JPH10237044A - New physiologically active substance nk26588, and its production and application - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject new substance capable of strongly suppressing the production of nitrogen monoxide, extremely useful as a nitrogen monoxide production inhibitor with expected lower side effects. SOLUTION: This substance is a physiologically active substance NK26588 expressed by the formula, its tautomer or its pharmaceutically permissible salt, and exhibits the following physicochemical properties: color and shape, white powder; molecular formula, C19 H29 NO2 ; molecular weight, 303; FAB-MS (Positive) m/z, 304(M+H)<+> ; melting point, 120-121 deg.C; solubility, soluble in dimethyl sulfoxide, methanol, ethyl acetate and chloroform, and insoluble in water; Rf value of thin layer chromatography, 0.61 [silica gel thin layer; chloroform-methanol (9:1) eluent]; etc. NK26588 can be produced by culturing a NK26588-producing strain belonging to Streptomyces in a nutrient medium, and isolating and collecting form the culture mixture.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a novel physiologically active substance NK2658 used as a nitric oxide production inhibitor.
8, its production method and its use.

[0002]

BACKGROUND OF THE INVENTION For decades, nitroglycerin has been administered to humans as a vasodilator in the treatment of cardiovascular disease. Recently, nitroglycerin administered in this way
It was revealed that it is converted into nitric oxide, a pharmacologically active metabolite in the body. Nitric oxide is an important component of vascular endothelium-derived relaxing factors (EDRFs) and is formed enzymatically from arginine as a normal metabolite. ED
RFs have been actively studied recently as being involved in the regulation of blood flow and vascular resistance. It has been found that in vivo, macrophages and nerve cells also produce nitric oxide in addition to vascular endothelium. It has been clarified that nitric oxide produced by macrophages has cytotoxic and cytostatic effects, and that nitric oxide produced by nerve cells is involved in memory as a signal transmitter.

[0003] More recently, it has been established that the enzyme that produces nitric oxide from arginine is nitric oxide synthase, which is of two different types, constitutive and inducible. Constitutive enzymes are present in normal endothelial cells and neurons. Nitric oxide production by constitutive enzymes in endothelial cells is thought to play a role in normal blood pressure regulation. In addition, the production of nitric oxide by constitutive enzymes in neurons plays a role in synaptic transmission plasticity, plays a role in memory and learning, and is also involved in the development of pathological conditions in cerebral ischemia etc. Can be considered. On the other hand, it was revealed that inducible nitric oxide synthase is induced by activated macrophages, and endothelial cells or vascular smooth muscle cells are also induced by various cytokines or a combination of cytokines. Severely life-threatening hypotension, often observed in sepsis or cytokine-induced shocks, is thought to be largely or entirely due to overproduction of nitric oxide by inducible nitric oxide synthase .

[0004] Therefore, these nitric oxide production inhibitors are used in septic shock [The Lancet, 338, 1
Pages 555 to 1557 and 1557 to 1558 (19
91)], arthritis (Ann. Rheum. Dis., Vol. 51, p. 1219 (1992)), stroke [Neur
Ossence Letters, Vol. 147, 15
9 (1992) and Science, Volume 265,
1883 (1994)], cerebral ischemia [Neuropha]
rmacol. 35, 1447 (199
4)], Parkinson's disease [Proc. Natl. Aca
d. Sci. USA, 88, 6368 (199
1)], pain [Br. Pharmacol. , 102nd
198 (1991)] and insulin-dependent diabetes [FEBS Letters, 299, 103 (1992)].

As the nitric oxide production inhibitor, ^NG -methyl-L-arginine (NMMA) [The Lancet,
338, 1555-1557 and 1557-1
558 (1991)] and dexamethasone [Hyp
ertension, Vol. 22, p. 34 (1993) and Proc. Natl. Acad. Sci. USA,
Vol. 92, p. 3473 (1995)].

[0006]

NMMA suppresses the production of nitric oxide as a nitric oxide synthase inhibitor, and dexamethasone suppresses the production of nitric oxide as a transcription inhibitor of the nitric oxide synthase gene. However, these compounds do not have satisfactory side effects. Therefore, an object of the present invention is to provide a physiologically active substance having a chemical structure different from that of the conventional one, and having a nitric oxide production inhibitory action expected to reduce side effects, a method for producing the same, and a use thereof. is there.

[0007]

Means for Solving the Problems As a result of intensive studies, the present inventors have found that the novel physiologically active substance NK26588 or a tautomer thereof or a pharmaceutically acceptable salt thereof has an inhibitory effect on nitric oxide production. I found it. To summarize the present invention, the first invention of the present invention is a novel physiologically active substance NK26.
588, comprising the following formula (1):

[0008]

Embedded image And a physiologically active substance NK26588 or a tautomer thereof or a pharmaceutically acceptable salt thereof.

The physical and chemical properties of NK26588 are as follows. NK26588 of physicochemical properties (1) Color and shape: white powder (2) Molecular _{formula: C 19 H 29 NO 2 (} 3) Molecular weight: 303 FAB-MS (Positi
ve) m / z 304 (M + H) ⁺ (4) Melting point: 120-121 ° C. (5) Ultraviolet absorption spectrum: shown in FIG. 1 of the accompanying drawings. (6) Infrared absorption spectrum: shown in FIG. 2 of the accompanying drawings. (7) Hydrogen nuclear magnetic resonance spectrum: FIG. 3 of the accompanying drawings
Shown in (8) Carbon nuclear magnetic resonance spectrum: FIG. 4 of the accompanying drawings
Shown in (9) Solubility: dimethyl sulfoxide, methanol,
It is soluble in ethyl acetate and chloroform and insoluble in water. (10) Rf value of thin layer chromatography: 0.61 Using a thin layer of silica gel (Art. 5715 manufactured by Merck), chloroform-methanol (9:
1) was used.

NK26588 exhibits pyridinol-pyridinone tautomerism, and any tautomer is included in the present invention. For example, the tautomer of NK26588 is represented by the following formula (2)

[0011]

Embedded image

And the following equation (3)

[0013]

Embedded image

[0014] These are NK26588
, According to a conventional method, for example, under a condition of a weak alkali such as sodium carbonate or sodium hydrogen carbonate. NK26588 and its tautomers may be in the form of their pharmaceutically acceptable salts, and include, for example, alkali metals such as sodium and potassium, and salts with hydrochloric acid and sulfuric acid. . The second invention of the present invention relates to a method for producing a novel physiologically active substance NK26588, in which NK26588-producing bacteria belonging to the genus Streptomyces are cultured in a nutrient medium, and the culture is expressed by the above formula (1). Physiologically active substance NK2
6588 is separated and collected. Next, the bacteriological properties of the NK26588-producing strain with the strain number NA 26588 will be described.

1. Morphological properties Observation after 2 weeks at 27 ° C. shows that the aerial mycelium is simply branched, its tip is wavy, and no ring-shaped branch is observed.
Neither sporangia nor zoospores are found. The spore surface is smooth, the spores are cylinder-shaped and the size is 0.4-0.
6 × 0.8 to 1.4 μm. In addition, spores are formed in a chain of 10 or more. 2. Growth in various media Table 1 below shows the growth state of each medium at 27 ° C for 2 weeks.

[0016]

[Table 1]

[0017] 3. Physiological properties Growth temperature range: 20-32 ° C Nitrate reduction: negative Starch hydrolysis (starch / inorganic salt agar medium): positive Coagulation of skim milk: negative Peptoneization of skim milk: positive Generation of melanin-like pigment: positive

4. Utilization of carbon source (on Pridham-Godribe agar medium) L-arabinose + D-xylose + D-glucose + D-fructose + sucrose + inositol + L-rhamnose + raffinose + D-mannitol +

5. Diaminopimelic acid in cell wall LL-diaminopimelic acid. To summarize the above,
In this strain, the cell wall is LL-diaminopimelic acid, and the types of menaquinone are mainly MK-9 (H ₆ ) and MK-9.
−9 (H ₈ ). According to the method of the International Streptomyces genus project (abbreviated as ISP), the spore-forming mycelium forms in Section Spir.
spore surface, the color of mature hyphae is gray color (Gray color-series),
It produces melanin-like pigments and presents brown soluble pigments in the medium. In addition, the color of the underlying mycelium exhibits a light yellow brown.
As a carbon source, L-arabinose, D-xylose, D-
-Utilize glucose, D-fructose, sucrose, inositol, L-rhamnose, raffinose, D-mannitol.

Based on the above properties, Bergey's Manual of Defect Bacteriology, edited by R.E. Buffanan and N.E. Gibbons (Bergey's Manual of Deterministic Bacteriology)
(Eminitive Bacteriology), 8th edition, 1974
26588 strain was found to belong to the genus Streptomyces. Therefore, the bacterium was used in Streptomyces sp. NA 2658.
No. 8 was named. The strain has been deposited with the National Institute of Bioscience and Human Technology, FERM P-16005. The NA26588 strain is liable to change its properties as seen in other actinomycetes. For example, NA
Even if the mutant strain is derived from the 26588 strain (naturally occurring or induced by irradiation, etc.), any Streptomyces bacterium having the ability to produce NK26588 can be used in the method of the present invention.

In the method of the present invention, the above-mentioned bacteria are cultured in a medium containing nutrients that can be used by ordinary microorganisms. As a carbon source, glucose, glycerin, galactose,
Syrup, dextrin, sucrose, starch, molasses,
Animal and vegetable oils can be used. As the nitrogen source, soybean flour, wheat, wheat germ, corn steep liquor, meat extract, peptone, yeast extract, ammonium sulfate, sodium nitrate, urea and the like can be used. In addition, if necessary,
It is advantageous to add inorganic salts capable of producing sodium, cobalt, chlorine, sulfuric acid, phosphoric acid, and other ions. In addition, organic and inorganic substances that assist the growth of bacteria and promote the production of the physiologically active substance NK26588 can be appropriately added.

As the culture method, a culture method under aerobic conditions,
In particular, the submerged culture method is suitable. The temperature suitable for culture is 15
３７37 ° C., but most often around 26-30 ° C. The production of the physiologically active substance NK26588 varies depending on the culture medium and culture conditions, but the accumulation reaches the maximum usually between 1 and 10 days in both shaking culture and tank culture. When the accumulated amount of the physiologically active substance NK26588 in the culture reaches the maximum, the culture is stopped and the target substance is isolated from the culture solution.

When the NK26588 obtained by the present invention is collected from the culture solution, it can be extracted and purified by appropriately combining ordinary separation means utilizing its properties. That is, after the culture solution is filtered, the cells can be extracted with an organic solvent such as methanol or acetone, then concentrated under reduced pressure, and further extracted with a water-immiscible organic solvent such as ethyl acetate.

In addition to the above-mentioned methods, known methods used for collecting fat-soluble substances, for example, adsorption chromatography, gel filtration chromatography, scraping from thin-layer chromatography, high-performance liquid chromatography, etc., are appropriately combined, or NK2 by repeating
6588 can be isolated purely.

Pharmaceutically acceptable salts of NK26588 and its tautomers can be produced by known methods, for example, by treating with a solution containing sodium hydroxide, potassium hydroxide and hydrochloric acid, sulfuric acid and the like. Can be obtained by

The third invention of the present invention relates to a nitric oxide production inhibitor comprising NK26588 or a pharmaceutically acceptable salt thereof and a pharmaceutical additive. Furthermore, the present invention relates to an inhibitor of the production of inducible nitric oxide synthase by the compound of the present invention. The pharmaceutical additive is not particularly limited, and commonly used additives can be used.

The proportion of the active ingredient (NK26588) in the composition cannot be said unconditionally because it varies depending on the dosage form and the like, but it can be used in a wide range from about 0.05 to 99%. About 1 to 50%, and about 1% to 60% for other preparations. The balance is a pharmaceutical additive.

NK26588 suppresses the production of nitric oxide as shown in the following test examples, but shows no toxicity. Therefore, NK26588 is extremely useful as a nitric oxide production inhibitor.

Normally, an effective amount of NK26588 is orally administered to warm-blooded animals including humans, or parenteral administration such as intravenous, intradermal or intramuscular administration, thereby suppressing the production of nitric oxide in the living body. can do. The dose varies depending on the subject to be administered, the administration route, and the like.
0.05 to 150 mg / kg / day, 0.5 to 100 mg
/ Kg / day, preferably 1 to 50 mg / kg / day.

Examples of the preparation for administration include conventionally used dosage forms. In the case of oral administration, tablets, granules, capsules and the like molded together with a pharmaceutical excipient such as a common excipient such as starch are used. In the case of parenteral administration, ordinary injections formulated with pharmaceutical additives such as physiological saline, dissolving agents and the like are used.

[0031]

As described in detail above, the present invention provides a novel physiologically active substance NK26588, which strongly inhibits the production of nitric oxide. Therefore, it is extremely useful as a nitric oxide production inhibitor.

[0032]

The NK26588 of the present invention will now be described by way of examples.
Production Examples and Preparation Examples are shown below. Example 1 As a primary seed medium and a secondary seed medium, glycerin 2.0%, glucose 1.0%, peptone (manufactured by Far East)
0.5%, meat extract 0.3%, yeast extract 0.3%, dipotassium hydrogen phosphate 0.05%, magnesium sulfate 0.0
5%, calcium carbonate 0.2%, silicone 0.01% as an antifoaming agent and Toho No. A medium consisting of 1 0.01% was used. The medium before sterilization was adjusted to pH 7.0 before use.

100 ml of the primary seed medium was dispensed into a 500 ml Erlenmeyer flask, sterilized at 120 ° C. for 20 minutes, and added to Streptomyces sp. NA 26588 strain (F
ERM P-16005) was inoculated with 1 to 2 platinum loops of the slant culture, and was inoculated with a rotary shaker at 27 ° C. and 200 rpm.
Culture was performed for one day to obtain a primary seed culture.

100 ml of the secondary seed medium was dispensed into a 500 ml Erlenmeyer flask, sterilized at 120 ° C. for 20 minutes, and transplanted with 2 ml of the primary seed culture at 200 ° C. at 27 ° C.
For 2 days using a rotary shaker for 2 minutes to obtain a secondary seed culture.

As a production medium, dextrin (Akadama N
o. 3) 2.0%, glucose 1.0%, galactose 1.0%, corn steep liquor (manufactured by Nissha).
A medium composed of 0%, 1.0% Pharmamedia, 0.05% magnesium sulfate, 0.2% calcium carbonate, and 0.0001% cobalt chloride was used. The medium before sterilization was adjusted to pH 7.0 before use.

20 liters of the production medium was charged into a 30 liter jar fermenter, steam-sterilized at 120 ° C. for 20 minutes, and then the secondary seed culture 20 obtained by the above method was used.
0 ml aseptically transplanted, 27 ° C, aeration rate 6 liters /
Culturing for 3 days while stirring at 210 rpm. After the completion of the culture, 18 liters of the culture solution was filtered to separate the culture filtrate and the cells.

To 3.5 kg of the wet cells, 10.5 liters of methanol was added, and the mixture was stirred well to extract the active ingredient, which was concentrated to remove methanol, and then the pH was adjusted to 6 with 6N hydrochloric acid.
And extracted with an equal volume of ethyl acetate. The ethyl acetate extract was concentrated to obtain 7.6 g of a brown oily substance. This crude substance was suspended in chloroform: methanol (25: 1) and then applied to an 800 ml column of silica gel 60 (Art. 7734 manufactured by Merck) previously filled with the same mixed solvent to elute the active ingredient with the same mixed solvent. And concentrated to dryness to give 150.8 mg of a yellow oil.

This crude substance was suspended again in chloroform: methanol (25: 1), applied to a silica gel 60/60 ml column previously filled with the same mixed solvent, and the active ingredient was eluted with the same mixed solvent and concentrated to dryness. This gave 74.1 mg of a pale yellow oil.

This crude substance was dissolved in 2 ml of methanol,
2 g of YMC-GEL (ODS-A60-200 / 60, manufactured by Yamamura Chemical Laboratory) was added and concentrated to dryness. Next, this was suspended in 50% methanol and then applied to a 135 ml column of YMC-GEL previously filled with 50% methanol, and the active ingredient was eluted by a linear gradient elution method of 50% methanol to 95% methanol, and concentrated. After removing methanol and freeze-drying, 14.8 mg of pure NK26588 white powder was obtained.

Using pure NK26588, an ultraviolet absorption spectrum, an infrared absorption spectrum, a hydrogen nuclear magnetic resonance spectrum and a carbon nuclear magnetic resonance spectrum were measured. These spectra are as shown in FIG. 1, FIG. 2, FIG. 3 and FIG.

Example 2 30 parts by weight of NK26588, 120 parts of crystalline lactose, 147 parts of crystalline cellulose and magnesium stearate 3
The part was tableted with a V-type mixer to obtain a tablet of 300 mg per tablet.

[0042]

[Test Examples] Test examples show that NK26588 has a nitric oxide production inhibitory effect and does not show toxicity.

Test Example 1 Inhibitory Effect of NK26588 on Nitric Oxide Production The inhibitory action of NK26588 on nitric oxide production was examined using macrophage cells. The method is Dennis
J. Stuhr and Michael A. Ma
rletta [Cancer RES. , 47th
Vol., 5590 (1987)].
First, J774.1 cells, a macrophage cell line, were seeded in wells and cultured for 2-3 hours to allow the cells to adhere to the wells. A test drug was added to the culture solution, and then endotoxin and interferon-γ were added as inducers of inducible nitric oxide synthase. 4 more in this state
After culturing for 8 hours, inducible nitric oxide synthase was induced. After 48 hours, it was determined by measuring the NO ^2-in the culture supernatant nitric oxide amount produced by the cell.
The amount of NO ²⁻ is determined by Laura G. et al. The method of Green et al. [A
nal. Biochem. 126, 131 (1
982)]. As a result, an IC ₅₀ value was determined from the volume-response curve, and the value was shown. In this assay, pure NK26588 inhibited nitric oxide production by 50% at a concentration of 2.0 μg / ml.

Test Example 2 Toxicity of NK26588 NK26588 was administered intraperitoneally to mice and the toxicity was examined. No toxicity was observed even at a dose of 100 mg / kg.

[Brief description of the drawings]

FIG. 1 shows an ultraviolet absorption spectrum of a 12.9 μg / ml methanol solution of NK26588.

FIG. 2 shows an infrared absorption spectrum of NK26588 in a potassium bromide tablet.

FIG. 3: 5 measured in heavy methanol of NK26588
1 shows a 00 MHz hydrogen nuclear magnetic resonance spectrum.

FIG. 4: 1 measured in heavy methanol of NK26588
1 shows a 25 MHz carbon nuclear magnetic resonance spectrum.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification code FI (C12N 1/20 C12R 1: 465) (C12P 17/12 C12R 1: 465)

Claims (6)

[Claims] [Claim 1] The following formula (1) NK26588 or a tautomer thereof or a pharmaceutically acceptable salt thereof, which is a compound represented by the formula: 2. NK2658 belonging to the genus Streptomyces
8 producers in a nutrient medium and NK2
The method for producing a physiologically active substance NK26588 according to claim 1, wherein 6588 is collected. 3. A nitric oxide production inhibitor comprising the NK26588 according to claim 1, or a tautomer thereof or a pharmaceutically acceptable salt thereof, and a pharmaceutical additive. 4. An inducible nitric oxide synthase production inhibitor comprising the NK26588 or the tautomer or a pharmaceutically acceptable salt thereof according to claim 1, and a pharmaceutical additive. 5. A pharmaceutical comprising the NK26588 according to claim 1, or a tautomer thereof or a pharmaceutically acceptable salt thereof, and a pharmaceutical additive. 6. Streptomyces sp. ( Strep)
tomyces sp. ) NA26588 strain or a mutant thereof.