JPH0262880A - Dithiodiketopiperazine compound - Google Patents

Abstract

NEW MATERIAL:A compound, expressed by the formula and having the follow ing physico-chemical properties. Melting point; 89-92 deg.C. Elementary analysis (%) (as C31H38O9N2S2); C, 57.67; H, 5.80; N, 4.50; S, 9.85. Molecular weight; field desorption mass spectrometry (FD-MS), m/z 647(M+H). Molecular formula; C31H38O9N2S2. Optical rotation; [alpha] =-179.6 (C=0.044, CHCl3). Solubility; insoluble in water and soluble in methanol, ethanol, ethyl acetate, benzene and chloroform. Color reaction; negative to ninhydrine and ferric chloride reac tion and positive to iodine reaction and sulfuric acid. Neutral. Light-yellow powder, etc. USE:A blood platelet agglutination inhibitor. PREPARATION:A strain of Ramichlovidium Schulzeri var. Schulzeri F-2440 (FERM P-10187) is cultured at 25-28 deg.C and pH6.8-7 for 5 days.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to dithiodiketopiperazine compounds useful as platelet aggregation inhibitors.

[Prior Art] A compound similar to the dithiodiketopiperazine compound of the present invention is Sporidesmin A (Sporidesmin A).
A) [Tetrahedron Letters
onLetters), page 3131, (196
6th year) Ko, Aranotin [Tetrahedron Letters (Ietra-hedron Le
tters), p. 4467, (1968)]
It has been known.

[Problems to be Solved by the Invention] An object of the present invention is to provide a novel compound that has an inhibitory effect on platelet aggregation.

[Means for Solving the Problems] The present inventors isolated a large number of bacterial strains from soil for the purpose of searching for new physiologically active substances, and as a result of conducting various studies on the cultures of the strains, certain strains of bacteria were discovered. The present invention has been completed by discovering that a new substance can be produced that has a strong inhibitory effect on platelet aggregation.

That is, the present invention is based on the following formula! and a dithiodiketopiperazine compound represented by the following formula (1) (hereinafter, the compound of formula (1) is referred to as PI-061, and the compound of formula (2) is referred to as PI-063.

).

The strain that produces the target substance of the present invention is a strain newly isolated by the present inventors from the soil of Kagiya-cho, Tokai City, Aichi Prefecture, and the microorganism name is "Lamicrhizoium schultzer".
Birschltour F-2440 (Ramichlor
idium 5chulzeri var.

'5chulzeri F-2440) J and Microorganism Deposit No. 10187 (FERM P
-10187) has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology as J. This strain can be cultured to obtain the target substance of the present invention.

The mycological properties of this strain are shown below.

1) Morphology This strain grows well on oatmeal agar medium, YpSs agar medium, etc., but conidial settlement is moderate. When a colony grown on an oatmilk agar medium is observed under a microscope, the hyphae are transparent and have septa, and the highly branched conidiophores are mainly perpendicular or slightly bent from the basal hyphae in the medium or on the surface of the medium. (30-150X2.2-3
．． 8μ), forming many small teeth at its tip 1
The conidia are single cells and are formed in a symbogeotype on the denticles. When observed with an electron microscope, the conidia are wave-shaped and the size is 5.
．． 2-9.5X 2.0-3.2μ, and its surface was smooth.

2) Properties on media Table 1 shows the results of macroscopic observation when cultured on various media at 25°C for 14 days.

(Hereinafter, blank space) 3) Physiological and ecological properties ■Optimal growth conditions The optimal growth conditions for this strain are YpSs medium with a pH of 5.
, 1 to 7.6, and the temperature is 15 to 25°C.

■Growth range The growth range of this strain is PH2, 7 in YpSs medium.
-9.2, temperature 12-30°C.

■Distinction between aerobic and anaerobic; From the morphological characteristics that are more than aerobic and the culture characteristics, it was suggested that this strain may belong to the genus Microrhizoium. Therefore, based on the above-mentioned properties, Illustrated encyclopedia of fungi, Yotoshi Uda-1 edited by Keisuke Tsubaki, (1
987) and The b by G. S. de Hoog.
Lack yeasts and allied Hy
homycetes 5t-udies Mycol
As a result of comparison with many known bacterial strains reported in 1977, this strain was found to be Ran+ich.
loridium 5chulzeri var, 5c
It showed properties closest to those of Hulzeri.

Based on the above results, this strain was identified as Ramirolidium Schulzer F-2440 (Ram1chl
oridiuco 5chulzari var, 5c
hulzeriF-2240).

The production of PI-061 and PI-063 of the present invention is carried out by culturing the present strain under aerobic conditions in a medium containing various nutritional substances, roughly in the same manner as in the production of general fermentation products.

A liquid medium is mainly used as the medium, and glucose, sucrose, molasses, starch, etc. are used alone or in combination as carbon sources. Nitrogen sources include meat extract, yeast extract,
Soy flour, polypeptone, etc. are used alone or in combination.

In addition, PI-061 and PI-0 help the growth of this strain.
Organic substances and inorganic salts that promote the production of 63 can be added as necessary. As an antifoaming agent, Adekanol,
Silicon or the like can be used.

As for the culture method, aerobic culture such as shaking culture and aerated agitation culture is suitable, and the pH is 4-8.25-30℃ for 2-6 days.
The culture is preferably carried out at pH 6, 8 to 7, and 0.25 to 28°C for 5 days.

PI-061 and PI-06 produced by this culture
3 can be isolated according to a general method for collecting fermentation products.

Since PI-061 and PI-063 are mainly accumulated in the culture solution, the following method is effective, for example.

That is, after the completion of culture, culture P is removed by centrifugation or filtration.
After obtaining a solution and bacterial cells, this P solution is adsorbed on an adsorption resin such as Diaion HP-20 (product name: manufactured by Mitsubishi Chemical Industries, Ltd.), and then P solution is absorbed with an organic solvent such as a lower alcohol or acetone.
I-061 and PI-063 are eluted. The bacterial cells are extracted with an organic solvent such as acetone.Then, this extract and the eluate from the adsorption resin are combined and concentrated, and then transferred to a water-insoluble organic solvent such as ethyl acetate, benzene, or chloroform.
Concentrate this to form a syrup. This syrup was dissolved again in an organic solvent such as ethyl acetate, benzene, or chloroform, and subjected to gel filtration using Sephadex LH-20 (trade name: manufactured by Pharmacia) and high performance liquid chromatography to obtain PI-061. and PI-0
63 can be purified and isolated.

PI-061 and PI-0 isolated by the above purification method
63 has the following physical and chemical properties.

PI-061: (a) Melting point 89-92°C (b) Elemental analysis value (as C31H3809N2S2) (theoretical value) C: 57.59%, H: 5.88%, N: 4.33%
S: 9.91% (actual value) C: 57.67%, H: 5.80%, N, 4.50%
S: 9.85% (e) Molecular weight F D-MS: m/z 647 (M+H) (d) Molecular formula % formula % (e) Optical rotation [α co”-179,6° (C=0.044 , c
HcI4) (f) Ultraviolet absorption spectrum The results of measurements in methanol are shown in FIG.

(g)'H-NMR spectrum The results measured at 400 MHz in deuterated chloroform are shown in FIG.

(h) Solubility in solvents Insoluble in water. Soluble in methanol, ethanol, ethyl acetate, benzene, and chloroform.

(i) Color reaction Ninhydrin, ferric chloride reaction: Negative iodine reaction, Sulfuric acid: Positive (j) Basic, acidic, neutral (k) Color and shape of the substance Pale yellow powder PI-063: (a) Elemental analysis value (as C29H3209N2S2)
(Theoretical value) C: 56.49%, H: 5.19%, N:
4.56% S: 10.39% (actual value) C: 56.66%, H: 5.32%, N:
4.62%S: 10.50% (b) Molecular weight FD-MS: m/z 617 (M+H)" (d) Molecular formula % Formula % (f) Ultraviolet absorption spectrum The results measured in acetonitrile are shown in Figure 3. show.

(g)'H-NMR spectrum The results measured at 400 MHz in deuterated chloroform are shown in FIG.

(h) Solubility in solvents Insoluble in water, soluble in methanol, ethanol, ethyl acetate, benzene, and chloroform.

(i) Color reaction Ninhydrin, ferric chloride reaction: negative iodine reaction, sulfuric acid: positive Effects] PI-061 and PI-063 of the present invention are novel physiologically active substances that have an excellent inhibitory effect on platelet aggregation caused by arachidonic acid, adenosine diphosphoric acid, and flagen, and are useful as therapeutic agents for thrombosis, etc. It is.

[Example] Next, the present invention will be specifically described with reference to Examples and Test Examples.

(Example) (1) 4% glucose, 1% polypeptone, 0.1% yeast extract in a jar fermenter container with a content capacity of 50R.
A medium consisting of 30 parts was added and sterilized. The sterilized medium was inoculated with 1.6% of the bacterial solution of this strain, which had been cultured with reciprocating shaking at 30°C for 4 days in a medium having the same composition as this, and cultured with stirring and aeration at 30°C for 4 days.

After culturing, 150R of the culture solution was filtered and separated into bacterial cells and P solution, and the bacterial cells were 20f! , and extracted with acetone.

The p solution was adsorbed on an adsorption resin [Diaion HP-20 (trade name; manufactured by Mitsubishi Chemical Industries, Ltd.) 5000af], washed with purified water 101, and the active substance was eluted with 8 parts of acetone. Combine the acetone extract derived from the bacterial cells and the acetone eluate derived from the P solution, and after distilling off the acetone, the residue is 2f! , twice with ethyl acetate. The ethyl acetate layer was dehydrated with anhydrous sodium sulfate and then concentrated to dryness to obtain 18 g of a brown oily substance.

(2) 18g of the obtained oily substance for 1000! ! was dissolved in chloroform and adsorbed onto a 1 fl column of silica gel column chromatography [Wafugel C-200 (trade name: manufactured by Wako Tsumugi Kogyo Co., Ltd.) prepared with chloroform. After washing with 2 fL of chloroform, elution was performed with chloroform-acetone (80:20), and 2 g of a brown oily substance was obtained by concentrating and drying. Sephadex LH-20 (trade name;
Gel filtration was performed using 500ae (manufactured by Pharmacia), and both fractions containing PI-061 and PI-063 were collected and concentrated to dryness to obtain 1.2 g of yellow powder. 1 to 2 g of the obtained crude powder was gel-filtered under Toyovar HW-40 (trade name: Toyo Soda Rui, 300d') to obtain 40 mg of PI-061 as a pale yellow powder and 3 m of PI-061 as a pale yellow oil.
g of PI-063 was obtained.

(Test example) Platelet aggregation inhibitory effect (1) Preparation method of platelets Collect blood from the common carotid artery of a male rabbit weighing 2.0 to 2.5 kg, and add 3.8% sodium citrate to 1/10th the blood volume. Add the solution, mix gently, and centrifuge at 1300Xg for 2 minutes.
And so. The precipitate was further centrifuged at 1600Xg for 10 minutes, and the resulting supernatant was collected as platelet-poor plasma (rP P P
).

(2) Preparation method of aggregation inducing agent Arachidonic acid (manufactured by Sigma) is diluted with ethanol and adenosine diphosphoric acid or collagen (manufactured by Kyoto Daiichi Kagaku Co., Ltd., Agrivac) is diluted with physiological saline to the required concentration.
An aggregation inducing agent containing each of the above components was prepared.

(3) Platelet aggregation inhibitory activity measurement method 200Pf1 PRP was taken, and after making the difference in absorbance with PPP constant, PI was adjusted to the required concentration with methanol.
-061 and PI-063 were added to PRP and stirred.

As a control, only methanol was used. Next, the above aggregation-inducing agent (final concentration: 0.1 mM for arachidonic acid, 10 μM for adenosine diphosphate, 10 μM for collagen)
Add /d) and measure the change in absorbance using Binary Bioscience Plaidlet Aggregation Tracer.
Using a type 4A platelet aggregometer, the nephelometric method of Born et al.
n, J, Physiol, vol. 168, no. 178
Page (1968)], and the 50% platelet aggregation inhibitory concentration (tC,
value) was calculated.

The results are shown in Table 2.

The 'H-NMR spectrum of PI-063 measured at MHz is shown.

Table 2

Claims (2)

[Claims] (1) Formula ▲Contains mathematical formulas, chemical formulas, tables, etc.▼ A dithiodiketopiperazine compound represented by (2) Formula ▲Contains mathematical formulas, chemical formulas, tables, etc.▼ A dithiodiketopiperazine compound represented by