LU85362A1 - NEW ANTITUMOR ANTIBIOTIC COMPLEX SAID BBM-1675 - Google Patents

Description

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Technological background i 1. Field of the invention

The present invention relates to novel anti-tumor antibiotics, as well as their production and isolation.

CD.MJ - 1 - SY-1742A

2. Known state of the art

The structure of the anti-tumor antibiotics forming the object of the invention has not yet been identified. Due to their particular physical, chemical and biological properties, the Applicant is nevertheless led to believe that the antibiotics BBM-1675 are new.

European patent publication No. 95154A1 * describes the fermentation of Actinomadura pulveraceus sp. nov. No. 6049 (ATCC 39100) for the production of anti-tumor antibiotics called WS 6049-A and WS 6049-B.

The structure of antibiotics WS 6049 has not yet been elucidated, but the characteristic properties stated for the antibiotics show that WS 6049A and WS 6049B may have a structural relationship with the antibiotics BBM-1675 which are the subject of the present invention. However, the spectroscopic results show that neither the WS 6049-A nor the WS 6049-B is identical to any of the BBM-1675 antibiotics of the invention. In addition, the producing organism described in the document ηβ 95154A1 can be clearly differentiated from Actinomadura verrucosospora used according to the invention by the color of its aerial mycelium on ISP media No. 2, 3 and 4, by its positive reaction of peptonization of milk and its ability to use D-fructose, D-mannitol, trehalose and cellulose. Summary Ä The present invention relates to a new j. anti-tumor antibiotic complex, hereinafter called BBM-1675, which is obtained by culture of a strain producing * BBM-1675 of Actinomadura verrucosospora preferably the strain Actinomadura verrucosospora H964-92 (ATGC 39334) or the strain Actinomadura verrucosospora A1327Y (ATCC 39638), or one of their mutants, in an aqueous nutritive medium containing assimilable sources of carbon and nitrogen 9 under nitrogen under aerobic conditions in submersion until production of a significant quantity of this com-xi »BBM-1675 complex by this organism in this culture medium, and possibly by isolation of the complex outside * of the culture medium. The invention also relates to the two major bioactive components of the complex * BBM-1675 called BBB-1675A ^ and A2 and the four minor bioactive components ^ of this complex called BBM-1675A3, A ^, B ^ and B ^. These components can be separated and purified according to conventional techniques of chromatography. The BBM-1675 complex and its bioactive components is antimicrobial and antitumor.

In the drawings:

Fig. 1 is the infrared absorption spectrum * of BBM-1675 A. partially purified (KBr pellet).

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Fig. 2 is the infrared absorption spectrum i 'of the partially purified BBM-1675 A £ (KBr pellet).

Fig. 3 is the infrared absorption spectrum of the BBM-1675 A3 (KBr pellet).

Fig. 4 is the infrared absorption spectrum of BBM-1675 A ^ (KBr pellet).

Fig. 5 is the proton magnetic resonance spectrum of BBM-1675 A ^ partially purified in CDC13 (60 MHz).

Fig. 6 is the magnetic resonance spectrum of the protons of BBM-1675 A ^ partially purified in DCDCl, (60 MHz).

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

Fig. 8 is the magnetic resonance spectrum of the protons of BBM-1675 A4 in CDC13 (60 MHz).

Fig. 9 is the infrared absorption spectrum of the purified BBM-1675 A ^ (KBr pellet).

Fig. 10 is the proton magnetic resonance spectrum of BBM-1675 A ^ purified in CDCl ^ (360 MHz).

Fig. 11 is the 13 magnetic resonance spectrum of C of BBM-1675 A ^ purified in CDCl ^ ^ (90.3 MHz).

Fig. 12 is the infrared absorption spectrum of the purified BBM-1675 A ^ (KBr pellet).

Fig. 13 is the magnetic resonance spectrum of the protons of the BBM-1675 A2 purified in CDCl ^ (360 MHz).

Fig. 14 is the magnetic resonance spectrum of BBM-1635 A ^ purified in CDC13 (90.3 MHz).

Detailed Description The invention relates to a new anti-tumor antibiotic complex called BBM-1675 and its preparation by fermentation of certain strains of Actino- ~. madura verrucosospora and more particularly of the strain Actinomadura verrucosospora H964-92 and of a mutant of this one called Actinomadura verrucosospora A1327Y. The above-mentioned initial strain was isolated from a soil sample collected at Pto Esperanza, Misiones, Argentina. A biologically pure culture of the organism has been deposited in the American Type Culture Collection, Washington, D.C. under the number ATCC 39334 from its permanent collection of microorganisms. Subsequently, the mutant strain A1327Y was obtained by conventional treatment of the strain H964-92 using nitrosoguanidine (NTG) and was deposited in the American Type Culture Collection under No. ATCC 39638.

? f As with many antibiotic-producing cultures, fermentation of Actinomadura verrucosospora strain * H964-92 or A1327Y results in the formation of a mixture or complex of substances. Two major bioactive components, namely BBM-1675 A ^ and A ^, and four minor bioactive components, namely BBM-1675 A ^, A ^, B ^ and B2, were separated from the complex BBM-1675 produced in during fermentation.

The BBM-1675 and its components BBM-1675 Αη, i *. , h ^,, A ^, B ^ and B2 show antimicrobial activity against a wide variety of microorganisms, especially Gram-positive bacteria.

, The BBM-1675 complex and its separate bioactive components also exhibit the phage-inducing properties of certain lysogenic bacteria. Two of the components, namely BBM-1675 A1 and A2, subjected in vivo to screening tests against different mouse tumor systems, have shown inhibitory activity against leukemia L-1210, leukemia P-388, melanoma B16 and Lewis lung carcinoma. BBM-1675 A ^ and A ^ also demonstrated activity against mouse P-388 leukemia.

The complex and its bioactive components can therefore be used as antimicrobial agents or antitumor agents to inhibit tumors in mammals.

The microorganism

The atinomycete strain No. H964-92 was isolated from a soil sample and prepared according to the usual techniques in the form of a biologically pure culture for the purpose of characterization.

The strain H964-92 forms on the aerial mycelium short chains of spores which take straight, flexuous or angular forms. The spores are 2 ^ spherical or oval and have a warty surface.

“" The aerial mycelium is scarce in most environments. The aerial mycelium is white and subsequently takes on a pinkish tinge or turns more bluish in certain agar media. The substrate mycelium is colorless or pale pink. growth temperature ranges from 15 ° C to 43 ° C.

CD.MJ - 5 -

The amino acid composition of the cell wall and the sugar composition of the complete cellular hydrolySat ** show that the strain H964-92 belongs to type IIIB of cell wall. Menaquinone has been identified as MK-9 (H,.) * MK-9 (HQ).

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On the basis of the main characteristics of morphology, culture and physiology, together with the characteristics of chemical composition of the cell wall, the strain H964-92 can be considered to belong to the genus Actinomadura.

Although. the original H964-92 strain gives rise to only moderate growth and to a rare aerial mycelium, a variant giving rise to good growth and better formation of the aerial mycelium was obtained by treatment of the H964 strain -92 using nitrosoguanidine (NTG). This variant, called strain A1327Y, facilitated the pursuit of taxonomic examination and was subsequently identified under the name Actinomadura verrucosospora.

Processes

The media and methods applied to determine the culture characteristics and the use of carbohydrates are those recommended by the International Streptomyces Project (Intl. J. Syst. Bacteriol. 16: 313-340, 1966). Additional media described by S.A. Waksman ^ (The Actinomycetes, volume 2) and G. M. Lvedemann ^ (Intl. J. Syst. Bacteriol. 21: 240-247, 1971) have also been used. The amino acid composition. of the cell wall and the sugar composition of the complete cell hydrolyzate were determined according to the techniques described by Becker et al. in Appl. Microbiol. 13: 236-243, 1965 and by Lechevalier and Lechevalier in The Actinomycetes.

Ed. H. Prauser, Jena, Gustav Fischer Verlag, pages 393-405, 1970, respectively. Menaquinone has been identified by mass spectrum analysis as indicated by Collins et al. in J. Gen. Microbiol. 100: 221-230, 1977 and the composition of menaquinone 'has been represented on the basis of the system described by

Yamada et al. in J. Gen, Appl. Microbiol. 23: • 331-335, 1977.

Morphology 'The strain H964-92 forms mycelium in stratum and aerial mycelium. The substrate mycelium is long, branched and not fragmented into short filaments. In the aerial mycelium, short chains of spores are formed in the monopod or at the end of the hypha. Whorled ramifications of spore chains are also observed near the tip of the hyphae.

These spore chains contain 2 to 10 chain spores and are straight, flexible or angular.

The spores have a warty surface and are spherical to elliptical (0.5-0.6 X 0.6-1.4 ^ µm) with rounded or pointed ends. After maturation, each spore is often separated by an empty sheath. Mobile spores, sporangia or sclerotic granules were not observed on any of the media examined.

Culture and physiology characteristics

The growth of the H964-92 strain is weak to moderate in chemically defined media and also in natural organic media. The formation of aerial mycelium is generally weak, but is moderate in oatmeal agar (ISP Ne3 medium), starch and inorganic salt agar (ISP No. 4 medium) and agar. by Bennett. Spontaneous variants without aerial mycelium appear very frequently. The aerial mycelium is white, but then turns pale pink in oatmeal agar, starch and inorganic salt agar and asparagine and glycerol agar (ISE Ne 5 medium). The color of the aerial mass then turns bluish after a long incubation (5 months) in oatmeal agar, asparagine and glycerol agar and * tyrosine agar. The substrate mycelium is colorless to yellowish in Czapek agar, tyrosine * agar, malt extract and yeast extract agar (ISP medium No. 2), iron agar , with yeast extract and peptone (ISP medium No. 6) and Bennett agar and is pinkish in asparagine and glucose agar and asparagine and glycerol agar. Melanoid pigments and other diffusible pigments are not formed. The ** variant ηβ A1327Y from the original strain forms * an aerial mycelium, mainly pale blue, and carries an abundant mass of aerial spores.

The H964-92 strain grows at 15 ° C, 28 ° C, 37 ° C and 43 ° C, but not at 10 ° C or 47 ° C. It is sensitive to 1% NaCl and resistant to lysozyme at 0.01 / e.

The culture and physiology characteristics of the producing strain are given in Tables 1 and 2 respectively. The use of carbon sources is explained in Table 3.

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Physiological characteristics of the H964-92 strain

Test Response Process and medium r Maximum temperature growth interval Bennett agar growth rature from 28 ° C to 37 ° C, moderate at 20 ° C and 43 ° C.

None at 10 ° C and 47 ° C

"Liquefaction of liquefied gelatin with gelatin peptone and glucose

Hydrolysis of starch agar, starch agar, donation box Reactions in uncoagulated and skimmed milk Difco skimmed milk fully peptonized

Pigment formation not produced tyrosine agar, yeast and peptone melanoid iron agar and yeast extract and tryptone broth Reduction of unreduced nitrate Nitrate and glucose broth of Czapek and nitrate broth, yeast extract and glucose Resistance to NaCl growth at 5% Agar with extract and less. No yeast and growth at T% tryptone

Lysozyme resistance, cross-agar with 0.01 / 6 sessence extract and yeast and tryp- minus. No tone growth at 0.1% -nj · * pH growth of 5.0 at Agar with extract. 9.5; no cross-yeast and tryp- sysis at 4.5 and 10.0 tone CD.MJ - 12 - TABLE 3

Use of carbon sources Strain H964-92

Actinomadura

Original verrucosospora variant strain A1327Y KCC A-0147

Glycerol + + + D (-) - Arabinose - - L (+) - Arabinose + + + î D-Xylose + + + D-Ribose + - - L-Rhamnose + + + D-Glucose + + + D-Galactose - - - D-Fructose + + + M-Mannose - ** L (-) - Sorbose -

Sucrose + + +

Lactose - - - "Cellobiose + + + Melibiose - - -

Trehalosis + + +

Raffinose - D (+) - Melezitosis - - -

Soluble starch + - + +

Cellulose + + +

Dulcitol -

Inositol + λ D-Mânnitol + + + D-Sorbitol - ï Salicme -

; Observation after 3 weeks of incubation at 28 ° C

Basic medium: Pridham-Gottlieb inorganic medium

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Composition of the cell wall and constituent sugars of the complete cell.

The purified cell wall of strain H964-92 contains mesodiaminopimelic acid, but * no glycine. The complete cellular hydrolyzate contains madurose (3-0-methyl-D-galactose), glu-. cose and ribose. Amino acids from the cell wall and sugars from the whole cell indicate that the H964-92 strain belongs to type III for the cell wall. The major components of mena-quinone are MK-9 (HC) and MK-9 (H_).

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Taxonomic position of strain H964-92

The H964-92 strain has the following major characteristics: (1) aerial spore chains: short, straight, flexible or angular.

** (2) Spores: warty surface (3) Aerial mycelium:. pinkish or bluish. (4) mycelium as a substrate: pink in certain environments. (5) Diffusible pigment: none.

(6) Mesophile. (7) Type III cell wall.

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(8) Menaquinone system: MK-9 (H ^) and MK-9 (H0).

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These major characteristics indicate that the H964-92 strain belongs to the genus Actinomadura.

Already known species of the genus Actinomadura have been isolated from mammals. Some strains have also been isolated from plant substances. However, many of the new species recently proposed have been isolated from soil samples. According to digital taxonomy and the review of Actinomadura and related actinomycetes made - by Goodfellow et al. in J. Gen. Microbiol. 112: i 95-111 (1979), most species of Actinomadura originating from the earth are classified in group no 7 among the 14 groups described. The H964-92 strain is very related to group 7 species. Nonomura and Ohara in J. Ferment. Technol. 49: 904-912 (1971) CD.MJ - 14 - i mention five saprophytic species of the genus Actinoma-dura and Nonomura [J. Ferment. Technol. 52: 71-77 (1974)] and Preobrazhenskaya et al. CtActinomycetes and Related Oraanisms 12: 30-38 (1977)] provide the keys to the identification and classification of Actino-madura species. Following the comparison with the descriptions of 30 species including organisms cited in patents, the strain H964-92 appears very similar to Actinomadura coerulea described in the article by Preobrazhenskaya et al. above and to Actinomadura verrucosospora described in the aforementioned articles from Nonomura.

The strain H964-92 compared directly to A. verrucosospora KCC A-0147 appears to be closely related to J \. verrucosospora by the characteristics * of morphology, culture and physiology. Therefore, the strain H964-92 is considered to be a new strain of Actinomadura verrucosospora.

It should be noted that for the production of BBM-1675, the present invention, although described in detail with reference to the particular strain Actinomadura verrucosospora H964-92 (ATGC 39334) and its mutant A1327Y (ATCC 39638-)., N is not limited to these microorganisms or microorganisms described in detail by the culture characteristics mentioned here. It is specifically intended that the invention applies to the strain H964-92 and to all of its variants and * natural and artificial mutants producing BBM-16 75.

h Production of antibiotics - The antibiotics BBM-1675 forming the subject of the invention can be obtained by culture of a strain producing BBM-1675 of Actinomadura verrucosospora. preferably a strain of Actinomadura verrucosospora having the identifying characteristics of ATCC No. 39334 or 39638, or a mutant of CD.MJ - 15 - the latter, in a conventional aqueous nutrient medium. The organism is cultivated in a nutritive medium containing nutritive sources known for actinomycetes, namely assimilable sources of carbon and nitrogen with possibly inorganic salts and other known growth factors. Aerobically submerged conditions are preferably maintained for the production of large quantities of the antibiotic, although for the production of limited quantities, surface and bottle cultures are also suitable. The general techniques applied to the culture of other actinomycetes are suitable for the purposes of the invention.

.The nutrient medium must contain an appropriate assimilable carbon source such as glycerol, L (+) -arabinose, D-xylose, D-ribose, L-rhamnose, D-glucose, D- fructose, sucrose, cellobiose, soluble starch, D-mannitol or inositol. Ammonium chloride, ammonium sulfate, urea, ammonium nitrate, sodium nitrate, etc. can be used as nitrogen sources either alone or in conjunction with organic nitrogen sources such as peptone, meat extract, yeast extract, corn steep liquor, soy flour, cottonseed meal, etc.

If necessary, the medium can also be supplemented with nutritive inorganic salts constituting sources of sodium, potassium, calcium, ammonium, phosphate, sulphate, chloride, bromide, carbonate, zinc, magnesium, manganese, cobalt,, iron, etc.

The production of the antibiotics BBM-1675 can be carried out at any temperature ensuring satisfactory growth of the producing organism, for example from 15 to 45 ° C., and is carried out with advantage to the see CD.MJ '- 16 - sinage of 27 at 32 ° C. Usually, production is optimal after incubation times of around 68 * to 180 hours, depending on whether the fermentation is carried out - b in shaken flasks; jar with agitator or fermenters.

"For work in fermenters, it is desirable to produce a vegetative inoculum in a nutrient broth by inoculating the culture broth using a culture on inclined agar or soil or using a lyophilized culture of the producing organism. An active inoculum obtained in this way is then aseptically transferred into the medium of the fermenter. The production of the antibiotic can be monitored by diffusion test on agar with paper discs in the presence of Staphvlococcus aureus 209P as an organism ^ d1 test.

Isolation and purification

At the end of the fermentation, the BBM-1675 complex can be isolated from the broth according to the usual techniques, for example solvent extraction. For example, the whole broth can be separated by filtration or centrifugation into a mycelial cake and a supernatant. The mycelial cake antibiotic can be isolated by suspending the solids in methanol, separating the insolubles by filtration and concentrating the methanolic extract. The active fraction of the supernatant broth can be collected by extraction in n-butanol. The aforementioned n-butanolic extract and me-thanolic extract can be combined and evaporated with azeotropy into an aqueous solution which deposits most of the antibiotic activity in the form of an oily solid. This can be dissolved in methanol and the solution can be filtered.

The filtrate is concentrated and added to a mixture of ethyl acetate and water. The crude BBM-1675 complex contained in the resulting organic extract can be precipitated from the solution by the addition of an antisolvent such as n-hexane.

The raw BBM-1675 complex is a mixture of different components comprising two major bioactive components, namely BBM-1675 A ^ and Ä2, and four minor bioactive components, namely BBM-1675, A ^, A ^, B ^ and Έ > 2 · These bioactive components can be separated and purified according to the usual techniques of chromatography. According to a method, the crude BBM-1675 complex is first dissolved in methanol and purified by chromatography on a column of Sephadex LH-20 from which it is eluted using methanol. This partially purified complex can then be chromatographed on a column of silica gel and eluted in fractions using chloroform added with metha- "V nol in increasing concentration to give BBM-1675 A ^, a mixture of BBM-1675 A2, A ^ and A ^ and a mixture of BBM-1675 B ^ and B2 The component can be further purified by chromatography on a column of Sephadex LH-20 from which it is eluted with methanol.

The mixture of A2 with A ^ and A ^ can be fractionated by chromatography on a column of Bondapak C ^ g (Waters Associates, Inc.) which is eluted with increasing concentrations of aqueous acetonitrile. The mixture of B ^ and B2 can be fractionated by chromatography on a column of silica gel eluted ^ with a mixture of chloroform and methanol. Further details relating to the preferred chromatographic separations are given in the examples below.

; Physico-chemical properties of BBM-1675 components

The six bioactive components of the BBM-1675 complex are distinguishable from each other using two CCM systems as illustrated in the following table CD.MJ - 18 - TABLE 4 CCM of the components of BBM-1675 r Values of Rf • Si02 * Silane

Component CHCl 3-CH3OH (5: 1 v / v) CH3CN-H20 (75:25 v / v) BBM-1675 h1 0.74 0.18 BBM-1675 A2 0.71 0.21 BBM-1675 A3 0, 72 0.28 BBM-1675 A4 0.71 0.78 BBM-1675 Βχ 0.63 0.23 BBM-1675 B2 0.60 0.16 * Phase C inversion silica gel, 0

J-O

The separation of BBM-1675 A2, A3 and A ^ is difficult by ordinary phase TLC, but is achievable by phase inversion TLC.

The different components of BBM-1675 have similar solubility and color reaction properties. For example, they are soluble in chloroform, ethyl acetate, acetone, ethanol and methanol, sparingly soluble in benzene and water and insoluble in n-hexane and carbon tetrachloride. They give positive reactions with ferric chloride, the reagents of Ehrlich and Tollens and negative responses in the tests of Sakaguchi, to ninhydrin and to the anthron.

The characteristic physicochemical properties of the components of BBM-1675 are given in table 5 below.

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The UV absorption maxima of the BBM-1675 components are observed at 253, 282 and 320 nm and are not displaced in acid or alkaline solution. The IR and RME spectra of BBM-1675 A2, A ^ and A ^ ~ are the subject of Figs. 1 to 4 and 5 to 8 respectively.

The 360 MHz RMP spectrum of BBM-1675 A ^ reveals an acetyl radical (Æ: 2.11 ppm), an N-CH ^ radical (2.52ppm), four OCH3 radicals (3.42, 3.80, 3 , 88 and 3.98 ppm) and ^ an exomethylene radical (4.57 and 5.48 ppm), in addition to two aromatic protons (7.50 and 8.59 ppm) and a NH proton (11.79 ppm). The 13C NMR spectrum of BBM-1675 Αχ includes 55 carbon signals including a triple intensity signal (6: 56.0 ppm, OCH ^). The formula 'gross assigned to BBM-1675 An is Cc_H_oN.0 ^ "on 1 57 72 4 32 based on nuclear magnetic resonance spectra 13

protons and C, micro-analysis and "molecular weight determination by HPLC and SMIS

(high performance liquid chromatography and secondary ion mass spectrometry).

Study of the structure of BBM-1675 A ^

By addition of 0.5 N-CH3OH HCl at room temperature, BBM-1675 A ^ loses its bioactivity and gives rise to a lipophilic chromophore substance (compound I) and to various unidentified fragments. .Composite I has a UV absorption similar to that of the antibiotic from which it comes, which suggests that Compound I retains the chromophore structure of BBM-1675 A ^. Two other chromophore fragments related to compound I are obtained by alkaline hydrolysis of BBM-1675 A ^: hydrolysis by KOH 0, O5N-CH3OH at 55 ° C for 1 hour leads to compound II having maxima d UV absorption at 252, 284, 297 (shoulder) and 322 nm, while the reaction in KOH 1N-CH30H gives an acid chromophore substance called compound III. The physicochemical properties of compounds I, II and CD.MJ - 21 - III are summarized in Table 6 below.

.TABLE 6

Properties of compounds I. II and III

Compound I Compound II Compound III 'M: 82 - 83 ° C 133 ° c 253-255 ° C

[o <] ^ 9 (c 0.2 CHC13): -100 ° 0 0

Gross formula: C ^ NO ^ C ^ H ^ NOg C ^ H ^ NOg UV ^ S0H nm (€): 244 (21.850) 252 (26.600) 248 (26.900) 276 (9400) 283 (11.200) 295 (14.400) 318 (6300) 297 (thick.8800) 310 (13.500). 322 (11.700) SM m / z: 457 (M +) 295 (M +) 281 (M +) 425 280 263 341 263 236 * 281 251 222 264 248 218

CCM

"(Xylene- * MEK-CH_0H = 5: 5 v / v): Rf 0.58 0.66 0.13 * MEK = methyl ethyl ketone

The spectroscopic data and the chemical transformation below provide information relating to the structure of compounds II and III. The 13 NMR spectra of C and H show the presence of four OCH- radicals, one CH_ radical, two radicals | <5 6 waters -C =, of two radicals> C = 0 and of a radical yNH ^ in compound II. The NMR spectrum of compound III is similar to that of compound II and differs from it only by the absence of one of the four OCHCH radicals observable in compound II. This difference, together with the acidic character of compound III, suggests that compound II is a methyl ester of compound III. Heated at reflux in IN methanolic KOH, compound II is quantitatively converted to compound III, while compound III is converted to compound II by reaction with diazomethane. The reaction of compound II with NaBH ^ in ethanol gives a reduction product (compound IV, M +: m / z 267) whose NMR spectrum reveals the existence of a radical -Ci ^ OH instead ™ ^ of the radical -COOCH ^ of compound II. By hydrogenation on palladium carbon, compound II gives a di-hydrodérivé (compound V, M +: m / z 297). The NMR spectrum of the protons of compound V is distinguished by a new doublet of methyl radical and by the absence of the signals of the exomethylene radical existing in compound II. In addition, one of the OCH ^ radicals appears at a higher field (6: 3.5Chppm) in the compound V.

These results indicate the presence of a radical -C = CH_,

1 L

0CH3 in compound II which is reduced by hydrogenation - to a radical "-CH-CH, I 6 och3 in compound V. Compound II heated in 1.5N methanolic HCl at 80 ° C for 3 hours gives a product of hydrolysis, the chromatography of which on a column of silica gel reveals the existence of a weakly basic compound (compound VI, M +: m / z 211). The IR spectrum and the physicochemical properties show that the compound VI contains a radical ΝΗ3 Comparison of the IR and NMR spectra of compound VI and of an authentic sample i reveals that it is methyl 4,5-dimethoxy-anthranilate. The structures of the compounds; It a VI therefore appear to be those indicated below. - after.

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

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MeOH ^ | [COOCH3. By reaction with 0.05N-CH30H KOH at 55 ° C., the compound I gives rise to a new chromophore fragment (compound VII, ci5H2iN07 'M +: m / z 323 ^ and to a sugar (compound VIII) The NMR spectrum of compound VII includes a singlet of C-CH3 and high field signals of two OCH3 radicals in addition to the weak field signals of three OCH3 radicals and signals of the two ΓΤ1 MT _ 0 Λ _ aromatic protons normally observed for compounds II, V and VI. Hydrolysis of compound VII with 1.5N methanolic HCl leads to compound VI identical to that already obtained from compound II.-After separation of compound VI, the hydrolyzate added with 2 , 4-dinitrophenylhydrazine precipitates a yellow solid identified as 2,4-dinitrophenylhydra-zone of pyruvic acid 11. Compound VII is therefore * 4,5-dimethoxy-N- (2 ', 2'-dimethoxypropionyl) Methyl anthranilate. Compound VIII does not include a molecular ion peak but includes the peak due to M-OCH ^ at m / z 131 in the mass spectrum, in agreement with the crude formula C7H1404 · The NMR spectrum indicates a 2,6-dideoxyhexopyranose structure for compound VIII. This proposition is confirmed by the - 13 NMR spectrum of C of compound I which comprises the si- ~ T gnaux of a C-CH ^ radical, of a -CH2 radical, of three O-CH <radicals and of a anomeric carbon atom in addition to 15 carbon signals attributable to compound VII. The Cg proton of the sugar appears in a weak field (6: 5.39 ppm, byte), which indicates that the C ^ -OH of the sugar is esterified by the carboxyl radical of the compound Vil. These results are summarized below.

s *

Structures of compounds I. VII and VIII Compound i compound VIII

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Compound VII

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The molecular weight of compound I (457) is approximately one third that of the complete molecule of BBM-1675 (proposed formula C57H72N4 ° 32 '· PM calcu_ lé = 1324). The partial structure of BBM-1675 A ^ is considered to be as follows: ί CH30 NH-CO-C = CH2 I Î> CH3 H.

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0

After the filing date of U.S. Patent Application No. 495,231, it was discovered that the BBM-1675 A ^ and A3 described above and obtained in accordance with Example 2 are not in fact not perfectly pure and that some of the characterizing properties used to define these components lack precision. After further chromatographic purifications described in more detail in Examples 3 and 6 below, BBM-1675 A ^ and were isolated in a purer form and completely characterized as described below. In addition, the elementary analysis of the components A ^ and A ^ was revised to take account of the presence of sulfur in these compounds and the retention times in HPLC were calculated for these two components. The corrected physicochemical properties of the components of BBM-1675 are summarized below.

BBM-1675

Description: white to pale yellow crystals; . PF. 156-158 ° (decomposition).

Elementary 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% 2 ί Ο: 28.31? Έ Ο: 27.62% Ο: 21.96% (by difference) (by difference) (by difference) „UV absorption spectrum: Instrument-Varian UV, Cary 219

Solvent - methanol 2 Concentration - 0.01356 g / 1 ^ (nm) Max absorbencies 320 12.4 280 ep (shoulder) 253 25.1 210 25.5

No significant modification by acids or bases. Optical rotation: solvent -CHC1- [<*] £ = -207 ° (C = 0.0351)

Optical rotation at a second analysis: [ot] D7 ° = “191 ° (C = °, 5, chc13)

Infrared absorption spectrum: see Fig. 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-1.

Mass spectrum:

Instrument - VG-ZAB-2F FAB-MS-thioglycerol

Ions in the molecular weight range (m / z): 1249, 1357, 1463; with addition of NaCl (m / z): 1271, 1379, 1485, 1597. t FAB-MS-MB (MB: matrix, P.M. 154); ions in the molecular weight range (m / z): 1249, 1283, 1403, 1555; with - addition of NaCl (m / z): 1249, 1271, 1303, 1425, 1483, 1577.

FAB-MS-glycerol-DMSO: ions in the molecular mass range (m / z): 1215, 1247, 1279, 1293, 1325, 1353; with addition of NaCl (m / z): 1215, 1237, 1247, 1269, 1325, 1347, 1375.

Instrument: Kratos MS-50 FAB-MS-thioglycerol;

Ions in the molecular weight range (m / z): 1357, 1463.

Molecular weight (based on the mass spectra above): P.M. = 1248.

Nuclear magnetic resonance spectra:

Instrument - WM360 Brucker Solvent: CDC1-.

1RMN: 360 MHzô (ppm): 11.75 (1H, s); 8.55 (1H, s); 7.45 (1H, s); 6.61 (1H, m); 6.23 (1H, bs); 6.17 (1H, bs); 5.93 (1H, d, J = 9.3); 5.82 (1H, d, J = 9.3); 5.7 (1H, si); 5.49 (1H, m); 5.45 (1H, d, J = 2.3); 5.38 (1H, bs); 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, bs); 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, ml); 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); 1.31 (3H, d, J = 6.3); 1.29 (3H, d, J = 6.3), 1.08 (6H) D.

See Fig. 10 NMR from 13c to 90.3 MHz

See Fig. 11

In the course of a separate experiment, the 13: C NMR spectrum of purified BBM-1675 A ^, measured on a sample dissolved in CDCl ^ (at 80 MHz), gives the main peaks indicated below.

13C NMR of BBM-1675 A (80 MHz in CDC1) X J '"Chemical shifts in ppm (multiplicity *) BBM-1675 A1 13.7 (q) 16.6 (q) 17.5 (q) 19, 3 (q) 22.2 (q) 22.6 (q) 23.4 (q) 29.0 (t) 34.0 (t) 35, l (t) 39.5 (t) 47.2 ( d) 52.5 (q) 55.6 (i) 56.0 (q) 57, l (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, l (i) 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) 103.8 (d) 107.6 (s) 112.5 (d) 123, l (d) 124.9 (d) s 130, l (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) 166.4is) 191, 8 (s) ♦ Multiplicity - q = quadruplet; d = doublet; i = uncertain; t = triplet; S = singlet.

rn. m.t _? q _ BBM-1675 Ä2

Description: white crystals; P.F. 147-149eC Elementary analysis: C: 52.71% H: 5.94% N: 3.94% S: 9.39% 0 (by difference): 28.01% s

UV absorption spectrum:. Instrument - Varian ÜV, Cary 219

Solvent: methanol Concentration: 0.02052 g / liter \ nax Absorbency 320 12.2 282 16.3 252 26.2 214 25.8

No significant modification by acids or bases. ‘Optical rotation: [ot] ^ = -179.4e (c = 0.5, CHCl ^)

Infrared spectrum: see Fig. 12 Beckman IR instrument 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 “1.

Mass spectrum: Instrument: VG-ZAB-2F FAB-MS-thioglycerol

Ions in the molecular weight range (m / z): 968, 1249, 1355, 1357, 1463, 1569; with addition of NaCl (m / z): 990, 1271, 1379, 1485, 1593 FAB-MS-MB (MB: matrix, p.m. 154); ions in the molecular weight range (m / z): 1249, 1403, 1419, 1555, 1571, 1587; with addition of NaCl (m / z): 1249, 1271, 1425, 1441, 1457, 1483, 1577.

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

CD.MJ - 30 - ί.

f ι ι ί

Molecular weight (based on the mass spectra above): apparent P.M. "= 1248 * Nuclear magnetic resonance spectrum: see Fig.13

Instrument: WM 360 Brucker Solvent: CDCl ^ '1H NMR 360 MHz <5 (ppm): 11.91 (1H, s); 8.62 (1H, s); 7.58 (1H, s); 6.56 (1H, m); 6.22 (1H, s); 6.15 (1H, bs); 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, si); 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, si), 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 (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).

13 C NMR at 90.3 MHz See Fig. 14

In a separate experiment, the 13 C NMR spectrum of the purified BBM-1675, measured on a sample dissolved in CDCl 4 (80 MHz), gives the main peaks indicated below.

BBM-1675 A2 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 36.1 77.1 77.7 78.1 78.3 83.3 86.2 88.4 90.4 97.2 98.3 99.1 99.5 4 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 CD.MJ - 31 - ti υ ο

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Thin layer chromatography (TLC) and high performance liquid chromatography (HPLC) of the components of BBM-1675 A. Study n ° 1 - Summary of TABLE 8 TLC and HPLC of the components of BBM-1675 CCM (Rf) HPLC (time of retention _ in minutes) _

SiCL * Silane Lichrosorb RP-18 CHCU-MeOH OLCN-Η, Ο CH_CN-MeOH-0, 1M CH-COONH.

(5: 1 v / v) (75:25 v / v) 0 (5: 2: 3 v / v) _ BBM-1675 Αχ 0.74 0.18 13.3 BBM-1675 A2 0.71 0, 21 17.3 BBM-1675 Â3 0.72 0.28 8.0 BBM-1675 A4 0.71 0.78 5.1 J- BBM-1675 Βλ 0.63 0.23 BBM-1675 B2 0.60 0 , 16

* C1Q phase inversion silica gel

B. Study No. 2 - TLC and HPLC for the purified components A ^ and A2_

CCM

Analtech GHLF Silica Gel Uniplates plates are used for all normal phase chromatographies. For the one-dimensional TLC we take 2.5 cm x 10 cm plates. They are developed in cylindrical glasses of 6.4 cm (outside diameter) by 12 cm containing 10 ml of eluent. For the two-dimensional TLC, 7.5 cm x 10 cm plates are used. The sample is placed in the lower left corner 1 cm above the edge. The plate is first developed in a tank (12.7 cm wide, 8.6 cm deep and 13 cm high) containing 50 ml of the first eluent.

The plate is then dried in air, it is rotated 90 ° in the opposite direction to that of the watch hands and it is developed in a second tank containing 50 ml en.MJ - 33 - of the second eluent.

For all phase inversion chromatographies, pre-coated analytical silica gel plates C-18 from Whatman are used. The 2.5 cm x 7.6 cm plates are developed in cylindrical glasses containing 10 ml of eluent.

The plates are observed in normal phase first in 254 nm UV light. The plates are then inserted into a glass cylinder (6.4 cm outside diameter x 12 cm) containing iodine crystals. The plates are examined again after approximately 2 minutes. The phase inversion plates are examined only in UV light at 254 nm. The zones are detected by looking for the extinction of the fluorescence of a dye fixed by im-_ _ pregnation.

Analytical HPLC

The following components are used to build an analytical HPLC system: Waters Associates Model 6000A solvent flow pump; Varian Varichrom model VUV-10 UV / visible detector set to 254 nm 0.1 DO; Fisher Recordai 5000 Series recorder; Waters Associates model 660 solvent programmer; Waters Associates U6K injector; ^ U-Bonda-pak C18 column (10 ^ u) Alltech (4.6 mm inside diameter x 25 cm) with Whatman Co. Pell ODS guard column (0.03-0.038 mm) (4.6 mm inside diameter x 5 cm). The components are connected with 316 stainless steel tube (outside diameter 1.6 mm, inside diameter 0.23 mm). The eluent flow rate is 2 ml / minute for all analyzes.

* Preparative HPLC

The following components are used to form a medium pressure liquid chromatography system: Fluid Metering, Inc. pump model RP-SY 2CSC FMI Lab; Fluid Metering pulse damper,

Inc. model PD-60LF FMI; 15 ml sample loop made of a polypropylene tube (external diameter 3.0 mm, internal diameter 1.5 mm) rolled on a cardboard tube (external diameter 8.65 cm); Glenco Sériés 3500 Universal LC columns; absorption and fluorescence detector with standard optical unit. 6 Instrument Specialties Co. model UA-5; instrumentation Specialties Co. shut off valve, model 590 and fractionation collector Instrumentation Specialties Co., model 328. The components are connected by polypropylene and Teflon tubing (outside diameter 3.0 mm, inside diameter 1.5 mm ) and Glenco connectors and taps or valves in the order shown.

Glenco 3500 Universal LC serial columns are packed using a dispersion of the absorbent, defined in the solvent indicated, applying the usual techniques. The empty space between the sedimented bed and the top of the tube is filled with standardized Ottawa sand. The eluent is pumped at a maximum flow rate without exceeding a back pressure of 4.14 bars (approximately −20 ml / minute).

Gradient elution

For all gradient elutions, a Glenco gradient elution apparatus is used which comprises two chambers of the same diameter, the same height and the same volume connected in tandem by a Teflon tap. One bedroom serves as a mixing chamber and. the other serves as a static tank. The least polar solvent is initially stored in the mixing chamber i. The most polar solvent is kept in the static chamber. Bars of magnetic stirrers coated with Teflon (1 cm x 3.7 cm) are introduced into the two chambers and they are trained using Thomas model 15 Magne-matic stirrers. 11- CD.MJ - 35 - pompe is pumped from the mixing chamber to the medium pressure HPLC system, passing through polypropylene tube (internal diameter 1.5 mm, external diameter 3.0 mm). As the eluent leaves the mixing chamber, the solvent in the static tank is allowed to freely replace it, which establishes a linear gradient of eluent.

*

TLC analysis of BBM-1675 A ^ and A ^. Table 9 below collates the Rf ob served from BBM-1675 A ^ and on plates in normal phase. The Rf is calculated by dividing the distance measured from the center of an area to the point of application of the sample by the distance measured from the front of the solvent to the point of application of the sample.

TABLE 9

Rf

System / compound BBM-1675 Aj BBM-1675 A ^ 4% methanol in chloroform 0.33 0.30 5% methanol in diethyl ether 0.39 50% acetone in Skellysolve B 0.38 0.31

Table 10 below collates the Rf ob served from BBM-1675 A ^ and A ^ on normal phase plates developed in two dimensions. The positions of the spots are given in Cartesian coordinates.

The X coordinate is the Rf in the second specified solvent system. The Y coordinate is the Rf in the first specified solvent system.

* TABLE 10 Rf

System / compound_ BBM-16 75 A ^ BBM-1675 A ^ 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, 029)

Table 11 below collates the Rf observed from BBM-1675 A ^ and 'A ^ on C-18 phase inversion TLC plates developed in binary eluents.

TABLE 11

RI

S £ S teme / compound_ BBM-1675 A ^ BBM-1675 A ^ 25% 0.5 M NaCl in acetonitrile 0.18 0.21 25% water in acetonitrile 0.00 0.00 'Table 12 below brings together the Rf ob served from BBM-1675 A ^ and A ^ on CCM phase inversion plates C-18 developed with ternary eluents.

TABLE 12 --- Rf _System / compound BBM-1675 A ^ BBM-1675 A ^ CH3CN: Methanol: 0.5M 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 î 40% : 40%: 20% 0.60 0.49 50%: 20%: 20% 0.42 0.34 60%: 20%: 20% 0.74 0.69 CH3CN: Methanol: water 40%: 30% : 30% 0.00 0.00 TABLE 12 (continued)

Rf _System / coropose_ BBM-1675 A1 BBM-1675 A ^

CH.CN: Methanol: 0.1 M NH.OAC

40%: 30%: 30% 0.32 0.22 v CE ^ CN: Methanol: 0.1M NaE ^ PO ^ 40%: 30%: 30% 0.00 0.00, HPLC analysis of BBM-1675 A ^ and A ^

BBM-1675 A ^ and BBM-1675 A2 were analyzed on C-18 phase inversion silica gel columns with single, binary and ternary eluents. The results collated in Tables 13, 14 and 15 are the K 'observed for these compounds. The quantity K 'is given by the following formula: T - T R o K - - o J where TD is the retention time measured from the mo-

R

ment from the injection to the top of the peak and Tq is the time of the empty volume.

TABLE 13

·. .K

System / compound BBM-1675 A ^ BBM-1675 A ^

Acetonitrile a to tetrahydrofuran - to a methanol 0.00 0.00 a = the compound is not eluted from the column TABLE 14 K '

System / compound_ BBM-1675 A ^ BBM-1675 A ^ 25% water in acetonitrile aa 25% methanol in water 1.25 1.25 a = the compound is not eluted from the column TABLE 15 Ternary eluents

System / compound_ BBM-1675 BBM-1675 A ^ CH ^ CN: Methanol: water 40% z 30%: 30% aa * CH-CN: Methanol: 0.1M NH.OAc 3 '4 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 ba = not elected b = not determined * Biological properties of the components of BBM-1675

The biological activity of the BBM-1675 components is determined with respect to different bacteria (Gram-positive, Gram-negative and acid-resistant) and different fungi according to the technique of dilution of the single to double on agar . Nutrient agar is used for Gram-positive and Gram-negative bacteria and N® 1001 medium (3% glycerol, 0.3% sodium L-glutamate, 0.2% peptone, 0.31 % Na ^ PO ^, 0.1% Κί ^ ΡΟ ^, 0.005% ammonium citrate, 0.001% MgSO ^ and 1.5% agar) for acid-resistant organisms. We take Sabouraud agar for mushrooms. As can be seen from Table 16, each of the six components of BBM-1675 (A ^, A3 »A4» B ^, B £) has a broad antimicrobial spectrum. BBM-1675 A ^, A2, A ^ and A ^ in particular are very active against Gram-positive bacteria.

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The pro-phage induction activity is determined on the lysogenic bacterium E. coli W1709 (λ) manifested by the components of BBM-1675 by applying the procedure of Lein et al. in Nature 196: 783-784 (1962). The counts are made on plate agar containing the test compound (T) and control plates (C). A T / C ratio between the counting results on the plates greater than 3.0 is considered to be significant and the lysogenic induction activity (ILB activity) is expressed by the minimum induction concentration of the compound examined. As shown in Table 17, the components of BBM-1675 have an intense ILB activity on lysogenic bacteria, suggesting that they may have anti-tumor activity.

TABLE '17

Lysoqene induction activity of the BBM-1675 components Antibiotic CIM (^ uq / ml) * BBM-1675 Αχ 0.0063.

BBM-1675 A2 0.0125 BBM-1675 A3 ^ 0.05 BBM-1675 A4 0.10 BBM-1675 0.10 BBM-1675 B2 0.20 * minimum induction concentration The anti-tumor activity of BBM-1675 A ^ and A2 manifests in different mouse tumors. P-388 lymphocytic leukemia, L-1210 lymphoid leukemia, melanotic melanoma B16 and Lewis lung carcinoma were implanted intraperitoneally with BDF .. male mice with an inoculum of 10.10, 5 x 10 ° and 10 ° cells per mouse, respectively. Graduated doses of the test compounds are administered to mice intraperitoneally 24 hours after inoculation of the tumor.

Administrations are performed once on the first day only, on days 1, 4 and 7 (q3d x 3), once a day for 9 days (qd 1 -> 9) or 11 days (qd 1 -> 11). The components A ^ and A ^ against leukemia P-388 are tested only according to the program q3d x 3 due to the insufficiency of the available reserves of these components.

i BBM-1675 A ^, »A ^ and A4 are dissolved in 0.9% physiological saline solution containing 10% dimethyl sulfoxide and chromomycin A ^ (Toyomycin, Takeda) is used as the reference compound. solution in 0.9% physiological saline solution. The number of dead and surviving animals in the treated and untreated mice is noted each day and the mean survival time (MST) CD.MJ - 42 - is calculated for each test group (T) and control group (C ). A T / C quotient equal to or greater than 125% indicates that a significant anti-tumor effect has been achieved. The results are collated in Tables 18 to 23. BBM-1675 A- ^ and A2 show very 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 A ^ in terms of the minimum effective dose. However, BBM-1675 A ^ and A ^ are less active than components A. or A0 against P-388 leukemia (Table 19). • 1 2 BBM-1675 A ^ and A2 are also active against leukemia L-1210 (table 21), melanoma B16 (table 22) and Lewis lung carcinoma (table 23). The toxicity of BBM-1675 A1 and A2 was determined on male ddY mice by intraperitoneal or intravenous administration, the BBM-1675 A ^ appearing to be approximately 10 times more toxic than the BBM-1675 A2 (table 24). The therapeutic indices of BBM-1675 A1 and A2 are 4 to 8 times and 8 to 20 times more favorable than those of chromomycin A, respectively, against leukemia P-388 (table 25). A second series of experiments is carried out by intravenous administration of the components of BBM-1675 against P-388 and L-1210 leukemias injected intravenously at 5 × 5 × 10 and 10 cells per mouse, respectively. For these latter experiments, the reference agent 5 is adriamycin, dissolved in physiological saline solution at 0.9% and administered on days 1, 4 and 7. The results are given in Tables 26 and 27. The components A ^ and A2 are superior to Adriamycin by the maximum value of T / C, the minimum effective dose and the range of activity.

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Chromomycin A ^ 0.81 0.41 0.23 TABLE 25

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The anti-tumor activity of BBM-1675 A ^ was also determined and in a second experiment against leukemia P-388, leukemia L-1210 and melanoma B16 in mice. The results are shown in Tables 28, 29 and 30. Details relating to the procedures applied for this purpose have been described in Cancer Chemother. Rep. 3: 1-87 (part 3), 1972.

TABLE 28

Effect of BBM-1675 A ^ and A ^ on P-388 leukemia. . Dose ip TSM CMP effect Survivogram day program Days% T / C g vants _'_ day 5 days 5 (30) * NSC 38270 qd.l- »9 400 13.0 163 -0.6 6/6 200 11 .0 138 -0.9 6/6 BBM-1675 Αχ day 1 51.2 20.0 250 -2.1 4/6 = · 6 ω '° 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 days 1, 5, 25.6 8.0 100 -1.8 6/6 9 12.8 13.5 169 -1.5 6/6 1 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 4 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 TABLE 28 (continued )

Effect of BBM-1675 A ^ and on leukemia P-388 t Dose ip TSM Effect CMP Surviv

Program Officer, ug / kg / day Days% T / C g vants ___._ day 5 d, 5 (30) - BBM-1675 A ^ qd 1 -> 9 12.8 tox. tox. tox. 1/6 DMSO —r was born in Ale. , r 6.4 6.0 75 -1.5 4/6 ser.phys. '' '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 day 1,256 tox. tox. tox. 0/6 128 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 days 1, 5, 128 tox. tox. tox. 0/6 ο 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 i 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

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TABLE 28 (continued)

Effect of BBM-1675 A ^ and A? on leukemia P-388 5 Aoent Proaramme Dose ip TSM CMP effect Surviv ^ 09, ug / kg / day Days% T / C g - vants _: _______ day 5 i.5 (30) * BBM-1675 A2 0.25 11.0 138 -0.4 6/6 DMSO -> qd 1 -> 9 64 tox. tox. tox. 1/6 ser.phys.

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

Ser. Control 8.0 - +0.5 10/10 phys.

Tumor inoculum: 10 ** ascites cells in irp implantation.

Host: CDF mouse ^ $

Tox. : <4/6 mice surviving day 5

Assessment: TSM = average survival time

Effect:% T / C = (TSM treated / TSM control) x lOO

Criterion:% T / C ^ 125 considered as significant antitumor activity.

^ * NSC 38270 = olivomycin A

CD.MJ - 60 - TABLE 29

Effect of BBM-1675 A ^ and h 2 on L-1210 leukemia. . _ Dose ip TSM Effect CMP Survi-, A9ent 'Program ^ / kg / ln ^ Jou!: S% T / cg vants __; _' _day 5 j.5 (30) BBM-1675 Αχ day 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 40.3 6/6 0.2 7.0 100 -0.5 5/6 0.1 7.0 100 40.8 5/6 - days 1, 5, 25.6 tox. tox. tox. 1/6 9 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 40.4 5/6 0.05 7.0 100 40.3 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 40.3 6 / 6 0.05 7.0 100 40.3 6/6 0.025 6.0 86 -0.6 6/6 TABLE 29 (continued)

Effect of BBM-1675 A ^ and A2 on leukemia L-1210 'Program Agent “P5®1? ^ O® Surviv • ^, ug / kg / m d Days% T / C g vants —----- day 5 ~ d.5 (30) BBM-1675 Ä £ day 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 A ^ and A ^ on melanoma B16

Agent Dose ip TSM CMP effect Survi-, ug (M) or days% T / C g vants' mg / kg / inj day 5 d.10 (61) BBM-1675 Αχ 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 A2 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

Phys. Ser. Control. 25.0 - -0.1 10/10 a One without tumor; TSM d.m.o. = 55.0 (220%),

Two without tumor; TSM d.m.o. = 46.0 (184%) tumor inoculum: 0.5 ml of a 10% ground material, ip Host: BDF mouse. * Administration: qd 1 -> 9

Tox. : <7/10 mice surviving day 10

Assessment: TSM = average survival time

Effect:% T / C = (TSM treated / TSM control) x 100

Criterion:% T / C ^ 125 considered to be significant antitumor activity

After further purification of BBM-1675 A ^ as shown in Example 6, samples of the compound purified against leukemia L-1210, leukemia P-388 and melanoma B16 are tested in mice.

The results are collated below.

TABLE 31

Effect of purified BBM-1675 A ^ on P-388 leukemia (Day 1 Administration)

Change- Survi-, Dose Route, pro- TSM T / C average means (rog / Kg / dose) gram days (%) in from d.5 weight _____lour 5 _ BBM-1675 A ^ 0.1024 ip, 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 -2.0 6/6 ”TABLE 31 (continued)

Effect of purified BBM-1675 A ^ on P-388 leukemia (Day 1 Administration)

Change-, Dose Route, pro- TSM T / C moderately- Survi-composition (mg / kg / dose) gram days {%) in high weight d.5 _ __ _ _ _ day 5 _ BBM-1675 A ^ 0.0256 ip, 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 ip, 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 (excipient) 1 x 10 0 ip, q4d x 3 11.0 100 -0.7 9/9 Host: female mouse CDF1 • g

Location and site: 1 x 10 cells, ip TABLE 32

Effect of purified BBM-16 75 A.) on leukemia L-1210 (Administration day 1)

Change- Survi-

Compound Dose Route, pro- TSM T / C average ('mg / kg / dose) gram days {%) in de j.5 weight __ _ _ _ _ day 5 _ BBM-1675 A ^ 0.1024 ip , 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 ip, 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 ip, 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 - (excipient) 1 x 10- ip, qd x 5 7.0 100 0.1 10/10 Host: mouse fQuelle CDF1

Layout and site: 1 x 106 cells, ip TABLE 33

Effect of purified BBM-1675 A ^ on melanoma B16 (Administration day 1)

Change- Survi- _, Dose Route, pro- TSM T / C average ave ^ (mg / kg / dose) gram days (%) in d.5 'weight _____day 5 _ * BBM-1675 Αχ 0.0064 ip, 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 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 5 0.0002 24.5 163 -2.4 10/10 0.0001 25.5 170 -2.3 10/10

Control (excipient) 0.5 ml ip, qd x 9 15.0 100 -0.3 10/10 ** BBM-1675 Αχ 0.0064 iv, q4dx 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 ip, 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 (excipient) 0.2 ml iv, q4d x 3 17.5 100 -0.4 10/10 Host: female mouse BDF1 ^ Implantation and site: 0.5 ml of ground material at 10%, ip ** Implantation and site: subcutaneous fragment

The above results show that the purified BBM-1675 A ^ has substantially the same antitumor properties as the less purified sample tested previously. This compound is exceptionally effective, because at a dose of 25 nanograms / kg administered 5 times a day, it has been shown to be effective against leukemia P-388 in mice.

"

BBM-1675 A ^ is effective against leukemia 0 P-388 and L-1210 when administered as a single injection on day 1, every four days in 3 injections or 5 times a day. Against melanoma B16, this compound is also effective in intravenous injection in animals carrying subcutaneous tumors and it is also effective in intraperitoneal injection in animals carrying tumors implanted ip. This ability of the active ingredient to reach a distant tumor is unusual among anti-tumor antibiotics.

As stated above, the components of BBM-1675 have intense antimicrobial activity and are useful for the curative treatment of infections caused by such microorganisms in mammals or other animals. In addition, these components can be used for conventional applications of antimicrobial agents, such as disinfection of medical and dental equipment.

Induction of prophage in bacteria. Lysogens and activity against tumors in mice reveal that the components of BBM-1675 * 5 are also therapeutically useful in inhibiting the development of tumors in mammals.

A subject of the invention is therefore also a method of therapeutic treatment of a host animal suffering from a microbial infection or a malignant tumor, according to which this animal is administered in CD.MJ - 67 - dose effective against microbe or tumor, BBM-1675 A ^,, A ^, B ^ or or a pharmaceutical composition of any of these.

According to another aspect, the subject of the invention is a pharmaceutical composition comprising in an amount effective against microbes or tumors of BBM-1675 A ^,, A ^, A ^, or B ^ in combination with a pharmaceutically acceptable excipient or diluent inert. These compositions can be prepared in any pharmaceutically acceptable form suitable for parenteral administration.

The preparations according to the invention intended for parenteral administration are in particular sterile aqueous or non-aqueous solutions, suspensions or emulsions. They can be prepared in the form of sterile solid compositions which can be dissolved in water, sterile saline or other sterile injectable medium immediately before administration.

It is obvious that the amount of antibiotics BBM-1675 which is preferred in practice varies with the nature of the component, the pharmaceutical form, the mode and the site of administration, the species to which the host belongs and the affection to treat.

Many factors which modify the influence of the active ingredient can be considered by the specialist, such as age, body weight, sex, diet, timing and route of administration, the rate of excretion. , the state of the host, the combination with other active ingredients, the sensitivity reactions and the severity of the disease. The administration can be carried out continuously or periodically within the limits of the maximum tolerated dose. The optimal dose for a given set of conditions. CD.MJ - 68 - can be assessed by the specialist using standard dose determination tests, taking into account the indications given above.

The examples below further illustrate the invention without limiting it.

EXAMPLE 1

BBM-1675 fermentation

The H964-92 strain of Actinomadura s is cultivated and maintained on an inclined agar containing 1% malt extract, 0.4% glucose, 0.4% yeast extract, 0.05% CaCO ^ and 1.6% agar. A well-formed culture on an inclined agar is used to inoculate a vegetative medium containing 3% starch - soluble, 3% dried yeast, 0.3% I ^ HPO ^, 0.1% KH2PO4, 0.05% of MgSO4.7H2O, 0.2% NaCl and 0.1% CaCO ^, the pH being adjusted to 7.0 before sterilization. The vegetative culture is incubated at 32 ° C. for 72 hours in a rotary shaker (250 revolutions per minute) and 5 ml of the culture are introduced into a 500 ml Erlenmeyer flask containing 100 ml of a fermentation medium. containing 3% cane molasses, 1% corn starch, 1% fish meal, 0.1% CaCO ^ and 0.005% CuS04.5H20 (pH adjusted to 7.0 before sterilization). Fermentation is carried out at 28 ° C for six days in the rotary shaker. The antibiotic activity of the fermentation broth is determined by agar diffusion from a paper disk against Staohlococcus aureus 209P as a standard organism. Antibiotic activity reaches a maximum of about 1 µg / ml after five days of fermentation.

"

The fermentation of BBM-1675 is also carried out in agitator fermenters. 500 ml of the inoculation culture prepared as above are introduced into 20 liter fermenters containing 10 liters r.n. m.t _ fi q _ of a fermentation medium having the same constitution as that used for fermentation in shaken flask. Fermentation is carried out at 32 ° C. with an air flow rate of 12 liters per minute and stirring at 250 revolutions per minute. Under these conditions, the production of antibiotics reaches a maximum of about 0.9 µg / ml after 68-76 hours of fermentation.

"'

Fermentation studies are also carried out in fermentation tanks. A seed culture is stirred for four days at 30 ° C in Erlenmeyer flasks containing vegetative medium which comprises 3% soluble starch, 3% dried yeast, 0.3% K2HPO4, 0.1 % of KH2PO4, 0.05% of MgSO4.7H2O, 0.2% of NaCl and 0.1% of CaCO 4. The seed culture is introduced into a 200 liter seed tank containing 130 liters of seed medium having the same constitution as above and the contents of the tank are stirred at 240 rpm at 30 ° C for 31 hours. The second seeding culture is used to inoculate 3000 liters of fermentation medium containing 1% corn starch, 3% cane molasses, 1% fish meal, 0.005% CuSO4.5H2O and '0 , 1% CaCO ^ * Production is carried out in tanks at 28 ° C at 164 revolutions per minute with aeration of 2000 liters / minute. The pH of the broth increases as the fermentation progresses and reaches a value of 7.7-7.8 after 170-180 hours, - time at which the maximum antibiotic activity amounts to 1.7 ^ ug / ml.

EXAMPLE 2

Isolation and purification of BBM-1675 components »

The collected fermentation broth (3000 liters, pH 7.8) is separated into a mycelial cake and a supernatant liquid using a Sharpless centrifuge. The mycelial cake is put on top. CD.MJ - 70 - pension in 1600 liters of methanol and the mixture is stirred for 1 hour. The insolubles are separated by filtration and the methanoli extract is concentrated only under vacuum, up to 43 liters. The activity contained in the supernatant is isolated by two extractions with n-butanol in an amount of 1000 liters each time. The extracts in n-butanol and the concentrated methanolic extract are combined and evaporated by azeotropy with occasional supply of water in an aqueous solution (20 liters) which deposits most of the antibiotic in the form of a oily solid. The solid is digested in 30 liters of methanol and the insolubles are separated by filtration. The methanolic extract is concentrated under vacuum to obtain 10 liters of a solution to which 40 liters of ethyl acetate and 30 liters of water are added. After 30 minutes of stirring, the organic layer is separated, dried over sodium sulfate and evaporated in vacuo to 4 liters. By adding the concentrate to 20 liters of n-hexane, a pale yellow solid is obtained which is the crude BBM-1675 complex (90.14 g, title: 55 μg / mg). This complex turns out to be at CCM a mixture of two major components, BBM-1675 A ^ and A ^, with a few minor components. They are separated and purified by repeated chromatography which is carried out in a cold room to avoid deterioration.

; The BBM-1675 complex (20 g) is dissolved in methanol (20 ml) and the solution is poured onto a column of Sephadex LH-20 (0 5.5 cm, height 85 cm).

The methanol solution is developed and the elution is monitored by biological assay against Staphylococcus aureus 209P. The active eluates are combined, concentrated in vacuo and lyophilized to obtain the BBM-1675 complex in the form of a semi-pure solid CD.MJ - 71 - i (4.19 g). The solid is then chromatographed on a column of silica gel (0 5.0 cm, height 50 cm) which is eluted with chloroform containing increasing amounts of methanol (1 -> 59 »v / v).

We gather the eluates according to their activity (quickly antibacterial (against J3. Aureus) and the CCM

(S1O2; CHClg-CH ^ OH = 5: 1 v / v), then they are concentrated under vacuum. First, almost homogeneous BBM-1675 A1 is eluted (production after evaporation: 351 mg) with i 2% methanol in chloroform, and then a mixture of BBM-1675 A2, Ag and A (507 mg), then a mix from BBM-1675 B (210 mg) with 3% methanol in chloroform. BBM-1675 A ^ is deposited on a column of Sephadex LH-20 (0 2.0 cm, height 80 cm) which is developed with methanol. The active fractions are concentrated in vacuo to dryness and the residual solid is crystallized in methanol to obtain colorless platelets of pure BBM-1675 A ^ (124 mg) (this product is the starting compound for example 3A). The BBM-1675 A2, Ag complex is separated and by chromatography on a Bondapak C ^ g column (Waters, 0 3.0 cm, height 50 cm). Elution is carried out with aqueous acetonitrile and the bioactive eluates are examined by TLC (Merck, silane, that is to say reverse phase silica gel C18: CHgCN-1 ^ O = 75:25 v / v ). The minor components A ^ (33 mg) and Ag (18 mg) are eluted successively in this order using 20% acetonitrile, then another major component A2 (301 mg) (this is the starting compound for Example 3B) with 50% acetonitrile.

The solid containing BBM-1675 B ^ and B2 is chromatographed on a silica gel column (0 3.0 cm, height 40 cm) with chloroform and methanol as developing solvent. The active fractions eluted with 4% methanol in CD.MJ - 72 - chloroform are combined and evaporated to obtain the; BBM-1675 'B ^ pure (7 mg). We elect another fraction. " active with 5% methanol to obtain after evaporation the BBM-1675 B2 (8 mg).

EXAMPLE 3 ïj ·

Further purification of BBM-1675 A ^ and A2 A. Purification of BBM-1675 A ^

A Glenco column with an inside diameter of 2.67 cm and a height of 75 cm is packed in the middle with a suspension of silica gel with octadecyl phase (C18) fixed by Baker (particle size 40 μm). ) in methanol. The column is connected to a medium pressure HPLC system and brought to equilibrium with 1500 ml of eluent (41.6% acetonitrile, 21.6% methanol, 36.8% acetate d 0.1 M ammonium). BBM-1675 A ^ partially purified (100.5 mg) obtained according to the purification technique of Example 2 is dissolved in 2 ml of acetonitrile and introduced into the sample loop. The sample is pumped into the column. The column is eluted using the above eluent, collecting 87 ml fractions. The eluates are monitored at 254 nm and 340 ma. Fractions 55 to 71 are pooled and extracted twice with 1500 ml volumes of chloroform. The chloroform is evaporated to dryness to collect 89.8 mg of residue C.

A Glenco column is packed with a diameter; 1.5 cm inside and 20 cm high at the middle

in a dispersion of 12 g of Woelm * ’silica gel (particle size 60 to 200 μm). We deposit the residue C

on the column in a chloroform solution. The column is eluted with 500 ml of chloroform in which a linear gradient up to 10% of methanol in chloroform is established, collecting fractions of 20-25 ml. After analysis by TLC on silica gel, CD.M.T - 73 - the fractions 6-9 are combined and evaporated to dryness to collect 73 mg of residue D.

A Glenco column with an internal diameter of 1.5 cm and a height of 20 cm is packed by means of a dispersion of 12 g of Woelm silica gel (gra-nulometry 63-200 μm) in Skellysolve B. The residue D is dissolved in approximately 2 ml of chloroform and b is deposited on the column. We move the chloro-. formed with 25 ml of skellysolve B. The column is eluted with 500 ml of Skellysolve B in which a linear gradient is established up to 60% acetone in Skellysolve B, collecting fractions of 28-25 ml. Fractions 19-23 are pooled and evaporated to dryness to collect 65.6 mg of pure BBM-1675 A ^.

This residue appears homogeneous in three TLC systems (5% methanol in chloroform; 5% methanol in ether and 50% acetone in Skellysolve B, on silica gel) and in HPLC (silica gel C-18: 41.5% acetonitrile, 21.5% methanol, 7.0% 0.1 M ammonium acetate).

Gel permeation chromatography with purified BBM-1675 A ^

A Pharmacia column with an inside diameter of 2.5 cm and a height of 45 cm is packed with a dispersion of Sephadex LH-20 in methanol and the chromatographic bed is adjusted to 33.4 cm. Purified BBM-1675 A1 (about 120 mg) is dissolved in; 2 ml of methanol and placed in a 2.5 ml sample tank. The sample is placed on the column and the elution is started with 1.75 ml per minute of methanol, collecting 10 ml fractions [Pharmacia Frac-100 fraction collector].

The eluate is monitored at 254 nm using an Isco UA-5 detector. We note that BBM-1675 A. ^ elutes for Ve / Vt from 0.79 to 0.91 (Ve = elution volume; Vt = vo- CD.MJ - 74 - lume du lit).

B. Purification of BBM-1675

A Glenco column with an inside diameter of 2.65 cm and a height of 75 cm is packed with a dispersion of silica gel with b-tadecyl phase (C18) fixed by Baker (particle size 40 μm). ) in methanol. The series is connected to a medium pressure HPLC system and brought to equilibrium = with 1500 ml of eluent (50% acetonitrile, 20% methanol, 30% ammonium acetate 0.1 M). Partially purified BBM-1675 (76.9 mg) as obtained by the method of Example 2 is dissolved in 2 ml of acetonitrile and introduced into the sample loop. The sample is pumped into the column. The column is eluted with the above eluent, collecting 87 ml fractions. The eluate is monitored at 254 and 340 nm. Fractions 31 to 38 are pooled and extracted twice with 500 ml aliquots of chloroform. The chloroform is evaporated to dryness to collect 65.8 mg of homogeneous BBM-1675.

‘The BBM-1675 is homogeneous in 2 CCM systems, a 2-dimensional CCM system and HPLC.

EXAMPLE 4 · ·

Preferred method of extracting BBM-16 75 Aj ^

The raw fermentation broth (6800 ml) obtained according to the general procedure of Example 1 is introduced into a polypropylene container (diameter 12 cm at the top and 10 cm at the bottom, height 37 cm), the bottom of which has a tap. There we are. 5 introduces an equal volume of chloroform. The mixture is stirred at the right speed with a pneumatic CRC agitator for 2 hours. About 4 liters (1.3 kg) of Dicalite (filter aid) are added and dispersed. The mixture is filtered on a layer of Dicalite deposited in a Buchner No. 12 funnel.

CD.MJ - 75 -

We collect it. filtrate in a 19 liter bottle fitted with a vacuum connection (Ace. N ° 5396-06).

The filter layer is washed with 2 liters of chloroform. The filtrate is introduced into a 20-liter separation funnel and the phases are left to separate. The lower phase is collected (chloroform).

s »

A Glenco tube of an in- diameter is packed; 2.5 cm thick and 40 cm high by means of a dispersion of 91 g of Woelm silica gel (particle size 63-200 μm). Using an FMI RPY-2CSD pump, the above chloroform phase is pumped through the column. The column is rinsed with 600 ml of fresh chloroform. This chloroform eluate is rejected. The column is then eluted with 600 ml of 105έ methanol in chloroform. The fraction thus eluted is evaporated to dryness to obtain 547 mg of residue A.

; The residue A is dissolved in 50 ml of chloroform. The chloroform solution is added to 20 g of Dicalite in a round bottom flask with a capacity of 1000 ml. A suspension is formed by adding approximately 200 ml of Skellysolve B. The solvents are removed on a rotary evaporator. The residue is dispersed in 300 ml of Skellysolve B. The dispersion is poured into an Ace chromatographic tube (part No. B5872-14) (internal diameter 41 mm, height 45.7 cm) using the following technique . A plug of glass wool is inserted into the constriction of the stopper between it and the column. A 1 cm layer of standardized Ottawa sand is deposited on the glass wool.

"

The air from the plug, the glass wool plug and the sand is removed by passing Skellysolve B under pressure (0.39 bar). The dispersion is then introduced into the column and it is left to form CD. MJ _ 7fi - a bed during pressure flow. The column is never allowed to dry out. After obtaining a stable bed in the column, a 2 cm layer of Ottawa sand is placed on top of the bed.

The bed is then eluted with another 600-700 ml of Skellysolve B. The bed is eluted with 500 ml of toluene. The toluene eluate is evaporated to dryness to collect 93 mg of residue B. By applying the procedure of Example 3, this partially purified BBM-1675 A ^ can be further purified.

EXAMPLE 5

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

The variant A1327Y, obtained from Actinomadura verrucosospora No. H964-92 by treatment with NTG, is used to inoculate a vegetative medium containing 2% soluble starch, 1% glucose, 0.5% yeast extract. , 0.5% NB amine type A and 0.1% CaCOg, the pH being adjusted to 7.0 before sterilization. The vegetative culture is incubated at 32 ° C for 4 days in a rotary shaker (250 rpm) and 5 ml of the culture are transferred into a 500 ml Erlenmeyer flask containing 100 ml of a medium of fermentation comprising 3% cane molasses, 1% corn starch, 1% fish meal, 0.005% CuSO ^ .Sf ^ O, 0.05% MgS04.7H20 and 0.1% CaC03, the pH being adjusted to; 7.0 before sterilization.

Fermentation is carried out at 28 ° C for s * 7 days on a rotary shaker. The tibiotic production reaches a maximum of about 1.5 µg / ml. EXAMPLE 6 /

Isolation and purification of the components of BBM-1675 The fermentation broth collected in Example 5 (3000 liters, pH 7.6) is separated into a CD.mj - 77 - mycelial cake and a supernatant, using '' a Sharpless centrifuge. The mycelial cake is stirred with 2000 liters of methanol for 1 hour and the insolubles are separated by filtration. The activity contained in the supernatant liquid is extracted into 1800 liters of n-butanol. The extracts in methanol and n-butanol are combined and concentrated by azeotropy with occasional addition of water to obtain an aqueous solution (20 liters) which deposits most of the antibiotic in the form of an oily solid . The mixture is stirred three times with 20 liters of ethyl acetate each time to extract the activity. The extracts are collected, the insolubles are separated by filtration and they are evaporated under vacuum to 4 liters. The concentrate is poured into 30 liters of n-hexane with stirring to obtain the crude BBM-1675 complex in the form of a pale yellow solid (81.7 g, title: 59 μg / mg). This complex turns out to be CCM and HPLC a mixture of two major components, namely BBM-1675 and Ä2, and a few minor components. They are separated and purified by a series of chromatographies which are carried out in a cold room to avoid deterioration.

The crude BBM-1675 complex (20 g) is dissolved in methanol (20 ml) and placed on a column of Sephadex LH-20 (0 5.5 cm, height 85 cm). The column is developed in methanol and the elution is monitored by biological assay against Staphylococcu ^ v * aureus 209P. The active eluates are pooled, concentrated in vacuo and lyophilized to obtain the solid, semi-pure BBM-1675 complex (4.86 g, titer: 203 µg / mg). The solid is then chromatographed on a silica gel column (0 3.0 cm, height 70 cm) using chloroform supplemented with a healthy amount of CD.MJ - 78 - methanol (1 to 5%) as development solvent. The eluates are collected on the basis of antibacterial activity against £> _. aureus and on the basis of .CCM (S1O2, CHCl3-MeOH = 5: 1, v / v) and they are concentrated under vacuum. BBM-1675 A ^ (425 mg after evaporation, title: 960 μg / mg) is eluted first with 2% methanol in chloroform, then a mixture of BBM-1675 A £, A ^ and A is eluted. ^ (732 mg, titer: 340 ^ ug / mg). and then the BBM-1675 B complex (200 mg, titer: 190 µg / mg) with 3% methanol in chloroform.

BBM-1675 A ^ above is rechromatographed on silica gel (column 0 2.2 cm × 44 cm) with 2% methanol in benzene. The bioactive eluates are examined by HPLC (Lichrosorb RP-18: CH3CN-MeOH-0, 1MCH3COONH4 5: 2: 3, v / v) and the fractions containing homogeneous BBM-1675 A1 are concentrated to dryness. The residual residue is crystallized from methanol (10 ml) to obtain colorless prisms of BBM-1675 A ^ (197 mg, titer: 1000 µg / mg).

The complex of BBM-1675 A3, A3 and A4 (537 mg) is separated by chromatography on a column of

Bondapak C18 (Waters, 0 2.0 cm, height 42 cm). Elution is carried out with aqueous acetonitrile and the bioactive eluates are examined by TLC (Merck, silane: CH ^ N-t ^ O = 75:25, v / v). The minor components BBM-1675 A ^ (45 mg, titer: 410 ^, ug / mg) and A3 (19 mg, titer: 300 ^ ug / mg) are eluted successively with 20% keto-nitrile, then a major component, namely BBM-1675 A2 (203 mg), with 50% acetonitrile.

. 'The fraction of BBM-1675 A' is crystallized from v 2 chloroform-n-hexane to obtain a deposit of clear colorless sticks (70 mg, title: 290 ^, ug / mg). The solid containing the mixture of BBM-1675 B is chromatographed on a column of silica gel (0 3.0 cm, height 40 cm) with chloroform and methanol as de-CD.MJ-79 solvent. The active fractions eluted with 4% methanol in chloroform are combined and evaporated to obtain pure BBM-16 75 B ^ (7 mg, title: 180 μg / mg). Another active fraction is eluted at a methanol concentration of 5% to obtain, by evaporation, BBM-1675 B £ (8 mg, title: 140 μg / mg).

s »CD.MJ - 80 -

Claims (23)

2. The anti-tumor antibiotic BBM-1675 which, in substantially purified form: (a) is present in white crystals; (b) is soluble in chloroform, ethyl acetate, acetone, ethanol and methanol, sparingly soluble in benzene and water and insoluble in n-hexane and carbon tetrachloride; - (c) gives a positive reaction with ferric chloride and the reagents of Ehrlich and Tollens and »a negative reaction in the tests of Sakaguchi, S? ninhydrin and anthrone; (d) has an infrared absorption spectrum (KBr) substantially as illustrated in FIG. 12; (e) in solution in CDClg, presents a proton magnetic resonance spectrum substantially like CD.MJ - 82 - as illustrated in FIG. 13; (f) has a melting point of about 147 to 149 ° C; 27 (g) has an optical rotation [“] β of -179.4e (c = 0.5, CHC13); (h) has an apparent molecular weight of 1248 as determined by mass spectroscopy; (i) has the approximate elementary composition 52.71% of carbon, 5.94% of hydrogen, 3.94% of nitrogen, 5 9.39% of sulfur and 28.01% (by difference) of oxygen ; (j) present on thin layer chromatography on silica gel an Rf of 0.71 with the solvent system CHCl ^ -CH ^ OH (5: 1 v / v) and present on thin layer chromatography on silica gel with phase inversion an Rf of 0.21 with the solvent system CH ^ CN-î ^ O (75:25 v / v); (k) in solution in methanol at a concentration of 0.02052 g / liter, presents the maximum absorption and absorptivities in the following ultraviolet: λ (nm) Max absorbtivities ___ 320 12.2 282 16, 3,252 26.2 214 25.8 without substantial modification by addition of an acid or a base; (l) exhibits, on high performance liquid chromatography, a retention time of 17.3 minutes on a column of silica gel with C18 phase inversion with the solvent system CH3CN-CH3OH-0.1M CH3COONH4 “(5: 2: 3 v / v); . (m) is effective in inhibiting the growth of bacteria and fungi of various species; (n) induces prophage in lysogenic bacteria, and (o) is effective in inhibiting the development of P-388 leukemia, L-1210 leukemia, B16 melanoma and Lewis lung carcinoma in mice. 3. The anti-tumor antibiotic BBM-1675 which: (a) is soluble in chloroform, ethyl acetate, acetone, ethanol and methanol, sparingly soluble in benzene and water and insoluble in n-hexane and carbon tetrachloride; (b) gives a positive reaction with ferric chloride and the Ehrlich and Tollens reagents and a negative reaction in the Sakaguchi, ninhydrin and anthrone tests; (c) has an infrared absorption spectrum (KBr) substantially as illustrated in FIG. 3; (d) in solution in CDCl 4, exhibits a proton magnetic resonance spectrum substantially as illustrated in FIG. 7; (e). has a melting point of about 125 to 127 ° C; 27 (f) has an optical rotation [(Χ] β of -161e (c = 0.5, CHC13); (g) has the approximate elementary composition 54.55% carbon, 6.46% hydrogen, 3 , 73% nitrogen, 7.49% sulfur and 27.77% (by difference) of oxygen; (h) presents, on thin layer chromatography on silica gel, an Rf of 0.72 with the solvent system CHCl ^ -CH ^ OH (5: 1 v / v) and presents, on thin layer chromatography on silica gel with phase inversion, an Rf of 0.28 with the solvent system CH ^ CN-ï ^ O - (75 : 25 v / v); (i) presents the absorption maxima in the ultranaviolet below after dissolution in methanol, HG1 0.01N-CH3OH and NaOH 0.01N-CH30H UV X nm (E ^) 253 (286) max 1 cm methanol 282 (158) 320 (122) / - * τ \ RJf TOA uv nm (EÎ *) 253 <287) max ± cm HCl 0,01N-CH3OH 282 (160) 320 (126) UV λ nm (E ^) 252 (280) max 1 cm c NaOH 0.01N-CH3OH 283 (162) 318 (120) * (j) present in high performance liquid chromatography a retention time of 8.0 minutes on one column C18 phase inversion silica gel with the solvent system CH3CN-CH3OH-0, IM CH3C00NH ^ (5: 2: 3 v / v); (k) is effective in inhibiting the growth of bacteria and fungi of various species; (l) induces prophage in lysogenic bacteria, and (m) is effective in inhibiting the development of P-388 leukemia in mice. 4. The anti-tumor antibiotic BBM-1675 which: (a) is soluble in chloroform, ethyl acetate, acetone, ethanol and methanol, sparingly soluble in benzene and water and insoluble in n-hexane and carbon tetrachloride; (b) gives a positive reaction with ferric chloride, the reagents of Ehrlich and Tollens and a negative reaction in the tests of Sakaguchi, with ninhydrin and with 1'anthron; (c) has an infrared absorption spectrum (KBr) substantially as illustrated in FIG. 4; s (d) in solution in CDC13, exhibits a proton magnetic resonance spectrum substantially as illustrated in FIG. 8; (e) has a melting point of about 123-126 ° C; 27 (f) has an optical rotation [οί] β of -176 ° (c = 0.5, CHC13); (g) has the approximate elementary composition 54.65% of carbon, 6.29% of hydrogen, 3.51% of nitrogen, 8.07% of sulfur and 27.48% (by difference) of oxygen; (h) present on thin layer chromatography on silica gel an Rf of 0.71 with the solvent system CHCl ^ -CH ^ OH (5: 1 v / v) and present on thin layer chromatography on silica gel with phase inversion an Rf of 0.78 with the solvent system CH ^ CN-t ^ O (75: 25 v / v); (i) presents the absorption maxima in the ultra violet below after dissolution in methanol, 0.01N-CH3OH HCl and NaOH 0.01N-CH3OH UV λ nm (E ^) 253 (257) max 1 cm methanol 282 (153) 320 (117) UV> nm (E **) 253 (258) max 1 cm HCl 0.01N-CH3OH. 282 (155) 320 (118) UVA nm (eJ *) 252 (266 ) max 1 cm 0.01N NaOH-CH3OH 283 (160) 318 (118) (j) present in high performance liquid chromatography a retention time of 5.1 minutes on a column of silica gel with phase inversion C ^ g with the solvent system CH ^ N-CHgOH-0.1M CH3C00NH4 (5: 2: 3 v / v); - (k) is effective in inhibiting the growth of bacteria and fungi of various species; 9 (l) induces prophage in lysogenic bacteria, and (m) is effective in inhibiting the development of P-388 leukemia in mice. rn m.t _ a fi _ f f 5. The anti-tumor antibiotic BBM-1675 B ^ which: (a) is soluble in chloroform, ethyl acetate, acetone, ethanol and methanol, sparingly soluble in benzene and water, and insoluble in n-hexane and carbon tetrachloride; (b) gives a positive reaction with ferric chloride and the reagents of Ehrlich and Tollens and a negative reaction to the tests of Sakaguchi, to ninhydrin and to the anthone; (c) a melting point of about 159 to 161 "C; (d) has an optical rotation of -171 ° (c = 0.5, CHC13); (e) present on thin layer chromatography on silica an Rf of 0.63 with the solvent system CHClj -CH '^ OH (5: 1 v / v) and presents, at thin layer chromatography on silica gel with phase inversion, an Rf of 0.23 with the system solvent CH ^ CN-i ^ O (75: 25 v / v); (f) shows the maximum absorption in the ultraviolet below after dissolution in methanol, HCl 0.01N-CH3OH and NaOH 0 , 01N-CH3OH ÜV λ nm (E? · *) 253 (225) max 1 cm methanol 282 (140) 320 (104) ÜV λ nm (E ^, 253 (225) max 1 cm) HCl 0.01N-CH3OH 282 (140) 320 (105) - ÜV Λ nm (E ^) 252 (236) max 1 cm NaOH 0.01N-CH3OH 283 (141) 318 (105) (g) is effective in inhibiting the growth of bacteria and fungi of various species, and CD.MJ - 87 - (h) induces prophage in lysogenic bacteria. 6. The anti-tumor antibiotic BBM-1675 B £ which: (a) is soluble in chloroform, ethyl acetate, acetone, ethanol and methanol, sparingly soluble in benzene and water and insoluble in n-hexane and carbon tetrachloride; (b) gives a positive reaction with ferric chloride and the reagents of Ehrlieh and Tollens and -, a negative reaction in the tests of Sakaguchi, with ninhydrin and with the anthone; (c) a melting point of about 156 to 159 ° C; 27 (d) has an optical rotation of -122e (c = 0.5, CHC13); (e) present on thin layer chromatography on ge-1 of silica an Rf of 0.60 with the solvent system CHClg-CH ^ OH (5: 1 v / v) and present on thin layer chromatography on gel of phase inversion silica an Rf of 0.16 with the solvent system Cf ^ CN-t ^ O (75:25 v / v); (f) presents the absorption maxima in the following ultraviolet after dissolution in methanol, 0.01N-CH3OH HCl and NaOH 0, OlN-CH3OH ÜV-X nm (E? *) 248 (212) max 1 cm methanol 279 (141) 318 (103) UV λ nm (E ^) 248 (210) max 1 cm HCl 0,01N-CH3OH 279 (140) 318 (103) ϋνλ nm (E ^) ΓΠ3.Χ X Crn oxo / OOO \ NaOH 0, ISO-CH_0H 248 (Z33) 0 278 (150) 318 (110) (g) is effective in inhibiting the growth of bacteria and fungi of various species, and AL (h) induces prophage in lysogenic bacteria. 7. Method for producing the anti-tumor antibiotic BBM-1675 A ^, characterized in that a strain of Actinomadura verrucosospora produc- is cultivated; of BBM-1675 A ^ in an aqueous nutrient medium containing assimilable sources of carbon and a-nitrogen under aerobic conditions under submersion until production of a significant quantity of BBM-1675 A ^ per l organism in the culture medium, then the BBM-1675 A ^ is isolated from the culture medium. 8. Method according to claim 7, characterized in that the producer organism of BBM-1675 has the identification characteristics of Actinomadura verrucosospora strain H964-92 (ATCC 39334), of Actinomadura verrucosospora strain A1327Y (ATCC 39638) or of a mutant of these. 9. Method for producing the anti-tumor antibiotic BBM-1675 A ^, characterized in that a strain of Actinomadura verrucosospora producing BBM-1675 A ^ is cultivated in an aqueous nutritive medium containing assimilable sources of carbon and nitrogen under aerobic conditions under submersion until a significant amount of BBM-1675 A ^ is produced by the organism in the culture medium, then the BBM-1675 A ^ <3u culture medium is isolated . 10. Method according to claim 9, characterized in that the organism producing BBM-1675 A ^ has the identifying characteristics of Actinomadura verrucosospora strain H964-92 (ATCC 39334), of Actinoma- - * dura verrucosospora strain A1327Y (ATCC 39638) or a mutant thereof. " 11. Method for producing the anti-tumor antibiotic BBM-1675 A ^, characterized in that a strain of Actinomadura verrucosospora producing BBM-1675 A ^ is cultivated in an aqueous nutritive medium CD.MJ - 89 - § f ! fi containing assimilable sources of carbon and nitrogen under aerobic aerosol conditions until production of a substantial amount of BBM-1675 A ^ per 1 Organism in the culture medium, then the BBM- is isolated 1675 A ^ of the culture medium. 12. Method according to claim 11, characterized in that the producer organism of BBM-1675 A ^ has the identifying characteristics of Actinomadura verrucosospora strain H964-92 (ATCC 39334), of Actinomadura verrucosospora strain A1327Y (ATCC 39638 ) or a mutant thereof. 13. Process for producing the anti-tumor antibiotic BBM-1675 A ^, characterized in that a strain of Actinomadura verrucosospora producing BBM-1675 A ^ is cultivated in an aqueous nutritious medium containing assimilable sources of carbon and nitrogen under aerobic conditions under submersion until a substantial amount of BBM-1675 A4 d is produced by the organism in the culture medium, then BBM-1675 A. is isolated from the culture medium. 4 14. Method according to claim 13, characterized in that the producer organism of BBM-1675 A ^ has the identifying characteristics of Actinomadura verrucosospora strain H964-92 (ATCC 39334), of Actinomadura verrucosospora strain A1327Y (ATCC 39638 ) or a mutant thereof. 15. Process for producing the anti-moral antibiotic C BBM-1675 B ^, characterized in that one cultivates a strain of Actinomadura verrucosospora produc--; of BBM-1675 B ^ in an aqueous nutrient medium containing assimilable sources of carbon and nitrogen under aerobic aerosol conditions until the body produces a substantial amount of BBM-1675 B1 the culture medium, then the BBM-1675 B ^ is isolated from the culture medium. * CD .MJ - Qfl - \ i J i i * 16. The process as claimed in claim 15, characterized in that the producer organism of BBM-1675 B1 has the identifying characteristics of Actinomadura verrucosospora strain H964-92 (ATCC 39334), of Actinomadura verrucosospora strain A1327Y (ATCC 39638 ) or a mutant thereof. 17. Method for producing the anti-tumor antibiotic BBM-1675 B2, characterized in that a strain of .Actinomadura verrucosospora producing BBM-1675 B2 is cultivated in an aqueous nutritive medium containing assimilable sources of carbon and nitrogen in the aerobic conditions under submersion until the body produces a significant amount of BBM-1675 B2 in the culture medium, then the BBM-1675 B2 is isolated from the culture medium. 18. Method according to claim 17, characterized in that the producer organism of BBM-1675 B2 has the identification characteristics of d1Actinomadura verrucosospora strain H964-92 (ATCC 39334), of Actinomadura verrucosospora strain A1327Y (ATCC 39638) or d 'a mutant of these. 19. A method of therapeutic treatment of a host animal carrying a microbial infection, characterized in that the host is administered, in an effective antimicrobial dose, BBM-1675 A ^, BBM-1675 A ^, BBM-1675 A-, BBM-1675 A., BBM-1675 B1 or BBM-1675 B2. 20. A method of therapeutic treatment of a host animal carrying a malignant tumor, characterized - in that> the host is administered, in a dose effective for inhibiting the tumor, BBM-16 75 A ^, BBM- 16 75 A2, BBM-1675 A3, BBM-1675 k ^, BBM-1675 B ± or BBM-1675 B2. 21. Pharmaceutical composition, characterized in that it comprises, in an antimicrobial amount r * r \ Mt QT ΐ effective, of BBM-1675 Αχ, of BBM-1675 A2, of BBM-1675 A ^, I of BBM-1675 A4 , BBM-1675 B ^ or BBM-1675 B2 together with a pharmaceutical excipient or diluent. 22. Pharmaceutical composition, characterized in that it comprises, in an amount effective for inhibiting a tumor, BBM-1675 A ^, BBM-1675 A ^, BBM-1675 A3, BBM-1675 A4, BBM-1675 B1 OR * BBM-1675 B2 together with a pharmaceutical excipient or diluent. 23. Biologically pure culture of the microorganism Actinomadura verrucosospora strain H964-92 (ATCC 39334), characterized in that it is capable of producing the antibiotic BBM-1675 in an iso-lable amount per culture in an aqueous nutritive medium containing assimilable sources carbon and nitrogen. 24. Biologically pure culture of the microorganism Actinomadura verrucosospora strain A1327Y (ATCC 39638), characterized in that it is capable of producing the antibiotic BBM-1675 in an iso-lable amount per culture in an aqueous nutritive medium containing assimilable sources of carbon. and nitrogen. u 9