LU82318A1 - NOVEL ANTITUMOR-ANTIBACTERIAL COMPLEX AND PROCESS FOR PRODUCING THE SAME - Google Patents

Description

. OC 51.o95

.-82 3 1 g ‘GRAND-DUCHY OF LUXEMBOURG

Patent No ............................................... .....

of . ..Ier ..... Apr i l 198o Minister

Title issued · from the National Economy and the Middle Classes

Industrial Property Service

^ ^ LUXEMBOURG

/ f, <ή? Invention Patent Application I. Request ...... SOGiétè .dite.: ..... RRTSTOL - MYERS .... C.QMP.MY., ...... 3.45 Park Avenue , at _________ (1) ...... NEW-YORK, ..... ”·. ¥ · ....... loo22, ..... Etatsr.U.nis ...... ...........................

....... pajg ~ ^ tons.ietttL ^ tecquea. ^ eJ ^ yÆeg # - j ^ iftSi8üilLjSBLÆialiAê-d! BJ ° ^ · !! · _______ (2> ...... dataire ... ..............................-................... .................................................. .................................................. .................................................. ................................

»Deposits this ....... first .... avxil ..... 19.QO ...... guatr. ^ R.vingt -------------- ----------------------------------------- (3) to ..... ..... 15 .......... ... hours, at the Ministry of National Economy and the Middle Classes, in Luxembourg: 1. this request for obtaining a patent d invention relating to: ........ "New ..... coiiip.lexe ..... antlfc.lMQ; j: .äl“ iän.fc.ib. ^^ (4> ... ........of production".*.................................. .................................................. .................................................. .................................................. ............

d ^ a / ^ £ 4 φφφφφφ φ / τ / φφ / φφ /, φ ΦΦφφφ / ΦΙφτφ / τΙ, / φφ / ϊ / φ / χΑ ^ / φφ / Α / //Α)/·.Τ/ ... .................................................. .................................................. .................................................. .................................................. ........ (5) 2. the delegation of power, dated .NEW — YORK .____________________ March 27, 198o ..............

3. description in French ------------ ._______________________________ of the invention in two copies; 4 ................ 8 ............. drawing boards, in two copies; 5. the receipt of the fees paid to the Luxembourg Registration Office on ......... 1st .... April .... l98o_____________________________________________________________________________________________________________________________________________________________________________________________________________ claims priority for the above patent application for one ( des) request (s) of e (6) .............— patent .____________________________ filed (s) er / /i)_________.^î^....Etats—Uni.sd / J ^ erects ..............

on _________ 2 .... April ..... 19.79 ........... (Na ....... 0.2.6 .., .. 4.88) .__________ (8) on behalf of ........ S .... ± n.SalesUT.S ________________________________________________________________________________ (9); elect domicile for him / her and, if designated, for his / her representative, in Luxembourg _____________________________. — 35.r ..... bld * ...... Royal ------------ ---------------------------------------------------------- ---------------------------------------------------------- ---------------------------------------------------------- ------------------- (io) requests the issuance of a patent for the subject described and represented in the abovementioned appendices, - with adjournment of this grant a month.

v Π. Deposit Minutes

The above patent application has been filed with the Ministry of National Economy and the Middle Classes, Industrial Property Service in Luxembourg, dated: April 1, 1980

Pr. The Minister f · ·> * / - at 3 p.m. ^ VV * of the National Economy) / / Aes Classes Moyenes, ^ rc ............. {/ (!: ! J filUS il λΑη 4 D. 51.o95

CLAIM OF PRIORITY

of the patent application / ö / i / ttftfcjfejq / W TO THE UNITED STATES OF AMERICA From APRIL 2, 1979 "Brief" filed in support of a request for

PATENT

at

Luxembourg

in the name of: BRISTOL-MYERS COMPANY

* for: "New antitumor-antibacterial complex and its F’ production process ".

B

S

The present invention relates to a novel anti-tumor-antibiotic complex and a method for producing it, collecting it and dividing it into components with biological activity.

“5 According to the attached spectral information and the available physicochemical properties, the antitumor-antibiotic complex of the present invention appears to be related by its structure to the group of quinoxaline antibiotics such as echinomycin (Dell et al. 10 J. Am. Chem. Soc., 97 ”2 ^ 97 (1975), quinomycins (Shoji * et al., J. Antibiotics, 14a, 335 (1961) and triostin C (The Merck Index, 9th edition, However, the anti-tumor-antibiotic complex differs from the above-mentioned antibiotics in the following aspects: 1. The complex of the invention and its components contain as chromophore a quinoline nucleus instead of a quinoxaline nucleus as in group of actinoleukin antibiotics.

2. The complex of the invention and its components do not contain sulfur, in contrast to the presence of a disulfide or thio-acetal bridge which is encountered in the structure of antibiotics of the actinoleukin type. .25 3 · The complex of the invention and its components display a relatively weak antimicrobial activity, compared to; actinoleukins which are powerful anti biotic antibacterials.

The present invention provides a novel anti-tumor-antibiotic complex called BBM-928. This complex, which contains at least six components, is prepared by culture of a strain producing BBM-928 of an actinomycete (ATCC 31 491) or a mutant of this actinomycete in an aqueous nutritive medium, under conditions aerobic immersion. The invention also relates to a method by which the BBM-928 complex is collected from the culture medium and divided into its components by countercurrent distribution and chromatography techniques.

U

2 The invention encompasses the BBM-928 complex and its components A, B, C, D, E and F endowed with biological activity. It relates in particular to components A, B, C and D of BBM-928 in their diluted forms, in the form of true concentrates and in the purified form.

In the accompanying drawings, Figure 1 shows the infrared absorption spectrum of BBM-928A in potassium bromide; Figure 2 shows the infrared absorption spectrum of BBM-928B in potassium bromide; Figure 3 shows the infrared absorption spectrum of BBM-928C in potassium bromide; Figure 4 shows the infrared absorption spectrum of BBM-928D in potassium bromide; FIG. 5 represents the magnetic resonance spectrum of the protons of BBM-928A in solution in deuterochloroform in the presence of TMS as internal standard, the determination being carried out by means of an NMR spectrometer at a frequency of 90 MHz; FIG. 6 represents the magnetic resonance spectrum of the protons of BBM-928B in solution in deuterochloroform in the presence of TMS as an internal standard, the determination being carried out by means of an NMR spectrum meter at a frequency of 90 MHz ; * 25 FIG. 7 represents the magnetic resonance spectrum of the protons of BBM-928C in solution in deuterium

rochloroform in the presence of TMS as internal standard, the determination being carried out using an NMR spectrometer

at a frequency of 90 MHz; 30 and FIG. 8 represents the magnetic resonance spectrum of the protons of BBM-928D in solution in deute-rochloroform in the presence of TMS as internal standard, the determination being carried out by means of an NMR spectrometer 35 at a frequency of 90 MHz ;

In Figures 1 to 4, the wave numbers are expressed in cm, the wavelengths are expressed in micrometers and the transmission is expressed as a percentage.

3 The invention provides a novel anti-tumor-antibiotic complex arbitrarily called BBM-928. It is believed that this complex may be related in structure to the group of actinoleukin antibiotics, and is obtained by fermentation of an as yet unidentified actinomycete strain. Any strain of actinomycete capable of forming the BBM-928 complex in a culture medium can be used, the producer microorganism which is preferred being the specific strain of actinomycete bearing the number G455-10 101 in the collection of Bristol-Banyu cultures. The micro-organism was isolated from a soil sample collected in the Philippine islands and was deposited in the United States of America; in the collection of typical cultures of microorganisms under the reference ATCC 31 491.

The novel anti-tumor-antibiotic complex of the present invention discussed herein contains at least six components called BBM-928A, B, C, D, E and F. The components A, B, C and D of the complex BBM-928 were isolated in crystalline form and the chemical structures of components A, B and C were identified. The complex of the invention and its individual components are endowed with antibacterial and antitumor properties. As far as antibacterial activity is concerned, the complex and its individual components are useful as nutritional supplements in animal nutrition and as therapeutic agents in the treatment of bacterial infections in mammals. In addition, antibiotics are useful for cleaning and sterilizing laboratory glassware and surgical instruments and can be used in conjunction with soaps, detergents and washing solutions for hygienic purposes. With regard to anti-tumor effects, the complex and its individual components are particularly useful against various intraperitoneally implanted tumors in mice.

Actinomycete specific strain N ° G455-101

A general description is given below of the microorganism which is appreciated for the production of the 4 μ antibiotic-antitumor complex BBM-928. Observations of the cultural, physiological and morphological characteristics of the organism have been made in accordance with conventional taxonomic methods (see, for example, Shirling and 15 collaborators, Int. J. Syst. Bacteriol. _16, 313 (1966) and

Lechevalier et al., Biol. Actinomycetes Related * Org. _n, 78 (1976).

Micromorphology -

The strain No. G455-101 forms both a mycelium 10 in the substrate and an aerial mycelium, and the mycelium in the substrate is well developed, elongated and branched (width 0.5-0.8 pm). There is no distinct fragmentation of the; mycelium of the substrate. Unlike ordinary species of

Streptomyces, strain G455-101 carries only a short or rudimentary aerial mycelium 15 or does not form mycelium at all in certain agar media. Short or long spore chains are produced in the aerial mycelium, these chains each comprising 2 to 50 oval spores (most include 5 to 20 spores). The spore chains 20 are rectilinear, sinuous or in the form of a loop. The spores have a spherical (0.3-0.4 pm), oval or cylindrical (0.3 x 1.5 - 3.0 pm) shape and their surface is smooth. Spores are often separated by empty hyphae. An amorphous vesicle resembling a sporangium which envelops a short, coiled chain of 125 spores is occasionally observed on the aerial mycelium.

Composition of the cell wall and sugars contained in the whole cell

The cell wall of strain G455-101 contains meso-diaminopimelic acid, but does not contain glycine. Whole cell hydrolyzate reveals the presence of glucose, mannose and madurose (3-0-methyl-D-; galactose). The data mentioned above regarding the cell wall composition and the sugars of the whole cell indicate that the strain G455-101 is an actinomycete whose cell wall belongs to type IIIB.

V! 5

Cultural and physiological characteristics

The strain G455-101 grows abundantly, it forms a pink or greyish pink aerial mycelium and it produces a reddish pigment insoluble in water in nutritionally rich agar media, for example agar with yeast extract. and malt extract and oatmeal agar. However, in agar containing mineral salts and starch, agar containing glycerol and asparagine and tyrosine agar, this strain has poor growth, it forms a rudimentary white or beige aerial mycelium and it produces a small amount of a reddish pigment. It does not produce melanoid pigment in peptone, yeast and iron agar and in tyrosine agar. It reduces nitrates to nitrites. It develops abundantly at 28 ° C, 37 ° C and 45 ° C, but does not develop at 10 ° C or 50 ° C. Pentoses and hexoses are suitably used by this strain. The cultural and physiological characteristics of the strain G455-101 are produced, respectively, in Tables I and II. The use of carbon sources is shown in Table III.

»6

TABLE I

Cultural characteristics of the strain M ° G455-101 * 1 * Czapek Cxx agar zero or rare growth (sucrose agar R dark pink „5 and nitrate) A white to pale pink D none 2. Broth with moderate growth tryptone, and with flaky extract, sedimented 10 yeast (ISP N ° 1) and not pigmented 3. Agar with abundant yeast C extract and R red extract dark red to malt brown (ISP N ° 2) reddish * 15 A abundant, grayish pink to purplish pink D none 4. Flour agar C abundant 2o oats (ISP N ° 3) R bright yellowish red A moderate, pink D grayish yellow 5. Agar with poor mineral salts C and to R light yellowish brown at 2 starch (ISP No. 4) dark red A rare, white to beige D none »30 6. Poor glycerol agar C; and with asparagine R yellowish pink to brown, (ISP N ° 5) reddish A rare, white D none * 7 TABLE I (Continued) 7. Peptone agar, poor C, pleated with reddish orange R extract bright yeast and iron A none 5 (ISP No. 6) Light yellowish orange 8. Agar with poor tyrosine C (ISP No. 7) R dark red A rare, white 10 D none. 9 · Poor glucose and C ammonium salt agar R reddish brown A rare, light gray 15 D none 10.Modern Bennett C agar R reddish brown A limited, greyish pink 20 D none * observed after incubation at 37 ° C during 3 weeks.

xx abbreviation? C - growth R - color on the back of the mycelium 25 of the substrate A - aerial mycelium * D - diffusible pigment> 8

TABLE II

Physiological characteristics of the strain N ° G455-101 Test Response Method and medium 5 Nitrite ^ - positive nitrate Inorganic medium: broth of Czapek with sucrose and nitrate

Nitrate £ —nitrate positive Organic medium; 0.5% yeast extract, 1.0% glucose, 0.5% KNO ^, "0.1% CaCO ^

Hydrolysis of weakly Aged medium of Luedemann casein in positive agar medium. 15 Coagulation of positive skim milk

Liquefaction of the negative 15% gelatin in a tryptone broth gelatin with yeast extract (medium 20 ISP N ° 1)

Positive H ^ S production Addition of 0.1% L- from L- cysteine to the cysteine tryptone broth and yeast extract (ISP medium No. 1) more

25 agar. The presence of H ^ S

is detected by means of a paper strip impregnated with a 10% aqueous solution of lead acetate 30 Formation of negative Peptone, melanoid yeast and iron agar (ISP No. 6) and agar with tyrosine (ISP N ° 7) s 35 Positive reaction Aqueous solution of H2O? catalase ά Reaction to positive Kovac reagent oxidase

Growth temperature Bennett agar abundant growth at 28, 37 and 45 ° C.

Poor growth at 20 ° C. No growth at 10 ° C and 50 ° C.

9

TABLE III

Use of carbon sources by the strain N ° G455-101 PG Lm 5 - - 1. Glycerol ++ + 2. D (-) - arabinose + + 3. L (+) - arabinose + + 4. D-xylose + + + 10 5. D-ribose ++ + 6. L-rhamnose ++ + 7- D-glucose + + 8. D-galactose ++ + 9. D-fructose ++ + 15 10. D-mannose ++ + 11. L (-) - sorbose 12. Sucrose 13 · Lactose -, ± 14. Cellobiose + + 20 15. Melibiosis -, + 16. Trehalose + + 17 · Raffinose 18. D (+) - melezitose 19. Soluble starch ^ + 25 20. Dulcitol 4 21. Inositol + 22. D-mannitol ++ +

B

23. D-sorbitol 24. Salicin 30 25. Cellulose + + 26. Chitin + + 27. Keratin + +

PG base medium: Pridham - Gottlieb inorganic medium, __ supplemented with 0.1% yeast extract 3b

Lm: Luedemann organic medium Incubation for 2 weeks at 37 ° C.

10

It should be noted that the production of the BBM-928 complex of the present invention is not limited to the specific specific strain of actinomycete No. G455-101 defined by the growth characteristics and the microscopic characteristics. data above. These characteristics are indicated for purposes of illustration only, and the invention contemplates the use of strains or mutants produced from the organism described above, by conventional means known to those skilled in the art. art, such as x-ray irradiation, ultraviolet irradiation, nitrogen mustard action, phage exposure, etc. means which are capable of producing the BBM-928 complex or individual components of this complex.

15 Preparation of the anti-tumor-antibiotic complex BBM-928

The process of the present invention for the production of the anti-tumor-antibiotic complex BBM-928 consists in culturing by fermentation the actinomycete strain No. G455-101 in an aqueous solution containing an assimilable carbon source and an assimilable nitrogen source. under aerobic immersion conditions, until appreciable anti-tumor-antibiotic activity has been imparted to said solution. Conventional fermentation methods are used to cultivate the strain of actino-, fungus No. G455-101. Media which are useful for the production of the antibiotic-antitumor agents of the present invention include an assimilable source of carbon such as starch, glucose, dextrin, maltose, lactose, sucrose, fructose, mannose, molasses, glycerol, etc. The nutrient medium must also contain an assimilable source of nitrogen such as a protein, a protein hydrolyzate, polypeptides, amino acids, a soluble corn extract, casein, urea, etc., as well as nutritive mineral salts which introduce anions and inorganic cations such as potassium, sodium, ammonium, calcium, sulfate, carbonate, phosphate, chloride, nitrate, etc.

11

Any temperature favorable for satisfactory growth of the organism can be used in the production of the BBM-928 complex. Temperatures from about 20 to 45 ° C are suitable, and the preferred temperature for optimizing growth of the organism is in the range of 28 to 34 ° C, the temperature range of 30 to 32 ° C being much appreciated. Maximum production of the BBM-928 complex is generally achieved in a period of approximately 4 to 6 days. Conventional methods are used in the fermentation period. For example, the preparation in small quantities is advantageously carried out in shaken flasks, or by surface cultures. The preparation in large quantities is preferably carried out under aerobic culture conditions in immersion, in sterile tanks. In the case of tank fermentation, a vegetative inoculum is first produced in a nutritious broth by inoculation of the broth culture with spores from the organism, so as to produce a young and active sowing culture which is then transferred under aseptic conditions into the medium contained in the fermentation tank. Aeration can be produced in vats and bottles by injecting sterile air through or onto the surface of the medium being fermented, stirring being continued in the vats by means of a mechanical stirrer. Foam-blocking agents, such as silicone oil, soybean oil and bacon oil, can be added, if desired.

Antibiotic levels in fermentation broth or BBM-928 complex extracts can be determined by the agar diffusion test using paper discs, the test microorganism being Sarcina lutea and agar nutritive being used as test medium. The pH is adjusted to 9.0 to obtain the optimal sensitivity of the test medium which is used to determine the optimal power of the broth.

The BBM-928 complex is isolated from the fermentation broth by conventional means, for example by solvent extraction methods. The purification is advantageously carried out by preparative methods of countercurrent distribution and chromatography, as described in detail in Examples 2 and 3 below, in order to obtain the components A, B, C, D, E and F of the BBM-928 complex.

Physicochemical properties of components A, B, C and D of the BBM-928 complex of Example 3

The individual components of the BBM-928 complex have similar characteristics of solubility and color reactions. For example, they are very soluble in chloroform and methylene chloride, slightly soluble in benzene, ethanol, methanol and n-butanol and insoluble in water and in n-hexane. Positive reactions are obtained with ferric chloride and Ehrlich reagent and negative reactions are obtained with Tollens reagent, Sakaguchi reagent and ninhydrin.

The characteristic physicochemical properties of the components of BBM-928 of Example 3 are reproduced in the following Table IV.

13

TABLE IV

Physico-chemical properties of components A, B, C and D

of the BBM-928 complex BBM-928

5 J g c D

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

C “Jd5 (os1, 10 CHC13 -27 ° -7 ^ ° -91 ° -13 °

Analysis (values 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 15 0: (by difference) 28.49 32.23 29.39 31.42 in nm (EJ *) max 1 cm 20 in EtOH 235 (586) 235 (570) 235 (638) 235 (550) 264 (415) 264 ( 400) 264 (442) 264 (380) 345 (165) 354 (163) 345 (173) 345 (155) in EtQH-HCl 234 (610) 234 (556) 234 (650) 234 (565) 25 264 (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) 30 330 (180) 330 (116) 330 (117) 330 (140) 383 (170) 383 (122) 383 (122) 383 (145)

Molecular weight (osmometry, in 35 CHC13) 1450 - 1470 14

The infrared spectrum and the nuclear magnetic resonance spectrum of components A, B, C and D are reproduced, respectively, in Figures 1 to 4 and in Figures 5 to 8 of the accompanying drawings. The nuclear magnetic resonance spectra of BBM-928A (Figure 5), BBM-928B (Figure 6) and BBM-928C (Figure 7) are very similar to each other, the only difference being the presence of acetyl groups in the components A (δ: 2.03 ppm, 2 molar equivalents) and B (δ: 2.05 ppm, 1 molar equivalent), 10 but not in C. By acetylation with acetic anhydride in pyridine, 2, 3 and 4 molar equivalents of acetyl group were introduced, respectively, into components A, B * and C of the BBM-928 complex. The three acetylation products thus obtained have identical properties with regard to thin layer chromatography and the UV, IR and NMR spectra, which indicates that component A of BBM-928 is a mono-acetylated derivative of the component. BBM-928B and a diacetylated derivative of the component BBM-928C.

The acid hydrolysis of component BBM-928A gives five fragments (I, II, III, IV and V) absorbing ultraviolet light, soluble in n-butanol and five water-soluble substances (NPS-1, 2, 3, 4 and 5) ninhy-drine positive. These latter substances were separated by chromatography on "Dowex 50 x 4" and identified as the following .25 amino acids:

Compounds Rf (S-123) * _Identification. NPS-1 0.72 β-hydroxy-N-methylvaline (HM-Val) NPS-2 0.49 glycine (Gly) 30 NPS-3 0.46 serine (Ser) NPS-4 0.45 sarcosine (Sar) NPS -5 0.23 not identified s thin layer chromatography, S-123 silica gel plate: 10% ammonium acetate - methanol - 10% ammonia solution (9: 10: 1)

The five fragments absorbing ultraviolet light indicated above are represented by the following formulas: 35; 15

Fragment I

CH 0 ^ OH C14H14N2 ° 5 3 r <TT nrr Mass spectrum: T π J j 20H m / e 306 (M +) H ^ '^ CO-HH-CH-COgH 227,233,251,345 nm CH „i 3

HH OH

| | Fragment II

0H, 0 * ___ OH 0-C0-0H-C (0H) 2 C20H25N3 ° 8 '2 ρττ Mass spectrum: I I I | 2 m / e 377 (M + -58) ^ / ^ IT'cO-IlH-OH-COjH λ ”2 ° Η: 229,234,261,345 nm

CH30 „.OH

J Fragment III

V ^ AcKEM) λ ^ ° Η = 230,235,262,345 nm t

Fragment IV

HO OH C10H7N04 ’Y II Mass spectrum:

Dd m / e 205 (M +) îf ^ COgH% Me0H: 228,260,354 nm ^ max

H ^ C ŒEL Fragment V

5 \ / 3 • C-OH ^ 23 ^ 30 ^ 4 ^ 9 CH 0 _ ^ OH O-CO-CH-H-CO-CH ^ HH ΛΜθ ° Η: 230,235,262,345 nm 3 i I> x I ch2 ch5 ch *

k- Jk. I

H C0-M-CH-C02H

16

During acid hydrolysis (HCl 6N)> fragments I, II, III and V give the following degradation products: 5 Conditions of hydrolysis

Fragment Closed tube, Reflux, 110 °, 20 hours 110 ° C, 3 hours 10 I IV, Ser II IV, Ser, HM-Val I, HM-Val

III IV

V - I, HM-Val, Sar 15 Ser: serine HM-Val: β-hydroxy-N-methylvaline

Sar: sarcosine Basic hydrolysis of BBM-928A or BBM-928C with decinormal sodium hydroxide at 25 ° C for 3 hours gives fragment VI (the abbreviations Ser, HM-Val and Sar have the definitions given below) above, Gly denotes glycine and the symbol "X" represents an unidentified group).

25 „! ._C0 ^ Ser-'X-M31y -) - Sar ^ HH-Val

’Fragment VI

30

By treating fragment VI with decinormal hydrochloric acid at 110 ° C for 1 hour, fragment VII is obtained plus the compound HM-Val.

CH30 H

l IL —CCH-Ser ^ X-Kîly ^ Sar

Fragment VII

17

Treatment of fragment VI with decinormal sodium hydroxide at 37 ° C for 40 hours gives fragment VIII plus fragment I.

5 X- ^ Gly ^ Sar ^ HM-Val

Fragment VIII

Treatment of fragment VII with decinormal sodium hydroxide at 37 ° C for 40 hours gives fragment IX plus fragment I.

X- »Gly- * Sar Fragment IX

The unidentified group (X) has the molecular formula CpjHgüIgC ^ (in peptide form) from micro-analysis of fragment IX and other peptide fragments containing (X) and spectral analysis, the results of which are summarized below.

20 Magnetic resonance Magnetic resonance of the 13C nuclei tlc] ue of the protons Attribution 30.14 (t) 2.36 ppm (2H, m) -CH ^ - 25 61.4 (d) -j 4.2-4, 5 (2H, m) 2 x -CHC (o or M) 61.7 (d) / 140.7 (d) 6.76 (1H, t) -CH = N- • 30 171 (s) - -CO - (amide)

-OH

NH

In accordance with the spectral data obtained for fragments VIII and IX and with the 360 MHz proton magnetic resonance spectrum of the Ap compound of the BBM-928 complex of Example 4, the unidentified amino acid portion (X) seems to be the best represented by the following tetrahydropyridazine structure: 18 CO-LA0h '5 (X): Based on the results of the degradation tests described above, the spectral data, microanalysis, and molecular weight determinations, we considers that the following structures best represent the components A, B and C of the BBM-928 complex: CH3 ° £ —v LA A * h0Ri N C0 + Ser * N— | C-> Gly * Sar-HBM-Val i ° 0 0 ho ^^ \ / v ^ och_ \ I "j il | 3

HM-Val "-Sar + GiyC. I

ÎT -Ser ^ CO ~ AnAA ^ ** ° - (> "R ^ R2 Ser: serine

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

BBM-928 CH H

4 19

Antimicrobial Activity The antimicrobial activity of the components of BBM-928 was determined against various species of bacteria and fungi by the method of serial dilution in nutrient agar at pH 7, using of Steer's multi-inoculation device. The size of the inoculum is normalized so as to use an aliquot of 0.0025 ml of test organisms containing approximately 10 ^ cells per ml for all bacteria and all fungi, except acid-fast bacteria. "In which a suspension of 10 cells / ml is used. The minimum inhibitory concentrations determined after incubation for about 18 hours at 37 ° C are shown in Table V. As shown in this table, the components of the BBM-928 complex are moderately to weakly active against Gram-positive bacteria and bacteria acid-resistant, but practically inactive against Gram-negative bacteria and fungi.

The prophage induction activity in a lysogenic bacterium (IBL) was determined for the components of the BBM-928 complex. No appreciable IBL activity was observed in the case of components A, B and C of the BBM-928 complex up to a concentration of 100 pg / ml.

9 20

TABLE V

In vitro antimicrobial activity of BBM-928 components against aerobic bacteria

BBM-928 components (concentration £ 0 (minimum inhibitory concentration in μς / ml)

BBRI Test organization A B C D E F

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

Sp-1 S. pyogenes S-23 6.3 12.5 25 50 50 12; 5 S1-1 S. lutea PCI 1001 6; 3 12? 5 50 50 50 25

Mf-1 flavus D12 12; 5 25 50 100 100 25

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

Bs-1 3 ^ subtills PCI 219 25 25 100 100 100 25

Bg-1 JL_ megaterium D2 25 25 50 100 100 25

Ba-3 B. anthracis A9504 6? 3 12; 5 25 50 50 12 ^ 5 M6-1 ÎL_ smsgmatis 607 D87 25 25 25 100 100 25

Mp-1 phlei D88 12; 5 12; 5 12; 5 50 '50 12; 5

Ec-1 coli NIBJ> 100> 100> 100> 100> 100> 100

Kp-1 IL_ pneumoniae D-ll> 100> 100> 100> 100> 100> 100

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

Pv-1 Ph_ vulgaris A9436> 100> 100> 100> 100> 100> 100

Pm-1 mirabilis A9554> 100> 100> 100> 100> 100> 100

Pg-1 P _; _ morganii A9553> 100> 100> 100> 100> 100> 100; Sm-1 marcescens A20019> 100> 100> 100> 100> 100> 100 - Al-1 A ^ faecalis ATCC 8750> 100> 100> 100> 100> 100> 100

Ca-1 (L_ arbicans IAM 4888> 100> 100> 100> 100> 100> 100

Cn-3 C; _ neoformans 100> 100 100> 100> 100> 100

H

21

Antitumor activity

Comparative tests between the components A, B, C and D of the BBM-928 complex and mitomycin C, from the point of view of antitumor activity, were carried out for the 5 tumors with intraperitoneal implantation: leukemia P388, leukemia L1210 , B16 melanoma, Lewis Lung carcinoma (LL) and I80 sarcoma ascites (S180). Test solutions of the components of BBM-928 in 0.9% sodium chloride containing 10% dimethyl sulfoxide, and of mitomycin C in 0.9% sodium chloride were administered once daily according to dosage programs ranging from a single single day treatment to multiple daily treatments. By varying the dosage, the minimum effective dose (DEM) is determined which gives an average survival time of the treated animals at least 1.5 times greater than the average survival time of a control group. This level of activity is considered to be an indication of significant antitumor activity. The results are reproduced in Table VI which also indicates the 20 calculated activity reports demonstrating that component A of the BBM-928 complex is significantly more active than is mitomycin C, the superiority of this component ranging from a factor 10 to 300 depending on the tumor strain and dosage. The intraperitoneal values of DL ^ 0 of the components A, B, C * 25 and D of the BBM-928 complex and of mitomycin C, determined, by the method of Van der Waerden, Arch. Expt. Path.

! Pharmak., 195? 389 (1940), are also shown in Table VI.

κ 22

TABLE VI

Antitumor activity and toxicity of components A, B, C and D of the BBM-928 complex and mitomycin C

5 Minimum effective dose (DEM, DL ™, route _ (rog / kg / day) __ intraperitoneal P388 L1210 B16 LL S180 (mg / kg / .d) 10 Treatment 8- BBM-9289A 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. 15 Mitomycin C 0.1 3 1 0.3 0.3 9.3

Treatment ^ BBM-928A 0.001 0.003 0.003 0.003 0.0001 0.013

Mitomycin C 0.1 0.3 0.3 0.3 0.03 1.4 20

Activity report (BBM-928A / mitomycin C)

Treatmenta 30 - 300 10 100

Treatment ^ 100 100 100 100 100 25 a. only one treatment the first day.

b. daily treatments from day 1 to day 9.

; Example 1

Production of the BBM-928 complex 30 Fermentation on agar

An inclined culture on a well-developed agar of the actinomycete species G455-101 is used to inoculate a 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 ^, 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 culture are transferred into a 500 ml Erlenmeyer flask containing 100 ml of fermentation medium containing 2% of soluble starch te 23 , 1% of "Pharmamedia", 0.003% of ΖηδΟ ^ .γτ ^ Ο and 0.4% of CaCO ^ · The production of the BBM-928 complex reaches its maximum after approximately 5 days of culture with shaking.

5 Fermentation in tanks

A seed culture is shaken for 4 days in Erlenmeyer flasks, and inoculated into 100 liters of germination medium composed of 2.0% oatmeal (Quaker Products, Australia), 0, 5% of glucose, 0.2% of dry yeast, 0.0008% of MnCl2.4H20, 0.0007% of CuSO ^ .ÏHgO, 0.0002% of ZnS0j | .7H20 and 0.0001% of FeS0j | .7H20 in a fermenter with a seed tank of • 200 liters, which is stirred at 200 rpm. at 30 ° C for 54 hours. A 15 liter portion of the seed culture is then inoculated into 170 liters of fermentation medium containing 2.0% soluble starch, 1.0 ί of "Pharmamedia" medium, 0.003% of ZnSO 4. 78 ^ 0 and 0.4% CaCO ^ in a 400 liter tank fermenter which is operated at 30 ° C at 200 rpm at an aeration speed of 20 150 l / min. The pH of the broth gradually increases as the fermentation progresses and it reaches 8.4-8.5 after 100-120 hours; at this time, the maximum potency of the antibiotic is reached for a concentration of 30 pg / ml.

- Example 2, Isolation of the BBM-928 complex by solvent extraction. The broth collected (170 liters, pH 8.5) obtained in Example 1 is filtered with a clarifying agent. Activity is detected in both the mycelial cake and the filtrate. The mycelial cake is extracted twice with a mixture of solvents formed from acetone and methanol (1: 1, twice 30 liters). The 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 40 liters of n-butanol each time. By concentration under reduced pressure of the combined n-butanolic extracts and lyophilization of the residue, 21.4 g of a crude solid are obtained. According to

H

24 analysis by thin layer chromatography, this substance consists of a complex containing three main components A, B and C, and three secondary components D, E and F, whose Rf values are indicated on; 5 following table VII.

TABLE VII

Thin layer chromatography of silica gel of the components of the BBM-928 complex

Rf values x 10 -

System N-118 xx System N-103 *** BBM-928A 0.71 0.48 BBM-928B 0.53 0.26 15 BBM-928C 0.27 0.07 BBM-928D 0.73 0.53 BBM -928E 0.56 0.34 BBM-928F 0.39 0.17 x detection by ultraviolet ray analyzer 20 (Shimadzu CS- 910) at 345 nm xx n-butanol - methanol - water (63: 27: 10 ) xxx xylene - methyl ethyl ketone - methanol (5: 5: 1)

Example 3 Purification of the BBM-928 Complex

The crude complex of Example 2 is purified using a preparative distribution apparatus against. current (Mitamura, 100 ml / tube) by means of a system. solvents comprising carbon tetrachloride, chloroform, methanol and water in the proportion of 5: 2: 5: 1. After 50 transfers, the contents of tubes No. 5 to 20 are combined and concentrated, giving 4.4 g of a pale yellow powder containing the components A, B, D, E and F. The mixture is dissolved in a low volume of chloroform and the solution is loaded on a column of silica gel "C-200" (500 ml) previously treated with ethyl acetate. The column is developed with ethyl acetate containing an increasing quantity of methanol (2-5% volume by volume) and the fractions are controlled by determination of the optical density at 345 nm. The secondary component D is eluted first with ethyl acetate and it is followed by the component A. The components E, B and F are then eluted in this order at a methanol concentration of 3% · Each 5 fraction containing the desired component is evaporated under reduced pressure and the residue is crystallized from a mixture of chloroform and methanol. Likewise, the crude preparation of component C is obtained from the content of tubes No. 21 to 35 resulting from the countercurrent sharing described above. The purification of component C is carried out by chromatography on silica gel and crystallization from a mixture of chloroform and methanol. The yields of the components, A, B, C, D, E and F have the following respective values: 988 mg, 420 mg, 848 mg, 130 mg, 119 mg and 114 mg. 15 Example 4

Subsequent Purification of Component A of BBM-928 Complex of Example 3 The thin layer chromatographic test of component BBM-928A of Example 3 (performed using a system formed from 10% methanol in toluene) indicates that the sample is not entirely homogeneous, because it contains an additional material which passes just before the component BBM-928A. The following steps were used to carry out the purification:, (1) Chromatography of the sample is carried out on silica gel using a linear gradient. ranging from chloroform to a methanol-chloroform mixture containing 6% methanol. The fractions eluted between the proportions of 2.4% and 3.3% of methanol in the methanol-chloro-form mixture (containing the component BBM-928A plus a certain amount of impurity) are combined for the operation next.

(2) A concave gradient is generated using three containers, the first two of which contain a mixture of methanol and toluene to 2% methanol and the third of which contains a mixture of methanol and toluene to 6% of methanol. The fractions collected at the end of the first stage of chromatography on a column of silica gel give, according to this gradient, a secondary component which is eluted first and which is closely followed by the purified component BBM-928A (hereinafter called after component BBM-928A).

sr

The molecular weight of the component BBM-928Ap, determined according to the mass spectrometric method by desorption in a field, is equal to 1427, which corresponds to the crude formula C ^ HygN 1 (molecular weight 1427.417). Elemental analysis (samples dried at 100 ° C for 18 hours) 10 Cl E% n% 0% a • Calculated for c64h78n14 ° 24: 53.85 5.51 13'74 26'90 found13: 52.47 5.48 13 , 81 28,24a 15 a. by difference b. average of three measurements.

Claims (13)

1, - I C-Hlly-Sar- * HM-Val °? "0 i HO Λ ππτ T 0" 0 HM-Val "-Sar * -Gly" -C j | j / —N. "- Ser <-C0 -L ^ i * ^ ΖοΛ_) wherein Ser is serine, Gly is glycine, Sar is sarcosine and HM-Val is β-hydroxy-N-methylvaline. 1. The antibiotic-anti-tumor 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 the ferric chloride reagent and the Ehrlich reagent and a negative reaction with the Tollens reagent, the Sakaguchi reagent and the ninhydrin reagent; (c) is an antitumor agent effective against P388 leukemia, L1210 leukemia, B16 melanoma, Lewis-Lung carcinoma and sarcoma 180 ascites by intraperitoneal implantation in mice $ (d) at a melting point 246-248 ° C; 20 (e) has a specific rotation [aj ^ equal to 27 ° (c = 1, CHC13); (f) has a molecular weight of 1427; (g) contains approximately, according to the elemental analysis, 52.47% of carbon, 5.48% of hydrogen, 13.81% of nitrogen and. 28.24% oxygen; (h) has, in the analysis by thin layer chromatography of silica gel, an Rf value of 0.71 with the mixture of solvents 30 formed of n-butanol, methanol and water in the proportion of 63 : 27: 10 and a value; of Rf 0.48 with the mixture of solvents formed from xylene, methyl ethyl ketone and methanol in the proportion of 5: 5: 1; (I) gives acidic water-soluble substances to ninhydrin by acid hydrolysis including 8-hydroxy-N-methylvaline, glycine, serine and sarcosine; 28 (j) gives, by basic hydrolysis, a fragment VI of formula: 2. The antibiotic-antitumor compound according to claim 1, characterized in that the amino acid portion X has the crude formula in the peptide form. 3 · The antibiotic-antitumor compound according to claim 2, characterized in that the amino acid portion X in the peptide form is the tetrahydro-pyridazine radical of formula: 30 o kA " 4. The antibiotic-antitumor compound according to claim 1, characterized in that it corresponds to the structure: 29 I ^ / V-OR 5 1 \ I C-M51y-> Sar ^ HM-Val t 3 ° i H0. ^ -V /> ^ OCH3 / HM-Val ^ SarKîly ^ C '1') —N "—SerKIO — i R o— \ \ H .- 5. The anti-tumor-antibiotic compound BBM-928B having a quinoline chromophore and giving, by acid hydrolysis, water-soluble components comprising serine, glycine, sarcosine and β-hydroxy-N-methylvaline, which has in its form essentially pure the following characteristics: (a) is 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 the ferric chloride reagent and the Ehrlich reagent and a negative reaction with the Tollens and Sakaguchi reagents and the ninhydrin reagent; (c) is an effective anti-tumor agent against P388 leukemia implanted intraperitoneally in mice; (d) has a melting point of 214-217 ° C; (e) has a specific rotation (aj ^ of -74 ° (e = 1, CHClg); 3 ° .ο (f) contains approximately, according to elementary analysis, 50.14 t of carbon, 5.29 % hydrogen, 12.34% nitrogen and 32.23% oxygen; 5 (g) has an Rf value of 0.53 in the thin layer chromatography of silica gel with the mixture of solvents n -butanol-methanol-water in the proportion of 63:27:10 and an Rf value of 0.26 with the mixture of solvents 10 formed from xylene, methyl ethyl ketone and methanol in the proportion of 5: 5: 1 5 (h) has an infrared absorption spectrum in potassium bromide corresponding substantially to the representation of FIG. 2; and (i) gives the dissolved state in deuterochloroform a magnetic resonance spectrum of protons corresponding substantially to the curve in Figure 6. 5 CH3 ° 'Yÿ * v "Y'% v" '0H A. —CCH-S er * X * Gly * Sar - "- EM-Val v N Fragment VI 10 in which Ser is serine, Gly is glycine , Sar is sarcosine, HM-Val is β-hydroxy-N-methylvaline and the symbol X represents an amino acid portion; 15 (k) has an infrared absorption spectrum in potassium bromide corresponding substantially to the representation given in FIG. 1; and (1) in the dissolved state in deuterochloroform, gives a spectrum of magnetic resonance of the protons corresponding substantially to the representation given in FIG. 5. 6. The compound according to claim 5, having the structure: CH30V ^ \ pH 25 XjOl s CO- ^ Ser + N— (i OCly + S Val tn 0 1 H0 ^ ^ ΩΓΗ HM-Val «* Sar <-Gly + C | TT 30 35 in which Ser is serine, Gly is glycine, Sar is sarcosine, HM-Val is e-hydroxy-N-methylvaline and R1 is an acetyl group 31 if j- 7. The anti-tumor-antibiotic compound BBM-928C having a quinoline chromophore and giving, by acid hydrolysis, water-soluble components comprising serine, glycine, sarcosine and β-hydroxy-N-methylvaline, which is in its form essentially pure the following characteristics: (a) is soluble in chloroform and methylene chloride, slightly soluble in benzene, ethanol, methanol and n-butanol and substantially insoluble in water and in n- hexane; (b) gives a positive reaction with the ferric chloride reagent and the Ehrlich reagent and a negative reaction with the reagent 15 Tollens and Sakaguchi and the ninhydrin reagent; (c) is an effective anti-tumor agent against leukemia P388, leukemia L1210, melanoma B16, Lewis-Lung carcinoma and sarcoma 180 ascites tumors implanted intraperitoneally in mice; (d) has a melting point of 244-248 ° C; (e) has a specific rotation (aj ^ pde -91 ° (c = 1, '25 CHC13); (f) has an approximate molecular weight of 1470; (g) contains approximately according to elementary analysis 51.77 % carbon, 5.29% hydrogen, 13.55% nitrogen and 29.39% oxygen; (h) has in the silica gel thin layer chromatography an Rf value of 0, 27 with a mixture of solvents formed from n-butanol, methanol and water in the proportion of 63:27:10 and an Rf value of 0.07 with the mixture of solvents formed from xylene, methyl ethyl ketone and of methanol in the proportion of 5: 5: 1; 32 (i) has an infrared absorption spectrum in potassium bromide corresponding substantially to the curve in FIG. 3; and (j) gives the dissolved state in the deuterochloroform a proton magnetic resonance spectrum corresponding substantially to the curve in Figure 7 · 8. The compound according to claim 7, corresponding to the structure: 10 CH, 0 ^ OH XjC ^ x f> '"N CO- ^ Ser- ^ N— ( 9. The anti-tumor-antibiotic compound BBM-928D '25 having the following characteristics: (a) is 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 the ferric chloride reagent and the Ehrlich reagent and a negative reaction with the Tollens and Sakaguchi reagents and the ninhydrin reagent; (c) is an anti-tumor agent effective against P388 leukemia with intraperitoneal implantation in mice 33 (d) has a melting point of 224 at 227 ° C; (e) has a specific rotation (aj ^ pde -13 ° (c = 1, CHC13); (f) contains approximately according to the elementary analysis 5.75 t of carbon, 5.25% of hydrogen, 12.58% nitrogen and 31.42% -, oxygen 5 (g) present in the chromatography on thin layer of silica gel an Rf value of 10 0.73 with the mixture of solvents formed of n- butanol, methanol and water in the proportion of 63:27:10 and an Rf value of 0.53 in the solvent mixture formed of xylene, methyl ethyl ketone and methanol in the proportion of 5: 5: 1 ; (h) has an infrared spectrum in potassium bromide corresponding substantially to the curve of Figure 4; and (i) gives the dissolved state in deuterochloroform a proton magnetic resonance spectrum corresponding substantially to the Figure 8 curve. 10 BBM-928D 0.73 0.53 BBM-928E 0.56 0.34 BBM-928F 0.39 0.17 10. A method of production by fermentation of the anti-tumor-antibiotic complex BBM-928, characterized in that it consists in cultivating a strain of actinomycetes having the characteristics of the strain No. G455-101 in an aqueous solution containing a source of assimilable carbon and a source of assimilable nitrogen under aerobic conditions in immersion until the BBM-928 30 complex produced confers appreciable anti-tumor-antibiotic activity to said solution. R2 ° / where Ser is serine, Gly is glycine, Sar is sarcosine, HM-Val is β-hydroxy-N-methylvaline and R ^ and R2 are acetyl groups. 11. The method of claim 10, characterized in that it further comprises a step of separation of the BBM-928 complex from the culture medium. 12. The method of claim 10, characterized in that it further comprises a step of dividing the BBM-928 complex into components A, B, C, D, E and F which have the following values of Rf in the chromatography on a thin layer of silica gel: 34; Rf values n-butanol-methanol- xylene-methylethyl-water (63:27:10) ketone-methanol 5 (5: 5: 1) BBM-928A 0.71 0.48 BBM-928B 0.53 0.26 BBM-928C 0.27 0.07 13 · Medicament, characterized in that it consists of or in that it contains an antitumor-antibiotic compound according to any one of claims 1 to 9- * *