KR0177585B1 - Antitumor Substance B E-13793C-producing strain - Google Patents

Abstract

A novel anti-tumor substance of formula I BE-13793C or a pharmaceutically acceptable salt thereof, and a novel microorganism belonging to the genus Streptoverticlium                 Streptoverticillium species BA-13793 or mutants thereof are described.

Formula I

The novel material is prepared by culturing the microorganism and its mutants and harvesting the anti-tumor material BE-13793C from the culture obtained.

Since the novel substance BE-13793C or a pharmaceutically acceptable salt thereof has anti-tumor activity, these substances may be used in mammalian tumors, including human tumors.                 It is useful for treatment.

Description

            Anti-tumor substance BE-13793C-producing strain         

The present invention relates to novel compounds useful in the medical field. More particularly, the invention provides novel antitumor effects by inhibiting the growth and proliferation of tumor cells.                 Substances, methods of making these new substances, uses of these substances, and the genus Streptorticillium, which provides these substances.                 It relates to a novel microorganism.

In the field of cancer chemotherapy, metabolites of various microorganisms such as bleomycin or adriamycin have been used clinically.                 come. However, many of these materials are not sufficiently effective against the various tumors that are clinically encountered, and also the ability of tumor cells for these agents to                 There has been a clear increase in resistance and has been a barrier to clinical use of them [see The Proceedings of the 47th Congress of                 the Japanese Cancer Association, pages 12-15, 1988].

In this situation, the need for continuous development of new antitumor agents is natural. In other words, to overcome the resistance of various forms of tumors to existing anti-tumor agents                 There is a strong need for materials that can be effective even if they do not respond to available anti-tumor agents.

The inventors of the present invention screened the metabolites of various microorganisms to study promising antitumor agents, and found that the antitumor activity of the novel compounds of the general formula                 It became. The present invention has been accomplished based on the above findings.

Accordingly, the present invention provides novel anti-tumor substances of formula (I) or a pharmaceutically acceptable salt thereof represented by BE-13793C.

In a further aspect, the present invention provides a process for preparing an anti-tumor material BE-13793C, the use of the BE-13793C material as an anti-tumor agent, and to produce a BE-13793C material                 The present invention relates to a novel microorganism belonging to the genus Streptoverticillium.

The physical and chemical properties of the novel anti-tumor substance BE-13793C of the present invention are as follows.

Physical and Chemical Properties of BE-13793C

Appearance: Yellowish orange amorphous solid or crystal.

Molecular Formula: C ₂₀ H ₁₁ N ₃ O ₄ .

Elemental Analysis: Calcd C, 67.23%; H, 3.10%

Found C, 67.21%; H, 3. 12%.

Melting point: No definite decomposition point (melting point) below 295 ℃.

Solubility: poorly soluble in water, soluble in methanol, easily soluble in organic solvents such as tetrahydrofuran or dimethylsulfoxide.

Acid / Neutral / Base Distinguishing: Acid.

R _f value: 0.45 (developing solvent: chloroform / methanol; 5: 1 v / v), (Kieselgel 60 F ₂₅₄ , manufactured by Merck).

Color reaction: Potassium permanganate: positive

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

Ultraviolet (UV) absorption spectrum (λ _max ^MeOH , nm): 245, 298, 307, 327, 400

Infrared (IR) Absorption Spectrum (ν _{cm -1} ^KBr ): 3430, 3270, 1743, 1710, 1590, 1485, 1408, 1335, 1290, 1245, 1060, 815, 800, 765

¹ H-NMR Spectrum (DMSO-d ₆ , δ ppm):

6.98 (2H, br d, J = 7.6 Hz), 7.13 (2H, t, J = 7.6 Hz), 8.42 (2H, br d, J = 7.6H z), 10.19 (2H, br                 s), 10.87 (1H, br s), 11.57 (2H, br s)

¹³ C-NMR spectrum (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)

Biological Activity of BE-13793C

In vitro activity tests are performed to assess the inhibitory activity of the antitumor substance BE-13793C on mouse tumor cells. In an in vitro anti-tumor assay using P388 tumor cells, firstly the solution obtained by dissolving the test substance in dimethylsulfoxide was treated with a cell culture medium containing 20% dimethylsulfoxide (DMSO-RPMI-1640 medium 20% v / v). Serial dilution). Then, the diluted solution 2㎕, and the tumor cells or 2x10 ⁴ 3x10 ⁴ cells culture medium (fetal calf serum -RPMI 1640 medium-10% v / v) 200㎕ containing. Each mixture is then incubated for 72 hours at 37 ° C. under 5% CO ₂ . Viable cells are then counted using a Coulter counter. This result is compared with control data. As a result, antitumor substance BE-13793C exhibits a potent inhibitory effect on P388 tumor cell proliferation. The concentration of the anti-tumor substance BE-13793C (IC ₅₀ ) which inhibits P388 / S tumor cell proliferation 50% is 0.7 μM and the 50% inhibitory concentration of P388 / V cell proliferation is 0.7 μM.

P388 / S cells are common mouse leukemia cells, and P388 / V cells are resistant to the antitumor substance vincristine P388                 Leukemia cells.

In addition, the antitumor substance BE-13793C inhibits P388 / A cell proliferation resistant to the antitumor agent adriamycin, its 50% proliferation inhibitory concentration (IC ₅₀ ) is 1.0 μM.

Compounds of the present invention, named BE-13793C, have antitumor effects against Erhrlich tumor cells (plural) implanted in mice. In this assay, 10 ⁶ tumor cells (lethal dose) per mouse are administered intraperitoneally. The test substance is then serially diluted and administered intraperitoneally. The results are summarized in Table 1.

The acute toxicity of the anti-tumor substance BE-13793C in female ICR mice was associated with death on day 5 with one intraperitoneal administration of 100 mg / kg of the substance.                 Not found.

As described above, the anti-tumor substance BE-13793C of the present invention significantly inhibits the proliferation of mouse cancer cells. Therefore, it is leukemia and lung, stomach, colon                 It is useful as a tumor therapeutic agent of a mammal including various tumors, such as a tumor.

In addition, the present invention is in the form of a pharmaceutical composition comprising a single form or any inert and pharmaceutically acceptable carrier with an effective amount of a compound of the present invention                 It relates to the use of a compound of the invention as an antitumor agent.

Such pharmaceutical compositions are prepared by mixing the compounds of the present invention with inert and pharmaceutically acceptable carriers, and for various administrations such as oral, parenteral or topical administration.                 It can be provided in the form. Suitable dosage forms include oral solid preparations (e.g. tablets, capsules, pills, powders, granules) and oral liquid preparations (e.g. solutions, suspensions,                 Emulsions). It may also be provided as a sterile composition dissolved immediately before use in sterile water, physiological saline or other sterile solvent for injection.

The content of the compound of the present invention in this composition is preferably 10 to 100% w / w.

Examples of certain pharmaceutically acceptable salts of the compounds of the invention include inorganic or organic bases such as sodium hydroxide, potassium hydroxide, sodium bicarbonate, triethylamine or                 Included are those obtained using 2-aminoethanol).

Clinically preferred dosages of the compounds of the present invention depend on the particular compound employed, the type of combination composition, frequency of administration, site of treatment, and the nature of the host and tumor.                 Follow. For example, the daily dose per adult is 10 to 500 mg orally and 10-100 mg for parenteral administration, preferably intravenously.                 Although the frequency of administration varies depending on the method of administration and the condition of the patient, in general the compounds of the invention are sufficient to be administered from one to five times a day.

The preparation method of BE-13793C is described below.

Microorganisms and mutants thereof used in the preparation of the anti-tumor substance BE-13793C of the present invention can be used as long as they can produce the anti-tumor substance BE-13793C.                 It is not limited. For example, microbial strains having the following bacteriological properties can be used.

1. Form

Microscopically, this strain has well-developed airborne hyphae that annularly forms at approximately regular intervals. It also forms five to eight second side chains,                 5 to 10 terminal spore chains are observed respectively.

Each spore is cylindrical (0.5 × 1 to 1.5 μm) and has a smooth surface.

No specific organs (eg spore sac, flagella spores or fungal nuclei) or segments of mycelium are observed.

2. Culture Characteristics

Table 2 shows the characteristics when incubated for 14 days in various agar plate medium at 28 ℃.

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

12 ℃: Poor growth and no hyphae in air.

20 ℃: Poor growth and no hyphae in air.

28 ° C: Good growth and good hyphae formation in air.

37 ° C: Good growth and poor mycelial formation.

45 ° C: No growth.

4. Physiological Features

(1) Liquefaction of gelatin: negative (glucose-peptone-gelatin medium).

(2) Hydrolysis of starch: positive (starch-inorganic salt agar medium).

(3) Coagulation and peptonation of skim milk: negative (skim milk medium).

(4) melanin pigment formation: negative.

(5) Resistance to saline: grown at a saline content of 4% W / V or less (yeast-malt-agar medium).

5. Utilization of carbon source

The following sugars are added to the Pridham-Gottlieb agar basal medium and the strains are incubated at 28 ° C. for 14 days. Table 3 shows this result.                 Indicates.

6. Amino Acid Composition of Cell Wall

LL-diaminopimelic acid and glycerin are detected.

These bacteriological properties indicate that this strain belongs to the genus Streptoverticillium. Bergey's Manual of Determinative                 Hosenkin no edited by Bacteriology 8th Edition (1974) and Japanese Society of Actinomycetes                 Dotei Jikken-ho], the strain is closely related to Streptoticillium mobaraense.                 It can be seen that it is related. However, this strain differs from the strain in that it uses raffinose and sucrose. In addition, streptoverticlium mobaraense,                 Unlike this strain, green agar is shown in agar medium. This fact suggests that this strain is novel. Thus, it is known as Streptoverticillium spp.                 Named BA-13793.

This strain was deposited on January 20, 1989 with the accession number FERM P-10489.                 Research Institute, Agency of Industrial Science and Technology, Ministry of                 International Trade and Industry, Japan) and on March 1, 1990, under the Butafest Treaty,                 It was transferred and deposited as FERM BP-2785.

For the purposes of the present invention, all variants and mutants of the antitumor substance BE-13793C-producing microorganism can be used. Mutants such as this                 Irradiation with X-ray or ultraviolet light from the strain, substances causing chemical mutations (e.g. nitrogen mustard, azaserine, nitrous acid, 2-aminopurine or                 Known as N-methyl-N'-nitro-N-nitrosoguanidine (NTG), or conventional transformation techniques (e.g. contact, transformation, transduction or conjugation with phage)                 Guided by technology.

In order to prepare the anti-tumor substance BE-13793C of the present invention, BE-13793C-producing strain BA-13793 was cultured in nutrient medium under aerobic conditions.                 A culture containing the antitumor substance BE-13793C is obtained. The nutritional substance contained in the medium may be one commonly used in culturing Actinomycetes.                 For example, the carbon source can be selected from commercially available glucose, glycerol, maltose, starch, sucrose, molasses or dextrin and mixtures thereof.                 Nitrogen sources include commercially available soybean powder, corn gluten meal, corn steep liquor, meat extract, yeast extract, cottonseed meal, peptone, wheat germ, fish meal, inorganic ammonium salt,                 Sodium nitrate and mixtures thereof. As inorganic salts, commercially available calcium carbonate, sodium chloride, potassium chloride, magnesium sulfate or various phosphates                 It can be used individually or in combination. In addition, traces of heavy metal salts (eg iron, copper, cobalt, molybdenum, manganese, zinc salts) can be used if necessary.                 In addition, when foaming occurs a lot, antifoaming agents such as various vegetable oils (eg, soybean oil or flax oil), higher alcohols (eg, octadecanol), and various silicone compounds                 It may optionally be added to the medium. In addition, as long as the strain is used to enhance the production of the anti-tumor substance BE-13793C, other media components (eg, boron compounds,                 3- (N-morpholino) propanesulfonic acid) can be used.

This strain can be cultured in the same manner as is generally used for the preparation of microbial metabolites. That is, solid culture or liquid culture can be used. Liquid                 In the case of culture, stationary culture, stirred culture, shaking culture or aeration culture can be performed, but shaking culture and aeration culture under agitation are particularly preferable. culture                 The temperature may range from 20 to 37 ° C., with a range of 25 to 30 ° C. preferred. The pH value of the medium is preferably in the range of 4-8. This culture is 24                 To 192 hours, preferably 48 to 120 hours.

The desired antitumor substance BE-13793C can be recovered from the culture by a separation method commonly used for recovery of microbial metabolites from the culture.                 Since BE-13793C is contained in the culture filtrate and the cells, conventional separation methods (e.g., solvent extraction,                 Purification by ion exchange chromatography, affinity chromatography, partition chromatography, or gel filtration). In addition, high performance liquids                 Chromatography and thin layer chromatography can be used.

Preferred separation and purification methods are as follows. First, the culture solution is centrifuged to recover the cells, and then extracted with an organic solvent such as methanol or acetone. this                 The extract is concentrated under reduced pressure, and the obtained concentrate is extracted with an organic solvent such as ethyl acetate. Concentrate this extract to give crude containing BE-13793C                 Obtain the product. This crude product is then purified by column chromatography using, for example, Sephadex LH-20. therefore,                 BE-13793C can be obtained in the form of a yellow orange crystalline material.

The following examples are merely intended to illustrate the invention in more detail, and are not intended to be limiting. The present invention is directed to all variations and examples of the examples given herein                 Based on the properties of BE-13793C described in the specification, all known preparations, concentrations, extractions and those applicable to those skilled in the art for BE-13793C                 It is contemplated that purification methods be included. EXAMPLE

Glucose 0.1%, Dextrin 2.0%, Corn gluten meal 1.0%, Fish meal 0.5%, Yeast extract 0.1%, Sodium chloride 0.1%, Magnesium sulfate 0.5%,                 Calcium chloride 0.05%, ferrous sulfate 0.0002%, cupric chloride 0.00004%, manganese chloride 0.00004%, cobalt chloride 0.00004%, zinc sulfate                 Culture comprising 0.00008%, sodium borate 0.00008%, ammonium molybdate 0.00024% and 3- (N-morpholino) propanesulfonic acid 0.5%                 Streptoverticlium cultured on agar slope medium in four 500 ml conical flasks each containing 100 ml of medium (pH 6.7)                 After inoculating BA-13793 strain, each flask is incubated for 72 hours on a rotary shaker (180 rpm) at 28 ° C. Recall 1 ml aliquot of the culture                 Inoculate 50 500 ml conical flasks each containing 100 ml of the mentioned medium for 120 hours on a rotary shaker (180 rpm) at 28 ° C.                 Incubate. The obtained culture solution (about 5 L) is filtered and the cells thus obtained are washed with 500 ml of deionized water. Subsequently, 2.5 liters of methanol was added thereto to                 The mixture is stirred at rt for 1 h. Methanol extract is obtained after filtration. The extraction with methanol is repeated. Methanol extract (about 5 L) combined                 Concentrate to 800 ml. The concentrate thus obtained is extracted with 3 liters of ethyl acetate and the ethyl acetate extract is concentrated to dryness. The residue thus obtained is                 Wash with 500 ml of chloroform. This yields 720 mg of crude product containing BE-13793C. This crude product was dissolved in 2 liters of methanol                 Concentrate. Filtration of the orange precipitate thus formed yields a product containing 546 mg of BE-13793C. Solvent in the product                 Dissolved in a mixture (methanol / tetrahydrofuran; 1: 1 v / v) and columned with Sephadex LH-20 (1.5 × 120 cm, Pharmacia)                 Chromatography develops with methanol / tetrahydrofuran (1: 1 v / v). The BE-13793C fraction thus obtained was concentrated to obtain 99 mg of BE-13793C.                 Obtained in the form of a yellow orange crystalline material.

Anti-tumor substance BE-13793C of the present invention is a tumor resistant to the anti-tumor agent as well as the proliferation of tumor cells that are not resistant to conventional anti-tumor agent                 Inhibit the proliferation of cells. Thus, the materials of the present invention are useful as therapeutic agents for tumors in mammals, including human tumors.

Claims (1)

Streptoverticillium sp. BA-13793 [FERM BP-2785] microorganism capable of producing the antitumor substance BE-13793C.