Antibiotic compounds marcellomycin and musettamycin

A novel anthracycline antibiotic complex designated herein as bohemic acid complex is produced by fermentation of Actinosporangium sp. A.T.C.C. 31127. The complex and two bioactive components designated marcellomycin and musettamycin exhibit antibiotic activity and inhibit the growth of various tumor systems in rodents.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
This invention relates to new anthracycline antibiotics and to their 
production and recovery. 
2. Description of the Prior Art 
A number of anthracycline glycosides have been described in the literature. 
Among them, daunomycin and adriamycin are particularly being watched with 
keen interest in the field of cancer chemotherapy and have already been 
applied clinically for human cancers. 
Preparation of adriamycin by fermentation of S. peuceticus var. caesius is 
disclosed in U.S. Pat. No. 3,590,028. Chemical conversion of daunomycin to 
adriamycin is taught in U.S. Pat. No. 3,803,124. 
Daunomycin (produced by fermentation of S. peuceticus in U.K. Pat. No. 
1,003,383) may be the same as Rhone-Poulenc's 13,057 R.P. (formerly 
rubidomycin and now daunorubicin; see U.K. Pat. No. 985,598, 1,188,262 and 
1,241,750 and U.S. Pat. No. 3,616,242) and is probably identical to Ciba's 
danubomycin disclosed in U.S. Pat. No. 3,092,550 and U.K. Pat. No. 
901,830. See also U.S. Pat. No. 3,686,163 on dihydrodaunomycin. 
Cinerubin A and cinerubin B, glycosides of the aglycone 
.epsilon.-pyrromycinone, are disclosed in U.K. Pat. No. 846,130 [see also 
U.S. Pat. No. 3,864,480 and Keller-Schierlein, et al., Antimicrobial 
Agents and Chemotherapy, page 68 (1970) and Chemical Abstracts, 54, 1466i 
(1960)]. 
The anthracycline glycoside carminomycin described in J. Antibiotics 
27:254-259 (1974), in West German Specification 2,362,707 and in J. Amer. 
Chem. Soc. 97(20):5955-5956 (1975) has been reported to be active against 
several animal tumor systems. 
Trypanomycin is described in Antimicrobial Agents and Chemotherapy 
1:385-391 (1972) as having strong antiprotozoal activity. It has an 
aglycone similar to but not identical with .epsilon.-pyrromycinone. 
The antibiotic pyrromycin disclosed in Chem. Ber. 92:1904-1909 (1959) 
contains the aglycone .epsilon.-pyrromycinone and the glycosidic sugar 
rhodosamine. 
For further illustrative and summary disclosures of anthracycline 
antibiotics see Index of Antibiotics from Actinomycetes, Hamao Umezawa, 
Editor-In-Chief, University Park Press, State College, Pennsylvania, 
U.S.A. (1967) as follows: 
______________________________________ 
Antibiotic Page Number 
______________________________________ 
Aklavin 111 
Cinerubin A 220 
Cinerubin B 221 
Danubomycin 242 
Daunomycin 243 
Pyrromycin 542 
Rhodomycin A,B 561 
Rubidomycin 574 
______________________________________ 
The textbook Antibiotics, Volume 1, Mechanism of Action, edited by David 
Gottlieb and Paul D. Shaw, Springer-Verlag New York, Inc., N.Y., N.Y. 
(1967) at pages 190-210 contains a review by A. DiMarco entitled 
Daunomycin and Related Antibiotics. 
Information Bulletin, No. 10, International Center of Information of 
Antibiotics, in collaboration with WHO, December, 1972, Belgium, reviews 
anthracyclines and their derivatives. 
SUMMARY OF THE INVENTION 
This invention relates to novel anthracycline antibiotic substances. More 
particularly, it relates to an anthracycline antibiotic complex designated 
herein as bohemic acid complex, said complex being produced by cultivating 
a bohemic acid-producing strain of Actinosporangium sp., most preferably 
Actinosporangium sp., A.T.C.C. 31127, in an aqueous nutrient medium 
containing assimilable sources of nitrogen and carbon under submerged 
aerobic conditions until a substantial amount of said bohemic acid complex 
is produced by said organism in said culture medium and optionally 
recovering the complex from the culture medium. Also provided by the 
present invention are two novel bioactive anthracycline components of 
bohemic acid complex, said components being prepared by extraction of the 
whole fermentation broth with a water-immiscible organic solvent followed 
by separation and isolation of the individual antibiotic compounds as by 
chromatographic procedures. The bohemic acid complex and its bioactive 
components designated herein as musettamycin and marcellomycin exhibit 
both antibacterial and antitumor activity. 
DETAILED DESCRIPTION 
Bohemic acid complex and its components marcellomycin and musettamycin may 
be produced by fermentation of a new member of the genus Actinosporangium 
designated Actinosporangium sp. strain C-36,145. The above organism was 
obtained from a soil sample taken from Ontario, Canada. A culture of the 
organism has been deposited in the American Type Culture Collection, 
Washington, D.C., and added to its permanent collection of microorganisms 
as A.T.C.C.31127. 
As in the case of many antibiotic-producing cultures, fermentation of 
Actinosporangium sp. strain C-36,145, A.T.C.C. 31127, results in the 
production of a mixture or complex of component substances. Two bioactive 
anthracycline components, musettamycin and marcellomycin, have been 
separated from the bohemic acid complex produced by the abovementioned 
organism. 
Musettamycin and marcellomycin have been determined to have the following 
structural formulae: 
##STR1## 
As shown, both of the above components are anthracycline glycosides of the 
aglycone .epsilon.-pyrromycinone. Musettamycin contains two glycosidic 
sugar units, i.e. 2-deoxy-L-fucose and L-rhodosamine, while marcellomycin 
contains two units of 2-deoxy-L-fucose and one L-rhodosamine unit. The 
structures of the components were determined by analysis of their infrared 
and nuclear magnetic resonance spectra and are in agreement with the 
physical data provided below. 
In the above formulae the dotted lines indicate that the bonded group is 
partially located below the plane of the ring to which it is attached. The 
spikes indicate that the attached group is positioned above the plane of 
the ring. 
The bohemic acid complex and the individual musettamycin and marcellomycin 
components form salts with both acids and bases, and the pharmaceutically 
acceptable salts of the complex and components are included within the 
scope of the present invention. Examples of such pharmaceutically 
acceptable salts include salts with stronger acids such as hydrochloric, 
hydrobromic, sulfuric, nitric and phosphoric and with metallic cations, 
e.g. alkali metal or alkaline earth metal cations such as sodium, 
potassium, calcium and magnesium. 
Preparation of the novel anthracycline antibiotics of the present invention 
is described below. 
THE MICROORGANISM 
The strain C-36,145 has the following morphological characteristics. The 
strain forms a sporangium-like body (false sporangium) on the tip of the 
sporophore, which is an agglomeration of a thickly coiled spore-chain. The 
spore-chain often interlaces with the neighboring aerial hyphae, and it 
develops to the sporangium-like structure covered by a viscid material. 
The sporangium-like body and aerial mycelium are formed on 
glucose-asparagine agar, tyrosine agar, yeast extract-malt extract agar 
and oat meal agar. In addition to the formation of a large number of false 
sporangia, there are also produced ordinary spore-chains, though much less 
in number, which form open spirals. Spores in the sporangium-like body are 
smooth in surface, ellipsoidal in shape and non-motile. Spores in the 
ordinary spore-chain are of oval shape and have a smooth or occasionally 
warty surface. Primary mycelium is branched, non-septated and 
non-fragmented. 
Table 1 reports the cultural properties obtained on various media. Strain 
C-36,145 grows well on most of the agar media tested, but the formation of 
aerial mycelium and the sporulation are somewhat slow. Mass color of the 
aerial mycelium is light greenish gray. Reverse side of the growth is 
reddish orange to red in glucose-asparagine agar, inorganic salts-starch 
agar, yeast extract-malt extract agar and oat meal agar. It produces 
melanoid pigment in tyrosine agar and peptone-yeast extract-iron agar. 
The physiological characteristics and carbohydrate utilization of strain 
C-36,145 are shown in Tables 2 and 3, respectively. The growth temperature 
ranged from 20.degree. to 37.degree. C. and no proliferation was seen at 
43.degree. C. 
Strain C-36,145 contains LL-diaminopimelic acid (LL-DAP) and glycine as 
characteristic amino acid components in the cell wall. Diagnostic 
carbohydrate was not present. 
The morphological, cultural and physiological characteristics of Strain 
C-36,145 are similar to those of the genus Streptomyces except for the 
formation of sporangium-like body. The cell wall composition is also 
similar to that of the type I group (Streptomyces type) according to the 
classification of Lechevalier and Lechevalier in Int. J. Syst. Bacteriol. 
20:435-443 (1970). The sporangium-like body of strain C-36,145 appears to 
be different from the normal sporangium produced by the well-defined 
sporangium-forming genera in that (1) the latter form small sporangia at 
an early stage of the growth which mature with time and (2) ordinary 
sporangia are usually not covered by a viscous material. 
Krassilnikov and Tsi-Shen proposed in 1961 a new genus of Actinosporangium 
in the family Actinoplanaceae (later transferred to the family 
Streptosporangiaceae) for the organism producing a spore mass which was 
very similar to sporangium (Isv. Akad. Navk. USSR, Ser. Biol., 113-116, 
1961). Subsequently, the sporangium-like body was found to be a viscid 
spore-forming mass and different from the real sporangium, and the genus 
Actinosporangium was placed in the family Streptomycetaceae on the basis 
of its morphological characteristics and the cell wall composition of the 
type I group. 
Thus, on the basis of all the available data, Strain C-36,145 is considered 
to be a new species of the genus Actinosporangium. It should be noted, 
however, that only two species in the genus Actinosporangium have been 
reported in the literature and hence the genus has not yet been fully 
established (see H. Prauser: The Actinomycetales. The Jena Intl. Sym. on 
Taxonomy. Sept., pp. 329-335, 1968. Veb. Gustav Fischer Verlag, JENA, 
1970). 
It is to be understood that the present invention is not limited to use of 
the particular Strain C-36,145 or to organisms fully answering the above 
descriptions. It is especially intended to include other bohemic 
acid-producing strains or mutants of the said organism which can be 
produced from the described organism by various means including 
x-radiation, ultraviolet radiation, treatment with nitrogen mustards, 
phage exposure, and the like. 
Table 1 
__________________________________________________________________________ 
Cultural Characteristics of Strain C-36,145 
Growth 
Reverse Color 
Aerial Mycelium 
Diffusible pigment 
Sucrose-nitrate 
Moderate 
Yellowish pink 
Scant, whitish 
None 
agar 
Glucose-asparagine 
Good Reddish orange 
Abundant, greyish 
None or reddish 
agar to red leaf orange 
Glycerol- Good Rose Moderate, light 
None or reddish 
asparagine agar grey to pale 
pink 
pink 
Inorganic salts- 
Moderate 
Reddish orange 
Poor, greyish 
Light orange, 
starch agar to deep red 
leaf partially 
light yellow 
Tyrosine agar 
Good Brown to deep 
Poor, greyish 
Dark brown 
purplish- 
leaf 
brown 
Nutrient agar 
Poor Colorless 
None None 
Yeast extract- 
Good Deep red 
Moderate, greyish 
Light brown 
malt extract leaf partially 
agar greyish pink 
Oat meal agar 
Moderate 
Vivid reddish 
Moderate, greyish 
Vivid orange 
orange leaf 
Peptone-yeast 
Moderate 
Black None Black 
extract-iron 
agar 
__________________________________________________________________________ 
Table 2 
__________________________________________________________________________ 
Physiological Characteristics of Strain C-36,145 
Tests Responses Methods and Materials Employed 
__________________________________________________________________________ 
Nitrate reduction in 
Strongly positive Czapek's sucrose nitrate broth 
inorganic medium 
Nitrate reduction in 
Strongly positive Nitrate medium, consisted of yeast, 
organic medium yeast extract 0.5%, glucose 1.0%, 
KNO.sub.3 0.5% and CaCO.sub.3 0.1%. 
Skim-milk agar 
Abundant growth. Negative hydrolysis. 
Leudeman's medium [Intl. J. Syst. 
Deep yellowish-red to deep reddish- 
Bacteriol. 21:240-247 (1971)] 
purple mycelial pigment. 
10% Skim-milk 
Brownish broth color. Reddish-orange 
solution ring growth. No peptonization nor 
coagulation. 
Gelatin slab 
Rapid and complete liquefaction. 
Basal medium: Yeast extract 0.4% 
malt extract 1.0% and glucose 
0.4%. 
Melanin formation 
Strongly positive Tyrosine agar and peptone-yeast 
extract-iron agar. 
Growth-temperature 
Optimal growth at 28.degree. C. Moderate 
Yeast extract-malt extract agar. 
growth at 20-37.degree. C. Slow growth at 
at 15.degree. C. No growth at 10.degree. C. and 
43.degree. C. 
NaCl tolerance 
Moderate growth at 0.5-4%-NaCl. 
Basal medium: 1%-yeast extract, 
No growth at 8%-NaCl. 
2%-soluble starch and 1.5% 
__________________________________________________________________________ 
agar. 
Table 3 
______________________________________ 
Carbohydrate Utilization 
of Strain C-36,145 
D(-)-Arabinose - 
L(+)-Arabinose ++ 
D-Xylose ++ 
D-Ribose ++ 
L-Rhamnose ++ 
D-Glucose ++ 
D(+)-Galactose ++ 
D-Fructose ++ 
D-Mannose ++ 
Sucrose ++ 
Maltose ++ 
Lactose ++ 
D(+)-Melibiose ++* 
Raffinose ++* 
D(+)-Melezitose - 
Soluble starch ++ 
Cellulose - 
Glycerol ++ 
Inositol ++ 
D-Mannitol ++* 
Sorbitol - 
Dulcitol - 
______________________________________ 
Basal medium: Pridham-Gottlieb medium 
*Rich production of reddish-orange pigment 
Preparation of Bohemic Acid Complex 
Bohemic acid complex may be produced by cultivating a bohemic 
acid-producing strain of Actinosporangium sp. having the characteristics 
of A.T.C.C. 31127 or a mutant thereof under submerged aerobic conditions 
in an aqueous nutrient medium. The organism is grown in a nutrient medium 
containing on assimilable carbon source, for example an assimilable 
carbohydrate. Examples of suitable carbon sources include sucrose, 
lactose, maltose, mannose, fructose, glucose, glycerol and soluble starch. 
The nutrient medium should also contain an assimilable nitrogen source 
such as fish meal, peptone, soybean flour, peanut meal, cotton seed meal 
or corn steep liquor. Nutrient inorganic salts can also be incorporated in 
the medium. Such salts may comprise any of the usual salts capable of 
providing sodium, potassium, ammonium, calcium, phosphate, sulfate, 
chloride, bromide, nitrate, carbonate or like ions. 
Production of the bohemic acid complex can be effected at any temperature 
conducive to satisfactory growth of the organism, e.g. 
20.degree.-37.degree. C., and is conveniently carried out at a temperature 
of about 27.degree. C. 
The medium normally is slightly alkaline, but the exact pH can be varied 
widely depending on the particular medium used. 
The fermentation may be carried out in Erlenmeyer flasks or in laboratory 
or industrial fermentors of various capacities. When tank fermentation is 
to be used, it is desirable to produce a vegetative inoculum in a nutrient 
broth by inoculating a small volume of the culture medium with the spore 
form of the organism. After obtaining an active inoculum in this manner, 
it is transferred aseptically to the fermentation tank medium for large 
scale production of the antibiotics. The medium used for the vegetative 
inoculum can be the same as that employed for larger fermentations, 
although other media can be employed. 
As is customary in aerobic submerged culture processes, sterile air is 
blown through the culture medium. Agitation may be maintained by means of 
agitators generally familiar to those in the fermentation industry. 
In general, optimum production of the bohemic acid complex is achieved 
after incubation periods of about 190-210 hours in stir-jar fermentors or 
tank fermentors. The course of the fermentation can be followed by 
assaying the fermentation medium from time to time against an organism 
susceptible to the bohemic acid complex, e.g. D. pneumoniae, St. pyogenes 
or S. aureus. 
The bohemic acid complex may be recovered from the fermentation medium by 
extraction with a water-immiscible organic solvent, preferably a polar 
organic solvent such as ethyl acetate, methyl isobutyl ketone or a 
(higher)alcohol (e.g. n-butanol), and most preferably methyl isobutyl 
ketone. The majority of the antibiotic activity is found in the broth, and 
thus the broth may be filtered prior to extraction. In the preferred 
procedure, however, the whole fermentation broth is extracted with the 
organic solvent at a pH of between about 7.5 and 8.5. The organic phase 
may then be filtered and dried to give the solid bohemic acid complex. 
Alternatively, the organic extract may be concentrated and the solid 
complex precipitated by dilution with a suitable antisolvent such as 
Skellysolve B. 
Separation and Isolation of Musettamycin and Marcellomycin 
The anthracycline antibiotics musettamycin and marcellomycin can be 
separated from each other and from the other components of bohemic acid 
complex by chromatography of solutions of bohemic acid complex on columns 
packed with a suitable adsorbent such as Sephadex LH 20 (trade name for 
dextran derivatives used as gel filtrants in organic solvents; 
manufactured by Pharmacia Fine Chemicals, Inc.). The bohemic acid complex 
components are then eluted from the adsorbent with a suitable organic 
solvent such as chloroform. Multiple fractions are collected and, with 
suitable dilution, their adsorbancies are determined at 490 m.mu.. The 
latter are plotted graphically against the corresponding fraction numbers 
to determine peaks for the components eluted from the column. The 
appropriate fractions, as determined from the elution sequence are 
combined and evaporated to give the solid individual antibiotics. The 
solids may be partially purified by recrystallization from a suitable 
organic solvent such as acetonitrile, chloroform-Skellysolve B or 
methanol. 
The musettamycin and marcellomycin components may be further purified by 
use of the high pressure liquid chromatography procedure described in 
detail in Examples 10 and 11. This procedure has been found to give 
extremely pure samples of the two antibiotics. 
Bohemic acid complex or its musettamycin and marcellomycin components can 
be converted by methods known per se to the pharmaceutically acceptable 
salts described above. 
Biological Activity Data 
The in vitro minimum inhibitory concentration (MIC) of musettamycin and 
marcellomycin were determined for a number of microorganisms using the 
standard tube dilution procedure. 
Table 4 
______________________________________ 
Antimicrobial Spectrum of 
Musettamycin and Marcellomycin 
Test Organism MIC in .mu.g/ml. 
______________________________________ 
Bacteria: Mar. Mus. 
Streptococcus pneumoniae 
A-9585 .03 .06 
Streptococcus pyogenes 
A-9604 .03 .06 
Staphylococcus aureus 
A-9497 1 0.5 
Staphylococcus aureus 
A-9537 1 1 
Escherichia coli 
A-15119 125 &gt;125 
Escherichia coli 
A-21780 32 32 
Klebsiella pneumoniae 
A-9977 125 &gt;125 
Proteus mirabilis 
A-9900 &gt;125 &gt;125 
Fungi: 
Candida albicans 
A-9540 125 125 
Candida tropicalis 
A-15051 125 63 
Candida krusei A-15052 125 125 
Cryptococcus neoformans 
A-1503 125 63 
Trichophyton A-9870 &gt;125 &gt;125 
mentagrophytes 
______________________________________ 
Mar.=Marcellomycin 
Mus.=Musettamycin 
The acute intraperitoneal LD.sub.50 in mice is 9.8 to 21.12 mg./kg for 
musettamycin and 6.35 to 10.56 mg./kg. for marcellomycin. 
The compounds of the present invention were also tested against various 
transplantable rodent tumer systems. Details of the methods used have been 
described in Cancer Chemoth. Reports 3:1-87 (Part 3), 1972. 
The first observation of tumor inhibitory effects was with the Walker 256 
carcinosarcoma implanted as a solid intramuscular tumor in rats. Treatment 
of the animals with fermentation broth containing bohemic acid complex 
caused 73% inhibition of tumor growth compared to untreated control 
tumors. 
Typical effects of therapy with partially purified musettamycin on two 
lymphatic leukemias in mice, i.e. P-388 and L-1210, are shown below in 
Table 5. Significant tumor inhibition over a 16 fold dose range was 
observed with P-388 leukemia. 
Table 5 
__________________________________________________________________________ 
Effect of Musettamycin on Transplanted Mouse Leukemias 
P-388 (ascitic) L-1210 (ascitic) 
Avg. wt. 
T/C Avg. wt. 
T/C 
Dose 
Difference 
Percent 
Survivors 
Difference 
Percent 
Survivors 
mg/kg 
(T-C, g.) 
MST Day 5 (T-C, g.) 
MST Day 5 
__________________________________________________________________________ 
6.4 -3.4 160 6/6 -2.5 140 6/6 
3.2 -3.0 145 6/6 -2.5 120 5/6 
1.6 -2.8 145 6/6 -1.3 133 6/6 
.8 -2.0 140 6/6 -1.1 107 6/6 
.4 -1.6 135 6/6 -- -- -- 
.2 -2.1 120 6/6 -- -- -- 
__________________________________________________________________________ 
Treatment: 
Single injection Day 1 intraperitoneally.? 
Evaluation:? 
T/C percent MST=median survival time in days: Treated/Control .times. 100 
T/C.gtoreq.125 considered significant prolongation of host survival. 
Purified musettamycin and marcellomycin (as prepared by the procedures 
described in Examples 10 and 11 below) were tested against L-1210 mouse 
leukemia and the results are shown in Table 6. These data indicate that 
marcellomycin is about four times as potent as musettamycin in its 
inhibitory effects on this tumor. 
The antibiotic compounds of the present invention including bohemic acid 
complex, its components musettamycin and marcellomycin, and salts and 
mixtures thereof, exhibit both antimicrobial and antitumor activity. The 
invention includes within its scope pharmaceutical compositions containing 
at least one of such antibiotic substances mentioned above with a 
compatible pharmaceutically acceptable carrier. The compositions may also 
contain other active antibacterial and/or antitumor agents. The 
compositions may be made up in any pharmaceutical form appropriate for the 
route of administration in question. Examples of such compositions include 
solid compositions for oral administration such as tablets, capsules, 
pills, powders and granules, liquid compositions for oral administration 
such as solutions, suspensions, syrups and elixers and preparations for 
parenteral administration such as sterile solutions, suspensions or 
emulsions. 
Table 6 
__________________________________________________________________________ 
Effect of Bohemic Acid Products 
on L-1210 Leukemia 
Effect 
Average 
Dose Day of 
Total MST MST Weight Survivors 
Compound 
mg/kg/day 
Treatment 
Injections 
Days %T/C Change in g. 
Day 5 
__________________________________________________________________________ 
Mus. 12.8 1 1 10.5 150 -0.6 6/6 
6.4 1 1 10.0 143 +0.5 6/6 
3.2 1 1 9.0 129 +0.3 6/6 
1.6 1 1 9.0 129 +0.3 6/6 
.8 1 1 8.5 121 +1.2 6/6 
.4 1 1 8.0 114 +1.3 6/6 
Mus. 6.4 1.fwdarw.5 
5 6.0 86 -1.5 5/6 
3.2 1.fwdarw.5 
5 11.0 157 -1.3 6/6 
1.6 1.fwdarw.5 
5 10.0 143 +0.3 6/6 
.8 1.fwdarw.5 
5 9.5 136 +0.4 6/6 
.4 1.fwdarw.5 
5 9.0 129 +0.6 6/6 
.2 1.fwdarw.5 
5 9.0 129 +1.8 6/6 
Mar. 12.8 1 1 10.0 143 -0.7 3/6 
6.4 1 1 11.0 157 -0.7 6/6 
3.2 1 1 11.0 157 -0.7 6/6 
1.6 1 1 10.0 143 +0.1 6/6 
.8 1 1 10.5 150 0 6/6 
.4 1 1 9.0 129 -0.8 6/6 
Mar. 6.4 1.fwdarw.5 
4 TOXIC 
TOXIC 
TOXIC 0/6 
3.2 1.fwdarw.5 
5 6.0 86 -1.2 3/6 
1.6 1.fwdarw.5 
4 6.0 86 -0.1 5/6 
.8 1.fwdarw.5 
5 10.0 143 -0.2 6/6 
.4 1.fwdarw.5 
5 9.0 129 0 6/6 
.2 1.fwdarw.5 
5 9.0 129 -1.1 6/6 
Control 
Saline 
1.fwdarw.5 
5 7.0 -- +3.0 10/10 
__________________________________________________________________________ 
Inoculum: 10.sup.6 ascitic cells, i.p. into BDF.sub.1 female mice 
Treatment: i.p. in 0.5 ml. volume 
Evaluation: MST = median survival time in days; %T/C = MST treated/MST 
control .times. 100. 
Criteria: T/C .gtoreq. 125 considered significant tumor inhibition 
(prolongation of host survival) 
Mus. = Musettamycin 
Mar. = Marcellomycin 
For use as an antibacterial agent the compositions are administered so that 
the concentration of active ingredient is greater than the minimum 
inhibitory concentration for the particular organism being treated. A 
suggested dosage for use as an antitumor agent in mammalian species is 2.5 
to 10 mg./M.sup.2 for a single injection intravenous treatment course with 
marcellomycin and 10 to 40 mg/M.sup.2 for a single intravenous treatment 
with musettamycin. 
The following examples serve to illustrate the invention without limiting 
it. .mu. STYRAGEL, phenyl/CORASIL and phenyl/PORASIL B are tradenames of 
Waters Associates, Inc. for gel permeation chromatographic packing 
materials produced by chemically bonding organo-silanes to silica. 
Sephadex LH-20 is the tradename of Pharmacia Fine Chemicals, Inc. for a 
modified cross-linked dextran used in adsorption and gel filtration 
chromatography.

EXAMPLE 1 
Shade-flask fermentation 
The organism Actinosporangium sp. Strain C-36,145 (A.T.C.C. 31127) is grown 
on an agar slant medium consisting of 2 g. D-glucose, 20 g. oatmeal, 2 g. 
soy peptone and 2 g. agar made up to one liter with distilled water. After 
at least 6 days growth at 27.degree. C., spores are transferred to a 500 
ml. Erlenmeyer flask containing 100 ml. of sterile medium consisting of 30 
g. D-glucose, 10 g. soybean flour, 10 g. Pharmamedia (Traders Oil Mill 
Co., Fort Worth, Texas) and 3 g. CaCO.sub.3 made up to one liter with 
distilled water. This vegetative culture is incubated at 27.degree. C. on 
a Gyrotary tier shaker (Model G53, New Brunswick Scientific Co., Inc.) set 
at 210 rev./min. describing a circle with a 5.1 cm. diameter. After 48 
hours four milliliters of culture are transferred to a 500 ml. Erlenmeyer 
flask containing 100 ml. of sterile production medium consisting of 50 g. 
glycerol, 20 g. soybean flour, 10 g. peanut meal and 10 g. CaCo.sub.3 made 
up to one liter with distilled water. The culture is incubated at 
27.degree. C. for 144 hours on a shaker set at 230 rev./min. At this time 
antibiotic activity consisting of the bohemic acid complex is found in the 
culture filtrate and mycelium. 
EXAMPLE 2 
Stir-jar fermentation 
Bohemic acid complex is produced in stir-jar fermentors with the use of a 
48 hour old vegetative culture as described in Example 1. Four hundred 
milliliters of culture is transferred to 10 liters of sterile production 
medium as described in Example 1 including 0.01% Hodag Fl silicone 
antifoam (Hodag Chemical Corp., Skokie, Ill.) contained in a 14 liter 
capacity stir-jar. The stir-jar is installed in a Fermentor Drive Assembly 
(Model FS-614, New Brunswick Scientific Co., Inc., New Brunswick, N.J.). 
The temperature is maintained at 27.degree. C., the air flow rate is 6 
liters/min. and the agitator is set at 300 r.p.m. Hodag Fl antifoam is fed 
automatically as required to control foaming. At approximately 210 hours 
the incubation is terminated and bohemic acid complex is found in the 
culture filtrate and the mycelium. 
EXAMPLE 3 
Tank fermentation 
A tank fermentor with 37.8 liters of sterile production medium (as in 
Example 1) is inoculated with 1.89 liters of vegetative culture (as 
prepared in Example 1), agitated with an impeller speed of 300 r.p.m., 
aerated at 85 liters/min. and incubated at 27.degree. C. for 190 hours. 
The bohemic acid complex is found in the culture filtrate and mycelium. 
EXAMPLE 4 
Tank fermentation 
A tank fermentor with 3028 liters of production medium (as in Example 1) is 
inoculated with 152 liters of vegetative culture (as prepared in Example 
1), agitated with an impeller speed of 155 r.p.m., aerated at 141.6 
liters/min. and incubated at 27.degree. C. for 190 hours. At this time the 
presence of bohemic acid complex is found in the culture filtrate and 
mycelium. 
EXAMPLE 5 
Isolation of bohemic acid complex 
Whole fermentation broth (7 liters) at its harvest pH 8.1, was stirred with 
about an equal volume of methyl isobutyl ketone for 20-30 min. A large 
amount of diatomaceous earth filter aid was then added and, after stirring 
thoroughly to mix this in, the mixture was filtered on a filter aid mat 
using vacuum suction. The filtrate separated into two phases of which the 
lower (aqueous) was discarded. The organic phase was evaporated under 
vacuum to a small volume (50-100 ml.) and diluted with Skellysolve B 
(tradename for a petroleum ether fraction of b.p. 60.degree.-68.degree. C. 
consisting essentially of n-hexane and sold by Skelly Oil Co.) to 
precipitate a dark red solid which was dried in vacuo to give 1.9 g. of 
bohemic acid complex. 
EXAMPLE 6 
Isolation of bohemic acid complex on a large scale 
Whole fermentation broth (3,000 liters) at pH 8.0-8.5 was chilled at 
25.degree. C. and agitated vigorously with 3,000 liters methyl isobutyl 
ketone for 30 min. at 20.degree.-30.degree. C. To the emulsion was added 
360 kg. filter aid and the mixture was stirred vigorously for another 
hour. It was then allowed to settle for about 30 min. after which 
2,300-2,500 liters of organic phase was decanted and chilled at 
0.degree.-10.degree. C. An additional 800 liters methyl isobutyl ketone 
was agitated with the mixture for 20 min., decanted after settling 30 
min., and combined with the chilled first fraction to give a total extract 
volume of 3,300-3,400 liters. This was polish filtered to give a final 
3,100-3,200 liter methyl isobutyl ketone extract free of solids and 
insoluble aqueous phase. The organic extract was vacuum concentrated at 
0.degree.-10.degree. C. to a final volume of 6 liters. This was added to 
60 liters Skellysolve B with stirring at 20.degree.-25.degree. C. The 
precipitated solids were collected on a Nutsche filter, washed with 10 
liters Skellysolve B and sucked dry to give 900-1,000 g. somewhat oily and 
dark red amorphous product. This was stirred with excess ether, filtered 
on a Buchner funnel, rinsed with additional ether, sucked dry and dried in 
vacuo to give 351 g. amorphous dark red bohemic acid complex. 
EXAMPLE 7 
Fractionation of bohemic acid complex 
Sephadex LH-20 soaked for 68 hours in chloroform was slurry packed into a 
Pharmacia SR 25/100 column (25 mm. I.D. .times. 100 cm. height) equipped 
with adjustable teflon tips at each end. The column was packed so as to be 
completely filled from tip to tip, an effective bed height of 90-95 cm. 
Bohemic acid complex (500 mg.) was dissolved in 10 ml. chloroform and 
applied to the column which was then allowed to develop with chloroform by 
downflow at a take-off rate of 1 ml./min. Eluted liquid was collected in 6 
ml. cuts in a fraction collector. Samplings of even numbered tubes were 
diluted 80-fold with chloroform and read in a Bausch and Lomb Spectronic 
20 colorimeter at 490 m.mu.. Four distinct bands of anthracycline pigments 
were noted as follows: 
______________________________________ 
Wgt. upon 
Tube No. Evaporation 
______________________________________ 
1-4 -- 
5-11 First Band 66 mg. 
12-14 -- 
15-21 Second Band 36 mg. 
22-35 -- 
36-44 Third Band 18 mg. 
45 -- 
46-57 Fourth Band 48 mg. 
58- -- 
______________________________________ 
The solids obtained were chromatographed on Brinkmann 60F254 silica gel 
thin layer plates using an 80:20 toluene:methanol solvent system. The 
first band to elute was shown by thin layer chromatography to be a complex 
inactive mixture. The second band gave a characteristic pinkish red zone 
at about R.sub.f =0.75. This fraction was also inactive and was found to 
comprise mainly .eta.-pyrromycinone. The third fraction to elute gave a 
single zone with R.sub.f .about.0.3. This was determined to be 
musettamycin and was highly active when tested on both the L-1210 and 
P-388 mouse leukemia systems. The final band to elute gave a zone at 
R.sub.f .about.0.3 and comprised mainly marcellomycin. This component also 
exhibited high activity when tested on the two mouse leukemias. Although 
both musettamycin and marcellomycin on thin-layer chromatography have 
R.sub.f values around 0.3, musettamycin moves slightly faster. When mixed, 
musettamycin and marcellomycin are resolved then into two very close zones 
of color. 
EXAMPLE 8 
Large scale fractionation 
A 6 inch (diameter) .times. 77 inch (height) glass column was equipped at 
the base with a no clog filter on top of which was placed a layer of glass 
wool followed by a circular polyethylene frit. The latter was cut to a 
diameter of 57/8 inches to allow for swelling in chloroform. Sephadex 
LH-20 (8.73 kg.) was stirred for 3 hours in chloroform, filtered, and the 
solid reslurried in chloroform and left to stand for 16 hours. The mixture 
was then agitated for 15 min. and loaded onto the column. A 30 g. sample 
of bohemic acid complex (prepared as in Example 6) was heated with 1.5 
liters of chloroform for 15 min. and then stirred for 16 hours. Upon 
filtration, 7.5 g. of undissolved material containing some activity was 
separated. (In later runs it was found that the sample could be completely 
dissolved in chloroform containing 30-40% methanol. Chromatographic 
results were the same with this procedure.) The filtrate was applied to 
the column and downward development begun with chloroform. A flow rate of 
16 ml./min. at takeoff was maintained throughout the run. An initial 
volume of 1,445 ml. of eluant was taken before color reached the bottom of 
the gel bed. At this point collection of 100 ml. fractions was initiated 
and continued until the liquor was quite light again, a total of 906 cuts. 
Aliquots of every fifth fraction were diluted and analyzed as described in 
Example 7. Four components were seen to have separated as indicated below. 
______________________________________ 
Wgt. upon 
Fraction Description Evaporation 
______________________________________ 
1-20 1st component, mixture*, 
6.74 g. 
inactive 
21-44 2nd component, mixture, 
4.18 g. 
inactive** 
45-53 trough - discarded 385 mg. 
54-70 3rd component, single 
1.45 g. 
compound-musettamycin, 
active 
71-75 trough - discarded 198 mg. 
76-110 4th component, mixture 
4.03 g. 
comprising mainly 
marcellomycin, active 
111-200 post cut 1.72 g. 
______________________________________ 
*as evidenced by thin layer chromatography 
**mainly .eta.-pyrromycinone 
EXAMPLE 9 
Partial purification of musettamycin 
The solid (421.4 mg.) obtained from the third component of Example 8 was 
dissolved in excess boiling chloroform and the solution filtered hot 
through fluted filter paper. The filtrate was boiled down to &lt;20 ml. on a 
steam bath. Skellysolve B was then added dropwise to the warm solution 
until the cloud point was reached followed by addition of several drops of 
chloroform. After being allowed to cool to ambient temperature, the 
mixture was placed in a freezer at -20.degree. C. overnight. The deep red 
crystalline platelets were collected and dried in vacuo to give 358 mg. 
musettamycin, m.p. 162.degree.-163.degree. C. 
EXAMPLE 10 
Purification of Musettamycin 
Crude ethylenediamine tetraacetic acid (EDTA) - washed musettamycin was 
passed over a four column bank of .mu.STYRAGEL (trademane for a gel 
filtrant column packing material used in gel permeation chromatography; 
the material consisting of small beads, 8-10 microns in diameter, composed 
of rigid, crosslinked polymers of styrene divinylbenzene; and manufactured 
by Waters Associates, Inc.) (500-100-100-100 A) with chloroform as a 
mobile phase at a flow rate of 0.7 ml./min. for an initial clean-up. Nine 
runs of 100 mg. each were made. The elution was monitored with the aid of 
a refractive index detector (Waters Associates) and in each run the main 
peak eluting at 37.5- 45 min. was collected. The main fraction from the 
nine runs was combined and evaporated to dryness in vacuo. The dark red 
amorphous solid (660 mg.) was dissolved in 22 ml. of a 45:55 solvent 
mixture of acetonitrile:0.01 M sodium acetate, pH 4.0. The mixture was 
centrifuged to remove a small amount of suspended material and the clear 
supernatant transferred to a sealable penicillin vial. A portion (1.25 
ml., .about. 37 mg.) of the dark red solution was loaded into a U6K 
injector (Waters Associates, Inc.) and the sample then loaded onto the 
column. The sample was chromatographed on a 1 meter .times. 4.6 mm. I.D. 
stainless steel column packed with phenyl/PORASIL B (tradename for a 
bonded phase material for partition chromatography manufactured by Waters 
Associates, Inc.; composed of a totally porous silica having liquid 
diphenyl dichlorosilane chemically bonded to the silica) (37-75 .mu. 
particle size). The mobile phase consisted of a 35:65 acetonitrile:0.01 M 
sodium acetate pH 4.0 mixture at a flow rate of 1.9-2.0 ml./min. The 
elution was monitored using a refractive index monitor. Fractions were 
collected at 75 sec. intervals with the aid of an automatic fraction 
collector (Scientific Manufacturing Industries). 120 tubes were collected 
and the column was washed at a flow rate of 3.0 ml./min. with methanol for 
15 min., water for 15 min. and acetonitrile:0.01 M sodium acetate pH 4 
mixture (35:65) for 15 min. The flow rate was reduced to 2.0 ml./min. and 
the system equilibrated for 5 min. prior to subsequent injection. A 25 
.mu.1 aliquot of the eluant in every fifth tube was chromatographed on an 
analytical system to check for composition prior to combination of the 
tubes. The analytical system was comprised of a 61 cm. .times. 2.1 mm. 
I.D. stainless steel column packed with phenyl/CORASIL (tradename for a 
bonded phase material for partition chromatography manufactured by Waters 
Associates, Inc.; consists of a solid glass bead which has been coated 
with a single layer of silica to which is bonded diphenyldichlorosilane) 
(37-50 .mu. particle size). The mobile phase was a 45:55 mixture of 
acetonitrile:0.01 M sodium acetate pH 4.0 at a flow rate of 0.7 ml./min. 
Eluant was monitored with a UV detector (LDC) at 254 nm. Tube combinations 
were made on the basis of the analytical chromatograms. Tubes 61-120 were 
found to contain the desired musettamycin. These tubes were combined and 
evaporated in vacuo (45.degree. C.). The dark red residues were flushed 
repeatedly with methanol and 2 .times. 100 ml. with methylene chloride. 
The residue was then triturated with 100 ml. chloroform and filtered to 
remove inorganic salts. The solid was then evaporated to dryness with dry 
N.sub.2 and dried under high vacuum over P.sub.2 O.sub.5 . 
Musettamycin as purified above is characterized by the following data: 
Description: dark red crystalline powder 
Molecular formula: C.sub.36 H.sub.45 O.sub.14 N 
Molecular weight: 715.76 
Elemental analysis: Theoretical: C, 60.41; H, 6.34; N, 1.95. Experimental: 
C, 60.27; H, 6.59; N, 1.99. Corrected for 0.83 H.sub.2 O and 0.83 residue: 
C, 60.27; H, 6.50; N, 1.99. 
Infrared spectrum: The infrared absorption spectrum of musettamycin (KBr 
pellet) shows major bands at the following wave lengths: 3480, 2970, 2930, 
2820, 2770, 1735, 1600, 1450, 1320, 1295, 1220, 1160, 1010 and 990 
cm.sup.-.sup.1. 
Ultraviolet spectrum: At a concentration of 0.013 g./liter in methanol 
musettamycin exhibits the following maxima and absorptivities: 233 m.mu., 
57.7; 256 m.mu., 33.2; 284 m.mu., 14.5; 466 m.mu., 14.3; 490 m.mu., 17.4; 
510 m.mu., 14.6; 524 m.mu., 12.9; and 570 m.mu., 3.3. 
Nuclear magnetic resonance spectra: 
a. 100 mHz pmr spectrum: At a concentration of 25 mg./0.5 ml. in 
CDCl.sub.3, the spectrum showed the following chemical shifts in ppm and 
pattern descriptions: 7.66, s; 7.32, s; 7.21, s (CDCl.sub.3); 5.50, m; 
5.24, m; 5.00, m; 4.48, quartet; 4.10, s; 3.68, s; 4.2 to 3.4, overlapping 
m's; 2.16, s; 2.5 to 1.3, overlapping m's and 1.3 to 0.9, overlapping 
doublets and triplets [s=singlet, m=multiplet]. 
b. 25 mHz cmr spectrum: At a concentration of 200 mg./ml. in CDCl.sub.3, 
the spectrum shows the following observed chemical shifts and intensities: 
(unnumbered peaks are due to solvent) 
______________________________________ 
Relative 
No. Intensity Freq. in Hz PPM 
______________________________________ 
1 34 4777.66 189.816 
2 34 4654.38 184.918 
3 60 4303.11 170.962 
4 57 4076.06 161.941 
5 39 3980.33 158.138 
6 45 3961.43 157.387 
7 57 3578.36 142.168 
8 44 3327.79 132.213 
9 52 3299.86 131.103 
10 32 3269.30 129.889 
11 36 3258.97 129.478 
12 32 3021.49 120.043 
13 42 2875.63 114.249 
14 42 2816.92 111.916 
15 37 2812.68 111.747 
16 30 2548.48 101.251 
17 42 2489.88 98.923 
72 1967.51 78.169 
78 1935.57 76.900 
72 1903.47 75.624 
18 27 1852.56 73.602 
19 87 1797.79 71.426 
20 36 1791.56 71.178 
21 34 1775.10 70.525 
22 33 1716.43 68.193 
23 31 1670.07 66.351 
24 30 1649.89 65.550 
25 29 1548.63 61.527 
26 33 1431.42 56.870 
27 64 1318.54 52.386 
28 69 1074.95 42.708 
29 18 847.88 33.686 
30 23 819.21 32.547 
31 29 805.76 32.013 
32 17 722.16 28.691 
33 42 449.34 17.852 
34 56 418.12 16.612 
35 60 165.37 6.570 
______________________________________ 
High pressure liquid chromatogram: The retention time for musettamycin was 
found to be 11 min. 20 sec. using Waters Associates, Inc. modular 
components operating at the following parameters: 61 cm. .times. 2.1 mm. 
phenyl/CORASIL support column; 45 to 55 ratio of acetonitrile to 0.01 M 
sodium acetate, pH 4; 0.7 ml./min. flow rate; 
254 m.mu. UV detector. 
Melting point: 210.degree.-212.degree. C. 
Solubility: Soluble in most organic solvents; will crystallize from 
methanol in higher concentrations at 20.degree. C.; insoluble in water, 
aliphatic hydrocarbons and ether. 
EXAMPLE 11 
Purification of marcellomycin 
Crude EDTA-washed marcellomycin was subjected to an initial clean-up using 
a four column bank of .mu. STYRAGEL (tradename for a gel filtrant column 
packing material used in gel permeation chromatography; the material 
consisting of small beads, 8-10 microns in diameter, composed of rigid, 
crosslinked polymers of styrene divinylbenzene; manufactured by Waters 
Associates, Inc.) (500-100-100-100 A) with chloroform as a mobile phase. 
The flow rate for this separation was 0.6-0.7 ml./min. and the elution was 
monitored with a refractive index detector (Waters Associates, Inc.). Ten 
runs of 100 mg. each were made and in each case the main peak eluting at 
26-32 min. was collected. The main fraction from the 10 runs was combined 
and evaporated to dryness in vacuo. The dark red amorphous solid was 
divided into twenty-five, 30-35 mg. fractions. Each was dissolved in 1.0 
ml. of a 45:55 acetonitrile: 0.01 M sodium acetate pH 4.0 solvent mixture 
immediately prior to loading into the chromatographic system consisting of 
a 1 meter .times. 4.6 mm. I.D. stainless steel column packed with 
phenyl/PORASIL B (tradename for a bonded phase material for partition 
chromatography manufactured by Waters Associates, Inc.; composed of a 
totally porous silica having liquid phase of diphenyl dichlorosilane 
chemically bonded to the silica) (37-75 .mu. particle size). The mobile 
phase was 45:55 acetonitrile:0.01 M sodium acetate pH 4.0 at a flow rate 
of 3.0 ml./min. The elution was monitored using a different refractive 
index monitor (Waters Associates, Inc.). Fractions were collected at 1.0 
min. intervals with the aid of an SMI (Scientific Manufacturing 
Industries) fraction collector. Fifty-nine tubes were collected and the 
column was washed at a flow rate of 4.0 ml./min. with acetonitrile and for 
15 min. with the mobile phase prior to a subsequent injection. The eluant 
contained in every fifth tube was chromatographed on an analytical system 
prior to combination of tubes. The analytical system was composed of a 61 
cm. .times. 2.1 mm. I.D. stainless steel column of phenyl/CORASIL 
(tradename for a bonded phase material for partition chromatography 
manufactured by Waters Associates, Inc.; consists of a solid glass bead 
which has been coated with a single layer of silica to which is chemically 
bonded diphenyldichlorosilane) (37-50 .mu. particle size). The mobile 
phase consisted of a 45:55 acetonitrile: 0.01 M sodium acetate pH 4.0 
solvent mixture with a flow rate of 0.68.degree.-0.7 ml./min. The eluant 
was monitored with a UV detector at 254 nm. Twenty-five .mu.l. of eluant 
was injected into this system. Tube combinations were made on the basis of 
the analytical chromatograms. In general, tubes 18-35 were found to 
contain exclusively the desired marcellomycin component. These tubes were 
combined and evaporated to dryness in vacuo at 45.degree. C. The residue 
was flashed repeatedly with methanol and 2 .times. 100 ml. with methylene 
chloride. The residue was triturated with 75 ml. of methylene chloride and 
filtered. The filtrate was evaporated in vacuo (45.degree. C.) to dryness. 
Dark red residue was then dried under high vacuum over P.sub.2 O.sub.5. 
Marcellomycin as purified above is characterized by the following data: 
Description: dark red amorphous powder 
Molecular formula: C.sub.42 H.sub.55 NO.sub.17 
Molecular weight: 845.90 
Elemental analysis: Theoretical: C, 59.64; H, 6.55; N, 1.65; O, 32.15. 
Experimental: C, 56.32; H, 6.64; N, 1.74. Corrected for H.sub.2 O and 
residue: C, 68.77; H, 6.77; N, 1.82. 
Infrared spectrum: The infrared absorption spectrum of marcellomycin (KBr 
pellet) shows major bands at the following wave lengths: 3450, 2960, 2940, 
2820, 2790, 1730, 1615, 1600, 1450, 1260, 1095, 1010 and 800 
cm.sup.-.sup.1. 
Ultraviolet spectrum: At a concentration of 0.013 g./l. in methanol, 
marcellomycin exhibits the following maxima and absorptivities: 233 m.mu., 
47.5; 256 m.mu. shoulder, 24.7; 284 m.mu. shoulder, 10.6; 490 m.mu., 15.8; 
410 m.mu. shoulder, 3.4; 465 m.mu. shoulder, 13.2; 480 m.mu. shoulder, 
14.7; 510 m.mu. shoulder, 12.5; 524 m.mu. shoulder, 10.6; and 580 m.mu. 
shoulder, 1.1. 
Nuclear magnetic resonance spectra: 
a. 100 mHz pmr spectrum: At a concentration of 25 mg./5 ml. in CD.sub.2 
Cl.sub.2, the spectrum showed the following chemical shifts in ppm and 
pattern descriptions: 7.63, s; 7.24, s; 5.52, m; 5.30, m; 5.33, m 
(CD.sub.2 Cl.sub.2); 4.94, m; 4.50, quartet; 4.30 to 3.90, overlapping 
m's; 3.69, s; 2.70 to 0.09, overlapping m's; and 2.19, s [s=singlet, 
m=multiplet]. 
b. 25 mHz cmr spectrum: At a concentration of 60 mg/2 ml. in CD.sub.2 
Cl.sub.2, the spectrum shows the following observed chemical shifts and 
intensities: (unnumbered peaks are due to solvent). 
______________________________________ 
Relative 
No. Intensity Freq. in Hz PPM 
______________________________________ 
1 38 3454.09 137.574 
2 31 3331.45 132.727 
3 56 2964.01 118.088 
4 37 2739.81 109.156 
5 49 2642.13 105.264 
6 37 2627.41 104.678 
7 52 2246.11 89.487 
8 30 1995.14 79.488 
9 46 1969.58 78.469 
10 38 1926.64 76.759 
11 35 1918.43 76.432 
12 37 1682.08 67.015 
13 26 1543.35 61.488 
14 32 1489.08 59.326 
15 29 1484.44 59.141 
16 45 1213.98 48.366 
17 51 1195.68 47.636 
18 55 1155.94 46.053 
19 50 768.18 30.605 
20 54 516.93 20.595 
21 91 455.17 18.134 
22 64 444.15 17.695 
23 43 433.12 17.256 
24 49 378.19 15.067 
25 54 348.77 13.895 
26 50 339.07 13.509 
27 57 305.61 12.176 
28 63 303.14 12.077 
29 49 206.56 8.229 
30 53 96.91 3.861 
67 54.60 2.175 
136 27.27 1.087 
175 0.01 0.000 
165 -27.03 -1.077 
67 -54.44 -2.169 
31 103 -264.55 -10.540 
32 40 -478.75 -19.074 
33 38 -491.67 -19.589 
34 50 -514.78 -20.509 
35 47 -533.08 -21.238 
36 32 -610.57 -24.326 
37 49 -899.27 -35.828 
38 67 -918.94 -36.611 
39 80 -926.83 -36.926 
40 79 -1202.93 -47.926 
______________________________________ 
High pressure liquid chromatogram: The retention time for marcellomycin was 
found to be 10 min. 24 sec. using Waters Associates, Inc. modular 
components operating at the following parameters: 61 cm. .times. 2.1 mm. 
phenyl/CORASIL support column; 45 to 55 ratio of acetonitrile to 0.01 M 
sodium acetate, pH 4; 0.68 to 7 ml./min. flow rate; 254 m.mu. UV detector. 
Melting Point: Softening at 134.degree.-135.degree. C.; gradual thickening, 
but does not melt up to 300.degree. C.