KR19980076024A - Selective thrombin inhibitors for oral administration - Google Patents

Abstract

The present invention relates to novel compounds useful as thrombin inhibitors. More specifically, the present invention provides a novel sulfonyl derivative represented by the following Chemical Formula 1, a method for preparing the same, and a blood coagulation containing the compound as an active ingredient, which exhibits excellent selectivity to thrombin and shows a potent thrombus formation inhibitory effect even by oral administration. Or a pharmaceutical composition for treating various thrombosis:

[Formula 1]

In the above formula,

Ar represents a substituted or unsubstituted aryl group,

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

R ₃ , R ₄ and R ₅ each independently represent hydrogen or lower alkyl.

Description

Selective thrombin inhibitors for oral administration

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

[Formula 1]

In the above formula,

Ar represents a substituted or unsubstituted aryl group,

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

R ₃ , R ₄ and R ₅ each independently represent hydrogen or lower alkyl.

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. Since there are various serine proteases similar to trypsin (typically plasmin) in the human body, especially in the blood, therefore, in developing thrombin inhibitors, these serine proteases, in particular trypsin It is very important to have less restraining properties.

Under these circumstances, extensive research has been conducted to develop selective thrombin inhibitors that effectively inhibit thrombin and have low inhibitory activity against trypsin.

Representative compounds developed as effective thrombin inhibitors include the first arylsulfonyl arginine-based compounds, Argatroban of formula (4) (US Pat. No. 4,258,192 and US 4201863).

[Formula 4]

This compound has been reported to have a 250-fold thrombin inhibitory effect compared to trypsin and has already been commercialized in Japan in 1990 (Biochemistry 1984, 23, 85-90). However, this compound is prepared through a very complicated synthetic process, and has the disadvantage of no activity for oral administration.

In addition, NAPAP of the following formula (5), a benzamidine-based arylsulfonyl compound, has been developed as a thrombin inhibitor. See J. Biol. Chem. 1991, 266, 20085-20093.

[Formula 5]

Meanwhile, the Ro46-6240 compound of formula 6, which has improved selectivity to thrombin relative to trypsin, has also been reported as a potent thrombin inhibitor, which shows the potential for development as an intravenous preparation, but has a short blood half-life, which is why it is an oral administration. There is no potential for development. See J. Med. Chem. 1994, 37, 3889-3901.

[Formula 6]

A representative compound reported as an effective thrombin inhibitor capable of oral administration is CVS-1123 of Chemical Formula 7, developed by Corvas. It has been reported that this compound can be orally administered to dogs and monkeys as well as to mice, but has a drawback of less than 50 times the selectivity for trypsin (Wo 9315756 and WO 9408941).

[Formula 7]

Therefore, the present inventors solve the disadvantages of the thrombin inhibitors of the prior art as described above to develop a compound that is relatively easy to synthesize, excellent in thrombin inhibitory activity, selectivity for thrombin relative to trypsin, and also can be administered orally Intensive research has been conducted for a long time. As a result, it was confirmed that the compound of formula 1 defined above can effectively achieve this object, and have completed the present invention.

Accordingly, an object of the present invention is to provide a novel thrombin inhibitor capable of oral administration and high selectivity and a method of preparing the same.

The present invention also relates to a composition for preventing blood clotting and treating various thrombosis containing the compound as an active ingredient.

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,

Ar represents a substituted or unsubstituted aryl group,

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

R ₃ , R ₄ and R ₅ each independently represent hydrogen or lower alkyl.

In the definition of the substituent for the compound of Formula 1, Ar is preferably substituted or unsubstituted by one or more substituents selected from the group consisting of halogen, lower alkyl, lower alkoxy, lower alkoxycarbonyl, aryl and dialkylamino. And a 6 to 10-membered aryl group optionally containing a nitrogen atom as a heteroatom. Specific examples of particularly preferred Ar include the following groups:

The term loweralkyl in the definition of the substituent of the compound of formula 1 according to the present invention means a straight or branched chain hydrocarbon radical having 1 to 4 carbon atoms, including methyl, ethyl, propyl, isopropyl, isobutyl, t-butyl, The term cycloalkyl means cyclic alkyl having 3 to 8 carbon atoms including cyclopentyl.

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

One.

2.

3.

4. (S) -3- [4- (N-methylamino) amidino-phenyl] -N-cyclopentyl-N-methyl-2- (5,6, 7,8-tetrahydronaphthalene-2-sul Phonyamino) -propionamide

5.

6.

7.

8.

9.

10. (S) -3- [4- (N-amino-N-methyl-amidino) -phenyl] -N-cyclopentyl-N-methyl-2- (4-propyl-benzenesulfonylamino) -propion amides

11.

12. (S) -3- [4- (N-amino-N-methyl-amidino) -phenyl] -N-cyclopentyl-N-methyl-2- (4-isobutyl- benzenesulfonylamino)- Propionamide

13. (S) -3- [4- (N-amino-N-methyl-amidino) -phenyl] -N-cyclopentyl-N-methyl-2- (5,6,7,8-tetrahydronaphthalene 2-sulfonylamino) -propionamide

14. (S) -3- [4- (N-amino-N-methyl-amidino) -phenyl] -N-cyclopentyl-N-methyl-2- (4-biphenyl-sulfonylamino) -propion amides

15. (S) -3- [4- (N-amino-N-methyl-amidino) -phenyl] -N-cyclopentyl-N-methyl-2- (6-methoxynaphthalene-2-sulfonylamino ) -Propionamide

16. (S) -3- [4- (N-amino-N-methyl-amidino) -phenyl] -N-cyclopentyl-N-methyl-2- (5-methyloxycarbonylnaphthalene-2-sul Phonyamino) -propionamide

17. (S) -3- [4- (N-amino-N-methyl-amidino) -phenyl] -N-cyclopentyl-N-methyl-2- (5-dimethylaminonaphthalene-1-sulfonylamino ) -Propionamide

18. (S) -3- [4- (N-amino-N-methyl-amidino) -phenyl] -N-cyclopentyl-N-methyl-2- (4-trifluoromethyl-benzenesulfonylamino ) -Propionamide

The compounds of formula 1 according to the invention may also form pharmaceutically acceptable salts. Such pharmaceutically acceptable salts include acids that form non-toxic acid addition salts containing pharmaceutically acceptable anions such as inorganic acids, such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrobromic acid, hydroiodic acid, tartaric acid, formic acid, Organic carbonic acid such as citric acid, acetic acid, trichloroacetic acid or trifluoroacetic acid, gluconic acid, benzoic acid, lactic acid, fumaric acid, maleic acid, etc., sulfonic acid such as methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid or naphthalenesulfonic acid Acid addition salts formed by and the like are included.

On the other hand, the compounds according to the invention may have an asymmetric carbon center and therefore may exist as racemic compounds, diastereomeric mixtures and individual diastereomers, all of these isomers being included within the scope of the invention.

The present invention relates to a process for preparing the compound of formula 1 as defined above.

According to the present invention, the compound of Formula 1 as defined above may be prepared by reacting a compound of Formula 2 with a compound of Formula 3.

[Formula 2]

[Formula 3]

Wherein Ar, R ₁ , R ₂ , R ₃ , R ₄ and R ₅ are as defined above.

Method for preparing a compound of Formula 1 as described above 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 a hydrazine derivative of Formula 3, which is a nucleophile. This reaction can preferably be carried out in the presence of a solvent. Examples of the solvent that can be preferably used for this purpose may be any organic solvent which does not adversely affect the reaction, but generally alcohol solvents such as methanol, ethanol, propanol and the like are preferably used.

In the present reaction, reaction conditions including reaction amount, reaction temperature, reaction time, and the like can be easily determined by those skilled in the art according to a specific reactant. In general, the reaction temperature may vary, but it is particularly preferable to carry out the reaction at 0 ℃ to 50 ℃. In addition, the reaction time generally takes 0.5 to 5 hours, but preferably performs the reaction for 1 to 2 hours.

After completion of the reaction, the product can be separated and purified by conventional post-treatment methods such as chromatography, recrystallization and the like.

The methylmercapto compound of formula (2) used as an intermediate in preparing the compound of formula (1) in Scheme 1 is first coupled to the C-terminus of the compound of formula (8) by R ₁ and R ₂ substituted amine group of the formula (9) The compound was obtained, and the N-terminal amino protecting group of the compound of Formula 9 was removed to obtain the compound of Formula 10, and then the compound of Formula 10 was reacted with sulfonyl chloride of ArSO ₂ Cl to prepare the N-terminal moiety. The introduction of a polyyl group affords a compound of formula 11, which is prepared by a method of saturating the nitrile compound with hydrogen sulfide in the presence of a base to produce a thioamide compound of formula 12 and methylating the thioamide compound of formula 12. Can:

[Formula 8]

[Formula 9]

[Formula 10]

[Formula 11]

[Formula 12]

Wherein Ar, R ₁ and R ₂ are as defined above and P represents a conventional amino protecting group such as benzyloxycarbonyl, allyloxycarbonyl, t-butoxycarbonyl or trityl group and the like.

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 ₁ and R ₂ is a substituted amine group of the first to the C- terminus of the compound of formula (8) in order to produce a methyl mercapto compound of formula 2 to obtain a compound of formula 9 . 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 (8) 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, and using it in a 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 (9) produced by the coupling reaction of compound of formula (8) then removes the N-terminal amino protecting group to yield the compound of formula (10). The compound of formula 10 is reacted with a sulfonyl compound of the general formula ArSO ₂ Cl to introduce a sulfonyl group to the N-terminal portion to obtain a compound of formula 11, and then the nitrile compound of formula 11 is a base, for example Saturation with hydrogen sulfide in the presence of pyridine and trimethylamine yields a thioamide compound of formula 12, which is methylated to yield 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 thrombin inhibitory effect of the compound of formula 1 according to the present invention is dissociation constant Ki using 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 dissociation constant Ki represents the degree of dissociation between the enzyme and the thrombin inhibitor compound, the smaller the dissociation constant value is, the greater the binding activity of the inhibitor to the enzyme, and thus, the inhibitory activity may be evaluated to be greater. 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 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.

On the other hand, in order to determine the selectivity for thrombin inhibition of the compound according to the present invention in the same manner as the method for measuring the thrombin inhibitory activity by measuring the inhibitory effect of the compound of formula (1) to trypsin Ki value then trypsin / Find the ratio of thrombin.

In this case, the test method for trypsin is the same as thrombin, except that N-benzoyl-valine-glycine-arginine para-nitroanilide hydrochloride (N-benzoyl-Val-Gly-Arg-p-nitroanilide hydrochloride) use.

As a result of measuring the inhibitory activity of the compound of Formula 1 according to the present invention with respect to thrombin and trypsin, the compound of the present invention not only has an excellent thrombin inhibitory effect but also has excellent selectivity of thrombin to trypsin, for example, Example 3 Selectivity of thrombin relative to trypsin of 4, 6, 4 and 6 was 2000 times, 1200 times and 5500 times, respectively, and the selectivity of the known thrombin inhibitors, agatroban and NAPAP, was only 250 or 50 times, respectively. I knew it was lost.

As mentioned above, the compound of formula 1 according to the present invention is a thrombin inhibitor which is excellent in selectivity to thrombin and also available by oral administration compared to known compounds. 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 a 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.

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 by administering oral preparations. 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 effect and thrombolytic effect, the active compound of formula 1 according to the present invention is one or more components selected from thrombolytic agents and platelet activity inhibitors It can be administered at the same time. Thrombolytic agents that can be administered in combination with a compound of the present invention in this manner may include t-PA, urokinase, streptokinase, and the like, and platelet activity inhibitors include aspirin, ticlopidin, Clopidrogel, 7E3 monoclonal antibody, and the like.

However, the preparations containing the compounds according to the invention for the purpose of the treatment and prevention of thrombi are not limited to those described above, and any preparations useful for the treatment and prevention of thrombi can be included.

The present invention is explained in more detail by the following examples and experimental examples, but the scope of the present invention is not limited in any way by these.

Preparation Example 1

Synthesis of Cyclopentyl-Methylamine Hydrochloride

After dissolving 10 ml (113 mmol) of cyclopentanone in 50 ml of methanol, 50 ml of water was added thereto. To this was added 7.6 g (113 mmol) of methylamine hydrochloride and 7.1 g (113 mmol) of sodium borocyanohydride (NaBH ₃ CN). The reaction solution was stirred for 12 hours while heating to reflux at pH 6, then cooled to 0 ° C and neutralized with 6N sodium hydroxide solution. After distilling under reduced pressure to remove methanol, the mixture was cooled to 0 ° C and adjusted to pH 2 with dilute hydrochloric acid. The reaction solution was washed three times with diethyl ether and the aqueous layer was adjusted to pH 11 again. 50 ml of dioxane was added thereto, and 37 g (169.5 mmol) of t-butyloxycarbonyl anhydride was added thereto. The reaction mixture was stirred at room temperature for 3 hours. After the reaction was completed, the reaction mixture was distilled under reduced pressure, the reaction solution was concentrated to about 30 ml, extracted with ethyl acetate, and washed with an aqueous solution of acid and base. The organic layer was dried over anhydrous magnesium sulfate, filtered and concentrated. The white solid obtained was purified using column chromatography. The obtained solid was dissolved in 60 mL of 4N hydrochloric acid-dioxane solution and stirred for 30 minutes. The solvent was removed under reduced pressure and dried in vacuo to yield 13.7 g (yield 90.5%) of the title compound.

¹ H NMR (CD ₃ OD, ppm) δ: 3.50 (m, 1H), 2.68 (s, 3H), 2.10 (m, 2H), 1.86-1.50 (m, 6H)

Preparation Example 2

synthesis

0.7 g (2.41 mmol) of (S) -3- (4-cyano-phenyl) -2- (butyloxycarbonyl-amino) -propionic acid was dissolved in 6 ml of dimethylformamide (DMF). After cooling to 0 ° C., 0.7 g of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and 0.4 g of 1-hydroxybenzotriazole hydrate (HOBT) were added until complete dissolution. Stirred. 0.4 g (2.96 mmol) of the compound obtained in Preparation Example 1 and 1.0 ml of N-methylmorpholine were added to this solution. The reaction solution was slowly raised to room temperature and stirred for 3.5 hours. After the reaction was completed, the product was distilled under reduced pressure to remove volatiles. The residue obtained was diluted with ethyl acetate and then washed sequentially with saturated aqueous sodium hydrogen carbonate solution, diluted hydrochloric acid and saturated brine. Dried over anhydrous sodium sulfate, filtered and concentrated. The residue was subjected to column chromatography [eluent: ethyl acetate / hexane (7/3, volume ratio)] to yield 0.65 g (yield 73.0%) of the title compound.

¹ H NMR (CDCl ₃ , ppm) δ: 7.61 (m, 2H), 7.32 (m, 2H), 5.48, 5.01-4.86, 4.12 (m, m, m, 3H), 2.75, 2.62 (s, s, 3H), 2.90-1.20 (m, 17H)

Mass (FAB, m / e): 372 (M + H)

Preparation Example 3

Synthesis of 4-propyl-benzenesulfonylchloride

1.5 ml (22.6 mmol) of chlorosulfonic acid were added to the reaction vessel, followed by cooling to 0 ° C. 1.0 mL (7.17 mmol) of propylbenzene was slowly added dropwise thereto. At this time, it was added dropwise while being careful not to rise the temperature 5 ℃ or more. All were added dropwise and then raised to room temperature and stirred for 3 hours. The reaction was terminated by cooling back to 0 ° C. and adding ice. Water and 40 ml of ethyl acetate each were added to the reaction solution, followed by extraction three times. The combined organic layers were dried over anhydrous magnesium sulfate, filtered and concentrated to give 1.3 g of a residue. The obtained compound was a mixture of 2-propylbenzenesulfonylchloride and 4-propylbenzenesulfonylchloride, and their ratio was 1: 2 by NMR analysis. Conversion of this yielded 0.86 g (55% yield) of the title compound.

¹ H NMR (CDCl ₃ , ppm) δ: 8.04 (dd, 2H), 7.49 (dd, 2H), 2.82 (t, 2H), 1.78 (m, 2H), 1.06 (t, 3H)

Mass (FAB, m / e): 219 (M + H)

Preparation Example 4

synthesis

0.97 g (2.61 mmol) of the compound obtained in Preparation Example 2 was dissolved in 10 ml of methanol, and then cooled to 0 ° C. Excess (30 equivalents) of acetyl chloride was slowly added thereto, and the temperature was raised to room temperature and stirred for 1 hour. Removal of all volatiles under reduced pressure yielded a white solid. The obtained solid was dried in vacuo, then dissolved in 10 ml of dimethylformamide, and 1.0 ml of N-methylmorpholine was added and stirred for 10 minutes. To this was added 0.7 g (3.21 mmol) of the mixture obtained in Preparation Example 3 and stirred for 1 hour. The solvent was removed by vacuum reduction, and extracted with dichloromethane and 40 ml of water three times. The combined organic layers were dried over anhydrous magnesium sulfate, filtered and concentrated. The obtained residue was separated by column chromatography [eluent: hexane / ethyl acetate (7/3, volume ratio)] to obtain 0.8 g (yield 82%) of the title compound.

¹ H NMR (CDCl ₃ , ppm) δ: 7.72 (dd, 2H), 7.65 (m, 2H), 7.34 (m, 4H), 5.86 (m, 1H), 4.63, 4.56, 4.39, 3.88 (m, m , m, m, 2H), 3.05 (m, 2H), 2.69 (m, 2H), 2.59, 2.40 (s, s, 3H), 1.76-1.04 (m, 10H), 1.00 (t, 3H)

Mass (FAB, m / e): 454 (M + H)

Example 1

synthesis

0.45 g (0.99 mmol) of the compound obtained in Production Example 4 was dissolved in 20 ml of pyridine, and added to a flask equipped with 1.0 ml of triethylamine. At this time, the flask was installed to slowly flow hydrogen sulfide (H ₂ S) gas into the solution through one branch and to flow gas from the other branch. The hydrogen sulfide gas was saturated while stirring the reaction solution 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 reaction solution was distilled under reduced pressure to remove volatiles and dried with a vacuum pump. 10 ml of acetonitrile and 0.25 ml of methane iodide (CH ₃ I) were added together to the obtained yellow solid, and the mixture was heated to reflux for 30 minutes. It was distilled under reduced pressure again to remove volatiles and dried with a vacuum pump. The residue was dissolved in 8 ml of anhydrous methanol and stirred. To this was added 0.04 ml (0.66 mmol) of 80% hydrazine hydrate (H ₂ NNH ₂ .H ₂ O) in three portions at 10 minute intervals. After the reaction was completed, the reaction solution was concentrated and the residue was purified by column chromatography [eluent: dichloromethane / methanol (95/5, volume ratio)] to give 0.19 g (yield 80%) of the title compound.

¹ H NMR (CD ₃ OD, ppm) δ: 7.66 (m, 4H), 7.42 (m, 2H), 7.33 (m, 2H), 4.58, 4.43, 4.05 (m, m, m, 2H), 3.04, 2.88 (m, m, 2H), 2.66 (m, 2H), 2.59, 2.46 (s, s, 3H), 1.70-1.10 (m, 10H), 0.93 (m, 3H)

Mass (FAB, m / e): 486 (M ⁺ +1)

Example 2

synthesis

1 g (1.96 mmol) was dissolved in 20 ml of methanol, and 0.31 ml (3 molar equivalents) of monomethylhydrazine (CH ₃ NHNH ₂ ) was added in 3 portions and added over 30 minutes. The reaction mixture was stirred at room temperature for 6 hours and then concentrated. The residue was purified by column chromatography [eluent: dichloromethane / methanol (10/1, volume ratio)] to give 410 mg (yield 41%) of the title compound.

¹ H NMR (CD ₃ OD, ppm) δ: 8.3 (1H, m), 7.9 (3H, m), 7.7 (1H, m), 7.67 (2H, m), 7.5 (2H, m), 7.2 (2H , m), 4.6, 4.45 (1H, m, m), 4.1, 3.9 (1H, m, m,), 2.9 (2H, m), 2.72 (3H, s), 2.35, 2.25 (3H, s, s ), 1.5-0.5 (8H, m)

Mass (FAB, m / e): 508 [M + H]

Example 3

Synthesis of Hydrochloride

1 g (1.96 mmol) was dissolved in 20 ml of methanol, and then 86 mg (3 molar equivalents) of 1-butoxycarbonyl-1-methyl-hydrazine [t-BOC (CH ₃ ) NNH ₂ ] was divided into three portions for 30 minutes. Was added over. The reaction mixture was stirred at room temperature for 6 hours and then concentrated, and the residue was purified by column chromatography to give 820 mg of the compound. The compound was again dissolved in 15 ml of methanol, cooled in an ice water bath, and 3 ml of acetyl chloride was added little by little. Stirred at the same temperature for 10 minutes, the ice-water bath was removed and stirred at room temperature for 20 minutes. Concentration after completion of the reaction gave 730 mg (yield 68%) of the title compound.

¹ H NMR (CD ₃ OD, ppm) δ: 8.32 (1H, m), 7.95 (3H, m), 7.77-7.6 (5H, m), 7.4 (2H, m), 4.65, 4.52 (1H, m, m), 4.2, 4.08 (1H, m, m), 3.0 (2H, m), 2.75 (3H, s), 2.56, 2.28 (3H, s, s), 1.6-0.5 (8H, m)

Mass (FAB, m / e): 508 [M + H]

Example 4

synthesis

0.3 g (0.58 mmol) was dissolved in 20 mL of methanol, and then 32.0 mg (0.75 mmol, 1.3 molar equivalent) of monomethylhydrazine (CH ₃ NHNH ₂ ) was added in 3 portions, and added over 30 minutes. The reaction mixture was stirred at room temperature for 6 hours and then concentrated. The residue was purified by column chromatography [eluent: dichloromethane / methanol (10/1, volume ratio)] to give 24 mg (yield 82%) of the title compound.

¹ H NMR (CD ₃ OD, ppm) δ: 7.6-7.4 (6H, m), 7.2 (1H, t), 4.6, 4.45, 4.1 (2H, m, m, m), 3.3 (3H, brs), 3.1 (1H, m), 2.9 (2H, m), 2.8 (4H, s), 2.6, 2.5 (3H, s, s), 1.9 (4H, brs), 1.5-0.5 (8H, m)

Mass (FAB, m / e): 512 [M + H]

Example 5

Synthesis of Hydrochloride

0.30 g (0.58 mmol) was dissolved in 20 ml of methanol, followed by 3 parts of 110 mg (0.75 mmol, 1.3 molar equivalents) of 1-butoxycarbonyl-1-methyl-hydrazine [t-BOC (CH ₃ ) NNH ₂ ]. Divided into and added over 30 minutes. The reaction mixture was stirred at room temperature for 1 hour and then concentrated, and the residue was purified by column chromatography to give 260 mg of the compound. The compound was again dissolved in 10 ml of methanol, cooled in an ice water bath, and 2 ml of acetyl chloride was added little by little. Stirred at the same temperature for 10 minutes, the ice-water bath was removed and stirred at room temperature for 20 minutes. The reaction was completed and concentrated to give 190 mg (65% yield) of the title compound.

¹ H NMR (CD ₃ OD, ppm) δ: 7.7 (2H, m), 7.5 (2H, m), 7.2 (3H, m), 4.6, 4.45, 4.1 (2H, m, dd, m), 3.1 ( 3H, brs), 3.0-2.9 (2H, m), 2.8 (4H, s), 2.5, 2.4 (3H, s, s), 1.9 (4H, brs), 1.6-1.1 (8H, m)

Mass (FAB, m / e): 512 [M + H]

Example 6

synthesis

The reaction was carried out in the same manner as in Example 2 to give the title compound in a yield of 85%.

¹ H NMR (CD ₃ OD, ppm) δ: 7.7 (2H, m), 7.6 (2H, d), 7.4 (2H, m), 7.3 (2H, m), 4.6, 4.5, 4.1 (2H, m, m, m), 3.3 (3H, s), 3.0 (1H, m), 2.9 (1H, m), 2.7 (2H, t), 2.6, 2.5 (3H, s, s), 1.7 (2H, m) , 1.6-1.1 (8H, m), 1.0 (3H, t)

Mass (FAB, m / e): 500 [M + H]

Example 7

Synthesis of Hydrochloride

The reaction was carried out in the same manner as in Example 3 to give the title compound in a yield of 66%.

¹ H NMR (CD ₃ OD, ppm) δ: 7.7-7.6 (4H, m), 7.4 (2H, m), 7.3 (2H, m), 4.6, 4.4, 4.0 (2H, m, m, m), 3.1 (3H, s), 3.0 (2H, m), 2.7 (2H, t), 2.5, 2.4 (3H, s, s), 1.7 (2H, m), 1.6-1.1 (8H, m), 1.0 ( 3H, t)

Mass (FAB, m / e): 500 [M + H]

Experimental Example 1 Inhibitory Activity of a Thrombin Inhibitor

The inhibitory activity against thrombin of the compound according to the present invention was measured according to the method described below.

To the 1.5 mL cuvette was added 1160 μl of 0.1 M Tris buffer (pH 7.8) containing 150 mM NaCl and 0.1% PEG8000 (polyethylene glycol, molecular weight about 8000). The substrate solution was prepared by dissolving Chromozyme TH in dimethyl sulfoxide (DMSO) at a concentration of 10 mM and diluting with the buffer solution to a concentration of 0.1 mM. 225 µl of the 0.1 mM substrate solution thus prepared was added to the cuvette. As an inhibitor solution, the 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 calculating the initial velocity Vi from the slope within 30 seconds of the initial reaction time in each spectrum, an inverse (1 / Vi) graph of the initial velocity versus the inhibitor concentration is shown. After calculating the first equation that satisfies the points on the graph, Ki can be calculated from the x-intercept of the equation using the Michaelis-Menten equation. The Km value used in this calculation was 5.2 μM, determined by varying the substrate concentration at a constant enzyme concentration.

Michaelis-Menten equation

In the enzyme scheme, [I] denotes the concentration of the inhibitor, [S] denotes the concentration of the substrate, Vmax denotes the maximum initial velocity, and Km denotes the Michaelis constant, which means 5.2 μM. .

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

That is, 1160 µl of buffer solution was added to a 1.5 ml volume cuvette, and 15 µl of 0.1 mg / ml human thrombin solution and 100 µl of inhibitor solution were added thereto and 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.

On the other hand, the inhibitory activity of the compound according to the present invention against trypsin was also measured as described for the case of thrombin.

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

The enzyme activity of the inhibitor according to the present invention, measured for thrombin and trypsin according to the method described above, was expressed as Ks value, and the selectivity to thrombin was also expressed as trypsin / thrombin. The results are as shown in Table 1 below.

TABLE 1

Inhibitor's ability to inhibit thrombin and trypsin

From the above experimental results, it can be seen that the compound of the present invention has superior selectivity to thrombin relative to trypsin, compared with known compounds.

Experimental Example 2: Pharmacokinetics

Experiment Method:

Male rats were fasted for 20 hours and then tested. Physiological saline was used to prepare a 1% (10 mg / ml) solution of the compound of Example 2 and then orally administered in an amount such that 30 mg of the compound per kg body weight of the rat was administered. Blood was collected at a predetermined time interval and blood drug concentrations were measured using high pressure liquid chromatography. In addition, the blood was immediately mixed with methanol and zinc sulfate, and the supernatant was measured for blood drug concentration at an ultraviolet wavelength of 231 nm.

Experiment result:

Table 2 below shows blood drug concentrations over time after oral administration of the compound of Example 2 in rats. The compound of Example 2 showed pharmacokinetic properties that the drug is absorbed upon oral administration and maintained in the blood for a long time.

TABLE 2

Blood Drug Concentration vs. Time of Oral Administration of Compound of Example 2 at 30 mg / kg in Mice

Minutes Blood concentration (ng / ml) Average concentration (+ error) (ng / mL) Rat-1 Rat-2 Rat-3 5 1145 341 700 729 (201) 10 2196 1182 1656 1678 (254) 20 1979 1585 1741 1679 (99) 40 1592 1313 1481 1462 (70) 60 1025 978 865 956 (41) 90 747 723 495 655 (70) 120 521 586 403 503 (46) 180 421 445 318 395 (34) 240 339 360 278 326 (21)

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, Ar represents a substituted or unsubstituted aryl group, R ₁ and R ₂ each independently represent lower alkyl or cycloalkyl, R ₃ , R ₄ and R ₅ each independently represent hydrogen or lower alkyl. The compound of claim 1, wherein Ar is unsubstituted or substituted by one or more substituents selected from the group consisting of halogen, lower alkyl, lower alkoxy, lower alkoxycarbonyl, aryl and dialkylamino, and optionally nitrogen as a heteroatom. Compounds representing 6 to 10-membered aryl groups containing atoms. A compound according to claim 2 wherein Ar represents one of the groups of the formula: The compound according to claim 1, wherein (S) -3- [4- (S)-(S) -3- [4- (N-amino-N-methyl-amidino) -phenyl] -N-cyclopentyl-N -Methyl-2- (4-biphenyl-sulfonylamino) -propionamide, (S) -3- [4- and a 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 a hydrazine derivative of Formula 3 [Formula 1] [Formula 2] [Formula 3] In the above formula, Ar represents a substituted or unsubstituted aryl group, R ₁ and R ₂ each independently represent lower alkyl or cycloalkyl, R ₃ , R ₄ and R ₅ each independently represent hydrogen or lower alkyl. A process according to 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.