JPS6136515B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention provides novel alkanamide derivatives, more specifically, the general formula []: [In the formula, R ¹ is a lower alkyl group, R ² is a cycloalkyl group, A is a methylene group or a carbonyl group, and n means an integer of 1 to 3]. The compound of the present invention has a platelet aggregation inhibiting effect, an anti-inflammatory effect, or a phosphodiesterase inhibitory effect, and is useful as an antithrombotic agent, an anti-inflammatory agent, an anti-ulcer agent, and an anti-asthmatic agent. In the above general formula [], the lower alkyl group includes, for example, methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, and the like. Examples of the cycloalkyl group include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, and the like. Representative compounds of the present invention include the following. N-methyl-N-cyclohexyl-4-[6-
(3,4-dihydrocarbostyryl)]butyric acid amide, N-methyl-N-cyclohexyl-5-[6-
(3,4-dihydrocarbostyryl)]valeramide, N-methyl-N-cyclohexyl-4-[6-
(3,4-dihydrocarbostyryl)]-4-oxobutyric acid amide, N-methyl-N-cyclohexyl-5-[6-
(3,4-dihydrocarbostyryl)]-5-oxovaleramide, N-methyl-N-cyclohexyl-3-[6-
(3,4-dihydrocarbostyryl)]propionic acid amide, N-butyl-N-cyclooctyl-4-[6-
(3,4-dihydrocarbostyryl)]butyric acid amide, N-ethyl-N-cyclohexyl-5-[6-
(3,4-dihydrocarbostyryl)]-5-oxovaleramide, N-methyl-N-cyclopropyl-4-[6-
(3,4-dihydrocarbostyryl)]-4-oxobutyric acid amide, N-isopropyl-N-cyclohexyl-5-
[6-(3,4-dihydrocarbostyryl)]valeramide. The compounds of the present invention can be produced by various methods, but
For example, as shown in the reaction formula below, it is produced by reacting a known carboxylic acid derivative [] with a known amine derivative []. [In the formula, R ¹ , R ² , A and n are the same as above] The method for producing the compound of the present invention will be explained in more detail according to the above reaction formula. The reaction between carboxylic acid [] and amine [] is
Conditions used in known amide bond forming reactions can be employed, and preferably, for example, a mixed acid anhydride method is used. That is, a carboxylic acid [ ] is reacted with an alkylhalocarboxylic acid to form a mixed acid anhydride, which is then reacted with an amine [ ]. Examples of the alkylhalocarboxylic acids used in the mixed acid anhydride method include methyl chloroformate, methyl bromoformate, ethyl chloroformate, ethyl bromoformate, isobutyl chloroformate, and the like. The mixed anhydride is obtained by the usual Schotten-Baumann reaction, and the compound represented by the general formula [] is produced by reacting it with an amine [] without isolation. The Schotten-Baumann reaction is carried out using basic compounds commonly used in the reaction, such as organic bases such as triethylamine, trimethylamine, pyridine, dimethylaniline, N-methylmorpholine, and inorganic bases such as potassium carbonate, sodium carbonate, potassium bicarbonate, and sodium bicarbonate. It is carried out in the presence of a base, and is generally carried out at a temperature of -20 to 100°C, preferably 0 to 50°C, for 5 minutes to 10 hours, preferably 5 minutes to 2 hours. In addition, the reaction between the obtained mixed acid anhydride and amine [] is carried out at -20 to 150°C, preferably at 10 to 50°C.
℃ for 5 minutes to 10 hours, preferably 5 minutes to 10 hours.
The test is carried out under conditions of 5 hours. This reaction is usually carried out using a suitable solvent, such as halogenated hydrocarbons such as methylene chloride, chloroform, and dichloroethane, aromatic hydrocarbons such as benzene, toluene, and xylene, and ethers such as diethyl ether, tetrahydrofuran, and dimethoxyethane. Esters such as methyl acetate and ethyl acetate, N/N-
dimethylformamide, dimethyl sulfoxide,
It is carried out using an aprotic polar solvent such as hexamethylphosphoric triamide. In the above method, the ratio of carboxylic acid [], alkylhalocarboxylic acid and amine [] to be used is usually equivalent to 1.5 molar ratios per carboxylic acid []. It may be used up to twice the mole. The compound of the general formula [] produced in this way is
It can be easily isolated and purified by conventional separation means. Examples of the separation means include solvent extraction, solvent dilution, recrystallization, liquid chromatography, and gas chromatography. EXAMPLES Next, the present invention will be explained in more detail with reference to Examples, but the present invention is not limited thereto. Example 1 0.7 g of 4-[6-(3,4-dihydrocarbostyryl)]butyric acid is suspended in 10 ml of dimethylformamide. Add and dissolve 0.4 g of triethylamine while stirring at room temperature. 0.5 g of isobutyl chloroformate is added dropwise to this while stirring under ice cooling, and the mixture is stirred at room temperature for 30 minutes. 0.4 g of N-methylcyclohexylamine was added dropwise to this, and the mixture was stirred at room temperature for 1 hour. The reaction solution was poured into water and extracted with ethyl acetate. The ethyl acetate solution is washed sequentially with dilute hydrochloric acid, saturated aqueous sodium bicarbonate, and saturated brine. Dry over sodium sulfate and evaporate ethyl acetate. The residue was recrystallized from ligroin to give colorless prismatic N-methyl-N-cyclohexyl-5-
0.7 g of [6-(3,4-dihydrocarbostyryl)]butyric acid amide is obtained. Melting point: 95-98°C Examples 2-3 The following compounds were obtained in the same manner as in Example 1 above. N-methyl-N-cyclohexyl-4[6-
(3,4-dihydrocarbostyryl)]-4-oxobutyric acid amide, colorless needle crystals (recrystallization solvent: ethanol), melting point 207-208°C N-methyl-N-cyclohexyl-5-[6-
(3,4-dihydrocarbostyryl)]-5-oxovaleric acid amide, pale yellow powder crystal (recrystallization solvent;
ethanol-ligroin), melting point 150-152° C. The results of pharmacological tests of the compound of the present invention are shown. <Pharmacological test 1> Inhibitory effect on cyclic adenosine monophosphate phosphodiesterase (C-AMP-PDE) This test was conducted in Biochimica et Biophysica Acta No.
Volume 429, pages 485-497 (1976) and Biochemistry
It is carried out according to the activity assay method described in Medicine, Vol. 10, pp. 301-311 (1974). That is, first, to the precipitated platelets obtained by centrifuging rabbit PRP at 3000 rpm for 10 minutes, 10 ml of a solution of 1 mmol of MgCl ₂ added to a 50 mmol Tris-HCl buffer with pH 7.4 was added to the platelets. Float the platelets, grind them with a Teflon potter type homogenizer, and then
Repeat freezing and thawing several times, then apply ultrasonic waves at 200 W for 300 seconds to disrupt the cells, and then ultracentrifuge at 100,000 G for 60 minutes to obtain the supernatant as a crude oxygen solution. Preliminary 50 mmol-Tris acetate buffer (PH6.0)
Pass 10 ml of the crude oxygen solution prepared above through a 1.5 x 20 cm DEAE-cellulose column buffered with
Wash and elute with 30 ml of 50 mmol Tris acetate buffer, and elute by applying a linear gradient to this buffer with 0 to 1 M sodium acetate-Tris acetate buffer (total eluate volume: about 300 ml). The flow rate is
The flow rate was 0.5 ml/min, and 5 ml of each fraction was collected. By the above operation, 100 μmol of high C-
It has weak activity at AMP substrate concentrations below 2 nmol/ml/min and at C-AMP substrate concentrations as low as 0.4 μmol.
Collect fractions with strong activity greater than 100 pmol/ml/min. This is called C-AMP-PDE. 0.1 ml of test compound aqueous solution of each concentration was determined in advance.
A total of 0.2 ml of a mixture containing 0.4 μmol of C-AMP (tritium C-AMP) at pH 8.0 and 40 mmol Tris-HCl buffer (containing 50 μg of bovine serum albumin and 4 mmol of _MgCl2 ) was added to the substrate solution. To this, add C-AMP-PDE solution with a constant concentration prepared above.
Add 0.2 ml and react at 30°C for 20 minutes to generate tritium 5-AMP from tritium C-AMP. Next, after immersing in boiling water for 2 minutes to stop the reaction, the reaction solution was cooled in ice water, 0.05 ml of snake venom (1 mg/ml) was added as 5'-nucleotidase, and the reaction was allowed to proceed for 10 minutes at 30°C. Converts 5′-AMP to tritium adenosine. The entire amount of the reaction solution obtained was transferred to a cation exchange resin [AG.50W×4200
~400 mesh (Bio-Rad product), column size 0.5 x 1.5 cm] to bind only the tritium adenosine added, and after washing with 6 ml of distilled water, 3N-
Elute with 1.5 ml of ammonia water. Add 10 ml of Triton-toluene type scintillator to the entire volume of this eluate.
PDE activity is measured by adding PDE and measuring tritiated adenosine produced using a liquid scintillation counter. PDE of each test compound measured according to the above method
The PDE inhibition rate (%) is calculated from the activity value (Vs) and the control value (Vc) (water not containing the test compound) using the following formula. PDE inhibition rate (%)=Vc-Vs/Vc×100 The obtained inhibitors (%) are shown in Table 1. Table 1 also shows the results of a similar test using the known papaverine as a control compound. Test compound: No.1: N-methyl-N-cyclohexyl-5-
[6-(3,4-dihydrocarbostyryl)]valeric acid amide No. 2: N-methyl-N-cyclohexyl-5-
[6-(3,4-dihydrocarbostyryl)]-4
-Oxobutyric acid amide No. 3: Papaverine (control drug)

[Table] <Pharmacological test 2> Platelet aggregation inhibitory effect was measured using an AG-type aggregometer [Bryston Manufacturing Company (Bryston
Manufacturing Co.)]. The blood sample collected from the rabbit is a mixture of sodium citrate and whole blood, with a mixing ratio of 1:9 (volume ratio).
It is. The sample was centrifuged at 1000 rpm for 10 minutes to collect platelet rich serum.
plasma (PRP)]. The resulting PRP was separated, and the remaining blood sample was further centrifuged at 3000 rpm for 15 minutes to collect serum with low platelet concentration [Platelet
poor plasma (PPP)]. The number of platelets contained in the PRP was determined using the Brecher-Clonkite method.
PRP was diluted with PPP to prepare a PRP sample containing 300,000 platelets/ ^mm2 for the adenosine diphosphate (ADP)-induced aggregation test, and 450,000/mm2 for the collagen-induced aggregation test. PRP samples containing platelets/mm ² were prepared. 0.6 ml of the PRP sample prepared above was added to 0.01 ml of a solution containing the compound to be tested at a predetermined concentration, and the mixture was placed in a constant temperature bath at a temperature of 37° C. for 1 minute. Next, 0.07 ml of ADP or collagen solution was added to the mixture. Measure the permeability of this mixture,
Changes in permeability were measured using an agglomerometer at a stirrer rotation speed of 1100 rpm. In this test ADP
Alternatively, Oren Veronal buffer (PH7.35) was used to prepare the collagen solution. the above
The ADP solution was adjusted to a concentration of 7.5 x 10 ^-5 M, and the collagen solution was prepared by adding 5 ml of the above buffer to 100 mg of collagen and grinding it, and the supernatant was used as a collagen inducer. Aspirin was used as a control substance in the ADP-induced aggregation test and the collagen-induced aggregation test. The aggregation inhibitory effect was measured as inhibition rate (%) with respect to the aggregation rate of the control. The aggregation rate is calculated according to the formula below. Aggregation rate = c-a/b-a x 100 where a: Permeability of PRP b: Permeability of PPP c: Permeability of PRP containing test compound and aggregation inducing agent Rabbit platelets induced with collagen or ADP Table 2 shows the inhibitory effect on aggregation. Note that the numerical values in Table 2 are inhibition rates (%). Moreover, test compounds No. 1 and 2 are the same as those in pharmacological test 1.

【table】

Claims (1)

[Claims] 1. General formula [In the formula, R ¹ is a lower alkyl group, R ² is a cycloalkyl group, A is a methylene group or a carbonyl group, and n means an integer of 1 to 3].