TRICYCLIC COMPOUND USED AS GPR84 ANTAGONIST

Abstract

The present invention relates to a tricyclic compound used as a GPR84 antagonist, and in particular relates to a tricyclic compound having a structure shown in formula I, and tautomers, stereoisomers, hydrates, solvates, pharmaceutically acceptable salts or prodrugs thereof; the definitions of the ring Cy, L1, R1 are as described in the present invention; the tricyclic compound has significant GPR84 antagonism, good pharmaceutical developability and high safety.

##STR00001##

Claims

1. A tricyclic compound, a tautomer, a stereoisomer, a hydrate, a solvate, a pharmaceutically acceptable salt, or a prodrug thereof, and the tricyclic compound has a structure of formula I: ##STR00025## wherein ##STR00026## ring Cy is L.sub.1 is absent or L.sub.1 is C.sub.1-C.sub.4 alkylene, C.sub.2-C.sub.4 alkenylene with one double bond, or C.sub.2-C.sub.4 alkynylene with one triple bond; R.sub.1 is C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkoxy, 3- to 6-membered cycloalkyl, or 4- to 6-membered heterocycloalkyl; the R.sub.1 is optionally substituted by R.sub.11; the R.sub.11 is a substituent selected from the following: halogen, cyano, hydroxyl, C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkoxy, C.sub.3-C.sub.6 cycloalkyl, C.sub.1-C.sub.6 haloalkyl, and C.sub.1-C.sub.6 haloalkoxy; when there is more than one substituent, R.sub.11 groups are the same or different substituents.

2. The tricyclic compound, the tautomer, the stereoisomer, the hydrate, the solvate, the pharmaceutically acceptable salt, or the prodrug thereof according to claim 1, wherein L.sub.1 is absent or L.sub.1 is CH.sub.2, CH?CH, or C?C.

3. The tricyclic compound, the tautomer, the stereoisomer, the hydrate, the solvate, the pharmaceutically acceptable salt, or the prodrug thereof according to claim 1, wherein R.sub.1 is 3- to 6-membered cycloalkyl.

4. The tricyclic compound, the tautomer, the stereoisomer, the hydrate, the solvate, the pharmaceutically acceptable salt, or the prodrug thereof according to claim 1, wherein R.sub.11 is a substituent selected from the following: halogen, cyano, C.sub.1-C.sub.6 alkyl, and C.sub.1-C.sub.6 alkoxy.

5. The tricyclic compound, the tautomer, the stereoisomer, the hydrate, the solvate, the pharmaceutically acceptable salt, or the prodrug thereof according to claim 1, wherein the tricyclic compound is: ##STR00027##

6. An intermediate B having a structure of ##STR00028## wherein Cy is as defined in claim 1; X is selected from: OTf, OTs, OMs, chlorine, bromine, or iodine.

7. A preparation method for the tricyclic compound, the tautomer, the stereoisomer, the hydrate, the solvate, the pharmaceutically acceptable salt, or the prodrug thereof according to claim 1, comprising the following step: 1) reacting the intermediate B with compound H-L.sub.1-R.sub.1 to obtain the tricyclic compound; wherein L.sub.1 and R.sub.1 are as defined in claim 1; intermediate B having a structure of ##STR00029## wherein Cy is as defined in claim 1; X is selected from: OTf, OTs, OMs, chlorine, bromine, or iodine.

8. The method according to claim 7, further comprising: 2) reacting the intermediate B with compound H-L.sub.1-R.sub.1 in the presence of a catalyst; and/or 3) reacting the intermediate B with compound H-L.sub.1-R.sub.1 under the protection of an inert gas; and/or 4) reacting the intermediate B with compound H-L.sub.1-R.sub.1 under an alkaline condition.

9. A pharmaceutical composition, comprising: the tricyclic compound, the tautomer, the stereoisomer, the hydrate, the solvate, the pharmaceutically acceptable salt, or the prodrug thereof according to claim 1; and a pharmaceutically acceptable carrier.

10.-11. (canceled)

12. A method for preventing and/or treating a disease related to GPR84 in a subject in need, comprising administering to the subject an effective amount of the tricyclic compound, the tautomer, the stereoisomer, the hydrate, the solvate, the pharmaceutically acceptable salt, the prodrug thereof according to claim 1.

13. The method according to claim 12, wherein the disease related to GPR84 is selected from: inflammatory diseases, pulmonary diseases, neuroinflammatory diseases, infectious diseases, autoimmune diseases, endocrine, metabolic diseases, and diseases related to impaired immune function.

14. The method according to claim 13, wherein the inflammatory disease is inflammatory bowel disease or vasculitis: the pulmonary disease is chronic obstructive pulmonary disease and/or pulmonary interstitial disease, and the pulmonary interstitial disease is preferably congenital pulmonary fibrosis or idiopathic pulmonary fibrosis; the autoimmune disease is rheumatoid arthritis.

15. A method for preventing and/or treating a disease related to GPR84 in a subject in need, comprising administering to the subject an effective amount of the pharmaceutical composition according to claim 9.

16. The method according to claim 15, wherein the disease related to GPR84 is selected from: inflammatory diseases, pulmonary diseases, neuroinflammatory diseases, infectious diseases, autoimmune diseases, endocrine, metabolic diseases, and diseases related to impaired immune function.

17. The method according to claim 16, wherein the inflammatory disease is inflammatory bowel disease or vasculitis; the pulmonary disease is chronic obstructive pulmonary disease and/or pulmonary interstitial disease, and the pulmonary interstitial disease is preferably congenital pulmonary fibrosis or idiopathic pulmonary fibrosis; the autoimmune disease is rheumatoid arthritis.

18. The tricyclic compound, the tautomer, the stereoisomer, the hydrate, the solvate, the pharmaceutically acceptable salt, or the prodrug thereof according to claim 1, wherein L.sub.1 is absent or L.sub.1 is C?C.

19. The tricyclic compound, the tautomer, the stereoisomer, the hydrate, the solvate, the pharmaceutically acceptable salt, or the prodrug thereof according to claim 1, wherein R.sub.1 is cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.

20. The tricyclic compound, the tautomer, the stereoisomer, the hydrate, the solvate, the pharmaceutically acceptable salt, or the prodrug thereof according to claim 19 wherein R.sub.1 is ##STR00030##

21. The method according to claim 8, wherein, the catalyst is a palladium catalyst and/or a copper catalyst; the palladium catalyst is selected from: Pd(PPh.sub.3).sub.2Cl.sub.2, Pd(OAc).sub.2, Pd(TFA).sub.2, PdCl.sub.2, Pd(PPh.sub.3).sub.4, and Pd.sub.2(dba).sub.3; the copper catalyst is a monovalent copper catalyst; more preferably, the copper catalyst is CuI; the inert gas is nitrogen, helium, neon, or argon.

22. The method according to claim 21, wherein, the palladium catalyst is Pd(PPh.sub.3).sub.2Cl.sub.2 or Pd(OAc).sub.2.

Description

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0096] The present disclosure is further illustrated below in conjunction with specific examples. It should be understood that the following description is only the most preferred embodiment of the present disclosure and should not be construed as limiting the scope of protection of the present disclosure. On the basis of a full understanding of the present disclosure, the experimental methods without indication of specific conditions in the following examples shall be implemented usually in accordance with conventional conditions or the conditions suggested by the manufacturer. Those skilled in the art can make non-essential modifications to the technical solutions of the present disclosure, and such modifications should be considered to be included in the scope of protection of the present disclosure.

[0097] In the following examples, DCM stands for dichloromethane: MeOH stands for methanol: TEA stands for triethylamine: DMF stands for N,N-dimethylformamide; EA stands for ethyl acetate.

Example 1: Preparation of Compound I-1

[0098] The synthetic route is as follows:

##STR00013##

Step 1: Synthesis of 2-((2-oxabicyclo[2.1.1]hexan-1-yl)methoxy)-9-(allyloxy)-6,7-dihydro-4H-pyrimido[6,1-?]isoquinolin-4-one

[0099] ##STR00014##

[0100] (2-Oxabicyclo[2.1.1]hexan-1-yl)methanol (0.23 g, 2.1 mmol) was dissolved in a solution of anhydrous DCM (100 mL) under nitrogen atmosphere at 0? C., then sodium hydride (0.081 g, 2.1 mmol, content of 60% in mineral oil) was added thereto, and the mixture was stirred for 15 minutes. 9-(Allyloxy)-2-chloro-6,7-dihydro-4H-pyrimido[6,1-?]isoquinolin-4-one (0.5 g, 1.6 mmol) was added thereto, and the reaction mixture was warmed to room temperature and stirred overnight. Saturated NH.sub.4Cl aqueous solution (50 mL) was added thereto to wash once. The organic layer was washed once with water (50 mL), dried over MgSO.sub.4, and concentrated under reduced pressure to obtain the crude product, and the crude product was purified by a silica gel column to obtain the product 2-((2-oxabicyclo[2.1.1]hexan-1-yl)methoxy)-9-(allyloxy)-6,7-dihydro-4H-pyrimido[6,1-?]isoquinolin-4-one (0.45 g, yield of 79%), LC-MS, M/Z (ESI): 367.2 [M+H].sup.+.

Step 2: Synthesis of 2-((2-oxabicyclo[2.1.1]hexan-1-yl)methoxy)-9-hydroxy-6,7-dihydro-4H-pyrimido[6,1-?]isoquinolin-4-one

[0101] ##STR00015##

[0102] Compound 2-((2-oxabicyclo[2.1.1]hexan-1-yl)methoxy)-9-(allyloxy)-6,7-dihydro-4H-pyrimido[6,1-?]isoquinolin-4-one (0.45 g, 1.23 mmol) was placed in a 100 mL single-necked flask, then DCM/MeOH (10 mL/10 mL) was added thereto, and K.sub.2CO.sub.3 (0.34 g, 2.46 mmol) and Pd(PPh.sub.3).sub.4 (0.071 g, 0.061 mmol) were added thereto at room temperature. The reaction mixture was stirred overnight at room temperature. The reaction mixture was detected by thin-layer chromatography. After the reaction was completed, the reaction solvent was concentrated to dryness to obtain the crude product, and the crude product was diluted with EA (20 mL). The organic phase was extracted three times with water (20 mL?3), and the aqueous phases were combined, and then the pH of the aqueous phases was adjusted to 4. The aqueous phase was then extracted three times with DCM (20 mL?3), and the organic phases were combined, washed once with saturated brine (20 mL?1), then dried, filtered, and concentrated to obtain the crude product 2-((2-oxabicyclo[2.1.1]hexan-1-yl)methoxy)-9-hydroxy-6,7-dihydro-4H-pyrimido[6,1-?]isoquinolin-4-one (0.35 g, yield of 87%), LC-MS, M/Z (ESI): 327.3[M+H].sup.+.

Step 3: Synthesis of 2-((2-oxabicyclo[2.1.1]hexan-1-yl)methoxy)-4-oxo-6,7-dihydro-4H-pyrimido[6,1-?]isoquinolin-9-yl trifluoromethanesulfonate

[0103] ##STR00016##

[0104] Compound 2-((2-oxabicyclo[2.1.1]hexan-1-yl)methoxy)-9-hydroxy-6,7-dihydro-4H-pyrimido[6,1-?]isoquinolin-4-one (0.35 g, 1.07 mmol) was dissolved in DCM (10 mL). 1,1,1-Trifluoro-N-phenyl-N-((trifluoromethyl)sulfonyl)methanesulfonamide (0.46 g, 1.28 mmol) and TEA (0.27 mL, 1.93 mmol) were added thereto, then the reaction mixture was stirred at room temperature for 5 hours, and the solvent was concentrated to dryness to obtain the crude product which was purified by a silica gel column (DCM: MeOH (V/V)=50:1 to 20:1) to obtain 2-((2-oxabicyclo[2.1.1]hexan-1-yl)methoxy)-4-oxo-6,7-dihydro-4H-pyrimido[6,1-?]isoquinolin-9-yl trifluoromethanesulfonate (0.31 g, yield of 63.4%), LC-MS, M/Z (ESI): 459.1 [M+H].sup.+.

Step 4: Synthesis of 2-((2-oxabicyclo[2.1.1]hexan-1-yl)methoxy)-9-(cyclopropylethynyl)-6,7-dihydro-4H-pyrimido[6,1-?]isoquinolin-4-one

[0105] ##STR00017##

[0106] Pd(PPh.sub.3).sub.2Cl.sub.2 (0.011 g, 0.016 mmol), CuI (0.012 g, 0.065 mmol), and 2-((2-oxabicyclo[2.1.1]hexan-1-yl)methoxy)-4-oxo-6,7-dihydro-4H-pyrimido[6,1-?]isoquinolin-9-yl trifluoromethanesulfonate (0.15 g, 0.33 mmol) were placed in a sealed tube under nitrogen atmosphere. Cyclopropylacetylene (0.11 g, 1.64 mmol) and triethylamine (0.17 g, 1.64 mmol) were dissolved in anhydrous DMF (3 mL), added to the sealed tube and stirred overnight at 60? C. The reaction mixture was detected by thin-layer chromatography. After the raw materials were completely reacted, the reaction mixture was concentrated to remove DMF to obtain the crude product, which was purified by a silica gel column (dichloromethane:methanol (V/V)=50:1 to 10:1) to obtain 2-((2-oxabicyclo[2.1.1]hexan-1-yl)methoxy)-9-(cyclopropylethynyl)-6,7-dihydro-4H-pyrimido[6,1-?]isoquinolin-4-one (I-1) (63.6 mg, yield of 51.9%).

[0107] LC-MS, M/Z (ESI): 375.2 [M+H].sup.+.

[0108] .sup.1H NMR (400 MHZ, CDCl.sub.3) ? 7.58 (d, J=8.2 Hz, 1H), 7.34 (dd, J=8.2, 1.5 Hz, 1H), 7.28 (d, J=1.0 Hz, 1H), 6.37 (s, 1H), 4.69 (s, 2H), 4.24-4.11 (m, 2H), 3.84 (d, J=8.8 Hz, 2H), 2.95 (dd, J=7.2, 4.8 Hz, 3H), 1.84 (dt, J=4.8, 4.3 Hz, 2H), 1.59 (dd, J=4.7, 1.8 Hz, 2H), 1.47 (tt, J=8.2, 5.1 Hz, 1H), 0.96-0.88 (m, 2H), 0.87-0.80 (m, 2H).

Example 2: Preparation of Target Compound I-2

[0109] ##STR00018##

[0110] The synthetic route of compound I-2 referred to the synthetic method of I-1, and (2-oxabicyclo[2.1.1]hexan-1-yl)methanol was replaced by (hexahydrofuro[3,2-b]furan-2-yl)methanol. After a similar four-step reaction, 9-(cyclopropylethynyl)-2-((hexahydrofuro[3,2-b]furan-2-yl)methoxy)-6,7-dihydro-4H-pyrimido[6,1-?]isoquinolin-4-one (I-2) was obtained. LC-MS, M/Z (ESI): 405.2 [M+H].sup.+.

Example 3: Preparation of Target Compound I-3

[0111] The synthetic route is as follows:

##STR00019##

Step 1: Synthesis of 9-(allyloxy)-2-((4-methyl-2-oxabicyclo[2.1.1]hexan-1-yl)methoxy)-6,7-dihydro-4H-pyrimido[6,1-?]isoquinolin-4-one

[0112] ##STR00020##

[0113] (4-Methyl-2-oxabicyclo[2.1.1]hexan-1-yl)methanol (0.23 g, 2.1 mmol) was dissolved in a solution of anhydrous DCM (100 mL) under nitrogen atmosphere at 0? C., then sodium hydride (0.081 g, 2.1 mmol, content of 60% in mineral oil) was added thereto, and the mixture was stirred for 15 minutes. 9-(Allyloxy)-2-chloro-6,7-dihydro-4H-pyrimido[6,1-?]isoquinolin-4-one (0.5 g, 1.6 mmol) was added thereto, and the reaction mixture was warmed to room temperature and stirred overnight. Saturated NH.sub.4Cl aqueous solution (50 mL) was added thereto to wash once. The organic layer was washed once with water (50 mL), dried over MgSO.sub.4, and concentrated under reduced pressure to obtain the crude product, and the crude product was purified by a silica gel column to obtain the product 9-(allyloxy)-2-((4-methyl-2-oxabicyclo[2.1.1]hexan-1-yl)methoxy)-6,7-dihydro-4H-pyrimido[6,1-?]isoquinolin-4-one (0.47 g, yield of 80%).

[0114] LC-MS, M/Z (ESI): 381.2 [M+H].sup.+.

Step 2: Synthesis of 2-((4-methyl-2-oxabicyclo[2.1.1]hexan-1-yl)methoxy)-9-hydroxy-6,7-dihydro-4H-pyrimido[6,1-?]isoquinolin-4-one

[0115] ##STR00021##

[0116] Compound 9-(allyloxy)-2-((4-methyl-2-oxabicyclo[2.1.1]hexan-1-yl)methoxy)-6,7-dihydro-4H-pyrimido[6,1-?]isoquinolin-4-one (0.45 g, 1.23 mmol) was placed in a 100 mL single-necked flask, then DCM/MeOH (10 mL/10 mL) was added thereto, and K.sub.2CO.sub.3 (0.34 g, 2.46 mmol) and Pd(PPh.sub.3).sub.4 (0.071 g, 0.061 mmol) were added thereto at room temperature. The reaction mixture was stirred overnight at room temperature. The reaction mixture was detected by thin-layer chromatography. After the reaction was completed, the reaction solvent was concentrated to dryness to obtain the crude product, and the crude product was diluted with EA (20 mL). The organic phase was extracted three times with water (20 mL?3), and the aqueous phases were combined, and then the pH of the aqueous phases was adjusted to 4. The aqueous phase was then extracted three times with DCM (20 mL?3), and the organic phases were combined, washed once with saturated brine (20 mL?1), then dried, filtered, and concentrated to obtain the crude product 2-((4-methyl-2-oxabicyclo[2.1.1]hexan-1-yl)methoxy)-9-hydroxy-6,7-dihydro-4H-pyrimido[6,1-?]isoquinolin-4-one (0.35 g, yield of 87%).

[0117] LC-MS, M/Z (ESI): 341.3 [M+H].sup.+.

Step 3: Synthesis of 2-((4-methyl-2-oxabicyclo[2.1.1]hexan-1-yl)methoxy)-4-oxo-6,7-dihydro-4H-pyrimido[6,1-?]isoquinolin-9-yl trifluoromethanesulfonate

[0118] ##STR00022##

[0119] Compound 2-((4-methyl-2-oxabicyclo[2.1.1]hexan-1-yl)methoxy)-9-hydroxy-6,7-dihydro-4H-pyrimido[6,1-?]isoquinolin-4-one (0.35 g, 1.07 mmol) was dissolved in DCM (10 mL). 1,1,1-Trifluoro-N-phenyl-N-((trifluoromethyl)sulfonyl)methanesulfonamide (0.46 g, 1.28 mmol) and TEA (0.27 mL, 1.93 mmol) were added thereto, then the reaction mixture was stirred at room temperature for 5 hours, and the solvent was concentrated to dryness to obtain the crude product, which was purified by a silica gel column (DCM: MeOH (V/V)=50:1 to 20:1) to obtain 2-((4-methyl-2-oxabicyclo[2.1.1]hexan-1-yl)methoxy)-4-oxo-6,7-dihydro-4H-pyrimido[6,1-?]isoquinolin-9-yl trifluoromethanesulfonate (0.31 g, yield of 63.4%). LC-MS, M/Z (ESI): 473.1 [M+H].sup.+.

Step 4: Synthesis of 2-((4-methyl-2-oxabicyclo[2.1.1]hexan-1-yl)methoxy)-9-(cyclopropylethynyl)-6,7-dihydro-4H-pyrimido[6,1-?]isoquinolin-4-one

[0120] ##STR00023##

[0121] Pd(PPh.sub.3).sub.2Cl.sub.2 (0.011 g, 0.016 mmol), CuI (0.012 g, 0.065 mmol), and 2-((4-methyl-2-oxabicyclo[2.1.1]hexan-1-yl)methoxy)-4-oxo-6,7-dihydro-4H-pyrimido[6,1-?]isoquinolin-9-yl trifluoromethanesulfonate (0.15 g, 0.33 mmol) were placed in a sealed tube under nitrogen atmosphere. Cyclopropylacetylene (0.11 g, 1.64 mmol) and triethylamine (0.17 g, 1.64 mmol) were dissolved in anhydrous DMF (3 mL), added to the sealed tube and stirred overnight at 60? ? C. The reaction mixture was detected by thin-layer chromatography. After the raw materials were completely reacted, the reaction mixture was concentrated to remove DMF to obtain the crude product, which was purified by a silica gel column (dichloromethane:methanol (V/V)=50:1 to 10:1) to obtain 2-((4-methyl-2-oxabicyclo[2.1.1]hexan-1-yl)methoxy)-9-(cyclopropylethynyl)-6,7-dihydro-4H-pyrimido[6,1-?]isoquinolin-4-one (I-3) (63.6 mg, yield of 51.9%).

[0122] LC-MS, M/Z (ESI): 389.2 [M+H].sup.+.

[0123] .sup.1H NMR (400 MHZ, CDCl.sub.3) ? 7.58 (d, 1H), 7.34 (dd, 1H), 7.28 (d, 1H), 6.37 (s, 1H), 4.69 (s, 2H), 4.24-4.11 (m, 2H), 3.63 (s, 2H), 2.95 (t, 2H), 1.70-1.61 (m, 4H), 1.47 (dd, 1H), 1.34 (s, 3H), 0.96-0.88 (m, 2H), 0.87-0.80 (m, 2H).

[0124] In the test examples of the present disclosure, a control compound was prepared with reference to the preparation method of compound 122 in the patent WO 2013/092791 A1, and the control compound has a structure as follows:

##STR00024##

Test Example 1: GPR84 Antagonistic Effect Determination Experiment

[0125] The antagonistic effect of the compound against GPR84 was determined in a CHO stably transfected cell line highly expressing the human GPR84 receptor. The stably transfected cells were cultured until 80% confluence; the cells were collected by trypsinization, counted, and then inoculated into a 384-well plate at 5 L/well. 10? compound working solution was prepared with 1? Stimulation Buffer. 1 ?L of 10? compound was added to the corresponding experimental well, centrifuged, and then incubated at 37? C. for 20 min; then 4 ?L of 2.5 ?M Forskolin & 200 nM 6-OAU solution were added thereto, centrifuged, and then incubated at 37? C. for 30 min. After the reaction was completed, the content of CAMP in the cells was quantified according to the method in the instructions of the cAMP assay kit (Perkin Elmer, Cat #TRF0263). The antagonistic effect (IC.sub.50 value) of the test compounds was calculated.

TABLE-US-00001 TABLE 1 Antagonistic effect of test compounds against GPR84 Test compound IC.sub.50 (nM) Control compound 76.46 I-1 27.23

[0126] The results show that the compound of the present disclosure has strong antagonistic effect against GPR84.

Test Example 2: Pharmacokinetic Experiment in Mice

[0127] For the pharmacokinetic experiment in mice, male ICR mice, 20 to 25 g, were used and fasted overnight. Three mice were taken and orally administered by gavage (3 mg/kg): blood was collected before the administration, and at 15 min, 30 min, 1 hour, 2 hours, 4 hours, 8 hours, and 24 hours after the administration. The blood samples were centrifuged at 6800 g for 6 min at 2 to 8? C., and plasma was collected and stored at ?80? C. 20 ?L of plasma at each time point was taken, and added with 200 ?L of methanol containing 100 ng/mL internal standard. The mixture was vortexed, mixed evenly, and then centrifuged at 18000 g for 7 min at 2 to 8? ? C. 200 ?L of the mixture was transferred to a 96-well injection plate for LC-MS/MS quantitative analysis. The main pharmacokinetic parameters were analyzed using WinNonlin 7.0 software with non-compartmental model.

TABLE-US-00002 TABLE 2 Results of pharmacokinetic tests in mice C.sub.max T.sub.max AUC.sub.0-t T.sub.1/2 Test compound (ng/mL) (hr) (h * ng/mL) (h) Control compound 4119.83 0.50 7283.23 3.81 I-1 (3 mg/kg) 14761 0.50 54389 1.03

[0128] The results show that the compound of the present disclosure has higher oral exposure in mice and good druggability.

Test Example 3: Pharmacokinetic Experiment in Dogs

[0129] For the pharmacokinetic test in dogs, 3 male Beagle dogs, 8 to 10 kg, were fasted overnight and orally administered 3 mg/kg by gavage. Blood was collected before the administration, and at 15 min, 30 min, 1 hour, 2 hours, 4 hours, 8 hours, and 24 hours after the administration; the blood samples were centrifuged at 6800 g for 6 min at 2 to 8? C., and plasma was collected and stored at ?80? C. The plasma at each time point was taken and added with an acetonitrile solution containing internal standard in 3 to 5 times the amount. The mixture was vortexed and mixed for 1 min, and centrifuged at 13000 rpm at 4? C. for 10 min. The supernatant was collected, added with water in 3 times the amount, and mixed. An appropriate amount of the mixture was taken for LC-MS/MS analysis. The main pharmacokinetic parameters were analyzed using WinNonlin 7.0 software with non-compartmental model.

[0130] Experimental results show that the compound of the present disclosure exhibits excellent pharmacokinetic properties in dogs.

Test Example 4: Pharmacokinetic Experiment in Rats

[0131] The pharmacokinetic properties of the control compound and the compound of the present disclosure in rats were determined according to the following experimental methods.

[0132] Three male SD rats were used at a dose of 2.5 mg/kg, the route of administration was by gavage, and the vehicle was 5% DMSO+10% Solutol+85% Saline. The rats were fasted overnight, and the time points of blood collection were before administration and at 15 min, 30 min, 1 hour, 2 hours, 4 hours, 8 hours, and 24 hours after administration. The blood samples were centrifuged at 6800 g for 6 min at 2 to 8? C. and plasma was collected and stored at ?80? ? C. 20 ?L of plasma at each time point was taken and added with 200 ?L of methanol containing 100 ng/mL internal standard. The mixture was vortexed, mixed evenly, and then centrifuged at 18000 g for 7 min at 2 to 8? C. 200 ?L of the mixture was transferred to a 96-well injection plate for LC-MS/MS quantitative analysis. The main pharmacokinetic parameters were analyzed using WinNonlin 7.0 software with non-compartmental model.

[0133] Experimental results show that the compound of the present disclosure exhibits excellent pharmacokinetic properties in rats.

Test Example 5: Plasma Protein Binding Rate of Compounds

[0134] The plasma protein binding rate of compounds was detected by equilibrium dialysis (HTDialysis, HTD 96b). The compound was prepared into a 0.5 ?M stock solution with DMSO, and then 25-fold diluted with 0.05 M sodium phosphate buffer as a working solution. A blank 96-well plate was taken and preloaded with 380 ?L of plasma per well. The plasma was then added with the working solution at 20 ?L/well and mixed well, with the compound at a final concentration of 1 ?M, containing 0.2% DMSO per well.

[0135] 100 ?L of 0.05 M sodium phosphate buffer was added to the reception-side of each dialysis chamber (HTD 96b), and then 100 ?L of plasma containing the compound was added to the supply-side. The dialysis chamber was covered with a plastic lid, shaken, and incubated at 37? C. for 5 hours.

[0136] After incubation, 25 ?L of samples were taken from each of the supply-side and the reception-side of the dialysis chamber and placed in the blank 96-well plate. An equal volume of plasma was added to each of the supply-side samples while an equal volume of 0.05 M sodium phosphate buffer was added to each of the reception-side samples, and the mixture in each well was mixed well. The 96-well plate was added with acetonitrile solution containing internal standard at 200 ?L per well, vortexed and shaken at 600 rpm for 10 min, and centrifuged at 5594 g for 15 min (Thermo Multifuge?3R). 50 ?L of the supernatant was then transferred to a new 96-well plate, and the samples were mixed with 50 ?L of ultra-pure water for LC-MS/MS analysis.

[0137] The plasma protein binding rate and free fraction were calculated using the following formulas: % binding rate=100?([supply-side concentration].sub.5h?[reception-side concentration].sub.5h)/[supply-side concentration].sub.5h. % free fraction=100?% binding rate

[0138] Experimental results show that the compound of the present disclosure has a higher free fraction in human plasma and has good druggability.

Test Example 6: Stability of Human Liver Microsomes

[0139] The stability of human liver microsomes of the control compound and the compound of the present disclosure was determined according to the following experimental method. The liver microsome stability test of the compound was detected by incubating the compound and human liver microsomes in vitro. First, the compound to be tested was prepared into a 10 mM stock solution in DMSO solvent, and then the compound was diluted to 0.5 mM with acetonitrile. Liver microsomes (Corning) were diluted with PBS into a microsome/buffer solution, and the solution was used to dilute 0.5 mM compound into a working solution, in which the concentration of the compound was 1.5 ?M, and the concentration of liver microsomes was 0.75 mg/mL. A deep-well plate was taken, 30 ?L of the working solution was added per well, and then 15 ?L of pre-heated 6 mM NADPH solution was added thereto to initiate a reaction, and the reaction was incubated at 37? C. At 0, 5, 15, 30, and 45 min of the incubation, 135 ?L of acetonitrile was added to the corresponding wells to terminate the reaction. After the reaction was terminated with acetonitrile at the last time point of 45 min, the deep-well plate was vortexed and shaken for 10 min (600 rpm), and then centrifuged for 15 min. After centrifugation, the supernatant was collected, and added with purified water in a ratio of 1:1 to perform LC-MS/MS detection. A ratio of a peak area of compound to a peak area of internal standard at each time point was obtained, and the peak area ratios of the compound at 5, 15, 30, and 45 min were compared with the peak area ratio at 0 min to calculate the remaining percentage of the compound at each time point. Tin was calculated by using Graphpad 5 software.

[0140] Experimental results show that the compound of the present disclosure exhibits good stability in human liver microsomes, slow metabolism, and good druggability.

[0141] Although the examples of the present disclosure are illustrated and described above, it can be understood that the above examples are illustrative and should not be construed as limiting the present disclosure. Those skilled in the art can make changes, modifications, substitutions, and variations based on the above examples within the scope of the present disclosure.