given in reference 1, and spectrum in refer-
ence 3. [a]" = —48.7° (c = 1 per cent in chloro-
form). Positive Fehling, Tollen, and silver mirror
tests. Cherry-red color with Elson-Morgan rea-
gent. Negative biuret, ninhydrin, Millon, Molisch,
orcinol, phloroglucinol, Pauly, FeCls , and Saka-
guchi tests. Stable to boiling at pH 2 to 9 for 10
minutes. Most stable at neutrality, less so at
acid pH, and relatively unstable at alkaline pH.
Precipitated by ammonium reineckate and picric
acid, but not flavianic acid or methyl orange.
C.25H44NO7 : C = 63.38' e; H = 9.14%; N = 2.91%;
molecular weight 477; or C32H.i409N: molecular
weight 586.4. No S, P, or halogen (1, 3).
Biological activity: Active on gram-positive
bacteria at 0.1 to 12.5 y.g per ml, including M.
smegrnatis. Less active (25 to 250 ixg per ml) on
other mycobacteria. Inactive on gram-negative
bacteria, filamentous fungi, yeasts, phage, and
viruses tested. Active in vivo against pneumo-
coccal and Borrelia duttonii infections (1, 3).
Cross-resistance with erythromycin-carbomycin
group (2).
Toxicitij: LD50 (mice) 1.5 gm per kg orally,
0.5 gm per kg subcutaneously (3). Mice tolerate
249 mg per kg intraperitoneally, 124.5 mg per kg
intravenously, and 41.5 mg per kg intracerebrally.
Fifty per cent are killed by 332 mg per kg intra-
peritoneally, 166 mg per kg intravenously, and
58 mg per kg intracerel^rally (1).
References:
1. Takahashi, B. J. Antil)iotics (Japan) 7.4:
149-154, 1954.
2. Nishimura, H. et al. Ann. Rept. Shionogi
Research Lab. 6: 278-285, 1956 (Chem.
Abstr. 51: 5193d, 1957).
3. Kuroya, M. and Nishimura, H. Japanese
Patent 7750, September 17, 1957.
.41boverticilliii
Produced by: Streptoniyces sp.
Chemical and physical properties: Colorless,
amorphous powder. Soluble in water and metha-
nol. Insoluble in acetone, esters, and ether. LTltra-
violet absorption spectrum maximum at 208 m^
(0.1 N HCl) or 218 mM (0.1 N NaOH). [at" =
— 33.5° (c = 1.0 per cent in water). Negative
Tollen, Molisch, Benedict, maltol, Elson-Morgan,
biuret. Millon, Sakaguchi, anthrone, and FeCls
tests. Yellow precipitate with Fehling's reagent;
positive ninhydrin. Stable to heating at 100°C
for 1 hour at acid and neutral pH. Does not yield
furfural on acid hydroly.sis. Reineckate: C =
32.03%,; H = 5.21%; N = 22.72%; Cr = 9.35%.
HCl: C = 40.58%; H = 6.48%; N = 18.12%,;
CI = 8.98';;,. No s.
Biological activity: Active on mycobacteria
(0.002 to 2.0 ng per ml). Very slightly active on
Sarcina liitea, B. suhtilis, and B. anthracis. Not
active on Staph, aureus or other l)a.cteria, fungi,
or yeasts tested.
Toxicity: LD,=,fi (mice) 50 to 100 mg per kg in-
travenously. No delayed toxicity.
Reference: 1. Maeda, K. et al. J. Antibiotics
(Japan) 11 A: 30-31, 1958.
Alioniycin
Produced by: Streptomyces acidomyceticus .
Remarks: This culture also produces actithiazic
acid. Culture belongs to the lavendulae group.
Method of extraction: Extracted from the my-
celium with hot methanol. Extract concentrated
in vacuo, adjusted to i)H 9.0, and extracted with
butanol. Solvent removed in vacuo, and acetone
added to the residual syrup to give aliomycin.
Can also be extracted from mature culture-
broths.
Chemical and physical properties: Pentaene.
Yellow powder. Soluble in distilled water, alka-
line water, glacial acetic acid, methyl Cellosolve,
ethylene glycol, methanol, ethanol and hot Inita-
nol, isoamyl alcohol, and dioxane. Insoluble in
water at pH 3.0, or in ether, benzene, ethyl ace-
tate, or acetone. Ultraviolet absorption spectrum
maxima at 321, 330, and 350 m^. (!ives a dark
purple color in concentrated H2SO4 . Positive
Fehling test. Slightly positive Molisch test.
Stalile at neutrality. Contains S and N, but no
halogen.
Biological activity: Active on fungi and yeasts
at 7.5 to 20 ixg per ml. Active on Trichomonas
foetus. Active on Yoshida sarcoma cells at 0.5
mg per ml. Activity partially reversed by cyste-
ine.
Toxicity: LD511 (crvide substance, mice) 45 mg
per kg intraperitoneally; 2.65 gm per kg orally.
Reference: 1. Igarashi, S. et al. J. Antibiotics
(Japan) 9B: 101-103, 1956.
A I
oiii^ cm
Produced by: Streptomyces sp. (1).
Method of extraction: Dried powdered mycelium
extracted with ethyl acetate containing about 1
184
DESCRIPTIONS OF ANTIBIOTICS
per cent tri ethyl amine, methanol-1 per cent tri-
ethylamine, acetone, or an ethyl acetate-acetone -
water mixture (5:1:0.5). Ethyl acetate-extracts
are concentrated in vacuo. To the resulting oily
brown residue is added a saturated solution of
tannin to precipitate impurities, and the whole
filtered. Water is added to the filtrate and mixed,
and the aqueous and organic phases separated.
To the aqueous phase NaCl is added to satura-
tion, to salt out an additional amount of the or-
ganic phase. Both organic phases are extracted
with methanol, extract concentrated in vacuo,
and lyophilized to give a brown oil (3).
Chemical and physical properties: Liglit brown
clear oil. Ultraviolet absorption spectnun maxi-
mum at 270 m/i, with a small peak at 350 niju
(methanol). Does not form active precipitates
with picric acid, p-toluenesulfonic acid, methyl
orange, ammonium reineckate, resorcinol, or pro-
caine HCl. Rf values in various systems of paper
chromatography are given, and are said to differ-
entiate the antibiotic from those given in the key
system of Ammann and (iottlieb (4). Unstable
at acid pH; most stable at pH (5.0; 50 per cent
inactivated after 3 hours at 100°C (3).
Biological activity: Active on Candida. Not
active on bacteria (2).
References:
1. Woznicka, W. et al. Med. Doswiadczalna i
Mikrobiol. 9: 57-62, 1957.
2. Woznicka, W. el al. Med. Do.swiadczalna i
Mikrobiol. 9:293-308,1957.
3. Woznicka, W. et al. Med. Doswiadczalna i
Mikrobiol. 9:441-450,1957.
4. Ammann, A. and Gottlieb, D. Apjil. Micro-
biol. 3: 181-18(j, 1955.
Allhioiii}! ciii
Produced by: Streptoniyces althioticus (I).
Method of extraction: Broth adjusted to pH (i.O
and filtered. Filtrate extracted with butyl acetate
or butanol. Extracts concentrated in vacuo. Cool-
ing of residue precipitates althiomycin. Recrys-
tallized from ethyl Cellosolve. Can be adsorbed
from broth on clay and eluted with aqueous ace-
tone. Can also be extracted with ethanol, acetone,
or ethyl acetate from mycelium.
Chemical and physical properties: White needles;
brown at r20-l(iO°C and decompose at 172-174°C.
Soluble in ethyl Cellosolve, dioxane, and pyri-
dine. Slightly soluble in acetone, methanol, buta-
nol, ethyl acetate, and butyl acetate. Insoluble
in water, ether, petroleum ether, and benzene.
[a];° = +20.3° (c = 1.33 per cent in methyl Cello-
solve). Ultraviolet absorption spectrum maxi-
mum at 220 to 223 m^ {E]'cm 810) and a shoulder
at 285 to 290 m^ (£^IL 210) in 0.03 .V HCl contain-
ing 1 per cent methyl Cellosolve, or at 235 ni/x
{E\fm 611) and 300 to 305 m^ {E\'L 317) in 0.03 N
NaOH containing 1 per cent methyl Cellosolve.
Infrared spectrum given in reference 1. Positive
ninhydrin and Tollen tests. Negative FeCU ,
Fehling, and Sakaguchi reactions. C15H14N4S2O6 :
C = 44.49%; H = 3.51%; N = 13.92%;
S = 14.77%. Stable at pH 5 to 7.
Biological activity: Active on gram-positive and
some gram-negative bacteria, ])ut not on myco-
bacteria, Pr. vulgaris, Ps. aeruginosa, fungi, or
yeasts. Active in mice on D. pneumoniae and Sal.
typhosa.
Toxicity: Mice tolerate 720 mg per kg int ra-
pe ritoneally.
Reference: 1. Yamaguchi, H. et al. J. Anti-
l)iotics (Japan) lOA: 195-200, 1957.
Aniaroniycin
Produced by: Streptoniyces flavochromogenes.
Synonym: Related to picromycin and griseo-
mycin.
Method of extraction: Broth, adjusted to pH
7.5, is extracted with 1^ volume of benzene. Ben-
zene is extracted three times with pH 2.0 HCl
solution. HCl solution adjusted to pH 5.7 and
back-extracted twice with benzene. Benzene is
dried in vacuo and the crude residue taken up in
hot CS2 . When cooled, white prisms precipitate
out. Recrystallization from ethanol.
Chemical and physical properties: Basic, bitter-
tasting prisms; m.p. 164.5-165°C. [a]" = +6.19°
(1 per cent in ethanol). Free base: C = 63.66%;
H = 8.73%; N = 3.0%; O = 24.61%. C25H39O7N.
Ultraviolet spectrum shows broad peak at ap-
proximately 220 m;Li. Infrared spectrum (CCU as
solvent) shows bands at 2.9, 3.4, 5.7, 6.1, 6.85,
7.2, 7.45, 7.85, 8.38, 8.60, 9.0, 9.25, 9.5, 10.15, 10.60,
and 11.3 m- Readily soluble in ether, chloroform,
and methanol. Soluble in benzene, toluene, ethyl
acetate, butanol, carbon tetrachloride, ethanol,
and warm carbon disulfide. Slightly soluble in
cold CS2 . Scarcely soluble in petroleum ether
and water. A yellow color develops when concen-
trated H2SO4 is added to an aqueous solution of
the antibiotic. Positive Fehling and Tollen tests.
Negative FeCla , Schiff, Sakaguchi, xanthopro-
teic, biuret, ninhydrin, and Molisch tests. Precipi-
tated from aciueous solution l)y picric acid and
reineckate salt; stable to heating at 100°C for 20
minutes at pH 2 and pH 7, and for 10 minutes at
pH 8.
Biological activity: Activity limited to gram-
DESCRIPTIONS OF ANTIBIOTICS
185
positive and a few gram-negative bacteria, such
as members of the genera Hemophilus and Bru-
cella. Not active on gram-negative bacteria, yeasts,
or fungi. Slightly active on some saprophytic
mycobacteria.
Toxieitij: LD50 (mice) 150 to 232 mg per kg in-
travenously.
Reference: 1. Hata, T. et al. J. Antibiotics
(Japan) 8A: 9-14, 1955.
Amice tin
Produced by: Streptomyces vinaceus-drappus (1,
3, 7), S. fasciculatis (1, 3), S. sacromyceticus (6),
S. sindenensis (8, 11), S. plicatus (12), and a
Streptomyces sp. resembling *S. griseus (9).
Synonyms: Sacromycin (9), allomycin (8), anti-
biotic D 13.
Remarks: An original description of this anti-
biotic indicated the presence of more than one
antibiotic in the broth of S. vinaceus-drappus.
It is possible that some of these were synonymous
with amicetin B (plicacetin) and bamicetin (7).
Method of extraction: I. Adsorbed from broth-
filtrate on a cation e.xchange resin (IRC-50, IR-
100, etc.) or on charcoal. Eluted from charcoal
with 10 per cent aqueous acid-acetone. Eluates
adjusted to pH 7 to 8 and freeze dried. Purified b}^
countercurrent distribution (water-methylene
chloride or water-butanol). Anhydrous crystals
can also be prepared by precipitation from anhy-
drous methanol (7).
II. Clarified broth extracted with butanol at
pH 8.5 to 9.5. Butanol extracted with dilute sul-
furic acid (final pH 2.0). This is repeated and
adjustment to pH 8.5 with seeding precipitates
the free base. Further purification is obtained by
treating a dilute HCl solution with activated
carbon, filtering, and reprecipitating the anti-
biotic from solution by adjusting to pH 8 to 8.5.
The slurry of hydrated needles is converted to a
granular, high melting point form by stirring at
60-65°C (3).
Chemical and physical properties: Amphoteric
substance. Base: colorless. Exists in two forms:
needles, m.p. 165-169°C, or granular, m.p. 244-
245°C. Soluble in aqueous mineral acid and alkali
and water-saturated butanol.. Slightly soluble in
water at 22°C. Ultraviolet maxima at 306 m/i
(Ell'tn 512) in 50 per cent aqueous ethanol; 272
niM (Ell, 283) and 325 m^ (£^11 412) in 50 per
cent ethanol-50 per cent 0.1 N NaOH; 304 van
{E\l°m 451) in 50 per cent ethanol-50 per cent 0.1
N HCl; 305 m/x (ElL 465) in water; 316 m^ (^IL
433) in 0.1 .V HCl; and 322 m^ (^5L 470) in 0.1
A" NaOH. Infrared spectrum given in reference 3.
[at* = +116.5° (c = 0.5 per cent in 0.1 A' HCl).
Stable in alkaline solution at >pH 8.0; stable in
acid. Rf values on paper chromatography given
in reference 3. C29H42N6O9 : C = 55.98%; H =
6.92%; N = 13.18%. Molecular weight about 640.
pKa' about 1.1, 7.0, and 10.4. Mild alkaline hy-
drolysis yields cytosamine. C18H32N4O6 . Acid
hydrolysis yields a salt of a base, cytimidine,
CisHitNsOj ; m.p. (of salt) 264-266°C. Structural
formula of amicetins (3, 4, 7, 10) given in Chapter
6. The dimethylamino sugar is amosamine. Hydro-
chloride: Fine white crystals; m.p. 190-192°C.
More water-soluble than amicetin. Methyl orange
and orange II react with amicetin to give water-
insoluble salts. Orange II salt: m.p. 204-206°C.
Biological activity: Both needles and granular
forms have similar microI)iological activity, being
active largely against gram -positive bacteria,
especially mycobacteria (2, 4). Active in protect-
ing mice infected with M. tuberculosis H37R.V (1).
Prolong survival time of mice with transmitted
leukemia (Line 82), but not in mice with two other
leukemia strains (5).
Toxicity: Citrate complex of amicetin: acute
LD50 (mice) about 90 mg per kg intravenously,
600 to 700 mg per kg subcutaneously. LD50 (rats)
about 200 mg per kg intravenously. Especially
toxic to guinea pigs, being 40 times as toxic as
streptomycin, but only ^10 as toxic as penicillin
given subcutaneously (2).
References:
1. McCormick, M. H. and Hoehn, M. M.
Antibiotics & Chemotherapy 3: 718-
720, 1953.
2. DeBoer, C. et al. J. Am. Chem. Soc. 75:
499, 1953.
3. Hinman, J. W. et al. J. Am. Chem. Soc.
75: 5864-5866, 1953.
4. Flynn, E. B.. et al. J. Am. Chem. Soc.
75: 5867-5871, 1953.
5. Burchenal, J. H. et al. Proc. Soc. Exptl.
Biol. Med. «6: 891-893, 1954.
6. Nisho, Y. et al. Japan. J. Bacteriol. 9:
600-601, 1954.
7. British Patent 708,686, May 5, 1954.
8. Tatsuoka, S. et al. Ann. Rept. Takeda
Research Lab. 13: 41-44, 1954.
9. Himmia, Y. et al. J. Antibiotics (Japan)
8A: 148-152, 1955.
10. Stevens, C. L. et al . J. Am. Chem. Soc.
78: 6212, 1956.
11. Nakazawa, K. and Fujii, S. Ann. Rept.
Takeda Research Lai). 16: 109-110, 1957.
12. Haskell, T. H. et al. J. Am. Chem. Soc.
80: 743-747, 1958.
186
DESCRIPTIONS OF ANTIBIOTICS
Aniiccliii H
Produced by: Streptornyces plicatus (2, 3).
Synonyms: Plicacetin, believed to be a pre-
cursor of amicetin (1); antibiotic C (2).
Method of extraction: Broth-filtrate extracted
with l-butanol at slightly alkaline pH values,
extract concentrated, then back-extracted into
water at pH 2.0. Could be precipitated from aque-
ous solutions with various aromatic azosulfonic
acid dj'es, or adsorbed on and eluted from IRC-
50. Precipitated from absolute methanol solution
with ether or by raising an aqueous solution to
pH 9.0 with NH4OH. Recrystallization from hot
water or dilute aciueous methanol gives crystal
form I; from ethyl acetate, crystal form II; and
from absolute ethanol, crystal form III (1).
Chemical and physical properties: Crystal I: ni.p.
182-184 °C; colorless needles. Crystal II: m.p.
160-163°C; colorless needles. Crystal III: m.p.
222-225°C; dense prisms. Base: [a]'^ = +181°
(c = 2.7 per cent in methanol). Soluble in dilute
acid, lower alcohols, chloroform, and methylene
chloride. Sparingly solulale in ethyl acetate, ether,
and cold water. Insoluble in benzene and petro-
leum ether. Ultraviolet absorption spectrum
maxima: 257 and 311.5 m/x (0.1 N HCl), 249 and
321 niM (pH 7.0, phosphate buffer), 329 m^ (0.1
N NaOH). pKa = 2.2, 7.0, and 10.9. Infrared spec-
trum of crystal III given in references 1 and 3.
Rf value = 0.86 (l-butanol saturated with 0.05
M pH 7.0 phosphate buffer) (3). Rf (n-butanol
and acetic acid) = 0.28 (2). Soluble in dilute acid.
Negative Molisch, biuret, Sakaguchi, ninhydrin,
FeCls , and Fehling tests. Positive Ehrlich and
Bratton-Marshall tests (both for arylamino
groups) (2). Gives insoluble precipitates with
picric, picrolonic, styphnic, and phosphotungstic
acid. A hydrochloric acid solution reacts with
nitrous acid giving a diazo compound (I). This
reacts with N-(l-naphthyl)ethylenediamine to
give a violet color with a maximum at 550 mju.
Alkaline hydrolysis products include cytosa-
mine. Acid hydrolysis product is p-aminoben-
zoylcytosine. CooHjsNsOt : C = 57.69%; H =
6.84%; N = 13.52% (1). Structural formula given
in Chapter 6.
Biological activity: Active on gram-positive bac-
teria, including mycobacteria. Negligible activity
on gram -negative bacteria and fungi. Has less
activity in vitro and in vivo on M. lubercidosis
H37Rv than amicetin or baniicetin (3).
References:
1. Sensi, P. et al. Antilnotics & Chemother-
apy 7: 645-052, 1957.
2. British Patent 707,332, April 14, 1954.
3. Haskell, T. R. et al. J. Am. Chem. Soc.
80: 743-747, 1958.
LiiiKloni'v cin
Produced by: Streptornyces sp. (1).
Synonym: Resembles valinomycin (1, 2).
Method of extraction: Broth and mycelium
treated with a filter-aid, the pH adjusted to 3.5
with concentrated HCl, and filtered. The solid
residue is extracted with methanol. Methanol
removed by evaporation in vacuo at 40°C; the
residual suspension diluted with water and freeze
dried. Solid extracted in a Soxhlet apparatus for
3 to 6 hours with petrol (b.p. 30-60°C). Petrol
evaporated to dryness, giving an oily residue
which partially crystallizes on standing. Recrys-
tallization from petrol or 50 per cent aqueous
ethanol. Increased yields can be obtained by
chromatographing benzene solutions of the dried
mother litpiors on silicic acid and developing
with benzene, then chloroform. In large fermen-
ters, most of the activity is in the broth (1).
Chemical and physical properties: Neutral,
colorless needles; m.p. 192 °C. [a]" = +19.2°
(c = 1.2 per cent in ethanol). Insoluble in water;
partially soluble in petrol; very soluble in organic
solvents. Sta))le. No characteristic absorption in
ultraviolet or visible light. Infrared absorption
spectrum given in reference 2. Hydrolysis in alco-
holic acid or alkali, followed by treatment with
concentrated HCl, gives two products, D(— )-
valine and D( — )-a-hydroxyisovaleric acid, both
biologically inactive. Rf values of 0.86 (paper
impregnated with ethylene glycol, with petrol,
b.p. 100-120°C as mobile phase); 0.90 (water-
saturated n-amyl alcohol); 0.36 (ethjd alcohol-
acetic acid-water, 3:1:6); and 0.89 (ethanol-
water, 2:3). C = 60.22%,; H = 8.62%; N = 7.06%;
C-Me = 31.7%. C40H68O12N4 . Chemical structure
shown at top of p. 187.
Biological activity: Active on yeasts and fila-
mentous fungi. Resistance does not develop
readily. Not active on bacteria (1).
References:
1. Taber, W. A. and Vining, L. C. Can. J.
:\Iicrobiol. 3: 953-965, 1957.
2. Vining, L. C. and Taber, W. A. Can. J.
Chem. 35: 1109-1116, 1957.
Amphoniyciii
Produced by: Streptornyces canus (1, 3), S. vio-
laceus (5), and a Streptornyces sp. resembling S.
lavendulae (5).
Synonym: Closely related to crystallomycin
and aspartocin.
DESCRIPTIONS OF ANTIBIOTICS
187
\ \ c-x-
Ha-
c
//.
N— C-
H II
O
<('
^n,
'O
o /
ff O /
/ ^ ^^
c;=o
I
XH
\ ^/c- C/f
W o \ ^^Z-
-C
\
\
c \
Method of extraction: Broth-filtrate adjusted to
pH 1.95 and filtered. Filtrate extracted with
n-butanol, washed with water at pH 2.0, then
back-extracted into water at pH 6.4 to 7.4. Pro-
cedure repeated (3). I. Water-extracts from
butanol treated with Darco G-60 at pH 5 to 7 and
eluted with butanol-saturated water (pH 9).
Re-extracted into butanol. Extract concentrated
in vacuo and ether added to precipitate ampho-
niA'cin. Reprecipitated from anhydrous ether (5).
II. Water-extracts freeze dried, taken up in
water, adjusted to pH 2.0 with phosphoric acid,
and extracted into butanol. Extract filtered and
decolorized with activated charcoal. Butanol
concentrated in vacuo. Amphomycin precipitated
on addition of excess ethyl acetate. Can also be
precipitated from an aqueous solution (pH 2.2)
\)y isoelectric precipitation at pH 3.4. III. Puri-
fied by precipitation as the reineckate or as the
calcium salt (reaction of calcium chloride with
Na amphomj^cin) (1, 3).
Chemical and physical properties: Amphoteric
polypeptide with an isoelectric point at 3.4 to 3.5.
White to off-white amorphous powder. Soluble in
water. Soluble in methanol as the acid form and
as the salt form. Soluble in higher alcohols con-
taining at least four carbon atoms, only in the
acid form. Insoluble in nonpolar solvents. End-
absorption (210 to 230 m^) of ultraviolet light.
Infrared spectrum given in reference 5. [ajc =
+7.5° (c = 1 per cent in water at pH 6.) Optical
activity decreases at higher or lower pH. Positive
biuret test. Negative ninhydrin, Sakaguchi, Mo
lisch, Ehrlich-Pauly, xanthoproteic, Adamkie-
wicz, Ehrlich, Liebermann, and Seliwanoff tests.
Stable in aqueous solution for at least 1 month
at room temperature. Rf = 0.09 on paper chroma-
tography (water-saturated butanol -collidine-/3-
naphthalenesulfonic acid, 98:2:1). C = 54.4%;
H = 7.2%; N = 14.2''r (for the free acid). Mini-
mal molecular weight 1400 to 1500. Acid hydroly-
sates contain aspartic acid, glycine, valine, pro-
line, and a fifth unidentified amino acid (1, 3, 5).
Biological activity: Active on gram -positive bac-
teria. Not active on gram-negative bacteria, C.
albicans, or Trichophyton mentagrophytes (1, 3).
Active in vivo (mice) on 1). pneumoniae (1, 3), B.
anthracis, Erysipelothrix rhusiopathiae (5), Try-
panosoma gambiense, and T. rhodesiense infections
(4).
Active on d()\vn\- mildew of cucinnher, Pseudo-
peronospora cubensis (0).
Toxicity: LD50 (mice) 177.8 mg per kg intra-
venously. LDo,, (dogs) 100 mg per kg intravenously
(Nasalt) (2).
Utilization: Possible u.se in topical application
in gram-positive infections.
References:
1. Heinemann, B. et al. Antibiotics & Chemo-
therapy 3: 1239-1242, 1953.
2. Tisch, D. E. et al. Antibiotics Ann. 1011-
1019, 1954-1955.
3. British Patent 73(i,325, September 7, 1955.
188
DESCRIPTIONS OF ANTIBIOTICS
4. Packchanian, A. Antibiotics cV: Chemo-
therapy 6: 084-691, IDoC.
5. Giolitti, G. et al. (!iorn. iiiicroWiol. ."}: TO-
SS, 1957.
6. Ark, P. A. and Thompson, J. B. Plant
Disease Reptr. 41: 452-454, 1957.
Anipliotericin A
Produced by: Streptomyces sp. The same culture
produces amphotericin B.
Method of extraction: Whole culture mixed with
an equal volume of isopropanol; pH adjusted to
10.5. After removal of the mycelium, solution is
concentrated in vacuo, witli formation of a ]ire-
cipitate. Precipitate washed with water and ace-
tone before drying. This crude concentrate con-
tains both amphotericin A and B. Crude material
slurried in a 2 per cent (weight per volume) meth-
anol solution of calcium chloride. Amphotericin
A is dissolved by the methanolic calcium chloride
solution and can be precipitated from this solution
by the addition of water. Repeating this latter
procedure yields crystalline amphotericin A.
Chemical and physical properties: Conjugated
tetraene. Slightly soluble (8 to 40 mg per ml) in
methanol, glacial acetic acid, and propylene
glycol. Soluble in N,N-dimethylformamide, meth-
anol-calcium chloride, and basic methanol to the
extent of 90 to 125 mg per ml. Insoluble in water,
ethanol, butanol, acetone, ethyl acetate, ether,
and benzene. Decomposition above 153°C. [«][, ' =
— 9.9° in 0.1 N methyl hydroxide-hydrochloric
acid. [a]f'' = +32° in acid dimethylformamide.
C = 60.3%; H = 8.4%; N = 1.7%. The ultraviolet
light absorption of amphotericin A is charac-
teristic of a conjugated tetraene, and is similar to
that of rimocidin and nystatin. Amphotericin A is
amphoteric and forms a water-soluble sodium salt.
Negative FeCls test; positive Alolisch test. Potas-
sium permanganate and bromine carljon tetra-
chloride solutions decolorized.
Biological activity: Active in vitro against fungi
but not against bacteria. Amphotericin A has a
wide spectrum of antifungal activity, being active
against both filamentous fungi and yeasts. Active
in vivo against experimental C albicans, Histo-
plasma capsulatum, and Cryptococcus neoformans
infections of mice. Absorbed after oral administra-
tion to mice.
Toxicity: LD50 (mice) 450 mg per kg intraperi-
toneally.
Reference: 1. Steinberg, B. A. ct al . Antil)i-
otics Ann. 574-591, 1955-1956.
Amphotericin B
Produced by: Streptotnyces nodosus (15). This
culture also produces amphotericin A (1).
Method of extraction: Whole culture mixed with
an equal volume of isopropanol; pH adjusted to
10.5. After removal of the mycelium, solution is
neutralized and concentrated in vacuo, with for-
mation of a precipitate. Precipitate washed with
water and acetone before drying. This crude con-
centrate contains both amphotericin A and B.
Crude material slurried in a 2 per cent (weight per
volume) methanol solution of calcium chloride.
Amphotericin B is not appreciably dis.solved in
this solvent. Previously undissolved material ex-
tracted with acidified N,N-dimethylformamide.
Methanol added to solution. Reaction of the form-
amide-methanol solution maintained at pH 5,
while water is added, resulting in precipitation of
amphotericin B. Repeating the latter procedure
yields crystalline amphotericin B (1).
Chemical and physical properties: Amphoteric
conjugated heptaene. Deep yellow prisms (from
dimethylformamide). Decomposes at >170°C.
Soluble in dimethyl sulfoxide and acetic N,N-di-
methylformamide (GO to 80 mg per ml). Slightly
soluble (0.2 to 4 mg per ml) in propylene glycol,
glacial acetic acid, and N,N-dimethylformamide.
Insoluble in water, methanol, ethanol, butanol,
acetone, ethyl acetate, ether, benzene, chloroform,
pyridine, and alcoholic KOH. X^^^^, 363, 382, 406.
Infrared spectrum is given in reference 1. [a]'„ " —
-33.6 (in 0.1 N methyl hydroxide-HCl) or +33.3
(in acid dimethylformamide). Positive Molisch,
KMn(J4 , and Br-CCl4 tests. Negative FeCls test.
C46HT3-75NO,s-2n : C = 57.59%; H = 8.0%,; N =
1.7%. Neutralization equivalent, 959 (perchloric
acid in acetic acid). Possibly contains a lactone
group. Hydrogenation product is colorless, bio-
logically inactive, tetradecahydroamphotericin B,
C46H87NO2 , having an infrared spectrum similar
to the parent polyene and which darkens at 160°C
but is not decomposed at 250°C. Prolonged acetol-
ysis of amphotericin B yields tri- and tetraacetates
of an amino desoxyhexose "mycosamine," CeHis-
NO4 , which is also obtained from nystatin (see
Chapter 6). Amphotericin B forms a ])oorly water-
soluble sodium salt (1, 2, 5, 9).
Biological activity: Active in vitro against fungi
but not against bacteria. Amphotericin B is more
active against yeasts and yeast -like fungi than
against filamentous fungi (1, 16). Active in tissue
culture at 0.1 to 1.0 Mg per ml against the patho-
genic form of Histoplasma capsulatum. Sporo-
trichium schenkii, Cryptococcus neoformans, C.
albicans, and Blastomyces dermatitidis (6). Cross-
resistance with nystatin but not pimaricin (17).
Active in vivo against experimental C. albicans, H.
capsulatum, Coccidioides immitis, Cr. neoformans.
DESCRIPTIONS OF ANTIBIOTICS
189
and Trichophyton luentagrophi/tes infections of
mice. Absorbed after oral administration (1, 7).
Active in rabbits against lihizopus oryzae infec-
tions (12), in hamsters on H. capsulatum (8), and
in mice on Mucor pusillus (14) and Paracoccidioides
brasiliensis (19). Has a protective but variable
curative effect in rabbits infected with Asper-
ijilhis finnigatiis (20).
Toxicity: LDso (mice) 280 mg per kg intraperi-
toneally (1). Possible renal toxicity in human
lieings (13).
Utilization: Sy.-^temic myco.ses. Coccidioidal and
cryptococcal meningitis (11). Cutaneous crj-pto-
coccosis, histoplasmosis, and candidiasis (8, 13,
18). Certain cases of histoplasmosis, blastomyco-
sis, and pulmonary coccidioidomycosis. Effective
in one case each of aspergillosis, ocular candidia-
sis, and chromoblastomycosis (Hormodendnan
pedrosoi) (18).
References:
1. Sternberg, T. H. et al. Antibiotics Ann.
566-591, 1955-1956.
2. Donovick, R. et al. (iiorn. micr()l:)iol. 2:
147-159, 1956.
3. Halde, C. et al. Antibiotics Ann. 123-127,
1956-1957.
4. Kozinn, P. J. et al. Antibiotics Ann. 128-
134, 1956-1957.
5. Dutcher, J. D. et al. Antil)iotics Ann.
866-869, 1956-1957.
6. Larch, H. W. et al. Antibiotics Ann. 918-
922, 1956-1957.
7. Halde, C. et al. J. Invest. Dermatol. 28:
217-232, 1957.
8. Baum, G. L. et al. Antibiotics & Chemo-
therapy. 7: 477-482, 1957.
9. Walters, I). R. ct al. J. Am. Chem. Soc.
79: 507()-5077, 1957.
10. Utz, J. P. et al. Antibiotics Ann. 65-70,
1957-1958.
11. Furcolow, M. L. and Rubin, H. 17th Conf.
on Chemotherapy Tuberc, Veterans
Admin. February 1958, pp. 309-310.
12. Chick, E. W. et al. Antibiotics & Chemo-
therapy 8: 394-399, 1958.
13. Crounse, R. G. and Lerner, A. B. A. M. A.
Arch. Dermatol. 77: 210-215, 1958.
14. Osswald, H. and Seeliger, H. P. R. Arz-
neimittel-Forsch. 8: 370-374, 1958.
15 Donovick, R. Personal communication.
October, 1958.
16. MuUer, W. H. Am. J. Botany 45: 183-190,
1958.
17. Sorensen, L. J. et al. Antibiotics Ann.
920-923, 1958-1959.
18. Costello, M. J, et al. A. M. A. Arch. Derma-
tol. 79: 184-193, 1959.
19. MacKinnon, J. E. Ann. fac. med. Monte-
video 43: 201-206, 1958 (Biol. Ab.'slr. 33:
2813, 1959).
20. Evans, J. H. and Baker, R. D. Antibiotics
& Chemotherapy 9: 209-213, 1959.
Angolamyclii
Produced by: Streptomyces eurythennus.
Method of extraction: Culture-filtrate extracted
with ethyl acetate at pH 8. Transferred into dilute
aqueous acetic acid. Re-extraction with ethyl
acetate after being made alkaline with Na^COs .
Purification by chromatography on alumina
(eluent: chloroform-methanol, 16:1). Crystallized
from ether. Can also be precipitated from a
warmed ether solution of the crude material by
seeding with the pure substance. Recrystallization
from benzene on the addition of ether.
Chemical and physical properties: Basic, lipo-
philic sul>stance, probably belonging to the
macrolide group (2). Two crystal forms: (a) color-
less crystals from benzene-ether; m.p. 133-136°C;
(b) colorless needles from diisopropyl ether; m.p.
165-168°C. Ultraviolet absorption spectrum maxi-
mum at 240 niM (log e = 4.16). Infrared spectrum
given in reference 1. [aju = —64° (c = 1.30 per
cent in chloroform). Negative Fischback-Levine
reaction for carbomycin. C49-.50H87.91O1SN. Acid
hydrolysis products include two sugars, neither of
which is desosamine.
Biological activity: Active on certain gram-
positive bacteria and Endainoeba histolytica. Not
active on gram-negative bacteria, mycobacteria,
or 3'easts. Some activity in vivo on Streptococcus
pyogenes infections in mice.
Toxicity: Mice tolerate 500 mg per kg subcu-
taneously.
References:
1. Corbaz, R. et al. Helv. Chim. Acta 38:
1202-1209, 1955.
2. Brink, N. G. and Harman, R. E. Quart.
Revs. (London) 12: 93-115, 1958.
Angus tiny ciiis
Produced by: Stieptoniyces hygroscopicus (1), S.
hygioscopicus var. angustniyceticus (4), and S.
hygroscopicus var. decoyimine (7).
Synonym: Related to psicofuranine.
Method of extraction: Broth adjusted to pH 7.6,
stirred with carbon, and filtered. Carbon eluted
with 80 per cent aqueous acetone at pH 2.0.
Eluates are neutralized and kept in the cold over-
190
DESCRIPTIONS OF ANTIBIOTICS
night. Impurities thus precipitated are discarded
and the acetone solution is concentrated in vacuo.
The concentrate is seeded and stored in the cold
(2), or freeze dried (1). Freeze -dried solid taken
up in hot ethanol, concentrated in vacuo, and
purified by chromatography on alumina (ben-
zene-ethanol, 1:1, as solvent and developer).
Crystallized from benzene-ethanol (1). Compo-
nent C is separated from A and B (see below) by
partition chromatography on cellulose (aqueous
n-butanol-1 per cent pyridine). A and B are sepa-
rated by countercurrent distribution (pyridine -
n-butanol-water, 1:100:100) (2). Repeated re-
crystallization from water (-1).
Chemical and physical properties: Angustmycin
is a mixture of three substances, one of which
(A) is biologically active. The other two, B and
C, are biologically inactive. B was identified as
adenine, a structural moiety of A and C. Angust-
mycin A: Basic substance. Hydrate: m.p. 128-
130°C, remelting at 164.5-165.5°C (decomposition)
(4). Soluble in water, methanol, ethanol, pyri-
dine, acetic acid, methyl Cellosolve, dimethyl-
formamide, and phenol. Sparingly soluble in
butanol and dioxane. Insoluble in ether, acetone,
chloroform, carbon disulfide, ethyl acetate, and
other organic solvents (2). Positive Molisch test.
Negative Fehling and Tollen tests (4). Ultra-
violet absorption spectrum maximum at 260 ni/n
(e = 17,100) in acidic or alkaline solution or
methanol (1, 2, 4). Infrared spectrum given in
reference 4. [a]" = +17.02° (c = 1.4 per cent in
dimethylformamide) (2); pK,, = 9.8. CuHu04N5-
H.,0. C" = 44.4%; H = 5.09%; N = 23.42%. Struc-
tural formula of angustmycin A (6-amino-9 (L-1 , -
2-fucopyranoseenyl)purine) (4) given in Chapter
6. Tetraacetate: needles; m.p. 187-188°C (4).
Benzoate: m.p. 115-116°C (3). Hemimethanolate:
needles; m.p. 172-174°C. Acid hydrolysis products
include adenines and "angustose" or L-2-keto-
fucopyranose. Angustose crystallizes as needles;
m.p. 115-116°C. [at' = +18° (c = 1 per cent in
ethanol), CeHioOs - Rf (butanol-acetie acid-
water, 4:1:5) = 0.40 (3, 4, 6). Angustmycin C:
needles; m.p. 202-204°C (decomposition), [a]^^ =
— 71.1° (c = 1.8 per cent in pyridine). Ultraviolet
absorption spectrum maximum (water) at 260
van {Eil'm 510). Optically inactive. Infrared spec-
trum given in reference 5. Positive Molisch test.
Negative ninhydrin reaction (2, 5). CnHisOsNs .
Structural formula of angustmycin C (6-amino-9
(/3-p-psicofuranosyl)purine) (5) given in Chapter
6. An antibiotic, Antibiotic U 9586, said to have
the same structure as angustmycin C (inactive)
was reported active on bacteria and tumors in
vivo (7).
Chemical and physical properties of Antibiotic
U 9586: 6-Amino-9-D-psicofuranosylpurine. Nee-
dles; m.p. 212-214° (decomposition). Ultraviolet
absorption spectrum maxima at 259 m^i (E'lcm
508) in 0.01 A' H2SO4 and 261 mn (E^ 530) in
0.01 A^ NaOH. Positive ammoniacal silver nitrate
and Jordon-Prj'de tests (ketohexoses) and Bene-
dict test (after hydrolysis). Negative Bial, nin-
hydrin, and Benedict (before hydrolysis) tests.
[a]^ = —537° (c = 1 per cent in dimethyl sulf-
oxide). CuHisNsOs : C = 44.25%; H = 5.10%;
N = 23.74%; O = 27.02%. Acid hydrolysis prod-
ucts give adenine and D-psicose. Structural for-
mula given in Chapter 6.
Biological activity: Angustmycin A inhibits
Mycobacterium 607 and M. phlei at 25 ng per ml.
Inactive against virulent M. tuberculosis H37Rv
at 100 ng per ml. No activity on other bacteria or
fungi (1).
Toxicity: Mice tolerate 2.5 gm per kg intraperi-
toneally.
References:
1. Yimtsen, H. et al. J. Antibiotics (Japan)
7A: 113-119, 1954.
2. Yiintsen, H. et al. J. Antibiotics (Japan)
9A: 195-201, 1956.
3. Yiintsen, H. J. Antibiotics (Japan) llA:
77-80, 1957.
4. Yiintsen, H. J. Antibiotics (Japan) llA:
233-243, 1957.
5. Yiintsen, H. J. Antibiotics (Japan) llA:
244-249, 1957.
6. Yiint.sen, H. and Yonehara, H. Bull. Agr.
Chem. Soc. Japan 21: 261-262, 1957.
7. Schroeder, W. and Hocksema, H. J. Am.
Chem. Soc. 81: 1767-1768, 1959.
Anisomycin
Produced by: Sireptoniyces griseolus (3, 4), S.
roseochromogenes (14), and a Streptonryces sp.
(3,4).
Synonyms: Antibiotic PA 106; Flagecidin.
Method of extraction: Broth-filtrate adjusted to
pH 9.0 and extracted countercurrently with
methyl isobutyl ketone. Back-extracted into
water at pH 2.0. This water is adjusted to pH
9.0 and extracted countercurrently with CCI4 .
On concentration of this extract, anisomycin
crystallizes out. Recrystallization from hot ethyl
acetate or water (2). Can also be extracted from
broth by diethyl ether, benzene, ethyl or butyl
acetate, butanol, or chloroform. May also be re-
covered and jjurified by adsorption on charcoal,
DESCRIPTIONS OF ANTIBIOTICS
191
treatment with ion exchange resins, and chroma-
tography on ahnnina. May be preciijitated from
a concentrated chloroform-extract by addition of
cyclohexane (3).
Chemical and physical properties: Basic sub-
stance. Long white needles; m.p. 140-141 °C. Can
be distilled in vacuo at a few degrees above its
melting point. Soluble in lower alcohols, esters,
ketones, chloroform, and dilute ac^ueous acids.
Moderately soluble in water. Low solubility in
ether, carbon tetrachloride, and hydrocarbons
(3, 4). X^'ST^ 224 mu (e = 10,800), 277 m^ U =
1800), and 283 m^ (e = IfiOO). Infrared data given
in reference 4. [a]'if = —30° (c = 1 per cent meth-
anol). Aqueous solutions lose jjotency slowly at
acid pH, more rapidly at alkaline pH. Powder is
stable C14H19NO4 • C = 63.51' t; H = 7.21^:^; N =
5.22%. Salts are very soluble in water (3, 4).
Biological activity: Active on protozoa (1), some
fungi, Candida stellatoides, C. albicans, and Sac-
charomyces cerevisiae. Slightly active on gram-
positive and gram-negative bacteria. Active in
vitro and in vivo (mice) on Trichomonas foetus
and T. vaginalis (3, 4, 7, 8). Some activity on
Endamoeba histolytica in vitro (1) and in guinea
pigs (5). Some activitj- on Nosenia disease in bees
(9). Active on powdery mildew of beans {Erysiphe
polygoni) (11), of roses (Sphaerotheca) (12), and
of wheat (E. graminis f. sp. triiici) (15). Also
active on downy mildew of lima beans (Phyto-
phthora phaseoli) (10) and broccoli (Peronospora
parasitica) (13).
Toxicity: LDjo (mice) 140 mg per kg intraven-
ously, 148 mg per kg orally, 400 mg per kg intra-
peritoneally. LDjo (rats) 72 mg per kg orally,
107 mg per kg intravenously, 345 mg per kg intra-
peritoneally. Causes emesis in cats and dogs.
Tolerated by monkeys in daily oral doses up to
64 mg per kg over a 32-week period (7).
Utilization: Effective, in a limited clinical
study, against vaginal infections with Tricho-
monas vaginalis (6).
References:
1. Lynch, J. E. et al. Antibiotics & Chemo-
therapy 4: 844-848, 1954.
2. Sobin, B. A. and Tanner, F. W.. Jr. J.
Am. Chem. Soc. 76: 4053, 1954.
3. Tanner, F. W. et al. U. S. Patent 2,691,618,
October 12, 1954.
4. Tanner, F. W., Jr. et al. Antibiotics Ann.
809-812, 1954-1955.
5. Lynch, J. E. et al. Antil)iotics Ann. 813-
819, 1954-1955.
6. Frye, W. W. et al. Antil)iotics Ann. 820-
823, 1954-1955.
7. Gardocki, J. F. el al. Antibiotics & Chemo-
therapy 5: 490-495, 1955.
8. Lynch, J. E. et al. Antibiotics & Chemo-
therapy 5: 508-514, 1955.
9. Katznelson, H. and Jamieson, C. A. Glean-
ings Bee Culture 83: 275-277, 1955.
10. Zaumeyer, W. J. and Webster, R. E. Phy-
topathology 46: 470, 1956.
11. Zaumej'er, W. J. Antibiotics Ann. 1015-
1018, 1955-1956.
12. Kirby,R. S. Plant Disease Reptr. 41:534-
535, 1957.
13. Natti, J. J. Plant Disease Reptr. 41:
780-788, 1957.
14. British Patent 768,364, February 13, 1957.
15. Powers, H. R., Jr. Phytopathology 48:
474-477, 1958.
Antibiotic l-81d-ls
Produced by: Streptouiyces albus.
Synonym: Has properties in common with cam-
phomycin.
Method of extraction: From 70 to 95 per cent of
the antibiotic is present in the mycelium. Myce-
lium separated from the broth and extracted with
methanol, ethyl acetate, n-butanol, diethyl ether,
benzene, chloroform, or methyl isobutyl ketone.
Extract concentrated by distillation under re-
duced pressure. Residual acjueous slurry extracted
with diethyl ether. Extract concentrated, then
treated with n-pentane. Residue which precipi-
tates is then dissolved in hot ethyl acetate, from
which, after addition of hexane and cooling, the
crude antibiotic precipitates. Recrystallized from
ethyl acetate-aliphatic hydrocarbons, dioxane-
water, ethanol-water, or chloroform-benzene mix-
tures.
Chemical and physical properties: Small colorless
needles; m.p. 140-141°C. Soluble in lower alcohols,
chloroform, ethyl acetate, dioxane, ether, and
acetone. Slightly soluble in benzene. Insoluble in
water, aliphatic hydrocarbons, cold 5 per cent
NaOH, and cold 5 per cent HCl. [0]^ = -29.8°
(c = 1.14 per cent in acetone). Ultraviolet absorp-
tion spectrum maxima at 244 and 284 m/x (aqueous,
acidic, or dry methanol). Addition of alkali shifts
the 284 niM peak to 274 m/u. Infrared absorption
spectrum given in reference 1. Heating with 5 per
cent NaOH causes a gas of basic pH to be given
off. Red in concentrated H0SO4 and lavender in
cold 85 per cent H3PO4 . Positive KMn04 test.
C38H63-65O12N: C = 63.10%; H = 8.87%; O =
26.48% ;N = 1.92%.
Biological activity: Active on D. pneumoniae and
Sarcina lutea at 6.3 Mg ppi" nil, Streptococcu.<i pyo-
192
DESCRIPTIONS OF ANTIBIOTICS
genes at 12.5 /xg j^er ml, and Staph, aureus. Strep-
tococcus agalactiae, and B. anthracis at 25 ng per
ml. Inactive on other bacteria. Very active on
fungi and yeasts. Complete control of tomato early
Might (Alternaria solani) by spraying at 80 ppm.
Active in vivo but not in vitro on influenza PR 8
virus in chick embryos.
Reference: 1. Herrmann, E. C. et al. U. S. Patent
2,805,185, September 3, 1957.
Antibiotic 1(» CM
Produced by: Strepionnjces sp. resemljling S.
albus.
Method of extraction: Broth adjusted to pH 2.0,
autoclaved, filtered, and treated with activated
carbon. Filtrate adjusted to pH 8.0. Adsorption
on activated carbon and elution with acidic etha-
nol. Eluate evaporated m vacuo. Purified by chro-
matography on a 1:1 mixture of Celite and acti-
vated carbon. Development with ethanol.
Chemical and physical properties: Soluble in
butanol; insoluble in ether. Reineckate soluble in
acetone; hydrochloride much less so. Ultraviolet
al)sorption spectrum maximum (water) at 252 ni/i,
with a shoulder at 260 to 280 m^, and an inflection
at 312 m/jL. Stable to autoclaving at pH 2.5 for 15
mi mites; less stable at neutral and alkaline pH.
Distinguished from amicetin, carbomycin, eryth-
romycin, griseomycin, methymycin, and pro-
actinomycin by paper chromatography. Rf = 0.12
(isobutylcarlMnol-n-nonanol-CCl4-propionic acid,
50:50:25:2).
Biological activity: Active on gram-positive
bacteria; less so on gram -negative l)acteria. No
activity on fungi or Clostridia. B. suhtilis, 1 to
>100 ng per ml; Staph, aureus, 1 to 2 jug per ml;
Sarcina lutea, 0.1 to 0.5 jug per ml; Neisseria ca-
tarrhalis, 2 jug per ml; Sal. schottDiuclU'i i , 50 yug
per ml; E. coli, 25 to >100 ng per ml; Mycobac-
teri}ini spp., 50 Mg per ml. Activity antagonized
by cysteine, sodium thioglycolate, hydrazine,
and hydroxylamine.
Toxicity: LD.io (crude powder) 500 to 700 mg per
kg intravenously.
Reference: 1. Sokolski, W. T. Thesis, Purdue
University, 1955.
Antibiotic 26/1
Produced by: Streptomyces globispurus.
Method of extraction: Extraction of the broth
and/or the mycelium with isobutanol at pH 7 to
8. Tenfold concentration of the extract under re-
duced pressure at 35 to 40°C. Standing 12 to 15
hours al 4°C permits the formation of a precipi-
tate. Further i)urification on anion exchanger
AV-10.
Chemical and physical properties: Heptaene,
weakly acidic, yellow, amorphous powder, crystal-
lized with (lifhculty. Poorly soluble in water below
pH 7.0. Solul)le in dimethylformamide and 80 per
cent diethylene glycol. Sparingly soluble in meth-
anol, ethanol, butanol, and acetone. Insoluble in
chloroform, benzene, toluene, petroleum ether,
ethyl acetate, and ethyl ether. Biological activity
lost rapidly at pH 4 to 5. Powder can be kept for
6 months in the refrigerator without drop in ac-
tivity. Alcoholic solutions give a violet color with
concentrated sulfuric acid. Negative biuret, xan-
thoproteic, and ninhydrin reactions. Ethanolic
solutions have light absorption maxima at 359, 380,
and 404 mju- Paper chromatographj^ (n-butanol-
acetic acid-water, 20:1:25) shows that the sub-
stance belongs to the candicidin-ascosin-tri-
chomycin group of heptaenes.
Biological activity: Active against filamentous
fungi and yeasts. No activity against bacteria.
Against fungi, the action is not only fungistatic
but also fungicidal.
Toxicity: LD.Mi(mice)9to 11 mg per kg intraperi-
toneally, 520 to 740 mg per kg, subcutaneously.
The most purified preparation has an intraperi-
toneal LD.50 of 35 to 60 mg per kg.
Reference: 1. Tsyganov, V. A. et al. Antihiotiki
4(1): 21-26, 1959."
Antibiotic 30-10
Produced by: Streptouiyces sp. (1).
Method of extraction: Broth-filtrate extracted
with benzene at pH 5.4. Extract concentrated to
dryness in vacuo (1, 2).
Chemical and physical properties: Crude sub-
stance: yellow-brown. Soluble in methanol, etha-
nol, butanol, amyl acetate, ether, benzene, and
chloroform. Scarcely soluble in petroleum ether
and water. Heat -stable at mildly acid pH (2).
Biological activity: Active on certain fungi {e.g.,
Alternaria solani, liotrytis bassiana, Gloeosporium.
nelumbii, Colletotrichum lagenarium, Fusarium
lini, Gibberella zeae, Penicillium glaucum, Rhizoc-
tonia solani, and Sclcrotinia) . Very slight to no
activity on yeasts and bacteria (1, 2).
References:
1. Nisikado, G. et al. Xogaku Kenk.\u 13:
63-72, 1955.
2. Nisikado, Y. et al. Ber. Ohara Inst, land-
wirtsch. Biol. Okayama Univ. 10: 229-240,
1956.
DESCRIPTIONS OF ANTIBIOTICS
193
Antibiotic 136
Produced by: Streptomyces lavendulae and an
nnidentified Streptomyces sp. (1).
Remarks: Streptothricin-like. One of the com-
ponents of broth (Fraction B) is slreptothricin
(1,2).
Method of extraction: Broth-filtrate treated with
carbon at pH 2.5. Adsorption on Folin Decalso
at pH 7.5 and ehition with 10 per cent aqueous
NH4CI. Eluate adsorbed on 13arco G-60 at pH
7.4 and eluted with 0.05 A' HCl in 50 per cent
aqueous methanol. Eluates concentrated in vacuo,
filtered, and antibiotic precipitated out on addi-
tion of acetone. ReiJrecipitated from methanol
with acetone. Can also be adsorbed on Super
Filtrol at pH 7.0 and eluted by acidic solutions
(pH 1.5 to 2.0) of the sulfates or hydrochlorides of
pyridine, diethylamine, or brucine (1).
Chemical and physical properties: Basic sul)-
stance. White amorphous powder. HCl salt: Sol-
uble in methanol and water. Sulfate: Precipitated
from aqueous solutions with methanol. Precipi-
tated by Ag"*", flavianic and picrolonic acids. Most
stable to heating at pH 2.0 (1). Broth contains
five components; purified substance contains at
least three, including streptothricin (2).
Biological activity: Active on gram-po.sitive and
gram-negative bacteria, mycobacteria, and fungi.
Most active at alkaline pH. Glucose decrea.ses ac-
tivity on Staph, albus and E. coli. No activity in
riro on pneinnococcal (Type I) infections in mice
(1).
Toxicity: LDso (mice) 0.2 mg per kg intrave-
nously or subcutaneously. Orally, mice tolerate
>94 mg per kg.
References:
1. Bohonos, N. el al. Arch. Biochem. 15: 215-
225, 1947.
2. Benedict, R. G. Botan. Rev. 19: 229-320,
1953.
Antibiotic 156
Produced by: Streptomyces sp. resembling S.
lavendulae.
Remarks: Supjiosed to belong to the strepto-
thricin group, but has no antifungal activity.
Method of extraction: Extraction like that of
streptomycin. Purified by alumina chromatog-
raphy.
Chemical and physical properties: Basic polypep-
tide. Sulfate: Amorphous substance. Decomposes
at 124°C. Soluble in water and methanol, [ajf =
-1-22.5 (c = 1.09 per cent in H2O). Positive biuret,
Fehling, Benedict, and silver nitrate tests. Nega-
tive maltol, Sakaguchi, and FeCU tests. Hydroly-
sis products include leucine, valine, proline, ser-
ine, and lysine. The peptide lysine-serine was also
[sohited. Helianthate: Plates; m.p. 280-28rC (de-
composition).
Biological activity: Active on gram-po.sitive and
gram-negative bacteria and mycobacteria. No
activity on fungi or viruses.
Toxicity: LD50 100 mg per kg (no route given).
Reference: 1. Kawamata, J. and Fujimoto, Y.
J. Antibiotics (Japan) 7B: 192, 1954.
.Antibiotic 146
Produced by: Nocardia mesenterica. -^
Synonym: Has properties in common with leuco-
mycin.
Method of extraction: See mesenterin.
Chemical and physical properties: Basic sub-
stance: white; m.p. 81-87°C. Ultraviolet light
absorption maxima at 230 to 231 m^ {Eum 369) and
at 280 m/x (E'lL 13.2) in ethanolic solutions, [aj^ =
82° (c = 0.5 per cent in ethanol). C = (30.47%;
H = 7.99%; N = 2.02%. Negative Fehling reac-
tion. Brown color upon addition of concentrated
sulfuric acid. A 90 per cent loss of activity upon
heating at 100°C for 30 minutes at pH 2.0. No loss
at pH 4.0 to 8.0.
Biological activity: Active in vitro against gram-
positive bacteria. No activity against gram-
negative bacteria.
'Toxicity: Low.
Reference: 1. Ueda, M. and Umezawa, H. J.
Antibiotics (Japan) 8A: I(i4-1B7, 1955.
Antibiotic .>»7/1.3
Produced by: An actinomycete closely related to
Streptomyces lavendulae.
Method of extraction: Adsorption on carbon at
pH 8; elution with methanol at pH 1.5 to 2 or
with 20 per cent aqueous acetone. Precipitation
with acetone after concentration of eluate. Fur-
ther purification by formation of a iterate which
is transformed into a hydrochloride.
Chemical and physical properties: Basic sub-
stance. Crude preparations of the hydrochloride
are hygroscopic, white powders. Soluble in water,
methanol, and ethanol; insoluble in ether, acetone,
chloroform, benzene, ethyl acetate, and butanol.
Very stable. Positive Pauly reaction; negative
maltol, Sakaguchi, ninhydrin, biuret, and Molisch
tests. Decolorization of permanganate solutions
and bromine water.
Biological activity: Active against gram-positive
and gram-negative bacteria. Active against fungi,
194
DESCRIPTIOXS OF ANTIBIOTICS
mainly strains of Candida. Effective in the treat-
ment of experimental candidiasis.
Toxicity: Nephrotoxic to animals.
Reference: 1. Trakhtenberg, D. M. et aJ . Anti-
biotiki 4(2): 9-13, 1959.
Antibiotic 721
Produced by: Streptomyces sp.
Method of extraction: Isolation procedure like
that for streptothricin. Purified by chromatog-
raphy on alumina with methanol as solvent, and
by countercurrent distribution (n-butanol-water).
Chemical and physical properties: Amber-reddish
substance. Very soluble in water and methanol;
solul)le in ethanol and in acetone, giving a dark
brown color like FeCl.-! . On acidification, the
brown-colored methanolic solution changes to
straw-yellow, then to dark salmon after a few mo-
ments. On adjusting to alkaline pH the color be-
comes brilliant yellow. Positive Molisch test.
Negative Schiff, Benedict, Millon, and Sakaguchi
tests. Negative Grove-Randell tests for carl)o-
mycin and erythromycin.
Biological activity: Active on gram-positive bac-
teria. Inactive on mycobacteria, Nocardia, gram-
negative bacteria (except Neisseria and Brucella),
and yeasts.
Toxicity: Mice tolerate 500 tng per kg subcu-
taneously.
Reference: 1. Albuquerque, M. M. et al. Rev.
inst. antibioticos 1: 89-94, 1958.
Antibiotic 1212
Produced by: Strains of blue-violet Strepto-
myces.
Synonym: Antibiotic 452-7.
Chemical and physical properties: Red-violet
substance, insoluble in water, soluble in alcohol.
Biological activity: Active against gram-positive
bacteria including Staph, aureus. Inactive in vitro
and in vivo against mouse encephalomyelitis virus
(Theiler's virus), experimental poliomyelitis in
cotton rats, and herpes virus (Min strain). In
chick embryos, marked inhibitory effect upon
types A, A-1, and B influenza virus. No virucidal
effect on influenza virus.
Toxicity: Mice tolerate up to 0.1 mg subcutane-
ously and 0.001 mg intracerebrally (doses given
per 8 to 10 gm mouse).
Reference: 1. Tiermanova, K. I. X'opros}- Vir-
usol. 4(1): 71-70, 1959.
.Antibiotic 1943
Produced by: Streptomyces sp.
Method of extraction: Broth-hit rate passed
through a carbo.xylic-type cation exchange resin
(Na+ form). Elution with 6 per cent HCl. Most
activity in the fraction at neutrality (I). Acid
fractions are neutralized, filtered, and added to I.
Clarified with Norit at acid pH. Filtrate neutra-
lized and retreated with Norit. Elution with 80
per cent aqueous methanol. Methanol distilled to
dryness. Residue dissolved in water and freeze
dried.
Chemical and physical properties: Crystalline.
Positive Sakaguchi, Molisch, and ninhydrin tests.
Negative maltol and biuret tests.
Biological activity: Active on Staph, aureus, B.
subtilis, Pr. vulgaris, and Sal. typhosa at 0.1 Mg
per ml; K. pneumoniae, E. coli, and A. aerogenes
at 1.0 Mg per ml; and B. cereus and Ps. aeruginosa
at 10 Mg per ml. Very weak activity on mycobac-
teria. Active in vivo on D. pneumoniae, Strepto-
coccus hemolyticus , and K. pneumoniae. Active on
Sal. typhimurium infections in mice.
Toxicity: Ototoxic to cats.
Reference: 1. Murray, F. J. et al. Antibiotics &
Chemotherapy 7: 345-348, 1957.
Antibiotic 2814Iv
Produced by: Streptomyces sp. belonging to the S.
reticuli group. This organism also produces netrop-
sin and a pentaene antifungal antibiotic.
Synonym: Probably identical to mycolutein.
Method of extraction: Mycelium extracted with
butanol. Crystallized from dimethylformamide-
water or chloroform-isopropanol.
Chemical and physical properties: Long yellow-
green needles or irregular yellow plates; m.p.
154-150°C. Very soluble in alcohols, ketones, es-
ters, ether, and benzene. Soluble in chloroform,
pyridine, and dimethylformamide. Poorly soluble
or insoluble in petroleum ether and water. Gives an
olive-green color in concentrated H2SO4 that rap-
idly changes to red-brown. Ultraviolet absorption
spectrum maxima at 255 and 345 niM- [alo = +44°
(c = 0.75 per cent in chloroform). C = (i6.52%;
H = 6.09%; N = 3.52%.
Biological activity: Weakly fungistatic. Inhil)its
P. nofatiim. P. glaucum. and .4. niger at >() Mg
per ml.
Toxicity: Very to.xic. Mice tolerate 1 mg per kg
subcutaneously, but 2 mg per kg is lethal.
Reference: 1. Thrum, H. Naturwissenschaften
46: 87, 1959.
Antibiotic 6270
Produced by: Streptomyces (Actinomyces) flavo-
chromogenes (1).
Synonym: Brazhiukova states that this anti-
DESCRIPTIONS OF AXTIlilOTICS
195
l)iotic" differs from echinoinyciii, !)ut is similar to
the echinomycin-like antibiotic of Berger. It may
also be related to actinoleukin (1).
Method of extraction: Isolated from mycelium
by extraction with acetone. Extract evaporated
off and residue extracted with chloroform. Chloro-
form concentrated. Precipitated from concentrate
by addition of petroleum ether. Chromatographed
on alumina from benzene. Crystallized from aceto-
nitrile (1 ).
Cheiuical and physical properties: Crystalline;
m.p. 210-215°C. Ultraviolet absorption spectrum
maximum at 320 mix, minimum at 285 m/x. C-^ii-
H37O6-7N6S; C = 65.19% {sic, probably 5(i.l9'f);
H = 6.47%; N = 13.66%; S = 5.56% Acid
hydrolysis products include serine, alanine, and
dimethylleucine, the first two in cqui molar
quantities, not exceeding 33 per cent. Boiling
with 3 per cent XaOH for 2 hours yields 1.3
moles of NH, (1).
Biological activity: Active at maximal tolerated
levels on Crocker sarcoma and lymplnjsarcoma and
Ehrlich's carcinoma in mice, and on sarcoma 45
(rats) (2).
Toxicity: At low do.ses, causes l)lood abnormali-
ties and changes in the size of the spleen (2).
References:
1. Brazhnikova, M. G. Abstr. Communs. Sym-
posium on Antilnotics. Prague, 1959, j)p.
140-141.
2. Shorin, V. A. Aljstr. Communs. Symposivmi
on Antibiotics. Prague, 1959, pp. 1(S5-186.
3. Rossolemo, (). K. et at. Antibiotiki K 6):
54-59, 1959.
Antibiotic T,(»«(» H.I'.
Produced by: Strcptomyces sp. resemliling S.
kitasaloensis.
Method of extraction: Broth-filtrate extracted
with butanol at acid pH. Solvent back-extracted
with water at pH 7 to 8. Aqueous extract adjusted
to acid pH and extracted with eth\'l acetate.
Chemical and physical properties: Acidic sub-
stance; m.p. 200-205°C (decomposition). Soluble
in alcohols, ethyl acetate, and pyridine. Salts solu-
ble in water. "(CeHsOsX,)^: C = 38.3%; H =
4.45%; O = 42.4%; N = 14.7%. pK„ = 6. Ultra-
violet absorption spectrum maxima at 227 mju
(£lL 532), 271 niM (^'IL 650), and 304 m/x <£'u'm
663). Optically inactive.
Biological activity: Very slightly active on gram-
positive and gram-negative bacteria. Not active
on mycobacteria. Active in vitro and in vivo (mice,
rats) on Trichomonas vaginalis. Active on Enda-
moeha histolytica.
Toxicity: Ll).iii (mice) 50 mg per kg subcutane-
ously, about 500 mg per kg orally.
Reference: 1. Despois, R. et al. Ciorn. micro-
biol. 2: 76-90, 1956.
Antibiotic AYF
Produced by: Strcptomyces aureofaciens. Culture
also produces tetracycline.
Synonynis: Probably synonymous with aureo-
facin and ayfactin.
Method of extraction: Mycelium slurried in n-
butanol, adjusted to pH 9.0 to 10.0, and filtered.
Extract washed with aciueous sochiun ethylene-
diaminetetraacetate at pH 9.5. Concentration of
butanol-extract precipitates antibiotic. May also
be extracted from the washed concentrated buta-
nol with water at pH 11.5 to 12.0. Acidification of
aqueous phase to pH 5.0 precipitates the anti-
biotic. Purified by washing with methyl isobutj'l
ketone-chloroform-acetone (9:9:2) and water at
])H 1.5. Separated into two substances by extrac-
tion with methanolic CaCU . Fraction B goes into
the CaCl> solution and precipitates out on addi-
tion of water. It is reprecipitated from the same
solution, then partitioned between butanol and a
2 i)er cent a([ue<)iis Na ethylenediaminetetraace-
tate solution, adjusted to pH 9.5 with NH4OH;
concentration of the butanol phase yields Frac-
tion B. Fraction A is crystallized from chmethyl-
formamide or dimethylacetamide by dilution with
water.
Chemical and phy^ictil properties: Weakly acidic
heptaenes. Proctiim A: Dark lirown crystalline
substance. Insolul)le in water and common or-
ganic solvents. Moderately soluble in dimethyl-
formamide (becoming more solulale in presence of
CaClj) and dimethylacetamide (solubility also
enhanced by CaCla). Soluble in pyridine and di-
methyl sulfo.xide. Ultraviolet absorption spectrum
maxima at 344, 363, 383 (E'lL 526) and 409 mn
(dimethylacetamide). C = 62.6%; H = 7.86%,;
X = 2.8% (Dumas) and 2.5' c (Kjeldahl). Fraction
B: Dark yellow crystalline substance. Soluble to
700 Mg ppi" nil in water at pH 3 to 10, to >2 per
cent in methanolic CaCl-2 , dimethylformamide,
pyridine, and dimethyl sulfoxide, and insoluble
in common organic solvents. Ultraviolet absorp-
tion spectrum the same as Fraction A except
-fi'icm 556 at 383 m/.i. Infrared spectrum given in
reference 1. C = (i2.4% ; H = 7.62%:; N = 2.8%
(Kjeldahl).
Biological activity: Active on yeasts and fungi.
Active in vivo (mice) against C. albicans infections.
Toxicity: Complex: LD.50 (mice) 3.82 mg per kg
intraperitoneally, >1000 mg per kg orally.
100
DESCRIPTIONS OF ANTIBIOTICS
Reference: 1. Kaplan, M. A. et al. Antibiotics
& Chemotherapy 8: 491-495, 1958.
Aiitibiotic A 6
Produced by: Streptoniyces sp. resembling S.
fradiae (1).
Method of extraction: I. Culture-l)roth treated
with carbon at pH 2.0 and filtered. Filtrate ad-
sorbed on carbon at pH 7.0. Elution with methanol
or 20 per cent aqueous acetone at pH 2.0. Addition
of acetone to eluate gives precipitate. Reprecipi-
tated from absolute methanol with ether, or from
water-methanol. Purification using an ion ex-
change resin (1). II. Broth-filtrate treated with
carbon as in I, adjusted to pH 7.2, and chroma-
tographed on IRC-50 (Na+ form). Eluted with 0.5
A^ HCl. Eluate adjusted to pH 5 to 6 and concen-
trated to dryness in vacuo. Precipitated from an
anhydrous methanol solution of residue on addi-
tion of ether. One component (Ab-I) purified by
chromatography on alumina (eluted with 50 per
cent methanol) and on IRC-50 (3).
Chemical and physical properties: Basic com-
pound. Complex: Contains two components. End-
absorption of ultraviolet light. Positive Sakaguchi
test. Negative biuret, FeCh , maltol, Schiff, Mil-
Ion, xanthoproteic, nitroprusside, and Fehling
tests. Helianthate: m.p. 203-205°C (decomposi-
tion). Reineckate: d.p. 285-29C°C.
Biological activity: Inhibits gram-positive and
gram-negative bacteria, including Pseudomonas
pyocyaneus. Active against soft rot of a variety of
vegetables caused by Bacillus carotovorus (2).
Toxicity: Mice tolerate 320 mg per kg intraperi-
toneally (3).
References:
1. Tatsumi, C. and Miyaura, D. J. PVrmenta-
tion Technol. 32: 1-7, 1954.
2. Tatsumi, C. and Miyaura, D. J. Fermenta-
tion Technol. 32: 3(34-366, 1954.
3. Miyaura, J. and Tatsumi, C. J. Fermenta-
tion Technol. 33: 533-535, 1955.
Antibiotic A 20
Produced by: Streptomyces sp.
Method of extraction: Adsorbed from cultiu'e-
filtrate on carbon (Kerozite) at pH 8.2. Eluted
with acetone containing 2 per cent concentrated
HCl. The eluate should be al)out neutral after
this operation; otherwise it is difficult to separate
the active substance. Eluate evaporated to dry-
ness under reduced pressure. Residue taken up in
methanol, filtered to remove insoluble impurities,
then precipitated with acetone. Precipitated as
the picrate. Conversion to sulfate.
Chemical and physical properties: Basic antil)i-
otic. Hydrochloride: Hygroscopic, amorphous,
cream-colored powder. Soluble in absolute metha-
nol. Slightly soluble in glacial acetic acid. Insolu-
ble in ethanol, isopropanol, n-butanol, acetone,
cliloroform, benzyl alcohol, benzene, 1,4-dioxane,
pyridine, and ethyl ether. Positive Sakaguchi,
glucosamine, Tollen, and Fehling (weak) tests.
Negative maltol and Molisch tests.
Biological activity: Active on gram-positive bac-
teria (0.52 to 20 Mg per ml); less active on myco-
bacteria (15 to 60 Mg per ml), gram-negative bac-
teria (3.9 to > 100.0 Mg per ml), and fungi (20 to
100 Mg per ml). Not active on Pseudomonas .
Toxicity: Has a retarded toxic action at low
doses; 50 mg per kg subcutaneously is lethal to
mice after 72 hours. In rats, 60 mg per kg is lethal
under the same conditions.
Reference: 1. Gongalves de Lima, O. et al. Anais
soc. biol. Pernamliuco 13: 3-9, 1955.
Antibiotic A 67
Produced by: Streptomyces sp. resembling S.
antibioticus.
Method of extraction: Partially extracted by n-
butanol. Adsorption on activated carbon and elu-
tion with 80 per cent methanol.
Chemical and physical properties: Neutral sub-
stance. Most stal)le at pH 7.2 to 7.5. Destroyed by
lioiling for 1 hour at pH 7.2. Soluble in water,
methanol, ethanol, and acetone. Unstable in non-
sterile sand.
Biological activity: Active on fungi, such as cer-
tain Pythium spp., Metarrhizium glutinosum, and
Aspergillus clavatus. Less active on other fungi,
such as Sclerotiniafructicola and Alternaria solani.
Very slightly active on certain bacteria and yeasts.
No activity on Rhizobium spp.
Reference: 1. CJregory, K. F. et al. Am. J.
Botany 39: 405-415, 1952.
Antibiotic A 116
Produced by: Streptomyces sp.
Method of extraction: Adsorbed from broth-fil-
trate on carbon (Kerozite) at pH 8.2. Eluted with
acidic methanol. Eluate concentrated to a syrup,
filtered to remove solids, and treated with ace-
tone to precipitate other impurities. Supernatant
evaporated to dryness. Residue taken up in metha-
nol and inactive residue centrifuged off. Precipi-
tated from methanol with acetone and li groin.
Operation repeated. Reprecipitated from metha-
nol with acetone to give Fraction C. The super-
natant from the precipitation of Fraction C evapo-
rated to dryness. Residue taken up in isopropanol
DESCRIPTIONS OF ANTIBIOTICS
197
and ])rec'ipitated with acetone. Methanolic solu-
tion centrit'uged to remove insoluble impurities,
and supernatant evaporated to dryness to give
Fraction A 10.
Chemical and physical properties: Fractions C
and A 10 differ only in their reaction with picric
acid : C forms a picrate ; A 10 does not . C is a hygro -
scopic cream-colored substance; A 10 is an amor-
phous, hygroscopic, gray-colored suljstance. A 10
and C: Positive Sakaguchi, Tollen, Benedict, and
Molisch tests. Negative maltol, Millon, biuret,
ninhydrin, glucosamine, and Schiff tests.
Biological activity: A 10: Active on one strain
of B. subtilis at 0.9 to 1.8 ^g Pf' n\\, but inactive
on a second. Active on micrococci, streptococci,
and Sarcina at 0.9 to 9.2 ng per ml. Very slightly
active (33 to 49 /xg per ml) on Neisseria caiarrhalis
and Brucella suis. Inactive on other gram-nega-
tive bacteria, mycobacteria, and Candida kritsei.
C: very moderate activity on certain gram-posi-
tive bacteria; inactive on certain strains on which
A 10 is active.
Toxicity: Mice tolerate 300 mg per kg subcu-
taneously.
Reference: 1. Gongalves de Linui, ( ). et al. Anais
soc. biol. Pernambuco 13: 125 129, 1955.
Antibiotic Ax 18
Produced by: Streptoniyccs recifensis (formerly
Nocardia recifei (2)).
Method of extraction: Precipitated from the cul-
ture-broth as the picrate. Conversion to the hydro-
chloride or sulfate, which are precipitated from
a methanolic solution on addition of ligroin. Re-
precipitated from absolute methanol. Conversion
to orange II salt, then to the sulfate (1).
Chemical and physical properties: Basic sub-
stance. Sulfate or hydrochloride: Amorphous, gray
substance, soluble in water and methanol. Insolu-
ble in acetone, ligroin, and ethyl ether. Positive
Sakaguchi, biuret, and ninhydrin tests. Negative
maltol test (1).
Biological activity: Very slightly active on a
strain of Staph, aureus (27.5 Mg ppr ml) :iii<l '»n B.
anthracis (55 Mg per ml) (1).
References :
1. Gongalves de Lima, (). et al. Anais soc. biol.
Pernambuco 13: 21-29, 1955.
2. Falcao de Morals, J. O. e< ai. Anais soc. biol.
Pernambuco 15:239-253, 1957.
Antibiotic E 129
Produced by: Streptomyces ostreogriseus (1, 5).
No description is given.
Synonyms: Ostreogrycin.
Certain antibiotic complexes, such as the
actinomycins, have a confused nomenclature be-
cause numerous groups of scientists have worked
with the complex in question, and each has intro-
duced a different set of terms for the components.
Only one scientific group has been responsible for
the characterization of the E 129 complex, but they
have introduced what appears to be two confusing
sets of designations. In their patent publication
(5), they refer to six components. A, B, C, D, E,
and Z. Component A is synonymous with the
previously described antibiotic PA 114A. Com-
ponent Z is reportedly synonymous with PA 11-lB.
They describe in some detail the component B,
said to be a new sultstance similar chemically to
A and Z. (This will be referred to below as "Patent
Component B.") In some of their other publica-
tions (2, 3), the E 129 complex was said to con-
tain three components, A, B, and G. Component
A w^as synonymous with PA 114A and staphylo-
mycin Mi . Component B was synonymous with
PA 114B, and may be presumed to be the same as
Z (above), l)ut different from staphylomycin S.
This Component B will hereafter be referred to as
''Other Component B." It was not known whether
Component Cx was the same as staphylomycin M2
because of the impurity of Mj . A comparison of
the data availal)le on Patent Component B and
Component (J does not make it possible to state
whether they are the same. They are therefore
treated separately below.
Method of extraction: Component G: Broth-fil-
trates extracted with ethyl acetate. Solvent re-
moved under reduced pressure. Purification by
fractional precipitation, chromatography, and
countercurrent distribution (aciueous methanol-
hydrocarbon solvent) (2). Patent Component B:
Separated from crude complex by chromatography
on alumina at pH 4.0 with ethylene dichloride as
solvent and developer, or ethylene dichloride-
petroleum ether (1:1) followed by a 3: 1 mixture as
developer. These fractions contain mainly Com-
ponent A. Chloroform fractions which follow con-
tain mainly B. A final methanol-ethyl acetate-
water elution gives a fraction containing all the
components. Petroleum ether added to the chloro-
form gives a precipitate of B. Further purified by
countercurrent distribution with one of the fol-
lowing systems: ethyl acetate-water-methanol
(4:2.5:1.5); or l)enzene-methanol-water (2:1:1),
(4:1:3), or (3:1:2) (5).
Chemical and physical properties: Component G:
White, homogeneous, amorphous solid. Highly
solul)le in moist polar solvents. Nearly insolulile
in water, light petroleum, or carljon tetrachloride
198
DESCRIPTIONS OF ANTIBIOTICS
III l)roniofonn, has little ultraviolet absorption
above 270 m^. Infrared data given in reference 2.
Positive FeCls (green) reaction. C35H48N4O9 (2).
Patent Component B: Solul)le in lower alcohols,
ketones, esters, methylene dichloride, acetic acid,
tlioxane, and dimethylformamide. Moderately
soluble in benzene; slightly soluble in water (2.7
mg per ml) and the lower ethers. Insoluble in
light petroleum (b.p. 60-80°C) and carbon tetra-
chloride. Ultraviolet absorption spectrum maxi-
mum at 215 niM (^IL 650) (ethanol). Infrared
spectrum given in reference 5. [a]; = —17.4°
(c = 0.4 per cent in methanol). C = 63.25%; H =
7.10%; N = 8.05%; O = 21.60%. Molecular weight,
650. From these figures the formula C34H46O9N4
may be calculated (5). All -patent components:
Data on Rf values on paper chromatography given
in reference 5.
Biological activity: Complex: Active in vitro on
gram-positive bacteria, including Staph, aureus,
Sarcina lutea, and B. subtilis (3). Most active at
pH 5.5 to 7.5; less active at pH 8.5. Resistance to
E 129 develops more slowly than to erythromycin,
spiramycin, novobiocin, or vancomycin. Partial
cross-re.sistance with erythromycin and spiramy-
cin (1). Patent Component B: Active on the same
organisms as the complex at 0.32 to 20 /xg per ml
(5). Complex: Active in mice on Streptococcus hemo-
lijticus /3 infections (3). Relationship of the compo-
nents: G mixed with eciual quantities of "Other
Component B" is six times as potent as A. Alone,
all three are relatively inactive. Mixtures of A
and ''Other Component 5".- B potentiates an equal
weight of A; any excess of A over B acts as an inert
diluent. Mixtures of "Other Component 7?" and G:
B potentiates an equal weight of G; any excess of
one factor over the other acts as an inert diluent.
Mixtures of A, "Other Component B," and G (not
containing a large excess of B over G) : Any B pres-
ent preferentially potentiates an equal weight of
G. Only when there is more B than G is any B
available to potentiate A (3).
Toxicity: Complex: Nontoxic to mice at thera-
peutic levels (2).
Utilization: As effective as erythromycin on
furunculosis (4).
References:
1. Garrod, L. P. and Waterworth, P. M. Brit.
Med. J. 2: 61-65, 1956.
2. Ball, S. et al. Biochem. J. 68: 24P, 1958.
3. Bes.sell, C. J. et al. Biochem. J. 6«: 24P-
25P, 1958.
4. Scott, A. and Waterworth, P. M. Brit . Med.
J. 2: 83-84, 1958.
5. Ball, S. and Hughes, I. W. British Patent
799,053, Julv 30, 1958.
Aiilihiolic K MM
Produced by: Streptomyces sp.
Method of extraction: Adsorption of active sub-
stance from broth-filtrate on lonex-C (H+) resin
at pH 7.2. Elution with 80 per cent aqueous ace-
tone. Eluate evaporated in vacuo. Aqueous residue
extracted with ethyl acetate at pH 7.2. Extract
evaporated to dryness in vacuo. Syrup dissolved
in ethanol and precipitated with cold water at
pH 2.0. Chromatographed on alimiimi from an
ether solution and eluted with ether.
Chemical and physical properties: Brownish
powder. Soluble in methanol, ethanol, butanol,
amyl alcohol, acetone, ethyl acetate, chloroform,
dichloroethylene, and ether. Insoluble in petro-
leum ether, benzene, and distilled water. Stable
to boiling for 20 minutes at pH 2.0 to 9.0. Ultra-
violet absorption maximum at 230 to 235 m/x (c =
0.1 per cent in aqueous methanol).
Biological activity: Not active on bacteria, fungi,
or yeasts except B. anlhracis (weak activity). Ac-
tive in tissue culture on influenza PR 8 virus, pos-
sibly inhibiting intracellular growth (1, 2); also
active on PR 8 in ovo but not in mice (3).
Toxicity: LD;,o (mice) 100 mg per kg iiitraperi-
toneally.
References:
1. Higo, N. et al. JaiKin. J. Microbiol. 1: 91-
97, 1957.
2. Miyakawa, J. et ul. Japan. J. Microbiol.
2: 53-62, 1958.
3. Hinuma, Y. et al. Japan. J. Microbiol. 2:
63-68, 1958.
Antibiotic EI5
Produced t)y: Streptomyces sp. having gray spores
and differing from S. griseus, S. lavendulae, and
the actinorul)in -producer.
Synonym: Possibly related to actinoruliin
(streptothricin-type substance) .
Method of extraction: Adsorbed from broth-ttl-
trate on carbon. p]hited with acid-methanol and
precipitated on addition of acetone. Purified l)y
chromatography on Decalso or on IRC-50. Elu-
ates concentrated in vacuo and precipitated with
acetone.
Chemical and physical properties: Acid salt:
White substance. Soluble in water and acid-
methanol. Insoluble in ether and acetone. Thermo-
stable (resists boiling).
Biological activity: Similar to actinorubin.
Strains of bacteria made resistant to EIr, were also
resistant to stre))tothricin, but the reverse was
not uniformly true.
DESCRIPTIONS OF ANTIBIOTICS
199
Toxicil!/: Similar to actinonihiii; less toxic than
streptothricin. Causes necrosis of the liver and
renal damage at high concentrations.
Reference: 1. Weiser, R. S. et al . Proc. Soc. Exptl.
BioL Med. 72: 283-287, 1949.
Antibiotic F 43
Produced by: Streptoniyces sp.
Sj/nnni/in: Related to actinolenkin and levomy-
cin.
Method of extraction: Broth hltered at pH 7.0,
and treated with acidic clay at pH 8.0. Elation
with 80 per cent aqueous acetone. Acetone re-
moved by evaporation; aqueous residue extracted
with butjd acetate at pH 8.0. Extract concentrated
to dryness in vacuo. Chromatographed on alumina
from a butyl acetate-ether (2:1) solution. De-
veloped with ethyl ether, followed by butyl ace-
tate and ethyl ether (1:1), and hnally butyl ace-
tate which elutes the antibiotic. Precipitated from
ethanol with petroleum ether. Recrystallized from
ethanol at — 10°C.
Chemical and physical properties: White plate-
lets; m.p. 2r2-214°C. Soluble in methanol,
ethanol, butanol, amyl alcohol, ethyl acetate, chlo-
roform, and dichloroethylene. Insoluble in petro-
leum ether, benzene, ether, and distilled water.
Positive Molisch (purple color) test. Negative
ninhydrin, biuret, xanthoproteic, Hopkins-Cole,
FehUng, Millon, and Fed., tests. C = 54.33%;
H = 5.95%; N = 14.13%. No S or halogens. Ultra-
violet absorption spectrum maxima at 243 m^
(EJL 425) and 320 to 325 niM (^IL 145) (c = 0.1
per cent in aqueous methanol). Infrared spectrum
given in reference 1. Stable to boiling at pH 2.0
to 9.0 for 20 minutes.
Biological activity: Very active on gram-positive
bacteria (<0.01 to 0.4 /xg per ml). Not active on
gram-negative bacteria, except Sal. enteritidis
(1.6 mg per ml) and Ps. aeruginosa. Not active on
most fungi and yeasts tested, except A. niger (6.3
Mg per ml), Sacch. cerevisiae (6.3 Mg per ml), and
Botrytis bassiana (1.6 Mg per ml). Active on PR 8
influenza virus in i'i7/'0, probably affecting the host
cell in some way (1, 2). Also active on PR 8 in oro,
but not in mice (3).
Toxicity: LD50 (mice) 0.6 mg per kg intraperi-
toneally, 1.2 mg per kg subcutaneously.
References:
1. Higo, N. ('/ al. Japan. J. Microliiol. I: 91-
97, 1957.
2. Miyakawa, T. et al. Japan. J. Microliiol.
2: 53-62, 1958.
3. Hinuma, Y. et al. Japan. J. Microbiol. 2:
63-68, 1958.
Antibiotic F 256
Produced by: Streptomyces sp.
Method of extraction: Broth-filtrate extracted
with n-butanol at pH 8.0. Extract evaporated in
vacuo with addition of water. Aqueous residue
extracted with ethyl ether at pH 8.0. Extract
concentrated to dryness in vacuo.
Chemical and physical properties: White powder,
soluble in methanol, ethanol, butanol, amyl alco-
hol, acetone, ethyl acetate, chloroform, dichloro-
ethylene, and ether. Insoluble in petroleum ether,
benzene, and distilled water. Stable to boiling
for 20 minutes at pH 2.0 to 9.0. Ultraviolet ab-
sorption maximum at 275 niM (c = 0.1 per cent in
aciueous methanol).
Biological activity: Very active on gram-posi-
tive bacteria. Not active on gram-negative bac-
teria, fungi, or yeasts, except .4. niger (3.2 Mg per
ml), Sacch. cerevisiae (12.5 Mg per ml), and Botrytis
bassiana (25 Mg per ml). Active on influenza PR 8
virus in tissue culture.
Toxicity: LD.so (mice) 1 mg per kg intraperito-
neall3^
Reference: 1. Higo. N. et al. Japan. J. Micro-
biol. 1: 91-97, 1957.
Antibiotic F 416
Produced by: Streptomyces sp.
Method of extraction: Broth-filtrate extracted
with ethyl acetate. Extract concentrated to drj--
ness in vacuo. Residue dissolved in ethanol and
precipitated with cold water at pH 2.0. Chromato-
graphed from ether-petroleum ether (9:1) solu-
tion on alumina. Developed with petroleum ether,
ether and petroleum ether (1:1), then ether. Ether
fraction evaporated to drj-ness, then precipitated
from an ether-petroleum ether (9:1) solution at
room temperature.
Chemical and physical properties: Colorless
needles; m.i). 114-117°C. Soluble in methanol,
ethanol, butanol, amyl alcohol, acetone, ethyl
acetate, chloroform, dichloroethylene, and ether.
Not soluble in petroleum ether, benzene, or dis-
tilled water. Negative Molisch, Fehling, Millon,
xanthoproteic, FeCls , biuret, and Hopkins-Cole
tests. Stable at pH 5.0 to 7.0 but not 2.0 or 9.0
when boiled 20 minutes. Ultraviolet absorption
spectrum showed onh' end-absorption (c = 0.1
per cent in aciueous methanol).
Biological activity: Not active on bacteria, ex-
cept Sarcina lutea (<0.05 Mg per ml) and Micro-
coccus citreus (<0.05 Mg per ml). Not active on
fungi and yeasts, except A. niger (3.2 Mg per ml),
Absidia orchitis (6.3 Mg per ml), and a Mycotorida
sp. (12.5 Mg per ml). Active in tissue culture on
200
DESCRIPTIONS OF ANTIBIOTICS
influenza PR 8 virus, proi)ably by direct inacti-
vation of free virus particles (2).
Toxicity: LDjo (mice) 40 mg per kg intraperi-
toneally.
References:
1. Higo, N. et al. Japan. J. Microliiol. 1: 91-
97, 1957.
* 2. Miyakawa, T. et al. Japan. J. Microbiol.
2: 53-62, 1958.
Antibiotic GB/229
Produced by: Streptomyces sp. possibly related
to S. rose us.
Method of extraction: Adsorl)ed on Darco G-60
from broth-filtrate at pH 6.0 to 6.6. Eluted with
dilute aqueous HCl (pH 3.0) and eluate lyophil-
ized. Precipitated from an aqueous solution on ad-
dition of acetone. Purified by salt interconversion.
Chemical and physical properties: Hydrochloride:
Very soluble in water. [ol\t> = —32.5° (in w^ater).
No absorption of ultraviolet light. Infrared spec-
trum not very characteristic. Positive ToUen and
Fehling tests; weakly positive ninhydrin and biu-
ret tests; negative Molisch and Schiff tests. N =
13.49%; CI = 20.4%. Stable l)etween pH 2.5 and
8.0 and at room temperature. Rf = 0.90 (3 per
cent ammonium chloride); = 0.15 (acetone-HoO,
1:1). The reineckate and helianthate are hardly
soluble in water, the picrate slightly soluble.
Biological activity: Active on gram-positive and
gram-negative bacteria, mycobacteria, fungi, and
yeasts at 1 to 20 )ug per ml. Active on Fs. aeruginosa
at 50 Mg per ml. Cross-resistance with neomycin
and streptomycin.
Toxicity: LD50 (rats) 20 mg per kg intrave-
nously.
Reference: 1. Rolland, G. et al. Rass. med.
sper. 3: 1-6, 1956.
Antibiotic J 4
Produced by: Streptomyces sp. belonging to the
S. fungicidicus "group G."
Method of extraction: Broth-filtrate extracted
with ethyl acetate at pH 4.0. Concentrated extract
passed through an alumina column, mixed with
water, and solvent evaporated off in vacuo. Yellow
precipitate dissolved in ether. Ether concentrated
to precipitate J4. Recrystallized from methanol
and chloroform.
Chemical and physical properties: White needles;
m.p. 175°C. Very soluble in alcohol and ether;
fairly soluble in ethyl acetate, acetone, and chloro-
form; insoluble in water. No characteristic ultra-
violet absorption spectrum. C = 66.79S'o; H =
6.48%; N = 10.33%; O = 17.40^.o. Negative nin-
hydrin, Sakaguchi, biuret, Millon, Molisch, Seli-
wanoff, FeCla , Tollen, and Fehling tests.
Biological activity: Active on gram-positive bac-
teria, including Mycobacterium avium.
Reference: 1. Taguchi, H. and Nakano, A. J.
Fermentation Technol. 35: 145-149, 1957.
Antibiotic K 125a
Produced by: Streptomyces sp. This organism
produces at least two antibiotics.
Chemical and physical properties: Yellow pow-
der; m.p. 194°C (decompoisition). Insoluble or
sparingly soluble in water, ether, ethyl acetate,
and acetone. Readily soluble in alcohols. Nega-
tive Sakaguchi, Millon, biuret, and FeCls tests.
Purple color in concentrated H2SO4 . Stable be-
tween pH and 8.
Biological activity: Primarily active on gram-
positive bacteria. Some gram-negative bacteria
and fungi also inhibited.
Reference: 1. Okuda, T. et al. J. Antibiotics
(Japan) 7B: 4-6, 1954.
.\ntibiotic L. A. 7017
Produced by: Streptomyces sp.
Method of extraction: Broth-filtrate extracted
with ethyl acetate at pH 2.0. Extracts distilled to
dryness in vacuo under N2 . Residue dissolved in
acetone and chromatographed on alumina. Active
fractions concentrated under N2 . Isopropyl ether
is added to concentrate, and mixture concentrated
in vacuo until a flocculent green-yellow precipitate
forms. Purification by countercurrent distribu-
tion (phosphate buffer pH 7.3 — Initanol, 1:1). Ad-
dition of petroleum ether to active fractions causes
activity to transfer to aqueous phase, which is
extracted at pH 2.5 with ethyl acetate. Extract
concentrated in vacuo, and antibiotic precipitated
on addition of isopropyl ether.
Chemical and physical properties: Acidic. Green-
yellow powder; m.p. 154-157°C (decomposition).
[a]f = —155° (c = 0.4 per cent in ethanol). C =
56.99%; H = 7.18%. No N, S, P, or halogens. Very
soluble in most organic solvents. Insoluble in di-
alkyl ethers, petroleum ether, and ligroin. Fairly
soluble in bicarbonate, with CO2 evolution, and in
alkaline or neutral buffers. Slightly soluble in
water. Decolorizes KMn04 . Does not absorb Br2
and gives a brown color with FeCh . Negative
Fehling test. pK' = 5.0 and pK" = 9.5. Ultravio-
let absorption spectrum maxima (methanol) at
280.5 mfj. {Elfra 470) and 430 m^ (jEIL 103). Infra-
red spectrum given in reference 1.
Biological activity: Active on gram-positive bac-
DESCRIPTIONS OF ANTIBIOTICS
201
teria. Not active on gram-negative bacteria or
yeasts.
Toxicity: LD50 (mice) 0.35 mg per kg intrave-
nously.
Reference: 1. Sensi, P. et al. Antiliiotics &
Chemotherapy 8: 241-244, 1958.
Antibiotic PA 86
Produced by: Streptomyces riniosus.
Method of extraction: Whole culture filtered with
a filter-aid at acid pH. Filtrate extracted with
Initanol at pH 7.8 to 9.0. Butanol evaporated in
vacuo with addition of water, and cooled to give
a precipitate. Taken up in hot methanol, filtered,
and water added. Solvent is evaporated off until a
solid precipitates. May also be extracted from
the broth-filtrate with benzyl or amyl alcohol.
Recrystallized from methanol -water or hot 50 per
cent aqueous acetone.
Chemical and physical properties: Tetraene.
White needles, small plates, or rosettes; m.p.
230-235°C (decomposition). Soluble in acjueous
alcohols. Low solubility in water, alcohols, ethers,
and ketones. Ultraviolet absorption spectrum
ma.xima at 279, 291, 304, and 318 m^. Infrared
spectrum given in reference 1. Optically inactive
in common solvents. Unstable in acid. C = 60.30% ;
H = 8.30%; N = 3.41%o. No S, P, or halogens.
Biological activity: Active on yeasts and fila-
mentous fungi.
Reference: 1. British Patent 719,878, December
8, 1954.
Anlihiolics PA 108, PA UliX, PA l.J.JB, and
PA 148
Produced by: Streptomyces sp.
Synonyms: Macrolide-type antiljiotics related to
spiramycin, narbomycin, leucomycin, borrclidin,
and angolamycin.
Method of extraction: Extraction of filtered broth
with ethyl acetate or methyl isobutyl ketone. Sol-
vent evaporated to dryness in vacuo. Further
purification by countercurrent distribution in the
system benzene-cyclohexane-95 per cent ethanol-
water, 5:5:8:2 by volume. Coefficient distribu-
tions in that system: PA 108 = 0.43; PA 133A =
1.50; PA 133B = 0.50; PA 148 = 0.17; carbomy-
cin = 0.41; carbomycin B = 0.67; erythromycin =
0.24; oleandomycin = 0.25.
Chemical and physical properties: PA 108:
m.p. 121-123°C. Proposed empirical formula:
CssHesNOu . [a]f = -36.8° (c = 1 per cent in
CHCI3). Light-absorption maximum at 279 m^
(£■10^289). PA 133 A: Amorphous material melts
at 87.2-88.0°C. Tentative empirical formula:
C.25H4,,N06 . [a]f = +39.6° (c = 0.5 per cent in
methanol). Light-absorption maxima at 226 m^
(Elfra 183) and at 275 m/x {Eu^ 9.0). PA 133B:
m.p. 99.8-101°C. Tentative empirical formula:
C25H45NOUJ . [a]n' = +22.5° (c = 0.5 per cent in
methanol). Light absorption maximum at 223
m^ (£'icm 184). PA 148: Amorphous material; melts
at 115-118°C. Tentative empirical formula:
CssHesNOu . [at^ = -69.3° (c = 0.5 per cent in
methanol). Light-absorption maximum at 238 m/x
(£•!;?,„ 153) with a shoulder at 280 m^- Infrared
absorption spectra and paper chromatograjihy
data given in reference 1.
Biological activity: PA 108: About as active as
carbomycin against B. subtilis in a cylinder plate
assay. PA 133A and PA 133B: About one fourth
as active as carbomycin against B. subtilis in a
cylinder plate assay.
Reference: 1. Murai, K. et al. Antil)iotics &
Chemotheraijy 9: 485-490, 1959.
Anlihiotic PA 114
Produced by: Streptomyces olivaceus (2). The
organism produced a nimiber of minor components
in addition to PA 114A, B, and B-3, described be-
low (4).
Synonyms: PA 114A is identical to E 129A and
staphylomycin Mi . PA 114B is the same as E
129B but differs from staphylomycin S (3).
Method of extraction: Broth-filtrate extracted
with methyl isol)utyl ketone or other solvents, in-
cluding ether, benzene, ethyl acetate, butanol, or
chloroform (1, 2). I'^xtract concentrated and
chilled with precipitation of 114A. Addition of
hexane to the supernatant gives a precipitate.
Precipitate taken up in methylene chloride. Im-
purities precipitated l)y addition of carbon tetra-
chloride and concentration. Addition of hexane
to the concentrate gives 114B. 114A is purified
by countercurrent distribution (benzene-metha-
nol-water, 2:1:1). 114A crystallizes from hot n-
butyl ethyl ketone or methanol-water. 114B and
114B-3 also purified by countercurrent distribu-
tion (toluene-methanol-water, 4:3:1). Further
purified by chromatography on silica gel followed
by redistribution countercurrently. Crystallized
from methanol or toluene-hexane (1,4).
Chemical and physical properties: Complex:
Amorphous yellow powder. Soluble in lower alco-
hols, chloroform, acetone, dioxane, Vjenzene, ether,
and methylene chloride. Sparingly soluble in
water and carbon tetrachloride. Insoluble in hex-
ane. Infrared spectrum data given in reference 2.
PA II4A: Neutral polypeptide. Colorless needles;
m.p. 200°C (decomposition). Ultraviolet absorp-
202
DESCRIPTIONS OF ANTIBIOTICS
tion spectrum maxima (methanol) at 220 to 230
m/i (^Ic'm 655) and 275 m^ (Elcm 200). Infrared ab-
sorption spectrum given in reference 1. [al^ =
— 207° (c = 0.5 per cent in methanol). Positive
FeClg (green), neutral permanganate, Br2in CCh ,
Tollen, 2,4-dinitrophenylhydrazine, and copper
acetate tests. Negative Fehling, Molisch, Millon,
and ninhydrin tests. C35H42N4O9 or C2.5H31N3O6 :
C = 03.7%; H = 6.48%; N = 8.61%. Molecular
weight, 525. PA II4B: Weakly acidic polypeptide.
Colorless tablets (from methanol) orneedles (from
toluene); m.p. 265°C (decomposition). Ultraviolet
absorption maxima (methanol) at 260 m/u (Eicm
217) and 305 m^ (£l?„> 105). [«]„' = -59.7° (c =
0.5 per cent in methanol). Infrared absorption
given in reference 1. Positive FeCU (red), neutral
permanganate, Bro in CCI4 tests. Negative Tollen,
2,4-dinitrophenylhydrazine, copper acetate, Feh-
ling, Molisch, Millon, and ninhydrin tests. CjoHes-
N,0,2 : C = 62.02%; H = 6.21%; N = 12.77%.
Molecular weight, 981. PA lUB-3: Polypeptide
differing slightly in amino acid content from PA
114B. Needles; m.p. 207-208°C. [«]" = -37.2°.
Ultraviolet absorption spectrum maxima (meth-
anol) 258 niM (Elcm 204.5) and 305 m^ (^11 95).
C = 62.77%o; H = 6.52%o; N = 12.61% (1, 2, 4).
Biological activity: PA 114A and 114B have a
synergistic action. PA 114B-3 and 114 A have a
marked synergistic action in vitro. PA 114 B-3
and 114B have a similar, but less marked syner-
gistic activity. Complex is active on gram-positive
bacteria, mycobacteria, and the genera Neisseria
and Hemophilus, but not against other gram-
negative bacteria. Not active on fungi. Some cross-
resistance with carbomycin and erythromycin,
but not other commonly used antibiotics. Very
slightly active (250 Mg per ml) on Endamoeba histo-
lytica. Resistance develops slowly and follows the
penicillin ])attern. In vitro activity not reduced
by NaCl, glucose, cysteine, thioglycoUate, urea,
or serum. Artificial mixtures of A and B are ac-
tive over a wide ratio of concentrations within the
limits of 20 to 80 per cent. Active in vivo on infec-
tions caused by Staph, aureus and Streptococcus
pyofjenes (mice). Somewhat active in ovo and in
mice on Rickettsia akari and the psittacosis organ-
ism. Alone, neither A nor B is active in vivo. Maxi-
mal protection with artificial mixtures is obtained
with the following combinations: PA 114A 80 to
95 per cent; PA 114B 5 to 20 per cent (1,2, 4). This
differs from E 129 complex.
Toxicity: Mice tolerate 200 to 400 mg per kg
orally and subcutaneously. Rabbits tolerate at
least 100 mg i)er kg intramuscularly' (1, 2).
References:
1. Celmer, W. D. ci a/. Antibiotics Ann. 437-
452, 1955-1956.
2. Sobin, B. A. et al. U. S. Patent 2,787,580,
April 2, 1957.
3. Ball, S. et al. Biochem. J. 68: 24P, 1958.
4. Hobbs, 1). C. and Celmer, W. D. Federation
Proc. 18: 246, 1959.
Antibiotic PA 128
Produced by: Streptomyces sp.
Method of extraction: Broth and mycelium ex-
tracted with n-butanol at pH 7.0 to 8.0. Clarified
extracts concentrated under reduced pressure with
addition of water. Purification of residue by coun-
tercurrent distribution (ligroin, b.p. 60-90°C, and
80 per cent aqueous methanol). Active fractions
concentrated to remove methanol, then extracted
with ether. Concentration of ether-extracts gives
crude substance. Recrystallization from ether.
Chemical and physical properties: Light yellow
rectangular plates; m.p. 143-144°C (decompo.si-
tion). Slightly soluble in water. Soluble in lower
alcohols, acetone, ethyl acetate, and chloroform.
Ultraviolet absorption maxima at 245 m^ (-E'lem
468) and 285 m/i (E\l'm 234). Infrared spectrum
given in reference 1. [a]f = —2.0° (c = 1 per cent
in methanol). Positive 2,4-dinitrophenylhydra-
zine, bromine water, and KMn04 tests. Negative
FeCls test. No color with aqueous NaOH or
H2SO4 .
Biological activity: Active in vitro on Tricho-
monas vaginalis and Endamoeba histolytica. Not
active on gram-positive or gram-negative bac-
teria or fungi. No activity in vivo.
Toxicity: Very toxic; 25 mg per kg daily for 5
days is lethal to mice.
Utilization: Possible utilization in studies in
vitro on protozoal metabolism.
Reference: 1. Rao, K. V. and Lynch, J. E. Anti-
biotics & Chemotherapy 8: 437-440, 1958.
Antibiotic PA 132
Produced by: Streptomyces sp.
Method of extraction: Broth, acidified to pH 2.5
with dilute H2SO4 , extracted with an organic
solvent such as chloroform, ether, or methyl iso-
butyl ether. Extract concentrated in vacuo. Con-
centrate chromatographed on acid-washed alu-
mina and developed with chloroform followed by
2.5 per cent methanol in CCI4 . Also purified l^y
countercurrent distribution (toluene -methanol -
water). Ether solution treated dropwise with
benzylamine with stirring. Yellow precipitate tri-
turated witli ethyl acetate-ether (1:9) to yield
DESCRIPTIONS OF ANTIBIOTICS
203
buff-colored henzylamine salt. A 50 per cent aque-
ous methanol solution of this salt is adjusted to
pH 2.5 and extracted with ether; this gives free
base on evaporation to dryness (1).
Chemical and physical properties: Labile, lac-
tonic acid (I) has a biologically active, stable,
crystalline monobenzj-lamine salt (II). It is a
colorless, amorphous powder which darkens on
standing, [afo = —161° (c = 1 per cent in metha-
nol). Ultraviolet absorption spectrum has one
peak at 218.5 ni/n (E'l^m 358). Positive permanga-
nate and bromine tests. Negative FeCh (yellow-
green), Fehling, 2,4-dinitrophenylhvdrazine, Tol-
len alcoholic silver nitrate, and sodium hypoiodite
tests. No S, N, or halogens. CieHis-aoOs : C =
64.67%; H = 6.29%, 2 methyl groups, but no
methoxyl group. pKa = 5.3 (50 per cent aqueous
ethanol). Neutral equivalent, 292. Soluble in most
organic solvents. Insoluljle in water. Infrared
spectrum given in reference 1. II: m.p. 128-131°C.
[a]v, = —130° (c = 1 per cent in methanol). Neu-
tral equivalent, 373. pKa = 8.9 (50 per cent aque-
ous ethanol). Infrared spectrum in reference 1.
Soluble in methanol. Slightly soluble in water and
ethyl acetate. Insoluble in he.xane and ether (1).
Biological activity: Active on gram-positive bac-
teria including Clostridium perfringetis, Strepto-
coccus pyogenes, D. pneumoniae, Erysipelothrix
rhusiopathiae . and Corynebacterium diphtheriae
(0.19 to 0.78 Mg per ml). Less active on Staph,
aureus strains resistant to other antibiotics. Mod-
erately active on some gram-negative bacteria,
including E. coli. Not active on mycobacteria.
Very active on Trichomonas vaginalis and Enda-
moeba histolytica. Slightly active on yeasts and
saprophytic fungi; moderately active on phyto-
pathogenic fungi. In vivo, toxicity is too great to
give effective doses (2).
Toxicity: LDso (mice) 12.5 mg per kg subcu-
taneously, 25 mg per kg orally (2).
Utilization: Possibly against plant di-sease (2).
References:
1. Koe, K. B. e/ a?. Antibiotic.-^ Ann. 672-075,
1956-1957.
2. English, A. R. et al. Antibiotics Ann. G76-
681, 1956-1957.
Antibiotic PA 147
Produced by: Streptomyces sp.
Method of extraction: Broth-filtrate extracted
with methyl isobutyl ketone. Extracts concen-
trated in vacuo. Back-extracted into pH 6.8 phos-
phate-acetic acid buffer. Buffer adjusted to pH 2
and e.xtracted with ethyl acetate. Extract concen-
trated in vacuo. Purified by chromatography on
alumina from ethyl acetate concentrate. Forms a
crystalline barium salt.
Chemical and physical properties: (3-Carbo.xy-
2,4-pentadienal lactol.) Ba salt: Faintly yellow,
crystalline powder. Cr-HioOe-BaHoO: C = 35.83%;
H = 3.12%; Ba = 34.03%; H^O =3.10%; CH3-C =
2.24% ; CH3CO = 3.08%. Optically inactive. Ultra-
violet absorption spectrum maximum at 272 mfx
{e = 2200). F/eeacrrf.'CeHeO.'i. Ultraviolet maximum
272mAi (e = 17200). Reacts with benzylamine to give
a purple-red solid. Structural formula of free acid
given in Chapter 6. H ydrogenation product: Color-
less oil; b.p. 120-125°C. Optically inactive.
Biological activity: Ba salt has weak activity
against Pasteurella niultocida (62.5 Mg per ml).
Even less active on other bacteria (mainly gram-
positive bacteria and Hemophilis). Not active on
yeasts (2).
Toxicity: Free acid: LD50 (micej 315 mg per kg
(route unknown) (2).
References:
1. Els, H. et al. J. Am. Chem. Soc. 80: 878-
880, 1958.
2. Sobin, B. A. Personal communication, 1958.
Antibiotic PA 150
Produced by: Streptomyces sp. (2).
Method of extraction: Mycelium extracted with
water-saturated butanol. Extract concentrated in
vacuo until antibiotic precipitates. Purification by
fractional precipitation from a methanolic calciiun
chloride solution with water, followed by conver-
sion to a crystalline salt {e.g., monosodium or
triethylamine sulfate double salt). Precipitation
of the crystalline amphoteric form from an aque-
ous alcohol solution of the salt by neutralization
with dilute acid or alkali and cooling (2).
Chemical and physical properties: Amphoteric
heptaene. Yellow substance. Antibiotic and salts
show gradual darkening and decomposition up to
260°C. Slightly soluble in pyridine and dimethyl-
formamide; less soluble in methanol, ethanol,
propanol, butanol, and dioxane. Insolul^le in
water, acetone, methyl isobutyl ketone, ethyl ace-
tate, chloroform, benzene, and methyl cyclohex-
ane. Solubility in alcohols enhanced by water.
Triethylamine sulfate and sulfate salts soluble as
above. HCl and monosodium salts more soluble
in polar solvents. Ultraviolet absorption spectrum
maxima at 340, 358, 377 (EiL 1033), and 397 m/x
(80 per cent aqueous methanol). [a]f,^ = +294°
(pyridine) or —34° (dimethylformamide-0.1 N
HCl). Po.sitive Fehling and 2,4-dinitrophenyl-
hydrazine tests. Weakly positive ninhydrin test.
Blue color in concentrated H2SO4 . Infrared spec-
204
DESCRIPTIONS OF ANTIBIOTICS
trum given in reference 2. C54H82N2O1S : C =
62.03%; H = 7.83%; N = 2.73%; C-CH3 = 5.48%.
No methoxyl or acetyl groups. Light- and heat-
labile. Most stable at neutrality. Sodium salt:
[Q.]f = -2590° (water). Hi/drochlonde: [ar]'f =
+ 140° (c = 0.4 per cent in dimethylformamide).
Biological activiti/: Active on yeasts and fila-
mentous fungi (1).
Toxicity: LD50 (mice) 2.25 mg per kg subcu-
taneously, and 14 mg per kg orally (1).
References:
1. English, A. R. and McBride. T. J. Antibi-
otics Ann. 893-896, 1957-1958.
2. Koe, B. K. et al. Antibiotics Ann. 897-
905, 1957-1958.
Antibiotic PA 153
Produced by: Streptomyces sp. (2).
Method of extraction: Same as antibiotic PA
150 (2).
Chemical and physical properties: Amphoteric
pentaene. Colorless needles e.xhibiting strong
gray-green fluorescence under ultraviolet light.
Solubilities and melting point same as PA 150.
Ultraviolet absorption spectrum maxima at 303,
317, 332, and 349 niM (fi'Lm 1445) (80 per cent aque-
ous methanol). Infrared spectrum given in refer-
ence 2. [ry\f = +398° (pyridine) or +353° (di-
methylformamide-0.1 A' HCl). Positive ninhydrin,
2,4-dinitrophenylhydrazine, and Fehling tests.
Violet color in concentrated H2SO4 . C.htHbiNOh :
C = 59.94%; H = 8.29%; N = 1.88%,; C-CH3 =
6.01%. No methoxyl or acetyl groups. Heat- and
light-labile. Most stable at pH 7 to 10. Sodium
suit: [a]f = +205° (methanol). Hydrochloride:
[a]'^ = +283° (c = 0.4 per cent in dimethylform-
amide).
Biological activity: Moderately active on yeasts
and filamentous fungi (1).
Toxicity: LDso (mice) >200 mg per kg subcu-
taneously and >400 mg per kg orally (1).
References:
1. English, A. R. and McBride, T. J. Antibi-
otics Ann. 893-896, 1957-1958.
2. Koe, B.K. etal. Antibiotics Ann. 897-905,
1957-1958.
Antibiotic PA 155A
Produced by: Streptomyces albus (2).
Method of extraction: Extraction of l)roth with
ethyl acetate. Solvent concentrated and treated
with an excess of heptane to precipitate the crude
antibiotic. Countercurrent distribution in the sys-
tem benzene-methanol-water (2:1:1). Active frac-
tions chromatographed on acid-washed alumina in
ethyl acetate. Elution with 2 to 5 per cent metha-
nol in ethyl acetate. Concentration of the solvent
gives a colorless crystalline solid (1).
Chemical and physical properties: Very weak
base. Suggested empirical formula: C14H15O2N3 .
Colorless rectangular prisms; in.]). 209-210°C.
[a]f = —214° (c = 2 per cent in methanol). Max-
ima of light absorption at 218, 273, 281, and 288
m^i. Spectrvmi similar to that of tryptophan.
Slightly soluble in water, benzene, and ether.
Moderately soluble in lower alcohols and acetone.
Negative FeCls , ninhydrin, and 2,4-dinitrophen-
ylhydrazine reactions. Deep blue color with
Ehrlich reagent in strong alkali. Bromine water
decolorized. Positive neutral permanganate reac-
tion. Infrared absorption spectrum given in ref-
erence 1. Crystalline picrate contains 2 moles of
picric acid to 1 mole of PA 155A. Stable for 1
hour at 100°C in the pH range 2.0 to 8.0. Not
stable at an alkaline pH (1).
Biological activity: Active against gram-positive
bacteria.
References:
1. Rao, K. V. Antibiotics & Chemotherapy
10: 312-315, 1960.
2. Marsh, W. S. et al. Antibiotics & Chemother-
apy 10: 316-319, 1960.
Antibiotic PA 166
Produced by: Streptomyces sp. (2).
Method of extraction: Same as antibiotic PA
150 (2).
Chemical and physical properties: Amphoteric
tetraene. Colorless needles. Same solubilities and
melting point as PA 150. Ultraviolet absorption
spectrum maxima at 291, 304 {E\f^ 1098), and 319
m/x (80 per cent acjueous methanol). Infrared
spectrum given in reference 2. Same test reactions
as PA 153. C3 5Hs:,NO,4 : C = 59.59%,; H = 7.66%;
N = 2.00%; C-CHs = 6.77%. No methoxyl or
acetyl groups. Light- and heat-labile. Most stable
from pH 7.0 to 10.0. Sodium salt: [a if = +194°
(methanol). Hydrochloride: [a]i, = +239° (di-
methylformamide) (2) .
Biological activity: Active on yeasts and fila-
mentous fungi. No activity in mice on C. albicans
(1).
Toxicity: LDsn (mice) >800 mg per kg subcu-
taneously, >1000 mg per kg orally. Nonirritating
topically in rabbits (1).
References:
1. English, A. R. and McBride, T. J. Antibi-
otics Ann. 893-896, 1957-1958.
2. Koe, B.K. et al. Antibiotics Ann. 817-905,
1957-1958.
DESCRIPTIONS OF ANTIBIOTICS
205
Antibiotic SAX 10
Produced by: Streptomyces aureits.
Synonyms: Resembles antitumor tuitil)iotic 289.
The authors (1) state that it is related to luteo-
mycin, but differs in ultraviolet absorption, solu-
bility, and biological activity.
Method of extraction: Agar plate cultures ex-
tracted with acetone or acidic water. Extracted
from the water with ethyl acetate, butanol, chloro-
form, or benzene. Recrystallized from benzene-
petroleum ether.
Chemical and physical piopciiics: Brown-orange
needles. Blackens at 150-160°C; no melting point
up to 250°C. Soluble in chloroform, acetone, meth-
anol, and benzene. Slightly .solul)le in water and
petroleum ether. Ultraviolet absorption spectrum
maxima at 215, 255, and 430 mn in 0.1 A' HCl.
Infrared absorption spectrimi given in reference
1. Negative ninhydrin, Molisch, xanthoproteic,
and Tollen tests. Purple color in 10 per cent so-
dium carbonate solution; deep orange in concen-
trated H2SO4 . Alcoholic solution turns purple on
addition of magnesium acetate. Data on paper
chromatographic behavior given in reference 1.
C29H33NO9 ± CH2: C = 64.78%; H = fi.43%;
N = 2.51%. No halogen or S. Hydrochloride:
Solul)le in methanol, acetone, and water. Spar-
ingly soluble in ether and benzene.
Biological activity: Active on gram-positive bac-
teria. Inactive on mycol)acteria, gram-negative
l)acteria, and fungi.
Reference: 1. Kinoshita, S. ami Nakayama, K.
J. Antibiotics (Japan) 9B: 319-323, 1956.
Antibiotic SKCC l.JTT
Produced by: Streptomyces sp.
Method of extraction: Extraction of culture-fil-
trate with benzene. Concentration in vacuo, back-
extraction with 0.05 A' HCl, lyophilization.
Chemical and physical properties: Red-brown
powder. Soluble in water, ethanol, and acetone.
Insoluble in ether or benzene. In aqueous solution,
yellow at acid or neutral reaction and purple at
alkaline ])H. Unstable at alkaline reaction. Is
stable for 10 minutes at 100°C, pH 3.5. Picrate
crystals melt with decomposition at 165-168°C.
Maximal light absorption of the picrate at 245 and
255 n\fx.
Biological activity: Active against gram-posi-
tive bacteria, but not active against E. coli and
fungi.
Toxicity: LD50 (mice) 5 mg per kg intraperi-
toneally.
Reference: 1. Reilly, H. C. Bacteriol. Proc.
26. 1952.
Antibiotic X 2(»6
Produced by: Streptomyces sp.
Method of extraction: Can be extracted from
both the mycelium and the broth-filtrate. Broth
extraction: extraction with butyl acetate, concen-
tration in vacuo, back-extraction with phosphate
buffer (pH 8.9). Mycelium extraction: extraction
with ethanol and methanol, concentration in
vacuo, extraction with butyl acetate, back-extrac-
tion with phosphate butter (pH 8.9). Further puri-
fication of l)otli extracts by chromatography on
alumina.
Chemical and physical properties: Colorless or-
ganic acid; m.p. 126-128°C. [aJc = +15.0° in
methanol. No characteristic absorption in the
ultraviolet. Unstable at acid reaction or in alkaline
solutions. Soluble in alcohols, esters, acetone,
ether, and petroleum ether. Insoluble in water
and alkali. C46-47H80-82O1;! .
Biological activity: Active in vitro against gram-
positive bacteria and mycobacteria. Not active in
vivo against bacterial and protozoan infections.
Toxicity: LD50 (mice) 11 mg per kg subcutane-
ously.
Reference: 1. Berger, J. et al. J. Am. Chem. Soc.
73: 5295-5298, 1951.
Aiiiibiotic X :u(i
Produced by: Streptomyces sp.
Synonyms: May be related to resist omycin.
Nucleus similar to that of the tetracyclines.
Method of extraction: Broth-filtrate extracted
with ether at pH 4.5. Evaporation of ether. Crys-
tallization from ethanol, methanol, or dimethyl-
formamide. Mycelial mat extracted in a Soxhlet
apparatus with petroleum ether to remove inac-
tive impurities, then with ether followed by chlo-
roform. Also extracted from the dried, powdered
mycelium with butanol. Butanol concentrated in
vacuo, filtered to remove inactive precipitates,
then evaporated to dryness. Residue dissolved in
0.1 X NaOH, filtered, then acidified to precipitate
the antibiotic.
Chemical ami physical properties: Bright yellow
needles; m.p. 330-331 °C (decomposition). Solul)le
in ether, chloroform, carljon tetrachloride, ethyl
acetate, n-butanol, ethanol, methanol, dimethyl-
formamide, and pyridine. Sparingly soluble in
water, acetone, benzene, and petroleum ether.
Alkaline solution is dark red. Positive FeCls test.
Negative 2,4-(linitroj)henylliy(lrazine reaction.
No N, halogen, or S. C-nR-^A), : C = 70.31%;
H = 5.01%; C-CH3 = 4.68%. Molecular weight
(Rast) 404 it 40. pKa = 7.66. Infrared spectrum
given in reference 1. Ultraviolet absorption spec-
206
DESCRIPTIONS OF ANTIBIOTICS
truni iiuixinia: in ethanol: 218 ni/u (log e 4.G6),
289 m/i (log e 4.G0), 370 niju (log e4.0G) with shoul-
ders at 273 niM (log e 4.39) and 305 niju (log « 4.48) ;
in 0.1 N ethanolic HCl: 269 m^ (log e 4.46), 292
niM (log 6 4.40), 321 ibm (log e 4.22), 340 ni/^ (log
e 4.22), and 370 m/u (log e 4.14), and a shoulder at
228 niM (log e 4.49) ; zn 0.7 N ethanolic NaOH: 225
niM (log 6 4.51), 256 mju (log e 4.43), 296 m^ (log
e 4.50), 385 niju (log e 4.34), and shoulders at 230
mn (log e 4.52), 320 mn (log « 4.31), and 345 m^
(log e 4.13). Monoacetate: m.p. 205-207°C. Triace-
tate: m.p. 248°C. Partial formula (one oxygen un-
placed) :
-CH3
OH
-CH=CH
OH O OH
-2H
-CH=CH
2A
Biological activity: Active mainly on gram-posi-
tive bacteria. Very slight activity on gram-nega-
tive bacteria and C. albicans (125 ^g per ml), but
active on Ps. pyocyaneus at 5 /ug per nd. Not ac-
tive on M. tuberculosis.
Toxicity: Mice tolerate 1 gm per kg orally.
Reference: 1. Vora, V. C. et al. J. Sci. Ind. Re-
search (India) 16C: 182-185, 1957.
Antibiotic X 464
Produced by: Streptomyces sp.
Method of extraction: Most of the antil)iotic pres-
ent in cells. Extraction from mycelium with meth-
anol, concentration in vacuo. Extraction of concen-
trate with butyl acetate, concentration in vacuo.
Extraction of residue with petroleum ether, con-
centration in vacuo. Residual oil partitioned be-
tween aqueous methanol and petroleiun ether. By
successive aqueous methanol extraction, most of
the activity is collected in the methanol, which is
concentrated in vacuo. Residue dissolved in ben-
zene and chromatographed over alumina.
Chemical and physical properties: Colorless or-
ganic acid; m.p. 170~172°C (decomposition). No
characteristic absorption in ultraviolet light.
C25H40O7 .
Biological activity: Active in vitro against gram-
positive bacteria and mycobacteria. Not active in
vivo against bacterial or protozoan infections.
Toxicity: LD50 (mice) 2.5 mg per kg intraperito-
neally.
Reference: 1. Berger, J. et al. J. Am. Chem. Soc.
73: 5295-5298, 1951.
Anliltiolic X 5.37A
Produced by: Streptomyces sp.
Method of extraction: Most of the antilnotic pres-
ent in the cells. Extraction of cell material with
butyl alcohol, concentration in vacuo, washing
with sodium carbonate, drying to solid. Extraction
of this solid in a Soxhlet apparatus with petroleum
ether. Concentration in vacuo; crystallization from
petroleum ether.
Chemical and physical properties: Colorless or-
ganic acid; m.p. 100-109°C. [aJo = —7.2° in metha-
nol. Maximal light al)sorption at 317 and 249 ni/u
in isopropyl alcohol. Soluble in organic solvents;
insoluble in water. Maximal light absorption of
the sodium salt at 308 and 245 mn. C34H62O8 .
Biological activity: Active in vitro against gram-
positive bacteria and mycobacteria. No activity
in vivo against bacterial or protozoan infections.
Toxicity: LD50 (mice) 40 mg per kg intraperi-
toneally.
Reference: 1 . Berger, J. et al. J. Am. Chem. Soc.
73: 5295-5298, 1951.
Aiitil>iotic of Cliaiidraselvliai'
Produced by: Streptomyces sp.
Method of extraction: Extraction from broth
with ethylene dichloride. Extract dried with
Na2S04 and concentrated to dryness under re-
duced pressvare at 40 °C.
Chemical and physical properties: Red substance.
Sparingly soluble in water; highly soluble in etha-
nol, butyl alcohol, and ethylene dichloride. Inac-
tivated at 60 °C and above.
Biological activity: Active on gram-negative and
gram-positive bacteria, including Ps. pyocyanea
(2 ij.g per ml) and mycobacteria (5 to 20 ^g per ml) .
Not active on fungi or actinomycetes.
Reference: 1. Chandrasekhar, S. Antiliiotics &
Chemotherapy 5: 742-743, 1955.
Antibiotic of Mukherjee
Produced by: Streptomyces sp. resembling the »S.
fiadiae-S. californicus group, but different from
the neomycin-producer.
Method of extraction: Adsorption on charcoal,
elution with SO per cent acetone (pH 2.2). Acetone
evaporated off in vacuo. Addition of acetone to the
residue precipitates the antibiotic. Recrystalliza-
tion from water.
Chemical and physical properties: Soluble in
water; insoluble in organic solvents. Stable at
100 °C for 30 minutes. Not affected by cysteine or
acid pH. Chromatography (n-butanol-piperidine
p-toluenesulfonic acid and methanol-water, 9:1)
DESCRIPTIONS OF ANTIBIOTICS
207
indicates that the antibiotic differs from strepto-
mycin, streptothricin, and neomycin. Forms a
heli ant hate.
Biological activity: Culture active on gram-posi-
tive and gram-negative bacteria (including strep-
tomycin-resistant E. coli), mycobacteria, and
fungi. Broth active against gram-positive and
gram-negative bacteria.
Reference: 1. ^lukherjee, S. K. et al. Indian J.
Pharm. 15: 281-282, 1953.
Antibiotic of Kollaiid
Produced by: Streptomyces sp.
Method of extraction: Extracted from broth with
ethyl acetate. Purification by chromatography on
alumina.
Chemical and physical properties: Acidic sub-
stance. Yellow powder. Soluble in methanol,
ethanol, and acetone. Insoluble in water. (Na salt
most soluble in water at pH 6.5 to 7.0.) Said to
differ from other known antibiotics in ultraviolet
absorption and Rf values on chromatography.
Biological activity: Active on gram-positive and
certain strains of gram-negative bacteria. Active
on mycobacteria, but not pathogenic yeasts or
fungi. No cross-resistance with clinicalh^ common
antibiotics. Active in vivo in protecting mice
against D. pneumoniae, Streptococcus faecalis, and
Pr. vulgaris infections.
Toxicity: LD50 (mice) 500 mg per kg intrave-
nously, >4 gm per kg orally.
Reference: 1. RoUand, G. et al. Rass. med. sper.
2: 321-322, 1955.
Antibiotic of Sackniann
Produced by: Streptomyces sp. resembling *S.
roseochromoyenes .
Method of extraction: Adsorption on activated
carbon, and elution with acidic methanol (pH 3.0).
Eluate neutralized and methanol removed by dis-
tillation in vacuo. Residue precipitated from a
solution in warm methanol by addition of anhy-
drous ether. Purified by hydrochloride — ' pic-
rate -^ hydrochloride salt conversion. Purified by
chromatography on alumina (acidic) with metha-
nol as solvent and developer. The fastest moving
zone, giving a bright blue fluorescence, is the anti-
biotic. Addition of anhydrous ether to the concen-
trate of the active fractions precipitates the anti-
l)iotic.
Chemical and physical properties: Polypeptide
with a reducing sugar moiety. White amorphous
powder. Becomes yellow at 144°C, brownish at
169°C, and chars at 235°C. Very soluble in water
and acidic (HCl to pH 3) methanol. Not soluble
in ether, acetone, isopropjd alcohol, or other or-
ganic solvents. Gives an orange color wliich is
quantitative with Weber's reagent. Positive Mol-
isch, Fehling, and biuret reactions. Negative
Sakaguchi, maltol, FeCls , ninhydrin, Millon,
Elson-^Iorgan, Hopkins-Cole, and xanthoproteic
tests.
Biological activity: Active on gram-positive bac-
teria; less active on gram-negative bacteria. Much
less active on streptococci than on staphylococci.
Toxicity: LD50 (mice) 150 to 160 mg per kg.
Nephrotoxic.
Reference: 1. Sackmann, F. Zentr. Bakteriol.
Parasitenk., Abt. II 109: 42-72, 1956.
Antifungal Antibiotic 757
Produced by: Streptomyces sp.
Method of extraction: Broth-filtrate extracted
with butanol at pH 9.0. Extract concentrated in
vacuo just until a fine precipitate is formed. Pre-
cipitate washed with petroleum ether and re-ex-
tracted into acjueous butanol (1:1). Butanol frac-
tion concentrated and new precipitate washed
with ether-acetone (4:1).
Chemical and physical properties: Heptaeue.
Amorphous yellow powder. Soluljle in pyridine
and NaOH solutions. Slightly soluble in methanol,
ethanol, and propylene glj'col. Scarcely soluble
in butanol. Insoluble in acetone, chloroform,
benzol, petroleum ether, and water. Photo -labile.
Precipitated by Group 2 metals from aciueous
solution. Relatively thermostable. Ultraviolet ab-
sorption spectrum maxima (methanol) at 361, 381,
and 404 m/i.
Biological activity: Active on yeasts and fungi.
Reduces number of seminal cells in germinal tissue
of mice and rabbits, and has antimitotic effects on
the glandular crypts of mouse intestine and root
meristem cells of Allium repa. No activity on
Ehrlich adenocarcinoma in mice.
Toxicity: LD50 (mice) 5 mg per kg intraperito-
neally, 60 mg per kg subcutaneously.
Reference: 1. Craveri, R. and Giolitti, G. Ann.
Microbiol. 7: 81-92, 1956.
Antifungal Antibiotic 7071 R. P.
Produced by: Streptomyces sp. resembling S.
kitasatoensis.
Method of extraction: Broth-filtrate extracted
with butanol. Concentration of the solvent pre-
cipitates the antibiotic. Recrystallization from
water-saturated butanol.
Chemical and physical properties: Tetraene; m.p.
275-280°C (decomposition). [a]l° = -f90° (c = 1
per cent in methanol) ; = -f80° (c = 1 per cent in
208
DESCRIPTIONS OF ANTIBIOTICS
pyridine). C = 58.3%; H = 8.0%; O = 31.5%);
N = 1.65%. Ultraviolet absorption spectrum max-
ima at 291 niM (E'lL 562), 304 m^ (£'lcm 863),
and 318 m^ (Elfm 783).
Biological activity: Active on yeasts and fvingi.
Not active on bacteria. More active than nj'statin.
Toxicity: LD50 (mice) 37 mg per kg svibcutane-
ously, 250 mg per kg orally.
Reference: 1. Despois, R. et nl. Giorn. micro-
biol. 2: 76-90, 1956.
Antifungal Anlihiolic A 228
Produced by: Streptomyces sp. (2).
Method of extraction: I. Filtered l)roth ex-
tracted with n-butanol (or chloroform) at pH 7.0.
Extract concentrated under reduced pressure;
diethyl or petroleum ether, ethyl acetate, or ace-
tone added to precipitate A 228 complex. Precipi-
tate slurried in ether, then dried in vacuo. II.
Adsorbed from the whole culture-broth on 1 per
cent magnesium trisilicate and 2 per cent siliceous
earth, and eluted with acetone, then 80 per cent
aqueous acetone. Eluates concentrated under re-
duced pressure at <35°C to remove the acetone.
Aqueous residue extracted with n-butanol. Ex-
tract concentrated under reduced pressure. Pre-
cipitated from the residue on addition of ether or
petroleum ether. Purified by countercurrent dis-
tribution (water: n-butanol-diethyl ether, 1:1.75).
Two fractions, A 228a and A 228b, are separated
by reversed phase partition chromatography on
Alloprene (chlorinated rubber containing 2 per
cent butanol) with n-butanol-saturated water as
solvent and developer. Fraction A 228a is ob-
tained in the earlier fractions, A 228b in the later
(2).
Chemical and physical properties: Complex, con-
taining two neutral heptaenes. Yellow-brown
substances. Both have the following characteris-
tics: Soluble in methanol, ethanol, and butanol.
Soluble in chloroform with loss of biological ac-
tivity. Very slightly soluble in water. Insoluble in
anhydrous acetone, diethyl ether, ethyl acetate,
and petroleum ether. Ultraviolet absorption spec-
trum maxima (aqueous ethanol) at 291, 304, 318,
332, and 350 m^; E]a.: (a) 150, 265, 475, 695, 700,
and (b) 160, 290, 520, 785, 805, respectively. Infra-
red data given in reference 1. Yellowish blue
fluorescence in ultraviolet light (aqueous solu-
tion). Decolorizes laromine water with the forma-
tion of a faintljr yellow or white precipitate. Dark
violet color in H2SO4 . Stable in powder form. In
aqueous solution, stable in the cold and compara-
tively stable at room temperature in the dark.
Photo-labile. Alcoholic solutions are more stable
than aqueous; more stable at neutrality than at
acid pH. C = about 60%; H = about 8%; N =
about 2%; S = 4%. No halogens (1, 2).
Biological activity: Active on yeasts and filamen-
tous fungi. Not active on bacteria, mycobacteria,
or actinomvcetes. Active on Trichomonas vaginalis
at 15 to 30 Mg per ml and Endamoeba histolytica at
60 to 120 jug per ml. No difference in antibiotic ac-
tivity between A 228a and A 228b (2).
Toxicity: Two of three mice were killed by an
intraperitoneal injection of 83 mg per kg of A
228a, but 41.5 mg per kg was tolerated. Mice also
tolerate 100 mg per kg of A 228b (same route) (2).
Not absorbed from the intestinal tract (1).
References:
1. Peynaud, E. and Lafourcade, S. Rev. fer-
mentations et ind. aliment. 8: 228-242,
1953.
2. Ball, S. et al. German Patent 942,047, April
26, 1956.
Antifungal Antibiotic J 44
Produced by: Streptomyces sp. belonging to <S.
fungicidicus "group G."
Method of extraction: Broth-filtrate extracted
with ethyl acetate at pH 4.0. Extract concentrated
in vacuo, and passed through a column of alumina.
Water added; solvent removed in vacuo. Precipi-
tate filtered off, filtrate adjusted to pH 4.0, and
extracted with ethyl acetate. Concentration of
extract to yellow syrup. Chromatography on
alumina with methanol as solvent and developer.
Active fractions concentrated. Antibiotic J 4A
precipitated from concentrate in the cold. Re-
crystallization from ether-methanol. Antilnotic
also present in mycelivnn,
Chemical and physical properties: White prisms;
m.p. 164-170°C. Soluble in ethyl acetate and diox-
ane. Fairly soluble in ethanol, ether, and water.
Sparingly soluble in chloroform and petroleum
ether. Ultraviolet absorption sijectrum maximimi
at 212 niju with a minor peak at 260 mju. C = 61 .47% ;
H = 7.41%; N = 5.15%; O = 25.93%. Negative
ninhydrin, Sakaguchi, biuret, Millon, Molisch,
Fehling, Seliwanoft", and P'eCls tests. Weakly
positive Tollen test. Gives orange color in 40 per
centHoSOj.
Biological activity: Active on fungi, including
Aspergillus and Penicillium, but less active on
Trichophyton. Not active on the yeasts tested.
Reference: 1. Taguchi, H. and Nakano, A. J.
Fermentation Technol. 35: 145-149, 1957.
Antifungal \ii I ibiotic J 4B
Produced by: Streptomyces sp. belonging to the
S. fungicidicus "group G."
DESCRIPTIONS OF ANTIBIOTICS
209
Method of extraction: Broth-filtrate extracted
first with ethyl acetate at pH 4.0 to remove anti-
fungal antibiotic J 4A and antibiotic J 4, then with
l)utanol at i)H 8.0. Extract concentrated in vacuo.
Residual solution taken up in methanol. Addition
of ether precipitates J 4B. Antil)iotic also ])resent
in mycelium.
Chemical and physical properties: Tetraene.
Yellow powder. Insoluble in ether, petroleum
ether, and chloroform. Positive Sakaguchi reac-
tion. Negative ninhydrin, biuret, Millon, Molisch,
Seliwanoff, and FeCl.3 tests; weakly positive ToUen
test. Gives a brown color in 40 per cent H2SO4 .
Ultraviolet absorption spectrum maxima at about
288, 303, and 311 m,j..
Biological activity: Active on yeasts and filamen-
tous fungi. Not active on bacteria.
Reference: 1. Taguchi, H. and Nakano, A. J.
Fermentation Technol. 35: 145-149, 1957.
Antifungal Antibiolifs <>f |{a<> anti I nia
Produced by: Streptoniyces s])]). related to S.
viridans (1).
Method of extraction: Extracted with n-butanol
from broths and mycelium (1).
Chemical and physical characteristics: Three an-
tibiotics, A, B, and C, have Rf values of 0.00, 0.33,
and 0.95, respectively, on paper chromatography
(benzene-acetic acid-water, 2:2:1). A and B have
ultraviolet absorption spectrum maxima at 3()(),
380, and 405 m/i, indicating that they are hep-
taenes. C has no characteristic absorption from
250 to 400 m« (1).
Biological activity: Active on yeasts and prol)a-
l)ly on other fungi. Not active on bacteria (1).
Reference: 1. Rao, P. L. N. and Uma, B. N.
Nature, London 182: 115-110, 1958.
Aniifnngal Heplaene F 17C
Produced by: Streptomyces cinnamomeus f. aza-
coluta.
Method of extraction: Extraction of m^'celium
with 95 per cent ethanol. Extract concentrated
in vacuo under nitrogen. Precipitate collected and
washed with water. Precipitate dissolved in n-
l)utanol-pyridine-water (1:1:2) and added to n-
butanol-water (7:8). Emulsion is centrifuged.
Concentration of the upper phase luider nitrogen
and in vacuo to three-fifths of the original volume.
The polyene precipitates and is washed and dried.
Chemical and physical properties: Amphoteric
heptaene. Amorphous yellow powder. Deep blue
color with sidfuric acid. Absor])tioii of light simi-
lar to candidin, with a maximum at 335 ni/i in
aqueous neutral solutions. In ethanol, peaks at
408, 383, 3(i5, and 347 m^i (weak) with a shoulder
at 320 m^.. Insoluble in ether, petroleum ether,
benzene, chloroform, ethyl acetate, and water.
Soluble in pyridine, methanol, and dimethyl sulf-
oxide. Slightly soluble in absolute ethanol and
acetone; solubility increases upon addition of
water to these solvents. Unstable: half -life of neu-
tral aqueous solutions 1 hour at 70°C, of neutral 95
per cent ethanolic solutions4 to 6 days at 4°C in the
dark. Paper chromatography suggests that F 17C
has one factor in common with ascosinand another
one with PA 150 and the antifungal antibiotic 757.
Biological activity: Active against yeasts and
filamentous fungi. Inactive against bacteria and
actinomycetes.
Reference: 1. Craveri, R. ct al. Antibiotics &
Chemotherajjv 10: 430-439, 19()0.
.\nlini^ cins
Produced by: Streptomyces sp. (1, 15, 18, 25,
27), S. kitasawaensis (17), and S. griseus (27).
Synonyms: Antimj-cin A, antimycin A 35 (1, 15),
antimycin A 102 (15), antipiriculin A (17), virosin
(19), antibiotic 720A (18). See also blastmycin.
Remarks: Like the actinomycins, the antimycin
complexes contain a number of closely related sul)-
stances. The complex ijroduced by one organism
may contain the same components as the complex
])roduced i)y another, but in different proportions.
One complex may contain components not found
in another.
Method of extraction: Brotli, adjusted to pH 9.0,
treated with Celite 503, filtered, and filtrate ad-
justed to pH 2.5 and retreated with Celite. Celite
washed with acidic water and eluted with 95 per
cent ethanol. Ethanol concentrated in vacuo, and
extracted successively with chloroform. Chloro-
form extract concentrated in vacuo to a dark oil.
Oil extracted with benzene. Benzene equilibrated
with an etiual volume of 70 per cent ethanol. Ben-
zene laj-er, after separation, concentrated in
vacuo to a dark oil. Oil stirred with petroleum
ether, and the brown solid that separates is ex-
tracted exhaustively with pctioleuni ctluM' in a
Soxhlet apparatus. Resulting light tan solid crys-
tallized from ether, and recrystallized from metha-
nol and l)enzene-petroleum ether. Further puri-
fication i)y countercurrent distribution and
partition chromatography (10, 23, 24).
Chemical and physical properties: All the anti-
mycin components have the following general
properties: Colorless substances. Soluble in meth-
anol, ethanol, acetone, ether, n-butanol, chloro-
form, and ethyl acetate. Slightly soluble in pe-
troleum ether, benzene, carbon disulfide, carbon
210
DESCRIPTIONS OF ANTIBIOTICS
tetrachloride. Insoluble in water, 5 per cent HCl,
and 5 per cent NaHCOg (3, 12, 15, 17 j. Ultraviolet
absorption spectrum maxima at about 225 to 228
and 320 to 330. Bathochromic shift on addition of
base. Infrared spectrum (the same for all com-
ponents isolated) given in references 12 and 24.
Positive hydroxamic, Millon, FeCU , Gibbs diazo,
Liebermann's nitroso, and KMn04 tests. No color
in concentrated H2SO4 - Negative Molisch, nin-
hydrin, Ehrlich, fuchsin aldehyde, 2,4-dinitro-
phenylhydrazine, chromotropic acid, pine splint,
and cyanogen bromide tests (3, 12, 17). Stable at
room temperature. Photosensitive (23). Mild alka-
line hydrolysis yields, among other products,
antimycic acid (CuHuOoN^) and N-(3-amino-
salicyloyl)-L-threonine (12, 13). Hydrolysis prod-
ucts also include a neutral, stable, colorless, pleas-
ant-smelling, water-insoluble oil, C16H28O4 (26),
believed to be:
CH3O R' R"
I II
CHsCHC— O-
R
Individual components of the antimycin com-
plexes all contain the antimycic acid residue, but
differ in the nature of the substitution in the neu-
tral fragment above (i.e., R, R', and R" = H or
alkyl group) (28). Two different structures have
been proposed for "antimycin," but for which
antimycin component is not clear (20, 26). One
complex (A 35) has been separated into five frac-
tions: Ai , A-2a , A-2b , A3 , and a minor fraction,
A4 (23). Another (A 102) contained almost 60 per
cent A3 , in addition to A, , A. , and A4 (15, 23).
Another complex was shown to contain the same
components as A 102 (24). Rf values of these vari-
ous components on paper chromatography in
different systems are given in references 23 and 24.
Antimycin A, : m.p. 149-150°C (23) or 147-148°C
(24). [a]u = +74.0° (in chloroform). C = 61.11%;
H = 7.32%; N = 5.03S7c. C28H4nNo09 (23, 24).
Antimycin A-2x '■ m.p. 147-148°C. C26H34O9N2.
Antimycin A^h : m.p. 168°C. C25H30O9N2 (23).
Antimycin A,, m.p. 170.5-171.5°C (23) or 167-
168°C. [aJD = +84.0° (in chloroform). C = 60.03%,;
H = 6.93%; N = 5.33%. C26H36N2O9 (23, 24).
Biological activity: Active against yeasts and
filamentous fungi. Very little activity against bac-
teria (1). Inhibits influenza virus in tissue culture
by action of the host tissue cells (8) or by direct
inactivation of the virus (21). Slightly active on
RC mammary carcinoma in mice (22). Selectively
inhibits an electron transport component acting
between succinic dehydrogenase and cytochrome
C in the succinoxidase system, and between di-
aphorase and cytochrome C in diphosphopyridine
nucleotide systems (4, 5, 9, 11). Partially inhibits
O2 uptake in diphosphopyridine nucleotide-
coupled oxidation of malate and D-glyceraldehyde
3-phosphate in certain organs (14). Some protec-
tive action on apple scab and tomato early blight
(2), Helminthosporium seedling blight of oats
(15), and rice blast {Piricidana oryzae) (17, 19).
Toxicity: Complexes A 35 and A 102: LD50 (mice)
0.9 mg per kg intravenously, 7.6 mg per kg intra-
peritoneally, and 25 mg i)er kg subcutaneously
(15, 26). LD50 (rats) 0.81 mg per kg intraperi-
toneally. LDo (rats) 12 mg per kg, and LDioo (rats)
30 mg per kg orally (11, 15). Toxic to certain in-
sects and spiders (6, 7). Not toxic to a variety of
plants when applied as a spray (100 units per ml)
(2). Greatest phytoto.xicity in most rapidly grow-
ing parts of plant, and most toxic in oil solution
(15).
Utilization: Useful in elucidation of metabolic
processes.
References:
1. Leben, C. and Keitt, G. W. Phytopath-
ology 38: 899-906, 1948.
2. Leben^ C. and Keitt, G. W. Phytopath-
ology 39: 529-540, 1949.
3. Dunshee, B. R. e/ «/. J. Am. Chem. Soc.
71: 2436-2437, 1949.
4. Ahmad, K. et al. Federation Proc. 8: 178,
1949.
5. Ahmad, K. et al. Arch. Biochem. 28:
281-294, 1950.
6. Kido, G. S. and Spyhalski, E. Science
112: 172-173, 1950.
7. Beck, S. D. J. Econ. Entomol. 43: 105,
1950.
8. Ackerman, W. W. J. Biol. Chem. 189:
421-428, 1951.
9. Potter, V. R. and Reif, A. E. J. Biol. Chem.
194: 287-297, 1952.
10. Schneider, H. G. et al. Arch. Biochem.
Biophys. 37:147-157,1952.
11. Reif, A. E. and Potter, V. R. Cancer Re-
search 13: 49-57, 1953.
12. Tener, G. M. et al. J. Am. Chem. Soc.
75: 1100-1104, 1953.
13. Tener, G. M. et al. J. Am. Chem. Soc.
75: 3623-3625, 1953.
14. Reif, A. E. and Potter, V. R. Arch. Bio-
chem. Biophys. 48: 1-6, 1954.
15. Lockwood, J. L. et al. Phytopathology
44: 438-446, 1954.
16. Leben, C. and Keitt, G. W. Antibiotics &
Chemotherapy 6: 191-193, 1956.
DESCRIPTIONS OF ANTIBIOTICS
211
17. Nakayama, K. et al. J. Antilnotics (Ja-
pan) 9A: 63-()6, 195().
IS. Sakagami, Y. et al. J. Antil)iotics (Japan)
9A: 1-5, 1956.
19. Nakazawa, K. Meeting Japan Antil)iotic
Research Assoc. 1953 (as given in refer-
ence 18).
20. Tener, G. M. Doctoral Dissertations U:
343, 1954 (as given in Velick, S. F. Ann.
Rev. Biochem. 25: 284, 1956).
21. Miyakawa, T. et al. Japan. J. Microliiol.
2: 53-62, 1958.
22. Tarnowski, G S. and Stock, C. C. Cancer
Research 18: (Suppl. I) 25, 1958.
23. Liu, W. C. Dissertation Abstr. Univer-
sity of Wisconsin, 19: 662, 1958.
24. Harada, Y. et al. J. Antilnolics (Japan)
11 A: 32-35, 1958.
25. Burger, J. Quoted in reference 2(i.
26. Strong, F. M. Topics in microbial chem-
istry. John Wiley and Sons, Inc., New
York, 1958, pp. 1-43.
27. Karasawa, K. et al. J. Gen. A])])l. Micro-
biol. 5: 13-20, 1959.
28. Van Tamelen, E. E. et al. J. Am. Chem.
Soc. 81: 750-751, 1959.
Anlini> coin
Produced by: Streptoiiiyces aureus (1).
Synonyms: Fungicidin RAW; antibiotic C 381.
Remarks: Following the original description (1)
of this antibiotic, the culture ceased to produce
the polyene. It was later found that production
of one of the components of the original complex
could be induced bj- the addition of high concen-
trations of CaCl-2 or AlgCle and mevalonic acid
to the culture medium (4, 5).
Method of extraction: Broth extracted with
butanol. Extract cooled to precipitate lipid im-
])urities, then concentrated to dryness in vacuo.
Residue washed with petroleum ether, then ace-
tone, and dried. Residue can also be taken up in
ethanol, filtered, and distilled to dryness. Pre-
cipitates from an ethanol solution on addition of
ice-cold ether (1).
Chemical and physical properties: Tetraene.
Original complex (see "Remarks'") contains two
active components, Rf values about 0.55 and 0.45
(ethanol- n-butanol-water, 1:5:5). The faster
moving component is called antimj'coin A (4, 5).
Complex: Soluble in water, ethanol, and physio-
logical saline solution. Insoluble in ether, chloro-
form, and acetone. Unstable at acid pH; most
stable at pH 7.0. Ultraviolet absorption spectrum
maxima at 290, 305, and 316 m/x (ethanol) (1, 4).
Ditl'ers from nystatin and rimocidin (1, 3). Ciude
antimycoin A: Brown or gray substance. Soluble
in pyridine, butanol, and dimethj-l sulfoxide (5).
Biological activity: Active on yeasts and fungi,
including Cryptococcus. Not active on bacteria or
actinomycetes. Prevents C. albicans infection and
has some activity on Histoplasma capsulatum in
ovo. Active in vivo (mice) on Coccidioides immitis
infections (1, 2).
Toxicity: LD50 (mice) 204 mg per kg intraperi-
toneally (1), >532 mg per kg subcutaneously (2).
References:
1. Raubitscheck, F. e/ a/. Antil)iotics cV Chem-
otherapy 2: 179-183, 1952.
2. Schwartz, J. A. et al. 11th Conf. Tul)erc.
Vet. Adm. 86-93, 1952.
3. Oroshnik, W. et al. Science 121: 147-149.
1955.
4. Schaffner, C. P. et al. Antiliiotics Ann.
869-873, 1957-1958.
5. Steinman, I. D. Thesis, Rutgers University,
1958.
Anliphlei Antibiotic I
Produced by: Streptomyces sp. resembling S.
aureus.
Method of extraction: Broth adjusted to pH 5.0
and filtered. Filtrate evaporated in vacuo to dry-
ness and extracted with methanol. Extract evap-
orated to dryness and re-extracted into methanol.
Addition of butanol in excess, concentration 171
vacuo of supernatant, and precipitation with ace-
tone. Reprecipitated from methanol with acetone.
Purified l\v conversion to helianthate, then hj'-
drochloride.
Chemical and physical properties: Basic sub-
stance. HCl salt: White amorphous powder. Heli-
anthate: Crystalline; m.p. 243-247°C. (decomposi-
tion). Negative l)iuret, xanthoproteic, Millon,
Sakaguchi, Vole sulfur, Hopkins-Cole, Molisch,
glucosamine, and maltol tests. Most stable to
boiling at pH 5 to 6; less stable at alkaline than
at acid pH. Soluble in anhydrous methanol.
Biological activity: Active mainly on M. phlei.
Less active on M. tuberculosis (human type), M.
smegmatis, and M. avium. Very slight activity on
B. subtilis and B. anthracis. No activity on gram-
negative bacteria.
Toxicity: Mice tolerate 10 to 20 mg of the crude
HCl salt intramuscularly and intravenously.
Reference: 1. Ouchi, N. Tohoku J. Exptl. Med.
55: 355-365, 1952.
Antiplilei Antibiotic II
Pioduced by: Streptomyces aureus (2).
Synonym: New antiphlei factor (2).
212
DESCRIPTIOXP OF ANTIBIOTICS
Method of extraction: Broth-filtrate evaporated
in vacuo at pH 5.0, dried, and extracted with an-
hydrous methanol. Addition of butanol to extract
precipitates impurities; subsequent addition of
acetone to the methanol i)recii)itates the anti-
biotic (1).
Chemical and physical properties: Crystalline
helianthate; m.p. 243-247°C (Ij.
Biological activity: Active on mycobacteria (1).
Not active on other bacteria (2).
Toxicity: Doses of 10 to 20 mg in mice (intra-
venously and intramuscularly) are nontoxic (1).
References:
1. Ouchi, N. Tohoku J. Exptl. Med. 51:144,
1951 .
2. Kurosawa, H. J. Antibiotics (Japan) 4:
183-193, 1951.
Antismegmatis Antibiotic
Produced by: Strepfomyces sp. resembling .S.
lavendulae.
Method of extraction: Concentration i)y ]jrecipi-
tation by cold.
Chemical and physical propeities: Heat-stable
at pH 7.0.
Biological activity: Most active, at an alkaline
reaction, against M. suieginatis and .1/. phlei. No
activity against bacteria, fungi, or a pathogenic
strain of M. bovis.
Reference: 1. Kelner, A. and Morton, H. E.
Proc. Soc. Exptl. Biol. Med. 6:i: 227-230, 1940.
.\iititunior Antibiotic 2559
Produced by: Streptomyces sp. (1) reseml)ling *S.
tanashiensis (luteomycin-producer) (3).
Synonym: Said to resemble luteomycin (1), but
differs in ultraviolet absorption spectrum, ele-
mentary analysis, and biological activity (3).
Method of extraction: I. Broth-filtrate extracted
with ethyl or butyl acetate at pH 7.0. Re-extracted
into water at pH 2.0. Process repeated twice using
less solvent each time. Adjusted to pH 5.0 and
freeze dried. II. Adsorption on diatomaceous
earth from water or butyl acetate. Eluted with
acetone (1).
Chemical and physical pioperties: Basic sub-
stance. Orange or reddish yellow powder. HCl
salt: Soluble in w^ater, methanol, ethanol, and
acetone. Sulfate: Orange or orange-brown crystals.
Soluble in water. Slightly soluble in ethanol and
acetone. Forms a picrate, reineckate, helianthate,
and citrate. Positive FeCls test. Negative ninhy-
drin, Molisch, and Sakaguchi tests. Indicator
properties: Changes from orange-yellow to purple
from pH 7.5 to 9.0. Ultraviolet absorption spec-
trum (at acid pH) maxima at 215, 257, and 430
niM (1). At alkaline pH, the peaks shift toward the
longer wave lengths. Sulfate: C = 48.21%; H =
5.40%; N = 2.21%; S = 4.97%. C26H33NOi2-H2S04 .
Most stable at pH 3 to 6. Less stable at alkaline
pH. Thermolabile (2). Acid hydrolysis product is
biologically active "teomycic acid," CnHssHOr ,
which is a green-black substance with vdtraviolet
absorption spectrum maxima at 258 to 261 m^
(£'lem485) and 355 to 356 m^ (fi'iMn 151) (methanol)
and no melting point up to 300°C. Other data given
in reference 5.
Biological activity: Active on gram-positive
bacteria, but less so than luteomycin. Very little
or no activity on fungi and gram-negative bac-
teria. Active on Toxoplasma gondii in vitro (4).
Active in vivo (rats) against Yoshida sarcoma (1).
Antimitotic effect (2). Kills HeLa cells at 10 Mg
per ml and causes disappearance of HeLa cells
in mitosis at 1.25 jug per ml (6). Teomycic acid
(hydrolysis jjroduct) moderately active on gram-
positive bacteria, including mycobacteria (3 to
50 ng per ml). No activity on fungi or gram-nega-
tive bacteria. No activity on Ehrlich ascites car-
cinoma (5).
Toxicity: MLD (mice) about 10 mg per kg in-
travenously (1).
Refetences:
1. Umezawa, H. cl al. J. Antil)iotics (Japan)
6A: 45-51, 1953.
2. Osato, T. et al. J. Antibiotics (Japan) 6A:
52-56, 1953.
3. Okami, Y. et al. J. Antiliiotics (Japan)
6A: 153-157, 1953.
4. Okami, Y. et al. J. Antibiotics (Japan)
8A: 126-131, 1955.
5. Nakamura, S. J. Antibiotics (Japan) 9A:
207-209, 1956.
6. Umezawa, H. Giorn. microl)iol. 2: 160-
193, 1956.
Antitumor Substance 1418 Al
Produced by: Streptomyces sp.
Remarks: Not sufficiently characterized to per-
mit differentiation from certain other antibiotics
having antitumor activity and ultraviolet spectra
of a similar nature. Authors (1) state that it differs
from cellocidin and lenamycin.
Method of extraction: Extraction of broth-filtrate
at pH 2.0 or 7.0 with butanol. Extract evaporated,
residual syrup washed with ether. Taken up in
benzene, concentrated, and filtered to remove
white precipitate of trans-cinnamic acid amide.
Subjected to countercurrent distribution (76 per
cent methanol-benzene-chloroform, 2:1:1).
DESCRIPTIONS OF ANTIBIOTICS
218
Cheniical and physical ptopertics: Powder. Sol-
uble ill methanol, ethanol, acetone, ethyl acetate,
chloroform, carbon tetrachloride, and benzene.
Slightly soluble in ether and ethyl Cellosolve.
Insoluble ill water. Ultraviolet absorption spec-
trum maxima at 217 ran (methanol) or at 225 and
337.5 m;u (alkaline aqueous solution). Infrared
spectrum given in reference 1. Rf values given in
reference 1 . Stable in the culture broth at pH 2 to
7, but unstable to pH 8.0.
Biological activity: Active on Ehrlich carcinoma
and HeLa cells in vitro. Inliibited increase of
ascites and prolonged survival of mice with Ehr-
lich ascites carcinoma.
Toxicity: LD50 2.5 to 5.0 mg per kg intrave-
nously. Mice tolerate 2.5 mg per kg intraperitone-
ally.
Reference: 1. Murase, AI. et al. J. Antiijiotics
(Japan) 12A: 75-80, 1959.
Aiilitiinior Aiilihiolic K ~'^
Produced by: Streptoniyces albulns. This culture
produces nystatin and two forms of cyclohexim-
ide, in addition to E 73.
Remarks: Physical and some chemical jjrojjerties
of E 73 are said to resemble cycloheximide and the
streptovitacins.
Chemical and physical propeities: Colorless
crystals. CnHooOgN. 3-[2-(3,5-Dimethyl-5-acet-
oxy-2-oxocyclohexyl) -2-hydroxyet hyl ] glutarim -
ide. Degradation products given in reference 2.
Structural formula:
CH3
./
O
CH3
)CHOHCHo
NH
OCOCH3 O
Biological activity: Active on sarcoma ISO in
mice (1).
References :
1. Rao, K. V. and CuUen, W. P. Abstr. 134th
Meeting Am. Chem Soc. 22 -O to 23-0,
1958.
2. Rao, K. V. Abstr. 134th Meeting Am. Chem.
Soc. 23-0, 1958.
3. Rao, K. V. et al. J. Am. Chem. Soc. «2:
1127-1132, 19(30.
Antivirubin
Produced by: Streptoniyces longispororuber (2).
Chemical and physical pioperties: Pigment (1).
Biological activity: Active against gram-positive
bacteria (2) and against influenza, vaccinia, and
tobacco mosaic viruses in vitro (1).
Toxicity: Said to be toxic (1).
References:
1. Germanova, K. I. Voprosy Virusol. 4(1):
71-7(3, 1959.
2. Tremina,G. A. Antibiotiki 1(4): 9-13, 1950.
A.scosin
Produced by: Streptoniyces canescus (1, 5).
Synonyms: Similar to trichomycin and candici-
din.
Method (if extraction : Mycelium extra.cted witli
methanol, ])yridinc, or ([uinoline (1). Broth ex-
tracted with l>utanol at pH 7 to 8. Heptane or
"Stoddard solvent" added to extract at pH 7 to
8, followed l)y solid NaHCO:; (8 gm per gallon);
pH adjusted to 9.5 to 10.5 with NaOH. Aciueous
layer cooled, then adjusted to ])H 4.0 to precipi-
tate ascosin (5).
Chemical and physical properties: Contains two
heptane components, A (ethanol-soluble) and B
(ethanol -insoluble). Component A: Ultraviolet
absorption spectrum maxima at 340, 358, 377, and
399 niM- Component B: maxima at 340, 358, 37G,
and 398 m/x (in ethanol). In water, the last three
peaks are flattened to form a plateau at 320 to
350 mju (4). Ciude substance: Orange-brown or
yellow-orange. Weakly acid, unstable. Soluble in
water-containing solvents such as pyridine, pico-
liiies, and quinoline. Slightly soluble in water,
dry pyridine, dry quinoline, phenol, methanol,
ethanol, butanol, formamide, ethyl acetate, n-bu-
tyl acetate, and amyl acetate. Scarcely soluble or
insoluble in petroleum ether, benzene, chloroform,
acetone, ether, dioxane, and acetic anhydride.
Soluble in, l>ut inactivated by H3PO4 , dipropyl
hydrogen phosphate, and aromatic sulfonic acids
(1, 5). Gives precipitates with Ag+, Ba++, Fe+++,
and aqueous, but not methaiiolic brucine. Intense
unstable l)lue color in 35 per cent H3PO4 reversed
liy dilution with water or methanol. Gives a green
color with HCl (5). Negative Molisch, Tollen,
Ehrlich, FeCls , ninhydrin, Benedict, and Saka-
guchi tests. Crude material has ultraviolet ab-
sorption sjiectrum maxima at 234, 240 (infl.), 288,
and 32() ni/n (infl.), in addition to those given above
(1). Infrared data given in reference 1 and spec-
trum in reference 5.
Biological activity: Active on yeasts and certain
filamentous fungi (e.g., P. spinulosum but not
P. patulum or P. chrysogenum; and A. niger but
not other aspergilli tested) (1). Active on the
yeast phase of Histoplasuia capsulalum . but not
214
DESCRITTIOXS OF AXTIBIOTICS
the mycelial phase (5). Active //( vivo (mice) on
experimental histoplasmosis l)ut not torulosis
(Cryptococcus neoformans) (1, 3). Antifungal ac-
tivity in vitro suppressed by unsaturated, l)ut not
saturated, fatty acids, and Tween 80 (2).
Toxicity: LDio (mice) 8.6 mg per kg (crude sub-
stance) (1), or 18 to 22 mg per kg intraperitone-
ally (5), 12.5 mg per kg intravenously. Intra-
venous administration accompanied by vestil)ular
disturbances and nerve cell degeneration in brain
nuclei (1).
Utilization: Active on tinea capitis in chiltlren
((i).
References:
1. Hickey, R. J. et al. Antibiotics A: Chemo-
therapy 2: 472-483, 1952.
2. Hickey, R. J. Arch. Biochem. Biophys.
46: 331-33(), 1953.
3. Emmons, C. W. and Haberman, R. T. Anti-
biotics ct Chemotherapy 3: 1204-1210,
1953.
4. Vining, L. C. e< a/. 8th Congr. intern, botan.,
Paris Vol. prelim. Sect. 24, 106-110, 1954.
5. Cohen, I. R. U. S. Patent 2,723,216, Novem-
ber 8, 1955.
6. Lubowe, I. I. et al. Antibiotics Ann. 135-
139, 1956-1957.
A spar loci II
Produced by: Streptoniyces griseus var. spiralis,
S. violaceus.
Synonym: Similar to amphomycin.
Method of extraction: To the culture are added
1 gm of calcium chloride per liter and some Hyflo
Super-Cel. pH adjusted to 5.0 to 5.5 with hydro-
chloric acid; the mixture then stirred and filtered.
Cake is washed with water and suspended in
water, the pH of which is adjusted to 9.7 to 10.
Insoluble materials discarded. Alkaline extract
adjusted to pH 1.0 to 2.0 and extracted with
n-butanol. Neutralized butanol is concentrated,
and calcium chloride added. pH adjusted to 5.0
to 5.5, and the white microcrj-stalline calcium
salt of aspartocin removed by centrifugation,
w-ashed with wet butanol, then washed with ace-
tone, and air dried (1).
Chemical and physical properties: Acidic pol}'-
peptide with a fatty acid moiety. C = 53.36%;
H = 7.51%; N = 13.36%; S = 0.42%,; CI = 0.07%;
ash = 0.62%:; NH. (Van Slyke) = 4.27%,. No
characteristic light-absorption spectra. [a]l =
+26.4° (c = 2.1 per cent in methanol). More than
50 mg per ml of aspartocin will go into solution
in methanol, ethanol, or glacial acetic acid. Dis-
solves slowly in water or n-butanol. Relatively
insoluble in acetone or ethyl acetate. More readily
.soluble in water at pH <3.0 or >3.6. At pH 3.3,
calcium chloride helps to dissolve the antibiotic,
and sodium chloride precipitates it. At pH 3, 6
per cent of the activity is left after 30 minutes at
1()()°C; at pH 5.0, 72 per cent of the activity re-
mains. Other pH values are intermediate. As-
l)artocin can be separated from amphomycin by
chromatography and electrophoresis. Positive
biuret test. Rapid uptake of bromine and de-
colorization of potassium permanganate. Negative
^Nlillon, xanthoproteic, Sakaguchi, tryptophan
(Tillman-Alt), sodium nitroprusside, Molisch, and
anthrone tests. Acid hydrolysates ninhydrin-posi-
tive and contain an oil which has the properties
of an unsaturated fatty acid. Paper chroma-
tography revealed seven ninhydrin-positive com-
ponents in the acid hydrolysate. Four were identi-
fied by bioassays and paper chromatography a.s^
aspartic acid (35 per cent), glycine (10 per cent),
L-proline (8 per cent), and L-valine (8 per cent).
Also characterized by the hydrolysates were:
D-a-pipecolic acid, a(L)iS-methyl aspartic acid,
and a-)3-diaminobutyric acid (4).
Biological activity: Active against gram-positive
l)ut not against gram-negative liacteria. Four to
eight times more active than amphomycin against
B. subtilis and Corynehacterium xerosis. Activity
reduced in vitro by inorganic phosphates, and in-
creased by calcium ions. Development of re-
sistance slow. Cross-resistance with amphomycin
(2). Effective in mice infected with Staph, aureus ^
Streptococcus pyogenes, and D. pneumoniae when
administered intraperitoneally, subcutaneously,
or intravenously; not effective orally (3).
Toxicity: LDso (mice) 110 mg per kg intraperi-
toneally, 200 mg per kg subcutaneously, 120 mg
per kg intravenously (3).
References:
1. Shay, A. J. et al . Antibiotics Ann. 194-
198, 1959-1960.
2. Kirsch, E. J. et al. Antibiotics Ann. 205-
212, 1959-1960.
3. Redin, G. S. and McCoy, M. E. Antibiotics
Ann. 213-219, 1959-1<)60.
4. Martin, J. H. et al. J. Am. Chem. Soc. 82:
2079, 1960.
Aiireofaciii
Produced by: Streptoniyces aureofaciens. This
culture also produces chlortetracycline.
Synonyms: Prol)ably the same as antibiotic
AYF and ayfactin.
Method of extraction: Mycelium extracted with
methanol. Extracts combined and concentrated
in vacuo to precipitate aureofacin.
Chemical and physical properties: Heptaene.
DESCRIPTIONS OF ANTIBIOTICS
215
Yellowish brown powder. Soluble in alcohols,
acetone, glacial acetic acid, methyl Cellosolve,
and ethylene glycol. Slightly soluble in benzene
and dioxane; insoluble in water at any pH, chloro-
form, esters, ether, and petroleum ether. Ultra-
violet absorption spectrum maxima at 359. 380,
and 402 m^. Slightly positive Molisch and Fehling
tests. Negative FeCU test. Blue-violet color in
concentrated H2SO4 . Unstable at acid pH. Does
not contain S.
Biological activity: Active on fungi and yeasts.
Activity affected by cysteine but not glucose.
Toxicity: LDoo (mice) 5 mg per kg intrai)eri-
toneally.
Reference: 1. Igarasi, S. et al. J. Antil)iotics
(Japan) 9B: 79-80, 1956.
Aiireolic Acid
Produced by: Streptomyces sp.
Method of e.r traction: Extraction of broth-filtrate
with a mixture of butanol and chloroform. Con-
centration of solvent in vacuo. Concentrate applied
to a column of Florisil which is washed with chlo-
roform to remove impurities. Elution of antibiotic
with 20 per cent methanol in chloroform. Concen-
tration in vacuo to amorphous yellow-tan powder.
Powder is dissolved in methanol; upon addition of
chloroform, crystallization of a magnesium salt
of the acid begins.
Chemical and physical properties: Weak acid,
yellow. Tentative formula of magnesium salt :
(C56-6oH96-io4029-3i)2 Mg. [a]n, = -f(iS° (1 per ceut
in methanol). Soluble in lower alcohols and ace-
tone; moderately soluble in water, ethyl acetate,
and ether. Negative Fehling and anthrone tests;
not decolorized by sodium hydrosulfide. Positive
FeCls test; positive test with tyrosine reagent.
Ultraviolet light-absorption maxima for the mag-
nesium salt in 0.01 A' NaOH at 235 and 280 ni/j.
Biological activity: Active against certain gram-
positive bacteria; limited activity against Tricho-
monas vaginalis. No activity against gram-nega-
tive bacteria, mycobacteria, fungi, or viruses.
Limited activity against Streptococcus pyogenes in
vivo.
Toxicity: LD50 (mice) 2.5 to 5 mg per kg intra-
venously. A do.se of 0.25 mg per kg is fatal to rab-
bits and dogs. Low oral toxicity, suggesting lack
of absorption.
Reference: 1. Grundy, W. E. et al. Antibiotics
& Chemotherapy 3: 1215-1220, 1953.
Aureotliricin
Produced by: Streptomyces thioluteus (6), S.
farcinicus (4), S. celluoloflavus (7), <S. cyanojiavus
(16), and other Streptomyces spp. (8, 15).
Synonym: Farcinicin (4).
Remarks: Has the nucleus 3-amino-5-methyl-
pyrrolin-4-ono-(4,3-D)-l,2-dithiole in common
with thiolutin (11). Reported to be produced
simultaneously with thiolutin by one of these
cultures (15).
Method of extraction: Ethyl acetate extract of
broth and nixcclium concentrated in vacuo, de-
hydrated with anhydrous Na2S04 , and chro-
matographed on an alumina column. Column de-
veloped with ethyl acetate-ether (1:1). Active
yellow fraction concentrated in vacuo until crys-
tals appear, then chilled for 24 hours. Recrystal-
lized from ethyl acetate (1, 11).
Chemical and physical properties: Golden-yellow
"thready" crystals (1). Sublimes at 200°C; de-
composes at 254°C (uncorrected) (2, 3) or m.p.
260-270°C (decomposition) (9). Insoluble in water;
slightly solui)le in ethyl acetate, butyl acetate,
acetone, benzol, ether, and ethanol. X„kix 248
{e = 6100), 312 (f = 3900), and 388 (e = 11,000).
Infrared spectrum data given in reference 5. Green
color in concentrated HCl after 24 hours. CaHm-
N2O2S2 (1, 2, 3, 5, 9). Structural formula (9) given
in Chapter 6. 3-Propionamido derivative of 3-
amino-5-methylpyrrolin-4-ono-(4,3-D)-l,2-dithi-
ole. Acid hydrolysis yields pyrrothine, CeHe-
N2OS2 , a weak amine isolated as the hydrochlo-
ride (pKa' 2.9), which gives a red color with
glutaconic aldehyde and is also a hydrolysis prod-
uct of thiolutin (11).
Biological activity: Active on gram-positive and
gram-negative bacteria (2), as well as a variety of
fungi and bacteria pathogenic for plants (13).
Inhibits growth of ascites cells of Ehrlich car-
cinoma in mice, but does not prolong the survival
period (12). Promotes chick growth at 15 mg per
kg of ration (10).
Toxicity: More toxic than chloramphenicol (2).
Kills HeLa cells at 2.5 /xg per ml (14).
References:
1. Maeda, K. Japan. Med. J. 2: 85-88, 1949.
2. Umezawa, H. et al. J. Antibiotics (Japan)
2: 107-111, 1949.
3. Maeda, K J. Antibiotics (Japan) 2: 795-
796, 1949.
4. Hata, T. et al. J. Antibiotics (Japan) 3:
312-325, 1950.
5. Celmer, W. D. et al. J. Am. Chem. Soc.
74: 6304-6305, 1952.
6. Okami, Y. Thesis, Hokkaido University,
1952 (as given in Washizu, F. et al. J.
Antibiotics (Japan) 7A:60, 1954).
7. Nishimura, H. et al. J. Antibiotics (Japan)
6A: 57-65, 1953.
216
DESCRIPTIONS OF ANTIBIOTICS
8. Maeda, K. J. Antibiotics (Japan) 6A:
137-138, 1953.
9. Celmer, W. D. and Solomons, LA. Anti-
biotics Ann. 022-625, 1953-1954.
10. Takahashi, T. et al. J. Antibiotics (Japan)
7A: 26, 1954.
11. Celmer, W. D. and Solomons, I. A. J. Am.
Chem. Soc. 77: 2861-2865, 1955.
12. Nitta, K. et al. J. Antibiotics (Japan)
8A: 120-125, 1955.
13. Koaze, Y. et al. J. Antibiotics (Japan)
9A: 89-96, 1956.
14. Umezawa, H. Giorn. microbiol. 2:160-
193, 1956.
15. Nakamura, M. et al. Ann. Rept. Takamine
Lab. 9:35-43,1957.
16. Funaki, M. et al. J. Antibiotics (Japan)
llA: 143-149, 1958.
Ayfactin
Produced by: Strains of Streptomyces aureofa-
ciens and S. viridifaciens which produce tetra-
cycline and/or chlortetracycline.
Synonyms: Antibiotic AYF and aureofacin are
probably identical with ayfactin.
Method of extraction: I. Whole broth acidified
to pH 2.0 and filtered with a filter-aid. Mycelium
adjusted to pH 9.0 to 10.0 with NH4OH and ex-
tracted with n-butanol. Extract washed with
water, adjusted to pH 7.0, and concentrated by
azeotropic distillation, preferably in vacuo. Ayfac-
tin precipitates on cooling or on addition of Skelly-
solve C. Purified by: (a) Slurrying with methyl
isobutyl ketone and water. Precipitate taken up
in pyridine, solution concentrated to dryness in
vacuo, and toluene added to precipitate ayfactin
from the residue. Precipitate dissolved in di-
methylformamide and filtered to remove insoluble
impurities. Precipitated from filtrate with Skelly-
solves, isopropanol, or toluene, (b) Slurrying with
aqueous acid, hot formamide, methanol, or ace-
tone. II. Mycelium obtained as in I; extracted
with acetone. Extract adjusted to pH 6.0 to pre-
cipitate ayfactin. III. Purified by suspending
crude substance in a water-n-butanol (1:1) mix-
ture, adjusting to pH 1.5 with stirring, and collect-
ing the active precipitate formed at the interface.
Butanol-phase extracted with water at pH 7.0.
Aqueous extract lyophilized to give ayfactin.
Butanol concentrated, and ayfactin precipitated
on addition of Skelh'solve C.
Chemical and physical properties: Crystalline
heptaene. Does not sublime in vacuo up to 200°C.
Nearly insoluble in water. Very soluble in n-buta-
nol, dimethylformamide, pj^ridine, morpholine,
and piperidine. Insoluble in ethyl acetate, ace-
tone, ether, methanol, chloroform, and HCl.
Ultraviolet absorption spectrum maxima (pyri-
dine) at 330, 348, 366, 388, and 410 ni/u- Infrared
spectrum given in reference 1. C = 64.85%; H =
7.68%; N = 3.01%. C25H36-36NO7 .
Biological activity: Probably active on fila-
mentous fungi and yeasts. Little antibacterial
activity. Active in protecting mice from C. albi-
cans infections. Not absorbed from the gastro-
intestinal tract.
Toxicity: LDsu (mice) 0.8 mg per kg intraperi-
toneally.
Reference: 1. British Patent 796,982, June 25,
1958.
Azalomycins
Produced by: Streptomyces hygroscopicus K5-4,
which also produces an antimycobacterial factor.
Synonyms: Azalomycin F has properties in
common with musariu and hygrostatin. Azaolo-
mycin B is similar to elaiophylin.
Method of extraction: I. Extraction of the wet
mycelial cake with acetone. Evaporation of ace-
tone in vacuo. Residue dissolved in methanol,
which is poured into 5 times its volume of ether.
The insoluble part is mainly azalomycin F; the
fraction soluble in the methanol-ether mixture is
mainly azalomycin B and the antimycobacterial
factor. II. Extraction of the l>roth-filtrate with
ethyl acetate yields azalomycin B; extraction
with butanol yields azalomycin F (1). Azalomycin
B, containing solvent solution from either the
mycelium or the broth, is concentrated to dryness
in vacuo, dissolved in ethyl acetate, washed with
2 per cent NaHCOs , 0.01 A' HCl, and water, and
concentrated in vacuo. Upon standing overnight
at 5-10°C, crystals of azalomycin B precipitate.
Recrystallization from aqueous alcohol or acetone
(1). Azalomycin F: Crude extract of azalomycin F
is dissolved in methanol and chromatographed
over a column of alumina. Elution with methanol
or 20 per cent aqueous methanol. Eluate is con-
centrated. Upon addition of acetone, the anti-
biotic precipitates. Recrystallization from meth-
anol -acetone solutions and finally from methanol
(1).
Chemical and physical properties: Azalomycin
B: Neutral compound. White, needle-shaped
crystals; m.p. 185-187°C (decomposition). C =
61.88%o; H = 8.72%,; OCH, = 10.12%,. Molecular
weight (Rast) 284. C,4H2405 • Molecular weight
272.33. [aif = -48° (c = 1 per cent in methanol).
Light-absorption maximum 252.5 ni/i in methanol.
Infrared absorption spectrum given in reference
DESCRIPTIONS OF ANTIBIOTICS
217
2. Soluble in methanol, ethanol, and chloroform.
Moderatel}^ soluble in acetone and ethyl acetate.
Slightly soluble in ether and benzene. Insoluble in
water and petroleum ether. Dark brownish color
in Tollen reaction, with no mirror. Green color in
Fehling test. Permanganate solution decolorized.
Molisch, anthrone, and FeCU tests negative.
Most stable at an acid pH (2). Azalomycin F :
Neutral compound. White needle-shaped crystals;
m.p. 125-127°C (decomposition). C = 60.41%;
H = 8.57%; N = 4.33%. C3oH.=,oOioN2 . Molecular
weight (Berger-Akiya) 600. Molecular weight
598.72. [q;]jj = +46 (c = 1 per cent in methanol).
Light-absorption maxima at 240 and 268 m^ in
methanol. Infrared absorption spectrum given in
reference 2. Soluble in methanol and ethanol.
Moderately soluble in 20 per cent aqueous acetone.
Slightly soluble in acidic water. Insoluble in alka-
line water, acetone, ethyl acetate, and chloroform.
Dark brown color in Tollen reaction. Brown color
in concentrated H2SO4 . Wine-color in concen-
trated HCl. Positive Molisch, anthrone, Fehling,
FeCls , ninhj'drin, Millon, and biuret tests. Posi-
tive ninhydrin reaction after 2 minutes of hy-
drolvsis with 5 .V HCl. Most stable at an alkaline
pH.'
Biological activity: Azaloinijcin B: Active in
ritro against gram-positive bacteria, including
Clostridia. Inactive against mvcobacteria, gram-
negative bacteria, and fungi. Azalomycin F: Active
in vitro against gram-positive bacteria and fungi,
including yeasts and Trichomonas vaginalis.
Toxicity: LD50 (mice): azalomycin B, 281 mg
per kg; azalomycin F, 25.9 mg per kg intraperi-
toneally.
References:
1. Arai, M. J. Antibiotics (Japan) 13A:
46-50, 19(J0.
2. Arai, M. J. Antibiotics (Japan) 13A:
51-56, 1960.
Azaserine
Produced by: Streptomyces fragilis (1). One or
more antibiotics in addition to azaserine are pro-
duced In' this culture (12).
Method of extraction: I. Broth-filtrates flash-
evaporated at <35°C. Concentrates extracted into
95 per cent ethanol and extracts filtered; chroma-
tographed on alumina at pH 5.0 to 6.0 or pH 7 to
8 and developed with graded quantities of water
in ethanol and finally water. Active eluates are
concentrated, adsorbed on carbon from 1 to 2 per
cent aciueous acetone or from water, and eluted
with 1 to 5 per cent acetone. Active fractions are
freeze dried. Crj'stallized from boiling 90 ])er cent
ethanol on cooling. Recrystallized from 90 per
cent methanol or 90 per cent ethanol (4).
Chemical and physical properties: O-Diazoace-
tyl-L-serine (5). Long light yellow-green needles;
m.p. 146-162°C (decomposition). Verj^ soluble in
water. Slight solubility in cold absolute methanol,
ethanol, and acetone increased by warming. Ultra-
violet absorption spectrum maximum at 250.5 m/x
(E\7m 1140) (pH 7.0 phosphate buffer). In 0.1 A'
NaOH, biological activity is destroyed and the
ultraviolet absorption maximum shifts to 252 m/n
{E\!'^ 1230). In 0.1 .V HCl, No is evolved, biologi-
cal activity is destroyed, and the ultraviolet ab-
sorption disappears (3,4). Infrared spectrum given
in reference 4. [a]""' = —0.5° (c = 8.46 per cent
in water at pH 5.18). Rotation in 2 A' HCl changes
until a constant value of [a]^ = +9.7° is reached.
pKa' = 8.55. Positive ninhjdrin, sodium |3-naph-
thoquinone-4-sulfonate, and ammonium silver
nitrate tests (4). Crystallographic data given in
reference 4. Most stable at pH 6 to 8 (4). C5H7N3O4.
C = 34.85%; H = 4.30%; N = 24.44%; O = 37.36%
(4). Structural formula (5) is given in Chapter 6.
Azaserine has l^een synthesized, and both the DL-
and D -forms prepared (6).
Biological activity: In vitro: Moderately active
on gram-positive and gram-negative bacteria,
mycobacteria, and fungi (2, 12). Amoebicidal to
Endamoeha histolytica at 50 to 100 pg per ml (22).
Active on Chlorella pyrenoidosa (27). Produces
greatly elongated, multinucleate, and nonseptate
filaments in E. coli at barely inhibitory levels and
higher (11). Induces formation of active phage
from lysogenic E. coli strain K-12 (17). The D-
form has no activity on Kloeckera brevis (6) or
other organisms (13). In vivo: A correlation be-
tween the activity of azaserine on K. brevis in
vitro and its activity on tumors in vivo was found
(2). Active on Plasmudiinn lophurae (chicks),
rickettsia of epidemic typhus (eggs), meningo-
pneumonitis virus (eggs), but not mycobacterial
or other viral infections (mice) (9). In mice: active
on 6C3HED lymphosarcoma (ascites and solid),
C3H-SX lymphoma (ascites and solid) (29); mod-
erately active on adenocarcinoma E0771, Patter-
son Ij-mphosarcoma, Mecca lymphosarcoma, sar-
coma ISO (ascitic and solid), Ehrlich ascites
carcinoma, and Krelis-2 ascites carcinoma; slightly
active on transmitted leukemia 82 and L1210 leu-
kemia (7, 19, 28). In rats: moderately active on
Walker carcinosarcoma 256, sarcoma R 39, Jen.^^en
sarcoma, and Murphy-Sturm lymphosarcoma;
slightly active on Flexner-Jobling carcinoma (8,
19, 28). Ascitic plasma cell neoplasm of mice
(70429) was initially inhibited l)ut later developed
218
DESCRIPTIONS OF ANTIBIOTICS
transmissible resistance (25). Inhibits purine syn-
thesis in normal and tumor cells of animals and E .
coli (20, 21). Inhibits incorporation of formate
into the nucleic acids of sarcoma 180, adenocar-
cinoma E0771, as well as normal tissues such as
the intestine and liver (mice) (8).
Toxicity: MLD (mice) 62 to 124 mg per kg in-
travenously (16); 25 mg per kg per day in rats
produces lesions of the pancreas, liver, and kid-
ney, as well as depletion of cellular elements in
the bone marrow, reticulocytopenia, and granulo-
cytopenia (10). Administered during the 8th to
12th day of gestation, may cause fetal resorption
and teratogenic effects in rats, but does not have
adverse effects on the mother even after five con-
secutive abortions, delay mating, or harm subse-
quent litters. The fetus is directly affected, not
the ovaries, placenta, or pituitary (24). In chick
embryos, azaserine given from the 3rd to 5th day
of incubation causes skeletal abnormalities and
developmental defects (2.3). Toxic to the canine
fetus (26). In human beings, causes mouth lesions,
anorexia, apathy, nau.sea, vomiting, and some
leukopenia (14). Growth of roots (but not shoots)
of cucumber, barley, and flax seedlings is inhibited
by <3 fig per ml (15).
Utilization: Some beneficial results in Hodgkin's
disease, chronic lymphatic leukemia, and acute
leukemia in children, liut protjably will be used, if
at all, only in combination with other drugs (14,
31).
References:
1. Stock, C. C. et al. Nature, London 17.'?:
71-72, 1954.
2. Ehrlich, J. et al. Nature, London 173: 72,
1951.
3. Bartz, Q. R. et al. Nature, London
72, 1954.
4. Fusari, S. A. et al. J. Am. Chem. Soc.
2878-2881, 1954.
5. Fusari, S. A. et al. ,1. Am. Chem. Soc. 76:
2881-2883, 1954.
6. Nicolaides, E. D. et al. J. Am. Chem. Soc.
76: 2887-2891, 1954.
7. Burchenal, J. H. et al. Proc. Soc. Exptl.
Biol. Med. 86: 891-893, 1954.
8. Skipper, H. E. et al. Federation Proc. 13:
298-299, 1954.
9. Ehrlich, J. et al. Federation Proc. 13: 351,
1954.
10. Sternberg, S. S. f/ a/. Federation Proc. 1.3:
444, 1954.
11. Maxwell, R. E. and Nickel, V. S. Science
120: 270-271, 1954.
12. Coffey, G. F. et al. Antibiotics tt Chemo-
therapy 4: 775-791, 1954.
173:
76:
13. Stock, C. C. et al. Al)str. 12.5th :\Ieeting
Am. Chem. Soc. 12M, 1954.
14. Ellison, R. R. et al. Cancer 7: 801-814,
1954.
15. Norman, A. G. Science 121: 213-214,
1955.
16. Nitta, K. e/ a/. J. Antibiotics (Japan) 8A:
120-125, 1955.
17. Gots, J. S. et al. Biochim. et Biophys.
Acta 17: 449-450, 1955.
18. Sugiura, K. (cjuoted in Stock, C, C. et al.
Acta Unio Intern, contra Cancrum 11:
186-193, 1955).
19. Sugiura, K. and Stock, C. C. Proc. Soc.
Exptl. Biol. Med. 88: 127-129, 1955.
20. Bennett, L. L., Jr. et al. Arch Biochem.
Biophys. 64: 423-436, 1956.
21. Tomisek, A. J. et al. Arch. Biochem. Bio-
phys. 64: 437-455, 1956.
22. Nakamura,M. Nature, London 178:1119-
1120, 1956.
23. Karnovsky, D. A. et al. Proc. Am. Assoc.
Cancer Research 2: 101, 1956.
24. Thiersch, J. B. Proc. Soc. Exptl. Biol.
Med. 94: 27-32, 1957.
25. Potter, M. and Law, L. W. J. Natl. Cancer
Inst. 18: 413-442, 1957.
26. Friedman, M. H. J. Am. Vet. Med. Assoc.
130: 159-162, 1957.
27. Tomiiiek, A. etal. Plant Physiol. 32:7-10,
1957.
28. Sugiura, K. Ann. N. Y. Acad. Sci. 76: 575-
585, 1958.
29. Sassenrath, E. N. Ann. N. Y. Acad. Sci.
76: 601-609, 1958.
30. Ammann, C. A. and Safferman, R. S. Anti-
biotics & Chemotherapy 8: 1-7, 1958.
31. Duvall, L. R. Cancer Chemotherapy Rept.
pp. 65-86, May, 1960.
Azoniyciii
Produced by: Nocardia mesenterica (2, 6), Strep-
tomyces eurocidicus (4), and a Streptomyces sp. re-
sembling S. eurocidicus. N. mesenterica also pro-
duces me.senterin and Antibiotic 446. S. eurocidicus
also produces tertiomycin and eurocidin. The
Streptomyces sp. resembling S. eurocidicus also
produces methymycin (9).
Synonym: Antibiotic llA (9).
Method of extraction: Broth-filtrate extracted
with ethyl acetate at pH 2.0; pH of extract ad-
justed to 7.0. Concentration in vacuo. Crude crys-
tals form on cooling. Recrystallization from meth-
anol or ethanol (1).
Chemical and physical properties: Acidic sub-
stance. White needle-shaped crystals (4) or color-
DESCRIPTIONS OF ANTIBIOTICS
219
less prisms (7); m.p. 2S1-284°C (decomposition)
(1, 4, 7). Soluble in aqueous NaOH and NH4OH
(j-ellow solutions). Slightly soluble in methanol,
ethanol, propylene glycol, acetone, ethyl acetate,
and butjd acetate. Almost insoluble in water, car-
bon disulfide, carbon tetrachloride, ether, and
petroleum ether (7). Ultraviolet alisorption maxi-
mum at 313 (E'JcL 680) or 31-4 mju (£^lcm 905) in
ethanol. Infrared spectrum given in reference 7.
Optically inactive in methanol. Negative Pauly,
Sakaguchi, FeCls , ninhydrin, biuret, Fehling,
^Nlolisch, and Millon tests. After catalytic hydro-
genation, gives positive Pauly reaction. No S or
halogen (1, 4, 7). At pH 2.0 to 8.0, 50 per cent of
the antibiotic activity lost at 100°C in 30 minutes
(4). C3H3N3O2 (3): C = 31.89%; H = 2.65%; N =
36.75% (7). 2-Nitro-imidazole. Structural formula
given in Chapter 6. Acetylaminoazomycin deriva-
tive: Fine needles; m.p. 278-279°C (decomposition)
(7).
Biological activity: Inhibits the growth of gram-
positive and gram-negative bacteria at 3 to 25
fxg per ml. Active on mjcobacteria. No antifungal
activity (1). Active on Trichomonas but not Eu-
glena gracilis or Tetrahymena geleii (8). Inhibits
ascites increase in mice with Ehrlich carcinoma
but does not prolong survival time (5).
Toxicity: LDso (mice) 80 mg per kg intrave-
nously (Ij.
References:
1. Maeda, K. et al. J. Antibiotics (Japan) ()A:
182, 1953.
2. Okami, Y. ct al. J. Antil)iotics (Japan) 7A:
53-54, 1954.
3. Nakamura, S. and Umezawa, H. J. Anti-
biotics (Japan) 8A: 66, 1955.
4. Osato, T. et al. J. Antibiotics (Japan) 8A:
105-109, 1955.
5. Nitta, K. et al. J. Antiliiotics (Japan) 8A:
120-125, 1955.
6. Ueda, M. and Umezawa, H. J. Antil)iotics
(Japan) 8.4: 164-167, 1955.
7. Nakamura, S. Pharm. Bull. (Tokyo) 3:
379-383, 1955.
8. Horie, H. J. Antibiotics (Japan) 9A: 168,
1956.
9. Taguchi, H. and Nakano, A. J. Fermenta-
tion Techno!. 3.5: 191-195, 1957.
Bacteriolytic Factors
Produced by: Various species of Streptomyces
■capable of producing enzymatic systems with the
capacity to cause the lysis of dead and living l)ac-
teria. Hence thej- are designated as bacteriolysins.
Actinomj'cetin, described previously, belongs to
this group of (■()ini)()unds. Theoretically these sub-
stances should l)e considered as antibiotics, since
they are produced by microorganisms and are able
to suppress the growth of and even to kill micro-
organisms.
Method of exiractiun: The best yield of l)acteri-
olysin is obtained on semisynthetic medium
containing casein hydrolysate as a source of ni-
trogen. Production rate is greater at 37°C than
at 30°C. The preparation can be concentrated by
precipitation with (NH4)..S04 , followed bj- cHaly-
sis of the dissolved precipitate.
Biological activity: Lysis of living bacteria
slower than that of dead cells. Highly fibrinolytic,
causing hydrolysis of extracts of M antigen from
Streptococcus pyogenes.
Reference: 1. Pakula, R. et al. Acta Microbiol.
Polon. 3: 363-371, 1954.
Baiiiicetiii
Produced by: Streptomyces plicatus (1).
Remarks: Produced simultaneoush' with amice-
tin and amicetin B (plicacetin) ; differs from ami-
cetin in having one less — CH-2 group in the glycosi-
dic moiety.
Synonym: Antil)iotic L) (1).
Method of extraction: Broth acidified and filtered.
Filtrate (pH 5.5) concentrated in vacuo. Concen-
trate extracted in a Podl)ielniak extractor with
1-butanol at pH 8.6. Extract back-extracted into
0.05 .V H0SO4 ; pH of acidic extract adjusted to
5.5. Concentration in vacuo. Residue extracted
into butanol at pH 8.6. Addition of acetone fol-
lowed by butanol-acetone (50:50) gives gelatinous
precipitate (I). A water suspension of I stirred
with acetone gives a granular precipitate (II),
which is a mixture of amicetin and bamicetin. II
suspended in dipotassiiun hydrogen phosphate,
adjusted to pH 8.2 with HCl, water added, and
mixture extracted with chloroform in a Porlbiel-
niak extractor. Chloroform-extracts contain the
amicetin; the aqueous solution (III), bamicetin.
Ill adjusted to pH 5.5 and concentrated in vacuo.
Residue extracted with butanol at pH 8.5. Butanol
concentrated. Addition of acetone, followed by
acidic butanol-acetone gives the hydrochloride.
Conversion to the ba.se, followed by treatment
with absolute ethanol to give the high melting
point form. The ethanol -insoluble powder (IV)
leached with water. Purification by dissolving IV
in dilute aciueous methanol, lyophilization, and
recrystallization from absolute ethanol (2, 3).
Chemical and physical properties: Dense white
microcrystals; m.p. 240-241 °C (decomposition).
[a]-;3 = +123° (c = 0.5 per cent in 0.1 A' HCl). Has
220
DESCRIPTIONS OP^ ANTIBIOTICS
the same ultraviolet and infrared absorption spec-
trum as amicetin. Bamicetin is more water-soluble
than amicetin. Can be ditTerentiated from amicetin
by X-ray diffraction pattern studies or counter-
current distribution (1-butanol-O.l M phosphate
buffer, pH 6.9). Anhydrous, high melting point
form less soluble in water than the hydrated form.
Rf value = 0.22 (1-butanol saturated with 0.05 .1/
pH 7.0 j)hosphate buffer). Negative Bratton-Mar-
shall and Ehrlich tests. C,8H4oN609 : C = 55.16%;
H = 6.8l7o; N = 13.62% (1). Acid hydrolysis
yields cytimidine and the monomethylamino-
glycoside (bamicetamine), Ci3H25N06 (1-3).
Biological activity: On a weight basis, has twice
as much activity as amicetin against E. coli (1).
Toxicity: Less irritating than amicetin when
administered subcutaneously to dogs (1).
References:
1. British Patent 707,332, April 14, 1954.
2. Haskell, T. H. el al. J. Am. Chem. Soc. 80:
743-747, 1958.
3. Haskell, T. H. J. Am. Chem. Soc. «(»: 747-
751, 1958.
Blasticidins
Produced by: Streptoniyces griseochronwgenes.
Method of extraction: Broth-filtrate extracted
with butanol at pH 4.0. Extract shaken with so-
dium bicarbonate solution, then water. Solvent
concentrated, then dried in vacuo. Residue ex-
tracted with methanol. Extract evaporated in
vacuo. Resulting powder extracted with ether.
Ether-insoluble residue named blasticidin A.
Petroleum ether added to the ether precipitates
crude blasticidin C. After concentration of the
petroleum ether to a syrup and distillation in
vacuo, a colorless liquid, blasticidin B, is obtained.
Purification of blasticidin C by chromatography
on alumina from chloroform solution, and elution
with ethanol.
Chemical and physical properties: A: Light yel-
low powder. Soluble in water, methanol, ethanol,
and water-saturated butanol. Insoluble in ben-
zene, ether, petroleinn ether, chloroform, and ace-
tone. Ultraviolet light-al)sorption spectrum shows
one peak at 216 m/i. B: Colorless liquid; li.p. 36°C
^t Kooo iimi Hg. Soluble in methanol, ethanol,
benzene, ether, acetone, petroleum ether, and
chloroform. Insoluble in water. ('.• Light reddish
brown powder. Soluble in ether, acetone, and
chloroform. Insoluble in water, benzene, and pe-
troleum ether. Broth activity stable to boiling for
55 minutes at pH 5.0 to 7.0; unstable when heated
above or below this range.
Biological activity: A, B, and C are moderately
active on a variety of fungi, including Firicularia
o/'i/zae. A is most active (<1 to lOO/ig per ml) (1,2).
References:
1. Fukunaga, K. et al. Bull. Agr. Chem. Soc.
Japan 19: 181-188, 1955.
2. Koaze, Y. et al. J. Antibiotics (Japan) 9A:
89-96, 1956.
Bla.siicidin S
Produced by: Streptoniyces griseochroniogenes.
Produced by same culture that forms l)lasticidins
A, B, and C.
Method of extraction: Broth-filtrate treated with
activated carbon at pH 4.0. Elution with 50 per
cent aqueous acetone. Eluate concentrated in
vacuo at 40°C. Oxalic acid added to the concen-
trate to remove the Ca++. Filtrate passed through
an 1R4B column to remove excess oxalate. Effluent
chromatographed on IR-50 (H"""). Elution with
acetone-1 N HCl, 1:1. Precipitation of antibiotic
on addition of acetone. Purification by chroma-
tography on alumina from a 90 per cent aqueous
methanol solution. Developed with 70 to 90 per
cent aqueous acetone and eluted with 50 per cent
aqueous acetone. Recrystallized from aciueous ace-
tone.
Physical and chemical properties: Free base:
White needles; m.p. 235-236°C. Soluble in water
and acetic acid. Insoluble in methanol, ethanol,
acetone, benzene, ether, ethyl acetate, butyl ace-
tate, chloroform, carbon tetrachloride, and methyl
ethyl ketone. Negative FeCl.'? , Fehling, Tollen, so-
dium nitroprusside, triphenyltetrazolium chlor-
ide, maltol, Millon, Ehrlich, Sakaguchi, Molisch,
biuret, xanthoproteic, and Graf ketone tests. Posi-
tive diazo, 2,4-dinitrophenylhydrazine, ammonia-
cal silver nitrate, ninhydrin, and Graf aldehj-de
tests. [aJn = +108.4° (c = 1 per cent in water).
C14H20O5N6 : C = 47.119o; H = 5.83%; N =
24.46%. Ultraviolet absorption spectrum maxi-
mum at 275 niM (ML 349) in 0.1 N HCl or 266 to
270 niM (£'lcm 266) in 0.1 A" NaOH. Infrared spec-
trum given in reference 1. Most stable at 100°C at
pH 5.0 to 7.0. Less stable at pH 4.0 than pH 2.0 or
pH 8.0 to 9.0. Melting points: HCl, 224-225°C
(decomposition); picrate, 200-202°C (decomposi-
tion) ; and helianthate, 224-225°C (decomposition).
Biological activity: Active on Pseudomonas (5 to
50 fig per ml), slightly active on other bacteria (50
to 100 fxg per ml), including mycobacteria (10 to
50 Mg per ml). Active on phytopathogenic fungi
(10 to 100 Hg per ml), including Piricularia oryzae
(5 to 10 Mg per ml).
Toxicity: LD50 (mice) 2.82 mg per kg intrave-
nously.
DESCRIPTIONS OF ANTIBIOTICS
221
Reference: 1. Takeuchi, S. et al. J. Antibiotics
(Japan) IIA: 1-5, 1958.
Blaslmj ciii
Produced by: Streptomijces blastmycelicns (1), and
other Streptoinyces spp. (5).
Synonyms: May be an isomer of antimycin in
A3 (5); may contain A3 itself as the major compo-
nent, with traces of A4 (-i).
Method of extraction: Filtered brotli extracted
with l)enzene. Mj'celium extracted with 80 per
cent aciueous acetone; acetone then evaporated.
Re.sidue extracted with benzene, and this benzene-
extract combined with the l^enzene from the broth.
After washing with phosphate buffer at pH 2.0
and evaporation in vacuo, an oily syrup is ol)-
tained. Syrup extracted with petroleum ether in a
Soxhlet apparatus. Precipitated from the petro-
leum ether solution. Recrystallized from benzene-
petroleum ether or ether-petroleum ether (1).
Chemical and physical properties: White needles;
m.p. 1(J7°C (1) or 168-169°C (3). Very soluble in
acetone, ethyl acetate, benzene, chloroform,
methyl isol)utyl ketone, and carbon tetrachloride;
soluble in methanol, ethanol, and ether; .slightly
soluble in n-hexane and cyclohexane; scarcely
soluble or insoluble in petroleum ether and water.
Ultraviolet absorption maxima at 225 irxfx (^icm
()25) and 321 m^u (E'lcm 116). Bathochromic shift
on addition of base, to 222 and 245 m^. Infrared
absorption spectrum given in reference 1. [a]^ =
+77.4° (c = 1 per cent in methanol). Positive
FeCls , diazo, hydroxamic acid, and biuret tests;
negative ninhydrin, Molisch, Fehling, Tollen,
Ehrlich, and Millon tests. C = 59.81'^.; H =
6.89%; N = 5.34S^b- No S, P, halogen, or ash.
C-.eHseN-jOg . Acetylation gives a l)iologically in-
active diacetyl derivative; white fibrous crystals;
m.p. 146-150°C. Mild alkaline hydrolysis gives
l)roducts including a neutral oil (blastmycinone),
b.p. 190-191 °C, and an acidic substance (blast-
mycic acid) (I), m.p. 141.5-142°C. Further alkaline
hydrolysis of I gives formic acid and antimycic
acid (structure: N-(3-aminosalicyloyl)-L-threo-
nine), which is also a degradation product of the
antimycins. Complete structure of blastmycin
(1, 3) is given in Chapter 6.
Biological activity: Active on certain finigi, in-
cluding Piricularia oryzae and P. grisea at 0.005
mg per ml. Also active on Gloeosporium lacticola,
Corticium centrifugus, Ophiostoma Jimbriata, Scle-
rotinia hydrophilum, but not aspergilli, penicillia,
fusaria, Elsinoe, or C. albicans (1). Not active on
bacteria. Inhibits anaerobic glycolysis of Ehrlich
ascites tumor cells. Blocks electron transport be-
tween cytochromes B and C (2).
Toxicity: LD50 (mice) 1.8 mg per kg intraperi-
toneally (1).
References:
1. Watanabe, K. ct al . J. Antil)iotics (Japan)
lOA: 39-45, 1957.
2. Lardy, H. A. et al. Arch. Biochem. Biophys.
78:' 587-597, 1958.
3. Yonehara, H. and Takeuchi, S. J.Anti-
l)iotics (Japan) llA: 254-263, 1958.
4. Liu, W. C. Thesis, University of Wisconsin,
1958.
5. Karasawa, K. et al. J. Gen. Appl. Micro-
biol. .1: 13-20, 1959.
B<
-lidi
Produced by: Streptomyces rochei and an uniden-
tified Streptoinyces sp. (1, 3).
Method of extraction: Broth-filtrate extracted
with butyl acetate at pH 7. Extract evaporated
to dryness. Residue purified by treatment with
bentonite clay in butyl acetate, then by extraction
into alkali from ether. Alkaline extract acidified
and extracted with benzene. Benzene concentrated
to precipitate the antibiotic. Recrystallized from
benzene. Can also be adsorbed onto bentonite clay
from benzene and eluted with methanol (1, 4).
Chemical and physical properties: Acidic sub-
stance; m.p. 145-146°C. Molecule may contain a
.site of conjugated unsaturation. Soluble in eth-
anol, isopropyl alcohol, and benzene, [a]'^ = —28°
(ethanol). Ultraviolet absorption spectrum maxi-
mum at 256 m/j. (£^i'Jn, 550) (isopropyl alcohol).
CosHjsOeN. Forms a crystalline methyl ester (m.p.
153-154°C) and a crystalline p-nitrobenzyl ester
(m.p. l(;i°C) (1, 4).
Biological activity: Active in vitro and in vivo
(mice) on Borrelia. Active in vitro on certain
micrococci and Sarcina lutea, but not active on
Staph, aureus, E. coli, or B. subtilis. Active on
Tetrahyniena geleii. Not active in vivo on other
bacterial, viral, or Treponema pallidum infections
in mice (1-4). Slightly active on carcinoma 1025
and grand epidermoid carcinoma (mice) (6).
Toxicity: LD.50 (mice) 74.7 mg per kg subcutane-
ously, 39.0 mg per kg intravenously. LD.5n (rat)
1.78 mg per kg subcutaneously (2). Poor growth
in rats fed borrelidin is partially overcome Ijy
addition of niacin or trj^ptophan to diet (5).
References:
1. Berger, J. et al. Arch Biochem. 22: 476-
478, 1949.
2. Buck, M. et al. Trans. N. Y. Acad. Sci. 11:
207-210, 1948-1949.
222
DESCRIPTIONS OF ANTIBIOTICS
3. Berger, J. and Goldberg, M. W. J. Clin.
Invest. 28: 1046, 1949.
4. JampoLsky, L. M. and Goldberg, M. W. J.
Clin. Invest. 28: 1046, 1949.
5. Cooperman, J. M. et. al. Proc. See. Exptl.
Biol. Med. 76: 18-20, 1951.
6. Sugiura, K. and Sugiura, M. M. Cancer
Research 18: (Suppl. 1) 290, 1958.
Bottroniycin
Produced by: Streptomyces bottropensis (1).
Synonym: B-Mycin (1).
Method of extraction: Broth-filtrate extracted
with butyl acetate. Extract concentrated azeo-
tropically. Concentrate washed with NaHCO?
solution and water, then extracted with phosphate
buffer (pH 2). Aqueous extract washed with ether
to remove butyl acetate; residual ether evaporated
and crude Ijottromycin precipitated by adjusting
to pH 9. Purification by chromatography on Flori-
Positive bromine test. Rf = 0.94 on paper chro-
matography (water-saturated butanol with 2.5
per cent acetic acid). Salicylate: White crystals;
m.p. 160-161 °C. Acetate: Amorphous; m.p. 138-
148°C (decomposition). Poorly soluble in water.
Hydrochloride: Cream-colored amorphous sub-
stance; m.p. 190-210°C (decomposition). Poorly
soluble in water. Acid hydrolysis products include
seven ninhydrin-positive substances, including
glycine, valine, and two unidentified crystalline
amino acids: (I) CioHuNO. (m.p. 176-177°C) and
(II) CeHgNaO.S (m.p. 197.5-201 .5°C). Degrada-
tion with boiling acetic anhydride yields two crys-
talline acetyl products: (III) C21H34N4O4 (m.p.
165-170°C) and (IV) C19H23N3O4S. Compound IV
is the methyl ester of an N-acetyl dipeptide com-
posed of I and II. I and II were identified as a-
amino-/3-phenylbutyric acid and |8-(2-thiazole)-/3-
alanine, respectively (3, 4). The structure of IV
is:
-CH(CH3)— CH^CONH— CH— CHo— COOCH3
"^ / NH C.
I a
c=o
s
N
CH3
sil with chloroform as solvent and developer, or
by means of precipitates formed with organic
acids such as salicylic, benzoic, acetic, picric, or
anthranilic acids (2). Can also be adsorbed on
carbon and eluted with acetone-HCl; on IRC-120
and eluted with 10 per cent NaCl; on Magnesol
and eluted with benzene-methanol; or on Florisil
and fractionally eluted with chloroform containing
7.5 per cent ethanol. Active fractions acidified with
0.1 A' H2SO4 , and organic solvent removed in
vacuo. Precipitated from aqueous residue by ad-
justing pH to 9 (1).
Chemical and physical properties: Weak sulfur-
containing base. Glittering, white amorphous
powder; m.p. 143-147°C (decomposition). CssHjg-
NtOsS: C = 58.94%; H = 7.78%; N = 12.53%,;
S = 3.88%. Molecular weight, about 770. pKa' =
about 6.5. Readily soluble in most organic solvents.
Insoluble in hexane, cyclohexane, and petroleum
ether. More soluble in ice water than water at
30°C. [a]f = -14.2° (c = 0.5 per cent in 96 per cent
ethanol). Ultraviolet absorption spectrum maxi-
mum at 203 ni/i and a weak shoulder at 240 m/j.
(1, 2). Infrared data given in reference 2. Less
stable at alkaline than acid pH (2). Negative nin-
hydrin test. One primary amino group (Van
iSlyke). No acetyl or N-alkyl groups. Two to
three C-methyl groups and one methoxyl group.
Bottroniycin contains this methyl ester grouping
and is biologically inactivated on its removal (5) .
Biological activity: Active on gram-positive bac-
teria and mycobacteria. Not active on gram-nega-
tive bacteria, except for Hemophilus, Neisseria,
and Brucella. No cross-resistance with carbomycin
or erythromycin.
References:
1. British Patent 762,736, December 5, 1956.
2. Waisvisz, J. M. et al. J. Am. Chem. Soc.
79: 4520-4521, 1957.
3. Waisvisz, J. M. e/ a?. J. Am. Chem. 8oc. 79:
4522-4524, 1957.
4. Waisvisz, J. M. et al. J. Am. Chem. Soc.
79: 4524-4527, 1957.
5. Waisvisz, J. M. and Van der Hoeven, M. G.
J. Am. Chem. Soc. 80: 383-385, 1958.
Boviiiocidiii
Produced- by: Streptomyces sp.
Synonym: /3-Nitropropionic acid.
Remarks: This comjiound had been isolated pre-
viously from plants and fungi.
Method of extraction: Broth-filtrate adjusted to
pH 2.5 and extracted with ethyl acetate. Back-
extraction into pH 9.0 water. After adjustment of
pH to 2.5, extraction with ethyl acetate. Ethyl
acetate washed with water and evaporated to dry-
DESCRIPTIONS OF ANTIBIOTICS
22?.
ness in vacuo. Residue dissolved in methyl alcohol;
decolorization with charcoal. Filtrate evaporated
to dryness, dissolved in anhydrous ethyl acetate,
and passed through a column of alumina. Column
washed with ethyl acetate, acetone, and methanol.
Elution with phosphate buffer at pH 6. Adjusted
to pH 3, extracted with ethyl acetate, washed with
water, and concentrated in vacuo. A precipitate
forms, which is dissolved in ether; petroleum ether
added gradually. Upon standing at room tempera-
ture, colorless crystals are obtained. Recrystalli-
z at ion from the same ether-petroleum ether mix-
ture.
Chemical and phijsical properties: Colorless crys-
tals; m.p. 66-67°C. Soluble in water, alcohols, ace-
tone, and ether. Moderateh^ soluble in benzene.
Insoluble in petroleum ether. C = 30.8-4%; H =
4.40%; N = 11.66%,. C.3H5O4N. Molecular weight
119.8 (foimd, Rast's method, 110). Light-absorp-
tion maximum at about 270 iw/x at pH 8.4. Infrared
absorption spectrum given in reference 1. Bovino-
cidin was found to be identical to |8-nitropropionic
acid.
Biological activity: Weakly active against -1/.
tuberculosis BCG. No activity against other myco-
bacteria, bacteria, and fungi tested. Biological
activity not reduced by /3-alanine.
Toxicity: LD50 (mice) 50 mg per kg intrave-
nously.
Reference: 1. Anzai, K. and Suzuki, S. J. Anti-
biotics (Japan) 13A: 133-136, 1960.
Broni tetracycline
Produced by: Certain Streptomyces aureofaciens
(chlortetracycline-producer) mutants. Produced
instead of chlortetracycline when bromide is pres-
ent in a Cl-deficient medium (3).
Method of extraction: Broth-filtrate acidified to
pH 2.0, mixed with Hyflo Super-Cel, and filtered.
BaCl-.-2H20 added to filtrate, pH adjusted to 8.5,
and cooled to 0°C. Solids extracted with butanol
at pH 2.0. Extract adjusted to pH 3.5 and concen-
trated in vacuo at <30°C. Concentrate adjusted
to pH 5.0; precipitate washed with ether and dried
(1). Separated from concurrently produced tetra-
cj^'cline by partition chromatography (butanol-
chloroform over stationary Celite-aqueous HCl)
(3). Purification by precipitation as HCl salt and
conversion to base by Vjringing an aqueous solu-
tion to pH 5.2 with triethylamine and cooling.
Crystallized from benzene (1).
Chemical and physical properties: Base: m.p.
170-1 72°C. [cx]l° = -196° (in 0.1 X HCl). Ultra-
violet absorption spectrum maxima (in 0.1 A HCl)
at 227 niM (e = 17915), 260 m^ (t = 17480), and
370 m/x (t = 9630) ; or (in 0.25 -Y NaOH) at 225
niM (e = 28215), 255 m^ (e = 15115), 285 m^ (e =
14610), and 345 mn (« = 7221). Infrared spectrum
is the same as that of chlortetracycline (1). More
stable than the other tetracyclines in distilled
water at 100°C for 15 minutes. CasHosOsNoBr (1,
2). Structural formula given in Chapter 6. HCl
salt: Bright ^-ellow crystals. Browns at 218°C;
decomposes at 235°C. Soluble in water to 1.36 per
cent at 25°C, in dry butanol to 0.038 per cent.
I)Ka = 3.4, 7.4, and 9.2. [a]„ = -205° (0.5 per cent
in 0.03 X aqueous HCl). Yields tetracycline on
catalytic hydrogenation. Has 77 per cent of the
absorption of chlortetracycline at 368 m^ (2, 3).
Biological activity: Qualitatively, antibacterial
activity resembles chlortetracycline. Has 95 per
cent of the activity of chlortetracycline on Staph,
aureus on a weight basis, and 90 per cent of the
activity on E. coli (3). Has same activity in vivo
as other tetracyclines (2).
Toxicity: LD50 (HCl salt, mice) 89 mg per kg
intravenously (2).
References:
1. Sensi, P. ct al. Farmaco (Pavia) 10: 337-
345, 1955.
2. Rolland, G. et al . Farmaco (Pavia) 1(1:
340-355, 1955.
3. Doerschuk, A. P. et al. J. Am. Chem. Soc.
78: 1508-1509, 1956.
B
rvanivcin
Produced by: Streptomyces hawaiiensis (1, 2).
Synonym: Thiactin (2).
Method of extraction: Extracted from whole
broth or V)roth-filtrate with methyl isobutyl ke-
tone, butanol, or chloroform. Purification b}- ad-
sorption on alumina or Florisil from chloroform;
washing with water, acetone, and chloroform;
and elution with a 5 per cent methanol-95 per cent
chloroform mixture or aciueous t-butyl alcohol.
Original methyl isobutyl ketone extract (in the
cold) can be processed ))y "flashing" into water
(cUstilling (71 vacuo with the addition of water
until no original solvent remains) and lyophiliz-
ing, or In- flashing into t-butyl alcohol. Recrys-
tallized by flashing a concentrated chloroform
solution into methyl isobutyl ketone, or by cooling
a warm methyl Cellosolve or dio.xane solution and
addition of 10 to 50 per cent water or acetone.
Forms salts with mineral acids or metals such as
calcium or sodium (1, 2).
Chemical and physical properties: Amphoteric,
white, crystalline, sulfur-containing polypeptide.
Darkens at about 205-224°C and melts at 223-
235°C. Soluble to the extent of >2 mg per ml in
224
DESCRIPTIONS OF ANTIBIOTICS
formamide, chloroform, methyl Cellosolve, pyri-
dine, glacial acetic acid, ethylene glycol mono-
methyl ether, 2 N HCl, warm butanol, warm di-
oxane, and hot amyl alcohol. Soluble to <2 mg
per ml in the following hot solvents: acetone,
benzene, amyl acetate, ethj-l acetate, dimethy-
oxyethane, and glycol; and in cold 0.1 N HCl.
Insoluble in water, ethanol, acetone, benzene,
ethyl acetate, and dimethoxyethane (1, 2). Ultra-
violet absorption spectrum maximum at 310 ni/u
(£JJL 125) (in 6 N H0SO4) with strong end-ab-
sorption below 250 m/^ (1) . Infrared spectrum given
in reference 2. Paper chromatographic behavior in
a variety of systems given in reference 2. [a]p =
— 68.5 to —69.5° (in chloroform). Stable to boiling
in aqueous solution for 1 minute. C = 51.9S^i; H =
5.59%; N = 16.6%; S = 9.5%. No halogen or P.
Molecular weight 1600 to 1750. Acid or BaOH hy-
drolysis products include cystine or cysteine,
threonine, a -alanine, glycine, and isoleucine.
Other unidentified ninhydrin-positive substances
are also present (1, 2).
Biological activity: Very active (0.002 to 0.5 ^g
per ml) on gram-positive bacteria and mycobac-
teria. Not active at 10 Mg per ml on gram-negative
bacteria. Inhibits C. albicans at 6.25 ng per ml.
Active in vivo (mice) on D. pneumoniae, Strepto-
coccus hemolyticus, and S. pyogenes, when given
intraperitoneally, intramuscularly, or orally (1,
2). No cross-resistance with many commonly used
antibiotics (3).
Toxicity: Mice tolerate 1 gm per kg intramuscu-
larly, and 2 gm per kg intraperitoneally (2). LD50
(mice) >1 gm per kg orally (1). Material injected
intramuscularly is absorbed very slowly (1).
References:
1. Cron, M. J. et al. Antibiotics & Chemo-
therapy 6: 63-67, 1956.
2. British Patent 790,521, February 12, 1958.
3. Jones, W. F., Jr. and Finland, M. Anti-
biotics & Chemotherapy 8: 387-391, 1958.
Bulging Factor
Produced by: Strepfoniyces sp. (2).
Method of extraction: Broth-filtrate treated with
charcoal at pH 1.5 and filtered. Filtrate adjusted
to pH 7.0, filtered, and retreated with charcoal.
Charcoal eluted with 0.1 N HCl in methanol or
75 per cent acetone in water. Precipitated from
methanolic eluate with ether. Acetone-eluate neu-
tralized with IRA-400 (0H~) and concentrated in
vacuo. Factor precipitated as reineckate (2).
Chemical and physical properties: Basic sub-
stance. Water-soluble. Insoluble in organic sol-
vents. Stable at acid but not alkaline pH (2).
Biological activity: Active on certain fungi, caus-
ing a pronounced bidging of the hyphal wall at
intervals alternating with normal hyphal growth.
Active only on fungi having chitinous cell walls
(fungi imperfecti, basidiomycetes, ascomycetes,
and zygomycetes), not those with cellulosic cell
walls (oomycetes). Believed to act specifically on
the chitin. Also active on the conidia of certain
fungi, such as those of Ophiostoma paradoxum,
which swell to 10,000 times their normal size. Not
active on the bacteria or viruses tested (1, 2).
References:
1. Rombouts, J. E. Proc. 6th Intern. Congr.
Microbiol. 5: 205-207, 1953.
2. Links, J. et al. J. Gen. Microbiol. 17: 596-
601, 1957.
Cabicidin
Produced by: Streptomyces gougeroti (1).
Method of extraction: No data, but probably sim-
ilar to other pentaene antibiotics.
Chemical and physical properties: Pentaene.
Colorless or light yellow columns; m.p. 225°C.
Soluble in methanol, propanol, butanol, acetone,
pyridine, ethylene glycol, proplylene glycol, and
glycerol. Slightly soluble in ethyl acetate and
methyl Cellosolve. Insoluble in diethyl ether,
petroleum ether, benzene, and water. Ultraviolet
absorption spectrum maxima at 320, 339, and 354
m/x (1 per cent methanolic solution). [a]l'^ = —135°
(methanol). Rotation changes to —87° on ex-
posure to ultraviolet light (1).
Biological activity: Active on yeasts and fungi
(1).
Reference: 1. Ogata, K. et al. Japanese Patent
9245, October 17, 1958.
Cacaoniycetiii
Produced by: Streptomyces sp. reseml)ling S.
cacaoi.
Method of extraction: Broth-filtrate extracted
with chloroform at pH 5.0. Chromatographed on
alumina and eluted with 95 per cent ethanol. Ex-
tract evaporated to dryness in vacuo. Residue ex-
tracted with acetone. Extract evaporated to dry-
ness .
Chemical and physical properties: Crude yellow
syrup. Slightly soluble in water. Soluble in many
organic solvents. Contains four components. Very
unstable at alkaline pH.
Biological activity: Active on filamentous fungi,
particularly ascomycetes, but not on yeasts. Very
slightly active on gram-positive bacteria and
Mycobacterium avium.
DESCRIPTIONS OF ANTIBIOTICS
225
Toxicity: MLD (mice) aboiil 300 m^ i)er kg sub-
cutaneously.
Reference: 1. Wakaki, S. (t al. J. Antibiotics
(Japan) 5:24-49,1952.
Caeriil«>ni> <-iii
Produced by: Streptomyces caendeus.
Method of extraction: Active filtrate extracted
with 34 volume of ether. Solvent evaporated to
dryne.ss in vacuo. Crude extract washed with cold
petroleum ether, dissolved in ethanol, clarified
with charcoal, and crystallized twice from eth-
anol. The crude extract can also be sublimed in
vacuo at 120°C. Sublimate crystallized from eth-
anol.
Chemical and physical properties: Colorless
needles; m.p. 175°C. Soluble in ethyl acetate, ace-
tone, ethanol, methanol, chloroform, and ether.
Sparingly soluble in benzene, petroleum ether, and
water. Insoluble in sodium ])icarbonate and car-
bonate solutions. Soluble in dilute sodium hydrox-
ide and hydrochloric acid. Alcoholic solutions give
a red color with ferric chloride. Deep red color
e formed when ferrous salts are added to mineral
acid solutions (reaction typical of a ,a'-dip3'ridj'l) .
Proposed empirical formula Ci2Hii02N3 , on the
basis of the analysis: C = 63.09%; H = 4.99%;
N = 18.48%; and molecular weights of 251 (Rast)
and 215 (isothermal distillation in acetone). No
terminal methyl groups (Kuhn-Roth). No acetyl
groups (saponification). One methoxy group pres-
ent (Zeisel) and one active hydrogen (Zerewiti-
noff). Amphoteric substance. pK^ = 4.38 and
pKb = 9.81. Infrared absorption spectrum given
in reference 1. Light-absorption ma.ximum at
about 235 m/x with shoulders at about 285 and 295
m^t.
Biological activity: Active against yeasts and
filamentous fungi at levels of 5 to 10 ng per ml.
Active against gram-positive and gram-negative
bacteria at levels of 10 to 100 Mg per ml.
Reference: 1. Funk, A. and Divekar, T. V. Can.
J. Microbiol. 5: 317-321, 1959.
Caniphoniycin
Produced by: Streptomyces rutgersensis var. caste-
larense (2).
Synonym: Has properties in common with anti-
biotic l-81d-ls.
Method of extraction: Agar culture extracted
with ether at room temperature. Extract evapo-
rated at <20°C in vacuo and residue taken up in
ether. Process repeated. Residue taken up in ab-
solute alcohol.
Chemical and physical properties: Resinous,
waxy, white or white-yellow powder. Basic; m.p.
20-22°C. (A higher m.p. of 142°C (2) or 149°C (1)
first reported was found (2) to be that of a decom-
position product.) Soluble in ether, methyl ace-
tate, amyl acetate, chloroform, benzene, butanol,
ethanol, and methanol, ^'ery slightly soluble in
water. Ultraviolet absorption spectrum shows a
progressive drop from 210 to 450 m^, with no
maxima. Positive Tollen test. Gives a precipitate
with Nessler's reagent. Negative Heller, Millon,
Fehling, Benedict, a-naphthol, biuret, Adam-
kiewicz, nitroprusside, and FeCl.-! tests. On heating
in concentrated HCI or H2SO4 , alcoholic solution
gives a green color which turns orange, then red-
dish. Heating in NaOH gives a red color which
clears somewhat on cooling and becomes colorless
on oxidation. Reductive properties. Contains C,
H, O, and N. Gives off NH.i when boiled in NaOH.
Has quinonic properties (1, 2).
Biological activity: Active mainly on gram-posi-
tive bacteria and mycobacteria. Slightly active on
certain strains of E. coli. Active at 5 /ig per ml on
Helminthosporium sativum and Neurospora crassa,
but not other fungi tested. Partially inactivated
by thiamin and niacin (2).
Toxicity: Mice tolerate 550 mg per kg, orally or
intravenously (1). Toxic to corn, oats, barley,
wheat, alfalfa, cabbage, tomato, carrots, and
willow twigs at 100 to 1000 ppm. Stimulates ger-
mination at 1 to 10 ppm (2).
References:
1. Cercos, A. P. Rev. arg. agron. 20: 53-02,
1953.
2. Cercos, A. P. Rev. invest, agr. (Buenos
Aires) 8: 263-283, 1954.
Candicidin
Produced by: Streptomyces griseus (1).
Remarks: Similar to trichomycin and ascosin.
Method of extraction: I. Culture (including
mycelium) adjusted to pH 2.5, stirred with Hyflo
Super-Cel, and filtered. Eluted from solids with
n-butanol. Extract treated with NaHCO.3 to re-
move pigmented impurities, then concentrated to
drj'ness in vacuo. Residue treated with petroleum
ether, taken up in water, then freeze dried. Puri-
fied by (a) treating with acetone to give Fraction
C, then extracting with 95 per cent ethanol to
yield Fractions A and B; (b) chromatography on a
cellulose powder column from a chloroform sus-
pension, elution with 95 per cent ethanol followed
by an ethylene glycol monomethyl ether-ethanol
mixture (1:1) to give Fractions A, B, and C; or
(c) partition (methanol-petroleum ether-chloro-
form-water, 3:1:3:2) in absence of O2 at pH 4.0.
226
DESCRIPTIONS OF ANTIBIOTICS
Upper phase concentrated, then extracted with
bntanol at pH 7.0. Forced into a small volume of
water by addition of petroleum ether to butanol
(5, 7). II. Broth adjusted to pH 3.5 to 4.0 and
filtered with a filter-aid. Cake extracted with
water-saturated n-butanol. Extract washed with
0.333 M NaHCOs , then with water at pH 7.0;
pentane added to separate out an aqueous layer.
To this aqueous layer are added aqueous washes
of the remaining butanol-pentane layer, and the
combined aqueous extracts are concentrated in
vacuo and lyophilized (13).
Chemical and physical properties: Conjugated
heptaene. Crude candicidin: Soluble in butanol,
glycerol, benzyl alcohol, ethylene glycol, and
ethylene glycol monomethyl ether. Partially sol-
uble in water, ethanol, chloroform, and acetone.
Insoluble in benzene, petroleum ether, carbon tet-
rachloride, xylene, carbon disulfide, ethylene di-
chloride, ether, and ethyl acetate (1). Three compo-
nents were demonstrated on paper chromatography
with Rf values of 0.00, 0.44, and 0.66 [methanol-
0.880 (sic; probably specific gravity) ammonia-
water, 20:1:4]. N = 1.58 to 2.16%. Nondialyz-
able (1, 7). Fraction A. Reddish brown substance.
Soluble in water, ethanol, butanol, ethylene gly-
col, and ethylene glycol monomethyl ether. In-
soluble in acetone (1). Ultraviolet absorption
spectra at 340, 360, 380, and 403 mju (ethanol) (5).
C = 62.9%; H = 9.6%; N = 4.7%,. At pH 7, with-
stands heating for 10 minutes at 60°C (1). Fraction
B: Greenish substance. Soluble in butanol, ethyl-
ene glycol, and ethylene glycol monomethyl ether.
Insoluble in water, ethanol, and acetone (1). Ul-
traviolet absorption spectrum maxima at 340, 362,
381, and 404 m^ (ethanol) (5). C = 57.8%; H =
9.9%; and N = 7.3% (1). Fraction C: Reddish
brown substance. Soluble in ethanol, butanol,
ethylene glycol, ethylene glycol monomethyl
ether, and acetone. Insoluble in water. Ultraviolet
absorption spectrum maxima at 358, 379, and 402
niM (1). Fractions A and B are different forms of
the same compound; A is the sodium salt of B.
Fraction C is probably a degradation product of
canchcidin. Candicidin contains an aromatic com-
ponent in its molecule, and is ninhydrin-positive
(with two amino groups). The molecule of candici-
din contains mycosamine and p-aminoacetophe-
none (14).
Biological activity: Active mainly on yeasts, but
also on certain filamentous fungi, including a
variety of plant pathogens. Fractions A and B
differ only quantitatively in antifungal activity.
Not active on bacteria, actinomycetes (including
Nocardia asteroides), or mycobacteria. Fraction C
is relatively inactive. A and B are most active at
pH 7 to 8 (100 times more active than at pH 5 to
6). In tissue culture, inhibits pathogenic (yeast)
stage of Histoplasvia capsulatum, Cryptococcits
neofornians, Candida albicans, and Blastomyces
dermatitidis. Protects against wheat and Poa
pratensis stem rust (Puccinia graminis) and infec-
tions by Cronarti\im rihicola (uredial stage). Some
control of snapdragon rust {Puccinia antirrhini)
and powder mildew of beans. Excellent control of
bean rust (Uromyces phaseoli) and brown rot of
peach (Monilinia fructicola) (1-4, 10-12, 15).
Toxicity: Crude candicidin: LD50 (mice) 663 mg
per kg subcutaneousl}-, 79 mg per kg intraperi-
toneally. Nontoxic to germination of pea seeds at
125 /ig per ml or less (1) . Candicidin A : LDjo (mice)
47 to 65 mg per kg intraperitoneally, 277 mg per
kg subcutaneously. Necrosis at injection site (sub-
cutaneous or intradermal). Conjuctiva of rabbits
and oral mucosa (human) not irritated by a 1 per
cent solution applied topically. Least inhibitory
dose (tissue culture) 20 to 80 jug per ml. Candici-
din B: LD50 (mice) 159 mg per kg subcutaneously,
53 mg per kg intraperitoneally (1, 2, 4).
Utilization: Vulvovaginitis (8). Intertriginous:
moniliasis (6).
References:
1. Lechevalier, H. et al. Mycologia 45: 155-
171, 1953.
2. Kligman, A. M. and Lewis, F. S. Proc. Soc.
Exptl. Biol. Med. 82: 399-404, 1953.
3. Alcorn, S. J\I. and Ark, P. A. Plant Disease
Reptr. :38: 705-709, 1954.
4. Hu, F. et al. Arch. Dermatol. Syphilol.
70: 1-15, 1954.
5. Vining, L. C. et al. 8th Congr. intern.
botan., Paris Vol. prelim. Sect. 24, 106-
110, 1954.
6. Franks, A. G. et al. J. Invest. Dermatol.
23: 75-76, 1954.
7. Vining, L. C. et al. Antibiotics Ann. 980-
987, 1954-1955.
8. Fox, J. L. Antibiotic :Med. 1: 349-350,
1955.
9. Oroshnik, W. ct al. Science 121: 147-149,
1955.
10. Ark, P. A., and Alcorn, S. :\I. Plant Dis-
ease Reptr. 40: 85-92, 1956.
11. Lar.sh, H. W. et al. Antibiotics Ann. 918-
922, 1956-1957.
12. Muller, W. H. Am. J. Botany 45:183-190,
1958.
13. Siminoff. P. U. S. Patent 2,872,373, Febru-
ary 3, 1959.
14. Borowski, E. Personal communication.
15. Lechevalier, H. Antibiotics Ann. 614-
618, 1959-1960.
DESCRIPTIONS OF ANTIBIOTICS
227
Caii(li<IiM
Produced by: Streptoniyces viridoflavus.
Synonyins: Originalh' the confusing designations
candidin A and candidin B referred to different
salt forms of the basic candidin molecule. Candi-
din may exist as different salt forms, depending on
the pH of isolation.
Method of extraction: Whole l)roth mixed with
Hyflo Super-Cel, adjusted to pH 4.8 with dilute
acid to precipitate the colloidally suspended can-
didin, and the whole filtered. Solids extracted with
butanol-methanol (4:1), water added until separa-
tion into phases begins, and pH adjusted to 7.0
with NH4OH. Organic pha.se concentrated //( vacuo
at low temperature vmtil precipitate forms. A
pyridine-glacial acetic acid (10:1) solution of this
precipitate is heated to 50°C, warm water added,
and the whole cooled. Precipitate which forms is
washed with acetone and crystallized from 90 per
3ent aqueous dioxane (8).
Chemical and physical properties: Amphoteric,
conjugated heptaene macrolide. Rosettes (from
aqueous dioxane) or needles. No melting point;
decomposes at >180°C. Soluble in dimethylfor-
mamide, pyridine, and dimethyl sulfoxide. Less
soluble in glacial acetic acid. Slightly soluble in
methanol, 70 per cent 1-propanol, water-saturated
butanol, and 90 per cent dioxane. Nearly or com-
pletely insoluble in ethanol, butanol, t-butanol,
dioxane, toluene, petroleum ether, sodium car-
bonate, and water. Ultraviolet absorption spec-
trum maxima at 365, 385 (^^I'cm 1600), and 408 m^
(alcohols). Shows a reflectance peak at 340 n\fi in
water suspension, a characteristic shared only by
amphotericin B among the better known hep-
taenes. Infrared spectrimi given in reference 5.
[a]'^ = +363°C (c = 0.3 per cent in dimethyl-
formamide), or +205° (c = 0.3 per cent in glacial
acetic acid). Positive bromine, KMn04 , ninhy-
drin, antimony trichloride, and sulfuric acid
chromophore (deep blue) tests. Negative ninhy-
drin (after hydrolysis), semicarbazide, Benedict,
Fehling, Tollen, Schiff, 2,4-dinitrophenylhydra-
zine, FeChi . and pine splint tests. Can be differ-
entiated by paper chromatography and by bio-
logical assay from the other well known heptaene
antibiotics (candicidin, ascosin, trichomycin, and
amphotericin B). Photo-, thermo-, and acid-labile.
Most stable at neutral and alkaline pH. Stable in
drj^ form at low temperatures vuider Nj . C46H7.3
OieN. Molecular w^eight 895. C = {\().(W; ; H =
9.76%; N = 1.65%. Contains four methyl groups,
one primary' amino group, and one carboxyl group.
Acid degradation products include mycosamine.
Lactone contains a series of secondary and ter-
tiary hydroxyl groups. N -acetylcandidi n,: m.p.
159-161°C. N -acetylcandidi n, brucine salt: m.p.
107-168°C. Hydrocandidins are biologically inac-
tive (1, 3-5, 8, 9).
Biological activity: Active on yeasts and fila-
mentous fungi, including plant pathogens. Com-
pletely suppresses all fungi in the fecal flora of
mice dosed orally, with a concomitant 1000-fold
increase in bacterial count. Active in mice with
Histoplasma capsulatiun and C. albicans infec-
tions. Most active on solid media at an alkaline
pH (about 8.0). N -acetylcandidi n: one-tenth as
active as candidin (1, 3, 4, 6, 9).
Toxicity: LDoo (mice) 7 to 36 mg per kg intra-
peritoneally, 1.5 mg per kg intravenously, and 30
mg per kg subcutaneously (with necrosis at injec-
tion site). Splenic atrophy observed after oral ad-
ministration of 100 mg per kg to mice (1, 3).
References:
1. Taber, W. et al. Antibiotics & Chemother-
apy 4: 455-461, 1954.
2. Taber, W. and Vining, L. C. Bacteriol.
Proc. 86, 1954.
3. Vining, L. C. et al. Antibiotics Ann. 980-
987, 1954-1955.
4. Oroshnik, W. et al. Science 121: 147-149,
1955.
5. \'ining, L. C. and Taber, W. A. Can. J.
Chem. ;U: 1163-1167, 1956.
6. Muller. W. H. Am. J. Botany 45: 183-190,
1958.
7. Solotorovsk\'. M. et al. Antibiotics &
Chemotherapy 8:304-371, 1958.
8. Borowski, E. Personal communication,
1959.
9. Lechevalier, H. unpublished data.
Candiniycin
Produced by: Strepto)n.yces echiniensis (1).
Synonyms: Closely related to ascosin and candi-
cidin (1).
Method of extraction: Extracted from broth and
mycelium; purification by chromatography (1).
Chemical and physical properties: Yellow. Sol-
uble in aciueous acetone, alcohol, glycol, and dilute
NaOH. Less soluble in water. Insoluble in ether,
benzol, acetone, and chloroform. Contains no
S or halogen. C = 57.17%; H = 8.18%; N = 1.70%.
Negative FeCU , ninhydrin, Molisch, Fehling
(cold), and Sakaguchi tests. Silver mirror test
positive in hot state. Addition of H2SO4 changes
the powder to a blue, then to a violet color. Ultra-
violet maxima (methanol) at 362, 382, and 406 m^-
Stable to acid and alkali pH (1).
228
DESCRIPTIONS OF ANTIBIOTICS
Biological activity: Very active on fungi, yeasts,
and Trichomonas vaginalis (1). Active in rats on
Yoshida sarcoma cells, but does not prolong sur-
vival time. Not active on Ehrlich carcinoma
(mice) (2).
Toxicity: LD50 5 mg per kg sul)cutaneously ;
animal not given.
References:
1. Shibata, M. et al. J. Antil)iotics (Japan)
7B: 168, 1954.
2. Aramaki, Y. et al. Ann. Rept. Takeda Re-
search Lab. 14: 60-91, 1955.
Carboniyciii
Produced by: Sireptomyces halstedii (1, 26) (also
produces carbomycin B), S. hygroscopicus (8), and
S. albireticuli (21).
Synonyms: Magnamycin; carljomycin A, anti-
biotic M4209 (9).
Methods of extraction: I. Broth-filtrate ex-
tracted with benzene, chloroform, amyl acetate,
trichloroethylene, butanol, or ether at pH 6.6 or
higher. Solvent: (A) Evaporated to dryness. Resi-
due yields crystals when taken up in a small
amount of isopropanol. Recrystallized from aque-
ous acetone or ethanol. (B) Solvent cHstilled off
in the presence of water. Watery residual solution:
(a) freeze dried and recrystallized from benzene-
petroleum ether or aqueous methanol; (b) chro-
matographed on silica gel from 10 per cent acetone
in benzene and developed with acetone-benzene
mixtures containing gradually increasing amounts
of acetone, the principal fraction being in the 30
per cent acetone in benzene; solvent evaporated
off; residue crystallized from 70 per cent hot aque-
ous isopropanol on addition of water; or (c) ex-
tracted with neutral benzene, extract concen-
trated; concentrate back-extracted into water at
pH 2.0 to 2.5; aqueous extract neutralized to give
free base (8, 9, 22). II. Broth-filtrate extracted
with methyl isobutyl ketone at pH 3.0. Extract
concentrated in vacuo and extracted with dilute
sulfuric acid (pH 2.0). Aqueous extract washed
with benzene, adjusted to pH 6.5, and extracted
with ether repeatedly. Ether evaporated to dry-
ness. Residue triturated in ethanol. Recrystallized
from methanol-water (10). III. Carbomycin
forms complexes with such aromatic solvents as
benzene, mesitylene, benzyl chloride, toluene,
ethyl benzene, naphthalene, chlorobenzene, and
toluene. Such complexes may be precipitated by
addition of hexane to a sufficiently pure solution of
the complex or V)y concentration in vacuo. The
complex may be cleaved to give the free base as
shown previously (24). These comple.xes may also
be separated as solids from the crude broth-ttltrate
if sufhcient complexing agent is added at pH 8.5
to 9.5. Cleavage is obtained with acid, or the com-
plex extracted into acetone or methanol and the
extract concentrated to remove the solvent. Base
precipitates on addition of water to the residue
(25).
Chemical and physical properties: Weakly basic
(26). Macrolide (28). Base: Various crystal forms
have been reported. Prisms; m.p. 207-209°C (open
capillary in oil bath) or 220-222°C (hot stage)
(22); slender white blunt-ended needles; m.p.
199. 5-200. 5°C (1); or rectangular plates or laths;
softens at 208-210°C; m.p. 210-216°C (decomposi-
tion) (9, 10). Very soluble in chloroform, ethyl
acetate, glacial acetic acid, 0.05 N HCl, amyl ace-
tate, Initanol, benzene, and acetone. Less soluble
in methanol, ethanol (25 mg per ml), ethyl ether,
and isopropanol (6.2 mg per ml). Insoluble in hot
or cold water, aqueous alkali, and petrolevmi ether
9, 22). Ultraviolet spectriuii maxima at 238 nifi
(£■!";, 185) and 327 m/x (E\ln 0.9) in absolute etha-
nol (10) or at 241 m/x (£'1L 158) in 2 per cent Na2-
HP()4 (22). Infrared spectrum given in references
10, 22, and 26. [a]t = -53° ± 3° (c = 0.3 per cent
in 0.05 N acetic acid), or —56.1° (c = 1 per cent in
chloroform) (10, 22). Positive 2,4-dinitrophenyl-
hydrazine, Bayer permanganate, bromine, and
eerie nitrate tests. Positive (10) or negative (22)
Fehling and Tollen tests. Violet color with Schiff's
fuchsin reagent (9). Violet color in 4 A^ HCl (10)
or red-violet with strong acid (5). Intense yellow
color in methanolic KOH (9). Negative ninhydrin,
Van Slyke, boric acid, and FeCls tests (10). Water-
insoluble precipitate with trichloroacetic acid (9).
No precipitate with silver nitrate or mercuric
chloride (22). Rf values in various systems given in
references 8 and 22. Most stable in aqueous solu-
tions at pH 5 to 7; less stable at pH 3 or 9. Crystals
stable for several months in the dark at room tem-
perature (10). C = 59.89%; H = 7.96%; N = 1.78%;
C— CHs = 10.58%; N— (CH3)2 = 2.94%; OCH.,
= 3.75% (12). No S, P, or halogen (5). Molecular
weight 810 to 866 (9, 12, 26). C^HejOieN. Struc-
tural formula is given in Chapter 6. Mild acid
hydrolysis yields, among other products, the 4-
isovaleryl methyl glycoside of the sugar mycarose.
The glycoside is an oily neutral substance; b.p
116°C (1.1 mm), nf 1.4493; [«]'„" = -10.7° (c =
9 per cent in chloroform). Mycarose itself is a
crystalline solid; m.p. 128-129°C. [a]f = -31.1°
(c = 4 per cent in water) (11). Strong acid hydroly-
sis yields miicaminose, CSH17NO4 . Hydrochlo-
ride: m.p. 115-116°C. [aif = -f-31° (c = 1 per cent
in water) (20). Methanolysis removes mycarose,
DESCRIPTIONS OF ANTIBIOTICS
229
yielding the unstable crystalline base carimbose,
C3oH47()i2N (28,29); m.p'. 188~189°C. [a];' = -24°
(c = 1 per cent in ethanol). Ultraviolet absorption
spectrum maximum at 240 ni/i (£'icm 196) and a
weak band at 285 ni/x (i'lcm 1-4) in phosphate
buffer at pH 6.0 (9, 12). Hydrogenation yields a
biologically active tetrahydroproduct: long color-
less needles, softens at 116°C, licjuifies at 118-
122°C. [a]f = -53° (c = 0.5 per cent in ethanol).
Ultraviolet absorption spectrum maximum at 330
m/i. Carbomycin forms the following salts: Hydro-
chloride: Amorphous substance (22); m.p. 157-
160°C (decomposition), softens at 155°C (9, 22) or
149-150°C (12). Soluble in water, methanol, and
ethanol. Insoluble in ethyl ether and hexane. Same
[a]° as carbomycin base. Sulfate: Crystalline;
m.p. 163-164°C (decomposition); shrinks at 158°C
(9). Diacetate: m.p. 149-151°C. Pentaacetate: m.p.
134-135°C. Thiosernicarbazone: m.p. 170-173°C.
Ultraviolet maxima at 230 m/x (^IL 218) and 268
m/i (Ei'L 268) (9, 12). Oxime: CrystaUine; m.p.
198-199°C (10). Periodate: White crystals; m.p.
134-135°C (decomposition) (10).
Biological activity: In vitro: Active on gram-
positive bacteria but not gram-negative bacteria,
except Heinophiliis. Active on Actinomyces israelii
and mycobacteria but not on Nocardia asteroides.
Not active on fungi. Active on pleuropneumonia-
like organisms (PPLO) (1, 2, 6, 10). Moderately
active on Endamoeba histolytica, Trypanosoma
cruzi, Leiskmania donovani; less active on T. rho-
desiense, Trichmonas vaginalis, and T. foetus (5).
Cross-resistance with erythromycin, oleandomy-
cin, spiramycin, and streptogramin (23), resist-
ance developing slowly in a stepwise pattern (1).
Activity luiaffected by serum (10). The highest
concentration permitting epithelial cell migration
in tissue culture is 190 /xg per ml (30). In vivo:
Active (in mice) on infections caused by Strepto-
coccus pyogenes, D. pneumoniae, Staph, aureus,
and Pasteurella multocida (4, 16). Antitoxoplasmic
activity (rabbits) (17). Active on PPLO causing
the air sac disease in ovo and in adult chickens
(10). Active on the following rickettsiae in eggs
and a variety of experimental animals; Rickettsia
prowazekii, R. mooseri, R. typhi, R. akari, R.
tsutsugamushi , R. rickettsii, R. conorii, Coxiella
burnetii, and North Queensland tick typhus. Also
active on the organisms causing psittacosis, lym-
phogranuloma venereum, himian and feline pneu-
monitis, bovine encephalomyelitis, ornithosis,
canine distemper, meningopneumonitis, and blue-
tongue (Sonora strain). Such rickettsiae and psit-
tacosis-like forms can be recovered from surviving
treated animals, indicating a static effect of the
drug. No activity on herpes simplex, rabies, vac-
cinia, or poliomyelitis type II (5, 7, 8, 10, 15, 18,
22). Prolongs survival time in mice infected with
Clostridium perfringens and C. histolyticum (31).
Anthelmintic activity against Aspicularis tetrap-
tera and Syphacia obvelata in mice (8). In-
creases growth rate of poultry and swine (1, 19).
Controls Rhizoctonia infection of lettuce (13).
Causes elongation of wheat roots in water solu-
tion (3).
Toxicity: LDso (mice) 550 mg per kg intrave-
nously, 900 to 1000 mg per kg intramuscularly,
2950 mg per kg subcutaneously, and 550 mg per
kg orally (1). LD50 (rats) 700 mg per kg intra-
venously, and >5000 mg per kg orally (10). Highly
toxic to guinea pigs (8). Eggs tolerate 10 mg per
embryo (15). Minimal dose causing inhibition of
mitosis in HeLa cells is 62.5 jug per "^1 (27); 1 to
10 ixg per ml has no effect on human spermatozoa
(5).
Utilization: Infections caused by gram-positive
organisms, especially staphylococci resistant to
other antibiotics. Amoebiasis. Veterinary medi-
cine. Less effective than penicillin (5, 14, 18).
References:
1. Tanner, F. W., Jr. et al. Antil)iotics &
Chemotherapy 2: 441-443, 1952.
2. Welch, H. et al. AntiVjiotics & Chemother-
apy 2: 693-696, 1952.
3. Barton, L. V. and MacNab, J. Contrib.
Boyce Thompson Inst. 17: 419-434, 1954.
4. English, A. R. et al. Antibiotics & Chemo-
therapy 3: 94-98, 1953.
5. Seneca, H. and Ides, D. Antibiotics &
Chemotherapy 3: 117-121, 1953.
6. P'usillo, M. H. et al. Antibiotics & Chemo-
therapy 3: 581-586, 1953.
7. Wong, S. C. et al. Antibiotics & Chemo-
therapy 3: 741-750, 1953.
8. Pagano, J. F. et al. Antibiotics & Chemo-
therapy 3: 899-902, 1953.
9. Dutcher, J. D. et cd. Antibiotics & Chemo-
therapy 3: 910-914, 1953.
10. Finlay, A. and Regna, P. P. 6th Intern.
Congr. Microbiol., Rome 58-72, 1953.
11. Regna, P. P. et al. J. Am. Chem. Soc. 75:
4625-4626, 1953.
12. Wagner, R. L. et al . J. Am. Chem. Soc.
75: 4684-4687, 1953.
13. Hilborn, M. T. Phytopathology 43: 475,
1953.
14. Hewitt, W. L. and Wood, J. P. New Engl.
J. Med. 249: 261-269, 1953.
15. Price, D. A. J. Am. Vet. Med. Assoc. 125:
199-202, 1954.
230
DESCRIPTIONS OF ANTIBIOTICS
16. Riser, J. S. and deMello, G. C. Proc. 58th
Ann. Meeting U. S. Livestock Sanitary
Assoc. 81-97, 1954.
17. Bogacz, J. Bull. soc. pathol. e.xotique 47:
903-913, 1954.
18. Hawley, G. E. and Downing, H. E. Anti-
biotics Ann. 336-340, 1954-1955.
19. Reynolds, W. M. et al. Antibiotics Ann.
510-515, 1954 1955.
20. Hochstein, F. A. and Regna, P. P. J. Am.
Chem. Soc. 77: 3353-3355, 1955.
21. Nakazawa, K. et al. J. Agr. Chem. Soc.
Japan 29: 661-664, 1955.
22. British Patent 738,537, October 12, 1955.
23. Jones, W. F. et al. Proc. Soc. Exjitl. Biol.
Med. 93: 388-393, 1956.
24. Tanner, F. W., Jr. et al. U. S. Patent
2,771,392, November 20, 1956.
25. Friedman, I. J. et al. U. S. Patent 2,792,330,
May 14, 1957.
26. Tanner, F. W., Jr. et al. U. S. Patent
2,796,379, June 18, 1957.
27. Nitta, K. Japan. J. Med. Sci. & Biol. 10:
277-286, 1957.
28. Woodward, R. B. Angew. Chem. 69:50-58,
1957.
29. Brink, N. G. and Harman, R. E. Quart.
Revs. (London) 12: 93-115, 1958.
30. Lawrence, J. C. Brit. J. Pharmacol. 14:
168-173, 1959.
31. Ryan, F. J. et al. J. Infectious Diseases
78: 223-231, 1946.
Carbomycin B
Produced by: Streptotnyces halstedii. This culture
also produces carbomycin (1).
Synonym: Magnamycin B.
Method of extraction: Methanol-w^ater mother
liquors from carbomycin recrystallizations are
diluted with a large volume of water to give a
precipitate. Dried precipitate taken up in anhy-
drous ethanol and stirred at room temperature for
8 hours. Carbomycin, crystallized by this proce-
dure, filtered off. Filtrate evaporated, dissolved in
acetone, and water added to turbidity. Carbo-
mycin B separated out overnight. Recrj'stallized
from a 5:1 acetone-water mixture and anhydrous
acetone (1).
Chemical and physical properties: Probably is an
Q:,|3,7,6-unsaturated ketone. Weakly basic sub-
stance. May be isomeric with carbomycin. Color-
less anisotropic plates, frequently hexagonal;
m.p. 140-144°C (decomposition); softens at 138°C.
Soluble at 20-25°C in methanol to 1.4 gm per ml;
ethanol 0.45 gm per ml; acetone 0.25 gni i)er ml;
benzene 0.15 gm per ml; ether 0.03 gm per ml; and
water, 0.1 to 0.2 mg per ml (1, 3). Ultraviolet ab-
sorption spectrum maximum at 278 niju (J^lcm 276)
(E = 25,000). Infrared spectrum given in refer-
ence 1. [a]; = —35° (c = 1 per cent in chloroform).
Buffered aqueous solutions (pH 5.0) have a half-
life of >3 months. Less stable at pH 3 or 10. C =
60.55%; H = 8.42%; N = 1.78%; O— CHg = 4.01%;
N— CH3 = 3.10%o- Equivalent weight 870. pKb =
7.56. C4i.4.>H67-69NOi5-i6 . Hvdrolysis lu methanoUc
HCl yields, like carbomycin, methyl mycarose
isovalerate. Vigorous acid hydrolysis yields, again
like carbomycin, mycaminose. Other evidence in-
dicates that carbomycin B may differ from carbo-
mycin in more than one double position, and may
contain one oxygen atom less (1). Hydrochloride:
White crystals. More soluble in water than the
base, but less so than carbomvcin A hydrochloride
(2).
Biological activity: Resembles carbomycin (1).
Stimulates rat growth without concomitant fecal
microflora changes, when administered intraperito-
neally, but has no growth-stimulatory effect and
produces marked changes in intestinal microflora
when given orally (2).
Toxicity: LD50 (mice) 300 mg per kg intrave-
nously. Other routes are comparable to carbo-
mycin (1).
References:
1. Hochstein, F. A. and Murai, K. J. Am.
Chem. Soc. 76: 5080-5083, 1954.
2. Dick, E. C. and Johansson, K. R. Anti-
biotics & Chemotherapy 7: 349-358, 1957.
3. Tanner, F. W. et al. U. S. Patent 2,785,104,
March 12, 1957.
Carcinoniycin
Produced by: Streptomyces carcinomycicus ,
Streptomyces sp. (4), S. gannmycicus (2), and *S.
ganmycicus (3).
Synonyms: Gannmycin; carzinomycin (4).
Method of extraction: Precipitated from broth-
filtrate with 50 per cent ZnCh . Precipitate ex-
tracted with Na2HP04 at 25°C. Zn3(P04)2 filtered
off and supernatant dialyzed against running wa-
ter at 0°C for 48 hours. One per cent NaCl is added
and the antibiotic reprecipitated bj^ addition of
acetone. Precipitate taken up in water and freeze
dried. Cannot be extracted with organic solvents.
Chemical and physical properties: Dark brown
powder.
Biological activity: Active against Ehrlich carci-
noma (ascites form) in mice. No activity on bac-
teria or fungi.
Toxicity: Mice tolera.te 500 mg per kg int raven-
DESCRIPTIONS OF ANTIBIOTICS
231
ously, intraperitoneally, intramuscularly, and
subcutaneously.
References:
1. Hosoya, S. Ann. Meeting Japan. Chemo-
therapy Soc. 3: 128-131, 1955.
2. Harada, Y. and Kiibo, S. J. Antit)iotics
(Japan) 9B: 160-167, 1956.
3. Harada, Y. and Tanaka, S. J. Antibiotics
(Japan) 9A: 113-117, 1956.
4. Umezawa, H. Giorn. micro])iol. 2: 160-193,
1956.
Cardiciii
Produced by: Xocardia sp.
Method of extraction: Mycelium extracted with
hot methanol; or acidified broth extracted with
butanol. Methanol concentrated /// vacuo, and
cardicin precipitated on addition of ether. I.
Dissolved in dilute NH4OH and reprecipitated on
acidification. II. Purification by extraction of an
acidified aqueous solution with butanol. Butanol
washed with NaHCO.'i and water, and step II re-
peated.
Chemical and physical properties: Stable. Acidic
form insoluble in water, saline solution, ether, and
acetone; soluble in methanol, ethanol, and hy-
drous butanol. Sodium salt insoluble in ether and
V)utanol; soluble in water, methanol, ethanol, and
large volumes of hydrous butanol.
Biological activity: Active in vitro against gram-
positive bacteria, fungi, and the following phages:
staphylo])hage, streptophage. Bacillus cereus
phage, B. megaterium phages, coliphage, and en-
terococcus phage. Active in eggs on PR 8, Lee,
and FM-1 strains of influenza virus.
Toxicity: 93 ^g injected intraperitoneally or
subcutaneously kills mice in 2 days.
Reference: 1. Machlowitz, R. A. et al. Anti-
biotics & Chemotherapy 3: 966-970, 1953.
C
arvoni^ cm
Produced by: Streptomyces filamentosus (1, 2).
Method of extraction: Extracted from broth-
filtrate with butyl or ethyl acetate. Extracted
from mycelium with alcohols, benzene, or petro-
leum ether (2).
Biological activity: Active on gram-positive bac-
teria, including mycobacteria. Less active on
gram-negative bacteria. Active on Yoshida sar-
coma (rats), increasing survival time and produc-
ing some cures (1, 2).
References :
1. Okami, Y. et id. J. Antibiotics (Japan)
6A: 153-157, 1953.
2. Yamamoto, T. and Umezawa, H. Japanese
Patent 396, January 26, 1955.
Carzinocidin
Produced by: Streptomyces kitasawaensis (2).
This culture also produces actinomycin A (3).
Method of extraction: I. Formation of a pre-
cipitate by adjusting the broth-filtrate to pH 5.0
and adding 5 per cent by volume of a 20 per cent
ZnClo solution. Precipitate extracted with 1 per
cent Na2lIP04 solution. To this solution at pH
7.0, 80 per cent saturated ammonium sulfate is
addetl. A precipitate forms, which is dissolved in
water and purified by dialysis. Addition of acetone
forms a precipitate of crude brown-black carzino-
cidin powder (1). II. Broth-filtrate adsorbed on
carbon at pH 2.4, and eluted with water at pH
8.0. Eluate concentrated at pH 4.4 and freeze
dried. Purified by extracting an aqueous solution
with butanol at pH 6.5; extract evaporated in
vacuo (3).
Chemical and physical properties: Polypeptide
containing cj'stine, lysine, glycine, and glutamic
acid. No crystals form; no definite melting point.
Soluble in alkaline water. Slightly soluble in 20
per cent acetone (pH 8.0), 20 per cent methanol,
20 per cent ethanol, 20 per cent pyridine, and bu-
tanol-saturated alkaline water. Insoluble in acidic
water. No specific ultraviolet absorption. Positive
xanthoproteic and Pauly reactions. "Pseudoposi-
tive" Millon reaction. Negative biuret, Adam-
kiewicz, Liebermann, H2SO4 , ninhydrin, diphen-
ylamine, cysteine, nitroprusside, Folin, anthrone,
Sakaguchi, and FeCla reactions. Unstable to heat,
especially at alkaline pH. C = 37.2%; H = 6.1%;
N = 12.2%; S = 3.5%. Molecular weight >6000
(3).
Biological activity: Slight activity against C.
albicans, Torula utilis, and Sacch. cerevisiae. Ac-
tive in mice against Ehrlich carcinoma, mainly
against the subcutaneous solid tvmior form. Slight
activity against Yoshida sarcoma (1, 3).
Toxicity: LD50 (mice) 4.7 mg per kg intraven-
ously, 43.5 mg per kg intraperitoneally, 20 mg per
kg subcutaneously. Cystine and methionine do
not decrease the toxicity of this substance in mice
(1,3).
References:
1. Harada, Y. et al. J. Antibiotics (Japan)
9A: 6-15, 1956.
2. Harada, Y. and Tanaka, S. J. Antibiotics
(Japan) 9A: 113-117, 1956.
3. Harada, Y. et al. Japanese Patents 789, 790,
and 791, February 18, 1959; and 898, Feb-
ruary 21, 1959.
232
DESCRIPTIONS OF ANTIBIOTICS
Carziiiophilin A
Produced by: Streptomyces sahachiroi (1, 7).
Synonytn: Carziiiophilin (3).
Method of extraction: Extraction of culture-
filtrate with organic solvents such as butyl ace-
tate, benzene, or chloroform at pH 6.0 to 7.0.
Back-extraction in water at pH 9.0 to 10.0. Aque-
ous extract either (a) freeze dried; (1)) precipitated
by addition of (NH4)2S04 , and precipitate purified
by extraction into acetone or butanol; or (c)
chromatographed on alumina at pH 5 to 8 and
eluted with alkaline aqueous acetone, pH 7 to 10
(1, 7). Broth-filtrate stirred with diatomaceous
earth or similar adsorbent at pH 6.5, and eluted
with methanol at pH 7 to 10. Eluate concentrated
in vacuo at about pH 7.0. Concentrate extracted
with organic solvents at pH 5 to 8, then back-
extracted into water at pH 7 to 10 and freeze
dried (7).
Chemical and physical properties: Acidic sub-
stance. Colorless needles. Darkens above 205°C;
m.p. 217-222°C (decomposition). Soluble in ace-
tone, chloroform, ethjd and butyl acetate, ben-
zene, dioxane, and aqueous alkali. Slightly soluble
or insoluble in water, methanol, ethanol, ether,
carbon tetrachloride, and petroleum ether
kin = +57.8° (chloroform). Ultraviolet absorp-
tion spectrum maxima: 218, 250, and 283 m/i (2
per cent NaHCO,, or methanol) ; 230 m/x {£{7^1 940)
and 283 m^ (^!cm 460) (0.1 N NaOH); 219 and 252
mp (ether); 210 and 330 ni/x (acetone); 270 niyu
(CHCI3); 242 and 378 m^ (CS.). Infrared data
given in reference 2. Positive l^romine, xantho-
proteic, diphenylamine, 2,4-dinitrophenylhydra-
zine, and sodium nitroprusside tests. Gives the
following reactions: Bayers test (KMn04 in
NaOH, green); ninhydrin (yellow), and anthrone
(yellow). Negative Molisch, Sakaguchi, Fehling,
Benedict, FeClj , and Tollen reactions. Forms
salts with alkalies. Biological activity destroyed
by thiourea, cysteine, methionine, hydroquinone,
vitamin B12 , H2O2 , and ultraviolet light. Thermo-
stable in dry state; unstable in aqueous solutions.
C = 59.78%; H = 5.17%; N = 6.93%. Molecular
weight 800 to 1200. Alkaline decomposition pro-
ducts include two crystalline substances: I.
m. p. 78-79°C; colorless needles; CuHis-uOj . II.
m. p. 177-180°C; colorless granular crystals;
C12-13H12-13O3 (1, 3, 4, 7).
Biological activity: Active in vitro against gram-
positive bacteria {Sarcina lutea) and mycobac-
teria. Limited activity against gram-negative
bacteria, except members of the genus Brucella
and K. pneumoniae (1). Active on Nocardia aster-
aides at 10 Mg per ml but not on protozoa (7). Not
active on fungi (1). Pure substance has about 20
times more activity on Yoshida sarcoma than
crude material (8). Active on ascitic forms of
Ehrlich carcinoma, hepatoma 7974, sarcoma 180,
and Krebs 2 carcinoma (1, 6). Certain concentra-
tions stimulate root tip growth of Allium cepa.
A very slight reduction in the number of mitotic
figures was noted at the highest concentration
used (8). Kills HeLa cells at 0.031 ng per ml (5).
Toxicity: LD50 (mice) 150 fxg per kg intraven-
ously (4). Subcutaneous or intramuscular adminis-
tration causes induration, necrosis, and ulceration
at site of injection. Leukopenia and urobilin in
urine also noted (2).
Utilization: Some effects on neoplastic disease
(2,7).
References:
1. Hata, T. et al. J. Antibiotics (Japan) 7A:
107-112, 1954.
2. Shimada, N. et al. J. Antibiotics (Japan)
8A: 67-76, 1955.
3. Kamada, H. et al. J. Antibiotics (Japan)
8A: 187-188, 1955.
4. Kamada, H. et al. Chemotherapy 4: 8,
1956.
5. Umezawa, H. Ciiorn. microbiol. 2: 160-193,
1956.
6. Sugiura, K. and Creech, H. J. Ann. N. Y.
Acad. Sci. 63: 962-976, 1956.
7. British Patent 777,287, June 19, 1957.
8. Ammann, C. A. and Safferman, R. S. Anti-
biotics & Chemotherapy 8: 1, 1958.
Catenuliii
Produced by: Streptomyces sp.
Synonyms: Closely related to hydroxymycin and
paromomycin.
Method of extraction: Adsorption on carbon or
precipitation as the salt (I) of eriochrome violet.
A methanolic solution of I treated with triethyl-
amine sulfate gives crude catenulin sulfate. Can
be crystallized as the helianthate or the p{p'-
hydroxyphenylazobenzene) sulfonate salt from
hot water.
Chemical and physical properties: Sulfate: In-
soluble in methanol. End-absorption of ultraviolet
light. Infrared spectrum said to be ch;iracteristic
of a polypeptide, [afj' = -|-51.9° (c = 1 per cent
in water). C = 31.45%; H = 6.15%; N = 7.92%;
SO4 = 28.12%. All the nitrogen is basic. Prolonged
acid hydrolysis yields a product tentatively identi-
fied as neamine by paper chromatography. Differ-
entiated from neomycins A and B and the strepto-
mycins by paper chromatography. Stable in
aciueous solution from pH 1.5 to 10.0.
DESCRIPTIONS OF ANTIBIOTICS
233
Biological activity: Active against mycobacteria,
7v. pneuDioniae, and B. subtilis. Cross-resistance
with neomycin and viomycin, but ciuantitatively
different from neomycin (3).
Toxicity: LD50 (mice) 125 mg per kg intraven-
ously. Causes neurotoxicity in cats. Toxic to duck-
weed (Lemna minor) at 1 ppm (2). Causes wheat
root tip ehmgation in taj) water sohition at 0.1
ppm (4).
References:
1. Davisson, J. W. Antibiotics & Chemotlier-
apy 2: 460-462, 1952.
2. Nickell, L. G. and Finhiy, A. C. J. Agr.
FoodChem. 2:178-182,1954.
3. Szybalski, W. and Bryson, V. Am. Rev.
Tuberc. 69: 267-269, 1954.
4. Barton, L. X. and MacNab, J. Contrib.
Boyce Thompson Inst. 17: 419, 1954.
Celesticelin
Produced by: Strepto)iiyces caelesfis (3).
Synonym: Antibiotic D 52 (3).
Method of extraction: Extraction of the filtered
broth with methylene chloride at pH 7.8. Concen-
tration of the extract. Addition of petroleum ether
to the concentrate precipitates crude celesticelin
as a tan solid. Oxalic acid or salicylic acid added
to the methanolic solutions of celesticelin. The
oxalate or salicylate formed is purified l)y re-
crystallization. Reconversion of the salts to the
amorphous free base accomplished by extracting
their aqueous solutions at pH 7.5 with methylene
chloride. Also can be extracted from broth with
ethyl or amyl acetate, or butanol. Extract concen-
trated. Concentrate added to hexane to give
amorphous celesticelin. Can also be precipitated
or isolated from the organic extract of l)roth as
the salt of an acid. Purification or initial extrac-
tion can also be accomplished by adsorption on
alumina, silica gel, activated clays, or acetic acid-
treated charcoal. Elution with a polar organic
solvent in which celesticelin is soluble (3).
Chemical and physical properties: Amphoteric,
colorless, amorphous substance. pKa of basic
group, 7.7; pKa of acidic group, 9.8. Soluble in
acidic and strongly basic aqueous solutions. In
soluble in water between pH 7 and 10. Soluble in
polar solvents, l)ut insoluble in ether or light hy-
drocarbons. Stable between pH 5 and 7 for 60
days or longer at 24°C. [afo for the free base =
4-126.6° (c = 0.5 per cent in chloroform). Maximal
vdtraviolet light absorption in 0.01 A^ alcoholic
potassium hydroxide at 248 and 341 m/x; in 0.01 N
alcoholic sulfuric acid at 240 and 310 m/x. Positive
FeCl-i , Molisch, and Ekkert tests. Formation of
white precipitates with bromine water, Millon's
reagent, and mercuric chloride. No precipitation
with silver nitrate or lead acetate. Negative Bene-
dict, ninhydrin, and iodoform tests. C = 54.87%;
H = 6.75%; N = 5.30%; S = 6.02%. Empirical
formula: C-24H:i6N209S. Hydrochloride: White semi-
crystalline powder. [a]o = -|-96.7° (c = 0.5 per
cent in water). Oxalate: White needles; m.p. 149-
154°C. [af^ = -1-106.4° (c = 0.5 per cent in water).
Indefinite benzenesulfonyl chloride test. Negative
nitroprusside, FeClg (brown), and l)romine in
CCI4 tests. Positive NaN.3-l2 test (for C— CH or
C^S). Salicylate: Crystals; m.p. 136-138°C.
[a]l^ = +90.2° (c = 0.5 per cent in water). All
salts have essentially the same ultraviolet spec-
trum as the base (3). Mild alkaline hydrolysis
yields salicylic acid and a basic product, Cn-
II32N2O7S, called desalicetin. Acid hydrolysis
yields, among other products, L-hygric acid and
a reducing amino sugar, CgHigHOe , and celestose.
Partial struct lu'e of celesticelin (2):
H S— CH.CH2— O— C- ,,
\ ^ \ /
c
/ \ OH
HOHC O
HOHC
C
CH-
CHOH-
CH,
CH, NH OCH3
I
C=0
L
CH:,-N/
Biohnjicul activity: Active in vitro against gram-
positive bacteria at the level of 0.19 to 12.5 /xg
per ml. Not active against gram-negative bac-
teria or fungi. Active in vivo against experimental
Streptococcus hemolyticus and Staph, aureus in-
fections. Inactive in vivo against M. tuberculosis
H37Rv and Newcastle and influenza viruses. Re-
ported to enhance the growth of animals and poul-
try. Active on plant disea.ses, including fire blight
of apple and pear trees, bacterial spot of tomatoes,
walnut blight, halo blight of beans, turf diseases,
mint rust, and cherry leaf spot. Active in vitro
on Xocardia asteroides (3).
Toxicity: LD50 (mice) 167 mg per kg (free base) ;
233 mg per kg (oxalate) intraperitoneally (3).
References:
1. DeBoer,C.et al. Antibiotics Ann. 831-841,
1954-1955.
234
DESCRIPTIONS OF ANTIBIOTICS
2. Hiiiman, J. W. and Hoeksema, H. Abstr.
129th Meeting Am. Chem. Soc. 17M-18AI,
1956.
3. British Patent 768,971, February 27, 1957.
Cellocidin
Produced by: Slreptomyces chibaensis (1).
Method of extraction: Broth-filtrate stirred with
activated carbon at pH 6.4 to 6.8. Elation with 80
per cent aqueous methanol. Concentration of elu-
ate in vacuo precipitates the antibiotic. Recrystal-
lization from hot aqueous methanol (1).
Chemical and physical properties: Acetylenedi-
carboxamide (2). Formula given in Chapter 6.
Melts at 216-218°C (decomposition). Slightly solu-
ble in water, methanol, ethanol, and acetone. In-
soluble in other organic solvents. Relatively stable
at acid pH and neutrality. Unstable at alkaline
pH, decomposing with evolution of ammonia
when boiled. Ultraviolet absorption spectrum
maximum at 299 m^ (£''1™ 290) in 0.1 N NaOH)
(1). Infrared spectrum given in reference 1. Cata-
lytic reduction product is succinamide. Major
acid hydrolysis product is C4H3O4CI (chlorofumaric
acid) (2).
Biological activity: Active on Micrococcus flavus
and E. coli at 10 ^g per ml, B. subtilis at 20 ^g per
ml, and Staph, aureus at 200 ng per ml. Active on
M. tuberculosis BCG at 3 /xg per ml. Active in vitro
on N.F. mouse sarcoma but not in vivo on Ehrlich
ascites carcinoma (1).
Toxicity: LD50 (mice) 11 mg per kg intrave-
nously (1).
References:
1. Suzuki, S. et al. J. Antibiotics (Japan)
llA: 81-83, 1958.
2. Suzuki, S. and Okuma, K. J. Antibiotics
(Japan) 11 A: 84-86, 1958.
Cellostalin
Produced by: Streptomyces cellostalicus (1).
Synonym: Similar to l)lasticidin S.
Method of extraction: Broth-filtrate passed
through an IRC-50 column (11+) and eluted with
80 per cent aqueous acetone (I), followed by 80
per cent acetone containing 0.2 N HCl (II). Frac-
tion I contains an unrelated unti-Sarcina anti-
biotic. Fraction II is adjusted to pH 6.0, concen-
trated in vacuo, and dried. Residue extracted with
anhydrous methanol, filtered, and dried in vacuo.
Final precipitation from acidic methanol with
acetone. Purified by chromatography on alumina
and Darco G-60 (1).
Chemical and physical properties: Basic sub-
stance. Very soluble in water; slightly soluble in
methanol; insoluble in ethanol, butaiiol, acetone,
ether, and other organic solvents. Forms a pic-
rate, reineckate, and phosphotungstate. Stable at
acid and neutral pH. Ultraviolet absorption spec-
trum maximum at 265 niyu (E\cm 115) (distilled
water). Infrared spectrum given in reference 1.
Positive ninhydrin, Sakaguchi, and Molisch tests.
Negative biuret, Millon, Fehling, maltol, FeCls ,
and Elson-Morgan tests. C = 22.10% (sic; proba-
bly 52.10%) ; H = 4.52%; N = 13.15%; O = 27.77%.
Sulfate: Colorless platelets. Reineckate: Crystal-
line substance; m.p. 156-166 °C (1).
Biological activity: Very slightly active (50 to
100 fig per ml) on certain gram-positive and gram-
negative bacteria, including Micrococcus citreus,
B. anthracis, Sarcina lutea, Salmonella, Ps. aerugi-
nosa, Pr. vulgaris, and Clostridium botulinum, but
not Staph, aureus, E. coli, or B. subtilis. Slightly
more active (6.3 to 100 fxg per ml) on yeasts and
fungi. Static action on Trichomonas vaginalis at
100 fig per ml (1). Active on Ehrlich ascites carci-
noma (1, 2).
Toxicity: LD50 (mice) about 15 mg per kg intra-
peritoneally (1).
References:
1. Hamada, S. Tohoku J. Ivxptl. Med. 67:
173-179, 1958.
2. Hamada, S. and Sato, S. Tohoku J. Ivxptl,
Med. 67: 181-186, 1958.
Cepliaioiiiyciii
Produced by: Streptomyces tanashiensis var.
cephalomyceticus .
Method of extraction: Adsorption on IRC-50 or
XE-64 resins (H"*"). Resins washed with water.
Elution with aciueous ammonia at pH 9.0. Pre-
cipitation of eluate at pH 3.6. Precipitate washed
with cold 2 per cent aqueous acetic acid, redis-
solved in water at pH 9.0, and centrifuged. The
supernatant adjusted to pH 5.1. The resulting
precipitate is purified further ]\v re])eating the
last series of operations.
Chemical and physical properties: Protein-like,
brownish, amorphous substance. Does not pass
through semipermeable membranes. Soluble in
water at pH values of less than 1 or more than 6.
Insoluble in organic solvents. Salted out of aciue-
ous solutions at three-fourths saturation with
(NH4)2S04 . It is precipitated by alum, picric
acid, and trichloroacetic acid. Positive Sakaguchi,
biuret, ninhydrin, diazo, and Folin tests. Negative
Fehling, Molisch, and xanthoproteic reactions. No
decoloration of bromine and permanganate solu-
tions. C = 55.39%; H = 6.66%; N = 9,93%. S, P,
and halogens not detected. Acid hydrolysates
DESCRIPTIONS OF ANTIBIOTICS
235
contain aspartic acid, glutamic acid, glycine,
threonine, alanine, tyrosine, valine, leucine, phen-
3'lalanine, arginine, and three unidentified nin-
hydrin-positive spots. Electrophoresis reveals the
])resence of three components. Strong end-absorp-
tion in ultraviolet light, with a shoulder at 255 to
260 niju. Infrared absorption curve given in refer-
ence 1.
Biological urtivity: Active against D. pneu-
moniae, Shigella dysenteriae, and Sacch. sake at
the level of 10 ng per ml. Not active on other bac-
teria and fungi tested. Active against strain Naka-
yama of Japanese B encephalitis virus in mice.
Toxicity: LD50 (mice) 31 mg per kg intrave-
nously, 55 mg per kg intraperitoneally, 161 mg per
kg subcutaneously, and more than 1000 mg per
kg orally.
Reference: 1. Matsumae, A. J. Antibiotics
(Japan) 13A: 143-154, 1960.
CerevioccicUii
Produced by: Streploniyces sp., closely related to
S. cacaoi.
Method of extraction: Absorbed on charcoal at
pH 4.3 and eluted with 80 per cent acjueous acetone,
pH 7.4. Eluate adjusted to pH 6.4 with HCl,
concentrated, and extracted with ethyl acetate.
The acetate is evaporated to dr\-ness and the resi-
due taken up in a small amoxuit of chloroform.
Filtered chloroform solution chromatographed
on alimiina, which is eluted first with chloroform
and then with methanol, the latter removing
another antibiotic present. Chloroform solutions
combined with ligroin, chromatographed on alu-
mina, and eluted with methanol. Methanol eluate
combined with the chloroform solution that has
been passed through the column and evaporated.
The antibiotic separates and is recrystallized from
hot ethyl alcohol.
Chemical and physical properties: Colorless
needles; m.p. 249-250°C (decomposition). Soluble
in methanol. Insoluble in ethanol, acetone, ethyl
acetate, ether, chloroform, and benzene. Sparingly
soluble in water. No characteristic ultraviolet
absorption spectrum. pK = 4.5 (50 jjcr cent meth-
anol). C22H39N5O4 . Calculated: C = 58.12%;
H = 8.74%; N = 16.0%,; Found: C = 57.76%;
H = 8.79%; N = 16.01%. Positive Janovsky nitro-
group reaction; negative Tollen, biuret, Fehling,
ninhydrin, Sakaguchi, maltol, and glucosamine
tests.
Biological activity: Active on certain yeasts. Not
active on representative bacteria, M. tuberculosis
607, or ('. albicans in concentrations of 10 ng per
ml.
Toxicity: Mice are killed l)y an intravenous in-
jection of 150 mg.
Reference: 1. Yamashita, S. et al. J. Antil)iotics
(Japan) 8A: 42-43, 1955.
Chartreusin
Produced by: Streptomyces chartreusis (1, 7),
Streptomyces sp. (1,8), Streptomyces sp. resembling
S. chartreusis (9), Streptomyces sp. resembling S.
viridis or S. viridochromogenes (3, 4, 6), and Strep-
tomyces I'iridochromogenes (2, 5).
Synonyms: Antibiotic X 465A (9, 10); antibiotic
747 (3); antibiotic 6A36 (2); antibiotic C 72 (8);
antibiotic 1293 (5).
Method of extraction: I. Mycelium extracted
with 80 per cent acetone; broth-filtrate extracted
with chloroform. Combined extracts concentrated
to dryness in vacuo. Crystallized from acetone.
Recrystallized from acetone-water. Sodivmi salt
crystallized from water (1). II. Mycelium ex-
tracted with aqueous butanol; broth-filtrate ex-
tracted with butanol-butyl acetate (1:1).
Extracted into water at pH 9.0 to 9.2, then back-
extracted into chloroform at pH 3.0 to 3.5. Con-
centrated extract purified by (a) removal of sol-
vent and recrystallization from benzene -ligroin;
or (b) removal of solvent, then chloroform solu-
tion of residue chromatographed on anhydrous
calcium hydrophosphate and eluted with acetone-
chloroform (1:3). Active fractions crystallized
from aqueous acetone or acetone-ligroin (2). III.
Broth-filtrate extracted with chloroform. Crude
residue from acetone crystallized from acetone or
aciueous acetone (6). IV. Broth and mj^celium
extracted with methylene chloride. Extract con-
centrated in vacuo, filtered, then reconcentrated.
Addition of ethanol to boiling solution precipi-
tates the antibiotic. Recrystallized from methyl-
ene chloride-ethanol (9).
Chemical and physical properties: Weakly acidic,
glucosidic substance. Anhydrous chartreusin: Thin
greenish yellow plates (3, fi, 9) or yellow prisms
(2); m.p. 184-187°C (2, 3, 6, 9). Comparison of
products from references 1, 3, 6, and 9 shows no
depression of mixed melting points. Other charac-
teristics are identical (9, 10). Soluble in chloro-
form, less so in acetone, slowly soluble in NaHCOg
solution. More soluble in dilute acid than water.
Soluble with inactivation in NaOH and Na2C03
solutions (3, 6). Ultraviolet absorption spectrum
maxima at 236, 266, 334, 380, 401, and 424 m^i (95
per cent ethanol) (6, 9). Slight differences in ultra-
violet spectrum were noted (1, 2). Infrared spec-
trum given in references 1 and 2. [q]„ = +127.5° ±
10° (c = 0.3 per cent in p\ii(line) or —36.2° ± 4°
236
DESCRIPTIOXS OF ANTIBIOTICS
(c = 0.3 per cent in glacial HAc). Gives yellow-
green color in alcoholic FeCls (6) and dark green
in FeCla and dimethjdformamide. Chartrexisin di-
hydrate: Yellow rhombic plates, m.p. 234-235°C
(crystallized from acetonitrile) (9); or greenish
yellow crystals, m.p. 180°C. Fluoresces in ultra-
violet light. Stable for several hours at pH 2 to 10
at 22°C (1). Pentabenzoate: m.p. 204-206°C (formed
with benzoyl chloride in i)yridine) (6). Penta-
acetyl derivative: m.p. 270°C. Alkali-insoluble (6).
Chartreusin decomposes both by heating to 250°C
and by acid hydrolysis to give a weakly acidic
aglycone, C19H10-12O6 . Acid hydrolysis products
also include D-fuco.se and D-digitalose. Aglycone:
Yellow needles, subliming at 260°C (crystallized
from toluene or pyridine) or m.p. 310-311 °C (6,
9). Soluble in aqueous alkali, but not in common
organic solvents. Negative flavonol test (9). Char-
treusin is believed to be a derivative of 2-phenyl-
naphthalene or 2,3-benzofluorene, containing a
lactone group, two phenolic hydroxyls, and two
o.xygens of undetermined nature. There is an ad-
ditional methyl or methylene group present, and
one of the phenolic groups is glycosidically bound
to a disaccharide chain composed of I)-fucose and
l)-digitalose (10). C = 59.49, 59.09, 59.89, or
59.71%; H = 5.43, 5.30, 5.19, or 5.26% (1, 2, 6, 9).
C32H34-36O14 (6, 9, 10) or CisHisOs (1, 2, 4).
Biological activity: Active in vitro on gram-posi-
tive bacteria and mycobacteria. Active on certain
gram-negative bacteria (Neisseria andHeniophilus).
Active on Xocardia asteroides, various Strepto-
niyces spp., and Actinomyces bovis. Active on vari-
ous bacteriophages. Slightly active (10 to 40 /zg
per ml) on influenza A(FM-l), influenza B (Lee),
HVJ (hemagglutinating virus of Japan or Sendai
virus) in contact tests. Active in tissue culture on
influenza PR 8. Not active on fungi or Tricho-
monas (1, 3, 7-9, 11). Not active in vivo on Strep-
tococcus hemolyticus, pneumococcus type I, Sal.
schottmuelleri, M. tuberculosis, C . albicans, Histo-
plasma capsulatuni , Trypanosoma equiperdum ,
Endamoeba histolytica, Syphacia obvelata, influ-
enza A or SK viruses, or sarcoma 180 (9).
Toxicity: Chartreusin: LDn (mice) 2500 mg per
kg subcutaneously (1). Xn chartreusin: LDso
(mice) 250 to 300 mg per kg intravenously (1-3).
Said to have a cumulative toxicity (1).
References :
1. Leach, B. Fj. et cd. J. Am. Chem. Soc. 75:
4011-4012, 1953.
2. Ishii, Y. et al. J. Antil)iotics (Japan) 8A:
96-99, 1955.
3. Ghoine, M. and Zavaglio, V. Giorn. micro-
V)iol. 1: 176-184, 1955.
4. Grein, A. et al. Giorn. microbiol. 1: 310-
315, 1955.
5. Shibata, M. et al. Ann. Kept. Takeda Re-
search Lab. 15: 45-48, 1956.
6. Arcamone, F. et al. Antibiotics & Chemo-
therapy 6: 283-285, 1956.
7. Calhoun, K. M. and Johnson, L. E. Anti-
biotics & Chemotherapy 6: 294-298, 1956.
8. Miyakawa, T. Virus 7: 394-399, 463, 1957.
9. Berger, J. et al. J. Am. Chem. Soc. 80:
1636-1638, 1958.
10. Sternbach, L. H. et al. J. Am. Chem. Soc.
80: 1639-1647, 1958.
11. Anzai, O. Virus 8: 174-181, 1958.
Chloramphenicol
Produced by: Streptomyces venezuelae (5, 26).
Also produced by S. phaeochromogenes var. chloro-
myceticus (0, 10), »S. omiyaensis (10), Streptomyces
sp. (22, 71), and by chemical synthesis (16).
Synonyms: Chloromycetin; sintomicetin; levo-
mycetin; synthomycin; antibiotic 8-44.
Method of extraction: lA. Acidified liroth-fil-
trate extracted with ethyl acetate, amyl acetate,
cyclohexanone, butanol, or methyl isobutyl ke-
tone. Extract solvent distilled off in vacuo. Resi-
due e.xtracted into diethyl ether or nitromethane.
Chromatographed in aluminum oxide. Solvent in
active fractions evaporated off; residue taken up
in water and washed with petroleum ether. Con-
centration of aciueous solution gives crystals.
Recrystallized from methylene dichloride, ethyl-
ene dichloride, and diethyl ether petroleimi ether
(1, 8, 17). IB. Broth-filtrate extracted with ethyl
acetate at pH 8.5 to 9.0. Extracts concentrated,
kerosene added, and the whole washed with 0.01
A^ H0SO4 , 5 per cent NaHCOs , and distilled water.
Solvent dried, then distilled off in vacuo at 37°C.
Cooling yields crystals (8). II. Broth-filtrate
extracted with ether. Extract evaporated to dry-
ness. Residue washed with hot benzene, leaving
crystals. Recrystallized from benzene-methanol.
Purified by chromatography on alumina from 30
per cent methanol in CHCI3 . Eluate concentrated
to dryness. Residue crystallized from chloroform-5
per cent methanol (3). III. Adsorbed from broth-
filtrate at pH 5.6 on activated carbon. Eluted with
acidic acetone. Eluate adjusted to pH 4.0 to 5.0
and solvent distilled off in vacuo. Taken up in
ether. Ether dried, concentrated, decolorized,
and evaporated to dryness. Residue taken up in
hot water. Antibiotic crystallizes out on drying
(17,22).
Chemical and physical properties: Neutral sub-
stance (1). Free base: Colorless needles or elon-
DESCRIPTIONS OF ANTIBIOTICS
237
gated plates; m.p. 149. 7-150. 7°C (corrected) (1)
or 144-147°C (3). Sublimes without deeompo.sition
in high vaciuun (15). Very soluble in methanol,
ethanol, l)utanol, jiropylene glycol (150.8 mg per
ml), acetone, ether, and amyl acetate (1, 3). Solu-
ble in cyclohexanone, methyl isolnityl ketone,
nitrobenzene, nitromethane, and ether. \'ery
sparingly soluble in water (2.5 mg per ml at 25°C),
acid, alkali, hot benzene, and chloroform (3). In-
soluble in petrolemn ether, cold benzene, cold 5
per cent sodium Incarbonate, and vegetable oils
(3, 8, 17, 18). Ultraviolet absorption spectrum
maximmn at 278 niju (£'1™ 298) (water or 0.1 A'
HCl) and 279.0 to 279.5 m/. (0.1 A' NaOH) (3).
[a]i^ = -25.5° (ethyl acetate) (1) or +18° (etha-
nol) (17). Positive KMnOj and ferrous hydroxide
tests (for oxidized nitrogen). Biuret test is not
characteristically positive; green crystals of a
copper salt deposit on standing (17). Negative
Sakaguchi, Pauly, FeCls , Molisch, Benedict
(boiling) (3, 8), thiosemicarbazone, boiling or cold
alcoholic silver nitrate, and periodic acid tests
(17). Stable at room temperature in ac[ueous solu-
tion at pH 2 to 9 for >24 hours; stable to boiling
for 5 hours (1). Crystallographic data given in
reference 40. C = 41.11%; H = 3.89%; N = 8.60%;
CI (nonionic) = 21.71% (1). C11H12O5N2CI, .
Molecular weight 323.1. Structural formula given
in Chapter 6. Acid or alkaline hydrolysis products
include dirhloroacelic acid and a basic substance,
C11H12N2O4 , having only ^loo the activity of
chloramphenicol against Shigella paradysenteriae
(17). Acetyl derivative of chloramphenicol : Crystal-
line; m.p. 141-142°C (17). A variety of derivatives
and analogues of chloramphenicol have been re-
ported (21 , 25, 29, 31 , 36, 41 , 57) . Many of these are
biologically active, but none is of greater in-
terest than the parent compovmd. Certain gen
eralizations concerning the relationship of biologi-
cal activity to structure in this group have been
made (42, 59).
Biological activity: In vitro: Active on gram-
positive and gram-negative bacteria, rickettsiae,
and psittacosis group (1, 3, 4, 19). Not active on
filamentous fungi or yeasts (4, 19). Active on Ac-
tinomyces israelii and Xocardia farcinica, but not
other pathogenic nocardias (23). Active on certain
pleuropneumonia-like organisms (38). Amebista-
tic to Endamoeba histolytica at 125 to 250 fj.g per
ml, depending on conditions of the test (28). Ac-
tive on Tetrahymena pyrijormis (54). At bacte-
riostatic concentrations, inhibits intracellular
growth of coliphage T-1 in E. coli (50). Not active
on gonococci phagocytized l)y HeLa cells (62),
but kills Brucella phagocytized by guinea pig
leucocytes (71). Inhil)its phagocytosis of Staph.
aureus by human polymorphonuclear neutrophils
at 15 Mg per ml (45). Prevents nitrate assimilation
by the fungus Scopulariopsis brevica)ilis grown in
static surface culture, but does not prevent as-
similation of other N-containing compounds or
nitrate when the culture is grown under submerged
conditions (70). Chloramphenicol inhibits synthe-
sis of protein and nucleic acids (67). d-Chloram-
phenicol is biologically inactive, but does not
interfere with the activity of chloramphenicol
(30). Chloramplienicol decomposition products
produced by the action of certain bacteria have
either a growth-stimulating effect on bacteria, or
interfere with the growth-inhibitory action of the
antibiotic (.3i)). In vivo: Active (in mice) on K.
pneumoniae (type A), Shigella paradysenteriae, D.
pneumoniae (type I), Streptococcus hemolyticus,
S. viridans, Borrelia novyi, Pasteurella multocida,
P. pestis, Sal. gallinarum, Malleomyces pseudo-
mallei, Clostridium tetani, and C. septicum. (4, 12,
14, 27, 47, 66). Slight activity on Treponema palli-
dum (rabbits) (19). Active at high doses on Enda-
moeba histolytica in rats; su])pressive but no cura-
tive effect in dogs (28). Active on Plasniodium
berghei (mice), P. gallinaceum (chicks), and P.
cathemerium (canary) (32). Interferes with the
killer action of Paramecium aurelta (37). Antitoxin
activity in mice against Sal. bareilly endotoxins
(58). Active on Rickettsia prowazekii (chick em-
bryo), R. orientalis (chick embryo and mice), R.
(ikari. R. maoscri, R. rickctt-^ii. li . tsutsugamushi ,
R. burncti, R. conorii. and the rickettsia that
causes North Queensland tick tyi)hus (1, 2, 13,
33). Active on jisittacosis, lymphogranuloma
venereiun, and mouse ]>neumonitis virus (2, 9,
52). Inactive on St. Louis and Japanese encepha-
litis viruses, variola, influenza A (PR 8), A-1, and
B, fixed rabies, distemper, Newcastle disease,
vaccinia, polio (Lansing and Y-SK), Theiler's
intestinal, miunps, chick bronchitis, and laryngo-
tracheitis viruses (2, 4, 19). Active on stone-fruit
virus in artificially' inoculated cucumliers, and on
toliacco mosaic virus in tomato seedlings when
the chloramphenicol is introduced by vacuum (51).
Inhibits the virus tumor of Rumex acetosa L (11).
Active on crown-gall tissue of tomato (53). Pro-
tects wheat seedlings from Xanthonionas translu-
cens infections when they are grown in a solution
containing the antibiotic (60). Active on sarcoma
180 in mice when tumor cell suspension is mixed
with the antibiotic before inoculation and a pellet
of the drug is implanted sidicutaneously 1 day
after inoculation with such tvmior cells (64). Pro-
longs motility and life-span of hvunan spermatozoa
238
DESCRIPTIONS OF ANTIBIOTICS
(63). When administered to the silkworm (Bonibiix
mori L), growth increases l)ut silk production
decreases (46). When fed to weanling pigs, weight
and feed efficiency increase (48). Prevents con-
tamination in grain fermentation (43). Preserves
beef (44) and fish (20).
Toxicity: LDso (mice) 150 mg per kg (1), 195.4
mg per kg (18), or 245 mg per kg (4) intravenously.
LDso (mice) 1320 mg per kg intraperitoneally,
and 2640 mg per kg orally (18). LDso (rats) 175.5
mg per kg intravenously (in 60 per cent propylene
glycol), or 279.4 mg per kg, same route (in 50 per
cent acetamide). LD.50 (rabbits) 117.0 mg per kg
intravenously (in 100 per cent propylene glycol)
(18). LDso (10-day-old chick embryo, allantoic
route) 4.8 mg per egg (68). Repeated parenteral
administration results in anemia in dogs (4), but
this was later reported to be indirectly a result of
malnutrition secondary to the anorexia produced
by the drug (56). Anemia, vmrelated to malnutri-
tion, was reported in ducks receiving chloram-
phenicol (55). Blood dyscrasias occur rarely in
certain human beings (35, 69), but no such re-
actions were noted in 2142 patients who received
massive doses of B complex and ascorbic acid
concurrently with the antibiotic (61). Plants and
plant cells: Transiently toxic to Alliian cepa root
cells at 1000 ppm (34). Causes chlorosis and re-
duction of dry weight in wheat seedlings grown
in 25 Mg per ml (60). Nontoxic at 125 ^g per ml to
normal tomato ph\nt tissue, but inhibits crown-
gall tissue of the same plant at this level (53).
Animal and tumor cells: Highest concentration
permitting epithelial cell migration in tissue cul-
ture is 4.0 mg per ml (72). Least injurious doses
to spleen of chick embryo and human skin cells
in tissue culture are 165 to 300 and 135 to 275 Mg
per ml, respectively (49). Minimal dose causing
degeneration of HeLa cells is 500 Mg per ml (65).
Utilization: Active on a variety of diseases
caused by gram-positive and gram-negative bac-
teria, spirochetes, rickettsiae, and certain viruses
(69).
References:
1. Ehrlich, J. et al. Science lOft: 417, 1947.
2. Smadel, J. E. and Jackson, E. B. Science
106: 418, 1947.
3. Gottlieb, I), et al. J. Bacteriol.
417, 1948.
4. Smith, R. M. et al. J. Bacteriol.
448, 1948.
5. Ehrlich, J. et al. J. Bacteriol. 56: 467-
477, 1948.
6. Umezawa, H. et al. Japan. Med. J. 1:
358-363, 1948.
409-
425-
7. Okami, V. Japan. Med. J. 1: 499-503.
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8. Bartz, Q. R. J. Biol. Chem. 172: 445-450.
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9. Smadel, J. E. and Jackson, E. B. Proc.
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11. Nickell, L. (i. Thesis, Yale Univ., 1949.
12. Gauld, R. L. et al. J. Bacteriol. 57: 349-
352, 1949.
13. Smadel, J. E. e/ a/. J. Immunol. 62:49-65,
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14. Thompson, P. E. and Dunn, M. C. Fed-
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15. Rebstock, M. C. et al. J. Am. Chem. Soc.
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71: 2463-2468, 1949.
17. Bartz, Q. R. U. S. Patent 2,483,871, Octo-
ber 4, 1949.
18. Gruhzit, O. M. et al. J. Clin. Invest. 28:
943-952, 1949.
19. McLean, I. W., Jr. et al. J. Clin. Invest.
28: 953-963, 1949.
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516, 1950.
23. Liftman, M. L. et al. Am. J. Clin. Pathol.
20: 1076-1078, 1950.
24. Fasal, P. J. Am. Med. Assoc. 144: 759,
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DESCRIPTIOXS OF ANTIBIOTICS
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35. Lewis, C. N. et al. Antibiotics & Chemo-
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41. Phillips, A. P. J. Am. Chem. Soc. 74:
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42. Collins, R. J. et al. J. Pharm. and Pharma-
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43. Day, W. H. et al. Abstr. r24th Meeting
Am. Chem. Soc. 23A, 1953.
44. Goldberg, H. S. et al. Food Technol. 7:
165-166, 1953.
45. Hemmer, M. L. et al. Antil)iotics & Chem-
otherapy 3: m^ni, 1953.
46. Murthy, M. R. V. and Sreenivasaya, Al.
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47. Kiser, J. S. and deMello, G. C. Proc. 58th
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530-536, 1954.
51. Kirkpatrick, H. C. and Lindner, R.
Phytopathology 44: 529-533, 1954.
52. Loosli, C. G. et al. Antibiotics Ann. 474-
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53. Klemmer, H. W. et al. Phytopathology
45: 618-625, 1955.
54. Gross, J. A. Biochim. et Biophys. Acta
18: 452-453, 1955.
55. Rigdon, R. H. et al. Antil)iotics & Chem-
otherapy 5: 38-44, 1955.
56. Reutner, T. F. et al. Antil)iotics & Chem-
otherapy 5:679-711,1955.
57. Rebstock, M. C. et al. J. Am. Chem. Soc.
77: 24-26, 1955.
58. Brunner, L. Zentr. Bakteriol. Parasitenk.,
Orig. 163: 13-30, 1955.
59. Hahn. F. E. et al. Antibiotics & Chemo-
therapy 6:531-543,1956.
60. Hagborg, W. A. F. Can. J. Microbiol. 2:
80-86, 1956.
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C.
61. Woolington, S. S. et al. Antibiotics Ann.
365-375, 1956-1957.
62. Thayer, J. U. et al. Antiliiotics Ann. 513-
517, 1956-1957.
63. Joel, C. A. and Kornhauser, S. Fertility
and Sterility 7: 430-439, 1956.
64. Bernfeld, P. and Inglis, N. R. Proc. Am.
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65. Nitta, K. Japan. J. Med. Sci. & Biol. 10:
277-286, 1957.
66. Hezebicks, M. M. and Nigg, C. Antil)iotics
& Chemotherapy 8: 543-560, 1958.
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with antimetabolic activity. Ciba Foun-
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68. Gentry, R. P. Avian Diseases 2: 76-82,
1958.
69. Woodward, T. 1'^. and Wisseman, C. L.
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168-173, 1959.
Clilorlelracj cliiie
Produced by: Streptuinyces atireofaciens (15)
and S. sayaniaensis (105).
SyiK.itiyin.'^: Aureomycin, biomycin, duomycin,
fiamycin, s,yntomycin, aureomykoin. (See also
tetracyclines.)
Method of e.rtrartion: lA. Whole broth adjusted
to pH 1.4, ammonivmi oxalate and Arquad 16
(50 per cent solution in isopropanol) added, pH
adjusted to 8.5, and the whole extracted with
methyl isobutyl ketone. Water added to extract,
and pH adjusted to 0.5. Crystals form on pro-
longed stirring. Purified by treatment with so-
dium hydrosulfide at pH 1.8 for 10 minutes, then
at pH 0.5 for 20 hours (137). Crystallized from hot
water on cooling and addition of HCl. Conversion
from hydrochloride to base: Substance slurried in
dimethylformamide. Addition of NaHCOj gives
precipitate. Toluene solution of precipitate azeo-
t Topically distilled. Cooling gives precipitate.
Recrystallized from benzene (88). IB. Whole
broth adjusted to pH 2.9 and filtered. Arquad C
(a commercial mixture of alkyl and alkenyltri-
methylammonium chlorides, principally dodecyl-
trimethylammonium chloride) and calcium chlo-
ride are added to filtrate, and pH adjusted to 9.0
to precipitate a chlortetracycline-organic base-
240
DESCRIPTIONS OF ANTIBIOTICS
bivalent metallic ion complex. Precipitate slurried
in water and a methanolic solution of the precipi-
tate adjusted to pH 2.5 to precipitate impvirities.
Solution adjusted to pH 7.0 to precipitate the
antibiotic (131). II. Adsorbed from broth-filtrate
on Florisil or charcoal columns. Developed with
acidic alcohol or acetone at pH <5.0 under ultra-
violet light. First band (blue) discarded; yellow
band which follows is active. Eluate concentrated
in vacuo. Concentrate taken uj) in butanol, re-
concentrated, and precipitated from concentrate
with absolute ether (15). III. Earth metals pres-
ent in, or added to the whole broth at pH 7.0 to
8.5 precipitate chlortetracycline as a salt . All
solids filtered off, and wet cake extracted with
n-l)utanol, isopropanol, or sec -butanol at pH 1.2
to 1.4 (adjusted with sulfuric or hydrochloric
acids). Extracts containing the sulfate shaken
with sodium chloride solution to salt out chlor-
tetracycline. (This has the effect of increasing the
distribution coefficient of chlortetracycline where
K = C solvent /C aqueous.) Solvent concentrated
under reduced pressure to incipient precipitation,
chilled, and pH adjusted to 0.8 to precipitate the
antibiotic. Extracts containing the hydrochloride
are concentrated under reduced pressure at 45-
55° C, added to/3-ethoxyethanol-ethanol-HCl, and
cooled (60-62, 121). Three purification procedures
are used: (a) Crude chlortetracycline dissolved in
such basic compounds as triethylamine, ammonia,
ethanolamine, or morpholine in a lower alcohol
(such as ethanol) or a lower alkoxy-lower alkanol
(2-ethoxyethanol or ethylene chlorohydrin), filter-
ing off insoluble impurities and acidifying (85).
(b) Aciueous solution of crude chlortetracycline
treated at pH 3.0 with an anionic sulfuric acid
derivative such as di-(2-ethylhexyl) sidfosuccinate
("Aerosol OT"). The salts thus formed are ex-
tracted into methyl isobutyl ketone, ethylene
dichloride, or n-propyl acetate, decolorized, then
concentrated under reduced pressure at <55°C
and acidified (86). (c) Heavy metal chelating
agents are used to sequester impurities before
I)recipitating the antibiotic as a salt. Such agents
include various aminopolycarboxylic acids, svich
as (ethvlenedinitrilo)tetraacetic acid and others
(132). ■
Chemical and phi/sical prupertiea: Ami)hoteric
(88). Free base: Yellow, acicidar to bladed crystals
(15, 88); m.p. 168-109°C (decomposition) (12).
Soluble in water to 0.5 to 0.6 mg per ml at 25°C.
Very soluble in aqueous solutions above pH 8.5,
dioxane, pyridine, Cellosolves, and carbitol (12,
15). Soluble in methanol, ethanol, butanol, ace-
tone, ethyl acetate, and benzene. Insoluble in
ether and petroleum ether. Ultraviolet absorption
spectrum maxima at 230, 262.5, and 367.5 vajj. (0.1
N HCl) ; at 255, 285, and 345 m^ (0.1 N NaOH)
(12) and at 230, 275, and 367.5 m,x (water) (35).
Infrared spectrum given in reference 15. [a]'^ =
— 275.0° (methanol) (12). Treatment with alco-
holic FeCls gives a green-brown color with re-
flected light and a reddish color with transmitted
light (12). Fluoresces intensely yellow in neutral
or slightly alkaline solution, changing to blue in
marked alkaline solution or after heating. No
fluorescence in acid solution (22). Alkaline fusion
products include 5-chlorosalicylic acid, dimethyl-
amine, and ammonia. Other degradation products
given in reference 40. Reductive dehalogenation
with 10 per cent palladium on charcoal and 1 mole
of triethylamine yields tetracycline (68). Acid
degradation products include biologically active
anhydrochlortetracycline (water is removed from
ring C) (104). Heating produces biologically in-
active, nontoxic isochlortetracycline (127, 128).
Chlortetracycline is 7-chloro-4-dimethylamino-
l,4,4a,5,5a,6,ll,12a-octahydro-3,6, -10,12,12a,
pentahydroxy -6 - methyl -1,11- dioxo - 2 - naphtha -
cenecarboxamide. C22H23N2O8CI: C = 55.10%;
H = 4.90%; N = 5.72%,; CI = 7.27%. Structural
formula (39, 88) given in Chapter 6. Forms com-
ple.xes with metal halides (41), and salts with
metals and acids. Hydrochloride: Clear vitreous
lemon-yellow tabular or orthorhombic crystals
(12, 15). Decomposes without melting at >210°C
(12, 20), or darkens at 208°C; m.p. 234-236°C (de-
composition) (88, 121). Soluble in water to 14 mg
per ml at 25°C (12). Soluble in methanol. Slightly
soluble in ethanol. Soluble in acetone to 0.13 mg
per ml at 25°C. Ultraviolet absorption spectrum
maxima at 229, 251, 265, and 370 m^i (pH 4.3, 0.1
M KH.2PO4 buffer); at 223, 240, 248, and 276 m^
(pH 8.9, 0.1 M K2HPO4 buffer) (15) and at 224 nip
(e = 29,480) 254 m/x (« = 14,635), 287 m^ (« =
14,378) and 346 m^ (e = 7,008) (in 0.25 N NaOH)
(106). [a]f = -235° (c = 1 per cent in water) (88).
Crystallographic data given in references 12, 15,
and 16. pKa' = 3.4, 7.4, and 9.2 (39). Most stable
at about pH 2.0. Stable at pH 1 to 10 at low tem-
peratures for 18 hours, but loses 50 per cent of
activity at 37°C overnight (3). In distilled water,
65 per cent of initial activity remains after heat-
ing at 100°C for 15 minutes. In pH 7.0 phosphate
buffer, 0.062 M, under the same conditions, only
0.44 per cent of the activity remains (107). Meth-
anolate: Yellow plates; m.p. 172-174°C (decom-
position). Very stable to drying (88). Ca salt:
Amorphous. Decomposes over a range of 200-
300°C with gradual darkening at lower tempera-
ture (121).
Biological activity: Bacteriostatic. Active on
DESCRIPTIONS OF ANTIBIOTICS
241
gram-positive and gram-negative t)acteria, certain
protozoa, rickettsiae, and the psittacosis group.
Not active on fungi. In vitro: Active on strepto-
cocci (0.3 to 1.25 ^lg per ml), diplococci (0.1 to
0.3 Mg per ml), staphylococci {<0.6 jug per ml;,
E. coli, Aerobacler aerogenes (<5.0 ^g per ml),
K. pneumoniae (1.0 to 5.0 ng per ml), Hemophihis
influenzae (2.0 ng per ml), Brucella suis and B.
abortus (<0.75/igpe/'ml), and Actinomyces israelii.
Not active at 20 /ig per ml on Pseudomonas or
Proteus (11). Active on pleuropneumonia-like
organisms (PPLO) (0.23 to 1.25 fxg per ml) (54).
Inhibits bacteria-free Tetrahymena and two color-
less flagellates at >0.1 ^g per nil. At 0.85 /xg per
ml early growth is stimulated (30). Promotes
growth of protozoa living on Pohjtomella at 10 to
50 ^lg per ml (31), and at a narrow range of con-
centration (about 0.1 Mg pf" i"l) ill a bacteria-
free culture. At higher concentrations, the pro-
tozoa are inhibited (30). Interferes with the killer
action of Paramecium aurelia (53). Active on
Endamoeba histolytica and T richomonas vaginalis
(14). Stimulates growth of ('. albicans at >0.1
mg per ml (65). Inhibits CO2 fixation in the dark
by Scenedesmus obliquus, but increases sucro.se
accumulation and stimulates photosynthesis ui)
to si.x times the normal rate at 1.5 X lO^-* M and
above (64). Inactive on free coliphage T3, l)ut
prevents adsor|)tion and growth of the phage if
(he host cell is j)reincubated with subbacterio-
static concentrations of the antibiotic. Infected
cells are more susceptible to the antibiotic than
normal cells (66). Activity adversely affected in
presence of serum (2). Growth inhibition of E.
coli is reversed by glycine, inosine, lumichrome,
and riboflavin; in the case of the latter, competi-
tively (67). Resistance develops relatively slowly
(2). Anhydrochlortetracycline, although having
only a fraction of the antibacterial activity of
chlortetracycline, is much more active than the
parent compound on actinomycetes (104). In vivo:
Active in various animals on Streptococcus pyo-
genes, S. hemolyticus, Staph, aureus, D. pneu-
moniae, B. anthracis, Erysipelothrix rhusiopathiae,
Pastetirella multocida. Listeria monocytogenes, K.
pneumoniae, Sal. typhosa. E. coli, Pr. vulgaris,
and Ps. pseudomallei (2, 4, 5, 11, 93, 135). Cell-
free preparations of ('. albicans, prepared by
sonic vibration, are lethal to mice when given in
combination with chlortetracycline, but innocu-
ous alone (130). Active on Endamoeba histolytica
infections in rats, dogs, and rabbits (37, 118),
Plasmodium cathemerium in the canary, and P.
berghei in mice (38). Some activity on Toxoplasma
gondii infections in mice (77). When administered
in the diet, it increases the susceptibility of Aedes
aegypti to /-•. gallinaceuni infections, but decreases
it in Anopheles (58) . Active in rats on experimental
polyarthritis (PPLO strain L4) (13), and in chick
embryos on a PPLO strain causing arthritis in
the goat (120). Active in eggs, mice, and guinea
pigs on rickettsiae of Rocky Mountain spotted
fever, murine, scrub. North Queensland tick and
epidemic typhus. North African tick bite fever,
Boutonneuse fever, Q fever, and rickettsial pox
(8, 28, 103). Active in mice on infections caused
by the following viruses: feline pneumonitis (but
not in vitro) (18), a gray lung fever (21), mouse
hepatitis (32), vaccinia (also active in vitro) (33),
Rous sarcoma [in vitro), tested in chickens (47),
herpes simplex {in vitro) (111) and the lympho-
granuloma-psittacosis group (8). Not active in
mice on influenza B, canine distemper, ral)ies
street virus, Newcastle disease virus, Venezuelan
equine encephalomyelitis, poliomyelitis (MEF-1
strain) (8, 52), myxoma virus (chick embryo),
fibroma, myxomatosis viruses (rabl)its) (78), or
Bittner's milk virus (78). Moderate activity on
cat ascarids; no activity on hookworms or tape-
worms (79). Anthelmintic activity in mice (57)
and horses (83). Protects against hemorrhagic
shock in dogs (54). May stimulate Walker car-
cinosarcoma (rat) (87). Enhances growth of the
following transplanted tumors: rat carcinoma
175-0, Crocker rat carcinoma, mouse sarcoma 180,
and mouse adenocarcinoma E0771 at 1.6 to 3.2
mg per 100 gm of body weight when given for 4
to 10 days. At 8 mg per 100 gm for 18 days, com-
pletely inhibits some 1- to 3-day transplants of
Crocker rat carcinoma, but not 10-day transplants
(27). No effect on mammary tumors (C3H mice)
or Lucke kidney adenocarcinoma (frogs) (78). In
])lants, chlortetracycline controls, by seed treat-
ment, black rot of rutabaga (Xanthomonas cam-
pestris) (92). Some control of tomato crown -gall
(Agrobacteriuui tumefacieus) (23). Active on bac-
teria-free gall tissue (112). Stimulates growth of
radish plants (72) and lupine root (Lupinus) (87).
Increased growth rate has been reported in the
following when chlortetracycline was added to the
diet: mice (81); protein-deficient (98) or vitamin-
deficient (99) rats; chicks (16); pullets (70); gos-
lings (46); geese (100); bobwhite quail (122); tur-
key poults (19); lambs (95); weanling (93) and
disease-free pigs (97); calves (69, 114); yearlings
(69); steers (126); horses (89); cats (91); dogs
(48); midernourished children (74); and young
men (90). .\lkali-degraded chlortetracycline has
no growth-promoting eft'ects in the chick (26).
Chlortetracycline is degraded to biologically in-
active isochlortetracycline in the rat intestinal
tract (133). Dietary chlortetracycline: (a) In-
242
DESCRIPTIOXS OF ANTIBIOTICS
creases: feed efficiency (46, 48, 95, %, 114, 124);
egg hatchability (70); lamb carcass grades (95);
utilization of calcium and phosphorus (chick) (42);
nitrogen retention (cockerels) (101), (calves)
(124); intestinal permeaV)ility to organic nitrogen
(rats) (75); serum carotenoid levels in chicks (45);
vitamin A deposition in vitamin A-deficient rats
(113) ; nicotinic acid levels in the liver (rats) (115) ;
intestinal synthesis of vitamins (rats) (108); blood
reducing sugar levels (calves) (102), (pigs) (80).
(b) Causes: toxic symptoms and death in guinea
pigs at levels causing growth stimulation in other
animals; believed caused by an increase in the
numbers of Listeria monocytogenes in the gut, and
subsequent disease symptoms (116). (c) Reduces:
intestinal weight in pigs (117) and chicks (123);
urinary nitrogen excretion in cockerels (101) and
dairy calves (125). (d) Has a sparing effect on:
vitamin B12 (chick) (16), (pigs) (73); nicotinic
acid and folic acid (chick) (25); thiamine (chick)
(44); pantothenic acid (pigs) (73); all vitamins
(rats) (108); tryptophan (chicks) (24); choline
(rats) (this activity was destroyed on heating)
(49) ; protein (rats) (98) ; manganese (chicks) (43).
Toxicity: LD50 (mice) 134 mg per kg (1) or 50
to 100 mg per kg (5, 107) intravenously, 3 to 4 gm
per kg subcutaneously (5). Mice tolerate 1500 mg
per kg orally, 200 mg per kg intraperitoneally (1).
LDoo (rats) 118 mg per kg intravenously (1), 13.55
gm per kg orally (119). LD.mi (chick embryo) 12.0
mg (134). Guinea pigs tolerate 300 mg per kg sub-
cutaneously, but are killed by 200 mg per kg in-
traperitoneally (1). Very toxic to guinea pigs
orally (116). Dogs are killed by a dose of 150 mg
per kg intravenously (5). Nonirritating at 1 per
cent to rabbit eyes (11). Fish and amphibians:
Toxic to guppies at 1 mg per ml in 10 to 30 min-
utes (59). At dilution of 1:10,000 to 1:25.000, in-
hibits tadpole metamorphosis (76). Insects: Toxic
to the granary weevil (Sitophilus granarius) and
the confused flour beetle (Tribolium confusum) at
0.3 to 0.5 gm ])er 14 gm of grain. At these levels
the lesser grain borer (Rhizopertha doniinica) is
stimulated reproductively (63). Kills the adults
of the rice weevil {Calandra oryzae) and the small
rice weevil (C sasakii) after exposure to 0.2 gm
in a jar for 2 days (84) . Plants: Toxic to the growth
of lentil and pea seedlings at dilutions of 5 X
10~\ and has a temporary blocking action on the
production of chlorophyll. Not toxic to rape
(Brassica napus) at 1 X 10^^ (55). Na-K chloro-
phyllin prevents characteristic chlorotic toxicity
symptoms in beans and cucumbers normally pro-
duced when the plants are sprayed with certain
concentrations of chlortetracycline. Cells: Least
injurious doses for spleen cells (from cliick em-
bryo) and human skin cells in tissue culture are
150 to 300 Mg per ml and 100 to 200 Mg per ml, re-
spectively (94). Cytotoxic to Allium cepa root
cells at 50 ppm (29). Nontoxic to normal plant
cells at 50 Mg per 'n'. although crown-gall cells
are inhibited at this level (112). Minimal dose
causing inhil)ition of HeLa cells is 125 Mg l>er ml
(129).
Utilization: Active against a variety of infec-
tions caused t)y gram-positive and gram-negative
bacteria, actinomycetes, rickettsiae, and psittaco-
sis group (6, 7, 9-11, 14, 17). It is widely used in
veterinary medicine. Used in feeds to augment
growth rate of poultry, calves, pigs, etc. Used in
preservation of foods: fish (109), poultry (82),
meat (110), milk (50), and spinach (133). Prevents
contamination in grain fermentation (71).
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68. Boothe, J. H. et al. J. Am. Chem. Soc.
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69. Perry, T. W. et al. Purdue I'niv. Agr.
Expt. St a. Mimeo A. H. 120, November
30, 1953.
Piatt, C. S. New Jersey Agr. Expt. Sta.
Bull. 769, 1953.
Day. W. H. et al. Abstr. 124th Meeting
Am. Chem. Soc. 23 A, 1953.
Coresi, R. and Girard, R. Bull. soc.
pharm. Bordeaux 92: 117, 1953.
73. Catron, D. V. et al. J. Animal Sci. 12:
51-61, 1953.
244
74.
75.
76.
DESCRIPTIONS OF ANTIBIOTICS
79,
80.
81.
82.
83.
84.
85.
86.
87.
88.
89.
90.
91.
92.
93.
94.
95.
96.
97.
98,
99
Scrimshaw, N. S. and (luznian, M. A,
INCAP Sci. Contrib. 1-29, Proc. Meet.
Natl. Vit. Found., New York, 1953.
Ferrando, R. e( al. Compt. rend 236:
1618-1620, 1953.
Sanfilippo, G. Boll. soc. ital. l)iol. .sper.
29: 1339-1342, 1953.
p]yles, D. E. and Coleman, N. Am. J.
Trop. Med. Hyg. 2: 64-69, 1953.
Ambrus, J. L. et al. Antibiotics & Chem-
otherapy 3: 16-22, 1953.
Brown, H. W. et al. Antibiotics tt Chem-
otherapy 3: 243-248, 1953.
Catron, D. V. e( al. Antibiotics & Chem-
otherapy 3: 571-577, 1953.
Mirone, L. Antil)iotics & Chemotherapy
3: 600-602, 1953,
Kersey, R. C. et al. Antibiotics Ann,
438,' 1953-1954.
Levine, N. D. Am. J. Vet. Researcli 14:
548-549, 1953.
Yasue, V. Rept. Ohara Inst. Agr. Biol.
42: 114-117, 1954.
Winterbottom, R. et al. U. S. Patent
2,671,806, March 9, 1954.
British Patent 717,281, Octol^er 27, 1954.
Costa, E. and Murtas, L. Arch. ital. sci.
farmacol. 3: 181-184, 1954.
Stephens, C. R. et al. J. Am. Chem. Soc.
76: 3568-3575, 1954.
Taylor, J. H. et al. Vet. Record 66:
744-748, 1954.
Haight, T. H. and Pierce, W. E. J. Lab.
Clin. Med. 44: 807-808, 1954.
Dickenson, C. D. and Scott, P. P. Brit.
J. Nutrition 8: 380-385, 1954,
Sutton, M. D. and Bell, W. Plant Disease
Rei)tr. 38: 547-552, 1954.
Kiser, J. S. and deMello, (i. C. Proc. 58th
Ann. Meeting U. S. Livestock Sanitary
Assoc. 81-97, 1954.
Pomerat, C. M. and Leake, C. D. Ann.
N. Y. Acad. Sci. 58: 1110-1124, 1954.
Hatfield, E. E. et al. J. Animal Sci. 13:
715-725, 1954.
Lasley, J. F. et al. Univ. Missouri Agr.
Expt. Sta. Bull. 543, 1954.
Hill, E. G. and Larson, X. L, Ann. Rept.
Hormel Inst. L^niv, Minn, 69-73, 1954-
1955.
Berry, M. E. antl Schuck, C. J. Nutrition
54: 271-284, 1954.
Schendel, H. E. and Johnson, B. C. J.
Nutrition 54: 461-468, 1954.
100. Amschler. J. W. et al. Bodenkultur 8:
43-49, 1954.
101. ^riiayer, R. H. and Heller, V. G. Poultry
Sci, 34: 97-102, 1955.
102. Voelker, H. H. et al. Antibiotics & Chem-
otherapy 5: 224-231, 1955.
103. Ormsbee, R. A. et al. J. Infectious Dis-
eases 96: 162-167, 1955.
104. Goodman, J. J. et al. J. Bacteriol, 69:
70-72, 1955,
105. Arishima, M. et al. J. Agr. Chem. Soc.
Japan 29: 810-817, 1955.
106. Sensi, P. et al. Farmaco (Pavia) 10:
337-345, 1955.
107. Rolland, G. et al. Farmaco (Pavia) 10:
346-355, 1955.
108. Baumann, C. A. 1st Intern. Conf. Uses
Antibiotics in Agr. 47-54, 1955.
109. Tarr, H. L. A. 1st Intern. Conf. Uses
Antibiotics in Agr. 199-209, 1955.
110. Deatherage, F. E. 1st Intern. Conf. Uses
Antibiotics in Agr. 211-222, 1955.
111. MacKneson, R. G. and Ormsby, H. L.
Am. J. Ophthalmol. 39: 689-691, 1955.
112. Klemmer, H. W. et al. Phytopathology
45: 618-625, 1955,
113. High, E. (;, Federation Proc. 14: 437.
1955,
114. Owen, F. G. et al. J. Dairy Sci. 38: 891-
900, 1955.
115. Halevy, S. et al. Brit. J. Nutrition 9:
57-62, 1955.
116. Roine, P. et al. Brit. J, Nutrition 9:
181-191, 1955.
117. Braude, R. et al. Brit. J. Nutrition 9:
363-368, 1955.
118. Thompson, P. E. et al. Antibiotics it
Chemotherapy 6: 337-350, 1956.
119. C'unningham, R. W. cpioted in Hines, L.
R. Antibiotics & Chemotherapy 6:
623-641, 1956.
120. Adler, H. E. et al. Cornell Vet. 46: 206-
216, 1956.
121. Starbird, E. E. and Pidacks, C. U. S.
Patent 2,763,681, September 18, 1956.
122. Mraz, F. R. f/ «/, Poultry Sci. 35:76-80,
1956.
123. Keeling, A, I), et al. Poidtry Sci, 35:
1150, 1956,
124. Price, J. D. et al. Poultry Sci, 35: 1165-
1166, 1956.
125. Hogue, D. E. et al. J. Animal Sci. 15:
788-793, 1956.
126. Beeson, W. M. et al. Purdue Univ. Agr.
Expt. Sta. Mimeo A. H. 116, 1956.
DESCRIPTIONS OF ANTIBIOTICS
245
127. Al)l)ey, A. et al. Antibiotics Ann. 831-
838, 195G-1957.
128. Shirk, R. J. et al. Antibiotics Ann. 843-
848, 1956-1957.
129. Nitta, K. Japan. J. Med. Sci. & Biol.
10: 277-286, 1957.
130. Roth, F. J., Jr. and Murphy, W. H., Jr.
Proc. Soc. Exptl. Biol. Med. 94: 530-
532, 1957.
131. British Patent 775,916, May 29, 1957.
132. British Patent 781,881, August 28, 1957.
133. Becker, R. F. et al. Antibiotics Ann.
229-235, 1957-1958.
134. Gentry, R. F. Avian Diseases 2: 70-82,
1958.
135. Hezebicks, M. M. and Xigg, C. Antibi-
otics & Chemotherapy 8: 543-560, 1958.
136. Ark, P. A. and Thompson, J. P. Plant
Disease Reptr. 42: 1203-1205, 1958.
137. Fox, S. M. et al. U. S. Patent 2,875,247,
February 24, 1959.
138. Kotchetkova, G. V. and Popoba, O. L.
Antibiotiki 1(4): 37-40, 1956.
CI
iroiiiiii
Frotluced by: Slieplumi/ces sp. reseml)ling S. anli-
bioticiis (1 ).
Method of e.vt faction: Broth-filtrate extracted
with liutanol or amyl alcohol at pH 6 to 7. Taken
up in methanol, and fractionally precipitated with
ether (2, 3).
Chemical and physical properties: Tetraene. Fine
white needles. Colors at 145-150°C, but does not
melt up to 220°C. Crude chromin soluble in meth-
anol, water-saturated butanol, chloroform, and
alkaline water. Crystalline chromin is not soluble
in most organic solvents or water. Soluble in
NaOH and acetic acid. Ultraviolet absorption
spectrum maxima at 281, 292.5, 305, and 320 m/x.
Infrared spectrum given in reference 3. Positive
Fehling test (on heating). Negative ninhydrin,
biuret, Molisch, Millon, FeCht , and Sakaguchi
tests. Questionably positive Tollen test. Most
stable at neutrality. C = 58.19^c; H = 7.81%;
N = 2.29%. No S or halogen (2, 3). Some informa-
tion on hydrolysis products given in reference 3.
Biological activity: Active on fungi and yeasts
at 0.16 to 2.5 Mg per ml. Not active on bacteria.
Activity reduced by human serum (2, 3).
Toxicity: LDjo (mice) 36 mg per kg intraperi-
toneally (3).
References:
1. Wakaki, S. et al. J. Antibiotics (Japan) 4:
357-362, 1951.
2. Wakaki, S. el al. J. Antibiotics (Japan) 5:
677-681, 1952.
3. Wakaki, S. ct al. J. Antibiotics (Japan)
()B: 247-250, 1953.
4. Katagiri, K. et al. Shionogi Kenkyusho
Nempo 7: 715-723, 1957.
Chroinoin veins
Produced by: Streptoinyces griseus.
Method of extraction: Separated and inirified l)y
chromatography and fractional precipitation.
Chemical and physical properties: Complex con-
taining five components, Ai to A5 . Previously re-
ported substances B and C were found to be
formed by transformati'on of A under various con-
ditions and with various agents. .43.' Bright yellow-
powder; m.p. 183°C (decomposition). Reputed to
have ultraviolet and infrared absorption spectra
differing from other antibiotics, [a]'," = —26° (c
= 1 per cent in ethanol).
Biological activity: As: Active on Staph, aureus
and B. subtilis at 0.10 and 0.05 jug per ml (2), re-
spectively and on other gram-positive bacteria.
Slightly active on mycobacteria. Active on vac-
cinia virus; slightly active on influenza virus (1).
Active in rats or mice on Yoshida sarcoma, Ehrlich
carcinoma, hepatoma AH 130, hepatoma MH 134,
sarcoma 180 (all ascites type), and leukemia SN 36.
Not active on nitromin-resistant AH 7974 (2).
Toxicity: A3: LD.50 (mice) 2.12 mg per kg intra-
peritoneally. Rats, hamsters, rabbits, cats, and
dogs die on administration of 0.25 to 1.0 mg per
kg, but tolerate 0.1 to 0.2 mg per kg. Mice tolerate
400 mg per kg orally (2). Toxic to chicken embryo
fibroblasts in vitro (1).
Utilization: Favorable effect in some human l)e-
ings with neoplastic disease (3).
References:
1. Aramaki, Y. et al. Repts. Takeda Research
Lab. 14: 60-91, 1955.
2. Tatsuoka, S. et al. Proc. Japan. Cancer
As.soc, 17th Meeting 23-24, 1958.
3. Okuniura, A. Proc. Japan. Cancer Assoc,
17th Meeting 25-26, 1958.
Clii
•y.sonivcin
Produced by: Streptomyces sp.
Synonym: Authors (1) state that the ultraviolet
spectnun shows similarities to aureolic acid.
Method of extraction: Extraction from filtrate
with organic solvents at acid or alkaline reaction.
Dried mycelium treated with Skellysolve C to
remove inactive pigments, then exhaustively ex-
tracted with l)()iliug ethvl acetate. Green-l^rown
246
DESCRIPTIONS OF ANTIBIOTICS
extract passed through Florisil cohimu.s to remove
most dark-colored impurities. While l)eing
shielded from bright daylight to prevent further
pigment formation, the columns are washed with
the same solvent to remove the active material.
Bright yellow eluate concentrated in vacuo, and
recrystallized by dissolving in hot pyridine and
adding ethanol or by dissolving in hot acetic acid
and adding water.
Chemical and physical properties: Slender green-
ish yellow needles or rods; m.p. 255-2(iO°C (decom-
position). Can be sublimed at 240°C without loss
of activity. Neutral substance, practically insol-
uble in water and petroleum ether; slightly soluble
in methanol, higher alcohols, and ethyl acetate;
and more soluble in pyridine, glacial acetic acid,
and dio.xane. Soluble in concentrated HCl and can
be recovered unchanged from it by adding water.
Dry crystals are photosensitive, and turn brown
on exposure to light. Stable from pH 3 to 7 and to
heating to 100°C. [a]^ = +16° (c = 1 per cent in
acetic acid). Ultraviolet maxima at 247 to 287 m^u
and a broad band between 390 to 400 m/x. Gives
inactive red solution in alkali. On hydrogenation
with platinum oxide as catalyst and glacial acetic
acid as solvent, 4 moles of hydrogen are taken up.
The colorless product, recrystallized from hot
ethanol, is devoid of biological activity and melts
at 208°C; ultraviolet maxima at 240 and 355 m/i.
Analysis of this hydrogenation product gave a
formula of C22H28O7 . Molecular weight 360. C =
65.46%; H = 6.96%; O = 27.47%,. From this anal-
ysis the tentative formula C22H20O7 is proposed
for chrysomycin, since the original compound
could not be analyzed satisfactorily.
Biological activity: Active against B. cereus
phage at 0.01 Mg per ml, and other phages, includ-
ing staphylophage, streptophage, enterococci
phage, and cholera phage at 0.2 mg per ml by the
paper-disc method. Phagocidal to five bacterial
viruses. Has antibacterial activity (active mainly
against gram-positive bacteria) and slight anti-
fungal activity.
Toxicity: Mice (20 gm) tolerate 2 mg in peanut
oil intraperitoneally; 5 mg by the same route pro-
duces transient paralysis and loss of appetite.
Reference: 1. Strelitz, F. et al. J. Bacteriol.
69: 280-283, 1955.
Ciniianiycin
Produced by: Streptoniyces cinnanwnieiis (1) f.
cinnamonieus (4).
Method of extraction: Broth-filtrate passed
through a column of IRC-50 (H+) and eluted with
0.1 N HCl. Neutralized to pH 6.0 with IR-4B.
Effluent concentrated in vacuo and freeze dried.
Solid dissolved in 80 per cent methanol (acpieous)
and chromatographed on alumina. Development
with aqueous methanol (1). Countercurrent dis-
tribution (n-butyl alcohol and a volatile ammo-
nium acetate buffer, 0.2 M, pH 5.4) indicates the
presence of two substances, one of which is unre-
lated to cinnamycin (2).
Chemical and physical properties: Cream-colored
powder. Basic polypeptide. Soluble in water, hy-
drated alcohols, and glacial acetic acid. Insolu-
ble in ether (1). Ultraviolet absorption spectrum
shows end-absorption at 230 m^t- Infrared spec-
trum given in reference 2. Acjueous solutions are
levorotatory. Stable at pH 2 to 9 for 30 minutes
at 92°C. Photo-stable. Not inactivated by pepsin
or trypsin (1). Dialyzes through cellophane. Iso-
electric point pH 5.0 (2). Contains N and S; no
halogen. Positive Sakaguchi and biuret tests.
Negative ninhydrin, FeCls , Molisch, maltol, Tol-
len, and reducing sugar tests. No sulfhydryl or
disulfide groups (1). Acidic and basic hydrolysis
products include aspartic acid, arginine, glutamic
acid, proline, phenylalanine, valine, mesolanthio-
nine, and /3-methyl lanthionine (C7H14N2O2S),
which has also been isolated from subtilin and
nisin (1, 2).
Biological activity: Active on gram-positive rods
and mycobacteria (5 to 55 fxg per ml) ; very active
on Clostridium botulinuni (0.085 Mg per ml); not
active on gram-positive cocci, gram-negative bac-
teria, or yeasts (1). Tested against phytopatho-
gens and compared with duramycin (4).
Toxicity: LDso (mice) 5 to 10 mg per kg intra-
peritoneally; 400 mg per kg or less is nontoxic
subcutaneously (3).
References:
1. Benedict, R. G. et al. Antibiotics & Chemo-
therapy 2: 591-594, 1952.
2. Dvonch, W. et al. Antibiotics & Chemo-
therapy 4: 1135-1142, 1954.
3. Ambrose, A. M. Antibiotics & Chemother
apy 4: 1242-1244, 1954.
4. Lindenfelser, L. A. et al . Antibiotics &
Chemotherapy 9: 690-695, 1959.
Cladomyciii
Produced by: Streptomyces lilacinus (2).
Method of extraction: Broth extracted with ethyl
acetate. Extract concentrated in vacuo at <40°C.
Precipitated from concentrate on addition of pe-
troleum ether. Taken up in ethyl acetate, washed
with HCl (pH 2.0) and NaOH (pH 10.0) solutions,
concentrated in vacuo at <40°C, and precipitated
as before. Purified by chromatography on a cellu-
DESCRIPTIONS OF ANTIBIOTICS
247
lose column. Eluted with nK'tluuiol (2, '.]). C!;tn
also he extracted from broth with ether or chloro-
form (1).
Chemical and physical properties: Dark red sub-
stance. Sohible in alcohol.s, acetic esters, acetone,
benzene, and chloroform. Insoluble in petroleum
ether and water. No specific ultraviolet absorption
spectrum. Contains no halogen (1-3).
Biulugical activity: Active at 0.0039 to 3.0 /ng per
ml against gram-positive and gram-negative bac-
teria, including m^ycobacteria, Pseudonionas, and
Proteus. Not active on fungi (1, 2).
Toxicity: LD50 (mice) 724.5 mg per kg intrave-
nously (1, 2).
References:
1. Nakazawa, K. ef al. J. Antibiotics (Japan)
9B: 81, 1956.
2. Nakazawa, K. et al. Ann. Rejjts. 4'aketla
Research Lab. 16: 111-115, 1957.
3. Japanese Patent 3241, April 30, 1959.
Coelicolorin
Produced by: Streptomyces coelicolor.
Method of extraction: Extraction of solid cul-
tures with acetone at acid pH. Precipitation of
active substance from acetone solution with water;
extraction with benzene. Further purification by
chromatography on alumina.
(^heuiical and physical properties: Indicator; red
up to pH 5, violet at pH 6 to 7, and t)lue higher
than pH 8. Soluble in water at alkaline reaction.
Very solul)le in acetone, ethyl acetate, and chloro-
form. Soluble in alcohol, methanol, benzene, and
ether. Insoluble in petroleum ether, m.p. 142-
14(5°C.
Biological activity: Active mainly against gram-
positive bacteria.
Toxicity: LDso (mice) about 500 mg per kg in-
traperitoneally.
Reference: 1. Hatsuta, V. J. Antibiotics (Ja-
pan) 2: 276-277, 1949.
Coerulonij ciii
Produced by: Streptomyces coerulescens.
Synonym: Similar to chartreusin.
Method of extraction: I. Precipitation from cul-
ture-filtrate at pH 2.5 to 3.0. Precipitate extracted
with acetone; extract concentrated. A brown
precipitate forms. Precipitate extracted with al-
cohol. Crystallization from alcohol yields crude
crystals contaminated with pigments; the pig-
ments are removed by chromatography of alco-
holic solution on aluminum oxide. The \'oll()w
acetonic eluate is concentrated and the antibiotic
crystallized from the alcohol. II. Extraction of
the culture mcdiuin with l)utanol, chloroform, or
ethyl acetate at neutrality, followed l)y further
purification as in previous method.
Chemical and physical properties: Crystals in
form of platelets. Darken and decompose at 181-
183°C. Molecular weight (Rast) 660. Moderately
soluble in chloroform, less soluble in acetone and
alcohol, and poorly soluble in water. At an alkaline
reaction, 20 mg will dissolve in 1 ml of water. No
N or S. Light-al)sorption maxima at 240, 270, 340,
380, 400, and 430 mfx.
Biological activity: Active against gram-positive
bacteria and phages (especially actinophages).
Active against infivienza virus.
Toxicity: In mice, 200 mg per kg are tolerated
subcutaneously, and 300 mg per kg orally.
Reference: 1. Brajnikova, M. G. ef al. Anti-
biotiki 2(6): 16-20, 1957.
CoUiiioinycin
Produced by: Streptomyces collinus. This organ-
ism also produces rubromycin (1 ) .
Method of extraction: See rubromycin.
Chemical anil physical properties: Amphoteric
substance. Orange prisms; m.p. 280-282°C. Mod-
erately soluble in chloroform, acetone, and diox-
ane; slightly soluble in ether and lower alcohols;
practically insoluble in i)etroleum ether, water,
and sodium bicarbonate. Na salt: Violet substance.
Red-violet in 2 A' NaOH. Ultraviolet absorption
maxima at 535 and 575 mn. Carmine-red in con-
centrated H2S()4 , with maxima at 480 and 525 mn.
A pyridine-methanol solution treated with tita-
nium trichloride turns olive-green, then red. A
yellow-red dioxane solution becomes pale yellow
on treatment with sodium hyposulfite. This color
reaction is reversed by standing in the air. Con-
tains no nitrogen. C = 59.41%; H = 4.01%; O =
34.83%,; O— CH:, = 16.2%,. Acetate: Yellow nee-
dles; m.p. 228-230°C. Benzoate: Lemon-yellow nee-
dles; m.p. 226°C. Prolonged heating in H2SO4-
containing acetone yields a decomposition
product: orange needles, m.p. 273-275°C, with
ultraviolet absorption maxima at 515 and 529 ni/x
(ether). Gives a red-violet color in NaHCO.-i (2).
Biological activity: Active on Staph, aureus (2).
References:
1. Lindenbein, W. Arch. Alikrobiol. 1": 361-
383, 1952.
2. Brockmanii, H. and Renneberg, K. H. Na-
turwis.senschaften 40: 166-167, 1953.
Cryplocidin
Produced by: Streptomyces sp.
Method of extraction: Mycelium extracted with
248
DESCRIPTIONS OF ANTIBIOTICS
methanol. Extract concentrated in mciin to pre-
cipitation. Precipitate dried, washed with aljso-
lute ethanol, and taken up in 90 per cent acetone.
Solution concentrated, filtered if necessary, and
reconcentrated to precipitate the antibiotic.
Chemical and physical properties: Hexaene.
Crystalline; m.p. 100-1 15°C (with foaming). Very
soluble in 0.01 iV NaOH and 80 per cent acetone;
soluble in methanol; scarcely soluble in water,
ethanol, and acetone. Ultraviolet absorption spec-
trum shows major maxima at 341 (E'lcin 380), 358
(£'lcm 585), and 380 m^ (£'icm 605), and minor peaks
at 290, 305, and 320 m//. Negative biuret, Saka-
guchi, Molisch, Fehling, ninhydrin, and FeCls
tests. Dark blue in H2SO4 . Yellow in acidic solu-
tion, slowly becoming brown at alkaline pH. Loses
40 per cent of activity in 0.01 .V NaOH at 100°C
for 5 minutes. Stable at the same pH for 5 hours
at 5°C. Paper chromatographic behavior given in
reference 1.
Biological activity: Active on Staph, aureus at
0.8 Mg per ml, B. subtilis at 12.5 ng per ml, Candida
iitilis at 1.2 /.tg per ml, and P. chrysogenuni at 6.0
fj.g per ml.
Toxicity: LD50 (mice) 135 mg per kg orally, >10
mg per kg intravenously.
Reference: 1. Sakamoto, J. M. J. J. Antibiotics
(Japan) 12A: 21-23, 1959.
Crystalloniyciii
Produced by: Streptomyces violaceoniger var.
cristallomicini (1).
Synonym: Similar to amphomycin. Crystallo-
mycin differs from amphomycin in that (a) it is
more active against experimental pneumococcal
infections, and (b) it has more antibacterial activ-
ity in liciuid media. Complete cross-resistance was
not observed between these two substances (1).
Toxicity: LD50 (mice) 124 mg per kg intrave-
nously, 109 mg per kg intraperitoneally, 220 mg
per kg subcutaneously, and over 1500 mg per kg
orally (2). Pharmacological data given in reference
2.
References:
1. Shorin, V. A. and Shapovalova, S. P. Anti-
biotiki 4(1): 77-81, 1959.
2. (ioldberg, L. E. Antilnoliki 1(1): 03-66,
1959.
Cyanonij ciii
Produced by: Streptomyces cyanoflavus.
Method of extraction: Extracted from broth-fil-
trate at neutral or alkaline pH with chloroform,
methylene chloride, or tetrachloroethane. Back-
extracted into acidic water. Mycelium extracted
two or three times with 0.1 A' HCl, then back-
extracted into chloroform at pH 8.5. Process re-
peated several times. Final acidic water extract
neutralized and cooled to precipitate cyanomycin.
Recrystallized from hot water.
Chemical and physical properties: Monoacidic
base. Possibly contains a cjuinone group. Dark
blue needles; m.p. r28°C (decomposition). Soluble
in water to 0.5 mg per ml; in hot water to 5 mg per
ml. Sokdile in lower alcohols, acetone, chloroform,
and methylene chloride. Slightly soluble in carbon
tetrachloride, ethyl acetate, benzene, and ether.
Insoluble in petroleum ether and cyclohexane. De-
composes in 0.1 A^ NaOH, but not in 0.1 N HCl.
pK = 4.98. Ultraviolet absorption spectrum max-
ima at 240 (£;'iL 550), 278 {E\"L 2140), and 384 m/x
(E^,n 780) in 0.1 N HCl; at 239 (^IL 760), 278
(£lL 1180), 310 {E'-^L 730), and 384 m/x (j5;1L 500)
in phosphate buffer at pH 5.0; at 238 (E'Icm 1010),
310 (£"1™, 1470), and 378 m,x (ETc^u 320) in carbonate
buffer at pH 9.0; at 239, 293, and 370 m^ in 0.1 .V
NaOH. Infrared spectrum given in reference 1.
Negative Fehling, biuret, ninhydrin, and FeCls
tests. Red solution in H2SO4 changes to green-
yellow with zinc powder; effect reversilile l\v H2O2
but not by bubbling air through the solution.
Aciueous solution changed to light blue with zinc
powder; change unaffected by atmospheric ()■.> ;
and l^ecomes colorless on addition of H2O2 .
Weakly alkaline solution reversibly changed to
light blue-green with H2O2 . Aqueous solution be-
comes yellow-l^rown with H2O2 ; and blue, then
colorless with zinc powder. Aciueous solution be-
comes yellow-brown on addition of NaHS()4 ,
then red with H2O2 . C1.5H12N2O2 : C = 69.22%;
H = 5.16' f ; N = 10.769f . Picrate: Purple-red nee-
dles; m.p. 157.5°C.
Biological activity: Active on E. coli at 4 ^g per
ml and Staph, aureus at 6 Mg per ml. Much less
active (10 to 00 fig per ml) on other bacteria tested.
Not active on fungi or yeasts.
Toxicity: Mice tolerate 25 mg per kg intrave-
nously.
Reference: 1. Funaki, M. et al . J. Antibiotics
(.lapan) UA: 143-149, 1958.
C> cloliexiniide
Produced by: Streptomyces griseus (1, 20, 77)
(also produces streptomycin or streptovitacin),
Streptomyces sp. related to S. virido- or olivochro-
mogenes (52), 5. nonrsei (53) (also produces nysta-
tin), Streptomyces sp. differing from S. griseus (71)
(also produces a stereoisomer of cycloheximide,
"naramycin B"), Streptomyces sp. (41), and iS.
albulus (culture produces (72) two forms of cyclo-
DESCRIPTIONS OF ANTIBIOTICS
249
hexiinide, nystatin, and antitunioi' antibiotic
E73).
Synonyms: Actidione (1), naramyoin A (71),
antibiotic A 67 (41).
Hrniarks: Substances having great 1>' inferior or
no antifungal activity, inadonc (50, 51) and
nonactin (52), have been isolated from cyclohexi-
mide-containing broths. Streptovitacin, an anti-
tumor substance, has also been isolated from
certain cycloheximide broths (77). A cyclohexi-
midedike substance (m.p. 105-106°C) that was one
third as active a cycloheximide was reported (31).
It may be similar to naramycin B (see below).
Method of extraction: I. Adsorbed on carl)on or
carbon-Celite at pH 2.0 or other acid pH from
broth-filtrate. Eluted with anhydrous or 80 per
cent acetone, or anhydrous methanol. Eluate dis-
tilled ofi', residual solution extracted with chloro-
form. Extract decolorized with carbon and evapo-
rated. Purified by countercurrent distribution
(benzene-water). Crystallized from amyl acetate
(1, 2), or from anhydrous ethyl ether on pouring
into petroleum ether. Further purified by chro-
matography on Darco (l-(iO-Celite, with 20 per
cent aqueous acetone as solvent and 60 to 100 per
cent acetone as developer. Crystallized from ac-
tive fractions on adding amyl acetate and seeding.
Recrystallized from hot water or 30 i^er cent
methanol (2). II. Broth-filtrate extracted with
amyl acetate. Mycelium extracted with acetone.
Extracts concentrated, then extracted with amyl
acetate. Amyl acetate-extracts combined and
concentrated in vacuo. Nonactin precipitates on
standing. Mother liciuor chromatographed on alu-
minum oxide and developed with chloroform-
met hanol (19:1). Further purified by countercur-
rent distribution (CCU-CHCL^-MeOH-water, 55:
55:75:25). Crystallized from ether by seeding; re-
crystallized from acetone-petroleum ether (52).
III. Adsorbed on carbon from broth-filtrates at
pH 5.5 to 6.0. P]luted with acetone at pH 4.0 to
4.5 followed by water. Eluates adjusted to pH 6
to 7, and acetone distilled off in vacuo. Aqueous
residue extracted into chloroform. Chloroform
neutralized and an oil which separates, removed.
Chloroform distilled in vacuo. Residue extracted
with water, clarified, and acetone added to 20 per
cent. Chromatography on Darco (j-60-Celite 545,
developed with 20 per cent acetone, and eluted
with aqueous acetone containing successively less
water. Active fractions are distilled in vacuo to
remove the acetone, extracted into chloroform,
and chloroform distilled off. Residue crystallized
from amy! acetate. Recrystallized from 30 per
cent methanol (53).
Chemical and physical properties: Neutral sub-
stance. Rectangular or square colorless plates;
m.p. 115-117°C (1, 2, 53). Very soluble in all com-
mon organic solvents except saturated hydrocar-
bons. Soluble to 2.1 gm per 100 ml of water and 7
gm per 100 ml of amyl acetate (2). Ultraviolet
absorption spectrum maximum at 287 m/x (e =
36.7) (4). Infrared data given in reference 4. [aYo =
-2.8° (c = 9.6 per cent in methanol) (1), or +6.8°
(c = 2 per cent in water) (2). pKa' = 11-2 (4).
Positive Fearon-Mitchell test (primary or sec-
ondary alcoholic hydro.xyl) (4). Crystals stable
for several hours at 100°C. Acjueous solutions
stable for several hoiu's at 60 but not 70°C (26).
C = 64.167c-; H = 8.17%; N = 5.13%- C15H23NO4 .
Structural formula (2, 4) is given in Chapter 6.
/3 - [2 - (3 ,5 - dimethyl -2 - oxocyclohexyl)-2-hydroxy-
ethyl} glutarimide. Inactivated by alkali at room
temperature (1), giving a fragrant volatile prod-
uct, d-2,4-dimethylcyclohexanone, as well as pro-
pionaldehyde-2,2-diacetic acid (4). Cycloheximide
diacetate: m.p. 148-149°C (2). [a]f = +24.6° (c =
3.7 per cent in methanol). Biologically inactive
(1). Monoxime: m.p. 203-204°C (2, 4). Semicarba-
zone: m.p. 182-183 °C (2, 4).
Biological activity: In vitro: Active on a variety
of plant pathogens at 0.125 to 20 Mg per ml, includ-
ing members of the phycomycetes, ascomycetes,
basidiomycetes, and Fungi Imperfecti (8). Most
active on yeasts of the Saccharomyces genus; less
so on Torula or Hansensia. Not active on Kloeckera
apiculata. Active on most alcoholic fermentation
processes; slightly active on respiration; not ac-
tive on lactic fermentation (31). Not active on
gram-positive or gram-negative bacteria (15).
Active on Tetrahymena geleii, Euglena gracilis var.
hacillaris (colorless), and Endamoeba histolytica
(25). Inhibits the growth of certain members of
Chlorophyceae, Xanthophyceae, and Bacillario-
phyceae at 50 ppm or less, but has no effect on
Myxophyceae (68). Active in vitro on influenza A
(PR 8) virus liy direct inactivation of the virus
particles (67). Partially inhibitory levels of cyclo-
heximide in a medium on which Allomyces arbus-
cula is grown give rise to the formation of male
and female gametangia on portions of the sporo-
phyte (22). Inhibits protein and nucleic acid syn-
thesis in Sacch. carlsbergensis. At minimal growth-
inhibitory levels, synthesis of desoxyribonucleic
acid and protein is completely inhibited, but ribo-
luicleic acid synthesis continues slowly for some
time ((id). In animals: Temporary clearance of E.
histolytica from macaques (13), rats, and dogs
(58). Inhibits growth of the following in mice,
rats, or hamsters: Miyono adenocarcinoma, Mecca
250
DESCRIPTIONS OF ANTIBIOTICS
lymphosarcoma (69), and Eagles KB epidermoid
carcinoma (76). Moderately inhiliits Bashford
carcinoma 63, adenocarcinoma E 0771, carcinoma
1025, Wagner and Ridgeway osteogenic sarcomas,
Harding-Passey melanoma, Flexner-Jobling car-
cinoma (69), and RC mammary carcinoma (70).
Slight inhibition of Crocker sarcoma 180 in mice
(32). Inhibition (at 1 mg per day) of weight in-
crease in mice inoculated with Ehrlich ascites
carcinoma, an effect which ceases with withdrawal
of the drug (40). In plants: Cycloheximide con-
trols: Powdery mildew of: beans {Erysiphe poly-
goni) (3) ; dewberry (Sphaerotheca humuli) (20) ;
onion (17); squash {Erysiphe cichoracearum) (19);
rose {Sphaerotheca pannosa var. rosae) (5); grape
{Uncimula necator) (49); and cherry {Podosphaero
oxyacanthae) (46). Leaf spot of cherry (9) and ar-
tificial infections with Coccomyces hiemalis of
cherry trees (34). Smut (covered) of oats {Ustilago
kolleri) (21) and wheat {Tillctia sp. (30) and T.
foetida (75)). Rust of: wheat (Puccinia graminis
var. tritici) (43); mint (Puccinia menthae) (17);
turf (stem rust, Puccinia graminis) (48) ; safflower
{P. carthami) (54) ; red cedar {Gymnosporangium
juniperi-virginianae) (45); white pine (63); apple
leaf (cedar rust) (42) ; and asparagus (semicar-
bazone used) (74). Turf diseases: Sclerotinia ho-
meocarpa and Hehninthosporium sp. infections
(18, 28); "fading out" {Curvularia lunata) (35);
leaf spot phase of "melting out" {Helmintho-
sporium vagans) (59). Miscellaneous: Sphacelotheca
sorghi infections of sorghum (38); Rhizoctonia and
V erticillium infections of potato stems (36) ; brown
rot of peach {Sclerotinia fructicola) (49) ; rot of
Delicious apples (73); oak wilt (Endoconidiophora
fagacearum) (44); wood-rotting fungi, including
Poria microspora (16); cucumber scab iCladospo-
rium cucunierinuin) (10); l)lack leg of cabbage
{Phoma lingam) ((iO) ; derrite disease of coffee
{Phyllosticta coffeicola) (61); Dactylium dendroides
mildew infection of mushrooms (56); post-harvest
decay {Botrytis and Rhizopus) of strawberries
(64). Other activity: At very low concentrations,
increases seed germination of oats and Madrid
sweet clover, sprout growth of oats, and top
growth of l)oth oats and clover. Added to soil,
increases bacterial and actinomycete population
and nodules on sweet clover, but decreases the
fungal population (33). Holds down mold count
on harvested black raspberries (27).
Toxicity: LDso (mice) 150 to 160 mg per kg in-
travenously (1) and subcutaneously (31); 375 mg
per kg orally (31). LD50 (guinea pigs) (iO mg per
kg subcutaneously; (rabbits) 17 mg per kg in-
travenously^; (cats) 4 mg per kg intraperitoneally;
(rats) 2.7 mg per kg subcutaneously, 5 mg per kg
oralh-, and 2.5 mg per kg intravenously (12, 31).
Intravenous injection of 1 mg per kg in dogs is
followed by vomiting within a few minutes (7).
Cycloheximide was found to be very repellent to
rats (12). Plants: Inhibits germination of the
following seeds b}' soaking for 4 hours (ppm) :
radish (100), pea (>100), wheat (100). Soaking for
30 minutes at 100 ppm has no effect on seed ger-
mination of cantaloupe, spinach, or cucumber
seeds (6). Toxic to tomato and bean plants at 1
ppm, wheat at 10 ppm, geranium and peach at 100
ppm; not toxic to strawberry at 1000 ppm (applied
as sprays) (14, 30). Stunts alfalfa seedlings at 0.4
yug per ml in a soil solution (29). pH of the spray
was found to be a factor in to.xicity of cyclohexim-
ide (37). Nontoxic when applied to cantaloupe
plants as a 4-ppm spray (47). Use of chlorophyll in
cycloheximide sprays reduces the toxicity of the
antibiotic on beans without affecting antifungal
activity (65). Injurious to roses at concentrations
of 0.1 to 0.4 ppm when sprayed on the young leaves
(42). Produces cytological aberrations, including
prophase inhibition, in Pisum sativum (39) and
Allium cepa (11) root cells, as well as in HeLa
cells at 12.5 /xg per ml (57). Human beings: Nausea
observed following cycloheximide injections in
human beings (7). Toxic by intrathecal route, but
not intravenously (23). Crystalline cycloheximide
and its concentrated solutions are highly irritating
to the skin, producing a reddening and sloughing
(62).
Utilization: Plant disease. Used commercially
for control of turf diseases and cherry leaf spot
(62). Usefulness of cycloheximide is reduced by
the fact that at effective therapeutic levels it is
often toxic to the host plant (55). Sweet wine
preservation (31). Therapeutic effect in one case
of cryptococcic meningo-encephalitis, and gave a
remission in one ca.se of coccidioidal meningitis
(24).
sTEREOisoMER.s OF CYCLOHEXIMIDE: Cyclohe.xim-
ide has four centers of asymmetry permitting
isomers.
Naramycin B
Produced by: Streptomyces sp. differing from S.
griseus. This culture also produced cycloheximide
'(71).
Chemical and physical properties: Colorless
plates; m.j). 109-1 10°C. Ultraviolet absorption
spectrum maximum at 292.5 m^u (log e, 1.49) and
a shoulder at 232 m/i (methanol). Infrared data
given in reference 71. [alo" = +50.2° (c = 2 per
DESCRIPTIONS OF ANTIBIOTICS
251
cent in methanol). Ci5Hv,04N: C = t)4.3',o; H =
7.80' r; N = 4.90%. More heat-stable than cyclo-
heximide. Acetate: m.p. 150.5-152°C. [a|n ' =
+02.5° (c = 2 per cent in methanol). Benzuate:
m.p. 159-160.5°C. [a]!; ' = +54.6° (c = 1 per cent
in methanol) (71 ).
Biological activity: Has 32 per cent of the activ-
ity of cycloheximide on Saccharontyces sake (71).
Inactone
(See remarks under cycloheximide.)
Chemical and physical properties: Colorless Hat
needles; m.p. 116°C. Ultraviolet absorption spec-
trum maximum at 330 m/j. {E{^„^ 50) (ethanol).
Infrared spectrum similar to that of cycloheximide
(51). [a]'^ = —55° (c = 2 per cent in water). Inac-
tone is one of eight possible dehydrocyclohexim-
ides having the following structure (50, 51):
CHs
CH
H.,
H.,C^^C=0
C C=o
/ \
C— CHOH-
-CH.
-HC< >NH
..CHC\^//
H
\ /
C C=o
H.,
Biological activity: None.
Nonactin (52)
Chemical and physical properties: Neutral sul)-
stance. Colorless needles; m.p. 147-148°C. Opti-
cally inactive. Ultraviolet absorption spectrum
maximum at 264 m/i (weak). Infrared spectrum
given in reference 52. Chemically unreactive sub-
stance. C30H48O9 .
Biological activity: Very little or none.
Isocyclohe.riinide
Method of extraction: Mother liquors of cyclo-
heximide crystallization batches evaporated uniler
reduced pressure. Extraction with chloroform of
aqueous concentrate. Chloroform-extract decolor-
ized with carbon and concentrated to syru]). Iso-
propanol added, and after storage for 3 months
at 5 to 10°C, crude isocyclohe.ximide collected.
Further purification by chromatography on car-
bon, followed by recrystallization.
Chemical and physical properties: Same infrared
light absorption spectnmi as cycloheximide l)ut
slight differences in the low cm~' regions; m.p.
95-102°C. [a]:' = +32° (c = 1.0 per cent in CH.-
OH).
Biological activity: Thirty per cent of the cyclo-
lieximide activity against Saccharomyces pastoria-
niis.
Toxicity: Thirty per cent of the intravenous
toxicity of cycloheximide in mice.
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CycloseiMiie
Produceil hi/: Streptomyce.s orehidaceus (5, 9, 35),
S. gar ij ijhal us strains (22), S. lavendulae strains
(2, 6, 21), S. roseochroiHogenes (1, 20), and S. naga-
sakiensis (28).
»Synont//».s.- Oxamycin (3), orientomycin (orient-
mycin), antil)iotic K 300, special sul)stance 2 (20),
DKSCniIPTIONS OF ANTIBIOTICS
258
;uitil)io(i<- 106-7 (22), ;uitil)iotic PA 94, seromycin,
ami antibiotic JN 21 (35).
Method of extraction: I. Ads()rl)e(l from culture-
filtrates on Dowex 2 (OH" form) or other strong
basic anion exchange resin and fluted with dilute
sulfuric acid. Eluate decolorized with charcoal at
pH 7.0. Cycloserine precipitated as the silver salt
on addition of silver nitrate at pH (15. Converted
to the base by decomposing an aqueous solution of
the salt with dilute HCl, and freeze drying. Crys-
tallized from water with alcohol or acetone (9, 35,
3(3). II. Broth clarified with charcoal, adjusted
to pH 1.5 to 3.0, and adsorbed on IR-120 (OH"
cycle). Elution with 0.1 to 0.2 N NH4OH. (a)
Chromatographed on Amberlite XE-98 (()H+
form) and eluted with 0.3 A^ acetic acid. Eluate
treated with charcoal, adjusted to pH 10.0 to 10.5,
concentrated, and treated with isoproi)anol,
ethanol, or acetone to separate out impurities.
Adjustment of supernatant to pH 6.0 with acetic
acid and cooling precipitated cycloserine. Crys-
tals dissolved in water and adjusted to pH 12
with KOH. Solution treated with ethanol and iso-
propanol and mixture filtered. Filtrate cooled,
atljusted to pH 5.8 with glacial acetic acid to re-
l)recipitate cyclo.serine (8, 23). (b) Chromato-
graphed on alumina with 50 per cent methanol as
solvent and developer. Concentration first precipi-
tates impurities, then cycloserine. Recrystallized
from hot methanol or from water on addition of
ethanol (2).
Chemical and phi/sical properties: Amphoteric
substance (0, 20). Fine white needles; m.p. 153-
156°C (decomposition) (5, 6, 8, 9), or 149-150°C
(29). Sulilimes in vacuo at 100°C (35). Very soluble
in water; soluble in methanol; slightly soluble in
acetone; very slightly soluble or insoluble in most
other organic solvents (2). Ultraviolet absorption
spectrum maximum at 22(5 m/x iEilm 402) (water,
jjH G.O) (8). Infrared spectrum given in references
1, 9, and 20. [a]f;^ = +112° (c = 5 per cent in 2 N
NaOH). [a]546i = 137° ± 2° (c = 5 per cent in 2
A^ NaOH). pK,^ = 4.4 to 4.5 and 7.3 to 7.4 (8, 9,
20). Exists in solution as a dipolar ion (9). Positive
FeCl.j test (2). Green color with cupric ions in
aqueous solution; reddish l)rown with iron; bio-
logical activity destroyed in Ijoth cases (35). De-
composes in glacial acetic acid (22). Rf = 0.4 (80
per cent ethanol-water), producing a brown-yel-
low color with ninhydrin. Highly dif'fusil)le (3).
Stable to heat (2). Stable to alkali; unstable at
acid or neutral pH (5). Crystallographic data
on the anhydrous and hydrous forms given in
reference 22. C = 35.4%; H = 5.98%; N = 26.9%,.
Equivalent weight, 101 to 104. Structural formula,
D-4-amino-3-isoxazolichnone (C-sHeNaOs) (8, 9) is
given in Chapter 6. Prolonged acid hydrolysis
yields DL-serine and hydroxylamine. In solution,
cyclo.serine dimerizes to 2,5-bis(aminoxymethyl)-
3,6-diketopiperazine; m.p. 190-200°C (decomposi-
tion) (8). Calcium salt: m.p. 215--221°C (decom-
position) (8, 22). Macjnesiuni salt: m.p. 224-228°C
(22). Silver salt: Square platelets (35). Cycloserine
and some of its analogues have been synthesized
(10, 30). L-4-A)nino-3-isoxazolidinone: m.p. 148-
149°C. [alf = -119° (c = 1 per cent in water) (29).
D-1 -Cycloserine (racemic): m.p. 136-137°C (29).
Biological activity: In vitro: Very limited (6.25
to 500 fig per ml) activity on gram-positive and
gram-negative bacteria. Most active on mycobac-
teria at pH 6.4 to 7.0. Activity otherwise unaffected
in a range of 4.5 to 8.0. Not active on fungi (4, 11,
14, 15). Active on bacteria-free Endamoeba histo-
lytica; 'loofh as active on E. histolytica in bac-
teria-containing cultures (25). Competitively
inhibits the growth factor for mycobacteria, myco-
bactin; cycloserine contains an iso-oxazole ring,
mycobactin an oxazole ring (7). Reversibly and
noncompetitively inhibits catalase activity in
mycobacteria, as well as purified beef liver cata-
lase. Also blocks purified peroxidase and the
peroxidase activity of tubercle bacilli (39). Block-
ing the amino group destroys the activity on E.
coli (32). In vivo: More active in vivo than could
be expected from in vitro tests. Active in mice on
infections caused by Hurrelia n(n'yi , Staph, aureus,
K. pneumoniae, Sal. schottmuelleri, E. coli, Ps.
aeruginosa. Streptococcus pyogenes, and D. pneu-
moniae (4). Active in mice, but not in vitro, on
Nocardia asteroides (37). Moderately active on
mouse leprosy (33). Active against tuberculosis
in monkeys, but has limited to no activity in mice,
guinea pigs, and rabbits because of differences in
attainable blood levels of the drug (16). Slightly
active in mice and eggs on Rickettsia )nooseri , and
in eggs on feline pneumonitis. Not active on SK
encephalomyelitis or swine influenza virus. Not
active in vivo on protozoa, except slightly on Plas-
modium, gallinaceuni (chickens) and Endamoeha
histolytica (rats). No anthelmintic activity (4).
d- Versus I- and h'acemic Cycloserine
d-Cycloserine is most active on bacteria; 1-cy-
closerine is almost inactive. The racemic mixture
has activity equal to the d-form, although it
contains only 50 per cent of tlie d-form (26). 1-Cy-
closerine is twice as active as the racemic mixture,
and 10 times as active as d-cycloserine against
human strains of M. tuberculosis (27). The race-
mate is more active than either the d- or 1-form
against pnevmiococcus type I, but not against Sal.
enteritidis (30). The racemic form is more active
254
DESCRIPTIONS OF ANTIBIOTICS
than the d-forin against E. coli in a synthetic
medium. The 1-form is inactive unless employed
in an asparagine-containing medium, in which it
is as active as the d-form (27). d-Cyclo.serine is
more active against tuberculosis in mice than the
dl- or 1 -forms (29).
Toxicity: LDso (mice) 1.81 to 2.5 gm per kg
intravenously, 2.87 to 4.3 gm per kg, intraperi-
toneally, 2.8 to >5 gm per kg subcutaneously,
and 5.29 ± 0.20 gm per kg orally. LDso (rat) >5.0
gm per kg subcutaneously and orally (18, 19).
Rats and mice show slight depression at 0.25
gm per kg orally; transient coma at 1 gm per
kg, and deep coma at 4 gm per kg. Four gm per kg
induce convulsive seizures in rabV)its (17). Four
hundred mg per kg per day are severely neurotoxic
to monkeys (31). Side reactions in human pa-
tients include lethargy, convulsions, and disori-
entation (13). Pyridoxine administered simulta-
neously is said to reduce the toxicity of cycloserine
(40).
Utilization: Used in tuberculosis as a last resort
in combination with other drugs (12, 34). Used for
certain other ])acterial infections (11, 24) and
leprosy (38).
References:
1. Kurosawa, H. J. Antibiotics (Japan) 4:
183-193, 1951.
2. British Patent 715,362, September 15, 1954.
3. Harris, D. A. et al. Antibiotics & Chemo-
therapy 5: 183-190, 1955.
4. Cuckler, A. C. et al. Antibiotics & Chemo-
therapy 5: 191-197, 1955.
5. Harned, R. L. et al. Antibiotics & Chemo-
therapy 5: 204-205, 1955.
6. Shull, G. M. and Sardinas, J. L. Anti-
biotics & Chemotherapy 5: 398-399,
1955.
7. Sutton, W. B. and Sanfield, L. Antibiotics
& Chemotherapy 5: 582-584, 1955.
8. Kuehl, F. A., Jr. et al. J. Am. Chem. Soc.
77: 2344-2345, 1955.
9. Hidy, P. H. et al. J. Am. Chem. Soc. 77:
2345-2346, 1955.
10. Stammer, C. H. et al . J. Am. Chem. Soc.
77: 2346-2347, 1955.
11. Welch, H. p/ «/. Antibiotic Med. 1:72-79,
1955.
12. Epstein, I. C. et al. Antibiotic .Med. I:
80-93, 1955.
13. Robinson, H. J. et al. Antibiotic Med. 1:
351-357, 1955.
14. Barclay, W. R. and Russe, H. Am. Rev.
Tu be r c . 72 : 236-24 1 , 1 955 .
15. Steenkon, W., Jr. and Wolinsky, E. Am.
Rev. Tuberc. 73: 539-546, 1956.
16. Conzelman, G. M., Jr. and Jones, R. K.
Am. Rev. Tuberc. 74: 802-806, 1956.
17. Robinson, H. J. et al. Am. Rev. Tuberc.
74: 972-976, 1956.
18. Anderson, R. C. et al. Antibiotics &
Chemotherapy 6: 360-368, 1956.
19. Spencer, J. N. and Payne, H. G. Antibiot-
ics & Chemotherapy 6: 708-717, 1956.
20. Shoji, J. J. Antibiotics (Japan) 9A: 164-
167, 1956.
21. Shibata, M. et al. Ann. Rept. Takeda Re-
search Lab. 15: 28-35, 1956.
22. British Patent 757,089, September 12, 1956.
23. British Patent 758,500, October 3, 1956.
24. Lillick, L. et al . Antil)iotics Ann. 158-
164, 1955-1956.
25. Nakamura, M. Experientia 13:29,1957.
26. Trivellato, E. and Concilio, C. Giorn. ital.
chemioterap. 4: 495 498, 1957.
27. Trivellato, E. Giorn. ital. chemioterap.
4: 499-503, 1957.
28. Aburaya, I. and Sugai, T. Chemotherapy
(Tokyo) 5: 3, 1957.
29. Serembe, M. and Ziliotto, C!. Minn. Med.
48: 4212-4224, 1957.
30. Plattner, P. A. et al. Helv. Chim. Acta
40: 1531-1552, 1947.
31. Storey, P. B. and McLean, R. L. Am. Rev.
Tuberc. 75: 514-516, 1957.
32. Trivellato, E. and Concilio, C. Bull. soc.
ital. biol. sper. 33: 463, 1957.
33. Chang, Y. T. Intern. J. Leprosy 25: 257-
261, 1957.
34. Storey, P. B. and McLean, R. L. Anti-
biotic Med. 4: 223-231, 1957.
35. British Patent 768,(X)7, February 13, 1957.
36. Harned, R. L. U. S. Patent 2,789,983,
April 23, 1957.
37. Sanford, J. P. e/ «/. Antibiotics Ann. 22-26,
1957-1958.
38. Tran-van-Bang. Bull. mem. soc. med. hop.
Paris 74: 256-258, 1958.
39. Andrejew, A. et al. Biochim. et Biophys.
Acta 30: 102-111, 1958.
40. Epstein, I. G. et al. Antibiotics Ann. 472-
481, 1958-1959.
41. Kurihara, T. and Chiba, K. Ann. Rept.
Tohoku Coll. Pharm. 3: 83-89, 1956.
42. Freerksen, E. et al. Antibiotica et Chemo-
ther a pi a 6: 303-396, 1959.
Oenietliyl tetracyclines
Produced bji: Streptouu/ces aiireofaciens (mutant
of the original Duggar chlortetracycline-pro-
ducer). I is produced in chloride-free media; II
in chloride-containing media (1).
DESCRIPTIONS OF ANTIBIOTICS
255
Synont/Ni.s of 7-chloro-G-demethly tetracycline:
Deinethylchlortetracycline, decloinycin, leder-
inycin.
Chemical and physical properties: I. 6-De-
methyltetracycline hydrochloride hemihydrate :
m.p. 203-209°C (decomposition). C21H24N2CIO8.5 :
C = 52.52%; H = 5.34%; N = 6.05%; CI = 7.51%;
H2O = l.m%. [a]f = -259° (c = 0.5 per cent in
0.1 jV H2SO4). Ultraviolet absorption spectrum
maxima essentially the same as the corresponding
(i-methylated tetracycline (1). II. 7-Chloro-6-
demethyltetracycline sesquihydrate : m.p. 174-
178°C (decomposition). C21H24N2CIO9.6 : C =
51.13%; H = 4.93%; N = 6.00%; CI = 7.39%;
H2O = 4.45%. [a]f = -258° (c = 0.5 per cent in
0.1 A^ H2SO4). Both I and II and their epimers
(synthetic) are considerably more resistant to
alkaline or acidic degradation than other tetra-
cj'clines. Ultraviolet absorption spectrum maxima
of II are essentially the same as corresponding
6-methvlated tetracyclines (1). Structural formu-
las(l):
R H OH H H H N(CH3)2
OH I
OH O
I: R = H
II: R = CI
Biulogical activity: I and II: 24 and 75 per cent,
respectively, of the activit}- of chlortetracycline
against Staph, anrens. II is also active against
Staphylococcus, Streptococcus, Klebsiella, and
pneumococcal infections in mice (2).
Toxicity: II has a low order of toxicity. Ab-
sorbed slowly from the gastrointestinal tract.
Produces very high serum concentrations, prob-
ably because it is cleared slowly by the kidney (1).
Utilization: P^ffective in the treatment of a
number of himian diseases (3).
References :
1. McCormick, J. R. D. et at. J. Am. Chem.
Soc. 79: 4561-4564, 1957.
2. Sweeney, W. M. et al. Antibiotics <fc Chemo-
therapy 9: 13-22, 1959.
3. Finland, M. et al . Antibiotics Ann. 375-
446, 1959-1960.
De.serloniycin
Produced by: Streptomyces ftarofungiiii . This
strain also produces flavofungin.
Method of extraction: Extracted with organic
solvents from broth and mycelium.
Chemical and physical properties: Snow-white,
glittering hexagonal crystals; m.p. 189-190°C.
Low solubility in water, absolute alcohols, ether,
and acetone; higher solubility in aqueous alcohols.
Positive bromine, KMn04 , ninhydrin, and Kuhn-
Roth (C-methyl) tests. Very soluble in neutral
aqueous solution. C33H60-62O14N. Does not con-
tain N — Me or acetyl groups. Gives hydrogenated
and acetylated products.
Biological activity: Active on gram -positive and
gram-negative liacteria (1 to 25 Mg per ml). Inhibits
leukemic and Ehrlich ascites carcinoma cells in
vitro. Cystostatic (10 yug per ml) and cytolytic (50
to 100 Mg per ml) on fibroblast, HeLa, and Crocker
sarcoma cells by tissue culture method.
Toxicity: LD50 (mice) 1.35 mg per kg intrave-
nously, 2.6 mg per kg intraperitoneally, 5.3 mg per
kg subcutaneously, and 12 to 15 mg per kg orally.
Reference: 1. Uri, J. el al. Nature, London
1«2: 401, 1958.
D
lazoinvcins
Produced by: Streptomyces ambofaciens.
Synonyms: Related to azaserine, DON, and
alazopeptin.
Method of extraction: Concentration of the fil-
tered broth to 5 to 10 per cent of original volume;
10 volumes of methanol added and the filtrate
concentrated. The aqueous concentrate passed
through a Dowex 1 acetate column. The effluent
contains 6-diazo-5-o.xo-l-norleucine (DON). The
cohnnn is eluted with 1 per cent phosphate buffer
at pH 7.0. Two active fractions are eluted. Frac-
tion A chromatographed on Celite or silica gel and
eluted with the system phosphate buffer-n-buta-
nol-isopropanol. The active component, diazo-
mycin A, converted to its lithium salt and crys-
tallized. Fraction B, which still contains some
diazomycin A, is purified by countercurrent dis-
tribution between phenol and water. Two peaks
are observed. Both fractions chromatographed on
Dowex 1 acetate resin. One of the fractions is
homogenous: diazomycin C. The other fraction
can be split into two components, diazomycin A
and a new component, diazomycin B.
Chemical and physical properties: Aliphatic
diazo compounds. Very labile at acidic pH values.
Most stable between pH 6.0 and 8.0. Light ab-
sorption spectrum maxima at 275 and 245 niju.
Extinction coefficients at 275 niyu: diazomycin A,
520; B, 550; C, 340; at 245 ni/.: A, 315; B, 340; C,
210. Ninhydrin reaction: A and C, light gray-blue;
B, intense blue. Solubility in methanol: A and C,
readily soluble; B, slightly soluble. Rf values (80
per cent isopropanol) : A, 0.6 to 0.7; B, 0.2 to 0.3;
256
DESCRIPTIONS OF ANTIBIOTICS
C, 0.8 to l.U. Infrared absorption .spectra given in
reference 1. Elementary analysis: A, C = 44.34%;
H = 5.08%; N = 17.22%. B, C = 43.15%; H =
5.59%; N = 19.22%. C, C = 46.03%,; H = 5.93%;
N = 23.82%.
Biological activity: Active against certain yeasts
and bacteria. B. subtilis used as the assay organ-
ism. Diazomycin B is the most active biologically,
then A, then C. Active in animals against sarcoma
180, adenocarcinoma 755, and to a lesser extent
against leukemia 1210.
Reference: 1. Rao, K. V. et al. Antiljiotics
Ann. 943-949, 1959-1960.
I ) i li > < I ros I re p I c> 111 N f i 1 1
Produced by: Catalytic hydrogenation of strep-
tomycin (1), Streptomijces humidus (23) (organism
also produces humidin), and Streptomyces sp. (32).
Synonym: Antibiotic 23572 (23).
Method of extraction: I. See references 1, 14,
and 23 for chemical details on hydrogenation. II.
Extracted from culture-filtrate with n-butanol (or
isoamyl alcohol) containing 3 per cent lauric acid,
at pH 7.5. Organic layer shaken with acpieous acid
at pH 2.0. Aqueous extract shaken with ether
(23). III. Crystalline sulfate is precipitated
from an acjueous solution (pH 5.5) on addition of
glycerol (or formamide). Heating to 50-60°C, addi-
tion of methanol to turbidity, temperature nuiin-
tained at 60°C, stirring (21).
Chemical and physical properties: In (Hhydro-
streptomycin, the aldehyde of streptomycin is re-
duced to the corresponding carbinol (see formula
in Chapter 6). Trihydrochloride: White powder or
fine needles containing methanol (lost on heating) ;
ni.]). 185-190°C (decomposition). Powder soluble
in methanol to 1 gm per ml; needles to 45 mg per
ml (1, 11). No characteristic absorption in ultra-
violet light. Infrared spectrum given in reference
23. [a]:? = -89.5° (c = 0.98 per cent in water) (1).
Crystallographic data given in references 11 and
14. Differs from streptomycin in these reactions:
(a) not inactivated by hydroxylamine or cysteine;
(b) not degraded to maltol in alkali; (c) more
stable in alkali; and (d) forms many complex
mixed acid salts but not the double CaClj salt
that streptomycin forms (1-3, 15). Positive Saka-
guchi test. Negative Fehling (boiling), phenol-
H2SO4 , and silver mirror tests. Slightly positive
Tollen test. CoiHjiNtOis^HCI: C = 36.5%; H =
0.21%,; N = 3.91%. Equivalent weight, 690. pKa'
= 7.7. Yields streptidine and dihydrostreptobiosa-
mine on alkaline hydrolysis (1, 3, 23). Sulfate:
Trapezoidal plates or small noidiygroscopic plate-
lets. Anhydrous salt: m.j). 25()°C or 255-265°C
(decomposition). Soluble in water, and moderately
soluble in aqueous nietlianol. [aYu = —88° (c = 1
per cent in water) (10, 11, 23). Crystallographic
data given in reference 11. Trihelianthate: Plates
(23); m.p. 215-225°C (decomposition) (1), 222-
225°C (23), or 224-330°C (decomposition) (3).
Reineckate: Needles. Sinter at 194°C; m.p. 204-
206°C (decomposition) (3). Forms metal chelates:
Dihydrostreptomycin-copper : Dark blue sub-
stance. Decomposes at 178-183°C (19).
Biological activity: In vitro and in vivo activity
is of the same order as streptomycin (2, 9) except
that against Salmonella it is one third to one
fourth as active in vitro, and in vivo it is even less
active (6). Dihydrostreptomycin has some rickett-
siostatic activity, but less than streptomycin (5).
In vivo: A dihydrostreptomycin-dependent strain
derived from a pathogenic strain of Sal. typhosa
was nonpathogenic for mice, but capable of stim-
ulating immunity (16). Inhibits the killer action
of Paramecium aurelia (13). Active on crown-gall
{Agrobacterium tumefaciens) of geranium (17).
Toxicity: Sulfate: LD50 (mice) 262.5 mg per kg
intravenously (4) , 1700 mg per kg intraperitoneally
(23), 1600 ± 108 mg per kg (6), 910 mg per kg (30),
or 1200 mg per kg (22), or 1800 mg per kg (23)
subcutaneously. LD50 (1 -week-old chicks) 743 mg
per kg intramuscularly; (4-week-old chicks) 1868
mg per kg, same route (20). LDso (10-day -old
chick embryos, allantoic cavity) 80.6 mg (29). As
in the case of streptomycin, the reduced toxicity
of the jjantothenate salt of dihydrostreptomycin
was reported. Pantothenate: LD50 (mice) 1550 mg
per kg (expressed as base) subcutaneously (22).
This was confirmed by some (24, 26) ; others dis-
agreed (25). Dihydrostreptomycin was reported
to have less severe vestibular toxicity than that of
streptomycin in cats (7) and in human beings (8,
27). However, it produces a delayed deafness
which is more serious than that produced by strep-
tomycin (12, 31). Nephrotoxic (8). Plants: Pro-
duces aiiochlorosis antl growth inhibition in l)arley
(18).
Utilization: Clinically effective in tuberculosis
and in numerous other infectious diseases due to
gram+ and gram— l)acteria (27, 28).
References:
1. Peck, R. L. et al. J. Am. Chem. Soc. 68:
1390-1391, 1946.
2. Bartz, Q. R. et al. .J. Am. Chem. Soc. 68:
2163-2166, 1946.
3. Fried, J. and Wintersteiner, O. J. Am.
Chem. Soc. 69: 79-86, 1947.
4. Donovick, R. and Rake, (i. J. Bacteriol.
53: 205-211, 1947.
5. Smadel, J. E. et al. J. Immunol. 57: 273-
284, 1947.
DESCRIPTIONS OF ANTIBIOTICS
257
G. Rake, G. et al. Am. Rev. Tuhere. 58:
479-480, 1948.
7. Edison, A. O. et al. Am. Rev. Tuhere. 58:
487-493, 1948.
8. Hobson, L. B. et al. Am. Rev. Tuhere. 58:
501-524, 1948.
9. Wak.sman, S. A., ed. Streptomycin; nature
and practical applications. The Williams
& Wilkins Co., Baltimore, 1949.
10. Solomons, J. A. and Regna, P. P. Science
109: 515, 1949.
11. Wolf, E. J. et al. Science 1(19: 515-516,
1949.
12. O'Connor, J. B. ct al. Am. Rev. Tuhere.
63: 312-324, 1951.
13. Williamson, M. et al. J. Biol. C'hem. 197:
7G3-770, 1952.
14. Wolf, F. J. U. S. Patent 2,594,245, April
22, 1952.
15. Bogert, V. V. and Solomons, I. A. J. Am.
Chem. Soc. 75: 2355-2351), 1953.
16. Reitman, M. and Iverson, W. P. Anti-
biotics Ann. 604-608, 1953-1954.
17. Janke, A. and (iranits, J. Zentr. Bakteriol.,
Parasitenk., Abt. 2 108: ()(i-75, 1954.
18. Signol, M. Compt. rend. soc. l)iol. 148:
64(5-648, 1954.
19. Foye, W. D. c/ a/. J. Am. Pharm. Assoc,
Sci. Ed. 44:261-263,1955.
20. Huebner, R. A. et al. Cornell Vet. 46:
219-222, 1956.
21. Katz, L. U. S. Patent 2,744,892, May 8,
1956.
22. Keller, H. et al. Arzneimittel-Forsch. 6:
61-66, 1956.
23. Belgian Patent 553,388, Decemlier 13, 1956.
24. Keller, H. et al. Antibiotics Atui. 549-553,
1956-1957.
25. Hawkins, J. E., Jr. et al . Antil)iotics Ann.
554-5(>3, 1956-1957.
26. Osterberg, A. C. et al. Antibiotics Ann.
564-573, 1956-1957.
27. Mihaly, J. P. et al. Antil)iotics Ann. (302-
608, 1957-1958.
28. Hewitt, W. L. et al. Antibiotics Ann.
609-613, 1957-1958.
29. (ientry, R. F. Avian Diseases 2: 7(i-82,
1958.
30. Brigham, R. S. and Nielsen, J. K. Anti-
biotics & Chemotherapy 8: 122-129,
1958.
31. Weinstein, L. and Ehrenkranz, N. J. Strep-
tomycin and dihydrostreptomycin. Med-
ical p]ncyclopedia, Inc., New York, 1958.
32. Kavanagh, F. Appl. Microl)i()l. 8: KiO-
l(j2, 19()0.
Dill y<lr«>(lesox_vs t rep toniyciii
Froiliiced hi/: This antibiotic is a chemical de-
rivative of streptomycin.
Chemical and physical properties: More stable
in solution than dihydrostreptomycin. Structural
formula given in Chapter 6.
Biological activity: Equally as active as dihydro-
streptomycin in vitro against a variety of bac-
teria; equally active in vivo against M. tuberculo-
sis. .Slightly less effective against other infections.
Toxicity: LD.m) (mice) 214 mg per kg (no route
given). This substance is reputed to be slightly
less toxic than dihydrostreptomycin. No vestibu-
lar or ototoxic symptoms were observed in hunum
beings treated with the drug.
Utilization: Treatment of tuberculosis.
Reference: 1. Obuchi, S. et al. J. Antibiotics
(Japan) IIA: 199-201, 1958.
l,6-Dihy(lro.\ypliena/;iiie
Produced by: Streptoniyces thioluteus.
Remarks: 1 ,6-Dihydroxyphenazine is also ob-
tained by the reduction of iodinin, an antibiotic
jjroduced by Pseudomonas iodinuui.
Methixt of e.rlrartion: Culture-filtrate extracted
with benzene at pll 6.5 to 7.0 or adjusted to pH 12,
filtered, and filtrate neutralized to give a precipi-
tate. Precipitate extracted with benzene. Benzene
extracted with 0.1 A^ NaOH, and extract neutral-
ized to give a yellow jjrecipitate. Precipitate re-
extracted with l)enzene. Benzene concentrated in
vacuo. Crystallized from dioxane or ethyl acetate
or piu'ified l)y svdilimation.
Chemical and physical properties: Golden-yellow
prisms; ni.p. 274°C. Very soluble in dioxane and
pyridine. Soluble in alcohols, acetone, ethyl ace-
tate, chloroform, l)enzene, ethyl ether, aqueous
sodium carl)onate, and NaOH. Insoluble in water,
aqueous sodium bicarbonate, and petroleum ether.
Ultraviolet absorption spectrum maxima at 272
(^ie™ ()350), 372 (^J^ni 245), and 440 to 445 mfi
iE\%, 165) (methanol), or 291 (EI^'^, 4300) and 520
to 530 niM (£'i'rm 180) (0.1 N NaOH). Infrared ab-
sorption spectrum given in reference 1. Puri)le in
alkali. i<>thanol solution turns green with FeCls ,
gives a blue precipitate with lead acetate. Gives a
green-blue precipitate with cupric sulfate and a
violet precipitate with silver nitrate. Ci'HsN-iOi :
C = 68.48%; H = 3.99%; N = 13.51%,. Structural
formula given in Chapter 6. Diacetyl derivative:
Light yellow needles; m.p. 235°C.
Biological activity: Active at 3 to 20 jug per ml on
certain yeasts and fungi. Not active on bacteria.
Reference: 1. Akabori, H. and Nakamura, M.
J. Antibiotics (Japan) 12A: 17-20, 1959.
258
DESCRIPTIONS OF AXTUilOTICS
DON (6-Diazo-5-«>xo-L-noiieiiciiiej
Produced by: StreptODiyces sp. similar to S. au-
reus and S. phaeochromogenes (4).
Method of extraction: Broth adjusted to pH 6.8,
filtered, and concentrated in vacuo. Concentrate
diluted tenfold with 95 per cent ethanol and fil-
tered. Filtrate purified by (a) adsorption on alu-
mina at pH 5.5 to 6.5 and elution with 25 per cent
aqueous alcohol; (b) carbon chromatography (1
per cent aqueous acetone as solvent and devel-
oper); and (c) crystallization of best carbon frac-
tion from acjueous alcohol or acetone (5).
Chemical and physical properties: Fine light
yellow-green needles. Decomposes at 145-155°C
with gas evolution. Very soluble in water, aqueous
methanol, ethanol, and acetone. Slightly soluble
in absolute alcohols. Ultraviolet absorption max-
ima at 274 (E'lcm 683) and 244 m^ (^'llm 376). No
spectral shift in alkali or acid. Loss of activity
against Torulopsis albida concomitant with loss
of ultraviolet absorption characteristics in 0.1 A'
alkali or 0.1 A' HCl (5). Infrared spectrum given
in reference 5. [a]^ = +21° (c = 5.4 per cent in
water). Positive ninhydrin and Tollen tests. Aque-
ous solution yields gas when treated with strong
acid. Rf values on paper chromatography given
in reference 5. Sensitive to extremes of pH and
heat. pKa' values of 2.1 and 8.95 in water. DON
has been synthesized and the DL- and D-isomers
prepared. C = 42.16%; H = 5.70%; N = 24.07%;
diazo N = 16.01%,. Alolecular weight, 171. CeHg-
N303(2,5, 14).
Biological activity: Antitumor sul)stance.
Slightly active on certain bacteria, j^easts, and
Erro equinus in eggs. Some activity (at toxic lev-
els) on Plasmodium lophurae infections in chicks
(4). Unlike azaserine, DON does not give rise to
long, nonseptate filaments in E. coli (11). Crocker
sarcoma 180 growth is "restrained" in mice bj^
oral or intraperitoneal treatment, but the tumor
growth potential is not affected (1). DON inhibits
Miyono adenocarcinoma, Ehrlich carcinoma,
Krebs 2 ascites carcinoma, carcinoma 1025, Ridg-
way osteogenic sarcoma, Mecca lymphosarcoma,
and leukemia L 1210. Slight inhibition of a variety
of other carcinomas. No effect on sarcoma T
241, Gardner lymphosarcoma, or Harding-Passey
melanoma (10, 13). Active on mast cell neoplasm
P 815, l)ut resistant sublines of this tumor can be
developed (12). DON is reportedly a better in-
hit)itor of mouse tumors; azaserine is superior in
rat tumors (15). Active on an ascitic plasma cell
neoplasm (70429) in mice (19).
Toxicity: LDoo (mice) 76 ± 14 mg per kg intra-
venously (4) ; 250 Mg per kg intraperitoneally in a
single daily dose for 7 days is tolerated by mice,
but with weight loss. Tumor-bearing mice are
more susceptible to DON toxicity than normal
mice (1). In single doses DON and azaserine have
similar toxicity, but DON is 50 to 100 times more
toxic in chronic toxicity tests with mice, rats, and
dogs (9). LDso of DON in eggs is about ^^oth that
of azaserine. DON or azaserine toxicity to eggs
overcome by immediate injection of adenine or
hypoxanthine following injection of DON or
aza.serine (7). Two doses of 0.5 mg per kg given
to mother rats between implantation and mid-
term cause complete litter destruction; the fetus
is affected directly, not the placenta, ovaries, or
pituitary. This effect can be overcome by adenine.
No cumulative toxicity or impairment of fertility
was noted in mother rats having repeated complete
destruction of litters. Subsequent offspring were
normal (17). Toxic symptoms in human beings
include oral soreness and ulceration, diarrhea, and
vomiting (18).
Utilization: Clinical trials in human beings indi-
cated transient or no activity on a variety of neo-
plastic diseases (3). Some activity has been re-
ported on Hodgkin's disease (16). Some evidence
of "temporary arrest" of neoplastic diseases in hu-
man beings (18, 20).
References:
1. Clarke, D. A. et al. Abstr. 129th Meeting
Am. Chem. Soc. 12M, 1956.
2. Westland, R. D. ef al. Abstr. 129th Meeting
Am. Chem. Soc. 14M, 1956.
3. Eidinoff, M. L. et al. Abstr. 130th Meeting
Am. Chem. Soc. 2C, 1956.
4. Ehrlich, J. et al. Antibiotics ct Chemo-
therapy 6: 487-497, 1956.
5. Dion. H. W. et al. J. Am. Chem. Soc. 78:
3075-3077, 1956.
6. Burchenal, J. H. and Dagg, M. K. Proc.
Am. Assoc. Cancer Research 2: 97, 1956.
7. Dagg, C. P. et al. Proc. Am. Assoc. Cancer
Research 2: 101, 1956.
8. Magi 11, G. B. et al. Proc. Am. Assoc. Can-
cer Research 2: 130, 1956.
9. Sternberg, S. S. ct al . Proc. Am. Assoc.
Cancer Research 2: 150, 1956.
10. Sugiura, K. and Sugiura-Schmid, M. Proc.
Am. Assoc. Cancer Research 2: 151, 1956.
11. Maxwell, R. E. and Nickel, Y. S. Antibio-
tics & Chemotherapy 7:81-89, 1957.
12. Potter, M. Ann. N. Y. Acad. Sci. 76:
630-642, 1958.
13. Burchenal, J. H. and Holmberg, E. A. D.
Ann. N. Y. Acad. Sci. 76: 826-837, 1958.
14. DeWald, H. A. and Moore, A. M. J. Am.
Chem. Soc. 80: 3941-3945, 1958.
15. Reilly, H. C. In Amino acids and peptides
DESCRIPTIONS OF ANTIBIOTICS
259
with antimetabolic activity. Ciba Foun-
dation Symposium. Little, Brown and
Company, Boston, 1958, pp. 62-74.
16. Krantz, S. et al . J. Natl. Cancer Inst.
22: 433-439, 1959.
17. Thiersch, J. B. Proc. Soc. E.xptl. Biol.
Med. 94: 33-25, 1957.
18. Magill, G. B. et al. Cancer 10: 1138-1156,
1957.
19. Potter, M. and Law, L. W J, Natl.
Cancer Inst. 18: 413-442, 1957.
20. Duvall, L. R. Cancer Chemotherapy
Rept. 7:86-98, 1960.
Dura 111 vein
Produced by: Streptoniyces cintuvnonieus f. azaco-
luta (1,3).
Synonym: Fraction B of antibiotic F 17(2). Cul-
ture-broths contain three or more antibiotics
(3) related to cinnamycin.
Method of extraction: Broth extracted with buta-
nol. Addition of heptane causes separation of an
aciueous layer containing most of the antibiotic.
Concentration in vacuo of watery layer, followed
by lyophilization. Purified by chromatography
on alumina at pH 4.7 with 80 ])er cent ethanol as
solvent and developer (2).
Chemical and physical properties: Slightly acidic
polypeptide. Soluble on heating in absolute metha-
nol, water-methanol, and water-ethanol. Forms a
crystalline picrate and alcoholate, and a helian-
thate. HCl: Solul)le in water, aqueou.s acetone,
methanol, and ethanol. Slightly soluble in alisolute
alcohols. Surface-active. Infrared data given in
reference 2. No ultraviolet absorption. {a]t~' =
— 6.4° (c = 3.9 per cent in water). No definite
melting point. Positive biuret and azide-iodine
tests. Negative FeCls , Benedict, Molisch, periodic
acid, Millon, nitroprusside, Pauly, Sakaguchi,
.xanthoproteic, and Hopkins-Cole tests. Stable to
heat at pH 3 to 9. Acid hydrolysates contain
lanthionine, /3-methylanthionine, aspartic acid,
glycine, glutamic acid, proline, valine, phenylala-
nine, and possibly ornithine and hydroxyproline.
Contains several free carljoxyl and at least one
free amino group. Picrate: m.p. 212-245°C (decom-
position). Insoluble in absolute methanol; soluble
in water-saturated butanol. C = 51.30%; H =
5.76%; N = 16.85%; S = 3.18% (2).
Biological activity: Active on gram -positive rods;
less active on fungi and yeasts (2).
References:
1. Pridham, T. G. et al. Phytopathology 46:
575-581, 1956.
2. Shotwell, O. L. et al. J. Am. Chem. Soc.
80: 3912-3915, 1958.
3. Lindenfelser, L. A. et al. U. S. Patent
2,865,815, December 23, 1958.
Ecliiiioniycin
Produced by: Streptomyces echinatus (2) and an
unidentified Streptomyces sp. (4).
Synonyms: Antibiotic X 948 (1, 4). May be
related to actinoleukin (5).
Method of extraction: Culture-filtrate extracted
at pH 7 to 8 with ethyl acetate; extract freed from
inactive bases and acids and concentrated in
vacuo. Active substance precipitated with petro-
leum ether and purified by chromatography over
alumina. Crystallization from methyl alcohol (3).
Chemical and physical properties: Weakly basic
(3). Colorless powder, m.p. 217-218°C (3); or
colorless prisms, m.p. 236-238°C (4). Soluble in 20
per cent HCl, but not in other dilute mineral acids.
Ultraviolet absorption spectrum maximum 243
ni/Li (log e = 3.81). Infrared spectrum given in
reference 3. [ajo = —310° (c = 0.86 per cent in
CHCI.3) (3). Negative tests for thio- and disulfide
groups (6). CosHstOtNtS (3, 4) or C,-,ij-62H6o-64-
O12N10S2 : C = 55.45%; H = 5.88%; N = 15.33%;
S = 5.27%; N-CH3 = 5.2%; C-CH3 = 2.75%.
Molecular weight, 1050 ± 50 or 1604 ± 30 (6).
Acid hydrolysis products include D -serine,
L-alanine, L-N-methyl valine, and N-methyl-N'-
phenylthiourea. Alkaline hydrolysis yields cjuin-
oxaline-2-carboxylic acid (3, 6). Probable struc-
tvu'al formula (6) :
.N,
CH3
CHs
H3C
\ /
CH3 CH
CH3
f^^^^ S— CO— NH— CH— CO— NH— CH— CO— N— C— CO— N CH— CO-O-CH,
^N^
S CH2
I I
CH2 s
CH,— O— CO— CH— N— CO — C— N— CO— CH-NH-CO-CH-NH-CO ^ ^
II II
HC CH3 CH3 CH3
/ \
HsC CH3
260
Di<:sc'uirTi()xy of antibiotics
BioUxjicdl (irtivitij: Active against gram-positive,
gram negative, and acid-fast bacteria, protozoa,
and viruses. Some caiicerolytic activity. Active
in vivo against Trypanosoma cfjuipcrdion and T.
brucci infections in mice (CJ)on about 0.30 mg per
kg subcutaneously). Local antit richomonal ac-
tivity (1).
Toxicity: Toxic to young chicks a1 0.01 per cent
in diet (4). LD50 (mice) 3.8 mg per kg subcutane-
ously, 0.75 mg per kg intra-abdominally, >2500
mg per kg orally (Ij, 0.4 mg i)er kg intraperitone-
ally (2).
References:
1. Schnitzer, R. J. Ann. N. Y. Acad. Sci. 00:
1090-1092, 1952.
2. Corbaz, R. et al. Helv. Chim. Acta 40:
199-204, 1957.
3. Keller-Schierlein, W. and Prelog, V. Helv.
Chim. Acta 40: 205-210, 1957.
4. Berger, J. et al. Experientia 1."}: 434-436,
1957.
5. Ishihara, S. et al. J. Antiljiotics (Japan)
UA: 160-161, 1958.
6. Keller-Schierlein, W. et al. Helv. Chim.
Acta 42: 305-322, 1959.
Echinomycin-like Antibiotic
Produced by: Streptoniyces sp. (1) differing from
echinomycin-i)rodvicer.
Synonym: Antibiotic X 1008 (1).
Method of extraction: Whole broth extracted with
butanol. Extract concentrated in vacuo. Anti-
biotic precipitated with petroleum ether. Suc-
cessively extracted into methylene chloride and
methanol, then crystallized from an ethanol-aceto-
nitrile mixture (1).
Chemical and physical properties: Cube-like
crystals; m.p. 209-216°C (decomposition), [aj^ =
— 282° (c = 1 per cent in chloroform), (lives a
melting point depression when mixed with echino-
mycin. Same ultraviolet spectrum as echinomycin.
Infrared spectrum differs in certain details from
that of echinomycin. C29H38O7N6S: C = 56.40%;
H = 6.52%; N = 13.69%; S = 5.08%. The side
chain attached to the quinoxaline residue probably
differs from echinomycin (1).
Biological activity: Same in vitro antibacterial
activity as echinomycin. No trypanocidal activity
in mice. Only jiartial cross-resistance with echino-
mycin.
Toxicity: Highly toxic (1).
Reference: 1. Berger, J. cl al. I'^xpericMitia I.'):
434-436, 1957.
Elirlicliin
Produced by: Streptomyces lavendulae.
Method of extraction: Culture-filtrate adjusted
to pH 2.0 with concentrated HCl. A dark brown
precipitate collected by centrifugation.
Chemical and physical properties: Stable at neu-
trality and alkaline pH. Nondialyzable. Inac-
tivated in vitro \>y horse serum. Unaffected \^y
tryptic digestion.
Biological activity: Inhibitory to influenza A
and influenza B /// vitro. Active in vivo against
influenza B. Inactive against bacteria, fungi,
Chlamydozoaceae, pox viruses, and bacterial
viruses.
Toxicity: LDn (mice) 100 mg per kg intra])eri-
toneally, 300 mg per kg subcutaneously.
Reference: 1. Groupe, V. et al. J. Immunol.
67: 471-482, 1951.
Elaioniycin
Produced by: Streptomyces gelaticiis (4) initially
identified as S. hepaticus (1, 3); Streptomyces sp.
(5).
Synomjm: Identical to or closely related to
hygroscopin A.
Method of extraction: I. Broth-filtrate extracted
with ethyl acetate at pH 7.0. Extract concentrated
in vacuo. Residue successively extracted with
water. Extract filtered, and extracted with petro-
leum ether at pH 7.0. Solvent removed in vacuo
and residual oil extracted with ether. Ether dis-
tilled off and oily residue subjected to molecular
distillation at pressures of <1 m at 60-61 °C. Puri-
fied by countercurrent distribution (n-heptane-
methanol-water containing 0.125 per cent (by
weight) Na2S04 ; 1.25:1.25:1). Active fractions
concentrated in vacuo at 37°C. Residue saturated
with NaCl and extracted with n-heptane. Extract
concentrated in vacuo and residue sulijected to
molecular distillation to give elaioniycin. II.
Residue from ethyl acetate-extract of Ijroth (see I)
extracted with n-heptane. Upper phase solvent
removed by evaporation in vacuo. Residue purified
by chromatography on HCl-washed alumina ad-
justed to pH 4.5, using multiple absorption col-
mnns in series, and n-heptane as solvent and
developer. Eluted with ether or methanol. Oily
residue after removal of solvent from active frac-
tions is subjected to molecidar distillation (1, 3).
Chemical and physical properties: Slightly yellow
oil. Soluble in all common organic solvents but
sparingly soluble in water. Ultraviolet absorption
spectrum maximum (methanol) 237.5 m/u (e =
11,000), remaining unchanged in polar and non-
DESCRIPTIONS OF ANTIBIOTICS
261
polar solvents and at acidic and alkaline pH in
aqueous solution. Peak gradually disappears in
0.1 A'^ NaOH. Infrared speotrvun given in reference
1. [aJc = +38.4° (c = 2.8 per cent in absolute
ethanol). Gives a deep purple color when placed
in light having a wave length of 256 n^fx. ni^ =
1.4798. Positive iodoform test. Negative FeCln ,
Benedict, ninhydrin, Sakaguchi, periodic acid,
xanthate (for OH~ groups), hydro.xamate (ester
groups), sodium nitroprusside (for methyl ke-
tone), and Ehrlich tests. Stable in neutral or
acidic aqueous solutions, but in 0.1 A' NaOH de-
composes into a yellow jiroduct of unknown char-
acter. C13H26N2O3 : C = 60.12%; H = 10.06%;
N = 10.95%. Molecular weight, 244. Acid hydrol-
ysis products include racemic «-hydroxyoctanoic
acid. Contains an aliphatic a,|8-unsaturated
azoxy group and exists in the l)-threo configura-
tion (1, 3, 6). Structural formula is given in Chap-
ter 6.
Bidhxjical artivilji: Active on human and bovine
varieties of M . (tthcn-nlosis but not on other types
of mycobacteria. Very slight activity on fungi.
Not active on bacteria. Not active in vivo (mice
and guinea pigs) on tuberculosis (2).
Toxicity: LD50 (mice) 43.7 mg per kg intra-
venously, 62.5 mg per kg subcutaneously (2).
References:
1. Haskell, T. H. et al. Antibiotics & Chemo-
therapy 4: 141-144, 1954.
2. Ehrlich, J. et al. Antibiotics & Chemo-
therapy 4: 338-342, 1954.
3. British Patent 730,341, May 18, 1955.
4. Anderson, L. E. et al. Antibiotics A: Chem-
otherapy 6: 100-115, 195C).
5. Ohkuma, K. et at. J. Antibiotics (Japan)
lOA: 224-225, 1957.
6. Stevens, C. L. ci at. J. Am. Chem. Soc.
80: 6088-6092, 1958.
Kluiopli,\ liii
Produced 1)1/: Strcptonn/cc.'i ntelariosporiis var.
nielanosporofaciens. The same organism ])roduces
melanosporin.
Synonym: Similar to azalomycin B.
Method of extraction: See melanosporin.
Chemical and physical properties: White crys-
tals; m.p. 178-183°C (decomposition), [a];" =
— 49° (in chloroform). Soluble in chloroform,
acetone, and ethyl acetate. Slightly soluble in
lower alcohols. Insoluble in water, ether, and
benzene. Tentative empirical formula: (CeHioO)),,.
Light-absorption maximum at 252 m/x. Infrared
absorjjtion spectr(un given in reference 1.
Biological activity: Active against gram positive
bacteria.
Toxicity: Mice tolerate 100 mg per kg intra-
peritoneally.
Reference: 1. Arcamone, F. M. et al. GioriK
mierobioi. 7: 2(17-2](i, 195i).
FIikIoitij fins
Produced by: Strcptomyces c/h/'/s (closely related
to S. hi/f/roscopicus) (7, 12), and Sti'cptomyces sj).
(2, 13).
Synonyms: Helixins A and B. Related to medi-
ocidin. A second, nonpolyenic, ether-soluble anti-
biotic (9-20F-1) present in cTidomycin l)r()ths may
be the same as helixiu C (2, 13).
Method of extraction: Mycelium and precipitate
from acidified broth extracted successively with
butanol. Extract evaporated with addition of
water. Residue treated with ether and boiling
benzene, then suspended in water and retreated
with ether. Aqueous layer (pH 8.5 to 9.0) centri-
fuged. Supernatant acidified to preci])itate dark
brown gummy antibiotic. Taken up in 0.05 A^
NaOH and precipitated with glacial acetic acid;
or, taken up in absolute alcohol and reprecipitated
with amyl acetate (2, 8). May also be extracted in
the same way as candidin and separated into com-
ponents A and B l)y covuitercurrent distribution
(ethyl acetate-n-propanol-0.1 .V aqueous NH4AC,
3:1:3) (13).
Chemical ami physical propciiiis: ('(Duplex:
Yellow-brown powder. Soluble in alcohols con-
taining 10 to 20 per cent water, methyl Cellosolve,
dimethylformamide, pyridine, glacial acetic acid,
0.2 A^ NaOH, or HCl. Soluble in water at pH 2.0
or below and at pH 7.0 or above; insoluble in water
at pH 4 to 6. Partiallj' soluble in methanol and
ethanol; sparingly soluble in dioxane. Insoluble
in ether, chloroform, benzene, ethyl acetate, ace-
tone, and other nonpolar solvents. Can be pre-
cipitated from aqueous solution by Na"*", Ca^""", or
Mg"'"+. Surface-active and forms emulsions easily.
Slowly diffusible. Low nitrogen content (about
3.7 per cent). Stable to acid and alkali and to auto-
claving at pH 7.0. pK,.,' = 2.5; pKa^ = 5.5 to 6.5;
pKa^ = 9. Contains two major components: A,
a tetraene; and B, a hexaene. A greater amount of
the tetraene was present in the helixin complex
than in the endomycin complex. The helixin I)
component is i)resent in varying quantities in
endomycin. Rf values for A and B are 0.04 and
0.37 (water-saturated n-butanol), 0.09 and 0.38
(water-saturated n-butanol on paper buffered
with 0.1 A' sodium phos])hate at pH 12.0). and
262
DESCRIPTIONS OF ANTIBIOTICS
0.30 and 0.70 (t-l)Utanol-\vater, 4:1). Endutnyrin
A: Ultraviolet absorption spectrum maxima at
about 292, 308, and 320 mjj. Endomynn B: LTltra-
violet absorption spectrum ma.xima at 338, 359,
and 380 m/x (13).
Biological activity: Active on fungi, bacteria,
and Trypanosoma cruzi. More active on yeast-like
fungi (0.25 to 10 ^g per ml) than on filamentous
fungi (10 to 13 /ng per ml). More active on gram-
positive than on gram-negative bacteria. Some
activity on T . cruzi infections in mice (2, 4). Active
(not fungicidal) on C. albicans in tissue culture
and lytic to Trichomonas vaginalis in vitro (5, 6).
Controls leaf rust of wheat (3), strawberry fruit
rot (Botrytis cinerea) (10), and turf brown jjatch
(Rhizoctonia solani) (9); provides partial protec-
tion against Pseudoperonospora cnbensis (11).
Some control of bean root rot (14) and downj^
mildew of broccoli {Peronospora parasitica (15)).
Antifungal activity enhanced l)y the neomycins
(16).
Toxicity: Mice tolerate 0.5 gm per kg but not
1 gm per kg (no route given) (2). The LID (least
injurious dose) to tissue cultures of chick heart,
spleen cells, and human skin are 100 to 200 jug
per ml, 100 to 200 ^g per ml, and 50 to 100 ^g per
ml, respectively (5, 6). Produces some al)normali-
ties in Allium cepa roots at 100 ppm (1). Nontoxic
as 10,000-ppm spray to wheat, tomato, or liean
plants (3).
Utilization: Control of plant diseases.
References:
1. Wilson, G. B. J. Heredity 41: 226-231,
1950.
2. Gottlieb, D. ct al. Phj'topathology 41:
393-400, 1951.
3. Anderson, H. W. and Gottlieb, D. Econ.
Botany 6: 294-308, 1952.
4. Packchanian, A. Am. J. Trop. Med. Hyg.
2: 243-253, 1953.
5. Hu, F. et al. A.M.A. Arch. Dermatol.
Syphilol. 70: 1-15, 1954.
(). Wilkins, J. R. and Hen.shaw, C. T. Exptl.
Parasitol. 3:417-424,1954.
7. Pomerat, C. M. and Leake, C. D. Ann. N.
V. Acad. Sci. 58: 1110-1124, 1954.
8. British Patent 705,622, May 17, 1954.
9. Shurtleff, M. C. Phytopathology 45: 186,
1955.
10. Horn, N. L. Phytopathology 46: 15, 1956.
11. Ark, P. A. and Thompson, J. P. Phyto-
l)athology 46: 634, 1956.
12. Tresner, H. D. and Backus, E. J. Appl.
Microbiol. 4: 243-250, 1956.
13. Vining, L. C. and Taber, W. A. Can. J.
Chem. 35: 1461-1466, 1957.
14.- Davison, A. D. and Vaughn, J. R. Plant
Disease Reptr. 41 : 432-435, 1957.
15. Natti, J. J. Plant Disease Reptr. U:
780-788, 1957.
16. Sokolski, W. T. and Burch, M. R. Anti-
biotics & Chemotherapy 10: 157-162,
1960.
Eiidoniyciii-Iike Complex, Ilelixiiis
Produced by: Streptomyces sp. (1).
Method of extraction: Precipitate formed in
filtered liroth at pH 3.0; extracted with ethanol.
Extract concentrated in vacuo; heli.xin precipitates
out as a red gum on addition of chloroform (1).
Gum dissolved in absolute ethanol, and water
added. Mixture filtered and extracted with ethyl-
ene dichloride to remove helixin C. Aqueous etha-
nol layer concentrated under reduced pressure
and extracted with diethyl ether following addi-
tion of NaHCOg . Aqueous layer acidified to pH
3.0 and precipitate filtered off and taken up in
absolute ethanol. Ethanol solution extracted with
ethyl acetate and filtered. Filtrate dried in vacuo.
By dissolving this preparation in 0.06 A' NH4OH
and extracting with 1:1 n-butanol-ethyl acetate,
helixin b is obtained, contaminated with a small
amount of helixin 1). The aciueous layer remaining
is extracted with n-butanol. Concentration of this
extract to dryness gives helixin A. Helixin B is
further purified l)y partition chromatography (3).
Chemical and physical properties: Helixin coni-
plcx: Contains four components. A, B, C, and D,
having approximate Rf values of 0.07, 0.42, 0.85,
and 0.68, respectively (butanol-ethyl acetate,
1:1) (3). Solul)le in ethanol, methanol, pyridine,
and glacial acetic acid. Slighth- soluble in n-buta-
nol, acetone, and chloroform. Insoluble in ether,
petroleum ether, benzene, ethyl acetate, and car-
bon tetrachloride. Negative Molisch, ninhydrin,
Hopkins-Cole, xantho})roteic, Millon, and FeCl.3
tests. More stable at alkaline pH; somewhat less
stable than endomycin (1). Most active at alkaline
pH (2). May have one or more components in
common with endomycin (3).
Biological actii^y: Active against yeasts and
fungi at 15 /ug per ml or less. Active on bacteria at
>30 Mg per ml (1). Control of Helminthosporium
vicioriae blight of oats and H. sativum seeding
blight of barley with seed treatment (greenhouse
tests). In the field, seed treatment with helixin B
controls wheat bunt, oat smut, and covered smut
of barley (4). Protective action against tomato
early blight (Alternaria solani) (2).
DESCRIPTIONS OF ANTIBIOTICS
2G.3
Toxicity: Helixin B toxic to tomato and cowpea
cuttings at 7.5 to 15 /ug per ml. Not toxic to whole
tomato plants at 90 /ig per ml when used as a water-
ing solution. No phytotoxic effects at 3 mg per
ml, applied as a spray on young bean, corn, cow-
pea, tomato, and wheat plants. Inhil)ition of seed
germination at 25 to 100 ^g per ml (2).
Utilization : Plant diseases caused hy fungi.
References:
1. Leben, C. et al. Alycologia H: 159-109,
1952.
2. Leben, C. and Keitt. (1. W. Phytopa-
thology 42: 168-170. 1952.
3. Smeby, R. R. et al. Phytopathology 12:
506-511, 1952.
4. Leben, C. et al. Phytopathology 43: 391-
394, 1953.
F]iiteroni> ciii
Produced by: Sireptoniyces alhireticuli (1, 3).
This culture also produces eurocidin, carl)omyciii,
and tertiomycin A (5).
Method of extraction: Extracted from culture-
broth with ethyl acetate at pH 2.0. Back-extracted
into Na-iCOs . Re-extracted into ethyl acetate at
acid pH (acidified with H.>S04). Recrystallized
from ethanol (2).
Chemical and physical properties: Acidic sub-
stance. Pale yellow crystals. Decomposes at 160-
162°C. Soluble in methanol, ethanol, ethyl acetate,
butyl acetate, and dioxane. Sparingly soluble in
water, acetone, and chloroform. Insoluble in
benzene, ether, and petroleum ether. Ultraviolet
absorption spectrum maximum at 300 to 320 mn
(ethanol). Optically inactive in methanol. Con-
tains carbonyl and C — OCHa groups. C = 38.21%;
H = 4.62%", N = 14.32%. CMsO-,^, . Releases
CO2 on addition of sodium bicarbonate (2-4).
Biological activity: Active on gram-negative
bacteria, including E. ccAi. Proteus, Serratia, Sal.
typhi, Shigella, V. cholerae, and Pseudonionas. Less
active on gram-positive bacteria, including Staph,
aureus, B. subtilis, Ps. aeruginosa, and myco-
bacteria. Active on certain viru.ses. Not active on
fungi. More active at acid than alkaline pH (2-4).
Toxicity: LD50 (mice) 135 to 138 mg per kg
intravenously (2, 4).
References:
1. Nakazawa, K. J. Agr. Chem. Soc. Japan
29: 647-649, 1955.
2. Nakazawa, K. J. Agr. Chem. Soc. Jajjan
29:659-661, 1955.
3. Shibata, M. Japanese Patent 4994, 1956.
4. Shibata, iVI. Japanese Patent 4995, 1956.
5. Miyake, A. et al. J. Antibiotics (Japan)
12A: 59-64, 1959.
El ythroniycin
Produced by: Streptomyces erythrcus (13).
Synonym: Erythromycin A.
Method of extraction: I. Broth-filtrate extracted
with amyl acetate or methyl isobutyl ketone at
pH 9.4. Back-extracted into water at pH 5.1.
Aciueous extract adjusted to pH 8.0, concentrated,
and readjusted to pH 9.5 to 11.0 to precipitate
erythromycin. Recrystallized from acetone -water
or petroleum ether (9, 13). II. Broth-filtrate de-
fatted with petroleum ether, adjusted to pH 8.5,
and extracted with ethyl acetate or butanol. Ex-
tracts evaporated to dryness in vacuo. Solid tritu-
rated with petroleum ether. Precipitated from
ethanol on addition of water and cooling. Re-
crystallized from ethanol and aqueous acetone to
give the "erythromycin acid addition salt." Also
isolated by absorption on acid-treated charcoal
and l)utanol elution. "Acid addition salt" sus-
pended in water, pH adjusted to 6.3, and washed
with CHCI3 . Aqueous layer extracted with amyl
acetate at pH 9.8. Extracts evaporated off in
vacuo. Residue recrystallized from aqueous etha-
nol (13). III. Crude powders, containing two or
more components of the complex, yield, on dis-
solving in nitromethane, warming, decolorizing,
and cooling, essentially pure erythromycin, leav-
ing the other components in the mother liquor.
Recrystallized from the same solvent and aqueous
acetone (37). IV. Crude erythromycin salt solu-
tion adjusted to pH 8.0 to 8.5, heated to 35-45°C,
stirred with acetone, ethanol, or isoi)ropanol and
a water-soluble salting-out agent such as NaCl.
The whole adjusted to pH 10 to 10.8, and heated
to 35-45°C to bring erythromycin into the organic
phase. Water added to tiu-bidity at 45°C and the
whole cooled to 15°C to give crystals (38).
Chemical and physical properties: Basic macro-
lide (1). Free base (hydrate or solvates from ace-
tone or chloroform): White needles of the hexag-
onal system or short rods; ni.]). 136-140°C or
134-136°C (15). If slow heating is continued, re-
solidifies, then melts again at 190-193°C (15). Very
soluble in alcohols, acetone, chloroform, aceto-
nitrile, and ethyl acetate. Moderately soluble in
ether, ethylene dichloride, and amyl acetate. Sol-
uble to 2 mg per ml in water (1, 13). Ultraviolet
absorj)tion spectrum maximum at 278 niyu (e = 27)
(15), 280 mfj. (e = 50, broad peak, pH 6.3) (1), or
288 to 289 niM (£'1™ 0.395) (13). Infrared spectrum
given in references 13 and 15. [afp = —78° (c =
1.99 i)er cent in ethanol) (1, 38). Separation from
264
DESCRIPTIONS OF ANTIBIOTICS
erythomyciii B by a variety of systems with paper
chromatography has been reported (18). Other
information on Rf values given in reference 13.
pK;/ = 8.6 to 8.8 (1, 15j. Cry.stallographic data
given in references 1, 13, and 16. Moderately stable
at pH 5.0 to 8.5; 75 per cent activity lost on boil-
ing for 1 hour; 50% lost after heating at 60°C for
5 minutes (3). C = 61.05%; H = 9.43%; N =
1.91%; C— CH, = 17.98% (13). After dra.stio acid
degradation, products include dimethylamine and
desosamine. Desosamine (3-dimethylamino-4-des-
oxy-5-methylaldopentose) (I): CsHnNO^HCl;
m.p. 183-184°C or 191-193°C. [a]f = +54.5° (c =
2 per cent in ethanol) (9, 15). Treatment with 0.3
A' HCl at 25°C yields a nitrogen-free sugar, cladi-
nose (II), a neutral oil, CsHif04 , and erythralos-
amine (III). Cladinose: Soluble in water, alcohols,
acetone, ether, and benzene; slightly soluble in
petroleum ether. Positive Tollen and iodoform
tests. Acid-labile (10, 25). Erythralosaniinc: Elon-
gated prisms; m.p. 206-207°C. Soluble in dilute
HCl, alcohols, acetone, ether, chloroform, and
benzene. Insoluble in water and dilute alkali.
Hydrolysis products include I (10, 15). Reduction
of erythromycin gives dihi/droerythroniycin, m.p.
133-135°C, C;nH69NOu (IV). Treatment with
HCl -methanol removed II, to give a substituted
polyhydroxylactone, C29H.55N()io (V), differing
from III. Acid hydrolysis of V gave dihydroeryth-
ronolide (VI) , C21H40O8 , in which the ketone group
of the aglycone of erythromycin (erythronolide)
has been reduced to a hydroxyl (24, 26). Structural
formula (C^HgtOisN) given in Chapter 6. Solvates:
Very stable to heating. Solvent easily removed on
addition of water (39). Hydrochloride: White
needles; m.p. 170-173°C. Very solul)le in lower
alcohols. Soluble to 40 mg per ml in water. Slightly
soluble in ethyl acetate, ether, amyl acetate, and
chloroform (5, 13). Infrared spectrum given in
reference 13. Acid addition salt (with unidentified
organic acid, see II under "Methods of Extrac-
tion"): White hexagonal needles; m.p. 82-83. 5°C
(13). A number of derivatives, none of which had
'activity greater than erythromycin, are reported
in references 31 and 32.
Biological activity: In vitro: Active on gram-
positive bacteria, mycol)actoria, corynebacteria,
Actinomyces israelii, Clostridia, and certain gram-
negative bacteria of the Heniophilns-Brucella
group (1-3, 0, 8). Not active on A', asteroides or
pathogenic fungi (8). Active on Endamoeba histo-
lytica only in the presence of bacteria, and Tricho-
monas vaginalis (1, 12). Active on pleuropneu-
monia-like organisms isolated from arthritic
goats (27). Most active at alkaline pH (3). Bac-
teriostatic or l)actericidal depending on concen-
tration of 1 he antibiotic and on the organism used.
Active only on multiplying bacteria (4). Cross-
resistance with carbomycin, spiramycin, oleando-
mycin, and streptogramin (22). In vivo: Active
(mice) on Streptococcus pyogenes, D. pneumoniae,
Hemophilus pertussis (only when organism was
injected intranasally, not intracerebrally), Co-
rynehacteriuni diphtheriae, Clostridium tetani,
Borrelia novyi, Leptospira icterohaemorrhayiae
(hamsters), and moderatel}' active on M. tubercu-
losis (1, 6, 7, 14, 29, 30). Active on Endamoeba
histolytica (rats) (12), Trichomonas vaginalis (1),
Trypanosoma equipcrdum (but not T. cruzi), and
toxoplasmosis in mice (1, 12). Active (chick em-
bryos) on Rickettsia proioazekii and R. typhi; mod-
erately active on R. rickettsii and R. akari; and
slightly active on Coxiella burnetii (7, 20). Active
(chick embryos) on psittacosis, meningopneumo-
nitis, lymphogranuloma venereum, and feline and
mouse pneumonitis agents (1, 6, 7, 21, 28), but
not on MM, Semiliki Forest, poliomyelitis (Lan-
sing type 2), influenza A (PR 8), or Ij'mphocytic
choriomeningitis viruses (7). Active (mice) on
oxyurids, Syphacia obvelata, and Aspicularis
let rapt era (12). Increases growth rate of swine,
chicks (11), and turkey poults (36).
Toxicity: Base: LD.,o (mice) 1800 to >2500 mg
per kg sul)cutaneously, 3112 ± 211 mg per kg
orally, and >700 mg per kg intraperitoneally
(1,5). LDou (rat) >3000 mg per kg orally, >2C00
mg per kg subcutaneously (5). LD50 (guinea pig)
413.4 ± 51.7 mg per kg intraperitoneally (5). A
delayed toxicity for guinea pigs was reported
(17). Pigs exhibiting toxic symptoms at as low a
dose as 1.5 mg per pig have no gross morphological
findings excejjt paleness and spleen shrinkage
(35). LDoo (hamster) 3018 ± 190 mg per kg orally
(5). Increasing toxicity to hamsters was noted
over a period following introduction ot the drug
into the laboratory- (30). Hydrochloride: LDso
(mice) 425.6 ± 15.7 mg per kg intravenously,
490.0 ± 30.4 mg per kg intraperitoneally, 1849 ±
89 mg per kg sul)cutaneously, and 2927 ± 162 mg
per kg orally (5). Nontoxic to carnation cuttings
at 120 ppm (23). Least injurious doses for human
skin and chick embryo spleen cells in tissue cid-
ture are 85 to 170 and 30 to 60 jug per ml, resjjcc-
tively (19). Highest concentration permitting
migration of epithelial cells in tissue culture is
2.5 mg per ml (40). Minimal dose inhibiting mitosis
ot HeLa cells is 1 to 2 mg per ml (33).
Utilization: Infections caused by gram-positive
bacteria, especially those in which penicillin can-
DESCRIPTIONS OF ANTIBIOTICS
265
not be used because of sensitivity of tlie patient
or resistance of the orji;anisni. Amoebiasis.
References:
1. McGuire, J. M. et al. Antibiotics & Chemo-
therapy 2: 281-283, 1952.
2. Welch, H. et al. Antibiotics & Chemo-
therapy 2: 693-696, 1952.
3. Haight, T. H. and Finland, M. Proc. Soc.
Exptl. Biol. Med. 81: 175-183, 1952.
4. Haight, T. H. and Finland, M. Proc. Soc.
Exptl. Biol. Med. 81: 188-193, 1952.
5. Anderson, R. C. et iil. J. Am. Pliarm.
Assoc . , Sci . Ed . 41: 555-559 , 1952 .
6. Heilman, F. R. et al. Proc. Staff Meetings
Mayo Clinic 27: 285-304, 1952.
7. Powell, H. M. et al. Antibiotics tt Chemo-
therapy 3:165-182,1953.
8. Fusillo, M. H. et al. Antibiotics & Chemo-
therapy 3: 581-586, 1953.
9. Clark, R. K. Antibiotics & Chemotherapy
3:663-671, 1953.
10. Hasbrouck, R. B. and (iarven, F. C. Anti-
biotics & Chemotherapy 3: 1040-1052,
1953.
11. Gerard, W. K. ct al. J. Agr. Food Chem.
1: 784-788, 1953.
12. McCowen, M. C. et al. Am. .J. Trop. .Med.
Hyg. 2: 212-218, 1953.
13. Bimch, R. L. and McGuire, J. M. U. S.
Patent 2,653,899, September 29, 1953.
14. Kiser, J. S. and deMello, G. C. Proc. 58th
Ann. Meeting U. S. Livestock Sanitary
Assoc. 81-97, 1954.
15. Flynn, E. G. et al. J. Am. Chem. Soc. 76:
3121 3131, 1954.
16. Rose, H. A. Anal. Chem. 2(»: 938-939,
1054.
17. Kaipainen, W. J. and Faine, S. Nature,
London 174:969-970,1954.
18. Sokolski, W. T. el al . J. Antibiotics (Ja-
pan) 4: 1057-1060, 1954.
19. Pomerat, C. M. and Leake, C. D. Ann. N.
Y. Acad. Sci. 58: 1110-1124, 1954.
20. Ormsbee, R. A. et al. J. Infectious Dis-
eases 96: 162-167, 1955.
21. Loosli, C. (;. el al. Antibiotics Ann. 474-
489, 1954-1955.
22. Jones, W. F. et al. Proc. Soc. Exptl. Biol.
Med. 93: 388-393, 1956.
23. Gasiorkiewicz, E. C. Plant Disease Reptr.
40; 421-423, 1956.
24. Sigal, M. V., Jr. et al. J. Am. Chem. Soc.
78: 388-395, 1956.
25. Wiley, P. F. and Weaver, O. J. Am. Chem.
Soc. 78: 808-810, 1956.
I. Am. Chem. Soc. 78:
Cornell Vet. 46: 206-
26. Gerzon, K. et al.
6396-6408, 195().
27. Adler, H. E. et al
216, 1956.
28. Erturk,0. Cornell Vet. 46:355-300,1956.
29. Anwar, A. A. and Tinner, T. B. Bull.
Johns Hopkins Hosp. 98: 85-101, 1956.
30. Cook, A. R. and Thompson, P. E. Anti-
biotics & C^hemotherapy 7: 425-434,
1957.
31. Clark, R, K., Jr. and Freifelder, M. Anti-
l)iotics & Chemotherapy 7: 483-486,
1957.
32. Clark, R. K., Jr. and Varner, E. L. Anti-
biotics & Chemotherapy 7:487-489, 1957.
33. Nitta, K. Japan. J. Med. Sci. & Biol. 10:
277-286, 1957.
34. Wiley, P. F. ct al. J. Am. Chem. Soc. 79:
6062-6070, 1957.
35. Tigertt, W. D. and Gochenour, W. S., Jr.
Nature, London 180: 1429-1430, 1957.
36. McGinnis, J. et al. Poultry Sci. 37: 810-
813, 1958.
37. Clark, R. K., Jr. U. S. Patent 2,823,203,
February 11, 1958.
38. Friedland, W. C. el al. U. S. Patent 2,833,
696, May 6, 1958.
39. Croley, 1). R. U. S. Patent 2,864,817, De-
cember 16, 1958.
40. Lawrence, J. C. Bril . J. Pharmacol. 11:
168-173, 1959.
Kryllironi_> oiii IJ
I'roditccd by: Sireploiui/ce.s ert/threii.s.
Synorif/in: Closely related to erythromycin.
Remarks: The i)roportions of the erythromycins
jjroducetl can be varied by changing the nitrogen
content of the culture media (5).
Method of extraction: I. Broth at pH 9.5 ex-
tracted with chloroform or amyl acetate, then
l)ack-extracted into 0.1 M phosphate buffer at pH
5.2. Extraction procedures repeated. Purified and
separated from erythromycin by column chro-
matography on powdered cellulose with 0.01 N
XH4OH, saturated with methyl isobutyl ketone
as develo]3er and eluant, or l>y countercurrent
distribution (acetone-methyl isobutyl ketone-0.1
A' i)hosphate buffer, pH 6.5, 1:20:20). Active frac-
tions concentrated in vacuo; extracted from the
aqueous solution with chloroform at pH 9.6 to
10.5. Chloroform evaporated oft", residue extracted
with ether, again eva])orated, and the antibiotic
crystallized from acetone (1,2). II. Amyl acetate
extract of broth back-extracted into aqueous
acetic acid at pH 5.0 to 6.5 and traces of amyl
2()t)
DESCRIPTIONS OF ANTIBIOTICS
acetate removed by distillation in vacuo. Salted
into acetone at pH 10 and precipitated from ace-
tone by addition of water. Crystallized from ace-
tone as a mixture of A and B. Mixture dissolved
in an aqueous solvent at pH 1.4. Standing at this
pH for 40 minutes destroys A. B is precipitated
on addition of NaOH, crystallized from water-
acetone, and recrystallized from dry acetone (7).
Chemical and physical properties: Basic sub-
stance. Rectangular plates; m.p. 198°C or 201-
203°C (uncorrected). Soluble in ether, acetone,
chloroform, ethyl acetate, and benzene. Sparingly
soluble in water. Ultraviolet absorption spectrum
maximum at 289 niju {E = 36.4). Infrared spectrum
given in reference 2. [a]f = — 78° (c = 2 per cent
in ethanol). pKa' = 8.8. Molecular weight, 730.
More stable to acid than erythromycin, a property
which permits their separation. Rf = 0.6 (metha-
nol-acetone-water, 19:6:75); erythromycin Rf =
0.7. Water-soluble acid salts: Hydrochloride, m.p.
149-150°C; Stearate, m.p. 54-57°C. Base: C^v-
H„NO,., : C = 62.08%; H = 9.56%; N = 1.99%;
C— CH, = 14.61%; O— CHs = 4.79%. Mild acid
hydrolysis products include: neutral oil, cladinose,
C8H16O4 , and two crystalline bases, one optically
active, C29H56NO9 (m.p. 119-121°C), and the other
optically inactive, C29H51NO8 (m.p. 239-240°C).
Strong acid hydrolysis produces desosamine,
CsHnNOs . Structural formula of erythromycin B
(1, 2, 4, 6, 7) given in Chapter 6.
Biological activity: Qualitatively, B has the same
antimicrobial activity as erythromycin, but is
only 75 to 85 per cent as active quantitatively.
Cross-resistance exists between A and B. Resist-
ance to B develops more readily than to A (2, 5).
Toxicity: Twice as toxic as erythromycin (3).
References:
1. Pettinga, C. W. et al. Abstr. 124th Meeting
Am. Chem Soc. 47 O, 1953.
2. Pettinga, C. W. et al. J. Am. Chem. Soc.
76: 569-571, 1954.
3. Sylvester, J. C. and Josselyn, L. E. Antibi-
'otics Ann. 283-285, 1954-1955.
4. Clark, R. K. and Taterka, M. Antibiotics &
Chemotherapy 5:206-211, 1955.
5. Grundy, W. E. et al. Antibiotics & Chemo-
therapy 5: 212-217, 1955.
6. Wiley, F. F. et al. J. Am. Chem. Soc. 79:
6070-6074, 1957.
7. Denison, F. W. et al. U. S. Patent 2,834,714.
May 13, 1958.
Erytliromyciii C
Produced by: Streptomyces erythreus. This cul-
ture also produces erythomycins A and B.
Method of cctracliun: Broth-filtrate extracted
with chloroform at pH 9.75. Extracts concentrated
in vacuo and chilled to precipitate erythromycin
and erythromj'cin B. Supernatant dried in vacuo,
dissolved in upper phase of an equilibrated methyl
isobutyl ketone -0.1 A' sodium phosphate buffer
(pH 6.5)-acetone system (20:20:1) and subjected
to countercurrent distribution to separate C from
erythromycin. Active fractions containing C ad-
justed to pH 9.75 and extracted with chloroform.
Extract dried over anhydrous Na2S04 , filtered,
and concentrated in vacuo to precipitate C.
Chemical and physical properties: Basic sub-
stance. Needle-shaped crystals; m.p. 121-125°C.
Can be separated from erythromycin by chroma-
tography' on cellulose, developed with 0.01 A'
NH4OH saturated with methyl isobutyl alcohol.
Soluble in chloroform, acetone, and ether. Rela-
tively insoluble in water. pKa' = 8.5. Ultraviolet
absorption spectrum maximum at 292 ni/i (E =
108). Infrared spectrum given in reference 1.
CseH^jNOu : C = 59.75%; H = 9.10%; N =
1.95%; O = 29.49%. Molecular weight, 730. Acid
methanolysis yields erythralosamine and a neutral
sugar, C7H14O4 . Although this formula is the same
as that of mycarose from carbomycin, the two
sugars are not the same. Erythromycin C differs
from erythromycin by the absence of the meth-
oxyl group in cladinose (see Chapter 6) .
Biological activity: Similar to erythromycin and
erythromycin B.
Reference: 1. Wiley, P. F. et al. J. Am. Chem.
Soc. 79: 6074-6077, 1957.
Etaniyciii (Viridogrisein)
Produced by: Streptomyces sp. resembling S.
lavendulae (1), S. griseus (2, 5), and S. griseoviri-
dus (5).
Synonyms: Antibiotic K 179 (11), viridogrisein.
Possible synonym: antibiotic 6613 (12, 13).
Remarks: Etamycin contains four amino acids
not previously found in nature (9).
Method of extraction: I. Broth extracted with
methyl isobutyl ketone or ethylene dichloride.
E.xtract concentrated and: (a) concentrate added
to 10 volumes of Skellysolve B, which forms a pre-
cipitate of crude etamycin. Crude etamycin dis-
solved in acetone and jjrecipitated as the hydro-
chloride; or (b) chromatographed on alumina
adjusted to pH 5.0 to 7.0 with HCl. Eluted with 50
per cent ethyl acetate, 40 per cent methanol, and
10 per cent water. Purification bj^ countercurrent
distribution (benzene-methanol-water-n-heptane-
(Na)2S04 , 7:10:6:6:0.125 by weight with respect
to water) (1, 2). II. Precipitates almost quanti-
DESCRIPTIONS OF ANTIBIOTICS
267
tatively from sohitions of ethanolic ;iimnonia as
the ammonium salt, leaving almost all impurities
in solution. This colorless, crystalline salt is un-
stable in the dry state and decomposes to the
pure antibiotic in vacuo (10).
Chemical and physical properties: Macrocyclic
peptide lactone, containing eight amino acids.
Weakly basic (1) or amphoteric (3). Base: Color-
less amorphous powder (10). Soluble in lower alco-
hols and ketone, benzene, chloroform, carbon
tetrachloride, carbon disulfide, ethyl acetate,
ether, 1 .V HCl, and 1 X NaOH. Slightly soluble
in water. Insoluble in petroleum ether (1, 9).
\^l° 350 (E\7n. 71) with a shoulder at 303 (^I?,„ 34)
(3). [a]f = +7.7° (c = 2 per cent in methanol) (1).
Positive tests for an active methylene group, pep-
tide bond, ester, and amide groupings (hydroxamic
acid test). Brownish red color with F'eCh test.
Weakly positive Folin-Ciocalteau and Bayer tests.
Negative ninhydrin and Sakaguchi tests (1, 3).
pKa' = 7.4 (10 per cent acjueous ethanol). Stable
at acid pH and neutrality; inactivated by alkali.
Esterihcation destroys microbiological activity.
Calculated formula: C44H620„N8-H20: C =
59.0%; H = 7.05%; N = 12.3%; N— Me = 5.35%.
Molecular weight, 800 to 900. Proposed structure
(9):
OH
negative bacteria and C. albicans. Active in vivo
against experiniental D. pneumomae and Staph,
aureus infections in mice and E. histolytica in-
fections in rats and dogs. Some antirickettsial
activity; no antiviral activity. Active against
bovine mastitis, infectious bronchitis of chicks,
and panleukemia of cats (1. 4, 6). Active in ovo
on pleuropneumonia-like organisms (avian) (7).
Intermediate activity on sarcoma 180 in mice (8).
Toxicity: LD50 (mice) 273.5 mg per kg intra-
peritoneally, >2000 mg per kg subcutaneously,
>3000 mg per kg orally. Etamycin produces a
reversible leukopenia in dogs (1). At certain con-
centrations, given orally or subcutaneously to
mice, nonedematous weight gain without increased
food intake was noted (4).
References:
1. Heinemann, B. et al. Antibiotics
728-744, 1954-1955.
2. Bartz, Q. R. et al. Antibiotics Ann.
783, 1954-1955.
3. Haskell, T. H. et al. Antibiotics
784-789, 1954-1955.
4. Ehrlich, J.e/oL AntiVnotics Ann. 790-805,
1954-1955.
5. Anderson, L. E. et al. Antibiotics & Chem-
otherapy 6: 100-115, 1956.
Ann.
777-
Ann.
i5-hy(lr(ixypir()lii lie acid
H3C
-Ml
O I CH— C XH
L-Threiminc^ ,.,^ ^,j^
O
l.euciue
L-d-l'hcnvlsa:
c=o
1
CH— X— C— CH— NH— C
! I
CH, CH3
L-.\laniiu'
Sarcosine
L-^ , X-l)iiiiethylleuciiie
Hydrochloride: m.p. 163-170°C (decomposition).
Soluble in methanol, ethanol, formamide, and in
water to the extent of 4 mg per ml. Slightly sol-
uble in acetone, ethyl acetate, and less polar sol-
vents. X*'mSc"304 {E\'^^86) with strong end-absorp-
tion at <250 (1).
Biological activity: Active in vitro against gram-
positive bacteria, including Clostridia, at a con-
centration of 0.04 to 2.5 fj.g per ml. Active on
Endanioeba histolytica. Inactive against mostgram-
6. Thompson, P. E. et al. Antibiotics &
Chemotherapy 6: 337-350, 1956.
7. Yamamoto, R. and Adler, H. E. Am. J.
Vet. Research 17: 538-542, 1956.
8. Field, J. B. et al. Cancer Research 18:
(Suppl. 1) 503, 1958.
9. Sheehan, J. C. et at. J. Am. Chem. Soc.
80: 3349-3355, 1958.
10. Arnold, R. B. et al. J. Chem. Soc. 4466-
4470, 1958.
268
DESCRIPTIONS OF ANTIBIOTICS
11. Magyar, K. ef al. Al)str. C'omimms. Sym-
posium on Antibiotics, Prague, pp. 26-27,
1959.
12. Kudinova, M. K. Antibiotiki 1(2): 29-33,
1959.
13. Brajnikova, M. G. e/ o/. Antibiotiki 4(4):
29-32, 1959.
Ett
usconivcin
Produced by: Streptomjces lucensis (1, 2).
Synonym: Antibiotic 1163 F. I. (2).
Method of extraction: Mycelium and culture-
filtrate extracted with n-butanol, methanol, etha-
nol, or isopropyl alcohol. Extract concentrated
in vacuo to give an active precipitate. Ether added
to the mother liquors precipitates a fvuiher active
fraction. Purified by washing with acetone, fol-
lowed 1)\ liot a<iueous isopropanol ; taking up in
anhydrous methanol containing CaClo . Precipi-
tated as the base on addition of water. Chroma-
tograi)hed on silica gel. Crystallized from water
saturated with butanol (1, 3).
Chemical and physical properties: Amphoteric
conjugated tetraene. White crystals. Browns,
gradually followed by decomposition at >150°C.
Soluble in dimethylformamide, pyridine, glacial
acetic acid, and by the formation of salts in acidic
or alkaline methanol. Moderately soluble in aque-
ous lower alcohols; slightly sokdjle in anhydrous
methanol and water; insoluble in acetone, chloro-
form, ether, benzene, and other nonpolar solvents.
Ultraviolet absorption spectrum maxima at 290,
305, and 317 ni/x (E\l'^ about 840, 1385, and 1170).
Infrared spectrum given in reference 3. [a\„ =
+49.8° (in methanol containing 0.1 A' HCl) ; +296°
(pyridine). Gives a red-brown color with sulfuric
acid. Positive KMn04 and bromine in carbon
tetrachloride tests. Negative FeCl;i and Molisch
(weak brown) tests. Stable in the dry state. Lal)ile
to heat, light, air, alkali, and acid. Most stal)le in
aqueous solution at pH 7.0 (3). Rf values in vari-
ous systems of paper chromatography given in
reference 3. Broth was reported to contain three
components, A, B, and C, all differing in their
physicochemical characteristics (2).
Biological activity: Moderately active on yeasts,
fungi, Trichomonas vaginalis, and Endanioeba his-
tolytica. Active in vivo (mice) on intestinal Candida
infections. Also active (rats) on Endanioeba nniris
and intestinal flagellates. Not active on bacteria
(1).
Toxicity: LDso (mice) 44.6 mg per kg intraven-
ously, 37.1 mg per kg intraperi1on(>ally, and 1263
mg per kg orally (1).
References:
1. Arcamone, F. et al. Giorn. microbio!. 4:
119-128, 1957.
2. DiMarco, A. and Ghione, M. (iiorn. ital.
chemioterap. 4: 451-461, 1957.
3. Arcamone, F. and Perego, M. Ann. chim.
(Rome) 49:345-351,1959.
Eiiliciii
Produced by: Streptomyces sp. closely related to
S. parvus. This organism also produces an actino-
mycin and a basic antibiotic which is active
against gram-positive and gram-negative bacteria.
Method of extraction: I. Adsorption on char-
coal, followed by elution with acidic alcohol. II.
Adsorption on cation exchange resins and elution
with acid. III. Precipitation of antibiotic from
culture-filtrate as the insoluble picrate. The crude
picrate extracted with methanol; addition of hy-
drochloric acid and ether to the methanolic ex-
tract precipitates eulicin hydrochloride. The crude
hydrochloride is passed through a column of
Duolite S-30 resin. Effluent from the column is ti-
trated with a solution of methyl orange, resulting
in the precipitation of the insoluble helianthate.
The helianthate is crystallized and recrystallized
from 80 per cent ethanol. Eulicin hydrochloride is
obtained by the addition of an excess of hj'dro-
chloric acid to methanolic solutions of the helian-
thate. The helianthic acid precipitate is removed
by filtration, and eidicin hydrochloride is pre-
cipitated by adcHtion of ether.
Chemical and physical properties: Basic sub-
stance. HCl salt: White hygroscopic powder.
Poorly defined infrared spectrum. Helianthate:
Sinters at 142°C; m.p. 154-156°C (decomposition).
Free base: Hygroscopic gummy substance decom-
posing with release of ammonia within a few hours
of preparation. Weakly dextrorotatory. Positive
Sakaguchi test. Alkaline hydrolysis products in-
clude 9-aminononanoic acid. Acid hydrolysis
products include 9-guanidinononanoic acid and
"eulicinine," the latter prepared as the helian-
thate; m.p. 155-158°C. Structure of (Miiicinine
(1,4):
NH
H,NCNH(CH2)8CH— CH(CH2)3NH2
I I
OH NH.
The structure of eulicin (C24H62O2N8) is given in
Chapter 6.
Biological activity: Active /// vitro on yeasts and
fungi at 0.018 to 5.0 ng, per ml, but only very
slightly active (121 ng, \wv ml) on C. albicans. Ac-
tive on X. asteroides at 2.3 jug per ml (1, 2). Active
DESCRIPTIONS OF ANTIBIOTICS
269
in mice against exjioriniental infections caused
bv B. ilniHdlitidls and ('ryptorocfiis )ieofannaiis
(1,3).
Toxicity: LDju (mice) 3 mg per kg intravenously,
17 mg per kg intraperitoneally, 12 mg per kg intra-
muscularly, and 40 mg per kg suhcutaneously (1).
References:
1. Charney, J.c/a/. Antibiotics Ami. 228-235,
1955-1956.
2. Muller, W. H. Am. J. Botany 45: 183-190,
1958.
3. Solotorovsky, M. cl nl . Antil)iotics & Chem-
otherapy 8: 3(14-371, 1958.
4. Harmon, R. E. ct al. J. Am. Chem. Soc.
80: 5173-5178, 1958.
Kiim\ oelin
Produced by: Strcptoniyces sp.
Method of extraction: Washed mycelium ex-
tracted with methanol, the extract concentrated
in vcK IK), and lyophilized. Residue extracted re-
peatedly with ethanol, and the ethanol extract
concentrated. Baryta water is athled to the con-
centrate until no more ])reci])itat ion occurs. Pre-
cipitate removed l)y centrit'ugation; excess
Ba(OH)2 precipitated l)y CO2 . An inactive i)re-
cipitate forms on adding an etiual volume of H2O,
and is filtered off. Eumycetin ])recipitated on
chilling for 24 to 48 hours. Recrystallized from
50 per cent ethanol.
Chemical and phi/t^ical properties: Colorless
needles; m.p. 148-150°C. Soluble in methanol,
ethanol, l)utanol, acetone, ether, chloroform, and
ethyl acetate. Sparingly soluble or insoluble in
water, 10 per cent HCl, and 10 per cent NaOH.
Negative biuret, Fehling, ninhydrin, Alillon,
Molisch, Liebermann-Burchard, Sakaguchi, and
Rosenheim tests. Positive FeCls and diazo tests.
No color produced by addition of concentrated
sulfuric or hydrochloric acids. Ultraviolet spec-
trum shows peak at 302 ni/i (c = 0.02 per cent in
methanol).
Biological activifi/: Active on fungi. Inhibits
after 72 hours: Tonila rubra, M jicodernia sp., and
P. chrysogenum at <0.05 pg per ml; Oidiuni luctis
at 1 /ug per ml; Aspergillus oryzae at 0.325 ^tg per
ml; Willia anomala at 0.5 jug per ml; Saccharoniy-
ces sake at 1.0 ng per ml; C. albicans at 10 fxg per
ml; and Trichophyton interdigitale at 0.15 /ig per
ml. Active against Xocardia and Streptoniyccs.
Not active against bacteria. Mycobacterium (107
inhibited by 100 /ug per ml. Activity against ('.
albicans affected by horse serum. No hemolytic
activity against rabbit red blood cells.
Toxicity: LDso (iu water with carboxymethyl-
cellulose) 3.0 mg per kg svd)cutaneously in mice,
2.2 mg per kg intraperitoneally.
Reference: 1. Aral, T. and Takamizawa, Y. J.
Antibiotics (Japan) 7A: lt)5-l(J8, 1954.
Eiiriniyciii
Produced by: Streptoniyccs sp.
Method of extraction: Clarification of broth at
pH 2.0 with 1 per cent "Kerozite" with agitation
for 1 hour. Active filtrate adjusted to pH 8.2 with
sodium hydroxide; the precipitate that forms is
filtered off. The filtrate treated with 1.5 per cent
Kerozite (2 hovu'S of agitation). Elution with acidic
methanol (0.37 per cent HCl). Neutralization of
the eluate with sodium hydroxide, and concentra-
tion in vacuo. Addition of acetone in excess gives
an inactive precipitate, and the antibiotic is pre-
cipitated as the picrate from the active layer. The
solid hydrochloride may be precipitated by pe-
troleum ether. Purification by chromatography
on alumina. Inactive impurities removed with
acidic methanol (10 p(>r cent formic acid) and the
antibiotic eluted with water.
Chemical and physical properties: Paper chroma-
tography (n-butanol -propionic acid-water, 4:1:5)
indicates three constituents, of which two are ac-
tive and ninhydrin-positive. Light cream-colored.
Sulfate very solul)le in water (hygroscopic), solu-
ble in methanol, slightly solul^le in ethanol, and
very slightly soluble in acetone. Practically in-
soluble in ether and ligroin. Precipitated as a pic-
rate, phosphotungstate, helianthate, and reineck-
ate. Positive ninhydrin, Sakaguchi, biuret, and
creatinine tests. Creatinine test more strongly
positive after acid hydrolysis. Ammonia is released
during alkaline hydrolysis, with loss of a guanidino
group. Negative maltol, Schifl', Fehling, and glu-
cosamine tests. A salmon color in 3.5 per cent HCl
is intensified by boiling; a pale yellow color in 10
per cent NaHCO.-j fades on boiling. Thermostable.
Little loss in activity noted after heating for 3
hotu's in 5 per cent BaOH over a steam bath, or
30 minutes at 100°C at pH 2.0. The principal sub-
stance, purified by chromatography on a cellulose
column and developed with n-butanol-acetic acid-
water (4:1.8:5), decomjjoses at 155°C (chloride)
andl30°C (sulfate).
Biological activity: Active on gram-positive and
gram-negative bacteria and mycol)acteria. Pro-
tects mice and partially protects rabbits against
infection with Staph, aureus, (iuinea pigs pro-
tected against infection with B. anthraci.'^.
Toxicity: LDo (mice) 2(X) mg per kg sul)cutane-
ously; 700 mg per kg (rabbits); and 300 mg per
kg (guinea pigs).
270
DESCRIPTIOXh! OF ANTIBIOTICS
Referenre: 1. Cionq'alve.s de Lima, O. ct al.
Anais soc. biol. Pernainl)ueo 12: 9-17, 1954.
Eurocidiii
Produced by: Streptomyces albireticitli (4), S.
eurocidicus (2, 3), and Streptomyces sp. (6). S.
albiretiodi produces carbomycin, tertiomycin A,
and enteromycin (10). S. eurocidicus also produces
a eurocidin-like antibiotic (EI-I), as well as azo-
mycin and the tertiomycins. E-I may be more like
"pentaene antifungal antibiotic I." A Strepto-
myces sp. (6) also produces a eurocidin-like sub-
stance (E-II) and an actinomycin.
Remarks: See also eurocidin-like antil)iotic.
Method of extraction: Eurocidin: Extracted from
myceliiun with methanol. Crystallized from aque-
ous methanol (5). E-I: Broth-filtrate extracted
with butanol. Extract evaporated to dryness in
vacuo. Residue taken up in methanol and precipi-
tated by adding ether. Small amount present in
mycelium extracted with methanol (3). E-II:
Methanol extracts of mycelium evaporated to
dryness in vacuo. Residue taken up in ethyl ace-
tate. Extract evaporated to dryness in vacuo.
Chromatographed on alumina from an acetone
solution and developed with benzene, acetone
(15 per cent )-benzene, followed l)y acetone (30
per cent) -benzene, then acetone. Antibiotic eluted
with 50 per cent methanol (6).
Chemical and physical properties: Pentaenes.
Eurocidin: Gray-white or cream-colored sub-
stance. Does not melt up to 300°C. Insoluljle or
sparingly soluble in water, butanol, methanol,
ethanol, ether, ethyl and butyl acetate, propylene
and ethylene glycol, and acetone. Soluble in acids,
alkalies, ])yridine, dimethyl sulfoxide, and hy-
drous organic solvents (1, 5, 7-9). Ultraviolet ab-
sorption spectrum maxima at 318, 332, and 350 m/j,
(ethanol) (1). [a]'^ = -200° (c = 0.25 per cent in
0.1 N HCI) and +22° (c = 0.25 per cent in 0.1 N
NaOH). Positive Fehling test. Negative biuret,
Molisch, and Sherivanov tests (5). Rf = 0.40 by
ascending paper chromatography (methanol-
butanol-water, 9:10:10) (8). C = 57.99%; H =
8.13%; N = 1.65%. No halogens or sulfur (5).
E-I: Soluble in water, methanol, and ethanol.
Very slightly soluble in acetone. Insoluble in ben-
zene, ether, and petroleum ether. Ultraviolet ab-
sorption spectrum maxima at 318, 333, and 351
m;u (3). E-II: White substance. Ultraviolet ab-
sorption spectrum maxima at 310 m^t (shoulder),
322.5 niM (E'lL 125), 338.5 mfx iE\°L 179), and 356.5
m/i (E\7,n 164) (6).
Biological aiiirity: Eurocidin: Active on yeasts,
filamentous fungi, and certain protozoa, including
Trichomonas rayinalis (1, 5). Active in mice on T.
vaginalis (9). E-I: Active on yeasts and fungi but
not on bacteria or A', asteroides (3). E-II: Active
on ('. albicans and ,4. niger at 25 to 50 ng per ml
(6).
Toxicity: Eurocidin: LDso (mice) 22 mg per kg
intraperitoneally (1). E-I: LDs,, (mice) 36 mg per
kg (no route given) (3).
References:
1. Nakazawa, K. 72nd Meeting Japan. Anti-
biotics Research Assoc. September, 1953.
2. Okami, Y. et al. J. Antibiotics (Japan)
7A: 98-103, 1954.
3. Utahara, R. et al. J. Antibiotics (Japan)
7A: 120-124, 1954.
4. Nakazawa, K. J. Agr. Chem. Soc. Japan
29: 647-649, 1955.
5. Nakazawa, K. J. Agr. Chem. Soc. Japan
29: 650-652, 1955.
6. Maeda, K. et al. J. Antiljiotics (Japan)
9A: 125-127, 1956.
7. Shibata, M. Japanese Patent 4995, 1956.
8. Steinman, I. D. Thesis, Rutgers Univer-
•sity, 1958.
9. Hamada, Y. Osaka Daigaku Igaku Zassi
7: 237-245, 1955.
10. Miyake, A. et al. J. Antil)iotics (Japan)
12A: 59-64, 1959.
Eurocidin -I ike An lihiolic
Produced by: Streptomyces sp. belonging to the
S. reticuli group. This organism also produces
netropsin and antibiotic 2814K.
Method of extraction: Mycelium extracted with
butanol.
Chemical and physical properties: Amorphous
yellow pentaene. Soluble in concentrated H2SO4 ,
giving a blue-violet color. Ultraviolet absorption
spectrum maxima at 317, 332 to 333, and 350 m^u.
Has nitrogen content and solubility properties
similar to eurocidin, but does not show the [a]'^ =
— 200° (0.1 A' HCI) given for eurocidin.
Biological activity: Active on Sacch . cerevisiae
at 1:400,000.
Reference: 1. Thrum, H. Nat urwissenschaften
46: 87, 1959.
Everici 11
Produced by: Probably a Streptomyces.
Method of extraction: Broth acidified with oxalic
acid to pH 2.0 and filtered. Broth neutralized with
NaOH and adsorbed on cation exchange resin.
Elution with 1.5 A' HCI. Neutralized eluate evapo-
rated to dryness under reduced pressure. Extrac-
tion of residue with methanol. Precipitation of
DESCRIPTIONS OF ANTIBIOTICS
271
crude evericin with acetone. Further purification
liy chromatography on charcoal.
Chemical and physical properties: Basic antibi-
otic. Unstable at 100°C. Hydrochloride salt hygro-
scopic. Sulfate not hygro.scopic. Sulfates of 370
units per mg (1 unit = 1 yug of streptomycin by
diffusion assay) were obtained. Evericin diffuses
more slowly than streptomycin in agar. Ultra-
violet al)sorption spectnnn shows end-absorption
and a characteristic shoulder at ajiproximately
240 ni/Lt. Maltol test negative. Negative FeCls and
Molisch tests. Positive Tollen, biuret, and nin-
hydrin reactions. On chromatography of the acid
hydrolysate on activated carbon, the first frac-
tions contain four atypical amino acids; subse-
c[uent fractions contain no amino acids l>ut gvumi-
dine. [»]„ = —20.1° (c = 1 percent in water.)
Biological activity: Bacterial spectrum similar
to streptomycin. Active against streptomycin-
resistant strains.
Toxicity: LDsu (mice) 10 to 12.5 mg per kg in-
travenously and subcutaneously.
Reference: 1. Bodanskj', M. Acta Chim. ."i:
237-241, 1953.
Exfoliatin
Produced by: Streptoniyces exfoliatiis (1).
Method of extraction: Broth at pH 7.0 extracted
with ethyl acetate. Extract distilled in vacuo.
Residual syrup washed with petroleum ether to
precipitate exfoliatin. Crystallized from hot etha-
nol on cooling (1 ).
Cheniicol and physical properties: Colorless
needles; m.p. 172°C. Soluble in ethanol, acetone,
chloroform, and ethyl acetate. Very slightly solu-
ble in petroleum ether, ether, and water. Positive
Molisch and FeCls tests. Negative Fehling, Tol-
len, and Liebermann-Burchard tests. C = 50.74%;
H = 6.10%; CI = 5.48%. C27H4nOi9Cl (1, 2).
Biological activity: Active on gram-positive bac-
teria. Less active on mycobacteria. Not active on
fungi or gram-negative bacteria, except Hemo-
philus (1 , 2).
Toxicity: LDsi, (mice) 500 mg per kg subcutane-
ously (1, 2).
References:
1. Umezawa, H. et al. Japan. J. Med. Sci. &
Biol. 5: 311-316, 1952.
2. Umezawa, H. et al. J. Antiljiotics (Japan)
5: 466, 1952.
Feriiiicidiii
Produced by: Streptomyces sp. similar to S.
griseolus.
Method of extraction: Filtered broth acidified to
pH 3.0 to 4.0, treated with activated carl)on, eluted
with 80 per cent acetone. Concentration of eluate
in vacuo followed by extraction with chloroform.
Chloroform removed under reduced pressure, and
the residue adsorbed on a carbon column. Column
washed with 20 per cent acetone, and fermicidin
eluted with 60 per cent acetone. Acetone evapo-
rated to dryness in vacuo, and the residue dissolved
in benzene, absorbed on alumina, and eluted with
1 per cent methanol containing benzene. Metha-
nol-benzene solution evaporated to dryness in
vacuo. Recrystallization from ether.
Chemical and physical properties: Colorless
needles; m.p. 96-98°C. Soluble in methanol,
ethanol, chloroform, benzene, and ethyl acetate.
Slightly soluble in water and ether. Insoluble in
petroleum ether. Aqueous solutions are weakly
acidic. [a]o = +52.3° (c = 0.62 per cent in water).
Stable for 1 hour at 100°C at pH 5 or less. Unstable
at alkaline reaction. Ultraviolet absorption maxi-
mum at 290 m^u.
Biological activity: Active on Sacch. forniosensis
and Sacch. pastorianus at 0.04 /xg per ml; Sacch.
cerevisiae at 0.1 /jg per ml; Hansenula anomala
and Torula rubra at 0.2 ng per ml; Candida krusei
at 0.5 ng per ml; and Trichomonas vaginalis at 0.2
fxg per ml. Active on influenza virus; not active
against l)acteria and filamentous fungi.
Toxicity: LD50 (mice) 180 mg per kg intrave-
nously; and (rats) 2 mg per kg.
References:
1. Igarasi, S. J. Antil)iotics (Japan) 7B: 221-
225, 1954.
2. Wada, S. et al. Chem. Abstr. 52: 167031,
1958.
Fervenulin
Produced by: Streptomyces fervens.
Method of extraction: Filtration of the broth at
pH 8.0. Filtrate extracted with l^ volume of
methyl chloride at pH 6.0. Concentration to an oil.
Extraction of the oil with the upper phase of the
solvent system acetone-n-hexane-water (5:3:1).
Concentration of the solvent yields crystalline
fervenulin. Pure fervenulin obtained by fractional
crystallization from the solvent system ethyl ace-
tate-acetone (3:1) or by countercurrent distritni-
tion in the solvent system benzene-methanol-
water (1:1:0.2).
Chemical and physical properties: Brilliant yellow
orthorhombic crystals; m.p. 178-179°C (decompo-
sition). Sulilimes 70°C at 10 fj. pressure. C =
43.83%; H = 3.73%; N = 35.99%; O = 17.27%.
Molecular weight (radiological), 189; vapor pres-
sure, 190; saponification, 186. Molecular weight,
272
DESCRIPTIONS OF ANTIBIOTICS
1!)3.17, with suggested empirical formula CtHt-
NoOj . Infrared alisorption .spectrum given in ref-
erence 1. Light absorption maxima at 239, 270 to
280, and 340 m^ in pH 7.85 pho.sphate buffer. At
pH 10 the maxima at 239 and 340 m^i are destroyed
but the maximum at 270 to 280 m^ remain-s un-
changed. Neutral compound, soluV)le in practically
all common organic solvents except hydrocarbons.
Soluble in water (2 mg per ml cold; 40 mg per ml
hot). Destroyed by alkalies. Not affected by 6 A'
HCl at 100°C for 40 hours. Positive copper svUfide
test. Negative ninhydrin, FeCls , Tollen, Benedict
iodoform, biiu'et, Hinslierg, antl Sakaguchi tests
(1).
Biolofiii-al activity: In vitro: Low tlegree of ac-
tivity against gram -positive and gram -negative
bacteria. Of 23 strains of bacteria tested, only
three were sensitive to less than 25 fxg per ml. Low
degree of activity against some fungi {Histoplasma
rapsulatuiii) and some protozoa (2). In vivo: Lim-
ited activity against trichomonas infections in
mice and hamsters. No activity in mice against
experimental infections caused by Streptococcus
hemolyticus, K. pneumoniae, Histoplasma capsula-
tum, and Cryptococcus neoformans (2). Inactive
against sarcoma 180, Ehrlich carcinoma ascites.
Walker adenocarcinoma, Murphy-Sturm lympho-
sarcoma, Jensen sarcoma, and Ouerin adenocar-
cinoma in mice or rats (2).
Toxicity: LD50 (mice) 65 mg i)er kg intraperito-
neally; (hamsters) 11.2 mg i)er kg (2).
References:
1. Eble,T.E. f/o/. Antibiotics Ami. 227-229,
1959-1960.
2. DeBoer, C. e/ fl/. Antibiotics Ann. 220-226,
1959-19()0.
Filipiii
Prtnliiccil Ijy: Streptomyces filipinensis (2, 8).
This culture also produces an unrelated antifungal
substance, which is chloroform-soluble (8).
Method of extraction: 1. I'^thyl acetate, ether, or
n-butanol extracts of filtered broth evaporated to
ifo volume, hexane or Skellysolve B added, and
residting precipitate washed with petroleum ether
and dried in vacuo. Extracted from mycelium with
methanol and butanol. Crystallized by trituration
with chloroform and i-ecrystallized from methanol
(2, 8). II. Broth mixed with diatomaceous earth,
filtered, and broth extracted with ethyl acetate.
Solvent extract concentrated in vocuo, and tilijjin
precipitated with petroleum ether. Crystallized
from chloroform (3).
Chemical and physical properties: 'i'ellow, neu-
tral conjugated pentaene. Fine needles. At 147°C
undergoes transition to second, partially degraded
form with m.p. 195-205°C (decomposition). Very
soluble in dimethylformamide and pyridine. Solu-
ble in methanol, ethanol, n-butanol, isopropyl
alcohol, t -butyl alcohol, ether, ethyl acetate, amyl
acetate, and glacial acetic acid. Nearly insoluble
in water, chloroform, 50 per cent ethanol, methyl-
ene chloride, and Skellysolve B. X*J^°x" 322 (£'1L
910), 338 {E^L 1360), and 355 (£lL 1330) with a
shoulder at 305. No shift in acid or alkali. Infrared
.spectrum given in reference '3. [aln = —148.3°
(c = 0.89 per cent in methanol). Positive Molisch
test. Negative ninhydrin, biuret, Benedict, Tol-
len, 2,4-dinitrophenylhydrazine, and FeCls tests.
Deep blue color in concentrated HCl or H2SO4 ,
Init not in HNOj . Thermolabile. Photosensitive
in Oj . Subject to auto-oxidation. Stable in dark
in air at 5°C. Seventy per cent inactivated by 10
per cent horse serum in a methanol solution. Rf
values on paper chromatography given in refer-
ences 2 and 3. Tentative empirical formula:
C32H;,40iii (11). Saponification equivalent, 574.
Filipin undergoes a nonreversible, autocata-
lytic degradation in concentrated niethanolic or
ethanolic solutions standing at 4°C, to a white,
biologically and optically inactive, crystalline
polyene, CsoHsoOio , m.p. 195-205°C, with an in-
frared spectrum nearly identical to filipin and
X^^ at 318 (EuL 1050), 303 (.^IL 1170), 290 (i/lL
770) and a sho\ilder at 281. Gives a wine-red color
in concentrated H2SO4 . Basic hydrolysis of filipin
yields a biologically inactive acid thought to be
derived from a lactone ring (2, 3, 10).
Biological activity: Active on fungi and yeasts;
slightly active on Trichomonas foetus. Not active
on bacteria. Naturally occurring fungal rot of pea
and tomato seeds (caused by penicillia, aspergilli,
and phycomycetes for the most part) almost com-
pletely suppressed bj' soaking seeds for 3 hours in
25 to 100 Mg per ml of filipin. Partial protection of
tomato plants from gray leaf spot (Stemphyliunt
solani) in greenhouse tests (1, 2). Active on the
damping-off stage of safflower root rot (Pythium
sp.) (5), downy mildew of cucumber {Pseudopero-
nospora cubensis) (6) and broccoli (Peronospora
parasitica) (7), black mold of onion, corn leaf
blight, collar rot of tomatoe.s, bitter rot of apples,
and tomato wilt (8). Improves emergence of cu-
cumber, vegetable marrow, and muskmelon (4).
Activity on Penicillium oxalicum reversed by
cholesterol (10). Intermediate activity on sarcoma
180 (mice) (8).
Toxicity: LDoo (mice) 17 mg per kg intrajieri-
toneally (8). No effect on pea and tomato seed
germination at 100 ^g per ml applied by soaking.
DESCRIPTIONS OF ANTIBIOTICS
273
No toxic eft'ect on young tomato plants sprayed
with 415 ng of filipin per ml of 25 per cent methanol
(1,2).
References:
1. Gottlieb, 1). et al. Plant Disease Reptr.
39: 219, 1955.
2. Ammann, A. et al . Phytopathology 45:
559-563, 1955.
3. Whitfield, G. B. et al. J. Am. Chem. Soc.
77: 4799-4801, 1955.
4. Wallen, V. R. and Bell, W. Plant Disease
Reptr. 40: 129-132, 1956.
5. Gattani, M. L. Plant Disease Reptr. 41:
160-164, 1957.
6. Ark, P. A. and Thompson, J. B. Plant
Disease Reptr. 41: 452-454, 1957.
7. Natti,J.J. Plant Disease Reptr. 41:780-
788, 1957.
8. British Patent 783,486, September 25, 1957.
9. Field, J. B. et al. Cancer Research 18:
(Suppl. 1) 492, 1958.
10. Sloneker, J. H. Thesis, University of Illi-
nois, 1958.
11. Tingstad, J. E. and Garrett, E. R. J. Am.
Pharm. Assoc, Sci. Ed. 49: 352-355, 1960.
Flavacid
Produced by: Streptoniyces sp. closely related to
»S. flavus.
Method of extraction: The mycelium of the Strep-
tomyces washed with water, dried, and crushed
into powder. Extraction of mycelium with 1:1
mixture of butyl acetate-acetone, or with ethyl
acetate. These solvents removed from the my-
celium a toxic antibiotic (D-substance), which is
discarded. Mycelium extracted with methanol and
discarded. Methanol evaporated in vacuo, and
the solid residue extracted with butanol at pH
5.0. Butanol-extract washed with acidic water
(pH 2.0) and adjusted to pH 7.0. After concentra-
tion of butanol to a small volume, flavacid is pre-
cipitated out with saturated alkaline methanol.
Yield: 500 to 800 mg per 100 gm of dry mycelium.
Chemical and physical properties: Hexaene (2):
Weak acid, active and unstable under acidic con-
ditions. Sodium salt: solul^le in methanol and
aciueous acetone and moderately soluble in water
and ethanol; slightly soluble in acetone and buta-
nol; insoluble in ether, ethyl acetate, chloroform,
and benzene. Indicator properties: green in acid
solutions, yellow at neutrality, red at alkaline
pH. Inactive at neutrality and at alkaline reac-
tion. Ninhydrin and FeCh reactions negative.
Ultraviolet absorption maxima at 335, 355, and 373
m;u (methanol); m.p. 102-105°C (1). A tetraene
(peaks at 293, 306, and 324 ni/x (ethanol)) is also
present in .small cjuantities, as with endomycin.
The hexaene from flavacid was shown to differ from
that of endomycin. Not enough of the tetraene
was present for comparison with the endomycin
tetraene (2).
Biological activity: Active //( vivo against gram-
positive bacteria (3 to 6 ng per ml); verj^ slight
activity against gram-negative bacteria. Active
against yeasts and filamentous fungi, including
Trichophyton and C. albicans. Inactivated by
human serum. Experimental systemic infections
of mice and guinea pigs with ('. albicans do not
respond to flavacid treatment. Oral administra-
tion of flavacid reduces the nvmil>er of C albicans
in the digestive tract.
Toxicity: LDso (mice) 50 mg per kg intraperito-
neally.
I'tilization: Some effect in the treatment of tri-
chophytosis in man, when applied topically. Ex-
cellent results obtained in the topical treatment
of Trichomonas vaginalis in human beings.
References:
1. Takahashi, J. J. Antibiotics (Japan) 6A:
117-121, 1953.
2. Vining, L. C. and Taber, W. A. Can. J.
Chem. 37: 1461-1466, 1957.
Flavensoniycin
Produced by: Streptoniyces sp. resembling S.
tanashiensis (2).
Synonym: Antibiotic 829 (1).
Method of extraction: Broth-filtrate extracted
with benzene at pH 7.5 to 8.0. Extract concen-
trated in vacuo until precipitate appears. Precipi-
tate washed with petroleum ether and extracted
with acetone. Acetone concentrated and petro-
leum ether added to precipitate crude flavenso-
mycin. Purification by chromatography on alu-
mina from a benzene solution, washing with ethyl
acetate, ethanol-ethyl acetate, and elution with
methanol (2, 3).
Chemical and physical properties: Pale yellow,
odorless tabular crystals; m.p. 152° ± 2°C. Solu-
ble in water, lower alcohols and acetates, benzene,
chloroform, pyridine, acetone, dioxane, and pro-
pylene glycol. Insoluble in ether, petroleum ether,
hexane, carbon tetrachloride, and carbon disul-
fide. Contains N, but no S or halogen. Ultraviolet
absorption spectrum maximum at 251 niyu (metha-
nol). Infrared data given in reference 2. Stable, in
dry form, in organic solvents and in neutral
atiueous solutions at <12°C. Positive Molisch,
Fehling, and Ehrlich (diazo) tests. Negative Tol-
274
DESCRIPTIONS OF ANTIBIOTICS
leu, Seliwunot'f, Millon, Liebermann, Sakagvichi,
biuret, and ninhydrin tests (1-3).
Biological activity: Most active on Saccharomy-
^es and Penicillium (0.5 /ig per ml). Less active (5
to 50 /xg per ml) on other fungi. No activity on bac-
teria. Active on insects (Miisca domestica and
Locusta migratoria) (2). Strongly inhibits mitosis
at pre-prophase in Allium cepa root cells (1).
Toxicity: LD50 (mice) 1 mg per kg intraperito-
neally, 2 mg per kg subcutaneously, and 25 mg per
kg orally (1, 2).
References :
1. Craveri, R. and Veronesi, U. Riv. liiol.
(Perugia) 49: 89-97, 1957.
2. Craveri R. and Giolitti, G. Nature, Lon
don 179: 1307, 1957.
3. Craveri, R. et al. Nuovi ann. igiene e micro-
biol. 9: 185-187, 1958.
Flaveolin
Produced by: Streptouiyccs sp. resembling iS.
flaveolus.
Method of extraction: Culture medium adjusted
to pH 2.0 and filtered. Filtrate decolorized with
charcoal at pH 2.0, then antibiotic adsorbed on
charcoal at pH 7.0. Elution with 80 per cent ace-
tone (pH 1). Acetone evaporated off in flash-
evaporator and Ca++ removed as oxalate. Filtrate
extracted with l)utanol at pH 8.0. Re-extraction
into water (pH 1.0). Separated from impurities by
countercurrent distribution (butanol and 0.067
M Sorensen's buffer at pH 5.5). Active fractions
transferred from acidic water into chloroform re-
peatedly. Precipitated from chloroform on addi-
tion of ether.
Chemical and physical properties: Basic sub-
stance. Possible ciuinonoid structure. HCl salt:
Yellowish powder. Soluble in water, methanol,
ethanol, and propanol. Scarcely soluble in acetone,
benzene, butanol, and chloroform. Insoluble in
ether, jjetroleum ether, and ethyl acetate. Lemon -
yellow in acidic solution, brown at neutrality, and
reddish at alkaline pH. Most stable at acid pH.
Decolorized by H2O2 in presence of NajCOs .
Positive Liebermann nitroso reaction and Pauly
tests. Negative FeCl,? , Millon, biuret, Sakaguchi,
ninhydrin, and Fehling (black color, no precipi-
tate) tests. Picrate: m.p. 126-128°C (decomposi-
tion). Reincckate:n\.Y>. 147-150°C (decomposition).
Base: Soluble in butanol, chloroform, and iso-
amjd alcohol. Not precipitated by methyl orange
or flavianic acid. Rf = 0.74 (wet butanol) or 0.83
{3 per cent NH^Cl) by paper chromatography.
Biological activity: Active on gram-positive and
gram-negative bacteria, mycobacteria, yeasts, and
fungi.
Toxicity: Mice tolerate 10 mg per kg intrave-
nously, but 50 mg per kg is lethal.
Reference: 1. Takahashi, B. J. Antibiotics
(Japan) 6: 11-20, 1953.
Flavocidin
Produced by: Streptomyces sp.
Synonym: Possibly related to antibiotic F 256.
Method of extraction: Activity present primarily
in the mycelium. Mycelium exhaustively extracted
with boiling methanol. Methanol concentrated,
and antibiotic precipitated on addition of water.
Recrystallized from boiling chloroform.
Chemical and physical properties: Colorless
needles; m.p. 144-145°C. Very soluble in methanol,
ethanol, acetone, ethyl acetate, and chloroform.
Soluble in l)enzene and ether; sparingly soluble in
water; insoluble in petroleum ether and ligroin.
tntraviolet absorption spectrum maximum at 275
niyu (£'icm 2.01) in methanol. Infrared spectrum
given in reference 1. [a]l = +94° (c = 1 per cent
in methanol). Positive l)romine-chloroform, nitro-
prusside, Molisch, diazo, and m-dinitrobenzene
tests. Negative ninhydrin test. Addition of vanil-
lin to a glacial acetic acid solution gives a red
color, indicating a phenolic hydroxyl group.
C.,4H5o-.mN09 : C = 65.20%; H = 9.12%; N =
2.05%. Molecular weight, 625 to 643.
Biological activity: Active on Micrococcus fiavus
at 0.003 Mgper ml and on Sarcina lutea. Not active
on other bacteria. Active on influenza virus in
vitro.
Toxicity: LDsn (mice) 15.6 to 31.25 mg per kg
intraperitonealh'.
Reference: 1. Shibata, M. et al. Ann. Rept.
Takeda Research Lab. 17: 16-18, 1958.
Flavofiingin
Produced by: Streptomyces flavofungini (2).
(This culture also produces desertomycin.) Strep-
tomyces sp. (These cultures also produce an anti-
bacterial antibiotic (4).)
Synonym: Antifungal antibiotic SA IX (1); said
to be related to mycoticin (4).
Method of extractiou: Present iii mycelium and
broth (2).
Chemical and physical properties: Fine yellow
to yellow-green needles (2, 4). Soluble with diffi-
culty in w^ater. Yellow fluorescence in viltraviolet
light. Ultraviolet absorption spectrum maxima
at 263 and 368 niyu (3). Forms a stable acetyl deriva-
tive (4). vSlow diffusibility in agar. Reversil)ly
bound by serum proteins (2).
Biological activity: Active on yeasts, filamentous
fungi, and .V. asteroides. Not active on bacteria,
except B. siibtilis and Staph, aureus at >100 /xg
DESCRIPTIONS OF ANTIBIOTICS
per nil. Active on ('. albicans in the mouse intes-
tinal tract (2). Not active on streptomycetes (4).
Toxicity: LDao (mice) 25 mg per kg intraperi-
toneally (2) ; also 40 to 50 mg per kg intraperitone-
ally, 2.5 to 4.0 mg per kg intravenously, and 750
to 1200 mg per kg orally (3). Toxic to conjunctiva
(3).
Utilization: Used tojjically. orally, and par-
enterally. Active on vaginal trichomoniasis (3).
References:
1. Uri, J. Acta Physiol. Acad. Sci. Hung. 11:
(suppl.) 103-104, 1957.
2. Bekesi, I. Nature, London 1«1: UOS, 1958.
3. Kelentey, B. et al. Ahstr. Conununs. Sym-
posium on Antil)iotics, Prague, pj). 67-68,
1959.
4. Uri, J. Arzneimittel-Forsch. 9: 175-181,
1959.
Fradicin
Produced by: Streptoniyces fradiac (1).
Synonym: Factor X (1).
Method of extraction : I. Broth-filtrate extracted
with n-butanol at pH 7.0. Kxtract concentrated
to dryness in vacuo. Residue taken up in 95 per
cent ethanol, concentrated, and added dropwise
to acetone. Solution concentrated //; vacuo and
added dropwise to petrole\mi ether. Supernatant
discarded. Residual oil dissolved in t-butanol and
lyophilized (1). H- Culture filtered with the aid
of Super-Cel. Cake extracted with isopropanol-
ethyl acetate (1:10). Extract (A) concentrated to
give either a precipitate or a tarry residue. Etha-
nol solution of such residues chromatographed on
Super Filtrol Celite, developed with ethanol, then
with 10 per cent (volume per volimie) ammonia
in ethanol (3). Active fractions concentrated and
jirecipitated by scratching the side of the flask
containing the concentrate. Extract A can also be
treated by adjusting the i)H to 5.8 and stirring
with Stoddard solvent to separate two phases.
Lower phase adjusted to pH 8.0 to 8.5 to precipi-
tate fradicin. Crystallized from hot ethylene di-
chloride on cooling. Further purified by chroma-
tography on Florisil with 1,4-dioxane as solvent
and developer (3).
Chemical and physical properties: Weak base.
Light green-yellow crj'stals. No definite melting
point. Darkens at about 180-300°C. Most soluble
in dioxane, propylene glycol, ethylene dichloride,
and other chlorinated hydrocarbon solvents. In-
soluble or very sparingly soluble in cyclohexane,
xylene, and petroleum ether. Slightly soluble in
water. Ultraviolet absorption spectrum maxima
at about 242 and 292 ni/x, with a shoulder at 265
mn (absolute methanol) (1). Lltraviolet spectrum
of this pre{)aration shows the presence of a con-
taminating hexaene, probably the same as the
biologically inactive hexaene later reported to be
produced by this strain of S. fradiae (8). {a]^^ =
+65° (c = 1.0 per cent in 1,4-dioxane). More
stable at neutrality than at pH 2.0. Neutral ec^uiv-
alent, 498 to 514. C30H34N4O4 : C = 70.29%; H =
6.59%; N = 10.86%; O— CH. = 11.95%. Molecvdar
weight, about 500 (Barger). Hydrochloride: Nee-
dles. Darken on exposure to light. Alkali fusion
yields a volatile product which gives positive
pine-splint and l%hrlich (pyrrol) tests (1-3).
Bioloyind (uiivitji: Active on yeasts and fungi.
Active on certain protozoa, such as Trypanosoma
cruzi and Endamoeba histolytica. Not active on
bacteria (1, 5, 7). Activity strong at pH >7.0;
greatly reduced at lower i)H (1, 2). Cysteine and
other reducing agents decrease antifungal activitj"
(4), as do oleate and Tween 80 (6).
Toxicity: LDjo (mice) 4 mg per kg intraperi-
toneally and orally. Irritating to rabbit skin at
50 Mg per gm of a hydroi)hilic ointment (5).
References:
1. Swart, E. A. et al. Proc. Soc. Exptl. Biol.
Med. 73: 376-378, 1950.
2. Hickey,R. J. andHidy, P. H. Science 113:
361-362, 1951.
3. Hidy, P. H. and Hickey, R. J. Arch. Bio-
chem. Biophys. 34: 67-71, 1951.
4. Waksman, S. A. et al. Bull. World Health
Organization 6: 163-173, 1952.
5. Schwartz, J. A. et al. Trans. 11th Veterans
Admin. Conf. Tuberc. 86-93, 1952.
6. Hickey, R. J. Arch. Biochem. Biophys.
46: 331-336, 1953.
7. Packchanian, A. Am. J. Trop. Med. Hyg.
2: 243, 1953.
8. Steinman, I. 1). Thesis, Rvitgers University,
1958.
Fiatliciii-like An libiotic
Produced by: Streptoniyces sp. differing from S.
fradiae and S. roseojlavus (mycelin-producer).
Remarks: Authors (1) believe that the antibiotic
may also resemble mycelin. May be contaminated
with, or actually be a hexaene.
Method of extraction: Mycelium extracted with
methanol. Extract washed with water and con-
centrated in vacuo. Ether added to the concentrate
to give a precipitate.
Chemical and physical properties: Dark yellow
powder. Soluble in butanol, methanol, ethanol,
and pyridine. Slightly soluble in water. Insoluble
in chloroform, benzene, acetone, ethyl acetate,
ether, and petroleum ether. Ultraviolet absorption
spectrum maxima at 243, 294, 335, 355, and 373 mix.
276
DESCRIPTIONS OF ANTIBIOTICS
Biuloguat uctwiUj: Active on Sacch . sake and
C. albicans.
Toxiciti/: LDsci (mice) 15 mg per k^ intraperi-
toneally.
Reference: 1. Utahara, R. et al. J. Antibiotics
(Japan) 12A: 73-74, 1959.
Fianij celiii
Pfdilnccd bi/: Streptomyces lavendiihic (1, 2, S).
This strain was later identified as S. j'rudiae (13).
Streptomyces sp. (6).
Synonyms: Soframycin, antibiotic K. F. 185
(6, 8), neomycin (14).
Method of extraction: I. Broth-tiltrate treated
with sodium hexametaphosphate, pH adjusted to
8.5, and adsorption on IRC-50. Flnted with aciue-
ous 5 per cent triethyhimine or dilute HCl (pH
2.0). Adsorbed on triethylamine-washed IRC-50
under different pH conditions and eluted with
aqueous acidic methanol; pH of eluate adjusted to
5 with triethylamine, and framycetin precipitated
with 40 per cent triethylamine sulfate in absolute
methanol. Purification by chromatography on
alumina. Crystallized as the picrate (1, 6, 8). II.
Broth-filtrate adsorbed on carbon at pH 8.2. Car-
bon washed, then eluted with methanol-HCl
(10: 1 ) , followed by methanol. Added to ethyl ether
to precipitate framycetin (1, 8). III. Whole
culture adjusted to pH 3 or heated to 35-50°C, and
filtered. Adsorbed on carbon or silica gel at pH 7,
atul eluted with acidic alcohol. Eluates purified by
treatment with carbon, and with IR-100 at pH 4.0,
followed by adsorption on IRC-50. Eluted with
acidic alcohol (pH 2.0). Further purified by double
decomposition between sodium p-(p'-hydroxy-
phenylazo)benzene sulfonate and a crude salt of
the antibiotic, and other salt interconversions (8).
Chemical ami physical properties: Complex,
containing three components (1). Base: m.p. about
195°C (decomposition). No characteristic absorp-
tion in ultraviolet light, [a]'-^ = +64° (c = 1 per
cent in water). Stable to autoclaving for 30 min-
utes at pH 8.4 and for 8 hours at 25°C at pH 2 to
10. C = 46.6%; H = 7.3%; N = 12.8%; O = 33.1%;
Van Slyke N = 11.35% (as nitrogen); acetyl in-
dex = 17.7%. C2:iH44N,;Oi:j . All hydroxyl groups
are reported to be in the form of secondary alco-
hols. Rf = 0.22 (water-saturated n-butanol-2 per
cent p-toluenesulfonic acid, Whatman No. 4 paper,
15 hours at 24°C (1, 8)). Hydrochloride: Amorphous
white powder. Soluble in water and aciueous
methanol. Insoluble in acetone, ether, and other
common organic solvents. [«]„ = +57° (c = 1 per
cent in water). Green color with the anthrone test.
Negative Elson -Morgan and Sakaguchi tests. All
N present is in the form of primary amino groups.
Acid liydrolysis or methanolysis products include
neamine, a diamine hexose (C6H12O3N2 or CeH^-
O4N2), and a pentose. The amino sugar gives posi-
tive ninhydrin, Elson-Morgan, and ammoniacal
AgNOs tests, but a negative Keller-Kiliani (tor
desoxy-2-hexoses) test. Rf = 0.17 (n-butanol-
acetic acid-water, 50:25:25). The dipicrate ot this
amino sugar has a m.p. of 126 °C. Pentose gives
positive Tollen and Seliwanoff tests, reduces
l)enzidene and tetrazolium, and gives a negative
Keller-Kiliani test (3, 6, 10). Picrate: m.p. 189°C
(decomposition, corrected). [0]^ = —34° (c = 0.5
per cent in methanol (1). Sulfate: Soluble in water.
Insoluble in methanol, acetone, and ethyl ether.
[a]u = +44"^ (c = 1 per cent in water) (8). p-ip'-
H yd rox yphen ylazo)benzene sulfonate: Crystals;
m.p. 250°C. Soluble in methanol. Scarcely soluble
in ethanol. Insoluble in water, l)utanol, acetone,
ether, and ])enzene (8).
Biological activity: Active on gram-positive and
gram-negative bacteria, including mycobacteria,
Proteus, and Pseudomonas spp. No cross-resistance
with streptomycin (5, 9, 11). Active in mice on
Staph, aureus, E. coli, Sal. typhosa, Sal. typkimur-
ium, Sal. paratyphi (A and B), D. pneumoniae, M.
tuberculosis var. hominis H37Rv, and B. anthracis
(8).
Toxicity: LDao (mice) 40 to 50 mg per kg intra-
venously, 250 to 275 mg per kg intraperitoneally,
450 mg per kg subcutaneously, and >5 gm per kg
orally. LDf,,) (rat) 450 mg per kg intramuscularly,
700 mg per kg subcutaneously (8). Toxic to the
eighth cranial nerve (7). Chronic toxicity tests
showed that isonicotinic acid administered to
guinea pigs with framycetin overcomes the toxic-
ity of the antibiotic at certain levels (4).
Utilization: Bowel sterilization (12). Pulmonary
disease (7).
References :
1. Hagemann, (1. T. Thesis, University of
Paris, 1952.
2. Decaris, L. J. Ann. pharm. franc. II:
44-46, 1953.
3. Pen^u, H. et al. Ann. pharm. franc. 11:
431-438, 1953.
4. Lutz, A. Compt.rend. 236:157-159,1953.
5. IaxXz. \. et al. Strasbourg med. 4:431-433,
1953.
6. Janot, .M. M. et al. Bull. soc. chim. France
21: 1458-1463, 1954.
7. Sors, C. and Trocme, Y. Presse med. 61:
364-365, 1954.
8. Rendu, H. et al. French Patent 1,051,202,
January 14, 1954.
9. Lutz, A. and Witz, Al. A. Compt. rend.
soc. biol. \W: 1467-1470, 1955.
DESCRIPTIONS OF ANTIBIOTICS
277
10. Saito, A. and Schaffiier, C. P. Resvunes 3rd
Intern. Congr. Biocheni. 98, 1955.
11. Lutz, A. and Hofferer, M. J. Rev. imnmnol.
19: 68-85, 1955.
12. Shidlovsky, B. A. el al. Antibiotics Ann.
118-121, 1955-1956.
13. Waksman, S. A. Neomycin. The Wil-
liams & Wilkins Co., Baltimore, 1958.
14. Rinehart, K. L., Jr. ct al. .). Am. Chem.
Soc. 82: 3938-3946, 19C.().
Fungicliroiiiiii
Produced bi/: Streptonujces cellulosne (1). This
strain also ])roduces an actinomycin.
Sniiini i/ni: l''ungichromatin , a closely related
compound ( 1 ).
Method of extraction: Ivxtracted from l>roth-
filtrate with ethyl or amyl acetate, or other organic
solvents, and from mycelium with methanol,
ethanol, or acetone. Extracts concentrated ///
rocuo to precipitate a fungichromin-actinomycin
mixture. Separated by repeated crystallization
from methanol.
Chemical and phi/sical properties: Conjugated
pentaene (3). Pale yellow crystals; m.p. 205-210°C
(uncorrected). Soluble in methanol, ethanol,
butanol, acetone, pyridine, and dimethylformam-
ide. Insoluble in water and aliphatic hydro-
carbons. Ultraviolet absorption spectrum maxima
at 322.5, 338.5, and 356.5 m/x, with a shoulder at
310 niyu (in organic solvents). Infrared data given
in reference 1. Violet color, changing to blue in
concentrated sulfuric acid. Positive ToUen (slow),
KMn04 , and bromine tests. Hydrogenation prod-
uct is a white, waxy solid, with no antifungal
activity. Mild acetylating conditions destroy
antibiotic activity but not characteristic ultra-
violet absorption. C = 60.93%; H = 8.65%; O =
30.42%. Molecular weight, 688.8. C,;,Hf,„0,., . Con-
tains 10 toll hydroxyl groups, and 3 to 4 C — CHs
groups, but no methoxyl or acetoxyl groups, N, S,
or halogens. Sodivmi periodate oxidation yields,
among other products, 2-methyl-2,4,6,8,10-
dodecapentaenedial (1, 3).
Biological activity: Active on yeasts and fungi:
C albicans, 6.25 to 12.5 ^g per ml; Blastomyces
ilermatitidis, 0.78 /xg per ml ; A. niger, 3 to 25 ng per
ml; Trichophyton mentagrophytes, 12.4 to 50 /ig per
ml; Fusariiun oxysporum, 10 jug per ml. Active on
l^each brown rot (Sclerotinia fructicola) (1,2).
Toxicity: LD.^r, (mice) 16.4 mg jjer kg intra-
peritoneally. Mice tolerate 1000 mg jjer kg orally
(1).
References:
1. Tytell, A. A. et al. Antiljiotics Ann
718, 1954-1955.
■16-
2. Szkolnik, M. and Hamilton, J. M. Plant
Disease Reptr. 41 : 289-292, 1957.
3. Cope, A. C. and Johnson, H. E. J. Am.
Chem. Soc. «(»: 1504-1506, 1958.
Fiisconiyeiii
Produced by: Streptoniyces fuscus.
Synoriy)ns: Related to flaveolin. Differentiated
from luteomycin and xanthomycin (1).
Method of extraction: Adsorbed from l)roth on
carbon or cation exchange resin. I'^luted with
acidified acetone, methanol, or ethanol.
Chemical and physical properties: Basic sulj-
stance. H ydrochloride: Decomposes at 180°C.
Soluble in methanol, ethanol, butanol, and ace-
tone. Insoluble in ether, ethjd acetate, l)utyl
acetate, lienzene, petroleum ether, chloroform,
and carbon tetrachloride. Negative Sakaguchi,
Molisch, Benedict, glucosamine, maltol, Fehling,
FeChi , and ninhydrin tests. End-absori)tion of
ultraviolet light .
Biological activity: Active on gram-positive
bacteria; less active on gram-negative bacteria.
Cross-resistance with streptomycin and strep-
tothricin.
Toxicity: Mice tolerate injections (no route
given) of 500 mg per kg.
References:
1. Ishida, N. and Miyazaki, J. J. Autilnotics
(Japan) 5:481-487,1952.
2. Hata, T. Japane.se Patent 5046, 1953.
Ganciditis
Produced by: Streptoniyces sp.
Method of extrac'idu : Filtered Ijroth treated with
activated carbon. i",lu1 ion with 50 per cent acetone
(pH 2.0). Eluate adjusted to pH 6.0, filtered, con-
centrated in vacuo, and filtered again. Evaporated
to dryness in vacuo and residue taken up in a small
volume of methanol. Addition of ether precipi-
tates crude gancidin. The antibiotic could also be
extracted with organic solvents at acid pH and re-
extracted into water at alkaline i)H. Purified and
fractionated by count ere vu-rent distril)ution
(chloroform-McIlvaine's Iniffer). With the buffer
at pH 4.25, three biologically active fractions are
obtained, I, II, and III. Fraction II is concen-
trated and redistril)uted countercurrently in the
above system. The major active fraction is taken
to dryness. An ether solution of the residue is con-
centrated to give gancidin A crystals. Fraction I
is redistributed countercurrently in the above
system with the buffer at pH 2.5. The fraction in
the first four tul)es is concentrated and ether added
to give gancidin W. (Jancidin W recrystallized
from chloroform. Fraction III contains two com-
ponents (1, 2).
278
DESCRIPTIONS OF AXTIlilOTICS
Chemical and physical properties: Gancidin A.-
Basic substance. Orange columnar crystals.
Changes to dark orange at 110°C, then to black at
290°C. Soluble in chloroform, methanol, ethanol,
acetone, and water. Slightly soluble in ethyl
acetate, benzene, carbon tetrachloride, and ether.
Insoluble in petroleum ether. Ultraviolet absorp-
tion spectrum maxima at 205 niyu (-Eh-'m 324), 265
mM {E^L 248), and 340 m^ {E'lL 28) in 0.1 .V
HCl. Infrared absorption spectrum data given in
reference 1. Positive Fehling test. Negative nin-
hydrin, 2,4-dinitrophenylhydrazine, and FeCls
tests. More stable to heat at acid than at alkaline
pH. C43H58-60N6O14 : C = 58.7%; H = 6.68%; N =
9.49%. Molecular weight, 1002 (Rast) or 883. Hy-
drochloride: Needles. Gancidin W: Neutral sub-
stance. White platelets; m.p. 163-164 °C. Soluble
in methanol, ethanol, and acetone. Slightly solu-
ble in ethyl acetate, carbon tetrachloride, ether,
and water. Insoluble in petroleum ether. Ultra-
violet absorption spectrum maximum at 206 niM
{Eum 296). Infrared data given in reference 1.
Negative biuret, ninhydrin, Ehrlich, Fehling,
Benedict, Tollen, Molisch, 2,4-dinitrophenyl-
hydrazine, ferrous hydroxide, FeCls , and Brj
tests. CuHn.i902N2 : C = 63.04%; H = 8.7%;
N = 13.65%. Molecular weight, 210 (2).
Biological activity: Later work did not confirm
early reports of good antitumor activity in vivo.
Gancidin A is said to resemble xanthomycin A in
certain respects (1,2). Gancidin A: Very active on
gram-positive bacteria. Active on Ehrlich ascites
carcinonui in vitro but not in vivo. Gancidin W:
No activity on B. subtilis. Very slight activity at
>5 mg per kg against Ehrlich ascites carcinoma
in mice (2).
Toxicity: Gancidin A: LUsu (mice) 80 yug per kg
intravenously. Gancidin W: LDjo (mice) 80 mg
per kg intravenously (2).
References:
1. Aiso, K. et al. J. Antibiotics (Japan) 9A:
97-101, 1956.
2. Wakaki, S. et al. J. Antibiotics (Japan)
llA: 150-155, 1958.
COOH
O— C CH— N
I I II
CH. CH. C— NHo
NH..
^N^
H
Geonij fill
Produced hy: Strains of StrcploiiiyccH .cantho-
phaeus (1, 3).
Synonym: St rPi)tothricin-like antibiotic.
Method of extraction: Broth-filtrate chroma-
tographed on IRC-50 (buffered with 0.2 M Na3P04
buffer at pH 6.5) and eluted with 0.5 \ HCl. Active
fractions adjusted to pH 6.0 with IR-4B, then
concentrated to dryness in vacuo. Adsorbed on
active carbon and eluted with weak H2SO4 (pH 2
to 3). Sulfate ions precipitated as Ba salt, and
antibiotic converted to an oily picrate. Purifica-
tion by salt conversion (hydrochloride — > heli-
anthate — + hydrochloride -^ base). Dried and
powdered myceliimi extracted with 1 ,V HCl-
methanol (1:1), then purified by adsori)tion on
IRC-50.
Chemical ami physical properties: Complex:
Contains four to six closely related components,
varying with the producing strain. Faintly yellow
powder. Ultraviolet absorption spectrum shows
end-absorption only (water). Infrared spectrum
given in reference 3. Positive ninhydrin and Elson-
Morgan tests. Weakly positive Sakaguchi test and
tests for carbohydrates and sugars. Light blue
color with biuret test. Negative maltol, FeCls ,
and 2,4-dinitrophenylhydrazine reactions. (CeHis-
02N2)s.iu : C = 49.48%; H = 8.91%; N = 20 to
25%. Acid hydrolysis products include NH;j ,
CO2 , L-/3-lysine, and an amino acid, geamine,
which is isomeric with or identical to streptolidine
(roseonine) from streptothricin, streptolin, and
roseothricin (see streptothricin-like antil)iotic).
Geamine: C6H12O3N4 . Geamine dihydrochloride:
Colorless needles; m.p. 208-213°C. [a\l = +57.5°
(c = 1.9 per cent in H2O). Geomycin may contain
a peptide moiety made up of glutamic acid, as-
partic acid, serine, threonine, glycine, and alanine
(2-4). It contains at least four /3-lysine residues
connected at the e-amino groups. (Jeamine is
bound by its hydroxyl to a sugar or amino sugar
moiety (R). Partial structvu-e (5):
NH,— CO— CH.— CH— [CH2].,
I I
NHo NH
I
CHoCO-
R
NH[CH2]:rCH-CH2-CO-NH[CH2]3CHCH2CO-NH[CH2]3CH-
NHo NH2 NH2
DESCRIPTIONS OF ANTIBIOTICS
279
Hclianthatc: Red platelets (from dilute methanol) ;
in. p. 2[)5-215°C (decomposition). Hydrochloride:
Hygroscopic, colorless powder. Soluble in water
and methanol. Less soluble in ethanol. Insoluble
in acetone, ethyl acetate, ether, and petroleum
ether, [a]^ = +16.0° ± 0.4° (c = 3.25 per cent in
water). Molecular weight, 1650 ± 165.
Biological activity: Active against gram-positive
and gram-negative bacteria. No effect on entero-
cocci, pneumococci, or Streptococcus viridans.
Active on Endamoeha histolytica in rats (2).
Toxicity: Nephrotoxic (2).
References:
1. Lindenbein, W. Arch. Mikrobiol. 17: 301-
383, 1952.
2. Brockmann, H. and Musso, H. Naturwiss-
enschaften 41:451-452,1954.
3. Brockmann, H. and Musso, H. Chem. Ber.
87: 1779-1799, 1954.
4. Brockmann, H. and Musso, H. Chem. Ber.
88: 648-661, 1955.
5. Brockmann, H. and Colin, R. Chem. Ber.
92: 114-130, 1959.
Graiialiciii
Produced by: A variety of Streptoun/ces olivaceus
which produces a soluble pigment.
Method of extraction: Broth-filtrate adjvisted to
1)H 3 with dilute HCl. Extracted with ethyl ace-
tate. Extract concentrated in vacuo. Addition of
petroleum ether precipitates crude granaticin.
Purified bj' chromatography on a cellulose column.
Cellulose suspended in benzol-saturated formam-
ide; column pretreated with a benzol solution of
8-hydroxyquinoline, followed by washing with
formamide-saturated benzol. A benzol solution of
crude granaticin applied to column. Elution with
formamide-saturated benzol. Four pigmented
substances, brown-red, violet-blue (granaticin),
blue, and orange are distinguished, in that order.
Eluate concentrated to dryness, dissolved in ethyl
acetate, and precipitated with petroleum ether.
Recrystallization from acetone.
Chemical and physical properties: Tricyclic
tetrahydroxyquinone dicarboxylic acid. Small
granular red crystals; m.p. 204-206°C (decomposi-
tion). Indicator properties: red at acid pH, blue at
alkaline pH. C22H.20O10 : C = 59.40%; H = 4.72%;
O = 35.95%; C—CH., = 7.31%. Crystallographic
data given in reference 1. Kuhn-Roth oxidation
yields more than 1 mole of acetic acid. Ultraviolet
absorption spectrum maxima (rectified alcohol):
223, 286 (496), 532, and 576 uim- Infrared spectrum
given in reference 1. On acetylation gives a tetra-
acetyl derivative, m.p. 242-243°C (gas generated
over 250°C). [a if," = +100° (c = 0.818 per cent in
chloroform). Absorption spectra given in refer-
ence 1.
Biological activity: Active on gram-positive bac-
teria and Trichomonas foetus. Slightly active on
Pasteurella pestis, Vibrio ro)n»ta, M. tuberculosis,
and Endamoeba histolytica.
Toxicity: 250 mg per kg subcutaneously is
lethal for mice.
Reference: 1. Corbaz, R. et al. Helv. Chim.
Acta 40: 12()2-12l)9, 1957.
(iras.serioniycin
Produced by: Streplomyces lavendulae (1) and
S. griseolavendus (2).
Method of extraction: Adsorbed from broth-
filtrates on charcoal at alkaline pH. Eluted with
80 per cent methanol at pH 2.0. Eluate concen-
trated in vacuo, taken up in anhydrous methanol,
and precipitated with acetone. Purified by adsorj)-
tion on an ion exchange resin, and by salt conver-
sion.
Chemical and physical properties: Basic sub-
stance belonging to the streptothricin group. Base:
Soluble in water, slightly solul)le in methanol and
ethylene glycol. Insoluble in other organic sol-
vents. No specific absorption in ultraviolet light.
Infrared spectrum given in reference 2. Positive
Molisch, Fehling, Tollen, and ninhydrin tests.
Negative biuret, Sakaguchi, xanthoproteic, Mil-
lon, Adamkiewicz, Liebermann, Neubauer, and
Fed,, tests. Hydrochloride: Faintly yellow powder.
Soluble in water and methanol; insoluble in or-
ganic solvents. Molecular weight, 610. Heiianthate:
m.p. 215-225°C (decomposition). Reineckate: m.)).
187-190°C (decomposition). C = 22.04%; H =
4.51%; N = 19.35%; Cr = 11.22%. No figures
given for S.
Biological activity: Active on gram-negative and
gram-positive bacteria, but not on cultures of
E. coli resistant to streptothricin, neomycin, or
grisein. Active on fungi and Sacch. cerevisiae, but
not on other yeasts. Delays death of silkworms
infected with silkworm jaundice virus (1, 2).
Toxicity: Silkworms tolerate injections of 5 jug
(2).
References:
1. Ueda, K. et al. J. Antibiotics (Japan) 8A:
91-95, 1955.
2. Sumiki, Y. et al. Japanese Patent 6296,
August 15, 1957.
Grisaniine
Produced by: Streplomyces griseoflavus.
Method of extraction: Broth-filtrate adjusted to
280
DESCRIPTIONS OF ANTIBIOTICS
pH 7.4 and stirred with 4 per cent acidic clay.
Clay filtered off and eluted with methanol -acetone
(1:1). Eluate concentrated in vacuo. Extraction
of residue with ethyl acetate at pH 7.5 to 8.0. The
solvent layer extracted with acidic water and
back-extracted with ethyl acetate at an alkaline
reaction. The solvent extract is passed through an
alumina column and eluted with a mixture of
methanol and ethyl acetate (1:1). Concentration
of eluate in vacuo and extraction of residue with an
ethyl acetate-ether (1:10) mixture. Crystallization
(needles) occurs in a few hours at room tempera-
ture .
Chemical and physical properties: Basic sub-
stance. Soluble in water, ethyl acetate, butyl
alcohol, and chloroform; slightly soluble in ether;
insoluble in petroleum ether, benzene, toluene,
and ligroin. Ultraviolet absorption maxima at 255
and 320 m/x in water. Melting point of free grisa-
mine is 165-170°C, and of its sulfate 175-180°C.
Negative ninhydrin, biuret, Fehling, Sakaguchi,
and FeCls tests. Weakly positive p-dimethylamine
aldehyde and sodium nitroprusside tests. C-jsNe-
HssOio : C = 55.62%; H = 6.33%; N = 11.58%.
Biological activity: Active on M. tuberculosis
strains 607, BCG, and H-2 (human type), and M.
phlei; very slightly active on B. subtilis and Staph,
aureus. Not active on gram-negative bacteria,
yeasts, or fungi tested.
Toxicity: 150 mg per kg intravenously is toler-
ated by mice; 160 mg per kg causes death within
1 week.
Reference: 1. Sawazaki, T. et al. J. Antibiotics
(Japan) 8A: 39-41, 1955.
Grisein
Produced by: Streptomyces griseus strains (1-4,
12) and S. subtropicus (8).
Synonyms: Antibiotic 3510 (3), antil)iotic A 1787
(12), albomycin (8, 14, 15). All are identical to, or
very closely related to grisein.
Remarks: Zahner et al. (15) recently called
sideromycins antibiotics that show cross resistance
with grisein and are antagoni?;ed in their action
upon gram-po.sitive (but not gram-negative) bac-
teria by ferrioxamines. They included in this group
of antibiotics grisein, albomycin, antibiotic 1787,
LA 5352, LA 5937, ferrimycin, and antibiotic 22765;
the first three are active upon both gram -positive
and gram -negative bacteria and the last four only
upon gram-positive bacteria. Ferrioxamines, pro-
duced by streptomycetes, form a new group of iron-
containing metabolites, with growth-stimulating
properties for a number of microorganisms. Bickel
et al. (16) proposed to include them, together with
some known substances, like ferrichrome, copro-
gen, and the terregens, which either contain or bind
iron, in a new class of growth factors, the sider-
amines. This biological property of the sideramines
is counteracted by iron-containing antibiotics
from streptomycetes, the sideromycins.
Method of extraction: Adsorption from broth-
filtrate on activated carbon at pH 7.5 to 8.0. Elu-
tion with aqueous pyridine, a-picoline, or ethanol
(5, 6). Eluate evaporated in vacuo and methanol
added to precipitate impurities. Addition of ether
precipitates grisein. Precipitate leached with
methanol, then treated with silver oxide to remove
impurities. Distributed between water and phenol-
chloroform mixtures l)y altering the pH of the
aqueous phase and the phenol concentration.
Reprecipitated from aqueous phase on addition of
isopropyl alcohol or ether. Purification by chroma-
tography on silica gel from a solution in 34 per
cent phenol-chloroform saturated with pH 4.6, 0.1
M citrate buffer; the solution is diluted in half
with chloroform before chromatography. Elution
with 17 per cent phenol -chloroform (6, 7).
Chemical and physical properties: Complex with
neutral or weakly acidic components (6, 11). Red
powder (1, 8). Insoluble in ether, chloroform,
absolute acetone and ethanol, and benzene.
Slightly soluble in acetone and 95 per cent ethanol.
Soluble in water and phenol (1, 6). Ultraviolet
absorption spectrum maxima at 265 m^u (-Biem 108)
and 420 m/x (^1^28.9) (6). Positive biuret test (8).
Negative Brady, Fehling, Tollen, Liebermann,
Sakaguchi, Alillon, Pauly, Molisch, Seliwanoff',
orcinol, phloroglucinol, and ninhydrin tests (6,
8). Mild deamination results in loss of antibac-
terial activity (8). Heat -stable (1). Moderately
stable to aqueous acid solution; inactivated by
methanolic HCl (6). C4oH6iNioO-2oSFe: N =
13.28%; amino N = 1.34% (6, 8) ; Fe = 4.50%
(11). Molecvdar weight, 1300 (8, 13) or 1090 (6).
Removal of iron causes a loss of color, the ultra-
violet peak at 420 ni/x, and reduction in biological
activity. These changes are reversed on re-addi-
tion of Fe to the molecule. Contains four compo-
nents, A, B, C, and D, with Rf values of 1.0, 0.75,
0.60, and 0.28 on paper chromatography (butanol-
acetic acid-water, 4:1:5). Component C breaks
down during purification with concomitant ap-
pearance of more A (10). Albomycin was first re-
ported to contain three active components and
two inactive ones which were degradation prod-
ucts of the first three (11). Electrophoretic studies,
however, showed the presence of one major com-
ponent making up 85 per cent of the complex, and
three other trace components (13). Grisein was
DESCRIPTIONS OF ANTIBIOTICS
281
rt'l)ortCHl lo form an anioi'i^hou!!! jncrute, hut no
helianthate or reineckate (6). Albomycin forms a
reineckate (11). No free C=S or ^^CSH groups.
Crude grLsein and albomycin hydrolysates con-
tain many amino acids, but purer preparations of
grisein contain only glutamic and an imidentified
amino acid; purer preparations of albomycin
(component D) contain only ornithine and serine
(6, 8, 11). An acid hydrolysis product of grisein is
3-methyluracil; m.p. 182-183°C (6):
NH-
OC
I
CH.N-
-CO
CH
CH
Biological activih/: Active on gram-positive and
gram-negative bacteria. No activity on ,1. aero-
genes, B. mycoides, Sal. tt/phosa, Pr. vulgaris, Ps.
aeruginosa, or fungi. Limited activity on myco-
bacteria (1, 10). Albomycin is active on .1. aero-
genes (8). Active in mice against Staph, aureus and
Sal. schottniuelleri infections (1). Development of
resistance is very rapid (1, 9). Cross-resistance
with viomycin. A is the most active component;
1) the least (10). Inactivated by excess Fe (2, 8),
hut not by cysteine or hydroxylamine (2).
Toxicity: Nontoxic. Albomycin well tolerated
by mice, rabbits, cats, and guinea pigs. No toxic
symptoms in clinical use (8).
Utilization: Used clinically in the T. S. S. K.
against coccal infections, pneumonia, meningitis,
peritonitis, relapsing fever, prostatitis, gonococcal
lu-ethritis, and other penicillin-resistant infections
(8).
References:
1. Reynolds, D. M. el al. Proc. Soc. Exptl.
Biol. Med. 64: 50-54, 1947.
2. Reynolds, I). M. and Waksman, S. A. J.
Bacteriol. 55:739-752, 1948.
3. Garson, W. and Waksman, S. A. Proc.
Natl. Acad. Sci. U. S. 34: 232, 1948.
4. Umezawa, \\. ct al. J. Antibiotics (Japan)
2B: 104-109, 1949.
5. Kuehl, F. A., Jr. and Chalet, L. U. S.
Patent 2,505,053, April, 1950.
6. Kuehl, F. A., Jr. et al. J. Am. Chem. Soc.
73: 1770-1773, 1951.
7. Kuehl, F. A., Jr. U. S. Patent 2,54(),2()7,
March, 1951.
8. Cause, G. F. Brit. Med. J. 2:1177-1179,
1955.
9. Garrod, L. P. and Waterworth, P. M. lirit .
Med. J. 2: 61-65, 1956.
10. Stapley, E. O. and Ormond, R. E. Science
125: 587-589, 1957.
11. Braznikova, M. G. et al. Biokhimiya 22:
111-117, 1957.
12. Thrum, H. Naturwissenschaften 44: 561-
562, 1957.
13. Samsonov, G. V. et al. Biokhimiya (Eng.
transl.) 23: 206-209, 1958.
14. Waksman, S. A. Science 125: 585-587,
1957.
15. Ziihner, H. et al. Arch. Mikrobiol. .36: 325
349, 1900.
16. Bickel, V. H. et al. Experientia 16.- 129-133,
1960.
Griseoflaviii
Produced hi/: Streptoniyces griscoflavus.
Method of extraction: Adsorption on acidic clay
at pH 2.0, elution with 80 per cent acetone at pH
7.0. Evaporation of acetone, aqueous solution
extracted with ethyl acetate, evaporation to dry-
ness. Brown powder dissolved in methanol, chro-
matography over aluminum oxide. Crystallization
from ethanol.
Chemical and physical properties: Colorless
crystals; ni.)). 210-215°C (decomposition). Soluble
in methanol, ethanol, propanol, phenol, acetic
acid, and alkaline water. Slightly soluble in water,
ethyl acetate, and butyl acetate. Insoluble in
ether, petroleum ether, benzene, and chloroform.
Negative biuret, ninhydrin, Sakaguchi, Molisch,
and FeChi tests.
Biological activity: Active primarily against
gram-positive bacteria; limitetl activity against
gram-negative bacteria and mycoliacteria. Strains
of staphylococci, Vihrio comma, and one Mycobac-
terium are the most sensitive organisms (1 to 2 ^g
per ml ) .
Toxicity: LD.51, (mice) >250 mg per kg intra-
peritoneally.
Reference: 1. Waga, Y. J. Antil)iotics (Japan)
6A: 66-72, 1953.
Griseoluteiii A
Produced by: Streptomyces griseoluteus (3).
Synonym: Griseolutein. The same organism also
produces griseolutein B.
Method of extraction: Broth-Hltrate extracted
with ethyl acetate at pH 2.0. Extract concentrated
//( vacuo. Concentrate chromatographed on alu-
mina and developed with ethyl acetate. Active
yellow fraction concentrated and cooled to precipi-
tate crystals. Recrystallized from ethyl acetate
(1). Crude powder containing both A and B is
dissolved in aqueous sodium bicarbonate (pH 7.2)
and solution extracted with ethyl acetate at pH
5.8. Most of B remains in the aciueous laj'er; A is
282
DKSCRII'TIOXS OF ANTIBIOTICS
transferred 1o the solvent. Solvent conoent rated
in vacuo. A is purified by countercurrent distribu-
tion (ethyl acetate-phosphate buffer, pH 5.8).
Crystallized from ethyl acetate. Recrystallized
from methanol (6).
Chemical and physical properties: Orange-yellow
needles; m.p. 194-197°C (decomposition). Ultra-
violet absorption spectrum maxima at 265 m^u
(£'}L 1980) and 362 m^ (E^L 330) (methanol).
Infrared spectrum given in reference 6. CnHu-
O6N2 : C = 59.51%; H = 4.36%; N = 8.02%;
O — CH3 = 7.3%. Insolul:>le in water, ether, and
benzene. Slightly soluble in ethyl acetate and
ethanol. Soluble, but destroyed, at alkaline reac-
tion. More stable at pH 2.0 than 7.0. Monomethijl
ester of A: Yellow crystals; m.p. 149°C. The struc-
ture of griseolutein A, l-methoxy-4-[(hydroxy-
acetoxy)methyl]-9-carboxyphenazine (6, 7), is
given in Chapter 6.
Biological activity: Active on gram-positive and
gram-negative bacteria (1). Activity affected by
cysteine and serum, Init not by cystine. More
active at acid than alkaline pH (2, 4).
Toxicity: >2 gm per kg or >500 mg per kg are
not to.xie to mice subcutaneously (2, 4).
References:
1. Umezawa, H. et al. J. Antibiotics (Japan)
4: 34-40, 1951.
2. Ogata, Y. J. Antibiotics (Japan) 4: 213-
218, 1951.
3. Okami, Y. J. Antibiotics (Japan) 5: 477-
480, 1952.
4. Ogato, T. et al. J. Antibiotics (Jai)an) 7A :
15-16, 1954.
5. Nakamura, S. et al. J. Antibiotics (Japan)
lOA: 265-266, 1957.
6. Nakamura, S. et al. J. Antibiotics (Japan)
12A: 55-58, 1959.
7. Yagishita, K. J. Antibiotics (Japan) 13A:
83-96, 1960.
Griseoluleiii li
Produced by: A mutant of the Streptomyces
griseoluteus culture that produces griseolutein A.
Method of extraction: Broth-filtrate extracted
with butyl acetate at pH 2.0. Extract concen-
trated in vacuo to precipitate griseolutein B. Crude
powder dissolved in dioxane, then concentrated
to give crystalline substance (2, 4). Puritted by
countercurrent distribution between pH 5.8 phos-
phate buffer and ethyl acetate. Acidification of
active fractions gives crystals. Recrystallized
from pyridine-dioxane-ether (7).
Chemical and physical properties: Contains a
phenazine nucleus (6). Yellow prisms. Browns at
al)out 160°C, darkens at 180°C, and chars at 220°C.
Solul)le in aciueous NaHCO.'s and pyridine. Spar-
ingly soluble in dioxane and methyl ethyl ketone.
Insoluble in toluene, ether, and butyl acetate.
Ultraviolet absorption spectrum maxima at 281 to
283 niM (EiL 296) and 342 to 344 niyu (J5}L 170)
(methanol). Infrared absorption spectrum given
in reference 1. [a\',i' = —6° (c = 0.7 per cent in
pyridine) (7). Infrared spectrum given in reference
4. Unstable in aciueous solution. C47HibObN2 (3).
The structure of griseolutein B (l-methoxy-4(a,/3-
dihydroxyethoxymeth3'l)phenazine - 9 - carboxylic
acid) (8-10) is given in Chapter 6. Diacetyl griseo-
lutein B: Light yellow prisms (7). Alkaline hy-
drolysis of this derivative gives griseoluteic acid
(7)."
Biological activity: Active on gram-positive and
gram-negative bacteria, mycobacteria, and pro-
tozoa (2, 3, 5). Antirickettsial activity (3). Only
slight cross-resistance between A and B. Active
in mice on Staph, aureus infections (2). Active on
Ehrlich carcinoma in mice (5). B has weaker anti-
bacterial activity than A, especially against Pr.
vulgaris (4). Diacetyl griseolutein B is biologically
active (8).
References:
1. Yagishita, K. et al. J. Antibiotics (Japan)
6A: 113-116, 1953.
2. Ogata, Y. et al. J. Antibiotics (Japan)
6A: 139, 1953.
3. Ogata, Y. Japan. J. Med. Sci. & Biol. 6:
493-501, 1953.
4. Osato, T. et al. J. Antibiotics (Japan)
7A: 15-16, 1954.
5. Umezawa, H. Giorn. microbiol. 2: 160-
193, 1956.
6. Nakamura, S. et al . J. Antibiotics (Japan)
lOA: 265-266, 1957.
7. Nakamura, S. Chem. Pharm. Bull. 6:539-
543, 1958.
8. Nakamura, S. Chem. Pharm. Bull. 6:
547-550, 1958.
9. Nakamura, S. J. Antibiotics (Japan) 12A:
26-27, 1959.
10. Yagashita, K. J. Antibiotics (Japan) 13A:
83-96, 1960.
Griseoiiiyciii
Produced by: Streptomyces griseohis (1, 3).
Synonyms: Griseomycin is similar or identical
to proactinomycin B, and is isomeric with picro-
mycin (6). Related to amaromycin (1) andlomycin
(2").
Method of extraction: Whole culture acidified to
pH 2.0 and hltered. Broth-iiltrate can be extracted
DESCRIPTIONS OF ANTIBIOTICS
283
at an alkaline pH l)y most organic solvents (ben-
zene, chloroform, ether, amyl acetate, and tolu-
ene). Back-extraction in water at pH 2.0. After
two or three purifications in this manner, concen-
tration of organic solvent solution permits crystal-
lization (1, 3).
Chemical and physical properties: Macrolide,
containing desosamine. White plate crystals, hav-
ing a bitter taste; m.p. 76-80°C (1) or 70-75°C (3).
Very soluble in most organic solvents, but only
slightly soluble in water. Infrared data given in
reference 3. [a^i^ = +32° (c = 1 per cent in CHCLi)
(1), or [a]o = -7.0° (CHCl:i) (3). Stable at room
temperature between pH 2 and 9, but rapidly
destroyed at 100°C. C28H48NO8 . Molecular weight,
530. Hydrochloride: m.p. 120°C. Very soluble in
water, ethanol, methanol, and chloroform. Poorly
soluble in other organic solvents. Precii)itated
from aqueous solutions by picric acid and Rei-
necke salt (1, 3, 6). Criseomycin reacts with
methyl iodide to form a ((uaternary salt: C29H51-
NOsI, m.p. 193-195°C. [«]„ = +31.0° (ethanol).
This salt has the same formula, melting point, and
infrared spectrum as the picromycin quaternary
salt formed with methyl iodide, but differs in rota-
tion (5).
Biological activity: Active mainly against gram-
positive bacteria, including mycobacteria. Active
against Neisseria. Inactive against most gram-
negative bacteria and C. albicans. Cross-resistance
in vitro between carbomycin, erythromycin, and
griseomycin. Not inactivated in vitro by human
serum or whole blood (1). Destroyed in the liver
(-t).
To.ririty: LDoo (mice) 1330 mg per kg subcutane-
ously, 210 mg per kg intraperitoneally. Orally, 2100
mg per kg is not toxic, (iriseomycin is absorbed
by the gastrointestinal wall (1). Nontoxic to
rabbits when applied cortically {()).
References:
1 . Van Dijck, P. J. et al. Antibiotics & ('hemo-
therapy 3: 1243-1240, 1953.
2. DeSomer, P. et al. Resumes Communs. Oth
Intern. Congr. Microbiol. 1: 161, 240, 1953.
3. Belgian Patent 522,647, September 30, 1953.
4. DeSomer, P. et al. Antibiotics & Chemo-
therapy 4: 546-550, 1954.
5. Vanderhaeghe, H. Cited in reference 4.
6. DeSomer, P. Giorn. microbiol. 2: 216-232,
1956.
Griseoviridin
Produced by: Streptomyres griseus and S. griseo-
viridus (1, 5). These same strains also produce
etamycin (viridogrisein) (1).
Method of extraction: Culture-broth adjusted to
pH 4.4 and filtered on Hyflo Super-Cel. Extraction
of the filtrate at pH 7.2 with 0.5 volume of ethylene
dichloride. Concentration of the extract in vacuo
yields a precipitate of crude griseoviridin, which
is added to boiling absolute methanol and filtered
on paper. To the filtrate, Nuchar C190N is added;
after refiuxing for 5 minutes, the suspension is
filtered through paper. Crystallization is allowed
to proceed by storing the filtrate at 8°C. The
crystals are washed with cold absolute methanol
and dried in vacuo (1). Crvstallized from pvridine
(2).
Chemical and physical properties: Neutral sub-
stance. Griseoviridin base: Plate crystals; m.p. 228-
230°C. Soluble in pyridine; moderately soluble in
lower alcohols; sparingly soluble in water and
nonpolar solvents. Ultraviolet absorption spec-
trvnii maximum at 220.5 ni/j (e = 44,000) and an
inflection at 277.5 mju (e = 1500). Infrared data
given in reference 2. [a]" = —237° (c = 0.5 per
cent in methanol). Contains no thiol, methoxyl, or
methylimino groups. Positive Baeyers and chro-
mate-nitric acid tests. Negative FeCh , Folin-
Ciocalteau, Sakaguchi, and Jacobs-Hoffman tests.
Acid hydrolysis products include cysteine, serine,
and an unidentified ninhydrin-positive substance
(3). C2.2H.>9()7N,S : C = 54.9%; H = 5.8%; O =
24.4%; N = 8.7%; S = 6.2%. Molecular weight,
485 to 490 (1-3). Griseoviridin crystallizes from
methanol in two different forms, depending on the
temperature of the solvent dtu-ing crystallization.
Form A (a hemimethanolate) is obtained at
>30°C; form B at <30°C. B loses solvent in air to
give A. Form A: Large crystals; m.p. 160°C.
[aJu = —237° (c = 0.5 per cent in methanol).
Crystallographic data given in reference 2. Reac-
tion product with HCl: Needles; m.p. about 180°C
(decomposition) (3). Partial structure of griseo-
viridin (4) :
CHs
N— C— S— CH,— CH— C—
/ I I
_C=0 N
/ \
o
\ II
N— CHo— [C5(OH)]— CH— C=CH— C—
/ I I
OH O O
I I II
CH.,CHCH2CH=CHC—
Biological activity: Active in vitro against a
variety of gram positive and gram-negative Ijac-
teria and actinomycetes at a level of 0.1 to 10 yug
284
DESCRIPTIONS OF ANTIIMOTICS
per ml, such us strains of .1. hovis, Br. siiis, ('lus-
tridium hemolijticum, Corynebacterium diphtheriae,
D. pneumoniae, E. coli, H. influenzae, Neisseria
catarrhalis, Sh. paradysenteriae, and Streptococcus
pyogenes. Among the nonsensitive bacteria are
strains of K. pneumoniae, Staph, aureus, Pr.
vulgaris, and Ps. aeruginosa. No activity against
fungi, Endamoeba histolytica, Trichomonas foetus,
Trypanosoma crnzi, or T. rhodesiense. Active
against H. pertussis in mice. Not active against
experimental tuberculosis of mice. Active against
bovine mastitis caused by Staph, aureus, Staph,
albus, or E. coli, and against infectious bronchitis
of chickens. Limited antirickettsial activity
against Miyagawanella ornithosis and M. psittacii
in mice, and Rickettsia prowazekii in eggs. Inactive
against viruses in embryonated eggs (1).
Toxicity: LD50 (mice) 75 mg per kg intrave-
nously, >100 mg per kg intraperitoneally, >100
mg per kg subcutaneously. Maximal tolerated dose
(mice) 50 mg per kg intraperitoneally. Not irritat-
ing when instilled into the udders of lactating
cows (1).
References :
1. Bartz, Q. R. et al. Antibiotics Ann. 777-
805, 1954-1955.
2. Ames, D. E. e? oi. J. Chem. Soc. 4260-4263,
1955.
3. Ames, D. E. and Bowman, R. E. J. Chem.
Soc. 4264-4270, 1955.
4. Ames, D. E. and Bowman, R. E. J. Chem.
Soc. 2925-2928, 1956.
5. Anderson, L. E. et al. Antibiotics & Chemo-
therapy 6: 100-115, 1956.
Grizin
Produced by: Streptomyces sp. of the S. griseus
group (1).
Synonyms: Grizemin, antibiotic lEM 1.
Method of extraction: Broth treated with char-
coal at pH 3. Filtrate adjusted to pH 4.5, refiltered,
and antibiotic adsorbed at pH 7.5. Elution with
40 to 50 per cent ethanol at pH 3. Neutralization
followed by concentration in vacuo. Purified by
formation of various crystalline salts, e.g., picrate
and helianthate (1).
Chemical and physical properties: Basic anti-
biotic. Helianthate: Brown powder; m.p. 194-
196°C (decomposition). Hydrochloride: White
powder. Soluble in water and methanol. Stable
as dry powder and to boiling for 10 minutes. Posi-
tive biuret, ninhydrin, and Bert rand tests. Test
for glucosamine positive. Negative Sakaguchi,
maltol, and histidine tests. N = 13.6 to 14.9%
(helianthate) in the form of a-amino nitrogen (1).
Probably a polypeptide.
Biological activity: Active on gram-negative and
gram-positive bacteria, yeasts, and fungi. Active
on dysentery in mice. Effective on angular leaf
spot of cotton, and lemon and mandarin necrosis
{Bacterium citriputeale). Prevents withering of
apricot {Bad. armeniaca). EfTective against a
lemon tree disease (Deuterophoma tracheiphilla)
(1).
Toxicity: Mice tolerate 0.5 mg per day for 6 days
(1).
Utilization: Dysentery in children (1).
Reference: 1. Krassilnikov, N. A. et al. Mikro-
biologiya 26: 418-425, 1957.
Grubilin
Produced by: Streptomyces sp.
Method of extraction: Isolated from the mycelium
with organic solvents.
Chemical and physical properties: Green-yellow
substance. Base: Insoluble in water and organic
solvents. Gives color reactions and an ultraviolet
absorption spectrum typical of a heptaene. Xa
salt: Water-soluble.
Biological activity: Active on yeasts and fila-
mentous fnngi; not active on bacteria or Strepto-
myces.
Toxicity: LD50 (mice) 15 mg per kg intrave-
nously, 30 mg per kg intraperitoneally, and >500
mg per kg subcutaneously.
Reference: 1. Uri, J. et al. Abstr. Communs.
Symposium on Antil)iotics, Prague. 46-47, 1959.
Heliomyciu
Produced by: Streptomyces flavochrouiogenes var.
heliomycini .
Synonyms: Resistomycin, antibiotic X 340.
Method of extraction: Mycelium washed and
pressed. Extracted with 3 volumes of technical
acetone per volume of wet mycelium. Extraction
repeated with one half the volume of acetone. To
the combined acetone-extracts, saturated barium
hydroxide is added until no further precipitation
occurs. The precipitated barium salt of heliomycin
is suspended in water. HCl added to bring the pH
to 3.0; this solubilizes barium as barium chloride,
which is removed by filtration. The precipitated
heliomycin is put in solution in acetone at pH 7.5
and impurities filtered off. Precipitation of yellow
crystalline heliomycin upon acidification to pH
3.0 with HCl. Recrystallization from acetone or
dioxane.
Chemical and physical properties: Yellow needle-
shaped crystals. Soluble in dioxane, acetone, ethyl
acetate, butyl and ethyl alcohols. Less soluble in
chloroform, ether, and benzene. Poorly soluble in
carl)on tetrachloride. The solubility in organic
l)i:SC'HIPTI()XS OF AXTIlilOTICS
285
solvents is iiKTeased by aciilific;itioii. IhsoIuIjIc in
wiiter unless strong alkalies are added; then it
gives a red color. Very stable except in aqueous
ail<aline solutions, such as 0.1 A' NaOH. Xo melt-
ing point; vipon heating over 100°C, it decomposes.
Molecular weight, 235 (Rast). Positive Millon,
sodium nitrite, and sodium nitr()])russide tests.
i'ositive Anchel test for i^henols and ciuinones. Xo
reduction of Fehling's reagent. Contains no X', S,
or P. Ala.ximal absorption of light at 2()9, 290, 320,
340, 370, 400, and 515 m/x (alcoholic solution).
Alkaline degradation is accompanied by changes
in light -absorption spectrum (given in reference
4).
liioloijii-dl tutivilij: Active on staphylococci and
iuHuenza virus (contact test). Slight activity on
influenza in mice (1). Active on tobacco mosaic
virus it\ vitro (2) and smalljjox vaccine (4). B.
injicoidcx used as assay organism (5).
Toxicity: LDso (mice) 25 mg jx'r kg intraperi-
toneally (6).
References:
1. Gause, G. F. Giorn. mici()l>iol. 2: l!)4-200,
1950.
2. Ukholina, R. S. Mikrobiologiya 27: 347-
350, 1958.
3. Brajhnikova, M. (i. et at. 2n(l All-Union
Conf. Antibiotics, Moscow, p. 9, 1957.
4. Kremer, V. E. Antibiotiki 4(6) : 59-(J3, 1959.
5. Brajhnikova, M. G. c/ «/.. Antibiotiki 3(2):
29-34, 1958.
0. Goldberg, L. E. Antil)i()tiki 5(1): 107-112,
19()().
Holoniycin
Frodticcd by: Streptomi/ces griseus.
Si/iiohijin: Belongs to the thiolutin aureothricin
series, differing from thiolutin oidy in the X — CHs
group.
Method of extraction: Broth-filtrate extracted
with ethyl acetate. E.xtract concentrated in vacuo.
Ghromatographed on aluminum oxide, washed
with absolute chloroform anfl chloroform metha-
nol (99:1), and eluted with chloroform-nu>t haiiol
(,97:3). Active fractions concentrated in vacuo to
an oily residue. Residue taken up in ethyl acetate;
the antibiotic precipitates after a short time. Re-
crystallized from methanol-ethyl acetate.
Chemical and physical properties: Des -X"^ -methyl -
thiolutin. X'eutral. Glittering orange-yellow
rhomi)ic crystals; m.p. 2G4-271°C (mixed melting
point with thiolutin, 240-250°C). Could be sub-
limed in vdciio. Soluble in organic solvents. Ultra-
violet absorption spectrum maxima (ethanol) at
about 390 m^x, with lesser peaks at about 303 and
250 mjj.. Infrared spectriuu given in reference 1.
Paper chr()nia(ograi)liic data given in reference 1.
C7H602X,S, : C = 39.25%; H = 2.79%; X =
13.07%; S = 29.77%; C— CH, = 7.04%; CH.CO =
21.38%. .\cid hydrolysis product is "holothin,"
C5H40Xi or des-X'-methylpyrrothine. Structural
formula of holoniycin given in Chapter G.
Biological oi-tivity: .\ctive on Streptococcus
pyogenes (1 ^g per ml), 1'. chidcrae, M . tuberculosis,
E. coli, Sal. schiilltuucllcn, and Klebsiella (10 yug
per m\); Staph, aurcu.^, Ps. aeruginosa, ('. albicans,
and Endouiyccs albicans (101 fxg per ml). Active on
Trichomonas foetu.K at 1 ^ug per ml and Endamoeba
histolytica at 10 /xg per nd. Holothin has some
antil)iotic activity.
Reference: 1. Ettlinger, L. et al. Helv. Chim.
Acta 42: 563-569, 1959.
il
iiniKlii
Produced by: Strcptoniyccs huuiidus, the strain
that also produces dihydrostreptomycin.
Method of extraction: Cells extracted with ace-
tone. Concentration of solvent under reduced
pressure. Aqueous concentrate acidified with HCl
aiul extracted with ethyl acetate. Back-extraction
with 1 A' XaOH. Upon neutralizatioji, crystalliza-
tion occurs.
Chemical ami physical properties: Colorless
platel(>ts; m.]). 145 14()°C. Tentative empirical
formula: {CvM-ni()i)„ ■ (a)i,'' = —6° (c = 1 i)er cent
in ethanol). [a]'v = —10° (c = 1 per cent in ace-
tone) and —8° (c = 1 per cent in dioxane). Light-
absorption maxinui at about 245 and 285 m/x.
Infrared absorjjtion sj)ectruni given in reference 1.
Soluble in acetone, dioxane, ami ethyl acetate.
Slightly soluble in n-butyl alcohol, ether, and
ethanol. Insoluble in methanol, benzene, water,
petroleum ether, and carbon tetrachloride. Orange
color with sulfuiic acid. Potassium permanganate
and bromine water decohji'ized. Xegative Fehling
reaction.
Biological activity: Active against certain fungi
and protozoa. Xo activity against bacteria. Active
against Sacch. cerevisiae but not against species of
Candida. More active at alkaline than at acid jjH.
Activity reduced by ascorbic acid but not l)y
cysteine.
Toxicity: LD50 (mice) 4.5 mg jjer kg intrajieri-
toneally, 54 mg per kg orally.
Reference: 1. Xakazawa, K. et al. J. Agr. Chem.
Soc. Japan 32: 713-716, 1958.
Ilv<i
i'ox\ iii\ cm
Produced by: Streptomyces paucisporogenes (2).
Synonyms: Antibiotic 4915 (2). Similar to cate-
nulin and ]iai'()momycin (4).
Method of cxtna-tion: Cultiu'e-filtrate adjusted
286
DESCRIPTIONS OF ANTIBIOTICS
to pH 6 to 7, calcium precipitated as the oxalate,
and the whole filtered. Filtrate adsorbed on IRC-
50 (Na+ phase), eluted with 1 ,V H0SO4 , and pre-
cipitated as the pentachlorophenol derivative.
Purified by conversion to the sulfate, followed by
fractional crystallization from an aciueous metha-
nolic solution of the /^-(/>'-hydroxyphenylazo)ben-
zene sulfonate (2).
Chemical and physical properties: Basic amino
polysaccharide. Base: White amorphous powtier.
Soluble in water and methanol. Insoluble in com-
mon organic solvents. [a]f = -|-63° ± 2° (c = 1
per cent in water). C20H47O15X5 . Molecular
weight, 610 (2, 3). Methanolysis in the presence of
HCl gives a product which was termed "pseudo-
neamine," CisHooOtN.-j , composed of 1,3-diamino-
4,5,6-trihydroxycyclohexane (meso) (also present
in neomycin and kanamycin) and D-glucosamine.
Two possible structures for pseudoneamine were
proposed :
OH
NH, H
H / \ OH
H OH\
NHo H
H
CH.OH
H
H
H
H ().
H \
H OH/
OH
NHo H
Sulfate: White amorphous powder. No charac-
teristic infrared or ultraviolet al)sorption spec-
trum, [a if," = -f 50-52° (c = 1 i)er cent in water)
and does not vary with pH. Rf = 0.4(1 on paper
chromatography (methanol 2 i)or cent aqueous
NaC'l, 2:1), paper impregnated with NaHSOj at
pH 2.4. Positive ninhydrin, diazo p-nitroaniline
(for primary amino groups), and periodic acid
tests. Negative nitroi)russide (secondary amino
groups), Fehling, Sakaguchi, Pauly, and I'llson-
Morgan tests. Furfural formed on treatment with
acid. Total N = ().2%; N (Van Slyke) = G.0%; N
(Sorensen) = 3%. p-(p' -Hydroxy phenylazobenzene
sulfonate: Orange crystals; m.p. 220°C (decom-
position). Thermostable from ])H 2 to 10. la]f:' =
+37° (c = 0.5 per cent in methanol) (2). N -Benzoyl
hydroxymycin: White needles; m.p. 232°C (decom-
position). [a\f = +36° ± 2° (c = 0.3 per cent in
methanol). Biologically inactive (2).
Biological activity: Active on gram-negative and
gram-positive bacteria and mycobacteria. Active
on Endamoeba histolytica at 5 to 10 jug per ml (2).
Weak activity in vitro on Trichomonas vaginalis
(1). Not affected by serum or glucose. Active on
mycobacteria resistant to streptomycin. Active in
mice on infections caused by Staph, aureus, Diplo-
coccus mucosus, K. pneumoniae, Sal. typhosa, E.
coli, and Ps. aeruginosa. Less active on D. pneu-
moniae and Streptococcus hemolyticus infections.
As active as streptomycin on tuberculosis in mice
and guinea pigs (2). Active in mice on T. vaginalis
(1). No antiviral activity in vivo.
Toxicity: LD50 (mice) 125 ± 5 mg per kg intra-
venously, 1020 =h 110 mg per kg subcutaneously.
Does not have neon\ycin-like toxicity (2).
References :
1. Vaisman, A. and Hamelin, A. Comjjf. rend.
247: l(i3-165, 1958.
2. Hagemann, (.J. et al . Ann. pharm. frang. 16:
585-596, 1958.
3. Bartos, M. J. Ann. pharm. frang. 16: 596-
600, 1958.
4. Schaffner, C. P. Personal communication.
Hy tli'oxyst rep toni vein
Produced by: Streptoini/ccs rubrireticuli (formerly
referred to as S. rcticuli) (1, 11). This strain also
produces rotaventin. S. griseocarneus (2, 3, 5, 6, 8).
These strains also produce an antifungal substance
in low yield (9). Streptoniyces sp. This strain differs
from the above, but belongs to the reticuli group
(12).
Synony}ns: Reticulin (1, 10), antil)iotic NA 232-
Ml (2).
Method of extraction: I. See I under strepto-
mycin. II. Culture-broth acidified, filtered, neu-
tralized, and adsorbed on IRC-50. Eluted with 0.5
A'^ H2SO4 . Eluate neutralized, concentrated, and
acetone added to give the crude substance. Puri-
fied by chromatography on Darco G-60-Celite 545
(2).
Chcniical and physical properties: Differ?, from
streptomycin by one oxygen atom present as a
hydroxyl group on the methyl group of the strep-
tose moiety (2) (see Chapter 6). Solul)lo in water,
ethanol, and dilute acids (9). [a]„ = -790° (2).
DESCRIPTIONS OF ANTIBIOTICS
287
Positive Sakaguchi, Elson-Morgan, Alolisch, and
Benedict tests (I). Negative biuret, ninhydrin,
Fehling, and maltol tests (2). Rf = 0.09 to 0.11
(n-butanol, 2 per cent piperidine, 2 per cent p-
toluene sulfonic acid monohydrate) (1). About
50 per cent inactivation at 100°C for 5 to 10 min-
utes (1). Trihydrochlonde: White substance (4).
[a Id = —95° (c = 1 per cent in water) (4). C =
35.6%; H = 5.95%; N = 13.9%; CI = 14.8%.
CaiHsgNyOnrSHCl. Helianthate: Reddish brown
crystals. Darken at about 220°C and char without
melting. X-ray diffraction pattern similar to
streptomycin helianthate (4). Yields a dihydro
derivative on catalytic hydrogenation (3, 8).
Alkaline hydrolysis products include pyromeconic
and isokojic acids, but no maltol (2, 10). Isokojic
acid (2-hydroxymethyl-3-hydro.\y-l,4 pyrone) :
m.p. 154-157°C. Ultraviolet absorption spectrum
maximum at 274 m^ (£"1™ 690) in 0.1 N HCl. Posi-
tive FeCls test (2, 3).
Biological activity: Similar to that of strepto-
mycin (1, 2). Inactivated by cysteine and reacti-
vated by iodine (1).
Toxicity: LDjo (mice) 154 mg per kg intrave-
nously, 865 to 948 mg per kg subcutaneously (2, 7).
Acute and chronic toxicity said to be essentially
the same as for streptomycin.
Utilization: None. Presents no known advantage
over streptomycin.
References :
1. Hosoya, S. et al. Japan. J. Exptl. Med.
20: 327-337, 1949.
2. Grundy, W. E. et al. Arch. Biochem. 28:
150-152, 1950.
3. Benedict, R. G. et al. Science 112: 77-78,
1950.
4. Stodola, F. H.e/aL J. Am. Chem. Soc. 73:
2290-2293, 1951.
5. Benedict, R. G. et al . J. Bacteriol. 62:
487-497, 1951.
6. Grundy, W. E. et al. Antibiotics & Chemo-
therapy 1: 309-317, 1951.
7. Ambrose, A. M. Proc. Soc. Exptl. Biol.
Med. 76: 466, 1951.
8. Benedict, R. G. and Stodola, F. H. U. S.
Patent 2,617,755, November 11, 1952.
9. Hosoya, S. et al. J. Antibiotics (Japan)
5:525-527, 1952.
10. Hosoya, S. et al. J. Antibiotics (Japan)
6A: 102, 1953 (abstr. of 6B: 61-66, 1953).
11. Hosoya, S. Quoted in Benedict, R. G.
Botan. Rev. 19: 229-320, 1953.
12. Nakazawa, K. et al. Ann. Rept. Takeda
Research Lab. 13: 67-77, 1954.
Hygroiiiyciii
Produced by: Streptoniyces hygroscopicu.'i (1) and
S. noboritoensis. The latter organism also produces
blastmycin and an antibiotic active on gram-
positive bacteria (7).
Synonym: Homomycin (7).
Method of extraction: I. Broth-filtrate satu-
rated with (NH4)2S04 and extracted with n-
butanol. Extract concentrated in vacuo, filtered,
and petroleum ether added to precipitate hygro-
mycin. Chromatographed on carbon from a 0.001 N
sulfuric acid solution and developed with an aciue-
ous solution containing 10 per cent n-butanol and
30 per cent acetone. Active fractions concentrated
in vacuo with addition of n-butanol to remove
water. Antibiotic precipitated on addition of
petroleum ether. Further purified by counter-
current distribution between n-butanol or n-amyl
alcohol and water-glacial acetic acid (2). II. Cul-
ture media acidified, stirred with acidic clay, and
filtered. Filtrate neutralized and stirred with
active carbon. Eluted from the carbon with 80
per cent aqueous acetone. Eluates concentrated
in vacuo and acetone added to the concentrate to
precipitate impurities. Concentrated in vacuo,
then lyophilized. Chromatographed on alumina
from methanol-ethanol (1:1) and developed with
acetone containing 20 per cent 0.5 N HCl. Further
purification by chromatography on silica gel with
water-saturated n-butanol as solvent and devel-
oper. Active fractions treated with carbon, con-
centrated in vacuo, and precipitated by adding
ether or petroleum ether. Final purification by
countercurrent distribution, first in an n-butanol-
0.25 M phosphate buffer (pH 4.6), then in n-bu-
tanol-ethyl acetate-water (1.2:0.5:1.9) (7).
Chemical and physical properties: Weakly acidic
substance (2). White powder. Gradually melts at
80-90°C. Colors above 155°C (7). Very soluble in
water and ethanol; essentially insoluble in less
polar solvents. Infrared absorption spectrum
given in reference 2. Ultraviolet absorption spec-
trum maxima in dilute acid at 214 m/u (Eum 416)
and 272 m^ (ElL 306) (2) ; in dilute alkali at 254
m,x (£'}L 350), 286 m^ (£"5^ 194), and 323 m,x
(E'lcm 116) (6); in water at 270 to 272 m,u (E'i'L
291) (3). [a]f = -126° (c = 1 per cent in water)
(2). Positive Folin-Ciocalteau, Fehling, Benedict,
diazo, iodoform, Nessler, ToUen, indole, and
carbazole tests. Negative FeCla , Ehrlich, nin-
hydrin, biuret, phloroglucinol, Seliwanoff, gluco-
samine, anthrone, Molisch (doubtful), and maltol
tests (2, 3, 7, 8). Acetylation product is biologically
inactive (2, 3). pKa' = 8.9 (water) (6). Rf = 0.63
in water-saturated n-butanol. Cannot be hvdro-
288
DESCRII'TIOXS OF ANTIBIOTICS
genated; j-ield^ a biologicall)- inactive tetraacetate
(7). Stable to boiling for 10 minutes at pH 3.7; less
stable at pH 9.0 and above (I) . 2 ,4-Dinitrophenijl-
hydrazone derivative: Yellow crystalline substance;
m.p. 154-156°C (6, 7). Acid hydrolysis products of
hygromycin include neo-inosamine-2 (5, (i). Alka-
line hydrolysis yields 3,4-dihydroxy-a-methylcin-
namic acid. The sugar in hygromycin is 5-keto-6-
desoxyarabohexose, C-i.iHigNOri : C = 53.85%;
H = 6.15%; N = 2.78%; O = 37.53%; C— Me =
2.56%. The possible structure of hygromycin (6)
is discussed in Chapter 6.
Biological activity: Active on certain gram-
positive and gram-negative bacteria, mycobac-
teria, and actinomycetes at 3 to 51 jug per ml (1).
Active on Endamoeba histolytica and Leptcjspira
pomona (11). Very active on pleuropneumonia-
like organisms (9). Activity on K. pneumoniae
inhibited by cysteine. Activity unaffected by pH.
Rate at which resistance develops depends on the
microorganism used. Active in mice on M. tubercu-
losis H37I{v, l)Ut only one third to one fifth the
activity of streptomycin. Also active in mice on
Streptococcus pyogenes and Borrelia novyi, and
moderately active on K. pneumoniae. Also active
against mouse meningopneumonitis infections.
Not active against viruses such as "MM" and
Semiliki Forest viruses (1, 7). Active on E. his-
tolytica (rats) and oxyurids (mice) (11).
Toxicity: Mice tolerate 2 gm per kg intrave-
nously and subcutaneously (7).
I'tilization: Prevention of bacterial decomposi-
tion of fish stickwater (4). Used in animal feeds
for prevention and cure of large round worms,
nodular worms, and whipworms (10).
References :
1. Pittenger, R. C. ct al. Antil)iotics &
Chemotherapy 3: 121)8-1278, 1953.
2. Mann, R. L. et al. Antibiotics & Chemo-
therapy 3: 1279-1282, 1953.
3. Sumiki, Y. et al. J. Antibiotics (Japan)
8A: 170, 1955.
4. Idler, D. R. et al. Appl. Microbiol. 3:
265-268, 1955.
5. Isono, K. et al. J. Antibiotics (Japan)
9A: 225, 1956.
6. Mann, R. L. and Woolf, 1). O. J. Am.
Chem. Soc. 79: 120-126, 1957.
7. Isono, K. et al. J. Antibiotics (Japan)
lOA: 21-30, 1957.
8. Namiki, M. et al . J. .\n1 il)iotics (Japan)
lOA: 160-170, 1957.
9. Wick, W. E. and Holmes, 1). H. Bacteriol.
Proc. 21-22, 1958.
10. Conrad, J. H. and Beeson, W. M. Purdue
Univ. Agr. Expt. Sta. Mimeo. AH 233,
May, 1958.
11. McCowen, M. C. et al. Antibiotics Ann.
883-886, 1956-1957.
12. Handy, A. H. et al. Poultry Sci. 36: 748-
754, 1957.
Hygromycin B
Produced by: Streptcmyces hygroscopicus.
Method of extraction: Broth treated with Amber-
lite IRC-50 (Na+ cycle). Elution with 0.1 .V HCl.
Eluate treated with carbon at pH 10.5. Hygro-
mycin B eluted with a mixture of concentrated
NH4()H, water, and acetone (1:3:6). Eluate con-
centrated and antibiotic precipitated with ace-
tone. Precipitate dissolved in methanol. Repre-
cipitation with ether. Further purification by
adsorption on Amberlite IRC-50 (Li+ cycle) and
elution with 29 per cent NH4OH (1).
Chemical and physical properties: Polyhydroxy
l)ase. Amorphous powder. Melts over wide range
about 180°C. C1.5H28N2O9-10. Very soluble in
water and methanol; essentially insoluble in less
polar solvents. Two titrable basic groups: pKa'
7.1 and pKa' 8.8. No absorption ot ultraviolet
light. Infrared data given in reference 1. Positive
anthrone and Molisch tests. Negative Benedict
and Fehling tests. Forms a crystalline p-(p'-
hydroxyphenylazo)benzenesulfonic acid salt.
Biological activity: Moderate activity on gram-
positive and gram-negative bacteria and fungi
(6.2 to 100 Mg per ml). Very active on helminths,
including ascarids, in swine (1). Increases growth
rate of baby pigs when added to the diet (2).
Utilization : .\nthelmintic.
References:
1. Mann, R. L. and Bromer, W. W. J. Am.
Chem. Soc. 80: 2714-2716, 1958.
2. Teague. H. S. and Rut ledge, E. A. Ohio
Agr. Expt. Sta. Mimeo 114, July, 1959.
H> fir*>niycin-like Aiitihiolic
Produced by: Streptomyces sp.
Synonyms: Antibiotic 1703-18B. Similar, Imt
not identical, to hygromycin (1).
Chemical and physical properties: Acid hy-
drolysis products include: neo-inosamine-2 (1)
and the 3,4-dihydroxy-a-methylcinnamic acid
amide of neo-inosamine-2. The latter is isomeric,
and possibly identical with, the corresponding
degradation i)roduct of hygromycin (2). Structure
of the degradation product:
DESCRIPTIONS OF ANTIBIOTICS
289
HO-
HO— I
OH
OH
References:
1. Patrick, J. B. el al. J. Am. Chom. Soc. T«:
2652, 1956.
2. Allen, i\. R., Jr. J. Am. Chem. Soc. 78:
5691-5692, 1956.
Hygroscopins
Produced by: Streptoniyces hyqroscopicus (1, 2).
Synonyms: Hygroscopiii A and elaiomycin are
similar or closely related.
Method of extraction: Broth extracted with
butyl acetate at pH 2.0. Extract washed with
sodium bicarbonate solution, then concentrated
in vacuo. Concentrate extracted with methanol.
Extract chromatographed on carbon to give three
fractions. Fraction I (eluted with methanol) gives
hygroscopin B on distillation. Fraction II (eluant,
methanol) contains hygroscopins A and B, which
are separated b}^ distillation or carbon chroma-
tography. Fraction III (eluant, ethyl acetate)
gives hygroscopin C (1,3).
Chemical and physical properties: H yi/roscopiii
A: Oil; b.p.„.„o3 64°C. [a]o^ = +84.7° (methanol).
Ultraviolet absorption spectrum maximum at 235
m;u (ethanol). Refractive index: no 1.4830. Ci:)H34-
OisN-j . Hygroscopin B: Oil; b.p.o.oos 70°C or b.p.d.o
108°C. [a\\* = -38.8° (methanol). Ultraviolet
absorption spectnun maximum at 233 m/i (etha-
nol). Refractive index: tiv 1.4935. Infrared spec-
trum given in reference 1. C10H2SO3N2 : C =
63.53%; H = 10.05%; N = 10.07%,. Molecvdar
weight, 290 ± 10. Hygroscopin (': Not charac-
terized (1 , 3).
Biological activity: Hygroscopin A: Active on
fungi, yeasts, influenza A virus (in tissue culture),
and M. tuberculosis var. hominis H37Rv. Tran-
siently active on Yoshida sarcoma (rats) at 1 mg
l)er kg. Hygroscopin B: Active on influenza A
virus (as above) but not on fungi, yeasts, or myco-
bacteria. Same activity as hygroscopin A on
Yoshida sarcoma.
Toxicity: Hygroscopin A: LD.^i (mice) 8.75 mg
per kg intraperitoneally. Hygroscopin B: Ll):,ii
(mice) 1928 mg per kg intraperitonealh*.
Eeferences:
1. Tatsuoka, S. et al. J. Antil)iotics (Japan)
7B: 329-332, 1954.
2. Nakazawa, K. et al. J. Agr. Chem. Soc.
Japan 28: 296-299, 1954.
3. Tatsuoka, S. et al. J. Antibiotics (Japan)
8A: 31, 1955.
Hygrostatin
Produced by: Streptomyces hygrostaticus.
Synonym: Similar to musarin.
Method of extraction: Mycelium collected and
washed with water. Extraction from mycelium
with methanol or acetone. Solvent concentrated.
Concentrate extracted with butanol at pH 6.5.
Precipitation from butanol with ether. Precipitate
dissolved in a 1:1 mixture of benzene and metha-
nol, and chromatographed over alumina. Elution
carried out by increasing the methanol content of
the mixture. Further purification by coiuiter-
current distribution in a chloroform-methanol-pH
7.0 phosphate buffer system (1:1:0.5). Active frac-
tions evaporated and dissolved in butanol. Upon
addition of ether, a precipitate forms, which then
is dried.
Chemical and physical properties: Pale yellow
powder. Decomposes 129-131 °C. Contains N but
no S or halogens. Very soluble in methanol and
pyridine. Soluble in ethanol, butanol, and iso-
l^ropanol. Slightly soluble in benzene, chloroform,
and acetone. Insoluble in ethyl acetate, ether,
petroleum ether, dioxane, and water. Dark red to
purple color with concentrated sulfuric acid.
Orange-brown to red-purple with concentrated
HCl. Negative biuret, FeChj , Benedict, ninhy-
drin, Fehling, and Tollen tests. Acid hydrolysate
also ninhydrin-negative. [a]o = -(-43° (c = 1.21
per cent in methanol). Light absorption maximum
at 240 niM (E\7m 360) and a shoulder at 255 to 270
nxfj.. Stable for 30 mimites at 100°C at neutrality.
Unstable at acid and alkaline pH values.
Biological activity: Active against filamentous
fungi, yeasts, and gram-i)ositive bacteria. Inactive
against gram-negative bacteria and Clostridia
Toxicity: LD50 (mice) 8.6 mg per kg intrave-
nously, 21.7 mg i)er kg intraperitoneally, 246.7 mg
per kg subcutaneously, and 530 mg per kg orally.
Necrosis at the site of injection. Autopsy shows
bleeding in the lungs. Nontoxic at 500 /xg per ml
when sprayed on plant leaves.
Reference: 1. Kojo, K. et al. Yakugaku Kenkyu
30: 654-664, 1958.
2<)0
DESCRIPTIONS OF ANTIBIOTICS
l-sorhodoiii veins
Produced by: Streptnnii/ces pnipiirasrens (1).
This culture also produces rhodomycius A and B.
Remarks: See rhodomycins.
Method of extraction: See rhodomycin A. Crude
isorhodomycin A from paper chromatography
taken up in a small amount of ethanol. A drop of
concentrated HCl added. Cooling gives crystals
(2).
Chemical and physical properties: Isorhodomycin
A: Dark red prisms; m.p. 220°C. Soluble in water
and low-molecular weight alcohols. Very slightly
soluble in benzene and chloroform. Insoluble in
ether and i)etroleum ether. Red fluorescence under
ultraviolet light. Ultraviolet light -absorption
spectrum maxima at about 235, 305, 525, 551, 563,
and 610 niyu (methanol). [q|606-7ro = +268° dz 30°
(c = 0.1 per cent in methanol). Cjo-iiHog-siOsN ■-
HCl: C = 54.25%; H = 6.84%; O = 27.92%; N =
3.12%; CI = 7.3%. Perchlorate: Thin red needles;
m.p. 177°C (2). Isorhodomycin B: Crimson-red
substance (3).
Biological activity: Active on Staph, aureus (2).
References :
1. Lindenbein, W. Arch. Mikrobiol. 17: 361-
383, 1952.
2. Brockmann, H. and Patt, P. Chem. Ber.
8a: 1455-1468, 1955.
Kanamycin A
Produced by: Streptomyces kanamyceticus.
Broths contain a second, butanol -soluble sub-
stance, active on B. subtilis (2), as well as kana-
mycin B (see next abstract). Rf values for the B.
subtilis factor, kanamycin A and B, respectively,
are 0, 0.1 to 0.26, and 0.21 to 0.37 on paper chro-
matography (2 per cent p-toluene sulfonic acid)
(9).
Method of extraction: I. Adsorbed from l)roth-
filtrates on IRC-50 (Na+ form), and eluted with
HCl. Eluate neutralized, diluted, and resorbed
on IRC-50 (regenerated with NH4OH). Eluted
with 0.2 X NH4OH; eluate concentrated in vacuo,
diluted with methanol, and adjusted to pH 8.0
to 8.2 to precipitate kanamycin sulfate. Repeated
recrystallization from methanol-water at pH 7.8
to 8.2. Converted to the base by treatment of an
aqueous solution of the sulfate with a strongly
basic ion exchange resin, concentration, and
crystallization with methanol-ethanol (6). II.
Broths treated with IRC-50 and eluted as in I.
Eluates adjusted to pH 6.0 to 8.0, evaporated
in vacuo, and lyophilized. Powder taken up in
methanol, filtered, and acetone added to give a
l)recipitate. Precipitated as the reineckate, then
converted to other salts (2, 3).
Chemical and physical properties: Tetraacidic
l)ase. Base: Soluble in water. Insoluble in non-
polar organic solvents (10). [afo = +146° (c = 1
percent in 0.1 X H)S04). Po.sitive ninhydrin, Mo-
lisch, and Elson-Morgan tests (6). The two latter
tests were at first mistakenly reported as being
negative (2). Blue-violet color with the biuret
test (24). Negative reducing sugar, Tollen, Saka-
guchi, and maltol tests (6). Stable; can be auto-
claved for 1 hour at 120°C in aqueous solution
with only 10 per cent loss of activity (10). Acid
hydrolysis products include 2-desoxystrept amine
(1 ,3-diamino-4,5,6-trihydroxycyclohexane, which
is also obtained from neamine) and two amino
sugars: 6-amino-6-desoxy-D-glucose and 3-amino-
3-desoxy-D-glucose (also known as kanosamine).
C = 44"8%; H = 7.5%; N = 11.3%. C.sHseN^Ou .
Structural formula (6, 7, 17) of kanamycin A given
in Chapter 6. Sulfate: White, irregular, prismatic
crystals (hydrate) (6) or plates (3). No melting
point; decomposes over a wide range above 250°C
(6). Soluble in water but insoluble in organic
solvents (2). [atf = +121° (c = 1 per cent in
H2O) (3). Hydrochloride: Hygroscopic, white,
amorphous powder. Very soluble in water, soluble
in methanol, and slightly soluble in ethanol. In-
soluble in acetone, ethyl acetate, butyl acetate,
ether, benzene, and petroleum ether. No charac-
teristic absorption maxima in ultraviolet light
(2). Infrared spectrum given in reference 2. [a],^ =
+ 103° (c = 1 per cent in H2O). More stable at pH
6 to 8 than pH 2.0 (2). Reineckate: Darkens at
191-193°C, decomposes at 211-213°C (24). Picrate:
Crystalline; m.p. 225-230°C (decomposition) (6).
Tetra-X -acetyl kanamycin: Crystalline; m.p. 250-
255°C (decomposition) (6). Characteristics of a
variety of Schiff bases formed by kanamycin
given in reference 6.
Biological activity: Active on gram-positive and
gram-negative bacteria, including actinomycetes
and mycobacteria. Relatively inactive on strepto-
cocci, diplococci, Clostridia, and Pseudomonas.
Not active on fungi (2, 18). Most active at alka-
line pH (18). Active /"« vivo (mice and guinea pigs)
on M. tuberculosis H37Rv, but is less effective
than isoniazid and slightly less so than strepto-
mycin. No cross-resistance with p-aminosalicylic
acid, cycloserine, streptovaricin, streptomycin,
or isoniazid. Partial cross-resistance with phleo-
mycin when E. coli is used as the test organism,
but not when Mycobacterium 607 is used. Cross-
resistance with streptothricin, neomycin, and
viomycin in vitro. Kanamycin causes fragilitj' of
DESCRIPTIONS OF ANTIBIOTICS
291
the cell wall in E. coU . Resistance to E. coti de-
velops slowly, in stepwise fashion. Resistance of
mycobacteria develops rapidly (3, 4, 9, 18). Active
in mice on D. pnnnnoin'ae, Sal. typhosa. Staph.
o///T».s, and Pr. Dilcians infections. Not active on
Streplococcus heinoli/ticiis (I, 2, 11). Preventive
action on mouse leprosy, leptospira in guinea pigs,
and Treponema pallidum infections in rabbits (8,
9). Not active on sarcoma 180 or lOhrlich carci-
noma (16).
Toxicity: Crystalline sulfate: LD50 (rats) 415 to
830 mg per kg intravenously; (rabbits) 225 to 300
mg per kg intravenously (3). LDsn (mice) 167.9
mg per kg intraperitoneally, 1648 mg per kg sub-
€Utaneously, and 316.3 mg per kg intravenously.
Doses greater than 10,000 mg per kg are tolerated
orally (poorly absorbed from the intestinal tract ).
Less vestibular toxicity to cats than streptomy-
cin; less auditory toxicity to rats than dihydro-
streptomycin. Much less nephrotoxicity to dogs
than neomycin (5). Nephrotoxic and ototoxic in
human beings, but does not have the delayed oto-
toxicity of neomycin (20).
Utilization: Active on certain diseases caused
by gram-positive and gram-negative organisms.
Can be given orally, parenterally, or topically
(19). Not considered effective in infections caused
by enterococci, pneumococci, or anaerobes (20).
Somewhat active on tuberculosis (21). Relief of
typhoid and Endamoeba histolytica carrier states
(22, 23) and can be used safely in patients allergic
to streptomycin (12). Possibly beneficial in cirrho-
sis (13). Bowel sterilization (14). Bacterial infec-
tions resistant to commonly used antibiotics (15).
References:
1. Takeuchi, T. et al. J. Antibiotics (Japan)
lOA: 107-114, 1957.
2. Umezawa, H. et al. J. Antibiotics (Japan)
lOA: 181-188, 1957.
3. Maeda, K. et al. J. .\iitil)iotics (Japan)
lOA: 228-231, 1957.
4. Steenken, W., Jr. et al. Trans. 17th Veter-
ans Admin. Conf. Chemotherapy Tuberc.
386-391, 1958.
5. Dickison, H. L. and Tisch, D. E. Trans.
17th Veterans Admin. Conf. Chemother-
apy Tuberc. 391-397, 1958.
6. Cron, M. J. (7 at. J. Am. Chem. Soc. »0:
752-753, 1958.
7. Cron, M. J. et al. J. Am. Chem. Soc. HO:
4741-4742, 1958.
8. Kawaguchi, Y. ct al. Japan. J. Microbiol.
2: 95-99, 1958.
9. Umezawa, H. Ann. N. Y. Acad. Sci. 76:
20-26, 1958.
10. Cron, M. J. et al. Ann. N. Y. Acad. Sci.
76: 27-30, 1958.
11. Hunt, G. A. and Moses, A. J. Ann. N. Y.
Acad. Sci. 76: 81-87, 1958.
12. Donomae, I. Ann. N. Y. Acad. Sci. 76:
166-187, 1958.
13. Chalmers, T. C. et al. Ann. N. Y. Acad.
Sci. 76: 188-195, 1958.
14. Cohn, I., Jr. Ann. N. Y. Acad. Sci. 76:
212-223, 1958.
15. Yow, E. M. and Monzon, O. T. Ann. N. Y.
Acad. Sci. 76: 372-390, 1958.
16. Sugiura, K. Ann. N. Y. Acad. Sci. 76:
575-585, 1958.
17. Umezawa, S. et al. J. Antibiotics (Japan)
llA: 162, 1958.
18. Morikubo, Y. J. Antibiotics (Japan) llA:
171-180, 1958.
19. Russo, J. J. and Movmtain, C. Antibiotics
Ann. 605, 1958-1959.
20. Finegold, S. M. et al. Antil)iotics Ann.
(506-622, 1958-1959.
21. Shapiro, M. and Hyde, L. Antibiotics
Ann. 708-710, 1958-1959.
22. Bettag, O. L. et al. Antibiotics Ann. 721-
724, 1958-1959.
23. Ruiz Sanchez, F. R. et al. Antibiotics Ann.
725-735, 1958-1959.
24. Japanese Patent 3749, May 18, 1959.
Kananiyciii B
Produced by: Streptomyces kanainyceticus. This
culture also produces kanamycin A and another
antibiotic (see kanamycin A).
Method of extraction: Isolated l)y countercurrent
distribution of salicylidene derivatives of the
mixture of kanamycins A and B (methanol-water-
chloroform-benzene, 5:4:2:1) and chromatog-
raphy on Amberlite XE-64 (NH/ form) with 0.08
A" NH4OH. Recrystallized from 90 i)er cent etha-
nol (1).
Chemical and physical properties: Basic sub-
stance. Decomposes over a wide range above
170°C. Soluble in water; slightly soluble in lower
alcohols; insoluble in nonpolar organic solvents.
Infrared data given in reference 1; spectrum is
said to resemble kanamycin A. [ajl = +135° (c =
0.63 per cent in H2O). Positive Molisch, Elson-
Morgan, and ninhydrin tests. Negative Fehling
and Benedict tests. C = 44.75%; H = 7.50%;
N = 12.55%. Yields Schiff bases with aromatic
aldehydes. Unlike kanamycin A, yields no fur-
fural after sulfvu-ic acid treatment. Acid hydroly-
sates contain desoxystrept amine, kanosamine,
and one unidentified ninhydrin -positive spot.
292
]:)E8CR1PTI0N.S OF ANTIBIOTICS
but no 6-gluco8amine (6-desoxy-<)-amino-D-glu-
cose) as with kanamycin A. N-acetyl kananij/rin B:
Decomposes gradually at 220-250°C. [«)" = +150°
(c = 0.42 per cent in water, 1).
Biological activity: Active on gram -positive and
gram-negative liacteria, including mycobacteria.
Almost 2 to 3 times as active as kanamycin A
against bacteria such as Aerobacter aerogenes, B.
cereus, B. subtilis, Br. bronchisepiica, E. coli, K.
pneumoniae, Staph, aureus, and Proteus spp. Not
active on Clostridia or ('. albicans, Salmonella,
or Serratia marcescens. Neither kanamycin A nor
B is very active on streptococci. B is less active
than A on mycobacteria (2).
Toxicity: More toxic to animals than kanamy-
cin A (3).
References:
1. Schmitz, H. et al. J. Am. Chem. See. »(»:
2911-2912, 1958.
2. Gourevitch, A. et al. Antil)iotics & Chemo-
therapy 8: 149-159, 1958.
3. Hubef, K. Quoted in Finegold, S. N. et al.
Antibiotics Ann. 606-622, 1958-1959.
Lagosin
Produced by: Streptnmyces sp. (1).
Synonym: Antibiotic A 246.
Method of extraction: Mycelium (60 per cent of
the activity) extracted with 80 per cent aqueous
acetone. Extract concentrated iu vacuo. A small
amount of n-butanol added to the aqueous residue
and the water removed by concentration in vacuo.
Precipitated from residual solution by addition of
diethyl ether in excess. Filtrate (40 per cent of the
activity) extracted with n-butanol. Extracts con-
centrated, then treated as above (1).
Chemical and physical properties: Macrocyclic
lactone, with pentaene chromophore. Crystalline
substance; m.p. about 235°C (decomposition).
[afn = -160° (c = 0.2 per cent in methanol).
Ultraviolet absorption spectnun maxima at 325,
340, and 358 m^ (J^lcm 1491). C41H66-70O14 . Perhy-
dro derivative: C^iHts-soOh ; m.p. 156-157°C.
[a]^" = +3.5° (c = 1.98 per cent in methanol)
(1-3). Structural formula given in Chapter 6 and
reference 4.
Biological activity: Active on yeasts and fila-
mentous fungi (1).
References:
1. Ball, S. et al. J. Gen. Microbiol. 17: 96-
103, 1957.
2. Dhar, M. L. ct al. Proc. Chem. Soc. 148-
149, 1958.
3. Dhar, M. L. et al. Proc. Chem. Soc. 154-
155, 1958.
4. Dhar, M. L. cl al. Proc. Chem. Soc. 310-
311, 1960.
Laveiiduliii
Produced by: Strcptomyces lavendulae (1).
Synonym: Streptothricin-like antibiotic.
Method of extraction: Like that for actinorubin.
Chemical and physical properties: Basic sub-
stance. HCl salt: White powder. Helianlhate:
Orange needles in clusters; m.p. 212-220°C (de-
composition). Soluble in 80 per cent aqueous
methanol; insoluble in 20 per cent methanol. C =
51.16%; H = 5.99%; N = 17.32%; S = 9.17%.
Probable empirical formula: C49H63O18N13S3 (1).
Major component in l)roth indistinguishable from
streptothricin on paper chromatography (wet
butanol-jo-toluenesulfonic acid) (4j.
Biological activity: Active on gram-positive and
gram-negative bacteria, including mycobacteria.
Slightly active on Trichophyton interdigitale (16
Mg per mlj. Cross-resistance with actinorul)in
and streptothricin (3).
Toxicity: LDiuo (mice) 28.5 mg per kg intra-
peritoneally. Toxic effects at therapeutic levels
(2).
References:
1. Junowicz-Kocholaty, R. and Kocholaty, W.
J. Biol. Chem. 168: 757-764, 1947.
2. Morton, H. E. Proc. Soc. Exptl. Biol. Med.
64: 327-331, 1947.
3. Kelner, A. and Morton, H. E. J. Bacteriol.
.i3: 695-704, 1947.
4. Benedict, R. G. Botan. Rev. 19: 229-320,
1953.
Lenaiiiyciii
Produced by: Strcptomyces sp.
Method of extraction: Broth adjusted to pH 4.0,
filtered, then extracted with n-l)utyl acetate. The
aqueous phase concentrated //; vacuo and the
gummy Inown sul)stance which precipitates on
addition of absolute ethanol is discarded. Ac|ue-
ous ethanolic filtrate treated with alumina, then
concentrated in vacuo with addition of absolute
methanol. Addition of acetone gives an inactive
white precipitate. Concentrate of the mother
liquor gives a crude precipitate of lenamycin on
standing in desiccator. Recrj'stallization from
methanol.
Chemical and physical properties: Organic acid
amide. Needles; m.p. 202-207°C (decomposition)
(d.p. 290-300°). Optically inactive. C = 40.89%;
H = 4.42%; N = 22.91%. No S, halogen, or met-
als. C4H4N2O2-.3 . Ultraviolet absorption spectrum
maximum at 216 niyu {E\7m 817). Infrared absorp-
DESCRII'TIONS OF ANTIBIOTICS
293
tion spectrum given in reference 1. Negative nin-
iiydrin, biuret, anthrone, FeCl.t , Elson-Morgan,
and Sakaguchi tests. No nitro or oxime groups.
Unstable to alkaline pH; more stable at acid pH.
Biological activity: Active on HeLa cells in tis-
sue culture (human cervical carcinoma cell).
Reference: 1. Sekizawa, Y. J. Biochem. (To-
kyo) 4.>: 150 H)2, 1958.
Leucoinyciii
Produced by: Streptoniyces kitatiatoensis strains
(2, 9) and Streptotnyces sp. (12).
Synonym: Antibiotic C (537 (12).
Method of extraction: I. Culture medium acidi-
fied, stirred with "Dicarite," and filtered. Filtrate
extracted with benzene at pH 7.2 to 9.0. Back-
extracted into water at pH 3 to 4. Extracted into
ether at pH 9.0. Extract evaporated to dryness.
Purified by treating a methanolic solution with
activated carbon, then chromatographing on
alumina from benzene, developed with acetone-
benzene (3:7). Precipitated as the tartrate. Broth
contains three components; mycelium, a fourth.
The three bases are demonstrable on countercur-
rent distribution (isopropyl ether-0.062 M phos-
phate buffer pH6.25, 1:1) (5).
Chemical and physical properties: Basic sub-
stances. Complex: Yellowish rhoml)oid crystals;
m.p. 126-129°C (1, 5). Soluble in alcohols, acetone,
ethyl acetate, butyl acetate, chloroform, ether,
and benzene. Slightly soluble in water. Insoluble
in petroleum ether. Ultraviolet absorption spec-
trum maximiun at 230 to 232 m^i (^lt"m 228) and a
weak band at 285 myu (£'i°cm 8.6) (ethanol) (4). In-
frared absorption spectrum given in reference 1.
[a]'o = —60.42° (c = 2 per cent in ethanol). pKi, =
7.5 (water). Positive Molisch, Schiff, Tollen, and
Seliwanoff tests. Negative Fehling, Benedict,
biuret, ninhydrin, Sakaguchi, glucosamine, xan-
thoproteic, FeClij , and maltol tests. Violet color
ill concentrated HCl, brown-purple in concen-
trated H2SO4 , and yellow in alkaline methanol.
Gives a precipitate with trichloroacetic acid (1,
4). C33-3sH54-6f,NO„.i.. : C = 61.32%; H = 8.61%;
N = 2.03%. Hydrochloride: White, column-shaped
crystals. Acetate: Crystalline substance; m.p.
135. 5°C. Biologically active. Tartrate: White
needles; m.p. 125°C. Very soluble in water, alco-
hols, and acetone. Slightly soluble in chloroform
and ether. Insoluble in benzene, petroleum ether,
dichloroethylene, and trichloroethylene. Aciueous
solution loses 28 per cent of its potency at room
temperature in 15 days (1). Base 0: m.p. 135°C.
C = 60.1%; H = 8.4%; O = 28.0%; N = 1.6%.
jiKi, = 7.2. Ultraviolet sj)ectrum maxima at 231
niM (Ell,, 333) and 279 m/x (^I'cm 5). Rf = 0.03
(stationary phase: disodium phosphate; mobile
phase: cyclohexane-methyl isobutyl ketone,
85:15). Base I: m.p. 142-143°C. [a]f = -76° (c =
1 per cent in methanol). C = 60.1%; H = 8.6%;
O = 28.5%; N = 1.7%. pK,, = 7.1. Ultraviolet
absorption spectrum maxima at 231 niyu (E'll,,, 296)
and 279 m^- {Eiln 2.5). Rf = 0.03 (above system).
Base II: m.p. 139°C. C = t)0.2%; H = 8.3%;
O = 29.1%; N = 1.5%. Ultraviolet absorption
spectrum juaxima at 231 mn (ET^,,, 330) and 279 m^t
{Ei%n 2.2). Rf = 0.15 (above system) (8).
Biological activity: Active on gram-positive bac-
teria and mycobacteria. Not active on gram-nega-
tive bacteria, except the Hemophilus-Neisseria
group. Not active on filamentous fungi or yeasts
(1). Cross-resistance with oleandomycin but not
erythromycin (11, 12). Less active at alkaline
than at acid pH. Active in vivo against Staph,
aureus, Streptococcus pyogenes, Clostridium welchii
(guinea i)ig), Borrelia duttonii (mice), Rickettsia
tsutsngamushi (mice), Rickettsia prowazekii (chick
embryos), lymphogranuloma (mice), and sheep
infectious pneumonia. Moderately active on
Treponema pallidum (guinea pigs and rabbits).
Not active on Trypanosoma evansi (mice) or rabies
virus (mice) (3, 7, 12). Addition to the soil in which
a cucumber seedling is growing doubles the curva-
ture caused by a one-sided growth of the hypocotyl
that occurs when one of the cotyledons is shaded
(6).
Toxicity: LDjo (mice) 650 mg per kg intrave-
nously, >800 mg per kg subcutaneously. Mice
tolerate >2 gm per kg of the tartrate orally (1).
Utilization: Effective in a case of chronic chole-
cystitis (10).
References:
1. Hata, T. et at. J. Antibiotics (Japan) 6A:
87-89, 1953.
2. Hata, T. e< rt/. J. Antil)iotics (Jai)an) 6A :
109-112, 1953.
3. Hata, T. et al. J. Antil)iotics (Japan) 6.4:
163-171, 1953.
4. Sano, Y. et al. J. Antibiotics (Japan) "A:
88-92, 1954.
5. Sano, Y. J. .Vntiliiotics (Japan) 7A: 93-
97, 1954.
6. Kribben, F. J. Naturwissenschaften 41:
144-145, 1954.
7. Hashimoto, T. et al. Japan. J. Bacteriol.
10: 787-790, 1955.
8. Despois, R. et al. Giorn. microliiol. 2:
76-90, 1956.
9. Nakamura, G. et al . J. Antibiotics (Japan)
9B: 213-217, 1956.
294
DESCRIPTIONS OF ANTIBIOTICS
10. Yoshida, R. and Tsununa, M. J. Aiiti
biotics (Japan) lOA: 177, 1957.
11. Welch, H. et at. Antibiotics Ann. 337-
341, 1958-1959.
12. Steinberg, B. A. et ul. Antibiotics Ann.
342-345, 1958-1959.
Leucomycin B
Produced by: Streptomyrcs kitasatoensis (1).
Remarks: Produced in broths coincidentally
with leucomycin.
Method of extrortion: Filtered Ijroth extracted
with benzene or butyl acetate at pH 8.0. Back-
extracted into dilute HCl (pH 4.0), washed with
butyl acetate, and adjusted to pH 8.0 with 1 N
NaOH. Alkaline solution extracted with benzene.
After concentration of the extract to a small vol-
ume, carbon is added. Elution from carbon with
hot benzene. Benzene solution concentrated ///
vacuo. Leucomycin B precipitated in the cold.
Recrystallized from atiueous ethanol. Differen-
tiated from leucomycin by paper chromatography
(ether) (1).
Chemical and physical properties: White needles;
m.p. 192-193°C. Basic compound. Relatively in-
soluble in water. Soluble in ether, acetone, chloro-
form, ethyl acetate, butyl acetate, and benzene.
Acid salts are water-soluble, [afo = —49.6° (c =
2 per cent in ethanol). C4iH69NOi6 : C = 59.34%;
H = 8.31%; N = 1.65%; O = 30.71%, l)y differ-
ence. Ultraviolet absorption spectrum maximum
at 232 niju (£'/cm 325). Infrared absorption spec-
trinn given in reference 1. Biologically active de-
rivatives include acetyl leucomycin B (I), the
thio.semicarbazone (II), 2,4-dinitrophenylhydra-
zone (III), and isonicotinic hydrazone (IV) (1).
Biological activity: Leucomycin B is active on
gram-positive bacteria and relatively inactive on
mycobacteria, gram-negative bacteria, fungi,
Candida, and Nocardia asteroides. Derivatives I,
II, III, and IV are active on gram-positive bac-
teria and mycobacteria.
Reference: 1. Sano, Y. J. Antibiotics (Japan)
9: 202-206, 1956.
Leucomycin- like Complex
Produced by: Strepiomyces sp. with some simi-
larities to <S. kitasatoensis.
Synonym: Antibiotic 6, 237 R.P.
Method of extraction: Broth-filtrate extracted
with amyl acetate at pH 8. Extract treated with
acidic water. Readjustment to alkaline pH and
extraction with ethylene dichloride. Evaporation
gives crude substance. Purification by counter-
current distribution (isopropyl ether-0.062 M
phosphate i)uft'er i)H 6.5, 1:1) and chromatog-
rajihy on alumina from benzene.
Chetnical and physical properties: Complex sub-
stance composed of two bases. Base I: m.p. 139-
140°C. [a]c. = —78° (c = 1 per cent in methanol).
C = 60.5%; H = 8.5%; O = 27.8%; N = 1.85%.
pKi, = 7.1. LHtraviolet absorption spectrum max-
ima at 231 mix (£'!";„, 311) and 279 m^i {El^ln, 6.3).
Rf = 0.03 (stationary phase: disodium phosphate;
mobile phase: cyclohexane-methyl isobutyl ke-
tone, 85:15). Base II: m.p. 144°C. [a]f = -75°
(c = 1 per cent in methanol). C = 60.5%; H =
8.4%; () = 29.3%; N = 1.6%. pK,, = 7.1. Ultra-
violet absorption spectrum maxima at 231 m/x
(E'l'L, 348) and 279 mM (EW, 2.3). Rf = 0.15 (sys-
tem given above). Leucomycin contains more
Base I than Ba.se II. The reverse is true in this
complex. A third base, present in the original
leucomycin, is not present in this complex.
Biological activity: Active on gram-positive bac-
teria. Not active on gram-negative bacteria, ex-
cept Neisseria, Pasteurella, etc. Cross-resistance
with erythromycin and carbomycin. Active in
mice on streptococcal, i)nevunococcal, and staph-
ylococcal infections.
Toxicity: LDso (mice) about 2 gm i)er kg sul)-
cutaneously, >5 gm per kg orally.
Reference: 1. Despois, R. et al. Giorn. micro-
])iol. 2: 76-90, 1956.
Levomycin
Produced by: Streptomyces sp.
Synonyms: Similar to actinoleukin and anti-
biotic F 43.
Method of extraction: l^xtracted from broth at
all pH values by ethyl acetate, n-butyl alcohol,
or ether. A pigmented impurity can be removed
by treatment of the concentrated ethyl acetate
extract in the cold with 0.01 A' NaOH. Precipi-
tated from washed and concentrated ethyl ace-
tate solutions by petroleum ether or n-hexane.
Purification by covmtercurrent distribution
(methanol-l)enzene-water, 5:5:1 ). Crystallization
of most active fraction from cold chloroform-
ethanol. Chromatograj^hy over silicic acid in
chlorof orm-methanol .
Chonical and physical properties: Colorless
prisms; m.p. 222-224°C. Co-H.^sN^Om : C =
54.05%; N = 13.90%; H = 6.35%. Very soluble in
chloroform and pyridine; less so in carbon tetra-
chloride, ethyl acetate, and hot alcohols. Slightly
soluble in ether, benzene, cold alcohols, acetone,
and (lioxane. Insoluble in water, petroleum ether,
5 per cent aqueous HCl, and NaOH. Soluble in
cold 6 -V HCl with slow decomposition. [a\u =
DESCRIPTIONS OF ANTIBIOTICS
295
— 290° (c = 2 per cent in acetone). Ultraviolet
absorption maxima at 318 iw/j. (Eilm 185) and 243
niju (Ei\n 1200). Infrared absorption spectrum
given in reference 1. Saponification equivalent,
427 to 489. Negative ninhydrin, biuret, Millon,
Hopkins-Cole, Pauly, Sakaguchi, maltol, Bene-
dict, ToUen, 2,4-dinitrophenylhydrazine, bro-
mine water, bromine (CCU), KMn()4, periodate,
methanolic FeCls , Molisch, zinc -ammonium
chloride, Zeisel alkoxyl, and hydro.xamic acid
tests. Positive pine-splint test. Treatment with
cold methanolic NaOH yields inactive levomycic
acid (m.p. 155-160°C) with an infrared spectrum
similar to levomycin. Vigorous alkaline and acid
hydrolyses reveal presence of at least four nin-
hydrin-positive components, a yellow fluorescent
l)igment, and a volatile acid. Possibly a peptide
with a chromophoric moiety.
Biological activity: Moderately active on gram-
positive and gram -negative bacteria and myco-
bacteria.
Toxicity: LDmo (mice) 44 mg per kg intrave-
nously.
Reference: 1. Carter, H. E. et al. Arch. Bio-
chem. Biophys. o.'i: 282-293, 1954.
Litniocidiii
Produced by: Nocardia cjianea iProactinomyccs
cyane us -antibioticus ) ( 1 ) .
Method of extraction: Water-extract of agar
culture acidified to pH 3.5 and treated with char-
coal. Elution with acidic acetone. Eluate evapo-
rated to dryness //; vacuo. Residue taken up in
ethanol, then precipitated by addition of water.
To an acidified (HCl) .solution, ether, then water,
is added luitil an acfueous phase separates, leaving
the antibiotic in the ether layer. Reprecipitated
from ethanol to give red form. Red form dissolved
in ethanol, neutralized, and ethanol evaporated
in vacuo to give the l)lue form (2).
Chemical and physical properties: Possibly re-
lated to anthocyanin pigments, but differing from
them in many respects. Red at acid pH, violet
at neutrality, and blue at alkaline pH. Red form:
m.p. 144-146°C. Slightly soluble in acidic water.
Soluble in ethanol, acetone, and alkaline water.
Ultraviolet absorption spectrum maxima at 460
to 480 mix, 510 to 530 m/x, and 560 to 570 m^. Dry
powder stable. Aqueous solutions more stable at
acid than alkaline pH. Positive FeCls test. Blue
precipitate with lead acetate. Decolorized by zinc
dust; color restored on exposure to air. Decolor-
ized by bisulfite; color not restored with strong
acid. Does not form salts with mineral acids.
Strong alkali at room temperature destroys the
l)iological activity' l)ut not the color. Heating with
20 per cent HCl for 10 hours at 80-90°C affects the
color, but not the activity. No carbohydrate
present. On alkaline hydrolysis gives two frac-
tions: an acid fraction giving a positive FeCls
test, ami an alkaline fraction which does not con-
tain phloroglucinol. Blue form: Very soluble in
water. Picrate: Red substance; m.p. 100-102°C
(2,3).
Bioloyical activity: Active on gram-positive bac-
teria and mycobacteria. Very slightly active on
gram-negative bacteria. Activity unaffected l)v
horse serum. Not active on Staph, aureus in mice
(1).
Toxicity: LDr,o (mice) al)out 50 mg i)er kg intra-
peritoneally (1 ).
References:
1. Cau.se, G. F. .J. Bacteriol. .il: 649-653,
1946.
2. Brazhnikova, M. G. J. Bacteriol. 51:
(j55-()57, 1946.
3. Paskhina, T. S. Biokhimiya (Engl, transl.)
21: 453-455, 1956.
Longisporiii
Produced by: Actinomyces iStreptomi/ces) longi-
sporus (1).
Method of extraction: Broth and mycelium ex-
tracted with chloroform. Mycelium re-extracted
four times with chloroform. Extracts evaporated
first at atmospheric pressure, then in vacuo. Resi-
due taken up in anhydrous ether, and treated by
passing over an alumina column. Ether taken to
dryness. Residue taken up in absolute alcohol or
petroleum ether (b.p. 60-80°C). Crystallization
occurs slowly in the cold. Recrystallized from
absolute alcohol, then petroleum ether (1).
Chemical and physical properties: Neutral sub"
stance. Large snow-white prisms; m.j). 99-101°C.
Soluble in chloroform, ethanol, aTid petroleum
ether. Insoluble in water, acid, or alkali, lajr, =
-|-2.62° (c = 5 per cent in chloroform). C =
66.44%; H = 9.03%; No N, S, or P. Molecular
weight, 651.2. CupHj^Oii, . Product of degradation
with alcoholic KOH is an acid, C11H1SO4 ; m.p.
64-65°C. Preliminary structural formula of longi-
sporin (1):
(CioHieO)— CO— O— (CioHieO)
O
CO-CC^H.eO.)-
co
I
■0
Biological activity: Active in vitro against myco-
bacteria. Active on other gram-positive bacteria
(1).
296
DESCRIPTIONS OF ANTIBIOTICS
Toxiriti/: Too toxic for clinical use. Hemolytic
(1).
Reference: 1. Men'shikov, G. P. and Rul)instein,
M. AI. Zhur. Olxshcliei Khini. 26: 2035-2039,
1956.
Lui'idin
Produced bi/: Streptoniyces linidiis (1).
Method of extraction: Adsorption on charcoal at
pH 6.0 to 6.5. Elution with a([ueou.s methanol at
pH 2.0 to 2.5. Precipitation with acetone. Further
purification bj' formation of picrate, which is
transformed into a hydrochloride (2).
Chemical and physical properties: Basic sub-
stance. Negative maltol and ninhydrin tests.
Positive Pauly and l)iuret tests. Faint Molisch
reaction (2).
Biological activity: Same general activity as
antibiotics of the streptothricin group. Active
against the virus of silkworm jaundice (2). Cul-
ture filtrates active in ovo against influenza A
virus (1).
Toxicity: Toxicity in animals is of the strep-
tothricin type (2).
References:
1. Krassilnikov, N. A. ct ol. Mikrobiologiya
26: 558, 1957.
2. Trakhtenberg, D. M. et al. Antil)iotiki
4(2): 9-13, 1959.
Lustericiii
Produced by: Streptomyces sp.
Method of extraction: Broth extracted with
ethyl acetate; mycelium with methanol. Extracts
concentrated in vacuo at low temperature. Ace-
tone is added to the residues, and the whole
filtered. Acetone removed from the filtrate by
vacuum distillation, and the residue extracted
with ethyl acetate. Chromatographed on Magne-
sol-I)icalite with acetone as solvent and devel-
oper. Active fractions concentrated /// vacuo and
crystallized from ethanol. Recrystallized from
hot ethanol.
Chemical and physical properties: Thin colorless
needles; m.p. 130°C. Soluble in methanol, ethanol,
acetone, ethjd acetate, and chloroform. Sparingly
soluble in petroleum ether. Insohible in water.
End-absorption in ultraviolet light. Infrared
spectrum given in reference 1. Optically inactive
in methanol. C40H64O13 : C = 63.87%; H = 8.71%.
Molecular weight, 780 ± 60.
Biological activity: Active on gram p(jsitive bac-
teria at 2 to 5 /.tg per ml and on mycobacteria at
20 to 50 ixg per ml. Active on C. albicans and P.
notatuui at 5 /ig i)er ml and Sacch. cerevisiae and
.1. niger at 50 ng per ml.
Toxicity: LDjo (mice) 125 to 150 mg per kg intra-
peritoneally.
Reference: 1. Shibata, M. et al. Ann. Rept.
Takeda Research Lai). 17: 19-22, 1958.
Liileomycin
Produced by: Streptomyces tanashiensis (resem-
bling S. aureus) (1, 6, 9); Streptomyces sp. (re-
sembling iS. tanashiensis) (3, 14); iS. flaveolus
(16); Streptomyces sp. (4,5).
Synonyms: Antibiotic H 2053 (12). Probably:
Special substance 3 (K 349) (3) and substance 1
(14).
Method of extraction: Broth-filtrate extracted
with ethyl or butyl acetate, chloroform, or ether
at pH 7.5 to 8.0. Back-extracted into water at
pH <3.0. Chromatographed on alumina from a
water-insoluble solvent and developed with a
water-soluble solvent. Crystallization occurs as
the active fractions are dehydrated (6), or bj-
addition of ether to an acetone solution (2).
Chemical and physical properties: Basic sub-
stance. Free base: Very soluble in benzene. Soluble
in water and common organic solvents. Ultra-
violet absorption spectrum maxima at 280 and
420 to 440 niM (0.1 A' HCl) or 270 to 280 and 500
ni/i (0.1 X NaOH) (6). Hydrochloride: Orange-
yellow rhomboid or needle crystals; m.p. 199-
200°C (decomposition). Very soluble in methanol,
acetone, benzyl alcohol, and methyl acetate. Less
solul)le in water and ethanol. Insoluble in ben-
zene, ether, butanol, l>utyl acetate, chloroform,
and petroleum ether. Ultraviolet absorption spec-
trum maxima at 270 to 290 m/j. and 420 to 430 n^/x
(0.1 V HCl) or at 270 to 280 m^ and >500 m/i (0.1
X NaOH). Negative xanthoproteic, biuret, ninhy-
drin, Molisch, Fehling, and Sakaguchi tests. Red-
brown color with FeCl:; . Decolorized by HjOi
in presence of NasCO.i . Orange-yellow at acid
pH, brownish yellow at neutrality, purple-red at
pH 7.8, and purple at pH >7.8. Rf = 0.70 by paper
chromatography (3 per cent aqueous NH4OH).
C = 54.29%; H = 6.09%; N = 2.95%; CI = 7.37%;
O = 29.3%. C2.iH29N09-HCl. Reineckate: Orange
needles; m.p. 200-205°C (decomposition) (1, 2, 4,
6, 10).
Biological activity: Active on gram-positive
bacteria and mycobacteria. Less active on gram-
negative bacteria, except for Hemophilus. Very
slightly active on fungi (2, 16). Activity reversed
by serum (1). Active in mice on Pneumococcus,
Brucella melitensis, Hemophilus pertussis, and
Leptospira autumnalis infections (4, 8). Transient
DESCRIPTIONS OF ANTIBIOTICS
•2\):
activity on Voshida sarcoma aiul ascites hepa-
toma (12, 15).
To.riciti/: Mice tolerate 6 mg per kg suhcutane-
ously or intravenously, and 108 mg per kg orally
References:
1. Hata, T. et al. Kitasato Arch. K.xptl. Med.
22: 229-242, 19-49.
2. Hata, T. et al. J. Antibiotics (Japan) 3:
313-325, 1950.
3. Kiiroya, M. et al. J. Antibiotics (Japan)
4: 363-366, 1951.
4. Hosoya, S. et al. Japan. J. I^.xptl. Med.
21: 411-417. 1951.
5. Ho.soya, S. et al. Japan. J. I^\i>tl. Med.
22: 313-316, 1952.
6. Hata, T. et al. J. Antil)iotics (Japan) 5:
529-534, 1952.
7. Sano, Y. J. Antibiotics (Japan) 5: 535-
538, 1952.
8. Nakase, Y. and Hata. T. J. Antiliiotics
(Japan) 5: 542-547, 1952.
9. Hata, T. et al. Kitasato Arch, l-^xptl. Med.
24: 447-457, 1952.
10. Sano, H. Kitasato Arch. Exptl. Med. 24:
459-467, 1952.
11. Nakase, Y. and Hata, T. Kitasato Arch.
Exptl. Med. 24: 469-480, 1952.
12. Hosoya, S. et al . J. Antil)iotics (Japan)
6.\: 42, 1953.
13. Osato, T. et al. J. Antil)iotics (Japan)
6A: 52-56, 1953.
14. Kamada, T. J. Antibiotics (Jajnin) 6A :
172-181, 1953.
15. Koga, F. J. Antibiotics (Japan) TV: 176,
1954.
16. Govorcin, B. Tehnicki Pregled Zagreb
8: 43 52, 1956.
.^l;iiiii«)si«l«>slfeptoniyciii
Produced hi/: Streptoitu/ces griseiis (1). This
organism also produces c.ycloheximide, strei)tocin,
and streptomycin (6).
Si/nont/iii: Streptomycin B (1).
Remarks: Mannosidostreptomycin is cleaved to
streptomycin by the enzyme mannosidostrejito-
mycinase (5).
Method of e.r traction: I'^xtracted from bi-(jtli
along with streptomycin. Separated by chroma-
tography on acid washed alumina with 50 per
cent methanol as solvent and developer. The
more firmly absorbed, less active fractions con-
tain mannosidostreptomycin. Crystallized as the
reineckate and converted to the hydrochloride
(1, 2). See alsf) II and \'I under streptomycin.
Chemical and physical properties: Trihi/dro-
chloride: White amorphous powder; m.p. 179-
182°C (decomposition) (1) or isotropic hexagonal
plates or prisms (dihydrate). [a]^ = —54.1°
(c = 1 per cent in water). pK'^, = 7.6. Anht/droiis
form: C = 38.25%; H = 6.22%; N = 11.31%;
CI = 12.52%. Co7H490nN7-3HCl (1, 2, 4). Tn-
reineckate: Large thin plates containing 8.10 per
cent water of crystallization. Anhydrous form:
m.p. 178-179°C (decomposition, corrected). Struc-
tural formula (7) :
CHjOH
OH
298
DESCRIPTIONS OF ANTIBIOTICS
Dihiidrostieptomycin B-HCl: m.p. 194-195°C
(decomposition, corrected), [a] ',', = —55° (c =
0.9 per cent in water) (2).
Biological activity: Antimicrol)ial activity' in
vitro and in vivo is qualitatively .similar to strepto-
mycin, but quantitatively less active on a weight
basis (1, 3, t>).
Toxicity: Intravenous toxicity similar to strep-
tomj^cin (4).
References:
1. Fried, J. and Titus, E. J. Biol. Chem. 168:
391-392, 1947.
2. Fried, J. and Stavely, H. E. J. Am. Chem.
Soc. 69: 1549-1550, 1947.
3. Rake, G. et al. Proc. Soc. Exptl. Biol. Med.
65: 107-112, 1947.
4. Heuser, L. J. ct al. J. Am. Chem. Soc. 70:
2833-2834, 1948.
5. Perlman, D. and Langlykko, A. F. J. Am.
Chem. Soc. 70: 3968-3969, 1948.
6. Waksman, S. A., ed. Streptomycin; nature
and practical applications. The Williams
& Wilkins Co., Baltimore, 1949.
7. Fried, J. and Stavely, H. E. J. Am. Chem.
Soc. 74:5461-5468, 1052.
Matanij ciii
Produced by: Streptoniyces niatensis (1).
Synonym: Very closely related to, and probably
the same as, althiomycin, differing only in the
reaction to Fehling test.
Method of extraction: Broth -fill rate extracted at
pH 6.5 to 7.0 with ethyl acetate. Extract concen-
trated in vacuo and cooled to give precipitate.
Precipitate taken up in boiling acetone, decolor-
ized, concentrated in vacuo and cooled. Recrystal-
lized from ethyl acetate to give a mixture of mata-
mycin and "Compound I," a closely related but
antibiotically inactive substance. Purification and
separation of the two compounds by countercur-
rent distribution (acetone-water-ethyl acetate,
2.5:2:2).
Cheniiccd and physical properties: Not strongly
acidic or basic. White crystalline substance; m.j).
173°C (decomposition). Very slightly soluble in
hot water, dioxane, methanol, ethanol, acetone,
and ethyl acetate. Insoluble in petroleum ether.
Ultraviolet absorption spectrum shows strong
absorption before 220 ni/x and a maximiun at 285
niM (ETcm 190) (methanol) or at 237 niyu (EHm
550) and 305 to 307 m^ (Elvira 270) (in 0.1 A' NaOH) .
Infrared spectrum given in reference 2. Unstable
at acid pH, being inactivated to the "Compound
I" also present in the fermentation broths. [a]o =
+36.6°; [a]«6 = +57.4°; [aUi = +65.4° (c = 0.11
per cent in methanol). Positive Tollen, PV'hling,
bromine, KMn()4 , 2,4-dinitrophenylhydrazine,
and ninhydrin (after acid hydrolysis) tests. Nega-
tive Fed;) , Sakaguchi, and Ehrlich diazo tests.
Does not diazotize with nitrous acid. Rf values on
chromatograijhy are given in reference 2. Acid
hydrolysis products include cysteine, glycine,
serine, alanine, questionably arginine, and two
unidentified ninhydrin-positive substances. C =
43.95%; H = 4.06%; N = 14.45%; S = 13.57%.
No halogen (2).
Biological activity: Active on gram-positive
bacteria at 0.5 to 5 ng per ml, E. coii at 50 fxg per
ml, and K. pneumoniae at 5 ng per ml. Inactive on
Ps. aeruginosa, Pr. vulgaris, and fungi (1).
References:
1. Margalith, P. et al. Antibiotics & Chemo-
therapy 9: 71-75, 1959.
2. Sensi, P. et al. Antibiotics & Chemotherapy
9: 76-80, 1959.
Mediocidiii
Produced by: Streptomyces mediocidicus (1).
Method of extraction: Mycelivun extracted with
acetone. Extract evaporated to drjmess in vacuo.
Residue extracted with methanol; ether added to
extract to give a precipitate (1).
Chemical and physical properties: Hexaene.
Yellow powder. Very soluble in methanol; soluble
in ethanol; slightly soluble in water and acetone;
insoluble in benzene, ether, and petroleum ether
(1). x!^^'.^" 339 to 340, 356 to 357, and 377 to 378 (1).
Biological activity: Active mainly on yeasts;
moderate activity against certain filamentous
fungi and gram-positive bacteria. Slight inhibition
of ascites development in mice inoculated with
Ehrlich carcinoma (1, 2).
Toxicity: LD50 (mice) = 2 mg per kg intra-
peritoneally (1). Minimal concentration causing
HeLa cell degeneration is 200 fxg per ml (3).
References:
1. Utahara, R. et al. J. Antibiotics (Japan)
7A: 98-103, 120-124, 1954.
2. Nitta, K. e/ cd. J. Antibiotics (Japan) 8A:
120-125, 1955.
3. Nitta, K. Japan. J. Med. Sci. & Biol. 10:
277-286, 1957.
IVIegacidin
Produced by: Streptomyces sp. resembling, but
not identical to, iS. fradiae.
Method of extraction: Broth extracted with
ethylene chloride. Extract concentrated in vacuo.
Residue taken up in benzene. Benzene shaken
with acetic acid and concentrated to drvness.
DESCRIPTIONS OF ANTIBIOTICS
299
Purification Ijy cliromatograpliy on alumina from
benzene. Elution with benzene, chloroform, and
chloroform-methanol (50:1) (active fraction). Re-
chromatographed on alumina and eluted with
chloroform. Subjected to countercurrent distribu-
tion (methanol-water-carbon tetrachloride-ehlo-
roform, 4:1:3:2). Water added to active fractions;
extracted with chloroform. E.xtract taken to dry-
ness. Residue extracted with ethyl acetate and
precipitated with ether. Recrystallization from
ethyl acetate-ether.
Chemical and phi/sical properties: Neutral sub-
stance. Colorless thin plates. C24H.38O10: C =
59.27%; H = 7.94%; active H = 0.42%; O— CH;i =
6.52%; C— CH, = 9.38%. Ultraviolet absorption
s])ectrum maximum at 217 niyu (log e = 3.94).
Infrared absorption spectrum given in reference
1. Positive FeCl.-i te.st. Hydrogenation product has
no ultraviolet absorption spectnun maximum at
217 nxfi. May contain three to foin- C — CHg groups,
and an Q:,/3-unsaturated carl)onyl group. Forms a
monoacetyl derivative: colorless crystals; m.p.
218.5-220°C.
Biological adivity: Very narrow r:inge of activ-
ity: B. megaieriian (0.1 to 1 jug per ml) and Strepto-
coccus pyogenes (10 ^g per ml). Other gram-positive
bacteria, including streptococci. Staph, aureus,
and mycobacteria, gram-negative l)acteria, and
yeasts are not affected. No activity on Strepto-
coccus pyogenes infection in mice.
Toxicity: Mice tolerate 0.5 gm per kg sul)cutane-
ously.
Reference: 1. Ettlinger, L. et at. Monatsh.
Cheni. J!«: 989-995, 1957.
Melanoniyciii
Produced by: Streptomyces utelanoyeues (2).
Method of extraction: Broth-filtrate adjusted to
pH 4.0 and passed through a column of IRC-50
resin (H+ form) and the active fracticjn eluted
with 1 to 2 .V NH4OH. Addition of 10 per cent
acetic acid to pH 3.0 produces a l)lackish ])recipi-
tate. An aqueous solution of this material (pH
8.0 with 4 per cent NaOH) is dialyzed against tap
water overnight, then lyophilized. Another
method of extraction involves the use of IR4B
(Cl~ form) resin and elution with aqueous alkali.
The antibiotic can be precipitated with zinc
chloride, potassium alum, ammonium sulfate.
trichloroacetic, picric, and phosphotungstic acids,
ammonium reineckate, and other agents. Not
precipitated by methyl orange.
Chemical and physical properties: Tasteless,
black-brownish, amorphous powder. Amphoteric.
Insoluble in acidic water and organic solvents.
Soluble in water at i)H 6 to 9 and in methanol at
acid or alkaline pH. Soluble with inartivation in
acetone, l)utanol, and ethanol containing more
than 10 per cent alkaline water. Nondializal)le.
Positive xanthoproteic, diazo, sodium nitroprus-
side, and Millon tests. Acid hydrolysis jjroducts
include phenylalanine, leucine, proline, alanine,
arginine, histidine, glutamic acid, and glj'cine.
Ultraviolet absorption spectrum shows an indis-
tinct shoulder at 260 to 270 mfi. Isoelectric point
about pH 3.0. Na melanomycin: C = 54.74%; H =
6.99%; N = 9.87%; Na = 1.9%; Kjeldahl N =
9.00%. Activity decreased by 50 per cent after
heating to 100°C for 10 minutes. Hemolyzes horse
red blood cells at >124 yug per ml (1).
Biological activity: Mild but definite effect
against Ehrlich carcinoma, lioth ascitic and sub-
cutaneous forms. No antibacterial or antifungal
activity (1). Anti-ascarid activity (ova) (3).
Toxicity: Maximal tolerated dose = 250 mg per
kg subcutaneously, 50 mg per kg intravenously,
and 125 mg i)er kg intraperitoneally (1).
References :
1. Sugawara, R. et ul. J. Antibiotics (.Japan)
1(»A: 133-137, 1957.
2. Sugawara, R. and Onuma, .M. J. .\ntiliiotics
(Japan) lOA: 138-142, 1957.
3. Takaoka, M. et al. J. Antibiotics (Japan)
llA: 134-137, 1958.
Melaiiosporiii
Produced by: Streptomyces mclanosporiis var.
melanosporofaciens.
Remarks: The same organism jM-oduces elaio-
phylin.
Method of extraction: Extraction of the mycelium
with n-butanol. Concentration of butanol to syrup.
Addition of ether to syrup yields a yellowish pre-
cipitate. White crystals of elaioj)hylin olitained
from the mother licpior, on standing. The precipi
tate contains melanosporin, elaioj^hylin, and other
antibiotics, including a polyene. Elaiophylin ex-
tracted with chloroform, followed by evaporation
and crystallization from acjueous ethanol. Melano-
sporin is left in the residue, which is suspeiuied in
anhydrous ethanol, stirred, and filtered, .\ddition
of ether to the ethanol precipitates impvu-e mela-
nosporin. Further purification by countercurrent
distril)Ution in cliloroform - methanol - w;it(M'
(2:2:1).
Chemical and physical properties: \'ery light
yellow amorphous solid; m.p. 132-134°C. Soluble
in alcohols and dimethylformamide. Insoluble in
acetone, chloroform, and ether, [ajj," = +30° (c =
1.578 per cent in methanol). Light -absorption
300
DESCRIPTIONS OF ANTIBIOTICS
maxiimun at 230 m^ in methanol. Tentative
empirical formula: CeoHinOaiNs . Yellow color,
turning brown with sulfuric acid. Brown, non-
specific Molisch test. Negative Fehling, FeCls,
and ninhydrin tests. Strong acid hj'drolysis yields
three ninhydrin-positive fragments. Infrared ab-
sorption spectrum given in reference 1.
Biological activity: Active against gram-i)ositive
bacteria and fungi.
Toxicity: LDso (mice) 15 mg per kg intraperi-
toneally, 350 mg per kg orally.
Reference: 1. Arcamone, F. M. et al. Giorn.
niicrol)iol. 7: 207-216, 1959.
Meseiileriii
Produced by: Nocardia iiiesentericd .
Method of extraction: Extraction of broth at pH
7.4 with butyl acetate. Concentration to small
volume under vacuum. Concentrate cooled; crude
crystals of azomycin precipitate and are removed.
The concentrate then extracted with water at pH
2.0. Aqueous layer adjusted to pH 8.0 and ex-
tracted with benzene. Evaporation of the benzene
leaves a crude white amorphous powder, which is
dissolved in methanol and treated with active
carbon. Filtrate evaporated to dryness and the
residue dissolved in benzene. Benzene solution
chromatographed over a column of alumina. Elu-
tion with benzene yields antil)iotic 440; further
elution with benzene and acetone yields mesen-
terin.
Chemical and physical properties: Basic, color-
less substance. Crystallized as needles; m.p. 122-
126°C. Strong end-absorption of ultraviolet light
in ethanolic solutions. C = 65.82%; H = 7.10%;
X = 8.66 to 8.44%. No halogen or S. Negative
ninhydrin, Millon, biuret, Tollen, Fehling, and
FeCl.i tests. Positive Molisch reaction. Very
soluble in methanol, ethanol, ethyl acetate, butyl
acetate, and ether. Soluble in benzene and acidic
water. Slightly soluble in water. Insoluble in
petroleum ether. A 10 per cent loss of activity at
pH 6.0 upon heating for 30 minutes at 100°C in
atjueous solution.
Biological activity: Active in vitro against gram-
positive bacteria, including mycobacteria. No
activity against gram-negative bacteria.
Toxicity: LD50 >100 mg per kg. (Route of in-
jection and animal used not reported.)
Reference: 1. Ueda, M. and ITmezawa, H. J.
Antibiotics (Japan) 8A: 1(;4-1()7, 1955.
Melhyniycin
Produced by: Streptoniyces sp. (1) and S. euro-
cidicus (10).
Synonym: Antibiotic 11 B (10).
Remarks: Belongs to the erythromycin-carbo-
mycin-proactinomycin group. Differs from picro-
mycin only in point of attachment of desosamine
to the lactone ring.
Method of extraction: Broth-filtrate extracted
with amyl acetate at pH 9.5. Back-extracted into
dilute sulfuric acid. Acid washed with chloroform
at pH 2.5, then extracted with it at pH 9.5. Con-
centration of extract and dilution of concentrate
with Skellysolve C gives needles of methymycin.
Recrystallized from absolute ethanol (1). Purified
by chromatography. Crystallized from ethyl
acetate (5).
Chetnicdl and physical properties: Macrolide (9).
Needles, m.p. 203-205°C (from ethyl acetate) ; or a
polymorphic modification (prisms from ethanol),
m.p. 195.5-197°C (5). Free base: Soluble in meth-
anol, acetone, chloroform, and dilute acids;
moderately solul)le in ethanol and ether; insoluble
in water and hexane. Ultraviolet absorption spec-
trum maxima at 223 to 225 m^t (e = 10,500) and at
322 mix (e = 47) (ethanol) (1, 5). Infrared spec-
trum given in reference 1. [a]~n = +61° (c = 0.7
per cent in methanol). Positive permanganate and
Br tests. Negative Molisch, biuret, ninhydrin, and
FeCls tests. pKi, = 5.7 (1,6). Rf values in various
systems (paper chromatography) given in refer-
ence 3. Sulfate: needles; m.p. 173-178°C. Soluble in
methanol and water (1). 2 ,^-Dinitrophenylhydra-
zone: Orange nee(\\eii;Vi\.\). 205-207°C (5). Quater-
nary salt with methyl iodide: C26H46NO7I; m.p.
190°C. [al, = +31.0° (ethanol) (7). Mild acid
hydrolysis products include desosamine (6), also
foimd in erythromycin. Oxidation with KMn04 in
acetone yields the lactone of ^S-hydroxy -«,«', 7-
trimethylpimelic acid. Both products are also
produced from narbomycin and picromycin (8).
Formula of methymycin: C25H43NO7 . C = 63.93%;
H = 9.28%; N = 2.84%; 2N— CH3 = 5.83%; 6C—
CH3 = 18.3% (5). Complete structure (6) given in
Chapter 6.
Biological activity: Active against certain strains
ot gram-i)ositive bacteria and a few strains of
gram-negative bacteria {Malleomyces mallei, Br.
suis, and Pasteurella tularensis). No antifungal
activity (1). Not active on Toxoplasma gondii
infections in mice (4).
Utilization : Suppressive but not curative effect
on clinical brucellosis (2).
References:
1. Donin, M. N. e^ a?. Antibiotics Ann. 179-
185, 1953-1954.
2. Max, G. C. and Mendez, D. Antibiotics &
Chemotherapy 4: 83-86, 1954.
DKSCRIPTIOXS OF AXTIBIO PIC'S
;;()i
3. Sokolski, W. T. (7 III. Antibiotics & C'hciiio-
therapy 4: 1057-10(30, 1954.
4. Paleiicia, L. et al. Rev. inst. saluliridad
y eufermedad trop. (Alex.) 14: lli-i-lK),
1954.
5. Djerassi, C. et al. J. Am. C'hein. Soc. ~i\:
1729-1732, 1956.
(5. Djerassi, C. and Zderic, J. A. J. Am. Chem.
Soc. 78: 6390-6395, 195(;.
7. DeSomer, P. Giorn. mic-rohioi. 2; 216-
232, 1956.
8. Anliker, R. et al. Helv. Chim. Acta 39:
1785-1790, 1956.
9. Woodward, R. B. Aiigew. Chom. 69: 50-
58, 1957.
10. Taguchi, H. and Nakano, A. J. Fermenta-
tion Technol. 35: 191-195, 1957.
Mi
am^cin
Prcxhii-etl hi/: Strept(n)nices sp. resembling (S.
aDihofaciens.
Remarks: Belongs to the eryt hrnnix cin-like
group. Culture also produces spiramycin.
Method of extraction: Broth extracted with chlo
roform, n-l)Utanol, or ethyl acetate at pH 8.5.
Purification l)y repeated extraction into water at
1)H 4.5 and back-extraction into solvent al pH
8.5, then fractional precipitation from a mixhirc
of ether-petroleum ether. Crystallized from ether.
Further purification by countercurrent distribu-
tion (ethyl acetate-0.1 .1/ phosphate bulTcr ])H
6.9).
('hei)iical ami phi/sicul properties: Soluble in
lower alcohols, dilute acids, chloroform, acetone,
ethyl acetate, and benzene. Slightly solul)le in
water, carbon disulfide, carbon tetrachloride, and
petroleum ether (40-60°C); m.p. 221-222°C (de-
composition). [a\f = —18° ((■ = 1 per cent in 0.02
N hydrochloric acidj. Optically inactive in 0.02 N
HCl. Ultraviolet absorption spectrum maxinuun
at 230 m^i (broad peak). Infrared absorption spec-
trum given in reference 1. Decolorizes 2 per cent
KMnOi in acetone in the cold but does not react
with Br in CCh . Positive Molisch and Elson-
Morgan tests. Negative ninhydrin test. Dissolves
in concentrated H2S()4 with a light yellow color
which fades on dilution with water. C = 61.45%;
H = 8.65'; c; N (Dumas) = 2.32%. Acid hydrolysis
with A^ HCl at 60°C for 24 hours yields two spots
when chromatographed on ])aper strijjs (water-
saturated 10 per cent acetic acid in n-bu(anol)
and sprayed with ammoniacal AgNO^ ; the spots
are identical to tho.se from erythromycin under
the same conditions (1). ProbaJjly a member of the
macrolide group (2).
liiiilixiical actirity: Active on gram-])osit ive l)ac
teria. Shows cross-resistance with erythromycin
and carbomycin. Protects mice against infections
with D. pneiDiKiiiKie.
T<).cicit!/: Mice tolerate 750 mg i^er kg intraperi-
toneally, and 580 mg per kg orally.
Referenees:
1. Schmitz, H. et al. Antibiotics <S[ Chenu)-
therapy 7: 37-39, 1957.
2. Brink, X . (1. and Harman, R. K. (^uart.
Revs. (London) 12:93-115,1958.
iMicrocins
Produced hi/: M icciDiKniosporit sp.
Method of extraction: Broth-tilt rate extracted
with ethyl acetate at i)H 2.0. Extract shaken with
pH 7.0 phosphate liuffer. l']thyl acetate layer con-
tains microcin .\. Bul'fer layer re-extracted into
ethyl acetate at ])H 2.0 to give microcin B.
Cheinieal and phi/sieal properties: Mierocui .\ :
Xeutral, reddish violet substance. Insoluble in
water. Microcin B: Acidic, yellowish red sub-
stance. Slightly soluble in water. Both sul)stances
give negative Moliscdi and FeCl:; tests.
Bioloijical actiritji: Active on Sacch . fornioscn.^is
and gram-positive bacteria. Less active on gram-
negative bacteria.
Toxicitji: LI);,(i (mice) 625 mg ])er kg intrave-
nously.
Reference: 1. Taira, T. and Fujii, S. J. Aiiti
biotics (Japan) 5: 185-187, 1952.
Micrimnmosporiii
Produced hi/: M icrontonospora s]). (1).
Method of extraction: Precipitated, with loss of
activity, l)y 50 to 90 per cent ethanol or acetone,
and without loss of activity by saturation of
broth-filtrate with (XH4)2S()4 to 50 to 75 per cent
(3). Chocolate-brown precipitate dissolved in 5
per cent XaCl solution and dialyzed against tap
water, then distilled water. Solution lyophilized.
Could also l)e adsorbed on Xorit, but not eluted
with aqueous buffer solutions or conunon organic
solvents (2).
Chemical ami phi/sical properties: Highly ])ig-
mented protein, jjrobably associated with a carbo-
hydrate moiety. Water-soluble. Insoluble in ether,
alcohol, or acetone. Destroyed l)y concentrated
acid, at high i)H, or by heat. Not destroyed by
pepsin or trypsin. Positive Molisch test. Negative
phloroglucinol, orcinol, and najihthoresorcinol
reactions. X = 6.7%. After acid hydrolysis, gives
5.5 per cent amino N (1-3).
Biological actirity: Active on gram-])osilive l)ac-
302
DESCRIPTIOX.^ OF ANTIBIOTICS
teria. Not active on B. cereus-»n/c(>idcs grouj). No
activitj' on gram-negative bacteria (1, 3).
References:
1. Waksman, S. A. e( al. Soil Sci. 54: 281-
2()(), 1942.
2. Waksman, S. A. el al. J. Bacteriol. 5.'?:
355-357, 1947.
3. Welsch, M. Rev. beige pathol. et med. exptl.
Suppl. II: 18, 1947.
Mikainyciii A
1^ rod need by: Slreptoniyees niilakdcnsls (1).
Synonyms: Mikamycin. The main component. s
of streptogramin and staphylomycin are thought
to be the same as those of mikamj'cin (1).
Method of exlraclion: Broth-filtrate extracted
with ethyl acetate at pH 6.0. Extract washed
with water, with dilute HCl at pH 2.0, and with
NaHCCJs at pH 7.4. Solvent layer concentrated in
vacuo at 27-30°C. Addition to a large volume of
petroleum ether precipitates mikamycin. Powder
dissolved in methanol, filtered, and solution con-
centrated. Concentrate washed with ether, and
extracted with chloroform. Chloroform solution
chromatographed on HNOs-treated alumina in
carbon tetrachloride. Washed with benzene and
ethyl acetate. Eluted with methanol. Active frac-
tions concentrated and precipitated from ether.
Purification by countercurrent distribution
(methanol - benzene - chloroform - water, 20:25:
15:7). The solvent of the active fractions evapo-
rated in racuu and the aqueous residue extracted
with chloroform. Extract concentrated to dryness.
Redistributed countercurrently (methanol-ben-
zene-chloroform-water, 27:28:12:8). Active frac-
tions crystallized from benzene (1).
Chemical and physical properties: Neutral. Yel-
lowish white crystalline substance; m.p. 178°C
(decomposition). C31H39O9N., : C = 62.08%; H =
().5S%; N (Dumas) = 7.34%. No halogens or S. Mo-
lecular weight, 617. [a]^ = —152° (c = 0.5 per cent
in methanol). Very soluble in chloroform, metha-
nol, ethanol, and acetone. Soluble in methylene
chloride, ethylene chloride, benzene, and ethyl ace-
tate. Slightly soluble in ether and water. Insolu-
ble in petroleum ether, carbon tetrachloride, and
hexane. Positive Fehling and diazo tests. Negative
maltol, glucosamine, ninhydrin, Millon, and
biuret tests. Black-green color with FeClj .
Black-brown precipitate with ToUen test (no
mirror). Green color and brown precipitate with
Benedict test. Labile to alkaline pH; most stable
at pH 4.0. Ultraviolet absorption spectrum (meth-
anol) maximvuii at 226 ni/x (£{'''1^ 624) and an in-
flection at 270 niM (^'llm 200). In 0.1 N NaOH-4
})or cent methanol, a maximvuu at 293 m^ (£'i"cm
462) shifts to 283 niju (^llm 201) after inactivation
at this pH. Acidification causes this peak at 283
niju to disappear. It reappears on readjustment to
alkaline pH. Staphylomycin and streptogramin
react similarly. P'orms a dinitrophenylhydrazone:
m.p. 160-165°C (decomposition). Infrared data
given in references 2 and 3. Paper chromatography
(3 per cent NH4CI) shows one spot (Rf = 0.59) for
mikamycin, two spots (Rf = 0.60 and 0.39) for strep-
togramin, and two spots (Rf = 0.61 and 0.41) for
staphylomycin (assay organism, Sarcina liitea).
Hydrogenation yields decahydroini kaiii yci n . which
has no absorption of ultraviolet light: white
plates; m.)). 94°C (decomposition). Contains
2C— CHs , IN CH.i , 2C = O, and 5C = C.
Monoacetafe: Biologically inactive, yellow-green
crystals; m.]). 138°C. Ultraviolet absorption spec-
trum maximum at 236 niyu ie = 42,347) and an
inflection at 270 m^ (e = 21,621). 2,^-Dimtro-
phenylhydrazone: Red crystals; m.p. 180-186°C
(decomposition). Acid hydrolysates of mikamycin
contain L-proline and glycine. An additional
weakly ninhydrin-positive spot was assigned to an
N-methyl-containing moiety. Mikamycin may
contain a chromophore attached to a peptide
chain (1 ).
Bioloyicid activity: Active on gram-jjositive
bacteria (0.1 to 55 yug per ml) ; less active on myco-
bacteria (20 to 28 Mg per ml) (1); not active on
gram-negative bacteria, fungi, or yeasts. Active
in mice on D. pneumoniae infections (1).
Toxicity: Mice tolerate 250 mg per kg inlraperi-
toneally (1).
References:
1. Arai, M. et al . J. Antibiotics (Japan) llA:
14-20, 1958.
2. Arai, M. et al. J. Antil)iotics (Japan) llA:
21-25, 1958.
3. Okabe, K. J. Antiliiotics (Japan) 12A:86-
89, 1959.
4. Watanabe, K. et al . J. Antibiotics (Japan)
12A: 112-113, 1959.
Mikamycin B
Produced by: Slreptoniyees niitakaensis.
Synotiyin: Similar to antiliiotic PA 114B.
Remarks: Mikamycin was described as probably
identical with streptogramin and antibiotic 899.
This compound shoidd now be called mikamycin
A, since another antibiotic has been found to be
produced l)y the same actinomycete and has l)een
called mikamycin B. As previously observed for
similar antibiotics, such as PA 114, staphylo-
DESCRIPTIONS OF ANTIBIOTICS
303
inycin, and antibiotic 129, a synergistic relation-
ship was found between mikamycins A and B {2).
Method of extraction: Extraction of l)roth-filtrate
with ethyl acetate. Concentration of the ethyl
acetate extract under vacuum to '20 vohune. A
I)recipitate forms which contains 48 per cent
mikamycin A and 6.5 per cent mikamycin B. Addi-
tion of 10 volumes of petroleum ether to the super-
natant gives a syrup containing 13 per cent mika-
mycin A and 18 per cent mikamycin B. Syrup
dissolved in methanol; addition of ether causes
formation of a precipitate, which is dried in vacuo.
Resulting powder dissolved in benzene and chro-
matographed over silicic acid. Cohmm washed
with chloroform, and elution carried out with
chloroform containing 5 per cent acetone. Frac-
tions of the eluate containing only mikamycin B
(Rf = 0.05, ascending paper chromatography with
3 per cent ammonium chloride) combined and con-
centrated to a syrup in vacuo. Syrup treated with
ether. A white insoluble powder contains 72 per
cent mikamycin B and less than 1 per cent mika-
mycin A. This powder extracted with ether in a
Soxhlet apparatus. Crude crystals of mikamycin
B precipitated from the ether solution. Recrystal-
lized from methanol.
Chemical and physical properties: Amj)hoteric,
white, platelet -shajjed crystals, which melt at
160°C, solidify above 180°C, and decompose at
262°C. [aln = -61.3° (c = 1.0 per cent in meth-
anol). Molecular weight (Signer), 865. C = 60%;
H = 6.43%; N = 12.53%. Suggested empirical
formula: C45H58Ns()ii . Light-absorption maxima
at 209, 260, and 305 m/i in methanol. Infrared al)-
sorption spectrum given in reference 1. Soluble in
chloroform, acetone, methyl isobutyl ketone,
l)enzene, ethyl acetate, ethanol, and methanol.
Slightly soluble to insoluble in ether, cyclohexane,
petroleum ether, and water. Salts are solul)le in
water. Positive FeCl.'i (brown-red) test. Mikamy-
cin A gives a dark green FeCls test. Almost nega-
tive ninhydrin reaction. Negative Ehrlich, biuret,
Fehling, and Tollen reactions. Stable at neutral
and acidic reactions (1).
Mikamycin is a macrocyclic peptide lactone
composed of the following seven amino acids: 3-
hydroxypicolinic acid, L-threonine, D-a-amino-«-
Ijutyric acid, L-proline, L-phenylglycine, L-4-oxo-
pipecolic acid, and L-p-dimethyl amino-N-methyl-
phenylalanine. This last amino acid had not been
l)reviously reported (4). The structure given in
reference (5) is closely related to those of etamycin
and staphylomycin S.
Biological activity: Active against gram-positive
bacteria. Mikamycins A and B have synergistic
activity in vitro and in vivo when there is 10 to 90
per cent of mikamycin B in the mixture (2). Mika-
mycin B inhibits Staph, aureus at 8 fxg per ml (A
at 2yug per ml ) ; mikamycin A inhibits Sarcina lutea
at 0.25 fjLg per ml (B at 4 /xg per ml) ; B. subtilis is
inhibited by mikamycin A at >64 ng per ml (B
at 8 ng per ml). Staph. a>n-eus exposed to mika-
mycin B becomes more sensitive to A, but the
reverse is not true (3).
Toxicity: Mice tolerate 350 ng per ml intraperi-
toneally (3).
References:
1. Watanabe, K. J. Antiiiiotics (Japan) 13A:
57-61 , 1960.
2. Watanabe, K. J. Antibiotics (.Japan) I3A:
62-69, 1960.
3. Watanabe, K. et al. J. Antibiotics (Japan)
12A: 112-113, 1959.
4. Watanabe, K. J. Antibiotics (Japan) 14A:
1-13, 1961.
5. Watanabe, K. J. Antibiotics (Japan) 14A:
14-17, 1961.
Miranij cin
Produced by: Strcptoniyces niirabilis.
Method of extraction: Unknown. Culture-broths
contain more than one antibiotic.
Chemical and physical properties: Heat -stable.
Biological activity: Active against gram -posi-
tive and gram-negative bacteria.
Toxicity: Said to be nontoxic.
References:
1. Ruschmann, (I. Pharmazie 7:542-550,639-
648, 823-831, 1952.
2. BcungJ. Pharmazie 13:305 310,1958.
M il<>ni\ cins
Produced by: Streplomyces caespitosus (2, 9) and
S. griseovinaceseus (3).
Remarks: In the hands of one group of investi-
gators (9), S. caespitosus produced a complex
differing somewhat from that originally described
(1). Further fractions other than those described
below have been reported, i)ut no details are
known (12).
Methods of extraction: Original description: Fil-
tered broth treated with activated carbon at
pH 6.0 to 6.5. Eluted with acetone. Acetone con-
centrated in vacuo and the residue extracted
with chloroform (I). Purple-red chloroform ex-
tract dehydrated with anhydrous Na2S()4 aiul
chromatographed on alumina. Colored fractions
included: yellow, blue-violet, pink and l)Iue, red-
violet, and purple. The latter two, mitomycins
A ami B, eluted with methanol or water. Puri-
304
DESCRIPTIONS OF ANTIBIOTICS
fiod l)y rechroniatographing on alumina from
chloroform, with chloroform or chloroform-ace-
tone as developer. Recrystallized from acetone
on addition of carbon tetrachloride in the cold.
Mitomycins A and B could also he separated by
countercurrent distribution (benzene-chloroform
and pH 7.2 phosphate buffer) (1). Mitomycin C
was also distinguished (4). The later workers used
a method of extraction essentially the same as
that given above except that "Fraction R" was
described as being left in the aqueous layer after
step I. This was extracted from the aqueous layer
with cyclohexanone, the extract concentrated,
and the substance precipitated with petroleum
ether. Chromatography gave active fractions de-
scribed as mitomycins A, B, and C(X), and frac-
tions X and Y (9).
Chemical ami physical properties: Mitomycins
A and B: Basic substances. Plates or needles.
Solu])le in acetone, methanol, ethanol, chloro-
form, methyl ethyl ketone, jjyridine, methyl
Cellosolve, n-butanol, ethylene dichloride, water,
ethyl and n-butyl acetate, ether, and cyclohexa-
nol. Slightly soluble or insoluble in xylene, car-
bon disulfide, carbon tetrachloride, ligroin, pe-
troleum ether, and cyclohexane. Infrared spectra
given in reference 1. Positive 2,4-dinitrophenyl-
hydrazine and bromine tests. Negative Elson-
Morgan, biuret, and anthrone tests. Mitomycin A:
m.p. 159-161 °C (decomposition). Soluble in ben-
zene, toluene, trichloroethylene, and nitroben-
zene. Light -absorption maxima at 215 ni/i {E\\m
234), 316 to 318 m^ {E^^ra .122), and 530 m/x (E\\n^
18.8) (in water) ; at 360 and 550 to 560 niyu (in 0.1
N NaOH); or at 235, 285, 335, and 430 m^ (in 0.1
A^ HCl). Red-violet color of mitomycin A fades
in Schiff's reagent, 0.1 A HCl, and 0.1 A NaOH;
changes to blue in concentrated H2SO4 and to
orange in concentrated HCl. Gives a green color
with Molisch reagent. C = 54.22%; H = 5.05%;
N = 11.68%. No CI or S. Mitomycin B: m.p. 182-
184°C (decomposition). Insoluble in benzene,
toluene, trichloroethylene, and nitrobenzene.
Light-absorption maxima at 220 ran {Ei'lm 117.5),
320 niM (ETl,n 55), and 550 m/x iEX\ru 9.9) (in water);
at 360 and 580 m^ (0.1 N NaOH) ; and at 232, 284,
335, and 430 to 440 m^ (0.1 A HCl). The violet
color of mitomycin B fades with Schiff's reagent
and 0.1 A' HCl; changes to blue with 0.1 A NaOH,
and from blue to green with concentrated H2SO4 .
Color changes from yellow to orange with concen-
trated HCl, and from green to brown with the
Molisch reagent (1). Mitomycin A -like: Red
crystals; m.p. 167-168°C. Light-absorption max-
ima at 216 niM {E^cm 485), 320 m^ {ETcm 255), and
520 ni/x (fi'icni 40) ill methanol. Pure substance re-
tains 93 per cent of its activity after 3 hours at
100°C. Less pure preparations are more labile.
C = 51.46%; H = 5.64%; N = 9.00%; O =
33.90%. Degradation products include cinnamic
acid amide. Mitomycin C (X): Bluish violet crys-
tals. No melting point up to 360°C. Soluble in
water, methanol, acetone, butyl acetate, and cy-
clohexanone. Sparingly soluble in benzene, carbon
tetrachloride, and ether. Insoluble in petroleum
ether. Ultraviolet absorption spectrum maxima
at 216 niyu (^llm 742), 360 m^ (£'}lm 742), and 560
m/.i (-fi'itm 0.06). Infrared spectrum given in refer-
ence 9. Positive Fehling, hydroxylamine hydro-
chloride, biuret, Ehrlich, FeClj , nitrous acid,
and Liebermann tests. Questionable positives:
bromine, 2,4-dinitrophenylhydrazine, and Janov-
sky reactions. Negative Benedict, Tollen, fuchsin,
ninhj'drin, Millon, and Raymond tests. Data on
paper chromatographic behavior given in refer-
ence 9. Bluish violet at alkaline pH, red at weakly
acid pH, and yellow at acid pH. Color changes
are reversil)le. Most stable at pH 6 to 7; labile to
acid and alkali. Photo-labile. Prolonged boiling
destroys biological activity and toxicity. Easily
oxidized, but not easily reduced. C = 53.84%;
H = 5.14%; X = 15.49%. C54H6iNi;sOi, (tenta-
tive). Chroinophore of C: Isolation procedure
given in reference 9. Red-brown crystals; m.p.
65-70°C (not distinct). Soluble in water, metha-
nol, ethanol, acetone, cyclohexanone, and diox-
ane. Ultraviolet absorption spectrum maxima at
215 niM (^'Hm 630) and 320 m^ (£'rcm 295) with a
shoulder at 260 mn. Exhibits the same color
changes as mitomycin C. Biologically inactive.
Fraction R: Brownish red amorphous powder.
Soluble in acetone, Viutanol, methanol, dioxane,
and alkaline water. Insoluble or slightly soluble
in ethyl acetate, butyl acetate, chloroform, car-
bon tetrachloride, ether, benzene, and water,
intraviolet absorption spectrum maxima at 215
and 315 mfi. Does not change color with pH.
Fraction Y: Orange crystals. Browns at 180°C.
Carbonizes at 240°C. Soluble in chloroform, ace-
tone, benzene, ether, and acidic acjueous solu-
tion. Insoluble or slightly soluble in benzene,
ether, and neutral or alkaline water. LTtraviolet
absorption spectrum maxima at 207, 237, 286, and
345 m^i (9). Violet in alkaline solution; yellow in
acidic. Considered a partial degradation product
of mitom>cin H.
Biological activity: Mitomycins A and B: Active
on gram -positive and gram-negative bacteria,
mycobacteria, and Nocardia asteroides. Not active
on fungi or yeasts. Mitomycin A is activ(> in a
DESCRIPTIONS OF ANTIBIOTICS
305
range from 0.0005 to 1.0 /ug per ml; B, from 0.05 to
5.0 /xg per ml. Partial cross-resistance with strep-
tomycin (1, 5). Inactive in mice on D. pneu-
moniae, Snl. enteritidis, and Leptospira ictero-
liaeiuonhagiae. Active on Rickettsia tsutsiujaniushi .
Inactive on MiyagawaneUa and viruses (6). Both
are active on Yoshida sarcoma in rats and P]hrlich
carcinoma (ascitic form) in mice (1, 4). Mitomy-
cins A and B are equally active against tumors;
C is said to be about one fourth to one fifth as
active. Some effect on the solid form of Khrlich
carcinoma (4). Mitomycin A-like: Same as mito-
mycins A and B (9). Mitomycin C: Same in vitro
activity as A and B (9), but only one twentieth
as active as A on B. subtilis (4). Active in vi:o on
D. pneumoniae, Sal. enteritidis, L. icterohaemor-
rhagiae, R. tsutsugamushi, and MiyagawaneUa
(sheep). Inactive on toxoplasmosis (mice) or in-
fections caused by other small viru.ses (5, 6). Anti-
ascarid activity (12). Active on the following neo-
plasms: sarcoma 180 (ascitic), Ehrlich carcinoma
(ascitic), adenocarcinoma EO 771, Bashford car-
cinoma 63, Miyono adenocarcinoma, carcinoma
1025, Wagner osteogenic sarcoma, Ridgeway
osteogenic sarcoma, Lewis lung carcinoma, Hard-
ing-Passey melanoma, glioma 26, Friend virus
leukemia, Flexner-Jobling carcinoma. Walker
carcinosarcoma 256, Gardiner lymphosarcoma,
Jen.sen sarcoma, Murphy-Sturm lymphosarcoma,
Crabb hamster sarcoma, Iglesias functional rat
adrenal tiunor, Iglesias functional rat ovarian
tumor, diploid and tetraploid Hirosaki sarcoma
(ascites and solid), Usubuchi sarcoma (ascitic),
sarcoma-3-B (ascitic), ascites hepatoma 7974,
Yoshida sarcoma, and mouse lymphatic leukemia
SN36 (7, 10, 13). May interfere with the de novo
synthesis of purines (11). Fraction R: Moderately
active on Ehrlich carcinoma (ascitic), but not on
bacteria (9). Fraction Y: Same as mitomycins A
and B (9).
Toxicity: Mitomycin A: LD.50 (mice) 1.0 to 1.5
mg per kg intravenously (5). Mitomycin A -like:
LD50 (mice) 2.5 mg per kg intravenously. Mito-
mycin C: More toxic for rats and hamsters than
for mice (13). LD.=,o (mice) 5 mg per kg (9) or 7.5
to 10.0 mg per kg (5) intravenously, 9 mg per kg
iiitraperitoneally (9). Toxicity also studied in rats
and rabbits (14). Fraction R: LD51) >500 mg per
kg (no route given) (9).
Utilization: Mitomycin C(X) has been reported
clinically effective in certain neoplastic diseases
(8, 12). "
References:
1. Hata, T. et al. J. Antil)iotics (Ja])an) 9A:
141-146, 1956.
2. Sugawara, R. and Hata, T. J. Antibiotics
(Japan) 9A: 147-151, 1956.
3. Japanese Patent 2,898, April 17, 1956.
4. Kanamori, H. et al. J. Antibiotics (Japan)
lOA: 120-127, 1957.
5. Matsmnae, A. et al. Japan. J. Microbiol.
1: 183-189, 1957.
6. Saito, Y. et al. Japan. J. Microbiol. 1:
191-196, 1957.
7. Usubuchi, I. et al. Cann 48: 447-448,
1957.
8. Sakai, K. Chemotherapy (Tokyo) 5: 322,
1957.
9. Wakaki, S. et al. Antibiotics & Chemo-
therajjy 8: 228-240, 1958.
10. Usubuchi, I. et al. Chemotheraijy (Tokvo)
6: 378-392, 1958.
11. Reilly, H. C. and Cappuccino, J. (1. Proc.
Am. Assoc. Cancer Research 2: 338, 1958.
12. Shiraha, Y. et al . Antibiotics Ann. 533-
540, 1958-1959.
13. Sugiura, K. Cancer Research 19: 438-
445, 1959.
14. Sokoloff, B., et al. (Irowth 24: 1-27. 1960.
Moldcidin A
Produced by: Sfreptomyces sp.
Remarks: Moldcidin B, another pentaene, has
been shown to be identical with pentamycin.
Method of extraction: Extraction of the mycelium
with methyl alcohol. Concentration of solvent in
vacuo to '10 volume. Precipitate collected and
dried. Extract dissolved in methyl alcohol, fil-
tered, water added. Upon concentration, crystals
precipitate.
Chemical and physical properties: Pentaene.
Light -absorption maxima at 324, 339, and 358 niyu.
Insoluble in butanol, ether, petroleum ether, ethyl
acetate, and 0.01 X HCl. Slightly soluble in water,
acetone, ethyl alcohol, and 0.1 A' NaOH. Soluble
in methyl alcohol, 80 per cent acetone, glacial
acetic acid, and pyridine. Positive ninhydrin re-
action. Negative biuret, IVhling, Molisch, Saka-
guchi, and FeCL tests. Slow melting between 180
and 230°C. No loss of activity of 1 mg per ml in
methyl alcohol at 5°C during 24 hours. Inactivated
by ultraviolet light. C = 55.87%; H = 8.84%; N =
1.50%.
Biological activity: Active against a numljer of
yeasts and filamentous fungi at levels of 0.5 to 30
/ig per ml. No activity against l)acteria. Active
against Trichomonas vaginalis.
Toxicity: \A):,» (mice) 10 mg per kg intrave-
nouslv.
30()
DESCRIPTIONS OF ANTIBIOTICS
Ctilization: Active again.st (\ albicans and T.
vaginalis when applied toi)ically in tlio vagina.
References:
1. Sakamoto, J. M. J. J. Antil)i()tics (Japan)
12 A: 169-172, 1959.
2. Ogawa, H. et al. J. .\ntil)iot ics (Japan)
13A: 353-355, 1960.
Mold in
Fnxluccd by: Streptoini/ccs phacochroniogenes
(1,2).
Method of extraction: Broth e.xtracted with ethyl
-acetate. Extract concentrated in vacuo. Syrup
washed with petroleum ether, water, and hot
water, and then dried. Residue dissolved in etha-
nol and jjrecipitated on addition of distilled water.
Antibiotic is also present in mycelium (1), which
can be extracted with ethanol (2).
Chemical and physical properties: Soluble in
ethanol and ethyl acetate. Slightly soluble in
petroleum ether, ether, and benzene. Almost in-
soluble in water. Positive Molisch and FeCls tests.
Negative biuret, Millon, ninhydrin, Tollen, Feh-
ling, and Sakaguchi tests (1, 2). Ultraviolet ab-
sorption spectrum maximum at 320 ni/u.
Biological activity: Active on yeasts (1).
Toxicity: MLD is 10 mg per kg intraperitoneally
(l).
References:
1. Maeda, K. et al. J. Antibiotics (Japan) 5:
465, 1952.
2. Maeda, K. et al. Japan. J. Med. Sci. &
Biol. 5: 327-339, 1952.
Monaniyciii
Produced by: Streptomyces jatnaicensis.
Method of extraction: Extraction of culture-fil-
trate or mycelium with ether or butanol. Evapora-
tion of solvent. Countercurrent distribution using
ethyl acetate-cyclohexane-methanol-water (12:10:
10:7) followed by countercurrent distribution
using light petroleum (b.p. 60-80°C) -methanol -
water (10:10:1). Chromatography on Amberlite
C. G. 4J. Crystallization from light petroleum.
Chemical and physical properties: Needles; m.p.
126 °C. Ba.se, giving a crystalline monohydrochlo-
ride; m.p. 187°C. [aln = -62° ± 5° (c = 0.9 per
cent in ethanol). Tentative empirical formula:
C22H.,6-.38N405 . Oue N-methyl and three C-methyl
groups. End-absorption of ultraviolet light. In-
frared spectrvnii shows no evidence of aromatic
structure but suggests the presence of an amide
linkage. No reaction with sodium metaperiodate
or with hydrogen in presence of ])latinum catalyst.
Stable al)ove pH 7.0.
Biological activity: Active against gram-positive
l)acteria. Not active against gram-negative bac-
teria. No cross-resistance with penicillin, chlor-
tetracycline, chloramphenicol, or sulfamethazine,
using Staph, aureus. Not inactivated by human
serum.
Toxicity: 850 mg per kg subcutaneously pro-
duces no vmfavorable effect in mice.
Reference: 1. Hassall, C. H. and Magnus, K. E.
Nature, London 181: 1223-1224, 1959.
.Monilin
Produced by: Streptomyces sp. (1), and S. sakai-
ensis (2).
Synonym: Similar to toyocamycin.
Method of extraction: Broth-filtrate extracted
with n-butanol or amyl alcohol at pH 11. Extract
washed with water and concentrated in vacuo until
a precipitate forms. Can also be adsorbed on char-
coal from broth and eluted with 90 per cent ace-
tone. Removal of the solvent by vacuum distilla-
tion gives an aqueous residue, which is treated as
above with butanol. Recrystallized from hot wa-
ter, ethanol, and methanol. Purified by counter-
current distribution or chromatography (1, 2).
Chemical and physical properties: Basic sub-
stance. White needles; m.p. 235-238°C (decomposi-
tion). Soluble in methanol, ethanol, butanol, and
acetone. Sparingly soluble in water. Insoluble in
ethyl acetate, toluene, butyl acetate, l)enzene,
petroleum ether, and ether. Ultraviolet absorption
spectrum maxima at 230 and 280 m;u in water. In-
frared spectrum given in reference 2. Positive
ninhydrin and Sakaguchi tests. CisHaoNeO.-j .
Molecular weight, 190 ± 10. C = 46.93%; H =
5.34%; N = 21.80%.
Biological activity: Active on C. albicans, C.
tropicalis, and C. parakrusei. Slighth' active on
Sacch. sake. Not active on ('. krusei or C. pseudo-
tropicalis. Not active on bacteria (1).
Toxicity: LDsa (mice) 3.94 mg per kg intraperi-
toneally (1, 2).
References:
1. Fujii, S. et al. Ann. Rept. Takeda Research
Lab. 14:8-10,1955.
2. Fujii, S. et al. Japanese Patent 5,899, August
3, 1957.
3Iusariii
Produced by: Actinomyces (Streptomyces) sp. (1)
having red mycelium. A variant of this culture
produces monamycin (4).
Method of crtraction: I. Broth extracted with
n-butanol at pH 7.0. f]xtract concentrated in
vacuo. Addition of ether to concentrate precipi-
tates the antibiotic. II. Addition of (NH4)2S04
to broth; precipitate extracted with cokl metha-
DESCRIPTIONS OF ANTIBIOTICS
307
nol. Extract concentrated. Addition of ether to
concentrate for precipitation. III. Mycelial ex-
tract (hot methanol) evaporated to dryness under
reduced pressure. Broth-filtrate acidified to pH
3.5 to 4.0 with dilute H.3PO4 . Resulting precipitate
and residue from mycelial extraction treated with
1 per cent neutral sodium phosphate buffer. Buffer
extracted with n-butanol; butanol evaporated to
dryness in vacuo at 45°C. Residue taken up in
methanol; concentration in vacuo. Addition of
ether to residual solution gives musarin (3).
Chemical and physical properties: Acid. Yellow-
ish or colorless substance. Sodium or potassivun
salts soluble in water, methanol, ethanol, and
butanol; insoluble in ether and acetone. Acid
precipitable with HCI, H.,P()4 , or H2SO4 from
neutral solution. Form? inactive precipitates with
BaCh , HgCls , or copper acetate. Free acid is
unstable as dry powder; sodium salt is stable.
Activity unaltered at pH 2 or 11 at room tempera-
ture for 30 minutes, but destroyed at 100°C under
these conditions. Sodium salt: Decomposes at
about 170°C without melting. [a]f = +35.1° ±
l.t)° (c = 1.21 per cent in methanol). Ultraviolet
absorption spectrum maxima at 240 m^u {E\"cm 375)
and 267 mn (E^lm 200) (ethanol). Free acid: C =
57.75%; H = 8.34%; N = 3.70%. C:ioHcnOnN, .
No S, P, or halogens. Equivalent weight about
5000. Negative Molisch, Millon, biuret, Salkowski,
Liebermann (steroids and cholesterol), and murex-
ide tests. No color with H2SO4 in acetic acid or
with I2 . Positive Axenfeld (protein) test (3). Fails
to pass a porcelain filter (2). Treatment with
methanol containing a few drops of methanolic
HCI gives an inactive product, soluble in acetone
and chloroform but insoluble in 1 per cent neutral
phosphate buffer, [a]^ = +32.2° ± 2.0° (c = 0.995
per cent in methanol) (3).
Biological activity: Active against certain fungal
l)lant pathogens including fusaria, gram-positive
i)acteria, and mycobacteria. Not active on gram-
negative bacteria (3).
References:
1. Meredith, C. H. Phytoi)athology 33: 403,
1943.
2. Thaysen, A. C. and Butlin, K. B. Nature,
London 156: 781-782, 1945.
3. Arnstein, H. R. V. et al. J. Gen. ^licrobiol.
2: 111-122, 1948.
4. Mfg. Chemist 22: 47, 1951.
.Mutoniycin
Produced by: Streptomyres utroolivaceus var.
mutomycini.
Method of extraction: The antibiotic is present
mainly in the mycelium liut can be extracted to a
les.ser extent from the broth. To extract from the
broth, the mycelium is filtered off and the litiuid
acidified with HCI to pH 3.0. A precipitate forms,
which is extracted with acetone (neutralized with
NaOH to pH 7.0). The acetone-extract is clarified
with 0.4 per cent charcoal and evaporated at 40°C.
During concentration, the antibiotic precipitates
out. Precipitate dissolved in chloroform at 45°C.
After concentration of the chloroform, the anti-
biotic is precipitated by addition of 5 volumes of
petroleum ether. Repeated crj^stallization from a
65:35 chloroform-benzene mixture. From the
mycelium, the antibiotic is extracted with acetone.
Acetonic extract jjurified further as indicated
above.
Chemical and physical properties: White powder
consisting of needle-shaped crystals; m.p. 141.5-
142.0°C. Insoluble in water, ether, 5 per cent so-
dium hj^droxide, sodium carbonate, and hydro-
chloric acid. Soluble in ethanol and acetone and
to a lesser extent in chloroform and benzene. Solu-
bility is increased in organic solvents by a reduc-
tion of the pH, but the antibiotic is less stable at
acid than at alkaline pH. Molecular weight
(Rast), 124. C = 65.5%; H = 9.1%; O = 25.5%.
Suggested empirical formula: C7H11.1...O2 . No
characteristic light alisoi-jjlion in the ultraviolet
range.
Biological activity: Active against respiratory-
deficient mutants of sta{)hylococci. Not active
against other liacteria. Slightly inhil)its Ehrlich
carcinoma in mice.
Toxicity: Mice tolerate single oral or subcu-
taneous injection of 0.5 to 1.0 gm per kg.
Reference: 1. Cause, C. F. Antibiotiki 4(3):
20-23, 1959.
M_> celiii
Produced by: Streptoniyces roseoflavus (1), and
possibly by S.fradiae (dextromycin-producer) and
iS. diastatochromogenes (3).
Method of extraction: Mycelium extracted with
methanol, ethanol, or acetone. Methanol-extract
concentrated in vacuo. Ethanol and Ba(OH)2
added to precipitate impurities. Ba"^"^ removed
with CO2 . Alcohol layer concentrated to precip-
itate mycelin. Chromatographed on alumina from
acetone (1).
Chemical and physical properties: Prisms. Black-
ens at 260°C and decomposes at 263°C. Soluble in
chloroform, butanol, methanol, ethanol, amy]
alcohol, acetone, and benzene. Insoluble in water,
ether, and petroleum ether. No N or S. Negative
Molisch test. Stable to heat and acid, and to alkali
in aqueous acetone solution (1).
Biological activity: Active on filamentous fungi;
308
DESCRIPTIONS OF ANTIBIOTICS
less active on yeasts. Not active on l)acteria (1).
More active at alkaline than acid pH. Somewhat
active topically against dermal Trichophyton
purpureum infections in guinea pigs (2).
Toxicitij: Reputedly too toxic for systemic use
(2).
Rejerences:
1. Aiso, K. et al. J. Antibiotics (Japan) 5:
217-219, 1952.
2. Aiso, K. et al. J. Antibiotics (Japan) 5:
488-491, 1952.
3. Igarashi, S. et al. J. Antibiotics (Japan)
9A: 226, 1956.
Mycelin-IMO
Produced hy: Streptomyces sp. resembling S.
diastatochromogenes.
Method of extraction: Present in both the myce-
lium and culture -filtrate. Isolated by absorption
on alumina or activated charcoal, and elution
with organic solvents.
Chernical and physical properties: Hexaene.
Yellow crystals. Decomposes at 214-222°C. Soluble
in acetone, alcohols, butyl acetate, and chloro-
form. Insoluble in water. Ultraviolet absorption
spectrum maxima (in methanol) at 243, 294 (320),
335, 355, and 373 m^. Infrared spectrum given in
reference 1. [cx]l = +70.0° ± 2.0° (c = 1 per cent
in 1,4-dioxane). Dark green color in concentrated
H2SO4 . Stable substance. Molecular weight, 345.
C = 71.29%; H = 5.96%; N = 11.31%. No S or
halogens.
Biological activity: Active on fungi and yeasts
at 0.5 to 5.0 Mg per ml.
Toxicity: LD50 (mice) 1.5 mg per kg intraperi-
toneally.
Reference: 1. Ogata, K. et al. Japanese Patent
5,898, 1957.
Mycetin
Produced by: Streptomyces violaceus.
Method of extraction: Extraction of dried, pul-
verized agar culture with a mixture of equal parts
of ethanol and ethylene dichloride (the latter can
be replaced by chloroform or benzene). Solvent
evaporated and substance dissolved in ethanol.
Chemical and physical properties: Intensely
violet in color, although uncertain whether active
substance is also colored. Thermostable.
Bwlogiccd activity: Active against gram-positive
bacteria, incluchng micrococci and streptococci,
corynebacteria, and spore -formers; also active
against mycobacteria. Proteins and pus depress
activity. Very limited, if any, activity in vivo.
\ References:
1. Krassilnikov, N. A. and Koreniako, A. I.
Mikrobiologiya 8: 673, 1939; 14: 80-85,
1945.
2. Fainshmidt, O. I. and Koreniako, A. I.
Biokhimiya 9: 147-153, 1944.
Mycolutein
Produced hy: Streptomyces sp.
Method of extraction: Extraction of the myce-
lium with methanol, concentration of the extract
in vacuo to one tenth its original volume. On
standing at 4°C a precipitate forms, which is ex-
haustively extracted with warm chloroform. Aftei
concentration of the chloroform-extract, addition
of petroleum ether results in the formation of a
light yellow precipitate. This precipitate is then
dissolved in a minimal amount of boiling metha-
nol. On cooling, crystals are formed. Recrystalliza-
tion from methanol and benzene.
Chemical and physical properties: Bright yellow
tabvdar crystals; m.p. 157-158°C. Absorption of
light at 254 ni/x (£;1L 680) and at 345 m^ {E^L 400)
in methanolic solution, [aif = -|-54° (c = 1 per cent
in chloroform). Countercurrent distribution (39
transfers) shows the substance to be homogeneous.
Soluble in chloroform, acetone, lower alcohols,
benzene, choxane, pyridine, and glacial acetic
acid. Soluble to a limited extent in ethyl acetate,
carl)on tetrachloride, diethyl ether, and petroleum
ether. Insoluble in water at all pH values. Decom-
posed by aqueous sodium hydroxide; dissolves in
concentrated sulfuric acid, giving an olive color
which turns rapidly to dark red. Stable in boiling
methanol for at least 10 minutes in the pH range
of 5.1 to 8.2, but rapid decomposition occurs at
higher and lower pH values. Decolorizes a solu-
tion of potassium permanganate in acetone and a
solution of bromine in methanol in the cold. Posi-
tive test for aromatic nucleus with anhydrous
aluminum chloride and chloroform. FeCl.-s test
negative. Infrared spectrum given in original
paper (1). C = 66.45%; H = 5.95%; N = 3.51%.
Freezing-point depression indicates a molecidar
weight of 417 in benzene and 495 in 1,4-dioxane.
Tentative empirical formula: C2iH-24N06 .
Biological activity: Mycolutein inhibits the
growth of Candida species, ('ryptococcus neo-
formans, Trichophyton species, and Microsporum
gypseuni at the level of 0.2 to 12.5 Mg per ml after
48 hours of incubation at 28°C on a medium con-
taining no sugar. The fungi gradually grow at
higher concentrations of the antibiotic upon pro-
longed incubation. The addition of sugars to the
DESCRIPTIONS OF ANTIBIOTICS
309
test agar prevents the action of the antil)iotic.
No activity against bacteria.
Toxicity: Mice are not killetl by intraperitoneal
administration of 5 mg per kg; 25 mg per kg by
the same route kill mice.
Reference: 1. Schmitz, H. and Woodside, R.
Antibiotics & Chemotherapy 5: 652-657, 1!)55.
Myconiyceliii
Produced bij: Strepfonii/ces arenae fgray-spored)
(1).
Method of extraction: Antibiotic present mostly
in mycelimn. Extraction may be carried ovit l)y:
I. Organic solvents, inchiding methanol, acetone,
butanol, or t-butanol, at pH 4.0 to 10.0. Ex-
tract concentrated to a watery residue, and
residue e.xtracted with chloroform at pH 2.0 to
10.0. Chloroform chromatograi)hed on (a) silica
gel, and antibiotic eluted with chloroform con-
taining increasing amounts of methanol; or (bj
Darco C-OO, and eluted with methanol (80 i)er
cent ) -chloroform (20 per cent) containing 0.1 .V
HCl. II. Ivxtraction with 50 to 80 per cent etha-
nol. Extract evaporated to an acpieous residue and
extracted with butanol at pH 2 to 4. Re-extraction
into water at pH 10 (1).
ChemicdJ and plujsical properties: Negatively
charged acitlic sul)stance. Countercurrent dis-
tribution studies (pH 7.0; water-butanol) indicate
presence of at least two substances, one more
soluble in water, the other in butanol. Soluble
in dilute NaOH, methanol, acetone, n-butanol,
and t-bvitanol. Alkali-metal salts soluble; precipi-
tate on acidification. Cu, Ca, Mg, and Pb salts
insoluble. Positive anthrone test. Negative Inuret
test. Molecular weight, about 10,000. Nondiffusi-
l)le; nondialyzable. Ultraviolet absorption spec-
tnun maximum at 260 ni/x (Eil'^ 70). Infrared
spectrum given in reference 1. Darkens at about
170°C; chars a little above 200°C. Contains C, H,
N, and <1 per cent S; no P. Precipitates with lead
acetate and may be regenerated on addition of
H2S or H2SO4 in acetone, methanol, or n-butanol
(1).
Biological activity: Active mainly on mycobacte-
ria. Moderately active on gram-positive bacteria.
Active on streptomycin- and streptothricin-
resistant Staph, aureus. Not active on gram-nega-
tive bacteria or C. albicans (1). No in vivo activity
on tuberculosis in guinea pigs (2).
References:
1. Grundy, W. E. et at. Canadian Patent
515,162, August 2, 1955.
2. CJrundy, W. E. Personal communication,
1958.
Mycoiii_\ ciii
Produced hi/: Xocardia acidophilus (10).
Method of extraction: Cooled broth-filtrate ex-
tracted at pH 2.5 with hexane in a Podbielniak
extractor. Re-extracted into cold 0.5 per cent pH
7.5 sodium phosphate buffer. Buffer stirred with
cold methylene chloride, then adjusted to pH 2.0.
Extract dehydrated, decolorized, and crystallized
from the treated extract on cooling with dry ice-
acetone. Recrystallized from methylene chloride
or acetone (8). Can also be extracted from broth
with ether or amyl acetate (1).
Chcuiii-iil and physical properties: Unsaturated
carl)oxylic acid. ( — )-3,5,7,8-n-tridecatetraene-
10, 12-diyiioic acid (9). Free acid: Colorless nee-
dles; m.p. 75°C (decomposes explosively). Soluble
in methylene chloride and acetone. Ultraviolet
absorption spectrum maxima at 267 ni/x (« =
67,000) and 281 m^ (e = 61,000) with an inflection
at 256 m/x (e = 35,000) (7, 8). Infrared absorption
spectnun given in reference 8. [a]f = —130° (c =
0.4 per cent in absolute ethanol). Highly unstable
to heat and oxidizing conditions. Half -life of crys-
tals at 27°C is 3 hours. Forms a precipitate with
alcoholic silver nitrate, which then darkens at
room temperature. Solution in li([ui(l ammonia
changes from yellow to green, to l>hie, to purple,
to red. Neutral eciuivalent, 198 (7, 8). Xa salt:
Relatively stable. Decomposes in acid (2). Methyl
ester: Crystalline; m.p. 44°C (decomposition).
Poorly sohibie in water. Biologically active.
CisHu.O., : C = 78.17%; H = 5.36%; C— CH, =
0.48%. Structure (7) given in Chapter 6. A rela-
tively stable, l)iologically active isomer of myco-
mycin, isomycomycin, has been prepared by treat-
ing a dilute aciueous solution of mycomycin with
dilute NaOH (1.0 A') at room temperature, then
treating the resulting precipitate with HCl at pH
2.0. Isomycomycin: 3,5-Tridecadiene-7,9,ll-tri-
3'noic acid (10). Long white needles. Decomposes
slowly when heated above 100°C. Soluble in ether,
ethanol, dioxane, ethyl acetate, and glacial acetic
acid. Slightly soluble in benzene, chloroform, and
carbon disulfide. Insoluble in hexane and petro-
leum ether. Ultraviolet absorption spectrum
maxima at 260 and 270 niyu with weak maxima at
290, 307, 327, and 348 ni/u. Infrared data given in
reference 10. C13H10O2 . Possible structural for-
mula given in Chapter 6.
Biological activity: Active on gram-positive and
gram-negative bacteria, mycobacteria, and fungi
310
DESCRIPTIONS OF ANTIBIOTICS
(2), but not on Fs. aeruginosa (4). Early reports of
activity against Mycobacteriiun tiiherciilasis H87Rv
in mice and hamsters (3) were not supported by
further work with highly purified preparations (6).
Isomycomyctn: Active on mycobacteria (10).
Toxicity: Mice tolerate 25 mg per kg intrave-
nously (4, 8). Guinea pigs tolerate subcutaneously
administered mycomycin verj^ poorly, but 2.5
mg per day for 35 days is nontoxic (5).
References:
1. Johnson, E. A. and Bur don, K. L. J. Bac-
teriol. 51: 281, 1947.
2. Johnson, E. A. Bacteriol. Proc. 68-69,
1949.
3. Hobby, G. 8th Veterans Admin. Strepto-
mycin Conf. 295-298, 1949.
4. Jenkins, D. E. 9th Veterans Admin. Strep-
tomycin Conf. 179-186, 1950.
5. Jenkins, D. E. 10th Veterans Admin. Conf.
Chemotherapy Tuberc. 286-287, 1951.
6. Jenkins, D. E. 11th Veterans Admin. Conf .
Chemotherapy Tuberc. 309-310, 1952.
7. Celmer, W. D. and Solomons, I. A. J. Am.
Chem. Soc. 74:1870-1871,1952.
8. Celmer, W. D. and Solomons, I. A. J. Am.
Chem. Soc. 74: 2245-2248, 1952.
9. Celmer, W. D. and Solomons, LA. J. Am.
Chem. Soc. 75: 1372-1376, 1953.
10. Celmer, W. 1). U. S. Patent 2,703,328,
March 1, 1955.
Mycoiliotlin
Produced by: Streptoniyces sp.
Method of extraction: Extracted from broth-
filtrate with chloroform, methyl isobutyl ketone,
or n-butanol, at pH 4.0 to 8.0. The mycelium-filter-
aid cake is eluted with methanol to recover a
minor portion of active component. Concentration
of the chloroform-extract precipitates a white,
antibiotically inactive substance mixed with red
crystals of mycorhodin. Crystallization from
methanol or acetone gives the inactive substance
as a precipitate. Mycorhodin recovered from the
mother liquors on addition of petroleum ether.
Recrystallized from hot methanol with water or
warm chloroform-ether (1:1). Purified by chroma-
tography on Super-Cel from chloroform-ether, and
by countercurrent distribution in a methanol-
water-chloroform-carl)on tetrachloride (3:1:1.5:4)
system or methanol-methyl isobutyl ketone-water
(1:1:1).
Chemical and physical properties: Indicator sub-
stance. Bright red needles; m.p. 200-202°C (de-
composition, corrected). Soluble in aqueous meth-
anol. Ultraviolet absorption spectrum maxima at
258 niM iB^L 426), 420 m^ (^'IL 85), and 471 m^
(filem 87) (95 per cent ethanol); or at 258 niju {E^i^^
563), 410 m^x (^IL 79), and 461 m^ (E'lL 82) (95
per cent ethanol-0.1 .V HCl); or at 595 m^ (^'um
136) (95 per cent ethanol-0.1 A' NaOH). Infrared
spectrum data given in reference 1. In aqueous
methanol, red-orange color changes to purple at
pH 8.10, and to deep blue at higher pH values.
Dark red-purple color in concentrated H2SO4 and
alcoholic FeCla . Brown color in KMn()4 . De-
colorized by H2O2 at alkaline but not at neutral
pH. A solution of the antibiotic lost l)oth indicator
and antibiotic properties when kept for 12 hours
at room temperature, but was stable under these
conditions at 6°C. Very stable to acid. Contains
no amino acids. Hydrolysis products include re-
ducing sugars. C = 58.7%; H = 5.2%; N = 2.1%.
Molecular weight, 635 to 698. Acetyl derivative:
Yellow crystals. Inferior antibiotic activity. Di-
potassium salt: Amorphous, l)lue salt. Water-
soluble. Stable.
Biological activity: Active on gram-positive
l)acteria and mycobacteria, but not on gram-
negative bacteria. Active in protecting and curing
mice with infections of D. pneumoniae and Staph,
aureus.
Toxicity: LD50 (mice) 170 mg per kg intraperi-
toneally, about 500 mg per kg intramuscularly,
and >1250 mg per kg orally. Produces severe
delayed toxicity symptoms, including blood ab-
normalities, in dogs in chronic toxicity studies.
Reference: 1. Misiek, M. et al. Antibiotics &
Chemotherapy 9: 280-285, 1959.
Mycospocidin
Produced by: Streptomyces bobiliae.
Method of extraction: Broth extracted with liuta-
nol; mycelium with methanol-pyridine (3:1).
Methanol -pyridine evaporated and extracted with
l)utanol. Combined butanol-extracts washed with
water at pH 8.0 and pH 2.0, and then evaporated
to about 1^0 volume. Addition of acetone to con-
centrate precipitates crude mycospocidin. Purifi-
cation by chromatographing a pyridine solution
on alumina suspended in methanol -benzene, wash-
ing with methanol-benzene (1:1), and eluting
with methanol-pyridine (1:1). Further purifica-
tion by countercurrent distribution (ethyl acetate-
pyridine-water, 5:1:5).
Chemical and physical properties: White crystal-
line powder; m.p. 233-234°C (decomposition).
[a]n = +56° (1.0 per cent in pyridine). Soluble in
pyridine and aqueous NaOH. Sparingly soluble
in methanol and ethanol. Insoluble in acetone,
ethyl acetate, benzene, chloroform, carbon tetra-
DESCRIPTIONS OF ANTIBIOTICS
311
chloride, ether, and petroleum ether. Ultraviolet
absorption spectrum maxima at 215 niju (£'l?m 215)
and 257 to 258 m^ (^Icm 89) (methanol). Infrared
.spectrum given in reference 1. Po.sitive diazo re-
action. Negative ninhydrin, biuret, ToUen, FeCl:i ,
and Fehling test.s. Stable to heating for 10 min-
ute.s at 100°C at pH 2.0 to 9.0. Rf value.s of 0.53
(water-saturated butanol) and 0.86 (phenol-water.
8:2). C = 54.215%; H = 7.57%; N = 0.43%. C-M,H,-y
N2O9 . Acid hydrolysi.s products include two nin-
hydrin-positive i)roducts, one of which may be
glycine.
Biological actinlj/: Active against gram-positive
l)acteria and mycobacteria; moderately active on
fiuigi. Not active on gram-negative bacteria. No
activity on the ascitic form of Mhrlich carcinoma
in mice.
Toxicity: LDjo (mice) 1 to 2 mg per kg intraperi-
toneally.
Reference: I. Nakamura, S. c/ a/. J. Antil>iotics
(Japan) 10.\ : 248-253, 1957.
M^
ttlii
Produced by: Streptomyces luvendttlae strains (2),
Streptomyces sp. (4).
Remarks: Two mycothricin complexes (A and B)
were described, each from a different S. lavendulae
strain and each varying in the number of compo-
nents present. Complex B contains streptothricin
and a component also present in ".streptothricin
VI"' (see streptothricin-like antibiotics). It also
•contains a component (IV) apparently unique to
the mycothricin complex (2, 4).
Method of extraction: I. Culture-broth treated
with Darco G-60 at pH 2.5. Filtrate adjusted to
pH 7.5 and adsorbed onto fresh Darco. Elution
with 75 per cent ethanol. Eluate adjusted to pH
7.0 with IR-45 (OH^ form), concentrated in vacuo,
and precipitated with acetone. Freeze dried from
an aqueous solution (2). II. Broth acidified to
pH 3 with acetic acid and treated with oxalic acid
to remove the contaminating Ca"*"*" and Mg++.
Filtered. Chromatographed at pH 6.0 on IRC-50
l)uffered with ammonium acetate at pH 6.5. Eluted
with 10 per cent acetic acid. Active fractions
treated by passing through a column of IR-45
(OH" phase) to reduce pH to 4.0, lyophic acid, aspartic acid,
serine, alanine, and i)roline.
Biological activity: Antitumor and antibacterial
activity. Active in mice on Ehrlich ascites car-
cinoma and in rats on sarcoma 45. Causes reduc-
tion of number of mitotic figures and necrosis in
cancer cells.
Toxicity: Said to be nontoxic.
/^e/erenre; 1. Derkach, V. S. Antibiotiki 2(5):
40-44, 1957.
Neoiiie thy 111 vein
Produced by: Streptomyces sp. This strain also
produces methymycin (1).
Remarks: Differs chemically from methymycin
only in location of one hydroxyl group (2).
Method of extraction: Chromatography of the
mother liquors during the isolation of methymy-
cin on alumina (deactivated with 10 per cent aque-
ous acetic acid). Ether used as solvent and 0.5 to
1.0 per cent methanolic ether as developer. Methy-
mycin is eluted first, followed by neomethymycin.
Crystallized from ether-hexane (2).
Chemical and physical properties: Macrolide;
m.p. 156-158°C. Isomeric with methj'mycin and
picromycin. [a]„ = +93° (CHCI3). Ultraviolet
absorption spectrum maximiuu (ethanol) at 227.5
niM (log e = 4.10). C.-^HwOtX: C = 63.75%; H =
9.04%; N = 3.07%; N^(CH,), = 5.9%; C-CH3 =
16.76%; — OCH;, = 0.0%. Neutral eciuivalent
(perchloric acid titration), 472. Acid hydrolysis
(HCl) yields desosamine HCl. (This is also a hy-
drolysis i)roduct of picromycin, erythromycin,
narbomycin, and methymycin.) Methylene solvate:
Large hexagonal plates; m.p. 154-156°C with a
gas given off at 135-140°C. [a\„ = +66° (ethanol).
Acetone solvate: Long crystals; m.p. 156-158°C.
Complete structure of neomethymycin (1, 2):
HO
N(CH,)2
CH3
OH CH.3 O CHs CH.i i CH3
I I II I III
CH;,CH— CH— CH— CH=CH— C— CH— CH,— CH— CH— CH
I I
o c=o
314
DESCRIPTIONS OF ANTIBIOTICS
Biological activity: YinuaUy identical to that of
methymycin, except that neomethymycin is 4
times more active on B. subtilis (2).
References:
1. Djerassi, C. and Halpern, D. J. Am. Chem.
Soc. 79: 2022-2023, 1957.
2. Djerassi, C. and Halpern, I). Tetrahedron
3: 255-268, 1958.
Neomycin
Produced by: Streptomyces fradiae, S. albogrise-
olus, Streptomyces sp. (2).
Synonyms: Similar to or clo.sely related to strep-
tothricins BI and BII, flavomycin, framycetin,
catenulin, dextromycin, colimycin (7), miserin
(6), mycerin, and antibiotic 956 (5).
Remarks: Among the various preparations com-
prising neomycin fractions, framycetin occupies
a prominent place. Since considerable literature
has accumulated concerning this preparation, it
is discussed separately.
Method of extraction: I. Clarification of broth
at pH 2.0 with charcoal. Adsorption of neomycin
at alkaline pH on charcoal; elution with acidic
ethanol or acetone. II. Adsorption of neomycin
on IRC-50 (resin eciuilibrated at pH 7 to 8 with
alkali); elution with aciueous HCl, H2SO4 , or
ammonia. Freeze dried. III. Alkylbenzene or
alkylnaphthalene sulfonic acid added to broth at
pH 3 to 5; the resulting sulfonic acid salt of neo-
mycin extracted with butanol. Re-extracted into
dilute mineral acid. Neutralization and removal
of excess mineral acid with Ba(0H)2 or Ca(0H)2
or with suitable ion exchange resin. Purification
(a) by conversion of one salt to another; i.e., neo-
mycin sulfate to the p-hydroxyazobenzene-p'-sul-
fonate salt to the purified hydrochloride; (b) by
suitable ion exchange resins (Amberlite XE-89
and Dowex 50 X16); (c) by acetone precipitation
of ash from an aqueous neomycin solution at pH
10.1 and subsetiuent acetone precipitation of
purified neomycin sulfate from the neutralized
filtrate; (d) by carbon chromatography. Separa-
tion of neomycins B and C l)y chromatography on
carbon or alumina (2).
Che7nical and physical properties: Neomycin is a
complex composed of two isomeric entities: neo-
mycin B and neomycin C.
Neomycin B
Free base: Colorless, amorphous solid, [a]^ =
+83° (c = 1 to 2 per cent in 0.2 A' HCl). Very sol-
uble in water; insoluble in ethanol or methanol.
Sulfate: Colorless, amorphous solid. [a]jj = +58°
(c = 0.5 per cent in water) or +56°. Same solu-
bility as free base. Base content about 68 per
cent. Reineckate: Pink platelets from aqueous
acetone. Insoluble in water; soluble in hot water
and acetone. N-Acetate: Colorless needles from
aciueous acetone; m.p. 200-205°C (decomposition);
softens at 185-190°C. [a]l = +62° (c = 0.4 per
cent in water). Acetyl content 32 per cent. Bio-
logically imictive; cannot be converted to free
neomycin.
Neomycin C
Free base: Colorless amorphous solid, [a]^ =
+ 121° (c = 1 to 2 per cent in 0.2 N H2SO4). Very
soluble in water; insoluble in ethanol or methanol.
Sulfate: Colorless, amorphous solid. [aJr, = +82°
(c = 0.5 per cent in water). Same solubility as free
base. Base content al)out 68 per cent. Helianthate:
Similar to neomycin B. N -Acetate: Colorless nee-
dles from aqueous acetone; melting point similar
to neomycin B N-acetate, but gives 10° depres-
sion in mixed melting point determination, [al^ =
-f90° (c = 0.4 per cent in water). Acetyl content
32%. Biologically inactive; cannot be converted
to free neomycin.
Neomycin Complex
Basic, white substance. Water-soluble; insoluble
in organic solvents. C2:iH46N60i3 . No typical
ultraviolet spectrum (2). Infrared spectrum given
in reference 1. Positive Molisch, carbazole, and
ninhydrin tests. Negative Elson-Morgan, Fehling,
and Tollen tests. No acidic, carbonyl, methoxyl,
guanidine, or free aldehyde groups. Crude neo-
mycin is stable from pH 2 to 9; highly purified
preparations stable to alkali only (1, 2). Acid
hydrolysis products include a nonreducing base,
neamine (neomycin A), C12H24-26N4O6 , and a
methylglycoside moiety.
Nea))une
Precipitates from alcoholic ammonia as fine
needles; m.p. 250-256°C (decomposition). [a]o =
+ 123° (c = 0.7 per cent in water). Infrared spec-
trum given in reference 2. Tentative structure of
neamine :
H H
C6H903(NH2)2---0
Diaminohexose
NH2
OH H
2- Desoxystreptamine
DESCRIPTIONS OF ANTIBIOTICS
315
Mcthtjtylycosides: These are termed methyl neo-
hiosaminides B and C, and can be differentiated
by paper chromatography and specific rotation.
P'ehling's and Tollen's reagents reduced. Contain
primary amino groups. C12H24N2O8 :
^ OCH3
C6H,03(NH2)2''
Diaminohexose
OH OH
D-Ribose
Chemically, neomycins B and C differ only in the
diaminohexose (neosamine) portion of the neo-
biosamine portion of their molecules (I). Neos-
amines B and C are isomeric (1). The structure of
methyl neobiosaminide C, neobiosamine C, and
neosamine C (2) :
CHjNHR'
Neosamine C
R' = H
Neobiosamine C: R = R' = H
Methyl neobiosamide C: R = CH3; R' = H
Tentative structure of neomycin:
CeHgOaCNHj), Q
neomycin and viomycin, streptothricin, strepto-
mycin, and the mild silver protein Argyrol. The
activity of neomycin B is greater than that of C,
and there are some qualitative differences between
their activities. Neomycin is active tn vivo against
a variety of infections, including those caused by
Neisseria intracellularis, K. pneumoniae, Fasten -
reiki mulfocida, H. influenzae, V. cholerae, and Pr.
vulgaris. It is also effective against salmonellosis,
plague, and anthra.x, but not promising against
tuberculosis or infections with gram-positive
cocci, except SUtph. aureus. Active on rickettsial
pox in pigs, and on amebiasis in rats (2). Anti-
fungal activity of endomycin enhanced by the
neomycins (4). Neamine has little biological ac-
tivity, being active mainly against gram-positive
bacteria.
Toxicity: Subcutaneous LDin (mice) of neomy-
cins B and C hydrochlorides are 220 and 290 mg
per kg, respectively. Intravenous LD50 of com-
mercial neomycin as sulfate (mostly neomycin B)
is 26.2 to 42.5 mg base per kg. Local toxicity is
slight. Nephrotoxic. Causes loss of hearing (2).
Neamine: LD.s,, (mice) 320 mg per kg intrave-
nously, 1250 mg per kg subcutaneously.
Utilization: Urinary infections refractory to
other antibiotics. Infantile tliarrhea. Intestinal
asepsis. Topical applications (.2).
References:
1. Waksman, S. A., ed. Neomycin. Rutgers
Univ. Press, New Brunswick, N. J., 1953.
2. Waksman, S. A., ed. Neomycin; its nature
and practical applications. The Williams
& Wilkins Co., Baltimore, 1958.
3. Rinehart, K. L., Jr. et al. J. Am. Chem.
Soc. 80: 6462, 1958.
4. Rinehart, K. L., Jr. and Woo, P. W. K. J.
Am. Chem. Soc. «(): 6463-6464, 1958.
5. Trai Joun-chen et al. AntilMotiki 3(2):
27-28, 1958.
H H
C6H802(NH2)2---0
NH,
OH H
Biological activity: Active on most gram-positive
and gram-negative rods, many gram-positive
cocci, mycobacteria, and actinomycetes. Slightly
active on certain algae and yeasts. Not active on
viruses, protozoa, filamentous fungi, or Clostridia.
Cross-resistance exists, in certain cases, between
6. Kotchetkova, Ci. \'. and Popoba,0. L. Anti-
liiotiki ] (4): 37-40, 1956.
7. (iause, G. F., ed. The antibiotic colimycin
and its clinical application. Moscow, 1959.
8. Sokolski, W. T. and Burch, M. R. Anti-
biotics & Chemotherapy 10: 157-162, 1960.
316
DESCRIPTIONS OF ANTIBIOTICS
Neonocardin
Produced hy: Xocaidia kuroishi (2).
Method of extraction: Broth containing mycelium
heated to 100°C for 20 minutes, then cooled and
filtered. Treated with diatomaceous earth at pH
2.0, then ad.'^orbed on activated carVjon at pH 5
to 7. Carbon washed with water, methanol, and
ether. Elution into 0.04 .V HCl-methanol. Addi-
tion of ether to eluate precipitates neonocardin.
Reprecipitated from absolute methanol with
ether. Can be extracted from the dried mycelium
with distilled water (3).
Chemical and physical properties: Hydrochloride:
Yellow-gray or grayish white powder (3). Found
to differ from other known antibiotics on paper
chromatography (4).
Biological activity: Culture-filtrate active on
gram-positive and gram-negative bacteria (1).
Toxicity: Mice tolerate 2 mg intraperitoneally
(3).
References:
1. Uesaka, I. J. Antil)iotics (Japan) 3:
27-34, 1950.
2. Uesaka, I. J. Antibiotics (Japan) 5:75-79,
1952.
3. Uesaka, I. J. Antibiotics (Japan) 5: 154-
159, 1952.
4. Ueda, S. and Uesaka, I. J. Antibiotics
(Japan) 5: 170-171, 1952.
Netropsin
Produced by: Streptoniyces netropsis (2), Sirepto-
myces sp. resembling S. netropsis (7, 12), Strepto-
niyces sp. (6, 13), S. amhofaciens (6), Streptoniyces
sp. belonging to the S. reticuli group (15), Strepto-
niyces sp. (16).
Synonyms: Antibiotic lA-887 (7), sinanomyciii
(12), congocidin (11, 13), antibiotic T 1384 (13),
antibiotic K 117 (16).
Method of extraction: I. Broth-filtrate stirred
with ammonium oxalate at pH 6.5 to precipitate
Ca"*"*", then stirred with orange II at pH 5.5 to
precipitate the antibiotic. Dye precipitate fil-
tered with Super-Cel, then dye dissociated from
the antibiotic by stirring with 80 per cent acetone-
20 per cent methanol containing methanolic tri-
ethylamine sulfate. Super-Cel-netropsin sulfate
mixture washed with acetone-methanol, then ex-
tracted with cold distilled water and BaCl-i to
give the HCl salt. Adjusted to pH 2.0 with H2SO4
to precipitate Ba"^+, then treatment with IR-4 ion
exchange resin to remove excess SOT- A saturated
aciueous solution of the amorphous product gives
crystals on standing (2). II. Broth-filtrate ad-
sorbed on IRC-50 cation exchange resin (eciuili-
brated to pH 7.5 with NaOH). Fluted with 0.46 iV
HCl or methanolic HCl. Active fractions adjusted
to pH 6.0 and excess Ca'*^^ removed as oxalate.
Filtrate concentrated in vacuo, then cooled to
give crystals of antibiotic. Recrystallized from
water or methanol. Purification on alumina (3, 11).
III. Adsorption from broth-filtrate on acidic
clay at pH 7.6 and extraction of myceliimi with
80 per cent acetone (pH 5.0). Elution from clay
with 80 per cent acetone (pH 2.0). Eluate concen-
trated in vacuo, adjusted to pH 2.0, and lyophil-
ized. Solid extracted with ethanol and pre-
cipitated on addition of ether. Purification by
chromatography on alumina from ethanol-meth-
anol (3:1) and development with methanol. Pre-
cipitated from active fractions with ether (7).
Chemical and physical properties: Unstable, di-
acidic base. HCl salt: Fine needles; m.p. 167-173°C
(decomposition) or long, thin, colorless, hydrated
prisms exhibiting oblique extinction. Soluble in
methanol and ethanol; moderately soluble in
water and water-saturated n-butanol. Insoluble
in almost all other nonpolar solvents. No optical
activity in water. Ultraviolet absorption spectrum
maxima at 295 m/. {EZn 423) and 238 niju (^llm 430) .
Infrared spectrum given in reference 2. Positive
Sakaguchi, Dragendorfi (N— CHu), Ehrlich alde-
hyde (3), Weber-Rose, and Bayer tests. Negative
ninhydrin, biuret, Tollen, Fehling, maltol, Mo-
lisch, fuchsinaldehyde, miu'exide, amino anti-
pyridine (phenol), 2,4-dinitrophenylhydrazine,
Elson-Morgan, FeCls , and Hanke-Koessler tests.
Inactivated by 0.2 A' NaOH in <2 hours at 25°C.
C = 42.9%; H = 5.78%; N = 28.1%; CI (total
and ionic) = 13.7% (2, 7, 10-12). Sulfate: Long
colorless needles; m.p. 224-225°C. CisH-ibNioOs-
i^H2S04 . More soluble in hot than in cold water.
Insoluble in common organic solvents. Ultraviolet
absorption spectrum maxima at 236 mn (£'iem 429)
and 297 m/x (Eum 436). Differentiated from strep-
tot hricin and streptomycin by paper chromatog-
raphy (water-saturated n-butanol containing 2
per cent p-toluenesulfonic acid). Hydrogenation
product is biologically inactive (1, 2, 7, 10). Pic-
rate: Sheaves of yellow needles; m.p. 205 or 232°C
(decomposition). Browns and sinters at 225°C
(1, 7, 10). Helianthate: Orange plates; m.p. 215°C
(decomposition) (10). :\Iild alkaline hydrolysis
gives either glycocyamidine or guanidinoacetic
acid, ammonia, and /3-[4-(4-amino-l-methyl-2-
pyrrolecarboxamido) - 1 -methyl -2 -pyrrolecarbox-
amido]propionamide (netropsinine). The latter
compound was also obtained from congocidin and
antibiotic T 1384 (10, 11, 13). Structure of netrop-
sin (13) given in Chapter 6. /3-[4-(4-guanidino-
DKSC^UPTIOXS OF ANTIBIOTIC'S
317
acet ainidiiio - 1 - methyl - 2 - pyrrolecarl^oxamido) -1 -
methyI-2-pyrrolecarboxamido]propionamide.
Biological activity: Active on gram -positive and
gram-negative bacteria, but not on Ps. aeruginosa.
Very slightly active on C. albifons (90 /ug i)er ml)
(2), P. chrysogennm, and Piriciilaria ori/zae (7).
Active on Trichomonas vaginalis and T. foetus in
vitro at 12 ug per ml (2, 5). Active on ChloreUa
pyrenoidosa at 10 ^g per ml (14). Has antiphage
activity (9j. Active on clothes moth larvae and
the black carpet beetle (1, 2). Xo cross-resistance
with chloramphenicol, streptomycin, or strepto-
thricin (2). Active in mice on vaccinial infections
(4). Conflicting reports on anti-influenza activity
(4, 12). No activity in vivo on feline pneumonitis,
western eciuine encephalomyelitis, or poliomyelitis
(4). Moderate inhibition of Mecca lymphosarcoma
(mice) and slight inhibition of carcinoma 1025
(mice) and Walker carcinosarcoma 256 (rats) (8).
Not active on l{!lirlich ascites carcinoma (12).
Toxicity: lA);,,, (mice) 17 mg per kg intrave-
nously, 70 mg per kg subcutaneously, and >3()0
mg jjer kg orally (1).
References:
1. Finlay, A. C. e< «/. J. Am. Chem. Soc. 73:
341-343, 1951.
2. Finlay, A. C. and Sotnn, B. A. U. S. Patent
2,586,782, 1952.
3. Desi)ois, R. and Ninet, L. Riass. commun.
6th Congr. Intern. Microbiol. I: 162-
163, 241-242, 1953.
4. Schabel, F. M., Jr. ct al . Proc. Soc. E.xptl.
Biol. Med. 83: 1-3, 1953.
5. Seneca, H. and Ides, D. Am. J. Trop.
Med. Hyg. 2: 1045-1049, 1953.
6. Pinnert-Sindico, S. Ann. iiist. Pasteur
87: 702-707, 1954.
7. Isono, K. c( al. J. Antibiotics (Japan)
8A: 19-21, 1955.
8. Sugiura, K. Cancer Research 3: (suppl.)
18-27, 1955.
9. Asheshov, I. N. et al. Cancer Research
3: (suppl.) 57-62, 1955.
10. van Tamelen, E. E. et al. J. Am. Cliem.
Soc. 78:2157-2159,1956.
11. Julia, M. and Joseph, N. Compt. rend.
243:961-964, 1956.
12. Watanabe, K. J. Antil)iotics (Japan) 9A:
102-107, 1956.
13. Waller, C. W. et al. J. Am. Chem. Soc.
79: 1265-1266, 1957.
14. Tomisek, A. et al. Plant Physiol. 32:
7-10, 1957.
15. Thrum, H. Naturwissenschaften 46: 87,
1959.
1(). Magyar, K. et al. Abstr. Commun. Sym-
posium on Antilnotics, Prague 26-27,
1959.
Niger Factor
Produced by: Streptoinyces sp. resembling S.
alhus. This culture produces si.x other antibiotics,
including myxoviromycin, toyocamycin, actino-
flocin, lutea factor, and two tetraenic antifungal
antibiotics (1, 2).
Synonyui: Said to l)e related to cvcloheximide
(1).
Method (if (xtractidii : I. Broth extracted with
chloroform at acid ])H. Chromatography of ex-
tract on a cellulose cohunn. Elution with 20 per
cent NH4CI. Purified l)y countercurrent distrilni-
tion (benzol -pH 7.0 phosphate buffer). Crude
powder treated with chloroform and crystallized
from amyl acetate on standing (1). II. Adsorbed
from broth on IRC-50 (H+ phase) at pH 7.8. Eluted
with aciueous 80 per cent acetone. Eluate ex-
tracted with chloroform at pH 5. Proceed as in
Step I (2).
Cheuiicdl and physical properties: Colorless crys-
tals. Ultraviolet absorption maximum at 258 m^.
Stable over a wide range of pH values (1 ).
Biological activity: Active on certain fungi and
yeasts, such as Willia anoiitala, Sacch. sake, and
.4 . niger. Not active on ('. albicans or Trichophyton
interdigitale (1). Antiviral activity against influ-
enza A.
Toxicity: LDm (mice) 160 mg per kg intrave-
nousl.y (1).
References:
1. Sato, K. and Katagiri, K. Chemotherapy
5: 182-183, 1957.
2. Katagiri, K. et al. Shionogi Kenkyusho
Nemjjo 7: 715-723, 1957.
Nigericiii
Produced by: Streptoniyces violaceoniger strains
and related Streptornyces spp. (1, 2).
Method of extraction: Precipitated from l)roth b}'
acidification to pH 2 to 3. Precipitate dissolved in
base. Crystallization as Na salt bj^ warming this
solution at pH 8.5 or above. Recrystallized from
hot metlianol on addition of water. Can also be
extracted from broth by n-butanol, Itutyl acetate,
or ethyl ether, or atlsorl)ed on charcoal and eluted
with methanol (1 ).
Chemical and physical properties: Acidic sub-
stance (CssHeaOii)- -Vfl salt: Colorless needles;
m.p. 246-254°C. Free acid and Na salt are slightly
soluble in water and soluble in lower alcohols.
318
DESCRIPTIONS OF ANTIBIOTICS
Free acid: CssHf^Ou : C = 65.42%; H = 9.90%.
Nasalt:i)—CB.z = 3.99%.
Biological activity: Active on gram-po.sitive bac-
teria (0.12 to 0.5 ng per ml), mycobacteria (0.5 to
4.0 Mg per ml), C. albicans (2 yug per ml). Tricho-
phyton mentagrophytes (16 yug per ml). Gram-nega-
tive bacteria are resistant to 64 /xg per ml.
Biological activity inhibited by K+. Inhibits
respiration and phosphate uptake in the presence
of several substrates; also inhibits mitochondrial
adenosinetriphosphatase. This inhiliition is de-
pendent in part on the str\ictural integrity of the
mitochondria (3).
Toxicity: LD50 (mice) 2.5 mg per kg intraperi-
toneally.
References:
1. Harned, R. L. et al. Antibiotics & Chemo-
therapy 1: 594-596, 1951.
2. Benedict, R. G. and Lindenfelser, L. A.
Given in Benedict, R. G. Botan. Rev.
19: 229-320, 1953.
3. Lardy, H. A. et al. Biochim. et Biophys.
Acta 78: 587-597, 1958.
Nironiycins
Produced by: Streptoniyces albus.
Synonyms: Closely related to cycloheximide and
fermicidin.
Method of extraction: Extraction of broth at pH
7.4 with butanol. Evaporation of butanol under
reduced pressure to syrup; washed with petroleum
ether. Resulting crude residue dissolved in water
and extracted with ethyl acetate; concentrated in
vacuo to syiup. Syrup washed with petroleum ether
and dried. Residue dissolved in butyl alcohol and
passed through an alumina column to remove im-
purities. Active effluent evaporated. Upon addi-
tion of benzene, white crystals appear and are re-
moved. Benzene solution evaporated. Residue
washed with petroleum ether and dried. Crude
powder dissolved in 20 per cent aqueous acetone
and chromatographed on carbon. Elution with
progressively drier acetone. Fractions active
against Saccii. sake combined and the acetone
evaporated. Active powder purihed by counter-
current distribution (benzene-water, 1:1, 30 trans-
fers). One active substance shows a peak at Tube
6 (niromycin A) and another at Tube 9 (niromy-
cin B). Niromycin A is further purified by repeat-
ing distribution, but is unstable and does not
crystallize. Niromycin B crystallized from ethyl
acetate (1).
Chemical and phi/sical properties: Xiro)nycin A:
Hygroscopic, white, amorphous, neutral sub-
stance; m.p. 98-105°C. Soluble in water, methanol,
ethanol, butanol, ethyl acetate, butyl acetate, ace-
tone, and chloroform. Slightly solul)Ie in benzene
and ether. Insoluble in petroleum ether. No spe-
cific ultraviolet light absorption. No optical rota-
tion (c = 1 per cent in ethanol). Positive Tollen
and 2,4-dinitrophenylhydrazine reactions. Nega-
tive ninhydrin, FeCls , Fehling, Benedict, Molisch,
biuret, and permanganate reactions (1). Niromy-
cin B: Neutral substance. White hygroscopic crys-
tals; m.p. 47-67°C. Same solubility properties,
color reactions, light absorption, and optical rota-
tion as niromycin A. C = 62.57%; H = 7.54%;
N = 5.3%. Infrared spectrum given in reference 1.
Semicarbazone: m.p. 175-1 76°C. C = 42.06%; H =
7.33%; N = 35.90%. 24-Dinitrophenylhydrazone:
m.p. 199-200°C (decomposition). C = 56.29%;
H = 5.53%; N = 15.16%. Suggested formula for
niromycin B: C14H21NO4 (1).
Biological activity: Both niromycins are active
against fungi and viruses, but not against bac-
teria. Examples of minimal inhiVntory concentra-
tions in ^g per ml: Hansenula anomala: A = 1.5,
B = 0.7; Sacch. cerevisiae: A = 0.35, B = 0.17;
Sacch. sake: A = 0.7, B = 0.35; A. niger: A = B =
>1000. In tissue cultures, niromycin A inhil)its
Newcastle disease virus at 0.01 ^g per ml ; nontoxic
to chick embryo cells at 6.25 jug per ml. Niromycin
B inhibits Newcastle disease virus at 0.036 /ug per
ml and shows no toxicity at 0.75 Mg per ml. Both
substances inhil)it multiplication of influenza
virus in chick embryos (2).
Toxicity: Niromycin A: LD50 (mice) 40 to GO mg
per kg intravenously. Niromycin B: LDsn (mice)
48 mg per kg, (rats) 1.8 mg per kg intravenously.
References:
1. Osato, T. ('/ a/. J. Antibiotics (Japan) 13A:
110-113, 1960.
2. Osato, T. e/ o/. J. Antibiotics (Japan) 13A:
97-109, 1960.
INitrosporin
Produced ly: Streptomyces nitrosporeus (1).
Method of extraction: I. Adsorption on charcoal
or cation exchange resin and elution with acidic
acetone or dilute HCl. II. Broth extraction by
ethyl ether, butyl or ethyl acetate at pH 7.0. Re-
extracted into water at pH 2.0. Back-extracted
into ethyl acetate at pH 7.0 and extract concen-
trated in vacuo. Chromatographed on alumina,
developed with ethyl acetate. Fractions pooled
and lyophilized. Solid taken up in water, warmed,
filtered, then cooled to precipitate crystals (1).
Chemical and physical properties: Basic white
crystalline substance. Browns at 115-120°C; m.p.
130-140°C. Soluble in ethanol and ethyl acetate.
DESCRIPTIONS OF ANTIBIOTICS
319
Slightly soluble in ether, l>enzene, ethylene di-
chloride, and acidic water. Insoluble in water.
Unstable to, and turning brown on, exposure to
air. Ultraviolet absorption spectnun maxima at
250 and 320 m^. Negative FeCl.3 , ninhydrin, biu-
ret, Molisch, and Fehling tests. CsoHssNaOe:
C = 60.34%; H = 6.41%; N = 7.88%. No S or
halogen (1).
Biological activity: Active on gram-positive bac-
teria; less so on gram-negative bacteria. Not ac-
tive on B. (u/thracis. Not active on fungi or myco-
bacteria (1).
Toxicity: LD50 (mice) 16 mg per kg intrave-
nously (1).
Reference: 1. Umezawa, H. and Takeuchi, T.
J. Antil)iotics (Japan) 5: 270-273, 1952.
Nocardaniiti
Produced by: Xocardia sj). similar to .Y. flavesccns
(1).
Method of extraction: Mycelium-containing cul-
ture-broth extracted with butanol. Extract con-
centrated to dryness in vacuo. Residue washed
and pulverized with ether, filtered, then taken up
in hot water. Crystallizes on cooling (1). Recrys-
tallized from water-saturated butanol or hot water
(2).
Chemical and physical properties: Reddish
needles (1,2); ni.]). 183-184°C. Soluble in boiling
methanol and dilute NaOH. Slightly solulile in
hot water (1). Optically inactive. Positive am-
moniacal silver nitrate and Fehling tests (2).
Hygroscopic (1). Cives a red-l)rown color with
FeCls (aqueous or alcoholic) and a green color
with copper chloride (1). Monoacetate: Fine
needles; m.p. 118°C. CuHieOeNo : C = 54.84%;
H = 6.90%; N = 11.34%; COCH, = 17.57% (2).
Base: CgHnO-jN.. (2). Structure of nocardamin
given in Chapter 6.
Biological activitij: Active on mycobacteria. Not
active on bacteria or fungi (2). Iiuictivated l)y
serum (1).
References :
1. Stoll, A. et al. Schweiz. Z. Pathol, u Bak-
teriol. 14: 225-233, 1951.
2. Stoll, A. et al. Helv. Chim. Acta 34: 862-
873, 1951.
Nocardiaiiiii
Produced by: Nocardia sp. (1).
Method of extraction: Filtered broth extracted
with ether. Extract dehydrated and evaporated to
dryness. Residue dissolved in chloroform-ether
(50:50) or benzene and chromatographed on alu-
mina. A reddish material eluted with chloroform-
ether or chloroform; recrystallized from methanol
(1).
Chemical and physical properties: Weak base (2).
C65-67H96-i04Oi.^N,s: C = 57.25%; H = 7.25%;
N = 18.3%. No S, P, or halogens. Red prisms;
m.p. 228-235 °C (decomposition). Soluble in chlo-
roform, glacial acetic acid, and pyridine;
moderately soluble in acetone, methanol, and
dilute acids; sparingly soluble in water and ether;
insoluble in petroleum ether, carbon disulfide, and
carbon tetrachloride. [a]f = —223° (c = 0.3 per
cent in methanol). Ultraviolet al)sorption spec-
trum maximum at 440 mn (log e = 4.52) (meth-
anol). Infrared spectrum given in reference 1.
Negative xanthine, Liebermann, Schiff, Ehrlich,
biuret, and hydroxamic acid tests. Acid hydroly-
sates give negative ninhydrin and Ehrlich tests,
but a methanolic solution of alkali fusion prod-
ucts to which concentrated HCI has been added
gives a positive Ehrlich test, indicating possible
presence of jjyrrole nuclei.
Biological activity: Active on gram-positive
bacteria; not active against gram-negative l)ac-
teria or mycobacteria.
References:
1. Bick, I. R. et al. Antibiotics & Chemo-
therapy 2: 255-258, 1952.
2. Cram, 1). J. (iivcTi in Benedict, R. G.
Botan. Rev. 19: 229 320, 1953.
\<K'ar<liii
Produced by: .Xocardia cocliaca (1).
Synonym: PossiV)le relationshij) with trehalosa-
mine.
Method of extraction: Broth-filtrate stirred with
charcoal. Eluted with ether-95 per cent ethaaol
(1:1). Evaporated to dryness. Mycelium extracted
with ether-ethanol (1:1). Production of the anti-
biotic (l)ut not the mycciiiun) greatly stimulated
t)y trehalose.
Chemical and physical properties: Crude sul)-
stance. Water-soluble and thermostable.
Biological activity: Active on certain strains of
mycobacteria, including M. tuberculosis var.
hominis H37Rv. Active in vivo on mycobacteria in
chick embryo, guinea pig, and mouse (1,2).
Toxicity: 25 ing of crude nocardin is more toxic
to mice than 1.5 mg of streptomycin, sul)cutane-
ously (2).
References:
1. Emmart, E. W. Am. Rev. Tuberc. .i6: 316-
333, 1947.
2. Emmart, E. W. et al. J. Bacteriol. .57:
505-514, 1949.
320
DESCRIPTIONS OF ANTIBIOTICS
i\ocar<l<>rii!)iii
Produced by: Xocardia narashinoensis (2).
Method of extraction: Extraction of the broth-
till rate with n-butanol at pH 2.0 to 3.0. Concentra-
tion of the solvent layer to syrup in vacuo. Syrup
extracted with acetone; again concentrated to
syrup. Extraction of the syrup with water; 10 per
cent NaOH added dropwise until a deep red pre-
cipitate forms. Precipitate collected by centrifu-
gation, washed with water, and dried. Nocardoru-
bin can also be extracted with acetone from the
mycelium.
Chemical and physical properties: Red at alkaline
reaction, yellow at acid pH. Alkaline form is
slightly soluble in dioxane, ethyl acetate, chloro-
form, and propylene glycol. It is almost insoluble
in most organic solvents and water. The acid form
is soluble in water and organic solvents. Carbo-
hydrate and protein tests negative. Turns black
at 180°C, but does not melt at 250°C. Aqueous
solutions stable for at least 2 months at 27°C.
Heating solutions at pH 2.0 reduces the activity
in 5 to 15 minutes at 60°C. At pH values above
5.8, this treatment does not destroy the antibiotic
activity.
Biological activity: Active against many gram-
positive bacteria in a concentration of 0.001 to
5.0 fig per ml. Also active against mycobacteria
and actinomycetes. Active against certain gram-
negative bacteria at a concentration of 20 fig per
ml. Activity against fungi low or ml. Limited
activity against D. pneumoniae in mice. The de-
velopment of resistance to nocardorul)in is slow.
Toxicity: LDo (mice) about 154 mg per kg; LDmn
about 230 mg per kg intraperitoneally.
References :
1. Aiso, K. et al. J. Antibiotics (Japan) "A:
1-6, 1954.
2. Endo, T. J. Antibiotics (Japan) 9A: 228,
1956.
Noforiiiicin
Produced by: Nocardia formica.
Synonym: Antibiotic MK 61.
Method of extraction: Adsorption from the cul-
ture-filtrate on activated carbon, and elution with
90 per cent acpieous methanol under acidic condi-
tions. Concentration by chromatography over
activated carbon or a cation exchange resin. The
salt-free concentrate evaporated until crystals
form (1).
Chemical and physical properties: Basic sub-
stance. Analysis of crystalline sulfate: CnH,'j4Nifi
Oo(S04)2; m.p. 265°C (decomposition) for the
hydrochloride. Water-solul)le. Dialyzable (1).
Stable in solutions up to pH 8.0. Among the hy-
drolysis products, ammonia and glutamic acid
have l)een identified. Nonreducing. No phenolic
groups (1).
Biological activity: Prolongs life of mice infected
with swine influenza, influenza A (PR 8), or in-
fluenza B (Lee). Treatment is effective whether
the material is administered subcutaneously,
intravenously, or orally. An effect is obtained
when the material is administered as early as 24
hours before, or as late as 24 hours afte
