Substituted cyclopenta[4,5]pyrrolo[1,2-a]pyrazines as bruton's tyrosine kinase inhibitors

Abstract

A compound of formula (I) as Bruton's tyrosine kinase inhibitor, a preparation method therefor, and an application thereof in the field of medicine. The Bruton's tyrosine kinase inhibitor may be used for preventing and/or treating a disease related to Bruton's tyrosine kinase mediation, such as an autoimmune disease, cancer, or an inflammatory disease. Through experimental research, the provided compound has high selectivity with respect to a Bruton's tyrosine kinase target, and can exhibit a uniquely advantageous medicinal effect in an in vivo animal experiment. ##STR00001##

Claims

1. A compound of formula (I): ##STR00044## or a pharmaceutically acceptable salt, stereoisomer, or tautomer thereof, wherein: R.sub.1 is halogen, CN, C.sub.1-6 alkyl, CF.sub.3, NH.sub.2, NHC.sub.1-6 alkyl, or OC.sub.1-6 alkyl; R.sub.2 is C.sub.1-3 alkyl; R.sub.3 is C.sub.1-3 alkyl; X is N; R is OH; R.sub.0 is H; A is ##STR00045## R.sub.4 is C.sub.1-6 alkyl; R.sub.5 is H or C.sub.1-3 alkyl; X.sub.1 is N; T is a nitrogen-containing 5- or 6-membered heterocycloalkyl, wherein the 5- or 6-membered heterocycloalkyl is substituted by one T.sub.1 substituent, one T.sub.2 substituent, or one T.sub.1 substituent and one T.sub.2 substituent; T.sub.1 is C.sub.1-6 alkyl; and T.sub.2 is C.sub.1-6 alkyl or 3- to 6-membered heterocycloalkyl.

2. The compound according to claim 1, or a pharmaceutically acceptable salt, stereoisomer, or tautomer thereof, wherein T is: ##STR00046##

3. The compound according to claim 2, or a pharmaceutically acceptable salt, stereoisomer, or tautomer thereof, wherein: T.sub.1 is CH.sub.3, CH.sub.2CH.sub.3, CH.sub.2CH.sub.2CH.sub.3, or CH(CH.sub.3).sub.2; and T.sub.2 is CH.sub.3, CH.sub.2CH.sub.3, CH.sub.2CH.sub.2CH.sub.3, CH(CH.sub.3).sub.2, or oxetanyl.

4. The compound according to claim 3, or a pharmaceutically acceptable salt, stereoisomer, or tautomer thereof, wherein T is: ##STR00047##

5. The compound according to claim 1, or a stereoisomer thereof, wherein the compound, or stereoisomer thereof, is selected from the group consisting of: ##STR00048## ##STR00049## ##STR00050## ##STR00051## or a pharmaceutically acceptable salt or tautomer thereof.

6. A pharmaceutical composition comprising at least one pharmaceutically acceptable carrier or diluent and a therapeutically effective amount of the compound according to claim 1, or a pharmaceutically acceptable salt, stereoisomer, or tautomer thereof.

7. A method for inhibiting Bruton's tyrosine kinase activity in a subject, wherein the method comprises administering to the subject in need thereof a therapeutically effective amount of the compound according to claim 1, or a pharmaceutically acceptable salt, stereoisomer, or tautomer thereof.

8. The method according to claim 7, wherein the subject has a Bruton's tyrosine kinase-mediated disease or disorder selected from the group consisting of an autoimmune disorder, cancer, and an inflammatory disease.

9. The method according to claim 8, wherein: (a) the cancer is selected from the group consisting of esophageal cancer, leukemia, lung cancer, lymphoma, pancreatic cancer, rectal cancer, and thyroid cancer; and (b) the autoimmune disorder or inflammatory disease is selected from the group consisting of an allergic reaction, arthritis, asthma, chronic obstructive pulmonary disease, Crohn's disease, inflammatory bowel disease, multiple sclerosis, organ transplant rejection, psoriasis, rhinitis, systemic lupus erythematosus, and thrombocytopenic purpura.

10. A method for inhibiting Bruton's tyrosine kinase activity in a subject, wherein the method comprises administering to the subject in need thereof a therapeutically effective amount of the pharmaceutical composition according to claim 6.

11. The method according to claim 10, wherein the subject has a Bruton's tyrosine kinase-mediated disease or disorder selected from the group consisting of an autoimmune disorder, cancer, and an inflammatory disease.

12. The method according to claim 11, wherein: (a) the cancer is selected from the group consisting of esophageal cancer, leukemia, lung cancer, lymphoma, pancreatic cancer, rectal cancer, and thyroid cancer; and (b) the autoimmune disorder or inflammatory disease is selected from the group consisting of an allergic reaction, arthritis, asthma, chronic obstructive pulmonary disease, Crohn's disease, inflammatory bowel disease, multiple sclerosis, organ transplant rejection, psoriasis, rhinitis, systemic lupus erythematosus, and thrombocytopenic purpura.

13. A process for preparing a compound of formula I-12: ##STR00052## wherein: R.sub.1 is halogen, CN, C.sub.1-6 alkyl, CF.sub.3, NH.sub.2, NHC.sub.1-6 alkyl, or OC.sub.1-6 alkyl; R.sub.2 is C.sub.1-3 alkyl; R.sub.3 is C.sub.1-3 alkyl; X is N; R.sub.0 is H; R.sub.4 is C.sub.1-6 alkyl; X.sub.1 is N; T is a nitrogen-containing 5- or 6-membered heterocycloalkyl, wherein the 5- or 6-membered heterocycloalkyl is substituted by one T.sub.1 substituent, one T.sub.2 substituent, or one T.sub.1 substituent and one T.sub.2 substituent; T.sub.1 is C.sub.1-6 alkyl; and T.sub.2 is C.sub.1-6 alkyl or 3- to 6-membered heterocycloalkyl; wherein the process comprises the following steps: (1) reacting a compound of formula I-1: ##STR00053## wherein: X.sub.1 is N; and T is a nitrogen-containing 5- or 6-membered heterocycloalkyl, wherein the 5- or 6-membered heterocycloalkyl is substituted by one T.sub.1 substituent, one T.sub.2 substituent, or one T.sub.1 substituent and one T.sub.2 substituent; with a reducing agent 1 selected from the group consisting of borane dimethyl sulfoxide, borane tetrahydrofuran, lithium aluminum hydride, and reducing iron powder, to provide a compound of formula I-2: ##STR00054## wherein: X.sub.1 is N; and T is a nitrogen-containing 5- or 6-membered heterocycloalkyl, wherein the 5- or 6-membered heterocycloalkyl is substituted by one T.sub.1 substituent, one T.sub.2 substituent, or one T.sub.1 substituent and one T.sub.2 substituent; (2) reacting the compound of formula I-2 provided in step (1) above with a compound of formula Int 3: ##STR00055## wherein: R.sub.4 is C.sub.1-6 alkyl; in the presence of a catalyst selected from the group consisting of 1,1-binaphthyl-2,2-bis(diphenylphosphine), [1,1-bis(diphenylphosphino)ferrocene]palladium dichloride, 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene, cuprous iodide, 2-dicyclohexylphosphine-2,4,6-triisopropylbiphenyl, palladium acetate, 1,10-phenanthroline, tetrakis(triphenylphosphine)palladium, and tris(dibenzylideneacetone)dipalladium, to provide a compound of formula I-3: ##STR00056## wherein: R.sub.4 is C.sub.1-6 alkyl; X.sub.1 is N; and T is a nitrogen-containing 5- or 6-membered heterocycloalkyl, wherein the 5- or 6-membered heterocycloalkyl is substituted by one T.sub.1 substituent, one T.sub.2 substituent, or one T.sub.1 substituent and one T.sub.2 substituent; (3) reacting the compound of formula I-3 provided in step (2) above with bis(pinacolato)diboron in the presence of a catalyst selected from the group consisting of 1,1-binaphthyl-2,2-bis(diphenylphosphine), [1,1-bis(diphenylphosphino)ferrocene]palladium dichloride, 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene, cuprous iodide, 2-dicyclohexylphosphine-2,4,6-triisopropylbiphenyl, palladium acetate, 1,10-phenanthroline, tetrakis(triphenylphosphine)palladium, and tris(dibenzylideneacetone)dipalladium, to provide a compound of formula I-4: ##STR00057## wherein: R.sub.4 is C.sub.1-6 alkyl; X.sub.1 is N; and T is a nitrogen-containing 5- or 6-membered heterocycloalkyl, wherein the 5- or 6-membered heterocycloalkyl is substituted by one T.sub.1 substituent, one T.sub.2 substituent, or one T.sub.1 substituent and one T.sub.2 substituent; (4) reacting the compound of formula I-4 provided in step (3) above with a compound of formula I-10: ##STR00058## wherein: R.sub.1 is halogen, CN, C.sub.1-6 alkyl, CF.sub.3, NH.sub.2, NHC.sub.1-6 alkyl, or OC.sub.1-6 alkyl; R.sub.2 is C.sub.1-3 alkyl; R.sub.3 is C.sub.1-3 alkyl; X is N; and R.sub.0 is H; in the presence of a catalyst selected from the group consisting of 1,1-binaphthyl-2,2-bis(diphenylphosphine), [1,1-bis(diphenylphosphino)ferrocene]palladium dichloride, 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene, cuprous iodide, 2-dicyclohexylphosphine-2,4,6-triisopropylbiphenyl, palladium acetate, 1,10-phenanthroline, tetrakis(triphenylphosphine)palladium, and tris(dibenzylideneacetone)dipalladium, to provide a compound of formula I-11: ##STR00059## wherein: R.sub.1 is halogen, CN, C.sub.1-6 alkyl, CF.sub.3, NH.sub.2, NHC.sub.1-6 alkyl, or OC.sub.1-6 alkyl; R.sub.2 is C.sub.1-3 alkyl; R.sub.3 is C.sub.1-3 alkyl; X is N; R.sub.0 is H; R.sub.4 is C.sub.1-6 alkyl; R.sub.1 is H or C.sub.1-3 alkyl; X.sub.1 is N; T is a nitrogen-containing 5- or 6-membered heterocycloalkyl, wherein the 5- or 6-membered heterocycloalkyl is substituted by one T.sub.1 substituent, one T.sub.2 substituent, or one T.sub.1 substituent and one T.sub.2 substituent; T.sub.1 is C.sub.1-6 alkyl; and T.sub.2 is C.sub.1-6 alkyl or 3- to 6-membered heterocycloalkyl; and (5) reacting the compound of formula I-11 provided in step (4) above with a reducing agent 2 selected from the group consisting of potassium borohydride, sodium borohydride, sodium cyanoborohydride, and sodium triacetylborohydride, to provide a compound of formula I-12: ##STR00060## wherein: R.sub.1 is halogen, CN, C.sub.1-6 alkyl, CF.sub.3, NH.sub.2, NHC.sub.1-6 alkyl, or OC.sub.1-6 alkyl; R.sub.2 is C.sub.1-3 alkyl; R.sub.3 is C.sub.1-3 alkyl; X is N; R.sub.0 is H; R.sub.4 is C.sub.1-6 alkyl; X.sub.1 is N; T is a nitrogen-containing 5- or 6-membered heterocycloalkyl, wherein the 5- or 6-membered heterocycloalkyl is substituted by one T.sub.1 substituent, one T.sub.2 substituent, or one T.sub.1 substituent and one T.sub.2 substituent; T.sub.1 is C.sub.1-6 alkyl; and T.sub.2 is C.sub.1-6 alkyl or 3- to 6-membered heterocycloalkyl.

14. The process of claim 13, wherein step (4) of the process further comprises: (4a) reacting a compound of formula I-5: ##STR00061## wherein: R.sub.2 is C.sub.1-3 alkyl; and R.sub.3 is C.sub.1-3 alkyl; with phosphorous oxychloride (POCl.sub.3), to provide a compound of formula I-6: ##STR00062## wherein: R.sub.2 is C.sub.1-3 alkyl; and R.sub.3 is C.sub.1-3 alkyl; (4b) reacting the compound of formula I-6 provided in step (4a) above with a compound of the following formula: ##STR00063## in the presence of diisopropylethanolamine (DIPEA), to provide a compound of formula I-7: ##STR00064## wherein: R.sub.2 is C.sub.1-3 alkyl; and R.sub.3 is C.sub.1-3 alkyl; (4c) reacting the compound of formula I-7 provided in step (4b) above with N-bromosuccinimide (NBS), to provide a compound of formula I-8: ##STR00065## wherein: R.sub.2 is C.sub.1-3 alkyl; and R.sub.3 is C.sub.1-3 alkyl; (4d) reacting the compound of formula I-8 provided in step (4c) above with (i) n-butyllithium (n-BuLi) or (ii) a catalyst selected from the group consisting of 1,1-binaphthyl-2,2-bis(diphenylphosphine), [1,1-bis(diphenylphosphino)ferrocene]palladium dichloride, 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene, cuprous iodide, 2-dicyclohexylphosphine-2,4,6-triisopropylbiphenyl, palladium acetate, 1,10-phenanthroline, tetrakis(triphenylphosphine)palladium, and tris(dibenzylideneacetone)dipalladium, to provide a compound of formula I-9: ##STR00066## wherein: R.sub.1 is halogen, CN, C.sub.1-6 alkyl, CF.sub.3, NH.sub.2, NHC.sub.1-6 alkyl, or OC.sub.1-6 alkyl; R.sub.2 is C.sub.1-3 alkyl; and R.sub.3 is C.sub.1-3 alkyl; and (4e) reacting the compound of formula I-9 provided in step (4d) above with a compound of the following formula: ##STR00067## wherein: R.sub.0 is H; in the presence of a catalyst selected from the group consisting of 1,1-binaphthyl-2,2-bis(diphenylphosphine), [1,1-bis(diphenylphosphino)ferrocene]palladium dichloride, 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene, cuprous iodide, 2-dicyclohexylphosphine-2,4,6-triisopropylbiphenyl, palladium acetate, 1,10-phenanthroline, tetrakis(triphenylphosphine)palladium, and tris(dibenzylideneacetone)dipalladium, to provide a compound of formula I-10: ##STR00068## wherein: R.sub.1 is halogen, CN, C.sub.1-6 alkyl, CF.sub.3, NH.sub.2, NHC.sub.1-6 alkyl, or OC.sub.1-6 alkyl; R.sub.2 is C.sub.1-3 alkyl; R.sub.3 is C.sub.1-3 alkyl; X is N; and R.sub.0 is H.

Description

DETAILED DESCRIPTION OF THE INVENTION

(1) To illustrate the purposes, technical solutions and advantages of the present application, the present application will be further described in detail with the following accompanying drawings and examples. Obviously, the described examples are only a part of the examples of the present application, rather than all of the examples. All other examples obtained by a person of ordinary skill in the art based on the examples in the present application are within the protection scope of the present application.

(2) The abbreviations used in the present application is summarized in Table 1.

(3) TABLE-US-00001 TABLE 1 Abbrevia- No. tion Name 1 XPhos 2-Dicyclohexylphosphine-2,4,6-triisopropylbiphenyl 2 Xantphos 4,5-Bis(diphenylphosphino)-9,9-dimethyloxanthene 3 BINAP 1,1-Binaphthyl-2,2-bis(diphenylphosphine) 4 DME Dimethyl ether 5 DMF N,N-Dimethylformamide 6 DIPEA N,N-Diisopropylethylamine 7 NMP N-Methylpyrrolidone 8 NBS N-Bromosuccinimide 9 DCM Dichloromethane 10 TEA Triethanolamine 11 LDA Lithium diisopropylamine 12 TFA Trifluoroacetic acid 13 TBAF Tetrabutylammonium fluoride

Example 1 (S)-9-fluoro-2-(3-(hydroxymethyl)-1-methyl-5-((5-(2-methyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-yl)amino)-6-oxo-1,6-dihydro-[3,4-bipyridin]-2-yl)-7,7-dimethyl-3,4,7,8-tetrahydro-2H-cyclopenta[4,5]pyrrolo[1,2-a]pyrazin-1(6H)-one

(4) ##STR00013## ##STR00014##

Step 1 tert-butyl (S)-3-methyl-4-(6-nitropyridin-3-yl)piperazine-1-carboxylate 1-2b

(5) Compound 1-2a (20 g, 100 mmol), Int-1 (24 g, 150 mmol) and potassium carbonate (50 g, 235 mmol) were dissolved in toluene (250 mL), and stirred thoroughly. Then, palladium acetate (0.68 g, 3 mmol) and BINAP (2.0 g, 3 mmol) were added. After that, the air in the reaction system was replaced three times with nitrogen, and the reaction was warmed to 90 C. and stirred for 3 h. The reaction was quenched by adding water, and extracted with dichloromethane. The organic phase was washed with water (100 mL) and saturated salt water (100 mL) in turn, dried over anhydrous sodium sulfate, filtered and concentrated. The resulting crude product was purified by column chromatography to obtain the title compound 1-2b (19.5 g, 58.7 mmol) with a yield of 58.7%.

(6) MS m/z (ESI): 323.2 [M+H].sup.+.

Step 2 (S)-5-(2-methylpiperazin-1-yl)pyridin-2-amine 1-2c

(7) Compound 1-2b (10.0 g, 32.2 mmol) was dissolved in a solution of hydrochloric acid in methanol (100 mL), then added with 20 mL of concentrated hydrochloric acid and stirred for 24 h. After that, a large amount of golden yellow solid was precipitated. The solid was collected by suction filtration, dissolved in dichloromethane and then dissociated in saturated potassium carbonate solution. After that, the reaction system was extracted with dichloromethane. The organic phase was washed with water (100 mL) and saturated salt water (100 mL) in turn, dried over anhydrous sodium sulfate, filtered and concentrated to obtain the title compound 1-2c (7.0 g, 31.5 mmol) with a yield of 98.0%.

(8) MS m/z (ESI): 223.1 [M+H].sup.+.

Step 3 (S)-2-methyl-1-(6-nitropyridin-3-yl)-4-(oxiran-3-yl)piperazine 1-2d

(9) Compound 1-2c (7.0 g, 31.5 mmol) was dissolved in THF (50 mL) and 2 mL of acetic acid was added followed by Int 2 (3.0 g, 41 mmol). The mixture was stirred at room temperature for 15 min, and then added with sodium borohydride acetate (10.0 g, 47.3 mmol) in batches. The reaction system was stirred at room temperature for 3 h, quenched with water and extracted with dichloromethane. The organic phase was washed with water (150 mL) and saturated salt water (150 mL) in turn, dried over anhydrous sodium sulfate, filtered and concentrated. The resulting crude product was purified by column chromatography (pure ethyl acetate) to obtain the title compound 1-2d (3.5 g, 12.5 mmol) with a yield of 40.0%.

(10) MS m/z (ESI): 279.1 [M+H].sup.+.

Step 4 (S)-5-(2-methyl-4-(oxiran-3-yl)piperazin-1-yl)pyridin-2-amine 1-2e

(11) Compound 1-2d (3.5 g, 12.5 mmol) was dissolved in methanol and stirred thoroughly. Then, palladium carbon (1.7 g, 16.0 mmol) was added. After that, the air in the reaction system was replaced three times with hydrogen balloons. The reaction was stirred at room temperature for about 12 h, and subjected to suction filtration. The filtrate was spin dried to obtain the crude product 1-2e (2.8 g, 11.3 mmol) with a yield of 70.5%. MS m/z (ESI): 249.1 [M+H].sup.+.

Step 5 (S)-5-bromo-1-methyl-3-((5-(2-methyl-4-(oxiran-3-yl)piperazin-1-yl)pyridin-2-yl)amino)pyridin-2(1H)-one 1-2f

(12) Compound 1-2e (2.8 g, 11.3 mmol) and Int 3 (3.2 g, 12.0 mmol) were dissolved in toluene (25 mL), and palladium acetate (0.27 g, 1.1 mmol), Xantphos (1.4 g, 2.2 mmol) and potassium carbonate (4.8 g, 34 mmol) were added under stirring conditions. The air in the reaction system was replaced three times with nitrogen. The reaction was performed at 105 C. for 3.0 h under reflux, cooled, quenched by adding saturated ammonium chloride solution, and extracted with dichloromethane. The organic phase was washed with water (100 mL) and saturated salt water (100 mL) in turn, dried over anhydrous sodium sulfate, filtered, concentrated, purified by column chromatography to obtain the title compound 1-2f (2.8 g, 6.5 mmol) with a yield of 57.1%.

(13) MS m/z (ESI): 434.2 [M+H].sup.+.

Step 6 (S)-1-methyl-3-((5-(2-methyl-4-(oxiran-3-yl)piperazin-1-yl)pyridin-2-yl)amino)-5-(4,4,5,5-tetramethyl-1,3,2-dioxol-2-yl)pyridin-2(1H)-one 1f

(14) Compound 1-2f (2.8 g, 6.5 mmol) was dissolved in THF (25 mL), added with 2.5 g of pinacol borate (B.sub.2pin.sub.2) and stirred thoroughly, and then added with Pd.sub.2(dba).sub.3 (0.19 g, 0.33 mmol), XPhos (0.62 g, 1.3 mmol) and potassium carbonate (4.8 g, 34 mmol). The air in the reaction system was replaced three times with nitrogen. The reaction was performed at 65 C. for 6.0 h, quenched by adding saturated ammonium chloride solution, and extracted with dichloromethane. The organic phase was washed with water (100 mL) and saturated salt water (100 mL) for several times, dried over anhydrous sodium sulfate, filtered, concentrated, purified by column chromatography (DCM:MeOH=98:2) to obtain the title compound 1f (1.4 g, 3.2 mmol) with a yield of 50%.

(15) MS m/z (ESI): 560.4 [M+H].sup.+.

Step 7 2-chloro-4,4-dimethylcyclopent-1-ene-1-formaldehyde 1-2

(16) In a 1 L reaction flask, compound 1-1 (20 g, 0.177 mmol) was dissolved in dichloromethane (350 ml), to which DMF (32.5 g, 0.44 mol) was added under ice bath, and then POCl.sub.3 (57.0 g, 0.375 mol, 2.1 eq) was slowly added dropwise to the above reaction system. After the reaction solution has been slowly warmed to 30 C. and stirred at room temperature for 30 min, 3,3-dimethylcyclopentanone (20 g, 0.178 mol) was slowly added dropwise to the above reaction solution. The reaction solution was gradually warmed and reacted under reflux for 5 h, cooled to room temperature, then quenched by adding K.sub.3PO.sub.4 (10% w aq, 300 ml) dropwise to the reaction solution under ice bath with stirring, and left standing. The aqueous phase was reverse extracted with dichloromethane (200 ml). The organic phase was combined and spin dried to obtain 30 g crude product, which was used directly in the next step.

Step 8 7,7-dimethyl-3,4,7,8-tetrahydro-2H-cyclopenta[4,5]pyrrolo [1,2-a]pyrazin-1(6H)-one 1a

(17) The crude product 1-2 obtained in the previous step and 2-piperazinone (18.5 g, 0.19 mol), NMP (300 ml) and DIPEA (46 g, 0.36 mol) were added to the reaction flask in turn, warmed to 120 C., and reacted for 5 h under nitrogen protection. The reaction completion was confirmed by LCMS. The reaction solution was cooled, and quenched by adding water (1000 ml). A large amount of solid was precipitated, and filtered, which was then slurried with petroleum ether:ethyl acetate (5:1, 200 ml), filtered and dried to obtain product 1-3.

(18) 1H NMR (400 MHz, CDCl.sub.3) 6.73-6.67 (m, 1H), 5.97 (s, 1H), 4.01-3.88 (m, 2H), 3.70-3.60 (m, 2H), 2.51-2.49 (m, 2H), 2.47 (q, J=0.7 Hz, 2H), 1.23 (s, 6H).

(19) MS m/z (ESI): 205 (M+1).sup.+

Step 9 9-bromo-7,7-dimethyl-3,4,7,8-tetrahydro-2H-cyclopenta[4,5]pyrrolo [1,2-a]pyrazin-1(6H)-one 1b

(20) Compound 1a (1 g, 4.9 mmol) was dissolved in dichloromethane (30 mL) and cooled to 20 C. NBS (1.05 g, 5.88 mmol) was added to the solution and the reaction solution was warmed to room temperature and stirred overnight. After the reaction was complete, the reaction solution was diluted with dichloromethane (50 mL) and washed with saturated aqueous Na.sub.2S.sub.2O.sub.3 solution. The organic phase was dried over anhydrous sodium sulfate, filtered and spin dried to obtain the crude product, which was purified by silica gel column chromatography to obtain compound 1b (960 mg) with a yield of 69%.

Step 10 9-fluoro-7,7-dimethyl-3,4,7,8-tetrahydro-2H-cyclopenta[4,5]pyrrolo [1,2-a]pyrazin-1(6H)-one 1c

(21) Compound 1b (560 mg, 2.0 mmol) was dissolved in tetrahydrofuran (15 mL) and then n-butyllithium (30 mL, 7.0 mmol) was added dropwise while stirring at 78 C. The resulting solution was stirred at 40 C. for 3 h and then cooled to 78 C., to which a solution of N-fluorobenzenesulfonimide (1.26 g, 4.0 mmol) in tetrahydrofuran (10 mL) was added dropwise. The resulting solution was stirred at room temperature for 3 h, quenched by adding water (15 mL) and extracted with ethyl acetate (502 mL). The combined organic phases were dried over anhydrous sodium sulphate, filtered and spin dried to obtain a crude product, which was purified by silica gel column chromatography to obtain compound 1c (310 mg) with a yield of 70%.

Step 11 4-chloro-2-(9-fluoro-7,7-dimethyl-1-oxo-1,3,4,6,7,8-hexahydro-2H-cyclopenta[4,5]pyrrolo[1,2-a]pyrazin-2-yl)nicotinaldehyde 1e

(22) Compound 1c (250 mg, 1.12 mmol), compound 1d (246.4 mg, 1.12 mmol), Pd.sub.2(dba).sub.3 (211 mg, 0.23 mmol), XantPhos (266 mg, 0.46 mmol) and potassium carbonate (309 mg, 2.24 mmol) were suspended in tetrahydrofuran (15 mL). The air in the reaction system was replaced twice with nitrogen. After that, the reaction solution was heated to 65 C. and stirred for 2 h. After the reaction was complete, the reaction solution was spin dried and the resulted residue was purified by silica gel column chromatography to obtain compound 1e (170 mg) with a yield of 42%.

Step 12 (S)-2-(9-fluoro-7,7-dimethyl-1-oxo-1,3,4,6,7,8-hexahydro-2H-cyclopenta[4,5]pyrrolo[1,2-a]pyrazin-2-yl)-1-methyl-5-((5-(2-methyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-yl)amino)-6-oxo-1,6-dihydro-[3,4-bipyridyl]-3-formaldehyde 1g

(23) Compound 1e (130 mg, 0.36 mmol), compound 1f (173 mg, 0.36 mmol), Pd(dppf)Cl.sub.2 (26.3 mg, 0.036 mmol) and potassium phosphate (114.5 mg, 0.54 mmol) were dissolved in a mixed solution of tetrahydrofuran (16 mL) and water (4 mL). The air in the reaction system was replaced twice with nitrogen. After that, the reaction solution was heated to 65 C. and stirred for 3 h. After the reaction was complete, the solution was spin dried, diluted with water (30 mL) and extracted with dichloromethane (302 mL). The combined organic phases were dried with anhydrous sodium sulfate, filtered and spin dried to obtain a crude product, which was purified by silica gel column chromatography to obtain compound 1g (40 mg) with a yield of 16%.

Step 13 (S)-9-fluoro-2-(3-(hydroxymethyl)-1-methyl-5-((5-(2-methyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-yl)amino)-6-oxo-1,6-dihydro-[3,4-bipyridin]-2-yl)-7,7-dimethyl-3,4,7,8-tetrahydro-2H-cyclopenta[4,5]pyrrolo[1,2-a]pyrazin-1(6H)-one

(24) Compound 1 g (40 mg, 0.06 mmol) was dissolved in a mixed solution of tetrahydrofuran (10 mL) and water (3 mL), added with K.sub.2HPO.sub.4 (10.4 mg, 0.06 mmol) and NaOH (2.4 mg, 0.06 mmol), followed by sodium borohydride (6.7 mg, 0.18 mmol). The reaction solution was stirred at room temperature for 2 h, diluted with water (20 mL) and extracted with dichloromethane (302 mL). The combined organic phases were dried over anhydrous sodium sulfate, filtered and spin dried to obtain a crude product, which was purified by silica gel column chromatography to obtain compound 1 (5 mg).

(25) 1H NMR (400 MHz, DMSO-d6) 8.61 (d, J=2.3 Hz, 1H), 8.48 (d, J=5.0 Hz, 1H), 8.44 (s, 1H), 7.83 (d, J=2.8 Hz, 1H), 7.45 (d, J=2.3 Hz, 1H), 7.40-7.32 (m, 2H), 7.24 (d, J=9.0 Hz, 1H), 4.94 (t, J=5.1 Hz, 1H), 4.55 (td, J=6.5, 2.9 Hz, 2H), 4.49-4.39 (m, 4H), 4.25-4.12 (m, 3H), 3.82 (d, J=11.1 Hz, 1H), 3.67 (d, J=5.5 Hz, 1H), 3.60 (s, 3H), 3.40 (q, J=6.2 Hz, 1H), 3.13-3.06 (m, 1H), 2.94 (t, J=8.9 Hz, 1H), 2.58-2.53 (m, 3H), 2.45 (s, 2H), 2.36-2.28 (m, 2H), 2.19 (t, J=9.1 Hz, 1H), 1.22 (s, 6H), 0.93 (d, J=6.3 Hz, 3H). MS m/z (ESI): 683[M+H].sup.+.

Example 2 (S)-9-chloro-2-(3-(hydroxymethyl)-1-methyl-5-((5-(2-methyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-yl)amino)-6-oxo-1,6-dihydro-[3,4-bipyridin]-2-yl)-7,7-dimethyl-3,4,7,8-tetrahydro-2H-cyclopenta[4,5]pyrrolo[1,2-a]pyrazin-1(6H)-one

(26) ##STR00015##

(27) A synthetic method similar to that of Example 1 was used, in which N-bromosuccinimide was replaced by N-chlorosuccinimide to obtain 9-chloro-7,7-dimethyl-3,4,7,8-tetrahydro-2H-cyclopenta[4,5]pyrrolo[1,2-a]pyrazin-1(6H)-one intermediate, which was directly used in reactions similar to that of Example 1 after step 3 to obtain the title product 2 with a yield of 13%.

(28) 1H NMR (400 MHz, DMSO-d6) 8.61 (d, J=2.3 Hz, 1H), 8.48 (d, J=5.0 Hz, 1H), 8.44 (s, 1H), 7.83 (d, J=2.8 Hz, 1H), 7.45 (d, J=2.3 Hz, 1H), 7.40-7.32 (m, 2H), 7.24 (d, J=9.0 Hz, 1H), 4.94 (t, J=5.1 Hz, 1H), 4.55 (td, J=6.5, 2.9 Hz, 2H), 4.49-4.39 (m, 4H), 4.25-4.12 (m, 3H), 3.82 (d, J=11.1 Hz, 1H), 3.67 (d, J=5.5 Hz, 1H), 3.60 (s, 3H), 3.40 (q, J=6.2 Hz, 1H), 3.13-3.06 (m, 1H), 2.94 (t, J=8.9 Hz, 1H), 2.58-2.53 (m, 3H), 2.42 (s, 2H), 2.38-2.29 (m, 2H), 2.19 (t, J=9.1 Hz, 1H), 1.21 (s, 6H), 0.93 (d, J=6.3 Hz, 3H). MS m/z (ESI): 699 (M+1).sup.+.

Example 3 (S)-2-(3-(hydroxymethyl)-1-methyl-5-((5-(2-methyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-yl)amino)-6-oxo-1,6-dihydro-[3,4-bipyridin]-2-yl)-7,7,9-trimethyl-3,4,7,8-tetrahydro-2H-cyclopenta[4,5]pyrrolo[1,2-a]pyrazin-1(6H)-one

(29) ##STR00016##

Step 1 7,7,9-trimethyl-3,4,7,8-tetrahydro-2H-cyclopenta[4,5]pyrrolo[1,2-a]pyrazin-1(6H)-one

(30) Compound 1a (0.5 g, 2.45 mmol) was dissolved in acetic acid (10 mL), and then added with hydroiodic acid (10 mL) and paraformaldehyde (0.5 g). The reaction solution was stirred under nitrogen protection at 25 C. for 3 h, and then added dropwise with hypophosphoric acid (50%, 0.8 mL) until the reaction solution was colourless. Saturated ammonium chloride was added under an ice bath. After that, the reaction solution was extracted with ethyl acetate (502 mL). The combined organic phases were dried over anhydrous sodium sulphate, filtered and spin dried to obtain a crude product, which was purified by silica gel column chromatography to obtain compound 3b (280 mg) with a yield of 52.4%.

(31) MS m/z (ESI): 215 (M+1).sup.+

(32) This step was followed by a similar reaction as in Example 1 after step 5 to produce the title product 3 with a yield of 19%.

(33) 1H NMR (400 MHz, DMSO-d6) 8.60 (d, J=2.4 Hz, 1H), 8.46-8.42 (m, 2H), 7.81 (d, J=2.8 Hz, 1H), 7.46 (d, J=2.4 Hz, 1H), 7.36-7.30 (m, 2H), 7.23-7.21 (m, 1H), 4.95 (t, J=5.2 Hz, 1H), 4.55-4.38 (m, 6H), 4.18-4.10 (m, 3H), 3.78-3.75 (m, 1H), 3.65 (s, 1H), 3.58 (s, 3H), 3.40-3.35 (m, 2H), 3.09-3.06 (m, 1H), 2.95-2.90 (m, 1H), 2.57-2.51 (m, 2H), 2.34-2.30 (m, 4H), 2.17 (s, 4H), 1.19 (s, 6H), 0.91 (d, J=6.4 Hz, 3H).

Example 4 (S)-2-(3-(hydroxymethyl)-1-isopropyl-5-((5-(2-methyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-yl)amino)-6-oxo-1,6-dihydro-[3,4-bipyridin]-2-yl)-7,7-dimethyl-3,4,7,8-tetrahydro-2H-cyclopenta[4,5]pyrrolo[1,2-a]pyrazin-1(6H)-one 4

(34) ##STR00017##

Step 1 3,5-dibromo-1-isopropylpyridin-2(1H)-one 4b

(35) Compound 4a (2.52 g, 10 mmol) was dissolved in DME (30 mL), and added with 2-iodopropane (2.04 g, 1.2 mmol) and caesium carbonate (3.9 g, 1.2 mmol). The reaction solution was refluxed at 80 C. for 3 h. After cooling, the reaction solution was diluted with ethyl acetate (60 mL) and washed with aqueous NaHCO.sub.3. The organic phase was dried with anhydrous sodium sulfate, filtered and spin dried to obtain the crude product, which was purified by silica gel column chromatography to obtain compound 4b (1.7 g) with a yield of 58%.

Step 2 (S)-5-bromo-1-isopropyl-3-((5-(2-methyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-yl)amino)pyridin-2(1H)-one 4d

(36) Compound 4b (1 g, 3.4 mmol), compound 4c (843 mg, 3.4 mmol), palladium acetate (152 mg, 0.68 mmol), Xantphos (786 mg, 1.36 mmol) and potassium carbonate (1.41 g, 10.2 mmol) were suspended in toluene (30 mL). The air in the reaction system was replaced twice with nitrogen. After that, the reaction solution was heated to 100 C. and stirred for 2 h. After the reaction was completed, the reaction solution was spin dried. The resulted residue was dissolved in dichloromethane (60 mL) and washed with saturated saline (30 mL). The organic phase was dried with anhydrous sodium sulfate, filtered and spin dried to obtain a crude product, which was purified by silica gel column chromatography to obtain compound 4d (670 mg) with a yield of 43%.

Step 3 (S)-1-isopropyl-3-((5-(2-methyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-yl)boranyl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2(1H)-one 4e

(37) Compound 1d (560 mg, 1.2 mmol), diboron pinacol ester (309 mg, 1.2 mmol), Pd.sub.2(dba).sub.3 (220 mg, 0.24 mmol), XPhos (228 mg, 0.48 mmol) and potassium acetate (235 mg, 2.4 mmol) were suspended in tetrahydrofuran (20 mL). The air in the reaction system was replaced twice with nitrogen. After that, the reaction solution was heated to 70 C. and stirred for 2 h. After the reaction was complete, the reaction solution was spin dried, and the resulted residue was purified by silica gel column chromatography to obtain compound 4e (500 mg) with a yield of 81%.

Step 4 (S)-2-((7,7-dimethyl-1-oxo-1,3,4,6,7,8-hexahydro-2H-cyclopenta[4,5]pyrrolo[1,2-a]pyrazin-2-yl)-1-isopropyl-5-((5-(2-methyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-yl)amino-6-oxo-1,6-dihydro-[3,4-bipyridyl]-3-formaldehyde 4f

(38) Compound 4e (506 mg, 1 mmol), compound 8a (343 mg, 1 mmol), Pd(dppf)Cl.sub.2 (73.1 mg, 0.1 mmol) and potassium phosphate (424 mg, 2.0 mmol) were dissolved in tetrahydrofuran (10 mL). The air in the reaction system was replaced twice with nitrogen. After that, the reaction solution was heated to 50 C. and stirred for 3 h. After the reaction was complete, the solution was spin dried, diluted with water (30 mL) and extracted with dichloromethane (502 mL). The combined organic phases were dried with anhydrous sodium sulfate, filtered and spin dried to obtain a crude product, which was purified by silica gel column chromatography to obtain compound 4g (350 mg) with a yield of 51%.

Step 5 (S)-2-(3-(hydroxymethyl)-1-isopropyl-5-((5-(2-methyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-yl)amino)-6-oxo-1,6-dihydro-[3,4-bipyridin]-2-yl)-7,7-dimethyl-3,4,7,8-tetrahydro-2H-cyclopenta[4,5]pyrrolo[1,2-a]pyrazin-1(6H)-one 4

(39) Compound 4g (250 mg, 0.36 mmol) was dissolved in a mixed solution of tetrahydrofuran (15 mL) and water (3 mL), added with K.sub.2HPO.sub.4 (62.6 mg, 0.36 mmol) and NaOH (14.4 mg, 0.36 mmol), followed by sodium borohydride (38 mg, 1 mmol). The reaction solution was stirred at room temperature for 2 h, diluted with water (30 mL) and extracted with dichloromethane (302 mL). The combined organic phases were dried over anhydrous sodium sulfate, filtered and spin dried to obtain a crude product, which was purified by silica gel column chromatography to obtain compound 4 (80 mg) with a yield of 32%.

(40) 1H NMR (400 MHz, DMSO-d6) 8.59 (d, J=2.2 Hz, 1H), 8.52-8.40 (m, 2H), 7.81 (d, J=3.0 Hz, 1H), 7.61 (d, J=2.3 Hz, 1H), 7.40-7.29 (m, 2H), 7.21 (d, J=9.0 Hz, 1H), 6.53 (s, 1H), 5.21 (p, J=6.8 Hz, 1H), 5.07 (s, 1H), 4.52 (td, J=6.4, 2.9 Hz, 2H), 4.40 (dt, J=21.9, 5.9 Hz, 4H), 4.18 (t, J=8.7 Hz, 3H), 3.87-3.73 (m, 1H), 3.65 (s, 1H), 3.36 (q, J=6.3 Hz, 1H), 3.14-3.00 (m, 1H), 2.91 (t, J=10.3 Hz, 1H), 2.61-2.48 (m, 2H), 2.39 (s, 2H), 2.28 (d, J=3.9 Hz, 2H), 2.14 (t, J=9.2 Hz, 1H), 1.34 (d, J=6.8 Hz, 6H), 1.27-1.12 (m, 7H), 0.89 (d, J=6.3 Hz, 3H).

(41) MS m/z (ESI): 693[M+H].sup.+.

Example 5 (S)-3-(7,7-dimethyl-1-oxo-1,3,4,6,7,8-hexahydro-2H-cyclopenta [4,5]pyrrolo[1,2-a]pyrazin-2-yl)-4-(hydroxymethyl)-5-(1-methyl-5-((5-(2-methyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-yl)amino)-6-oxo-1,6-dihydropyridin-3-yl) benzonitrile

(42) ##STR00018## ##STR00019##

Step 1 3,5-dibromo-4-methylbenzoyl chloride 5b

(43) 3,5-Dibromo-4-methylbenzoic acid (5.0 g, 17.0 mmol, 1.0 eq) was dissolved in DCM (50 ml), added with oxalyl chloride (7.2 ml, 85.0 mmol, 5.0 eq) and DMF (2 drops) dropwise under cooling in an ice water bath, and reacted overnight at room temperature. The resultant was dried by evaporation under reduced pressure to obtain crude product 5b, which is used directly in the next reaction step.

Step 2 3,5-dibromo-4-methylbenzamide 5b

(44) Compound 5b (5.3 g, 17.0 mmol, 1.0 eq) was dissolved in anhydrous THF (5 ml), added with 25% ammonium hydroxide solution and reacted at room temperature for 30 min. The resultant was extracted with ethyl acetate (50 ml). The organic layer was washed with saline and dried over anhydrous sodium sulfate in turn, and concentrated to dryness to obtain a white solid (4.9 g) 5c.

(45) MS m/z (ESI): 293 [M+1].sup.+

Step 3 3,5-dibromo-4-methylbenzonitrile 5d

(46) Compound 5c (4.6 g, 15.7 mmol), triphenylphosphine oxide (45 mg, 0.16 mmol) and TEA (6.5 ml, 47.1 mmol) were introduced into DCM (100 ml). Under cooling in an ice water bath, oxalyl chloride (2.7 ml, 31.4 mmol) was added. The reaction system was warmed to room temperature, reacted for 30 min, concentrated and purified by flash column to obtain a white solid (HHT0130-180-1A, 3.0 g).

(47) MS m/z (ESI): 275 [M+1]+

Step 4 3,5-dibromo-4-(bromomethyl)benzonitrile 5e

(48) Compound 5d (1.0 g, 3.6 mmol, 1.0 eq) was dissolved in DCM (30 ml) and water (6 ml). NBS (0.64 g, 3.6 mmol) and hydrogen peroxide (0.12 g, 3.6 mmol) were added sequentially. The reaction was refluxed for 4 h, and cooled to room temperature. The organic layer was separated, dried over anhydrous sodium sulphate, concentrated and purified by flash column to obtain a white solid (1.1 g).

(49) MS m/z (ESI): 354 [M+1].sup.+

Step 5 2,6-dibromo-4-cyanobenzyl acetate 5f

(50) Compound 5e (1.0 g, 2.8 mmol, 1.0 eq) was dissolved in DMF (10 ml), and added with potassium acetate (0.33 g, 3.4 mmol, 1.2 eq). The reaction was performed at 80 C. for 1 h, cooled to room temperature, diluted with ethyl acetate (50 ml), washed sequentially with water (20 ml) and saline (15 ml3), dried over anhydrous sodium sulfate, concentrated and purified by flash column (PE to PA/EA=10:1) to obtain a white solid (HHT0130-158-1A, 590 mg).

(51) MS m/z (ESI): 333 [M+1].sup.+

Step 6 2-bromo-4-cyano-6-(7,7-dimethyl-1-oxo-1,3,4,6,7,8-hexahydro-2H-cyclopenta[4,5]pyrrolo[1,2-a]pyrazin-2-yl)benzyl acetate 5g

(52) Compound 5f (540 mg, 1.6 mmol, 1.5 eq) and 7,7-dimethyl-3,4,7,8-tetrahydro-2H-cyclopenta[4,5]pyrrolo[1,2-a]pyrazin-1(6H)-one (7) (0.22 g, 1.1 mmol, 1.0 eq) were introduced into anhydrous 1,4-dioxane (30 ml), and sequentially added with cesium carbonate (0.72 g, 2.2 mmol, 2.0 eq), Pd.sub.2(dba).sub.3 (0.10 g, 0.11 mmol, 0.1 eq) and Xantphos (0.13 g, 0.22 mmol, 0.2 eq). The reaction was refluxed for 8 h, dried by evaporation under reduced pressure, and added with ethyl acetate (50 ml). The resultant was washed sequentially with water (20 ml) and saline (20 ml), concentrated and purified by flash column to obtain a yellow solid (290 mg).

(53) MS m/z (ESI): 457 [M+1].sup.+

Step 7 (S)-3-(7,7-dimethyl-1-oxo-1,3,4,6,7,8-hexahydro-2H-cyclopenta[4,5]pyrrolo[1,2-a]pyrazin-2-yl)-4-(hydroxymethyl)-5-(1-methyl-5-((5-(2-methyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-yl)amino)-6-oxo-1,6-dihydropyridin-3-yl) benzonitrile 5

(54) Compound 5g (100 mg, 0.22 mmol, 1.0 eq) and (S)-1-methyl-3-((5-(2-methyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-yl)amino)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2(1H)-one (0.11 g. 0.22 mmol, 1.0 eq) were introduced into a mixture of n-butanol (20 ml) and water (4 ml), and sequentially added with potassium phosphate (93 mg, 0.44 mmol), Pd.sub.2(dba).sub.3 (20 mg, 0.022 mmol) and XPhos (21 mg, 0.044 mmol). The reaction was refluxed for 8 h, dried by evaporation under reduced pressure, and added with ethyl acetate (50 ml). The resultant was washed sequentially with water (20 ml) and saturated saline (20 ml), concentrated and purified by flash column to obtain a near white solid (60 mg).

(55) MS m/z (ESI): 689 [M+1].sup.+

(56) 1H NMR (400 MHz, DMSO-d6) 8.56 (t, J=2.5 Hz, 1H), 8.44 (s, 1H), 7.94 (d, J=1.7 Hz, 1H), 7.83 (d, J=2.9 Hz, 1H), 7.79 (d, J=1.7 Hz, 1H), 7.40-7.33 (m, 2H), 7.24 (d, J=9.0 Hz, 1H), 6.51 (s, 1H), 5.09 (q, J=4.2 Hz, 1H), 4.60-4.35 (m, 6H), 4.17 (dq, J=25.9, 7.8, 6.8 Hz, 3H), 3.92-3.81 (m, 1H), 3.67 (s, 1H), 3.58 (s, 3H), 3.39 (q, J=6.2 Hz, 1H), 3.09 (d, J=12.1 Hz, 1H), 2.93 (t, J=9.7 Hz, 1H), 2.56 (d, J=3.8 Hz, 2H), 2.41 (s, 2H), 2.32 (d, J=3.8 Hz, 2H), 2.19 (d, J=10.0 Hz, 1H), 1.21 (s, 6H), 0.92 (dd, J=6.4, 2.3 Hz, 3H).

Example 6 (S)-2-(7,7-dimethyl-1-oxo-1,3,4,6,7,8-hexahydro-2H-cyclopenta [4,5]pyrrolo[1,2-a]pyrazin-2-yl)-3-(hydroxymethyl)-4-(1-methyl-5-((5-(2-methyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-yl)amino)-6-oxo-1,6-dihydropyridin-3-yl) benzonitrile

(57) ##STR00020##

Step 1 2-bromo-4-chloro-3-formylbenzonitrile 6b

(58) Compound 6a (5.00 g, 23.1 mmol) was dissolved in THF (50 mL), added with LDA (15.0 mL, 30.1 mmol) dropwise at 78 C. and stirred for 0.5 h at 78 C. DMF (1.89 mL, 25.5 mmol) was added dropwise at 78 C. and stirred for 2 h at 78 C. The reaction completion was confirmed by LCMS. The reaction was quenched with saturated NH.sub.4Cl solution (50 mL) and extracted with EA (50 mL3). The organic phases were combined, washed with saturated saltine (50 mL), dried over anhydrous Na.sub.2SO.sub.4, filtered and concentrated. The residue was subjected to column chromatography (PE/EA=100/1 to 1/100) to obtain a white solid compound 6b (0.60 g, yield 10%).

(59) LCMS (ESI-MS) m/z: 245.9 (M+H).sup.+.

Step 2 2-bromo-4-chloro-3-(hydroxymethyl)benzonitrile 6c

(60) Compound 6b (0.60 g, 2.46 mmol) was dissolved in anhydrous MeOH (10 mL). NaBH.sub.4 (0.19 g, 4.92 mmol) was added at 0 C. The reaction system was brought back to room temperature and stirred for 2 h. The reaction completion was confirmed by LCMS. The reaction solution was quenched with water (20 mL) and extracted with EA (20 mL3). The organic phases were combined, washed with saturated saltine (20 mL), dried over anhydrous Na.sub.2SO.sub.4, filtered and concentrated. The residue was subjected to column chromatography (PE/EA=100/1 to 1/100) to obtain a white solid compound 6c (0.50 g, yield 82%).

(61) LCMS (ESI-MS) m/z: 247.9 (M+H).sup.+.

Step 3 2-bromo-6-chloro-3-cyanobenzyl acetate 6d

(62) Compound 6c (0.50 g, 2.03 mmol) was dissolved in DCM (5 mL). TEA (0.53 mL, 4.06 mmol) and acetyl chloride (0.22 mL, 3.05 mmol) were added under an ice bath. The reaction was stirred for 2 h at room temperature. The reaction completion was confirmed by LCMS. The reaction solution was quenched with water (10 mL) and extracted with DCM (10 mL3). The organic phases were combined, washed with saturated saltine (10 mL), dried over anhydrous Na.sub.2SO.sub.4, filtered and concentrated. The residue was subjected to column chromatography (PE/EA=100/1 to 1/100) to obtain a white solid compound 6d (0.45 g, yield 77%).

(63) LCMS (ESI-MS) m/z: 287.9 (M+H).sup.+.

Step 4 6-chloro-3-cyano-2-(7,7-dimethyl-1-oxo-1,3,4,6,7,8-hexahydro-2H-cyclopenta[4,5]pyrrolo[1,2-a]pyrazin-2-yl)benzyl acetate 6e

(64) Compound 6d (0.45 g, 1.56 mmol) was dissolved in dioxane (5 mL), to which compound 5 (0.32 g, 1.56 mmol), Pd(AcO).sub.2 (35 mg, 0.16 mmol), Xantphos (90 mg, 0.16 mmol) and Cs.sub.2CO.sub.3 (1.02 g, 3.13 mmol) were added. The reaction was refluxed for 2 h under N.sub.2 protection. The reaction completion was confirmed by LCMS. The reaction solution was added with water (10 mL) and extracted with EA (10 mL3). The organic phases were combined, washed with saturated saltine (10 mL), dried over anhydrous Na.sub.2SO.sub.4, filtered and concentrated. The residue was subjected to column chromatography (PE/EA=100/1 to 1/100) to obtain a pale yellow solid compound 6e (0.21 g, yield 33%).

(65) LCMS (ESI-MS) m/z: 412.2 (M+H).sup.+.

Step 5 (S)-3-cyano-2-(7,7-dimethyl-1-oxo-1,3,4,6,7,8-hexahydro-2H-cyclopenta[4,5]pyrrolo[1,2-a]pyrazin-2-yl)-6-(1-methyl-5-((5-(2-methyl-4-(oxa-3-yl)piperazin-1-yl)pyridin-2-yl)amino)-6-oxo-1,6-(dihydropyridin-3-yl)benzyl acetate 6f

(66) Compound 6e (0.16 g, 0.39 mmol) was dissolved in dioxane (3 mL), to which compound 7 (0.19 g, 0.39 mmol), Pd(dppf)Cl.sub.2 (28 mg, 0.039 mmol) and K.sub.3PO.sub.4 (0.16 g, 0.78 mmol) were added. The reaction was performed with microwave for 2 h under N.sub.2 protection. The reaction completion was confirmed by LCMS. The reaction solution was added with water (10 mL) and extracted with EA (10 mL3). The organic phases were combined, washed with saturated saltine (10 mL), dried over anhydrous Na.sub.2SO.sub.4, filtered and concentrated. The residue was subjected to column chromatography (PE/EA=100/1 to 1/100) to obtain a brown compound 6f (80 mg, yield 28%).

(67) LCMS (ESI-MS) m/z: 367.1 (M+H).sup.+.

Step 6 (S)-2-(7,7-dimethyl-1-oxo-1,3,4,6,7,8-hexahydro-2H-cyclopenta[4,5]pyrrolo[1,2-a]pyrazin-2-yl)-3-(hydroxymethyl)-4-(1-methyl-5-((5-(2-methyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-yl)amino)-6-oxo-1,6-dihydropyridin-3-yl) benzonitrile 5

(68) Compound 6f (80 mg, 0.11 mmol) was dissolved in MeOH (2 mL) and an aqueous solution of NaOH (1 mL, 0.22 M) was added. The reaction was stirred for 2 h at room temperature. The reaction completion was confirmed by LCMS. The reaction solution was diluted with water (5 mL) and extracted with DCM (5 mL3). The organic phases were combined, washed with saturated saltine (5 mL), dried over anhydrous Na.sub.2SO.sub.4, filtered and concentrated. The residue was subjected to a reversed phase column chromatography (H2O/MeCN=20/1 to 1/20) and a normal phase column chromatography (DCM/EA=100/1 to 1/100) to obtain a white solid compound 6 (10 mg, yield 13%).

(69) LCMS (ESI-MS) m/z: 689.5 (M+H).sup.+.

(70) 1H NMR (400 MHz, CDCl.sub.3) 8.59 (s, 1H), 7.95-7.86 (m, 2H), 7.77 (d, J=4.0 Hz, 1H), 7.56-7.52 (m, 2H), 6.85-6.81 (m, 2H), 4.72-4.47 (m, 5H), 4.39-4.34 (m, 1H), 4.17-3.96 (m, 3H), 3.70 (s, 2H), 3.67-3.29 (m, 4H), 3.12-3.07 (m, 2H), 2.60-2.03 (m, 7H), 1.30-1.25 (m, 9H), 0.96-0.86 (m, 3H).

Example 7 (S)-4-(7,7-dimethyl-1-oxo-1,3,4,6,7,8-hexahydro-2H-cyclopenta [4,5]pyrrolo[1,2-a]pyrazin-2-yl)-3-(hydroxymethyl)-2-(1-methyl-5-((5-(2-methyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-yl)amino)-6-oxo-1,6-dihydropyridin-3-yl) benzonitrile

(71) ##STR00021##

Step 1 4-bromo-2-chloro-3-formylbenzonitrile 7b

(72) Compound 7a (4.00 g, 18.5 mmol) was dissolved in THF (50 mL), added with LDA (12.0 mL, 24.1 mmol) dropwise at 78 C. and stirred for 0.5 h at 78 C. DMF (1.51 mL, 20.4 mmol) was added dropwise at 78 C. and stirred for 2 h at 78 C. The reaction completion was confirmed by LCMS. The reaction was quenched with saturated NH.sub.4Cl solution (50 mL) and extracted with EA (50 mL3). The organic phases were combined, washed with saturated saltine (50 mL), dried over anhydrous Na.sub.2SO.sub.4, filtered and concentrated. The residue was subjected to column chromatography (PE/EA=100/1 to 1/100) to obtain a white solid compound 2 (0.45 g, yield 10%).

(73) LCMS (ESI-MS) m/z: 243.9 (M+H+).

Step 2 2-chloro-4-(7,7-dimethyl-1-oxo-1,3,4,6,7,8-hexahydro-2H-cyclopenta [4,5]pyrrolo[1,2-a]pyrazin-2-yl)-3-formylbenzonitrile 7c

(74) Compound 7b (0.45 g, 1.84 mmol) was dissolved in DMF (3 mL), to which compound 1a (0.32 g, 1.84 mmol), Pd.sub.2(dba).sub.3 (0.17 g, 0.18 mmol), Xantphos (0.10 g, 0.18 mmol) and Cs.sub.2CO.sub.3 (1.20 g, 3.69 mmol) were added. The reaction was refluxed for 2 h under N.sub.2 protection. The reaction completion was confirmed by LCMS. The reaction was quenched with water (10 mL) and extracted with EA (10 mL3). The organic phases were combined, washed with saturated saltine (10 mL), dried over anhydrous Na.sub.2SO.sub.4, filtered and concentrated. The residue was subjected to column chromatography (PE/EA=100/1 to 1/100) to obtain a pale yellow solid compound 7c (0.14 g, yield 21%).

(75) LCMS (ESI-MS) m/z: 368.2 (M+H).sup.+.

Step 3 (S)-4-(7,7-dimethyl-1-oxo-1,3,4,6,7,8-hexahydro-2H-cyclopenta [4,5]pyrrolo[1,2-a]pyrazin-2-yl)-3-(formyl)-2-(1-methyl-5-((5-(2-methyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-yl)amino)-6-oxo-1,6-dihydropyridin-3-yl)benzonitrile 7d

(76) Compound 7c (0.14 g, 0.38 mmol) was dissolved in dioxane (3 mL), to which compound 1f (0.18 g, 0.38 mmol), Pd(dppf)Cl.sub.2 (27 mg, 0.038 mmol) and K.sub.3PO.sub.4 (0.16 g, 0.76 mmol) were added. The reaction was performed with microwave for 2 h under N.sub.2 protection. The reaction completion was confirmed by LCMS. The reaction was quenched by adding water (10 mL) and extracted with EA (10 mL3). The organic phases were combined, washed with saturated saltine (10 mL), dried over anhydrous Na.sub.2SO.sub.4, filtered and concentrated. The residue was subjected to column chromatography (PE/EA=100/1 to 1/100) to obtain a brown compound 7d (20 mg, yield 7%).

(77) LCMS (ESI-MS) m/z: 687.5 (M+H).sup.+.

Step 4 (S)-4-(7,7-dimethyl-1-oxo-1,3,4,6,7,8-hexahydro-2H-cyclopenta [4,5]pyrrolo[1,2-a]pyrazin-2-yl)-3-(hydroxymethyl)-2-(1-methyl-5-((5-(2-methyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-yl)amino)-6-oxo-1,6-dihydropyridin-3-yl) benzonitrile 7

(78) Compound 7d (20 mg, 0.029 mmol) was dissolved in MeOH (2 mL) to which NaBH.sub.4 (2.2 mg, 0.058 mmol) was added. The reaction was stirred for 2 h at room temperature. The reaction completion was confirmed by LCMS. The reaction solution was quenched by adding water (5 mL) and extracted with DCM (5 mL3). The organic phases were combined, washed with saturated saltine (5 mL), dried over anhydrous Na.sub.2SO.sub.4, filtered and concentrated. The residue was subjected to a reversed phase column chromatography (H2O/MeCN=20/1 to 1/20) and a normal phase column chromatography (DCM/EA=100/1 to 1/100) to obtain a white solid compound 7 (2 mg, yield 10%).

(79) LCMS (ESI-MS) m/z: 689.5 (M+H).sup.+.

(80) 1H NMR (400 MHz, CDCl.sub.3) 8.39 (s, 1H), 7.88-7.76 (m, 3H), 7.36-7.29 (m, 2H), 6.81 (s, 2H), 5.38-5.30 (m, 2H), 4.71 (s, 3H), 4.53-4.21 (m, 4H), 4.17-3.99 (m, 2H), 3.70-3.49 (m, 6H), 3.07 (s, 1H), 2.56-2.50 (m, 3H), 2.23-2.19 (m, 2H), 2.03-1.98 (m, 2H), 1.70-1.60 (m, 5H), 0.97-0.86 (m, 6H).

Example 8 2-(3-(hydroxymethyl)-4-(7-((5-(4-methylpiperazin-1-yl)pyridin-2-yl)amino)furo[3,2-b]pyridin-5-yl)pyridin-2-yl)-7,7-dimethyl-3,4,7,8-tetrahydro-2H-cyclopenta[4,5]pyrrolo[1,2-a]pyrazin-1(6H)-one

(81) ##STR00022## ##STR00023##

Step 1 4-chloro-2-(7,7-dimethyl-1-oxo-1,3,4,6,7,8-hexahydro-2H-cyclopenta [4,5]pyrrolo[1,2-a]pyrazin-2-yl)nicotinaldehyde 8a

(82) Compound 1a (1.0 g, 4.9 mmol), 2-bromo-4-chloronicotinaldehyde (1.1 g, 4.9 mmol), palladium acetate (60 mg, 0.25 mmol), Xantphos (140 mg, 0.25 mmol), caesium carbonate (3.2 g, 10 mmol) and dioxane (20 ml) were added sequentially to a 100 ml reaction flask. The air in the reaction system was replaced 3 times with nitrogen. After that, the reaction solution was heated to 85 C., and reacted with stirring under nitrogen protection for 5 h. It was confirmed by LCMS detection that the raw materials were almost completely reacted. The reaction solution was cooled to room temperature and extracted with DCM/MeOH (10:1, 150 ml2). The organic phase was dried, spin dried and separated on a column to obtain a pale yellow solid compound 2 (800 mg, yield: 47%).

(83) LCMS (ESI-MS) m/z: 344.2 (M+H).sup.+.

(84) 1H NMR (400 MHz, DMSO-d6) 9.81 (s, 1H), 8.55 (d, J=5.4 Hz, 1H), 7.51 (d, J=5.4 Hz, 1H), 6.58 (s, 1H), 4.26-4.15 (m, 4H), 2.55 (s, 2H), 2.38 (s, 2H), 1.18 (s, 6H).

Step 2 2-(4-chloro-3-(hydroxymethyl)pyridin-2-yl)-7,7-dimethyl-3,4,7,8-tetrahydro-2H-cyclopenta[4,5]pyrrolo[1,2-a]pyrazine-1(6H)-one 8b

(85) Compound 8a (700 mg, 2.0 mmol) was dissolved in a mixture of dichloromethane (10 ml) and methanol (10 ml). Under ice bath conditions, sodium borohydride (160 mg, 4.0 mmol) was then added to the reaction system in batches. After the reaction was stirred for 2 h at room temperature, the reaction completion of the raw materials was confirmed by LCMS detection. The reaction was quenched by adding saturated ammonium chloride solution to the reaction solution. The organic phase was dried and spin dried to obtain a white solid compound 8b (700 mg, yield: 100%).

(86) LCMS (ESI-MS) m/z: 346.2 (M+H).sup.+.

Step 3 methyl (4-chloro-2-(7,7-dimethyl-1-oxo-1,3,4,6,7,8-hexahydro-2H-cyclopenta[4,5]pyrrolo[1,2-a]pyrazin-2-yl)pyridin-3-yl)acetate 8c

(87) Compound 8b (550 mg, 1.6 mmol) was dissolved in dichloromethane (10 ml). Under ice bath condition, triethylamine (900 mg, 9 mmol) and acetic anhydride (1.6 g, 16 mmol, 10 eq) were added dropwise to the reaction system, which was kept at room temperature with stirring overnight. The reaction completion was confirmed by LCMS. Water and dichloromethane were added to the reaction solution. The organic phase was dried, spin dried, purified through a column to obtain a white solid compound 8c (600 mg, yield: 97%).

(88) LCMS (ESI-MS) m/z: 388.2 (M+H).sup.+.

Step 4 (3-(acetoxymethyl)-2-(7,7-dimethyl-1-oxo-1,3,4,6,7,8-hexahydro-2H-cyclopenta[4,5]pyrrolo[1,2-a]pyrazin-2-yl)pyridin-4-yl)boronic acid 8d

(89) Compound 8c (370 mg, 1.0 mmol), Pd(dppf)(OAc).sub.2 (73 mg, 0.1 mmol), XPhos (47 mg, 0.1 mmol), potassium acetate (300 mg, 3.0 mmol), diboron (500 mg, 2.0 mmol) and dioxane were added sequentially to a reaction flask. The air in the reaction system was replaced 3 times with nitrogen. After that, the reaction system was heated to 70 C., and reacted for 4 h. It was confirmed by LCMS detection that the raw materials were almost completely reacted. After the reaction solution was cooled to room temperature, water and ethyl acetate were added to the reaction solution. The organic phase was dried and concentrated to dryness to obtain the crude product 8d (500 mg, purity: 80%), which was used directly in the next reaction step.

(90) LCMS (ESI-MS) m/z: 398.3 (M+H).sup.+.

Step 5 5-chloro-2-(triisopropylsilyl)furano[3,2-b]pyridine 8f

(91) Compound 8e (3.0 g, 11.7 mmol), triisopropylsilylacetylene (4.3 g, 24 mmol), cuprous iodide (200 mg, 1.1 mmol), PdCl.sub.2(PPh3).sub.2 (700 mg, 1.0 mmol), dioxane (50 ml) and triethylamine (50 ml) were added sequentially to a reaction flask. The air in the reaction system was replaced 3 times with nitrogen. After that, the reaction solution was heated to 50 C. and reacted for 2 h. The reaction completion was confirmed by LCMS. After the reaction was cooled to room temperature, the reaction solution was diluted with water and ethyl acetate. The organic phase was dried and concentrated to dryness to obtain a pale green solid compound (3.6 g, yield: 100%).

(92) LCMS (ESI-MS) m/z: 310.2 (M+H).sup.+.

(93) 1H NMR (400 MHz, Chloroform-d) 7.69 (dd, J=8.6, 1.0 Hz, 1H), 7.17 (d, J=8.6 Hz, 1H), 7.11 (d, J=0.9 Hz, 1H), 1.44-1.34 (m, 3H), 1.12 (d, J=7.4 Hz, 18H).

Step 6 5-chloro-7-iodo-2-(triisopropylsilyl)furano[3,2-b]pyridine 8 g

(94) Compound 7 (3.1 g, 10 mmol) was dissolved in anhydrous tetrahydrofuran (20 ml), and cooled to a temperature of 65 C. N-butyllithium (13 mmol, 1.3 eq) was added dropwise to the reaction system and stirred for 1 h. Then, iodine (3.8 g, 15 mmol) dissolved in anhydrous tetrahydrofuran (10 ml) was added dropwise to the above reaction system. The reaction was stirred at low temperature for 2 h. The reaction completion was confirmed by LCMS. The reaction was quenched by adding saturated ammonium chloride solution and sodium sulphite solution to the reaction solution. Ethyl acetate and water were added. The mixture was stirred and left standing for further separating. The organic phase was dried and concentrated to dryness to obtain a yellow solid compound 8g (4.0 g, yield: 92%).

(95) 1H NMR (400 MHz, Chloroform-d) 7.59 (s, 1H), 7.19 (s, 1H), 1.40 (ddd, J=14.8, 8.1, 6.9 Hz, 3H), 1.14 (d, J=7.5 Hz, 18H).

Step 7 5-chloro-N-(5-(4-methylpiperazin-1-yl)pyridin-2-yl)-2-(triisopropylsilyl)furano[3,2-b]pyridin-7-amine 8h

(96) Compound 8g (560 mg, 1.2 mmol), 5-(4-methylpiperazin-1-yl)pyridin-2-amine (250 mg, 1.30 mmol) (Shanghai WuXi AppTec New Drug Development Co., Ltd.), palladium acetate (40 mg, 0.18 mmol), Xantphos (40 mg, 0.07 mmol), cesium carbonate (1.2 g, 3.6 mmol) and dioxane (15 ml) were added sequentially to the reaction system. The air in the reaction system was replaced 3 times with nitrogen. After that, the reaction was carried out at 85 C. for 4 h. It was confirmed by LCMS detection that the raw materials were almost completely reacted. The reaction solution was cooled, and diluted with water and ethyl acetate. The organic phase was dried and concentrated to dryness and separated on a column to obtain a yellow solid compound 8h (500 mg, yield: 78%).

(97) LCMS (ESI-MS) m/z: 500.4 (M+H).sup.+.

(98) 1H NMR (400 MHz, Chloroform-d) 8.08 (d, J=2.9 Hz, 1H), 7.77 (s, 1H), 7.31 (dd, J=8.9, 3.0 Hz, 1H), 7.08 (s, 1H), 7.03 (dd, J=8.9, 0.7 Hz, 1H), 6.91 (s, 1H), 3.34 (s, 4H), 2.84 (s, 4H), 2.53 (s, 3H), 1.42-1.35 (m, 3H), 1.13 (d, J=7.4 Hz, 18H).

Step 8 methyl (2-(7,7-dimethyl-1-oxo-1,3,4,6,7,8-hexahydro-2H-cyclopenta[4,5]pyrrolo[1,2-a]pyrazin-2-yl)-4-(7-((5-(4-methylpiperazin-1-yl)pyridin-2-yl)amino)-2-(triisopropylsilyl)furano[3,2-b]pyridin-5-yl)pyridin-3-yl)acetate 8i

(99) Compound 8h (80 mg, 0.16 mmol), compound 8d (100 mg, crude product, 1.0 eq), K.sub.3PO.sub.4 (110 mg, 0.5 mmol), Pd(dppf)Cl.sub.2 (12 mg, 0.016 mmol), DME (5 ml) and water (1.5 ml) were added sequentially to a reaction flask. The air in the reaction system was replaced 3 times with nitrogen. After that, the reaction system was heated to 80 C. and reacted for 4 hours. The reaction solution was cooled to room temperature, and diluted with water and ethyl acetate. The organic phase was dried and concentrated to dryness to obtain a crude product. An oily compound 8i (30 mg, purity: 80%) was obtained by preparation plate separation.

Step 9 2-(3-(hydroxymethyl)-4-(7-((5-(4-methylpiperazin-1-yl)pyridin-2-yl) amino)-2-(triisopropylmethylsilyl)furano[3,2-b]pyridin-5-yl)pyridinyl-2-yl)-7,7-dimethyl-3,4,7,8-tetrahydro-2H-cyclopenta[4,5]pyrrolo[1,2-a]pyrazine-1(6H)-one 8j

(100) Compound 8i (30 mg, purity of 80%) was dissolved in tetrahydrofuran (6 ml) and water (2 ml) and lithium hydroxide (24 mg, 1.0 mmol) was added to the reaction system, which was stirred at room temperature overnight. The reaction completion was confirmed by LCMS. Water and ethyl acetate were added to the reaction solution. The organic phase was dried and concentrated to dryness to obtain the crude product 8j, which was used directly in the next reaction step.

(101) LCMS (ESI-MS) m/z: 775.6 (M+H)+.

Step 10 2-(3-(hydroxymethyl)-4-(7-((5-(4-methylpiperazin-1-yl)pyridin-2-yl) amino)furo[3,2-b]pyridin-5-yl)pyridin-2-yl)-7,7-dimethyl-3,4,7,8-tetrahydro-2H-cyclopenta[4,5]pyrrolo[1,2-a]pyrazin-1(6H)-one 8

(102) Compound 8j (25 mg, 80% purity) was dissolved in tetrahydrofuran (5 ml) and TBAF (1.0 M, 0.2 ml) was added dropwise to the reaction system under ice bath conditions and the reaction was continued with stirring for 2 h. The reaction completion was confirmed by LCMS. Ethyl acetate and saturated ammonium chloride solution were added to the reaction solution. The organic phase was dried, concentrated to dryness and subjected to preparation plate separation to obtain a pale yellow solid compound 8 (10 mg, yield: 60%).

(103) LCMS (ESI-MS) m/z: 619.4 (M+H).sup.+.

(104) 1H NMR (400 MHz, DMSO-d6) 11.00 (s, 1H), 8.61-8.51 (m, 2H), 8.11 (d, J=3.0 Hz, 1H), 7.59-7.55 (m, 1H), 7.51 (d, J=5.0 Hz, 1H), 7.37 (q, J=5.1, 4.7 Hz, 2H), 7.23 (d, J=5.1 Hz, 2H), 7.12 (d, J=5.8 Hz, 1H), 6.56 (s, 1H), 5.32 (t, J=4.9 Hz, 1H), 4.43 (d, J=8.8 Hz, 1H), 4.27 (d, J=12.3 Hz, 1H), 4.21 (t, J=5.8 Hz, 1H), 3.92 (d, J=12.0 Hz, 1H), 3.80 (s, 1H), 3.14 (d, J=8.5 Hz, 3H), 2.79 (d, J=4.1 Hz, 2H), 2.58 (d, J=8.0 Hz, 1H), 2.42 (s, 1H), 1.99 (dd, J=8.5, 6.5 Hz, 2H), 1.22 (s, 6H).

Example 9 2-(3-(hydroxymethyl)-4-(7-((5-(4-(oxetan-3-yl) piperazin-1-yl)pyridin-2-yl)amino)furo[3,2-b]pyridin-5-yl)pyridin-2-yl)-7,7-dimethyl-3,4,7,8-tetrahydro-2H-cyclopenta[4,5]pyrrolo[1,2-a]pyrazin-1(6H)-one

(105) ##STR00024##

Step 1 tert-butyl 4-(6-nitropyridin-3-yl)piperazine-1-carboxylate 9b

(106) Compound 9a (2.5 g, 12.3 mmol), tert-butyl piperazine-1-carboxylate (1.9 g, 10 mmol), palladium acetate (110 mg, 0.5 mmol), BINAP (310 mg, 0.5 mmol, 0.05 eq), K.sub.3PO.sub.4 (6.3 g, 30 mmol, 3.0 eq) and toluene (20 ml) were added sequentially to a 100 ml reaction flask. The air in the reaction system was replaced 3 times with nitrogen. After that, the reaction solution was heated to 95 C., and reacted with stirring under nitrogen protection for 5 h. It was confirmed by LCMS detection that the raw materials were almost completely reacted. The reaction solution was cooled to room temperature and extracted with DCM/MeOH (10:1, 150 ml2). The organic phase was dried, spin dried and separated on a column to obtain the pale yellow solid compound 9b (1.8 g, yield: 58%).

(107) 1H NMR (400 MHz, DMSO-d6) 8.21 (d, J=3.0 Hz, 1H), 8.14 (d, J=9.2 Hz, 1H), 7.43 (dd, J=9.3, 3.1 Hz, 1H), 3.57-3.38 (m, 8H), 1.39 (s, 9H).

Step 2 1-(6-nitropyridin-3-yl)piperazine 9c

(108) Compound 9b (1.0 g, 3.2 mmol) was dissolved in dichloromethane (10 ml) under ice bath conditions, and TFA (8 ml) was added dropwise to the reaction system. After the reaction was stirred for 2 h at room temperature, the complete reaction of the raw materials was confirmed by TLC detection. The reaction solution was directly spin dried to obtain the yellow oily compound 9c, which was used directly in the next reaction step.

Step 3 1-(6-nitropyridin-3-yl)-4-(oxetan-3-yl)piperazine 9d

(109) Compound 9c (1.2 g, 10 mmol) was dissolved in methanol (20 ml) and 3-oxetanone (720 mg, 10 mmol), zinc chloride (870 mg, 6.4 mmol) and sodium cyanoborohydride (400 mg g, 6.4 mmol) were added to the reaction system at room temperature and stirred for 4 h at 50 C. The reaction completion was confirmed by LCMS. Water and dichloromethane were added to the reaction solution. The organic phase was dried, spin dried, purified through a column to obtain a yellow solid compound 9d (800 mg, yield: 94%).

(110) 1H NMR (400 MHz, Chloroform-d) 8.15 (d, J=9.1 Hz, 1H), 8.12 (d, J=3.0 Hz, 1H), 7.19 (dd, J=9.2, 3.0 Hz, 1H), 4.70 (t, J=6.5 Hz, 2H), 4.62 (t, J=6.1 Hz, 2H), 3.57-3.52 (m, 1H), 3.50-3.45 (m, 4H), 2.53-2.48 (m, 4H).

Step 4 5-(4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-amine 9e

(111) Compound 9d (800 mg, 3.0 mmol) was dissolved in methanol (20 ml). Palladium carbon (10% w, 100 mg) was added to the reaction system. The air in the reaction system was replaced 3 times with nitrogen. After that, the reaction was continued with stirring for 4 h. TLC showed that the raw materials were completely reacted. The reaction solution was filtered through diatomaceous earth and spin dried to obtain a white solid compound 9e (700 mg, yield: 98%)

(112) LCMS (ESI-MS) m/z: 235.2 (M+H).sup.+.

Step 5 5-chloro-N-(5-(4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-yl)-2-(triisopropylsilyl)furano[3,2-b]pyridin-7-amine 9g

(113) Compound 9e (800 mg, 1.8 mmol), compound 9f (420 mg, 1.8 mmol, 1.0 eq), palladium acetate (20 mg, 0.09 mmol), Xantphos (50 mg, 0.09 mmol), cesium carbonate (1.1 g, 3.6 mmol) and dioxane (20 ml) were added sequentially to a reaction flask. The air in the reaction system was replaced 3 times with nitrogen. After that, the reaction was heated to 85 C. and reacted for 4 h. LCMS showed that the raw materials were completely reacted. The reaction solution was cooled, and diluted with water and ethyl acetate. The organic phase was dried and concentrated to dryness and separated on a column to obtain a yellow solid compound 9g (800 mg, yield: 82%).

(114) LCMS (ESI-MS) m/z: 542.4 (M+H).sup.+.

Step 6 methyl (2-(7,7-dimethyl-1-oxo-1,3,4,6,7,8-hexahydro-2H-cyclopenta[4,5]pyrrolo[1,2-a]pyrazin-2-yl)-4-(7-((5-(4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-yl)amino)-2-(triisopropylsilyl)furano[3,2-b]pyridin-5-yl)pyridin-3-yl)acetate 9h

(115) Compound 9 g (150 mg, 0.28 mmol), (3-(acetoxymethyl)-2-(7,7-dimethyl-1-oxo-1,3,4,6,7,8-hexahydro-2H-cyclopenta[4,5]pyrrolo[1,2-a]pyrazin-2-yl)pyridin-4-yl)boronic acid (200 mg. purity: 87%), Pd(dppf)Cl.sub.2 (20 mg, 0.028 mmol), K.sub.3PO.sub.4 (180 mg, 0.84 mmol), dioxane (6 ml) and water (2 ml) were added sequentially to a reaction flask. The air in the reaction system was replaced 3 times with nitrogen. After that, the reaction was heated to 80 C. and reacted for 4 h. The reaction solution was separated and purified by preparation plate separation to obtain compound 9h (20 mg, purity: 90%, yield: 10%).

(116) LCMS (ESI-MS) m/z: 859.6 (M+H)+.

Step 7 2-(3-(hydroxymethyl)-4-(7-((5-(4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-yl)amino)-2-(triisopropylsilyl)furano[3,2-b]pyridin-5-yl)pyridin-2-yl-7,7-dimethyl-3,4,7,8-tetrahydro-2H-cyclopenta[4,5]pyrrolo[1,2-a]pyrazine-1(6H)-one 9i

(117) Compound 9h (20 mg, purity: 90%) was dissolved in tetrahydrofuran (6 ml) and water (2 ml), and lithium hydroxide (24 mg, 1.0 mmol) was added to the reaction solution, which was reacted at room temperature overnight. The reaction completion was confirmed by TLC. The reaction solution was diluted with ethyl acetate and water. The organic phase was dried and then spin dried to obtain a crude product 9i, which was used directly in the next reaction step.

Step 8 2-(3-(hydroxymethyl)-4-(7-((5-(4-(oxetan-3-yl) piperazin-1-yl)pyridin-2-yl)amino)furo[3,2-b]pyridin-5-yl)pyridin-2-yl)-7,7-dimethyl-3,4,7,8-tetrahydro-2H-cyclopenta[4,5]pyrrolo[1,2-a]pyrazin-1(6H)-one 9

(118) Compound 9i (20 mg, 90% purity) was dissolved in tetrahydrofuran (5 ml) and TBAF (1.0 M, 0.2 ml) was added dropwise to the reaction system under ice bath conditions and the reaction was continued with stirring for 2 h. The reaction completion was confirmed by LCMS. Ethyl acetate and saturated ammonium chloride solution were added to the reaction solution. The organic phase was dried, concentrated to dryness and subjected to preparation plate separation to obtain a pale yellow solid compound 9 (5 mg, yield: 60%).

(119) LCMS (ESI-MS) m/z: 661.4 (M+H).sup.+.

(120) 1H NMR (400 MHz, DMSO-d6) 9.68 (s, 1H), 8.52-8.48 (m, 2H), 8.30 (d, J=2.2 Hz, 1H), 7.96 (d, J=3.0 Hz, 1H), 7.47-7.40 (m, 2H), 7.21 (d, J=9.0 Hz, 1H), 7.17 (s, 1H), 7.10 (d, J=2.2 Hz, 1H), 6.63 (s, 2H), 6.52 (s, 1H), 5.32-5.28 (m, 2H), 5.18-5.14 (m, 1H), 4.54 (t, J=6.5 Hz, 2H), 4.44 (t, J=6.0 Hz, 2H), 4.39 (t, J=5.5 Hz, 2H), 4.17 (t, J=5.3 Hz, 2H), 3.11 (t, J=5.0 Hz, 4H), 2.39 (d, J=7.8 Hz, 4H), 1.97 (d, J=7.7 Hz, 4H). 1.20 (s, 6H).

Example 10 9-fluoro-2-(3-(hydroxymethyl)-1-methyl-5-((5-(4-(oxetan-3-yl) piperazin-1-yl)pyridin-2-yl)amino)-6-oxo-1,6-dihydro-[3,4-bipyridin]-2-yl)-7,7-dimethyl-3,4,7,8-tetrahydro-2H-cyclopenta[4,5]pyrrolo[1,2-a]pyrazin-1(6H)-one

(121) ##STR00025##

(122) A synthetic method similar to that of Example 1 was used, in which tert-butyl (S)-3-methylpiperazine-1-carboxylate was replaced by tert-butylpiperazine-1-carboxylate to obtain the title product 10 with a yield of 19%.

(123) 1H NMR (400 MHz, DMSO-d6) 8.61 (d, J=2.3 Hz, 1H), 8.48 (d, J=5.0 Hz, 1H), 8.44 (s, 1H), 7.83 (d, J=2.8 Hz, 1H), 7.45 (d, J=2.3 Hz, 1H), 7.40-7.32 (m, 2H), 7.24 (d, J=9.0 Hz, 1H), 4.93 (t, J=5.1 Hz, 1H), 4.52 (t, J=6.5 Hz, 2H), 4.50-4.33 (m, 4H), 4.31-4.12 (m, 3H), 3.89-3.79 (m, 1H), 3.60 (s, 3H), 3.53-3.40 (m, 1H), 3.08 (t, J=5.0 Hz, 4H), 2.57 (q, J=8.9 Hz, 2H), 2.42-2.35 (m, 6H), 1.22 (s, 6H).

(124) LCMS (ESI-MS) m/z: 668.8 (M+H).sup.+.

Example 11 2-(5-((5-(2-oxa-7-azaspiro[3.5]nonan-7-yl)pyridin-2-yl) amino)-3-(hydroxymethyl)-1-methyl-6-oxo-1,6-dihydro-[3,4-bipyridin]-2-yl)-9-fluoro-7,7-dimethyl-3,4,7,8-tetrahydro-2H-cyclopenta[4,5]pyrrolo[1,2-a]pyrazin-1(6H)-one

(125) ##STR00026##

(126) A synthetic method similar to that of Example 1 was used, in which tert-butyl (S)-3-methylpiperazine-1-carboxylate was replaced by 2-oxa-7-azaspiro[3.5]nonane to obtain the title product 11 with a yield of 26%.

(127) 1H NMR (400 MHz, DMSO-d6) 8.61 (d, J=2.3 Hz, 1H), 8.48 (d, J=5.0 Hz, 1H), 8.44 (s, 1H), 7.83 (d, J=2.8 Hz, 1H), 7.45 (d, J=2.3 Hz, 1H), 7.40-7.32 (m, 2H), 7.24 (d, J=9.0 Hz, 1H), 4.93 (t, J=5.1 Hz, 1H), 4.52 (t, J=6.5 Hz, 2H), 4.50-4.33 (m, 4H), 4.31-4.12 (m, 3H), 3.89-3.79 (m, 1H), 3.60 (s, 3H), 3.08 (t, J=5.0 Hz, 4H), 2.57 (q, J=8.9 Hz, 2H), 2.42-2.35 (m, 2H), 1.65-1.43 (m, 4H), 1.22 (s, 6H).

(128) LCMS (ESI-MS) m/z: 653.8 (M+H).sup.+.

Example 12 9-fluoro-2-(3-(hydroxymethyl)-1-methyl-5-((5-(2-methyl-2,7-diazaspiro[3.5]nonan-7-yl)pyridin-2-yl)amino)-6-oxo-1,6-dihydro-[3,4-bipyridin]-2-yl)-7,7-dimethyl-3,4,7,8-tetrahydro-2H-cyclopenta[4,5]pyrrolo[1,2-a]pyrazin-1(6H)-one

(129) ##STR00027##

(130) A synthetic method similar to that of Example 1 was used, in which tert-butyl (S)-3-methylpiperazine-1-carboxylate was replaced by 2-methyl-2,7-diazaspiro[3.5]nonane to obtain the title product 12 with a yield of 29%.

(131) 1H NMR (400 MHz, DMSO-d.sub.6) 8.61 (d, J=2.3 Hz, 1H), 8.48 (d, J=5.0 Hz, 1H), 8.44 (s, 1H), 7.83 (d, J=2.8 Hz, 1H), 7.45 (d, J=2.3 Hz, 1H), 7.40-7.32 (m, 2H), 7.24 (d, J=9.0 Hz, 1H), 4.93 (t, J=5.1 Hz, 1H), 4.52 (t, J=6.5 Hz, 2H), 4.31-4.12 (m, 3H), 3.89-3.79 (m, 1H), 3.60 (s, 3H), 3.08 (t, J=5.0 Hz, 4H), 2.57 (q, J=8.9 Hz, 2H), 2.42-2.35 (m, 6H), 2.21 (s, 3H), 1.65-1.43 (m, 4H), 1.22 (s, 6H).

(132) LCMS (ESI-MS) m/z: 666.8 (M+H).sup.+.

Example 13 9-fluoro-2-(5-((5-(2-hydroxy-7-azaspiro[3.5]nonan-7-yl)pyridin-2-yl)amino)-3-(hydroxymethyl)-1-methyl-6-oxo-1,6-dihydro-[3,4-bipyridin]-2-yl)-7,7-dimethyl-3,4,7,8-tetrahydro-2H-cyclopenta[4,5]pyrrolo[1,2-a]pyrazin-1(6H)-one

(133) ##STR00028##

(134) A synthetic method similar to that of Example 1 was used, in which tert-butyl (S)-3-methylpiperazine-1-carboxylate was replaced by 2-methyl-2,7-diazaspiro[3.5]nonane to obtain the title product 13 with a yield of 17%.

(135) 1H NMR (400 MHz, DMSO-d6) 1H NMR (400 MHz, DMSO-d6) 8.61 (d, J=2.3 Hz, 1H), 8.48 (d, J=5.0 Hz, 1H), 8.44 (s, 1H), 7.83 (d, J=2.8 Hz, 1H), 7.45 (d, J=2.3 Hz, 1H), 7.40-7.32 (m, 2H), 7.24 (d, J=9.0 Hz, 1H), 4.94 (t, J=5.1 Hz, 1H), 4.55 (td, J=6.5, 2.9 Hz, 2H), 4.29-4.19 (m, 1H), 4.25-4.12 (m, 3H), 3.82 (d, J=11.1 Hz, 1H), 3.67 (d, J=5.5 Hz, 1H), 3.60 (s, 3H), 3.40 (t, J=6.2 Hz, 4H), 2.45 (s, 2H), 2.36-2.28 (m, 2H), 2.09-1.75 (m, 8H), 1.22 (s, 6H).

(136) LCMS (ESI-MS) m/z: 667.8 (M+H).sup.+.

Example 14 9-fluoro-2-(3-(hydroxymethyl)-1-methyl-5-((5-(4-methylpiperazin-1-yl)pyridin-2-yl)amino)-6-oxo-1,6-dihydro-[3,4-bipyridin]-2-yl)-7,7-dimethyl-3,4,7,8-tetrahydro-2H-cyclopenta[4,5]pyrrolo[1,2-a]pyrazin-1(6H)-one

(137) ##STR00029##

(138) A synthetic method similar to that of Example 1 was used, in which tert-butyl (S)-3-methylpiperazine-1-carboxylate was replaced by 1-methylpiperazine to obtain the title product 14 with a yield of 21%.

(139) .sup.1H NMR (400 MHz, DMSO-d.sub.6) 8.61 (d, J=2.3 Hz, 1H), 8.48 (d, J=5.0 Hz, 1H), 8.44 (s, 1H), 7.83 (d, J=2.8 Hz, 1H), 7.45 (d, J=2.3 Hz, 1H), 7.40-7.32 (m, 2H), 7.24 (d, J=9.0 Hz, 1H), 4.94 (t, J=5.1 Hz, 1H), 4.39 (d, J=5.1 Hz, 2H), 3.58 (s, 3H), 3.08 (t, 4H), 2.41-2.30 (m, 11H), 2.39-2.19 (m, 4H), 1.22 (s, 6H).

(140) LCMS (ESI-MS) m/z: 626.7 (M+H).sup.+.

Example 15 9-fluoro-2-(3-(hydroxymethyl)-1-methyl-5-((4-(4-(oxetan-3-yl) piperazin-1-yl)phenyl)amino)-6-oxo-1,6-dihydro-[3,4-bipyridin]-2-yl)-7,7-dimethyl-3,4,7,8-tetrahydro-2H-cyclopenta[4,5]pyrrolo[1,2-a]pyrazin-1(6H)-one

(141) ##STR00030##

(142) A synthetic method similar to that of Example 1 was used, in which tert-butyl (S)-3-methylpiperazine-1-carboxylate was replaced by tert-butylpiperazine-1-carboxylate and 5-chloro-2-nitropyridine was replaced by 1-bromo-4-chlorobenzene to obtain the title product 15 with a yield of 19%.

(143) .sup.1H NMR (400 MHz, DMSO-d.sub.6) 8.61 (d, J=2.3 Hz, 1H), 8.48 (d, J=5.0 Hz, 1H), 8.44 (s, 1H), 7.83 (d, J=2.8 Hz, 1H), 7.45 (d, J=2.3 Hz, 1H), 7.26 (d, J=9.0 Hz, 2H), 7.04 (d, J=9.0 Hz, 2H), 4.93 (t, J=5.1 Hz, 1H), 4.52 (t, J=6.5 Hz, 2H), 4.50-4.33 (m, 4H), 4.31-4.12 (m, 3H), 3.89-3.79 (m, 1H), 3.60 (s, 3H), 3.53-3.40 (m, 1H), 3.08 (t, J=5.0 Hz, 4H), 2.57 (q, J=8.9 Hz, 2H), 2.42-2.35 (m, 6H), 1.22 (s, 6H).

(144) LCMS (ESI-MS) m/z: 667.8 (M+H).sup.+.

Example 16 9-fluoro-2-(3-(hydroxymethyl)-1-methyl-5-((6-(4-(oxetan-3-yl) piperazin-1-yl)pyridin-3-yl)amino)-6-oxo-1,6-dihydro-[3,4-bipyridin]-2-yl)-7,7-dimethyl-3,4,7,8-tetrahydro-2H-cyclopenta[4,5]pyrrolo[1,2-a]pyrazin-1(6H)-one

(145) ##STR00031##

(146) A synthetic method similar to that of Example 1 was used, in which tert-butyl (S)-3-methylpiperazine-1-carboxylate was replaced by tert-butylpiperazine-1-carboxylate and 5-chloro-2-nitropyridine was replaced by 2-bromo-5-chloropyridine to obtain the title product 16 in a yield of 23%.

(147) 1H NMR (400 MHz, DMSO-d6) 8.60 (d, J=2.3 Hz, 1H), 8.47 (d, J=5.0 Hz, 1H), 8.41 (s, 1H), 7.82 (d, J=2.9 Hz, 1H), 7.44 (d, J=2.3 Hz, 1H), 7.37-7.30 (m, 2H), 7.21 (d, J=9.0 Hz, 1H), 4.93 (t, J=5.1 Hz, 1H), 4.52 (t, J=6.5 Hz, 2H), 4.50-4.33 (m, 4H), 4.31-4.12 (m, 3H), 3.89-3.79 (m, 1H), 3.60 (s, 3H), 3.53-3.40 (m, 1H), 3.08 (t, J=5.0 Hz, 4H), 2.57 (q, J=8.9 Hz, 2H), 2.42-2.35 (m, 6H), 1.22 (s, 6H).

(148) LCMS (ESI-MS) m/z: 668.8 (M+H).sup.+.

Example 17 (S)-9-bromo-2-(3-(hydroxymethyl)-1-methyl-5-((5-(2-methyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-3-yl)amino)-6-oxo-1,6-dihydro-[3,4-bipyridin]-2-yl)-7,7-dimethyl-3,4,7,8-tetrahydro-2H-cyclopenta[4,5]pyrrolo[1,2-a]pyrazin-1(6H)-one

(149) ##STR00032##

(150) A synthetic method similar to that of Example 1 was used, and the intermediate 1b was synthesized to obtain the title product 17 with a yield of 29%.

(151) 1H NMR (400 MHz, DMSO-d6) 8.59 (d, J=2.3 Hz, 1H), 8.46 (d, J=5.0 Hz, 1H), 8.42 (s, 1H), 7.82 (d, J=2.9 Hz, 1H), 7.43 (d, J=2.3 Hz, 1H), 7.38-7.30 (m, 2H), 7.24-7.20 (m, 1H), 4.93 (t, J=5.1 Hz, 1H), 4.62-4.32 (m, 6H), 4.21 (d, J=10.2 Hz, 3H), 3.82 (d, J=6.4 Hz, 1H), 3.65 (d, J=5.9 Hz, 1H), 3.59 (s, 3H), 3.43-3.33 (m, 1H), 3.08 (d, J=11.5 Hz, 1H), 2.93 (t, J=9.9 Hz, 1H), 2.69-2.50 (m, 3H), 2.37-2.23 (m, 4H), 2.17 (t, J=9.7 Hz, 1H), 1.21 (d, J=2.9 Hz, 6H), 0.91 (d, J=6.3 Hz, 3H). LCMS (ESI-MS) m/z: 743.7 (M+H)+.

Example 18 (S)-2-(3-(hydroxymethyl)-1-methyl-5-((5-(2-methyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-yl)amino)-6-oxo-1,6-dihydro-[3,4-bipyridin]-2-yl)-7,7-dimethyl-1-oxo-1,3,4,6,7,8-hexahydro-2H-cyclopenta[4,5]pyrrolo[1,2-a]pyrazine-9-carbonitrile

(152) ##STR00033## ##STR00034##

Step 1 7,7-dimethyl-1-carbonyl-1,3,4,6,7,8-hexahydro-2H-cyclopenta[4,5]pyrrolo[1,2-a]pyrazine-9-carbonitrile

(153) Compound 1b (0.5 g, 1.77 mmol) was dissolved in toluene (10 mL). Copper(I) cyanide (0.16 g, 1.77 mmol) and tetrakis(triphenylphosphine)-palladium (50 mg) were added. The reaction solution was stirred for 3 h at 100 C. under nitrogen protection. After cooling, the reaction was washed by adding saturated saline and extracted with ethyl acetate (502 mL). The combined organic phases were dried over anhydrous sodium sulfate, filtered and spin dried to obtain a crude product, which was purified by silica gel column chromatography to obtain compound 18a (280 mg) with a yield of 69.1%.

(154) MS m/z (ESI): 230 (M+1).sup.+

(155) This step was followed by a similar reaction as in Example 1 after step 5 to produce the title product 18 with a yield of 19%.

(156) 1H NMR (400 MHz, DMSO-d6) 8.59 (d, J=2.4 Hz, 1H), 8.48 (d, J=5.2 Hz, 1H), 8.44 (s, 1H), 7.81 (d, J=2.8 Hz, 1H), 7.40-7.34 (m, 3H), 7.24-7.22 (m, 1H), 4.99 (s, 1H), 4.55-4.38 (m, 6H), 4.30-4.28 (m, 3H), 3.90 (s, 1H), 3.67-3.65 (m, 1H), 3.59 (s, 3H), 3.38-3.34 (m, 1H), 3.09-3.05 (m, 1H), 2.95-2.90 (m, 1H), 2.66-2.65 (m, 2H), 2.31-2.13 (m, 6H), 1.22 (s, 6H), 0.91 (d, J=6.4 Hz, 3H).

(157) LCMS (ESI-MS) m/z: 689.8 (M+H).sup.+.

Example 19 (S)-2-(3-(hydroxymethyl)-1-methyl-5-((5-(2-methyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-yl)amino)-6-oxo-1,6-dihydro-[3,4-bipyridin]-2-yl)-7,7-dimethyl-9-(trifluoromethyl)-3,4,7,8-hexahydro-2H-cyclopenta[4,5]pyrrolo[1,2-a]pyrazine-1(6H)-one

(158) ##STR00035##

Step 1 7,7-dimethyl-9-(trifluoromethyl)-3,4,7,8-tetrahydro-2H-cyclopenta[4,5]pyrrolo [1,2-a]pyrazin-1(6H)-one

(159) Compound 1a (0.5 g, 2.45 mmol) was dissolved in DMF (10 mL) and diiododifluoromethane (1.48 g, 4.9 mmol) was added. The reaction solution was stirred for 18 h under strong light and nitrogen protection. After cooling, the reaction was washed by adding saturated saline and extracted with ethyl acetate (502 mL). The combined organic phases were dried over anhydrous sodium sulfate, filtered and spin dried to obtain a crude product, which was purified by silica gel column chromatography to obtain compound 19a (180 mg) with a yield of 27%.

(160) MS m/z (ESI): 230 (M+1).sup.+

(161) This step was followed by a similar reaction as in Example 1 after step 5 to produce the title product 19 with a yield of 16%.

(162) .sup.1H NMR (400 MHz, Methanol-d.sub.4) 7.38 (d, J=1.9 Hz, 1H), 7.25 (d, J=1.8 Hz, 1H), 7.07 (d, J=7.6 Hz, 1H), 6.85 (dd, J=7.7, 1.6 Hz, 1H), 6.65 (d, J=1.5 Hz, 1H), 6.09 (s, 1H), 4.47 (s, 2H), 3.90 (d, J=11.0 Hz, 2H), 3.36 (d, J=11.7 Hz, 2H), 3.17-3.01 (m, 6H), 2.81 (s, 3H), 2.76 (d, J=7.2 Hz, 2H), 2.50 (s, 1H), 2.46 (s, 3H), 2.33 (s, 3H), 2.25 (s, 3H), 2.12 (s, 3H), 1.97 (td, J=13.4, 12.8, 4.0 Hz, 2H), 1.81 (d, J=13.9 Hz, 2H), 1.78 (d, J=12.5 Hz, 2H), 1.67-1.53 (m, 3H), 1.28-1.22 (m, 4H), 0.86 (t, J=7.0 Hz, 3H).

(163) LCMS (ESI-MS) m/z: 732.8 (M+H).sup.+.

Example 20 (S)-9-ethoxy-2-(3-(hydroxymethyl)-1-methyl-5-((5-(2-methyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-yl)amino)-6-oxo-1,6-dihydro-[3,4-bipyridyl]-2-yl)-7,7-dimethyl-3,4,7,8-tetrahydro-2H-cyclopenta[4,5]pyrrolo[1,2-a]pyrazin-1(6H)-one

(164) ##STR00036##

Step 1 9-ethoxy-7,7-dimethyl-3,4,7,8-tetrahydro-2H-cyclopenta[4,5]pyrrolo [1,2-a]pyrazin-1(6H)-one

(165) Compound 1b (0.5 g, 1.77 mmol) was dissolved in dichloromethane (10 mL) and sodium ethoxide (0.16 g, 1.77 mmol) was added. The reaction solution was sealed under nitrogen protection and reacted with stirring at 50 C. for 18 h. After cooling, the reaction was washed by adding saturated saline and extracted with ethyl acetate (502 mL). The combined organic phases were dried over anhydrous sodium sulfate, filtered and spin dried to obtain a crude product, which was purified by silica gel column chromatography to obtain compound 20a (210 mg) with a yield of 47.8%.

(166) MS m/z (ESI): 248.3 (M+1).sup.+

(167) This step was followed by a similar reaction as in Example 1 after step 5 to produce the title product 20 with a yield of 30%.

(168) .sup.1H NMR (400 MHz, Methanol-d.sub.4) 7.38 (d, J=1.9 Hz, 1H), 7.25 (d, J=1.8 Hz, 1H), 7.07 (d, J=7.6 Hz, 1H), 6.85 (dd, J=7.7, 1.6 Hz, 1H), 6.65 (d, J=1.5 Hz, 1H), 6.09 (s, 1H), 4.47 (s, 2H), 4.07 (m, 2H), 3.90 (d, J=11.0 Hz, 2H), 3.36 (d, J=11.7 Hz, 2H), 3.19-3.01 (m, 6H), 2.81 (s, 3H), 2.76 (d, J=7.2 Hz, 2H), 2.50 (s, 1H), 2.41 (s, 3H), 2.33 (s, 3H), 2.23 (s, 3H), 2.14 (s, 3H), 1.98 (td, J=13.4, 12.8, 4.0 Hz, 2H), 1.82 (d, J=13.9 Hz, 2H), 1.77 (d, J=12.5 Hz, 2H), 1.69-1.56 (m, 3H), 1.38 (m, 2H), 1.27-1.21 (m, 4H), 0.86 (t, J=7.0 Hz, 3H).

(169) LCMS (ESI-MS) m/z: 708.9 (M+H).sup.+.

Example 21 (S)-2-(3-(hydroxymethyl)-1-methyl-5-((5-(2-methyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-3-yl)amino)-6-oxo-1,6-dihydro-[3,4-bipyridin]-2-yl)-9-methoxy-7,7-dimethyl-3,4,7,8-tetrahydro-2H-cyclopenta[4,5]pyrrolo[1,2-a]pyrazin-1(6H)-one

(170) ##STR00037##

(171) A synthetic method similar to that of Example 20 was used, in which sodium ethoxide was replaced by sodium methanol to obtain the title product 21 with a yield of 35%.

(172) .sup.1H NMR (400 MHz, Methanol-d.sub.4) 7.38 (d, J=1.9 Hz, 1H), 7.25 (d, J=1.8 Hz, 1H), 7.07 (d, J=7.6 Hz, 1H), 6.85 (dd, J=7.7, 1.6 Hz, 1H), 6.65 (d, J=1.5 Hz, 1H), 6.09 (s, 1H), 4.47 (s, 2H), 3.90 (d, J=11.0 Hz, 2H), 3.83 (s, 3H), 3.36 (d, J=11.7 Hz, 2H), 3.19-3.01 (m, 6H), 2.81 (s, 3H), 2.76 (d, J=7.2 Hz, 2H), 2.50 (s, 1H), 2.41 (s, 3H), 2.33 (s, 3H), 2.23 (s, 3H), 2.14 (s, 3H), 1.98 (td, J=13.4, 12.8, 4.0 Hz, 2H), 1.82 (d, J=13.9 Hz, 2H), 1.77 (d, J=12.5 Hz, 2H), 1.69-1.56 (m, 3H), 1.27-1.21 (m, 4H), 0.86 (t, J=7.0 Hz, 3H).

(173) LCMS (ESI-MS) m/z: 694.8 (M+H).sup.+.

Example 22 (S)-2-(3-(hydroxymethyl)-1-methyl-5-((5-(2-methyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-3-yl)amino)-6-oxo-1,6-dihydro-[3,4-bipyridin]-2-yl)-7,7-dimethyl-9-propoxy-3,4,7,8-tetrahydro-2H-cyclopenta[4,5]pyrrolo[1,2-a]pyrazin-1(6H)-one

(174) ##STR00038##

(175) A synthetic method similar to that of Example 20 was used, in which sodium ethoxide was replaced by sodium propanol to obtain the title product 22 with a yield of 26%.

(176) .sup.1H NMR (400 MHz, Methanol-d.sub.4) 7.38 (d, J=1.9 Hz, 1H), 7.25 (d, J=1.8 Hz, 1H), 7.07 (d, J=7.6 Hz, 1H), 6.85 (dd, J=7.7, 1.6 Hz, 1H), 6.65 (d, J=1.5 Hz, 1H), 6.09 (s, 1H), 4.47 (s, 2H), 3.99 (m, 2H), 390 (d, J=11.0 Hz, 2H), 3.36 (d, J=11.7 Hz, 2H), 3.19-3.01 (m, 6H), 2.81 (s, 3H), 2.76 (d, J=7.2 Hz, 2H), 2.50 (s, 1H), 2.41 (s, 3H), 2.33 (s, 3H), 2.23 (s, 3H), 2.14 (s, 3H), 1.98 (td, J=13.4, 12.8, 4.0 Hz, 2H), 1.82 (d, J=13.9 Hz, 2H), 1.77 (d, J=12.5 Hz, 2H), 1.72 (m, 2H), 1.69-1.56 (m, 3H), 1.27-1.21 (m, 4H), 0.99 (m, 3H), 0.86 (t, J=7.0 Hz, 3H).

(177) LCMS (ESI-MS) m/z: 722.9 (M+H).sup.+.

Example 23 (S)-9-cyclopropoxy-2-(3-(hydroxymethyl)-1-methyl-5-((5-(2-methyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-yl)amino)-6-oxo-1,6-dihydro-[3,4-bipyridin]-2-yl)-7,7-dimethyl-3,4,7,8-tetrahydro-2H-cyclopenta[4,5]pyrrolo[1,2-a]pyrazin-1(6H)-one

(178) ##STR00039##

(179) A synthetic method similar to that of Example 20 was used, in which sodium ethoxide was replaced by sodium cyclopropanolate to obtain the title product 23 with a yield of 17%.

(180) .sup.1H NMR (400 MHz, Methanol-d.sub.4) 7.38 (d, J=1.9 Hz, 1H), 7.25 (d, J=1.8 Hz, 1H), 7.07 (d, J=7.6 Hz, 1H), 6.85 (dd, J=7.7, 1.6 Hz, 1H), 6.65 (d, J=1.5 Hz, 1H), 6.09 (s, 1H), 4.47 (s, 2H), 390 (d, J=11.0 Hz, 2H), 3.37 (m, 3H), 3.19-3.01 (m, 6H), 2.81 (s, 3H), 2.76 (d, J=7.2 Hz, 2H), 2.50 (s, 1H), 2.41 (s, 3H), 2.33 (s, 3H), 2.23 (s, 3H), 2.14 (s, 3H), 1.98 (td, J=13.4, 12.8, 4.0 Hz, 2H), 1.82 (d, J=13.9 Hz, 2H), 1.77 (d, J=12.5 Hz, 2H), 1.69-1.56 (m, 3H), 1.27-1.21 (m, 4H), 0.86 (t, J=7.0 Hz, 3H), 0.58 (m, 2H), 0.34 (m, 2H).

(181) LCMS (ESI-MS) m/z: 720.9 (M+H).sup.+.

Example 24 (S)-2-(3-(hydroxymethyl)-1-methyl-5-((5-(2-methyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-yl)amino)-6-oxo-1,6-dihydro-[3,4-bipyridin]-2-yl)-9-isopropoxy-7,7-dimethyl-3,4,7,8-tetrahydro-2H-cyclopenta[4,5]pyrrolo[1,2-a]pyrazin-1(6H)-one

(182) ##STR00040##

(183) A synthetic method similar to that of Example 23 was used, and sodium ethoxide was replaced by sodium isopropylate to obtain the title product 24 with a yield of 25%.

(184) .sup.1H NMR (400 MHz, Methanol-d.sub.4) 7.38 (d, J=1.9 Hz, 1H), 7.25 (d, J=1.8 Hz, 1H), 7.07 (d, J=7.6 Hz, 1H), 6.85 (dd, J=7.7, 1.6 Hz, 1H), 6.65 (d, J=1.5 Hz, 1H), 6.09 (s, 1H), 4.47 (s, 2H), 4.69 (m, 1H), 3.90 (d, J=11.0 Hz, 2H), 3.36 (d, J=11.7 Hz, 2H), 3.19-3.01 (m, 6H), 2.81 (s, 3H), 2.76 (d, J=7.2 Hz, 2H), 2.50 (s, 1H), 2.41 (s, 3H), 2.33 (s, 3H), 2.23 (s, 3H), 2.14 (s, 3H), 1.98 (td, J=13.4, 12.8, 4.0 Hz, 2H), 1.82 (d, J=13.9 Hz, 2H), 1.77 (d, J=12.5 Hz, 2H), 1.69-1.56 (m, 3H), 1.31 (m, 6H), 1.27-1.21 (m, 4H), 0.99 (m, 3H), 0.86 (t, J=7.0 Hz, 3H).

(185) LCMS (ESI-MS) m/z: 722.9 (M+H).sup.+.

Example 25 (S)-9-amino-2-(3-(hydroxymethyl)-1-methyl-5-((5-(2-methyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-yl)amino)-6-oxo-1,6-dihydro-[3,4-bipyridin]-2-yl)-7,7-dimethyl-3,4,7,8-tetrahydro-2H-cyclopenta[4,5]pyrrolo[1,2-a]pyrazin-1(6H)-one

(186) ##STR00041##

Step 1 9-amino-7,7-dimethyl-3,4,7,8-tetrahydro-2H-cyclopenta[4,5]pyrrolo [1,2-a]pyrazin-1(6H)-one

(187) Compound 1b (0.5 g, 1.77 mmol) was dissolved in a solution of ammonia in methanol (10 mL). The reaction solution was stirred at 80 C. for 3 h under nitrogen protection in a sealed tube. After cooling, the reaction was washed by adding saturated saline and extracted with ethyl acetate (502 mL). The combined organic phases were dried over anhydrous sodium sulfate, filtered and spin dried to obtain a crude product, which was purified by silica gel column chromatography to obtain compound 25a (156 mg) with a yield of 40.1%.

(188) MS m/z (ESI): 219 (M+1).sup.+

(189) This step was followed by a similar reaction as in Example 1 after step 5 to produce the title product 25 with a yield of 11%.

(190) .sup.1H NMR (400 MHz, Methanol-d.sub.4) 7.38 (d, J=1.9 Hz, 1H), 7.25 (d, J=1.8 Hz, 1H), 7.07 (d, J=7.6 Hz, 1H), 6.85 (dd, J=7.7, 1.6 Hz, 1H), 6.65 (d, J=1.5 Hz, 1H), 6.09 (s, 1H), 5.82 (s, 2H), 4.47 (s, 2H), 3.90 (d, J=11.0 Hz, 2H), 3.36 (d, J=11.7 Hz, 2H), 3.19-3.01 (m, 6H), 2.81 (s, 3H), 2.76 (d, J=7.2 Hz, 2H), 2.50 (s, 1H), 2.41 (s, 3H), 2.33 (s, 3H), 2.23 (s, 3H), 2.14 (s, 3H), 1.98 (td, J=13.4, 12.8, 4.0 Hz, 2H), 1.82 (d, J=13.9 Hz, 2H), 1.77 (d, J=12.5 Hz, 2H), 1.69-1.56 (m, 3H), 1.28-1.21 (m, 4H), 0.88 (t, J=7.0 Hz, 3H).

(191) LCMS (ESI-MS) m/z: 679.8 (M+H).sup.+.

Example 26 (S)-2-(3-(hydroxymethyl)-1-methyl-5-((5-(2-methyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-yl)amino)-6-oxo-1,6-dihydro-[3,4-bipyridin]-2-yl)-9-methylamino-7,7-dimethyl-3,4,7,8-tetrahydro-2H-cyclopenta[4,5]pyrrolo[1,2-a]pyrazin-1(6H)-one

(192) ##STR00042##

(193) A synthetic method similar to that of Example 25 was used, in which the solution of ammonia in methanol was replaced by methylamine hydrochloride to obtain the title product 26 with a yield of 24.6%.

(194) .sup.1H NMR (400 MHz, Methanol-d.sub.4) 7.38 (d, J=1.9 Hz, 1H), 7.25 (d, J=1.8 Hz, 1H), 7.07 (d, J=7.6 Hz, 1H), 6.85 (dd, J=7.7, 1.6 Hz, 1H), 6.65 (d, J=1.5 Hz, 1H), 6.09 (s, 1H), 4.47 (s, 2H), 3.90 (d, J=11.0 Hz, 2H), 3.36 (d, J=11.7 Hz, 2H), 3.19-3.01 (m, 6H), 2.81 (s, 3H), 2.76 (d, J=7.2 Hz, 2H), 2.72 (s, 2H), 2.50 (s, 1H), 2.41 (s, 3H), 2.33 (s, 3H), 2.23 (s, 3H), 2.14 (s, 3H), 1.98 (td, J=13.4, 12.8, 4.0 Hz, 2H), 1.82 (d, J=13.9 Hz, 2H), 1.77 (d, J=12.5 Hz, 2H), 1.69-1.56 (m, 3H), 1.28-1.22 (m, 4H), 0.88 (t, J=7.0 Hz, 3H).

(195) LCMS (ESI-MS) m/z: 693.9 (M+H).sup.+.

Example 27 (S)-9-bromo-2-(3-(hydroxymethyl)-1-methyl-5-((5-(2-methyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-3-yl)amino)-6-oxo-1,6-dihydro-[3,4-bipyridin]-2-yl)-7,7-dimethyl-3,4,7,8-tetrahydro-2H-cyclopenta[4,5]pyrrolo[1,2-a]pyrazin-1(6H)-one-9-deuterium

(196) ##STR00043##

Step 1 7,7-dimethyl-3,4,7,8-tetrahydro-2H-cyclopenta[4,5]pyrrolo[1,2-a]pyrazin-1(6H)-one-9-deuterium

(197) Compound 1b (0.5 g, 1.77 mmol) was dissolved in tetrahydrofuran. The reaction solution was added with sodium deuterated borohydride under nitrogen protection and stirred for 3 h in an ice bath. The reaction was washed by adding saturated saline and extracted with ethyl acetate (502 mL). The combined organic phases were dried over anhydrous sodium sulfate, filtered and spin dried to obtain a crude product, which was purified by silica gel column chromatography to obtain compound 27a (160 mg) with a yield of 44.2%.

(198) MS m/z (ESI): 206.3 (M+1).sup.+

(199) This step was followed by a similar reaction as in Example 1 after step 5 to produce the title product 26 with a yield of 21%.

(200) 1H NMR (400 MHz, DMSO-d6) 8.64 (d, J=22.6 Hz, 2H), 8.47 (d, J=5.0 Hz, 1H), 7.92 (s, 1H), 7.45 (dd, J=9.7, 2.6 Hz, 2H), 7.36-7.24 (m, 2H), 4.83-4.60 (m, 4H), 4.40 (q, J=11.7 Hz, 3H), 4.23-4.04 (m, 3H), 3.79 (d, J=11.5 Hz, 1H), 3.56 (s, 3H), 3.18-3.05 (m, 3H), 2.54 (d, J=8.8 Hz, 2H), 2.42 (s, 2H), 1.18 (d, J=5.6 Hz, 8H), 0.90 (s, 3H).

(201) MS m/z (ESI): 666.8 [M+H].sup.+.

(202) Biological Experimental Examples:

(203) Experiment 1: Study on the Activity of the Compound Inhibiting BTK Kinase

(204) The BTK kinase inhibitory activity of the compound was determined using a mobility shift method with the reaction system (kinase solution, ATP, kinase substrate2). The compound was co-incubated with the enzyme for 30 minutes, and then the conversion rate was read using the Caliper EZ reader. The compound inhibition rate was determined using the calculation formula and the data was fitted using Graphpad to derive the IC.sub.50 value for the compounds of this application.

(205) Test Result

(206) The compounds of the present application have a significant inhibitory effect on BTK kinase with a 50% inhibition concentration of BTK<0.75 nM, and their selectivity is better than ibrutinib.

(207) TABLE-US-00002 TABLE 2 Study on the activity of the compound inhibiting BTK kinase Compound No. BTK (IC.sub.50, nM) H01 0.23 H02 0.59 H03 0.14 H04 0.26 H05 0.69 H06 0.41 H07 0.38 H08 0.23 H09 0.71 H10 0.21 H11 0.26 H12 0.49 H13 0.18 H14 0.27 H15 0.68 H16 0.17 H17 0.73 H18 0.65 H19 0.52 H20 0.55 H21 0.38 H22 0.74 H23 0.51 H24 0.65 H25 0.44 H26 0.46 Ibrutinib 0.78
Experiment 2. Inhibitory Effect of the Compound on Cell Proliferation
1. Experimental Aims and Methods

(208) This experiment was to determine the effect of the compound on cell proliferation.

(209) To determine the inhibitory effect of the compound on the proliferation of tumour cells, tumour cells (A20 5E6 cells) were cultured to a certain amount and seeded into 96-well plates. Different concentrations of the compound were added to the plates according to the experimental requirements and the inhibitory effect on tumour cells was observed after 1 h, 2 h, 4 h, 8 h and 24 h. The absorbance value was measured after the addition of CCK8 and 2 h incubation to determine the inhibitory effect of the compound on cell proliferation. The IC.sub.50 value of the compound was obtained by data fitted with Graphpad.

(210) 2. Result of the Experiment

(211) The compounds of this application can effectively inhibit the proliferation of tumour cells and are more effective than ibrutinib.

(212) TABLE-US-00003 TABLE 3 Inhibitory effect of the compound on cell proliferation Compound No. IC.sub.50 (M) H01 0.024 H02 0.023 H03 0.061 H04 0.026 H05 0.023 H06 0.031 H07 0.040 H08 0.029 H09 0.031 H10 0.021 H11 0.013 H12 0.013 H13 0.019 H14 0.047 H15 0.023 H16 0.047 Ibrutinib 0.087
Experiment 3. Relieving Effect of the Compound on Rheumatoid Arthritis
1. Aim and Method of the Experiment

(213) The aim of this experiment was to test the effect of the compound on a type 11 collagen-induced arthritis model in rats. The rats were inoculated and molded by intradermal immune injection in root of tail twice on day 0 and day 7, and were clinically scored. Animals that were successfully modelled (clinical score>3 or more) were randomly assigned to each dosing group for therapeutic administration. At the same time, various indicators were tested, including clinical score, toe volume measurement (twice a week), etc.

(214) 2. Result of the Experiment

(215) The compounds of this application can effectively relieve arthritis and reduce toe volume of the animals, and alleviate the progression of arthritis 14 days after administration, which showed superior effect over ibrutinib.

(216) Experiment 4: Effect of the Compound on Lymphoma

(217) 1. Aim and Method of the Experiment

(218) The aim of this experiment was to test the effect of the compound on lymphoma. A20 5E6 cells were cultivated, and inoculated subcutaneously into SCID nude mice. Tumour volumes were measured twice a week. After the tumour species had grown to a certain volume, the successfully molded animals were randomly allocated to each dose administration group for therapeutic administration. At the same time, the changes in tumor size and body weight were observed every day.

(219) 2. Result of the Experiment

(220) The compounds of this application can effectively reduce the size of solid tumours in animals, alleviate tumour progression and improve body weight in animals, which showed better results than ibrutinib.

(221) Experiment 5. Effect of the Compound on Acute Pneumonia

(222) 1. Aim and Method of the Experiment

(223) The aim of this experiment was to test the effect of the compounds on acute pneumonia.

(224) The animals were anesthetized with chloral hydrate, and then administered lipopolysaccharide to obtain chemically induced acute pneumonia model. Animals were divided into control and model groups. Bronchoalveolar lavage fluid and lung tissues were collected at various time periods after lipopolysaccharide inhalation, and the animals were monitored for lung injury and other indicators by a respiratory function monitor. After tissue collection, tissue sections and staining were performed, and cell count and corresponding inflammatory factors were measured in bronchoalveolar lavage fluid.

(225) 2. Result of the Experiment

(226) After administration, the compounds of this application can effectively improve the inflammatory cell infiltration shown in HE staining compared to the control group. Lymphocytes, monocytes and neutrophils in bronchoalveolar lavage fluid were significantly reduced compared to the control group. IL-1p levels in tissue and blood were also significantly reduced compared to the control group. The effect of the compounds of the present application was superior than ibrutinib.

(227) Experiment 6. Effect of the Compound on Systemic Lupus Erythematosus

(228) 1. Aim and Method of the Experiment

(229) The aim of this experiment was to test the pharmacodynamic effects of the test compound on spontaneous systemic lupus erythematosus in MRL/lpr mice.

(230) The serum of MRL/lpr mice was collected after 7 days of adaptation, and the contents of antinuclear antibodies in blood were measured. The mice were grouped for administration. Blood samples were collected at weeks 4, 8, 16 and 20 to determine anti-nuclear antibodies, anti-single stranded deformable DNA antibodies, anti-histone, urea nitrogen, creatinine and urine protein in blood tests. Results of the efficacy of the compound on systemic lupus erythematosus were observed.

(231) 2. Result of the Experiment

(232) The compounds of the present application can effectively reduce the content of relevant indicators in the blood of animals and effectively alleviate the progression of systemic lupus erythematosus in animals.

(233) Experiment 7. Inhibition Effect of the Compound on hERG Potassium Channel Current

(234) 1. Aim and Method of the Experiment

(235) The aim of this experiment was to apply electrophysiological manual membrane clamp to detect the effect of the compound on hERG potassium channel.

(236) The hERG potassium ion channel was overexpressed in HEK293 cells, which was incubated in a 37 C. 5% CO.sub.2 incubator using a medium consisting of DMEM/150% fetal bovine serum/1% penicillin-streptomycin. During the experiment, the cells were transferred to a cell bath embedded in an inverted microscope stage, perfused with extracellular fluid, and stabilized for 5 minutes. The experiment could be started after the cells precipitated. Membrane currents were recorded using a HEKA EPC-10 membrane clamp amplifier and PATCHMASTER acquisition system (HEKA Instruments Inc., D-67466 Lambrcht, Pfalz, Germany).

(237) The experiments were performed in whole-cell recording mode, with current values recorded according to preset electrophysiological stimulation protocol. Perfusion was performed and recorded using the drugs to be tested, in a concentration ranging from low to high. Data were collected using PATCHMASTER V2X60 (HEKA Instruments Inc., D-67466 Lambrecht, Pfalz, Germany) and Origin 8.5 (OriginLab Corporation, Northampton, MA) software was used for analysis and statistics.

(238) 2. Result of the Experiment

(239) The compounds of the present application have hERG IC50>40 M, with a relatively good cardiac safety.

(240) Experiment 8. Pharmacokinetic Result of the Compound

(241) 1. Aim and Method of the Experiment

(242) The aim of this experiment was to test the content of the compound in plasma and tissue.

(243) The content of the compound was measured in plasma and tissues before administration, and 15 mins, 30 mins, 1 hr, 2 hr, 4 hr, 8 hr, 12 hr, 24 hr and 48 hr after administration to SD rats.

(244) 2. Result of the Experiment

(245) T.sub.1/2 on the rats was 3-4 h.

(246) Experiment 9. Result of Acute Toxicity Experiment on the Compound

(247) 1. Aim and Method of the Experiment

(248) The aim of this experiment was to test the toxic effect of the compound on mice.

(249) Mice were administered with different doses of the compounds at a single time and observed for 14 days. Death, poisoning reaction, weight change, diet, appearance, and behavior of the animals were recorded. At the end, the animals were dissected, and organs were taken. Histopathological examination was performed.

(250) 2. Result of the Experiment

(251) The compounds of the present application have an LD50>1000 mg/kg and is safe. Compared to the control mice, no weight or behavioural abnormalities were observed in the administered mice within 14 days from the date of administration, and no significant organ lesions were found on endpoint autopsy. The compounds of the present application did not show significant toxicity. Blood analysis did not reveal any obvious abnormal indicators.

(252) Experiment 10. Pre-Experiment Result of Chronic Toxicity of the Compound

(253) 1. Aim and Method of the Experiment

(254) The aim of this experiment was to test the toxic effect of the compounds on rats after long-term administration.

(255) Rats were administered with the compounds in different high doses for 21 days. The body weight and related physiological signs of the animals were observed every day, and death, poisoning reactions, weight changes, diet, appearance and behavior of the animals were recorded. After 21 days, the animals were dissected. Blood samples were collected for routine blood tests, blood biochemistry and coagulation tests. Organs were taken for histopathological examination.

(256) 2. Result of the Experiment

(257) The compounds of the present application have an LD50>100 mg/kg and is relatively safe. Compared to the animals in the control group, no abnormal weight, behavior and physiological changes were observed in the animals in the drug administration group within 21 days from the date of administration, and no obvious organ lesions were found in the endpoint anatomy. The compounds of the present application did not show obvious toxicity. Blood analysis did not show any abnormality of relevant indicators in animals in the administration group.

(258) The above are only the preferred examples of the present application and are not intended to limit the present application. Any modifications, equivalent substitutions and improvements made within the spirit and principle of the present application should be included in the protection scope of the present application.