JP2002155036A - New antifungal compound and method for producing the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new compound useful as a medicine, especially an antifungal agent. SOLUTION: Q73453 substance represented by formula (I) or its pharmaceutically acceptable salt. A method for producing the Q73453 substance, characterized by culturing a microorganism belonging to the genus Pseudallescheria and then isolating the Q73453 substance from the culture product. The method for producing the Q73453 substance, wherein the microorganism is Pseudallescheria ellipsoidea CBS128. 78.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a novel fermentation product useful as a medicine, especially as an antifungal agent, a method for producing the same, and a medicine therefor.

[0002]

2. Description of the Related Art Rapid progress in research and development of antibacterial agents since the 1950's and their widespread use have led to the elimination of many bacterial infectious diseases. On the other hand, in recent years, opportunistic infections have become a major problem. Therapeutic agents for fungal infections, which account for the majority of opportunistic infections, include amphotericin B, griseofulvin,
Five drugs of nystatin, ketoconazole and fluconazole are used. However, due to frequent use of fluconazole, the emergence of fluconazole-resistant bacteria has been reported, and amphotericin B, which is the only effective therapeutic agent for azole-based drug-resistant bacteria, is concerned about side effects such as nephrotoxicity.

[0003]

There is still a continuing need to create effective antifungal compounds.

[0004]

Means for Solving the Problems The present inventors have conducted intensive studies on substances produced by many naturally occurring microorganisms, and found that microorganisms belonging to the genus Pseudoareseria produce substances having antifungal activity. I found something to do. Then, the microorganism was cultured, and the novel antifungal compound was successfully isolated from the culture, thereby completing the present invention. That is, the present invention relates to Q73 represented by the following formula (I).
453 or a pharmaceutically acceptable salt thereof.

[0005]

Embedded image Also, the present invention provides a method for producing a Q73453 substance, comprising culturing a microorganism belonging to the genus Pseudoareseria and capable of producing the Q73453 substance, and isolating and collecting the Q73453 substance from the culture. About. Examples of microorganisms belonging to the genus Pseudoareseria include commercially available Pseudoareseria ellipsoidia
CBS 128.78 strain (Centraalbureau Voor Schimmelc
ulturesBaarn-Delft; The Netherlands) is preferred. Further, the present invention relates to a medicament, particularly an antifungal agent, comprising a Q73453 substance or a pharmaceutically acceptable salt thereof as an active ingredient.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION The Q73453 substance of the present invention or a pharmaceutically acceptable salt thereof has an asymmetric carbon atom and a double bond, and therefore has a stereoisomer (racemate, optical isomer, Diastereomers) and geometric isomers (cis or trans). Accordingly, the compounds of the present invention include a mixture or isolated forms of these stereoisomers or geometric isomers. Further, the present invention relates to Q7
Also included are various hydrates and solvates of the 3453 substance or a pharmaceutically acceptable salt thereof, and crystals thereof, and polymorphs thereof. The compounds of the present invention also include all compounds that are metabolized in vivo and converted into the aforementioned Q73453 substance or a pharmaceutically acceptable salt thereof, so-called prodrugs. The group forming the prodrug of the present invention includes Prog. Med. 5: 2157-2161.
(1985) and groups described in Hirokawa Shoten 1990, “Development of Pharmaceuticals,” Vol. 7, Molecular Design 163-198.

The compound of the present invention may form a salt. There is no particular limitation as long as it is a pharmaceutically acceptable salt. Specific examples of the acid addition salt include inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, phosphoric acid and the like. , Formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid,
Acid addition salts with organic acids such as citric acid, methanesulfonic acid, ethanesulfonic acid, aspartic acid, glutamic acid, and the like. The salt with a base is a salt with an inorganic or organic base, specifically, an inorganic base such as sodium, potassium, magnesium, calcium, aluminum, an organic base such as methylamine, ethylamine, ethanolamine, lysine, or the like. Examples thereof include salts with a basic amino acid such as ornithine.

(Production method) As the microorganism used for producing the Q73453 substance of the present invention, any microorganism can be used as long as it belongs to the genus Pseudoareseria and has an ability to produce the Q73453 substance. Preferably, a commercially available Pseudoareseria ellipsoidia CB
S128.78 strain.

[0009] The cultivation is carried out according to a general microorganism culturing method. The medium used for the culture may be any medium as long as the microorganism used can grow, and a synthetic medium, a semi-synthetic medium, or a natural medium can be used. As nutrients to be added to the medium, known nutrients for microorganisms can be used. For example, commercially available molasses,
Glucose, maltose, fructose, mannitol, potato starch, corn starch, dextrin,
Carbohydrates such as soluble starch or fats and oils, fats and the like, as a nitrogen source, commercially available peptones, meat extracts, corn steep liquor, cottonseed meal, peanut flour,
Soy flour, yeast extract, NZ-amine, wheat germ, caseins, fish meal, and inorganic or organic nitrogen sources such as sodium nitrate and ammonium nitrate are used.

The metal salts include Na, K, Mg, Ca, Z
Lead sulfates such as n, Fe, Mn, Co, and Cu, hydrochlorides, nitrates, phosphates, carbonates, and the like can be added as necessary. If necessary, valine, leucine, isoleucine, phenylalanine, tryptophan, methionine, lysine, arginine, glutamic acid, aspartic acid and other amino acids, vitamins, oleic acid, methyl oleate, lard oil,
A secondary metabolite production promoting substance such as silicone oil or a surfactant or an antifoaming agent can be appropriately used. In addition to these, any additives can be used as long as they can be used by the bacteria producing the compound of the present invention and are useful for producing the compound of the present invention. The culturing method may be the same as the culturing method for producing general antibiotics, and the culturing method may be solid culturing or liquid culturing. In the case of liquid culture, any of stationary culture, shaking culture, and stirring culture may be performed, but aeration and stirring culture is particularly desirable. As the culture conditions, the culture temperature is a temperature at which the production bacterium can grow and produce the compound of the present invention, that is,
It can be suitably applied in the range of 10 to 40 ° C, but preferably about 24 to 28 ° C. The pH can be appropriately applied in the range of 4 to 9, but is preferably 6 to 7. The cultivation time varies depending on various conditions, usually 1 to 30
The compound of the present invention is accumulated in the medium in about a day.

In order to isolate the target compound from the culture, conventional extraction and purification means used for isolating a metabolite of a microorganism can be appropriately used. The substance in the culture may be a culture solution as it is, or a culture filtrate obtained by centrifuging or adding a filter aid to the culture to obtain a culture filtrate, which is an organic compound that is immiscible with water such as ethyl acetate, chloroform, benzene, and toluene. Add solvent and extract. Further, the culture solution is brought into contact with an appropriate carrier, the produced substance in the filtrate is adsorbed, and then the substance is extracted by eluting with a suitable solvent. For example, Amberlite XAD-2, Diaion HP-20, Diaion CHP-20P, or Diaion SP-8
The substance is adsorbed by contact with a porous adsorption resin such as 50. Next, the substance is eluted using a mixture of water and an organic solvent such as methanol, ethanol, acetone and acetonitrile. The fraction containing the substance can be obtained by gradually or continuously increasing the mixing ratio of the organic solvent from a low concentration to a high concentration.

Next, the substance-containing fraction obtained by each of the above procedures is subjected to column chromatography using silica gel, ODS, etc., centrifugal liquid separation chromatography, ODS
It can be further separated and purified by a conventional method such as high performance liquid chromatography (HPLC) using the above method. The compound of the present invention obtained as described above can be subjected to a conventional salt formation reaction, if desired, to obtain a pharmaceutically acceptable salt. Stereoisomers and geometric isomers can also be obtained by a known chemical isomerization method or a separation method (for example, a method of treating with an acid or a base, isomerizing and then performing optical resolution or the like).

[0013] The pharmaceutical composition of the present invention containing one or more of the Q73453 substance of the present invention or a pharmaceutically acceptable salt thereof as an active ingredient is a pharmaceutical carrier commonly used in the art. , Excipients and the like, and can be prepared by a commonly used method. Oral administration by tablets, pills, capsules, granules, powders, liquids, etc., or injections such as intravenous and intramuscular injections, suppositories, eye drops, eye ointments, transdermal solutions, ointments, Any form of parenteral administration using a transdermal patch, an inhalant, a transmucosal solution, a transmucosal patch, or the like may be used.

As the solid composition for oral administration according to the present invention, tablets, powders, granules and the like are used. In such a solid composition, the one or more active substances comprise at least one inert excipient, such as lactose,
It is mixed with mannitol, glucose, hydroxypropylcellulose, microcrystalline cellulose, starch, polyvinylpyrrolidone, magnesium aluminate metasilicate and the like. The composition may contain an inert additive, for example, a lubricant such as magnesium stearate, a disintegrant such as sodium carboxymethyl starch, and a solubilizing agent according to a conventional method. The tablets or pills may be coated with sugar coating or a gastric or enteric coating, if necessary.

[0015] Liquid compositions for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, elixirs and the like, and are generally inert solvents such as purified water, Contains ethanol. This composition contains, besides the inert solvent, auxiliaries such as solubilizers, wetting agents and suspending agents,
It may contain sweetening agents, flavoring agents, fragrances and preservatives. Injections for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Aqueous solvents include, for example, distilled water for injection and physiological saline. Examples of the non-aqueous solvent include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, alcohols such as ethanol, and polysorbate 80 (trade name). Such compositions may also contain isotonic agents, preservatives, wetting agents, emulsifiers, dispersants, stabilizers, and solubilizing agents. These are sterilized by, for example, filtration through a bacteria retaining filter, blending of a bactericide or irradiation. In addition, these can be used by preparing a sterile solid composition, dissolving and suspending in sterile water or a sterile injection solvent before use.

Usually, in the case of oral administration, 0.1 to 500 mg orally and 0.01 to 100 mg parenterally are appropriate for an adult per day, and these are administered once or more than once a day. The dose is appropriately determined depending on the individual case in consideration of symptoms, age, sex, and the like. Since the dose varies under various conditions, a dose smaller than the above dose range may be sufficient.

[0017]

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples. Example 1 10 g glucose, 20 g potato starch, 5 g polypeptone,
Medium containing yeast extract 5g, calcium carbonate 4g, distilled water 1L
(pH 7.0) was dispensed in 100 ml aliquots into 500 ml navel conical flasks and sterilized at 120 ° C. for 20 minutes. Pseudoadescheria grown well on potato dextrose agar
Ellipsoidia (Pseudallescheria ellipsoidea) CB
The S128.78 strain was scraped, inoculated, and shake-cultured at 24 ° C. and 200 rpm for 4 days. The above culture solution was inoculated at a rate of 3% into four similarly prepared culture media and shake-cultured at 24 ° C. and 200 rpm for 3 days to obtain a seed culture solution. Next, as a production medium, brown rice (60 g) was placed in a 500 ml Erlenmeyer flask.
200 media containing 60 mg of strike extract, 60 mg of dipotassium phosphate, 60 mg of sodium tartrate and 100 ml of distilled water were prepared and sterilized twice at 120 ° C. for 20 minutes. Seed culture in this medium
Inoculation was performed at a rate of 3%, and stationary culture was performed at 24 ° C. for 20 days.

80% of the 20 L culture thus cultured
100 L of acetone water was added and stirred for 3 hours. Thereafter, 5% Radiolite # 600 (Showa Chemical Co., Ltd.) was added and the mixture was filtered, and acetone was distilled off from the filtrate under reduced pressure. This concentrated solution was subjected to two-layer partition extraction with an equal volume of ethyl acetate. The obtained ethyl acetate layer was concentrated to dryness under reduced pressure to obtain 1.8 g of an extract. The obtained extract was subjected to SSC-OD
ODS flash column chromatography using S-7515-12A (Senshu Kagaku KK) was performed. The elution of ODS flash column chromatography was performed using methanol
This was carried out with a mixed solvent of water, and the content of methanol was gradually increased. That is, first add the mixed solvent (60:40) to 100
Then, 100 ml of (70:30), (80:20), (90:10), and (100: 0) were sequentially flowed and eluted. As a result, methanol-water (90:10)
The desired activity was observed in the eluted fraction of. The obtained active fraction was concentrated to dryness under reduced pressure, and Sephadex LH-20
(Amersham Pharmacia Biotech Co., Ltd.) 30φ × 90
It was subjected to 0 mm column chromatography. Use methanol for elution, one using a fraction collector.
It was fractionated by ml. The obtained active fraction, fraction 44-
46 was concentrated to dryness under reduced pressure to give 210 mg of the active substance. The obtained active substance was finally fractionated using high performance liquid chromatography. High performance liquid chromatography
L-column ODS as a column
φ × 250mm), acetonitrile-water (90: 1
Using the mixed solution of 0), the mixture was flowed at an outflow rate of 7 ml / min. Detection was performed at a detection wavelength of 210 nm, and activity was observed in the peak eluted at 18.9 minutes. The peak was collected and concentrated under reduced pressure to isolate 7.2 mg of the substance Q73453-A.

The isolated Q73453-A material had the following physicochemical properties: (1) Color and shape: white amorphous translucent solid. (2) Classification of acidic, neutral and basic: neutral. (3) Solubility: soluble in methanol, but hardly soluble in water and hexane. (4) Ultraviolet absorption spectrum (solvent: methanol): 2
The absorption maximum is shown at 80, 226 and 202 nm (FIG. 1). (5) Molecular weight: 345.53 (6) Molecular formula: C ₂₂ H ₃₅ NO ₂ (7) HPLC analysis Column: L-column ODS (Chemicals Inspection Association; 4.6φ)
Elution solvent: mixed solution of acetonitrile-water (90:10) Outflow rate: 0.7 ml / min Detection wavelength: 210 nm Detection time: 10.6 minutes (8) ¹ H-NMR spectrum (400 MHz, CDCl ₃ ): (No. (Fig. 2) (9) ¹³ C-NMR spectrum (100 MHz, CDCl ₃ ): (Fig. 3) From the above physicochemical properties, the chemical structural formula of the Q73453-A substance is as follows with the double bond in the trans form It was decided to be.

Embedded image

[0020]

The substance Q73453 of the present invention or a pharmaceutically acceptable salt thereof is a human pathogen, Candida.
It shows antifungal activity against Albicans and the like, and is useful as a medicine, especially as an antifungal agent. The antifungal activity of the compound of the present invention was confirmed by the following pharmacological test.

Test Example 1 Test for Antifungal Activity The antifungal activity against Candida albicans ATCC 10231 was measured by a microfluidic dilution method (Kume Hikaru, Yamazaki Toshikazu, Clinical and Microorganisms, Vol. 21, No. 5, pp. 573-580, 1994). It was measured as the minimum inhibitory concentration (MIC) according to it. Q73453 of the present invention
-A material had a MIC of 0.16 mg / ml.

[0022]

[Brief description of the drawings]

FIG. 1 is an ultraviolet absorption spectrum of Q73453-A substance.

FIG. 2: ¹ H-NMR spectrum of Q73453-A substance (400
MHz, CDCl ₃ ).

FIG. 3: ¹³ C-NMR spectrum of Q73453-A substance (10
0 MHz, CDCl ₃ ).

[0023]

──────────────────────────────────────────────────の Continuation of the front page (51) Int.Cl. ⁷ Identification symbol FI theme coat ゛ (Reference) C12R 1: 645) C12R 1: 645) (72) Inventor Yoji Yamaguchi 1-1-8 Shozuzawa, Itabashi-ku, Tokyo Yamanouchi Pharmaceutical Co., Ltd. (72) Inventor Masato Watanabe 1-1-8 Shozusawa, Itabashi-ku, Tokyo Yamanouchi Pharmaceutical Co., Ltd. (72) Nami Shinmura 3-17-1, Hasune, Itabashi-ku, Tokyo Yamanouchi Pharmaceutical Co., Ltd. F term (reference) 4B064 AE01 BA04 BD02 BE09 BG01 BH04 BH05 CA05 CE07 CE08 CE10 DA01 4C206 AA01 AA02 AA04 FA11 KA01 KA19 MA01 MA04 NA14 ZB35 4H006 AA01 AA03 AB29 BN30 BR10 BU36

Claims (5)

[Claims] 1. A Q73453 substance represented by the following formula (I) or a pharmaceutically acceptable salt thereof. Embedded image 2. Pseudallescheri
a) culturing a microorganism belonging to the genus and capable of producing the Q73453 substance according to claim 1, and isolating and collecting the Q73453 substance from the culture;
Production method of 73453 substance. 3. Pseudallescheri
a) The microorganism belonging to the genus is Pseudallescheria ellipsoidea CBS1
3. The method according to claim 2, wherein the strain is 28.78 strains. 4. A medicament comprising the Q73453 substance according to claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient. 5. The medicament according to claim 4, which is an antifungal agent.