TRIAZINE DIONE DERIVATIVE, PREPARATION METHOD THEREFOR AND APPLICATION THEREOF IN MEDICINE

Abstract

Provided are a triazine dione derivative, a preparation method therefor and an application thereof in medicine. Specifically, provided are a triazine dione derivative represented by general formula (I), a preparation method therefor, a pharmaceutical composition containing the derivative and a use thereof as a therapeutic agent, particularly a use in preparing a myosin inhibitor and a use in preparing a drug for treating hypertrophic cardiomyopathy (HCM) or heart diseases having pathophysiological features related to HCM.

##STR00001##

Claims

1. A compound of general formula (I) or a pharmaceutically acceptable salt thereof: ##STR00119## wherein: ring A is selected from the group consisting of cycloalkyl, heterocyclyl, aryl and heteroaryl; R.sup.1 is selected from the group consisting of halogen, alkyl, alkenyl, alkynyl, alkoxy, haloalkyl, haloalkoxy, cyano, amino, nitro, hydroxy, hydroxyalkyl, C(O)R.sup.6, C(O)OR.sup.7, S(O).sub.tR.sup.8, S(O).sub.tNR.sup.9R.sup.10, C(O)NR.sup.9R.sup.10, NR.sup.9R.sup.10 and ##STR00120## R.sup.2 are identical or different and are each independently selected from the group consisting of a hydrogen atom, halogen, alkyl, alkenyl, alkynyl, alkoxy, haloalkyl, haloalkoxy, cyano, amino, nitro, hydroxy, hydroxyalkyl, C(O)R.sup.6, C(O)OR.sup.7, S(O).sub.tR.sup.8, S(O).sub.tNR.sup.9R.sup.10, C(O)NR.sup.9R.sup.10 and NR.sup.9R.sup.10; alternatively, R.sup.1 and one adjacent R.sup.2, or two adjacent R.sup.2, fuse with ring A to form cycloalkyl or heterocyclyl, wherein the cycloalkyl or heterocyclyl is optionally substituted with one or more substituents selected from the group consisting of a hydrogen atom, halogen, alkyl, haloalkyl, alkoxy, haloalkoxy, cyano, amino, nitro and hydroxy; L.sub.2 is selected from the group consisting of a covalent bond, (CH.sub.2).sub.r, C(O), NR.sup.a, an oxygen atom and a sulfur atom; R.sup.a is selected from the group consisting of a hydrogen atom, alkyl, haloalkyl, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl; ring C is selected from the group consisting of cycloalkyl, heterocyclyl, aryl and heteroaryl; R.sup.5 are identical or different and are each independently selected from the group consisting of a hydrogen atom, halogen, alkyl, alkenyl, alkynyl, alkoxy, haloalkyl, haloalkoxy, cyano, amino, nitro, hydroxy, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl; R.sup.3a is selected from the group consisting of halogen, alkyl, alkenyl, alkynyl, alkoxy, haloalkyl, haloalkoxy, cyano, amino, nitro, hydroxy, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl, wherein the alkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl are each independently optionally substituted with one or more substituents selected from the group consisting of halogen, alkoxy, haloalkoxy, cyano, amino, nitro and hydroxy; R.sup.3b is a hydrogen atom; R.sup.10 is alkyl or ##STR00121## wherein the alkyl is optionally substituted with one or more substituents selected from the group consisting of halogen, alkoxy, haloalkoxy, cyano, amino, nitro and hydroxy; L.sub.1 is a covalent bond or (CH.sub.2).sub.r; ring B is selected from the group consisting of cycloalkyl, heterocyclyl, aryl and heteroaryl; R.sup.4 are identical or different and are each independently selected from the group consisting of a hydrogen atom, halogen, alkyl, alkenyl, alkynyl, alkoxy, haloalkyl, haloalkoxy, oxo, cyano, nitro, hydroxy, hydroxyalkyl, C(O)R.sup.6, C(O)OR.sup.7, S(O).sub.tR.sup.8, S(O).sub.tNR.sup.9R.sup.10, C(O)NR.sup.9R.sup.10, cycloalkyl, (CH.sub.2).sub.r-cycloalkyl, heterocyclyl, (CH.sub.2).sub.r-heterocyclyl, aryl, (CH.sub.2).sub.r-aryl, heteroaryl and (CH.sub.2).sub.r-heteroaryl; R.sup.6 are identical or different at each occurrence and are each independently selected from the group consisting of a hydrogen atom, alkyl, haloalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl, wherein the alkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl are each independently optionally substituted with one or more substituents selected from the group consisting of halogen, alkyl, alkenyl, alkynyl, alkoxy, haloalkyl, haloalkoxy, cyano, amino, nitro, hydroxy and hydroxyalkyl; R.sup.7 are identical or different at each occurrence and are each independently selected from the group consisting of a hydrogen atom, alkyl, alkenyl, alkynyl, haloalkyl, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl; R.sup.8 are identical or different at each occurrence and are each independently selected from the group consisting of a hydrogen atom, alkyl, alkenyl, alkynyl, haloalkyl, hydroxyalkyl, hydroxy, cycloalkyl, heterocyclyl, aryl and heteroaryl; R.sup.9 and R.sup.10 are identical or different at each occurrence and are each independently selected from the group consisting of a hydrogen atom, alkyl, alkenyl, alkynyl, haloalkyl, hydroxyalkyl, cycloalkyl, (CH.sub.2).sub.r-cycloalkyl, heterocyclyl, (CH.sub.2).sub.r-heterocyclyl, aryl, (CH.sub.2).sub.r-aryl, heteroaryl and (CH.sub.2).sub.r-heteroaryl; alternatively, R.sup.9 and R.sup.10, together with the nitrogen atom to which they are attached, form heterocyclyl, and the heterocyclyl is optionally substituted with one or more substituents selected from the group consisting of halogen, alkyl, oxo, alkenyl, alkynyl, alkoxy, haloalkyl, haloalkoxy, cyano, amino, nitro, hydroxy, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl; p is 0, 1, 2, 3, 4, 5 or 6; r is 0, 1, 2, 3, 4, 5 or 6; m is 0, 1, 2, 3 or 4; s is 0, 1, 2, 3, 4, 5 or 6; and t is 0, 1 or 2.

2. The compound of general formula (I) or the pharmaceutically acceptable salt thereof according to claim 1, being a compound of general formula (I-1) or a pharmaceutically acceptable salt thereof: ##STR00122## wherein: ring A, R.sup.0, R.sup.1, R.sup.2, R.sup.3a, R.sup.3b and m are as defined in claim 1.

3. The compound of general formula (I) or the pharmaceutically acceptable salt thereof according to claim 1, wherein ring A is selected from the group consisting of 3- to 8-membered cycloalkyl, 3- to 12-membered heterocyclyl, 6- to 10-membered aryl and 5- to 10-membered heteroaryl.

4. The compound of general formula (I) or the pharmaceutically acceptable salt thereof according to claim 1, being a compound of general formula (II) or a pharmaceutically acceptable salt thereof: ##STR00123## wherein: R.sup.0, R.sup.1, R.sup.2, R.sup.3a, R.sup.3b and m are as defined in claim 1.

5. The compound of general formula (I) or the pharmaceutically acceptable salt thereof according to claim 1, being a compound of general formula (II-1) or a pharmaceutically acceptable salt thereof: ##STR00124## wherein: R.sup.0, R.sup.1, R.sup.2, R.sup.3, R.sup.3b and m are as defined in claim 1.

6. The compound of general formula (I) or the pharmaceutically acceptable salt thereof according to claim 1, wherein R.sup.4 is selected from the group consisting of C.sub.1-6 alkyl, 3- to 8-membered cycloalkyl and 3- to 12-membered heterocyclyl.

7. The compound of general formula (I) or the pharmaceutically acceptable salt thereof according to claim 1, wherein R.sup.1 is selected from the group consisting of halogen, C.sub.1-6 alkyl, C.sub.1-6 alkoxy, C.sub.1-6 haloalkyl, C.sub.1-6 haloalkoxy and ##STR00125## L.sub.2 is a covalent bond or an oxygen atom; ring C is selected from the group consisting of 3- to 8-membered cycloalkyl, 3- to 12-membered heterocyclyl, 6- to 10-membered aryl and 5- to 10-membered heteroaryl; R.sup.5 are identical or different and are each independently selected from the group consisting of a hydrogen atom, halogen, C.sub.1-6 alkyl, C.sub.1-6 alkoxy, C.sub.1-6 haloalkyl, C.sub.1-6 haloalkoxy and C.sub.1-6 hydroxyalkyl; p is 0, 1, 2, 3, 4, 5 or 6; R.sup.2 are selected from the group consisting of a hydrogen atom, halogen, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.1-6 alkoxy, C.sub.1-6 haloalkyl and C.sub.1-6 haloalkoxy; alternatively, R.sup.1 and one adjacent R.sup.2, or two adjacent R.sup.2, fuse with ring A to form 3- to 8-membered cycloalkyl or 3- to 12-membered heterocyclyl.

8. The compound of general formula (I) or the pharmaceutically acceptable salt thereof according to claim 1, wherein R.sup.1 is selected from the group consisting of C.sub.1-6 alkyl, C.sub.1-6 haloalkoxy and ##STR00126## L.sub.2 is a covalent bond or an oxygen atom; ring C is selected from the group consisting of cyclopropyl, tetrahydroftranyl and pyridinyl; R.sup.5 are identical or different and are each independently selected from the group consisting of a hydrogen atom, halogen and C.sub.1-6 alkyl; p is 0, 1 or 2; R.sup.2 are identical or different and are each independently a hydrogen atom or halogen; alternatively, R.sup.1 and one adjacent R.sup.2 fuse with ring A to form cyclobutyl, tetrahydrofuranyl, cyclopentyl and cyclohexyl.

9. The compound of general formula (I) or the pharmaceutically acceptable salt thereof according to claim 1, wherein R.sup.3, is selected from the group consisting of halogen, C.sub.1-6 alkyl, C.sub.1-6 alkoxy, C.sub.1-6 haloalkyl, C.sub.1-6 haloalkoxy and C.sub.1-6 hydroxyalkyl.

10. A compound selected from: ##STR00127## ##STR00128## ##STR00129## ##STR00130## or a pharmaceutically acceptable salt thereof.

11. A method for preparing a compound of general formula (I) or a pharmaceutically acceptable salt thereof, comprising: ##STR00131## conducting a nucleophilic substitution reaction of a compound of general formula (IA), or a salt thereof, with a compound of general formula (V) to give the compound of general formula (I) or the pharmaceutically acceptable salt thereof; wherein: R.sup.w is a leaving group; ring A, R.sup.0, R.sup.1, R.sup.2, R.sup.3a, R.sup.3b and m are as defined in claim 1.

12. A pharmaceutical composition comprising the compound of general formula (I) or the pharmaceutically acceptable salt thereof according to claim 1, and one or more pharmaceutically acceptable carriers, diluents or excipients.

13. A method for inhibiting myosin, comprising administering to a patient in need thereof a therapeutically effective amount of the pharmaceutical composition according to claim 12.

14. A method for treating a disease or condition, comprising administering to a patient in need thereof a therapeutically effective amount of the pharmaceutical composition according to claim 12, wherein the disease or condition is selected from the group consisting of diastolic heart failure with preserved ejection fraction, ischemic heart disease, angina pectoris, restrictive cardiomyopathy, diastolic dysfunction, hypertrophic cardiomyopathy (HCM), heart failure with preserved ejection fraction (HFpEF), heart failure with mid-range ejection fraction (HFmREF), valvular diseases, aortic stenosis, inflammatory cardiomyopathy, Leffler endocarditis, endomyocardial fibrosis, infiltrative cardiomyopathy, hemochromatosis, Fabry disease, glycogen storage disease, congenital heart defect, tetralogy of Fallot, left ventricular hypertrophy, refractory angina and Chagas disease.

15. The compound of general formula (I) or the pharmaceutically acceptable salt thereof according to claim 1, wherein ring A is phenyl.

16. The compound of general formula (I) or the pharmaceutically acceptable salt thereof according to claim 1, wherein R.sup.3a is C.sub.1-6 alkyl.

17. The compound of general formula (I) or the pharmaceutically acceptable salt thereof according to claim 1, wherein R.sup.1 is selected from the group consisting of C.sub.1-6 alkyl, C.sub.1-6 haloalkoxy and ##STR00132## L.sub.2 is a covalent bond or an oxygen atom; ring C is selected from the group consisting of 3- to 6-membered cycloalkyl, 3- to 6-membered heterocyclyl and 5- or 6-membered heteroaryl; R.sup.3 are identical or different and are each independently selected from the group consisting of a hydrogen atom, halogen, C.sub.1-6 alkyl, C.sub.1-6 alkoxy, C.sub.1-6 haloalkyl, C.sub.1-6 haloalkoxy and C.sub.1-6 hydroxyalkyl; p is 0, 1 or 2.

18. The compound of general formula (I) or the pharmaceutically acceptable salt thereof according to claim 1, wherein R are identical or different and are each independently a hydrogen atom or halogen.

19. The compound of general formula (I) or the pharmaceutically acceptable salt thereof according to claim 1, wherein R.sup.1 and one adjacent R.sup.2, or two adjacent R.sup.2, fuse with ring A to form 3- to 6-membered cycloalkyl or 3- to 6-membered heterocyclyl.

20. The method according to claim 14, wherein the disease or condition is hypertrophic cardiomyopathy (HCM).

Description

DETAILED DESCRIPTION

[0200] The present disclosure is further described below with reference to examples below, which are not intended to limit the scope of the present disclosure.

EXAMPLES

[0201] The structures of the compounds were determined by nuclear magnetic resonance (NMR) spectroscopy and/or mass spectrometry (MS). NMR shifts () are given in 10.sup.6 (ppm). NMR analyses were performed on a Bruker AVANCE-400 nuclear magnetic resonance instrument, with deuterated dimethyl sulfoxide (DMSO-d.sub.6), deuterated chloroform (CDCl.sub.3) and deuterated methanol (CD.sub.3OD) as solvents and tetramethylsilane (TMS) as an internal standard.

[0202] MS analyses were performed on an Agilent 1200/1290 DAD-6110/6120 Quadrupole MS liquid chromatography-mass spectrometry system (manufacturer: Agilent; MS model: 6110/6120 Quadrupole MS), Waters ACQuity UPLC-QD/SQD (manufacturer: Waters, MS model: Waters ACQuity Qda Detector/Waters SQ Detector) and THERMO Ultimate 3000-Q Exactive (manufacturer: THERMO, MS model: THERMO Q Exactive).

[0203] High performance liquid chromatography (HPLC) analyses were performed on Agilent HPLC 1200DAD, Agilent HPLC 1200VWD and Waters HPLC e2695-2489 high performance liquid chromatographs.

[0204] Chiral HPLC analyses were performed on an Agilent 1260 DAD high performance liquid chromatograph.

[0205] Preparative HPLC was performed on Waters 2767, Waters 2767-SQ Detecor2, Shimadzu LC-20AP and Gilson-281 preparative chromatographs.

[0206] Preparative chiral chromatography was performed on a Shimadzu LC-20AP preparative chromatograph.

[0207] The CombiFlash preparative flash chromatograph used was CombiFlash Rf200 (TELEDYNE ISCO).

[0208] Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plates, 0.15-0.2 mm layer thickness, were adopted for thin-layer chromatography (TLC) analysis and 0.4-0.5 mm layer thickness for TLC separation and purification.

[0209] Silica gel column chromatography generally used 200- to 300-mesh silica gel (Huanghai, Yantai) as the carrier.

[0210] The mean inhibition of kinase and the IC.sub.50 value were determined on a NovoStar microplate reader (BMG, Germany).

[0211] The known starting materials of the present disclosure may be synthesized using or according to methods known in the art, or may be purchased from ABCR GmbH & Co. KG, Acros Organics, Aldrich Chemical Company, Accela ChemBio Inc., Chembee Chemicals, and other companies.

[0212] In the examples, the reactions could all be conducted in an argon atmosphere or a nitrogen atmosphere unless otherwise specified.

[0213] The argon atmosphere or nitrogen atmosphere means that the reaction flask is connected to a balloon containing about 1 L of argon or nitrogen.

[0214] The hydrogen atmosphere means that the reaction flask is connected to a balloon containing about 1 L of hydrogen.

[0215] Parr 3916EKX hydrogenator, Qinglan QL-500 hydrogenator or HC2-SS hydrogenator was used in the pressurized hydrogenation reactions.

[0216] Hydrogenation reactions generally involve 3 cycles of vacuumization and hydrogen purging.

[0217] A CEM Discover-S 908860 microwave reactor was used in microwave reactions.

[0218] In the examples, a solution refers to an aqueous solution unless otherwise specified.

[0219] In the examples, the reaction temperature is room temperature, i.e., 20 C. to 30 C., unless otherwise specified.

[0220] The monitoring of the reaction progress in the examples was conducted by thin-layer chromatography (TLC). The developing solvent for reactions, the eluent system for column chromatography purification and the developing solvent system for thin layer chromatography included: A: n-hexane/ethyl acetate system, and B: dichloromethane/methanol system. The volume ratio of the solvents was adjusted according to the polarity of the compound, [0221] or by adding a small amount of basic or acidic reagents such as triethylamine and acetic acid.

Example 1

6-(((S)-1-(2-Fluoro-5-(((S)-tetrahydrofuran-3-yl)oxy)phenyl)ethyl)amino)-3-isopropyl-1,3,5-triazine-2,4(1H,3H)-dione 1

[0222] ##STR00100##

Step 1

3-Isopropyl-6-(1H-pyrazol-1-yl)-1,3,5-triazine-2,4(1H,3H)-dione 1c

[0223] 2-Isocyanatopropane 1a (4.06 g, 47.71 mmol, Shanghai Titan Scientific Co., Ltd.) and 1H-pyrazole-1-carboximidamide hydrochloride 1b (6.66 g, 45.44 mmol, Bide Pharmatech Ltd.) were dissolved in N,N-dimethylacetamide (35 mL). The solution was cooled to 10 C., and 1,8-diazabicycloundec-7-ene (12.00 g, 47.65 mmol) was added dropwise over 5 min. The reaction was stirred in an ice bath for another 30 min. N,N-Carbonyldiimidazole (9.80 g, 68.08 mmol) was added under ice bath. The mixture was cooled to 5 C., and 1,8-diazabicycloundec-7-ene (17.14 g, 68.06 mmol) was added dropwise over 10 min. The reaction was stirred in an ice bath for another 1 h. 2 N hydrochloric acid (132 mL) was added dropwise at room temperature over 30 min. The mixture was filtered, and the filter cake was collected and dried in vacuo to give the title product 1c (3.30 g, yield: 32.9%).

[0224] MS m/z (ESI): 222.0 [M+1].

Step 2

(S)-2-Fluoro-5-((tetrahydrofuran-3-yl)oxy)benzaldehyde 1e

[0225] 4-Fluoro-3-formylphenylbenzeneboronic acid 1d (10.00 g, 59.55 mmol, Hanhai Chemical Co., Ltd.) and (S)-tetrahydrofuran-3-ol (15.80 g, 179.33 mmol, Accela ChemBio Inc.) were dissolved in dichloromethane (100 mL), and copper acetate (22.00 g, 121.13 mmol), pyridine (10.00 g, 126.42 mmol) and triethylamine (13.00 g, 128.71 mmol) were added. The mixture was stirred at room temperature for 24 h, filtered, and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography with eluent system A to give the title product 1e (1.00 g, yield: 8.0%).

[0226] MS m/z (ESI): 211.0 [M+1].

Step 3

(R)N-(2-Fluoro-5-(((S)-tetrahydrofuran-3-yl)oxy)benzylidene)-2-methylpropane-2-sulfinamide 1f

[0227] Compound 1e (1.00 g, 4.76 mmol) and (R)-2-methylpropane-2-sulfinamide (580 mg, 4.78 mmol, Bide Pharmatech Ltd.) were dissolved in dichloromethane (20 mL), and cesium carbonate (1.90 g, 5.83 mmol) was added. The mixture was stirred at room temperature for 16 h, filtered, and concentrated under reduced pressure to give the crude title product 1f (1.40 g, yield: 93.9%). The crude product was directly used in the next step without being purified.

[0228] MS m/z (ESI): 313.9 [M+1].

Step 4

(R)N((S)-1-(2-Fluoro-5-(((S)-tetrahydrofuran-3-yl)oxy)phenyl)ethyl)-2-methylpropane-2-sulfinamide 1g

[0229] The crude compound 1f (100 mg, 0.32 mmol) was dissolved in dichloromethane (5 mL), and the system was purged with nitrogen three times. The reaction was cooled to 60 C., and a 3 M solution of methylmagnesium bromide in 2-methyltetrahydrofuran (0.22 mL, 0.66 mmol, Shanghai Titan Scientific Co., Ltd.) was added dropwise. The reaction was stirred at room temperature for 5 h in a nitrogen atmosphere. A saturated ammonium chloride solution (10 mL) was added, and the organic phase was separated. The aqueous phase was extracted with ethyl acetate (20 mL2). The organic phases were combined, washed with a saturated sodium chloride solution (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography with eluent system B to give the title product 1g (105 mg, yield: 99.9%).

[0230] MS m/z (ESI): 330.0 [M+1].

Step 5

(S)-1-(2-Fluoro-5-(((S)-tetrahydrofuran-3-yl)oxy)phenyl)ethan-1-amine hydrochloride 1h

[0231] Compound 1g (105 mg, 0.32 mmol) was dissolved in ethanol (5 mL). The solution was cooled to 0 C., and thionyl chloride (101 mg, 0.85 mmol, Shanghai Hushi Chemical Co., Ltd.) was added dropwise. The reaction was stirred at room temperature for 1h. The reaction mixture was concentrated under reduced pressure to give the crude title product 1h (83 mg, yield: 99.5%). The crude product was directly used in the next step without being purified.

[0232] MS m/z (ESI): 208.9 [M16].

Step 6

6-(((S)-1-(2-Fluoro-5-(((S)-tetrahydrofuran-3-yl)oxy)phenyl)ethyl)amino)-3-isopropyl-1,3,5-triazine-2,4(1H,3H)-dione 1

[0233] Compound 1c (70 mg, 0.32 mmol) and compound 1h (83 mg, 0.32 mmo) were dissolved in N-methylpyrrolidone (8 mL), and triethylamine (64 mg, 0.63 mmol) was added. The mixture was stirred at 120 C. for 16 h and purified by high performance liquid chromatography (Boston Phlex C18 15030 mm, 5 m; elution system: water (10 mmol of ammonium bicarbonate), acetonitrile, 20-95% acetonitrile; 20 min of gradient elution; flow rate: 30 mL/min) to give the title product 1 (6 mg, yield: 5.0%).

[0234] MS m/z (ESI): 379.0 [M+1].

[0235] .sup.1H NMR (500 MHz, DMSO-d.sub.6) 10.50 (brs, 1H), 7.66 (brs, 1H), 7.11 (m, 1H), 6.92 (m, 1H), 6.84 (m, 1H), 5.22 (m, 1H), 4.96 (m, 1H), 4.80 (m, 1H), 3.89-3.71 (m, 4H), 2.19 (m, 1H), 2.02-1.88 (m, 1H), 1.39 (d, 3H), 1.29 (d, 6H).

Example 2

6-(((S)-1-(2-Fluoro-5-(((R)-tetrahydrofuran-3-yl)oxy)phenyl)ethyl)amino)-3-isopropyl-1,3,5-triazine-2,4(1H,3H)-dione 2

[0236] ##STR00101##

Step 1

(R)-2-Fluoro-5-((tetrahydrofuran-3-yl)oxy)benzaldehyde 2a

[0237] Compound 1d (10 g, 59.5 mmol) and (R)-tetrahydrofuran-3-ol (10 g, 113 mmol, Bide Pharmatech Ltd.) were dissolved in dichloromethane (100 mL), and pyridine (10 g, 126 mmol, adamas), triethylamine (13 g, 129 mmol, adamas) and anhydrous copper acetate (23 g, 127 mmol, Bide Pharmatech Ltd.) were added. The reaction was stirred for 24 h. The reaction mixture was filtered and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography with eluent system A to give the title product 2a (1.9 g, yield: 15.2%).

[0238] MS m/z (ESI): 211.0 [M+1].

Step 2

(R)N-(2-Fluoro-5-(((R)-tetrahydrofuran-3-yl)oxy)benzylidene)-2-methylpropane-2-sulfinamide 2b

[0239] Compound 2a (1.9 g, 9.04 mmol) and (R)-2-methylpropane-2-sulfinamide (1.1 g, 9.08 mmol, adamas) were dissolved in dichloromethane (30 mL), and cesium carbonate (3.6 g, 11.4 mmol) was added. The reaction was stirred for 16 h. The reaction mixture was filtered and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography with eluent system A to give the crude title product 2b (2.8 g, yield: 98.8%).

[0240] MS m/z (ESI): 313.9 [M+1].

Step 3

(R)N((S)-1-(2-Fluoro-5-(((R)-tetrahydrofuran-3-yl)oxy)phenyl)ethyl)-2-methylpropane-2-sulfinamide 2c

[0241] The crude compound 2b (300 mg, 0.96 mmol) was dissolved in dichloromethane (10 mL), and the system was purged with nitrogen three times. The reaction was cooled to 60 C., and a 3 M solution of methylmagnesium bromide in 2-methyltetrahydrofuran (0.67 mL, 2.01 mmol, Shanghai Titan Scientific Co., Ltd.) was added dropwise. The reaction was stirred at room temperature for 5 h in a nitrogen atmosphere. A saturated ammonium chloride solution (10 mL) was added, and the organic phase was separated. The aqueous phase was extracted with ethyl acetate (20 mL2). The organic phases were combined, washed with a saturated sodium chloride solution (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography with eluent system B to give the title product 2c (315 mg, yield: 99.9%).

[0242] MS m/z (ESI): 330.0 [M+1].

Step 4

(S)-1-(2-Fluoro-5-(((R)-tetrahydrofuran-3-yl)oxy)phenyl)ethylamine hydrochloride 2d

[0243] Compound 2c (315 mg, 0.96 mmol) was dissolved in ethanol (6 mL). The solution was cooled to 0 C., and thionyl chloride (303 mg, 2.55 mmol, Shanghai Hushi Chemical Co., Ltd.) was added dropwise. The reaction was stirred at room temperature for 1h. The reaction mixture was concentrated under reduced pressure to give the crude title product 2d (250 mg, yield: 99.9%). The crude product was directly used in the next step without being purified.

[0244] MS m/z (ESI): 208.9 [M16].

Step 5

6-(((S)-1-(2-Fluoro-5-(((R)-tetrahydrofuran-3-yl)oxy)phenyl)ethyl)amino)-3-isopropyl-1,3,5-triazine-2,4(1H,3H)-dione 2

[0245] Compound 1c (164 mg, 0.74 mmol) and compound 2d (250 mg, 0.96 mmo) were dissolved in N-methylpyrrolidone (8 mL), and triethylamine (75 mg, 0.74 mmol) was added. The mixture was stirred at 120 C. for 16 h and purified by high performance liquid chromatography (Boston Phlex C18 15030 mm, 5 m; elution system: water (10 mmol of ammonium bicarbonate), acetonitrile, 20-95% acetonitrile; 20 min of gradient elution; flow rate: 30 mL/min) to give the title product 2 (30 mg, yield: 10.7%).

[0246] MS m/z (ESI): 379.0 [M+1].

[0247] .sup.1H NMR (500 MHz, DMSO-d.sub.6): 10.52 (brs, 1H), 7.27 (brs, 1H), 7.13 (m, 1H), 6.92-6.83 (m, 2H), 5.20 (m, 1H), 4.97 (m, 1H), 4.79 (m, 1H), 3.88-3.73 (m, 4H), 2.20 (m, 1H), 1.95 (m, 1H), 1.39 (d, 3H), 1.29 (d, 6H).

Example 3

3-Isopropyl-6-(((1S)-1-(3-((tetrahydrofuran-3-yl)oxy)phenyl)ethyl)amino)-1,3,5-triazine-2,4(1H,3H)-dione 3

[0248] ##STR00102##

Step 1

Tetrahydrofuran-3-yl methanesulfonate 3b

[0249] Tetrahydrofuran-3-ol 3a (2.0 g, 22.7 mmol, Accela ChemBio Inc.) and triethylamine (3.4 g, 33.7 mmol) were dissolved in dichloromethane (20 mL), and methanesulfonyl chloride (2.84 g, 24.9 mmol, Sinopharm Chemical Reagent Co., Ltd.) was added at 0 C. The mixture was reacted at room temperature for 24 h. Water (100 mL) was added, and extraction was performed with dichloromethane (20 mL2). The organic phases were combined and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography with eluent system A to give the title product 3b (3.1 g, yield: 82.2%).

[0250] .sup.1H NMR (500 MHz, CDCl.sub.3) 5.34 (m, 1H), 4.06-3.89 (m, 4H), 3.06 (s, 3H), 2.29-2.24 (m, 2H).

Step 2

3-((Tetrahydrofuran-3-yl)oxy)benzaldehyde 3c

[0251] Compound 3b (700 mg, 4.21 mmol) and 3-hydroxybenzaldehyde (500 mg, 4.09 mmol, Accela ChemBio Inc.) were dissolved in N,N-dimethylformamide (10 mL), and potassium carbonate (850 mg, 6.16 mmol) was added. The mixture was reacted at 90 C. for 16 h. The reaction mixture was diluted with water (50 mL) and extracted with ethyl acetate (20 mL2). The organic phases were combined and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography with eluent system A to give the title product 3c (710 mg, yield: 90.2%).

[0252] MS m/z (ESI): 193.0 [M+1].

Step 3

(R)-2-Methyl-N-(3-((tetrahydrofuran-3-yl)oxy)benzylidene)propane-2-sulfinamide 3d

[0253] Compound 3c (710 mg, 3.69 mmol) and (R)-2-methylpropane-2-sulfinamide (450 mg, 3.71 mmol) were dissolved in dichloromethane (10 mL), and cesium carbonate (1.5 g, 4.60 mmol) was added. The reaction was stirred for 16 h. The reaction mixture was filtered and concentrated. The resulting residue was purified by silica gel column chromatography with eluent system A to give the title product 3d (1.08 g, yield: 99.0%).

[0254] MS m/z (ESI): 296.0 [M+1].

Step 4

(R)-2-Methyl-N-((1S)-1-(3-((tetrahydrofuran-3-yl)oxy)phenyl)ethyl)propane-2-sulfinamide 3e

[0255] Compound 3d (500 mg, 1.69 mmol) was dissolved in dichloromethane (10 mL). The solution was cooled to 60 C. in a nitrogen atmosphere, and a 3 M solution of methylmagnesium bromide in 2-methyltetrahydrofuran (1.2 mL, 3.6 mmol) was added dropwise. The reaction was warmed to room temperature and stirred for 16 h. A saturated aqueous ammonium chloride solution (20 mL) was added at 0 C., and the organic phase was separated. The aqueous phase was extracted with ethyl acetate (20 mL2). The organic phases were combined, washed with a saturated sodium chloride solution (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography with eluent system A to give the title product 3e (490 mg, yield: 92.9%).

[0256] MS m/z (ESI): 312.1 [M+1].

Step 5

(1S)-1-(3-((Tetrahydrofuran-3-yl)oxy)phenyl)ethylamine hydrochloride 3f

[0257] Compound 3e (490 mg, 1.57 mmol) was dissolved in ethanol (5.0 mL), and thionyl chloride (220 mg, 1.85 mmol) was added at 0 C. The mixture was stirred at room temperature for 1 h. The reaction mixture was concentrated under reduced pressure to give the crude title product 3f (390 mg, 102%). The crude product was directly used in the next step without being purified.

[0258] MS m/z (ESI): 208.1 [M+1].

Step 6

3-Isopropyl-6-(((1S)-1-(3-((tetrahydrofuran-3-yl)oxy)phenyl)ethyl)amino)-1,3,5-triazine-2,4(1H,3H)-dione 3

[0259] Compound 1c (150 mg, 0.68 mmol) and compound 3f (211 mg, 0.87 mmo) were dissolved in N-methylpyrrolidone (6 mL), and triethylamine (69 mg, 0.68 mmol) was added. The mixture was stirred at 120 C. for 16 h and purified by high performance liquid chromatography (Boston Phlex C18 15030 mm, 5 m; elution system: water (10 mmol of ammonium bicarbonate), acetonitrile, 20-95% acetonitrile; 20 min of gradient elution; flow rate: 30 mL/min) to give the title product 3 (40 mg, yield: 16.4%).

[0260] MS m/z (ESI): 361.0 [M+1].

[0261] .sup.1H NMR (500 MHz, DMSO-d.sub.6) 10.45 (brs, 1H), 7.28-7.25 (m, 2H), 6.92-6.88 (m, 2H), 6.81 (m, 1H), 5.02-4.98 (m, 2H), 4.81 (m, 1H), 3.90-3.74 (m, 4H), 2.19 (m, 1H), 1.92 (m, 1H), 1.40 (d, 3H), 1.29 (d, 6H).

Example 4

(S)-3-Isopropyl-6-((1-(3-((6-methylpyridin-3-yl)oxy)phenyl)ethyl)amino)-1,3,5-triazine-2,4(1H,3H)-dione 4

[0262] ##STR00103##

Step 1

3-((6-Methylpyridin-3-yl)oxy)benzaldehyde 4b

[0263] (3-Formylphenyl)boronic acid 4a (800 mg, 5.34 mmol, Accela ChemBio Inc.) and 6-methylpyridin-3-ol (300 mg, 275 mmol, Bide Pharmatech Ltd.) were dissolved in dichloromethane (6.0 mL), and triethylamine (560 mg, 5.54 mmol), pyridine (440 mg, 5.56 mmol) and anhydrous copper acetate (1.0 g, 5.51 mmol, Bide Pharmatech Ltd.) were added. The reaction was stirred for 24 h. The reaction mixture was filtered through celite, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography with eluent system A to give the title product 4b (410 mg, yield: 69.9%).

[0264] MS m/z (ESI): 214.0 [M+1].

Step 2

(R)-2-Methyl-N-(3-((6-methylpyridin-3-yl)oxy)benzylidene)propane-2-sulfinamide 4c

[0265] Compound 4b (410 mg, 1.92 mmol) and (R)-2-methylpropane-2-sulfinamide (235 mg, 1.94 mmol, adamas) were dissolved in dichloromethane (6 mL), and cesium carbonate (760 mg, 2.33 mmol) was added. The reaction was stirred for 16 h. The reaction mixture was filtered and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography with eluent system A to give the title product 4c (560 mg, yield: 92.0%).

[0266] MS m/z (ESI): 317.1 [M+1].

Step 3

(R)-2-Methyl-N((S)-1-(3-((6-methylpyridin-3-yl)oxy)phenyl)ethyl)propane-2-sulfinamide 4d

[0267] Compound 4c (500 mg, 1.58 mmol) was dissolved in dichloromethane (10 mL). The reaction was cooled to 60 C. in a nitrogen atmosphere, and a 3 M solution of methylmagnesium bromide in 2-methyltetrahydrofuran (1.1 mL, 3.3 mmol, Shanghai Titan Scientific Co., Ltd.) was added dropwise. The reaction was warmed to room temperature and stirred for 16 h. A saturated aqueous ammonium chloride solution (20 mL) was added at 0 C., and the organic phase was separated. The aqueous phase was extracted with ethyl acetate (20 mL2). The organic phases were combined, washed with a saturated sodium chloride solution (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography with eluent system A to give the title product 4d (480 mg, yield: 91.3%).

[0268] MS m/z (ESI): 333.1[M+1].

Step 4

(S)-1-(3-((6-Methylpyridin-3-yl)oxy)phenyl)ethylamine hydrochloride 4e

[0269] Compound 4d (250 mg, 0.75 mmol) was dissolved in ethanol (6 mL). The solution was cooled to 0 C., and thionyl chloride (238 mg, 2.00 mmol, Shanghai Hushi Chemical Co., Ltd.) was added dropwise. The reaction was stirred at room temperature for 1 h. The reaction mixture was concentrated under reduced pressure to give the crude title product 4e (199 mg, yield: 99.9%). The crude product was directly used in the next step without being purified.

[0270] MS m/z (ESI): 229.0 [M+1].

Step 5

(S)-3-Isopropyl-6-((1-(3-((6-methylpyridin-3-yl)oxy)phenyl)ethyl)amino)-1,3,5-triazine-2,4(1H,3H)-dione 4

[0271] Compound 1c (129 mg, 0.58 mmol) and compound 4e (200 mg, 0.76 mmo) were dissolved in N-methylpyrrolidone (6 mL), and triethylamine (59 mg, 0.58 mmol) was added. The mixture was stirred at 120 C. for 16 h and purified by high performance liquid chromatography (Boston Phlex C18 15030 mm, 5 m; elution system: water (10 mmol of ammonium bicarbonate), acetonitrile, 20-95% acetonitrile; 20 min of gradient elution; flow rate: 30 mL/min) to give the title product 4 (4 mg, yield: 1.8%).

[0272] MS m/z (ESI): 382.0 [M+1].

[0273] .sup.1H NMR (500 MHz, DMSO-d.sub.6) 10.46 (brs, 1H), 8.24 (s, 1H), 7.40-7.34 (m, 3H), 7.27 (m, 1H), 7.13 (m, 1H), 7.05 (s, 1H), 6.86 (m, 1H), 5.03 (m, 1H), 4.80 (m, 1H), 2.45 (s, 3H), 1.39 (d, 3H), 1.29 (d, 6H).

Example 5

(S)-3-Isopropyl-6-((1-(3-(trifluoromethoxy)phenyl)ethyl)amino)-1,3,5-triazine-2,4(1H,3H)-dione 5

[0274] ##STR00104##

Step 1

(R)-2-Methyl-N-(3-(trifluoromethoxy)benzylidene)propane-2-sulfinamide 5b

[0275] 3-(Tri fluoromethoxy)benzaldehyde 5a (1.0 g, 5.26 mmol, Bide Pharmatech Ltd.) and (R)-2-methylpropane-2-sulfinamide (640 mg, 5.28 mmol) were dissolved in dichloromethane (20 mL), and cesium carbonate (2.1 g, 6.44 mmol) was added. The reaction was stirred for 16 h. The reaction mixture was filtered and concentrated. The resulting residue was purified by silica gel column chromatography with eluent system A to give the title product 5b (1.45 g, yield: 94.0%).

[0276] MS m/z (ESI): 294.0 [M+1].

Step 2

(R)-2-Methyl-N((S)-1-(3-(trifluoromethoxy)phenyl)ethyl)propane-2-sulfinamide 5c

[0277] Compound 5b (500 mg, 1.70 mmol) was dissolved in dichloromethane (10 mL). The solution was cooled to 60 C. in a nitrogen atmosphere, and a 3 M solution of methylmagnesium bromide in 2-methyltetrahydrofuran (1.2 mL, 3.6 mmol) was added dropwise. The reaction was warmed to room temperature and stirred for 16 h. A saturated aqueous ammonium chloride solution (20 mL) was added at 0 C., and the organic phase was separated. The aqueous phase was extracted with ethyl acetate (20 mL2). The organic phases were combined, washed with a saturated sodium chloride solution (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography with eluent system A to give the title product 5c (490 mg, yield: 92.9%).

[0278] MS n/z (ESI): 310.0 [M+1].

Step 3

(S)-1-(3-(Trifluoromethoxy)phenyl)ethylamine hydrochloride 5d

[0279] Compound 5c (250 mg, 0.81 mmol) was dissolved in ethanol (10 mL). The solution was cooled to 0 C., and thionyl chloride (256 mg, 2.15 mmol, Shanghai Hushi Chemical Co., Ltd.) was added dropwise. The reaction was stirred at room temperature for 1h. The reaction mixture was concentrated under reduced pressure to give the crude title product 5d (195 mg, yield: 99.9%). The crude product was directly used in the next step without being purified.

[0280] MS m/z (ESI): 188.8 [M16].

Step 4

(S)-3-Isopropyl-6-((1-(3-(trifluoromethoxy)phenyl)ethyl)amino)-1,3,5-triazine-2,4(1H,3H)-dione 5

[0281] Compound 1c (119 mg, 0.54 mmol) and compound 5d (195 mg, 0.81 mmo) were dissolved in N-methylpyrrolidone (6 mL), and triethylamine (54 mg, 0.53 mmol) was added. The mixture was stirred at 120 C. for 16 h and purified by high performance liquid chromatography (Boston Phlex C18 15030 mm, 5 m; elution system: water (10 mmol of ammonium bicarbonate), acetonitrile, 20-95% acetonitrile; 20 min of gradient elution; flow rate: 30 mL/min) to give the title product 5 (30 mg, yield: 15.6%).

[0282] MS m/z (ESI): 359.0 [M+1].

[0283] .sup.1H NMR (500 MHz, DMSO-d.sub.6) 10.49 (brs, 1H), 7.68 (brs, 1H), 7.48 (m, 1H), 7.40-7.35 (m, 2H), 7.25 (m, 1H), 5.10 (m, 1H), 4.81 (m, 1H), 1.42 (d, 3H), 1.29 (d, 6H).

Example 6

(S)-6-((1-(5-Cyclopropyl-2-fluorophenyl)ethyl)amino)-3-isopropyl-1,3,5-triazine-2,4(1H,3H)-dione 6

[0284] ##STR00105##

Step 1

5-Cyclopropyl-2-fluorobenzaldehyde 6b

[0285] 5-Bromo-2-fluorobenzaldehyde 6a (10.00 g, 49.26 mmol, Bide Pharmatech Ltd.) and cyclopropylboronic acid (6.35 g, 73.93 mmol, Accela ChemBio Inc.) were dissolved in 1,4-dioxane (100 mL) and water (5 mL), and [1,1-bis(diphenylphosphino)ferrocene]dichloropalladium complexed with dichloromethane (2.42 g, 2.96 mmol) and tripotassium phosphate (27.15 g, 128.07 mmol) were added. The system was purged with nitrogen three times, and the mixture was stirred at 100 C. for 16 h, filtered, and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography with eluent system A to give the title product 6b (7.00 g, yield: 86.6%).

[0286] .sup.1H NMR (500 MHz, CDCl.sub.3) 10.32 (s, 1H), 7.52 (m, 1H), 7.31 (m, 1H), 7.05 (m, 1H), 1.91 (m, 1H), 1.01-0.97 (m, 2H), 0.70-0.66 (m, 2H).

Step 2

(R)N-(5-Cyclopropyl-2-fluorobenzylidene)-2-methylpropane-2-sulfinamide 6c

[0287] Compound 6b (1.00 g, 6.09 mmol) and (R)-2-methylpropane-2-sulfinamide (738 mg, 6.09 mmol, Bide Pharmatech Ltd.) were dissolved in dichloromethane (20 mL), and cesium carbonate (2.38 g, 7.30 mmol) was added. The reaction was stirred for 16 h. The reaction mixture was filtered and concentrated under reduced pressure to give the crude title product 6c (1.62 g, yield: 99.5%). The crude product was directly used in the next step without being purified.

[0288] MS m/z (ESI): 268.0 [M+1].

Step 3

(R)N((S)-1-(5-Cyclopropyl-2-fluorophenyl)ethyl)-2-methylpropane-2-sulfinamide 6d

[0289] The crude compound 6c (1.62 g, 6.06 mmol) was dissolved in dichloromethane (10 mL), and the system was purged with nitrogen three times. The reaction was cooled to 60 C., and a 3 M solution of methylmagnesium bromide in 2-methyltetrahydrofuran (4.25 mL, 12.75 mmol, Shanghai Titan Scientific Co., Ltd.) was added dropwise. The reaction was stirred at room temperature for 5 h in a nitrogen atmosphere. A saturated ammonium chloride solution (10 mL) was added, and the organic phase was separated. The aqueous phase was extracted with ethyl acetate (40 mL2). The organic phases were combined, washed with a saturated sodium chloride solution (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography with eluent system B to give the title product 6d (1.70 g, yield: 99.0%).

[0290] MS m/z (ESI): 284.0 [M+1].

Step 4

(S)-1-(5-Cyclopropyl-2-fluorophenyl)ethylamine hydrochloride 6e

[0291] Compound 6d (300 mg, 1.06 mmol) was dissolved in ethanol (10 mL). The solution was cooled to 0 C., and thionyl chloride (335 mg, 2.82 mmol, Shanghai Hushi Chemical Co., Ltd.) was added dropwise. The reaction was stirred at room temperature for 1 h. The reaction mixture was concentrated under reduced pressure to give the crude title product 6e (228 mg, yield: 99.9%). The crude product was directly used in the next step without being purified.

[0292] MS m/z (ESI): 179.9 [M+1].

Step 5

(S)-6-((1-(5-Cyclopropyl-2-fluorophenyl)ethyl)amino)-3-isopropyl-1,3,5-triazine-2,4(1H,3H)-dione 6

[0293] Compound 1c (156 mg, 0.71 mmol) and compound 6e (190 mg, 0.88 mmo) were dissolved in N-methylpyrrolidone (8 mL), and triethylamine (71 mg, 0.71 mmol) was added. The mixture was stirred at 120 C. for 16 h and purified by high performance liquid chromatography (Boston Phlex C18 15030 mm, 5 m; elution system: water (10 mmol of ammonium bicarbonate), acetonitrile, 20-95% acetonitrile; 20 min of gradient elution; flow rate: 30 mL/min) to give the title product 6 (50 mg, yield: 21.3%).

[0294] MS m/z (ESI): 333.0 [M+1].

[0295] .sup.1H NMR (500 MHz, DMSO-d.sub.6) 10.48 (brs, R H), 7.20 (brs, 1H), 7.12 (m, 1H), 7.06 (m, 1H), 6.98 (m, 1H), 5.22 (m, 1H), 4.80 (m, 1H), 1.92 (m, 1H), 1.40 (d, 31H), 1.29 (d, 6H), 0.95-0.91 (m, 2H), 0.65-0.62 (in, 2H).

Example 7

(S)-6-((1-(Bicyclo[4.2.0]octan-1(6),2,4-trien-3-yl)ethyl)amino)-3-isopropyl-1,3,5-triazine-2,4(1H,3H)-dione 7

[0296] ##STR00106##

Step 1

(R)N-(Bicyclo[4.2.0]oct-1(6),2,4-trien-3-ylmethylene)-2-methylpropane-2-sulfinamide 7b

[0297] Bicyclo[4.2.0]octa-1(6),2,4-triene-3-carbaldehyde 7a (2.9 g, 22.0 mmol, prepared by the method disclosed in step 1 on pp. 512-513 of the specification in the patent application WO2019023147A1) and (R)-2-methylpropane-2-sulfinamide (2.8 g, 23.0 mmol) were dissolved in dichloromethane (40 mL), and cesium carbonate (8.6 g, 26.4 mmol) was added. The reaction was stirred for 16 h. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to give the crude title product 7b (5.7 g). The crude product was directly used in the next step without being purified.

[0298] MS m/z (ESI): 236.1 [M+1].

Step 2

(R)N((S)-1-(Bicyclo[4.2.0]oct-1(6),2,4-trien-3-yl)ethyl)-2-methylpropane-2-sulfinamide 7c

[0299] To a solution of the crude compound 7b (2.8 g, 12.1 mmol) in anhydrous dichloromethane (80 mL), a 3 M solution of methylmagnesium bromide in methyltetrahydrofuran (8.1 mL, 24.2 mmol, Shanghai Titan Scientific Co., Ltd.) was added dropwise at 50 C. The mixture was reacted at room temperature for 16 h in a nitrogen atmosphere. A saturated aqueous ammonium chloride solution (50 mL) was added, and extraction was performed with dichloromethane (50 mL2). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography with eluent system A to give the title product 7c (2.0 g, yield: 66.2%).

[0300] MS m/z (ESI): 252.1 [M+1].

Step 3

(S)-1-(Bicyclo[4.2.0]octan-1(6),2,4-trien-3-yl)ethylamine hydrochloride 7d

[0301] Compound 7c (500.0 mg, 2.0 mmol) was dissolved in methanol (3 mL), and a 4 M solution of hydrogen chloride in 1,4-dioxane (2 mL) was added dropwise. The reaction was stirred for 1 h. The reaction mixture was concentrated under reduced pressure to give the crude title product 7d (366.0 mg). The crude product was directly used in the next step without being purified.

[0302] MS m/z (ESI): 131.1 [M16].

Step 4

(S)-6-((1-(Bicyclo[4.2.0]octan-1(6),2,4-trien-3-yl)ethyl)amino)-3-isopropyl-1,3,5-triazine-2,4(1H,3H)-dione 7

[0303] Compound 1c (182 mg, 0.82 mmol) and compound 7d (182 mg, 0.99 mmo) were dissolved in N-methylpyrrolidone (6 mL), and triethylamine (83 mg, 0.82 mmol) was added. The mixture was stirred at 120 C. for 16 h and purified by high performance liquid chromatography (Boston Phlex C18 15030 mm, 5 m; elution system: water (10 mmol of ammonium bicarbonate), acetonitrile, 20-95% acetonitrile; 20 min of gradient elution; flow rate: 30 mL/min) to give the title product 7 (30 mg, yield: 12.1%).

[0304] MS m/z (ESI): 301.0 [M+1].

[0305] .sup.1H NMR (500 MHz, DMSO-d.sub.6) 10.46 (brs, 1H), 7.22 (brs, 1H), 7.15 (m, 1H), 7.10-7.01 (m, 2H), 4.96 (m, 1H), 4.80 (m, 1H), 3.14-3.08 (m, 4H), 1.39 (d, 3H), 1.29 (d, 6H).

Example 8

(S)-6-((1-(2,3-Dihydrobenzofuran-6-yl)ethyl)amino)-3-isopropyl-1,3,5-triazine-2,4(1H,3H)-dione 8

[0306] ##STR00107##

Step 1

(R)N-((2,3-dihydrobenzofuran-6-yl)methylene)-2-methylpropane-2-sulfinamide 8b

[0307] 2,3-Dihydrobenzofuran-6-carbaldehyde 8a (1.0 g, 6.8 mmol, Jiangsu Aikon Biopharmaceutical R&D Co., Ltd.) and (R)-2-methylpropane-2-sulfinamide (860.0 mg, 7.1 mmol, Shanghai Titan Scientific Co., Ltd.) were dissolved in dichloromethane (40 mL), and cesium carbonate (2.6 g, 8.1 mmol) was added. The reaction was stirred for 16 h. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to give the crude title product 8b (1.8 g). The crude product was directly used in the next step without being purified.

[0308] MS m/z (ESI): 252.0 [M+1].

Step 2

(R)N((S)-1-(2,3-Dihydrobenzofuran-6-yl)ethyl)-2-methylpropane-2-sulfinamide 8c

[0309] To a solution of the crude compound 8b (1.7 g, 6.8 mmol) in anhydrous dichloromethane (45 mL), a 3 M solution of methylmagnesium bromide in methyltetrahydrofuran (4.9 mL, 14.6 mmol) was added dropwise at 50 C. The mixture was reacted at room temperature for 16 h in a nitrogen atmosphere. A saturated aqueous ammonium chloride solution (30 mL) was added, and extraction was performed with dichloromethane (30 mL2). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography with eluent system A to give the title product 8c (1.6 g, yield: 90.6%).

[0310] MS m/z (ESI): 268.1 [M+1].

Step 3

(S)-1-(2,3-Dihydrobenzofuran-6-yl)ethylamine hydrochloride 8d

[0311] Compound 8c (534.0 mg, 2.0 mmol) was dissolved in methanol (3 mL), and a 4 M solution of hydrogen chloride in 1,4-dioxane (2 mL) was added dropwise. The reaction was stirred for 1 h. The reaction mixture was concentrated under reduced pressure to give the crude title product 8d (400.0 mg). The crude product was directly used in the next step without being purified.

[0312] MS m/z (ESI): 147.1 [M16].

Step 4

(S)-6-((1-(2,3-Dihydrobenzofuran-6-yl)ethyl)amino)-3-isopropyl-1,3,5-triazine-2,4(1H,3H)-dione 8

[0313] Compound 1c (168 mg, 0.76 mmol) and compound 8d (186 mg, 0.93 mmo) were dissolved in N-methylpyrrolidone (8 mL), and triethylamine (77 mg, 0.76 mmol) was added. The mixture was stirred at 120 C. for 16 h and purified by high performance liquid chromatography (Boston Phlex C18 15030 mm, 5 m; elution system: water (10 mmol of ammonium bicarbonate), acetonitrile, 20-95% acetonitrile; 20 min of gradient elution; flow rate: 30 mL/min) to give the title product 8 (15 mg, yield: 6.2%).

[0314] MS m/z (ESI): 317.0 [M+1].

[0315] .sup.1H NMR (500 MHz, DMSO-d.sub.6) 10.45 (brs, 1H), 7.21-7.16 (m, 2H), 6.80-6.74 (m, 2H), 4.95 (m, 1H), 4.80 (m, 1H), 4.52-4.49 (m, 2H), 3.14-3.11 (m, 2H), 1.39 (d, 3H), 1.29 (d, 6H).

Example 9

(S)-6-((l-(2,3-Dihydro-1H-inden-5-yl)ethyl)amino)-3-isopropyl-1,3,5-triazine-2,4(1H,3H)-dione 9

[0316] ##STR00108##

Step 1

(R)N-(1-(2,3-Dihydro-1H-inden-5-yl)ethylidene)-2-methylpropane-2-sulfinamide 9b

[0317] To a solution of 1-(2,3-dihydro-1H-inden-5-yl)ethan-1-one 9a (1.0 g, 6.3 mmol, TCI (Shanghai) Co., Ltd.) and (R)-2-methylpropane-2-sulfinamide (1.1 g, 8.8 mmol, Shanghai Titan Scientific Co., Ltd.) in anhydrous tetrahydrofuran (20 mL), a 1 M solution of chlorotriisopropoxytitanium in hexane (7.5 mL, 7.5 mmol, Shanghai Titan Scientific Co., Ltd.) was added. The reaction was stirred at 65 C. for 16 h. A saturated aqueous sodium bicarbonate solution (30 mL) was added, and extraction was performed with ethyl acetate (50 mL2). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography with eluent system A to give the title product 9b (570.0 mg, yield: 34.7%).

[0318] MS m/z (ESI): 264.0 [M+1].

Step 2

(R)N((S)-1-(2,3-Dihydro-1H-inden-5-yl)ethyl)-2-methylpropane-2-sulfinamide 9c

[0319] To compound 9b (570 mg, 2.2 mmol) in anhydrous tetrahydrofuran (10 mL), a 1 M solution of L-selectride in tetrahydrofuran (3.5 mL, 3.5 mmol, Shanghai Titan Scientific Co., Ltd.) was added dropwise at 78 C. The mixture was reacted at 0 C. for 1 h. A saturated aqueous ammonium chloride solution (20 mL) was added, and extraction was performed with ethyl acetate (30 mL2). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give the crude title product 9c (570 mg). The crude product was directly used in the next step without being purified.

[0320] MS m/z (ESI): 266.1 [M+1].

Step 3

(S)-1-(2,3-Dihydro-1H-inden-5-yl)ethylamine hydrochloride 9d

[0321] The crude compound 9c (570 mg, 2.2 mmol) was dissolved in methanol (3 mL), and a 4 M solution of hydrogen chloride in 1,4-dioxane (2 mL) was added dropwise. The reaction was stirred for 1 h. The reaction mixture was concentrated under reduced pressure to give the crude title product 9d (430.0 mg). The crude product was directly used in the next step without being purified.

[0322] MS m/z (ESI): 145.1 [M16].

Step 4

(S)-6-((1-(2,3-Dihydro-1H-inden-5-yl)ethyl)amino)-3-isopropyl-1,3,5-triazine-2,4(1H,3H)-dione 9

[0323] Compound 1c (170 mg, 0.77 mmol) and compound 9d (186 mg, 0.94 mmo) were dissolved in N-methylpyrrolidone (8 mL), and triethylamine (78 mg, 0.77 mmol) was added. The mixture was stirred at 120 C. for 16 h and purified by high performance liquid chromatography (Boston Phlex C18 15030 mm, 5 m; elution system: water (10 mmol of ammonium bicarbonate), acetonitrile, 20-95% acetonitrile; 20 min of gradient elution: flow rate: 30 mL/min) to give the title product 9 (30 mg, yield: 12.4%).

[0324] MS m/z (ESI): 315.0 [M+1].

[0325] .sup.1H NMR (500 MHz, DMSO-d.sub.6) 10.45 (brs, 1H), 7.20-7.17 (m, 3H), 7.08 (m, 1H), 4.98 (m, 1H), 4.80 (m, 1H), 2.85-2.80 (m, 4H), 2.03-1.97 (m, 2H), 1.39 (d, 3H), 1.29 (d, 6H).

Example 10

[0326] (S)-3-Isopropyl-6-((1-(5,6,7,8-tetrahydronaphthalen-2-yl)ethyl)amino)-1,3,5-triazine-2,4(1H,3H)-dione 10

##STR00109##

Step 1

(R)-2-Methyl-N-(1-(5,6,7,8-tetrahydronaphthalen-2-yl)ethylidene)propane-2-sulfinamide 10b

[0327] To a solution of 1-(5,6,7,8-tetrahydronaphthalen-2-yl)ethan-1-one 10a (2.0 g, 11.5 mmol, Alfa Aesar (Tianjin) Chemical Co., Ltd.) and (R)-2-methylpropane-2-sulfinamide (2.1 g, 17.3 mmol, Shanghai Titan Scientific Co., Ltd.) in anhydrous tetrahydrofuran (20 mL), tetraethyl titanate (4.0 g, 17.3 mmol, Energy Chemical) was added. The reaction was stirred at 65 C. for 16 h. A saturated aqueous sodium bicarbonate solution (60 mL) was added, and extraction was performed with ethyl acetate (100 mL2). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography with eluent system A to give the title product 10b (2.6 g, yield: 81.6%).

[0328] MS m/z (ESI): 278.0 [M+1].

Step 2

(R)-2-Methyl-N((S)-1-(5,6,7,8-tetrahydronaphthalen-2-yl)ethyl)propane-2-sulfinamide 10c

[0329] Compound 10b (350 mg, 1.26 mmol) was dissolved in tetrahydrofuran (10 mL), and the system was purged with nitrogen three times. The reaction was cooled to 78 C., and a 1 M solution of L-selectride in tetrahydrofuran (2.02 mL, 2.02 mmol, Shanghai Titan Scientific Co., Ltd.) was added dropwise. The reaction was stirred at 0 C. for 1 h in a nitrogen atmosphere. A saturated ammonium chloride solution (10 mL) was added, and the organic phase was separated. The aqueous phase was extracted with ethyl acetate (40 mL2). The organic phases were combined, washed with a saturated sodium chloride solution (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography with eluent system B to give the title product 10c (340 mg, yield: 96.4%).

[0330] MS m/z (ESI): 280.0 [M+1].

Step 3

(S)-1-(5,6,7,8-Tetrahydronaphthalen-2-yl)ethylamine hydrochloride 10d

[0331] Compound 10c (340 mg, 1.22 mmol) was dissolved in ethanol (10 mL). The solution was cooled to 0 C., and thionyl chloride (290 mg, 2.43 mmol, Shanghai Hushi Chemical Co., Ltd.) was added dropwise. The reaction was stirred at room temperature for 1 h. The reaction mixture was concentrated under reduced pressure to give the crude title product 10d (257 mg, yield: 99.8%). The crude product was directly used in the next step without being purified.

[0332] MS m/z (ESI): 158.9 [M16].

Step 4

(S)-3-Isopropyl-6-((1-(5,6,7,8-tetrahydronaphthalen-2-yl)ethyl)amino)-1,3,5-triazine-2,4(1H,3H)-dione 10

[0333] Compound 1c (271 mg, 1.23 mmol) and compound 10d (257 mg, 1.47 mmo) were dissolved in N-methylpyrrolidone (8 mL), and triethylamine (124 mg, 1.23 mmol) was added. The mixture was stirred at 120 C. for 16 h and purified by high performance liquid chromatography (Boston Phlex C18 15030 mm, 5 m; elution system: water (10 mmol of ammonium bicarbonate), acetonitrile, 20-95% acetonitrile; 20 min of gradient elution; flow rate: 30 mL/min) to give the title product 10 (40 mg, yield: 9.9%).

[0334] MS m/z (ESI): 329.0 [M+1].

[0335] .sup.1H NMR (500 MHz, DMSO-d.sub.6) 10.48 (brs, 1H), 7.23 (brs, 1H), 7.04-6.99 (m, 3H), 4.94 (m, 1H), 4.80 (m, 1H), 2.71-2.63 (m, 4H), 1.75-1.69 (m, 4H), 1.37 (d, 3H), 1.29 (d, 6H).

Example 11

(S)-6-((1-(5-Fluoro-2,3-dihydrobenzofuran-6-yl)ethyl)amino)-3-isopropyl-1,3,5-triazine-2,4(1H,3H)-dione 1l

[0336] ##STR00110## ##STR00111##

Step 1

Methyl 5-fluorobenzofuran-6-carboxylate 11b

[0337] 6-Bromo-5-fluorobenzofuran 11a (3.20 g, 14.88 mmol, prepared by the method of synthesizing intermediate A1.2b on page 36 of the specification in the patent application WO2017219948A1) was dissolved in methanol (50 mL), and [1,1-bis(diphenylphosphino)ferrocene]dichloropalladium complexed with dichloromethane (1.26 g, 1.49 mmol) and N,N-diisopropylethylamine (3.01 g, 29.75 mmol) were added. The system was purged with carbon monoxide gas three times, and the reaction was stirred at 70 C. for 40 h. The reaction mixture was filtered and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography with eluent system A to give the title product 11b (1.50 g, yield: 51.9%).

[0338] MS m/z (ESI): 194.8 [M+1].

Step 2

Methyl 5-fluoro-2,3-dihydrobenzofuran-6-carboxylate 11c

[0339] Compound 11b (1.50 g, 7.73 mmol) was dissolved in methanol (50 mL), and 10% palladium on carbon hydrogenation catalyst (wet) was added. The system was purged with hydrogen three times, and the reaction was stirred for 16 h. The reaction mixture was filtered and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography with eluent system A to give the title product 11c (1.37 g, yield: 90.4%).

[0340] MS m/z (ESI): 196.8 [M+1].

Step 3

(5-Fluoro-2,3-dihydrobenzofuran-6-yl)methanol 11d

[0341] Compound 11c (1.37 g, 7.0 mmol) was dissolved in a tetrahydrofuran (30 mL) solution, and a 2 M solution of lithium borohydride in tetrahydrofuran (34.9 mL, 69.8 mmol) was added dropwise. The reaction was stirred at room temperature for 16 h. The reaction was quenched by addition of methanol (5 mL) in an ice bath, and the reaction mixture was adjusted to pH 6 with 1 M hydrochloric acid and extracted with ethyl acetate (30 mL2). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography with eluent system A to give the title product 11d (1.05 g, yield: 89.4%).

[0342] MS m/z (ESI): 190.0 [M+22].

Step 4

5-Fluoro-2,3-dihydrobenzofuran-6-carbaldehyde 11e

[0343] Compound 11d (1.05 g, 6.24 mmol) was dissolved in a dichloromethane (20 mL) solution, and Dess-Martin oxidant (3.97 g, 9.36 mmol) was added. The mixture was reacted at room temperature for 2 h. The reaction was quenched by addition of saturated sodium thiosulfate (20 mL) and saturated sodium bicarbonate (20 mL) in an ice bath, and extraction was performed with dichloromethane (30 mL2). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography with eluent system A to give the title product 11e (1.03 g, yield: 99.3%).

[0344] MS m/z (ESI): 167.0 [M+1].

Step 5

(R)N-((5-Fluoro-2,3-dihydrobenzofuran-6-yl)methylene)-2-methylpropane-2-sulfinamide 11f

[0345] Compound 11e (1.30 g, 7.82 mmol) and (R)-2-methylpropane-2-sulfinamide (1.42 g, 11.72 mmol, Bide Pharmatech Ltd.) were dissolved in dichloromethane (20 mL), and cesium carbonate (1.58 g, 14.09 mmol) was added. The reaction was stirred for 16 h. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to give the crude title product 11f (2.80 g). The crude product was directly used in the next step without being purified.

[0346] MS m/z (ESI): 270.0 [M+1].

Step 6

(R)N((S)-1-(5-Fluoro-2,3-dihydrobenzofuran-6-yl)ethyl)-2-methylpropane-2-sulfinamide 11g

[0347] To a solution of the crude compound 11f (2.80 g, 10.40 mmol) in anhydrous dichloromethane (45 mL), a 3 M solution of methylmagnesium bromide in methyltetrahydrofuran (6.93 mL, 20.79 mmol) was added dropwise at 50 C. The mixture was reacted at room temperature for 2 h in a nitrogen atmosphere. A saturated aqueous ammonium chloride solution (30 mL) was added, and extraction was performed with dichloromethane (30 mL2). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by high performance liquid chromatography (Boston Phlex C18 15030 mm, 5 m; elution system: water (10 mmol of ammonium bicarbonate), acetonitrile, 20-95% acetonitrile; 20 min of gradient elution; flow rate: 30 mL/min) to give the title product 11g (1.50 g, yield: 50.56%).

[0348] MS m/z (ESI): 286.0 [M+1].

Step 7

(S)-1-(5-Fluoro-2,3-dihydrobenzofuran-6-yl)ethylamine hydrochloride 11h

[0349] Compound 11g (300 mg, 1.05 mmol) was dissolved in ethanol (10 mL), and thionyl chloride (250 mg, 2.10 mmol) was added dropwise. The reaction was stirred for 1 h. The reaction mixture was concentrated under reduced pressure to give the crude title product 11h (228 mg). The crude product was directly used in the next step without being purified.

[0350] MS m/z (ESI): 164.9 [M16].

Step 8

(S)-6-((1-(5-Fluoro-2,3-dihydrobenzofuran-6-yl)ethyl)amino)-3-isopropyl-1,3,5-triazine-2,4(1H,3H)-dione 1l

[0351] Compound 1c (232 mg, 1.05 mmol) and compound 11h (228 mg, 1.05 mmo) were dissolved in N-methylpyrrolidone (6 mL), and triethylamine (106 mg, 1.05 mmol) was added. The mixture was stirred at 120 C. for 16 h and purified by high performance liquid chromatography (Boston Phlex C18 15030 mm, 5 m; elution system: water (10 mmol of ammonium bicarbonate), acetonitrile, 20-95% acetonitrile; 20 min of gradient elution; flow rate: 30 mL/min) to give the title product 1l (90 mg, yield: 25.7%).

[0352] MS m/z (ESI): 334.9 [M+1].

[0353] .sup.1H NMR (500 MHz, DMSO-d.sub.6) 10.52 (brs, 1H), 7.28 (brs, 1H), 7.08 (d, 1H), 6.75 (m, 1H), 5.14 (m, 1H), 4.79 (m, 1H), 4.53-4.50 (m, 2H), 3.16-3.13 (m, 2H), 1.38-1.37 (d, 3H), 1.30-1.28 (d, 6H).

Example 12

(S)-6-((1-(2-Fluoro-5-((6-methylpyridin-3-yl)oxy)phenyl)ethyl)amino)-3-isopropyl-1,3,5-triazine-2,4(1H,3H)-dione 12

[0354] ##STR00112##

Step 1

2-Fluoro-5-((6-methylpyridin-3-yl)oxy)benzaldehyde 12a

[0355] Compound 1d (61.55 g, 363.25 mmol) and 6-methylpyridin-3-ol (20.00 g, 183.28 mmol, Bide Pharmatech Ltd.) were dissolved in dichloromethane (800 mL), and triethylamine (37.02 g, 366.53 mmol), pyridine (28.99 g, 366.50 mmol) and anhydrous copper acetate (66.58 g, 366.57 mmol, Bide Pharmatech Ltd.) were added. The reaction was stirred for 24 h. The reaction mixture was filtered through celite, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography with eluent system A to give the title product 12a (8.00 g, yield: 18.9%).

[0356] MS m/z (ESI): 231.9 [M+1].

Step 2

(R)N-(2-Fluoro-5-((6-methylpyridin-3-yl)oxy)benzylidene)-2-methylpropane-2-sulfinamide 12b

[0357] Compound 12a (8.00 g, 34.60 mmol) and (R)-2-methylpropane-2-sulfinamide (6.29 g, 51.90 mmol) were dissolved in dichloromethane (200 mL), and cesium carbonate (20.30 g, 62.27 mmol) was added. The reaction was stirred for 16 h. The reaction mixture was filtered and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography with eluent system A to give the title product 12b (8.20 g, yield: 70.9%).

[0358] MS m/z (ESI): 334.9 [M+1].

Step 3

(R)N((S)-1-(2-Fluoro-5-((6-methylpyridin-3-yl)oxy)phenyl)ethyl)-2-methylpropane-2-sulfinamide 12c

[0359] Compound 12b (8.20 g, 24.52 mmol) was dissolved in dichloromethane (100 mL). The reaction was cooled to 60 C. in a nitrogen atmosphere, and a 3 M solution of methylmagnesium bromide in 2-methyltetrahydrofuran (17.2 mL, 51.49 mmol, Shanghai Titan Scientific Co., Ltd.) was added dropwise. The reaction was warmed to room temperature and stirred for 5 h. A saturated aqueous ammonium chloride solution (100 mL) was added at 0 C., and the organic phase was separated. The aqueous phase was extracted with ethyl acetate (80 mL2). The organic phases were combined, washed with a saturated sodium chloride solution (100 mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by high performance liquid chromatography (Boston Phlex C18 15030 mm, 5 m; elution system: water (10 mmol of ammonium bicarbonate), acetonitrile, 20-95% acetonitrile; 20 min of gradient elution; flow rate: 30 mL/min) to give the title product 12c (2.10 g, yield: 24.4%).

[0360] MS m/z (ESI): 349.0[M1].

Step 4

(S)-1-(2-Fluoro-5-((6-methylpyridin-3-yl)oxy)phenyl)ethylamine hydrochloride 12d

[0361] Compound 12c (1.10 g, 3.14 mmol) was dissolved in ethanol (12 mL). The solution was cooled to 0 C., and thionyl chloride (747 mg, 6.28 mmol) was added dropwise. The reaction was stirred at room temperature for 1 h. The reaction mixture was concentrated under reduced pressure to give the crude title product 12d (887 mg, yield: 99.9%). The crude product was directly used in the next step without being purified.

[0362] MS m/z (ESI): 247.0 [M+1].

Step 5

(S)-6-((1-(2-Fluoro-5-((6-methylpyridin-3-yl)oxy)phenyl)ethyl)amino)-3-isopropyl-1,3,5-triazine-2,4(1H,3H)-dione 12

[0363] Compound 1c (694 mg, 3.14 mmol) and compound 12d (887 mg, 3.14 mmo) were dissolved in N-methylpyrrolidone (12 mL), and triethylamine (317 mg, 3.13 mmol) was added. The mixture was stirred at 120 C. for 16 h and purified by high performance liquid chromatography (Boston Phlex C18 15030 mm, 5 m; elution system: water (10 mmol of ammonium bicarbonate), acetonitrile, 20-95% acetonitrile; 20 min of gradient elution; flow rate: 30 mL/min) to give the title product 12 (480 mg, yield: 38.3%). MS m/z (ESI): 399.9 [M+1].

[0364] .sup.1H NMR (500 MHz, DMSO-d.sub.6) 10.53 (brs, 1H), 8.22 (d, 1H), 7.34-7.21 (m, 4H), 7.12 (m, 1H), 6.95 (m, 1H), 5.22 (m, 1H), 4.79 (m, 1H), 2.45 (s, 3H), 1.41 (d, 3H), 1.29 (d, 6H).

Example 13

(S)-6-((1-(2-Fluoro-5-methylphenyl)ethyl)amino)-3-isopropyl-1,3,5-triazine-2,4(1H,3H)-dione 13

[0365] ##STR00113##

Step 1

(R)N((S)-1-(2-Fluoro-5-methylphenyl)ethyl)-2-methylpropane-2-sulfinamide 13b

[0366] (R)N-(2-Fluoro-5-methylbenzylidene)-2-methylpropane-2-sulfinamide 13a (6.90 g, 28.59 mmol, prepared by the method of synthesizing intermediate 3B on page 56 of the specification in the patent application WO2020092208A1) was dissolved in dichloromethane (100 mL), and the system was purged with nitrogen three times. The reaction was cooled to 60 C., and a 3 M solution of methylmagnesium bromide in 2-methyltetrahydrofuran (19.1 mL, 57.18 mmol) was added dropwise. The reaction was stirred at room temperature for 2 h in a nitrogen atmosphere. A saturated ammonium chloride solution (100 mL) was added, and the organic phase was separated. The aqueous phase was extracted with ethyl acetate (80 mL2). The organic phases were combined, washed with a saturated sodium chloride solution (100 mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by high performance liquid chromatography (Boston Phlex C18 15030 mm, 5 m; elution system: water (10 mmol of ammonium bicarbonate), acetonitrile, 20-95% acetonitrile; 20 min of gradient elution; flow rate: 30 mL/min) to give the title product 13b (5.60 g, yield: 76.1%).

[0367] MS m/z (ESI): 258.0 [M+1].

Step 2

(S)-1-(2-Fluoro-5-methylphenyl)ethylamine hydrochloride 13c

[0368] Compound 13b (670 mg, 2.60 mmol) was dissolved in ethanol (10 mL). The solution was cooled to 0 C., and thionyl chloride (620 mg, 5.21 mmol) was added dropwise. The reaction was stirred at room temperature for 1 h. The reaction mixture was concentrated under reduced pressure to give the crude title product 13c (493 mg, yield: 99.9%). The crude product was directly used in the next step without being purified.

[0369] MS m/z (ESI): 153.9 [M+1].

Step 3

(S)-6-((1-(2-Fluoro-5-methylphenyl)ethyl)amino)-3-isopropyl-1,3,5-triazine-2,4(1H,3H)-dione 13

[0370] Compound 1c (712 mg, 3.22 mmol) and compound 13c (493 mg, 2.60 mmo) were dissolved in N-methylpyrrolidone (10 mL), and triethylamine (326 mg, 3.22 mmol) was added. The mixture was stirred at 120 C. for 16 h and purified by high performance liquid chromatography (Boston Phlex C18 15030 mm, 5 m; elution system: water (10 mmol of ammonium bicarbonate), acetonitrile, 20-95% acetonitrile; 20 min of gradient elution; flow rate: 30 mL/min) to give the title product 13 (400 mg, yield: 40.6%).

[0371] MS m/z (ESI): 306.9 [M+1].

[0372] .sup.1H NMR (500 MHz, DMSO-d.sub.6) 10.42 (brs, 1H), 7.22-7.05 (m, 4H), 5.21 (m, 1H), 4.79 (m, 1H), 2.28 (s, 3H), 1.40 (d, 3H), 1.29 (d, 6H).

Example 14

(S)-6-((1-(2-Fluoro-5-((6-methylpyridin-3-yl)oxy)phenyl)ethyl)amino)-3-(tetrahydro-2H-pyran-4-yl)-1,3,5-triazine-2,4(1H,3H)-dione 14

[0373] ##STR00114##

Step 1

4-Isocyanatotetrahydro-2H-pyran 14b

[0374] To bis(trichloromethyl)carbonate (11.9 g, 40.0 mmol, Shanghai Titan Scientific Co., Ltd.) in anhydrous dichloromethane (30 mL), a solution of tetrahydro-2H-pyran-4-amine 14a (10.0 g, 100.0 mmol, Accela ChemBio Inc.) and N,N-diisopropylethylamine (28.4 g, 220.0 mmol, Shanghai Titan Scientific Co., Ltd.) in anhydrous dichloromethane (120 mL) was slowly added dropwise at 15 C. The mixture was reacted at room temperature for 2 h. The reaction mixture was concentrated under reduced pressure to give the crude title product 14b. The crude product was directly used in the next step without being purified.

Step 2

6-(1H-Pyrazol-1-yl)-3-(tetrahydro-2H-pyran-4-yl)-1,3,5-triazine-2,4(1H,3H)-dione 14c

[0375] To a solution of compound 1b (10.5 g, 95.2 mmol) and the crude compound 14b in anhydrous N,N-dimethylacetamide (120 mL), 1,8-diazabicyclo[5.4.0]undec-7-ene (15.2 g, 100.0 mmol, Accela ChemBio Inc.) was slowly added dropwise at 10 C. The reaction was stirred at 0 C. for 1h. Subsequently, carbonyldiimidazole (23.2 g, 142.8 mmol, Bide Pharmatech Ltd.) and 1,8-diazabicyclo[5.4.0]undec-7-ene (21.7 g, 142.8 mmol, Accela ChemBio Inc.) were added to the reaction at 0 C. The reaction was stirred at 60 C. for 16 h. The reaction mixture was concentrated under reduced pressure, and dichloromethane was added to the resulting residue. The mixture was stirred and filtered, and the filter cake was collected and dried under reduced pressure to give the title product 14c (16.6 g, yield over two steps: 63.4%).

[0376] MS m/z (ESI): 264.1 [M+1].

Step 3

(S)-6-((1-(2-Fluoro-5-((6-methylpyridin-3-yl)oxy)phenyl)ethyl)amino)-3-(tetrahydro-2H-pyran-4-yl)-1,3,5-triazine-2,4(1H,3H)-dione 14

[0377] Compound 14c (148.3 mg, 0.7 mmol) and compound 12d (198.0 mg, 0.7 mmo) were dissolved in N-methylpyrrolidone (2 mL), and N,N-diisopropylethylamine (452.6 mg, 3.5 mmol) was added. The mixture was reacted in a microwave reactor at 140 C. for 2 h and purified by high performance liquid chromatography (Sharpsil-T Prep C18 5 m 30150 mm; elution system: water (10 mM ammonium bicarbonate), acetonitrile, acetonitrile was increased from 16% (v/v) to 36% (v/v) over 20 min, 30 mL/min; detection wavelengths: 214 & 254 nm) to give the title product 14 (95.0 mg, yield: 30.7%).

[0378] MS m/z (ESI): 442.1 [M+1].

[0379] .sup.1H NMR (500 MHz, DMSO-d.sub.6) 8.20 (s, 1H), 7.79 (brs, 1H), 7.30 (m, 1H), 7.23-7.19 (m, 2H), 7.14 (m, 1H), 6.92 (m, 1H), 5.26 (m, 1H), 4.66 (m, 1H), 3.87-3.83 (m, 2H), 3.32-3.25 (m, 3H), 2.51-2.44 (m, 3H), 2.41 (s, 31H), 1.42-1.33 (m, 4H).

Example 15

(S)-6-((1-(3-((6-Methylpyridin-3-yl)oxy)phenyl)ethyl)amino)-3-(tetrahydro-2H-pyran-4-yl)-1,3,5-triazine-2,4(1H,3H)-dione 15

[0380] ##STR00115##

[0381] Compound 14c (337.0 mg, 1.3 mmol) and compound 4e (338.9 mg, 0.7 mmo) were dissolved in N-methylpyrrolidone (4 mL), and N,N-diisopropylethylamine (827.3 mg, 6.4 mmol) was added. The mixture was reacted in a microwave reactor at 140 C. for 2 h and purified by high performance liquid chromatography (Sharpsil-T Prep C18 5 m 30150 mm; elution system: water (10 mM ammonium bicarbonate), acetonitrile, acetonitrile was increased from 16% (v/v) to 36% (v/v) over 20 min, 30 mL/min; detection wavelengths: 214 & 254 nm) to give the title product 15 (166.0 mg, yield: 30.6%).

[0382] MS m/z(ESI):424.1 [M+1].

[0383] .sup.1H NMR (500 MHz, DMSO-d.sub.6) 8.24 (d, 1H), 7.66 (brs, 1H), 7.36-7.33 (m, 2H), 7.26 (d, 1H), 7.14 (d 1H), 7.05 (s, 1H), 6.85 (d 1H), 5.04 (m, 1H), 4.66 (m, 1H), 3.89-3.85 (m, 2H), 3.31-3.27 (m, 3H), 2.51-2.47 (m, 3H), 2.45 (s, 3H), 1.41-1.38 (m, 4H).

Example 16

(S)-6-((1-(2-Fluoro-5-methylphenyl)ethyl)amino)-3-(tetrahydro-2H-pyran-4-yl)-1,3,5-triazine-2,4(1H,3H)-dione 16

[0384] ##STR00116##

[0385] Compound 14c (425 mg, 1.61 mmol) and compound 13c (297 mg, 1.94 mmo) were dissolved in 1,4-dioxane (10 mL), and the reaction was stirred at 120 C. for 16 h. The reaction mixture was concentrated under reduced pressure and purified by high performance liquid chromatography (Boston Phlex C18 15030 mm, 5 m; elution system: water (10 mmol of ammonium bicarbonate), acetonitrile, 20-95% acetonitrile; 20 min of gradient elution; flow rate: 30 mL/min) to give the title product 16 (310 mg, yield: 55.1%).

[0386] MS m/z (ESI): 349.0 [M+1].

[0387] .sup.1H NMR (500 MHz, DMSO-d.sub.6) 10.50 (brs, 1H), 7.28-7.05 (m, 4H), 5.22 (m, 1H), 4.64 (m, 1H), 3.90-3.86 (m, 2H), 3.33-3.27 (m, 2H), 2.54-2.42 (m, 2H), 2.28 (s, 3H), 1.45-1.39 (m, 5H).

Example 17

(S)-3-Cyclohexyl-6-((1-(2-fluoro-5-methylphenyl)ethyl)amino)-1,3,5-triazine-2,4(1H,3H)-dione 17

[0388] ##STR00117##

Step 1

3-Cyclohexyl-6-(1H-pyrazol-1-yl)-1,3,5-triazine-2,4(1H,3H)-dione 17b

[0389] Cyclohexyl isocyanate 17a (8.97 g, 71.66 mmol, Shanghai Titan Scientific Co., Ltd.) and compound 1b (10.00 g, 68.22 mmol) were dissolved in N,N-dimethylacetamide (50 mL). The reaction was cooled to 10 C., and 1,8-diazabicycloundec-7-ene (17.18 g, 68.22 mmol) was added dropwise over 5 min. The reaction was stirred in an ice bath for another 30 min. Subsequently, N,N-carbonyldiimidazole (14.73 g, 102.33 mmol) was added under ice bath. The reaction was cooled to 5 C., and 1,8-diazabicycloundec-7-ene (25.77 g, 102.33 mmol) was added dropwise over 10 min. The reaction was stirred in an ice bath for another 1h. 2 N hydrochloric acid (197 mL) was added dropwise at room temperature over 30 min. The mixture was filtered, and the filter cake was collected and dried in vacuo to give the title product 17b (16.00 g, yield: 89.8%).

[0390] MS m/z (ESI): 262.0 [M+1].

Step 2

(S)-3-Cyclohexyl-6-((1-(2-fluoro-5-methylphenyl)ethyl)amino)-1,3,5-triazine-2,4(1H,3H)-dione 17

[0391] Compound 17b (680 mg, 2.60 mmol) and compound 13c (392 mg, 2.56 mmo) were dissolved in 1,4-dioxane (10 mL), and the reaction was stirred at 120 C. for 16 h. The reaction mixture was concentrated to dryness and purified by high performance liquid chromatography (Boston Phlex C18 15030 mm, 5 m; elution system: water (10 mmol of ammonium bicarbonate), acetonitrile, 20-95% acetonitrile; 20 min of gradient elution; flow rate: 30 mL/min) to give the title product 17 (420 mg, yield: 46.6%).

[0392] MS m/z (ESI): 347.0 [M+1].

[0393] .sup.1H NMR (500 MHz, DMSO-d.sub.6) 10.45 (brs, 1H), 7.27-7.05 (m, 4H), 5.21 (m, 1H), 4.38 (m, 1H), 2.28 (s, 3H), 2.23-2.14 (m, 2H), 1.75-1.72 (m, 2H), 1.58 (m, 1H), 1.50-1.47 (m, 2H), 1.41-1.40 (d, 3H), 1.28-1.18 (m, 2H), 1.07 (m, 1H).

Biological Evaluation

[0394] The present disclosure is further described and explained below with reference to test examples, but these examples are not intended to limit the scope of the present disclosure.

Test Example 1: Inhibitory Effects of the Compounds Disclosed Herein on ATPase Activity of Myosin

[0395] The method below was used to determine the inhibitory effects of the compounds disclosed herein on the ATPase activity of myosin, and the experimental method is briefly described below: [0396] 1. Materials and instruments [0397] 1. Myocardial actin (Cytoskeleton, AD99) [0398] 2. Myosin motor protein S 1 fragment (Cytoskeleton, CS-MYSO3) [0399] 3. ATP (Sigma, A7699-IG) [0400] 4. UltraPure 1 M Tris-HCl buffer, pH 7.5 (Thermo, 15567027) [0401] 5. CytoPhos phosphate assay biochem kit (Cytoskeleton, BK054) [0402] 6. Magnesium chloride solution (Sigma, 68475-100ML-F) [0403] 7. Potassium chloride solution (Sigma, 60142-100ML-F) [0404] 8. EGTA (Sigma, E3889-100G) [0405] 9. 96-well plate (Corning, 3697) [0406] 10. U-bottom 96-well plate (Corning, 3795) [0407] 11. Microplate reader (BMG, PHERAstar) [0408] 12. Constant-temperature incubator (BOXUN, SPX-100B-Z)

II. Procedures

[0409] Myocardial actin (1.61 M) and myosin motor protein S 1 fragment (0.07 M) were mixed with different concentrations of small-molecule compounds (initial concentration of 100 M, serially diluted 3-fold to 9 concentrations), and the plate was incubated at 37 C. for 1 h. Then 120 M ATP was added and the plate was incubated at 37 C. for 2 h. Finally, the assay solution in the CytoPhos phosphate assay biochem kit was added to each well (70 L/well), and the plate was incubated at room temperature for 10 min. The OD readings at the wavelength of 650 nM were taken on a microplate reader. The Pi amount was calculated according to the standard curve. The data were processed using GraphPad software. An inhibition curve was plotted according to the compound concentrations and the corresponding inhibition rates, and the concentration at which the inhibition rate was 50%, i.e., the IC.sub.50 value, was calculated. The experimental results are detailed in Table 1.

TABLE-US-00002 TABLE 1 The inhibitory activity of the compounds disclosed herein against myosin ATPase Example No. IC.sub.50(M) 1 2.41 2 2.55 3 5.11 4 1.66 5 1.98 6 0.45 7 1.33 8 3.75 9 0.68 10 0.62 11 1.39 12 0.81 13 1.00 14 0.35 15 1.13 16 1.06

[0410] Conclusion. The compounds disclosed herein have good inhibitory effects on myosin ATPase.

Test Example 2: Pharmacokinetic Evaluation of the Compound Disclosed Herein in Beagles

1. Abstract

[0411] With beagles as test animals, the plasma concentrations of the test compounds were measured by LC/MS/MS at different time points after intragastric administration and intravenous injection. The pharmacokinetic performance of the compound disclosed herein was studied in beagles and its pharmacokinetic profile was evaluated.

2. Experimental Protocol

2.1. Experimental Compounds

[0412] The compound of Example 16, compound MYK-461

##STR00118##

Example 1 of WO2014205223A1).

2.2. Experimental Animals

[0413] Pharmacokinetics of the compound of Example 16 in beagles: 8 beagles, an equal number of males and females, divided into 2 groups of 4, provided by Shanghai Medicilon Inc. Pharmacokinetics of compound MYK-461 in beagles: 6 beagles, male, divided into 2 groups of 3, provided by Shanghai Medicilon Inc.

2.3. Preparation of Compound Solutions

[0414] A certain amount of the compound of Example 16 was measured out, and 5% DMSO, 30% PG, 30% PEG400 and 35% normal saline were added to prepare a clear solution.

[0415] A certain amount of compound MYK-461 was measured out, and 5% DMSO, 20% PG, 20% PEG400 and 55% normal saline were added to prepare a clear solution.

2.4. Administration

[0416] Beagles were fasted overnight and then given compounds by intragastric administration and intravenous injection at doses of 2 mg/kg and 0.5 mg/kg, respectively, and at volumes of 5 mL/kg and 2 mL/kg, respectively.

3. Procedures

[0417] 1.0-mL blood samples were collected from the jugular veins or forelimb veins of the animals in the intragastric administration group before administration and 0.25 h, 0.5 h, 1.0 h, 2.0 h, 4.0 h, 6.0 h, 8.0 h, 12.0 h and 24.0 h after administration, placed into EDTA-K2 anticoagulant tubes, and centrifuged at 10,000 rpm for 5 min (4 C.), and plasma was isolated within 1 h and stored at 80 C. before analysis. The blood collection to centrifugation process was performed under ice bath conditions. Three hours after administration, feeding was resumed.

[0418] Blood samples were collected from the animals in the intravenous injection group before administration and 5 min, 0.25 h, 0.5 h, 1.0 h, 2.0 h, 4.0 h, 8.0 h, 12.0 h and 24.0 h after administration and processed as in the intragastric administration group.

[0419] Determination of plasma concentrations in beagles after intragastric administration and intravenous injection of different concentrations of test compounds: 30 L of beagle plasma at each time point post-dose was mixed with an internal standard solution (internal standard for the compound of Example 16: 100 ng/mL warfarin; internal standard for compound MYK-461: 100 ng/mL tolbutamide) and 300 L of methanol; the mixture was vortexed for 1 min and centrifuged at 18,000 g for 7 min, and 200 L of the supernatant was transferred to a 96-well plate; 1 L of the supernatant was taken for LC/MS/MS analysis.

4. Pharmacokinetic Parameters

[0420]

TABLE-US-00003 TABLE 2 The pharmacokinetic parameters of the compound disclosed herein in beagles Area under Apparent Plasma curve Half- Clearance volume of concentration AUC life Residence CL/F distribution Route of Dose Cmax (ng/ T.sub.1/2 time (mL/min/ Vz/F Compound administration (mg/kg) (ng/mL) mL*h) (h) MRT(h) kg) (mL/kg) Example Intravenous 0.5 1077 716 5.6 7.5 10.4 5022 16 injection Intragastric 2 1537 3102 7.5 9.5 9.26 6050 administration MYK-461 Intravenous 0.5 215 1376 47.2 72.2 1.63 6308 injection Intragastric 2 1437 8719 38.9 59.2 1.48 3897 administration

[0421] Conclusion: The compound of Example 16 of the present disclosure demonstrated a good absorption profile in beagles. In addition, the compound of Example 16 of the present disclosure has a significantly shorter T.sub.1/2. Compound MYK-461 has a longer T.sub.1/2, so the accumulation is more serious clinically, and the clinical administration needs to be constantly adjusted, which increases the medication risk. Reducing T.sub.1/2 can clinically reduce or prevent drug accumulation in the body, favoring the determination of dosages for patients and avoiding the risks posed by accumulation. It is clear that the compound of Example 16 of the present disclosure has significant pharmacokinetic advantages over compound MYK-461.

Test Example 3: Pharmacokinetic Evaluation of the Compound Disclosed Herein in Cynomolgus Monkeys

I. Abstract

[0422] With cynomolgus monkeys as test animals, the plasma concentrations of the test compounds were measured by LC/MS/MS at different time points after intragastric administration and intravenous injection. The pharmacokinetic performance of the compound disclosed herein was studied in cynomolgus monkeys and its pharmacokinetic profile was evaluated.

2. Experimental Protocol

2.1. Experimental Compounds

[0423] The compound of Example 16 and compound MYK-461.

2.2. Experimental Animals

[0424] Pharmacokinetics of the compound of Example 16 in cynomolgus monkeys: 8 cynomolgus monkeys, an equal number of males and females, divided into 2 groups of 4, provided by Shanghai Medicilon Inc.

[0425] Pharmacokinetics of compound MYK-461 in cynomolgus monkeys: 6 cynomolgus monkeys, male, divided into 2 groups of 3, provided by Shanghai Medicilon Inc.

2.3. Preparation of Compound Solutions

[0426] A certain amount of the compound of Example 16 was measured out, and 5% DMSO, 30% PG, 30% PEG400 and 35% normal saline were added to prepare a clear solution.

[0427] A certain amount of compound MYK-461 was measured out, and 5% DMSO, 20% PG, 20% PEG400 and 55% normal saline were added to prepare a clear solution.

2.4. Administration

[0428] Cynomolgus monkeys were fasted overnight and then given compounds by intragastric administration and intravenous injection at doses of 2 mg/kg and 0.5 mg/kg, respectively, and at volumes of 5 mL/kg and 2 mL/kg, respectively.

3. Procedures

[0429] 1.0-mL blood samples were collected from the forelimb veins of the animals in the intragastric administration group before administration and 0.25 h, 0.5 h, 1h, 2 h, 4 h, 6 h, 8 h, 12 h and 24 h after administration, placed into EDTA-K2 anticoagulant tubes, and centrifuged at 10,000 rpm for 5 min (4 C.), and plasma was isolated within 1 h and stored at 80 C. before analysis. The blood collection to centrifugation process was performed under ice bath conditions. Three hours after administration, feeding was resumed, and ad libitum access to water was given.

[0430] Blood samples were collected from the animals in the intravenous injection group before administration and 5 min, 0.25 h, 0.5 h, 1.0 h, 2.0 h, 4.0 h, 8.0 h, 12.0 h and 24.0 h after administration and processed as in the intragastric administration group.

[0431] Determination of plasma concentrations in cynomolgus monkeys after intragastric administration and intravenous injection of different concentrations of test compounds: 20 L of cynomolgus monkey plasma at each time point post-dose was mixed with an internal standard solution (internal standard for the compound of Example 16: 10 ng/mL verapamil; internal standard for compound MYK-461: 100 ng/mL camptothecin) and 400 L of methanol; the mixture was vortexed for 1 min and centrifuged at 18,000 g for 7 min, and 200 L of the supernatant was transferred to a 96-well plate: 2 L of the supernatant was taken for LC/MS/MS analysis.

4. Pharmacokinetic Parameters

[0432]

TABLE-US-00004 TABLE 3 The pharmacokinetic parameters of the compound disclosed herein in cynomolgus monkeys Area under Apparent Plasma curve Half- Clearance volume of concentration AUC life Residence CL/F distribution Route of Dose Cmax (ng/ T.sub.1/2 time (mL/min/ Vz/F Compound administration (mg/kg) (ng/mL) mL*h) (h) MRT (h) kg) (mL/kg) Example Intravenous 0.5 1659 1022 5.6 6.8 7.46 3579 16 injection Intragastric 2 553 2405 10.3 12.5 11.8 10263 administration MYK-461 Intravenous 0.5 222 976 27.3 42.8 5.02 8203 injection Intragastric 2 563 3521 78.3 114 2.34 11369 administration

[0433] Conclusion: The compound of Example 16 of the present disclosure demonstrated a good absorption profile in cynomolgus monkeys. In addition, the compound of Example 16 of the present disclosure has a significantly shorter T.sub.1/2. Compound MYK-461 has a longer T.sub.1/2, so the accumulation is more serious clinically, and the clinical administration needs to be constantly adjusted, which increases the medication risk. Reducing T.sub.1/2 can clinically reduce or prevent drug accumulation in the body, favoring the determination of dosages for patients and avoiding the risks posed by accumulation. It is clear that the compound of Example 16 of the present disclosure has significant pharmacokinetic advantages over compound MYK-461.

Test Example 4: Toxicokinetic Evaluation of 14-Day Repeated Intragastric Administration of the Compound Disclosed Herein to SD Rats

1. Abstract

[0434] With SD rats as test animals, the plasma concentrations of the test compounds and the concentrations of the original forms of the compounds in the administration solutions were measured by LC/MS/MS at different time points after intragastric administration. The toxicokinetic performance of the compound disclosed herein was studied in SD rats and its toxicokinetic profile was evaluated.

2. Experimental Protocol

2.1 Test Compounds

[0435] The compound of Example 16 and compound MYK-461.

2.2. Experimental Animals

[0436] 24 SD rats, an equal number of males and females, evenly divided into 6 groups of 4, an equal number of males and females in each group, provided by Vital River Laboratory Animal Technology Co., Ltd.

2.3. Preparation of Compound Solutions

[0437] A certain amount of the compound of Example 16 was measured out, and 15% PEG400 and 85% (10% TPGS+1% HPMC K100LV) were added to prepare a pale yellow homogenous suspension.

[0438] A certain amount of compound MYK-461 was measured out, and 0.5% MC was added to prepare a colorless clear solution.

2.4. Administration

[0439] The compound of Example 16 was intragastrically administered at doses of 5 mg/kg, 15 mg/kg and 30 mg/kg, at a volume of 10 mL/kg.

[0440] Compound MYK-461 was administered at doses of 0.5 mg/kg, 1.5 mg/kg and 3 mg/kg, at a volume of 10 mL/kg.

3. Procedures

[0441] 0.2-mL blood samples were collected from the orbit 0.5 h, 1.0 h, 2.0 h, 4.0 h, 8.0 h and 24.0 h after administration on day 1, and before administration and 0.5 h, 1.0 h, 2.0 h, 4.0 h, 8.0 hi, 24.0 h after administration on day 7 and day 14, placed into EDTA-K2 anticoagulation tubes, and centrifuged at 10,000 rpm for 1 min (4 C.), and plasma was isolated within 1 h and stored at 20 C. before analysis. The blood collection to centrifugation process was performed under ice bath conditions. Two hours after administration, feeding was resumed.

[0442] Determination of plasma concentrations in SD rats after intragastric administration of different concentrations of test compounds: 20 L of SD rat plasma at each time point post-dose was mixed with 50 L of an internal standard solution (internal standard for the compound of Example 16: 100 ng/mL verapamil; internal standard for compound MYK-461: 100 ng(mL camptothecin) and 200 L of acetonitrile; the mixture was vortexed for 5 min and centrifuged at 3700 rpm for 10 min, and 1 L of the supernatant was taken for LC/MS/MS analysis.

4. Toxicokinetic Parameters

[0443]

TABLE-US-00005 TABLE 4 The toxicokinetic parameters of the compound disclosed herein in SD rats Apparent Plasma Area under Half- Clearance volume of concentration curve life Residence CL/F distribution Dose Cmax AUC T.sub.1/2 time (mL/min/ Vz/F Compound (mg/kg) Date (ng/mL) (ng/mL*h) (h) MRT(h) kg) (mL/kg) Example 5 Day 1 5735 30026 4.8 5.0 3.06 1265 16 Day 7 4473 28136 4.9 6.0 2.99 1279 Day 14 4858 29780 5.1 6.2 2.80 1239 15 Day 1 16125 80443 5.1 5.4 3.08 1369 Day 7 16800 70916 6.5 6.7 3.58 2129 Day 14 14125 82906 5.6 6.4 2.98 1445 30 Day I 18725 104640 5.6 6.4 4.72 2257 Day 7 21550 120552 5.9 6.7 4.14 2146 Day 14 20000 139432 5.6 6.9 3.52 1689 MYK-461 0.5 Day 1 40.2 491 13.3 19.0 12.9 13778 Day 7 51.7 888 14.7 21.3 7.46 8412 Day 14 89.3 1558 32.9 47.3 3.06 5861 1.5 Day 1 140 2577 19.1 28.2 6.39 9561 Day 7 269 5181 20.5 30.2 3.58 4802 Day 14 333 6320 26.2 38.5 2.57 5027 3.0 Day 1 297 5213 28.5 43.1 4.91 8931 Day 7 628 12549 190 275 1.97 4355 Day 14 767 15985 46.5 68.1 1.3 3802

[0444] Conclusion: The compound of Example 16 of the present disclosure did not show significant accumulation in SD rats after 14 days of repeated intragastric administration, while compound MYK-461 did, which increased the medication risk. It is clear that the compound of Example 16 of the present disclosure has significant toxicokinetic advantages over compound MYK-461.

Test Example 5: Identification of Reactive Metabolites of the Compound Disclosed Herein in Human Liver Microsomes

[0445] The identification of reactive metabolites of the compound disclosed herein in human liver microsomes was carried out using the experimental method below:

I. Materials and Instruments

[0446] 1. Phosphate-buffered saline (purchased from Shanghai Sangon) [0447] 2. NADPH (ACROS, A2646-71-1) [0448] 3. Human liver microsome (Corning Gentest, Cat No. 452161, Lot No. 905002) [0449] 4. Thermo UHPLC-Q-Exactive Orbitrap mass spectrometer (Thermo Fisher Scientific) [0450] 5. Acquity BEH C.sub.18 column, 2.1100 mm, 1.7 m (Waters, USA) [0451] 6. Positive control compound (diclofenac).

II. Experimental Compounds

[0452] The compound of Example 16 and compound MYK-461.

III. Procedures

[0453] 1. Preparation of test compound solutions: A proper amount of a test compound was precisely measured out and dissolved in a proper amount of DMSO. After the solution was well mixed, a 30 mM stock solution was obtained. A 10 mM stock solution was 10-fold diluted with 50% acetonitrile/water (v/v) to obtain a 3.0 mM working solution 1. The 3.0 mM working solution 1 was 10-fold diluted with PBS to obtain a 300 M working solution 2, and the solution was stored at 4 C. before use. [0454] 2. Preparation of phosphate-buffered saline: Proper amounts of K.sub.2HPO.sub.4 and KH.sub.2PO.sub.4 were measured out and dissolved in 4 L of pure water to prepare a 100 mM buffer, and the pH was then adjusted with phosphoric acid or sodium hydroxide to 7.4. [0455] 3. Preparation of liver microsome solutions: Proper amounts of liver microsome stock solutions of various species (20 mg/mL) were measured out and diluted with the 100 mM phosphate-buffered saline (pH 7.4) to prepare 1.43 mg/mL microsome solutions. [0456] 4. Preparation of NADPH cofactor solution: Proper amounts of NADPH and magnesium chloride were measured out and dissolved in a proper amount of the 100 mM phosphate-buffered saline (pH 7.4) so the concentrations of NADPH and magnesium chloride were 10 mM and 30 mM, respectively, and the solutions were stored for later use. [0457] 5. Preparation of glutathione (GSH) solution: A proper amount of glutathione was measured out and dissolved in a proper amount of the 100 mM phosphate-buffered saline (pH 7.4) so the concentration of GSH was 50 mM, and the solution was stored for later use. [0458] 6. The incubation system is shown below:

TABLE-US-00006 Liver microsomal protein concentration 1 mg/mL Species Human Test compound concentration 30 M NADPH concentration 1.0 mM MgCl.sub.2 concentration 3.0 mM GSH concentration 5 mM Incubation medium 100 mM PBS pH of system 7.4 Incubation temperature 37 C. Length of incubation 60 min Incubation volume 200 L Positive control Diclofenac (10 M)

[0459] To a 1.5 mL centrifuge tube, 20 L of the 300 M working solution 2 was precisely transferred and 140 L of a 1.43 mg/mL liver microsome solution was then added so the concentration of liver microsomal protein in the incubation system was 1 mg/mL. After 20 L of 10 mM NADPH solution and 20 L of 50 mM GSH solution were added, the tube was placed into a 37 C. constant-temperature incubator for shaking incubation, and a timer was set. After 60 min of incubation, the incubated sample was taken out of the incubator, and 1000 L of ice-cold acetonitrile solution was added. The reaction was stopped, and the sample was left to stand at room temperature for 10 min and then centrifuged at 12,000 rpm for 10 min. All the supernatant was transferred to a centrifuge tube and concentrated to dryness in vacuo at 37 C. The residue was reconstituted with 200 L of 25% acetonitrile/water solution and centrifuged at 12,000 rpm for 10 min. The supernatant was transferred to a 96-well plate, and 5 L of it was pipetted for LC/MS analysis. For a blank sample, 20 L of PBS was added instead of working solution 2. For an NCF sample, 20 L of PBS was added instead of GSH solution. Positive control diclofenac (10 M) was tested in the same way as the test compound. The collected data were processed and analyzed using Xcalibur software. According to the exact molecular weight, tandem mass spectrometry fragments were analyzed to see if the compound disclosed herein would be metabolically activated to produce reactive metabolites.

TABLE-US-00007 TABLE 5 Identification of reactive metabolites of the compound disclosed herein in human liver microsomes Compound Result Example 16 Negative MYK-461 Positive

[0460] Conclusion: In the experiment, no glutathione (GSH) conjugate associated with the compound of Example 16 of the present disclosure was detected, but a GSH conjugate associated with compound MYK-461 was detected. Therefore, the compound of Example 16 of the present disclosure is safer than compound MYK-461.