CA1241282A - Bbm-1675, a new antitumor antibiotic complex - Google Patents

Abstract

ABSTRACT

A novel antibiotic complex designated herein as BBM-1675 complex is produced by fermentation of certain novel strains of Actinomadura verrucosospora. The complex may be separated into two major components, BBM-1675 A1 and A2, and four minor components, BBM-1675 A3, A4, B1 and B2, and such components exhibit both antimicrobial activity and antitumor activity.

Description

I ~2~}Z

SUE

BACKGROUND OF THE INVENTION
, _ .
1. Field of the Invention This invention relates to new anti tumor antibiotic substances and to their production and recovery.

2. Description of the Prior Art The anti tumor antibiotic compounds of the present invention have not yet been identified in terms of structure. In view of their unique physical, chemical and biological properties, however, applicants believe that the BBM-1675 antibiotics are novel substances.

European Patent Publication No. Allah discloses fermentation of Actinomadura pulverizes spy nova No. 6049 (ATTICS
39100) to produce anti tumor antibiotics designated WE 6049-A and WE 6049-B. The structures of the WE 6049 antibiotics have not yet been elucidated, but the characterizing properties given for the antibiotics indicate that WE 6049-A and WE 6049-B may be related in structure to the BBM-1675 antibiotics of the present invention. Spectral data show, however, that neither WE AYE
nor WE 6049B is identical to any of applicants' BBM-1675 components. Moreover, the producing organism described in European Patent Application Publication No. Allah may be clearly differentiated from Actinomadura verrucosospora employed in the present invention in the color of its aerial Muslim on IS Medium Nos. 2, 3 and 4, in its positive milk peptonization and in its positive utilization of D-fructose, D-mannitol, trowels and cellulose.

SUMMARY OF THE INVENTION

There is provided by the present invention a new anti tumor antibiotic complex designated herein as BBM-1675, said ~241282 complex being produced by cultivating a BBM-1675-producing strain of Actinomadura verrucosospora, most preferably Actinomadura verrucosospora strain H964-92 (ATTICS 39334) or Actinomadura verrucosospora strain AYE (ATTICS 39638), or a mutant thereof, in an aqueous nutrient medium containing assimilable sources of carbon and nitrogen under submerged aerobic conditions until a substantial amount of said BBM-1675 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 major bioactive components of BBM-1675 complex designated as BBM-1675Al and A and four minor bioactive combo-newts of said complex designated BBM-1675A3, A, By and By. The components may be separated and purified by conventional chromatographic procedures. The BBM-1675 complex and its bioactive components exhibit both antimicrobial and anti tumor activity.

DESCRIPTION O_ THE DRAWINGS
IG. 1 shows the infrared absorption spectrum of partially purified BBM-1675 Al ~KBr pellet).
IG. 2 shows the infrared absorption spectrum of partially purified BBM-1675 A (KBr pellet).
IG. 3 shows the infrared absorption spectrum of BBM-1675 A
(KBr pellet).
IG. 4 shows the infrared absorption spectrum of BBM-1675 A
(KBr pellet).
IG. 5 shows the proton magnetic resonance spectrum of partially purified BBM-1675 Al in CDC13 (60 MHz).
IG. 6 shows the proton magnetic resonance spectrum of partially purified BBM-1675 A in CDC13 (60 MHz).
IG. 7 shows the proton magnetic resonance spectrum of lo 1282 BBM-1675 A in CDC13 (60 MHz).
IG. 8 shows the proton magnetic resonance spectrum of BBM-1675 A in CDC13 (60 MHz).
IG. 9 shows the infrared absorption spectrum of purified BBM-1675 Al (KBr pellet).
IG. 10 shows the proton magnetic resonance spectrum of purified BBM-1675 Al in CDC13 (360 MHz).
IG. 11 shows the 13C magnetic resonance spectrum of purified BBM-1675 Al in CDC13 (90.3 MHz).
IG. 12 shows the infrared absorption spectrum of purified BBM-1675 A (Cry pellet).
IG. 13 shows the proton magnetic resonance spectrum of purified BBM-1675 A in CDC13 (360 MHz).
IG. 14 shows the 13C magnetic resonance spectrum of purified BBM-1675 A in CDC13 (90.3 MHz).

DETAILED DESCRIPTION

This invention relates to a novel anti tumor antibiotic complex designated herein as BBM-1675 and to its preparation by fermentation of certain strains of Actinomadura verrucosospora, most particularly Actinomadura verrucosospora strain H964-92 and a mutant thereof designated Actinomadura verrucosospora strain AYE. The above-mentioned parent strain was isolated from a soil sample collected at Pro Esperanza, Mushiness, Argentina. A
biologically pure culture of the organism has been deposited with the American Type Culture Collection, Washington, DO and added to its permanent collection of microorganisms as ATTICS 39334.
Subsequently, the mutant strain AYE was obtained by convent tonal nitrosoguanidine (NAG) treatment ox strain H964-92 and was it 1 282 deposited with the American Type Culture Collection as ATTICS 39638.

As in the case of many antibiotic-producing cultures, fermentation of Actinomadura verrucosospora strain H964-92 or train AYE results in the production of a mixture or complex of component substances. Two major bioactive components, BBM-1675 Al and A, and four minor bioactive components, BBM-1675 A, A, By and By, have been separated from the BBM-1675 complex produced during the fermentation process.

BBM-1675 and its components BBM-1675 Al, A, A, A, B
and I exhibit antimicrobial activity against a broad spectrum of microorganisms including especially gram-positive bacteria. The BBM-1675 complex and separated bioactive components thereof also exhibit phase inducing properties in lysogenic bacteria. Two of the components, BBM-1675 Al and A, have been submitted to in viva screening against various mouse tumor systems and demonstrate inhibitory activity against L-1210 leukemia, P-388 leukemia, B16 melanoma and Lewis lung carcinoma. BBM-1675 A and A have been shown to exhibit activity against mouse P-388 leukemia. The complex and its bioactive components, therefore, may be used as antimicrobial agents or as anti tumor agents for inhibiting mammalian tumors The Microorganism The actinomycete Strain No. H964-92 was isolated from a soil sample and prepared by conventional procedures as a biologic gaily pure culture for characterization. Strain H964-92 forms on the aerial Muslim short spore-chains which show straight, phloxes or hooked shapes. The spores are spherical or oval-shaped and have a warty surface. Aerial Muslim is poorly formed on most media. The aerial mass color is white which later turns to a pinkish shade, or further changes to a bluish color in some ajar media. The color of substrate Muslim is colorless or pale pink. The growth temperature ranges from 15C to 43~C. The cell-wall amino acid composition and whole cell hydrolyzate sugar ~2412~32 components show that strain H964-92 belongs to cell wall Type IIIB. The menaquinone was identified as MCKEE) MCKEE).

Based on the major morphological, cultural and physic-logical characteristics along with the chemical cell-wall composition characteristics, strain H964-92 can be classified as belonging to the genus Actinomadura.

Although the original strain H964-92 gave only moderate growth and bore scant aerial Mazola, a variant showing good growth and improved aerial Muslim formation was obtained by NAG
(nitrosoguanidine) treatment of H964 92. The variant, designated strain AYE, facilitated further taxonomical investigation and was subsequently identified as Actinomadura verrucosospora.

Methods The media and procedures used for examining cultural characteristics and carbohydrate utilization were those recomb mended by the International Streptomyces Project (Into. J. Cyst.
Bacterial. 16: 313-340, 1966). Additional media described by SPA. Waksman (The Actinomycetes, Vol. 2) and G. M. Lvedemann (Into. J. Cyst. Bacterial. _: 240-247, 1971) were also used.
The cell wall-amino acid composition and whole cell hydrolyzate sugar components were analyzed according to the methods described by Becker, et at. in Apply Microbial. 13: 236-243, 1965 and by Lechevalier and Lechevalier in The Actinomycetes, Ed. H. Praiser, Jean, Gustav Fischer Verlag, pp. 393-405, 1970, respectively.
The menaquinone was identified by mass spectral analysis according to the procedures ox Collins et at. in J. con.
Microbial. 100: 221-230, 1977, and the menaquinone composition was represented based on the system described by Yamaha et at. in J. con. Apply Microbial. _ : 331~335, 1977.

Morphology Strain H96g-92 forms both substrate and aerial Mazola.
The substrate Muslim is long, branched and not fragmented into ~241Z82 short filaments. In the aerial Muslim, short spore-chains are formed monopodially or at the hyphal tip. Whorl-like branches of spore-chain are also observed nearby the hyphal tip. These spore-chains contain 2 to 10 spores in a chain and are straight, phloxes or hooked in shape. The spores have a warty surface and are spherical to elliptical (0.5-0.6 X 0.6-1.4 em) in shape with rounded or pointed ends. After maturation each spore is often separated with empty sheath. Motile spores, springy or sclerotic granules are not seen in any media examined.

Cultural and Physiological Characteristics Growth of strain H964-92 is poor to moderate in both chemically defined media and natural organic media. Formation of aerial Muslim is generally poor but is moderate in oat meal ajar WISP No. 3 medium), inorganic salts-starch ajar WISP No. 4 medium) and Bennett's ajar. Spontaneous variants which lack aerial Muslim occur at high frequency. The color of aerial Muslim is white which later turns to pale pink in oat meal ajar, inorganic salts-starch ajar and glycerol-asparagine ajar lisp No. 5 medium). The aerial mass color further changes to a bluish color after long incubation (5 months) in oat meal ajar, glycerol-asparagine ajar and Tarzan ajar. The color of sub-striate Muslim is colorless to yellowish in Czapek's ajar, Tarzan ajar, yeast extract-malt extract ajar WISP No. 2 medium), peptone-yeast extract-iron ajar WISP No. 6 medium) and Bennett's ajar, and is a pinkish color in glucose-asparagine ajar and glycerol-asparagine ajar. Melinda and other diffusible pigments are not produced. A variant No. AYE, which was obtained from the original strain, forms predominantly pale blue aerial Muslim and bears abundant aerial spore mass.

Strain H964-92 grows at 15C, 28C, 37C and 43C, but not at Luke or at 47C. It is sensitive to Nail at 7%, and resistant to lysozyme at 0.01%.

24~282 The cultural and physiological characteristics of the producing strain are shown in Tables 1 and 2, respectively. The utilization of carbon sources is shown in Table 3.
Table 1 Cultural Characteristics Ox Strain H964-92 (original strain ATTICS 39334 and variant AYE

strain No. H964-92 Actinomadu~a Original Strain verrucosospora (AXE 39334) Variant No. AYE gCC A-0147 rottenest extract G: abundant, phloxes, moderate, eloccose, moderate, iloccose, ajar WISP No. I sediment Ed and not sediment Ed and not sediment Ed and not pigmented pigmented pigmented Sucrose-nitrate ajar G: moderate poor poor ICzapek's ajar) R: colorless colorless colorless A: scant: light gray no or scant pinkish no or scant; pale (264), to pale pink white (9) blue (18S) (7) D: none none none Glucose-asparagine ajar G: moderate poor poor R: white (263) to deep colorless colorless yellowish pink (27) A: no or very scant: no or very scant; no or very scant;
pale pink (7) white white D: none none none Glycerol-asparagine ajar G: poor to mediate moderate moderate WISP No. S) R: colorless to light light yellowish light yellowish pink yellowish pink tea) pink ~28) (28) to deep yellow-is pink (27) A: poor; light yellow- moderate white to moderate; white to is pink (pa), await light pink (4) strong pink (2) S months light bluish gray (190) D: none none none Inorganic salts-starch G: abundant moderate moderate ajar WISP No. 4) R: yellowish white light yellowish pink light yellowish pink (92) (28) (28) A: abundant; light pink moderate; light bluish abundant; pale blue (4) to pinkish gray gray (190) (18S) (10) D; none none none ~yrosine ago G: moderate moderate moderate WISP No. 7) R: yellowish White t92) strong yellowish pink strong yellowish (26) pink (26) A: poor; light yellow- moderate; white to moderate: White to is pink (28), very light pink (4) light pink (4) later (S months) partially light bluish gray (190) D: none none none ~2~Z8Z
g Table 1 - count ' d Nutrient ajar G: poor to moderate poor poor R: pale yellow I colorless to pal colorless to pale pink I pink (7) A: none none none D: none none none Yeast extract-malt G: abundant abundant abundant extract ajar R: pale yellow ~89) strong yellowish strong yellowish WISP No. 2) pink (26) pink ~26) A: poor: white (263) poor; white to pale poor: white to pale pink I pink I
D: none none none Oat meal ajar G: moderate poor poor WISP No. 3) R: colorless to pale pale yellowish pale yellowish pink I pink ~31) pink ~31) A: poor; pinkish very scant; vivid very scant; vivid white to) to light pale blue ~184) pale blue bluish gray ~190) D: none none none Bennett's ajar G: abundant abundant abundant R: grayish yellow ~90~ strong yellowish strong yellowish pink ~25~ pink ~26) A: oddity; white ~263) moderate: pale yellow- none to yellowish white is pink (31) and ~92) bluish white ~189) D: none none none Peptone-yeast extract- G: moderate abundant abundant iron ajar WISP NO. 6) R: colorless colorless colorless A: none none none D: none none none Observed after incubation at 37-C for 3 weeks.
Abbreviation: G - Growth; R - Reverse color; A Aerial Muslim; D - Diffusible pigment Jo Color and number in parenthesis follow the color standard in jelly Lo Do Judd;
ISCC-N95 color-name charts illustrated with Sauntered Colors. DO Dept. of Comma Cur. 553, Washington, DO Nov., 1975'.

124:1Z82 Table 2 Physiological Characteristics of Strain H964-92 Test Response Method and Medium Range of temperature Maximal growth at Bennett's ajar for growth 28C to 37C.
Moderate at 20C
and 43C. No growth at 10C and 47C.

Gelatin liquefaction Liquefied Glucose-peptone-gelatin medium Starch hydrolysis Hydrolyzed Starch ajar plate Reactions in skimmed Not coagulated Disco skimmed milk milk and completely peptonized formation of Melinda Not produced Tarzan ajar, peptone-yeast-pigment iron ajar and tryptone-yeast extract broth.

Nitrate reduction Not reduced Czapek's glucose-nitrate broth and glucose-yeast extract-nitrate broth Resistance to Nail Growth at I or Tryptone-yeast extract ajar less. No growth at 7%.

Lysozyme Resistant. Growth Tryptone-yeast extract ajar at 0.01% or less.
No growth at 0.1%.

pi Growth in 5.0 to Tryptone-yeast extract ajar 9.5. No growth at 4.5 and 10Ø

~2~2~32 Table 3 Utilization of Carbon Sources Strain No H964-92 Actinomadura Original Variantverrucosospora strain No. A_327YKCC A-0147 Glycerol + + +
D~-~-Arabinose - - -L(+)-Arabinose + + +
D-Xylose + + +
Drubs +
L-Rhamnose + + +
D-Glucose + + +
D-Galactose - - -D-Fructose + + +
D-Mannose - - -L(-)-Sorbose Sucrose + + +
Lactose Syllabus + + +
Melibiose Trowels + + +
Ruffians D(+)-Melezitose Soluble starch + + +
Cellulose + + +
Dulcitol Instill +
D-Mannitol + + +
D-Sorbitol Salicin Observed after incubation at 28C for 3 weeks Basal medium: Pridham-Gottlieb inorganic medium :~Z41Z82 Cell-Wall Composition and Whole Cell Sugar Components Purified cell-wall of strain H964-92 contains Mazda-aminopimelic acid but lacks Gleason. The whole cell hydrolyzate shows the presence of Madras (3-O-methyl-D-galactose), glucose and rubs. The cell-wall amino acid and whole cell sugar components indicate that strain H964-92 is placed in cell-wall Type IIIB. Two major components of menaquinone were identified as MCKEE) and MCKEE).

Taxonomic Position of Strain H964-92 Strain H964-92 has the following major characteristics:
(1) Aerial spore-chains: short, straight, phloxes or hooked in shape. I Spores: warty surface. (3) Aerial Muslim: pinkish or bluish color. (4) Substrate Muslim: pinkish in some media.
(5) Diffusible pigment: none. (6) Misfile. (7) Cell-wall Type IIIB. (8) Menaquinone system: MCKEE) and MCKEE).

These major characteristics indicate that strain H964-92 is placed in the genus Actinomadura. Early species of the genus Actinomadura were isolated from mammals. Some strains were also obtained from plant materials. However, many of the new species proposed recently were isolated from soil. According to the numerical taxonomy and review of the Actinomadura and related actinomycetes by Gadfly et at. in J. con. Microbial.
112: 95-111 (1979), most Actinomadura species of soil origin are classified into Cluster No. 7 among the 14 clusters described.
Strain No. H964-92 is most related to the species of Cluster 7.
Nonomura and O'er in J. Ferment. Tuitional. OWE 904-912 (1971) reported five saprophytic species of the genus Actinomadura and Nonomura [J. Ferment. Tuitional. 52: 71-77 (1974)] and Preobrazhenskaya et at. [Actinomycetes and Related Organisms 12:
30-38 (1977)] published the keys for identification and classic ligation of the Actinomadura species. As a result of comparison with the descriptions of 30 species including organisms disclosed in patents, strain H964-92 appears most similar to Actinomadura Charlie described in the Preobrazhenskaya et at. reference above ~;~4~82 and to Actinomadura verrucosospora described in the Nonomura references cited above.

Strain No. H964-92 was directly compared with A.
verrucososeora strain KCC A-0147 and was found to be closely related to A. verrucosospora in the morphological, cultural and physiological characteristics. Thus, strain H964-92 is classified as a new strain of Actinomadura verrucosospora.

It is to be understood that for the production of B3M-1675, the present invention, though described in detail with reference to the particular strain Actinomadura verrucosospora strain H964-92 (ATTICS 39334) and the mutant strain thereof designated strain AYE (ATTICS 39638), is not limited to these microorganisms or to microorganisms fully described by the cultural characteristics disclosed herein. It is specifically intended that the invention embrace strain H964-92 and all natural and artificial BBM-1675-producing variants and mutants thereof.

Antibiotic Production The BBM-1675 antibiotics of the present invention may be prepared by cultivating a BBM-1675-producing strain of Actinomadura verrucosospora, preferably a strain of Actinomadura verrucosospora having the identifying characteristics of ATTICS
39334 or ATTICS 39638, or a mutant thereof, in a conventional aqueous nutrient medium. The organism is grown in a nutrient medium containing known nutritional sources for actinomycetes, i.e. assimilable sources of carbon and nitrogen plus optional inorganic salts and other known growth factors. Submerged aerobic conditions are preferably employed for the production of large quantities of antibiotic, although for production of limited amounts, surface cultures and bottles may also be used.
The general procedures used for the cultivation of other action-Maoists are applicable to the present invention.

12~12~32 The nutrient medium should contain an appropriate assimilable carbon source such as glycerol, L(~)-arabinose, D-xylose, Drubs, L-rhamnose, D-glucose, D-fructose, sucrose, syllabus, soluble starch, D-mannitol or instill. As nitrogen sources, ammonium chloride, ammonium sulfate, urea, ammonium nitrate, sodium nitrate, etc. may be used either alone or in combination with organic nitrogen sources such as petunia, meat extract, yeast extract, corn steep liquor, soybean powder, cotton seed flour, etc. There may also be added, if necessary, nutrient inorganic salts to provide sources of sodium, potassium, calcium, ammonium, phosphate, sulfate, chloride, bromide, carbonate, zinc, magnesium, manganese, cobalt, iron, and the like.

Production of the BBM-1675 antibiotics can be effected at any temperature conducive to satisfactory growth of the producing organism, e.g. 15-45C, and is conveniently carried out at a temperature of around 27-32C. Ordinarily, optimum product lion is obtained after incubation periods of from about 68-180 hours, depending on whether shake-flask, stir-jar or tank ferment station is employed. When tank fermentation is to be carried out, it is desirable to produce a vegetative inoculum in a nutrient broth by inoculating the broth culture with a slant or soil culture or a lyophilized culture of the producing organism After obtaining an active inoculum in this manner, it is trays-furred aseptically to the fermentation tank medium. Antibiotic production may be monitored by the paper disc-agar diffusion assay using Staphylococcus Ayers 209P as the test organism.

Isolation and Purification When fermentation is complete, the BBM-1675 complex may be obtained from the broth by conventional isolation procedures, e.g. solvent extraction. Thus, for example, the whole broth may be separated by filtration or centrifugation into mycelial cake and broth supernatant. Antibiotic in the mycelial cake may be recovered by suspending the cake in methanol, filtering off insoluble materials and concentrating the methanolic extract.
Activity in the broth supernatant may be recovered by extraction ` 12412~32, with n-butanol. The above-mentioned n-butanol and methanol extracts may then be combined and evaporated azeotropically to an aqueous solution which deposits most of the antibiotic activity as an oily solid. The solid may then be dissolved in methanol and the solution filtered. Filtrate is concentrated and added to a mixture of ethyl acetate and water. The resulting organic extract contains the crude BBM-1675 complex which may be precip-stated from solution by addition of an anti solvent such as Nixon.

The crude BBM-1675 complex is a mixture of several components including two major bioactive components, BBM-1675 A
and A, and four minor bioactive components, BBM-1675 A, A, By and By. These bioactive components may be separated and purified by conventional chromatographic procedures. In one procedure the crude BBM-1675 complex is first dissolved in methanol and purified by Sephadex L~-20 column chromatography using methanol as the eluding solvent. This partially purified complex may then be chromatographed on a silica gel column and eluded in a stops manner using chloroform plus an increasing concentration of methanol to provide BBM-1675 Al, a mixture of BBM-1675 A, A
and A and a mixture of BBM-1675 By and By. The Al component may be further purified by Sephadex*LH-20 column chromatography using methanol as the eluding solvent. The mixture of A, A and A
may be separated by chromatography on a column of Bondapak*C18 (Waters Associates, Inc.) using increasing concentrations of aqueous acetonitrile as the eluant. The mixture of By and By components may be separated by silica gel column chromatography using a mixture of chloroform and methanol as the eluding solvent. Further details of the preferred chromatographic separation procedures are provided in the examples which follow.

Physico-chemical Properties of 8BM-1675 Components The six bioactive components of BBM-1675 complex are distinguishable from each other by two TLC systems as shown in the following Table.
* trade marks.

I."- Jo 1~41Z~32 Table 4 TLC of BBM-1675 Components Of Values Sue * Silenced Component CHC13-CH30H(5:1 v/v) CHICANO v/v) BBM-1675 Al 0. 74 0.18 BBM-1675 A 0.71 0. 21 BBM-1675 A 0.72 0.28 BBM-1675 A 0.71 0.78 BBM-1675 By 0.63 0.23 BBM-1675 By 0.60 0.16 * C18 reverse phase silica gel Separation of BBM-1675 A, A and A was difficult by ordinary phase TLC systems but could be achieved by a reverse phase TLC.

The individual BBM-1675 components show volubility and color reactions similar to each other. For example, they are soluble in chloroform, ethyl acetate, acetone, ethanol and methanol, slightly soluble in Bunsen and water, and insoluble in Nixon and carbon tetrachloride. They give positive reactions with ferris chloride, Ehrlich and Talons reagents but negative responses in Sakaguchi, ninhydrin and enthrone tests.

Characteristic physico-chemical properties of BBM-1675 components are shown in the Table 5 below.

Table S
Phy~icc~-Chemical Properties of BBM~1675 Components ~D~1-167~ Al A A A I 32 .`~eltinq point (doe) :156 _ 158~C147 _ 149 C 125 _ 127~C 123 _ 126~C lS9 _ 161DC 156 _ 159C
ED I 0.5 ICKY : -191 -179.~- -161 -176 -171- -122-Anti. Pound (e) C: 51.52 53.81 55.00 53.67 H: 5.81 6.31 6.52 6.35 N: 4.02 3.82 3.57 3.45 (by difference 0: 38.65 36.06 34.91 36.53 W ox Noah inch : 253 (325) 253 (281) 253 (286) 253 (25~) 253 ~225) 248 (212) 282 ~195) 282 (172~ 282 (158) 282 (153) 282 (140) 279 (141)320 (143) 320 (128~ 320 (122) 320 (117) 320 (104) 318 (103) inane liCl-C~30N: 253 (323) 253 (276) 253 (287) 253 (258) 253 (225) 248 (210) 282 (192) 282 (167) 282 (160) 282 (loss) 282 (140) 2~9 (140) 320 (144) 320 (128) 320 (126) 320 (118) 320 (105) 318 (103) in 0.01N NdOi~-CH30~1 : 252 (325) 252 (289) 252 (280) 252 (266) 252 (236) 248 (233) 283 tl-t2) 283 (171) 283 (162) 283 (160) 282 (141) 278 (150) 318 (136) 318 (122) 318 (120) 318 (118) 318 (105) 318 (110) Sol. wt.
(app~o:dmate value) : 1 300 1 100 1 100 1 400 (Gel HPLC, Fink GEL-101) The US absorption maxima of BBM-1675 components were observed at 253, 282 and 320 no, which did not shift in acidic or alkaline solution. The IT and PER spectra of BBM-1675 Al, A, A
and A are shown in Fugues 1-4 and Fugues 5-8 respectively. The 360 MHz PER of BBM-1675 Al indicated one acutely (~:2.11 Pam), one N-CH3(2.52 Pam), four OUCH, 3.80, 3.88 and 3.98 Pam) and one exomethylene (4.57 and 5.48 Pam) groups, along with two aromatic (7.50 and 8.59 Pam) and one NH (11.79 Pam protons. The CUR
spectrum of BBM-1675Al exhibited 55 carbon signals including a triple intensity signal (~:56.0 Pam, OUCH). The molecular formula of BBM-1675Al is deduced to be C57H72N4O32 based on proton and C NOR spectra, microanalysis and molecular weight determination by HPLC and SIMS (secondary ion mass spectrometer).

~Z41Z82 Structural Study of_BBM-1675 Al Upon treatment with OZONE HCl-CH30H at room temperature, BBM-1675Al loses its bioactivity and affords a lipophilic chrome-phone substance (compound I) along with several unidentified fragments. Compound I shows US absorption similar to that of parent antibiotic suggesting that compound I retains the chrome-phonic structure of BBM-1675Al. Two other chromcphoric fragments related to compound I are obtained by alkaline hydrolysis of BBM-1675Al: hydrolysis with 0.05N COUCH at 55C for one hour yields compound II having US absorption maxima at 252, 284, 297 (shoulder) and 322 no, while the reaction in lo COUCH affords an acidic chromophore substance designated compound III.
Physico-chemical properties of compounds I, II and III are summarized in Table 6 below.

Tale 6 Prooertie- of Compounds I, II and III

Compound I Compound II Compound III

Mop. : 82 _ 83 C 133 2~3 _ 255 C

ED (c OOZE ~HC13) : ^100~ o O

Molecular formula : C2l~3lNolo 14~17 6 C13~15N6 W ~CN3ON no (~) : 244 (21,850) 25i (26,600) 248 (26,900) Sax 276 (9,400) 283 (11,200) 295 (14,400) 318 (6,300) Shea) 310 (13,500) . 322 (11,700) MS m/z : 457 to ) 295 (M ) 281 (M ) Sal 251 222 LO

tXylene-~MEX-CC3ON-5:5:1 TV : Of 0.58 0.66 0.13 MIX methyl ethyl kitten 12~12~32 Structural information about compounds II and III was provided from the following spectral data and chemical transform motion. The 13C and proton NOR indicated the presence of four OUCH, one SHEA, seven -C=, two KIWI and one NO groups in compound II. The NOR spectra of compound III was similar to those of II, differing only in the absence of one of the four OUCH groups observed for compound II. This difference, together with the acidic nature of compound III, suggested that compound II is a methyl ester of compound III. Wren heated under reflex with lo methanolic KOCH, compound II was quantitatively converted to compound III, while compound III was converted to compound II
by treatment with diazomethane. Treatment of compound II with Nub in KIWI gave a reduction product (compound IV, M my 267) which showed a SHEA group in the NOR in place of the -COUCH group of compound II. Upon hydrogenation over palladium on charcoal, compound II afforded a dihydroderivative (compound V, M my 297). The proton NOR spectrum of compound V exhibited a new doublet methyl signal and the absence of the exomethylene group present in compound II. Furthermore, one of the OUCH
groups appeared at higher field (I: 3.50 Pam) in compound V.
These results indicated the presence of a -C=CH2 ooze group in compound II which was reduced by hydrogenation to OUCH

group in compound V. Compound II was heated with 1. SUN methanolic hydrogen chloride at 80C for 3 hours and the hydrolyzate chrome-to graphed on a silica gel column to afford a weakly basic compound (compound VI, M: m/z 211). The IT spectrum and physico-chemical properties indicated that compound VI contained an NH2 group. Compound VI was identified as methyl 4,5-dimethoxy-anthranilate by comparative IT and NOR studies with an authentic sample. Consequently, the structures of compounds II-VI were determined as shown below.

Structures of compounds II, III, IV, V and VI

Compound II Compound III

SHEA SHEA KOCH

SHEA COUCH SHEA OH

Ahab \ Com~ound_IV

Pd/C C 3 H CO I
H Moe \ C~30 SHEA

\ / Compound V
Cc,m~ound VI SHEA

SHEA COOK SHEA COOK

1241~82 Upon treatment with 0. 05N COUCH at 55C, compound I
was split into a new chromophoric fragment (compound VII, C15H21N07, M: m/z 327) and a sugar (compound VIII). The NOR
spectrum of VII exhibited one singlet C-CH3 and two high-field OUCH groups in addition to three low-field OUCH and two aromatic protons commonly observed for compounds II, V and VI. further hydrolysis of VII with 1. ON methanolic hydrogen chloride gave compound VI which was identical with that previously obtained from II. After removal of VI, the hydrolyzate was treated with 2, 4-dinitrophenylhydrazine to precipitate yellow solid which was identified as 2,4-dinitrophenylhydrazone of pyruvic acid. Thus, compound VII is methyl 4,5-dimethoxy-N-(2',2'-dimethoxypropionyl)-anthranilate. Compound VIII did not show the molecular ion peak but did show the MESSIAH peak at m/z 131 in the mass spectrum in agreement with the molecular formula of C7H1404. The NOR
spectrum indicated 2,6-dideoxyhexopyranose structure for compound VIII. The assignment was supported by the 13C-NMR of compound I
which gave rise to one C-CH3, one -SHEA, three OUCH< and one anomeric carbon in addition to 15 carbon signals assignable to compound VII. The C3 proton of the sugar appeared at low-field ( I: 5.33 Pam, octet) revealing that KIWI of the sugar was esterified by the carboxyl group of VII. The above results are summarized below:

~24i2~3~

Structures of Communed I, VII and VIII

Compound I ompo~nd VII Compound VI
OOZE _ _ C~3~ 1 3 SCHICK C~33 C 3 No 01~ OH 3 OUCH 3 C~3 0 COOK 3 SHEA Compound VIII I

3 CUB 3~0H
L I 3 -CH3-CO-COO~

The molecular weight of compound I (457) accounts for about one-third of the entire molecule of BBM~1675Al (proposed formula C57H72N4032; caulked MY = 1324). The partial structure of BBM-1675 Al is considered to be as follows:

NOAH - CO- I C~2 OUCH
SHEA OWE

o JO

12~128Z

Subsequently, it was discovered that components BBM-1675 Al and A described above and produced according to Example 2 below were in fact not completely pure and that certain of the characterizing properties used to define such components were inaccurate. Following additional chromatographic purification procedures as described more fully in Examples 3 and 6 below, 8BM-i675 Al and A were isolated in more purified form and fully characterized as described below. Also, the elemental analysis data for components A and A was revised to show the presence of sulfur in these compounds and HPLC retention times were calculated for these two components. Summarized below are the revised physico-chemical properties of the BBM-1675 components.

Description: white to pale yellow crystals;
my 156-158 (doe.

Elemental analysis:

Analysis 1 Annuluses Average C: Sly% C: 52.74% C. 52.17 H: 6.31% H: 5.99 H: 6.15 N: 5.31% N: 3.94% N: 4.63~
S: 8.47% S: 9.71% S: 9.09%
O try difference% O (by difference% O (by difference%

Ultraviolet absorption spectrum: Instrument-Varian US, Cry 219 Solvent - methanol Concentration - 0.01356 g/l maxim absorptivities 320 12.4 280 so (shoulder) 253 25.1 210 25.5 ~L2~1282 No significant change with acid or base Optical rotation: Solvent - SCHICK
[ ]24 = -207 (C=0.0351) A second analysis showed the following optical rotation:

[ ED = -191 (C=005, SCHICK).

Infrared absorption spectrum: See FIG. 9 Major absorption bands (KBr):c 985, 1015, 1970, 1110, 1150, 1210, 1250, 1308, 1380, 1405, 1446, 1520, 1592, 1608, 1668, 1715, 2920, 2960, 3360, 3440, cam Mass spectra: Instrument - VG-ZAB-2F
FAB-MS-thioglycerol Molecular mass range ions (m/z): 1249, 1357, 1463; with the addition of Nail (m/z): 1271, 1379, 1485, 1597.
FAB-MS-MB (MB:matrix, mow. 154); Molecular mass range ions (m/z): 1249, 1283, 1403, 1555; with the addition of Nail (m/z): 1249, 1271, 1303, 1425, 1483, 1577.
FAB-MS-glycerol-DMSO: Molecular mass range ions (m/z): 1215, 1247, 1279, 1293, 1325, 1353; with addition of Nail (m/z): 1215, 1237, 1247, 1269, 1325, 1347, 1375.
Instrument: Crypts MS-50 FAB-MS-thioglycerol; Molecular mass range ions (m/z): 1357, 1463.

Molecular weight (based on above-described apparent MY = 1248 mass spectral data):

~241Z~;~

Nuclear Magnetic Resonance Spectra: Instrument - WOMB Bucker Solvent: CDC13 NOR: 360 MHz Pam 11.75 (lo, s); 8.55 (lo, s); 7.45 lo s); 6.61 (lo, m); 6.23 (lo, bus); 6.17 I bus); 5.93 (lo, d, J=9.3); 5.82 (lo, d, J=9.3); 5.7 (lo, bus);
5.49 (lo, m); 5.45 I d, J=2.3); 5.38 (lo, bus); 4.95 (lo, d, J=10.2); 4.64 (OH, m);

4.54 (lo, d, J=2.3); 4.2 (lo, s); 4.15-3.35 !26-28H) [4.10 (lo, m); 4.02 (lo, bus); 3.95 (OH, s); 3.85 (OH, s), 3.79 (OH, s); 3.46 (lo, m); 3.40 (OH, s)]; 2.82-2.70 (OH, bum) 2.50 (OH, s), 2.47 (lo, m); 2.38-2.22 OH
2.12 lo m); 2.11 (OH, s); 1.60-1.05 (22H) [1.39 (OH, d, J=6.3); 6.31 (OH, d, J=6.3);
1.29 (OH, d, J=6.3), 1.08 (OH)]

See FIG. 10 C NOR: 90.3 MHz See FIG. 11 In a separate test the 13C NOR spectrum of purified BBM-1675 Al was determined for a sample dissolved in CDC13 (80 MHz). Major peaks are indicated below.

CUR of BBM-1675A (80 MHz in CDCL ) Chemical shift in Pam (Multiplicity*) BBM-1675A1 13.7(~) 16.6(q) 17.5(q) 19.8(q) 22.2(q) 22.6(q) 23.4(q) 29.0(t) 34.0(t) 35.1(t) 39.5(t) 47.2(d) 52.5(q) 55.6(u) 56.0(q) 57.1(d) 62.4(t) 64.5(d) 67.7(d) 68.2(d) 68.8(t) 69.2(d) 69.6(t) 70.2(d) 71.9(d) 76.0(d) 76.6(d) 77.1(u) 77.3(d~ 83.4(s) 86.6(d) 88.4(s) 90.5(t) 97.2(d) 98.3(s) 99.0(d) ~2~12~

99.6(d) 103.8(d) 107.6(s) 112.5(d) 123.1(d) 124.9(d) 130.1(d) 131.5(s) 134.9(s) 136.7(s) 144.0(s) 147.2(s) 153.8(s) 154.4(s) 155.0(sJ 160.7(s) 166.4(s) 191.8(s) multiplicity - q = quartet; d = doublet; u = uncertain t = triplet; s = singlet Description: white crystals; my 147-149C

Elemental analysis: C: 52.71%
H: 5.94%
N: 3 D 9 4%
S: 9.39%
Oboe difference): 28.01%

Ultraviolet absorption spectra: Instrument - Variant US, Cry 219 Solvent: methanol Concentration: 0.02052 g/l Max (no) absorptivities 320 12.2 282 16.3 252 26.2 214 25.8 No significant change with acid or base.

Optical rotation: [and = -179.4 (c 0.5, SCHICK) Infrared spectra: See FIG. 12 Instrument: Beckman IT Model 4240 ~l2~1Z8;~

Major absorption bands (KBr~: 950, 1015, 1070, 1100, 1155, 1213, 1250, 1313, 1375, 1405, 1~50, 1520, 15~5, 1610, 1685, 1735, 2940, 2980, 3440, Cal.

Mass Spectra: Instrument: VG-ZAB-2F
FAB-MS-thioglycerol Molecular mass range ions (m/z): 968, 1249, 1355, 1357, 1463, 1569; with the addition of Nail my 990, 1271, 1379, 1485, 1593 FAB-MS-M~ (MY: matrix, mow. 154); Molecular mass range ions (m/z): 1249, 1403, 1419, 1555, 1571, 1587; with the addition of Nail (m/z): 1249, 1271, 1425, 1441, 1457, 1483, 1577.
FAB-MS-glycerol-DMSO; Molecular mass range ions (m/z): 1215, 1231, 1247, 1263, 1279, 1293, 1309, 1325, 1326, 1341, 1353, 1363.

Molecular weight : apparent MY = 1248 (based on above-described mass spectral data) Nuclear Magnetic : See Fig 13 Resonance Spectra Instrument: WE 3S0 Bucker Solvent : CDC13 H NOR 360 MHz Pam 11.91 (lo, s); 8.62 (lo, s); 7.58 (lo, s); 6.56 (lo, m); 6.22 (lo, s); 6.15 (lo, bus); 5.91 (lo, d, J=9.6);

5.83 (lo, d, J=9.6); 5.70 (lo, m); 5.45 (lo, d, J=2.2); 5.44 (lo, s), 5.34 (lo, bus); 4.95 (lo, d, J=10.2); 4.75 (lo, m); 4.65 (lo, d, J=6.8); 4.54 (lo, d, J=2.2); 4.47 (lo, m);
4.18 (lo, s); 4.10 (lo, bus), 4.05-3.50 (20-24H); [3.96 (OH, s); 3;87 (OH, s); 3.77 (OH, s)]; 3.46 (lo, m); 3.39 (OH, s); 2.79 (lo, m); 2073 (OH, m); 2.50 OH s); 2.50 ~Z4~ZE32 I
(lo, m); 2.38-2.22 (OH); 2.14 (lo, m); 2.10 (OH, s); 1.98 (OH, m); 1.65-1.45 (6-8H); 1.38 (OH, d, J=6.0); 1.34 (OR, d, Joy); 1.22 (OH, d, J=6.8); 1.10 (OH).
13C NOR 90.3 MHz See FIG. 14 In a separate test the 13C NOR spectrum of purified BBM-1675A2 was determined for a sample dissolved in CDC13 (80 MHz). Major peaks are indicated below.

BBM-1675A2 13.7 16.9 17.5 19.8 22.3 22.7 23.4 33.1 34.1 35.1 39.3 47.6 52.6 55.7 56.0 56.1 57.6 62.4 64.5 64.9 65.9 68~3 69.2 69.7 71.9 73.6 75.8 76.1 77.1 77.7 78.1 78.3 83.3 86.2 88.4 90.4 97.2 98.3 99.1 99.5 99.6 103.8 107.1 112.4 123.2 124.B 129.9 137.3 144.1 154.2 154.5 160.9 167.9 192.2 Tale 7 Phy~ico-chemical Properties of 8BM-1675 A, A, By, By A A By By netting point (doe) : 125 - 127C123 - 126-C159 - 161C 156 - 159-C
lid (c 0.5, SCHICK): -161C -176C -171C -122-C
Anal. Found (~) : C: 54.55 54.65 ~:6.46 6.29 N:3.73 3.51 5:7.49 8.07 W McCoy no (Elm ) : 253 (286) 253 ~257) 253 (225) 248 (212~
in neon 282 (158) 282 (153)282 (140)279 (141) 320 (122) 320 (117)320 (104)318 (103) in 0.01N HCl-MeOH : 253 (287) 253 (258) 253 (225) 248 (210) 282 (160) 282 (155)282 (140)2~9 (140) 320 (126) 320 (118)320 (105)318 (103) in 0.01N Nemo : 252 (280) 252 (266) 252 (236) 248 (233) 283 (162) 283 (160)283 (141)278 (150) 318 (120) 318 (118)318 (EYE (110) 12~ 2 Thin-layer Chromatography (TLC) and High Performance Liquid Chromatography (HPLC) Data on BBM-1675 Components -A. Study No. 1 - Summary Table 8 TLC and HPLC of BBM-1675 components TLC (Of) HPLC (retention time, in minutes) Sue *Silenced Lichrosorb RP-18 CHC13-MeOH CHICANO CH3CN-MeOH-0.lM CHICANO
(5:1 TV (7~:25 V/v), (5:2:3 v/v) BBM-1675A1 0.74 0.18 13.3 BBM-1675A2 0.71 0.21 17.3 BBM-1675A3 0.72 0.28 8.0 BBM-1675A4 0. 71 0. 78 5.1 BBM-1675B1 0.63 0.23 BBM-1675B2 0.60 0.16 * C18 reverse phase silica gel B. Study No. 2 - TLC and HPLC for purified Al and A components TLC Chromatography Anal tech GULF Silica Gel Uniplates were used for all normal phase chromatography. Plates measuring 2.5 cm x 10 cm were used for one-dimensional TLC. These were developed in glass cylinders measuring 6.4 cm (old.) by 12 cm and containing 10 ml 1241~2~32 of eluant. en measuring 7.5 cm x 10 cm were used for two dimensional TLC. The sample was applied to the lower left hand corner 1 cm from the edges. The plate was developed first in a tank (12.7 cm wide, 8~6 cm deep, 13 cm high) containing 50 ml of the first eluant. The plate was then air dried, rotated 90~
counterclockwise, and developed in a second tank containing 50 ml of the second eluant.

Whatman*analytical precoated C-18 silica gel plates were used for all reverse phase chromatography. Plates measuring 2.5 cm x 7.6 cm were developed in glass cylinders measuring 10 ml of eluant.

Normal phase plates were viewed under 254 no us light first. The plates were then inserted into a glass cylinder (6.4 cm old. by 12 cm) containing It crystals. The plates were then reexamined after approximately 2 minutes. Reversed phase plates were visualized under 254 no us light only. Zones were detected by looking for quenching of the fluorescence of an impregnated dye.

Analytical HPLC

The following components were used to construct an analytical HPLC system: Waters Associates Model AYE Solvent Delivery System pump; Variant Varichrom Model VIVA uv/vis Detector set at 254 no 0.1 ODE Fisher Record Al Series 5000 Recorder; Waters Associates Model 660 Solvent Programmer; Waters*
Associates Model U6K injector; All tech*, ~-Bondapak*C18 (10~) column (4.6 mm I'd. x 25 cm) with a Whatman*Co. Poll OHS*
(0.03-0.038 mm) guard column (4.6 mm I'd. x 5 cam The components were connected with 316 stainless steel tubing (1.6 mm old. - 0.23 mm I'd.). fluent was pumped at 2 ml/min for all analysis.
* trade marks.

12412~2 Preparative HPLC

The following components were used to construct a medium pressure liquid chromatography system: Fluid Metering, Inc. Model ROSY 2CSC FMI*Lab Pump; Fluid Metering, Inc. Model PD-60LF FMI Pulse Dampener; a 15 ml sample loop constructed of polypropylene tubing (3.0 mm old. x 1.5 mm I'd.) wrapped around a cardboard tube (8.65 cm old.); Glenco Series 3500 Universal LO
columns; Instrument Specialties Co. Model UA-5 Absorbance/Fluor-essence Monitor with a Type 6 optical unit; Instrumentation Specialties Co.*Model 590 Flow Interrupter Valve; and an Instrumentation Specialties Co.*Model 328 Fraction Collector.
The components were connected with polypropylene and Teflon tubing (3.0 mm old. x 1.5 mm I'd.) and Glenco*multifit connectors and valves in the order listed.

The Glenco*series 3500 Universally Columns were slurry packed with the defined adsorbent in the designated solvent using standard techniques. The void between the settled bed and tube top was filled with standard Ottawa sand. fluent was pumped at a maximum rate which would not exceed 60 psi back pressure (approx-irately 20 ml/min).

Gradient Elusion A Glenco*gradient elusion apparatus consisting of two chambers of equal diameter, height and volume connected in tandem with a Teflon valve was used for all gradient elusions. One chamber served as a mixing chamber and one as a static reservoir.
The less polar solvent was initially held in the mixing chamber.
The more polar solvent was held in the static chamber. Teflon coated magnetic stirring bars (1.0 x 3.7 cm) were placed in both chambers and driven by Thomas Model 15 Magne-matic*stirrers.
fluent was pumped from the mixing chamber to the medium pressure HPLC system through polypropylene tubing (1.5 mm ID x 3.0 mm OX).
As eluant was removed from the mixing chamber, the solvent in the static reservoir was allowed to freely replace it, thus creating a linear gradient of eluant.
* trade marks.
so ' 12~12~2 TLC ANALYSIS OF BBM-1675A and A

Summarized in Table 9 below are the observed Of values for BBM-1675Al and A on normal phase plates. Of is calculated by dividing the measured distance of the center of a zone from the point of sample application by the measured distance of the solvent front from the point of sample application.

Table 9 Of System/Compound BBM-1675Al BBM-1675A2 4% methanol in chloroform 0.33 0.30 5% methanol in deathly ether 0.39 50% acetone in Skellysolve B 0.38 0.31 Summarized in Table 10 below are the observed Of values of BBM-1675Al and A on normal phase plates developed in two dimensions. The position of the spots are expressed in Cartesian coordinates. The X coordinate is the Of values of the second listed solvent system. The Y coordinate is the Of values of the first listed solvent system.

Table 10 Of System/Compound BBM-1675Al BBM-1675A2 4% methanol in chloroform us 5% methanol in deathly ether (0.34, 0.33) (0.28, C.23) 4% methanol in chloroform us 50% acetone in Skellysolve B (0.33, 0.29) ~Z4~282 Summarized in Table 11 below are the observed Of values for BBM-1675Al and A on C-18 reversed phase TLC plates developed in binary eluants.

Table 11 System/Compound BBM-1675Al BBM-1675A2 25% 0.5 M Nail in acetonitrile 0.18 0.21 25~ waxer in acetonitrile 0.00 0.00 Summarized in Table 12 below are the observed Of values of BBM-1675Al and A on C-18 reversed phase TLC plates developed with ternary eluants.

Table 12 Of System/Compound R3M-1675Al BBM-1675A2 Acetonitrile : Methanol 0.5M Nail 80% : 10% : 10% 1.00 1.00 60% : 10~ : 30% 0.57 0.50 40% : 30% : 30% 0.32 0.22 30~ : 50% : 20% 0.44 0.33 50% : 30% : 20% 0.62 0.54 40% : 40% : 20% 0.60 0.49 50% : 20% : 20% 0.42 0.34 60% : 20% : 20% 0.74 0.69 Acetonitrile : Methanol : water 40% : 30% : 30% 0.00 0.00 Acetonitrile : Methanol : Old Nikko 40% : 30% r 30% 0.32 0.22 12¢:~2~32 Acetonitrile : Methanol : Old Nope 40% : 30~ : 30% 0.00 0.00 HPLC ANALYSIS OF BB~S-1675A AND A

BBM-1675Al and BBM-1675A2 were assayed using single, binary, and ternary eluants on C-18 reversed phase silica gel columns. Summarized in Tables 13, 14 and 15 below are the observed K' values for these compounds. The K' was calculated using the following formula:

K' = Tryout . To where TRY is the retention time measured from time of injection to peak apex and To is the void volume time.

Table 13 System/Compound BBM-1675Al BBM-1675A2 Acetonitrile a a Tetrahydrofuran a a Methanol 0.00 0.00 a= compound did not elude from column.

Table 14 System/Compound BBM-1675Al BBM-1675A2 25% water in acetonitrile a a 25% methanol in water 1.25 1. 25 a= compound did not elude from column.

~2412~3~

Table 15 Ternary Eluants System Compound BBM-1675Al BBM-1675A2 Acetonitrile : Methanol : water 40% : 30% : 30% a a Acetonitrile : Methanol : Old Nikko 40% : 30% : 30% 1.7 3.0 50% : 20% : 30% 3.8 6.5 43.3% :23.3% : 33.3% 6.1 b 42.5% :22.5% : 35.0% I b 41.5% :21.5% : 37.0% 9.7 b a= did not elude b= not determined Biological Properties of BBM-16?5 Components Antimicrobial activity of the BBM-1675 components was determined for a variety of bacteria (gram-positive, gram-negative and acid fast and fungi by the serial two-fold ajar dilution method. Nutrient ajar medium was used for gram-positive and gram-negative bacteria and No. 1001 medium (3%
glycerol, 0.3% sodium L-glutamate, 0.2% petunia, 0.31% Nope, 0.1% KH2PO4, 0.005% ammonium citrate, 0.001% McCoy and 1.5% ajar) for acid-fast organisms. Subbreed ajar medium was used for fungi. As shown in Table 16, each of the six BBM-1675 components tall A, A, A, By, By) showed a broad spectrum of antimicrobial activity. BBM-1675 Al, A, A and A in particular were highly active against gram-positive bacteria.

1241~32 ~36-Table 16 AntimicrobiR1 Activity of 58M-1675 Components _ MIX on ~cq/ml _ __ _ _ _ Strain BBM-1675 A 2-~~-3 --~~~ - A By 32 S. Ayers 209P~0.0008 0.0063 0.0063 0.0125 0.012 0.0063 S. Ursa Smith <0.00080.0031 0.0063 0.0125 0.012 0.012 3. subtilis PI 21g ooze 0.0125 0.0125 0.05 OOZE
M. lutes 10010.0016 0.0063 0.0125 0.0125 0.1 0.1 M. flavus <0.0008 0.0016 0.0063 0.0125 0.025 0.025 My~ob~cterium 607 0.05 0.1 NT NT 0.05 0.025 3. golf NUDGE 0.1 0.8 1.6 3.1- 0.8 3.1 X. pneumonia Dull 0.40.8 1.6 3.1 0.8 0.8 P. archness D15 0.81.6 1.6 3.1 3.1 3.1 C. albicans IAMB 4888 0.40.4 1.6 6.3 3.1 1.6 C. neoformans 1.6 3.1 1.6 6.3 6.3 12.5 A second antimicrobial test was carried out on purified Al and A (as prepared in En. 3 below) and on components A and A. Data is summarized below.

MIX by AD ~mcg/ml) Strain ~BM-1675 Al A A 4 s. awry 209P <0.0008 0.0063 0.0063 0.012 S Ursa Smith <0.0008 0.0031 0.0063 0.012 3. subtilis PI 219 <0.0008 0.05 0.012 0.025 M. lutes 1001 0.0016 0.0063 0.012 0.05 M. flavus <0.0008 0.0016 0.0063 0.012 vcobacterium 607 OOZE 0.1 0.16 0.16 I. golf NINE 0.1 0.8 1.6 3.1 X. Newman Dull 0.4 0.8 1.6 3.1 P. aeNqinosa DlS 0.8 1.6 3.1 3.1 3. fr~gilis Aye 0.2 1.6 0.2 0.4 C. diffusely Aye 0.4 0.8 0.05 0.4 C. Perfrinqens Aye OOZE 0.8 0.4 0.4 C. ~lbican~ lam 4888 Owe 0.4 1.6 6.3 C. neoformans1.6 3.1 1.6 6.3 The activity of prophage induction in lysogenic bee-terbium _ golf Wow (~) was determined for BBM-1675 components according to the method of loin et at. in Nature 196: 783-784 (1962). The plaque count was made on ajar plates containing test material (T) and control plate (C). A T/C ratio of the plaque counts of greater than 3.0 was considered significant and the lysogenic induction activity (ILL activity) was expressed as the minimum inducible concentration of the test compound. As shown in Table 17, BBM-1675 components showed strong ILL activity in ~?,4128~
I
the lysogenic bacteria, thus suggesting that they may possess anti tumor activity.

Table 17 Lysogenic Induction Activity of BBM-1675 Components Antibiotic MICE (mcg/ml) BBM-16 75 Al 0.00 63 BBM-1675 A 0.0125 BBM-1675 A I. I
BBM-16 75 A 0.10 BBM-1675 By 0.10 BBM-1675 82 0.2~

* minimum inducible concentration The anti tumor activity of BBM-1675 Al and A was determined in various mouse tumor systems. Lymphocytic leukemia P-388, lymphoid leukemia L-1210, melanotic melanoma B16 and Lewis lung carcinoma were implanted intraperitoneally into male BDFl mice at an inoculum size of 106, 105, 5 x 105 and 106 cells per mouse, respectively. Graded doses of test compounds were admix-sistered to the mice intraperitoneally 24 hours after the tumor inoculation. The treatments were given once on the first day only, on day 1, 4 and 7 (q3d x 3), once daily for 9 days Ed 1-~9) or 11 days (Ed 1-~11). Components A and A were tested only against P-388 leukemia by a q3d x 3 schedule due to short supply of material.

BBM-1675 Al, A, A and A were dissolved in 0.9%
saline containing 10% dim ethyl sulfoxide, and chromomycin A
(Toyomycin, Tweaked) employed as a reference compound was disk solved in 0.9% saline. Death or survival of the treated and non-treated mice was recorded daily, and the median survival time (MST ) was calculated for each of the test (T) and control (C) groups. A T/C value equal to or greater than 125% indicates that a significant anti tumor effect was achieved. The results are ~Z41~32 shown in Tables 18 through 23. BBM~1675 Al and A showed extremely potent anti tumor activity against P-388 leukemia with a maximum T/C value of 160~. They are approximately 100 to 3000 times more active than chromomycin A in terms of minimum effective dose. BBM-1675 A and A, however, were less active than component Al ox A against P-388 leukemia (Table 19).
BBM-1675 Al and A were also active against L-1210 leukemia (Table 21), B16 melanoma (Table 22) and Lewis lung carcinoma (Table 23). The toxicity of BBM-1675 Al and A was determined in male dry mice by intraperitoneal or intravenous administration;
BBM-1675 Al was about 10 times more toxic than BBM-1675 A (Table 24). The therapeutic indices of BBM-1675 Al and A were 4 to 8 and 8 to 20 times better than those of chromomycin A, respectively, in the P-388 leukemia system (Table 25). Second experiments were carried out by intravenous administration of BBM-1675 components against P-388 and L-1210 leukemia, which were inoculated intravenously at 5 x 105 and 104 cells per mouse, respectively. In these experiments, adriamycin was used as a reference agent, which was dissolved in 0.9% saline and administered on days 1, 4 and 7. The results are shown in Tables 26 and 27. Both components Al and A were superior to adriamycin in terms of maximum T/C value, minimum effective dose and activity range.

12~12~32 Table 18 Effect of BBM-1675 Coy onents on P-388 Leukemia P
(Day 1 treatment) Average wt.
Dose, ip~ST T/Cchange on Survivors on (mg~kg/day*)(days) I) day 5 go day 5 day 45 BBM-1675 Al 0.0319.0 -2.6 5/5 0/5 0.0119.0 Jo -1.0 5/5 0/5 0.00318.0 -1.0 5/5 0/5 0.00120.0 ~;1-1.4 5/5 0/5 0.0003 16.0 0.0 5/5 0/5 0.0001 15.0120 -0.2 5/5 0/5 0.00003 14.0112 -0.2 5/5 0~5 BYWAY A 0~3 S.048 --aye 3J5 0~5 Owe 20.0 (~)-1.8 5/5 0/5 0.0318.0 (I -1.4 5/5 0/5 0.0117.0 /~)0.6 5/5 0/5 0.00317.0 (I -0.4 5/5 0/5 0.00116.0 (I owe 5/5 0/5 0.0003 15.0120 0.0 5/5 0/5 0.0001 14.0llZ 0.0 5/5 0/5 Chromomycin A 1 19.0~) -0.2 4/5 0 /5 Owe 17.0 ~)+0.6 5/5 0/5 Owe 16.0 (~+0.8 5/5 0/5 0.0314.0 1120.0 5/5 0/5 0.0114.0 112-0.2 5/5 0/5 Vehicle - 12.5 _+0.1 10/10 0/10 * day 1, imp.
Circle indicates a significant anti tumor activity.

~Z~1128Z

Table 1 9 Effect of BBM-1675 Components on P-388 leukemia (Days 1, 4 and 7 treatment) Average wt.
Dose, ipMST T/Cchange on Survivors on (mq/kq/day~)~days) day 5 (~) day 45 BBM-1675 Al 0.03 7.056 -2.85/5 0/5 0.01lg.O _~-1.0 5/50/5 0.00319.0 ~-0.6 5/50/5 0.00116.0 ~)-0.6 5/50/5 0.0003 17.0(~ -0.45/5 o/5 0.0001 16.0~! -0.25/5 0/5 0.00003 14.0112 +0.4 5/5 0/5 ~BM-1675 A 0 tweaks. - - 2/5 0/5 0.1 11.0 88-1.0 5/50/5 0.0318.0 (~)-1.2 5/50/5 OWE 118~0 ~7-0.4 5/50/5 0.00318.0 ~-0.4 owe 0.00117.0 (~-0.4 5/50/5 0.0003 16.0(~ -0.45/5 o/5 0.0001 16.0~;) -0.25/5 0/5 0.00003 15.0120 -0.45/5 0/5 ~BM-1675 A 0.0117.5(~ +0.2 4/4 0/4 0.00115.0 120+0.6 4/40/4 0.0001 13.5108 +0.6 4/4 0/4 BBM-1675 A 0.0116.5~) +0.2 4/4 0/4 Owe L 14.0112 +0.4 4/4 0/4 0.0001 12.5100 +0.6 4/4 0/4 Chromon~cin A 0.318.0 Jo +0.6 5/5 1/5 0.1 18.0 (~;)+0.6 5/50/5 0.0317.0 (~)-0.2 5/50/5 0.0114.0 1120.0 5/50/5 Vehicle 12.5 -+0.4 10/10 0/10 days 1, 4 and 7, imp.
Circle indicates a significant anti tumor activity.

~24~2~3Z

Table 20 fact of BBM-1675 Collponents on P-388 leukemia (Ed 1~9 treatment) Average wt.
Dose, it MST T/Cchange on Survivors on (mg/ky/day*) (days) (%) I (g) day 5 day 45 BBM-1675 Pal 0.01 7.0 56 -1.8 5/5 0/5 0.003 13.0 104 -1.0 5/5 0/5 0.001 19.0 I -1.2 5/5 0/5 0.0003 19.0~) -0.8 5/5 0/5 Owe 18.0(~ Owe 5/5 0/5 Owe - 16.0(~ ~0.2 5/5 0/5 0.00001 16.0(~) -0.2 5/5 0/5 BBM-1675 A 0.1 6.0 48 -2.2 4/5 0/5 0.03 13.0 104-1~4 5/5 0/5 OWE 18.0 ) -1.0 5~5 0/5 0.003 18.0 (I -0.6 5/5 0/5 OWE 18.0 I;) -0.8 5/5 0/5 0.0003 17.0~;) -0.4 5/5 0/5 Owe 16.0(~) -0.4 5/5 0/5 0.00003 15.0120 -0.6 5/5 0/5 0.00001 15.0120 +0.4 5/5 0/5 Chromomycin A 0.39.072 -2.0 5/5 0/5 0.1 18.0 ~0.4 5/5 0/5 0.03 18.0 (I 0.0 5/5 0/5 0.01 15.0 120 -0.2 5/5 0/5 0.003 13.0 104 -0.2 5/5 0/5 Vehicle - 12.5 - +0.4 10/10 0/10 * Ed 1-~9, imp.
Circle indicates a significant anti tumor activity.

124~Z~2 Table 21 Effect of BBM-1675 Components on L-1210 Leukemia Average wt.
Dose MST T/C change on Survivors on (mg/kg/day~)(days) t%) day 5 (g) day 5 day 45 BBM-1675 Al 0.003 14.5 --1.7 6/6 0/6 0.001 12.0 -0.5 6/6 1/6 Owe 12.0 (~) +0.3 6/6 0/6 0.0001 11.0 116 +1.0 6/6 0/6 33M-1675 A 0~03 10.5 111 lo 6/6 0/6 0.01 13.5 (I> -1.2 6/6 0~6 Owe 13.0 I) -0.2 6/6 0/6 0.001 11.0 116 +1.3 6/6 0/6 0.0003 10.5 111 fly 6/6 0/6 ChromomycinA3 0.3 8.5 89 -1.2 6/6 0/6 0.1 11.5 121 +1.2 6/6 0/6 0.03 11.0 116 +1.2 6/6 0/6 0.0 1 11.0 116 +1.3 6/6 0/S
0.003 10.0 105 +1.5 6/6 0/6 Vehicle - 9.5 +1.4 12/12 0/12 Ed 1--9,i.p.
Circle indicates a significant anti tumor activity.

z8Z

Table 2 2 Effect of BBM-1675 Components on B16 Melanoma Average wt.
Dose MSTT/C change on Survivor on day S (~) day 5 day 45 BBM-1675 Al 0.003 10.0 61 -0.7 6/6 0/6 0.001 31.5 0.0 6/6 0/6 0.0003 40.5 ~0.3 6/6 0/6 0.0001 27.0 +0.8 6/6 0/6 0.00003 22.0~ +1.8 6/6 0/6 0.00001 18.0109 +2.2 6/6 0/6 BBM-1675 A 0 03 11.0 67 -0.8 6/6 0/6 0.01 26.5 +0.3 6/6 0/6 0.003 29.5 +0.2 6/6 0/6 0.001 26.0 +0.8 6/6 0/6 0.0003 22.0 +0.2 6/6 0/6 0.0001 18.0109 +0.2 I 0/6 0.00003 17.0103 +1.7 6/6 0/6 Chromomycin A 0.1 25.5 ~+2.3 6/6 0/6 0.03 23.0 +2.2 6/6 0/6 0.01 21.0 +2.3 6/6 0/6 0.003 18.0109 +2.2 6/6 0/6 Vehicle - 16.5 - +2.1 12/12 0/12 Ed 1~9, imp.
Circle indicates a significant anti tumor activity.

lZ4 1Z82 Table 23 Effect of B~M-1675 Components on Lewis lung Carcinoma Average wt.
Dose MET T/Cchange on Survivors on (mg/kg/day~)(days) (~) day S (g) day 5 day 45 8BM-1675 Al 0.00310.091 --1.7 5/6 0/6 0.00131.5 I) -0.7 6/6 1/6 0.000321.5 -0.7 6/6 0/6 0.000121.0 (~+1.0 6/6 0/6 0.00003 13.011~3 +1.0 6/6 0/5 0.000~1 11.5105 +1.0 6/6 0/6 B8M-1675 A 0-03 10.091 -1.8 6~Ç 0/6 0.01 25.5 I -1.7 6/6 0/6 0.00328.5 (I 0.0 6/6 1/6 O .00117.0 (~)-0.3 6/6 0/6 O .0003 15.0(~) +1.2 6/6 0/6 0.000110.5 95~0.5 6/6 0/6 0.00003 11.0100 +0.8 6/6 0/6 Chromomycin A 0.1 21.5 +1.2 6/6 1/6 O .0317.0 (~+1.7 5/5 0/5 0.01 17.0 (~)+1.5 6/6 0/6 0.00311.5 Lucy 6/6 0/6 Vehicle - 11.0 - +0.8 12/12 1/12 Ed 1~11, imp.
Circle indicates a significant anti tumor activity.

J,Z4lz8~

Table 24 Toxicity of BBM-1675 Components LD50 (m~/kg/day) Multiple dose Single dose (Ed 1~9) - pi ivy_ - imp.
BBM-1675 Al 0.019 0.010 0.00046 BBM-1675 A 0.18 0.10 0.0072 Chromomycin A 0.81 0.41 0.23 Tubule therapeutic Indices Loomed _ Singled 1}9 L1210 B16 LO
BBM-1675 Al 63 ~46 2 15 5 Chromomycin A 8 inactive 23 23 * minimum effective dose 1~4i282 Table 26 Effect of BBM-1675 Components on Intravenously Implanted P- 3 8 8 Leukemia a Average wt.
Dose MST T/Cchange on survivors on (mg/Xg/day~)(days) I day 5 (q) day 5 day 45 BBM-1675 Al 0.019.5 106 -1.7 6/6 0/6 O . 003 14 . O -O . 3 6/6 0~6 - 0.001 11.5 +0.3 6/6 0/6 0.0003 9.0 100 +0.3 6/6 0/6 BBM-1675 A 0.17.0 78 -3.7 6/6 0/6 0.03 15 . O -1 . 0 6/6 0/6 O . 01 12 . O -q . 5 6/6 0/6 0.003 9.0 100 ~1.0 6/6 0/6 Adriamycin 30 lox. - - 0/6 0/6 10 9 . O 100-1 . 5 6~6 0/6 3 12.0 +0.7 6/6 0/6 1 9.0 100+1.7 6/6 0/6 -Vehicle - 9-0 +1.7 12/12 0/12 days 1, 4 and 7, i . v.
Circle indicates a significant anti tumor activity.

12~ :12B~:

To b 1 e 2 7 Effect of ~BM-1675 Components on Intravenously Implanted L-1210 Leukemia Average wt.
Dose MST T/C change on Survivors on (mq/kq/dav~)(days) (96) day 5 (q) day 5 BBM-1675 Al 0.008 9.5 119 -2.0 4/6 0/6 0.00414.0 -0.2 6~6 0/6 0.00213.0 +0.2 6/6 0/6 0.0019.5 - 119 ~0.8 6/6 0/6 OOZE 9.0 113 +0.8 6/6 0/6 BBM-1675 A 0.063 11.0 I -1.8 6/6 0/6 0.03214.0 (I l0.2 6/6 0/6 0.01610.5 +0.8 6/6 0/6 0.0088.0 100 +1.2 6/6 0/6 0.0048.0 100 +0.8 6/6 0/6 Adrianycin 16 lox. - - 2/6 0/6 8 12.0 (~) +0.2 6/6 0/6 4 9.0 113 +1.5 6/6 0/6 2 8.0 100 ~1.7 6/6 0/6 Vehicle - 8.0 - +1.412/12 0/12 days 1, 4 and 7, ivy.
Circle indicates a significant anti tumor activity.

Anti tumor activity of components BBM-1675 Al and A was also determined by a second test against P-388 leukemia, L-1210 leukemia and B16 melanoma in mice. Results of these tests are shown below in Tables 28, 29 and 30. Details of the methods used in these tests have been described in Cancer Chemother. Rep. 3:
1-87 (Part 3), 1972.

Table 28 Effect of 3BM-1675 Al and A on P-388 Leukemia Effect AWN, Treatment Dose, IT MY MST go Survivors material Schedule~q/kq/dayDavs T/C do do (30) SKYE 38270 qdl~9 400 13.0 163 -0.6 6/6 200 11.0 138 -0.9 6/6 Molly do 51.2 20.0 250 -2.1 4/6 Dylan 25.6 18.0 225 -1.8 6/6 12.8 16.5 206 -1.1 6/6

6.4 13.0 163 +0.1 6/6 3.2 12.0 150 -0.3 6/6 1.6 11.0 138 -0.3 6/6 0.8 10.5 131 0 S/6 I 10.0 125 +0.4 6/6 0.2 10.0 125 +0.3 6/6 0.1 10.0 125 0 6/6 do 5 L 9 25.6 8.0 100 -1.8 6/6 12.8 13.5 169 -1.5 6/6 6.4 16.5 206 -0.8 6/6 3.2 16.0 200 -0.8 6/6 1.6 15.5 194 +0.3 6/6 0.8 12.5 156 +0.3 6/6 I 12.0 150 -0.1 6/6 0.2 11.5 144 +0.2 6/6 0.1 12.0 150 +0.8 6/6 0.05 10.0 125 +0.8 6/6 qdl 912.8 FOX FOX TO 1/6 6.4 6.0 75 -1.5 4/6 3.2 13.0 163 -1.2 6/6 1.6 1~.5 181 -1.6 6/6 0.8 16.5 206 -2.3 6/6 0.4 16.0 200 -0.9 6/6 0.2 15.0 188 -0.8 5/5 0.1 13.0 163 -0.4 6/6 0.05 12.0 150 +0.1 6/6 0.025 12.0 150 -0.7 6/6 issue Table 28 keynoted 3BM-1675A2 do 256 TO TO TO 0/6 DMSO-saline 128 12.5 156 -3.5 4~6 64 27.0 338 -1.9 6/6 32 26.0 3Z5 -2.0 6/6 16 16.0 200 -1.8 6/6 B 15.5 194 -1.9 6~6 4 15.0 lay -0.7 6/6 2 12.0 150 -0.5 6/6 1 12.0 150 0 6/6 0.5 10.0 125 +0.2 6/6 do 5 9128 TO TO TO 0/6 32 TO TO -1.3 2/6 ., 16 24.5 306 -1.3 5/5 8 17.5 Zl9 -1.1 6/6 4 15.0 188 0 6/6 2 15.0 188 +0.1 6/6 1 12.5 156 -0.4 6/6 0.5 12.0 150 -0.4 6/6 abeam A .. -DMSO~s~lin~ . 0.25 , Lowe 138 -0.4 6/6 qdl'9 64 TO TO TO 1/6 32 6.0 75 I 4/6 16 8.0 100 ~.1.9 6/6 8 15.5 194 -1.3 6/6 4 1~.0 213 -1.8 6/6 2 15.0 188 -1.1 6/6 1 14.0 175 -0.5 6/6 0.5 14.0 175 -0.6 6/6 0.25 12.0 150 -0.1 6/6 0.125 12.0 150 ~0.1 6/6 Control Saline 8.0 - ~0.5 10/10 .
Tumor inoculum: 106 Auschwitz cells implanted imp.
Not : Cal I mice.
To : c4/6 mice alive on do Evaluation : MST median survival time.
Effect : T/C - (MST treated/YST control) x 100.
Criteria : T/C >125 considered significant antitvmor activity ^ SKYE 38270 - olivomycin A

Table 29 Effect of 8BM-1675 Al and A on L-1210 Luke EffectAWC.
Treatment Dose, IPMST MY em Survivors on Material Schedule _ ~g/kq/in]Dav3 T/C do do (30) 3BM-1675 Al do 51.212.0171 -1.1 5~6 25.6 7.0100-2.3 6/6 12.8 9.0129-1.1 5/6 6.4 9.5136-0.5 6/6 3.2 6.086 -1.7 6~6 1.6 7.0100-0.8 6/6 0.8 ~.0114-0.4 6/6 0.4 7.0100~0.3 6/6 0.2 7.0100-0.5 5/6 0.1 7.0100+0.8 S/6 do 5 9 25.6 TOXTOX TO 1/6 12.8 9.0129-1.8 6/6 6.4 9.0129-0.8 6/6 3.2 8.0114-1.9 6/6 1.6 8.5121 0 6/6 0.8 8.0114-0.4 6/6 0.4 7.5107-1.3 6/6 0.2 8.0114 0 6/6 0.1 8.0114+0.4 5/6 0.05 7.0100~0.3 6/6 Ed 1-9 12.8 TOXTOX-2.4 3/6 6.4 8.0114-1.6 6/6 3.2 8.0114-1.7 6/6 lob 9.0129-2.1 6/6 0.8 8.5121-1.6 6/6 0.4 8.0114-1.0 6/6 0.2 8.0114-0.5 5/6 0.1 7.0100+0.3 6/6 0.05 7.0100+0.3 6/6 0.025 6.086 -0.6 6/6 BBM-1675 A do 256 TOYTOX TO 0/6 128 7.0100-1.8 5/6 64 7.5107-1.3 4/6 32 8.0114-2.2 5/6 16 7.0100-2.3 6/6 8 9.5136-1.4 6/6 4 8.51'1-1.1 6/6 2 8.0114-0.8 6/6 1 8.0114 0 6/6 0.5 8.0114-0.1 6/6 ~2~1ZS2 Table 3 0 Effect of B8M-1675 Al and A on B16 Melanoma .
Effect AWN
Dose, IT MST MST gym Survivors on Material I (m) or mg/kg/inj Days T/C do _ d.l0 ~61) BBM-1675 Al 3.2M TO TO -1.8 2/10 1.6 16.0 64 -lob 10/10 0.8 34.5 168 -1.8 10/10 0.4 56.5 226 -0.9 10/10(2)b 0.2 47.0 188 -0.7 10/10 0.1 37.0 148 -0.4 19/10 BBM-1675 A 16M13.0 52 -2.1 10/10 8 29.5 118 -2.0 10/10 4 43.5 174 -1.1 10/10 2 50.5 202 -2.1 10/10(3)b 1 3.5 140 -1.0 10/10 0.5 38.0 152 -1.1 10/10 Control Saline 25.0 - -0.1 10/10 a Only one without tumor; MST duo = 55.0 (220~), -b Two without tumor; MST duo = 46.0 (184~) Tumor inoculum: 0.5 ml of a 10~ brie, it Yost : BDFl I mice.
treatment : Ed 1~9 To : <7/10 mice alive on d.10 Evaluation : MST = median survival time Effect : T/C = (MST treated~MST control) x 100.
Criteria , : T/C 125 considered significant anti tumor activity.

~2'~1Z8Z

After further purification of BBM-1675Al according to Example 6, samples of the purified compound were tested against L-1210 leukemia, P-388 leukemia and B16 melanoma in mice.
Results of these tests are shown below.

Table 31 Effect of Purified D8M-1675Al on P383 Leukemia (Day 1 Treatment) Average wt.
Dose MSTT/C change on Survivors on Compound (mq/kq/dose)R~ute, schedule Days I day 5 day 5 3BM-1675A1 0.1024 imp., Ed x 1 TO TO 0/6 0.0512 17.5 159 -1.8 4/6 0.0256 16.5 150 -Z.6 6/6 0.0128 17.5 15~ -1.4 I
0.0064 15.5 141 -2.2 6/6 0.0032 15.5 141 -2.5 6/6 0.0016 16.5 150 -1.0 6/6 0.0003 15.0 136 -1.2 6/6 0.0004 15.0 136 -2.0 6/6 0.0256 imp., q4d x 3: TO TO -1.5 1/6 0.0128 10.0 91 -2.5 S/6 0.0064 17.5 lS9 -1.9 6/6 0.0032 17.0 lS5 -0.8 6/6 0.0016 17.0 155 -2.0 6/6 0.0008 15.0 136 -1.7 6/6 0.0004 15.0 136 -0.4 6/6 0.0002 13.0 118 -0.8 6/6 0.0001 13.0 118 -1.3 6/6 0.00005 13.5 123 -1.0 6/6 0.0128 imp., Ed x 5; TO TO 0/6 0.0064 TO TO ~3.6 3/6 0.0032 17.5 155 -2.2 S/6 0.0016 14.5 132 -2.0 6/6 0.0008 loss 1~1 -2.2 6/6 0.0004 16.0 145 -2.8 6/6 0.0002 17.0 155 -1.3 6/6 0.0001 14.0 lZ7 -1.6 5/6 0.00005 15.0 136 -1.6 6/6 0.000025 15.0 136 -1.0 6/6 control vehicle) 1 x 106 imp., q4d x 3: 11.0 100 -0.7 9/9 Host: CDFl female mice Implant level and site: 1 x 10 cells, imp.

1'~41282 Table 32 Effect of Purified yule on L-1210 Leukemia Do 1 treatment) Average wt.
Dose MST arc change on Survivors on Compound (mg/kg/dose~ Route, schedule Days I) do 5 do S
MOE 0.1024 imp., Ed x 1 TO TO 1/6 0.0512 TO DO -2.0 ù/6 0.0256 8.0 114 -2.9 4/6 0.0128 11.0 157 -2.0 6/6 0.0064 11.0 15, -1.9 6/6 0.0032 10.0 143 -2.0 6/6 0.0016 loud 143 -2.6 5/6 0.0008 8.0 114 -0.4 6/6 0.0256i.p., q4dx 3 TO FOX --2.3 2/6 0.0128 10.5 lo -1.7 6/6 Ode 11.0 157 -1.8 6/6 0.003" 11.0 157 -1.4 6/6 0.0016 10.5 150 -1.9 6/5 0.0008 9.0 129 -0.6 6/6 0.0004 8.5 121 -0.7 6/6 0.0002 8.0 114 -0.5 6/6 0.0128i.p., ye x 5 TO TO -aye 2/6 0.0064 7.0 100 -1.8 5/6 0.0032 11.5 164 -1.0 6/6 0.0016 11 . 0 15 T -1 . 5 6/6 0.0008 10.0 143 -1 . 6 5/6 0.0004 8.5 121 -0.4 6/6 0.0002 8.5 121 0.1 6/6 0.0001 8.5 121 0.0 6/6 Control (vehicle) 1 x 106i.p., Ed x 5 7,0 100 0.1 10/10 East: CDFl female mice Implant level and site: 1 x 106 cells, imp.

1;Z82 Table 33 Effect of Purified 3BM-1675Al on 316 Melanoma day 1 reagent Dose MS~T/C Averaq~ wt. Survivor on Compound (m~/k~/dose) Route, schedule Days I) day S do 5 3BM-1675A1 0.0064 imp., q4d x 3 16.5 110 -3.8 8/10 0.0032 22.5lS0 -3.010/10 0.0016 25.0167 -1.910/10 0.0008 22.0147 -2.310/10 0.0004 24.0160 -1.9 9/10 0.0016 imp., Ed x 927.0 180 -3.7 10/10 0.0008 27.0180 -2.910/10 0.0004 26.0173 -2.310/10 0.0002 24.5163 -2.410/10 0.0001 25.5170 -2.310/10 Control (vehicle) OHS My imp., go x 9 15.0 100 I 3 10/10 ~3BM-1675A1 0.0064 ivy., god x 3 lS.0 86 -4.7 10/10 0.0032 32.5186 -2.110/10 0.0016 26.0149 -1.410/10 0.0008 24.0137 -0.410/10 0.0004 24.5140 -0.010/10 0.0064 imp., q4d x 318.0 103 -2.7 10/10 0.0032 23.0131 -1.410/10 0.0016 24.0137 -0.710/10 0.0008 25.5146 -0.810/10 0.0004 21.5123 -1.410/10 Control (vehicle) 0.2 ML ivy., q4d x 3 17.5 100 ~0.4 10/10 Yost: 3DFl female vice Implant level and site: OUZEL 10~ ROY, imp.
implant level and site: Fragment, sac.

~Z~Z~32 The above screening data indicates that the purified BBM-1675Al component has substantially the same anti tumor properties as the less purified sample screened previously. The compound has exceptionally high potency since activity has been demonstrated at a dose of 25 nanograms/kg on a daily times 5 schedule against P-388 leukemia in mice. On tests against P-388 and L-1210 leukemia, BBM-1675Al is effective whether given as a single injection, day 1, every fourth day for 3 injections, or daily times 5. Against B16 melanoma the compound was equally effective given intravenously to animals bearing subcutaneous tumors as when it was given intraperitoneally to animals bearing it tumor implants. This property of successful pharmacologic delivery of a drug to a tumor at a distant site is unusual among anti tumor antibiotics.

As shown above the BBM-1675 components possess potent antimicrobial activity and are thus useful in the therapeutic treatment of mammals and other animals for infectious diseases caused by such microorganisms. Additionally the components may be utilized for other conventional applications of antimicrobial agents such as disinfecting medical and dental equipment.

The induction of prophage in lysogenic bacteria and the activity shown against mouse tumor systems indicate that the BBM-1675 components are also therapeutically useful in inhibiting the growth of mammalian tumors.

The present invention, therefore, provides a method for therapeutically treating an animal host affected by a microbial infection or by a malignant tumor which comprises administering to said host an effective antimicrobial or tumor-inhibiting dose of BBM-1675 Al, A, A, A, By or By, or a pharmaceutical combo-session thereof.

In another aspect, the present invention provides a pharmaceutical composition which comprises an effective antimicrobial or tumor-inhibiting amount of BBM-1675 Al, A, A, A, By or By in combination with an inert pharmaceutically 12412~32 acceptable carrier or delineate. These compositions may be made up in any pharmaceutical form appropriate for parenteral ad minis-traction.

Preparations according to the invention for parenteral administration include sterile aqueous or Nancy solutions, suspensions or emulsions. They may also be manufactured in the form of sterile solid compositions which can be dissolved in sterile water, physiological saline or some other sterile inject-able medium immediately before use.

It will be appreciated that the actual preferred amounts of the BBM-1675 antibiotics used will vary according to the particular component, the particular composition formulated, the mode of application and the particular situp! host and disease being treated. Many factors that modify the action of the drug will be taken into account by those skilled in the art, for example, age, body weight, sex, diet, time of administration, route of administration, rate of excretion, condition of the host, drug combinations, reaction sensitivities and severity of the disease. Administration can be carried out continuously or periodically within the maximum tolerated dose. Optimal applique-lion rates for a given set of conditions can be ascertained by those skilled in the art using conventional dosage determination tests in view of the above guidelines.

The following examples are provided for illustrative purposes only and are not intended to limit the scope of the invention.

Example 1 Fermentation of BBM-1675 Actinomadura strain No. H964-92 was grown and maintained on an ajar slant containing 1% malt extract, 0.4%
glucose, 0.4% yeast extract, 0.05% Cook and 1.6~ ajar. A
well-grown ajar slant was used to inoculate vegetative medium lZ41~8~

containing I soluble starch, 3% dry yeast, 0.3% K2HPO4, 0.1%
KH2PO4, 0.05% McCoy OWE, 0.2% Nail and 0.1% Cook, the pi being adjusted to pi 7.0 before sterilization. The vegetative culture was incubated at 32C for 72 hours on a rotary shaker (250 rum) and 5 ml of the growth was transferred into a 500-ml Erlenmeyer flask which contained 100 ml of fermentation medium composed of 3% cane molasses, 1% corn starch, 1% fish meal, 0.1% Cook and 0.005~ Quiz SHEA (pi 7.0 before sterilization). The fermentation was carried out at 28C for six days on the rotary shaker. The antibiotic activity in the fermentation broth was determined by the paper-disc ajar diffusion using Staphylococcus Ayers 209P as the test organism. The antibiotic potency reached a maximum of about 1 mcg/ml aster five days fermentation.

Fermentation of BBM-1675 was also performed in stir-jar fermenters. Five hundred milliliters of inoculum growth as prepared above was transferred into 20-liter jar fermenters containing 10 liters of fermentation medium which consisted of the same ingredients as used in the shake flask fermentation.
The fermentation was carried out at 32C with an aeration rate of 12 liters/minute and agitation at 250 rum. Under these condo-lions, the antibiotic production reached a maximum of about 0.9 mcg/ml after 68-76 hours of fermentation.

Fermentation studies were also carried out in ferment-lion tanks. A seed culture was shaken for four days at 30C in Erlenmeyer flasks containing vegetative medium consisting of 3%
soluble starch, 3% dry yeast, 0.3% K2HPO4, 0.1% KH2PO4, 0.05%
McCoy OWE, 0.2% Nail and 0.1% Cook. The seed culture was inoculated to a 200-liter seed tank containing 130 liters of seed medium having the same composition as above, and the seed tank was stirred at 240 rum at 30C for 31 hours. The second seed culture was used to inoculate 3,000 liters of fermentation medium containing 1% corn starch, 3% cane molasses, 1% fish meal, 0.005%
Quiz OWE and 0.1% Cook. The production tank was operated at 28C at 164 rum with an aeration rate of 2,000 liters/minute.
The broth pi gradually rose with the progress of fermentation and sluice I
reached 7.7-7.8 after 170-180 hours, when a peak antibiotic activity of 1.7 mcg/ml was produced.

Example 2 Isolation and Purification of BBM-1675 Components The harvested fermentation broth (3,000 liters, pi 7.8) was separated to mycelial cake and superannuate with the aid of a Sharpness centrifuge. The mycelial cake was suspended in 1,600 liters of methanol and the mixture stirred for one hour. The insoluble materials were filtered off and the methanolic extract was concentrated on vacua to 43 liters. The activity contained in the broth superannuate was recovered therefrom by extraction with two l,000-liter portions of n-butanol. The n-butanol extracts and concentrated methanol extract were combined and evaporated azeotropically by occasional additions or water to an aqueous solution (20 liters) which deposited most of the antibiotic activity as an oily solid. The solid was digested in 30 liters of methanol and the insoluble were removed by filtration. The methanol extract was then concentrated in vacua to a 10-liter solution, to which was added 40 liters of ethyl acetate and 30 liters of water. After being stirred for 30 minutes, the organic layer was separated, dried over sodium sulfate and evaporated in vacua to 4 liters. Addition of the concentrate into 20 liters of Nixon afforded a pale yellow solid of crude BBM-1675 complex (90.14 g, potency: 55 mcg/mg). The complex was shown in TLC to be a mixture of two major components, BBM-1675 Al and A, and several minor ones. They were separated and purified by repeated chromatographies which were performed in a cooled room to prevent deterioration.

The BBM-1675 complex (20 g) was dissolved in methanol (20 ml) and charged on a column of Sephadex L~-20 (I 5.5 x 85 cm). The column was developed with methanol and the elusion monitored by bioassay using Staphylococcus Ayers 209P. The active equates were combined, concentrated in vacua and Lyon falsehood to give a semi-pure solid of BBM-1675 complex ~4.13 g).

124i282 The solid was then chromatographed on a column of silica gel (I
OWE x 50 cm) using chloroform plus an increasing amount (1-~5%
v/v) of methanol as eluants.

Equates were pooled on the basis of antibacterial activity (vs. S. Ayers) and TLC (Sue; CHC13-CH30H = 5:1 v/v) and concentrated _ vacua. Nearly homogeneous BBM-1675 Al (yield after evaporation: 351 my) was eluded first with 2% methanol in chloroform and then a mixture of BBM-1675 A, A and A (507 my) followed by BBM-1675 B mixture (210 my) with 3% methanol in chloroform. The solid of BBM-1675 Al was applied on a column of Sephadex L~-20 (I 2.0 x 80 cm) which was developed with methanol.
The active fractions were concentrated in vacua to dryness and the residual solid was crystallized from methanol to afford colorless plates of pure BBM-1675 Al (124 my) (this material is starting material for Example PA.). The complex BBM-1675 A, A
and A was separated by chromatography on a column of Bondapak C18 (Waters, 3.0 x 50 cm). Elusion was carried out with aqueous acetonitrile and the bioactive equates were examined by TLC (Merck, *Silenced: CHICANO = 75:25 v/v). The minor components A (33 my) and A (18 my) were eluded successively in that order with 20% acetonitrile followed by another major component A (301 my) (this material is starting material for Example 3B) with 50% acetonitrile.

The solid containing BBM-1675 By and By was chrome-to graphed on a column of silica gel (I 3.0 x 40 cm) with sheller-form and methanol as the developing solvent. The active fractions eluded with 4% methanol in chloroform were combined and evaporated to afford pure BBM-1675 By (7 my). Another active fraction was eluded at 5% methanol concentration, which upon evaporation afforded BBM-1675 By (8 my).

*C18 reverse phase silica gel ~Z41Z8Z

Example 3 Further Purification of BBM-1675 Al and A

A. Purification of BBM-1675Al A 2.67 cm I'd. x 75 cm Glenco column was slurry packed with Baker bonded phase octadecyl (C18~ silica gel (40 micron particle size) in methanol. The column was connected into a medium pressure HPLC system and equilibrated with 1.5 1 of eluant (~1.6% acetonitrile - 21.6% methanol - 36.8% 0.1 M ammonium acetate). Partially purified BBM-1675Al (100.5 my) obtained according to the purification procedure of Example 2 was dissolved in 2 ml of acetonitrile and drawn into the sample loop.
The sample was pumped onto the column. The column was eluded with the above eluant collecting 87 ml fractions. The eluant was monitored at 254 no and 340 no. Fractions 55 through 71 were pooled and extracted twice with 1500 ml allocates of chloroform.
The chloroform was evaporated to dryness to yield 89.8 my of residue C.

A 1.5 cm I'd. x 20 cm Glenco column was slurry packed with 12 g of Whelm silica gel (60-200 micron particles). Residue C was applied to the column in a chloroform solution. The column was eluded with a 500 ml linear gradient of chloroform to 10%
methanol in chloroform collecting 20-25 ml fractions. After analysis by TLC on silica gel, fractions 6-9 were pooled and evaporated to dryness to yield 73 my of residue D.

A 1.5 cm I'd. x 20 cm Glenco column was slurry packed with 12 g Whelm silica gel (63-200 micron particles) in Smelly-solve B. Residue D was dissolved in approximately 2 ml of SCHICK
and applied to the column. The chloroform was displaced with 25 ml of Skellysolve B. The column was then eluded with a 500 ml linear gradient of Skellysolve B to 6096 acetone in Skellysolve B
collecting 28-25 ml fractions. Fractions 19-23 won e pooled and evaporated to dryness to yield 65.6 my of pure BBM-1675Al.

~.~41282 This residue was homogeneous in three TLC systems I
methanol in chloroform; 5% methanol if. ether; and 50% acetone in Skellysolve B on silica gel) and HPLC (C-18 silica gel -41.5%
acetonitrile:21.5% methanol% 0.1 M ammonium acetate).

Gel Permeation Chromatography with purified B3M-1675Al A 2.5 cm I'd. x 45 cm Pharmacia column was slurry packed with Sephadex LH-20 in methanol and adjusted to a 33.4 cm chromatography bed. Purified BBM-1675Al (approximately 120 my) was dissolved in 2 ml of methanol and transferred to a 2.5 ml sample reservoir. The sample was applied to the column and elusion commenced at 1.75-ml/min with methanol collecting 10 ml fractions [Pharmacia Frock fraction collector]. The eluant was monitored at 254 no with an Disco UA-5 detector. BBM-1675A
was observed to elude at Vet of 0.79 to 0.91 (Vie= elusion volume; Vet= bed volume).

B . Purify cation of BBM-1675A2 A 2.65 cm I'd. x 75 cm Glenco column was slurry packed with Baker bonded phase octadecyl (C18) silica gel (40 micron particle size) in methanol. The column was connected into the medium pressure HPLC system and equilibrated with 1.5 1 of eluant (50% acetonitrile - 20% methanol - 30% 0.1 M ammonium acetate).
Partially purified BBM-1675A2 (76.9 my) as obtained by the procedure of Example 2 was dissolved in 2 ml of acetonitrile and drawn into the sample loop. The sample was pumped onto the column. The column was eluded with the above eluant collecting 87 ml fractions. The eluant was monitored at 254 and 340 no.
Fractions 31 through 38 were pooled and extracted twice with 500 ml allocates of chloroform. The chloroform was evaporated to dryness to yield 65.8 my of homogeneous BBM-1675A2.

BBM-1675A2 was homogeneous in 2 TLC systems, one 2-d TLC analysis and HPLC.

12~1282 Example 4 Preferred extraction process for BBM-1675A

Raw fermentation (6.8 1) obtained according to the general procedure of Example 1 was transferred to a polypropylene bucket (12 cm d, top; 10 cm d, bottom; 37 cm high) equipped with a faucet at the bottom. An equal volume of chloroform was added.
The mixture was stirred at a good rate with a CXC-air driven stirrer for 2 hours. Approximately 4 1 (1.3 kg) of Dicalite (filter aid) was added and allowed to mix in. The mixture was filtered on a Dicalite pad which was held in a No. 12 Buchner funnel. The filtrate was collected in a 19 1 solution bottle equipped with a vacuum take-off (Ace. No. 5396-06). The mat was washed with 2 liters of chloroform. The filtrate was transferred to a 20 1 separator funnel and the phases allowed to separate.
The lower phase (chloroform) was removed.

A 2.5 cm I'd. x 40 cm Glenco tube was slurry packed with 91 g of Whelm silica gel (63-200 micron particles). Using an FMI RPY-2CSD pump, the above chloroform phase was pumped through the column. The column was rinsed with 600 ml of fresh chloroform. The chloroform eluant was discarded. The column was then eluded with 600 ml of 10% methanol in chloroform. This eluant was evaporated to dryness to yield 547 my of residue A.

Residue A was dissolved into 50 ml of chloroform. The chloroform solution was added to 20 g of Dicalite in a 1 1 round bottom flask. A slurry was created by adding approximately 200 ml of Skellysolve B. The solvents were moved in a rotatory evaporator. The residue was slurries in 300 ml of Skellysolve B.
The slurry was packed into an Ace flask chromatography tube (Part No. B5872-14) (41 mm id x 45.7 cm) by the following procedure. A
glass wool plug was inserted into the throat of the stop cock between the cock and the column tube. A 1 cm layer of standard Ottawa sand was added above the glass wool. The stopcock, glass wool and sand bed were purged of air by passing a pressurized (5.7 psi) flow of Skellysolve B through them. The slurry was then added to the column and allowed to form a packed 12~1Z82 bed under pressurized flow. The column was never allowed to go dry. After a stable column bed was obtained, a 2 cm layer of Ottawa sand was added onto the top of the bed. The bed was then eluded with an additional 600-700 ml of Skellysolve B. The bed was eluded with 500 ml of Tulane. The Tulane eluant was evaporated to dryness to yield 93 my of residue B. This partially purified BBM-1675Al may then be further purified according to the procedure of Example 3.

Example 5 Fermentation of BBM-1675 complex using variant HOWE

A variant strain AYE, which was obtained by NAG
treatment of Actinomadura verrucosose~ strain No. H964-92, was used to inoculate vegetative medium containing 2% soluble starch, 1% glucose, 0.5~ yeast extract, 0.5% NB-amine type A and 0.1~
Cook, the pi being adjusted to 7.0 before sterilization. The vegetative culture was incubated at 32C for four days on a rotary shaker (250 rum) and 5 ml of the growth was transferred into a 500 ml Erlenmeyer flask which contained 100 ml of fermentation medium composed of 3% cane molasses, 1% corn starch, 1% fish meal, 0.005% Quiz OWE, 0.05% McCoy OWE and 0.1% Cook, the pi being adjusted to 7.0 before sterilization.

The fermentation was carried out at 28C for 7 days on the rotary shaker. The antibiotic production reached a maximum of cay 1.5 mcg/ml.

Example 6 Isolation and Purification of BBM-1675 Components The harvested fermentation broth from En. 5 (3,000 L, pi 7.6) was separated to mycelial cake and superannuate by using a Sharpness centrifuge. The mycelial cake was stirred with 2,000 L
of methanol for one hour and the insoluble materials were removed by filtration. The activity contained in the broth superannuate was extracted therefrom with 1,800 L of n-butanol. The methanol and n-butanol extracts were combined and concentrated azeotropically 124i2~2 by occasional additions of water to an aqueous solution (20 L) which deposited most of the antibiotic activity as an ugly solid.
The mixture was shaken three times with 20 L each of ethyl acetate to extract the activity. The extracts were pooled, filtered to remove the insoluble and evaporated in vacua to 4 L.
Addition of the concentrate into 30 of Nixon under stirring afforded pale yellow solid of crude BBM-1675 complex (81.7 g, potency: 59 mcg/mg). The complex was shown by TLC and HPLC to be a mixture of two major components, BBM-1675 Al and A and several minor ones. They were separated and purified by a series of chromatography which were carried out in a cold room to prevent deterioration.

The crude BBM-1675 complex (20 g) was dissolved in methanol (20 ml) and charged on a column of Sephadex LH-20 (I 5.5 x 85 cm). The column was developed with methanol and the elusion monitored by bioassay using Staphylococcus Ayers 209P. The active equates were pooled, concentrated in vacua and lyophilized to give a semi-pure solid of BBM-1675 complex (4.86 g, potency:
203 mcg/mg). The solid was then chromatographed on a column of silica gel (I 3.0 x 70 cm) using chloroform and an increasing amount (1-5%) of methanol as developing solvents. The equates were pooled on the basis of antibacterial activity against S.
Ayers and TLC (Sue, CHC13-MeOH = 5:1, v/v) and concentrated in vacua. BBM-1675 Al (425 my after evaporation, potency: 960 mcg/mg) was eluded first with 2% methanol in chloroform and then a mixture of BBM-1675 A, A and A (732 my, potency: 340 mcg/mg) followed by BBM-1675 B complex (200 my, potency: 190 mcg/mg) with I methanol in chloroform. The above BBM-1675 Al was rechromato-graphed on silica gel (column: 2.2 x 44 cm) with 2% methanol in Bunsen. The bioactive equates were examined by HPLC (Lichrosorb RP-18: CH3CN-MeOH-O.lMCH3COONH4 = 5:2:3, v/v) and the fractions containing homogeneous BBM-1675 Al evaporated in vacua to dryness. The residual solid was crystallized from methanol (10 ml) to give colorless prisms of BBM-1675 Al (197 my, potency:
1,000 mcg/mg).

~2412~2 The complex of BBM-1675 A, A and A (537 my) was separated by column chromatography on Bondapak C18 (Waters, 2.0 x 42 cm). Elusion was carried out with aqueous acetonitrile and the bioactive equates were examined by TLC (Merck, silenced:
CHICANO = 75:25, v/v). The minor components BBM-1675 A (45 my, potency: 410 mcg/mg) and A (19 my, potency: 300 mcg/mg) were eluded successively with 20~ acetonitrile followed by a major component, BB~-1675 A (203 my) with 50~ acetonitrile. The BBM-1675 A fraction was crystallized from chloroform-n-hexane to deposit colorless rods (70 my, potency: 290 mcg/mg). The solid containing BBM-1675 B mixture was chromatographed on a column of silica gel (I 3.0 x 40 cm) with chloroform and methanol as developing solvent. the active fractions eluded with I methanol in chloroform were pooled and evaporated to afford pure BBM-1675 By (7 my, potency: 180 mcg/mg). Another active fraction was eluded at 5% methanol concentration, which upon evaporation afforded BBM-1675 By (8 my, potency: 140 mcg/mg).

Claims (21)

1. The process for the production of the antitumor antibiotic BBM-1675 which comprises cultivating a BBM-1675 producing strain of Actinomadura verrucosospora in an aqueous nutrient medium containing assimilable sources of carbon and nitrogen under submerged aerobic conditions until a substantial amount of BBM-1675 is produced by said organism in said culture medium.

2. The process for the production of the antitumor antibiotic BBM-1675 Al which comprises cultivating a BBM-1675 Al-producing strain of Actinomadura verrucosospora in an aqueous nutrient medium containing assimilable sources of carbon and nitrogen under submerged aerobic conditions until a substantial amount of BBM-1675 Al is produced by said organism in said culture medium and then recovering BBM-1675 Al from the culture medium.

3. The process according to Claim 2 wherein the BBM-1675 Al-producing organism has the identifying characteristics of Actinomadura verrucosospora strain H964-92 (ATCC 39334), Actinomadura verrucosospora strain A1327Y (ATCC 39638), or a mutant thereof.

4. The process for the production of the antitumor antibiotic BBM-1675 A2 which comprises cultivating a BBM-1675 A2-producing strain of Actinomadura verrucosospora in an aqueous nutrient medium containing assimilable sources of carbon and nitrogen under submerged aerobic conditions until a substantial amount of BBM-1675 A2 is produced by said organism in said culture medium and then recovering BBM-1675 A2 from the culture medium.

5. The process according to Claim 4 wherein the BBM-1675 A2-producing organism has the identifying characteristics of Actinomadura verrucosospora strain H964-92 (ATCC 39334), Actinomadura verrucosospora strain A1327Y (ATCC 39638), or a mutant thereof.

6. The process for the production of the antitumor antibiotic BBM-1675 A3 which comprises cultivating a BBM-1675 A3-producing strain of Actinomadura verrucosospora in an aqueous nutrient medium containing assimilable sources of carbon and nitrogen under submerged aerobic conditions until a substantial amount of BBM-1675 A3 is produced by said organism in said culture medium and then recovering BBM-1675 A3 from the culture medium.

7. The process according to Claim 6 wherein the BBM-1675 A3-producing organism has the identifying characteristics of Actinomadura verrucosospora strain H964-92 (ATCC 39334), Actinomadura verrucosospora strain A1327Y (ATCC 39638), or a mutant thereof.

8. The process for the production of the antitumor antibiotic BBM-1675 A4 which comprises cultivating a BBM-1675 A4-producing strain of Actinomadura verrucosospora in an aqueous nutrient medium containing assimilable sources of carbon and nitrogen under submerged aerobic conditions until a substantial amount of BBM-1675 A4 is produced by said organism in said culture medium and then recovering BBM-1675 A4 from the culture medium.

9. The process according to Claim 8 wherein the BBM-1675 A4-producing organism has the identifying characteristics of Actinomadura verrucosospora strain H964-92 (ATCC 39334), Actinomadura verrucosospora strain A1327Y (ATCC 39638), or a mutant thereof.

10. The process for the production of the antitumor antibiotic BBM-1675 B1 which comprises cultivating a BBM-1675 B1-producing strain of Actinomadura verrucosospora in an aqueous nutrient medium containing assimilable sources of carbon and nitrogen under submerged aerobic conditions until a substantial amount of BBM-1675 B1 is produced by said organism in said culture medium and then recovering BBM-1675 B1 from the culture medium.

11. The process according to Claim 10 wherein the BBM-1675 B1-producing organism has the identifying characteristics of Actinomadura verrucosospora strain H964-92 (ATCC 39334), Actinomadura verrucosospora strain A1327Y (ATCC 39638), or a mutant thereof.

12. The process for the production of the antitumor antibiotic BBM-1675 B2 which comprises cultivating a BBM-1675 B2-producing strain of Actinomadura verrucosospora in an aqueous nutrient medium containing assimilable sources of carbon and nitrogen under submerged aerobic conditions until a substantial amount of BBM-1675 B2 is produced by said organism in said culture medium and then recovering BBM-1675 B2 from the culture medium.

13. The process according to Claim 12 wherein the BBM-1675 B2-producing organism has the identifying characteristics of Actinomadura verrucosospora strain H964-92 (ATCC 39334), Actinomadura verrucosospora strain A1327Y (ATCC 39638), or a mutant thereof.

14. The antitumor antibiotic BBM-1675 Al which in substantially purified form:

(a) appears as white to pale yellow crystals;

(b) is soluble in chloroform, ethyl acetate, acetone, ethanol and methanol, slightly soluble in benzene and water, and insoluble in n-hexane and carbon tetrachloride;

(c) gives a positive reaction with ferric chloride, Ehrlich and Tollen's reagents and a negative reaction in Sakaguchi, ninhydrin and anthrone tests;

(d) exhibits an infrared absorption spectrum (KBr) substan-tially as shown in FIG. 9;

(e) when dissolved in CDC13 exhibits a proton magnetic resonance spectrum substantially as shown in FIG. 10;

(f) has a melting point in the range of about 156-158°C;

(g) has an optical rotation of [.alpha.]?7 = -191° (c 0.5, CHC13);

(h) has an apparent molecular weight of 1248 as determined by mass spectroscopy;
(i) has an approximate elemental composition of 52.1796 carbon, 6.15% hydrogen, 4.63% nitrogen, 9.0996 sulfur and 27.96% (by difference) oxygen;

(j) exhibits in silica gel thin layer chromatography an Rf value of 0.74 with the solvent system CHC13-CH3OH (5:1 v/v) and exhibits in reverse phase silica gel thin layer chromatography an Rf value of 0.18 with the solvent system CH3CN-H2O (75:25 v/v);

(k) when dissolved in methanol at a concentration of 0.01356 g/1 exhibits the following ultraviolet absorption maxima and absorptivities:

.lambda.Amax (nm) absorptivities 320 12.4 280 shoulder 253 25.1 210 25.5 with no significant change upon addition of acid or base;

(1) exhibits a high performance liquid chromatography retention time of 13.3 minutes with a C18 reversed phase silica gel column and the solvent system CH3CN-CH3OH-0.1M-CH3COONH4 (5:2:3 v/v);

(m) is effective in inhibiting the growth of various bacteria and fungi;

(n) induces prophage in lysogenic bacteria; and (o) is effective in inhibiting the growth of P-388 leukemia, L-1210 leukemia, B16 melanoma and Lewis lung carcinoma in mice.

15. The antitumor antibiotic BBM-1675 A2 which in substantially purified form:

(a) appears as white crystals;

(b) is soluble in chloroform, ethyl acetate, acetone, ethanol and methanol, slightly soluble in benzene and water, and insoluble in n-hexane and carbon tetrachloride;

(c) gives a positive reaction with ferric chloride, Ehrlich and Tollen's reagents and a negative reaction in Sakaguchi, ninhydrin and anthrone tests;

(d) exhibits an infrared absorption spectrum (KBr) substan-tially as shown in FIG. 12;

(e) when dissolved in CDC13 exhibits a proton magnetic resonance spectrum substantially as shown in FIG. 13;

(f) has a melting point in the range of about 147-149°C;

(g) has an optical rotation of [.alpha.]?7 = - 179.4° (c 0.5, CHC13);

(h) has an apparent molecular weight of 1248 as determined by mass spectroscopy;

(i) has an approximate elemental composition of 52.71%
carbon, 5.94% hydrogen, 3.94% nitrogen, 9.39% sulfur and 28.01% (by difference) oxygen;

(j) exhibits in silica gel thin layer chromatography an Rf value of 0.71 with the solvent system CHC13-CH3OH (5:1 v/v) and exhibits in reverse phase silica gel thin layer chroma-tography an Rf value of 0.21 with the solvent system CH3CN-H2O (75:25 v/v);

(k) when dissolved in methanol at a concentration of 0.02052 g/1 exhibits the following ultraviolet absorption maxima and absorptivities:

.lambda.max (nm) absorptivities 320 12.2 282 16.3 282 26.2 214 25.8 with no significant change upon addition of acid or base;

(1) exhibits a high performance liquid chromatography retention time of 17.3 minutes with a C18 reversed phase silica gel column and the solvent system CH3CN-CH3OH-0.1M
CH3COONH4 (5:2:3 v/v);

(m) is effective in inhibiting the growth of various bacteria and fungi;
(n) induces prophage in lysogenic bacteria; and lo) is effective in inhibiting the growth of P-388 leukemia, L-1210 leukemia, B16 melanoma and Lewis lung carcinoma in mice.

16. The antitumor antibiotic BBM-1675 A3 which:

(a) is soluble in chloroform, ethyl acetate, acetone, ethanol and methanol, slightly soluble in benzene and water, and insoluble in n-hexane and carbon tetrachloride;

(bJ gives a positive reaction with ferric chloride, Ehrlich and Tollen's reagents and a negative reaction in Sakaguchi, ninhydrin and anthrone tests;

~c) exhibits an infrared absorption spectrum (KBr) substan-tially as shown in FIG. 3, (d) when dissolved in CDC13 exhibits a proton magnetic resonance spectrum substantially as shown in FIG. 7;

te) has a melting point in the range of about 125-127°C;

(f) has an optical rotation of [.alpha.]?7 = - 161° (c 0.5, CHC13);

(g) has an approximate elemental composition of 54.55%
carbon, 6.46% hydrogen, 3.73% nitrogen, 7.49% sulfur and 27.77% (by difference) oxygen;

(h) exhibits in silica gel thin layer chromatography an Rf value of 0.72 with the solvent system CHC13-CH3OH (5:1 v/v) and exhibits in reverse phase silica gel thin layer chroma-tography an Rf value of 0.28 with the solvent system CH3CN-H2O (75:25 v/v);

(i) exhibits the following ultraviolet absorption maxima when dissolved in methanol, 0.01N HC1-CH3OH and 0.01N
NaOH-CH3OH

UV .lambda.max nm (E??m) : 253 (286) in methanol 282 (158) 320 (122) UV .lambda.max nm (E??m) : 253 (287) in 0.01N HC1-CH3OH 282 (160) 320 (126) UV .lambda.max nm (E??m) : 252 (280) in 0.01N NaOH-CH3OH 283 (162) 318 (120) ;

(j) exhibits a high performance liquid chromatography retention time of 8.0 minutes with a C18 reversed phase silica gel column and the solvent system CH3CN-CH3OH-0.1M
CH3COONH4 (5:2:3 v/v);

(k) is effective in inhibiting the growth of various bacteria and fungi;

(1) induces prophage in lysogenic bacteria; and (m) is effective in inhibiting the growth of P-388 leukemia in mice.

17. The antitumor antibiotic BBM-1675 A4 which:

(a) is soluble in chloroform, ethyl acetate, acetone, ethanol and methanol, slightly soluble in benzene and water, and insoluble in n-hexane and carbon tetrachloride;

(b) gives a positive reaction with ferric chloride, Ehrlich and Tollen's reagents and a negative reaction in Sakaguchi, ninhydrin and anthrone tests;

(c) exhibits an infrared absorption spectrum (KBr) substan-tially as shown in FIG. 4;

(d) when dissolved in CDC13 exhibits a proton magnetic resonance spectrum substantially as shown in FIG. 8;

(e) has a melting point in the range of about 123-126°C;

(f) has an optical rotation of [.alpha.]?7 - 176° (c 0.5, CHC13);

(g) has an approximate elemental composition of 54.65%
carbon, 6.29% hydrogen, 3.51% nitrogen, 8.07% sulfur and 27.48% (by difference) oxygen;

(h) exhibits in silica gel thin layer chromatography an Rf value of 0.71 with the solvent system CHC13-CH3OH (5:1 v/v) and exhibits in reverse phase silica gel thin layer chroma-tography an Rf value of 0.78 with the solvent system CH3CN-H2O (75:25 v/v);

(i) exhibits the following ultraviolet absorption maxima when dissolved in methanol, 0.01N HC1-CH3OH and 0.01N
NaOH-CH30H

UV .lambda.max nm (E??m) : 253 (257) in methanol 282 (153) 320 (117) UV .lambda.max nm (E??m) : 253 (258) in 0.01N HC1-CH3OH 282 (155) 320 (118) UV .lambda.max nm (E??m) : 252 (266) in 0.01N NaOH-CH3OH 283 (160) 318 (118);

(j) exhibits a high performance liquid chromatography retention time of 5.1 minutes with a C18 reversed phase silica gel column and the solvent system CH3CN-CH3OH-0.1M
CH3COONH4 (5:2:3 v/v);

(k) is effective in inhibiting the growth of various bacteria and fungi;

(1) induces prophage in lysogenic bacteria; and (m) is effective in inhibiting the growth of P-388 leukemia in mice.

18. The antitumor antibiotic BBM-1675 B1 which:

(a) is soluble in chloroform, ethyl acetate, acetone, ethanol and methanol, slightly soluble in benzene and water, and insoluble in n-hexane and carbon tetrachloride;

(b) gives a positive reaction with ferric chloride, Ehrlich and Tollen's reagents and a negative reaction in Sakaguchi, ninhydrin and anthrone tests;

(c) has a melting point in the range of about 159-161°C;

(d) has an optical rotation of [a]?7 - 171° (c 0.5, CHC13);

(e) exhibits in silica gel thin layer chromatography an Rf value of 0.63 with the solvent system CHC13-CH3OH (5:1 v/v) and exhibits in reverse phase silica gel thin layer chromatography an Rf value of 0.23 with the solvent system CH3CN-H2O (75:25 v/v);

(f) exhibits the following ultraviolet absorption maxima when dissolved in methanol, 0.01N HC1-CH3OH and 0.01N
NaOH-CH3OH

UV .lambda.max nm (E??m) : 253 (225) in methanol 282 (140) 320 (104) UV .lambda.max nm (E??m) : 253 (225) in 0.01N HC1-Ch3OH 282 (140) 320 (105) UV .lambda.max nm (E??m) : 252 (236) in 0.01N NaOH-CH3OH 283 (141) 318 (105);

(g) is effective in inhibiting the growth of various bacteria and fungi; and (h) induces prophage in lysogenic bacteria.

19. The antitumor antibiotic BBM-1675 B2 which:

(a) is soluble in chloroform, ethyl acetate, acetone, ethanol and methanol, slightly soluble in benzene and water, and insoluble in n-hexane and carbon tetrachloride;

(b) gives a positive reaction with ferric chloride, Ehrlich and Tollen's reagents and a negative reaction in Sakaguchi, ninhydrin and anthrone tests;

(c) has a melting point in the range of about 156-159°C;

(d) has an optical rotation of [.alpha.]?7 - 122° (c 0.5, CHCl3);

(e) exhibits in silica gel thin layer chromatography an Rf value of 0.60 with the solvent system CHCl3-CH3OH (5:1 v/v) and exhibits in reverse phase silica gel thin layer chroma-tography an Rf value of 0.16 with the solvent system CH3CN-H2O (75:25 v/v);

(f) exhibits the following ultraviolet absorption maxima when dissolved in methanol, 0.01N HCl-CH3OH and 0.01N
NaOH-CH3OH

UV .lambda.max nm (E??m) : 248 (212) in methanol 279 (141) 318 (103) UV .lambda.max nm (E??m) : 248 (210) in 0.01N HC1-CH3OH 279 (140) 318 (103) UV .lambda.max nm (E??m) : 248 (233) in 0.01N NaOH-CH3OH 278 (150) 318 (110) ;

(g) is effective in inhibiting the growth of various bacteria and fungi, and (h) induces prophage in lysogenic bacteria.

20. A biologically pure culture of the microorganism Actinomadura verrucosospora strain H964-92 (ATCC 39334), said culture being capable of producing the antibiotic BBM-1675 in a recoverable quantity upon cultivation in an aqueous nutrient medium containing assimilable sources of carbon and nitrogen.

21. A biologically pure culture of the microorganism Actinomadura verrucosospora strain A1327Y (ATCC 39638), said culture being capable of producing the antibiotic BBM-1675 in a recoverable quantity upon cultivation in an aqueous nutrient medium containing assimilable sources of carbon and nitrogen.