JPH0231055B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to an anticoagulant containing ^N2 -arylsulfonyl-L-argininamide or a salt thereof as an active ingredient. More specifically, (2R, 4R)-1-[N ² -(3-methyl-1,
The present invention relates to an anticoagulant containing 2,3,4-tetrahydro-8-quinolinesulfonyl)-L-arginyl]-4-methyl-2-piperidinecarboxylic acid and/or its salts as an active ingredient. N ² -Arylsulfonyl-L- has antithrombin action and is effective as an excellent therapeutic agent for thrombosis.
Regarding argininamides, a patent application has already been filed in 1983.
No. 4529, but the present invention provides an anticoagulant containing as an active ingredient a compound that has a special three-dimensional structure not reported therein and has particularly excellent antithrombin action. It is. The anticoagulant of the present invention is represented by the following formula (). (In the above formula (), * indicates an asymmetric carbon, (2R,
4R) is an optical isomer. ) As mentioned above, the ^N2 -arylsulfonyl-L-argininamide () and its salts of the present invention have a highly specific inhibitory effect on thrombin in the mammalian body, and have almost no toxicity. It is useful as a diagnostic agent for measuring thrombin in blood or in the treatment and prevention of thrombosis. The compounds are also useful as platelet aggregation inhibitors. By measuring the fibrinogen clotting time, the antithrombin effect of N ² -arylsulfonyl-L-arginine amide () of the present invention was determined from the known antithrombin agent N ² -(p-tolylsulfonyl)-L-arginine methyl. Ester (hereinafter
TAME). The test was conducted as follows. Beef fibrinogen (corn fraction 1)
A solution of 150 mg of Cohn Fraction 1 (manufactured by Armor) dissolved in 40 ml of Borate Saline Buffer (PH7.4).
Mix 0.8 ml and 0.1 ml of borate saline buffer (control sample) or sample solution under ice cooling, and then add 5 units/ml of thrombin (reagent manufactured by Mochida Pharmaceutical Co., Ltd.).
Add 0.1ml under ice-cooling, mix well and immediately bring to 25°C.
Transfer to a constant temperature bath. A stopwatch was started the moment it was placed in the thermostatic bath, and the time until fibrin was observed was measured. When no sample was added (control experiment), the solidification time was 50-55 seconds. The experimental results are shown in Table 1. In Table 1, "concentration that doubles the coagulation time" refers to the concentration of the active ingredient required to prolong the coagulation time from 50 to 55 seconds in the control experiment to 100 to 110 seconds. For TAME, the concentration that doubled the clotting time was 1100 μM. When the solution of ^N2 -arylsulfonyl-L-argininamide () of the present invention is administered intravenously to animals, the high antithrombin effect in the circulating blood is
3 hours, the elimination half-life of the antithrombin agent of the present invention in the circulation was approximately 60 minutes. The physiological conditions of the experimental animals (rats, rabbits, and dogs) were maintained in good condition. The decrease in platelet counts caused by continuous thrombin infusion in experimental animals was suppressed by co-injection of the inhibitor of the present invention. The acute toxicity value (LD ₅₀ ) of this inhibitor when administered intravenously to mice (male, 20 g) is approximately 250 mg/Kg.
It's weight. I ₅₀ = 0.02 μM of the compound of the present invention, whereas the other optical isomer (2S, 4S)-1-[N ² -
(3-methyl-1,2,3,4-tetrahydro-
8-quinolinesulfonyl)-L-arginyl]-
4-Methyl-2-piperidinecarboxylic acid is I ₅₀ =
It is 70 μM, which is about 2000 times more effective in the present invention. The method for producing the compound is shown below. The compound of the present invention can be produced by various methods, for example, the method described in Japanese Patent Application No. 53-4529 can be used. The most preferred method will be explained below. The reaction route is as follows. (In the formula, R represents a lower alkyl group.) (In the formula, R represents a lower alkyl group) (In the formula, R represents a lower alkyl group) (In the formula, R represents a lower alkyl group) In the above formula, () is (2R,4R)-4-methyl-
It is a lower alkyl ester of 2-carboxypiperidine, and * in the formula represents an asymmetric carbon atom. R ³ and R ⁴ represent a hydrogen atom or a protecting group for a guanidide group, such as a nitro group, a tosyl group, a trityl group, or an oxycarbonyl group. At least one of R ³ and R ⁴ must be a protecting group. R ⁵ is a protecting group for a 2-amino group, and examples of the protecting group include a benzyloxycarbonyl group and a tert-butoxycarbonyl group. The N ² -arylsulfonyl-L-argininamide () of the present invention is produced by hydrogenolysis of N ^G -substituted-N ² -arylsulfonyl-L-argininamide (). As the protecting groups R ³ and R ⁴ , a nitro group or an oxycarbonyl group is particularly preferable. These groups can be removed very easily by hydrogenolysis. Examples of the oxycarbonyl group include benzyloxycarbonyl group and p-nitrobenzyloxycarbonyl group. Hydrogenolysis produces methanol, ethanol, acetic acid,
Using a hydrogen activation catalyst such as nickel, palladium, platinum, ruthenium or rhodium in an inert solvent such as tetrahydrofuran or dioxane, in a hydrogen atmosphere at 0°C to 200°C, preferably room temperature to
React within the range of 150℃. The reaction time varies depending on the solvent or catalyst used or the amount used, but the reaction is completed in 2 to 120 hours. Hydrogen pressure is 1-100
A range of Kg/cm ² is good. When reducing the 3-methyl-8-quinolyl group at the same time, it can be carried out by appropriately selecting a method such as increasing the reaction time or increasing the amount of catalyst. In this case, the end point of the reaction can be determined by measuring the amount of hydrogen absorbed. After the reaction is completed, the catalyst is filtered and the solvent is distilled to obtain N ² -arylsulfonyl-L-argininamide (). It can be purified by recrystallization from diethyl ether-tetrahydrofuran, diethyl ether-methanol, water-ethanol, ethanol, etc., or by chromatography using silica gel or alumina in some cases. Lower alkyl esters of (2R,4R)-4-alkyl-2-piperidinecarboxylic acid () can be easily produced from (2R,4R)-4-alkyl-2-piperidinecarboxylic acid and lower alcohols by conventional methods. (2R,4R)-4-alkyl-2-piperidinecarboxylic acid is obtained by adding L-tartaric acid to 4-alkyl-2-piperidinecarboxylic acid (racemic form) in ethanol,
(2R,4R)-4-alkyl-2-
A 1:1 addition salt of piperidinecarboxylic acid and L-tartaric acid is obtained. The three-dimensional structure of this product was determined from X-ray analysis. N ² -arylsulfonyl-L-argininamide () forms salts with various inorganic or organic acids or bases. The N ² -arylsulfonyl-L-argininamide () obtained in the above reaction can be isolated in free form or in salt form. By reacting the free base with the desired acid
An acid addition salt of N ² -arylsulfonyl-L-argininamide () can be obtained. The above acids include hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, sulfuric acid, phosphoric acid, acetic acid, citric acid, maleic acid, succinic acid, lactic acid, tartaric acid, gluconic acid, benzoic acid, methanesulfonic acid. acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid and the like. Similarly, a base addition salt of compound () can be obtained by reacting free compound () with the desired base. Examples of such bases include sodium hydroxide, potassium hydroxide, ammonium hydroxide, triethylamine, procaine, dibenzylamine, 1-ephenamine, N,N'-dibenzylethylenediamine, and N-ethylpiperidine. Free amides can be regenerated by treating these salts with bases or acids. The anticoagulant of the present invention is administered alone or in combination with a pharmaceutically acceptable carrier to mammals, including humans. Its composition is determined by the compound's solubility, chemical properties, route of administration, dosage regimen, etc. For example, when a compound is administered parenterally by intra-intravenous, intravenous, or subcutaneous injection, it is used as a sterile solution with the addition of saline or other solutes such as glucose to make the solution isotonic. The compounds may also be administered orally in the form of tablets, capsules or granules containing suitable excipients such as starch, lactose, sucrose and the like. In addition, the compound is added with sugar, corn syrup, flavoring, coloring, etc., dehydrated, molded, solidified, and used as a lozenge such as a troche or lozenge. When administered orally as a solution, colorants and flavoring agents are added. The optimal dosage for humans of the therapeutic agent of the present invention is determined by a physician depending on the administration method, the type of compound, and the patient's condition. Large doses are required to achieve the same effect with oral administration as with parenteral administration. Therapeutic doses are generally 1 to 50% of the active ingredient administered parenterally.
mg/Kg/day, orally 1 to 250 mg/Kg/day. Next, the method for producing the compound will be explained in more detail using Examples, but the present invention does not exceed the gist thereof.
The present invention is not limited to the compounds synthesized in these Examples. Note that the present invention also includes pharmaceutical compositions containing the compound of the present invention as an active ingredient. Such compositions are in the form as described above, and particularly the present invention includes compositions in a single dosage format. Example (A) Separation of trans and cis isomers from ethyl 4-methyl-2-piperidine carboxylate The cis and trans isomers were separated by vacuum distillation. Trans form Boiling point 85~7℃/7mmHg Cis form Boiling point 107~8℃/5mmHg (B) Optical resolution of trans form Ethyl 4-methyl-2-piperidinecarboxylate (trans form) racemic form in excess concentrated hydrochloric acid Boiling for an hour yields 4-methyl-2-piperidinecarboxylic hydrochloride. 4-Methyl-2-piperidinecarboxylic hydrochloride was added to an ion exchange resin (Diaion SK-112,
Product name, manufactured by Mitsubishi Chemical Industries, Ltd.)
Methyl-2-piperidinecarboxylic acid racemate is obtained. 143.2 g of this racemate is boiled in 2900 ml of 95% ethyl alcohol, 150 g of L-tartaric acid is added, and after cooling, the precipitated salt is filtered to obtain 145.9 g of crude crystals. When the crude crystals are recrystallized from 1000 ml of 90% ethyl alcohol, (2R,4R)-4-methyl-2-piperidinecarboxylic acid-L-tartrate is obtained. The physical properties of this product are as follows. Melting point 183.9-185.0℃ [α] ²⁶ _D = +4.4゜ (C = 10g/100mlH ₂ O) Elemental analysis value C ₁₁ H ₁₉ NO ₈ C H N Calculated value 45.05 6.53 4.77 Actual value 45.12 6.48 4.70 X-ray diffraction Accordingly, this compound is (2R, 4R) -
4-Methyl-2-piperidinecarboxylic acid and L-
It is a 1:1 compound of tartaric acid. Using 2000ml of ion exchange resin (Diaion SK112) washed with water, this compound was eluted with a 3% aqueous ammonium hydroxide solution, and the solvent was distilled off, resulting in 63.0g of (2R,4R)-4-methyl-2-
Piperidine carboxylic acid was obtained in powder form. The physical properties of this compound recrystallized from ethyl alcohol-water are shown below. Melting point 275.0-277.8℃ [α] ¹⁸ _D = -18.0゜ (C = 10g/100ml2N-HCl) Elemental analysis value C ₇ H ₁₃ NO ₂ as C H N Calculated value 58.72 9.15 9.78 Actual value 58.80 9.09 9.71 Optical antipode Concentrate and dry the mother liquor containing
It was treated in the same manner as described above to obtain 64.6 g of (2S,4S)-4-methyl-2-piperidinecarboxylic acid. The physical property values are as follows. Melting point 275.0~277.8℃ [α] ²⁴ _D = +17.8° (C = 10g/100ml/2N-
HCl) Elemental analysis value C ₇ H ₁₃ NO ₂ C H N Calculated value 58.72 9.15 9.78 Actual value 58.82 9.10 9.69 (C) Synthesis of ethyl (2R, 4R)-4-methyl-2-piperidinecarboxylate (2R, 4R) )-4-Methyl-2-piperidinecarboxylic acid 51.6 g in anhydrous ethyl alcohol 30
Thionyl chloride 128.6 with stirring below ℃
g was added dropwise, and the mixture was allowed to react at room temperature for 1 hour and then for another 1 hour under reflux. The solvent was distilled off and the residue was dissolved in benzene.
Dissolve in 500 ml, wash with 100 ml of 5% potassium carbonate and 200 ml of saturated saline, and dry over anhydrous sodium sulfate. When the benzene is distilled off and the remaining liquid is vacuum distilled, 57.4g of ethyl (2R, 4R) -
4-Methyl-2-piperidine carboxylate was obtained. The physical properties are as follows. Boiling point 83-85℃/7mmHg [α] ²² _D = -24.0゜ (C = 5g/100mlC ₂ H ₅ OH) Elemental analysis value C ₉ H ₁₇ NO ₂ as C H N Calculated value 63.13 10.00 8.18 Actual value 63.20 9.96 8.12 Example (A) Ethyl (2R, 4R)-1-[N ^G -nitro-
N ² -(tert-butoxycarbonyl)-L-arginyl]-4-methyl-2-piperidinecarboxylate N ^G -nitro- ^{N 2} -(tert-butoxycarbonyl)-L-arginine 28.3 g in 450 ml of anhydrous tetrahydrofuran The solution dissolved in -20℃
While stirring, add triethylamine 9
g then 12.2 g of isobutyl chloroformate
Add. After 10 minutes, more ethyl (2R, 4R) -
4-Methyl-2-piperidine carboxylate
Add 15.2 g and stir the mixture for 10 minutes at -20°C. At this point the reaction mixture was warmed to room temperature and
The solvent is evaporated off and the residue is dissolved in 400 ml of ethyl acetate. It is then washed successively with 200 ml of water, 100 ml of 5% sodium bicarbonate solution, 100 ml of 10% citric acid solution, and 200 ml of water, and the ethyl acetate solution is dried over anhydrous sodium sulfate. 31.3g when ethyl acetate is distilled off
(47.5%) of ethyl (2R, 4R) -1- [N ^G -
Nitro-N ² -(tert-butoxycarbonyl)-
L-Arginyl]-4-methyl-2-piperidinecarboxylate was obtained in the form of syrup. IR (KBr): 3300, 1730, 1680cm ^-1 (B) Ethyl (2R, 4R) -1 - (N ^G - Nitro - L
-Arginyl)-4-methyl-2-piperidinecarboxylate hydrochloride Ethyl (2R,4R)-1-[N ^G -nitro-
Dissolve 30 g of N ² -(tert-butoxycarbonyl)-L-arginyl]-4-methyl-2-piperidinecarboxylate in 30 ml of ethyl acetate, and add 80 ml of 10% anhydrous hydrogen chloride-ethyl acetate at 0°C with stirring. Add. After 3 hours, 200 ml of anhydrous ethyl ether was added to the solution, resulting in a viscous oil. This material was filtered and washed with anhydrous ethyl ether to give ethyl (2R,
4R)-1-(N ^G -nitro-L-arginyl)
-4-Methyl-2-piperidinecarboxylate hydrochloride was obtained as an amorphous solid. (C) Ethyl (2R, 4R)-1- [N ^G -nitro-
N ² -(3-methyl-8-quinolinesulfonyl)
-L-arginyl]-4-methyl-2-piperidinecarboxylate ethyl (2R,4R)-1-[N ^G -nitro-L
-Arginyl)-4-methyl-2-piperidinecarboxylate hydrochloride 25g in chloroform
18.5 g of triethylamine and 14.7 g of 3-methyl-8-quinolinesulfonyl chloride were dissolved in 200 ml and maintained at 5° C. under stirring, followed by stirring at room temperature for 3 hours. After stirring, the solution is washed twice with 50 ml of water and dried over anhydrous sodium sulfate. After removing the solvent by evaporation, using 50g of silica gel and washing with chloroform, the concentration was 3%.
Elution was performed with methanol-chloroform. When the eluted solvent was distilled off, 32.5g (92.1%) of ethyl (2R, 4R)-1-[N ^G -nitro-N ² -(3-
Methyl-8-quinolinesulfonyl)-L-arginine]-4-methyl-2-piperidinecarboxylate was obtained as a solid. IR (KBr): 3250, 1725, 1640cm ^-1 (D) (2R, 4R) - 1 - [N ^G - Nitro - N ² - (3
-Methyl-8-quinolinesulfonyl)-L-arginyl]-4-methyl-2-piperidinecarboxylic acid ethyl (2R,4R)-1-[N ^G -nitro-
N ² -(3-methyl-8-quinolinesulfonyl)
-L-Arginyl]-4-methyl-2-piperidinecarboxylate 30g and ethanol 100ml
and 100 ml of a 1N aqueous sodium hydroxide solution, and stirred at room temperature for 24 hours. then 1N
Neutralize with hydrochloric acid and concentrate to 70ml. The solution was brought to pH 11 with 1N sodium hydroxide and diluted with ethyl acetate.
100ml, then wash with 100ml of chloroform, 1N
Acidify with hydrochloric acid. Filter the formed precipitate and add 20% water
Wash with 27g (95%) of (2R,4R)-1
―[N ^G -Nitro-N ² -(3-methyl-8-quinolinesulfonyl)-L-arginyl]-4-
Methyl-2-piperidinecarboxylic acid was obtained. Melting point 211-213℃ IR (KBr): 3280, 1720, 1620 cm ^-1 Elemental analysis value C ₂₃ H ₃₁ N ₇ O ₇ C H N Calculated value 50.26 5.69 17.84 Actual value 50.05 5.45 17.45 (E) (2R, 4R )-1-[N ² -(3-methyl-1,
2,3,4-tetrahydro-8-quinolinesulfonyl)-L-arginyl]-4-methyl-2
―Piperidinecarboxylic acid (2R, 4R)―1― [N ^G ―Nitro―N ² ―
(3-methyl-8-quinolinesulfonyl)-L
-Arginyl]-4-methyl-2-piperidinecarboxylic acid 3.0g in ethanol 40ml and acetic acid 10ml
0.3 g of 5% Pd/C was added, and the mixture was shaken at 80° C. for 4 hours under a hydrogen pressure of 50 kg/cm ² . The catalyst is then removed by filtration and the solvent is removed by evaporation. From the resulting viscous oil 30 ml of chloroform and saturated sodium bicarbonate solution
Extract the chloroform layer with 30 ml of water and 30 ml of water.
After washing with ml of water, chloroform was removed by evaporation, and the resulting crude crystals were recrystallized from ethanol.
(94%) of (2R,4R)-[N ² -(3-methyl-1,2,3,4-tetrahydro-8-quinolinesulfonyl)-L-arginyl]-4-methyl-2-piperidinecarboxylic acid was gotten. Melting point 188-191℃ NMR 100MHz Solvent CD ₃ OH δ value 6.5 (Triplet 1H) 7.1 (Doublet 1H) 7.4 (Doublet 1H) Elemental analysis value C ₂₃ H ₃₆ N ₆ O ₅ S C H N Calculated value 54.31 7.13 16.52 Actual measurement Value 54.01 6.98 16.61 Hydrous alcohol (alcohol content 15%)
The monohydrate of the above compound is obtained by recrystallization from . The N ² -arylsulfonyl-L-argininamide obtained in this example is shown in Table 1 along with its anti-coagulant activity.

[Table] * indicates an asymmetric carbon.
Production Example 1 Tablet for Oral Administration Tablets were obtained by a conventional method using the ingredients shown in Table 2 below.

[Table] Production Example 2 Capsules for Oral Administration Using the same compound of the present invention as in Production Example 1, the ingredients shown in the table below were mixed and filled into gelatin capsules to prepare capsules.

[Table] Production Example 3 Sterile Solution for Injection The components shown in the table below were mixed to form a solution and sterilized to prepare a solution for intravenous injection.

【table】

Claims (1)

[Claims] 1 The following formula () (In the above formula (), * indicates an asymmetric carbon, (2R,
4R) is an optical isomer. ) An anti-blood coagulant containing N ² -arylsulfonyl-L-argininamide and/or its salts as an active ingredient.