JPH0912468A - Novel substance DQ90C, method for producing the same, and nerve cell process regeneration factor containing the DQ90C - Google Patents

Abstract

(57) [Summary] (Modified) [Purpose] To provide a substance which is a small molecule and has a neurite regenerating effect. [Structure] A novel substance DQ90C represented by the formula (I), a method for producing the same, and a nerve cell process regeneration factor containing the substance as an active ingredient.

Description

Detailed Description of the Invention

[0001]

The present invention relates to a novel substance DQ90C,
The present invention relates to a method for producing the same and a nerve cell process regenerating factor containing DQ90C as an active ingredient.

2. Description of the Related Art Nerve growth factor (hereinafter referred to as NGF)
Is a polypeptide having a molecular weight of 140,000, which is necessary for differentiation, growth and function maintenance of sensory and sympathetic nerves. This NGF is α,
It consists of three subunits, β and γ, of which β
Since the subunit alone expresses NGF activity, β-
Also called NGF. NGF has the effect of extending neurites and the effect of regulating the production of neurotransmitters, and it has been proved in vitro that it has a regenerating effect on nerve cells of old animals [Age, Volume 8, Page 19 (198
5)], and because of these effects, it is one of the ones that have recently attracted attention as anti-dementia drugs. However, NGF cannot pass through the blood-brain barrier due to its molecular size, and cannot be a therapeutic agent for external administration. On the other hand, PC-12 cells, which are cell lines cloned from rat adrenal medullo pheochromocytoma, are known to stop growth and differentiate into cells with neurites by adding NGF. . Therefore, PC-1
Two cells are often used as a model system for neural differentiation. In addition to NGF, it was investigated using these cells that fibroblast growth factor and interleukin 6 also induce process elongation. Recently, staurosporine, a low-molecular substance derived from a microorganism [Neurochemistry, Vol. 26, , P. 200 (1987)]
And lactacystin (OM-6519 substance) [lactacys
tin: S. Omura et al., J. Antibiotics, 44, 113 (1991)], [S. Omura et al., J. Antibiotics, 44.
Vol. 117 (1991)] and Japanese Patent Application Laid-Open No. 3-98594 also show that neurite outgrowth of PC-12 cells is similarly caused. Besides these, Neuro 2A
Substances such as PS-990 substance having neurite outgrowth action on cells [S. Toki et al., J. Antibiotics, 47, 1175 (1994)] are also shown, but DQ90
Substance C has a different molecular formula from these known compounds and is clearly distinguished.

Further, as a compound similar in structure to the novel substance DQ90C according to the present invention, a proteolytic enzyme inhibitor tyrostatin [tyrostatin: K. Oda et al., Agric. B is used.
iol.Chem., 53, 405 (1989)], but there is no description about the PC-12 cell neurite regenerating activity of the substance, and the substance is clearly distinguished from the DQ90C substance in its molecular formula. It As a similar proteolytic enzyme inhibitor, a leupeptin analog Ac-Leu-Leu showing an action of enhancing NGF effect on PC-12 cells.
-Nle-al [Y. Saito et al., Neuroscience Letters,
89, 102 (1988)], etc.
The DQ90C substance has a different molecular formula from these known compounds and is clearly distinguished. Considering its use as an anti-dementia drug, it has a molecular size (small molecule) that can cross the blood-brain barrier.
It is extremely important to provide a substance that is less toxic and exhibits a neurite outgrowth action at a lower concentration as a solution to human medical and social problems.

[0003]

SUMMARY OF THE INVENTION It is an object of the present invention to provide a substance which is a small molecule and has a neurite regenerating effect.

The present inventors completed the present invention by isolating a novel substance DQ90C having a neurite outgrowth activity from a culture solution of a strain belonging to the genus Streptomyces. The present invention provides a novel substance D represented by the formula (I)
Provides Q90C.

[0004]

Embedded image

The present invention also provides a neuronal process regenerating factor containing the compound DQ90C as an active ingredient. The present invention also provides a method for producing a DQ90C substance, which comprises culturing a DQ90C substance-producing bacterium belonging to the genus Streptomyces and collecting the DQ90C substance from the culture.
As the DQ90C substance-producing bacterium of the present invention, any bacterium belonging to the genus Streptomyces and having a DQ90C substance-producing ability can be used. Specifically, Streptmyces halstedii 27
The 23-SV2 strain (hereinafter referred to as "DQ-90 strain") can be advantageously used. DQ-90 strain The DQ-90 strain found by the present inventors as a strain of the genus Streptomyces having the DQ90C substance-producing ability has the following contents.

1) Origin and Deposit No. DQ-90 strain is an actinomycete isolated from a soil sample collected at Hakone Pass in Shizuoka Prefecture. It has been deposited and has obtained the number of "Micromachine Research Deposit No. 5135" (FERM BP-5135). 2) Mycological Properties The mycological properties of the DQ-90 strain are as follows. The characterization of actinomycete DQ-90 strain is "Patent Office Industry-Specific Examination Standards"
Was carried out according to the method described in. The basal hypha of this strain is not broken. The aerial hyphae form the main axis, and 10 to 50 or more straight or curved spore chains are formed at the tips branched irregularly therefrom. This spore chain is a bird's nest (ne
St-like) dense lumps are formed. Spores are non-motile, cylindrical or oblong, width 0.3-0.5 μm, length
From 1.0 to 1.2 μm, the spore surface is smooth. Sclerotia, sporangia and other special forms are not observed. The cell wall chemotype is type I, which contains LL diaminopimelic acid. The culture properties are shown in Table 1. The flora color on the surface of the community belongs to the gray series. The back side color is unclear and does not change with pH. The diffusible pigments were light orange to light brownish gray.

[0007]

[Table 1] Table 1 Culture properties Medium Bacterial surface color of colony surface Backside color of colony Diffusible pigment sucrose, no aerial hyphae, light brown grey, no nitrate agar (3ec) Glucose / As gray series light brown, pale orange paragine agar (2fe) (4le to 4ni) (4ea) ) Glycerin ・ As Gray series Light tea taste Gray Light tea taste Gray Paragin Agar (2fe) (4ge) (4ec) Inorganic salt / starch agar Gray series Light tea taste gray None (5fe) (4ge) Tyrosine agar Gray series Light yellow brown None ( cd ~ (3gc ~ 3le) Nutrient agar No aerial hypha No pale yellow (2db) Yeast / malt agar gray series Reddish brown Light brown (5fe) (6le ~ 6li) (4gc) Oatmeal agar Gray series Light brown to brown Akira Chami gray (5fe) (4lg~4ni) (5ec ) * () in the color harmony Maniyuaru (container copolyarylene configuration of America, 1950) color plate U De. The physiological properties are shown in Table 2. This strain is mesophilic and uses assimilation ability of carbon source glucose, arabinose, xylose, and fructose. Based on the morphological characteristics and cell wall chemotype of this strain, it is located in the genus Streptmyces (hereinafter abbreviated as S.).

[0008]

[Table 2] Table 2 Physiological properties Growth temperature range 20-37 ℃ Optimum temperature 20-30 ℃ Melanin pigment Tyrosine agar-Peptone yeast iron agar ± Tryptone yeast broth-Hydrolysis of starch + Liquefaction of gelatin-Peptonization of skim milk powder-Coagulation of skim milk powder + Reduction of nitrate + Assimilation of carbon source D-glucose + L-arabinose + D-xylose + D-fructose + sucrose + L-rhamnose-raffinose-i-inositol- D -mannite-

[0009] Based on the above-mentioned various properties, "Bacterial name approval list,
1980 ”and subsequent S.R. We searched for species of the genus and selected closely related species. S. When the diagnostic properties of S. nigrifaciens were selected, this strain and S. nigrifaciens were selected. Nigrifaciens ( S.nigrif
The characteristics of aciens ) are slightly different in the assimilation ability of carbon source, but the others are in good agreement (Table 3). However
Bergey's Manual of Systematic Bacteriology Vol.
4, Williams et al. Niglifaciens was introduced into S. It is known as a synonym for S.halstedii . Therefore, this strain 2723-SV2 is a strain of Streptmyces halstedii ( Streptmyces halstedii ) 2.
723-SV2 strain.

[0010]

[Table 3] Table 3 Comparison between this strain and related species This strain Streptomyces Streptomyces 2732-SV2 Niglifasciens Halsteady spore chain morphology Straight / curved ＋ ＋ spore surface smooth ＋ ＋ ＋ bacterial color gray ＋ ＋ ＋ reverse color unclear ＋ ＋ ＋ pH diffusive Pigmentation + +-pH sensitivity --- Melanin pigment production --- Hydrolysis of starch + + + Reduction of nitrate + + + Growth temperature 10 ° C --- -37 ° C + + + 45 ° C ----Assimilation of carbon source arabinose + + + xylose + + + inositol - - - mannitol - + - rhamnose - - - raffinose - - - sucrose + - -

Cultivation method of DQ90C substance-producing bacterium DQ90C substance belongs to the genus Streptomyces
It can be produced by aerobically culturing the 0C substance-producing bacterium in an appropriate medium and separating and purifying the target product from the culture. The medium is a medium containing nutrients that can be utilized by ordinary microorganisms. As the nutrient source, known nutrients conventionally used for cultivation of actinomycetes can be used. Specifically, as the carbon source, glucose, starch syrup, dextrin,
Starch, molasses, fats and oils can be used. As the nitrogen source, organic substances such as soybean flour, wheat germ, cottonseed meal, corn steep liquor, meat extract, peptone, yeast extract and inorganic substances such as ammonium sulfate and ammonium nitrate can be used. If necessary, sodium, potassium, calcium, magnesium, cobalt, chlorine,
Inorganic salts capable of producing phosphoric acid, sulfuric acid and other ions can be added. In addition, organic and inorganic substances that help the growth of bacteria and promote the production of DQ90C substance can be appropriately added.

As the culture method, the aerobic liquid culture method is most suitable as in the generally used antibiotic production method. A suitable temperature for culturing is 20 to 30 ° C., but in most cases, culturing is performed at around 27 ° C. The production of the DQ90C substance varies depending on the medium and the culture temperature, but in liquid culture, the maximum accumulation is usually reached in 1 to 3 days. Thus, the target substance is isolated and purified from the culture in which the DQ90C substance has accumulated. Method for Purifying DQ90C Substance In collecting the DQ90C substance obtained from the present invention from a culture, a conventional separation means utilizing its properties, for example, filtration, centrifugation, dialysis, concentration, drying, freezing,
Adsorption, desorption, a method utilizing the difference in solubility in various organic solvents, means such as chromatography and the like can be used alone or in combination to extract and purify. For example,
The synthetic adsorbent HP-20 (manufactured by Mitsubishi Chemical Co., Ltd.), ODS column chromatography, MPLC, and HPLC can be appropriately combined and carried out. The DQ90C substance thus obtained had the following physicochemical properties, and as a result of analysis of various spectrum data, it was found to have the chemical structure represented by the above formula (I).

Physicochemical properties (1) Appearance: White powder (2) Melting point: 152-154 ° C. (3) Molecular formula: C ₂₈ H ₃₇ N ₃ O ₅ (4) High resolution mass spectrum (m / z): 496 .2817 (M + H) ⁺ measured value: 496.2813 calculated value (5) specific rotation: -12.0. (C = 0.025, methanol) (6) Solubility: Soluble in methanol and dimethyl sulfoxide, but insoluble in water and chloroform. (7) Ultraviolet absorption spectrum λ max nm (in methanol): 260 (1000), 269 (1400), 278 (1500)

(8) Infrared absorption spectrum (KBr disk method) Shown in FIG. : 3277, 2960 to 2850, 1736, 1635, 1540 to 1440, 1388, 1216 (cm ⁻¹ ) (9) ¹ H NMR spectrum (500 MHz, heavy dimethyl sulfoxide). Shown in FIG. (10) ¹³ C NMR spectrum (125 MHz, heavy dimethyl sulfoxide) Shown in FIG.

Biological Activity of DQ90C Substance Rat Adrenal Medullary Cell P of DQ90C Substance According to the Present Invention
The neurite regeneration effect on C-12 cells will be described. First, the PC-12 cells were placed in a Dulbecco's modified Eagle medium containing 10% fetal bovine serum according to a conventional method, and 37
The cells were grown to a semi-confluent state at 5 ° C. in a 5% carbon dioxide atmosphere. This is then mixed with 50 ng / ml NGF
And in Dulbecco's modified Eagle's medium containing 3% fetal bovine serum, a cell concentration of 1/2 to 1/5, preferably 1 /
The cells were subcultured at a concentration of 4 cells and cultured at 37 ° C. in a 5% carbon dioxide gas atmosphere for 24 hours. Then, the NGF-treated cells were
The sample was added by suspending it in Dulbecco's modified Eagle medium containing 1% fetal bovine serum, seeding it on a 24-well collagen-coated plate. After 24 hours, morphological changes of cells were observed by a microscope and evaluated.

As a result, the effective neurite regeneration activity concentration of the DQ90C substance on PC-12 cells was 0.05 to
It was 0.5 μg / ml. Since the DQ90C substance of the present invention has a neurite regenerating effect on rat adrenal medullocytoma PC-12 cells, it is considered to be useful as a therapeutic agent for neuropathy, for example, an anti-dementia drug.

INDUSTRIAL APPLICABILITY According to the present invention, a novel compound DQ90C having a neurite regeneration action and useful as a therapeutic agent for neuropathy, for example, an anti-dementia drug could be provided. Next, the present invention will be described by way of examples.

[0017]

EXAMPLES Example 1 1) Preparation of Seed Mother 1 The medium used was prepared by dissolving the components having the following composition in 1 liter of water and adjusting the pH to 7.2. Soluble starch 10.0 g Molasses 10.0 g Polypeptone 10.0 g Meat extract 10.0 g Dispensed 15 ml of the above medium into a large test tube and sterilized, and then sterilized Streptomyces Halstein 2723-SV2 strain (hereinafter DQ-90 strain) from slant. 1 platinum loop inoculation, 27 ℃
Seed mother 1 was cultured for 1 day with shaking.

2) Preparation of Seed Mother 2 The medium used was prepared by dissolving the components having the following composition in 1 liter of water and adjusting the pH to 7.0. Glycerin 20.0 g Molasses 10.0 g Casein 5.0 g Polypeptone 1.0 g Calcium carbonate 4.0 g 100 ml of the above medium was added to a 500 ml Erlenmeyer flask (7
Bottle), sterilized, and then seed seed 1 (1 ml each)
Was inoculated and cultured with shaking at 27 ° C. for 1 day. 3) Culture The culture medium used is the one shown in 2). This medium 2
7 liters were placed in a 50 liter jar fermenter, sterilized, and then seeded with the seed mother 2 (total of 7), and stirring speed was 200 rpm. The culture was carried out with aeration and stirring at 27 ° C. for 1 day under the conditions of an aeration rate of 30 liters / minute.

4) Purification of DQ90C substance After culturing under the above-mentioned conditions, the culture supernatant obtained by centrifuging 80 liters of the culture broth was adsorbed on a "Diaion HP-20" (manufactured by Mitsubishi Chemical Corporation) column. This with water, then 30%
After washing with methanol, 70% methanol was passed through, and the obtained 70% methanol solution was concentrated to dryness. The crude active fraction thus obtained was applied to an MPLC LiChroprep C18 (MERCK) column to obtain a fraction containing the DQ90C substance.
After further concentration of this fraction, HPLC TSKgel-80TM ODS (TOS
(OH) column to obtain 3 mg of DQ90C substance.

[Brief description of the drawings]

FIG. 1 is an infrared absorption spectrum of DQ90C substance.

FIG. 2 is a 500 MHz ¹ H NMR spectrum of a DQ90C substance in a heavy dimethyl sulfoxide solution.

FIG. 3 is a 125 MHz ¹³ C NMR spectrum of DQ90C material in a solution of heavy dimethyl sulfoxide.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Reference number within the agency FI technical display location C12R 1: 465) C07M 7:00 (72) Inventor Nobuaki Tsuge 1-Mirikaeicho, Higashiosaka City, Osaka Prefecture 5-7 House Food Co., Ltd. (72) Inventor Yoji Hirao 1-5-7 Mikitei-cho, Higashiosaka City, Osaka Prefecture House Food Co., Ltd.

Claims (3)

[Claims] 1. A novel substance DQ90 represented by the formula (I).
C. Embedded image 2. A neurite regenerating factor comprising the compound DQ90C according to claim 1 as an active ingredient. 3. A DQ90C belonging to the genus Streptomyces
A method for producing a DQ90C substance, which comprises culturing a substance-producing bacterium and collecting the DQ90C substance from the culture.