KR100377557B1 - Selective thrombin inhibitors with acyl guanidine group - Google Patents

Abstract

The present invention relates to a novel selectivity and orally administrable novel thrombin inhibitor of formula (1), a preparation method thereof, and a pharmaceutical composition for preventing blood coagulation and thrombosis containing the same as an active ingredient:

Wherein R ¹ is hydrogen, R ₂ NCOCO-, RCO-, RSO _2- , ROCO-, R ₂ NCOCH _2- , RNHSO _2- , ROSO ₂ -or ROCOCH _2- , and R is hydrogen, 1 to Lower alkyl of 8, aryl of 6 to 10 carbon atoms, arylalkyl of 5 to 12 carbon atoms, heteroaryl of 4 to 10 carbon atoms, heteroarylalkyl of 5 to 11 carbon atoms, or cycloalkyl of 3 to 8 carbon atoms, R ² is hydrogen, fluorine, chlorine, bromine, iodine, hydroxy or methoxy, and X and X 'are each independently hydrogen, fluorine, chlorine, bromine, iodine, formyl, cyano, hydroxy or methoxy As substituted at the meta- or para- position, n is 0, 1 or 2, and m is 2 or 3.

Description

Selective thrombin inhibitors with acyl guanidine group

The present invention relates to a novel thrombin inhibitor, and more particularly, a highly selective and orally administrable thrombin inhibitor of formula (1), a preparation method thereof, and a medicament for preventing blood coagulation and treating thrombosis containing the same as an active ingredient. To a pharmaceutical composition.

[Formula 1]

Wherein R ¹ is hydrogen, R ₂ NCOCO-, RCO-, RSO _2- , ROCO-, R ₂ NCOCH _2- , RNHSO _2- , ROSO ₂ -or ROCOCH _2- , and R is hydrogen, 1 to Lower alkyl of 8, aryl of 6 to 10 carbon atoms, arylalkyl of 5 to 12 carbon atoms, heteroaryl of 4 to 10 carbon atoms, heteroarylalkyl of 5 to 11 carbon atoms, or cycloalkyl of 3 to 8 carbon atoms, ,

R ² is hydrogen, fluorine, chlorine, bromine, iodine, hydroxy or methoxy,

X and X 'are each independently hydrogen, fluorine, chlorine, bromine, iodine, formyl, cyano, hydroxy or methoxy, substituted at the meta- or para- position,

n is 0, 1 or 2,

m is 2 or 3.

Many complex enzymatic reactions are known to be involved in the coagulation process. The final stage of the coagulation process involves the conversion of prothrombin to thrombin. Thrombin produced at this stage activates platelets, converts fibrinogen to fibrin, and the like. Fibrin is converted into a polymer by a polymerization reaction and crosslinked by activated blood factor XIII to become insoluble coagulation. Thrombin also acts to activate blood factors V and VIII, which participate in the coagulation process, further accelerating the coagulation response. Thus, thrombin inhibitors can act as effective anticoagulants, while inhibiting platelet activity and preventing fibrin production and stabilization. Therefore, there has been a search for a method for preventing blood coagulation and treating various thrombosis by developing a substance capable of inhibiting thrombin activity for a long time.

However, to be used as an effective anticoagulant, "selectivity" that inhibits only thrombin is essential. The reason for this is that there are various serine proteases having an active site structure similar to thrombin in the human body, so that the thrombin inhibitor lacking selectivity causes side effects such as bleeding. In general, the selectivity of a thrombin inhibitor is determined by comparing the inhibitory ability to thrombin and the trypsin, a representative enzyme of serine protease. Under these circumstances, extensive research has been conducted to develop selective thrombin inhibitors that effectively inhibit thrombin and at the same time have low inhibitory activity against trypsin. In addition, as the need for prophylactic anticoagulant as well as the treatment of various diseases caused by blood clots, the focus is on the research and development of oral thrombin inhibitors that are easy to use in addition to injections.

With representative compounds developed as effective thrombin inhibitors. First, there may be mentioned arylsulfonyl arginine-based compound, a half (Argatroban, K _i = 1.9nM) to ahgateu of formula 1c (U.S. Patent No. 4,258,192 and No. 4,201,863 No.).

The compound has been reported to have a 250-fold thrombin inhibitory effect compared to trypsin and has already been commercialized as an injectable preparation in Japan in 1990 (Biochemistry 1984, 23, 85-90).

In addition, as a thrombin inhibitor, a benzamidine-based arylsulfonyl compound, NAPAP of Formula 1d, has been developed, which is easy to synthesize and has an effective thrombin inhibitor (K _i = 6.0 nM). There is a problem that it is only about twice (J. Biol. Chem. 1991. 266, 20085-20093).

Meanwhile, Ro46-6240 compound of formula 1e with improved selectivity compared to trypsin has been reported as a potent thrombin inhibitor (K _i = 0.2 nM), which shows the possibility of development as an intravenous drug, but has a short half-life in blood and is administered orally. There is no potential for development as a zero (J. Med. Chem. 1994, 37, 3889-3901).

An orally administrable and effective thrombin inhibitor is CVS-1123 of Formula 1f (K _i = 1.4 nM) developed by Corvas.

The compound has been reported to be orally administered in dogs and monkeys as well as in mice, but the disadvantage is that the selectivity to trypsin is 50 times or less (International Patent Publications WO 9315756 and WO 9408941).

Meanwhile, as a thrombin inhibitor developed by Akzo Nobel (IC ₅₀ = 34 nM), the compound of Formula 1g has a disadvantage in that it has low selectivity for trypsin (IC ₅₀ = 4 nM) and cannot be orally administered (International Publication). Patent WO 9730073 and 15 ^th EFMC International Symposium on Medicinal Chemistry, Edinburgh, 1998, UK.

Therefore, the present inventors have intensively researched to develop a novel compound which is not only excellent in thrombin inhibitory activity but also has high selectivity for thrombin compared to trypsin and is orally administrable. It has been confirmed that the present invention is completed and the present invention has been completed.

Accordingly, an object of the present invention is firstly to provide a novel selectivity and orally administrable novel thrombin inhibitor of Formula 1, and secondly, to provide a method for preparing the compound, and thirdly, to provide a blood containing the compound as an active ingredient. It is to provide a pharmaceutical composition for the prevention of coagulation and treatment of thrombosis.

The present invention provides a compound of formula 1, pharmaceutically acceptable salts, hydrates, solvates and isomers thereof:

[Formula 1]

Wherein R ¹ is hydrogen, R ₂ NCOCO-, RCO-, RSO _2- , ROCO-, R ₂ NCOCH _2- , RNHSO _2- , ROSO ₂ -or ROCOCH _2- , and R is hydrogen, 1 to Lower alkyl of 8, aryl of 6 to 10 carbon atoms, arylalkyl of 5 to 12 carbon atoms, heteroaryl of 4 to 10 carbon atoms, heteroarylalkyl of 5 to 11 carbon atoms, or cycloalkyl of 3 to 8 carbon atoms, ,

R ² is hydrogen, fluorine, chlorine, bromine, iodine, hydroxy or methoxy,

X and X 'are each independently hydrogen, fluorine, chlorine, bromine, iodine, formyl, cyano, hydroxy or methoxy, substituted at the meta- or para- position,

n is 0, 1 or 2,

m is 2 or 3.

In the compound, R is hydrogen, lower alkyl having 1 to 8 carbon atoms, aryl having 6 to 10 carbon atoms or arylalkyl having 5 to 12 carbon atoms, R ² is hydrogen, and X and X 'are hydrogen. Preferably, R ¹ is RSO _2- , R ₂ NCOCH ₂ -or RNHSO _2- , R is hydrogen, lower alkyl of 1 to 3 carbon atoms, aryl of 6 carbon atoms or arylalkyl of 5 to 12 carbon atoms and more preferably, n is 1 and m is 2. Representative examples of the compound,

tert-butyl- (1R) -1-benzhydryl-2-{(2S) -2-[({4-[(diaminomethylene) amino] -4-oxobutyl} amino) carbonyl] pyrrolidinyl } -2-oxoethylcarbamate;

(2S) -N- {4-[(diaminomethylene) amino] -4-oxobutyl} -1-{(2R) -2-[(methylsulfonyl) amino] -3,3-diphenylpropano Il} -2-pyrrolidinecarboxamide;

(2S) -1-[(2R) -2- (acetylamino) -3,3-diphenylpropanoyl] -N- {4-[(diaminomethylene) amino] -4-oxobutyl} -2 -Pyrrolidinecarboxamide;

(2S) -1-[(2R) -2-amino-3,3-diphenylpropanoyl] -N- {4-[(diaminomethylene) amino] -4-oxobutyl} -2-pyrroli Dine carboxamide;

tert-butyl- (1R) -1-benzhydryl-2- {2-[({4-[(diaminomethylene) amino] -oxobutyl} amino) carbonyl] -1-piperidinyl} -2 Oxoethyl carbamate;

2-[((1R) -1-Benzhydryl-2-{(2S) -2-[({4-[(diaminomethylene) amino] -4-oxobutyl} amino) carbonyl] pyrrolidinyl } -2-oxoethyl) amino] acetic acid;

(2S) -1-{(2R) -2-[(benzylsulfonyl) amino] -3,3-diphenylpropanoyl} -N- {4-[(diaminomethylene) amino] -4-oxo Butyl} -2-pyrrolidinecarboxamide;

tert-butyl- (1R) -1-benzhydryl-2-{(2S) -2-[({3-[(diaminomethylene) amino] -3-oxopropyl} amino) carbonyl] pyrrolidinyl } -2-oxoethylcarbamate;

(2S) -N- {3-[(diaminomethylene) amino] -3-oxopropyl) -1-{(2R) -2-[(methylsulfonyl) amino] -3,3-diphenylpropano Il} -2-pyrrolidinecarboxamide;

(2S) -N- {3-[(diaminomethylene) amino] -3-oxopropyl} -1-{(2R) -2-[(ethylsulfonyl) amino] -3,3-diphenylpropano Il} -2-pyrrolidinecarboxamide;

(2S) -1-{(2R) -2-[(benzylsulfonyl) amino} -3,3-diphenylpropanoyl} -N- {3-[(diaminomethylene) amino] -3-oxo Propyl} -2-pyrrolidinecarboxamide;

(2S) -N- {3-[(diaminomethylene) amino] -3-oxopropyl} -1-{(2R) -3,3-diphenyl-2-[(propylsulfonyl) amino] propano Il} -2-pyrrolidinecarboxamide;

N- {3-[(diaminomethylene) amino] -3-oxopropyl} -1-{(2R) -2-[(methylsulfonyl) amino] -3,3-diphenylpropanoyl} -2 Piperidinecarboxamide;

(2S) -N- {3-[(diaminomethylene) amino] -3-oxopropyl} -1-{(2R) -2-[(methylsulfonyl) amino] -3,3-diphenylpropano Il} -2-azetidinecarboxamide;

N- {4-[(diaminomethylene) amino] -4-oxobutyl} -1-{(2R) -2-[(methylsulfonyl) amino] -3,3-diphenylpropanoyl} -2 Piperidinecarboxamide; or

(2S) -1-{(2R) -2-[(aminosulfonyl) amino] -3,3-diphenylpropanoyl} -N- {3-[(diaminomethylene) amino] -3-oxo Propyl} -2-pyrrolidine carboxamide.

Among the compounds listed above,

(2S) -N- {4-[(diaminomethylene) amino] -4-oxobutyl} -1-{(2R) -2-[(methylsulfonyl) amino] -3,3-diphenylpropano Il} -2-pyrrolidinecarboxamide;

(2S) -1-{(2R) -2-[(benzylsulfonyl) amino] -3,3-diphenylpropanoyl} -N- {4-[(diaminomethylene) amino] -4-oxo Butyl} -2-pyrrolidinecarboxamide;

tert-butyl- (1R) -1-benzhydryl-2-{(2S) -2-[({3-[(diaminomethylene) amino] -3-oxopropyl} amino) carbonyl] pyrrolidinyl } -2-oxoethylcarbamate;

(2S) -N- {3-[(diaminomethylene) amino] -3-oxopropyl) -1-{(2R) -2-[(methylsulfonyl) amino] -3,3-diphenylpropano Il} -2-pyrrolidinecarboxamide;

(2S) -N- {3-[(diaminomethylene) amino] -3-oxopropyl} -1-{(2R) -2-[(ethylsulfonyl) amino] -3,3-diphenylpropano Il} -2-pyrrolidinecarboxamide;

(2S) -1-{(2R) -2-[(benzylsulfonyl) amino} -3,3-diphenylpropanoyl} -N- {3-[(diaminomethylene) amino] -3-oxo Propyl} -2-pyrrolidinecarboxamide;

(2S) -N- {3-[(diaminomethylene) amino] -3-oxopropyl} -1-{(2R) -3,3-diphenyl-2-[(propylsulfonyl) amino] propano Il} -2-pyrrolidinecarboxamide; or

(2S) -1-{(2R) -2-[(aminosulfonyl) amino] -3,3-diphenylpropanoyl} -N- {3-[(diaminomethylene) amino] -3-oxo Propyl} -2-pyrrolidinecarboxamide is particularly preferred.

The present invention also relates to (a) reacting an alanine derivative of formula (2) with a proline methylester hydrochloride of formula (3) or an analog thereof to hydrolyze to obtain a carboxylic acid derivative of formula (4),

Coupling a carboxylic acid derivative of formula 4 with an amine derivative of formula 5 to obtain an ester derivative of formula 6,

Reacting the ester derivative of formula 6 directly with guanidine or reacting with guanidine by removing the ROCO- group from the ester derivative of formula 6, or removing the ROCO- group from the ester derivative of formula 6 and removing sulfonylated or acylated guanidine By reaction with, to obtain a compound of formula 1a;

(b) coupling an azetidine carboxylic acid derivative of formula 7 with an amine derivative of formula 5 to obtain an ester derivative of formula 8,

Removing the ROCO- group from the ester derivative of formula 8 and coupling with an alanine derivative of formula 2 to obtain an ester derivative of formula 9,

React the ester derivative of formula 9 directly with guanidine or react with guanidine by removing the ROCO- group from the ester derivative of formula 9, or remove the ROCO- group from the ester derivative of formula 9 and remove sulfonylated or acylated guanidine By reacting with a compound of formula 1b to obtain a compound of formula 1b:

[Formula 1a]

[Formula 1b]

[Formula 2]

[Formula 3]

[Formula 4]

[Formula 5]

[Formula 6]

[Formula 7]

[Formula 8]

[Formula 9]

Wherein W is hydrogen, R ₂ NCOCO-, RCO-, RSO _2- , ROCO-, RNHSO ₂ -or ROSO _2- , R and m are the same as defined above and n 'is 1 or 2.

The present invention also provides a pharmaceutical composition for preventing blood coagulation and thrombosis, characterized in that it contains the compound as an active ingredient. The pharmaceutical composition is preferably formulated as an injectable or oral preparation.

Hereinafter, the present invention will be described in detail.

Lower alkyl herein means straight or branched chain alkyl including methyl, ethyl, isopropyl, isobutyl, t-butyl.

In addition, abbreviations used herein are defined to have the following meanings.

boc (or Boc): butoxycarbonylPh: phenyl

Me: Methyl Et: Ethyl

n-Pr: n-propylAc: acetyl

Ms: Methylsulfonyl Bn: Benzyl

diphe: diphenylAla: alanine

Pro: Proline Arg: Arginine

Aze: L-2-azetidine carboxylic acidPip: DL-Pipecolic acid

Representative compounds according to the present invention include the compounds shown in Tables 1a to 1d.

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, citric acid Organic carbonic acid such as acetic acid, trichloroacetic acid, 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, and the like. Acid addition salts formed by

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

The invention also includes hydrates, solvates of compounds of formula 1 and prodrugs that are converted to compounds of formula 1.

Representative examples of methods for preparing compounds of Formula 1 in the present invention are shown in Schemes 1 and 2 below.

Scheme 1 shows the synthesis of D- (diphe) Ala-Pro-Arg derivative compounds, wherein N-boc-D- (diphe) Ala-OH (Formula 2a) is substituted with proline methyl ester hydrochloride or an analog thereof (Formula 3) The reaction is carried out and hydrolyzed to obtain a carboxylic acid derivative (Formula 4a). This is then coupled with the amine derivative (Formula 5) to obtain an ester derivative (Formula 6a). When the ester derivative (Formula 6a) is directly reacted with guanidine, the final compound of Formula 1a 'is obtained. The boc group is removed from the ester derivative (Formula 6a) to obtain a compound of Formula 6a', and then reacted with guanidine to react with guanidine. The final compound is obtained. When the compound of Formula 6a ″ obtained by acylating or sulfonylating the compound of Formula 6a ′ is reacted with guanidine, the final compound of Formula 1a ″ ′ is obtained.

Scheme 2 shows the synthesis of a D- (diphe) Ala-Aze-Arg derivative compound. First, N-boc-Aze (Formula 7b) was coupled with an amine derivative (Formula 5b) to obtain an ester derivative (Formula 8b). The boc group is then removed and coupled with N-boc-D- (diphe) Ala-OH (Formula 2a) to give a compound of Formula 9b. The boc group is removed from the compound of formula 9b and sulfonylated to obtain a compound of formula 9b 'and then reacted with guanidine to obtain a final compound of formula 1b'.

Known coupling reagents that can be used for the coupling reaction of amino groups include dicyclohexylcarbodiimide (DCC), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide (EDC), bis- ( 2-oxo-3-oxazolidinyl) -phosphinic acid chloride (BOP-Cl), diphenylphosphoryl azide (DPPA), isobutylchloroformate and the like, but are not limited to these.

As mentioned above, the compound of Formula 1 according to the present invention has superior selectivity to thrombin and is orally administered as compared with known compounds. Therefore, the compound of the present invention is useful for the prevention of blood coagulation and treatment of thrombosis, the present invention provides a pharmaceutical composition for preventing blood coagulation and thrombosis containing the compound of formula (1) as an active ingredient.

The total daily dose 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 between 0.001 and 10 mg / kg body weight, but the specific dose level for a particular patient is determined by the specific compound or body weight to be used. May vary with sex, health status, diet, time of administration, method of administration, rate of excretion, drug mixture and severity of disease.

The compounds of the present invention can be administered in preparations for injection or oral administration 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 the known art. Solvents that can be used include water, Ringer's solution or isotonic NaCl solution, and sterile fixed oils are commonly used as a solvent or suspending medium. Any non-irritating fixed oil may be used for this purpose, including mono, diglycerides, and fatty acids such as oleic acid are used in the preparation of injectables. Solid dosage forms for oral administration may be capsules, tablets, pills, powders, or granules, 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 compound 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 advantages 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 administered by oral administration. The results of pharmacokinetic experiments demonstrate that oral administration of the pharmaceutical composition of the present invention confirms that the concentration of the drug is characterized by long-term maintenance in the blood. Therefore, the compound of the present invention is more useful in that it can be effectively used as a preparation for oral administration, unlike conventional thrombin inhibitors.

On the other hand, in the case of obtaining the desired anticoagulant effect and thrombolytic effect by clinically administering the compound of the present invention, the compound of the formula (1) according to the present invention simultaneously with at least one component selected from thrombolytic agents and platelet activity inhibitors May be administered. Thrombolytic agents that can be administered in combination with a compound of the present invention may include t-PA, urokinase, streptokinase, and the like, and platelet activity inhibitors include aspirin, ticlopidin , Clopidrogel, 7E3 monoantibody, and the like. However, preparations containing the compounds according to the invention for the purpose of the treatment and prevention of thrombosis are not limited to those described above, and any preparations useful for the treatment and prevention of thrombosis may be included.

EXAMPLE

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.

Hereinafter, the reagents used in the examples will be described by the following abbreviations.

DMF: Dimethylformamide

THF: Tetrahydrofuran

TEA: Triethylamine

DIPEA: Diisopropylethylamine

EDC: 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride

HOBt: hydroxybenzotriazole

[Production example]

Production Example 1-1 (2S) -1-{(2R) -2-[(tert-butoxycarbonyl) amino] -3,3-diphenylpropanoyl} -2-pyrrolidinecarboxyl Acid synthesis

Boc-D- (diphe) Ala-OH (3.01 g, 8.82 mmol), proline methylester hydrochloride (1.75 g, 1.2 equiv), EDC (2.54 g, 1.5 equiv), HOBt (1.79 g, 1.5 equiv) approx. The solution was dissolved in 40 ml of DMF, maintained at 0 ° C., and then stirred at room temperature for one day after adding TEA (4.92 ml, 4.0 equiv). Concentrated at low pressure and the residue was dissolved in ethyl acetate and then extracted-dried-condensed by conventional methods. Separation-purification using column chromatography (30% ethyl acetate / hexanes) gave 3.88 g (yield: 97%) of the title compound as a pale yellow oil.

The obtained compound was dissolved in 50 ml of a mixed solvent (THF: MeOH: H ₂ O = 3: 2: 1), and lithium hydroxide (0.51 g, 1.4 equivalents) was added thereto, followed by stirring at room temperature for 3 hours. Neutralized with 6N HCl (2.2 mL), concentrated at low pressure, the residue was dissolved in ethyl acetate and extracted-dried-concentrated by conventional methods to yield 3.76 g of the title compound as a white powder quantitatively.

¹ H-NMR (CDCl ₃ , 500 MHz) δ 7.30-7.15 (m, 10H), 5.12 (m, 2H), 4.36 (m, 1H), 4.14 (m, 1H), 3.75 (m, 1H), 2.70 ( m, 1H), 2.22 (m, 1H), 1.81 (m, 1H), 1.53 (m, 1H), 1.33 (s, 9H).

Production Example 1-2 (2S) -1-{(2R) -2-[(tert-butoxycarbonyl) amino] -3,3-diphenylpropanoyl} -2-piperidinecarboxyl Synthesis of Acids (Diastereoisomers)

The title compound was obtained in a similar manner to Preparation Example 1-1.

¹ H-NMR (CDCl ₃ , 500 MHz) δ 7.38-7.19 (m, 10H), 5.55-5.04 (m, 2H), 5.19 (m, 0.5H), 4.61 (m, 0.5H), 4.44 (m, 1H ), 4.01 and 3.83 (br d, 1H), 3.09 and 2.50 (m, 1H), 2.16 and 1.88 (m, 1H), 1.69-1.20 (m, 14H).

Preparation Example 2-1 Synthesis of γ-Aminobutyryl Acid Methyl Ester Hydrochloride

2,2-dimethoxypropane (175ml, 1.42mol) was added to γ-aminobutyryl acid (10.3g, 0.10mol), and then c-HCl (15ml) was added and stirred at room temperature for one day. After concentration at low pressure, the residue was dissolved in about 20 ml of methanol, and then, 200 ml of dimethyl ether was added, and the resulting precipitate was filtered and washed with dimethyl ether. Recrystallization with methanol and dimethyl ether gave 14.6 g (yield: 95%) of white crystals.

¹ H-NMR (D ₂ O, 300 MHz) δ 3.74 (s, 3H), 3.07 (t, J = 7.6 Hz, 2H), 2.55 (t, J = 7.3 Hz, 2H), 1.99 (m, 2H).

Preparation Example 2-2 Synthesis of β-alanine Methyl Ester Hydrochloride

Thionyl chloride (29.4 g, 18.0 mL, 2.2 equiv) was slowly added to 200 mL of methanol at -78 ° C. After stirring at this temperature for 30 minutes, β-alanine (10 g, 0.112 mol) was added thereto, followed by stirring for one day while slowly raising the temperature to room temperature. Concentration at low pressure, 200 ml of dimethyl ether was added to the residue, and the precipitate formed by trituration was filtered and dried to obtain 14.9 g (yield: 95%) of white powder.

¹ H-NMR (D ₂ O, 500 MHz) δ 3.79 (s, 3H), 3.34 (t, J = 6.4 Hz, 2H), 2.86 (t, J = 6.4 Hz, 2H).

Preparation Example 3-1 Methyl 4-{[((2S) -1-{(2R) -2-[(tert-butoxycarbonyl) amino] -3,3-diphenylpropanoyl} pyrroli Synthesis of diyl) carbonyl] amino} butanoate

Compound (546 mg, 1.245 mmol) of Preparation Example 1-1, γ-aminobutyryl acid methyl ester hydrochloride (230 mg, 1.2 equivalents) of Preparation Example 2-1, EDC (310 mg, 1.3 equivalents), HOBt (219 mg, 1.3 equivalents) ) Was dissolved in about 10 mL of DMF, maintained at 0 ° C., and then DIPEA (0.65 mL, 3.0 equivalents) was added thereto, followed by stirring at room temperature for one day. Concentrate at low pressure to dissolve the residue in ethyl acetate and extract-dry-concentrate in conventional manner. Separation-purification using tube chromatography (40% ethyl acetate / hexanes) gave 603 mg (yield: 90%) of the title compound as a white foam.

¹ H-NMR (CDCl ₃ , 300 MHz) δ 7.40-7.05 (m, 11H), 4.93 (br, 1H), 4.85 (dd, 1H), 4.38 (d, J = 11.4 Hz, 1H), 4.29 (d, J = 8.0Hz, 1H), 3.64 (s, 3H), 3.58 (m, 1H), 3.28 (m, 1H), 3.10 (m, 1H), 2.51 (m, 1H), 2.30 (m, 2H), 2.07 (m, 1H), 1.80 (m, 2H), 1.59 (m, 3H), 1.36 (s, 9H).

Preparation Example 3-2 Methyl 3-{[((2S) -1-{(2R) -2-[(tert-butoxycarbonyl) amino] -3,3-diphenylpropanoyl} pyrroli Synthesis of diyl) carbonyl] amino} propanoate

The title compound was obtained in a similar manner to Preparation Example 3-1.

¹ H-NMR (CDCl ₃ , 500 MHz) δ 7.40-7.15 (m, 10H + NH), 4.94 (d, 1H), 4.88 (m, 1H), 4.37 (d, J = 11.4 Hz, 1H), 4.25 ( d, J = 8.25 Hz, 1H), 3.66 (s, 3H), 3.61 (m, 1H), 3.53 (m, 1H), 3.34 (m, 2H), 2.54 (m, 2H), 2.46 (m, 1H ), 2.08 (m, 1H), 1.70 (m, 1H), 1.44 (m, 1H), 1.35 (s, 9H), 1.27 (m, 1H).

Preparation Example 3-3 Methyl 4-{[((2S) -1-{(2R) -2-[(tert-butoxycarbonyl) amino] -3,3-diphenylpropanoyl} piperidi Synthesis of nil) carbonyl] amino} butanoate (diastereomer)

The title compound was obtained in a similar manner to Preparation Example 3-1.

¹ H-NMR (CDCl ₃ , 500 MHz) δ 7.40-7.05 (m, 11H), 5.20-4.70 (m, 2H), 4.55-4.18 (m, 2H), 3.69 and 3.63 (two s, 3H), 3.80- 2.90 (m, 3H), 2.40-2.20 (m, 4H), 1.90-0.80 (m, 7H), 1.35, 1.34 and 1.32 (three s, 9H).

Preparation Example 3-4 Methyl 3-{[((2S) -1-{(2R) -2-[(tert-butoxycarbonyl) amino] -3,3-diphenylpropanoyl} piperidi Synthesis of nil) carbonyl) amino} propanoate (diastereomer)

The title compound was obtained in a similar manner to Preparation Example 3-1.

¹ H-NMR (CDCl ₃ , 500 MHz) δ 7.40-7.15 (m, 10H + NH), 5.20-4.95 (m, 2H), 4.50-4.13 (m, 2H), 3.69 and 3.65 (two s, 3H), 3.65-3.15 (m, 3H), 2.95 (m, 1H), 2.57 (m, 2H), 2.38 (m, 2H), 1.50-0.86 (m, 4H), 1.35 and 1.34 (two s, 9H).

Production Example 4-1 Methyl 4-[({(2S) -1-[(2R) -2-amino-3,3-diphenylpropanoyl] pyrrolidinyl} carbonyl) amino] butanoate Synthesis of

6 ml of a 30% trifluoroacetic acid / dichloromethane solution was added to the compound of Preparation Example 3-1 (300 mg, 0.558 mmol) at 0 ° C. and stirred at room temperature for 1.5 hours. Concentrated at low pressure, the residue was dissolved in ethyl acetate, washed with saturated sodium bicarbonate, saturated sodium chloride, and dried under reduced pressure to give 238 mg (yield: 98%) of the title compound as a white foam.

¹ H-NMR (CDCl ₃ , 300 MHz) δ 7.40-7.15 (m, 10H), 4.26 (m, 3H), 3.65 (s, 3H), 3.48 (m, 1H), 3.35-3.12 (m, 2H), 2.64 (m, 1H), 2.33 (m, 2H), 2.10 (m, 1H), 1.80-1.20 (m, 6H).

Production Example 4-2 to 4-4

The following compounds were used in the reaction without removing boc groups in a similar manner and without purification.

Production Example 5-1 Methyl 4-[({(2S) -1-[(2R) -2- (acetylamino) -3,3-diphenylpropanoyl] pyrrolidinyl} carbonyl) amino] Synthesis of Butanoate

Compound (71 mg, 0.161 mmol) and DIPEA (67 mg, 3.0 equivalents) of Preparation Example 4-1 were dissolved in 3 ml of dichloromethane, followed by addition of acetyl chloride (31 mg, 1.5 equivalents). After stirring for one day at room temperature, extraction-drying-condensation concentration was carried out in a conventional manner to obtain a residue, which was separated and purified by column chromatography (80% ethyl acetate-hexane-ethyl acetate) to yield 61 mg (yield: 80%). The title compound was obtained as a pale yellow oil.

Production Example 5-2 2-{[(1R) -1-Benzhydryl-2-((2S) -2-{[(4-methoxy-4-oxobutyl) amino] carbonyl} pyrroli Synthesis of diyl) -2-oxoethyl] amino} acetic acid

3 ml of acetonitrile were added to the compound of Preparation Example 4-1 (127 mg, 0.290 mmol), benzyl bromoacetate (104 mg, 1.5 equiv) and anhydrous potassium carbonate (82 mg, 2.0 equiv), followed by stirring at room temperature for one day. Extract, dry, and decompression concentrate in a conventional manner to obtain a residue, which was separated-purified by column chromatography (80% ethyl acetate-hexane-ethyl acetate) to give 61 mg (yield: 80%) of the title compound as a pale yellow oil. Got it. Concentrated at low pressure, the residue was dissolved in ethyl acetate, washed with sodium chloride solution and dried-concentrated. This was separated-purified by column chromatography (60% ethyl acetate-hexane-ethyl acetate) and 142 mg (yield: 84%) of BnO ₂ CCH ₂ NH-D- (diphe) Ala-Pro-NH (CH ₂ ) ₃ CO ₂ Me was obtained.

The compound (142 mg, 0.242 mmol) was dissolved in 10 ml of ethanol and Pd / C (Degussa type E101, 10%, 50 mg) was added, followed by hydrogenation for 2 hours. The reaction mixture was passed through celite, washed with methanol and concentrated to give 109 mg (yield: 91%) of the title compound.

Production Example 5-3 Methyl 4-{[((2S) -1-{(2R) -2-[(methylsulfonyl) amino] -3,3-diphenylpropanoyl} pyrrolidinyl) car Synthesis of Bonyl] amino} butanoate

The compound of Preparation Example 4-1 (75 mg, 0.171 mmol) and DIPEA (86 mg, 4.0 equiv) were dissolved in 4 mL of THF and MsCl (25 mg, 1.3 equiv) was added thereto. After stirring for one day at room temperature, extraction-drying-concentrating-concentration-concentrated-in-concentration-produced-results, and separated-purified by column chromatography (60% ethyl acetate-hexane-ethyl acetate) to yield 80 mg (yield: 91%). The title compound was obtained as a pale yellow oil.

¹ H-NMR (CDCl ₃ , 300 MHz) δ 7.45-7.05 (m, 10H), 6.64 (br, 1H), 5.07 (d, J = 8.4 Hz, 1H), 4.86 (m, 1H), 4.35 (d, J = 11.1 Hz, 1H), 4.13 (m, 1H), 3.65 (s, 3H), 3.59 (m, 1H), 3.26 (m, 2H), 2.85 (s, 3H), 2.58 (m, 1H), 2.33 (m, 2H), 2.04 (m, 1H), 1.80 (m, 3H), 1.58 (m, 2H).

Preparation Example 5-4 Methyl 4-{[((2S) -1-{(2R) -2-[(benzylsulfonyl) amino] -3,3-diphenylpropanoyl} pyrrolidinyl) car Synthesis of Bonyl] amino} butanoate

The title compound was obtained in a similar manner to Preparation Example 5-3.

¹ H-NMR (CDCl ₃ , 300 MHz) δ 7.40-7.05 (m, 15H), 6.70 (br, 1H), 4.90 (m, 1H), 4.77 (d, J = 8.25 Hz, 1H), 4.33 (d, J = 11.1 Hz, 1H), 4.22 (m, 1H), 4.12 (ABq, J = 38.4, 13.6 Hz, 2H), 3.62 (s, 3H), 3.59 (m, 1H), 3.21 (m, 2H), 2.55 (m, 1H), 2.30 (m, 2H), 2.04 (m, 1H), 1.78 (m, 3H), 1.40 (m, 2H).

Production Example 5-5 Methyl 3-{[((2S) -1-{(2R) -2-[(methylsulfonyl) amino] -3,3-diphenylpropanoyl} pyrrolidinyl) car Synthesis of Bonyl] amino} propanoate

The title compound was obtained in a similar manner to Preparation Example 5-3.

¹ H-NMR (CDCl ₃ , 500 MHz) δ 7.40-7.20 (m, 10H), 6.87 (br, 1H), 5.06 (d, J = 8.2 Hz, 1H), 4.88 (m, 1H), 4.34 (d, J = 11.0 Hz, 1H), 4.17 (d, J = 6.0 Hz, 1H), 3.68 (s, 3H), 3.60 (m, 1H). 3.45 (m, 2H), 2.83 (s, 3H), 2.62 (m, 1H), 2.50 (m, 2H), 2.03 (m, 1H), 1.77 (m, 1H), 1.42 (m, 2H).

Production Example 5-6 Methyl 3-{((2S) -1-{(2R) -2-[(ethylsulfonyl) amino] -3,3-diphenylpropanoyl} pyrrolidinyl) carbonyl ] Synthesis of amino} propanoate

The title compound was obtained in a similar manner to Preparation Example 5-3.

¹ H-NMR (CDCl ₃ , 500 MHz) δ 7.40-7.20 (m, 10H), 6.91 (br, 1H), 4.97 (d, J = 8.7 Hz, 1H), 4.84 (m, 1H), 4.33 (d, J = 11Hz, 1H), 4.19 (d, J = 6.4Hz, 1H), 3.67 (s, 3H), 3.62 (m, 1H), 3.45 (m, 2H), 2.96-2.80 (m, 2H), 2.62 (m, 1H), 2.52 (m, 2H), 2.03 (m, 1H), 1.75 (m, 1H), 1.42 (m, 2H), 1.13 (t, J = 7.3 Hz, 3H).

Production Example 5-7 Methyl 3-{[((2S) -1-{(2R) -2-[(benzylsulfonyl) amino] -3,3-diphenylpropanoyl} pyrrolidinyl) car Synthesis of Bonyl] amino} propanoate

The title compound was obtained in a similar manner to Preparation Example 5-3.

¹ H-NMR (CDCl ₃ , 500 MHz) δ 7.45-7.10 (m, 15H), 6.95 (br, 1H), 4.95 (dd, J = 11.5, 8.25 Hz, 1H), 4.78 (d, J = 8.25 Hz, 1H), 4.32 (d, 1H), 4.17 (m, 1H), 4.08 (ABq, J = 67.1, 13.3 Hz, 2H), 3.63 (m, 1H), 3.61 (s, 3H), 3.48-3.36 (m , 2H), 2.60 (m, 1H), 2.47 (m, 2H), 2.03 (m, 1H).

Production Example 5-8 Methyl 3-{[((2S) -1-{(2R) -3,3-diphenyl-2-[(propylsulfonyl) amino] propanoyl} pyrrolidinyl) car Synthesis of Bonyl] amino} propanoate

The title compound was obtained in a similar manner to Preparation Example 5-3.

¹ H-NMR (CDCl ₃ , 500 MHz) δ 7.42-7.18 (m, 10H), 6.92 (br, 1H), 4.99 (d, J = 9.15 Hz, 1H), 4.84 (m, 1H), 4.33 (d, J = 11.45 Hz, 1H), 4.20 (m, 1H), 3.67 (s, 3H), 3.62 (m, 1H), 3.44 (m, 2H), 2.88-2.73 (m, 2H), 2.61 (m, 1H ), 2.52 (t, 2H).

Production Example 5-9 Methyl 4-{[((2S) -1-{(2R) -2-[(methylsulfonyl) amino] -3,3-diphenylpropanoyl} piperidinyl) car Synthesis of Bonyl] amino} butanoate (diastereomer)

The title compound was obtained in a similar manner to Preparation Example 5-3.

¹ H-NMR (CDCl ₃ , 500 MHz) δ 7.45-7.20 (m, 10H), 6.80-6.55 (two br s, 1H), 5.35-5.00 (m, 2H), 4.80-4.05 (m, 2H), 3.69 And 3.64 (two s, 3H), 3.70-2.70 (m, 3H), 2.82 (s, 3H), 2.40-2.20 (m, 4H), 1.85-0.85 (m, 7H).

Production Example 5-10 Methyl 3-{[(2S) -1-{((2R) -2-[(methylsulfonyl) amino] -3,3-diphenylpropanoyl} piperidinyl) car Synthesis of Bonyl] amino} propanoate (diastereomer)

The title compound was obtained in a similar manner to Preparation Example 5-3.

¹ H-NMR (CDCl ₃ , 500 MHz) δ 7.45-7.20 (m, 10H), 6.81 and 6.70 (two br s, 1H), 5.35-5.00 (m, 2H), 4.50-4.00 (m, 2H), 3.71 And 3.66 (two s, 3H), 3.80-3.00 (m, 3H), 2.81 (s, 3H), 2.85-2.20 (m, 5H), 1.95-0.85 (m, 4H).

Preparation Example 6-1 Synthesis of tert-butyl (2S) -2-{[(3-methoxy-3-oxopropyl) amino] carbonyl} -1-azetidinecarboxylate

The title compound was obtained in a similar manner to Preparation Example 3.

¹ H-NMR (CDCl ₃ , 500 MHz) δ 4.58 (t, J = 7.3 Hz, 1H), 3.88 (q, J = 8.25 Hz, 1H), 3.77 (m, 1H), 3.68 (s, 3H), 3.55 (m, 2H), 2.57 (m, 2H), 2.42 (m, 2H), 1.45 (s, 9H).

Production Example 6-2 Methyl 3-{[((2S) -1-{(2R) -2-[(tert-butoxycarbonyl) amino] -3,3-diphenylpropanoyl} azeti Synthesis of diyl) carbonyl] amino} propanoate

The title compound was obtained in a similar manner to Preparation Examples 3 and 4.

¹ H-NMR (CDCl ₃ , 500 MHz) δ 7.86 (br, 1H), 7.40-7.20 (m, 10H), 4.93 (d, J = 8.25 Hz, 1H), 4.67 (dd, J = 8.25, 11.0 Hz, 1H), 4.38 (m, 1H), 4.32 (d, J = 11.0 Hz, 1H), 3.99 (m, 1H), 3.69 (s, 3H), 3.50 (m, 2H), 2.95 (m, 1H), 2.54 (m, 2H), 2.26 (m, 1H), 1.92 (m, 1H), 1.34 (s, 9H).

Production Example 6-3 Methyl 3-{[((2S) -1-{(2R) -2-[(methylsulfonyl) amino] -3,3-diphenylpropanoyl} azetidinyl) car Synthesis of Bonyl] amino} propanoate

The title compound was obtained in a similar manner to Preparation Examples 4 and 5-3.

¹ H-NMR (CDCl ₃ , 500 MHz) δ 7.60 (br, 1H), 7.40-7.25 (m, 10H), 5.09 (d, J = 8.25 Hz, 1H), 4.55 (dd, J = 8.25, 11 Hz, 1H ), 4.39 (m, 1H), 4.33 (d, J = 11 Hz, 1H), 4.01 (m, 1H), 3.70 (s, 3H), 3.49 (m, 2H), 3.07 (m, 1H), 2.75 ( s, 3H), 2.51 (t, J = 6.4 Hz, 2H), 2.29 (m, 1H), 1.95 (m, 1H).

EXAMPLE

Example 1 tert-butyl- (1R) -1-benzhydryl-2-{(2S) -2-[({4-[(diaminomethylene) amino] -4-oxobutyl} amino) carb Synthesis of Bonyl] pyrrolidinyl} -2-oxoethylcarbamate (Compound 1)

Methyl 4-{[((2S) -1-{(2R) -2-[(tert-butoxycarbonyl) amino] -3,3-diphenylpropanoyl} pyrrolidinyl of Preparation Example 3-1 ) Carbonyl] amino} butanoate (67 mg, 0.125 mmol) and guanidine hydrochloride (60 mg, 5.0 equiv) were dissolved in 4 mL of degassed DMF and NaH (23 mg, 4.5 equiv) was added. The mixture was stirred at 60 ° C. for one day and then concentrated at low pressure, and the residue was first separated-purified by column chromatography (20% methanol-dichloromethane). Secondary purification was carried out with RP-HPLC (λ = 232 nm, 15 ml / min, CH ₃ CN: H ₂ O: TFA = 45: 55: 0.1, Microsorb C ₁₈ 80-220-C8), followed by lyophilization to 42 mg (yield). : 51%) as a white powder.

^OneH-NMR (DMSO-d₆, 500 MHz) δ 11.36 (s, 1H), 8.24 (br s, 4H), 7.55 (t, J = 5.5 Hz, 1H), 7.40-7.10 (m, 11H), 5.10 (m, 1H), 4.34 (d, J = 11.5 Hz, 1H)), 3.81 (m, 1H), 3.63 (m, 1H), 3.10-3.00 (m, 3H), 2.39 (m, 2H), 1.80-1.10 (m, 6H), 1.27 ( s, 9H).

HR-MASS calculated: M + H ⁺ (C ₃₀ H ₄₁ N ₆ O ₅ ): 565.3138. Found: 565.3152.

[Examples 2 to 15]

In a similar manner to Example 1, the compounds listed in Tables 2A and 2C were obtained.

Example 16 (2S) -1-{(2R) -2-[(aminosulfonyl) amino] -3,3-diphenylpropanoyl} -N- {3-[(diaminomethylene) amino ] -3-Oxopropyl} -2-pyrrolidinecarboxamide (Compound 16)

Compound 8 (102 mg, 0.185 mmol) obtained from Example 8 was dissolved in 5 ml of saturated HCl / ethyl acetate, stirred at room temperature for 1 hour and concentrated under low pressure. 4 ml of dichloromethane and TEA (136 µl, 5.0 equiv.) Were added to the residue, followed by 2.0 M chlorosulfamide / dichloromethane (ClSO ₂ NH ₂ , 100 µl, 1.0 equiv.), Followed by stirring for 3 hours. Concentration at low pressure gave the residue primary separation-purification by column chromatography (20% methanol-dichloromethane). This was secondarily purified by RP-HPLC (λ = 232 nm, 15 mL / min, CH ₃ CN: H ₂ O: TFA = 30: 70: 0.1, Microsorb C ₁₈ 80-220-C8) and lyophilized to 9 mg (yield). : 8%) as a white powder.

¹ H-NMR (DMSO-d ₆ , 500 MHz) δ 11.18 (s, 1H), 8.35-8.05 (br s, 4H), 7.83 (m, 1H), 7.45-7.15 (m, 11H), 6.52 (s, 2H), 4.83 (m, 2H), 4.25 (d, J = 11.5 Hz, 1H), 3.80 (m, 1H), 3.66 (m, 1H), 3.30-3.20 (m, 2H), 2.94 (m, 1H ), 2.55 (m, 2H), 1.70-1.30 (m, 4H).

HR-MASS calculated: M + H ⁺ (C ₂₄ H ₃₂ N ₇ O ₅ S): 530.2186, found: 530.2192.

Experimental Example

Experimental Example 1 Inhibitory Activity of a Thrombin Inhibitor

The thrombin inhibitory effect of the compound of formula 1 according to the present invention is described in Methods in Enzymology Vol. 80 pp341-361; Biochemistry 27 pp 2144-2151, 1988] was determined by determining the dissociation constant Ki value using the following formula.

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

[E]: concentration of enzyme not bound to inhibitor

[I]: concentration of inhibitor not bound to enzyme

[EI]: concentration of the combination of enzyme and inhibitor

Since the dissociation constant Ki indicates the degree of dissociation between the enzyme and the thrombin inhibitor, the smaller the dissociation constant, the greater the binding capacity of the inhibitor to the enzyme. This dissociation constant can be obtained by reacting with a specific substrate that develops upon hydrolysis under the action of thrombin and measuring the degree of color development as a function of time according to spectrophotometry.

In this experimental example, chromozyme TH (Chromozyme TH: Tosyl-Gly-Pro-Arg-4-nitroanilide acetate) was used as a substance for the coloration upon hydrolysis under the action of thrombin. Chromozyme TH is hydrolyzed by thrombin to produce yellow para-nitroanilide. Therefore, the thrombin inhibitory activity of the compound according to the present invention can be measured by measuring the amount of para-nitroaniline produced by the change in absorbance with time. That is, the activity of the enzyme can be measured from the change in absorbance, which may be directly related to the ability of the thrombin inhibitor to inhibit the enzyme activity.

In order to determine the selectivity for thrombin inhibition to trypsin of the compound according to the present invention was carried out in the same manner as the method for measuring the thrombin inhibitory activity was measured by Ki value to determine the inhibitory effect of the compound of formula 1 to trypsin and then trypsin / thrombin The ratio of was calculated. At this time, the test method for trypsin is performed in the same manner as thrombin, but N-benzoyl-valine-glycine-arginine-para-nitroanilide hydrochloride (N-benzoyl-Val-Gly-Arg-p-nitroanilide hydrochloride) as a substrate Was used.

Specifically, the ability to inhibit thrombin activity of the compounds according to the present invention was measured as follows.

1160 μl of 0.1M Tris buffer (pH 7.8) containing 150 mM NaCl and 0.1% PEG 8000 (polyethylene glycol, molecular weight about 8,000) was added to a 1.5 mL cuvette. 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 inhibitor compound according to the present invention was dissolved in dimethyl sulfoxide to 10 mg / ml and diluted with the buffer solution at a concentration of 0.1 mg / ml, 0.01 mg / ml, and 0.001 mg / ml. The solution was taken up to the amount of 3, and the total volume was added to the cuvette with 100 μl of Tris buffer solution.

15 μl of human thrombin dissolved in the Tris buffer solution at a concentration of 0.1 mg / ml was added to the cuvette containing the reaction solution at room temperature to initiate the enzymatic hydrolysis reaction. The amount of para-nitroanilide produced by the reaction for two minutes from the moment of addition of the enzyme was monitored by the change in absorbance at 381 nm, showing the continuous spectrum of reaction time versus absorbance. The above experiments were performed on different inhibitor concentrations to obtain continuous spectra.

After obtaining the initial velocity Vi from the slope within the initial 30 seconds of the reaction time in each spectrum, an inverse graph of the initial velocity versus the inhibitor concentration (1 / Vi) is shown. After calculating the first equation satisfying the points on the graph, Ki was calculated from the x-intercept of the equation using the enzyme reaction equation. The K _m 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 compounds according to the invention against trypsin was also measured as described above.

As substrate, a 20 μM solution of N-benzoyl-Val-Gly-Arg-p-nitroanilide hydrochloride was used, and the inhibitor was in the range of 0-120 μg. Various concentrations were used at. In addition, trypsin was dissolved in 0.1N HCl to make 45 ㎍ / ㎖ with the Tris buffer solution just before the experiment 40 ul was used. In the same way as for the thrombin, the total volume of the reaction solution was set to 1.5 ml and the same method was used. The K _m value used for the Ki calculation was also determined by the same method, which was 20.2 μM.

The enzymatic activity of the inhibitor according to the present invention measured for thrombin and trypsin according to the method described above is expressed in Ki values, and the selectivity to thrombin is expressed in trypsin / thrombin. The results are shown in Table 3 [Inhibition ability of thrombin and trypsin of the compound according to the present invention].

Experimental Example 2 Inhibitory Effect on Thrombus Formation in Animals

Inhibition of the compounds according to the present invention against thrombus formation was measured in animal models. Animals used were Sprague Dawley, weighing 250-300 g, which were bred using commercial standard feed at a temperature of 20-22 ° C and 12-hour contrast in the laboratory of laboratory of LG Chem Institute of Technology. Four were used. First, rats were anesthetized by intraperitoneal injection of urethane at a dose of 1.3 g / kg body weight. The abdomen of the rat was excised to expose the inferior vena cava, and then sutures were applied to the vein of the lower part and 1.6 cm below the left renal vein. The compound of the example was injected through the left femoral vein at a dose of 5 mg / kg body weight and 5 minutes later, thromboplastin was also injected through the femoral vein. Subsequently, the sutures laid down after 30 seconds were ligated. Fifteen minutes after the suture was ligated, the veins were removed and the thrombus produced was taken and weighed.

The thrombus formation inhibitory effect of the compound according to the present invention was calculated by comparing the degree of thrombus formation of the control group administered with the same volume of 10% HPCD (hydroxypropyl β-cyclodextrin) solution, and the results are shown in Table 4 below. Thrombus formation inhibitory effect].

Experimental Example 3 Pharmacokinetics of Compounds According to the Present Invention

The absorption effect upon oral administration of the compound according to the present invention was determined by measuring blood drug concentration according to the following experimental method. Male rats and dogs were fasted for 18 hours each and used for the experiment. A 1% (10 mg / ml) solution of the example compound was prepared using an appropriate dissolution aid and then administered orally. Blood was collected at a predetermined time interval, followed by liquid extraction with methylene chloride and back extraction with dilute hydrochloric acid solution to measure blood drug concentration by high pressure liquid chromatography (HPLC). As a result, some example compounds showed a significantly higher oral absorption in animals. As an example, the results of Example 9 compounds are given in Table 5 [Example 9 compound oral administration to rats at a concentration of 30 mg / kg over time] and 6 [Example 9 compounds to dogs at 10 mg / kg] In the case of oral administration in concentration, blood drug concentration with time] is shown.

Table 5 shows that when the compound of Example 9 was administered orally to rats, it was absorbed into the gastrointestinal tract and detected in the blood at high concentrations up to 2 hours after administration. From Table 6, the compound of Example 9 was also orally administered to dogs. Although the dose was 1/3 of the dose compared to that of the rat, the blood concentration peak of the absorbed drug was similar to that of the rat.

Since the thrombin inhibitor according to the present invention is highly selective and orally administrable, it can be usefully used for preventing blood clotting and treating thrombosis.

Claims (5)

Compounds of Formula 1, pharmaceutically acceptable salts, hydrates, solvates and isomers thereof: [Formula 1] Wherein R ¹ is hydrogen, R ₂ NCOCO-, RCO-, RSO _2- , ROCO-, R ₂ NCOCH _2- , RNHSO _2- , ROSO _2- , ROCOCH _2- , R is hydrogen, 1 to Lower alkyl of 8, aryl of 6 to 10 carbon atoms, arylalkyl of 5 to 12 carbon atoms, heteroaryl of 4 to 10 carbon atoms, heteroarylalkyl of 5 to 11 carbon atoms, or cycloalkyl of 3 to 8 carbon atoms, , R ² is hydrogen, fluorine, chlorine, bromine, iodine, hydroxy or methoxy, X and X 'are each independently hydrogen, fluorine, chlorine, bromine, iodine, formyl, cyano, hydroxy or methoxy, substituted at the meta- or para- position, n is 0, 1 or 2 and m is 2 or 3. The compound of claim 1, wherein R is hydrogen, lower alkyl of 1 to 8 carbon atoms, aryl of 6 to 10 carbon atoms, or arylalkyl of 5 to 12 carbon atoms, R ² is hydrogen, and X and X 'are hydrogen compound. 3. R ¹ is RSO _2- , R ₂ NCOCH ₂ -or RNHSO _2- , R is hydrogen, lower alkyl of 1 to 3 carbon atoms, aryl of 6 carbon atoms or aryl of 5 to 12 carbon atoms Alkyl, n is 1 and m is 2. 6. The method according to any one of claims 1 to 3, tert-butyl- (1R) -1-benzhydryl-2-{(2S) -2-[({4-[(diaminomethylene) amino] -4-oxobutyl} amino) carbonyl] pyrrolidinyl } -2-oxoethylcarbamate; (2S) -N- {4-[(diaminomethylene) amino] -4-oxobutyl} -1-{(2R) -2-[(methylsulfonyl) amino] -3,3-diphenylpropano Il} -2-pyrrolidinecarboxamide; (2S) -1-[(2R) -2- (acetylamino) -3,3-diphenylpropanoyl] -N- {4-[(diaminomethylene) amino] -4-oxobutyl} -2 -Pyrrolidinecarboxamide; (2S) -1-[(2R) -2-amino-3,3-diphenylpropanoyl] -N- {4-[(diaminomethylene) amino] -4-oxobutyl} -2-pyrroli Dine carboxamide; tert-butyl- (1R) -1-benzhydryl-2- {2-[({4-[(diaminomethylene) amino] -oxobutyl} amino) carbonyl] -1-piperidinyl} -2 Oxoethyl carbamate; 2-[((1R) -1-Benzhydryl-2-{(2S) -2-[({4-[(diaminomethylene) amino] -4-oxobutyl} amino) carbonyl] pyrrolidinyl } -2-oxoethyl) amino] acetic acid; (2S) -1-{(2R) -2-[(benzylsulfonyl) amino] -3,3-diphenylpropanoyl} -N- {4-[(diaminomethylene) amino] -4-oxo Butyl} -2-pyrrolidinecarboxamide; tert-butyl- (1R) -1-benzhydryl-2-{(2S) -2-[({3-[(diaminomethylene) amino] -3-oxopropyl} amino) carbonyl] pyrrolidinyl } -2-oxoethylcarbamate; (2S) -N- {3-[(diaminomethylene) amino] -3-oxopropyl) -1-{(2R) -2-[(methylsulfonyl) amino] -3,3-diphenylpropano Il} -2-pyrrolidinecarboxamide; (2S) -N- {3-[(diaminomethylene) amino] -3-oxopropyl} -1-{(2R) -2-[(ethylsulfonyl) amino] -3,3-diphenylpropano Il} -2-pyrrolidinecarboxamide; (2S) -1-{(2R) -2-[(benzylsulfonyl) amino} -3,3-diphenylpropanoyl} -N- {3-[(diaminomethylene) amino] -3-oxo Propyl} -2-pyrrolidinecarboxamide; (2S) -N- {3-[(diaminomethylene) amino] -3-oxopropyl} -1-{(2R) -3,3-diphenyl-2-[(propylsulfonyl) amino] propano Il} -2-pyrrolidinecarboxamide; N- {3-[(diaminomethylene) amino] -3-oxopropyl} -1-{(2R) -2-[(methylsulfonyl) amino] -3,3-diphenylpropanoyl} -2 Piperidinecarboxamide; (2S) -N- {3-[(diaminomethylene) amino] -3-oxopropyl} -1-{(2R) -2-[(methylsulfonyl) amino] -3,3-diphenylpropano Il} -2-azetidinecarboxamide; N- {4-[(diaminomethylene) amino] -4-oxobutyl} -1-{(2R) -2-[(methylsulfonyl) amino] -3,3-diphenylpropanoyl} -2 Piperidinecarboxamide; or (2S) -1-{(2R) -2-[(aminosulfonyl) amino] -3,3-diphenylpropanoyl} -N- {3-[(diaminomethylene) amino] -3-oxo Propyl} -2-pyrrolidinecarboxamide. The method of claim 4, wherein (2S) -N- {4-[(diaminomethylene) amino] -4-oxobutyl} -1-{(2R) -2-[(methylsulfonyl) amino] -3,3-diphenylpropano Il} -2-pyrrolidinecarboxamide; tert-butyl- (1R) -1-benzhydryl-2-{(2S) -2-[({3-[(diaminomethylene) amino] -3-oxopropyl} amino) carbonyl] pyrrolidinyl } -2-oxoethylcarbamate; (2S) -1-{(2R) -2-[(benzylsulfonyl) amino] -3,3-diphenylpropanoyl} -N- {4-[(diaminomethylene) amino] -4-oxo Butyl} -2-pyrrolidinecarboxamide; (2S) -N- {3-[(diaminomethylene) amino] -3-oxopropyl) -1-{(2R) -2-[(methylsulfonyl) amino] -3,3-diphenylpropano Il} -2-pyrrolidinecarboxamide; (2S) -N- {3-[(diaminomethylene) amino] -3-oxopropyl} -1-{(2R) -2-[(ethylsulfonyl) amino] -3,3-diphenylpropano Il} -2-pyrrolidinecarboxamide; (2S) -1-{(2R) -2-[(benzylsulfonyl) amino} -3,3-diphenylpropanoyl} -N- {3-[(diaminomethylene) amino] -3-oxo Propyl} -2-pyrrolidinecarboxamide; (2S) -N- {3-[(diaminomethylene) amino] -3-oxopropyl} -1-{(2R) -3,3-diphenyl-2-[(propylsulfonyl) amino] propano Il} -2-pyrrolidinecarboxamide; or (2S) -1-{(2R) -2-[(aminosulfonyl) amino] -3,3-diphenylpropanoyl} -N- {3-[(diaminomethylene) amino] -3-oxo Propyl} -2-pyrrolidinecarboxamide.