FI67403C - ADJUSTMENT OF ANTIBODY FRAMEWORK - Google Patents

Description

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Patent- och registrarstyralsan Ammuu ικΐφΐ odi ucLskrift · * puMiceratf 30.11.o4 (32) (33) (31) , New York, New York 10022, USA (72) Hideo Koshiyama, Tokyo, Fumihide Sakai, Yokohama,

Hiroaki Ohkuma, Tokyo, Japan-Japan (jP) (74) Oy Kolster Ab (54) Method for the preparation of antibiotic compounds against tumors -Förfarande för framställning av antibiotföreningar med antitumöreffekt

The invention relates to a process for the preparation of the novel antitumor antibiotic compounds BBM-928A, BBM-928B, BBM-928C and BBM-928D and a BBM-928 complex containing them, wherein (A) BBM-928A has the following properties: (a) it is soluble chloroform and methylene chloride, some benzene, ethanol, methanol and n-butanol, and is substantially insoluble in water and n-hexane; (b) it reacts positively with ferric chloride and Ehrlich reagents and reacts negatively with Tollens, Sakaguchi and ninhydrin reagents; (c) it is an effective antitumor agent against the following tumors implanted intraperitoneally in mice: P388 leukemia, L1210 leukemia, B16 melanoma, Lewis lung carcinoma and sarcoma 180 ascites; (d) it has a melting point of 246-248 ° C; 67403 - 25 o (e) its specific rotation is / * 7D - “27 (c 1, CHCl 3); (f) it has a molecular weight of 1427; (g) its Elemental Analysis is about 52.47% carbon, 5.48% hydrogen, 13.81% nitrogen and 28.24% oxygen; (h) its Rf value in silica gel thin layer chromatography is 0.71 for the solvent system n-butanol-methanol-water (63:27:10) and 0.48 for the solvent system xylene-methyl ethyl ketone-methanol (5: 5: 1); (i) acid hydrolysis yields water-soluble nin-hydrin-positive substances such as β-hydroxy-N-methylvaline, glycine, serine and sarcosine; (j) by base hydrolysis, a fragment of the formula “Tr ^ r08

Ser-> X ^ Gly- ^ s · Sar- ^ HM-Val wherein Ser is serine, Gly is glycine, Sar is sarcosine, HM-Val is β-hydroxy-N-methylvaline and X is an amino acid group of the gross formula in peptide form is C ^ Hg ^ C ^; (k) its infrared spectrum in potassium bromide is substantially shown in Figure 1; and (l) dissolved in deuterated chloroform, it has substantially the proton NMR spectrum shown in Figure 5; (B) BBM-928B has a quinoline chromophore and acid hydrolysis yields water-soluble components such as serine, glycine, sarcosine and p-hydroxy-N-methylvaline, and in substantially pure form has the following properties: (a) it is soluble in chloroform and methylene chloride; somewhat in benzene, ethanol, methanol and n-butanol, and is substantially insoluble in water and n-hexane; (b) it reacts positively with ferric chloride and Ehrlich reagents and reacts negatively with Tollens, Sakaguchi and ninhydrin reagents; (c) it is effective against P 388 leukemia implanted intraperitoneally in mice; (d) it has a melting point of 214-217 ° C; (e) its specific rotation is = -74 ° (c 1, CHCl 3); 3 67403 (f) its Elemental Analysis is about 50.14% carbon, 5.29% hydrogen, 12.34% nitrogen and 32.23% oxygen; (g) its Rf value in silica gel thin layer chromatography is 0.53 for the solvent system n-butanol-methanol-water (63:27:10) and 0.26 for the solvent system xylene-methyl ethyl ketone-methanol (5: 5: 1); (h) its infrared spectrum in potassium bromide is substantially shown in Figure 2; and (i) when dissolved in deuterated chloroform, it has substantially the proton NMR spectrum shown in Figure 6; (C) BBM-928C has a quinoline chromophore and acid hydrolysis yields water-soluble components such as serine, glycine, sarcosine and p-hydroxy-N-methylvaline, and in substantially pure form has the following properties: (a) it is soluble in chloroform and methylene chloride, some benzene, ethanol, methanol and n-butanol, and is substantially insoluble in water and n-hexane; (b) it reacts positively with the ferric chloride and Ehrlich reagents and reacts negatively with the Tollens, Sakaguchi and ninhydrin reagents; (c) it is effective against the following tumors implanted intraperitoneally in mice: P388 leukemia, L1210 leukemia, B16 melanoma, Lewis lung carcinoma and sarcoma 180 ascites; (d) it has a melting point of 244-248 ° C; (e) its specific rotation is / ot_7p5 = -91 ° (c1, CHCl3); (f) it has a molecular weight of about 1470; (g) its Elemental Analysis is about 51.77% carbon, 5.29% hydrogen, 13.55% nitrogen and 29.39% oxygen; (h) its R 2 value in silica gel thin layer chromatography is 0.27 for the solvent system n-butanol-methanol-water (63:27:10) and 0.07 for the solvent system xylene-methyl ethyl ketone-methanol (5: 5: 1); (i) its infrared spectrum in potassium bromide is substantially shown in Figure 3; and (j) dissolved in deuterated chloroform, it has substantially the proton NMR spectrum shown in Figure 7; and (D) BBM-928D has the following properties: 4 67403 (a) it is soluble in chloroform and methylene chloride, to some extent in benzene, ethanol, methanol and n-butanol, and is substantially insoluble in water and n-hexane; (b) it reacts positively with ferric chloride and Ehrlich reagents and reacts negatively with Tollens, Sakaguchi and ninhydrin reagents; (c) it is effective against P388 leukemia implanted intraperitoneally in mice; (d) it has a melting point of 224-227 ° C; (e) it has a specific rotation of λ 7 D = 13 13 ° (c 1, CHCl 3); (f) its Elemental Analysis is about 50.75% carbon, 5.25% hydrogen, 12.58% nitrogen and 31.42% oxygen; (g) its R 2 value in silica gel thin layer chromatography is 0.73 for the solvent system n-butanol-methanol-water (63:27:10) and 0.53 for the solvent system xylene-methyl ethyl ketone-methanol (5: 5: 1); (h) its infrared spectrum in potassium bromide is substantially shown in Figure 4; (i) dissolved in deuterated chloroform, it has substantially the proton NMR spectrum shown in Figure 8.

Based on the spectra shown and the available physicochemical properties, the antitumor antibiotic compounds in question are close in structure to quinoxaline antibiotics such as ecinomycin, Dell et al., J. Am. Chem. Soc., 97, 2497 (1975), quinomycins,

Shoji et al., J. Antibiotics, 14a, 335 (1961), and triostine C, The Merck Index, 9th Edition, 9399. However, the antitumor antibiotic compounds now described differ from these antibiotics as follows: 1. The antibiotic complex now prepared and its components contain a quinoline group as a chromophore instead of an actinoleukine group instead of quinoxaline contained in antibiotics.

2. The antibiotic complex now prepared and its components do not contain sulfur, whereas actinoleukin antibiotics instead have disulfide or thioacetal bridges.

3. The antibiotic complex now prepared and its components are relatively weakly antimicrobial compared to actinoleukins, which have a strong antibacterial effect.

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The novel antitumor antibiotic compounds BBM-928A, BBM-928B, BBM-928C and BBM-928D and the BBM-928 complex containing them are prepared according to the invention by culturing Actino-mycetes strain ATCC 31491 in an aqueous solution containing an assimilable carbon source and an assimilable carbon source. a nitrogen source, under submerged aerobic conditions, until the solution containing the formed BBM-928 complex shows substantial antitumor antibiotic activity, after which the BBM-928 complex is recovered from the culture medium and, if desired, BBM-928A, BBM-928B, BBM-928C and BBM-928C -928D is separated. After recovery of the BBM-928 complex, the bioactive components contained in the complex can be separated using countercurrent distribution and chromatography techniques.

Figure 1 shows the infrared absorption spectrum of BBM-928A in KBr.

Figure 2 shows the infrared absorption spectrum of BBM-928B in KBr.

Figure 3 shows the infrared absorption spectrum of BBM-928C in KBr.

Figure 4 shows the infrared absorption spectrum of BBM-928D in KBr.

Figure 5 is a proton magnetic resonance spectrum of BBM-928A dissolved in deuterated chloroform as determined by NMR spectrometry, frequency 90 MHz, using TMS as an internal standard.

Figure 6 is a proton magnetic resonance spectrum of BBM-928B dissolved in deuterated chloroform as determined by NMR spectrometry, frequency 90 MHz, using TMS as an internal standard.

Figure 7 is a proton magnetic resonance spectrum of BBM-928C dissolved in deuterated chloroform as determined by NMR spectrometry, frequency 90 MHz, using TMS as an internal standard.

Figure 8 is a proton magnetic resonance spectrum of BBM-928D dissolved in deuterated chloroform as determined by NMR spectrometry, frequency 90 MHz, using TMS as an internal standard.

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The new BBM-928 complex is believed to be structurally close to actinoleukin antibiotics and has been prepared by culturing strain G455-101 of an actinomycete microorganism deposited in the Bristol-Banyu culture collection. The microorganism was isolated from a soil sample from the Philippines and deposited with the American Type Culture Collection under the accession number ATCC 31491.

The new antitumor antibiotic complex contains at least six components designated BBM-928A, B, C, D, E and F. Components A, B, C and D of BBM-928 are isolated as crystals and the structures of components A, B and C has been identified. The complex and its various components have antibacterial activity against tumors. Due to its antibacterial activity, the complex and its components can be used as additives in animal feed and as therapeutic agents in the treatment of bacterial infections in mammals. In addition, antibiotics can be used to clean and sterilize laboratory glassware and surgical instruments, and in combination with soaps, detergents, and washing solutions for cleaning purposes. The complex and its components have been shown to have antitumor activity, especially against various tumors implanted intraperitoneally in mice.

Actinomycete strain No. G455-101

The following is a general description of a microorganism producing the antibiotic complex BBM-928 against tumors. Observations were made on the culture conditions and physiological and morphological characteristics of the micro-organism according to standard taxonometric methods, cf. e.g., Shirling et al.,

Int. J. Syst. Bacteriol. 16, 313 (1966) and Lechevalier et al., Biol. Actinomycetes Related Org. 11, 78 (1976).

Micromorphology - Strain G455-101 forms both culture medium and aerial mycelium, and the culture medium mycelium is well developed, long and branched (thickness 0.5-0.8 μm). No clear fragment formation is observed in the culture medium mycelium. Unlike common Streptomyces species, strain G455-101 has only short, atrophied air mycelium, and on some agar media it does not form them at all. The Ilmarih mast has short and long spore chains containing 2-50 oval 7 67403 spores (mostly 5-20 spores) in the chain. The spore chains are straight, curved or loop-like in shape. The spores are spherical in shape (0.3 to 0.4 μm), oval or cylindrical (0.3 x 1.5 to 3.0 μm) and have a smooth surface. Spores are often separated by empty hyphae. An amorphous sporangin-like vesicle is sometimes observed in the aerial mycelium, from which short helical spore chains develop.

Cell wall composition and whole cell sugar components - The cell wall of strain G455-101 contains meso-diaminopimelic acid but does not contain glycine. The whole cell hydrolyzate contains glucose, mannose and madurose (3-O-methyl-D-galactose). The cell wall composition and whole cell sugar components indicate that strain G455-101 belongs to the cell wall type IIIB of actinomycetes.

Cultivation and physiological properties - Strain G455-101 grows abundantly, forming a pink or greyish pink aerial mycelium and producing a reddish water-insoluble pigment in nutrient-rich agar media such as yeast extract-malt extract agar and oatmeal agar. However, its growth is weak in starch agar containing inorganic salts, glycerol-asparagine agar, and tyrosine agar, where it forms a white or beige atrophied air mycelium and produces a small amount of reddish pigment. Peptone-yeast iron agar and tyrosine agar do not form melanoid pigment. Nitrate is reduced to nitrate. It grows profusely at 28 ° C, 37 ° C and 45 ° C, but does not grow at 10 ° C or 50 ° C. The strain makes good use of pentoses and hexoses. The culture characteristics of strain G455-101 are shown in Table 1 and the physiological properties in Table 2. The utilization of carbon sources is shown in Table 3.

table 1

Cultivation characteristics of strain G455-101A

XX

1. Czapek agar G no growth or weak growth (sucrose nitrate) '.

R dark rose red A white or pink D none 2. Tryptone yeast extract broth moderate growth, fluffy (ISP No. 1), sedimented, no pigment 8 67403 3. Yeast extract - malt extract agar G rich (ISP No. 2) „..

R deep reddish-brown A abundant, greyish light red to purple D none 4. Oatmeal agar (ISP No. 3) G abundant R strong yellowish red A moderate, pink D greyish-yellow 5. Inorganic salts - G weak starch - G weak starch from brownish brown to dark red A weak, white or beige D none 6. Glycerol-asparagine agar G weak (ISP No. 5) R yellowish pink to reddish A weak, white D none 7. Peptone-yeast extract-iron G weak, corrugated agar (ISP No. 5) 6) R strong reddish orange A is not D light yellowish orange 8. Tyrosine agar (ISP No. 7) G weak R dark red A weak, white D is not 9. Glucose-ammonium salt.lat- G weak a <3ar r reddish brown A weak, light gray D none 10. Bennet1 in agar G moderate R reddish brown A limited, greyish pink D not 9 67403 χ observations made after three weeks incubation at 37 ° C xx Abbreviations: G - k rowth R - substraattirihmaston underside of color A - D aerial mycelium - a diffusible pigment

Table 2

Physiological properties of strain G455-101

Experiment Result Method and medium

Nitrate nitrile positive inorganic medium: Czapek sucrose nitrate broth

Nitrate to nitrite positive organic medium: 0.5% yeast extract, 1.0% glucose, 0.5% KNO3, 0.1% CaCO3

Casein hydrolysis weakly Luedemann1 in agar medium positive in agar medium

Positive coagulation of skimmed milk

Gelatin liquefaction-negative tryptone-yeast extract broth (ISP

medium No. 1) + 15% Gela tine

Production of H2S by positive tryptone-yeast extract (ISP

cysteine # 1 medium) + 0.1% L-cysteine + agar. H2S was detected on a paper strip containing 10% lead acetate solution

Melanoid formation-negative peptone-yeast extract-iron agar (ISP No. 6) and tyrosine agar (ISP No. 7)

Catalase reaction positive H2O2 ~ aqueous solution

Oxidase reaction positive Kovacs reagent

Growth temperature abundant growth on Bennet agar at 28, 37 and 45 ° C Poor growth at 20 ° C. Does not grow at 10 ° C or 50 ° C 10 67403

Table 3

Utilization of carbon sources of strain G455-101 PG Lm 1. Glycerol ++ + 2. D (-) - arabinose + + 3. L (+) - arabinose + + 4. D-xylose ++ + 5. D-ribose ++ + 6. L-rhamnose ++ + 7. D-glucose + + 8. D-galactose ++ + 9. D-fructose ++ + 10. D-mannose ++ + 11. L (-) - sorbose 12. Sucrose 13. Lactose - 14. Cellobiose + + 15. Melibiosis 16. Trehalose + + 17. Raffinose 18. D (+) - melecitose 19. Soluble starch + 20. Dulsitol 21. Inositol + 22. D-mannitol ++ + 23. D-sorbitol 24. Salicin 25. Cellulose + + 26. Chitin + + 27. Wheytin + +

Base medium PG: Pridham-Gottlieb inorganic medium + 0.1% yeast extract LM: Luedemann's organic medium Incubation for two weeks at 37 ° C.

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Preparation of the antitumor antibiotic complex BBM-928

To prepare the antitumor antibiotic, BBM-928 complex, an actinomycete strain having the characteristics of strain G455-101 is cultured in an aqueous solution containing an assimilable carbon source and an assimilable nitrogen source under said submerged aerobic conditions until the BBM-92 complex is produced. received antibiotic activity against tumors. Conventional fermentation methods are used to cultivate the actinomycete strain G455-101. In the preparation of antitumor antibiotics, media containing, for example, starch, glucose, dextrin, maltose, lactose, sucrose, fructose, mannose, molasses, glycerol, etc. are used as sources of assimilable carbon. , amino acids, corn broth, casein, urea, etc., and inorganic nutrient salts to give inorganic anions and cations such as potassium, sodium, ammonium, calcium, sulfate, carbonate, phosphate, chloride, nitrate, and the like.

Any culture temperature at which the organism grows satisfactorily can be used to produce the BBM-928 complex. The culture can be performed at about 20-45 ° C, with a temperature range of 28-34 ° C being preferred for optimal growth and 30-32 ° C being most preferred. The maximum yield of the BBM-928 complex is usually obtained in about 4-6 days. Conventional methods are used for fermentation. Small amounts are prepared, for example, in conventional shake flasks or surface cultures. The preparation of larger amounts is preferably performed under submerged aerobic culture conditions in sterile tanks. In tank fermentation, a vegetative inoculum is first produced in the culture broth by inoculating the spores of the organism into the culture broth to obtain a young active inoculum culture, which is then aseptically transferred to the medium in the fermentation tank. Aeration in the tank and bottles can be performed by introducing sterile air into the fermentation medium 12 67403 while stirring the contents of the tank with a mechanical stirrer. If necessary, a defoamer such as silicone oil, soybean oil or lard oil can be used.

The anti-biotic level of the fermentation broth or BBM-928 complex extract can be determined by a paper disc agar diffusion test using the test organism Sareina lute and the test medium as nutrient agar. To achieve the optimum sensitivity of the test system used to determine the best performance of the broth, its pH is adjusted to 9.0.

The BBM-928 complex is isolated from the fermentation broth by conventional methods such as solvent extraction. Purification is conveniently performed by preparative countercurrent distribution and chromatography methods, as described in more detail in Examples 2 and 3 below, to give BBM-928 components A, B, C, D, E and F.

Physicochemical Properties of Components A, B, C and D of Example 3 BBM-928 The individual components of BBM-928 all have similar solubility properties and color reactions. For example, they are readily soluble in chloroform and methylene chloride, to some extent in benzene, ethanol, methanol and n-butanol, and are insoluble in water and n-hexane. Ferric chloride and Ehrlich reagents give positive reactions and Tollens, Sakaguchi and ninhydrin reagents give negative reactions.

Typical physicochemical properties of the BBM-928 components of Example 3 are shown in Table 4.

J

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Table 4 Physicochemical properties of BBM-928 components A, B, C and D _ BBM-928__

A B C D

Melting point 246-248 ° C 214-217 ° C 244-248 ° C 224-227 ° C

mp & p5 (c 1, CHCl 3) -27 ° -74 ° -91 ° -13 °

Analysis found C: 53.19 50.14 51.77 50.75 H: 5.40 5.29 5.29 5.25 N: 12.92 12, 34 13.55 12.58 (difference) O: 28.49 32.23 29, 39 31.42 \ naks' <E Ϊ L>

In EtOH 235 (586) 235 (570) 235 (638) 235 (550) 264 (415) 264 (400) 264 (442) 264 (380) 345 (165) 345 (163) 345 (173) 345 (155) )

In EtOH-HCl 234 (610) 234 (556) 234 (650) 234 (565) 264 (410) 264 (446) 264 (442) 264 (405) 345 (165) 349 (188) 345 (173) 345 (165)

In EtOH-NaOH 230 (564) 230 (530) 230 (580) 230 (650) 256 (763) 256 (775) 256 (704) 256 (930) 330 (180) 330 (116) 330 (117) 330 (140) 383 (170) 383 (122) · 83 (122) 383 (145)

Mol- p.

(osmometer, CHCl 3) 1.450 - 1.470

The infrared (IR) and NMR spectra of components A, B, C and D are shown in Figures 1-4 and 5-8, respectively. The NMR spectra of BBM-928A (Fig. 5), BBM-928B (Fig. 6) and BBM-928C (Fig. 7) are quite similar, with the only difference in the acetyl groups of components A and B (A, & : 2.03 ppm, 2 molecules) (B, &: 2.05 ppm, 1 molecule), which is not present in component C. By acetylation with acetic anhydride in pyridine BBM-928, 2, 3 and 4 molar equivalents of acetyl groups were added to components A, B and C, respectively. The acetylated compounds thus obtained had identical properties in thin-layer chromatography, UV, IR and NMR spectra, indicating that BBM-928A is a monoacetyl derivative of BBM-928B and a diacetyl derivative of BBM-928C.

Acid hydrolysis of BBM-928A yielded five n-butanol-soluble, UV-absorbing fragments (I, II, III, IV, and V) and five water-soluble, ninhydrin-positive substances (NPS-1, 2, 3, 4, and 5). . The latter were separated by chromatography (Dowex 50 x 4) and identified as the following amino acids:

Compound Rf (S-123) x Identification NPS-1 0.72-hydroxy-N-methyl medium (HM-Val) NPS-2 0.49 glycine (Gly). NPS-3 0.46 serine (Ser) NPS-4 0.45 sarcosine (Sar) NPS-5 0.23 (not specified) x TLC, silica gel plate S-123: 10% ammonium acetate-methanol-10% ammonia (9: 10: 1)

The above-mentioned UV-absorbing fragments were shown to have the following structures:

OH Fragment I

| CH-OH H H 0 O 2 14 M 14 N 2 O N CO-NH-GH-CO-H, 1 MS: m / e 306 (M) M MeOH; 227 233 251 345

Max 15 67403

CH ^ Fragment II

NH OH C20H25N3O8O-CO-CH-C (CH3) 2 MS; m / e 377 (M + -58)

If I ch2 CO-NH-CH-CO2H λϋ ^ Η; 229 · 234> 261 * 345 nm

Fragment III

CH 0 OH XMeOH: 230, 235, 262, 345 nm (NPS-5)

Fragment IV

C10H7NO4 δ0 \ ΓΧν ^ / ^ γ / ° Η MS: m / e 205 (M +) k

H, C CH, Fragment V

3w 3 C-OH C H N 0 * ^ 23H30 4 9

CH 0 H 0 -CO-CH «N-CO-CH2NH

3 T T CH 2 CH 3 CH 3 X 2 H: 230, 235, 262, 345 nm

N CO-M-CH-CO 2 H

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Acid hydrolysis of fragments I, II, III and V (6-n HCl) gave the following decomposition products:

The hydrolysis conditions

Fragment Sealed tube Heating, 110 °, 20 h reflux 110 ° C, 3 h I IV, Ser II IV, Ser, HM-Val I, HM-Val

III

V I, HM-Val, Sar

Ser: serine HM-Val: β-hydroxy-N-methylvaline Sar: sarcosine Base hydrolysis of BBM-928A or BBM-928C with 0.1 N NaOH at 25 ° C for 3 hours gave fragment VI ( the abbreviations Ser, HM-Val and Sar denote as defined above, Gly is glycine and the symbol "X" denotes an undefined group) oh (_lj ^ ^ _CCH'Ser -'- X- + Gly - * - Sar - * 'HM-Val

Fragment VI

Treatment of fragment VI with 1.1 N HCl at 110 ° C for 1 hour gave fragment VII + HM-Val.

0H

^ —CCH-Ser- ^ X-KJly ^ Sar 67403

Fragment VII

Treatment of fragment VI with 0.1 N NaOH at 37 ° C for 40 hours gave fragment VIII + fragment I.

X -> Gly -> Sar -> HM —Ä / al

Fragment VIII

Treatment of fragment VII with 0.1 N NaOH at 37 ° C for 40 hours gave fragment IX + fragment I.

X - »Gly -> Sar

Fragment IX

The undefined group X has the gross formula C5HgN2O2 (in peptide form) based on the microanalysis and specific analyzes of fragment IX and other X-containing fragments shown in the table below.

13 C-NMR Proton NMR Group 30.14 (t) 2.36 ppm (2H, m)? CH 2 44.2 - 4.5 (2H, m) 2 x -CH <^ 61/7 (d) ^ (0 or N) 140.7 (d) 6.76 (1H, t) -CH = N-171 (s) - -CO- (amide)

-OH

NH

Based on the spectral results of fragments VIII and IX and the proton NMR (360 MHz) of BBM-928Ap of Example 4, the undefined amino acid group X can best be represented by the following tetrahydropyridazine structure:

OH

(X)

IB

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Based on the experiments, spectra, microanalyses and molecular weight determinations performed on the degradation products described above, the structures of BBM-928A, B and C can best be described by the following formula:

_ OH

y ^) ~ 0h C0- "Ser- * N— / OGly-Sar- * HM-Val

0 On ^ HO

1 »; 1 3 HM-Val «-Sar * -Gly-C v | V-n * ^ er <-C0 R2 R2 Ser 5 serine

Gly: glycine BBM-928 A Ac Ac Sar: sarcosine BBM-928 B Ac H HM-Val: /} - hydroxy-N-methyl- BBM-928 C H H livalin

Antimicrobial Activity The antimicrobial activity of the BBM-928 components was determined by the agar dilution series method on nutrient agar at pH 7 against a variety of bacteria and fungi using a Steer multi-environment device. The inoculum size was standardized to 0.0025 ml of a test organism suspension containing about 10 cells / ml, except for acid-fast bacteria, which had a suspension of 106 cells / ml. Mini-male concentrations (MICs) were determined by overnight incubation at 37 ° C and are shown in Table 5. The results show that the components of BBM-928 are moderately active or weakly active against gram-positive and acid-fast bacteria. but practically inactive against grara-negative bacteria and fungi.

19 67403 Propagation induction activity of BBM-928 components in lysogenic bacteria (ILB) was determined. With BBM-928 components A, B and C, up to concentrations of 100 pg / ml, vbitu does not show substantial ILB activity.

Table 5 Antimicrobial activity of BBM-928 components against aerobic bacteria in vitro BBRE BBM-928 components (MIC ^ g / ml)

Code of the test organism_ X ö 5 E-F—

Sa-1 S. aureus 209P 12.5 25 50 100 100 25

Sp-1 S. pyogenes S-23 6.3 12.5 25 50 50 · 12.5 S1-1 S. lutea PCI 1001 6.3 12.5 50 50 50 25

Mf-1 M. flaws D12 12.5 25 50 100 100 25

Cr-1 xerosis 53K-1 25 50> 100> 100> 100 50

Bs-1 B ^ subtllis PCI 219 25 25 100 100 100 25

Bg-1 B ^ megaterium D2 25 25 50 100 100 25

Ba-3 B. anthracis A9504 6.3 12.5 25 50 50 12.5 M6-1 M. smegmatis 607 D87 25 25 25 100 100 25

Mp-1 M. phlei D88 12.5 12.5 12.5 50 50 12.5

Ec-1 _E; _ coli NIHJ> 100> 100> 100> 100> 100> 100

Kp-i K £ _ pneumoniae D-ll> 100> 100> 100> 100> 100> 100

Pa-3 aeruginosa A9930> 100> 100> 100> 100> 100> 100

Pv-1 P ^ vulgaris A9436> 100> 100> 100> 100> 100> 100

Po-1 Ll mirabilis A9554> 100> 100> 100> 100> 100> 100

Pg-1 P ^ _ morganii A9553> 100 '> 100> 100> 100> 100> 100

Sm-1 marcescens A20019> 100> 100> 100> 100> 100> 100

Al-1 A. faecalls ATCC 8750> 100> 100> 100> 100> 100> 100

Ca-1 albicans IAM 4888> 100> 100> 100> 100> 100> 100

The antitumor activity of Cn-3 Cneoformans 100> 100 100> 100> 100> 100 20 67403 BBM-928 components A, B, C and D and mitomycin C was studied by intraperitoneal implantation of the following in mice: P388 leukemia, L1210 leukemia, B16 melanoma , Lewis lung carcinoma (LL) and sarcoma 180 as-praise (S180). Test solutions of BBM-928 components and mitomycin C in 0.9% saline containing 10% dimethyl sulfoxide were administered to experimental animals once daily with a dosing regimen comprising one to several daily treatments. By varying the doses, the minimum effective dose (MED) was determined at which the mean survival time of the treated animals was at least 1.25 times that of the control group. This level of activity is considered a significant measure of antitumor activity. The results are summarized in Table 6, which also calculates activity ratios showing that BBM-928A is clearly more active than mitomycin C, with an activity ratio of 10-300 depending on tumor times. Intraperitoneal LD50 values for BBM-928 components A, B, C and D and mitomycin C were determined by the method described by Van der Waerden, Arch. Exp. Path. Pharmak., 195, 389 (1940). LD ^ g values are also shown in Table 6.

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Table 6 Antitumor activity and toxicity of BBM-928 components A, B and C and mitomycin C.

Minimum effective dose (MED, mg / kg / day) ^ D50 '*' p 'P388 L1210 B16 LL S180 (mg / kg / day) Treatment3 BBM-928A 0.003> 0.1 0.003 0.03 0.003 0.13 BBM- 928B 0,1 - - - 0,18 BBM-928C ----- 0,81 BBM-928D 0f003 - - - 0,083

Mitomycin C 0.1 3 1 0.3 0.3 9.3 Treatments BBM-928A 0.001 0.003 0.003 0.003 0.0001 0.013

Mitcmycin C 0.1 0.3 0.3 0.3 0.03 1.4

Activity ratio (BBM-928A / bitanysilnl C) Processing3 30 - 300 10 100 · Treatments 100 100 100 100 100 a. Single treatment on day 1 b. Daily treatments on days 1-9

Example 1 Production of BBM-928 complex

Agar Fermentation - A well-grown agar culture of strain G455-101 (ATOC 31491) belonging to the actinomycetes was used to inoculate growth medium containing 2% soluble starch, 1% glucose, 0.5% Pharmamedia, 0.5% yeast extract, 0.5% NZ-amine (type A) and 0.1% CaCO 3, and the pH of which was adjusted to 7.2 before sterilization. The seed culture was incubated for 72 hours at 32 ° C on a rotary shaker (250 rpm) and 5 ml of growth medium was then transferred to a 67403 500 ml Erlenmeyer flask containing 100 ml of fermentation medium containing 2% soluble starch, 1% Pharmamedia, 0.003% ZnSo 2 · 7H 2 O and 0.4% CaCO 2. In shake culture, the maximum yield of the BBM-928 complex was usually obtained in about 5 days.

Tankkl fermentation - The seed culture was shaken in Erlenmeyer flasks for 4 days, and the culture was then inoculated with culture medium (100 L) in a 200 liter tank fermentor containing 2.0% oatmeal (Quaker Products, Australia), 0.5% glucose, 0, 2% dry yeast, 0.0008% MnCl 2 * 4H 2 O 0/0007% CuSO 4 * 7H 2 O, 0.0002% ZnSO 2 • 7H 2 O and 0.0001% FeSO 2 · 7H 2 O, and the culture broth was stirred at 200 rpm at 30 ° C 54 hours. Thereafter, 15 liters of seed culture was used to inoculate 170 liters of fermentation medium containing 2.0% soluble starch, 1.0% Pharmamedia, 0.003% ZnSO 2 H 2 O and 0.4% CaCO 2 in a 400 liter tank fermentor, and the fermentation medium was mixed at 200 rpm. At ° C and conducting air at 150 rpm. As the fermentation continued, the pH of the broth gradually increased and reached a value of 8.4 to 8.5 in 100-120 hours, when the amount of antibiotic had reached its maximum value of 30 μg / ml.

Example 2 Isolation of BBM-928 complex by solvent extraction

The growth broth prepared in Example 1 (170 L, pH 8.5) was filtered using a filter aid. Activity was observed in both mycelium cake and filtrate. The mycelium cake was extracted twice with acetone-methanol (1: 1, 30 1 x 2). The extracts were combined and evaporated under reduced pressure, and the resulting aqueous concentrate was extracted with n-butanol. The broth filtrate was extracted twice with n-butanol (40 L x 2). The combined n-butanol extracts were evaporated under reduced pressure and lyophilized to give the crude product (21.4 g). According to thin layer chromatography, it was a complex containing three main components A, B, and C and minor components D, E, and F, the Rf values of which are shown in Table 7 below.

23 67403

Table 7

Silica gel TLC of BBM-928 components

Rf values

System N-118xx System N ~ 103XXX

BBM-928A 0.71 0.48 BBM-928B 0.53 0.26 BBM-928C 0.27 0.07 BBM-928D 0.73 0.53 BBM-92 8E 0.56 0.34 BBM-928F 0 .39 0.17 x was detected by UV detector (Shimadzu CS-910) at 345 nm xx n-butanol-methanol-water (63:27:10) xxx xylene-methyl ethyl ketone-methanol (5: 5: 1)

Example 3 Purification of the BBM-928 complex

The crude complex obtained in Example 2 was purified on a preparative countercurrent distributor (Mitamura, 100 ml / tube) using a carbon tetrachloride-chloroform-methanol-water solvent system (5: 2: 5: 1). After 50 transfers, the contents of tubes 5-20 were combined and evaporated to give a pale yellow powder (4.4 g) containing components A, B, D, E and F. This mixture was dissolved in a small amount of chloroform, and the solution was applied to a silica gel column pretreated with ethyl acetate (silica gel C-200, 500 ml). The column was eluted with ethyl acetate containing increasing amounts of methanol (2-5%, v / v) and the optical density of the fractions was monitored at 345 nm. First, the minor component D eluted with ethyl acetate and then component A. Components E, B and F eluted with a solvent mixture containing 3% methanol, respectively. Each fraction containing the various components was evaporated under reduced pressure, and the residue was crystallized from chloroform-methanol. From tubes 21-35, crude component C was obtained by saunas with a countercurrent-bending apparatus as described above. Purification of component C was performed by chromatography on silica gel and crystallization from a chloroform-methanol mixture. The yields of components A, B, C, D, E and 24 67403 F were 988 mg, 420 mg, 843 mg, 130 mg, 119 mg and 114 mg, respectively.

Example 4

Further purification of the BBM-928A component obtained in Example 3

Thin layer chromatographic analysis of the BBM-928A component obtained in Example 3 (toluene-methanol 9: 1 system was used) showed that it was not completely homogeneous but contained a substance slightly above the chromatographic run. To purify component A, the following steps were performed: (1) The sample was chromatographed on silica gel using a linear gradient of chloroform-6% methanol in chloroform. Fractions eluting with methanol concentrations of 2.4 to 3.3% (containing fractions containing BBM-928A and some impurities) were taken to the next purification step.

(2) Using three vessels, the first two containing 2% methanol in toluene and the third 6% methanol in toluene, a concave gradient was obtained. The material obtained in Step 1 was chromatographed (silica gel column) with this gradient, eluting first the minor component and immediately followed by the pure BBM-928A component (referred to herein as BBM-928A).

The molecular weight of BBM-928A was determined on a Field-Desorption Thomas spectrometer and was 1427, corresponding to the gross formula C64H78N14 ° 24 (mo4-ethylPa ^ no 1427,417).

Elemental analysis (samples dried at 100 ° C for 18 hours) from C64H78N14024: calculated: C 53.85 H 5.51 N 13.74 Oa 26.90 obtained * 5: c 52.47 H 5.48 N 13.81 28 , 24a a. Calculated from the difference b. Mean of the three determinations

Claims (1)

A process for preparing antitumor antibiotic compounds BBM-928A, BBM-928B, BBM-928C and BBM-928D and a complex containing BBM-928 containing them, wherein (A) BBM-928A has the following properties: (a) it is soluble in chloroform and methylene chloride, some benzene, ethanol, methanol and n-butanol, and is substantially insoluble in water and n-hexane; (b) it reacts positively with ferric chloride and Ehrlich reagents and negatively reacts with Tollens, Sakaguchi and ninhydrin reagents; (c) it is an effective antitumor agent against the following tumors implanted intraperitoneally in mice: P388 leukemia, L1210 leukemia, B16 melanoma, Lewis lung carcinoma and sarcoma 180 ascites; (d) it has a melting point of 246-248 ° C; (e) it has a specific rotation capacity of λ * -7 ° -27 ° (c 1, CHCl 3); (f) it has a molecular weight of 1427; (g) its Elemental Analysis is about 52.47% carbon, 5.48% hydrogen, 13.81% nitrogen and 28.24% oxygen; (h) its R 2 value in silica gel thin layer chromatography is 0.71 for the solvent system n-butanol-methanol-water (63:27:10) and 0.48 for the solvent system xylene-methyl ethyl ketone-methanol (5: 5: 1); (i) acid hydrolysis yields water-soluble ninhydrin-positive substances such as N, N-hydroxy-N-methylvaline, glycine, serine and sarcosine; (j) by base hydrolysis it gives a fragment of formula CH3 <Y ^ \ ^ WHH VI N __CO— * Ser * X— * Gly> Sar— »HM-Or where Ser is serine, Gly is glycine, Sar is sarcosine, HM -Val is β-hydroxy-N-methylvaline and X is an amino acid group of formula 26 67403 in peptide form CgHg ^ C ^; (k) its infrared spectrum in potassium bromide is substantially shown in Figure 1; and (l) dissolved in deuterated chloroform, it has substantially the proton NMR spectrum shown in Figure 5; (B) BBM-928B has a quinoline chromophore and acid hydrolysis yields water-soluble components such as serine, glycine, sarcosine and hydroxy-N-methylvaline, and in substantially pure form has the following properties: (a) it is soluble in chloroform and methylene clotide; benzene, ethanol, methanol and n-butahol, and is substantially insoluble in water and n-hexane; (b) it reacts positively with ferric chloride and Ehrlich reagents and reacts negatively with Tollens, Sakaguchi and ninhydrin reagents; (c) it is effective against P 388 leukemia implanted intraperitoneally in mice; (d) it has a melting point of 214-217 ° C; (e) its specific rotation is / λ max = -74 ° (c 1, CHCl 3); (f) its Elemental Analysis is about 50.14% carbon, 5.29% hydrogen, 12.34% nitrogen and 32.23% oxygen; (g) its Rf value in silica gel thin layer chromatography is 0.53 for the solvent system n-butanol-methanol-water (63:27:10) and 0.26 for the solvent system xylene-methyl ethyl ketone-methanol (5: 5: 1); (h) its infrared spectrum in potassium bromide is substantially shown in Figure 2; and (i) when dissolved in deuterated chloroform, it has substantially the proton NMR spectrum shown in Figure 6; (C) BBM-928C has a quinoline chromophore and acid hydrolysis yields water-soluble components such as serine, glycine, sarcosine and hydroxy-N-methylvaline, and in substantially pure form has the following properties: (a) it is soluble in chloroform and methylene chloride; benzene, ethanol, methanol and n-butanol, and is substantially insoluble in water and n-hexane; 27 67403 (b) it gives a positive reaction with ferric chloride and Ehrlich reagents and a negative reaction with Tollens, Sakaguchi and ninhydrin reagents; (c) it is effective against the following tumors implanted intraperitoneally in mice: P388 leukemia, L1210 leukemia, B16 melanoma, Lewis lung carcinoma and saikoma 180 ascites; (d) it has a melting point of 244-248 ° C; (e) its specific rotation is /V(.7 ^ 5 = -91 ° (c1, CHCl3); (f) it has a molecular weight of about 1470; (g) its Elemental Analysis is about 51.77% carbon, 5.29% hydrogen, 13.55% nitrogen and 29.39% oxygen, (h) its Rf value in silica gel thin layer chromatography is 0.27 for the solvent system n-butanol-methanol-water (63:27:10) and 0.07 for the solvent system xylene methyl ethyl ketone- methanol (5: 5: 1), (i) its infrared spectrum in potassium bromide is substantially shown in Figure 3, and (j) when dissolved in deuterated chloroform it has substantially the proton NMR spectrum shown in Figure 7, and (D) BBM-928D has the following properties: (a) it is soluble in chloroform and methylene chloride, to some extent in benzene, ethanol, methanol and n-butanol, and is substantially insoluble in water and n-hexane, (b) it reacts positively with ferric chloride and Ehrlich reagents and reacts negatively with With Tollens, Sakaguchi and ninhydrin reagents (c) it is an effective intraperitoneal prevented P388 implanted in mice against leukemia; (d) it has a melting point of 224-227 ° C; (O) its specific rotation is = "13 (c 1, CHCl 3); (f) its Elemental Analysis is about 50.75% carbon, 5.25% hydrogen, 12.58% nitrogen and 31.42% oxygen; (g) its Rf value in silica gel thin layer chromatography is 0.73 for the solvent system n-butanol-methanol-water (63:27:10) and 0.53 for the solvent system xylene-methyl ethyl ketone-methanol (5: 5: 1); 28 6 7 4 0 3 (h) its infrared spectrum in potassium bromide is substantially shown in Figure 4, and (i) when dissolved in deuterated chloroform it has substantially the proton NMR spectrum shown in Figure 8, characterized in that Actinomycetes strain ATCC 31491 is cultured in an aqueous solution containing an assimilable carbon source; an assimilable nitrogen source, under submerged aerobic conditions, until the solution containing the formed BBM-928 complex shows substantial antitumor, antibiotic activity, after which the BBM-928 complex is recovered from the culture medium and, if desired, BBM-928A, BBM-928B, BBM-928B, BBM-928B, C and BBM-928D are separated. 29 Patent application 6 74 0 3 For use in the preparation of antibiotic preparations BBM-928A, BBM-928B, BBM-928C and BBM-928D as well as the BBM-928 complex in the form of a solid, anti-tumor effect, colors (A) for BBM (eg) chloroform and methylene chloride, benzene, ethanol, methanol and n-butanol, and other olefins and n-hexane; (b) a positive reaction with ferric chloride and Ehrlich reagents and a negative reaction with Tollens, Sakaguchi and ninhydrin reagents; (c) having effective antitumor mice up to and including intraperitoneally implanted tumors: P388 leukemia, L1210 leukemia, B16 melanoma, Lewis lung carcinoma and sarcoid 180 ascites; (d) a melting point of 246- * 248 ° C; (e) the specific age of the reaction is about 27 ° (c 1, CHCl 3); (f) the molecular weight is 1427, · (g) the elemental analysis is about 52.47% mole, 5.48% whey, 13.81% whey and 28.24% sugar; (h) the Rf content was flash chromatographed on silica gel with 0.71 ml of a n-butanol-methanol solution (63:27:10) and 0.48 with a xylene methyl ketone-methanol (5: 5: 1); (i) vidyrohydrolys erhälles frän den vattenlösliga nin-hydridin-positiva ämnen, säsom ^ -hydroxy-N-methylvalin, glycin, serin och sarcosine? (j) by bashydrolysis of fractions and moieties of the formula OH Ser-3 X- ^ Gly-> Sar ^ HM — or a serine serine, a glycine glycine, a sarcosine, an HM-valine or N-methylvaline and X are amino groups, especially the peptide form of the gross form (s) in the infrared spectrum of potassium bromide and the active compound of Figure 1; and (1) chloroform is deuterated with the proton NMR spectrum of Figure 5; (B) said BBM-928B having a quinoline chromophore and via a hydrolysis of the hydrogen component, ash, serine, glycine, sarcosine and / or hydroxy-N-methylvaline, and having a preferred form of the compound; (a) a solution of chloroform and methylene chloride, benzene, ethanol, methanol and n-butanol, and optionally hydrogenated n-hexane; (b) a positive reaction with ferric chloride and an Ehrlich reagent and a negative reaction with a Tollens, Sakaguchi and ninhydrogen reagent; (c) is effective in moth intraperitoneal implantation of P388 leukemia; (d) a melting point of 214-217 ° C (e) a specific melting point of -74 ° (c 1, CHCl 3); (f) elemental analysis of about 50.14% col, 5.29% fertilizer, 12.34% kväve and 32.23% syre; (g) chromatographed on silica gel with 0.53 g of n-butanol-methanol-water (63:27:10) and 0.26 g of xylene-methylethyl-methanol (5: 5: 1); (h) the infrared spectrum of potassium bromide is shown in Figure 2; and (i) lost chloroform in the form of a proton NMR spectrum; (C) a BBM-928C with a quinoline chromophore and a hydrolysed hydrogen component, such as serine, glycine, sarcosine and β-hydroxy-N-methylvaline, and a hydrogenated formulation; (a) a solution of chloroform and methylene chloride, benzene, ethanol, methanol and n-butanol, and optionally hydrogenated n-hexane; 67403 31 (b) a positive reaction with ferric chloride and Ehrlich reagents and a negative reaction with Tollens, Sakaguchi and ninhydrin reagents; (c) having an effective effect on moss intraperitoneally implanted tumors: P3881 leukemia, L1210 leukemia, B16 melanoma, Lewis lung carcinoma and sarcoma 180 ascites; (d) a melting point of 244-248 ° C; (O) the specificity of the reaction form is / * ^ D = -91 (c 1, CHCl 3); (f) having a molecular weight of about 1470; (g) an elemental analysis of about 51.77% col, 5.29% claim, 13.55% yield and 29.39% syrup; (h) the residue was chromatographed on silica gel with 0.27 ml of a n-butanol-methanol solution (63:27:10) and 0.07 ml of a xylene methylmethylketone-methanol (5: 5: 1); (i) the infrared spectrum of potassium bromide is shown in Figure 3; (j) lost chloroform in the form of a proton NMR spectrum; (D) BBM-928D comprises the following: (a) a solution of chloroform and methylene chloride, benzene, ethanol, methanol and n-butanol, and other olefins and n-hexane; (b) a positive reaction with ferric chloride and an Ehrlich reagent and a negative reaction with a Tollens, Sakaguchi and ninhydrin reagent, (c) a positive reaction with moss intraperitoneally implanted P388 leukemia; (d) mp 224-227 ° C; (e) the specificity of the reaction is δ = -13 ° (c 1, CHCl 3); (f) the elemental analysis comprises about 50.75% col, 5.25% yield, 12.58% yield and 31.42% sugar; (g) the residue was chromatographed on silica gel with 0.73 g of n-butanol-methanol-water (63:27:10) and 0.53 with xylene-methyl ethyl ketone (5: 5: 1);