Carbostyril derivatives as anti-thrombotic agents

Abstract: A pharmaceutical composition containing at least one member selected from the group consisting of carbostyril derivatives represented by general formula (I) and salts thereof and being useful as a remedy for thrombosis, especially a platelet agglutination inhibitor, a platelet mass dissociating agent and a cerebrovascular circulation ameliorant, and further as a phosphodiesterase inhibitor. In formula (I) R¹ represents hydrogen or lower alkyl which may be substituted by hydroxyl; and R² represents phenylated lower alkyl which may be substituted by 1 to 3 substituents selected from the group consisting of lower alkoxy groups and halogen atoms on the benzene ring, pyridylated lower alkyl or a group -A-NR³R⁴ wherein A represents lower alkylene and R³ and R⁴ may be the same with or different from each other and each represents lower alkyl or phenyl.

Description: Technical Field The present invention relates to the use of a carbostyril derivative or a salt thereof for preparing a medicament for inhibiting platelet aggregation. Background Art A carbostyril derivative having the general formula (1): wherein R¹ is hydrogen or a lower alkyl group optionally having hydroxy group as a substituent; R² is a phenyl(lower alkyl) group optionally having 1 to 3 substituents selected from the group consisting of a lower alkoxy group and a halogen atom on the phenyl ring, pyridyl(lower alkyl) group, or a group of the formula -A-NR³R⁴ (wherein A is a lower alkylene group, R³ and R⁴ are the same or different and represent a lower alkyl group or phenyl group), or a salt thereof, is a known compound which is described in, for example, EP -A-355583 to be useful as cardiotonics. These compounds are also known to be useful as antihistamines (Japanese Patent First Publication No. 8319/1981). In addition, among the compounds represented by the above general formula, 6-[3-(3,4-dimethoxybenzylamino-2-hydroxypropoxy)-2-(1H)-quinoline compound (the compound of the formula (1) wherein R¹ is hydrogen, R² is 3,4-dimethoxybenzyl group; OPC-18790) has an inotropic activity and is useful as an agent for treating heart failure (The Japanese Journal of Pharmacology, The 64th Annual Meeting, March 1991, Kobe, Japan and International Congress of New Drug Development, Aug. 1991, Seoul, Korea). Further, JP-A-55129268 discloses a carbostyril derivative of the formula: wherein R¹ is a lower alkenyl group or phenyl group; R² is hydrogen atom, a lower alkenyl group or a lower alkyl group, and the bond between 3- and 4-positions in the carbostyril nucleus is single bond or double bond, or an acid addition salt thereof (which compounds are having central nervous depressant and antihistaminic activities and are useful as preanesthetic, sleep producing drug, central muscle relaxant, a drug used before operation, anti-schizophrenia, sedatives, anti-anxiety, anti-manic-depressives, and antihistaminics); and JP-A-57014574 discloses that the compounds of the formula: wherein R¹ is a lower alkyl group, R² is a cycloalkyl group or pyridyl group, and A is a lower alkylene group, have platelet aggregation inhibitory activity, phosphodiesterase inhibitory activity, antiulcer activity, cardiotonic activity (inotropic activity), antiinflammatory activity, and hypotensive activity and are useful as a drug for prophylaxis and treatment of thrombosis, and as phosphodiesterase inhibitor, antiulcer agent, cardiotonics, antiinflammatory agent, and hypotensive agent. The present inventors have intensively studied the above-mentioned carbostyril derivative and a salt thereof, and as a result, have found that said compound shows an excellent platelet aggregation inhibitory activity, cerebral blood flow increasing activity and platelet aggregate dissociation activity, and is useful as an agent for treating thrombosis. Thus, the present invention has been completed. Disclosure of the Invention That is, the present invention relates to the use of the carbostyril derivatives represented by the above general formula (1) and salt thereof for preparing a medicament for inhibiting platelet aggregation. Said medicament is further useful as ameliorant of cerebral circulation and platelet aggregate dissociation agent. The carbostyril derivatives having the above general formula (1) and the salt thereof have an excellent platelet aggregation inhibitory activity, cerebral blood flow increasing activity, platelet aggregate dissociation activity, thromboxane A₂ antagonistic activity, and the like. Accordingly, said carbostyril derivatives and the salt thereof can be used suitably as an agent for prevention and treatment of thrombosis such as cerebral apoplexy, cerebral infarction, myocardial infarction, etc., ameliorants of cerebral circulation, and an agent for treatment of glaucoma. Each group in the above general formula (1) is shown more specifically as follows: The lower alkyl group includes a straight chain or branched chain alkyl group having 1 to 6 carbon atoms such as methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, pentyl, hexyl, etc. The phenyl(lower alkyl) group optionally having 1 to 3 substituents selected from the group consisting of a lower alkoxy group and a halogen atom on the phenyl ring includes a phenylalkyl group optionally having 1 to 3 substituents selected from the group consisting of a straight chain or branched chain alkoxy group having 1 to 6 carbon atoms and a halogen atom on the phenyl ring wherein the alkyl moiety is a straight chain or branched chain alkyl group having 1 to 6 carbon atoms, such as benzyl, 2-phenylethyl, 1-phenylethyl, 3-phenylpropyl, 4-phenylbutyl, 1,1-dimethyl-2-phenylethyl, 5-phenylpentyl, 6-phenylhexyl, 2-methyl-3-phenylpropyl, di-phenylmethyl, 2,2-diphenylethyl, 2-(3-methoxyphenyl)ethyl, 1-(4-methoxyphenyl)ethyl, 2-methoxybenzyl, 3-(2-ethoxyphenyl)propyl, 4-(3-ethoxy-phenyl)butyl, 1,1-dimethyl-2-(4-ethoxyphenyl)ethyl, 5-(4-iso-propoxyphenyl)pentyl, 6-(4-hexyloxyphenyl)hexyl, 3,4-dimethoxybenzyl, 3,4,5-trimethoxybenzyl, 2,5-dimethoxybenzyl, 2-chlorobenzyl, 2-(3-bromophenyl)ethyl, 1-(4-chlorophenyl)ethyl, 3-(4-fluorophenyl)propyl, 4-(2,3-dichlorophenyl)butyl, 1,1-dimethyl-2-(2,4-dibromophenyl)ethyl, 5-(3,4-difluorophenyl)pentyl, 6-(2,4,6-trichlorophenyl)hexyl, 2-methyl-3-(2-fluoro-phenyl)propyl, 2-chloro-4-methoxybenzyl, 3-methoxybenzyl, 4-methoxybenzyl, 3-chlorobenzyl, 4-chlorobenzyl, and the like. The pyridyl(lower alkyl) group includes those pyridylalkyl groups wherein the alkyl moiety is a straight chain or branched chain alkyl group having 1 to 6 carbon atoms, such as (3-pyridyl)-methyl, (2-pyridyl)methyl, 2-(4-pyridyl)ethyl, 1-(2-pyridyl)ethyl, 3-(2-pyridyl)propyl, 4-(2-pyridyl)ethyl, 1,1-dimethyl-2-(3-pyridyl)ethyl, 5-(2-pyridyl)pentyl, 6-(4-pyridyl)hexyl, and the like. The lower alkylene group includes a straight chain or branched chain alkylene group having 1 to 6 carbon atoms such as methylene, ethylene, trimethylene, 2-methyltrimethylene, 2,2-dimethyltrimethylene, 1-methyltrimethylene, methylmethylene, ethylmethylene, tetramethylene, pentamethylene, hexamethylene, and the like. The lower alkyl group optionally having hydroxy group as a substituent includes a straight chain or branched chain alkyl group having 1 to 6 carbon atoms optionally having hydroxy group as a substituent, such as methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, pentyl, hexyl, hydroxymethyl, 2-hydroxyethyl, 1-hydroxyethyl, 3-hydroxypropyl, 4-hydroxybutyl, 1,1-dimethyl-2-hydroxyethyl, 5-hydroxypentyl, 6-hydroxyhexyl, 2-methyl-3-hydroxypropyl, and the like. The carbostyril derivative represented by the general formula (1) of the present invention can easily be converted into an acid addition salt by treating with a pharmaceutically acceptable acid. Said acid includes, for example, an inorganic acid such as hydrochloric acid, sulfuric acid, phosphoric acid, hydrobromic acid, etc., an organic acid such as oxalic acid, maleic acid, fumaric acid, malic acid, tartaric acid, citric acid, benzoic acid, etc. Among the carbostyril derivative represented by the general formula (1) of the present invention, those compounds having an acidic group can easily be converted into a salt by treating with a pharmaceutically acceptable basic compound. Said basic compound includes, for example, sodium hydroxide, potassium hydroxide, calcium hydroxide, sodium carbonate, potassium hydrogen carbonate, and the like. The present invention includes, of course, an optical isomer. The compound of the general formula (1) is usually used in the form of the conventional pharmaceutical preparation. The pharmaceutical preparation is prepared using conventional diluents or vehicles such as fillers, thickening agents, binders, wetting agents, disintegrators, surfactants, lubricants, etc. The pharmaceutical preparation may be in various dosage forms depending on the therapeutical purpose and such dosage form includes typically tablets, pills, powders, solutions, suspensions, emulsions, granules, capsules, suppositories, injections (solutions, suspensions, etc.), and the like. For formulating into tablets, the carriers known in the field can widely be used, including, for example, vehicles such as lactose, sucrose, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, crystalline cellulose, silic acid, etc.; binders such as water, ethanol, propanol, simple syrup, a glucose solution, a starch solution, a gelatin solution, carboxymethyl-cellulose, shellac, methylcellulose, potassium phosphate, polyvinylpyrrolidone, etc.; disintegrators such as dried starch, sodium alginate, agar powder, laminaran powder, sodium hydrogen carbonate, calcium carbonate, polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, stearic acid monoglyceride, starch, lactose, etc.; disintegration inhibiting agents such as sucrose, stearin, cacao butter, hydrogenated oil, etc.; absorption promoting agents such as quaternary ammonium salt, sodium lauryl sulfate, etc.; wetting agents such as glycerol, starch, etc.; absorbants such as starch, lactose, kaolin, bentonite, colloidal silic acid, etc.; lubricants such as purified talc, stearate, boric acid powder, polyethylene glycol, etc., and the like. In addition, tablets may also be in the form of the conventional coated tablet, including sugar coated tablets, gelatin coated tablets, enteric coated tablets, film coated tablets, or double-layered tablets, multilayer tablets. For formulating into pills, the carriers known in the field can widely be used, including, for example, vehicles such as glucose, lactose, starch, cacao butter, hydrogenated vegetable oil, kaolin, talc, etc.; binders such as acacia powder, powdered tragacanth, gelatin, ethanol, etc.; disintegrators such as laminaran, agar, etc., and the like. For formulating into suppositories, the carriers known in the field can widely be used, including, for example, such as polyethylene glycol, cacao butter, higher alcohols, esters of higher alcohols, gelatin, semisynthesized glyceride, etc. For preparation of injections, the solutions or suspensions are sterilized and are preferably isotonic to blood. For formulating into these solutions, pills and suspensions, any conventional diluent known in the field can be used, including, for example, water, ethyl alcohol, propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, polyoxyethylene sorbitan fatty acid esters, and the like. In this case, the pharmaceutical preparation of the present invention may contain salt, glucose or glycerol in an amount sufficient for preparing the isotonic solution. The pharmaceutical preparation may also contain conventional solubilizing agents, buffers, anesthetizing agents and the like. Said pharmaceutical preparation may further contain optionally coloring agents, preservatives, flavoring agents, perfumes, sweetening agents, etc. and other drugs. The amount of the compound of the general formula (1) contained in the pharmaceutical preparation of the present invention is not particularly limited and selected from the wide range and usually from 1 to 70 % by weight, preferably from 1 to 30 % by weight based on the total amount of the composition. The administration route of the pharmaceutical preparation of the present invention is not particularly limited and determined depending on dosage forms, age and sex or other conditions of patients, severity of the disease, and the like. For example, tablets, pills, solutions, suspensions, emulsions, granules and capsules are orally administered. The injections are intravenously administered solely or together with the conventional auxiliary liquid such as glucose, amino acid, etc. or, if necessary, administered intramuscularly, intradermally, subcutaneously or intraperitoneally. The suppositories are administered intrarectally. The dosage amount of the pharmaceutical preparation of the present invention is suitably determined depending on usage, age, sex or other conditions of patients, severity of the disease, and the like. The amount of the compound of the general formula (1) as the active ingredient is usually administered in an amount of about 0.1 to 10 mg per 1 kg of body weight per day. The dosage unit preferably contains the active ingredient in an amount of 1 to 200 mg. Best Mode for Carrying out the Invention Pharmacological test results and Examples of Preparations are shown below. Pharmacological test 1 The platelet aggregation inhibitory activity of test compounds was measured in accordance with the method of Born et al. [J. Physiol., London, 162 , 67, (1962)] using a platelet aggregator [Platelet Aggregation Tracer manufactured by Niko Biosceince K.K.]. To 9 volumes of blood taken from human beings was added 1 volume of 3.8 % citric acid and this sample was centrifuged at 1100 rpm for 10 minutes to give a platelet rich plasma (PRP). The remaining sample was further centrifuged at 3000 rpm for 15 minutes to give a platelet poor plasma (PPP). The platelets in the PRP obtained above was counted using Coulter Counter, Coulter electronics inc. and diluted to 300000 platelets/µl with the PPP to prepare a PRP solution. A cell for measurement of aggregation was charged with a solution containing a fixed concentration of test compounds (1 to 2 µl) and the PRP solution prepared above (200 µl) and warmed at 37°C for 1 minute. Then, to the cell was added a suspension of adenosine diphosphate (ADP, manufactured by Sigma) or collagen (Collagen Reagent Horm, manufactured by Hormon-Chemie) to induce platelet aggregation and the transparency of the cell was measured and a platelet aggregation curve was prepared. The final concentration of the above ADP and the collagen was adjusted to 14 to 16 µM and 1 to 4 µg/ml, respectively. Maximum Aggregation Rate (MAR) of platelets was calculated from the platelet aggregation curve by the following formula: MAR = (b-a)/(c-a) x 100 wherein (a) represents the transparency of PRP measured in the similar test, (b) represents the transparency of the PRP solution containing the test compound and the aggregation inducer at the time of maximum change in the above test, and (c) represents the transparency of the PPP solution measured in the similar test. MAR for control with no addition of the test compound was also calculated, and based on this value, the platelet aggregation inhibitory rate (%) of each test compound at various concentrations in the above test was calculated from the following formula: Inhibitory rate = [1- (MAR of PRP with addition of test compound)/(MAR of PRP with no addition of test compound)] x 100 Pharmacological test 2 (not part of the invention) C-AMP phosphodiesterase inhibitory activity of the compound of the present invention was measured in accordance with the method described in Biochimica et Biophysica Acta, Vol. 484, pp398 - 407 (1977) and Biochemical Medicine, Vol. 10, pp301 - 311 (1974). That is, the heart taken out from dog was put in a solution (pH 6) containing 2-mercaptoethanol (3.75 mmol) in Tris-acetate buffer (50 mmol) at 4°C and the blood vessels and the like were removed. The heart was minced in 3 volumes of the buffer and ground with a homogenizer. After repeating freezing and thawing twice, the product was centrifuged at 105000 x g to give a supernatant which was used as a crude enzyme solution. The crude enzyme solution (10 ml) was passed through DEAE-cellulose column buffered with a solution (pH 6) containing 2-mercaptoethanol (3.75 mmol) in Tris-acetate buffer (50 mmol) and washed and eluted with the same buffer (30 ml). Elution was conducted with sodium acetate-Tris-HCl buffer by the linear gradient method at a flow rate of 0.5 ml/minute to give fractions of each 5 ml (total amount of elution: about 300 ml). Thereby, there was obtained a fraction which showed a weak activity of not more than 2 nmol/ml/minute at a higher concentration of C-AMP substrate of 100 µmol and a strong activity of not less than 100 pmol/ml/minute at a lower concentration of C-AMP substrate of 0.4 µmol and this fraction was used as C-AMP phosphodiesterase. An aqueous solution (0.1 ml) containing various concentrations of test compound was mixed with Tris-HCl buffer (pH 8.0, 40 mmol, containing calf serum albumin 50 µg and MgCl₂ 4 mmol) containing C-AMP (tritium C-AMP)(0.4 µmol) to prepare a substrate solution (0.2 ml). To the above substrate solution was added the C-AMP phosphodiesterase solution (0.2 ml) and the mixture was reacted at 30°C for 20 minutes to convert tritium C-AMP into tritium 5'-AMP. After the reaction solution was dipped into a boiling water to quench the reaction, it was cooled in ice-water. Thereto was added snake venom (1 mg/ml)(0.05 ml) and the reaction was conducted at 30°C for 10 minutes to convert the tritium 5'-AMP into tritium adenosine. The reaction solution was passed through a cation exchange resin to absorb the tritium adenosine, which was washed with distilled water and eluted with 3N aqueous ammonia (1.5 ml). The eluate was measured for the produced tritium adenosine using the conventional liquid scintillation counter and thereby the phosphodiesterase activity was measured. From the above results, the phosphodiesterase activity of the test compound at various concentrations (Vs) was obtained, and using this value and the activity value of control (water without the test compound), a phosphodiesterase inhibitory rate (%) was calculated in accordance with the following formula: Phosphodiesterase inhibitory rate (%) = (Vc - Vs)/Vc x 100 Pharmacological test 3 A hybrid dog was anesthetized with pentobarbital (30 mg/kg i.v.) and the chest was opened under artificial respiration. The topical blood flow (the blood flow in the cerebellum and the brain) was determined in the following manner. That is, about 600 million colored microspheres were introduced into the left atrium and then various amounts of the test compound was continuously administered intravenously over 30 minutes. After administration of each dosage, about 600 million colored microspheres having a distinctive color for each dosage were administered at the left atrium. Thereafter, the cerebellum and the brain were taken out and treated with an alkali to extract the colored microspheres from the tissue. The collected colored microspheres were counted for each color under a microscope and a local blood flow was calculated by comparing with the colored microspheres in arterial blood using the following formula: Qm = (Cm x Qr)/Cr wherein Qm means the topical blood flow, Cm means the number of microspheres per 1 g of the tissue, Qr means a rate of collection of arterial blood, and Cr means the number of microspheres. Increasing rate of blood flow (%) was calculated in accordance with the following formula: Increasing rate of blood flow (%) = [(Qmd - Qmc)/Qmc] x 100 wherein Qmd means the topical blood flow when the test compound was administered, and Qmc means the topical blood flow when the test compound was not administered. Pharmacological test 4 In the same manner as in the above test 1, the obtained PRP solution (200 µl) was put in cell for measurement of aggregation and the cell was warmed at 37°C for 1 minute. Thereto was added ADP or collagen suspension (final concentration of 14 to 16 µM and 1 to 4 µg/ml, respectively) without addition of the test compound to form aggregates of platelet. MAR was determined after stable transparency was obtained. This MAR was referred to as "MAR-1". Then, the test compound was added and MAR was determined from the transparency after 5 minutes. This MAR was referred to as "MAR-2". Platelet aggregate dissociation rate (%) was calculated in accordance with the following formula: Platelet aggregate dissociation rate (%) = [(MAR-1) - (MAR-2)]/(MAR-1) x 100 The test compounds used in the above tests are as follows: Test Compound No. 1. 6-[3-(N-methyl-N-benzylamino)-2-hydroxypropoxy]carbostyril 2. 6-[3-(3-methoxybenzyl)amino-2-hydroxypropoxy]carbostyril hydrochloride 3. 6-[3-(4-chlorobenzylamino)-2-hydroxypropoxy]carbostyril hydrochloride 4. 6-{3-[N-methyl-N-(2-pyridylmethyl)amino]-2-hydroxypropoxy}carbostyril 5. 6-[3-{N-methyl-N-[2-(N-methylanilino)ethyl]amino}-2-hydroxypropoxy]carbostyril 6. 6-[3-(4-methoxybenzylamino)-2-hydroxypropoxy]carbostyril hydrochloride 7. 6-[3-(3,4-dimethoxybenzyl)amino-2-hydroxypropoxy]carbostyril 8. 6-[3-(3-chlorobenzyl)amino-2-hydroxypropoxy]carbostyril 9. 6-[3-(3,4-diethoxybenzyl)amino-2-hydroxypropoxy]carbostyril 10. 6-{3-[N-(2-hydroxyethyl)-N-(4-fluorobenzyl)amino]-2-hydroxypropoxy}carbostyril The results of Pharmacological tests 1, 2, 3 and 4 are shown in the following Tables 1, 2, 3 and 4, respectively. Test compound No. Phosphodiesterase inhibitory rate (%) Conc. (mol) 10⁻⁴ 10⁻⁵ 2 2.32 48.63 3 32.46 86.11 7 15.27 72.39 9 50.63 89.07 10 23.90 74.63 Test comp. No. Organ where blood flow was measured Dosage (µg/kg/min) Increasing rate of blood flow (%) 7 Cerebellum 3 15.2 ± 11.5 10 29.7 ± 11.5 Brain 3 25.9 ± 7.0 10 57.8 ± 10.9 Preparation Example 1 6-[3-(3-Methoxybenzyl)amino-2-hydroxypropoxy] carbostyril hydrochloride 5 mg Starch 132 mg Stearate magnesium 18 mg Lactose 45 mg Total 200 mg According to the usual method, tablets each containing the above components were prepared. Preparation Example 2 6-[3-(3,4-Dimethoxybenzyl)amino-2-hydroxypropoxy]-carbostyril 500 mg Polyethylene glycol (molecular weight: 4000) 0.3 g Sodium chloride 0.9 g Polyoxyethylene sorbitan monooleate 0.4 g Sodium metabisulfite 0.1 g Methylparaben 0.18 g Propylparaben 0.02 g Distilled water for injection 100 ml The above parabens, sodium metabisulfite and sodium chloride were dissolved in the above distilled water at 80°C with stirring. The obtained solution was cooled to 40°C. The compound of the present invention, polyethylene glycol and polyoxyethylene sorbitan monooleate were dissolved successively in the solution and thereto was added the distilled water for injection to adjust to the final volume. The obtained solution was sterilized by filtration with a suitable filter paper and each 1 ml was poured into an ample to prepare injections.

Claims: Use of carbostyril derivatives having the general formula: wherein R¹ is hydrogen or a lower alkyl group optionally having hydroxy group as a substituent; R² is a phenyl(lower alkyl) group optionally having 1 to 3 substituents selected from the group consisting of a lower alkoxy group and a halogen atom on the phenyl ring, pyridyl(lower alkyl) group, or a group of the formula -A-NR³R⁴ (wherein A is a lower alkylene group, R³ and R⁴ are the same or different and represent a lower alkyl group or phenyl group), or a salt thereof for preparing a medicament for inhibiting platelet aggregation. The use of claim 1, wherein the medicament is an ameliorant of cerebral circulation. The use of claim 1, wherein the medicament is an agent for platelet aggregate dissociation. The use of claim 1, wherein the medicament is an agent for treating thrombosis. The use according to any of claims 1-4, wherein the carbostyril derivative is 6-[3-(3-methoxybenyzl)amino-2-hydroxypropoxy]carbostyril. The use according to any of claims 1-4, wherein the carbostyril derivative is 6-[3-(3,4-dimethoxybenzyl)amino-2-hydroxypropoxy]carbostyril.

Inventors: FUJIOKA TAKAFUMI, TERAMOTO SHUJI, TOMINAGA MICHIAKI, YABUUCHI YOICHI, FUJIOKA, TAKAFUMI, TERAMOTO, SHUJI, TOMINAGA, MICHIAKI, YABUUCHI, YOICHI
Applicants: OTSUKA PHARMA CO LTD, OTSUKA PHARMACEUTICAL CO., LTD.
Application year: 1992
Main classification: A61K31
IPC codes: C07D215, A61P43, A61P7, A61K31