KR19980075902A - Selective thrombin inhibitors for oral administration - Google Patents

Abstract

The present invention relates to novel compounds useful as thrombin inhibitors. More specifically, the present invention exhibits potent thrombogenic inhibitory effect by selectively inhibiting thrombin, and is also effective by oral administration. Novel sulfonyl derivatives of the following formula (1), preparation methods thereof, and blood coagulations containing the compound as an active ingredient A pharmaceutical composition for the prophylaxis or treatment of various thrombosis:

[Formula 1]

In the above formula,

X, Y and Z each independently represent CH ₂ , CH, NH, N, O or S, or

X represents carbonyl and Y and Z may be bonded in a fused form with substituted phenyl,

R ₁ and R ₂ each independently represent lower alkyl or cycloalkyl,

R ₃ represents hydrogen, lower alkyl or amino.

Description

Selective thrombin inhibitors for oral administration

The present invention relates to novel compounds useful as thrombin inhibitors. More specifically, the present invention provides a novel sulfonyl derivative represented by the following Chemical Formula 1, which shows effective selective thrombin inhibitory effect even by oral administration, a preparation method thereof, and a method for preventing blood coagulation or treating various thrombosis containing the compound as an active ingredient. Regarding the pharmaceutical composition:

[Formula 1]

In the above formula,

X, Y and Z each independently represent CH ₂ , CH, NH, N, O or S, or

X represents carbonyl and Y and Z may be bonded in a fused form of substituted phenyl,

R ₁ and R ₂ each independently represent lower alkyl or cycloalkyl,

R ₃ 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 fibrinogens 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. do. Thrombin also plays a role in activating the 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 (typically 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 developed as effective thrombin inhibitors include the first arylsulfonyl arginine-based compounds, Argatroban of formula (4) (US Pat. No. 4,258,192 and US 4201863).

[Formula 4]

This compound has been reported to have a 250-fold thrombin inhibitory effect compared to trypsin and has already been commercialized in Japan in 1990 (Biochemistry 1984, 23, 85-90). 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 (5), a benzamidine-based arylsulfonyl compound, has been developed as a thrombin inhibitor. See J. Biol. Chem. 1991, 266, 20085-20093.

[Formula 5]

On the other hand, Ro46-6240 compound of formula (6), which has improved selectivity to thrombin relative to trypsin, has been reported as a powerful thrombin inhibitor, which shows the potential for development as an intravenous infusion, but has a short blood half-life. There is no possibility (see J. Med. Chem. 1994, 37, 3889-3901).

[Formula 6]

Recently, some piperazide compounds have been reported to be orally administrable in rats, but these compounds also have low selectivity for thrombin relative to trypsin (WO 94/18185).

The present inventors have been intensively researching for a long time to develop compounds that are relatively easy to synthesize, have significantly improved selectivity for thrombin relative to trypsin, and are also orally administered. As a result, it was confirmed that the compound of formula 1 defined above 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 and treating various thrombosis containing the compound as an active ingredient.

The invention is explained in more detail below.

The present invention relates to a compound represented by the following formula (1), a pharmaceutically acceptable salt and isomer thereof.

[Formula 1]

In the above formula,

X, Y and Z each independently represent CH ₂ , CH, NH, N, O or S, or

X represents carbonyl and Y and Z may be bonded in a fused form of substituted phenyl,

R ₁ and R ₂ each independently represent lower alkyl or cycloalkyl,

R ₃ represents hydrogen, lower alkyl or amino.

The term loweralkyl in the above definitions for substituents of compounds of formula 1 according to the invention means straight-chain or branched hydrocarbon radicals having 1 to 4 carbon atoms, including methyl, ethyl, propyl, isopropyl, isobutyl, t-butyl The term cycloalkyl means cyclic alkyl having 3 to 8 carbon atoms including cyclopentyl.

Preferred compounds of formula (I) of the present invention are X, Y and Z each independently represent CH ₂ , CH, N, O or S, X represents carbonyl and Y and X substituted phenyl in the form of fused And R ₁ and R ₂ each independently represent methyl or cyclopentyl, and R ₃ is a compound representing hydrogen or amino.

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

One.

2. (S) -3- [4- (Amino-imino-methyl) -phenyl] -2- (benzothiazole-5-sulfamoylamino) -N-cyclopentyl-N-methyl-propionamide

3.

4.

5. Methyl Ester

6. Methyl Ester

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 present invention 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 below.

[Formula 2]

[Formula 3]

Wherein X, Y, Z, R ₁ , R ₂ and R ₃ are as defined above.

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

Scheme 1

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 which does not adversely affect the reaction, but generally alcohol solvents such as methanol, ethanol, propanol and the like are preferably used.

In the present reaction, reaction conditions including reaction amount, reaction temperature, reaction time, and the like can be easily determined by those skilled in the art according to a 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 post-treatment 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 is a methylmercapto compound, for example, by coupling the compound of Formula 7 with an amine substituted with R ₁ and R ₂ The compound of formula 11 was obtained by introducing a sulfonyl group at the N-terminus by obtaining a compound of formula 8 and removing the amino protecting group from the compound of formula 8 and reacting the compound of formula 9 with the sulfonyl derivative of formula 10. After obtaining, the compound of formula 11 can be prepared by saturation with hydrogen sulfide in the presence of a base to give a thioamide compound of formula 12, and methylation of the resulting compound of formula 12.

[Formula 7]

[Formula 8]

[Formula 9]

[Formula 10]

[Formula 11]

[Formula 12]

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

The method for preparing the compound of Formula 2 as described above may be represented by the following Scheme 2.

Scheme 2

As shown in Scheme 2, in order to prepare the compound of Formula 2, R ₁ and R ₂ substituted amine groups are first coupled to the C-terminus of the compound of Formula 7 to obtain a compound of Formula 8.

Known coupling reagents that can be used for the coupling reaction of compounds of formula 7 include dicyclohexylcarbodiimide (DCC), 3-ethyl-3 '-(dimethylamino) -propylcarbodiimide (EDC), bis- (2-oxo-3-oxazolidinyl) -phosphinic acid chloride (BOP-Cl), diphenylphosphoryl azide (DPPA), and the like, but are not limited thereto.

In addition, the carboxylic acid compound of the formula (7) 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 necessary to form amide bonds by coupling reactions with amines or to form ester bonds by coupling reactions 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 (8) produced by the coupling reaction of a compound of formula (7) with an amine group is then removed by an conventional method to remove the N-terminal amino protecting group to obtain a compound of formula (9). The compound of formula 9 is reacted with a sulfonylchloride compound of formula 10 to introduce a sulfonyl group to the N-terminal portion of the compound to obtain a compound of formula 11, and then the nitrile compound of formula 11 is combined with pyridine and trimethylamine. Saturation with hydrogen sulfide in the presence of the same base yields a thioamide compound of formula 12, which is subsequently methylated to afford the desired compound of formula 2. To methylate the compound of formula 12, methylating agents such as methane iodide, dimethylsulfate ((CH ₃ ) ₂ SO ₂ ) or methyltriplate (CH ₃ OTf) can be used.

As mentioned above, the compound of formula 1 according to the present invention selectively acts on thrombin and shows potent thrombogenic inhibitory effect. 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 blood clotting 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 may be used. Specific compounds, body weight of each patient, sex, health status, diet, time of administration, method of administration, rate of excretion, drug mixture 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.

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. Thrombolytic agents that can be administered in combination with a compound of the present invention in this manner may include t-PA, urokinase, streptokinase, and the like, and platelet activity inhibitors include aspirin, ticlopidin, Clopidrogel, 7E3 monoclonal antibody, 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.

Preparation Example 1

synthesis

6.0 ml (81.2 mmol) of chlorosulfonic acid were added to the reaction vessel, followed by cooling to 0 deg. 500 µl (4.6 mmol) of benzothiazole was slowly added dropwise thereto. After dropwise addition, the temperature was raised to room temperature, followed by stirring for 1 hour, followed by stirring for 1 hour while heating to 145 ° C. The reaction was stopped by cooling back to room temperature and adding ice. Water and 40 ml of ethyl acetate were added to the reaction solution, followed by extraction three times. The organic layers were combined, dried over anhydrous magnesium sulfate, filtered and concentrated to obtain 1.63 g of benzothiazolesulfonyl chloride.

0.56 g (1.0 mmol) of (S) -3- (4-cyano-phenyl) -2- (butyloxycarbonyl-amino) -N-methylpropionamide was dissolved in 15 ml of dimethylformamide (DMF). And 0.28 ml of N-methylmorpholine were added. Cooled to 0 ° C and the benzothiazolesulfonylchloride obtained in the above reaction was dissolved dropwise in 10 ml of dimethylformamide and slowly added dropwise. After all were added dropwise, the temperature was raised to room temperature and stirred for 1 hour. After the reaction was completed, the product was distilled under reduced pressure to remove volatiles. The residue was diluted with ethyl acetate and washed sequentially with saturated aqueous sodium hydrogen carbonate solution, diluted hydrochloric acid and saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated. The residue was subjected to column chromatography [eluent: ethyl acetate / hexane (1/1, volume ratio)] to yield 0.62 g (2. total yield of 28.7%) of the title compound. The obtained compound was in the form of a mixture of two structural isomers in which sulfonyl groups are bonded to 2- and 4- positions of the phenyl ring, respectively, and the concentration ratio of the two structural isomers was 4: 3 when analyzed by liquid chromatography.

¹ H NMR (CDCl ₃ , ppm) δ: 9.2, 9.1 (m, m, 1H), 8.5, 8.4, 8.2, 7.9 (m, m, m, m, 2H), 7.5, 7.3 (m, m, 4H ), 6.6, 5.9 (m, m, 1H), 4.8, 4.7, 4.6, 4.5, 4.3, 3.8 (m, m, m, m, m, m, 2H), 2.6-2.30 (m, 4H), 2.1 (m, 2H), 1.8-0.8 (m, 8H)

Mass (FAB, m / e): 469 (M ⁺ +1)

Example 1

synthesis

0.21 g (0.43 mmol) of the compound obtained in Preparation Example 1 was dissolved in 15 ml of pyridine and added to a flask equipped with a branch. 1 ml of triethylamine was added thereto. Hydrogen sulfide (H ₂ S) gas was slowly introduced into the solution from one branch of the flask, and the gas was flowed from the other branch. Hydrogen sulfide gas was saturated in the solution with stirring for about 20 minutes. The color of the solution changed from colorless to green and gradually dark brown. The flask was sealed with a rubber stopper and left at room temperature for 3 days. After the reaction was completed, the mixture was distilled under reduced pressure to remove volatiles and dried with a vacuum pump. 10 ml of acetonitrile and 0.264 ml of methane iodide (CH ₃ I) were added together to the obtained yellow solid, and the mixture was heated to reflux for 1 hour. It was distilled under reduced pressure again to remove volatiles and dried with a vacuum pump. The residue was dissolved in 10 ml of acetonitrile and stirred. To this was added 0.03 ml (0.6 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 preparative liquid chromatography to obtain the title compound as two isomers each having a sulfonyl group bonded to the 2- and 4- positions of the phenyl ring. Yield 37.1%) was obtained.

¹ H NMR (CD ₃ OD, ppm) δ: 9.6 (s, 1H), 8.6 (q, 1H), 8.2 (q, 1H), 8.0 (q, 1H), 7.6 (q, 2H), 7.4 (q , 2H), 4.8, 4.6, 4.4, 4.3 (m, m, m, m, 2H), 3.2, 3.1 (m, m, 2H), 2.7, 2.5 (s, s, 3H), 1.8-1.20 (m , 8H)

Mass (FAB, m / e): 501 (M ⁺ +1)

¹ H NMR (CD ₃ OD, ppm) δ: 9.7 (s, 1H), 8.5 (q, 1H), 8.1 (q, 1H), 7.9 (q, 1H), 7.5 (q, 2H), 7.2 (q , 2H), 4.9, 4.6, 4.3, 4.1 (m, m, m, m, 2H), 3.4, 3.0 (m, m, 2H), 2.6, 2.3 (s, s, 3H), 1.9-1.3 (m , 8H)

Mass (FAB, m / e): 501 (M ⁺ +1)

Example 2

synthesis

The reaction was carried out in the same manner as in Example 1 except that the title compound was heated under reflux in a methanol solvent using ammonium acetate instead of hydrazine hydrate to give the title compound two isomers in which the sulfonyl group is bonded to the 2- and 4- positions of the phenyl ring, respectively. As a result, 0.07 g (yield 33.4%) was obtained.

¹ H NMR (CD ₃ OD, ppm) δ: 9.5 (s, 1H), 8.6 (q, 1H), 8.2 (q, 1H), 7.9 (q, 1H), 7.7 (q, 2H), 7.5 (q , 2H), 4.8, 4.6, 4.4, 4.3 (m, m, m, m, 2H), 3.2, 3.0 (m, m, 2H), 2.7, 2.5 (s, s, 3H), 1.8-1.20 (m , 7H), 1.1, 0.9 (m, m, 1H)

Mass (FAB, m / e): 486 (M ⁺ +1)

¹ H NMR (CD ₃ OD, ppm) δ: 9.6 (s, 1H), 8.8 (q, 1H), 8.1 (q, 1H), 7.7 (q, 1H), 7.5 (q, 2H), 7.2 (q , 2H), 4.7, 4.6, 4.3, 4.2 (m, m, m, m, 2H), 3.3, 3.1 (m, m, 2H), 2.8, 2.6 (s, s, 3H), 1.7-1.0 (m , 7H), 1.0, 0.8 (m, m, 1H)

Mass (FAB, m / e): 486 (M ⁺ +1)

Preparation Example 2

synthesis

10.0 mL (50.1 mmol) of chlorosulfonic acid was dissolved in 10 mL of chloroform, and then cooled to 0 ° C. 2.0 g (4.6 mmol) of 3,4-methylenedioxybenzene was dissolved in 2 ml of dimethylformamide (DMF) and slowly added dropwise thereto. After all were added dropwise, the mixture was stirred at 0 ° C. for 10 minutes and ice was added to stop the reaction. Water and 40 ml of ethyl acetate were added to the reaction solution, and extracted three times. The organic layers were combined, dried over anhydrous magnesium sulfate, filtered and concentrated to obtain 1.63 g of benzo [1,3] dioxol-5-sulfonyl chloride.

After dissolving 1.8 g (3.1 mmol) of (S) -3- (4-cyano-phenyl) -2- (butyloxycarbonyl-amino) -N-methylpropionamide in 10 ml of dimethylformamide (DMF) The reaction was carried out in the same manner as in Preparation Example 1, using the benzo [1,3] dioxol-5-sulfonylchloride obtained above to obtain 1.3 g of the title compound (26.7 total yield of 66.7%).

¹ H NMR (CD ₃ OD, ppm) δ: 7.5 (q, 2H), 7.4-7.2 (m, 3H), 7.1 (q, 1H), 6.8 (q, 1H), 6.1 (d, 2H), 5.7 (m, 1H), 4.6, 4.5, 4.3, 3.9 (m, m, m, m, 2H), 3.0 (m, 2H), 2.6, 2.4 (s, s, 3H), 1.8-1.5 (m, 8H ), 1.4 (m, 1H), 1.1 (m, 1H)

Mass (FAB, m / e): 424 (M ⁺ +1)

Example 3

synthesis

The reaction was carried out in the same manner as in Example 1 using 1.3 g (1.8 mmol) of the compound obtained in Preparation Example 2 to obtain 0.61 g (yield 69.8%) of the title compound.

¹ H NMR (DMSO, ppm) δ: 9.6 (s, 1H), 8.6 (q, 1H), 8.2 (q, 1H), 8.0 (q, 1H), 7.6 (q, 2H), 7.4 (q, 2H ), 4.8, 4.6, 4.4, 4.3 (m, m, m, m, 2H), 3.2, 3.1 (m, m, 2H), 2.7, 2.5 (s, s, 3H), 1.8-1.20 (m, 8H )

Mass (FAB, m / e): 488 (M ⁺ +1)

Example 4

synthesis

The reaction was carried out in the same manner as in Example 3 except that 0.43 g (yield 50.5%) of the title compound were obtained by heating under reflux in a methanol solvent using ammonium acetate instead of hydrazine hydrate.

¹ H NMR (DMSO, ppm) δ: 9.6 (s, 1H), 8.6 (q, 1H), 8.2 (q, 1H), 8.0 (q, 1H), 7.6 (q, 2H), 7.4 (q, 2H ), 4.8, 4.6, 4.4, 4.3 (m, m, m, m, 2H), 3.2, 3.1 (m, m, 2H), 2.7, 2.5 (s, s, 3H), 1.8-1.20 (m, 8H )

Mass (FAB, m / e): 473 (M ⁺ +1)

Preparation Example 3

Synthesis of 9-oxo-9H-fluorene-1-carboxylic acid methyl ester

1.0 g (4.46 mmol) of 9-fluorene was dissolved in 25 ml of methanol, and then a catalytic amount of sulfuric acid was added. The reaction mixture was stirred while heating to reflux for 1 hour, and extracted three times with 40 ml of ethyl acetate solution and sodium bicarbonate aqueous solution. The organic layers were combined, dried over anhydrous magnesium sulfate, filtered and concentrated to give the desired title compound quantitatively. Obtained in yield.

¹ H NMR (CDCl ₃ , ppm) δ: 7.9, 7.7, 7.6-7.1 (m, m, m, 7H), 3.5 (s, 3H)

Mass (FAB, m / e): 227 (M ⁺ +1)

Preparation Example 4

Synthesis of Methyl Ester

Using the compound obtained in Preparation Example 3, the reaction was carried out according to the same method as Preparation Example 1 to obtain 0.36 g (yield 12.5%) of the title compound.

¹ H NMR (CDCl ₃ , ppm) δ: 8.1-7.8 (m, 2H), 7.8-7.4 (m, 6H), 7.3 (m, 2H), 6.3 (m, 1H), 4.8-4.6, 4.4 (m , m, m, 2H), 4.0, 3.9 (s, m, 5H), 3.1-2.9 (m, 2H), 2.6, 2.5 (s, s, 3H), 1.8-1.2, 1.1, 0.9 (m, m , m, 8H)

Mass (FAB, m / e): 560 (M ⁺ +1)

Example 5

Synthesis of Methyl Ester

The reaction was carried out in the same manner as in Example 1 using the compound obtained in Preparation Example 4, to obtain 0.06 g (41% yield) of the title compound.

¹ H NMR (CD ₃ OD, ppm) δ: 8.1-7.6 (m, 8H), 7.5-7.1 (m, 3H), 5.2 (m, 1H), 4.7, 4.5 (m, m, 2H), 4.2 ( m, 2H), 4.0 (s, 3H), 3.1-2.9 (m, 3H), 2.7, 2.5 (s, s, 3H), 2.4 (m, 1H), 2.1-0.9 (m, 8H)

Mass (FAB, m / e): 592 (M ⁺ +1)

Example 6

Synthesis of Methyl Ester

The reaction was carried out in the same manner as in Example 5, but by heating under reflux in a methanol solvent using ammonium acetate instead of hydrazine hydrate to obtain 0.05 g (yield 34.6%) of the title compound.

¹ H NMR (CD ₃ OD, ppm) δ: 8.2 (br, 1H), 8.6-7.9, 7.9-7.7 (m, m, 8H), 7.5-7.3 (m, 2H), 4.7, 4.5, 4.3 (m , m, m, 2H), 4.1 (m, 2H), 4.0 (s, 3H), 3.1-2.7 (m, 4H), 2.5, 2.4 (s, s, 3H), 2.0-0.9 (m, 8H)

Mass (FAB, m / e): 577 (M ⁺ +1)

Experimental Example 1 Inhibitory Activity of a Thrombin Inhibitor

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 (pH 7.8) containing 150 mM NaCl and 0.1% PEG8000 (polyethylene 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 0.1 mg / ml, 0.01 mg / ml, and 0.001 mg / ml. The solution was taken between 10 μg and added to the cuvette in a total volume of 100 μl with Tris buffer.

At room temperature, 15 μl of thrombin dissolved in the Tris buffer solution at a concentration of 0.1 mg / ml was added to the cuvette containing the reaction solution. 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 calculating the initial velocity Vi from the slope within 30 seconds of the initial reaction time in each spectrum, an inverse (1 / Vi) graph of the initial velocity versus the inhibitor concentration 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 5.2 μM, determined 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 means 5.2 μM. . Table 1 shows the enzyme activity inhibitory ability of each inhibitor measured for thrombin as Ki value.

TABLE 1

Inhibitor's ability to inhibit thrombin

Compound (Example Number) Ki value (nM) Compound (Example Number) Ki value (nM) One 9.7 2 78.6 3 1.9 4 29.9 5 108.2 6 2160

As can be seen from the results described in Table 1, the compounds according to the present invention show an excellent inhibitory effect of several nM on thrombin.

Experimental Example 2: Pharmacokinetics

Experiment Method:

Male rats were fasted for 20 hours and then tested. Physiological saline was used to prepare a 1% (10 mg / ml) solution of the compound of Example 3, followed by oral administration. Blood was collected at predetermined time intervals, and immediately mixed with methanol and zinc sulfate, and then blood plasma concentrations were measured at an ultraviolet wavelength of 231 nm using high pressure liquid chromatography (HPLC).

Experiment result:

The measured results of blood drug concentration over time after oral administration of the compound of Example 3 are shown in Table 2 below. As can be seen from the results in Table 2, the compound of Example 3 showed pharmacokinetic properties that the drug is absorbed upon oral administration and maintained in the blood for a long time.

TABLE 2

Blood Drug Concentration with respect to the Time of Oral Administration of the Compound of Example 3 at 30 mg / kg in Mice

Minutes Blood concentration (ng / ml) Average concentration (+ error) (ng / mL) Rat-1 Rat-2 Rat-3 5 775 2446 961 1394 (916) 10 1066 2877 1505 1816 (945) 20 980 1086 625 897 (241) 40 405 1812 500 906 (786) 60 198 744 529 490 (275) 90 157 290 161 203 (75) 120 145 162 168 158 (12) 180 142 79 164 128 (44) 240 137 49 68 85 (46)

Claims (8)

A compound represented by the following formula (1), a pharmaceutically acceptable salt thereof, and an isomer thereof: [Formula 1] In the above formula, X, Y and Z each independently represent CH ₂ , CH, NH, N, O or S, or X represents carbonyl and Y and Z may be bonded in a fused form of substituted phenyl, R ₁ and R ₂ each independently represent lower alkyl or cycloalkyl, R ₃ represents hydrogen, lower alkyl or amino. The compound of claim 1, wherein X, Y and Z each independently represent CH ₂ , CH, N, O or S, or X represents carbonyl and Y and X may be bonded in a fused form. R ₁ and R ₂ each independently represent methyl or cyclopentyl, and R ₃ represents hydrogen or amino. The compound according to claim 2, wherein (S) -2- (benzothiazole-5-sulfonylamino) -N-cyclopentyl-3- [4- (hydrazino-imino-methyl) -phenyl] -N-methyl -Propionamide, (S) -3- [4- (amino-imino-methyl) -phenyl] -2- (benzo [1,3] dioxol-5-sulfonylamino) -N-cyclopentyl-N -Methyl-propionamide, methylester and (S) -7- {2-methylester. 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] In the above formula, X, Y and Z each independently represent CH ₂ , CH, NH, N, O or S, or X represents carbonyl and Y and Z may be bonded in a fused form of substituted phenyl, R ₁ and R ₂ each independently represent lower alkyl or cycloalkyl, R ₃ represents hydrogen, lower alkyl or amino. The process according to claim 4, wherein the reaction is carried out in the presence of a solvent. The method of claim 5 wherein the solvent is an alcohol. A thrombin inhibitor composition comprising a compound of formula 1 according to claim 1 as an active ingredient together with a pharmaceutically acceptable carrier. The composition of claim 7 formulated as an oral dosage form.