HU193928B - Process for preparing anticarcinogen antibiotics bbm-1675 - 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

The present invention relates to a process for the preparation of antibiotics having antitumor activity.

The structure of the antitumor antibiotics of the present invention has not yet been elucidated. Given the unique physical, chemical, and biological properties, the antibiotics of the present invention, BBM-1675, are expected to be novel compounds.

No. 95154A1. European Patent Publication Publication No. 6,198,198, discloses Actinomadura pulveraceus sp. nov. No. 6049 (ATCC 39100), which results in the anti-tumor antibiotics WS 6049-A and WS 6049-B. The structure of WS 6049 has not been elucidated so far, but based on the particular characteristics of the antibiotics it can be concluded that the antibiotics WS 6049-A and WS 6049-B may be related to the antibiotics of the present invention designated BSM-1675. In any event, the spectral data indicate that neither WS 6049-A nor WS 6049-B is the same as BBM-1675 of this application. In addition, spectral data indicate that neither the WS 6049-A nor the WS 6049-B is the same as the reported BBM-1675 component. In addition, No. 95154A1. The generic strain described in European Patent Application Publication No. 4,282,195 is clearly distinguishable from Actinomadura verrucosospora of the present invention in terms of the aerial mycelium on ISP media 2, 3 and 4, both positive milk peptonization and D-fructose, D-mannitol. , trehalose and cellulose.

The present invention provides a novel class of antitumor antibiotic compounds, designated BBM-1675, which is produced by culturing an Actinomadura verrucosospora strain that produces ΒΒΜ-1675, most preferably Actinomadura verrucosospora H964-92 (ATCC ), or Actinomadura verrucosospora A1327Y (ATCC 39638), or a mutant thereof, in an aqueous medium containing a digestible source of carbon and nitrogen under aerobic conditions, as long as the microorganism in question is the appropriate amount of said medium. -1675, and if desired, the product is isolated from the culture medium. The process of the present invention provides two major bioactive components of the BBM-1675 group of compounds, designated BBM-1675 A, and A ₂ , and four other minor components, designated BBM-1675 A ₃ , A ₄ , B, and B. ₂ . The individual components can be separated and purified by conventional chromatography. The BBM-1675 group of compounds and some of its bioactive components exhibit both antibacterial and antitumor activity. 2

The present invention relates to the preparation of a novel class of antibiotic-like compounds having antitumor activity, designated herein as ΒΒΜ-1675. By fermentation of certain strains of Actinomadura verrucosospora, in particular Actinomadura verrucosospora strain H964-92 and a mutant of Actinomadura verru cosospora A 1327Y. The above-mentioned parent strain was isolated from a soil sample collected in Puerto Esperanza (Misiones, Argentina). The biologically pure culture of the organism was deposited in the American Type Culture Collection, Washington, D.C., which classified it as a permanent microorganism collection under ATCC 39334. Subsequently, the A1327Y mutant strain was obtained by standard nitrosoguanidine treatment of an H964-92 strain and deposited in the American Type Culture Collection under the accession number ATCC 39638.

As with many antibiotic producing strains, fermentation of Actinomadura verrucosospora H964-92 and A1327Y results in a mixture or group of products. Two essential biologically active components, BBM-1675 A ₁ and A ₂ , and four minor biological components, BBM-1675 A ₃ , A ₄ , B, and B _{2, were} isolated from the BBM-1675 group produced by fermentation.

The A ₂ , A ₃ , A ₄ , B, and B ₂ components of BBM-1675 and BBM-1675 exhibit antibacterial activity against a broad range of microorganisms, particularly Gramm-positive pathogens. The BBM-1675 group of compounds and its isolated bioactive components are also capable of exerting a phage-inducing effect in lysogenic bacteria. Two of the components, BBM-1675 A and A _{2, were} screened in vivo for various mouse tumor systems and showed inhibitory activity in L-1210 leukemia, P-388 leukemia, B16 melanoma and Lewis lung cancer. BBM-1675 A ₃ and A ₄ inhibited P-388 mouse leukemia. The compound group and its bioactive components are therefore useful as antibacterial agents and antitumor agents for inhibiting mammalian tumors.

The H964-92 strain was isolated from a soil sample and transformed into a biologically pure culture by standard procedures. The aerial mycelium of strain H964-92 forms short spore chains that show straight, curled or hooked shapes. The spores are spherical or oval and have a warty surface. In most media the aerial mycelium develops poorly. The mass of the aerial mycelium is white, which subsequently turns pink or later turns blue on certain agar agar media. The mycelial substrate is colorless to light pink. The culture temperature is between 15 ° C and 43 ° C. The amino acid composition of cell-2193928 wall and the sugar components of whole cell hydrolyzate indicate that strain H964-92 belongs to cell type IIIb. Menaquinone was identified as MK-9 (H ₆ ) · MK-9 (H ₈ ).

Based on the major morphological, culturing and physiological characteristics, together with the chemical composition of the cell wall, strain H964-92 can be classified into the genus Actinomadura.

Although the original strain H964-92 showed only moderate growth and sparse aerial mycelium, a variant with nitrosoguanidine treatment was obtained which showed good growth ability and improved aerial mycelium formation. The variant, designated A1327Y strain, facilitated further taxonomic research and was later identified as Actinomadura verrucosospora strain.

The culture media and methods used to examine the breeding characteristics and carbohydrate utilization were as recommended by the International Streptomyces Project (ISP) (International J. Syst. Bacteriol. 16, 313-340, 1966). Other media described by S. A. Waksman (The Actinomycetes, Vol. 2) and G. M. Levedemann (Inti. J. Syst. Bacteriol. 21, 240-247, 1971) were also used. The amino acid composition of the cell wall and the sugar components of the whole cell hydrolyzate are described by Becker et al. (Appl. Microbiol. 13, 236-243, 1965) and Lechevalier and Lechevalier (The Actinomycetes, H. Prauser ed., Jena, Gustav Fischer Verlag, 393-405). , 1970). Menaquinone was determined by mass spectrometric analysis as described by Collins et al., J. Gen. Microbiol. 100, 221-230 (1977) and the composition of menaquinone was described by Yamada et al. (J. Gen. Appl. Microbiol. 23, 331-335, 1977).

Strain H964-92 forms both substrate and aerial mycelium. The substrate mycelium is long, branched, and does not fold into short fibers. In the aerial mycelium, short spore chains are formed monopodically or a

Table I on the tip of fungi (hyphae). In addition to the hyphae type, spore chains similar to spiral branches are also observed. These chains have from 2 to 10 spores each, straight, curly or hooked in shape. The spores have a warty, spherical or oval surface (0.5-0.6x0.6-1.4 μπι) with rounded or pointed ends. Upon maturation, "often spores are separated by empty shells.

No moving spores, spores or hardened granules were found in the test material.

The strain H964-92 and chemically defined, as well as natural organic táptalajo15 ° k ⁿ mild to moderate degree grows. Aerial mycelia are generally poor but already moderate on oatmeal agar (ISP # 3), inorganic salt-starch agar (ISP # 4) and Bennett's agar. Spontaneous variants lacking the aerial mycelium are common. The aerial mycelium is white in color, which subsequently turns light pink on oatmeal agar, inorganic salt-starch agar or glycerol-asparagine agar (ISP # 5). The color of the aerial mycelium mass, after a long culture time (5 months) turns blue on oatmeal agar, glycerol-asparagine agar and tyrosine agar. Layer color of substrate micelle 30 on Czapek agar, tyrosine agar, starch extract malate extract agar (ISP)

No. 2 medium), peptone starch extract iron agar (ISP # 6) and Bennett agar on a colorless to yellowish glucose-asparagine agar and glycerol-asparagine agar. Melanoid or other diffusible pigments are not produced. The strain A1327Y from the original strain forms a predominantly pale blue aerial mycelium and has an abundance of air spores.

H964-92 at 15 ° C, 28 ° C, 37 ° C and

They grow at 43 ° C, but no longer at 10 ° C or 47 ° C. At a concentration of 7%, sodium chloride is sensitive and at a concentration of 0.01%, it is resistant to lyso ₄₅ zym.

The breeding and physiological characteristics of the producing strain are shown in Table I and Table II. is shown in Table. The use of coal sources is described in Annex III. Table.

Breeding characteristics of strain H964-92 (variant of original ATCC 39334 and A1327Y (ATCC 39638))

Strain 11964-92 in Actinomadura

--------------------------- verrucosospora

Original strain Λ1327Υ variant ---------------- (ATCC 39334) (ATCC 39638) KCC A-0147

Tripton Yeast Extract Agar (ISP # 1)

N: abundant, fluffy, sedimentary and non-pigmented moderate, flaky, sedimentary and non-pigmented moderate, flaky, sedimentary and non-pigmented

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Table I (continued)

Czapek agar N: Moderate Sz: colorless LM: sparse; light gray (264) light pink DP: None weak colorless or sparse; whitish pink (9) none weak colorless or sparse, light blue (185) no Glucose-aspartic N: Moderate weak weak gin agar Sz: White (263) colorless colorless dark yellow to pink (27) LM: None or na- no or no no or no very sparse; clear very sparse; Fe very sparse; Fe pink Er Er DP: None no no Glycerol aszpa- N: mild to weak moderate moderate ragin agar (ISP # 5) sékeltig Sz: colorless vi- light yellowish light yellowish soft yellowing pink (28) from pink (28) to fat (28) LM: weak; light yellow-pink moderate; Fe dark yellowish-pink (27) moderate; white strong ro- (28) for 5 months clear from the body to fat (2) light blue-gray when kept light (190) DP: None pink (4) no no inorganic N: Abundant moderate moderate salts-starch W: Yellowish-white (92) light yellowish light yellowish agar (ISP # 4) pink (28) pink (28) LM: abundant; _; clear moderate; vilá- bőgéges; vilá- from pink (4) gos bluish gray gos blue (185) gray to pink (10) DP: None (190) no no Tirarin agar N: Moderate moderate moderate (ISP # 7) W: Yellowish-white (92) very yellowish very yellowish LM: weak; clear pink (26) moderate; Fe pink (26) moderate; Fe yellowish pink clear from the body clear from the body (28), later to pink (4) to pink (4) (5 months later) Partially Light Blue / Gray (190) DP: None no no Nutrient Agar Nutrient N: mild to moderate- weak weak soil Celtic Sz: light yellow colorless vi- colorless LM: None no soft pink (7) to light pink (7) none DP: None no no

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7 Table I 8 (continuation) Yeast N: Abundant copious copious malt extract W: Light yellow (89) very yellowish very yellowish agar (ISP # 2) fat color (26) pink (26) LM: weak; white weak; white weak; white- (263) light rose from clear light · to color (7) to fat (7) DP: None no no Oatmeal agar N: Moderate weak weak (ISP # 3) Sz: colorless vi- light yellowish light yellow · light pink (7) pink (31) pink (31) LM: weak; whitish very sparse; very sparse; from pink (9) bright light bright light light blue to gray (190) blue (184) blue (184) DP: None no no Bennett's agar N: Abundant copious copious W: Grayish (90) strong yellowing strong yellowing fat color (26) fat color (26) LM: Moderate; white- moderate; vilá- no from (263) yellowish gos yellow roses to white (92) color (31) and bluish white (189) DP: None no no Peptone-yeast N: Moderate no no extract-iron agar Sz: colorless colorless colorless (ISP # 6) LM: None no no DP: None no no

Notes:

1. Observations were performed in cultures for 3 weeks at 37 ° C.

2. Abbreviations: N = Growth, Sz = Color, 40

LM = air mycelium, DP = diffusible pigment.

II. Type 1:

3. Color designations and numerals in parentheses follow ISCC-NBS Color Standards, issued by Kelly, K.L. and Judd D.B. (issued by U.S. Department of Commerce 553, Washington, D.C., Nov. 1975).

a t

Physiological characteristics of strain H964-92

Test

Response

Method and culture medium

Limits of breeding temperature

Dispensing of gelatin

Hydrolysis of starch Reactions in skimmed milk

Formation of melanoid pigment

Maximum increase from 28 ° C to 37'C. Moderate at 20 ° C and 43 ° C. Treasure growth at 10 ° C and 47 ° C.

Megfolyósodott

Hydrolyzed Not coagulated and completely converted to peptone

It doesn't produce

Bennett's agar

Glucose peptone gelatin medium

Starch agar plate with Difco skimmed milk

Tyrosine agar, peptone yeast agar and

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Nitrate reduction

Resistance to NaCl

lysozyme

PH

II. Table (continued)

He did not reduce it

Growth up to 5 ^ or below. At 7% there is no growth

Tough. An increase of less than 0.01%. There is no growth at 0.1%.

Growth between 5.0 and 9.5, no increase at 4.5 and 10.0.

tripton yeast extract medium Czapek medium and glucose yeast extract nitrate medium Tripton yeast extract agar

Tripton yeast extract agar.

Tripton yeast extract agar

III. Spreadsheet

Utilization of coal sources

H964-92 Actinomadura ------------------- verrucosospora

Original tribe A1327Y variant KKC A-0147 Glycerol + + + D (-) - arabinose - - - L (+) "arabinose + + + D-xylose + + + D-ribose + - - L-rhamnose + + + D-glucose + + + D-galactose - - - D-fructose + + + D-mannose - - - L (-) sorbose - - - saccharose + + + lactose - - - cellobiose, + + melibiose - - - trehalose + + raffinose - - - D (-) - melezitose - - - soluble spy opener + + + cellulose T + + dulcitol - - - inositol + - - D-mannitol + + D-sorbitol - - - salicin - - - Explanation:

Observations were made from cultures maintained at 28 ° C for 3 weeks.

The basic medium was Pridham-Gottlieb's inorganic medium.

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The purified cell wall of strain H964-92 contains mesodiaminopimellic acid but lacks glycine. Madurose (3-0-methyl-D-galactose), glucose and ribose are present in the whole cell hydrolyzate. Cell wall amino acids and whole cell sugar components indicate that strain H964-92 belongs to cell type IIIb. Two menokinone components were identified to a greater extent: MK-9 (H ₆ ) and MK-9 (H ₈ ).

From a taxonomic point of view, the main features of strain H964-92 are: (1) chains of air spores: short, straight, curled or hooked; (2) the surface of the spores is warty; (3) the aerial mycelium is pink in some media; (4) does not form diffusible pigments; (5) mesophilic; (6) has a cell wall of type IIIb; (7) menaquinone system: MK-9 (H ₆ ) and MK-9 (H ₈ ).

These main characteristics indicate that the strain H964-92 can be classified into the genus Actínomadura. Early varieties of the genus Actínomadura were isolated from mammals. Certain strains were also obtained from plant materials. In addition, many of the newly found varieties have been isolated from the soil. Goodfellow et al. (J. Gen. Microbiol. 112, 95-111 (1973)), according to the neumerical taxonomy and review of Actinomadura genus and related radiation fungi, most of the soil-derived Actinomadura cultivars can be classified into 7 of the 14 described groups. . The H964-92 strain is very closely related to the Group 7 varieties. Nonomura and Ohara (J. Fermen. Technol., 49, 904-912 (1971)) reported five saprophytic species of the genus Actinomadura and Nonomura (J. Ferment. Technol., 52, 71-77 (1974)) and Preobrazenskaya et al. (Actinomyces and Related Organisms 12, 30-38 (1977)) disclosed the keys necessary for identification and taxonomy of the genus Actinomadura. Based on comparative studies of 30 different varieties, including those disclosed in patents, strain H964-92 appears to be very similar to Actinomadura coerulea in Preobrazenskaya et al., Cited above, and in Act. madura verrucospora.

The H964-92 strain was directly compared with the strain A. verrucosospora KKC A-0147 and was found to be very closely related to the A. verrucosospora strain in terms of morphology, breeding and physiology. Thus, strain H964-92 can be classified as a new strain of the species Actinomadura verrucosospora.

It is understood that the present invention, although detailed in the description of a specific strain of Actinomadura verrucosospora, strain H964-92 (ATCC 39334) and its mutant, strain A1327Y (ATCC 39638), is not limited to the production of BBM-1675. these micro-organisms or those micro-organisms which have been fully described with respect to the culture characteristics disclosed herein. It is a particular intention that the invention encompasses strain H964-92 and all natural variants and mutants thereof which produce natural and artificial BBM-1675 compounds.

The BBM-1675 antibiotics of the present invention are prepared by culturing any BBM-1675 antibiotic producing strain of Actinomadura verrucosospora, preferably a strain of Actinomadura verrucosospora identified by the characteristics of an ATCC 39334 or ATCC 39638 or a mutant or a mutant thereof. medium. The organism can be cultured on any medium containing nutrient sources known to the fungi, so that various sources of carbon and nitrogen can be used, and in addition, inorganic salts and known growth factors can be used as desired. A submerged aerobic culture method is preferred for the production of a large amount of the antibiotic, although surface cultures or bottles may also be used for the production of a limited amount. In the process of the present invention, all operations applicable to the cultivation of other radiation fungi can be used.

The medium must contain an appropriately absorbable carbon source such as glycerol, L (- j -) - arabinose, D-xylose, D-ribose, L-rhamnose, D-glucose, D-fructose, sucrose, cellobiose, soluble starch, D-mannitol or inositol. Nitrogen sources include ammonium chloride, ammonium sulfate, urea, ammonium nitrate, sodium nitrate, and the like. it may be used either individually or in combination with an organic nitrogen source such as peptone, meat extract, yeast extract, corn jam, soybean meal, cotton seed meal, and the like. Some inorganic salts such as sodium, potassium, calcium, ammonium, phosphate, sulphate, chloride, bromide, carbonate, zinc, magnesium, manganese, cobalt can be added to the medium if necessary. , iron salt and the like.

The production of BBM-1675 antibiotics can be carried out at any temperature that leads to a satisfactory growth of the producing organism, e.g. 15 ° C to 45 ° C, and suitably 27 ° C to 32 ° C. Generally, the best yield is achieved within a growing time of 68 to 180 hours, depending on whether fermentation is carried out using shaker flasks, cucumber bottles or tanks. If tank fermentation is to be carried out, it is desirable to first produce an inoculum by inoculating the producing organism with a suitable inoculum into the desired medium. After obtaining an active inoculum in this manner, it is inoculated under aseptic conditions as shown in Figure 7

-7193928 into fermentation tank medium. The progress of antibiotic production can be monitored by a paper disk agar diffusion method using Staphylococcus aureus strain 209P as a test organism.

Upon completion of the fermentation, BBM-1675 can be recovered from the fermentation broth by standard isolation techniques, e.g. liquid extraction. For example, the whole fermentation broth may be separated into mycelial filter cake and supernatant fermentation broth by filtration or centrifugation. The antibiotic remaining in the filter cake can be recovered by suspending it in methanol and concentrating the methanolic extract. The active ingredients in the supernatant of the fermentation broth can be recovered by n-butanol extraction. The above-mentioned n-butanol and methanol extracts can also be combined and azeotropically distilled to obtain an aqueous solution from which the active antibiotics precipitate as an oily solid. The solid was dissolved in methanol and filtered. The filtrate was concentrated and added to ethyl acetate-water. The resulting organic extract contains the crude BBM-1675 complex which can be precipitated from solution by addition of a non-solvent such as n-hexane.

The crude BBM-1675 group of compounds is a mixture of several components, of which the major bioactive components are BBM-1675 A and A ₂ , and the four less bioactive components are BBM-1675 A ₃ , A ₄ , B, and B ₂ . These bioactive components can be isolated and purified by conventional chromatography. In one method, the crude BBM-1675 complex was first dissolved in methanol and then purified by Sephadex LH-20 column chromatography eluting with methanol. This partially purified complex is further purified by chromatography on a column of silica gel followed by stepwise elution with chloroform and increasing concentrations of methanol. The result: BBM-1675 A, a mixture of BBM-1675 A ₂ , A ₃ and A ₄ and BBM-1675

A mixture of B and B ₂ . Component A can be further purified by Sephadex LH-20 column chromatography and elution with methanol. Mixtures of A ₂ , A ₃ and A _{4 were} separated by chromatography on a Bondapak C ₁₈ column (Waters Associates, Inc.) eluting with increasing concentrations of aqueous acetonitrile. The mixture of components B and B ₂ can 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 can be found in the Examples.

The six bioactive components of the BBM-1675 group of compounds can be distinguished by two thin layer chromatography techniques, as shown in Figure IV. Table.

ARC. T h

TLC for BBM-1675 Components - Rf values

component Silica CHC1 ₃ -CH ₃ OÍ1 / 5: 1 v / v C ₁₈ reverse phase silica gel CH ₃ CN-H ₂ 0/75: 25 v / v BBM-1675 Ai 0.74 0.18 BBM-1675 A ₂ She; 71 0.21 BBM-1675 A ₃ 0.72 0.28 BBM-1675 A ₄ 0.71 0.78 BBM-1675 Bi 0.63 0.23 BBM-1675 B ₂ 0.60 0.16

Separation of BBM-1675 A ₂ , A ₃ and A ₄ was difficult with ordinary phase thin-layer chromatography systems, but could be achieved with phase-reversed thin-layer chromatography. θθ

The unique BBM-1675 components show similar solubilities and color reactions. For example, they are soluble in chloroform, ethyl acetate, acetone, ethanol and methanol, are slightly soluble in benzene and 65-8 water, and are insoluble in n-hexane and carbon tetrachloride. They give a positive reaction with ferric chloride, Ehrlich and Tollen, but negative for Sakaguchi, ninhydrin and anthron.

The typical physicochemical properties of the components of the BBM-1675 complex are summarized in Table V.

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The UV absorption maxima of the BBM-1675 components were found at 253, 282 and 320 nm, which were not shifted in either acidic or alkaline media. The IR and proton resonance spectra of BBM-1675 A, A ₂ , A ₃ and A _{4 are shown in} Figures 1-4. 5 to 8 and FIGS. Figures. The 360 MHz proton resonance spectrum of BBM-1675 A contained one acetyl group (delta: 2.1 ppm, one> N-CH ₃ group (2.52 ppm), four -OCH ₃ groups (3.42, 3.80, 3 , 88 and 3.98 ppm) and exo methylene (4.57 and 5.48 ppm), together with two aromatic (7.50 and 8.59 ppm) and one -NH (11.79 ppm) proton. -1675 a, ¹³ C magnetic resonance spectrum showed 55 carbon signals, including a triple-intensity signal (delta 56 ppm, _OCH3). the BBM-1675, molecular chemical formula C ₅₇ H ₇₂ N ₄ O ₃₂ proton resonance and ¹³ C magnetic resonance spectra, and elemental microanalysis and high pressure liquid chromatography and secondary ion mass spectrometry.

BBM-1675 A, at room temperature with 0.5 N HCl-CH ₃ OH, loses its biological activity and results in a lipophilic chromophore (Compound I) with various unidentified fragments. Compound (I) exhibits UV absorption similar to the resulting antibiotic, suggesting that Compound (I) retains the structure of BBM-1675A, chromoform. Alkaline hydrolysis of BBM-1675 A yields two other chromophore fragments: hydrolysis of 0.05 N KOH-CH ₃ OH at 55 ° C for one hour gives compound II with UV absorption maxima of 252, 284, 297 (shoulder). ) and 322 mm, while reaction with 1 N KOH-CH ₃ OH yields an acidic chromophore material called Compound (III).

A summary of the physicochemical properties of compounds (I), (II) and (III) is given in Table VI. Table.

VI. Spreadsheet The (I), Properties of Compounds (II) and (III) Compound (I) Compound (II) (III) Mp 82-83 ° C I33 ° C 253-255 ° C (c.0,2 CHC1 ₃₎ -100 0 0 Molecular formula C ₂ iH ₃ ιΝΟχo CiuHi ₇ NO ₆ Ci ₃ H ₁₅ NO ₆ UV lambda ^CH3 ° ^H , .nm (£) max 244 (21.85C) 252 (26,600) 248 (26,900) 276 (9,400) 283 (11,200) 295 (14,400) 318 (6,300) 297 (including 8,800) 310 (13,500) MS m / z 457 (M ⁺ ) 295 (M +) 281 (M ⁺ ) 425 280 263 341 263 236 281 251 222 264 248 218 Vékonyrétegkromato- Rf 0.58 0.66 0.13

graph (xylene-methyl-ethyl-ketone-methanol = = 5: 5: 1 v / v)

Information on the structure of compounds (II) and (III) was obtained from the following spectral data and chemical conversion. A ^{, 3} C and proton nuclear resonance showed the presence of four -OCH ₃ groups, one = CH ₂ group, seven -C = atom and one> NH group in compound (II). NMR spectrum of the compound (III) are similar to compound (II) includes, and there was only one difference if one of the four θθ _-OCH3 group detected in the (II) compound lacking. This difference, in addition to the acidic nature of Compound III, supported the assumption that Compound II was the methyl ester of Compound III. After refluxing with 1 N methanolic KOH at reflux temperature, compound (II) was quantitatively converted to compound (III) and treated with diazomethane (III) to compound (11). By reaction of (II) in ethanol, sodium borohydride in a reduced product (compound (IV), M ^+: m / z 267) were obtained, that the NMR spectrum -COOCH ₃ group in place of the compound (II) is a -CH ₂ OH radical shown. After palladium-on-carbon hydrogenation, the dihydro derivative of compound (II) was obtained (compound V, M ⁺ : m / z 297). The proton resonance spectrum of compound (V) contained a new doublet methyl signal, while

The absence of an exomethylene group in compound -10193928. In addition, one of the '-OCH3 groups appears at a higher level (delta: 3.50 ppm) in Compound (V). These results are one

-C = CH ₂ OCH ₃ indicate the presence of the group (II) compound, comprising the hydrogenation - CH - CH ₃ l

OCH was reduced to _{3 in} compound (V).

Compound (II) was heated in a 1.5N methanolic hydrochloric acid solution at 80 ° C for 3 hours and the hydrolyzate was chromatographed on a silica gel column to give a slightly basic product (Compound VI, M ⁺ : m / z 211). The IR spectrum and physicochemical properties showed that compound (VI) contains an NH ₂ group.

Compound (VI) was identified as methyl 4,5-dimethoxy anthranilate by comparative IR and NMR tests performed on a valid sample. As a result, the structure of compounds (II) to (VI) was determined as shown in formula (1).

Compound (I) was treated with 0.05 N KOH-CH ₃ OH at 55 ° C on a new chromophore portion of compound (VII), C ₁₅ H ₂₁ NO ₇ , M ⁺ : m / z 327) and a sugar (( Compound VIII). In the NMR spectrum of Compound VII, one C-CH ₃ singlet and two -OCH ₃ groups (high-lying) can be found along with two low-lying ÖCH ₃ groups and two aromatic protons, which are usually found in (II), (V) and ( VI). Further hydrolysis of compound (VII) with 1.5N methanolic hydrochloric acid gave compound (VI) which was identical to that obtained previously from compound (II). After removal of compound (VI), the hydrolyzate was treated with 2,4-dinitrophenylhydrazine, which precipitated a yellow solid, which was pyruvic acid.

It was identified as 2,4-dinitrophenyl hydrazone. Thus, Compound (VII) methyl-4,5-dimethoxy-N- (2'2'-dimethoxy-propionyl) -anthran Elemental analysis:

1. Analysis 2. Analysis see. The compound (VIII) showed the molecular ion peak, but the released mass spectrum of m / z _M-OCH3 131nél peak in good agreement with the C ₇ H ₄ O _I4 összegképlettel. Nuclear Magnetic Resonance Spectrum S showed the structure of compound (VIII) to be 2,6-dideoxyhexopyranose. This assumption was also supported by the ¹³ C magnetic resonance spectrum of Compound (I), which has one -C-CH ₃ group, one -CH ₂ - group, three -O-CH 2 groups and one anomeric carbon atom besides the 15 carbon signal, which are attributable to compound (VII). The C ₃ sugar proton appeared at a low level (delta: 5.39 ppm, octet) revealing that the C ₃ -OH group of the sugar was esterified with the carboxyl group of compound (VII). The above are illustrated in formula 2.

The molecular weight (457) of Compound (I) corresponds to one third of the total BBM-1675 Aj molecule (suggested formula: C ₅₇ H ₇₂ N ₄ O ₃₂ ; calcined molecular weight: 1324). The putative partial structure of BBM-1675 A is illustrated by formula (Ia).

No. 495,231. Following the filing of our US patent application, it was found that the BBM-1675 A and A ₂ components described above, when prepared according to Example 2, are in fact not completely pure and that some of the characteristic data used to determine these compounds are inaccurate. Additional chromatographic purification procedures, which are described in more detail in the following Examples 3 and 6, have resulted in the preparation of BBM-1675 A ₁ and A _{2 in} a purer form and characterized as follows. We also reviewed the ₃ and ₄ having the elemental composition analysis, which showed the presence of sulfur is being represented, and the two component retention values were determined with high performance liquid chromatography as well. The revised physico-chemical properties of the BBM-1675 components are as follows.

BBM-1675A,

Description: White to light yellow crystals;

156-158 ° C.

Average

C: 51.60%

B: 6.31%

N: 5.31%

S: 8.47%

0. (Difference):

28.31%

C: 52.74%

H: 5.99%

N: 3.94%

S: 9.71% (difference):

27.62%

C: 52.17%

H, 6.15%

N: 4.63%

S: 9.09% (difference):

27,96%.

-11ϊ93928

22

Ultraviolet Absorption Spectrum: Instrument Varian UV, Cary 219

Solvent - methanol Concentration - 0.01356 g / l.

lambda / nn / max

absorbance

320

280

253

210

12.4 shoulders

25.1

25.5

There is no significant change with the acid or base.

Optical Rotation: Solvent - CHCl ₃ [α] ²⁴ = -207 ° (C = 0.0351) 2q

A second analysis showed the following optical rotation: [α] ²⁷ = -191 ° (C = 0.5 CHCL _? )

Infrared absorption spectrum: see Figure 9. 25

The major absorption bands (KBr) are:

985, 1015, 1070, 1110, 1150, 1210, 1250,

1308, 1380, 1405, 1446, 1520, 1592, 1608,

1668, 1715, 2920, 2960, 3360, 3440 cm ^-1

Mass Spectrum: Instrumentation - VG - ZAB - 2F FAB-MS-Thioglycerol Molecular Ions Range:

(m / z): 1249, 1357, 1463;

With NaCl (m / z): 1271, 1379, 1485,

1597th

FAB-MS-MB (MB: matrix, 154 molecular weight);

Molecular ion range (m / z): 1249,

1283, 1403, 1555; Addition of NaCl (m / z): ₄₀

1249, 1271, 1303, 1425, 1483, 1577.

FAB-MS-Glycerol-DMSO: Range of molecular ions (m / z): 1215, 1247, 1279, 1293,

1325, 1353; NaCl added (m / z): 1215,

1237, 1247, 1269, 1325, 1347, 1375.

Device: Kratos MS-50 ⁴⁵

FAB-MS-Glycerol-DMSO: Molecular Ionization (m / z): 1357, 1463.

Molecular Weight Apparent Molecular Weight: 1248 ₅₀ (Based on Mass Spectrometry Data Above)

NMR Spectrum: Device - WM360 Brucker "

Solvent: CDCl ₃ NMR: 360 MHz delta (ppm): 11.75 (1H, s);

8.55 (1H, s); 7.45 (1H, s)

6.61 (1H, m); 6.23 (1H, broad s); 6.17 (1H, br s ^ü); 5.93 (1H, d, J = 9.3);

5.82 (1H, d, J = 9.3), 5.7 (1H, broad s); 5.49 (1H, m); 5.45 (1H, d, J = 2.3);

5.38 (1H, broad s); 4.95 65 (1H, d, J = 10.2); 4.64 (2 H, 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, broad s); 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, broad m); 2.50 (3H, s); 2.47 (1H, m); 2.38-2.22 (5H); 2.12 (1H, m); 2.11 (3H, s); 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.

¹ C NMR: 90.3 MHz.

See below. Fig.

In a separate study, the effect of the purified BBM-1675 ³ C NMR spectrum of a sample, which was dissolved in ₃ (80 MHz) in CDCl. The major peaks are given below.

BBM-1675 Aj ¹³ C Magnetic Resonance Spectrum (80 MHz in CDCl 3)

Chemical shift in ppm (Multiplicity)

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) 90.5 (T) 97.2 (D) 98.3 (S) 99.0 (D) 99.6 (D) 103.1 8 (d) 107.1 3 (s) 112.5 (D)

-12193928

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) 5

160.7 (s) 166.4 (s) 191.8 (s).

Multiplicity: q = quartet, d = doublet, u = uncertain, t = triplet, s = singlet.

BBM-1675 A ₂

Description: White crystals, m.p. 147-149 ° C.

Elemental Analysis: C: 52.71%

H: 5.94%

N: 3.94%

S '9 39%

O: 28.01% (difference)

Ultraviolet Absorption Spectra: Instrument - Varian

UV, Cary 219 20

Solvent: methanol Concentration: 0.02052 g / lambda _max / nm / Absorbances

------------------------------------ ₂₅

320 12.2

282 16.3

252 26.2

214 25.8

There is no significant change with the acid or base.

Optical rotation: [α] ²⁷ = -179.4 ° (c = 0.5, CHCL ₃ ).

Infrared spectra: see Figure 12. 35 Instrument: Beckman IR Model 4240 Principal Absorption Bands (KBr): 950, 1015,

1070, 1100, 1155, 1213, 1250, 1313, 1375, 1405,

1450, 1520, 1595, 1610, 1685, 1735, 2940, 2980,

3440 cm ¹ . 40

Mass Spectrum: Instrument: VG-ZAB-2F FAB-MS-Thioglycerol Range of Molecular Ions (m / z):

968,1249,1355, 1357, 1463, 1569; Replies ₄₅ by addition of NaCl (m / z): 990, 1271, 1379, 1485. FAB-MS-MB (MB: matrix, mw 154); MolekuVII. Table Ia range (m / z): 1249, 1403, 1419, 1555, 1571, 1587; NaCl (m / z): 1249, 1271, 1425, 1441, 1457, 1483, 1577.

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

Molecular Weight apparent molecular weight: 1248 (based on mass spectrometry data as described above)

NMR spectrum: see Figure 13.

Device: WM 360 Brucker Solvent: CDC1 ₃ Ή NMR 360 MHz delta (ppm)

11.91 (1H, s); 8.62 (1H, s); 7.58 (1H, s); 6.56 (1H, m); 6.22 (1H, s); 6.15 (1H, broad s); 5.91 (1H, d, J = 9.6); 5.83 (1H, d, J = 9.6); 5.70 (1H, m), 5.45 (1H, d, J = 2.2); 5.44 (1H, s); 5.34 (1H broad s); 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, bs); 4.05 3.50 (20-24H); 3.96 (3H, s); 3.87 (3H, s); 3.77 (3H, s); 3.46 (1H, m); 3.39 (3H, s); 2.79 (1H, m); 2.73 (2 H, m); 2.5 (3H, s); 2.50 (1H, m); 2.38-2.22 (3H); 2.14 (1H, m); 2.10 (3H, s); 1.98 (2 H, 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).

¹³ C NMR 90.3 MHz.

See Figure 14.

In a separate study, we prepared 80 Mhz ⁱ³ C magnetic resonance spectrum of the purified sample is dissolved in _{CDCl3 two.} The main peaks are given below:

BBM-1675 A ₂

13.7 16.9 17.5 19.8

34.1 35.1 39.3 47.6

57.6 62.4 64.5 64.9 71.9 73.6 75.8 76.1 83.3 86.2 88.4 90.4

99.6 103.8 107.1 112.4

144.1 154.5 160.9 167.9

22.3 22.7 23.4 33.1 52.6 55.7 56.0 56.1 65.9 68.3 69.2 69.7

77.1 77.7 78.1 78.3

97.2 98.3 99.1 99.5

123.2 124.8 129.9 137.3

192.2

Physico-chemical properties of BBM-1675 A3, Αι +, Βχ, B2

A3 Al »Bi B2

melting point 125-127 ° C 123-126'C 159-161 ° C 156- -159 ° C [alV ^{c °> ⁵ > ^chc1 3> -161 ° C -176®C -171 ° C -122 ° C Elementary Analysis C: 54.55 54.65 m 11: 6.46 6.29 N 3.73 3.51 S: 7.49 8.07 UV lambda nm 253 (286) 253 (257) 253 (225) 248 (212) taste ^max (E, ^ in methanol: 1 cm) 282 (158) 282 (153) 282 (140) 279 (141) 320 (122) 320 (117) 320 (104) 318 (103) 0.01 N in HCl-MeOH: 253 (287) 253 (258) 253 (225) 248 (2.0) 282 (160) 282 (155) 282 (140) 279 (140) 320 (126) 320 (118) 320 (105) 318 (103) 0.01 in nNaOH-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)

-13193928

26

Thin Layer Chromatography (TLC) and High Performance Liquid Chromatography (NTFK) Data for BBM Components A. No. 1 Study Summary

VIII. Spreadsheet

BBM-1675 components TLC and NTFK data

CTR (Rf)

NTFK (retention time, minutes)

Silica gel CHCl ₃ -MeOII (5: 1 v / v) C18 reversed silica gel CH ₃ CN-H ₂ 0 (75:25 v / v) Lichtosorb RP-18 _CH3 CN-MeOH-O, 1 m ₃ 0Η 000ΝΗ ₄ (5: 2: 3 v / v) BBM-1675 0.74 0.18 13.3 BBM-1675A ₂ 0.71 0.21 17.3 BBM-1675A ₃ 0.72 0.28 8.0 BBM-1675Ait 0.71 0.78 5.1 BBM-1675Bi 0.63 0.23 - BBM-1675B ₂ 0.60 0.16 · "

B. No. 2 Study ₂ - TLC and NTFK data for purified A and A ₂ components

TLC

Analtech GHLF Silica Gel Uniplate plates were used for all normal phase chromatography. The plates used for the one-dimensional VRK were 2.5 x 10 cm. These were developed in 6.4 cm diameter 12 cm high glass cylinders containing 10 ml of eluent. For two-dimensional TLC studies, 7.5 x 10 cm plates were used. The sample was applied at the lower left corner 1 cm from the edges. The plate was first developed in 50 ml first eluent containers (12.7 cm wide, 8.6 cm deep and 13 cm high). The plate was then air dried, rotated 90 ° counterclockwise, and recovered in a second container containing 50 ml of the second eluent.

Whatman's pre-coated C-18 silica gel plates were used for phase-reversed chromatography. The 2.5 cm x 7.6 cm plates were developed in glass cylinders containing 10 ml of eluent.

Normal phase plates were first examined under 254 nm UV light. The plates were then placed in glass cylinders (6.4 cm diameter, 12 cm height) containing J ₂ crystals. After about two minutes, the plates were re-examined. Phase-reversed plates were examined under 254 nm UV light only. The zones were detected by looking for quenching of the fluorescence of an impregnated dye. Analytical high performance liquid chromatography

The system is composed of the following components: Waters Associates 6000A Fluid Transfer System Pump, 14

Varian Varichrom operating VUV-10 UV / visible detector set to OD 254 nm 0.1, Fisher Recorded streak _"n 5000 has típusjelzésű recorder of Waters Associates Model 660 solvent programmer of Waters Associates Model U6K injector according Alltech, μ column μ -Bondapak C _18/10 (4.6 mm i.d. x 25 cm) Whatman Co. Pell ODS (from 0.03 to 0.038 mm) and guard column (4.6 i.D. x 5 cm). The components were connected with a 316 stainless steel tube (1.6mm outer diameter - 0.13mm inner diameter). The eluent was passed at 2 ml / min for all assays.

Preparative high pressure liquid chromatography

A medium-pressure liquid chromatography system was used in the next edit alcohol ⁴ 5 lake elements: Fluid Metering Inc. RP-SY 2CSC FMI laboratory pump, Fluid Metering, Inc. FMI PD-60 LF pulse wetting polypropylene tube (3.0 mm outer diameter,

5q F5mm inner diameter) made of 8.65mm outer diameter cardboard tube twisted sample loop, Glenco 3500 Series Universal LC Columns, Instrument Specialties Co. UA-5 Type Absorbance / Fluorescence Monitor with Type ₆ Optical Unit, Instrumentation Specialities Co. 590 Flow Interrupt Valve and an Instrumentation Specialties Co. 328 Fraction Collector. Components with polyethylene and Teflon tube (3.0mm outside diameter,

1.5 mm internal diameter) and Glenco Multifit switches and valves are connected in the order listed.

Glenco 3500 Universal LC columns were filled with slurry of the designated absorbent with the specified solvent 65 in a conventional manner. The settled bed and

-14193928 The free space between the tip of the tube was filled with standard Ottawa sand. The eluent was pressed through at a pressure of not more than 4.2 atm (about 20 mL / min).

Gradient elution

All gradient elution assays were performed on a Glenco gradient elution device, consisting of two chambers of the same diameter, height, and void volume connected in series by a Teflon valve. One chamber served as a mixing chamber and the other as a static container. The less polar solvent was originally stored in the static chamber. Teflon-coated magnetic stirrers (1.0 x 3.0 cm) were placed in both chambers and were Thomas's Type 15 Magnet mat

IX. Various mixers were rotated. The eluent was printed from the mixing chamber on a polypropylene tube (3.0 mm outer diameter, 1.5 mm inner diameter) into a medium pressure liquid chromatography system. As the eluent was removed from the mixing chamber, it was replaced by the free flow of solvent in the static chamber to form a linear gradient of the eluent.

Analysis of BBM-1675 A and A ₂

The following section IX. Table 4 summarizes the Rf values of BBM-1675 A and A _{2 in} normal phase plates. The Rf value was calculated by dividing the distance from the sample application point to the center of the zone by the distance from the sample application point to the solvent front.

fever

System / Compound Rf

BBM-1675 Ai BBM-1675 A ₂

4% methanol in chloroform 57a methanol in diethyl ether 50% acetone in Skellysolve B

0.33 0.30

0.39

0.38 0.31

The following Table X summarizes the results for BBM-1675 A and A ₂ on normal phase plates, which were obtained in two dimensions. The position of the patches is Cartesian coordinate. It is expressed in terms of notes. The X coordinate is the Rf of the second solvent system listed. The Y coordinate is the Rf of the first solvent system listed.

z a t

System / Compound Rf

BBM-1675 Ai BBM-1675 A ₂

4% methanol in chloroform (see above)

57a methanol in diethyl ether (0.34, 0.33) (0.28, 0.23) 47a methanol in chloroform (see above)

50% Acetone Skellysolve (0.33, 0.29)

B in

The following, XI. Table 4 summarizes the Rf values observed on BBM-1675 A and A ₂ phase reversed TLC plates. The plates were developed in two 55-component eluents.

XI. Spreadsheet

System / Compound

Rf

BBM-1675 Ai BBM-1675 A ₂

25% in 0.5 m NaCl in acetonitrile 0.18

25% water in acetonitrile 0.00

0.21

0.00

-15193928

The following XII. Tables 1 and ₂ are measured on BBM-1675 Aj and A ₂ C-18 reversed TLC plates

Specifies the Rf values for which discs were recovered in a ternary eluent.

XII. Spreadsheet

System / Compound Rf

BBM-1675 Αχ BBM-1675 A ₂ acetonitrile: methanol: 0.5 m 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 Oh, 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 acetonitrilmetanol 0.1 m NHi ^ OAc 40% 30% 30% 0.32 0.22 acetonitrile methanol: 0.1 m NaFfaPCfa 40% 30% 30% 0.00 0.00

High Pressure Liquid Chromatography (NNFK) for BBM-1675 A and A ₂

BBM-1675 A, and BBM-1675 A _z were tested on a C-18 phase reversed silica gel column using one, two and three component eluents. The observed K 'values are shown below in XIII, XIV. Table summarizes. The K 'values were calculated using the following formula ³⁰ :

tz> TR-T _O ^K “T _o where TR is the retention curve measured from injection to the highest peak and T _{o is} the empty volume time.

XIII. Spreadsheet

System / compound k '

BBM-1675 Ai BBM-1675 A ₂

Acetonitrile a

Tetrahydrofuran a

Methanol 0.00 0.00

New match:

a = the compound was not soluble in the column.

XIV. Spreadsheet

J

System / Compound K

BBM-1675 Ai BBM-1675 A ₂

25% water in acetonitrile a

25% methanol in water 1.25 1.25

Comment:

a = the compound was not soluble in the column.

-16193928

XV. Spreadsheet

System / compound k '

BBM-1675 Al BBM-1675 Al ₂ acetonitrile: methanol: water

40% 30% 30% the the acetonitrile methanol: 0.1 m NH. OAc 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

Comment:

a = not eluted, b = not determined.

Biological properties of BBM-1675 components

The antimicrobial efficacy of BBM-1675 components on bacteria (Gram positive, Gram negative and acid resistant) and certain fungi was determined by the double agar dilution method. For Gram positive and Gram negative bacteria agar medium was used; medium (3% glycerol, 0.3% sodium L-glutamate, 0.2% peptone, 0.31% Na ₂ HPO ₄ , 0.1% KH ₂ PO ₄ , 0.005% ammonium citrate, 0.001% magnesium sulfate and 1.5% agar). Sabouraud agar medium was used for the fungi. As shown in FIG. Table 6 shows that the six components of BBM1675 (A 1, A ₂ , A ₃ , A ₄ , B 1, B ₂ ) showed a broad spectrum of antibacterial activity. BBM-1675 A, A ₂ , A ₃ and A ₄ showed particularly potent activity against Gram positive pathogens.

XVI. Spreadsheet

Antimicrobial activity of BBM-1675 components

Minimum inhibitory concentration (mcg / ml)

Tribe BBM-1675 Ah! THE 2 ₃ au bi b ₂ S. aureus 209P ΖΟ, ΟΟΟδ 0.0063 0.0063 .0125 0,012 0.0063 S. aureus Smith z.0,0008 0.0031 0.0063 .0125 0,012 0,012 B. subtilis PCI 219 z0,0008 0.05 .0125 .0125 0.05 0.05 M. luteus 1001 0.0016 0.0063 .0125 .0125 0.1 Q, 1 M. fiavus ζΟ, ΟΟΟδ 0.0016 0.0063 .0125 0,025 0,025 Mycobacterium 607 0.05 0.1 - - 0.05 0,025 E. coli NIHJ 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 also performed with purified A and A ₂ (a

Prepared according to Example 3) and the components A ₃ and A ₄ . Your data will be summarized below.

-17193928

33 34 Minimum inhibition concentration by agar diffusion assay (mcg / ml) Tribe BBM-1675 Αχ A ₂ A ₃ A _u

S. aureus 209P Z = 0.0008 0.0063 0.0063 0,012 S. aureus Smith z 0.0008 0.0031 0.0063 Q O12 E. subtilis PCI 219 Z0,0008 0.05 0,012 0,025 M. luteus 1001 0.0016 0.0063 0,012 0.05 M. flavus Z0,0008 0.0016 0.0063 0,012 Mycobacterium 607 0.05 0.1 0.16 0.16 E. coli NIHJ 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. fragilis A20928 0.2 1.6 0.2 0.4 C. difficile A21675 0.4 0.8 0.05 0.4 C. perfringens A9635 0.05 0.8 0.4 0.4 C. albicans IÁM 4888 0.4 0.4 1.6 6.3 C. neoformans 1.6 3.1 1.6 6.3

The value of induction of prophage activity on BBM-1675 components on lysogenic E. coli W1709 (lambda) by Lein et al; (Naturre 196, 783-784 (1962)). Platelet counts were performed on agar plates containing test substance (T) and control platelet (C). Platelet counts were found to be significant at T / C above 3.0 and expressed as lysogen induction activity (ILB activity) at the minimum concentration of test compound inducing induction.

As in the XVII. Table B1 shows the BBM-1675 components showing strong ILB activity in lysogen bacteria, suggesting their anti-tumor activity.

XVII. Spreadsheet

Lysogen induction activity of BBM-1675 components

Antibiotic Induction Minimum Concentration (mcg / ml)

BBM-1675 Αχ 0.0063 BBM-1675 A ₂ .0125 BBM-1675 A ₃ 0.05 BBM-1675 A ₄ 0, 10 BBM-1675 Bi 0:10 BBM-1675 B ₂ 0.20

The antitumor activity of BBM-1675 A and A ₂ was determined on various mouse tumor systems. Lymphocytic leukemia (P-388), lymphoid leukemia (1-1210), melanotic melanoma (B-16) and Lewis lung carcinoma were implanted intraperitoneally in BDF, male mice by inoculation of 10®, 10 ^s , 5 χ 10® and 10® cells, in the order of the above list. After 24 hours, the test compounds were administered spleen tube ⁶⁰ typical doses following tumor cell implantation the mice intraperitoneáüsan. Treatment is given only once on the first day, days 1, 4, and 7 (q3d x 3), once daily for 9 days (qd 1-9) or 11 days (qd 1- 11). Components A ₃ and A ₄ were tested for P-388 leukemia only with q3d x 3 administration due to the limited supply of material.

BBM-1675 A, A ₂ , A ₃ and A _{4 were} dissolved in physiological saline containing 10% dimethylsulfoxide. Chromomycin A ₃ (Toyomycin, Takeda) was also used as a reference substance dissolved in physiological saline. Death or survival of treated and untreated mice was recorded daily and the median survival time (MST) calculated for each treated (T) and untreated (C) groups. T / C values greater than or equal to 125% indicate a significant anti-tumor 18193928 effect. The results were reported in XVII-XXIII. tables. BBM-1675 A and A ₂ showed extremely potent antitumor activity against P-388 leukemia with a maximum T / C of 160%. These are 100 to 3000 times more potent than chromomycin A _{3 for} the 5 lowest effective doses. BBM-1675 A ₃ and A ₄ , on the other hand, were less effective on components A and A ₂ than P-388 leukemia (Table XIX).

BBM-1675 A and A ₂ were also effective against L-1210 leukemia (Table XXI),

B16 melanoma (Table XXII) and Lewis lung carcinoma (Table XXIII). Toxicity of BBM-1675 A and A _{2 to} male ddY mice was determined by intraperitoneal or intravenous administration; BBM-1675 A, about ten times more toxic than BBM-1675

A ₂ (Table XXIV). The therapeutic indices of BBM-1675 A and A _{2 were} 4-8 times and 8-20 times better than chromomycin A ₃ when tested on the P-388 leukemia system (Table XXV). A second set of assays was performed by intravenous administration of BBM-1675 components to P-388 and L-1210 leukemia induced by intravenous inoculation of 5 x 10 ⁵ and 10 ⁴ cells per mouse. In these experiments, adriamycin was used as a comparator, which was administered on days 1, 4 and 7 in physiological saline. The results were published in XXVI. and XXVII. tables illustrate. Both A 1 and A ₂ outperform adriamycin in terms of maximum T / C values and minimum effective dose and spectrum width.

XVIII. Spreadsheet

Effect of BBM-1675 Components on P-388 Leukemia (Day 1 Treatment)

Dose, ip (mg / kg / day) Period (days) (T / C) (%) Weight change on day 5 (G) Survivors Day 5 Day 45 BBM-1675 Al 0.03 19.0 © -2.6 5/5 0/5 0.01 19.0 Q52) -1.0 5/5 0/5 0,003 18.0 $ B4 -1.0 5/5 0/5 0,001 20.0 <360) -1.4 5/5 0/5 0.0003 16.0 0.0 5/5 0/5 0.0001 15.0 120 -0.2 5/5 0/5 0.00003 14.0 1 12 -0.2 5/5 0/5 BBM-1675 A _{2 is} 0.3 6.0 48 -3.8 3/5 0/5 0.1 20.0 (Í6Q) -1.8 5/5 0/5 0.03 18.0 Q44) -1.4 5/5 0/5 0.01 17.0 Ö2Ö -0.6 5/5 0/5 0,003 17.0 -0.4 5/5 0/5 Q 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 A ₃ 1 19.0 05 ^ -0.2 4/5 0/5 0.3 17.0 (J36) +0.6 5/5 0/5 0.1 16.0 (j2fp +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 Carrier - 12.5 - +0.1 10/10 0/10

Note: + = Day 1, intraperitoneally.

Boxed numbers indicate significant antitumor activity.

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XIX. Blackboard z. a t

Effect of BBM-1675 Companions on P-388 Leukemia (Day 1, Day 4 and Day 7 Treatment)

Dose, ip (mg / kg / day) ⁴ Period (days) t / c: (2) Weight change is Day 5 (8) Survivors Day 5 Day 45 BBM-1675 Ai 0.03 7.0 56 -2.8 5/5 0/5 0.01 19.0 (15¾ -1.0 5/5 0/5 0,003 19.0 ¢ 5¾ -0.6 5/5 0/5 0,001 16.0 (Γ2 © -0.6 5/5 0/5 0.0003 17.0 ¢ 136) -0.4 5/5 0/5 0.0001 16.0 ($ 12) -0.2 5/5 0/5 0.00003 14.0 112 +0.4 5/5 0/5 BBM-1675 a ₂ 0.3 Tox. - - 2/5 0/5 0.1 11.0 88 -1.0 5/5 0/5 0.03 18.0 (ΓΪΦ -1.2 5/5 0/5 0.01 18.0 -0.4 5/5 0/5 0,003 18.0 (144) -0.4 5/5 0/5 0,001 17.0 (13q -0.4 5/5 0/5 0.0003 16.0 (Tiíy -0.4 5/5 0/5 0.0001 16.0 028) -0.2 5/5 0/5 0.00003 15.0 120 -0.4 5/5 0/5 BBM-1675 Dig 0.01 17.5 +0.2 4/4 0/4 0,001 15.0 120 + O 6 4/4 0/4 0.0001 13.5 108 +0.6 4/4 0/4 BBM-1675 THE" 0.01 16.5 +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 A ₃ 0.3 18.0 +0.6 5/5 1/5 0.1 18.0 Q44J + ö _f 6 5/5 0/5 0.03 17.0 6 36) -0.2 5/5 0/5 0.01 14.0 12 0.0 5/5 0/5 Carrier - 12.5 - +0.4 10/10 0/10

Comment:

+ = Days 1, 4 and 7, i.p.

Boxed numbers indicate significant antitumor activity.

XX. Spreadsheet

Effect of BBM-1675 Components on P-388 Leukemia (qd 1 to 9 treatments)

Dose, ip Duration T / C Weight Loss Survivors Day 45 (mg / kg / day) ⁴ (days) (X) Day 5

Day 5 (g)

BBM-1675 Ai 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 05¾ -1.2 5/5 0/5 0.0003 19.0 052) -0.8 5/5 0/5 0.0001 18.0 (144) 0.0 5/5 0/5 0.00003 16.0 jpg) +0.2 5/5 0/5 0.00001 16.0 <T2 £ S) -0.2 5/5 0/5 BBM-1675 A ₂ 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 -1.0 5/5 0/5 0,003 18.0 044) -0.6 5/5 0/5 0,001 18.0 Q44) -0.8 5/5 0/5 0.0003 17.0 Q3Ö -0.4 5/5 0/5 0.0001 16.0 Q2fp -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 A ₃ 0.3 9.0 72 -2.0 5/5 0/5 0.1 18.0 ^ 4¾) +0.4 5/5 0/5 0.03 18.0 ¢ 44) 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 Carrier - '2.5 - +0.4 10/10 0/10

Comment:

+ qd 1 -> 9, i.p.

Boxed numbers indicate significant antitumor activity.

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40

XXI. Spreadsheet

Effect of BBM-1675 components on L-1210 leukemia

Dose, ip. (Mg / kg / day) + Duration T / C Weight change ag Day 5 (G) Survivors Day 5 Day 45 (days) (X) BBM-1675 Ai 0,003 14.5 (I53) 1.7 6/6 0/6 0,001 12.0 26 ¢) -0.5 6/6 1/6 0.0003 12.0 +0.3 6/6 0/6 0.0001 11.0 116 + 1.0 6/6 0/6 BBM-1675 A ₂ 0.03 10.5 111 -1.5 6/6 0/6 0.01 13.5 U42) -1.2 6/6 0/6 0,003 13.0 (&) -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 Carrier - 9.5 - + 1.4 12/12 0/12

Comment:

+ = qd 1 -> 9, ip.

Boxed numbers indicate significant antitumor activity. XXII. Spreadsheet

Effect of BBM-1675 components on B16 melanoma

Dose, ip. Duration T / C Weight Changes Survivors Day 45 (mg / kg / day) · * · (days) (£) to Day 5

Day 5 (g)

BBM-1675 Ai 0,003 10.0 61 0.7 6/6 0/6 0,001 31.5 0.0 6/6 0/6 0.0003 40.5 (24 ^ +0.3 6/6 0/6 0.0001 27.0 (P54) +0.8 6/6 0/6 0.00003 22.0 + 1.8 6/6 0/6 0.00001 18.0 109 +2.2 6/6 0/6 BBM-1675 a ₂ 0.03 11.0 67 -0.8 6/6 0/6 0.01 26.5 (Í6l) +0.3 6/6 0/6 0,003 29.5 fl79> +0.2 6/6 0/6 0,001 26.0 (Ί58) +0.8 6/6 0/6 0.0003 22.0 Q3§ +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 A ₃ 0.1 25.5 (155) +2.3 6/6 0/6 0.03 23.0 39 ¢) +2.2 6/6 0/6 0.01 21.0 (Γ27) +2.3 6/6 0/6 0,003 18.0 1Ó9 +2.2 6/6 0/6 Carrier - 16.5 - +2.1 12/12 0/12

Comment:

+ = qd 1 -> 9, ip.

Boxed numbers indicate significant antitumor activity.

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42

XXIII. Spreadsheet

Effect of BBM-1675 components on Lewis lung carcinoma

Dose, ip. Duration T / C Weight Change- Survivors Day 45 (mg / kg / day) ⁺ (days) (%) Day 5

Day 5 (g)

BBM-1675 A? 0,003 10.0 91 1.7 5/6 0/6 0,001 31.5 0.7 6/6 1/6 0.0003 21.5 -0.7 6/6 0/6 0.0001 21.0 Q9J) + 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-1675 A ₂ 0.03 10.0 91 1.8 6/6 0/6 0.01 25.5 (G3 ^) -1.7 6/6 0/6 0,003 28.5 @ 59) 0.0 6/6 1/6 0,001 17.0 -0.3 6/6 0/6 0.0003 15.0 Q3 $ + 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 A ₃ 0.1 21.5 + 1.2 6/6 1/6 0.03 17.0 + 1.7 5/5 0/5 0.01 17.0 Q55) + 1.5 6/6 0/6 0,003 11.5 105 + 1.7 6/6 0/6 Carrier - 11.0 - +0.8 12/12 4/12

New match:

+ = qd 1 -> 11, ip.

Boxed numbers indicate significant antitumor activity.

XXIV. Spreadsheet

Toxicity of BBM-1675 components

LD ₅₀ / mg / kg / day / One gift ζ Multiple doses (qd 1 -> 9) ip arc. ip BBM-1675 Αχ 0.019 0.010 0, 00046 BBM-1675 A ₂ 0.18 0.10 She, 0072 Chromomycin A ₃ 0.81 0.41 She, 23 XXV. T á b fever Therapeutic indices ^LD 50 / minimally effective dose P-388 Single qd 1 -> 9 L1210 B16 Lewis BBM-1675 Αχ 63 46 2 15 5 BBM-1675 A ₂ 180 72 2 24 24 Chromomycin A ₃ 8 8 effect- 23 23

maybe

-2 243

XXVI. Spreadsheet

Effect of BBM-1675 Components on Intravenously Implanted P-388 Leukemia

Dose, ip. Duration T / C Weight Change Over 45 days

(Mg / kg / day) (days) (%) is Day 5 (g) people 5 days BBM-1675 Αχ 0.01 9.5 106 1.7 6/6 0/6 0,003 14.0 -0-, third 6/6 0/6 0,001 11.5 +0.3 6/6 0/6 0.0003 9.0 100 +0.3 6/6 0/6 BBM-1675 A ₂ 0.1 7.0 78 -3.7 6/6 0/6 0.03 15.0 © -i, o 6/6 0/6 0.01 12.0 -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 +0.7 6/6 0/6 1 9.0 100 + 1.7 6/6 0/6 Carrier - 9.0 - + 1.7 12/12 0/12

New match:

+ = Days 1, 4 and 7, iv.

Boxed numbers indicate significant antitumor activity.

XXVII. Spreadsheet

Effect of BBM-1675 Components on Intravenously Implanted L-1210 Leukemia

Dose, ip. Duration T / C Weight Changes- Over- 45 days (mg / kg / day) + (days) (%) of live weight

5th on the sun (G) 5 days BBM-1675 Ai 0,008 9.5 119 -2.0 4/6 0/6 0,004 14.0 cg) -0.2 6/6 0/6 0,002 13.0 63 ¢) +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-1675 A ₂ 0,063 11.0 -1.8 6/6 0/6 0.032 14.0 © +0.2 6/6 0/6 0,016 10.5 @) +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 @> +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 Carrier - 8.0 - + 1.4 12/12 0/12

Comment:

+ = Days 1, 4 and 7, iv.

Boxed numbers indicate significant antitumor activity.

-23193928

D The antitumor activity of BBM-1675 A and A ₂ components was also tested in another test against P-388 leukemia, L-1210 leukemia and Biti melaloma in mice. The results of these tests are shown in Tables XXVIII, XXIX below. and Tables XXX. Details of the methods used in these assays are provided by Cancer Chemother. Rep. 3, 1-87 (part 3), 1972.

XXVIII. Spreadsheet

BBM-1675 Ai and _A2 effect P-388 leukemia

Material Treatment I.p. dose Medium Medium Weight change survival change Survivors at 5. days (30) program / Ig / kg / day Sun effect% T / C on day 5 NSC 38270 qd1-9 400 13.0 163 -0.6 6/6 200 11.0 138 -0.9 6/6 BBM-1675 Ai d.1 51.2 20.0 250 "2.1 4/6 DMSO physiotherapy 25.6 18.0 225 1.8 6/6 logical kitchen 12.8 16.5 206 1.1 6/6 saline solution 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.1,5-9 25.6 8.0 100 -1.8 6/6 12.8 13.5 169 -1.5 6/6 6.4 16.5 206 -0.8 6/6 3.2 16.0 200 -0.8 6/6 1.6 15.5 194 +0.3 6/6 0.8 12.5 156 +0.3 6/6 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-1675 A ₂ d.1 256 Tox. Tox. Tox. 0/6 DMSO physiotherapy 128 12.5 156 -3.5 4/6 logical kitchen 64 27.0 338 -1.9 6/6 saline solution 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.1,5-9 128 Tox. Tox. Tox. 0/6 64 Tox. Tox. Tox. 0/6

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48

XXVIII: Table

Material Treatment program (continuation) Median survival effect% T / C Weight change in Figure 5. on the sun Survivors at 5. days (30) Ip dose ug / kg / day Medium survival Sun 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 BBM-1675 A ₂ 0.5 12.0 150 -0.4 6/6 DMSO-Physio-qdl-9 0.25 11.0 138 -0.4 6/6 logical kitchen 64 Tox. Tox. Tox. 1/6 saline solution 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 fiz. Paper of 8.0 - +0.5 10/10

belly

Tumor implantation: 10 ⁵ asc (with cellular ip implantation

Host animal: CDFi male mouse

Toxic: 4/6 mouse live at 5

Evaluation: Median survival time

Effect:% T / C = median survival time / control survival time x 100

Conditions: 7, T / C = 125 is classified as a characteristic anticancer effect NSC 38270 = olivomycin A

XXIX. Spreadsheet

Effect of BBM-1675 A and A ₂ on L-1210 leukemia

Material Handicrafts- I.p. dose Medium Medium Weight change on day 5 Survivors on day 5 (30) this program pg.kg/inj. survival Sun survival effect 7 T / C BBM-1675 A _x d., 1st 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 1 14 -0.4 6/6 9.4 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

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XXXI. Spreadsheet

Effect of purified BBM-1675 Al on P-388 leukemia

Compound Dose (Mg / kg / dose) Dosage Method Medium T / C Average weight change is Day 5 Survivors on the 5th na · pon and beat survival (%) days BBM-1675 Ai 0.1024 · i.p., qd x 1 Tox. Tox. 0/6 .0512 17.5,159 -1.8 4/6 0.0256 16.5,150 -2.6 6/6 .0128 17.5,159 -1.4 6/6 0.0064 15.5,141 -2.2 6/6 .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 -2.0 6/6 0.0256 · i.p., qd4 x 3 Tox. Tox. -1.5 1/6 .0128 10.0 91 -2.5 5/6 0.0064 17.5,155 -1.9 6/6 .0032 17.0 155 -0.8 6/6 0.0016 1.7.0 155 -2.0 6/6 0.0008 15.0 136 1.7 6/6 0.0004 15.0 136 -0.4 6/6 0.0002 13.0118 -0.8 6/6 0.0001 13.0118 1.3 6/6 0.00005 13.5 123 -1.0 6/6 .0128 i-Fri, qd x 5 Tox. Tox. 0/6 0.0064 Tox. Tox. -3.6 3/6 .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 (carrier) 1 x 10 ⁶ i-Fri, q4d x 3 11.0 100 -0.7 9/9 host Animal : CDFi female mouse Level and location of inoculation: 1 x 1C I ⁶ cells, ip XXX. T á b 1, Azat the : BBM-1675 A, and Aa, effect on B16 melanoma Material The dose i.p. Medium Medium Weight Changes Survivors pg / m or survival survival on day 5, day 5 (30) mg / kg / inj. Sun effect% T / C BBM-1675 The _{L is} 3.2M Tox. Tox. -1.8 2/10 1.6 16.0 64 -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-1675 A ₂ 16M 13.0 52 -2.1 10/10 8 29.5 118 -2.0 10/10 4 43.5 174 -1.1 10/10

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52

XXX. Table (continued)

Material The dose i.p. g / m or mg / kg / inj. Medium survival Sun Median survival effect% T / C Weight change on day 5 Survivors on day 5. (30) 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 fiziológi- 25.0 - 0.1 10/10

and saline a = only one tumor; median survival time d.m.o. = 55.0 (220%) b = no two tumors; median survival time d.m.o. = 46.0 (184%) Tumor implantation; 0.5 ml i.p. 10% suspension

Host Animal: BDFx male mouse

Treatment: qd 1 - 9

Toxic: 7/10 mice live on day 10

Evaluation: Median survival time

Effect:% T / C = median survival time treated / median survival time control / 100 x

Conditions:% T / O125 is considered a significant anticancer effect.

Following purification of BBM-1675 A, further purified according to Example 6, samples of the purified compound were analyzed for L-1210 leukemia, P-388 leukemia.

XXXI. The effect of T? And B16 on melanoma was investigated in mice. The results of the assays are shown below.

fever

Effect of purified BBM-1675 Al on P-388 leukemia

Compound

Dosage Dosage regimen Medium (mg / kg / dose) and schedule survival days

T / C Average Survivors (%) Weight Loss - Day 5 to Day 5

.1024 · i.p., qd x 1 Tox. Tox. 0/6 .0512 17.5 159 -1.8 4/6 0.0256 16.5 150 -2.6 6/6 .0128 17.5 159 -1.4 6/6 0.0064 15.5 141 -2.2 6/6 .0032 15.5 141 -2.5 6/6 0.0016 16.5 150 -i, o 6/6 0.0008 15.0 136 -1.2 6/6 0.0004 15.0 136 2.0 6/6 0.0256 · i.p., qd4 x 3 Tox. Tox. -1.5 1/6 .0128 10.0 91 -2.5 5/6 0.0064 17.5 155 -1.9 6/6 .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 .0128 i.P ·, qd x 5 Tox. Tox. 0/6 0.0064 Tox. Tox. -3.6 3/6

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54

XXXI. Table (continued)

Compound Dose Dosage Medium T / C Mean Survivors (mg / kg / dose) and rate of survival (%) Weight loss - Day 5 Day

Day 5

.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 1 χ 10 ⁶ i.p., q4ú x 3 11.0 100 -0.7 9/9

(carrier)

Host Animal: CDFi female mouse

Level and location of inoculation: χ 10 ⁶ cells, ip XXXII. Spreadsheet Cleaned BBM-1675 Effect of Ai L- ' 1210 leukemia (First Day Treatment) Compound Dose Dosage Medium T / C Average survivors (Mg / kg / dose) and beat survival (%) súlyválto- 5. Sun See Figure 5. on the sun BBM-1675 Αχ .1024 i.p., qd x 1 tox. Tox. 1/6 .0512 Tox. Tox. -2.0 0/6 0.0256 8.0 114 -2.9 4/6 .0128 11.0 157 -2.0 6/6 0.0064 11.0 157 -1.9 6/6 .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 .0128 10.5 150 -1.7 6/6 0.0064 11.0 157 -1.8 6/6 .0032 1 1.0 157 -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 .0128 i.p., qd x 5 tox. Tox. -2.8 2/6 0.0064 7.0 100 -1.8 5/6 .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 1 χ 10 ⁶ i.p., qd x 5 7.0 100 0.1 10/10

(carrier)

Host Animal: CDFi female mouse

Inoculation level and location: 1 x 10 ⁶ cells, ip

-28193928

XXXIII. Spreadsheet

Effect of purified BBM-1675 Ai on methanol B16 (Day 1 treatment)

Compound Dose (Mg / kg / dose) Dosage regimen and rate Medium T / C Mean Survival (Z) Weightloss- Survivors on day 5 Sun debts on day 5 BBM-1675 Ai 0.0064 · i.p., q4d x 3 16.5 110 -3.8 8/10 .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 ip, 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 0.5m · i.p., qd x 9 15.0 100 -0.3 10/10 (carrier) ⁺ * BBM- 0.0064 arc., q4d x. · 3 15.0 86 -4.7 10/10 1675 Αχ .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 .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 (carrier) 0.2 ML ARC., tj4d x 3 17.5 100 -0.4 10/10 Animal hosts! BDPi female mouse

+ Inoculation level and correct 0.5 HL ΌΖ-oa suspension, i.p. ++ Inoculation level and correct fragment, s.c.

The above screening data show that the purified BBM-1675 A component 40 has substantially the same antitumor properties as the less purified sample previously screened. The component is extremely high in efficacy as it is 25 nanograms / kg in mice administered ⁵ times daily 5 times daily against P-388 leukemia. BBM-1675 A, P-388, and L-1210, when tested for leukemia, are effective for either gg on the first day of one injection, every three days on three injections, or five times daily. The compound is effective against B16 melanoma in animals which have been injected intravenously with tumors subcutaneously and intraperitoneally with tumors implanted ip. This property, namely the ability to reach a distant tumor pharmacologically efficiently, is completely unusual for anticancer antibiotics. θθ

As described above, the BBM-1675 components have potent antimicrobial activity and are therefore useful in the treatment of diseases caused by such microorganisms in mammals and other animals. Further, application of the components of ⁶⁵ sek can be used in customary antibacterial agent with other conventional purposes such as disinfecting medical and dental instruments.

Prophage induction in lysogenic bacteria and activity against murine tumor systems indicate that BBM-1675 components are also useful in inhibiting the growth of tumors in mammals which are pharmaceutically useful.

The present invention also encompasses the manufacture of a pharmaceutical composition comprising BBM-1675 A, A ₂ , A ₃ , A ₄ , B, or B _{2 in} an inert, pharmaceutically acceptable carrier or diluent having an antimicrobial or antitumor activity. . These pharmaceutical compositions may be formulated in any dosage form suitable for parenteral administration.

The compositions of the invention for parenteral administration are sterile aqueous or non-aqueous solutions, suspensions, or emulsions. They may also be prepared in sterile solid form which is dissolved in sterile distilled water, physiological saline or any other sterile injectable medium immediately prior to use.

-29193928

It will be understood that the preferred dosage of the BBM1675 antibiotics used will vary depending upon the particular components, the composition of the composition, the mode of administration, the site, the subject and the disease. Those skilled in the art will consider many factors that alter the effect of the drug, such as age, weight, sex, meal, time of treatment, route of administration, degree of clearance, condition treated, drug co-factors, sensitivity and disease. severity. Dosage may be continuous or intermittent with the maximum tolerated dose. The optimum dosage will be determined by one of ordinary skill in the art in the given circumstances, taking into account the foregoing guidelines, and conventional dosage adjustments.

The following examples illustrate the invention without limiting it.

Example 1

Fermentation of BBM-1675

The H-964-92 Actinomadura strain was grown and maintained on sloped agar containing 1% malt extract, 0.4% glucose, 0.4% yeast extract, 0.05% calcium carbonate and 1.6% agar. A vegetative medium containing 3% soluble starch, 3% dry yeast, 0.3% dipotassium hydrogen phosphate, 0.1% potassium dihydrogen phosphate, 0.05% magnesium sulfate was selected for inoculation with good growth slant agar. H ₂ O), containing 0.2% sodium chloride and 0.1% calcium carbonate and adjusted to pH 7 before sterilization. The vegetative culture was grown at 32 ° C for 72 hours. Rotate on a rotary shaker (250 rpm) and place 5 ml of the culture in a 500 ml Erlenmeyer flask containing 100 ml of 3% cane molasses, 1% corn starch, 1% fishmeal, 0.1% calcium carbonate and 0.005% It was a sterile medium containing CuSO ₄ .5H ₂ O and adjusted to pH 7 before sterilization. The fermentation was carried out at 28 ° C for 6 days on a rotary shaker. The antibiotic activity of the fermentation broth was determined by paper disk agar diffusion using Staphylococcus aureus 209P as a test organism. Maximum antibiotic activity of about 1 mcg / ml was reached after 5 days of fermentation.

Fermentation of BBM-1675 was also performed in a stirred cucumber bottle fermenter. 500 ml of the inoculum grown as described above was transferred to a 20 liter cucumber fermenter containing 10 liters of sterile medium of the same composition as used above for the shake culture. Fermentation was carried out at 32 ° C with 12 liters / min aeration and 250 rpm. Under these conditions, the maximum antibiotic production was 0.9 mcg / ml after 68-76 hours of fermentation.

Fermentation experiments were also carried out in fermentors. Spore culture for 4 days at 30 ° C from 3% soluble starch, 3% dry yeast, 0.3% K ₂ HPO ₄ , 0.1% KH ₂ PO ₄ , 0.05% MgSO ₄ . The mixture was shaken on a vegetative medium consisting of 7 H ₂ O, 0.2% NaCl and 0.1% CaCO ₃ . The inoculum culture was inoculated into a 200 L inoculum fermenter containing 130 L of medium of the same composition and the fermentor was stirred at 240 rpm for 30 hours at 30 ° C. This culture was used to inoculate a 3000 1 of culture medium with it, containing 1% of corn starch, 3% cane molasses, 1% fish meal, 0.005% CuSO ₄ .5 H ₂ O and CaCO ₃ 0.1% of the total. The fermentation vessel was stirred at 28 ° C at 164 rpm with aeration of 2000 rpm. As the fermentation progressed, the pH of the medium gradually increased, reaching 7.7-7.8 after 170-180 hours, producing a peak of 1.7 mcg / ml of antibiotic activity.

Example 2

Isolation and purification of BBM-1675 components

The resulting fermentation broth (3000 liters, pH 7.8) was transferred to a Sharpless centrifuge έβ the mycelium separated from the supernatant. The mycelial cake was suspended in 1,600 l of methanol and the mixture was stirred for one hour. Insoluble materials were filtered off and the methanolic extract was concentrated in vacuo to 43 liters. The active substance in the culture supernatant was recovered by extraction twice with 1000 liters of n-butanol. The n-butanol extract and the concentrated methanol extract were combined and subjected to azeotropic distillation with occasional addition of water. Finally, 20 liters of an aqueous residue were obtained from which most of the antibiotic activity was recovered as an oily solid. The solid was digested with 30 L of methanol and the insoluble residue was filtered off. The methanolic extract was then concentrated in vacuo to 10 liters, to which was added 40 liters of ethyl acetate and 30 liters of water. After stirring for 30 minutes, the organic phase was separated, dried over sodium sulfate and concentrated in vacuo to 4 liters. Addition of this concentrate to 20 liters of n-hexane gave a crude BBM-1675 complex (90.14 g, potency: 55 mcg / mg) as a light yellow solid. TLC showed the product to be a mixture of two major components, BBM-1675 A, and A ₂ , and some minor components. These were separated and purified by repeated chromatography in a cold room to prevent decomposition.

20 g of BBM-1675 complex was dissolved in 20 ml of methanol and Sephadex LH-20 (diameter

5.5 x 85 cm). The column was eluted with methanol and the elution verified by biological assay using Staphylococcus aureus 209P. The eluate containing the active substance 30193928 was combined, concentrated in vacuo and lyophilized to give the pure solid BBM-1675 complex (4.19 g). The solid was then chromatographed on a silica gel column (5.0 x 50 cm) using chloroform and increasing volumes (1-5% v / v) methanol as eluent.

The eluates were collected by antimicrobial activity (against S. aureus) and by thin layer chromatography (SiO ₂ ; CHCl ₃ -CH ₃ OH = 5: 1 v / v) and concentrated in vacuo. Almost homogeneous BBM-1675 A 1 was eluted first with 2% methanol in chloroform (351 mg post-evaporation yield), and then a mixture of BBM-1675 A ₂ , A ₃ and A ₄ (507 mg) was added, followed by 3% methanol chloroform to a mixture of BBM-1675 B components (210 mg). Solid BBM-1675A1 was loaded onto a Sephadex LH-20 column (2.0 x 80 cm) eluted with methanol. The active fractions were evaporated to dryness in vacuo and the resulting solid was crystallized from methanol to give pure BBM-1675A (124 mg) as a plate (starting material from Example 3A). The BBM-1675 A ₂ , A ₃ and A ₄ complexes were separated by chromatography on a Bondapak C ₁₈ (Waters, 3.0 x 50 cm) column. Elution was carried out with aqueous acetonitrile and the bioactive eluates were examined by thin layer chromatography (Merck _C18 reverse phase silica gel, CH ₃ CN-H ₂ O = 75: 25 v / v). The smaller components A ₄ (33 mg) and A ₃ (18 mg) were eluted sequentially with 20% acetonitrile, followed by elution with 50% acetonitrile, another major component, A ₂ (this material is 3B). (starting material of example.

The solid containing BBM-1675 Β, and B ₂ was chromatographed on a silica gel column (diameter: 3.0 x 40 cm) using chloroform and methanol as eluent. The active fractions eluted with 4% methanol in chloroform were combined and evaporated to give pure BBM-1675B1 (7 mg). At a concentration of 5% methanol, another active fraction could be extracted which yielded BBM-1675 B ₂ (8 mg).

Example 3

Further purification of BBM-1675 A and A ₂

A. Purification of BBM-1675 A

A Glenco column (2.67 cm internal diameter, 75 cm high) was filled with Baker solid phase (C18) silica slurry (40 micron particle size) suspended in methanol. The column was connected to a medium pressure liquid chromatography system and equilibrated with 1.5 L of eluent (41.6% acetonitrile, 21.6% methanol, 36.8% 0.1 molar ammonium acetate). The partially purified BBM-1675A1 (100.5 mg) obtained in Example 2 was dissolved in 2 ml of acetonitrile and aspirated into the sample loop. The sample is pressed into the column. The column was eluted with the above eluent, collecting 87 ml fractions. The eluent was monitored between 254 nm and 340 nm. Fractions 55-71 were combined and extracted twice with 1.500 mL chloroform. The chloroform was evaporated to give 89.8 mg of C residue.

A Glenco column of 1.5 cm internal diameter and 20 cm long was filled with a slurry of 12 g Woelm silica gel (particle size 60200 microns). Chloroform solution of the C residue was applied to the column. The column was eluted with a linear gradient of 500 mL of chloroform to 10% methanol in chloroform to collect 20-25 mL fractions. After thin layer chromatography on silica gel, fractions 6-9 were combined and evaporated to dryness to give 73 mg of D-residue.

A 1.5 cm internal diameter and 20 cm long Glenco column was filled with a slurry of 12 g Woelm silica gel (63200 micron particle size) in Skellysolve B. The D residue was dissolved in about 2 mL of CHCl ₃ and applied to the column. Chloroform was displaced with 25 ml of Skellysolve B. The column was then eluted with a linear gradient of 500 mL Skellysolve B to 60% acetone Skellysolve B to collect 28-25 mL fractions. Fractions 19-23 were combined and evaporated to dryness to give 65.6 mg of pure BBM-1675A1.

This residue was assayed by three thin layer chromatography systems (5% methanol in chloroform; 5% methanol in ether and 50% acetone in Skellysolve B on silica gel) and high performance liquid chromatography (C-18 silica gel-41.5% acetonitrile: 21.5% methanol: 37.0% (0.1 molar ammonium acetate) were uniform.

Gel permeation chromatography was purified with BBM-1675 A

A 2.5 cm internal diameter Pharmacia column of 2.5 cm internal diameter was filled with Sephadex LH-20 slurry suspended in methanol and adjusted to a 33.4 cm chromatography bed. Purified BBM-1675 [mu] L (about 120 mg) is dissolved in 2 mL of methanol and placed in a 2.5 mL sample container. The sample was loaded onto the column and eluted at 1.75 mL / min with 10 mL of methanol (Pharmacia Frac-100 fraction collector). The eluent was monitored at 254 nm with an Isco UA-5 detector. BBM-1675 A was observed to elute at a Ve / Vt of 0.79-0.91 (Ve = elution volume: Vt = bed volume).

B. Purification of BBM-1675 A ₂

A Glenco column (2.65 cm internal diameter, 75 cm long) was filled with Baker solid phase octadecyl (C18) silica slurry (40 micron particle size) suspended in methanol. The column was connected to a medium pressure high pressure liquid chromatography 31

-31193928 and 1.5 L of eluent (50% acetonitrile - 20% methanol - 30% 0.1 molar ammonium acetate). Partially purified BBM-1675 A ₂ (76.9 mg) obtained in Example ₂ was dissolved in 2 ml of acetonitrile and aspirated into the sample loop. The sample is printed in the column. The column was eluted with the above eluent and the 87 ml fractions were collected. The eluent was monitored at 254 and 340 nm. Fractions 31-38 were combined and extracted with chloroform (2 x 500 mL). Evaporation of the chloroform gave 65.8 mg of homogeneous BBM-1675 A ₂ .

The BBM-1675 A _{2 has been shown to be consistent in} two thin layer chromatography systems, a 2 d thin layer chromatography system and a high performance liquid chromatography system.

Example 4

BBM-1675 A is the preferred extraction process

6.8 liters of the fermentation broth obtained by the general procedure described in Example 1 is placed in a polyethylene bucket (diameter 12 cm, bottom 10 cm, height 37 cm) with a stopcock. An equal volume of chloroform was added. The mixture was stirred at ambient temperature for 2 hours with an air-driven CRC mixer. About 4 liters (1.3 kg) of Dicalite (filtration aid) were added and allowed to mix. The mixture was filtered through a Decalite filter bed 12 on a Büchner funnel. The filtrate was collected in a 19L liquid bottle fitted with a vacuum aspirator, and the filter cake was washed with 2 L chloroform. The filtrate was placed in a 20 liter separatory funnel and allowed to separate. The lower phase (chloroform) was removed.

A Glenco tube of 2.5 cm internal diameter and 40 cm high was filled with a slurry of 91 g of Woelm silica gel (63-200 micron particle size). Using the FMI RPY-2CSD pump, the former chloroform phase was passed through the column. The column was rinsed with 600 mL of fresh chloroform. Eluting chloroform was discarded. The column was then eluted with 600 ml of 10% methanol in chloroform. This was evaporated to dryness to give 547 mg of A residue.

The residue A is dissolved in 50 ml of chloroform. This solution was added to 20 g of Decalite in a 1 liter round-bottomed flask. About 200 ml of Skellysolve B is added to form a slurry. The solvents were evaporated in a Rotavapor. The residue is slurried with 300 ml of Skellysolve B. The slurry was filled into an Ace vial chromatography tube (part B5872-14) (41 mm internal diameter x 45.7 cm) by the following procedure. Insert a plug of glass wool in the neck of the shut-off valve between the valve and the column tube. Standard Ottawa sand is placed in a 1 cm layer over the glass wool. The shut-off valve, the glass wool pond and the sand bed are de-aerated under pressure (0.4 atm) by pressing Skellysolve B. Subsequently, the slurry is placed in the column, ensuring that it provides a stable bed at overflow. We never let the column dry. Once a stable bed was formed in the column, a layer of Ottawa sand was placed on top of it. The bed was then eluted with an additional 600-700 mL of Skellysolve B. The next elution was with 500 mL of toluene. The toluene eluant was evaporated to dryness to give 93 mg of residue B. This partially purified BBM-1675 A can be further purified by the procedure described in Example 3.

Example 5

Fermentation of BBM-1675 complex with H964-92-A1327Y variant

Actinomadura verrucosospora H964-92 strain was inoculated with NTG-treated A1327Y variant medium containing 2% soluble starch, 1% glucose, 0.5% yeast extract, 0.5% NB-type A and 0.1% CaCO ₃ , pH before sterilization

We set it to 7. The vegetative culture was fermented on a rotary shaker (250 rpm) for four days at 32 ° C, and 5 ml of the culture was inoculated into a 500 ml bottle of Rlenmayer containing 100 ml of a 3% cane molasses, 1% corn starch, 1% fish meal, 0.005% CuSO ₄ . 5 H ₂ O, 0.05% MgSO ₄ . Of 7 H ₂ O and 0.1% of CaCO _3, produced 7 before sterilization pH medium.

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

Example 6

Isolation and purification of BBM-1675 components

The fermentation broth prepared according to Example 5 (3.000 L, pH 7.6) was selected by a Sharpless centrifuge for mycelial cake and supernatant. The mycelial cake was stirred with 2000 liters of methanol for one hour and insoluble materials were filtered off. The active ingredient is extracted from the supernatant with 1.800 l of n-butanol. The methanol and n-butanol extracts are combined and sometimes azeotropically distilled by addition of water. An aqueous solution (20 L) is obtained, from which most of the active ingredient precipitates as an oily solid. The mixture was extracted three times with 20 L of ethyl acetate to recover the active ingredient. The extracts were combined, filtered to remove insoluble material and concentrated in vacuo to 4 liters. Pouring the concentrate with stirring into 30 L of n-hexane gave a light yellow crude complex (81.7 g; effect: 59 mcg / mg). According to TLC and NNFK studies, the mixture contains two major components, BBM-1675 A, and A ₂ and some minor components. They were separated and purified by a series of chromatography in cold room to prevent decomposition, 32193928.

The crude BBM-1675 complex (20 g) was dissolved in methanol (20 mL) and applied to a column of Sephadex LH-20 (5.5 mm x 85 cm). The column was eluted with methanol and eluted by biological assay using Staphylococcus aureus 209P. The drug-containing eluates were collected, evaporated in vacuo, and lyophilized to give semi-pure solid BBM-1675 complex (4.86 g, efficacy: 203 mcg / mg). The solid was then passed through a silica gel column (3.0 x 70). cm) using chloroform and increasing amounts (15%) of methanol as developing solvent. Showed S. aureus for antibacterial activity on the basis of the combined eluates was performed by TLC (SiO _2, CHC1 ₃ -MeOH 5: 1, v / v) and concentrated in vacuo. BBM-1675 Aj was eluted first (425 shirts after evaporation; potency: 960 mcg / mg), then a mixture of BBM-1675 A ₂ , A ₃ and A ₄ (732 mg; potency: 340 mcg / mg), which was BBM-1675 Complex B was followed (200 mg; potency: 190 mcg / mg) with chloroform containing 3% methanol. The above BBM-1675A1 was rechromatographed on silica gel (column: 2.2 mm x 44 cm) with 2% methanol in benzene. Bioactive eluates were assayed with NNFK (Lichtosorb RP-18: CH ₃ CN-MeOH-01 mole CH ₃ COONH ₄ = 5: 2: 3, v / v) and fractions containing the homogeneous BBM-1675 Α l were vacuum-dried. evaporated. The residual solid was crystallized from methanol (10 mL) to give colorless prisms BBM-1675 197 (197 mg, efficacy 1000 mg / mg).

The BBM-1675 A ₂ , A ₃ , and A ₄ complex (537 mg) was separated by column chromatography on a Bondapak C ₁₈ column (Waters, 2.0 x 42 cm). Elution was performed with aqueous acetonitrile and the eluates were assayed with TLC (Merck, phase reversed, CH ₃ CN-H ₂ O = 75:25, v / v). The smaller components were eluted sequentially. BBM 1675 A ₄ (45 mg, potency: 410 mcg / mg) and A ₃ (19 mg, potency: 300 mcg / mg) were eluted with 20% acetonitrile followed by 50% acetonitrile. eluted with nitrile, then with 50% acetonitrile, a larger component was BBM-1675 A ₂₄ (203 mg). Fractions BBM-1675 A _{2 were} crystallized from chloroform-n-hexane to give colorless bars (70 mg, efficacy: 290 mcg / mg). The solid containing BBM-1675 B was chromatographed on a silica gel column (3.0 x 40 cm) with chloroform and methanol as developing agents; the active fractions eluted with 4% methanol in chloroform were collected and evaporated to give BBM1675 Β (7 mg, efficacy · 180 mcg / mg). Another active fraction was eluted with 5% methanol to give BBM-1675 B ₂ (8 mg, efficacy: 140 mcg / mg) after evaporation.

Claims (4)

A process for preparing BBM-1675 antibacterial and antitumor antibiotic complex and its components A, A ₂ , A ₃ , A ₄ , B and B ₂ - melting point of component A, 156-158 ° C, refractive index [α] D ⁷ (c 0.5, _CHCl3); -191 °, typical UV spectra maxima (nm _x Kn ^ OH in CH ₃ 253 (325) 282 (195) 320 (143); 0.01 N in HCl-CH ₃ OH: 253 (323), 282 (192), 320 (144), 0.01 N in NaOH-CH ₃ OH: 252 (325), 283 (172) 318 (136), estimated molecular weight (HPLC gel, Finepak GEL-101): 1300 - melting point of component A ₂ : 147-149 ° C, refractive index [α] ²⁷ (c 0.5, CHCl ₃ ) -179.4 °, Characteristic bands of UV spectrum (k / ntj / U m / l) in CH ₃ OH: 253 (281), 282 (172), 320 (128), 0.01 in NHCl-CH2OH: 253 (276), 282 (167), 320 (128), 0.01 n in NaOH-CH ₃ OH: 252 (289), 283 (171), 318 (122), estimated molecular weight (HPLC gel, Finepak GEL-101): 1100 - melting point of component A ₃ : 125-127 ° C, refractive index [α] ²⁷ (c 0.5, CHCl ₃ ) -161 °, Characteristics of UV maxima (^ ma.tUm / 1cm): OH in CH _3: 253 (286), 282 (158), 320 (122), 0.01n in HCl-CH ₃ OH: 253 (287), 282 (160), 320 (126), 0.01 n in NaOH-CH ₃ OH: 252 (280), 283 (162), 318 (120), estimated molecular weight (HPLC gel, Finepak GEL-101): 1100 - melting point of Compound A ₄ : 123-126 ° C, refractive index [α] ²⁷ (c 0.5, CHCl ₃ ) -176 °, Characteristic bands of the UV spectrum (λ _{max x} nm /%) in CH ₃ OH: 253 (257), 282 (153), 320 (117), 0.01 N in HCl-CH ₃ OH: 253 (258), 282 (155), 320 (118), 0.01 n in NaOH-CH ₃ OH: 252 (266), 283 (160), 318 (118), estimated molecular weight (HPLC gel, Finepak GEL-101): 1400 - melting point 159-161 of component B ° C, refractive index [α] ²⁷ (c 0.5, CHCl ₃ ) -171 °, Characteristics of UV maxima (X _m, nmax / Í%) in _CH3 OH: 253 (225), 282 (140), 320 (105), 0.01 N in HCl-CH ₃ OH: 253 (225), 282 (140), 320 (105), 0.01N NaOH was CHjOH: 252 (236), 282 (141), 318 (105) - 3 B ₂ component mp I56-159 ° C, the refractive index is [a] ²⁷ (c 0.5, CHC1 ₃ ) -122 °, Characteristics of UV maxima (Xmaxnm / I '& n) in _CH3 OH: 248 (212), 279 (141), 318 (103), 0.01 N in HCl-CH ₃ OH: 248 (210), 279 (140), 318 (103), 0.01 N in NaOH-CH ₃ OH: 248 (233), 278 (150), 318 (110) -, -33193928, characterized in that Actinomadura verrucosospora H964-92 (ATCC 39334) or Actinomadura verrucosospora A1327Y (ATCC 39638) or mutants thereof were assayed in an aqueous medium containing assimilable carbon and nitrogen sources, aerated, optionally selected and purified for its components. (Priority: May 15, 1984) The method of claim 1, wherein the strain Actinomadura verrucosospora H964-92 (ATCC 39334) or a mutant thereof is used. (Priority: May 16, 1983) 3. A process for the preparation of an antibacterial and antitumor pharmaceutical composition, wherein any of the BBM-1675 components produced by the method of claim 1 is formulated for parenteral administration. (Priority: May 15, 1984) A process for the preparation of an antibacterial and antitumor pharmaceutical composition, wherein any of the BBM-1675 components produced by the method of claim 2 is formulated for parenteral administration. (Priority: May 16, 1983) 17 sheets drawing lnt.Cl ₄ C 12 P 1/06; The 61 K 35/74 Arabic ₍ . Partially flour-based BBM-1675 A <(in KBr pastilles) for in-red absorption spectrum Wavelength in microns