Compound having effect of inhibiting platelet aggregation and salt thereof, and composition for preventing or treating thrombotic diseases, containing same

Abstract

The present invention relates to a novel compound having an effect of inhibiting platelet aggregation and a salt thereof and, more specifically, to: a novel platelet aggregation inhibitor specifically inhibiting shear stress-induced platelet aggregation; a pharmaceutical composition containing the same as an active ingredient; and a preparation method therefor.

Claims

1. A compound represented by the following Formula (I) or (II), or a pharmaceutically acceptable salt or stereoisomer thereof: ##STR00028## wherein, R.sub.1 is hydroxy or C.sub.1-C.sub.10 alkoxy; X is N or O; R.sub.2 is (CH.sub.2).sub.p-5- to 12-membered heterocycle-(CH.sub.2).sub.pC.sub.6-C.sub.12 aryl, 5- to 12-membered heterocycle, (CH.sub.2).sub.pNHC(O)C.sub.6-C.sub.12 aryl, CHR.sub.4R.sub.5, 5- to 12-membered heteroaryl, C.sub.6-C.sub.12 aryl, C.sub.6-C.sub.12 aryl-O-5- to 12-membered heteroaryl, or (CH.sub.2).sub.p-5- to 12-membered heteroaryl, provided that when R.sub.2 is (CH.sub.2).sub.p-5- to 12-membered heteroaryl, the heteroaryl is not pyrazine, pyridine or indole, and when R.sub.2 is 5- to 12-membered heteroaryl, the heteroaryl is not benzothiazole; wherein p is an integer of 1 to 10; R.sub.4 and R.sub.5 are each independently C.sub.1-C.sub.6 alkoxycarbonyl or CH.sub.2-5- to 12-membered heteroaryl; wherein said heterocycle and heteroaryl may contain 1 to 3 heteroatoms selected from N, O and S; said aryl can be substituted with 1 to 4 substituents selected from the group consisting of aminocarbonyl, nitro, nitrile, C.sub.1-C.sub.6 alkylaminocarbonyl, and hydroxy-C.sub.1-C.sub.6 alkyl and said heterocycle and heteroaryl is substituted with 1 to 4 substituents selected from the group consisting of halogen, oxo, aminocarbonyl, nitro, C.sub.1-C.sub.6 alkoxy, nitrile, C.sub.1-C.sub.6 alkylamino carbonyl, hydroxyl, and hydroxyl-C.sub.1-C.sub.6 alkyl; R.sub.3 is hydrogen; when X is O, R.sub.3 does not exist; or when X is N, X taken together with R.sub.2 and R.sub.3 may form a 5- to 12-membered heterocycle containing 1 to 3 heteroatoms selected from O, N and S; wherein said heterocycle can be substituted with C.sub.6-C.sub.12 aryl; a 6- to 12-membered heteroaryl containing 1 to 3 heteroatoms selected from O, N and S, which is unsubstituted or substituted with halogen; or OCHR.sub.6R.sub.7; and wherein R.sub.6 and R.sub.7 are each independently C.sub.6-C.sub.12 aryl or a 6- to 10-membered heteroaryl containing 1 to 3 heteroatoms selected from N, O and S, both of which are unsubstituted or substituted with halogen, provided that when X is N, X taken together with R.sub.2 and R.sub.3 forms a 6-membered heterocycle, said heterocycle is substituted with C.sub.6-C.sub.12 aryl; a 6- to 12-membered heteroaryl containing 1 to 3 heteroatoms selected from O, N, and S, which is unsubstituted or substituted with halogen or OCHR.sub.6R.sub.7.

2. A compound which is selected from the following group: (6,7-Dihydrothieno[3,2-c]pyridin-5(4H)-yl)(3,4-dihydroxyphenyl)methanone; (3,4-Dihydroxyphenyl)(4-(5-fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)methanone; N-((4-(4-Fluorobenzyl)morpholin-2-yl)methyl)-3,4-dihydroxybenzamide; 3,4-Dihydroxy-N-(2-oxotetrahydrothiophen-3-yl)benzamide; N, N-(Nonan-1,9-diyl)bis(3,4-dihydroxybenzamide); (4-((4-Chlorophenyl)(pyridin-2-yl)methoxy)piperidin-1-yl)(3,4-dihydroxyphenyl)methanone; (S)-(4-((4-Chlorophenyl)(pyridin-2-yl)methoxy)piperidin-1-yl)(3,4-dihydroxyphenyl)methanone; (S)-Methyl-2-(3,4-dihydroxybenzamido)-3-(1H-indol-3-yl)propanoate; 4-(3,4-Dihydroxybenzamido)-1-methyl-3-propyl-1H-pyrazole-5-carboxamide; 2-Methyl-4-oxo-4H-pyran-3-yl 3,4-dihydroxybenzoate; (3,4-Dihydroxyphenyl)(4-phenylpiperazin-1-yl)methanone; (6,7-Dihydrothieno[3,2-c]pyridin-5(4H)-yl)(4-hydroxy-3-methoxyphenyl)methanone; 2-Ethyl-4-oxo-4H-pyran-3-yl 4-hydroxy-3-methoxybenzoate; 2-Methyl-4-oxo-4H-pyran-3-yl 4-hydroxy-3-methoxybenzoate; 4-Hydroxy-3-methoxy-N-(4-methoxy-2-nitrophenyl)benzamide; 4-(4-(4-Hydroxy-3-methoxybenzamido)phenoxy)-N-methylpicolinamide; 4-((4-Chlorophenyl)(pyridin-2-yl)methoxy)piperidin-1-yl)(4-hydroxy-3-methoxyphenyl)methanone; 4-Hydroxy-N-((3-hydroxy-5-(hydroxymethyl)-2-methylpyridin-4-yl)methyl)-3-methoxybenzamide; (6,7-Dihydrothieno[3,2-c]pyridin-5(4H)-yl)(2,5-dihydroxyphenyl)methanone; (4-((4-Chlorophenyl)(pyridin-2-yl)methoxy)piperidin-1-yl)(2,5-dihydroxyphenyl)methanone; N-((4-(4-Fluorobenzyl)morpholin-2-yl)methyl)-2,5-dihydroxybenzamide; (2,5-Dihydroxyphenyl)(4-(5-fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)methanone; N-(3,4-Dimethoxyphenethyl)-2,5-dihydroxybenzamide; 2,5-Dihydroxy-N-(2-(thiophen-2-yl)ethyl)benzamide; and 2,5-Dihydroxy-N-(2-oxotetrahydrothiophen-3-yl)benzamide, or a pharmaceutically acceptable salt or stereoisomer thereof.

3. A pharmaceutical composition comprising a compound according to claim 1, or a pharmaceutically acceptable salt or stereoisomer thereof.

4. A pharmaceutical composition comprising a compound according to claim 2, or a pharmaceutically acceptable salt or stereoisomer thereof.

5. A method for the treatment of a thrombotic disease, comprising: administering to a subject in need thereof an effective amount of a compound of claim 1, or a pharmaceutically acceptable salt or stereoisomer thereof.

6. The method of claim 5, wherein the thrombotic disease is selected from the group consisting of pulmonary embolism, thrombotic phlebitis, deep vein thrombosis, portal thrombosis, angina pectoris, arteriosclerosis and cerebral infarction.

7. A method for the treatment of a thrombotic disease, comprising: administering to a subject in need thereof an effective amount of a compound of claim 2, or a pharmaceutically acceptable salt or stereoisomer thereof.

8. The method of claim 7, wherein the thrombotic disease is selected from the group consisting of pulmonary embolism, thrombotic phlebitis, deep vein thrombosis, portal thrombosis, angina pectoris, arteriosclerosis and cerebral infarction.

Description

SPECIFIC EMBODIMENTS OF THE INVENTION

(1) Hereinafter, in order to facilitate understanding of the present invention, examples will be presented. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited to the following examples,

EXAMPLES

(2) Preparation of Experiments and Instruments

(3) 1. Analytical Instruments

(4) The following instruments were used to identify the structure of the product obtained in this experiment. The nuclear magnetic resonance spectrum (1H-NMR) was used with 300 MHz or 400 MHz, and the solvents were CDCl.sub.3 and DMSO-d6. The coupling constant (J) was expressed in Hz. The mass spectrum (manufacturer: JEOL/model name: JMS-AX 505 wA spectrometer; or manufacturer: JEOL/model name: JMS-HX/HX 110A spectrometer) was used according to the manufacturer's manual and expressed in m/z form.

(5) 2. TLC and Column Chromatography

(6) Silica gel (Merck F254) from Merck was used for thin layer chromatography (TLC) and silica (Merck EM 9385, 230-400 mesh) was used for column chromatography. In addition, to confirm the separated substances on TLC, the plate was monitored under UV lamp (at 254 nm), or the plate was immersed in anisaldehyde and potassium permanganate (KMnO.sub.4) color development reagents, followed by heating.

(7) 3. Reagents Used

(8) The reagents used in this experiment were purchased from Sigma-Aldrich, Lancaster, or Fluka, and the solvents used in the reaction were purchased from Sigma-Aldrich, Merck or Junsei Chemical Co. (Japan). The first grade reagents were used without purification. THF used as the solvent was prepared by heating under reflux over sodium metal in the presence of benzophenone in an argon stream until it turned blue. Dichloromethane (CH.sub.2Cl.sub.2) was prepared by adding CaH.sub.2 in an argon stream and heating under reflux. Ethyl acetate and hexane were heated under reflux in an argon stream and purified.

Preparation Example 1: Preparation of 3,4-Diacetoxybenzoic Acid

(9) 10.0 g of PCA was added to a mixture of 150 mL of purified water and 36.1 mL of TEA, and 18.4 mL of acetic anhydride was added dropwise at 20 C. or lower. The mixture was stirred at room temperature overnight. Hydrochloric acid was added thereto to adjust the pH to 3.0, and the mixture was stirred at room temperature for 1 hour. The resulting solid was filtered, washed with purified water, and dried at 40 C. to obtain 9.2 g of the title compound as a white solid.

(10) Yield: 59.5%

(11) .sup.1H NMR (400 MHz, CDCl.sub.3) 8.05 (dd, J=2.0 and 8.4 Hz, 1H), 7.97 (d, J=2.0 Hz, 1H), 7.35 (d, J=8.8 Hz, 1H), 2.35 (s, 3H), 2.34 (s, 3H)

Preparation Example 2: Preparation of 4-Acetoxy-3-methoxybenzoic Acid

(12) Vanillic acid (10.0 g) was added to a mixture of 150 mL of purified water and 16.5 mL of TEA, and 8.4 mL of acetic anhydride was added dropwise at 20 C. or lower. The mixture was stirred at room temperature overnight. Hydrochloric acid was added thereto to adjust the pH to 3.0, and the mixture was stirred at room temperature for 1 hour. The resulting solid was filtered, washed with purified water and dried at 40 C. to obtain 10.7 g of the title compound as an ocher-colored solid.

(13) Yield: 85.5%

(14) .sup.1H NMR (400 MHz, CDCl.sub.3) 7.78 (dd, J=6.4 and 8.0 Hz, 1H), 7.73 (d, J=2.0 Hz, 1H), 7.16 (d, J=8.4 Hz, 1H), 3.93 (s, 3H), 2.37 (s, 3H)

Example 1: (6,7-Dihydrothieno[3,2-c]pyridin-5(4H)-yl)(3,4-dihydroxyphenyl)methanone

(15) 0.5 g of 3,4-diacetoxybenzoic acid obtained in Preparation Example 1 was added to DCM 10 mL, and the mixture was stirred at 10 C. 0.66 g of PCl.sub.5 was added at 10 C. or lower, the mixture was stirred for 2 hours while maintaining the temperature at 0 to 10 C., and 10 ml of purified water was added for extraction. The aqueous layer was discarded, and the organic layer was dried over Na.sub.2SO.sub.4 and filtered. The filtrate was concentrated, 10 mL of DCM was added, cooled to 0 C., and 0.33 g of 4,5,6,7-tetrahydrothieno[3,2,c]pyridine hydrochloride was added. 0.44 mL of TEA was added dropwise while maintaining the temperature at 0 C., the temperature was gradually raised to room temperature, and the mixture was stirred for 2 hours. DCM phase was concentrated and 10 mL of EtOAC and 10 mL of purified water were added for layer separation. The aqueous layer was further extracted with 10 mL of EtOAc, the residual aqueous layer was discarded, and the organic layer was concentrated. 5 mL of MeOH, 5 mL of purified water and 2.9 mL of TEA were added to the concentrate, and the mixture was refluxed for 4 hours. The MeOH phase was concentrated, 10 mL of EtOAc and 10 mL of purified water were added, and the layers were separated. The organic layer was concentrated, and sonicated for 5 minutes after the addition of a small amount of DCM. The resulting solid was filtered and washed with a small amount of DCM to obtain 0.38 g of the title compound as a white solid.

(16) Yield: 65.7%

(17) .sup.1H NMR (400 MHz, DMSO-d.sub.6) 9.30 (brs, 2H), 7.35 (d, J=4.0 Hz, 1H), 6.88 (brs, 1H), 6.84 (s, 1H), 6.77 (d, J=4.0 Hz, 1H), 4.64 (s, 1H), 3.96 (s, 1H), 3.87 (s, 1H), 2.89 (s, 1H), 2.81 (s, 1H)

Example 2: (3,4-Dihydroxyphenyl)(4-(5-fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)methanone

(18) 0.5 g of 3,4-diacetoxybenzoic acid obtained in Preparation Example 1 was added to DCM 10 mL, and the mixture was stirred at 10 C. 0.66 g of PCl.sub.5 was added at 10 C. or lower, the mixture was stirred for 2 hours while maintaining the temperature at 0 to 10 C., and 10 ml of purified water was added to extraction. The aqueous layer was discarded and the organic layer was dried over Na.sub.2SO.sub.4 and filtered. The filtrate was concentrated, 10 mL of DCM was added, cooled to 0 C., and 0.48 g of 6-fluoro-3-(4-piperidinyl)-1,2-benzisoxazole hydrochloride was added. 0.44 mL of TEA was added dropwise while maintaining the temperature at 0 C., the temperature was gradually raised to room temperature, and the mixture was stirred for 2 hours, DCM phase was concentrated, and 10 mL of EtOAC and 10 mL of purified water were added for extraction. The aqueous layer was further extracted with 10 mL of EtOAc, the residual aqueous layer was discarded, and the organic layer was concentrated. 5 mL of MeOH, 5 mL of purified water and 2.9 mL of TEA were added to the concentrate, and the mixture was refluxed for 4 hours. The MeOH phase was concentrated, 10 mL of EtOAc and 10 mL of purified water were added, and the layers were separated. The organic layer was concentrated and sonicated for 5 mines after the addition of a small amount of DCM. The resulting solid was filtered and washed with a small amount of DCM to obtain 0.42 g of the title compound as a white solid.

(19) Yield: 56.1%

(20) .sup.1H NMR (400 MHz, DMSO-d.sub.6) 9.23 (brs, 2H), 8.07 (dd, J=5.6 and 8.8 Hz, 1H), 7.71 (dd, J=2.0 and 9.2 Hz, 1H), 7.30 (td, J=2.0 and 9.2 Hz, 1H), 6.84 (d, J=1.6 Hz, 1H), 6.766.75 (m, 2H), 4.19 (brs, 2H), 3.533.46 (m, 2H), 3.12 (brs, 1H), 2.102.07 (m, 2H), 1.831.73 (m, 1H)

Example 3: N-((4-(4-Fluorobenzyl)morpholin-2-yl)methyl)-3,4-dihydroxybenzamide

(21) 3.0 g of 3,4-diacetoxybenzoic acid obtained in Preparation Example 1 was added to DCM 60 mL, and the mixture was stirred at 10 C. 3.9 g of PCl.sub.5 was added at 10 C. or lower, the mixture was stirred for 2 hours while maintaining the temperature at 0 to 10 C., and 60 ml of purified water was added for layer separation. The aqueous layer was discarded and the organic layer was dried over Na.sub.2SO.sub.4 and filtered. The filtrate was concentrated, added with 60 mL of DCM, cooled to 0 C., and 2.7 g of 3-aminomethyl-4-(4-fluorobenzyl)morpholine was added. 5.2 mL of TEA was added dropwise while maintaining the temperature at 0 C., the temperature was gradually raised to room temperature, and the mixture was stirred for 2 hours. DCM phase was concentrated and 60 mL of EtOAC and 60 mL of purified water were added for extraction. The aqueous layer was further extracted with 60 mL of EtOAc, the aqueous residual layer was discarded, and the organic layer was concentrated. 3 mL of MeOH, 3 mL of purified water and 17.5 mL of TEA were added to the concentrate, and the mixture was refluxed for 4 hours. The MeOH phase was concentrated and 60 mL of EtOAc and 60 mL of purified water were added for extraction. The organic layer was concentrated, purified by flash column chromatography, and vacuum-dried to obtain 2.9 g of the title compound as a foam.

(22) Yield: 63.8%

(23) .sup.1H NMR (400 MHz, DMSO-d.sub.6) 9.45 (brs, 1H), 9.11 (brs, 1H), 8.18 (t, J=5.6 Hz, 1H), 7.33 (dd, J=5.6 and 8.4 Hz, 2H), 7.27 (d, J=1.6 Hz, 1H), 7.197.11 (m, 3H), 6.75 (d, J=8.4 Hz, 1H), 3.77 (d, J=10.8 Hz, 1H), 3.593.40 (m, 4H), 3.293.17 (m, 2H), 2.73 (d, J=11.2 Hz, 1H), 2.55 (d, J=11.2 Hz, 1H), 2.04 (t, J=10.4 Hz, 1H), 1.82 (d, J=10.4 Hz, 1H)

Example 4: 3,4-Dihydroxy-N-(2-oxotetrahydrothiophen-3-yl)benzamide

(24) To 10 mL of DMF were added 0.5 g of protocatechuic acid, 0.68 g of EDC, 0.48 g of HOBt, 1.4 mL of TEA and 0.55 g of homosystein thiolactone hydrochloride, and the mixture was stirred at 60 to 80 C. for 4 hours. 10 mL of EtOAc and 10 mL of purified water were added to the reaction mixture and the layers were separated. The aqueous layer was extracted once with 10 mL of EtOAc and the aqueous layer was discarded. The organic layer was washed three times with 10 mL of purified water, dried over Na.sub.2SO.sub.4, and filtered. The filtrate was concentrated by distillation under reduced pressure and purified by flash column chromatography to obtain 0.28 g of the title compound as a white solid.

(25) Yield: 34.1%

(26) .sup.1H NMR (400 MHz, DMSO-d.sub.6) 9.52 (brs, 1H), 9.17 (brs, 1H), 8.42 (d, J=8.0, 1H), 7.29 (d, J=2.0, 1H), 7.20 (dd, J=2.0 and 8.4, 1H), 6.77 (d, J=8.8, 1H), 4.79 (sex, J=7.6, 1H), 3.473.29 (m, 2H), 2.462.22 (m, 2H)

Example 5: N,N-(Nonan-1,9-diyl)bis(3,4-dihydroxybenzamide)

(27) 0.5 g of 3,4-diacetoxybenzoic acid obtained in Preparation Example 1 was added to DCM 10 mL, and the mixture was stirred at 10 C. 0.66 g of PCl.sub.5 was added at 10 C. or lower, the mixture was stirred for 2 hours while maintaining the temperature at 0 to 10 C., and 10 ml of purified water was added for layer separation. The aqueous layer was discarded and the organic layer was dried over Na.sub.2SO.sub.4 and filtered. The filtrate was concentrated, added with 10 mL of DCM, cooled to 0 C., and 0.13 g of 1,9-diaminononane was added. 0.44 mL of TEA was added dropwise while maintaining the temperature at 0 C., the temperature was gradually raised to room temperature, and the mixture was stirred for 2 hours. DCM was concentrated and 10 mL of EtOAC and 10 mL of purified water were added for layer separation. The aqueous layer was extracted with 10 mL of EtOAc, the aqueous layer was discarded, and the organic layer was concentrated. 5 mL of MeOH, 5 mL of purified water and 2.9 mL of TEA were added to the concentrate, and the mixture was refluxed for 4 hours. The MeOH solution was concentrated, 10 mL of EtOAc and 10 mL of purified water were added, and the layers were separated. The organic layer was concentrated under reduced pressure and purified by flash column chromatography to obtain 0.65 g of the title compound as a white solid.

(28) Yield: 71.9%

(29) .sup.1H NMR (400 MHz, DMSO-d.sub.6) 9.40 (brs, 2H), 9.08 (brs, 2H), 8.10 (t, J=5.6 Hz, 2H), 7.26 (d, J=2.4 Hz, 2H), 7.17 (dd, J=2.0 and 8.0 Hz, 2H), 6.74 (d, J=8.0 Hz, 2H), 3.09 (q, J=6.8 Hz, 4H), 1.47 (t, J=6.0 Hz, 4H), 1.27 (s, 10H)

Example 6: (4-((4-Chlorophenyl)(pyridin-2-yl)methoxy)piperidin-1-yl)(3,4-dihydroxyphenyl)methanone

(30) 0.5 g of 3,4-diacetoxybenzoic acid obtained in Preparation Example 1 was added to DCM 10 mL, and the mixture was stirred at 10 C. 0.66 g of PCl.sub.5 was added at 10 C. or lower, the mixture was stirred for 2 hours while maintaining the temperature at 0 to 10 C., and 10 ml of purified water was added for layer separation. The aqueous layer was discarded and the organic layer was dried over Na.sub.2SO.sub.4 and filtered. The filtrate was concentrated, added with 10 mL of DCM, cooled to 0 C., and 0.57 g of 2-[(4-chlorophenyl)(4-piperidinyloxy)methyl]pyridine was added. 0.44 mL of TEA was added dropwise while maintaining the temperature at 0 C., the temperature was gradually raised to room temperature, and the mixture was stirred for 2 hours. DCM phase was concentrated and 10 mL of EtOAC and 10 mL of purified water were added for layer separation. The aqueous layer was further extracted with 10 mL of EtOAc, the residual aqueous layer was discarded, and the organic layer was concentrated. 5 mL of MeOH, 5 mL of purified water and 2.9 mL of TEA were added to the concentrate, and the mixture was refluxed for 4 hours. The MeOH phase was concentrated, 10 mL of EtOAc and 10 mL of purified water were added, and the layers were separated. The organic layer was concentrated under reduced pressure and purified by flash column chromatography to obtain 0.72 g of the title compound as a foam.

(31) Yield: 78.1%

(32) .sup.1H NMR (400 MHz, DMSO-d.sub.6) 9.25 (brs, 2H), 8.47 (d, J=4.8 Hz, 1H), 7.81 (td, J=1.6 and 7.6 Hz, 1H), 7.57 (d, J=8.0 Hz, 1H), 7.447.24 (m, 5H), 6.806.67 (m, 3H), 5.70 (s, 1H), 3.76 (brs, 1H), 3.66 (brs, 2H), 3.19 (brs, 2H), 1.85 (brs, 2H), 1.53 (brs, 2H)

Example 7: (S)-(4-((4-Chlorophenyl)(pyridin-2-yl)methoxy)piperidin-1-yl)(3,4-dihydroxyphenyl)methanone

(33) 0.5 g of 3,4-diacetoxybenzoic acid obtained in Preparation Example 1 was added to DCM 10 mL, and the mixture was stirred at 10 C. 0.66 g of PCl.sub.5 was added at 10 C. or lower, the mixture was stirred for 2 hours while maintaining the temperature at 0 to 10 C., and 10 ml of purified water was added for layer separation. The aqueous layer was discarded and the organic layer was dried over Na.sub.2SO.sub.4 and filtered. The filtrate was concentrated, 10 mL of DCM was added, cooled to 0 C., and 0.57 g of (s)-2-[(4-chlorophenyl)(4-piperidinyloxy)methyl]pyridine was added. 0.44 mL of TEA was added dropwise while maintaining the temperature at 0 C., the temperature was gradually raised to room temperature; and the mixture was stirred for 2 hours. DCM phase was concentrated and 10 mL of EtOAC and 10 mL of purified water were added for layer separation. The aqueous layer was further extracted with 10 mL of EtOAc, the residual aqueous layer was discarded, and the organic layer was concentrated. 5 mL of MeOH, 5 mL of purified water and 2.9 mL of TEA were added to the concentrate, and the mixture was refluxed for 4 hours. The MeOH phase was concentrated, 10 mL of EtOAc and 10 mL of purified water were added, and the layers were separated. The organic layer was concentrated under reduced pressure and purified by flash column chromatography to obtain 0.47 g of the title compound as a foam.

(34) Yield: 51.0%

(35) .sup.1H NMR (400 MHz, CDCl.sub.3) 8.50 (brs, 2H), 7.72 (t, J=6.8 Hz, 1H), 7.52 (d, J=7.6 Hz, 1H), 7.387.21 (m, 5H), 6.84 (s, 1H), 6.72 (s, 2H), 5.65 (s, 1H), 3.91 (brs, 1H), 3.69 (brs, 2H), 3.40 (brs, 1H), 3.31 (brs, 1H), 1.73 (brs, 4H)

Example 8: (S)-Methyl-2-(3,4-dihydroxybenzamido)-3-(1H-indol-3-yl)propanoate

(36) To 10 mL of DMF were added 0.5 g (3.24 mmol) of protocatechuic acid, 0.68 g (3.57 mmol) of EDC, 0.48 g (3.57 mmol) of HOBt and 1.58 mL (11.35 mmol) of TEA and 1.0 g (3.89 mmol) of D-tryptophan methylester hydrochloride, and the mixture was stirred at 60 to 80 C. for 4 hours. 10 mL of EtOAc and 10 mL of purified water were added to the reaction mixture and the layers were separated. The aqueous layer was extracted once with 10 mL of EtOAc and the aqueous layer was discarded. The organic layer was washed three times with 10 mL of purified water, dried over Na.sub.2SO.sub.4 and filtered. The filtrate was concentrated with distillation under reduced pressure and purified by flash column chromatography to obtain 0.37 g of the title compound as a foam.

(37) Yield: 33.6%

(38) .sup.1H NMR (400 MHz, DMSO-d.sub.6) 10.84 (s, 1H), 9.51 (s, 1H), 9.14 (s, 1H), 8.42 (d, J=7.2 Hz, 1H), 7.54 (d, J=7.6 Hz, 1H), 7.33 (d, J=8.0 Hz, 1H), 7.26 (d, J=2.0 Hz, 1H), 7.217.19 (m, 2H), 7.086.97 (m, 2H), 6.75 (d, J=8.0 Hz, 1H), 4.63 (td, J=5.6 and 7.6 Hz, 1H), 3.61 (s, 3H), 3.273.21 (m, 2H)

Example 9: 4-(3,4-Dihydroxybenzamido)-1-methyl-3-propyl-1H-pyrazole-5-carboxamide

(39) 0.5 g of 3,4-diacetoxybenzoic acid obtained in Preparation Example 1 was added to DCM 10 mL, and the mixture was stirred at 10 C. 0.66 g of PCl.sub.5 was added at 10 C. or lower, the mixture was stirred for 2 hours while maintaining the temperature at 0 to 10 C., and 10 ml of purified water was added for layer separation. The aqueous layer was discarded and the organic layer was dried over Na.sub.2SO.sub.4 and filtered. The filtrate was concentrated, added with 10 mL of DCM, cooled to 0 C., and 0.41 g of 4-amino-1-methyl-3-propyl-1H-pyrazole-5-carboxamide hydrochloride was added. 0.44 mL of TEA was added dropwise while maintaining the temperature at 0 C., the temperature was gradually raised to room temperature, and the mixture was stirred for 2 hours. DCM phase was concentrated and 10 mL of EtOAC and 10 mL of purified water were added for layer separation. The aqueous layer was further extracted with 10 mL of EtOAc, the aqueous layer was discarded, and the organic layer was concentrated. 5 mL of MeOH, 5 mL of purified water and 2.9 mL of TEA were added to the concentrate, and the mixture was refluxed for 4 hours. The MeOH phase was concentrated, 10 mL of EtOAc and 10 mL of purified water were added, and the layers were separated. The organic layer was concentrated by distillation under reduced pressure to give a residue in the form of foam. The residue was crystallized in a mixed solvent of EtOAC/DCM to obtain 0.21 g of the title compound as an off-white solid.

(40) Yield: 31.4%

(41) .sup.1H NMR (400 MHz, CDCl.sub.3) 9.83 (s, 1H), 9.69 (brs, 1H), 9.29 (brs, 1H), 7.38 (d, J=2.0 Hz, 1H), 7.34 (dd, J=2.0 and 8.0 Hz, 1H), 6.84 (d, J=8.4 Hz, 1H), 3.94 (s, 3H), 1.56 (sextet, J=7.2 Hz, 2H), 1.17 (t, J=7.2 Hz, 2H), 0.85 (t, J=7.2 Hz, 3H)

Example 10: 2-Methyl-4-oxo-4H-pyran-3-yl 3,4-dihydroxybenzoate

(42) 0.5 g of 3,4-diacetoxybenzoic acid obtained in Preparation Example 1 was added to DCM 10 mL, and the mixture was stirred at 10 C. 0.66 g of PCl.sub.5 was added at 10 C. or lower, the mixture was stirred for 2 hours while maintaining the temperature at 0 to 10 C., and 10 ml of purified water was added for layer separation. The aqueous layer was discarded and the organic layer was dried over Na.sub.2SO.sub.4, and filtered. The filtrate was concentrated, added with 10 mL of DCM, cooled to 0 C., and 0.24 g of maltol was added. 0.44 mL of TEA was added dropwise while maintaining the temperature at 0 C., the temperature was gradually raised to room temperature, and the mixture was stirred for 2 hours, DCM phase was concentrated and 10 mL of EtOAC and 10 mL of purified water were added for layer separation. The aqueous layer was further extracted with 10 mL of EtOAc, the aqueous layer was discarded, and the organic layer was concentrated. 5 mL of MeOH, 5 mL of purified water and 2.9 mL of TEA were added to the concentrate, and the mixture was refluxed for 4 hours. The MeOH phase was concentrated, 10 mL of EtOAc and 10 mL of purified water were added, and the layers were separated. The organic layer was concentrated and sonicated for 5 minutes after addition of a small amount of DCM. The resulting solid was filtered and washed with a small amount of DCM to obtain 0.40 g of the title compound as a white solid.

(43) Yield: 72.6%

(44) .sup.1H NMR (400 MHz, CDCl.sub.3) 9.79 (brs, 2H), 8.19 (d, J=5.6 Hz, 1H), 7.477.44 (m, 2H), 6.89 (d, J=8.8 Hz, 1H), 6.46 (d, J=6.0 Hz, 1H), 2.26 (s, 3H)

Example 11: (3,4-Dihydroxyphenyl)(4-phenylpiperazin-1-yl)methanone

(45) 0.5 g of 3,4-diacetoxybenzoic acid obtained in Preparation Example 1 was added to DCM 10 mL, and the mixture was stirred at 10 C. 0.66 g of PCl.sub.5 was added at 10 C. or lower, the mixture was stirred for 2 hours while maintaining the temperature at 0 to 10 C., and 10 ml of purified water was added for layer separation. The aqueous layer was discarded and the organic layer was dried over Na.sub.2SO.sub.4 and filtered. The filtrate was concentrated, added with 10 mL of DCM, cooled to 0 C., and 0.31 g of 1-phenylpiperazine was added. 0.44 mL of TEA was added dropwise while maintaining the temperature at 0 C., the temperature was gradually raised to room temperature, and the mixture was stirred for 2 hours. DCM phase was concentrated and 10 mL of EtOAC and 10 mL of purified water were added, followed by layer separation. The aqueous layer was further extracted with 10 mL of EtOAc, the aqueous layer was discarded, and the organic layer was concentrated. 5 mL of MeOH, 5 mL of purified water and 2.9 mL of TEA were added to the concentrate, and the mixture was refluxed for 4 hours. The MeOH phase was concentrated, 10 mL of EtOAc and 10 mL of purified water were added, and the layers were separated. The organic layer was concentrated by distillation under reduced pressure and purified by flash column chromatography to obtain 0.28 g of the title compound as a white solid.

(46) Yield: 45.2%

(47) .sup.1H NMR (400 MHz, CDCl.sub.3) 9.35 (s, 1H), 9.21 (s, 1H), 7.247.21 (m, 2H), 6.966.75 (m, 6H), 3.62 (brs, 4H), 3.14 (brs, 4H)

Example 12: (6,7-Dihydrothieno[3,2-c]pyridin-5(4H)-yl)(4-hydroxy-3-methoxyphenyl)methanone

(48) 0.5 g of 4-acetoxy-3-methoxybenzoic acid obtained in Preparation Example 2 was added to DCM 10 mL, and the mixture was stirred at 10 C. 0.74 g of PCl.sub.5 was added at 10 C. or lower, the mixture was stirred for 2 hours while maintaining the temperature at 0 to 10 C., and 10 ml of purified water was added for layer separation. The aqueous layer was discarded and the organic layer was dried over Na.sub.2SO.sub.4 and filtered. The filtrate was concentrated, added with 10 mL of DCM, cooled to 0 C., and 0.4 g of 4,5,6,7-tetrahydrothieno[3,2,c]pyridine hydrochloride was added. 0.5 mL of TEA was added dropwise while maintaining the temperature at 0 C., the temperature was gradually raised to room temperature, and the mixture was stirred for 2 hours. DCM phase was concentrated and 10 mL of EtOAC and 10 mL of purified water were added for layer separation. The aqueous layer was further extracted with 10 mL of EtOAc, the aqueous layer was discarded, and the organic layer was concentrated. 5 mL of MeOH, 5 mL of purified water and 3.3 mL of TEA were added to the concentrate, and the mixture was refluxed for 4 hours. The MeOH phase was concentrated, and the resulting solid was filtered and washed with purified water to obtain 0.55 g of the title compound as a white solid.

(49) Yield: 79.9%

(50) .sup.1H NMR (400 MHz, DMSO-d.sub.6) 9.48 (s, 1H), 7.35 (d, J=4.4 Hz, 1H), 7.01 (d, J=1.6 Hz, 1H), 6.936.90 (m, 2H), 6.83 (d, J=8.0 Hz, 1H), 4.60 (s, 1H), 3.79 (s, 3H), 3.74 (brs, 2H), 288 (t, J=4.8 Hz, 2H)

Example 13: 2-Ethyl-4-oxo-4H-pyran-3-yl 4-hydroxy-3-methoxybenzoate

(51) 0.5 g of 4-acetoxy-3-methoxybenzoic acid obtained in Preparation Example 2 was added to DCM 10 mL, and the mixture was stirred at 10 C. 0.74 g of PCl.sub.5 was added at 10 C. or lower, the mixture was stirred for 2 hours while maintaining the temperature at 0 to 10 C., and 10 ml of purified water was added thereto, followed by layer separation. The aqueous layer was discarded and the organic layer was dried over Na.sub.2SO.sub.4 and filtered. The filtrate was concentrated, 10 mL of DCM was added, cooled to 0 C., and 0.31 g of ethylmaltol was added. 0.5 mL of TEA was added dropwise while maintaining the temperature at 0 C., the temperature was gradually raised to room temperature, and the mixture was stirred for 2 hours. DCM was concentrated and 10 mL of EtOAC and 10 mL of purified water were added, followed by layer separation. The aqueous layer was extracted with 10 mL of EtOAc, the aqueous layer was discarded, and the organic layer was concentrated. 5 mL of MeOH, 5 mL of purified water and 3.3 mL of TEA were added to the concentrate, and the mixture was refluxed for 4 hours. The MeOH was concentrated, 10 mL of EtOAc and 10 mL of purified water were added, and the layers were separated. The organic layer was distilled under reduced pressure and purified by flash column chromatography to obtain 0.34 g of the title compound as a white solid.

(52) Yield: 49.2%

(53) .sup.1H NMR (400 MHz, DMSO-d.sub.6) 10.24 (s, 1H), 8.23 (d, J=5.2 Hz, 1H), 7.63 (d, J=7.6 Hz, 1H), 7.53 (s, 1H), 6.95 (d, J=8.0 Hz, 1H), 6.48 (d, J=5.6 Hz, 1H), 3.85 (s, 3H), 2.62 (q, J=6.8 Hz, 2H), 1.15 (t, J=7.2 Hz, 3H)

Example 14: 2-Methyl-4-oxo-4H-pyran-3-yl 4-hydroxy-3-methoxybenzoate

(54) 0.5 g of 4-acetoxy-3-methoxybenzoic acid obtained in Preparation Example 2 was added to DCM 10 mL, and the mixture was stirred at 10 C. 0.74 g of PCl.sub.5 was added at 10 C. or lower, the mixture was stirred for 2 hours while maintaining the temperature at 0 to 10 C., and 10 ml of purified water was added thereto, followed by layer separation. The aqueous layer was discarded and the organic layer was dried over Na.sub.2SO.sub.4 and filtered. The filtrate was concentrated, 10 mL of DCM was added, cooled to 0 C., and 0.27 g of maltol was added. 0.5 mL of TEA was added dropwise while maintaining the temperature at 0 C. the temperature was gradually raised to room temperature, and the mixture was stirred for 2 hours. DCM was concentrated and 10 mL of EtOAC and 10 mL of purified water were added, followed by layer separation. The aqueous layer was extracted with 10 mL of EtOAc, the aqueous layer was discarded, and the organic layer was concentrated. 5 mL of MeOH, 5 mL of purified water and 3.3 mL of TEA were added to the concentrate, and the mixture was refluxed for 4 hours. The MeOH was concentrated, and the resulting solid was filtered and washed with purified water to obtain 0.35 g of the title compound as a white solid.

(55) Yield: 53.2%

(56) .sup.1H NMR (400 MHz, DMSO-d.sub.6) 10.24 (s, 1H), 8.21 (d, J=5.6 Hz, 1H), 7.63 (d, J=8.4 Hz, 1H), 7.53 (s, 1H), 6.95 (d, J=8.0 Hz, 1H), 6.48 (d, J=5.6 Hz, 1H), 3.85 (s, 3H), 2.83 (s, 3H)

Example 15: 4-Hydroxy-3-methoxy-N-(4-methoxy-2-nitrophenyl)benzamide

(57) 0.5 g of 4-acetoxy-3-methoxybenzoic acid obtained in Preparation Example 2 was added to DCM 10 mL, and the mixture was stirred at 10 C. 0.74 g of PCl.sub.5 was added at 10 C. or lower, the mixture was stirred for 2 hours while maintaining the temperature at 0 to 10 C., and 10 ml of purified water was added thereto, followed by layer separation. The aqueous layer was discarded and the organic layer was dried over Na.sub.2SO.sub.4 and filtered. The filtrate was concentrated, 10 mL of DCM was added, cooled to 0 C., and 0.40 g of 4-methoxy-2-nitroaniline was added. 0.5 mL of TEA was added dropwise while maintaining the temperature at 0 C., the temperature was gradually raised to room temperature, and the mixture was stirred for 2 hours. DCM was concentrated and 10 mL of EtOAC and 10 mL of purified water were added, followed by layer separation. The aqueous layer was extracted with 10 mL of EtOAc, the aqueous layer was discarded, and the organic layer was concentrated. 5 mL of MeOH, 5 mL of purified water and 3.3 mL of TEA were added to the concentrate, and the mixture was refluxed for 4 hours. The MeOH was concentrated, 10 mL of EtOAc and 10 mL of purified water were added, and the layers were separated. The organic layer was distilled under reduced pressure and purified by flash column chromatography to obtain 0.54 g of the title compound as a red solid.

(58) Yield: 71.3%

(59) .sup.1H NMR (400 MHz, DMSO-d.sub.6) 10.32 (s, 1H), 9.81 (brs, 1H) 7.63 (d, J=8.8, 1H), 7.527.46 (m, 3H), 7.35 (dd, J=2.8 and 8.8, 1H), 3.86 (s, 1H), 3.85 (s, 3H)

Example 16: N-(3-Ethynylphenyl)-4-hydroxy-3-methoxybenzamide

(60) 0.5 g of 4-acetoxy-3-methoxybenzoic acid obtained in Preparation Example 2 was added to DCM 10 mL, and the mixture was stirred at 10 C. 0.74 g of PCl.sub.5 was added at 10 C. or lower, the mixture was stirred for 2 hours while maintaining the temperature at 0 to 10 C., and 10 ml of purified water was added thereto, followed by layer separation. The aqueous layer was discarded and the organic layer was dried over Na.sub.2SO.sub.4 and filtered. The filtrate was concentrated, 10 mL of DCM was added, cooled to 0 C., and 0.25 g of 3-aminophenylacetylene was added. 0.5 mL of TEA was added dropwise while maintaining the temperature at 0 C., the temperature was gradually raised to room temperature, and the mixture was stirred for 2 hours, DCM was concentrated and 10 mL of EtOAC and 10 mL of purified water were added, followed by layer separation. The aqueous layer was extracted with 10 mL of EtOAc, the aqueous layer was discarded, and the organic layer was concentrated. 5 mL of MeOH, 5 mL of purified water and 3.3 mL of TEA were added to the concentrate, and the mixture was refluxed for 4 hours. The MeOH was concentrated, 10 mL of EtOAc and 10 mL of purified water were added, and the layers were separated. The organic layer was distilled under reduced pressure. To the resulting solid was added a small amount of IPA and stirred to obtain 0.30 g of the title compound as a white solid.

(61) Yield: 47.2%

(62) .sup.1H NMR (400 MHz, DMSO-d.sub.6) 10.07 (s, 1H), 9.75 (brs, 1H), 7.92 (d, J=1.2 Hz, 1H), 7.80 (dd, J=1.2 and 8.4 Hz, 1H), 7.547.50 (m, 2H), 7.36 (t, J=8.0 Hz, 1H), 7.10 (dd, J=1.2 and 8.0 Hz, 1H), 6.90 (d, J=8.0 Hz, 1H), 4.18 (s, 1H), 3.87 (s, 3H)

Example 17: 4-(4-(4-Hydroxy-3-methoxybenzamido)phenoxy)-N-methylpicolinamide

(63) 0.5 g of 4-acetoxy-3-methoxybenzoic acid obtained in Preparation Example 2 was added to DCM 10 mL, and the mixture was stirred at 10 C. 0.74 g of PCl.sub.5 was added at 10 C. or lower, the mixture was stirred for 2 hours while maintaining the temperature at 0 to 10 C., and 10 ml of purified water was added thereto, followed by layer separation. The aqueous layer was discarded and the organic layer was dried over Na.sub.2SO.sub.4 and filtered. The filtrate was concentrated, 10 mL of DCM was added, cooled to 0 C., and 0.55 g of 4-(4-aminophenoxy)-N-methylpicolinamide was added. 0.5 mL of TEA was added dropwise while maintaining the temperature at 0 C., the temperature was gradually raised to room temperature, and the mixture was stirred for 2 hours, DCM was concentrated and 10 mL of EtOAC and 10 mL of purified water were added, followed by layer separation. The aqueous layer was extracted with 10 mL of EtOAc, the aqueous layer was discarded, and the organic layer was concentrated. 5 mL of MeOH, 5 mL of purified water and 3.3 mL of TEA were added to the concentrate, and the mixture was refluxed for 4 hours. The MeOH was concentrated. 10 mL of EtOAc and 10 mL of purified water were added, and the layers were separated. The organic layer was distilled under reduced pressure and purified by flash column chromatography to obtain 0.48 g of the title compound as a light yellow solid.

(64) Yield: 51.3%

(65) .sup.1H NMR (400 MHz, CDCl.sub.3) 8.60 (s, 1H), 8.39 (d, J=5.6, 1H), 8.10 (q, J=5.2, 1H), 7.717.65 (m, 3H), 7.52 (d, J=2.0, 1H), 7.43 (dd, J=2.0 and 8.4, 1H), 7.046.90 (m, 4H), 6.73 (brs, 1H), 3.78 (s, 3H), 3.01 (d, J=5.2, 3H)

Example 18: 4-((4-Chlorophenyl)(pyridin-2-yl)methoxy)piperidin-1-yl)(4-hydroxy-3-methoxyphenyl)methanone

(66) To 10 mL of DMF were added 0.5 g of vanillic acid, 0.51 g of EDC, 0.44 g of HOBt, 1.44 mL of TEA and 0.99 g of 2-[(4-chlorophenyl)(4-piperidinyloxy)methyl]pyridine, and the mixture was stirred at 60 to 80 C. for 4 hours. 10 mL of EtOAc and 10 mL of purified water were added to the reaction mixture and the layers were separated. The aqueous layer was extracted once with 10 mL of EtOAc and the aqueous layer was discarded. The organic layer was washed three times with 10 mL of purified water, dried over Na.sub.2SO.sub.4 and filtered. The filtrate was distilled under reduced pressure and purified by flash column chromatography to obtain 0.40 g of the title compound as a pale yellow solid.

(67) Yield: 31.6%

(68) .sup.1H NMR (400 MHz, DMSO-d.sub.6) 9.41 (brs, 1H), 8.47 (d, J=4.0 Hz, 1H), 7.81 (t, J=7.0, 1H), 7.57 (d, J=8.0 Hz, 1H), 7.446.94 (m, 6H), 6.846.67 (m, 3H), 5.71 (s, 1H), 3.87 (s, 3H), 3.743.69 (m, 2H), 3.37 (brs, 2H), 1.85 (brs, 2H), 1.72 (brs, 2H)

Example 19: 4-Hydroxy-N-((3-hydroxy-5-(hydroxymethylmethyl)-2-methylpyridin-4-yl)methyl)-3-methoxybenzamide

(69) To 10 mL of DMF were added 0.5 g of vanillic acid, 0.51 g of EDC, 0.44 g of HOBt, 1.44 mL of TEA and 0.72 g of pyridoxoamine 2HCl, and the mixture was stirred at 60 to 80 C. for 4 hours, 10 mL of EtOAc and 10 mL of purified water were added to the reaction mixture and the layers were separated. The aqueous layer was extracted once with 10 mL of EtOAc and the aqueous layer was discarded. The organic layer was washed three times with 10 mL of purified water, dried over Na.sub.2SO.sub.4 and filtered. The filtrate was distilled under reduced pressure and purified by flash column chromatography to obtain 0.62 g of the title compound as a pale yellow solid.

(70) Yield: 65.6%

(71) .sup.1H NMR (400 MHz, DMSO-d.sub.6) 10.50 (s, 1H), 9.76 (s, 1H), 9.18 (t, J=5.6, 1H), 7.29 (s, 1H), 7.47 (d, J=2.0, 1H), 7.42 (dd, J=2.0 and 8.4, 1H), 6.83 (d, J=8.4, 1H), 5.25 (t, J=5.2, 1H), 4.67 (d, J=4.8, 2H), 4.48 (d, J=6.0, 2H), 3.82 (s, 3H), 2.35 (s, 3H)

Example 20: (6,7-Dihydrothieno[3,2-c]pyridin-5(4H)-yl)(2,5-dihydroxyphenyl)methanone

(72) To 10 mL of DMF were added 0.5 g of gentisic acid, 0.93 g of EDC, 0.66 g of HOBt, 1.35 mL of TEA and 0.62 g of 4,5,6,7-tetrahydrothieno[3,2,c] pyridine hydrochloride, and the mixture was stirred at 60 to 80 C. for 4 hours. 10 mL of EtOAc and 10 mL of purified water were added to the reaction mixture and stirred. The resulting solid was filtered, refluxed in 10 mL of EtOAc for 1 h and then filtered. The filtrate was washed with a small amount of EtOAc to obtain 0.38 g of the title compound as an apricot-colored solid.

(73) Yield: 42.6%

(74) .sup.1H NMR (400 MHz, DMSO-d.sub.6) 9.08 (s, 1H), 8.93 (s, 1H), 7.34 (brs, 1H), 7.35 (d, J=4.4 Hz, 1H), 7.01 (d, J=1.6 Hz, 1H), 6.936.90 (m, 2H), 6.83 (d, J=8.0 Hz, 1H), 4.60 (s, 1H), 3.79 (s, 3H), 3.74 (brs, 2H), 2.86 (t, J=4.8 Hz, 1H)

Example 21: (4-((4-Chlorophenyl)(pyridin-2-yl)methoxy)piperidin-1-yl)(2,5-dihydroxyphenyl)methanone

(75) To 10 mL of DMF were added 0.5 g of gentisic acid, 0.68 g of EDC, 0.48 g of HOBt, 1.4 mL of TEA and 1.08 g of 2-[(4-chlorophenyl)(4-piperidinyloxy)methyl]pyridine, and the mixture was stirred at 60 to 80 C. for 4 hours. 10 mL of EtOAc and 10 mL of purified water were added to the reaction mixture and the layers were separated. The aqueous layer was extracted once with 10 mL of EtOAc and the aqueous layer was discarded. The organic layer was washed three times with 10 mL of purified water, dried over Na.sub.2SO.sub.4 and filtered. The filtrate was distilled under reduced pressure and purified by flash column chromatography to obtain 0.78 g of the title compound as a foam.

(76) Yield: 54.9%

(77) .sup.1H NMR (400 MHz, DMSO-d.sub.6) 9.05 (s, 1H), 8.89 (s, 1H), 8.47 (dd, J=0.8 and 4.0, 1H), 7.81 (td, J=1.6 and 8.0, 1H), 7.57 (d, J=7.6, 1H), 7.437.25 (m, 5H), 6.676.60 (m, 2H), 6.46 (d, J=2.8, 1H), 5.69 (s, 1H), 3.93 (brs, 1H), 3.64 (p, J=4.4, 1H), 3.14 (brs, 2H), 1.85 (brs, 1H), 1.551.52 (m, 2H)

Example 22: N-((4-(4-Fluorobenzyl)morpholin-2-yl)methyl)-2,5-dihydroxybenzamide

(78) To 10 mL of DMF were added 0.5 g of gentisic acid, 0.68 g of EDC, 0.48 g of HOBt, 1.4 mL of TEA and 0.73 g of 3-aminomethyl-4-(4-fluorobenzyl)morpholine, and the mixture was stirred at 60 to 80 C. for 4 hours. 10 mL of EtOAc and 10 mL of purified water were added to the reaction mixture and the layers were separated. The aqueous layer was extracted once with 10 mL of EtOAc and the aqueous layer was discarded. The organic layer was washed three times with 10 mL of purified water, dried over Na.sub.2SO.sub.4 and filtered. The filtrate was distilled under reduced pressure and purified by flash column chromatography to obtain 0.80 g of the title compound as a foam.

(79) Yield: 68.5%

(80) .sup.1H NMR (400 MHz, CDCl.sub.3) 7.317.27 (m, 2H), 7.057.00 (m, 4H), 6.896.88 (m, 2H), 3.963.67 (m, 4H), 3.493.33 (m, 3H), 2.79 (d, J=11.6, 1H), 2.69 (d, J=11.6, 1H), 2.241.91 (m, 2H)

Example 23: (2,5-Dihydroxyphenyl)(4-(5-fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)methanone

(81) To 10 mL of DMF were added 0.5 g of gentisic acid, 0.68 g of EDC, 0.48 g of HOBt, 1.4 mL of TEA and 0.83 g of 6-fluoro-3-(4-piperidinyl)-1,2-benzoisoxazole hydrochloride, and the mixture was stirred at 60 to 80 C. for 4 hours. 10 mL of EtOAc and 10 mL of purified water were added to the reaction mixture and the layers were separated. The aqueous layer was extracted once with 10 mL of EtOAc and the aqueous layer as discarded. The organic layer was washed three times with 10 mL of purified water, dried over Na.sub.2SO.sub.4 and filtered. The filtrate was distilled under reduced pressure and purified by flash column chromatography to obtain 0.64 g of the title compound as a white solid.

(82) Yield: 55.4%

(83) .sup.1H NMR (400 MHz, DMSO-d.sub.6) 9.07 (s, 1H), 8.92 (s, 1H), 8.02 (dd, J=5.6 and 8.8, 1H), 7.70 (dd, J=1.6 and 8.8, 1H), 6.706.54 (m, 3H), 4.57 (brs, 1H), 3.613.45 (m, 3H), 3.09 (brs, 2H), 2.06 (brs, 2H), 1.80 (brs, 2H)

Example 24: N-(3,4-Dimethoxyphenethyl)-2,5-dihydroxybenzamide

(84) To 10 mL of DMF were added 0.5 g of gentisic acid, 0.68 g of EDC, 0.48 g of HOBt, 1.4 mL of TEA and 0.6 mL of 3,4-dimethoxyphenethylamine, and the mixture was stirred at 60 to 80 C. for 4 hours. 10 mL of EtOAc and 10 mL of purified water were added to the reaction mixture and the layers were separated. The aqueous layer was extracted once with 10 mL of EtOAc and the aqueous layer was discarded. The organic layer was washed three times with 10 mL of purified water, dried over Na.sub.2SO.sub.4 and filtered. The filtrate was distilled under reduced pressure and purified by flash column chromatography to obtain 0.73 g of the title compound as a white solid.

(85) Yield: 71.0%

(86) .sup.1H NMR (400 MHz, DMSO-d.sub.6) 11.63 (brs, 1H), 9.00 (brs, 1H), 8.72 (t, J=5.2, 1H), 7.22 (d, J=2.8, 1H), 6.886.71 (m, 5H), 3.72 (s, 3H), 3.71 (s, 3H), 3.50 (q, J=6.8, 2H), 2.78 (t, J=7.6, 2H)

Example 25: 2,5-Dihydroxy-N-(2-(thiophen-2-yl)ethyl)benzamide

(87) To 10 mL of DMF were added 0.5 g of gentisic acid, 0.68 g of EDC, 0.48 g of HOBt, 1.4 mL of TEA and 0.4 mL of thiophene-2-ethylamine, and the mixture was stirred at 60 to 80 C. for 4 hours. 10 mL of EtOAc and 10 mL of purified water were added to the reaction mixture and the layers were separated. The aqueous layer was extracted once with 10 mL of EtOAc and the aqueous layer was discarded. The organic layer was washed three times with 10 mL of purified water, dried over Na.sub.2SO.sub.4 and filtered. The filtrate was distilled under reduced pressure and purified by flash column chromatography to obtain 0.56 g of the title compound as a white solid.

(88) Yield: 65.6%

(89) .sup.1H NMR (400 MHz, DMSO-d.sub.6) 11.62 (brs, 1H), 9.03 (brs, 1H), 8.83 (t, J=5.2, 1H), 7.34 (dd, J=1.2 and 4.8, 1H), 7.23 (d, J=3.2, 1H), 6.976.85 (m, 3H), 6.73 (d, J=8.8, 1H), 3.53 (q, J=6.8, 2H), 3.08 (t, J=6.8, 2H)

Example 26: 2,5-Dihydroxy-N-(2-oxotetrahydrothiophen-3-yl)benzamide

(90) To 10 mL of DMF were added 0.5 g of gentisic acid, 0.68 g of EDC, 0.48 g of HOBt, 1.4 mL of TEA and 0.55 g of homosystein thiolactone hydrochloride, and the mixture was stirred at 60 to 80 C. for 4 hours. 10 mL of EtOAc and 10 mL of purified water were added to the reaction mixture and the layers were separated. The aqueous layer was extracted once with 10 mL of EtOAc and the aqueous layer was discarded. The organic layer was washed three times with 10 mL of purified water, dried over Na.sub.2SO.sub.4 and filtered. The filtrate was distilled under reduced pressure and purified by flash column chromatography to obtain 0.21 g of the title compound as a white solid.

(91) Yield: 25.6%

(92) .sup.1H NMR (400 MHz, DMSO-d.sub.6) 11.33 (brs, 1H), 9.06 (brs, 1H), 8.92 (d, J=8.0, 1H), 7.24 (d, J=3.2, 1H), 6.89 (dd, J=2.8 and 8.8, 1H), 6.77 (d, J=8.8, 1H), 4.84 (sex, J=5.6, 1H), 3.503.25 (m, 2H), 2.562.26 (m, 2H)

(93) TABLE-US-00001 TABLE 1 Inhibitory effect of shear stress-induced Example Compound platelet aggregation 1 20~30% 2 20~30% 3 30% or more 4 30% or more 5 20~30% 6 20~30% 7 20~30% 8 20~30% 9 0 20~30% 10 20~30% 11 30% or more 12 20~30% 13 20~30% 14 30% or more 15 20~30% 16 30% or more 17 20~30% 18 30% or more 19 0 20~30% 20 30% or more 21 20~30% 22 30% or more 23 20~30% 24 20~30% 25 20~30% 26 20~30%

Experimental Example

Experimental Example 1: Measurement of Inhibitory Effect of Shear Stress-Induced Platelet Aggregation

(94) 1-1. Measurement of Platelet Aggregation Inhibitory Effect Using Human Platelet Rich Plasma (PRP)

(95) Blood was collected from the veins of healthy male volunteers who had not taken any medication for more than 2 weeks. All related studies were conducted under the approval of the Seoul National University Institutional Review Board (IRB No. 1305/001-016). During the whole process of study the use of glass containers or glass pipettes was avoided, and the experiments were performed at room temperature. To separate platelet rich plasma (PRP), the blood was collected with 3.2% sodium citrate as an anticoagulant. The whole blood was centrifuged at 150 g for 15 minutes to obtain the supernatant (PRP), and the residue was centrifuged at 2,000 g for 20 minutes to obtain platelet poor plasma (PPP). The number of platelets of the PRP thus obtained was counted with an optical microscope using a hemacytometer. PRP was diluted with PPP so as to include 310.sup.8 platelets per 1 ml, and then used in the experiments. PRP was put on a cone-plate viscometer (RotoVisco 1, Thermo Fischer Scientific, USA), and shear stress was applied to the PRP at 37 C. for 3 minutes at a shear rate of 10,800 s.sup.1. In order to evaluate candidate substances, prior to the induction of platelet aggregation, 598.8 l of PRP was treated with 1.2 l of 25 M candidate substance and incubated at 37 C. for 3 minutes using a thermomixer. After applying the shear stress, 20 l of PRP was fixed in 280 l of a suspension buffer containing 0.5% glutaraldehyde (134 mM NaCl, 2.9 mM KCl, 1.0 mM MgCl.sub.2.6H.sub.2O, 10.0 mM HEPES, 5.0 mM dextrose, 12.0 mM NaHCO.sub.3, 0.34 mM Na.sub.2HPO.sub.4, 0.3% BSA, pH 7.4). Then, the number of single platelets in the suspension was counted using a hemacytometer. The degree of inhibition by the candidate substance was calculated according to the following formula (1). The degree of inhibition was determined by the method described below. No shear stress-applied group served as a control, and only shear stress-applied group served as positive control.
Degree of inhibition (%)=[(1A/B)100]/[(1A/C)100]Formula 1

(96) A: Number of single platelets in the shear stress-applied sample after treatment with candidate substance

(97) B: Number of single platelets in the control group with no shear stress applied

(98) C: Number of single platelets in the positive control group with only shear stress applied

(99) TABLE-US-00002 TABLE 2 Platelet aggregation inhibitory effect of candidate substances in PRP Example Degree of inhibition (%, Mean SEM) 1 23 2 12 25 6 13 26 6 14 34 5 15 24 8 20 30 5 23 28 3

(100) As shown in Table 2 above, it was confirmed that the effect of inhibiting shear stress-induced platelet aggregation was 30% or more in Example 14 and Example 20 (each 25 M). In addition, it was confirmed that the inhibitory effect was 20 to 30% in Example 1, Example 12, Example 13, Example 15, and Example 23 (each 25 M).

(101) 1-2. Measurement of Platelet Aggregation Inhibitory Effect Using Human Washed Platelets (WP)

(102) To separate human washed platelets (WP), blood was collected from the veins of healthy males using acid-citrate-dextrose (ACD) as an anticoagulant. At the time of blood collection, platelet activation was inhibited by treatment with 1 M of prostaglandin E.sub.1 (PGE.sub.1). The collected blood was centrifuged at 150 g for 15 minutes to obtain PRP from the supernatant, which was centrifuged at 500 g for 10 minutes to obtain platelets. The platelets were suspended in and washed with a washing buffer (134 mM NaCl, 2.9 mM KCl, 1.0 mM MgCl.sub.2.6H.sub.2O, 10.0 mM HEPES, 5.0 mM dextrose, 12.0 mM NaHCO.sub.3, 0.34 mM Na.sub.2HPO.sub.4, 10% ACD, 0.3% bovine serum albumin, 1 M PGE.sub.1, pH 7.4), and then re-centrifuged at 400 g for 10 minutes. The platelets thus obtained were suspended in the suspension buffer. The number of platelets in WP were counted using a hemacytometer. The platelets were diluted with the suspension buffer so as to include 310.sup.8 platelets per 1 ml, and CaCl.sub.2 was added to give a final concentration of 2 mM and then used in the experiments. vWF was added to WP to give a final concentration of 10 g/ml. WP was put on a cone-plate viscometer, and shear stress was applied to the WP at 37 C. for 3 minutes at a shear rate of 10,800 s.sup.1. In order to evaluate efficacy of candidate substances, prior to the induction of platelet aggregation, WP was treated with 10, 25, and 50 M of candidate substances and incubated at 37 C. for 3 minutes using a thermomixer. After applying the shear stress, 20 l of WP was fixed in 280 l of the suspension buffer containing 0.5% glutaraldehyde. Then, the number of single platelets in the suspension was counted using a hemacytometer. The degree of inhibition by the candidate substance was calculated according to formula (1). No shear stress-applied group served as control, and only shear stress-applied group served as a positive control.

(103) TABLE-US-00003 TABLE 3 Platelet aggregation inhibitory effect of candidate substances in WP Example Degree of inhibition (%, Mean SEM) 1 10 M 4 3.5 25 M 23 1.2 50 M 27 1.7 12 10 M 10 2.1 25 M 30 0.9 50 M 33 2.1 13 10 M 12 3.8 25 M 29 4.1 50 M 34 2.7 14 10 M 8 1.5 25 M 27 2.3 50 M 39 0.6 15 10 M 14 2.5 25 M 26 4.0 50 M 36 3.2 20 10 M 18 1.5 25 M 42 4.2 50 M 46 2.1

(104) As shown in Table 3 above, it was confirmed that Example 1, Example 12, Example 13, Example 14, Example 15 and Example 20 inhibited shear stress-induced platelet aggregation in WP in a dose-dependent manner.

Experimental Example 2: Evaluation of Drug Efficacy in Arterial Thrombosis Model

(105) Vehicle (DMSO: Tween 80: DW=1:2:17) or candidate substance (25 mg/kg) was orally administered to overnight-fasted male Sprague-Dawley rats (250 to 300 g). 30 minutes after administration, the rats were anesthetized by intraperitoneal injection of urethane (1.25 g/kg). The anesthetized animals were restrained on the operating table, and the body temperature was maintained using a heating pad during the entire operating procedure. An incision was made around the neck of the animals to carefully expose the right carotid artery, and the adipose tissues attached to the blood vessels were carefully removed. Doppler flow probe (0.5 mm-diameter, MA0.5PSB, Transonic System Inc., USA) was fixed to the exposed carotid artery, and a doppler flow-meter (TS420, Transonic System Inc., USA) was connected thereto. 60 minutes after oral administration, a piece of Whatman No. 1 filter paper (2 mm1 mm) soaked with 50% FeCl.sub.3 solution was attached to the underlying blood vessels to which the probe was fixed. The filter paper was removed after 10 minutes, and the change of blood flow due to thrombus formation in the carotid artery was measured for 60 minutes after the time point of removal. The occlusion time was defined as the time at which the blood flow became zero more than one minute for the first time.

(106) TABLE-US-00004 TABLE 4 Thrombogenesis inhibitory effect of candidate substances Example Time of thrombogenesis (second) Fold (vs. Control) Control 356 38 1 936 120 2.6 12 665 77 1.9 13 752 69 2.1 14 615 54 1.7 15 816 78 2.3 20 2122 265 6.0 23 674 92 1.9

(107) As shown in Table 4 above, it was confirmed that the candidate substances exhibiting the effect of 2.5 times or more as compared with the control in the iron chloride (FeCl.sub.3)-induced thrombogenesis model are Example 1 and Example 20.

Experimental Example 3: Measurement of Thrombosis Inhibitory Effect of Candidate Substances in the Carotid Artery Shear Stress Model

(108) Vehicle (0.5% methylcellulose), clopidogrel (8 mg/kg), aspirin (50 mg/kg) or a candidate substance was orally administered to male Sprague-Dawley rats (250 to 300 g). 2 hours after administration, the rats were anesthetized by ketamine/rompun (ketamine/xylazine) cocktail 0.1 ml/100 g. Except for the normal control group, the right cervical skin of the experimental animals was incised to expose the common carotid artery. A surgical tube with a length of 1 mm and an inside diameter of 0.58 mm was inserted into the exposed carotid artery and tied with a single thread. The exposed site and the operation site were treated with an anti-adhesion agent, and then sutured for the animals to recover. After the surgery, vehicle, clopidogrel, aspirin or a candidate substance was orally administered to the rats twice a day for 3 days. On the fourth day, 2 hours after the final administration, urethane (100 mg/300 l/100 g) was administered intraperitoneally to the rats to induce anesthesia, and the operation site was opened to separate a total of 1 cm of the blood vessel, 5 mm above and 5 mm below the surgical tube. The separated blood vessel was perfused with 0.2 ml of 0.9% saline at 0.3 ml/min to remove the remaining blood, and then maintained in 1 ml of protein lysis buffer (NaOH 2 g, Na.sub.2CO.sub.3 0.1 g in 500 ml D.W). After collected, the blood vessel thrombus was double-boiled in boiling water together with the protein lysis buffer. 200 l of the reaction solution (10 ml bicinchoninic acid solution+200 l 4% aqueous solution of copper sulfate) was added to 10 l of the heated thrombus, and the mixture was reacted at 37 C. for 30 minutes and quantified with an ELISA reader (562 nm).

(109) TABLE-US-00005 TABLE 5 Thrombosis inhibitory effect of candidate substances in the carotid artery shear stress model Substance Dose Amount of thrombus administered (mg/kg) Operation (mg, Mean SEM) Normal C. x 0.005 0.002 Negative C. 0.124 0.007 Clopidogrel 8 0.067 0.004 Aspirin 50 0.092 0.007 Example 1 25 0.065 0.005 Example 12 25 0.069 0.007 Example 13 25 0.064 0.006 Example 14 25 0.066 0.006 Example 15 25 0.070 0.006 Example 20 25 0.068 0.005 Example 23 25 0.071 0.006

(110) As shown in Table 5 above, it was confirmed that in the animals each orally administered with Example 1, Example 12, Example 13, Example 14, Example 15, Example 20, and Example 23, the shear stress-induced thrombogenesis was significantly, remarkably inhibited compared with the control group. This suggested that the degree of inhibition of the candidate substances against the thrombogenesis is as good as comparable to that of clopidogrel when compared with the group administered with clopidogrel, which is the most commonly used antiplatelet agent.
Degree of inhibition (%)=100[(CA)100/(BA)]Formula 2

(111) A: Amount of thrombus in Normal Control

(112) B: Amount of thrombus in Negative Control

(113) C: Amount of thrombus in candidate substance treatment

Experimental Example 4: Measurement of Inhibitory Effect of Candidate Substances for Chemical Agonist (Physiological Agonist)-Induced Platelet Aggregation

(114) To investigate whether the candidate substance has inhibition selectivity for shear-stress-induced platelet aggregation, which is by a physical stimulus, the inhibitory effect of the candidate substance was investigated in is experiment was conducted by activating platelets with physiological agonistsi.e., thrombin, collagen and ADPto study the inhibitory effect of the candidate substance.

(115) Human PRP (598.8 l) was treated with 1.2 l of the candidate substance per concentration (25, 100, 250 M) and reacted at 37 C. for 3 minutes using a thermomixer. Then, PRP (495 l) was put in the aggregometer cuvette and preincubated for 1 minute, followed by treatment with platelet aggregation-inducing reagent, thrombin (0.6-0.8 U/ml), collagen (2-5 g/ml), or ADP (adenosine diphosphate, 15-20 M) at the minimum concentration causing maximum aggregation. The degree of aggregation of the platelets was measured by a turbidity change using a lumi-aggregometer (Chrono-Log Co., USA). The turbidity of the PRP was considered as 0%, and the turbidity of the PPP was considered as 100%. During the measurement, the reaction mixture was continuously stirred at 1,000 rpm with a silicone-coated magnetic stir bar. The reaction was observed for 5 minutes for thrombin or ADP, and 6 minutes for collagen.

(116) TABLE-US-00006 TABLE 6 Results of measurement of the degree of aggregation by thrombin Degree of platelet aggregation Example (%, Mean SEM) Thrombin 83.5 6.5 1 25 M 88.0 2.1 100 M 88.7 6.1 250 M 78.7 1.8 12 25 M 99.5 7.5 100 M 95.0 12.0 250 M 88.5 4.5 13 25 M 92.5 5.5 100 M 95.0 12.0 250 M 88.5 4.5 14 25 M 81.3 2.5 100 M 84.0 0.6 250 M 86.0 4.6 15 25 M 95.5 3.5 100 M 100.5 4.5 250 M 98.0 3.0 20 25 M 84.0 3.2 100 M 82.3 3.7 250 M 82.0 15.0 23 25 M 96.0 4.0 100 M 88.5 6.5 250 M 93.5 1.5 DTI.sup.1) 2 M 11.5 1.2 .sup.1)DTI: Direct Thrombin Inhibitor

(117) As shown in Table 6 above, it was confirmed that when compared with the group treated with thrombin only, the candidate substances did not show the platelet aggregation inhibitory effect up to 250 M. In contrast, it was confirmed that the platelet aggregation was inhibited when the positive control DTI was treated.

(118) TABLE-US-00007 TABLE 7 Results of measurement of the degree of aggregation by collagen Degree of platelet aggregation Example (%, Mean SEM) Collagen 82.6 1.2 1 25 M 84.3 3.5 100 M 80.7 10.2 250 M 84.3 3.8 12 25 M 83.0 4.0 100 M 80.0 2.0 250 M 87.5 3.5 13 25 M 90.0 3.0 100 M 79.5 0.5 250 M 85.5 3.5 14 25 M 82.0 1.7 100 M 80.0 1.7 250 M 92.0 2.0 15 25 M 86.0 0.0 100 M 84.5 4.5 250 M 83.5 3.5 20 25 M 82.3 5.8 100 M 81.3 3.5 250 M 87.7 2.0 23 25 M 82.0 5.0 100 M 84.5 4.5 250 M 83.5 2.5

(119) As shown in Table 7 above, it was confirmed that when compared with the group treated with collagen only, the candidate substances did not show the platelet aggregation inhibitory effect up to 250 M.

(120) TABLE-US-00008 TABLE 8 Results of measurement of the degree of aggregation by ADP Degree of platelet aggregation Example (%, Mean SEM) ADP 76.0 1.3 1 25 M 77.5 3.5 100 M 69.0 6.0 250 M 78.0 7.0 12 25 M 76.0 3.0 100 M 67.5 2.5 250 M 77.0 12.0 13 25 M 75.5 1.5 100 M 75.0 1.0 250 M 71.5 6.5 14 25 M 74.5 2.5 100 M 75.0 3.0 250 M 63.5 5.5 15 25 M 70.5 2.5 100 M 68.5 3.5 250 M 67.0 4.0 20 25 M 73.0 2.0 100 M 69.0 1.0 250 M 66.5 2.5 23 25 M 73.5 1.5 100 M 66.0 1.0 250 M 65.0 0.0 Clopidogrel 40 M 18.3 3.4

(121) As shown in Table 8 above, it was confirmed that when compared with the group treated with ADP only, the candidate substances did not show the platelet aggregation inhibitory effect up to 250 M. In contrast, it was confirmed that the platelet aggregation was inhibited when the positive control clopidogrel was treated.

Experimental Example 5: Evaluation of Cytotoxicity of Candidate Substances

(122) 5-1. Evaluation of Cytotoxicity of Candidate Substances Using EA.hy926 Cell Line

(123) Experiments were conducted to determine the cytotoxicity of candidate substances in human vascular endothelial cells (EA.hy926). EA.hy926 was passaged in DMEM (Dulbecco's Minimum Essential Medium) supplemented with 10% fetal bovine serum (FBS) at 5% CO.sub.2/37 C. The cells were cultured in a 96 well plate at 110.sup.4 cells/well for 48 hours, washed twice with DMEM and then cultured for 24 hours. Candidate substances were prepared at final concentrations of 25 and 100 M, applied to the wells at 200 l per well and cultured for 24 hours. MTT assay was used to measure the absorbance at 570 nm after colorization in violet.

(124) TABLE-US-00009 TABLE 9 Results of evaluation of cytotoxicity of candidate substances in the EA.hy926 cell line Example Concentration (M) Cell viability (%) 1 25 92.2 2.8 100 94.2 1.9 12 25 95.1 1.9 100 97.2 2.8 13 25 100.1 1.9 100 94.2 2.0 14 25 99.6 1.7 100 99.5 1.6 15 25 100.8 2.5 100 94.9 3.3 20 25 99.7 3.2 100 97.7 2.2 23 25 104.3 1.8 100 99.9 1.9

(125) As shown in Table 9 above, it was confirmed that the candidate substances did not show toxicity up to 100 M in the Ea.hy926 cell line.

(126) 5-2. Evaluation of Cytotoxicity of Candidate Substances Using Human Platelets

(127) Leakage of lactate dehydrogenase (LDH) from the platelets was measured by a spectro-photometry method. 1.2 l of the test substance was added to 598.8 l of PRP, and the mixture was reacted at 37 C. for 3 minutes using a thermomixer. After the reaction, 100 l of the sample was centrifuged at 12,000 g for 2 minutes, and 80 l of the supernatant was taken and refrigerated until evaluation. The evaluation was performed within 24 hours. The control group (positive control) was treated with 50 M digitonin for 1 hour. 25 l of the sample was added to 1 ml of a pre-warmed Tris-EDTA NADH solution (56 mM Tris(hydroxymethyl) aminomethane, 5.6 mM EDTA, 0.17 mM -NADH pH 7.4) and reacted for 5 minutes at 37 C. Then, 100 l of a 14 mM pyruvate solution previously heated at 37 C. was added to the reaction mixture, and absorbance was immediately measured at a wavelength of 339 nm for 1 minute using a spectrophotometer (UV-Vis spectrophotometer, UV-2201, Shidamadzu, Japan). The rate of decrease in absorbance means the rate of oxidation of NADH, indicating the activity of LDH liberated from platelets. Total activity of LDH was measured by inducing lysis of platelets with 0.3% Triton X-100. The basal level of LDH (Control) was measured in plasma. The activity of each sample was measured as a percentage of the total activity of LDH.

(128) TABLE-US-00010 TABLE 10 Results of evaluation of cytotoxicity of candidate substances in platelets Example LDH leakage (%, Mean SEM) Digitonin 50 M 80.4 1.7 1 25 M 2.7 0.5 100 M 2.4 0.2 250 M 2.7 0.5 12 25 M 2.3 0.5 100 M 3.6 0.1 250 M 3.6 0.8 13 25 M 2.2 0.4 100 M 4.0 1.2 250 M 4.0 0.3 14 25 M 4.8 0.2 100 M 5.6 0.9 250 M 4.2 0.9 15 25 M 2.7 0.1 100 M 3.2 0.4 250 M 3.1 1.3 20 25 M 4.1 0.7 100 M 5.2 0.4 250 M 3.5 1.2 23 25 M 1.8 0.1 100 M 2.2 1.3 250 M 3.2 0.5

(129) As shown in Table 10 above, it was confirmed that the candidate substances did not show toxicity up to 250 M in the platelets.

(130) 5-3. Evaluation of Cytotoxicity of Candidate Substances Using Human Liver Cells

(131) Experiments were conducted to evaluate cytotoxicity in human liver cells (HepG2). HepG2 cells, which are human liver carcinoma cell lines, were cultured in DMEM (Dulbecco's minimum essential medium) supplemented with 10% FBS (fetal bovine serum) and 1% Penicillin/Streptomycin at 37 C. and 5% CO.sub.2. The cells were cultured in a 48 well plate at 410.sup.4 cells/well for 24 hours and then used in the experiments. The HepG2 cells were treated with the candidate substances (250 M) for 18 hours, and then the medium was removed and WST-1 was added, followed by shading for 3 hours for reaction. After 3 hours, the supernatant was taken and absorbance was measured at 450 nm. Cell viability was calculated by comparing the absorbance measured after adding WST-1 to HepG2 cells not treated with the candidate substances. Acetaminophen (40 mM) was used as a control.

(132) TABLE-US-00011 TABLE 11 Results of evaluation of cytotoxicity of 250 M of candidate substances in HepG2 cell line Example Cell vaibility (%) Acetaminophen 35.3 3.5 1 91.7 2.0 12 89.0 2.3 13 98.3 5.5 14 87.3 2.7 15 89.7 2.3 20 87.7 2.6 23 82.7 5.5

(133) As shown in Table 11 above, it was confirmed that the candidate substances did not show hepatotoxicity up to 250 M in the HepG2 cell line.

Experimental Example 6: Measurement of Plasma Coagulation Time

(134) In order to separate plasma, blood was collected with 3.2% sodium citrate as an anticoagulant. Whole blood was centrifuged at 2000 g for 20 minutes to obtain plasma. After treating the plasma with the test substances for 3 minutes, plasma coagulation time was measured. For plasma coagulation time, Activated Partial Thromboplastin Time (aPTT) and Prothrombin Time (PT) were measured using BBL Fibrometer (Becton Dickinson, Cockeysville, Md.). For measurement of aPTT, the plasma was treated with aPTT reagents in fibrometer cup and reacted at 37 C. for 3 min. After the reaction, CaCl.sub.2 was added and blood coagulation time was measured immediately. For the measurement of PT, PT reagent was added to warmed plasma, and blood coagulation time was measured immediately. DTI, known to prolong plasma coagulation time, was used as a control.

(135) TABLE-US-00012 TABLE 12 Results of measurement of plasma coagulation time - aPTT Example Time (sec) Control 25.5 0.8 1 25 M 23.8 1.9 100 M 24.8 2.0 250 M 26.7 0.8 12 25 M 24.3 0.1 100 M 26.7 1.0 250 M 27.2 2.0 13 25 M 24.7 1.0 100 M 26.6 1.8 250 M 27.5 1.7 14 25 M 27.0 0.4 100 M 26.7 0.2 250 M 26.3 1.7 15 25 M 26.3 2.3 100 M 26.6 1.0 250 M 29.3 0.5 20 25 M 24.9 1.0 100 M 25.3 0.6 250 M 25.8 0.9 23 25 M 26.2 0.2 100 M 29.5 1.2 250 M 26.1 0.2 DTI 2 M 86.6 2.4

(136) As shown in Table 12 above, it was confirmed that the candidate substances did not significantly change the aPTT points compared with Control, which indicates that the candidate substances did not affect aPTT.

(137) TABLE-US-00013 TABLE 13 Results of measurement of plasma coagulation time - PT Example Time (sec) Control 10.4 0.2 1 25 M 10.8 0.5 100 M 10.7 0.8 250 M 11.0 0.1 12 25 M 10.9 0.1 100 M 11.0 0.5 250 M 10.5 0.3 13 25 M 11.0 0.3 100 M 10.6 0.2 250 M 10.8 0.3 14 25 M 10.1 0.3 100 M 10.1 0.0 250 M 11.1 0.1 15 25 M 10.7 0.0 100 M 10.6 0.2 250 M 10.9 0.1 20 25 M 10.8 0.4 100 M 10.2 0.4 250 M 11.2 0.4 23 25 M 10.3 0.1 100 M 10.7 0.2 250 M 10.8 0.3 DTI 2 M 47.1 1.8

(138) As shown in Table 13 above, it was confirmed that the candidate substances did not significantly change the PT points compared with Control, which indicates that the candidate substances did not affect PT.

Experimental Example 7: Evaluation of Bleeding Side Effect (Bleeding Time) in Experimental Animals

(139) Male Sprague-Dawley rats (250 to 300 g) were fasted overnight and then were orally administered with the candidate substances at each concentration. After 1 hour, the rats were anesthetized by intraperitoneal injection of urethane (1.25 g/kg). The 3 mm portion of the tail tip of the rats was cut and carefully wiped every 30 seconds. Bleeding time was measured in hours until no more blood was drawn, and when the bleeding lasted longer than 30 minutes, the bleeding time was recorded as 30 minutes.

(140) TABLE-US-00014 TABLE 14 Example Bleeding time (min) Control 6.2 0.4 1 25 M 6.0 0.9 100 M 6.5 0.9 12 25 M 6.3 0.6 100 M 6.5 0.3 13 25 M 6.5 0.6 100 M 7.2 0.3 14 25 M 6.7 0.9 100 M 6.3 0.6 15 25 M 7.3 0.7 100 M 6.2 0.6 20 25 M 6.7 0.6 100 M 7.7 0.4 Aspirin 25 M 9.5 0.8 100 M 15.1 0.8 Clopidogrel 2.5 M 14.3 1.6 25 M 26.8 1.6

(141) As shown in Table 14 above, bleeding, which is expected to be a side effect of the candidate substances, was found to be lower than that of clopidogrel and aspirin as control substances. It was confirmed that all of the compounds of the Examples did not show side effects of bleeding.

Experimental Example 8: Evaluation of Bleeding Side Effect by Repeated Administration

(142) Candidate substances (50 mg/kg), clopidogrel (15 mg/kg), or aspirin (100 mg/kg) was each orally administered to ICR mouse (male, 35 to 40 g) once daily for 7 days. One hour after the last administration, the mice were anesthetized by breathing, and the 4 mm portion from the tail tip of the mice was cut. Then, the tail tip of the mice was immersed in a transparent 15-ml tube containing the pre-prepared 37 C. saline. The time when blood was not spread any more and the bleeding stopped was measured.

(143) TABLE-US-00015 TABLE 15 Example Dose (mg/kg) Bleeding time (min) Control 1.6 0.3 Clopidogrel 15 35.4 2.9 Aspirin 100 12.3 0.7 Example 13 50 1.6 0.4 Example 14 50 1.6 0.3

(144) As shown in Table 15 above, the time taken to stop bleeding at the clinical dose exceeded 35 minutes in the case of clopidogrel, and thus the bleeding time was delayed by about 22 times compared with the control group. In the case of aspirin, the time taken to stop bleeding exceeded 12 minutes, and thus the bleeding time was delayed by about 7.6 times. However, when the compounds of Example 13 and Example 14 were repeatedly administered once a day for 7 days, the time taken to stop bleeding was almost the same as that of the control, and the bleeding time was about 1 minute and 30 seconds. Therefore, almost no bleeding side effect was observed for the candidate substances, and it was confirmed that the candidate substances are a new antiplatelet agent candidate that overcomes the bleeding side effect problem of the existing antiplatelet agents.

Experimental Example 9: 2-Week Repeat-Dose Toxicity Study

(145) After oral administration of vehicle (0.5% methylcellulose) or the candidate substances at low dose, medium dose, and high dose (100, 300, 1000 mg/kg) to ICR mice (female, male, 18 to 26 g) once a day for 2 weeks, the NOAEL values of the candidate substances were calculated by observing and recording mortality, body weight change, clinical observations, food and drinking water consumption, gross necropsy findings, histopathological evaluation of abnormal organ, and organ weights.

(146) TABLE-US-00016 TABLE 16 Example NOAEL (No observed adverse effect level) Example 13 >1000 mg/kg Example 14 >1000 mg/kg

(147) As shown in Table 16 above, when the compounds of Example 13 or Example 14 were orally administered at doses of 100, 300 and 1000 mg/kg once a day for 2 weeks, no dead animals were observed, and no abnormal findings were observed in body weight change, clinical observations, food and drinking water consumption, gross necropsy findings, and organ weights. Therefore, it was confirmed that the NOAEL value of the candidate substances might be 1000 mg/kg or more.

Experimental Example 10: Statistics

(148) The significance of the test results was assessed as significant when p was 0.05 or less via the Student's t-test and the one-way ANOVA test.