WO1995004041A1 - Carbazole compound - Google Patents

Abstract

A compound represented by formula (I) which has antioxidant and cell death inhibitor activities and is useful for treating cerebral, cardiac and hepatic diseases, arteriosclerosis, and so forth.

Description

 Technical Information

 TECHNICAL FIELD The present invention relates to a compound having an antioxidant action and a cell death inhibitory action. Background art

 As a related compound having the same action as the compound of the present invention, a compound described in JP-A-3-227971 is known, but it is structurally unstable. Disclosure of the invention

 The present inventors have isolated a large number of strains from soil and conducted various studies on cultures of the strains.As a result, the inventors have found that compounds produced by certain strains have strong antioxidant activity and cell death inhibitory activity. They have found and completed the present invention. That is, the present invention provides a formula

で表わされる化合物である （以下、 これを C S— 7 9と称する。 ） 。

(Hereinafter, this is referred to as CS-79.)

The strain that produces CS-79 is a strain newly isolated from the soil collected by the present inventors in Matsumoto City, Nagano Prefecture, and has the microorganism name “Streptomyces ex fo l iatus 24 19—S VT 2J and deposited under Microorganisms Deposit No. “Micro-organism Deposit No. 12707 (FERM P-12707)” with the National Institute of Advanced Industrial Science and Technology.

 The bacteriological properties of this strain are shown below. '

 1) Form

 The basal hypha of this strain is not broken.

 The aerial hyphae form the main axis, thereby forming a curved or looped sporangium consisting of 10 to 50 or more at the irregularly branched tip. The spores are non-motile, cylindrical or oblong in shape, 0.6-0.8 m wide, 0.8-1.0 m long, and the spore surface is smooth.

 Sclerotia, sporangia and other special forms are not observed.

 The cell wall chemical type is type I.

 2) Growth on the medium

 Table 27 shows the results of visual observation when cultured for 14 days at 27 on various media.

1

 The flora color on the colony surface is a gray series, and the reverse color is unclear, slightly changing at pH.

The diffusible dye was light brown.

【table 1】

() Indicates the color code of the Color Harmony Manual (Container Corporation, America, 1950).

 3) Physiological properties

 ① Growth temperature range

The strain was grown in a range of 2 0 4 5 ° C, the optimum temperature for growth because in 2 0 3 7 ₀

 ②Biochemical properties

 a) Aerobic and anaerobic: aerobic

 b) Liquefaction of gelatin: +

c) Coagulation of skim milk: 1 d) Peptidation of skim milk: 1

 e) Starch hydrolysis: +

 f) Formation of melanin-like pigment: +

 g) Cell wall type: Type I ''

 ③ Use of carbon source

 (On Puri Doham-Godrib agar)

 Uses: glucose, arabinose, xylose, fructos, ramnose, raffinose, inositol, mannite

 Do not use: sucrose

Based on the above properties, species of the genus Streptomyces (hereinafter abbreviated as S.) described in the “List of Approved Bacteria Names 1980” and the list of valid names thereafter. , And two closely related species were selected. Comparison of the diagnostic properties of S. nash virensis (S. nash V i 11 e_ns_is—) and _S. Only the assimilation of carbon sources is different.

[Table 2]

太-Byeon, c = J Reno Α, Α 7 Reno Γ

2 4 1 9 S V T? / No reneno / \ * one's 7 "へ 7 no spore spore form curved 4- 丄 10 loops + +

Spore surface smooth + 4-t t ^ gray 4-byeon

 M ^ unclear + red / orange r pH sensitivity,

 Ten

Diffusible pigment raw + Byeon

 ρ Η ΐ ΐ t

 Byon +

Melani w ^ ¹ ^ 4- Starch hydrolysis + Byeon

S Xiaotate Root + 4- 1 Growth temperature at 10

 3 7 at 4- +

4 5 in +

Assimilation of coal cable sources

 Arabinose + + + quidylose + + 1 inositol +-

 Mannit +

 Rhamnose + + Raffi North + One

 Sucrose

S. soft skulls have morphology, surface color, pH sensitivity of diffusible dyes, carbon Source assimilation is different. Therefore, this strain is most similar to S. nasivirensis, but is described in “Bergey's Manual of Systematic B acteriology Vol. According to Williams et al. (Williams, et. A 1.), S. nasibirensis is a synonym of S. exfoliatus. Therefore, the present strain 24 19— SVT 2 was identified as one strain contained in S. ixhorietus, and S. tomvcesexfo 1 iatus (Stre. Tomvcesexfo 1 iatus) was identified. Called SVT 2 shares

O o

 Production of CS-79 is carried out by culturing the strain under aerobic conditions in a medium containing various nutrients, in a manner similar to the production of general fermentation products. As a carbon source, glucose, lactose, starch, etc. are used alone or in combination. As a nitrogen source, meat extract, automeal, yeast extract, soybean powder, polybebutone, etc. are used alone or in combination. In addition, organic and inorganic salts that promote the growth of the strain and promote the production of CS-79 can be added as necessary. As the defoaming agent, Adekinol, silicon, or the like can be used.

 Aerobic culture such as shaking culture and aeration / agitation culture is suitable for the culture method, and is performed at pH 4 to 8, 24 to 30 for 2 to 6 days, preferably at pH 6 to 7, 24 to 27. Incubate in C for 2-3 days.

 The CS-79 produced by this culture can be isolated according to a general method for collecting S-produced products. Since C S _ 79 is accumulated in the satellite, for example, the following method is effective.

 That is, after completion of the culture, the S-isomer is eluted with an organic solvent such as acetate by centrifugation or filtration.

Then, after condensing the Satsuma extract, it is transformed into a water-insoluble organic solvent such as ethyl acetate, benzene, or cro-form, and concentrated to form a syrup. Repeat this syrup with benzene, ethyl acetate, acetate, methanol, The compound of the present invention can be purified and isolated by dissolving it in an organic solvent such as n-port form and subjecting it to silica gel column chromatography, gel filtration power chromatography, and high-performance liquid power chromatography.

 The physicochemical properties of CS-79 obtained by the above purification method are shown below.

(a) Elemental analysis:

Found (%) C 7 4. 6 6 , H 6. 7 9, N 4. 2 5 theory (%) C 7 4. 7 8 , H 6. 82, N 4. 1 5 (C2lH 2 3N 〗 Calculated as 03)

 (b) Mass spectrometry value:

 High resolution F A B—MS / z 3 3 8.17 5 7 (M + H) 10 Theoretical 3 3 8.1 7 5 6

 (c) Molecular weight: 3 3 7

 (d) Melting point: 144 to 1 45 ° C

 (e) Specific rotation:

 Measurement is impossible due to coloring

 (f) UV absorption spectrum:

 M e 0 H Medium

 λ nax 2 30 nm (ε = 3 2 2 0 0)

 2 6 7 η m (ε = 2 9 7 0 0)

 4 2 5 η m (ε = 54 0 0)

 0. 0 1 Ν N a O H—MeO H

 λ nax 24 2 nm (ε = 3 0 4 0 0)

 2 87 nm (ε = 2 8 0 0 0)

 4 7 0 n m (ε = 8 1 0 0)

 (g) Infrared absorption spectrum:

 Figure 1 shows the results determined in the KBr lock.

 (h) — NMR spectrum:

FIG. 2 shows the results measured at 500 MHz in els—DMSO. (i) ¹³ C—NMR spectrum: FIG. 3 shows the results of measurement at 125 MHz in ds-DMS O. (j) Solubility in solvents:

 DMS 0, easily soluble in DMF

Soluble M e OH, E t OH, CH C 1 3, E t OA c

 n-hexane, poorly soluble in water

 (k) Color reaction:

 Positives: H2S 04, eord

 (1) Basic, acidic or neutral:

BRIEF DESCRIPTION OF THE FIGURES

 FIG. 1 shows the infrared absorption spectrum of CS-79 measured in KBr tablets.

FIG. 2 shows the ¹ H-NMR spectrum of CS-79 measured at 500 MHz in d 6 -DMSO.

FIG. 3 shows the ¹³ C-NMR spectrum of CS-79 measured at 125 MHz in cls-DMS0. BEST MODE FOR CARRYING OUT THE INVENTION

 Next, the production method of the compound of the present invention will be described in more detail with reference to examples. Example

 (1) 500 ml of a liquid medium containing 2.5 g of dextrin, 2.1 g of soybean flour, 0.2 g of dried yeast, and 0.4 g of calcium carbonate per 100 ml 100 ml of a triangular flask was sterilized, sterilized, and inoculated with two strains of Streptomyces ixexolitus 24 19 SVT, followed by rotation culture at 27 ° C for 48 hours.

Then, in a 50 L jar fermenter containing 30 L of a sterile medium having the same composition as the seed medium was inoculated with 500 ml of the above-described seed culture, which had been precultured, and aerated for 27, 48 hours. Agitation culture was performed. After completion of the culture, 30 L of the culture solution was separated into a supernatant and cells, and then 15 L of acetone was added to the cells and extracted. The acetone extract was extracted twice with 3 L of ethyl acetate after the acetone was distilled off. The ethyl acetate fraction was dehydrated with anhydrous sodium sulfate, concentrated, and then added to the concentrated solution with 100 ml of n-butyl ether to obtain 5.5 g of a yellow precipitate.

(2) The precipitate obtained in 1 above was dissolved in 15 ml of black-mouthed form and adsorbed on a silica gel-filled column (volume: 500 ml, solvent: black-mouthed form). After washing with 1 L of black-mouthed form, the fraction eluted with the mixed solvent of black-mouthed form-methanol (50: 1) was removed. Then, black hole Holm - methanol Honoré (2 0: 1) in a mixed solvent performs elution to give a yellow substance 2. 2 g by concentrated to dryness _P

 The obtained sample was subjected to gel filtration using Sephadex LH-20 (trade name, manufactured by Pharmacia) prepared with chloroform-methanol (1: 1), and the active fractions were collected and concentrated to dryness. 300 mg of CS-79 was obtained as a powder. Industrial applicability

 Since the compound of the present invention has an antioxidant effect and a cell death inhibitory effect, it is useful for brain diseases, heart diseases, liver diseases and arteriosclerosis diseases.

 For this purpose, the compound of the present invention is orally or parenterally administered in the form of tablets, pills, capsules, granules, injections, etc., manufactured according to conventional formulation techniques. be able to. In each of the above formulations, usual additives such as efficacious agents, tying agents, pH adjusters, dissolving agents, emulsifying agents, and suspending agents are used.

Hi can be.

 The administration of the compound of the present invention to a subject can vary depending on the age of ^, the type and condition of the disease, and the like. 100 to 500 mg can be administered in 1 to several divided doses o

Next, the antioxidant action and the cell death inhibitory action of the compound of the present invention will be described with reference to test examples. The result is shown. Test example 1 (antioxidant action)

 (1) Microsomal preparation method ''

 The liver of a 10-week-old male male rat was taken, homogenized, centrifuged at 3 ° C. and 30000 rpm for 10 minutes, and the supernatant was taken. The supernatant was collected, centrifuged at 100,000 rpm for 10 minutes, and the supernatant was collected. This was further centrifuged at 4 ° C and 300,000 rpm for 60 minutes, and the sedimented fraction was collected. The buffer (0. OSS mol Zl tris-hydrochloric acid, 0.174m0 It was composed of 1/1 Shirayidari potassium ρ ml 7.4) 70 ml was added to obtain a microsome solution.

 (2) Antioxidant activity measurement

 After mixing 0.5 ml of the microsomal solution, 0.75 ml of the above buffer solution and 0.05 ml of the sample (1% methanol solution of the compound of the present invention), L-ascorbic acid (0.05 ml) was added. 0 1 511 0 1/1) was allowed to react for 0.5 hour with 0.5 calories. The reaction was stopped by the addition of 0.5 ml of 20% trifluoroacetic acid, and the mixture was centrifuged at 300 rpm for 10 minutes, and the supernatant was collected. To this was added 0.5 ml of 0.67% thiobarbituric acid.

 After reacting at 100 ° C for 20 minutes, the absorbance at 530 nm was measured and used as an index of antioxidant action. The IC5B value of CS-79 was 0.22 / M. Test example 2 (Suppression of hippocampal CA A1 cell shedding by gerbil gerbils)

 The study was performed according to the method described in "Basic and clinical" (Vol. 24, NO.4, -3δ page 9, 199 iV-).

1E55-80 g of male gerbils were grouped into 8 groups per group, and 3 ^ rivers. The gerbil was anesthetized with ether and fixed in a dorsal position. After local infusion with xylocaine, both cervical jugular veins were exposed by cutting the midline of the neck and carefully separated from the nearby vagus nerve. Clip the artery with an aneurysm clip for 3 minutes, then clip Was removed and the skin was sutured. Sham-operated animals were treated similarly except that they were not occluded. In gerbils anaesthetized with ether, 7 days later, the brain was perfused from the left ventricle with 10% formalin buffer, and the hippocampal region was cut coronally into slices of 3-4 mm / cm2 and paraffin wrapped After embedding, sections were made according to the conventional method. The slide was stained with hematoxylen, eosin and cresyl violet. The drug was suspended in 5% arabic gum and administered intraperitoneally immediately after re-opening of ischemia. Ischemic neuropathy was evaluated on a scale of 0-3.

 0 (-): Normal nerve cell

 1 (+): damage of several nerve cells

 (One or several neuronal disorders)

 2 (++): Many neuronal disorders

 3 (+ + +): Most neuronal damage In this screening system, one (normal neuron) and + (mild neuropathy) have a protective effect on neuronal death when more than 50%. I judge.

[Table 3]

Dose Neuropathy incidence (%)

 mg / kg, i.p.) + + + + + + Control 1-100 0 0 0 Control 2-0 0 0 100 Compound 100 25 37.5 25 12.5

Control 1—A group in which the cervical vein was exposed and the skin was sewn without being tied for 3 minutes Control 2—A group in which the carotid artery was exposed and ligated for 3 minutes, and then the skin was sutured. MK—80, an NMDA (N-methyl D-aparlarate) antagonist with a reported cytoprotective effect The result of 1 is described. The test was performed according to the same method as described above. MK-801 was dissolved in physiological saline and administered intraperitoneally immediately after ischemia for 3 minutes.

[Table 4]

Dose Neuropathy incidence (%)

 (mg / kg, i.p.) + ÷-J L Control 1 1 100 0 0 0 Control 2 1 0 12.5 25 62.5

MK-8 0 1 5 0 62.5 12.5 25

MK—801: (+) — 5—methyl 1 0, 11—dihid 5H—dibenzo [a, d] cycloheptene 1-5, 10—imin CS-79, which is a pedestal of the present invention, exhibited MK-80 action.

Claims

The scope of the claims 1 set H ₃ C

Compound _c represented by