KR100373373B1 - Selective thrombin inhibitor - Google Patents

Abstract

PURPOSE: A novel compound as a selective thrombin inhibitor having improved selectivity to thrombin versus trypsin is provided to prevent blood coagulation and treat thrombosis by thrombus formation inhibiting activity. CONSTITUTION: The selective thrombin inhibitor is provided by the novel sulfonyl derivatives having the formula 1: wherein R1 is hydroxyl, an alkyloxy, amino or an alkylamino; each of R2 and R3 is independently a lower alkyl or a cycloalkyl; R4 is hydrogen, a lower alkyl or amino; and n is an integer of 1 to 5. The pharmaceutical composition for preventing blood coagulation and treating thrombosis comprises the sulfonyl derivatives of the formula 1 as an active ingredient with pharmaceutically acceptable carriers.

Description

Selective thrombin inhibitor

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

[Formula 1]

Where

R ₁ represents hydroxyl, alkyloxy, amino or alkylamino,

R ₂ and R ₃ each independently represent lower alkyl or cycloalkyl,

R ₄ represents hydrogen, lower alkyl or amino,

n is an integer from 1 to 5,

(n=1~5) 부위에 추가로 메틸기를 치환제로 가질 수 있다.

The (n = 1-5) site | part can have a methyl group further as a substituent.

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. The thrombin produced in this process activates platelets, converts fibrinogen to fibrin, etc., wherein the 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 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 can be synthesized only through a very complicated synthesis process.

In addition, as a thrombin inhibitor, a benzamidine-based arylsulfonyl compound (NAPAP) of formula (5) was developed, which is not only easy to synthesize but also an effective thrombin inhibitor, which has only about 50 times the thrombin inhibitory effect with trypsin. See J. Biol. Chem. 1991, 266, 20085-20093.

[Formula 5]

The present inventors have been intensively researching for a long time to develop compounds that are relatively easy to synthesize and have significantly improved selectivity for thrombin relative to trypsin. 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.

It is therefore an object of the present invention to provide novel thrombin inhibitors with high selectivity to thrombin and methods for their preparation.

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 described in detail below.

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

[Formula 1]

Where

R ₁ represents hydroxyl, alkyloxy, amino or alkylamino,

R ₂ and R ₃ each independently represent lower alkyl or cycloalkyl,

R ₄ represents hydrogen, lower alkyl or amino,

n is an integer from 1 to 5,

(n= 1 ~ 5) 부위에 추가로 메틸기를 치환제로 가질 수 있다.

In addition to the (n = 1-5) moiety, a methyl group may be included as a substituent.

In the above definitions for the substituents of the compounds of the formula (I) according to the invention the term "alkyl" means a straight or branched chain hydrocarbon radical having 1 to 8 carbon atoms, the term "lower alkyl" means methyl, ethyl, propyl, isopropyl, iso Butyl, a straight or branched chain hydrocarbon radical having 1 to 4 carbon atoms including t-butyl, and the term "cycloalkyl" means cyclic alkyl having 3 to 8 carbon atoms including cyclopentyl.

Preferred compounds of formula (I) of the invention wherein R ₁ represents hydroxy or lower alkyloxy, R ₂ and R ₃ each independently represents methyl or cyclopentyl, R ₄ represents hydrogen, methyl or amino, n is It is a compound which shows the integer of 1-5.

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

1. (S) -6- [4- {1- (Cyclopentyl-methyl-carbamoyl) -2- [4- (hydrazino-imino-methyl) -phenyl] -ethylsulfamoyl} -phenyl]- Hexanophosphate Methyl Ester

2. (S) -6- [4- {1- (Cyclopentyl-methyl-carbamoyl) -2- [4- (hydrazino-imino-methyl) -pentyl] -ethylsulfamoyl} -phenyl]- Hexanophosphate

3. (S) -6- {4- [2- [4- (Amino-imino-methyl) -phenyl] -1- (cyclopentyl-methyl-carbamoyl) -ethylsulfamoyl] -phenyl} -hexa Old mountains

4. (S) -6- [4- {1- (Cyclopentyl-methyl-carbamoyl) -2- [4- (imino-methylamino-methyl) -phenyl] ethylsulfamoyl} -phenyl] -hexa Aged acid methyl ester

5. (S) -6- [4- {1- (Cyclopentyl-methyl-carbamoyl) -2- [4- (imino-methylimino-methyl) -phenyl] -ethylsulfamoyl} -phenyl] Hexanophosphate

6. (S) -3- [4- {1- (Cyclopentyl-methyl-carbamoyl) -2- [4- (hydrazino-imino-methyl) -phenyl] -ethylsulfamoyl} -phenyl]- 2-methyl-propionic acid methyl ester

7. (S) -3- [4- {1- (Cyclopentyl-methyl-carbamoyl) -2- [4- (hydrazino-imino-methyl) -phenyl] -ethylsulfamoyl} -phenyl]- 2-Methyl-propionic acid

8. (S)-[4- {1- (Cyclopentyl-methyl-carbamoyl) -2- [4- (hydrazino-imino-methyl) -phenyl] -ethylsulfamoyl} -phenyl] -methyl acetate ester

9. (S)-[4- {1- (Cyclopentyl-methyl-carbamoyl) -2- [4- (hydrazino-imino-buckwheat) -phenyl] -ethylsulfamoyl} -phenyl] -acetic acid

10. (S)-{4-[(2- [4- (Amino-imino-methyl) -phenyl] -1- (cyclopentyl-methyl-carbamoyl) -ethylsulfamoyl] -phenyl} -methyl acetate ester

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 isomeric forms are 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 derivative of Formula 2 with an amine derivative of Formula 3 below.

[Formula 2]

[Formula 3]

H ₂ NR ₄

Wherein R ₁ , R ₂ , R ₃ , R ₄ and n 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 workup methods such as chromatography, recrystallization and the like.

The methylmercapto compound of Formula 2 used as an intermediate in the preparation of the compound of Formula 1 in Scheme 1, for example, by coupling the compound of Formula 6 with an amine substituted with R ₂ and R ₃ to obtain a compound of Formula 7 And a compound of formula (8) obtained by removing an amino protecting group from the compound of formula (7) with a sulfonyl derivative of formula (9) to introduce a sulfonyl group at the N-terminus to obtain a compound of formula (10). Compound 10 may be prepared by saturation with hydrogen sulfide in the presence of a base to give the thioamide compound of Formula 11 and methylation of the resulting compound of Formula 11.

[Formula 6]

[Formula 7]

[Formula 8]

[Formula 9]

[Formula 10]

[Formula 11]

Wherein R ₁ , R ₂ , R ₃ and n are as defined above and P is benzyloxycarbonyl. Conventional amino protecting groups such as allyloxycarbonyl, t-butoxycarbonyl or trityl group and the like.

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 ₂ and R ₃ substituted amine groups are first coupled to the C-terminus of the compound of Formula 6 to obtain a compound of Formula 7.

Known coupling reagents that can be used for the coupling reaction of compounds of formula 6 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 represented by the formula (6) 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 that 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 (7) produced by the coupling reaction of a compound of formula (6) 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 (8). The compound of formula 8 was reacted with a sulfonyl chloride compound of formula 9 to introduce a sulfonyl group to the N-terminal portion of the compound to obtain a compound of formula 10, and then the nitrile compound of formula 10 with pyridine and trimethylamine Saturation with hydrogen sulfide in the presence of the same base yields a thioamide compound of formula 11, 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 (11).

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 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 of 6-phenyl-hexanophosphate methyl ester

1.0 g (5.20 mmol) of 6-phenylhexanoic acid was dissolved in 250 ml of methanol, and 15 µl of sulfuric acid was added thereto, followed by stirring under heating to reflux for 1 hour. An aqueous solution of sodium hydrogen carbonate 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 give the title compound in a quantitative yield.

Preparation Example 2

Synthesis of (S) -6- {4- [2- (4-cyano-phenyl) -1- (cyclopentyl-methyl-carbamoyl) -ethylsulfamoyl] -phenyl} -hexanophosphate methyl ester

1.7 mL (24.6 mmol) of chlorosulfonic acid was added to the reaction vessel, followed by cooling to 0 ° C. To this was slowly added dropwise 1.07 g (5.20 mmol) of 6-phenylhexanophosphate methyl ester obtained in Production Example 1. At this time, it was added dropwise while being careful not to rise the temperature 5 ℃ or more. After dropwise addition, the temperature was raised to room temperature and stirred for 1 hour. The reaction solution was cooled to 0 ° C. again and ice was added to stop the reaction. Water and 40 ml of ethyl acetate were added to the reaction solution, followed by extraction three times. The combined organic layers were dried over anhydrous magnesium sulfate, filtered and concentrated to yield 1.63 g of 6- (4-chlorosulfonylphenyl) -hexanophosphate methyl ester. It was.

3.01 g (5.35 mmol) of (S) -3- (4-cyano-phenyl) -2- (t-butyloxycarbonyl-amino) -N-methylpropionamide was dissolved in 10 ml of dimethylformamide (DMF). After the addition, 1.4 ml of N-methylmorpholine was added. The reaction solution was cooled to 0 ° C, and 6- (4-chlorosulfonylphenyl) -hexanophosphate methyl ester obtained above was dissolved in 10 ml of dimethylformaldehyde and slowly added dropwise thereto. After all were added dropwise, the temperature was raised to room temperature and stirred for 1 hour. After the reaction was completed, the reaction solution 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, then dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by column chromatography [eluent: ethyl acetate / hexane (1/1, volume ratio)] to obtain 0.33 g of the title compound (2. total yield 11.7%).

Example 1

(S) -6- [4- {1- (cyclopentyl-methyl-carbamoyl) -2- [4- (hydrazino-imino-methyl) -phenyl] -ethylsulfamoyl} -phenyl] -hexanoin Synthesis of Acid Methyl Ester

0.33 g (0.6122 mmol) of the compound obtained in Preparation Example 2 was dissolved in 10 ml of pyridine and added to a flask equipped with a branch. 0.5 ml of triethylamine was added thereto. Hydrogen sulfide (H ₂ S) gas was allowed to flow slowly into the solution through one branch of the flask and the gas flowed out 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. 15 ml of acetonitrile and 0.23 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 0.06 ml (1.07 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: chloroform / methanol (6/1, volume ratio)] to give 0.19 g (54.2%) of the title compound.

Example 2

(S) -6- [4- {1- (cyclopentyl-methyl-carbamoyl) -2- [4- (hydrazino-imino-methyl) -phenyl] -ethylsulfamoyl} -phenyl] -hexanoin Acid synthesis

0.25 g (0.437 mmol) of the compound obtained in Example 1 was dissolved in 10 mL of a water / methanol (3/1) solution, followed by addition of 0.055 mg (1.3116 mmol) of lithium hydroxide hydrate (LiOH.H 2 O). The reaction solution was stirred at room temperature for 4 hours and then distilled under reduced pressure to remove the solvent. The residue was purified by preparative high pressure liquid chromatography to give 0.11 g (45% yield) of the title compound.

Example 3

(S) -6- {4- [2- [4- (amino-imino-methyl) -phenyl] -1- (cyclopentyl-methyl-carbamoyl) -ethylsulfamoyl] -phenyl} -hexanophosphoric acid Synthesis of

The reaction was carried out in the same manner as in Example 1, except that 0.6 g of a methyl ester compound was obtained by heating under reflux in a methanol solvent using ammonium acetate instead of hydrazine hydrate, and 0.6 g (1.0778 mmol) of the obtained compound was carried out. In the same manner as in Example 2, 0.33 g (yield 56.3%) of the title compound was obtained.

Example 4

(S) -6- [4- {1- (cyclopentyl-methyl-carbamoyl) -2- [4- (imino-methylamino-methyl) -phenyl] -ethylsulfamoyl} -phenyl] -hexanoin Synthesis of Acid Methyl Ester

The reaction was carried out in the same manner as in Example 1, except that 0.39 g (yield 30.7%) of the title compound was obtained using a 2.0 M methylamine solution dissolved in methanol instead of hydrazine hydrate.

Example 5

(S) -6- [4- {1- (cyclopentyl-methyl-carbamoyl) -2- [4- (imino-methylamino-methyl) -phenyl] -ethylsulfamoyl} -phenyl] -hexanenoyne Acid synthesis

The reaction was carried out in the same manner as in Example 2 using the compound obtained in Example 4, to obtain 0.12 g (yield 31.52%) of the title compound.

Preparation Example 3

Synthesis of 2-methyl-3-phenyl-propionic acid methyl ester

1.0 g (6.090 mmol) of α-methylhydrocinnamic acid was dissolved in 25 ml of methanol, and 15 µl of sulfuric acid was added thereto, followed by stirring under heating to reflux for 1 hour. An aqueous sodium hydrogen carbonate solution 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 the title compound in quantitative yield.

Preparation Example 4

Synthesis of 3- {4- [2- (4-cyano-phenyl) -1- (cyclopentyl-methyl-carbamoyl) -ethylsulfamoyl] -phenyl} -2-methyl-propionic acid methyl ester

Using the compound obtained in Preparation Example 3, the reaction was carried out in the same manner as in Preparation Example 2, to obtain 1.3 g (21.1 total yield of 41.1%) of the title compound.

Example 6

(S) -3- [4- {1- (cyclopentyl-methyl-carbamoyl) -2- [4- (hydrazino-imino-methyl) -phenyl] -ethylsulfamoyl} -phenyl] -2- Synthesis of Methyl-propionic Acid Methyl Ester

The reaction was carried out in the same manner as in Example 1 using the compound obtained in Preparation Example 2 to obtain 0.21 g (yield 44.7%) of the title compound.

Example 7

(S) -3- [4- {1- (cyclopentyl-methyl-carbamoyl) -2- [4- (hydrazino-imino-methyl) -phenyl] -ethylsulfamoyl} -phenyl] -2- Synthesis of Methyl-propionic Acid

The reaction was carried out in the same manner as in Example 2 using the compound obtained in Example 6 to obtain 0.045 g (25% yield) of the title compound.

Preparation Example 5

Synthesis of {4- [2- (4-cyano-phenyl) -1- (cyclopentyl-methyl-carbamoyl) -ethylsulfamoyl] -phenyl} -acetic acid methyl ester

The reaction was carried out in the same manner as in Preparation Example 2 using phenylacetic acid methyl ester to obtain 0.25 g of the title compound (2% total yield 11%).

Example 8

Synthesis of (S)-[4- {1-cyclopentyl-methyl-carbamoyl) -2- [4- (hydrazino-imino-methyl) -phenyl] -ethylsulfamoyl} -phenyl] -acetic acid methylester

The reaction was carried out in the same manner as in Example 1 using 0.32 g of the compound obtained in Preparation Example 5, to obtain 0.15 g (yield 43.9%) of the title compound.

Example 9

Synthesis of (S)-[4- {1- (cyclopentyl-methyl-carbamoyl) -2- [4- (hydrazino-imino-methyl) -phenyl] -ethylsulfamoyl-phenyl] -acetic acid

The reaction was carried out in the same manner as in Example 2 using the compound obtained in Example 8, to obtain 0.33 g (yield 23%) of the title compound.

Example 10

Synthesis of (S)-{4- (2- [4- (amino-imino-methyl) -phenyl] -1- (cyclopentyl-methyl-carbamoyl) -ethylsulfamoyl] -phenyl} -acetic acid methyl ester

The reaction was carried out in the same manner as in Example 1 using the compound obtained in Preparation Example 5, but by heating and refluxing in methanol solvent using ammonium acetate instead of hydrazine hydrate to obtain 0.12 g (yield 31%) of the title compound. .

Experimental Example 1 Inhibitory Activity of a Thrombeam 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. In other words, in the present invention, para-gen is 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, 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 to a concentration of 0.1 mM. 225 µl of the 0.1 mM substrate solution thus prepared was added to the cuvette. As the 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. Taken between 10 μg and 100 μl total volume with Tris buffer and added to the cuvettes.

At room temperature, 15 µl of human 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 two minutes from the moment of addition of the enzyme was monitored by the change in absorbance at 381 nm to show the 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 following enzyme reaction (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, [1] represents the concentration of the inhibitor, [S] represents the concentration of the substrate, Vmax means the maximum initial velocity, and Km represents the Michaelis constant, which means 5.2 μM. . Table 1 shows the enzyme activity inhibitory capacity of each inhibitor measured for thrombin as Ki value.

TABLE 1

Inhibitors' ability to inhibit torombin

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 against thrombin.

Claims (7)

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

Where R ₁ represents hydroxyl, straight or branched chain alkyloxy of 1 to 8 carbon atoms, amino or straight or branched chain alkylamino of 1 to 8 carbon atoms, R ₂ and R ₃ each independently represent linear or branched alkyl having 1 to 4 carbon atoms or cycloalkyl having 3 to 8 carbon atoms, R ₄ represents hydrogen, straight or branched chain alkyl or amino of 1 to 4 carbon atoms, n is an integer from 1 to 5,

The (n = 1-5) site | part can have a methyl group further as a substituent. The compound of claim 1, wherein R ₁ represents hydroxy or straight or branched chain alkyloxy having 1 to 4 carbon atoms, R ₂ and R ₃ each independently represent methyl or cyclopentyl, and R ₄ is hydrogen, methyl or amino And n is an integer of 1-5. The compound of claim 2, wherein (S) -6- [4- {1- (cyclopentyl-methyl-carbamoyl) -2- [4- (hydrazino-imino-methyl) -phenyl] -ethylsulfamoyl} -Phenyl-hexanophosphoric acid methyl ester, (S) -6- [4- {1- (cyclopentyl-methyl-carbamoyl) -2- [4- (hydrazino-imino-methyl) -phenyl] -ethyl Sulfamoyl} -phenyl] -hexanophosphoric acid, (S) -6-14- [2- [4- (amino-imino-methyl) -phenyl] -1- (cyclopentyl-methyl-carbamoyl) -ethyl Sulfamoyl] -phenyl} -hexanophosphoric acid, (S) -6- [4-11- (cyclopentyl-methyl-carbamoyl) -2- [4- (imino-methylamino-methyl) -phenyl] ethyl Sulfamoyl} -phenyl] -hexanophosphoric acid methyl ester, (S) -6- [4- {1- (cyclopentyl-methyl-carbamoyl) -2- [4- (imino-methylimino-methyl) -Phenyl] -ethylsulfamoyl} -phenyl] -hexanophosphoric acid, (S) -3- [4- {1- (cyclopentyl-methyl-carbamoyl) -2- [4- (hydrazino-imino- Methyl) -phenyl-ethylsulfamoyl} -phenyl] -2-methyl-propionic acid methyl ester, (S) -3- [4- {1- (cyclopentyl-methyl-car Moyl) -2- [4- (hydrazino-imino-methyl) -phenyl] -ethylsulfamoyl} -phenyl] -2-methyl-propionic acid, (S)-[4- {1- (cyclopentyl-methyl -Carbamoyl) -2- [4- (hydrazino-imino-methyl) -phenyl] -ethylsulfamoyl} -phenyl] -acetic acid methyl ester, (S)-[4- {1- (cyclopentyl-methyl -Carbamoyl) -2- [4- (hydrazino-imino-methyl) -phenyl] -ethylsulfamoyl} -phenyl] -acetic acid and (S)-{4-[(2- [4- (amino- Imino-methyl) -phenyl] -1- (cyclopentyl-methyl-carbamoyl) -ethylsulfamoyl] -phenyl} -acetic acid methylester. A method of preparing a compound of formula 1 and salts thereof by reacting a methylmercapto compound of formula 2 with an amine derivative of formula [Formula 1]

[Formula 2]

[Formula 3] H ₂ NR ₄ Where R ₁ represents hydroxyl, straight or branched alkyloxy of 1 to 8 carbon atoms, amino or straight or branched alkylamino of 1 to 8 carbon atoms, R ₂ and R ₃ each independently represent linear or branched alkyl having 1 to 4 carbon atoms or cycloalkyl having 3 to 8 carbon atoms, R ₄ represents hydrogen, straight or branched chain alkyl or amino of 1 to 4 carbon atoms, n is an integer from 1 to 5,

(n = 1 to 5) may further have a methyl group. 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 composition for preventing coagulation or treating thrombosis, which comprises a compound of formula 1 according to claim 1 as an active ingredient together with a pharmaceutically acceptable carrier.