JPH0733395B2 - Antibiotic TAN-928 and method for producing the same - Google Patents

Description

TECHNICAL FIELD The present invention relates to a novel antibiotic TAN-928 and salts thereof useful as a therapeutic agent for fungal infections, a process for producing them, and TAN.
-928 Producing bacteria.

BACKGROUND ART The novel antibiotic TAN-928 of the present invention is a novel antibiotic based on its physicochemical and biological data, and such an antifungal antibiotic has not been reported yet.

Problems to be Solved by the Invention Chemotherapy by administration of antifungal agents is mainly performed for infectious diseases caused by fungi, but conventional antifungal agents often have strong side effects. The long-term or large-scale administration causes a bacterial replacement phenomenon or the emergence of resistant bacteria (resistance phenomenon), which has become a major problem in the current field of chemotherapy for fungal infections. In order to solve these problems, there is a need in the art for novel antifungal agents that have weak side effects and are clinically effective.

Means for Solving Problems The present inventors have conducted exploratory research for separating microorganisms from a large number of soil samples and obtaining antifungal agents from their metabolites. As a result, it was found that certain filamentous fungi produce antibiotics with antifungal activity in culture, which was isolated purely from the culture and, due to its physicochemical and biological properties, It was confirmed that the antibiotic was a new substance, and it was named antibiotic TAN-928.

The present inventors have completed the present invention as a result of further research based on these findings.

That is, the present invention relates to (1) the antibiotic TAN-928 or a salt thereof, and (2) the genus Trichoderma.
Culturing in a medium a microorganism having the ability to produce -928,
The present invention relates to a method for producing the antibiotic TAN-928 or a salt thereof, which comprises collecting and collecting the antibiotic TAN-928 in a culture.

Antibiotic TAN-928 is used as a bacterium producing the antibiotic TAN-928.
Any strain can be used as long as it produces Trichoderma aureoviride M-161 (hereinafter referred to as M-16).
The strain named "(sometimes abbreviated as 1 bacterium)" or a strain related to it is one example that is most effectively used.
The M-161 bacterium shows the following culture findings. This observation was made by observing each culture surface at 28 ° C for 2 weeks. The values in parentheses () in the table are the Color Harmony Manual 4th Edition (Continer Corporation of America 1958).
It is the description of the color name by (year).

The growth pH range of M-161 bacteria is pH 2.7 to pH 10.0, and the optimum pH is pH 5.0.
~ PH 6.0. Also, the growth temperature range is 11.5 ℃ ~ 39.0
℃, the optimum growth temperature is 30 ℃ ~ 33 ℃. The M-161 bacterium forms a ring-shaped phialide (Phialide) and is oval in shape and has a smooth surface and a size of 3.5 to 6 μm × 2.5 to 6 μm (average 3.8 × 3.2.
(μm) Phialospore.

As a result of trying to compare with the existing strains based on the morphological characteristics, culture findings, and physiological properties of the M-161 bacterium, this bacterium was identified as Trichoderma aureoviride, and Trichoderma aureoviride M-161 was identified. I named it. Trichoderma Aureobilide M-161 is Showa 62
Contract number I from the Fermentation Research Institute (IFO) from May 26, 2014
It was deposited as FO 32063 and deposited on June 6, 1987 by the Institute of Microbial Technology (FRI), Ministry of International Trade and Industry, under the deposit number FERM P-9402, and the deposit was converted to a deposit under the Budapest Treaty. Consigned number FERM
It is stored in the same laboratory as BP-1881.

As a general property of microorganisms, mycological properties easily change,
Trichoderma aureobilide M-161 is no exception, and various mutant strains can be easily obtained. However, even these mutants can be used in the method of the present invention as long as they do not lose the property of producing the antibiotic TAN-928. Of course, it does not matter whether those mutations are derived from natural causes or artificially performed using various mutagens (for example, ultraviolet rays, X-rays, radiation, nitrosoguanidine, etc.).

In culturing in the method of the present invention, a medium containing a carbon source that can be assimilated by microorganisms, a digestible nitrogen source, inorganic salts and the like is generally used. If necessary, trace nutrients, growth promoting substances, precursors and other trace effective substances may be added to the medium. Generally, carbon sources that can be assimilated by microorganisms include glucose, sucrose, molasses, starch, dextrin, and glycerin, and digestible nitrogen sources include meat extract, soybean powder, corn steep liquor, peptone, casein, cottonseed meal. Etc., and inorganic nitrogen compounds such as nitrates and ammonium compounds, all of which are effectively utilized. The culture may be performed by a surface culture method, but is usually a deep aeration stirring culture method.
In the case of the deep aeration agitation culture method, the properties of the medium are preferably near neutral, and the temperature during culture is preferably maintained at 20 to 36 ° C, preferably 24 to 30 ° C. However, the culture conditions such as the culture composition, the liquidity of the medium, the culture temperature, and the number of agitation should be appropriately adjusted and selected so as to obtain a preferable result depending on the kind of the strain to be used and external conditions. Needless to say.

To harvest the antibiotic TAN-928 of the present invention from the culture,
As shown in Examples described later, in consideration of the physicochemical properties of the substance, a separation / purification means usually used for collecting microbial metabolites is appropriately used. For example, an extraction method with a solvent, a method utilizing a difference in solubility with impurities, or chromatography is used alone or in combination. As the carrier used in chromatography, conventional inorganic and organic carriers such as silica gel, alumina, activated carbon, cellulose, high-porous resin, dextran gel and the like can be used.

In order to collect the antibiotic TAN-928 produced in the culture, it is necessary to extract it with a solvent such as methanol or acetone from the culture supernatant and the cultured cells obtained by previously separating by centrifugation or filtration. The obtained bacterial cell extracts were combined, and after being adsorbed on a column of a synthetic adsorbent, for example, Diaion HP-20 (manufactured by Mitsubishi Kasei), the column was developed stepwise with hydrous methanol to obtain an active fraction. After collecting and concentrating, the concentrated solution is made weakly acidic and extracted with an organic solvent such as n-butanol, i-butanol and ethyl acetate. After the obtained extract is concentrated, an organic solvent such as diethyl ether is added to obtain an elementary powder, which is then subjected to silica gel column chromatography. The column is developed with a mixed solvent system of ethyl acetate-isopropyl alcohol-water, fractions showing a single spot by thin layer chromatography described later are collected and concentrated, and the residue is treated with a mixed solvent of ethanol-ethyl acetate or the like. As a result, TAN-928 can be obtained as a white powder.

The physicochemical properties of TAN-928 (free form) obtained in Example 2 described later are as follows.

1) Shape: White powder 2) Elemental analysis value (%): Measured value Calculated value ^* C 59.79 59.54 H 8.00 8.08 N 2.01 2.04 S 4.52 4.68 ( ^* Calculated as including 1 mol of water) 3) Measured molecular weight: Di SI / MS method for sodium salt m / z 712 (M + H) ⁺ 4) Specific rotation: ▲ [α] ²⁴ _D ▼ -48.0 ° (c = 0.52, in methanol) 5) UV absorption spectrum: Terminal absorption (methanol) Middle) 6) Infrared absorption spectrum (main peak in KBr tablet method, cm
^-1 ): 3430,2970,2880,1750,1700,1630,1500,1460,1375,
1340,1260,1110,1055,980,950,885,840,765,625,585.

7) ¹³ C nuclear magnetic resonance (NMR) spectrum (100 MHz, heavy DMSO
Medium, δppm): The following signals are observed.

174.9,165.8,144.5,134.6,131.8,128.2,80.1,73.0,63.
0,62.5,60.5,55.5,55.2,54.5,42.1,40.2,39.4,35.1,34.
7,33.1,32.4,31.9,29.7,28.9,28.0,26.8,20.5,20.3,19.
7,19.5,19.4,18.8,17.3,15.8.

8) Solubility: Easily soluble; Methanol, dimethylsulfoxide Slightly soluble; Water, ethyl acetate, n-hexane 9) Color reaction: Positive; Phosphomolybdic acid, potassium permanganate, iodine, Greig-Riebach, Ehrlich's reagent Coagulation positive ; ninhydrin negative; Sakaguchi, Burton, Dragendorff's reagent 10) an acidic, neutral, basic distinction: amphoteric 11) thin layer chromatography (TLC) (carrier, silica gel glass plate 60F _254, 0.25 mm, West Germany Merck Made): 12) High Performance Liquid Chromatography (HPLC) Carrier: INERTSIL CDS5 (4.6 × 150mm) Solvent system: 75% Methanol-0.01M phosphate buffer (pH 6.3) Flow rate: 1 ml / min Retention time (t _R ): 7.4 min 13) ¹ H NMR spectrum (90 MHz, in deuterated methanol): shown in FIG.

TAN-928 obtained in Example 2 described below was treated with 6N-HCl to form 110
Β-Hydroxy-L when hydrolyzed by heating at ℃ for 10 hours
- generation of leucine and H ₂ SO ₄ was observed, whereas, TAN-92
8 to 0.1 M phosphate buffer: 50% methanol (1: 1 (V /
V)) (pH8.0) and hydrolyzed at 80 ℃ for 7 hours, C
A sterol having a molecular formula of ₂₈ H ₄₂ O ₅ was obtained. The physicochemical properties of this substance are as follows.

1) Elemental analysis value: C, 67.99; H, 9.06 Calculated value (as C ₂₈ H ₄₂ O ₅ .2H ₂ O): C, 67.99; H, 9.37 2) ¹³ C NMR spectrum (75 MHz, in deuterated methanol, δpp
m): 16.89,18.19,20.13,20.47,21.49,21.90,29.48,30.2
5,31.84,33.88,34.31,34.74,36.33,40.22,41.23,41.94,
44.30,57.47,57.53,62.51,65.25,65.43,69.15,130.20,1
33.85,135.98,146.95,178.03 Based on the physicochemical properties shown above, TAN-928 was judged to be a novel antibiotic having a sterol skeleton. Its structural formula is shown by the following formula.

Further, since TAN-928 has an acidic functional group, a pharmaceutically acceptable salt such as a metal salt such as sodium salt, potassium salt, calcium salt or an amine salt such as ammonium salt can be prepared by a method known per se. Can be formed.

Next, the antibacterial action of TAN-928 will be described. Filter paper disc (Toyo Seisakusho, diameter 8 mm) 1000 g / ml of TAN-928 free form
After soaking in methanol solution and air-drying for a certain period of time to eliminate methanol, paste this on agar-containing agar plates of various fungi and bacteria, incubate at a specified concentration for a specified time, and then suppress the growth that occurred around the filter disk. The diameter of the circle is shown in Table 1. In the table, "0" indicates that no inhibition circle was found. The medium used is as follows.

A: Sabouraud dextrose agar B: Trypticase soy agar (Becton Dickinson, USA) As is clear from Table 1, the antibiotic TAN-928 exhibits antibacterial activity against certain fungi. TAN-928 dose
No acute toxicity was observed when the mice were intraperitoneally administered at 200 mg / kg.

As is clear from these data, it can be said that TAN-928 exhibits antibacterial activity against fungi and is a mildly toxic antibiotic for mammals. Therefore, TAN-928 can be used for the treatment of fungal infections in humans, livestock and poultry.

For this treatment, TAN-928 can be formulated into various dosage forms of pharmaceutical compositions according to known formulation techniques.
For example, TAN-928 is usually 01-10 W / V%, preferably 0.5-
A liquid agent dissolved in distilled water at a concentration of 5 W / V%, or petroleum jelly or lanolin as the base, and TAN-928 is usually 0.
As an ointment containing 2 to 100 mg, preferably 2 to 50 mg,
It can be used for sterilization and disinfection of human or animal skin or mucous membrane.

The antibiotic TAN-928 is also considered to be a promising compound as a synthetic intermediate for new drugs.

As is clear from the chemical and biological properties described above, TAN-928 is a novel antibiotic.

EXAMPLES Next, the present invention will be described in more detail with reference to Examples.
The present invention is not limited to this. Percentages refer to weight / volume% unless otherwise stated.

Example 1 From 2.0% glucose, 3.0% soluble starch, 1.0% corn steep liquor, 1.0% defatted soybean powder, 0.5% peptone, 0.3% sodium chloride, 0.5% calcium carbonate (pH 7 adjusted) to 3 volumes of Sakaguchi Kolben After injecting 500 ml of broth medium, sterilize and add Trichoderma aureobilide M-
From slope culture of 161 (IFO 32063, FERM BP-1881) strain 1
After inoculating the platinum loop, 120 round trips / minute on a reciprocal shaker at 28 ℃
It was cultured for 48 hours. Actol (defoamer, manufactured by Takeda Pharmaceutical Co., Ltd.) in the above medium composition in a 50-volume stainless steel tank.
Prepared and sterilized medium 30 containing 0.05% and inoculated it with 500 ml of the whole culture of Sakaguchi Korben that had been cultivated previously, and aerated 30
The seed culture was obtained by submerged culture at 28 ° C for 48 hours at a stirring rate of 280 rpm for 1 minute.

Glucose 1.0%, dextrin 4.0%, raw soybean powder 0.5%, peptone 0.5%, yeast extract 0.2%, manganese sulphate 0.05%, potassium monophosphate in a 200-volume stainless steel tank.
Medium (pH 7.0) consisting of 0.1% and calcium carbonate 0.5% 120
After the preparation, the seed culture solution 6 was inoculated into the sterilized one, and cultured at 24 ° C for 5 days at an aeration rate of 120 / min and a stirring rate of 200 rpm.

Example 2 Hyflo Supercell (Johns Manville, USA) was added to the culture solution (150) obtained by culturing two 200-volume stainless steel tanks by the method of Example 1 and filtered, and the obtained filtrate was obtained. After separation, methanol (129) is added to the solid containing cells and stirred for 30 minutes. After stirring, the methanol extract (127) obtained by filtration was
It is passed through a column of Diaion HP-20 (20, Mitsubishi Kasei Kogyo, Japan) together with the culture filtrate (129) obtained by filtration. Column is 30% (V / V) methanol water (80)
After washing with, it was developed with 80% (V / V) methanol water (138). After concentrating the eluate containing the active ingredient to 21, adjust the pH to 4.
6 was extracted twice with i-butanol (10), the i-butanol layers were combined and concentrated, and treated with ether to obtain a crude powder (58.5 g).

The obtained crude powder was mixed with silica gel (100 g) and then subjected to column chromatography on silica gel (500 g, manufactured by Merck & Co., West Germany). The column was washed with ethyl acetate-isopropyl alcohol-water (10: 1: 0.5,4.0) and ethyl acetate-
After developing with isopropyl alcohol-water (5: 1: 0.5,4.0) and collecting and concentrating the active fraction (4.0) showing a single spot by the above-mentioned thin layer chromatography, the residue was mixed with ethanol-ethyl acetate. By treating with a solvent twice, a purified white powder (462 mg) of TAN-928 (free form) was obtained.

EFFECTS OF THE INVENTION The novel antibiotic TAN-928 and its salt of the present invention exhibit antibacterial activity against fungi, and can be administered to humans and other animals orally, parenterally or externally to treat these fungal infections. Can be advantageously used for the treatment of

[Brief description of drawings]

FIG. 1 shows the infrared absorption spectrum (KBr method) of the antibiotic TAN-928 obtained in Example 2, and FIG. 2 shows the ¹ H nuclear magnetic resonance spectrum (90 MHz, in deuterated methanol).

Claims (2)

[Claims] 1. A formula Antibiotic TAN-928 or its salt shown by. 2. An antibiotic TAN-928 belonging to the genus Trichoderma.
A method for producing an antibiotic TAN-928 or a salt thereof, which comprises culturing a microorganism having the ability to produce TAN in a medium, allowing the antibiotic TAN-928 to be produced and accumulated in the culture, and collecting this.