JP2954700B2 - Compound MS-347 - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a novel bioactive substance having a vasodilatory effect
MS-347.

2. Description of the Related Art As a substance having a similar structure to the compound MS-347 of the present invention, Aspergillus sydow (Aspergillus sydow)
i) produces Sydowinin B [Agricultural and biological chemistry
Biological Chemistry) 39 , 2337-2340 (1975)], but the literature does not describe its biological activity. Further, Aspergillus used in the present invention.
SP SPC16640 is not only MS-347, Sydowinin
B also produces and accumulates, but Sydowinin B has no vasodilator effect.

SUMMARY OF THE INVENTION An object of the present invention is to provide a novel bioactive substance having a vasodilatory effect.

Means for Solving the Problems The present inventor has found that a substance obtained by culturing a microorganism newly isolated from soil is a novel substance having a vasodilatory effect, and completed the present invention.

According to the present invention, the following structural formula Can be provided. Hereinafter, the compound is referred to as MS-347.

MS-347 can be obtained by culturing a microorganism belonging to the genus Aspergillus.

 Hereinafter, the present invention will be described in detail.

 The physicochemical properties of MS-347 are as follows.

(1) Properties: yellow powder (2) Molecular weight: 316 (3) Molecular formula: C ₁₆ H ₁₂ O ₇ (4) Elemental analysis Analytical value C: 60.57% H: 3.78% Calculated value as C ₁₆ H ₁₂ O ₇ C: 60.76% H: 3.83% (5) High-resolution EIMS spectrum (m / z) Analytical value: 316.0594 (M ⁺ ) Calculated value as C ₁₆ H ₁₂ O ₇ : 316.0582 (6) Melting point: 253-256 ° C. (7) UV absorption spectrum (measured in acetonitrile) λmax (nm) 344 (ε3,600), 280 (ε27,000), 216
(Ε19,000) No change due to acidity or alkalinity (8) Infrared absorption spectrum (measured by KBr method) ν (cm ⁻¹ ) 3473,1732,1655,1595,1473,1053 (9) EIMS spectrum (m / z ) 316 (M ⁺ ), 300,284,268,258 (10) ¹³ C-NMR spectrum (100 MHz, DMSO-d ₆ ) δ (ppm) 180.0 (s), 166.5 (s), 159.8 (s), 1
59,4 (s), 154.9 (s), 153.2 (s), 138.4 (d), 12
5.7 (d), 112.3 (s), 108.9 (d), 108.2 (s), 104.
5 (d), 62.2 (t), 56.7 (s), 56.0 (d), 52.6
(Q) (11) ¹ H-NMR spectrum (400 MHz, DMSO-d ₆ ) δ (ppm) 12.03 (1 H, br.s), 7.22 (1 H, dd, J = 9.9,
3.8Hz), 7.03 (1H, d, J = 1.3Hz), 6.94 (1H, dd, J = 9.9,
1.5Hz), 6.81 (1H, d, J = 1.3Hz), 5.55 (1H, t, J = 5.8H)
z), 4.58 (2H, d, J = 5.8Hz), 4.34 (1H, dd, J = 3.8,1.5H
z), 3.74 (3H, s) (12) Color reaction: Positive for iodine and anisaldehyde reactions, Gibbs reaction (13) Solubility Solubility: Benzene, chloroform, methanol, n-butanol, ethyl acetate, acetone Insoluble in dimethyl sulfoxide: hexane, water MS-347 was found to be a novel compound from the above data.

 The above data was measured with the following equipment.

Elemental analysis: Yanagimoto CHN coder MT-5 EIMS (including high resolution): Hitachi M-80B spectrometer Melting point: Yanagimoto micro-melting point analyzer UV absorption spectrum: Hitachi 200-20 spectrometer Red outside absorption spectrum: JEOL JIR-RFX 3001 type spectrometer NMR spectrum: Bruker AM400 spectrometer Next, the Rf values of MS-347 thin layer chromatography using various developing agents are shown in Table 1. Detection was performed by irradiation with ultraviolet light at 253.7 nm.

Developing: room temperature, rising method, developing time: 20 to 40 minutes Next, the vasodilatory effect of MS-347 will be described with experimental examples.

Experimental Example Contraction-suppressing action in rabbit thoracic aorta specimen After extracting the thoracic aorta of a rabbit (male, body weight 2-3 kg), a ring specimen about 3 mm in width was prepared, and a Krebs-Henseleit solution (composition: NcCl 118 mM) , KCl
4.75 mM, CaCl ₂ 2.54 mM, MgSO ₂ 1.19 mM, NaHCO ₃ 12.5 m
M, KH ₂ PO ₄ 1.19 mM and glucose 10.0 mM) were suspended at a load of 2 g in a Magnus tube. 37 ° C inside the Magnus tube
And a gas mixture (95% O ₂ + 5% CO ₂ ) was vented. The generated tension was measured isometrically using an isometric transducer (FD pickup TB-612T, manufactured by Nihon Kohden) and recorded on an ink pen recorder.

Specimens were allowed to stabilize for more than 60 minutes before inducing contraction with 40 mM KCl. When the contraction was stabilized, MS-347 was added to Magnus to examine the inhibitory effect on KCl contraction. Table 2 shows the results.

As is apparent from Table 2, MS yield was 40 mM KCl.
-347 showed a contraction inhibitory action.

Since MS-347 has a vasodilatory effect as shown in the above experimental examples, it is expected to be used as an antihypertensive agent, a therapeutic agent for ischemic heart disease, an agent for improving peripheral and cerebral circulation, and the like.

 Next, a method for manufacturing MS-347 will be described.

MS-347 can be obtained by culturing a microorganism capable of producing MS-347 in a medium, producing and accumulating MS-347 in the culture, and collecting MS-347 from the culture.

Examples of the MS-347-producing microorganism include a wild-type strain, a mutant strain obtained by mutating a wild-type strain by an artificial mutation method, for example, ultraviolet irradiation, X-ray irradiation, treatment with a mutagen, or a naturally-occurring mutant strain. Any strain can be used as long as it has -347 productivity. As a specific example, Aspergillus sp.
p.) SPC-16640 strain (hereinafter referred to as SPC-16640 strain). The SPC-16640 strain is a fungus newly isolated by the present inventors, and its bacteriological properties are as follows.

Macroscopic observation When cultivated at 25 ° C using malt extract agar medium,
The diameter of the colony reaches 52 to 57 mm on the seventh day of culture. The colonies are white fluffy, with a light grayish green at the center. The back has a lemon color. Secretes a pale lemon pigment into the medium.

When cultured on a potato-glucose agar medium at 25 ° C., the diameter of the colonies reaches 59 to 63 mm on the seventh day of culture. The colonies are white fluffy, with a grayish blue at the center. The back has a dull reddish-brown color. Secrets a dark wine brown pigment in the medium.

When cultured at 25 ° C. using Tupec yeast extract agar medium, the diameter of colonies reaches 74 to 78 mm on the seventh day of culture.
The settlement is white fluffy and pale grayish green at the center. The back has a cream color.

When cultured at 25 ° C. using Tuapec yeast extract agar medium supplemented with 20% sucrose, the diameter of the colony was 78 to 7 days after culture.
Reaches 80mm. The settlement is fluffy and has a dark green-blue color, and its surroundings are white. The back surface has a pale yellow-green color.

The optimal growth temperature of this strain is 13-42 ° C, and it grows best around 35 ° C. Optimum growth at pH 2 to 9 where it can grow
pH is 5-6.

Light microscopic observation of the present strain when cultured on Tuapec yeast extract agar medium The mycelium has partition walls and is colorless, smooth and well-branched. Hyphae do not become bundles. The conidiophores rise from the mycelium, and the conidiophores are smooth, colorless, about 100 μm in length and 5 in width.
9.59.5 μm. The conidial head has a cylindrical shape. At the tip of the conidiophore, a colorless apical sac of 15 to 21 μm in diameter is formed, and a large number of overlying phialides are formed in the upper three quarters or more. The phialide grows singly and has a length of 5 to 6.5 μm and a width of 1.5 to 2.5 μm at the widest part. No metres are observed. The ontogeny of the conidia is endogenous budding, and the conidia form from the tip of the phialide and form a chain. Phialo-type conidia are unicellular, spherical or sub-spherical and have a diameter of 2-2.5.
μm, slightly punctate and colorless on the surface, appearing blue-green when assembled. This strain is the anamorph described above.
Only the teleomorph is not observed.

Based on the mycological properties described above, the taxonomic position of the bacterium was described as "The Genera of Funjay Sporrating in Pure Culture 2nd Edition (The Genera of Fu
ngi Sporulating in Pure Culture, 2nd ed.Cramer, Vadu
z, JAvon Arx, 1974 years) the results of the search in accordance with ", this strain, Aspergillus sp. (Aspergillus s
p.). The present inventors have defined this strain as " Aspergillus sp. SPC
-16640 "and deposited under the Budapest Treaty with the Institute of Microbial and Industrial Technology, National Institute of Advanced Industrial Science and Technology on November 16, 1990 as FERM BP-3163.

For culturing microorganisms, a usual culturing method used for culturing fungi 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 contains a carbon source, a nitrogen source, an inorganic substance, etc. which can be assimilated by fungi.

As the carbon source, glucose, fructose, sucrose, lactose, stabilose, starch, dextrin, mannose, maltose, molasses, carbohydrates such as mashed potato, organic acids such as citric acid, malic acid, acetic acid, fumaric acid, methanol, Alcohols such as ethanol, hydrocarbons such as methane, ethane and n-propane, amino acids such as glutamic acid, glycerol, and cottonseed oil are used.

Examples of the nitrogen source include ammonium salts such as ammonium chloride, ammonium sulfate, ammonium nitrate, and ammonium phosphate, aspartic acid, glutamine, cystine,
Amino acids such as alanine, urea, malt extract, peptone, meat extract, yeast extract, dried yeast, corn steep leaker, soy flour, cottonseed oil, soy casein, casamino acid, pharmamedia, soluble vegetable protein, vegetables and fruits Juice is used.

As inorganic substances, potassium dihydrogen phosphate, disodium hydrogen phosphate, magnesium phosphate, magnesium sulfate, ferrous sulfate, manganese sulfate, cobalt sulfate, zinc sulfate, ammonium molybdate, aluminum potassium sulfate, barium carbonate, calcium carbonate , Cobalt chloride,
Magnesium chloride, potassium chloride, sodium chloride and the like are used.

In addition, if necessary, a substance that promotes or stabilizes the growth of cells or the production of MS-347, such as a vitamin (for example, pantothenic acid) and a pH buffer (for example, 3- (N-morpholino) propanesulfonic acid), is added to the medium. be able to.

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

The culture can be carried out by shaking culture, aeration stirring culture, etc.
It is carried out at a temperature of 40 ° C. and near neutral pH. After culturing for 3 to 10 days, the accumulation of MS-347 reaches the maximum, and the culture is completed.

When isolating and purifying MS-347 produced and accumulated in the culture, a method for isolating and purifying a normal microorganism-producing physiologically active substance from the culture is applied. Extraction of bacterial components with solvents such as acetone and methanol, filtration, removal of bacterial cells by centrifugation, etc., distribution with an appropriate solvent system, adsorption resin, silica gel, modified silica gel, alumina, cellulose, diatomaceous earth, magnesium silicate, gel MS by adsorption / desorption treatment of active substance by column chromatography or thin-layer chromatography using a filtering agent, etc.
-347 can be isolated and purified.

One example of isolating MS-347 from a culture is as follows.

The culture and the cells are separated by filtering or centrifuging the culture. An appropriate organic solvent such as methanol or acetone is added to the obtained cells, and the mixture is sufficiently stirred, and then filtered or centrifuged again to obtain an extract. After water is added to the obtained extract to dilute it appropriately, the extract is treated with an adsorption resin, for example, Diaion HP-20 (manufactured by Mitsubishi Kasei Co., Ltd.) to adsorb the active substance on the resin. The eluate is then eluted with a suitable solvent such as methanol and the eluate is concentrated under reduced pressure. After adding water as needed to this concentrated liquid, an appropriate solvent which is not miscible with water, for example, ethyl acetate or n-butanol is added to extract the active substance.
After the extract is concentrated to dryness under reduced pressure, it is suspended in a small amount of methanol, and methanol-insoluble matter is recovered by filtration. The obtained methanol-insoluble matter is dissolved in a suitable developing solvent, for example, 0.
Dissolve 5% methanol / chloroform and perform silica gel column chromatography by a conventional method. If necessary, silica gel column chromatography is repeated using a solvent system having the same or different composition. The fractions containing MS-347 are collected, concentrated to dryness under reduced pressure, dissolved in a suitable solvent such as acetone, and crystallized to perform MS-34.
7 is obtained as a yellow powder. The detection of MS-347 in the above purification step is performed by performing thin-layer chromatography using a silica gel plate containing a fluorescent agent, and then irradiating with ultraviolet light of 253.7 nm.

 Examples will be described below.

As Example 1 Inoculum Aspergillus sp (Aspergillus
sp.) SPC-16640 (FERM BP-3163) was used. A medium (pH 6.5) composed of 0.2 dl / dl of V-8 vegetable juice (manufactured by Campbell) and 3.0 g / dl of dextrin as a seed medium
Was used. The bacterium was inoculated into a large test tube containing 10 m of a seed medium having the above composition, and cultured at 25 ° C. with shaking until the bacterium grew sufficiently. 5 m of the seed culture 10 m thus obtained
Was added to an Erlenmeyer flask (300 m) containing 50 m of a seed medium having the above composition, and cultured with shaking at 25 ° C. for 2 days. Seed culture solution 5m obtained in this way, 50m
Erlenmeyer flask (300m containing production medium)
). The production medium was glucose 2.0 g / dl, mashed potato raw material (Snow Brand Milk Products Co., Ltd.) 4.0 g / dl, peptone (Kyokuto Pharmaceutical Co., Ltd.) 0.5 g / dl, potassium dihydrogen phosphate
A medium (pH 6.0) having a composition of 0.5 g / dl and magnesium phosphate octahydrate 0.05 g / dl was used. Fermentation culture at 25 ° C
For 6 days with shaking.

After completion of the culture, the culture (5.5) was filtered to obtain bacterial cells. Add 5.5 methanol to the cells,
After sufficiently stirring, the cells were removed again by filtration. After adding an equal amount of water to the obtained methanol extract, the mixture was passed through a column filled with 1 Diaion HP-20 to adsorb the active substance. After washing the column with 3 50% methanol, the active substance was eluted from the column with 3 methanol. After concentrating the methanol fraction under reduced pressure, water was added to 200 m, and the mixture was extracted four times using 200 m each of ethyl acetate. The ethyl acetate layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain 4.7 g of a brown oil. This oil was suspended in 200 m of methanol, and the suspension was filtered to obtain a methanol insoluble (1.0 g). This methanol-insoluble material was supplied to the upper end of a column packed with 200 m of Wakogel C-200 (manufactured by Wako Pure Chemical Industries, Ltd.), and developed with chloroform (1) and then chloroform containing methanol to 0.5%. All fractions eluted with chloroform containing methanol to 0.5% were collected and concentrated to dryness to obtain 598 mg of a yellow powder. This powder was dissolved in 300 m of acetone, and then left at 4 ° C. for crystallization. The crystals were separated by filtration and dried to obtain 422 mg of a yellow powder of MS-347.

In the above purification step, MS-347 was detected by performing thin-layer chromatography using a silica gel plate containing a fluorescent agent (Silica Gel 60F ₂₅₄ , manufactured by Merck), and then irradiating with ultraviolet light at 253.7 nm.

Effects of the Invention According to the present invention, a novel compound MS having a vasodilator effect
-347 can be obtained.

──────────────────────────────────────────────────の Continuation of the front page (51) Int.Cl. ⁶ identification code FI C12R 1:66) Examiner Koichi Niimi (58) Investigated field (Int.Cl. ⁶ , DB name) C12P 17/18 C07D 493 / 04 CA (STN) REGISTRY (STN) WPI (DIALOG) BIOSIS (DIALOG)

Claims (1)

(57) [Claims] 1. A compound MS-347 represented by the following structural formula.