Bicyclic compound as a caspase inhibitor

Abstract

A compound represented by formula (I), a pharmaceutically acceptable salt or tautomer thereof, and an application of the compound as a caspase inhibitor. ##STR00001##

Claims

1. A compound represented by formula (I), a pharmaceutically acceptable salt or tautomer thereof, ##STR00056## wherein, ring A is selected from a 5- or 6-membered heteroaryl group, which is optionally substituted with R; ring B is selected from phenyl or C.sub.3-6 cycloalkyl, which is optionally substituted with R; R is selected from halogen, OH, NH.sub.2, or a C.sub.1-3 alkyl group optionally substituted with 1, 2 or 3 R.sup.1; R.sup.1 is selected from F, Cl, Br, I, OH, NH.sub.2, NH(CH.sub.3) or N(CH.sub.3).sub.2.

2. The compound, the pharmaceutically acceptable salt or tautomer thereof according to claim 1, wherein a heteroatom of ring A is independently selected from O, S or N, and number of the heteroatom of ring A is selected from 1, 2 or 3.

3. The compound, the pharmaceutically acceptable salt or tautomer thereof according to claim 1, wherein ring A is selected from oxazolyl, isoxazolyl, imidazolyl, thiazolyl, 1,2,4-oxadiazolyl, 1,3,4-oxadiazolyl or pyrazolyl.

4. The compound, the pharmaceutically acceptable salt or tautomer thereof according to claim 1, wherein ring A is selected from ##STR00057##

5. The compound, the pharmaceutically acceptable salt or tautomer thereof according to claim 1, wherein ring B is selected from phenyl or cyclohexyl, which is optionally substituted with R.

6. The compound, the pharmaceutically acceptable salt or tautomer thereof according to claim 1, wherein R is selected from F, Cl, Br, I, OH, NH.sub.2, or Me or Et optionally substituted with 1, 2 or 3 R.sup.1.

7. The compound, the pharmaceutically acceptable salt or tautomer thereof according to claim 6, wherein R is selected from F, Cl, Br, I, OH, NH.sub.2 or CF.sub.3.

8. The compound, the pharmaceutically acceptable salt or tautomer thereof according to claim 1, wherein ring B is selected from ##STR00058##

9. The compound, the pharmaceutically acceptable salt or tautomer thereof according to claim 1, wherein ##STR00059## is selected from ##STR00060## and ring B is optionally substituted with R.

10. The compound the pharmaceutically acceptable salt or tautomer thereof according to claim 1, wherein ##STR00061## is selected from ##STR00062##

11. The compound, the pharmaceutically acceptable salt or tautomer thereof according to claim 10, wherein ##STR00063## selected from ##STR00064##

12. The compound, the pharmaceutically acceptable salt or tautomer thereof according to claim 1, wherein ##STR00065## is selected from ##STR00066##

13. The compound, the pharmaceutically acceptable salt or tautomer thereof according to claim 1, wherein the compound is represented by the following formulae, ##STR00067## ##STR00068## ##STR00069##

14. A pharmaceutical composition comprising a therapeutically effective amount of the compound, the pharmaceutically acceptable salt or tautomer thereof according to claim 1, and a pharmaceutically acceptable carrier or excipient.

15. A method of treating caspase receptor related diseases in a mammal, comprising administering a therapeutically effective amount of the compound, the pharmaceutically acceptable salt or tautomer thereof according to claim 1 to the mammal in need thereof.

16. The compound, the pharmaceutically acceptable salt or tautomer thereof according to claim 6, wherein R is selected from F, Cl, Br, I, OH, NH.sub.2, or Me optionally substituted with 1, 2 or 3 R.sup.1, and wherein R.sup.1 is selected from F, Cl, Br or I.

17. The compound, the pharmaceutically acceptable salt or tautomer thereof according to claim 1, wherein R is selected from F, Cl, Br, I, OH, NH.sub.2, or Me or Et optionally substituted with 1, 2 or 3 F.

18. The compound, the pharmaceutically acceptable salt or tautomer thereof according to claim 17, wherein R is selected from F, Cl, Br, I, or Me optionally substituted with 1, 2 or 3 F.

19. The compound, the pharmaceutically acceptable salt or tautomer thereof according to claim 7, wherein R is selected from F, Cl, Br, I or CF.sub.3.

20. The compound, the pharmaceutically acceptable salt or tautomer thereof according to claim 1, wherein the compound is selected from a compound of formula (II), ##STR00070##

Description

EXAMPLES

(1) The present application is described in detail below by way of examples, but is not intended to be construed as limitation. The present application has been described in detail herein, and the specific embodiments thereof are disclosed. Various changes and modifications made to the embodiments of the present application will be apparent to persons skilled in the art, without departing from the spirit and scope of the present application.

Preparation Example 1: Preparation of Compound 1-a

(2) ##STR00019##
Step 1: Synthesis of Compound A-3

(3) Compound A-1 (30.00 g, 92.78 mmol, 1.00 eq) and 4-methylmorpholine (15.02 g, 148.45 mmol, 16.33 mL, 1.60 eq) were dissolved in tetrahydrofuran (468 mL) under the protection of nitrogen at −10° C. Compound A-2 (19.01 g, 139.17 mmol, 18.28 mL, 1.50 eq) was slowly added dropwise thereto, and stirred for 40 min while maintaining the temperature at −10° C. The reaction mixture was filtered, and the filter cake was washed with tetrahydrofuran (200 mL). The combined filtrate was poured into a three-necked flask and kept at the temperature of 0° C. A CH.sub.2N.sub.2-diethyl ether solution (370 mL) was added into the flask under the protection of nitrogen, further stirred at 0° C. for 20 minutes, followed by being heated to 20° C. and stirred for another 2 hours. The reaction mixture was then further cooled to 0° C. and treated with HBr (30 mL, 35% acetic acid solution), and then the mixture was stirred at 0° C. for 15 min, followed by being heated to 20° C. and stirred for another 45 min. After the reaction was completed, the reaction mixture was extracted by using ethyl acetate (500 mL) and water (400 mL), and separated. The organic phase was further washed with water (400 mL), saturated sodium bicarbonate solution (400 mL) and saturated saline (400 mL). It was dried over anhydrous sodium sulfate and then concentrated to give a crude product, which was purified by column chromatography to give a colorless oily compound A-3 (30.00 g, yield: 76%).

(4) Step 2: Synthesis of Compound A-5

(5) Compound A-3 (25.00 g, 62.46 mmol, 1.00 eq) and A-4 (12.45 g, 74.95 mmol, 1.20 eq) were dissolved in DMF (350.00 mL). KF (14.52 g, 249.84 mmol, 5.85 mL, 4.00 eq) was added thereto under the protection of nitrogen, and then the reaction was stirred at 20° C. for 15 hours. After the reaction was completed, 500 mL of ethyl acetate was added thereto, and it was washed with saturated sodium bicarbonate solution (350 mL), water (350 mL) and saturated saline (350 mL). The organic phase was dried over anhydrous sodium sulfate, and concentrated to give crude product, which was purified by column chromatography (petroleum ether:ethyl acetate=1:0-3:1) to give compound A-5 (18.00 g, yield: 56%).

(6) Step 3: Synthesis of Compound A-6

(7) Compound A-5 (9.50 g, 19.57 mmol, 1.00 eq) was added into a mixed solvent of methanol (30.00 mL) and tetrahydrofuran (30.00 mL), and then sodium borohydride (2.96 g, 78.28 mmol, 4.00 eq) was added thereto while maintaining the temperature at 0° C., and after the addition was completed, the reaction mixture was stirred at 25° C. for 1 hour. After the reaction was completed, the reaction mixture was added into water (200 mL), and NH.sub.4Cl (200 mL, aq, 10%) was added thereto, and then extracted with ethyl acetate (500 mL*3). The combined organic phase was washed with water (500 mL) and saline (500 mL), dried over anhydrous sodium sulfate, and filtered to give a colorless oily compound A-6 (9.00 g, crude), which was directly used in the next reaction without purification.

(8) Step 4: Synthesis of Compound A-7

(9) Compound A-6 (9.00 g, 18.46 mmol, 1.00 eq) was dissolved in methanol (500.00 mL), and Pd—C (10%, 2.5 g) was added thereto. The mixture was replaced 3 times with hydrogen and maintained at a pressure of 15 psi, and the mixture was stirred for 4 hours while maintaining the temperature at 25° C. After the reaction was completed, filtration and concentration was performed to give a yellow oily compound A-7 (6.10 g, crude), which was directly used in the next reaction without purification.

(10) Step 5: Synthesis of Compound A-9

(11) Compound A-7 (6.10 g, 17.2 mmol, 1.00 eq) and compound A-8 (3.85 g, 17.2 mmol, 1.00 eq) were dissolved in ethyl acetate solution (100 mL), and then T.sub.3P (16.42 g, 25.8 mmol, 1.50 eq, 50% ethyl acetate solution) and DIPEA (4.44 g, 34.4 mmol, 2.0 eq) were successively added thereto, and stirred at 25° C. for 4 hours. The reaction solution was added with water (50 mL) for quenching and separation. The organic phase was washed once with saturated sodium bicarbonate solution (50 mL), water (50 mL) and saturated saline (50 mL), respectively. The organic phase was dried over anhydrous sodium sulfate and concentrated to give a crude product. The crude product was purified by column chromatography (ethyl acetate:petroleum ether=1:20-1:2) to give the product, compound A-9 (3.50 g, yield: 36.5%); LCMS m/z=581.2 [M+Na]+.

(12) Step 6: Synthesis of Compound 1-a

(13) Compound A-9 (3.30 g, 5.91 mmol, 1.00 eq) was dissolved in a mixed solvent of methanol (33.0 mL) and THE (33.0 mL), and Pd—C (10%, 330 mg) was added thereto. The mixture was replaced three times with hydrogen and maintained at a pressure of 15 psi, and the mixture was stirred for 2 hours while maintaining the temperature at 25° C. After the reaction was completed, filtration and concentration was performed to give yellow oily compound A (2.18 g, 4.50 mmol, yield: 76.2%), which was directly used in the next reaction without purification; LCMS m/z=425.2 [M+H].sup.+.

Preparation Example 2: Preparation of Compound 1-e (Referring to the Preparation Route of Compound 1-a, Replacing Compound A-1 with the Chiral Enantiomer Thereof)

(14) TABLE-US-00001 Compound No. Structure MS(m/z) [M + H].sup.+ 1-e 0 425.2

Example 1: (S)-3-((S)-2-(5-(2-chlorophenyl)isoxazol-3-carboxamido) propionamido)-4-oxo-5-(2,3,5,6-tetrafluorophenoxy)pentanoic Acid (Compound 1)

(15) ##STR00021##
Step 1: Synthesis of Compound 1-c

(16) Compound 1-b (100.00 mg, 447.21 μmol, 1.00 eq) was dissolved in dichloromethane (10.00 mL), and compound NMM (135.71 mg, 1.34 mmol, 147.51 μL, 3.00 eq), HOBt (82.78 mg, 612.67 μmol, 1.37 eq), EDCl (117.45 mg, 612.67 μmol, 1.37 eq) and compound 1-a (189.79 mg, 447.21 μmol, 1.00 eq) were added thereto. The reaction solution was stirred at 25° C. for 4 hours. After the reaction was completed, the reaction solution was directly concentrated to give a crude product, which was purified by flash silica-gel column chromatography (petroleum ether:ethyl acetate=4:1) to give a colorless oily compound 1-c. LCMS m/z=652.3 [M+Na].sup.+.

(17) Step 2: Synthesis of Compound 1-d

(18) Compound 1-c (220.00 mg, 349.22 μmol, 1.00 eq) was dissolved in dichloromethane (10.00 mL), and iodobenzene diacetate (435.31 mg, 1.35 mmol, 3.87 eq) and TEMPO (54.92 mg, 349.22 μmol, 1.00 eq) were added thereto. The reaction solution was stirred at 25° C. for 15 hours. After the reaction was completed, dichloromethane (20 mL) was added to the reaction solution, and the solution was successively washed with water (20 mL), saturated sodium bicarbonate solution (20 mL) and saline (20 mL). The organic phase was dried over anhydrous Na.sub.2SO.sub.4, filtered and concentrated to give a crude product. The crude product was purified by flash silica-gel column chromatography (petroleum ether:ethyl acetate=7:3) to give a light yellow oily compound 1-d. LCMS m/z=650.1 [M+Na].sup.+.

(19) Step 3: Synthesis of Compound 1

(20) Compound 1-d (210.00 mg, 334.41 μmol, 1.00 eq) was dissolved in dichloromethane (4.00 mL), and trifluoroacetic acid (1.54 g, 13.51 mmol, 1.00 mL, 40.39 eq) was added thereto. The reaction solution was stirred at 25° C. for 3 hours. After the reaction was completed, the reaction solution was concentrated to give a crude product. The crude product was purified by prep-HPLC (in the condition of trifluoroacetic acid), and freeze-dried to give compound 1. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 12.47 (br s, 1H), 9.01 (br s, 1H), 8.67 (br s, 1H), 7.96 (dd, J=2.26, 7.28 Hz, 1H), 7.72 (d, J=7.39 Hz, 1H), 7.48-7.65 (m, 3H), 7.38 (s, 1H), 5.02-5.42 (m, 2H), 4.62 (d, J=6.02 Hz, 1H), 4.40-4.54 (m, 1H), 2.73-2.85 (m, 1H), 2.52-2.65 (m, 1H), 1.37 (d, J=7.03 Hz, 3H); LCMS m/z=572.1 [M+H].sup.+.

Examples 2-17: The Compounds of Examples 2-17 were Prepared by Referring to the Synthetic Route of Example 1 and Replacing Compound 1-b with Different Intermediate Acids

(21) TABLE-US-00002 Example Structure of intermediate acid Structural formula Example 2 Example 3 Example 4 Example 5 Example 6 0 Example 7 Example 8 Example 9 Example 10 Example 11 0 Example 12 Example 13 Example 14 Example 15 Example 16 0 Example 17

Example 18: The Compound of Example 18 was Prepared by Referring to the Synthetic Route of Example 1 and Replacing Compound 1-a with Intermediate 1-e

(22) TABLE-US-00003 Example intermediate Structural formula Example 18

(23) The NMR and MS data of Examples 2-13 and Examples 17-18 were as follows:

(24) TABLE-US-00004 MS (m/z) Example .sup.1H NMR [M + H].sup.+ Example 2 .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.82 (d, J = 7.03 Hz, 1H), 554.1 8.66 (br d, J = 7.78 Hz, 1H), 8.44 (s, 1H), 7.78 (br d, J = 7.28 Hz, 2H), 7.39-7.65 (m, 4H), 5.15-5.32 (m, 2H), 4.63 (q, J = 6.69 Hz, 1H), 4.45 (quin, J = 7.09 Hz, 1H), 2.75-2.83 (m, 1H), 2.58 (dd, J = 6.78, 16.81 Hz, 1H), 1.38 (d, J = 7.03 Hz, 3H) Example 3 .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.70 (d, J = 7.53 Hz, 1H), 537.2 8.11 (brd, J = 5.52 Hz, 1H), 8.03 (d, J = 7.03 Hz, 2H), 7.85 (s, 1H), 7.40-7.60 (m, 4H), 5.15-5.35 (m, 2H), 4.59-4.70 (m, 1H), 4.48 (quin, J = 7.15 Hz, 1H), 2.73- 2.83 (m, 1H), 2.60 (dd, J = 6.53, 17.07 Hz, 1H), 1.35 (d, J = 7.03 Hz, 3H) Example 4 .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.65 (d, J = 7.53 Hz, 1H), 537.1 7.80 (d, J = 7.03 Hz, 2H), 7.52-7.60 (m, 1H), 7.47 (br t, J = 7.53 Hz, 2H), 7.33-7.40 (m, 1H), 5.14-5.35 (m, 2H), 4.64 (q, J = 6.53 Hz, 1H), 4.46 (t, J = 7.03 Hz, 1H), 2.73- 2.83 (m, 1H), 2.59 (dd, J = 6.53, 17.07 Hz, 1H), 1.35 (d, J = 7.03 Hz, 3H) Example 5 .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 13.64-13.96 (m, 1H), 555.1 8.64 (d, J = 5.52 Hz, 1H), 7.81-8.31 (m, 2H), 7.21-7.66 (m, 6H), 5.13-5.35 (m, 2H), 4.63 (d, J = 5.02 Hz, 1H), 4.42-4.51 (m, 1H), 2.78 (dd, J = 5.77, 16.81 Hz, 1H), 2.54-2.65 (m, 1H), 1.35 (d, J = 7.03 Hz, 3H) Example 6 .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 12.49 (br s, 1H), 8.91 571.9 (d, J = 6.52 Hz, 1H), 8.66 (d, J = 7.53 Hz, 1H), 7.97 (d, J = 8.53 Hz, 2H), 7.65 (d, J = 8.53 Hz, 2H), 7.50-7.62 (m, 1H), 7.43 (s, 1H), 5.17-5.32 (m, 2H), 4.62 (q, J = 6.53 Hz, 1H), 4.46 (quin, J = 7.03 Hz, 1H), 2.80 (dd, J = 6.02, 17.07 Hz, 1H), 2.59 (dd, J = 6.53, 17.07 Hz, 1H), 1.36 (d, J = 7.53 Hz, 3H) Example 7 .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 12.49 (br s, 1H), 8.75 538.0 (s, 1H), 8.70 (d, J = 7.53 Hz, 1H), 8.26 (d, J = 6.53 Hz, 1H), 8.01-8.09 (m, 2H), 7.48-7.69 (m, 4H), 5.25 (q, J = 17.73 Hz, 2H), 4.65 (d, J = 6.53 Hz, 1H), 4.45-4.58 (m, 1H), 2.73-2.85 (m, 1H), 2.61 (dd, J = 6.02, 16.56 Hz, 1H), 1.38 (d, J = 7.03 Hz, 3H) Example 8 .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 12.49 (br s, 1H), 9.17 538.0 (br s, 1H), 8.68 (br s, 1H), 7.93 (dd, J = 3.01, 6.53 Hz, 2H), 7.69 (s, 1H), 7.48-7.63 (m, 4H), 5.09-5.44 (m, 2H), 4.62 (d, J = 4.52 Hz, 1H), 4.39-4.53 (m, 1H), 2.80 (dd, J = 6.02, 16.06 Hz, 1H), 2.59 (d, J = 3.51 Hz, 1H), 1.37 (d, J = 7.03 Hz, 3H) Example 9 .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 12.47 (br s, 1H), 8.89 538.0 (br s, 1H), 8.68 (br s, 1H), 7.89-7.98 (m, 2H), 7.50- 7.65 (m, 4H), 7.38 (s, 1H), 5.12-5.40 (m, 2H), 4.63 (br s, 1H), 4.41-4.54 (m, 1H), 2.79 (dd, J = 6.53, 15.56 Hz, 1H), 2.61 (d, J = 4.02 Hz, 1H), 1.37 (d, J = 7.03 Hz, 3H) Example .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 12.46 (br s, 1H), 8.91 572.0 10 (br s, 1H), 8.65 (br s, 1H), 8.04 (s, 1H), 7.86-7.96 (m, 1H), 7.53-7.67 (m, 3H), 7.51 (s, 1H), 5.22 (br s, 2H), 4.64 (br s, 1H), 4.37-4.55 (m, 1H), 2.78 (dd, J = 5.52, 15.56 Hz, 1H), 2.55-2.66 (m, 1H), 1.37 (d, J = 7.03 Hz, 3H) Example .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 12.31 (br s, 1H), 8.97- 605.9 11 9.08 (m, 1H), 8.69 (dd, J = 7.78, 15.81 Hz, 1H), 8.01 (d, J = 7.53 Hz, 1H), 7.89 (s, 2H), 7.80-7.87 (m, 1H), 7.49- 7.67 (m, 1H), 7.16 (s, 1H), 5.13-5.39 (m, 2H), 4.64 (dt, J = 7.03, 13.55 Hz, 1H), 4.41-4.51 (m, 1H), 2.71- 2.86 (m, 1H), 2.55-2.66 (m, 1H), 1.37 (d, J = 7.03 Hz, 3H) Example .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 12.48 (br s, 1H), 9.01 556.0 12 (br s, 1H), 8.69 (br s, 1H), 8.01 (t, J = 7.03 Hz, 1H), 7.38-7.77 (m, 4H), 7.22 (d, J = 2.51 Hz, 1H), 5.11-5.37 (m, 2H), 4.63 (br s, 1H), 4.40-4.53 (m, 1H), 2.79 (dd, J = 5.52, 16.56 Hz, 1H), 2.56-2.65 (m, 1H), 1.37 (d, J = 7.53 Hz, 3H) Example .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 9.60 (br s, 1H), 8.70 (br 539.0 13 s, 1H), 8.09 (dd, J = 1.25, 7.78 Hz, 2H), 7.49-7.72 (m, 4H), 5.23 (br s, 2H), 4.40-4.74 (m, 2H), 2.79 (dd, J = 6.02, 16.31 Hz, 1H), 2.53-2.63 (m, 1H), 1.40 (d, J = 7.03 Hz, 3H) Example .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 9.22 (br d, J = 6.27 Hz, 572.1 17 1H), 8.67 (br s, 1H), 7.75 (dd, J = 1.51, 7.53 Hz, 1H), 7.68 (dd, J = 1.00, 7.78 Hz, 1H), 7.48-7.63 (m, 4H), 5.20 (br s, 2H), 4.65 (br s, 1H), 4.38-4.51 (m, 1H), 2.70-2.85 (m, 1H), 2.55-2.69 (m, 1H), 1.36 (d, J = 7.28 Hz, 3H). Example .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 12.47 (br s, 1H), 8.85- 572.0 18 9.04 (m, 1H), 8.60-8.70 (m, 1H), 7.89-7.99 (m, 1H), 7.71 (d, J = 7.41 Hz, 1H), 7.48-7.64 (m, 3H), 7.34-7.40 (m, 1H), 5.12-5.38 (m, 2H), 4.51-4.69 (m, 1H), 4.31- 4.50 (m, 1H), 2.72-2.85 (m, 1H), 2.53-2.66 (m, 1H), 1.37 (d, J = 7.03 Hz, 3H).

Experimental Example 1: Assay of In Vitro Inhibitory Activity of Compounds on Caspase

(25) Experimental Purpose:

(26) Caspase Inhibitor Screening Kit for BioVision was used in this experiment to test the inhibitory activity of the test compounds on Caspase.

(27) Experimental Material:

(28) 1) Kit:

(29) Caspase-1 Inhibitor Screening Kit (BioVision #K151-100)

(30) Caspase-3 Inhibitor Screening Kit (BioVision #K153-100)

(31) Caspase-8 Inhibitor Screening Kit (BioVision #K158-100)

(32) Note: Each caspase enzymatic experiment uses the reagents in corresponding kit thereof. Each enzyme was dissolved in 550 μl of the corresponding 2× reaction buffer, sub-packed and stored at −80° C., respectively.

(33) 2) Black 384-well plate (PerkinElmer #6007279)

(34) 3) Instrument: Multi-function microplate reader Molecular Devices (Model: SpectraMax M2e)

(35) Experimental Method:

(36) 1) The compound was diluted to a 200*test concentration with DMSO via the multiple dilution, then to a 2*test concentration with ddH.sub.2O, and added to a 384-well experimental plate at 12.5 μl per well. Test compounds and control compounds were tested at 6 concentration points, test concentrations ranging from 1000 nM to 0.32 nM. The ddH.sub.2O containing 1% DMSO was added to the 0% inhibition control well, and a high concentration of the control compound was added to the 100% inhibition control well (final concentration: 5 μM).

(37) 2) 2× reaction buffer containing 10 mM DTT was prepared. The enzyme caspase stock solution was diluted 5-fold with 2× reaction buffer containing 10 mM DTT and added to a 384-well experimental plate at 6.25 μl per well. After being mixed, the enzyme and compounds were incubated at 37° C. for 30 minutes.

(38) 3) The fluorogenic substrate of the enzyme caspase was diluted 5-fold with 2× reaction buffer containing 10 mM DTT, and then added to a 384-well experimental plate at 6.25 μl per well. The total reaction volume was 25 μl, the final concentration of the substrate was 50 μM, and the final concentration of DMSO was 0.5%. After the substrate was added, the 384-well experimental plate was incubated at 37° C. for 30 minutes.

(39) 4) The fluorescence intensity (excitation light wavelength was 400 nm, emission light wavelength was 505 nm) was measured by using a multi-function microplate reader. Fluorescence intensity was used to calculate the inhibitory effect of the compounds on Caspase. GraphPad Prism software was used for fitting compound inhibition curves and calculating IC.sub.50 values.

(40) Experimental Results:

(41) The experimental results of the test compounds were shown in Table 1.

(42) TABLE-US-00005 TABLE 1 Test results of enzymatic activities of test compounds Compound No. Caspase-1 Caspase-3 Caspase-8 Example 1 4.6 13.0 10.3 Example 2 5.3 29.6 24.6 Example 3 4.8 24.0 25.9 Example 4 4.1 11.4 11.7 Example 5 3.9 13.7 16.8 Example 6 6.0 24.5 19.9 Example 7 6.9 20.2 30.0 Example 8 5.5 21.8 18.0 Example 9 14.2 44.4 23.2 Example 10 6.6 22.0 16.3 Example 11 9.2 30.0 22.6 Example 12 6.7 21.0 17.9 Example 17 4.2 9.5 31.4 Example 18 7.5 26.1 62.1
Experimental Conclusion:

(43) As can be seen from the above Table 1, the compounds of the present application can have good inhibitory activity on Caspase.

Experimental Example 2: Mouse Pharmacokinetic Study

(44) Experimental Purpose:

(45) This experiment intended to investigate the pharmacokinetics in plasma and liver of male C57BL/6J mice after oral administration of the compounds.

(46) Experimental Method:

(47) Mice were randomly divided into three groups (3 male mice per group). The compound was formulated into the specified formulation. Oral formulations may be clear or uniform suspensions. Animals were intragastrically administered with a given dose of the preparation, respectively.

(48) Whole blood samples were collected from animals through jugular vein puncture at 3 time points of 30 minutes, 2 hours, and 6 hours after administration, approximately 25 μL per sample; while the liver was collected at each time point.

(49) The plasma samples were added to centrifuge tubes containing anticoagulant, and centrifuged at 4° C., 3000 g for 15 min, and the supernatant plasma was taken and quickly frozen on dry ice and then stored in a refrigerator at −70±10° C. until LC-MS/MS analysis was performed.

(50) The blood outside the liver was sopped up with absorbent paper, and the weight of the liver was weighed, and then placed in liquid nitrogen to freeze. MeOH/15 mM PBS (1:2) at a volume-weight ratio of 1:5 was added thereto, and then the homogenization was performed at 14000 rpm for 2 minutes. It was then stored in a refrigerator at −70±10° C. until LC-MS/MS analysis was performed.

(51) Data Processing:

(52) The plasma drug concentration data for the compounds were processed with the non-compartment model using WinNonlin™ Version 6.3.0 (Pharsight, Mountain View, Calif.) pharmacokinetic software. The maximal concentration (C.sub.max), maximal concentration time (T.sub.max) and quantitative end time were directly obtained from the plasma concentration-time chart.

(53) The following pharmacokinetic parameters were calculated by using logarithmic linear trapezoidal method: elimination phase half-life (T.sub.1/2); the in vivo mean residence time of the drug from point 0 to the last time point (MRT.sub.0-last); the in vivo mean residence time of the drug from point 0 to infinite time (MRT.sub.0-ifn); the area under time-plasma concentration curve from point 0 to the last time point (AUC.sub.0-last); the area under time-plasma concentration curve from point 0 to infinite time (AUC.sub.0-inf).

(54) For individual plasma concentrations less than BQL, the one occurred before T.sub.max was calculated as 0, and the one occurred after T.sub.max was directly excluded. All parameters and ratios were reported in the forms of three significant digits.

(55) The pharmacokinetic parameters of this experiment were calculated according to the theoretical blood collection times and the theoretical administration concentrations in the protocol. The deviations between the actual administration concentrations and the theoretical concentrations were within the range of ±20%. The deviations between the actual blood collection times and the theoretical blood collection times were in conformity with the relevant SOP (the points within 1 hour after administration were within the range of ±1 min, and the others were within 5% of the theoretical time).

(56) Experimental Results:

(57) The experimental results of the test compounds were shown in Table 2.

(58) TABLE-US-00006 TABLE 2 Pharmacokinetic study of the test compounds Compound No. IDN-6556 Example 1 0.5 h   Plasma drug concentration (nM) 435 2523 Liver drug concentration (nmol/kg) 8600 46440 2 h Plasma drug concentration (nM) 10.9 112 Liver drug concentration (nmol/kg) 930 3126 6 h Plasma drug concentration (nM) 5.79 12.8 Liver drug concentration (nmol/kg) 230 3252 plasma drug-time curve (nM .Math. h) 314 1795 liver drug-time curve (nM .Math. h) 9347 48442 Area ratio of plasma/liver drug-time curves 30 25
Experimental Conclusion:

(59) It can be seen from the above Table 2 that, the reference compound IDN-6556 had a relatively small amount of liver exposure at different time points, and the area of the liver drug-curve was lower. The liver exposure of the compound shown in Example 1 can have significant improvement compared to IDN-6556, with about 5 times higher.

(60) At the same time, Example 1 also maintained a comparable area ratio of liver/plasma drug-time curves. If drugs were used to treat liver diseases, high liver exposures of drugs make it possible for us to reduce the dose to be administered.

Experimental Example 3: Pharmacodynamic Study in Mice

(61) Experimental Purpose:

(62) The therapeutic effects of the test compound IDN-6556 and the compound of Example 1 in the CCl.sub.4-induced chronic liver fibrosis model of male C57BL/6 mice were tested.

(63) Experimental Method:

(64) Male C57BL/6 mice were randomly divided into 7 groups: pseudo model group (group 1), model group (group 2, solvent p.o., q.d), IDN-6556 (group 3, 3 mg/kg, p.o., bid), IDN-6556 (group 4, 10 mg/kg, p.o., bid), Example 1 (group 5, 3 mg/kg, p.o., bid), Example 1 (group 6, 10 mg/kg, p.o., bid)), Example 1 (group 7, 20 mg/kg, p.o., bid). CCl.sub.4 was formulated into a CCl.sub.4-olive oil mixed solution according to the doses by using olive oil, modeling is performed by orally administration three times a week for 4 weeks; and the pseudo model group was orally administered with the same volume of olive oil alone. Compounds were orally administrated from the day of modeling, twice a day for 28 days, while the pseudo model group and the model group were orally administered with an equal volume of drug solvent. On the next day after the last administration, the animals were fasted for 6 hours, followed by euthanizing, and the livers were collected. Liver tissues were fixed in 10% formalin solution for histopathological analysis.

(65) Experimental Results:

(66) Table 3 showed pathology assay score of liver. It can be seen from this table that the different dose groups of IND-6556 and Example 1 all can significantly improve liver tissue damage caused by CCl.sub.4, and in addition, Example 1 also significantly inhibited the formation of liver fibrosis compared with the model group (p<0.01).

(67) TABLE-US-00007 TABLE 3 Pathology assay of liver (mean ± sem) Total score of Inflammatory Ballooning Percentage of Group liver injury cell infiltration change score liver fibrosis (%) Group 1 0.00 ± 0.00   0.00 ± 0.00   0.00 ± 0.00   0.88 + 0.03 Group 2 2.57 ± 0.29.sup.###  2.00 + 1.20.sup.###  0.50 + 0.14.sup.###   .sup. 1.95 + 0.09.sup.### Group 3 1.60 ± 0.07*** 1.55 + 0.06*  0.05 + 0.04*** 1.84 + 0.07 Group 4 1.43 ± 0.12*** 1.35 + 1.10*** 0.08 + 0.04*** 1.86 + 0.12 Group 5 1.13 ± 0.06*** 1.13 + 0.06*** 0.00 ± 0.00*** .sup. 1.61 + 0.05.sup.$$ Group 6 1.20 ± 0.07*** 1.15 + 0.07*** 0.00 ± 0.00*** .sup. 1.62 + 0.07.sup.$ Group 7 1.12 ± 0.04*** 1.10 + 0.04*** 0.00 ± 0.00*** 1.69 + 0.10 One-way ANOVA: .sup.###p < 0.001 vs. group 1; *p < 0.05 vs. group 2; ***p < 0.001 vs. group 2 T-test: .sup.$p < 0.05 vs. group 2; .sup.$$p < 0.01 vs. group 2.
Experimental Conclusion:

(68) The results demonstrated that liver fibrosis of C57BL/6 mice was successfully induced by oral administration of CCl.sub.4. Different doses of IDN-6556, oral administration twice a day for 28 days, can significantly inhibit CCl.sub.4-induced liver tissue damage, especially inflammatory cell infiltration; however, no clear inhibitory effect on liver fibrosis was observed. Different doses of Example 1, oral administration twice a day for 28 days, can significantly inhibit CCl.sub.4-induced liver tissue damage, particularly inflammatory cell infiltration, while significant inhibitory effect on liver fibrosis can be observed. Overall, the pharmacodynamic action of Example 1 was superior to IND-6556 in this model.