Patent Publication Number: US-2022213119-A1

Title: Thienoheterocyclic derivative, preparation method therefor and medical use thereof

Description:
TECHNICAL FIELD 
     The disclosure belongs to the field of medicine and relates to a thienyl fused heterocyclic derivative, preparation method therefor, and medical application thereof. Specifically, the present disclosure relates to a thienyl fused heterocyclic derivative represented by formula (I), a preparation method for preparing the same, a pharmaceutical composition comprising the same, and a use as an ERK inhibitor for treating ERK mediated diseases and disorders or inhibiting the MAPK-ERK signaling pathway. 
     BACKGROUND 
     The proliferation, differentiation, metabolism and apoptosis of normal cells are strictly regulated by cell signal transduction pathways in vivo. Mitogen activated protein kinase (MAPK) plays a very important role in signal transduction pathway. Extracellular signal regulated kinase (ERK) is a member of MAPK family. Through the RAS-RAF-MEK-ERK pathway, the exogenous stimulation signal is transmitted to the ERK, and the activated ERK is transferred into the nucleus to regulate the activity of transcription factors, thereby regulating the biological functions of cells such as proliferation, differentiation and apoptosis, or to participate in the regulation of cell morphology and the redistribution of cytoskeleton by phosphorylating cytoskeleton components in the cytoplasm. 
     RAS and RAF gene mutations cause the continuous activation of the MAPK-ERK signal pathway, which promotes the malignant transformation and abnormal proliferation of cells, and finally produces tumors (Roberts P J et al., Oncogene, 2007, 26(22), 3291-3310). The combination of a MEK inhibitor and a B-RAF inhibitor can further improve the effect of the B-RAF inhibitor in inhibiting tumor growth, and significantly improve the disease-free progression and overall survival rate of melanoma patients with BRAFV600E and v600k mutations (Frederick DT et al., Clinical Cancer Research, 2013, 19(5), 1225-1231). Although the combination of B-RAF/MEK inhibitors can inhibit tumor, their curative effect is short. Most patients will develop drug resistance within 2-18 months, and the tumor will further deteriorate. The mechanism of resistance to B-RAF/MEK inhibitors is very complex, which is mostly related to the reactivation of the ERK signaling pathway (Smalley I et al., Cancer Discovery, 2018, 8(2), 140-142). Thus, the development of new ERK inhibitors is effective not only for patients with mutations in MAPK signaling pathway, but also for patients with resistance to B-RAF/MEK inhibitors. 
     B-RAF/MEK inhibitors not only inhibit tumor growth, but also regulate the immune microenvironment of tumor. B-RAF/MEK inhibitor can enhance the expression of tumor specific antigen, improve the recognition and killing of tumors by antigen-specific T cells, and promote the migration and infiltration of immune cells. In animal models, after treatment with B-RAF/MEK inhibitors, the expression of PD-L1 in tumor tissues increased. When combined with antibodies to checkpoint molecules (such as PD-1 antibody and CTLA4 antibody), it showed a better effect of inhibiting tumor growth than B-Raf/MEK inhibitors used alone (Boni A et al., Cancer Research, 2010, 70(13), 5213-5219). Studies have shown that ERK inhibitosr are similar to B-RAF/MEK inhibitors, and their combination with checkpoint antibodys can regulate the tumor microenvironment, improve the function of cytotoxic T cells, and achieve the effect of inhibiting tumor growth. 
     At present, many compounds have been developed. Among them, BVD-523 developed by Biomed Valley Discoveries is in the clinical phase II, MK-8353 developed by Merck and Astex-029 developed by Astex are in the clinical phase I. Relevant patents include WO1999061440A1, WO2001056557A2, WO2001056993A2, WO2001057022A2, WO2002022601A1, WO2012118850A1, WO2013018733A1, WO2014179154A2, WO2015103133A1, WO2016192063A1, WO2017180817A1, and WO2018049127A1. 
     SUMMARY OF THE INVENTION 
     The object of the present disclosure is to provide a compound of formula (I): 
     
       
         
         
             
             
         
       
     
     or a stereoisomer, tautomer, mesomer, racemate, enantiomer, diastereomer thereof, or mixture thereof, or a pharmaceutically acceptable salt thereof, 
     wherein: 
     R 1  is selected from the group consisting of hydrogen atom, halogen, alkyl, alkoxy, haloalkyl, haloalkoxy, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl and heteroaryl; 
     R 2  is identical or different, and each is selected from the group consisting of hydrogen atom, halogen, alkyl, alkoxy, haloalkyl, haloalkoxy, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl and heteroaryl; 
     R 3  is selected from the group consisting of hydrogen atom, halogen, alkyl, alkoxy, haloalkyl, haloalkoxy, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl and heteroaryl, wherein the alkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl are each optionally further substituted by one or more substituents selected from the group consisting of alkyl, alkoxy, oxo, halogen, amino, cyano, nitro, hydroxy, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl; 
     R 4  is selected from the group consisting of hydrogen atom, halogen, alkyl, alkoxy, haloalkyl, haloalkoxy, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl and heterocyclyl; 
     R 5  is identical or different, and each is selected from the group consisting of hydrogen atom, halogen, alkyl, alkoxy, haloalkyl, haloalkoxy, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl and heteroaryl; 
     R 6  is selected from the group consisting of hydrogen atom, halogen, alkyl, alkoxy, haloalkyl, haloalkoxy, hydroxy and hydroxyalkyl; 
     L is a bond or alkylene, wherein the alkylene is optionally further substituted by one or more substituents selected from the group consisting of alkyl, alkoxy, oxo, halogen, amino, cyano, nitro, hydroxy, hydroxyalkyl, aminoalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl; 
     R 7  is selected from the group consisting of hydrogen atom, halogen, alkyl, alkoxy, haloalkyl, haloalkoxy, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl and heteroaryl, wherein the alkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl are each optionally further substituted by one or more substituents selected from the group consisting of alkyl, alkoxy, oxo, halogen, amino, cyano, nitro, hydroxy, hydroxyalkyl, haloalkyl, haloalkoxy and aminoalkyl; 
     n is 1, 2 or 3; and 
     m is 0, 1 or 2. 
     In some embodiments of the present disclosure, the compound of formula (I), or the stereoisomer, tautomer, mesomer, racemate, enantiomer, diastereomer thereof, or mixture thereof, or the pharmaceutically acceptable salt thereof, is a compound of formula (I-P1) or formula (I-P2): 
     
       
         
         
             
             
         
       
     
     or a stereoisomer, tautomer, mesomer, racemate, enantiomer, diastereomer thereof, or mixture thereof, or a pharmaceutically acceptable salt thereof, wherein: 
     L, R 1  to R 7 , m and n are as defined in the compound of formula (I). 
     In some embodiments of the present disclosure, in the compound of formula (I), or the stereoisomer, tautomer, mesomer, racemate, enantiomer, diastereomer thereof, or mixture thereof, or the pharmaceutically acceptable salt thereof, n is 1 or 2. 
     In some embodiments of the present disclosure, the compound of formula (I), or the stereoisomer, tautomer, mesomer, racemate, enantiomer, diastereomer thereof, or mixture thereof, or the pharmaceutically acceptable salt thereof, is a compound of formula (II): 
     
       
         
         
             
             
         
       
     
     or a stereoisomer, tautomer, mesomer, racemate, enantiomer, diastereomer thereof, or mixture thereof, or a pharmaceutically acceptable salt thereof, wherein: 
     z is 0, 1, 2, 3 or 4; and L, R 1  to R 7  and m are as defined in the compound of formula (I). 
     In some embodiments of the present disclosure, the compound of formula (I), or the stereoisomer, tautomer, mesomer, racemate, enantiomer, diastereomer thereof, or mixture thereof, or the pharmaceutically acceptable salt thereof, is a compound of formula (II-P1) or formula (II-P2): 
     
       
         
         
             
             
         
       
     
     or a stereoisomer, tautomer, mesomer, racemate, enantiomer, diastereomer thereof, or mixture thereof, or a pharmaceutically acceptable salt thereof, wherein: 
     L, R 1  to R 7 , m or z are as defined in the compound of formula (II). 
     In some embodiments of the present disclosure, in the compound of formula (I), or the stereoisomer, tautomer, mesomer, racemate, enantiomer, diastereomer thereof, or mixture thereof, or the pharmaceutically acceptable salt thereof, the L is an alkylene, wherein the alkylene is optionally further substituted by one or more substituents selected from the group consisting of alkyl, alkoxy, oxo, halogen, amino, cyano, nitro, hydroxy, hydroxyalkyl and aminoalkyl; preferably further substituted by hydroxyalkyl; and more preferably, L is —CH(R 8 )—, wherein R 8  is C 1-6  hydroxyalkyl. 
     In some embodiments of the present disclosure, the compound of formula (I), or the stereoisomer, tautomer, mesomer, racemate, enantiomer, diastereomer thereof, or mixture thereof, or the pharmaceutically acceptable salt thereof, is a compound of formula (III): 
     
       
         
         
             
             
         
       
     
     or a stereoisomer, tautomer, mesomer, racemate, enantiomer, diastereomer thereof, or mixture thereof, or a pharmaceutically acceptable salt thereof, wherein: 
     R 8  is selected from the group consisting of alkyl, alkoxy, oxo, halogen, amino, cyano, nitro, hydroxy, hydroxyalkyl, aminoalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl; 
     R 1  to R 3 , R 5 , R 7 , m and z are as defined in the compound of formula (II). 
     In some embodiments of the present disclosure, the compound of formula (I), or the stereoisomer, tautomer, mesomer, racemate, enantiomer, diastereomer thereof, or mixture thereof, or the pharmaceutically acceptable salt thereof, is a compound of formula (III-P 1) or formula (III-P2): 
     
       
         
         
             
             
         
       
     
     or a stereoisomer, tautomer, mesomer, racemate, enantiomer, diastereomer thereof, or mixture thereof, or a pharmaceutically acceptable salt thereof, wherein: 
     R 8  is selected from the group consisting of alkyl, alkoxy, oxo, halogen, amino, cyano, nitro, hydroxy, hydroxyalkyl, aminoalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl; 
     R 1  to R 3 , R 5 , R 7  and z are as defined in the compound of formula (III). 
     In some embodiments of the present disclosure, the compound of formula (I), or the stereoisomer, tautomer, mesomer, racemate, enantiomer, diastereomer thereof, or mixture thereof, or the pharmaceutically acceptable salt thereof, is a compound of formula (IV): 
     
       
         
         
             
             
         
       
     
     or a stereoisomer, tautomer, mesomer, racemate, enantiomer, diastereomer thereof, or mixture thereof, or a pharmaceutically acceptable salt thereof, wherein: 
     R 1  to R 8 , m and z are as defined in the compound of formula (III); and n is as defined in the compound of formula (I). 
     In some embodiments of the present disclosure, the compound of formula (I), or the stereoisomer, tautomer, mesomer, racemate, enantiomer, diastereomer thereof, or mixture thereof, or the pharmaceutically acceptable salt thereof, is a compound of formula (IV-P1) or formula (IV-P2): 
     
       
         
         
             
             
         
       
     
     or a stereoisomer, tautomer, mesomer, racemate, enantiomer, diastereomer thereof, or mixture thereof, or a pharmaceutically acceptable salt thereof, wherein: 
     R 1  to R 8 , m and z are as defined in the compound of formula (III); and n is as defined in the compound of formula (I). 
     In some embodiments of the present disclosure, in the compound of formula (I), or the stereoisomer, tautomer, mesomer, racemate, enantiomer, diastereomer thereof, or mixture thereof, or the pharmaceutically acceptable salt thereof, R 3  is selected from the group consisting of C 1-6  hydroxyalkyl, 3 to 6 membered heterocyclyl and 5 or 6 membered heteroaryl, wherein the C 1-6  hydroxyalkyl, 3 to 6 membered heterocyclyl and 5 or 6 membered heteroaryl are each optionally further substituted by one or more substituents selected from the group consisting of C 1-6  alkyl, C 1-6  alkoxy, halogen, amino, cyano, hydroxy, and C 1-6  hydroxyalkyl. 
     In some embodiments of the present disclosure, in the compound of formula (I), or the stereoisomer, tautomer, mesomer, racemate, enantiomer, diastereomer thereof, or mixture thereof, or the pharmaceutically acceptable salt thereof, R 3  is a heterocyclyl, wherein the heterocyclyl is optionally further substituted by one or more substituents selected from the group consisting of alkyl, alkoxy, oxo, halogen, amino, cyano, nitro, hydroxy and hydroxyalkyl. 
     In some embodiments of the present disclosure, in the compound of formula (I), or the stereoisomer, tautomer, mesomer, racemate, enantiomer, diastereomer thereof, or mixture thereof, or the pharmaceutically acceptable salt thereof, R 3  is a heteroaryl, wherein the heteroaryl is optionally further substituted by one or more substituents selected from the group consisting of alkyl, alkoxy, oxo, halogen, amino, cyano, nitro, hydroxy and hydroxyalkyl. 
     In some embodiments of the present disclosure, in the compound of formula (I), or the stereoisomer, tautomer, mesomer, racemate, enantiomer, diastereomer thereof, or mixture thereof, or the pharmaceutically acceptable salt thereof, R 7  is an aryl, wherein the aryl is optionally further substituted by one or more substituents selected from the group consisting of alkyl, alkoxy, oxo, halogen, amino, cyano, nitro, hydroxy and hydroxyalkyl; preferably, R 7  is a phenyl, wherein the phenyl is optionally further substituted by one or more substituents selected from the group consisting of C 1-6  alkyl, C 1-6  alkoxy, halogen, amino, cyano, hydroxy and C 1-6  hydroxyalkyl. 
     In some embodiments of the present disclosure, in the compound of formula (I), or the stereoisomer, tautomer, mesomer, racemate, enantiomer, diastereomer thereof, or mixture thereof, or the pharmaceutically acceptable salt thereof, R 8  is selected from the group consisting of alkyl, alkoxy, oxo, halogen, amino, cyano, nitro, hydroxy, hydroxyalkyl and aminoalkyl, preferably hydroxyalkyl or aminoalkyl; and more preferably C 1-6  hydroxyalkyl. 
     In some embodiments of the present disclosure, in the compound of formula (I), or the stereoisomer, tautomer, mesomer, racemate, enantiomer, diastereomer thereof, or mixture thereof, or the pharmaceutically acceptable salt thereof, R 1  is selected from the group consisting of hydrogen atom, halogen, alkyl, alkoxy, haloalkyl, haloalkoxy, hydroxy and hydroxyalkyl, preferably hydrogen atom, halogen or alkyl, more preferably hydrogen atom or alkyl; and most preferably hydrogen atom or C 1-6  alkyl. 
     In some embodiments of the present disclosure, in the compound of formula (I), or the stereoisomer, tautomer, mesomer, racemate, enantiomer, diastereomer thereof, or mixture thereof, or the pharmaceutically acceptable salt thereof, R 1  is an alkyl, and R 4  is a hydrogen atom; preferably, R 1  is a C 1-6  alkyl, and R 4  is a hydrogen atom. 
     In some embodiments of the present disclosure, in the compound of formula (I), or the stereoisomer, tautomer, mesomer, racemate, enantiomer, diastereomer thereof, or mixture thereof, or the pharmaceutically acceptable salt thereof, R 2  is selected from the group consisting of hydrogen atom, halogen, alkyl, alkoxy, haloalkyl, haloalkoxy, hydroxy and hydroxyalkyl, preferably hydrogen atom, halogen or alkyl; more preferably hydrogen atom, halogen or C 1-6  alkyl; and most preferably halogen. 
     In some embodiments of the present disclosure, in the compound of formula (I), or the stereoisomer, tautomer, mesomer, racemate, enantiomer, diastereomer thereof, or mixture thereof, or the pharmaceutically acceptable salt thereof, R 5  is identical or different, and each is selected from the group consisting of hydrogen atom, halogen, alkyl, alkoxy, haloalkyl, haloalkoxy, hydroxy and hydroxyalkyl; preferably hydrogen atom or alkyl; and more preferably hydrogen atom or C 1-6  alkyl. 
     In some embodiments of the present disclosure, in the compound of formula (I), or the stereoisomer, tautomer, mesomer, racemate, enantiomer, diastereomer thereof, or mixture thereof, or the pharmaceutically acceptable salt thereof, R 6  is selected from the group consisting of hydrogen atom, halogen, alkyl, alkoxy, haloalkyl, haloalkoxy, hydroxy and hydroxyalkyl; preferably hydrogen atom or alkyl; and more preferably hydrogen atom. 
     Typical compounds of the present disclosure include, but are not limited to: 
                                 Example           No.   Structure and name of the compound                   1                     
   1                   (S)-2-(2-(5-Chloro-2-((tetrahydro-2H-pyran-4-yl)amino)           pyrimidin-4-yl)-4-oxo-6,7-dihydrothieno[3,2-c]pyridin-5(4H)-           yl)-N-(1-(3-chlorophenyl)-2-hydroxyethyl)acetamide                2                     
   2                   (S)-2-(2-(5-Chloro-2-((tetrahydro-2H-pyran-4-yl)amino)           pyrimidin-4-yl)-6,6-dimethyl-4-oxo-4,6-dihydro-5H-thieno           [2,3-c]pyrrol-5-yl)-N-(1-(3-chlorophenyl)-2-hydroxyethyl)           acetamide               3-P1                     
   3-P1                   (S)-2-(2-(5-Chloro-2-((tetrahydro-2H-pyran-4-yl)amino)           pyrimidin-4-yl)-4-oxo-6,7-dihydrothieno[3,2-c]pyridin-5(4H)-           yl)-N-((S)-2-hydroxy-1-(m-methylphenyl)ethyl)propionamide               3-P2                     
   3-P2                   (R)-2-(2-(5-Chloro-2-((tetrahydro-2H-pyran-4-yl)amino)           pyrimidin-4-yl)-4-oxo-6,7-dihydrothieno[3,2-c]pyridin-5(4H)-           yl)-N-((S)-2-hydroxy-1-(m-methylphenyl)ethyl)propionamide               4-P1                     
   4-P1                   (S)-2-(2-(5-Chloro-2-((tetrahydro-2H-pyran-4-yl)amino)           pyrimidin-4-yl)-4-oxo-6,7-dihydrothieno[3,2-c]pyridin-5(4H)-           yl)-N-((S)-1-(3-chlorophenyl)-2-hydroxyethyl)propionamide               4-P2                     
   4-P2                   (R)-2-(2-(5-Chloro-2((tetrahydro-2H-pyran-4-yl)amino)           pyrimidin-4-yl)-4-oxo-6,7-dihydrothieno[3,2-c]pyridin-5(4H)-           yl)-N-((S)-(3-chlorophenyl)-2-hydroxyethyl)propionamide                5                     
   5                   2-(2-(5-Chloro-2-((tetrahydro-2H-pyran-4-yl)amino)           pyrimidin-4-yl)-6,6-dimethyl-4-oxo-4,6-dihydro-5H--           thieno[2,3-c]pyrrol-5-yl)-N-((S)-1-(3chlorophenyl)-2-           hydroxyethyl)propionamide                6                     
   6                   (5)-N-(1-(3-Chlorophenyl)-2-hydroxyethyl)-2-(4-oxo-2-(2-           ((tetrahydro-2H-pyran-4-yl)amino)pyrimidin-4-yl)-6,7-           dihydrothieno[3,2-c]pyridin-5(4H)-yl)acetamide                7                     
   7                   (S)-N-(2-Hydroxy-1-(m-methylphenyl)ethyl)-2-(2-(5-methyl-           2-((1-methyl-1H-pyrazol-5-yl)amino)pyrimidin-4-yl)-4-oxo-           6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl)acetamide               8-P1                     
   8-P1                   (S)-N-((S)-2-Amino-1-(3-chlorophenyl)ethyl)-2-(2-(5-chloro-           2-((tetrahydro-2H-pyran-4-yl)amino)pyrimidin-4-yl)-4-oxo-           6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl)propionamide               8-P2                     
   8-P2                   (R)-N-((S)-2-Amino-1-(3-chlorophenyl)ethyl)-2-(2-(5-chloro-           2-((tetrahydro-2H-pyran-4-yl)amino)pyrimidin-4-yl)-4-oxo-6,7-           dihydrothieno[3,2-c]pyridin-5(4H)-yl)propionamide               9-P1                     
   9-P1                   (S)-2-(2-(5-Chloro-2-((tetrahydro-2H-pyran-4-yl)amino)           pyrimidin-4-yl)-4-oxo-6,7-dihydrothieno[3,2-c]pyridin-5(4H)-           yl)-N-((S)-1-(3-fluoro-5-methoxyphenyl)-2-hydroxyethyl)           propionamide               9-P2                     
   9-P2                   (R)-2-(2-(5-Chloro-2-((tetrahydro-2H-pyran-4-yl)amino)           pyrimidin-4-yl)-4-oxo-6,7-dihydrothieno[3,2-c]pyridin-5(4H)-           yl)-N-((S)-1-(3-fluoro-5-methoxyphenyl)-2-hydroxyethyl)           propionamide               10-P1                     
   10-P1                   (S)-N-((S)-1-(3-Fluoro-5-methoxyphenyl)-2-hydroxyethyl)-2-(2-           (5-methyl-2-((1-methyl-1H-pyrazol-5-yl)amino)pyrimidin-4-yl)-           4-oxo-6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl)propionamide               10-P2                     
   10-P2                   (R)-N-((S)-1-(3-Fluoro-5-methoxyphenyl)-2-hydroxyethyl)-2-(2-           (5-methyl-2-((1-methyl-1H-pyrazol-5-yl)amino)pyrimidin-4-yl)-           4-oxo-6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl)propionamide               11                     
   11                   (R)-N-((S)-1-(3-Fluoro-5-methoxyphenyl)-2-hydroxyethyl)-2-(4-           oxo-2-(2-((tetrahydro-2H-pyran-4-yl)amino)pyrimidin-4-yl)-6,7-           dihydrothieno[3,2-c]pyridin-5(4H)-yl)-propionamide 11               12                     
   12                   (R)-2-(2-(5-Chloro-2-(((S)-1-hydroxypropan-2-yl)amino)           pyrimidin-4-yl)-4-oxo-6,7-dihydrothieno[3,2-c]pyridin-5(4H)-           yl)-N-((S)-1-(3-fluoro-5-methoxyphenyl)-2-hydroxyethyl)           propionamide 12               13                     
   13                   (R)-2-(2-(5-Chloro-2-(((S)-1-hydroxypropan-2-yl)amino)           pyrimidin-4-yl)-4-oxo-6,7-dihydrothieno[3,2-c]pyridin-5(4H)-           yl)-N-((S)-2-hydroxy-1-(m-methylphenyl)ethyl)           propionamide 13               14                     
   14                   (R)-2-(2-(5-Chloro-2-((1-methyl-1H-pyrazol-5-yl)amino)           pyrimidin-4-yl)-4-oxo-6,7-dihydrothieno[3,2-c]pyridin-5(4H)-           yl)-N-((S)-1-(3-fluoro-5-methoxyphenyl)-2-hydroxyethyl)           propionamide 14               15                     
   15                   (R)-N-((S)-1-(3-Fluoro-5-methoxyphenyl)-2-hydroxyethyl)-2-           (2-(2-((1-methyl-1H-pyrazol-5-yl)amino)pyrimidin-4-yl)-4-           oxo-6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl)propionamide 15               16                     
   16                   (R)-N-((S)-1-(3-Fluoro-5-methoxyphenyl)-2-hydroxyethyl)-2-           (2-(5-methyl-2-((tetrahydro-2H-pyran-4-yl)amino)pyrimidin-4-           yl)-4-oxo-6,7-dihydrothieno[3,2-c]pyridin-5-(4H)-yl)           propionamide 16               17                     
   17                   (R)-N-((S)-1-(3-Fluoro-5-methoxyphenyl)-2-hydroxyethyl)-2-(2-           (2-(((S)-1-hydroxypropan-2-yl)amino)-5-methylpyrimidin-4-yl)-           4-oxo-6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl)           propionamide 17                    
or a tautomer, mesomer, racemate, enantiomer, diastereomer, prodrug, mixture thereof, or a pharmaceutically acceptable salt thereof.
 
     In another aspect, the present disclosure relates to a compound of formula (IA) or formula (IA-P1) or formula (IA-P2): 
     
       
         
         
             
             
         
       
     
     or a stereoisomer, tautomer, mesomer, racemate, enantiomer, diastereomer thereof, or mixture thereof, or a pharmaceutically acceptable salt thereof, 
     wherein: R 1  to R 5 , m and n are as defined in the compound of formula (I). 
     In some embodiments of another aspect of the present disclosure, the compound of formula (IA) is a compound of formula (IIA) or formula (IIA-P1) or formula (IIA-P2): 
     
       
         
         
             
             
         
       
     
     or a stereoisomer, tautomer, mesomer, racemate, enantiomer, diastereomer thereof, or mixture thereof, or a pharmaceutically acceptable salt thereof, 
     wherein: R 1  to R 5 , m and z are as defined in the compound of formula (II). 
     In some embodiments of another aspect of the present disclosure, the compound of formula (IA) is a compound of formula (IIIA) or formula (IIIA-P1) or formula (IIIA-P2): 
     
       
         
         
             
             
         
       
     
     or a stereoisomer, tautomer, mesomer, racemate, enantiomer, diastereomer thereof, or mixture thereof, or a pharmaceutically acceptable salt thereof, 
     wherein: R 1  to R 3 , R 5 , m and z are as defined in the compound of formula (III). 
     Typical compounds of formula (IA) of the present disclosure include, but are not limited to: 
                                 Ex-           ample           No.   Structure and name of the compound                  1h                     
   1h                   2-(2-(5-Chloro-2-((tetrahydro-2H-pyran-4-yl)amino)pyrimidin-4-           yl)-4-oxo-6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl)acetic acid               2f                     
   2f                   2-(2-(5-Chloro-2-((tetrahydro-2H-pyran-4-yl)amino)pyrimidin-           4-yl)-6,6-dimethyl-4-oxo-4,6-dihydro-5H-thieno[2,3-c]pyrrol-5-yl)           acetic acid               3e                     
   3e                   2-(2-(5-Chloro-2-((tetrahydro-2H-pyran-4-yl)amino)pyrimidin-           4-yl)-4-oxo-6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl)           propanoic acid               5f                     
   5f                   2-(2-(5-Chloro-2-((tetrahydro-2H-pyran-4-yl)amino)pyrimidin-4-           yl)-6,6-dimethyl-4-oxo-4,6-dihydro-5H-thieno[2,3-c]pyrrol-5-yl)           propanoic acid               6e                     
   6e                   2-(4-Oxo-2-(2-((tetrahydro-2H-pyran-4-yl)amino)pyrimidin-4-           yl)-6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl)acetic acid               7f                     
   7f                   2-(2-(5-Methyl-2-((1-methyl-1H-pyrazol-5-yl)amino)pyrimidin-           4-yl)-4-oxo-6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl)acetic acid               10b                     
   10b                   2-(2-(5-Methyl-2-((1-methyl-1H-pyrazol-5-yl)amino)pyrimidin-           4-yl)-4-oxo-6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl)           propanoic acid               11d                     
   11d                   2-(4-Oxo-2-(2-((tetrahydro-2H-pyran-4-yl)amino)pyrimidin-           4-yl)-6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl)propanoic            acid 11d               12c                     
   12c                   2-(2-(5-Chloro-2-(((S)-1-hydroxypropan-2-yl)amino)pyrimidin-           4-yl)-4-oxo-6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl)           propanoic acid 12c               14c                     
   14c                   2-(2-(5-Chloro-2-((1-methyl-1H-pyrazol-5-yl)amino)pyrimidin-           4-yl)-4-oxo-6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl)           propanoic acid 14c               15e                     
   15e                   2-2-(2-((1-Methyl-1H-pyrazol-5-yl)amino)pyrimidin-4-yl)-4-oxo-           6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl)propanoic acid 15e               16e                     
   16e                   2-(2-(5-Methyl-2-((tetrahydro-2H-pyran-4-yl)amino)pyrimidin-           4-yl)-4-oxo-6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl)propanoic            acid 16e                                                         2-(2-(5-Methyl-2-(((S)-1-hydroxypropan2-yl)amino)pyrimidin-           4-yl)-4-oxo-6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl)           propanoic acid                    
or a tautomer, mesomer, racemate, enantiomer, diastereomer, prodrug, mixture thereof, or a pharmaceutically acceptable salt thereof.
 
     In another aspect, the present disclosure relates to a method for preparing the compound of formula (I), comprising a step of: 
     
       
         
         
             
             
         
       
     
     subjecting a compound of formula (IA) and a compound of (IB) to a condensation reaction in the presence of an alkaline reagent to obtain the compound of formula (I), 
     wherein: the alkaline reagent is preferably N,N-diisopropyl ethylamine; and 
     R 1  to R 7 , L, m and n are as defined in the compound of formula (I). 
     In another aspect, the present disclosure relates to a method for preparing the compound of formula (I-P1), comprising a step of: 
     
       
         
         
             
             
         
       
     
     subjecting a compound of formula (IA-P1) and a compound of (IB) to a condensation reaction in the presence of an alkaline reagent to obtain the compound of formula (I-P1), wherein: R 1  to R 7 , L, m and n are as defined in the compound of formula (I). 
     In another aspect, the present disclosure relates to a method for preparing the compound of formula (I-P2), comprising a step of: 
     
       
         
         
             
             
         
       
     
     subjecting a compound of formula (IA-P2) and a compound of (IB) to a condensation reaction in the presence of an alkaline reagent to obtain the compound of formula (I-P2), wherein: R 1  to R 7 , L, m and n are as defined in the compound of formula (I). 
     Another aspect of the present disclosure relates to a method for preparing the compound of formula (II), comprising a step of: 
     
       
         
         
             
             
         
       
     
     subjecting a compound of formula (IIA) and a compound of (IB) to a condensation reaction in the presence of an alkaline reagent to obtain the compound of formula (II), 
     wherein: the alkaline reagent is preferably N,N-diisopropyl ethylamine; z is 0, 1, 2, 3 or 4; and 
     R 1  to R 7 , L and m are as defined in the compound of formula (I). 
     In another aspect, the present disclosure relates to a method for preparing the compound of formula (II-P1), comprising a step of: 
     
       
         
         
             
             
         
       
     
     subjecting a compound of formula (IIA-P1) and a compound of (IB) to a condensation reaction in the presence of an alkaline reagent to obtain the compound of formula (II-P1), 
     wherein: the alkaline reagent is preferably N,N-diisopropyl ethylamine; and 
     R 1  to R 7 , L, m and z are as defined in the compound of formula (II). 
     In another aspect, the present disclosure relates to a method for preparing the compound of formula (II-P2), comprising a step of: 
     
       
         
         
             
             
         
       
     
     subjecting a compound of formula (IIA-P2) and a compound of (IB) to a condensation reaction in the presence of an alkaline reagent to obtain the compound of formula (II-P2), 
     wherein: the alkaline reagent is preferably N,N-diisopropyl ethylamine; and 
     R 1  to R 7 , L, m and z are as defined in the compound of formula (II). 
     In another aspect, the present disclosure relates to a method for preparing the compound of formula (III), comprising a step of: 
     
       
         
         
             
             
         
       
     
     subjecting a compound of formula (IIIA) and a compound of (IIIB) to a condensation reaction in the presence of an alkaline reagent to obtain the compound of formula (III), 
     wherein: the alkaline reagent is preferably N,N-diisopropyl ethylamine; and 
     R 1  to R 3 , R 5 , R 7 , R 8 , m and z are as defined in the compound of formula (III). 
     In another aspect, the present disclosure relates to a method for preparing the compound of formula (III-P1), comprising a step of: 
     
       
         
         
             
             
         
       
     
     subjecting a compound of formula (IIIA-P1) and a compound of (IIIB) to a condensation reaction in the presence of an alkaline reagent to obtain the compound of formula (III-P 1), 
     wherein: the alkaline reagent is preferably N,N-diisopropyl ethylamine; and R 1  to R 3 , R 5 , R 7 , R 8 , m and z are as defined in the compound of formula (III). 
     In another aspect, the present disclosure relates to a method for preparing the compound of formula (III-P2), comprising a step of: 
     
       
         
         
             
             
         
       
     
     subjecting a compound of formula (IIIA-P2) and a compound of (IIIB) to a condensation reaction in the presence of an alkaline reagent to obtain the compound of formula (III-P2), 
     wherein: the alkaline reagent is preferably N,N-diisopropyl ethylamine; and 
     R 1  to R 3 , R 5 , R 7 , R 8 , m and z are as defined in the compound of formula (III). 
     In another aspect of the present disclosure relates to a method for preparing the compound of formula (IV) —  comprising a step of 
     
       
         
         
             
             
         
       
     
     subjecting a compound of formula (IA) and a compound of (IIIB) to a condensation reaction in the presence of an alkaline reagent to obtain the compound of formula (IV), 
     wherein: the alkaline reagent is preferably N,N-diisopropyl ethylamine; and 
     R 1  to R 8 , m and n are as defined in the compound of formula (IV). 
     In another aspect, the present disclosure relates to a method for preparing the compound of formula (IV-P1), comprising a step of: 
     
       
         
         
             
             
         
       
     
     subjecting a compound of formula (IA-P1) and a compound of (IIIB) to a condensation reaction in the presence of an alkaline reagent to obtain the compound of formula (IV-P 1), 
     wherein: the alkaline reagent is preferably N,N-diisopropyl ethylamine; and 
     R 1  to R 8 , m and n are as defined in the compound of formula (IV). 
     In another aspect, the present disclosure relates to a method for preparing the compound of formula (IV-P2), comprising a step of: 
     
       
         
         
             
             
         
       
     
     subjecting a compound of formula (IA-P2) and a compound of (IIIB) to a condensation reaction in the presence of an alkaline reagent to obtain the compound of formula (IV-P2), 
     wherein: the alkaline reagent is preferably N,N-diisopropyl ethylamine; 
     R 1  to R 8 , m and n are as defined in the compound of formula (IV). 
     In another aspect, the present disclosure relates to a pharmaceutical composition, comprising a therapeutically effective amount of the compound of formula (I) or formula (II) or formula (III) or formula (IV) or the tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or the pharmaceutically acceptable salt thereof of the present disclosure, and one or more pharmaceutically acceptable carriers, diluents or excipients. 
     The present disclosure further relates to a use of the compound of formula (I) or formula (II) or formula (III) or formula (IV) or the tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or the pharmaceutically acceptable salt thereof, or the pharmaceutical composition comprising the same in the preparation of a medicament for inhibiting ERK. 
     The present disclosure further relates to a use of the compound of formula (I) or formula (II) or formula (III) or formula (IV) or the tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or the pharmaceutically acceptable salt thereof, or the pharmaceutical composition comprising the same in the preparation of a medicaments for treating or preventing cancer, inflammation, or other proliferative diseases, preferably in the preparation of a medicament for treating or preventing cancer; wherein the cancer is selected from the group consisting of melanoma, liver cancer, kidney cancer, lung cancer (such as non-small cell lung cancer or small cell lung cancer), nasopharyngeal carcinoma, colorectal cancer, pancreatic cancer, cervical cancer, ovarian cancer, breast cancer, bladder cancer, prostate cancer, leukemia, head and neck squamous cell carcinoma, carcinoma of uterine cervix, thyroid cancer, lymphoma, sarcoma, neuroblastoma, brain tumor, myeloma (such as multiple myeloma), astrocytoma, and glioma. 
     The present disclosure also relates to a method for inhibiting ERK, comprising a step of administering to a patient in need thereof a therapeutically effective amount of the compound of formula (I) or formula (II) or formula (III) or formula (IV) or the tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or the pharmaceutically acceptable salt thereof, or the pharmaceutical composition comprising the same. 
     The present disclosure also relates to a method for treating or preventing ERK-mediated diseases, comprising a step of administering to a patient in need thereof a therapeutically effective amount of the compound of formula (I) or formula (II) or formula (III) or formula (IV) or the tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or the pharmaceutically acceptable salt thereof, or the pharmaceutical composition comprising the same. 
     The present disclosure also relates to a method for treating or preventing cancer, inflammation, or other proliferative diseases, preferably cancer, comprising a step of administering to a patient in need thereof a therapeutically effective amount of the compound of formula (I) or formula (II) or formula (III) or formula (IV) or the tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or the pharmaceutically acceptable salt thereof, or the pharmaceutical composition comprising the same; wherein the cancer is selected from the group consisting of melanoma, liver cancer, kidney cancer, lung cancer (such as non-small cell lung cancer or small cell lung cancer), nasopharyngeal carcinoma, colorectal cancer, pancreatic cancer, cervical cancer, ovarian cancer, breast cancer, bladder cancer, prostate cancer, leukemia, head and neck squamous cell carcinoma, carcinoma of uterine cervix, thyroid cancer, lymphoma, sarcoma, neuroblastoma, brain tumor, myeloma (such as multiple myeloma), astrocytoma, and glioma. 
     The present disclosure further relates to the compound of formula (I) or formula (II) or formula (III) or formula (IV) or the tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or the pharmaceutically acceptable salt thereof, or the pharmaceutical composition comprising the same, for use as a medicament. 
     The present disclosure further relates to the compound of formula (I) or formula (II) or formula (III) or formula (IV) or the tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition comprising the same, for use as an ERK inhibitor. 
     The present disclosure further relates to the compound of formula (I) or formula (II) or formula (III) or formula (IV) or the tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition comprising the same, for use in treating or preventing ERK-mediated diseases. 
     The present disclosure further relates to the compound of formula (I) or formula (II) or formula (III) or formula (IV) or the tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or the pharmaceutically acceptable salt thereof, or the pharmaceutical composition comprising the same, for use in treating or preventing cancer, inflammation, or other proliferative diseases, preferably in treating or preventing cancer; wherein the cancer is selected from the group consisting of melanoma, liver cancer, kidney cancer, lung cancer (such as non-small cell lung cancer or small cell lung cancer), nasopharyngeal carcinoma, colorectal cancer, pancreatic cancer, cervical cancer, ovarian cancer, breast cancer, bladder cancer, prostate cancer, leukemia, head and neck squamous cell carcinoma, carcinoma of uterine cervix, thyroid cancer, lymphoma, sarcoma, neuroblastoma, brain tumor, myeloma (such as multiple myeloma), astrocytoma, and glioma. 
     The active compound can be prepared in a form suitable for administration by any appropriate route, and the active compound is preferably in a unit dose form, or in a form in which the patient can self-administer in a single dose. The unit dose of the compound or composition of the present disclosure can be expressed in the form of tablets, capsules, cachets, bottled syrups, powders, granules, lozenges, suppositories, regenerated powders or liquid formulations. 
     The dosage of the compound or composition used in the treatment method of the present disclosure will generally vary according to the severity of the disease, the weight of the patient, and the relative efficacy of the compound. However, as a general guide, a suitable unit dose can be 0.1 to 1000 mg. 
     In addition to the active compound, the pharmaceutical composition of the present disclosure can also comprise one or more auxiliaries including a filler (diluent), binder, wetting agent, disintegrant, excipient and the like. Depending on the administration mode, the composition can comprise 0.1 to 99% by weight of the active compound. 
     The pharmaceutical composition comprising the active ingredient can be in a form suitable for oral administration, for example, a tablet, troche, lozenge, aqueous or oily suspension, dispersible powder or granule, emulsion, hard or soft capsule, syrup or elixir. An oral composition can be prepared according to any known method in the art for the preparation of pharmaceutical composition. Such a composition can comprise one or more ingredient(s) selected from the group consisting of sweeteners, flavoring agents, colorants and preservatives, in order to provide a pleasing and palatable pharmaceutical formulation. The tablet contains the active ingredient in admixture with nontoxic, pharmaceutically acceptable excipients suitable for the manufacture of tablets. 
     An aqueous suspension comprises an active ingredient in admixture with excipients suitable for the manufacture of an aqueous suspension. The aqueous suspension can also comprise one or more preservatives such as ethyl paraben or n-propyl paraben, one or more colorants, one or more flavoring agents, and one or more sweeteners. 
     An oil suspension can be formulated by suspending the active ingredient in a vegetable oil or mineral oil. The oil suspension can contain a thickener. The aforementioned sweeteners and flavoring agents can be added to provide a palatable formulation. 
     The active ingredient in admixture with the dispersants or wetting agents, suspending agents or one or more preservatives can be prepared as dispersible powders or granules suitable for the preparation of an aqueous suspension by adding water. Suitable dispersants or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, such as sweeteners, flavoring agents and colorants, can also be added. These compositions can be preserved by adding an antioxidant such as ascorbic acid. 
     The pharmaceutical composition of the present disclosure can also be in the form of an oil-in-water emulsion. 
     The pharmaceutical composition can be in the form of a sterile injectable aqueous solution. Acceptable vehicles or solvents that can be used are water, Ringer&#39;s solution or isotonic sodium chloride solution. The sterile injectable formulation can be a sterile injectable oil-in-water micro-emulsion in which the active ingredient is dissolved in the oil phase. For example, the active ingredient is dissolved in a mixture of soybean oil and lecithin. The oil solution is then added into a mixture of water and glycerol, and processed to form a micro-emulsion. The injectable solution or micro-emulsion can be introduced into a patient&#39;s bloodstream by local bolus injection. Alternatively, the solution and micro-emulsion are preferably administrated in a manner that maintains a constant circulating concentration of the compound of the present invention. In order to maintain this constant concentration, a continuous intravenous delivery device can be used. An example of such a device is Deltec CADD-PLUS. TM. 5400 intravenous injection pump. 
     The pharmaceutical composition can be in the form of a sterile injectable aqueous or oily suspension for intramuscular and subcutaneous administration. Such a suspension can be formulated with suitable dispersants or wetting agents and suspending agents as described above according to known techniques. The sterile injectable formulation can also be a sterile injectable solution or suspension prepared in a nontoxic parenterally acceptable diluent or solvent. Moreover, sterile fixed oils can easily be used as a solvent or suspending medium. 
     The compound of the present disclosure can be administered in the form of a suppository for rectal administration. These pharmaceutical compositions can be prepared by mixing the drug with a suitable non-irritating excipient that is solid at ordinary temperatures, but liquid in the rectum, thereby melting in the rectum to release the drug. Such materials include cocoa butter, glycerin gelatin, hydrogenated vegetable oils, mixtures of polyethylene glycols with various molecular weights and fatty acid esters of polyethylene glycols. 
     It is well known to those skilled in the art that the dosage of a drug depends on a variety of factors including but not limited to, the following factors: activity of a specific compound, age of the patient, weight of the patient, general health of the patient, behavior of the patient, diet of the patient, administration time, administration route, excretion rate, drug combination and the like. In addition, the optimal treatment, such as treatment mode, daily dose of the compound of formula (I) or the type of pharmaceutically acceptable salt thereof can be verified by traditional therapeutic regimens. 
     Term Dedinitions 
     Unless otherwise stated, the terms used in the specification and claims have the meanings described below. 
     The term “alkyl” refers to a saturated aliphatic hydrocarbon group, which is a straight or branched chain group comprising 1 to 20 carbon atoms, preferably an alkyl comprising 1 to 12 carbon atoms, and more preferably an alkyl comprising 1 to 6 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2,3-dimethylpentyl, 2,4-dimethylpentyl, 2,2-dimethylpentyl, 3,3-dimethylpentyl, 2-ethylpentyl, 3-ethylpentyl, n-octyl, 2,3-dimethylhexyl, 2,4-dimethylhexyl, 2,5-dimethylhexyl, 2,2-dimethylhexyl, 3,3-dimethylhexyl, 4,4-dimethylhexyl, 2-ethylhexyl, 3-ethylhexyl, 4-ethylhexyl, 2-methyl-2-ethylpentyl, 2-methyl-3-ethylpentyl, n-nonyl, 2-methyl-2-ethylhexyl, 2-methyl-3-ethylhexyl, 2,2-diethylpentyl, n-decyl, 3,3-diethylhexyl, 2,2-diethylhexyl, and various branched isomers thereof. More preferably, the alkyl group is a lower alkyl comprising 1 to 6 carbon atoms, and non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl and the like. The alkyl can be substituted or unsubstituted. When substituted, the substituent group(s) can be substituted at any available connection point. The substituent group(s) is preferably one or more groups independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, thiol, hydroxy, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocyclylthio and oxo. 
     The term “alkylene” refers to a saturated linear or branched aliphatic hydrocarbon group having two residues derived from the removal of two hydrogen atoms from the same carbon atom or two different carbon atoms of the parent alkane. It is a linear or branched alkylene comprising 1 to 20 carbon atoms, preferably 1 to 12 carbon atoms, and more preferably 1 to 6 carbon atoms. Non limiting examples of alkylene include, but are not limited to, methylene (—CH 2 —), 1,1-ethylidene (—CH(CH 3 )—), 1,2-ethylidene (—CH 2 CH 2 —), 1,1-propylene (—CH(CH 2 CH 3 )—), 1,2-propylene (—CH 2 CH(CH 3 )—), 1,3-propylene (—CH 2 CH 2 CH 2 —), 1,4-butylene (—CH 2 CH 2 CH 2 CH 2 —) and the like. The alkylene can be substituted or unsubstituted. When substituted, the substituent group(s) can be substituted at any available connection point. The substituent group(s) is preferably one or more groups independently optionally selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, thiol, hydroxy, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio and oxo. 
     The term “alkoxy” refers to an —O-(alkyl) and —O-(unsubstituted cycloalkyl) group, wherein the alkyl is defined as above. Non limiting examples of alkoxy include methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentoxy, and cyclohexoxy. The alkoxy can be optionally substituted or unsubstituted. When substituted, the substituent group(s) is preferably one or more groups independently selected from the group consisting of hydrogen atom, halogen, alkyl, alkoxy, haloalkyl, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl and heteroaryl. 
     The term “cycloalkyl” refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent group comprising 3 to 20 carbon atoms, preferably 3 to 12 carbon atoms, more preferably 3 to 8 (for example 3, 4, 5, 6, 7 or 8) carbon atoms, and most preferably 3 to 6 (for example 3, 4, 5 or 6) carbon atoms. Non-limiting examples of monocyclic cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl, cyclooctyl and the like, preferably cycloalkyl. Polycyclic cycloalkyl includes a cycloalkyl having a spiro ring, fused ring or bridged ring. 
     The term “spiro cycloalkyl” refers to a 5 to 20 membered polycyclic group with individual rings connected through one shared carbon atom (called a spiro atom), wherein the rings can contain one or more double bonds, but none of the rings has a completely conjugated a-electron system. The spiro cycloalkyl is preferably a 6 to 14 membered spiro cycloalkyl, and more preferably a 7 to 10 membered spiro cycloalkyl. According to the number of the spiro atoms shared between the rings, the spiro cycloalkyl can be divided into a mono-spiro cycloalkyl, di-spiro cycloalkyl, or poly-spiro cycloalkyl, and the spiro cycloalkyl is preferably a mono-spiro cycloalkyl or di-spiro cycloalkyl, and more preferably a 4-membered4-membered, 4-membered5-membered, 4-membered6-membered, 5-membered5-membered, or 5-membered6-membered mono-spiro cycloalkyl. Non-limiting examples of spiro cycloalkyl include: 
     
       
         
         
             
             
         
       
     
     The term “fused cycloalkyl” refers to a 5 to 20 membered all-carbon polycyclic group, wherein each ring in the system shares an adjacent pair of carbon atoms with another ring, one or more rings can contain one or more double bonds, but none of the rings has a completely conjugated π-electron system. The fused cycloalkyl is preferably a 6 to 14 membered fused cycloalkyl, and more preferably a 7 to 10 membered fused cycloalkyl. According to the number of membered rings, the fused cycloalkyl can be divided into a bicyclic, tricyclic, tetracyclic or polycyclic fused cycloalkyl, and the fused cycloalkyl is preferably a bicyclic or tricyclic fused cycloalkyl, and more preferably a 5-membered5-membered, or 5-membered6-membered bicyclic fused cycloalkyl. Non-limiting examples of fused cycloalkyl include: 
     
       
         
         
             
             
         
       
     
     The term “bridged cycloalkyl” refers to a 5 to 20 membered all-carbon polycyclic group, wherein every two rings in the system share two disconnected carbon atoms, the rings can have one or more double bonds, but none of the rings has a completely conjugated π-electron system. The bridged cycloalkyl is preferably a 6 to 14 membered bridged cycloalkyl, and more preferably a 7 to 10 membered bridged cycloalkyl. According to the number of membered rings, the bridged cycloalkyl can be divided into a bicyclic, tricyclic, tetracyclic or polycyclic bridged cycloalkyl, and the bridged cycloalkyl is preferably a bicyclic, tricyclic or tetracyclic bridged cycloalkyl, and more preferably a bicyclic or tricyclic bridged cycloalkyl. Non-limiting examples of bridged cycloalkyl include: 
     
       
         
         
             
             
         
       
     
     The cycloalkyl ring includes the above cycloalkyl (including monocycloalkyl, spiro cycloalkyl, fused cycloalkyl and bridged cycloalkyl) fused to the ring of aryl, heteroaryl or heterocyclyl, wherein the ring bound to the parent structure is cycloalkyl. Non-limiting examples include indanyl, tetrahydronaphthyl, benzocycloheptyl and the like, and preferably benzocyclopentyl, tetrahydronaphthyl. 
     The cycloalkyl can be optionally substituted or unsubstituted. When substituted, the substituent group(s) is preferably one or more groups independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, thiol, hydroxy, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocyclylthio and oxo. 
     The term “heterocyclyl” refers to a 3 to 20 membered saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent group, wherein one or more ring atoms are heteroatoms selected from the group consisting of N, O, S, S(O) and S(O) 2 , but excluding —O—O—, —O—S— or —S—S— in the ring, with the remaining ring atoms being carbon atoms. Preferably, the heterocyclyl has 3 to 12 ring atoms wherein 1 to 4 (for example 1, 2, 3, or 4) atoms are heteroatoms; preferably, 3 to 8 (for example 3, 4, 5, 6, 7 or 8) ring atoms wherein 1 to 3 atoms are heteroatoms; and preferably 3 to 6 ring atoms wherein 1 to 3 atoms are heteroatoms. Non-limiting examples of monocyclic heterocyclyl include azetidinyl, pyrrolidinyl, imidazolidinyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydrothienyl, dihydroimidazolyl, dihydrofuranyl, dihydropyrazolyl, dihydropyrrolyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl and the like, and preferably tetrahydropyranyl, piperidinyl, pyrrolidinyl. Polycyclic heterocyclyl includes a heterocyclyl having a spiro ring, fused ring or bridged ring. 
     The term “spiro heterocyclyl” refers to a 5 to 20 membered polycyclic heterocyclyl group with individual rings connected through one shared atom (called a spiro atom), wherein one or more ring atoms are heteroatoms selected from the group consisting of N, O, S, S(O) and S(O) 2 , with the remaining ring atoms being carbon atoms, where the rings can contain one or more double bonds, but none of the rings has a completely conjugated π-electron system. The spiro heterocyclyl is preferably a 6 to 14 membered spiro heterocyclyl, and more preferably a 7 to 10 membered Spiro heterocyclyl. According to the number of the spiro atoms shared between the rings, the spiro heterocyclyl is divided into a mono-spiro heterocyclyl, di-spiro heterocyclyl, or poly-spiro heterocyclyl, and the spiro heterocyclyl is preferably a mono-spiro heterocyclyl or di-spiro heterocyclyl, and more preferably a 4-membered4-membered, 4-membered5-membered, 4-membered6-membered, 5-membered5-membered, or 5-membered6-membered mono-spiro heterocyclyl. Non-limiting examples of spiro heterocyclyl include: 
     
       
         
         
             
             
         
       
     
     The term “fused heterocyclyl” refers to a 5 to 20 membered polycyclic heterocyclyl group, wherein each ring in the system shares an adjacent pair of atoms with another ring, wherein one or more rings can contain one or more double bonds, but none of the rings has a completely conjugated a-electron system, and wherein one or more ring atoms are heteroatoms selected from the group consisting of N, O, S, S(O) and S(O) 2 , with the remaining ring atoms being carbon atoms. The fused heterocyclyl is preferably a 6 to 14 membered fused heterocyclyl, and more preferably a 7 to 10 membered fused heterocyclyl. According to the number of membered rings, the fused heterocyclyl can be divided into a bicyclic, tricyclic, tetracyclic or polycyclic fused heterocyclyl, and the fused heterocyclyl is preferably a bicyclic or tricyclic fused heterocyclyl, and more preferably a 5-membered5-membered or 5-membered6-membered bicyclic fused heterocyclyl. Non-limiting examples of fused heterocyclyl include: 
     
       
         
         
             
             
         
       
     
     The term “bridged heterocyclyl” refers to a 5 to 14 membered polycyclic heterocyclyl group, wherein every two rings in the system share two disconnected atoms, wherein the rings can have one or more double bonds, but none of the rings has a completely conjugated π-electron system, and wherein one or more ring atoms are heteroatoms selected from the group consisting of N, O, S, S(O) and S(O) 2 , with the remaining ring atoms being carbon atoms. The bridged heterocyclyl is preferably a 6 to 14 membered bridged heterocyclyl, and more preferably a 7 to 10 membered bridged heterocyclyl. According to the number of membered rings, the bridged heterocyclyl can be divided into a bicyclic, tricyclic, tetracyclic or polycyclic bridged heterocyclyl, and the bridged heterocyclyl is preferably a bicyclic, tricyclic or tetracyclic bridged heterocyclyl, and more preferably a bicyclic or tricyclic bridged heterocyclyl. Non-limiting examples of bridged heterocyclyl include: 
     
       
         
         
             
             
         
       
     
     The heterocyclyl ring includes the above heterocyclyl (including mono-heterocyclyl, fused heterocyclyl, spiro heterocyclyl and bridged heterocyclyl) fused to the ring of aryl, heteroaryl or cycloalkyl, wherein the ring bound to the parent structure is heterocyclyl. 
     Non-limiting examples thereof include: 
     
       
         
         
             
             
         
       
     
     and the like. 
     The heterocyclyl can be optionally substituted or unsubstituted. When substituted, the substituent group(s) is preferably one or more groups independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, thiol, hydroxy, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocyclylthio and oxo. 
     The term “aryl” refers to a 6 to 20 membered all-carbon monocyclic ring or polycyclic fused ring (i.e. each ring in the system shares an adjacent pair of carbon atoms with another ring in the system) having a conjugated π-electron system, preferably a 6 to 10 membered aryl, and more preferably a 6 membered aryl, for example, phenyl and naphthyl. The aryl ring can be fused to the ring of heteroaryl, heterocyclyl or cycloalkyl, wherein the ring bound to the parent structure is aryl ring. Non-limiting examples thereof include: 
     
       
         
         
             
             
         
       
     
     The aryl can be substituted or unsubstituted. When substituted, the substituent group(s) is preferably one or more groups independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, thiol, hydroxy, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio and heterocyclylthio. 
     The term “heteroaryl” refers to a 5 to 20 membered heteroaromatic system comprising 1 to 4 (for example 1, 2, 3, or 4) heteroatoms selected from the group consisting of O, S and N. The heteroaryl is preferably a 5 to 10 (for example 5, 6, 7, 8, 9 or 10) membered heteroaryl comprising 1 to 3 heteroatoms, and more preferably a 5 or 6 membered heteroaryl comprising 1 to 3 heteroatoms. Non-limiting examples include: for example, pyrazolyl, imidazolyl, furyl, thienyl, thiazolyl, oxazolyl, pyrrolyl, triazolyl, tetrazolyl, pyridyl, pyrimidinyl, thiadiazolyl, pyrazinyl and the like. The heteroaryl ring can be fused to the ring of aryl, heterocyclyl or cycloalkyl, wherein the ring bound to the parent structure is heteroaryl ring. Non-limiting examples thereof include: 
     
       
         
         
             
             
         
       
     
     The heteroaryl can be optionally substituted or unsubstituted. When substituted, the substituent group(s) is preferably one or more groups independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, thiol, hydroxy, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio and heterocyclylthio. 
     The term “alkylthio” refers to a —S-(alkyl) and —S-(unsubstituted cycloalkyl) group, wherein the alkyl is defined as above. Non limiting examples of alkylthio include methylthio, ethylthio, propylthio, butylthio, cyclopropylthio, cyclobutylthio, cyclopentylthio, and cyclohexylthio. The alkylthio can be optionally substituted or unsubstituted. When substituted, the substituent group(s) is preferably one or more groups independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, thiol, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio and heterocycloalkylthio. 
     The term “cycloalkyloxy” refers to a —O-cycloalkyl group, wherein the cycloalkyl is as defined above. 
     The term “haloalkyl” refers to an alkyl group substituted by halogen(s), wherein the alkyl is as defined above. 
     The term “haloalkoxy” refers to an alkoxy group substituted by halogen(s), wherein the alkoxy is as defined above. 
     The term “hydroxyalkyl” refers to an alkyl group substituted by hydroxy(s), wherein the alkyl is as defined above. 
     The term “aminoalkyl” refers to an alkyl group substituted by amino(s), wherein the alkyl is as defined above. 
     The term “hydroxy” refers to an —OH group. 
     The term “halogen” refers to fluorine, chlorine, bromine or iodine. 
     The term “amino” refers to a —NH 2  group. 
     The term “cyano” refers to a —CN group. 
     The term “nitro” refers to a —NO 2  group. 
     The term “aldehyde” refers to a —C(O)H group. 
     The term “carboxy” refers to a —C(O)OH group. 
     The term “alkoxycarbonyl” refers to a —C(O)O(alkyl) or —C(O)O(cycloalkyl) group, wherein the alkyl and cycloalkyl are as defined above. 
     “Optional” or “optionally” means that the event or circumstance described subsequently can, but need not, occur, and such a description includes the situation in which the event or circumstance does or does not occur. For example, “the heterocyclyl optionally substituted by an alkyl” means that an alkyl group can be, but need not be, present, and such a description includes the situation of the heterocyclyl being substituted by an alkyl and the heterocyclyl being not substituted by an alkyl. 
     “Substituted” refers to one or more hydrogen atoms in a group, preferably up to 5 (for example 1, 2, 3, 4 or 5), and more preferably 1 to 3 hydrogen atoms, independently substituted by a corresponding number of substituents, wherein each substituent has independent options (that is, the substituents can be identical or different). It goes without saying that the substituents only exist in their possible chemical position. The person skilled in the art is able to determine whether the substitution is possible or impossible by experiments or theory without excessive effort. For example, the combination of amino or hydroxy having free hydrogen and carbon atoms having unsaturated bonds (such as olefinic) may be unstable. 
     The term “pharmaceutical composition” refers to a mixture of one or more of the compounds described herein or physiologically/pharmaceutically acceptable salts or prodrugs thereof with other chemical components, and other components such as physiologically/pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to facilitate administration of a compound to an organism, which is conducive to the absorption of the active ingredient so as to show biological activity. 
     A “pharmaceutically acceptable salt” refers to a salt of the compound of the present disclosure, which is safe and effective in mammals and has the desired biological activity. 
     The compound of the present disclosure can also comprise isotopic derivatives thereof. The term “isotopic derivatives” refers to compounds that differ in structure only in the presence of one or more isotopically enriched atoms. For example, a compound having the structure of the present disclosure except replacing hydrogen with “deuterium” or “tritium”, or replacing fluorine with an  18 F-fluorine labeling ( 18 F isotope), or replacing carbon with  11 C-,  13 C-, or  14 C-enriched carbon ( 11 C-,  13 C-, or  14 C-carbon labeling;  11 C-,  13 C-, or  14 C-isotope) is within the scope of the present disclosure. Such compounds can be used, for example, as analytical tools or probes in biological assays, or as tracers for in vivo diagnostic imaging of disease, or as tracers for pharmacodynamics, pharmacokinetics or receptor studies. Deuterated compounds can generally retain activity comparable to non-deuterated compounds, and when deuterated at certain specific sites, the resulting compounds can achieve better metabolic stability, thereby obtaining certain therapeutic advantages (such as increased in vivo half-life or reduced dosage requirements). 
     For drugs or pharmacologically active agents, the term “therapeutically effective amount” refers to a sufficient amount of a drug or agent that is non-toxic but can achieve the desired effect. The determination of the effective amount varies from person to person, depending on the age and general condition of the recipient, and also on the specific active substance. The appropriate effective amount in a case can be determined by the person skilled in the art according to routine experiments. 
     Synthesis Method of the Compound of the Present Disclosure 
     In order to achieve the object of the present disclosure, the present disclosure applies the following technical solutions: 
     
       
         
         
             
             
         
       
     
     A method for preparing the compound of formula (I) or the tautomer, mesomer, racemate, enantiomer, diastereomer thereof, or mixture thereof, or the pharmaceutically acceptable salt thereof according to the present disclosure, comprises the following step of: 
     subjecting a compound of formula (IA) and a compound of (IB) to a condensation reaction in the presence of an alkaline reagent to obtain the compound of formula (I), wherein: R 1  to R 7 , L, m and n are as defined in the compound of formula (I). 
     
       
         
         
             
             
         
       
     
     A method for preparing the compound of formula (I-P1) or the tautomer, mesomer, racemate, enantiomer, diastereomer thereof, or mixture thereof, or the pharmaceutically acceptable salt thereof according to the present disclosure, comprises the following step of: 
     subjecting a compound of formula (IA-P1) and a compound of (IB) to a condensation reaction in the presence of an alkaline reagent to obtain the compound of formula (I-P1), wherein: R 1  to R 7 , L, m and n are as defined in the compound of formula (I). 
     
       
         
         
             
             
         
       
     
     A method for preparing the compound of formula (I-P2) or the tautomer, mesomer, racemate, enantiomer, diastereomer thereof, or mixture thereof, or the pharmaceutically acceptable salt thereof according to the present disclosure, comprises the following step of: 
     subjecting a compound of formula (IA-P2) and a compound of (IB) to a condensation reaction in the presence of an alkaline reagent to obtain the compound of formula (I-P2), wherein: R 1  to R 7 , L, m and n are as defined in the compound of formula (I). 
     
       
         
         
             
             
         
       
     
     A method for preparing the compound of formula (II) or the tautomer, mesomer, racemate, enantiomer, diastereomer thereof, or mixture thereof, or the pharmaceutically acceptable salt thereof according to the present disclosure, comprises the following step of:  
     subjecting a compound of formula (IIA) and a compound of (IB) to a condensation reaction in the presence of an alkaline reagent to obtain the compound of formula (II), wherein: the alkaline reagent is preferably N,N-diisopropyl ethylamine; z is 0, 1, 2, 3 or 4; and R 1  to R 7 , L and m are as defined in the compound of formula (I). 
     
       
         
         
             
             
         
       
     
     A method for preparing the compound of formula (II-P1) or the tautomer, mesomer, racemate, enantiomer, diastereomer thereof, or mixture thereof, or the pharmaceutically acceptable salt thereof according to the present disclosure, comprises the following step of:  
     subjecting a compound of formula (IIA-P1) and a compound of (IB) to a condensation reaction in the presence of an alkaline reagent to obtain the compound of formula (II-P1), wherein: the alkaline reagent is preferably N,N-diisopropyl ethylamine; and R 1  to R 7 , L, m and z are as defined in the compound of formula (II). 
     
       
         
         
             
             
         
       
     
     A method for preparing the compound of formula (II-P2) or the tautomer, mesomer, racemate, enantiomer, diastereomer thereof, or mixture thereof, or the pharmaceutically acceptable salt thereof according to the present disclosure, comprises the following step of: 
     subjecting a compound of formula (IIA-P2) and a compound of (IB) to a condensation reaction in the presence of an alkaline reagent to obtain the compound of formula (II-P2), wherein: the alkaline reagent is preferably N,N-diisopropyl ethylamine; and R 1  to R 7 , L, m and z are as defined in the compound of formula (II). 
     
       
         
         
             
             
         
       
     
     A method for preparing the compound of formula (III) or the tautomer, mesomer, racemate, enantiomer, diastereomer thereof, or mixture thereof, or the pharmaceutically acceptable salt thereof according to the present disclosure, comprises the following step of:  
     subjecting a compound of formula (IIIA) and a compound of (IIIB) to a condensation reaction in the presence of an alkaline reagent to obtain the compound of formula (III), wherein: the alkaline reagent is preferably N,N-diisopropyl ethylamine; and R 1  to R 3 , R 5 , R 7 , R 8 , m and z are as defined in the compound of formula (III). 
     
       
         
         
             
             
         
       
     
     A method for preparing the compound of formula (III-P1) or the tautomer, mesomer, racemate, enantiomer, diastereomer thereof, or mixture thereof, or the pharmaceutically acceptable salt thereof according to the present disclosure, comprises the following step of:  
     subjecting a compound of formula (IIIA-P1) and a compound of (IIIB) to a condensation reaction in the presence of an alkaline reagent to obtain the compound of formula (III-P1), wherein: the alkaline reagent is preferably N,N-diisopropyl ethylamine; and R 1  to R 3 , R 5 , R 7 , R 8 , m and z are as defined in the compound of formula (III). 
     
       
         
         
             
             
         
       
     
     A method for preparing the compound of formula (III-P2) or the tautomer, mesomer, racemate, enantiomer, diastereomer thereof, or mixture thereof, or the pharmaceutically acceptable salt thereof according to the present disclosure, comprises the following step of:  
     subjecting a compound of formula (IIIA-P2) and a compound of (IIIB) to a condensation reaction in the presence of an alkaline reagent to obtain the compound of formula (III-P2), wherein: the alkaline reagent is preferably N,N-diisopropyl ethylamine; and R 1  to R 3 , R 5 , R 7 , R 8 , m and z are as defined in the compound of formula (III). 
     
       
         
         
             
             
         
       
     
     A method for preparing the compound of formula (IV) or the tautomer, mesomer, racemate, enantiomer, diastereomer thereof, or mixture thereof, or the pharmaceutically acceptable salt thereof according to the present disclosure, comprises the following step of:  
     subjecting a compound of formula (IA) and a compound of (IIIB) to a condensation reaction in the presence of an alkaline reagent to obtain the compound of formula (IV), wherein: the alkaline reagent is preferably N,N-diisopropyl ethylamine; and R 1  to R 8 , m and n are as defined in the compound of formula (IV). 
     
       
         
         
             
             
         
       
     
     A method for preparing the compound of formula (IV-P1) or the tautomer, mesomer, racemate, enantiomer, diastereomer thereof, or mixture thereof, or the pharmaceutically acceptable salt thereof according to the present disclosure, comprises the following step of:  
     subjecting a compound of formula (IA-P1) and a compound of (IIIB) to a condensation reaction in the presence of an alkaline reagent to obtain the compound of formula (IV-P1), wherein: the alkaline reagent is preferably N,N-diisopropyl ethylamine; and R 1  to R 8 , m and n are as defined in the compound of formula (IV). 
     
       
         
         
             
             
         
       
     
     A method for preparing the compound of formula (IV-P2) or the tautomer, mesomer, racemate, enantiomer, diastereomer thereof, or mixture thereof, or the pharmaceutically acceptable salt thereof according to the present disclosure, comprises the following step of:  
     subjecting a compound of formula (IA-P2) and a compound of (IIIB) to a condensation reaction in the presence of an alkaline reagent to obtain the compound of formula (IV-P2), wherein: the alkaline reagent is preferably N,N-diisopropyl ethylamine; R 1  to R 8 , m and n are as defined in the compound of formula (IV); and the condensation agent used in the condensation reaction is preferably 2-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate. 
     The alkaline reagent in the above reactions includes organic bases and inorganic bases. The organic bases include, but are not limited to, triethylamine, N,N-diisopropylethylamine, n-butyllithium, lithium diisopropylamide, potassium acetate, sodium tert-butoxide and potassium tert-butoxide. The inorganic bases include, but are not limited to, sodium hydride, potassium phosphate, sodium carbonate, sodium acetate, potassium acetate, potassium carbonate, cesium carbonate, sodium hydroxide, lithium hydroxide and potassium hydroxide; preferably, N,N-diisopropylethylamine. 
     The above reactions are carried out in a solvent. The solvent used includes, but is not limited to: acetic acid, methanol, ethanol, n-butanol, toluene, tetrahydrofuran, dichloromethane, petroleum ether, ethyl acetate, n-hexane, dimethyl sulfoxide, 1,4-dioxane, ethylene glycol dimethyl ether, water or N,N-dimethylformamide and the mixture thereof, preferably N,N-dimethylformamide. 
     The condensation agent in the above condensation reactions includes, but is not limited to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N,N′-dicyclohexyl carbodiimide, N,N,N′,N′-diisopropyl carbodiimide, O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate, 1-hydroxybenzotriazole, 1-hydroxy-7-azobenzotriazole, O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate, 2-(7-azobenzotriazole)-N,N,N′,N′-tetramethyluronium hexafluorophosphate, 2-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate, benzotriazol-1-yloxytri s(dimethylamino)phosphonium hexafluorophosphate and benzotriazol-1-yl-oxytripyrrolyl phosphate hexafluorophosphate, preferably 2-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate. 
     The details of one or more embodiments of the present disclosure are set forth in the above specification. Although any methods and materials similar or identical to those described herein can be used to perform or test the present disclosure, the preferred methods and materials are described below. Through the specification and claims, other features, purposes and advantages of the present disclosure will be apparent. In the specification and claims, unless the context clearly indicates otherwise, the singular form includes the plural referent. Unless otherwise defined, all technical and scientific terms used herein have the general meanings as understood by the person of ordinary skill in the art to which the present disclosure belongs. All patents and publications cited in the specification are incorporated by reference. The following examples are provided to more fully illustrate the preferred embodiments of the present disclosure. These examples should not be construed as limiting the scope of the present disclosure in any way, and the scope of the present disclosure is defined by the claims. 
    
    
     DETAILED DESCRIPTION 
     EXAMPLES 
     The structures of the compounds were identified by nuclear magnetic resonance (NMR) and/or mass spectrometry (MS). NMR shifts (δ) are given in 10 −6  (ppm). NMR is determined by a Bruker AVANCE-400 machine. The solvents for determination are deuterated-dimethyl sulfoxide (DMSO-d 6 ), deuterated-chloroform (CDCl 3 ) and deuterated-methanol (CD 3 OD), and the internal standard is tetramethylsilane (TMS). 
     MS was determined by an Agilent 1200/1290 DAD-6110/6120 Quadrupole MS liquid chromatograph/mass spectrometer (manufacturer: Agilent, MS model: 6110/6120 Quadrupole MS), waters ACQuity UPLC-QD/SQD (manufacturer: waters, MS model: waters ACQuity Qda Detector/waters SQ Detector), THERMO Ultimate 3000-Q Exactive (manufacturer: THERMO, MS model: THERMO Q Exactive). 
     High performance liquid chromatography (HPLC) was determined on an Agilent HPLC 1200DAD, Agilent HPLC 1200VWD and Waters HPLC e2695-2489 high pressure liquid chromatograph. 
     Chiral HPLC was determined on an Agilent 1260 DAD high performance liquid chromatograph. 
     Preparative HPLC was carried out on Waters 2545-2767, Waters 2767-SQ Detecor2, Shimadzu LC-20AP and Gilson GX-281 preparative chromatographs. 
     Chiral preparation was carried out on a Shimadzu LC-20AP preparative chromatograph. 
     CombiFlash rapid preparation instrument used was Combiflash Rf200 (TELEDYNE ISCO). 
     Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plate was used as the thin layer silica gel chromatography (TLC) plate. The dimension of the silica gel plate used in TLC was 0.15 mm to 0.2 mm, and the dimension of the silica gel plate used in product purification was 0.4 mm to 0.5 mm. 
     Yantai Huanghai 200 to 300 mesh silica gel was generally used as a carrier for silica gel column chromatography. 
     The average kinase inhibition rates and IC 50  values were determined by a NovoStar microplate reader (BMG Co., Germany). 
     The known starting materials of the present disclosure can be prepared by the known methods in the art, or can be purchased from ABCR GmbH &amp; Co. KG, Acros Organics, Aldrich Chemical Company, Accela ChemBio Inc., Dan Chemical Company etc. 
     Unless otherwise stated, the reactions can be carried out under argon atmosphere or nitrogen atmosphere. 
     Argon atmosphere or nitrogen atmosphere means that a reaction flask is equipped with an argon or nitrogen balloon (about 1 L). 
     Hydrogen atmosphere means that a reaction flask is equipped with a hydrogen balloon (about 1 L). 
     Pressurized hydrogenation reaction was performed on a Parr 3916EKX hydrogenation instrument and a Qinglan QL-500 hydrogen generator or HC2-SS hydrogenation instrument. 
     In hydrogenation reactions, the reaction system was generally vacuumed and filled with hydrogen, and the above operation was repeated three times. 
     CEM Discover-S 908860 type microwave reactor was used in microwave reactions. 
     Unless otherwise stated in the examples, the solution refers to an aqueous solution. 
     Unless otherwise stated in the examples, the reaction temperature is room temperature from 20° C. to 30° C. 
     The single configuration in the examples can be confirmed by the synthesis from chiral raw materials or single crystal structure. 
     The reaction process in the examples was monitored by thin layer chromatography (TLC). The developing solvent used in the reactions, the eluent system in column chromatography and the developing solvent system in thin layer chromatography for purification of the compounds included: A: dichloromethane/methanol system, B: n-hexane/ethyl acetate system, C: petroleum ether/ethyl acetate system, D: acetone, E: dichloromethane/acetone system, F: ethyl acetate/dichloromethane system, G: ethyl acetate/dichloromethane/n-hexane, H: ethyl acetate/dichloromethane/acetone. The ratio of the volume of the solvent was adjusted according to the polarity of the compounds, and a small quantity of alkaline reagent such as triethylamine or acidic reagent such as acetic acid could also be added for adjustment. 
     Example 1 
     (S)-2-(2-(5-Chloro-2-((tetrahydro-2H-pyran-4-yl)amino)pyrimidin-4-yl)-4-oxo-6,7-dihydro thieno[3,2-c]pyridin-5(4H)-yl)-N-(1-(3-chlorophenyl)-2-hydroxyethyl)acetamide 1 
     
       
         
         
             
             
         
       
       
         
         
             
             
         
       
     
     Step 1 
     tert-Butyl 2-(2-bromo-4-oxo-6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl)acetate 1c 
     Compound 2-bromo-6,7-dihydrothieno[3,2-c]pyridin-4(5H)-one 1a (600 mg, 2.58 mmol) was dissolved in 10 mL of tetrahydrofuran, followed by the addition of 1 M a solution of lithium hexamethyldisilazide in tetrahydrofuran (3.8 mL, 3.87 mmol). The reaction solution was stirred at −78° C. for 1 hour, followed by the addition of tert-butyl 2-chloroacetate 1b (622 mg, 4.13 mmol). The reaction solution was slowly warmed up to room temperature and reacted for 15 hours. 30 mL of water was added, and the reaction solution was extracted with ethyl acetate (20 mL×3). The organic phase was washed with saturated sodium chlorate solution, dried over anhydrous sodium sulfate, and then concentrated under reduced pressure. The residue was purified by silica gel column chromatography with developing solvent system C to obtain the title compound 1c (900 mg), yield: 99%. 
     MS m/z (ESI): 346.5 [M+1]. 
     Step 2 
     tert-Butyl 2-(2-(2, 5-dichloropyrimidin-4-yl)-4-oxo-6, 7-dihydrothieno[3 ,2-c]pyridin-5(4H)-yl)acetate 1e 
     2,4,5-Trichloropyrimidine 1d (200 mg, 1.09 mmol), hexamethyldistannane (375 mg, 1.09 mmol), lithium chloride (65 mg, 1.09 mmol), and tetrakis(triphenylphosphine)palladium (163 mg, 0.15 mmol) were mixed and suspended in 6 mL of dioxane, and then the reaction solution was heated to 100° C. and stirred for 8 hours. The reaction solution was cooled to room temperature, followed by the addition of compound 1c (85 mg, 0.25 mmol), cuprous iodide (30 mg, 0.16 mmol) and bis(triphenylphosphine)palladium (II) dichloride (43 mg, 0.07 mmol). The reaction solution was heated to 105° C. and stirred for 3 hours. 5 mL of saturated potassium fluoride solution was added, and the reaction solution was stirred for 2 hours, and then extracted with ethyl acetate. The organic phase was concentrated under reduced pressure. The residue was purified by thin layer chromatography with developing solvent system C to obtain the title compound 1e (38 mg), yield: 25.3%. 
     MS m/z (ESI): 414.3 [M+1]. 
     Step 3 
     tert-Butyl 2-(2-(5-chloro-2((tetrahydro-2H-pyran-4-yl)amino)pyrimidin-4-yl)-4-oxo-6,7-dihydrothien o[3,2-c]pyridin-5(4H)-yl)acetate 1g 
     Compound 1e (38 mg, 0.09 mmol) was dissolved in 2 mL tetrahydrofuran solvent, followed by the addition of tetrahydro-2H-pyran-4-amine 1f (18 mg, 0.18 mmol, Shanghai Bide Pharmatech Ltd.) and N,N-diisopropyl ethylamine (60 mg, 0.45 mmol). The reaction solution was reacted at 120° C. in microwave reactor for 1 hour, and then concentrated under reduced pressure. The residue was purified by thin layer chromatography with developing solvent system C to obtain title compound 1g (30 mg), yield: 68.2%. 
     MS m/z (ESI): 479.3 [M+1]. 
     Step 4 
     2-(2-(5-Chloro-2-((tetrahydro-2H-pyran-4-yl)amino)pyrimidin-4-yl)-4-oxo-6,7-dihydrothie no[3,2-c]pyridin-5(4H)-yl)acetic acid 1h 
     Compound 1g (18 mg, 37 μmol) was dissolved in 2 mL of dichloromethane, followed by the addition of trifluoroacetic acid (0.3 mL). The reaction solution was stirred for 2 hours, and then concentrated under reduced pressure to obtain the crude title compound 1h, which was directly used in the next step. 
     MS m/z (ESI): 423.3 [M+1]. 
     Step 5 (S)-2-(2-(5-Chloro-2((tetrahydro-2H-pyran-4-yl)amino)pyrimidin-4-yl)-4-oxo-6,7-di hydrothieno[3 ,2-c]pyridin-5(4H)-yl)-N-(1-(3-chlorophenyl)-2-hydroxyethyl)acetamide 1 
     The crude compound 1h (18 mg, 42 μmol), (S)-2-amino-2-(3-chlorophenyl) ethan-1-ol 1i (14 mg, 81 Shanghai Bide Pharmatech Ltd.), 2-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (15 mg, 1.09 mmol), and N,N-diisopropyl ethylamine (27 mg, 200 μmol) were dissolved in 2 mL of N,N-dimethyl formamide, and the reaction solution was stirred for 2 hours. The title compound 1 (5 mg) was obtained by preparation and purification, yield: 20.4%. 
     MS m/z (ESI): 576.3 [M+1]. 
       1 H NMR (400 MHz, CD 3 OD): δ 8.45 (s, 1H), 8.22 (s, 1H), 7.49-7.52 (m, 1H), 7.20-7.22 (m, 3H), 4.98-4.99 (m, 1H), 3.97-4.01 (m, 3H), 3.88-3.91 (m, 4H), 3.72-3.75 (m, 4H), 3.57-3.61 (s, 2H), 1.96-1.98 (m, 2H), 1.58-1.61 (m, 2H). 
     Example 2 
     (S)-2-(2-(5-Chloro-2-((tetrahydro-2H-pyran-4-yl)amino)pyrimidin-4-yl)-6,6-dimethyl-4-ox o-4,6-dihydro-5H-thieno[2,3-c]pyrrol-5-yl)-N-(1-(3-chlorophenyl)-2-hydroxyethyl)acetami de 2 
     
       
         
         
             
             
         
       
       
         
         
             
             
         
       
     
     Step 1 
     6,6-Dimethyl-5,6-dihydro-4H-thieno[2,3-c]pyrrol-4-one 2b 
     6,6-Dimethyl-5,6-dihydro-4H-thieno[2,3-c]pyrrol-4-one 2a (180 mg, 1 mmol, prepared according to the method disclosed in patent application “WO2016106029”) was dissolved in 10 mL of N,N-dimethyl formamide, followed by the addition of sodium hydrogen (82 mg, 2.1 mmol) at 0° C., and the reaction solution was stirred for 1 hour. Methyl chloroacetate (233 mg, 2.1 mmol) was added, and the reaction solution was stirred overnight. Ethyl acetate (150 mL) was added, and the reaction solution was washed with water. The organic phase was dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography with developing solvent system C to obtain the title compound 2b (130 mg), yield: 51%. 
     MS m/z (ESI): 240.1 [M+1]. 
     Step 2 
     Methyl 2-(2-bromo-6,6-dimethyl-4-oxo-4,6-dihydro-5H-thieno[2,3-c]pyrrol-5-yl)acetate 2c 
     Compound 2b (130 mg, 0.54 mmol) was dissolved in 10 mL of acetonitrile, followed by the addition of N-bromosuccinimide (97 mg, 0.54 mmol) at 0° C. The reaction solution was stirred for 16 hours, and then concentrated to dryness by rotary evaporation. The residue was purified by silica gel column chromatography with developing solvent system C to obtain the title compound 2c (120 mg), yield: 69%. 
     MS m/z (ESI): 318.2 [M+1]. 
     Step 3 
     Methyl 2-(2-(2,5-dichloropyrimidin-4-yl)-6,6-dimethyl-4-oxo-4,6-dihydro-5H-thieno[2,3-c]pyrrol-5-yl)acetate 2d 
     Compound 2c (110 mg, 0.35 mmol) was dissolved in 20 mL of dioxane, followed by the addition of 4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bis(1,3,2-dioxaborolane) (210 mg, 0.83 mmol), potassium acetate (100 mg, 1.0 mmol), and [1,1′-bis(diphenylphosphino)ferrocene]palladium (II) dichloride (25 mg, 0.03 mmol). After the addition was completed, the reaction system was purged with argon three times, and stirred at 90° C. for 2 hours. The reaction solution was cooled to room temperature, followed by the addition of 2,4,5-trichloropyrimidine (95 mg, 0.52 mmol), anhydrous potassium carbonate (72 mg, 0.52 mmol), and tetrakis(triphenylphosphine)palladium (40 mg, 0.03 mmol). 0.3 mL of water was added dropwise. After the addition was completed, the reaction system was purged with argon three times, and stirred at 90° C. for 2 hours. The reaction solution was cooled to room temperature and filtered. The filtrate was concentrated to obtain compound 2d (60 mg). The product was directly used in the next step without purification. 
     MS m/z (ESI): 386.1 [M+1]. 
     Step 4 
     Methyl 2-(2-(5-chloro-2-((tetrahydro-2H-pyran-4-yl)amino)pyrimidin-4-yl)-6,6-dimethyl-4-oxo-4, 6-dihydro-5H-thieno[2,3-c]pyrrol-5-yl)acetate 2e 
     The crude compound 2d (60 mg, 0.16 mmol) was dissolved in 5 mL tetrahydrofuran, followed by the addition of tetrahydropyran-4-amine (24 mg, 0.24 mmol) and N,N-diisopropyl ethylamine (100 mg, 1.0 mmol). The reaction solution was reacted at 100° C. in microwave reactor for 1 hour, and then concentrated under reduced pressure. The residue was purified by silica gel column chromatography with developing solvent system C to obtain the title compound 2e (30 mg), yield: 43%. 
     MS m/z (ESI): 451.1 [M+1]. 
     Step 5 
     2-(2-(5-Chloro-2-((tetrahydro-2H-pyran-4-yl)amino)pyrimidin-4-yl)-6,6-dimethyl-4-oxo-4, 6-dihydro-5H-thieno[2,3-c]pyrrol-5-yl)acetic acid 2f 
     Compound 2e (30 mg, 0.06 mmol) was dissolved in 3 mL of methanol and 0.5 mL of water, followed by the addition of lithium hydroxide (16 mg, 0.7 mmol), and the reaction solution was reacted for 1 hour. 1 M diluted hydrochloric acid was added dropwise, and the reaction solution was adjusted to pH 3. The title crude compound 2f (29 mg) was obtained after concentration. The product was directly used in the next step without purification. 
     MS m/z (ESI): 437.2 [M+1]. 
     Step 6 
     (S)-2-(2-(5-Chloro-2-((tetrahydro-2H-pyran-4-yl)amino)pyrimidin-4-yl)-6,6-dimethyl-4-ox o-4,6-dihydro-5H-thieno[2,3-c]pyrrol-5-yl)-N-(1-(3-chlorophenyl)-2-hydroxyethyl)acetami de 2 
     The crude compound 2f (29 mg, 0.06 mmol) was dissolved in 5 mL of N,N-dimethyl formamide, followed by the addition of 2-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (15 mg, 0.06 mmol), compound 1i (11 mg, 0.06 mmol, Shanghai Bide Pharmatech Ltd.), and N,N-diisopropyl ethylamine (100 mg, 1.0 mmol). The reaction solution was reacted overnight, and then filtered. The residue was purified by silica gel column chromatography with developing solvent system A to obtain 2 (5 mg), yield: 13%. 
     MS m/z (ESI): 590.1 [M+1]. 
       1 H NMR (400 MHz, CD 3 OD): δ 8.30 (s, 2H), 7.41-7.28 (m, 4H), 4.99-4.97 (m, 1H), 4.25 (d, 2H), 4.02-4.00 (m, 3H), 3.80-3.78 (m, 2H), 3.76-3.58 (m, 2H), 1.66-1.58 (m, 2H), 1.66-1.58 (m, 8H). 
     Examples 3-P1 and 3-P2 
     (S)-2-(2-(5-Chloro-2-((tetrahydro-2H-pyran-4-yl)amino)pyrimidin-4-yl)-4-oxo-6,7-dihydro thieno[3,2-c]pyridin-5(4H)-yl)-N-((S)-2-hydroxy-1-(m-methylphenyl)ethyl)propionamide 3-P1 
     (R)-2-(2-(5-Chloro-2-((tetrahydro-2H-pyran-4-yl)amino)pyrimidin-4-yl)-4-oxo-6,7-dihydro thieno[3,2-c]pyridin-5(4H)-yl)-N-((S)-2-hydroxy-1-(m-methylphenyl)ethyl)propionamide 3-P2 
     
       
         
         
             
             
         
       
       
         
         
             
             
         
       
       
         
         
             
             
         
       
     
     Step 1 
     tert-Butyl 2-(2-bromo-4-oxo-6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl)propionate 3b 
     Compound 1a (800 mg, 3.44 mmol) was dissolved in 10 mL of tetrahydrofuran, followed by the addition of 1 M a solution of lithium hexamethyldisilazide in tetrahydrofuran (5.2 mL, 5.20 mmol). The reaction solution was stirred at −78° C. for 1 hour, followed by the addition of tert-Butyl 2-bromopropionate 3a (622 mg, 4.13 mmol, Tokyo Chemical Industry (Shanghai) Co., Ltd.). The reaction solution was slowly warmed up to room temperature, and then reacted for 15 hours. 30 mL of water was added, and the reaction solution was extracted with ethyl acetate (20 mL×3). The organic phase was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and then concentrated under reduced pressure. The residue was purified by silica gel column chromatography with developing solvent system C to obtain the title compound 3b (1.10 g), yield: 88.6%. 
     MS m/z (ESI): 360.2 [M+1]. 
     Step 2 
     tert-Butyl 2-(2-(2,5-dichloropyrimidin-4-yl)-4-oxo-6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl)propion ate 3c 
     Compound 1d (200 mg, 1.09 mmol), hexarnethyldiscannane (375 mg, 1.09 mmol), lithium chloride (65 mg, 1.09 mmol), tetrakis(triphenylphosphine)palladium (163 mg, 0.15 mmol) were mixed and suspended in 6 mL of dioxane, and then the reaction solution was heated to 100° C. and stirred for 8 hours. The reaction solution was cooled to room temperature, followed by the addition of compound 3b (115 mg, 0.32 mmol), cuprous iodide (30 mg, 0.16 mmol), and bis(triphenylphosphine) palladium (II) dichloride (43 mg, 0.07 mmol). The reaction solution was heated to 105° C. and stirred for 3 hours. Saturated potassium fluoride solution was added, and the reaction solution was stirred for 2 hours, and then extracted with ethyl acetate. The organic phases were combined and concentrated under reduced pressure. The residue was purified by thin layer chromatography with developing solvent system C to obtain the title compound 3c (50 mg), yield: 31.3%. 
     MS m/z (ESI): 428.3 [M+1]. 
     Step 3 
     tert-Butyl 2-(2-(5-chloro-2-((tetrahydro-2H-pyran-4-yl)amino)pyrimidin-4-yl)-4-oxo-6, 7-dihydro thieno[3,2-c]pyridin-5(4H)-yl)propionate 3d 
     Compound 3c (50 mg, 0.12 mmol) was dissolved in 2 mL tetrahydrofuran, followed by the addition of 4-aminotetrahydropyran if (23 mg, 0.24 mmol) and N,N-diisopropyl ethylamine (75 mg, 0.58 mmol). The reaction solution was reacted at 120° C. in microwave reactor for 1 hour, and then concentrated under reduced pressure. The residue was purified by thin layer chromatography with developing solvent system C to obtain the title compound 3d (50 mg), yield: 86.8%. 
     MS m/z (ESI): 493.3 [M+1]. 
     Step 4 
     2-(2-(5-Chloro-2-((tetrahydro-2H-pyran-4-yl)amino)pyrimidin-4-yl)-4-oxo-6,7-dihydro thieno[3,2-c]pyridin-5(4H)-yl)propanoic acid 3e 
     Compound 3d (20 mg, 40 μmol) was dissolved in 2 mL of dichloromethane, followed by the addition of trifluoroacetic acid (0.7 mL), and the reaction solution was stirred for 2 hours. The reaction solution was concentrated under reduced pressure to obtain the crude title compound 3e (40 mg). The product was directly used in the next step without purification. 
     MS m/z (ESI): 437.3 [M+1]. 
     Step 5 
     (S)-2-(2-(5-Chloro-2-((tetrahydro-2H-pyran-4-yl)amino)pyrimidin-4-yl)-4-oxo-6,7-dihydro thieno[3,2-c]pyridin-5(4H)-yl)-N-((S)-2-hydroxy-1-(m-methylphenyl)ethyl)propionamide 3-P1 
     (R)-2-(2-(5-Chloro-2-((tetrahydro-2H-pyran-4-yl)amino)pyrimidin-4-yl)-4-oxo-6,7-dihydro thieno[3,2-c]pyridin-5(4H)-yl)-N-((S)-2-hydroxy-1-(m-methylphenyl)ethyl)propionamide 3-P2 
     The crude compound 3e (40 mg, 91 μmol), (S)-2-amino-2-(m-methylphenyl) ethan-l-ol 3f (27 mg, 180 Shanghai Bide Pharmatech Ltd.), 2-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (40 mg, 106 μmol), and N,N-diisopropyl ethylamine (59 mg, 456 μmol) were dissolved in 2mL N,N-dimethyl formamide. The reaction solution was stirred for 2 hours, and then concentrated under reduced pressure. The residue was purified by preparative HPLC (instrument model: Gilson 281, chromatographic column: Sharpsil-T, Prep 21.2*150 mm, 5 μm, C18; mobile phase: A-water (0.1% trifluoroacetic acid), B-acetonitrile; flow rate: 30 mL/min; column temperature: room temperature) to obtain the title compounds 3-P1 and 3-P2 (11 mg, 9 mg). 
     3-P1 Single Configuration Compound (Shorter Retention Time) 
     MS m/z (ESI): 570.1 [M+1]. 
     HPLC analysis: Retention time 11.21 min, purity: 98.2% (chromatographic column: Sharpsil-T, Prep 21.2*150 mm; 5 μm; mobile phase: A-water (0.1% trifluoroacetic acid), B-acetonitrile, gradient ratio: A 5%-95%). 
       1 H NMR (400 MHz, CD 3 OD): δ 8.45 (s, 1H), 8.31 (s, 1H), 7.03-7.19 (m, 4H), 5.36-5.39 (m, 1H), 4.96-4.98 (m, 1H), 3.97-4.01 (m, 3H), 3.74-3.77 (m, 2H), 3.52-3.56 (m, 1H), 3.51-3.53 (m, 3H), 3.01-3.03 (m, 2H), 2.25 (s, 3H), 2.02 (d, 2H), 1.59-1.63 (m, 2H), 1.44 (d, 3H). 
     3-P2 Single Configuration Compound (Longer Retention Time) 
     MS m/z (ESI): 570.3 [M+1]. 
     HPLC analysis: Retention time 12.14 min, purity: 96.2% (chromatographic column: Sharpsil-T, Prep 21.2*150 mm; 5 μm; mobile phase: A-water (0.1% trifluoroacetic acid), B-acetonitrile, gradient ratio: A 5%-95%). 
       1 H NMR (400 MHz, CD 3 OD): δ 8.46 (s, 1H), 8.27 (s, 1H), 7.07-7.23 (m, 4H), 5.32-5.35 (m, 1H), 4.96-4.98 (m, 1H), 3.97-4.01 (m, 3H), 3.73-3.76 (m, 4H), 3.52-3.56 (m, 2H), 3.22-3.25 (m, 1H), 3.15-3.17 (m, 1H), 2.33 (s, 3H), 2.01-2.03 (m, 2H), 1.59-1.63 (m, 2H), 1.46 (d, 3H). 
     Examples 4-P1 and 4-P2 
     (S)-2-(2-(5-Chloro-2-((tetrahydro-2H-pyran-4-yl)amino)pyrimidin-4-yl)-4-oxo-6,7-di hydrothieno[3,2-c]pyridin-5(4H)-yl)-N-((S)-1-(3-chlorophenyl)-2-hydroxyethyl)propionami de 4-P1 
     (R)-2-(2-(5-Chloro-2((tetrahydro-2H-pyran-4-yl)amino)pyrimidin-4-yl)-4-oxo-6,7-di hydrothieno[3,2-c]pyridin-5(4H)-yl)-N-((S)-(3-chlorophenyl)-2-hydroxyethyl)propionamid e 4-P2 
     
       
         
         
             
             
         
       
       
         
         
             
             
         
       
     
     The crude compound 3e (53 mg, 121 μmol), compound 1i (50 mg, 240 μmol), 2-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (75 mg, 180 μmol), and N,N-diisopropyl ethylamine (78 mg, 600 μmol) were dissolved in 2 mL of N,N-dimethyl formamide. The reaction solution was stirred for 2 hours, and then concentrated under reduced pressure. The residue was purified by preparative HPLC (instrument model: Gilson 281; chromatographic column: Sharpsil-T, Prep 21.2*150 mm, 5 μm, C18; mobile phase: A-water (10 mM ammonium bicarbonate), B-acetonide; flow rate: 30 mL/min; column temperature: room temperature) to obtain the title compounds 4-P1 and 4-P2 (15 mg, 15 mg). 
     4-P1 Single Configuration Compound (Shorter Retention Time) 
     MS m/z (ESI): 590.3 [M+1]. 
     HPLC analysis: Retention time 10.75 min, purity: 98.6% (chromatographic column: Sharpsil-T, Prep 21.2*150 mm, 5 μm; mobile phase: A-water (10 mM ammonium bicarbonate), B-acetonitrile, gradient ratio: A 5%-95%). 
       1 H NMR (400 MHz, CD 3 OD): δ 8.43 (s, 1H), 8.26 (s, 1H), 7.35 (s, 1H), 7.22-7.24 (m, 3H), 5.30-5.35 (m, 1H), 4.98-4.99 (m, 1H), 3.95-3.97 (m, 3H), 3.76-3.78 (m, 2H), 3.70-3.72 (m, 1H), 3.55-3.57 (m, 3H), 3.04-3.06 (m, 2H), 2.00-2.02 (m, 2H), 1.58-1.62 (m, 2H), 1.44 (d, 3H). 
     4-P2 Single Configuration Compound (Longer Retention Time) 
     MS m/z (ESI): 590.3 [M+1]. 
     HPLC analysis: Retention time 11.75 min, purity: 99.1% (chromatographic column: Sharpsil-T, Prep 21.2*150 mm, 5 μm; mobile phase: A-water (10 mM ammonium bicarbonate), B-acetonitrile, gradient ratio: A 5%-95%). 
       1 H NMR (400 MHz, CD 3 OD): δ 8.40 (s, 1H), 8.23 (s, 1H), 7.39 (s, 1H), 7.24-7.27 (m, 3H), 5.30-5.34 (m, 1H), 4.98-4.99 (m, 1H), 4.00-4.02 (m, 3H), 3.76-3.78 (m, 4H), 3.50-3.53 (m, 2H), 3.22-3.26 (m, 1H), 3.13-3.15 (m, 1H), 2.00-2.02 (m, 2H), 1.58-1.62 (m, 2H), 1.43 (d, 3H). 
     Example 5 
     2-(2-(5-Chloro-2-((tetrahydro-2H-pyran-4-yl)amino)pyrimidin-4-yl)-6,6-dimethyl-4-oxo-4,6-dihydro-5H-thieno[2,3-c]pyrrol-5-yl)-N-((S)-1-(3-chlorophenyl)-2-hydroxy ethyl)propionamide 5 
     
       
         
         
             
             
         
       
       
         
         
             
             
         
       
     
     Step 1 
     tert-Butyl 2-(6,6-dimethyl-4-oxo-4,6-dihydro-5H-thieno[2,3-c]pyrrol-5-yl)propionate 5b 
     6,6-Dimethyl-5,6-dihydro-4H-thieno[2,3-c]pyrrol-4-one 5a (1 g, 6 mmol, prepared according to the method disclosed in the patent application “WO2016106029”) was dissolved in 10 mL of N,N-dimethyl formamide, followed by the addition of sodium hydrogen (458 mg, 12 mmol) at 0° C., and the reaction solution was stirred at 0° C. for 1 hour. tert-Butyl 2-bromopropionate (1.9 g, 9 mmol) was added, and the reaction solution was stirred overnight. Ethyl acetate (200 mL) was added, and the reaction solution was washed with water. The organic phase was dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by thin layer chromatography with developing solvent system A to obtain the title compound 5b (1.2 g), yield: 67.9%. 
     MS m/z (ESI): 296.1 [M+1]. 
     Step 2 
     tert-Butyl 2-(2-bromo-6,6-dimethyl-4-oxo-4, 6-dihydro-5H-thieno[2,3-c]pyrrol-5-yl)propionate 5c 
     Compound 5b (1.2 g, 4.0 mmol) was dissolved in 20 mL of acetonitrile, followed by the addition of N-bromosuccinimide (730 mg, 4.0 mmol) at 0° C. The reaction solution was stirred for 16 hours, and then concentrated. The residue was purified by thin layer chromatography with developing solvent system A to obtain the title compound 5c (1.2 g), yield: 78.9%. 
     MS m/z (ESI): 374.2 [M+1]. 
     Step 3 
     tert-Butyl 2-(2-(2,5-dichloropyrimidin-4-yl)-6,6-dimethyl-4-oxo-4,6-dihydro-5H-thieno[2,3 ]pyrrol-5-yl)propionate 5d 
     Compound 5c (374 mg, 1 mmol) was dissolved in 20 mL dioxane, followed by the addition of 4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bis(1,3,2-dioxaborolane) (609 mg, 2.4 mmol), potassium acetate (294 mg, 3.0 mmol) and [1,1′-bis(diphenylphosphino)ferrocene] platinum (II) dichloride (73 mg, 0.1 mmol). After the addition was completed, the reaction system was purged with argon three times, and stirred at 90° C. for 2 hours. The reaction solution was cooled to room temperature, followed by the addition of 2,4,5-trichloropyrimidine (275 mg, 1.5 mmol), anhydrous potassium carbon (72 mg, 0.52 mmol), and tetrakis(triphenylphosphine)palladium (207 mg, 1.5 mmol). 1 mL of water was added dropwise. After the addition was completed, the reaction system was purged with argon three times and stirred at 90° C. for 2 hours. The reaction solution was cooled to room temperature and filtered. The filtrate was concentrated, and the residue was purified by thin layer chromatography with developing solvent system C to obtain compound 5d (150 mg), yield: 34%. 
     MS m/z (ESI): 442.1 [M+1]. 
     Step 4 
     tert-Butyl 2-(2-(5-chloro-2-((tetrahydro-2H-pyran-4-yl)amino)pyrimidin-4-yl)-6,6-dimethyl-4-oxo-4, 6-dihydro-5H-thieno[2,3-c]pyrrol-5-yl)propionate 5e 
     The crude compound 5d (150 mg, 0.34 mmol) was dissolved in 5 mL of tetrahydrofuran, followed by the addition of compound if (52 mg, 0.51 mmol) and N,N-diisopropyl ethylamine (100 mg, 1.0 mmol). The reaction solution was reacted at 100° C. in microwave reactor for 1 hour, and then concentrated under reduced pressure. The residue was purified by thin layer chromatography with developing solvent system A to obtain compound 5e (80 mg), yield: 46.5%. 
     MS m/z (ESI): 407.1 [M+1]. 
     Step 5 
     2-(2-(5-Chloro-2-((tetrahydro-2H-pyran-4-yl)amino)pyrimidin-4-yl)-6,6-dimethyl-4-oxo-4,6-dihydro-5H-thieno[2,3-c]pyrrol-5-yl)propanoic acid 5f 
     Compound 5e (80 mg, 0.16 mmol) was dissolved in 5 mL of dichloromethane, followed by the dropwise addition of 1 mL of trifluoroacetic acid. The reaction solution was reacted for 1 hour, and then concentrated to obtain the title compound 5f (71 mg), yield: 100%. 
     MS m/z (ESI): 450.8 [M+1]. 
     Step 6 
     2-(2-(5-Chloro-2-((tetrahydro-2H-pyran-4-yl)amino)pyrimidin-4-yl)-6,6-dimethyl-4-oxo-4,6-dihydro-5H-thieno[2,3-c]pyrrol-5-yl)-N-((S)-2-hydroxy-1-(m-methylphenyl)ethyl) propionamide 5 
     Compound 5f (71 mg, 0.16 mmol) was dissolved in 5 mL of N,N-dimethyl formamide, followed by the addition of 2-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (44 mg, 0.19 mmol), compound 3f (24 mg, 0.16 mmol) and N,N-diisopropyl ethylamine (41 mg, 0.3 mmol). The reaction solution was reacted overnight, and then concentrated. The residue was purified by thin layer chromatography with solvent system A to obtain compound 5 (15 mg), yield: 16%. 
     MS m/z (ESI): 583.8 [M+1]. 
       1 H NMR (400 MHz, CD 3 OD): δ 8.32 (s, 2H), 7.25-7.02 (m, 4H), 4.99-4.97 (m, 1H), 4.36-4.27 (m, 1H), 4.05-3.95 (m, 3H), 3.81-3.70 (m, 2H), 3.63-3.50 (m, 2H), 2.30 (d, 3H), 2.05-1.95 (m, 2H), 1.78-1.74 (m, 3H), 1.68-1.57 (m, 8H). 
     Example 6 
     (S)-N-(1-(3-Chlorophenyl)-2-hydroxyethyl)-2-(4-oxo-2-(2-((tetrahydro-2H-pyran-4-yl) amino)pyrimidin-4-yl)-6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl)acetamide 6 
     
       
         
         
             
             
         
       
       
         
         
             
             
         
       
     
     Step 1 
     tert-Butyl 2-(2-(2-(methylthio)pyrimidin-4-yl)-4-oxo-6, 7-dihydrothieno[3 ,2-c]pyridin-5 (4H)-yl)acetat e 6b 
     2-(Methylthio)-4-(n-butyltin)pyrimidine 6a (60 mg, 0.14 mmol, Tokyo Chemical Industry (Shanghai) Co., Ltd.), compound lc (50 mg, 0.14 mmol), cuprous iodide (10 mg, 0.05 mmol), and bis(triphenylphosphine)palladium (II) dichloride (20 mg, 0.03 mmol) were heated to 105° C. and stirred for 3 hours. 5 mL of saturated potassium fluoride solution was added, and the reaction solution was stirred for 2 hours, and then extracted with ethyl acetate. The organic phases were combined and concentrated under reduced pressure. The residue was purified by thin layer chromatography with developing solvent system C to obtain the title compound 6b (15 mg), yield: 26.5%. 
     MS m/z (ESI): 392.3 [M+1]. 
     Step 2 
     tert-Butyl 2-(2-(2-(methylsulfonyl)pyrimidin-4-yl)-4-oxo-6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl)a cetate 6c 
     Compound 6b (15 mg, 0.04 mmol) was dissolved in 2 mL of dichloromethane, followed by the addition of m-chloroperoxobenzoic acid (19 mg, 0.09 mmol). The reaction solution was reacted for 3 hours, and then washed with saturated sodium thiosulfate solution and saturated sodium chloride solution. The organic phases were combined, dried and concentrated under reduced pressure to obtain the title compound 6c (20 mg). The product was directly used in the next step without purification. 
     MS m/z (ESI): 424.3 [M+1]. 
     Step 3 
     tert-Butyl 2-(4-oxo-2-(2-((tetrahydro-2H-pyran-4-yl)amino)pyrimidin-4-yl)-6,7-dihydrothieno[3 ,2-c]p yridin-5(4H)-yl)acetate 6d 
     The crude compound 6c (10 mg, 23 μmol) was dissolved in 2 mL of tetrahydrofuran, followed by the addition of compound if (11 mg, 0.108 mmol) and N,N-diisopropyl ethylamine (15 mg, 0.108 mmol). The reaction was carried out in microwave reactor at 120° C. for 1 hour, and then concentrated under reduced pressure. The residue was purified by thin layer chromatography with developing solvent system C to obtain the title compound for 6d (5 mg), yield: 47.6%. 
     MS m/z (ESI): 445.3 [M+1]. 
     Step 4 
     2-(4-Oxo-2-(2-((tetrahydro-2H-pyran-4-yl)amino)pyrimidin-4-yl)-6,7-dihydrothieno[3 ,2-c] pyridin-5(4H)-yl)acetic acid 6e 
     Compound 6d (5 mg, 11 μmol) was dissolved in 1 mL of dichloromethane, followed by the addition of trifluoroacetic acid (0.3 mL). The reaction was stirred for 2 hours, and then concentrated under reduced pressure to obtain the crude title compound 6e (10 mg). 
     The product was directly used in the next step without purification. 
     MS m/z (ESI): 389.3 [M+1]. 
     Step 5 
     (S)-N-(1-(3-Chlorophenyl)-2-hydroxyethyl)-2-(4-oxo-2-(2-((tetrahydro-2H-pyran-4-yl) amino)pyrimidin-4-yl)-6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl)acetamide 6 
     The crude compound 6e (5 mg, 13 μmol), compound li (5 mg, 25 μmol), 2-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (10 mg, 13 μmol), and N,N-diisopropyl ethylamine (8 mg, 60 μmol) were dissolved in 1 mL of N,N-dimethyl formamide, and the reaction solution was stirred for 2 hours. The title compound 6 (4 mg) was obtained by preparation and purification, yield: 50.1%. 
     MS m/z (ESI): 542.3 [M+1]. 
       1 H NMR (400 MHz, CD 3 OD): δ 7.50 (s, 2H), 7.37-7.42 (m, 5H), 5.10-5.13 (m, 1H), 4.59 (s, 1H), 4.48 (t, 2H), 3.92-3.95 (m, 2H), 3.78-3.80 (m, 2H), 3.49-3.51 (m, 2H), 3.01 (s, 4H), 1.35-1.37 (m, 4H). 
     Example 7 
     (S)-N-(2-Hydroxy-1-(m-methylphenyl)ethyl)-2-(2-(5-methyl-2-((1-methyl-1H-pyrazol-5-yl) amino)pyrimidin-4-yl)-4-oxo-6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl)acetamide 7 
     
       
         
         
             
             
         
       
       
         
         
             
             
         
       
     
     Step 1 
     4-Chloro-5-methyl-2-(methyl sulfonyl)pyrimidine 7b 
     Compound 4-chloro-5-methyl-2-(methylthio)pyrimidine 7a (500 mg, 2.86 mmol, Tokyo Chemical Industry (Shanghai) Co., Ltd.) was dissolved in 10 mL of dichloromethane, followed by the addition of m-Chloroperoxobenzoic acid (1.270 g, 6.3 mmol). The reaction solution was stirred for 2 hours, and then washed with saturated sodium thiosulfate solution and saturated brine, dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain the crude title compound 7b (510 mg). The product was directly used in the next step without purification. 
     MS m/z (ESI): 207.2 [M+1]. 
     Step 2 
     4-Chloro-5-methyl-N-(1-methyl-1H-pyrazol-5-yl)pyrimidin-2-amine 7d 
     N-(1-methyl-1H-pyrazol-5-yl)formamide 7c (270 mg, 2.15 mmol, prepared according to the method disclosed in patent application “WO2017/80979”) was dissolved in N,N-dimethyl formamide, followed by the addition of sodium hydride (60%, 250 mg, 6.5 mmol) at 0° C. The reaction solution was stirred for 0.5 hours, followed by the addition of compound 7b (445 mg, 2.15 mmol). The reaction solution was further reacted for 2 hours. 20 mL of water was added, and the reaction solution was extracted with ethyl acetate. The organic phases were combined and concentrated under reduced pressure. The residue was purified by thin layer chromatography with developing solvent system C to obtain the title compound 7d (240 mg), yield: 49.7%. 
     MS m/z (ESI): 224.3 [M+1]. 
     Step 3 
     tert-Butyl 2-(2-(5-methyl-2-((1-methyl-1H-pyrazol-5-yl)amino)pyrimidin-4-yl)-4-oxo-6,7-dihydrothie no[3,2-c]pyridin-5(4H)-yl)acetate 7e 
     Compound 7d (80 mg, 0.36 mmol), hexamethyldistannane (140 mg, 0.43 mmol), lithium chloride (21 mg, 0.35 mmol) and tetrakis(triphenylphosphine)palladium (61 mg, 0.05 mmol) were mixed and suspended in 6 mL of dioxane, and then the reaction solution was heated to 100° C. and stirred for 8 hours. The reaction solution was cooled to room temperature, followed by the addition of compound lc (80 mg, 0.24 mmol), cuprous iodide (22 mg, 0.12 mmol), bis(triphenylphosphine)palladium (II) dichloride (32 mg, 0.06 mmol). The reaction solution was heated to 105° C. and stirred for 3 hours. Saturated potassium fluoride solution was added, and the reaction solution was stirred for 2 hours, and then extracted with ethyl acetate. The organic phases were combined and concentrated under reduced pressure. The residue was purified by thin layer chromatography with developing solvent system C to obtain the title compound 7e (50 mg), yield: 45.5%. 
     MS m/z (ESI): 455.3 [M+1]. 
     Step 4 
     2-(2-(5-Methyl-2-((1-methyl-1H-pyrazol-5-yl)amino)pyrimidin-4-yl)-4-oxo-6,7-dihydrothi eno[3,2-c]pyridin-5(4H)-yl)acetic acid 7f 
     Compound 7e (25 mg, 55 μmol) was dissolved in 2 mL of dichloromethane, followed by the addition of trifluoroacetic acid (0.7 mL). The reaction solution was stirred for 2 hours, and then concentrated under reduced pressure to obtain the crude title compound 7f, which was directly used in the next step. 
     MS m/z (ESI): 399.3 [M+1]. 
     Step 5 
     (S)-N-(2-Hydroxy-1-(m-methylphenyl)ethyl-2-(2-(5-methyl-2-((1-methyl-1H-pyrazol-5-yl) amino)pyrimidin-4-yl)-4-oxo-6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl)acetamide 7 
     The crude compound 7f (22 mg, 55 μmol), compound 3f (16 mg, 105 μmol), 2-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (38 mg, 106 μmol) and N,N-diisopropyl ethylamine (57 mg, 450 μmol) were dissolved in 2 mL of N,N-dimethyl formamide, and the reaction solution was stirred for 2 hours. The title compound 7 (15 mg) was obtained by preparation and purification, yield: 51.0%. 
     MS m/z (ESI): 532.5 [M+1]. 
       1 E1 NMR (400 MHz, CD 3 OD): δ 8.25 (s, 1H), 7.90 (s, 1H), 7.78 (s, 1H), 7.05-7.19 (m, 4H), 6.36 (s, 1H), 4.04-4.07 (m, 1H), 4.53 (s, 3H), 4.29 (s, 2H), 3.76-3.79 (m, 4H), 3.15-3.19 (m, 2H), 2.44 (s, 3H), 2.32 (s, 3H). 
     Examples 8-P1 and 8-P2 
     (S)-N-((S)-2-Amino-1-(3-chlorophenyl)ethyl)-2-(2-(5-chloro-2-((tetrahydro-2H-pyran-4-yl) amino)pyrimidin-4-yl)-4-oxo-6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl)propionamide 8-P1 
     (R)-N-((S)-2-Amino-1-(3-chlorophenyl)ethyl)-2-(2-(5-chloro-2-((tetrahydro-2H-pyran-4-yl) amino)pyrimidin-4-yl)-4-oxo-6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl)propionamide 8-P2 
     
       
         
         
             
             
         
       
       
         
         
             
             
         
       
     
     Step 1 
     N-((S)-2-Azido-1-(3-chlorophenyl)ethyl)-2-(2-(5-chloro-2-((tetrahydro-2H-pyran-4-yl)ami no)pyrimidin-4-yl)-4-oxo-6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl)propionamide 8b 
     The crude compound 3e (43 mg, 98 μmol), (S)-2-azido-1-(3-chlorophenyl)ethan-1-amine hydrochloride 8a (40 mg, 200 prepared according to the method disclosed in patent application US2016362407A1), 2-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (75 mg, 200 μmol), and N,N-diisopropyl ethylamine (63 mg, 490 μmol) were dissolved in 2 mL of N,N-dimethyl formamide. The reaction solution was stirred for 2 hours, and then concentrated under reduced pressure. The residue was purified by thin layer chromatography with developing solvent system C to obtain the title compound 8b (40 mg), yield: 66.0%. 
     MS m/z (ESI): 615.3 [M+1]. 
     Step 2 
     (S)-N-((S)-2-Amino-1-(3-chlorophenyl)ethyl)-2-(2-(5-chloro-2-((tetrahydro-2H-pyran-4-yl) amino)pyrimidin-4-yl)-4-oxo-6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl)propionamide 8-P1 
     (R)-N-((S)-2-Amino-1-(3-chlorophenyl)ethyl)-2-(2-(5-chloro-2-((tetrahydro-2H-pyran-4-yl) amino)pyrimidin-4-yl)-4-oxo-6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl)propionamide 8-P2 
     Compound 8b (40 mg, 95 μmol) was dissolved in 2 mL methanol solution, followed by the addition of palladium carbon hydrogenation catalyst (wet) (10%, 27 mg, 120 μmol). The reaction solution was stirred for 1 hour under hydrogen atmosphere. The reaction solution was filtered and concentrated under reduced pressure. The residue was purified by preparative HPLC (instrument model: Gilson 281; chromographic column: Sharpsil-T, Prep 30*150 mm, 5 μm, C18; mobile phase: A-water (0.1% trifluoroacetic acid), B-acetonide; flow rate: 30 mL/min; column temperature: room temperature) to obtain the title compounds 8-P1 and 8-P2 (18 mg, 18 mg). 
     8-P1 Single Configuration Compound (Shorter Retention Time): 
     MS m/z (ESI): 589.3 [M+1]. 
     HPLC analysis: Retention time 15.06 min, purity: 96.8% (chromatographic column: Sharpsil-T, Prep 30*150 mm, 5 μm; mobile phase: A-water (0.1% trifluoroacetic acid), B-acetonitrile, gradient ratio: A 5%-95%). 
       1 H NMR (400 MHz, CD 3 OD): δ 8.45 (s, 1H), 8.28 (s, 1H), 7.30-7.41 (m, 4H), 5.30-5.32 (m, 1H), 5.16-5.19 (m, 1H), 3.97-4.01 (m, 3H), 3.70-3.72 (m, 1H), 3.52-3.56 (m, 3H), 3.35-3.38 (m, 2H), 3.08-3.10 (m, 1H), 3.00-3.02 (m, 1H), 2.02-2.04 (m, 2H), 1.63-1.66 (m, 2H), 1.46 (d, 3H). 
     8-P2 Single Configuration Compound (Longer Retention Time): 
     MS m/z (ESI): 589.3 [M+1]. 
     HPLC analysis: Retention time 15.91 min, purity: 98.2% (chromatographic column: Sharpsil-T, Prep 30*150 mm, 5 μm; mobile phase: A-water (0.1% trifluoroacetic acid), B-acetonitrile, gradient ratio: A 5%-95%). 
       1 H NMR (400 MHz, CD 3 OD): δ 8.41 (s, 1H), 8.27 (s, 1H), 7.32-7.45 (m, 4H), 5.25-5.29 (m, 1H), 4.99-5.01 (m, 1H), 3.97-4.01 (m, 3H), 3.76-3.87 (m, 2H), 3.52-3.55 (m, 2H), 3.34-3.36 (m, 2H), 3.18-3.20 (m, 2H), 2.01-2.03 (m, 2H), 1.63-1.66 (m, 2H), 1.51 (d, 3H). 
     Examples 9-P1 and 9-P2 
     (S)-2-(2-(5-Chloro-2-((tetrahydro-2H-pyran-4-yl)amino)pyrimidin-4-yl)-4-oxo-6,7-dihydr othieno[3,2-c]pyridin-5(4H)-yl)-N-((S)-1-(3-fluoro-5-methoxyphenyl)-2-hydroxyethyl)prop ionamide 9-P1 
     (R)-2-(2-(5-Chloro-2-((tetrahydro-2H-pyran-4-yl)amino)pyrimidin-4-yl)-4-oxo-6,7-dihydr othieno[3,2-c]pyridin-5(4H)-yl)-N-((S)-1-(3-fluoro-5-methoxyphenyl)-2-hydroxyethyl)prop ionamide 9-P2 
     
       
         
         
             
             
         
       
       
         
         
             
             
         
       
     
     The crude compound 3e (53 mg, 121 μmol), compound 9a (33 mg, 180 μmol), 2-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (75 mg, 180 μmol), and N,N-diisopropyl ethylamine (78 mg, 600 μmol) were dissolved in 2 mL of N,N-dimethyl formamide. The reaction solution was stirred for 2 hours, and then concentrated under reduced pressure. The residue was purified by preparative HPLC (separation conditions: chromographic column: Sharpsil-T prep 30*150 mm, 5 μm, C18; mobile phase: A-water (0.1% TFA), B-acetonide; column temperature: room temperature, flow rate: 30 mL/min). The corresponding components were collected and concentrated under reduced pressure to obtain the title compounds 9-P1 and 9-P2 (20 mg, 20 mg). 
     9-P1 Single Configuration Compound (Shorter Retention Time): 
     MS m/z (ESI): 604.1 [M+1]. 
     Preparative HPLC: Retention time 19.20 min, purity: 98.8% (chromatographic column: Sharpsil-T Prep 30*150 mm; 5 μm; C18; mobile phase: A-water (0.1% TFA), B-acetonitrile; column temperature: room temperature, flow rate: 30 mL/min); 
       1 H NMR (400 MHz, CD 3 OD): δ 8.40 (s, 1H), 8.26 (d, 1H), 6.73 (s, 1H), 6.68 (d, 1H), 6.57 (d, 1H), 5.35-5.32 (m, 1H), 4.96-4.93 (m, 1H), 3.99-3.96 (m, 3H), 3.76-3.74 (m, 6H), 3.58-3.56 (m, 3H), 3.08-3.05 (m, 2H), 2.01-1.99 (m, 2H), 1.63-1.60 (m, 2H), 1.45 (d, 3H). 
     9-P2 Single Configuration Compound (Longer Retention Time): 
     MS m/z (ESI):604.1 [M+1]; 
     Preparative HPLC: Retention time 20.800 min, purity: 99.2% (chromatographic column: Sharpsil-T Prep 30*150 mm; 5 μm; C18; mobile phase: A-water (0.1% TFA), B-acetonitrile; column temperature: room temperature, flow rate: 30 mL/min); 
       1 H NMR (400 MHz, CD 3 OD): δ 8.42 (s, 1H), 8.25 (d, 1H), 6.75 (s, 1H), 6.68 (d, 1H), 6.59 (d, 1H), 5.36-5.32 (m, 1H), 4.94-4.92 (m, 1H), 4.01-3.98 (m, 3H), 3.77-3.74 (m, 7H), 3.58-3.56 (m, 2H), 3.25-3.23 (m, 1H), 3.14-3.12 (m, 1H), 2.02-1.99 (m, 2H), 1.66-1.63 (m, 2H), 1.45 (d, 3H). 
     Examples 10-P1 and 10-P2 
     (S)-N-((S)-1-(3-Fluoro-5-methoxyphenyl)-2-hydroxyethyl)-2-(2-(5-methyl-2-((1-methyl-1H-pyrazol-5-yl)amino)pyrimidin-4-yl)-4-oxo-6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl)propi onamide 10-P1 
     (R)-N-((S)-1-(3-Fluoro-5-methoxyphenyl)-2-hydroxyethyl)-2-(2-(5-methyl-2-((1-methyl-1 H-pyrazol-5-yl)amino)pyrimidin-4-yl)-4-oxo-6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl)pro pionamide 10-P2 
     
       
         
         
             
             
         
       
       
         
         
             
             
         
       
       
         
         
             
             
         
       
     
     Step 1 
     tert-Butyl 
     2-(2-(5-methyl-2-((1-methyl-1H-pyrazol-5-yl)amino)pyrimidin-4-yl)-4-oxo-6,7-dihydrothie no[3,2-c]pyridin-5(4H)-yl)propionate 10a 
     Compound 7d (80mg, 0.36 mmol), hexamethyldistannane (140 mg, 0.43 mmol), lithium chloride (21 mg, 0.35 mmol), tetrakis(triphenylphosphine)palladium (61 mg, 0.05 mmol) were mixed and suspended in 5 mL of dioxane, and then the reaction solution was heated to 105° C. and stirred for 8 hours. The reaction solution was cooled to room temperature, followed by the addition of compound 3b (130 mg, 0.36 mmol), cuprous iodide (35 mg, 0.18 mmol), and bis(triphenylphosphine)palladium (II) dichloride (49 mg, 0.07 mmol). The reaction solution was heated to 105° C. and stirred for 3 hours. Saturated potassium fluoride solution was added, and the reaction solution was stirred for 2 hours, and then extracted with ethyl acetate. The organic phases were combined and concentrated under reduced pressure. The residue was purified by thin layer chromatography with developing solvent system C to obtain the title compound 10a (40 mg), yield: 23.1%. 
     MS m/z (ESI): 469.1 [M+1]. 
     Step 2 
     2-(2-(5-Methyl-2-((1-methyl-1H-pyrazol-5-yl)amino)pyrimidin-4-yl)-4-oxo-6,7-dihydrothi eno[3,2-c]pyridin-5(4H)-yl)propanoic acid 10b 
     Compound 10a (40 mg, 85 μmol) was dissolved in 1 mL of dichloromethane, followed by the addition of trifluoroacetic acid (0.5 mL). The reaction solution was stirred for 2 hours, and then concentrated under reduced pressure to obtain the crude title compound 10b (35 mg), which was directly used in the next step. 
     MS m/z (ESI): 413.2 [M+1]. 
     Step 3 
     (S)-N-((S)-1-(3-Fluoro-5-methoxyphenyl)-2-hydroxyethyl)-2-(2-(5-methyl-2-((1-methyl-1H-pyrazol-5-yl)amino)pyrimidin-4-yl)-4-oxo-6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl)propi onamide 10-P1 
     (R)-N-((S)-1-(3-Fluoro-5-methoxyphenyl)-2-hydroxyethyl)-2-(2-(5-methyl-2-((1-methyl-1 H-pyrazol-5-yl)amino)pyrimidin-4-yl)-4-oxo-6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl)pro pionamide 10-P2 
     The crude compound 10b (35 mg, 85 μmol), compound 9a (18.9 mg, 102 μmol), 2-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (38 mg, 106 μmol), and N,N-diisopropyl ethylamine (57 mg, 450 μmol) were dissolved in 2 mL of N,N-dimethyl formamide. The reaction solution was stirred for 2 hours, and then concentrated under reduced pressure. The residue was purified by preparative HPLC with the separation conditions (chromographic column: Gemini 20*250 mm, 5 μm, C18; mobile phase: A-water (10 mmol ammonium acetate), B-acetonide; column temperature: room temperature; flow rate: 18 mL/min). The corresponding components were collected and concentrated under reduced pressure to obtain the title compounds 10-P1 and 10-P2 (10 mg, 10 mg). 
     10-P1 Single Configuration Compound (Shorter Retention Time): 
     MS m/z (ESI): 580.2[M+1]; 
     HPLC analysis: Retention time 10.83min, purity: 98.3% (chromatographic column: Sharpsil-T, Prep 21.2*150 mm, 5 μm; mobile phase: A-water (10 mM ammonium bicarbonate), B-acetonitrile); 
       1 H NMR (400 MHz, CDCl 3 ): δ 8.25 (s, 1H), 7.89 (s, 1H), 7.53 (d, 1H), 7.21 (d, 1H), 6.76 (s, 1H), 6.58-6.54 (m, 2H), 6.49 (d, 1H), 6.38 (d, 1H), 5.41-5.31 (m, 2H), 5.03-5.00 (m, 1H), 3.89-3.85 (m, 2H), 3.82 (s, 3H), 3.69 (s, 3H), 3.62-3.60 (m, 2H), 3.49-3.47 (m, 2H), 2.47 (s, 3H), 1.32 (d, 3H). 
     10-P2 Single Configuration Compound (Longer Retention Time): 
     MS m/z (ESI):580.2 [M+1]; 
     HPLC analysis: Retention time 14.93min, purity: 98.7% (chromatographic column: Sharpsil-T, Prep 21.2*150 mm, 5 μm; mobile phase: A-water(10 mM ammonium bicarbonate), B-acetonitrile); 
       1 H NMR (400 MHz, CDCl 3 ): δ 8.25 (s, 1H), 7.87 (s, 1H), 7.50 (d, 1H), 7.15 (d, 1H), 6.84 (s, 1H), 6.66-6.62 (m, 2H), 6.55 (d, 1H), 6.37 (d, 1H), 5.37-5.31 (m, 2H), 5.03-5.01 (m, 1H), 3.89-3.86 (m, 2H), 3.82 (s, 3H), 3.79 (s, 3H), 3.73-3.69 (m, 2H), 3.15-3.08 (m, 2H), 2.46 (s, 3H), 1.32 (d, 3H). 
     Example 11 
     (R)-N-((S)-1-(3-Fluoro-5-methoxyphenyl)-2-hydroxyethyl)-2-(4-oxo-2-(2-((tetrahydro-2H-pyran-4-yl)amino)pyrimidin-4-yl)-6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl)-propionamid e 11 
     
       
         
         
             
             
         
       
       
         
         
             
             
         
       
     
     Step 1 
     tert-Butyl 2-(2-(2-chloropyrimidin-4-yl)-4-oxo-6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl)propionate 11b 
     Compound 3b (2.0 g, 5.55 mmol), bis(pinacolato)diboron (4.2 g, 16.5 mmol), cuprous iodide (1.0 g, 5.55 mmol), tributylphosphine (1.9 g, 9.4 mmol) and potassium tert-butanol (934 mg, 8.3 mmol) were mixed and suspended in 30 mL tetrahydrofuran. Under argon atmosphere, the reaction solution was heated to 60° C. and stirred for 40 minutes. The reaction solution was cooled to room temperature and filtered through diatomaceous earth, followed by the addition of compound 2,4-dichloropyrimidine 11a (804 mg, 5.4 mmol), [1,1′-bis(diphenylphosphino)ferrocene]palladium (II) dichloride dichloromethane complex (457 mg, 0.54 mmol), sodium carbonate (1.1 g, 10.7 mmol), and 5 mL of water. Under argon atmosphere, the reaction solution was heated to 60° C. and stirred for 2 hours. Saturated sodium chloride solution was added, and the reaction solution was stirred for 2 hours, and then extracted with ethyl acetate. The organic phases were combined and concentrated under reduced pressure. The residue was purified by thin layer chromatography with developing solvent system C to obtain the title compound 11b (430 mg), yield: 20.2%. 
     MS m/z (ESI): 392.1 [M+1]. 
     Step 2 
     tert-Butyl 2-(4-oxo-2-(2-((tetrahydro-2H-pyran-4-yl)amino)pyrimidin-4-yl)-6,7-dihydrothieno[3 ,2-c]p yridin-5(4H)-yl)propionate 11c 
     Compound 11b (430 mg, 1.1 mmol) was dissolved in 10 mL of tetrahydrofuran, followed by the addition of 4-aminotetrahydropyran 1f (331 mg, 3.1 mmol) and N,N-diisopropyl ethylamine (423 mg, 3.3 mmol). The reaction solution was reacted at 110° C. in microwave reactor for 2 hours, and then concentrated under reduced pressure. The residue was purified by thin layer chromatography with the developing solvent system C to obtain the title compound 11c (150 mg), yield: 30.0%. 
     MS m/z (ESI): 459.2 [M+1]. 
     Step 3 
     2-(4-Oxo-2-(2-((tetrahydro-2H-pyran-4-yl)amino)pyrimidin-4-yl)-6,7-dihydrothieno[3,2-c] pyridin-5(4H)-yl)propanoic acid 11d 
     Compound 11c (115 mg, 0.25 mmol) was dissolved in 20 mL of dichloromethane, followed by the addition of trifluoroacetic acid (0.7 mL). The reaction solution was stirred for 2 hours, and then concentrated under reduced pressure to obtain the crude title compound 11d (100 mg). The product was directly used in the next step without purification. 
     MS m/z (ESI): 403.2 [M+1]. 
     Step 4 
     (R)-N-((S)-1-(3-Fluoro-5-methoxyphenyl)-2-hydroxyethyl)-2-(4-oxo-2-(2-((tetrahydro-2H-pyran-4-yl)amino)pyrimidin-4-yl)-6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl)-propionamide 11 
     The crude compound 11d (230 mg, 0.57 mmol), compound 9a (211 mg, 1.1 mmol), 2-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (270 mg, 1.1 mmol), and N,N-diisopropyl ethylamine (150 mg, 1.1 mmol) were dissolved in 10 mL of N,N-dimethyl formamide. The reaction solution was stirred for 2 hours, and then concentrated under reduced pressure. The residue was purified with preparative HPLC (instrument model: waters, chromatographic column: X-bridge Prep 30*150 mm, 5 μm, C18; mobile phase: A-water (10 mM ammonium acetate), B-acetonide; flow rate: 30 mL/min, column temperature: room temperature). At retention time 17.32 min, compound 11 (110 mg, compound in a single configuration with longer retention time) was obtained, yield: 30.7%. 
     MS m/z (ESI): 570.2 [M+1]. 
       1 H NMR (400 MHz, CDCl 3 ): δ 8.26 (s, 1H), 7.87 (s, 1H), 7.72 (d, 1H), 6.83 (d, 1H), 6.69 (d, 2H), 6.53 (d, 1H), 5.41 (dd, 2H), 5.07 (d, 1H), 4.03 (d, 3H), 3.88 (dd, 3.7 Hz, 1H), 3.81 (d, 5H), 3.70 (dt, 1H), 3.58 (t, 2H), 3.24-3.11 (m, 1H), 3.04 (dt, 1H), 2.16-1.97 (m, 3H), 1.61 (dt, 2H), 1.45 (d, 3H). 
     Example 12 
     (R)-2-(2-(5-Chloro-2-(((S)-1-hydroxypropan-2-yl)amino)pyrimidin-4-yl)-4-oxo-6,7-dihydr othieno[3,2-c]pyridin-5(4H)-yl)-N-((S)-1-(3-fluoro-5-methoxyphenyl)-2-hydroxyethyl)prop ionamide 12 
     
       
         
         
             
             
         
       
       
         
         
             
             
         
       
     
     Step 1 
     tert-Butyl 2-(2-(5-chloro-2-(((S)-1-hydroxypropan-2-yl)amino)pyrimidin-4-yl)-4-oxo-6,7-dihydrothie no[3,2-c]pyridin-5(4H)-yl)propionate 12b 
     Compound 3c (60 mg, 0.14 mmol) was dissolved in 2 mL of tetrahydrofuran, followed by the addition of (S)-2-aminopropan-1-ol 12a (21 mg, 0.28 mmol) and N,N-diisopropyl ethylamine (75 mg, 0.58 mmol). The reaction solution was reacted at 120° C. in microwave reactor for 1 hour, and then concentrated under reduced pressure. The residue was purified by thin layer chromatography with developing solvent system C to obtain the title compound 12b (35 mg), yield: 53.5%. 
     MS m/z (ESI): 467.3 [M+1]. 
     Step 2 
     2-(2-(5-Chloro-2-(((S)-1-hydroxypropan2-yl)amino)pyrimidin-4-yl)-4-oxo-6,7-dihydrothie no[3,2-c]pyridin-5(4H)-yl)propanoic acid 12c 
     Compound 12b (30 mg, 65 μmol) was dissolved in 2 mL of dichloromethane, followed by the addition of trifluoroacetic acid (0.7 mL). The reaction solution was stirred for 2 hours, and then concentrated under reduced pressure to obtain the crude title compound 12c (27 mg). The product was directly used in the next step without purification. 
     MS m/z (ESI): 411.3 [M+1]. 
     Step 3 
     (R)-2-(2-(5-Chloro-2-(((S)-1-hydroxypropan-2-yl)amino)pyrimidin-4-yl)-4-oxo-6,7-dihydr othieno[3,2-c]pyridin-5(4H)-yl)-N-((S)-1-(3-fluoro-5-methoxyphenyl)-2-hydroxyethyl)prop ionamide 12 
     The crude compound 12c (27 mg, 65 μmol), compound 9a (24 mg, 130 μmol, Shanghai Bide Pharmatech Ltd.), 2-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (22 mg, 93 μmol), and N,N-diisopropyl ethylamine (42 mg, 324 μmol) were dissolved in 2 mL of N,N-dimethyl formamide. The reaction solution was stirred for 2 hours, and then concentrated under reduced pressure. The residue was purified with preparative HPLC (instrument model: Gilson, chromatographic column: Sharpsil-T Prep 30*150 mm, 5 μm, C18; mobile phase: A-water (10 mM ammonium acetate), B-acetonide; flow rate: 30 mL/min, column temperature: room temperature). At retention time 14.75 min, compound 12 (15 mg, 39.5%, compound in a single configuration with longer retention time) was obtained. 
     MS m/z (ESI): 578.2[M+1]. 
       1 H NMR (400 MHz, CD 3 OD): δ 8.44 (s, 1H), 8.25 (s, 1H), 6.74 (s, 1H), 6.70 (d, 1H), 6.59 (d, 1H), 5.34-5.32 (m, 1H), 4.96-4.93(m, 1H), 4.08-4.05 (m, 1H), 3.78-3.75(m, 7H), 3.63-3.60 (m, 2H), 3.26-3.23 (m, 1H), 3.14-3.11 (m, 1H), 1.45 (d, 3H), 1.26 (d, 3H). 
     Example 13 
     (R)-2-(2-(5-Chloro-2-(((S)-1-hydroxypropan-2-yl)amino)pyrimidin-4-yl)-4-oxo-6,7-dihydr othieno[3,2-c]pyridin-5(4H)-yl)-N-((S)-2-hydroxy-1-(m-methylphenyl)ethyl)propionamide 
     
       
         
         
             
             
         
       
     
     The crude compound 12c (145 mg, 352 μmol), compound 3f (80 mg, 529 μmol, Shanghai Bide Pharmatech Ltd.), 2-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (200 mg, 528 μmol), and N,N-diisopropyl ethylamine (273 mg, 2.1 mmol) were dissolved in 2 mL of N,N-dimethyl formamide. The reaction solution was stirred for 2 hours, and concentrated under reduced pressure. The residue was purified with preparative HPLC (instrument model: waters, chromatographic column: X-bridge Prep 30*150 mm, 5 μm, C18; mobile phase: A-water (10 mM ammonium acetate), B-acetonide; flow rate: 30 mL/min, column temperature: room temperature). At retention time 13.97 min, compound 13 (30 mg, 15.6%, compound in a single configuration with longer retention time) was obtained. 
     MS m/z (ESI): 544.2[M+1]. 
       1 H NMR (400 MHz, CD 3 OD): δ 8.44 (s, 1H), 8.24 (s, 1H), 7.22 (t, 1H), 7.18 (d, 2H), 7.08 (d, 1H), 5.34-5.31 (m, 1H), 4.97-4.94 (m, 1H), 4.07-4.03 (m 1H), 3.76-3.74 (m, 4H), 3.63-3.60 (m, 2H), 3.26-3.23 (m, 1H), 3.13-3.10 (m, 1H), 2.33(s, 3H), 1.43 (d, 3H), 1.28 (d, 3H). 
     Example 14 
     (R)-2-(2-(5-Chloro-2-((1-methyl-1H-pyrazol-5-yl)amino)pyrimidin-4-yl)-4-oxo-6,7-dihydr othieno[3,2-c]pyridin-5(4H)-yl)-N-((S)-1-(3-fluoro-5-methoxyphenyl)-2-hydroxyethyl)prop ionamidel4 
     
       
         
         
             
             
         
       
       
         
         
             
             
         
       
     
     Step 1 
     tert-Butyl 2-(2-(5-chloro-2-((1-methyl-1H-pyrazol-5-yl)amino)pyrimidin-4-yl)-4-oxo-6,7-dihydrothie no[3 ,2-c]pyridin-5 (4H)-yl)propionate 14b 
     Compound 3c (240 mg, 0.56 mmol) was dissolved in 5 mL of dioxane, followed by the addition of 1-methyl-1H-pyrazol-5-amine 14a (108 mg, 1.11 mmol), tris(dibenzylideneacetone)dipaladium (76 mg, 0.08 mmol), 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene (64 mg, 0.11 mmol), and cesium carbonate (365 mg, 1.12 mmol). The reaction solution was reacted at 100° C. in microwave reactor for 1 hour, and then concentrated under reduced pressure. The residue was purified by thin layer chromatography with developing solvent system C to obtain the title compound 14b (40 mg), yield: 14.9%. 
     MS m/z (ESI): 489.3 [M+1]. 
     Step 2 
     2-(2-(5-Chloro-2-((1-methyl-1H-pyrazol-5-yl)amino)pyrimidin-4-yl)-4-oxo-6,7-dihydrothie no[3,2-c]pyridin-5(4H)-yl)propanoic acid 14c 
     Compound 14b (18 mg, 36 μmol) was dissolved in 1.5 mL of dichloromethane, followed by the addition of trifluoroacetic acid (0.5 mL). The reaction solution was stirred for 2 hours, and then concentrated under reduced pressure to obtain the crude title compound 14c (15 mg). The product was directly used in the next step without purification. 
     MS m/z (ESI): 433.1 [M+1]. 
     Step 3 
     (R)-2-(2-(5-Chloro-2-((1-methyl-1H-pyrazol-5-yl)amino)pyrimidin-4-yl)-4-oxo-6,7-dihydr othieno[3,2-c]pyridin-5(4H)-yl)-N-((S)-1-(3-fluoro-5-methoxyphenyl)-2-hydroxyethyl)prop ionamide 14 
     The crude compound 14c (120 mg, 277 μmol), compound 9a (66 mg, 356 Shanghai Bide Pharmatech Ltd.), 2-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (158 mg, 415 μmol), and N,N-diisopropyl ethylamine (214 mg, 1.6 mmol) were dissolved in 2 mL of N,N-dimethyl formamide. The reaction solution was stirred for 2 hours, and then concentrated under reduced pressure. The residue was purified with preparative HPLC (instrument model: waters, chromatographic column: X-bridge Prep 30*150 mm, 5 μm, C18; mobile phase: A-water (10 mM ammonium acetate), B-acetonide; flow rate: 30 mL/min, column temperature: room temperature). At retention time 17.69 min, the title compound 14 (30 mg, 18.1%, compound in a single configuration with longer retention time) was obtained. 
     MS m/z (ESI): 600.2[M+1]. 
       1 H NMR (400 MHz, CD 3 OD): δ 8.45 (d, 2H), 7.47 (s, 1H), 6.75 (s, 1H), 6.70 (d, 1H), 6.59 (d, 1H), 6.39 (s, 1H), 5.35-5.32(m, 1H), 4.97-4.94 (m, 1H), 3.78-3.73(m, 10H), 3.25-3.23 (m, 1H), 3.14-3.11 (m, 1H), 1.44 (d, 3H). 
     Example 15 
     (R)-N-((S)-1-(3-Fluoro-5-methoxyphenyl)-2-hydroxyethyl)-2-(2-(2-((1-methyl-1H-pyrazol-5-yl)amino)pyrimidin-4-yl)-4-oxo-6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl)propionamide 
     
       
         
         
             
             
         
       
       
         
         
             
             
         
       
       
         
         
             
             
         
       
     
     Step 1 
     4-Chloro-N-(1-methyl-1H-pyrazol-5-yl)pyrimidin-2-amine 15b 
     N-(1-Methyl-1H-pyrazol-5-yl)formamide 7c (324.82 mg, 2.60 mmol, prepared according to the method disclosed in patent application “WO2017/80979”) was dissolved in 15 mL of N,N-dimethyl formamide, followed by the addition of sodium hydride (60%, 311.47 mg, 7.79 mmol) at 0° C. The reaction solution was stirred for 0.5 hours, followed by the addition of compound 4-chloro-2-(methylsulfonyl)pyrimidine 15a (500 mg, 2.60 mmol). The reaction solution was further reacted for 2 hours. 20 mL of water was added, and the reaction solution was extracted with ethyl acetate (20 mL×3). The organic phases were combined and concentrated under reduced pressure. The residue was purified by thin layer chromatography with developing solvent system C to obtain the title compound 15b (270 mg), yield: 49.6%. 
     MS m/z (ESI): 210.3 [M+1]. 
     Step 2 
     tert-Butyl 2-(4-oxo-2-(4,4,5,5-tetramethyl-1,3 ,2-dioxaborolan-2-yl)-6,7-dihydrothieno[3 ,2-c]pyridin-5 (4H)-yl)propanoate 15c 
     Under argon atmosphere, compound 3b (2.0 g, 5.5 mmol) was dissolved in 70 mL tetrahydrofuran, followed by the addition of 4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-di(1,3,2-dioxaborolane) (3.9 g, 15.5 mmol), cuprous iodide (1.1 g, 5.5 mmol), tri-n-butyl phosphine (1.1 g, 5.5 mmol) and potassium tert-butoxide (934 mg, 8.3 mmol) successively. The reaction solution was stirred at 60° C. for 1 hour, and then cooled and filtered through diatomaceous earth. The filtrate was concentrated, and the residue was purified by column chromatography with eluent system 
     C to obtain the title compound 15c (1.5 g), yield: 66.3%. 
     MS m/z (ESI): 352.2 [M-55]. 
     Step 3 
     tert-Butyl 2-2-(2-((1-methyl-1H-pyrazol-5-yl)amino)pyrimidin-4-yl)-4-oxo-6,7-dihydrothieno[3,2-c]p yridin-5(4H)-yl)propanoate 15d 
     Under argon atmosphere, compound 15c (520 mg, 1.28 mmol), pre-prepared compound 15b (90 mg, 0.42 mmol), [1,1′-bis(diphenylphosphino)ferrocene]palladium (II) dichloride dichloromethane complex (72 mg, 0.085 mmol), and sodium carbonate (91 mg, 0.86 mmol) were suspended in 10 mL of 1,4-dioxane and 2 mL of water. The reaction solution was heated to 60° C. and stirred for 1 hour. The reaction solution was cooled and filtered through diatomaceous earth. The filtrate was collected and extracted with ethyl acetate (20 mL×3). The organic phases were combined and concentrated under reduced pressure. The residue was purified by column chromatography with eluent system A to obtain the title compound ig (100 mg), yield: 51.2%. 
     MS m/z (ESI): 455.1 [M+1]. 
     Step 4 
     2-2-(2-((1-Methyl-1H-pyrazol-5-yl)amino)pyrimidin-4-yl)-4-oxo-6,7-dihydrothieno[3,2-c]p yridin-5(4H)-yl)propanoic acid 15e 
     Compound 15d (120 mg, 264 μmol) was dissolved in 2 mL of dichloromethane, followed by the addition of trifluoroacetic acid (0.7 mL). The reaction solution was stirred for 2 hours, and then concentrated under reduced pressure to obtain the crude title compound 15e (100 mg). The product was directly used in the next step without purification. 
     MS m/z (ESI): 399.0 [M+1]. 
     Step 5 
     (R)-N-((S)-1-(3-Fluoro-5-methoxyphenyl)-2-hydroxyethyl)-2-(2-(2-((1-methyl-1H-pyrazol-5-yl)amino)pyrimidin-4-yl)-4-oxo-6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl)propionamide 15 
     The crude compound 15e (100 mg, 250 μmol), compound 9a (55 mg, 296 μmol, Shanghai Bide Pharmatech Ltd.), 2-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (114 mg, 300 μmol), and N,N-diisopropyl ethylamine (194 mg, 1.5 mmol) were dissolved in 3 mL of N,N-dimethyl formamide. The reaction solution was stirred for 2 hours, and then concentrated under reduced pressure. The residue was purified with preparative HPLC (instrument model: waters, chromatographic column: Sharpsil-T, Prep 30*150 mm, 5 μm, C18; mobile phase: A-water (0.1% TFA), B-acetonide; flow rate: 30 mL/min, column temperature: room temperature), washed with saturated sodium bicarbonate solution and concentrated to dryness by rotary evaporation. At retention time 14.29 min, compound 15 (20 mg, 14.1%, compound in a single configuration with longer retention time) was obtained. 
     MS m/z (ESI): 566.2[M+1]. 
       1 H NMR (400 MHz, CD 3 OD): δ 8.41 (d, 1H), 8.07 (s, 1H), 7.56 (s, 1H), 7.33 (d, 1H), 6.75 (s, 1H), 6.70 (d, 1H), 6.59 (d, 1H), 6.49 (s, 1H), 5.34-5.32(m, 1H), 4.96-4.94 (m, 1H), 3.80-3.73(m, 6H), 3.71-3.68 (m, 4H), 3.25-3.22 (m, 1H), 3.12-3.09 (m, 1H), 1.44 (d, 3H). 
     Example 16 
     (R)-N-((S)-1-(3-Fluoro-5-methoxyphenyl)-2-hydroxyethyl)-2-(2-(5-methyl-2-((tetrahydro-2 H-pyran-4-yl)amino)pyrimidin-4-yl)-4-oxo-6,7-dihydrothieno[3,2-c]pyridin-5-(4H)-yl)prop ionamidel6 
     
       
         
         
             
             
         
       
       
         
         
             
             
         
       
     
     Step 1 
     tert-Butyl 2-(4-oxo-2-(4,4,5,5-tetramethyl-1,3 ,2-dioxaborolan-2-yl)-6,7-dihydrothieno[3 ,2-c]pyridin-5-yl)propanoate 16a 
     Under argon atmosphere, compound 3b (2 g, 5.56 mmol) was dissolved in 50 mL of THF, followed by the addition of 4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-di(1,3,2-dioxaborolane) (3.9 g, 15.4 mmol), cuprous iodide (1 g, 5.3 mmol), tributylphosphine (1.1 g, 5.4 mmol) and [1,1′-bis(diphenylphosphino)ferrocene]palladium (II) dichloride (109 g, 0.15 mmol)successively. The reaction solution was stirred at 60° C. for 40 minutes, and then cooled and filtered through diatomaceous earth. The filtrate was concentrated, and the residue was purified by column chromatography with eluent system C to obtain the title compound 16a (2 g), yield: 88.4%. 
     MS m/z (ESI): 408.1 [M+1]. 
     Step 2 
     tert-Butyl 2-(2-(2-chloro-5-methyl-pyrimidin-4-yl)-4-oxo-6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl)p ropanoate 16c 
     Under argon atmosphere, the mixture of compound 16a (2 g, 4.9 mmol), 2,4-dichloro-5-methylpyrimidine 16b (1.6 g, 9.8 mmol), tetrakis(triphenylphosphine)palladium (681 mg, 0.6 mmol) and sodium carbonate (1 g, 9.4 mmol) were suspended in 30 mL of 1,4-dioxane and 4 mL of water , and then the reaction solution was heated to 60° C. and stirred for 3 hours. The reaction solution was cooled and filtered through diatomaceous earth. The filtrate was collected and extracted with ethyl acetate (20 mL×3). The organic phases were combined and concentrated under reduced pressure. The residue was purified by column chromatography with eluent system A to obtain the title compound 16c (1.5 g), yield: 74%. 
     MS m/z (ESI): 408.3 [M+1]. 
     Step 3 
     tert-Butyl 2-(2-(5-methyl-2-((tetrahydro-2H-pyran-4-yl)amino)pyrimidin-4-yl)-4-oxo-6,7-dihydrothie no[3,2-c]pyridin-5(4H)-yl)propanoate 16d 
     Compound 16c (531 mg, 1.4 mmol) was dissolved in 5 mL of tetrahydrofuran, followed by the addition of compound if (143 mg, 1.4 mmol) and N,N-diisopropyl ethylamine (546 mg, 4.2 mmol). The reaction solution was reacted in microwave reactor at 100° C. for 4 hours, and then concentrated under reduced pressure. The residue was purified by thin layer chromatography with developing solvent system A to obtain the title compound 16d (400 mg), yield: 64.3%. 
     MS m/z (ESI): 473.2 [M+1]. 
     Step 4 
     2-(2-(5-Methyl-2-((tetrahydro-2H-pyran-4-yl)amino)pyrimidin-4-yl)-4-oxo-6,7-dihydrothie no[3,2-c]pyridin-5(4H)-yl)propanoic acid 16e 
     Compound 16d (200 mg, 0.4 mmol) was dissolved in 2 mL of dichloromethane, followed by the addition of trifluoroacetic acid (0.7 mL). The reaction solution was stirred for 2 hours, and then concentrated under reduced pressure to obtain the crude title compound 16e (175 mg). The product was directly used in the next step without purification. 
     MS m/z (ESI): 417.2 [M+1]. 
     Step 5 
     (R)-N-((S)-1-(3-Fluoro-5-methoxyphenyl)-2-hydroxyethyl)-2-(2-(5-methyl-2-((tetrahydro-2 H-pyran-4-yl)amino)pyrimidin-4-yl)-4-oxo-6,7-dihydrothieno[3,2-c]pyridin-5-(4H)-yl)prop ionamide 16 
     The crude compound 16e (200 mg, 0.5 mmol), compound 9a (90 mg, 0.5 mmol, Shanghai Bide Pharmatech Ltd.), 2-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (113 mg, 0.5 mmol), and N,N-diisopropyl ethylamine (186 mg, 1.4 mmol) were dissolved in 2 mL of N,N-dimethyl formamide. The reaction solution was stirred for 2 hours, and then concentrated under reduced pressure. The residue was purified with preparative HPLC (instrument model: waters, chromatographic column: X-bridge Prep 30*150 μm, 5 μm, C18; mobile phase: A-water (10mM ammonium acetate), B-acetonide; flow rate: 30 mL/min, column temperature: room temperature). At retention time 16.60 min, the title compound 16 (30 mg, 39.5%, compound in a single configuration with longer retention time) was obtained. 
     MS m/z (ESI): 584.2 [M+1]. 
       1 H NMR (400 MHz, CD 3 OD): δ 8.15 (s, 2H), 6.76-6.59 (m, 3H), 5.35-5.33 (m, 1H), 4.97-4.93(m, 1H), 4.04-4.01 (m, 3H), 3.82-3.73(m, 7H), 3.58-3.56 (m 2H), 3.34-3.21 (m, 2H), 2.50 (s, 3H), 2.06-2.02 (m, 2H), 1.71-1.70 (m, 2H), 1.48 (d, 3H). 
     Example 17 
     (R)-N-((S)-1-(3-Fluoro-5-methoxyphenyl)-2-hydroxyethyl)-2-(2-(2-(((S)-1-hydroxypropan-2-yl)amino)-5-methylpyrimidin-4-yl)-4-oxo-6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl)prop ionamide 17 
     
       
         
         
             
             
         
       
     
     In accordance with the method of Example 16, compound 1f was replaced with 12a. The final product was purified by preparative HPLC (instrument model: Gilson, chromatographic column: Sharpsil-T, Prep 30*150 mm, 5 μm, C18; mobile phase: A-water (10 mM ammonium acetate), B-acetonide; flow rate: 30 mL/min; column temperature: 
     room temperature). At retention time 14.10 min, the title compound 17 (30 mg, compound in a single configuration with longer retention time) was obtained. 
     MS m/z (ESI): 558.2[M+1]. 
       1 H NMR (400 MHz, CD 3 OD): δ 8.13 (s, 1H), 7.91 (s, 1H), 6.76 (s, 1H), 6.71 (d, 1H), 6.62 (d, 1H), 5.37-5.32 (m, 1H), 4.97-4.94 (m, 1H), 4.13-4.10 (m, 1H), 3.81(s, 3H), 3.78-3.73 (m, 4H), 3.65-3.58 (m, 2H), 3.26-3.15 (m, 2H), 2.41 (s, 3H), 1.47 (d, 3H), 1.28 (d, 3H). 
     Biological Evaluation 
     Test Example 1: ERK1 Enzyme Activity Test 
     1. Test Purpose 
     The purpose of this experiment is to detect the inhibitory ability of the compounds on the ERK1 enzyme activity and evaluate the activity of the compounds in vitro according to IC 50 . The ADP-Glo™ Kinase Assay Kit was used in this experiment. Under the action of enzyme, the substrate was phosphorylated and ADP was produced at the same time. The ADP-Glo Reagent was added to remove unreacted ATP in the reaction system. The kinase detection reagent was used to detect the ADP produced by the reaction. In the presence of the compound, the inhibition rate of the compound was calculated by measuring the signal value. 
     2. Experimental Method 
     Enzyme and substrate preparation: ERK1 (1879-KS-010, R&amp;D) and substrate (AS-61777, anaspec) were prepared to be 0.75 ng/μl and 100 μM respectively in the buffer. The enzyme solution and substrate solution were then prepared into a mixed solution at a volume ratio of 2:1 for later use. ATP was diluted to 300 μM with the buffer. The compounds were dissolved in DMSO (dimethyl sulfoxide, Shanghai Titan Scientific Co., Ltd.) to prepare a stock solution with an initial concentration of 20 mM, and then Bravo was used to prepare solutions of the compounds. Finally, a 3 μL of a mixed solution of enzyme and substrate, and 1 μL of different concentrations of the compound (the initial concentration is 50 μM, 4-fold dilution) were added to each well of the 384-well plate, and the plate was incubated at 30° C. for 10 minutes, and finally 1 μL of 300 μM ATP solution was added to each well, and the plate was incubated at 30° C. for 2 hours. Then 5 μL of ADP-Glo was added, and the plate was incubated at 30° C. for 40 minutes. Then 10 μL of Kinase detection buffer was added, and the plate was incubated at 30° C. for 40 minutes. The 384-well plate was taken out and placed in a microplate reader (BMG labtech, PHERAstar FS), and the chemiluminescence was measured by the microplate reader. 
     3. Data Analysis 
     The data were processed and analyzed with Microsoft Excel, Graphpad Prism 5. The IC 50  values of the compounds were obtained, and the results are shown in the table below. 
     
       
         
           
               
               
               
             
               
                   
                   
               
               
                   
                 Example No. 
                 IC 50  (nM) 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                   
                 9-P2 
                 19 
               
               
                   
                 10-P2 
                 32 
               
               
                   
                 11 
                 5 
               
               
                   
                 12 
                 24 
               
               
                   
                 14 
                 27 
               
               
                   
                 15 
                 4 
               
               
                   
                 16 
                 3 
               
               
                   
                 17 
                 12 
               
               
                   
                   
               
               
                   
                 Conclusion: The compounds of the present disclosure had a significant inhibitory effect on ERK1 enzyme activity. 
               
            
           
         
       
     
     Test Example 2: ERK2 Enzyme Activity Test 
     1. Test Purpose 
     The purpose of this experiment is to detect the inhibitory ability of the compounds on the ERK2 enzyme activity and evaluate the activity of the compounds in vitro according to IC 50 . The ADP-Glo™ Kinase Assay Kit was used in this experiment. Under the action of enzyme, the substrate was phosphorylated and ADP was produced at the same time. The ADP-Glo Reagent was added to remove unreacted ATP in the reaction system. The kinase detection reagent was used to detect the ADP produced by the reaction. In the presence of the compound, the inhibition rate of the compound was calculated by measuring the signal value. 
     2. Experimental Method 
     Enzyme and substrate preparation: ERK2 (1230-KS-010, R&amp;D) and substrate (Custom peptides, Gill Biochemical) were prepared to be 0.75 ng/μl and 1500 μM in the buffer (40 mM Tris, 20 mM MgCl 2 , 0.1 mg/mL BSA, 50 μM DTT). The enzyme solution and substrate solution were then prepared into a mixed solution at a volume ratio of 2:1 for later use. ATP was diluted to 500 μM with the buffer. The compounds were dissolved in DMSO (dimethyl sulfoxide, Shanghai Titan Scientific Co., Ltd.) to prepare a stock solution with an initial concentration of 20 mM, and then Bravo was used to prepare solutions of the compounds. Finally, a 3 μL of a mixed solution of enzyme and substrate, and 1 μL of different concentrations of the compound (the initial concentration is 50 μM, 4-fold dilution) were added to each well of the 384-well plate, and the plate was incubated at 30° C. for 10 minutes, and finally 1 μL of 500 μM ATP solution was added to each well, and the plate was incubated at 30° C. for 2 hours. Then 5 μL of ADP-Glo was added, and the plate was incubated at 30° C. for 40 minutes. Then 10 μL of Kinase detection buffer was added, and the plate was incubated at 30° C. for 40 minutes. The 384-well plate was taken out and placed in a microplate reader (BMG labtech, PHERAstar FS), and the chemiluminescence was measured by the microplate reader. 
     3. Data Analysis 
     The data were processed and analyzed with Microsoft Excel, Graphpad Prism 5. The IC 50  values of the compounds were obtained, and the results are shown in the table below. 
     
       
         
           
               
               
               
             
               
                   
                   
               
               
                   
                 Example No. 
                 IC 50  (nM) 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                   
                 1 
                 1 
               
               
                   
                 2 
                 3 
               
               
                   
                 3-P1 
                 203 
               
               
                   
                 3-P2 
                 4 
               
               
                   
                 4-P1 
                 434 
               
               
                   
                 4-P2 
                 6 
               
               
                   
                 5 
                 22 
               
               
                   
                 6 
                 29 
               
               
                   
                 7 
                 3 
               
               
                   
                 9-P1 
                 410 
               
               
                   
                 9-P2 
                 3 
               
               
                   
                 10-P2 
                 5 
               
               
                   
                 11 
                 2 
               
               
                   
                 12 
                 2 
               
               
                   
                 13 
                 9 
               
               
                   
                 14 
                 6 
               
               
                   
                 15 
                 3 
               
               
                   
                 16 
                 1 
               
               
                   
                 17 
                 2 
               
               
                   
                   
               
               
                   
                 Conclusion: The compounds of the present disclosure had a significant inhibitory effect on ERK2 enzyme activity. 
               
            
           
         
       
     
     Test Example 3: In Vitro Proliferation Inhibition Test of Compounds on Colo205 Tumor Cells 
     1. Test Purpose 
     The purpose of this experiment is to test the inhibitory activity of the compounds on the proliferation of Colo205 cells (CCL-222, ATCC) in vitro. The cells were treated with different concentrations of compounds in vitro. After 3 days of culture, the cell proliferation was detected using CTG (CellTiter-Glo® Luminescent Cell Viability Assay, Promega, catalog number: G7573) reagent. The in vitro activity of the compounds was evaluated according to the IC 50  value. 
     2. Experimental Method 
     In the following, taking the in vitro proliferation inhibition test method of Colo205 cells as an example, the method in the present disclosure for testing the in vitro proliferation inhibitory activity of the compounds of the present disclosure is described. This method is also applicable to, but not limited to, the in vitro proliferation inhibitory activity test on other tumor cells. 
     Colo205 cells were digested, centrifuged and then resuspended. The single cell suspension was mixed well, and the density of viable cells was adjusted to 5.0×10 4  cells/ml with cell culture medium (RPMI1640+2% FBS), and 95 μl/well was added to a 96-well cell culture plate. Only 100 μl medium was added to the peripheral wells of the 96-well plate. The culture plate was incubated in an incubator for 24 hours (37° C., 5% CO 2 ). 
     The compound was dissolved in DMSO (dimethyl sulfoxide, Shanghai Titan Scientific Co., Ltd.) and prepared into a stock solution with an initial concentration of 20 mM. The initial concentration of the small molecule compound is 2 mM, and then 4-fold diluted into to 9 points, and the tenth point is DMSO. Another 96-well plate was taken, 90 μl of cell culture medium (RPMI1640+2% FBS) was added to each well, then 10 μl of different concentrations of the test sample was added to each well, the mixture was mixed well, and then 5 μl of different concentrations of the test sample was added to the cell culture plate, and each sample has two duplicate holes. The culture plate was incubated in the incubator for 3 days (37° C., 5% CO 2 ). The 96-well cell culture plate was taken out, 50 μl CTG solution was added to each well, and the plate was incubated for 10 minutes at room temperature. In a microplate reader (BMG labtech, PHERAstar FS), chemiluminescence was measured with the microplate reader. 
     3. Data Analysis 
     The data were processed and analyzed with Microsoft Excel, Graphpad Prism 5. The results are shown in the table below: 
     
       
         
           
               
               
               
             
               
                   
                   
               
               
                   
                 Example No. 
                 IC 50  (nM) 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                   
                 1 
                 8 
               
               
                   
                 3-P2 
                 26 
               
               
                   
                 4-P2 
                 29 
               
               
                   
                 7 
                 38 
               
               
                   
                 9-P2 
                 18 
               
               
                   
                 10-P2 
                 50 
               
               
                   
                 11 
                 16 
               
               
                   
                 12 
                 5 
               
               
                   
                 13 
                 26 
               
               
                   
                 14 
                 14 
               
               
                   
                 15 
                 49 
               
               
                   
                 17 
                 17 
               
               
                   
                   
               
               
                   
                 Conclusion: The compounds of the present disclosure had a significant inhibitory effect on the proliferation of Colo205 tumor cells. 
               
            
           
         
       
     
     Pharmacokinetic Evaluation 
     Test Example 4. Pharmacokinetics Assay of the Compounds of the Present Disclosure in Mice 1. Abstract 
     Mice were used as the test animal. The drug concentration in plasma at different time points was determined by LC/MS/MS method after administration of compound 3-P2, compound 4-P2, compound 9-P2, compound 10-P2, compound 12 and compound 15. The pharmacokinetic behavior of the compounds of the present disclosure in mice was studied, and the pharmacokinetic characteristics were evaluated. 
     2. Test Protocol 
     2.1. Test Samples 
     Compound 3-P2, Compound 4-P2, Compound 9-P2, Compound 10-P2, Compound 12 and Compound 15. 
     2.2. Test Animals 
     54 of C57 mice (female) were equally divided into 6 groups, which were purchased from Shanghai Jiesijie Laboratory Animal Co., Ltd., with the animal production license number: SOCK (Shanghai) 2013-0006. 
     2.3 Preparation of the test compound 
     A certain amount of compound was weighted, followed by the addition of 5% volume of DMSO and 5% Tween 80 to dissolve it, then followed by the addition of 90% normal saline to prepare a colorless and clear solution of 0.1 mg/mL. 
     2.4 Administration 
     After an overnight fast, C57 mice were intragastrically administered the test compound at an administration dose of 2 mg/kg and an administration volume of 0.2 ml/10 g. 
     3. Process 
     The mice were intragastrically administered the test compounds. 0.1mL of blood was taken before the administration and at 0.25, 0.5, 1.0, 2.0, 4.0, 6.0, 8.0, 11.0, 24.0 hours after the administration. The blood was placed in a heparinized test tube, and centrifuged at 3500 rpm for 10 minutes. The plasma was separated and stored at −20° C. 
     The content of the test compound in the plasma of mice after injection administration of the test compound at different concentrations was determined: 25 μl of mouse plasma at each time point after the administration was taken, followed by the addition of 50 μl of the internal standard camptothecin solution (100 ng/mL) and 200 μl of acetonitrile. The resulting solution was vortex-mixed for 5 minutes, and centrifuged for 10 minutes (4000 rpm). 4μl of the supernatant was taken from the plasma samples for LC/MS/MS analysis. 
     4. Results of pharmacokinetic parameters 
     The pharmacokinetic parameters of the compounds of the present disclosure are shown below: 
     
       
         
           
               
               
            
               
                   
               
               
                   
                 Pharmacokinetics assay in mice 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                   
                   
                 Apparent 
               
               
                   
                 Plasma 
                 Area under 
                   
                 Residence 
                   
                 distribution 
               
               
                   
                 Concentration 
                 curve 
                 Half Life 
                 Time 
                 Clearance 
                 volume 
               
               
                   
                 Cmax 
                 AUC 
                 T1/2 
                 MRT 
                 CLz/F 
                 Vz/F 
               
               
                 No. 
                 (ng/mL) 
                 (ng/mL * h) 
                 (h) 
                 (h) 
                 (mL/min/kg) 
                 (mL/kg) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 3-P2 
                 708 
                 914 
                 0.945 
                 1.16 
                 36.5 
                 2985 
               
               
                 4-P2 
                 376 
                 364 
                 1.02 
                 1.14 
                 91.5 
                 8092 
               
               
                 9-P2 
                 1477 
                 1816 
                 4.49 
                 1.85 
                 18.4 
                 7136 
               
               
                 10-P2 
                 1003 
                 1829 
                 1.26 
                 1.64 
                 18.2 
                 1993 
               
               
                 12 
                 1476 
                 1602 
                 2.65 
                 1.05 
                 20.8 
                 4766 
               
               
                 15 
                 1070 
                 1832 
                 1.11 
                 1.85 
                 18.2 
                 1755 
               
               
                   
               
               
                 Conclusion: The compounds of the present disclosure were well absorbed, and had a significant pharmacokinetic advantage.