HU212160B - Process for producing antitumour compound be-13793c - Google Patents

Abstract

FIELD OF THE INVENTION The present invention relates to a novel antineoplastic agent BE-13793C for the preparation of a pharmaceutically acceptable salt of formula (I). The new material is produced by a new microorganism and mutants of the genus Streptoverticillium. Preferably, the novel compound increases the range of substances that can be used in chemotherapy for cancer for which the resistance is not yet present. H Ο. N. O / H H 1 OH OH (1) HU 212 160 B Scope of the description: 6 pages (including 1 sheet)

Description

The present invention relates to the preparation of a novel compound of interest in the medical field. More particularly, the present invention relates to the production of a novel substance which inhibits the growth and proliferation of cancer cells, thereby exerting an anti-cancer effect.

Many microbial metabolites, such as bleomycin or adriamycin, are used in clinical chemotherapy for cancer. However, many of these agents are not effective enough in most clinical cancers, and the development of tumor cell resistance to these drugs, for which more and more data are available, affects their clinical use (Proceedings of the 47th Congress of the Japanese Cancer Association, pp. 12- 15, 1988).

Under these circumstances, there is, of course, a constant need for new anticancer agents, so there is a great need for a substance that does not develop resistance to known anticancer agents in various types of cancer and which is effective even in cases where the currently available anticancer agents agents are not effective.

A number of microbial metabolites have been investigated for potential anticancer agents. As a result, we have found a compound of formula (I) which has excellent antitumor activity. This recognition forms the basis of the invention.

The present invention therefore relates to a process for the preparation of a novel antitumor agent and its pharmaceutically acceptable salts of formula I-BE-13793C. The material can be used as an anticancer agent and is a novel process. a microorganism belonging to the genus Streptoverticillitim is used. which produces BE-13793C.

The novel anticancer agent BE-13793C of the present invention is characterized by the following physicochemical properties:

Appearance: yellow-orange amorphous or crystalline solid.

The molecular formula of the molecule is C20H11N3O4.

Elemental analysis:

Found: C, 67.23; H, 3.10%;

Found: C, 67.21; H, 3.12%.

Melting point: No decomposition or melting point up to 295 ° C.

Solubility: slightly soluble in water, soluble in methanol and very soluble in tetrahydrofuran or dimethylsulfoxide.

Acidity / neutrality / basicity: acidic.

_Rf value: 0.45 (silica gel 60 F ₂₅₄ Merck adsorbent, and chloroform: methanol = 5: silica 1 v / v mixture of).

Color reaction: potassium permanganate: positive.

Mass Spec (FAB-MS) (m / z): 357 [M] ⁺ .

Ultraviolet (UV) absorption spectrum (MeOH; λ ™, nm): 245, 298, 307, 327, 400.

Infrared (IR) Absorption Spectrum (KBr; v, cm ^-1 ): 3430. 3270, 1743, 1710, 1590, 1485, 1408, 1335. 1290, 1245, 1060, 815, 800, 765.

1 H-NMR (DMSO-d ₆ , δ ppm): 6.98 (2H, broad d, J

7.6 Hz); 7.13 (2H, t, J = 7.6 Hz); 8.42 (2H, broad d,

J = 7.6 Hz); 10.19 (2H, bs); 10.87 (1H, broad s); 11.57 (2H, bs).

³ C NMR (DMSO-d ₆ , δ ppm): 110.9 (d); 115.2 (d);

115.5 (s); 119.7 (s); 120.6 (s); 123.0 (s); 128.7 (s);

130.0 (s); 143.3 (s); 171.2 (s).

The biological effect of BE-13793C

In vitro mouse mouse cells were assayed for inhibitory activity of BE-13793C anticancer agent. For the in vitro assay with P388 tumor cells, the test substance was first dissolved in dimethyl sulfoxide and serially diluted from the resulting solution in 20% (v / v) DMSO-RPMI-1460 media. Two microliters of the dilution was then added to 200 microliters of cell culture medium (10% v / v bovine embryo serum = RPMI-1640) containing 2X10 ⁴ or 3X10 ⁴ tumor cells. The mixtures were then incubated at 37 ° C in the presence of 5% v / v CO2 for 72 hours. Live cells were counted using a Coulter counter. The result was compared with control data and it was found that anticancer agent BE-13793C exerted an intense inhibitory effect on the growth of P388 tumor cells. The anticancer agent BE-13793C has an IC ₅₀ , i.e. a concentration that causes 50% inhibition of the growth of P388 / S tumor cells from 0.7 microns and 0.7 microns for P388 / V cells.

P388 / S cells are commonly used mouse leukemia cells, while P388 / V cells represent a strain of P388 leukemia cells that have developed resistance to anticancer vincristine.

In addition, the anticancer agent BE-13793C also inhibited the growth of P388 / A cells, which in turn became resistant to the anti-cancer adriamycin; in this case, the 50% inhibitory concentration (IC ₅₀ ) is 1.0 microns.

The compound of the invention, BE-13793C, also exhibited antitumor activity against transplanted murine Ehrlich tumor cells (ascites type). In this study, 10 ⁶ tumor cells [lethal dose] per mouse were administered intraperitoneally. The test substance was serially diluted and administered intraperitoneally. Table 1 summarizes the results.

/. spreadsheet

Effect of BE-13793C on Ehrlich Ascites Cancer

Material Dose, ip ³ (mg / kg, injection) MST ⁴ (days) MST ⁵ ' ⁶ (% T / C) BE-13793C 50 28.6 218 20 26.4 202 8 16.0 122 control 0 13.1 100

Footnote:

1. Inoculum: 10 ⁶ Ehrlich ascites cancer cell, intraperitoneally

2. Host: Female ICR mouse

HU 212 160 B

3. Treatment Scheme: BE-13793C was administered intraperitoneally from day one to day 10, once daily.

MST: Mean survival time in days.

5.% T / C: (treated MST / control MST) x 100.

Criterion 6: When% T / C 5 125, the test compound is considered to have significant antitumor activity at the given dose.

Concerning the acute toxicity of BE-13793C anticancer agent in female ICR mice, there was no mortality up to day 5 when administered intraperitoneally at 100 mg / kg once daily.

As mentioned above, the antitumor compound BE-13793C of the present invention remarkably inhibits the growth of murine cancer cells. Therefore, it can be used in medicine as a valuable drug for the treatment of mammalian tumors such as leukemia and other cancers such as lung, stomach and colon cancers.

The invention also encompasses the use of a compound of the invention as an anticancer agent in the form of a pharmaceutical composition comprising an effective amount of the compound of the invention, optionally in an inert and pharmaceutically acceptable carrier or carriers.

Such a pharmaceutical composition can be prepared by combining the compound of the invention with inert and pharmaceutically acceptable carriers and various dosage forms, such as those for oral, parenteral or topical application. Suitable dosage forms are, for example, solid oral preparations (tablets, capsules, pills, powders, granules) and liquid oral preparations (e.g. solutions, suspensions, emulsions). In addition, sterile compositions prepared with sterile water, physiological saline or other sterile injectable solvent prior to use may also be mentioned. The composition may contain from 10 to 100% w / w of the compound of the invention.

The compound of the present invention may be used in any of its salts, as long as it is pharmaceutically acceptable. Examples of such salts are those derived from inorganic or organic bases such as sodium hydroxide, potassium hydroxide, sodium bicarbonate, triethylamine or 2-aminoethanol.

The clinically preferred dose of the compound of the invention will vary depending upon the compound employed, the type of material used to formulate the composition, the frequency of administration, the site of treatment, the host and the characteristics of the tumor. For example, the daily dose for an adult human is between 10 and 500 mg for oral administration and between 10 and 100 mg for parenteral, preferably intravenous. Although the frequency of administration will depend on the route of administration and the condition of the patient, it will generally be appropriate to administer the compound of the invention 1-5 times daily.

The following is a process for preparing BE-13793C. No restriction can be imposed on the microorganisms and their mutants used in the production of the anticancer agent BE-13793C as long as the

BE-13793 are capable of producing anticancer material. For example, strains of the microorganism having the following bacteriological characteristics may be used.

Morphological characteristics:

Under the microscope, the strain shows well-developed aerial mycelia, which form screws at almost constant intervals. In addition, 5-8 secondary branches are observed, each with 5-10 terminal spore chains.

The spores are cylindrical in shape (0.5x1-0.5x1.5 micrometers) and have a smooth surface.

No specific organ (eg sporangium, flagella spore, sclerotium) or fragmentation of the hyphae is observed.

Breeding characteristics:

Table 2 shows the characteristics of the culture on different agar plates at 28 ° C for 14 days.

Table 2

Medium Growth of aerial um Color of the basal mycelium Soluble color material yeast- extract- malt- extract- agar (ISP-2) very good, flat weak, cotton- white clear- brown no oatmeal- agar (ISP-3) very good, flat weak. cotton- white yellow no starch opener- szervet- flax salt agar (1SP-4) very good, flat good, dear Pot-szür- késfehér pale sárgásna- ranch no glycerol· aspartic gin-agar (1SP-5) very good, outstanding good, powdery yellowish-white sárgásna- ranch no peptone- yeast- extract- iron agar (ISP-6) very good, wrinkled good, powdery greyish white pale- brown no tyrosine agar (ISP-7) very good, outstanding good, powdery yellowish-white clear- brown no nutrient agar very good, flat good, powdery white pale yellowish- brown no sucrose dextrose, a nitrogen Rat-agar weak- few colorless no glucose aspartic gin-agar weak few sárgásna- ranch no

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3. Growth temperature (yeast extract-malt extract-agar medium, 14 days):

° C: poor growth and no aerial mycelium formation.

° C: poor growth and no aerial mycelium formation.

° C: Good growth and good aerial mycelium formation.

° C: Good growth but poor aerial mycelium formation.

° C: no growth.

4. Physiological characteristics:

(1) Gelatin liquefaction on glucose-peptone-gelatin medium: negative.

(2) Starch hydrolysis on starch inorganic salt agar medium: positive.

(3) Coagulation and peptonizing effect on skimmed milk: negative (medium: skimmed milk).

(4) Production of melanoid pigments: negative.

(5) Resistance to table salt in yeast extract malate extract agar medium: Increased salinity below 4% w / v.

5. Exploitation of carbon sources:

The following sugars were added to Pridham-Gottlieb agar medium and the strain was cultured at 28 ° C for 14 days. The results are shown in Table 3.

Table 3

D-glucose + D-Xylose - L-arabinose + L-rhamnose + D-fructose + D-galactose + raffinose + D-mannitol - inositol + salicin - saccharose -

Comment:

+: good recovery;

±: doubtful recovery:

no recovery.

6. Amino acid composition of the cell wall:

LL-diaminopimellic acid and glycine were detected.

These bacteriological characteristics suggest that the strain belongs to the genus Streptoverticillium. Relevant literature, including Bergey's Manual of Determinative Bacteriology 8th Edition (1974) and Hosenkin no Dotei Jikkenho (The Society for Actinomycetes, Japan) discloses that this strain is closely related to Streptoverticillium mobaraense-nck. However, it differs in raffinose and sucrose utilization. In addition, unlike the strain of the present invention, it forms a green mycelium on Streptoverticillium mobaraense agar medium. These facts indicate that the strain of the invention is new. so this Streptoverticillium sp. We named it BA-13793.

This strain was deposited in the Fermentation Research Institute, the Ministry of International Trade and Industry, Japan, where FERM P-10489 was converted into a deposit under the Budapest Convention by FERM BP-2785. number.

All variants and mutants of the microorganism producing the antitumor agent BE-13793C can be used for the purposes of the invention. Such mutants are known from the original strain, such as X-rays or ultraviolet light, chemical mutagens (e.g. nitrogen mustard, azaserine, nitric acid, 2-aminopurine or N-methyl-N'-nitro-N-nitrosoguanidine). ) or by routine transformation procedures (e.g., phage contact, transformation, transduction or conjugation).

To produce the BE-13793C antitumor agent of the present invention, the BA-13793-producing strain BE-13793C is cultured in an aerobic medium; A culture containing the anticancer agent BE-13793C is obtained. The medium may include nutrients that are commonly used to cultivate Actinomycetes. For example, the carbon source may be selected from commercially available glucose, glycerol, maltose, starch, sucrose, molasses, dextrin and mixtures thereof. Examples of nitrogen sources include commercially available soybean meal, corn germ meal, corn jam, meat extract, yeast extract, cotton seed meal, peptone, wheat germ, fish meal, inorganic ammonium salts, sodium nitrate, and mixtures thereof. Inorganic salts include commercially available calcium carbonate, sodium chloride, potassium chloride, magnesium sulfate and various phosphates, alone or in combination. In addition to the above, trace amounts of salts of heavy metals such as iron, copper, cobalt, molybdenum, manganese and zinc may also be used. Moreover, when significant foaming occurs, various vegetable oils such as soybean oil or linseed oil, higher alcohols such as octadecanol and various silicone compounds may optionally be added to the medium. In addition, any other medium component, such as boron compounds, 3- (N-morpholino) propanesulfonic acid, which can be used by the strain to promote the production of anticancer agent BE-13793C, may be used.

The strain can be cultured by the same procedures as those commonly used to produce microbial metabolites. Thus, solid or liquid culture can be used. In the case of liquid culture, a mixed, shaken or immersed aerobic culture mode may be selected, with shaking and immersed aerobic culture being particularly preferred. The culture is preferably at 20 and 37 ° C, preferably at 25 and 30 ° C

EN 212 160 B. The pH of the medium is preferably between 4 and 8. The culture is continued for 24 to 192 hours, preferably 48 to 120 hours.

The desired antitumor agent BE-13793C can be extracted from the culture using standard techniques for isolation of microbial metabolites. Because BE-13793C is present in both fermentation broth and cells, methods for purification from fermentation broth and cells can be combined, such as solvent extraction, ion exchange chromatography, affinity chromatography, partition chromatography or gel filtration. In addition, high performance liquid chromatography and thin layer chromatography methods can be used.

The separation and purification are preferably carried out as follows. The culture is first centrifuged to separate the cells and then extracted with an organic solvent such as methanol or acetone. The extract was concentrated under reduced pressure and the concentrate was extracted with an organic solvent such as ethyl acetate. This extract was evaporated to give a crude product containing ΒΕΙ3793C. The crude product is then purified by column chromatography on, for example, Sephadex LH-10 adsorbent. Thus, BE-13793C is obtained as a yellow-orange crystalline solid.

The following example is intended to illustrate further details of the invention and is not intended to be limiting. The present invention is to be understood as embracing all possible modifications of the example, as well as all known methods of preparation, evaporation, extraction and purification, which those skilled in the art, having regard to the properties of BE-13793C herein, work.

Example

Four 100 ml to 100 ml 6.7 pH 6.7 media containing 0.1% glucose, 2.0% dextrin, 1.0% wheat germ, 0.5% fish meal, 0.1% yeast extract in 500 ml conical flasks , 0.1% sodium chloride, 0.05% magnesium sulfate, 0.05% calcium chloride, 0.0002% iron (II) sulfate, 0.00004% copper (II) chloride, 0.00004 % manganese chloride, 0.00004% cobalt chloride, 0.00008% zinc sulfate, 0.00008% sodium borate, 0.00024% ammonium molybdate and 0.5% 3- (N-morpholino) propanesulfonic acid Contains Streptoverticillium BA-13793, inoculated on an oblique agar. Each flask was then incubated on a rotary shaker (180 rpm) at 28 ° C for 72 hours. 1 ml aliquots of the culture were inoculated into 100 ml of the above-mentioned medium in 50 500 ml conical flasks and incubated on a rotary shaker (180 rpm) at 28 ° C for 120 hours. The resulting culture, which was ca. 5 L, filtered and the filtered cells washed with 500 mL deionized water. Methanol (2.5 L) was then added and the mixture was stirred at room temperature for 1 hour. Filtration gave a methanolic extract. This methanol extraction is repeated. The combined approx. Extract 5 liters of methanolic extracts with ca. Concentrate to 800 ml. The resulting concentrate was extracted with 3 L of ethyl acetate and the ethyl acetate extract was evaporated to dryness. The residue was washed with 500 ml of chloroform. 720 mg of crude product containing BE-13793C are obtained. This crude product was dissolved in methanol (2 L) and the solution was concentrated. The resulting orange precipitate was filtered off to give 546 mg of product containing ΒΕΙ 3793C. This material was dissolved in a 1: 1 by volume mixture of methanol and tetrahydrofuran and chromatographed on a column of Sephadex LH-20 (1.5x120 cm - Pharmacia) eluting with a 1: 1 mixture of methanol and tetrahydrofuran. The fractions containing BE-13793C-1 were evaporated to give BE-13793C (99 mg) as a yellow-orange crystalline solid.

The antitumor agent γ 3793C of the present invention inhibits the growth of not only tumor cells that do not exhibit resistance to current anticancer agents, but also those that have developed resistance to said antitumor agents. Thus, the compound of the invention is a valuable drug for treating cancerous diseases in mammals, including humans.

Claims (2)

CLAIMS 1. A process for the preparation of an antitumor agent or a pharmaceutically acceptable salt thereof, having the formula I-BE-13793C, comprising culturing a microorganism or a mutant of the genus Streptoverticillium which produces the BE-13793C anticancer agent. the anticancer agent produced by BE-13793C is isolated and optionally converted to a pharmaceutically acceptable salt. 2. The method of claim 1, wherein the Streptoverticillium sp. Deposited under FERM BP-2785. The strain BA-13793 or a mutant thereof is cultured.