NL8001868A - COMPOUNDS WITH ANTITUMOR AND ANTIBACTERIAL EFFECT; METHOD FOR PREPARING THEREOF PREPARATIONS WITH ANTITUMOR AND ANTIBACTERIAL EFFECT. - Google Patents

Description

i VO 307

Compounds with anti-tumor and antibacterial activity; method for the preparation thereof preparations with anti-tumor and anti-bacterial activity.

The invention relates to a new anti-tumor-anti-biotic complex as well as methods of preparing, recovering and separating into biologically active compounds.

Based on currently available spectral data and physicochemical properties, the anti-tumor antibiotic complex of the 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 C1961) and triostine C, 10 The Merck Index, 9th Ed., 9399. However, the anti-tumor antibiotic complex differs from these antibiotics in the following aspects: 1. The complex according to the invention and its components contain a quinoline nucleus as chromophore instead of quinoxaline as in the actinoleukin group of antibiotics.

2. The complex of the invention and its components do not contain sulfur, in contrast to the presence of disulfide or thioacetal bridges in the structure of actinoleukin antibiotics.

3. The complex of the invention and its components exhibit a relatively weak antimicrobial activity compared to the actinoleukins which are strong antibacterial antibiotics.

The invention provides a new anti-tumor antibiotic complex, which will be commonly referred to as BBM-928. The complex containing at least six components is prepared by culturing a ΒΒΓ1-92Β producing strain of actinomycetes (ATCC 31491) or a mutant thereof in an aqueous nutrient medium using submerged aerobic conditions under 800 1 868-2. The invention also relates to a method of recovering the BBM-928 complex from the culture medium and separating the complex into its biologically active components by countercurrent distribution and chromatography techniques.

The invention extends to the BBM-928 complex and its biologically active components BBM-928 A, B, C, D, E and F. The invention particularly relates to the components BBM-928 A, B, C, and D in dilute forms, as true concentrates and in purified form.

Fig. 1 is the infrared absorption spectrum of BBM-928 A in potassium bromide.

Fig. 2 is the infrared absorption spectrum of BBM-928 B in potassium bromide.

Fig. 3 is the infrared absorption spectrum of BBM-928 C in potassium bromide.

Fig. 4 is the infrared absorption spectrum of BBM-928 D in potassium bromide.

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

Figure 6 shows the proton magnetic resonance spectrum of BBM-928 B dissolved in deuterated chloroform using 25 TMS as an internal standard, determined with an NMR spectrometer at a frequency of 90 MHz.

Fig. 7 shows the proton magnetic resonance spectrum of BBM-928 C dissolved in deuterated chloroform using TMS as an internal standard, determined with an NMR spectrometer at a frequency of 90 MHz.

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

The invention relates to a new anti-tumor anti-80 0 1 8 68 • F * - 3 - biotic complex arbitrarily designated here as BBM-928.

The complex is believed to be structurally related to the actino-leukin group of antibiotics and is obtained by fermentation of a hitherto unidentified strain of actinomy-5 cells. Any actinomycete strain that can form BBM-928 in a culture medium can be used, with an actinomycete species of microorganisms of strain No.G455-101 being preferred in the Bristol-Banyu culture set. The micro-organism was isolated from a soil sample collected in the Philippines and deposited with the US type culture collection of micro-organisms ATCC as ATCC 31491.

The novel anti-tumor antibiotic complex of the invention has at least six components, designated BBM-928 A, B, C, O, E, and F. Components A, B, C, and D of the BBM-928 complex are isolated in crystalline form, the chemical structures of components A, B and C have been identified. The complex (and the individual components) of the invention have anti-tumor and antibacterial properties. With regard to the antibacterial activity, the complex and its individual components are useful for addition to animal feed and as therapeutic agents for treating bacterial infection in mammals In addition, the antibiotics can be used to clean and sterilize laboratory glassware and surgical instruments and used in combination with soaps, detergents and washing solutions for sanitary purposes As far as anti-tumor effects are concerned, the complex and the individual components thereof particularly useful for use against a variety of intraperitoneal implanted mouse tumors.

Actinomycetes sport jstam Mo_.C! _455-1Q1.

A general description will now be given of the microorganism which is preferably used for preparing the antibiotic-anti-tumor complex BBM-928. Observations were made of the culture, physiological and morphological properties of the organism according to standard taxonomic methods, for which reference can be made, for example, to Shirling et al ..

Int. J. Syst. Bacteriol. 16, 313 (1966] and Lechevalier et al., Biol.

ö η n 1 ft fift.-4-

Actinomycetes Related Org. 11, 78 (1976).

Micromorphology:

Strain No. G455-1Q1 forms both substrate and aerial mycelia, and the substrate mycelellum is well developed, long and branched (a width of 0.5 - 0.8 µm). Separate fragmentation of the substrate mycelium is not observed. Unlike common Strepto myces, strain G455-101 bears only short or vestigial aerial mycelia, or does not form at all in certain agar media. Short or long spore chains are formed in the aerial mycelium, which contains 2-10 50 oval spores in a chain (usually 5-20 spores).

Track chains are straight, tortuous, or looped. The spores are spherical (0.3 - 0.4 ym), oval or cylindrical (0.3 x 1.5 - 3.0 ym) in shape and have a smooth surface. Spores are often separated by empty hyphae. An amorphous sporangium-like vesicle encasing a short wound spore chain is occasionally observed on the aerial mycelium.

Composition yan_the_celwand_en_suikercgmpongnten of the_ whole_cel:

The cell wall of strain G455-101 contains meso-diamin-pimelic acid but does not contain glycine. Whole cell hydrolyzate shows the presence of glucose, mannose and madurose (3-O-methyl-D-galactose). The above cell wall composition and whole cell sugar components demonstrate that strain G455-101 is an actinomycete species with cell wall type IIIB.

! Sbi§§! Lz_ë ^ physi0i0g -------- 2®! 2§?!)? PB§n: 25 Stem (3455-101 grows abundantly, forms pink or grayish pink air mycelium and produces a reddish, not water soluble pigment in high nutrient agar media, eg yeast extract malt extract agar and oat flour agar. Inorganic salts starch agar, glycerol asparagine and tyrosine agar, however, gives poor growth, produces white or beige rudimentary air mycelium reddish pigment Melanoid pigment is not produced in peptone yeast iron agar and tyrosine agar Nitrate is reduced to nitrite It grows abundantly at 28 ° C, 37aC and 45 ° C but does not grow at 10 ° C or 50 ° C .

Pentoses and hexoses are well used by the strain. The culture and physiological properties of strain G 455-1C1 800 1 8 68 - * i - 5 - are shown in Tables A and B. The use of carbon sources is shown in Table C.

Table A

%

Growing properties of strain G455-101 5 1. Czapek's agar G little or no growth (sucrose - nitrate agar) r donKsr „3rd A white to light pink D no 10 2. Trypton - yeast extract-moderate growth, floccose, broth (ISP No.l) sedimented and non-pigmented 3. yeast extract - malt extract- G abundant 15 a No. ar No.2] R deep red to reddish brown A abundant, grayish pink to puperrose D no 20 4. oatmeal agar. CISP No.3) G abundant R strong yellowish red A moderate rose D grayish yellow 25 5. inorganic salts - starch G poor flour agar (ISP No.4] R light tan to dark red A little, white to both - 0 none 30 6.glycerol asparagine agar G poor (ISP No. 5) R yellowish pink to reddish brown A little, white D none 800 1 8 68 35 - 6 -

Table A (continued) 7. Peptone Yeast Extract Iron Agar G Bad, Pleated CISP No. 6) D. . . ...

R strong reddish A none 5 D light yellow-orange 8. tyrosine agar (ISP No.7) G bad R dark red A little, white 10 D none 9. glucose-ammonium salt agar G bad R red brown A little, light gray 15 0 none 10. Bennett Js agar G moderate R reddish brown A limited, grayish pink 20 D no x observed after incubation at 37 ° C for 3 weeks xx abbreviations: G - growth R - color backside of substrate mycelium 25 A - air mycelium D - diffusible pigment

Table B

Physiological properties of strain G455-1Q1 30 Test___________________ Response __With _gn ^ medium nitrite from nitrate positive inorganic medium: Czapek's sucrose nitrate stock nitrite from nitrate positive organic medium: 0.5% yeast extract, 1.0% glucose, 0.5% 35 KN03, 0.1% CaCO3 8001868 1 t - 7 -

Table B (continued) case hydrolysis in weak positive Luedemann's agar medium agar medium 5 skim milk coagulation positive gelatin liquid test negative 15% gelatin in tryptone yeast extract broth (ISP No. 1 medium) i-^ S formation from L-cyst positive L-cysteine (0 , 1%] added ine to trypton yeast extract broth (ISP No. 1 medium) plus agar f ^ S detection with a paper strip containing 10% aq lead acetate solution

formation of melanoxid negative peptone yeast iron agar (ISP

No. S) and tyrosine agar (ISP

No. 7)

Catalase reaction positive at ^ ¨ ° Pl ° ss * nS

oxidation reaction positive Kovacs' reagent growth temperature abundant Bennett's agar 7n growth at

28, 37 and 45 ° C

Bad growth at 20 ° C.

No growth at 10 and 50 ° C

Table C

Use of carbon sources for strain G455-101 PG Lm 1. glycerol ++ + 2. D (-) - arabinose + + 30 3. L (+) - arabinose + + 4. D-xylose ++ + 5. D-ribose ++ + 6. L-rhamnose ++ + 7.0-glucose + + 35 8. D-galactose ++ + 9. D-fructose ++ + unn18 68 - a -

Table C (continued) 10. D-mannose + + ++ + 11 .. LC -) - sorbose 12 .. sucrose 5 13 .. lactose · 1. ~.

14. cellobiose + + 15. melibiosis -, + 16. trehalose + + 17. raffinose 10 18. D (+) - melezitose - 19. soluble starch + 20. duloitol 21. inositol + 22. D-mannitol ++ + 23. D-sorbitol 24. salicin 25 .. cellulose + + 26. chitin + + 27. Keratin + + 20 Basal medium PG: Pridham - Gottlieb's inorganic medium, supplemented with 0.1% yeast extract.

Lm; Luedemann's Organic Medium Incubation for 2 weeks at 37 ° C.

Obviously, the preparation of the BBM-928 complex according to the invention is not limited to the use of the particular actinomycete strain G455-101 described by the above growth and microscopic properties. These properties are given for illustrative purposes only, and the invention therefore contemplates the use of strains or mutants obtained from the above-described organism by known methods such as exposure to X-rays, ultraviolet rays, nitrogen mustard, phage exposure, and the like, including BBM- 928 complex or individual components thereof can be prepared. The method of the invention for preparing the anti-tumor antibiotic BBM-928 complex comprises culturing by fermen-8001868 *% -9-titration of actinomycetes strain G455-101 in an aqueous solution. contains an assimilable carbon source and an assimilable nitrogen source under submerged aerobic conditions until substantial anti-tumor and antibiotic activity is imparted to the solution. Conventional fermentation methods are used for the cultivation of actinomycetes strain G455-1Q1. Media suitable for preparing the antibiotic anti-tumor agents of the invention contain an assimilable carbon source such as starch, glucose, dextrin, maltose, lactose, sucrose, fructose, mannose, molasses, glycerol, and the like. The nutrient medium also preferably contains an assimilable nitrogen source such as protein, protein hydrolyzate, polypeptides, amino acids, corn soak water, casein, urea and the like as well as nutritional inorganic salts containing inorganic anions and cations such as potassium, sodium, ammonium, calcium, sulfate, carbonate, phosphate, chloride, nitrate and the like. "

In the preparation of the ΒΒΠ-92Β complex, any temperature that leads to a satisfactory growth of the organism can be used. Temperatures ranging from about 20 to about 45 ° C are useful, preferably optimizing growth of the organism from about 28 to about 34 ° C. Most preferred is a temperature range of 30 - 32 ° C. A maximum preparation of the BBM-928 complex is generally reached after about 4-6 days. Known methods are used in the fermentation period.

The preparation of small amounts is e.g. suitably performed in shake flasks or with surface cultures. The preparation of large quantities is preferably carried out in other immersed aerobic culture conditions in sterile vessels. In vessel fermentation, a vegetative seed preparation is first prepared in a feed broth by inoculating the broth culture with a spore of the organism to obtain a young active seed culture which is then aseptically transferred into the fermentation broth. Aeration in barrels and bottles can be achieved by forcing sterile air through or on the surface of the fermentation medium 8001868-10, further agitation in the barrels being obtained using a mechanical stirrer. Anti-foaming agents such as silicone oil, soybean oil and pork lard oil can be added as needed. Antibiotic levels in the fermentation broth or the extracts of BBM-928 complex can be determined by paper disc agar diffusion analysis using Sarcina lutea as test organism and using nutrient agar as the analysis medium. The pH is adjusted to 9.0 for optimum sensitivity of the analysis system used 10 ƒ to determine the optimal broth strength.

The BBM-928 complex is isolated from the fermentation broth in a known manner, e.g. with solvent extraction processes. The purification is preferably carried out by preparative countercurrent distribution and chromatographic processes, as will be further explained in Examples II and III, to obtain BBM-928 components A, B, C, 0, E and F.

Physical Chemical Properties of Compounds 928 Component A C, D, and D of Example III: Individual components of BBM-928 show solubility and color reactions similar to each other. They are e.g. readily soluble in chloroform and methylene chloride, slightly soluble in benzene, ethanol, methanol and n-butanol and insoluble in water and n-hexane. Positive reactions with iron (III) chloride and Ehrlich reagent are obtained, with negative reactions to Tollens, akaguchi and ninhydrates.

characteristic physicochemical properties of BBM-928 components of Example III are shown in Table D.

Table D

38 Physico-chemical properties of BBM-928 components

A, B, C and D.

BBM-928

A B C D

Melting point, ° C 246 - 248 214 - 217 244 - 248 224 - 227 35 (c, 1, CHCl3) -27 ° -74 ° -91 ° -13 ° 8001868 - 11 -

Table D (continued)

Analysis; found C: 53.19 50.14 51.77 50.75 H: 5.40 5.29 5.29 5.25 5 N: 12.92 12.34 13.55 12.58 (by difference] 0: 28.49 32.23 29.39 31.42 in nm (E ^ 3 'max 1 cm in EtDH 235 (586] 235 (570] 235 (638] 235 (5503 264 (415] 264) 4003 264 (442] 264 (380] 10 345 (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) 15 i in EtOH-NaGH 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) 20 Molar Weight (Osmometer, in CHCl 3) 1,450-1,470

Infrared (IR) and Nuclear Magnetic Resonance (NMR) spectra of components A, B, C and D are shown in Figures 1 - 4 and 5 - 8. The NMR spectra of BBM-928 A (Figure 5), BBM -928 B Figure B), and BBM-928 C (Figure 7) are very similar with the only difference being the presence of acetyl groups in component A (J: 2.03 ppm, 2 mol equivalent) and B (X : 2.05 ppm, 1 mole equivalent) but not in 30 ° C. In acetylation with acetic anhydride in pyridine, 2, 3 and 4 mole equivalents of acetyl group in resp. BBM-928 components A, B and C introduced. The three acetylation products thus obtained showed identical properties in TLC, UV, IR and NMR spectra, showing that BBM-928 A is a monoacetyl derivative of BBM-928 B 35 and a diacetyl derivative of BBM-928 C, 8001868-12 -

Acid hydrolysis of BBM-928 A gave 5 n-butanol soluble UV absorbing fragments (I - V) and five water soluble nin hydrin positive materials CNPS-1 - NPS-5]. The latter materials were separated by Dowex 50x4 chromatography and identified as the following amino acids:

Compound Rf CS-123) * Identification NPS-1 0.72 ^ -hydroxy-N-methylvaline (HM-Val) NPS-2 0.49 glycine (Gly] 10 NPS-3 0.46 serine CSer) NPS-4 0 45 sarcosine (Sar) NPS-5 0.23 not established x TLC, silica gel plate S-123: 10% ammonium acetate - methanol - 10% ammonia solution 15 C9: 10: 1), The five UV absorbing fragments were found in the following structures to have:

Fragment I: formula 1 C14H14N2D6 20 MS / m / e 306 (M +) ^ MeOH 227 233 25 345 nm max

Fragment II: formula 2 C20H25N3 ° 8 25 MS: m / e 377 CM + -58) ^ MeOhi 22g 234 345 nm max

Fragment III: formula 3 λΜβ0Η: 230, 235, 262, 345 nm max 30

Fragment IV: formula 4 C10H7ND4 MS: m / e 205 CM +) MeOlH; 22fli 260 354 nm max 35 8001868 - 13 -

Fragment V: formula 5 C23H30N4 ° 9 A Ms0H; 230, 235, 262, 345 nm max 5

In acid hydrolysis (SN HCl], fragments I, II, III and V gave the following degradation products:

Hydrolysis conditions * reflux tube dense 1 Fragment 11Q ° C, 2Q hours 11Q ° C, 3 hours_ I IV, Ser II IV, Ser, HM-Val I, HM-Val

III IV

15 V - I, HM-Val, Sar

Ser: Serine HM-Val: 3-hydroxy-N-methylvaline

Sar: sarcosine

Basic hydrolysis of BBM-928 A or BBM-928 C with 0.1N NaOH

for 3 hours at 25 ° C gave fragment VI (abbreviations Ser, HM-Val, and Sar as above, while Gly represents glycine and the symbol X represents an unknown portion] of formula 6 of the formula sheet.

Treatment of fragment VI with Q, 1N HCl for 1 hour at 110 ° C gave fragment VII of formula 7 plus HM-Val.

Treatment of fragment VI with 0.1N NaOH for 40 hours at 37 ° C gave fragment VIII of formula 8 plus fragment I.

Treatment of fragment VII with Q, 1N NaOH for 40 hours at 37 ° C gave fragment IX of formula 9 plus fragment I.

The unknown portion CX) has a molecular formula C5HgN202 (in peptide form) based on microanalysis of fragment IX and other peptide fragments containing (X] and the spit language analysis below.

35 9001868 C-NMR proton NMR assigned to 30.14 Cti 2.36 ppm (2H, m) -Cl - - 14 -13 61.4 Cd) 1 4.2 - 4.5 C2H, m) 2 x -CH ^ 61.7 Cd] j '(0 or N) 5 140.7 Cd) 6.76 C1H, t) -CH «N- 171 Cs) - -CO- (amide)

-OH

NH

10 According to spectral data for fragments VIII and IX

and a 360 MHz proton NMR of ΒΒΜ-92Θ A ^ of Example IV, showed that the unknown amino acid portion (X) is best represented by the tetrahydro-pyridazine structure of formula 10.

Based on the results of the decomposition tests, spectral data, microanalysis and molecular weight determinations described above, BBM-928 A, B and C are believed to be best represented by formula 11 of the formula sheet, wherein R 1 R 2 Ser: serine " Gly: glycine 20 BBM-928 A Ac Ac Sar: sarcosine BBM-928 B Ac H HM-Val: β-hydroxy-N-methyl- BBM-928 C Η H Valine

AntimicrobiSle_effect:

The antimicrobial activity of BBM-928 components was determined. against a variety of bacteria and fungi by the agar dilution series method in nutrient agar at a pH of 7 using Steer's multi-grafting equipment. The graft size was standardized to apply 0.0025 3 4 3 cm test organisms containing approximately 10 cells / cm for all 30 bacteria and fungi except acid resistant bacteria using 6 3 for a 10 cells / cm suspension . The minimum inhibitory concentrations (MIC) determined after an overnight incubation at 37 ° C are shown in Table E. This shows that BBM-928 components are moderately to weakly effective against gram-positive and acid-resistant bacteria, but are practically inactive against gram-negative bacteria and fungi.

The activity of propagen 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 µg / cm.

Table E

In vitro antimicrobial activity of BBM-928 components against aerobic bacteria BBRI-BBM-928 components CMIC in µg / cm ^)

10 code Test organism A B C D s 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 Srlutea PCI 1001 6.3 12.5 50 50 50 25

Mf-1 M. flavus D12 12.5 25 50 100 100 25 15 Cr-1 C. xerosis 53K-1 25 50> 100> 100> 100 50

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 M. smegmatis 607D87 25 25 25 100 ~ 100 25 20 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-1 K. pneumoniae D-11> 100> 100> 100> 100> 100> 100

Pa-3 P.aeruginosa A9930> 100> 100> 100> 100> 100> 100

Pv-1 P.vulgaris A9436> 100> 100> 100> 100> 100> 100 25 Pm-1 P.mirabilis A9554> 100> 100> 100> 100> 100> 100

Pg-1 P.morganii A9553> 100> 100> 100> 100> 100> 100

Sm-1 S.marcescens A2001S> 100> 100> 100> 100> 100> 100

Al-1 A. faecalis ATCC8750> 100> 100> 100> 100> 100> 100

Ca-1 C.arbicans IAM4888> 100> 100> 100> 100> 100> 100 30 Cn-3 C.neoformans 100> 100> 100> 100> 100> 100

Antitum Contractivity:

Comparative study of BBM-928 components A, B, C and D with mitomycin C for anti-tumor activity was performed with the tumors intra-peritoneally implanted: P388 leukemia, L1210 leukemia,

80 0 1 80S

- 16/17 - B16 melanoma, Lewis Lung (LL) carcinoma, and sarcoma 180 ascites (S180). Test solutions of the 9-928 components in 0.9% brine, containing 10% dimethyl sulfoxide and mitomycin C in 0.9% brine, were administered once per day according to dosing schedules ranging from a single one-day treatment to multiple daily treatments. Varying the dose, the minimum effective dose (MED) was determined, which gave a mean survival value for the treated animals that was at least 1.25 times the size of a control group. This activity level is considered to be a measure of a significant anti-tumor activity. The results are shown in Table F, along with calculated efficacy ratios, showing that ΒΒΙΊ-928 A is significantly stronger than mitomycin C by a factor of 10-300, depending on the tumor strain and dosing schedule. Intrape-15 ritoneal LD ^ g values for ΒΒΙΊ-928 components A, B, C and D and mitomycin C, determined by the method of Van der Waerden, Arch. Expt. Path. Pharmak., 195, 389 (1940) are also shown in Table F.

Table F

20 Anti-tumor activity and toxicity of BBM-928 A,

B, C and D and mitomycin C.

Treatment Minimum effective dose (MED, mg / kg / day) LD ^ g, i.p. ling (a) P38B L121Q B16 LL S180 (mg / kg / day) BBM-928A 0.003> 0.1 0.003 0.03 0.003 0.13 25 BBM-928B 0.1 - - - 0.18 BBM-928C - 0 .81 BBM-928D 0.003 - - - 0.083 mitomycin CO. 13 1 0.3 0.3 9.3

Treated 30 ml 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 1.4

Activity ratio (BBM-928A to mitomycin C) 35 Treatment (a) 30 - 300 10 100 8001868 - 18 -

Table F (continued)

Treatment (b) 100 100 100 100 100 a. Simple treatment on day 1.

5 b. daily treatments from day 1 to day 9.

Example_I

Preparation_of_BBM; 928_cgmgle> <

Agar fermentation - A well grown agar slope of a strain of Actinomycetes, G455-101, was used to inoculate vegetative medium containing 2% soluble starch, 1% glucose, 0.5% pharmaceutical media, 0.5% yeast extract, 0.5 % NZ amine (type A) and 0.1% CaCOg, adjusting the pH to 7.2 before sterilization.

The seed culture was incubated for 72 hours at 32 ° C on a 3 rotary shaker (250 rpm), and 5 cm of the grown material was transferred to a 500 cm Erlenmeyer flask containing 100 3 cm cm fermentation medium consisting of 2 % soluble starch, 1% pharmaceutical media, 0.003% ZnSO ^ .7Η ^ 0 and 0.4% CaCOg. The preparation of the BBM-928 complex generally reached its maximum after about 5 days of shaking culture.

20 Barrel Fermentation - A seed culture was shaken in 3 ERlenmeyer flasks for 4 days and seeded in 100 dm germination medium, consisting of 2.0% oatmeal (Quaker Products, Australia), 0.5% glucose, 0.2% dry yeast, 0.0008 % MnC ^^ h ^ O, 0.0007% CuSO ^^ I ^ Q, 0.0002% ZnSO ^ .fl ^ O and 0.0001% FeSO ^ .71 ^ 0 in a 200 dm ^ entvatfer-25 mentor that stirred at 200 rpm for 3 hours at 30 ° C. A 15 dm portion of the seed culture was then titrated in 3 170 dm fermentation broth containing 2.0% soluble starch, 1.0% pharmaceutical 3 'media, 0.003% ZnSO 2 H 2 O and 0.4% CaCO 2. contained in a 400 dm vessel fermenter operated at 200 rpm at 30 ° C with an aeration rate of 150 dm per minute. The pH of the broth gradually increased with the progression of the fermentation and reached 8.4-8.5 after 100-120 hours, at which time an antibiotic strength peak of 30 µg / cm was obtained.

35 8001868 - 19 -

y ° orbseld_II

Isolation of_BBM ^ 28_cgrn £ le> <_ by oglosm ^ part extraction: 3

170 dm, pH 8.5, of the broth collected from Example I was filtered with a clarifier; Efficacy was found in both the mycerial cake 5 and the filtrate. The mycial cake was extracted twice with a solvent mixture of acetone and methanol Cl: 1, 30 dm, 2 times). The extracts were combined and evaporated under reduced pressure to obtain an aqueous concentrate extracted with π-butanol.

The broth filtrate was extracted twice with n-butanol (C2 x 40 dm). Concentration of the combined n-butanol extracts under reduced pressure and lycilization of the residue gave 21.4 g of a crude solid. According to a thin layer chromatography analysis, this material was a complex consisting of 3 main components A, B and C, and three secondary components D, E and F with Rf values as shown in Table G.

Table G

Silica gel TLC from BBM-928 components

RF values x 20 System N-118 xx System N-103 xxx BBM-928A 0.71 0.48 BBM-928B 0.53 0.26 BBM-928C 0.27 0.07 BBM-928D 0.73 0. 53 25 BBM-928E 0.56 0.34 BBM-928F 0.39 0.17 x: detection with UV scanner CShimadzu CS-910) at 345 nm xx: n-butanol - methanol - water C63: 27: 10) xxx : xylene - methyl ethyl ketone - methanol (5: 5: 1)

30 Example III

? yiy? iiQg_y§D_§0öl9? 8_cgmgl§x.

The crude complex of Example II was purified by a 3 preparative countercurrent distribution equipment (Mitamura, 100 cm per tube) using a solvent system of carbon-35 tetrachloride - chloroform - methanol - water (5: 2: 5: 1). After 800 l 68 - 20 - 50 passes, tubes 5-20 were combined and concentrated to give 4.4 g of a pale yellow powder containing the components. at A, B, D, E and F. This mixture was dissolved in a small amount of chloroform and placed on a column of silica gel C-200 3 5 C500 cm 3, which had been previously treated with ethyl acetate. The column was developed with ethyl acetate with an increasing amount of methanol (2 - 5% by volume 3 and the fractions were followed with an optical density at 345 nm. The component D was first eluted with ethyl acetate followed by component A. The Components E, B and F were then eluted in that order at a methanol concentration of 3% Each fraction containing the appropriate component was evaporated under reduced pressure and the residue was crystallized from chloroform-methanol Also a crude preparation of component C obtained from tubes 21 - 35 of the countercurrent distribution described above .. Purification of component C was carried out by silica gel chromatography and crystallization from chloroform-methanol. Yields for components A, B, C, D, E and F were 980 mg, 420 mg, 848 mg, 130 mg, 119 mg and 114 mg, respectively.

20 Preview_iy

Further purification_yan ^ compgnent_BBri-92SA_of_example ^ III

Thin layer chromatography analysis of the ΒΒΙΊ-928Α component of Example III (using a system consisting of 10% methanol in toluene) showed that the sample was not completely homogeneous. It contained additional material just before the BBM- 928A component was run The following steps were performed for purification.

(13 The sample was chromatographed on silica gel using a linear gradient from chloroform to 6% methanol-chloroform. Fractions eluted between 2.4 and 3.3% methanol-chloroform (containing the BBN-928A component plus contain some impurities] were pooled for the next step.

(23 A concave gradient was created using three vessels containing 2% methanol tololene in the first two and 6% metha 3001868-21 nol toluene in the third. The mixture of step (1) chromatographically over a silica gel column treated with this gradient gave a side component that was eluted first, closely followed by the purified BBM-928A component Chier designated 5 ΒΒΙΊ-928Α).

P

The molecular weight of BBM-928Ap, determined by field dispersion mass spectrometry, was 1427, corresponding to an empyric formula of Cg ^ H ^ N ^ O ^ (molecular weight 1427,417).

Elemental analysis (samples dried at 100 ° C for 18 hours): calculated for C 25 H 15 gN Q Q 53 53, H 5.51, N 13.74, 0a: 26.90.

found: C 52.47, H 5.48, N 13.81, 0: 28.243 a. by difference b. average of three determinations.

8001868

Claims (5)

1. Anti-tumor antibiotic compound BBM-928A with the following properties: a. Soluble in chloroform and methylene chloride, slightly soluble in benzene, ethanol, methanol and n-butanol, and practically insoluble in water and n-hexane b. gives a positive reaction with iron (III) chloride and Ehrlich reagent and a negative reaction with Tollens, Sakaguchi and ninhydrin reagent; c. is an effective anti-tumor agent against P388 leukemia, 11210 leukemia, B16 melanoma, Lewis Lung carcinoma, sarcoma 180 ascitep tumors, implanted intraperitoneally in the mouse; d. has a melting point of 246-248 ° C; 25 e. has a specific rotation of ZH7D = 27 ° (c 1, EHClg); f. has a molecular weight of 1427; 15 g. has an elemental composition of about 52.47% carbon, 5.48% hydrogen, 13.81% nitrogen and 28.24% oxygen; h. Silica gel thin layer chromatography shows an R 1 value of 0.71 with the solvent system n-butanol-methanol-water (63: 27: 10); and an Rp value of 0.48 with the solvent system xylene - methyl ethyl ketone - methanol (5: 5: 1); i. on acid hydrolysis, water-soluble ninhydrin gives positive materials, including / 3-hydroxy-N-methylvaline, glycine, serine and sarcosine; j. in basic hydrolysis, fragment VI of formula 6, wherein 25 represents ser setin, Gly represents glycine, Sar represents sarcosine, HM-Ual / 3-hydroxy-N-methylvaline and the symbol X represents an amino acid portion; k. has an infrared absorption spectrum in potassium bromide substantially according to Fig. 1; and 1. upon deposition in deuterated chloroform, gives a proton NMR spectrum substantially as shown in FIG. 5. The anti-tumor antibiotic according to claim 1, wherein the amino acid portion 8001868-23 acid X has the empirical formula C_HcN_CL in peptide form. Antitumor antibiotic according to claim 2, characterized in that the amino acid portion X in peptide form is the tetrahydropyridazine group of formula 10. The anti-tumor antibiotic of claim 1, of formula 11, wherein Ser represents serine, Gly represents glycine, Sar represents sarcosine, HM-Val / 3-hydroxy-N-methylvaline, and and R 2 are acetyl. 5. Anti-tumor antibiotic compound 8BP1-928B with a quinoline chromophore which gives water-soluble components, including serine, glycine, sarcosine and / 3-hydroxy-N-methylvaline, with acid hydrolase, and which in substantially pure form has the following properties: a soluble in chloroform and methylene chloride, slightly soluble in benzene, ethanol, methanol and n-butanol, and practically insoluble in water and n-hexane b. gives a positive reaction with iron (III) chloride and Ehriich reagent and a negative reaction with Tollens, Sakaguchi and nin-hydrien reagent; 20 c. is an effective anti-tumor agent against P388 leukemia implanted intraperitoneally in the stomach; d. has a melting point from 214 - 217¾ e. has a specific rotation of = * -74 ° C (1, CHCl 3); f. has an elemental composition of about 50.14% carbon, 5.29% hydrogen, 12.34% nitrogen, and 32.23% oxygen; g. shows a R ^ value of 0.53 with the n-butanol - methanol - water solvent system (S3: 27; 19], and an R ^ value of 0.26 with the xylene - methyl ethyl ketone - methanol solvent system (5: 5) in silica gel thin layer chromatography 30 h has an infrared absorption spectrum in potassium bromide substantially as shown in Figure 2 and when dissolved in deuterated chloroform it gives a proton NMR spectrum substantially as shown in Figure 6. The compound of claim 5 having the structure of Formula 11, wherein Ser represents serine, Gly represents glycine, Sar 8001868 * - 24 - represents sarcosine, HM-Val / 3-hydroxy-N-methylvaline, R ^ acetyl, and R2 is hydrogen. 7. Anti-tumor antibiotic compound BBM-928C with a quinoline neophromophore which, in acid hydrolysis, gives water-soluble components including serine, glycine, sarcosine, and / 3-hydroxy-N-methylvaline, and in substantially pure form has the following properties: a. soluble in chloroform and methylene chloride, slightly soluble in benzene, ethanol, methanol and n-butanol, and practically insoluble in water and n-hexane; b. gives a positive reaction with iron [III] chloride and Ehrlich reagent and a negative reaction with Tollens, Sakaguchi and ninhydrin reagent c. is an effective anti-tumor agent against B388 leukemia, L1210 leukemia, BIS melanoma, Lewis Lung carcinoma, and sarcoma 180 ascii tumors implanted intraperitoneally in the mouse; d. has a melting point of 244 - 248 ° C; e. has a specific rotation = -91 ° C (1, CHCl 3); f. has a molecular weight of about 1470; 20 g. has an elemental composition of about 51.77% carbon, 5.29% hydrogen, 13.55% nitrogen, and 29.39% oxygen, · h. Silica gel thin layer chromatography shows an R ^ value of 0.27 with a solvent system n-butanol - methanol - water (63: 27: 10], and an R ^ value of 0.07 with the solvent system xylene - methyl ethyl ketone - methanol (5: 5: 1); i. has an infrared absorption spectrum in potassium bromide substantially as shown in Figure 3; and j. when dissolved in deuterated chloroform, it gives a proton NMR spectrum mainly as shown in Figure 7. A compound according to claim 7, having the structure of formula 11, wherein Ser represents serine, Gly represents glycine, Sar represents sarcosine, Htl-Val / 3-hydroxy-N-methylvaline, R 1 and R 1 represent hydrogen. 9. Antitumar antibiotic BBM-928D having the following properties: too 1868 - 25 - a. Soluble in chloroform and methylene chloride, slightly soluble in benzene, ethanol, methanol and n-butanol, and practically insoluble in water and n-hexane b . gives a positive reaction with iron (III] chloride and Ehrlichf 5 reagent and a negative reaction with Tollens, Sakaguchi and ninhydrin reagent c. is an effective anti-tumor agent against intraperitoneally implanted P388 leukemia in the mouse; d. has a melting point of 224 - 227 ° C 10 e. Has a specific rotation of L «3% = -13 ° (c 1, CHCl 3); f. Has an elemental composition of about 50.75% carbon, 5.25% hydrogen, 12.58% nitrogen, and 31.42% oxygen; g.has a silica gel thin layer chromatography an R ^ value of 0.73 with the solvent system n-butanol - methanol - water C63: 27; 10], and an R 2 value of 0.53 with the solvent system xylene - methyl ethyl ketone - methanol (5: 5: 1]; h. Has an infrared spectrum in potassium bromide substantially according to FIG. 4; and i. dissolving in deuterated chloroform a proton NMR spectrum mainly according to fig. 8. 10. Fermentation process for preparing anti-tumor antibiotic BBM-928 complex, wherein a strain of Actinomycetes with the properties of strain G455-101 is grown in an aqueous solution containing an assimilable carbon source and an assimilable nitrogen source under submerged aerobic conditions, until the BBM-928 complex obtained imparts significant anti-tumor and antibiotic activity to the solution. The method of claim 10, wherein the BBM-928 complex is additionally recovered from the culture medium. The method of claim 10, wherein the BBM-928 complex is additionally separated into components A, B, C, D, E, and F, which components exhibit the following R 1 values in silica gel thin layer chromatography: 8001868 ψ - 26 - R_P ~ values π-butanol-methanol-xylene-methyl-ethyl-water (63:27:10) Ketone-methanol (5: 5: 1) ^ 1 - I '·· "BBM-928A 0.71 0.48 5 BBM-928B 0.53 0.26 BBM-928C 0.27 0.07 BBM-928D 0.73. 0.53 BBM-928E .0.56 0.34 BBM-928F 0.39 0.17 10. 13. Antitumar activity and / or antibiotic activity preparation, characterized in that it contains the BBH-928 complex or one of its components . 8001868