Novel serotonin inhibitors and pharmaceutical compositions containing them, and microbiological processes and organisms for the production thereof

Abstract: Novel physiologically active substances KS-501 and KS-502 of the formula : wherein R is hydrogen or carboxyl having inhibitory activity on serotonin release from platelets and/or platelet aggregation are produced by culturing a microorganism of the genus Sporothrix .

Description: This invention relates to compounds capable of inhibiting serotonin release from platelets and/or platelet aggregation, a process for preparing the same, and a pharmaceutical composition containing the same. It is already known that physiologically active substances capable of inhibiting serotonin release from platelets, and/or platelet aggregation, can be obtained by the culture of certain microorganisms. For example, the following compunds, all of microbiological origin, have been reported: Pyrrothines Chem. Pharm. Bull. 28 , 3157 - 3162 (1980) thiolutin : R = CH₃ aureothricin : R = CH₂CH₃ isobutyropyrrothine: R = CH(CH₃)₂ WF-5239 J. Antibiot. 37 , 469 - 474 (1984) WF-30581 J. Antibiot. 37 , 1153 - 1160 (1984) WF-30581A: R = CH₂CH₂CH₃ WF-30581B: R = CH₂CH₂CH₂CH₃ KS-290IIi-2 Japanese Published Unexamined Patent Application No. 195689/86 Staurosporine Agric. Biol. Chem. 50 , 2723 - 2727 (1986) Results of the recent research of the function of serotonin released from platelets suggest that serotonin released from platelets contributes to platelet aggregation and contraction of vessels, especially cerebral vessels. Therefore, substances inhibiting serotonin release from platelets, as well as substances inhibiting platelet aggregation, are considered to be useful as antithrombotic agents, especially those for cerebral thrombosis. According to the present invention, two novel physiologically active substances, herein identified as KS-501 and KS-502, have been discovered having serotonin release inhibiting activity, and platelet aggregation inhibiting activity, and which are therefore potentially useful as anti-thrombotic agents, especially for cerebral thrombosis. These compounds are produced by culturing microorganisms of the genus Sporothrix in a culture medium until KS-501 and/or KS-502 are accumulated in the culture, and recovering KS-501 and/or KS-502 therefrom. The physicochemical properties of KS-501 and KS-502 are given below. KS-501 State: white powder Melting point: 149 to 152°C Specific rotation: [α] 23 / D -53° (c=0.3, methanol) Solubility: Readily soluble: chloroform, ethyl acetate, acetonitrile, methanol, n-butanol, acetone, tetrahydrofuran, dioxane, dimethylsulfoxide, pyridine, monoethanolamine, acetic acid, alkaline water Insoluble: hexane, carbon tetrachloride, benzene, neutral water, acidic water Colour reaction: Positive in reactions with iodine, anisaldehyde and ferric chloride; negative in reaction with aniline-phthalic acid and ninhydrin and Reiden-Smith reactions. Ultraviolet absorption spectrum: Acidic and neutral methanol solution: λ max (ε) 254 nm (6760), 278 nm (5560, sh) Alkaline methanol solution: λ max (ε) 234 nm (16100, sh), 292 nm (11500) Infrared absorption spectrum: KBr 3380, 2960, 2924, 2852, 1722, 1608, 1586, 1466, 1323, 1264, 1168, 1149, 1129, 1064, 996 cm⁻¹ Mass spectrum: SIMS, positive m/z 605 (M+H)⁺, 443, 235 ¹H-NMR (400 MHz, CD₃OD, δ): 6.58 (1H, d, J=2.1Hz), 6.55-6.50 (3H, m), 6.38 (1H, d, J=2.1Hz), 5.55 (1H, d, J=1.8Hz), 4.28 (1H, dd, J=3.9, 1.8Hz), ca. 4.1 (2H), 3.76 (1H, br t, J=6.4Hz), 3.65 and 3.61 (AB in ABX, J AB =11.2, J AX =5.8, J BX =7.0Hz), 2.65 (2H, br dd), 2.56 (2H, br dd), ca. 1.6 (4H, m), 1.2-1.4 (16H), 0.90 (3H, t, J=6.9Hz), 0.88 (3H, t, J=7.1Hz) ¹³C-NMR (100 MHz, CD₃OD, δ): 168.8, 161.1, 159.3, 157.2, 153.1, 146.7, 144.7, 116.3, 114.0, 113.8, 111.1, 108.4, 107.5, 102.0, 85.5, 83.6, 78.6, 72.2, 64.4, 36.8, 34.9, 33.0 (2), 32.6, 32.4, 30.6, 30.3 (3), 23.7 (2), 14.4 (2) Rf values in thin layer chromatography of KS-501 with various developing agents are shown in Table 1. The detection was carried out under ultraviolet lamp at 253.7 nm. Silica gel thin layer chromatography of KS-501 Developing Agent Rf Value Chloroform : methanol = 7 : 3 (V/V) 0.49 Chloroform : methanol : conc. ammonia water = 19 : 5 : 1 (V/V/V) 0.08 Acetone 0.51 Thin layer : silica gel 60 F₂₅₄ plate (Merck Inc., 5628) Development: room temperature, ascending method, 15 to 30 minutes KS-502 State : white powder Melting point : 119 - 120°C Specific rotation : [α] 23 / D -45° (c=0.3, methanol) Solubility: Readily soluble : methanol, n-butanol, acetone, tetrahydrofuran, dioxane, dimethylsulfoxide, pyridine, monoethanolamine, acetic acid, alkaline water Soluble : chloroform, ethyl acetate, acetonitrile Insoluble : hexane, carbon tetrachloride, benzene, neutral water, acidic water Color reaction : Positive in reactions with iodine, anisaldehyde and ferric chloride; negative in reaction with anilinephthalic acid and ninhydrin and Reiden-Smith reactions. Ultraviolet absorption spectrum: Acidic and neutral methanol solution: λ max (ε) 252 nm (14100), 282 nm (6600, sh), 300 nm (5400, sh) Alkaline methanol solution: λ max (ε) 234 nm (18900, sh), 298 nm (24800) Infrared absorption spectrum: KBr 3450, 2972, 2940, 2872, 1724, 1595, 1463, 1432, 1375, 1343, 1245, 1161, 1135, 1062 cm⁻¹ Mass spectrum: SIMS, negative m/z 647(M-1)⁻¹, 392, 251 ¹H-NMR (400 MHz, CD₃OD, δ): 6.60 (1H, d, J=2.3Hz), 6.58 (1H, d, J=2.1Hz), 6.51 (1H, d, J=2.3Hz), 6.38 (1H, d, J=2.1Hz), 5.54 (1H, d, J=1.8Hz), 4.27 (1H, dd), ca. 4.1 (2H), 3.76 (1H, br t, J=5.9Hz), 3.65 and 3.61 (2H, AB in ABX, J AB =11.0, J AX =5.6, J BX =7.0Hz), 3.09 (2H, br dd), 2.65 (2H, br dd), ca. 1.6 (4H, m), 1.2-1.4 (16H), 0.88 (3H, t, J=6.6Hz), 0.87 (3H, t, J=6.7Hz) ¹³C-NMR (100 MHz, CD₃OD, δ): 176.6, 168.2, 164.3, 161.2, 157.3, 154.6, 149.6, 144.8, 116.0, 115.7, 115.6, 111.1, 108.6, 108.4, 102.0, 85.5, 83.5, 78.6, 72.2, 64.4, 36.5, 34.9, 33.2, 33.1, 33.0, 32.6, 31.0, 30.7, 30.5, 30.3, 23.74, 23.70, 14.46, 14.43 Rf values in thin layer chromatography of KS-502 with various developing agents are shown in Table 2. The detection was carried out under ultraviolet lamp at 253.7 nm. Silica gel thin layer chromatography of KS-502 Developing Agent Rf Value Chloroform : methanol = 7 : 3 (V/V) 0.31 Chloroform : methanol : conc. ammonia water = 19 : 5 : 1 (V/V/V) 0.00 Acetone 0.12 Thin layer : silica gel 60 F₂₅₄ plate (Merck Inc., 5628) Development: room temperature, ascending method, 15 to 30 minutes From the physico-chemical properties described above, KS-501 and KS-502 are considered to have the following structural formula. KS-501 :R=H KS-502 :R=COOH KS-501 and KS-502 have an inhibitory activity on serotonin release from platelets and KS-501 also has an inhibitory activity on platelet aggregation. Compounds similar to KS-502, R=COOH, but where the sugar moiety comprises a 6-membered pyranosyl ring, rather than a 5-membered furanosyl ring are disclosed in FR-A-2579599. Those compounds are alleged to have inhibitory TPI activity towards adenosine cyclic-3'5'-monophosphate phosphodiesterase. The process for preparing KS-501 and KS-502 is described below. KS-501 and/or KS-502 can be prepared by culturing a microorganism belonging to the genus Sporothrix and having an ability to produce KS-501 and/or KS-502 in a medium until KS-501 and/or KS-502 are accumulated in the culture, and recovering KS-501 and/or KS-502 from the culture. Any microorganism can be employed as a KS-501 and/or KS-502 producing microorganism, so long as it belongs to the genus Sporothrix and has an ability to produce KS-501 and/or KS-502. A preferred example is Sporothrix sp. KAC-1985 strain (hereinafter referred to as KAC-1985) isolated by the present inventors from fallen leaves collected in Yamakita-cho, Ashigarakami-gun, Kanagawa Prefecture, Japan. KAC-1985 strain has the following mycological characteristics. (1) Cultural characteristics Cultural characteristics of the strain on malt extract agar medium and potato-glucose agar medium are as follows. The growth is relatively slow. By culturing at 20°C for 30 days, a diameter of colony reaches 29 to 33 mm. The colony shows a dome-like shape. The surface and the back side of the colony are both white or creamy. A hypha has a septum and extends in and on the medium. The hypha has a diameter of 1 to 6 µm and is colorless, smooth and well branched. Sometimes fusion of hyphae is observed. A conidiophore is colorless and smooth, has a length of 4.5 to 20 µm and a width of 1.5 to 2.5 µm, and sometimes has septum. A conidium symodially grows at the end of conidiophore and sometimes the conidiophore extends after the formation of conidium. The conidium is a single cell, which is colorless, smooth and long oval, and has a length of 4 to 7 µm, or in a few cases, to 10 µm and a width of 1 to 2 µm. No complete generation of this strain is observed. (2) Physiological properties Growth temperature: 5 - 25°C Optimum growth temperature: 15 - 25°C Growth pH: 3 - 11 Optimum growth pH: 4 - 9 Based on the foregoing mycological properties, identification of KAC-1985 was made according to The Genera of Fungi Sporulating in Pure Culture, 2nd ed Cramer, Vaduz, J.A. von Arx, 1974. As the result, this strain was identified as a strain belonging to Sporothrix sp. The strain was named Sporothrix sp. KAC-1985 and deposited with the Fermentation Research Institute, Agency of Industrial Science and Technology under FERM BP-1278 on February 4, 1987. Ordinary culturing procedures employed for the culturing of fungi are applicable to the culturing in the present invention. Either a natural medium or a synthetic medium can be used, so long as it contains appropriate amounts of carbon source, nitrogen source, inorganic matters, etc. which are assimilable by the microorganism used. As the carbon source, carbohydrates such as glucose, fructose, Stabirose, sucrose, lactose, starch, dextrin, mannose, maltose, molasses and an instant mashed potato; organic acids such as citric acid, malic acid, acetic acid and fumaric acid; amino acids such as glutamic acid; glycerol, cotton seed oil, etc. can be used. As the nitrogen source, ammonium salts such as ammonium chloride, ammonium sulfate, ammonium nitrate and ammonium phosphate; amino acids such as aspartic acid, glutamine, cystine and alanine; urea, malt extract, peptone, meat extract, yeast extract, dry yeast, corn steep liquor, soybean powder, cotton seed cake, soybean casein, Casamino acids, Pharmamedia, soluble vegetable protein, vegetable or fruit juice, etc. can be used. As the inorganic matters, potassium dihydrogen phosphate, disodium hydrogen phosphate, magnesium sulfate, ferrous sulfate, manganese sulfate, cobalt sulfate, zinc sulfate, calcium panthothenate, ammonium molybdate, aluminium potassium sulfate, barium carbonate, calcium carbonate, cobalt chloride, sodium chloride, magnesium phosphate, etc. can be used. Furthermore, substances capable of promoting the propagation of microbial cells or the production of KS-501 and/or KS-502 such as vitamins and thiamine may be added to the medium, if required. When the microorganism to be used requires specific substances for the growth, it is necessary to add the substances to the medium. Culturing is carried out at a temperature of 15 to 25°C at pH around neutrality by shaking culture, aeration-stirring culture, etc. Maximum accumulation of KS-501 and/or KS-502 can be attained by culturing for 3 to 15 days, and the culturing is completed. To isolate and recover KS-501 and/or KS-502 accumulated in the cells, ordinary procedures for the recovery of a physiologically active substance from cells can be employed. That is, KS-501 and/or KS-502 can be isolated by collection of the cells through filtration, centrifugation, etc.; extraction from the cells with organic solvents such as methanol and acetone, etc.; partition with water or organic solvents; and adsorption and desorption of the active substance by column chromatography or thin layer chromatography using adsorptive resins, silica gel, silanized silica gel, alumina, cellulose, diatomaceous earth, magnesium silicate, gel filtering agents, etc. An example of the process for isolating KS-501 and/or KS-502 from the cells is given below. The cells are collected by filtration or centrifugation of the culture. An organic solvent such as methanol is added to the obtained cells. After thoroughly stirring the mixture, the cells are separated from the mixture by filtration or centrifugation again. The obtained filtrate or supernatant is concentrated under reduced pressure to remove the solvent and to obtain an aqueous solution. Then, a water-immiscible solvent such as ethyl acetate is added to the aqueous solution to conduct extraction. The extract is concentrated under reduced pressure, and subjected to repetitions of silica gel chromatography using a mixed solvent of chloroform-methanol as a developing solvent. The methanol content is stepwise increased to elute KS-501 and/or KS-502. In the case of the isolation of KS-501, fractions containing KS-501 are combined and concentrated under reduced pressure and the concentrate is dissolved in a small quantity of chloroform. The solution is allowed to stand at 0°C for crystallization. The thus obtained crystals are recrystallized by the same procedure to give white powders of KS-501. In the case of the isolation of KS-502, fractions containing KS-502 resulting from silica gel chromatography described above are combined and concentrated under reduced pressure and the concentrate is subjected to column chromatography using Sephadex LH-20 (product of Pharmacia Fine Chemicals, Inc.). Fractions containing KS-502 are combined and concentrated to dryness under reduced pressure to give white powders of KS-502. During the said purification, detection of KS-501 and/or KS-502 is carried out by silica gel thin layer chromatography followed by the iodine reaction or ultraviolet irradiation at 253.7 nm. KS-501 and KS-502 inhibit serotonin release from platelets and/or platelet aggregation and thus are expected to be useful as anti-thrombotic agents, especially those for cerebral thrombosis. Thus, according to a further feature of the present invention, there is provided a pharmaceutical composition comprising, as active ingredient, effective amounts of KS-501 and/or KS-502 usually in association with at least one pharmaceutically acceptable carrier or excipient. The pharmaceutical composition may be administered through oral or parenteral route (for example, injection, application, and inhalation). KS-501 and/or KS-502 can be administered as such, but generally administered in the form of tablets, pills, powder, granules, capsules, suppository, injection, etc. Conventional pharmaceutically acceptable carriers can be used for medical compositions of this invention. They include lactose, dextrose, sucrose, sorbitol, mannitol, glucose, cellulose, cyclodextrin, talc, starch, methylcellulose, gelatin, arabic gum, polyethylene glycol, carboxymethylcellulose, hydroxypropylcellulose, sodium benzoate, sodium hydrogensulfite, aluminium stearate, magnesium stearate, mineral oil, vegetable oil, white Vaseline®, liquid paraffin, etc., and can be appropriately selected in view of the kind of preparations. Certain specific embodiments of the present invention are illustrated by the following examples. Example 1 Sporothrix sp. KAC-1985 was used as the seed strain. The strain was inoculated into 40 mℓ of a seed medium (pH 6.0, prior to sterilization) containing 1.0 g/dℓ glucose, 0.5 g/dℓ peptone (product of Kyokuto Pharmaceutical Industry Co., Ltd.), 0.5 g/dℓ dry yeast Ebios (product of Asahi Breweries, Ltd.), 0.2 dℓ/dℓ V-8 Vegetable Juice (product of Campbell Co.) and 0.3 g/dℓ calcium carbonate in a 300 mℓ-Erlenmyer flask and subjected to shaking culture at 25°C until the strain sufficiently grew. Then, 10 mℓ of the seed culture was inoculated into 100 mℓ of a fermentation medium having the following composition. Composition of the fermentation medium: 1.0 g/dℓ glucose, 0.5 g/dℓ peptone, 0.5 g/dℓ dry yeast Ebios, 0.2 dℓ/dℓ V-8 Vegetable Juice, 0.2 dℓ/dℓ apple juice (product of Meijiya) and 0.5 g/dℓ calcium carbonate (pH 6.0, prior to sterilization). Culturing was carried out with shaking at 25°C for 12 days. After the completion of culturing, 1.3 ℓ of the culture was centrifuged at 7000 rpm using RPR-9-2 Model Rotor (product of Hitachi Ltd.). Then, 1.3 ℓ of methanol was added to the cells, and after thorough stirring, the cells were removed by filtration. The thus obtained methanol extract was concentrated under reduced pressure to remove methanol and to obtain about 100 mℓ of an aqueous solution. After being adjusted to pH 2 with 2N hydrochloric acid, the solution was extracted with 100 mℓ each of ethyl acetate three times. The ethyl acetate layer was concentrated under reduced pressure and the concentrate was dissolved in a small quantity of chloroform. The solution was charged to a column packed with 250 mℓ of silica gel C-200 (product of Wako Pure Chemical Industries, Ltd.) suspended in chloroform and successively eluted with 500 mℓ each of chloroform, 5% methanol/chloroform (V/V) and 10% methanol/chloroform (V/V) as developing solvents. The eluted fractions were taken in 18 g portions, and KS-501 was eluted in fraction Nos. 121 to 136. These fractions were combined and concentrated under reduced pressure, and the concentrate was then dissolved in a small quantity of chloroform. The solution was charged to a column packed with 100 mℓ of silica gel C-300 (Wako Pure Chemical Industries, Ltd.) suspended in chloroform and successively eluted with 200 mℓ of chloroform, 300 mℓ of 5% methanol/chloroform (V/V), 300 mℓ of 7% methanol/chloroform (V/V) and 300 mℓ of 9% methanol/chloroform (V/V) as developing solvents. The eluted fractions were taken in 18 g portions, and KS-501 was eluted in fraction Nos. 38 to 59. These fractions were combined and concentrated to give 315 mg of a pale yellow oily substance. The oily substance was dissolved in about 5 mℓ of chloroform and crystallized at 0°C. The obtained crystals were dissolved again in chloroform and recrystallized at 0°C to give 192 mg of KS-501 as white powders. During the foregoing steps, KS-501 was detected by silica gel thin layer chromatography followed by the iodine reaction or ultraviolet irradiation at 253.7 nm. Example 2 Sporothrix sp. KAC-1985 was cultured in the same manner as in Example 1. After the completion of culturing, 1.3 ℓ of the culture was centrifuged at 7000 rpm using RPR-9-2 Model Rotor. Then, 1.3 ℓ of methanol was added to the cells, and after thorough stirring, the cells were removed by filtration. The thus obtained methanol extract was concentrated under reduced pressure to remove methanol and to obtain about 100 mℓ of an aqueous solution. After being adjusted to pH 2 with 2N hydrochloric acid, the solution was extracted with 100 mℓ each of ethyl acetate three times. The ethyl acetate layer was concentrated under reduced pressure and the concentrate was dissolved in a small quantity of chloroform. The solution was charged to a column packed with 250 mℓ of silica gel C-200 suspended in chloroform and successively eluted with 500 mℓ each of chloroform, 5% methanol/chloroform (V/V), 10% methanol/chloroform (V/V) and 20% methanol/chloroform (V/V) as developing solvents. The eluted fractions were taken in 18 g portions, and KS-502 was eluted in fraction Nos. 160 to 201. These fractions were combined and concentrated under reduced pressure, and the concentrate was then dissolved in a small quantity of methanol. The solution was charged to a column packed with 100 mℓ of Sephadex LH-20 (product of Pharmacia Fine Chemicals Inc., suspended in methanol and eluted with methanol as a developing solvent. The eluted fractions were taken in 4.4 mℓ portions, and KS-502 was eluted in fraction Nos. 19 to 28. These fractions were combined and concentrated to dryness under reduced pressure to give 92 mg of KS-502 as white powders. During the foregoing steps, KS-502 was detected by silica gel thin layer chromatography followed by the iodine reaction or ultraviolet irradiation at 253.7 nm. The inhibitory activities of KS-501 and KS-502 on serotonin release from platelets and/or platelet aggregation are explained below, referring to experimental examples. Experimental Example 1 Effect on serotonin release from rabbit platelets (1) Method 9.25 vol of blood was sampled from the carotid artery of a white rabbit and placed in a FALCON 2070 tube (product of Falcon, Bector Dickinson) containing 0.75 vol of 77 mM ethylenediaminetetraacetic acid (EDTA). The thus obtained blood was centrifuged at 200 x g for 15 minutes to obtain platelet rich plasma (referred to as PRP hereinafter). This PRP was incubated with [2-¹⁴C]-serotonin (2 µCi/100 mℓ PRP) at 37°C for an hour to incorporate [2-¹⁴C]-serotonin therein. Then, the PRP was centrifuged at 650 x g for 15 minutes to obtain platelet precipitates. After washing with Tris-buffered Physiological saline (pH 7.4, containing 1 mM EDTA), the platelet precipitates were suspended in Ca⁺⁺-free Tyrode's solution to reach a final density of 10⁹ cells/mℓ. To 0.475 mℓ of the thus prepared [2-¹⁴C]-serotonin-labeled platelet suspension was added 5 µℓ of a solution of the test compound. After incubation at 37°C for 3 minutes, platelet activating factor (final concentration: 10⁻⁹M) was added as a stimulant, followed by incubation for further 3 minutes. 0.3 mℓ of the reaction solution was immediately taken and poured into 30 µℓ of ice-cold 0.1 mM formaldedhyde5 mM EDTA solution to terminate the reaction. After centrifugation at 3,000 rpm at 0°C for 10 minutes, 250 µℓ of the supernatant was taken and the radioactivity was measured with a liquid scintillation counter. (2) Results Compound Concentration (µg/mℓ) Rate of inhibition of serotonin release (%) KS-501 3.0 10.2 10.0 27.1 30.0 66.4 KS-502 10.0 2.7 30.0 28.9 50.0 51.4 As shown in Table 3, KS-501 and KS-502 inhibited serotonin release from rabbit platelets, depending on the concentration. Experimental Example 2 Effect on platelet aggregation of rabbit platelets (1) Method 8.5 vol of blood was sampled from the carotid artery of a white rabbit and placed in a FALCON 2070 tube (product of Falcon, Bector Dickinson) containing 1.5 vol of citrate-dextrose solution (71 mM citric acid, 85 mM sodium citrate and 111 mM dextrose). The thus obtained blood was centrifuged at 200 x g for 15 minutes to obtain PRP. This PRP was centrifuged at 650 x g for 10 minutes to obtain platelet precipitates. After washing with Ca⁺⁺-free Tyrode's solution containing 2 mM ethyleneglycol bis(2-aminoethylether) tetraacetic acid (EGTA), the platelet precipitates were again centrifuged at 650 x g for 10 minutes and washed. The thus obtained platelet precipitates were suspended in Ca⁺⁺-free Tyrode's solution containing 0.25% bovine serum albumin to reach a density of 5 x 10⁸ cells/mℓ. After the washed platelet suspension was allowed to stand at room temperature for about 30 minutes, 1 mM CaCl₂ or 1 mM EGTA was added thereto and the mixture was allowed to stand for further 30 minutes. Then, platelet aggregation was measured. The aggregation of platelets was determined using an aggregometer, AGGRETEC TE-500 (product of Erma Optical Co., Ltd.) to which the Born method was applied. First, 10 µℓ of a test compound solution was added to 0.2 mℓ of the washed platelet suspension, while stirring at 1,000 rpm and at 37°C. After incubation at 37°C for 3 minutes, 10 µℓ of thrombin (final concentration: 0.05 U/mℓ) or platelet activating factor (final concentration: 10⁻¹⁰ M) was added as a stimulant to initiate the reaction. The rate of inhibition of platelet aggregation of the test compound was calculated from the maximum aggregation rate obtained by the use of the test compound and the maximum aggregation rate obtained by the use of only the solvent used for dissolving the test compound, according to the following equation. A : Maximum aggregation rate of the solvent alone B : Maximum aggregation rate of the test compound (2) Results Compound Stimulant IC 50 (µg/mℓ) KS-501 Thrombin 57 Platelet activating factor 38 As shown in Table 4, KS-501 inhibited platelet aggregation stimulated with thrombin and platelet activating factor. This invention also extends to the microorganism Sporothrix sp KAC-1985 (FERM BP-1278) in substantially biologically pure form, and KS-502 producing mutants and variants thereof including KS-501 and KS-502 producing genetically engineered mutants and transformants thereof, and the use thereof in the production of KS-501 and/or KS-502. Also included within this invention are pharmaceutically active salts of KS-502.

Claims: The compound identified herein as KS-501, that compound being characterised by the following physicochemical and other characteristics: Physical state : white powder Melting point : 149 to 152°C Specific rotation : [α] 23 / D -53° (c=0.3,methanol) Solubility : Readily soluble: chloroform, ethyl, acetate, acetonitrile, methanol, n-butanol, acetone, tetrahydrofuran, dioxane, dimethylsulfoxide, pyridine, monoethanolamine, acetic acid, alkaline water Insoluble: hexane, carbon tetrachloride, benzene, neutral water, acidic water Colour reaction : Positive in reactions with iodine, anisaldehyde and ferric chloride; negative in reaction with aniline-phthalic acid and ninhydrin and Reiden-Smith reactions. Ultraviolet absorption spectra : Acidic and neutral methanol solution: λ max (ε) 254 nm (6760), 278 nm (5560, sh) Alkaline methanol solution: λ max (ε) 234 nm (16100, sh), 292 nm (11500) Infrared absorption spectrum: KBr 3380, 2960, 2924, 2852, 1722, 1608, 1586, 1466, 1323, 1264, 1168, 1149, 1129, 1064, 996 cm⁻¹ Mass spectrum : SIMS, positive m/z 605 (M+H)⁺, 443, 235 ¹H-NMR (400 MHz, CD₃OD,δ): 6.58 (1H, d, J=2.1Hz), 6.55-6.50 (3H, m), 6.38 (1H, d, J=2.1Hz), 5.55 (1H, d, J=1.8Hz), 4.28 (1H, dd, J=3.9, 1.8Hz), ca 4.1 (2H), 3.76 (1H, br t, J=6.4Hz), 3.65 and 3.61 (AB in ABX, J AB =11.2, J AX =5.8, J BX =7.0Hz), 2.65 (2H, br dd), 2.56 (2H, br dd), ca. 1.6 (4H, m), 1.2-1.4 (16H), 0.90 (3H, t, J=6.9Hz), 0.88 (3H, t,J=7.1Hz) ¹³C-NMR (100 MHz, CD₃OD, δ): 168.8, 161.1, 159.3, 157.2, 153.1, 146.7, 144.7, 116.3, 114.0, 113.8, 111.1, 108.4, 107.5, 102.0, 85.5, 83.6, 78.6, 72.2, 64.4, 36.8, 34.9, 33.0 (2), 32.6, 32.4, 30.6, 30.3 (3), 23.7 (2), 14.4 (2) and Rf values in thin layer chromatography, detection at 253.7 nm: Developing Agent Rf Value Chloroform:methanol = 7:3 (V/V) 0.49 Chloroform:methanol:conc.ammonia water = 19:5:1 (V/V/V) 0.08 Acetone 0.51 Source : obtainable from the culture of microorganisms of the genus Sporothrix , specifically Sporothrix sp. KAC-1985 (FERM BP-1278). Pharmaceutical activity : exhibits inhibitory activity on serotonin release from platelets and platelet aggregation. The compound identified herein as KS-502, that compound being characterized by the following physicochemical and other characteristics: Pyhsical state : white powder Melting point : 119 to 120°C Specific rotation : [α] 23 / D -45° (c=0.3,methanol) Solubility : Readily soluble: methanol, n-butanol, acetone, tetrahydrofuran, dioxane, dimethylsulfoxide, pyridine, monoethanolamine, acetic acid, alkaline water Soluble: chloroform, ethyl acetate, acetonitrile Insoluble: hexane, carbon tetrachloride, benzene, neutral water, acidic water Colour reaction : Positive in reactions with iodine, anisaldehyde and ferric chloride; negative in reaction with aniline-phthalic acid and ninhydrin and Reiden-Smith reactions. Ultraviolet absorption spectra : Acidic and neutral methanol solution: λ max (ε) 252 nm (14100), 282 nm (6600, sh), 300 nm (5400, sh) Alkaline methanol solution: λ max (ε) 234 nm (18900, sh), 298 nm (24800) Infrared absorption spectrum : KBr 3450, 2972, 2940, 2872, 1724, 1595, 1463, 1432, 1375, 1343, 1245, 1161, 1135, 1062 cm⁻¹ Mass spectrum : SIMS, negative m/z 647 (M-1)⁻¹, 392, 251 ¹H-NMR (400 MHz, CD₃OD,δ): 6.60 (1H, d, J=2.3Hz), 6.58 (1H, d, J=2.1Hz), 6.51 (1H, d, J=2.3Hz), 6.38 (1H, d, J=2.1Hz), 5.54 (1H, d, J=1.8Hz), 4.27 (1H, dd), ca. 4.1 (2H), 3.76 (1H, br t, J=5.9Hz), 3.65 and 3.61 (2H, AB in ABX, J AB =11.0, J AX =5.6, J BX =7.0Hz), 3.09 (2H, br dd), 2.65 (2H, br dd), ca. 1.6 (4H, m), 1.2-1.4 (16H), 0.88 (3H, t, J=6.6Hz) 0.87 (3H, t, J=6.7Hz) ¹³C-NMR (100 MHz, CD₃OD,δ): 176.6, 168.2, 164.3, 161.2, 157.3, 154.6, 149.6, 144.8, 116.0, 115.7, 115.6, 111.1, 108.6, 108.4, 102.0, 85.5, 83.5, 78.6, 72.2, 64.4, 36.5, 34.9, 33.2, 33.1, 33.0, 32.6, 31.0, 30.7, 30.5, 30.3, 23.74, 23.70, 14.46, 14.43 and Rf values in thin layer chromatography, detection at 253.7 nm: Developing Agent Rf Value Chloroform:methanol = 7:3 (V/V) 0.31 Chloroform:methanol:conc.ammonia water = 19:5:1 (V/V/V) 0.00 Acetone 0.12 Source : obtainable from the culture of microorganisms of the genus Sporothrix , specifically Sporothrix sp. KAC-1985 (FERM BP-1278). Pharmaceutical activity : exhibits inhibitory activity on serotonin release from platelets. Compounds of the formula: wherein R is hydrogen or carboxyl, and pharmaceutically active salts thereof. A process for the preparation of a compound as claimed in claim 1, 2 or 3, which comprises culturing a microorganism belonging to the genus Sporothrix and having an ability to produce the said compound in a culture medium, allowing the compound to accumulate therein, and recovering the accumulated compound from the culture. A process according to claim 4, wherein the microorganism used is Sporothrix sp. KAC-1985 (FERM BP-1278). A pharmaceutical composition comprising an active ingredient having platelet serotonin release and/or platelet aggregation inhibiting activity in admixture with a pharmaceutically acceptable carrier or excipient, characterised in that the said ingredient exhibiting platelet serotonin release and/or platelet aggregation inhibiting activity is a compound as claimed in claim 1, 2 or 3 or a pharmaceutically acceptable salt thereof. In substantially biologically pure form, the microorganism Sporothrix sp. KAC-1985 (FERM BP-1278), and KS-501 and KS-502 producing mutants and variants thereof. The use of Sporothrix sp. KAC-1985 (FERM BP-1278) and KS-501 and KS-502 producing mutants and variants thereof in the preparation of KS-501 and/or KS-502.

Inventors: ANDO KATSUHIKO C O PATENT DEPA, KASE HIROSHI C O PATENT DEPART, KAWAMOTO ISAO C O PATENT DEPAR, NAKANISHI SATOSHI C O PATENT D, SANO HIROSHI C O PATENT DEPART, YAMADA KOJI C O PATENT DEPARTM, YASUZAWA TORU C O PATENT DEPAR, ANDO, KATSUHIKO C/O PATENT DEPARTMENT, KASE, HIROSHI C/O PATENT DEPARTMENT, KAWAMOTO, ISAO C/O PATENT DEPARTMENT, NAKANISHI, SATOSHI C/O PATENT DEPARTMENT, SANO, HIROSHI C/O PATENT DEPARTMENT, YAMADA, KOJI C/O PATENT DEPARTMENT, YASUZAWA, TORU C/O PATENT DEPARTMENT
Applicants: KYOWA HAKKO KOGYO KK, KYOWA HAKKO KOGYO CO., LTD.
Application year: 1988
Main classification: C07H15
IPC codes: C07H15, C12P19