DK153501B - PROCEDURE FOR PREPARING AN ANTITUMOR ANTIBIOTIC COMPLEX - Google Patents

Description

DK 153501 B

The present invention relates to a process for the preparation of a novel antitumor antibiotic complex called BBM-928, or its components BBM-928 A, B, C, D, E and F, wherein BBM-928 A, B and C have the formula 05

CHO 3 ° H

3 / ~ "V" 0Ri is! 5: V / Avv'N ^^ C0 - * - Ser + N— (I C-KJly-S ar + HM-V a 1

0 "O -I HO x .OCH

io f o ~ o HM-Val ^ SarKily ^ C | 11 - N Ser * -CO where Ser represents serine, Gly glycine, Sar sarcosine, HM-Val β-hydroxy- Λ N-methylvaline, and R 1 and R 2 represent acetyl for BBM-928A, R represents 2 12 acetyl, and R represents hydrogen for BBM-928B and R and R represent hydrogen for BBM-928C.

Based on available spectral data and available physicochemical properties, the antitumor antibody complex of the present invention appears to be structurally related to the quinoxaline group of antibiotics such as echinomycin, Dell, et al., J. Am. Chem. Soc., 97, 2497 (1975), quinomycins, Shoji, et al., J. Antibiotics, 14A, 335 (1961), and Triostin C, Merck Index, 9th edition, 9399. However, the antitumor antibiotic complex of the invention differs from these 25 antibiotics in the following respects: 1. The present complex and components thereof contain a quinoline core as a chromophore rather than quinoxaline as in the actinoleukin group of antibiotics.

2. The present complex and components thereof do not contain sulfur as opposed to the presence of a disulfide or thioacetal bridge in the structure of actino-leukin antibiotics.

3. The present complex and components thereof show relatively weak antimicrobial activity over the actin leukukins which are potent antibacterial antibiotics.

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The process is characterized by culturing Actinomycetes ATCC 31491 or a mutant thereof with substantially the same properties as that in aqueous solution containing an assimilable carbon source and an assimilable nitrogen source under submerse aerobic conditions, 05 and recovering the BBM-928 complex from the culture medium and, if as desired, BBM-928 separates the complex into its components A, B, C, D, E and F.

Referring now to the drawing, where

FIG. Figure 1 shows the infrared absorption spectrum of BBM-928A in 10 potassium bromide;

FIG. 2 shows the infrared absorption spectrum of BBM-928B in potassium bromide;

FIG. Figure 3 shows the infrared absorption spectrum of BB.M-928C in potassium bromide; 4 shows the infrared absorption spectrum of BBM-928D in potassium bromide;

FIG. Figure 5 shows the nuclear magnetic resonance spectrum of BBM-928A dissolved in deuterated chloroform using TMS as the internal standard determined with an NMR spectrometer at a frequency 20 of 90 MHz;

FIG. Figure 6 shows the nuclear magnetic resonance spectrum of BBM-928B dissolved in deuterated chloroform using TMS as the internal standard determined with an NMR spectrometer at a frequency of 90 MHz; Figure 7 shows the nuclear magnetic resonance spectrum of BBM-928C dissolved in deuterated chloroform using TMS as the internal standard determined with an NMR spectrometer at a frequency of 90 MHz;

FIG. 8 shows the nuclear magnetic resonance spectrum of BBM-30 928D dissolved in deuterated chloroform using TMS as the internal standard determined with an NMR spectrometer at a frequency of 90 MHz.

The strain designated Actinomycetes strain G455-101 in the Bristol-Banyu culture collection was isolated from a soil sample collected in 35 Philippines and it is deposited in the American Type Culture Collection of Microorganisms as ATCC 31491.

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The aforementioned antitumor antibiotic complex has at least six components called BBM-928A, B, C, D, E and F. Components A, B, C and D of the BBIVI-928 complex have been isolated in crystalline form with identified chemical structures for Components A, B and C. The complex (and individual components) has anti-tumor antibacterial properties. Because of antibacterial activity, the complex and individual components thereof are useful as supplements for animal feed and as therapeutic agents for the treatment of bacterial infections in mammals and humans. Furthermore, 1 ° these antibiotics are useful for cleaning and sterilizing laboratory glassware and surgical instruments, and they can be used in combination with soaps, detergents and washing solutions for sanitary purposes. Because of antitumor effects, the complex and individual components thereof are particularly valuable against a variety of intraperitoneally implanted mouse tumors.

Description of strain No. G455-101 of the genus Actinomycetes

The following is a general description of the preferred microorganism for the preparation of the antibiotic anti-tumor complex BBM-20 g28. Observations of cultivation, physiological and morphological conditions were made for the organism according to standard taxonomic methods, e.g. Shirling, et al., Int. J. Syst. Bacteriol.

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

25 Mi chromorphology - Strain No. G455-101 forms both substrate and aerial mycelium, and the substrate mycelium is well-developed, long, and branched (0.5-0.8 μ in width). Clear fragmentation of the substrate mycelium is not observed. Unlike usual strains of Streptomyces, strain G455-101 has only short or rudimentary air mycelium or 50 does not form anything in certain agar media. Short or long spore chains are produced in the aerial mycelium, which contains 2-50 oval spores in a chain (usually 5-20 spores). Track chains are straight, curved or loop-shaped. The spores are spherical (0.3-0.4 μ), oval or cylindrical (0.3 x 1.5 - 3.0 μ) in shape and have a smooth surface. Spores are often 25 separated by empty hypers. An amorphous sporangium-like vesicle enclosing a short twisted spore chain is occasionally observed in the air mycelium.

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Composition of cell wall and whole cell sugar components - The cell wall of strain G455-101 contains meso-diaminopimelic acid but lacks glycine. Whole-cell hydrolyzate shows the presence of glucose, mannose and madurose (3-O-methyl-D-galactose). The aforementioned 05 cell wall composition and whole cell sugar components indicate that strain G455-101 is an actinomycetes species of cell wall type IIIB.

Cultural and Physiological Properties - Strain G455-101 grows vigorously, produces pink or grayish pink aerial mycelium and produces a reddish water-insoluble pigment in nutrient-rich agar media such as yeast extract malt extract agar and oatmeal agar. However, in inorganic salt starch agar, glycerol asparagine agar and tyrosine agar, it produces poor growth, forming white or beige rudimentary aerial mycelium and producing small amounts of reddish pigment. Melanoid pigment is not produced in peptone yeast iron agar and tyrosine 15 agar. Nitrate is reduced to nitrite. It grows strongly at 28 ° C, 37 ° C and 45 ° C, but does not grow at 10 ° C or at 50 ° C. Pentoses and hexoses are well utilized by the tribe. Cultural and physiological properties of strain G455-101 are shown in Figs. Tables I and II. The utilization of carbon sources is shown in Table III.

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Table I

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Cultural properties of strain No. G455-101 * 05 1. Czapek's agar G ** within or cranial growth

(sucrose nitrate agar) m9en or KnaP growthX

^ dark pink ^ white to pale pink

D

ω none 10 2. Trypton yeast extract substrate (ISP # 1) moderate growth, fluffy, sedimented and not pigmented 3. Yeast extract malt extract agar (ISP # 2) strong ^^ deep red to reddish brown ^ strong, greyish pink to light bluish pink ^ none 20 4. Oatmeal agar (ISP no. 3) 0 G strong ^ strong yellowish-red ^ moderate, pink 25 D grayish-yellow

5. Inorganic salts-starch- G

agar (ISP # 4) rings slightly yellowish-brown to dark red ^ button, white to beige 30 D · w none 6. Glycerol asparagine agar o (ISP # 5) ® low yellowish pink to reddish brown 33 ^ button, white ^ none

Table I (continued) 6

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7. Pepton Yeast Extract Iron- G Rings, Folded Agar (ISP No. 6) R Strong Reddish Orange 05 A None D Light Yellowish Orange 8. Tyrosine Agar (ISP No. 7) G Rings R Dark Red A Button, White D none 9. Glucose-ammonium salts- G low 15 agar R reddish brown A button, light gray D none 20 10. Bennett's agar G moderate R reddish brown A limited, grayish pink D none 25 * observed after incubation at 37 ° C for 3 weeks.

** Abbreviations: G - growth R - backing color for substrate mycelium A - aerial mycelium D - diffusible pigment.

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Table II

Physiological properties of strain No. G455-101

Test_ Reaction Method and medium 05 Nitrite from nitrate positive inorganic medium: Czapek's sucrose nitrate substrate

Nitrite from nitrate positive organic medium: 0.5% yeast extract, 1.0% glucose, 0.5% KN03, 0.1% CaCO3 .jq Casein hydrolysis in slightly positive Luedemann's agar medium agar medium .......... .........

Skim milk positive coagulation

Gelatin displacement negative 15% gelatin in tryptone yeast extract substrate (ISP No. 1 medium)

HpS preparation from positive L-cysteine (0.1%) added to L - cysteine try pton - yeast xtra substrate (ISP # 1 medium) plus agar. HpS detected with paper stripper containing 10% 2Q aqueous lead acetate solution.

Formation of melanoid negative peptone yeast iron agar (ISP # 6) and tyrosine agar (ISP # 7).

Catalase reaction positive H2 ° 2 water'9 solution.

Oxidase reaction positive Kovacs1 reagent Growth temperature vigorous growth Bennett's agar at 28, 37 and 45 ° C. Poor growth at 20 ° C.

No growth at 10 ° C and 50 ° C.

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Table III

Exploitation of Carbon Sources for Strain No. G455-101 PG Lm 05 1. Glycerol ++ + 2. D (-) - Arabinose + + 3. L (+) - Arabinose + + 4. D-Xylose ++ + 5. D- Ribose ++ + 10 6. L-Rhamnose ++ + 7. D-Glucose + + 8. D-Galactose ++ + 9. D-Fructose ++ + 10. D-Mannose ++ + 15 11. L- ( -) - Sorbose 12. Sucrose 13. Lactose -, _ + 14. Cellobiose + + 15. Melibiosis -, _ + 20 16. Trehalose + + 17. Raffinose 18. D (+) - Melezitosis 19. Soluble starch + 20. Dulcitol - 25 21. Inositol + 22. D-Mannitol ++ + 23. D-Sorbitol 24. Salicin - 25. Cellulose + + 30. 26. Chitin ++ 27. Keratin ++

Base medium PG: Pridham-Gottlieb's inorganic medium, containing 0.1% yeast extract.

35 Lm: Luedemann's organic medium

Incubation for 2 weeks at 37 ° C.

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In the method according to the application, mutants produced from the organism described above can also be used by conventional means, such as exposure to X-rays, ultraviolet radiation, nitrogen mustard gas, phage exposure and the like, which have substantially the same properties as this one and which are capable of producing the BBM-928 complex

Preparation of antitumor antibiotic BBM-928 complex

Media which can be used to prepare the subject anti-biotic antitumor agents include an assimilable carbon source such as starch, glucose, dextrin, maltose, lactose, sucrose, fructose, mannose, molasses, glycerol and the like. The nutrient medium should also contain an assimilable nitrogen source such as protein, protein hydrolyzate, polypeptides, amino acids, corn molding liquid, casein, urea and the like, as well as inorganic nutrient salts providing inorganic anions and cations such as potassium, sodium, ammonium, calcium, sulfate, carbonate, phosphate, chloride, nitrate and the like.

In the preparation of the BBM-928 complex, any temperature can be used which contributes to a satisfactory growth of the organism.

20 Temperatures varying from approx. 20 ° to 45 ° C can be used, with a preferred temperature for optimum growth of the organism ranging from approx. 28 ° to 34 ° C with a temperature range of 30 ° to 32 ° C being the most preferred. Generally, maximum production of the BBM-928 complex is achieved over approx. 4 to approx. 6 days. Conventional methods 25 are used during the fermentation period. Eg. the preparation of small quantities is conveniently carried out in shaking flasks or by surface cultures. Preparation of large quantities is preferably carried out under submerse aerobic culture conditions in sterile tanks. In tank fermentation, a vegetative inoculum is first prepared in a nutrient substrate by grafting the substrate culture with a spore from the organism to form a young, active seed culture, which is then aseptically transferred to the fermentation tank medium. Aeration in tanks and flasks can be achieved by forcing sterile air through or onto the fermentation medium with further agitation in tanks using a mechanical propeller.

55 Anti-foaming agents such as silicone oil, soybean oil and fatty oil can be added as needed.

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DK 153501B

The antibiotic content of the fermentation substrate or extracts of BBM-928 complex can be determined by paper slice agar diffusion assay using Sarcina lutea as test organism and nutrient agar as sample medium. The pH is adjusted to 05 9.0 for optimum sensitivity of the sample system used to determine optimal substrate strength.

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

Physicochemical Properties of BBM-928 Components A, B, C and D Individual components of BBM-928 exhibit uniform dissolve similarity and color reactions. They are e.g. readily soluble in chloroform and methylene chloride, poorly soluble in benzene, ethanol, methanol and n-butanol and insoluble in water and n-hexane. Positive reactions with ferric chloride and Ehrlich's reagents are obtained by negative reactions to Toliens, Sakaguchi and ninhydrin.

Characteristic physicochemical properties of BBM-928 components are shown in Table IV.

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Table IV

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Physicochemical properties of BBM-928 components A, B, C and D

05 _BBM-928_

A B C D

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

[<x] p5 (c1, CHCl3) -27 ° -74 ° -91 ° -13 ° 10

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 (by difference) : 28.49 32.23 29.39 31.42 λ in nm (E 1-6) max 1 cm 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) 'n 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) 383 (122) 383 (145) 25

Molecular Weight (Osmometer, in CHCl3) 1.450 - 1.470 30 35 DK 153501 B f s, 12

Infrared (IR) and nuclear magnetic resonance (NMR) spectra for components A, B, C and D are shown in Figs. FIG. 1-4 and FIG. 5-8 of the drawing. The NNIR spectra of BBM-928A (Fig. 5), q5 BBM-928B (Fig. 6) and BBM-928C (Fig. 7) are very similar, the only difference being the presence of acetyl groups in components A (δ: 2 , 03 ppm, 2 mol equiv) and B (δ: 2.05 ppm, 1 mol equiv) but not in C. By acetylation with acetic anhydride in pyridine, 2, 3 and 4 molar equivalents of acetyl group were introduced into respectively. BBM-928 components A, B and C. The three acetylation products thus obtained showed identical properties on TLC, 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 ri-butanol-soluble, UV-absorbing fragments (I, II, III, IV and V) as well as five water-soluble ninhydrin positive substances (NPS-1, 2, 3, 4 and 5). The latter substances were separated by Dowex 50 x 4 chromatography and identified as the following amino acids: 2q Compounds Rf (S-123) * Identification NPS-1 0.72 β-hydroxy-N-methylvaline (HM-Val) NPS-20 , 49 glycine (Gly) NPS-3 0.46 serine (Ser) 25 NPS-4 0.45 sarcosine (Sar) NPS-5 0.23 (not determined) * TLC, silica gel plate S-123: 10% ammonium acetate - methanol - 10% ammonia solution (0: 10: 1)

The five UV absorbent fragments mentioned above have been shown to have the following structures:

—0H Fragment I

H 2 ° H ° ΑΛ 6 ° CO-NH-GH-CO-H + + MS: m / e 306 (M) λΜβΟΗ; 227 233 251 345 ^

Max DK 153501B f 3 13

Fragment II

NH OH C20H25N3 ° 8 OH '', O-CO-CH-C (CH3) 2 MS; m / e 377 (h -58)

H: 229, 234, 261, 345 nm

Fragment III

OH MeOH

CH O λ: 230, 235, 262, 345

Owl3 max C0- (NPS-5)

Fragment IV CinH7NO.

OH 10 7 4 H0'Y ^ Vvvjj / ^ / MS: m / e 205 (M +) rn o XMe0H: 228, 260, 354 ran "bU2n max

H, C CH, Fragment V

V

0H · C23H30-V9

0-C0-CH-H-CH 2 C 0? H

I CH_ CH. CH, Xn: 230, 235, 262, 345 nm L jL I 2 3 3 max

C0-NH-CH-CO2H

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By acid hydrolysis (6N HCl), fragments I, II, III and V gave the following degradation products:

Hydrolysis conditions_ 05 closed tube reflux

Fragment nO ° C, 20 hours 110 ° C, 3 hours I IV, Ser II IV, Ser, HM-Val I, HM-Val

III

10 V - I, HM-Val, Sar

Ser: serine. HM-Val: β-hydroxy-N-methyl valine

Sar: sarcosin 15

Base hydrolysis of BBM-928A or BBM-928C with 0.1N NaOH at 25 ° C for 3 hours yields fragment VI (the abbreviations Ser, HM-Val and Sar are as indicated above, Gly represents glycine and the symbol "X, J indefinite part).

20

OH

lNJts ^ - CCH-Ser- ^ X-> Giy-> Sar ^ HM-Val 25 Fragment VI

Treatment of fragment VI with 0.1N HCl at 110 ° C for 1 hour gives fragment VII plus HM-Val.

. OH

-CO + Ser + X ^ HSly Sar

Fragment VII

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

X-KBly-s-Sar- ^ HM-Val 05

Fragment VIII

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

10 X-M3ly- »Sar

Fragment IX

The indefinite moiety (X) has a molecular formula CgHgNgO4 (in peptide form) based on microchannel analysis of fragment IX and other peptide fragments containing (X) and spectral analysis as summarized below.

20 C-NMR Proton NMR Determination 30.14 (t) 2.36 ppm (2H, m) -CH2 ~ 61.4 (d), 4.2-4.5 (2H, m) 2x -CH ^ T ^ 61.7 (d) * e, 'N; 140.7 (d) 6.76 (1H, t) -CH = N- 171 (s) - -CO- (amide)

-OH

NH

According to the spectral data for fragments VIII and IX as well as a 360 MHz proton NMR for BBM-928A in Example 4, the unpaired amino acid moiety (X) seems to be best assigned to the following tetrahydro-pyridazine structure: 35 I '

DK 153501 B

16 CO-

ll T

05 (X) 10 Based on the results of the degradation experiments, spectral data, microanalysis and molecular weight determinations described above, the following structures are best represented as representing BBM-928A, B and C: 15 LI LL * _ / - ° Ri C0 + Ser * N-i OHHLy + Sar + flM-Val 20 0 go £ HM-Val-f-Sar-i-Gly + ix \ | 3 \ —N <-Ser + C0 Λϋ / \ / 25

Rg Ser: serine

Gly: glycine 30 BBM-928A Ac Ac Sar: sarcosine BBM-928B Ac H HM-Val: β-hydroxy-N-methylvaline

BBM-928C Η H

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Antimicrobial activity

The antimicrobial activity of BBM-928 components was determined against a variety of bacteria and fungi by the serial agar dilution method 05 in nutrient agar at pH 7 using Steer's multi-grafting apparatus. The inoculum size was standardized to deliver a 0.0025 ml aliquot of test organisms containing ca. 10 cells per cell. ml for all bacteria and fungi with the exception of acid-resistant bacteria, for which 10 cells per cell were used. ml of suspension. The 10 minimum inhibitory concentrations (MIK) were determined after overnight incubation at 37 ° C and are shown in Table V. As can be seen, BBM-928 components are moderately to weakly active against gram - positive and acid-resistant bacteria, but practical. taken inactive against gram-negative bacteria and fungi.

The activity of prophage induction in lysogenic bacterium (ILB) was determined for BBM-928 components. No significant ILB activity was detected with BBM-928 components A, B and C up to a concentration of 100 pg / ml.

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Table V

In vitro antimicrobial activity of BBM-928 components against aerobic bacteria 05 BBR1 BBM-928 components (MIK in pg / ml)

Code Test organism A B C D 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

Sl-1 S. lutea PCI 1001 6.3 12.5 50 50 50 25

Mf-1 flavus D12 12.5 25 50 100 100 25

Cr-1 C2_ xerosis 53K-1 25 50> 100> 100> 100 50 15 Bs-1 B ^ subtilis 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 fL. smegmatis 607 D87 25 25 25 100 100 25

Mp-1 M. phlei D88 12.5 12.5 12.5 50 50 12.5 20 Ec-1 E; _ coli NOT> 100> 100> 100> 100> 100> 100

Kp-1 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

Pm-1 P ^ mirabilis A9554> 100> 100> 100> 100> 100> 100 Pg-1 morganii A9553> 100> 100> 100> 100> 100> 100

Sm-1 Si ^ marcescens A20019> 100> 100> 100> 100> 100> 100

Al-1 A ^ _ faecalis ATCC 8750> 100> 100> 100> 100> 100> 100

Ca-1 C ^ _ arbicans IAM 4888> 100> 100> 100> 100> 100> 100

Cn-3 C. neoformans 100> 100 100> 100> 100> 100 30 --------------------------------- ---- 35

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The antitumor activity

Comparative studies of BBM-928 components A, B, C and D with mitomycin C for antitumor activity were performed with the intraperitoneally implanted tumors: P388 leukemia, L121X) leukemia, B16.05 melanoma, Lewis pulmonary carcinoma (LL) and sarcoma 180.ascites (S180). Test solutions of BBM-928 components in 0.9% saline containing 10% dimethyl sulfoxide and mitomycin C in 0.9% saline were administered once daily according to dosing schedules ranging from a single one-day treatment to multiple daily treatments. When varying the dosage, the minimum effective dose (MED) was determined, which caused an average survival time of the treated animals at least 1.25 times greater than that of a control group. This level of activity is considered a measure of significant antitumor activity. Results are shown in Table VI together with calculated activity ratios illustrating that BBM-928A is significantly more active than mitomycin C with a factor of 10 to 300, depending on the tumor strain and dosing schedule. Intraperitoneal LD ^ values for BBM-928 components A, B, C and D as well as mitotycin C determined by the method of Van der Waerden, Arch. Expt. Path. Pharmak., 195: 389 (1940) is also shown in Table VI.

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Table VI

Antitumor activity and toxicity of BBM-928 A, B, C and D and Mitomycin C

05

Minimum effective dose (MED, mg / kg / day) LD -,, i.p.

P388 L1210 B16 LL SI 80 (md / 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 0.003 - - - 0.083

Mitomycin C 0.1 3 1 0.3 0.3 9.3

Treatment BBM-928A 0.001 0.003 0.003 0.003 0.0001 0.013 Mitomycin C 0.1 0.3 0.3 0.3 0.03 0.03 1.4

Activity Ratio (BBM-928A vs. Mitomycin C

Treatment3 30 - 300 10 100

Treatment0 100 100 100 100 100 a. Single treatment 1st day 20 b. Daily treatments 1st to 9th day 25 30 35

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Example 1

Preparation of BBM-928 Complex φ5 Agar Fermentation. A well grown agar slant culture of a strain of Actinomycetes ATCC 31491 is used for grafting of vegetative 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 with pH adjusted to 7.2 before sterilization. The seed culture is incubated at 32 ° C for 72 hours in a rotary shaker (250 rpm), and 5 ml of the growth medium is transferred to a 500-ml Erlenmeyer flask: containing 100 ml of fermentation medium composed of 2% soluble starch, 1% Pharmamedia, 0.003 % ZnSO4.7H2O and 0.4% CaCOg · Production of BBM-928 complex generally reaches the maximum after approx. 5 days ^ shake culture.

Tank fermentation - A seed culture is shaken for 4 days in Erlenmeyer flasks and seeded in 100 liters of propagating medium composed of 2.0% oatmeal (Quaker Products, Australia), 0.5% glucose, 0.2% dry yeast, 0.0008% MnCl2, 4H2O, 0.0007% CuSO4.7H2O, 0.0002% ZhSO4.7H2O, and 2q 0.0001% FeSC> 4.7H2Q in a 200-liter graft tank fermenter stirred at 200 rpm at 30 ° C for 54 hours . A 15-liter portion of the seed culture is then seeded on 170 liters of fermentation medium containing 2.0% soluble starch, 1.0% Pharmamedia, 0.003% ZnSO4.7H2O and 0.4% CaCOg in a 400-liter tank fermenter operating at 30 ° C at 2g 200 rpm with an aeration rate of 150 liters / min. The pH of the substrate gradually increases with progressive fermentation and reaches 8.4-8.5 after 100-120 hours, at which time a maximum antibiotic strength of 30 pg / ml is achieved.

Example 2

Isolation of BBM-928 complex by solvent extraction

Extracted substrate (170 liters, pH 8.5) from Example 1 is filtered 2g with a clarifier. The activity is determined in both the mycelial cake and the filtrate. The mycelial cake is extracted twice with a solvent mixture of acetone and methanol (1: 1, 30 liters x 2). Extracts DK 153501 B; 22 are combined and evaporated under reduced pressure to form an aqueous concentrate extracted with n-butanol. The substrate filtrate is extracted twice with n-butanol (40 liters x 2). Concentration of the combined n-butanol extracts under reduced pressure and lyophilization 05 of the residue gives a crude solid (21.4 g). According to thin-layer chromatographic study, this material is a complex consisting of three major components A, B and C as well as three minor components D, E and F with Rf values as listed in Table VII below.

Table VII

Silica gel TLC of BBM-928 components _Rf values * _ 15 System N-118 ** System N-103 *** BBM-928A 0.71 0.48 BBM-928B 0.53 0.26 BBM-928C 0, 27 0.07 20 BBM-928D 0.73 0.53 BBM-928E 0.56 0.34 BBM-928F 0.39 0.17 * Detection with UV Scanner (Shimadzu CS-910) at 345 25 nm ** n-butanol - methanol - water (63:27:10) *** xylene - methyl ethyl ketone - methanol (5: 5: 1)

Example 3 30

Purification of BBM-928 complex

The crude complex of Example 2 is purified by a preparative countercurrent distribution apparatus (Mitamura, 100 ml / tube) using a carbon tetrachloride-chloroform-methanol-water solvent system (5: 2: 5: 1) . After 50 transfers, pipe no.

5-20 and concentrated to give a light yellow powder (4.4 g)

DK 153501B

23 containing components A, B, D, E and F. This mixture is dissolved in a small amount of chloroform and loaded onto a column of silica gel C-200 (500 ml) which is pretreated with ethyl acetate. The column is developed by ethyl acetate with an increasing amount of methanol (2-5%, 05 V / V) and fractions determined for optical density at 345 nm. The minor component D is first eluted with ethyl acetate followed by component A. Components E, B and F are then eluted in this order at 3% methanol concentration. Each fraction containing the appropriate component is evaporated under reduced pressure and the residue 10 is crystallized from chloroform-methanol. Similarly, a crude composition of component C is obtained from tube # 21-35 from the countercurrent distribution described above. Purification of component C is carried out by silica gel chromatography and crystallization from chloroform-methanol. The yields for components A, B, C, D, E and F are respectively. 988 mg, 420 15 mg, 848 mg, 130 mg 119 mg and 114 mg.

Example 4

Further purification of component BBM-928A from Example 3 20

Thin-layer chromatographic examination of BBM-928A component of Example 3 (using a system consisting of 10% methanol in toluene) showed that the sample was not completely homogeneous, containing additional material running just ahead of the BBM-928A component. . The following steps were taken for purification.

(1) The sample is chromatographed on silica gel using a linear gradient of chloroform to 6% methanol-chloroform. Fractions eluting between 2.4 and 3.3% methanol-chloroform (containing the BBM-928A component plus some contaminants) are mixed for the next 30 steps.

(2) A concave gradient is formed using three containers containing 2% methanol-toluene in the first two and 6% methanol-toluene in the third. The mixture of step 1, chromatographed (silica gel column) on this gradient, yields a smaller component which first elutes closely followed by the purified BBM-928A component (herein called BBM-928A).

P

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The molecular weight of BBM-928A determined by Field Desorption Mass

P

Spectrometry is 1427, corresponding to an empirical formula of ^ 54 ^ 73 ^ -14 ^ 24 (molecular weight 1427,417).

Elemental analysis (samples dried at 100 ° C for 18 hours).

05 C Η N Oa

Calcd for C44H7gg 4 (-) 24: 53.85 5.51 13.74 26.90

Found: 52.47 5.48 13.81 28.24b 10 a. By difference.

b. Average of three provisions.

15 20- 25 30 35

Claims (1)

Process for preparing an antitumor antibiotic complex called βΒΜ-928, or its components BBM-928 A, B, C, D, 05. and F, wherein BBM-928 A, B and C have the formula OH “3 ° υ j ~ \ _ T ί X i J- ° \ CO ^ Ser + N— ^ Λ I C-KJly ^ S ar + HM-Val 10 0 ..I i HO x ✓OCH «to SO 3 HM-V al- ** S arKJly ^ | wherein Ser represents serine, Gly glycine, Sar sarcosine, HM-Val β-hydroxy-N-methylvaline, and R 1 and R 2 represent acetyl for BBM-928A, R 1 represents acetyl, and R 2 represents hydrogen for BBM-928B, and R 1 and R 2 represent hydrogen for BBM-928C, CHARACTERIZED BY culturing Actinomycetes ATCC 31491 or a mutant thereof having substantially the same properties as that in aqueous solution containing an assimilable carbon source and an assimilable nitrogen source under submerse aerobic conditions, and recover the BBM-928 complex from the culture medium and, if desired, separate the BBM-928 complex into its components A, B, C, D, E and F.