JPH0725878A - Platelet aggregation inhibitor pi-334 - Google Patents

Abstract

PURPOSE: To provide the subject novel compounds having inhibiting activity to platelet aggregation of adenosine diphosphate and useful in the treatment of thrombosis or the like.

CONSTITUTION: The compound of the formula having following characteristics: elementary analysis value; measured value (%) C-57.02, H-5.87, N-4.68, S-9.98; molecular formula; C₃₀H₃₆N₂O₉S₂; molecular weight: 632; melting point: 143 to 146°C; solubility; soluble in chloroform, methanol or the like; slightly soluble in n-bexane and benzen, and insoluble in water; color reaction; positive to I₂ and H₂SO₄, and negative to ninhydrine and FeCl₃; distinction among basicity, acidity and neutral: weak acidity, is obtained by culturing a novel microorganism, Ramichloridum-schulzeri-var. shulzeri-TF-0381 (FERM-P-13661), preferably under an aerobic condition at a pH or 7 to 8 and a temperature of 25 to 28°C for 5 days, and separating and purifying said culture media by the known method.

COPYRIGHT: (C)1995,JPO

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a novel compound having an inhibitory action on platelet aggregation.

[0002]

2. Description of the Related Art Compounds which are structurally similar to the compounds of the present invention and have an inhibitory action on platelet aggregation are not known.

[0003]

An object of the present invention is to provide a novel physiologically active substance having a platelet aggregation inhibitory action.

[0004]

The present inventors have isolated a large number of strains from soil and plants to achieve the above object,
As a result of various studies on the culture of the strain, it was found that a compound produced by a certain strain has a strong inhibitory effect on platelet aggregation, and the present invention has been completed.

That is, the present invention uses the formula

[0006]

A compound represented by the following formula (hereinafter referred to as PI-334).

[0008] strain to produce a novel physiologically active substance PI-334 of the present invention is a strain which the present inventors have newly isolated, the name of the microorganism "Ramichloridium schulzeri var. Sc
hulzeri TF-0381 ”and microorganism deposit number FERM
P-13661 has been deposited at the Institute of Biotechnology, Institute of Industrial Science and Technology.

The mycological properties of this strain are shown below. 1) Morphology This strain grows moderately on a plate medium such as potato-glucose agar medium, oatmeal agar medium, YpSs agar medium, LCA (Miura) agar medium, and spore formation is oatmeal agar. Medium, YpSs agar medium, LCA (Miura)
Good on agar media. When a colony formed by the strain of this strain on Oatmeal agar medium after culturing at 20 ° C. for 14 days is observed by an optical microscope, the hyphae are highly branched with a septum, and the conidia stalks are basal hyphae. Or it is a simple growth from aerial hyphae and is upright or slightly bent. The surface is smooth and the middle part is a slightly thick wall with a grayish brown color, which is clearly distinguishable from mycelium, but most of them become thin and indistinguishable toward the base. The length is 60 to 170 μm, and the diameter is 2.0 to 3.
0 μm. Symposio-type conidia are single-grown at the tip of 10 to 25 μm, and a large number of dentate processes are observed after conidia shedding. Conidia is 1 cell, surface is smooth, almost colorless, the base is slightly pointed, and the size is (4.2) to 5.8.
It is 11.8 × 2.0 to 3.6 μm. In addition, culture 3
Although extended to a week, no sexual reproductive organ formation was observed.

2) Growth state on medium The following Table 1 shows the results of macroscopic observation when the cells were cultured on various mediums at 20 ° C. for 14 days. For the color display, the systematic color names of the JIS Standards Color Book (1985) of the Japanese Standards Association were cited.

[0011]

[Table 1]

3) Physiological properties Growth temperature range and optimum temperature The strain of this strain is 13 in a Sabouraud liquid medium of pH 6.0.
It grows in the range of ~ 33 ° C, and the optimum temperature is 26-29 ° C. Growth pH range and optimum pH This strain has a pH of 4 to 1 at 26 ° C. in YpSs liquid medium.
It grows in the range of 0 and the optimum pH is 4-6.

4) Distinction between aerobic and anaerobic; aerobic

From the above morphological characteristics and culture properties, it is clear that this strain is a strain of the genus Ramichloridium in the subphylum Incomplete, and GS De Hoog, Stu
dies in Mycology, No.15, pages 1-140, (1977)
It was compared with many known strains reported in. As a result, this strain was Ramichloridium schulzeri var. Schu .
It was revealed that the strain showed the closest properties to lzeri , and this strain was designated as " Ramichloridium schulzeri var. schulzeri TF-038.
1 ”.

The production of PI-334 is carried out in the same manner as in the case of producing a general fermented product, and Ra in a medium containing various nutrients is used.
It is carried out by culturing michloridium schulzeri var. schulzeri TF-0381 strain under aerobic conditions. A liquid medium is mainly used as a medium, and glucose, sucrose, waste sugar concentrate, starch and the like are used alone or as a mixture as a carbon source. As a nitrogen source, meat extract, oatmeal,
Yeast extract, soybean flour, polypeptone and the like are used alone or in combination. In addition, PI-33 helps the growth of this strain
If necessary, an organic substance and an inorganic salt that accelerate the production of 4 can be added. As the defoaming agent, adecanol, silicone, or the like can be used. Aerobic culture such as shaking culture and aeration and agitation culture is suitable for the culture method.
3-10, 11-32 ° C for 3-6 days, preferably pH
Incubate at 7-8, 25-28 ° C for 5 days.

The PI-334 produced by this culture can be isolated according to a general method for collecting a fermentation product. For example, the following method is effective. That is, after completion of the culture, the culture solution is extracted with an organic solvent such as acetone. Next, this extract is concentrated and then redissolved in a non-water-soluble organic solvent such as ethyl acetate, benzene or chloroform, and concentrated to give a syrup. PI-334 is purified and isolated by again dissolving this syrup in an organic solvent such as benzene, ethyl acetate, acetone, methanol, chloroform and subjecting it to silica gel column chromatography, gel filtration column chromatography and high performance liquid column chromatography. be able to.

PI-33 obtained by the above purification method
The physicochemical properties of 4 are shown below. (A) Elemental analysis value: measured value (%) C 57.02, H 5.87, N 4.
68, S 9.98 theoretical value (%) C 56.95, H 5.73, N 4.
42, S 10.14 (C₃₀H₃₆N₂O₉S₂(B) Mass spectrum: FAB mass spectrum m / z 631 (MH)^- (C) Molecular formula: C₃₀H₃₆N₂O₉S₂ (D) Molecular weight: 632 (e) Melting point: 143 to 146 ° C. (f) Specific optical rotation: [α]_D ^{twenty five}: + 326 ° (c = 0.01, CHCl₃) (G) Ultraviolet absorption spectrum: λmax nm (ε) 208 (34000), 225 (4)
1000) (h) Infrared absorption spectrum: measured by neat method
The results are shown in FIG. (I)¹H-NMR spectrum: CDCl₃Medium, 400
The result measured in MHz is shown in FIG. (J)¹³C-NMR spectrum: CDCl₃Medium, 100
The result measured at MHz is shown in FIG. (K) Solubility in solvent: chloroform, ethyl acetate
Soluble in methanol and methanol, sparingly soluble in n-hexane and benzene insoluble in water (l) Color reaction: Positive: I₂, H₂SO_Four, Negative: Ninhydrin FeCl₃  (M) Basic, acidic, neutral: weakly acidic

The compound of the present invention has an excellent inhibitory effect on platelet aggregation caused by adenosine diphosphate.
It is useful as a therapeutic drug for thrombosis.

[0018]

EXAMPLES The present invention will be described more specifically below with reference to examples and test examples. Example (1) 100 ml using a 300 ml Erlenmeyer flask
Glucose per 2 g, polypeptone 0.5 g, yeast extract 0.2 g, monopotassium phosphate 0.1 g, magnesium sulfate 0.05 g, Ramichloridium schu in sterile liquid medium
lzeri var. schulzeri TF-0381 strain was inoculated at 26 ° C,
It was shake-cultured for 96 hours and used as a seed culture. Next, 200l
Using a volume jar fermenter, 120 ml of a sterile medium having the same composition as the seed culture was inoculated with 1200 ml of the seed culture solution,
The cells were aerobically cultured at 26 ° C. for 120 hours with stirring. After completion of the culture, centrifugation was performed to separate the cells and the supernatant. The bacterial cells were extracted with 28 l of acetone, and the acetone was distilled off.
It was adsorbed on 1 HP-20. Then, elution was carried out with 12 l of acetone, the eluate was distilled off under reduced pressure, and the residue was extracted twice with 28 l of ethyl acetate. The ethyl acetate fraction was dehydrated with anhydrous sodium sulfate and then concentrated to dryness to obtain 57.4 g of a brown oily substance. (2) The oily substance obtained in the preceding item 1 was added to acetonitrile 10
It was dissolved in 0 ml and the filtrate was mixed with ODS column chromatography (volume 0.8
l) was performed. Collect 60% acetonitrile elution fractions 7
49 mg of active fraction was obtained.

(3) 749 mg of the above oily substance was dissolved in methanol and used as a sample for high performance liquid column chromatography conducted under the following conditions. Column size 20φ × 250 mm Carrier ODS silica gel (manufactured by Senshu Scientific Co., Ltd.) Solvent composition 49.5% Acetonitrile, 49.5
% Water 1% THF Flow rate 18.0 ml / min Temperature 50 ° C. Detection wavelength 215 nm Device Waters M-600 Retention time 15.3 to 15.9 min.
g brown substance was obtained. (4) The brown substance of the preceding paragraph was dissolved in methanol to prepare a sample for high performance chromatography performed under the following conditions. Column size 21.5φ × 500 mm Carrier GS-310P (manufactured by Asahi Kasei) Solvent composition 100% methanol Flow rate 6.0 ml / min Temperature 50 ° C. Detection wavelength 215 nm Equipment Waters M-600 Holding time 23.1-25 .1 minute fraction was collected and 5 mg
PI-334 was obtained.

Test Example (Inhibitory Action on Platelet Aggregation) (1) Platelet Preparation Method Blood was collected from the common carotid artery of a male rabbit having a body weight of 2.0 to 2.5 kg, which was 1/10 volume of 3.8. % Sodium citrate solution was added, mixed gently, and centrifuged at 1300 × g for 2 minutes, and the supernatant was used as platelet-rich plasma (hereinafter referred to as PRP). The precipitate was further centrifuged at 1600 × g for 10 minutes, and the obtained supernatant was used as platelet poor plasma (hereinafter referred to as PPP). (2) Method for Preparing Aggregation Inducing Agent In advance, adenosine nitric acid (Kyoto Daiichi Kagaku
Agriback) was diluted with physiological saline to a required concentration to prepare an aggregation inducer. (3) Platelet aggregation inhibitory activity measurement method After making the difference in absorbance with 200 μl of PPP constant, PI-334 prepared at a required concentration with methanol was used as PRP.
And stirred. As a control, only methanol was used.
Then, an aggregating agent (final concentration: 5 μM) was added, and the change in absorbance was measured using a turbidimetric method such as Bone using a Platelet Aggregation Tracer 4A type platelet aggregometer from Nikko Bioscience. J. Physiol., No. 168
Vol., Pp. 178 (1968)], P
The 50% platelet aggregation inhibitory concentration (IC ₅₀ value) of I-334 was determined. As a result, IC ₅₀ values of PI-334 is 2.0 ×
It was 10 ^-5 M.

[Brief description of drawings]

FIG. 1 shows an infrared absorption spectrum of the compound of the present invention measured by a neat method.

FIG. 2 shows the ¹ H-NMR spectrum of the compound of the present invention measured in CDCl ₃ at 400 MHz.

FIG. 3 shows a ¹³ C-NMR spectrum of the compound of the present invention measured in CDCl ₃ at 100 MHz.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Mari Kishimoto 3-24-1 Takada, Toshima-ku, Tokyo Inside Taisho Pharmaceutical Co., Ltd. (72) Terumi Kagamien 3-24-1 Takada, Toshima-ku, Tokyo Taisho Pharmaceutical Co., Ltd. (72) Inventor Akira Kawashima 3-24-1 Takada, Toshima-ku, Tokyo Taisho Pharmaceutical Co., Ltd. (72) Inventor Kazuki Hanada 3-24-1 Takada, Toshima-ku, Tokyo Taisho Yaku Co., Ltd.

Claims (1)

[Claims] 1. A formula The compound represented by.