NO155780B - PROCEDURE FOR THE PREPARATION OF ANTITUMOR AND ANTIBIOTIC COMPLEX. - Google Patents

Description

The present invention relates to the production of a new antitumor and antibiotic complex.

Based on available spectral data and available physicochemical properties, it appears that the subject anti-turmo and antibiotic complex is structurally related to the quinoxaline group of antibiotics such as echinomycin, Dell et al., in "J.Am.Chem.Soc." , 97, 2497 (1975), kinomycins, Shoji et al. in "J. Antibiotics", 14A, 335 (1961) and triostin C, "Merck Index", 9th edition, 9399. However, the present antitumor antibiotic complex differs from these antibiotics in the following respects: 1. The present complex and components thereof contains a quinoline nucleus as a chromophore instead of quinoxaline as in the actinoleukin group of antibiotics. 2. The complex is present and its components do not contain sulfur in contrast to the presence of a disulfide or thioacetal bridge in the structure of actinoleukin antibiotics. 3. There is a complex and its components show relatively weak antimicrobial activity compared to the actinoleukins, which are powerful antibacterial antibiotics.

The hitherto unknown antitumor antibiotic complex in question is called BBM.928. The complex containing at least 6 components is produced by growing a BBM-928-producing strain of actinomycetes (ATCC 31491) or a mutant thereof in aqueous nutrient medium using submerged aerobic conditions. The invention also relates to a method for extracting the BBM.928 complex from the culture medium and separating the complex into bioactive components by countercurrent distribution and chromatography techniques.

The scope of the invention includes both the BBM-928 complex and the bioactive components BBM-928 A, B, C, D, E and F. The invention relates in particular to the components BBm-928 A, B,

D and D in diluted forms, as actual concentrates and

in purified form.

According to this, the present invention relates to a method for the production of an antitumor and antibiotic BBM-928 complex containing one or more bioactive components BBM-928A, BBM-928B, BBM-928C, BBM-928D, BBM-928E and BBM-928F , where the components A, B, C and D can be specified by the formula:

in which

Ser: serine

Gly : glycine

Sar : sarcosine

NM-Val: -hydroxy-N-methylvaline,

1 2

and in the case of BBM-928A, R and R are acetyl, in the case of BBM-928B, R 1 is acetyl and R 2 is hydrogen, when gel-12

where BBM-928C is R and R hydrogen, and in the case of BBM-928D R 1 is acetyl and R 2 is C2H^CO, which components exhibit the following R^ values by silica gel thin-layer chromatography in:

and this method is characterized by fermenting actinomycetes ATCC 31491 in an aqueous solution containing an assimilable carbon source and an assimilable nitrogen source

source under submerged aerobic conditions, until the formed BBM--928 complex gives the solution substantial antitumor activity, after which, if desired, the complex is separated into its components.

The invention shall be explained in more detail with reference to the drawings in which:

Fig. 1 shows the IR absorption spectrum for BBM-928A in KBr.

Fig. 2 shows the IR absorption spectrum for BBM-928B in KBr.

Fig. 3 shows the IR absorption spectrum for BBM-928C in KBr.

Fig. 4 shows the IR absorption spectrum for BBM-928D in KBr.

Fig. 5 shows the NMR spectrum of BBM-928A dissolved in deuterated chloroform without the use of TMS as an internal standard, determined with an NMR spectrometer at a frequency of 90 MHz. Fig. 6 shows the NMR spectrum of BBM-928B dissolved in deuterated chloroform using TMS as internal standard, determined by an NMR spectrometer at a frequency of 90 MHz. Fig. 7 shows the NMR spectrum of BBM-928C 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 NMR spectrum of BBM-928D dissolved in deuterated chloroform using TMS as internal standard, determined with an NMR spectrometer at a frequency of 90 MHz.

The previously unknown anti-tumor and antibiotic complex in question, which is arbitrarily called BBM-928, is believed to be structurally related to the actinoleukin group of antibiotics, and it is formed by fermentation of a hitherto unidentified strain of actinomycetes. Any strain of actinomycetes capable of producing BBM-928 in a culture medium may be used, the preferred producing microorganism being designated actinomycetes species strain No. G455-101 in the Bristol-Banyu culture collection. The microorganism was isolated from a soil sample collected in the Philippines and it is deposited in the American Type Culture Collection of Microorganisms as ATCC 31491.

As mentioned, the antitumor and antibiotic complex in question has at least 6 components called 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 with identified chemical structures of components A, B and C. The complex (and individual components) has antitumor and antibacterial properties. With regard to antibacterial activity, the complex and individual components thereof are applicable as nutritional supplements to animal feed and as therapeutic agents for the treatment of bacterial infections in mammals and humans. Furthermore, 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. With regard to the antitumor effect, the complex and individual components thereof are particularly valuable against a number 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 production of the antibiotic antitumor complex BBM-928. Observations were made of the cultivation, physiological and morphological conditions of the organism in accordance with standard taxonomic methods, e.g. SHirling et al., in "Int. J. Syst. Bacteriol. 16, 313 (1966) and Lechevalier et al., in "Biol. Actinomycetes Related Org., 11, 78 (1976).

Micromorphology: Strain no. G455-101 forms both substrate-

and the aerial mycelium and the substrate mycelium are well developed, long and branched with a width of 0.5 - 0.8 µm. Clear fragmentation of the substrate mycelium is not observed. Unlike common strains of Streptomyces, strain G455-101 has only short or rudimentary aerial mycelia or forms none in certain agar media. Short or long spore chains are produced in the aerial mycelium containing 2-50 oval spores in a chain, mostly 5-20 spores. The spore chains are straight, curved or loop-shaped. The tracks are spherical (0.3 - 0.4

um) , oval or cylindrical (0.3 x 1.5 - 3.0 um in shape and smooth surface. The spores are often separated by empty hyphae. An amorphous sporangium-like vesicle enclosing a short snophid spore chain is occasionally observed on the aerial mycelium .

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 former cell wall composition and whole cell sugar components indicate that strain G455-101 is an actinomycete species of cell wall type 111B.

Cultivational and physiological characteristics: Strain G455-101 grows vigorously, forms pale pink or greyish 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 gives little growth, forms white or beige-colored rudimentary aerial mycelium, and produces small amounts of reddish pigment. Melanoid pigment is not produced in peptone-yeast-iron-agar and tyrosine agar. Nitrate reduces to nitrite. It grows vigorously 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. Cultivational and physiological characteristics of strain G455-101 are shown in Tables I and II, respectively. The utilization of carbon sources is shown in Table III.

It must be understood that the production of the BBM in question

928 complex is not limited to the particular actinomycetes strain No. G455-101 described above with respect to growth and microscopy characteristics. These properties are only given as an illustration and the invention includes the use of strains or mutants produced from the above-described organism using conventional means such as exposure to X-ray radiation, ultraviolet radiation, nitrogen mustard, phage exposure, etc., which are capable of producing The BBM-928 complex or individual components thereof.

Preparation of antitumor antibiotic BBM-928 complex. The method according to the present invention for the production of the antitumor and antibiotic BBM-928 complex comprises cultivation by fermentation of actinomycetes strain no. G455-101 in an aqueous solution containing an assimilable carbon source and an assimilable nitrogen source under submerged aerobic conditions, until the solution is substantially antitumor and antibiotic activity. Conventional fermentation methods are used when cultivating actinomycetes strain no. G455-101. Media that can be used for the production of the antibiotic and antitumor agents in question 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 slurry, casein, urea, etc., as well as inorganic nutrient salts that provide inorganic anions and cations, such as potassium, sodium, ammonium, calcium, sulphate, carbonate, phosphate, chloride, nitrate etc.

In the preparation of the BBM-928 complex, any temperature can be used which contributes to a satisfactory growth of the organism. Temperatures varying from approx. 20 - approx. 45°C can be used, as a preferred temperature for optimal growth of the organism varies from approx. 28 - 34°C with a temperature range from 30 - 32°C as the most preferred. Maximum production of the BBM-928 complex is usually achieved

during approx. 4 - 6 days. Conventional methods are used during the fermentation period. E.g. the production of small quantities is carried out appropriately in shake flasks or by means of surface cultures. Production of large quantities is preferably carried out under highly aerobic cultivation conditions in sterile tanks. In tank fermentation, a vegetative inoculum is first prepared in a nutrient substrate by inoculating the substrate culture with a spore from the organism to form a young, active inoculum, which is then transferred aseptically 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 the tanks using a mechanical stirrer. Antifoam agents such as silicone oil, soybean oil and fatty oil can be added as needed.

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

The BBM-928 complex is isolated from the fermentation substrate

using conventional methods, such as solvent extraction. Purification is conveniently carried out by pre-parative countercurrent distribution and chromatography methods to be described in more detail below in Examples 2 and 3, to form the BBM-928 components A, B, C, D, E and F.

Physico-chemical properties of BBM-928 components A, B,

C and D in example 3.

Individual components of BBM-928 exhibit uniform solubility and color reactions. There are e.g. easily soluble in chloroform and methylene chloride, sparingly 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 with Tollens, Sakaguchi and ninhydrin.

Characteristic physicochemical properties of the BBM-928 components of Example 3 are shown in Table IV.

IR spectrum and NMR spectra for components A, B, C and D are shown in fig. 1-4 and 5-8 respectively. The NMR spectra for BBM-928A in Fig. 5, BBM-928B in fig. 6 and BBM-928C in fig.

7 are very similar to each other, the only difference being the presence of acetyl groups in component A (6:2.03 ppm, 2 mole equivalents) and B (6:2.05 ppm, 1 mole equivalent), but not in C. By acetylation with acetic anhydride in pyridine, 2, 3 and 4 molar equivalents of acetyl groups were introduced into the BBM-928 component A, B and C respectively. The three acetylation products thus obtained showed identical properties in the 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 n-butanol-soluble, UV-absorbing fragments (I, II, III, IV, 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 were identified as the following amino acids:

S-123: 10% ammonium acetate - methanol - 10% ammonium-alkaline solution (9:10:1).

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

On acid hydrolysis, 6N HC1, fragments I, II, III and V gave the following decomposition products:

Ser: serine

HM-Val : B-hydroxy-N-methylvaline

Sar : sarcosine.

Base hydrolysis of BBM-928A or BBM-928C with 0.1N NaOH at 25°C for 3 hours gives fragment VI (abbreviations Ser, HM-Val and Sar are as above, Gly denotes glycine and the symbol "X" denotes an undetermined moiety ).

Fragment VI

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

Fragment VII

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

Fragment VIII

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

Fragment IX

The undetermined part (X) has a molecular formula C-H^N-O., (on

5 6 2 2 c peptide form) based on microanalysis of fragment IX and other peptide fragments containing (X) and spectral analysis as summarized below.

According to spectral data for fragments VIII and IX as well as a 360 MHz proton NMR for BBM-928Ap in Example 4, the undetermined amino acid part (A) seems best to be assigned the following tetrahydropyridazine structure:

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

Ser: serine

Gly : glycine

Sar : sarcosine

HM-Val : ε-hydroxy-N-methylvaline

Antimicrobial activity

The antimicrobial activity of the BBm-928 components was determined against a variety of bacteria and fungi by the serial agar dilution method in nutrient agar at pH 7 using Steer's multi-inoculation apparatus. The inoculum size was standardized to the addition of 0.0025 mis proportion of test organisms containing approx. 10 4 cells per ml for all bacteria and fungi with the exception of acid-resistant bacteria for which a 10^ cell per ml of suspension. The minimal inhibitory concentrations, MICs, were determined after overnight incubation at 37°C and are shown in Table V. As can be seen, the 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 the BBM-928 components. No significant ILB activity was detected with BBM-928 components A, B and

C up to a concentration of 100 ug/ml.

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,

L1210 leukemia, B16 melanoma, Lewis lung carcinoma (LL)

and sarcoma 180 acites (S180). Sample 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 dosage schedules that varied from a single daily treatment to multiple daily treatments. By varying the dosage, the minimum effective dose, MED, was determined, which resulted in an average survival time for the treated animals of at least 1.25 times greater than for a control group. This level of activity is considered a measure of significant antitumor activity. The results are shown in Table VI together with the calculated activity ratios which show that BBM-928A is markedly more active than mitomycin C by a factor of 10 - 300, depending on the tumor strain and dosage scheme. Intraperitoneal LD<-q values of BBM-928 components A, B, C and D and mitomycin C, determined by the method according to Van der Waerden in "Arch. Expt. Path, Pharmak.", 195, 389 (1940 ) is also shown in the table

WE.

Example 1.

Preparation of BBM-928 complex

Agar fermentation: A well-grown agar slant culture of a strain of Actinomycetes species G455-101 was used for inoculation 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^ with the pH 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 medium was transferred to a 500 ml Erlenmeyer flask containing 100 ml of fermentation medium composed of 2% soluble starch, 1% Pharmamedia, 0.03% ZnSO^'7H 2 O and 0.4% CaCO^. The production of BBM-928 complex usually reaches a maximum after approx. 5 days shaking cultivation.

Tank fermentation: A seed culture is shaken for 4 days in Erlen-meyer flasks and inoculated onto 100 1 propagation medium composed of konieos salt, 2.0% oat flour, 0.5% glucose, 0.2% dry yeast, 0.0008% MnCl2.4H20, 0.0007% CuS04.7H20, 0.0002% ZnS04.7H20 and 0.0001% FeS04-7H20 in a 200 liter seeding tank fermenter which is stirred at 200 rpm. at 30°C for 54 hours. A 15 liter portion of the seed culture is then inoculated onto 180 liters of fermentation medium containing 2.0% soluble starch, 1.0% Pharmamedia, 0.003% ZnS04.7H20 and 0.4% CaC03 in a 400 liter tank fermenter operating at 30°C at 200 rpm. with an aeration rate of 150 l/min. The pH of the substrate gradually rises as fermentation progresses and reaches 8.4 - 8.5 after 100 - 120 hours, at which time a maximum antibiotic potency of 30 ug/ml is achieved.

Example 2

Isolation of BBM-928 complex by solvent extraction.

Recovered substrate, 170 1 with pH 8.5 from example 1 is filtered with a clarifying agent. The activity is determined both in the mycelial cake and the filtrate. The mycelial cake is extracted twice with a solvent mixture of acetone and methanol (1:1,

30 1 x 2). The extracts are combined and evaporated under a reduction

pressure to obtain an aqueous concentrate which is extracted with n-butanol. The substrate filtrate is extracted twice with n-butanol (40 1x2). Concentration of the combined n-butanol extracts under reduced pressure and lyophilization of the residue gives an impure solid in an amount of 21.4 g. According to thin-layer chromatographic investigations, this material is a complex consisting of three main components A, B and C as well as three minor components D , E and F with Rf values as listed in Table VII below.

Example 3

Purification of the BBM-928 complex

The impure coplex from Example 2 is purified by means of a preparative countercurrent distribution apparatus (Mitamura, 100 ml/tube) using a solvent system of carbon tetrachloride-chloroform-methanol-water (5:2:5:1). After 50 transfers, tubes Nos. 5 - 20 are combined and concentrated to obtain a pale yellow powder in an amount of 4.4 g, containing components A, B, D, E and F. This mixture is dissolved in a small amount of chloroform and is loaded onto a column of silica gel C-200 (500 ml) which is reserved with ethyl acetate. The column is developed using ethyl acetate with an increasing amount of methanol (2-5% v/v) and fractions are determined for optical density at 345 nm. The smaller 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 crystallized from chloroform-methanol. Correspondingly, an impure preparation of component C is obtained from tubes no. 21 - 3 5 from the countercurrent distribution described above. Purification of component C is carried out by silica gel chromatography and crystallization from chloroform-methane. The yields for component A,

B, C, D and E respectively F are 988 mg, 420 mg, 848 mg, 130

mg, 119 mg and 114 mg respectively.

Example 4.

Further purification of component BBM-928A from Example 3. Thin layer chromatographic examination of the BBM-938 component from Example 3 (using a system consisting of 10% methanol in toluene) showed that the sample was not completely homogeneous as it contained additional material which ran just ahead of the BBM-928A component. The following steps were taken for purification: (1) The sample was 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 contamination)

was mixed to the next step.

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

The molecular weight of BBM-928Ap was determined by field desorption mass spectrometry to be 1427, which corresponds to an empirical formula <C>64<H>78<N>i4°24 (<m>oleq weight 1427, 417).

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

Claims (1)

1. Process for the production of an antitumor and antibiotic BBM-928 complex containing one or more bioactive components BBM-928A, BBM-928B, BBM-928C, BBM-928D, BBM-928E and BBM-928F, where the components A, B, C and D can be specified by the formula in which Ser: serine Gly : glycine Sar: : sarcosine HM-Val : B-hydroxy-N-methylvaline, and in the case of BBM-928A R 1 and R 2 are acetyl, in the case of BBM-928B R 1 is acetyl and R 2 hydrogen, in the case of gel-12 where BBM-298C is R and R hydrogen, and in the case of BBM-928D R<1> is acetyl and R<2> is C2H5CO, which components exhibit the following R^ values by silica gel thin-layer chromatography: characterized by fermenting actinomycetes ATCC 31491 in an aqueous solution containing an assimilable carbon source and an assimilable nitrogen source under submerged aerobic conditions, until the formed BBM-928 complex gives the solution significant antitumor activity, after which, if desired, the complex is separated into its components.