JPH03227971A - Carbazole compound - Google Patents

Abstract

NEW MATERIAL:A carbazole compound expressed by the formula (R<1> is acetyl, N<1>-isobutyl-ureido-carbonyl; R<2> is n-bentyl, n-hexyl, n-heptyl or isoheptyl). USE:The compound expressed by the formula has antioxidizing action and is a medicine useful for brain disease, heart disease, lever disease and arteriosclerosis, etc. PREPARATION:Streptomyces cyaneus 2007 SVT1 is used as a producing germ strain and cultured in a culture medium containing various kind of nutrient matters under aerobic conditions to provide the novel compound. As the culture medium, a liquid culture medium is used and as a carbon source, glucose, sucrose, etc., is used and as a nitrogen source, meat extract, oat meal, etc., is used and further as necessary organic and inorganic salt are added to the culture medium. As a defoaming agent, adekanol, etc., is used. Aerobic culture is suitable as the culture method and the culture is especially preferably carried out at pH6-7 and 24-27 deg.C for 4 day.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to carbazole compounds having antioxidant activity.

PRIOR ART There are no known compounds related to the compounds of the present invention that have similar effects.

Problems to be Solved by the Invention An object of the present invention is to provide a compound with a new structure that has an antioxidant effect.

Means for Solving the Problems In order to achieve the above object, the present inventors isolated a large number of bacterial strains from soil and conducted various studies on the cultures of the strains. They discovered that it has an oxidizing effect and completed the present invention.

(In the formula, R1 represents an acetyl group or an N'-isobutyl-ureido-carbonyl group, and R2 represents an n-pentyl group, n
-Hexyl group, n-hebutyl group or isohebutyl group. ) is a compound represented by

The strain producing the compound of the present invention is a strain newly isolated by the present inventors from the soil of Takasakifu, Gunma Prefecture, and the microorganism name is Streptomyces cyaneus 20.
07SVI, and microorganism deposit number “Feikoken Bacteria No. 11”
No. 234 (FERM P-11234) and has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology.

The mycological properties of this strain are shown below.

[1] Morphological properties The basal hyphae of this strain do not divide. Aerial hyphae form spiral spore chains on the main axis or branches, consisting of 10 to 50 or more. Spores are nonmotile, oblong or cylindrical;
0.5-04 μm x 0.7-1.0 μm, exhibits a typical thorn-like surface on inorganic salt/starch agar, and a variety of surfaces ranging from a smooth surface or an incomplete thorn-like shape to a typical thorn-like surface on yeast/malt agar. .

No sclerotia, sporangia, or other special forms are observed. The cell wall chemotype is type ■.

[2] Culture properties Table 1 shows the results of visual observation when cultured on various media at 27°C for 12 weeks.

No. ] table America 1950) color standard code [3 physiological properties (1) Growth temperature range Grows in the range of 10-45°C.

It is 0C.

(2) Distinction between aerobic and anaerobic; (3) Liquefaction of gelatin; (4) Coagulation of skim milk; (Peptonization of ω skim milk; (6) Hydrolysis of starch; (7) Production of melanin-like pigments; a) Tyrosine agar b) Peptone yeast iron agar (8) Assimilation of carbon sources; D-glucose-arabinose D-xylose D-fructose Sucrose L-rhamnose Lanoinose Optimum temperature is 25-30 Positive, negative, positive, negative Positive Positive Positive Positive Positive Positive Positive Inositol Positive D-Mannitol Positive Based on the two morphological characteristics, this strain is placed in the genus Streptomyces. Based on the above properties, "Bacterial Name Approved List J (1980)"
Effective group Listyo and "Bergey's Manual of Bacterial Systematics, Volume 4"
As a result of comparison with many known bacterial strains reported in Stematic Bacteriology) J.
ces cyaneus).

Based on the above results, it was determined that this strain is the same species as Streptomyces cyaneus, and this strain was named Streptomyces cyaneus 2007SVT.

The production of the compounds of the present invention is carried out by culturing the present bacterial 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, honey, starch, etc. are used singly or in combination as secondary sources. As the nitrogen source, meat extract, oatmeal, yeast extract, soybean flour, polypeptone, etc. are used alone or in combination.

In addition, organic substances and inorganic salts that aid the growth of the present strain and promote the production of the compound of the present invention may be added as necessary. As the antifoaming agent, adecanol, silicone, etc. can be used.

Aerobic culture such as shaking culture or aerated agitation culture is suitable for culturing, and the culture is carried out at pH 4 to 8.24 to 30°C for 3 to 6 days, preferably at pH 6 to 7.24 to 27°C for 4 days. .

The compound of the present invention produced by this culture can be isolated according to a general method for collecting fermentation products.

That is, after completion of the culture, the bacterial cells and the supernatant are separated by centrifugation or filtration, and the compound of the present invention accumulated in the bacterial cells is extracted with an organic solvent such as a lower alcohol or acetone. The organic solvent is distilled off under reduced pressure, the residue is extracted with a water-insoluble solvent such as benzene, ethyl acetate, chloroform, etc., and this is concentrated and dried.

The syrup obtained in this operation is dissolved again in an organic solvent such as benzene, chloroform, acetone, or methanol,
The compound of the present invention can be purified and isolated by subjecting it to silica gel column chromatography, gel filtration column chromatography, and high performance liquid column chromatography.

Effects of the Invention Since the compounds of the present invention have antioxidant effects, they are useful for treating brain diseases, heart diseases, liver diseases, arteriosclerosis, and the like.

EXAMPLES Next, the present invention will be specifically explained with reference to Examples and Test Examples.

(Example) (1) pH consisting of 2.5% soluble starch, 1.5% soybean flour, 0.2% dry yeast and 0.4% calcium carbonate
100 i of the liquid medium of No. 7 was placed in a Erlenmeyer flask of No. 500 and sterilized at 120° C. and 2 atm for 20 minutes. Next, Stosib 1 to Myces cyaneus 2007 SVI+ strains were inoculated and rotary cultured at 27°C for 48 hours.

After the completion of the culture, the obtained 60I! The culture solution was centrifuged to obtain bacterial cells. After extracting the bacterial cells with 1oIl of acetone,
Acetone was distilled off under reduced pressure and the residue was 2f! , twice with ethyl acetate. The obtained ethyl acetate layers were combined and concentrated to dryness to obtain 8 g of brown syrup.

This syrup was dissolved in 10 d of a mixed solvent of n-hexane-ethyl acetate (3:1), and a mixed solvent of the same composition was adsorbed on a column (capacity: 600 d) filled with silica gel. Elute with a solvent of the same composition and collect 1 fraction of 20 m
The fractions No. 60 to No. 80 were collected and concentrated to dryness to obtain 138 mg of crude powder.

(2) The crude powder obtained in (1) was dissolved in 5 d of a mixed solvent of chloroform-methanol (1:1), and the mixture was prepared with a mixed solvent of the same composition. 42M). The active fractions eluted from 180 to 210 d were collected and concentrated to dryness to obtain 58 mg of crude powder.

(3) The crude powder obtained in (2) was dissolved in 5-methanol, and Toyovar HW4OF (
Gel filtration was performed using a product manufactured by Toyo Soda Co., Ltd. (capacity: 80 d).

Active fractions eluted from 440 to 50M were collected and concentrated to dryness to obtain 23 mg of crude powder.

(4> The crude powder obtained in (3) was dissolved in methanol 1d and used as a sample for high performance liquid chromatography performed under the following conditions.

Column size: 10φX 250mm Support:
ODS silica gel (trade name: Senshu Pack, manufactured by Senshu Scientific Co., Ltd.) Solvent composition: 80% methanol, 20% water Flow rate:
3 mg/min Detection: Uv absorption 254 nm A fraction with a retention time of 9 to 11 minutes was collected, and a compound of formula (I) in which R1 is an acetyl group and R2 is an n-pentyl group (hereinafter referred to as compound 1) was obtained. ), 3 mg of R
2 mg of a compound in which 1 is an acetyl group and R2 is an isohebutyl group (hereinafter referred to as compound 2) was obtained, and the retention time was 2.
A fraction of 0.8 to 21.5 minutes was collected to obtain 2 mg of a compound (hereinafter referred to as compound 3) in which R1 is an acetyl group and R2 is an n-hebutyl group in formula (I), A fraction with a retention time of 24 to 26 minutes was collected, and in the formula (I>, R1 is an N'-isobutyl-ureido-carbonyl group and R2 is n
- 2 mg of a compound having a hexyl group (hereinafter referred to as compound 4) was collected, and a fraction with a retention time of 50 to 51 minutes was collected. In formula (I), R' is N'-isobutyl-ureido-carbonyl 4 mg of a compound in which R2 is an isohebutyl group (hereinafter referred to as compound 5) was obtained, and the retention time was 5.
A fraction of 4 to 56 minutes was collected, and in formula (I), R1 was N.
6 mg of a compound in which "-isobutyl-ureido-carbonyl is an n-hebutyl group (hereinafter referred to as compound 6)"
I got it.

The physicochemical properties of these compounds are shown in Table 2.

Table 2] FAB mass spectrum measurement 2 Measured in methanol 1 In addition, 'H-NMR spectra of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5 and Compound 6 (measured at 400 MHz in deuterated chloroform) ) are shown in FIG. 1, FIG. 2, FIG. 3, FIG. 4, FIG. 5, and FIG. 6, respectively.

(Test Example) Antioxidant Effect Test (1) Preparation of Microsomes The liver of a 10-week old male Wistar rat was taken, homogenized, centrifuged at 4°C and 3000 rpm for 10 minutes, and the supernatant was collected. This supernatant was heated at 4°C for 10
The mixture was centrifuged at 000 rpm for 10 minutes, and the supernatant was collected. This was further centrifuged at 4°C and 30,000 rpm for 60 minutes, the precipitate fraction was collected, and a buffer solution (0.025 mo
l/R. l-Lis-hydrochloric acid, 0. 174mol/
R. Consists of potassium chloride. pH 7, 4) 70d was added to prepare a microsomal solution.

(2> Antioxidant effect measurement microsomal solution 0.5d, the above buffer solution 0.75rne
and sample (1% methanol solution of the compound of the present invention) 0
．． After mixing 05-, L-ascorbic acid 2- (0.0015 mol/It) was mixed with 0.05-. 5ml
The mixture was added and reacted at 30°C for 1 hour. The reaction was stopped by adding 0.5-20% trichloroacetic acid, centrifuged at 3000 rpm for 10 minutes, and the supernatant was collected. 0.6 to this
7% thiobarbic acid 0,5- was added.

After reacting at 100°C for 20 minutes, absorbance at 530 nm was measured and used as an index of antioxidant activity.

The results are shown in the table as ICso values.

Table of contents

[Brief explanation of drawings]

Figure 1 shows the 'H-NMR spectrum of compound 1 measured at 400 MHz in deuterated chloroform, and Figure 2 shows the 'H-NMR spectrum of compound 2 measured at 400 MHz in deuterated chloroform.
The MR spectrum is shown in Figure 3 in deuterated chloroform at 400 °C.
Figure 4 shows the 'H-NMR spectrum of compound 3 measured at 400 MHz in deuterated chloroform, and Figure 5 shows the 'H-NMR spectrum of compound 4 measured at 400 MHz in deuterated chloroform. 'H of compound 5
-NMR spectrum is shown in Fig. 6 in deuterium chloroporm.
The 'H-NMR spectrum of compound 6 measured at 00 MHz is shown.

Claims (1)

[Claims] (1) Formula ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ (In the formula, R^1 represents an acetyl group or N'-isobutyl-ureido-carbonyl group, and R^2 represents an n-pentyl group or n-hexyl group. , n-heptyl group or isoheptyl group).