GB2050384A - Antitumor antibacterial agents - Google Patents

Abstract

An antitumor antibacterial complex having a quinoline nucleas referred to as BBM-928 is described which is produced by microbiological fermentation under controlled conditions using a species of actinomycetes. The bioactive components of the complex have pronounced antitumor activity and are moderately to weakly active against gram-positive and acid fast bacteria but practically inactive against gram- negative bacteria and fungi. Based on median survival time, the component designated BBM-928A is substantially more active than mitomycin C by a factor of 10 to 300 fold against intraperitoneally implanted mouse tumors.

Description

SPECIFICATION Antitumor antibacterial agents This invention relates to a new antitumor antibiotic complex and to processes for the production thereof, the recovery and separation into bioactive components.

Based on present spectral data and available physico-chemical properties, and antitumor antibiotic 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), andtriostin C, The Merck Index, 9th Edition, 9399. However, the antitumorantibiotic complex of the present invention differs from these antibiotics in the following respects: 1. The present complex and components thereof contain a quinoline nucleus as a chromophore instead of quinoxaline as in the actinoleukin group of antibiotics.

2. The present complex and components thereof do not contain sulfur in contrast to the presence of disulfide or thioacetal bridge in the structure of actinoleukin antibiotics.

3. The present complex and components thereof show relatively weak antimicrobial activity compared to the actinoleukins which are potent antibacterial antibiotics.

A new antitumor antibiotic complex designated BBM-928 is provided by the present invention. The complex, which contains at least six components, is prepared by cultivating a BBM-928 producing strain of actinomycetes (ATCC 31491) or a mutant thereof in an aqueous nutrient medium employing submerged aerobic conditions. This invention also deals with a process for recovering the BBM-928 complex from the culture medium and the separation of the complex into its bioactive components by countercurrent distribution and chromatographic techniques.

The present invention includes within its scope the BBM-928 complex and bioactive components BBM-928 A, B, C, D, E, and F thereof. It is particularly concerned with components BBM-928 A, B, C, and D in dilute forms, as true concentrates and in purified form.

This invention provides a new antitumor antibiotic complex arbitrarily designated herein as BBM-928. The complex is believed to be structurally related to the actinoleukin group of antibiotics and is formed by the fermentation of an as yet unidentified strain of actinomycetes. Any strain of actinomycetes capable of forming BBM-928 in a culture medium may be used with the preferred producing microorganism designated actinomyctes species strain No. G455-101 in the Bristol-Banyu culture collection. The microorganism was isolated from a soii sample collected in the Philippine Islands and has been deposited with the American type culture collection of microorganisms at ATCC 31491.

The novel antitumor antibiotic complex of the present invention referred to herein has at least six components designated BBM-928A, B,C,D,E and F. Components A, B, C and D of the BBM-928 complex have been isolated in crystalline form and the chemical structures of components A, B and C identified. The complex and the individual component thereof have antitumor antibacterial properties. With respect to antibacterial activity, the complex and individual components thereof are useful as nutritional supplements in animal feeds and as therapeutic agents in treating bacterial infection in mammals. Additionally, the antibiotics are useful in cleaning and sterilizing laboratory glassware and surgical intruments and may be used in combination with soaps, detergents and wash solutions for sanitation purposes.Regarding antitumor effects, the complex and individual components thereof are particularly useful against a variety of intraperitoneally implanted mouse tumors.

The identification of components A, B, C and D of the complex BBM-928 has been made with reference to the infrared absorption and proton magnetic resonance, spectra thereof. These spectra are given in the accompanying drawings, in which: Figure 1 is the infrared absorption spectrum of BBM-928A in potassium bromide.

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

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

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

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

Figure 6 shows the proton magnetic resonance spectrum of BBM-20 928B dissolved in deuterated chloroform using TMS as internal standard determined with an NMR spectrometer at a frequency of 90 MHz.

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

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

Actinomycetes Species Strain No. G455- 101 The following is a general description of the preferred microorganism producing the antibiotic antitumor complex BBM-928. Observations were made of the cultural, physiological, and morphological features of the organism in accordance with standard taxonomic methods, e.g., Shining, et al., Int. J. Syst. Bacteriol. 16, 313 (1966) and Lechevalier, et al., Biol. Actinomycetes Related Org. 11,78 (1976).

Micromorphology.- Strain No. G455-101 forms both substrate and aerial mycelia, and the substrate mycelium is well-developed, long and branched (0.5-0.8 y in width). Distinct fragmentation of the substrate mycelium is not seen. Unlike ordinary species of Streptomyces, strain G455- 101 bears only short or rudimental aerial mycelia, or does not form any in some agar media. Short or long spore-chains are produced in the aerial mycelium, which contain 2-50 oval spores in a chain (mostly 5-20 spores).

Spore-chains are straight, flexuous or looped in shape. The spores are spherical (0.3-0.4 tut), oval or cylindrical (0.3 x 1.5 - 3.0 ) in shape and have smooth surface. Spores are often separated by empty hyphae. An amorphous sporangium-like vesicle which envelops short coiled spore-chain is observed occasionally on the aerial mycelium.

Cell Wall Composition 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, manose and madurose (3-0-methyl-D-galactose). The aforementioned cell wall composition and whole cell sugar components indicate that strain G455- 101 is an actinomycetes species of cell wall type IlIB.

Cultural and Physiological Characteristics.- Strain G455- 101 grows abundantly, forms pink or greyish pink aerial mycelium, and produces a reddish water-insoluble pigment in the nutritionally rich agar media, such as yeast extract - malt extract agar and oat meal agar. However, in inorganic salts - starch agar, glycerol asparagine agar and tyrosine agar, it gives poor growth, forms white or beige rudimental aerial mycelium, and produces small amount of 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., 37"C., and 45"C., but does not grow at 10"C. or at 50"C. Pentoses and hexoses are well utilized by the strain. Cultural and physiological characteristics of strain G455-101 are shown in Tables 1 and 2, respectively. Utilization of carbon sources is shown in Table 3.

TABLE 1 Cultural Characteristics of Strain No. G455- 101* 1. Czapek's agar no no or scant growth (Sucrose - nitrate agar) R dark rose A white to pale pink D none 2. Tryptone - yeast extract moderate growth, floccose, broth (ISP No. 1) sedimented and not pigmented 3. Yeast extract malt extract G abundant agar (ISP No. 2) R deep red to reddish brown A abundant, greyish pinkto purplish pink D none 4. Oat meal agar (ISP No.3) G abundant R strong yellowish red A moderate, pink D greyish yellow 5. Inorganic salts - starch G poor agar (ISP No. 4) light yellowish brown to dark red A scant, white to beize D none 6.Glycerol-asparagine agar G poor (ISP No. 5) R yellowish pink to reddish brown A scant, white D none TABLE 1 - Continued 7. Peptone-yeast extract-iron G poor, plicate agar (ISP No.6) R strong, reddish orange A none D light yellowish orange 8. Tyrosine agar (ISP No.7) g poor R dark red A scant, white D none 9. Glucose-ammonium salts G poor agar R reddish brown A scant, light grey D none 10.Bennet's agar G moderate R reddish brown A restricted, greyish pink D none * observed after incubation at 37"C. for 3 weeks **Abbreviation: G-Growth R - Reverse color of substrate mycelium A-Aerial mycelium D - Diffusible pigment TABLE 2 Physiological Characteristics of Strain No. G455- 101 Test Response Method and Medium Nitrite from nitrate Positive Inorganic medium:Czapek's sucrose nitrate broth Nitrite from nitrate Positive Organic medium: 0.5% yeast extract, 1.0% glucose, 0.5% KNO3, 0.1% CaCO3 Casein hydrolysis Weakly positive Luedemann's agar medium in agar medium Skimmed milkcoagu- Positive lation Gelatin liquefaction Negative 15% gelatin in tryptone-yeast extract broth (ISP No. 1 medium) H2S production from Position L-Cysteine (0.1%) added to L-cysteine tryptone-yeast extract broth (ISP No. 1 medium) plus agar.

H25 detected with a paper strip containing 10% aq.

lead-acetate solution.

Formation of melanoid Negative Peptone-yeast-iron agar (ISP No. 6) and tyrosine agar (ISP No. 7).

Catalase reaction Positive H202 aq. solution Oxidase reaction Positive k'ovacs' reagent Growth temperature Abundant growth Bennett's agar at 28,37 and 45 C. Poor growth at 20 C. No growth at 10 C. and 50 C.

TABLE 3 Utilization of Carbon Sources for Strain No. 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. Melibiose - + 16. Trehalose + + 17. Raffinose 18. D(+)-Melezitose 19. Solubie starch + 20. Dulcitol 21. Inositol + + 22. D-Mannitol ++ + 23. D-Sorbitol 24. Salicin 25. Cellulose + + 26. Chitin + + 27.Keratin + + Basal medium PG: Pridham - Gottlieb's inorganic medium, supplemented with 0.1% yeast extract LM: LM: Luedemann's organic medium Incubation for 2 weeks at 37 C It is understood that production of the BBM-928 complex of the present invention is not limited to the particular actinomycetes species strain No. G455-101 described by the above growth and microspcopic characteristics. These characteristics are given for illustrative purposes only and the invention contemplates use of strains or mutants produced from the above-described organism by conventional means known to the art such as exposure to X-ray radiation, ultraviolet radiation, nitrogen mustard, phage exposure, and the like, which are capable of producing the BBM-928 complex or individual components thereof.

Preparation ofAntitumorAntibiotic BBM-928 Complex The process of the present invention for producing the anti-tumor antibiotic BBM-928 complex comprises cultivating by fermentation actinomycetes strain No. G455-101 in an aqueous solution containing an assimilable carbon source and an assimilable nitrogen source under submerged aerobic conditions until substantial antitumor antibiotic activity is imparted to said solution. ConventiOnal fermentation methods are employed in cultivating actinomycetes strain No. G455-101. Media which are useful of the production of the antibiotic antitumor agents of the instant invention include an assimilable source of carbon 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 hydrolysate, polypeptides, amino acids, corn steep liquor, casein, urea and the like as well as nutrient inorganic salts which provide inorganic anions and cations such as potassium, sodium, ammonium, calcium, sulfate, carbonate, phosphate, chloride, nitrate, and the like.

In producing the BBM-928 complex, any temperature conducive to satisfactory growth of the organism may be employed. Temperatures ranging from about 20 to 45"C. are operable with a preferred temperature for optimizing growth of the organism ranging from about 28O to 340C. with a temperature range of 30 to 32 most preferred. Maximum production of the BBM-928 complex is generally obtained in about 4 to 6 days.

Conventional methods are employed in the fermentation period. For example, preparation of small amounts is conveniently carried out in shake flasks or by surface cultures. Preparation of large amounts is preferably carried out under submerged aerobic culture conditions in sterile tanks. With tank fermentation a vegetative inoculum is first produced in a nutrient broth by innoculating the broth culture with a spore from the organism to provide a young active seed culture which is then aseptically transferred to the fermentation tank medium. Aereation in tanks and bottles may be provided by forcing sterile air through or onto the service of the fermenting medium with further agitation in tanks provided by a mechanical impeller.

Antifoaming agents such as silicone oil, soybean oil and lard oil may 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 assay using Sarcina Lutea as a test organism and employing nutrient agar as the assay medium. The pH is adjusted 9.0 for optimal sensitivity of the assay system which is used to determine optimum broth potency.

The BBM-928 complex is isolated from the fermentation broth by conventional means such as solvent extraction procedures. Purification is conveniently carried out by preparative countercurrent distribution and chromatographic procedures as more fully described in Examples 2 and 3 below to provide BBM-928 components A, B, C, D, E, and F.

Physico-Chemical Properties of BBM-928 Components A, B, C, and D of Example 3 Individual components of BBM-928 show solubility and color reactions similar to each other. For example, they are 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 ferric chloride and Ehrlich reagents are obtained with negative reactions to Tollens, Sakaguchi and ninhydrin.

Characteristic physico-chemical properties of BBM-928 components of Example 3 are shown in Table 4.

TABLE 4 Physico-chemical 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 [al25 (c 1, CHCI3) -27" -74" -91" -13" Anal. 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) 0: 28.49 32.23 29.39 31.42 Bmax in nm (E1%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) in EtOH-HCI 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) Mol. wt.

(Osmometer, in CHCI3)1,450 - 1,470 Infrared (IR) and nuclear magnetic resonance (NMR) spectra of components A, B, C and Dare shown in Figure 1 to 4 and Figures 5 to 8, respectively, of the accompanying drawings. The NMR spectra of BBM-928A (Figure 5), BBM-928B (Figure 6) and BBM-928C (Figure 7) are very similar to each other with the only difference being the presence of acetyl groups in components A (8 : 2.03 ppm. 2 mol equivalent) and B (b: 2.05 ppm, 1 mol equivalent) but not in C. Upon acetylation with acetic anhydride in pyridine 2, 3 and 4 molar equivalents of acetyl group were introduced to BBM-928 components A, B and C, respectively.The three acetylation products thus obtained showed identical properties in 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 afforded five n-butanol-soluble,UV-absorbing fragments (I,II,lll,IV and V) and 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: Compounds Rf (S-1 23)* Identification NPS-1 0.72 f3-hydroxy-N-methyl- valine (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 assigned) *TLC, silica gel plate S-123: 10% ammonium acetate - methanol - 10% amonia solution (9:10:1) The five UV-absorbing fragments referred to above are shown to have the following structures:

Fragment I C14H14N2O6 MS: m/e 306 (Mt) MeOH : 227, 233, 251, 345 nm max

Fragment II C20H25N308 MS: m/e 377 (M±58) #MeOH : 229, 234, 261, max 345 nm

Fragment III #max MeOH : 230, 235, 262, 345 nm max Fragment IV C10H7NO4 MS : m/e 205 (M+) #MeOH max: 228, 260, 354 nm max

Upon acid hydrolysis (6N HCI), Fragments I, II, III and V gave the following degradation products: Hydrolysis condition sealed tube refluxed Fragment 110 , 20 hrs 110 C, 3 hrs IV, Ser II IV, Ser, HM-Val I, HM-Val Ill IV V - i, HM-Val, Sar Ser: serine HM-Val : ss-hydroxy-N-methylvaline Sar: sarcosine Base hydrolysis of BBM-928A or BBM-928C with 0.1 N NaOH at 25 C for 3 hour provides Fragment VI (abbreviations Ser, HM-Val, and Sar are as stated above with Gly representing glycine and the symbol "X" representing an unassigned moiety)

Fragment Vl Treating Fragment VI with 0.1 N HCI at 1 lOoC. for 1 hr. provides Fragment VII plus HM-Val.

Fragment VII Treating Fragment VI with 0.1 N NaOH at 37 C. for 40 Hr. provides Fragment VIII plus Fragment I.

X-Gly-Sar-HM-Val Fragment VIII Treating Fragment VII with 0.1 N NaOH at 37 C. for 40 hr. provides Fragment IX plus Fragment I.

X~Gly~Sar Fragment IX The unassigned moiety (X) has a molecular formula of C5H6N202 (in peptide form) based on microanalysis of Fragment IX and other peptide fragments containing (X) and spectral analysis summarized below.

13C-NMR Proton-NMR Assignment 30.14 (t) 2.36 ppm (2H, m) -CH2 61.4(d)} 4.2-4.5 (2H, m) 2x-CH 61.7(d) (OorN) 140.7 (d) 6.76(1H,t) -CH=N 171 (s) - -CO- (amide) -OH NH According to spectral data for Fragments VIII and IX and a 360 MHz proton NMR of BBM-928Ap of Example 4, the unassigned amino acid moiety (X) appears to be best represented by the following tetrahydropyridazine structure:

Based on the results of the above-described degradation experiments, spectral data, microanalysis and molecular weight determinations, the following structures are believed to best represent BBM-928A, B and C :

R1 R2 Ser : serine Gly : glycine BEM-928 A Ac Ac Sar : sarcosine BBM-928 B Ac H HM-Val : 0-hydroxy-N-methylvaline BBM-928 C H H Anti.s7icrobialAcvitity Antimicrobial activity of BBM-928 components was determined against a variety of bacteria and fungi by serial agar dilution method in nutrient agar at pH 7 using Steer's multi-inoculating apparatus. The inoculum size was standardized to apply a 0.0025-ml aliquot of test organisms containing approximately 104 cell/ml for all bacteria and fungi except acid-fast bacteria for which a 106 cell/ml suspension was used. Minimum inhibitory concentrations (MIC), determined after overnight incubation at 37 C., are shown in Table 5. As seen therein, BBM-928 components are moderately to weakly active against gram-positive and acid fast bacteria but practically 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 demonstrated with BBM-928 components A, B and C up to a concentration of 100 mcg./ml.

TABLE 5 In Vitro antimicrobial Activity of BBM-928 Components Against Aerobic Bacteria BBRI BBM-928 Components (MIC in mcg/ml) Code Test Organism A B C D E F Sa-1 S. aureus 209P 12.5 15 50 100 100 25 Sp-1 S. pyogenes S-23 6.3 12.5 15 50 50 12.5 Sl-1 5. lute PCI 1001 6.3 12.5 50 50 50 25 Mf-1 M.flavusDl2 12.5 25 50 100 100 25 Cr-1 Cxerosis53K-1 25 50 > 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. anthracisA9504 6.3 12.5 25 50 50 12.5 M6-1 M. smegmatis 607 D87 25 25 25 100 100 25 Mp-1 M.phIeiD88 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. vulgarisA9436 > 100 > 100 > 100 > 100 > 100 > 100 Pm-1 P. mirabilis A9554 > 100 > 100 > 100 > 100 > 100 > 100 Pg-1 P. morganiiA9553 > 100 > 100 > 100 > 100 > 100 > 100 Sm-1 S. 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 AntitumorActivity Comparative testing of BBM-928 components A, B, C, and D with mitomycin C for antitumor activity was carried out with the intraperitoneally implanted tumors:P388 leukemia, L1210 leukemia, B16 melanoma, Lewis Lung (LL) carcinoma, and sarcoma 180 ascites (S180). Test solutions of the BBM-928 components in 0.9% saline containing 10% dimethylsulfoxide and mitomycin C in 0.9% saline were administered once a day according to dosing schedules ranging from a single one-day treatment to multiple daily treatments. By varying dosage, the minimum effective dose (MED) was determined which provided a median survival time value for the treated animals at least 1.25 times greater than a control group. This level of activity is considered to be a measure of significant antitumor activity. Results are shown in Table 6 along with calculated activity ratios illustrating that BBM-928A is markedly more active than mitomycin C by a factor of 10 to 300 depending upon the tumor strain and dosing schedule.Intraperitoneal LD50 values of BBM-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 6.

TABLE 6 Antitumor Activity and Toxicity of BBM-928 A, B, C and D and Mitomycin C Minimum Effective Dose (MED, mg/kg/day) LD50, i.p.

P388 -1210 B16 LL S180 (mg/kg/day) Treatmenta 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 Treatmentb 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 vs. Mitomycin C) Treatment a 30 - 300 10 100 Treatment b 100 100 100 100 100 a. single treatment on day 1.

b. daily treatments from day 1 to day 9.

EXAMPLE 1 Production ofBBM-928 Complex AgarFermentation. - A well-grown agar slant of a strain of Actinomycetes species G455-101 is used to inoculate vegetative medium containing 2% soluble starch, 1% glucose, 0.5% Pharmamedia, 0.5% yeast extract, 0.5% NZ-amine (Type A) and 0.1% CaCO3 with the pH being adjusted to 7.2 before sterilization. The seed culture is incubated at 32 C for 72 hours on a rotary shaker (250 rpm), and 5 ml. of the growth 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% CaCO3.The production of the BBM-928 complex generally reaches maximum after about 5 days shaking culture.

Tank Fermentation. - A seed culture is shaken for 4 days in Erlenmeyer flasks and inoculated to 100 liters of germination medium composed of 2.0% oat meal (Quaker Products, Australia), 0.5% glucose, 0.2% dry yeast, 0.0008% MnCI24H2O, 0.0007% CuSO47H2O, 0.0002% ZnSO4-7H2O and 0.0001% FeSO47H2O in a 200 liter seed tank fermentor which is stirred at 200 rpm. at 30"C. for 54 hou rs.A 15-liter portion of the seed culture is then inoculated to 170 liters of fermentation medium containing 2.0% soluble starch, 1.0% Pharmamedia, 0.003% ZnSO4.7H2O and 0.4% CaCO3 in a 400-liter tank fermentorwhich is operated at 30 C. at 200 rpm with an aeration rate of 150 liters min. The broth pH gradually increases with the progress of fermentation and reaches 8.4 - 8.5 after 100 - 120 hours at which time a peak antibiotic potency of 30 mcg/ml is obtained.

EXAMPLE 2 Isolation ofBBM-928 Complex by Solvent Extraction Harvested broth (170 liters, pH 8.5) from Example 1 is filtered with a clarifying agent. Activity is found in both the mycerial cake and filtrate. The mycerial cake is extracted twice with a solvent mixture of acetone and methanol (1:1,30 liter x 2). Extracts are combined and evaporated under reduced pressure to give an aqueous concentrate which is extracted with n-butanol. The broth filtrate is extracted twice with n-butanol (40 liter x 2). Concentration of the combined n-butanol extracts under reduced pressure and lyophylization of the residue provides a crude solid (21.4 g).).According to thin layer chromatography assay, this material is a complex consisting of three major components, A, B and C, and three minor components, D, E and F having Rf values as set forth in Table 7 below.

TABLE 7 Silica gel TLC of BBM-928 Components Rf Values System N-118** System N-103*** 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-928E 0.56 0.34 BBM-928F 0.39 0.17 *detection by UV scanner (Shimadzu CS-910) at 345 nm **n-butanol - methanol - water (63 : 27:10) ***xylene - methylethylketone - methanol (5 : 5:1) EXAMPLE 3 Purification ofBBM-928 Complex The crude complex of Example 2 is purified by a preparative counter current distribution apparatus (Mitamura, 100 ml/tube) using a solvent system of carbon tetrachloride - chloroform - methanol - water (5 : 2 : 5: 1).After 50 transfers, tube Nos. 5 through 20 are combined and concentrated to give pale yellow powder (4.4 g.) containing components A, B, D, E and F. This mixture is dissolved in a small amount of chloroform and charged on 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%, WV) and fractions monitored by optical density at 345 nm. Minor component D is eluted first with ethyl acetate followed by component A.

Components E, B and F are eluted next in that order at 3% methanol concentration. Each fraction containing the appropriate component is evaporated under reduced pressure and the residue crystallised from chloroform - methanol. Likewise, crude preparation of component C is obtained from tube Nos. 21 through 35 of the above-described counter current distribution. Purification of component C is carried out by silica gel i:hromatography and crystallization from chloroform-methanol. Yields for components A, B, C, D, E and F are, respectively 988 mg., 420 mg., 848 mg., 130 mg., 119 mg., and 114 mg.

EXAMPLE 4 Further Purification of Component BBM-928A of Example 3 Thin layer chromatography assay of the BBM-928A component of Example 3 (employing a system consisting of 10% methanol in toluene) indicated the sample was not completely homogenous in that it contained additional material running just ahead of the BBM-928A component. The following steps were carried out to effect 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 contaminant) are composited for the next step.

2. A concave gradient is generated using three vessels containing 2% methanol toluene in the first two and 6% methanol-toluene in the third. The composite from Step 1 chromatographed (silica gel column) on this gradient provides a minor component which elutes first closely followed by the purified BBM-928A component (referred to herein as BBM-928Ap).

Molecular weight of BBM-928Ap, as determined by Field Desorption Mass Spectrometry, is 1427 corresponding to an empirical formula of C64H78N14024 (MW 1427.417).

Elemental Analysis (Samples dried at 100 Cfor 18 hours).

C H N oa Caiculated for C64H78N14024: 53.85 5.51 13.74 26.90 Foundb: 52.47 5.48 13.81 28.24a a. By difference.

b. Average of three determinations.

Claims (17)

1. The antitumor antibiotic compound BBM-928A which has the following characteristics: (a) soluble in chloroform and methylene chloride, slightly soluble in benzene, ethanol, methanol and n-butanol, and substantially insoluble in water and n-hexane; (b) gives a positive reaction with ferric chloride and Ehrlich reagents and a negative reaction with Tollens, Sakaguchi and ninhydrin reagents; (c) is an effective antitumor agent against P388 leukemia, L1210 leukemia, B16 melanoma, Lewis Lung carcinoma, and sarcoma 180 ascitestumours intraperitoneally implanted in the mouse; (d) has a melting point of 246-248 C.; (e) has a specific rotation of [a]D5 = 27 (c 1, CHCI3); (f) has a molecular weight of 1427;; (g) has an approximate elemental composition of 52.47% carbon, 5.48% hydrogen, 13.81% nitrogen, and 28.24% oxygen; (h) exhibits in silica gel thin layer chromatography an Rf value of 0.71 with the solvent system n-butanol-methanol-water (63:27:10), and a Rf value 0.48 with the solvent system xylene-methylethylketonemethanol (5:5::1); (i) affords on acid hydrolysis water-soluble ninhydrin positive substances including beta-hydroxy-N methyl-valine, glycine, serine, and sarcosine; (j) affords on base hydrolysis Fragment VI

Fragment Vl wherein Ser is serine, Gly is glycine, Sar is sarcosine, HM-Val is beta-hydroxy-N-methylvaline and the symbol X represents an amino acid moiety; (k) has an infrared absorption spectrum in potassium bromide substantially as shown in Figure 1; and (I) when dissolved in deuterated chloroform gives a proton NMR spectrum substantially as shown in Figure 5.

2. The antitumor antibiotic as claimed in claim 1 wherein the amino acid moiety X has the empirical formula C5H6N202 in the peptide form.

3. The antitumor antibiotic as claimed in claim 2 wherein the amino acid moiety X in the peptide form is the tetrahydropyridazine radical

4. The antitumour antibiotic compound as claimed in claim 1 which has the structure

wherein Ser is serine, Gly is glycine, Saris sarcosine, HM-Val is beta-hydroxy-N-methylvaline and R1 and R2 are acetyl.

5. The antitumor antibiotic compound BBM-928B which has a quinoline chromophore and which affords, on acid hydrolysis, water soluble components including serine, glycine, sarcosine, and beta-hydroxy-N methyl-valine, which in its substantially pure form has the following characteristics:: (a) soluble in chloroform and methylene chloride, slightly soluble in benzene, ethanol, methanol, and n-butanol, and substantially insoluble in water and h n-hexane; (b) gives a positive reaction with ferric chloride and Ehrlich reagents and a negative reaction with Tollens, Sakaguchi, and ninhydrin reagents; (c) is an effective antitumor agent against P388 leukemia intraperitoneally implanted in the mouse; (d) has a melting point of 214-21 7'C; (e) has a specific rotation of [aiD25 = -74 (c 1, CHCI3); (f) has an approximate elemental composition of 50.14% carbon, 5.29% hydrogen, 12.34% nitrogen, and 32.23% oxygen;; (g) exhibits in silica gel thin layer chromatography an Rf value of 0.53 with the solvent system n-butanol-methanol-water (63:27:10), and a Rf value of 0.26 with the solvent system xylenemethylethylketone-methanol (5:5:1); (h) has an infrared absorption spectrum in potassium bromide substantially as shown in Figure 2; and (i) when dissolved in dueterated chloroform gives a proton NMR spectrum substantially as shown in Figure 6.

6. The compound as claimed in claim 5 which has the structure:

wherein Ser is serine, gly is glycine, Sar is sarcosine, HM-Val is beta-hydroxy-N-methylvaline and R1 is acetyl.

7. The antitumor antibiotic compound BBM-928C which has a quinoline chromophore and which affords, on acid hydrolysis, water soluble components including serine, glycine, sarcosine, and beta-hydroxy-Nmethyl-valine, which in its substantially pure form has the following characteristics: (a) soluble in chloroform and methylene chloride, slightly soluble in benzene, ethanol, methanol and n-butanol, and substantially insoluble in water and n-hexane; (b) gives a positive reaction with ferric chloride and Ehrlich reagents and a negative reaction with Tollens, Sakaguchi and ninhydrin reagents; (c) is an effective antitumor agent against P388 leukemia, L1210 leukemia, B16 melanoma, Lewis Lung carcinoma, and sarcoma 180 ascites tumors intraperitoneally implanted in the mouse; (d) has a melting point of 244-248 C;; (e) has a specific rotation of [a]25 = -91" (c 1, CHCl3); (f) has an appropriate molecular weight of 1470; (g) has an approximate elemental composition of 51.77% carbon, 5.29% hydrogen, 13.55% nitrogen, and 29.39% oxygen; (h) exhibits in silica gel thin layer chromatography an Rf value of 0.27 with a solvent system n-butanol-methanol-water (63:27:10), and a Rf value of 0.07 with the solvent system xylenemethylethylketone-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 gives a proton NMR spectrum substantially as shown in Figure 7.

8. The compound as claimed in claim 7 which has the structure:

wherein Ser is serine, Gly is glycine Saris sarcosine, and HM-Val is beta-hydroxy-N-methylvaline.

9. The antitumor antibiotic BBM-928D having the following characteristics (a) soluble in chloroform and mthylene chloride, slightly soluble in benzene, methanol and n-butanol, and substantially insoluble in water and n-hexane; (b) gives a positive reaction with ferric chloride and Ehrlich reagents and a negative reaction with Tollens, Sakaguchi and ninhydrin reagents; (c) is an effective antitumor agent against intraperitoneally implanted P388 leukemia in the mouse; (d) has a melting point of 224-227 C; (e) has a specific rotation of [a]D5 = -13"(e 1,CHC13); (f) has an approximate elemental composition of 50.75% carbon, 5.25% hydrogen, 12.58% nitrogen, and 31.42% oxygen;; (g) exhibits in silica gel thin layer chromatography a Rf value of 0.73 with the solvent system n-butanol-methanol-water (63:27:10), and a Rf va lue of 0.53 with the solvent system xylenemethylethylketone-methanol (5:5:1); (h) has an infrared spectrum in potassium bromide substantially as shown in Figure 4; and (i) when dissolved in dueterated chloroform gives proton NMR spectrum substantially as shown in Figure 8.

10. A fermentation process for the production of the antitumor antibiotic BBM-928 complex which process comprises cultivating a strain of actinomycetes having the characteristics of strain No. G455-101 in an aqueous solution containing an assimilable carbon source and an assimilable nitrogen source under submerged aerobic conditions until the BBM-928 complex produced imparts substantial antitumor antibiotic activity to said solution.

11. A process as claimed in claim 10 which includes the further step of recovering the BBM-928 complex from the culture medium.

12. A process as claimed in claim 10 or claim 11 which includes the further step of separating the BBM-928 complex into components A,B,C,D,E, and F, wherein said components exhibit the following Rf values in silica gel thin layer chromatography: Rf Values n-butanol-mathanol- xylene-methylethylketone water (63:27:10) methanol (5:5:1) BBM-928A 0.71 0.48 BBM-928E 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

13. A process as claimed in claim 10 substantially as hereinbefore described.

14. The antibiotic complex BBM-928 whenever prepared by a process as claimed in claim 10, or claim 11 or claim 13.

15. A process as claimed in claim 12 substantially as hereinbefore described.

16. The components A,B,C,D,E and F of the antibiotic complex BBM-928 when separated by a process as claimed in claim 12 or claim 15.

17. The use of BBM-928 complex, or one of the components A,B,C,D,E or F as an antitumor agent.