Sultam compound and application method thereof

Abstract

Provided are a sultam compound having isocitrate dehydrogenase 1 (IDH1) inhibitory activity as represented by formula I or pharmaceutically-acceptable salts, solvates or hydrates thereof, a preparation method thereof, and a pharmaceutical composition containing the compound. The compound or the pharmaceutically-acceptable salts, solvates or hydrates thereof, and the pharmaceutical composition containing the compound can be used to treat IDH1 mutation-induced cancers. ##STR00001##

Claims

1. A compound represented by formula I or a pharmaceutically acceptable salt thereof, ##STR00110## wherein, X is CH.sub.2 or NR.sup.5; R.sup.1 is C.sub.3-6 cycloalkyl or 3- to 6-membered heterocycloalkyl containing 1 to 2 heteroatoms selected from the group consisting of N, O and S, which may be optionally substituted with one or more groups R.sup.6; R.sup.2 is phenyl or 5- to 6-membered heteroaryl containing 1 to 2 heteroatoms selected from the group consisting of N, O and S, which may be optionally substituted with one or more groups R.sup.7; R.sup.3 is phenyl, 5- to 6-membered heteroaryl containing 1 to 2 heteroatoms selected from the group consisting of N, O and S, phenyl CH.sub.2—, or 5- to 6-membered heteroaryl CH.sub.2— containing 1 to 2 heteroatoms selected from the group consisting of N, O and S, which may be optionally substituted with one or more groups R.sup.8; R.sup.4 is halogen, amino, hydroxyl, C.sub.1-3 haloalkyl or C.sub.1-6 alkyl; R.sup.5 is hydrogen or C.sub.1-6 alkyl; R.sup.6 is halogen, amino, hydroxyl, cyano, C.sub.1-3 haloalkyl, C.sub.1-6 alkyl or C.sub.3-6 cycloalkyl; R.sup.7 is halogen, amino, hydroxyl, cyano, C.sub.1-3 haloalkyl, ##STR00111##  C.sub.1-6 alkyl or C.sub.3-6 cycloalkyl; or R.sup.7 forms a O.fwdarw.N coordination linkage with N atom in R.sup.2; R.sup.8 is halogen, amino, hydroxyl, cyano, C.sub.1-3 haloalkyl, C.sub.1-6 alkyl, C.sub.3-6 cycloalkyl, C.sub.2-6 alkenyl or C.sub.2-6 alkynyl; R.sup.9 is H, C.sub.1-6 alkyl, C.sub.3-6 cycloalkyl, 3- to 6-membered heterocycloalkyl containing 1 to 2 heteroatoms selected from the group consisting of N, O and S, phenyl, or 5- to 6-membered heteroaryl containing 1 to 2 heteroatoms selected from the group consisting of N, O and S, which may be optionally substituted with one or more groups R.sup.10; R.sup.10 is halogen, amino, hydroxyl, cyano, C.sub.1-3 haloalkyl, C.sub.1-6 alkyl or C.sub.3-6 cycloalkyl; m is 0 or 1.

2. The compound or the pharmaceutically acceptable salt thereof according to claim 1, wherein, X is CH.sub.2 or NR.sup.5; R.sup.1 is C.sub.3-6 cycloalkyl or 3- to 6-membered heterocycloalkyl containing 1 to 2 heteroatoms selected from the group consisting of N, O and S, which may be optionally substituted with one or more groups R.sup.6; R.sup.2 is phenyl or 5- to 6-membered heteroaryl containing 1 to 2 heteroatoms selected from the group consisting of N, O and S, which may be optionally substituted with one or more groups R.sup.7; R.sup.3 is phenyl, 5- to 6-membered heteroaryl containing 1 to 2 heteroatoms selected from the group consisting of N, O and S, phenyl CH.sub.2—, or 5- to 6-membered heteroaryl CH.sub.2— containing 1 to 2 heteroatoms selected from the group consisting of N, O and S, which may be optionally substituted with one or more groups R.sup.8; R.sup.4 is halogen, amino, hydroxyl, C.sub.1-3 haloalkyl or C.sub.1-6 alkyl; R.sup.5 is hydrogen or C.sub.1-6 alkyl; R.sup.6 is halogen, amino, hydroxyl, cyano, C.sub.1-3 haloalkyl, C.sub.1-6 alkyl or C.sub.3-6 cycloalkyl; R.sup.7 is halogen, amino, hydroxyl, cyano, C.sub.1-3 haloalkyl, aminosulfonyl, N-substituted aminosulfonyl, C.sub.1-6 alkyl or C.sub.3-6 cycloalkyl; R.sup.8 is halogen, amino, hydroxyl, cyano, C.sub.1-3 haloalkyl, C.sub.1-6 alkyl, C.sub.3-6 cycloalkyl, C.sub.2-6 alkenyl or C.sub.2-6 alkynyl; m is 0 or 1.

3. The compound or the pharmaceutically acceptable salt thereof according to claim 1, wherein the compound is represented by formula II, ##STR00112## wherein, X is CH.sub.2 or NR.sup.5; R.sup.1 is C.sub.3-6 cycloalkyl or 3- to 6-membered heterocycloalkyl containing 1 to 2 heteroatoms selected from the group consisting of N, O and S, which may be optionally substituted with one or more groups R.sup.6; R.sup.2 is phenyl or 5- to 6-membered heteroaryl containing 1 to 2 heteroatoms selected from the group consisting of N, O and S, which may be optionally substituted with one or more groups R.sup.7; R.sup.3 is phenyl, 5- to 6-membered heteroaryl containing 1 to 2 heteroatoms selected from the group consisting of N, O and S, phenyl CH.sub.2—, or 5- to 6-membered heteroaryl CH.sub.2— containing 1 to 2 heteroatoms selected from the group consisting of N, O and S, which may be optionally substituted with one or more groups R.sup.8; R.sup.5 is hydrogen or C.sub.1-6 alkyl; R.sup.6 is halogen, amino, hydroxyl, cyano, C.sub.1-3 haloalkyl, C.sub.1-6 alkyl or C.sub.3-6 cycloalkyl; R.sup.7 is halogen, amino, hydroxyl, cyano, C.sub.1-3 haloalkyl, ##STR00113##  C.sub.1-6 alkyl or C.sub.3-6 cycloalkyl; or R.sup.7 forms a O.fwdarw.N coordination linkage with N atom in R.sup.2, R.sup.7 is halogen, amino, hydroxyl, cyano, C.sub.1-3 haloalkyl, C.sub.1-6 alkyl, C.sub.3-6 cycloalkyl, C.sub.2-6 alkenyl or C.sub.2-6 alkynyl; R.sup.9 is H, C.sub.1-6 alkyl, C.sub.3-6 cycloalkyl, 3- to 6-membered heterocycloalkyl containing 1 to 2 heteroatoms selected from the group consisting of N, O and S, phenyl, or 5- to 6-membered heteroaryl containing 1 to 2 heteroatoms selected from the group consisting of N, O and S, which may be optionally substituted with one or more groups R.sup.10; R.sup.10 is halogen, amino, hydroxyl, cyano, C.sub.1-3 haloalkyl, C.sub.1-6 alkyl or C.sub.3-6 cycloalkyl.

4. The compound or the pharmaceutically acceptable salt thereof according to claim 3, wherein, X is CH.sub.2 or NR.sup.5; R.sup.1 is C.sub.3-6 cycloalkyl or 3- to 6-membered heterocycloalkyl containing 1 to 2 heteroatoms selected from the group consisting of N, O and S, which may be optionally substituted with one or more groups R.sup.6; R.sup.2 is phenyl or 5- to 6-membered heteroaryl containing 1 to 2 heteroatoms selected from the group consisting of N, O and S, which may be optionally substituted with one or more groups R.sup.7; R.sup.3 is phenyl, 5- to 6-membered heteroaryl containing 1 to 2 heteroatoms selected from the group consisting of N, O and S, phenyl CH.sub.2—, or 5- to 6-membered heteroaryl CH.sub.2— containing 1 to 2 heteroatoms selected from the group consisting of N, O and S, which may be optionally substituted with one or more groups R.sup.8; R.sup.5 is hydrogen or C.sub.1-6 alkyl; R.sup.6 is halogen, amino, hydroxyl, cyano, C.sub.1-3 haloalkyl, C.sub.1-6 alkyl or C.sub.3-6 cycloalkyl; R.sup.7 is halogen, amino, hydroxyl, cyano, C.sub.1-3 haloalkyl, aminosulfonyl, N-substituted aminosulfonyl, C.sub.1-6 alkyl or C.sub.3-6 cycloalkyl; R.sup.8 is halogen, amino, hydroxyl, cyano, C.sub.1-3 haloalkyl, C.sub.1-6 alkyl, C.sub.3-6 cycloalkyl, C.sub.2-6 alkenyl or C.sub.2-6 alkynyl.

5. The compound or the pharmaceutically acceptable salt thereof according to claim 1, X is CH.sub.2, NH or N(CH.sub.3).

6. The compound or the pharmaceutically acceptable salt thereof according to claim 1, R.sup.5 is hydrogen, methyl, ethyl, propyl, isopropyl, butyl, isobutyl or tert-butyl.

7. The compound or the pharmaceutically acceptable salt thereof according to claim 1, R.sup.1 is cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, pyrrolidinyl or piperidyl, which may be optionally substituted with one or more groups R.sup.6.

8. The compound or the pharmaceutically acceptable salt thereof according to claim 1, R.sup.6 is F, Cl or Br.

9. The compound or the pharmaceutically acceptable salt thereof according to claim 1, R.sup.2 is phenyl, furyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, pyridyl, pyrimidyl, pyridazinyl, pyrazinyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, tetrazolyl or triazinyl, which may be optionally substituted with one or more groups R.sup.7.

10. The compound or the pharmaceutically acceptable salt thereof according to claim 1, wherein R.sup.2 is phenyl, furyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, pyridyl, pyrimidyl, pyridazinyl, pyrazinyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, tetrazolyl or triazinyl, which may be optionally substituted with one or more groups R.sup.7; R.sup.7 is F, Cl, Br, cyano, monofluoromethyl, difluoromethyl, trifluoromethyl, monofluoroethyl, difluoroethyl, trifluoroethyl, tetrafluoroethyl, pentafluoroethyl, monochloromethyl, dichloromethyl, trichloromethyl, aminosulfonyl or N-substituted aminosulfonyl.

11. The compound or the pharmaceutically acceptable salt thereof according to claim 1, R.sup.7 is F, Cl, Br, cyano, monofluoromethyl, difluoromethyl, trifluoromethyl, monofluoroethyl, difluoroethyl, trifluoroethyl, tetrafluoroethyl, pentafluoroethyl, monochloromethyl, dichloromethyl, trichloromethyl, or ##STR00114## or R.sup.7 forms a O.fwdarw.N coordination linkage with N atom in R.sup.2.

12. The compound or the pharmaceutically acceptable salt thereof according to claim 1, R.sup.9 is H, furyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, pyridyl, pyrimidyl, pyridazinyl, pyrazinyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, tetrazolyl or triazinyl, which may be optionally substituted with one or more groups R.sup.10.

13. The compound or the pharmaceutically acceptable salt thereof according to claim 1, R.sup.3 is phenyl, furyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, pyridyl, pyrimidyl, pyridazinyl, pyrazinyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, tetrazolyl, triazinyl, benzyl, furanylmethylene, thienylmethylene, pyrrolylmethylene, pyrazolylmethylene, imidazolylmethylene, pyridylmethylene, pyrimidinylmethylene, pyridazinylmethylene, pyrazinylmethylene, thiazolylmethylene, isothiazolylmethylene, oxazolylmethylene, isoxazolylmethylene, tetrazolylmethylene or triazinylmethylene, which may be optionally substituted with one or more groups R.sup.8.

14. The compound or the pharmaceutically acceptable salt thereof according to claim 1, R.sup.8 is F, Cl, Br, cyano, ethynyl, 1-propynyl or 1-butynyl.

15. The compound or the pharmaceutically acceptable salt thereof according to claim 1, wherein the compound is as follows: ##STR00115## ##STR00116## ##STR00117## ##STR00118## ##STR00119## ##STR00120## ##STR00121## ##STR00122## ##STR00123##

16. A method for treating IDH1 mutation-induced cancers comprising administering a therapeutically effective amount of the compound or the pharmaceutically acceptable salt thereof according to claim 1 to a patient in need thereof, wherein the IDH1 mutation has R132X mutation; and the IDH1 mutation-induced cancers are selected from the group consisting of glioblastoma, myelodysplastic syndrome, myeloproliferative neoplasm, acute myelogenous leukemia, sarcoma (preferably chondrosarcoma, fibrosarcoma), melanoma, non-small cell lung cancer, bile duct cancer or angioimmunoblastic non-Hodgkin's lymphoma.

17. A pharmaceutical composition, which comprises a therapeutically effective amount of the compound or the pharmaceutically acceptable salt thereof according to claim 1 and one or more pharmaceutically acceptable carriers or excipients.

18. The compound or the pharmaceutically acceptable salt thereof according to claim 7, R.sup.1 is ##STR00124##

19. The compound or the pharmaceutically acceptable salt thereof according to claim 9, R.sup.2 is ##STR00125##

20. The compound or the pharmaceutically acceptable salt thereof according to claim 13, R.sup.3 is ##STR00126##

Description

DESCRIPTION OF FIGURES

(1) FIG. 1 shows the inhibitory results of the compound in Example 2 and control AG-120 directed to 2-HG in IDH1-mutated HT-1080 cells.

(2) FIG. 2 shows the inhibitory results of the compound in Example 2 and control AG-120 directed to 2-HG outside IDH1-mutated HT-1080 cells.

(3) FIG. 3 shows the inhibitory results of the compound in Example 2 and control AG-120 directed to 2-HG in IDH1-mutated U87 cells.

DETAILED DESCRIPTION

(4) The following specific examples are provided to enable those skilled in the art to more clearly understand and practice the application. They should not be deemed as limiting the scope of the application, but are merely illustrations and typical representatives of the application. Those skilled in the art should understand that there are other synthetic routes for preparing the compounds of the present application, and ones provided below are non-limiting examples.

(5) Unless otherwise stated, the temperature is Celsius temperature. The reagents were purchased from commercial suppliers such as Sinopharm Chemical Reagent Beijing Co., Ltd., Alfa Aesar, or Beijing J&K Scientific Co., Ltd., and the like, and these reagents can be directly used without further purification, unless otherwise stated.

(6) Unless otherwise stated, the following reactions were carried out in an anhydrous solvent, under a positive pressure of nitrogen or argon gas, or using a drying tube. The reaction flasks were equipped with a rubber diaphragm so as to add substrates and reagents by a syringe. The glassware was dried in an oven and/or dried by heating.

(7) Unless otherwise stated, the purification by column chromatography was performed with silica gel (200-300 mesh) produced by Qingdao Haiyang Chemical Co., Ltd. The separation by preparative thin layer chromatography was performed by using thin layer chromatography silica gel prefabricated plates (HSGF254) manufactured by Yantai Chemical Industry Research Institute. MS was measured by using Thermo LCQ Fleet type (ESI) liquid chromatography-mass spectrometer. The optical rotation was measured by using SWG-3 automatic polarimeter from Shanghai Shenguang Instrument Co., Ltd.

(8) Unless otherwise stated, NMR data (.sup.1H-NMR) were taken at 400 Hz by using an equipment from Varian. The solvents used for NMR include CDCl.sub.3, CD.sub.3OD, D.sub.2O, DMSO-d and the like, and tetramethylsilane (0.00 ppm) was used as a baseline or residual solvent was used as a baseline (CDCl.sub.3: 7.26 ppm; CD.sub.3OD: 3.31 ppm; D.sub.2O: 4.79 ppm; DMSO-d.sub.6: 2.50 ppm). Upon indicating peak shape diversity, the following abbreviations represent different peak shapes: s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), br (broad), dd (doublet of doublets), dt (doublet of triplets). If a coupling constant is given, the unit thereof is Hertz (Hz).

(9) Unless otherwise indicated, the absolute configuration of the chiral center is not indicated for the title compounds in some Examples of the present application, and a mixture of all enantiomers was obtained during the preparation of such compounds. Although the enantiomers cannot be separated by an ordinary column chromatography, it does not mean that there are no enantiomers for such compounds.

Example 1: (S)—N—((R)-1-(2-chlorophenyl)-2-((3,3-difluorocyclobutyl)amino)-2-oxoethyl)-2-(4-cyanopyridin-2-yl)-N-(5-fluoropyridin-3-yl)-isothiazolidine-3-carboxamide 1,1-dioxide

(10) ##STR00040##

Step A: dimethyl L-homocysteinate dihydrochloride

(11) ##STR00041##

(12) Under stirring in an ice bath, thionyl chloride (10.64 g, 89.4 mmol) was added dropwise into a suspension of L-homocysteine (8.0 g, 29.8 mmol) in methanol. The solution is gradually clear. After the addition was completed, the reaction solution was stirred for 10 min, followed by removing the ice bath, and stirred again at room temperature overnight. The solvent was removed, so as to give dimethyl L-homocysteinate dihydrochloride (10.6 g, yield 100%).

(13) .sup.1H-NMR (400 MHz, DMSO-d.sub.6): δ=8.79 (s, 6H), 3.75 (s, 6H), 2.95-2.80 (m, 4H), 2.52-2.47 (m, 2H), 2.20-2.10 (m, 4H).

Step B: methyl (S)-2-amino-4-chlorosulfonylbutyrate hydrochloride

(14) ##STR00042##

(15) Under stirring in an ice bath, chlorine gas was introduced into a mixed solution of dimethyl L-homocysteinate dihydrochloride (10.6 g, 29.8 mmol) in ethanol (40 mL) and chloroform (80 mL) for 20 minutes, generating a white solid. The reaction solution was filtered and washed with chloroform, to give methyl (S)-2-amino-4-chlorosulfonylbutyrate hydrochloride (7.5 g, yield 100%).

(16) .sup.1H-NMR (400 MHz, DMSO-d.sub.6): δ=13.46 (s, 1H), 8.57 (s, 2H), 3.66 (s, 3H), 3.18-2.95 (m, 2H), 2.52-2.45 (m, 1H), 2.22-1.97 (m, 2H).

Step C: methyl (S)-isothiazolidine-3-carboxylate 1,1-dioxide

(17) ##STR00043##

(18) Under stirring in an ice-salt bath, a solution of triethylamine in chloroform was added dropwise into a suspension of methyl (S)-2-amino-4-chlorosulfonylbutyrate hydrochloride (4.5 g, 17.85 mmol) in chloroform. After the addition of the solution of triethylamine in chloroform was completed, the ice-salt bath was removed. It was stirred overnight at room temperature and the solvent was removed. Then it was filtered with diatomite and washed with ethyl acetate. The solvent was removed to give a light yellow oil, namely methyl (S)-isothiazolidine-3-carboxylate 1,1-dioxide (3.2 g, yield 100%).

(19) .sup.1H-NMR (400 MHz, CDCl.sub.3): δ=4.98 (s, 1H), 4.21 (dd, J=8.3, 4.6 Hz, 1H), 3.84 (s, 3H), 3.30-3.11 (m, 1H), 3.09-2.90 (m, 1H), 2.90-2.73 (m, 1H), 2.60 (ddd, J=18.4, 8.9, 4.7 Hz, 1H).

Step D: (S)-isothiazolidine-3-carboxylic acid 1,1-dioxide

(20) ##STR00044##

(21) Under stirring in an ice bath, a suspension of lithium hydroxide was added dropwise into a solution of methyl (S)-isothiazolidine-3-carboxylate 1,1-dioxide (2.4 g, 13.4 mmol) in methanol-tetrahydrofuran, reacting overnight, to which 1N hydrochloric acid was added dropwise, so as to make the pH less than 5. The solvent was removed. The residue was filtered and washed with methanol. The solvent was removed to give (S)-isothiazolidine-3-carboxylic acid 1,1-dioxide (2.2 g, yield 100%).

(22) .sup.1H-NMR (400 MHz, CDCl.sub.3): δ=3.85-3.75 (m, 1H), 3.10-2.85 (m, 2H), 2.55-2.30 (m, 2H).

Step E: (3S)—N-(1-(2-chlorophenyl)-2-((3,3-difluorocyclobutyl)amino)-2-oxoethyl)-N-(5-fluoropyridin-3-yl)-isothiazolidine-3-carboxamide 1,1-dioxide

(23) ##STR00045##

(24) At room temperature, 3-amino-5-fluoropyridine (57 mg, 0.508 mmol) and o-chlorobenzaldehyde (72 mg, 0.512 mmol) were dissolved in methanol and stirred for 30 min. (S)-isothiazolidine-3-carboxylic acid 1,1-dioxide (84 mg, 0.508 mmol) was then added into the mixed solution, stirred for 10 min, followed by adding 1,1-difluoro-3-isocyanocyclobutane (prepared according to the method described in patent CN103097340, 60 mg, 0.508 mmol), and stirred overnight. The solvent was removed and the residue was separated by silica gel column chromatography (PE:EA=1:1), to give the desired product (60 mg, yield 22%).

(25) m/z=517 [M+H].sup.+.

Step F: (S)—N—((R)-1-(2-chlorophenyl)-2-((3,3-difluorocyclobutyl)amino)-2-oxoethyl)-2-(4-cyanopyridin-2-yl)-N-(5-fluoropyridin-3-yl)-isothiazolidine-3-carboxamide 1,1-dioxide

(26) ##STR00046##

(27) (3S)—N-(1-(2-chlorophenyl)-2-((3,3-difluorocyclobutyl)amino)-2-oxoethyl)-N-(5-fluoropyridin-3-yl)-isothiazolidine-3-carboxamide 1,1-dioxide (60 mg, 0.116 mmol), 2-bromo-4-cyanopyridine (22 mg, 0.139 mmol), cuprous iodide (28 mg, 0.147 mmol), N,N′-dimethylethylenediamine (13 mg, 0.147 mmol) and cesium carbonate (95 mg, 0.29 mmol) were added into a sealed tube reactor, dioxane (8 mL) was added thereto, nitrogen gas was introduced thereto for 5 min and the tube was sealed. They were reacted overnight at 80° C. After the starting materials were consumed, the solvent was removed and then column chromatography (PE:EA=1:1) was performed, to give a racemic product, which was then subjected to thin layer chromatography (DCM:EA=8:1). (S)—N—((R)-1-(2-chlorophenyl)-2-((3,3-difluorocyclobutyl)amino)-2-oxoethyl)-2-(4-cyanopyridin-2-yl)-N-(5-fluoropyridin-3-yl)-isothiazolidine-3-carboxamide 1,1-dioxide (7 mg, yield 10%) was obtained.

(28) .sup.1H-NMR (400 MHz, CDCl.sub.3): δ=8.56 (d, J=5.1 Hz, 1H), 8.37 (s, 1H), 7.64 (s, 1H), 7.43 (d, J=8.1 Hz, 1H), 7.30-7.05 (m, 6H), 6.35 (s, 1H), 6.20 (s, 1H), 4.65-4.55 (m, 1H), 4.22-4.10 (m, 1H), 3.70-3.60 (m, 1H), 3.42-3.33 (m, 1H), 2.95-2.75 (m, 2H), 2.60-2.50 (m, 2H), 2.35-2.15 (m, 2H).

(29) m/z=619 [M+H].sup.+.

(30) HPLC conditions: chiral column: CHIRALPAK®IC-3 column (25 cm); mobile phase: n-hexane/ethanol=85/15; flow rate: 0.8 mL/min; column temperature: 40° C.; wavelength/time: 210 nm, 20 min; retention time: that of the title compound in Example 1 is 17.60 min.

Example 2: (S)—N—((S)-1-(2-chlorophenyl)-2-((3,3-difluorocyclobutyl)amino)-2-oxoethyl)-2-(4-cyanopyridin-2-yl)-N-(5-fluoropyridin-3-yl)-isothiazolidine-3-carboxamide 1,1-dioxide

(31) ##STR00047##

(32) In Step F of Example 1, (S)—N—((S)-1-(2-chlorophenyl)-2-((3,3-difluorocyclobutyl)amino)-2-oxoethyl)-2-(4-cyanopyridin-2-yl)-N-(5-fluoropyridin-3-yl)-isothiazolidine-3-carboxamide 1,1-dioxide was isolated by thin layer chromatography.

(33) .sup.1H-NMR (400 MHz, CDCl.sub.3): δ=8.47 (d, J=5.1 Hz, 1H), 8.31 (s, 1H), 8.08 (d, J=8.1 Hz, 1H), 7.62 (s, 1H), 7.30-6.75 (m, 6H), 6.56 (s, 1H), 6.22 (s, 1H), 4.80-4.70 (m, 1H), 4.40-4.30 (m, 1H), 3.75-3.65 (m, 1H), 3.40-3.33 (m, 1H), 3.10-2.98 (m, 2H), 2.60-2.40 (m, 4H).

(34) m/z=619 [M+H].sup.+.

(35) HPLC conditions: chiral column: CHIRALPAK®IC-3 column (25 cm); mobile phase: n-hexane/ethanol=85/15; flow rate: 0.8 mL/min; column temperature: 40° C.; wavelength/time: 210 nm, 20 min; retention time: that of the title compound in Example 2 is 17.91 min.

Example 3: (S)—N—((R)-1-(2-chlorophenyl)-2-((3,3-difluorocyclobutyl)amino)-2-oxoethyl)-2-(4-cyanopyridin-2-yl)-N-(3,5-difluorophenyl)-isothiazolidine-3-carboxamide 1,1-dioxide

(36) ##STR00048##

Step A: (3S)—N-(1-(2-chlorophenyl)-2-((3,3-difluorocyclobutyl)amino)-2-oxoethyl)-N-(3,5-difluorophenyl)-isothiazolidine-3-carboxamide 1,1-dioxide

(37) ##STR00049##

(38) Referring to Step E in Example 1, the reaction material 3-amino-5-fluoropyridine was replaced with 3,5-difluoroaniline to give the desired product of (3S)—N-(1-(2-chlorophenyl)-2-((3,3-difluorocyclobutyl)amino)-2-oxoethyl)-N-(3,5-difluorophenyl)-isothiazolidine-3-carboxamide 1,1-dioxide (yield 13%).

(39) m/z=534 [M+H].sup.+.

Step B: (S)—N—((R)-1-(2-chlorophenyl)-2-((3,3-difluorocyclobutyl)amino)-2-oxoethyl)-2-(4-cyanopyridin-2-yl)-N-(3,5-difluorophenyl)-isothiazolidine-3-carboxamide 1,1-dioxide

(40) ##STR00050##

(41) Referring to Step F in Example 1, (S)—N—((R)-1-(2-chlorophenyl)-2-((3,3-difluorocyclobutyl)amino)-2-oxoethyl)-2-(4-cyanopyridin-2-yl)-N-(3,5-difluorophenyl)-isothiazolidine-3-carboxamide 1,1-dioxide was obtained with a yield of 35%.

(42) .sup.1H-NMR (400 MHz, CDCl.sub.3): δ=8.54 (d, J=5.0 Hz, 1H), 7.67 (s, 1H), 7.43-6.76 (m, 8H), 6.11 (s, 1H), 6.01 (s, 1H), 4.80-4.72 (m, 1H), 4.16-4.2 (m, 1H), 3.68-3.65 (m, 1H), 3.38-3.34 (m, 1H), 2.80-2.98 (m, 2H), 2.58-2.54 (m, 2H), 2.28-2.30 (m, 2H).

(43) m/z=636 [M+H].sup.+.

Example 4: (S)—N—((S)-1-(2-chlorophenyl)-2-((3,3-difluorocyclobutyl)amino)-2-oxoethyl)-2-(4-cyanopyridin-2-yl)-N-(3,5-difluorophenyl)-isothiazolidine-3-carboxamide 1,1-dioxide

(44) ##STR00051##

(45) In Step B of Example 3, the title compound was isolated by thin layer chromatography with a yield of 40%.

(46) .sup.1H-NMR (400 MHz, CDCl.sub.3): δ=8.45 (d, J=5.0 Hz, 1H), 7.67 (s, 1H), 7.35-6.66 (m, 8H), 6.46 (s, 1H), 5.93 (d, J=6.8, 1H), 4.85-4.83 (m, 1H), 4.40-4.23 (m, 1H), 3.72-3.69 (m, 1H), 3.37-3.34 (m, 1H), 3.03-2.99 (m, 2H), 2.60-2.20 (m, 4H).

(47) m/z=636 [M+H].sup.+.

Example 5: (S)—N—((R)-1-(2-chlorophenyl)-2-((3,3-difluorocyclobutyl)amino)-2-oxoethyl)-2-(4-cyanopyridin-2-yl)-N-(3-trifluoromethylphenyl)-isothiazolidine-3-carboxamide 1,1-dioxide

(48) ##STR00052##

Step A: (3S)—N-(1-(2-chlorophenyl)-2-((3,3-difluorocyclobutyl)amino)-2-oxoethyl)-N-(3-trifluoromethylphenyl)-isothiazolidine-3-carboxamide 1,1-dioxide

(49) ##STR00053##

(50) Referring to Step E in Example 1, the reaction material 3-amino-5-fluoropyridine was replaced with 3-trifluoromethylaniline to give the target product, (3S)—N-(1-(2-chlorophenyl)-2-((3,3-difluorocyclobutyl)amino)-2-oxoethyl)-N-(3-trifluoromethylphenyl)-isothiazolidine-3-carboxamide 1,1-dioxide, with a yield of 50%.

(51) .sup.1H-NMR (400 MHz, CDCl.sub.3): δ=8.10 and 8.00 (s, 0.5 and 0.5H), 7.58-6.60 (m, 7H), 6.55 and 6.39 (s, 0.5 and 0.5H), 5.90 (s, 1H), 5.23 and 5.18 (d, J=7.2, 0.5 and 0.5H), 4.40-4.30 (m, 1H), 4.00-3.90 (m, 1H), 3.11-2.96 (m, 4H), 2.59-2.26 (m, 4H).

(52) m/z=566 [M+H].sup.+.

Step B: (S)—N—((R)-1-(2-chlorophenyl)-2-((3,3-difluorocyclobutyl)amino)-2-oxoethyl)-2-(4-cyanopyridin-2-yl)-N-(3-trifluoromethylphenyl)-isothiazolidine-3-carboxamide 1,1-dioxide

(53) ##STR00054##

(54) Referring to Step F in Example 1, (S)—N—((R)-1-(2-chlorophenyl)-2-((3,3-difluorocyclobutyl)amino)-2-oxoethyl)-2-(4-cyanopyridin-2-yl)-N-(3-trifluoromethylphenyl)-isothiazolidine-3-carboxamide 1,1-dioxide was obtained with a yield of 16%.

(55) .sup.1H-NMR (400 MHz, CDCl.sub.3): δ=8.54 (d, J=5.0 Hz, 1H), 8.40 (brs, 1H), 7.64-7.05 (m, 9H), 6.32 (m, 2H), 4.68-4.65 (m, 1H), 4.22-4.04 (m, 1H), 3.64-3.59 (m, 1H), 3.34-3.28 (m, 1H), 2.98-2.65 (m, 2H), 2.60-2.43 (m, 2H), 2.38-2.15 (m, 2H).

(56) m/z=668 [M+H].sup.+.

Example 6: (S)—N—((S)-1-(2-chlorophenyl)-2-((3,3-difluorocyclobutyl)amino)-2-oxoethyl)-2-(4-cyanopyridin-2-yl)-N-(3-trifluoromethylphenyl)-isothiazolidine-3-carboxamide 1,1-dioxide

(57) ##STR00055##

(58) In Step B of Example 5, (S)—N—((S)-1-(2-chlorophenyl)-2-((3,3-difluorocyclobutyl)amino)-2-oxoethyl)-2-(4-cyanopyridin-2-yl)-N-(3-trifluoromethylphenyl)-isothiazolidine-3-carboxamide 1,1-dioxide was isolated by thin layer chromatography with a yield of 23%.

(59) .sup.1H-NMR (400 MHz, CDCl.sub.3): δ=8.46 (d, J=5.0 Hz, 1H), 8.14 (s, 1H), 7.69-6.81 (m, 9H), 6.45 (s, 1H), 5.88-5.80 (m, 1H), 4.80-4.65 (m, 1H), 4.40-4.25 (m, 1H), 3.82-3.65 (m, 1H), 3.40-3.25 (m, 1H), 3.10-2.90 (m, 2H), 2.70-2.40 (m, 4H).

(60) m/z=668 [M+H].sup.+.

Example 7: (S)—N—((R)-1-(2-chlorophenyl)-2-((3,3-difluorocyclobutyl)amino)-2-oxoethyl)-2-(4-cyanopyridin-2-yl)-N-(3-cyano-5-fluorophenyl)-isothiazolidine-3-carboxamide 1,1-dioxide

(61) ##STR00056##

Step A: (3S)—N-(1-(2-chlorophenyl)-2-((3,3-difluorocyclobutyl)amino)-2-oxoethyl)-N-(3-cyano-5-fluorophenyl)-isothiazolidine-3-carboxamide 1,1-dioxide

(62) ##STR00057##

(63) Referring to Step E in Example 1, the starting material 3-amino-5-fluoropyridine was replaced with 3-fluoro-5-cyanoaniline to give the target product with a yield of 17%.

(64) m/z=541 [M+H].sup.+.

Step B: (S)—N—((R)-1-(2-chlorophenyl)-2-((3,3-difluorocyclobutyl)amino)-2-oxoethyl)-2-(4-cyanopyridin-2-yl)-N-(3-cyano-5-fluorophenyl)-isothiazolidine-3-carboxamide 1,1-dioxide

(65) ##STR00058##

(66) Referring to Step F in Example 1, (S)—N—((R)-1-(2-chlorophenyl)-2-((3,3-difluorocyclobutyl)amino)-2-oxoethyl)-2-(4-cyanopyridin-2-yl)-N-(3-cyano-5-fluorophenyl)-isothiazolidine-3-carboxamide 1,1-dioxide was obtained with a yield of 15%.

(67) .sup.1H-NMR (400 MHz, CDCl.sub.3): δ=8.83 (d, J=6.0 Hz, 1H), 8.57 (d, J=5.0 Hz, 1H), 7.66-7.12 (m, 8H), 6.29 (s, 1H), 6.16 (s, 1H), 4.63-4.61 (m, 1H), 4.21-4.10 (m, 1H), 3.70-3.60 (m, 1H), 3.40-3.25 (m, 1H), 3.30-2.82 (m, 2H), 2.61-2.42 (m, 2H), 2.40-2.20 (m, 2H).

(68) m/z=643 [M+H].sup.+.

Example 8: (S)—N—((S)-1-(2-chlorophenyl)-2-((3,3-difluorocyclobutyl)amino)-2-oxoethyl)-2-(4-cyanopyridin-2-yl)-N-(3-cyano-5-fluorophenyl)-isothiazolidine-3-carboxamide 1,1-dioxide

(69) ##STR00059##

(70) In Step B of Example 7, the title compound was isolated by thin layer chromatography with a yield of 20%.

(71) .sup.1H-NMR (400 MHz, CDCl.sub.3): δ=8.47-6.85 (m, 10H), 6.50 (d, J=9.77 Hz, 1H), 5.97 (s, 1H), 4.75-4.73 (m, 1H), 4.40-4.30 (m, 1H), 3.80-3.65 (m, 1H), 3.42-3.27 (m, 1H), 3.15-2.30 (m, 6H).

(72) m/z=643 [M+H].sup.+.

Example 9: (S)—N—((R)-1-(2-chlorophenyl)-2-((3,3-difluorocyclobutyl)amino)-2-oxoethyl)-2-(4-cyanopyridin-2-yl)-N-(3,4-difluorophenyl)-isothiazolidine-3-carboxamide 1,1-dioxide

(73) ##STR00060##

Step A: (3S)—N-(1-(2-chlorophenyl)-2-((3,3-difluorocyclobutyl)amino)-2-oxoethyl)-N-(3,4-difluorophenyl)-isothiazolidine-3-carboxamide 1,1-dioxide

(74) ##STR00061##

(75) Referring to Step E of Example 1, the starting material 3-amino-5-fluoropyridine was replaced with 3,4-difluoroaniline to give the target product with a yield of 18%.

(76) m/z=534 [M+H].sup.+.

Step B: (S)—N—((R)-1-(2-chlorophenyl)-2-((3,3-difluorocyclobutyl)amino)-2-oxoethyl)-2-(4-cyanopyridin-2-yl)-N-(3,4-difluorophenyl)-isothiazolidine-3-carboxamide 1,1-dioxide

(77) ##STR00062##

(78) Referring to Step F of Example 1, (S)—N—((R)-1-(2-chlorophenyl)-2-((3,3-difluorocyclobutyl)amino)-2-oxoethyl)-2-(4-cyanopyridin-2-yl)-N-(3,4-difluorophenyl)-isothiazolidine-3-carboxamide 1,1-dioxide was obtained with a yield of 30%.

(79) .sup.1H-NMR (400 MHz, CDCl.sub.3): δ=8.55 (s, 1H), 7.62 (s, 1H), 7.40-7.15 (m, 8H), 6.43 (s, 1H), 6.27 (s, 1H), 4.70-4.68 (m, 1H), 4.15-4.11 (m, 1H), 3.61-3.53 (m, 1H), 3.32-3.26 (m, 1H), 2.89-2.69 (m, 2H), 2.57-2.53 (m, 2H), 2.28-2.11 (m, 2H).

(80) m/z=636 [M+H].sup.+.

Example 10: (S)—N—((S)-1-(2-chlorophenyl)-2-((3,3-difluorocyclobutyl)amino)-2-oxoethyl)-2-(4-cyanopyridin-2-yl)-N-(3,4-difluorophenyl)-isothiazolidine-3-carboxamide 1,1-dioxide

(81) ##STR00063##

(82) In Step B of Example 9, the title compound was isolated by thin layer chromatography with a yield of 35%.

(83) .sup.1H-NMR (400 MHz, CDCl.sub.3): δ=8.47-8.43 (m, 1H), 7.66 (s, 1H), 7.35-6.82 (m, 8H), 6.44 (d, J=5.78, 1H), 5.97 (d, J=6.57, 1H), 4.81-4.79 (m, 1H), 4.33-4.32 (m, 1H), 3.73-3.69 (m, 1H), 3.36-3.33 (m, 1H), 3.02-2.99 (m, 2H), 2.61-2.40 (m, 4H).

(84) m/z=636 [M+H].sup.+.

Example 11: (S)—N—((R)-1-(2-chlorophenyl)-2-((3,3-difluorocyclobutyl)amino)-2-oxoethyl)-2-(4-cyanopyridin-2-yl)-N-(3-fluorophenyl)-isothiazolidine-3-carboxamide 1,1-dioxide

(85) ##STR00064##

Step A: methyl (S)-2-(4-cyanopyridin-2-yl)isothiazolidine-3-carboxylate 1,1-dioxide

(86) ##STR00065##

(87) Methyl (S)-isothiazolidine-3-carboxylate 1,1-dioxide (prepared by Step C of Example 1, 200 mg, 1.11 mmol), 2-bromo-4-cyanopyridine (204 mg, 1.11 mmol), cuprous iodide (105 mg, 0.55 mmol), N,N′-dimethylethylenediamine (98 mg, 1.11 mmol) and cesium carbonate (723 mg, 2.22 mmol) were added into a sealed tube reactor, dioxane (8 mL) was added thereto, nitrogen gas was introduced thereto for 5 min and the tube was sealed. They were reacted overnight at 80° C. After the starting materials were consumed, the solvent was removed and then separation by column chromatography (PE:EA=1:1) was performed. The target product methyl (S)-2-(4-cyanopyridin-2-yl)isothiazolidine-3-carboxylate 1,1-dioxide (230 mg, yield 74%) was obtained.

(88) .sup.1H-NMR (400 MHz, CDCl.sub.3): δ=8.40 (dd, J=5.2, 0.8 Hz, 1H), 7.69 (t, J=1.0 Hz, 1H), 7.19 (dd, J=5.2, 1.0 Hz, 1H), 5.01 (dd, J=8.0, 3.6 Hz, 1H), 3.78 (s, 3H), 3.64-3.55 (m, 1H), 3.48-3.42 (m, 1H), 2.95-2.84 (m, 1H), 2.65-2.52 (m, 1H).

Step B: (S)-2-(4-cyanopyridin-2-yl)isothiazolidine-3-carboxylic acid 1,1-dioxide

(89) ##STR00066##

(90) Under stirring in an ice bath, a suspension of lithium hydroxide was added dropwise into a solution of methyl (S)-2-(4-cyanopyridin-2-yl)isothiazolidine-3-carboxylate 1,1-dioxide (116 mg, 0.41 mmol) in methanol-tetrahydrofuran, reacting overnight. After the reaction was finished, it was diluted with 10 mL water, and extracted with ethyl acetate to remove impurities. The aqueous phase was added dropwise with 1 N hydrochloric acid to make the pH thereof less than 5, and then extracted with ethyl acetate. The solvent was removed to give (S)-2-(4-cyanopyridin-2-yl)isothiazolidine-3-carboxylic acid 1,1-dioxide (103 mg, yield 94%).

(91) .sup.1H-NMR (400 MHz, DMSO-d.sub.6): δ=13.5 (s, 1H), 8.54 (d, J=5.0, 1H), 7.51 (dd, J=3.74, 4.76 Hz, 1H), 7.45 (s, 1H), 4.95-4.90 (m, 1H), 3.75-3.60 (m, 2H), 2.85-2.72 (m, 1H), 2.46-2.38 (m, 1H).

Step C: (S)—N—((R)-1-(2-chlorophenyl)-2-((3,3-difluorocyclobutyl)amino)-2-oxoethyl)-2-(4-cyanopyridin-2-yl)-N-(3-fluorophenyl)-isothiazolidine-3-carboxamide 1,1-dioxide

(92) ##STR00067##

(93) Referring to Step E in Example 1, the starting material (S)-isothiazolidine-3-carboxylic acid 1,1-dioxide was replaced with (S)-2-(4-cyanopyridin-2-yl)isothiazolidine-3-carboxylic acid 1,1-dioxide to give the target product, (S)—N—((R)-1-(2-chlorophenyl)-2-((3,3-difluorocyclobutyl)amino)-2-oxoethyl)-2-(4-cyanopyridin-2-yl)-N-(3-fluorophenyl)-isothiazolidine-3-carboxamide 1,1-dioxide, with a yield of 30%.

(94) .sup.1H-NMR (400 MHz, CDCl.sub.3): δ=8.56 (d, J=5.0 Hz, 1H), 7.62 (s, 1H), 7.39 (d, J=8.1 Hz, 1H), 7.21-6.99 (m, 8H), 6.43 (s, 1H), 6.23 (s, 1H), 4.73 (dd, J=6.5, 3.1 Hz, 1H), 4.20-4.05 (m, 1H), 3.57 (dd, J=20.0, 11.9 Hz, 1H), 3.27 (dd, J=11.9, 3.5 Hz, 1H), 2.84-2.72 (m, 2H), 2.55 (dd, J=14.9, 9.3 Hz, 2H), 2.28-2.13 (m, 2H).

(95) m/z=618 [M+H].sup.+.

Example 12: (S)—N—((S)-1-(2-chlorophenyl)-2-((3,3-difluorocyclobutyl)amino)-2-oxoethyl)-2-(4-cyanopyridin-2-yl)-N-(3-fluorophenyl)-isothiazolidine-3-carboxamide 1,1-dioxide

(96) ##STR00068##

(97) In Step C of Example 11, the title compound was isolated by thin layer chromatography with a yield of 33%.

(98) .sup.1H-NMR (400 MHz, CDCl.sub.3): δ=8.46 (m, 1H), 7.67 (d, J=8.8 Hz, 1H), 7.63 (s, 1H), 7.22-6.84 (m, 8H), 6.47 (d, J=3.6, 1H), 6.08 (s, 1H), 4.82 (d, J=6.1 Hz, 1H), 4.33 (m, 1H), 3.68-3.60 (m, 1H), 3.40-3.28 (m, 1H), 3.10-2.98 (m, 2H), 2.68-2.38 (m, 4H).

(99) m/z=618 [M+H].sup.+.

Example 13: (S)—N—((R)-1-(2-chlorophenyl)-2-(cyclohexylamino)-2-oxoethyl)-2-(pyrimidin-2-yl)-N-(3-fluorophenyl)-isothiazolidine-3-carboxamide 1,1-dioxide

(100) ##STR00069##

Step A: (S)—N-(1-(2-chlorophenyl)-2-(cyclohexylamino)-2-oxoethyl)-N-(3-fluorophenyl)-isothiazolidine-3-carboxamide 1,1-dioxide

(101) ##STR00070##

(102) Referring to Step E in Example 1, the reaction materials 2,2-difluorocyclobutyl isocyanide and 3-amino-5-fluoropyridine were replaced with cyclohexyl isocyanide and 3-fluoroaniline. The target product was obtained with a yield of 81%.

(103) .sup.1H-NMR (400 MHz, CDCl.sub.3): δ=7.70-6.86 (m, 8H), 6.50-6.25 (m, 1H), 5.41-5.35 (m, 1H), 5.25-5.10 (m, 1H), 4.05-4.95 (m, 1H), 3.90-3.80 (m, 1H), 3.12-2.90 (m, 2H), 2.65-1.00 (m, 12H).

(104) m/z=508 [M+H].sup.+.

Step B: (S)—N—((R)-1-(2-chlorophenyl)-2-(cyclohexylamino)-2-oxoethyl)-2-(pyrimidin-2-yl)-N-(3-fluorophenyl)-isothiazolidine-3-carboxamide 1,1-dioxide

(105) ##STR00071##

(106) Referring to Step F in Example 1, (S)—N—((R)-1-(2-chlorophenyl)-2-(cyclohexylamino)-2-oxoethyl)-2-(pyrimidin-2-yl)-N-(3-fluorophenyl)-isothiazolidine-3-carboxamide 1,1-dioxide was obtained with a yield of 26%.

(107) .sup.1H-NMR (400 MHz, CDCl.sub.3): δ=8.61 (d, J=4.9 Hz, 2H), 7.75 (brs, 1H), 7.42-7.37 (m, 2H), 7.26-7.21 (m, 2H), 7.01-7.07 (m, 1H), 7.02-6.96 (m, 2H), 6.85 (brs, 1H), 6.12 (s, 1H), 5.71 (d, J=8.4 Hz, 1H), 4.75 (d, J=5.6 Hz, 1H), 3.76-3.68 (m, 2H), 3.34-3.30 (m, 1H), 2.53-2.46 (m, 2H), 1.86-1.78 (m, 2H), 1.52-1.62 (m, 4H), 1.24-1.30 (m, 2H), 1.12-0.73 (m, 2H).

(108) m/z=586 [M+H].sup.+.

Example 14: (S)—N—((S)-1-(2-chlorophenyl)-2-(cyclohexylamino)-2-oxoethyl)-2-(pyrimidin-2-yl)-N-(3-fluorophenyl)-isothiazolidine-3-carboxamide 1,1-dioxide

(109) ##STR00072##

(110) In Step B of Example 13, the title compound was isolated by thin layer chromatography with a yield of 30%.

(111) .sup.1H-NMR (400 MHz, CDCl.sub.3): δ=8.57 (d, J=4.9 Hz, 2H), 7.74 (s, 1H), 7.43-6.88 (m, 7H), 6.49 (s, 1H), 5.40 (d, J=7.9 Hz, 1H), 4.79 (s, 1H), 3.84-3.75 (m, 2H), 3.33-3.30 (m, 1H), 2.62-2.37 (m, 2H), 1.86-1.78 (m, 2H), 1.52-1.62 (m, 4H), 1.24-1.30 (m, 2H), 1.12-0.73 (m, 2H).

(112) m/z=586 [M+H].sup.+.

Example 15: (S)—N—((R)-1-(2-chlorophenyl)-2-(cyclohexylamino)-2-oxoethyl)-2-(4-cyanopyridin-2-yl)-N-(3-fluorophenyl)-isothiazolidine-3-carboxamide 1,1-dioxide

(113) ##STR00073##

(114) Referring to Step F in Example 1, the target product was obtained from (S)—N-(1-(2-chlorophenyl)-2-(cyclohexylamino)-2-oxoethyl)-N-(3-fluorophenyl)-isothiazolidine-3-carboxamide 1,1-dioxide (prepared by Step A of Example 13) with a yield of 39%.

(115) .sup.1H-NMR (400 MHz, CDCl.sub.3): δ=8.56 (d, J=5.0 Hz, 1H), 7.70 (s, 1H), 7.67 (s, 1H), 7.35-6.99 (m, 8H), 6.12 (s, 1H), 5.70 (d, J=7.4 Hz, 1H), 4.75-4.74 (m, 1H), 3.76-3.56 (m, 2H), 3.34-3.36 (m, 1H), 2.66-2.43 (m, 2H), 1.85-1.71 (m, 2H), 1.62-1.56 (m, 4H), 1.28-1.24 (m, 2H), 1.12-0.85 (m, 2H).

(116) m/z=610 [M+H].sup.+.

Example 16: (S)—N—((S)-1-(2-chlorophenyl)-2-(cyclohexylamino)-2-oxoethyl)-2-(4-cyanopyridin-2-yl)-N-(3-fluorophenyl)-isothiazolidine-3-carboxamide 1,1-dioxide

(117) ##STR00074##

(118) In Step B of Example 15, the title compound was isolated by thin layer chromatography with a yield of 39%.

(119) .sup.1H-NMR (400 MHz, CDCl.sub.3): δ=8.46 (d, J=5.0 Hz, 1H), 7.75-6.88 (m, 10H), 6.45 (s, 1H), 5.36-5.30 (m, 1H), 4.82-4.81 (m, 1H), 3.82-3.74 (m, 2H), 3.34-3.32 (m, 1H), 2.67-2.65 (m, 1H), 2.45-2.40 (m, 1H), 1.97-1.94 (m, 2H), 1.74-1.59 (m, 4H), 1.38-1.24 (m, 2H), 1.17-1.01 (m, 2H).

(120) m/z=610 [M+H].sup.+.

Example 17: (S)—N—((R)-1-(2-chlorophenyl)-2-(cyclohexylamino)-2-oxoethyl)-2-(4-fluoropyridin-2-yl)-N-(3-fluorophenyl)-isothiazolidine-3-carboxamide 1,1-dioxide

(121) ##STR00075##

(122) Referring to Step F in Example 1, (S)—N-(1-(2-chlorophenyl)-2-(cyclohexylamino)-2-oxoethyl)-N-(3-fluorophenyl)-isothiazolidine-3-carboxamide 1,1-dioxide (prepared by Step A of Example 13) was coupled with 2-bromo-4-fluoropyridine to give the target product with a yield of 25%.

(123) .sup.1H-NMR (400 MHz, CDCl.sub.3): δ=8.32 (dd, J=8.4, 5.9 Hz, 1H), 7.45-6.73 (m, 10H), 6.03 (s, 1H), 5.73 (s, 1H), 4.88-4.68 (m, 1H), 3.76-3.62 (m, 2H), 3.29 (ddd, J=12.0, 6.7, 2.6 Hz, 1H), 2.67-2.39 (m, 2H), 1.85-1.75 (m, 2H), 1.62-1.56 (m, 2H), 1.32-1.25 (m, 3H), 1.05-0.91 (m, 3H).

(124) m/z=603 [M+H].sup.+.

Example 18: (S)—N—((S)-1-(2-chlorophenyl)-2-(cyclohexylamino)-2-oxoethyl)-2-(4-fluoropyridin-2-yl)-N-(3-fluorophenyl)-isothiazolidine-3-carboxamide 1,1-dioxide

(125) ##STR00076##

(126) In Step B of Example 17, the title compound was isolated by thin layer chromatography with a yield of 25%.

(127) .sup.1H-NMR (400 MHz, CDCl.sub.3): δ=8.26 (s, 1H), 7.37-6.72 (m, 10H), 6.45 (s, 1H), 5.37 (d, J=8.1, 1H), 4.83 (d, J=7.7, 1H), 3.83-3.70 (m, 2H), 3.29 (ddd, J=12.0, 6.7, 2.6 Hz, 1H), 2.65-2.62 (m, 1H), 2.42-2.25 (m, 1H), 1.94-1.91 (m, 2H), 1.68-1.56 (m, 2H), 1.32-1.25 (m, 3H), 1.05-0.91 (m, 3H).

(128) m/z=603 [M+H].sup.+.

Example 19: (3S)—N-(1-(2-chlorophenyl)-2-(cyclohexylamino)-2-oxoethyl)-N-(3-fluorophenyl)-2-(4-ethynylpyridin-2-yl)isothiazolidine-3-carboxamide 1,1-dioxide

(129) ##STR00077##

Step A: 2-bromo-4-(2,2-dibromovinyl)pyridine

(130) ##STR00078##

(131) At 0° C., triphenylphosphine (4.23 g, 16.13 mmol) was added to a solution of carbon tetrabromide (2.68 g, 8.08 mmol) in dichloromethane, stirring the same for 5 min, followed by adding 2-bromo-4-aldehyde pyridine (0.50 g, 2.69 mmol) in methanol, warming up the same to room temperature and continuing to stir for a further 30 min. After being completed, the reaction was stopped by adding water. It was extracted with ethyl acetate (30 mL×3) and the organic phase was dried over anhydrous sodium sulfate. The product (140 mg, yield 15%) was obtained by isolating through silica gel column chromatography (PE:EA=10:1).

(132) .sup.1H-NMR (400 MHz, CDCl.sub.3): δ=8.39-8.38 (m, 1H), 7.63-7.62 (m, 1H), 7.40-7.38 (m, 2H).

Step B: 2-bromo-4-((trimethylsilyl)ethynyl)pyridine

(133) ##STR00079##

(134) At −78° C., 2.4M n-butyllithium (358 μL, 0.86 mmol) was added dropwise to a solution of 2-bromo-4-(2,2-dibromovinyl)pyridine (140 mg, 0.41 mmol) in tetrahydrofuran. After stirring for 30 min, trimethylchlorosilane (53 μL, 0.61 mmol) was further added thereto. They were stirred at −78° C. for 1 h, then warmed up to room temperature and further stirred for 30 min. After being completed, the reaction was stopped by adding water. It was extracted with ethyl acetate (30 mL×3) and the organic phase was dried over anhydrous sodium sulfate. The product (30 mg, yield 29%) was obtained by isolating through silica gel column chromatography (PE:EA=20:1).

(135) .sup.1H-NMR (400 MHz, CDCl.sub.3): δ=8.32-8.30 (m, 1H), 7.52-7.51 (m, 1H), 7.27-7.24 (m, 1H), 0.28-0.25 (m, 9H).

Step C: (3S)—N-(1-(2-chlorophenyl)-2-(cyclohexylamino)-2-oxoethyl)-N-(3-fluorophenyl)-2-(4-ethynylpyridin-2-yl)isothiazolidine-3-carboxamide 1,1-dioxide

(136) ##STR00080##

(137) At 80° C., in 1,4-dioxane (8 mL), (S)—N-(1-(2-chlorophenyl)-2-(cyclohexylamino)-2-oxoethyl)-N-(3-fluorophenyl)-isothiazolidine-3-carboxamide 1,1-dioxide (prepared by Step A of Example 13, 60 mg, 0.12 mmol), 2-bromo-4-[(trimethylsilyl)ethynyl]pyridine (30 mg, 0.12 mmol), cuprous iodide (12 mg, 0.06 mmol), N,N′-dimethylethylenediamine (13 μL, 0.12 mmol) and cesium carbonate (77 mg, 0.24 mmol) were stirred overnight. After the reaction was completed, the mixture was filtered and the mother liquor was concentrated. The title compound (5 mg, yield 7%) was obtained by isolating through silica gel column chromatography (PE:EA=1:1).

(138) .sup.1H-NMR (400 MHz, CDCl.sub.3): δ=8.36-8.30 (m, 1H), 7.73 (m, 1H), 7.55 (m, 1H), 7.46-7.30 (m, 2H), 7.24-6.88 (m, 6H), 6.46 and 6.03 (s, 1H), 5.74 and 5.39 (d, J=6.4 Hz, 1H), 4.80 and 4.75 (m, 1H), 3.81-3.66 (m, 3H), 3.31-3.28 (m, 1H), 2.62-2.49 (m, 2H), 2.05-1.55 (m, 6H), 1.15-0.84 (m, 4H).

(139) m/z=609 [M+H].sup.+.

Example 20: (3S)—N-(1-(2-chlorophenyl)-2-(cyclohexylamino)-2-oxoethyl)-2-(4-cyanopyridin-2-yl)-N-(3-(N-(4-cyanopyridin-2-yl)aminosulfonyl)phenyl)-isothiazolidine-3-carboxamide 1,1-dioxide

(140) ##STR00081##

Step A: (3S)—N-(1-(2-chlorophenyl)-2-(cyclohexylamino)-2-oxoethyl)-2-N-(3-aminosulfonyl)-isothiazolidine-3-carboxamide 1,1-dioxide

(141) ##STR00082##

(142) Referring to Step E in Example 1, the starting material 3-fluoro-5-aminopyridine was replaced with 3-aminobenzenesulfonamide, so as to give the target product with a yield of 53%.

(143) m/z=569 [M+H].sup.+.

Step B: (3S)—N-(1-(2-chlorophenyl)-2-(cyclohexylamino)-2-oxoethyl)-2-(4-cyanopyridin-2-yl)-N-(3-(N-(4-cyanopyridin-2-yl)aminosulfonyl)phenyl)-isothiazolidine-3-carboxamide 1,1-dioxide

(144) ##STR00083##

(145) Referring to Step F of Example 1, the title compound was obtained with a yield of 25%.

(146) .sup.1H-NMR (400 MHz, CDCl.sub.3): δ=8.60-6.60 (m, 14H), 6.41-6.39 (s, 1H), 6.18-5.56 (s, 1H), 5.44-5.36 (m, 1H), 4.62-4.59 (m, 1H), 3.90-3.61 (m, 2H), 3.40-3.33 (m, 1H), 2.60-2.24 (m, 2H), 2.00-0.80 (m, 10H).

(147) m/z=773 [M+H].sup.+.

Example 21: (S)—N-(1-(2-chlorophenyl)-2-(cyclohexylamino)-2-oxoethyl)-2-(3-cyanophenylethyl)-N-(3-fluorophenyl)-isothiazolidine-3-carboxamide 1,1-dioxide

(148) ##STR00084##

Step A: methyl (S)-2-(3-cyanophenylethyl-2-yl)isothiazolidine-3-carboxylate 1,1-dioxide

(149) ##STR00085##

(150) Methyl (S)-isothiazolidine-3-carboxylate 1,1-dioxide (179 mg, 1.0 mmol), 3-cyanobenzyl bromide (392 mg, 2.0 mmol), tetrabutylammonium iodide (37 mg, 0.1 mmol) were dissolved in DMF (3 mL). The reaction solution was stirred overnight at room temperature, diluted by adding water, extracted with ethyl acetate, dried, filtered and concentrated. The crude product was obtained by isolating through silica gel column chromatography to give the target product (260 mg, yield 88%)

(151) .sup.1H-NMR (400 MHz, CDCl.sub.3): δ=7.66 (s, 1H), 7.65-7.59 (m, 2H), 7.47 (t, J=7.6 Hz, 1H), 4.41 (q, J=15.6 Hz, 2H), 3.85-3.82 (m, 1H), 3.63 (s, 3H) 3.40-3.25 (m, 1H), 3.23-3.16 (m, 1H), 2.66-2.42 (m, 2H).

Step B: (S)-methyl 2-(3-cyanophenylethyl-2-yl)isothiazolidine-3-carboxylic acid 1,1-dioxide

(152) ##STR00086##

(153) Referring to Step B in Example 11, the target product was obtained with a yield of 75%.

(154) .sup.1H-NMR (400 MHz, CDCl.sub.3): δ=7.81 (s, 1H), 7.77-7.54 (m, 3H), 4.35 (s, 2H), 3.98-3.96 (m, 1H), 3.38-3.26 (m, 2H), 2.66-2.50 (m, 1H), 2.49-2.26 (m, 1H).

Step C: (S)—N-(1-(2-chlorophenyl)-2-(cyclohexylamino)-2-oxoethyl)-2-(3-cyanophenylethyl)-N-(3-fluorophenyl)-isothiazolidine-3-carboxamide 1,1-dioxide

(155) ##STR00087##

(156) Referring to Step E in Example 1, the title compound was obtained with a yield of 79%.

(157) .sup.1H-NMR (400 MHz, CDCl.sub.3): δ=7.80-6.75 (m, 12H), 6.41 and 6.37 (s, 0.5 and 0.5H), 5.41 and 5.35 (m, 0.5 and 0.5H), 4.62 and 4.53 (d, J=16, 0.5 and 0.5H), 4.15 and 4.04 (d, J=15.2, 16, 0.5 and 0.5H), 3.96-3.78 (m, 1H), 3.65-3.60 (m, 1H), 3.45-3.40 (m, 1H), 3.18-3.02 (m, 1H), 2.40-2.20 (m, 2H), 2.02-1.80 (m, 2H), 1.79-1.45 (m, 4H), 1.40-1.02 (m 4H).

(158) m/z=623 [M+H].sup.+.

Example 22: (S)—N—((R)-1-(2-chlorophenyl)-2-((4,4-difluorocyclohexyl)amino))-2-oxoethyl)-2-(4-cyanopyridin-2-yl)-N-(3-fluorophenyl)-isothiazolidine-3-carboxamide 1,1-dioxide

(159) ##STR00088##

Step A: (3S)—N-(1-(2-chlorophenyl)-2-((4,4-difluorocyclohexyl)amino)-2-oxoethyl)-N-(3-fluorophenyl)isothiazolidine-3-carboxamide 1,1-dioxide

(160) ##STR00089##

(161) Under stirring at room temperature, in MeOH (6.0 mL), 2-chloro-benzaldehyde (85 mg, 0.605 mmol) and 3-fluoroaniline (67 mg, 0.605 mmol) were mixed for 30 min. (S)-isothiazolinone-3-carboxylic acid 1,1-dioxide (150 mg, 0.908 mmol) was added and the reaction mixture was stirred for 10 min, followed by adding 1,1-difluoro-4-isocyanocyclohexane (88 mg, 0.605 mmol), and stirring overnight at room temperature. The solvent was removed under vacuum. (3S)—N-(1-(2-chlorophenyl)-2-((4,4-difluorocyclohexyl)amino)-2-oxoethyl)-N-(3-fluorophenyl)isothiazolidine-3-carboxamide 1,1-dioxide (114 mg, yield 34.7%), which was directly used in the next step, was obtained by isolating through silica gel column chromatography.

(162) m/z=544 [M+H].sup.+.

Step B: (S)—N—((R)-1-(2-chlorophenyl)-2-((4,4-difluorocyclohexyl)amino))-2-oxoethyl)-2-(4-cyanopyridin-2-yl)-N-(3-fluorophenyl)-isothiazolidine-3-carboxamide 1,1-dioxide

(163) ##STR00090##

(164) (3S)—N-(1-(2-chlorophenyl)-2-((4,4-difluorocyclohexyl)amino)-2-oxoethyl)-N-(3-fluorophenyl)isothiazolidine-3-carboxamide 1,1-dioxide (114 mg, 0.210 mmol), 2-bromo-4-cyanopyridine (47 mg, 0.252 mmol), cuprous iodide (21 mg, 0.11 mmol), N,N′-dimethylethylenediamine (19 mg, 0.21 mmol) and cesium carbonate (206 mg, 0.63 mmol) were added into a sealed tube reactor, dioxane (8 mL) was added thereto, nitrogen gas was introduced thereto for 5 min, the tube was sealed and the reaction was carried out at 80° C. overnight. After the solvent was removed, the column chromatography (PE:EA=1:1) was performed to give a racemic product, which was then subjected to thin layer chromatography (DCM:EA=8:1), so as to give the pure chiral compound (S)—N—((R)-1-(2-chlorophenyl)-2-((4,4-difluorocyclohexyl)amino))-2-oxoethyl)-2-(4-cyanopyridin-2-yl)-N-(3-fluorophenyl)-isothiazolidine-3-carboxamide 1,1-dioxide (39 mg, yield 28.7%).

(165) .sup.1H-NMR (400 MHz, CDCl.sub.3): δ=8.55 (d, J=4.7 Hz, 1H), 7.67 (s, 1H), 7.39 (d, J=7.7 Hz, 1H), 7.33-7.18 (m, 5H), 7.07 (dd, J=24.5, 16.7 Hz, 2H), 6.95-6.69 (m, 1H), 6.10 (s, 1H), 5.70 (d, J=5.6 Hz, 1H), 4.76 (d, J=6.9 Hz, 1H), 3.72-3.70 (m, 2H), 3.36-3.31 (m, 1H), 2.58-2.54 (m, 2H), 2.07-1.64 (m, 5H), 1.41-1.17 (m, 3H).

(166) m/z=646 [M+H].sup.+.

Example 23: (S)—N—((S)-1-(2-chlorophenyl)-2-((4,4-difluorocyclohexyl)amino)-2-oxoethyl)-2-(4-cyanopyridin-2-yl)-N-(3-fluorophenyl)-isothiazolidine-3-carboxamide 1,1-dioxide

(167) ##STR00091##

(168) In Step B of Example 22, (S)—N—((S)-1-(2-chlorophenyl)-2-((4,4-difluorocyclohexyl)amino)-2-oxoethyl)-2-(4-cyanopyridin-2-yl)-N-(3-fluorophenyl)-isothiazolidine-3-carboxamide 1,1-dioxide (40 mg, yield 29.4%) was obtained by isolating through thin layer chromatography.

(169) .sup.1H-NMR (400 MHz, CDCl.sub.3): δ=8.46 (s, 1H), 7.71-7.68 (m, 2H), 7.33 (d, J=13.3 Hz, 1H), 7.08-7.01 (m, 6H), 6.45 (s, 1H), 5.43 (d, J=7.6 Hz, 1H), 4.81 (d, J=7.2 Hz, 1H), 3.98-3.93 (m, 1H), 3.75-3.70 (m, 1H), 3.34 (s, 1H), 2.63 (s, 1H), 2.46 (s, 1H), 2.11-2.05 (m, 4H), 1.89-1.81 (m, 2H), 1.63-1.35 (m, 2H), 0.88 (s, 1H).

(170) m/z=646 [M+H].sup.+.

Example 24: (S)—N—((S)-1-(2-chlorophenyl)-2-((3,3-difluorocyclobutyl)amino)-2-oxoethyl)-2-(4-cyanopyridin-2-yl)-N-(3-fluorophenyl)-1,2,5-thiadiazolidine-3-carboxamide 1,1-dioxide

(171) ##STR00092##

Step A: methyl (S)-2-(benzylamino)-3-hydroxypropionate

(172) ##STR00093##

(173) Under stirring at room temperature, benzaldehyde (13.6 g, 128.6 mmol) and anhydrous sodium sulfate (6.0 g) were added into a 500 mL round-bottomed flask, which contained a solution of the mixture of L-serine methyl ester hydrochloride (20 g, 128.6 mmol) and triethylamine (13 g, 128.6 mmol) in dichloromethane. They were stirred to react overnight at room temperature. After the reaction was completed, it was filtered and concentrated. The concentrated residue was dissolved by adding methanol, and sodium borohydride (4.86 g, 128.6 mmol) was carefully added in portions under an ice bath. They were reacted for 1 hour at room temperature. Methanol was removed, and it was diluted with dichloromethane, washed with saturated aqueous solution of sodium bicarbonate, extracted with dichloromethane for three times. The combined organic phase was washed with brine, dried over sodium sulfate, filtered and concentrated under vacuum. Methyl (S)-2-(benzylamino)-3-hydroxypropionate (20.6 g, yield 77%) was obtained.

(174) .sup.1H-NMR (400 MHz, CDCl.sub.3): δ=7.38-7.26 (m, 5H), 4.48 (d, J=12.8 Hz, 1H), 3.8-3.72 (m, 6H), 3.61 (dd, J=10.8, 10.8 Hz, 1H), 3.54 (dd, J=6.8, 6.4 Hz, 1H).

Step B: methyl (S)-2-(benzyl(N-(tert-butoxycarbonyl)sulfonyl)amino)-3-hydroxypropionate

(175) ##STR00094##

(176) Under stirring in an ice bath, triethylamine (7.25 g, 71.69 mmol) and tert-butyl chlorosulfonyl carbamate (10.3 g, 47.79 mmol) were added into a solution of methyl (S)-2-(benzylamino)-3-hydroxypropionate (10.0 g, 47.79 mmol) in dichloromethane. They were reacted with stirring overnight at room temperature. After the reaction was completed, it was diluted by adding dichloromethane, quenched by adding water, extracted with dichloromethane for three times. The combined organic phase was washed with brine, dried over sodium sulfate, filtered and concentrated under vacuum. Methyl (S)-2-(benzyl(N-(tert-butoxycarbonyl)sulfonyl)amino)-3-hydroxypropionate (6.43 g, yield 35%) was obtained by isolating through column chromatography.

(177) .sup.1H-NMR (400 MHz, CDCl.sub.3): δ=7.44-7.28 (m, 6H), 4.67 (dd, J=14.3, 6.1 Hz, 2H), 4.56 (d, J=15.6 Hz, 1H), 4.00 (d, J=7.2 Hz, 2H), 3.69 (s, 3H), 2.84 (s, 1H), 1.48 (s, 9H).

Step C: (S)-2-tert-butyl-4-methyl-5-benzyl-1,2,5-thiadiazolidine-2,4-dicarboxylate 1,1-dioxide

(178) ##STR00095##

(179) Under stirring in an ice bath, DIAD (4.0 g, 19.84 mmol) was added into a solution of methyl (S)-2-(benzyl(N-(tert-butoxycarbonyl)sulfonyl)amino)-3-hydroxypropionate (6.42 g, 16.54 mmol) and triphenylphosphine (5.2 g, 19.84 mmol) in dichloromethane. They were reacted for 2 h at room temperature. After the reaction was completed, it was diluted with dichloromethane, quenched with water, extracted with dichloromethane for three times. The combined organic phase was washed with brine, dried over sodium sulfate, filtered and concentrated under vacuum. The target compound (5.77 g, yield 94.3%) was obtained by isolating through column chromatography.

(180) .sup.1H-NMR (400 MHz, CDCl.sub.3): δ=7.43-7.28 (m, 5H), 4.54 (d, J=14.4 Hz, 1H), 4.44 (d, J=14.4 Hz, 1H), 4.07-4.00 (m, 1H), 3.89 (t, J=2.9 Hz, 2H), 3.72 (s, 3H), 1.55 (s, 9H).

Step D: (S)-2-tert-butyl 4-methyl-1,2,5-thiadiazolidine-2,4-dicarboxylate 1,1-dioxide

(181) ##STR00096##

(182) Under stirring at room temperature, 10 wt % palladium carbon (10 g) was added into a solution of (S)-2-tert-butyl-4-methyl-5-benzyl-1,2,5-thiadiazolidine-2,4-dicarboxylate 1,1-dioxide in dichloromethane (2.97 g, 8.02 mmol). The gas was exchanged 10 times by circulating water pump. Under the protection of hydrogen gas, they were reacted overnight at room temperature. After the reaction was completed, it was filtered and concentrated. (S)-2-tert-butyl 4-methyl-1,2,5-thiadiazolidine-2,4-dicarboxylate 1,1-dioxide (1.49 g, yield 66%) was obtained by isolating through column chromatography.

(183) .sup.1H-NMR (400 MHz, CDCl.sub.3): δ=5.17 (d, J=8.4 Hz, 1H), 4.39 (dd, J=16, 7.6 Hz, 1H), 4.18 (dd, J=10.0, 2.4 Hz, 1H), 3.88-3.83 (m, 4H), 1.52 (s, 9H).

Step E: (S)-5-(tert-butoxycarbonyl)-1,2,5-thiadiazolidine-3-carboxylic acid 1,1-dioxide

(184) ##STR00097##

(185) Under stirring in an ice bath, lithium hydroxide monohydrate (342 mg, 14.27 mmol) was added to a solution of (S)-2-tert-butyl 4-methyl-1,2,5-thiadiazolidine-2,4-dicarboxylate 1,1-dioxide (800 mg, 2.854 mmol) in 10 mL methanol/water (the volume ratio being 5/1). They were reacted overnight at room temperature. After the reaction was completed, methanol was removed by rotary evaporation. The system was adjusted to a pH less than 3 with 4N HCl and extracted with ethyl acetate for three times. The combined organic phase was washed with brine, dried over sodium sulfate, filtered and concentrated under vacuum. (S)-5-(tert-butoxycarbonyl)-1,2,5-thiadiazolidine-3-carboxylic acid 1,1-dioxide (759 mg, yield 100%) was obtained by isolating through column chromatography.

(186) .sup.1H-NMR (400 MHz, CDCl.sub.3): δ=5.39-5.33 (m, 1H), 4.46 (s, 1H), 4.23 (dd, J=10, 5.6 Hz, 1H), 3.98 (dd, J=9.6, 7.6 Hz, 1H), 1.50 (s, 9H).

Step F: (3S)—N-(1-(2-chlorophenyl)-2-((3,3-difluorocyclobutyl)amino)-2-oxoethyl)-N-(3-fluorophenyl)-5-tert-butoxycarbonyl-1,2,5-thiadiazolidine-3-carboxamide 1,1-dioxide

(187) ##STR00098##

(188) Under stirring at room temperature, in MeOH (6.0 mL), 2-chloro-benzaldehyde (400 mg, 2.85 mmol) and 3-fluoroaniline (317 mg, 2.85 mmol) were mixed and stirred for 30 min. (S)-5-(tert-butoxycarbonyl)-1,2,5-thiadiazolidine-3-carboxylic acid 1,1-dioxide (759 mg, 2.85 mmol) was added thereto and the reaction mixture was stirred for 10 min. Then, 1,1-difluoro-4-isocyanocyclobutane (333 mg, 2.85 mmol) was added thereto and the reaction mixture was stirred overnight at room temperature. The solvent was removed under vacuum. (3S)—N-(1-(2-chlorophenyl)-2-((3,3-difluorocyclobutyl)amino)-2-oxoethyl)-N-(3-fluorophenyl)-5-tert-butoxycarbonyl-1,2,5-thiadiazolidine-3-carboxamide 1,1-dioxide (578 mg, yield 32.9%) was obtained by isolating through column chromatography.

(189) m/z=617 [M+H].sup.+.

Step G: (3S)—N-(1-(2-chlorophenyl)-2-((3,3-difluorocyclobutyl)amino)-2-oxoethyl)-2-(4-cyanopyridin-2-yl)-N-(3-fluorophenyl)-5-tert-butoxycarbonyl-1,2, 5-thiadiazolidine-3-carboxamide 1,1-dioxide

(190) ##STR00099##

(191) (3S)—N-(1-(2-chlorophenyl)-2-((3,3-difluorocyclobutyl)amino)-2-oxoethyl)-N-(3-fluorophenyl)-5-tert-butoxycarbonyl-1,2,5-thiadiazolidine-3-carboxamide 1,1-dioxide (570 mg, 0.924 mmol), 2-bromo-4-cyanopyridine (186 mg, 1.10 mmol), cuprous iodide (88 mg, 0.462 mmol), N,N′-dimethylethylenediamine (82 mg, 0.924 mmol) and cesium carbonate (903 mg, 2.772 mmol) were added into a sealed tube reactor, dioxane (8 mL) was added thereto, nitrogen gas was introduced thereto for 5 min and the tube was sealed to react overnight at 80° C. The solvent was removed. (3S)—N-(1-(2-chlorophenyl)-2-((3,3-difluorocyclobutyl)amino)-2-oxoethyl)-2-(4-cyanopyridin-2-yl)-N-(3-fluorophenyl)-5-tert-butoxycarbonyl-1,2,5-thiadiazolidine-3-carboxamide 1,1-dioxide (190 mg, yield 28.6%) was obtained by column chromatography (PE:EA=1:1).

(192) m/z=719 [M+H].sup.+.

Step H: (S)—N—((S)-1-(2-chlorophenyl)-2-((3,3-difluorocyclobutyl)amino)-2-oxoethyl)-2-(4-cyanopyridin-2-yl)-N-(3-fluorophenyl)-1,2,5-thiadiazolidine-3-carboxamide 1,1-dioxide

(193) ##STR00100##

(194) Under stirring in an ice bath, trifluoroacetic acid (2.0 mL) was added into a solution of (3S)—N-(1-(2-chlorophenyl)-2-((3,3-difluorocyclobutyl)amino)-2-oxoethyl)-2-(4-cyanopyridin-2-yl)-N-(3-fluorophenyl)-5-tert-butoxycarbonyl-1,2,5-thiadiazolidine-3-carboxamide 1,1-dioxide (190 mg, 0.264 mmol) in dichloromethane. Under the protection of nitrogen gas, they were reacted overnight at room temperature. After the reaction was completed, it was concentrated. (S)—N—((S)-1-(2-chlorophenyl)-2-((3,3-difluorocyclobutyl)amino)-2-oxoethyl)-2-(4-cyanopyridin-2-yl)-N-(3-fluorophenyl)-1,2,5-thiadiazolidine-3-carboxamide 1,1-dioxide (7 mg, yield 4.3%) was obtained by isolating through column chromatography.

(195) .sup.1H-NMR (400 MHz, CDCl.sub.3): δ=8.43 (t, J=5.6 Hz, 1H), 7.63 (d, J=8.1 Hz, 1H), 7.56 (s, 1H), 7.40-7.27 (m, 2H), 7.18 (d, J=6.1 Hz, 2H), 7.10-6.83 (m, 4H), 6.47 (d, J=6.7 Hz, 1H), 6.04 (d, J=6.3 Hz, 1H), 5.74 (d, J=28.7 Hz, 1H), 4.83 (s, 1H), 4.25 (d, J=39.4 Hz, 1H), 3.77 (d, J=9.7 Hz, 1H), 3.68-3.46 (m, 1H), 3.17-2.90 (m, 2H), 2.45 (m, 2H).

(196) m/z=619 [M+H].sup.+.

Example 25: (3S)—N-(1-(2-chlorophenyl)-2-((3,3-difluorocyclobutyl)amino)-2-oxoethyl)-2-(4-cyanopyridin-2-yl)-N-(3-fluorophenyl)-5-methyl-1,2,5-thiadiazolidine-3-carboxamide 1,1-dioxide

(197) ##STR00101##

Step A: methyl (S)-2-benzyl-1,2,5-thiadiazolidine-3-carboxylate 1,1-dioxide

(198) ##STR00102##

(199) Under stirring in an ice bath, trifluoroacetic acid (15.0 mL) was added into a solution of (S)-2-tert-butyl 4-methyl-5-benzyl-1,2,5-thiadiazolidine-2,4-dicarboxylate 1,1-dioxide (2.8 g, 7.559 mmol) in dichloromethane. Under the protection of nitrogen gas, they were reacted overnight at room temperature. After the reaction was completed, it was concentrated. Methyl (S)-2-benzyl-1,2,5-thiadiazolidine-3-carboxylate 1,1-dioxide (1.54 g, yield 75.5%) was obtained by isolating through column chromatography.

Step B: methyl (S)-2-benzyl-5-methyl-1,2,5-thiadiazolidine-3-carboxylate 1,1-dioxide

(200) ##STR00103##

(201) Under stirring at room temperature, methyl iodide (629 mg, 4.44 mmol) was added to a solution of methyl (S)-2-benzyl-1,2,5-thiadiazolidine-3-carboxylate 1,1-dioxide (600 mg, 2.22 mmol) and potassium carbonate (920 mg, 6.66 mmol) in DMF (6 mL). Under the protection of nitrogen gas, they were reacted overnight at room temperature. After the reaction was completed, it was diluted with ethyl acetate, quenched by adding water and extracted with ethyl acetate for three times. The combined organic phase was washed with brine, dried over sodium sulfate, filtered and concentrated under vacuum. Methyl (S)-2-benzyl-5-methyl-1,2,5-thiadiazolidine-3-carboxylate 1,1-dioxide (616 mg, yield 97.6%) was obtained by isolating through column chromatography.

(202) .sup.1H-NMR (400 MHz, CDCl.sub.3): δ=7.40-7.31 (m, 5H), 4.48 (d, J=1.2 Hz, 2H), 3.86 (dd, J=8, 7.6 Hz, 1H), 3.66 (s, 3H), 3.54 (dd, J=10, 9.6 Hz, 1H), 3.35 (dd, J=10, 10 Hz, 1H), 2.77 (s, 3H).

Step C: methyl (S)-5-methyl-1,2,5-thiadiazolidine-3-carboxylate 1,1-dioxide

(203) ##STR00104##

(204) Under stirring at room temperature, 10 wt % palladium carbon (300 mg) was added into a solution of methyl (S)-2-benzyl-5-methyl-1,2,5-thiadiazolidine-3-carboxylate 1,1-dioxide (616 mg, 2.166 mmol) in dichloromethane. The gas was exchanged 10 times by circulating water pump. Under the protection of hydrogen gas, they were reacted overnight at room temperature. After the reaction was completed, it was filtered and concentrated. Methyl (S)-5-methyl-1,2,5-thiadiazolidine-3-carboxylate 1,1-dioxide (342 mg, yield 81.4%) was obtained by isolating through column chromatography.

(205) .sup.1H-NMR (400 MHz, CDCl.sub.3): δ=5.19-5.13 (m, 1H), 4.26-4.20 (m, 1H), 3.88 (s, 3H), 3.63 (dd, J=10.4, 10.0 Hz, 1H), 3.55 (dd, J=10.0, 10.0 Hz, 1H), 2.72 (s, 3H).

Step D: (S)-5-methyl-1,2,5-thiadiazolidine-3-carboxylic acid 1,1-dioxide

(206) ##STR00105##

(207) Under stirring in an ice bath, lithium hydroxide monohydrate (210 mg, 8.755 mmol) was added to a solution of methyl (S)-5-methyl-1,2,5-thiadiazolidine-3-carboxylate 1,1-dioxide (340 mg, 1.751 mmol) in 10 mL methanol/water (the volume ratio being 5/1). They were reacted overnight at room temperature. After the reaction was completed, methanol was removed by rotary evaporation. The system was adjusted to a pH less than 3 with 4N HCl and extracted with ethyl acetate for three times. The combined organic phase was washed with bine, dried over sodium sulfate, filtered and concentrated under vacuum. (S)-5-methyl-1,2,5-thiadiazolidine-3-carboxylic acid 1,1-dioxide (315 mg, yield 100%) was obtained by isolating through column chromatography.

Step E: (3S)—N-(1-(2-chlorophenyl)-2-((3,3-difluorocyclobutyl)amino)-2-oxoethyl)-N-(3-fluorophenyl))-5-methyl-1,2,5-thiadiazolidine-3-carboxamide 1,1-dioxide

(208) ##STR00106##

(209) Under stirring at room temperature, 2-chloro-benzaldehyde (156 mg, 1.11 mmol) and 3-fluoroaniline (124 mg, 1.11 mmol) were mixed in MeOH (6.0 mL) for 30 min. (S)-5-methyl-1,2,5-thiadiazolidine-3-carboxylic acid 1,1-dioxide (200 mg, 1.11 mmol) was added thereto and the reaction mixture was stirred for 10 min. Then, 1,1-difluoro-4-isocyanocyclobutane (130 mg, 1.11 mmol) was added thereto and the reaction mixture was stirred overnight at room temperature. The solvent was removed under vacuum. (3S)—N-(1-(2-chlorophenyl)-2-((3,3-difluorocyclobutyl)amino)-2-oxoethyl)-N-(3-fluorophenyl))-5-methyl-1,2,5-thiadiazolidine-3-carboxamide 1,1-dioxide (240 mg, yield 41.0%) was obtained by isolating through silica gel column chromatography.

(210) .sup.1H-NMR (400 MHz, CDCl3): δ=7.63-7.57 (m, 1H), 7.42 (d, J=8.0 Hz, 0.5H), 7.32 (d, J=7.6 Hz, 0.5H), 7.29-7.20 (m, 1H), 7.14-6.92 (m, 4H), 6.78-6.6 (m, 1H), 6.55 (s, 0.5H), 6.34 (s, 0.5H), 6.14 (d, J=6.4 Hz, 0.5H), 6.01 (d, J=6.4 Hz, 0.5H), 5.72-5.52 (m, 1H), 4.35-4.29 (m, 1H), 4.20-4.06 (m, 1H), 3.56-3.41 (m, 1H), 3.09-2.80 (m, 3H), 2.66 (s, 3H), 2.61-2.31 (m, 2H).

(211) m/z=531 [M+H].sup.+.

Step F: (3S)—N-(1-(2-chlorophenyl)-2-((3,3-difluorocyclobutyl)amino)-2-oxoethyl)-2-(4-cyanopyridin-2-yl)-N-(3-fluorophenyl)-5-methyl-1,2,5-thiadiazolidine-3-carboxamide 1,1-dioxide

(212) ##STR00107##

(213) (3S)—N-(1-(2-chlorophenyl)-2-((3,3-difluorocyclobutyl)amino)-2-oxoethyl)-N-(3-fluorophenyl))-5-methyl-1,2,5-thiadiazolidine-3-carboxamide 1,1-dioxide (140 mg, 0.264 mmol), 2-bromo-4-cyanopyridine (91 mg, 0.291 mmol), cuprous iodide (50 mg, 0.132 mmol), N,N′-dimethylethylenediamine (42 mg, 0.264 mmol) and cesium carbonate (441 mg, 0.792 mmol) were added into a sealed tube reactor, dioxane (8 mL) was added thereto, nitrogen gas was introduced thereto for 5 min and the tube was sealed to react overnight at 80° C. The solvent was removed. (3S)—N-(1-(2-chlorophenyl)-2-((3,3-difluorocyclobutyl)amino)-2-oxoethyl)-2-(4-cyanopyridin-2-yl)-N-(3-fluorophenyl)-5-methyl-1,2,5-thiadiazolidine-3-carboxamide 1,1-dioxide (81 mg, yield 48.5%) was obtained through column chromatography (PE:EA=1:1).

(214) .sup.1H-NMR (400 MHz, CDCl.sub.3): δ=8.55-8.47 (m, 1H), 7.90-7.65 (m, 1H), 7.51 (d, J=5.2 Hz, 0.5H), 7.34 (d, J=6.4 Hz, 0.5H), 7.29-7.16 (m, 4H), 7.14 (m, 4H), 6.75-6.70 (m, 1H), 6.55 (d, J=4.4 Hz, 1H), 6.16 (d, J=12 Hz, 1H), 4.95-4.73 (m, 1H), 4.19-4.11 (m, 1H), 3.53-3.44 (m, 1H), 3.34 (t, J=7.8 Hz, 1H), 3.0-2.81 (m, 4H), 2.41-2.31 (m, 2H).

(215) m/z=633 [M+H].sup.+.

Example 26: (3S)—N-(1-(2-chlorophenyl)-2-(cyclohexylamino)-2-oxoethyl)-2-(4-cyanopyridin-2-yl)-N-(3-aminosulfonylphenyl)-isothiazolidine-3-carboxamide 1,1-dioxide

(216) ##STR00108##

(217) Referring to Step E in Example 1, the reaction materials 2,2-difluorocyclobutyl isocyanide, 3-amino-5-fluoropyridine and (S)-isothiazolidine-3-carboxylic acid 1,1-dioxide were replaced with cyclohexyl isocyanide, 3-aminobenzenesulfonamide and (S)-2-(4-cyanopyridin-2-yl)isothiazolidine-3-carboxylic acid 1,1-dioxide (prepared in Step B of Example 11), respectively. The title compound was obtained with a yield of 36%.

(218) .sup.1H-NMR (400 MHz, CDCl.sub.3): δ=8.58 (d, J=5.0 Hz, 1H), 8.14-6.84 (m, 12H), 6.52 (s, 1H), 5.53 (m, 1H), 4.76 (m, 1H), 3.88-3.78 (m, 1H), 3.80-3.65 (m, 1H), 3.39-3.36 (m, 1H), 2.69-2.63 (m, 1H), 2.50-2.42 (m, 1H), 1.95-1.90 (m, 2H), 1.74-1.59 (m, 4H), 1.38-1.24 (m, 2H), 1.17-0.94 (m, 2H).

(219) m/z=671 [M+H].sup.+.

Example 27: 3-((S)—N—((S)-1-(2-chlorophenyl)-2-((3,3-difluorocyclobutyl)amino)-2-oxoethyl)-2-(4-cyanopyridin-2-yl)-1,1-dioxoisothiazolidine-3-carboxamido)-5-fluoropyridine-1-oxide

(220) ##STR00109##

(221) (S)—N—((S)-1-(2-chlorophenyl)-2-((3,3-difluorocyclobutyl)amino)-2-oxoethyl)-2-(4-cyanopyridin-2-yl)-N-(5-fluoropyridin-3-yl)-isothiazolidine-3-carboxamide 1,1-dioxide (50 mg, 0.081 mmol, prepared in Example 2) and 3-chloroperoxybenzoic acid (28 mg, 0.16 mmol) were added to dichloromethane (10 mL) and they were stirred overnight at room temperature. The solvent was removed by vacuum concentration, and the residue was isolated by silica gel thin layer chromatography (EA) to give 3-((S)—N—((S)-1-(2-chlorophenyl)-2-((3,3-difluorocyclobutyl)amino)-2-oxoethyl)-2-(4-cyanopyridin-2-yl)-1,1-dioxoisothiazolidine-3-carboxamido)-5-fluoropyridine-1-oxide (22 mg, yield 43%).

(222) .sup.1H-NMR (400 MHz, CDCl.sub.3): δ 8.72 (s, 0.5H), 8.13 (s, 0.5H), 8.48-8.41 (m, 1H), 7.94 (s, 1H), 7.80-7.68 (m, 1H), 7.41 (d, J=8.0 Hz, 1H), 7.26-7.15 (m, 4H), 7.05-6.95 (m, 1H), 6.49 (s, 1H), 6.01 (s, 0.5H), 5.90 (s, 0.5H), 4.86-4.82 (m, 1H), 4.34 (s, 1H), 3.78-3.68 (m, 1H), 3.45-3.35 (m, 1H), 3.10-3.00 (m, 2H), 2.61-2.38 (m, 4H).

(223) m/z=635 [M+H].sup.+.

(224) Bioactivity Experiments

(225) Enzyme Assay:

(226) Resazurin is a traditional redox dye, and after a redox reaction, it can be reduced from a blue resazurin without fluorescence to a pink fluorescent substance, resorufin, which can be measured and quantified with relative fluorescence unit (RFU) of fluorophotometer (E.sub.x=530-570 nm, Em=590-620 nm). At present, resazurin is widely used for determining the viability of bacteria, cells, etc. and the enzyme activity detection of oxidoreductase. We detected the decrease of cofactor NADPH to determine the inhibitory activity of a compound against IDH1m and detected the generation of cofactor NADPH to determine the inhibitory activity of a compound against IDH WT. The compound was pre-incubated with IDH1m and NADPH, and then the reaction was initiated by adding α-KG and performed for certain time under a linear condition. Then, diaphorase (lipoamide dehydrogenase) and the corresponding substrate resazurin were added thereto for detection. Lipoamide dehydrogenase terminated the IDH1m reaction by decreasing the available cofactor NADPH, which oxidized NADPH to NADP, and reduced resazurin to high fluorescent resorufin. The amount of the remaining cofactor NADPH after a specific reaction time was quantified via an easily detectable fluorophore.

(227) The compound was pre-incubated with IDH-WT and NADP, and then the reaction was initiated by adding isocitric acid, diaphorase (lipoamide dehydrogenase) and the corresponding substrate resazurin, and performed for certain time under a linear condition, and followed by detecting the amount of fluorescent substance. NADP was reduced to NADPH in this experiment, and the latter reduced resazurin to high fluorescent resorufin under the action of lipoamide dehydrogenase. The amount of the generated cofactor NADPH after a specific reaction time was quantified via a detectable fluorophore, so as to calculate the inhibitory effect of the compound on IDH-WT.

(228) The specific operation was as follows: 2.5 μl of the compound diluted in a 3-fold gradient was added to a 384-well plate, followed by adding 5 μl of the reaction buffer (20 mM Tris-HCl, pH 7.5; 150 mM NaCl; 10 mM MgCl.sub.2; 0.4 mg/mL BSA (Bovine Serum Albumin) and 2 mM DTT (dithiothreitol)) containing 40 nM IDH1 (R132H/R132C) and 20 μM NADPH. Then, the above test mixture was incubated at 23° C. for 16 hours, and then 2.5 μl of the reaction buffer containing 4 mM α-KG was added to initiate the reaction. After they were incubated for 60 minutes at room temperature, 5 μl of the termination mixture (0.4 U/ml diaphorase and 20 μM resazurin) formulated with the reaction buffer was added to convert resazurin to resorufin, so as to measure the amount of the remaining NADPH. After incubating at 23° C. for 10 minutes, fluorescence values were determined through Flexstation 3 at Ex535/Em595. The enzyme activity of each compound was respectively determined at 12 concentrations, and the data were calculated using the software GraFit6.0 (Erithacus Software) to obtain the IC.sub.50 value of each compound.

(229) 2-HG Determination:

(230) In the presence of 2-HG, phosphoglycerate dehydrogenase PHGDH can reduce NAD to NADPH, and the latter may be quantitatively determined by lipoamide dehydrogenase and the substrate thereof, resazurin.

(231) HT-1080 cell is a human fibrosarcoma cell line with an IDH1 mutation (R132C). U87 cell is a human glioblastoma cell line with an IDH1 mutation (R132H). They were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, 100 units/mL penicillin and 0.1 mg/mL streptomycin.

(232) Cells was digested with trypsin, and inoculated into a 6-well plate at a density of 5×10.sup.5, and cultured overnight in an incubator at 37° C. Next day, the test compound was added (the final concentration of DMSO is 0.1%) thereto and cultured for another 24 hours. Culture medium of each sample was sucked out and centrifuged at 1000 rpm for 10 min. The supernatant was sucked out to detect the content of 2-HG therein. Additionally, cells were washed with PBS (phosphate buffered saline), digested with trypsin and collected. After the collected cells were washed with PBS for one time, the determination of intracellular 2-HG content was performed.

(233) The method for determining the intracellular 2-HG was as follows: cells were re-suspended with 300 μL reaction buffer (40 mM Tris-HCl, pH 8.5; 150 mM NaCl) and disrupted by ultrasonication. They were centrifuged for 10 min at 12,000 rpm and 4° C. to remove insoluble substances. 25 μL supernatant was sucked out to determine the protein concentration by a BCA kit. Another 200 μL supernatant was transferred to a new group of centrifuge tubes, each of which was added with 4 μL of 3 M HCl, placed at room temperature for 5 min and centrifuged at 12,000 rpm for 5 min at room temperature. 100 μL supernatant was sucked out and transferred to a 96-well “V” bottom plate, and 3.6 μL of 2 M Tris base (tromethamine) was added to each well, it was placed at room temperature for 5 min and centrifuged at 12,000 rpm for 2 min. The pH was approximately equal to 8.0 through detection by pH test paper.

(234) Preparation of standard curve of 2-HG: 2-HG stock solution was diluted to 500 μM with reaction buffer, and then 200 μL was taken therefrom for a 2-fold gradient dilution, 10 concentrations in total. The following operations were same as described above, including the steps for acid treatment and alkali neutralization.

(235) The aforementioned samples, the test cell samples or standard samples, were diluted in 5 folds, and then 5 μL of each sample was taken therefrom and added to a 384-well plate. 10 μL of the detection mixture (8 μM PHGDH (phosphoglycerate dehydrogenase); 0.5 mM NAD; 0.1 U/ml diaphorase and 10 μM resazurin) was added to each well, and they were reacted for 60 min at 23° C. Fluorescence values were determined through Flexstation 3 at Ex535/Em595.

(236) The measured fluorescence values were compared after being corrected with the protein concentrations of the corresponding samples.

(237) The method for determining extracellular 2-HG was as follows: 500 μL of each culture medium supernatant was taken. 10 μL of 3 M HCl was added into each tube and placed for 5 min at room temperature. Then, 18 μL of 2 M Tris base was added into each tube and placed for 5 min at room temperature. It was centrifuged at 12,000 rpm for 2 min. The pH was approximately equal to 8.0 detected by pH test paper. Preparation of standard curve of 2-HG: 2-HG stock solution was diluted to 500 μM with complete medium, and then 500 μL was taken therefrom for a 2-fold gradient dilution, 10 concentrations in total. The following operations were same as described above, including the steps for acid treatment and alkali neutralization. The aforementioned samples, the test culture supernatant samples or standard samples, were diluted in 5 folds, and then 5 μL was taken therefrom and added to a 384-well plate. 10 μL of the detection mixture (8 μM PHGDH; 0.5 mM NAD; 0.1 U/mL diaphorase and 10 μM resazurin) was added to each well and reacted for 60 min at 2300. Fluorescence values were determined through Flexstation 3 at Ex535/Em595.

(238) The selected compounds prepared as described above were analyzed according to the biological methods herein, and the results are as follows:

(239) 1. The inhibitory activities (IC.sub.50) of the compounds against IDH1 mutants (R132H and R132C) were shown in Table 1.

(240) TABLE-US-00001 TABLE 1 Example No. IDH1 (R132H) IC.sub.50 (nM) IDH1 (R132C) IC.sub.50 (nM) 1 <10000 — 2 <20 <20 3 <500 — 4 <20 — 5 <1000 — 6 <20 — 7 <500 — 8 <20 — 9 <1000 — 10 <20 — 11 <500 — 12 <20 <20 13 <1000 — 14 <20 — 15 <500 — 16 <20 — 17 <10000 — 18 <100 — 19 <1000 — 20 <20 — 21 <10000 — 22 <1000 — 23 <20 — 24 <20 — 25 20 — 26 <20 — 27 20 — Note: — means not determined.

(241) 2. The inhibitory results of the compound in Example 2 on 2-HG in IDH1-mutated HT-1080 cells were shown in FIG. 1. The inhibitory results of the compound in Example 2 on 2-HG outside IDH1-mutated HT-1080 cells were shown in FIG. 2.

(242) 3. The inhibitory results of the compound in Example 2 on 2-HG in IDH1-mutated U87 cells were shown in FIG. 3.

(243) 4. The inhibitory activities (IC.sub.50) of the compound in Example 2 on IDH1-mutated U87 cells were shown in Table 2 below.

(244) TABLE-US-00002 TABLE 2 Compound U87/IDH1 (R132H) IC.sub.50 (nM) AG-120 46 The compound in Example 2 6.7

(245) Pharmacokinetic Experiments

(246) Male SD rats were from Beijing Vital River Laboratory Animal Technology Co., Ltd., and divided into groups (3 rats per group). The rats were intragastrically administered with the test sample suspension (5 mg/kg) via a single peroral administration, respectively. The animals were fasted overnight before this study. The fasting time period was from 10 hours before administration to 4 hours after administration. Blood samples were taken at 0.25, 0.5, 1, 2, 4, 6, 8 and 24 hours after administration. After the rats were anesthetized with isoflurane using an anesthesia machine for small animal, and then 0.3 mL whole blood samples were taken from the fundus venous plexus. The blood samples were placed in heparin anticoagulant tubes, and centrifuged for 5 min at 4° C. and 4000 rpm. The plasma was transferred to centrifuge tubes, and stored at −80° C. until analysis. The samples in plasma were extracted through protein precipitation. The liquid extract was analyzed by LC-MS/MS, wherein HPLC conditions were as follows: flow rate 0.4 mL/min; mobile phase A: water/formic acid (99.9/0.1, v/v); mobile phase B: acetonitrile/formic acid (99.9/0.1, v/v); injection volume: 5 μL; column temperature: RT; autosampler temperature: RT; run time: 2.5 min.

(247) PK data of the compound in Example 12 and AG-120 were shown in Table 3:

(248) TABLE-US-00003 TABLE 3 Example 12 AG-120 Gender of rats Male Male Oral dose (mg/kg) 5 5 T.sub.1/2(hr) 10.7 3.28 Tmax(hr) 4.0 0.67 Cmax(ng/mL) 556 719 AUC.sub.INF.sub.—.sub.obs(hr*ng/mL) 10567 6315 Formulation of dosage forms 0.5% MC, 0.2% Tween80

(249) From the PK data, it can be known that the compound in Example 12 had a drug exposure in plasma far higher than that of AG-120 at the same oral dose. The half-life of the compound in Example 12 was up to 10.7 hours and pharmacokinetic properties thereof were significantly superior to that of AG-120.