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THIAZOLE-LACTAM-SPIROHETEROCYCLIC COMPOUNDS AND APPLICATIONS THEREOF

20240309018 ยท 2024-09-19

    Inventors

    Cpc classification

    International classification

    Abstract

    Thiazole-lactam-spiroheterocyclic compounds and applications thereof in the preparation of drugs for treating related diseases. Specifically disclosed are a compound as represented by formula (I) and pharmaceutically acceptable salts thereof.

    ##STR00001##

    Claims

    1. A compound represented by formula (I) or a pharmaceutically acceptable salt thereof, ##STR00029## wherein R.sub.1 and R.sub.2 are each independently selected from H and C.sub.1-3 alkyl, wherein the C.sub.1-3 alkyl is optionally substituted with 1, 2 or 3 R.sub.a; R.sub.4, R.sub.5, R.sub.6, and R.sub.7 are each independently selected from H, F, Cl, Br, I and C.sub.1-3 alkyl, wherein the C.sub.1-3 alkyl is optionally substituted with 1, 2 or 3 R.sub.c; n is 0 or 1; m is 1 or 2; ring A is selected from pyrazolyl and tetrahydropyranyl, wherein the pyrazolyl and tetrahydropyranyl are optionally substituted with 1, 2 or 3 R.sub.d; R.sub.a and R.sub.c are each independently selected from D, F, Cl, Br and I; R.sub.a is selected from F, Cl, Br, I, C.sub.1-3 alkyl and C.sub.1-3 alkoxy, wherein the C.sub.1-3 alkyl and C.sub.1-3 alkoxy are optionally substituted with 1, 2 or 3 R; R is selected from F, Cl, Br and I.

    2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein the compound has one or more of the following definitions; i) R.sub.1 and R.sub.2 are each independently selected from H, CH.sub.3 and CH.sub.2CH.sub.3, wherein the CH.sub.3 and CH.sub.2CH.sub.3 are optionally substituted with 1, 2 or 3 R.sub.a; ii) R.sub.1 and R.sub.2 are each independently selected from H, CH.sub.3, CHF.sub.2, CD.sub.3 and CH.sub.2CH.sub.3; iii) R.sub.4, R, R.sub.6, and R.sub.7 are each independently selected from H, F, Cl, Br, I and CH.sub.3, wherein the CH.sub.3 is optionally substituted with 1, 2 or 3 R.sub.c; iv) R.sub.4, R.sub.5, R.sub.6 and R.sub.7 are each independently selected from H, F, Cl, Br, I and CH.sub.3; v) R.sub.a is selected from F, Cl, Br, I, CH.sub.3 and OCH.sub.3, wherein the CH.sub.3 and OCH.sub.3 are optionally substituted with 1, 2 or 3 R; vi) R.sub.d is selected from F, Cl, Br, I, and CH.sub.3, wherein the CH.sub.3 is optionally substituted with 1, 2 or 3 R; vii) R.sub.d is selected from CH.sub.3 and OCH.sub.3; viii) R.sub.d is selected from CH.sub.3; ix) ring A is selected from ##STR00030## wherein the ##STR00031## are optionally substituted with 1, 2 or 3 R.sub.d; x) ring A is selected from ##STR00032## wherein the ##STR00033## is optionally substituted with 1, 2 or 3 R.sub.d; xi) ring A is selected from ##STR00034## xii) ring A is selected from ##STR00035## xiii) the structural moiety ##STR00036## is selected from ##STR00037##

    3.-10. (canceled)

    11. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein the compound is selected from ##STR00038## wherein R.sub.2 is as defined in claim 1; R.sub.6 and R.sub.7 are as defined in claim 1.

    12. A compound or a pharmaceutically acceptable salt thereof, wherein the compound is ##STR00039##

    13. A crystal form A of WX001, which is characterized by an X-ray powder diffraction pattern having characteristic diffraction peaks at 2? angles of: 10.2080?0.2000?, 18.8429?0.2000?, 20.6217?0.2000?; ##STR00040##

    14. The crystal form A of WX001 according to claim 13, wherein the X-ray powder diffraction pattern has characteristic diffraction peaks at 2? angles of 10.2080?0.2000?, 18.8429?0.2000?, 20.6217?0.2000?, 25.0767?0.2000?, 25.4797?0.2000?; alternatively, wherein the X-ray powder diffraction pattern has characteristic diffraction peaks at 2? angles of 10.2080?0.2000?, 15.2687?0.2000?, 17.6747?0.2000?, 18.8429?0.2000? 20.6217?0.2000?, 21.0531?0.2000?, 25.0767?0.2000?, 25.4797?0.2000?; alternatively, wherein the X-ray powder diffraction pattern has characteristic diffraction peaks at 2? angles of 10.2080?0.2000?, 14.4684?0.2000?, 15.2687?0.2000?, 17.6747?0.2000?, 18.8429?0.2000?, 20.6217?0.2000?, 21.0531?0.2000?, 21.5713?0.2000?, 22.0420?0.2000? 22.4540?0.2000?, 25.0767?0.2000?, 25.4797?0.2000?; alternatively, wherein the X-ray powder diffraction pattern has characteristic diffraction peaks at 2? angles of 10.2080?, 10.4856?, 14.4684?, 15.0133?, 15.2687?, 15.9518?, 16.6214?, 17.6747?, 17.9514?, 18.4703?, 18.8429?, 19.1531?, 20.6217?, 21.0531?, 21.2894?, 21.5713?, 22.0420?, 22.4540?, 25.0767?, 25.4797?, 26.3255?, 26.9544?; alternatively, wherein the crystal form A of WX001 has an XRPD pattern substantially as shown in FIG. 1.

    15.-18. (canceled)

    19. The crystal form A of WX001 according to claim 13, which has a differential scanning calorimetry curve having an onset of an endothermic peak at 241.0?3.0? C.; alternatively, which has a DSC curve as shown in FIG. 2.

    20. (canceled)

    21. The crystal form A of WX001 according to claim 13, which has a thermogravimetric analysis curve having a weight loss of up to 0.83% at 150.0?3.0? C.; alternatively, which has a TGA curve as shown in FIG. 3.

    22. (canceled)

    23. A method of treating a solid tumor in a subject in need thereof, comprising administering to the subject the compound according to claim 1 or a pharmaceutically acceptable salt thereof.

    24. The crystal form A of WX001 according to claim 19, which has a thermogravimetric analysis curve having a weight loss of up to 0.83% at 150.0?3.0? C.

    25. The crystal form A of WX001 according to claim 24, which has a TGA curve as shown in FIG. 3.

    26. A method of treating a solid tumor in a subject in need thereof, comprising administering to the subject the compound according to claim 12 or a pharmaceutically acceptable salt thereof.

    27. A method of treating a solid tumor in a subject in need thereof, comprising administering to the subject the crystal form A of WX001 according to claim 13.

    28. A composition, comprising the compound according to claim 1 or a pharmaceutically acceptable salt thereof.

    29. A composition, comprising the compound according to claim 12 or a pharmaceutically acceptable salt thereof.

    30. A composition, comprising the crystal form A of WX001 according to claim 13.

    Description

    BRIEF DESCRIPTION OF THE DRAWINGS

    [0104] FIG. 1: the XRPD pattern of the crystal form A of WX001 using Cu-K? radiation:

    [0105] FIG. 2: the DSC curve of the crystal form A of WX001:

    [0106] FIG. 3: the TGA curve of the crystal form A of WX001;

    [0107] FIG. 4: the DVS pattern of the crystal form A of WX001;

    [0108] FIG. 5: Tumor growth curves of human melanoma A375 in model animal after administration of solvent and WX001 respectively;

    [0109] FIG. 6: Rate of weight change in model animal of human melanoma A375 during the administration.

    DETAILED DESCRIPTION OF THE INVENTION

    [0110] The present disclosure is described in detail below by means of examples. However, it is not intended that these examples have any disadvantageous limitations to the present disclosure. The present disclosure has been described in detail herein, and embodiments are also disclosed herein. It will be apparent to those skilled in the art that various changes and modifications may be made to the embodiments disclosed herein without departing from the spirit and scope disclosed herein.

    Reference Example 1

    [0111] ##STR00022## ##STR00023##

    Step 1: Synthesis of Compound A-1-2.

    [0112] To a reaction flask were added A-1-1 (150 g, 635.36 mmol, 1 eq), calcium chloride (70.51 g, 635.36 mmol, 1 eq), tetrahydrofuran (500 mL) and ethanol (1000 mL). Sodium borohydride (48.07 g, 1.27 mol, 2 eq) was added under nitrogen, and the mixture was reacted at 20? C. for 15 hours. After the reaction was completed, the reaction solution was concentrated under reduced pressure. The concentrate was diluted with 15% citric acid aqueous solution (4000 mL), and extracted with ethyl acetate (4000 mL?3). The organic phases were combined, washed with saturated brine (2000 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to dryness to give a crude product. The crude product was purified by column chromatography to give A-1-2. .sup.1H NMR (400 MHZ, DMSO-d.sub.6) ? (ppm)=7.47 (s, 1H), 5.58 (br s, 1H), 4.52 (s, 2H).

    Step 2: Synthesis of Compound A-1-4.

    [0113] To a reaction flask were added A-1-2 (102 g, 525.64 mmol, 1 eq) and 2-methyltetrahydrofuran (1000 mL). The atmosphere was replaced with nitrogen gas. The mixture was cooled to ?70? C., and lithium diisopropylamide (2 M, 525.64 mL, 2.0 eq) was slowly added dropwise. The mixture was stirred at ?70? C. for 30 minutes. Then, a solution of A-1-3 (138.18 g. 788.46 mmol, 1.5 eq) in 2-methyltetrahydrofuran (400 mL) was slowly added dropwise, and the mixture was reacted at ?70? C. for another 1 hour. After the reaction was completed, the reaction solution was quenched with saturated aqueous ammonium chloride solution (2000 mL), and extracted with ethyl acetate (2000 mL?4). The layers were separated. The organic phases were combined, washed with saturated brine (1000 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to dryness to give a crude product. The crude product was first purified by column, and then purified by slurrying with methyl tert-butyl ether to give A-1-4. .sup.1H NMR (400 MHZ, DMSO-d.sub.6) ? (ppm)=6.41 (s, 1H), 5.43 (t, J=5.6 Hz, 1H), 5.00-4.84 (m, 4H), 4.38 (d, J=5.6 Hz, 2H), 1.12 (s, 9H).

    Step 3: Synthesis of Compound A-1-5.

    [0114] To a reaction flask were added A-1-4 (50 g, 135.39 mmol, 1 eq), azodicarboxylic acid dipiperidide (40.99 g, 162.47 mmol, 1.2 eq) and tetrahydrofuran (500 mL). The atmosphere was replaced with nitrogen gas. The mixture was cooled to 0? C., and a solution of tributylphosphine (32.87 g, 162.47 mmol, 40.09 mL, 1.2 eq) in tetrahydrofuran (100 mL) was slowly added dropwise. The mixture was reacted at 0? C. for 1 hour. After the reaction was completed, water (500 mL) and saturated brine (500 mL) were added to the reaction solution in sequence. The mixture was extracted with ethyl acetate (500 mL?2). The layers were separated. The organic phases were combined, washed with saturated brine (300 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to dryness to give a crude product. The crude product was slurried with 500 mL of methyl tert-butyl ether, and filtered. The filtrate was collected, and concentrated to give a crude product. The crude product was slurried with 50 mL of n-hexane, and filtered. The filter cake was collected and dried to give A-1-5. .sup.1H NMR (400 MHZ, DMSO-d.sub.6) ? (ppm)=5.29 (d, J=7.5 Hz, 1H), 4.88-4.75 (m, 3H), 4.60 (d, J=12.9 Hz, 1H), 4.22 (d, J=12.9 Hz, 1H), 1.26 (s, 9H).

    Step 4: Synthesis of Compound A-1-6.

    [0115] To a reaction flask were added A-1-5 (29 g, 82.55 mmol, 1 eq), tetrahydrofuran (250 mL) and water (50 mL). The atmosphere was replaced with nitrogen gas. Iodine (2.10 g, 8.26 mmol, 1.66 mL, 0.1 eq) was added, and the mixture was reacted at 50? C. for 18 hours. Then additional iodine (2.10 g, 8.26 mmol, 1.66 mL, 0.1 eq) was added, and the mixture was reacted at 50? C. for another 6 hours. After the reaction was completed, a solution of crude A-1-6 was obtained and used directly in the next step.

    Step 5: Synthesis of Compound A-1-7.

    [0116] To the solution containing crude A-1-6 was added sodium carbonate (17.50 g, 165.11 mmol, 2 eq). The atmosphere was replaced with nitrogen gas. Di-tert-butyl carbonate (27.03 g, 123.83 mmol, 28.45 mL, 1.5 eq) was added. The mixture was reacted at 20? C. for 12 hours. After the reaction was completed, the reaction solution was poured into water (200 mL), and then extracted with ethyl acetate (300 mL?3). The layers were separated. The organic phases were combined, washed with saturated brine (300 mL?3), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to give a crude product. The crude product was purified by column chromatography to give A-1-7. .sup.1H NMR (400 MHZ, DMSO-d.sub.6) ? (ppm)=5.46 (d, J=5.9 Hz, 1H), 5.32 (d, J=6.3 Hz, 1H), 4.64 (d, J=6.1 Hz, 1H), 4.55 (d, J=5.8 Hz, 1H), 4.49 (d, J=9.4 Hz, 2H), 1.53 (s, 9H).

    Step 6: Synthesis of Compound A-1-8.

    [0117] To a reaction flask were added A-1-7 (26.5 g, 76.32 mmol, 1 eq), glacial acetic acid (1.37 g, 22.90 mmol, 1.31 mL, 0.3 eq) and acetonitrile (260 mL). The atmosphere was replaced with nitrogen gas. The mixture was heated to 50? C., and a solution of sodium chlorite (32.48 g, 305.28 mmol, 85% purity, 4 eq) in water (70 mL) was added dropwise. After the addition was completed, the mixture was reacted at 50? C. for another 12 hours. Then, additional sodium chlorite (8.97 g, 99.21 mmol, 1.3 eq) and glacial acetic acid (458.31 mg, 7.63 mmol, 436.49 ?L, 0.1 eq) were added, and the mixture was reacted at 50? C. for another 6 hours. After the reaction was completed, the reaction solution was quenched with aqueous saturated sodium sulfite solution (150 mL), and water (90 mL) was added. The mixture was left to stand, and the organic phase was separated. The aqueous phase was extracted with ethyl acetate (90 mL). The combined organic phase was washed with saturated brine (90 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated to give a crude product. The crude product was slurried with ethyl acetate: n-hexane (1:5, 120 mL) with stirring, and filtered. The filter cake was collected and dried to give A-1-8. .sup.1H NMR (400 MHZ, CDCl.sub.3) ? (ppm)=5.61 (d, J=6.6 Hz, 2H), 4.76 (d, J=6.6 Hz, 2H), 1.66 (s, 9H).

    Step 7: Synthesis of Compound A-1.

    [0118] To a dried reaction flask were added A-1-8 (10 g, 27.68 mmol, 1 eq) and dichloromethane (100 mL). Trifluoroacetic acid (41.04 g, 359.90 mmol, 26.65 mL, 13 eq) was added at 0? C. The mixture was reacted at 0? C. for 0.5 hours. After the reaction was completed, the reaction solution was slowly poured into aqueous saturated sodium bicarbonate solution (1000 mL), and adjusted to pH of 7?8. The mixture was extracted with dichloromethane (1000 mL?3). The layers were separated. The organic phases were combined, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to give A-1. .sup.1H NMR (400 MHZ, DMSO-d.sub.6) ? (ppm)=9.59 (s, 1H), 4.99-4.78 (m, 4H).

    Reference Example 2

    [0119] ##STR00024##

    Step 1: Synthesis of Compound B-1-2.

    [0120] To a reaction flask were added sodium hydroxide (590.8 g, 14.8 mol, 1.05 eq), ice water (20 L), and B-1-1 (2000.00 g, 14.07 mol, 1 eq). Then methyl iodide (2495.80 g, 17.59 mol, 1.25 eq) was added and the mixture was reacted at 25? C. for 2 hours. After the reaction was completed, 6N glacial hydrochloric acid aqueous solution was slowly added into the reaction flask to adjust the pH to 6?7. The mixture was stirred for 0.5 hours, and filtered. The filter cake was collected. Acetonitrile (500 mL) was added to the filter cake. The mixture was stirred for 0.5 hours, and filtered. The filter cake was collected, and dried by baking to give B-1-2. .sup.1H NMR (400 MHZ, DMSO-d.sub.6) ? (ppm)=12.69 (br s, 1H), 7.74 (br s, 1H), 2.45 (s, 3H), 1.86 (s, 3H).

    Step 2: Synthesis of Compound B-1-3.

    [0121] To a reaction flask were added acetonitrile (15 L) and B-1-2 (1500.00 g, 9.60 mol, 1 eq) at 25? C. Then phosphorus oxychloride (1840.00 g, 12.0 mol, 1.25 eq) was added. The mixture was slowly heated to 62? C., and reacted at 62? C. for 12 hours. The reaction solution was poured into water (10.5 L), and solid sodium bicarbonate was added to adjust the pH to 6-7. The mixture was extracted with ethyl acetate (10.5 L), and the layers were separated to give an organic phase. The organic phase was washed with saturated brine (7.5 L), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to give B-1-3. .sup.1H NMR (400 MHZ, DMSO-d.sub.6) ? (ppm)=8.54 (s, 1H), 2.50 (s, 3H), 2.22 (s, 3H).

    Step 3: Synthesis of Compound B-1.

    [0122] To a reaction flask were added B-1-3 (100 g, 572.57 mmol, 1 eq), water (24.76 g, 1.37 mol, 24.76 mL, 2.4 eq) and acetonitrile (1000 mL). The atmosphere was replaced with nitrogen gas. Sodium iodide (571.59 g, 3.81 mol, 6.66 eq) and trimethylchlorosilane (186.61 g, 1.72 mol, 218.00 mL, 3 eq) were added in sequence, and the mixture was reacted at 20? C. for 14 hours. After the reaction was completed, dichloromethane (800 mL) and water (1200 mL) were added to the reaction solution in sequence. Then solid sodium bicarbonate was added to adjust the pH to 6-7. The layers were separated, and the aqueous phase was extracted once with dichloromethane (500 mL). The organic phases were combined, washed successively with aqueous saturated sodium sulfite solution (500 mL) and saturated brine (500 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to give a crude product. n-Heptane (0.5 L) was added to the crude product, and the mixture was stirred for 1 hour. The mixture was filtered, and the filter cake was collected to give B-1. .sup.1H NMR (400 MHZ, DMSO-d.sub.6) ? (ppm)=8.34 (s, 1H), 2.48 (s, 3H), 2.21 (s, 3H).

    Example 1

    [0123] ##STR00025##

    Synthetic Route:

    [0124] ##STR00026##

    Step 1: Synthesis of WX001-2

    [0125] To a reaction flask were added A-1 (500 mg, 1.92 mmol, 1 eq), WX001-1 (427.54 mg, 2.30 mmol, 1.2 eq) and NN-dimethylformamide (3 mL). The atmosphere was replaced with nitrogen gas. Cesium carbonate (935.92 mg, 2.87 mmol, 1.5 eq) was added, and the mixture was reacted at 25? C. for 16 hours. After the reaction was completed, the reaction solution was poured into water (20 mL), and the mixture was extracted with ethyl acetate (30 mL?3). The layers were separated. The organic phases were combined, washed with saturated brine (30 mL?3), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to give a crude product. The crude product was purified by thin layer chromatography on silica gel plate to give WX001-2. LCMS (m/z): 366, 368 [M+H].sup.+.

    Step 2: Synthesis of WX001-3

    [0126] In a pre-dried reaction flask, the atmosphere was replaced with nitrogen, and wet palladium on carbon (0.1 g, 819.15 ?mol, 10% purity, 1 eq) and ethanol (20 mL) were added. Then WX001-2 (300 mg, 819.15 ?mol, 1 eq) was added. The atmosphere was replaced with nitrogen three times, and the mixture was stirred to react at 50? C. and 50 Psi for 24 hours. After the reaction was completed, the reaction solution was filtered, and the filtrate was concentrated under reduced pressure to give a crude product. The crude product was purified by thin layer chromatography on silica gel plate to give WX001-3. .sup.1H NMR (400 MHZ, CDCl.sub.3) ? (ppm)=8.96 (s, 1H), 7.58 (t, J=7.6 Hz, 1H), 7.19 (d, J=7.6 Hz, 1H), 7.09 (d, J=7.5 Hz, 1H), 5.26 (d, J=7.6 Hz, 2H), 5.14 (s, 2H), 4.80 (d, J=7.6 Hz, 2H), 2.55 (s, 3H).

    Step 3: Synthesis of WX001-4

    [0127] To a dried reaction flask were added WX001-3 (60 mg, 208.81 ?mol, 1 eq), tetrahydrofuran (1 mL) and zinc chloride solution (0.7 M, 298.31 ?L, 1 eq). The mixture was cooled to ?78? C., and lithium hexamethyldisilazide (1 M, 417.63 ?L, 2 eq) was added. The mixture was reacted at 20? C. for 1 hour to give reaction solution 1.

    [0128] A mixture of B-1 (55.57 mg, 208.81 ?mol, 1 eq) and tetrakis(triphenylphosphine)palladium (7.24 mg, 6.26 ?mol, 0.03 eq) in NN-dimethylacetamide (1 mL) was heated to 50? C. under nitrogen, and then reaction solution I was added dropwise. After the addition was completed, the mixture was reacted at 50? C. for another 1 hour. After the reaction was completed, the reaction solution was poured into water (5 mL), and then extracted with dichloromethane (30 mL?3). The layers were separated. The organic phases were combined, washed with saturated brine (30 mL?3), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to give a crude product. The crude product was purified by thin layer chromatography on silica gel plate to give WX001-4. LCMS (m/z): 426.0 [M+H].sup.+.

    Step 4: Synthesis of WX001-5

    [0129] To a reaction flask were added WX001-4 (100 mg, 235.00 ?mol, 1 eq), acetonitrile (1 mL) and water (0.5 mL). The atmosphere was replaced with nitrogen gas three times, and potassium monopersulfate (288.95 mg, 470.01 ?mol, 2 eq) was added. The mixture was reacted at 20? C. for 14 hours. After the reaction was completed, the reaction solution was poured into a saturated sodium thiosulfate aqueous solution (5 mL), and the mixture was extracted with dichloromethane (30 mL?3). The layers were separated. The organic phases were combined, washed successively with saturated aqueous sodium bicarbonate solution (20 mL) and saturated brine (30 mL?3), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to give a crude product. The crude product was purified by thin layer chromatography on silica gel plate to give WX001-5. LCMS (m/z): 458.0 [M+H].sup.+.

    Step 5: Synthesis of WX001

    [0130] To a dried reaction flask were added WX001-5 (40 mg, 87.43 ?mol, 1 eq), C-1 (16.98 mg, 174.85 ?mol, 2 eq) and tetrahydrofuran (0.5 mL). The atmosphere was replaced with nitrogen gas. The mixture was cooled to 0? C., and lithium hexamethyldisilazide (1 M, 166.11 ?L, 1.9 eq) was added dropwise. The mixture was reacted at 0? C. for 1 hour. After the reaction was completed, the reaction solution was poured into water (5 mL), and the mixture was extracted with dichloromethane (30 mL?3). The layers were separated. The organic phases were combined, washed with saturated brine (30 mL?3), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to give a crude product. The crude product was purified by preparative high performance liquid phase chromatography (column: Waters Xbridge BEH C18 100*25 mm*5 um; mobile phase: [water (10 mM ammonium bicarbonate)-acetonitrile]: B (acetonitrile) %: 20%-50%, 10 min) to give WX001. .sup.1H NMR (400 MHZ, DMSO-d.sub.6) ?=9.67 (br s, 1H), 8.62 (s, 1H), 7.64 (t, J=7.7 Hz, 1H), 7.43 (d, J=1.8 Hz, 1H), 7.14 (dd, J=2.9, 7.7 Hz, 2H), 6.37 (d, J=1.5 Hz, 1H), 5.12 (d, J=7.2 Hz, 2H), 5.02 (s, 2H), 4.85 (d, J=7.2 Hz, 2H), 3.74 (s, 3H), 2.57 (s, 3H), 2.42 (s, 3H): LCMS (m/z): 475.0 [M+H].sup.+.

    Example 2: Preparation of the Crystal Form A of WX001

    [0131] ##STR00027##

    Step 1: Synthesis of Compound I-1-3.

    [0132] Tetrahydrofuran (12 L) and I-1-1 (1200 g, 5.69 mol) were added into a reaction kettle. Tetramethylethylenediamine (661.59 g, 5.69 mol) was slowly added into the reaction kettle. The atmosphere was replaced with nitrogen gas. The mixture was cooled to ?70? C. (internal temperature). Lithium diisopropylamide (2 M, 6.83 L) was slowly added dropwise, and the mixture was stirred at ?70? C. for 0.5 hours. A solution of I-1-2 (1.76 kg, 9.39 mol) in tetrahydrofuran (4.8 L) was slowly added dropwise (1.5 hours), and the mixture was reacted at ?70? C. for 1 hour. After the reaction was completed, water (6 L) was added dropwise to the reaction solution to quench the reaction mixture. The reaction bottle was washed with water (2.4 L). The mixture was combined, and stirred. The mixture was allowed to warm up to 10? C. The layers were separated, and the aqueous phase was extracted with ethyl acetate (6 L). The remaining aqueous phase was adjusted to pH 3?4 with potassium bisulfate aqueous solution (15.6 L), and then extracted three times with ethyl acetate (6 L). The organic phases were combined and washed with saturated brine (6 L). The organic phase was dried with 1200 g of anhydrous sodium sulfate (m/m=1:1), and concentrated under reduced pressure at 45? C. to give a crude product. Dichloromethane (2.4 L) and isopropyl ether (7.2 L) were added to the crude product and the mixture was stirred at room temperature for half an hour. The mixture was filtered and the filter cake was collected to give I-1-3. .sup.1H NMR (400 MHZ, DMSO-d.sub.6) ?=13.18 (br s, 1H), 6.20 (br s, 1H), 4.93-4.82 (m, 4H), 1.09 (s, 9H).

    Step 2: Synthesis of Compound I-1-4.

    [0133] Dichloromethane (1330 mL), I-1-3 (1330 g, 1.99 mol, crude product), and 4-dimethylaminopyridine (41.37 g, 338.61 mmol) were added to the reaction kettle in sequence. N,N-carbonyldiimidazole (419.86 g, 2.59 mol) was added in batches, and the mixture was reacted at 50? C. for 36 hours. After the reaction was completed, 2 N hydrochloric acid aqueous solution (5.32 L) was added to the reaction solution to adjust the pH to 3-4. The mixture was stirred for 0.5 hours, and then water (5.32 L) was added. The mixture was concentrated to remove the organic solvent. The residue was filtered to give a filter cake. The filter cake was stirred with sodium bicarbonate aqueous solution (5.32 L) for 0.5 hours, then filtered, and washed with water (2.66 L). The filter cake was collected. Absolute ethanol (10.64 L) was added to the crude product. The mixture was stirred for 2 hours, and filtered. The filter cake was washed with absolute ethanol (1.3 L). The filter cake was collected and dried to give I-1-4. .sup.1H NMR (400 MHZ, DMSO-d.sub.6) ?=9.58 (br s, 1H), 4.94-4.84 (m, 4H).

    Step 3: Synthesis of Compound I-1-6.

    [0134] To a reaction flask were added I-1-4 (283 g, 1.03 mol), cesium carbonate (505.36 g, 1.55 mol) and N,N-dimethylformamide (2800 mL). The atmosphere was replaced with nitrogen gas. I-1-5 (221.24 g, 1.19 mol) was added, and the mixture was reacted at 20? C. for 12 hours. After the reaction was completed, the reaction solution was slowly poured into ice water (14 L). The mixture was stirred for 1 hour, and filtered. The filter cake was collected. Methyl tert-butyl ether (5 L) was added to the crude product and the mixture was stirred for 2 hours, and filtered. The filter cake was collected, and dried to give I-1-6. .sup.1H NMR (400 MHZ, DMSO-d.sub.6) ?=7.63 (t, J=7.7 Hz, 1H), 7.13 (dd, J=2.8, 7.7 Hz, 2H), 5.04 (d, J=7.4 Hz, 2H), 4.98 (s, 2H), 4.86 (d, J=7.4 Hz, 2H), 2.41 (s, 3H).

    Step 4: Synthesis of Compound I-1.

    [0135] Three reactions were carried out in parallel. To a reaction flask were added I-1-6 (183 g, 468.60 mmol) and tetrahydrofuran (2745 mL). The atmosphere was replaced with nitrogen gas. Diisopropylethylamine (181.69 g. 1.41 mol) and diethyl phosphite (194.14 g. 1.41 mol) were slowly added dropwise at 20? C., and the mixture was reacted at 40? C. for 16 hours. After the reaction was completed, the reaction solution was diluted with water (915 mL), and the mixture was extracted with dichloromethane (1830 mL*3). The organic phase was washed with saturated brine (915 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated at 45? C., and dried to give a crude product. The crude product was added to a mixed solvent of methyl tert-butyl ether and n-hexane (total 1098 mL, volume ratio 1:5), and the mixture was stirred for 1 hour. The mixture was filtered, and the filter cake was collected and dried. The filter cake was added to water (5400 mL), and the mixture was stirred for 1 hour, and filtered. The filter cake was collected, and dried to give I-1. .sup.1H NMR (400 MHZ, DMSO-d.sub.6) ?=9.32 (s, 1H), 7.64 (t, J=7.6 Hz, 1H), 7.13 (t, J=8.4 Hz, 2H), 5.10 (d, J=7.4 Hz, 2H), 4.99 (s, 2H), 4.81 (d, J=7.5 Hz, 2H), 2.42 (s, 3H).

    ##STR00028##

    Step 5: Synthesis of Compound I-3.

    [0136] To two reaction flasks were respectively added a solution of I-1 (120 g, 403.05 mmol) and zinc chloride (0.7 M, 575.79 mL) in tetrahydrofuran (1200 mL). The atmosphere was replaced with nitrogen gas. The mixture was cooled to 0? C., and lithium hexamethyldisilazide (1 M, 806.11 mL) was slowly added dropwise. The mixture was allowed to warm up to 20? C., and then stirred for 1 hour to give reaction solution 1. To another reaction flask were added B-1 (107.25 g, 403.05 mmol), tetrakis(triphenylphosphine)palladium (13.97 g, 12.09 mmol) and N,N-dimethylformamide (600 mL), and the reaction solution was heated to 50? C. to give reaction solution 2. Reaction solution 1 was slowly added dropwise into reaction solution 2, and the mixture was reacted at 50? C. for 1 hour. After the reaction was completed, the reaction mixture was quenched with 0.1M ethylenediamine tetraacetic acid disodium salt (10800 mL) and stirred for 30 min. n-Heptane (4800 mL) was added and the mixture was stirred for 0.5 hours. The mixture was filtered, and the filter cake was collected and dried to give a crude product. The crude product was slurried with ethanol (7200 mL) at 20? C. for 2 hours, and filtered. The filter cake was collected, and dried to give I-3. .sup.1H NMR (400 MHZ, CDCl.sub.3) ?=8.54 (s, 1H), 7.54 (t, J=7.2 Hz, 1H), 7.19 (d, J=7.4 Hz, 1H), 7.07 (d, J=7.3 Hz, 1H), 5.29 (d. J=7.5 Hz, 2H), 5.11 (s, 2H), 4.84 (d, J=7.6 Hz, 2H), 2.73 (s, 3H), 2.66 (s, 3H), 2.52 (s, 3H).

    Step 6: Synthesis of Compound I-4.

    [0137] To a reaction flask were added I-3 (120 g, 282.00 mmol), acetonitrile (110 mL) and water (550 mL). The atmosphere was replaced with nitrogen gas. Potassium monopersulfate (329.40 g, 535.81 mmol) was added, and the mixture was reacted at 30? C. for 12 hours. After the reaction was completed, 200 mL of ice-water mixture was added to the reaction solution. Then saturated sodium bicarbonate aqueous solution and saturated sodium thiosulfate aqueous solution were added (600 mL each), followed by 600 mL of water. The mixture was filtered, and the filter cake was collected and dried to give a crude product. 600 mL of absolute ethanol was added to the crude product, and the mixture was stirred for 1 hour. The mixture was filtered, and the filter cake was collected to give I-4. .sup.1H NMR (400 MHZ, CDCl.sub.3) ?=8.90 (s, 1H), 7.55 (t, J=7.7 Hz, 1H), 7.20 (d, J=7.7 Hz, 1H), 7.06 (d, J=7.7 Hz, 1H), 5.31 (d, J=7.6 Hz, 2H), 5.11 (s, 2H), 4.84 (d, J=7.6 Hz, 2H), 3.44 (s, 3H), 2.92 (s, 3H), 2.50 (s, 3H).

    Step 7: Synthesis of the crystal form A of WX001.

    [0138] To a reaction flask were added I-4 (47 g, 102.73 mmol), C-1 (25.94 g, 267.09 mmol), dichloromethane (470 mL) and tetrahydrofuran (470 mL). The atmosphere was replaced with nitrogen gas. Lithium hexamethyldisilazide (1 M, 246.54 mL, 2.4 eq) was added dropwise at ?5? C. (controlling the internal temperature at ?5-3? C.), and the mixture was reacted at 0? C. for 0.5 hours. After the reaction was completed, the mixture was quenched with deionized water (470 mL). The mixture was concentrated to remove organic solvent, and filtered. The filter cake was collected. The filter cake was stirred with deionized water (1000 mL) at room temperature for 30 minutes, and filtered. The filter cake was collected. The filter cake was stirred with acetonitrile (1000 mL) at room temperature for 30 min, and filtered. The filter cake was collected to give the crystal form A of WX001. .sup.1H NMR (400 MHZ, CDCl.sub.3) ?=8.44 (s, 1H), 7.60-7.50 (m, 2H), 7.18 (d, J=7.4 Hz, 1H), 7.06 (d, J=7.6 Hz, 1H), 6.88 (s, 1H), 6.42 (d, J=1.9 Hz, 1H), 5.27 (d, J=7.6 Hz, 2H), 5.12 (s, 2H), 4.85 (d, J=7.6 Hz, 2H), 3.85 (s, 3H), 2.69 (s, 3H), 2.52 (s, 3H). The XRPD pattern of the crystal form A of WX001 is shown in FIG. 1: the DSC curve of the crystal form A of WX001 is shown in FIG. 2; and the TGA curve of the crystal form A of WX001 is shown in FIG. 3.

    Example 3: Polymorph Screening of WX001

    1. Gas-Solid Penetration

    [0139] About 20 mg of the crystal form A of WX001 in each portion was weighed into a 3-mL vial, and about 4 mL of solvent was added to a 20-mL vial. The 3-mL (open) vial was placed in the 20-mL vial, and the 20-mL vial was sealed. The samples adsorbed the solvent and partially dissolved, or were allowed to stand at room temperature for 7 days, and then the solids were collected and tested by XRPD. The test results are shown in Table 7.

    TABLE-US-00007 TABLE 7 Summary of the gas-solid penetration test Solvent Test results MeOH Crystal form A Acetone Crystal form A EtOAc Crystal form A MTBE Crystal form A ACN Crystal form A Toluene Crystal form A 1,4-Dioxane Crystal form A H.sub.2O Crystal form A

    2. Gas-Liquid Diffusion

    [0140] About 20 mg of the crystal form A of WX001 in each portion was weighed into a 3-mL vial, and dissolved in 1.2?1.8 mL of solvent. About 4 mL of anti-solvent was added to a 20-mL vial. The 3-mL (open) vial containing a clear solution was placed in the 20-mL vial, and then the 20-mL vial was sealed and allowed to stand at room temperature. The resulting solid was collected and tested by XRPD. The test results are shown in Table 8.

    TABLE-US-00008 TABLE 8 Summary of the gas-liquid penetration test Solvent Anti-solvent Test results CHCl.sub.3 Acetone Crystal form A DMSO EtOAc Crystal form A* Toluene Crystal form A* *indicates that the solid was obtained by volatilization at room temperature.

    3. Slow Volatilization

    [0141] 15?20 mg of the crystal form A of WX001 was weighed into a 3-mL vial, and dissolved in 1.0?3.0 mL of solvent. The vial was sealed with a sealing film, and 4 pinholes were punched in the sealing film. The solution was left to stand at room temperature to slowly volatilize. The resulting solid was collected and tested by XRPD. The test results are shown in Table 9.

    TABLE-US-00009 TABLE 9 Summary of the slow volatilization test Solvent (v/v) Test results MeOH/DCM (1:1) Crystal form A THF/H.sub.2O (1:1) Crystal form A CHCl.sub.3 Crystal form A 1,4-dioxane Crystal form A

    4. Slow Cooling

    [0142] 15?35 mg of the crystal form A of WX001 in each portion was weighed into a 3-mL vial, and dissolved in 1.0?3.0 mL of solvent. The solution was stirred and equilibrated at 50? C. for about 3.5 hours, and then filtered. The supernatant was collected. The resulting supernatant was placed in a biochemical incubator, cooled from 50? C. to 5? C. at 0.1? C./min, and then maintained at a constant temperature of 5? C. The precipitated solids were collected and tested by XRPD. The test results are shown in Table 10.

    TABLE-US-00010 TABLE 10 Summary of the slow cooling test Solvent (v/v) Test results CHCl.sub.3 Crystal form A* THF/H.sub.2O (1:1) Crystal form A* ACN/H.sub.2O (1:1) Crystal form A* *indicates that the sample was clear at 5? C. and ?20? C. and the solid was obtained by volatilization at room temperature.
    5. Stirring with Temperature Cycle

    [0143] About 25 mg of the crystal form A of WX001 in each portion was weighed into an HPLC vial, and 0.5 mL of solvent was added respectively. The resulting suspension was subjected to a temperature cycle program (the sample was heated to 50? C. and then cooled to 5? C. at a rate of 0.1? C./min; then this cycle was repeated; finally, the sample was maintained at 5? C.) with magnetic stirring (1000 rpm). The solids were collected by centrifugation, and tested by XRPD. The test results are shown in Table 11.

    TABLE-US-00011 TABLE 11 Summary of the test of stirring with temperature cycle Solvent (v/v) Test results MeOH Crystal form A MIBK Crystal form A EtOAc Crystal form A THF/H.sub.2O (1:1) Crystal form A ACN/H.sub.2O (1:1) Crystal form A DMAc/H.sub.2O (1:1) Crystal form A

    6. Stirring of Suspension at Room Temperature

    [0144] About 25 mg of the crystal form A of WX001 in each portion was weighed into an HPLC vial, and 0.5 mL of solvent was added respectively. The obtained turbid liquid was magnetically stirred (1000 rpm) at room temperature for 3 days. The solids were collected by centrifugation, and tested by XRPD. The test results are shown in Table 12.

    TABLE-US-00012 TABLE 12 Summary of the test of stirring of suspension at room temperature Solvent (v/v) Test results EtOH Crystal form A EtOAc Crystal form A THF Crystal form A DCM Crystal form A n-Heptane Crystal form A H.sub.2O Crystal form A EtOH/H.sub.2O (0.97:0.03, a.sub.w~0.2) Crystal form A EtOH/H.sub.2O (0.93:0.07, a.sub.w~0.4) Crystal form A EtOH/H.sub.2O (0.86:0.14, a.sub.w~0.6) Crystal form A Solvent (v/v) Test results EtOH/H.sub.2O (0.71:0.29, a.sub.w~0.8) Crystal form A ACN Crystal form A

    7. Stirring of Suspension at 50? C.

    [0145] About 25 mg of the crystal form A of WX001 in each portion was weighed into an HPLC vial, and 0.5 mL of solvent was added respectively. The obtained suspension was magnetically stirred (1000 rpm) at 50? C. for 3 days. The solids were collected by centrifugation, and tested by XRPD. The test results are shown in Table 13.

    TABLE-US-00013 TABLE 13 Summary of the test of stirring of suspension at 50? C. Solvent (v/v) Test results IPA Crystal form A Acetone/H.sub.2O (1:1) Crystal form A IPAc Crystal form A MTBE Crystal form A 2-MeTHF Crystal form A 1,4-dioxane Crystal form A CHCl.sub.3/n-heptane (1:1) Crystal form A Toluene Crystal form A DMSO/H.sub.2O (1:1) Crystal form A ACN Crystal form A

    8. Addition of Anti-Solvent

    [0146] About 15 mg of the crystal form A of WX001 was respectively weighed into a 20 mL vial, and 0.7?1.0 mL of solvent was added to completely dissolve the solid. The anti-solvent was added dropwise to the clear solution while stirring (1000 rpm) until solid precipitated or until the total volume of anti-solvent reached 10 mL. Samples without solid precipitation were stirred at 5? C. until solid precipitated. Clear samples were then stirred at ?20? C. until solid precipitated. Still clear samples were then volatilized at room temperature. The precipitated solid was separated and tested by XRPD. The results are shown in Table 14.

    TABLE-US-00014 TABLE 14 Summary of the test of addition of anti-solvent Solvent (v/v) Anti-solvent Test results CHCl.sub.3 MeOH Crystal form A* Acetone Crystal form A EtOAc Crystal form A MTBE Crystal form A 2-MeTHF Crystal form A ACN Crystal form A n-Heptane Crystal form A Toluene Crystal form A MeOH/DCM (1:1) MIBK Crystal form A IPAc Crystal form A MTBE Crystal form A THF/H.sub.2O (1:1) H.sub.2O Crystal form A NMP H.sub.2O Crystal form A DMAc H.sub.2O Crystal form A *indicates that the sample was clear at room temperature and the solid was obtained by stirring at 5? C.

    Example 4: Study on Hygroscopicity of the Crystal Form A of WX001

    Materials of the Assay:

    [0147] SMS DVS Advantage dynamic vapor sorption apparatus

    Method of the Assay:

    [0148] 10?30 mg of the crystal form A of WX001 was weighed and put into a DVS sample pan for testing.

    Results of the Assay:

    [0149] The DVS pattern of the crystal form A of WX001 is shown in FIG. 4, ?W=0.1134%.

    Conclusion of the Assay:

    [0150] The weight increase of the crystal form A of WX001 by moisture absorption at 25? C. and 80% RH was 0.1134%, indicating that the crystal form A of WX001 has almost no hygroscopicity.

    Example 5: Stability Test of the Crystal Form A of WX001

    [0151] 12 portions of samples of the crystal form A of WX001 were weighed in parallel, and each portion was about 5 mg. The sample was placed at the bottom of an HPLC vial to form a thin layer. The samples were placed in a chamber at a constant temperature and humidity of 60? C./75% RH conditions and in a 92.5% RH desiccator. The bottles were sealed with a sealing film. Some small holes were punched in the sealing film to ensure that the samples were fully in contact with the ambient air. The caps of the sample bottles placed at 60? C. under light and light-shielding conditions (samples under light-shielding conditions were wrapped in tin foil) were tighten. The test results are shown in Table 15 below:

    TABLE-US-00015 TABLE 15 Results of the solid stability test of the crystal form A of WX001 Test conditions Conditions for taking points Crystal form 0 day Crystal form A 60? C. 5 days Crystal form A 10 days Crystal form A 92.5% RH 5 days Crystal form A 10 days Crystal form A Visible light.sup.# Reaching illumination of Crystal form A 1.2E+06 Lux .Math. hrs Control group of Taking points simultaneously Crystal form A light-shielding with visible light group Visible light + Reaching illumination of 200 Crystal form A UV.sup.# W .Math. hrs/m.sup.2 Control group of Taking points simultaneously Crystal form A light-shielding with visible light + UV group 60? C./75% RH 1 month Crystal form A 2 months Crystal form A 3 months Crystal form A .sup.#ICH conditions

    [0152] Conclusion: the crystal form A of WX001 has good stability.

    Assay Example 1. Assay of In Vitro Kinase Activity

    1. Purpose of the Assay:

    [0153] The ability of the compound to inhibit ERK1 and ERK2 kinase activity was measured.

    2. Assay Buffer:

    [0154] 20 mM Hepes (pH 7.5), 10 mM MgCl.sub.2, 1 mM ethylenebis(oxyethylenenitrilo)tetraacetic acid (EGTA), 0.02% Brij35, 0.02 mg/mL bovine serum albumin (BSA), 0.1 mM Na.sub.3VO.sub.4, 2 mM dithiothreitol (DTT), 1% DMSO.

    3. Processing of Compound:

    [0155] The assay compound was dissolved in 100% DMSO to prepare a stock solution of specific concentration. The compound was serially diluted in DMSO solution using Integra Viaflo Assist smart pipette.

    4. Method of the Assay;

    [0156] 1) The substrate MBP was prepared in freshly prepared reaction buffer; [0157] 2) ERK1 (or ERK2) kinase was added to the above-mentioned MBP solution and mixed gently; [0158] 3) The compound dissolved in 100% DMSO was added to the kinase reaction system using ultrasound technology (Echo550; nanoliter range), and the mixture was incubated at room temperature for 20 minutes; [0159] 4) 33P-ATP (specific concentration of 10 ?Ci/?L) was added to the reaction system, and the reaction was started at this time; [0160] 5) The mixture was incubated at room temperature for 2 hours; [0161] 6) The amount of radioactivity was detected by filter-binding method; [0162] 7) ERK1 (or ERK2) kinase activity was calculated as the ratio of the remaining kinase activity in the assay sample to the kinase activity of the control group (treated by DMSO). Curve was fitted using Prism (GraphPad software) and IC.sub.50 values were calculated. [0163] 5. The assay results are shown in Table 16 and Table 17:

    TABLE-US-00016 TABLE 16 Result of ERK1 kinase activity assay ERK1 Compound IC.sub.50 (nM) WX001 1.4

    [0164] Conclusion: The compound of the present disclosure exhibits excellent inhibitory activity against ERK1 kinase.

    TABLE-US-00017 TABLE 17 Result of ERK2 kinase activity assay ERK2 Compound IC.sub.50 (nM) WX001 0.54

    [0165] Conclusion: The compound of the present disclosure exhibits excellent inhibitory activity against ERK2 kinase.

    Assay Example 2. Assay of In Vitro Cell Proliferation Inhibition

    1. Purpose of the Assay:

    [0166] The ability of the compound to inhibit the proliferation of HT29 tumor cells was measured.

    2. Processing of Compound:

    [0167] The assay compound was dissolved in 100% DMSO to prepare 10 mM stock solution.

    3. Method and Step of the Assay:

    [0168] 1) UV light of a biological safety cabin was turned on, and 30 minutes were counted down; [0169] 2) In a 37? C. water bath, RPMI1640 medium and trypsin were preheated; [0170] 3) After completion of the UV irradiation, the biological safety cabin was opened. The preheated medium, trypsin and phosphate buffered saline (PBS), etc. were wiped with alcohol and placed in the biological safety cabin; [0171] 4) HT29 cells were removed from the incubator, and the old medium was removed in biological safety cabin. 10 ml of PBS was added. The mixture was shaken gently, and then PBS was removed; [0172] 5) 1.5 ml of preheated 0.25% trypsin was added. The culture vessel was shaken horizontally so that the trypsin evenly covered the cells at the bottom, and placed in an incubator for 2 minutes; [0173] 6) Cell digestion was stopped with complete medium, and the cell suspension was pipetted to homogeneity and counted; [0174] 7) According to the result of cell counting, the density of cell suspension was adjusted to 1500 cells per well, and the cell suspension was seeded at 50 ?l per well; [0175] 8) The stock solution of the compound was serially diluted in DMSO solution, and the compound was added to the cell plate using Tecan; [0176] 9) The compound-added cell plate and CellTiterGlo were equilibrated at room temperature, and 25 microliters of CellTiterGlo was then added to each well. The cell plate was shaken for 1-2 minutes and then allowed to stand for 10 minutes. The signal value was then detected. The data were analyzed using XL-Fit, and the IC.sub.50 of each compound was calculated.

    4. The Assay Result is Shown in Table 18:

    [0177]

    TABLE-US-00018 TABLE 18 Result of in vitro cell activity assay HT29 Compound IC.sub.50 (nM) WX001 66

    [0178] Conclusion: The compound of the present disclosure exhibits excellent inhibitory activity on HT29 cell proliferation.

    Assay Example 3. In Vivo PK Study in Mice

    1. Purpose of the Assay:

    [0179] Female BALB/c mice were used as assay animals to determine the blood concentration of the compound and evaluate the pharmacokinetic behavior after a single administration.

    2. Procedure of the Assay:

    [0180] Four healthy adult female BALB/c mice were selected, wherein 2 mice were in an intravenous injection group and 2 mice were in an oral group. The vehicle in the intravenous injection group was 5% DMSO+95% (20% HP-?-CD). The compound to be assayed was mixed with an appropriate amount of vehicle for intravenous injection, vortexed and sonicated to prepare a clear solution of 0.5 mg/mL. The clear solution was filtered by a microporous membrane, and then ready for use. The vehicle in the oral group was 5% DMSO+95% (20% HP-?-CD). The compound to be assayed was mixed with the vehicle, vortexed and sonicated to prepare a solution of 0.3 mg/mL. Mice were administered 1 mg/kg intravenously or 3 mg/kg orally, and then whole blood was collected for a certain period. Plasma was prepared. The drug concentration was analyzed by LC-MS/MS method, and the pharmacokinetic parameters were calculated by Phoenix WinNonlin software (Pharsight, USA).

    [0181] Note: DMSO: dimethyl sulfoxide: HP-?-CD: hydroxypropyl-?-cyclodextrin.

    3. The Assay Result is Shown in Table 19:

    [0182]

    TABLE-US-00019 TABLE 19 Results of the PK assay of the compound Oral Com- C.sub.max DNAUC Vd.sub.ss Cl (mL/ T.sub.1/2 pound (nM) F % (nM .Math. h/mpk) (L/kg) min/kg) (h) WX001 4790 154 2935 1.23 18.9 1.1 Note: C.sub.max is maximum concentration; F % is oral bioavailability; DNAUC is AUC.sub.PO/Dose, AUC.sub.PO is oral exposure, and Dose is drug dose; Vd.sub.ss is volume of distribution; Cl is clearance rate; T.sub.1/2 is half-life; and NA is not available.

    [0183] Conclusion: The compound of the present disclosure exhibits excellent oral exposure and bioavailability.

    Assay Example 4. Solubility Study

    1. Purpose of the Assay:

    [0184] The solubility of the compound was determined to evaluate the solubility property of the compound.

    2. Assay Solution:

    [0185] 1) Buffer A (pH 2.0): 50 mM phosphate buffer, pH 2.0; Buffer B (pH 6.5): 50 mM phosphate buffer, pH 6.5: Buffer C (pH 7.4): 50 mM phosphate buffer, pH 7.4; [0186] 2) Preparation of standard solution: [0187] a) 50% acetonitrile solution and 50% buffer solution were mixed to obtain a diluent; [0188] b) 10 mM (10 ?L/compound) compound stock solution was added to the diluent (490 ?L/compound) to obtain 200 ?M detection standard solution; [0189] c) The 200 ?M UV detection standard solution was diluted with 10-fold and 200-fold volume of diluents to obtain 20 ?M and 1 ?M UV standard solutions, respectively; [0190] d) UV standard solutions of 1 ?M, 20 UM, and 200 ?M were used as standard solutions for solubility assays.

    3. Method of the Assay;

    [0191] 1) The compound was dissolved in DMSO to prepare a 10 mM stock solution. Amiodarone hydrochloride, carbamazepine, and chloramphenicol were used as controls in the solubility assay; [0192] 2) The stock solutions of the assay compound and the control (10 ?L each) were put into a 96-well plate, and 490 ?L of three different dissolution media (buffer A, B, C) were added respectively. The pH of the corresponding solubility solutions was 2.0, 6.5 and 7.4, respectively. The theoretical maximum concentration of the assay compound is 200 ?M with 2% DMSO; [0193] 3) The plate was shaken in a shaker at room temperature (25?2? C.) at 600 rpm for 24 hours; [0194] 4) 200 ?L of the solution was pipetted from the 96-well plate, filtered with suction by a vacuum suction filtration device, and transferred into a new 96-well plate as an assay sample; [0195] 5) The compound concentration was determined using HPLC-UV. HPLC conditions are as shown in Table 20:

    TABLE-US-00020 TABLE 20 HPLC conditions Assay HPLC-UV detection method Instrument Agilent 1200 Mobile A: Water + 0.37% trifluoroacetic acid phase B: Acetonitrile + 0.19% trifluoroacetic acid Column Waters Xbridge RP-C18 (2.1 ? 50 mm, 5 ?m) Time Flow rate (min) B % (mL/min) Proportion 0.00 5 1.0 2.00 90 1.0 2.50 90 1.0 3.01 5 1.0 4.00 5 1.0 [0196] 6) Three UV standard solutions from low concentration to high concentration (1 ?M, 20 ?M, 200 ?M) were injected into HPLC, and then the assay sample of the compound to be assayed was injected; [0197] 7) The UV chromatographic peaks were integrated, and the solubility of the sample was calculated.

    4. The Assay Results are Shown in Table 21:

    [0198]

    TABLE-US-00021 TABLE 21 Assay results of compound solubility Solubility at different pH Compound pH = 2.0 pH = 6.5 pH = 7.4 WX001 199.5 ?M 2.94 ?M 2.64 ?M

    [0199] Conclusion: The compound of the present disclosure has good solubility under different pH conditions.

    Assay Example 5. Assay of In Vivo Efficacy in Mouse Model of Human Melanoma A375

    1. Purpose of the Assay:

    [0200] The anti-tumor effect of WX001 was evaluated using a subcutaneous xenograft tumor model of human melanoma A375 cells in nude mouse.

    2. Assay Animal:

    [0201] Species: mouse [0202] Strain: BALB/c nude mouse [0203] Age: 6-8 weeks old [0204] Gender: female [0205] Body weight: 18-22 grams [0206] Supplier: Vital River Laboratory Animal Technology Co., Ltd.

    3. Environment for Rearing:

    [0207] Animals were reared in IVC (independent air supply system, and constant temperature and humidity) cages (3 animals per cage) in SPF grade of animal room at a temperature of 20-26? C. and a humidity of 40-70%;

    [0208] Cage: The cage was made of polycarbonate, and had a volume of 375 mm?215 mm?180 mm. The bedding material was corncob, which was replaced once a week:

    [0209] Food: Assay animals had free access to food (dry pelleted food sterilized by irradiation) throughout the assay period:

    [0210] Drinking water: Assay animals had free access to sterilized water:

    [0211] Cage identification: The animal information card for each cage should indicate the number, gender, strain, date of receipt, assay numbering of administration schedule, group, and start date of the assay of animals in the cage:

    [0212] Animal identification: Assay animals were identified by ear tags.

    4. Assay Procedure:

    [0213] 1) Assay cells and culture: Human melanoma A375 cells were cultured in monolayer in vitro. The culture conditions were DMEM medium plus 10% fetal bovine serum, and a 37? C. 5% CO.sub.2 incubator. Routine digestion with trypsin-EDTA was performed twice a week for passage. When the cell saturation was 80%-90% and the amount reached the requirement, the cells were harvested, counted, and inoculated: [0214] 2) Tumor tissue inoculation and grouping: 0.1 mL (5?10.sup.5) of A375 cells were subcutaneously inoculated into the right armpit of each mouse. When the average tumor volume reached 170 mm.sup.3, the animals were randomly divided into 4 groups and the administration was started. The grouping and administration schedule of the assay were shown in Table 22.

    TABLE-US-00022 TABLE 22 Grouping and administration schedule of assay animals Number of Dosage Cycle of Route and frequency of Group animals Drug (mg/kg) administration administration 1 6 Solvent 21 days Oral administration (PO), control twice daily (BID) (Vehicle) 2 6 WX001 12.5 21 days Oral administration (PO), twice daily (BID) 3 6 WX001 25 21 days Oral administration (PO), twice daily (BID) 4 6 WX001 50 21 days Oral administration (PO), twice daily (BID) [0215] 3) Daily observation of assay animals: The development of this assay protocol and any modifications were evaluated and approved by the Institutional Animal Care and Use Committee (IACUC). The use and welfare of assay animals were carried out in accordance with the regulations of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). Animals were monitored daily for health and death. Routine examinations included observation of tumor growth and the effects of drug treatment on the animals' daily behavior such as behavioral activities, food and water intake (visual inspection only), weight changes (weight measurements twice a week), appearance signs or other abnormalities. Animal deaths and side effects in each group were recorded based on the number of animals in each group. [0216] 4) Formulation of assay compound [0217] a) Vehicle group: 5% DMSO+95% (20% HP-?-CD). [0218] b) Assay compound group: A quantitative amount of the assay compound was weighed in a formulation bottle. A corresponding volume of DMSO was added and then the mixture was vortexed to obtain a clear solution. A corresponding volume of 20% HP-?-CD was added and then the mixture was vortexed to obtain a homogeneous suspension. [0219] 5) Tumor measurement and assay indicator: [0220] a) Tumor diameter was measured twice a week with a vernier caliper. The calculation formula of tumor volume was: TV=1/2?a?b.sup.2, wherein a and b represent the long and short diameters of tumor, respectively: [0221] b) The tumor-inhibitory efficacy of the compound was evaluated by TGI (%). TGI (%) reflected the inhibition rate of tumor growth. TGI (%) was calculated as follows: TGI (%)={[1?(average tumor volume at the end of administration of a treatment group-average tumor volume at the beginning of administration of this treatment group)]/(average tumor volume at the end of treatment in a solvent control group-average tumor volume at the beginning of treatment in the solvent control group)}?100%.

    5. Assay Results:

    [0222] 1) As shown in Table 23 and FIG. 5, in the subcutaneous xenograft tumor model of human melanoma A375 cells in nude mouse, WX001 could inhibit tumor growth in a dose-dependent manner until day 21 after oral administration, at three doses of 12.5 mg/kg, 25 mg/kg and 50 mg/kg, with the TGI of 45%, 58% and 102% respectively. [0223] 2) The body weight of assay animals was used as a reference index for indirect determination of drug toxicity. As shown in FIG. 6, when administered to the 21st day, the body weight of all animals in the solvent control group and WX001 group did not decrease significantly, and the tolerance was good.

    TABLE-US-00023 TABLE 23 Results of in vivo efficacy assay in mouse A375 model Drug TGI WX001 (12.5 mg/kg, PO, BID) 45% WX001 (25 mg/kg, PO, BID) 58% WX001 (50 mg/kg, PO, BID) 102%

    [0224] Conclusion: WX001 can inhibit tumor growth in a dose-dependent manner at three doses of 12.5 mg/kg, 25 mg/kg and 50 mg/kg. During the administration, the body weight of animals is not observed to decrease significantly, and the tolerance is good.

    Assay Example 6. In Vivo PK Study in SD Rats

    1. Purpose of the Assay:

    [0225] Male SD rats were used as assay animals. After a single administration, the plasma concentration of the compound was measured and the pharmacokinetic behavior was evaluated.

    2. Procedure of the Assay:

    [0226] Six healthy adult male SD rats were selected, wherein 3 rats were in an intravenous injection group and 3 rats were in an oral group. The vehicle in the intravenous injection group was 5% DMSO+95% (20% HP-?-CD). The compound to be assayed was mixed with an appropriate amount of vehicle for intravenous injection, vortexed and sonicated to prepare a clear solution of 0.2 mg/mL. The clear solution was filtered by a microporous membrane, and then ready for use. The vehicle in the oral group was 5% DMSO+95% (20% HP-?-CD). The compound to be assayed was mixed with the vehicle, vortexed and sonicated to prepare a solution of 1 mg/mL. SD rats were administered 1 mg/kg intravenously or 10 mg/kg orally, and then whole blood was collected for a certain period. Plasma was prepared. The drug concentration was analyzed by LC-MS/MS method, and the pharmacokinetic parameters were calculated by Phoenix WinNonlin software (Pharsight, USA).

    [0227] Note: DMSO: dimethyl sulfoxide: HP-?-CD: hydroxypropyl-?-cyclodextrin.

    3. The Assay Results are Shown in Table 24:

    [0228]

    TABLE-US-00024 TABLE 24 Results of the PK assay of the compound Oral Com- C.sub.max DNAUC Vd.sub.ss Cl (mL/ T.sub.1/2 pound (nM) F % (nM .Math. h/mpk) (L/kg) min/kg) (h) WX001 2500 49% 1445.79 1.08 13.4 3.77 Note: C.sub.max is maximum concentration; F % is oral bioavailability; DNAUC is AUC.sub.PO/Dose, AUC.sub.PO is oral exposure, and Dose is drug dose; Vd.sub.ss is volume of distribution; Cl is clearance rate; T.sub.1/2 is half-life.

    [0229] Conclusion: The compound of the present disclosure exhibits excellent oral exposure and bioavailability.

    Assay Example 7. In Vivo PK Study in Cynomolgus Monkeys

    1. Purpose of the Assay;

    [0230] Male cynomolgus monkeys were used as assay animals. After a single administration, the plasma concentration of the compound was measured and the pharmacokinetic behavior was evaluated.

    2. Procedure of the Assay;

    [0231] Five healthy adult male cynomolgus monkeys were selected, wherein 2 animals were in an intravenous injection group, and 3 animals were in an oral group. The vehicle in the intravenous injection group was 5% DMSO+95% (20% HP-?-CD). The compound to be assayed was mixed with an appropriate amount of vehicle for intravenous injection, and dissolved with stirring to prepare a clear solution of 0.4 mg/mL. The clear solution was filtered by a microporous membrane, and then ready for use. The vehicle in the oral group was 5% DMSO+95% (20% HP-?-CD). The compound to be assayed was mixed with the vehicle, and dissolved with stirring to prepare a solution of 0.3 mg/mL. Cynomolgus monkeys were administered 1 mg/kg intravenously or 3 mg/kg orally, and then whole blood was collected for a certain period. Plasma was prepared. The drug concentration was analyzed by LC-MS/MS method, and the pharmacokinetic parameters were calculated by Phoenix WinNonlin software (Pharsight, USA).

    [0232] Note: DMSO: dimethyl sulfoxide: HP-?-CD: hydroxypropyl-?-cyclodextrin.

    3. The Assay Results are Shown in Table 25:

    [0233]

    TABLE-US-00025 TABLE 25 Results of the PK assay of the compound Oral Com- C.sub.max DNAUC Vd.sub.ss Cl (mL/ T.sub.1/2 pound (nM) F % (nM .Math. h/mpk) (L/kg) min/kg) (h) WX001 921 50% 1152.35 1.98 16.1 2.41 Note: C.sub.max is maximum concentration; F % is oral bioavailability; DNAUC is AUC.sub.PO/Dose, AUC.sub.PO is oral exposure, and Dose is drug dose; Vd.sub.ss is volume of distribution; Cl is clearance rate; T.sub.1/2 is half-life.

    [0234] Conclusion: The compound of the present disclosure exhibits excellent oral exposure and bioavailability.

    Assay Example 8. In Vivo PK Study in Beagle Dogs

    1. Purpose of the Assay:

    [0235] Male beagle dogs were used as assay animals. After a single administration, the plasma concentration of the compound was measured and the pharmacokinetic behavior was evaluated.

    2. Procedure of the Assay:

    [0236] Five healthy adult male beagle dogs were selected, wherein 2 were in an intravenous injection group, and 3 were in an oral group. The vehicle in the intravenous injection group was 5% DMSO+95% (20% HP-?-CD). The compound to be assayed was mixed with an appropriate amount of vehicle for intravenous injection, and dissolved with stirring to prepare a clear solution of 0.4 mg/mL. The clear solution was filtered by a microporous membrane, and then ready for use. The vehicle in the oral group was 5% DMSO+95% (20% HP-?-CD). The compound to be assayed was mixed with the vehicle, and dissolved with stirring to prepare a solution of 0.3 mg/mL. Cynomolgus monkeys were administered 1 mg/kg intravenously or 3 mg/kg orally, and then whole blood was collected for a certain period. Plasma was prepared. The drug concentration was analyzed by LC-MS/MS method, and the pharmacokinetic parameters were calculated by Phoenix WinNonlin software (Pharsight, USA).

    [0237] Note: DMSO: dimethyl sulfoxide: HP-?-CD: hydroxypropyl-?-cyclodextrin.

    3. The Assay Results are Shown in Table 26:

    [0238]

    TABLE-US-00026 TABLE 26 Results of the PK assay of the compound Oral Com- C.sub.max DNAUC Vd.sub.ss Cl (mL/ T.sub.1/2 pound (nM) F % (nM .Math. h/mpk) (L/kg) min/kg) (h) WX001 1769 60% 2307.32 1.29 10.1 3.48 Note: C.sub.max is maximum concentration; F % is oral bioavailability; DNAUC is AUC.sub.PO/Dose, AUC.sub.PO is oral exposure, and Dose is drug dose; Vd.sub.ss is volume of distribution; Cl is clearance rate; T.sub.1/2 is half-life.

    [0239] Conclusion: The compound of the present disclosure exhibits excellent oral exposure and bioavailability.

    Assay Example 9. hERG Assay

    1. Purpose of the Assay:

    [0240] A fully automated patch-clamp method was used to assay the effect of the compound on hERG potassium channel (human Ether-a-go-go Related Gene potassium channel) current.

    2. Method of the Assay:

    2.1 Cell Preparation

    [0241] CHO-hERG cells were cultured in a 175 cm.sup.2 culture flask. When cells grew to a density of 60?80%, the culture medium was removed. Cells were washed once with 7 mL of PBS (Phosphate Buffered Saline), and then 3 mL of Detachin was added for digestion. After the digestion was completed, 7 mL of culture medium was added for neutralization, and then the mixture was centrifuged. The supernatant was aspirated, and then 5 mL of culture medium was added to resuspend the cells and ensure that the cell density was 2?5?10.sup.6/mL.

    2.2 Solution Preparation

    [0242] Extracellular solution formulation (mM): 140 NaCl, 5 KCl, 1 CaCl.sub.2), 1.25 MgCl.sub.2, 10 HEPES and 10 Glucose: the formulation was adjusted to pH of 7.4 with NaOH.

    [0243] Intracellular solution formulation (mM): 140 KCl, 1 MgCl.sub.2, 1 CaCl.sub.2), 10 EGTA and 10 HEPES: the formulation was adjusted to pH of 7.2 with KOH.

    2.3 Electrophysiological Recording Process

    [0244] The single-cell high-impedance sealing and whole-cell pattern formation processes were all automatically performed by a Qpatch instrument. After obtaining the whole-cell recording mode, the cells were clamped at ?80 mV. The cells were applied successively with a pre-voltage of ?50 mV for 50 milliseconds and a depolarizing stimulus of ?40 mV for 5 seconds, then repolarized to ?50 mV for 5 seconds, and then back to ?80 millivolts. This voltage stimulation was applied every 15 seconds, and recorded for 2 minutes. The extracellular solution was then given, and recorded for 5 minutes. Then a drug administration process started. The compound concentration started from the lowest assay concentration, and each assay concentration was given for 2.5 minutes. After all concentrations were given successively, a positive control compound 3 M Cisapride was given. At least 3 cells were assayed for each concentration (n?3).

    2.4 Compound Preparation

    [0245] 20.00 mM mother liquor of the compound was diluted with DMSO. 10 ?L of the mother liquor of the compound was added to 20 ?L of DMSO solution, and serially diluted 3-fold to 6 DMSO concentrations. 4 ?L of the compound with 6 DMSO concentrations was respectively added to 396 ?L of the extracellular solution, and serially diluted 100-fold to 6 intermediate concentrations. 80 ?L of the compound with 6 intermediate concentrations was respectively added to 320 ?L of the extracellular solution, and serially diluted 5-fold to the final concentration to be assayed. The highest assayed concentration was 40 ?M, and there was a total of 6 concentrations: 40, 13.3, 4.4, 1.48, 0.494 and 0.165 ?M, respectively. The DMSO content in the final assay concentration did not exceed 0.2%. This concentration of DMSO had no effect on the hERG potassium channel. All dilutions in the compound preparation were performed by a Bravo instrument.

    2.5 Data Analysis

    [0246] Assay data were analyzed by GraphPad Prism 5.0 software.

    2.6 Quality Control

    [0247] Environment: Humidity 20?50%, temperature 22?25? C. [0248] Reagent: The used assay reagents were purchased from Sigma, with a purity of >98%. [0249] Assay data in the report must meet the following standards: [0250] Whole cell sealing impedance >100 M? [0251] Tail current amplitude >300 pA [0252] Pharmacological parameters:

    [0253] The inhibitory effect of Cisapride with multiple concentrations on the hERG channel was measured as a positive control.

    3. The Assay Result is Shown in Table 27:

    [0254]

    TABLE-US-00027 TABLE 27 Assay result of the compound against hERG Compound IC50 (?M) WX001 >40

    [0255] Conclusion: The compound of the present disclosure has a weak inhibitory effect on hERG potassium channel current, thereby lowering the risk of cardiotoxicity and improving safety.

    Assay Example 10. Assay of Plasma Protein Binding (PPB)

    1. Purpose of the Assay:

    [0256] The binding degree of the assay compound to human/mouse/rat/canine/monkey plasma albumin was studied.

    2. Procedure of the Assay:

    [0257] 1) Matrix preparation: on the day of the assay, plasma was thawed in cold water and centrifuged at 3220 rpm for 5 min to remove all clots. The pH of the resulting plasma was measured and adjusted to 7.4?0.1 using 1% phosphoric acid or IN sodium hydroxide as needed. [0258] 2) Dilution procedure for the assay compound: the assay compound was dissolved in dimethyl sulfoxide (DMSO) to prepare stock solutions with concentrations of 10 mM and 2 mM, respectively. A 40 ?M working solution was prepared by diluting 2 ?L of stock solution (2 mM) with 98 ?L of DMSO. A 400 ?M working solution of the control compound was prepared by diluting 10 ?L of stock solution with 240 ?L of DMSO. The working solution of the compound (5 ?L) was mixed well with a blank matrix (995 ?L) at a ratio of 1:200 to prepare a loading matrix.

    3) Analysis Steps:

    [0259] a) An equal volume of 30 ?L of loading matrix (n=2) was transferred to a sample collection plate to prepare a time 0 (TO) sample for residue determination. The sample was immediately matched with the corresponding blank buffer to a final volume of 60 ?L, and the volume ratio of plasma to buffer in each well was 1:1. Then, 60 ?L of 4% H.sub.3PO.sub.4 in H.sub.2O and 480 ?L of stop solution containing the internal standard were added to the TO sample of the assay compound. They were then stored with other samples at 2-8? C. for further processing. [0260] b) The remaining plasma samples were pre-incubated in a carbon dioxide incubator at 37?1? C. for 30 min. Protein-free samples (F samples) and samples loaded with matrix (230 ?L) were all transferred into polycarbonate tubes (n=2) and ultracentrifuged at 37? C. and 155,000?g (35,000 rpm) for 4 h. [0261] c) To prepare T samples (assay samples), an additional matrix-containing sample was transferred to a separate 96-well plate (sample incubation plate) and incubated at 37? C. for 4 h. [0262] d) At the end of centrifugation, 30 ?L of protein-free samples and 30 ?L of T samples were transferred from the second layer of the supernatant (below the top layer) to a new sample collection plate. Each sample was mixed with the corresponding blank buffer or matrix to a final volume of 60 ?L with a matrix: buffer volume ratio of 1:1. 60 ?L of 4% H.sub.3PO.sub.4 aqueous solution and 480 ?L of stop solution (with internal standard) were added to all samples. The mixture was centrifuged at 4000 rpm for 20 min and 100 ?L of supernatant from each sample was analyzed by LC-MS/MS.

    3. The Assay Results are Shown in Table 28:

    [0263]

    TABLE-US-00028 TABLE 28 Assay results of the plasma protein binding of the compound Plasma protein binding rate (unbound %) Compound Human Mouse Rat Canine Monkey WX001 15.9% 11.7% 8.4% 14.3% 14.2%

    [0264] Conclusion: The compound of the present disclosure has moderate plasma protein binding.