JPS61234786A - Wf-20714 substance and production thereof - Google Patents

Abstract

NEW MATERIAL:WF-20714 Substance having the following physical and chemi cal properties. Colorless platy crystal: amphoteric substance; melting point: 261-263 deg.C (decomposition); [alpha]D<20>=+0.8 (c=1.0, H2O); elemental analysis: C 44.28%, H 5.57%, N 22.86%; mass analysis (SIMS): m/z=243(M+1); solubility: soluble in water, insoluble in methanol, ethanol, acetone, or ethyl acetate; color reaction: positive in ninhydrin, iodine, phosphomolybdic acid, potassium perman ganate, Pauly's reagent and negative in Molish reaction, 2,4- dinitrophenylhydrazine, and Dragendorff's reagent; etc. USE:An inhibitor of blood platelet aggregation. PREPARATION:For example, a microorganism [e.g., Myrothecium verrucaria F-20714 strain (FERM P-8174), etc.] is capable of producing WF-20714 substance is cultivated in a proper medium preferably by submerged culture at about 25-30 deg.C for 50-300 hours.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] This invention provides a novel WF-
It concerns 20,714 substances and is used in the medical field.

[Purpose of the invention]

The present invention relates to a novel WF-20714 substance.More specifically, the present invention provides a novel WF-20714 substance and its salts having platelet aggregation inhibiting activity, and a method for producing them.

[Structure of the invention]

This invention provides WF-207 having the following physical and chemical properties.
14 substances, their salts, and their production methods.

0 Color and properties: Colorless plate-like crystals ■ Dimphoteric substance with distinction between basic, acidic, and neutral ■ Melting point: 261
~263℃ (decomposition) [4] Specific luminosity: [α] Bas = 10.8° (c = 1.0
H2O).

■Elemental analysis value: C, 44,28%; H, 5,57%;
N, 22.86% ■Mass spectrometry value (SIMS): m/z=243 (M+1)
[7] Ultraviolet absorption spectrum: λ nm (ε) = 212 (5300) aX ■Infrared absorption nuvector: v KBr: 3400. 1660, 1610,
1580.

aX 1420, 1360, 1340, 1100.

940 礪■ 1H-Nuclear Magnetic Resonance Absorption Nuvector: δ (heavy water; internal standard tetramethylsilane): 3,06
(LH, dd, , T=13.2, 5.61(Z
), 3.08 (2H, d, J=6.9Hz
), 3.12 (IH, dd, Jmi, 13
, 2, 6.9Hz), 3.51(LH, t
, J=6.9Hz).

3,81(LH, d(1, J=5.9, 5.6
Hz), 7.24 (IH.

'd, J=1.3Hz), 8.45(LH,
d.
α), 63.4(d)+xx7.2(d), x3o. s
(s), 134. z(d), 173.4(S), 17
8,9(S)I)pm O Solubility: Soluble: Water-insoluble: Methanol, ethanol, acetone.

Ethyl acetate O color reaction: Positive: Ninhydrin. Iodine, phosphomolybdic acid, potassium permanganate, plagiarism reagent negative: silver nitrate, ferric chloride. Morish reaction +2.4
-Dinitrophenylhydrazine, Dragend/L/7 reagent O thin layer chromatography: Rf value (I)/n-butano/L/: ethanol: chloroform: aqueous ammonia = 4:'? :2:(II) f acetonitrile:water=6:4(I[D/n-butanol:acetic acid:water=2:l:*) Little sister manufactured by Mary Co.) WF-20714 of this invention manufactured by Eastman Company The substance may be a WF-20714 substance-producing bacterium belonging to the genus Myrocesium.
It can be produced by culturing -20714 substance-producing bacteria in a medium and separating and collecting the WF-20714 substance from the resulting culture.

Among the WF-20714 substance-producing bacteria used in this invention, the strain newly isolated and collected by the inventors from a soil sample collected in Yakushima, Kagoshima Prefecture (numbered as strain F-207) is as shown below. It has good mycological properties.

Characteristics on various media Table 1 shows the characteristics of the growth state when cultured for two weeks at 25°C on malt extract agar medium and potato-glucose agar medium. For color names, we used the Japan Color Research Institute's "Color Standard J."

Table I Culture characteristics of strain F-20714 on various media Strain F-20714 can grow in the range of 5 to 31°C, and the optimal growth temperature is 22 to 27°C.0 Also, the growth pH range of this strain The pH is between 4 and 10, and the optimum growth pH is between pH 5 and 6.

1''-20714 strain was not observed to exhibit sexual reproduction on various media, but abundant asexual reproduction consisting of conidiophores with densely packed mats of hyphae was formed. Conidiophores forms a layer on the upper part of the conidia, and the conidiation mode is phialoid, an endobud type.

Based on these morphological characteristics, strain F-20714 seems to belong to the genus Myrocesium, which is a fungus deuteromycetes.

Conidia consist of densely packed vegetative hyphae, and are heterogeneous in size, with a diameter of 50 to 500 μm, a height of 50 to 100 μm, and adjacent conidia aggregate to become even larger (1n or more in diameter).
do. Conidiophores are formed in parallel on the upper part of the conidiophore, are colorless, smooth, and have 2 to 4 branches that repeat several times. The apical part of the conidiophore has 3 to 5 phialides whorled on each branch, and is 10 to 15 μm long and 2 to 3.5 μm wide. Conidia are light green, smooth, boat-shaped to lens-shaped, with a slightly pointed tip and a papillary cut at the base, 6-8 μm long, 2.5-4 μJ wide, and 1-2 vacuoles. Motsu. Conidia formed from neighboring phialides aggregate to form a conidial stock, finally forming a dark green agglomerate with a diameter of 0.5 to 3 pellets on the conidium. The vegetative hyphae are colorless, smooth, slightly branched, and have septate walls.

Hyphal cells are rod-shaped or cylindrical, 1 to 3.5 μm wide, and no chlamydospores are formed.

Based on these characteristics, strain F-20714 is a strain of Eris (M,
B, ElliS) Dematiaceous Hyph
omycetes, C, M, Eng.

Key, 1971), page 555. −
7') -su (Myrothecium verru)
caria (Albertini et Schwei
nitz) Dl, mar ex Fri, es). Therefore, we investigated the characteristics of this strain described above and tested it [Ikones MiOrO-fungOrum (C0neS MiOrO-fungOrum)].
A Matsushima LeOtOrllll, 1
975), p. 100], Uda [Illustrated Encyclopedia of Fungi (Part 2), Kodansha, Tokyo, 1978, p. 10'71], etc., and as a result, there was a close match. Strain and identification Myrocesium b/l/IV carya-F-20714 (MyrO
theOium verrucaria F-20714
) was named.

This Milocesium Fist Pei IVI Reka'J7・F-2071
The four strains were deposited at the Institute of Microbial Technology, Agency of Industrial Science and Technology as Microtechnology Research Institute Deposit No. g/7'1 on May 5, 1985.

The WF-20714 substance-producing bacteria used in this invention can be treated with irradiation such as X-rays or ultraviolet rays, such as nitrogen mustard, azaserine, nitrous acid, 2-aminopurine, N-methyl-N'-nitro-N- The production ability of WF-20714 substance can be increased by commonly used bacterial mutation treatment methods such as treatment with mutagenic agents such as nitronedine (NTG), 7age contact, transformation, transduction, and conjugation. can.

WF-20714 produced by culturing WF-20714 substance-producing bacteria belonging to the genus Myrocesium in a medium.
In principle, the production of substances follows the cultivation methods of general microorganisms, but deep culture methods using liquid media are usually advantageous. The medium used for culturing may be any medium containing a nutrient source utilized by the WF-20714 substance-producing bacteria belonging to the genus Myrocesium. That is, a synthetic medium, a semi-synthetic medium, or a natural medium is used, and the medium composition is such that carbon sources such as olecose, sucrose, maletose, glycerin, starch, and liquefied starch are used, and nitrogen sources include: For example, meat extract, casein hydrolyzate, peptone, gluten meal, corn meal, cottonseed flour, soybean flour, corn steep liquor, peanut powder,
Wheat germ, dried yeast, yeast extract, urea, ammonium sulfate, etc. are used. In addition, inorganic salts such as disodium hydrogen sulfate, potassium dihydrogen sulfate, magnesium chloride, magnesium sulfate, calcium carbonate, sodium iodide, and cobalt chloride hexahydrate are also added to the medium as necessary. .

In addition, when foaming is significant during culturing, for example, vegetable oils such as soybean oil and linseed oil, octadecanol, tetradecanol, etc.
Antifoaming agents such as higher alcohols such as heptatool and silicone compounds may be added as appropriate.

The appropriate culture temperature is around 25-30°C, and good results are often obtained if seed culture is carried out as appropriate as the culture volume increases. The culture time for the main culture is suitably about 50 to 300 hours, and the culture time may be further extended as the medium becomes more concentrated.

The culture conditions described above are applied by selecting the optimum conditions for each depending on the characteristics of the production strain used.

Next, since the WF-20714 substance produced during culture usually accumulates in the liquid contained in the culture, it is generally removed by centrifugation, filtration, etc. to remove the bacterial cells and the filtrate. (
The supernatant liquid is separated, purified, and collected from the liquid by means used in the production of general antibiotics. That is, liquid conversion, treatment with resins such as anion exchange resins, cation exchange resins, nonionic adsorption resins, treatment with adsorbents such as activated carbon, silicic acid, silica gel, alumina, cellulone, etc., crystallization, By combining or repeatedly applying methods such as recrystallization in any order, the target substance WF-20714 can be separated and purified.

The WF-20714 substance obtained in free form is a base (e.g., sodium hydroxide, potassium hydroxide, hydroxide!
A desired salt can be obtained by reacting with methane, sodium bicarbonate, potassium carbonate, triethylamine, dicyclohexylamine, etc.) or an acid (for example, hydrochloric acid, sulfuric acid, formic acid, acetic acid, trichloroacetic acid, maleic acid, etc.).

〔Effect of the invention〕

The substance WF-20714 and its pharmaceutically acceptable salts, which are the target substances of this invention, have a platelet aggregation inhibiting effect, as is clear from the following tests, and are useful as medicines for humans and animals.

Test (platelet aggregation inhibitory effect test): (a) Test method Washed rabbit platelet suspension (platelet count: 5 x 105
/μl＞CBritish Journal of Hematology (Bri, tj-sh Journal of
Haematolo-g3') Volume 19, Nos. 7-17
(1970)] was added with a predetermined amount of WF-20714 substance, and while stirring, phospholipase C (PLO), a platelet aggregating agent, was added to PLO.
was added so that the concentration was 0.08 unit/ml.

Platelet aggregation was measured by turbidimetry by recording the change in light transmittance of the washed platelet suspension during aggregation.

The activity of the WF-20714 substance was expressed as the Co value, ie, the concentration required to inhibit platelet aggregation by 50%.

(1)) Test result C56: 1.1 /If/1nI! The substance WF-20714 and its pharmaceutically acceptable salts, which are the object substances of this invention, can be mixed with a conventional organic or inorganic solid or liquid carrier suitable for oral, parenteral or external use, and then prepared in a conventional pharmaceutical preparation. It can be used in the form of formulations, for example solid formulations such as capsules, tablets, granules or half-opens, semi-solid formulations such as ointments or liquid formulations such as solutions or emulsions.

In addition, the above-mentioned formulation may appropriately contain conventional additives such as stabilizers, wetting agents, and emulsifiers.

The dosage of WF-20714 substance and pharmaceutically acceptable salt depends on the patient's age, weight, symptoms, etc., but is usually administered in the range of about 0.1 to 1100 OIR per mouth.

〔Example〕

The present invention will be explained below with reference to Examples.

Example 1 Soluble starch 1.0%, corn starch 1.0%,
Glucose 160%, Cottonseed flour 1.0%, Dry yeast 1.0
%, cornsteep liquor 1.0% and calcium carbonate 0.2% (pH 6.0).
Pour 1 ooml into 4 bottles of 00M triangular 7 Ranuko 1.1
Sterilize for 30 minutes at 20°C. These were inoculated with one platinum loopful of Myrocesium bell/l//'Ju7 F-20714 strain (Feikoken Bacteria t$, No. I/7'7) from the slant, and shaken at 25°C for 4 days. Cultivate the chicken.

2.0% Soluble Starch, 2.0% Glecone, 2.0% Corn Steep Liquor, 0.0% Peanut Powder.
5%, begton 0.5%, dried yeast 0.5%, and calcium suboxide 0.2% (pH 6.5) were placed into four 304-volume jar fermenters each containing 201
Sterilize at 20°C for 30 minutes. Add the above seed culture solution to these.
Inoculate 6% each and culture at 25°C for 5 days (aeration rate zo
1 minute, internal pressure 1.0 Kg/ari! % stirring 25or
pm).

The obtained culture solution is filtered using Radiolight (trade name: manufactured by Showa Kagaku Kogyo Co., Ltd.) to obtain solution F (381). Solution F is adsorbed on SK to IB (H type) resin (trade name: manufactured by Mitsubishi Chemical Industries, Ltd.) (81), washed with water (241), and then eluted with a 1M aqueous pyridine solution (326). The eluate was diluted with diethylaminoethyl se7 y 7' y
DEAE-Sephadex A-25 (
Off type) (Product name: Manufactured by Fine Chemical Co., Ltd.) (31)
After washing with water (151), it is eluted and fractionated with O, IN hydrochloric acid. Both parts containing the target substance were combined and neutralized with a 6N aqueous sodium hydroxide solution, and then the carboxyme f)V
-* 7 y 7” 7 ri)l (CM-Seph
adex) C-25 (H+ type) (product name: manufactured by Fluin Chemical Co., Ltd.) (1,54) and water (4,5Al)
After sequential washing with O, OINlN hydrochloric acid, 51)
Elute with 0.03N hydrochloric acid (31). The eluate was adjusted to pH 9.0 with IN aqueous sodium hydroxide solution and applied to an activated carbon column (11). Elute and separate with water, and combine the fractions containing the target substance and concentrate under reduced pressure to 20 ml. 9C) Ethanol (2 one) was added to C while heating, and the precipitated crystals were separated and dried to obtain WF-20714 substance (1,5
3j') is obtained.

Claims (1)

[Claims] (1) WF-20714 substance and its salts having the following physical and chemical properties. [1] Color and properties: colorless plate-like crystals [2] Basic, acidic, neutral: amphoteric substance [3] Melting point: 261-263°C (decomposition) [4] Specific luminosity: [α]^2 ^0_D=+0.8°(c
=1.0H_2O) [5] Elemental analysis value: C, 44.28
%; H, 5.57%; N, 22.86% [6] Mass spectrometry value (SIMS): m/z = 243 (M+
1) [7] Ultraviolet absorption spectrum: λ^(H_2)^O_m_a_xnm(ε)=212(
5300) [8] Infrared absorption spectrum: ν^K^B^r_m_a_x: 3400, 1660, 1
610, 1580, 1420, 1360, 1340, 1
100, 940=cm^-^1 [9]^1H-nuclear magnetic resonance absorption spectrum: δ (heavy water;
internal standard tetramethylsilane): 3.06 (1H, dd
, J=13.2, 5.6Hz), 3.08 (2H, d,
J=6.9Hz), 3.12 (1H, dd, J=13.
2, 6.9Hz), 3.51 (1H, t, J=6.9H
z), 3.81 (1H, dd, J=6.9, 5.6Hz
), 7.24 (1H, d, J = 1.3Hz), 8.45
(1H, d, J=1.3Hz) ppm[10]^1^3
C-Nuclear magnetic resonance absorption spectrum: δ (heavy water; internal standard tetramethylsilane): 28.4 (t), 47.6 (t)
, 54.2(d), 63.4(d), 117.2(d)
, 130.8(s), 134.2(d), 173.4(
s), 178.9 (s) ppm (11) Solubility: Soluble: Water-insoluble: Methanol, ethanol, acetone, ethyl acetate (12) Color reaction: Positive: Ninhydrin, iodine, phosphomolybdic acid, permanganate Potassium, Pauli reagent negative: silver nitrate, ferric chloride, Molisch reaction, 2,4
-Dinitrophenylhydrazine, Dragendorff's reagent (13) Thin layer chromatography: Rf value (I) n-butanol: ethanol: chloroform:
Ammonia water = 4:7:2: 7 (II) Acetonitrile: water = 6:4 (III) n-butanol: acetic acid: water = 2:1:1 *) Manufactured by Merck * *) Manufactured by Eastman (2) ) WF-20714 substance-producing bacteria belonging to the genus Myrocesium are cultured in a medium, and WF-20714 is extracted from the culture.
A method for producing a WF-20714 substance, which comprises collecting the substance. (3) The method for producing the WF-20714 substance according to claim 2, wherein the WF-20714 substance-producing bacterium belonging to the genus Myrocesium is Myrocesium berucaria.