JP3053867B2 - Compound MS-444 - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a compound MS-444 produced by a microorganism belonging to the genus Micromonospora and having a vasodilating action.

BACKGROUND ART The compound MS-444 of the present invention is a novel substance, and there is no report of a substance having the same structure.

As substances having a structure similar to MS-444, the following two substances are described in Journal of Chemical Research, Synopsis.
opses) pp. 300-301, 1979 and Courte Radi de
Compasses Rendus des Seances de l'Academie des Sci
ence, Serie C), vol. 274, pp. 1410-1412, 1972, but there is no report on its biological activity.

DISCLOSURE OF THE INVENTION In order to obtain a novel vasodilator, a study was conducted on a large number of microorganism products, and as a result, a substance obtained by culturing a microorganism newly isolated from soil in a culture medium was found to be a vasodilator. It has been found that this is a novel physiologically active substance having an action.

According to the present invention, compound MS-444 represented by the following formula and having a vasodilator action can be provided.

Hereinafter, the physicochemical properties of the compound MS-444 of the present invention will be described in detail.

Properties: yellow powder Molecular weight: 230 Molecular formula: C ₁₃ H ₁₀ O ₄ mass spectrometry (EIMS method): m / z: 230 (M ⁺ ) High-resolution EIMS spectrum: Observed value 230.0596 Calculated value 230.0579 (C ₁₃ H ₁₀ O ₄ ) Melting point: 155 ° C (decomposition) Ultraviolet absorption spectrum (in methanol): λmax (nm):
203 (ε = 17,000), 245 (ε = 12,000), 319 (ε = 6,00
0), 387 (ε = 3,800) Infrared absorption spectrum (KBr tablet method): ν (cm ⁻¹ ): 338
8,1620,1604,1564,1473,1327,1300,1273,1257 ¹ H-NMR spectrum (400 MHz, acetone-d ₆ , internal standard TM
S): δ (ppm): 12.60 (1H, s), 8.28 (1H, s), 7.53 (1H, t, J
= 1.7Hz), 7.13 (1H, d, J = 8.8Hz), 6.69 (1H, dt, J = 8.
8,0.9 Hz), 3.93 (2H, dd, J = 1.7, 0.9 Hz), 2.68 (3H, s) ¹³ C-NMR spectrum (100 MHz, acetone-d ₆ , internal standard T
MS): δ (ppm): 189.7 (s), 159.2 (s), 158.6 (s), 147.
8 (s), 138.0 (d), 128.0 (s), 124.6 (d), 122.9
(S), 118.2 (s), 117.5 (s), 116.4 (d), 20.4
(T), 14.6 (q) Color reaction: Positive to color reaction with 50% sulfuric acid, iodine or anisaldehyde Solubility: Soluble in acetone, ethyl acetate, methanol, chloroform, hexane and dimethyl sulfoxide, carbon tetrachloride and Insoluble in water The physicochemical data were measured with the following equipment.

Mass spectrum: Hitachi M-80B mass spectrometer Ultraviolet absorption spectrum: Shimadzu Corporation UV-2200 spectrophotometer Red external absorption spectrum: JEOL JIR-RFX3001 infrared spectrophotometer NMR spectrum: Bruker AM400 nuclear magnetic resonance apparatus Melting point: Yanagimoto Mfg. Micro melting point analyzer From the above data, MS-444 was found to be a novel compound.

Next, MS-444 with various developing agents of the following experiments 1 to 3 was used.
Table 1 shows the Rf values of the thin layer chromatography. Detection was performed by an iodine reaction or an ultraviolet irradiation method at 253.7 nm.

Experiment 1 thin layer; Kieselgel 60F ₂₅₄ (Merck, Art.5628) developing solvent; chloroform: acetone = 95: 5 deployment method; room temperature rise method, for 15-30 minutes Experiment 2 thin layer; Kieselgel 60F ₂₅₄ (Merck Ethyl acetate: hexane = 1: 1 Development method: room temperature, rising method, 15 to 30 minutes Experiment 3 Thin layer; RP-18F ₂₅₄ s (Merck, Art. 13724) Development solvent ; 60% acetone / water developing method; room temperature, rising method, 15-60 minutes Next, the vasodilatory effect of MS-444 will be described using test examples.

Test Example 1 Vascular contraction-suppressing action in rabbit thoracic aorta specimen The abdominal midline of an inbred white rabbit (male, weighing 2 to 3 kg) was incised, the aorta was cut out from the abdomen at a length of about 2 to 2.5 cm, and the width was 3 to 3 cm. A 4 mm spiral strip was made. Both ends were ligated with silk thread, the lower end was connected to a fixed bar, the upper end was connected to a tension transducer (TB-612T, manufactured by Nihon Kohden), and suspended at an initial tension of 1.5 g. A Krebs-Henzeleit solution (NaCl 6.92 g / l, KCl
_{0.35g / l, MgSO 4 · 7H} 2 O 0.29g / l, CaCl 2 · 2H 2 O 0.37g / l, K
H ₂ PO ₄ 0.16 g / l, NaHCO ₃ 2.1 g / l, glucose 1.0 g / l] and passed 95% O ₂ and 5% CO ₂ gas. Specimens were allowed to stabilize for 1 to 2 hours before they were used for experiments.

As a vasoconstrictor, potassium chloride was added to the Magnus tube to a final concentration of 20 mM. The induced contraction reaction was isometrically recorded on a polygraph (Nihon Kohden AM-6000) via a tension transducer. MS-44
4 was dissolved in dimethyl sulfoxide so as to be 30 mg / ml, and appropriately diluted with Krebs-Hanselite solution,
Added to the Magnus tube 30 minutes prior to potassium chloride application.

Table 2 shows the ratio of shrinkage height (shrinkage ratio) when MS-444 was added at each concentration, assuming that the shrinkage height without MS-444 was 100%.

As is evident from Table 2, it was confirmed that MS-444 has an action of suppressing contraction of the isolated blood vessel.

MS-444 is obtained by culturing a microorganism belonging to the genus Micromonospora and having the ability to produce MS-444 in a medium, producing and accumulating MS-444 in a culture solution, and collecting MS-444 from the culture. be able to.

Examples of the MS-444 producing microorganism include micromonomers such as a wild strain or a mutant strain obtained by mutating a wild strain by an artificial mutation method, for example, ultraviolet irradiation, X-ray irradiation, treatment with a mutagenic agent, or a naturally mutated mutant strain. MS belonging to the genus Spora
Any microorganism can be used as long as it has -444 production ability. As a particularly preferred example, Micromonospora sp.
pora sp.) NK-3091 strain (hereinafter referred to as NK-3091 strain).

The characteristics of the NK-3091 strain in morphology, growth on various media, and physiological properties are as follows.

I. Morphology The NK-3091 strain is a commonly used agar medium, having a septum and forming a branched base hypha. No formation of aerial hyphae and no characteristic division of the underlying hyphae were observed.
Also, no formation of sporangia and sclerotia is found.

One spore is formed in a spore stalk that is simply branched from the basal hypha, and its morphology is spherical. Mature spores have a size of 0.
5 to 0.7 μm, the surface of which may be smooth or have wart-like projections.

II. Growth state on various media The NK-3091 strain shows normal or vigorous growth on commonly used synthetic and natural media, and the underlying mycelium shows orange or brown. The medium may produce a brown colored soluble pigment.

The characteristics of growth and color when cultured on various media at 28 ° C. for 14 days are shown below. The color display is Color Harmon
y Color classification according to Manual (Container Corporation of America) was followed.

1. Glucose-asparagine agar medium; growth; normal color of base mycelium; dark crag tan (4pg) soluble pigment; slight production, light brown 2. Glycerol-asparagine agar medium growth; slightly poor color of base mycelium; Yellow (3ia) Soluble pigment; None 3. Sucrose / nitrate agar medium Growth; Slightly poor Color of base mycelium; Coctan (4ie) Soluble pigment; Slightly produced, light brown 4. Starch / inorganic salt agar medium Growth; Good Color of base mycelium; sepia brown (3pn) soluble pigment; slightly produced, light brown 5. Tyrosine agar medium Growth; normal Color of base mycelium; bright melon yellow (3ia) soluble pigment; none 6. Nutrient agar medium Growth; poor color of base mycelium; tile red (5ne) soluble pigment; slightly produced, light brown 7. Malt extract / yeast extract agar medium Growth; normal color of base mycelium Dark brown (4pn) soluble pigment; production, dark brown 8. Oatmeal agar medium Growth; somewhat good Color of base mycelium; orange rust (4pe) Soluble pigment; slight production, light brown III. Physiological properties NK-3091 The physiological properties of the strain are shown below. The growth temperature range describes the results after culturing for 6 days, and the others describe the results after culturing for 2 to 3 weeks.

(1) Utilization of carbon source; A basal medium having the following composition was used.

The NK-3091 strain is L-arabinose, D-xylose,
D-glucose, sucrose and raffinose assimilate, but D-fructose, rhamnose, inositol,
D-mannitol does not assimilate.

(2) Action on milk: Coagulation, liquefaction (3) Starch hydrolysis action: Yes (4) Growth temperature range: 9 ° C to 40 ° C (5) Melanoid pigment formation: Predetermined medium (tyrosine agar medium, (Peptone yeast iron agar medium).

(6) Liquefaction action of gelatin: Yes IV. Cell wall composition Alanine, glutamic acid, meso, etc. were obtained from the cell wall prepared by the method of Kawamoto et al. [Journal of Bacteriology (J. Bacteriol.), 146 , 527 (1981)]. -Diaminopimelic acid and glycine were detected.

As described above, spores are formed only on the basal hypha and the cell wall is type II (meso-diaminopimelic acid, glycine)
Therefore, this strain is classified into the genus Micromonospora among actinomycetes. (Edited by the Actinomycetes Society of Japan, Identification Experiment Method for Actinomycetes, 1988 edition) Therefore, this strain was designated as Micromonospora sp .
omonospora sp.) Named NK-3091 and filed with the National Institute of Microbial Industry, National Institute of Advanced Industrial Science and Technology on October 29, 1991
No. 3623 (FERM BP-3623).

In culturing the microorganism, a usual culturing method used for culturing actinomycetes is applied. As the medium to be used, any of a natural medium and a synthetic medium can be used as long as the medium appropriately contains a carbon source, a nitrogen source, an inorganic substance, and the like which can be used by bacteria.

As the carbon source, glucose, sucrose, stabilose, starch, dextrin, mannose, maltose, carbohydrates such as molasses, citric acid, malic acid, acetic acid,
Organic acids such as fumaric acid, alcohols such as methanol and ethanol, hydrocarbons such as methane, ethane, propane and n-paraffin, amino acids such as glutamic acid and glycerol are used.

As a nitrogen source, ammonium salts such as ammonium chloride, ammonium sulfate, ammonium nitrate and ammonium phosphate, aspartic acid, glutamine, cystine,
Amino acids such as alanine, urea, peptone, meat extract,
Yeast extract, dried yeast, corn steep liquor, soybean flour, soluble vegetable protein, cottonseed meal, soybean casein, casamino acid, pharmamedia and the like are used.

Inorganic substances include potassium hydrogen phosphate, potassium dihydrogen phosphate, sodium dihydrogen phosphate, magnesium phosphate, magnesium sulfate, ferrous sulfate, manganese sulfate, copper sulfate, cobalt sulfate, zinc sulfate, calcium pantothenate, molybdenum Ammonium phosphate, aluminum potassium sulfate, barium carbonate, calcium carbonate, cobalt chloride,
Salt or the like is used.

In addition, if necessary, a substance that promotes the growth of cells or the productivity of MS-444, such as vitamins such as thiamine, can be added to the medium.

If the microorganism used requires a specific substance, it is necessary to add what is needed for growth.

The culture is performed by shaking culture, aeration stirring culture, etc.
The reaction is carried out at a pH of about neutral at 15 to 35 ° C.

After 3 to 15 days of culture, the accumulation of MS-444 reaches a maximum.

When the MS-444 accumulated in the culture is isolated and collected from the culture, a method of collecting a normal microorganism-producing physiologically active substance from the culture is applied. That is, acetone,
Extraction of bacterial components with organic solvents such as methanol, removal of bacterial cells by filtration, centrifugation, etc., adsorption resin, silica gel, silanized silica gel, reverse layer silica gel, aluminum, cellulose, diatomaceous earth, magnesium silicate, gel filtration MS-444 can be isolated by adsorption / desorption treatment of the active substance by column chromatography or thin layer chromatography using an agent, an ion exchange resin, or the like, or by partitioning with an appropriate solvent system.

One example of isolating MS-444 from a culture solution is as follows.

The cells are separated by filtering or centrifuging the culture. The obtained cells are treated with a suitable solvent such as methanol or acetone to obtain a cell extract.
The solvent is removed by concentrating the obtained bacterial cell extract under reduced pressure to obtain an aqueous solution. Next, a water-immiscible solvent such as ethyl acetate or butyl acetate is added to the aqueous solution to extract the active substance. After the extract is concentrated under reduced pressure, silica gel column chromatography is repeated to purify the active substance. As an elution solvent, an appropriate solvent such as chloroform, ethyl acetate, methanol, acetone or the like is used alone or in combination. The eluate containing the active substance is concentrated under reduced pressure, and then the active substance is further purified by repeated reverse _iABL silica gel column chromatography. As the elution solvent, acetone,
A suitable solvent such as methanol or water is used alone or in combination. The fraction containing MS-444 is collected, concentrated and dried under reduced pressure to give a yellow powder of MS-444.

Detection of MS-444 during the purification step is performed by thin layer chromatography using silica gel containing a fluorescent agent (Kieselgel F ₂₅₄ , manufactured by Merck) by an iodine reaction or an ultraviolet irradiation method at 253.7 nm.

BEST MODE FOR CARRYING OUT THE INVENTION Example 1 As an inoculum, Micromonospora sp.
ospora sp.) NK-3091, as a first-class medium, glucose 1 g / dl, soluble starch 1 g / dl, bactotripton (manufactured by Difco) 0.5 g / dl, yeast extract 0.5 g / dl, meat extract 0.3 g
A medium having a composition of / dl and calcium carbonate 0.2 g / dl (pH 7.2) was used. One platinum loop of the inoculum was inoculated into 10 ml of the above-mentioned type I medium placed in a thick tester, and cultured with shaking at 28 ° C for 5 days (type I culture).

10 ml of the culture solution obtained by the first type culture was inoculated into 50 ml of the second type medium in a 300 ml Erlenmeyer flask. The composition of the second type medium is the same as the composition of the first type medium. The second seed culture was performed at 28 ° C. for 2 days.

50 ml of the obtained second seed culture was inoculated into 50 ml of the main fermentation medium in a 300 ml Erlenmeyer flask. As main fermentation medium, glucose 2.5g / dl, corn steep liquor 1.5
g / dl, Soluble vegetable protein 1g / dl, cottonseed oil 0.5g / dl, cobalt chloride hexahydrate 1mg / dl, magnesium phosphate octahydrate 50mg / dl (pH 7.0) Using. The main fermentation culture was performed by shaking culture at 28 ° C. for 6 days.

The cells were obtained by centrifuging 15 liters of the fermentation end solution. 7.5 liters of the separated cells were added to methanol, and the mixture was stirred and filtered. After concentrating the obtained methanol extract under reduced pressure to obtain an aqueous solution, 1500 ml of ethyl acetate was added to 50 ml of an aqueous solution.
Extraction was performed three times by adding 0 ml each. A red-brown solid (3.35 g) obtained by concentrating the ethyl acetate layer under reduced pressure was dissolved in a small amount of acetone, a small amount of diatomaceous earth was added, and the residue was dried under reduced pressure using chloroform containing 1% methanol. 1 liter packed silica gel column Wako gel C-20
0 (manufactured by Wako Pure Chemical Industries, Ltd.), and the active substance was eluted with a solvent (3 liter) having the same composition.

Collect all the eluate and concentrate to dryness under reduced pressure to give 2.53 g
A red-orange solid was obtained. This substance was dissolved in a small amount of acetone, and a small amount of diatomaceous earth was added.
Silica gel column packed with chloroform containing 5% acetone Wakogel C-200 (Wako Pure Chemical Industries) 5
The solution was placed on the top of 00 ml and eluted with a solvent (2 liters) of the same composition.

When the eluate was collected in 20 g portions, MS-444 was obtained from fraction No. 37.
Eluted at 84. After collecting these fractions, the same silica gel column chromatography was further repeated,
The obtained eluate was concentrated under reduced pressure and concentrated to dryness to obtain 377 m
g of a yellow powder were obtained. This was dissolved in a small amount of acetone, and a small amount of YMC-ODS gel 60-260 / 70 (manufactured by Yamamura Chemical Laboratory Co., Ltd.) was added and dried under reduced pressure. The gel was placed on the upper end of 200 ml and eluted with a solvent (1 liter) having the same composition.

When the eluate was collected in 10 g increments, MS-444 was obtained from fraction number 24.
Eluted at 28. Collect these fractions and further
YMC-ODS column chromatography was repeated, and the obtained eluate was concentrated under reduced pressure to dryness to obtain a yellow powder.
233 mg of MS-444 were obtained.

INDUSTRIAL APPLICABILITY According to the present invention, a compound MS-44 having a vasodilator effect
4 can be provided.

──────────────────────────────────────────────────続 き Continuing on the front page (51) Int.Cl. ⁷ Identification symbol FI // (C12N 1/20 C12R 1:29) (72) Inventor Yuzuru Matsuda Examiner 1-2-7, Nukii Minamimachi, Koganei-shi, Tokyo Uchida Junko (56) Reference US Pat. No. 4,585,760 (US, A) Phytochemistry, 26 (9), p. 2499-2500 (1987) Chem. Soc. C, 1967 (10), p. 949-952 (58) Field surveyed (Int. Cl. ⁷ , DB name) C07D 307/92 A61K 31/343 A61P 9/08 C12N 1/20 C12P 17/04 BIOSIS (DIALOG) CA (STN) REGISTRY (STN )

Claims (6)

(57) [Claims] 1. A compound MS-444 represented by the following formula: 2. A method for producing a compound according to claim 1, wherein the microorganism is a microorganism belonging to the genus Micromonospora having the ability to produce the compound according to claim 1 and the compound is obtained from the culture. . 3. The microorganism belonging to the genus Micromonospora is Micromonospora sp. NK-30.
The production method according to claim 2, which is 91 strains (FERM BP-3623). [4] a pharmaceutical comprising the compound according to the above [1]; 5. A vasodilator comprising the compound according to claim 1. 6. The microorganism Micromonospora sp.
omonospora sp.) NK-3091 strain (FERM BP-3623).