GB2141425A - BBM-1675, A new antitumor antibiotic complex - Google Patents

Abstract

An 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-1675A1 and A2, and four minor components, BBM-1675 A3, A4, B1 and B2, and such components exhibit both antimicrobial activity and antitumor activity.

Description

SPECiFICATION BBM-1675, a new antitumor antibiotic complex Background of the invention 1. Field of the invention This invention relates to new antitumor antibiotic substances and to their production and recovery.

2. Description of the prior art The antitumor 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. 95154A1 discloses fermentation of Actinomadura pulveraceus sp. nov.

No. 6049 (ATCC 39100) to produce antitumor antibiotics designated WS 6049-A and WS 6049-B. The structures of the WS 6049 antibiotics have not yet been elucidated, but the characterizing properties given for the antibiotics indicate that WS 6049-A and WS 6049-B may be related in structure to the BBM-1675 antibiotics of the present invention. Spectral data show, however, that neither WS 6049A nor WS 6049B is identical to any of applicants' BBM-1675 components. Moreover, the producing organism described in European Patent Application Publication No. 95154A1 may be clearly differentiated from Actinomadura verrucosospora employed in the present invention in the color of its aerial mycelium on ISP Medium Nos. 2, 3 and 4, in its positive milk peptonization and in its positive utilization of D-fructose, D-mannitol, trehalose and cellulose.

Summary of the invention There is provided by the present invention a new antitumor antibiotic complex designated herein as BBM-1675, said complex being produced by cultivating a BBM-1675-producing strain of Actinomadura verrucosospora most preferablyActinomadura verrucosospora strain H964-92 (ATCC 39334) orActinoma dura verrucosospora strain A1327Y (ATCC 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 1675A1 and A2 and four minor bioactive components of said complex designated BBM-1 675A3, A4, B1 and B2. The components may be separated and purified by conventional chromatographic procedures. The BBM-1675 complex and its bioactive components exhibit both antimicrobial and antitumor activity.

Description ofthe drawings Figure 1 shows the infrared absorption spectrum of partially purified BBM-1675 A1 (KBr pellet).

Figure 2 shows the infrared absorption spectrum of partially purified BBM-1 675 A2 (KBr pellet).

Figure 3 shows the infrared absorption spectrum of B BBM-1675 A3 (KBr pellet).

Figure 4 shows the infrared absorption spectrum of BBM-1675 A4 (KBr pellet).

Figure 5 shows the proton magnetic resonance spectrum of partially purified BBM-1675 A1 in CDC13 (60 MHz).

Figure 6 shows the proton magnetic resonance spectrum of partially purified BBM-1675 A2 in CDCl3 (60 MHz).

Figure 7 shows the proton magnetic resonance spectrum of BBM-1675 A3 in CDC13 (60 MHz).

Figure 8 shows the proton magnetic resonance spectrum of BBM-1675 A4 in CDC13 (60 MHz).

Figure 9 shows the infrared absorption spectrum of purified BBM-1 675 A1 (KBr pellet).

Figure 10 shows the proton magnetic resonance spectrum of purified BBM-1675 A1 in CDC13 (360 MHz).

Figure 11 shows the 13c magnetic resonance spectrum of purified BBM-1675 A1 in CDC13 (90.3 MHz).

Figure 12 shows the infrared absorption spectrum of purified BBM-1675 A2 (KBr pellet).

Figure 13 shows the proton magnetic resonance spectrum of purified BBM-1 675 A2 in CDC13 (360 MHz).

Figure 14 shows the 13C magnetic resonance spectrum of purified BBM-1 675 A2 in CDCI3 (90.3 MHz).

Detailed description This invention relates to a novel antitumor antibiotic complex designated herein as BBM-1675 and to its preparation by fermentation of certain strains of Actinomadura verrucosospora, most particularly Actinomadura verrucosopora strain H964-92 and a mutant thereof designated Actinomadura verrucosospora strain A1327Y. The above-mentioned parent strain was isolated from a soil sample collected at Pto Esperanza, Misiones, Argentina. A biologically pure culture of the organism has been deposited (28th March, 1983) with the American Type Culture Collection, Washington, D.C. and added to its permanent collection of microorganisms as ATCC 39334.Subsequently, the mutant strain A1327Y was obtained by conventional nitrosoguanidine (NTG) treatment of strain H964-92 and was deposited with the American Type Culture Collection as ATCC 39638 on 21st March, 1984.

As in the case of many antibiotic-producing cultures, fermentation of Actinomadura verrucosospora strain H964-92 or strain A1327Y results in the production of a mixture or complex of component substances. Two major bioactive components, BBM-1675 A1 and A2, and four minor bioactive components, BBM-1675 A3, A4, B1 and B2, have been separated from the BBM-1675 complex produced during the fermentation process.

BBM-1675 and its components BBM-1675 A1, A2, A3, A4, B1 and B2 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 phage inducing properties in lysogenic bacteria. Two of the components, BBM-1675 A1 and A2, have been submitted to in vivo 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 A3 and A4 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 antitumor 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 biologically pure culture for characterization. Strain H964-92 forms on the aerial mycelium short spore-chains which show straight, flexuous or hooked shapes. The spores are spherical or oval-shaped and have a warty surface. Aerial mycelium 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 agar media. The color of substrate mycelium is colorless or pale pink. The growth temperature ranges from 15"C to 430C.The cell-wall amino acid composition and whole cell hydrolyzate sugar components show that strain H964-92 belongs to cell wall Type Ills. The menaquinone was identified as MK-9(H6) MK-9(H8).

Based on the major morphological, cultural and physiological 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 mycelia, a variant showing good growth and improved aerial mycelium formation was obtained by NTC (nitrosoguanidine) treatment of H964-92. The variant, designated strain A1327Y, 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 recommended by the International Streptomyces Project (Inc. J. Syst Bacterial 16: 313-340, 1966).

Additional media described by S. A. Waksman (TheActinomycetes, Vol. 2) and G. M. Lvedemann (Int J.

Syst Bacteriol. 21: 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 al. in Appl.

Microbiol. 13: 236-243, 1965 and by Lechevalier and Lechevalier in TheActinomycetes, Ed. H. Prauser, Jena, Gustav Fischer Verlag, pp. 393-405, 1970, respectively. The menaquinone was identified by mass spectral analysis according to the procedure of Collins et al. in J. Gen. Microbiol. 100: 221-230, 1977, and the menaquinone composition was represented based on the system described by Yamada et al. in J. Gen. Appi.

Micrnbiol 23: 331-335, 1977.

Morphology Strain H964-2 forms both substrate and aerial mycelia. The substrate mycelium is long, branched and not fragmented into short filaments. In the aerial mycelium, 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, flexuous or hooked in shape. The spores have a warty surface and are spherical to elliptical (0.5-0.6 x 0.6-1.4 Am) in shape with rounded or pointed ends.

After maturation each spore is often separated with empty sheath. Motile spores, sporangia 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 mycelium is generally poor but is moderate in oat meal agar (ISP No. 3 medium), inorganic salts-starch agar (ISP No. 4 medium) and Bennett's agar. Spontaneous variants which lack aerial mycelium occur at high frequency. The color of aerial mycelium is white which later turns to pale pink in oat meal agar, inorganic salts-starch agar and glycerol-asparagine agar (ISP No. 5 medium). The aerial mass colorfurther changes to a bluish color after long incubation (5 months) in oat meal agar, glycerol-asparagine agar and tyrosine agar. The color of substrate mycelium is colorless to yellowish in Czapek's agar, tyrosine agar, yeast extract-malt extract agar (ISP No. 2 medium), peptone-yeast extract-iron agar (ISP No. 6 medium) and Bennett's agar, and is a pinkish color in glucose-asparagine agar and glycerol-asparagine agar.

Melanoid and other diffusible pigments are not produced. A variant No. A1327Y, which was obtained from the original strain, forms predominantly pale blue aerial mycelium and bears abundant aerial spore mass.

Strain H964-92 grows at 1 5 C, 280C, 37"C and 43"C, but not at 10C or at 47"C. It is sensitive to NaCI at 7%, and resistant to lysozyme at 0.01%.

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 of Strain H964-92 (original strain A TCC 39334 and variant A 1327Y) Strain No. H964-92 Actinomadura Original Strain verrucosospora (A TCC 39334) Variant No. A 1327Y KCCA-0147 Tryptone-yeast extract G: abundant, floccose, moderate, floccose, moderate, floccose, agar (ISP No. 1) sedimented and not sedimented and not sedimented and not pigmented pigmented pigmented Sucrose-nitrateagar G: moderate poor poor (Czapek's agar) R: colorless colorless colorless A: scant; light gray no or scant; pinkish no or scant; pale (264), to pale pink white (9) blue (185) (7) D: none none none Glucose-asparagine agar G: moderate poor poor R: white (263) to deep colorless colorless yellowish pink (277) A: no or very scant; no or very scant; no or very scant; pale pink (7) white white D:none none none Glycerol-asparagine agar G: poor to moderate moderate moderate (ISP No. 5) R: colorless to light light yellowish light yellowish pink yellowish pink (28) pink (28) (28 to deep yellow ish pink (27) A: poor; light yellow- moderate; white to moderate; white to ish pink (28), after light pink (4) strong pink (2) 5 months light bluish gray (190) D: none none none Inorganicsalts-starch G: abundant moderate moderate agar (ISP 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) (185) (10) D: none none none Tyrosineagar G: moderate moderate moderate (ISP No.7) R: yellowish white (92) strong yellowish pink strong yellowish (26) pink (26) A: poor; light yellow- moderate;white to moderate; white to ish pink (28), very light pink (4) light pink (4) later (5 months) partially light bluish gray (190) D: none none none Nutrient agar G: poorto moderate poor poor R: pale yellow (89) colorless to pale colorless to pale pink(7) pink(7) A: none none none D: none none none TABLE 1 - cont'd Strain No. H964-92 Actinomadura Original Strain verrucosospora (A TCC39334) Variant No.A 1327Y KCCA-0147 Yeast extract-malt G: abundant abundant abundant extract agar R: pale yellow (89) strong yellowish strong yellowish (ISP No.2) pink (26) pink (26) A: poor; white (263) poor; white to pale poor; white to pale pink (7) pink (7) D: none none none Oat meal agar G: moderate poor poor (ISP No.3) R: colorless to pale pale yellowish pale yellowish pink (7) pink(31) pink (31) A: poor; pinkish very scant; vivid very scant; vivid white (9) to light pale blue (184) pale blue (184) bluish gray (190) D: none none none Bennett's agar G: abundant abundant abundant R: grayish yellow (90) strong yellowish strong yellowish pink (26) pink (26) A: moderate; white (263) moderate; pale yellow- none to yellowish white ish pink (31) and (92) bluish white (189) D: none none none Peptone-yeast extract- G: moderate abundant abundant iron agar (ISP 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 mycelium; D = Diffusible pigment Color and number in parenthesis follow the color standard in "Kelly, K. L. & D. B. Judd; ISCC-NBS color-name charts illustrated with Centroid Colors. U.S. Dept. of Comm. Cir. 553, Washington, D.C., Nov., 1975".

KCC = Kaken Culture Collection of Kaken Chemical Company TABLE 2 Physiological Characteristics of Strain H964-92 Test Response Method and Medium Range of temperature Maximal growth at Bennett's agar for growth 28"C to 37"C.

Moderate at 20"C and 43"C. No growth at 10"C and 47"C.

Gelatin liquefaction Liquefied Glucose-peptone-gelatin medium Starch hydrolysis Hydrolyzed Starch agar plate Reactions in skimmed Not coagulated Difco skimmed milk milk and completely peptonized Formation of melanoid Not produced Tyrosin agar, peptone-yeastpigment iron agar and tryptone-yeast extract broth.

Nitrate reduction Not reduced Czapek's glucose-nitrate broth and glucose-yeast extract nitrate broth Resistance to NaCI Growth at 5% or Tryptone-yeast extract agar less. No growth at 7%.

Lysozyme Resistant. Growth Tryptone-yeast extract agar at 0.01% or less.

No growth at 0.1%.

pH Growth in 5.0 to Tryptone-yeast extract agar 9.5. No growth at 4.5 and 10.0.

TABLE 3 Utilization of Carbon Sources Strain No, H964-92 Actinomadura Original Variant verrucosospora strain No. A 1327Y KCC A-0147 Glycerol + + + D(-)-Arabinose - L(+)-Arabinose + + + D-Xylose + + + D-Ribose + L-Rhamnose + + + D-Glucose + + + D-Galactose - D-Fructose + + + D-Mannose - L(-)-Sorbose Sucrose + + + Lactose - - Cellobiose + + + Melibiose - Trehalose + + + Raffinose - D(+)-Melezitose Soluble starch + + + Cellulose + + + Dulcitol - - Inositol + - D-Mannitol + + + D-Sorbitol - Salicin - Observed after incubation at 28"C for 3 weeks Basal medium:Pridham-Gottlieb inorganic medium Cell-wall composition and whole cell sugar components Purified cell-wall of strain H964-92 contains mesodiaminopimelic acid but lacks glycine. The whole cell hydrolyzate shows the presence of madurose (3-O-methyl-D-galactose), glucose and ribose. The cell-wall amino acid and whole cell sugar components indicate that strain H964-92 is placed in cell-wall Type lllB. Two major components of menaquinone were identified as MK-9(H6) and MK-9(H8).

Taxonomic position ofstrain H964-92 Strain H964-92 has the following major characteristics: (1) Aerial spore-chains: short, straight, flexuous or hooked in shape. (2) Spores: warty surface. (3) Aerial mycelium: pinkish or bluish color. (4) Substrate mycelium: pinkish in some media. (5) Diffusible pigment: none. (6) Mesophile. (7) Cell-wall Type Ills. (8) Menaquinonesystem: MK-9(H6) and MK-9(H8).

These major characteristics indicate that strain H964-92 is placed in the genusActinomadura. 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 theActinomadura and related actinomycetes by Goodfellow et al. in J.

Gen. Microbiol. 112: 96-111 (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 Ohara in J. Ferment Technol. 49: 904-912 (1971) reported five saprophytic species of the genus Actinomadura and Nonomura [J. Ferment Technol. 52:71-77(1974)] and Preobrazhenskaya petal.

[Actinomycetes and Related Organisms 12: 30-38 (1977)] published the keys for identification and classification 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 coerulea described in the Preobrazhenskaya et al. reference above and to Actinomadura verrucosospora described in the Nonomura references cited above.

Strain No. H964-92 was directly compared with A. verrucosospora strain KCC A-01 47 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 BBM-1675, the present invention, though described in detail with reference to the particular strain Actinomadura verrucosospora strain H964-92 (ATCC 39334) and the mutant strain thereof designated strain A1327Y (ATCC 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-producting 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 ofActinomadura verrucosospora having the identifying characteristics of ATCC 39334 or ATCC 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 antibiotics, although for production of limited amounts, surface cultures and bottles may also be used. The general procedures used for the cultivation of other actinomycetes are applicable to the present invention.

The nutrient medium should contain an appropriate assimilable carbon source such as glycerol, L(+)-arabinose, D-xylose, D-ribose, L-rhamnose, D-glucose, D4ructose, sucrose, cellobiose, soluble starch, D-mannitol or inositol. 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 peptone, 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-1 675 antibiotics can be effected at any temperature conducive to satisfactory growth of the producing organism, e.g. 15-45"C, and is conveniently carried out at a temperature of around 27-32"C. Ordinarily, optimum production is obtained after incubation periods of from about 68-180 hours, depending on whether shake-flask, stir-jar or tank fermentation 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 transferred aseptically to the fermentation tank medium. Antibiotic production may be monitored by the paper disc-agar diffusion assay using Staphylococcus aureus 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. Antiobiotic 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 with n-butanol. The above-mentioned n-butanol and methanol extracts may then be combined and evaporated azeotropicallyto 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 precipitated from solution by addition of an antisolvent such as n-hexane.

The crude BBM-1675 complex is a mixture of several components including two major bioactive components, BBM-1675 As and A2, and four minor bioactive components, BBM-1675 A3, A4, B, and B2. These bioactive components may be separated and purified by conventional chromatographic procedures. In one procedure the crude BBM-1 675 complex is first dissolved in methanol and purified by Sephadex LH-20 column chromatography using methanol as the eluting solvent. This partially purified complex may then be chromatographed on a silica gel column and eluted in a stepwise manner using chloroform plus an increasing concentration of methanol to provide BBM-1675 A1, a mixture of BBM-1675 A2, A3 and A4 and a mixture of BBM-1675 B1 and B2.The A1 component may be further purified by Sephadex LH-20 column chromatography using methanol as the eluting solvent. The mixture of A2, A3 and A4 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 B1 and B2 components may be separated by silica gel column chromatography using a mixture of chloroform and methanol as the eluting solvent. Further details of the preferred chromatographic separation procedures are provided in the examples which follow.

Physico-chemical properties of BBM-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.

TABLE 4 TLC of BBM-1675 Components Rf Values SiO2 * Silanized Component CHCl3-CH3OH(5; 1 vlv) CH3CN-H2O(75:25 v/v) BBM-1675A1 0.74 0.18 BBM-1675A2 0.71 0.21 BBM-1675A3 0.72 0.28 BBM-1675A4 0.71 0.78 BBM-1675 B1 0.63 0.23 BBM-1675 B2 0.60 0.16 * C18 reverse phase silica gel Separation of BBM-1675 A2, A3 and A4 was difficult by ordinary phase TLC systems but could be achieved by a reverse phase TLC.

The individual BBM-1675 components show solubility and color reactions similar to each other. For example, they are soluble in chloroform, ethyl acetate, acetone, ethanol and methanol, slightly soluble in benzene and water, and insoluble in n-hexane and carbon tetrachloride. They give positive reactions with ferric chloride, Ehrlich and Tollen's reagents but negative responses in Sakaguchi, ninhydrin and anthrone tests.

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

TABLE 5 Physico-Chemical Properties of BBM-1675 Components BBM-1675A1 A2 A3 A4 B, B2 Melting point (dec) 156 ~158 C 147 ~ 149 C 125127'C 123 ~ 126 C 159 ~ 161 C 156 ~ 159 C (α)D27 (c 0.5, CHCl3) -191 -179A' -161" -176" -171" -122" Anal.Found (%), 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) O: 38.65 36.06 34.91 36.53 (UV Amax nm(El1Scm) in CH3OH 253 (325) 253 (281) 253 (286) 253 (257) 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) in 0.01 N HCl-CH3OH 253 (323) 253 (276) 253 (287) 253 (258) 253 (225) 248 (210) 282(192) 282 (167) 282(160) 282 (155) 282 (140) 279 (140) 320(144) 320 (128) 320(126) 320 (118) 320 (105) 318(103) in 0.01 N NaOH-CH3OH 252 (325) 252 (289) 252 (280) 252 (266) 252 (236) 248 (233) 283 (172) 283 (171) 283 (162) 283 (160) 282 (141) 27 & 150) 318(1-36) 318(122) 318 (120) 318(118) 318(105) 318(110) Mo. wt.

(approximate value) 1,300 1,100 1,100 1,400 (Gel HPLC, Finepak (GEL-101) The UV absorption maxima of BBM-1675 components were observed at 253, 282 and 320 nm, which did not shift in acidic or alkaline solution. The IR and PMR spectra of BBM-1675 A1, A2, A3 and A4 are shown in Figures 1-4 and Figures 5-8 respectively. The 360 MHz PMR of BBM-1675 A1 indicated one acetyl (3:2.11 ppm), one N-CH3(2.52 ppm), four OCH3(3.42, 3.80, 3.88 and 3.98 ppm) and one exomethylene (4.57 and 5.48 ppm) groups, along with two aromatic (7.50 and 8.59 ppm) and one NH (11.79 ppm) protons. The CMR spectrum of BBM-1675A1 exhibited 55 carbon signals including a triple intensity signal (8:56.0 ppm, OCH3).

The molecular formula of BBM-1675A1 is deduced to be C57H72N4032 based on proton and 13C NMR spectra, micronalysis and molecular weight determination by HPLC and SIMS (secondary ion mass spectrometry).

Structural study of BBM- 1675A, Upon treatment with 0.5N HCI-CH30H at room temperature, BBM-1 675A1 loses its bioactivity and affords a lipophilic chromophore substance (compound I) along with several unidentified fragments.Compound I shows UV absorption similar to that of parent antibiotic suggesting that compound I retains the chromophoric structure of BBM-1 675A1. Two other chromophoric fragments related to compound I are obtained by alkaline hydrolysis of BBM-1 675A1: hydrolysis with 0.05N KOH-CH3OH at 55"C for one hour yields compound II having UV absorption maxima at 252,284,297 (shoulder) and 322 nm, while the reaction in 1 N KOH-CH30H affords an acidic chromophore substance designated compound Ill. Physico-chemical properties of compounds I, II and III are summarized in Table 6 below.

TABLE 6 Properties of Compounds I, Il and 111 Compound I Compound Compound lil M.p. 82 - 83"C 133" 253 - 255"C [(x]D9(c 0.2 CHCIg) -100" 0 0 Molecular formula C21H31N010 C14H17N06 C13H15NOs UV A CH30H nm (E) 244 (21,850) 252 (26,600) 248 (26,900) max 276 (9,400) 283 (11,200) 295(14,400) 318 (6,300) 297 (sh8,800) 310 (13,500) 318 (6,300) 322(11,700) MS m/z 457 (M+) 295 (M+) 281 (M+) 425 280 263 341 263 236 281 251 222 264 248 218 TLC (Xylene-*MEK-CH30H=5:5:1 v/v) :Rf0.58 0.66 0.13 * MEK = methyl ethyl ketone Structural information about compounds II and lil was provided from the following spectral data and chemical transformation. The 13C and proton NMR indicated the presence of four OCH3, one =CH2, seven two > C=O and one > NH groups in compound 11. The NMR spectra of compound Ill was similar to those of II, differing only in the absence of one of the four OCH3 groups observed for compound II. This difference, together with the acidic nature of compound Ill, suggested that compound II is a methyl ester of compound III. When heated under reflux with 1 N methanolic KOH, compound II was quantitatively converted to compound III, while compound Ill was converted to compound II by treatment with diazomethane.

Treatment of compound II with NaBH4 in C2H5OH gave a reduction product (compound IV, M+:m/z 267) which showed a -CH2OH group in the NMR in place of the -COOCH3 group of compound II. Upon hydrogenation over palladium on charcoal, compound II afforded a dihydroderivative (compound V, M+:m/z 297). The proton NMR 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 OCH3 groups appeared at higher field (8: 3.50 ppm) in compound V. These results indicated the presence of a

group in compound II which was reduced by hydrogenation to

group in compound V.Compound II was heated with 1 SN methanolic hydrogen chloride at 80"C for 3 hours and the hydrolyzate chromatographed on a silica gel column to afford a weakly basic compound (compound VI, M+: m/z 211). TheIR 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 IR and NMR studies with an authentic sample. Consequently, the structures of compounds Il-VI were determined as shown below.

Structures of compounds 11, III, IV, Vand Vl

Compound II Compound III CR //CH2 CH30 S XOCH3 - cH3 H-CC CH3 COOCH3 - \SOCH. OOH U300H NaBH4 Comound IV \ \ Compound IV - Pd/C CH3 +/MeOH 2Pd/c < H20H Compound V Compound VI . CR CH3 CH3 NH2 CR3 NH-CO-CR I OCR3 00CR CR3O 3 CH3ON 3 COOCH CH3 OOCH3 CH3 COOCH3 Upon treatment with 0.05N KOH-CH3OH at 55"C, compound I was split into a new chromophoricfragment (compound VII, CX5H21NO7, M+ : m/z 327) and a sugar (compound VIII). The NMR spectrum of VII exhibited one singlet C-CH3 and two high-field OCH3 groups in addition to three low-field OCH3 and two aromatic protons commonly observed for compounds II, V and VI. Further hydrolysis of VII with 1 SN 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 Vill did not show the molecular ion peak but did show the M-OCH3 peak at m/z 131 in the mass spectrum in agreement with the molecular formula of C7H,404. The NMR spectrum indicated 2,6-dideoxyhexopyranose structure for compound VI II. The assignment was supported by the 13C-NMR of compound I which gave rise to one C-CH3, one -CH2, three O-CH < and one anomeric carbon in addition to 15 carbon signals assignable to compound VII. The C3 proton of the sugar appeared at low-field (5: 5.39 ppm, octet) revealing the C3-OH of the sugar was esterified by the carboxyl group of VIII. The above results are summarized below: Structures of compound 1, VII and Vlil

Compound I Compound VII Compound V:: CCa 1 OCs OC 3 Cz3a OCE3 / C330NH2 o'=83 08-/CHJOH~I\ MjOlMI-CO-t-CBj 308) 3i3 8) Co O'=83 oC ca3N OOCs3 3 C8 '=83 Ca CoC83 Ca a13 Compound VIII 08 Os C8 8 0C53 3-COCooH The molecular weight of compound 1(457) accounts for about one-third of the entire molecule of BBM-1675A1 (proposed formula C57H72N4032; calc'd MW = 1324).The partial structure of BBM-1675 A1 is considered to be as follows:

Subsequent to the U.S. filing date of parent application Serial No. 495,231, it was discovered that components BBM-1 675 A1 and A2 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, BBM-1675 As and A2 were isolated in more purified form and fully characterized as described below. Also, the elemental analysis data for components A3 and A4 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.

BBM- 1675A t Description: white to pale yellow crystals; mp 156-158"(dec.) Elemental analysis: Analysis 1 Analysis 2 Average C: 51.60% 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 (by difference):28.31% 0 (by difference): 27.62% 0 (by difference):27.96% Ultraviolet absorption spectrum:Instrument-Varian UV, Cary 219 Solvent- methanol Concentration = 0.01356 g/l AmaxrnmJ absorptivities 320 12.4 280 sh (shoulder) 253 25.1 210 25.5 No significant change with acid or base Optical rotation: Solvent-CHCl3 [a]D4 = -207 (C=0.0351) A second analysis showed the following optical rotation: [&alpha;]D27 = -191" (C=0.5, CHCl3).

Infrared absorption spectrum: See Figure 9 Major absorption bands (KBr): 985,1015,1070,1110,1150,1210,1250,1308,1380,1405,1446,1520,1592,1608, 1668,1715,2920,2960,3360,3440, cm-' Mass spectra: Instrument - VG-ZAB-2F FAB-MS-thioglycerol Molecular mass range ions (m/z): 1249,1357, 1463; with the addition of NaCI (m/z): 1271,1379,1485, 1597.

FAB-MS-MB (MB:matrix, m.w. 154); Molecular mass range ions (m/z): 1249, 1283, 1403, 1555; with the addition of NaCI (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 NaCI (m/z): 1215,1237,1247,1269, 1325, 1347,1375.

Instrument: Kratos MS-50 FAB-MS-thioglycerol; Molecular mass range ions (m/z): 1357,1463.

Molecular weight (based on above-described apparent MW = 1248 mass spectral data): Nuclear Magnetic Resonance Spectra: Instrument - WM360 Brucker Solven: CDCl3 'NMR: 360 MHz8(ppm): 11.75 (1H,s); 8.55 H, s); 7.45 (1H, s); 6.61 (1H, m); 6.23(1 H, brs); 6.17 H, brs); 5.93 (1H, d, J=9.3); 5.82(1 H, d, J=9.3); 5.7 (1H, brs); 5.49 (1H, m); 5.45 (1H, d, J=2.3); 5.38 (1H, brs); 4.95 (1H, d, J=10.2); 4.64 (2H, m); 4.54 (1H, d, J=2.3); 4.2 (1H, s); 4.15-3.35 (26-28H) [4.10 (1H, m); 4.02 (1H, brs); 3.95 (3H, s); 3.85 (3H, s), 3.79 (3H, s); 3.46 (1H, m); 3.40 (3H, s)]; 2.82-2.70 (3H, brm); 2.50 (3H, 5), 2.47 (1H, m); 2.38-2.22 (5H); 2.12 (1H, m); 2.11 (3H, 5); 1.60-1.05 (22H) [1.39 (3H, d, J=6.3); 6.31 (3H, d, J=6.3); 1.29 (3H, d, J=6.3), 1.08 (6H)] See Figure 10 13C NMR: 90.3 MHz See Figure 11 In a separate test the 13C NMR spectrum of purified BBM-1675 A1 was determined for a sample dissolved in CDCI3 (80 MHz). Major peaks are indicated below.

CMR of BBM- 1675A, (80 MHz in CDCL3) Chemical shift in ppm (Multiplicity*) BBM-1675A1 13.7(q) 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) 89.5(t) 97.2(d) 98.3(s) 99.0(d) 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(s) 160.7(s) 166.4(s) 191.8(s) *Multiplicity - q = quartet; d = doublet; u = uncertain t = triplet; s = singlet BBM- 1675A2 Description: white crystals; mp 147-149"C Elemental analysis: C: 52.71% H: 5.94% N: 3.94% S: 9.39% O(by difference): 28.01% Ultraviolet absorption spectra: Instrument-Varian UV. Cary 219 Solvent: methanol Concentration: 0.02052 g/l AmaX (nm) absorptivities 320 12.2 282 16.3 252 26.2 214 25.8 No significant change with acid or base.

Optical rotation: [D27 = - 179.4" (c 0.5, CHC13) Infrared spectra: See Figure 12 Instrument: Beckman IR Model 4240 Major absorption bands (KBr): 950,1015,1070,1100, 1155,1213,1250,1313, 1375, 1405, 1450, 1520, 1595, 1610, 1685, 1735,2940,2980,3440, cm-l.

Mass spectra: Instrument: VG-ZAB-2F FAB-MS-thioglycerol Molecular mass range ions (m/z): 968, 1249, 1355, 1357, 1463, 1569; with addition of NaCI (m/z): 990, 1271,1379,1485,1593 FAB-MS-MB (MB: matrix, m.w. 154); Molecular mass range ions (m/z): 1249, 1403, 1419, 1555, 1571, 1587; with the addition of NaCI (m/z): 1249, 1271, 1425, 1441,1457,1483,1577.

FAB-MS-glycercol-DMSO; Molecular mass range ions (m/z): 1215, 1231, 1247, 1263, 1279,1293, 1309, 1325, 1326, 1341, 1353, 1369.

Molecularweight: apparent MW = 1248 (based on above-described mass spectral data) Nuclear Magnetic: See Figure 13 Resonance Spectra Instrument: WM 360 Bruckner Solvent: CDCI3 'H NMR 360 MHz 3(ppm): 11.91 (1H, s); 8.62 (1H, s); 7.58 (1H, s); 6.56 (1H, m); 6.22 (1H, s); 6.15 (1H, brs); 5.91 (1H, d, J=9.6); 5.83 (1H, d, J=9.6); 5.70 H, m); 5.45 (1H, d, J=2.2); 5.44(1H, s), 5.34(1H, brs); 4.95 (1H, d, J=10.2); 4.75 (1H, m); 4.65 (1H, d, J=6.8); 4.54 (1H, d, J=2.2); 4.47 (1H, m); 4.18(1H, s); 4.10 (1H, brs), 4.05-3.50 (20-24H); [3.96 (3H, s); 3.87 (3H, s); 3.77 (3H, s)] 3.46 (1 H, m); 3.39 (3H, s); 2.79 (1H, m); 2.73 (2H, m); 2.50 (3H, s); 2.50 (1H, m); 2.38-2.22 (3H); 2.14 (1H, m); 2.10 (3H, s); 1.98 (2H, m); 1.65-1.45 (6-8H); 1.38 (3H, d, J=6.0); 1.34 (3H, d, J=6.0); 1.22 (3H, d, J=6.8); 1.10 (6H).

13C NMR 90.3 MKz See Figure 14 In a separate test the 13C NMR spectrum of purified BBM-1675A2 was determined for a sample dissolved in CDCI3 (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 99.6 103.8 107.1 112.4 123.2 124.8 129.9 137.3 144.1 154.2 154.5 160.9 167.9 192.2 TABLE 7 Physico-chemical Properties of BBM- 1675 A3, A4, B,, B2 A3 A4 B1 B2 Melting point(dec): 125-127"C 123-126"C 159-161"C 156-159"C [&alpha;]D27(c0.5, CHCl3): -161"C -176"C -171"C -122"C Anal.Found (%): C: 54.55 54.65 H: 6.46 6.29 N: 3.73 3.51 S: 7.49 8.07 UV AmaX nm (E11,,'m): 253 (286) 253 (257) 253 (225) 248 (212) in MeOH 282 (158) 282 (153) 282 (140) 279 (141) 320(122) 320(117) 320(104) 318(103) in 0.01 N HCI-MeOH: 253 (287) 253 (258) 253 (225) 248 (210) 282(160) 282(155) 282(140) 279(140) 320(126) 320(118) 320(105) 318(103) in 0.01 N NaOH-MeOH: 252 (280) 252 (266) 252 (236) 248 (233) 283(162) 283(160) 283(141) 278(150) 318(120) 318(118) 318(105) 318(110) Thin-layerchromatography (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 (Rf) HPLC fretention time, in minutes SiO2 *Silanized Lichrosorb RP-18 CHCI3-MeOH CH3CN-H20 CH3CN-MeOH-0.1M CH3COONH4 85:1 v/v) 75:25 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 A1 and A2 components TLC Chromatography Analtech GHLF 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 (o.d.) by 12 cm and containing 10 ml of eluant. Plates 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 (12.7cm wide, 8.6cm deep, 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 nv uv light first. The plates were then inserted into a glass cylinder (6.4 cm o.d. by 12 cm) containing 12 crystals. The plates were then reexamined after approximately 2 minutes. Reversed phase plates were visualized under 254 nm uv 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 6000A Solvent Delivery System pump; Varian Varichrom Model VUV-10 uv/vis Detector set at 254 nm 0.1 OD; Fisher Recordal Series 5000 Recorder; Waters Associates Model 660 Solvent Programmer; Waters Associates Model U6K injector; Alltech, ll-Bondapak C18 (10) column (4.6 mm i.d. x 25 cm) with a Whatman Co. Pell ODS (0.03-0.038 mm) guard column (4.6 mm i.d. x 5 cm). The components were connected with 316 stainless steel tubing (1.6 mm o.d. - 0.23 mm i.d.). Eluant was pumped at 2 ml/min for all analysis.

Preparative HPLC The following components were used to construct a medium pressure liquid chromatography system: Fluid Metering, Inc. Model RP-SY 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 o.d. x 1.5 mm i.d.) wrapped around a cardboard tube (8.65 cm o.d.); Glenco Series 3500 Universal LC columns; Instrument Specialties Co. Model UA-5 Absorbance/Fluoresence Monitor with a Type 6 optical unit; Instrumentation Specialities 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 o.d. x 1.5 mm i.d.) and Glenco multifit connectors and valves in the order listed.

The Glenco series 3500 Universal LC 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. Eluant was pumped at a maximum rate which would not exceed 60 psi back pressure (approximately 20 ml/min).

Gradient elution A Glenco gradient elution apparatus consisting of two chambers of equal diameter, height and volume connected in tandem with a Teflon valve was used for all gradient elutions. 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 3.7cm) were placed in both chambers and driven by Thomas Model 15 Magne-matic stirrers. Eluant was pumped from the mixing chamber to the medium pressure HPLC system through polypropylene tubing (1.5 mm ID x 3.0 mm OD). 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.

TLC analysis ofBBM-1675A, andA2 Summarized in Table 9 below are the observed Rf values for BBM-1675A1 and A2 on normal phase plates.

Rf 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 Rf SystemlCompound BBM- 1675A1 BBM- 1675A2 4% methanol in chloroform 0.33 0.30 5% methanol in diethyl ether 0.39 50% acetone in Skellysolve B 0.38 0.31 Summarized in Table 10 below are the observed Rf values of BBM-1675A1 and A2 on normal phase plates developed in two dimensions. The position of the spots are expressed in Cartesian coordinates. The X coordinate is the Rf values of the second listed solvent system. The Y coordinate is the Rf values of the first listed solvent system.

TABLE 10 Rf SystemlCompound BBM- 1675A7 BBM- 1675A2 4% methanol in chloroform vs 5% methanol in diethyl ether (0.34, 0.33) (0.28, 0.23) 4% methanol in chloroform vs 50% acetone in Skellysolve B (0.33, 0.29) Summarized in Table 11 below are the observed Rf values for BBM-1675A1 and A2 on C-18 reversed phase TLC plates developed in binary eluants.

TABLE 11 Rf SystemlCompound BBM- 1675A, BBM- 1675A2 25% 0.5 M NaCI in acetonitrile 0.18 0.21 25% water in acetonitrile 0.00 0.00 Summarized in Table 12 below are the observed Rf values of BBM-1675A1 and A2 on C-18 reversed phase TLC plates developed with ternary eluants.

TABLE 12 Rf SystemlCompound BBM- 1675A1 BBM- 1675A2 Acetonitrile : Methanol : 0.5 NaCL 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 0.1M NH40Ac 40% : 30% : 30% 0.32 0.22 Acetonitrile : Methanol : 0.1M NaH2PO4 40% : 30% : 30% 0.00 0.00 HPLC analysis of BBM- 1675A, andA2 BBM-1675A1 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' - TR-To To where TR is the retension time measured from time of injection to peak apex and To is the void volume time.

TABLE 13 K' SystemlCompound BBM- 1675A1 BBM- 1675A2 Acetonitrile a a Tetrahydrofuran a a Methanol 0.00 0.00 a = compound did not elutefrom column.

TABLE 14 K' SystemlCompound BBM- 1 675A, BBM- 1 675A2 25% water in acetonitrile a a 25% methanol in water 1.25 1.25 a = compound did not elute from column.

TABLE 15 Ternary Eluants K' SystemlCampound BBM- 1675A, BBM- 1675A2 Acetonitrile : Methanol : water 40% 30% 30% a a Acetonitrile : Methanol : 0.1M NH40Ac 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% 7.8 b 41.5% 21.5% 37.0% 9.7 b a = did not elute b = not determined Biological properties of BBM- 1675 components Antimicrobial activity of the BBM-1675 components was determined for a variety of bacteria (grampositive, gram-negative and acid-fast) and fungi by the serial two-fold agar dilution method.Nutrient agar medium was used for gram-positive and gram-negative bacteria and No.1001 medium (3% glycerol,0.3% sodium L-glutamate, 0.2% peptone, 0.31% Na2HPO4, 0.1% KH2PO4, 0.005% ammonium citrate, 0.001% MgSO4 and 1.5% agar) for acid-fast organisms. Sabouraud agar medium was used for fungi. As shown in Table 16, each of the six BBM-1675 components (A1, A2, A3, A4, B1, B2) showed a broad spectrum of antimicrobial activity. BBM-1 675 A1, A2, A3 and A4 in particular were highly active against gram-positive bacteria.

TABLE 16 Antimicrobial Activity of BBM-1675 Components MIC in mcgiml Strain BBM-1675Ar A2 A3 A4 B, B2 S. aureus 209P < 0.0008 0.0063 0.0063 0.0125 0.012 0.0063 S. aureus Smith < 0.0008 0.0031 0.0063 0.0125 0.012 0.012 B. subtilis PCI 219 < 0.0008 0.05 0.0125 0.0125 0.05 0.05 M. luteus 1001 0.0016 0.0063 0.0125 0.0125 0.1 0.1 M. flavus < 0.0008 0.0016 0.0063 0.0125 0.025 0.025 Mycobacterium 607 0.05 0.1 NT NT 0.05 0.025 E. coliNIHJ 0.1 0.8 1.6 3.1 0.8 3.1 K. pneumoniae D11 0.4 0.8 1.6 3.1 0.8 0.8 P. aeruginosa D15 0.8 1.6 1.6 3.1 3.1 3.1 C. albicans IAM 4888 0.4 0.4 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 A1 and A2 (as prepared in Ex. 3 below) and on components A3 and A4.Data is summarized below.

MIC by ADT (mcg/ml) Strain BBM-1675A, A2 A3 A4 S. aureus 209P < 0.0008 0.0063 0.0063 0.012 S. aureus Smith < 0.0008 0.0031 0.0063 0.012 B. subtilis PCI 219 < 0.0008 0.05 0.012 0.025 M. luteus 1001 0.0016 0.0063 0.012 0.05 M. flavus < 0.0008 0.0016 0.0063 0.012 Mycobacterium 607 0.05 0.1 0.16 0.16 E. coliNIHJ 0.1 0.8 1.6 3.1 K. pneumoniae D11 0.4 0.8 1.6 3.1 P. aeruginosa D15 0.8 1.6 3.1 3.1 B. fragilisA20928 0.2 1.6 0.2 0.4 C. difficileA21675 0.4 0.8 0.05 0.4 C. perfringens A9635 0.05 0.8 0.4 0.4 C. albicans lAM 4888 0.4 0.4 1.6 6.3 C. neoformans 1.6 3.1 1.6 6.3 The activity of prophage induction in lysogenic bacterium E. coliW1709 (A) was determined for BBM-1675 components according to the method of Lein et al. in Nature 196: 783-784 (1962). The plaque count was made on agar 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 (lLB activity) was expressed as the minimum inducible concentration of the test compound. As shown in Table 17, BBM-1675 components showed strong ILB activity in the lysogenic bacteria, thus suggesting that they may possess antitumor activity.

TABLE 17 Lysogenic Induction Activity of BBM-1675 Components Antibiotic MIC* (mcg/mI) BBM-1 675 A1 0.0063 BBM-1675 A2 0.0125 BBM-1675A3 0.05 BBM-1675 A4 0.10 BBM-1675 B1 0.10 BBM-1675 B2 0.20 * minimum inducible concentration The antitumor activity of BBM-1675 A1 and A2 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 BDF1 mice at an inoculum size of 106, 105, 5 x 105 and 106 cells per mouse, respectively. Graded doses of test compounds were administered 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 (qd 1 < 9) or 11 days (qd 1 < 11). Components A3 and A4 were tested only against P-388 leukemia by a q3d x 3 schedule due to short supply of material.

BBM-1675 A1, A2, A3 and A4 were dissolved in 0.9% saline containing 10% dimethyl sulfoxide, and chromomycin A3 (Toyomycin, Takeda) employed as a reference compound was dissolved 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. AT/C value equal to or greater than 125% indicates that a significant antitumor effect was achieved. The results are shown in Tables 18 through 23. BBM-1675 A1 and A2 showed extremely potent antitumor activity against P-388 leukemia with a maximum T/C value of 160%. They are approximately 100 to 3000 times more active than chromomycin A3 in terms of minimum effective dose. BBM-1675 A3 and A4, however, were less active than component A1 or A2 against P-388 leukemia (Table 19). BBM-1 675 A1 and A2 were also active against L-1 210-leukemia (Table 21), B16 melanoma (Table 22) and Lewis lung carcinoma (Table 23). The toxicity of BBM-1675 A1 and A2 was determined in male ddY mice by intraperitoneal or intravenous administration; BBM-1675 A1 was about 10 times more toxic than BBM-1675 A2 (Table 24). The therapeutic indices of BBM-1675 A1 and A2 were 4 to 8 and 8 to 20 times better than those of chromomycin A3, 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 leukemias, 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 A1 and A2 were superior to adriamycin in terms of maximum TIC value, minimum effective dose and activity range.

TABLE 18 Effect of BBM-1675 Components on P-388 Leukemia (Day 1 treatment) Averagewt Dose, ip MST TIC change on Survivors on {mglkgiday*) (days) (%) day 5(g) day5 day45 BBM-1675A1 0.03 19.0 152 -2.6 5/5 0/5 0.01 19.0 152 -1.0 5/5 0/5 0.003 18.0 144 -1.0 5/5 0/5 0.001 20.0 160 -1.4 5/5 0/5 0.0003 16.0 128 0.0 5/5 0/5 0.0001 15.0 120 -0.2 5/5 0/5 0.00003 14.0 112 -0.2 5/5 0/5 BBM-1675 A2 0.3 6.0 48 -3.8 3/5 0/5 0.1 20.0 160 -1.8 5/5 0/5 0.03 18.0 44 -1.4 5/5 0/5 0.01 17.0 -0.6 5/5 0/5 0.003 17.0 136 -0.4 5/5 0/5 0.001 16.0 128 0.0 5/5 0/5 0.0003 15.0 120 0.0 5/5 0/5 0.0001 14.0 112 0.0 5/5 0/5 Chromomycin A3 1 19.0 152 -0.2 4/5 0/5 0.3 17.0 136 +0.6 5/5 0/5 0.1 16.0 128 +0.8 5/5 0/5 0.03 14.0 112 0.0 5/5 0/5 0.01 14.0 112 -0.2 5/5 0/5 Vehicle - 12.5 - +0.1 10/10 0/10 * day 1, i.p.

Circle indicates a significant antitumor activity.

TABLE 19 Effect of BBM-1675 Components on P-388 Leukemia (Days 1, 4 and 7 treatment) Average wt.

Dose, i.p MST TIC change on Survivors on mg/kg/day*) (days)(%) day 5(g) day 5 day 45 BBM-1675A1 0.03 7.0 56 -2.8 5/5 0/5 0.01 19.0 152 -1.0 5/5 0/5 0.003 19.0 152 -0.6 5/5 0/5 0.001 16.0 (m) -0.6 5/5 0/5 0.0003 17.0 (m) -0.4 5/5 0/5 0.0001 16.0 128 -0.2 5/5 0/5 0.00003 14.0 112 +0.4 5/5 0/5 BBM-1675A2 0.3 tox. - - 2.5 0/5 0.1 11.0 88 -1.0 5/5 0/5 0.03 18.0 144 -1.2 5/5 0/5 0.01 18.0 144 -0.4 5/5 0/5 0.003 18.0 144 -0.4 5/5 0/5 0.001 17.0 136 -0.4 5/5 0/5 0.0003 16.0 128 -0.4 5/5 0/5 0.0001 16.0 128 -0.2 5/5 0/5 0.00003 15.0 120 -0.4 5/5 0/5 BBM-1675A3 0.01 17.5 62) +0.2 4/4 0/4 0.001 15.0 120 +0.6 4/4 0/4 0.0001 13.5 108 +0.6 4/4 0/4 BBM-1675A4 0.01 16.5 132 +0.2 4/4 0/4 0.001 14.0 112 +0.4 4/4 0/4 0.0001 12.5 100 +0.6 4/4 0/4 Chromomycin A3 0.3 18.0 44 +0.6 5/5 1/5 0.1 18.0 144 +0.6 5/5 0/5 0.03 17.0 136 -0.2 5/5 0/5 0.01 14.0 112 0.0 5/5 0/5 Vehicle - 12.5 - +0.4 10/10 0/10 *days1,4and7,i.p.

Circle indicates a significant antitumor activity.

TABLE 20 Effect of BBM-1675 Components on P-388 Leukemia (qd 1- > 9 treatment) Average wt.

Dose, ip MST TIC change on Survivors on (mg/kg/day*) (days) (%) day 5 (g) day 5 day 45 BBM-1675A1 0.01 7.0 56 -1.8 5/5 0/5 0.003 13.0 104 -1.0 5/5 0/5 0.001 19.0 152 -1.2 5/5 0/5 0.0003 19.0 152 -0.8 5/5 0/5 0.0001 18.0 144 0.0 5/5 0/5 0.00003 16.0 (m) +0.2 5/5 0/5 0.00001 16.0 -0.2 5/5 0/5 BBM-1675A2 0.1 6.0 48 -2.2 4/5 0/5 0.03 13.0 104 -1.4 5/5 0/5 0.01 18.0 144 -1.0 5/5 0/5 0.003 18.0 144 -0.6 5/5 0/5 0.001 18.0 144 -0.8 5/5 0/5 0.0003 17.0 136 -0.4 5/5 0/5 0.0001 16.0 -0.4 5/5 0/5 0.00003 15.0 120 -0.6 5/5 0/5 0.00001 15.0 120 +0.4 5/5 0/5 Chromomycin A3 0.3 9.0 72 -2.0 5/5 0/5 0.1 18.0 6 +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 * qd 1 < 9 i.p.

Circle indicates a significant antitumor activity.

TABLE 21 Effect of BBM-1675 Components on L-1210 Leukemia Average wt.

Dose MST TIC change on Survivors on (mglkgiday*) days (%) day 5(g) day 5 day 45 BBM-1 675 A1 0.003 14.5 153 -1.7 6/6 0/6 0.001 12.0 -0.5 6/6 1/6 0.0003 12.0 126 +0.3 6/6 0/6 0.0001 11.0 116 +1.0 6/6 0/6 BBM-1675A2 0.03 10.5 111 -1.5 6/6 0/6 0.01 13.5 142 -1.2 6/6 0/6 0.003 13.0 137 -0.2 6/6 0/6 0.001 11.0 116 +1.3 6/6 0/6 0.0003 10.5 111 +1.0 6/6 0/6 Chromomycin A3 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 0.01 11.0 116 +1.3 6/6 0/6 0.003 10.0 105 +1.5 6/6 0/6 Vehicle - 9.5 - +1.4 12/12 0/12 * qd 1e0, i.p.

Circle indicates a significant antitumor activity,.

TABLE 22 Effect of BBM-1675 Components on B16 Melanoma Average wt.

Dose MST TIC change on Survivors on (mg/kg/day*) (days) (%) day 5 (g) day 5 day 45 BBM-1675A1 0.003 10.0 61 -0.7 6/6 0/6 0.001 31.5 191 0.0 6/6 0/6 0.0003 40.5 24 +0.3 6/6 0/6 0.0001 27.0 164 +0.8 6/6 0/6 0.00003 22.0 133 +1.8 6/6 0/6 0.00001 18.0 109 +2.2 6/6 0/6 BBM-1675A2 0.03 11.0 67 -0.8 6/6 0/6 0.01 26.5 161 +0.3 6/6 0/6 0.003 29.5 (i) +0.2 6/6 0/6 0.001 26.0 158 +0.8 6/6 0/6 0.0003 22.0 133 +0.2 6/6 0/6 0.0001 18.0 109 +0.2 6/6 0/6 0.00003 17.0 103 +1.7 6/6 0/6 Chromomycin A3 0.1 25.5 155 +2.3 6/6 0/6 0.03 23.0 139 +2.2 6/6 0/6 0.01 21.0 127 +2.3 6/6 0/6 0.003 18.0 109 +2.2 6/6 0/6 Vehicle - 16.5 - +2.1 12/12 0/12 * qd 1- > 9, i.p.

Circle indicates a significant antitumour activity.

TABLE 23 Effect of BBM-1675 Components on Lewis Lung Carcinoma Averagewt Dose MST TIC change on Survivors on {mglkgiday*) (days) (%) day 5(g) day5 day 45 BBM-1675A1 0.003 10.0 91 -1.7 5/6 0/6 0.001 31.5 286 -0.7 6/6 1/6 0.0003 21.5 195 -0.7 6/6 0/6 0.0001 21.0 191 +1.0 6/6 0/6 0.00003 13.0 118 +1.0 6/6 0/6 0.00001 11.5 105 +1.0 6/6 0/6 BBM-1675A2 0.03 10.0 91 -1.8 6/6 0/6 0.01 25.5 232 -1.7 6/6 0/6 0.003 28.5 259 0.0 6/6 1/6 0.001 17.0 155 -0.3 6/6 0/6 0.0003 15.0 136 +1.2 6/6 0/6 0.0001 10.5 95 +0.5 6/6 0/6 0.00003 11.0 100 +0.8 6/6 0/6 Chromomycin A3 0.1 21.5 195 +1.2 6/6 1/6 0.03 17.0 155 +1.7 5/5 0/5 0.01 17.0 155 +1.5 6/6 0/6 0.003 11.5 105 +1.7 6/6 0/6 Vehicle - 11.0 - +0.8 12/12 1/12 * qd 1- > 11, i.p.

Circle indicates a significant antitumor activity.

TABLE 24 Toxicity of BBM-1675 Components LD50 fmglkgiday) Multiple dose Single dose (qd 1 < 9) i.p i.v. i.p.

BBM-1675A1 0.019 0.010 0.00046 BBM-1675A2 0.18 0.10 0.0072 Chromomycin A3 0.81 0.41 0.23 TABLE 25 Therapeutic Indices LD501MED* P-388 Single qd 1- > 9 L1210 B16 LL BBM-1675A1 63 > 46 2 15 5 BBM-1765A2 180 72 2 24 24 Chromomycin A3 8 8 inactive 23 23 * mininum effective dose TABLE 26 Effect of BBM- 1675 Components on Intravenously Implanted P-388 Leukemia Average wt.

Dose MST TIC change on Survivors on (mg/kg/day*) (days) (%) day 5(g) day5 day 45 BBM-1675 A1 0.01 9.5 106 -1.7 6/6 0/6 0.003 14.0 156 -0.3 6/6 0/6 0.001 11.5 (m) +0.3 6/6 0/6 0.0003 9.5 100 +0.3 6/6 0/6 BBM-1675A2 0.1 7.0 78 -3.7 6/6 0/6 0.03 15.0 167 -1.0 6/6 0/6 0.01 12.0 133 -0.5 6/6 0/6 0.003 9.0 100 +1.0 6/6 0/6 Adriamycin 30 tox. - - 0/6 0/6 10 9.0 100 -1.5 6/6 0/6 3 12.0 133 +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 1,4and 7, i.v.

Circle indicates a significant antitumor activity.

TABLE 27 Effect on BBM-1675 Components on Intravenously Implanted L-1210 Leukemia Average wt.

Dose MST TIC change on Survivors on fmglkgiday*J (days) (%) day 5(g) day 5 day 45 BBM1675A1 0.008 9.5 119 -2.0 4/6 0/6 0.004 14.0 ( -0.2 6/6 0/6 0.002 13.0 16 +0.2 6/6 0/6 0.001 9.5 119 +0.8 6/6 0/6 0.0005 9.0 113 +0.8 6/6 0/6 BBM-1675A2 0.63 11.0 138 -1.8 6/6 0/6 0.032 14.0 175 +0.2 6/6 0/6 0.16 10.5 (t) +0.8 6/6 0/6 0.008 8.0 100 +1.2 6/6 0/6 0.004 8.0 100 +0.8 6/6 0/6 Adriamycin 16 tox - 2/6 0/6 8 12.0 150 +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 - +1.4 12/12 0/12 *days 1,4and7, i.v.

Circle indicates a significant antitumor activity.

Antitumor activity of components BBM-1675 A1 and A2 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 BBM-1675 A7 and A2 on P-388 Leukemia Effect A WC, Treatment Dose, IP MST MST gm Survivors Material Schedule Fglkgiday Days % TIC d.5 d.5(30) * NSC38270 qd 1 < 9 400 13.0 163 -0.6 6/6 200 11.0 138 -0.9 6/6 BBM-1675A1 d.1 51.2 20.0 250 -2.1 4/6 DMSO~saline 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 6/6 0.4 10.0 125 +0.4 6/6 0.2 10.0 125 +0.3 6/6 0.1 10.0 125 0 6/6 d.1,5 & 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 0.4 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 qd1 < 9 12.8 TOX TOX TOX 1/6 6.4 6.0 75 -1.5 4/6 3.2 13.0 163 -1.2 6/6 1.6 14.5 181 -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 BBM-1675A2 d.1 256 TOX TOX TOX 0/6 DMSOesaline - 128 12.5 156 -3.5 4/6 64 27.0 338 -1.9 6/6 32 26.0 325 -2.0 6/6 16 16.0 200 -1.8 6/6 8 15.5 194 -1.9 6/6 4 15.0 188 -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 d.1,5 & 128 TOX TOX TOX 0/6 64 TOX TOX TOX 0/6 32 TOX TOX -1.3 2/6 16 24.5 306 -1.3 5/5 8 17.5 219 -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 TABLE 28 - cont'd BBM-1675 A2 DMSOesaline 0.25 11.0 138 -0.4 6/6 qd1 < 9 64 TOX TOX TOX 1/6 32 6.0 75 -2.9 4/6 16 8.0 100 -1.9 6/6 8 15.5 194 -1.3 6/6 4 17.0 213 -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 ascites cells implanted i.p.

Host : CDF1 9 mice.

Tox . < 4/6 mice alive on d.5 Evaluation : MST = median survival time.

Effect : %T/C = (MSTtreated/MSTcontrol) x 100.

Criteria : % T/C 2 125 considered significant antitumor activity.

* NSC 38270 = olivomycin A TABLE 29 Effect on BBM-1675 A1 and A2 on L-1210 Leukemia Effect A WC, Treatment Dose, IP MST MST gm Survivors on Material Schedule Aglkglinj Days % TIC d.5 d.5(30) BBM-1675A1 d.1 51.2 12.0 171 -1.1 5/6 25.6 7.0 100 -2.3 6/6 12.8 9.0 129 -1.1 5/6 6.4 9.5 136 -0.5 6/6 3.2 6.0 86 -1.7 6/6 1.6 7.0 100 -0.8 6/6 0.8 8.0 114 -0.4 6/6 0.4 7.0 100 +0.3 6/6 0.2 7.0 100 -0.5 5/6 0.1 7.0 100 +0.8 5/6 d.1,5 & 25.6 TOX TOX TOX 1/6 12.8 9.0 129 -1.8 6/6 6.4 9.0 129 -0.8 6/6 3.2 8.0 114 -1.9 6/6 1.6 8.5 121 ' 0 6/6 0.8 8.0 114 -0.4 6/6 0.4 7.5 107 -1.3 6/6 0.2 8.0 114 0 6/6 0.1 8.0 114 +0.4 5/6 0.05 7.0 100 +0.3 6/6 qd 1 < 9 12.8 TOX TOX -2.4 3/6 6.4 8.0 114 -1.6 6/6 3.2 8.0 114 -1.7 6/6 1.6 9.0 129 -2.1 6/6 0.8 8.5 121 -1.6 6/6 0.4 8.0 114 -1.0 6/6 0.2 8.0 114 -0.5 5/6 0.1 7.0 100 +0.3 6/6 0.5 7.0 100 +0.3 6/6 0.025 6.0 86 -0.6 6/6 BBM-1675A2 d.1 256 TOX TOX TOX 0/6 128 7.0 100 -1.8 5/6 64 7.5 107 -1.3 4/6 32 8.0 114 -2.2 5/6 16 7.0 100 -2.3 6/6 8 9.5 136 -1.4 6/6 4 8.5 121 -1.1 6/6 2 8.0 114 -0.8 6/6 1 8.0 114 0 6/6 0.5 8.0 114 -0.1 6/6 TABLE 30 Effect of BBM-1675 At and A2 on B16 Melanoma Effect A WC Dose, IP MST MST gm Survivors on Material g (m) or mg/kg/inj Days % TIC d.5 d. 10(61) BBM-1675A1 3.2M TOX TOX -1.8 2/10 1.6 16.0 168 -1.8 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 10/10 BBM-1675A2 16M 13.0 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 0.5 140 -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 d.m.o. = 55.0 (220%), b Two without tumor; MST d.m.o. = 46.0 (184%) Tumor inoculum : 0.5 ml of a 10% brei, p Host : BDF1 ? mice.

Treatment : qd 1- > 9 Tox : #7/10 mice alive on d.10 Evaluation : MST = median survival time Effect : %T/C = (MSTtreated/MSTcontrol) x 100.

Criteria : nC 2 125 considered significant antitumor activity.

After further purification of BBM-1675A1 according to ExampleS, 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 BBM-1675At on P388 Leukemia (Day 1 Treatment) Average vvt.

Dose MST TIC change on Survivors on Compound (mglkgldosel Route, schedule Days (O/oJ day5 day5 BBM-1675A1 0.1024 i.p., qd x 1 TOX TOX 0/6 0.0512 17.5 159 -1.8 4/6 0.0256 16.5 150 -2.6 6/6 0.0128 17.5 159 -1.4 6/6 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 0.0008 15.0 136 -1.2 6/6 0.0004 15.0 136 -2.6 6/6 0.0256 i.p., q4d x 3; TOX TOX -1.5 1/6 0.0128 10.0 91 -2.5 5/6 0.0064 17.5 159 -1.9 6/6 0.0032 17.0 155 -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 i.p., qd x 5;TOX TOX 0/6 0.0064 TOX TOX -3.6 3/6 0.0032 17.5 155 -2.2 5/6 0.0016 14.5 132 -2.0 6/6 0.0008 15.5 141 -2.2 6/6 0.0004 16.0 145 -2.8 6/6 0.0002 17.0 155 -1.3 6/6 0.0001 14.0 127 -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 10e i.p., q4d x 3; 11.0 100 -0.7 9/9 Host: CDF1 female mice Implant level and site: 1 x 106 cells, i.p.

TABLE 32 Effect of Purified BBM-1675At on L-1210 Luekemia (Day 1 Treatment) Average wt.

Dose MST TIC change on Survivors on Compound {mglkgidose) Route, schedule Days (O/o) day 5 day 5 BBM-1675A1 0.1024 i.p., qd x 1 TOX TOX 1/6 0.0512 TOX TOX -2.0 0/6 0.0256 8.0 114 -2.9 4/6 0.0128 11.0 157 -2.0 6/6 0.0064 11.0 157 -1.9 6/6 0.0032 10.0 143 -2.0 6/6 0.0016 10.0 143 -2.6 5/6 0.0008 8.0 114 -0.4 6/6 0.0256 i.p., q4d x 3 TOX TOX -2.3 2/6 0.0128 10.5 150 -1.7 6/6 0.0064 11.0 157 -1.8 6/6 0.0032 11.0 157 -1.4 6/6 0.0016 10.5 150 -1.9 6/6 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 0.0128 i.p., qd x 5 TOX TOX -2.8 2/6 0.0064 7.0 100 -1.8 5/6 0.0032 11.5 164 -1.0 6/6 0.0016 11.0 157 -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 106 i.p., qd x 5 7.0 100 0.1 10/10 Host: CDF1 female mice Implant level and site: 1 x 106 cells, i.p.

TABLE 33 Effect of Purified BBM-1675A1 on B16 Melanoma (Day 1 Treatment) Average wt.

Dose MST TIC change on Survivors on Compound (mglkgldoseJ Route, schedule Days 1%1 day5 day5 *BBM-1675A1 0.0064 i.p., q4d x 3 16.5 110 -3.8 8/10 0.0032 22.5 150 -3.0 10/10 0.0016 25.0 167 -1.8 10/10 0.0008 22.0 147 -2.3 10/10 0.0004 24.0 160 -1.8 9/10 0.0016 i.p., qd x 9 27.0 180 -3.7 10/10 0.0008 27.0 180 -2.9 10/10 0.0004 26.0 173 -2.3 10/10 0.0002 24.5 163 -2.4 10/10 0.0001 25.5 170 -2.3 10/10 Control (vehicle) 0.5 ML i.p., qd x 9 15.0 100 -0.3 10/10 **BM-1675A1 0.0064 i.v., q4d x 3 15.0 86 -4.7 10/10 0.0032 32.5 186 -2.1 10/10 0.0016 26.0 149 -1.4 10/10 0.0008 24.0 137 -0.4 10/10 0.0004 24.5 140 -0.0 10/10 0.0064 i.p., q4d x 3 18.0 103 -2.7 10/10 0.0032 23.0 131 -1.4 10/10 0.0016 24.0 137 -0.7 10/10 0.0008 25.5 146 -0.8 10/10 0.0004 21.5 123 -1.4 10/10 Control (vehicle) 0.2 ML i.v., q4d x 3 17.5 100 -0.4 10/10 Host: BDF1 female mice *Implant level and site: 0.5ML 10% BREI, i.p.

**Implant level and site: Fragment, s.c.

The above screening data indicates that the purified BBM-1675A1 component has substantially the same antitumor 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 leukemias, BBM-1675A1 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 ip tumor implants. This property of successful pharmacologic delivery of a drug to a tumor at a distant site is unusual among antitumor 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 ortumor-inhibiting dose of BBM-1675 A1, A2, A3, A4, Bi or B2, or a pharmaceutical composition thereof.

In another aspect, the present invention provides a pharmaceutical composition which comprises an effective antimicrobial or tumor-inhibiting amount of BBM-1675 A1, A2, A3, A4, B1 or B2 in combination with an inert pharmaceutically acceptable carrier or diluent. These compositions may be made up in any pharmaceutical form appropriate for parenteral administration.

Preparations according to the invention for parenteral administration include sterile aqueous or non-aqueous 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 injectable 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 situs, 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 application 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 agar slant containing 1% malt extract, 0.4% glucose, 0.4% yeast extract, 0.05% CaCO3 and 1.6% agar. A well-grown agar slant was used to inoculate vegetative medium containing 3% soluble starch, 3% dry yeast, 0.3% K2HPO4, 0.1% KH2PO4, 0.05% MgSO4-7H2O,0.2% NaCI and 0.1% CaCO3, the pH being adjusted to pH 7.0 before sterilization.The vegetative culture was incubated at 32"C for 72 hours on a rotary shaker (250 rpm) 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% CaCO3 and 0.005%CuSO4-5H2O (pH 7.0 before sterilization). The fermentation was carried out at 28"C for six days on the rotary shaker. The antibiotic activity in the fermentation broth was determined by the paper-disc agar diffusion using Staphylococcus aureus 209P as the test organism. The antibiotic potency reached a maximum of about 1 mcg/ml after five days fermentation.

Fermentation of BBM-1675 was also performed in stir-jar fermenters. Five hundred mililiters 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 320C with an aeration rate of 12 liters/minute and agitation at 250 rpm. Under these conditions, 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 fermentation tanks. A seed culture was shaken for four days at 30"C in Erlenmeyer flasks containing vegetative medium consisting of 3% soluble starch, 3% dry yeast, 0.3% K2HPO4, 0.1% KH2PO4, 0.05% MgSO47H2O, 0.2% NaCI and 0.1% CaCO3. 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 rpm at 30"C 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% CuSO4-5H2O and 0.1% CaCO3.The production tank was operated at 28"C at 164 rpm with an aeration rate of 2,000 liters/minute. The broth pH gradually rose with the progress of fermentation and 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, pH 7.8) was separated to mycelial cake and supernate with the aid of a Sharpless 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 in vacuo to 43 liters. The activity contained in the broth supernate was recovered therefrom by extraction with two 1,000-liter portions of n-butanol. The n-butanol extracts and concentrated methanol extracts were combined and evaporated azeotropically by occasional additions of water to an aqueous solution (20 liters) which deposited most the antibiotic activity as an oily solid. The solid was digested in 30 liters of methanol and the insolubles were removed by filtration.The methanol extract was then concentrated in vacuo 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 vacuo to 4 liters. Addition of the concentrate into 20 liters of n-hexane 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 A1 and A2, 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 LH-20 ( 5.5 x 85 cm). The column was developed with methanol and the elution monitored by bioassay using Staphylococcus aureus 209P. The active eluates were combined, concentrated in vacuo and lyophilized to give a semi-pure solid of BBM-1675 complex (4.19 g). The solid was then chromatographed on a column of silica gel (p5 5.0 x 50 cm) using chloroform plus an interesting amount (15%vN) of methanol as eluants.

Eluates were pooled on the basis of antibacterial activity (vs. S. aureus) and TLC (SiO2; CHCl3-CH3OH = 5:1 v/v and concentrated in vacuo. Nearly homogeneous BBM-1675 A1 (yield after evaporation: 351 mg) was eluted first with 2% methanol in chloroform and then a mixture of BBM-1 675 A2, A3 and A4 (507 mg) followed by BBM-1675 B mixture (210 mg) with 3% methanol in chloroform. The solid of BBM-1675 A1 was applied on a column of Sephadex LH-20 ( 2;0 x 80 cm) which was developed with methanol. The active fractions were concentrated in vacuo to dryness and the residual solid was crystallized from methanol to afford colorless .plates of pure BBM-1675 A1 (124 mg) (this material is starting material for Example 3A.).The compled BBM-1675 A2, A3 and A4 was separated by chromatography on a column of Bondapak C18 (Waters, Z 3.0 x 50 cm). Elution was carried out with aqueous acetonitrile-and the bioactive eluates were examined by TLC (Merck, *Silanized: CH3CN-H2O = 75:25 v/v). The minor components A4 (33 mg) and A3 (18 mg) were eluted successively in that order with 20% acetonitrile followed by another major component A2 (301 mg) (this material is starting material for Example 3B) with 50% acetonitrile).

The solid containing BBM-1675 B1 and B2 was chromatographed on a column of silica gel ( 3.0 x 40 cm) with chloroform and methanol as the developing solvent. The active fractions eluted with 4% methanol in chloroform were combined and evaporated to afford pure BBM-1675 B1 (7 mg). Another active fraction was eluted at 5% methanol concentration, which upon evaporation afforded BBM-1675 B2 (8 mg).

*C18 reverse phase silica gel Example 3 Further purification of BBM- 1675A 1 and A2 A. Purification of BBM- 1675A, A2.67 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 (41.6% acetonitrile - 21.6% methanol - 36.8% 0.1 M ammonium acetate).

Partially purified BBM-1675A1 (100.5 mg) 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 eluted with the above eluant collecting 87 ml fractions. The eluant was monitored at 254 nm and 340 nm. Fractions 55 through 71 were pooled and extracted twice with 1500 ml aliquots of chloroform.

The chloroform was evaporated to dryness to yield 89.8 mg of residue C.

A 1.5 cm i.d. x 20 cm Glenco column was slurry packed with 12 g of Woelm silica gel (60-200 micron particles). Residue C was applied to the column in a chloroform solution. The column was eluted 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 mg of residue D.

A 1.5 cm i.d. x 20 cm Glenco column was slurry packed with 12 g Woelm silica gel (63-200 micron particles) in Skellysolve B. Residue D was dissolved in approximately 2 ml of CHCl3 and applied to the column. The chloroform was displaced with 25 ml of Skellysolve B. The column was then eluted with a 500 ml linear gradient of Skellysolve B to 60% acetone in Skellysolve B collecting 28-25 ml fractions. Fractions 19-23 were pooled and evaporated to dryness to yield 65.6 mg of pure BBM-1 675A1.

This residue was homogeneous in three TLC systems (5% methanol in chloroform; 5% methanol in ether; and 50% acetone in Skellysolve B on silica gel) and HPLC (C-18 silica gel -41.5% acetonitrile:21 .5% methanol:37.0%0.1 M ammonium acetate).

Gel permeation chromatography with purified BBM- 1675A, 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-1675A1 (approximately 120 mg) was dissolved in 2 ml of methanol and transferred to a 2.5 ml sample reservoir. The sample was applied to the column and elution commenced at 1.75 ml/min with methanol collecting 10 ml fractions [Pharmacia Frac-100 fraction collector].

The eluant was monitored at 254 nm with an Isco UA-5 detector. BBM-1675A1 was observed to elute at VeNt of 0.79 to 0.91 (Ve= elution volume; Vt= bed volume).

B. Purification ofBBM-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 1.51 of eluant (50% acetonitrile - 20% methanol - 30% 0.1 M ammonium acetate).

Partially purified BBM-1675A2 (76.9 mg) 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 eluted with the above eluant collecting 87 ml fractions. The eluant was monitored at 254 and 340 nm.

Fractions 31 through 38 were pooled and extracted twice with 500 ml aliquots of chloroform. The chloroform was evaporated to dryness to yield 65.8 mg of homogeneous BBM-1675A2.

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

Example 4 Prefered extraction process for BBM- 1675A t 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 CRC-air driven stirrer for 2 hours. Approximately 41(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 Buchnerfunnel. 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 separatory 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 Woelm 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 eluted with 600 ml of 10% methanol in chloroform. This eluant was evaporated to dryness to yield 547 mg of residue A.

Residue A was dissolved into 50 ml of chloroform. The chloroform solution was added to 20 g of Dicalite in a 11 round bottom flask. A slurry was created by adding approximately 200 ml of Skellysolve B. The solvents were moved in a rotary evaporator. The residue was slurried 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 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 eluted with an additional 600-700 ml of Skellysolve B. The bed was eluted with 500 ml of toluene. The toluene eluant was evaporated to dryness to yield 93 mg of residue B. This partially purified BBM-1 675A1 may then be further purified according to the procedure of Example 3.

Example 5 Fermentation of BBM- 1675 complex using variant H964-92-A 1327Y A variant strain Al 327Y, which was obtained by NTG treatment of Actinomadura verrucosospora 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% CaCO3, the pH being adjusted to 7.0 before sterilization.The vegetative culture was incubated at 32"C for four days on a rotary shaker (250 rpm) 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% CuSO45H20, 0.05% MgSO4.7H20 and 0.1% CaCO3, the pH being adjusted to 7.0 before sterilization.

The fermentation was carried out at 280C for 7 days on the rotary shaker. The antibiotic production reached a maximum of ca. 1.5 mcg/ml.

Example 6 Isolation and purification of BBM- 1675 components The harvested fermentation broth from Ex. 5 (3,000 L, pH 7.6) was separated to mycelial cake and supernate by using a Sharpless 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 supernate was extracted therefrom with 1,800 L of n-butanol. The methanol and n-butanol extracts were combined and concentrated azeotropically by occasional additions of water to an aqueous solution (20 L) which deposited most of the antibiotic activity as an oily solid. The mixture was shaken-three times with 20 Leach of ethyl acetate to extract the activity. The extracts were pooled, filtered to remove the insolubles and evaporated in vacuo to 4 L.Addition of the concentrate into 30 L of n-hexane 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 A1 and A2 and several minor ones. They were separated and purified by a series of chromatographies 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 (a 5.5 x 85 cm). The column was developed with methanol and the elution monitored by bioassay using Staphylococcus aureus 209P. The active eluates were pooled, concentrated in vacuo 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 ( 3.0 x 70 cm) using chloroform and an increasing amount (1-5%) of methanol as developing solvents. The eluates were pooled on the basis of antibacterial activity against S. aureus and TLC (SiO2, CHC13-MeOH = 5:1, v/v) and concentrated in vacuo. BBM-1675 An (425 mg after evaporation, potency: 960 mcg/mg) was eluted first with 2% methanol in chloroform and then a mixture of BBM-1675 A2, A3, and A4 (732 mg, potency: 340 mcg/mg) followed by BBM-1675 B complex (200 mg, potency: 190 mcg/mg) with 3% methanol in chloroform. The above BBM-1675 A1 was rechromatographed on silica gel (column: a 2.2 x 44 cm) with 2% methanol in benzene. The bioactive eluates were examined by HPLC (Lichrosorb RP-18: CH3CN-MeOH-0.1 MCH3COONH4 = 5:2:3, v/v) and the fractions containing homogeneous BBM-1 675 A1 evaporated in vacuo to dryness. The residual solid was crystallized from methanol (10 ml) to give colorless prisms of BBM-1675 A1 (197 mg, potency: 1,000 mcg/mg).

The complex of BBM-1675 A2, A3 and.A4 (537 mg) was separated by column chromatography on Bondapak C18 (Waters, a 2.0 x 42 cm). Elution was carried out with aqueous acetonitrile and the bioactive eluates were examined by TLC (Merck, silanized: CH3CN-H2O = 75:25, v/v). The minor components BBM-1675 A4 (45 mg, potency: 410 mcg/mg) and A3 (19 mg, potency: 300 mcg/mg) were eluted successively with 20% acetonitrile followed by a major component, BBM-1675 A2 (203 mg) with 50% acetonitrile. The BBM-1675 A2 fraction was crystallized from chloroform-n-hexane to deposit colorless rods (70 mg, potency: 290 mcg/mg). The solid containing BBM-1675 B mixture was chromatographed on a column of silica gel (a 3.0 x 40 cm) with chloroform and methanol as developing solvent The active fractions eluted with 4% methanol in chloroform were pooled and evaporated to afford pure BBM-1675 B1 (7 mg, potency: 180 mcg/mg). Another active fraction was eluted at 5% methanol concentration, which upon evaporation afforded BBM-1675 B2 (8 mg, potency: 140 mcg/mg).

Claims (36)

1. The antitumor antibiotic BBM-1 675 A1 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) substantially as shown in Figure 9; (e) when dissolved in CDC13 exhibits a proton magnetic resonance spectrum substantially as shown in Figure 10; (f) has a melting point in the range of about 156-158"C;; (g) has an optical rotation of [o],,27 = -191" (e 0.5, CHC13); (h) has an apparent molecular weight of 1248 as determined by mass spectroscopy; (i) has an approximate elemental composition of 52.17% carbon, 6.15% hydrogen, 4.63% nitrogen, 9.09% 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 CHCl3-CH3OH (5:1 v/v) and exhibits in reverse phase silica gel thin layer chromatography an Rfvalue of 0.18 with the solvent system CH3CN-H20 (75:25 v/v); (k) when dissolved in methanol at a concentration of 0.01356 g/l exhibits the following ultraviolet absorption maxima and absorptivities:: Xmax (nm) absorptivities 320 12.4 280 shoulder 253 25.1 210 25.5 with no significant change upon addition of acid or base; (I) 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-CH30H-0.1 M-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.

2. 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) substantially as shown in Figure 12; (e) when dissolved in CDC13 exhibits a proton magnetic resonance spectrum substantially as shown in Figure 13; (f) has a melting point in the range of about 147-149"C; (g) has an optical rotation of [(X]D7 = - 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 CHCl3-CH3OH (5:1 v/v) and exhibits in reverse phase silica gel thin layer chromatography an Rf value of 0.21 with the solvent system CH3CN-H20 (75:25 v/v); (k) when dissolved in methanol at a concentration of 0.02052 g/l exhibits the following ultraviolet absorption maxima and absorptivities:: Xmax (nm) absorptivities 320 12.2 282 16.3 282 26.2 214 25.8 with no significant change upon addition of acid or base; (I) 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-0.1 M 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.

3. 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; (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) substantially as shown in Figure 3; (d) when dissolved in CDCl3 exhibits a proton magnetic resonance spectrum substantially as shown in Figure 7; (e) has a melting point in the range of about 125-127"C; (f) has an optical rotation of [(X]D7 = - 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-CH30H (5:1 v/v) and exhibits in reverse phase silica gel thin layer chromatography an Rf value of 0.28 with the solvent system CH3CN-H20 (75::25 v/v); (i) exhibits the following ultraviolet absorption maxima when dissolved in methanol, 0.01 N HCl-CH3OH and 0.01N NaOH-CH3OH UV Amax nm (E1i'cm) 253 (286) in methanol 282 (158) 320(122) UV Xmax nm (E1 cm) 253 (287) in0.01N HCI-CH30H 282(160) 320(126) UV Xmax nm (Ea cm) 252 (280) in 0.01 N 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-0.1 M CH3COONH4 (5:2::3 v/v); (k) is effective in inhibiting the growth of various bacteria and fungi; (I) induces prophage in lysogenic bacteria; and (m) is effective in inhibiting the growth of P-388 leukemia in mice.

4. 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) substantially as shown in Figure 4; (d) when dissolved in CDCl3 exhibits a proton magnetic resonance spectrum substantially as shown in Figure 8; (e) has a melting point in the range of about 123-1 26 C; (f) has an optical rotation of [(I]D7 - 176'(c0.5,CHCl3);; (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 CHCl3-CH3OH (5:1 v/v) and exhibits in reverse phase silica gel thin layer chromatography 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, 0.01 N HCl-CH3OH and 0.01 N NaOH-CH3OH UV Xmax nm (E 1cm1%) 253 (257) in methanol 282 (153) 320 (117) UV Xmax nm (E1i'c'm) 253 (258) in 0.01 N HCI-CH30H 282(155) 320(118) UV Xmax nm (Ea /c m) 252 (266) in 0.01 N 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-CH30H-0.1 M CH3COONH4 (5:2:3 v/v); (k) is effective in inhibiting the growth of various bacteria and fungi; (I) induces prophage in lysogenic bacteria; and (m) is effective in inhibiting the growth of P-388 leukemia in mice.

5. 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]027 - 171 (c 0.5, CHCl3); (e) exhibits in silica gel thin layer chromatography an Rf value of 0.63 with the solvent system CHCl3-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, 0.01 N HCI-CH3OH and 0.01N NaOH-CH3OH UV Xmax nm (Es c m) 253 (225) in methanol 282 (140) 320(104) UV #max nm (E1i'c'm) 253 (225) in 0.01 N HCl-CH3OH 282 (140) 320 (105) UV Amax nm (E1 c m) 252 (236) in 0.01 N 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.

6. 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 [st]jD7 - 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 chromatograpy an Rfvalue of 0.16 with the solvent system CH3CN-H20 (75::25 v/v); (f) exhibits the following ultraviolet absorption maxima when dissolved in methanol,0.01N HCl-CH3OH and 0.01 N NaOH-CH3OH Us may nm (E11"c'm) 248 (212) in methanol 279(141) 318(103) UV Amax nm (En cm) 248 (210) in 0.01N HCI-CH3OH 279 (140) 318(103) UV Xmax nm (E11,7c'm) 248 (233) in 0.01 N 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.

7. The process for the production of the antitumor antibiotic BBM-1675 A1 which comprises cultivating a BBM-1675 A1-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 A1 is produced by said organism in said culture medium and then recovering BBM-1675 A1 from the culture medium.

8. The process according to Claim 7 wherein the BBM-1675 A1-producing organism has the identifying characteristics of Actinomadura verrucosospora strain H964-92 (ATCC 39334),Actinomadura verrucosospora strain Al 327Y (ATCC 39638), or a mutant thereof.

9. 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 aqeuous 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.

10. The process according to Claim 9 wherein the BBM-1675 A2-producing organism has the identifying characteristics ofActinomadura verrucosospora strain H964-92 (ATCC 39334),Actinomadura verrucosospora strain Al 327Y (ATCC 39638), or a mutant thereof.

11. The process for the production of the antitumor antibiotic BBM-1675 A3 which comprises cultivating a BBM-1675 A3-producing strain ofActinomadura 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.

12. The process according to Claim 11 wherein the BBM-1675 A3-producing organism has the identifying characteristics of Actinomadura verrucosospora strain H964-92 (ATCC 39334),Actinomadura verrucosospora strain Al 327Y (ATCC 39638), or a mutant thereof.

13. The process for the production of the antitumor antibiotic BBM-1 675 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.

14. The process according to Claim 13 wherein the BBM-1675 A4-producing organism has the identifying characteristics of Actinomadura verrucosospora strain H964-92 (ATCC 39334),Actinomadura verrucosospora strain Al 327Y (ATCC 39638), or a mutant thereof.

15. 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.

16. The process according to Claim 15 wherein the BBM-1675 B1-producing organism has the identifying characteristics of A ctinomadura verrucosospora strain H964-92 (ATCC 39334), A ctinomadura verrucosospora strain Al 327Y (ATCC 39638), or a mutant thereof.

17. 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 cqntaining 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.

18. The process according to Claim 17 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.

19. A method for therapeutically treating an animal host affected by amicrnbial infection which comprises administering to said host an effective antimicrobial dose of BBM-1675 A1, BBM-l675 A2, BBM-1675 A3, BBM-1675 A4, BBM-1675 B1 or BBM-1675 B2.

20. A method for therapeutically treating an animal host affected by a malignant tumor, which comprises administering to said host an effective tumor-inhibiting dose of BBM-1675 A1, BBM-1675 A2, BBM-1675 A3, BBM-1675A4, BBM-1675 B, or BBM-1675 B2.

21. A pharmaceutical composition comprising an effective antimicrobial amount of BBM-1675 Al, BBM-1675 A2, BBM-1675 A3, BBM-1675 A4, BB-1675 B1 or BBM-1675 B2 in combination with a pharmaceutical carrier or diluent.

22. A pharmaceutical composition comprising an effective tumor-inhibiting amount of BBM-1675 A1, BBM-1675 A2, BBM-1675 A3, BBM-1675 A4, BBM-1675 B1 or BBM-1675 B2 in combination with a pharmaceutical carrier or diluent.

23. 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.

24. 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.

25. Chemical compound BBM-1 675 Al obtained by cultivating Actinomadura verrucosospora strain H964-92 (ATCC 39334) or A1327Y (ATCC 39638).

26. Chemical compound BBM-1 675 A2 obtained by cultivating Actinomadura verrucosospora strain H964-92 (ATCC 39334) or Al 327Y (ATCC 39638).

27. Chemical compound BBM-1 675 A3 obtained by cultivating Actinomadura verrucosospora strain H-964-92 (ATCC 39334) or A1327\/ (ATCC 39638).

28. Chemical compound BBM-1 675 o obtained by cultivating Actinomadura verrucosospora strain H964-92 (ATCC 39334) or Al 327Y (ATCC 39638).

29. Chemical compound BBM-1675 B1 obtained by cultivating Actinomadura verrucosospora strain H964-92 (ATCC 39334) or A1327Y (ATCC 39638).

30. Chemical compound BBM-1675 B2 obtained by cultivating Actinomadura verrucosospora strain H964-92 (ATCC 39334) orA1327Y (ATCC 39638).

31. Chemical compound BBM-1675A1.

32. Chemical compound BBM-1675A2.

33. Chemical compound BBM-1675A3.

34. Chemical compound BBM-1675A4.

35. Chemical compound BBM-1675 B1.

36. Chemical compound BBM-1675 B2.