KR20010002891A - Thrombin inhibitor which has aryl or heteroaryl group and process of preparation thereof - Google Patents

Abstract

PURPOSE: A thrombin repressor having aryl or hetero-aryl group that has high absorbility when administered orally is provided to prevent blood coagulation or to cure thrombosis. CONSTITUTION: A thrombin repressor containing aryl or hetero-aryl group, pharmacologically acceptable salt, hydrate, isomer thereof are characterized by formula (1), in the formula, n is an integer of 0-2, m is an integer of 0-3, A is H, -SO2R¬1, -CO2R¬2, -(CH2)1-3CO2H or-COR¬1, here R¬1 is C1-C6 alkyl, C3-C7 cycloalkyl, aralkyl, aryl, -NHR¬3(R¬3 is H or C1-C4 alkyl), R¬2 is C1-C6 alkyl, C3-C7 cycloalky, aralkyl, aryl, B is phenyl, naphthyl, biphenyl, heteroaryl, C1-C6 alkyl, C3-C7 cycloalkyl, C5-C12 dicycloalkyl, C11-C16 tricycloalkyl which is substituted or unsubstituted by at least one substituent selected from the group of C1-C4 alkyl, halogen, hydroxy, C1-C4 alkyloxy, -CO2-C1-4 alkyl, amino or B is -CH(R¬4)2, R¬4 is phenyl, hetero-aryl, C1-C4 alkyl, C3-C7 cycloalkyl which is substituted or unsubstituted by at least one substituent selected from the group of C1-C4 alkyl, halogen, hydroxy, C1-C4 alkyloxy, -CO2-, C1-C4 alkyl, amino, C is aryl group or hetero-aryl group of five membered ring or six membered ring, which is substituted or unsubstituted by at least one substituent selected from the group of C1-C4 alkyl, halogen, hydroxy, amino.

Description

Thrombin inhibitor which has aryl or heteroaryl group and process of preparation

The present invention relates to a novel thrombin inhibitor and a method for preparing the same, and more particularly, a compound represented by the following general formula (1) having excellent antithrombotic inhibitory effect and high oral absorption, pharmaceutically acceptable salts, hydrates thereof, It relates to solvates or isomers 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.

[Formula 1]

In the above formula,

n represents an integer of 0 to 2,

m represents an integer of 0 to 3,

A is hydrogen, -SO₂R^One, -CO₂R²,-(CH₂)_1-3CO₂H or -COR^OneIndicates,

From here

R ¹ represents C _1-6 alkyl, C _3-7 cycloalkyl, aralkyl, aryl, or —NHR ³ (R ³ is hydrogen or C _1-4 alkyl),

R ² represents C _1-6 alkyl, C _3-7 cycloalkyl, aralkyl, or aryl,

B is phenyl, naph unsubstituted or substituted by one or more substituents selected from the group consisting of C _1-4 alkyl, halogen, hydroxy, C _1-4 alkyloxy, —CO ₂ -C _1-4 alkyl and amino Butyl, biphenyl, heteroaryl, C _1-6 alkyl, C _3-7 cycloalkyl, C _{5-12 dicycloalkyl} , C _11-16 tricycloalkyl, or —CH (R ⁴ ) ₂ ; Wherein R ⁴ is unsubstituted or substituted by one or more substituents selected from the group consisting of C _1-4 alkyl, halogen, hydroxy, C _1-4 alkyloxy, —CO ₂ -C _1-4 alkyl and amino Phenyl, heteroaryl, C _1-4 alkyl, C _3-7 cycloalkyl.

C represents a 5 or 6 membered aryl or hetero aryl group unsubstituted or substituted by one or more substituents selected from the group consisting of C _1-4 alkyl, halogen, hydroxy, and amino.

The term "alkyl" in the definition of the substituent of the compound of formula 1 according to the invention, when used alone or in combination with "alkyloxy" means a straight or branched chain hydrocarbon radical, respectively, and the term "cycloalkyl" It means cyclic alkyl including cyclohexyl.

The term "aryl" refers to a 6-14 membered monocyclic, bicyclic or tricyclic aromatic group, and the term "heteroaryl" refers to monocyclic or non-containing heteroatoms selected from oxygen, nitrogen and sulfur. It means a cyclic aromatic group.

The term "dicycloalkyl" refers to a saturated ring having two or more atoms in common and consisting of two rings, and the term "tricycloalkyl" refers to a saturated ring consisting of three rings.

In general, it is known that various complex enzyme reactions are involved in the coagulation process. The final step involves the conversion of prothrombin to thrombin. Thrombin generated in this process activates platelets, converts fibrinogens to fibrin, and the like, and the fibrin is converted into a polymer by a polymerization reaction and crosslinked by activated blood factor XIII to become insoluble coagulation. Thrombin also plays a role in activating the blood factors V and VIII involved in the coagulation process, further accelerating the coagulation reaction. Thus, inhibitors of thrombin can act as an effective anticoagulant, while inhibiting platelet activity and preventing fibrin production and stabilization, thereby preventing and treating various thrombosis.

Heparin, the most commonly used anticoagulant for injection, is based on a mechanism that first binds to antithrombin III and then inhibits thrombin. Therefore, heparin is not only effective in arterial thrombosis but also has low stability associated with bleeding. In addition, coumarin, which is most commonly used as an oral anticoagulant, is a vitamin K antagonist, which is very late (6-24 hours) after taking it, and, like heparin, bleeding cannot be ruled out. There is a difficulty to observe.

The study of small molecule thrombin inhibitors has been very popular as a thrombotic therapeutic agent, and Argatroban of Formula 2 has already been commercialized in Japan in 1990 (see US4258192; US4201863; Biochemistry 1984, 23, 85-90). . However, this compound is used only as an injection and has no activity for oral administration.

The emergence of small molecule thrombin inhibitors began with the study of the structure of D-Phe-Pro-Arg, which mimics the sequence of fibrinogen, a natural substrate of thrombin, and the tripeptide derivatives based on this have been studied the most. Earlier studies have found that many compounds contain argininal at the P1 position, and compounds of formula 3 (Epegatran: Effegatran) are representative and proved to be effective thrombin inhibitors (see Eli Lilly, J. Med). Chem. 1990, 33, 1729; Circulation 1994, 90, I-231).

D-Phe-Pro-Agmatine and its analogs, which lack aldehydes or similar nucleophilic groups at the P1 position, were then studied, typically Inogatrran of Formula 4 and Melagatran (Astra, WO9311152, 1993; Thromb. Haemost. 1995, 73, 1325; WO9429336, 1994; Thromb. Haemost. 1998, 79, 110). These thrombin inhibitors are being developed as anticoagulants for injection by Astra of Switzerland.

Accordingly, the present inventors intensively studied to develop a new thrombin inhibitor having excellent thrombin inhibitory activity and improving each of the disadvantages of the compounds of Formulas 2 to 5, and as a result, the compounds of Formula 1 defined above As a thrombin inhibitor containing heteroaryl or an aryl group such as low basic imidazole, it has been found that the concentration absorbed into the blood upon oral administration meets the intended purpose and the present invention has been completed.

It is an object of the present invention to provide a compound of formula (I), a pharmaceutically acceptable salt, hydrate, solvate or isomer thereof that not only has excellent thrombin inhibitory activity but also has excellent absorption upon oral administration:

[Formula 1]

In the above formula,

n represents an integer of 0 to 2,

m represents an integer of 0 to 3,

A is hydrogen, -SO₂R^One, -CO₂R²,-(CH₂)_1-3CO₂H or -COR^OneIndicates,

From here

R ¹ represents C _1-6 alkyl, C _3-7 cycloalkyl, aralkyl, aryl, or —NHR ³ (R ³ is hydrogen or C _1-4 alkyl),

R ² represents C _1-6 alkyl, C _3-7 cycloalkyl, aralkyl, or aryl,

B is phenyl, naph unsubstituted or substituted by one or more substituents selected from the group consisting of C _1-4 alkyl, halogen, hydroxy, C _1-4 alkyloxy, —CO ₂ -C _1-4 alkyl and amino Butyl, biphenyl, heteroaryl, C _1-6 alkyl, C _3-7 cycloalkyl, C _{5-12 dicycloalkyl} , C _11-16 tricycloalkyl, or —CH (R ⁴ ) ₂ ; Wherein R ⁴ is unsubstituted or substituted by one or more substituents selected from the group consisting of C _1-4 alkyl, halogen, hydroxy, C _1-4 alkyloxy, —CO ₂ -C _1-4 alkyl and amino Phenyl, heteroaryl, C _1-4 alkyl, C _3-7 cycloalkyl.

C represents a 5 or 6 membered aryl or hetero aryl group unsubstituted or substituted by one or more substituents selected from the group consisting of C _1-4 alkyl, halogen, hydroxy, and amino.

In the above definition of the substituent of the compound of formula 1 according to the invention, the term "alkyl" when used in combination with "alkyloxy" means a straight or branched chain hydrocarbon radical, the term "cycloalkyl" means cyclohexyl Means including cyclic alkyl.

The term "aryl" refers to a 6-14 membered monocyclic, bicyclic or tricyclic aromatic group, and the term "heteroaryl" refers to monocyclic or non-containing heteroatoms selected from oxygen, nitrogen and sulfur. It means a cyclic aromatic group.

The term "dicycloalkyl" refers to a saturated ring having two or more atoms in common and consisting of two rings, and the term "tricycloalkyl" refers to a saturated ring consisting of three rings.

Representative compounds of Formula 1 according to the present invention include the following compounds.

1. t-butyl (1R) -1-benzyl-2-[(2S) -2-({[3- (1H-imidazol-4-yl) -2-propynyl] amino} carbonyl) pyrroli Dinyl] -2-oxoethylcarbamate;

2. t-butyl (1R) -1-benzyl-2-oxo-2-[(2S) -2-({[3- (4-pyridinyl) -2-propynyl] amino} carbonyl) pyrroli Dinyl] ethyl carbamate;

3. (2S) -1-[(2R) -2-amino-3-phenylpropaneoyl] -N- [3- (4-pyridinyl) -2-propynyl] -2-pyrrolidinecarboxamide Trifluoroacetic acid;

4. t-butyl (1R) -1-benzyl-2-oxo-2-[(2S) -2-({[3- (1H-pyrazol-4-yl) -2-propynyl] amino} carbox Carbonyl) pyrrolidinyl] ethyl carbamate;

5. t-butyl (1R) -1-benzhydryl-2-[(2S) -2-({[3- (1H-imidazol-4-yl) -2-propynyl] amino} carbonyl) Pyrrolidinyl] -2-oxoethylcarbamate;

6. t-butyl (1R) -1-benzhydryl-2-[(2S) -2-({[3- (1-methyl-1H-imidazol-5-yl) -2-propynyl] amino } Carbonyl) pyrrolidinyl] -2-oxoethylcarbamate;

7. (2S) -1-{(2R) -2-[(benzylsulfonyl) amino] -3,3-diphenylpropaneoyl} -N- [3- (1H-imidazol-4-yl)- 2-propynyl] -2-pyrrolidinecarboxamide;

8. (2S) -N- [3- (1H-imidazol-4-yl) -2-propynyl] -1-{(2R) -2-[(methylsulfonyl) amino] -3,3- Diphenylpropane oil} -2-pyrrolidinecarboxamide;

9. (2S) -1-[(2R) -2- (acetylamino) -3,3-diphenylpropaneoyl] -N- [3- (1H-imidazol-4-yl) -2-propynyl ] -2-pyrrolidinecarboxamide;

10. Methyl (1R) -1-benzhydryl-2-[(2S) -2-({[3- (1H-imidazol-4-yl) -2-propynyl] amino} carbonyl) pyrroli Dinyl] -2-oxoethylcarbamate;

11. (2S) -1-{(2R) -2-[(aminosulfonyl) amino] -3,3-diphenylpropaneoyl} -N- [3- (1H-imidazol-4-yl)- 2-propynyl] -2-pyrrolidinecarboxamide;

12. t-butyl (1R) -2,2-dicyclohexyl-1-{[(2S) -2-({[3- (1H-imidazol-4-yl) -2-propynyl] amino} Carbonyl) pyrrolidinyl] carbonyl} ethylcarbamate;

13. (2S) -1-[(2R) -2-Amino-3,3-dicyclohexylpropaneoyl] -N- [3- (1H-imidazol-4-yl) -2-propynyl]- 2-pyrrolidinecarboxamide;

14. Methyl (1R) -2,2-dicyclohexyl-1-{[(2S) -2-({[3- (1H-imidazol-4-yl) -2-propynyl] amino} carbonyl ) Pyrrolidinyl] carbonyl} ethyl carbamate;

15. 2-({(1R) -1- (cyclohexylmethyl) -2-[(2S) -2-({[3- (1H-imidazol-4-yl) -2-propynyl] amino} Carbonyl) pyrrolidinyl] -2-oxoethyl} amino) acetic acid;

16. (2S) -N- [3- (6-amino-3-pyridinyl) -2-propynyl] -1-{(2R) -2-[(methylsulfonyl) amino] -3,3- Diphenylpropane oil} -2-pyrrolidinecarboxamide

The compounds of the general 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 R or S isomers, racemic compounds, diastereomeric mixtures and individual diastereoisomers, all of these isomeric forms being included within the scope of the invention. do.

The compound of formula 1 according to the present invention is an aryl halide or heteroaryl halide corresponding to the acetylene compound of formula 6, which corresponds to C of formula 1 at room temperature to elevated temperature, preferably from 25 to 70 ° C. in the presence of copper iodide and palladium catalyst The final compound may be prepared by reacting with the compound. Palladium catalysts that can be used at this time include bis (triphenylphosphine) palladium (II) chloride, tetrakis (triphenylphosphine) palladium (0), palladium (II) acetate and the like, but is limited to these no.

In the above formula

n, m, A, and B are as defined above.

On the other hand, the compound of Formula 6 used as a starting material in the method for preparing a compound of Formula 1 is prepared by the deprotection reaction, coupling reaction, amide bond reaction with propazylamine of the dipeptide compound of Formula 7 do or,

Wherein n, m and B are as defined above and P is an aminoprotecting group.

It can be manufactured and used according to the method shown in Scheme 1 or 2.

First, in the acetylene compound represented by Chemical Formula 6, n is 0, m is 2, and B is -CH (Ph) ₂ , according to the method of Scheme 1 below using an amino acid compound having a protecting group at the N-terminus as a starting material. Formation of dipeptide compounds, deprotection grouping, coupling reactions, hydrolysis and amide linkage reactions, or the formation of dipeptide compounds, hydrolysis, amide bond reactions, deprotection grouping and coupling according to Scheme 2 It can be prepared by the reaction:

In the above formula,

EDC stands for 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride,

HOBT stands for 1-hydroxybenzotriazole,

DMF stands for dimethylformamide and is used hereinafter with the same meaning,

P represents an aminoprotecting group.

Known coupling agents that can be used for the coupling reaction in the reaction include dicyclohexylcarbodiimide (DCC), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide (EDC), 1,1 ' -Carbodiimides such as dicarbonyldiimidazole (CDI) are used in a mixed state with 1-hydroxybenzotriazole (HOBT), or bis- (2-oxo-3-oxazolidinyl) -force Finic acid chloradide (BOP-Cl), diphenylphosphoryl azide (DPPA) and the like can be used, but are not limited thereto. In addition to dimethylformamide, conventional reactions such as dimethylacetamide, tetrahydrofuran, methylene chloride, chloroform, and the like may be used in the reaction, but are not limited thereto.

The carboxylic acid compound used in the amide coupling reaction in the above reaction may be used as it is, but it may be 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. have. 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-nor Butene-2 ', 3'-dicarboxyimide-derived esters, 2,4,5-trichlorophenol-derived esters, and the like, but are not limited to these.

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

Ki = [E] x [I] / [EI]

In the above formula

[E] is the concentration of enzyme that is not bound to the inhibitor,

[I] is the concentration of inhibitor that is not bound to the enzyme

[EI] is the concentration of the enzyme and inhibitor combination.

Since the dissociation constant Ki indicates the degree of dissociation between the enzyme and the thrombin inhibitor compound, the smaller the dissociation constant value, the greater the binding of the inhibitor to the enzyme, and thus the higher the inhibitory activity. These dissociation constants can be obtained by reacting with a specific substrate that exhibits 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 that is colored when hydrolyzed under the action of thrombin. Chromozyme TH is hydrolyzed by thrombin to produce a 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 a result of measuring the inhibitory activity of the compound of Formula 1 according to the present invention with respect to thrombin, it was confirmed that the compound of the present invention exhibited excellent thrombin inhibitory activity (see Experimental Example 1). In addition, the compound of formula 1 has the advantage that can exhibit an inherent effect even by oral administration (see Experimental Example 2). Therefore, the compounds of the present invention can be usefully used for the prevention of blood clotting and the treatment of thrombosis.

Another object of the present invention is to provide a pharmaceutical composition for preventing blood clotting and treating thrombosis, which contains a compound of formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient together with a pharmaceutically acceptable carrier.

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 0.001-10 mg / kg body weight, but specific dose levels for individual patients may be used. It may vary depending on the specific compound, the weight of the patient, sex, health condition, diet, the time of administration of the drug, the method of administration, the rate of excretion, the mixing of the drug and the severity of the disease.

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

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

Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. 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 compound of formula 1 according to the invention with one or more inert diluents such as sucrose, lactose, starch and the like and a carrier such as a lubricant such as magnesium stearate, a disintegrant, a binder and the like. .

One of the great features of the compound of formula 1 according to the present invention is that the pharmaceutical composition containing the same has an effect even when formulated orally as an oral preparation. Results of pharmacokinetic experiments demonstrated that oral administration of the pharmaceutical composition of the present invention confirms that the concentration of the drug is maintained in the blood for a long time. Therefore, it is more useful in that it can be effectively used as an oral preparation, unlike conventional thrombin inhibitors.

On the other hand, when clinically administering the compound of the present invention to obtain the desired anticoagulant 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 will be described in more detail based on the following Examples and Experimental Examples. However, these Examples and Experimental Examples are only for better understanding of the present invention, and the scope of the present invention is not limited by them in any sense.

Explanations of abbreviations and terms used in the names of compounds are as follows.

Boc: tert-Butoxycarbonyl

Bn: Benzyl

Cha: Cyclohexylalanine

Dcha: Dicyclohexylalanine

Dpa: Diphenylalanine

Imd: Imidazole

Phe: Phenylalanine

Pro: Proline

TFA: Trifluoroacetic acid

Tr: Trityl

Example 1

t-butyl (1R) -1-benzyl-2-[(2S) -2-({[3- (1H-imidazol-4-yl) -2-propynyl] amino} carbonyl) pyrrolidinyl] Synthesis of 2-oxoethylcarbamate hydrochloride

a) Preparation of Boc-D-Phe-Pro-NHCH ₂ CCH

1.5 g (4.1 mmol) of Boc-D-Phe-Pro-OH was dissolved in 50 mL of dimethylformamide. After cooling to 0 ° C. 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, 1.6 g (EDC, 8.2 mmol) and 1-hydroxybenzotriazole hydrate, 0.8 g (HOBT, 6.2 mmol) Was added and stirred until it dissolved completely. To this solution was added 0.42 mL (H ₂ NCH ₂ CCH, 6.2 mmol) and 2.9 mL (20.8 mmol) N-methylmorpholine. Slowly raised to room temperature and stirred for 2 hours. After completion of the reaction, distillation under reduced pressure was performed to remove volatiles. The resulting residue was diluted with ethyl acetate and washed sequentially with saturated aqueous sodium hydrogen carbonate solution, diluted hydrochloric acid, and saturated brine. Dried over anhydrous magnesium sulfate, filtered and concentrated. The residue was purified by column chromatography using ethyl acetate / hexane (1/1 volume ratio) eluent to afford 1.17 g (yield: 73%) of the title compound.

¹ H NMR (CDCl ₃ ): 1.48 (s, 9H), 1.60 (m, 2H), 1.85 (m, 1H), 2.20 (s, 1H), 2.27 (m, 1H), 2.60 (q, 1H), 3.01 (q, 2H), 3.62 (m, 1H), 3.90 (m, 1H), 4.08 (m, 1H), 4.50 (m, 2H), 5.28 (m, 1H), 7.17-7.48 (m, 5H)

FAB MS: 400 [M + 1] ⁺

b) Preparation of Boc-D-Phe-Pro-NHCH ₂ CC-4- (1-Tr) -imd

0.1 g (0.25 mmol) of the compound obtained in Preparation a) was dissolved in 10 mL of dimethylformamide, and 0.11 g (0.25 mmol) of 4-iodo-1-trityl-1H-imidazole was added to tetrakis (triphenylphosphate). Pin) palladium (0) 15 mg [Pd (PPh ₃ ) ₄ , 0.01 mmol], 5 mg copper iodide (CuI, 0.02 mmol) and 0.35 mL (2.5 mmol) triethylamine were added together and 15 at 50 ° C. under a nitrogen stream. Stir for hours. After completion of the reaction, the mixture was concentrated by distillation under reduced pressure. The residue was purified by column chromatography using ethyl acetate / hexane (3/1 volume ratio) eluent to afford 0.11 g (yield: 60%) of the title compound.

¹ H NMR (CDCl ₃ ): 1.40 (s, 9H), 1.55 (m, 1H), 1.78 (m, 1H), 1.91 (m, 1H), 2.40 (m, 1H), 2.70 (m, 1H), 2.92 (m, 2H), 3.60 (m, 2H), 4.28 (m, 2H), 4.59 (m, 1H), 7.07-7.53 (m, 22H).

FAB MS: 708 [M + 1] ⁺

c) Preparation of Boc-D-Phe-Pro-NHCH ₂ CC-4-imd.HCl

0.11 g (0.15 mmol) of the compound obtained in Preparation b) and 21 μl (0.28 mmol) of trifluoroacetic acid were dissolved in 2 mL of dichloromethane, followed by addition of 54 μl of triisopropylsilane (i-Pr ₃ SiH, 0.28 mmol). After stirring at room temperature for 2 hours. After completion of the reaction, the mixture was distilled under reduced pressure, concentrated, and purified by HPLC to obtain trifluoroacetic acid salt of the title compound. This was dissolved in ethyl acetate and hydrochloric acid saturated in ethyl acetate was added dropwise to obtain a precipitate. It was filtered and dried to give 61 mg (yield: 81%) of the title compound.

¹ H NMR (CDCl ₃ ): 1.49 (s, 9H), 1.52-1.72 (m, 2H), 1.80 (m, 1H), 2.20 (m, 1H), 2.53 (m, 1H), 3.00 (m, 2H ), 3.62 (m, 1H), 4.19 (m, 2H), 4.48 (d, 2H), 5.45 (s, 1H), 7.10-7.28 (m, 5H), 7.48 (s, 1H), 7.61 (s, 1H).

FAB MS: 466 [M + 1] ⁺

Example 2

t-butyl (1R) -1-benzyl-2-oxo-2-[(2S) -2-({[3- (4-pyridinyl) -2-propynyl] amino} carbonyl) pyrrolidinyl] Synthesis of Ethyl Carbamate

The reaction was carried out in the same manner except in preparation b), except that 4-bromopyridine hydrochloride was used instead of 4-iodo-1-trityl-1H-imidazole, to obtain the title compound ( Yield: 79%).

¹ H NMR (CDCl ₃ ): 1.43 (s, 9H), 1.62 (m, 1H), 1.78 (m, 1H), 1.85 (m, 1H), 2.29 (m, 1H), 2.60 (m, 1H), 3.01 (m, 2H), 3.64 (m, 1H), 4.06-4.37 (m, 2H), 4.50 (m, 2H), 5.22 (m, 1H), 7.12-7.41 (m, 6H), 7.58 (s, 1H), 8.54 (s, 2H).

FAB MS: 477 [M + 1] ⁺

Example 3

(2S) -1-[(2R) -2-amino-3-phenylpropaneoyl] -N- [3- (4-pyridinyl) -2-propynyl] -2-pyrrolidinecarboxamide trifluor Synthesis of Roroacetate

0.13 g (0.27 mmol) of the compound obtained in Example 2 was dissolved in trifluoroacetic acid and stirred at room temperature for 30 minutes. After completion of the reaction, the mixture was concentrated by distillation under reduced pressure. The residue was purified by column chromatography using dichloromethane / methanol (9/1 volume ratio) eluent to afford 81 mg (yield: 50%) of the title compound.

¹ H NMR (CDCl ₃ ): 1.31 (m, 1H), 1.69 (m, 2H), 1.99 (m, 1H), 2.35 (m, 1H), 2.83 (m, 1H), 2.91 (m, 1H), 3.01 (m, 1H), 3.85 (m, 1H), 4.24 (m, 1H), 4.32 (m, 1H), 4.58 (m, 1H), 7.18-7.49 (m, 6H), 7.78 (s, 1H) , 8.60 (d, 2 H).

FAB MS: 377 [M + 1] ⁺

Example 4

t-butyl (1R) -1-benzyl-2-oxo-2-[(2S) -2-({[3- (1H-pyrazol-4-yl) -2-propynyl] amino} carbonyl) Synthesis of Pyrrolidinyl] ethyl Carbamate

The reaction was carried out in the same manner, except that in preparation b) of Example 1, 4-iodopyrazole was used instead of 4-iodo-1-trityl-1H-imidazole, to obtain the title compound ( Yield: 64%).

¹ H NMR (CDCl ₃ ): 1.40 (s, 9H), 1.51-1.74 (m, 2H), 1.83 (m, 1H), 2.20 (m, 1H), 2.69 (m, 1H), 3.01 (m, 2H ), 3.74 (m, 1H), 4.16 (m, 2H), 4.62 (m, 2H), 5.74 (d, 1H), 7.12-7.35 (m, 5H), 7.66 (d, 2H).

FAB MS: 466 [M + 1] ⁺

Example 5

t-butyl (1R) -1-benzhydryl-2-[(2S) -2-({[3- (1H-imidazol-4-yl) -2-propynyl] amino} carbonyl) pyrroli Dinyl] -2-Oxoethylcarbamate hydrochloride synthesis

a) Preparation of Boc-D-Dpa-Pro-NHCH ₂ CCH

The reaction was carried out on Boc-D-Dpa-Pro-OH (J. Med. Chem. 1997, 40, 1565) in the same manner according to Preparation a) of Example 1, to obtain the title compound (yield: 87 %).

¹ H NMR (CDCl ₃ ): 1.23-1.52 (m, 12H), 1.71 (m, 1H), 2.12 (s, 1H), 2.47 (m, 1H), 3.67 (m, 1H), 3.82 (m, 1H ), 4.11 (m, 1H), 4.32 (m, 2H), 4.91 (m, 2H), 7.10-7.52 (m, 10H),

FAB MS: 476 [M + 1] ⁺

b) Preparation of Boc-D-Dpa-Pro-NHCH ₂ CC-4-imd.HCl

The reaction was carried out for the compound obtained in Preparation a) in the same manner according to Preparation b, c) of Example 1 to give the title compound (Yield: 74%).

¹ H NMR (CDCl ₃ ): 1.23-1.49 (m, 11H), 1.70 (m, 1H), 2.12 (m, 1H), 2.57 (m, 1H), 3.68 (m, 1H), 4.10 (m, 2H ), 4.27 (d, 1H), 4.36 (d, 1H), 4.88 (m, 1H), 5.44 (m, 1H), 7.05-7.68 (m, 12H).

FAB MS: 542 [M + 1] ⁺

Example 6

t-butyl (1R) -1-benzhydryl-2-[(2S) -2-({[3- (1-methyl-1H-imidazol-5-yl) -2-propynyl] amino} carbox Carbonyl) pyrrolidinyl] -2-oxoethylcarbamate

0.1 mg (0.17 mmol) of the compound Boc-D-Dpa-Pro-NHCH ₂ CC-4-imd.HCl obtained in Example 5 was dissolved in 1 mL of dichloromethane, lowered to 0 ° C., and 35 mg of trimethyloxonium tetrafluoroborate. (Me ₃ OBF ₄ , 0.24 mmol) was added. After 20 minutes, concentrated under reduced pressure and purified by HPLC to give 60 mg (yield: 63%) of the title compound.

¹ H NMR (CDCl ₃ ): 1.25-1.68 (m, 12H), 1.70 (m, 1H), 2.52 (m, 1H), 3.65 (m, 4H), 4.11 (m, 1H), 4.35 (m, 3H ), 4.85 (m, 1 H), 4.98 (m, 1 H), 7.08-7.72 (m, 12 H).

FAB MS: 556 [M + 1] ⁺

Example 7

(2S) -1-{(2R) -2-[(benzylsulfonyl) amino] -3,3-diphenylpropaneoyl} -N- [3- (1H-imidazol-4-yl) -2- Propynyl] -2-pyrrolidinecarboxamide hydrochloride

a) Preparation of BnSO ₂ -D-Dpa-Pro-NHCH ₂ CCH

5 g (10.5 mmol) of Boc-D-Dpa-Pro-NHCH ₂ CCH obtained in Preparation a) of Example 5 were dissolved in 100 mL of methanol, to which 10 mL of acetylchloride was added dropwise. After 4 hours, the product was distilled under reduced pressure to remove volatiles and dried in vacuo to obtain 4 g of a compound having a Boc group removed (yield: 93%). 0.2 g (0.48 mmol) of this compound was taken up in 1 mL of dichloromethane, cooled to 0 ° C., and then 0.11 g of benzylsulfonylchloride (BnSO ₂ Cl, 0.57 mmol) and 0.23 mL (1.44 mmol) of triethylamine were added thereto. . After 2 hours, the mixture was distilled under reduced pressure to remove volatiles, and the residue was purified by column chromatography, eluting with ethyl acetate / hexane (2/1 volume ratio), to obtain 0.2 g (yield: 80%) of the title compound.

¹ H NMR (CDCl ₃ ): 1.34 (m, 2H), 1.45 (m, 1H), 1.79 (m, 1H), 2.10 (s, 1H), 2.61 (m, 1H), 3.64 (m, 1H), 3.91 (m, 2H), 4.18 (d, 1H), 4.24 (m, 2H), 4.37 (d, 1H), 4.91-5.18 (m, 2H), 7.18-7.50 (m, 15H).

FAB MS: 530 [M + 1] ⁺

b) Preparation of BnSO ₂ -D-Dpa-Pro-NHCH ₂ CC-4-imd.HCl

The reaction was carried out for the compound obtained in Preparation a) in the same manner according to Preparation b, c) of Example 1, to obtain the title compound (Yield: 35%).

¹ H NMR (CDCl ₃ ): 1.37 (m, 1H), 1.50 (m, 1H), 1.75 (m, 1H), 1.88 (m, 1H), 2.76 (m, 1H), 3.65 (m, 1H), 3.89-4.30 (m, 5H), 4.41 (m, 1H), 4.83 (m, 1H), 7.10 (m, 1H), 7.13-7.57 (m, 14H), 7.75 (m, 1H), 8.34 (m, 1H).

FAB MS: 596 [M + 1] ⁺

Example 8

(2S) -N- [3- (1H-imidazol-4-yl) -2-propynyl] -1-{(2R) -2-[(methylsulfonyl) amino] -3,3-diphenyl Propane oil} -2-pyrrolidinecarboxamide hydrochloride

In Preparation a) MeSO ₂ -D-Dpa-Pro-NHCH ₂ CCH was obtained using methylsulfonylchloride instead of benzylsulfonylchloride (yield: 90%), and for this compound The same reaction was carried out according to preparation b, c) to give the title compound (yield: 30%).

¹ H NMR (CDCl ₃ ): 1.43 (m, 2H), 1.81 (m, 1H), 1.99 (m, 1H), 2.70 (m, 1H), 2.83 (s, 3H), 3.68 (m, 1H), 4.15 (m, 3H), 4.44 (m, 1H), 4.97 (m, 1H), 7.05-7.61 (m, 12H).

FAB MS: 520 [M + 1] ⁺

Example 9

(2S) -1-[(2R) -2- (acetylamino) -3,3-diphenylpropaneoyl] -N- [3- (1H-imidazol-4-yl) -2-propynyl]- Synthesis of 2-pyrrolidinecarboxamide hydrochloride

In Preparation a) of Example 7, acetyl chloride (MeCOCl) was used instead of benzylsulfonylchloride to give MeOC-D-Dpa-Pro-NHCH ₂ CCH (yield: 85%), and for the compound of Example 1 The same reaction was carried out according to preparation b, c), to obtain the title compound (yield: 41%).

¹ H NMR (CDCl ₃ ): 1.44 (m, 2H), 1.73 (m, 4H), 2.02 (m, 1H), 2.55 (m, 1H), 3.80 (m, 1H), 4.01 (dd, 1H), 4.11 (dd, 1H), 4.20 (d, 1H), 4.49 (d, 1H), 5.10 (dd, 1H), 7.05-7.40 (m, 12H).

FAB MS: 484 [M + 1] ⁺

Example 10

Methyl (1R) -1-benzhydryl-2-[(2S) -2-({[3- (1H-imidazol-4-yl) -2-propynyl] amino} carbonyl) pyrrolidinyl] Synthesis of 2-oxoethylcarbamate hydrochloride

In Preparation a) MeO ₂ CD-Dpa-Pro-NHCH ₂ CCH was obtained using methylchloroformate (MeOCOCl) instead of benzylsulfonylchloride (yield: 88%) in Example a) for this compound. The same reaction was carried out according to preparation b, c) of 1 to give the title compound (yield: 38%).

¹ H NMR (CD ₃ OD): 1.48 (m, 1H), 1.63 (m, 1H), 1.80 (m, 2H), 2.97 (m, 1H), 3.55 (s, 3H), 3.82 (m, 1H) , 4.03 (m, 1H), 4.19 (m, 2H), 4.36 (dd, 1H), 5.15 (dd, 1H), 7.15-7.48 (m, 12H).

FAB MS: 500 [M + 1] ⁺

Example 11

(2S) -1-{(2R) -2-[(aminosulfonyl) amino] -3,3-diphenylpropaneoyl} -N- [3- (1H-imidazol-4-yl) -2- Propynyl] -2-pyrrolidinecarboxamide hydrochloride

a) Preparation of NH ₂ SO ₂ -D-Dpa-Pro-OMe

2.13 g (HCO ₂ H, 45 mmol) of formic acid was slowly added to a solution of 6.36 g of chlorosulfonyl isocyanate (OCNSO ₂ Cl, 45 mmol) in dichloromethane (25 mL) and stirred at room temperature for 1 hour. The solution was boiled for 3 hours, and then cooled to obtain a 1.8 N sulfamoyl chloride (H ₂ NSO ₂ Cl) solution. After cooling 2.5 g (6.47 mmol) of dichloromethane (100 mL) in D-Dpa-Pro-OMe.HCl to 0 ° C., 1.8 N sulfamoylchloride solution (6 mL) was added, followed by 2.7 mL of triethylamine. At this time, adjust the solution to be slightly basic and check with pH paper. After completion of the reaction, the solution was diluted with dichloromethane (40 mL), washed with brine, dried over anhydrous magnesium sulfate, filtered and concentrated. The residue was purified by column chromatography using ethyl acetate / hexane (2/1 volume ratio) eluent to afford 1.88 g (yield: 67%) of the title compound as purified.

¹ H NMR (CDCl ₃ ): 1.38 (m, 1H), 1.74 (m, 3H), 2.70 (m, 1H), 3.67 (s, 3H), 4.17 (m, 1H), 4.34 (d, 1H), 4.75 (m, 1H), 4.95 (dd, 1H), 5.11 (s, 2H), 5.29 (d, 1H), 7.18 (m, 6H), 7.20-7.38 (m, 4H).

FAB MS: 431 [M + 1] ⁺

b) Preparation of NH ₂ SO ₂ -D-Dpa-Pro-OH.

1.88 g (4.36 mmol) of the compound obtained in Preparation a) were suspended in 150 mL of methanol, to which 0.5 N aqueous lithium hydroxide solution (40 mL) was added, followed by stirring for 6 hours. Acidified to pH ~ 2 with 1N aqueous hydrochloric acid solution, concentrated by distillation under reduced pressure, and extracted three times with 100 mL of ethyl acetate. The extracted solution was washed with brine, dried over anhydrous magnesium sulfate, filtered and concentrated to give 1.5 g of the title compound (yield: 83%).

¹ H NMR (CD ₃ OD): 1.43 (m, 1H), 1.72-1.83 (m, 3H), 2.87 (m, 1H), 3.75 (m, 1H), 4.05 (m, 1H), 4.29 (d, 1H), 4.95 (d, 1H), 5.11 (s, 2H), 7.24 (m, 6H), 7.33 (m, 2H), 7.40 (m, 2H).

FAB MS: 417 [M + 1] ⁺

c) Preparation of NH ₂ SO ₂ -D-Dpa-Pro-NHCH ₂ CC-4-imd.HCl

The reaction was carried out for the compound obtained in Preparation b) in the same manner as in Example 1 to obtain the title compound (yield: 35%).

¹ H NMR (CDCl ₃ ): 1.49 (m, 2H), 1.8 (m, 1H), 2.07 (m, 1H), 2.75 (m, 1H), 3.68 (m, 1H), 4.07 (m, 1H), 4.15 (m, 1H), 4.32 (m, 2H), 5.10 (m, 1H), 5.25 (s, 2H), 6.95-7.64 (m, 12H).

FAB MS: 521 [M + 1] ⁺

Example 12

t-butyl (1R) -2,2-dicyclohexyl-1-{[(2S) -2-({[3- (1H-imidazol-4-yl) -2-propynyl] amino} carbonyl Synthesis of Pyrrolidinyl] carbonyl} ethylcarbamate Hydrochloride

The reaction was carried out on Boc-D-Dcha-Pro-OH (J. Med. Chem. 1997, 40, 1565) in the same manner according to Preparation a) of Example 1 to Boc-D-Dcha-Pro-NHCH ₂ CCH was obtained and the same reaction was carried out on the compound according to preparation b, c) of Example 1 to yield the title compound (yield: 32%).

¹ H NMR (CDCl ₃ ): 1.02-1.81 (m, 32H), 1.97-2.25 (m, 4H), 3.57 (m, 1H), 4.10 (m, 2H), 4.55 (m, 2H), 5.36 (m , 1H), 7.30 (s, 1H), 7.76 (s, 1H), 8.72 (s, 1H).

FAB MS: 554 [M + 1] ⁺

Example 13

(2S) -1-[(2R) -2-amino-3,3-dicyclohexylpropaneoyl] -N- [3- (1H-imidazol-4-yl) -2-propynyl] -2- Synthesis of Pyrrolidine Carboxamide Trifluoroacetate

0.10 g (0.17 mmol) of the compound obtained in Example 12 was dissolved in trifluoroacetic acid and stirred at room temperature for 30 minutes. After completion of the reaction, the mixture was concentrated by distillation under reduced pressure. The residue was purified by column chromatography using dichloromethane / methanol (9/1 volume ratio) eluent to afford 75 mg (yield: 83%) of the title compound.

¹ H NMR (CD ₃ OD): 1.03-1.48 (m, 11H), 1.52-1.92 (m, 12H), 1.98-2.25 (m, 4H), 3.63 (m, 1H), 3.78 (m, 1H), 4.18-4.50 (m, 3H), 5.12 (m, 1H), 7.65 (s, 1H), 8.64 (s, 1H).

FAB MS: 454 [M + 1] ⁺

Example 14

Methyl (1R) -2,2-dicyclohexyl-1-{[(2S) -2-({[3- (1H-imidazol-4-yl) -2-propynyl] amino} carbonyl) pi Synthesis of Lolidinyl] Carbonyl} ethylcarbamate Hydrochloride

The same reaction was carried out according to Preparation a) of Example 7 on Boc-D-Dcha-Pro-NHCH ₂ CCH obtained in Example 12, but using MeO ₂ CD-Dcha using methylchloroformate instead of benzylsulfonylchloride. -Pro-NHCH ₂ CCH was obtained (yield: 85%) and the same reaction was carried out on this compound according to preparation b, c) of Example 1 to give the title compound (yield: 33%).

¹ H NMR (CD ₃ OD): 1.02-1.45 (m, 12H), 1.52-1.83 (m, 11H), 1.95-2.22 (m, 4H), 3.59 (m, 1H), 3.64 (s, 3H), 4.02 (m, 1H), 4.28 (m, 1H), 4.36 (m, 1H), 4.61 (m, 1H), 5.07 (m, 1H), 7.73 (s, 1H), 8.88 (s, 1H).

FAB MS: 512

Example 15

2-({(1R) -1- (cyclohexylmethyl) -2-[(2S) -2-({[3- (1H-imidazol-4-yl) -2-propynyl] amino} carbonyl Synthesis of Pyrrolidinyl] -2-oxoethyl} amino) acetic acid

a) Preparation of t-BuO ₂ CCH ₂ -D-Cha-Pro-OMe

0.3 g (0.94 mmol) of D-Cha-Pro-OMe.HCl was dissolved in 3 mL of acetonitrile, 0.21 mL of t-butyl bromoacetate (BrH ₂ CCO ₂ t-Bu, 1.41 mmol) and 0.65 mL (4.7 of triethylamine) mmol) was added and stirred for 36 h. After completion of the reaction, the mixture was concentrated by distillation under reduced pressure. The residue was purified by column chromatography using ethyl acetate / hexane (1/2 volume ratio) eluent to afford 0.28 g (yield: 76%) of the title compound.

¹ H NMR (CDCl ₃ ): 0.75-2.13 (m, 24H), 2.45 (m, 2H), 3.21-3.57 (m, 5H), 3.70 (s, 3H), 4.12 (m, 1H), 4.58 (m , 1H).

FAB MS: 397 [M + 1] ⁺

b) Preparation of t-BuO ₂ CCH ₂ -D-Cha-Pro-NHCH ₂ CC-4- (1-Tr) -imd

0.28 g (0.7 mmol) of the compound obtained in Preparation a) was obtained in the same manner as in Preparation 11), to yield 0.25 g of t-BuO ₂ CCH ₂ -D-Cha-Pro-OH (yield: 94%). For this compound, 0.22 g (yield 31%) of the title compound were obtained by the same method as Preparation a, b) of Example 1.

¹ H NMR (CDCl ₃ ): 0.70-2.37 (m, 26H), 3.22 (m, 2H), 3.38 (m, 2H), 3.58 (m, 1H), 4.10 (m, 1H), 4.24 (m, 2H ), 4.55 (m, 1 H), 7.05-7.65 (m, 17 H).

FAB MS: 728 [M + 1] ⁺

c) preparation of HO ₂ CCH ₂ -D-Cha-Pro-NHCH ₂ CC-4-imd trifluoroacetic acid salt

0.22 g (0.30 mmol) of the compound obtained in Preparation b) was dissolved in trifluoroacetic acid and stirred at room temperature for 30 minutes. After completion of the reaction, the mixture was concentrated by distillation under reduced pressure. The residue was purified by column chromatography using dichloromethane / methanol (9/1 volume ratio) eluent to afford 0.11 mg (yield: 69%) of the title compound.

¹ H NMR (CDCl ₃ ): 1.03 (m, 2H), 1.25-1.91 (m, 12H), 2.02 (m, 2H), 2.28 (m, 1H), 3.53 (m, 1H), 3.84 (m, 3H ), 4.28 (m, 2H), 4.43 (m, 2H), 5.11 (m, 1H), 7.66 (s, 1H), 8.69 (s, 1H).

FAB MS: 430 [M + 1] ⁺

Example 16

(2S) -N- [3- (6-amino-3-pyridinyl) -2-propynyl] -1-{(2R) -2-[(methylsulfonyl) amino] -3,3-diphenyl Synthesis of propanoyl} -2-pyrrolidinecarboxamide

5-Bromo-2 instead of 4-iodo-1-trityl-1H-imidazole in preparation b) of Example 1 for the compound MeSO ₂ -D-Dpa-Pro-NHCH ₂ CC obtained in Example 8 The reaction was carried out in the same manner, except that aminopyridine was used (yield: 35%).

¹ H NMR (CDCl ₃ ): 1.40 (m, 2H), 1.68 (m, 1H), 1.94 (m, 1H), 2.58 (m, 1H), 2.77 (s, 3H), 3.56 (m, 1H), 4.02 (m, 3H), 4.22 (m, 1H), 4.79 (m, 1H), 6.32 (m, 1H), 7.05-7.61 (m, 12H), 7.92 (s, 1H).

FAB MS: 522 [M + 1] ⁺

Experimental Example 1: Thrombin Inhibitory Activity

The inhibitory capacity for thrombin activity of the compounds according to the invention was measured as described below.

To the 1.5 mL cuvette was added 1160 μl of 0.1 M Tris buffer (pH 7.8) containing 150 mM NaCl and 0.1% PEG8000 (polyethylene glycol, molecular weight about 8,000). As an inhibitor solution, the 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, or 0.001 mg / ml. Taken between 0 and 10 μg and added to the cuvette with a total volume of 100 μl with Tris buffer. The substrate solution was prepared by dissolving chromozyme TH in dimethylsulfoxide (DMSO) at a concentration of 10 mM and diluting with the buffer solution to a concentration of 0.1 mM.

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

Velocity V was determined by measuring the slope in each spectrum, and then an inverse (1 / V) graph of velocity versus inhibitor concentration was shown. After calculating the linear equation that satisfies the points on the graph, Ki can be calculated from the X-intercept of the equation using the enzyme reaction equation. The Km value used in this calculation was 5.2 μM, determined by varying the substrate concentration at a constant enzyme concentration.

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-Val-Gly-Arg-p-nitroanilide hydrochloride was used, and the inhibitor was 0 to Various concentrations were used within the range of 120 μg. In addition, trypsin was dissolved in 0.1 N HCl, and made 40 ㎍ / ㎖ with the Tris buffer solution immediately before the experiment 40 ul was used. As in the experiment for thrombin, the total volume of the reaction solution was set to 1.5 ml and in the same manner. The Km value used for the calculation of Ki was determined by the same method, which was 20.2 μM.

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

Inhibitory ability to thrombin and trypsin Example Compound Thrombin Ki (nM) Trypsin Ki (nM) Example Compound Thrombin Ki (nM) Trypsin Ki (nM) One 1590 - 10 17 20000 2 38000 - 11 0.87 19000 3 86000 - 12 29 22000 4 5450 - 13 12 41000 5 25 24000 14 15 25000 6 127 - 15 480 - 7 3.0 24000 16 250 - 8 2.7 25000 Inogartran 15 9 208 - Agatrovan 25

As a result of measuring the inhibitory activity of the compound of formula (I) according to the present invention for thrombin and trypsin, it was confirmed that the compound of the present invention has an excellent inhibitory activity against thrombin and excellent selectivity compared to trypsin. In particular, example compounds 7, 8, and 11 have superior effects compared to the known athrombin inhibitors of agatroban of formula (2) and inogatran of formula (4).

Experimental Example 2: Pharmacokinetics

The absorption effect upon oral administration of the compound of formula 1 according to the present invention was determined by measuring blood drug concentration according to the following experimental method.

The male rats were fasted for 20 hours and then tested. Physiological saline was used to prepare a 1% (10 mg / ml) solution of the Example compound followed by oral administration. Blood was collected at a predetermined time interval, mixed with methanol at a ratio of 3: 1 (v / v), and the supernatant was measured at 205 nm by using high pressure liquid chromatography (HPLC).

As a result, most of the example compounds according to the present invention showed high oral absorption tendency in animals. As an example, the results of Example 10 compounds are shown in Table 2 below. Table 2 shows the pharmacokinetic properties of the high bioavailability while maintaining the high concentration of the drug of the example absorbed in the rat for a long time in the blood.

Blood Drug Concentration and Bioavailability with Oral or Intravenous Administration of Example 10 in Rats Oral administration (30mg / kg) Intravenous administration (10mg / kg) Cmax (mg / mL) 11.8 - Tmax (min) 60 - T _1/2 (min) - 40 AUC (mg.min / mL) 1382 788 F (%) 58

Cmax: peak blood concentration

Tmax: time of peak blood concentration

T _1/2 : blood half-life

AUC: Integration over time of blood concentration

F: bioavailability

The thrombin inhibitor according to the present invention is excellent in thrombin inhibitory activity and orally administrable, and thus may be usefully used for preventing blood clotting and treating thrombosis.

Claims (11)

A compound of Formula 1, a pharmaceutically acceptable salt, hydrate, solvate or isomer thereof: [Formula 1] In the above formula, n represents an integer of 0 to 2, m represents an integer of 0 to 3, A is hydrogen, -SO₂R^One, -CO₂R²,-(CH₂)_1-3CO₂H or -COR^OneIndicates, From here R ¹ represents C _1-6 alkyl, C _3-7 cycloalkyl, aralkyl, aryl, or —NHR ³ (R ³ is hydrogen or C _1-4 alkyl), R ² represents C _1-6 alkyl, C _3-7 cycloalkyl, aralkyl, or aryl, B is phenyl, naph unsubstituted or substituted by one or more substituents selected from the group consisting of C _1-4 alkyl, halogen, hydroxy, C _1-4 alkyloxy, —CO ₂ -C _1-4 alkyl and amino Butyl, biphenyl, heteroaryl, C _1-6 alkyl, C _3-7 cycloalkyl, C _{5-12 dicycloalkyl} , C _11-16 tricycloalkyl, or —CH (R ⁴ ) ₂ ; Wherein R ⁴ is unsubstituted or substituted by one or more substituents selected from the group consisting of C _1-4 alkyl, halogen, hydroxy, C _1-4 alkyloxy, —CO ₂ -C _1-4 alkyl and amino Phenyl, heteroaryl, C _1-4 alkyl, C _3-7 cycloalkyl. C represents a 5 or 6 membered aryl or hetero aryl group unsubstituted or substituted by one or more substituents selected from the group consisting of C _1-4 alkyl, halogen, hydroxy, and amino. The compound of claim 1, wherein A is -SO₂R^One, -CO₂R², -CH₂CO₂H or -COR^OneWhere R is^OneSilver c_1-6Alkyl, C_3-7Cycloalkyl, aryl or -NHR³(R³Is hydrogen or C_1-4Alkyl) and R²C_1-6Alkyl, C_3-7Compounds representing cycloalkyl, aralkyl or aryl. The compound of claim 1, wherein B represents phenyl, C _3-7 cycloalkyl, or —CH (R ⁴ ) ₂ , unsubstituted or substituted by one or more substituents selected from the group consisting of halogen, hydroxy and amino, wherein In which R ⁴ represents phenyl, heteroaryl, C _1-4 alkyl or C _3-7 cycloalkyl unsubstituted or substituted by one or more substituents selected from the group consisting of halogen, hydroxy and amino. The compound of claim 1, wherein C is phenyl, pyridine, pyrimidine, imidazole, pyrazole, thiazole unsubstituted or substituted by one or more substituents selected from the group consisting of C _1-4 alkyl, hydroxy, and amino. , Pyrrole, thiophene, or oxazole. The compound of claim 1, wherein n represents an integer of 1, m represents an integer of 1 to 3, and A represents -SO₂R^One, -CO₂R², -CH₂CO₂H or -COR^OneWhere R is^OneSilver c_1-3Alkyl or -NHR³(R³Is hydrogen or C_1-4Alkyl) and R²C_1-3Alkyl, B is phenyl unsubstituted or substituted by halogen, C_3-7Cycloalkyl or -CH (R⁴)₂Where R is⁴Represents phenyl or cyclohexyl, and C is C_1-2A compound representing imidazole, pyridine, pyrazole, or thiazole substituted or unsubstituted by one or more substituents selected from the group consisting of alkyl and amino. The method of claim 5, t-butyl (1R) -1-benzyl-2-[(2S) -2-({[3- (1H-imidazol-4-yl) -2-propynyl] amino} carbonyl) pyrrolidinyl] 2-oxoethyl carbamate; t-butyl (1R) -1-benzyl-2-oxo-2-[(2S) -2-({[3- (4-pyridinyl) -2-propynyl] amino} carbonyl) pyrrolidinyl] Ethyl carbamate; (2S) -1-[(2R) -2-amino-3-phenylpropaneoyl] -N- [3- (4-pyridinyl) -2-propynyl] -2-pyrrolidinecarboxamide trifluor Roacetic acid; t-butyl (1R) -1-benzyl-2-oxo-2-[(2S) -2-({[3- (1H-pyrazol-4-yl) -2-propynyl] amino} carbonyl) Pyrrolidinyl] ethyl carbamate; t-butyl (1R) -1-benzhydryl-2-[(2S) -2-({[3- (1H-imidazol-4-yl) -2-propynyl] amino} carbonyl) pyrroli Dinyl] -2-oxoethylcarbamate; t-butyl (1R) -1-benzhydryl-2-[(2S) -2-({[3- (1-methyl-1H-imidazol-5-yl) -2-propynyl] amino} carbox Carbonyl) pyrrolidinyl] -2-oxoethylcarbamate; (2S) -1-{(2R) -2-[(benzylsulfonyl) amino] -3,3-diphenylpropaneoyl} -N- [3- (1H-imidazol-4-yl) -2- Propynyl] -2-pyrrolidinecarboxamide; (2S) -N- [3- (1H-imidazol-4-yl) -2-propynyl] -1-{(2R) -2-[(methylsulfonyl) amino] -3,3-diphenyl Propanoyl} -2-pyrrolidinecarboxamide; (2S) -1-[(2R) -2- (acetylamino) -3,3-diphenylpropaneoyl] -N- [3- (1H-imidazol-4-yl) -2-propynyl]- 2-pyrrolidinecarboxamide; Methyl (1R) -1-benzhydryl-2-[(2S) -2-({[3- (1H-imidazol-4-yl) -2-propynyl] amino} carbonyl) pyrrolidinyl] 2-oxoethyl carbamate; (2S) -1-{(2R) -2-[(aminosulfonyl) amino] -3,3-diphenylpropaneoyl} -N- [3- (1H-imidazol-4-yl) -2- Propynyl] -2-pyrrolidinecarboxamide; t-butyl (1R) -2,2-dicyclohexyl-1-{[(2S) -2-({[3- (1H-imidazol-4-yl) -2-propynyl] amino} carbonyl ) Pyrrolidinyl] carbonyl} ethyl carbamate; (2S) -1-[(2R) -2-amino-3,3-dicyclohexylpropaneoyl] -N- [3- (1H-imidazol-4-yl) -2-propynyl] -2- Pyrrolidinecarboxamide; Methyl (1R) -2,2-dicyclohexyl-1-{[(2S) -2-({[3- (1H-imidazol-4-yl) -2-propynyl] amino} carbonyl) pi Rollidinyl] carbonyl} ethylcarbamate; 2-({(1R) -1- (cyclohexylmethyl) -2-[(2S) -2-({[3- (1H-imidazol-4-yl) -2-propynyl] amino} carbonyl ) Pyrrolidinyl] -2-oxoethyl} amino) acetic acid; And (2S) -N- [3- (6-amino-3-pyridinyl) -2-propynyl] -1-{(2R) -2-[(methylsulfonyl) amino] -3,3-diphenyl Propanoyl} -2-pyrrolidinecarboxamide. A thrombin inhibitor composition comprising a compound of formula 1 as defined in claim 1 together with a pharmaceutically acceptable carrier. The composition of claim 7 formulated as an oral dosage form. A compound of formula [Formula 6] Wherein n, m, A and B are as defined in claim 1. A compound of formula (6) as defined in claim 9 is reacted with an arylhalide or heteroaryl halide compound of CX (where C is as defined in claim 1 and X is halogen) in the presence of a copper iodide and a palladium catalyst A process for preparing a compound of formula 1 as defined in claim 1. [Formula 6] Wherein n, m, A and B are as defined in claim 1. [Formula 1] Wherein n, m, A, B and C are as defined in claim 1. 1) deprotecting the dipeptide compound of formula (7) having a protecting group at the N-terminus, or deprotecting the compound to A′-X (where A ′ is the same as A as defined in claim 1 except for hydrogen) , X is a halogen), and then reacted with a halide of amide, followed by an amide bond reaction with propazylamine, 2) The dipeptide compound is subjected to an amide bond reaction with propazylamine, followed by a deprotection reaction or the coupling reaction. A process for preparing the compound of formula 6 as defined in claim 9. [Formula 7] Wherein n, m and B are as defined in claim 1 and P is an aminoprotecting group. [Formula 6] Wherein n, m, A and B are as defined in claim 1.