KR100245806B1 - Orally administrable selective thrombin inhibitors - Google Patents

Abstract

The present invention relates to a novel selective thrombin inhibitor of formula (1), a method for preparing the same, and a pharmaceutical composition for inhibiting thrombin containing the active ingredient, which is effective even when used for oral administration.

[Formula 1]

Wherein R ¹ and R ² each independently represent hydrogen, alkyl or alkyloxy, R ³ and R ⁴ each independently represent lower alkyl or cycloalkyl, and R ⁵ represents hydrogen, lower alkyl or amino.

Description

Selective thrombin inhibitors for oral administration

The present invention relates to a novel selective thrombin inhibitor of formula (1) which is effective even when used for oral administration, a preparation method thereof and a pharmaceutical composition for thrombin inhibition containing the compound as an active ingredient.

[Formula 1]

Wherein R ¹ and R ² each independently represent hydrogen, alkyl or alkyloxy, R ³ and R ⁴ each independently represent lower alkyl or cycloalkyl, and R ⁵ represents hydrogen, lower alkyl or amino.

In general, it is known that various complex enzyme reactions are involved in the coagulation process. And the last step involves the reaction of converting prothrombin to thrombin. Thrombin produced in this process activates platelets, converts fibrinogen to fibrin, etc. At this time, the produced fibrin is converted into a polymer by a polymerization reaction and crosslinked by activated blood factor XIII to insoluble coagulation. Becomes Thrombin also plays a role in activating blood factors V and VIII involved in the coagulation process, further accelerating the coagulation reaction. Therefore, the inhibitor of thrombin acts as an effective anticoagulant, and can inhibit platelet activity and prevent fibrin production and stabilization, thus developing a new substance that can inhibit thrombin activity for a long time, thereby preventing blood clotting and preventing various thrombosis. Methods for treatment have been sought.

However, simply being able to inhibit thrombin is limited to use as an effective anticoagulant. The reason is that since thrombin is a serine protease similar to trypsin, an effective thrombin inhibitor has a high inhibitory effect on trypsin. In the development of thrombin inhibitors, serine proteolytic enzymes, especially trypsin, are relatively less resistant because of the diverse presence of trypsin-like serine proteases (typical plasmin) in the human body, particularly in the blood. It is very important to have the nature to do.

Under these circumstances, extensive research has been conducted to develop selective thrombin inhibitors that effectively inhibit thrombin and have low inhibitory activity against trypsin.

Representative compounds that have been developed as effective thrombin inhibitors include the first arylsulfonyl arginine-based compounds, Argatroban of Formula 10 (see, for example, US Pat. Nos. 4,251,921 and 4201863).

[Formula]

This chemical has been reported to inhibit thrombin by 250 times better than trypsin and has already been commercialized as an injectable preparation in Japan in 1990 (Biochemistry 1984, 23, pp85-90). However, this compound has the disadvantage that it is not active at all for oral administration and is synthesized through a very complicated process.

In addition, NAPAP of the following formula 11, a benzamidine-based arylsulfonyl compound, has been developed as a thrombin inhibitor, which is not only easy to synthesize but also has an effective thrombin inhibitory effect compared to trypsin by 50 times. See J. Biol. Chem. 1991, 266, pp 20085-20093.

[Formula 11]

On the other hand, Ro 46-6240 compound of formula (12), which has improved selectivity compared to trypsin, has been reported as a powerful thrombin inhibitor, which shows the possibility of development as an intravenous preparation, but has a short blood half-life and thus the possibility of development as an oral administration. Is not present. See J. Med. Chem. 1994, 37, pp 3889-3901.

[Formula 12]

Representative compounds developed as an effective thrombin inhibitor capable of oral administration include CVS-1123 of Formula 12 developed by Corvas Corporation. It has been reported that this compound can be orally administered to dogs and monkeys as well as to mice, but has a disadvantage of poor selectivity for trypsin (WO 9315756, WO 9408941).

[Formula 13]

Therefore, the present inventors have conducted a long-term intensive study to develop a compound that is relatively easy to synthesize and exhibits an effective thrombin inhibitory activity, and has a markedly improved selectivity for thrombin compared to trypsin, and is also orally administrable. It was confirmed that the compound of formula 1 according to the present invention can effectively achieve this object and have completed the present invention.

Accordingly, an object of the present invention is to provide a novel thrombin inhibitor capable of oral administration and high selectivity and a method of preparing the same.

The present invention also relates to a composition for preventing blood clotting or treating various thrombosis, containing the compound as an active ingredient.

Hereinafter, the present invention will be described in detail.

The present invention relates to compounds represented by the following formula (1), pharmaceutically acceptable salts and isomers thereof.

[Formula 1]

Wherein R ¹ and R ² each independently represent hydrogen, alkyl or alkyloxy, R ³ and R ⁴ each independently represent lower alkyl or cycloalkyl, and R ⁵ represents hydrogen, lower alkyl or amino.

In the above definitions for the substituents of the compounds of the formula (1) according to the invention, the term "alkyl" when used alone or in combination with "alkyloxy" is methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl , Straight or branched chain hydrocarbon radicals of 1 to 8 carbon atoms, such as octyl or isomers thereof, and the term "lower alkyl" refers to straight or branched chains of 1 to 4 carbon atoms such as methyl, ethyl, propyl, butyl, or isomers thereof By hydrocarbon radical, the term "cycloalkyl" means cyclicalkyl having 3 to 8 carbon atoms including cyclopentyl.

Preferred compounds of formula (1) of the present invention wherein R ¹ and R ² each independently represent hydrogen, C ₁ -C ₅ alkyl or C ₁ -C ₃ alkyloxy, and R ³ and R ⁴ are each independently C ₁ -C ₃ alkyl or C ₃ -C ₅ cyclicalkyl, R ⁵ is C ₁ -C ₃ alkyl or amino.

Particularly preferred compounds of formula (I) of the present invention are those in which R ¹ and R ² each independently represent hydrogen, methyl, ethyl, propyl, butyl or isomers thereof, pentyl or isomers thereof or ethyloxy, and R ³ and R ⁴ are each independently Represents methyl, ethyl or cyclopentyl, and R ⁵ is a compound representing methyl or amino.

Representative examples of the compound of formula 1 according to the present invention include the following compounds:

1. (S) -3- (4-amiderazono-phenyl) -N-cyclopentyl-N-methyl-2- (4-propyl-benzenesulfonylamino) propionamide

2. (S) -3- (4-amiderazono-phenyl) -N-cyclopentyl-N-methyl-2- (4-butyl-benzenesulfonylamino) propionamide

3. (S) -3- (4-amiderazono-phenyl) -N-cyclopentyl-N-methyl-2- (4-t-butyl-benzenesulfonylamino) propionamide

4. (S) -3- (4-amiderazono-phenyl) -N-cyclopentyl-N-methyl-2- (3,4-diethyl-benzenesulfonylamino) propionamide

5. (S) -3- (4-amiderazono-phenyl) -N-cyclopentyl-N-methyl-2- (3-methyl-4-propyl-benzenesulfonylamino) propionamide

6. (S) -3- (4-amiderazono-phenyl) -N-cyclopentyl-N-methyl-2- (4-isoamyl-benzenesulfonylamino) propionamide

7. (S) -3- (4-amiderazono-phenyl) -N-cyclopentyl-N-methyl-2- (4-ethyl-benzenesulfonylamino) propionamide

8. (S) -3- (4-amiderazono-phenyl) -N-cyclopentyl-N-methyl-2- (3-methyl-4-ethyl-benzenesulfonylamino) propionamide

9. (S) -3- (4-amiderazono-phenyl) -N-cyclopentyl-N-ethyl-2- (4-isobutyl-benzenesulfonylamino) propionamide

10. (S) -3- [4- (amino-methylimino-methyl) -phenyl] -N-cyclopentyl-N-methyl-2- (4-propyl-benzenesulfonylamino) propionamide

11. (S) -3- (4-amiderazono-phenyl) -N-cyclopentyl-2- (4-isobutyl-benzenesulfonylamino) -N-methyl-propionamide

12. (S) -3- [4- (Amino-methylimino-methyl) -phenyl] -N-cyclopentyl-2- (4-isobutyl-benzenesulfonylamino) -N-methyl-propionamide

13. (S) -3- (4-amiderazono-phenyl) -N-cyclopentyl-2- (2-isobutyl-benzenesulfonylamino) -N-methyl-propionamide

14. (S) -3- (4-amiderazono-phenyl) -N-cyclopentyl-2- (4- (2,2-dimethyl-propyl) -benzenesulfonylamino) -N-methyl-propionamide

15. (S) -3- [4- (Amino-methylimino-methyl) -phenyl] -N-cyclopentyl-2- (4- (2,2-dimethyl-propyl) -benzenesulfonylamino)- N-methyl-propionamide

16. (S) -3- (4-amiderazono-phenyl) -N-cyclopentyl-N-methyl-2- (4-ethyloxy-benzenesulfonylamino) -propionamide

The compounds of formula 1 according to the invention may also form pharmaceutically acceptable salts. Such pharmaceutically acceptable salts include acids that form non-toxic acid addition salts containing pharmaceutically acceptable anions such as inorganic acids, such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrobromic acid, hydroiodic acid, tartaric acid, formic acid, Organic carbonic acid such as citric acid, acetic acid, trichloroacetic acid or trifluoroacetic acid, gluconic acid, benzoic acid, lactic acid, fumaric acid, maleic acid, etc., sulfonic acid such as methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid or naphthalenesulfonic acid Acid addition salts formed by and the like are included.

On the other hand, the compounds according to the invention may have an asymmetric carbon center and therefore may exist as racemic compounds, diastereomeric mixtures and individual diastereomers, all of these isomers being included within the scope of the invention.

The invention also relates to a process for preparing the compound of formula 1 as defined above.

According to the present invention, the compound of Formula 1 as defined above may be prepared by reacting a methylmercapto compound of Formula 2 with an amine derivative of Formula 3:

[Formula 2]

[Formula 3]

H ₂ NR ⁵

Method for preparing a compound of formula 1 according to the present invention can be represented by the following scheme 1.

Scheme 1

Wherein R ¹ , R ² , R ³ , R ⁴ and R ⁵ are as defined above.

As shown in Scheme 1, the compound of Formula 1 according to the present invention may be prepared by reacting a methylmercapto compound of Formula 2 with an amine derivative of Formula 3, which is a nucleophile.

This reaction can preferably be carried out in the presence of a solvent. Examples of the solvent that can be preferably used for this purpose may be any organic solvent that does not adversely affect the reaction, but generally alcohol solvents such as methanol, ethanol, propanol and the like are preferably used.

The reaction conditions, including the amount of the reactants in the present reaction, the reaction temperature, the reaction time and the like can be easily determined by those skilled in the art according to the specific reactant. In general, the reaction temperature may vary, but it is particularly preferable to carry out the reaction at 0 ℃ to 50 ℃. In addition, the reaction time generally takes 0.5 to 5 hours, but preferably performs the reaction for 1 to 2 hours.

After completion of the reaction, the product can be separated and purified by conventional workup methods such as chromatography, recrystallization and the like.

The methylmercapto compound of formula (2) used as an intermediate in preparing the compound of formula (1) in Scheme 1 may, for example, couple a compound of formula ⁴ with an amine substituted with R ³ and R ⁴ to obtain a compound of formula 5 After removing the amino protecting group from the compound of formula 5, the compound of formula 6 is reacted with a sulfonyl derivative of formula 7 to introduce a sulfonyl group at the N-terminus to obtain a compound of formula 8, Can be prepared by saturating with hydrogen sulfide in the presence of a base to produce a thioamide compound of formula 9, and methylating the resulting compound of formula 9.

[Formula 4]

[Formula 5]

[Formula 6]

[Formula 7]

[Formula 8]

[Formula 9]

Wherein R ¹ , R ² , R ³ , R ⁴ and R ⁵ are as defined above and P is conventional such as benzyloxycarbonyl, allyloxycarbonyl, t-butoxycarbonyl or trityl group and the like. Phosphorus amino protecting group.

Method for preparing a compound of Formula 2 as described above can be represented by the following scheme 2.

Scheme 2

As shown in Scheme 2, in order to prepare the compound of Formula 2, R ³ and R ⁴ substituted amine groups are first coupled to the C-terminus of the compound of Formula 4 to obtain a compound of Formula 5.

Known coupling reagents that can be used for the coupling reaction of compounds of formula 4 include dicyclohexylcarbodiimide (DCC), 3-ethyl-3 '-(dimethylamino) -propylcarbodiimide (EDC), bis- (2-oxo-3-oxazolidinyl) -phosphinic acid chloride (BOP-C1), diphenylphosphoryl azide (DPPA), and the like, but are not limited thereto.

In addition, the carboxylic acid compound of the formula (4) used in this coupling reaction may be used as it is, but is preferably converted into a reactive derivative thereof, such as an acid halide and other activated ester derivatives, and used in the coupling reaction to promote the reaction. You can. Activated derivatives of carboxylic acids are required to form amide bonds by coupling with amines or to form ester bonds by coupling with alcohols. Such reactive derivatives include conventional derivatives which may be prepared by conventional methods in the art, for example acid halides include acid chlorides, and activated esters include methoxycarbonyl chloride, isobutyloxycarbonyl Anhydrides of carboxylic acids derived from alkoxycarbonyl halides and coupling reagents such as chlorides, N-hydroxyphthalimide-derived esters, N-hydroxysuccinimide-derived esters, N-hydroxy-5- Norbornene-2 ', 3'-dicarboxyimide-derived esters, 2,4,5-trichlorophenol-derived esters and the like, but are not limited to these.

The compound of formula 5, produced by the coupling reaction of a compound of formula 4 with an amine group, is then removed by conventional methods to remove the N-terminal amino protecting group to afford the compound of formula 6. The compound of formula 6 is reacted with a sulfonylchloride compound of formula 7 to introduce a sulfonyl group at the N-terminal portion of the compound to obtain a compound of formula 8, and then the nitrile compound of formula 8 is combined with pyridine and trimethylamine Saturation with hydrogen sulfide in the presence of the same base yields a thioamide compound of formula 9, which is subsequently methylated to afford the desired compound of formula 2. Methylating agents such as methane iodide, dimethylsulfate ((CH ₃ ) ₂ SO ₂ ) or methyltriplate (CH ₃ OTf) may be used to methylate the compound of formula (9).

As mentioned above, the novel compound of Formula 1 according to the present invention has excellent selectivity and shows effective thrombin inhibitory effect by oral administration compared with known compounds. Therefore, the compounds of the present invention are useful for the prevention of coagulation and for the treatment of thrombosis.

Accordingly, another object of the present invention is to provide a pharmaceutical composition for preventing coagulation and treating thrombosis, which contains the compound of Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

The total daily dose to be administered to the host in a single dose or in separate doses when administering a compound of the present invention for clinical purposes is preferably in the range of 0.001 mg to 10 mg per kg of body weight, but the specific dose level for a particular patient is used. Specific compounds, body weight of each patient, sex, health status, diet, time of administration, method of administration, rate of excretion, drug mixing and the severity of the disease may vary.

The compounds of the present invention can be administered in injectable and oral formulations as desired.

Injectable preparations, such as sterile injectable aqueous or oleaginous suspensions, can be prepared using suitable dispersing agents, wetting agents, or suspending agents according to known techniques. Pharmaceutically acceptable solvents that can be used include water, Ringer's solution and isotonic NaCl solution and sterile fixed oils are conventionally employed as a solvent or suspending medium. Any non-irritating fixed oil may be used for this purpose, including mono-, diglycerides, and also fatty acids such as oleic acid are used in the preparation of injectables.

The solid dosage form for oral administration may be in the form of capsules, tablets, pills, powders and granules, and particularly, capsules and tablets are useful. Tablets and pills are preferably prepared with enteric agents. Solid dosage forms can be prepared by mixing the active compounds of formula 1 according to the invention with one or more inert diluents such as sucrose, lactose, starch and the like and carriers such as lubricants such as magnesium stearate, disintegrants, binders and the like.

One of the great features of the compound of formula (1) according to the present invention is that the pharmaceutical composition containing the same shows an excellent effect even when formulated orally by oral formulation, which is a rat The results of pharmacokinetic experiments demonstrated that oral administration of the pharmaceutical composition of the present invention confirmed that the concentration of the drug had a characteristic of being maintained in the blood for a long time. Therefore, it is more useful in that it can be effectively used as an oral preparation, unlike the conventional thrombin inhibitor.

In addition, in the course of performing pharmacokinetic experiments, it was confirmed that the compound according to the present invention did not exhibit acute toxicity against mammals such as mice and dogs, and achieved the desired purpose.

On the other hand, when clinically administering the compound of the present invention to obtain the desired anticoagulant effect and thrombolytic effect, the active compound of formula 1 according to the present invention is one or more components selected from thrombolytic agents and platelet activity inhibitors It can be administered at the same time. In this way, the thrombolytic release that can be administered in combination with the compound of the present invention may include t-PA, urokinase, streptokinase, and the like, and platelet activity inhibitors include aspirin, ticlopidin. , Clopidrogel, 7E3 monoantibody, and the like.

However, the preparations containing the compounds according to the invention for the purpose of the treatment and prevention of thrombi are not limited to those described above, and any preparations useful for the treatment and prevention of thrombi can be included.

The present invention is explained in more detail by the following examples and experimental examples, but the scope of the present invention is not limited in any way by these.

[Production Example 1]

Synthesis of Cyclopentyl-Methylamine

10 ml (113 mmol) of cyclopentanone were dissolved in 50 ml of methanol, and then 50 ml of water was added thereto. 7.6 g (113 mmol) of methylamine hydrochloride was added to the reaction mixture, and then 7.1 g (113 mmol) of sodium borocyanohydride (NaBH ₃ CN) was added thereto, followed by stirring for 12 hours while heating to reflux at a pH of 6 conditions. . The reaction solution was cooled to 0 ° C. and the pH was adjusted to neutral with 6N sodium hydroxide solution. Methanol was removed under reduced pressure, cooled to 0 ° C., adjusted to pH 2 with dilute hydrochloric acid and washed three times with diethyl ether. The aqueous layer was adjusted back to pH 11. 50 ml of dioxane and 37 g (169.5 mmol) of butyloxycarbonyl anhydride were added thereto and stirred at room temperature for 3 hours. After the reaction was completed, the reaction solution was distilled under reduced pressure, concentrated to a total volume of about 30ml, extracted with ethyl acetate, washed with an acid, aqueous base solution. The organic layer was dried over anhydrous magnesium sulfate, filtered and concentrated to obtain a white solid obtained by column chromatography [eluent: ethyl acetate / hexane (7: 3, v / v)]. The obtained solid was dissolved in 60 mL of 4N hydrochloric acid-dioxane solution, stirred for 30 minutes, the solvent was removed under reduced pressure and dried in vacuo to yield 13.7 g (yield: 90.5%) of the title compound.

¹ H nmR (CD ₃ OD, ppm) δ: 3.50 (m, 1H), 2.68 (s, 3H), 2.10 (m, 2H), 1.86-1.50 (m, 6H)

[Production Example 2]

Synthesis of (S) -3- (4-cyanophenyl) -N-cyclopentyl-2- (butyloxycarbonylamino) -N-methyl-propionamide

0.7 g (2.41 mmol) of (S) -3- (4-cyanophenyl) -2- (butyloxycarbonylamino) propionic acid was dissolved in 6 ml of dimethylformamide (DMF) and then cooled to 0 ° C. 0.7 g of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and 0.4 g of 1-hydroxybenzotriazole hydrate (HOBT) were added to the solution and stirred until complete dissolution. 0.4 g (2.96 mmol) of the compound obtained in Preparation Example 1 and 1.0 ml of N-methylmorpholine were added to the solution, and the reaction temperature was gradually raised to room temperature and stirred for 3.5 hours. After the reaction was completed, the reaction solution was distilled under reduced pressure to remove volatiles, and the obtained residue was diluted with ethyl acetate, and then washed with saturated aqueous sodium hydrogen carbonate solution, diluted hydrochloric acid and saturated brine. After washing, the anhydrous sodium sulfate was dried, filtered and concentrated, and the residue was purified by column chromatography [eluent: ethyl acetate / hexane (7: 3, v / v)] to give 0.65 g of the title compound (yield: 73.0%) was obtained.

¹ H nm R (CDCl ₃ , ppm) δ: 7.61 (m, 2H), 7.32 (m, 2H), 5.48, 5.01-4.86, 4.12 (m, m, m, 3H), 2.75, 2.62 (s, s , 3H), 2.90-1.20 (m, 17H)

Mass (FAB, m / e): 372 (M ⁺ +1)

[Manufacture example 3]

Synthesis of 4-propyl-benzenesulfonylchloride

1.5 mL (22.6 mmol) of chlorosulfonic acid was placed in a reaction vessel and cooled to 0 ° C. 1.0 ml (7.17 mmol) of propylbenzenes were slowly added dropwise thereto, taking care not to increase the temperature by 5 ° C or more. After the addition was completed, the reaction solution was raised to room temperature and stirred for 3 hours. The reaction solution was cooled to 0 ° C. again and ice was added to terminate the reaction. 40 ml of water and ethyl acetate were added to the reaction mixture, and extracted three times. The organic layers were combined, dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated to give 1.3 g of a solution. The obtained compound was a mixture of 2-propylbenzenesulfonylchloride and 4-propylbenzenesulfonylchloride, and the ratio thereof was 1: 2 by nmR analysis. In terms of this, the yield of the title compound was 0.86 g (yield: 55%).

¹ H nm R (CDCl ₃ , ppm) δ: 8.04 (dd, 2H), 7.49 (dd, 2H), 2.82 (t, 2H), 1.78 (m, 2H), 1.06 (t, 3H)

Mass (FAB, m / e): 219 (M ⁺ +1)

[Production Example 4]

Synthesis of 3- (4-cyanophenyl) -N-cyclopentyl-N-methyl-2- (4-propyl-benzenesulfonylamino) -propionamide

0.97 g (2.61 mmol) of the compound obtained in Preparation Example 2 was dissolved in 10 ml of methanol, and then cooled to 0 ° C. An excess of acetyl chloride (30 equivalents) was slowly added thereto, and the mixture was raised to room temperature and stirred for 1 hour. All volatiles were removed under reduced pressure to give a white solid which was dried in vacuo and then dissolved in 10 ml of DMF. 1.0 ml of N-methylmorpholine was added thereto and stirred for 10 minutes. 0.7 g (3.21 mmol) of the mixture obtained in Preparation Example 3 was added to the reaction mixture, followed by stirring for 1 hour. The solvent was removed under reduced pressure, and extracted three times with 40 ml of dichloromethane and water. The combined organic layers were dried over anhydrous magnesium sulfate, filtered and the filtrate was concentrated. The obtained solid was separated and purified by column chromatography [eluent: hexane / ethyl acetate (7: 3, v / v)] to yield 0.8 g (yield: 82%) of the title compound.

¹ H nm R (CDCl ₃ , ppm) δ: 7.72 (dd, 2H), 7.65 (m, 2H), 7.34 (m, 4H), 5.86 (m, 1H), 4.63, 4.56, 4.39, 3.88 (m, m, m, m, 2H), 3.05 (m, 2H), 2.69 (m, 2H), 2.59, 2.40 (s, s, 3H), 1.76-1.04 (m, 10H), 1.00 (t, 3H)

Mass (FAB, m / e): 454 (M ⁺ +1)

Example 1

Synthesis of (S) -3- (4-amiderazono-phenyl) -N-cyclopentyl-N-methyl-2- (4-propyl-benzenesulfonylamino) propionamide

0.45 g (0.99 mmol) of the compound obtained in Preparation Example 4 was dissolved in 10 ml of pyridine and placed in a flask equipped with 1.0 ml of triethylamine. Hydrogen sulfide (H ₂ S) gas was slowly flowed into the solution from one branch of the flask, and the gas was flowed out from the other branch. The hydrogen sulfide gas was saturated in the solution with stirring for about 10 minutes. The color of the solution changed from colorless to green and gradually dark brown. The flask was closed with a rubber stopper and left at room temperature for 3 days to complete the reaction, followed by distillation under reduced pressure to remove volatiles and dried with a vacuum pump. To the obtained yellow solid, 10 ml of acetonitrile and 0.25 ml of methane iodide (CH ₃ I) were added together and heated to reflux for 30 minutes. It was distilled under reduced pressure again to remove volatiles, dried with a vacuum pump, and the residue was dissolved in 8 ml of anhydrous methanol and stirred. To this was added 0.04 ml (0.66 mmol) of 80% hydrazine hydrate (H ₂ NNH ₂ .H ₂ O) in three portions at 10 minute intervals. After the reaction was completed, the reaction solution was concentrated and the residue was purified by column chromatography [eluent: dichloromethane / methanol (95: 5, v / v)] to give 0.19 g (yield: 80%) of the title compound. It was.

¹ H nmR (CD ₃ OD, ppm) δ: 7.66 (m, 4H), 7.42 (m, 2H), 7.33 (m, 2H), 4.58, 4.43, 4.05 (m, m, m, 2H), 3.04 , 2.88 (m, m, 2H), 2.66 (m, 2H), 2.59, 2.46 (s, s, 3H), 1.70-1.10 (m, 10H), 0.93 (m, 3H)

Mass (FAB, m / e): 486 (M ⁺ +1)

Production Example 5

Synthesis of 4-butyl-benzenesulfonylchloride

The reaction was carried out according to the same method as Preparation Example 3 except for using butylbenzene instead of propylbenzene to obtain 0.6 g (yield: 50%) of the title compound.

[Manufacture example 6]

Synthesis of 3- (4-cyanophenyl) -N-cyclopentyl-N-methyl-2- (4-butylbenzenesulfonylamino) -propionamide

The reaction was carried out in the same manner as in Preparation Example 4 using the compound obtained in Preparation Example 5, to obtain 0.5 g (yield: 80%) of the title compound.

¹ H nmR (CDCl ₃ , ppm) δ: 7.2-7.7 (m, 8H), 5.7 (m, 1H), 4.6 (m, 0.5H), 4.5 (m, 0.5H), 4.4 (m, 0.5H ), 3.8 (m, 0.5H), 2.9-3.0 (m, 2H), 2.6-2.7 (m, 2H), 2.5 (s, 1.4H), 2.3 (s, 1.6H), 0.9-1.7 (m, 15H)

Mass (FAB, m / e): 468 (M ⁺ +1)

Example 2

Synthesis of (S) -3- (4-amiderazono-phenyl) -N-cyclopentyl-N-methyl-2- (4-butyl-benzenesulfonylamino) -propionamide

The reaction was carried out in the same manner as in Example 1 using the compound obtained in Preparation Example 6, to obtain 0.11 g (yield: 40%) of the title compound.

¹ H nmR (CD ₃ OD, ppm) δ: 7.4-7.8 (m, 8H), 4.7 (m, 0.5H), 4.5-4.6 (m, 1H), 4.2 (m, 0.5H), 3.0-3.2 (m, 2H), 2.8 (m, 2H), 2.7 (s, 1.6H), 2.6 (s, 1.4H), 1.0-1.8 (m, 15H)

Mass (FAB, m / e): 500 (M ⁺ +1)

[Manufacture example 7]

Synthesis of 3- (4-cyanophenyl) -N-cyclopentyl-N-methyl-2- (4-t-butyl-benzenesulfonylamino) -propionamide

The reaction was carried out according to the same method as Preparation Example 4, except that 4-t-butylbenzenesulfonylchloride was used instead of 4-propyl-benzenesulfonyl chloride to obtain 0.52 g (yield: 82%) of the title compound. It was.

¹ H nmR (CDCl ₃ , ppm) δ: 7.2-7.8 (m, 8H), 5.8 (m, 1H), 4.6 (m, 0.5H), 4.5 (m, 0.5H), 4.4 (m, 0.5H ), 3.8 (m, 0.5H), 2.9-3.0 (m, 2H), 2.5 (s, 1.3H), 2.3 (s, 1.7H), 0.9-1.7 (m, 17H)

Mass (FAB, m / e): 468 (M ⁺ +1)

Example 3

Synthesis of (S) -3- (4-amiderazono-phenyl) -N-cyclopentyl-N-methyl-2- (4-t-butyl-benzenesulfonylamide) -propionamide

Using the compound obtained in Preparation Example 7, the reaction was carried out in the same manner as in Example 1 to obtain 0.12 g (yield: 42%) of the title compound.

¹ H nmR (CD ₃ OD, ppm) δ: 7.5-7.9 (m, 8H), 4.7 (m, 0.5H), 4.6 (m, 0.5H), 4.4 (m, 0.5H), 4.2 (m, 0.5H), 3.1-3.2 (m, 2H), 2.7 (s, 1.5H), 2.6 (s, 1.5H), 1.2-1.9 (m, 17H)

Mass (FAB, m / e): 500 (M ⁺ +1)

[Manufacture example 8]

Synthesis of 3- (4-cyanophenyl) -N-cyclopentyl-N-methyl-2- (3,4-diethyl-benzenesulfonylamino) -propionamide

The reaction was carried out according to the same method as Preparation Example 4, except that 3,4-diethyl-benzenesulfonylchloride was used instead of 4-propyl-benzenesulfonyl chloride, and 0.54 g (yield: 83%) of the title compound was obtained. Obtained.

¹ H nmR (CDCl ₃ , ppm) δ: 7.1-7.6 (m, 7H), 5.8 (m, 1H), 4.4-4.7 (m, 1H), 4.4 (m, 0.5H), 3.7 (m, 0.5 H), 2.9-3.1 (m, 2H), 2.7 (m, 4H), 2.6 (s, 1.4H), 2.3 (s, 1.6H), 0.9-1.7 (m, 14H)

Mass (FAB, m / e): 468 (M ⁺ +1)

Example 4

Synthesis of (S) -3- (4-amiderazono-phenyl) -N-cyclopentyl-N-methyl-2- (3,4-diethyl-benzenesulfonylamino) -propionamide

Using the compound obtained in Preparation Example 8, the reaction was carried out according to the same method as in Example 1 to obtain 0.13 g (yield: 44%) of the title compound.

¹ H nmR (CD ₃ OD, ppm) δ: 7.3-7.8 (m, 7H), 4.6 (m, 0.5H), 4.6 (m, 1H), 4.1 (m, 0.5H), 2.9-3.1 (m , 2H), 2.7-2.8 (m, 4H), 2.6 (s, 1.6H), 2.5 (s, 1.4H), 1.1-1.8 (m, 14H)

Mass (FAB, m / e): 500 (M ⁺ +1)

[Manufacture example 9]

Synthesis of 3-methyl-4-propyl-benzeneselfonylchloride

The reaction was carried out according to the same method as Preparation Example 3 except for using 2-propyltoluene instead of propylbenzene to obtain 0.7 g (yield: 40%) of the title compound.

[Production Example 10]

Synthesis of 3- (4-cyanophenyl) -N-cyclopentyl-N-methyl-2- (3-methyl-4-propyl-benzenesulfonylamino) -propionamide

Using the compound obtained in Preparation Example 9, the reaction was carried out according to the same method as Preparation Example 4 to obtain 0.4 g (yield: 65%) of the title compound.

¹ H nmR (CDCl ₃ , ppm) δ: 7.1-7.6 (m, 7H), 5.8 (m, 1H), 4.5-4.7 (m, 1H), 4.4 (m, 0.5H), 3.8 (m, 0.5 H), 2.9-3.1 (m, 2H), 2.6-2.7 (m, 4H), 2.3-2.4 (s, 6H), 0.9-1.7 (m, 13H)

Mass (FAB, m / e): 468 (M ⁺ +1)

Example 5

Synthesis of (S) -3- (4-amiderazono-phenyl) -N-cyclopentyl-N-methyl-2- (3-methyl-4-propyl-benzenesulfonylamino) -propionamide

Using the compound obtained in Preparation Example 10, the reaction was carried out according to the same method as in Example 1 to obtain 0.09 g (yield: 43%) of the title compound.

¹ H nmR (CD ₃ OD, ppm) δ: 7.3-7.8 (m, 7H), 4.7 (m, 0.5H), 4.5 (m, 1H), 4.1 (m, 0.5H), 2.9-3.1 (m , 2H), 2.7 (m, 2H), 2.6 (s, 1.5H), 2.5 (s, 1.5H), 2.4 (s, 3H), 1.0-1.8 (m, 13H)

Mass (FAB, m / e): 500 (M ⁺ +1)

[Production Example 11]

Synthesis of 4-isoamyl-benzenesulfonylchloride

The reaction was carried out according to the same method as Preparation Example 3 except for using isoamylbenzene instead of propylbenzene to obtain 1.43 g (yield: 86%) of the title compound.

Example 6

Synthesis of (S) -3- (4-amiderazono-phenyl) -N-cyclopentyl-N-methyl-2- (4-isoamyl-benzenesulfonylamino) -propionamide

The reaction was carried out according to the same method as Preparation Example 4 using the compound obtained in Preparation Example 11, to obtain Intermediate (S) -3- (4-cyanophenyl) -N-cyclopentyl-N-methyl-2- (4 0.60 g (1.25 mmol) of isoamyl-benzenesulfonylamino) -propionamide was obtained and reacted according to the same method as in Example 1 to obtain 0.45 g (yield: 71%) of the title compound.

¹ H nmR (CD ₃ OD, ppm) δ: 7.4-7.8 (m, 8H), 4.7 (m, 0.5H), 4.5-4.6 (m, 1H), 4.2 (m, 0.5H), 3.0-3.2 (m, 2H), 2.8 (m, 2H), 2.7 (s, 1.6H), 2.6 (s, 1.4H), 1.0-1.8 (m, 17H)

Mass (FAB, m / e): 514 (M ⁺ +1)

[Manufacture example 12]

Synthesis of 4-ethyl-benzenesulfonylchloride

The reaction was carried out according to the same method as Preparation Example 3 except for using ethylbenzene instead of propylbenzene to obtain 0.84 g (yield: 81%) of the title compound.

Example 7

Synthesis of (S) -3- (4-amiderazono-phenyl) -N-cyclopentyl-N-methyl-2- (4-ethyl-benzenesulfonylamino) -propionamide

The reaction was carried out according to the same method as Preparation Example 4 using the compound obtained in Preparation Example 12, to obtain Intermediate (S) -3- (4-cyanophenyl) -N-cyclopentyl-N-methyl-2- (4 0.41 g (0.93 mmol) of -ethyl-benzenesulfonylamino) -propionamide was obtained and reacted according to the same method as in Example 1 to obtain 0.18 g (yield: 41%) of the title compound.

¹ H nmR (CD ₃ OD, ppm) δ: 7.4-7.8 (m, 8H), 4.65 (m, 0.5H), 4.45-4.55 (m, 1H), 4.14 (m, 0.5H), 2.95-3.2 (m, 2H), 2.8 (m, 2H), 2.65 (s, 1.6H), 2.55 (s, 1.4H), 0.9-1.8 (m, 17H)

Mass (FAB, m / e): 472 (M ⁺ +1)

[Production Example 13]

Synthesis of 3-methyl-4-ethyl-benzenesulfonylchloride

The reaction was carried out according to the same method as Preparation Example 3, except that ethylbenzene was used instead of propylbenzene to obtain 3.2 g of a mixture of the title compound and 4-methyl-3-ethyl-benzenesulfonyl chloride (yield: 99%). Obtained.

Example 8

Synthesis of (S) -3- (4-amiderazono-phenyl) -N-cyclopentyl-N-methyl-2- (3-methyl-3-ethyl-benzenesulfonylamino) -propionamide

The reaction was carried out according to the same method as in Preparation Example 4 using the compound obtained in Preparation Example 13, to obtain Intermediate (S) -3- (4-cyanophenyl) -N-cyclopentyl-N-methyl-2- (3 -Methyl-4-ethyl-benzenesulfonylamino) -propionamide and 3- (4-cyanophenyl) -N-cyclopentyl-N-methyl-2- (4-methyl-3-ethyl-benzenesulfonylamino 0.5 g (1.10 mmol) of a mixture of) -propionamide was obtained and reacted according to the same method as in Example 1, to obtain 0.35 g (yield: 65%) of the mixture of the title compound and the regioisomer.

¹ H nmR (CD ₃ OD, ppm) δ: 7.3-7.75 (m, 7H), 4.65 (m, 0.5H), 4.45-4.55 (m, 1H), 4.1 (m, 0.5H), 2.9-3.2 (m, 2H), 2.75 (m, 2H), 2.64, 2.55 (d, d, 3H), 2.45 (m, 3H), 1.1-1.8 (m, 11H)

Mass (FAB, m / e): 486 (M ⁺ +1)

Production Example 14

Synthesis of Cyclopentyl-ethylamine Hydrochloride

The reaction was carried out according to the same method as Preparation Example 1 except for using ethylamine hydrochloride instead of methylamine hydrochloride to obtain 3.0 g (yield: 88%) of the title compound.

[Production Example 15]

Synthesis of (S) -3- (4-cyanophenyl) -N-cyclopentyl-2- (butyloxycarbonylamino) -N-ethyl-propionamide

The reaction was carried out according to the same method as Preparation Example 2 using the compound obtained in Preparation Example 14 instead of cyclopentyl-methylamine hydrochloride to obtain 0.92 g (yield: 36%) of the title compound.

¹ H nm R (CDCl ₃ , ppm) δ: 7.3-7.75 (m, 4H), 5.6-5.4 (m, 1H), 4.9 (m, 0.5H), 4.8 (m, 0.5H), 4.59 (m, 0.5H), 4.05 (m, 0.5H), 3.3-2.95 (m, 2H), 2.1-0.9 (m, 20H)

Mass (FAB, m / e): 386 (M ⁺ +1)

[Production Example 16]

Synthesis of 4-isobutyl-benzenesulfonylchloride

The title compound was obtained in the same manner as in Preparation Example 3, except that 4-isobutylbenzene was used instead of propylbenzene (yield: 86%).

Example 9

Synthesis of (S) -3- (4-amiderazono-phenyl) -N-cyclopentyl-N-ethyl-2- (4-isobutyl-benzenesulfonylamino) -propionamide

Using the compound obtained in Preparation Example 15 and the compound obtained in Preparation Example 16, the reaction was carried out according to the same method as Preparation Example 4 to obtain Intermediate (S) -3- (4-cyanophenyl) -N-cyclopentyl- 0.46 g (0.95 mmol) of N-ethyl-2- (4-isobutyl-benzenesulfonylamino) -propionamide was obtained, and the reaction was carried out according to the same method as in Example 1 to 0.35 g (yield: 72%) of the title compound. ) Was obtained.

¹ H nmR (CD ₃ OD, ppm) δ: 7.25-7.82 (m, 8H), 4.65 (m, 0.5H), 4.4 (m, 0.5H), 4.18 (m, 0.5H), 4.05 (m, 0.5H), 2.9-3.35 (m, 4H), 2.61 (m, 2H), 1.25-1.8 (m, 8H), 0.9-1.1 (m, 9H)

Mass (FAB, m / e): 514 (M ⁺ +1)

Example 10

Synthesis of (S) -3- [4- (amino-methylimino-methyl) -phenyl] -N-cyclopentyl-N-methyl-2- (4-propyl-benzenesulfonylamino) -propionamide

The reaction was carried out in the same manner as in Example 1, except that 2.0M methylamine solution dissolved in methanol was used instead of hydrazine hydrate to obtain 68 mg (yield: 28%) of the title compound.

¹ H nmR (CD ₃ OD, ppm) δ: 7.66 (m, 4H), 7.43 (m, 2H), 7.33 (m, 2H), 4.59, 4.43, 4.09 (m, m, m, 2H), 3.10 (s, 3H), 3.05, 2.89 (m, m, 2H), 2.65 (m, 2H), 2.60, 2.46 (s, s, 3H), 1.70-1.10 (m, 10H), 0.93 (t, 3H)

Mass (FAB, m / e): 485 (M ⁺ +1)

Example 11

Synthesis of (S) -3- (4-amiderazono-phenyl) -N-cyclopentyl-2- (4-isobutyl-benzenesulfonylamino) -N-methyl-propionamide

The reaction was carried out according to the same method as in Preparation Example 3, except that isobutylbenzene was used instead of propylbenzene, to synthesize 4-isobutyl-benzenesulfonylchloride, which was prepared in Preparation Example 4 and 4-propyl-benzenesulfur. 58% of intermediate (S) -3- (4-cyanophenyl) -N-cyclopentyl-N-methyl-2- (4-isobutyl-benzenesulfonylamino) -propionamide Obtained in yield. This reaction was carried out in the same manner as in Example 1, to obtain 0.11 g (yield: 96%) of the title compound.

¹ H nmR (CD ₃ OD, ppm) δ: 7.65 (m, 4H), 7.44 (m, 2H), 7.32 (m, 2H), 4.61, 4.45, 4.09 (m, m, m, 2H), 3.04 , 2.89 (m, m, 2H), 2.60, 2.47 (s, s, 3H), 2.55 (m, 2H), 1.90 (m, 1H), 1.74-1.10 (m, 8H), 0.91 (d, 6H)

Mass (FAB, m / e): 500 (M ⁺ +1)

Example 12

Synthesis of (S) -3-[(4-amino-methylimino-methyl) -phenyl] -N-cyclopentyl-2- (4-isobutyl-benzenesulfonylamino) -N-methyl-propionamide

The reaction was carried out according to the same method as Example 11 except for using 2.0M methylamine solution dissolved in methanol instead of hydrazine hydrate to give 87 mg (yield: 76%) of the title compound.

¹ H nmR (CD ₃ OD, ppm) δ: 7.65 (m, 4H), 7.39 (m, 2H), 7.27 (m, 2H), 4.59, 4.41, 4.08 (m, m, m, 2H), 3.08 (s, 3H), 3.03, 2.85 (m, m, 2H), 2.58, 2.42 (s, s, 3H), 2.49 (m, 2H), 1.85 (m, 1H), 1.60-1.10 (m, 8H) , 0.87 (d, 6H)

Mass (FAB, m / e): 499 (M ⁺ +1)

Example 13

Synthesis of (S) -3- (4-amiderazono-phenyl) -N-cyclopentyl-2- (2-isobutyl-benzenesulfonylamino) -N-methyl-propionamide

The reaction was carried out according to the same method as in Preparation Example 3, except that isobutylbenzene was used instead of propylbenzene, to synthesize 2-isobutyl-benzenesulfonylchloride, which was prepared in Preparation Example 4 and 4-propyl-benzenesulfur. 7.1% of intermediate (S) -3- (4-cyanophenyl) -N-cyclopentyl-2- (2-isobutyl-benzenesulfonylamino) -N-methyl-propionamide Obtained in yield. This reaction was carried out in the same manner as in Example 1, obtaining 0.34 g (yield: 79%) of the title compound.

¹ H nmR (CD ₃ OD, ppm) δ: 7.96 (m, 1H), 7.72 (m, 2H), 7.70-7.25 (m, 5H), 4.62, 4.52, 4.46, 4.06 (m, m, m, m, 2H), 3.18-2.73 (m, m, 4H), 2.57 (s, 3H), 2.12 (m, 1H), 1.74-1.10 (m, 8H), 0.98 (m, 6H)

Mass (FAB, m / e): 500 (M ⁺ +1)

Example 14

Synthesis of (S) -3- (4-amiderazono-phenyl) -N-cyclopentyl-2- (4- (2,2-dimethyl-propyl) -benzenesulfonylamino) -N-methyl-propionamide

The reaction was carried out according to the same method as Preparation Example 3 except for using 2,2-dimethyl-propylbenzene instead of propylbenzene to synthesize 4- (2,2-dimethyl-propyl) -benzenesulfonylchloride, In the preparation example 4, this compound was used instead of the 4-propyl-benzenesulfonyl chloride to prepare the intermediate (S) -3- (4-cyanophenyl) -N-cyclopentyl-2- (4- (2,2-dimethyl- Propyl) -benzenesulfonylamino) -N-methyl-propionamide was obtained in 54% yield. This reaction was carried out in the same manner as in Example 1, to obtain 0.13 g (yield: 93%) of the title compound.

¹ H nmR (CD ₃ OD, ppm) δ: 7.65 (m, 4H), 7.43 (m, 2H), 7.29 (m, 2H), 4.61, 4.45, 4.10 (m, m, m, 2H), 3.08 , 2.89 (m, m, 2H), 2.61, 2.50 (s, s, 3H), 2.57 (m, 2H), 1.80-1.10 (m, 8H), 0.89 (s, 9H)

Mass (FAB, m / e): 514 (M ⁺ +1)

Example 15

(S) -3- [4- (amino-methylimino-methyl) -phenyl] -N-cyclopentyl-2- (4-2,2-dimethyl-propyl) -benzenesulfonylamino) -N-methyl Synthesis of Propionamide

The reaction was carried out according to the same method as Example 14 except for using 2.0M methylamine solution dissolved in methanol instead of hydrazine hydrate to obtain 0.23 g (yield: 82%) of the title compound.

¹ H nmR (CD ₃ OD, ppm) δ: 7.68 (m, 4H), 7.45 (m, 2H), 7.30 (m, 2H), 4.61, 4.47, 4.12 (m, m, m, 2H), 3.12 (s, 3H), 3.09, 2.90 (m, m, 2H), 2.64, 2.50 (s, s, 3H), 2.58 (m, 2H), 1.80-1.10 (m, 8H), 0.95 (s, 9H)

Mass (FAB, m / e): 513 (M ⁺ +1)

[Production Example 17]

Synthesis of 4-ethyloxy-benzenesulfonylchloride

The title compound was obtained in the same manner as in Preparation Example 3, except that 4-ethyloxybenzene was used instead of propylbenzene (yield: 65%).

[Production Example 18]

Synthesis of 3- (4-cyanophenyl) -N-cyclopentyl-N-methyl-2- (4-ethyloxy-benzenesulfonylamino) -propionamide

Using the compound obtained in Preparation Example 17, the reaction was carried out according to the same method as Preparation Example 4 to obtain 0.5 g (yield: 72%) of the title compound.

¹ H nmR (CDCl ₃ , ppm) δ: 7.6 (m, 4H), 7.4 (d, 2H), 6.9 (d, 2H), 5.9 (s, 1H), 4.0-4.7 (m, 4H), 3.1 (m, 2H), 2.6 (s, 1.6H), 2.5 (s, 1.4H), 1.1-1.9 (m, 11H)

Mass (FAB, m / e): 455 (M ⁺ +1)

Example 16

Synthesis of (S) -3- (4-amiderazono-phenyl) -N-cyclobentyl-N-methyl-2- (4-ethyloxy-benzenesulfonylamino) -propionamide

Using the compound obtained in Preparation Example 18, the reaction was carried out according to the same method as in Example 1 to obtain 0.19 g (yield: 43%) of the title compound.

¹ H nm R (CD ₃ OD, ppm) δ: 7.6-7.8 (m, 4H), 7.5 (d, 2H), 6.9 (d, 2H), 4.1-4.7 (m, 4H), 2.9-3.1 (m , 2H), 2.65 (s, 1.6H), 2.6 (s, 1.4H), 1.2-1.8 (m, 11H)

Mass (FAB, m / e): 487 (M ⁺ +1)

Reference Example 1

Inhibitory Activity of Thrombin Inhibitors

The enzyme inhibitory effect of the compound thrombin inhibitor according to the present invention was determined by the dissociation constant Ki measured spectrophotometrically using a substrate that reacts with the enzyme to produce color. That is, in the present invention, para-produced using chromozyme TH (tosyl-Gly-Pro-Arg-4-nitroanilide acetate) which is hydrolyzed by thrombin to produce yellow para-nitroanilide. The amount of nitroanilides is measured by the change in absorbance over time. From this rate, the activity of each enzyme can be measured and the inhibitory capacity of the inhibitor can be measured.

Inhibition ability on the thrombin activity of the compound according to the present invention was specifically measured by the method described below.

To the 1.5 mL cuvette was added 1160 μl of 0.1 M Tris buffer solution (pH 7.8) containing 150 mM NaCl and 0.1% PEG8000 (forylethylene glycol, molecular weight about 8000). The substrate solution was prepared by dissolving Chromozyme TH in dimethyl sulfoxide (DMSO) at a concentration of 10 mM and diluting with the buffer solution to a concentration of 0.1 mM. 225 µl of the 0.1 mM substrate solution thus prepared was added to the cuvette. As an inhibitor solution, the inhibitor compound according to the present invention was dissolved in dimethyl sulfoxide to 10 mg / ml, and then diluted with the buffer solution to make a concentration of 0.1 mg / ml, 0.01 mg / ml, and 0.001 mg / ml. The solution was taken between 0 and 10 μg, and the total volume was added to the cuvette with 100 μl of Tris buffer solution.

15 µl of bovine thrombin dissolved in the Tris buffer solution at a concentration of 0.1 mg / ml was added to the cuvette containing the reaction solution at room temperature, and the enzymatic hydrolysis reaction was started. The amount of para-nitroanilide produced by the reaction for 2 minutes from the moment of addition of the enzyme was monitored by the change in absorbance at 381 nm to show a continuous spectrum of reaction time versus absorbance. The above experiments were performed for different inhibitor concentrations to obtain continuous spectra.

After obtaining the initial velocity Vi from the slope within the initial 30 seconds of the reaction time in each spectrum, an inverse graph of the initial velocity versus the inhibitor concentration (1 / Vi) is shown. After calculating the first equation that satisfies the points on the graph, Ki can be calculated from the x-intercept of the equation using the Michaelis-Menten equation. The Km value used in this calculation was 8.3 μM, which was obtained by varying the substrate concentration at a constant enzyme concentration.

Michaelis-Menten equation

In the enzyme scheme, [I] denotes the concentration of the inhibitor, [S] denotes the concentration of the substrate, Vmax denotes the maximum initial velocity, and Km denotes the Michaelis constant, which is 8.3 μM. . Table 1 shows the enzyme activity inhibitory ability of each inhibitor measured for thrombin as Ki value.

The rate constant Ks used the same solution as that used to obtain the Ki value at the same concentration, but the experimental method was different as follows.

That is, 1160 µl of buffer solution was added to a 1.5 ml volume cuvette, 15 µl of 0.1 mg / ml bovine thrombin solution and 100 µl of inhibitor solution were added thereto, and the mixture was left at room temperature for 15 minutes, followed by adding 225 µl of 0.1 mM substrate solution. The change in absorbance over time was monitored for minutes. The inclination of the part showing a straight line in the obtained continuous spectrum is measured and represented by Vs. This experiment was run at several inhibitor concentrations to obtain Vs values at each inhibitor concentration, showing a graph of 1 / Vs versus inhibitor concentration. After obtaining a linear equation satisfying the points on the graph, the Ks value was determined from the x-section using the enzyme reaction equation.

On the other hand, the inhibitory activity of the compound according to the present invention against trypsin was also measured as described for the case of thrombin.

N-benzoyl-Val-Gly-Arg p-nitroanilide hydrochloride (N-benzoyl-Val-Gly-Arg p-nitroanilide hydrochloride) was used as a substrate and the inhibitor was in the range of 0 to 120 µg. Various concentrations were used. In addition, trypsin was dissolved in 0.1N HCl solution was made to 45 ㎍ / ㎖ concentration with the Tris buffer solution immediately before the experiment 40μl was used. Like the experiment for thrombin, the total volume of the reaction solution was set to 1.5 ml and the same method was used. The K _M value used for calculating the Ki value was also determined by the same method, which was 20.2 μM.

According to the method described above, the inhibitory ability of each inhibitor according to the present invention measured for thrombin and trypsin was expressed as Ki value and Ks value, and the selectivity to thrombin was expressed as trypsin / thrombin. The results are as shown in Table 1 below.

TABLE 1

Inhibitor's ability to inhibit thrombin and trypsin

From the above experimental results, the compound of the present invention exhibits excellent thrombin inhibitory activity. In particular, the compounds of Examples 1, 11, and 14 have a selectivity to thrombin relative to trypsin of 8400, 18000, and 10500 times, respectively. It can be seen that the compounds of Trovan) and 11 (NAPAP) are significantly superior to those of 250 and 50 times, respectively.

Reference Example 2

Pharmacokinetic Experiment

Experiment Method:

Male rats were fasted for 24 hours before testing. Physiological saline was used to prepare a 1% (10 mg / ml) solution of the compound of Example 1, followed by oral administration. Blood was collected at predetermined time intervals, and then immediately mixed with methanol and zinc sulfate, and then the high blood liquid chromatography (HPLC) for the supernatant was measured for blood serum concentrations at an ultraviolet wavelength of 231 nm.

Experiment result :

The measured results of blood drug concentration over time after oral administration of the compound of Example 1 are shown in Table 2 below. As can be seen from the results shown in Table 2, unlike the known thrombin inhibitor agatroban, the compound of Example 1 showed pharmacokinetic properties that the drug is absorbed upon oral administration and maintained in the blood for a long time.

TABLE 2

Concentration of blood drug over time when oral administration of the compound of Example 1 at 30 mg / kg concentration in rat

Claims (9)

A compound represented by the following formula (1), a pharmaceutically acceptable salt thereof, and an isomer thereof: [Formula 1]

Wherein R, R ¹ and R ² are each independently hydrogen, it represents an alkyl or alkyloxy of C _1-8 of C _1-8, R ³ and R ⁴ are independently alkyl or C _3- C for _1-4 respectively ₈ represents cycloalkyl, and R ⁵ represents hydrogen, C _1-4 alkyl or amino. The compound of claim 1, wherein R ¹ and R ² each independently represent hydrogen, C ₁ -C ₅ alkyl or C ₁ -C ₃ alkyloxy, and R ³ and R ⁴ are each independently C ₁ -C ₃ alkyl or C ₃ -C ₅ cycloalkyl, R ⁵ represents C ₁ -C ₃ alkyl or amino. The compound of claim 2, wherein R ¹ and R ² each independently represent hydrogen, methyl, ethyl, propyl, butyl or an isomer thereof, pentyl or an isomer thereof or ethyloxy, and R ³ and R ⁴ are each independently methyl, ethyl Or cyclopentyl and R ⁵ represents methyl or amino. (S) -3- (4-amide-razono-phenyl) -N-cyclopentyl-N-methyl-2- (4-propyl- benzenesulfonylamino) propionamide according to claim 1, (S)- 3- (4-amiderazono-phenyl) -N-cyclopentyl-N-methyl-2- (4-butyl-benzenesulfonylamino) propionamide, (S) -3- (4-amiderazono-phenyl ) -N-cyclopentyl-N-methyl-2- (4-t-butyl-benzenesulfonylamino) propionamide, (S) -3- (4-amiderazono-phenyl) -N-cyclopentyl-N -Methyl-2- (3,4-diethyl-benzenesulfonylamino) propionamide, (S) -3- (4-amiderazono-phenyl) -N-cyclopentyl-N-methyl-2- (3 -Methyl-4-propyl-benzenesulfonylamino) propionamide, (S) -3- (4-amiderazono-phenyl) -N-cyclopentyl-N-methyl-2- (4-isoamyl-benzenesulfur Ponylamino) propionamide, (S) -3- (4-amiderazono-phenyl) -N-cyclopentyl-N-methyl-2- (4-ethyl-benzenesulfonylamino) propionamide, (S)- 3- (4-amiderazono-phenyl) -N-cyclopentyl-N-methyl-2- (3- Methyl-4-ethyl-benzenesulfonylamino) propionamide, (S) -3- (4-amiderazono-phenyl) -N-cyclopentyl-N-methyl-2- (4-isobutyl-benzenesulfonyl Amino) propionamide, (S) -3- [4- (amino-methylimino-methyl) -phenyl] -N-cyclopentyl-N-methyl-2- (4-propyl-benzenesulfonylamino) propionamide , (S) -3- (4-amiderazono-phenyl) -N-cyclopentyl-2- (4-isobutyl-benzenesulfonylamino) -N-methyl-propionamide, (S) -3- [ 4-amino-methylimino-methyl) -phenyl] -N-cyclopentyl-2- (4-isobutyl-benzenesulfonylamino) -N-methyl-propionamide, (S) -3- (4-amide Lazono-phenyl) -N-cyclopentyl-2- (2-isobutyl-benzenesulfonylamino) -N-methyl-propionamide, (S) -3- (4-amiderazono-phenyl) -N- Cyclopentyl-2- (4- (2,2-dimethyl-propyl) -benzenesulfonylamino) -N-methyl-propionamide, (S) -3- [4- (amino-methylimino-methyl)- Phenyl] -N-cyclopentyl-2- (4- (2,2-dimethyl-pro ) -Benzenesulfonylamino) -N-methyl-propionamide and (S) -3- (4-amidelazono-phenyl) -N-cyclopentyl-N-methyl-2- (4-ethyloxy-benzenesulfur A compound selected from the group consisting of fonylamino) -propionamide. A method of preparing a compound of Formula 1 and salts thereof, characterized by reacting a methylmercapto compound of Formula 2 with an amine derivative of Formula 3: [Formula 1]

[Formula 2]

[Formula 3] H ₂ NR ⁵ Wherein R, R ¹ and R ² are each independently hydrogen, it represents an alkyl or alkyloxy of C _1-8 of C _1-8, R ³ and R ⁴ are independently alkyl or C _3- C for _1-4 respectively ₈ represents cycloalkyl, and R ⁵ represents hydrogen, C _1-4 alkyl or amino. A process according to claim 5, wherein the reaction is carried out in the presence of a solvent. The method of claim 6 wherein the solvent is an alcohol. A thrombin inhibitor composition containing a compound of formula 1 according to claim 1 as an active ingredient together with a pharmaceutically acceptable carrier. The composition of claim 8 formulated as an oral dosage form.