JPH06184181A - Substance am6222, its production and use thereof - Google Patents

Abstract

PURPOSE:To obtain a new compound useful as an endothelin antagonistic agent, etc., used for preventing and treating diseases such as hypertension, arteriosclerosis, Reynaud disease or asthma. CONSTITUTION:The substance AM6222A, AM6222B or AM6222C or salt thereof. Furthermore, the substance AM6222A has the following physical properties: color and state of the substance white powder; distinction of acidity, facility and neutrality: an acidic substance; specific rotatory power: [alpha]<23>D-57 deg.+ or -10 deg. (c =0.4, methanol); molecular formula: C52H70O10N2; molecular weight: 882 fast atom bombardment (FAB) mass spectrum [(M+H)<+> 883.5066]; elementary analysis: calculated (%): C, 70.72; H, 7.99 and N, 3.17; found (%): C, 70.81; H, 8.02 and N, 3.16; solubility: soluble in methanol, dimethylsulfoxide and alkaline water and insoluble in water, benzene and n-hexane; UV spectrum: 218, 266 and 300-310 (nm)(main absorption peaks); IR spectrum: shown in the figure. The substance AM6222A is obtained by culturing a microorganism, belonging to the genus Stachybotrys and capable or producing the substance AM6222A (FERM P-13344) under aerobic conditions.

Description

Detailed Description of the Invention

[0001]

This invention relates to AM6222A substance,
The present invention relates to an AM6222B substance, an AM6222C substance or a salt thereof (hereinafter also collectively referred to as an AM6222 substance), a production method thereof and an application thereof.

[0002]

BACKGROUND OF THE INVENTION Endothelin is a peptide consisting of 21 amino acids and is produced by endothelial cells of humans, pigs, etc., and is known to have a strong vasoconstrictor action and a persistent and strong pressor action. The action is known to occur by binding to receptors on vascular smooth muscle [Na
332, Volume 332, 411-415, 198.
8 years; FEBS Letters, Volume 231, 440
-444, 1988; Biochem. Bio
phys. Res. Commun. , Volume 154, 86
8-875, 1988]. Endothelin is considered to be one of the causative agents of hypertension, acute renal failure, myocardial infarction and cerebral vasospasm [The Lancet, 11
79-1182, 1988; Eur. J. Ph
armacol, Volume 162, pages 353-358,
1989]. Furthermore, endothelin has an action of contracting small intestine, bronchus, and uterine smooth muscle as well [FEBS Le
tters, vol. 247, pages 337-340, 19
1989; Biochem. Biophys. Res. C
ommun. , Volume 159, Pages 317-323, 1
989]. Recently, it was shown that endothelin antiserum is effective in myocardial infarction model and acute renal failure model, and the involvement of endothelin in these pathological conditions was demonstrated [JP-A-3-128329; JP-A-3128329].
-128330]. Thus, there are many reports on endothelin, including cerebral vasospasm after subarachnoid hemorrhage,
The close involvement in the pathology of the three major ischemic diseases of myocardial infarction and acute renal failure has been proved [protein and nucleic acid enzyme, No. 36, 2399-2406, 1991].

Therefore, a substance which inhibits the binding of endothelin to the endothelin receptor is expected to be useful as a drug by antagonizing the above physiological action of endothelin. However, in the current state of the art, no effective drug has been developed to completely prevent or treat even these individual ischemic diseases, let alone endocrine which is one of these etiological factors. No drug has yet been developed that can suppress and thus combat ischemic disease.

[0004]

The present invention has been made in view of the current state of the art, and an object of the present invention is to develop a substance having an excellent endothelin antagonism. Is. Further, an object of the present invention is to utilize endothelin antagonism to treat hypertension, angina, cardiomyopathy, arteriosclerosis, heart diseases such as myocardial infarction, Rayno's disease, cerebral artery spasm, and cerebral vasospasm after subarachnoid hemorrhage. It is intended to develop a novel drug capable of effectively preventing or treating such stroke attacks, asthma such as bronchoconstriction, acute renal failure and the like.

[0005]

The present inventors extensively screened existing compounds for substances that antagonize the action of endothelin, but did not achieve the object. Therefore, the present inventors focused their attention on fermentation products of microorganisms, and as a result of searching various microorganisms, the substance of interest of the present invention was produced in the culture solution of the incomplete fungus M6222 strain isolated from the cultivated land in Yamanashi Prefecture. It was discovered that the substance was purified and isolated, and the physicochemical properties thereof were studied in detail. As a result, it was confirmed that the substance was a novel substance that was unknown in the past, and this substance was newly identified as AM6222A substance, AM6222B substance and AM6222C substance. The substance was named, and as a result of further research, an industrial production method thereof was established, and an endothelin antagonism was also confirmed, and the present invention was completed.

That is, the present invention provides AM6222A substances, AM6222B substances, and AM6 substances having the following physicochemical properties.
The present invention relates to 222C substances, salts thereof, methods for producing them, and use thereof as endothelin antagonists. Physicochemical properties of AM6222A substance (a) Color and state of substance White powder (b) Distinction between acidic, basic and neutral Acid substance (c) Specific optical rotation [α] _D ²³ −57 ° ± 10 ° (c = 0.4, methano
(D) Molecular formula C ₅₂ H ₇₀ O ₁₀ N ₂ (e) Molecular weight 882 FAB mass spectrum [(M + H) ⁺ : 88
3.5066] (f) Elemental analysis Calculated value (%) C, 70.72; H, 7.99; N,
3.17 Found (%) C, 70.81; H, 8.02; N,
3.16 (g) Solubility Soluble in methanol, dimethylsulfoxide and alkaline water; insoluble in water, benzene and n-hexane. (H) Color reaction The iodine vapor reaction and potassium permanganate decolorization reaction are positive, and the ferric chloride reaction, ninhydrin reaction, and Molish reaction are negative. (I) Rf value about 0.60, Kieselgel 60F manufactured by Merck & Co., Inc.
₂₅₄ used Developing solvent: chloroform / methanol / acetic acid (10:
3: 0.1) (j) Ultraviolet absorption spectrum As shown in FIG. 1, the result of measurement with a methanol or acidic methanol solution λ _max nm (E _{1 cm} ^1% ) 218 (800), 266 (1
75), 300-310 (60) Results measured with alkaline methanol solution λ _max nm (E _{1 cm} ^1% ) 222 (698), 274 (1)
50), 310-325 (60) (k) Infrared absorption spectrum As shown in FIG. 2, the main wavenumbers (cm ⁻¹ ) in KBr tablets are as follows.

3400, 2950, 1670, 147
0,1350,1080 (l) ¹ H-NMR spectrum d ₆ - ¹ H-NMR as measured in dimethyl sulfoxide
The spectrum (400 MHz) is shown in FIG. (M) ¹³ C-NMR spectrum d ₆ - ¹³ C-NMR were measured in dimethyl sulfoxide
The signal in the spectrum (100 MHz) is found below (ppm).

172.7 (s), 168.2 (s), 1
67.4 (s), 155.9 (s), 155.8
(S), 153.7 (s), 153.6 (s), 13
4.0 (s), 133.2 (s), 116.8 (s),
116.3 (s), 112.2 (s), 111.8.
(S), 101.0 (d), 100.7 (d), 97.
9 (s), 97.8 (s), 73.5 (d), 73.4.
(D), 53.4 (d), 46.6 (t), 43.8.
(T), 41.7 (s), 41.7 (s), 41.3
(T), 39.3 (d), 39.3 (d), 37.3
(S), 37.3 (s), 36.5 (d), 36.4
(D), 31.6 (t), 31.6 (t), 30.7
(T), 30.6 (t), 28.6 (q), 28.6
(Q), 28.4 (t), 27.1 (t), 24.8
(T), 24.8 (t), 23.8 (t), 23.8
(T), 23.3 (t), 22.3 (q), 22.3
(Q), 20.4 (t), 20.4 (t), 15.9
(Q), 15.8 (q), 15.5 (q), 15.4
(Q) Physicochemical properties of AM6222B substance (a) Color and state of substance White powder (b) Distinction between acidic, basic and neutral Acid substance (c) Specific rotation [α] _D ²³ −64 ° ± 10 ° (C = 0.4, methano-
(D) Molecular formula C ₅₂ H ₇₀ O ₁₁ N ₂ (e) Molecular weight 898 FAB mass spectrum [(M + H) ⁺ : 89
9.5026] (f) Elemental analysis calculated value (%) C, 69.46; H, 7.85; N,
3.12 Found (%) C, 69.22; H, 7.76; N,
2.97 (g) Solubility Soluble in methanol, dimethylsulfoxide and alkaline water; insoluble in water, benzene and n-hexane. (H) Color reaction The iodine vapor reaction and potassium permanganate decolorization reaction are positive, and the ferric chloride reaction, ninhydrin reaction, and Molish reaction are negative. (I) Rf value of about 0.30 Kieselgel 60F manufactured by Merck & Co., Inc.
₂₅₄ used Developing solvent: chloroform / methanol / acetic acid (10:
3: 0.1) (j) Ultraviolet absorption spectrum As shown in FIG. 4, the result measured with a methanol or acidic methanol solution λ _max nm (E _{1 cm} ^1% ) 218 (785), 265 (1
75), 300-310 (60) Results measured with alkaline methanol solution λ _max nm (E _{1 cm} ^1% ) 222 (690), 274 (1)
50), 310-325 (60) (k) Infrared absorption spectrum As shown in FIG. 5, main wavenumbers (cm ⁻¹ ) in KBr tablets are as follows.

3400, 2950, 1660, 147
0,1350,1080 (l) ¹ H-NMR spectrum d ₆ - ¹ H-NMR as measured in dimethyl sulfoxide
The spectrum (400 MHz) is shown in FIG. (M) ¹³ C-NMR spectrum d ₆ - ¹³ C-NMR were measured in dimethyl sulfoxide
The signal in the spectrum (100 MHz) is found below (ppm).

172.8 (s), 168.2 (s), 1
67.3 (s), 155.8 (s), 155.8
(S), 153.7 (s), 153.6 (s), 13
4.0 (s), 133.3 (s), 116.7 (s),
116.3 (s), 112.3 (s), 111.8.
(S), 100.8 (d), 100.8 (d), 97.
8 (s), 97.7 (s), 77.7 (d), 73.4.
(D), 64.9 (d), 53.5 (d), 46.6
(T), 43.8 (t), 42.9 (s), 41.8.
(S), 41.3 (t), 39.3 (d), 38.7.
(D), 38.1 (s), 37.3 (s), 36.4
(D), 36.3 (d), 32.9 (t), 31.8.
(T), 31.6 (t), 30.7 (t), 30.6
(T), 28.9 (q), 28.6 (q), 28.4
(T), 27.1 (t), 24.9 (t), 23.8.
(T), 23.3 (t), 22.3 (q), 22.0
(Q), 20.4 (t), 20.2 (t), 16.8
(Q), 15.8 (q), 15.4 (q), 15.4
(Q) Physicochemical properties of AM6222C substance (a) Color and state of substance White powder (b) Distinction between acidic, basic and neutral Acid substance (c) Specific rotation [α] _D ²³ −65 ° ± 10 ° (C = 0.4, methano-
(D) Molecular formula C ₅₂ H ₇₀ O ₁₁ N ₂ (e) Molecular weight 898 FAB mass spectrum [(M + H) ⁺ : 89
9.5067] (f) Elemental analysis Calculated value (%) C, 69.46; H, 7.85; N,
3.12 Found (%) C, 69.22; H, 7.62; N,
3.02 (g) Solubility Soluble in methanol, dimethylsulfoxide and alkaline water; insoluble in water, benzene and n-hexane. (H) Color reaction The iodine vapor reaction and potassium permanganate decolorization reaction are positive, and the ferric chloride reaction, ninhydrin reaction, and Molish reaction are negative. (I) Rf value of about 0.43 made by Merck Kieselgel 60F
₂₅₄ used Developing solvent: chloroform / methanol / acetic acid (10:
3: 0.1) (j) Ultraviolet absorption spectrum Results measured with methanol or acidic methanol solution as shown in FIG. 7 λ _max nm (E _{1 cm} ^1% ) 218 (770), 265 (1)
70), 300-310 (60) Results measured with alkaline methanol solution λ _max nm
(E _1cm ^1% ) 222 (658), 275 (142), 3
10-325 (58) (k) Infrared absorption spectrum As shown in FIG. 8, the main wave numbers (cm ⁻¹ ) in KBr tablets are as follows.

3400, 2950, 1660, 147
0,1350,1080 (l) ¹ H-NMR spectrum d ₆ - ¹ H-NMR as measured in dimethyl sulfoxide
The spectrum (400 MHz) is shown in FIG. (M) ¹³ C-NMR spectrum d ₆ - ¹³ C-NMR were measured in dimethyl sulfoxide
The signal in the spectrum (100 MHz) is found below (ppm).

172.7 (s), 168.2 (s), 1
67.3 (s), 155.9 (s), 155.7
(S), 153.7 (s), 153.6 (s), 13
4.1 (s), 133.2 (s), 116.8 (s),
116.2 (s), 112.3 (s), 111.9.
(S), 100.9 (d), 100.8 (d), 97.
9 (s), 97.5 (s), 77.6 (d), 73.4.
(D), 64.8 (d), 53.4 (d), 46.5
(T), 43.8 (t), 42.9 (s), 41.7.
(S), 41.3 (t), 39.3 (d), 38.7.
(D), 38.0 (s), 37.2 (s), 36.5
(D), 36.1 (d), 32.7 (t), 31.7.
(T), 31.7 (t), 30.7 (t), 30.7
(T), 28.8 (q), 28.6 (q), 28.4
(T), 27.1 (t), 24.8 (t), 23.8
(T), 23.3 (t), 22.3 (q), 21.9.
(Q), 20.4 (t), 20.2 (t), 16.7
(Q), 15.8 (q), 15.5 (q), 15.3
(Q)

As a compound having the same molecular formula as the AM6222A substance, conventionally, a uridine-based oligoribonucleotide compound [Acta Chem. Scand. ，
No. 38, pp. 657-671, 1984] is known. While the uridine oligoribonucleotide compound contains a sugar, the AM6222A compound of the present compound shows that the color reaction and infrared absorption spectrum of
It is distinguished by the fact that it is a sugar-free compound. As compounds having the same molecular formula as the AM6222B and AM6222C substances, crown ether epoxy compounds having four methyl groups [JP-A-58-57378].
It has been known. On the other hand, the present compound AM6222
The B and AM6222C materials are distinguished by being compounds having eight methyl groups. Therefore AM6222A
The substances, AM6222B substance and AM6222C substance, were clearly distinguished from the known substances from the above properties, and were confirmed to be novel substances.

The following is the end of the AM6222 substance of the present invention.
The serine antagonism will be described.Radioligand binding assay (A) Preparation of crude receptor membrane fraction. Endothelin radio receptor measurement
Infant thoracic aortic smooth muscle-derived A-10 cells (ATCC CR
The membrane fraction of L-1476) was used. That is, A-10 fine
10% fetal bovine serum (Gibco) for cell culture, 1
00 μg / ml streptomycin sulfate and 100 units
D-EME medium containing knit / ml penicillin G (Dainippon
(Pharmaceutical company) was used. 75 cm for A-10 cells²Culture
Approximately 5x10 on the Rusco (manufactured by Falcon)^FiveSow, C
O₂In the incubator (37 ° C, 5% CO₂)
During the culture, the medium is changed once and confluent (2.5 x 1
0 ⁶Cells (5 days). Remove the culture solution
After leaving, PBS solution (0.02% KCl, 0.02% KH
₂PO_Four, 0.29% Na₂HPO_Four・ 12H₂O,
Wash the cells once with 0.8% NaCl; pH 7.4)
It was Then add 1 mM EGTA-containing PBS solution to the cell
Scrape cells with a scraper and spin at 1,000 rpm for 1
After centrifugation for 0 minute, the cell fraction was collected as a precipitate. This sink
50 mM Tris buffer for binding assay [50 mM
Tris / HCl, 10μM CaCl₂, 10 μM Mg
Cl₂, 100 μM PMSF (Phenylmeth)
ylsulfonyl Fluoride; Wako Pure Chemical Industries
2 μM leupeptin (Wako Pure Chemical Industries, Ltd.)
1 μM phosphoramidon (Wako Pure Chemical Industries, Ltd.)
0.1% bovine serum albumin (Wako Pure Chemical Industries, Ltd.)
Made); pH 7.4], frozen and thawed, homogenizer
-(Biotron BT type, manufactured by Ikeda Rika Co., Ltd.)
I'm monetized. Homogenate 31,000 rpm,
Centrifuged at 4 ° C for 60 minutes. Combine the resulting pellets
Resuspend in 30 ml of 50 mM Tris buffer for assay
And centrifuged at 2,000 rpm at 4 ° C for 10 minutes. So
The supernatant liquid of the above was dispensed as a crude receptor membrane fraction,
It was stored and thawed as needed before use.

(B) ¹²⁵ I-endothelin binding assay. ¹²⁵ I-endothelin-1 (1.35x10 ^-11 M)
(Amersham, Japan; specific activity 74TBq
/ Mmol) was reacted with 100 μl of crude receptor membrane fraction in 50 mM Tris buffer for binding assay and sample in a final volume of 200 μl at 37 ° C. for 3 hours. The amount of non-specific binding was measured in the presence of 1 × 10 ⁻⁷ M endothelin-1 (manufactured by Peptide Institute Co., Ltd.). After the reaction, Sell Harvester (manufactured by Skatron, Mode 17,0)
20 type), and the reaction mixture was filtered through a glass filter (manufactured by Skatron, 11,731 filter-mat). The filter is then washed with 3 ml of saline.
Washed twice. The radioactivity remaining on the filter is gamma-
It measured with the counter- (Aloka make, ARC-600 type).

The AM6222A substance, the AM6222B substance and the AM6222C substance were ¹²⁵ I to the receptor of the rat fetal thoracic aortic smooth muscle-derived A-10 cell membrane fraction.
-Inhibited endothelin binding. The inhibition value (I
C ₅₀ ) is 21 μg / ml, 25 μg / ml,
It was 26 μg / ml. AM6 on endothelin-induced rabbit aortic contraction response
Effect of 222 substance The thoracic aorta was extracted from a male albino rabbit (11 weeks old), the intima was stripped off, and cut into pieces of about 25 mm. After excision of adipose tissue, these arterial fragments (width 2 mm, length 2
5 mm) with 30 ml of Krebs-Henseleit
A Magnus tube filled with a nutrient solution was suspended by applying 1 g of tension. The generated tension is transferred to the isometric transducer (O
DC by Rientec, T7-8-240 type)
Strain Amplifier (Nichiden Saneisha,
6M92) and recorded on a recorder. During this period, the temperature was kept at 37 ° C and the mixed gas (95% O _2, 5% CO 2
₂ ) was made into small bubbles and aerated. When 1x10 ^-8 M endothelin-1 was added and a certain aortic contraction reaction was obtained, AM6222 substance 1x10 ^-5 M was added to determine the relaxation reaction of blood vessels, and each reaction of the test drug with the maximum contraction being 100% The rate (%) was calculated.

Endoseri using rabbit thoracic aortic preparation
3x10 for evoked contraction response ^-FiveAM6 at M concentration
222A substance, AM6222B substance and AM6222C
The relaxation rate (%) of the substance is 84%, 70% and 6 respectively.
It was 8%. Thus AM6222A substance, AM6
222B substance and AM6222C substance are excellent endose
It was confirmed to have a phosphorus antagonism.

From the results of the above-mentioned test, the AM6222 substance shows a vasodilatory action in humans as a drug that antagonizes the vasoconstrictor action involving endothelin, and therefore,
Hypertension such as peripheral circulatory insufficiency, angina, cardiomyopathy, arteriosclerosis, heart disease such as myocardial infarction, Rayno's disease, cerebral artery spasm,
It is useful as a preventive or therapeutic agent for cerebral ischemia, stroke attacks such as cerebral spasm after subarachnoid hemorrhage, asthma such as bronchoconstriction, and diseases such as acute renal failure.

The AM6222 substance according to the present invention is M6
Produced from 222 strains. The M6222 strain was isolated from cultivated soil collected in Yamanashi Prefecture. The mycological properties are as follows. (1) Growth condition in each medium

(A) Potato glucose agar medium (PD
A). In the case of culture at 25 ° C for 1 week, the diameter is 12-13m.
It will be 22-26 mm in 2 weeks. After 2 weeks, the flora is slightly thin and the central part is swollen. It is initially white, but it is light gray (1B1) to gray (1E) due to the formation of conidia.
1) The surface is often undulated by aerial hyphae and becomes velvet to slightly fluffy. The peripheral part is blunt to sawtooth. A gray-orange (5B5) water-soluble dye is produced in the medium. The back side is brown (6E5,6) to gray-orange (6B
5, 6), the color becomes lighter toward the periphery.

(B) Potato ginseng agar medium (PC
A). In case of culture at 25 ° C for 1 week, the diameter is 7-10m
m, 26 to 29 mm in 2 weeks. After 2 weeks, the flora becomes thin and flat. At first white hyphae spread thinly and become colored by the formation of conidia. The central part is slightly raised by aerial hyphae, slightly rough fluffy, light gray (1B
1) to gray (1E1). In other cases, dark brown (6F5) small dots due to the conglomerate agglomerate are densely present. The periphery is white with no conidia formation and is full-scale. A slightly pale brown water-soluble pigment is emitted in the medium. The center of the back side is light brown (5D6), and the others are light gray (1B1).

(C) A wort agar medium (MA). The settlement has a diameter of 12 mm in the case of culturing at 25 ° C and one week,
It becomes 22 to 25 mm in a week. Two weeks later, the central part of the flora was swelled like a lens due to aerial hyphae, and was orange-white (5A
2) or orange gray (5B2). Others are slightly lighter, from light gray (6D4) to gray-orange (6B4), and become lighter toward the periphery. The surface becomes a fine velvet shape. The peripheral part has a blunt tooth shape. No water-soluble pigment is produced. The back side is dark brown (6F4,5) to brown-orange (6C).
4, 5), the color becomes lighter toward the periphery. The color tone is described by Mezuen Handbook of Color (Me
then Handbook of Color)
It was done based on ¹⁾ .

(2) Physiological properties The pH of this bacterium that can grow is 3.4 to 11.2, and the optimum growth pH is 4.4 to 10.4. The temperature at which this fungus can grow is 13 to 37 ° C, and the optimum growth temperature is 25.0 to 3
It is 2.7 ° C.

(3) Morphological features under a microscope No sexual generation is formed. Colorless, smooth surface, width 2.2-5.
Conidia stalks stand up from 0 μm hyphae. Conidia stalks are scattered, formed singly, and no branches are seen. The base of the conidia peduncle is slightly or slightly bulging. The width narrows toward the top, but the tip is slightly spherical. It forms a few septums, the walls are smooth, but wart-like granules are sometimes seen above the handle. Length is 38-10
The width is 0 μm and the width is 3.0 to 5.0 μm. Fear rides are 6 to 12 walnuts on conidia peduncle. It has a club-like shape and bends slightly inward. No color can be seen. Length of smooth surface 8 ~ 11μ
m, width 4.0 to 5.0 μm. Conidia are endogenous from the top of the phialide. Conidia are slightly oval, oval or boat-shaped, and have an olive brown color. The base is raised like a hump and is slightly cut or cut. The surface is smooth and sometimes bumpy. Monocellular, with two oil vesicles inside. Conidia are 7.5-10 in length
(13.7) μm, width 3.5 to 4.5 (5.0) μm, forming a large viscous conidia mass at the tip of the phialide.

(4) Identification and Deposit of Microorganism The present bacterium is an incomplete fungus because no sexual generation is formed. Conidia belong to the incomplete filamentous fungal net because they are formed on the conidia stalks generated from hyphae. Furthermore, the conidia formation mode is the phialophore type, the conidia are brown, monocellular, and do not link and form a viscous conidial agglomerate at the tip of the phialide.
Fear Ride has 6 to 12 stalks on top of conidia peduncle and has a club-like shape, so Stachybotrys ( Stach)
^2,3) belonging to the genus ybotrys ).

At present, about 30 species of Stachybotrys are known. 16 of them are known to have egg-shaped, oval-shaped or boat-shaped conidia. Particularly, as a species similar to this bacterium, Stachybotrys albipes ( S. albipe)
s ), Stachybotrys sansevier area ( S. sans
Evieriae), Stachybotrys Pas -... Bisupora (S Parvispora), Stachybotrys, Jikuroa (S Dichroa), Stachybotrys, Burebiusukurusu (S Breviusculus), Stachybotrys cha -. sag (S but chartarum) and the like, S. albipes , S. sanse
Vierae differs from this strain in that the bacterium forms a hump-like conidia, and S. The parvispora conidia of size, different in that conidia of the smooth surface can be seen, S. dich
The roa conidiophores of length, different in terms of conidia of size, S. It differs from Brevius culus in the width of conidia, and S. It was different from chartarum in that the conidia stalk was not formed in the symposium and that many of the conidia had smooth surfaces, and no species consistent with this bacterium were found. Therefore, this production strain was designated as Stachybotrys espi-M62.
22 ( Stachybotrys sp. M6222)
I named it. A freeze-dried sample of this strain was deposited at the Institute of Microbial Science and Technology, the Agency of Industrial Science and Technology, under the number FERMP-13344. (Deposit date = December 18, 1992)

Reference 1) Kornerup. A, and J. H. Wans
cher; Methen Handbook of
Color, 3rd ed . Eyere Methue
n, London, 1978 2) Ando Katsuhiko; Fungal isolation and taxonomy Stachyb
Otrys genus, antibacterial and antifungal. Volume 19, Pages 597-607
J., 1991 3) Jong, S. et al. C, and E.C. E. Davis;
Contribution to the know
edge of Stachybotrys and M
emnoniella in culture. Myc
Otaxon 3 , 409-485, 1976 AM622
It should be understood that the production of the two substances is not limited to the use of the particular microorganisms described herein merely for purposes of illustration. The present invention includes AM6222 including artificial mutants and natural mutants that can be obtained from the microorganisms described by X-ray irradiation, ultraviolet irradiation, N-methyl-N'-nitro-N-nitrosoguanidine, 2-aminopurine and the like. It also includes the use of all mutants capable of producing the substance.

The AM6222 substance according to the present invention is a substance-producing bacterium belonging to the genus Stachybotrys (for example, Stac
hybotorys sp. M6222) can be produced by inoculating into a nutrient medium containing a carbon and nitrogen source capable of assimilating and culturing under aerobic conditions (eg, shaking culture, aeration and stirring culture). As the carbon source, glucose, dextrin, sucrose, fructose, glycerin, starch, maltose, molasses and the like are used alone or as a mixture. As nitrogen source, soybean powder,
Cottonseed meal, Corn steep liquor, Meat extract, Peptone, Wheat germ, Yeast extract, Oatmeal, Gluten meal, Fish meal, Ammonium salt (for example, ammonium sulfate, ammonium nitrate, ammonium phosphate, etc.) Urea,
Amino acids and the like are used alone or as a mixture. If necessary, inorganic salts such as the following may be added to the medium: sodium chloride, potassium chloride, calcium carbonate, sodium carbonate, potassium carbonate, sodium phosphate, potassium phosphate, magnesium salt, copper salt. , Cobalt salt, iron salt, zinc salt, manganese salt, etc. When the medium is significantly foamed, liquid paraffin, animal oil, mineral oil, silicone, etc. may be added if necessary.

The culturing method may be the same as the method for producing a general microbial metabolite, and may be solid culture or liquid culture. In the case of liquid culture, any of static culture, stirring culture, shaking culture, aeration culture and the like may be carried out, but shaking culture or deep aeration stirring culture is particularly preferable. The culturing temperature may be appropriately changed within the range in which the present AM6222 substance-producing bacterium produces the present substance, but it is usually 13 to 37 ° C, preferably 25 to 30 ° C. The preferable pH of the medium is in the range of 4 to 8, and the culturing time is usually 1 to 8 days, although it varies depending on the culturing conditions and the culturing amount.

In order to collect the desired AM6222 substance from the culture, the separation means usually used for collecting from the culture of the metabolite produced by the microorganism is appropriately used.
Since the AM6222 substance is mainly present in the culture filtrate, it can be separated from the culture filtrate by a conventional separation means such as a solvent extraction method.
The purification can be performed by an ion exchange resin method, an adsorption or partition chromatography method, a gel filtration method or the like alone or in combination. In addition, high performance liquid chromatography
And thin layer chromatography can also be used for extraction and purification.

The separation and purification of the AM6222 substance is carried out by appropriately utilizing the known method as described above, but it may be carried out as follows, for example. First, the culture solution is centrifuged to obtain a centrifugal supernatant. The obtained supernatant is extracted with an organic solvent such as ethyl acetate under acidic conditions, and the extract is concentrated. The concentrated residue was subjected to alumina column chromatography, and eluted with a mixed solvent of chloroform, ethyl acetate, methanol, acetone, acetic acid, water and the like alone or in an appropriate combination to separate the AM6222 substance, and further to a silica gel column if necessary. Chromatography, elute with a mixed solvent of chloroform, methanol, acetic acid, etc., collect and concentrate the desired fractions, add a solvent such as hexane to generate a precipitate, and then collect on a glass filter. By drying under reduced pressure, AM622
Two white powders are obtained. If necessary, the AM6222 substance can be further separated by a separating means such as silica gel column chromatography to obtain the AM6222A substance, the AM6222B substance, the A substance.
You may isolate and purify it as M6222C substance or each salt. As described above, the AM6222 substance can be treated by a conventional method such as treatment with an alkali metal such as sodium and potassium, an alkaline earth metal such as magnesium and calcium, a hydroxide or carbonate thereof, or ammonium hydroxide, in addition to release. It may be used in the form of an acceptable salt, and these salts are also included in the scope of the present invention.

The AM6222 substance used as the endothelin antagonist of the present invention is AM6222A substance,
Any one of AM6222B substance, AM6222C substance and salts thereof, or a mixture thereof may be used, and the AM6222 substance is mixed with an inorganic or organic carrier which is commonly used as an active ingredient thereof, and a solid, In addition to oral administration agents in semi-solid or liquid form, they are formulated into parenteral administration agents such as external preparations.

The administration method may be either oral administration or parenteral administration, and examples of parenteral administration include intramuscular injection, intravenous injection, nasal administration, vaginal administration and transdermal administration. Examples of the preparation for oral administration include tablets, powders, pills, granules, soft capsules, fine granules, powders, emulsions, suspensions, syrups and elixirs. Formulations for parenteral administration include injections,
Examples include ointments, lotions, tonics, sprays, suspensions, oils, emulsions and suppositories. To formulate the active ingredient of the present invention, a conventional method may be used.
Excipients, colorants, flavors, preservatives, stabilizers, buffers, suspensions, etc., tonicity agents, wetting agents, auxiliaries, emulsifiers, absorption accelerators and other commonly used additives are used as appropriate.

The dose of the pharmaceutical composition according to the present invention varies depending on its type, type of disease to be treated or prevented, administration method, patient's age, patient's symptoms, treatment time, etc. Is an active ingredient per adult per day (AM6222A substance, AM6222B substance, AM62
22C substance or salts thereof) 0.1 to 50 mg / kg
And 0.1 to 50 mg / k for intramuscular administration
0.5 to 100 mg for oral administration
It is administered in the range of / kg. In addition, this substance AM6222
A, AM6222B or AM6222C substance was orally administered to mice at a dose of 100 mg / kg of body weight. No particular toxicity was observed for any of the substances, and safety was confirmed.

[0035]

EXAMPLES The present invention will now be described in more detail with reference to examples, but the present invention is not limited thereto.

[0036]

Example 1 Glucose 1%, Dextrin 1%, I-
Strut extract 0.5%, casein hydrolyzate 0.5%, CaC
A medium composed of O ₃ 0.1% and Celite 1% (pH before sterilization
6.5) to 4 500 ml Erlenmeyer flasks, 100 m each
It was dispensed in increments of 1 and sterilized at 115 ° C for 15 minutes. In these, Stachybotrys sp. M6222 (FE
Slope cultures of RM P-13344) were inoculated with one platinum loop each and cultured at 26 ° C for 72 hours in a rotary shaker (200 rpm).

Separately, glucose 2%, peptone 1%, co-
Nusty-preca-1%, KH ₂ PO ₄ 0.2%, Mg
10 l of a medium (pH 6.5 before sterilization) having a composition of 0.1% SO ₄ .7H ₂ O and 1% celite was dispensed into 100 500 ml Erlenmeyer flasks, 100 ml each, and sterilized at 115 ° C for 15 minutes. Then, 4 ml each of the above-mentioned preculture was inoculated into 100 Erlenmeyer flasks of 500 ml containing 100 ml of the above medium, and cultured at 26 ° C for 72 hours on a rotary shaker (200 rpm).

[0038]

Example 2 Diatomaceous earth was added to the culture obtained in Example 1 and filtered. Ethyl acetate (5 l) was added to the obtained filtrate (8 l), the pH was adjusted to 2 with 1N hydrochloric acid, and the mixture was extracted with ethyl acetate. Water (2 L) was added to the obtained ethyl acetate layer and the pH was adjusted to 9 with 1N ammonia water, and the target substance was dissolved in the aqueous layer. Ethyl acetate (1.3 l) was added to the obtained aqueous layer to adjust the pH to 2, the mixture was extracted with ethyl acetate, and the extract was concentrated under reduced pressure to obtain 9 g of a crude substance. Dissolve this in methanol,
Alumina column (400 m
The column was washed with 80% methanol solution (1.6 l). Then, gradient elution was carried out using 80% methanol solution and 50% methanol solution (8 l) to fractionate 400 ml each, and AM6222A and AM6
Fraction containing 222C substance (fraction No5 to No1
1) and a fraction containing AM6222B substance (fractions No14 to No17) were obtained.

Fractions No5 to No11 were concentrated under reduced pressure, and the obtained concentrated liquid was extracted with ethyl acetate at pH 2.
Concentrate and pre-form chloroform-methanol-acetic acid (10:
1.2: 0.1) silica gel column (40
0 ml) and developed with the same mixed solvent, chloroform-
T using a system of methanol-acetic acid (10: 3: 0.1)
Only the fractions showing an Rf value of 0.60 by LC were collected and concentrated under reduced pressure, and a hexane solution was added to cause precipitation.
The precipitate was collected on a glass filter and dried under reduced pressure to obtain 120 mg of a single AM6222A substance as a white powder. Also, only fractions showing Rf value 0.43 were collected and concentrated, and hexane precipitation was performed in the same manner to obtain a single AM
56 mg of a white powder of 6222C substance was obtained.

Fractions No. 14 to No. 17 were concentrated under reduced pressure, the obtained concentrated liquid was extracted with ethyl acetate at pH 2, the extract was concentrated, and chloroform-methanol was used in advance.
It was applied to a silica gel column (150 ml) prepared with acetic acid (10: 2: 0.1) and developed with the same mixed solvent. Only fractions showing an Rf value of 0.30 were collected by TLC using a system of chloroform-methanol-acetic acid (10: 3: 0.1), concentrated under reduced pressure, and a hexane solution was added to cause precipitation. This precipitate was collected on a glass filter and dried under reduced pressure to obtain 39 mg of a single AM6222B substance as a white powder.
Got

[0041]

Example 3 AM6222A substance 2 obtained in Example 2
00 mg was dissolved in 10 ml of 4N ammonia water and freeze-dried to obtain 204 mg of white powder as an ammonium salt of AM6222A substance.

[0042]

Example 4 AM6222A substance 5 produced in Example 2
A 10% hydroxypropyl cellulose solution was added to 0 g, 80 g of lactose, 16 g of cone starch and 8 g of carboxymethyl cellulose and kneaded. To this, 1 g of magnesium stearate was added and mixed well, and the mixture was compressed by a compression tablet machine to give AM6222A per tablet.
1000 tablets containing 50 mg of substance were produced.

[0043]

EXAMPLE 5 2.5 g of AM6222A substance prepared in Example 3, 4.5 g of salt, 2.5 g of chlorobutanol,
Dissolve 0.5 g of sodium hydrogen carbonate in distilled water to obtain 50
Make 0 ml and dispense 1 ml each into ampoules, then inject 5
00 pieces were manufactured.

[0044]

The present invention is based on the AM6222A substance, AM62.
22B substance, AM6222C substance and salts thereof, these substances are novel pharmacologically active substances which have hitherto been unknown, exhibit excellent endothelin antagonism, and are drugs, for example, hypertension such as peripheral circulatory insufficiency, Angina,
Cardiomyopathy, arteriosclerosis, heart disease such as myocardial infarction, Reino-
It is useful as a preventive or therapeutic agent for disease, cerebral ischemia, cerebral artery spasm, stroke attacks such as cerebral vasospasm after subarachnoid hemorrhage, asthma such as bronchoconstriction, and acute renal failure.

Further, according to the present invention, a method for producing the above substance using a microorganism has been established.

[Brief description of drawings]

FIG. 1 shows an ultraviolet absorption spectrum of AM6222A substance in methanol.

FIG. 2 shows the infrared absorption spectrum of AM6222A substance in KBr tablets.

FIG. 3 shows ¹ H-NMR (400 MHz) of AM6222A substance measured in d ₆ -dimethylsulfoxide.
The spectrum is shown.

FIG. 4 shows an ultraviolet absorption spectrum of AM6222B substance in methanol.

FIG. 5 shows an infrared absorption spectrum of AM6222B substance in KBr tablets.

FIG. 6 shows ¹ H-NMR (400 MHz) of AM6222B substance measured in d ₆ -dimethylsulfoxide.
The spectrum is shown.

FIG. 7 shows an ultraviolet absorption spectrum of AM6222C substance in methanol.

FIG. 8 shows an infrared absorption spectrum of AM6222C substance in KBr tablets.

FIG. 9: ¹ H-NMR (400 MHz) of AM6222C substance measured in d ₆ -dimethylsulfoxide.
The spectrum is shown.

Claims (3)

[Claims] 1. An AM6222A substance, an AM6222B substance, and an AM, which have the following physicochemical properties:
6222C substance or a salt thereof. Physicochemical properties of AM6222A substance (a) Color and state of substance White powder (b) Distinction between acidic, basic and neutral Acid substance (c) Specific optical rotation [α] _D ²³ −57 ° ± 10 ° (c = 0.4, methano
(D) Molecular formula C ₅₂ H ₇₀ O ₁₀ N ₂ (e) Molecular weight 882 FAB mass spectrum [(M + H) ⁺ : 88
3.5066] (f) Elemental analysis Calculated value (%) C, 70.72; H, 7.99; N,
3.17 Found (%) C, 70.81; H, 8.02; N,
3.16 (g) Solubility Soluble in methanol, dimethylsulfoxide and alkaline water; insoluble in water, benzene and n-hexane. (H) Color reaction The iodine vapor reaction and potassium permanganate decolorization reaction are positive, and the ferric chloride reaction, ninhydrin reaction, and Molish reaction are negative. (I) Rf value 0.60, Kieselgel 60F, manufactured by Merck Ltd.
₂₅₄ used Developing solvent: chloroform / methanol / acetic acid (10:
3: 0.1) (j) Ultraviolet absorption spectrum Main absorption peaks (nm) in methanol are as follows. 218, 266, 300-310 (k) Infrared absorption spectrum The main wave numbers (cm ⁻¹ ) in KBr tablets are as follows. 3400, 2950, 1670, 1470, 1350,
1080 (l) ¹ H-NMR spectrum d ₆ - ¹ H-NMR as measured in dimethyl sulfoxide
The spectrum (400 MHz) is shown in FIG. (M) ¹³ C-NMR spectrum d ₆ - ¹³ C-NMR were measured in dimethyl sulfoxide
The signal in the spectrum (100 MHz) is found below (ppm). 172.7 (s), 168.2 (s), 167.4
(S), 155.9 (s), 155.8 (s), 15
3.7 (s), 153.6 (s), 134.0 (s),
133.2 (s), 116.8 (s), 116.3
(S), 112.2 (s), 111.8 (s), 10
1.0 (d), 100.7 (d), 97.9 (s), 9
7.8 (s), 73.5 (d), 73.4 (d), 5
3.4 (d), 46.6 (t), 43.8 (t), 4
1.7 (s), 41.7 (s), 41.3 (t), 3
9.3 (d), 39.3 (d), 37.3 (s), 3
7.3 (s), 36.5 (d), 36.4 (d), 3
1.6 (t), 31.6 (t), 30.7 (t), 3
0.6 (t), 28.6 (q), 28.6 (q), 2
8.4 (t), 27.1 (t), 24.8 (t), 2
4.8 (t), 23.8 (t), 23.8 (t), 2
3.3 (t), 22.3 (q), 22.3 (q), 2
0.4 (t), 20.4 (t), 15.9 (q), 1
5.8 (q), 15.5 (q), 15.4 (q) Physicochemical properties of AM6222B substance (a) Color and state of substance White powder (b) Distinction between acidic, basic and neutral Acid substance (C) Specific optical rotation [α] _D ²³ −64 ° ± 10 ° (c = 0.4, methano-
(D) Molecular formula C ₅₂ H ₇₀ O ₁₁ N ₂ (e) Molecular weight 898 FAB mass spectrum [(M + H) ⁺ : 89
9.5026] (f) Elemental analysis Calculated value (%) C, 69.46; H, 7.85; N,
3.12 Found (%) C, 69.22; H, 7.76; N,
2.97 (g) Solubility Soluble in methanol, dimethylsulfoxide and alkaline water; insoluble in water, benzene and n-hexane. (H) Color reaction The iodine vapor reaction and potassium permanganate decolorization reaction are positive, and the ferric chloride reaction, ninhydrin reaction, and Molish reaction are negative. (I) Rf value 0.30 manufactured by Merck & Co., Kieselgel 60F
₂₅₄ used Developing solvent: chloroform / methanol / acetic acid (10:
3: 0.1) (j) Ultraviolet absorption spectrum Main absorption peaks (nm) in methanol are as follows. 218, 265, 300-310 (k) Infrared absorption spectrum The main wave numbers (cm ⁻¹ ) in KBr tablets are as follows. 3400, 2950, 1660, 1470, 1350,
1080 (l) ¹ H-NMR spectrum d ₆ - ¹ H-NMR as measured in dimethyl sulfoxide
The spectrum (400 MHz) is shown in FIG. (M) ¹³ C-NMR spectrum d ₆ - ¹³ C-NMR were measured in dimethyl sulfoxide
The signal in the spectrum (100 MHz) is found below (ppm). 172.8 (s), 168.2 (s), 167.3
(S), 155.8 (s), 155.8 (s), 15
3.7 (s), 153.6 (s), 134.0 (s),
133.3 (s), 116.7 (s), 116.3
(S), 112.3 (s), 111.8 (s), 10
0.8 (d), 100.8 (d), 97.8 (s), 9
7.7 (s), 77.7 (d), 73.4 (d), 6
4.9 (d), 53.5 (d), 46.6 (t), 4
3.8 (t), 42.9 (s), 41.8 (s), 4
1.3 (t), 39.3 (d), 38.7 (d), 3
8.1 (s), 37.3 (s), 36.4 (d), 3
6.3 (d), 32.9 (t), 31.8 (t), 3
1.6 (t), 30.7 (t), 30.6 (t), 2
8.9 (q), 28.6 (q), 28.4 (t), 2
7.1 (t), 24.9 (t), 23.8 (t), 2
3.3 (t), 22.3 (q), 22.0 (q), 2
0.4 (t), 20.2 (t), 16.8 (q), 1
5.8 (q), 15.4 (q), 15.4 (q) Physicochemical properties of AM6222C substance (a) Color and state of substance White powder (b) Distinction between acidic, basic and neutral Acid substance (C) Specific optical rotation [α] _D ²³ −65 ° ± 10 ° (c = 0.4, methano-
(D) Molecular formula C ₅₂ H ₇₀ O ₁₁ N ₂ (e) Molecular weight 898 FAB mass spectrum [(M + H) ⁺ : 89
9.5067] (f) Elemental analysis Calculated value (%) C, 69.46; H, 7.85; N,
3.12 Found (%) C, 69.22; H, 7.62; N,
3.02 (g) Solubility Soluble in methanol, dimethylsulfoxide and alkaline water; insoluble in water, benzene and n-hexane. (H) Color reaction The iodine vapor reaction and potassium permanganate decolorization reaction are positive, and the ferric chloride reaction, ninhydrin reaction, and Molish reaction are negative. (I) Rf value 0.43 made by Merck & Co., Kieselgel 60F
₂₅₄ used Developing solvent: chloroform / methanol / acetic acid (10:
3: 0.1) (j) Ultraviolet absorption spectrum Main absorption peaks (nm) in methanol are as follows. 218, 265, 300-310 (k) Infrared absorption spectrum The main wave numbers (cm ⁻¹ ) in KBr tablets are as follows. 3400, 2950, 1660, 1470, 1350,
1080 (l) ¹ H-NMR spectrum d ₆ - ¹ H-NMR as measured in dimethyl sulfoxide
The spectrum (400 MHz) is shown in FIG. (M) ¹³ C-NMR spectrum d ₆ - ¹³ C-NMR were measured in dimethyl sulfoxide
The signal in the spectrum (100 MHz) is found below (ppm). 172.7 (s), 168.2 (s), 167.3
(S), 155.9 (s), 155.7 (s), 15
3.7 (s), 153.6 (s), 134.1 (s),
133.2 (s), 116.8 (s), 116.2
(S), 112.3 (s), 111.9 (s), 10
0.9 (d), 100.8 (d), 97.9 (s), 9
7.5 (s), 77.6 (d), 73.4 (d), 6
4.8 (d), 53.4 (d), 46.5 (t), 4
3.8 (t), 42.9 (s), 41.7 (s), 4
1.3 (t), 39.3 (d), 38.7 (d), 3
8.0 (s), 37.2 (s), 36.5 (d), 3
6.1 (d), 32.7 (t), 31.7 (t), 3
1.7 (t), 30.7 (t), 30.7 (t), 2
8.8 (q), 28.6 (q), 28.4 (t), 2
7.1 (t), 24.8 (t), 23.8 (t), 2
3.3 (t), 22.3 (q), 21.9 (q), 2
0.4 (t), 20.2 (t), 16.7 (q), 1
5.8 (q), 15.5 (q), 15.3 (q) 2. AM6222A belonging to the genus Stachybotrys
AM6222A substance, AM6222 substance characterized by culturing a substance, an AM6222B substance or an AM6222C substance producing bacterium to produce an AM6222A substance, an AM6222B substance or an AM6222C substance, and collecting these substances
Method for producing substance B, AM6222C substance or salts thereof. 3. An endothelin antagonist comprising one or more substances selected from the group consisting of AM6222A substance, AM6222B substance, AM6222C substance and salts thereof as an active ingredient.