JPS59179092A - 3(2-substituted-oxazol-5-yl)indole, its salt and their preparation - Google Patents

Abstract

PURPOSE:A newly isolated microorganism in Streptoverticillium is cultured to produce the titled novel substance with an activity of inhibiting blood platelet coagulation. CONSTITUTION:A novel strain, Streptoveticillium waxmanii No.30581 (FERM- 7013) has been isolated. The cultivation of the strain in a usual culture medium at 25-30 deg.C for 50-100hr gives a novel compound, 3(2-substituted-oxazol-5-yl)indole or its salt. In the formula, R is propyl or butyl. The products have an action of inhibiting blood platelet coagulation.

Description

DETAILED DESCRIPTION OF THE INVENTION This invention provides novel 3-(2-substituted-oxazole-5-
il) relating to indole and its salts.

More specifically, this invention 8I-1 relates to a novel 3-(2-substituted-oxazol-5-yl)indole having platelet aggregation inhibiting activity.

3-(2-substituted-oxazol-5-yl)indole, which is the target substance of this invention, is represented by the following general formula (% formula %) (wherein R means propyl or butyl). Examples of the salt of 3-(2-substituted-oxacyl-5-yl)indole include acid addition salts such as hydrochloride.

The inventor of this invention discovered that a newly grown plant from the soil (1.
They discovered that (2-substituted-oxazol-5-yl)indole can be produced, elucidated its chemical structure, and completed this invention.

(1) Process for producing 3-(2-substituted-oxazol-5-yl)indole 3-(2-substituted-oxazol-5-yl)indole can be produced from Streptparticillium soxmani, for example. 3-(
It can be produced by culturing a bacterium that produces 2-substituted-oxazol-5-yl)indole according to a conventional method.

3-(2-substituted-oxacy/l) used in this invention
/-5-il) Among the indole-producing bacteria, the strain (numbered A 30581) newly isolated from soil collected by the inventor of this invention in Wajima City, Ishikawa Prefecture is shown below. It has mycological properties.

■Various media (Growth status in Table 1 using 10 types of media shown in Table 1, 30°C) For colonies cultured for 40 days, growth status of the colony, color of the colony;
We observed the color of the back side of the colony and the formation of soluble pigments.

As a result, when released on a medium with good growth, the color of the aerial mycelium was grayish or reddish.No production of soluble pigment was observed in the 10 types of media used.

■ Morphological characteristics The wood fungus Ita is applied to oatmeal agar medium, yeast extract/1 bud extract agar medium, scooch/inorganic salt agar medium, etc.
'ii L. After growing at 30°C for 14 days, the morphology was observed and observed using an optical microscope 1 and an electronic W1 microscope.

The results are shown below.

ID spore-forming cocoon thread nucleation method: Whorl division technique + 1 = j Morphology of spore-forming hyphae, size of linear or wavy spores 20.7-0.8X1.5-1.8, l
i, 1 spore surface: Smooth spore release, 10~20 sporophyte iq life (ke [〆l'': aerial hyphal epiflagella j, presence or absence of staticia, not observed. presence or absence of sclerotia: observed). Physiological characteristics raw B:' C+ (-H□ll quality is mainly due to shading (5
Gottlieb
) (International Journal of Systematic Bacteriology, 16th Spring, 313th
-340 pages, 1966).

Growth temperature range: 14°C to 42°C optimal temperature
=31°C to 358C growth pH range, 3
~95 Optimum pH 7 Nitrate reduction ・Hydrolysis of positive starch ・Coagulation of positive milk ・Peptonization of sterile milk 2. Production of weakly positive melanin pigment ・Liquification of positive gelatin: Negative production of hydrogen sulfide: Physiological characteristics of the 30581 strain are that it produces melanin, reduces nitrate, and weakly peptonizes milk.

■ Cell wall composition Cell wall component CD Becker et al. (Applied Microbiology 12th Spring, No. 421-
423 pages, 1964) and Yamaguchi (Jihye-Narob.
Bacteriology Vol. 89, pp. 444-453, 19
The analysis was performed according to the method of 1965).

As a result, strain A 30581 contained LL' as a cell wall component.
- Contains diaminopinolic acid.

(2) Assimilation of the carbon source Assimilation of the carbon source was carried out according to the method of Pridham and Gottlieb (Journal Op.), Cteorology No. 56, pp. 107-114, 1948). Judgment is 30°
C, carried out after 14 days of incubation.

Glycerin + D-xylose - Sodium citrate Lactose - D-7 lactose Rhamnose ± maltose - Sodium succinate + Inulin ± Inositol Raffinose - D-Galactose + L-arabi/-su - D-Glucose + Mannitol + D - Mannose + Sucrose - Cellulose - D-trehalose Chitin - Sodium acetate - +: Assimilate. ±: Weakly assimilated, -: Formation of spores without assimilation The branching method of hyphae forms whorls, cell wall components + cLL
- Wood fungi "FA" to Streptocarticillium by adding diaminopimelic acid.

Therefore, the above characteristics are summarized in the report of Schuling and Gottlieb (Inc. National Journal of Systematic Bacteriology Vol. 18, No. 69-189).
Pages 279-'392, 1968; Ibid. No. 1
9@, pp. 391-512, 1969) or Locci et al.
agia 17'4, pp. 1-60, 1969)
We searched for Streptparticillium fucusmani, which seemed to be a good match, and conducted a detailed search. The result 1. (The strain fM 30581 matched very well with Streptparticillium waxmani. Therefore, the water bottle was
30581 (5 trepto-vertical)
,ivm waksm,ar+ii IG 30581
) was identified.

This is Streptocartillium waxmani.

Streptparticillium used in this invention
3-(2-Substituted-oxacyl-5-yl)indole production-1 For example, Streptparticillium waxmani A30581 bacteria may be treated with irradiation such as X-rays or ultraviolet rays, such as nitrogen mustard, hot serine, etc. Nitrous acid, 2-aminopurine, N-methyl-N'-
The 3-(2-substituted-oxazole -5-yl)indole production capacity, can be enhanced by one 3-(2-substituted-
Oxacyl-5-yl)indole is produced by culturing 3-(2-substituted-oxazol-5-yl)indole-producing bacteria belonging to the genus Sleptparticillium in a medium. In principle, the culture method is based on the culture method for general microorganisms, but the deep culture method using a liquid medium is usually advantageous. A medium was used, and the medium composition was as follows: Topo-g/l/cose, sucrose, glycerin, texturin, and starch hiko were used as carbon sources; Gluten Neil, Cornmeal, Cottonseed Meal, Soybean Flour, Beannut Meal, Corn Stibliquer, 7
17: An organic or inorganic nitrogen source such as dried Mflh, ammonium acetate, ammonium phosphate, or urea can be used. Carbonates of metals such as lucium, sulfates of gold such as potassium dihydrogen phosphate and dipotassium hydrogen phosphate, and chlorides of metals such as bugnesium chloride are added as appropriate. When foaming is significant during culturing, antifoaming agents such as higher alcohols, vegetable oils, silicone compounds, etc. may be added. Among these antifoam agents, vegetable oil may be used as a carbon source. 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 main culture is 5
Approximately 0 to 100 hours is appropriate, and the culture time may be further extended as the medium becomes more concentrated.

The culture conditions described above are selected and applied depending on the characteristics of the production strain used.

The compounds accumulated in the culture in this way are mainly present in the culture solution, so after removing the bacterial cells by centrifugation 111i or filtration, the filtrate is used for the production of general antibiotics. Separated, collected, and purified by means. Namely, vacuum condensation, freeze drying, solvent extraction, liquid conversion, such as anion exchange resin, cation exchange resin, nonionic adsorption resin, etc. (treatment with sealant, e.g. activated carbon, silicic acid,
Treatment with adsorbents such as silica gel, cellulose, alumina, crystallization, recrystallization, etc. can be used alone or in combination in any order, and the effective substances can be separated by repeated exposures. , collection, and purification.

In addition, when using, for example, the above-mentioned Streptparticillicum waxmani lr 30581 bacterium as a producing bacterium of 3-(2-substituted-oxazol-5-yl)indole, the compound of the general formula (I), which is the object compound of this invention, is used. 3-(2-iirf-substituted-oxazolium-5-
3-(2-methyloxazol-5-yl)indole or 8-indole other than indole is produced. These 3
-(2-propyloxazol-5-yl)indole, 3-(2-butyloxazol-5-yl)indole and 3-(2-methyloxazol-5-yl)indole can be prepared using, for example, an N3 gel column ( The chemical properties of f2+3-(2-substituted monooxazol-5-yl)indole can be determined by reciprocal (manufactured by Kagaku Co., Ltd.). Physicochemical I'i-f of 2-substituted-ogidazol-5-yl)indoles
When i' is shown, the following jη is obtained.

]) 3-(2-propyloxazol-5-yl)indole ■Appearance: Colorless monomorphic crystal ■Physical properties, basic substance ■Melting point: 128-13-0°C ■Molecule H1・226 (by mass spectrum) ( S, 1
Original kl'i analysis: (as C, 4H, 4N20) Total! A
-Bean paste C74, 31, H6, 24, Ima 12゜38 (
%, 1%? in value C74, 45, H6, 35, N 1
2.40t%) Φ) Ultraviolet absorption spectrum λ-r-9/-/1'': 224 (ε-24000) 1
) 1aλ 266 (ε-1' 5400 ) 282 (shoulder) (ε-13000) 298 (shoulder) (ε-1120 (1) ■Infrared absorption spectrum 1125, 1013. 1000. 918. 75
0. 745cIn' ■ 1H nuclear magnetic resonance spectrum: δ (ppm) (CDCf3):' 1.01 (3H, t
, J=7Hz).

1.87 (2H, m), 2.83 (2H, t, J=
7Hz).

7.10-7.95 (5H, m), 9.58 (IH
, Broad 3) Solubility in solvents: Easily soluble in methanol, ethanol, chloroform, ethyl acetate qd: Insoluble in benzene: Hexane, petroleum ether, water 0 color reaction.

Ehrlich reaction: Positive Sericum sulfate reaction: Positive Ninhydrin reaction: Negative 2) 3-(2-butyloxazol-5-yl)indole 0) Appearance: Colorless needle-shaped crystals Q) Physical properties: Basic substance ■Melting point: 123-125° C (4) Molecule 4jJ5:240 (by mass spectrum) ■Elemental analysis: (as C+5H162''20) Calculated value C74,97, H6,71, N11.66t%) Experimental value C74,82, H6,59, N11. 65f%)
■Ultraviolet absorption spectrum varnish 1'-''・224 (ε-22200) aX 266 (ε-144'0O) 282 (eyebrow) (ε=12200) 298 (shoulder) (ε-10400) ■Infrared absorption spectrum 7・-”+ 3125. 1630. 1612.
1570°1aX 1120, 1009. 920. 912. 775
．． 739 cm-1 (■1H nuclear magnetic resonance spectrum: δ (ppm) (CDCV, ): 0.96 (3H,
t, J=7Hz).

1.45 (2H, m), 1.80 (2H, m),
2.84 (2H, t+J=7Hz), 7.10-
7.92 (5H, m), 9.10 (IH.

Broad S) ■Solubility in solvents: Easy soluble: methanol, ethanol, chloroform, ethyl acetate Poorly soluble: benzene Insoluble: hexane, petroleum ether, water [Co., Ltd.] Color reaction/Ehrlich reaction: Positive Sericum sulfate reaction: Positive ninhydrin reaction , negative results and other studies indicate that the substances related to this invention produced by Streptparticillicum fuchsmanino C30581, 1. It turned out to be butyloxazol-5-yl)indole.

(3) The biological properties of 3-(2-substituted-oxacyl-5-yl)indole are as follows.

■ In vitro platelet aggregation inhibition reaction method.

Collect blood from the ear artery or carotid artery of a rabbit (Japanese white native) into a test tube prepared with 38% sodium citrate equivalent to 10% of the blood vessel. (10°C), remove the upper layer using a polyethylene beetroot tube with a Komagome pipette.
This refers to platelet-containing plasma (hereinafter referred to as PRP).

Platelet ~ (collection reaction Q) is carried out as follows: Cell PRP (400,000 ce11.s/, nff1)" 33
” and 0.02 me of 3-(2-substituted-oxazol-5-yl)indole solution were mixed, 0.05 me of the following aggregation inducer was added to this, and the change in absorbance was measured by 5I.
ENCO Tutorial Sample - Aggreg Make (DP-2)
4DE) (measured at a temperature of 37°C, 7 stirrings, and 101,000 rpm.

Aggregation inducing agent (d) Sort according to the following.

a) Arachidonic acid (manufactured by Sigma) 500 py/me Hcile J: raw W diet for cormorants
Water Teki'? and use it.

l:)) Collagen (derived from bovine Achilles lung) (manufactured by Tokyo Kasei), physiological saline 10 me (/'C Collagen 200 ”
9 was added, cooled, and then subjected to ultrasonic treatment ('5/4.8 minutes), and the thick lumps were extracted and the supernatant was used. (For practical use, dilute with physiological saline to an appropriate concentration.

Results/Table 2.

Table 2 Next, the present invention will be explained with reference to examples.

Example 1 I of 3% starch, 02% glucose, 1% cottonseed flour, 1% gluten meal, 1% dried yeast, 0.2% calcium carbonate
Dispense 100 me of the medium from J and f into ten 500 me corrugated flasks and heat at 120°C for 20 minutes iA] K
It was infected. One platinum loop of a slant culture of Streptparticillium sosoxmani strain 30581 was inoculated into each medium, and cultured with shaking at 30'C for 3 days.

Separately, add 200 e volume of medium 160I with the following composition: starch 3%, glucose 02%, cottonseed powder] candy, gluten meal 0.5'%, dry yeast fJJ 0.5%, calcium carbonate 0.2 olo (pH 7,0). After pouring into a stainless steel furnace and sterilizing at 120°C for 20 minutes,
A total of 1 stone of 7 cultures was inoculated, cultured at 30°C for 4 days, and after Sappo cultivation was completed, diatom ±3k17 was added to the culture and filtered. Approximately 301 acetone was added to the obtained bacterial cells, stirred for 10 minutes, filtered, and the bacterial cells were extracted again with 301 f' acetone. The extracts were combined and concentrated under reduced pressure. The resulting aqueous solution) pH at night was 7. OK, adjust, extract twice with about 101 ethyl acetate, concentrate the extract under reduced pressure,
An oily substance was obtained. Dissolve this in a small amount of chloroform,
It was subjected to silica gel column chromatography. The column was first flushed with hexane, and then the active substance was eluted with xane:pH'l:ethyl acid (1.1). The active section is under reduced pressure'c! It was evaporated to dryness, and the residue was applied to a reverse phase 1i S gel column (
(manufactured by Nippon Seikagaku Co., Ltd.). The active fraction was eluted from the column with methanol. One fraction of the eluted active substance was concentrated to dryness under reduced pressure, and crystallized from benzene:hexane (1:1). The resulting crystals contained three components: 3-(2-probyloxazol-5-yl)indole, 3-(2-butyloxazole)indole, and 3-(2-butyloxazole)indole. (
1,5P) 150 mq of this crystal was subjected to liquid chromatography using a Quarters Associate Micro Bondapak C-18 column. (Mobile phase: 50% Mek/-L)
This yielded 27 mg of 3-(2-propyloxazol-5-yl)indole, 14 mq of 3-(2-f-thyloxazol-5-yl)indole and 3-(2-
methyloxazol-5-yl)indo-/L/5
mg (mp, 204-205°C) was obtained.

41 2-

Claims (3)

[Claims] (1) 3-(2-substituted-oxazol-5-yl)indole or its salt represented by the general formula (wherein R is propyl or nibbutyl). (2) Producing 3-(2-substituted-oxazol-5-yl)indole represented by the general formula (wherein R means propyl or butyl), which belongs to Streptparticillicum 11. The bacteria are cultured, and 3-(
3-(2-
Process for producing substituted-oxazol-5-yl) indole 1 or its salts. (3) A planar platelet aggregation inhibitor containing 3-(2-substituted-oxazol-5-yl)indole or a salt thereof represented by the general formula (wherein R means propyl or butyl) as an active ingredient.