KR100361827B1 - Heterocyclicalanine derivative useful as thrombin inhibitor - Google Patents

Abstract

PURPOSE: A heterocyclicalanine derivative useful as a thrombin inhibitor is provided. The heterocyclicalanine derivative can be administered orally, and strongly inhibits the thrombin formation. CONSTITUTION: A heterocyclicalanine derivative represented by the formula(1), pharmaceutically acceptable salts thereof and isomers thereof are provided, wherein X and Y are independently O, S, NH, N or CH; R1 is substituted or unsubstituted aryl; R2 and R3 are independently lower alkyl or cycloalkyl; and R4 is hydrogen, lower alkyl or amino; and the heterocyclicalanine derivative is selected from the group consisting of (S) - (S) - 3 - £5 - amidino - thiopen - 2 - yl| - N - cyclopentyl - N - methyl - 2 - (4 - propyl - benzensulfonylamino) - propion amide, (S) - 5 - £2 - £5 - amidino - thiopen - 2 - yl| - 1 - (cyclopentyl - methyl - carbamoyl) - ethylsulfamoyl| - naphthalene - 1 - carboxylic acid methylester, (R,S) - 3 - (2 - amidinothiazol - 4 - yl) - N - cyclopentyl - N - methyl - 2 - (2 - naphthalenesulfonylamino) - propionamide and (R,S) - 3 -. A process for preparing the compound of the formula(1) comprises reacting methylmercapto compound of the formula(2) with amine derivatives of the formula(3).

Description

Heterocyclic Alanine Derivatives Useful as Thrombin Inhibitors

The present invention relates to novel compounds useful as thrombin inhibitors. More specifically, the present invention is a novel heterocyclic alanine derivative represented by the following Chemical Formula 1 having a powerful effect in inhibiting thrombogenesis, a method for preparing the same, and a medicament for preventing blood coagulation or treating various thrombosis containing the compound as an active ingredient. Regarding the composition:

[Formula 1]

In the above formula,

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

R ¹ represents substituted or unsubstituted aryl,

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

R ⁴ 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. The thrombin produced in this process activates platelets, converts fibrinogen to fibrin, and the like. The produced fibrin is polymerized by polymerization 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). Agatroban was already commercialized in Japan in 1990 (Biochemistry 1984, 23, 85-90). However, this compound is not only active for oral administration but also has the disadvantage of being effective in treating venous thrombosis as an injection but weak in arterial thrombosis.

[Formula 4]

Representative compounds that can be orally administered and a powerful thrombin inhibitor are compounds of the following structural formula 5, which is being developed by LG Chem, Inc., which has a very good absorption rate in animals (or about 60% in dogs). However, in order to fully inhibit thrombus formation in animal experiments, the effect needs to be further improved (see EP-739886A).

[Formula 5]

Accordingly, the present inventors have been intensively researching structural modification of the compound of Formula 5 for a long time by developing a compound having excellent thrombin inhibitory activity and ultimately effective in inhibiting thrombus formation. 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, it is an object of the present invention to provide a novel thrombin inhibitor having a possibility of oral administration and having a strong effect on inhibiting thrombus formation and a method for 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.

Hereinafter, the present invention will be described in more detail.

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,

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

R ¹ represents substituted or unsubstituted aryl,

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

R ⁴ represents hydrogen, lower alkyl or amino.

The term "lower alkyl" in the definition of the substituent of the compound of formula 1 according to the invention means a straight or branched chain hydrocarbon radical having 1 to 4 carbon atoms, including methyl, ethyl, propyl, isopropyl, isobutyl, t-butyl And the term "cycloalkyl" means cyclic alkyl having 3 to 8 carbon atoms including cyclopentyl, and the term "substituted or unsubstituted aryl" means 6 to 6 unsubstituted or substituted by one or more substituents as appropriate. A 10-membered aryl group.

Preferred compounds of formula (I) of the present invention represent X, O, S or N, Y represents CH when X is O and S, S represents X when X is N, R ¹ is substituted or unsubstituted Phenyl or naphthyl, R ² and R ³ each independently represent methyl or cyclopentyl, and R ⁴ is hydrogen, methyl or amino.

Particularly preferred compounds of formula (1) of the present invention are those wherein X represents O, S or N, Y represents CH when X is O and S, S represents X when X is N, and R ¹ represents lower alkyl or Phenyl or naphthyl unsubstituted or substituted by lower alkoxycarbonyl, R ² and R ³ each independently represent methyl or cyclopentyl, and R ⁴ is hydrogen, methyl or amino.

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

One.

2.

3.

4.

5. Methyl Ester

6.

7.

8.

9.

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 asymmetric carbon centers 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 compound of Formula 2 with an amine compound of Formula 3 below.

[Formula 2]

[Formula 3]

Wherein X, Y, R ¹ , R ² , R ³ and R ⁴ 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 the scheme 1 is first coupled to the C-terminus of the compound of formula (6) by R ² and R ³ substituted amine group of the formula (7) The compound was obtained and the N-terminal amino protecting group of the compound of Formula 7 was removed to obtain the compound of Formula 8, and then the compound of Formula 8 was reacted with the sulfonylchloride compound of R ¹ SO ₂ Cl to form the N-terminal Introducing a sulfonyl group at the site affords a compound of formula 9, and the nitrile compound of formula 9 is saturated with hydrogen sulfide in the presence of a base to give a thioamide compound of formula 10, which methylates the thioamide compound of formula 10 It can be prepared by the method:

[Formula 6]

[Formula 7]

[Formula 8]

[Formula 9]

[Formula 10]

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

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

[Scheme 2]

As shown in Scheme 2, to couple the first ring R ² and R ³ is a substituted amine group of the first to the C- terminus of the compound of formula (6) in order to produce a methyl mercapto compound of formula 2 to obtain a compound of formula 7 . Known coupling reagents that can be used for this coupling reaction 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 to these.

In this reaction, the carboxylic acid compound of the formula (6) 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.

The compound of formula (7) produced by the coupling reaction of compound of formula (6) then removes the N-terminal amino protecting group to yield the compound of formula (8). The compound of formula 8 was reacted with a sulfonylchloride compound of the general formula R ¹ SO ₂ Cl to introduce a sulfonyl group to the N-terminus to obtain a compound of formula 9, and then the nitrile compound of formula 9 was substituted with a base, For example, saturation with hydrogen sulfide in the presence of pyridine and trimethylamine yields a thioamide compound of formula 10, and the thioamide compound of formula 10 is methylated to give a methylmercapto compound of formula 2. Methylating agents which may preferably be used for the methylation of compounds of formula 12 include methane iodide, dimethylsulfate ((CH ₃ ) ₂ SO ₂ ), methyltriplate (CH ₃ OTf) and the like.

The amino acid compound of formula (6) used as starting material in preparing the compound of formula (2) in Scheme 2 also, for example, by reacting the compound of formula (11) with N-bromosuccinimide to obtain a compound of formula (12), The compound of formula 12 is reacted with diethylacetamidomalonate in the presence of sodium ethoxide, followed by hydrolysis and decarbonation to give an N-acetylamino acid compound of formula 13, which removes the compound of formula 13 Acetylation can yield the compound of formula 14, which can be prepared by introducing an amino-protecting group at the N-terminus of the compound.

[Formula 11]

[Formula 12]

[Formula 13]

[Formula 14]

Wherein X and Y are as defined above.

The method for preparing the compound of Formula 6 as mentioned above may be represented by the following Scheme 3.

Scheme 3

According to Scheme 3, a compound of Formula 11 is first reacted with N-bromosuccinimide (NBS) to obtain a bromo compound of Formula 12. At this time, in order to promote the radical reaction, benzoyl peroxide or AIBN (α, α'-azobisisobutyronitrile) or the like is used as a catalyst. The resulting compound of formula 12 is reacted with diethylacetamidomalonate in the presence of sodium ethoxide (NaEOt) according to the method described in J. Biol. Chem. 1991, 243, 3238-3247. The reaction product is hydrolyzed and decarboxylated to give the desired N-acetylamino acid compound of formula (13). This amino acid compound was subjected to deacetylation using an acylase enzyme as a catalyst according to the method described in J. Am. Chem. Soc. 1989, 111, 6354-6364; EP-0508220 A1. L-amino acid compound of formula (14) having optical activity is isolated and obtained, and a protecting group is attached to the N-terminus of this compound to obtain a compound of formula (6).

The thrombin inhibitory effect of the compound of formula 1 according to the present invention is determined by the following formula according to the method described in Methods in Enzymology V. 80, p341-361; Biochemistry 27, p2144-2151, 1988 Measure by determining the value.

Ki = [E] · [I] / [EI]

In the above formula, [E] is the concentration of the enzyme not bound to the inhibitor, [I] is the concentration of the inhibitor not bound to the enzyme, and [EI] is the concentration of the enzyme and inhibitor binding.

Since the inhibitory constant Ki represents the degree of dissociation between the enzyme and the thrombin inhibitor compound, the smaller the inhibitory constant value, the greater the binding activity of the inhibitor to the enzyme, and thus, the inhibitory activity may be evaluated to be greater. Such inhibitory constant can be obtained by reacting with a specific substrate showing color development when hydrolyzed by the action of thrombin and measuring the color development as a function of time according to spectrophotometry.

In the present invention, chromozyme TH (Chromozyme TH: tosyl-Gly-Pro-Arg-4-nitroanilide acetate) is used as a substance for developing a thrombin under hydrolysis. Hydrolysis of chromozyme TH with thrombin produces yellow para-nitroanilide. Therefore, the thrombin inhibitory activity of the compounds according to the invention can be measured by measuring the amount of para-nitroanilides produced by the change in absorbance over time. That is, the activity of the enzyme can be measured from the change in absorbance, which can be directly related to the ability of the thrombin inhibitor to inhibit the enzyme activity.

As mentioned above, the compound of the formula (1) according to the present invention, like the known compounds, is an excellent thrombin inhibitor and a potential thrombin inhibitor by oral administration. Accordingly, the compounds of the present invention are useful for preventing blood clotting 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, although specific dosage levels for individual patients may be used. The specific compound, the weight of the patient, sex, health status, diet, administration time, administration method, excretion rate, drug mixture and the severity of the disease may be changed.

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.

As the solid dosage form for oral administration, capsules, tablets, pills, powders and granules may be used, and capsules and tablets are particularly 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 of 5-bromomethyl-thiophene-2-carbonitrile

9.9 g (80.5 mmol) of 5-methyl-thiophene-2-carbonitrile and 15.0 g (84.3 mmol) of N-bromosuccinimide (NBS) were added to the reaction vessel together with 200 ml of carbon tetrachloride, and 0.23 g of benzoyl peroxide. (0.95 mmol) was added in three portions and heated to reflux. After reacting for 3 hours, 1.4 g of N-bromosuccinimide (NBS) was added. Benzoyl peroxide was added to the reaction mixture at 3 hour intervals and reacted for 12 hours. After cooling to 0 ° C., the insoluble solid was filtered off. The filtered solution was diluted with 400 ml of dichloromethane, washed twice with saturated sodium hydrogen carbonate solution, dried over anhydrous sodium sulfate, filtered and concentrated. The residue was subjected to column chromatography [eluent: ethyl acetate / hexane (1/35, volume ratio)] to give 14.0 g (yield 85.9%) of the title compound.

¹ H NMR (CDCl ₃ , ppm) δ: 7.50 (d, 1H), 7.13 (d, 1H), 4.68 (s, 2H)

Mass (FAB, m / e): 203 (M ⁺ +1)

Preparation Example 2

Synthesis of 2-acetylamino-3- (5-cyano-thiophen-2-yl) -propionic acid

14.0 g (69.3 mmol) of the compound obtained in Preparation Example 1, 15.4 g (69.5 mmol) of diethylacetamidomalonate and 70 ml of dioxane were added to a reaction vessel to prepare a solution of 70 ml of anhydrous ethanol and 1.8 g of sodium. And slowly added to the reaction vessel over 30 minutes. At this time, after reacting for 2 hours while heating under reflux, 140 mL of 1N sodium hydroxide was added thereto. The reaction mixture was heated to about 80 ° C. for 4 hours, cooled to room temperature, and concentrated under reduced pressure. The pH of the residue was adjusted to 10 with sodium hydroxide and washed twice with ethyl acetate. The aqueous layer was adjusted to pH 1 with dilute hydrochloric acid, extracted four times with dichloromethane / tetrahydrofuran (3/1 by volume), dried over anhydrous magnesium sulfate, filtered and concentrated to give 9.3 g of the title compound (yield 56). %) Was obtained.

¹ H NMR (CDCl ₃ , ppm) δ: 7.60 (d, 1H), 7.00 (d, 1H), 4.70 (m, 1H), 3.50 (m, 1H), 3.30 (m, 1H), 2.00 (s, 3H)

Mass (FAB, m / e): 239 (M ⁺ +1)

Preparation Example 3

synthesis

9.3 g (39.1 mmol) of the compound obtained in Preparation Example 2 were dissolved in water, and then adjusted to pH 6.5. 500 mg of acylase was added thereto and reacted at 36 ° C. for 3 days. The enzyme was filtered and adjusted to pH 1 with 6N hydrochloric acid. The reaction solution was washed four times with dichloromethane / tetrahydrofuran (3/1 by volume) and the aqueous layer was adjusted to pH 10 with 6N sodium hydroxide. The same amount of dioxane was added to the aqueous layer, 6.0 g (27.5 mmol) of t-butoxycarbonyl anhydride was added, followed by reaction at room temperature for 12 hours, and concentrated under reduced pressure. Again pH was adjusted to 10 with 6N sodium hydroxide. The reaction solution was washed twice with ethyl acetate, the aqueous layer was adjusted to pH 1 with 6N hydrochloric acid, extracted six times with dichloromethane / tetrahydrofuran (3/1, volume ratio), the organic layers were combined, and dried over anhydrous magnesium sulfate, Filtered and concentrated. The residue obtained was dried under vacuum to give 3.0 g (10.1 mmol) of intermediate (S) -3- (5-cyano-thiophen-2-yl) -2- (t-butoxycarbonylamino) -propionic acid. Obtained. The obtained intermediate compound was dissolved in dimethylformamide together with 2.0 g (14.8 mmol) of cyclopentyl-methylamine hydrochloride and 1.6 g (11.8 mmol) of 1-hydroxybenzotriazole hydrate (HOBT). After cooling the resulting solution to 0 ° C., 2.9 g (15.1 mmol) of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and 3.3 ml (30.0) of N-methylmorpholine (NMM) Mmol). After all the reactants were dissolved, the reaction mixture was heated to room temperature for 3 hours. Concentrated under reduced pressure to remove volatiles and diluted with ethyl acetate. It was washed sequentially with saturated sodium bicarbonate solution, dilute hydrochloric acid and saturated sodium chloride solution, dried over anhydrous magnesium sulfate, filtered and concentrated. The obtained residue was subjected to column chromatography [eluent: ethyl acetate / hexane (3/7, volume ratio)] to give 2.3 g (yield 16%) of the title compound.

¹ H NMR (CDCl ₃ , ppm) δ: 7.42 (d, 1H), 6.84 (t, 1H), 5.55 (m, 1H), 4.84, 4.20 (m, m, 2H), 3.28, 3.10 (m, m , 2H), 2.80 (d, 3H), 1.90-1.20 (m, 17H)

Mass (FAB, m / e): 378 (M ⁺ +1)

Preparation Example 4

synthesis

0.60 g (1.59 mmol) of the compound obtained in Preparation Example 3 was dissolved in 5 ml of ethyl acetate, and then cooled to 0 ° C. The solution was stirred dropwise while slowly using an excess amount of hydrochloric acid solution dissolved in ethyl acetate. After reacting for 1 hour, the reaction mixture was dried under reduced pressure and dried in vacuo. The residue obtained was dissolved in dimethylformamide and then 0.53 mL (4.82 mmol) N-methylmorpholine was added and stirred for 5 minutes. 0.46 g (2.01 mmol) of 2-naphthalenesulfonyl chloride was added to the reaction solution, followed by stirring at room temperature for 2 hours. The volatiles were removed under reduced pressure, diluted with dichloromethane and washed with water. The organic layer was dried over anhydrous magnesium sulfate, filtered and concentrated. The residue obtained was purified by column chromatography [eluent: ethyl acetate / hexane (4/6, volume ratio)] to yield 0.65 g (88% yield) of the title compound.

¹ H NMR (CDCl ₃ , ppm) δ: 8.31 (d, 1H), 8.00-7.50 (m, 6H), 7.38 (d, 1H), 6.86 (s, 1H), 6.00 (m, 1H), 4.55, 4.38, 4.11, 3.87 (m, m, m, m, 2H), 3.10 (m, 2H), 2.40 (d, 3H), 1.70-0.50 (m, 8H)

Mass (FAB, m / e): 468 (M ⁺ +1)

Example 1

synthesis

0.64 g (1.37 mmol) of the compound obtained in Production Example 4 was dissolved in 10 ml of pyridine and added to a flask equipped with 0.8 ml of triethylamine. Hydrogen sulfide (H ₂ S) gas was slowly flowed into the solution through one branch of the flask, and the gas was flowed out from the other branch. The reaction mixture was saturated with hydrogen sulfide gas with stirring for about 10 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. To the obtained yellow solid, 10 ml of acetonitrile and 0.26 ml (4.18 mmol) of methane iodide (CH ₃ I) were added together and 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 5 ml of methanol and stirred. 0.32 g (4.15 mmol) of ammonium acetate was added thereto, and the mixture was heated to reflux for 3 hours. After the reaction was completed, the reaction solution was concentrated and purified by HPLC to give 0.27 g (41% yield) of the title compound.

¹ H NMR (CD ₃ OD, ppm) δ: 8.36 (s, 1H), 8.00 (m, 3H), 7.80 (t, 1H), 7.65 (m, 3H), 7.06 (d, 1H), 4.70, 4.54 (m, m, 1H), 4.26, 4.14 (m, m, 1H), 3.15, 3.11 (m, m, 2H), 2.67, 2.38 (s, s, 3H), 1.70-0.60 (m, 8H)

Mass (FAB, m / e): 485 (M ⁺ +1)

Example 2

synthesis

The reaction was carried out in the same manner as in Example 1 using 0.093 g (0.20 mmol) of the compound obtained in Preparation Example 4, except that 0.12 ml (0.24 mmol) of a methylamine 2.0M solution dissolved in methanol instead of ammonium acetate was used. The reaction was carried out at room temperature while being added in three equal parts every 20 minutes. This gave 0.051 g (51% yield) of the title compound.

¹ H NMR (CD ₃ OD, ppm) δ: 8.35 (s, 1H), 8.00 (m, 3H), 7.80 (m, 1H), 7.65 (m, 3H), 7.05 (d, 1H), 4.69, 4.54 (m, m, 1H), 4.25, 4.15 (m, m, 1H), 3.21, 3.10 (m, m, 2H), 3.05 (s, 3H), 2.70, 2.39 (s, s, 3H), 1.70- 0.60 (m, 8H)

Mass (FAB, m / e): 499 (M ⁺ +1)

Example 3

synthesis

The reaction was carried out in the same manner as in Example 1 using 0.093 g (0.20 mmol) of the compound obtained in Preparation Example 4, but instead of ammonium acetate, 0.02 ml (0.33 mmol) of 80% hydrazine hydrate was divided into three equal parts every 20 minutes. The reaction was performed at room temperature while adding thereto. This gave 0.09 g (yield 90%) of the title compound.

¹ H NMR (CD ₃ OD, ppm) δ: 8.35 (d, 1H), 7.99 (m, 3H), 7.79 (m, 1H), 7.66 (m, 2H), 7.32 (m, 1H), 6.85 (dd) , 1H), 4.63, 4.50 (m, m, 1H), 4.22, 4.03 (m, m, 1H), 3.20, 3.05 (m, m, 2H), 2.55, 2.34 (s, s, 3H), 1.60- 0.60 (m, 8H)

Mass (FAB, m / e): 500 (M ⁺ +1)

Preparation Example 5

synthesis

The reaction was carried out in the same manner as in Preparation Example 4 using 0.15 g (0.48 mmol) of the compound obtained in Preparation Example 3, but purified using 4-propyl-benzenesulfonylchloride instead of 2-naphthalenesulfonylchloride. 0.16 g (73% yield) of the title compound were obtained.

¹ H NMR (CDCl ₃ , ppm) δ: 7.70 (d, 2H), 7.44 (t, 1H), 7.28 (d, 2H), 6.88 (s, 1H), 6.00 (dd, 1H), 4.57, 4.49, 4.35, 3.92 (m, m, m, m, 2H), 3.13 (m, 2H), 2.68 (m, 2H), 2.58 (d, 3H), 1.80-0.80 (m, 13H)

Mass (FAB, m / e): 460 (M ⁺ +1)

Example 4

synthesis

Using 0.16 g (0.35 mmol) of the compound obtained in Preparation Example 5, the reaction was carried out in the same manner as in Example 1 to obtain 0.046 g (yield 28%) of the title compound.

¹ H NMR (CD ₃ OD, ppm) δ: 7.90-7.60 (m, 3H), 7.35 (m, 2H), 7.07 (m, 1H), 4.56, 4.45, 4.15 (m, m, m, 2H), 3.15 (m, 2H), 2.71, 2.52 (s, s, 3H), 2.65 (m, 2H), 1.80-0.80 (m, 13H)

Mass (FAB, m / e): 477 (M ⁺ +1)

Preparation Example 6

Synthesis of Methyl Ester

The reaction was carried out in the same manner as in Preparation Example 4 using 0.15 g (0.48 mmol) of the compound obtained in Preparation Example 3, except for 5-chlorosulfonyl-naphthalene-1-carboxylic acid methyl ester instead of 2-naphthalenesulfonylchloride. 0.21 g (84% yield) of the title compound was purified using.

¹ H NMR (CDCl ₃ , ppm) δ: 9.20 (d, 1H), 8.84 (t, 1H), 8.27 (m, 2H), 7.72 (t, 1H), 7.63 (t, 1H), 7.18 (d, 1H), 6.62 (d, 1H), 6.09 (dd, 1H), 4.46, 4.35, 4.28, 3.87 (m, m, m, m, 2H), 4.03 (s, 3H), 3.02 (m, 2H), 2.52, 2.46 (s, s, 3H), 1.80- 0.90 (m, 8H)

Mass (FAB, m / e): 526 (M ⁺ +1)

Example 5

Synthesis of Methyl Ester

The reaction was carried out in the same manner as in Example 1 using 0.20 g (0.38 mmol) of the compound obtained in Preparation Example 6 to obtain 0.082 g (yield 40%) of the title compound.

¹ H NMR (CD ₃ OD, ppm) δ: 9.08 (m, 1H), 8.84 (m, 1H), 8.22 (m, 2H), 7.69 (m, 2H), 7.45 (t, 1H), 6.83 (m , 1H), 4.52, 4.36, 4.12 (m, m, m, 2H), 4.00 (s, 3H), 3.10 (m, 2H), 2.70, 2.55 (s, s, 3H), 1.80-0.80 (m, 8H)

Mass (FAB, m / e): 543 (M ⁺ +1)

Preparation Example 7

Synthesis of 2- (t-butyloxycarbonylamino) -N-cyclopentyl-N-methyl-acetamide

22 g of N-methylmorpholine and 13.7 g of isopropylchloroformate were added while maintaining a solution of 17.5 g of N- (t-butyloxycarbonyl) glycine in 200 ml of tetrahydrofuran while being cooled at -20 ° C. After stirring for 1 min, 13.5 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, and diluted with ethyl acetate to the residue from which the solvent was removed under reduced pressure, followed by washing 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 give 23.8 g of the title compound.

¹ H NMR (CDCl ₃ , ppm) δ: 4.95 (m, 1H), 4.00 (d, 1H), 3.93 (d, 1H), 2.85 (d, 3H), 1.91-1.39 (m, 17H)

Preparation Example 8

synthesis

2.1 g of diisopropylamine and 2.33 g of N, N, N ', N'-tetramethylethylenediamine were added to 100 ml of tetrahydrofuran. The solution was cooled to -78 ° C, and 13 ml of butyllithium (1.6 M solution) was added. Slowly added and stirred for 30 minutes, and 2.56 g of the compound obtained in Preparation Example 7 was added to the reaction solution. The reaction solution was raised to room temperature over 1 hour, cooled to -78 ° C again, and 1.88 g of ethyl-5-chloromethyl-2-furancarboxylate was added thereto, followed by raising to room temperature over 2 hours. The solvent was removed from the reaction solution under reduced pressure, ethyl acetate was added to the residue, the mixture was diluted, and washed sequentially with 1N aqueous hydrochloric acid solution and saturated aqueous sodium bicarbonate solution. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure, and then the residue was purified by column chromatography [eluent: ethyl acetate / hexane (3/7, volume ratio)] to give 2.15 g of the purified title compound.

¹ H NMR (CDCl ₃ , ppm) δ: 7.05 (s, 1H), 6.17 (s, 1H), 4.82 (m, 2H), 4.0 (q, 2H), 2.97 (m, 2H), 2.71 (d, 3H), 1.87-0.72 (m, 20H)

Mass (FAB, m / e): 409 (M ⁺ +1)

Preparation Example 9

synthesis

2.04 g of the compound obtained in Preparation Example 8 was added to 30 ml of a hydrochloric acid solution saturated in ethyl acetate, 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 1.5 g of N-methylmorpholine and 1.1 g of 2-naphthalenesulfonyl chloride were added to the dissolved solution, followed by stirring at room temperature for 3 hours. After removal of the solvent under reduced pressure, the residue was diluted with methyl acetate and washed sequentially with IN aqueous hydrochloric acid solution and saturated aqueous sodium bicarbonate solution. The organic layer was dried over anhydrous sodium sulfate, filtered and dried in vacuo to yield 2.45 g of the title compound.

¹ H NMR (CDCl ₃ , ppm) δ: 8.50 (d, 1H), 8.0-7.68 (m, 8H), 7.00 (d, 1H), 6.30 (d, 1H), 6.08 (dd, 1H), 4.78 ( q, 1H), 4.32 (q, 2H), 3.97 (q, 1H), 3.00 (m, 2H), 2.41 (d, 3H), 1.68-0.50 (m, 11H)

Mass (FAB, m / e): 499 (M ⁺ +1)

Preparation Example 10

synthesis

30 ml of methanol and 20 ml of ammonia water were added to 2.05 g of the compound obtained in Preparation Example 9, and the reaction solution was stirred at room temperature for 10 hours, and then dried under vacuum. The dried residue was dissolved in 10 ml of chloroform and 10 ml of phosphorus oxychloride and heated to reflux for 3 hours. The reaction solution was cooled and concentrated under reduced pressure, and then the residue was purified by column chromatography [eluent: ethyl acetate / hexane (7/3, volume ratio)] to give 1.35 g of the title compound.

¹ H NMR (CDCl ₃ , ppm) δ: 8.41 (d, 1H), 8.08-7.59 (m, 7H), 6.95 (d, 1H), 6.61 (dd, 1H), 4.78-4.50 (m, 1H), 4.45 (m, 1H), 3.02 (m, 2H), 2.52 (d, 3H), 2.75-0.60 (m, 8H)

Mass (FAB, m / e): 452 (M ⁺ +1)

Preparation Example 11

synthesis

The reaction was carried out in the same manner using 1.47 g of 4-chloromethyl-2-methylthiothiazole instead of ethyl 5-chloromethyl-2-furancarboxylate in Preparation Example 8 to obtain 1.71 g of the title compound.

¹ H NMR (CDCl ₃ , ppm) δ: 6.82 (s, 1H), 5.60 (d, 1H), 5.15-4.79 (m, 2H), 3.03 (m, 2H), 2.73 (d, 3H), 2.67 ( s, 3H), 1.96-0.78 (m, 17H)

Mass (FAB, m / e): 368 (M ⁺ +1)

Preparation Example 12

synthesis

Using 1.6 g of the compound obtained in Preparation Example 11, the reaction was carried out in the same manner as in Preparation Example 9, to obtain 1.95 g of the title compound.

¹ H NMR (CDCl ₃ , ppm) δ: 8.49-7.69 (m, 7H), 6.95 (d, 1H), 6.20 (d, 1H), 4.75 (m, 1H), 4.30 (m, 1H), 3.01 ( m, 2H), 2.68 (d, 3H), 2.51 (d, 3H), 1.70-0.60 (m, 8H)

Mass (FAB, m / e): 490 (M ⁺ +1)

Preparation Example 13

synthesis

1.8 g of the compound obtained in Preparation Example 12 was suspended in 20 ml of methanol and 20 ml of distilled water, cooled to 4 ° C, and 7 g of oxone was added three times and stirred at room temperature for 3 hours. The reaction solution was concentrated under reduced pressure, diluted with ethyl acetate and the residue was washed with saturated brine. The organic layer was dried over anhydrous sodium sulfate, filtered and dried under vacuum. 20 ml of dimethyl sulfoxide was added to the dried residue, and the reaction solution to which 0.3 g of cyano sodium was added was heated to 150 ° C., stirred for 30 minutes, and cooled to room temperature. The reaction solution was diluted with ethyl acetate and washed three times with saturated brine, and then the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The concentrated solution was subjected to column chromatography [eluant: ethyl acetate / hexane (7/3, volume ratio)] to give 1.1 g of the title compound.

¹ H NMR (CDCl ₃ , ppm) δ: 8.45 (m, 8H), 6.29 (dd, 1H), 4.64 (m, 1H), 4.35 (m, 1H), 3.15 (m, 2H), 2.61 (d, 3H), 1.90-0.65 (m, 8H)

Mass (FAB, m / e): 469 (M ⁺ +1)

Example 6

synthesis

The reaction was carried out in the same manner as in Example 1 using 0.5 g of the compound obtained in Preparation Example 10 to obtain 0.18 g of the title compound.

¹ H NMR (CD ₃ OD, ppm) δ: 8.53-7.40 (m, 9H), 6.71 (d, 1H), 4.78 (m, 1H), 4.31 (m, 1H), 3.18 (m, 2H), 2.62 (d, 3H), 1.82-0.72 (m, 8H)

Mass (FAB, m / e): 469 (M ⁺ +1)

Example 7

synthesis

The reaction was carried out in the same manner as in Example 3 using 0.5 g of the compound obtained in Preparation Example 10 to obtain 0.27 g of the title compound.

¹ H NMR (CD ₃ OD, ppm) δ: 8.81-7.51 (m, 9H), 6.68 (d, 1H), 4.52 (m, 1H), 4.21 (m, 1H), 3.31 (m, 2H), 2.57 (d, 3H), 1.79-0.84 (m, 8H)

Mass (FAB, m / e): 484 (M ⁺ +1)

Example 8

synthesis

The reaction was carried out in the same manner as in Example 1 using 0.5 g of the compound obtained in Preparation Example 13 to obtain 0.14 g of the title compound.

¹ H NMR (CD ₃ OD, ppm) δ: 8.49-7.71 (m, 8H), 4.84 (m, 1H), 4.36 (m, 1H), 3.49 (m, 2H), 2.65 (d, 3H), 1.87 -0.75 (m, 8H)

Mass (FAB, m / e): 485 (M ⁺ +1)

Example 9

synthesis

The reaction was carried out in the same manner as in Example 3 using 0.5 g of the compound obtained in Preparation Example 13 to obtain 0.23 g of the title compound.

¹ H NMR (CD ₃ OD, ppm) δ: 8.62-7.83 (m, 8H), 4.72 (m, 1H), 4.63 (m, 1H), 3.20 (m, 2H), 2.57 (d, 3H), 1.72 -0.83 (m, 8H)

Mass (FAB, m / e): 500 (M ⁺ +1)

Experimental Example 1 Inhibitory Activity of a Thrombin Inhibitor

The inhibitory activity against thrombin of the compound according to the present invention was measured as the thrombin inhibition equilibrium constant (Ki).

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 thrombin inhibitor compound according to the present invention was dissolved in dimethyl sulfoxide to a concentration of 10 mg / ml, and then diluted with the buffer solution at concentrations of 0.1 mg / ml, 0.01 mg / ml, and 0.001 mg / ml. After the preparation was taken so that the amount of inhibitor was between 0 and 10 μg, the total volume was added to the cuvette with 100 μl of Tris buffer solution.

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 obtaining the initial velocity Vi from the slope within 30 seconds of the initial reaction time in each spectrum, an inverse graph of the initial velocity versus the inhibitor concentration (1 / Vi) is shown. After calculating the first equation that satisfies the points on the graph, the inhibitory constant 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, [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 equilibrium constant Ks used the same solution as the same solution used to obtain 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 left at room temperature for 15 minutes. Then, 225 µl of 0.1 mM substrate solution was added thereto. 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 various 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.

Thrombin inhibitory activity of the inhibitor according to the present invention measured according to the above-described method is shown in Table 1 as Ki value and Ks value.

TABLE 1

Inhibitory Activity of the Compounds of the Invention on Thrombin

Compound (Example Number) Inhibitory activity against thrombin Compound (Example Number) Inhibitory activity against thrombin One Ki = 4.6 nM 6 Ki = 45.7 nM 2 Ki = 45 nM 7 Ki = 181 nM 3 Ki = 200 nM 8 Ki = 204 nM 4 Ks = 0.62 nM 9 Ki = 430 nM 5 Ki = 24.3 nM

From the above experimental results, it can be seen that the compound of the present invention exhibits high inhibitory activity on thrombin.

Experimental Example 2 Inhibition of Thrombosis in Animals

Inhibition ability on the thrombus formation of the compound according to the present invention was measured according to the method described below.

Experiment Method:

Animals were male Sprague Dawley rats of 250-300 g body weight, which were bred using commercial standard feed at a temperature of 20-22 ° C, light and dark at 12-hour intervals. . First, rats were anesthetized by intraperitoneal injection of urethane at a dose of 1.3 g / kg body weight. The abdomen of the rat was excised to expose the inferior vena cava, and then sutures were placed over the vein just below the left renal vein and 1.6 cm below. The compound of Example 4 was injected at a dose of 1 mg / kg body weight through the left femoral vein and 5 minutes later, thromboplastin was also injected through the femoral vein. Subsequently, the sutures placed over 30 seconds later were ligated. Fifteen minutes after the suture was ligated, the veins were removed and the thrombus generated was taken and weighed.

Experiment result:

Inhibition of venous thrombogenesis by the test drug was calculated by comparing the degree of thrombus formation of the control group to which the same dose of 10% HPCD (hydroxypropyl β-cyclodextrin) solution was administered. As a result, the compound of Example 4, the test drug, inhibited the thrombogenesis by about 60% compared to the control group, and the result was about 1.5 times higher than that of the compound of Formula 5, a known compound described above. It shows an inhibitory effect.

Claims (9)

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

In the above formula, X and Y each independently represent O, S, NH, N or CH, R ¹ represents substituted or unsubstituted aryl, R ² and R ³ each independently represent lower alkyl or cycloalkyl, R ⁴ represents hydrogen, lower alkyl or amino. 2. A compound according to claim 1, wherein X represents O, S or N, Y represents CH when X is O and S, S represents S when X is N, and R ¹ is substituted or unsubstituted phenyl or Naphthyl, R ² and R ³ each independently represent methyl or cyclopentyl, and R ⁴ represents hydrogen, methyl or amino. The compound of claim 2, wherein X represents O, S or N, Y represents CH when X is O and S, S represents X when X is N, and R ¹ is lower alkyl or lower alkoxycarbonyl Phenyl or naphthyl unsubstituted or substituted by R ² and R ³ each independently represent methyl or cyclopentyl, and R ⁴ represents hydrogen, methyl or amino. A compound according to claim 1, wherein (S)-(S) -3- [5-amidino-thiophen-2-yl] -N-cyclopentyl-N-methyl-2- (4-propyl-benzenesulfonylamino ) -Propionamide, (S) -5- [2- [5-amidino-thiophen-2-yl] -1- (cyclopentyl-methyl-carbamoyl) -ethylsulfamoyl] -naphthalene-1-carboxylic acid Methyl ester, (R, S) -3- (2-amidinothiazol-4-yl) -N-cyclopentyl-N-methyl-2- (2-naphthalenesulfonylamino) -propionamide and (R, S)-3- Compound selected from the group consisting. 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 and Y each independently represent O, S, NH, N or CH, R ¹ represents substituted or unsubstituted aryl, R ² and R ³ each independently represent lower alkyl or cycloalkyl, R ⁴ represents hydrogen, lower alkyl or amino. The method of claim 5 wherein the reaction is carried out in the presence of a solvent. The method of claim 6 wherein the solvent is an alcoholic 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. The composition of claim 8 formulated as an oral dosage form.