JPH11158109A - 2h-indene spiro-(3'-cyclohexene) compound - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide the subject new compound having excellent chymase inhibiting action and antimycotic action and useful as pharmaceuticals. SOLUTION: This invention discloses the compound expressed by the formula, its tautomer, its steric isomer, compounds produced by treating the above compounds with aqueous ammonia or an ammonium salt, their salts and hydrates. The compound of the formula can be produced by culturing a microbial strain capable of producing the compound of the formula [e.g. a microbial strain belonging to the genus Stachybotrys such as Stachybotrys parvispora Hughes RF-10131 (FERM P-16449)] in a culture medium composition under the culture medium condition of conventional fermentation production process (the culture temperature is preferably 15-32 deg.C and the initial pH is 6-8) for several days to several weeks and separating the compound from the culture product by conventional process for the separation of fermentation product.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a novel compound and a medicament containing the same, particularly to a chymase inhibitor and / or an antifungal agent containing a novel 2H-indene spiro- (3'-cyclohexene) compound.

[0002]

2. Description of the Related Art Human chymase is a neutral serine protease having a molecular weight of about 30,000, isolated in 1990, and is synthesized, stored and secreted mainly in mast cells, and is used in the heart, blood vessels,
It has been found to be present on the skin. Its main effect is the production of angiotensin II. Conventionally,
It was thought that angiotensin converting enzyme (hereinafter, abbreviated as ACE) acts on the production of angiotensin II, but recently, ACE acts on the production of angiotensin II in the human heart. It has been revealed that only about 10 to 15% is involved, and more than 80% is caused by the action of human chymase (Circulation Research).
Research) Vol. 66, p. 883, 1990
Year, Journal of Biological Chemistry (Journal of Biological Ch
mistry), vol. 266, p. 17173, 19
1991). For this reason, chymase inhibitors are attracting attention as a new type of therapeutic agent for cardiovascular disorders. Chymase has also been implicated in promoting histamine release from mast cells (see Journal of Biochemistry).
y) 103, 820-822, 1988),
The inhibitors hold promise as potential new types of anti-inflammatory and anti-allergic agents.

[0003] In addition, human chymase has various activities, such as promotion of macrophage foam cell formation in vitro, production of active collagenase from procollagenase, and limitation of extracellular matrix such as collagen, fibronectin and vitronectin. Degradation, thrombin and Ig
It has already been clarified that it has an action such as limited decomposition of G. It is also known pathologically that chymase activity is elevated in blood vessels after balloon injury and in hearts with cardiomyopathy. From such findings, it can be predicted that chymase is also involved in arteriosclerosis, inflammation, rheumatism and the like.

To date, International Patent Application Publication No. 93/2
5574, 95/27053, 95/2705
No. 5 discloses a peptidic chymase inhibitor. Further, as non-peptide chymase inhibitors, imidazolidine derivatives are disclosed in WO 96/04248, and triazine derivatives are disclosed in EP 713876. Further, as a chymase inhibitor having a similar structure to the compound of the present application, Japanese Patent Application No. 8-261687.
There is a number.

[0005]

At present, only a small number of non-peptide chymase inhibitors have been found as described above, and the physiological function of chymase has been elucidated.
Furthermore, for the development of clinically useful drugs, non-peptide chymase inhibitors having strong activity have been demanded.

[0006]

The present inventors have means for solving the problem] is a type of mold, Stachybotrys Parubisupora fuse (Sta
bathrooms parvispora Hugh
es) A compound having strong human chymase inhibitory activity was found in the culture of the RF-10131 strain, and the compound was isolated and purified to complete the present invention. Furthermore, they have found that the compounds of the present invention also have an antifungal effect.

That is, the present invention provides a compound represented by the formula (Ia):

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Which provides a compound represented by the formula (I), a tautomer thereof, a stereoisomer thereof, a compound obtained by treating them with aqueous ammonia or an ammonium salt, a pharmaceutically acceptable salt thereof or a hydrate thereof. is there. Further, the present invention provides a compound (I-I) wherein the compound treated with the above-mentioned ammonia water or ammonium salt has the following physical properties.
g), providing a pharmaceutically acceptable salt thereof or a hydrate thereof. Melting point: 132-135 ° C (decomposition) [α] _D ²⁵ = ± 0 ° (c = 0.5, methanol) Elemental analysis: C 73.88%, H 7.35%, N 3.37% FABMS, m / z; 422 HR- FABMS, m / z Found: 422.2331 IR (KBr) cm ^-1 ; 3287, 1657, 1640, 1611, 1545, 1524,
1425, 1273, 1228,1053 UV (MeOH), λ _max nm (E _1% ^1cm ); 216 (678), 276 (22
6), 368 (356) ¹ H NMR (CDCl ₃ , 300 MHz) δ: 1.52 (6H, s-like), 1.56
(6H, s-like), 1.94 (3H, d, J = 1.2 Hz), 2.31 (2H, m),
2.82 (2H, m), 3.94 (3H, s), 4.80 (2H, t-like), 6.23
(1H, d, J = 1.2 Hz), 6.27 (1H, br.s), 7.06 (1H, d,
J = 7.8 Hz) 7.43 (1H, t-like, J = 8.1 Hz), 8.40 (1H, br.s), 12.9
2 (1H, br.s) ¹³ C NMR (CDCl ₃ , 50 MHz) δ: 18.06, 19.21, 25.77, 3
6.94, 37.06, 55.80, 56.46, 82.79, 114.51, 118.95, 1
22.61, 129.65, 131.38, 131.54, 133.87, 135.78, 155.
13, 166.29

Further, the present invention provides a pharmaceutical composition characterized by containing a compound (Ia), a tautomer thereof, a stereoisomer thereof, a pharmaceutically acceptable salt thereof or a hydrate thereof. Products, in particular, chymase inhibitors and / or antifungal agents. The present invention also provides a pharmaceutical composition comprising the compound (Ig), a pharmaceutically acceptable salt thereof, or a hydrate thereof, specifically a chymase inhibitor and / or an antifungal agent. I do. In yet another embodiment, Stachyboli ( Stachyb)
otrys ), and cultivating a microorganism capable of producing the compound (Ia), a tautomer or a stereoisomer thereof, and culturing the compound, the tautomer or the stereoisomer thereof from the obtained culture. The present invention relates to a method for producing a compound (Ia), a tautomer thereof, a stereoisomer thereof, a pharmaceutically acceptable salt thereof or a hydrate thereof, which comprises collecting a body. In addition, Stachybotrys parvispora ( S
tachybotrys parvispora ) and a microorganism capable of producing the compound (Ia), a tautomer or a stereoisomer thereof, more specifically, a stachybotrys parvispora fuse RF-10131 ( Stach).
ybotrys parvispora Hughes
RF-10131).

The compound (Ia) of the present invention includes two tautomers (Ib) and (Ic) and (Id)
There are three stereoisomers, (Ie) and (If), and their separation is very difficult.

[0011]

Embedded image

The present invention provides compounds (Ia) to (I-
f) (hereinafter sometimes collectively referred to as compound (IA)), pharmaceutically acceptable salts thereof, and hydrates thereof. Compound (Ig), which is a derivative of compound (IA), obtained by treating compound (IA) with aqueous ammonia or an ammonium salt, a pharmaceutically acceptable salt thereof, or a water thereof. Also included are compounds (hereinafter, (IA) and (Ig) may be collectively abbreviated as compound (I)).

The method for producing the compound (I) of the present invention will be described below. In this method, a microorganism capable of producing the compound (IA) of the present invention is cultured under the medium composition and medium conditions used for normal fermentation production, and the compound (IA) is isolated and collected by a method of separating and collecting a normal fermentation product. ) Is isolated. Compound (I) of the present invention
Examples of microorganisms that can produce -A) include microorganisms belonging to the genus Stachybotrys, for example, Stachybotrys parvispora fuse RF-10131 strain. Stachybotrys Parvispora Fuse RF-10131
The strain had the following morphological properties.

The colonies on the cone mill agar medium are as follows:
It grows a little slower, its surface becomes moist, and it turns greenish black as conidia form. The conidiophores are solitary or branched and stand upright from the medium. The vegetative mycelium is colorless, buried in the medium,
The surface is smooth, has a width of 1.0 to 1.5 μm, is composed of multiple cells, and is branched. Each conidiophore is colorless and consists of 3-4 cells, height 65.0-100.0 μm, width 4.
0 to 7.0 μm, narrows toward the top, and becomes 2.5 to 3.0 μm in the phialide-adhered portion. The surface is smooth or granular, and the upper part is particularly strong. Fialide is a single cell with a height of 7.0 to 10.5 μm and a width of 3.5 to 4.5 μm, and has a club-like shape, a smooth surface, and a 4
~ 9 occur. Conidia are cylindrical single cells 5.5-6.5 μm in length and 2.0-3.0 μm in width, have an olive color, have a smooth or slightly rough surface, and have a spherical, wet gloss on phialide. It is formed as a conidium mass.

The colonies on the potato dextrose agar medium grow well, exhibit a grayish-yellow brown color, produce a yellow-brown soluble pigment, and form aerial hyphae. 10-35 ° C
And the optimum growth temperature is 23-30 ° C.
Since this strain does not form a sexual reproductive organ and has the property of forming conidia as a spherical wet shiny conidia mass at the apex of the phialide at the tip of the conidiophore, it is an incomplete bacterium belonging to the genus Stachybotrys It is determined that there is. These properties are described as Dermatiaseuus Hyphomycetes.
es), Commonwealth Mycological Institute, Kew, Slay, England (Commo)
nweth Mylogical Insti
Tute, Kew, Surrey, England),
542-544, 1971; More Dermatias Theeus Hyfomisates (More Derm)
atiaseus Hyphomycetes) Commonwealth Mycological Institute, Kew, Slay, England (Commonwealt)
h Mylogical Institute, K
ew, Surrey, England), pp. 463-
464, 1976; Matsushima Mycological Memoris
al Memoirs), No. 1-8, 1980-1
995; Mycological Papers
medical Papers) No. 48, pp. 74-7
6, 1952; Transactions of the British Mycology Society (Tran
actions of the British My
theoretical Society) 78, 3rd, 5th
56-559, 1982; Journal of the Antibacterial and Antifungal
Agents (Journal of the Ant)
ibacterial and Antifungal
Agents) 19, 11, 597-607,
As a result of comparison with known species of the genus Stachybotrys described in literatures such as 1991, the shape, size, surface structure, and other properties of conidia indicate that Stachybotrys parvispora fuse 1952 ( Stachybotrys p.
arvispora Hughes 1952).

This strain was deposited with the National Institute of Advanced Industrial Science and Technology (1-1-3 Higashi, Tsukuba, Ibaraki Prefecture) under the accession number "FERM P-16449" in 1997 (1992).
7 years) has been deposited since September 29.

As the medium for producing the compound (IA) of the present invention, any of a synthetic medium and a natural medium can be suitably used as long as it contains a carbon source, a nitrogen source and an inorganic salt. If necessary, vitamins or other nutrients may be added as appropriate.

Examples of the carbon source include sugars such as glucose, maltose, fructose, sucrose and starch; alcohols such as glycerol and mannitol;
One or more kinds may be appropriately selected and used from general carbon sources such as amino acids such as glycine, alanine and asparagine, and fats and oils such as soybean oil and olive oil in consideration of the assimilation of microorganisms. Nitrogen sources include soy flour, corn steep liquor, beef extract, peptone, yeast extract,
Amino acid mixture, organic nitrogen-containing compounds such as fish meal or ammonium salts, inorganic nitrogen compounds such as nitrates and the like,
One or more kinds may be appropriately selected and used in consideration of the assimilation property of the microorganism. As the inorganic salt, for example, calcium carbonate, sodium chloride, potassium chloride, magnesium sulfate, copper sulfate, manganese chloride, zinc sulfate, cobalt chloride, and various phosphates may be added as needed. Further, an antifoaming agent, for example, vegetable oil, polypropylene glycol or the like can be added as needed.

The culturing temperature can be appropriately changed within a range in which the microorganism grows and produces the compound (IA) of the present invention, but it is preferably 15 ° C to 32 ° C, more preferably 25 ° C to 32 ° C.
30 ° C. The initial pH is preferably around 6 to 8, and the culture time is usually about several days to several weeks. When the production amount of the compound (IA) of the present invention reaches a retrievable amount, preferably the maximum, the culture is continued. It should just end. The culture method is solid-phase culture,
Any of the commonly used methods such as aeration and agitation culture can be suitably used.

The method of separating and collecting the fermentation product from the culture includes filtration, centrifugation, various ion exchange resins and other active adsorption, chromatography, extraction with various organic solvents, and the like. A material separation and purification method can be used. For example, cells isolated and purified from a culture may be extracted with an organic solvent such as ethyl acetate, acetone, or methyl ethyl ketone, and then separated and purified by a combination of silica gel column chromatography and thin layer chromatography.

The compound (I-
A) can be led to various derivatives by a conventional method. For example, compound (IA) is dissolved in a suitable solvent,
The compound (Ig) can be obtained by treatment with aqueous ammonia or an ammonium salt. As a solvent, methanol,
A water-miscible solvent such as acetonitrile, tetrahydrofuran, and acetone is preferable, and the ammonium salt is preferably a salt with a weak acid such as ammonium carbonate, ammonium hydrogen carbonate, and ammonium acetate. The present invention includes pharmaceutically acceptable and pharmaceutically acceptable salts of compound (I), such as alkali metal salts such as sodium and potassium, and alkaline earth metal salts such as calcium and barium. These salts can be formed using commonly used reactions. The present invention also includes a hydrate of the compound (I), and one molecule of the compound (I) may be coordinated with an arbitrary number of water molecules.

Compound (I), a pharmaceutically acceptable salt thereof or a hydrate thereof has strong chymase inhibitory activity and / or
Alternatively, since it has antifungal activity, it can be administered to humans and other animals as a pharmaceutical or veterinary drug. Specific examples of chymase inhibitors include myocardial infarction, cardiac hypertrophy, heart failure, cardiomyopathy, hypertension, intimal thickening after PTCA (percutaneous coronary angioplasty), peripheral circulatory disorders, vasculitis, and atherosclerosis It is effective in treating chronic cardiovascular disorders such as diabetic and non-diabetic renal disorders and allergic diseases such as inflammation, allergy, rheumatism and atopy. As an antifungal agent, it is effective for the treatment of deep mycosis such as visceral candidiasis, pulmonary aspergillosis and dermatomycosis.

When compound (I), a pharmaceutically acceptable salt thereof or a hydrate thereof is administered as a medicine, it can be administered orally or parenterally. Oral administration may be carried out according to a conventional method and prepared and administered in a commonly used form such as tablets, granules, powders, capsules, pills, solutions, suspensions, syrups, buccals, and sublinguals. For parenteral administration, any commonly used dosage form, such as injections such as intramuscular administration, suppositories, transdermal absorption agents, inhalants, etc., can be suitably administered, but intravenous administration is particularly preferred.

The pharmaceutical composition of the present invention comprises various pharmaceutical additives such as excipients, binders, wetting agents, disintegrants, lubricants and diluents suitable for the final dosage form in an effective amount of the active ingredient. Can be mixed and prepared as needed. In the case of an injection, a preparation may be prepared by sterilizing with an appropriate carrier.

Specifically, the excipients include lactose, sucrose,
Glucose, starch, calcium carbonate or crystalline cellulose, etc .; binders such as methylcellulose, carboxymethylcellulose, hydroxypropylcellulose, gelatin or polyvinylpyrrolidone; disintegrants: carboxymethylcellulose, sodium carboxymethylcellulose, starch, sodium alginate, agar powder or lauryl Lubricants such as sodium sulfate include talc, magnesium stearate and macrogol. Cocoa butter, macrogol, methylcellulose, or the like can be used as a suppository base. In addition, when prepared as a liquid or emulsion, suspension injection, a solubilizing agent usually used,
Suspending agents, emulsifiers, stabilizers, preservatives, isotonic agents and the like may be appropriately added, and in the case of oral administration, a flavoring agent, a fragrance and the like may be added.

The dose of the chymase inhibitor is desirably determined in consideration of the patient's age, body weight, administration route, type and degree of disease, and the like. Usually 1 μg to 1000 mg /
The dose may be administered once to several times per kg / day. Also,
When administered parenterally, 0.1 μg to 100 mg / kg / day usually varies depending on the administration route.
It may be administered in one to several doses. The dosage as an antifungal agent is also preferably set in consideration of the patient's age, weight, administration route, type and degree of disease, etc.
When administered orally to humans, the dose is usually 1
In the case of parenteral administration of μg to 200 mg / kg / day, 0.1 μg to 20 mg / kg / day may be administered once to several times each.

[0027]

EXAMPLES The present invention will be described in more detail with reference to the following Examples, but it should not be construed that the present invention is limited thereto. Example 1 Production of Compound (IA) (1) (1) Fermentation Step To 200 ml of broth obtained by boiling 200 g of potato, 10 g of glucose and 15 g of agar were added and sterilized. And This was coated with Stachybotrys parvispora fuse RF-10131 and cultured at 28 ° C. for 10 days. 500 ml of physiological saline was added to the 20 obtained Petri dishes, and the mixture was pulverized while containing agar to prepare a bacterial solution. Each 8 ml of this bacterial solution was inoculated into 96 500 ml Mayer flasks containing brown rice medium (brown rice 25 g, Difco yeast extract 0.25 g, tap water 50 ml) autoclaved at 121 ° C. for 30 minutes, and incubated at 28 ° C. for 26 days. Stationary culture was performed.

(2) Separation Step The fermented products in each of the Meyer flasks obtained in the fermentation step were collected, added with 40 L of acetone, and extracted with stirring. After suction filtration, the solvent in the filtrate containing acetone is distilled off under reduced pressure, and the aqueous layer of the residue is extracted with 15 L of ethyl acetate. The ethyl acetate layer was concentrated to dryness to obtain a crude fraction (94 g). A part (40 g) of this crude fraction was purified by silica gel column chromatography (Merck Kieselgel 60, 180 g, 230-400 mesh, toluene: acetone (9: 1)).
Toluene: acetone (9: 1) was eluted with a total of 1,800 ml, and the first 800 ml was collected to obtain an A fraction, and the two fractions were combined and evaporated under reduced pressure to obtain a B fraction (16.0 g). Was.

The B fraction was again subjected to silica gel column chromatography (Merck Kieselgel 60, 120 g, 230
-400 mesh, chloroform) to obtain a C fraction (5.0 g). Furthermore, silica gel column chromatography (Merck Kieselgel 60, 100 g, 230
Purified with ４００400 mesh, toluene, toluene: acetonitrile (97: 3), (94: 6). The fractions eluted with toluene: acetonitrile (97: 3) and (94: 6) were collected, the solvent was distilled off under reduced pressure, and the D fraction (1.8) was obtained.
1 g). The D fraction (1.81 g) was subjected to silica gel column chromatography (Merck Kieselgel 60, 1).
10 g, 230-400 mesh, chloroform). Fractions of 15 grams each were collected, and fractions 69 to 81 were collected and the solvent was distilled off under reduced pressure. Diethyl ether (2 ml) was added to the residue to recrystallize the compound (I-
A) (307 mg) was obtained. In this (IA), at least one of the compounds (Ia) and (Ie), (Ib), (Ic) and (Id) and (I- The presence of either or both compounds of f) was confirmed by ¹ HNMR.

Compound (IA) (physicochemical properties) Properties: colorless needle crystals Solubility: soluble in chloroform, acetone, ethyl acetate, methanol Slightly soluble in n-hexane Insoluble in water Melting point: 105-105 107 ° C. [α] _D ²³ = ± 0 ° (c = 0.5, methanol) Elemental analysis: (as C ₂₆ H ₃₀ O ₅ ) Calculated; C: 73.91%, H: 7.16% C: 73.69%, H: 7.22% FABMS, m / z: 423 [M + H] ⁺

IR, λ _max ^KBr cm ^-1 : 3520-34
00, 1725, 1696, 1648, 1629, 16
02, 1485, 1275, 1250, 1080, 10
54,967 UV (methanol), nm (ε): 222 (31, 70
0), 255 (sh, 13, 400), 315 (5, 1
00)

(Ia) and / or (Ie) ¹ H NMR (CDCl ₃ , 600 MHz) δ: 2.127
(3H, d, J = 1.1 Hz), 3.801 (1H,
d, J = 10 Hz), 3.956 (3H, s), 5.6
28 (1H, d, J = 10 Hz), 6.455 (1H,
q, J = 1.1 Hz), 7.137 (1H, d, J =
8.0Hz), 7.292 (1H, d, J = 7.8H)
z), 7.438 (1H, t, J = about 8 Hz) ¹³ CNMR (CDCl ₃ , 150 MHz) δ: 20.5
1,55.92,58.17,75.73,78.9
1,116.13,116.86,130.39,13
1.51, 136.73, 143.29, 157.4
1, 163.54, 194.33, 194.83, 20
9.51

(Ib) ¹ H NMR (CDCl ₃ , 600 MHz) δ: 2.023
(3H, d, J = 1.3 Hz), 3.939 (3H,
s), 6.325 (1H, q, J = 1.3 Hz), 6.
578 (1H, d, J = 7.9 Hz), 7.016 (1
H, d, J = 7.8 Hz), 7.537 (1H, t, J)
= About 8 Hz), 10.392 (1H, s) ¹³ C NMR (CDCl ₃ , 150 MHz) δ: 20.3
0, 55.75, 58.36, 110.23, 111.
68, 117.24, 121.83, 122.40, 1
35.18, 142.82, 161.38, 166.1
7, 183.75, 188.44, 197.40, 20
1.21

(Id) and / or (If) ¹ H NMR (CDCl ₃ , 600 MHz) δ: 2.117
(3H, d, J = 1.1 Hz), 3.332 (1H,
d, J = 11 Hz), 3.967 (3H, s), 5.9
74 (1H, d, J = 11 Hz), 6.453 (1H,
q, J = 1.1 Hz), 7.147 (1H, t, J = about 8 Hz), 7.259 (1H, d, J = about 8 Hz),
7.428 (1H, t, J = about 8 Hz) ¹³ CNMR (CDCl ₃ , 150 MHz) δ: 20.5
1,55.84,58.76,73.03,80.6
3,116.27,116.99,129.48,13
1.42, 136.09, 143.29, 157.4
5,162.04, 191.82, 193.31, 20
8.63

(Ic) ¹ H NMR (CDCl ₃ , 600 MHz) δ: 1.897
(3H, d, J = 1.3 Hz), 3.945 (3H,
s), 5.936 (1H, d, J = 1.3 Hz), 6.
663 (1H, q, J = about 8 Hz), 7.032 (1
H, d, J = 7.8 Hz), 7.556 (1H, t, J)
= About 8 Hz), 10.390 (1H, s) ¹³ C NMR (CDCl ₃ , 150 MHz) δ: 18.4
5, 53.05, 55.84, 111.91, 113.
97, 117.31, 122.09, 129.68, 1
35.43, 142.44, 154.83, 161.3
8, 182.73, 188.51, 195.84, 20
1.40

In addition to the above, the following signals are observed. ¹ H NMR (CDCl ₃ , 600 MHz) δ: 1.466
(Br.s-like), 1.539 (br.s-li
ke), 1.566 (br. s-like), 1.57
2 (br.s-like), 1.617 (br.s-l)
ike), 1.644 (br.s-like), 1.6
56 (br.s-like), 1.702 (br.s-like)
like), 2.20-2.92 (m), 4.67-
5.05 (m) ¹³ C NMR (CDCl ₃ , 150 MHz) δ: 17.8
6, 17.89, 17.99, 18.04, 25.7
6,25.81,25.88,34.95,35.6
9, 36.42, 38.00, 38.34, 38.7
4,116.28,117.28,117.51,11
7.98, 118.65, 119.10, 134.4
1,134.72,134.93,137.50,13
7.55

Example 2 Production of Compound (IA) (2) 1 g of water was added to 200 g of potato, and after boiling, 10 g of glucose and 15 g of agar were added to a solution filtered with gauze to prepare a PG agar plate medium. Then apply Stachybotrys Parvispora fuse RF-10131 and apply at 28 ° C, 1
Cultured for 4 days. Agar plates grown on the entire surface are 6 mm in inner diameter.
A stainless steel pipe was used to make an agar piece.
Twelve agar pieces were crushed in physiological saline, and glucose 2%, polypeptone (Nippon Pharmaceutical) 1%, meat extract (Difco) 0.3%, yeast extract (Difco)
(Manufactured by Co., Ltd.) and a 2 L Meyer flask containing 800 ml of a medium containing 0.1% of sodium chloride. The inoculated flask was cultured at 28 ° C. at 180 rotations per minute for 4 days to obtain a seed. The above-described inoculum was placed in a 50-L jar fermenter containing 30 L of a PC medium consisting of 4% of potato starch, 0.5% of CA-1 (powder-made by Nippon Clea) and 0.03% of adecanol LG-121 (made by Asahi Denka Kogyo). Was inoculated and cultured at 28 ° C. for 60 days at 60 rotations per minute under aeration at 20 L / min for 14 days.

Similarly, another jar fermenter was cultured, and 38 L of the fermentation end solution collected from the two jar fermenters was centrifugally filtered to separate an aqueous layer and cells. 10 L of acetone
After filtration, acetone was distilled off under reduced pressure to obtain 4 L of an aqueous layer. 4 L of water was added thereto, followed by extraction with 8 L of ethyl acetate. The ethyl acetate layer was concentrated under reduced pressure to obtain 96 g of a crude substance. This crude substance was dissolved in 200 mL of toluene, applied to a φ12 cm column packed with 1 kg of silica gel (70 to 230 mesh, manufactured by Merck), eluted with a mixed solution of toluene and acetonitrile (95: 5), and fractionated by 400 mL. Fractions 8 to 14 containing (IA) were collected and concentrated under reduced pressure. The oil was crystallized from diethyl ether (I-
A) 2.61 g of crystals were obtained.

Example 3 Preparation of Compound (Ig) To a solution of 80 mg (0.19 mmole ) of (IA) in acetonitrile (12 ml) was added a 0.05 mmol aqueous ammonium bicarbonate solution (4 ml). Stirred for .5 hours. Acetonitrile was distilled off under reduced pressure, and diethyl ether (2 ml) was added to the obtained residue, followed by recrystallization to obtain 54 mg of a compound (Ig).

Appearance: yellow crystals Solubility: soluble in ethyl acetate, acetone, chloroform, methanol, ethanol Slightly soluble in n-hexane Insoluble in water Melting point: 132-135 ° C. (decomposition) [α] _D ²⁵ = ± 0 ° (c = 0.5, methanol) Elemental analysis: Calculated for C ₂₆ H ₃₁ NO ₄ ; C: 74.08%, H: 7.41%, N: 3.32% Found: C: 73 .88%, H: 7.35%, N: 3.37% FABMS, m / z: 422 [M + H] ⁺ HR-FABMS, m / z: C ₂₆ H ₃₂ NO ₄ Calculated; 422.2332 Value; 422.2331 [M + H] ⁺

IR (KBr) cm ^-1 : 3287,16
57, 1640, 1611, 1545, 1524, 14
25, 1273, 1228, 1053 UV (MeOH), λ _max nm (E _1% ^{1 cm} ): 216 (6
78), 276 (226), 368 (356) ¹ H NMR (CDCl ₃ , 300 MHz) δ: 1.52
(6H, s-like), 1.56 (6H, s-like)
e), 1.94 (3H, d, J = 1.2 Hz), 2.3.
1 (2H, m), 2.82 (2H, m), 3.94 (3
H, s), 4.80 (2H, t-like), 6.23.
(1H, d, J = 1.2 Hz), 6.27 (1H, b
r. s), 7.06 (1H, d, J = 7.8 Hz),
7.43 (1H, t-like, J = 8.1 Hz),
8.40 (1H, br.s), 12.92 (1H, b.
r. s) ¹³ C NMR (CDCl ₃ , 50 MHz) δ: 18.0
6, 19.21, 25.77, 36.94, 37.0
6,55.80,56.46,82.79,114.5
1,118.95,122.61,129.65,13
1.38, 131.54, 133.87, 135.7
8,155.13,166.29

From the above data and X-ray analysis, (I-
The structure of g) is assumed to be as follows.

Embedded image

Test Example 1 Chymase Enzyme Inhibitory Activity (1) Preparation of Compound (IA) Compound (IA) was prepared from dimethyl sulfoxide (DMS).
O) to give a concentration of 10 mg / ml. The DMSO concentration brought in for the activity measurement was 1%. (2) Measurement of chymase inhibitory activity Buffer solution (0.1 M Tris-HCl, 1.8 M NaCl
Compound (I-) dissolved in DMSO in pH 8.0)
A) and the method of Urata et al. (Journal of Biological Chemistry (Journal of Biolo)
medical Chemistry) Vol. 265, No. 22
348 to 22357), and treated at 37 ° C. for 30 minutes.
c-Ala-Ala-Pro-Phe-pNA (BACHEM Feinchemikalen
AG)) at a concentration of 0.5 mM.
The enzyme reaction was carried out. After the reaction, the absorbance of the solution (405
nm) was measured, and the inhibition rate was calculated. (3) Results The compound (IA) had a 50% inhibitory concentration (IC ₅₀ ) of human chymase inhibitory activity of 1.8 μM, indicating that the compound (IA) had chymase inhibitory activity.

Test Example 2 Antifungal Activity (1) Method for Preparing Compound (I) The compounds of the present invention were all prepared using dimethyl sulfoxide (DMS).
O) to give a concentration of 10 mg / ml. (2) Measurement of activity Candida albicans Ca-15 ( Candida
albicans ), Aspergillus fumigatus
MA ( Aspergillus fumigatus)
MA) (Sabouraud dextrose broth supplemented with 10% fetal bovine serum (Sabouraud)
about 2 × 10 ⁸ cfu
/ Ml and stored at -80 ° C)
To East Nitrogen Base (Yeast N)
(Itrogen Base; YNB, manufactured by Difco)
It was diluted to 1 × 10 ⁵ cfu / ml with the medium, and 100 μl was dispensed into each well of a 96-well microplate. The compound of the present invention is diluted with this bacterial solution to prepare a two-fold dilution series,
Growth inhibition was examined.

(3) Determination of culture conditions and results The determination of growth inhibition was determined by culturing at 30 ° C. for 24 hours and then measuring the absorbance at 595 nm using an autoreader (BIO-RAD). 50% of the absorbance of
The concentration that inhibits the growth of bacteria is defined below as the minimum effective concentration (M
(IC ₅₀ ). Table 1 shows the results.

[0046]

[Table 1] Table 1 shows that the compound (I) of the present invention has antifungal activity.

Formulation Example 1 Compound (IA) 3 mg Gelatin 150 mg Phenol 4 mg The above components were dissolved in an appropriate amount of purified water for injection to prepare an injection solution.

[0048]

As is clear from the above test examples, compound (I) exhibits chymase inhibitory activity and / or antifungal activity. Therefore, compound (I), a pharmaceutically acceptable salt thereof or a hydrate thereof is very useful as a therapeutic agent for chronic cardiovascular disorders, a therapeutic agent for allergic diseases and / or an antifungal agent.

[Brief description of the drawings]

FIG. 1 is a graph showing an IR spectrum of compound (IA).

FIG. 2 is a graph showing ¹ HNMR spectrum of compound (IA).

FIG. 3 is a graph showing a ¹³ C NMR spectrum of compound (IA).

FIG. 4 is a graph showing an IR spectrum of compound (Ie).

FIG. 5 is a graph showing ¹ HNMR spectrum of a compound (Ig).

FIG. 6 is a graph showing a ¹³ C NMR spectrum of compound (Ig).

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification code FI C12R 1: 645)

Claims (12)

[Claims] 1. A compound of the formula (Ia): In the compound represented by, its tautomer, its stereoisomer,
Compounds obtained by treating them with aqueous ammonia or ammonium salts, pharmaceutically acceptable salts thereof, or hydrates thereof. 2. A compound represented by the formula (Ia) according to claim 1, a tautomer, a stereoisomer, a pharmaceutically acceptable salt or a hydrate thereof. 3. A compound (Ig), a pharmaceutically acceptable salt or a hydrate thereof, wherein the compound treated with the aqueous ammonia or ammonium salt according to claim 1 has the following physical properties. Melting point: 132-135 ° C (decomposition) [α] _D ²⁵ = ± 0 ° (c = 0.5, methanol) Elemental analysis: C 73.88%, H 7.35%, N 3.37% FABMS, m / z; 422 HR- FABMS, m / z Found: 422.2331 IR (KBr) cm ^-1 ; 3287, 1657, 1640, 1611, 1545, 1524,
1425, 1273, 1228,1053 UV (MeOH), λ _max nm (E _1% ^1cm ); 216 (678), 276 (22
6), 368 (356) ¹ H NMR (CDCl ₃ , 300 MHz) δ: 1.52 (6H, s-like), 1.56
(6H, s-like), 1.94 (3H, d, J = 1.2 Hz), 2.31 (2H, m),
2.82 (2H, m), 3.94 (3H, s), 4.80 (2H, t-like), 6.23
(1H, d, J = 1.2 Hz), 6.27 (1H, br.s), 7.06 (1H, d,
J = 7.8 Hz), 7.43 (1H, t-like, J = 8.1 Hz), 8.40 (1H, br.
s), 12.92 (1H, br.s) ¹³ C NMR (CDCl ₃ , 50 MHz) δ: 18.06, 19.21, 25.77, 3
6.94, 37.06, 55.80, 56.46, 82.79, 114.51, 118.95, 1
22.61, 129.65, 131.38, 131.54, 133.87, 135.78, 155.
13, 166.29 4. It contains a compound represented by the formula (Ia) according to claim 1, a tautomer, a stereoisomer, a pharmaceutically acceptable salt thereof or a hydrate thereof. A pharmaceutical composition comprising: 5. A pharmaceutical composition comprising the compound according to claim 3, a pharmaceutically acceptable salt thereof or a hydrate thereof. 6. A compound represented by the formula (Ia) according to claim 1, a tautomer thereof, a stereoisomer thereof, a pharmaceutically acceptable salt thereof or a hydrate thereof. A chymase inhibitor. 7. A chymase inhibitor comprising the compound according to claim 3, a pharmaceutically acceptable salt thereof or a hydrate thereof. 8. A compound represented by the formula (Ia) according to claim 1, a tautomer, a stereoisomer, a pharmaceutically acceptable salt or a hydrate thereof. An antifungal agent characterized by the following. 9. An antifungal agent comprising the compound according to claim 3, a pharmaceutically acceptable salt thereof or a hydrate thereof. 10. Stachybotris ( Stachybotris)
lys ), and culturing a microorganism capable of producing a compound represented by the formula ( Ia ) according to claim 1, a tautomer or a stereoisomer thereof, and culturing the compound from the obtained culture. 2. The compound according to claim 1, wherein the tautomer or its stereoisomer is collected, the tautomer, its stereoisomer, their pharmaceutically acceptable salt or their hydration. Method of manufacturing a product. 11. Stachybotrys parvispora ( St)
achybotrysparvispora ),
A microorganism capable of producing the compound represented by the formula (Ia) according to claim 1, a tautomer or a stereoisomer thereof. 12. A stachybotrys Parvispora fuse RF-10131 ( Stachybotries)
parvispora Hughes RF-1013
The microorganism according to claim 11, which is 1).