Nitrogen-containing fused heterocyclic compounds of n-sulfonamide and use thereof

Abstract

The present disclosure discloses a class of nitrogen-containing fused heterocyclic compounds of N-sulfonamide and the use thereof. The nitrogen-containing fused heterocyclic compounds of N-sulfonamide of the present disclosure are compounds of formula I, isotope-labeled compounds thereof, solvates thereof, pharmaceutically acceptable salts thereof, solvates of the pharmaceutically acceptable salts thereof. Such compounds can promote the binding of p53 Y220C mutant with DNA and possess better inhibitory activity against the proliferation of cancer cells. ##STR00001##

Claims

1. A nitrogen-containing compound of formula I, an isotope-labeled compound thereof, a solvate thereof, a pharmaceutically acceptable salt thereof, or a solvate of the pharmaceutically acceptable salt thereof; ##STR00038## wherein X.sup.1, X.sup.2, and X.sup.3 are each independently selected from CH and N; R.sup.1, R.sup.2, R.sup.3, R.sup.4, and R.sup.5 are each independently selected from hydrogen and deuterium; R.sup.6 is (CH.sub.2).sub.nR.sup.7; n is 0, 1, 2, or 3; R.sup.7 is selected from C.sub.1-C.sub.6 alkyl, deuterated C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 haloalkyl, and C.sub.3-C.sub.6 cycloalkyl.

2. The compound of formula I, the isotope-labeled compound thereof, the solvate thereof, the pharmaceutically acceptable salt thereof, or the solvate of the pharmaceutically acceptable salt thereof according to claim 1, wherein the compound of formula I is represented by formula Ia; ##STR00039##

3. The compound of formula I, the isotope-labeled compound thereof, the solvate thereof, the pharmaceutically acceptable salt thereof, or the solvate of the pharmaceutically acceptable salt thereof according to claim 1, wherein X.sup.1 is N; X.sup.2 is CH or N; X.sup.3 is CH; or, X.sup.1 is N; X.sup.2 and X.sup.3 are CH.

4. The compound of formula I, the isotope-labeled compound thereof, the solvate thereof, the pharmaceutically acceptable salt thereof, or the solvate of the pharmaceutically acceptable salt thereof according to claim 1, wherein R.sup.6 is (CH.sub.2).sub.nR.sup.7; n is 0 or 1; R.sup.7 is selected from C.sub.1-C.sub.3 alkyl, deuterated C.sub.1-C.sub.3 alkyl, C.sub.1-C.sub.3 haloalkyl, and C.sub.3-C.sub.6 cycloalkyl.

5. The compound of formula I, the isotope-labeled compound thereof, the solvate thereof, the pharmaceutically acceptable salt thereof, or the solvate of the pharmaceutically acceptable salt thereof according to claim 1, wherein R.sup.6 is (CH.sub.2).sub.nR.sup.7; n is 0 or 1; R.sup.7 is selected from CH.sub.3, CD.sub.3, CF.sub.3, and cyclopropyl.

6. The compound of formula I, the isotope-labeled compound thereof, the solvate thereof, the pharmaceutically acceptable salt thereof, or the solvate of the pharmaceutically acceptable salt thereof according to claim 1, wherein R.sup.1, R.sup.2, and R.sup.3 are deuterium; R.sup.4 and R.sup.5 are hydrogen; or, R.sup.1, R.sup.2, and R.sup.3 are hydrogen; R.sup.4 and R.sup.5 are deuterium.

7. The compound of formula I, the isotope-labeled compound thereof, the solvate thereof, the pharmaceutically acceptable salt thereof, or the solvate of the pharmaceutically acceptable salt thereof according to claim 1, wherein formula I is formula Ib or Ic; ##STR00040## wherein in formulas Ib and Ic, ##STR00041## R.sup.1, R.sup.2, R.sup.3, R.sup.4, and R.sup.5 are each independently selected from hydrogen and deuterium; R.sup.6 is (CH.sub.2).sub.nR.sup.7; n is 0 or 1; R.sup.7 is selected from C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 haloalkyl, and C.sub.3-C.sub.6 cycloalkyl.

8. The compound of formula I, the isotope-labeled compound thereof, the solvate thereof, the pharmaceutically acceptable salt thereof, or the solvate of the pharmaceutically acceptable salt thereof according to claim 7, wherein in formulas Ib and Ic, ##STR00042## is selected from OCH.sub.3 and OCD.sub.3; R.sup.4 and R.sup.5 are selected from hydrogen; R.sup.6 is (CH.sub.2).sub.nR.sup.7; n is 0 or 1; R.sup.7 is selected from CH.sub.3, CD.sub.3, CF.sub.3, and cyclopropyl.

9. The compound of formula I, the isotope-labeled compound thereof, the solvate thereof, the pharmaceutically acceptable salt thereof, or the solvate of the pharmaceutically acceptable salt thereof according to claim 1, wherein the compound of formula I is selected from the following compounds: ##STR00043## ##STR00044## ##STR00045## ##STR00046## ##STR00047## ##STR00048## ##STR00049## an enantiomer thereof, or a mixture of the two in any ratio.

10. A pharmaceutical composition comprising the compound of formula I, the isotope-labeled compound thereof, the solvate thereof, the pharmaceutically acceptable salt thereof, or the solvate of the pharmaceutically acceptable salt thereof according to claim 1, and a pharmaceutical excipient.

11. A method for promoting the binding of p53 Y220C mutants with DNA, wherein the method comprises: administering to a patient a therapeutically effective amount of the compound of formula I, the isotope-labeled compound thereof, the solvate thereof, the pharmaceutically acceptable salt thereof, or the solvate of the pharmaceutically acceptable salt thereof according to claim 1.

12. A method for promoting the binding of p53 Y220C mutants with DNA, wherein the method comprises: administering to a patient a therapeutically effective amount of the pharmaceutical composition according to claim 10.

13. A method for the treatment of gastric cancer, wherein the method comprises: administering to a patient a therapeutically effective amount of the compound of formula I, the isotope-labeled compound thereof, the solvate thereof, the pharmaceutically acceptable salt thereof, or the solvate of the pharmaceutically acceptable salt thereof according to claim 1.

14. A compound of formula a-11 or a-32; ##STR00050## wherein X is halogen; R.sup.8 is an amino protecting group; X.sup.1, X.sup.2, and X.sup.3 are each independently selected from CH and N; R.sup.1, R.sup.2, R.sup.3, R.sup.4, and R.sup.5 are each independently selected from hydrogen and deuterium; R.sup.6 is (CH.sub.2).sub.nR.sup.7, n is 0, 1, 2, or 3; R.sup.7 is selected from C.sub.1-C.sub.6 alkyl, deuterated C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 haloalkyl, and C.sub.3-C.sub.6 cycloalkyl.

15. The compound of formula a-11 or a-32 according to claim 14, wherein the compound of formula a-11 or a-32 is any one of the following compounds; ##STR00051##

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows the tumor growth curves of the control group and each treatment group in the efficacy study of NUGC-3 human gastric cancer xenograft mice model described in test example IX;

(2) FIG. 2 shows the curves of relative mice body weight change in the control group and each treatment group in the efficacy study of NUGC-3 human gastric cancer xenograft mice model described in test example IX;

(3) FIG. 3 shows the tumor growth curves of the control group and each treatment group in the efficacy study of NUGC-3 human gastric cancer xenograft rat model described in test example X;

(4) FIG. 4 shows the curves of relative rat body weight change in the control group and each treatment group in the efficacy study of NUGC-3 human gastric cancer xenograft rat model described in test example X.

DETAILED DESCRIPTION OF THE INVENTION

(5) The present disclosure is further described through examples below, but is not limited to the scope of the examples provided. Unless otherwise stated, the actual operations disclosed in the present disclosure will adopt conventional techniques of organic synthesis, cell biology, cell culture, and molecular biology within the technical scope of the art.

(6) In each example, .sup.1HNMR was recorded by a BRUKER AVANCE NEO 400 MHZ, JNM-ECZ400s nuclear magnetic resonance instrument, and the chemical shift was expressed in (ppm); Liquid chromatography-mass spectrometry (LCMS) was recorded by a Shimadzu LC-20AD, Agilent 1260, and Agilent 1200 mass spectrometer; Preparative HPLC separation was performed using a WATERS Autop, Shimadzu LC20AR liquid chromatograph.

(7) TABLE-US-00001 BrettPhos Pd G3 Methanesulfonato(2-Dicyclohexylphosphino-3,6-dimethoxy-2,4,6-tri- i-propyl-1,1-bipheny)(2-amino-1,1-biphenyl-2-yl)palladium(II) BrettPhos Pd G4 Methanesulfonato(2-Dicyclohexylphosphino-3,6-dimethoxy-2,4,6-tri- i-propyl-1,1-biphenyl)(2-methylamino-1,1-biphenyl-2- yl)palladium(II) CDI N,N-Carbonyldiimidazole DBU 1,8-Diazabicyclo[5,4,0]undec-7-ene DCM Dichloromethane DIBAL-H Diisobutylaluminium hydride DMF N,N-Dimethylformamide DMP Dess-Martin periodinane DMSO Dimethyl sulfoxide EtOAc Ethyl acetate EDCI 1-Ethyl-(3-dimethylaminopropyl)carbonyldiimide hydrochloride HATU 2-(7-Azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphate MTBE tert-Butyl methyl ether MeOH Methanol NaBH.sub.3CN Sodium cyanoborohydride Na.sub.2SO.sub.4 Sodium sulfate NMP N-Methyl pyrrolidone Pd(dppf)Cl.sub.2 [1,1-Bis(diphenylphosphino) ferrocene]dichloropalladium(II) PE Petroleum ether Ruphos 2-Dicyclohexylphosphino-2,6-di-iso-propoxy-1,1-biphenyl SGC Silica gel column chromatography TBAF Tetrabutylammonium fluoride TEA Triethylamine THF Tetrahydrofuran

Preparation of Intermediates Int-1 and Int-1-7

(8) ##STR00024##

Step 1: Ethyl 8-bromoindolizine-2-carboxylate

(9) A mixture of 3-bromo-2-methylpyridine Int-1-1 (500 mg, 2.91 mmol), ethyl 3-bromopyruvate (850 mg, 4.36 mmol), and sodium bicarbonate (561 mg, 6.69 mmol) in butanone (5 mL) was stirred at 85 C. for 16 hours, then concentrated to dryness, and purified by SGC (0 to 10% EtOAc added to PE) method to obtain ethyl 8-bromoindolizine-2-carboxylate Int-1-2 (190 mg, 24.4% yield) as a gray solid. LCMS calculated for C.sub.11H.sub.11BrNO.sub.2 [M+H].sup.+: m/z=268.0/270.0; found: 267.9/269.9; .sup.1H NMR (400 MHZ, CDCl.sub.3) 7.88 (d, J=1.6 Hz, 1H), 7.87-7.83 (m, 1H), 7.03-6.99 (m, 1H), 6.95 (d, J=6.8 Hz, 1H), 6.43 (t, J=7.2 Hz, 1H), 4.37 (q, J=7.2 Hz, 2H), 1.40 (t, J=6.8 Hz, 3H).

Step 2: Ethyl 8-bromo-3-formylindolizine-2-carboxylate

(10) A solution of ethyl 8-bromoindolizine-2-carboxylate Int-1-2 (5.34 g, 19.9 mmol) in DCM (130 mL) was added dropwise to a solution of phosphorus oxychloride (5.19 g, 33.8 mmol) in DMF (130 mL) at 0 C. The mixture was stirred at 20 C. for 1 hour, then was slowly quenched with saturated sodium bicarbonate aqueous solution (300 mL) and extracted with DCM (200 mL). The separated organic layers were combined, dried over Na.sub.2SO.sub.4, filtered, and concentrated to dryness. The residue was purified by SGC (0 to 10% EtOAc added to PE) to obtain ethyl 8-bromo-3-formylindolizine-2-carboxylate Int-1-3 (10.6 g, crude product) as a yellow colloid. LCMS calculated for C.sub.12H.sub.11BrNO.sub.3 [M+H].sup.+: m/z=296.0/298.0; found: 295.9/297.9.

Step 3: Ethyl 8-bromo-3-(2,2,2-trifluoroethyl)indolizine-2-carboxylate

(11) A mixture of ethyl 8-bromo-3-formylindolizine-2-carboxylate Int-1-3 (8.50 g, 15.8 mmol) and 2,2-difluoro-2-triphenylphosphaniumylacetate (11.2 g, 31.5 mmol) in DMF (120 mL) was stirred for 2 hours at 60 C. TBAF (47.3 mL, 47.3 mmol, 1M in THF) was added to the mixture. The reaction mixture was stirred at 60 C. for 2 hours, then diluted with water (100 mL) and extracted with MTBE (100 mL3). The organic layers were combined, dried over Na.sub.2SO.sub.4, filtered, and concentrated to dryness under reduced pressure. The residue was purified by SGC (0 to 5% EtOAc added to PE) method to obtain ethyl 8-bromo-3-(2,2,2-trifluoroethyl)indolizine-2-carboxylate Int-1-4 (2.40 g, 43.4% yield) as a white solid. LCMS calculated for C.sub.13H.sub.12BrF.sub.3NO.sub.2 [M+H].sup.+: m/z=350.0/352.0; found: 349.9/351.9; .sup.1H NMR (400 MHZ, CDCl.sub.3) 7.89 (d, J=7.2 Hz, 1H), 7.12 (s, 1H), 7.05 (d, J=6.8 Hz, 1H), 6.56 (t, J=7.2 Hz, 1H), 4.40 (q, J=7.2 Hz, 2H), 4.25 (q, J=10.0 Hz, 2H), 1.42 (t, J=7.2 Hz, 3H).

Step 4: (8-Bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)methanol

(12) DIBAL-H (35.1 mL, 35.1 mmol, 1M in toluene) was added to a mixture of ethyl 8-bromo-3-(2,2,2-trifluoroethyl)indolizine-2-carboxylate Int-1-4 (4.10 g, 11.7 mmol) in THF (120 mL). The reaction mixture was stirred at 10 C. for 3 hours to obtain a yellow solution. The reaction mixture was poured into saturated ammonium chloride aqueous solution (200 mL), then water (200 mL) was added thereto, and the mixture was extracted with EtOAc (150 mL3). The organic layers were combined, dried over Na.sub.2SO.sub.4, filtered, and concentrated to dryness under reduced pressure. The residue was purified by SGC (0 to 5% EtOAc added to PE) method to obtain (8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)methanol Int-1-5 (3.55 g, 98.4% yield) as a white solid. LCMS calculated for C.sub.11H.sub.10BrF.sub.3NO [M+H].sup.+: m/z=308.0/310.0; found: 307.9/309.9; .sup.1H NMR (400 MHZ, CDCl.sub.3) 7.85 (d, J=7.2 Hz, 1H), 7.00 (d, J=6.8 Hz, 1H), 6.70 (br s, 1H), 6.50 (t, J=7.2 Hz, 1H), 4.84 (s, 2H), 3.83 (q, J=10.4 Hz, 2H).

Step 5: 8-Bromo-3-(2,2,2-trifluoroethyl)indolizine-2-carbaldehyde

(13) DMP (7.33 g, 17.2 mmol) was added to a solution of (8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)methanol Int-1-5 (3.55 g, 11.5 mmol) in DCM (400 mL) at 0 C. The reaction system was warmed to room temperature and stirred for 2 hours. The reaction mixture was poured into saturated sodium bicarbonate aqueous solution (20 mL), then was added with water (200 mL) and extracted with DCM (100 mL3). The organic layers were combined, dried over Na.sub.2SO.sub.4, filtered, and concentrated to dryness under reduced pressure. The residue was purified by SGC (0 to 7% EtOAc added to PE) method to obtain 8-bromo-3-(2,2,2-trifluoroethyl)indolizine-2-carbaldehyde Int-1-6 (2.70 g, 76.5% yield) as a white solid. LCMS calculated for C.sub.11H.sub.8BrF.sub.3NO [M+H].sup.+: m/z=306.0/308.0; found: 305.9/307.9.

Step 6: 8-Bromo-2-ethynyl-3-(2,2,2-trifluoroethyl)indolizine

(14) At 0 C., 8-bromo-3-(2,2,2-trifluoroethyl)indolizine-2-carbaldehyde Int-1-6 (500 mg, 1.63 mmol) was added to a mixture of dimethyl (1-diazo-2-oxopropyl)phosphonate (470 mg, 2.45 mmol) and potassium carbonate (451 mg, 3.27 mmol) in methanol (15 mL). The reaction system was warmed to room temperature and stirred for 16 hours. The reaction mixture was diluted with water (50 mL) and extracted with EtOAc (50 mL3). The organic layers were combined, dried over Na.sub.2SO.sub.4, filtered, and concentrated to dryness under reduced pressure. The residue was purified by SGC (0 to 3% EtOAc added to PE) method to obtain 8-bromo-2-ethynyl-3-(2,2,2-trifluoroethyl)indolizine Int-1 (380 mg, 77.0% yield) as a white solid. LCMS calculated for C.sub.12H.sub.8BrF.sub.3N [M+H].sup.+: m/z=302.0/304.0; found: 301.9/303.9; .sup.1H NMR (400 MHZ, CDCl.sub.3) 7.81 (d, J=7.2 Hz, 1H), 7.03 (d, J=7.2 Hz, 1H), 6.79 (s, 1H), 6.52 (t, J=7.2 Hz, 1H), 3.83 (q, J=10.0 Hz, 2H), 3.25 (s, 1H).

Step 7: 3-(8-Bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-ol

(15) Under nitrogen atmosphere, tetrakis(acetonitrile)copper(I) hexafluorophosphate (123 mg, 0.33 mmol) and tributylphosphine (268 mg, 1.32 mmol) were dissolved in toluene (15 mL), and the reaction mixture was stirred at 70 C. for 30 minutes. Then 8-bromo-2-ethynyl-3-(2,2,2-trifluoroethyl)indolizine Int-1 (1.00 g, 3.31 mmol) and formaldehyde aqueous solution (0.18 mL, 6.62 mmol, 36% to 38% content) were added thereto. The mixture was stirred at 70 C. overnight. After the completion of the reaction detected by TLC, the mixture was concentrated under vacuum. The residue was purified by SGC (0 to 15% EtOAc added to PE) method to obtain 3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-ol Int-1-7 (843 mg, 76.7% yield) as a white solid. LCMS calculated for C.sub.13H.sub.10BrF.sub.3NO [M+H].sup.+: m/z=332.0/334.0; found: 332.3/334.3; .sup.1H NMR (400 MHZ, DMSO-d.sub.6) 8.38 (d, J=7.1 Hz, 1H), 7.19 (d, J=7.1 Hz, 1H), 6.68 (t, J=7.1 Hz, 1H), 6.61 (s, 1H), 5.34 (t, J=6.0 Hz, 1H), 4.34 (d, J=6.0 Hz, 2H), 4.20-4.11 (m, 2H).

(16) Cpd-C: 4-((3-(8-(((3S,4R)-3-Fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-3-methoxy-N-methylbenzamide

(17) ##STR00025##

Step 1: 4-Amino-3-methoxy-N-methylbenzamide

(18) HATU (18.8 g, 49.5 mmol) was added to a solution of 4-amino-3-methoxybenzoic acid Cpd-C-1 (7.52 g, 45.0 mmol), methylamine solution (6.71 g, 54.0 mmol, 25% content), and triethylamine (18.8 mL, 135 mmol) in THF (45 mL) at 0 C. The reaction was stirred at 20 C. for 2 hours. The reaction mixture was concentrated under reduced pressure. The residue was purified by SGC (0 to 4% MeOH added to DCM) method to obtain 4-amino-3-methoxy-N-methylbenzamide Cpd-C-2 (6.08 g, 75.0% yield) as a white solid. LCMS calculated for C.sub.9H.sub.13N.sub.2O.sub.2 [M+H].sup.+: m/z=181.1; found: 181.2.

Step 2: 4-((3-(8-Bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-3-methoxy-N-methylbenzamide

(19) Formaldehyde aqueous solution (497 mg, 5.96 mmol, 36% to 38% content) and cuprous bromide (567 mg, 3.97 mmol) were added to a mixture of 8-bromo-2-ethynyl-3-(2,2,2-trifluoroethyl)indolizine Int-1 (600 mg, 1.99 mmol) and 4-amino-3-methoxy-N-methylbenzamide Cpd-C-2 (716 mg, 3.97 mmol) in 1,4-dioxane (12 mL). The reaction was stirred under microwave irradiation at 100 C. for 1 hour. The residue was purified by SGC (0 to 30% EtOAc added to DCM) method to obtain 4-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-3-methoxy-N-methylbenzamide Cpd-C-3 (480 mg, 48.9% yield) as a yellow solid. LCMS calculated for C.sub.22H.sub.20BrF.sub.3N.sub.3O.sub.2 [M+H].sup.+: m/z=494.1/496.1; found: 494.2/496.2.

Step 3: tert-Butyl (3S,4R)-3-fluoro-4-((2-(3-((2-methoxy-4-(methylcarbamoyl)phenyl)amino)prop-1-yn-1-yl)-3-(2,2,2-trifluoroethyl)indolizin-8-yl)amino)piperidine-1-carboxylate

(20) Under argon atmosphere, a mixture of 4-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-3-methoxy-N-methylbenzamide Cpd-C-3 (1.35 g, 2.73 mmol), tert-butyl (3S,4R)-4-amino-3-fluoropiperidine-1-carboxylate (1.19 g, 5.46 mmol), BrettPhos Pd G4 (0.50 g, 0.55 mmol), Ruphos (0.25 g, 0.55 mmol), and cesium carbonate (1.78 g, 5.46 mmol) in 1,4-dioxane (50 mL) was stirred at 100 C. for 16 hours. The reaction mixture was diluted with EtOAc (200 mL), washed with water (100 mL) and saturated brine (100 mL), dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated under reduced pressure. The residue was purified by SGC (0 to 50% EtOAc added to DCM) to obtain tert-butyl (3S,4R)-3-fluoro-4-((2-(3-((2-methoxy-4-(methylcarbamoyl)phenyl)amino)prop-1-yn-1-yl)-3-(2,2,2-trifluoroethyl)indolizin-8-yl)amino)piperidine-1-carboxylate Cpd-C-4 (1.00 g, 58.0% yield) as a yellow solid. LCMS calculated for C.sub.32H.sub.38F.sub.4N.sub.5O.sub.4 [M+H].sup.+: m/z=632.3; found: 632.5.

Step 4: 4-((3-(8-(((3S,4R)-3-Fluoropiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-3-methoxy-N-methylbenzamide

(21) HCl (7.92 mL, 31.7 mmol, 4N in 1,4-dioxane) was added to a solution of tert-butyl (3S,4R)-3-fluoro-4-((2-(3-((2-methoxy-4-(methylcarbamoyl)phenyl)amino)prop-1-yn-1-yl)-3-(2,2,2-trifluoroethyl)indolizin-8-yl)amino)piperidine-1-carboxylate Cpd-C-4 (1.00 g, 1.58 mmol) in DCM (16 mL) at 0 C. The mixture was warmed to room temperature and stirred for 1 hour. The mixture was concentrated to dryness, diluted with water (30 mL), and the pH was adjusted to 7 to 8 with saturated sodium bicarbonate solution. The aqueous phase was extracted with DCM (100 mL2), and the combined organic phases were dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated to obtain 4-((3-(8-(((3S,4R)-3-fluoropiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-3-methoxy-N-methylbenzamide Cpd-C-5 (450 mg, crude product) as a yellow solid. The crude product was used directly in the next step reaction. LCMS calculated for C.sub.27H.sub.30F.sub.4N.sub.5O.sub.2 [M+H].sup.+: m/z=532.2; found: 532.4.

Step 5: 4-((3-(8-(((3S,4R)-3-Fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-3-methoxy-N-methylbenzamide

(22) Sodium cyanoborohydride (160 mg, 2.54 mmol) was added to a solution of 4-((3-(8-(((3S,4R)-3-fluoropiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-3-methoxy-N-methylbenzamide Cpd-C-5 (450 mg, 0.85 mmol), acetic acid (0.02 mL, 0.42 mmol), and formaldehyde aqueous solution (212 mg, 2.54 mmol, 36% to 38% content) in methanol (20 mL) at room temperature, and the reaction mixture was stirred for 1 hour at room temperature. The pH was adjusted to 7 to 8 by dropwise adding saturated sodium bicarbonate solution to the reaction mixture. The aqueous phase was extracted with EtOAc (100 mL3). The organic phases were combined and washed with saturated brine (50 mL3), dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated. The residue was subjected to preparative purification to obtain 4-((3-(8-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-3-methoxy-N-methylbenzamide Cpd-C (119 mg, 25.2% yield) as a white solid. LCMS calculated for C.sub.28H.sub.32F.sub.4N.sub.5O.sub.2 [M+H].sup.+: m/z=546.2; found: 546.2; .sup.1H NMR (400 MHZ, DMSO-d.sub.6) 8.08 (d, J=4.6 Hz, 1H), 7.57 (d, J=6.9 Hz, 1H), 7.41 (dd, J=8.2, 1.7 Hz, 1H), 7.34 (d, J=1.7 Hz, 1H), 6.91 (s, 1H), 6.74 (d, J=8.3 Hz, 1H), 6.51 (t, J=7.2 Hz, 1H), 5.90 (t, J=6.2 Hz, 1H), 5.85 (d, J=7.5 Hz, 1H), 5.54 (d, J=8.4 Hz, 1H), 4.81 (d, J=49.8 Hz, 1H), 4.23 (d, J=6.2 Hz, 2H), 3.92 (q, J=10.7 Hz, 2H), 3.83 (s, 3H), 3.54 (d, J=27.9 Hz, 1H), 3.02 (t, J=10.5 Hz, 1H), 2.80 (d, J=11.3 Hz, 1H), 2.75 (d, J=4.5 Hz, 3H), 2.28-2.14 (m, 4H), 2.07 (t, J=11.2 Hz, 1H), 1.97 (ddd, J=24.0, 12.0, 3.2 Hz, 1H), 1.67 (dd, J=12.7, 2.7 Hz, 1H).

Example 1: 5-((3-(8-(((3S,4R)-3-Fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-methoxy-N-(methylsulfonyl)picolinamide (Cpd-1, i.e., Compound 1)

(23) ##STR00026## ##STR00027##

Step 1: 6-Methoxy-5-nitropicolinic acid

(24) A mixture of 6-chloro-5-nitropicolinic acid Cpd-1-1 (4.00 g, 19.7 mmol) and cesium carbonate (12.8 g, 39.4 mmol) in DMSO/MeOH (40 mL, 1:1) was stirred at 60 C. for 12 hours. LCMS detection showed that the starting material was completely consumed and the desired compound was detected. The reaction mixture was poured into icy water (50 mL), adjusted to pH=5 with 1N HCl, and extracted with EtOAc (30 mL3). The organic layers were combined, dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated to dryness under reduced pressure. The residue was purified by SGC (0 to 5% MeOH added to DCM) method to obtain 6-methoxy-5-nitropicolinic acid Cpd-1-2 (2.80 g, 71.7% yield, yellow solid). LCMS calculated for C.sub.7H.sub.7N.sub.2O.sub.5 [M+H].sup.+: m/z=199.0; found: 199.1.

Step 2: 6-Methoxy-N-(methylsulfonyl)-5-nitropicolinamide

(25) A mixture of 6-methoxy-5-nitropicolinic acid Cpd-1-2 (3.20 g, 16.2 mmol), methanesulfonamide (1.54 g, 16.2 mmol), EDCI (4.65 g, 24.3 mmol), and 4-dimethylaminopyridine (3.94 g, 32.4 mmol) in DMF (30 mL) was stirred at room temperature for 8 hours. LCMS detection showed that the starting material was completely consumed and the desired compound was detected. The reaction mixture was poured into water (100 mL) and extracted with EtOAc (30 mL3). The organic layers were combined, washed with saturated brine (30 mL), dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated to dryness under reduced pressure. The residue was purified by SGC (0 to 5% MeOH added to DCM) method to obtain 6-methoxy-N-(methylsulfonyl)-5-nitropicolinamide Cpd-1-3 (2.20 g, 49.5% yield, yellow solid). LCMS calculated for C.sub.8H.sub.10N.sub.3O.sub.6S [M+H].sup.+: m/z=276.0; found: 276.1.

Step 3: 5-Amino-6-methoxy-N-(methylsulfonyl)picolinamide

(26) Under hydrogen atmosphere, a mixture of palladium on carbon (1.60 g, 5% content) and 6-methoxy-N-(methylsulfonyl)-5-nitropicolinamide Cpd-1-3 (800 mg, 2.91 mmol) in EtOAc (15 mL) was stirred at room temperature for 2 hours. LCMS detection showed that the starting material was completely consumed and the desired compound was detected. The reaction mixture was filtered and concentrated to dryness under reduced pressure. The residue was purified by SGC (0 to 5% MeOH added to DCM) method to obtain 5-amino-6-methoxy-N-(methylsulfonyl)picolinamide Cpd-1-4 (500 mg, 70.1% yield, yellow solid). LCMS calculated for C.sub.8H.sub.12N.sub.3O.sub.4S [M+H].sup.+: m/z=246.1; found: 246.0.

Step 4: 8-Bromo-2-(3-((tert-butyldimethylsilyl)oxy)prop-1-yn-1-yl)-3-(2,2,2-trifluoroethyl)indolizine

(27) tert-Butyldimethylchlorosilane (68.0 mg, 451 mol) was added portionwise to a solution of 3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-ol Int-1-7 (100 mg, 302 mol) and imidazole (61.3 mg, 901 mol) in DCM (5 mL) at 0 C. The reaction mixture was stirred at 0 C. for 1 hour and then filtered to remove the solid. The filtrate was concentrated under reduced pressure to obtain a residue, which was purified by SGC (0 to 30% EtOAc added to PE) method to obtain 8-bromo-2-(3-((tert-butyldimethylsilyl)oxy)prop-1-yn-1-yl)-3-(2,2,2-trifluoroethyl)indolizine Cpd-1-5 (134 mg, 99.7% yield, yellow solid). LCMS calculated for C.sub.19H.sub.24BrF.sub.3NOSi [M+H].sup.+: m/z=446.1/448.1; found: 446.0/448.0.

Step 5: 2-(3-((tert-Butyldimethylsilyl)oxy)prop-1-yn-1-yl)-N-((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)-3-(2,2,2-trifluoroethyl)indolizin-8-amine

(28) Under argon atmosphere, a mixture of 8-bromo-2-(3-((tert-butyldimethylsilyl)oxy)prop-1-yn-1-yl)-3-(2,2,2-trifluoroethyl)indolizine Cpd-1-5 (134 mg, 301 mol), (3S,4R)-3-fluoro-1-methylpiperidin-4-amine (79.3 mg, 601 mol), BrettPhos Pd G3 (54.4 mg, 60.0 mol), and cesium carbonate (195 mg, 598 mol) in THF (10 mL) was stirred at 100 C. for 3 hours. The reaction mixture was cooled to room temperature, filtered, and concentrated under reduced pressure. The residue was purified by SGC (0 to 7% methanol added to DCM) method to obtain 2-(3-((tert-butyldimethylsilyl)oxy)prop-1-yn-1-yl)-N-((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)-3-(2,2,2-trifluoroethyl)indolizin-8-amine Cpd-1-6 (82.1 mg, 54.9% yield, white solid). LCMS calculated for C.sub.25H.sub.36F.sub.4N.sub.3OSi [M+H].sup.+: m/z=498.3; found: 498.0.

Step 6: 3-(8-(((3S,4R)-3-Fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-ol

(29) TBAF (0.20 mL, 0.20 mmol, 1M in THF) was added dropwise to a solution of 2-(3-((tert-butyldimethylsilyl)oxy)prop-1-yn-1-yl)-N-((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)-3-(2,2,2-trifluoroethyl)indolizin-8-amine Cpd-1-6 (82.1 mg, 165 mol) in THF (5 mL) at 0 C. The reaction mixture was stirred at 0 C. for 1 hour. LCMS detection showed that the starting material was completely consumed and the desired compound was detected. The reaction mixture was quenched with saturated sodium bicarbonate aqueous solution (50 mL) and extracted with EtOAc (50 mL3). The organic layers were combined, dried over Na.sub.2SO.sub.4, filtered, and concentrated to dryness under reduced pressure. The residue was purified by SGC (0 to 5% MeOH added to DCM) method to obtain 3-(8-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-ol Cpd-1-7 (57.0 mg, 90.2% yield, white solid). LCMS calculated for C.sub.19H.sub.22F.sub.4N.sub.3O [M+H].sup.+: m/z=384.2; found: 384.1.

Step 7: 3-(8-(((3S,4R)-3-Fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)propiolaldehyde

(30) A solution of pyridine sulfur trioxide (780 mg, 4.90 mmol) in DMSO (1 mL) was added dropwise to a solution of 3-(8-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-ol Cpd-1-7 (625 mg, 1.63 mmol), DMSO (1.20 mL, 16.3 mmol), and diisopropylethylamine (1.50 mL, 8.15 mmol) in DCM (20 mL) at 0 C. The reaction mixture was stirred at 0 C. for 0.5 hours. LCMS detection showed that the starting material was completely consumed and the desired compound was detected. The reaction mixture was quenched with saturated sodium bicarbonate aqueous solution (100 mL) and extracted with EtOAc (100 mL3). The organic layers were combined, dried over Na.sub.2SO.sub.4, filtered, and concentrated to dryness under reduced pressure. The residue was purified by SGC (0 to 5% MeOH added to DCM) method to obtain 3-(8-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)propiolaldehyde Cpd-1-8 (550 mg, 88.6% yield, yellow solid). LCMS calculated for C.sub.19H.sub.20F.sub.4N.sub.3O [M+H].sup.+: m/z=382.2; found: 382.1.

Step 8: 5-((3-(8-(((3S,4R)-3-Fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-methoxy-N-(methylsulfonyl)picolinamide

(31) A mixture of 3-(8-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)propiolaldehyde Cpd-1-8 (50.0 mg, 131 mol), 5-amino-6-methoxy-N-(methylsulfonyl)picolinamide Cpd-1-4 (38.6 mg, 157 mol), and tetraisopropyl titanate (112 mg, 393 mol) in THF (1 mL) was stirred at 100 C. for 1 hour. The mixed solution was then cooled to 0 C., slowly added with MeOH (0.5 mL) and sodium cyanoborohydride (12.4 mg, 197 mol), and the reaction mixture was warmed to room temperature and stirred for another 10 minutes. LCMS detection showed that the starting material was completely consumed and the desired compound was detected. The reaction mixture was poured into icy water (5 mL) and extracted with EtOAc (20 mL3). The organic layers were combined, washed with saturated sodium bicarbonate aqueous solution (20 mL), dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated under reduced pressure. The residue was purified by SGC (0 to 3% MeOH added to DCM) method to obtain a crude product as a yellow solid, which was subjected to preparative purification to obtain 5-((3-(8-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-methoxy-N-(methylsulfonyl)picolinamide Cpd-1 (14.8 mg, 18.5% yield, yellow solid). LCMS calculated for C.sub.27H.sub.31F.sub.4N.sub.6O.sub.4S [M+H].sup.+: m/z=611.2; found: 611.9; .sup.1H NMR (400 MHZ, CDCl.sub.3) 9.70 (s, 1H), 7.85 (d, J=8.0 Hz, 1H), 7.32 (d, J=7.0 Hz, 1H), 6.98 (d, J=8.0 Hz, 1H), 6.53 (t, J=7.2 Hz, 1H), 6.48 (s, 1H), 5.81 (d, J=7.3 Hz, 1H), 5.18 (t, J=5.9 Hz, 1H), 4.85 (d, J=49.1 Hz, 1H), 4.29 (d, J=6.0 Hz, 2H), 4.16 (d, J=9.4 Hz, 1H), 4.03 (s, 3H), 3.68 (q, J=10.1 Hz, 2H), 3.59-3.43 (m, 1H), 3.41 (s, 3H), 3.24 (t, J=11.0 Hz, 1H), 2.94 (d, J=11.0 Hz, 1H), 2.37-2.21 (m, 4H), 2.16 (t, J=11.1 Hz, 1H), 2.02 (d, J=10.0 Hz, 1H), 1.93 (td, J=12.2, 3.5 Hz, 1H).

Example 2: 5-((3-(8-(((3S,4R)-3-Fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d.SUB.3.)-N-(methylsulfonyl)picolinamide (Cpd-2, i.e., Compound 2)

(32) ##STR00028##

Step 1: 6-(Methoxy-d.SUB.3.)-5-nitropicolinic acid

(33) Under nitrogen atmosphere, a mixture of 6-chloro-5-nitropicolinic acid Cpd-1-1 (20.0 g, 98.7 mmol), cesium carbonate (96.5 g, 296 mmol), and deuterated methanol (4.27 g, 118 mmol) in DMSO (200 mL) was stirred at 60 C. for 16 hours. After the completion of the reaction detected by LCMS, the mixture was diluted with water (100 mL) and the pH was adjusted to 3 with 1N HCl. The mixture was filtered to collect the precipitate, and the filter cake was dried under vacuum to obtain 6-(methoxy-d.sub.3)-5-nitropicolinic acid Cpd-2-1 (12.0 g, crude product, yellow solid). The crude product was used directly in the next step reaction. LCMS calculated for C.sub.7H.sub.4D.sub.3N.sub.2O.sub.5 [M+H].sup.+: m/z=202.1; found: 201.9.

Step 2: Methyl 6-(methoxy-d.SUB.3.)-5-nitropicolinate

(34) Under nitrogen atmosphere, a mixture of 6-(methoxy-d.sub.3)-5-nitropicolinic acid Cpd-2-1 (12.0 g, 59.7 mmol) and cesium carbonate (58.4 g, 179 mmol) in DMSO (120 mL) was stirred at 0 C. for 0.5 hours. Iodomethane (9.32 g, 179 mmol) was then added, and the resulting reaction mixture was stirred at 0 C. for 2 hours. After the completion of the reaction detected by LCMS, the reaction mixture was poured into icy ammonium chloride aqueous solution (1000 mL) and extracted with EtOAc (1000 mL2). The combined organic layers were dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated to obtain a crude product. The residue was purified by SGC (0 to 20% EtOAc added to PE) method to obtain methyl 6-(methoxy-d.sub.3)-5-nitropicolinate Cpd-2-2 (10.0 g, 70.1% yield, yellow solid). LCMS calculated for CH.sub.6D.sub.3N.sub.2O.sub.5 [M+H].sup.+: m/z=216.1; found: 215.9.

Step 3: Methyl 5-amino-6-(methoxy-d.SUB.3.)picolinate

(35) Methyl 6-(methoxy-d.sub.3)-5-nitropicolinate Cpd-2-2 (10.0 g, 46.5 mmol) was dissolved in EtOAc (100 mL), then palladium on carbon (9.90 g, 5% content) was added thereto, and the resulting mixture was stirred for 2 hours at room temperature under hydrogen atmosphere. LCMS detection showed that the starting material was completely consumed and the desired compound was detected. The reaction mixture was filtered and concentrated to obtain methyl 5-amino-6-(methoxy-d.sub.3)picolinate Cpd-2-3 (8.20 g, 90.5% yield, yellow solid). LCMS calculated for C.sub.8H.sub.8D.sub.3N.sub.2O.sub.3 [M+H].sup.+: m/z=186.1; found: 186.2; .sup.1H NMR (400 MHZ, DMSO-d.sub.6) 7.53 (d, J=8.0 Hz, 1H), 6.85 (d, J=8.0 Hz, 1H), 5.88 (s, 2H), 3.75 (s, 3H).

Step 4: 3-(8-Bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)propiolaldehyde

(36) DMP (17.4 g, 41.0 mmol) was slowly added portionwise to a solution of 3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-ol Int-1-7 (6.80 g, 20.5 mmol) in DCM (120 mL) in an ice bath. Then the mixture was stirred at room temperature for 2 hours. The reaction was quenched with saturated sodium bicarbonate aqueous solution (50 mL) and extracted with DCM (30 mL3). The organic phases were combined and concentrated under vacuum. The residue was purified by SGC (0 to 15% EtOAc added to PE) method to obtain 3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)propiolaldehyde Int-2 (5.70 g, 84.3% yield, gray solid). LCMS calculated for C.sub.13H.sub.8BrF.sub.3NO [M+H].sup.+: m/z=330.0/332.0; found: 330.2/332.2; .sup.1H NMR (400 MHz, DMSO-d.sub.6) 9.47 (s, 1H), 8.49 (d, J=7.1 Hz, 1H), 7.28 (d, J=7.1 Hz, 1H), 6.90 (s, 1H), 6.78 (t, J=7.1 Hz, 1H), 4.31 (q, J=10.7 Hz, 2H).

Step 5: Isopropyl 5-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d.SUB.3.)picolinate

(37) Under nitrogen atmosphere, a solution of 3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)propiolaldehyde Int-2 (200 mg, 606 mol), methyl 5-amino-6-(methoxy-d.sub.3)picolinate Cpd-2-3 (135 mg, 730 mol), and tetraisopropyl titanate (517 mg, 1.82 mmol) in THF (2 mL) was stirred at 100 C. for 1 hour. The reaction mixture was cooled to 25 C., then added with MeOH (2 mL) and sodium cyanoborohydride (57.1 mg, 0.91 mmol), and stirred for another 10 minutes. LCMS detection showed that the starting material was completely consumed and the desired compound was detected. The reaction mixture was diluted with water (20 mL) and extracted with EtOAc (20 mL3). The combined organic layers were washed with saturated brine (50 mL), dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated. The residue was purified by SGC (0 to 25% EtOAc added to PE) method to obtain isopropyl 5-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d.sub.3)picolinate Cpd-2-4 (190 mg, 59.4% yield, yellow solid). LCMS calculated for C.sub.23H.sub.19D.sub.3BrF.sub.3N.sub.3O.sub.3 [M+H].sup.+: m/z=527.1/529.1; found: 526.9/529.0.

Step 6: Isopropyl 5-((3-(8-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d.SUB.3.)picolinate

(38) Under nitrogen atmosphere, a solution of isopropyl 5-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d.sub.3)picolinate Cpd-2-4 (170 mg, 322 mol), (3S,4R)-3-fluoro-1-methylpiperidin-4-amine (85.1 mg, 645 mol), Brettphos Pd G3 (58.3 mg, 64.3 mol), and cesium carbonate (210 mg, 644 mol) in 1,4-dioxane (2 mL) was stirred at 100 C. for 2 hours in a sealed tube. LCMS detection showed that the starting material was completely consumed and the desired compound was detected. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure. The crude product was subjected to preparative purification to obtain isopropyl 5-((3-(8-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d.sub.3)picolinate Cpd-2-5 (90.0 mg, 48.1% yield, yellow solid). LCMS calculated for C.sub.29H.sub.31D.sub.3F.sub.4NO.sub.3 [M+H].sup.+: m/z=579.3; found: 579.0.

Step 7: 5-((3-(8-(((3S,4R)-3-Fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d.SUB.3.)picolinic acid

(39) A mixture of isopropyl 5-((3-(8-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d.sub.3)picolinate Cpd-2-5 (40.0 mg, 69.2 mol) and sodium hydroxide (8.28 mg, 207 mol) in THF (0.4 mL), MeOH (0.2 mL), and water (0.1 mL) was stirred at room temperature for 16 hours. LCMS detection showed that the starting material was completely consumed and the desired compound was detected. The reaction mixture was concentrated under reduced pressure, diluted with water, adjusted to pH=5 with diluted HCl, and filtered. The residue was purified by SGC (0 to 3% MeOH added to DCM) method to obtain a crude product, which was then subjected to preparative purification to obtain 5-((3-(8-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d.sub.3)picolinic acid Cpd-2-6 (5.50 mg, 15.0% yield, white solid). LCMS calculated for C.sub.26H.sub.25D.sub.3F.sub.4N.sub.5O.sub.3 [M+H].sup.+: m/z=537.2; found: 537.0; .sup.1H NMR (400 MHz, CD.sub.3OD) 7.64 (d, J=7.3 Hz, 1H), 7.36 (d, J=6.9 Hz, 1H), 6.94 (d, J=7.8 Hz, 1H), 6.56 (s, 1H), 6.42 (t, J=7.2 Hz, 1H), 5.79 (d, J=7.3 Hz, 1H), 4.90 (s, 1H), 4.19 (s, 2H), 3.69 (q, J=10.5 Hz, 2H), 3.62-3.54 (m, 1H), 3.32-3.23 (m, 1H), 3.01 (d, J=12.2 Hz, 1H), 2.62-2.48 (m, 1H), 2.46-2.39 (m, 1H), 2.37 (s, 3H), 2.00-1.87 (m, 2H).

Step 8: 5-((3-(8-(((3S,4R)-3-Fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d.SUB.3.)-N-(methylsulfonyl)picolinamide

(40) A solution of 5-((3-(8-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d.sub.3)picolinic acid Cpd-2-6 (210 mg, 392 mol) and CDI (127 mg, 784 mol) in THF (8 mL) was stirred at 70 C. for 1 hour under argon atmosphere and then cooled to room temperature. Methanesulfonamide (74.5 mg, 784 mol) was added to the reaction mixture and stirred for 10 minutes, then DBU (120 L, 789 mol) was added thereto and stirred for another 1 hour at 70 C. The mixture was concentrated, then water (30 mL) was added, and the aqueous phase was extracted with EtOAc (50 mL3). The combined organic phases were washed with brine (50 mL3), dried over anhydrous Na.sub.2SO.sub.4, filtered, and the filtrate was concentrated under vacuum. The residue was subjected to preparative purification to obtain 5-((3-(8-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d.sub.3)-N-(methylsulfonyl)picolinamide Cpd-2 (50.0 mg, 20.8% yield, white solid). LCMS calculated for C.sub.27H.sub.28D.sub.3F.sub.4N.sub.6O.sub.4S [M+H].sup.+: m/z=614.2; found: 614.4; .sup.1H NMR (400 MHZ, DMSO-d.sub.6) 10.85 (s, 1H), 7.67 (d, J=8.0 Hz, 1H), 7.58 (d, J=6.9 Hz, 1H), 7.07 (d, J=8.1 Hz, 1H), 6.91 (s, 1H), 6.74 (s, 1H), 6.51 (t, J=7.2 Hz, 1H), 5.86 (d, J=7.5 Hz, 1H), 5.56 (d, J=8.4 Hz, 1H), 4.83 (d, J=49.6 Hz, 1H), 4.28 (d, J=6.0 Hz, 2H), 4.05 (s, 1H), 3.95 (q, J=10.7 Hz, 2H), 3.65-3.46 (m, 1H), 3.06 (t, J=11.0 Hz, 1H), 2.84 (d, J=11.5 Hz, 1H), 2.37-2.23 (m, 1H), 2.22 (s, 3H), 2.13 (t, J=11.3 Hz, 1H), 2.04-1.92 (m, 1H), 1.75-1.64 (m, 1H).

Example 3: 5-((3-(8-(((3S,4R)-3-Fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-4-methoxy-N-(methylsulfonyl)pyrimidine-2-carboxamide (Cpd-3, i.e., Compound 3)

(41) ##STR00029##

Step 1: tert-Butyl (tert-butoxycarbonyl)(2-chloro-4-methoxypyrimidin-5-yl)carbamate

(42) Under nitrogen atmosphere, a mixture of 2-chloro-4-methoxypyrimidin-5-amine Cpd-3-1 (5.00 g, 3.13 mmol), di-tert-butyl dicarbonate (14.4 g, 6.57 mmol), triethylamine (15.8 g, 15.7 mmol), and 4-dimethylaminopyridine (383 mg, 3.13 mmol) in THF (50 mL) was stirred at 40 C. for 1 hour. LCMS detection showed that the starting material was completely consumed and the desired compound was detected. The reaction mixture was diluted with water (100 mL) and extracted with EtOAc (50 mL3). The combined organic layers were washed with saturated brine (50 mL), dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated. The residue was purified by SGC (0 to 5% EtOAc added to PE) method to obtain tert-butyl (tert-butoxycarbonyl)(2-chloro-4-methoxypyrimidin-5-yl)carbamate Cpd-3-2 (4.80 g, 42.5% yield, white solid). LCMS calculated for C.sub.15H.sub.23ClN.sub.3O.sub.5 [M+H].sup.+: m/z=360.1; found: 360.2.

Step 2: Methyl 5-((tert-butoxycarbonyl)amino)-4-methoxypyrimidine-2-carboxylate

(43) Under carbon monoxide atmosphere, a mixture of tert-butyl (tert-butoxycarbonyl)(2-chloro-4-methoxypyrimidin-5-yl)carbamate Cpd-3-2 (4.30 g, 11.9 mmol), Pd(dppf)Cl.sub.2 (1.72 g, 2.38 mmol), and triethylamine (3.61 g, 35.7 mmol) in MeOH (43 mL) was stirred at 100 C. for 16 hours. LCMS detection showed that the starting material was completely consumed and the desired compound was detected. The reaction mixture was filtered and concentrated under reduced pressure. The residue was diluted with water (100 mL) and extracted with EtOAc (100 mL3). The combined organic layers were washed with saturated brine (50 mL), dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated. The residue was purified by SGC (0 to 20% EtOAc added to PE) method to obtain methyl 5-((tert-butoxycarbonyl)amino)-4-methoxypyrimidine-2-carboxylate Cpd-3-3 (2.00 g, 59.0% yield, white solid). LCMS calculated for C.sub.12H.sub.18N.sub.3O.sub.5 [M+H].sup.+: m/z=284.1; found: 284.1.

Step 3: 8-Bromo-2-(3-bromoprop-1-yn-1-yl)-3-(2,2,2-trifluoroethyl)indolizine

(44) At room temperature, 3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-ol Cpd-1-7 (2.00 g, 6.02 mmol) and triphenylphosphine (3.16 g, 12.0 mmol) were dissolved in DCM (30 mL) and stirred for 10 minutes, then carbon tetrabromide (3.00 g, 9.03 mmol) was added thereto. The reaction mixture was stirred at room temperature for 2 hours. LCMS detection showed that the starting material was completely consumed and the desired compound was detected. The reaction mixture was diluted with water (50 mL) and extracted with EtOAc (50 mL3). The combined organic layers were washed with saturated brine (50 mL), dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated. The residue was purified by SGC (0 to 10% EtOAc added to PE) method to obtain 8-bromo-2-(3-bromoprop-1-yn-1-yl)-3-(2,2,2-trifluoroethyl)indolizine Cpd-3-4 (1.55 g, 53.0% yield, white solid). LCMS calculated for C.sub.13H.sub.9Br.sub.2F.sub.3N [M+H].sup.+: m/z=393.9/395.9/397.9; found: 394.0/396.0/398.0.

Step 4: Methyl 5-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)(tert-butoxycarbonyl)amino)-4-methoxypyrimidine-2-carboxylate

(45) Under nitrogen atmosphere, a mixture of 8-bromo-2-(3-bromoprop-1-yn-1-yl)-3-(2,2,2-trifluoroethyl)indolizine Cpd-3-4 (1.37 g, 3.46 mmol), methyl 5-((tert-butoxycarbonyl)amino)-4-methoxypyrimidine-2-carboxylate Cpd-3-3 (980 mg, 3.46 mmol), and cesium carbonate (1.88 g, 13.8 mmol) in acetonitrile (20 mL) was stirred at 40 C. for 12 hours. LCMS detection showed that the starting material was completely consumed and the desired compound was detected. The reaction mixture was cooled to room temperature and filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by SGC (0 to 25% EtOAc added to PE) method to obtain methyl 5-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)(tert-butoxycarbonyl)amino)-4-methoxypyrimidine-2-carboxylate Cpd-3-5 (1.60 g, 65.1% yield, white solid). LCMS calculated for C.sub.25H.sub.25BrF.sub.3N.sub.4O.sub.5 [M+H].sup.+: m/z=597.1/599.1; found: 597.2/599.2.

Step 5: Methyl 5-((tert-butoxycarbonyl)(3-(8-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-4-methoxypyrimidine-2-carboxylate

(46) Under atmosphere, a mixture of methyl 5-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)(tert-butoxycarbonyl)amino)-4-methoxypyrimidine-2-carboxylate Cpd-3-5 (1.35 g, 2.26 mmol), (3S,4R)-3-fluoro-1-methylpiperidin-4-amine (448 mg, 3.39 mmol), Brettphos Pd G3 (205 mg, 226 mol), Ruphos (211 mg, 452 mol), and cesium carbonate (1.47 g, 4.52 mmol) in THF (20 mL) was stirred at 95 C. for 2 hours in a sealed tube. LCMS detection showed that the starting material was completely consumed and the desired compound was detected. The reaction mixture was cooled to room temperature and filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by SGC (0 to 5% MeOH added to DCM) method to obtain methyl 5-((tert-butoxycarbonyl)(3-(8-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-4-methoxypyrimidine-2-carboxylate Cpd-3-6 (1.20 g, crude product, yellow solid). LCMS calculated for C.sub.31H.sub.37F.sub.4N.sub.6O.sub.5 [M+H].sup.+: m/z=649.3; found: 649.2.

Step 6: 5-((tert-Butoxycarbonyl)(3-(8-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-4-methoxypyrimidine-2-carboxylic acid

(47) At room temperature, methyl 5-((tert-butoxycarbonyl)(3-(8-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-4-methoxypyrimidine-2-carboxylate Cpd-3-6 (1.20 g, 1.85 mmol) was dissolved in a mixed solvent of THF (8 mL), MeOH (8 mL) and water (2 mL), and lithium hydroxide (443 mg, 18.5 mmol) were added thereto. The system was stirred at room temperature for 2 hours. LCMS detection showed that the starting material was completely consumed and the desired compound was detected. The reaction mixture was adjusted to pH=4 with 1N HCl, diluted with water (20 mL), and extracted with DCM (20 mL3). The combined organic phases were dried over anhydrous Na.sub.2SO.sub.4 and concentrated under reduced pressure to obtain 5-((tert-butoxycarbonyl)(3-(8-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-4-methoxypyrimidine-2-carboxylic acid Cpd-3-7 (400 mg, crude product, yellow solid). LCMS calculated for C.sub.30H.sub.35F.sub.4N.sub.6O.sub.5 [M+H].sup.+: m/z=635.3; found: 635.5.

Step 7: tert-Butyl (3-(8-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)(4-methoxy-2-((methylsulfonyl)carbamoyl)pyrimidin-5-yl)carbamate

(48) A solution of 5-((tert-butoxycarbonyl)(3-(8-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-4-methoxypyrimidine-2-carboxylic acid Cpd-3-7 (260 mg, 410 mol) and CDI (133 mg, 820 mol) in DMF (4 mL) was stirred at 90 C. for 2 hours. The reaction mixture was cooled to room temperature, and then methanesulfonamide (78.0 mg, 820 mol) and DBU (206 mg, 820 mol) were added thereto. The reaction mixture was stirred at 90 C. for another 2 hours. LCMS detection showed that the starting material was completely consumed and the desired compound was detected. The reaction mixture was cooled to room temperature, diluted with water (50 mL), and extracted with DCM (50 mL3). The combined organic layers were washed with saturated brine (50 mL), dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated. The residue was purified by Prep-HPLC to obtain tert-butyl (3-(8-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)(4-methoxy-2-((methylsulfonyl)carbamoyl)pyrimidin-5-yl)carbamate Cpd-3-8 (100 mg, crude product, yellow solid). LCMS calculated for C.sub.31H.sub.38F.sub.4N.sub.7O.sub.6S [M+H].sup.+: m/z=711.3; found: 711.6.

Step 8: 5-((3-(8-(((3S,4R)-3-Fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-4-methoxy-N-(methylsulfonyl)pyrimidine-2-carboxamide

(49) Trifluoroacetic acid (1 mL) was added to a solution of tert-butyl (3-(8-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)(4-methoxy-2-((methylsulfonyl)carbamoyl)pyrimidin-5-yl)carbamate Cpd-3-8 (100 mg, 141 mol) in DCM (3 mL) at 0 C. The reaction mixture was slowly warmed to room temperature and stirred for 2 hours. LCMS detection showed that the starting material was completely consumed and the desired compound was detected. The reaction mixture was added dropwise to a mixture of saturated sodium bicarbonate solution (10 mL) and icy water (15 mL), and extracted with DCM (10 mL3). The combined organic layers were washed with saturated brine (50 mL), dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated. The residue was purified by Prep-HPLC to obtain 5-((3-(8-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-4-methoxy-N-(methylsulfonyl)pyrimidine-2-carboxamide Cpd-3 (8.05 mg, 8.8% yield, yellow solid). LCMS calculated for C.sub.26H.sub.30F.sub.4N.sub.7O.sub.4S [M+H].sup.+: m/z=612.2; found: 612.2; .sup.1H NMR (400 MHz, DMSO-d.sub.6) 8.05 (s, 1H), 7.60 (d, J=6.8 Hz, 1H), 6.93 (s, 1H), 6.80-6.70 (m, 1H), 6.52 (t, J=7.2 Hz, 1H), 5.86 (s, 1H), 5.64 (d, J=8.3 Hz, 1H), 4.87 (d, J=52.8 Hz, 1H), 4.32 (d, J=6.1 Hz, 2H), 4.05 (s, 3H), 4.02-3.90 (m, 2H), 3.60 (d, J=10.3 Hz, 1H), 3.23 (s, 3H), 3.18-3.08 (m, 1H), 2.91 (d, J=10.9 Hz, 1H), 2.28 (s, 3H), 2.05-1.94 (m, 1H), 1.78-1.67 (m, 1H), 1.23 (s, 2H).

Example 4: N-(Cyclopropylsulfonyl)-5-((3-(8-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d.SUB.3.)picolinamide (Cpd-4, i.e., Compound 4)

(50) ##STR00030##

Step 1: N-(Cyclopropylsulfonyl)-6-(methoxy-d.SUB.3.)-5-nitropicolinamide

(51) At room temperature, cyclopropylsulfonamide (1.33 g, 10.9 mmol) and triethylamine (3.32 g, 32.8 mmol) were sequentially added to a solution of 6-(methoxy-d.sub.3)-5-nitropicolinic acid Cpd-2-1 (2.20 g, 10.9 mmol) and 2-chloro-1-methylpyridinium iodide (3.35 g, 13.1 mmol) in dichloromethane (40 mL). The reaction mixture was stirred at 40 C. for 3 hours. LCMS detection showed that the starting material was completely consumed and the desired compound was detected. The reaction mixture was concentrated under reduced pressure, and the residue was diluted with water (100 mL). The pH was adjusted to 2 with 1N HCl aqueous solution. The precipitated solid was filtered, washed with water (100 mL), dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated to obtain N-(cyclopropylsulfonyl)-6-(methoxy-d.sub.3)-5-nitropicolinamide Cpd-4-1 (1.00 g, crude product, yellow solid). The crude product was used directly in the next step reaction. LCMS calculated for C.sub.10H.sub.7D.sub.3N.sub.3O.sub.6S [MH].sup.: m/z=303.1; found: 303.2.

Step 2: 5-Amino-N-(cyclopropylsulfonyl)-6-(methoxy-d.SUB.3.)picolinamide

(52) At room temperature, a mixture of N-(cyclopropylsulfonyl)-6-(methoxy-d.sub.3)-5-nitropicolinamide Cpd-4-1 (1.00 g, 3.29 mmol, crude product) and palladium on carbon (350 mg, 5% content) in THF (15 mL) and MeOH (15 mL) was stirred for 16 hours under hydrogen atmosphere. LCMS detection showed that the starting material was completely consumed and the desired compound was detected. The reaction mixture was filtered and concentrated under reduced pressure to obtain 5-amino-N-(cyclopropylsulfonyl)-6-(methoxy-d.sub.3)picolinamide Cpd-4-2 (730 mg, crude product, yellow solid). The crude product was used directly in the next step reaction. LCMS calculated for C.sub.10H.sub.9D.sub.3N.sub.3O.sub.4S [MH].sup.: m/z=273.1; found: 273.3.

Step 3: 5-((3-(8-Bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-N-(cyclopropylsulfonyl)-6-(methoxy-d.SUB.3.)picolinamide

(53) 3-(8-Bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)propiolaldehyde Int-2 (500 mg, 1.51 mmol), 5-amino-N-(cyclopropylsulfonyl)-6-(methoxy-d.sub.3)picolinamide Cpd-4-2 (395 mg, 1.44 mmol), and acetic acid (91.0 mg, 1.51 mmol) were dissolved in MeOH (6 mL) and DCM (4 mL), and the system was stirred at room temperature for 16 hours. Then NaBH.sub.3CN (286 mg, 4.54 mmol) was added thereto, and the system was stirred for another 0.5 hours. LCMS detection showed that the starting material was completely consumed and the desired compound was detected. The reaction mixture was diluted with water (40 mL) and filtered to collect the filter cake. The filter cake was purified by slurrying with methanol to obtain 5-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-N-(cyclopropylsulfonyl)-6-(methoxy-d.sub.3)picolinamide Cpd-4-3 (560 mg, 62.8% yield, yellow solid). LCMS calculated for C.sub.23H.sub.16BrD.sub.3F.sub.3N.sub.4O.sub.4S [MH].sup.: m/z=586.1/588.1; found: 586.2/588.2.

Step 4: N-(Cyclopropylsulfonyl)-5-((3-(8-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d.SUB.3.)picolinamide

(54) Under argon atmosphere, a mixture of 5-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-N-(cyclopropylsulfonyl)-6-(methoxy-d.sub.3)picolinamide Cpd-4-3 (560 mg, 950 mol), (3S,4R)-3-fluoro-1-methylpiperidin-4-amine (252 mg, 1.90 mmol), BrettPhos Pd G3 (86.3 mg, 100 mol), and cesium carbonate (620 mg, 1.90 mmol) in THF (13 mL) and NMP (1.3 mL) was stirred at 100 C. for 5 hours in a sealed tube. LCMS detection showed that the starting material was completely consumed and the desired compound was detected. The reaction mixture was diluted with water (50 mL) and extracted with EtOAc (20 mL3). The organic layers were combined and washed with water (40 mL) and saturated brine (40 mL), dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated under reduced pressure. The residue was purified by Prep-HPLC to obtain the product N-(cyclopropylsulfonyl)-5-((3-(8-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d.sub.3)picolinamide Cpd-4 (42.4 mg, 6.80% yield, yellow solid). LCMS calculated for C.sub.29H.sub.30D.sub.3F.sub.4N.sub.6O.sub.4S [M+H].sup.+: m/z=640.2; found: 640.1; .sup.1H NMR (400 MHZ, DMSO-d.sub.6) 10.88 (s, 1H), 7.68 (d, J=8.0 Hz, 1H), 7.58 (d, J=7.2 Hz, 1H), 7.08 (d, J=8.0 Hz, 1H), 6.92 (s, 1H), 6.76 (t, J=6.0 Hz, 1H), 6.51 (t, J=7.2 Hz, 1H), 5.86 (d, J=7.2 Hz, 1H), 5.55 (d, J=8.0 Hz, 1H), 4.83 (d, J=49.2 Hz, 1H), 4.28 (d, J=6.0 Hz, 2H), 3.99-3.91 (m, 2H), 3.62-3.49 (m, 1H), 3.13-3.01 (m, 2H), 2.84-2.82 (m, 1H), 2.33-2.21 (m, 1H), 2.23 (s, 3H), 2.15-2.10 (m, 1H), 2.03-1.93 (m, 1H), 1.71-1.67 (m, 1H), 1.19-1.15 (m, 2H), 1.09-1.04 (m, 2H).

Example 5: N-((Cyclopropylmethyl)sulfonyl)-5-((3-(8-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d.SUB.3.)picolinamide (Cpd-5, i.e., Compound 5)

(55) ##STR00031##

Step 1: N-((Cyclopropylmethyl)sulfonyl)-6-(methoxy-d.SUB.3.)-5-nitropicolinamide

(56) At room temperature, (cyclopropylmethyl)sulfonamide (1.69 g, 12.5 mmol) and triethylamine (3.62 g, 35.8 mmol) were sequentially added to a mixture of 6-(methoxy-d.sub.3)-5-nitropicolinic acid Cpd-2-1 (2.40 g, 11.9 mmol) and 2-chloro-1-methylpyridinium iodide (3.35 g, 13.1 mmol) in DCM (80 mL). The reaction mixture was stirred at 40 C. for 3 hours. LCMS detection showed that the starting material was completely consumed and the desired compound was detected. The reaction mixture was concentrated under reduced pressure, and the residue was diluted with water (100 mL), adjusted to pH=2 with 1N HCl aqueous solution, and extracted with DCM (100 mL3). The organic layers were combined, washed with water (50 mL) and saturated brine (50 mL), dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated under reduced pressure. The residue was purified by SGC (0 to 5% MeOH added to DCM) to obtain N-((cyclopropylmethyl)sulfonyl)-6-(methoxy-d.sub.3)-5-nitropicolinamide Cpd-5-1 (1.10 g, 29.0% yield, white solid). LCMS calculated for C.sub.11H.sub.9D.sub.3N.sub.3O.sub.6S [MH].sup.: m/z=317.1; found: 316.9.

Step 2: 5-Amino-N-((cyclopropylmethyl)sulfonyl)-6-(methoxy-d.SUB.3.)picolinamide

(57) At room temperature, a mixture of N-((cyclopropylmethyl)sulfonyl)-6-(methoxy-d.sub.3)-5-nitropicolinamide Cpd-5-1 (1.10 g, 3.46 mmol) and palladium on carbon (550 mg, 10% content) in THF (20 mL) and MeOH (30 mL) was stirred for 16 hours under hydrogen atmosphere. LCMS detection showed that the starting material was completely consumed and the desired compound was detected. The reaction mixture was filtered and concentrated under reduced pressure to obtain 5-amino-N-((cyclopropylmethyl)sulfonyl)-6-(methoxy-d.sub.3)picolinamide Cpd-5-2 (970 mg, crude product, yellow-white solid). The crude product was used directly in the next step reaction. LCMS calculated for C.sub.11H.sub.11D.sub.3N.sub.3O.sub.4S [MH].sup.: m/z=287.1; found: 287.1.

Step 3: 5-((3-(8-Bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-N-((cyclopropylmethyl)sulfonyl)-6-(methoxy-d.SUB.3.)picolinamide

(58) A mixture of 3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)propiolaldehyde Int-2 (500 mg, 1.51 mmol), 5-amino-N-((cyclopropylmethyl)sulfonyl)-6-(methoxy-d.sub.3)picolinamide Cpd-5-2 (416 mg, 1.44 mmol), acetic acid (86.6 mg, 1.44 mmol), and Na.sub.2SO.sub.4 (1.02 g, 7.21 mmol) in MeOH (20 mL) and 1,2-dichloroethane (20 mL) was stirred at room temperature for 16 hours. Then NaBH.sub.3CN (181 mg, 2.89 mmol) was added thereto, and the system was stirred for another 0.5 hours at room temperature. LCMS detection showed that the starting material was completely consumed and the desired compound was detected. The reaction mixture was diluted with water (40 mL) and extracted with DCM (100 mL3). The organic layers were combined, washed with water (50 mL) and saturated brine (50 mL), dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated under reduced pressure. The residue was purified by SGC (0 to 70% EtOAc added to PE) method to obtain 5-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-N-((cyclopropylmethyl)sulfonyl)-6-(methoxy-d.sub.3)picolinamide Cpd-5-3 (390 mg, 44.9% yield, yellow solid). LCMS calculated for C.sub.24H.sub.20BrD.sub.3F.sub.3N.sub.4O.sub.4S [M+H].sup.+: m/z=602.1/604.1; found: 602.3/604.3.

Step 4: N-((Cyclopropylmethyl)sulfonyl)-5-((3-(8-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d.SUB.3.)picolinamide

(59) Under argon atmosphere, a mixture of 5-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-N-((cyclopropylmethyl)sulfonyl)-6-(methoxy-d.sub.3)picolinamide Cpd-5-3 (390 mg, 650 mol), (3S,4R)-3-fluoro-1-methylpiperidin-4-amine (171 mg, 1.29 mmol), BrettPhos Pd G3 (58.7 mg, 60.0 mol), and cesium carbonate (422 mg, 1.29 mmol) in DMF (12 mL) was stirred at 100 C. for 5 hours. LCMS detection showed that the starting material was completely consumed and the desired compound was detected. The reaction mixture was diluted with water (50 mL), adjusted to pH=8 with 1N HCl aqueous solution, and extracted with EtOAc/THF (2:1, 30 mL3). The organic layers were combined and washed with water (40 mL) and saturated brine (40 mL), dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated under reduced pressure. The residue was purified by Prep-HPLC to obtain the product N-((cyclopropylmethyl)sulfonyl)-5-((3-(8-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d.sub.3)picolinamide Cpd-5 (81.2 mg, 19.1% yield, white solid). LCMS calculated for C.sub.30H.sub.32D.sub.3F.sub.4N.sub.6O.sub.4S [M+H].sup.+: m/z=654.3; found: 654.7; .sup.1H NMR (400 MHZ, DMSO-d.sub.6) 10.86 (s, 1H), 7.66 (d, J=8.0 Hz, 1H), 7.58 (d, J=7.2 Hz, 1H), 7.07 (d, J=8.0 Hz, 1H), 6.92 (s, 1H), 6.77 (t, J=6.0 Hz, 1H), 6.51 (t, J=7.2 Hz, 1H), 5.86 (d, J=7.2 Hz, 1H), 5.55 (d, J=8.0 Hz, 1H), 4.83 (d, J=49.2 Hz, 1H), 4.28 (d, J=6.0 Hz, 2H), 3.98-3.90 (m, 2H), 3.62-3.48 (m, 1H), 3.43 (d, J=7.2 Hz, 2H), 3.08-3.03 (m, 1H), 2.86-2.82 (m, 1H), 2.33-2.21 (m, 1H), 2.23 (s, 3H), 2.15-2.09 (m, 1H), 2.03-1.93 (m, 1H), 1.72-1.66 (m, 1H), 1.09-1.00 (m, 1H), 0.59-0.54 (m, 2H), 0.34-0.30 (m, 2H).

Example 6: 5-((3-(8-(((3S,4R)-3-Fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d.SUB.3.)-N-((2,2,2-trifluoroethyl)sulfonyl)picolinamide (Cpd-6, i.e., Compound 6)

(60) ##STR00032##

Step 1: N-(2,4-Dimethoxybenzyl)-2,2,2-trifluoroethane-1-sulfonamide

(61) At room temperature, 2,2,2-trifluoroethane-1-sulfonyl chloride Cpd-6-1 (750 mg, 4.11 mmol) was added dropwise to a solution of 2,4-dimethoxybenzylamine (1.37 g, 8.22 mmol) in THF (20 mL), and the system was stirred for 1 hour. LCMS detection showed that the starting material was completely consumed and the desired compound was detected. The reaction mixture was concentrated under reduced pressure, and the resulting residue was purified by SGC (0 to 1% MeOH added to DCM) method to obtain N-(2,4-dimethoxybenzyl)-2,2,2-trifluoroethane-1-sulfonamide Cpd-6-2 (1.20 g, 93.2% yield, white solid). LCMS calculated for C.sub.11H.sub.14F.sub.3NNaO.sub.4S [M+Na].sup.+: m/z=336.1; found: 336.1.

Step 2: 2,2,2-Trifluoroethane-1-sulfonamide

(62) N-(2,4-Dimethoxybenzyl)-2,2,2-trifluoroethane-1-sulfonamide Cpd-6-2 (1.45 g, 4.63 mmol) was dissolved in DCM (15 mL), and trifluoroacetic acid (15 mL) was slowly added thereto, and then the system was stirred for 1 hour at room temperature. LCMS detection showed that the starting material was completely consumed and the desired compound was detected. The reaction mixture was concentrated under reduced pressure to obtain 2,2,2-trifluoroethane-1-sulfonamide Cpd-6-3 (851 mg, 99.1% yield, purple solid). The crude product was used directly in the next step reaction. 1H NMR (400 MHZ, DMSO-d.sub.6) 7.50 (s, 2H), 4.26 (q, J=10.0 Hz, 2H).

Step 3: Methyl 5-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d.SUB.3.)picolinate

(63) A mixture of 3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)propiolaldehyde Int-2 (700 mg, 2.33 mmol), methyl 5-amino-6-(methoxy-d.sub.3)picolinate Cpd-2-3 (432 mg, 2.33 mmol), and acetic acid (280 mg, 4.67 mmol) in MeOH (20 mL) was stirred at room temperature for 16 hours. Then NaBH.sub.3CN (440 mg, 7.00 mmol) was added thereto, and the system was stirred for another 2 hours at room temperature. LCMS detection showed that the starting material was completely consumed and the desired compound was detected. The reaction mixture was diluted with water (40 mL) and extracted with DCM (100 mL3). The organic layers were combined, washed with water (50 mL) and saturated brine (50 mL), dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated under reduced pressure. The residue was purified by SGC (0 to 70% EtOAc added to PE) method to obtain methyl 5-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d.sub.3)picolinate Cpd-6-4 (625 mg, 53.7% yield, yellow solid). LCMS calculated for C.sub.21H.sub.15D.sub.3BrF.sub.3N.sub.3O.sub.3 [M+H].sup.+: m/z=499.1/501.1; found: 499.0/501.0.

Step 4: 5-((3-(8-Bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d.SUB.3.)picolinic acid

(64) Methyl 5-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d.sub.3)picolinate Cpd-6-4 (300 mg, 600 mol) and sodium hydroxide (72.1 mg, 1.80 mmol) were dissolved in a mixed solvent of THF (9 mL), MeOH (6 mL), and water (1 mL), and the system was stirred at 50 C. for 1 hour. LCMS detection showed that the starting material was completely consumed and the desired compound was detected. The reaction mixture was concentrated under reduced pressure, diluted with water (20 mL), and adjusted to pH=5 with 1N HCl. The solid was collected by filtration and dried to obtain 5-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d.sub.3)picolinic acid Cpd-6-5 (246 mg, 84.4% yield, white solid). LCMS calculated for C.sub.20H.sub.13D.sub.3BrF.sub.3N.sub.3O.sub.3 [M+H].sup.+: m/z=485.1/487.1; found: 485.0/487.0.

Step 5: 5-((3-(8-Bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d.SUB.3.)-N-((2,2,2-trifluoroethyl)sulfonyl)picolinamide

(65) Tricthylamine (0.21 mL, 1.48 mmol) was added to a mixture of 5-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d.sub.3)picolinic acid Cpd-6-5 (240 mg, 490 mol), 2,2,2-trifluoroethane-1-sulfonamide Cpd-6-3 (242 mg, 1.48 mmol), and 2-chloro-1-methylpyridinium iodide (316 mg, 1.24 mmol) in DCM (40 mL) at 0 C. The reaction mixture was stirred at 0 C. for 30 minutes. LCMS detection showed that the starting material was completely consumed and the desired compound was detected. The reaction mixture was diluted with DCM (100 mL) and washed sequentially with 0.5N HCl aqueous solution (50 mL), saturated sodium bicarbonate aqueous solution (50 mL), and saturated brine (50 mL), dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated under reduced pressure. The residue was purified by SGC (0 to 8% MeOH added to DCM) method to obtain 5-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d.sub.3)-N-((2,2,2-trifluoroethyl)sulfonyl)picolinamide Cpd-6-6 (160 mg, 51.3% yield, white solid). LCMS calculated for C.sub.22H.sub.15D.sub.3BrF.sub.6N.sub.4O.sub.4S [M+H].sup.+: m/z=630.0/632.0; found: 629.9/631.9.

Step 6: 5-((3-(8-(((3S,4R)-3-Fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d.SUB.3.)-N-((2,2,2-trifluoroethyl)sulfonyl)picolinamide

(66) Under argon atmosphere, a mixture of 5-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d.sub.3)-N-((2,2,2-trifluoroethyl)sulfonyl)picolinamide Cpd-6-6 (160 mg, 250 mol), (3S,4R)-3-fluoro-1-methylpiperidin-4-amine (67.0 mg, 510 mol), BrettPhos Pd G3 (23.0 mg, 30.0 mol), and cesium carbonate (165 mg, 510 mol) in THF (13 mL) and NMP (1.3 mL) was stirred at 100 C. for 4 hours in a sealed tube. LCMS detection showed that the starting material was completely consumed and the desired compound was detected. The reaction mixture was diluted with water (50 mL) and extracted with EtOAc (20 mL3). The organic layers were combined and washed with water (40 mL) and brine (40 mL), dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated under reduced pressure. The residue was purified by SGC (0 to 8% MeOH added to DCM) to obtain the product 5-((3-(8-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d.sub.3)-N-((2,2,2-trifluoroethyl)sulfonyl)picolinamide Cpd-6 (14.0 mg, 8.09% yield, white solid). LCMS calculated for C.sub.28H.sub.27D.sub.3F.sub.7N.sub.6O.sub.4S [M+H].sup.+: m/z=682.2; found: 682.5; .sup.1H NMR (400 MHZ, DMSO-d.sub.6) 10.53 (s, 1H), 7.64 (dd, J=16.0, 6.9 Hz, 2H), 7.01 (d, J=7.5 Hz, 1H), 6.93 (s, 1H), 6.53 (t, J=6.4 Hz, 1H), 6.50-6.33 (m, 1H), 5.90 (d, J=7.1 Hz, 1H), 5.81 (d, J=6.4 Hz, 1H), 5.09 (d, J=47.1 Hz, 1H), 4.70-4.33 (m, 2H), 4.26 (d, J=4.2 Hz, 2H), 3.97 (d, J=10.4 Hz, 2H), 3.89-3.76 (m, 1H), 2.73 (s, 3H), 2.49-2.45 (m, 2H), 2.26-2.16 (m, 1H), 1.97-1.88 (m, 1H).

Example 7: N-(Cyclopropylsulfonyl)-5-((3-(8-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-methoxypicolinamide (Cpd-7, i.e., Compound 7)

(67) ##STR00033##

Step 1: 6-Chloro-N-(cyclopropylsulfonyl)-5-nitropicolinamide

(68) At room temperature, cyclopropylsulfonamide (3.59 g, 29.6 mmol) and triethylamine (7.49 g, 74.1 mmol) were sequentially added to a mixture of 6-chloro-5-nitropicolinic acid Cpd-1-1 (5.00 g, 24.7 mmol) and 2-chloro-1-methylpyridinium iodide (7.57 g, 29.6 mmol) in DCM (50 mL). The reaction system was stirred at 40 C. for 3 hours. LCMS detection showed that the starting material was completely consumed and the desired compound was detected. The reaction mixture was concentrated under reduced pressure, and the residue was diluted with water (100 mL). The pH was adjusted to 2 with 1N HCl aqueous solution. The precipitated solid was filtered, washed with water, and dried to obtain 6-chloro-N-(cyclopropylsulfonyl)-5-nitropicolinamide Cpd-7-1 (8.90 g, crude product, white solid). The crude product was used directly in the next step reaction. LCMS calculated for C.sub.9H.sub.7ClN.sub.3O.sub.5S [MH]=: m/z=304.0/306.0; found: 303.9/305.9.

Step 2: N-(Cyclopropylsulfonyl)-6-methoxy-5-nitropicolinamide

(69) At room temperature, a solution of sodium methoxide (16.2 mL, 87.3 mmol, 5.4 mol/L) in methanol was added dropwise to a solution of 6-chloro-N-(cyclopropylsulfonyl)-5-nitropicolinamide Cpd-7-1 (8.90 g, 29.1 mmol) in methanol (90 mL). The reaction mixture was stirred at room temperature for 30 minutes. LCMS detection showed that the starting material was completely consumed and the desired compound was detected. The reaction mixture was quenched with 1N HCl aqueous solution, and the pH was adjusted to 2. The precipitated solid was filtered, washed with water, and dried to obtain N-(cyclopropylsulfonyl)-6-methoxy-5-nitropicolinamide Cpd-7-2 (6.70 g, crude product, white solid). The crude product was used directly in the next step reaction. LCMS calculated for C.sub.10H.sub.10N.sub.3O.sub.6S [MH].sup.: m/z=300.0; found: 300.0.

Step 3: 5-Amino-N-(cyclopropylsulfonyl)-6-methoxypicolinamide

(70) At room temperature, a mixture of N-(cyclopropylsulfonyl)-6-methoxy-5-nitropicolinamide Cpd-7-2 (6.70 g, 22.2 mmol) and palladium on carbon (3.30 g, 5% content) in THF (200 mL) and MeOH (130 mL) was stirred for 16 hours at room temperature under hydrogen atmosphere. LCMS detection showed that the starting material was completely consumed and the desired compound was detected. The reaction mixture was filtered and concentrated under reduced pressure to obtain 5-amino-N-(cyclopropylsulfonyl)-6-methoxypicolinamide Cpd-7-3 (6.20 g, crude product, white solid). The crude product was used directly in the next step reaction. LCMS calculated for C.sub.10H.sub.12N.sub.3O.sub.4S [MH].sup.: m/z=270.1; found: 270.1.

Step 4: 5-((3-(8-Bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-N-(cyclopropylsulfonyl)-6-methoxypicolinamide

(71) A mixture of 3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)propiolaldehyde Int-2 (1.00 g, 3.03 mmol), 5-amino-N-(cyclopropylsulfonyl)-6-methoxypicolinamide Cpd-7-3 (820 mg, 3.03 mmol), and acetic acid (180 mg, 3.03 mmol) in MeOH (15 mL) was stirred at room temperature for 16 hours. Then NaBH.sub.3CN (570 mg, 9.09 mmol) was added thereto, and the system was stirred for another 2 hours at room temperature. LCMS detection showed that the starting material was completely consumed and the desired compound was detected. The reaction mixture was quenched with saturated sodium bicarbonate aqueous solution (50 mL) and extracted with EtOAc (20 mL3). The organic layers were combined and washed with water (40 mL) and saturated brine (40 mL), dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated under reduced pressure. The residue was purified by slurrying with dichloromethane to obtain 5-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-N-(cyclopropylsulfonyl)-6-methoxypicolinamide Cpd-7-4 (500 mg, 28.2% yield, yellow solid). LCMS calculated for C.sub.23H.sub.21BrF.sub.3N.sub.4O.sub.4S [M+H].sup.+: m/z=585.0/587.0; found: 585.3/587.3.

Step 5: N-(Cyclopropylsulfonyl)-5-((3-(8-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-methoxypicolinamide

(72) Under argon atmosphere, a mixture of 5-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-N-(cyclopropylsulfonyl)-6-methoxypicolinamide Cpd-7-4 (500 mg, 850 mol), (3S,4R)-3-fluoro-1-methylpiperidin-4-amine (226 mg, 1.71 mmol), BrettPhos Pd G3 (77.4 mg, 10.0 mol), and cesium carbonate (557 mg, 1.71 mmol) in THF (15 mL) and NMP (1.5 mL) was stirred at 100 C. for 5 hours in a sealed tube. LCMS detection showed that the starting material was completely consumed and the desired compound was detected. The reaction mixture was diluted with water (50 mL) and extracted with EtOAc (20 mL3). The organic layers were combined and washed with water (40 mL) and saturated brine (40 mL), dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated under reduced pressure. The residue was purified by Prep-HPLC to obtain the product N-(cyclopropylsulfonyl)-5-((3-(8-(((3S,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-methoxypicolinamide Cpd-7 (46.1 mg, 8.3% yield, yellow solid). LCMS calculated for C.sub.29H.sub.33F.sub.4N.sub.6O.sub.4S [M+H].sup.+: m/z=637.2; found: 637.1; .sup.1H NMR (400 MHZ, DMSO-d.sub.6) 10.83 (s, 1H), 7.68 (d, J=6.8 Hz, 1H), 7.58 (d, J=6.8 Hz, 1H), 7.08 (d, J=8.4 Hz, 1H), 6.92 (s, 1H), 6.76 (t, J=6.0 Hz, 1H), 6.51 (t, J=7.2 Hz, 1H), 5.86 (d, J=7.6 Hz, 1H), 5.55 (d, J=8.4 Hz, 1H), 4.83 (d, J=48.6 Hz, 1H), 4.28 (d, J=6.4 Hz, 2H), 4.06 (s, 3H), 4.00-3.91 (m, 2H), 3.62-3.49 (m, 1H), 3.11-3.03 (m, 2H), 2.84-2.82 (m, 1H), 2.33-2.21 (m, 1H), 2.23 (s, 3H), 2.15-2.09 (m, 1H), 2.03-1.93 (m, 1H), 1.71-1.67 (m, 1H), 1.19-1.15 (m, 2H), 1.10-1.04 (m, 2H).

Example 8: 5-((3-(8-(((3R,4S)-3-Fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d.SUB.3.)-N-(methylsulfonyl)picolinamide (Cpd-8, i.e., Compound 8)

(73) ##STR00034##

Step 1: 6-Chloro-N-(methylsulfonyl)-5-nitropicolinamide

(74) 2-Chloro-1-methylpyridinium iodide (95.0 g, 372 mmol) and methanesulfonamide (35.0 g, 368 mmol) were added to a suspension of 6-chloro-5-nitropicolinic acid Cpd-1-1 (50.0 g, 247 mmol) in dichloromethane (500 mL) with stirring. After the system was stirred for 10 minutes, triethylamine (100 g, 988 mmol) was added dropwise thereto. Then the reaction mixture was heated to reflux and reacted for about 5 hours. After being cooled and concentrated, the residue was diluted with water (250 mL) and adjusted to pH=2 with concentrated HCl. A large amount of yellow solid precipitated. The mixture was stirred at room temperature overnight, subjected to suction filtration, and the filter cake was rinsed with water (250 mL). The filter cake was dried at 50 C. under vacuum to obtain 6-chloro-N-(methylsulfonyl)-5-nitropicolinamide Cpd-8-1 (68.0 g, 98.5% yield, yellow solid). LCMS calculated for C.sub.7H.sub.5ClN.sub.3O.sub.5S [MH].sup.: m/z=278.0; found: 278.1.

Step 2: 6-(Methoxy-d.SUB.3.)-N-(methylsulfonyl)-5-nitropicolinamide

(75) Under argon atmosphere, 6-chloro-N-(methylsulfonyl)-5-nitropicolinamide Cpd-8-1 (45.0 g, 161 mmol) and deuterated methanol (17.4 g, 482 mmol) were dissolved in anhydrous NMP (450 mL), then the system was cooled to 15 C. to 0 C., and a solution of 1M potassium tert-butoxide in tetrahydrofuran (338 mL, 338 mmol) was added dropwise thereto. Then the system temperature was controlled at 10 C. to 0 C., and the reaction was stirred for 40 minutes, then 4N HCl (135 mL) was added dropwise to quench the reaction. The system was concentrated under reduced pressure, and water (1.62 L) was added dropwise thereto with stirring. A large amount of yellow solid was produced and filtered. The filter cake was rinsed with water (500 mL) and methyl tert-butyl ether (500 mL), and the filter cake was recrystallized with acetonitrile-water to obtain the product 6-(methoxy-d.sub.3)-N-(methylsulfonyl)-5-nitropicolinamide Cpd-8-2 (30.0 g, 67.0% yield, yellow solid). LCMS calculated for C.sub.8H.sub.7D.sub.3N.sub.3O.sub.6S [M+H].sup.+: m/z=279.0; found: 279.2.

Step 3: 5-Amino-6-(methoxy-d.SUB.3.)-N-(methylsulfonyl)picolinamide

(76) 6-(Methoxy-d.sub.3)-N-(methylsulfonyl)-5-nitropicolinamide Cpd-8-2 (30.0 g, 108 mmol) was mixed with THF (600 mL) and acetic acid (3.24 g, 53.9 mmol), and stirred to form a solution, then methanol (600 mL) was added thereto. After argon replacement, palladium on carbon (3.00 g, 10% content) was added thereto, and argon was replaced with hydrogen, and then the reaction was stirred at room temperature for 12 hours. The reaction mixture was filtered through diatomite, and the filter cake was rinsed with THF. The filtrate was combined and concentrated. The residue was dried to obtain the product 5-amino-6-(methoxy-d.sub.3)-N-(methylsulfonyl)picolinamide Cpd-8-3 (26.0 g, 97.1% yield, off-white solid), which was directly used in the next step without further treatment. LCMS calculated for C.sub.8H.sub.9D.sub.3N.sub.3O.sub.4S [M+H].sup.+: m/z=249.1; found: 249.2.

Step 4: 8-Bromo-2-(3-((2-(methoxy-d.SUB.3.)-6-((methylsulfonyl)carbamoyl)pyridin-3-yl)amino)prop-1-yn-1-yl)-3-(2,2,2-trifluoroethyl)indolizine

(77) A mixed solvent of methanol (100 mL) and dichloromethane (100 mL) was added to 5-amino-6-(methoxy-d.sub.3)-N-(methylsulfonyl)picolinamide Cpd-8-3 (7.20 g, 29.0 mmol) and acetic acid (3.14 g, 58.0 mmol). When the mixture became clear, Int-2 (10.1 g, 30.5 mmol) was added thereto. The system was stirred at 10 C. to 15 C. for 16 hours. A large amount of turbidity was produced in the system, and then sodium cyanoborohydride (6.00 g, 87.1 mmol) was added thereto, and the system was stirred for another 2 hours. The system was concentrated, added with methanol (100 mL), added dropwise with water (100 mL) at room temperature, then stirred at room temperature for 2 hours, and filtered. The filter cake was rinsed with water (50 mL) and methyl tert-butyl ether (100 mL), and dried under vacuum with an oil pump to obtain 8-bromo-2-(3-((2-(methoxy-d.sub.3)-6-((methylsulfonyl)carbamoyl)pyridin-3-yl)amino)prop-1-yn-1-yl)-3-(2,2,2-trifluoroethyl)indolizine Cpd-8-4 (12.9 g, 79.0% yield, off-white solid). LCMS calculated for C.sub.21H.sub.16D.sub.3BrF.sub.3N.sub.4O.sub.4S [M+H].sup.+: m/z=562.0/564.0; found: 562.2/564.2.

Step 5: 5-((3-(8-(((3R,4S)-3-Fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d.SUB.3.)-N-(methylsulfonyl)picolinamide

(78) Under argon atmosphere, a mixture of 8-bromo-2-(3-((2-(methoxy-d.sub.3)-6-((methylsulfonyl)carbamoyl)pyridin-3-yl)amino)prop-1-yn-1-yl)-3-(2,2,2-trifluoroethyl)indolizine Cpd-8-4 (1.80 g, 3.20 mmol), (3R,4S)-3-fluoro-1-methylpiperidin-4-amine (500 mg, 3.80 mmol), BrettPhos Pd G3 (600 mg, 662 mol), and cesium carbonate (3.44 g, 10.6 mmol) in THF (20 mL) and DMF (10 mL) was stirred at 70 C. for 3 hours. The system was cooled and concentrated, and the residue was purified by silica gel column chromatography (MeOH:DCM=1:24) to obtain the product 5-((3-(8-(((3R,4S)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d.sub.3)-N-(methylsulfonyl)picolinamide Cpd-8 (830 mg, 42.3% yield, off-white solid). LCMS calculated for C.sub.27H.sub.28D.sub.3F.sub.4N.sub.6O.sub.4S [M+H].sup.+: m/z=614.2; found: 614.4; .sup.1H NMR (400 MHZ, DMSO-d.sub.6) d 10.83 (s, 1H), 7.67 (d, J=8.0 Hz, 1H), 7.58 (d, J=6.9 Hz, 1H), 7.07 (d, J=8.1 Hz, 1H), 6.92 (s, 1H), 6.75 (t, J=6.1 Hz, 1H), 6.52 (t, J=7.1 Hz, 1H), 5.86 (d, J=7.5 Hz, 1H), 5.56 (d, J=8.4 Hz, 1H), 4.84 (d, J=49.3 Hz, 1H), 4.28 (d, J=6.1 Hz, 2H), 3.95 (q, J=10.8 Hz, 2H), 3.56 (d, J=28.9 Hz, 1H), 3.07 (t, J=11.2 Hz, 1H), 2.85 (d, J=11.1 Hz, 1H), 2.40-2.09 (m, 5H), 2.05-1.91 (m, 1H), 1.69 (dd, J=13.2, 4.0 Hz, 1H).

Example 9: 5-((3-(8-(((3S,4S)-3-Fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d.SUB.3.)-N-(methylsulfonyl)picolinamide (Cpd-9, i.e., Compound 9)

(79) ##STR00035##

Step 1: 5-((3-(8-(((3S,4S)-3-Fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d.SUB.3.)-N-(methylsulfonyl)picolinamide

(80) Under argon atmosphere, a mixture of 5-((3-(8-bromo-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-N-(methylsulfonyl)-6-(methoxy-d.sub.3)picolinamide Cpd-8-4 (1.80 g, 3.20 mmol), (3S,4S)-3-fluoro-1-methylpiperidin-4-amine (400 mg, 3.02 mmol), BrettPhos Pd G3 (600 mg, 662 mol), and cesium carbonate (3.44 g, 10.6 mmol) in THF (20 mL) and DMF (10 mL) was stirred at 70 C. for 3 hours. The system was cooled and concentrated, and the residue was purified by silica gel column chromatography (MeOH:DCM=1:24) to obtain the product 5-((3-(8-(((3S,4S)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d.sub.3)-N-(methylsulfonyl)picolinamide Cpd-9 (500 mg, 26.9% yield, brown solid). LCMS calculated for C.sub.27H.sub.28D.sub.3F.sub.4N.sub.6O.sub.4S [M+H].sup.+: m/z=614.2; found: 614.4; .sup.1H NMR (400 MHZ, DMSO-d.sub.6) 10.85 (s, 1H), 7.68 (d, J=8.0 Hz, 1H), 7.55 (d, J=6.9 Hz, 1H), 7.07 (d, J=8.1 Hz, 1H), 6.78 (s, 1H), 6.74 (d, J=7.8 Hz, 1H), 6.51 (t, J=7.2 Hz, 1H), 5.86 (d, J=7.5 Hz, 1H), 5.74 (d, J=8.3 Hz, 1H), 4.57 (dtd, J=49.6, 9.3, 4.7 Hz, 1H), 4.28 (d, J=6.2 Hz, 2H), 3.95 (q, J=10.7 Hz, 2H), 3.49 (d, J=4.2 Hz, 1H), 3.10 (p, J=5.4 Hz, 1H), 2.77-2.62 (m, 1H), 2.24 (s, 3H), 2.19-1.90 (m, 3H), 1.61-1.39 (m, 1H).

Example 10: 5-((3-(8-(((3R,4R)-3-Fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d.SUB.3.)-N-(methylsulfonyl)picolinamide (Cpd-10, i.e., Compound 10)

(81) ##STR00036##

Step 1: 5-((3-(8-(((3R,4R)-3-Fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d.SUB.3.)-N-(methylsulfonyl)picolinamide

(82) Under argon atmosphere, a mixture of 8-bromo-2-(3-((2-(methoxy-d.sub.3)-6-((methylsulfonyl)carbamoyl)pyridin-3-yl)amino)prop-1-yn-1-yl)-3-(2,2,2-trifluoroethyl)indolizine Cpd-8-4 (1.80 g, 3.20 mmol), (3R,4R)-3-fluoro-1-methylpiperidin-4-amine (500 mg, 3.78 mmol), BrettPhos Pd G3 (600 mg, 662 mol), and cesium carbonate (3.44 g, 10.6 mmol) in THF (20 mL) and DMF (10 mL) was stirred at 70 C. for 3 hours. The system was cooled and concentrated, and the residue was purified by silica gel column chromatography (MeOH:DCM=1:24) to obtain the product 5-((3-(8-(((3R,4R)-3-fluoro-1-methylpiperidin-4-yl)amino)-3-(2,2,2-trifluoroethyl)indolizin-2-yl)prop-2-yn-1-yl)amino)-6-(methoxy-d.sub.3)-N-(methylsulfonyl)picolinamide Cpd-10 (1.12 g, 57.0% yield, brown solid). LCMS calculated for C.sub.27H.sub.28D.sub.3F.sub.4N.sub.6O.sub.4S [M+H].sup.+: m/z=614.2; found: 614.4; .sup.1H NMR (400 MHZ, DMSO-d.sub.6) 10.89 (s, 1H), 7.68 (d, J=8.0 Hz, 1H), 7.55 (d, J=6.9 Hz, 1H), 7.08 (d, J=8.1 Hz, 1H), 6.81-6.78 (m, 2H), 6.51 (t, J=7.1 Hz, 1H), 5.86 (d, J=7.4 Hz, 1H), 5.74 (d, J=8.3 Hz, 1H), 4.57 (dtd, J=49.6, 9.2, 4.8 Hz, 1H), 4.28 (d, J=6.1 Hz, 2H), 3.95 (q, J=10.7 Hz, 2H), 3.59-3.41 (m, 1H), 3.10 (dt, J=10.9, 5.7 Hz, 1H), 2.77-2.59 (m, 1H), 2.25 (s, 3H), 2.17-1.83 (m, 3H), 1.62-1.38 (m, 1H).

(83) Biological Evaluation

(84) I. Biochemical Assay: Test of Compounds Promoting the Binding of p53 Y220C Mutant With DNA

(85) Homogeneous time-resolved fluorescence (HTRF) assay was used to measure the reactivation effect of compounds on the p53 Y220C mutant. The recombinant His-tagged p53 Y220C (94-312) used for HTRF assays was expressed in E. coli and purified to the purity of 90% through Ni-NTA column. The biotin-labeled DNA used for HTRF assays was synthesized by Pharmaron Inc, with the specific sequence of 5-ATTAGGCATGTCTAGGCATGTCTAGG-3 (SEQ ID NO: 1).

(86) The binding of recombinant His-tagged p53 Y220C protein and biotin-labeled DNA was measured using fluorescence resonance energy transfer (FRET). For FRET assays, binding between p53 mutants and DNA sequences was measured by detecting the fluorescence of the interaction between an anti-His antibody (Cisbio, 61HI2KLA) conjugated to Eu and d.sub.2-conjugated streptavidin (Cisbio, 610SADLF) bound to a biotin-labeled DNA molecule.

(87) Compounds were prepared into 2 mM stock solutions and serially diluted into 10 concentrations at a 1:3 ratio in DMSO. Each 200-fold working concentration compound was sequentially diluted to 4-fold working concentration. 4 L of compound solution was transferred line by line to a 384-well assay plate using Echo, containing 2 replicates per column. 4 L of p53 solution was added to the assay plate. 4 L of biotin DNA solution was added to the assay plate, and then 4 L of assay solution (His-Eu antibody and streptavidin-d.sub.2) was added to each well of the assay plate. The assay plate was incubated overnight and protected from light. Fluorescence was read on BMG (BMG LRBTECH).

(88) The ratio of each well was calculated (ratio_665 nm/615 nm-ratio_background). The activity % was calculated as follows:

(89) $C=\left(\mathrm{Ave\_Ac}-\mathrm{Ave\_Ba}\right)/\left(\mathrm{Ave\_Dc}-\mathrm{Ave\_Bd}\right)$$R\mathrm{data}=\left(A-\mathrm{Ave\_Ba}-C*D\right)/\left(D-\mathrm{Ave\_Bd}\right)*\left(\mathrm{Ave\_Dc}-\mathrm{Ave\_Bd}\right)$$\mathrm{Activity}\%=R\mathrm{data}/\mathrm{Ave\_VC}*100.$ A: fluorescence intensity of the sample at 665 nm; D: fluorescence intensity of the sample at 615 nm; Ba: fluorescence intensity of the plate background at 665 nm; Bd: fluorescence intensity of the plate background at 615 nm; Dc: fluorescence intensity of HIS-Eu background at 615 nm; Ac: fluorescence intensity of streptavidin-d.sub.2 background at 665 nm; VC: DMSO treatment group.

(90) SC.sub.150 was calculated using Graphpad 8.0 to fit the logarithm of activity % and compound concentration to a nonlinear regression (dose response-variable slope).

(91) TABLE-US-00002 TABLE 1 Activity of compounds promoting the binding of p53 Y220C mutant with DNA Compound number SC.sub.150 (nM) Cpd-A 10 Cpd-B 15 Cpd-C 4.6 Cpd-1 1.0 Cpd-2 0.58 Cpd-3 0.74 Cpd-4 0.35 Cpd-5 1.1 Cpd-6 1.7 Cpd-7 0.64

(92) Cpd-A is selected from WO2021061643A1; Cpd-B is selected from WO2022213975A1;

(93) ##STR00037##

(94) The above data show that the representative compounds of the present disclosure have good activity for promoting the binding of p53 Y220C mutants with DNA, and are obviously superior to the reference compounds.

(95) II. Cellular Assay: Test of Anti-Proliferation Effect on NUGC-3 Cells

(96) The anti-proliferation effect was assessed against the human gastric adenocarcinoma cell line based on the activity of dehydrogenase in cells, which showed good correlation with the number of living cells. Cell line NUGC-3 (JCRB, JCRB0822) was cultured in RPMI 1640 (Invitrogen, 11875-085) medium supplemented with 10% (v/v) fetal bovine serum (BI, 04-002-1A), according to the standard instructions of the American Type Culture Collection. The cell line was authenticated by short tandem repeat profiling.

(97) In the proliferation assay, 1000 NUGC-3 cells were seeded in a 96-well flat clear bottom, TC-treated plate (Corning, 3903) in 200 L of medium per well, and allowed to recover overnight in culture medium. Compounds were added with increasing concentration, with DMSO treatment serving as a positive control. After 5 days of treatment, the plate was equilibrated to room temperature for 30 minutes, and the number of living cells was measured by the addition of the Cell Counting Kit-8 reagent (MCE, HY-K0301). Absorbance was measured on an Enspire (Perkin elme). The relative viability of each group was presented as the percentage change relative to the positive control group and then fit to a four-parameter logit non-linear curve using the program XLFit (IDBS)

(98) TABLE-US-00003 TABLE 2 Anti-proliferation activity of compounds on NUGC-3 cells Compound number NUGC-3 IC.sub.50 (M) Cpd-A 0.78 Cpd-B 0.36 Cpd-C 0.23 Cpd-1 0.10 Cpd-2 0.058 Cpd-4 0.097 Cpd-5 0.081 Cpd-6 0.19 Cpd-7 0.070 Cpd-8 0.34 Cpd-9 0.55 Cpd-10 0.90

(99) The above data show that the representative compounds of the present disclosure have good inhibitory activity on the proliferation of NUGC-3 cells, and are obviously superior to the reference compounds.

(100) III. Evaluation of Drug-Drug Interactions Related to Metabolic Enzymes

(101) (i) Test of the Inhibitory Activity on CYP Isoenzymes at a Single Concentration

(102) 1. Experimental Purpose:

(103) To test the inhibitory effect of compounds on CYP isoenzymes at a single concentration.

(104) 2. Experimental Instrument and Reagents:

(105) Liver Microsome:

(106) TABLE-US-00004 Species Gender Supplier Cat. No. Batch Storage Human Mix Corning 452117 38298 80 C.
Reagents:

(107) TABLE-US-00005 Compound Supplier Cat. No. Storage Terfenadine Sigma-Aldrich T9652 4 C. Tolbutamide Sigma-Aldrich T0891 4 C. Potassium hydrogen phosphate Sinopharm 20032117 Room temperature Reduced nicotinamide adenine Shanghai ABCONE N99640- 20 C. dinucleotide phosphate Biotechnology Co., Ltd. 100MG Phenacetin Sigma-Aldrich 101690303 Room temperature Diclofenac Sigma-Aldrich D6899-10G Room temperature Mephenytoin Glpbio GC14486 20 C. Dextromethorphan Sigma-Aldrich D9684-5G Room temperature Midazolam National Institute for Food PVJT-0H9Z Room and Drug Control temperature Testosterone Adamas reagent 171265 Room temperature Bupropion Cayman Chemical 10488 20 C. Company Amodiaquine Abmole M5412 20 C. B-Naphthoflavone Sigma-Aldrich N5757-1G 4 C. Sulfaphenazole Sigma-Aldrich S0758-1G 4 C. Benzylnirvanol Shanghai Yuanye Bio- Y43177-5 Room Technology Co., Ltd. mg temperature Quinidine Internation-Laboratory- 1311468-5G Room USA temperature Ketoconazole Sigma-Aldrich K1003- 4 C. 100MG
3. Experimental Methods:

(108) 8.71 g of K.sub.2HPO.sub.4 was dissolved in 950 mL of water. The pH was adjusted to 7.4 with HCl solution. The mixture was adjusted to a final volume of 1000 mL with water. The buffer was filtered through a 0.45 m filter. The buffer was stored in a refrigerator at 4 C. for future use. Terfenadine/tolbutamide (1 mg/mL each) stock solutions were prepared with DMSO. The above stock solution was diluted with acetonitrile to produce a quenching solution containing 5/10 ng/ml (terfenadine/tolbutamide).

(109) Positive Control Working Solutions:

(110) TABLE-US-00006 Stock Working Stock solution solution Incubation solution Total CYP concentration concentration concentration volume volume enzyme Inhibitor (mM) (mM) (M) (L) (L) Format 1A2 -Naphtho- 10 2 10 10 50 5 in 1 flavone 2C9 Sulfaphenazole 10 2 10 10 2C19 Benzylnirvanol 5 1 5 10 2D6 Quinidine 10 2 10 10 3A4/5 Ketoconazole 10 2 10 10 Ketoconazole 2 2 10 50 50 Single
Substrate Working Solutions:

(111) TABLE-US-00007 Stock Working Stock solution solution Incubation solution Total CYP concentration concentration concentration volume volume enzyme Substrate (mM) (mM) (M) (L) (L) Format 1A2 Phenacetin 250 50 50 2 10 5 in 1 2C9 Diclofenac 50 10 10 2 2C19 Mephenytoin 225 45 45 2 2D6 Dextromethorphan 25 5 5 2 3A4/5 Midazolam 12.5 2.5 2.5 2 Testosterone 50 50 50 5 5 Single

(112) Liver microsomes were thawed in a 37 C. water bath prior to use. Liver microsome working solution (final concentration of 0.5 mg/mL) was prepared. 5 mM NADPH working solution was prepared with phosphate buffered saline. The above stock solution was prepared into a 2 mM working solution with DMSO.

(113) 238.5 L of liver microsome working solution was added to a 1.1 mL tube. 1.5 L of test compound/control working solution/DMSO was added and mixed well by pipetting several times. The mixture was pre-incubated in water bath with shaking at 37 C. for 5 minutes. After the pre-incubation, 60 L of NADPH working solution was added and mixed well by pipetting several times. The mixture was incubated in water bath with shaking at 37 C. for 10 minutes. Immediately after incubation, 500 L of quenching solution was added and vortexed for 1 minute. All samples were centrifuged at 4,000 rpm for 15 minutes at 4 C. 300 L of the above supernatants were aliquoted for further LC-MS/MS analysis.

(114) 4. Data Processing Methods and Results:

(115) Inhibition rate (% inhibition) is calculated using metabolite formation of the test compound or the control compound compared to the matrix control.

(116) TABLE-US-00008 TABLE 3 Inhibitory activity of compounds on CYP isoenzymes* Inhibition rate (%) Compound CYP3A4/5 CYP3A4/5 number CYP1A2 CYP2C9 CYP2C19 CYP2D6 (Midazolam) (Testosterone) Cpd-A 12 38 42 5.5 83 ND Cpd-B 0.56 21 31 3.6 61 75 Cpd-C 18 43 67 9.5 79 ND Cpd-1 NI 6.7 NI 1.6 9.8 3.7 Cpd-2 NI 8.9 8.4 7.2 8.7 NI *Final compound concentration is 10 M; ND: not detected; NI: no inhibition

(117) The above data show that the compounds of the present disclosure have relatively weak inhibitory activity on CYP isoenzymes, and the risk of drug-drug interaction is significantly lower than that of the reference compounds.

(118) (ii) Determination of the Multi-Concentration Inhibitory Activity Curve of CYP3A4/5 enzymes

(119) 1. Experimental Purpose:

(120) To determine the concentration inhibitory activity curve of compounds on CYP3A4/5 enzymes.

(121) 2. Experimental Instrument and Materials:

(122) Liver Microsome:

(123) TABLE-US-00009 Species Gender Supplier Cat. No. Batch Storage Human Mix Corning 452117 38298 80 C.
Reagents:

(124) TABLE-US-00010 Compound Supplier Cat. No. Storage Terfenadine Sigma-Aldrich T9652 4 C. Tolbutamide Sigma-Aldrich T0891 4 C. Potassium hydrogen Sinopharm 20032117 Room phosphate temperature Reduced Shanghai ABCONE N99640-100MG 20 C. nicotinamide Biotechnology Co., Ltd. adenine dinucleotide phosphate Midazolam National Institute for Food PVJT-0H9Z Room and Drug Control temperature Testosterone Adamas reagent 171265 Room temperature Ketoconazole Sigma-Aldrich K1003-100MG 4 C.
3. Experimental Methods:

(125) 8.71 g of K.sub.2HPO.sub.4 was dissolved in 950 mL of water. The pH was adjusted to 7.4 with HCl solution. The mixture was adjusted to a final volume of 1000 mL with water. The buffer was filtered through a 0.45 m filter. The buffer was stored in a refrigerator at 4 C. for future use. Terfenadine/tolbutamide (1 mg/mL each) stock solutions were prepared with DMSO. The above stock solution was diluted with acetonitrile to produce a quenching solution containing 5/10 ng/ml (terfenadine/tolbutamide).

(126) Positive Control Working Solutions:

(127) TABLE-US-00011 Stock Working Stock solution solution Incubation solution Total CYP concentration concentration concentration volume volume enzyme Inhibitor (mM) (mM) (M) (L) (L) 3A4/5 Ketoconazole 10 2 10 10 50 Ketoconazole 2 2 10 50 50
Substrate Working Solutions:

(128) TABLE-US-00012 Stock Working Stock solution solution Incubation solution Total CYP concentration concentration concentration volume volume enzyme Substrate (mM) (mM) (M) (L) (L) 3A4/5 Midazolam 12.5 2.5 2.5 2 10 Testosterone 50 50 50 5 5

(129) Liver microsomes were thawed in a 37 C. water bath prior to use. Liver microsome working solution (final system concentration of 0.5 mg/mL) was prepared. 5 mM NADPH working solution was prepared with phosphate buffered saline. The above stock solution was prepared into a 2 mM working solution with DMSO. The above stock solution was diluted with acetonitrile (4-fold dilution, 6 non-zero concentrations).

(130) 238.5 L of liver microsome working solution was added to a 1.1 mL tube. 1.5 L of test compound/control working solution/DMSO was added and mixed well by pipetting several times. The mixture was pre-incubated in water bath with shaking at 37 C. for 5 minutes. After the pre-incubation, 60 L of NADPH working solution was added and mixed well by pipetting several times. The mixture was incubated in water bath with shaking at 37 C. for 10 minutes. Immediately after incubation, 500 L of quenching solution was added and vortexed for 1 minute. All samples were centrifuged at 4,000 rpm for 15 minutes at 4 C. 300 L of the above supernatants were aliquoted for further LC-MS/MS analysis.

(131) 4. Data Processing Methods and Results:

(132) The half-maximal inhibitory concentration (IC.sub.50) was calculated using Graph Pad Prism.

(133) TABLE-US-00013 TABLE 4 Inhibitory activity of compounds on CYP3A4/5 enzymes Compound CYP3A4/5 IC.sub.50 (M) CYP3A4/5 IC.sub.50 (M) number (Midazolam) (Testosterone) Cpd-A 2.3 0.89 Cpd-B 5.8 3.2 Cpd-C 3.8 ND Cpd-1 >50 >50 Cpd-2 >50 >50 ND: Not detected

(134) The above data show that the representative compounds of the present disclosure have no obvious inhibitory activity on CYP3A4/5 enzymes, and the risk of drug-drug interaction is significantly lower than that of the reference compounds.

(135) IV. In Vitro Metabolic Stability Evaluation: Study on the Metabolic Stability of Liver Microsomes:

(136) 1. Experimental Purpose:

(137) To evaluate the metabolic stability of the test compound in human/rat/mouse liver microsomes.

(138) 2. Experimental Reagents and Materials:

(139) Reagents:

(140) TABLE-US-00014 Storage Reagent name Manufacturer Cat. No. conditions Terfenadine Sigma-Aldrich T9652 2 to 8 C. Tolbutamide Sigma-Aldrich T0891 2 to 8 C. K.sub.2HPO.sub.4 SCR 20032117 RT Reduced nicotinamide adenine ACROS 328742500 20 C. dinucleotide phosphate (NADPH)
Materials:

(141) TABLE-US-00015 Experimental Gender Manufacturer Cat. No. Storage Human liver Mixed Corning 452117 80 C. Rat liver Male BioreclamationIVT M00001 80 C. Mouse liver Male BioreclamationIVT M00501 80 C.
3. Experimental Methods:

(142) The test compound and the positive control compound (dextromethorphan as a control compound) were dissolved respectively in DMSO to prepare a stock solution with a concentration of 10 mM, and the above stock solution was diluted into 200 M working solution with DMSO. 8.709 g of potassium hydrogen phosphate (K.sub.2HPO.sub.4) was dissolved in 950 mL of water, and the pH value of the solution was adjusted to 7.4 with hydrochloric acid, and then the volume was adjusted to 1000 mL with water. After filtration using a 0.22 m filter membrane, the mixture was stored in a 4 C. refrigerator for later use. After liver microsomes of various species (protein concentration of 20 mg/mL) were thawed in a 37 C. water bath, the liver microsomes were diluted with a phosphate buffer respectively to obtain a liver microsome working solution with protein concentration of 0.629 mg/mL. 5 mM NADPH solution was prepared using the above phosphate buffer for later use. 1 mg/mL stock solution of terfenadine/tolbutamide was prepared with DMSO and then diluted with a 50% methanol/50% acetonitrile mixed solution to a reaction stop solution containing 5/10 ng/mL (terfenadine/tolbutamide) internal standard.

(143) 238.5 L of liver microsome working solutions of various species were added to a 1.1 mL microtube, and 1.5 L of the test compound working solution or the positive control compound (dextromethorphan) working solution (200 M) was added respectively, mixed well, and pre-incubated in a 37 C. water bath for 5 minutes. 60 L of NADPH solution was added to start the reaction, and after mixing well, at the time points of 0, 5, 15, 30, and 60 minutes after the reaction, 30 L of the reaction mixture was respectively pipetted and added to 300 L of the reaction stop solution. Samples from all time points were vortexed vigorously for 1 minute and centrifuged at 4000 rpm for 15 minutes at 4 C. 100 L of the supernatant was added to 100 L of pure water and mixed well for LC-MS/MS analysis.

(144) 4. Data Processing Methods:

(145) The slope (ke) was determined by plotting the natural logarithm of the percentage of compound remaining against time, and the Tin and intrinsic clearance (CL.sub.int) were calculated based on the first-order kinetic formula:

(146) The compound remaining rate is calculated as follows:

(147) $\%\mathrm{remaining}\mathrm{rate}=\frac{\mathrm{ratio}\mathrm{of}\mathrm{compound}\mathrm{peak}\mathrm{area}\mathrm{to}\mathrm{internal}\mathrm{standard}\mathrm{peak}\mathrm{area}\mathrm{at}\mathrm{each}\mathrm{time}\mathrm{point}}{\mathrm{ratio}\mathrm{of}\mathrm{compound}\mathrm{peak}\mathrm{area}\mathrm{to}\mathrm{internal}\mathrm{standard}\mathrm{peak}\mathrm{area}\mathrm{at}\mathrm{initial}T0\mathrm{time}\mathrm{point}}100$

(148) Intrinsic clearance CL.sub.int (L/min/mg protein)=0.693*1000/T.sub.1/2/protein concentration (0.5 mg protein/mL)

(149) TABLE-US-00016 TABLE 5 Metabolic stability of compounds in human/rat/mouse liver microsomes In vitro Cl.sub.int Compound In vitro (L/min/mg number Species t.sub.1/2 (min) protein) Cpd-A Human 41 33.6 Rat 30 46.1 Mouse 27 50.7 Cpd-C Human 49 28.3 Rat 23 61.5 Mouse 17 83.9 Cpd-1 Human 136 7.3 Rat 28 35.0 Mouse 72 13.7 Cpd-2 Human >256 <5.4 Rat 119 11.7 Mouse 110 12.6

(150) The above data show that the representative compounds of the present disclosure have excellent metabolic stability in the liver microsomes of various species and are obviously superior to the reference compounds.

(151) V. In Vitro Metabolic Stability Evaluation: Metabolic Stability Test in Hepatocytes

(152) 1. Experimental Purpose:

(153) To determine the metabolic stability of compounds in human/rat/mouse hepatocytes.

(154) 2. Experimental materials:

(155) TABLE-US-00017 Species Strain Gender Supplier Human N/A Mix BioIVT Rat Sprague Dawley Male BioIVT Mouse ICR/CD-1 Male BioIVT
3. Experimental Design
3.1 Preparation of Compound Working Solution

(156) The test compound and the control drug verapamil powder were prepared into a high concentration stock solution using DMSO, and diluted to a 100 M working solution with 50% acetonitrile/water before use, with final concentrations of 1 M for the test compound and verapamil.

(157) 3.2 Preparation of Hepatocytes

(158) 1) 49.5 mL of Williams' Medium E and 0.5 mL GlutaMAX were mixed as the incubation solution. The hepatocyte recovery solution and the incubation solution were pre-warmed in a 37 C. water bath for at least 15 minutes prior to use. 2) A tube of cryopreserved hepatocytes was taken, ensuring that the hepatocytes were still in a cryogenic frozen state prior to resuscitation. The hepatocytes were quickly placed in a 37 C. water bath and gently shaken until all ice crystals were completely dispersed, sprayed with 70% ethanol and transferred to a biosafety cabinet. 3) The contents of the hepatocyte tube were poured into a centrifuge tube containing 50 mL of recovery medium, which were centrifuged at 100 g for 10 minutes. After centrifugation, the recovery medium was aspirated, and sufficient incubation medium was added to obtain a cell suspension having a cell density of approximately 1.510.sup.6 cells/mL. 4) Cellometer Vision was used to count hepatocytes and determine the density of viable cells. The survival rate of hepatocytes must be greater than 75%. The hepatocyte suspension was diluted with incubation medium to a viable cell density of 0.510.sup.6 cells/mL. 5) A portion of hepatocyte suspension with a density of 0.510.sup.6 cells/mL was boiled in water for 5 minutes to inactivate as a negative control. After cells were inactivated, substrate conversion mediated by non-cellular enzymes could be easily investigated.
3.3 Experimental Methods 1) 198 L of the suspension of live or inactivated cells was transferred to a 96-well deep-well plate, which was then placed on a vortexer and preheated in an incubator for 10 minutes. Live cells were incubated in duplicate, while inactivated cells were incubated in single. 2) 2 L of 100 M test compound or verapamil was added to each well for the reaction initiation, and the deep-well plate was placed back on the incubator vortexer. 3) 25 L of the suspension were taken from the incubated samples at 0, 15, 30, 60, 90, and 120 minutes, respectively, and added to 150 L of acetonitrile containing internal standards (200 nM alprazolam, 200 nM labetalol, 2 M ketoprofen, and 200 nM caffeine) to terminate the reaction. The mixture was vortexed for 10 minutes and centrifuged at 3220 g and 4 C. for 30 minutes. After centrifugation was completed, 100 L of the supernatant and 100 L of ultra-pure water were mixed well for UPLC-MS/MS analysis detection.
4. Data Analysis

(159) All data calculations were performed using Microsoft Excel software. Peak areas were detected by extracting the ion chromatogram. The in vitro half-life (t.sub.1/2) of the parent drug was detected by linear fitting of the natural logarithm of the percentage of parent drug eliminated with time.

(160) The in vitro half-life (t.sub.1/2) was calculated by the slope:

(161) $\mathrm{in}\mathrm{vitro}{t}_{1/2}=0.693/k$

(162) In vitro intrinsic clearance (unit L/min/10.sup.6 cells) was calculated using the following formula:

(163) $\mathrm{in}\mathrm{vitro}{\mathrm{CL}}_{\mathrm{int}}=\mathrm{kV}/N$ V=incubation volume per well (0.2 mL); N=number of cells per well (0.110.sup.6 cells)

(164) TABLE-US-00018 TABLE 6 Metabolic stability of compounds in human/rat/mouse hepatocytes Compound In vitro In vitro Cl.sub.int number Species t.sub.1/2 (min) (L/min/10.sup.6 cells) Cpd-A Human 61 22.7 Rat 73 19.0 Mouse 164 8.5 Cpd-B Human 82 16.8 Rat 22 63.8 Mouse 7 197.9 Cpd-C Human 75 18.4 Rat 88 15.7 Mouse 44 31.8 Cpd-1 Human >511 <2.7 Rat >511 <2.7 Mouse >511 <2.7 Cpd-2 Human >511 <2.7 Rat >511 <2.7 Mouse >511 <2.7

(165) The above data show that the representative compounds of the present disclosure have excellent metabolic stability in human/rat/mouse hepatocytes and are obviously superior to the reference compounds.

(166) VI. Cardiac Safety Evaluation: HERG Potassium Channel Inhibitory Activity Test

(167) 1. Experimental Purpose:

(168) The manual patch-clamp technique was used to evaluate whether test compounds have potential inhibitory effects on the voltage-gated potassium channel hERG. This experiment was conducted to detect the effect of compounds in five concentrations on hERG channel current, obtaining a dose-effect curve and calculating the IC.sub.50.

(169) 2. Experimental Instrument and Reagents:

(170) TABLE-US-00019 Experimental materials Supplier (Cat. No.) 6 cm cell culture dish Shanghai Yes Service Biotech, Inc. (150462) 3.5 cm cell culture dish Shanghai Yes Service Biotech, Inc. (150460) Dialyzed fetal bovine serum Shanghai Bohan Biotechnology Co., Ltd. (BS-0005-500) DMSO Beijing Solarbio Science & Technology Co., Ltd. (D8371) DMEM medium Thermo Fisher Scientific (China) Co., Ltd. (10569044) HEPES Thermo Fisher Scientific (China) Co., Ltd. (15630080) Trypsin Thermo Fisher Scientific (China) Co., Ltd. (12604) Phosphate buffered saline (without Beijing Solarbio Science & Technology Co., Ltd. (D1040) calcium and magnesium ions) Penicillin-streptomycin solution Thermo Fisher Scientific (China) Co., Ltd. (15140122) MEM non-essential amino acid Thermo Fisher Scientific (China) Co., Ltd. (11140050) solution Geneticin (G418) Thermo Fisher Scientific (China) Co., Ltd. (11811031) Blasticidin Thermo Fisher Scientific (China) Co., Ltd. (R21001) Polylysine Thermo Fisher Scientific (China) Co., Ltd. (P4832) Dofetilide Beijing Express Technology Development Co., Ltd. (D525700) Doxycycline Sigma-Aldrich (Shanghai) Trading Co., Ltd. (D9891) Carbon dioxide incubator Thermo Fisher Scientific (China) Co., Ltd. (Thermo371) Glass electrode drawing machine Japan NARISHIGE Company (PC-10 Puller) Micromanipulator positioner Qingdao Sources Optics Co., Ltd. (MC1000e) Perfusion system American ALA Company (VM8 gravity drug delivery system) Vacuum pump German Chemvak Company (V300) Osmometer Ashern Company (Osmo310)
3. Experimental Methods:

(171) Cell line and cell culture: HEK293 cell line stably expressing the hERG ion channel (Cat. No.: K1236) was purchased from Invitrogen Company. The cell line was cultured in a medium containing 85% DMEM, 10% dialyzed fetal bovine serum, 0.1 mM non-essential amino acid solution, 100 U/mL penicillin-streptomycin solution, 25 mM HEPES, 5 g/mL blasticidin, and 400 g/mL geneticin. When the cell density grows to 40% to 80% of the bottom area of the culture dish, the cells were digested with trypsin and passaged three times per week. Prior to the experiment, cells were cultured in a 3.5 cm culture dish at a density of 510.sup.5, induced with 1 g/mL doxycycline for 48 hours, and then the cells were digested and seeded on glass slides for subsequent manual patch clamp experiments.

(172) Solution preparation: extracellular fluid (in mM): 132 sodium chloride, 4 potassium chloride, 3 calcium chloride, 0.5 magnesium chloride, 11.1 glucose, and 10 HEPES (pH adjusted to 7.35 with sodium hydroxide). Intracellular fluid (in mM): 10 EGTA, 10 HEPES, 10 potassium chloride, 10 sodium chloride, and 110 potassium fluoride (pH adjusted to 7.2 with potassium hydroxide). The osmotic pressure of the solution was controlled between 280 to 300 mOsmol/kg. The solution was filtered and stored at 4 C. prior to use.

(173) Test compound solution preparation: Following the standard operating procedure, test compounds were dissolved in DMSO and prepared into a stock solution with a final concentration of 10 mM. The stock solution was gradiently diluted with DMSO as the solvent in a 1:3 ratio into three other intermediate concentration solutions at concentrations of (mM): 3.33, 1.11, and 0.37, respectively. Prior to the start of the experiment, the test compound gradient intermediate solution was diluted with extracellular fluid again in a 1:1000 ratio into working solutions of a series of concentrations, and the working solution of 30 M was prepared into a working solution in a 1:333.33 ratio using a stock solution of 10 mM, and the final concentrations of which were (M): 30, 10, 3.33, 1.11, and 0.37, respectively. The DMSO content in the working solution was 0.1 to 0.3% (v/v). 5 working solutions with different concentration gradients of 30, 10, 3.33, 1.11, and 0.37 M were used to determine the potential inhibitory effect of compounds on hERG channels and to fit the dose-effect curve and calculate IC.sub.50.

(174) Experimental procedure: A coverslip loaded with HEK293 cells in a culture dish was placed in the perfusion chamber of the microscope stage. A suitable cell was placed in the center of the field of view under an Olympus IX71 or IX73 inverted microscope, and the tip of the glass electrode was found using a 10 objective lens, and placed in the center of the field of view. Then the electrode was moved downward using a micromanipulator, while the coarse focus screw was adjusted to slowly bring the electrode closer to the cell. On the point of approaching the cell, the objective lens was switched to a 40 objective lens for observation, and an electrode was gradually brought closer to the surface of the cell using a micromanipulator for fine adjustment. The negative pressure was given to form a seal between the electrode tip and the cell membrane with a resistance higher than 1 G. The transient capacitive current Cfast was compensated in the voltage clamp mode. The brief negative pressure was then applied repeatedly to rupture the membrane, eventually forming a whole-cell recording mode. Under the condition that the membrane potential was clamped at 60 mV, the slow capacitive current Cslow, the cell membrane capacitance (Cm), and the input membrane resistance (Ra) were compensated respectively. After the cells were stable, the clamping voltage was changed to 90 mV, the sampling frequency was set at 20 kHz, and the filtering frequency was 10 kHz. The condition for detecting the leakage current was that the clamping voltage changed to 80 mV and the time duration was 500 ms. The hERG current test method was as follows: A 4.8-second depolarizing command voltage was applied to bring the membrane potential from 80 mV to +30 mV, and then a 5.2-second repolarizing voltage was instantly applied to lower the membrane potential to 50 mV to remove channel inactivation, thereby allowing the hERG tail current to be observed. The peak value of the tail current was the magnitude of the hERG current. The hERG current used to detect the test compound was continuously recorded for 120 seconds before administration to evaluate the stability of the hERG current generated by the test cells. Only stable cells within the acceptable range of the evaluation criteria were allowed to proceed to the subsequent compound testing. Test of the inhibitory effect of the test compound on hERG current: First, the hERG current measured in the extracellular fluid containing 0.1% DMSO was used as the detection baseline. After the hERG current had been stable for at least 5 minutes, the solution containing the test compound was sequentially perfused around the cells from a low concentration to a high concentration. After each perfusion was completed, approximately 5 minutes were waited to allow the compounds to fully act on the cells, and hERG currents were synchronously recorded. The last 5 hERG current values were recorded after the currents to be recorded tended to stabilize, and the average value was taken as the final current value at a specific concentration. After testing the compound, 450 nM dofetilide was added to the same cell, and the current was completely inhibited, serving as a positive control for that cell. At the same time, the positive compound dofetilide was tested before and after the end of the test drug experiment using the same patch clamp system for synchronous detection to ensure the reliability and sensitivity of the entire detection system. The above test steps were repeated on two separate test cells (n=2).

(175) 4. Data Processing Methods and Results:

(176) Data quality control standards: The initial seal resistance was greater than 1 G; The series resistance was less than 15 M and the voltage error of the series resistance was less than 5 mV; The leakage current at the detection voltage was less than 50% of the current value under this condition; The tail current was greater than the plateau current of the pre-pulse, with an initial tail current value greater than 250 pA; The membrane breakthrough resistance Ra was less than 15 M; The decay rate of the tail current per minute was less than 2.5%.

(177) Data analysis: Only data that meets the above criteria could be analyzed according to the following steps.

(178) Note: Data are output by PatchMaster software.

(179) After being infused with blank solvent or compound gradient solutions, stable 5 consecutive current values were obtained, and the average value was calculated, respectively, as tail current.sub.blank and tail current.sub.compound.

(180) The current suppression percentage is calculated by the following formula.

(181) $\mathrm{tail}\mathrm{current}\mathrm{inhibition}\mathrm{rate}=\left(1-\frac{\mathrm{tail}{\mathrm{current}}_{\mathrm{compound}}-\mathrm{tail}{\mathrm{current}}_{\mathrm{positive}\mathrm{drug}}}{\mathrm{tail}{\mathrm{current}}_{\mathrm{blank}}-\mathrm{tail}{\mathrm{current}}_{\mathrm{positive}\mathrm{drug}}}\right)100$

(182) The dose-effect curve was fitted by Graphpad Prism 8.0 software and the IC.sub.50 value was calculated.

(183) TABLE-US-00020 TABLE 7 Inhibitory activity of compounds on hERG Compound number hERG IC.sub.50 (M) Cpd-A 19 Cpd-B 5.5 Cpd-C 1.4 Cpd-1 >30 Cpd-2 >30

(184) The above data show that the representative compounds of the present disclosure have a lower risk of potential cardiotoxicity caused by hERG inhibition, and their safety is obviously superior to that of the reference compounds.

(185) VII. In Vivo Pharmacokinetic Study in Mice

(186) 1. Experimental Purpose:

(187) To test the pharmacokinetics of compounds in CD-1 mice.

(188) 2. Experimental Instrument and Materials:

(189) Animals: CD-1 mice (male)

(190) Instrument:

(191) TABLE-US-00021 Instrument Instrument name Manufacturer number API4000 Qtrap-Shimadzu 20AT AB Sciex/Shimadzu TW-017-0002 Large benchtop refrigerated Eppendorf TW-007-0017 centrifuge Electronic balance Sartorius TW-001-0013 Pipetting workstation Sptlabtech TW-013-0016 Vortex oscillator DRAGON LAB TW-005-0013 High-throughput tissue grinder Ningbo Scientz TW-011-0002 Biotechnology Co., Ltd.
Reagents:

(192) TABLE-US-00022 Name Manufacturer Cat. No. Batch number Acetonitrile Sigma-Aldrich 34851 F222M7L201 Methanol Sigma-Aldrich 75851G WXBD8013V DMSO Fisher Scientific D159-4 186403 Formic acid Sigma-Aldrich F112034 G2122206 PBS Shanghai Basal Media B310KJ H211017
Vehicle:

(193) Intravenous injection: 40% HP--CD in water; intragastric administration: 0.2% HPC+0.5% Tween80 in water.

(194) 3. Experimental Methods:

(195) 6 male mice. Fasting treatment was performed in the evening before the administration, with free access to water. During the experiment, the mice were allowed to eat and drink freely, and administered by intravenous injection or gavage. After administration, the animal status was observed, and abnormal behaviors were recorded. Blood was collected from the orbit at 0.0833, 0.25, 1, 2, 4, 8, and 24 hours after intravenous injection, and at 0.25, 0.5, 1, 2, 4, 6, 8, and 24 hours after intragastric administration. 20 L of plasma sample was taken and added with 250 L of acetonitrile (containing dexamethasone as an internal standard) to precipitate proteins. The mixture was centrifuged at 4 C. and 4000 rpm for 20 minutes, and 180 L of the supernatant was taken and mixed with 180 L of an aqueous solution containing 0.1% formic acid in a 96-well plate. Then, 10 L of the sample was taken for LC-MS/MS detection.

(196) 4. Data Processing Methods and Results:

(197) The standard curve was established using the internal standard method, with the theoretical standard curve concentration as the X-axis and the peak area ratio as the Y-axis (peak area of the test compound/peak area of internal standard). Linear regression method (weighting factor 1/X2) was used, R.sup.2>0.9900. Unknown samples were calculated by the standard curve. Pharmacokinetic parameters were calculated by the non-compartmental analysis model of WinNonlin 8.2 software and were presented in a report, including Cl.sub.int, C.sub.max, AUC, etc.

(198) TABLE-US-00023 TABLE 8 Pharmacokinetic parameters of compounds in CD-1 mice IV 1 mg/kg PO 10 mg/kg Compound Cl.sub.int AUC.sub.0-t C.sub.max AUC.sub.0-t number (mL/min/kg) (hr*M) (M) (hr*M) Cpd-A 18 1.7 2.2 10.4 Cpd-2 15 1.7 5.9 15.7

(199) The above data show that the representative compound of the present disclosure has a lower clearance and a higher oral exposure in mice, and is superior to the reference compound.

(200) VIII. In Vivo Pharmacokinetic Study in Rats

(201) 1. Experimental Purpose:

(202) To test the pharmacokinetics of compounds in SD rats.

(203) 2. Experimental Instrument and Materials:

(204) Animal: SD rats (male)

(205) Instrument:

(206) TABLE-US-00024 Instrument Instrument name Manufacturer number API4000 AB Sciex/Shimadzu TW-017-0002 Qtrap-Shimadzu 20AT Large benchtop Eppendorf TW-007-0017 refrigerated centrifuge Pipetting workstation Sptlabtech TW-013-0016 Vortex oscillator DRAGON LAB TW-005-0013 High-throughput Ningbo Scientz TW-011-0002 tissue grinder Biotechnology Co., Ltd.
Reagents:

(207) TABLE-US-00025 Name Manufacturer Cat. No. Batch number Acetonitrile Sigma-Aldrich 34851 WXBD8733V Methanol Sigma-Aldrich 75851G P2478457 DMSO Fisher Scientific D4540 BCCH3267 Formic acid Sigma-Aldrich F112034 B2320164 PBS Shanghai Basal Media B310KJ K211007
Vehicle:

(208) Intravenous injection: 40% HP--CD in water; intragastric administration: 0.2% HPC+0.5% Tween80 in water.

(209) 3. Experimental Methods:

(210) 6 male rats. Fasting treatment was performed in the evening before the administration, with free access to water. During the experiment, the rats were allowed to eat and drink freely, and administered by intravenous injection or gavage. After administration, the animal status was observed, and abnormal behaviors were recorded. Blood was collected via jugular vein puncture at 0.0833, 0.25, 1, 2, 4, 8, and 24 hours after intravenous injection; Blood was collected via jugular vein puncture at 0.25, 0.5, 1, 2, 4, 6, 8, and 24 hours after intragastric administration. 50 L of plasma sample was taken and added with 500 L of acetonitrile (containing dexamethasone as an internal standard) to precipitate proteins. The mixture was centrifuged at 4 C. and 4000 rpm for 20 minutes, and 300 L of the supernatant was taken and mixed with 300 L of Watsons aqueous solution in a 96-well plate. Then, 5 L of the sample was taken for LC-MS/MS detection.

(211) 4. Data Processing Methods and Results:

(212) The standard curve was established using the internal standard method, with the theoretical standard curve concentration as the X-axis and the peak area ratio as the Y-axis (peak area of the test compound/peak area of internal standard). Linear regression method (weighting factor 1/X2) was used, R.sup.2>0.9900. Unknown samples were calculated by the standard curve. Pharmacokinetic parameters were calculated by the non-compartmental analysis model of WinNonlin 8.2 software and were presented in a report, including Cl.sub.int, C.sub.max, AUC, etc.

(213) TABLE-US-00026 TABLE 9 Pharmacokinetic parameters of compounds in SD rats IV 1 mg/kg PO 10 mg/kg Compound Cl.sub.int AUC.sub.0-t Cmax AUC.sub.0-t number (mL/min/kg) (hr*M) (M) (hr*M) Cpd-A 7.1 4.2 3.2 35.8 Cpd-2 3.3 8.4 8.9 58.4

(214) The above data show that the representative compound of the present disclosure has a lower clearance and a higher oral exposure in rats, and is obviously superior to the reference compound.

(215) IX. In Vivo Efficacy Study in Human Gastric Cancer NUGC-3 Xenograft Mouse Model

(216) 1. Experimental Purpose:

(217) To test the in vivo anti-tumor activity of compounds in the subcutaneous human gastric cancer NUGC-3 xenograft model in female NUNU mice.

(218) 2. Experimental Materials:

(219) Animals: 6 to 8 weeks old female NUNU mice;

(220) Reagents:

(221) TABLE-US-00027 Reagent name Supplier Cat. No. Batch number Penicillin-streptomycin Gibco 15240-062 2441816 double antibody Trypsin-EDTA Gibco 25200-072 2756239 RPMI-1640 Gibco C22400500BT 6123180 DPBS Corning 21-031-CVC 21031095 FBS Dongling FBSV500 A22JV Matrigel Corning 354234 3012001 Glacial acetic acid Alfa Aesar 36289 U02F788 Tween80 Sigma P1754 WXBD4333V Dimethyl sulfoxide Leyan 1083196 Lg0913304311 (DMSO) Solutol Sigma 42966 BCCJ5912 Cpd-A vehicle: 0.2% HPC, 0.5% Tween80 in water (w/v/v); Cpd-2 vehicle: 5% DMSO/10% Solutol HS 15/85% pH 4.650.10 acetate buffer.
3. Experimental Protocol:

(222) Cell culture: NUGC-3 cells were cultured in RPMI-1640 medium containing 10% FBS.

(223) Molding and grouping: NUGC-3 cells in the exponential growth phase (510.sup.6) were harvested, resuspended in 0.2 mL of PBS and Matrigel (1:1), and then inoculated subcutaneously into the right flank of NUNU mice to establish a subcutaneous xenograft model. Mice were observed daily for healthy status and tumor growth after cell inoculation. On the 5th day after inoculation, 36 animals were selected, grouped, and the tumor volume of the animals in the group was approximately 150.0 mm.sup.3. The study was divided into 6 groups, with 6 mice in each group, and the administration was performed on the same day (see Table 10).

(224) 4. Experimental Design:

(225) Table 10 is the grouping and administration information for the efficacy experiment. Mice in the G1 group were orally administered vehicle control (5% DMSO/10% Solutol HS 15/85% pH 4.650.10 acetate buffer) once a day for a total of 19 days. Mice in the G2 group were orally administered Cpd-A once a day at a dose of 50 mg/kg for a total of 19 days. Mice in the G3 group were orally administered Cpd-A once a day at a dose of 100 mg/kg for a total of 19 days. Mice in the G4 group were orally administered Cpd-2 once a day at a dose of 25 mg/kg for a total of 19 days. Mice in the G5 group were orally administered Cpd-2 once a day at a dose of 50 mg/kg for a total of 19 days. Mice in the G6 group were orally administered Cpd-2 once a day at a dose of 100 mg/kg for a total of 19 days.

(226) TABLE-US-00028 TABLE 10 In vivo efficacy study design of NUGC-3 xenograft mouse model Administration Administration frequency Dose volume Administration (post Group N Compound (mg/kg) (mL/kg) route grouping) G1 6 Vehicle 10 PO QD*19 Days G2 6 Cpd-A 50 10 PO QD*19 Days G3 6 Cpd-A 100 10 PO QD*19 Days G4 6 Cpd-2 25 10 PO QD*19 Days G5 6 Cpd-2 50 10 PO QD*19 Days G6 6 Cpd-2 100 10 PO QD*19 Days
5. Experimental Observation and Result Analysis:

(227) Experimental observation: The health status and mortality of the animals were monitored daily. Routine checks included observing the effect of tumor growth and drug treatment on animals' normal behavior such as mobility, food and water consumption, body weight gain/loss, eye/hair matting and any other abnormal effect. The number of deaths and side effects of the animals in the group were recorded based on the number of the animals in each group. Clinical symptoms observed during the trial were recorded in the raw data. Tumor volume calculation formula: Tumor volume (mm.sup.3)=0.5(tumor long diametertumor short diameter.sup.2). During the research process, the entire administration process as well as tumor and body weight measurements were conducted in a Laminar Flow Cabinet.

(228) Data processing: Percentage of tumor growth inhibition TGI (%): TGI %=(1T/C)100%; Wherein C is the tumor volume C.sub.tC.sub.0 for the control group, C.sub.0 is the average tumor volume of the control group at the time of grouping, and C.sub.t is the average tumor volume of the control group after treatment; T is the tumor volume T.sub.tT.sub.0 for the treatment group, T.sub.0 is the average tumor volume of the treatment group at the time of grouping, and T.sub.t is the average tumor volume of the treatment group after treatment.

(229) Statistical analysis: Tumor volume and animal body weight results are expressed in MeanSEM. Tumor volumes between different groups were statistically compared and analyzed. All statistical analyses were completed in GraphPad Prism 8.0. The one-way ANOVA method was used to compare whether there was a significant difference in tumor volume or tumor weight between groups, where p0.05 was considered as having no significant difference, p<0.05 was considered as having a significant difference, and p<0.001 was considered as having an extremely significant difference.

(230) 6. Experimental Results:

(231) The tumor growth curves of the control group and each experimental group in the efficacy study of the human gastric cancer NUGC-3 xenograft model in mice are as shown in FIG. 1, and the relative body weight changes of the mice in the model are as shown in FIG. 2. The average tumor volume, percentage of tumor growth inhibition TGI (%), and comparison results of animals in each group at the end of the experiment are shown in Table 11.

(232) On the 19th day after grouping, the average tumor volume of G1 (Vehicle) group was 1482.2 mm.sup.3, and the average tumor volumes of G2 (Cpd-A, 50 mg/kg, QD), G3 (Cpd-A, 100 mg/kg, QD), G4 (Cpd-2, 25 mg/kg, QD), G5 (Cpd-2, 50 mg/kg, QD), and G6 (Cpd-2, 100 mg/kg, QD) groups were 1208.1 mm.sup.3, 399.1 mm.sup.3, 615.1 mm.sup.3, 202.2 mm.sup.3, and 102.5 mm.sup.3, respectively. The corresponding percentages of tumor growth inhibition (TGI) were 20.6%, 81.3%, 65.1%, 96.1%, and 103.6%, respectively. Statistical analysis showed that the average tumor volume in G3, G4, G5, and G6 groups was significantly smaller than that in the Vehicle group (p<0.05 for all); Wherein the difference in the average tumor volume between G5 and G6 groups and the Vehicle group was extremely significant (p<0.001 for both). The above results show that the reference compound Cpd-A exhibits good anti-tumor efficacy in the subcutaneous NUGC-3 mouse model at a high dose of 100 mg/kg, while the representative compound of the present disclosure Cpd-2 exhibits good anti-tumor efficacy at each test dose in this model and the activity has a dose dependency. At the same time, at a lower dose (such as 50 mg/kg, QD), Cpd-2 (TGI=96.1%) has better tumor growth inhibitory activity than Cpd-A (TGI=81.3%) at twice of the dose (100 mg/kg, QD), indicating that Cpd-2 has better anti-tumor efficacy than Cpd-A.

(233) TABLE-US-00029 TABLE 11 In vivo efficacy study results of NUGC-3 xenograft mouse model Tumor volume TGI (%).sup.a Group (mm.sup.3) (at 19 days) (at 19 days) p value.sup.b G1: Vehicle 1,482.2 333.8 G2: Cpd-A, 50 mg/kg 1,208.1 320.9 20.6 0.8127 G3: Cpd-A, 100 mg/kg 399.1 106.3 81.3 0.0037 G4: Cpd-2, 25 mg/kg 615.1 157.6 65.1 0.0243 G5: Cpd-2, 50 mg/kg 202.2 57.9 96.1 0.0006 G6: Cpd-2, 100 mg/kg 102.5 42.1 103.6 0.0003 Note: .sup.aTGI (%) = [1 (T.sub.19 T.sub.0)/(V.sub.19 V.sub.0)] 100 .sup.bp value of comparative analysis of the average tumor volume of each administration group and the average tumor volume of the Vehicle in the G1 group
X. In Vivo Efficacy Study in Human Gastric Cancer NUGC-3 Xenograft Rat Model
1. Experimental Purpose:

(234) To test the in vivo anti-tumor activity of compounds in the subcutaneous human gastric cancer NUGC-3 xenograft model in female B-Rag2/IL2rg KO SD rats.

(235) 2. Experimental Materials:

(236) Animals: 7 to 9 weeks old female B-Rag2/IL2rg KO SD rats;

(237) Reagents:

(238) TABLE-US-00030 Batch Reagent name Supplier Cat. No. number Phosphate buffered BasalMedia B310KJ L211110 saline (PBS) RPMI-1640 medium BasalMedia L210KJ D211212 Trypsin BasalMedia S310KJ B121201 Fetal bovine serum ExcellBio FSP500 012C-0530A Penicillin-streptomycin BasalMedia S110JV H121101 double antibody 100 Matrigel CORNING 354234 3012001 Solutol HS15 MeiluneBio MB1809 A0413D Acetic acid Taicang Hushi NA 20180322 Sodium acetate Sigma-Aldrich S2889 WXBD3974v Ultrapure water Guangzhou NA NA Watsons DMSO Sigma-Aldrich V900090 WXBF1310V Vehicle: 5% DMSO/10% Solutol HS 15/85% pH 4.65 0.10 acetate buffer;
3. Experimental Protocol:

(239) Cell culture: NUGC-3 cells were cultured in RPMI-1640 medium containing 10% FBS.

(240) Molding and grouping: NUGC-3 cells in the exponential growth phase (110.sup.7) were harvested, resuspended in 0.2 mL of PBS and Matrigel (1:1), and then inoculated subcutaneously into the right flank of B-Rag2/IL2rg KO SD rats to establish a subcutaneous xenograft model. Rats were observed daily for healthy status and tumor growth after cell inoculation. On the 10th day after inoculation, 24 animals were selected, grouped, and the tumor volume of the animals in the group was approximately 199.0 mm.sup.3. The study was divided into 4 groups, with 6 rats in each group, and the administration was performed on the same day (see Table 12).

(241) 4. Experimental Design:

(242) Table 12 is the grouping and administration information for the efficacy study. Rats in the G1 group were orally administered vehicle control (5% DMSO/10% Solutol HS 15/85% pH 4.650.10 acetate buffer) once a day for a total of 28 days. Rats in the G2 group were orally administered Cpd-2 once a day at a dose of 6.25 mg/kg for a total of 28 days. Rats in the G3 group were orally administered Cpd-2 once a day at a dose of 12.5 mg/kg for a total of 28 days. Rats in the G4 group were orally administered Cpd-2 once a day at a dose of 25 mg/kg for a total of 28 days.

(243) TABLE-US-00031 TABLE 12 In vivo efficacy study design of NUGC-3 xenograft rat model Administration Administration dose volume Administration Administration Group N Compound (mg/kg) (mL/kg) route frequency G1 6 Vehicle 10 PO QD 28 days G2 6 Cpd-2 6.25 10 PO QD 28 days G3 6 Cpd-2 12.5 10 PO QD 28 days G4 6 Cpd-2 25 10 PO QD 28 days
5. Experimental Observation and Result Analysis:

(244) Experimental observation: The health status and mortality of the animals were monitored daily. Routine checks included the effect of tumor growth and drug treatment on animals' normal behavior such as mobility, food and water consumption, body weight gain/loss, eye/hair matting and any other abnormal effect. The number of deaths and side effects of the animals in the group were recorded based on the number of the animals in each group. Clinical symptoms observed during the trial were recorded in the raw data. Tumor volume calculation formula: Tumor volume (mm.sup.3)=0.5(tumor long diametertumor short diameter.sup.2). During the research process, the entire administration process as well as tumor and body weight measurements were conducted in a Laminar Flow Cabinet.

(245) Data processing: Percentage of tumor growth inhibition TGITV (%): TGITV %=(1T/C)100%; Wherein C is the tumor volume C.sub.tC.sub.0 for the control group, C.sub.0 is the average tumor volume of the control group at the time of grouping, and C.sub.t is the average tumor volume of the control group after treatment; T is the tumor volume T.sub.tT.sub.0 for the treatment group, T.sub.0 is the average tumor volume of the treatment group at the time of grouping, and T.sub.t is the average tumor volume of the treatment group after treatment.

(246) Statistical analysis: Tumor volume and animal body weight results are expressed in MeanSEM. Tumor volumes between different groups were statistically compared and analyzed. All statistical analyses were completed in https://d.sub.2k.bio/Efficacy/Invivo. The wilcox.test nominal method was used to compare whether there was a significant difference in tumor volume or tumor weight between groups, where p0.05 was considered as having no significant difference, p<0.05 was considered as having a significant difference, and p<0.01 was considered as having an extremely significant difference.

(247) 6. Experimental Results:

(248) The tumor growth curves of the control group and each experimental group in the efficacy study of the human gastric cancer NUGC-3 xenograft model in rats are as shown in FIG. 3, and the relative body weight changes of the rats in the model are as shown in FIG. 4. The average tumor volume, percentage of tumor growth inhibition TGI (%), and comparison results of animals in each group at the end of the experiment are shown in Table 13.

(249) On the 28th day after grouping, the average tumor volume of G1 (Vehicle) group was 2946.2 mm.sup.3, and the average tumor volumes of G2 (Cpd-2, 6.25 mg/kg, QD), G3 (Cpd-2, 12.5 mg/kg, QD), and G4 (Cpd-2, 25 mg/kg, QD) groups were 395.0 mm.sup.3, 119.2 mm.sup.3, and 70.1 mm.sup.3, respectively. The corresponding percentages of tumor growth inhibition (TGI) were 92.9%, 102.9%, and 104.7%, respectively. Statistical analysis showed that the difference in the average tumor volume between G2, G3, and G4 groups and the Vehicle group was extremely significant (p<0.01 for all). The above results show that the representative compound of the present disclosure Cpd-2 exhibits good anti-tumor efficacy in the subcutaneous NUGC-3 xenograft rat model at each test dose, and the activity has a dose dependency.

(250) TABLE-US-00032 TABLE 13 In vivo efficacy study results of NUGC-3 xenograft rat model Tumor volume (mm.sup.3) (Mean SEM) TGI.sub.TV (%) Group (at 28 days) (at 28 days) p value.sup.a G1: Vehicle, QD 28 days, p.o. 2946.2 167.4 / / G2: Cpd-2, 6.25 mg/kg, QD 28 days, p.o. 395.0 63.1 92.9 0.0022 G3: Cpd-2, 12.5 mg/kg, QD 28 days, p.o. 119.2 10.2 102.9 0.0022 G4: Cpd-2, 25 mg/kg, QD 28 days, p.o. 70.1 14.9 104.7 0.0022 Note: p value of comparative analysis of the average tumor volume of each administration group and the average tumor volume of the Vehicle in the G1 group.