JPH08198888A - Dihydrophenazine derivative - Google Patents

Abstract

PURPOSE: To obtain the subject new derivative useful for the therapy of inflammation, ischemic disorders, etc., as an antioxidizing agent or an agent for inhibiting the toxicity of glutamic acid against neurocytes by culturing an actinomyces belonging to the genus Streptomyces and capable of producing the dihydrophenazine derivative. CONSTITUTION: The new dihydrophenazine derivative is represented by the formula, has an action for inhibiting the toxicity of glutamic acid against neurocytes, and is useful as the therapeutic medicine for diseases such as inflammation, rheumatoid arthritis and autoimmune diseases related to active oxygen, skin diseases caused by radiations, diseases such as Perkinson's disease, heart ischemic disorder and brain ischemic disorder. This compound is obtained by culturing an actinomyces strain [e.g. Streptomyces purpeofuscus 2887-SVS2 strain (FERM P-14717)]having an ability to produce the dihydrophenazine derivative in a culture medium, separating the product from the culture product, and subsequently purifying the culture product.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel dihydrophenazine derivative, a strain capable of producing the same, and a drug using the same.

[0002]

BACKGROUND OF THE INVENTION L-Glutamic acid is one of the natural amino acids that make up proteins, but it is known to have toxicity to nerve cells. If a substance having an action of suppressing this glutamate toxicity is obtained, it is expected that the substance can be used as a drug for activating or improving brain metabolism. In addition, diseases thought to involve active oxygen include inflammation, rheumatoid arthritis, autoimmune diseases, radiation-induced skin diseases, Parkinson's disease, and ischemic disorders of the heart and brain. If an antioxidant that can appropriately remove active oxygen is obtained, it is expected that the antioxidant can be used as a therapeutic agent for these diseases.

By the way, most of the many microorganisms known in microbiology live in soil. Most of the microorganisms that produce useful substances such as antibiotics, especially actinomycetes, are also derived from soil. The work of finding microorganisms that produce new and useful substances from soil has been continued. For example, Japanese Unexamined Patent Publication No. 64-22861 discloses the following compound produced from a strain belonging to the genus Streptomyces.

[0004]

Embedded image

According to the description of Japanese Patent Laid-Open No. 64-22861, the above compound has a function of removing superoxide. Also, Tetrahedron Letters 32 , No.7, 94
3-946 (1991) discloses the following compounds produced from strains belonging to the genus Streptomyces.

[0006]

[Chemical 6]

The R is H or CH ₃ . According to the description in the above article, the compound exhibits a function as a scavenger of radicals such as vitamin E.

[0008]

SUMMARY OF THE INVENTION The present inventors have discovered a new actinomycete strain belonging to the genus Streptomyces from soil. This strain was deposited on December 22, 1994 at the Institute of Biotechnology, Industrial Technology Institute of Industrial Technology. The deposit number is FERM P-
14717. When the present inventor conducted research on this strain, it was found that this strain produces a novel dihydrophenazine derivative represented by the following formula.

[0009]

[Chemical 7]

Regarding the dihydrophenazine derivative,
Further research has revealed that this substance has an inhibitory effect on glutamate toxicity and an antioxidant effect.

[0011]

DETAILED DESCRIPTION OF THE INVENTION First, a novel dihydrophenazine derivative will be described. As the chemical structure, as shown in the above formula, the hydroxyl group at the 1-position of α-L-rhamnose having a pyranose structure and the carboxyl group of the carboxyldihydrophenazine derivative are ester-bonded. The ester bond can be hydrolyzed and recombined.
Therefore, by decomposing this compound into α-L-rhamnose and a carboxyldihydrophenazine derivative, and esterifying the carboxyldihydrophenazine derivative, which is a decomposition product, with another sugar, to synthesize another kind of dihydrophenazine derivative. You can also The physicochemical properties of the dihydrophenazine derivative are as follows.

Shape: orange powder Melting point: 63.0-64.0 ° C. Specific rotation: [α] _D ²⁰ = -139.8 ° ( C = 0.00
6, MeOH) Molecular formula: C ₃₆ H ₄₀ N ₂ O ₇ Molecular weight: 612.2841 (actual value, HRFAB-M
S) 612.2835 (calculated value) Solubility: Soluble in MeOH, CH ₃ Cl, DMSO, acetone Insoluble in hexane

The above molecular formula and molecular weight were determined by FAB mass spectrum using m-nitrobenzyl alcohol as a matrix. This compound is FAB
The mass spectrum showed a (M) ⁺ peak at m / z 612. FIG. 1 is a graph showing an ultraviolet-visible absorption spectrum. The measurement is carried out in methanol and the scanning speed is 120.0 nm / min. Bandpass is 2.00
nm. As shown in FIG. 1, 232 nm (26
900), 246 nm (25800), 298 nm (1
9000), 368 nm (3300) and 495 nm
There is an absorption peak at (9700). No change in absorption peak due to acid or alkali was observed. Figure 2
It is a graph which shows an infrared absorption spectrum. The measurement is KB
r The tablet method was used. In the infrared absorption spectrum, hydroxyl group (3450 cm ⁻¹ ), secondary amine (333
Absorptions from 0 cm ⁻¹ ), ketones (1680 cm ⁻¹ ) and esters (1680 cm ⁻¹ , 1260 cm ⁻¹ ) were observed. FIG. 3 is a graph showing a ¹ H-NMR spectrum. The measurement was carried out in heavy acetone at 500 MHz. FIG. 4 is a graph showing a ¹³ C-NMR spectrum. The measurement was carried out in heavy acetone at 500 MHz.

The Rf value of thin layer chromatography (chloroform / methanol = 10/1) was 0.42. The above dihydrophenazine derivatives have so far only been obtained by culturing strains discovered by the present inventor. Next, this strain will be described.

Deposit number FERM P deposited on December 22, 1994 at the Institute of Biotechnology, Institute of Biotechnology, AIST.
The strain 14717 was isolated from a soil sample collected in Bunkyo-ku, Tokyo. This strain is an actinomycete belonging to the genus Streptomyces. In terms of morphology, the basic hyphae do not divide. The aerial mycelium forms a short main axis, and branches irregularly in a straight or curved shape longer than that,
Form one or more spore chains. Spores are non-motile, cylindrical or oblong and have a width of 0.5-0.
It is 8 μm. Also, the spore surface is smooth. No sclerotium, sporangia or other special morphology is observed. The cell wall chemotype is type I. A micrograph of this strain is shown in Figure 5 (100
4 times) and FIG. 6 (about 12000 times). Note that FIG.
The length of the white bar shown in is 500 nm. Next, the strains are (1) sucrose / nitrate agar medium, (2) glucose / asparagine agar medium, (3) glycerin / asparagine agar medium, (4) inorganic salt / starch agar medium, (5) tyrosine agar medium, (6) Nutrient agar medium,
The results of culturing in (7) yeast-malt agar medium and (8) oatmeal agar medium are shown. Table 1 shows the culturing properties on the various media mentioned above.

[0016]

[Table 1] Table 1 ──────────────────────────────────── Culture color of the surface of the culture medium Backside color of village Diffusible dye ───────────────────────────────────── (1) Gray series (d ) Light brown (5cb) None (2) Gray series (3fe) Dark brown (6pl-6pn) Light orange (5ca) (3) Gray series (d) Dark red Brown to dark brown (6pl-6pn) Light orange (4ea) (4) Gray series (3fe to 5fe) Dark brown (6nl) Light red (6l / 2gc) (5) Gray series (5fe) Dark red orange (6le) Pinkish white (5cb) (6) No mycelia Light brown (3ec ~ 3dc) None (7) Gray series (5fe) Brown to dark brown (5ni ~ 5pn) Dark orange (5ic) (8) Gray series (3fe ~ 5fe) Gray red brown (6l / 2ni) Light red (7gc) ───────────────────────────────────── Note: Brackets are colored -Indicates the color code of the Harmony Manual (Container Corporation of America, 1950).

As shown in Table 1, the flora color on the surface of the colony is gray series. The back side color is dark reddish brown or dark brown. The diffusible dye is recognized as light orange to light red or dark orange. The hue of these dyes did not change with pH changes. Furthermore, physiological properties and assimilability of carbon source are shown in Table 2 below.

[0018]

[Table 2] Table 2 ──────────────────────────────────── Physiological properties and carbon source Assimilation ──────────────────────────────────── Growth temperature range 20-37 ℃ Optimum temperature 30-37 ℃ Production of melanin pigment: Tyrosine agar + Peptone yeast iron agar + Tryptone yeast broth + Starch hydrolysis + Gelatin liquefaction + Defatted milk peptone + Nonfat milk coagulation − Nitrate reduction + Carbon Source assimilation: D-glucose + L-arabinose + D-xylose + D-fructose-sucrose-L-rhamnose-raffinose- i -inositol-D-mannitol + ───────────── ────────────── ─────────

As described above, this strain is mesophilic, and glucose, arabinose, xylose, and mannitol are used as the assimilation ability of carbon source. Based on the morphological characteristics and cell wall chemotype of this strain, this strain is located in the genus Streptomyces. Based on the above-mentioned properties (spore chain morphology, spore surface, microbial color, backside color, diffusible dye, assimilation ability of carbon source, etc.), "Bacterial name approval list, 1980" and valid name list We searched for Streptomyces spp. Described in 1. and selected related species. When the diagnostic properties of Streptomyces purpeofuscus are selected, as shown in Table 3 below, the properties of this strain and Streptomyces purpeofuscus are different except for the assimilation ability of carbon sources (only mannite differs). , Match well.

[0020]

[Table 3] Table 3 ──────────────────────────────────── Comparative items This strain Streptomyces・ Perpea phoscus ──────────────────────────────────── Spore chain morphology: straight or curved + + spores Surface: Smooth ++ Microbiota color: Gray ++ Back color: Red / orange ++ pH sensitivity − − Diffusible dye: production ++ pH sensitivity − − Melanin pigment production ++ Starch hydrolysis ++ nitrate reduction + + Growth temperature: 10 ° C --37 ° C + + 45 ° C + + Assimilation ability of carbon source: arabinose + + xylose + + inositol --- mannitol + -rhamnose --- raffinose --- sucrose --- fructose --- galactose + ＋ ───────────── ────────────────────────

As described above, since this strain is most similar to Streptomyces perpeofuscus, it was deposited under the species name. The label of the deposited strain (display for identification given by the depositor) is 2887-SVS2. The above-mentioned dihydrophenazine derivative can be produced by aerobically culturing a producing bacterium such as this strain in an appropriate medium and collecting the target product from the culture. The medium is composed of nutrient sources available to the dihydrophenazine derivative-producing bacterium. Glycerol is preferred as the carbon source. Molasses, casein and polypeptone can be preferably used as the nitrogen source. Inorganic salts can be added if necessary. A suitable antifoaming agent (eg, silicone) may be added to suppress foaming during fermentation.

For large-scale production of the dihydrophenazine derivative, it is preferable to employ aerobic submerged culture conditions. This condition is similar to the culture condition for mass production of other common bioactive substances. For small-scale production, shaking culture in a flask can be adopted. Alternatively, the culture may be performed using a jar fermenter. In that case,
In order to avoid growth delay in the production process, it is preferable to use vegetative cells of microorganisms for inoculation into the fermenter.
To this end, a relatively small amount of medium is inoculated with microbial cells and the inoculated medium is cultivated to produce vegetative cells of the microorganism,
The cultured vegetative cells are then preferably transferred to a fermentor.
The medium for producing vegetative cells may be different than the medium for producing the dihydrophenazine derivative.

The stirring and aeration of the culture mixture can be carried out in the usual way. A propeller or similar mechanical stirring device can be used for stirring. You may stir by rotation or shaking of a fermentor. Various pump devices can be used for ventilation. Alternatively, the medium may be aerated by passing sterile air. The fermentation temperature is generally 10 to 40 ° C, preferably 20 to 30 ° C. The fermentation time is generally 50 to 200 hours. Fermentation time varies depending on fermentation conditions and scale. After fermentation is complete, the dihydrophenazine derivative can be recovered from the culture broth by a variety of conventional recovery and purification methods. As the recovery method, solvent extraction, chromatography or recrystallization can be adopted. Solvent extraction and recrystallization uses a suitable solvent for the dihydrophenazine derivative. You may use together 2 or more types of solvents.

The dihydrophenazine derivative is generally found in cultured bacterial cell components. Therefore, after extracting the cells obtained by centrifuging or filtering the culture solution with an appropriate solvent,
It is preferable to carry out a purification treatment of the dihydrophenazine derivative. Acetone or methanol can be used as the solvent. Purification of dihydrophenazine derivatives is
It can be carried out by treating the extract according to a conventional method. For example, after removing the solvent from the extract by evaporation or distillation, re-extraction with an appropriate solvent (eg, ethyl acetate) and drying to dryness can be repeated to obtain a residue containing the dihydrophenazine derivative. Using this as a crude product, a dihydrophenazine derivative can be purified by a conventional purification method such as silica gel chromatography or HPLC fractionation.

With respect to microorganisms other than this strain, the dihydrophenazine derivative-producing strain can be isolated from the natural world by a conventional method. Specifically, it can be carried out in the same manner as the method for isolating an antibiotic-producing bacterium. Further, this strain may be subjected to irradiation or treatment for inducing other mutations to improve the productivity of the dihydrophenazine derivative. The biosynthesis of dihydrophenazine derivatives is presumed to involve many genes, similar to the antibiotics produced by actinomycetes. With the development of genetic recombination technology, genetic manipulation has become possible for biosynthesis of such substances. Therefore, the gene involved in the biosynthesis of the dihydrophenazine derivative of this strain can be introduced into another strain to allow the resulting transformant to produce the dihydrophenazine derivative.

The dihydrophenazine derivative obtained as described above exhibits an inhibitory effect on glutamate toxicity. Also,
The dihydrophenazine derivative also has an antioxidant effect. Therefore, the dihydrophenazine derivative of the present invention is useful as a glutamate toxicity inhibitor or an antioxidant. The dihydrophenazine derivative of the present invention has a stronger inhibitory effect on glutamic acid toxicity than that of conventionally known compounds, and is several n
M can suppress glutamate toxicity by about 50%. A known compound having an inhibitory effect on glutamate toxicity suppresses nerve cell death due to mild cerebral ischemia and activates brain metabolism. Therefore, the dihydrophenazine derivative of the present invention is also effective as a therapeutic agent for cerebral ischemic disorders such as cerebral infarction and cerebrovascular dementia. It has been suggested that glutamic acid exhibits toxicity by various mechanisms, and it is said that free radicals such as active oxygen are involved in the expression in any system. Therefore, it is known that antioxidants such as vitamin E suppress glutamate toxicity. The dihydrophenazine derivative of the present invention also exhibits a similar antioxidant effect. Therefore, the dihydrophenazine derivative of the present invention can be used for diseases involving active oxygen, such as inflammation,
It is also effective as a therapeutic drug for rheumatoid arthritis and autoimmune diseases.

[0027]

【Example】

[Example 1] PCI medium containing "fermentation" starch (1%), polypeptone (1%) and meat extract (1%) (pH before sterilization)
7.0) 15 ml of the test tube was inoculated with 1 platinum loop of slant culture of Streptomyces perpeofuscus 2887-SVS2 strain, and shake culture was carried out at 27 ° C. for 3 days. Then, 2 ml of this preculture was added to glycerin (2
%), Molasses (1%), casein (0.5%), polypeptone (0.1%) and calcium carbonate (0.4
%) Containing 100 ml of a medium containing a hump 5
Six 00 ml Erlenmeyer flasks were inoculated and shake cultured at 27 ° C. for 2 days. 600 ml of seeds prepared as above
Was inoculated into 60 liters of production medium containing glycerin (2%), molasses (1%), casein (0.5%), polypeptone (0.1%) and calcium carbonate (0.4%). The culturing was carried out in a jar fermenter while aerating at 30 liters per minute and stirring at 400 rpm while performing fermentation at 27 ° C. for 3 days.

"Isolation and purification" 60 liters of the culture broth was centrifuged, and the obtained cells were extracted with an equal amount of acetone.
After acetone was removed by concentration, the mixture was extracted with ethyl acetate under the condition of pH 3.0. The organic phase was concentrated under reduced pressure, washed with hexane, and the insoluble fraction was subjected to silica gel chromatography (WAKOGEL C-100, 500 ml). The column was washed with hexane / ethyl acetate = 2/1 (1.5 liter) and eluted with chloroform / methanol = 20/1 (1.5 liter). The active fractions were collected and concentrated under reduced pressure, then the reverse phase silica gel column (Senshu ODS-SS-1020-T, 5
(00 ml) and eluted with 90% methanol. The obtained active fraction was concentrated under reduced pressure and then subjected to reverse phase silica gel column (Senshu Pak. PEGASIL ODS, diameter 20 mm × 25.
HP in a solvent system of 90% methanol.
LC fractionation was performed. The activity peak was concentrated under reduced pressure to give the dihydrophenazine derivative of the present invention as a red powder.
0 mg was obtained.

[Example 2] "Glutamate toxicity inhibitory effect on nerve cells" In a glutamate toxicity inhibitory test, a hybridoma of mouse neuroblastoma and rat retinal nerve cells (N18-RE-10) was used.
5 cells) were used. When a high concentration of glutamic acid (1 to 10 mM) was added to this hybridoma, cell death due to oxidative stress was observed due to inhibition of intracellular uptake of cystine (Neuron 2 , 1547 (1989) and J. Pharmaco.
l. Exp. Ther, 250 , 1132 (1989)). The effect of the dihydrophenazine derivative of the present invention on this glutamate-induced cell death was examined. 10% FCS and HAT (hypo
xanthine 0.1 mM, aminopterin 40 nM, thymid
6.25 in a 96-well microplate containing Dulbecco's modified MEM medium containing 0.14 mM of ine (manufactured by Sigma).
Hybridoma cells were inoculated at a density of × 10 ³ cells / cm ³ . After 24 hours, 10 mM glutamic acid and the dihydrophenazine derivative of the present invention were added. After addition of glutamic acid, the cells were further cultured for 24 hours, and then the lactate dehydrogenase (LDH) activity contained in the cells and the medium was measured. The LDH release rate was calculated according to the following formula to evaluate glutamate toxicity.

[0030]

[Equation 1] LDH release rate = {LDH activity in medium / (intracellular + LDH activity in medium)} × 100

The measurement results are shown in FIG. In FIG. 7, the horizontal axis represents the concentration of the dihydrophenazine derivative of the present invention, and the vertical axis represents LDH.
It is a graph which plotted the measurement result as a release rate.

[Example 3] "Inhibition of lipid peroxidation" From the liver of male Wistar rats, the method of Ernster, L. et al. (J. Cell Biol. 15 , 541 (1
Microsomes were prepared according to 962)). Liver microsomes (protein amount 0.5 mg), 0.2 M Tris-hydrochloric acid buffer solution (pH 7.4), 2 mg / ml ADP, DMSO solution of test substance (dihydrophenazine derivative of the present invention) and 0.5 mg / ml NADPH Reaction solution 0.
5 ml was incubated for 1 hour at 37 ° C. The reaction vessel was transferred to ice water, and 0.05 ml of 0.5% butylhydroxytoluene-ethanol solution was immediately added to stop the reaction, and further 0.25 ml of 8.1% SDS, 1.75 ml of 20% acetic acid, 0.8%. Thiobarbituric acid solution (pH
3.5) 1.5 ml was added. The reaction vessel was transferred into boiling water and heated for 1 hour to develop color. After cooling, 4 ml of n-butanol was added and shaken, followed by centrifugation (3000 r).
pm, 10 minutes), and the absorbance of the supernatant at 530 nm was measured to determine the inhibition rate of the test substance against the lipid peroxidation reaction. The results are shown in Table 4.

[0033]

[Table 4] Table 4 ──────────────────────────────────── Concentration suppression rate IC ₅₀ (M ) ──────────────────────────────────── 10 ⁻⁷ M −0.5% 10 ⁻⁶ M 31.5% 1.5 × 10 ^-6 10 ^-5 M 95.0% 10 ^-4 M 99.1% ───────────────────────── ─────────────

[0034]

INDUSTRIAL APPLICABILITY As described above, the dihydrophenazine derivative of the present invention exhibits a glutamate toxicity inhibitory action and an antioxidant action. Therefore, the dihydrophenazine derivative of the present invention has an effect as a glutamate toxicity inhibitor or an antioxidant.

[Brief description of drawings]

FIG. 1 is a graph showing an ultraviolet-visible absorption spectrum.

FIG. 2 is a graph showing an infrared absorption spectrum.

FIG. 3 is a graph showing a ¹ H-NMR spectrum.

FIG. 4 is a graph showing a ¹³ C-NMR spectrum.

FIG. 5 is a photomicrograph (100 ×) of the strain.

FIG. 6 is a photomicrograph (about 12000 times) of the strain.

FIG. 7 is a graph showing the measurement results of glutamate toxicity.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display area A61K 31/71 ABG ABR ABS ADD AED C12N 1/20 A 8828-4B // C12P 19/58 (C12N) 1/20 C12R 1: 465) (C12P 19/58 C12R 1: 465)

Claims (6)

[Claims] 1. A dihydrophenazine derivative represented by the following formula. Embedded image 2. A strain of actinomycetes belonging to the genus Streptomyces and having an ability to produce a dihydrophenazine derivative represented by the following formula. Embedded image 3. A strain of actinomycetes, which belongs to the genus Streptomyces and has been deposited at the Institute of Biotechnology, Institute of Biotechnology, Accession No. FERM P-14717. 4. A method of producing L-glutamic acid for mammalian nerve cells, which is produced from a strain of actinomycetes belonging to the genus Streptomyces and deposited under the accession number FERM P-14717 at the Institute of Biotechnology, Institute of Biotechnology, Institute of Industrial Science and Technology. A substance that has the effect of suppressing toxicity. 5. A glutamate toxicity inhibitor comprising a dihydrophenazine derivative represented by the following formula. Embedded image 6. An antioxidant comprising a dihydrophenazine derivative represented by the following formula. [Chemical 4]