COMPOUND AS SHP2 INHIBITOR AND USE THEREOF

Abstract

The present invention falls within the field of medical chemistry and relates to a class of compounds as SHP2 inhibitors and the use thereof. Specifically, the present invention provides a compound represented by formula (I), or an isomer, a pharmaceutically acceptable salt, a solvate, a crystal or a prodrug thereof, a preparation method therefor, a pharmaceutical composition comprising the compounds, and the use of the compounds or the composition for treating a disease mediated by SHP2.

Claims

1. A compound represented by general formula (I), or an isomer, a pharmaceutically acceptable salt, a solvate, a crystal or a prodrug thereof, ##STR00167## wherein, L is absent or selected from —NH—, —S— and —O—; X is selected from CH.sub.2, NH, O and S; Y is selected from CH and N; R.sup.1 and R.sup.2 are independently selected from hydrogen, halogen, hydroxyl, alkyl, haloalkyl, hydroxyalkyl, alkoxy, haloalkoxy, hydroxyalkoxy, nitro, carboxyl, cyano, amino, monoalkylamino, alkylacylamino, alkylacyl, aminoacyl, alkylaminoacyl, bisalkylamino and cycloalkyl; ring Cx is selected from aryl, heteroaryl, cycloalkyl and heterocyclyl, which are optionally substituted with one or more R.sup.3, wherein R.sup.3 is selected from halogen, hydroxy, alkyl, haloalkyl, hydroxyalkyl, alkoxy, haloalkoxy, hydroxyalkoxy, nitro, carboxyl, cyano, amino, monoalkylamino, alkylacylamino, alkylacyl, alkylsulfonyl, aminoacyl, alkylaminoacyl, bisalkylamino, alkenyl, alkynyl, cycloalkyl, heterocyclyl, substituted heterocyclyl, cycloalkylamino, cycloalkylacyl, heterocyclylacyl, substituted heterocyclylacyl, cycloalkylaminoacyl, cycloalkylacylamino, alkylsulfonyl, alkylaminosulfonyl, alkylsulfonamido, cycloalkylsulfonamido, bisalkylphosphoryl and oxo group; and ring Cy is selected from ##STR00168## wherein R.sup.4 is selected from hydrogen, halogen, hydroxy, alkyl, haloalkyl, hydroxyalkyl, alkoxy, haloalkoxy, hydroxyalkoxy, nitro, carboxyl, cyano, amino, monoalkylamino, alkylacylamino, alkylacyl, aminoacyl, alkylaminoacyl, bisalkylamino and cycloalkyl.

2. The compound or an isomer, a pharmaceutically acceptable salt, a solvate, a crystal or a prodrug thereof according to claim 1, wherein Cx is selected from C.sub.6-12 aryl, C.sub.5-12 heteroaryl, C.sub.3-6 cycloalkyl and C.sub.3-6 heterocyclyl, which are substituted with one or more R.sup.2, wherein R.sup.2 is selected from halogen, hydroxy, alkyl, haloalkyl, hydroxyalkyl, alkoxy, haloalkoxy, hydroxyalkoxy, nitro, carboxyl, cyano, amino, monoalkylamino, alkylacylamino, alkylacyl, alkylsulfonyl, aminoacyl, alkylaminoacyl, bisalkylamino, alkenyl, alkynyl, cycloalkyl, heterocyclyl, substituted heterocyclyl, cycloalkylamino, cycloalkylacyl, heterocyclylacyl, substituted heterocyclylacyl, cycloalkylaminoacyl, cycloalkylacylamino, alkylsulfonyl, alkylsulfonamido, bisalkylphosphoryl and oxo group.

3. The compound or an isomer, a pharmaceutically acceptable salt, a solvate, a crystal or a prodrug thereof according to claim 1, wherein R.sup.3 is selected from halogen, hydroxy, C.sub.1-6 alkyl, halogenated C.sub.1-6 alkyl, hydroxy C.sub.1-6 alkyl, C.sub.1-6 alkoxy, halogenated C.sub.1-6 alkoxy, hydroxy C.sub.1-6 alkoxyl, nitro, carboxyl, cyano, amino, mono-C.sub.1-6 alkylamino, C.sub.1-6 alkylacylamino, C.sub.1-6 alkylacyl, C.sub.1-6 alkylsulfonyl, aminoacyl, C.sub.1-6 alkylaminoacyl, bis-C.sub.1-6 alkylamino, C.sub.2-10 alkenyl, C.sub.2-10 alkynyl, C.sub.3-8 cycloalkyl, C.sub.3-8 heterocyclyl, substituted C.sub.3-8 heterocyclyl, C.sub.3-8 cycloalkylamino, C.sub.3-8 cycloalkylacyl, C.sub.3-8 heterocyclylacyl, substituted C.sub.3-8 heterocyclylacyl, C.sub.3-8 cycloalkylaminoacyl, C.sub.3-8 cycloalkylacylamino, C.sub.1-6 alkylsulfonyl, C.sub.1-6 alkylaminosulfonyl, C.sub.1-6 alkylsulfonamido, C.sub.3-8 cycloalkylsulfonamido, bisC.sub.1-6 alkylphosphoryl and oxo group.

4. The compound or an isomer, a pharmaceutically acceptable salt, a solvate, a crystal or a prodrug thereof according to claim 1, wherein general formula (I) has a structure represented by general formula (Ia), ##STR00169## wherein Cx, L, X, Y and R.sup.4 have the definitions set forth in claim 1.

5. The compound or an isomer, a pharmaceutically acceptable salt, a solvate, a crystal or a prodrug thereof according to claim 1, wherein general formula (I) has a structure represented by general formula (Ib), ##STR00170## wherein Cx, L, X, Y and R.sup.4 have the definitions set forth in claim 1.

6. The compound or an isomer, a pharmaceutically acceptable salt, a solvate, a crystal or a prodrug thereof according to claim 1, wherein general formula (I) has a structure represented by general formula (Ic), ##STR00171## wherein Cx, L, X, Y and R.sup.4 have the definitions set forth in claim 1.

7. The compound or an isomer, a pharmaceutically acceptable salt, a solvate, a crystal or a prodrug thereof according to claim 6, wherein Cx is selected from ##STR00172## wherein n is 1, 2 or 3, and R.sup.3 has the definition set forth in claim 1.

8. The compound or an isomer, a pharmaceutically acceptable salt, a solvate, a crystal or a prodrug thereof according to claim 1, wherein the compound is selected from the group consisting of: ##STR00173## ##STR00174## ##STR00175## ##STR00176## ##STR00177## ##STR00178## ##STR00179## ##STR00180## ##STR00181## ##STR00182## ##STR00183## ##STR00184## ##STR00185##

9. A pharmaceutical composition comprising the compound or an isomer, a pharmaceutically acceptable salt, a solvate, a crystal or a prodrug thereof according to claim 1, and a pharmaceutically acceptable carrier.

10. A method for treating a SHP2-mediated disease, comprising administering a therapeutically effective amount of the compound or an isomer, a pharmaceutically acceptable salt, a solvate, a crystal or a prodrug thereof according to claim 1 to a subject in need thereof.

11. A method for treating a SHP2-mediated disease, comprising administering a therapeutically effective amount of the pharmaceutical composition according to claim 9 to a subject in need thereof.

12. The method according to claim 10, wherein the disease is a proliferative disease, a metabolic disease or a hematological disease.

13. A method for treating a SHP2-mediated disease, comprising administering a therapeutically effective amount of the compound or an isomer, a pharmaceutically acceptable salt, a solvate, a crystal or a prodrug thereof according to claim 8 to a subject in need thereof.

14. The method according to claim 13, wherein the disease is a proliferative disease, a metabolic disease or a hematological disease.

Description

DETAILED DESCRIPTION

[0089] Hereinafter, the present disclosure will be further described in detail below in conjunction with examples, but not limited to these examples. Unless otherwise specified, the materials used in the following examples are all commercially available.

EXAMPLES

Example 1: (S)-6-amino-2-(1-amino-1,3-dihydrospiro[indene-2,4′-piperidin]-1′-yl)-3-methyl-5-((2-(trifluoromethyl)pyridin-3-yl)thio)pyrimidin-4(3H)-one

[0090] ##STR00028##

Step 1 Synthesis of tert-butyl 1-oxo-1,3-dihydrospiro[tert-indene-2,4′-piperidine]-1′-carboxylate

[0091] ##STR00029##

[0092] Into a 1 L flask, tert-butyl N,N-bis(2-chloroethyl)carbamate (40 g, 0.165 mol), 1-indanone (21.84 g, 0.165 mol) and anhydrous N,N-dimethylformamide (400 mL) were added. Under the protection of nitrogen gas, sodium hydride (16.52 g, 0.41 mol) was added in batches in an ice bath. The mixture was stirred in the ice bath for 1 hour, then at room temperature for 1 hour, and finally at 60° C. for 8 hours. After completion of the reaction, water (20 mL) was added dropwise into the reaction system in the ice bath to quench the reaction. The solvent was then removed by distillation under reduced pressure. To the residue, ethyl acetate (400 ml) was added. The ethyl acetate phase was washed with water (200 mL) and then saturated brine (200 mL). Then the ethyl acetate phase was dried over anhydrous sodium sulfate, removed of solvent and purified with column chromatography, to obtain 13.87 g of target product. ESI-MS m/z 324.2 (M+Na).sup.+.

Step 2 Synthesis of tert-butyl (R,Z)-1-((tert-butylsulfinyl)imino)-1,3-dihydrospiro[indene-2,4′-piperidine]-1′-carboxylate

[0093] ##STR00030##

[0094] Into a 1 L flask, tert-butyl 1-oxo-1,3-dihydrospiro[tert-indene-2,4′-piperidine]-1′-carboxylate (13.87 g, 46.08 mmol), (R)-(+)-tert-butylsulfinamide (45 g, 369 mmol), tetraethyl titanate (115 ml, 553 mmol) and anhydrous tetrahydrofuran (300 mL) were added. After introducing protective nitrogen gas into the reaction flask, the mixture was stirred at 90° C. for 24 hours. After being cooled to the room temperature, the reaction solution was poured into water (500 mL). A large amount of white solid was precipitated. The insolubles were filtered off, and the filter cake was washed with ethyl acetate (100 mL, three times). The filtrates were combined, and stranded for layer separation, to separate out the organic phase. The organic phase was washed with water (200 mL) and then saturated brine (200 mL). Then the organic phase was dried over anhydrous sodium sulfate, removed of solvent and purified with column chromatography, to obtain 7 g of the title compound. ESI-MS m/z 405.2 [M+H].sup.+.

Step 3 Synthesis of tert-butyl (S)-1-(((R)-tert-butylsulfinyl)amino)-1,3-dihydrospiro[tert-indene-2,4′-piperidine]-1′-carbo xylate

[0095] ##STR00031##

[0096] Into a 500 ml flask, tert-butyl (R,Z)-1-((tert-butylsulfinyl)imino)-1,3-dihydrospiro[indene-2,4′-piperidine]-1′-carboxylate (7 g, 17.3 mmol) and anhydrous tetrahydrofuran (140 mL) were added. After the introduction of protective nitrogen gas, a tetrahydrofuran solution of lithium borohydride (2M, 10.4 mL) was added dropwise under an ice bath. After the addition, the mixture was stirred for 3 hours in an ice bath. After completion of the reaction, water (5 mL) was added into the reaction system under an ice bath to quench the reaction, and tetrahydrofuran was distilled off under high pressure. After that, ethyl acetate (200 mL) was added into the residual liquid. The ethyl acetate phase was washed with water (100 mL, two times) and then saturated brine (100 mL). Then the ethyl acetate phase was dried over anhydrous sodium sulfate, removed of solvent by distillation under high pressure, and purified with column chromatography to obtain 3 g of the title compound. ESI-MS m/z 407.2 [M+H].sup.+.

Step 4 Synthesis of (R)—N—((S)-1,3-dihydrospiro[indene-2,4′-piperidine]-1-yl)-2-methylpropane-2-sulfinamide

[0097] ##STR00032##

[0098] Into a 150 mL flask, tert-butyl (S)-1-(((R)-tert-butylsulfinyl)amino)-1,3-dihydrospiro[tert-indene-2,4′-piperidine]-1′-carboxylate (3 g, 7.39 mmol) and dichloromethane (60 mL) were added. At room temperature, the reaction system was added with trifluoroacetic acid (8 mL, 110.8 mmol) and reacted for 2 hours with stirring. The reaction system was added with a saturated sodium bicarbonate aqueous solution to adjust the pH to a weak alkaline. After the reaction system was layer separated, a dichloromethane phase was separated out, the water phase was extracted with dichloromethane (100 mL), and the dichloromethane phase was combined. The combined dichloromethane phase was dried over anhydrous sodium sulfate and removed of solvent, to obtain 1.42 g of the title compound. ESI-MS m/z 307.2 [M+H].sup.+.

Step 5 Synthesis of (R)—N—((S)-1′-(4-amino-1-methyl-6-oxo-1,6-dihydropyrimidin-2-yl)-1,3-dihydrospiro[indene-2,4′-piperidin]-1-yl)-2-methylpropane-2-sulfinamide

[0099] ##STR00033##

[0100] 6-Amino-3-methylpyrimidine-2,4(1H,3H)-dione (280 mg, 2 mmol), a Carter condensing agent BOP (1.770 g, 4 mmol), 1,8-diazabicycloundec-7-ene (3.040 g, 20 mmol), (R)—N—((S)-1,3-dihydrospiro[indene-2,4′-piperidine]-1-yl)-2-methylpropane-2-sulfinamide (0.610 g, 2 mmol) and anhydrous N, N-dimethylformamide (10.0 mL) were added into a single neck flask, to perform a reaction under the protection of argon gas at room temperature. After completion of the reaction, the mixture was extracted with water and ethyl acetate, removed of solvent, and purified with column chromatography, to obtain 790 mg of the title compound. ESI-MS m/z 430.2 [M+H].sup.+.

Step 6 Synthesis of (R)—N—((S)-1′-(4-amino-5-iodo-1-methyl-6-oxo-1,6-dihydropyrimidin-2-yl)-1,3-dihydrospiro[indene-2,4′-piperidine]-1-yl)-2-methylpropane-2-sulfinamide

[0101] ##STR00034##

[0102] (R)—N—((S)-1′-(4-amino-1-methyl-6-oxo-1,6-dihydropyrimidin-2-yl)-1,3-dihydrospiro[in dene-2,4′-piperidin]-1-yl)-2-methylpropane-2-sulfinamide (130 mg, 0.3 mmol), N-iodosuccinimide (75 mg, 0.33 mmol) and anhydrous N, N-dimethylformamide (1.0 mL) were added a single neck flask, and stirred under the protection of argon gas at room temperature. After completion of the reaction, the solvent was removed, and 240 mg of the title compound was obtained. ESI-MS m/z 556.2 [M+H].sup.+

Step 7 Synthesis of 2-ethylhexyl 2-(3-((2-(trifluoromethyl)pyridin-3-yl)thio)propanoate

[0103] ##STR00035##

[0104] 3-Bromo-2-trifluoromethylpyridine (5.000 g, 22.10 mmol), tris(dibenzylideneacetone)dipalladium (1.012 g, 1.11 mmol), 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene (1.279 g, 2.21 mmol), 2-ethylhexyl 3-mercaptopropionate (5.308 g, 24.31 mmol), N,N-diisopropylethylamine (5.713 g, 44.20 mmol) and anhydrous 1,4-dioxane (50.0 ml) were added a 250 ml single neck flask, replaced with argon three times, and heated up to 110° C. The mixture was reacted overnight until the reaction was completed as detected by LC-MS. The mixture was suction filtrated, removed of solvent by distillation under high pressure, and purified with column chromatography, to obtain 6.269 g of the title compound at a yield of 78.15%. ESI-MS m/z 364.2 [M+H].sup.+.

Step 8 Synthesis of 2-(trifluoromethyl)pyridine-3-thiol

[0105] ##STR00036##

[0106] Into a 100 ml flask, 2-ethylhexyl 2-(3-((2-(trifluoromethyl)pyridin-3-yl)thio)propanoate (3.074 g, 8.47 mmol) and anhydrous tetrahydrofuran (30.0 ml) were added. The mixture was cooled at −78° C. and replaced with nitrogen gas three time, and then added dropwise with a tetrahydrofuran solution of potassium tert-butoxide (1M, 25.41 ml). After the addition, the reaction system was stirred for 1 hour in an ice bath, until the reaction was completed as detected by LC-MS. The mixture was removed of solvent by distillation under high pressure, dissolved into water (30 ml), transferred into a separatory funnel, and added with diethyl ether (30 ml). The mixture was shaken and left to stand for layer separation and liquid separation. The water phase was added with a saturated citric acid solution (10 ml) to adjust pH to a weak acidity, and extracted with ethyl acetate (30 ml) for 3 times. Organic phases were combined, extracted with saturated brine (50 ml), and dried over anhydrous sodium sulfate, removed of solvent by distillation under high pressure, and purified with column chromatography, to obtain 1.500 g of the title compound at a yield of 98.68%. ESI-MS m/z 180.1 [M+H].sup.+.

Step 9 Synthesis of (R)—N—((S)-1′-(4-amino-1-methyl-6-oxo-5-((2-(trifluoromethyl)pyridin-3-yl)thio)-1,6-dihydro pyridin-2-yl)-1,3-dihydrospiro[indene-2,4′-piperidine]-1-yl)-2-methylpropane-2-sulfinamide

[0107] ##STR00037##

[0108] (R)—N—((S)-1′-(4-amino-5-iodo-1-methyl-6-oxo-1,6-dihydropyrimidin-2-yl)-1,3-dihydro spiro[indene-2,4′-piperidine]-1-yl)-2-methylpropane-2-sulfinamide (240 mg, 0.43 mmol), 2-(trifluoromethyl)pyridine-3-thiol (81 mg, 0.45 mmol), cuprous iodide (16 mg, 0.086 mmol), tetra methyldiethylamine (20 mg, 0.172 mmol), potassium phosphate (274 mg, 1.29 mmol) and anhydrous 1,4-dioxane (10 mL) were added into a single neck flask, heated up to 100° C. under an oxygen gas atmosphere, and stirred. After completion of the reaction, the resultant was removed of solvent and purified with column chromatography to obtain 170 mg of the title compound. MS m/z 607.3 [M+H].sup.+.

Step 10 Synthesis of (S)-6-amino-2-(1-amino-1,3-dihydrospiro[indene-2,4′-piperidin]-1′-yl)-3-methyl-5-((2-(trifluoromethyl)pyridin-3-yl)thio)pyrimidin-4(3H)-one

[0109] ##STR00038##

[0110] (R)—N—((S)-1′-(4-amino-1-methyl-6-oxo-5-((2-(trifluoromethyl)pyridin-3-yl)thio)-1,6-di hydropyridin-2-yl)-1,3-dihydrospiro[indene-2,4′-piperidine]-1-yl)-2-methylpropane-2-sulfinamide (170 mg, 0.28 mmol), hydrogen chloride solution in 1,4-dioxane (1 mL) and anhydrous methanol (1 mL) were added into a single neck flask, and stirred under the protection of argon gasroom temperature. After completion of the reaction, the resultant was removed of solvent and added with ethyl acetate (20 mL). The solution was basified to pH 10 with saturated sodium carbonate solution. The organic phase was separated. The aqueous phase was extracted with ethyl acetate and dried over anhydrous sodium sulfate, removed of solvent and purified with column chromatography, to obtain 12 mg of the title compound. ESI-MS m/z 503.2[M+H].sup.+. .sup.1H NMR (400 MHz, CD.sub.3OD) δ 8.25-8.24 (m, 1H), 7.44-7.42 (m, 1H), 7.34-7.27 (m, 2H), 7.11 (m, 3H), 3.88 (s, 1H), 3.56-3.53 (m, 2H), 3.34 (s, 3H), 3.18-3.04 (m, 3H), 2.70-2.67 (m, 1H), 1.93-1.78 (m, 2H), 1.53-1.50 (m, 1H), 1.30-1.34 (m, 1H).

Example 2: (S)-6-amino-2-(5-amino-5,7-dihydrospiro[cyclopentadieno[b]pyridine-6,4′-piperidine]-1′-yl)-3-methyl-5-((2-(trifluoromethyl)pyridin-3-yl)thio)pyrimidin-4(3H)-one

[0111] ##STR00039##

[0112] The title compound was prepared by using the same method as that in Example 1, except that the starting material 1-indanone was replaced with 6,7-dihydro-5H-cyclopentadieno[b]pyridin-5-one. ESI-MS m/z 504.2[M+H].sup.+. .sup.1H NMR (400 MHz, CD.sub.3OD) δ 8.6-8.7 (m, 2H), 8.45-8.40 (m, 1H), 8.10-7.96 (m, 1H), 7.60-7.52 (m, 1H), 7.41-7.38 (m, 1H), 4.5 (s, 1H), 3.72-3.50 (m, 2H), 3.4-3.0 (m, 4H), 3.13-3.01 (m, 3H), 1.95-1.80 (m, 2H), 1.71-1.50 (m, 2H).

Example 3: (S)-6-amino-2-(1-amino-1,3-dihydrospiro[indene-2,4′-piperidine]-1′-yl)-3-isopropyl-5-((2-(trifluoromethyl)pyridin-3-yl)thio)pyrimidin-4(3H)-one

[0113] ##STR00040##

[0114] The title compound was prepared by using the same method as that in Example 1, except that intermediate 6-amino-3-methylpyrimidine-2,4(1H,3H)-dione was replaced with 6-amino-3-isopropylpyrimidine-2,4(1H,3H)-dione. ESI-MS m/z 531.2[M+H].sup.+. .sup.1H NMR (400 MHz, CD.sub.3OD) δ 8.24 (d, 1H), 7.40-7.28 (m, 3H), 7.20-7.10 (m, 3H), 4.49-4.41 (m, 1H), 4.05 (s, 1H), 3.58-3.46 (m, 2H), 3.13 (t, 2H), 3.04 (d, 1H), 2.81 (d, 1H), 1.91-1.79 (m, 2H), 1.56-1.42 (m, 8H).

Example 4: (S)-6-amino-2-(1-amino-1,3-dihydrospiro[indene-2,4′-piperidine]-1′-yl)-3-methyl-5-((6-amino-2-chloropyridin-3-yl)thio)pyrimidin-4(3H)-one

[0115] ##STR00041##

[0116] The title compound was prepared by using the same method as that in Example 1, except that intermediate 3-bromo-2-trifluoromethylpyridine was replaced with 5-bromo-6-chloropyridin-2-amine. ESI-MS m/z 483.8[M+H].sup.+. .sup.1H NMR (400 MHz, CD.sub.3OD) δ 7.33 (s, 1H), 7.18 (s, 1H), 6.97-6.95 (d, J=8.0 Hz, 2H), 6.35-6.33 (d, J=8.0 Hz, 1H), 6.20 (s, 1H), 3.88 (s, 1H), 3.28 (s, 3H), 3.03-2.99 (m, 3H), 2.67-2.60 (m, 1H), 1.99-1.78 (m, 3H), 1.54-1.51 (d, J=8.0 Hz, 1H), 1.24 (s, 2H).

Example 5: (S)-6-amino-2-(1-amino-1,3-dihydrospiro[indene-2,4′-piperidine]-1′-yl)-3-methyl-5-((3-cyano-2-fluorophenyl)thio)pyrimidin-4(3H)-one

[0117] ##STR00042##

[0118] The title compound was prepared by using the same method as that of Example 2, except that 3-bromo-2-trifluoromethylpyridine was replaced with 3-bromo-2-fluorobenzonitrile. ESI-MS m/z 478.2[M+H].sup.+. .sup.1H NMR (400 MHz, CD.sub.3OD) δ 8.31 (s, 1H), 7.68-7.61 (m, 2H), 7.29-7.25 (m, 1H), 7.19-7.10 (m, 1H), 3.92 (s, 1H), 3.56-3.33 (m, 2H), 3.29 (s, 3H), 3.07-3.05 (d, J=8.0 Hz, 2H), 2.75-2.71 (d, J=16.0 Hz, 2H), 1.92-1.83 (m, 3H), 1.58-1.54 (d, J=16.0 Hz, 1H), 1.19-1.15 (d, J=16.0 Hz, 1H).

Example 6: (S)-6-amino-2-(1-amino-1,3-dihydrospiro[indene-2,4′-piperidine]-1′-yl)-3-methyl-5-((2-meth ylpyridin-3-yl)thio)pyrimidin-4(3H)-one

[0119] ##STR00043##

[0120] The title compound was prepared by using the same method as that in Example 1, except that 3-bromo-2-trifluoromethylpyridine in the example was replaced with 3-bromo-2-methylpyridine. ESI-MS m/z 448.9[M+H].sup.+. .sup.1H NMR (400 MHz, CD.sub.3OD) δ 8.13 (s, 1H), 7.36-7.31 (m, 1H), 7.18-7.16 (d, J=8.0 Hz, 3H), 7.09-7.101 (m, 2H), 3.87 (s, 1H), 3.56-3.48 (m, 2H), 3.30 (s, 3H), 3.08-2.94 (m, 3H), 2.63-2.59 (d, J=16.0 Hz, 1H), 2.50 (s, 3H), 1.92-1.76 (m, 2H), 1.55-1.52 (d, J=12.0 Hz, 1H), 1.10 (s, 1H).

Example 7: (S)-6-amino-2-(1-amino-1,3-dihydrospiro[indene-2,4′-piperidine]-1′-yl)-3-methyl-5-((4-(trifluoromethyl)pyrimidin-5-yl)thio)pyrimidin-4(3H)-one

[0121] ##STR00044##

[0122] The title compound was prepared by using the same method as that in Example 1, except that 3-bromo-2-trifluoromethylpyridine in Example 1 was replaced with 5-bromo-4-(trifluoromethyl)pyrimidine. ESI-MS m/z 504.1[M+H].sup.+. .sup.1H NMR (400 MHz, CD.sub.3OD) δ 9.09 (s, 1H), 8.35-8.45 (m, 1H), 7.29-7.38 (m, 1H), 7.20 (m, 3H), 3.94 (s, 1H), 3.45-3.60 (m, 2H), 3.20-3.35 (m, 3H), 3.11-3.02 (m, 3H), 2.68-2.64 (d, J=16.0 Hz, 1H), 1.92-1.77 (m, 2H), 1.55-1.52 (m, J=12.0 Hz, 1H), 1.22-1.19 (d, J=12.0 Hz, 1H).

Example 8: (S)-6-amino-2-(1-amino-1,3-dihydrospiro[indene-2,4′-piperidine]-1′-yl)-3-methyl-5-((2-(trifluoromethyl)pyridin-3-yl)thio)pyrimidin-4(3H)-one

[0123] ##STR00045##

[0124] The title compound was prepared by using the same method as that in Example 1, except that 3-bromo-2-trifluoromethylpyridine was replaced with 3-chloro-4-iodopyridin-2-amine. ESI-MS m/z 484.2[M+H].sup.+. .sup.1H NMR (400 MHz, CD.sub.3OD) δ 7.60-7.65 (m, 1H), 7.35-7.30 (m, 1H), 7.25-7.15 (m, 2H), 6.20-6.10 (m, 1H), 5.80-6.00 (m, 1H), 3.88 (s, 1H), 3.28 (s, 3H), 3.10-3.00 (m, 3H), 2.60-2.65 (m, 1H), 1.85-1.75 (m, 2H), 1.51-1.54 (m, 2H), 1.24-1.20 (m, 2H).

Example 9: (S)-3-((4-amino-2-(1-amino-1,3-dihydrospiro[indene-2,4′-piperidine]-1′-yl)-1-methyl-6-oxo-1,6-dihydropyrimidin-5-yl)thio)-2-chlorobenzonitrile

[0125] ##STR00046##

[0126] The title compound was prepared by using the same method as that in Example 1, except that 3-bromo-2-trifluoromethylpyridine was replaced with 3-bromo-2-chlorobenzonitrile. ESI-MS m/z 493.2[M+H].sup.+. .sup.1H NMR (400 MHz, CD.sub.3OD) δ 7.60-7.65 (m, 1H), 7.41-7.30 (m, 2H), 7.25-7.15 (m, 2H), 7.07-7.00 (m, 2H), 3.85 (s, 1H), 3.30 (s, 3H), 3.10-3.00 (m, 3H), 2.60-2.65 (m, 1H), 1.85-1.75 (m, 2H), 1.50-1.60 (m, 2H), 1.15-1.30 (m, 2H).

Example 10: (S)-3-((4-amino-2-(1-amino-1,3-dihydrospiro[indene-2,4′-piperidin]-1′-yl)-1-methyl-6-oxo-1,6-dihydropyrimidin-5-yl)thio)-N-methylbenzenesulfonamide

[0127] ##STR00047##

[0128] The title compound was prepared by using the same method as that in Example 1, except that 3-bromo-2-trifluoromethylpyridine was replaced with 3-bromo-N-methylbenzenesulfonamide. ESI-MS m/z 527.2[M+H].sup.+. .sup.1H NMR (400 MHz, CD.sub.3OD) δ 7.61-7.10 (m, 5H), 7.15-7.00 (m, 2H), 7.0-6.5 (m, 1H), 3.88 (s, 1H), 3.40 (s, 3H), 3.31 (s, 3H), 3.15-3.00 (m, 3H), 2.55-2.65 (m, 1H), 1.85-1.65 (m, 2H), 1.45-1.56 (m, 2H), 1.20-1.30 (m, 2H).

Example 11: (S)-6-amino-2-(1-amino-1,3-dihydrospiro[indene-2,4′-piperidin]-1′-yl)-5-(((2-amino-5-chloropyridin-4-yl)thio)-3-methylpyrimidin-4(3H)-one

[0129] ##STR00048##

[0130] The title compound was prepared by using the same method as that in Example 1, except that 3-bromo-2-trifluoromethylpyridine was replaced with 4-bromo-5-chloropyridin-2-amine. ESI-MS m/z 484.2[M+H].sup.+. .sup.1H NMR (400 MHz, CD.sub.3OD) δ 7.80-7.70 (m, 1H), 7.40-7.30 (m, 1H), 7.25-7.15 (m, 2H), 6.20-6.10 (m, 1H), 5.85-6.03 (m, 1H), 3.88 (s, 1H), 3.28 (s, 3H), 3.10-3.00 (m, 3H), 2.60-2.65 (m, 1H), 1.85-1.75 (m, 2H), 1.51-1.54 (m, 2H), 1.24-1.20 (m, 2H).

Example 12: Synthesis of (S)-6-amino-2-(1-amino-1,3-dihydrospiro[indene-2,4′-piperidin]-1′-yl)-5-((3-(dimethylphosphoryl)phenyl]thio)-3-methylpyrimidin-4(3H)-one

[0131] ##STR00049##

[0132] The title compound was prepared by using the same method as that in Example 1, except that 3-bromo-2-trifluoromethylpyridine was replaced with (3-bromophenyl)dimethylphosphine oxide. ESI-MS m/z 510.2[M+H].sup.+. .sup.1H NMR (400 MHz, CD.sub.3OD) δ 7.61-7.30 (m, 5H), 7.15-7.00 (m, 3H), 3.88 (s, 1H), 3.40 (s, 3H), 3.31 (s, 3H), 3.15-3.00 (m, 3H), 2.55-2.65 (m, 1H), 2.10-1.85 (m, 2H), 1.45-1.56 (m, 2H), 1.35 (s, 6H), 1.00-0.89 (m, 2H).

Example 13: Synthesis of (S)-6-amino-2-(1-amino-1,3-dihydrospiro[indene-2,4′-piperidine]-1′-yl)-3-methyl-5-((2-(methylsulfonyl)phenyl)thio)pyrimidin-4(3H)-one

[0133] ##STR00050##

[0134] The title compound was prepared by using the same method as that in Example 1, except that 3-bromo-2-trifluoromethylpyridine was replaced with 1-bromo-2-(methylsulfonyl)benzene. ESI-MS m/z 512.2[M+H].sup.+. .sup.1H NMR (400 MHz, CD.sub.3OD) δ 7.65-7.60 (m, 1H), 7.60-7.53 (m, 1H), 7.40-7.30 (m, 3H), 7.10-7.20 (m, 3H), 4.00 (s, 1H), 3.38 (s, 3H), 3.10-3.00 (m, 3H), 2.95 (s, 3H), 2.75-2.65 (m, 1H), 2.10-1.85 (m, 2H), 1.60-1.45 (m, 2H), 1.30-1.10 (m, 2H).

Example 14: (S)-6-amino-2-(1-amino-1,3-dihydrospiro[indene-2,4′-piperidin]-1′-yl)-3-methyl-5-((3-(meth ylsulfonyl) phenyl)thio)pyrimidin-4(3H)-one

[0135] ##STR00051##

[0136] The title compound was prepared by using the same method as that in Example 1, except that 3-bromo-2-trifluoromethylpyridine was replaced with 1-bromo-3-(methylsulfonyl)benzene. ESI-MS m/z 512.2[M+H].sup.+. .sup.1H NMR (400 MHz, CD.sub.3OD) δ 8.00-7.90 (m, 1H), 7.38-7.24 (m, 3H), 7.24-7.12 (m, 4H), 4.00 (s, 1H), 3.38 (s, 3H), 3.35 (s, 3H), 3.10-2.95 (m, 3H), 2.70-2.61 (m, 1H), 2.10-1.85 (m, 2H), 1.60-1.45 (m, 2H), 1.30-1.10 (m, 2H).

Example 15: (S)-2-((4-amino-2-(1-amino-1,3-dihydrospiro[indene-2,4′-piperidine]-1′-yl)-1-methyl-6-oxo-1,6-dihydropyrimidin-5-yl)thio)-N-methylbenzenesulfonamide

[0137] ##STR00052##

[0138] The title compound was prepared by using the same method as that in Example 1, except that 3-bromo-2-trifluoromethylpyridine was replaced with 2-bromo-N-methylbenzenesulfonamide. ESI-MS m/z 527.2[M+H].sup.+. .sup.1H NMR (400 MHz, CD.sub.3OD) δ 8.38-8.50 (m, 2H), 8.05-7.85 (m, 1H), 7.31-7.70 (m, 5H), 4.00 (s, 1H), 3.35 (s, 3H), 3.10-2.95 (m, 3H), 2.70-2.61 (m, 1H), 2.52 (s, 3H), 2.10-1.85 (m, 2H), 1.60-1.45 (m, 2H), 1.30-1.10 (m, 2H).

Example 16: (S)-3-((4-amino-2-(5-amino-5,7-dihydrospiro[cyclopenta[b]pyridine-6,4′-piperidine]-1′-yl)-1-methyl-6-oxo-1,6-dihydropyrimidin-5-yl)thio)-2-chlorobenzonitrile

[0139] ##STR00053##

[0140] The title compound was prepared by using the same method as that of Example 2, except that 3-bromo-2-trifluoromethylpyridine was replaced with 3-bromo-2-chlorobenzonitrile. ESI-MS m/z 494.1[M+H].sup.+. .sup.1H NMR (400 MHz, CD.sub.3OD) δ 8.50 (s, 1H), 8.00-7.90 (m, 1H), 7.71-7.65 (m, 1H), 7.41-7.35 (m, 1H), 7.35-7.30 (m, 1H), 7.00-7.10 (m, 1H), 4.45 (s, 1H), 3.71-3.60 (m, 2H), 3.35 (s, 3H), 3.25-2.96 (m, 4H), 2.00-1.82 (m, 2H), 1.62-1.51 (m, 2H).

Example 17: (S)—N-(4-((4-amino-2-(1-amino-1,3-dihydrospiro[indene-2,4′-piperidine]-1′-yl)-1-methyl-6-oxo-1,6-dihydropyrimidin-5-yl)thio)-3-chloropyridin-2-yl)cyclopropanecarboxamide

[0141] ##STR00054##

[0142] The title compound was prepared by using the same method as that in Example 1, except that 3-bromo-2-trifluoromethylpyridine was replaced with N-(4-bromo-3-chloropyridin-2-yl) cyclopropanecarboxamide. ESI-MS m/z 552.2[M+H].sup.+. .sup.1H NMR (400 MHz, d6-DMSO) δ 10.45-10.28 (s, 1H), 8.16-8.03 (m, 1H), 7.41-7.76 (m, 2H), 7.41-7.12 (m, 4H), 6.97-6.71 (m, 2H), 6.71-6.65 (m, 1H), 3.95-3.84 (s, 1H), 3.52 (s, 3H), 3.08-2.93 (m, 4H), 2.65-2.51 (m, 3H), 2.23-2.09 (m, 2H), 1.98-1.73 (m, 3H), 1.61-1.46 (m, 1H), 1.28-1.08 (m, 2H).

Example 18: (S)-6-amino-2-(1-amino-1,3-dihydrospiro[indene-2,4′-piperidine]-1′-yl)-5-((2-(difluoromethyl)pyridine-4-yl)thio)-3-methylpyrimidin-4(3H)-one

[0143] ##STR00055##

[0144] The title compound was prepared by using the same method as that in Example 1, except that 3-bromo-2-trifluoromethylpyridine was replaced with 4-bromo-2-(difluoromethyl)pyridine. ESI-MS m/z 484.9 [M+H].sup.+. .sup.1H NMR (400 MHz, d6-DMSO) δ 8.42 (dd, J=16.6, 1H), 7.32 (m, 2H), 7.17 (m, 4H), 6.65-7.04 (m, 3H), 3.91-3.81 (s, 1H), 3.54 (dd, J=29.3, 14.1 Hz, 2H), 3.31 (s, 3H), 2.96-3.16 (m, 2H), 2.66-2.57 (m, 1H), 1.96-1.85 (m, 1H), 1.85-1.74 (m, 1H), 1.55 (d, J=12.8 Hz, 1H), 1.24 (m, 1H), 1.15 (d, J=12.8 Hz, 1H).

[0145] According to the synthesis method of Example 1 of the present disclosure, the compound of examples 19-41 was prepared by using different commercially available raw materials. The characterization parameters of theses compounds are set forth in Table 1.

TABLE-US-00001 TABLE 1 ESI-MS Ex- m/z ample Structure of compound [M + H].sup.+ 19 [00056] 492.2 20 [00057] 525.2 21 [00058] 493.2 22 [00059] 545.1 23 [00060] 506.2 24 [00061] 520.2 25 [00062] 533.2 26 [00063] 534.2 27 [00064] 499.1 28 [00065] 509.2 29 [00066] 551.2 30 [00067] 535.3 31 [00068] 526.1 32 [00069] 489.2 33 [00070] 509.2 34 [00071] 509.2 35 [00072] 534.2 36 [00073] 475.3 37 [00074] 491.2 38 [00075] 616.3 39 [00076] 602.2 40 [00077] 616.3 41 [00078] 485.2 42 [00079] 562.1 43 [00080] 576.1 44 [00081] 588.1

Example 45: (R)-1′-(8-(((2-amino-3-chloropyridin-4-yl)thio)imidazo[1,2-c]pyrimidin-5-yl)-3H-spiro[1-benzofuran-2,4-piperidine]-3-amine

[0146] ##STR00082##

Step 1: Synthesis of 2-(2-fluorophenyl)-1,3-dithiane

[0147] ##STR00083##

[0148] In a 1000 mL three-necked flask, 2-fluorobenzaldehyde (20.0 g, 161.15 mmol) was dissolved in dichloromethane (340 mL). At room temperature and under protection of nitrogen gas, the mixture was added with propanethiol (16 mL, 159.54 mmol) and iodine (1.2 g, 4.83 mmol) to perform a reaction at room temperature for 18 h. After completion of the reaction, the reaction solution was poured into sodium thiosulfate aqueous solution (180 mL, 0.4 M), and then poured into sodium hydroxide aqueous solution (150 mL, 40%). The mixture was stirred for 10 min, extracted with dichloromethane (500M1) for three times, washed with saturated brine once, dried over anhydrous sodium sulfate, concentrated and then separated with column chromatography, to obtain 19.3 g of a white solid with a yield of 56%. ESI-MS m/z: 215.1[M+H].sup.+.

Step 2: Synthesis of tert-butyl 4-(2-(2-fluorophenyl)-1,3-dithio-2-yl)-4-hydroxypiperidine-1-carboxylate

[0149] ##STR00084##

[0150] In a 1 L three-neck flask, 2-(2-fluorophenyl)-1,3-dithiane (19.3 g, 90.25 mmol) was dissolved in anhydrous tetrahydrofuran (250 mL). Under protection of nitrogen gas, the reaction system was cooled to −20° C., and slowly added dropwise with lithium diisopropylamide (45 mL, 90.25 mmol). After the addition, the reaction was continued for 30 min. Then, the reaction system was cooled to −78° C. Tert-butyl 4-oxopiperidine-1-carboxylate dissolved in 180 mL of anhydrous tetrahydrofuran was added dropwise into the reaction system. After completion of the reaction, saturated ammonium chloride aqueous solution was added to quench the reaction. The resultant was extracted with ethyl acetate (300 mL) for 3 times. Organic phases were combined, washed with saturated brine once. The resulting organic phase was dried, concentrated and then separated by column chromatography, to obtain a white-like solid 27.5 g with a yield of 74%, ESI-MS m/z: 414.0 [M+H].sup.+.

Step 3: Synthesis of tert-butyl 4-(2-fluorobenzoyl)-4-hydroxypiperidine-1-carboxylate

[0151] ##STR00085##

[0152] In a 100 mL single neck flask, tert-butyl 4-(2-(2-fluorophenyl)-1,3-dithio-2-yl)-4-hydroxypiperidine-1-carboxylate (3.9 g, 9.43 mmol) were dissolved in dichloromethane (60 mL), and then pyridine (1.1 mL, 14.05 mmol), water (10 mL), pyridinium tribromide (4.5 g, 1.49 mmol) and tetrabutylammonium bromide (0.3 g, 0.94 mmol) were added. The mixture was stirred at room temperature for 24 h. After completion of the reaction, the reaction solution was poured into 250 mL of water, stirred for 10 min at room temperature, extracted with dichloromethane (200 mL) for 3 times. The organic phase was backwashed once with saturated sodium chloride aqueous solution. The resulting organic phase was dried, concentrated and then separated by column chromatography, to obtain 2.91 g of a yellow crude product with a yield of 90%. ESI-MS m/z: 324.2 [M+H].sup.+.

Step 4: Synthesis of tert-butyl 3-oxo-3H-spiro[benzofuran-2,4′-piperidine]-1′-carboxylate

[0153] ##STR00086##

[0154] In a 500 ml single neck flask, tert-butyl 4-(2-fluorobenzoyl)-4-hydroxypiperidine-1-carboxylate (20.4 g, 63.08 mmol) was dissolved in anhydrous tetrahydrofuran (200 mL), and potassium tert-butoxide (10.6 g, 94.7 mmol) was added. Under protection of nitrogen gas, the mixture was heated up to 70° C. to perform a reaction for 2 h. After completion of the reaction, the reaction solution was poured into 800 mL of water, stirred for 10 min at room temperature, and extracted with ethyl acetate (500 mL) for 3 times. The resulting organic phase was washed with saturated brine once, dried over anhydrous sodium sulfate, concentrated, and separated by column chromatography, to obtain 10.7 g of a white solid with a yield of 70%. ESI-MS m/z: 304.1 [M+H].sup.+.

Step 5: Synthesis of tert-butyl (R,Z)-3-((tert-butylsulfinyl)imino)-3H-spiro[benzofuran-2,4′-piperidine]-1′-carboxylate

[0155] ##STR00087##

[0156] In a 500 mL single neck flask, tert-butyl 3-oxo-3H-spiro[benzofuran-2,4′-piperidine]-1′-carboxylate (10.7 g, 35.27 mmol) was dissolved in anhydrous 2-methyltetrahydrofuran (100 mL), and R-tert-butylsulfinamide (12.82 g, 105.8 mmol) and titanium tetraethoxide (37 mL, 176.35 mmol) were added. Under protection of nitrogen gas, the mixture was heated up to 90° C. to perform a reaction overnight. After completion of the reaction, the reaction solution was poured into water (800 mL) and then poured into ethyl acetate (800 mL). The mixture was stirred at room temperature for 15 min, and filtrated with diatomite. The filtrate was washed with saturated sodium chloride aqueous solution once, dried, concentrated, and separated with column chromatography, to obtain 9.17 g of title product with a yield of 97%. ESI-MS m/z: 407.1 [M+H].sup.+.

Step 6: Synthesis of tert-butyl (R)-3-(((R)-(tert-butylsulfinyl)amino]-3H-spiro[benzofuran-2,4′-piperidine]-1′-carboxylate

[0157] ##STR00088##

[0158] In a 500 ml three neck flask, tert-butyl (R,Z)-3-((tert-butylsulfinyl)imino)-3H-spiro[benzofuran-2,4′-piperidine]-1′-carboxylate (9.17 g, 22.56 mmol) was dissolved in anhydrous tetrahydrofuran (100 mL). The mixture was cooled to 0° C., and added with lithium borohydride (2.0M, 18 mL, 35.43 mmol), to perform a reaction at 0° C. for 1 h. After completion of the reaction, the reaction solution was added slowly with saturated ammonium chloride aqueous solution. The mixture was extracted with 300 mL of ethyl acetate for 3 times, washed with saturated sodium chloride aqueous solution once, dried, and concentrated, and separated by column chromatography, to obtain 6.1 g of title product with a total yield of 70%. ESI-MS m/z: 409.1 [M+H].sup.+.

Step 7: Synthesis of (R)-2-methyl-N—((R)-3H-spiro[benzofuran-2,4′-piperidine]-3-yl)propane-2-sulfinamide

[0159] ##STR00089##

[0160] In a 100 mL single neck flask, tert-butyl (R)-3-(((R)-(tert-butylsulfinyl)amino]-3H-spiro[benzofuran-2,4′-piperidine]-1′-carboxylate (0.25 g, 0.61 mmol) was dissolved in anhydrous dichloromethane (10 mL), and trifluoroacetic acid (0.5 mL) was added. to perform a reaction at room temperature for 1 h. After completion of the reaction, saturated sodium bicarbonate solution was added until pH was 7-8. The resultant was extracted with 60 mL of ethyl acetate. The organic phase was washed with saturated brine, and concentrated to obtain the title product. ESI-MS m/z: 309.1 [M+H].sup.+.

Step 8: Synthesis of 5-bromo-2-chloro-N-(2,2-dimethoxyethyl)pyrimidin-4-amine

[0161] ##STR00090##

[0162] 5-Bromo-2,4-dichloropyrimidine (15 g, 66 mmol) and 150 mL of ethanol were added into a 250 mL three neck flask and cooled to 0° C. Under the protection of argon gas. The mixture was added with 2,2-dimethoxyethane-1-amine (13.94 g, 132.8 mmol), and stirred for 10 min, and added with triethylamine (13.4 g, 132.8 mmol) to perform a reaction overnight at room temperature. After completion of the reaction, the resultant was concentrated to remove ethanol, and added with 300 mL of ethyl acetate. The mixture was washed with water twice, washed with saturated sodium chloride solution, concentrated and dried, to obtain a white solid 20.1 g with a yield of 64%. ESI-MS m/z: 296.2 [M+H].sup.+.

Step 9: Preparation of 8-bromoimidazo[1,2-c]pyrimidin-5-ol

[0163] ##STR00091##

[0164] 5-Bromo-2-chloro-N-(2,2-dimethoxyethyl) pyrimidin-4-amine (5 g, 16.86 mmol) was added into a 250 ml single neck flask, and concentrated sulfuric acid (40 ml) was slowly added under an ice bath. After the addition, the mixture was reacted at 65° C. for 2 h. After the reaction was completed as detected by LC-MS and TLC, the reaction solution was poured into ice water, adjusted to a pH of about 6 with saturated sodium bicarbonate solution, and extracted with dichloromethane/ethanol (10:1) 500 mL for 3 times. The organic phase was washed with saturated brine once, dried over anhydrous sodium sulfate, and concentrated to obtain 1.5 g of a yellow solid product with a yield of 41%. MS (ESI) m/z: 214.2/216.2[M+H].sup.+.

Step 10: Preparation of 8-bromo-5-chloroimidazo[1,2-c]pyrimidine

[0165] ##STR00092##

[0166] 8-Bromoimidazo[1,2-c]pyrimidin-5-ol (750 mg, 3.50 mmol) was added into a 100 ml single neck flask, and then phosphorus oxychloride (13 ml, 140 mmol) and N,N-diisopropylethylamine (0.5 ml) were added. The mixture was reacted at 110° C. for 3 h under protection of nitrogen gas. After completion of the reaction, the reaction solution was concentrated, adjusted to a pH of about 6 with saturated sodium bicarbonate solution, and extracted with ethyl acetate for 3 times. Organic phases were combined, washed with saturated brine once, dried over anhydrous sodium sulfate and concentrated to obtain 0.5 g of a yellow-brown solid product with a yield of 61%, MS (ESI) m/z: 232.2[M+H].sup.+.

Step 11 Preparation of (R)—N—((R)-1′-(8-bromoimidazo[1,2-c]pyrimidin-5-yl)-3H-spiro[benzofuran-2,4′-piperidine]-3-yl))-2-methylpropane-2-sulfinamide

[0167] ##STR00093##

[0168] In a 100 mL single neck flask, 8-bromo-5-chloroimidazo[1,2-c]pyrimidine (150 mg, 0.645 mmol) was dissolved in anhydrous N,N-dimethylformamide (15 ml), and (R)-2-methyl-N—((R)-3H-spiro[benzofuran-2,4′-piperidine]-3-yl)propane-2-sulfinamide (199 mg, 0.645 mmol) and N,N-diisopropylethylamine (0.533 ml, 3.23 mmol) were added. The mixture was reacted at 110° C. for 1 h. After the reaction was completed as detected by LC-MS and TLC, 100 mL of ethyl acetate was added. The water phase was extracted with for 3 times. The organic phase was washed with saturated brine for three times, dried over anhydrous sodium sulfate, concentrated, and subjected to column chromatography, to obtain 50 mg of a product with a yield of 15%, ESI-MS m/z: 504.2/506.2 [M+H].sup.+.

Step 12: 2-ethylhexyl 2-(2-(3-amino-3-chloropyridin-4-yl)thio)propanoate

[0169] ##STR00094##

[0170] 3-Chloro-4-iodopyridin-2-amine (5 g, 19.65 mmol), 2-ethylhexyl 3-mercaptopropionate (5.4 ml, 23.58 mmol), tris(dibenzylideneacetone)dipalladium (1.1 g, 1.18 mmol), 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene (1.1 g, 1.97 mmol), N,N-diisopropylethylamine (6.5 ml, 39.3 mmol) were dissolved in 50 ml of dioxane. The mixture was reacted under protection of nitrogen gas at 110° C. for 2 h. After completion of the reaction, the resultant was filtered with diatomite. The filtrate was concentrated, and purified with a column (ethyl acetate/petroleum ether 30%-50%) to obtain 6.64 g of a white-like solid with a yield of 98%. MS (ESI) m/z: 345.0[M+H].sup.+.

Step 13: Sodium salt of 2-amino-3-chloropyridine-4-thiol

[0171] ##STR00095##

[0172] 2-Ethylhexyl 2-(2-(3-amino-3-chloropyridin-4-yl)thio)propanoate (0.5 g, 1.45 mmol) was dissolved in 10 ml of anhydrous tetrahydrofuran, and added dropwise with sodium ethoxide (20% wt. 0.5 ml, 1.52 mmol) under protection of nitrogen gas at room temperature. After 40 min of reaction, 15 ml of dichloromethane was added to the reaction system. A large amount of precipitation was precipitated. The resultant was ultrasonicated for 5 min and filtered. The filter cake was washed with dichloromethane, and then dried, to obtain 0.26 g of a white-like solid with a yield of 80%. MS (ESI) m/z: 161.1[M+H].sup.+.

Step 14 Preparation of (R)—N—((R)-1′-(8-((2-amino-3-chloropyridin-4-yl)-thio)imidazo[1,2-c]pyrimidin-5-yl)-3H-spiro[benzofuran-2,4′-piperidine]-3-yl)-2-methylpropane-2-sulfinamide

[0173] ##STR00096##

[0174] In a 100 ml single neck flask, (R)—N—((R)-1′-(8-bromoimidazo[1,2-c]pyrimidin-5-yl)-3H-spiro[benzofuran-2,4′-piperidine]-3-yl))-2-methylpropane-2-sulfinamide (50 mg, 0.099 mmol) was dissolved in 1,4-dioxane (10 ml), and then 2-amino-3-chloropyridine-4-thiol (30 mg, 0.198 mmol), 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene (30 mg, 0.059 mmol), tris(dibenzylideneacetone)dipalladium (30 mg, 0.029 mmol) and N,N-diisopropylethylamine (0.5 ml, 0.297 mmol) were added. The mixture was reacted at 100° C. for 1 h. After the reaction was completed as detected by LC-MS and TLC, the reaction solution was concentrated, and purified by column chromatography, to obtain 50 mg of a product with a yield of 86%. ESI-MS m/z: 584.1 [M+H]+.

Step 15 Preparation of (R)-1′-(8-(((2-amino-3-chloropyridin-4-yl)thio)imidazo[1,2-c]pyrimidin-5-yl)-3H-spiro[1-benzofuran-2,4-piperidine]-3-amine

[0175] ##STR00097##

[0176] In a 100 mL reaction flask, (R)—N—((R)-1′-(8-((2-amino-3-chloropyridin-4-yl)-thio)imidazo[1,2-c]pyrimidin-5-yl)-3H-spiro[benzofuran-2,4′-piperidine]-3-yl)-2-methylpropane-2-sulfinamide (50 mg, 0.085 mmol) was dissolved in 1,4-dioxane (15 ml), and then hydrochloric acid/dioxane solution (3 ml) was slowly added dropwise. After the addition, the mixture was reacted for 1 hour. After the reaction was completed as detected by LC-MS and TLC, the reaction solution was concentrated and separated by preparative HPLC, to obtain 9 mg of a white solid product with a yield of 21%. .sup.1H NMR (400 MHz, DMSO) δ 8.05 (s, 1H), 7.92 (s, 1H), 7.64-7.50 (m, 2H), 7.43 (d, J=7.2 Hz, 1H), 7.29-7.13 (m, 2H), 6.94 (t, J=7.3 Hz, 1H), 6.87 (d, J=7.9 Hz, 1H), 6.33 (s, 2H), 5.83 (d, J=5.2 Hz, 1H), 5.33 (s, 1H), 4.33 (s, 1H), 4.05 (d, J=13.3 Hz, 1H), 3.96 (d, J=13.2 Hz, 1H), 3.53 (d, J=12.6 Hz, 2H), 1.40-1.30 (m, 2H), 0.85 (d, J=6.8 Hz, 2H). ESI-MS m/z: 480.2 [M+H].sup.+.

Example 46: (S)-1′-(8-((2-amino-3-chloropyridin-4-yl)thio)imidazo[1,2-c]pyrimidin-5-yl)-5,7-dihydrospiro[cyclopenta[b]pyridine-6,4′-piperidine]-5-amine

[0177] ##STR00098##

Step 1: Preparation of 2,2′-(benzylazadiyl)bis(ethane-1-ol)

[0178] ##STR00099##

[0179] Diethanolamine (10 g, 95 mmol) was dissolved in 150 mL of acetonitrile, and then benzyl bromide (19.5 g, 114 mmol) and potassium carbonate (25.8 g, 190 mmol) were added. The mixture was reacted at 90° C. overnight, until the raw material was reacted completely as detected by TLC. The resultant was concentrated, extracted with water and ethyl acetate, and washed with saturated sodium chloride solution. The organic layer were collected and dried with anhydrous sodium sulfate, and purified by column chromatography, to obtain 15.8 g of light yellow oil with a yield of 85%. LC-MS m/z: 196 [M+H].sup.+.

Step 2: Preparation of N-benzyl-2-bromo-N-(2-bromoethyl)ethan-1-amine

[0180] ##STR00100##

[0181] 2,2′-(benzylazadiyl)bis(ethan-1-ol) (0.8 g, 4.1 mmol) was dissolved in 10 mL of toluene, and then phosphorus tribromide (2.2 g) was added dropwise. The mixture was reacted at 100° C. under reflux overnight. After the reaction was completed as detected by TLC, the resultant was concentrated to remove toluene, adjusted to a pH of 9 with saturated sodium bicarbonate solution, and extracted with ethyl acetate, to obtain an organic phase. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure, to obtain a crude product. The crude product was purified by column to obtain the title product. LC-MS m/z: 320/322 [M+H].sup.+.

Step 3: Preparation of 1′-benzylspiro[cyclopenta[b]pyridine-6,4′-piperidine]-5-(7H)-1-one

[0182] ##STR00101##

[0183] Sodium hydride (1.2 g, 30 mmol) was suspended in 10 mL of anhydrous N,N-dimethylformamide, and then a solution of 6,7-dihydro-5H-cyclopenta[b]pyridin-5-one (2 g, 15 mmol) in 20 mL of N,N-dimethylformamide was added dropwise at 0° C. After the addition, the mixture was raised to room temperature to perform a reaction for 1.5 h. Then, a solution of N-benzyl-2-bromo-N-(2-bromoethyl)ethan-1-amine (4.8 g, 15 mmol) in 20 mL of N,N-dimethylformamide was added dropwise again. After the addition, the mixture was continued to react at room temperature for 1.5 h, until the reaction was completed as detected by TLC. The reaction was quenched by addition of water. The resultant was extracted with dichloromethane, spin-dried, and extracted with water and ethyl acetate. The organic layer were collected, and dried with anhydrous sodium sulfate, and purified by column chromatography, to obtain 1.7 g of a purple-black solid with a yield of 38.5%. LC-MS m/z: 293.2 [M+1].sup.+.

Step 4: Preparation of tert-butyl 5-oxo-5,7-dihydrospiro[cyclopenta[b]pyridine-6,4′-piperidine]-1′-carboxylate

[0184] ##STR00102##

[0185] The intermediate 1′-benzylspiro[cyclopenta[b]pyridine-6,4′-piperidine]-5-(7H)-1-one (1.73 g, 5.9 mmol) was dissolved in 20 mL of 1,2-dichloroethane, and then 1-chloroethyl chloroformate (3.2 mL, 29.6 mmol) was added. The mixture was raised to 60° C. to perform a reaction overnight. The mixture was removed of 1,2-dichloroethane through rotary evaporation and the residue was dissolved in methanol (20 mL). The mixture was raised to 80° C. to perform reaction for 3 h under reflux. The mixture was removed of methanol through rotary evaporation and the residue was dissolved in 20 mL of dichloromethane, and added with N,N-diisopropylethylamine (2.9 mL, 17.5 mmol) and di-tert-butyl dicarbonate (2.7 mL, 11.7 mmol). The mixture was reacted at room temperature for 1 h, until the reaction was completed as detected by TLC. The resultant was extracted with water and dichloromethane. The organic layer was separated out, dried with anhydrous sodium sulfate and spin-dried, and purified by column chromatography, to obtain 0.7 g of light yellow oil with a yield of 39.1%. LC-MS m/z: 303.2 [M+H]+.

Step 5: Preparation of tert-butyl (R,Z)-5-((tert-butylsulfinyl)imino)-5,7-dihydrospiro[cyclopenta[b]pyridine-6,4′-piperidine]-1′-carboxylate

[0186] ##STR00103##

[0187] The intermediate tert-butyl 5-oxo-5,7-dihydrospiro[cyclopenta[b]pyridine-6,4′-piperidine]-1′-carboxylate (0.6 g, 1.98 mmol) was dissolved in 2-methyltetrahydrofuran (10 mL), and then tetraethyl titanate (2.26 g, 9.93 mmol) and R-(+)-tert-butylsulfinamide (0.72 g, 5.96 mmol) were added. The mixture was reflux at 90° C. overnight. After the reaction was completed as detected by TLC, the resultant was extracted with water and ethyl acetate, and filtered with diatomite to remove insolubles. The organic was washed with saline solution. The organic layer were collected and dried with anhydrous sodium sulfate, and purified with column chromatography, to obtain 0.42 g of a light yellow solid with a yield of 52.2%. LC-MS m/z: 405.6 [M+H].sup.+.

Step 6: Preparation of tert-butyl (S)-5-(((R)-(tert-butylsulfinyl)amino)-5,7-dihydrospiro[cyclopenta[b]pyridine-6,4′-piperidine]-1′-carboxylate

[0188] ##STR00104##

[0189] The intermediate tert-butyl (R,Z)-5-((tert-butylsulfinyl)imino)-5,7-dihydrospiro[cyclopenta[b]pyridine-6,4′-piperidine]-1′-carboxylate (0.42 g, 1.03 mmol) was dissolved in 10 mL of anhydrous tetrahydrofuran, and then lithium borohydride solution (0.8 mL, 1.6 mmol) was added dropwise at 0° C. The mixture was continued to react at 0° C. for 0.5 h. After the reaction was completed as detected by TLC, the reaction was quenched through addition of saturated ammonium chloride solution. The resultant was extracted with ethyl acetate, concentrated, and then purified by column chromatography, to obtain 0.15 g of a light yellow solid with a yield of 35.5%. LC-MS m/z: 408.2 [M+H].sup.+.

Step 7: Preparation of (R)—N—((S)-5,7-dihydrospiro[cyclopenta[b]pyridine-6,4′-piperidine]-5-yl)-2-methylpropane-2-sulfinamide

[0190] ##STR00105##

[0191] The intermediate tert-butyl (S)-5-(((R)-(tert-butylsulfinyl)amino)-5,7-dihydrospiro[cyclopenta[b]pyridine-6,4′-piperidine]-1′-carboxylate (150 mg, 0.36 mmol) was dissolved in 5 mL of dichloromethane, and then 1 mL of trifluoroacetic acid was added. The mixture was reacted at room temperature for 0.5 h with stirring. After the reaction was completed as detected by TLC, the resultant was neutralized with saturated sodium bicarbonate, extracted with ethyl acetate, dried over anhydrous sodium sulfate, and spin-dried. The resulting product was used in the next reaction directly. LC-MS m/z: 308.2 [M+H].sup.+

Step 8: Preparation of (R)—N—((S)-1′-(8-bromoimidazo[1,2-c]pyrimidin-5-yl)-5,7-dihydrospiro[cyclopenta[b]pyridine-6,4′-piperidin]-5-yl)-2-methylpropane-2-sulfinamide

[0192] ##STR00106##

[0193] This intermediate was synthesized in the same way as that in Step 11 of Example 45, except that the raw material (S)-2-methyl-N—((R)-3H-spiro[benzofuran-2,4′-piperidine]-3-yl)propane-2-sulfinamide was replaced with (R)—N—((S)-5,7-dihydrospiro[cyclopenta[b]pyridine-6,4′-piperidine]-5-yl)-2-methylpropane-2-sulfinamide. LC-MS m/z: 503.2/505.2 [M+H].sup.+.

Step 9: Preparation of (R)—N—((S)-1′-(8-bromoimidazo[1,2-c]pyrimidin-5-yl)-5,7-dihydrospiro[cyclopenta[b]pyridine-6,4′-piperidine]-5-yl)-2-methylpropane-2-sulfinamide

[0194] ##STR00107##

[0195] The target molecule was synthesized by using (R)—N—((S)-1′-(8-bromoimidazo[1,2-c]pyrimidin-5-yl)-5,7-dihydrospiro[cyclopenta[b]pyridine-6,4′-piperidine]-5-yl)-2-methylpropane-2-sulfinamide and 2-amino-4-bromo-3-chloropyridine as raw materials according to the method of Example 45. LC-MS m/z: 583.2 [M+H].sup.+.

Step 10: Synthesis of (S)-1′-(8-((2-amino-3-chloropyridin-4-yl)thio)imidazo[1,2-c]pyrimidin-5-yl)-5,7-dihydrospiro[cyclopenta[b]pyridine-6,4′-piperidine]-5-amine

[0196] ##STR00108##

[0197] The title compound was synthesized by using (R)—N—((S)-1′-(8-bromoimidazo[1,2-c]pyrimidin-5-yl)-5,7-dihydrospiro[cyclopenta[b]pyridine-6,4′-piperidine]-5-yl)-2-methylpropane-2-sulfinamide as raw materials according to Step 15 of Example 45. .sup.1H NMR (400 MHz, DMSO) δ 8.34 (d, J=3.8 Hz, 1H), 8.03 (s, 1H), 7.84 (s, 1H), 7.70 (d, J=7.4 Hz, 1H), 7.62-7.49 (m, 2H), 7.20 (s, 1H), 6.32 (s, 2H), 5.80 (d, J=5.3 Hz, 1H), 3.99 (s, 3H), 3.14 (d, J=16.3 Hz, 2H), 2.81 (d, J=16.3 Hz, 1H), 2.13-1.82 (m, 3H), 1.65 (d, J=13.0 Hz, 1H), 1.41-1.13 (m, 3H). LC-MS m/z: 479.2[M+H].sup.+.

Example 47: (R)-1′-(8-(((2-amino-3-fluoropyridin-4-yl)thio)imidazo[1,2-c]pyrimidin-5-yl)-3H-spiro[benzofuran-2,4′-piperidin]]-3-amine

[0198] ##STR00109##

[0199] The title compound was prepared according to the method of Example 45, except that 2-amino-4-bromo-3-chloropyridine was replaced with 2-amino-4-bromo-3-fluoropyridine.

[0200] 1H NMR (400 MHz, DMSO) δ 8.19 (s, 1H), 8.03 (s, 1H), 7.90 (s, 1H), 7.60 (s, 1H), 7.43 (dd, J=18.3, 6.2 Hz, 2H), 7.26-7.17 (m, 1H), 6.96-6.80 (m, 2H), 6.70-6.56 (m, 1H), 6.23 (s, 1H), 5.91 (s, 1H), 5.32 (s, 1H), 3.50 (s, 4H), 2.06-1.78 (m, 4H). 1.0 (d, J=6.8 Hz, 1H). ESI-MS m/z: 464.2 [M+H].sup.+.

Example 48: (R)-1′-(8-(2,3-dichlorophenyl)-7-methylimidazo[1,2-c]pyrimidin-5-yl)-3H-spiro[benzofuran-2,4′-piperidine]-3-amine

[0201] ##STR00110##

Step 1: Synthesis of 8-bromo-5-chloro-7-methylimidazo[1,2-c]pyrimidine

[0202] ##STR00111##

[0203] The title compound 8-bromo-5-chloro-7-methylimidazo[1,2-c]pyrimidine was synthesized according to the method in steps 8-10 of Example 45, except that 5-bromo-2,4-dichloropyrimidine was replaced with 5-bromo-2,4-dichloro-6-methylpyrimidine. ESI-MS m/z: 246.2 [M+H].sup.+.

Step 2: Synthesis of (R)—N—((R)-1′-(8-bromo-7-methylimidazo[1,2-c]pyrimidin-5-yl)-3H-spiro[benzofuran-2,4′-piperidine]-3-yl)-2-methylpropane-2-sulfinamide

[0204] ##STR00112##

[0205] The title product was synthesized according to the method in step 11 of Example 45, except that 8-bromo-5-chloroimidazo[1,2-c]pyrimidine was replaced with 8-bromo-5-chloro-7-methylimidazo[1,2-c]pyrimidine. ESI-MS m/z: 518.2/520.2 [M+H].sup.+.

Step 3: Synthesis of (R)—N—((R)-1′-(8-(2,3-dichlorophenyl)-7-methylimidazo[1,2-c]pyrimidin-5-yl)-3H-spiro[benzofuran-2,4′-piperidine]-3-yl)-2-methylpropane-2-sulfinamide

[0206] ##STR00113##

[0207] Into a 100 ml single neck flask, (R)—N—((R)-1′-(8-bromo-7-methylimidazo[1,2-c]pyrimidin-5-yl)-3H-spiro[benzofuran-2,4′-piperidine]-3-yl))-2-methylpropane-2-sulfinamide (233 mg, 0.451 mmol), dichlorobenzeneboronic acid (94 mg, 0.496 mmol), potassium carbonate (174 mg, 1.35 mmol), N,N-dimethylacetylamide (10 ml) and water (0.5 mL) were added, and then tetrakis(triphenylphosphine)palladium (70 mg, 0.06 mmol) was added. The mixture was reacted for at 110° C. 3 h under the protection of argon gas. After completion of the reaction, the mixture was extracted with 30 mL of water and 60 mL of ethyl acetate. Organic phases were combined, washed with water and saturated sodium chloride solution, and concentrated, and purified by column chromatography, to obtain 100 mg of the target product with a yield of 38%. ESI-MS m/z: 584.2 [M+H].sup.+.

Step 4: Synthesis of (R)-1′-(8-(2,3-dichlorophenyl)-7-methylimidazo[1,2-c]pyrimidin-5-yl)-3H-spiro[benzofuran-2,4′-piperidine]-3-amine

[0208] ##STR00114##

[0209] In a 100 mL reaction flask, (R)—N—((R)-1′-(8-(2,3-dichlorophenyl)-7-methylimidazo[1,2-c]pyrimidin-5-yl)-3H-spiro[benzofuran-2,4′-piperidine]-3-yl))-2-methylpropane-2-sulfinamide (100 mg, 0.172 mmol) was dissolved in anhydrous dioxane (8 mL), and added with hydrochloric acid/dioxane solution (4 mL). After completion of the reaction, the resultant was concentrated, to obtain 21 mg of title product with a yield of 25.6%. 1H NMR (400 MHz, DMSO) δ 7.79 (s, 1H), 7.74-7.73 (d, J=7.7 Hz, 1H), 7.57-7.44 (m, 2H), 7.41-7.36 (dd, J=14.5, 7.4 Hz, 2H), 7.17-7.15 (t, J=7.5 Hz, 1H), 6.91-6.87 (t, J=7.3 Hz, 1H), 6.81-6.79 (d, J=7.9 Hz, 1H), 4.18 (s, 1H), 3.89-3.78 (dd, J=29.6, 13.0 Hz, 2H), 3.44 (s, 2H), 2.20 (d, J=13.1 Hz, 1H), 2.14 (s, 3H), 2.08 (s, 1H) 2.02-1.92 (dd, J=25.3, 11.8 Hz, 3H), 1.86-1.83 (d, J=13.6 Hz, 1H). ESI-MS m/z: 480.2 [M+H]+.

Example 49: (R)-1′-(8-(2-chloro-3-methoxyphenyl)-7-methylimidazo[1,2-c]pyrimidin-5-yl)-3H-spiro[benzofuran-2,4′-piperidine]-3-amine

[0210] ##STR00115##

[0211] The title compound was prepared by using the same method as that of Example 48, except that the intermediate 2,3-dichlorobenzeneboronic acid was replaced with 2-chloro-3-methoxyphenylboronic acid. 1H NMR (400 MHz, DMSO) δ 7.76 (s, 1H), 7.45 (s, 1H), 7.43-7.31 (m, 2H), 7.26-7.10 (m, 2H), 6.96 (d, J=7.3 Hz, 1H), 6.88 (t, J=7.2 Hz, 1H), 6.79 (d, J=7.8 Hz, 1H), 4.16 (s, 1H), 3.92 (s, 3H), 3.80 (dd, J=28.6, 12.5 Hz, 2H), 2.15 (d, J=22.1 Hz, 5H), 2.07-1.75 (m, 4H), 1.23 (s, 2H). ESI-MS m/z: 476.2 [M+H]+.

Example 50: (R)-1′-(8-(3-chloro-2-((cyclopropylamino)pyridin-4-yl)imidazo[1,2-c]pyrimidin-5-yl)-3H-spiro[spiro[benzofuran-2,4′-piperidine]-3-amine

[0212] ##STR00116##

Step 1: Preparation of (R)—N—((R)-1′-(8-(3-chloro-2-((cyclopropylamino)pyridin-4-yl)imidazo[1,2-c]pyrimidin-5-yl)-3H-spiro[benzofuran-2,4′-piperidine]-3-yl)-2-methylpropane-2-sulfinamide

[0213] ##STR00117##

[0214] In a 100 ml single neck flask, the product in step 11 of Example 45 (R)—N—((R)-1′-(8-bromoimidazo[1,2-c]pyrimidin-5-yl)-3H-spiro[benzofuran-2,4′-piperidine]-3-yl))-2-methylpropane-2-sulfinamide (200 mg, 0.396 mmol) was dissolved in 1,4-dioxane (20 ml), and then 3-chloro-2-(cyclopropyl-2-azayl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaboran-2-yl)pyridine (128 mg, 0.436 mmol), tetrakis(triphenylphosphine)palladium (23 mg, 0.019 mmol), and potassium carbonate (164 mg, 1.19 mmol) were added. The mixture was reacted at 100° C. overnight. After the reaction was completed as detected by LC-MS and TLC, the resultant was subjected to suction filtration to remove insolubles, and ethyl acetate and water were added. The water phase was extracted for 3 times, and the organic phase was washed with saturated brine for 3 times. The resulting organic phase was dried over anhydrous sodium sulfate, concentrated, and separated by column chromatography, to obtain 90 mg of a product with a yield of 38%. ESI-MS m/z: 592.2 [M+H].sup.+.

Step 2: Preparation of (R)-1′-(8-(3-chloro-2-((cyclopropylamino)pyridin-4-yl)imidazo[1,2-c]pyrimidin-5-yl)-3H-spiro[benzofuran-2,4′-piperidine]-3-amine

[0215] ##STR00118##

[0216] In a 100 ml single neck flask, (R)—N—((R)-1′-(8-(3-chloro-2-((cyclopropylamino)pyridin-4-yl)imidazo[1,2-c]pyrimidin-5-yl)-3H-spiro[benzofuran-2,4′-piperidine]-3-yl)-2-methylpropane-2-sulfinamide (90 mg, 0.152 mmol) was dissolved in 1,4-dioxane (20 ml), and hydrochloric acid/dioxane (2 ml) was slowly added dropwise. After the addition, the mixture was reacted for 1 hour. After the reaction was completed as detected by LC-MS and TLC, the reaction solution was concentrated and separated by preparative HPLC, to obtain 10 mg of a white solid product with a yield of 13%. .sup.1H NMR (400 MHz, DMSO) δ 8.11 (d, J=4.9 Hz, 1H), 7.87 (s, 1H), 7.76 (d, J=9.2 Hz, 1H), 7.62 (s, 1H), 7.44 (d, J=7.1 Hz, 1H), 7.29-7.15 (m, 2H), 6.99-6.90 (m, 1H), 6.87 (d, J=8.0 Hz, 1H), 6.75 (d, J=4.9 Hz, 1H), 6.67 (s, 1H), 4.35 (s, 1H), 3.95 (d, J=12.3 Hz, 1H), 3.86 (d, J=12.7 Hz, 1H), 2.79 (s, 1H), 2.29-2.18 (m, 2H), 2.02 (dd, J=28.9, 11.4 Hz, 3H), 1.24 (s, 2H), 0.72 (d, J=5.0 Hz, 2H), 0.57 (s, 2H). ESI-MS m/z: 488.2 [M+H].sup.+.

Example 51: (R)-6-(3-amino-3H-spiro[benzofuran-2,4′-piperidine]-1′-yl)-3-(3-chloro-2-((cyclopropylamino)pyridin-4-yl)-5-methyl-2,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one

[0217] ##STR00119##

Step 1: 6-hydrazino-3-methylpyrimidine-2,4(1H,3H)-dione

[0218] ##STR00120##

[0219] In a 500 mL three-necked flask, 6-chloro-3-methylpyrimidine-2,4(1H,3H)-dione (10.0 g, 62.3 mmol) was dissolved in ethanol (200 mL), and then hydrazine hydrate (9.17 mL, 187.0 mmol) was added under protection of nitrogen gas at room temperature. The mixture was heated to react at 80° C. for 1 h, and then reacted at room temperature for another 1 h. After the reaction was completed as detected by LC-MS and TLC, the reaction solution was filtered. The filter cake was washed with 100 mL of ethanol, dried to obtain 13.2 g of crude product with a yield of 100%. ESI-MS m/z: 157.1[M+H].sup.+.

Step 2: (E)-6-((4-methoxybenzyl)diazenyl)-3-methylpyrimidine-2,4(1H,3H)-dione

[0220] ##STR00121##

[0221] In a 250 mL three-necked flask, 6-hydrazino-3-methylpyrimidine-2,4(1H,3H)-dione (9.7 g, 62.30 mmol) was dissolved in methanol (50 mL). After replacement by nitrogen gas, 4-methoxybenzaldehyde (9.1 mL, 74.76 mmol) was added. The mixed solution was reacted at room temperature for 30 min, and then the reaction solution was diluted with methanol (45 mL), to continue the reaction at room temperature for 30 min. After the reaction was completed as detected by LC-MS and TLC, the reaction solution was filtered. The filter cake was washed with 100 mL of methanol and dried, to obtain 16.7 g of a light yellow solid with a yield of 98%. ESI-MS m/z: 275.1[M+H].sup.+.

Step 3: 3-(2,3-dichloropyridin-4-yl)-2-(4-methoxybenzyl)-5-methyl-2,7-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4,6(5H)-dione

[0222] ##STR00122##

[0223] In a 100 mL single neck flask, (E)-6-((4-methoxybenzyl)diazenyl)-3-methylpyrimidine-2,4(1H,3H)-dione (0.47 g, 1.71 mmol) was dissolved in N,N-dimethylformamide/isopropanol (2:1, 9 mL), and then piperidine (0.15 mL, 1.67 mmol) and 2,3-dichloropyridine-4-carbaldehyde (0.3 g, 1.71 mmol) were added. The mixture was reacted at 85° C. for 1.5 h. After the reaction was completed as detected by LC-MS and TLC, the reaction solution was poured into 5 mL of water, added with 5 mL of ethyl acetate. The mixture was stirred for 10 min at room temperature, and then extracted with ethyl acetate for 3 times. The organic phase was washed with saturated sodium chloride aqueous solution once, dried, concentrated, and separated by column chromatography, to obtain 0.459 g of a yellow solid with a yield of 62%. ESI-MS m/z: 432.2 [M+H].sup.+.

Step 4: 3-(3-chloro-2-(cyclopropylamino)pyridin-4-yl)-2-(4-methoxybenzyl)-5-methyl-2,7-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4,6(5H)-dione

[0224] ##STR00123##

[0225] 3-(2,3-Dichloropyridin-4-yl)-2-(4-methoxybenzyl)-5-methyl-2,7-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4,6(5H)-dione (0.459 g, 1.06 mmol) was dissolved in cyclopropylamine (4 mL). After replacement by nitrogen gas, the mixture was reacted in microwave at 150° C. for 10 min. After completion of the reaction, the resultant was extracted with 50 mL of ethyl acetate for 3 times, washed with saturated brine once, dried over anhydrous sodium sulfate, and concentrated, and purified by column chromatography, to obtain 0.394 g of a brown solid with a yield of 82%. ESI-MS m/z: 453.2 [M+H].sup.+.

Step 5: (R)—N—((R)-1′-(3-(3-chloro-2-((cyclopropylamino)pyridin-4-yl)-2-(4-methoxybenzyl)-5-methyl-4-oxo-4,5-dihydro-2H-pyrazolo[3,4-d]pyrimidin-6-yl)-3H-spiro[benzofuran-2,4′-piperidine]-3-yl)-2-methylpropane-2-sulfinamide

[0226] ##STR00124##

[0227] In a 100 mL single neck flask, 3-(3-chloro-2-((cyclopropylamino)pyridin-4-yl)-2-(4-methoxybenzyl)-5-methyl-2,7-dihydro-4H-pyrazolo[3,4-d]pyrimidine-4-,6(5H)-dione (0.394 g, 0.869 mmol) was dissolved in anhydrous N, N-dimethylformamide (3 ml), and then the product in step 7 of Example 1 (R)-2-methyl-N—((R)-3H-spiro[benzofuran-2,4′-piperidine]-3-yl)propane-2-sulfinamide (0.295 g, 0.957 mmol), Castros condensing agent BOP (0.769 g, 1.738 mmol), and 1,8-diazabicycloundec-7-ene (0.6 mL, 4.345 mmol) were added. The mixture was replaced with nitrogen gas and then reacted at room temperature overnight. After completion of the reaction, saturated ammonium chloride aqueous solution was added to the reaction solution to quench the reaction. The resulting reaction solution was extracted with 60 mL dichloromethane for 3 times. The organic phase was washed with saturated sodium chloride aqueous solution once, dried, concentrated, and then separated by column chromatography, to obtain 0.345 g of a product with a yield of 53%. ESI-MS m/z: 743.1 [M+H].sup.+.

Step 6: Synthesis of (R)-6-(3-amino-3H-spiro[benzofuran-2,4′-piperidin]-1′-yl)-3-(3-chloro-2-((cyclopropylamino)pyridin-4-yl)-5-methyl-2,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one

[0228] ##STR00125##

[0229] In a 100 mL single neck flask, (R)—N—((R)-1′-(3-(3-chloro-2-((cyclopropylamino)pyridin-4-yl)-2-(4-methoxybenzyl)-5-methyl-4-oxo-4,5-dihydro-2H-pyrazolo[3,4-d]pyrimidin-6-yl)-3H-spiro[benzofuran-2,4′-piperidine]-3-yl)-2-methylpropane-2-sulfinamide (0.28 g, 0.377 mmol) was dissolved in anhydrous dichloromethane solution, and then trifluoroacetic acid (1.1 mL) and para toluene sulfonic acid (0.03 mL) were added dropwise. The mixture was reacted at room temperature for 10 min. After the reaction was completed as detected by LC-MS and TLC, the reaction solution was concentrated directly. ESI-MS m/z: 623.1 [M+H].sup.+. The concentrated reaction solution was dissolved in dioxane, and then added dropwise with 2 mL of hydrochloric acid-dioxane solution. The mixture was reacted at room temperature for 30 min. After the reaction was completed as detected by LC-MS and TLC, the reaction solution was directly concentrated and separated, to obtain 10 mg of white powder with a yield of 3.8%. 1H NMR (400 MHz, DMSO) δ 11.81 (s, 1H), 8.32 (d, J=5.1 Hz, 1H), 7.83 (d, J=4.9 Hz, 1H), 7.80 (d, J=8.7 Hz, 1H), 7.01 (m, 1H), 6.93-9.92 (m, 2H), 3.46 (s, 2H), 3.36 (s, 3H), 3.15 (s, 1H), 3.13 (s, 1H), 2.89 (m, 2H), 2.83 (m, 2H), 2.28 (s, 1H), 2.25 (m, 2H), 1.93 (m, 2H), 0.91 (m, 2H), 0.64 (m, 2H). ESI-MS m/z: 519.2 [M+H].sup.+.

Example 52: (S)-6-(5-amino-5,7-dihydrospiro[cyclopenta[b]pyridine-6,4′-piperidin]-1′-yl)-3-(3-chloro-2-((cyclopropylamino)pyridin-4-yl)-5-methyl-2,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one

[0230] ##STR00126##

Step 1: Synthesis of (R)—N—((S)-1′-(3-(3-chloro-2-((cyclopropylamino)pyridin-4-yl)-2-(4-methoxybenzyl)-5-methyl-4-oxo-4,5-dihydro-2H-pyrazolo[3,4-d]pyrimidin-6-yl)-5,7-dihydrospiro[cyclopenta[b]pyridine-6,4′-piperidin]-5-yl)-2-methylpropane-2-sulfinamide

[0231] ##STR00127##

[0232] In a 100 mL single neck flask, 3-(3-chloro-2-((cyclopropylamino)pyridin-4-yl)-2-(4-methoxybenzyl)-5-methyl-2,7-dihydro-4H-pyrazolo[3,4-d]pyrimidine-4,6(5H)-dione (0.106 g, 0.234 mmol) was dissolved in anhydrous DMF, and then (R)—N—((S)-5,7-dihydrospiro[cyclopenta[b]pyridine-6,4′-piperidine]-5-yl)-2-methyl propane-2-sulfinamide (0.079 g, 0.257 mmol), Castros condensing agent BOP (0.21 g, 0.468 mmol), and 1,8-diazabicycloundec-7-ene (0.16 mL, 1.17 mmol) were added in sequence. The mixture was replaced and protected with nitrogen gas and reacted overnight at room temperature. After completion of the reaction, saturated ammonium chloride aqueous solution was added to quench the reaction. The resultant was extracted with 60 mL of dichloromethane for 3 times, washed with saturated sodium chloride aqueous solution once, dried, concentrated, and subjected to column chromatography to obtain 44 mg of a weak polar isomer and 61 mg of a strong polar isomer with a yield of 61%. ESI-MS m/z: 742.1 [M+H].sup.+.

Step 6: Synthesis of (S)-6-(5-amino-5,7-dihydrospiro[cyclopenta[b]pyridine-6,4′-piperidine]-1′-yl)-3-(3-chloro-2-(cyclopropylamino)pyridin-4-yl)-5-methyl-2,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one

[0233] ##STR00128##

[0234] In a 100 mL single neck flask, (R)—N—((S)-1′-(3-(3-chloro-2-((cyclopropylamino)pyridin-4-yl)-2-(4-methoxybenzyl)-5-methyl-4-oxo-4,5-dihydro-2H-pyrazolo[3,4-d]pyrimidin-6-yl)-5,7-dihydrospiro[cyclopenta[b]pyridine-6,4′-piperidine]-5-yl)-2-methylpropane-2-sulfinamide (0.044 g, 0.059 mmol) was dissolved in 5 mL of anhydrous dichloromethane solution, and then trifluoroacetic acid (0.18 mL) and para toluene sulfonic acid (0.005 mL) were added dropwise. The mixture was reacted at room temperature for 10 min. After completion of the reaction, the reaction solution was concentrated. ESI-MS m/z: 622.1 [M+H]+. The concentrated reaction solution was dissolved in dioxane, and then 2 mL of hydrochloric acid-dioxane solution was added dropwise. The mixture was reacted at room temperature for 30 min. After completion of the reaction, the reaction solution was directly concentrated and purified to obtain 5 mg of the title product with a yield of 17%. 1H NMR (400 MHz, DMSO) δ 11.85 (s, 1H), 8.47 (d, J=5.3 Hz, 1H), 7.99 (d, J=5.1 Hz, 1H), 7.78 (d, J=8.9 Hz, 1H), 7.55 (m, 1H), 6.97 (t, 1H), 3.73 (s, 1H), 3.37 (s, 3H), 3.14 (m, 3H), 3.01 (m, 4H), 2.41 (s, 1H), 2.36 (s, 1H), 2.23 (m, 2H), 2.02 (m, 3H), 1.93 (m, 2H), 1.56 (m, 2H). ESI-MS m/z: 518.2 [M+H].sup.+.

Example 53: (R)-6-amino-2-(3-amino-3H-spiro[benzofuran-2,4′-piperidine]-1′-yl)-3-methyl-5-((2-(trifluoromethyl)pyridin-3-yl)thio)pyrimidin-4(3H)-one

[0235] ##STR00129##

Step 1: 6-amino-5-bromo-3-methylpyrimidin-2,4(1H,3H)-dione

[0236] ##STR00130##

[0237] In a 250 mL three-necked flask, 6-amino-3-methylpyrimidine-2,4(1H,3H)-dione (1.0 g, 7.08 mmol) was dissolved in N,N-dimethylformamide (50 mL). The mixture was replaced with nitrogen gas, and then N-bromosuccinimide (1.39 g, 7.79 mmol) was added at room temperature. The mixture was reacted at room temperature overnight. After the reaction was completed by detection, the reaction solution was filtered. The filter cake was washed with petroleum ether (50 mL) and dried, to obtain 0.9 g of a white solid with a yield of 25%. ESI-MS m/z: 220.1[M+H].sup.+.

Step 2: Synthesis of (R)—N—((R)-1′-(4-amino-5-bromo-1-methyl-6-oxo-1,6-dihydropyrimidin-2-yl)-3H-spiro[benzofuran-2,4′-piperidine]-3-yl)-2-methylpropane-2-sulfinamide

[0238] ##STR00131##

[0239] In a 100 mL three-necked flask, 6-amino-5-bromo-3-methylpyrimidine-2,4 (1H, 3H)-dione (0.43 g, 1.96 mmol) was dissolved in N,N-dimethylformamide (5 mL), and then (R)-2-methyl-N—((R)-3H-spiro[benzofuran-2,4′-piperidine]-3-yl)propane-2-sulfinamide (0.6 g, 1.96 mmol), Castros condensing agent BOP (1.74 g, 3.92 mmol) and 1,8-diazabicyclo[5.4.0]undec-7-ene (1.5 mL, 9.8 mmol) were added in sequence. The mixture was replaced with nitrogen gas and reacted at room temperature for 2.5 days. After the reaction was completed by detection, the resultant was extracted with ethyl acetate (100 mL) for 3 times, washed with saturated sodium chloride aqueous solution once, dried, concentrated and purified, to obtain 0.4 g of a crude product with a yield of 60%. ESI-MS m/z: 510.2/512.2[M+H].sup.+.

Step 3: Synthesis of (R)—N—((R)-1′-(4-amino-1-methyl-6-oxo-5-((2-(trifluoromethyl)pyridin-3-yl)thio)-1,6-dihydropyrimidin-2-yl)-3H-spiro[benzofuran-2,4′-piperidine]-3-yl)-2-methylpropane-2-sulfinamide

[0240] ##STR00132##

[0241] In a 100 mL single neck flask, (R)—N—((R)-1′-(4-amino-5-bromo-1-methyl-6-oxo-1,6-dihydropyrimidin-2-yl)-3H-spiro[benzofuran-2,4′-piperidine]-3-yl)-2-methylpropane-2-sulfinamide (0.4 g, 0.78 mmol) was dissolved in 1,4-dioxane (10 mL), and then 2-(trifluoromethyl)pyridine-3-thiol (0.4 g, 0.78 mmol), cuprous iodide (0.03 g, 0.16 mmol), tetramethylethylenediamine (0.05 mL, 0.31 mmol), potassium phosphate (0.5 g, 2.34 mmol) and phenanthroline (0.06 g, 0.31 mmol) were added. The mixture was reacted at 100° C. for 1.5 h. After the reaction was completed by detection, the resultant was extracted with ethyl acetate (100 mL) for 3 times, washed with saturated sodium chloride aqueous solution once, dried, concentrated, and subjected to column chromatography, to obtain 0.181 g of the title product with a yield of 29%. ESI-MS m/z: 609.1 [M+H].sup.+.

Step 4: Synthesis of (R)-6-amino-2-(3-amino-3H-spiro[benzofuran-2,4′-piperidine]-1′-yl)-3-methyl-5-((2-(trifluoromethyl)pyridin-3-yl)thio)pyrimidin-4(3H)-one

[0242] ##STR00133##

[0243] N-(1′-(4-amino-1-methyl-6-oxo-5-((2-(trifluoromethyl)pyridin-3-yl)thio)-1,6-dihydropyrimidin-2-yl)-3H-spiro[benzofuran-2,4′-piperidine]-3-yl)-2-methylpropane-2-sulfinamide (0.181 g, 0.30 mmol) was dissolved in dioxane (2 mL). The mixture was replaced by nitrogen gas, and then added dropwise with hydrochloric acid-dioxane solution (2 mL). After the reaction was completed as detected by LC-MS and TLC, the resultant was directly concentrated and separated by preparative chromatography, to obtain 15 mg of the title product (white). .sup.1H NMR (400 MHz, DMSO) δ 8.41 (d, J=3.3 Hz, 1H), 7.56-7.48 (m, 1H), 7.42 (d, J=8.1 Hz, 1H), 7.36 (d, J=7.1 Hz, 1H), 7.21-7.13 (m, 1H), 6.89 (t, J=7.2 Hz, 1H), 6.79 (d, J=7.9 Hz, 1H), 5.32 (s, 2H), 4.19 (s, 1H), 3.34 (s, 3H), 2.12-1.68 (m, 8H), 1.2 (s, 2H). ESI-MS m/z: 505.2 [M+H].sup.+.

Example 54: (S)-5-((5-(1-amino-1,3-dihydrospiro[indene-2,4′-piperidine]-1′-yl)imidazo[1,2-c]pyrimidin-8-yl)thio)-6-chloro-1-methylpyridin-2(1H)-one

[0244] ##STR00134##

Step 1: Synthesis of tert-butyl 1-oxo-1,3-dihydrospiro[indene-2,4′-piperidine]-1′-carboxylate

[0245] Under protection of nitrogen gas, sodium hydride (4.12 g, 103.25 mmol) was dissolved in anhydrous tetrahydrofuran (100 mL), and then slowly added dropwise with a solution of 2,3-dihydro-1H-inden-1-one (5.45 g, 41.30 mmol) in tetrahydrofuran at 0° C. The mixture was stirred at room temperature for 0.5 hours. After that, a solution of tert-butyl bis(2-chloroethyl)carbamate (10.0 g, 41.30 mmol) in tetrahydrofuran was slowly added at 0° C. dropwise. After the addition, the mixture was reacted at 60° C. overnight. After the reaction was completed as detected by LC-MS and TLC, ethyl acetate and water were added. The water phase was extracted for 3 times. The organic phase was dried over anhydrous sodium sulfate, concentrated, and separated by column chromatography, to obtain the title compound (3.50 g, yield: 31.80%). ESI-MS m/z: 246.20 [M+H].sup.+

Step 2: Synthesis of tert-butyl (R,Z)-1-((tert-butylsulfinyl)imino)-1,3-dihydrospiro[indene-2,4′-piperidine]-1′-carboxylate

[0246] ##STR00135##

[0247] Under protection of nitrogen gas, tert-butyl 1-oxo-1,3-dihydrospiro[indene-2,4′-piperidine]-1′-carboxylate (0.50 g, 1.66 mmol), (R)-2-methylpropane-2-sulfinamide (0.80 g, 6.64 mmol) and tetraethyl titanate (3.03 g, 2813.mmol) were dissolved in anhydrous tetrahydrofuran (50 mL). The mixture was reacted at 90° C. overnight. After the reaction was completed as detected by LC-MS and TLC, the reaction was quenched with saturated sodium bicarbonate. The resultant was filtered with diatomite and added with ethyl acetate and water. The water phase was extracted for 3 times. The organic phase was dried with anhydrous sodium sulfate, concentrated, and subjected to column chromatography, to obtain the title product (0.30 g, yield: 44.78%). ESI-MS m/z: 305.20 [M+H]+

Step 3: Synthesis of tert-butyl (S)-1-(((R)-tert-butylsulfinyl)amino)-1,3-dihydrospiro[indene-2,4′-piperidine]-1′-carboxylate

[0248] ##STR00136##

[0249] Tert-butyl (R,Z)-1-((tert-butylsulfinyl)imino)-1,3-dihydrospiro[indene-2,4′-piperidine]-1′-carboxylate (1.0 g, 2.47 mmol) was dissolved in anhydrous tetrahydrofuran (20 mL). Under protection of nitrogen gas, lithium borohydride solution (2.0M, 3.70 mmol) was slowly added dropwise at 0° C. The mixture was stirred at room temperature. After the reaction was completed as detected by TLC and LCMS, the reaction was quenched with saturated ammonium chloride at 0° C., and then added with ethyl acetate and water. The water phase was extracted with 3 times. The organic phase was dried with anhydrous sodium sulfate, concentrated, and separated by column chromatography, to obtain the title product (0.7 g, yield: 44.78%). ESI-MS m/z: 307.20 [M+H].sup.+

Step 4: Synthesis of (R)—N—((S)-1,3-dihydrospiro[indene-2,4′-piperidine]-1-yl)-2-methylpropane-2-sulfinamide

[0250] ##STR00137##

[0251] Tert-butyl (S)-1-(((R)-tert-butylsulfinyl)amino)-1,3-dihydrospiro[indene-2,4′-piperidine]-1′-carboxylate (0.20 g, 0.49 mmol) was dissolved in dichloromethane (5 mL), and then added with trifluoroacetic acid (2.5 mL). After the addition, 60° C. stirred at room temperature. After the reaction was completed as detected by TLC, the resultant was adjusted to a pH of 7-9 with sodium bicarbonate, extracted with ethyl acetate, washed with water and saturated sodium chloride, dried and concentrated, to obtain the title product, which was directly used in the next reaction (0.35 g, yield: 100%). ESI-MS m/z: 307.1 [M+H]+

Step 5: Synthesis of (S)-5-((5-(1-amino-1,3-dihydrospiro[indene-2,4′-piperidine]-1′-yl)imidazo[1,2-c]pyrimidin-8-yl)thio)-6-chloro-1-methylpyridin-2 (1H)-one

[0252] ##STR00138##

[0253] The title compound was prepared by using the same method as that of Example 45, except that 4-bromo-3-chloropyridin-2-amine was replaced with 5-bromo-6-chloro-1-methylpyridin-2 (1H)-one.

[0254] 1H NMR (400 MHz, CDCl3) δ 7.66 (s, 1H), 7.64-7.55 (m, 2H), 7.47 (s, 1H), 7.35 (s, 1H), 7.22 (s, 2H), 6.47 (d, J=9.4 Hz, 1H), 5.30 (d, J=40.2 Hz, 2H), 4.06 (s, 1H), 3.79 (d, J=13.5 Hz, 2H), 3.72 (s, 3H), 3.22 (dd, J=24.2, 12.2 Hz, 2H), 3.11 (d, J=15.6 Hz, 1H), 2.76 (d, J=15.5 Hz, 1H), 2.12-1.90 (m, 3H), 1.47 (d, J=12.8 Hz, 2H). ESI-MS m/z: 493.20 [M+H]+

[0255] According to the synthesis method of Example 45 or 48, the compounds of examples 55-74 was synthesized by using different commercially available raw materials. The characterization parameters of these compounds are set forth in Table 2:

TABLE-US-00002 TABLE 2 ESI-MS ex m/z ample structure of compound [M + H].sup.+ 55 [00139] 502.2 56 [00140] 501.3 57 [00141] 465.4 58 [00142] 464.2 59 [00143] 444.2 60 [00144] 467.1 61 [00145] 505.2 62 [00146] 452.1 63 [00147] 477.2 64 [00148] 495.1 65 [00149] 500.2 66 [00150] 462.3 67 [00151] 460.1 68 [00152] 505.2 69 [00153] 523.3 70 [00154] 498.3 71 [00155] 503.1 72 [00156] 500.1 73 [00157] 521.4 74 [00158] 520.2

Example 75: (R)-2-(5-(3-amino-3H-spiro[benzofuran-2,4′-piperidine]-1′-yl)-7-methylimidazo[1,2-c]pyrimidine-8-yl)-3-fluorophenol

[0256] ##STR00159##

[0257] The title compound was prepared by using the same method as that of Example 48, except that the intermediate of 2,3-dichlorobenzeneboronic acid was replaced with 2-fluoro-6-hydroxyphenylboronic acid, .sup.1H NMR (400 MHz, DMSO) δ 9.74 (s, 1H), 7.74 (s, 1H), 7.67-7.53 (m, 5H) 7.45 (d, J=11.8 Hz, 1H), 7.25 (dd, J=17.3, 8.3 Hz, 1H) 6.97-6.84 (m, 1H), 6.83-6.70 (m, 1H), 3.84 (dd, J=50.0, 21.6 Hz, 2H), 3.57 (s, 3H), 2.20 (d, J=23.4 Hz, 2H), 2.02 (dd, J=25.3, 17.6 Hz, 2H), 1.24 (s, 4H). ESI-MS m/z: 446.1 [M+H].sup.+

Example 76: (R)-6-amino-2-(3-amino-3H-spiro[benzofuran-2,4′-piperidine]-1′-yl)-3-methyl-5-((4-(trifluoromethyl)pyrimidin-5-yl)thiopyrimidin-4(3H)-one

[0258] ##STR00160##

[0259] The title compound was prepared by using the same method as that of Example 53, except that the intermediate 3-bromo-2-(trifluoromethyl)pyridine was replaced with 5-bromo-4-(trifluoromethyl)pyrimidine. .sup.1H NMR (400 MHz, DMSO) δ 9.09 (s, 1H), 8.43 (s, 1H), 7.34 (d, J=7.0 Hz, 1H), 7.15 (t, J=7.5 Hz, 1H), 7.01 (s, 2H), 6.91-6.84 (m, 1H), 6.77 (d, J=8.1 Hz, 1H), 4.43 (s, 2H), 4.11 (s, 1H), 3.30 (s, 3H), 3.23 (d, J=11.3 Hz, 4H), 1.99-1.66 (m, 4H). ESI-MS m/z: 506.2 [M+H].sup.+

Example 77: (S)-5-((1H pyrazolin[3,4-b]pyridine-4-yl)thio)-6-amino-2-(1-amino-1,3-dihydrospiro[indene-2,4′-piperidine]-1′-yl)-3-methylpyrimidin-4(3H)-one

[0260] ##STR00161##

[0261] The title compound was prepared by using the same method as that in Example 1, except that 3-bromo-2-trifluoromethylpyridine was replaced with bromo-1H-pyrazoline[3,4-b]pyridine. ESI-MS m/z 475.2 [M+H]+. .sup.1H NMR (400 MHz, d6-DMSO) δ 13.80-13.50 (m, 1H), 8.28-8.18 (m, 1H), 8.13-8.06 (m, 1H), 7.41-7.31 (m, 1H), 7.15-7.25 (m, 3H), 6.90-6.70 (s, 3H), 6.64-6.47 (m, 2H), 4.01-3.90 (m, 1H), 3.51-3.41 (s, 3H), 3.10-3.01 (m, 2H), 2.93-2.85 (m, 1H), 2.60-2.69 (m, 1H), 1.95-1.72 (m, 2H), 1.60-1.42 (m, 2H), 1.27-1.14 (m, 2H).

[0262] According to the synthesis method of Example 1 of the present disclosure, the compounds of examples 78-81 was synthesized by using different commercially available raw materials. The characterization parameters of these compounds are shown in Table 3:

TABLE-US-00003 TABLE 3 ESI-MS ex m/z ample structure of compound [M + H].sup.+ 78 [00162] 562.1 79 [00163] 560.2 80 [00164] 546.2 81 [00165] 572.2

Comparative Example 1

[0263] With reference to the method for compound 45 disclosed in WO2018/172984 (PCT/IB2018/051973), the compound represented by the following formula (Compound A) was prepared and identified by hydrogen and mass spectrometry.

##STR00166##

Experimental Example 1 Cell Proliferation Inhibition Test

[0264] 1. Experimental Materials

[0265] Test compounds: The compounds prepared in examples and comparative example of the present disclosure was tested. Each compound was formulated with DMSO into 20 mM. The concentrations of the compounds acting on NCI-H358 cells were 100 μM, 25 μM, 6.25 μM, 1.56 μM, 0.391 μM, 0.098 μM, 0.024 μM, 0.006 μM, 0.0015 μM, and 0.00038 μM successively.

[0266] Human non-small cell lung cancer cells NCI-H358 were purchased from the American Type Culture Collection (ATCC).

[0267] Reagents: The CCK-8 proliferation inhibition detection kit was purchased from Jiangsu KeyGEN BioTECH Co., Ltd. The CKX41 inverted microscope was purchased from Olympus, Japan. The multi-mode microplate reader was purchased from Molecular Devices, USA. The cell culture incubator was purchased from Thermo, USA.

[0268] 2. Experimental Methods

[0269] 2.1 Cell Culture

[0270] Cell recovery: A NCI-H358 cell cryogenic vial was taken out from a liquid nitrogen tank, placed in water bath at 37° C., and shaked gently to allow it to thaw. The thawed cryogenic vial was disinfected with alcohol cotton, and opened to aspirate the cell fluid into a centrifuge tube. To the centrifuge tube, 1 mL of serum-containing complete medium was added and well-mixed. The centrifuge tube was placed into a centrifuge, for centrifugation at 1000 rpm for 5 min. Then the supernatant was discarded, and complete medium was added to completely dissipate and resuspend the cells through repeated pipetting. The cells were seeded in petri dishes at appropriate concentrations and cultured in a CO.sub.2 incubator at 37° C., with 5% CO.sub.2, 95% humidified air.

[0271] Cell passage: After the cells grew to about 80-90% confluence, the original culture medium (1640 culture medium+10% FBS+1% penicillin-streptomycin+1 mM sodium pyruvate) was discarded, and 1 mL of PBS was added to wash away the residual medium and then discarded. 1 mL of trypsin digestion solution was added to digest for 1-2 min. When it was observed under the microscope that the pseudopodia of the cells shrinked and became round but the cells did not fall off into pieces, the trypsin was discarded and the digestion was terminated with 1-2 mL of complete medium. The cell suspension was collected by gently pipetting, and centrifuged at 1000 rpm for 5 min. After discarding the supernatant, the cells was resuspended in complete medium, seeded in petri dishes at the required density, and then cultured in a CO.sub.2 incubator at 37° C., with 5% CO.sub.2, 95% humidified air. Depending on cell growth, the culture medium was changed or passaged every 2-3 days.

[0272] 2.2 Experimental Steps:

[0273] After NCI-H358 cell passage, the cells were resuspended in fresh culture medium (1640 culture medium+3% FBS+1% penicillin-streptomycin+1 mM sodium pyruvate), and counted. The cells were seeded into a 96-well culture plate at a density of 1.5×10.sup.4 cells/mL, with each well added 100 μL thereto (ie, 1.5×10.sup.3 cells/well). After 24 h, 100 μL of fresh culture medium containing different drug concentrations (2×) was added to the old culture medium. The final concentrations of the compounds were 100 μM, 25 μM, 6.25 μM, 1.56 μM, 0.391 μM, 0.098 μM, 0.024 μM, 0.006 μM, 0.0015 μM, and 0.00038 μM, with each concentration group set two replicate wells. The cells were calculated in the incubator again for 168 h. The culture medium in wells was discarded as thoroughly as possible, and 100 μL of culture medium containing CCK-8 (CCK-8:culture medium=1:10) was added. After a certain time of culture in the incubator, the 96-well plate was taken out of the incubator, and allowed to equilibrate at room temperature for 5 min. The absorbance (OD value) at 450 nm was detected in a multi-mode plate reader, and the cell proliferation inhibition rate was calculated. The calculation formula was Inhibition (%)=100−(OD.sub.experiment well−OD.sub.blank well)/(OD.sub.solvent control well−OD.sub.blank well)×100. According to different drug concentrations and their corresponding inhibition rates, IC.sub.50 curves were plotted using GraghPad 5.0 software, to analyze the data and to obtain final IC.sub.50. The experimental results are set forth in Table 4.

TABLE-US-00004 TABLE 4 NIH358 cell NIH358 cell NIH358 cell proliferation proliferation proliferation inhibition inhibition inhibition example IC.sub.50 (nM) example IC.sub.50 (nM) example IC.sub.50 (nM) 1 1.6 45 3.9 51 6.0 4 5.6 46 8.5 53 4.9 6 2.8 47 16.7 compound 18.3 A 8 3.3 48 12 9 1.5 49 13.3

[0274] As can be seen from the above experiments, the compounds of the present disclosure exhibited good inhibitory activity on NCI-H358 cells, and are very promising to be a therapeutic agent for non-small cell lung cancer.

Experimental Example 2 Pharmacokinetic Experiment

[0275] 1. Experimental Materials

[0276] Compounds: The compounds of the present disclosure prepared in the above examples were tested. The drug vehicle was Captisol/50 mM sodium acetate with pH 4.6 (10%/90%, w/v %). Oral drugs were formulated into a 0.5 mg/mL clear solution. Intravenous drug were formulated into a 0.1 mg/mL clear solution.

[0277] Animals: Male BALB/c mice, SPF grade, were purchased from Shanghai SIPPR-Bk Lab Animal Co., Ltd, with a weight of 18-20 g, and adapted for 2-3 days before experiments.

[0278] Instrument: API4500-type Triple Quadrupole LC/MS System, from AB, United States, equipped with electrospray ion source (ESI) and dual pumps LC-30AD; SIL-30AC autosampler; CTO-30AC column oven; DGU-20A3R degasser; AnalystQSA01.01 chromatography workstation; Milli-Q ultrapure water device (Millipore Inc); QilinbeierVortex-5 shaker; HITACHI CF16RX II desktop high-speed freezing centrifuge.

[0279] 2. Experimental Method

[0280] (1) There were 3 mice per group. The compounds of the examples of the present disclosure were given by intragastric administration (I.G.) at 10 mg/kg, or the compounds of the examples of the present disclosure were given by intravenous (IV) administration at 1 mg/kg.

[0281] (2) At 5 min, 15 min, 30 min, 1 h, 2 h, 6 h, 10 h, and 24 h after the intragastric administration or intravenous administration, blood was collected from the orbital venous plexus in heparinized EP tubes (0.6 ML), and centrifuged at 8000 rpm/min for 5 min. The upper plasma was taken, frozen at −20° C. and analyzed by LC-MS/MS.

[0282] (3) Plasma drug concentration-time curves were plotted according to blood drug concentration data obtained from the above-mentioned step. Pharmacokinetic parameters were calculated by using WinNonlin software. The experimental results are shown in Table 5.

TABLE-US-00005 TABLE 5 routes of dose Example Example Example administration (mg/kg) index 45 48 53 I.G. 10 T.sub.1/2 (h) 7.3 15.6 3.0 C.sub.max (ng/mL) 1586.7 1893.3 1393.3 AUC.sub.(0-t) 19360.2 24157.4 6522.8 (h × ng/mL) AUC.sub.(0-∞) 21634.7 36969.5 7406.1 (h × ng/mL) F (%) 78.3 81.2 74.3

[0283] The pharmacokinetics of Compound A was determined using the method of Experimental Example 2. The experimental results showed that the bioavailability (F) of Compound A was 51.2%, which was significantly lower than that of the compounds of the present disclosure.

[0284] The experimental results show that the compounds of the present disclosure have good half-life T.sub.1/2, area under the curve AUC and bioavailability F, as well as good oral absorption and exposure, so they are suitable for finished medicine.

[0285] Although the present disclosure has been described in detail above, it will be understood by these skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit and scope of the invention. The scope of the present invention is not limited to the detailed description above, but should be attributed to the claims.