JP2844854B2 - FR901308 substance and method for producing the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION INDUSTRIAL APPLICATION This invention relates to a novel FR901308 substance. More specifically, the present invention relates to a FR having antibacterial and antitumor activity.
The present invention relates to a substance and a pharmaceutically acceptable salt thereof, a production method thereof, and a pharmaceutical composition containing them and a method of using the same.

[Arrangement of the Invention] The AR901308 substance is, for example, Arthroderm
a) It can be produced by culturing FR901308-producing bacteria belonging to the genus in a nutrient medium.

Microorganisms Microorganisms that can be used for the production of FR901308 substances are FR901308 substance-producing bacteria belonging to the genus Arsloderma. Among them, Arsloderma.sp F-10239 is newly isolated from soil collected in Tsukuba City, Ibaraki Prefecture.

This newly isolated Alsloderma.sp F-10239
Is a depositary number of the International Depositary Organization based on the Budapest Treaty, and the Institute of Microbial Industry and Technology (1-1-3 Higashi, Yatabe-cho, Tsukuba, Ibaraki Prefecture, 305 Ibaraki, Japan) has a deposit number of No. 2475 (FERM BP-2475). ) (Deposit date: 1989)
June 14, 2008).

Bacteria producing the new FR901308 substance are irradiated with X-rays, ultraviolet rays,
N-methyl-N'-nitro-N-nitrosoguanidine,
By conventional means such as treatment with 2-aminopurine
FR901308 can increase the production of substances.

The strain suppressively spreads on various media and forms a yellowish white to pale yellow colony. The strain, by culturing one month or more on oatmeal agar medium or YpSs agar medium, and Fuseiko嚢果the aerial mycelium was Deki mixed woven loosely (teleomorph), the genus Chrysosporium (Chryso
anamorph belonging to sporium ). The morphology, culture and physiological properties of the strain are shown below.

Table 1 shows the culture characteristics on various media. When inoculated on the center of potato dextrose agar medium and cultured at 25 ° C. for 14 days, the growth was repressive and expanded to 2.0-2.5 cm in diameter. The surface of the colony was flat, felt-like, white to yellowish white or pale yellow. The back of the settlement was light yellow to bright yellow. Conidia structures are slightly formed. When the same culture was performed on an oatmeal agar medium, the growth was more suppressive (1.5 to 2.0 cm in diameter). The colony surface was flat or raised, flocculent, white to yellowish-white. No anamorph was formed. One month after the start of the culture, ascocarps and conidia were abundantly formed on this medium.

The morphological characteristics were observed based on the growth on oatmeal agar medium and cornmeal agar medium. The ascocarp was Gimmnoascus-type, superficial, subspherical, white to yellowish-white, and 300-1000 μm in diameter including appendages. When cultured for a long period of time, adjacent ascocarps aggregate and grow to a diameter of 2000 μm. The ascocarp is composed of interwoven aerial hyphae and does not differentiate much. The ascocarp mycelium is colorless to pale yellow, with septum, straight or slightly bent, and bifurcated. The constituent cells of the ascocarp mycelium are rough (granular), cylindrical, thin-walled,
The size was (1.5-) 2-3 × 12-22 μm. The tip of the ascocarp mycelium often becomes a long, smooth, tapered appendix. This accessory is 1.5-2μm wide
And is straight, bent or loose spiral. Asci are scattered in ascos, are single-walled ascids, vanishing, subglobular to oval, endogenous 8 ascospores, 6-8 (-10) ×
5.5-7.5 (−8) μm. Ascospores are colorless, but when assembled they show yellow, smooth, 1 cell, 2.5-3.5
× 1.5-2 μm. It is usually oblate (flat sphere) to discoid, but when immature, it often has a shallow groove on the central equator.

The strain F-10239 forms a chrysosporium-type anamorph consisting of an aleurio-type conidia and a segmental-type conidia.
The aurelio-type conidia formed at the tip of the short branch of the vegetative hypha or directly on the side of the vegetative hypha are one cell, rough, pear-shaped or subspherical, cut at the base, and 2.
5-4 (-5) x 3-5 (-6.5) m. On the other hand, the segmented conidium formed inside the vegetative hypha is one cell,
The surface was smooth or rough, cylindrical to barrel, usually cut at both ends, and was 1.5-3.5 × 5-17 μm. The vegetative mycelium has septa, is colorless, smooth, and branched. Mycelial cells are cylindrical to filamentous and 2-10 μm wide. Chlamydospores are not formed.

The F-10239 strain can grow at 10 to 30 ° C, and the optimum growth temperature is 22 to 27 ° C (measured on a potato dextrose agar medium). This strain also grows at pH 4-11,
The optimum growth pH was pH 6-8 (Difco YM broth [D
ifco Co. Ltd .: YM broth]).

The characteristics of Table 1 were observed after culturing at 25 ° C. for 14 days. The description of the color tone was made based on the Muthuen Handbook of Color (6).

From the above characteristics, the F-10239 strain is an ascomycetous genus Arthroderma Currey ex Berkeley emend.Wei
tzman, McGinnis, Padhye et Ajello 1986) (1), (2), (3), (4), (5). Therefore, this producer strain was transformed into Arsloderma sp.
10239 ( Arthroderma sp, F-10239).

(1) Apinis AE: Mycol. Papers, 96: 1-56, 1964. (2) Padhye, AAand JW Carmichael: Can. J. Bot., 49:
1525-1540, 1971 (3) Benny, GLand JW Kimbrough: Mycotaxon, 12: 1-
91,1980. (4) Currah, RS: Mycotaxon, 24: 1-216, 1985. (5) Weitzman, I., MRMcGinnis, AAPadye, and L. Aje
llo: Mycotaxon, 25: 505-518,1986. (6) A. Kornarup and JHWanscher: Methuen Handbook
of Color, Third ed., Methuen, London, 1983 FR901308 substance FR901308 substance belongs to the genus Arsloderma FR901308
It is produced by culturing a substance-producing bacterium under aerobic conditions (eg, shaking culture, submerged culture, etc.) in a nutrient medium containing available carbon and nitrogen sources. The medium is
It may be a synthetic, semi-synthetic or natural medium.

Preferred carbon sources include glucose, mannose,
Glycerin, molasses, starch, starch hydrolyzate, and the like, and preferred nitrogen sources include meat extract, casein hydrolyzate, peptone, gluten powder, corn meal,
Cottonseed flour, soy flour, corn steep liquor, dried yeast,
Examples include ammonium phosphate, ammonium sulfate, and urea. In addition, metal phosphates, chlorides, and other salts, such as disodium hydrogen phosphate, potassium dihydrogen phosphate, calcium carbonate, ferrous sulfate, magnesium sulfate, copper sulfate, zinc sulfate, manganese chloride, and chloride Inorganic salts such as magnesium may also be added. If significant foaming is encountered during fermentation, an appropriate amount of a defoamer such as a vegetable oil, for example soybean oil, linseed oil, or a higher alcohol, for example, octadecanol, may be added.

 Culture may be performed at about 30 ° C. for 50 to 200 hours.

The optimal conditions for the culture are selected from the above fermentation conditions according to the characteristics of the microorganism strain used.

Most of the FR901308 substance thus produced in the culture solution is extracellular, so the culture solution is first filtered, and the filtrate is extracted with a suitable solvent such as ethyl acetate.

The desired compound is isolated and purified from the resulting mixture by procedures commonly used for the production of common antibiotics. For example, concentration under reduced pressure, lyophilization, solvent extraction, pH adjustment, treatment with anion exchange resin, cation exchange resin, nonionic adsorbent resin, etc., treatment with adsorbents such as activated carbon, silicic acid, silica gel, alumina, crystallization, Operations such as recrystallization may be employed alone or in appropriate combination.

The FR901308 substance produced in the culture can be isolated in its free form, but also in the form of a pharmaceutically acceptable salt, if desired. To isolate the substance in the form of a pharmaceutically acceptable salt, the desired compound obtained from the filtrate extract is treated with an inorganic base such as an alkali metal compound (eg, sodium hydroxide, potassium hydroxide, etc.), alkaline earth Bases such as organic bases (eg, triethylamine, dicyclohexylamine, etc.) or inorganic acids (eg, hydrochloric acid, sulfuric acid, phosphoric acid, etc.) or organic acids (eg, formic acid, etc.) Acetic acid, p-toluenesulfonic acid, citric acid, oxalic acid, etc.). Thereby, the corresponding salt of FR901308 substance can be obtained.

The salt of the FR901308 substance thus obtained can be reconverted into a free FR901308 substance by a conventional method known per se.

FR901308 substance is a new substance and has the following physicochemical properties.

Physicochemical properties of FR901308 substance: Appearance: Colorless (or white) prismatic substance Properties of substance: Neutral Color reaction: Positive-Cerium sulfate, sulfuric acid, iodine vapor, Dragendorff reaction Negative-Ninhydrin, chloride Each reaction of ferrous iron, Ehrlich, and Maurisch Solubility: soluble-chloroform, methanol, ethanol slightly soluble-ethyl acetate, acetone insoluble-hexane, water Melting point: 182 to 187 ° C Specific light intensity: ▲ [α] ²³ _D ▼ -112 ゜ (c = 1.0, CHCl ₃ ) Ultraviolet absorption spectrum: terminal absorption (methanol) Molecular formula: C ₂₇ H ₃₆ N ₄ O ₆ Elemental analysis (%): (as C ₂₇ H ₃₆ N ₄ O ₆ ) Calculated value: C63.26, H7.08, N10.93 Analytical value: C62.71, H7.05, N10.71 Molecular weight: FAB-MS m / z513 (M + H) ^{+ 1} H nuclear magnetic resonance spectrum: (400 MHz, CDCl ₃ −CD ₃ OD (2: 1)) δ 7.33−7.20 (5H, m) 5.02 (1H, dd, J = 10 and 6 Hz) 4.73 (1H, broad d, J = 7Hz) 4.28 (1H, t, J = 8Hz) 4.00 (1H, q, J = 7Hz) 3.72 (1H, m) 3.47 (1H, dd, J = 5 and 2.5Hz) 3.21 (1H, dd, J) = 13 and 10Hz) 3.06−2.95 (3H, m) 2.88 (1H, dd, J = 6 and 2.5Hz) 2.52−2.28 (3H, m) 2.08 (1H, m) 1.81-1.55 (6H, m) 1.59 ( 3H, d, J = 7Hz) 1.40-1.28 (4H, m) ¹³ C nuclear magnetic resonance spectrum: 207.9 (s) 174.7 (s) 174.0 (s) 172.9 (s) 171.5 (s) 136.1 (s) 128.6 (d ) X2 128.2 (d) X2 126.5 (d) 57.5 (d) 54.7 (d) 53.8 (d) 53.3 (d) 52.7 (d) 46.2 (t) 45.8 (t) 36.3 (t) 35.7 (t) 28.9 (t 28.1 (t) 25.0 (t) 24.3 (t) 24.0 (t) 22.3 (t) 14.9 (q) [Effect of the Invention] The FR901308 substance and its pharmaceutically acceptable salt are as follows:
It has antibacterial and antitumor activity and is useful as an antimicrobial and antitumor agent for humans and animals.

To demonstrate the utility of the FR901308 substance and its pharmaceutically acceptable salts, pharmacological test data is provided below.

(1) Antibacterial activity The target compound FR901308 has an antibacterial spectrum and shows activity against fungi including genus Aureobasidium and Geotricum.

Minimum Inhibitory Concentration (MIC) The MIC test was carried out using normal agar for the antibacterial test and Sabouraud agar for the antifungal test, followed by incubation at 30 ° C. for 24 hours, using the usual serial agar dilution method. The MIC value is represented by the minimum concentration (μg / ml) of FR901308 substance that inhibits the growth of microorganisms. The results are shown in Table 2.

(2) Antitumor activity 1) Test 1 Inhibition of human lung adenocarcinoma A549 cell proliferation in vitro In each well, fetal bovine serum, penicillin (50 units)
/ ml) and streptomycin (in minimal essential medium 100μ of 50 [mu] g / ml) Dulbecco supplemented with 5 × 10 ³ pieces of A5
A cytotoxicity test was performed using a microtiter plate containing 49 cells. The cells were incubated at 37 ° C. for 4 days and a colorimetric MTT (tetrazolium) assay was performed according to the method described in Mosmann (Journal of Immunological Methods 65: 55-63, 1983). . MTT [3- (4,5-dimethylthiazole-
2-yl) -2,5-diphenyltetrazolium bromide, manufactured by Sigma) was dissolved in PBS at a concentration of 5 mg / ml, and sterilized by filtration to remove a small amount of insoluble residues. After culturing A549 cells, this MTT solution (per 100μ of medium)
10μ) was added to all assay wells and the plates were further incubated at 37 ° C for 4 hours. Acidic isopropanol (100 μl of 0.04 NHCl isopropanol solution) was added to all wells and mixed well to dissolve the dark blue crystals. After all the crystals are dissolved, the plate is placed on a two-wavelength microplate photometer (MTP-100, Corona Electric Co., Ltd.)
Katsuta City, Japan) with reference wavelength 630nm, 550nm
Read in. The target compound of the present invention was dissolved and diluted in Dulbecco's minimum essential medium, and added to the cancer cell culture to a final concentration of 30 μg / ml or less. The results are shown in Table 3.

2) Test 2 Inhibition of human mammary adenocarcinoma MCF-7 cell growth in vitro 5 × in Dulbecco's minimum essential medium 100 μl supplemented with 10% fetal bovine serum, penicillin (50 units / ml) and streptomycin (50 μg / ml). 10 ³ of MCF-7 cells was performed cytotoxicity test using a microtiter plate containing the wells. Cells were incubated for 4 days at 37 ° C. in a 5% CO 2, 95% CO 2 incubator (wet) and colorimetric MTT assays were performed as described in Test 1. Test compounds were dissolved and diluted in Dulbecco's minimal essential medium and added to cancer cell cultures to a final concentration of 30 μg / ml or less. The results are shown in Table 4.

Pharmaceutical compositions of the present invention include powders, fine granules, granules, tablets,
Dragees, microcapsules, capsules, suppositories, solutions, suspensions, emulsions, syrups and other conventional pharmaceutical forms can be used. If desired, diluents or disintegrants (eg, sucrose, lactose, starch, crystalline cellulose, low-substituted hydroxypropylcellulose, synthetic aluminum silicate, etc.), binders (eg, cellulose, methylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, Polypropylpyrrolidone,
Polyvinylpyrrolidone, gelatin, gum arabic, polyethylene glycol and the like), coloring agents, sweeteners, lubricants (such as magnesium stearate) and the like may be incorporated into the composition.

The dose of the composition of the present invention varies depending on the age, weight, condition and the like of the patient, but is generally 100 mg to 10 g as a target compound or a pharmaceutically acceptable salt thereof, preferably 1 g to 5 g on the same basis. It is administered orally at daily dose levels at intervals of 1 to 3 times daily. A typical unit dose is 50 mg, 10
It may be 0 mg, 200 mg, 500 mg, 1 g, etc., but is not limited thereto.

[Examples] In order to further explain the present invention, examples are shown below.

Example 4% sucrose, 2% Pharmamedia, 1%
Dried yeast, 1% peptone (Kyokuto Pharmaceutical Co., Ltd.), 0.1%
2% potassium dihydrogen phosphate, 0.2% calcium carbonate,
A medium (160 ml) containing 0.1% of Twin 80 (polyoxyethylene sorbitan monooleate) was added to three 500 ml
Each flask was poured and sterilized at 120 ° C. for 30 minutes. One platinum loop of Alsloderma.sp F-10239 was inoculated into each medium and cultured on a rotary shaker at 220 rpm and 7.5 cm in amplitude for 6 days. The resulting culture is 2% modified starch, 2%
Scroll, 1% chicken meat-and-bone meal, 0.5% dry yeast, 0.2% potassium dihydrogen phosphate, 0.2% magnesium sulfate heptahydrate, 0.2% calcium carbonate, 0.001% zinc sulfate heptahydrate Medium, containing 0.05% adecanol (LG109, Asahi Denka Kogyo Co., Ltd.) and 0.05% silicone (KM-70, Shin-Etsu Chemical Co., Ltd.), and sterilized in advance at 120 ° C. for 30 minutes for 30 jars. Inoculate into fermenter, incubate at 30 ° C, 20 / min aeration and 200r
Culture was carried out for 7 days with stirring at pm.

After completion of the culture, diatomaceous earth (2 kg) was added to the culture solution of the two jar fermenters, and the mixture was filtered. The filtrate (30) was extracted with ethyl acetate 15. This extraction operation was performed three times, and the extracts were combined. The extract was concentrated to 2 under reduced pressure. After dehydration with anhydrous sodium sulfate, the ethyl acetate solution was further concentrated under reduced pressure.
(70-230 mesh, Merck) and mixed with 200 ml, and the mixture was suspended in methanol. After evaporation of the solvent, the resulting dry powder was subjected to column chromatography on silica gel 60 (300 ml) which had been pre-packed with n-hexane. The column was n-hexane (1), n-hexane-ethyl acetate (1: 1, 1),
Ethyl acetate (1), ethyl acetate-acetone (2: 1, 1
) And ethyl acetate-acetone (1: 1, 1). The target compound was eluted with ethyl acetate-acetone (2: 1) and ethyl acetate-acetone (1: 1). The fractions containing the desired compound were combined and concentrated under reduced pressure to give an oily residue. The oil was dissolved in 20 ml of dichloromethane and subjected to column chromatography on silica gel 60 (200 ml, 70-230 mesh, Merck) filled with a mixture of dichloromethane and methanol (40: 1). The column was diluted with dichloromethane-methanol (40:
1) After washing with 600 ml, elution was performed with 600 ml of the same solvent. The objective compound-containing fractions were combined and concentrated under reduced pressure to obtain a pale yellow powder. This pale yellow powder was dissolved in acetonitrile and stored overnight at 4 ° C. to give 660 mg of FR901308 substance as colorless crystals.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ identification symbol FI // (C12P 1/02 C12R 1: 645) Examiner Tetsuo Tsukanaka (58) Investigated field (Int.Cl. ⁶ , DB name) ) C12P 1/02

Claims (4)

(57) [Claims] 1. FR901308 substance having the following physicochemical properties and a pharmaceutically acceptable salt thereof: Appearance: Colorless (or white) prism crystals Property of substance: Neutral Color reaction: Positive-cerium sulfate, sulfuric acid , Iodine vapor, and Dragendorff reactions Negative-ninhydrin, ferric chloride, Ehrlich, Maurish reactions Solubility: soluble-chloroform, methanol, ethanol slightly soluble-ethyl acetate, acetone insoluble-hexane, water Melting point: 182 to 187 ° C Specific light intensity: ▲ [α] ²³ _D ▼ -112 ゜ (c = 1.0, CHCl ₃ ) Ultraviolet absorption spectrum: Terminal absorption (methanol) Molecular formula: C ₂₇ H ₃₆ N ₄ O ₆ elemental analysis _{(%) :( C 27 H 36} N 4 as O ₆₎ calculated: C63.26, H7.08, N10.93 analysis: C62.71, H7.05, N10.71 minutes child weight: FAB −MS m / z 513 (M + H) ^{+ 1} H nuclear magnetic resonance spectrum: (400 MHz, CDCl ₃ −CD ₃ OD (2: 1)) δ 7.33−7.20 (5H, m) 5.02 (1H, dd, J = 10 and 6 Hz) 4.73 (1H, broad d, J = 7Hz) 4.28 (1H, t, J = 8Hz) 4.00 (1H, q, J = 7Hz) 3.72 (1H, m) 3.47 (1H, dd, J = 5 and 2.5Hz) 3.21 (1H, dd, J) = 13 and 10Hz) 3.06−2.95 (3H, m) 2.88 (1H, dd, J = 6 and 2.5Hz) 2.52−2.28 (3H, m) 2.08 (1H, m) 1.81-1.55 (6H, m) 1.59 ( 3H, d, J = 7Hz) 1.40-1.28 (4H, m) ¹³ C nuclear magnetic resonance spectrum: (100 MHz, CDCl ₃ -CD ₃ OD (2: 1)) δ 207.9 (s) 174.7 (s) 174.0 (s ) 172.9 (s) 171.5 (s) 136.1 (s) 128.6 (d) X2 128.2 (d) X2 126.5 (d) 57.5 (d) 54.7 (d) 53.8 (d) 53.3 (d) 52.7 (d) 46.2 (t 45.8 (t) 36.3 (t) 35.7 (t) 28.9 (t) 28.1 (t) 25.0 (t) 24.3 (t) 24.0 (t) 22.3 (t) 14.9 (q) 2. A method for culturing a FR901308 substance producing bacterium belonging to the genus Arsuloderma in a medium, and isolating the FR901308 substance or a pharmaceutically acceptable salt thereof from the culture solution.
Method for producing FR901308 substance or a pharmaceutically acceptable salt thereof. 3. An antimicrobial agent comprising an FR901308 substance or a pharmaceutically acceptable salt thereof as an active ingredient. 4. An antitumor agent comprising an FR901308 substance or a pharmaceutically acceptable salt thereof as an active ingredient.