KR19980077253A - Selective thrombin inhibitor - Google Patents

Abstract

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 exhibits potent thrombogenic inhibition by selectively inhibiting thrombin, a method for preparing the same, and a method for preventing blood coagulation or treating various thrombosis containing the compound as an active ingredient. It relates to a pharmaceutical composition for:

[Formula 1]

In the above formula,

R ₁ represents substituted or unsubstituted naphthalene.

Description

Selective thrombin inhibitor

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 high selectivity to thrombin and potent antithrombotic effect, a preparation method thereof, and a method for preventing blood coagulation or treating various thrombosis containing the compound as an active ingredient. It relates to a pharmaceutical composition for:

[Formula 1]

In the above formula,

R ₁ represents substituted or unsubstituted naphthalene.

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 the formula (8) (see US 4258192 and US 4201863).

[Formula 8]

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 can be prepared only through a very complicated synthetic process, and has the disadvantage that there is no activity for oral administration.

In addition, NAPAP of the following formula (9), a benzamidine-based arylsulfonyl compound, was developed as a thrombin inhibitor, which is not only easy to synthesize but also has a problem that the thrombin inhibitory effect is only 50 times that of trypsin, despite being an effective thrombin inhibitor. See J. Biol. Chem. 1991, 266, 20085-20093.

[Formula 9]

Meanwhile, Ro46-6240 compound of formula 10, which has improved selectivity to thrombin relative to trypsin, has also been reported as a potent thrombin inhibitor, which shows the possibility of development as an intravenous infusion, but has a short blood half-life and thus development as an oral administration. There is no possibility (see J. Med. Chem. 1994, 37, 3889-3901).

[Formula 10]

In addition, recently, certain piperazide-based compounds show inhibitory activity against thrombin and these compounds have been reported to be orally administrable in mice, but the selectivity to thrombin relative to trypsin is very low (WO 94/18185).

The present inventors have been intensively researched for a long time to develop a compound that is relatively easy to synthesize, has excellent thrombin inhibitory activity, and has 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 explained in more 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]

In the above formula,

R ₁ represents substituted or unsubstituted naphthalene.

Preferred compounds of the formula (1) of the present invention are compounds in which R ₁ represents unsubstituted naphthalene.

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

One.

2.

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, Acid addition salts formed by citric acid, acetic acid, trichloroacetic acid or trifluoroacetic acid, gluconic acid, benzoic acid, organic carbonic acid such as lactic acid, fumaric acid, maleic acid, etc., sulfonic acid such as methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid or naphthalenesulfonic acid, etc. This includes.

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 is reduced to an azido compound of Formula 2 to produce an amine compound of Formula 3, and the resulting amine compound of Formula 3 is aminoiminomethylsulfonic acid ( It can be prepared by reacting with).

[Formula 2]

[Formula 3]

Wherein R ₁ is 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 Chemical Formula 1 according to the present invention reduces the azido compound of Chemical Formula 2, which is an intermediate, to generate an amine compound of Chemical Formula 3, and the amine compound of Chemical Formula 3 is converted to aminoiminomethylsulfonic acid ( ) To produce the desired compound of formula (I).

The reduction reaction of the compound of formula (2) can be carried out using any of the conventional reduction methods, but is preferably carried out in the presence of an activated palladium-carbon catalyst in the presence of a methanol solvent.

The amine compound of formula (3) obtained by reduction of the compound of formula (2) is subsequently reacted with aminoiminomethylsulfonic acid to produce the desired compound of formula (1). In this 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 5 to 20 hours, but preferably performs the reaction for 6 to 12 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 azido compound of Formula 2 used as an intermediate in preparing the compound of Formula 1 in Scheme 1, for example, by reacting a diethylacetamido malonate compound of Formula 4 with azidobromomethylbenzene The compound can be prepared by reacting the compound of formula 5 with cyclopentyl-methylamine to produce the compound of formula 6, and then reacting the compound of formula 6 with a sulfonylchloride derivative of formula 7.

[Formula 4]

[Formula 5]

[Formula 6]

[Formula 7]

Wherein R ₁ is as defined above and P represents a conventional amino protecting group such as benzyloxycarbonyl, allyloxycarbonyl, t-butoxycarbonyl or trityl group and the like.

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

Scheme 2

According to Scheme 2, an azido compound such as azidobromomethylbenzene was introduced to the C-α position of the malonate derivative of Formula 4, followed by hydrolysis, decarbonation, and an amino protecting group to prepare a compound of Formula 5. The amine group is then coupled to prepare a compound of formula (6). The desired intermediate compound of formula (2) can be prepared by deprotecting the amino group of the compound of formula (6) and reacting with the sulfonylchloride compound of formula (7) to form the sulfonamido group at the N-position.

Known coupling reagents that can be used for the coupling reaction of the compound of formula 5 with the amine group in Scheme 2 include dicyclohexylcarbodiimide (DCC), 3-ethyl-3 '-(dimethylamino) -propylka Bodyimide (EDC), bis- (2-oxo-3-oxazolidinyl) -phosphonic acid chloride (BOP-Cl), diphenylphosphoryl azide (DPPA), and the like, but are not limited to these; . In this reaction, the carboxylic acid compound of the formula (5) may be used as it is, but preferably, the reaction can be promoted by converting it into a reactive derivative thereof, such as an acid halide and other activated ester derivatives, to be used in the coupling reaction. 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.

As mentioned above, the compound of formula 1 according to the present invention has a selective inhibitory effect on thrombin, and thus is useful for preventing blood coagulation and for treating 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, sex, health status, diet, time of administration, method of administration, rate of excretion, drug mixture and severity of disease may be appropriately determined.

The compounds of the present invention may preferably be formulated in injectable preparations.

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.

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 of Cyclopentyl-Methylamine Hydrochloride

10 ml of cyclopentanone was dissolved in 50 ml of methanol, and then 50 ml of water was added thereto. 7.6 g of methylamine hydrochloride was added thereto, and 7.1 g of sodium borocyanohydride (NaBH ₃ CN) was added thereto. The reaction solution was stirred for 12 hours while heating to reflux at pH 6. After removing the methanol under reduced pressure, the mixture was cooled to 0 deg. The reaction solution was washed three times with diethyl ether and the aqueous layer was again cooled to 0 ° C. and adjusted to pH 11 with 6N sodium hydroxide solution. 50 ml of dioxane was added thereto, and 37 g of t-butyloxycarbonyl anhydride was added thereto, followed by stirring at room temperature for 3 hours. After the reaction was completed, the reaction solution was distilled under reduced pressure to remove the solvent and concentrated to about 30ml. The residual solution was extracted with ethyl acetate and washed with an acid and an aqueous base solution. The organic layer was dried over anhydrous magnesium sulfate, filtered and concentrated. The white solid obtained was purified using column chromatography. The obtained solid was dissolved in 60 mL of 4N hydrochloric acid-dioxane solution and stirred for 30 minutes. The solvent was removed under reduced pressure and dried in vacuo to yield 13.7 g 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)

Preparation Example 2

Synthesis of 3-azido-bromomethylbenzene

A solution of 10.7 g of meta-toluidine dissolved in 200 ml of tetrahydrofuran and 20 ml of distilled water was cooled to 0 ° C, and then 58 ml of concentrated hydrochloric acid was added. 9.6 g of sodium nitrite dissolved in 100 ml of distilled water was added dropwise to the reaction solution over 30 minutes, followed by further stirring for 30 minutes, and then 9.1 g of sodium azide was added and stirred for 1 hour. 500 ml of diethyl ether was added to the reaction solution, washed three times with saturated brine, and the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. 200 ml of chloroform was added to the concentrated solution, and 17.8 g of N-bromosuccinimide and 2.4 g of benzoyl peroxide were added to the dissolved solution, followed by heating to reflux for 5 hours. The reaction solution was cooled to room temperature to remove insoluble matters and concentrated under reduced pressure, and then the residue was purified by column chromatography [eluent: hexane] to give 8.9 g of the title compound.

¹ H NMR (CDCl ₃ , ppm) δ: 7.62 (s, 1H), 7.43 (d, 1H), 7.37 (d, 1H), 7.09 (t, 1H), 4.63 (s, 2H)

Preparation Example 3

Synthesis of 4-azido-bromomethylbenzene

The reaction was carried out in the same manner as in Preparation Example 2 using 10.7 g of para-toluidine to obtain 9.1 g of the title compound.

¹ H NMR (DMSO-d ₆ , ppm) δ: 7.49 (d, 2H), 7.07 (d, 2H), 4.58 (s, 2H)

Mass (FAB, m / e): 213 (M ⁺ +1)

Preparation Example 4

Synthesis of (S) -3- (3-azidophenyl) -2- (tert-butoxycarbonylamino) -propionic acid

8.68 g of diethylacetamidomalonate and 8.48 g of the compound obtained in Preparation Example 2 were added dropwise to a solution of 150 ml of anhydrous ethanol, 8.17 g of a 28% sodium methoxide solution at room temperature over 1 hour. 3.2 g of caustic soda dissolved in 50 ml of distilled water while heating and refluxing was added dropwise over 1 hour, and further stirred for 5 hours. The reaction solution was cooled, the solvent was removed under reduced pressure, 100 ml of distilled water and 100 ml of ethyl acetate were added to the residue, and the layers were separated. The aqueous layer was adjusted to pH 2, and the resulting solid was filtered. The filtered solid was added to 200 ml of distilled water, adjusted to pH 6.5, dissolved, and then 0.2 g of acylate was added and stirred at 37 ° C. for 3 days. 10 g of butyloxycarbonyl anhydride was slowly added while removing insoluble matters in the reaction solution, adjusting to pH 2, washing twice with ethyl acetate, and maintaining and adjusting the aqueous layer to pH 9 again. After the reaction was completed, the reaction solution was washed with ethyl acetate and the aqueous layer was adjusted to pH 2, and the resulting solid was filtered and dried under vacuum to give 4.9 g of the title compound.

¹ H NMR (DMSO-d ₆ , ppm) δ: 7.94 (s, 1H), 7.61 (d, 1H), 7.48 (d, 1H), 7.21 (t, 1H), 4.33 (s, 1H), 3.24 ( m, 1H), 3.06 (m, 1H), 1.45 (s, 9H)

Mass (FAB, m / e): 307 (M ⁺ +1)

Preparation Example 5

Synthesis of (S) -3- (4-azidophenyl) -2- (tert-butoxycarbonylamino) -propionic acid

Using 8.48 g of the compound obtained in Preparation Example 3, the reaction was carried out in the same manner as in Preparation Example 4, to obtain 4.7 g of the title compound.

¹ H NMR (DMSO-d ₆ , ppm) δ: 7.78 (d, 2H), 7.59 (d, 2H), 7.21 (d, 1H), 4.21 (s, 1H), 3.19 (m, 1H), 2.94 ( m, 1H), 1.32 (s, 9H)

Mass (FAB, m / e): 307 (M ⁺ +1)

Preparation Example 6

Synthesis of (S) -3- (3-azidophenyl) -2- (tert-butoxycarbonylamino) -N-cyclopentyl-N-methyl-propionamide

3.06 g of the compound obtained in Preparation Example 4 was dissolved in 50 ml of tetrahydrofuran, while 2.2 g of N-methylmorpholine and 1.37 g of isopropylchloroformate were added while maintaining the solution at -20 ° C., followed by stirring for 30 minutes. 1.35 g of the compound obtained in Preparation Example 1 was added. The reaction solution was raised to room temperature over 1 hour to complete the reaction, the solvent was removed under reduced pressure, ethyl acetate was added to the residue, and the residue was washed with 1N aqueous hydrochloric acid solution and saturated aqueous sodium bicarbonate solution. The organic layer was dried over anhydrous sodium sulfate, filtered and dried under vacuum to afford 3.71 g of the title compound.

¹ H NMR (CDCl ₃ , ppm) δ: 7.63 (s, 1H), 7.42 (m, 3H), 5.33 (m, 1H), 4.93 (m, 1H), 3.11 (m, 2H), 2.64 (d, 3H), 1.88-1.03 (m, 17H)

Mass (FAB, m / e): 388 (M ⁺ +1)

Preparation Example 7

Synthesis of (S) -3- (4-azidophenyl) -2- (tert-butoxycarbonylamino) -N-cyclopentyl-N-methyl-propionamide

Using 3.06 g of the compound obtained in Preparation Example 5, the reaction was carried out in the same manner as in Preparation Example 6, to obtain 3.58 g of the title compound.

¹ H NMR (CDCl ₃ , ppm) δ: 7.58 (d, 2H), 7.30 (d, 2H), 5.49 (m, 1H), 4.89 (m, 1H), 3.08 (m, 2H), 2.70 (d, 3H), 1.95-1.20 (m, 17H)

Mass (FAB, m / e): 388 (M ⁺ +1)

Preparation Example 8

synthesis

3.5 g of the compound obtained in Preparation Example 6 was added to 50 ml of a hydrochloric acid solution saturated in ethyl acetate, and stirred at room temperature for 1 hour, and the reaction solution was concentrated and dried under reduced pressure. 50 ml of tetrahydrofuran was added to the dried residue, and 2.1 g of N-methylmorpholine and 2.05 g of 2-naphthalenesulfonyl chloride were added to the solution, which was then stirred at room temperature for 5 hours. The solvent was removed under reduced pressure, ethyl acetate was added to the residue, the mixture was diluted, and then washed sequentially with 1N aqueous hydrochloric acid solution and saturated aqueous sodium hydrogencarbonate solution. The organic layer was dried over anhydrous sodium sulfate, filtered and dried under vacuum to afford 3.98 g of the title compound.

¹ H NMR (CDCl ₃ , ppm) δ: 8.83 (d, 1H), 8.09-6.75 (m, 10H), 6.05 (m, 1H), 4.51 (m, 1H), 4.01 (m, 1H), 2.99 ( m, 2H), 2.35 (d, 3H), 1.62-0.45 (m, 8H)

Mass (FAB, m / e): 478 (M ⁺ +1)

Preparation Example 9

synthesis

Using 3.41 g of the compound obtained in Preparation Example 7, the reaction was carried out in the same manner as in Preparation Example 8 to obtain 3.51 g of the title compound.

¹ H NMR (CDCl ₃ , ppm) δ: 8.38 (d, 1H), 8.0-6.87 (m, 10H), 5.92 (d, 1H), 4.51 (m, 1H), 3.88 (m, 1H), 2.98 ( m, 2H), 2.37 (d, 3H), 1.78-0.51 (m, 8H)

Mass (FAB, m / e): 478 (M ⁺ +1)

Example 1

synthesis

0.2 g of activated palladium-carbon was added to a solution of 2.1 g of the compound obtained in Preparation Example 8 in 20 ml of methanol, and charged with hydrogen gas under atmospheric pressure, followed by stirring at room temperature for 10 hours. After the reaction was completed, the reaction solution was filtered to remove insoluble matters, 2 g of aminoiminomethylsulfonic acid was added to the filtrate, and the mixture was stirred at room temperature for 10 hours. The reaction solution was concentrated under reduced pressure and the concentrate was separated and purified by HPLC to give 1.1 g of the title compound.

¹ H NMR (CD ₃ OD, ppm) δ: 8.50-7.19 (m, 11H), 4.61 (m, 1H), 4.19 (m, 1H), 2.98 (m, 2H), 2.48 (d, 3H), 1.71 -0.69 (m, 8H)

Mass (FAB, m / e): 494 (M ⁺ +1)

Example 2

synthesis

The reaction was carried out in the same manner as in Example 1 using 3 g of the compound obtained in Preparation Example 9 to obtain 2.05 g of the title compound.

¹ H NMR (CD ₃ OD, ppm) δ: 8.47-7.09 (m, 11H), 4.58 (m, 1H), 4.22 (m, 1H), 3.01 (m, 2H), 2.51 (d, 3H), 1.91 -0.73 (m, 8H)

Experimental Example 1 Inhibitory Activity of a Thrombin Inhibitor

The enzymatic inhibitory effect of the thrombin inhibitor, a compound of the present invention, was determined by the rate constants Ki and Ks measured spectrophotometrically using a substrate that reacts with an enzyme to produce color. The rate constant Ki value indicates the degree to which the inhibitor inhibits the enzyme activity when the enzyme, the substrate and the inhibitor are added simultaneously. The rate constant Ks value indicates the rate at which the enzyme and the inhibitor are reacted for a predetermined time before the hydrolysis of the substrate. The value obtained by measuring Hydrolysis of thrombin, a substrate, chromozyme TH (tosyl-Gly-Pro-Arg-4-nitroanilide acetate) produces para-nitroanilide. The amount of yellow para-nitroanilide thus produced was measured as a change in absorbance over time. From this rate the activity of the enzyme can be measured and the inhibitory capacity of the inhibitor can be measured.

The inhibitory ability of the inhibitor according to the present invention to thrombin was measured by the following method.

1160 μl of 0.1M Tris buffer (pH 7.8) containing 150 mM NaCl and 0.1% PEG8000 (polyethylene glycol, molecular weight about 8000) was added to a 1.5 mL cuvette. The substrate solution was prepared by dissolving chromozyme TH, a pigment generating substrate, 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. After taking between 10 μg and 100 μl of total volume with Tris buffer, the solution was added to a cuvette containing buffer and substrate.

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 following enzyme reaction. 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. .

The rate constant Ks was used in the same concentration as the same solution used to obtain the Ki, but the experimental method is as follows.

That is, 1160 µl of buffer solution was added to a 1.5 ml volume cuvette, and 15 µl of 0.1 mg / ml human thrombin solution and 100 µl of inhibitor solution were added thereto, and the mixture was left at room temperature for 15 minutes, followed by 225 µl of 0.1 mM substrate solution. The change in absorbance over time was monitored for 2 minutes with addition. 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.

As a substrate, a 20 μM solution of N-benzoyl-valine-glycine-arginine para-nitroanilide hydrochloride was used, and inhibitors were used at various concentrations in the range of 0 to 120 μg. In addition, trypsin was dissolved in 0.1N HCl was made to 45 ㎍ / ㎖ with the Tris buffer solution immediately before the experiment and 40 ul was used. As in the experiment for thrombin, the total volume of the reaction solution was set to 1.5 ml and the same method was used. The Km value used for the calculation of Ki was determined by the same method, which was 20.2 μM.

The inhibitory capacity of the inhibitor according to the present invention and the selectivity to thrombin (trypsin / thrombin) measured for thrombin and trypsin according to the method described above are shown in Table 1 below.

TABLE 1

Inhibitor's ability to inhibit thrombin and trypsin

Compound (Example Number) Inhibition of thrombin Inhibitory ability to trypsin Selectivity (Trypsin / Trombin) One Ks = 39.3 nM 2 Ks = 3.1 nM Ks = 23522 nM 3313

As can be seen from the results shown in Table 1, the compound of formula 1 according to the present invention shows an excellent inhibitory effect on thrombin, and also the selectivity of thrombin relative to trypsin is significantly superior to known compounds.

Claims (6)

A compound represented by the following formula (1), a pharmaceutically acceptable salt thereof, and an isomer thereof: [Formula 1] In the above formula, R ₁ represents substituted or unsubstituted naphthalene. A compound according to claim 1, wherein R ₁ represents unsubstituted naphthalene. The compound of claim 2 selected from the group consisting of: The azido compound of formula 2 is reduced to produce an amine derivative of formula 3, and the resulting compound of formula 3 is aminoiminomethylsulfonic acid ( A process for preparing a compound of formula (1) and salts thereof: [Formula 1] [Formula 2] [Formula 3] In the above formula, R ₁ represents substituted or unsubstituted naphthalene. 5. A process according to claim 4, wherein the reduction of formula (2) is carried out using a palladium-carbon catalyst activated in the presence of a methanol solvent. A thrombin inhibitor composition comprising a compound of formula 1 according to claim 1 as an active ingredient together with a pharmaceutically acceptable carrier.