Tetra-fused-ring lactam compound as well as intermediate and application thereof

Abstract

The invention discloses a tetra-fused-ring lactam compound as well as an intermediate and application of the tetra-fused-ring lactam compound. The tetra-fused-ring lactam compound is a compound as shown in a formula I or a pharmaceutically acceptable salt of the tetra-fused-ring lactam compound. The compound disclosed by the invention has a good MK2 inhibition effect and a good patent medicine prospect.

Description

Technical Field

[0001] This invention relates to the pharmaceutical field, specifically disclosing a tetracyclic lactam compound, its intermediate and its application of a compound of formula (I) and / or its pharmaceutically acceptable salt, a pharmaceutical composition and a method for preparing the same, and the use of the compound for treating diseases caused by protein kinase 2 activated by mitogen-activated protein kinase. Background Technology

[0002] Mitogen-activated protein kinase 2 (MAPKAPK2 or MK2) is expressed in various human tissues, with higher expression levels in the heart, skeletal muscle, and kidneys. At rest, MK2 resides in the nucleus and often forms a complex with p38. Upon phosphorylation of p38, MK2 is activated, and both are translocated to the cytoplasm to regulate the phosphorylation of various downstream substrates, such as heat shock proteins (HSP-25 / 27), leukocyte-specific protein-1 (LSP-1), 5-lipoxygenase (ALOX5), LIM kinase (LIMK), and cofilin. MK2 mediates multiple p38MAPK-dependent cellular responses and is an essential protein kinase for LPS-induced release of cytokines (TNF-α, IL-6, and IL-1β). It also plays a crucial role in the formation of filopodia in response to extracellular stimuli, leading to cell adhesion and migration.

[0003] HSP27 is one of the earliest discovered substrates of MK2. Its biological functions include regulating the NF-κβ pathway and modulating proteasome degradation, thereby enhancing the degradation of specific proteins. Among these functions, its interaction with actin and intermediate filaments is widely reported in the literature. It prevents non-covalent filament-filament interactions that form intermediate filaments and protects actin filaments from breakage, preserving focal contacts fixed to the cell membrane.

[0004] The role of MK2 in inflammation is mainly through phosphorylation of RNA-binding proteins (RBPs), such as zinc finger protein 36TTP (Tristetraprolin), human antigen R (Hu-antigen R, HuR), and adenine-uridine rich element RNA binding factor AUF1 (AUF1), which in turn regulates the transcriptional stability and post-translational levels of related cytokines and proteins, thereby influencing the occurrence and development of inflammation.

[0005] TTP (zinc finger protein 36) regulates the stability and translation of TNF-α mRNA. The instability of TNF-α and other cytokine mRNAs stems from the enrichment of adenine and uridine rich elements (AREs) in their untranslated 3'UTR regions. In normal cells, TTP binds to the 3'UTR regions of these mRNAs, mediating the sustained degradation of the associated cytokines. LPS-activated MK2 phosphorylation of TTP, followed by binding to the 14-3-3 protein, unblocks the degradation of the associated mRNA, allowing for stable transcriptional expression.

[0006] HuR is overexpressed in various tumor cell types. In normal cells, HuR is located in the nucleus, and its cytoplasmic-nuclear transport is crucial for mRNA stability and expression. MK2 can induce cytoplasmic HuR accumulation, thereby affecting the expression of intercellular adhesion molecules ICAM-1 and IL-8 in acute inflammation. In the cytoplasm, HuR enhances the stability and translation of target mRNAs by binding to them.

[0007] AUF1 is not only a central regulator of immune and inflammatory responses, but also participates in regulating the stability of various mRNAs related to tumorigenesis and development. Due to its tissue specificity, AUF1 also plays an important role in viral replication. By binding to mRNA AREs, AUF1 can regulate mRNA half-life. In most cases, AUF1 promotes mRNA degradation, while in other cases it plays a stabilizing role, thus achieving pleiotropic regulation of gene expression. The p38MAPK-MK2-HSP27 signaling axis promotes the proteasome degradation of AUF1.

[0008] In summary, the p38 / MK2 signaling pathway primarily regulates mRNA stability and translation through specific post-transcriptional mechanisms, participating in the biosynthesis and release of cytokines (TNF-α, IL-6, IL-1β, IFN-γ). MK2 has become a target for various chronic inflammatory diseases, such as rheumatoid arthritis, asthma, inflammatory bowel disease, atherosclerosis, and neuroinflammation.

[0009] Quinoline thiophene molecules derived from the benzothiophene structure have exhibited micromolar-level MK2 inhibitory efficacy, while also demonstrating high selectivity for cyclin-dependent kinase 2 (CDK2). For example, PF-3644022. Building upon PF-3644022, WO2016044463 discloses another class of covalently irreversibly binding MK2 inhibitors.

[0010] In addition, other quinoline thiophene skeleton molecules have been reported, such as WO2014149164, WO2009010488, WO2018170204, WO2018170200, WO2018170201, as well as similar quinoline furan skeleton WO 2023025298 and quinoxaloline thiophene skeleton WO2024044731, etc.

[0011] However, there are currently no MK2 inhibitors on the market, and the compounds disclosed in this patent have not been published in any literature, and these compounds exhibit specific MK2 inhibitory effects. Summary of the Invention

[0012] To overcome the limitation of the limited types of MK2 inhibitors in the prior art, this invention provides a tetracyclic lactam compound, its intermediate, and its applications. The five-membered nitrogen-containing cyclothiophene compound of this invention is completely different from existing technologies and exhibits excellent MK2 inhibitory effects.

[0013] The present invention solves the above-mentioned technical problems through the following technical solutions.

[0014] This invention provides a compound of formula I or a pharmaceutically acceptable salt thereof.

[0015]

[0016] in:

[0017] R c It is hydrogen or C 1-6 alkyl;

[0018] R1 and R2 are each independently hydrogen and C. 1-6 Alkyl or C 1-6 Alkoxy, the C 1-6 Alkyl groups and the C 1-6 The alkoxy group may optionally be surrounded by one or more R groups. R-1 replace;

[0019] Each R R-1 Independently hydroxyl, amino, or halogen;

[0020] X is S, O, CH2, or NH;

[0021] M1, M2 and M3 are each independently NH, C or CH;

[0022] Ring A is phenyl, 5-6 heteroaryl, or 6-membered heteroaryl cyclic C, and ring C is 5-6-membered heteroaryl or 5-6-membered heterocyclic alkyl. The heteroatom in the heteroaryl or heterocyclic alkyl group is one or more of N, O, or S, and the number is 1, 2, 3, or 4.

[0023] R3, R4, and R5 are independently hydrogen, halogen, cyano, or C. 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 Alkyne group, -C(=O)-NR6R7, -C(=O)-OR6, -C(=O)-R6, -NR6R7, -C 1-6 Alkylene-NR6R7 or The C mentioned 1-6 Alkyl groups and the C 1-6 The alkoxy group may optionally be surrounded by one or more R groups. R-2 replace:

[0024] Each R R-2 Independently hydroxyl, deuterium, amino, halogen, oxo group (=O), -S (=O)2-C 1-6 Alkyl, C 1-6 Alkyl or C 1-6 Alkoxy;

[0025] Each R6 and each R7 is independently either hydrogen or C. 1-6 Alkyl, the C 1-6 Alkyl groups may optionally be surrounded by one or more R groups. R-3 replace;

[0026] Each R R-3 Independently selected as hydroxyl, amino, or halogen;

[0027] M4 is a connecting bond, -C = O-, O, NH, C 1-6 Alkylene or C 2-6 Ethyne group;

[0028] n is 0, 1, 2, or 3;

[0029] Ring B is C 3-6 Cycloalkyl, 3-10-membered heterocycloalkyl, 3-6-membered heterocyclic alkenyl, phenyl or 5-6-membered heteroaryl;

[0030] The heteroatoms in the 3-6 heterocyclic alkyl group, the 3-6 heterocyclic alkenyl group, and the 5-6 heteroaryl group are independently one or more of N, O, or S, and the number is 1, 2, 3, or 4.

[0031] R b Hydroxyl group, halogen, oxo group (=O), C 1-6 Alkyl or C 1-6 Alkoxy, the C 1-6 Alkyl groups may optionally be surrounded by one or more R groups. R-4 replace;

[0032] Each R R-4 Independently selected as hydroxyl, amino, or halogen;

[0033] R a Halogen, cyano, C 1-6 Alkyl, C 1-6 Halogenated alkyl or C 1-6 Alkoxy;

[0034] m can be 0, 1, or 2.

[0035] In some schemes, for

[0036] In some schemes, R1 is C 1-6 Alkyl or C 1-6 Alkoxy, the C 1-6 Alkyl groups and the C 1-6 The alkoxy group may optionally be surrounded by one or more R groups. R-1 replace.

[0037] In some embodiments, the compound represented by Formula I is a compound represented by general formula II-1, II-2, or II-3:

[0038]

[0039] The definitions of each group in formula II-1, II-2 or II-3 are the same as those described above.

[0040] In some schemes,

[0041] In formula II-1, when one of the groups R3, R4, and R5 is M4 is C 1-6 Alkylene, where ring B is a 3-10 membered heterocyclic alkyl group, and when the heteroatom in the 3-10 membered heterocyclic alkyl group contains N, the 3-10 membered heterocyclic alkyl group contains at most one N, and the other two groups are independently halogen, cyano, and C. 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 Alkyne group, -C(=O)-NR6R7, -C(=O)-OR6, -C(=O)-R6, -NR6R7 or -C 1-6 Alkylene-NR6R7;

[0042] When one of the groups R3, R4, and R5 is When M4 is the connecting bond and ring B is a 5-6 membered heteroaryl group, the heteroatoms in the 5-6 membered heteroaryl group are independently N and / or O, and the number is 1 or 2.

[0043] In formula II-2, when one of the groups R3, R4, and R5 is H, another group is a halogen, and the remaining group is C. 1-6When alkyl, the C 1-6 Alkyl groups are covered by one or more R R-2 replace;

[0044] When one of the groups R3, R4, and R5 is When M4 is the connecting bond, ring B is a 5-6 membered heteroaryl group, and when the heteroatom in the 5-6 membered heteroaryl group contains N, the 5-6 membered heteroaryl group contains at most one N atom.

[0045] In some schemes, the general formula II-1 is general formula II-1a, II-1b, II-1c, II-1d, II-1e, II-1f, or II-1g:

[0046]

[0047]

[0048] In Formulas II-1a to II-1g, the definitions of each group are the same as those described in any one of the present invention.

[0049] Preferably, in general formula II-1a, R3 is a halogen; one of the groups in R4 and R5 is... The other group is hydrogen, halogen, C 1-6 Alkyl or C 1-6 Alkoxy;

[0050] M4 is the connecting key, and ring B is C. 3-6 Cycloalkyl or 3-10 membered heterocycloalkyl, where n is 0, 1, 2 or 3, R b Hydroxyl, halogen, C 1-6 Alkyl or C 1-6 Alkyl group.

[0051] In some schemes, the general formula II-2 is general formula II-2a, II-2b, II-2c, II-2d, II-2e, II-2f, or II-2g:

[0052]

[0053] In formulas II-2a to II-2g, the definitions of each group are the same as those described in any one of the present invention.

[0054] In some schemes, the general formula II-3 is general formula II-3a, II-3b, II-3c, II-3d, II-3e, II-3f, or II-3g:

[0055]

[0056]

[0057] In formulas II-3a to II-3g, the definitions of each group are the same as those described in any one of the present invention.

[0058] In formulas II-1, II-2, II-3, II-1a to II-1g, II-1a to II-1g, II-3a to II-3g, R3 is a halogen, such as Cl.

[0059] In some schemes, the 5-6 heteroaryl group in ring A and the 5-6 heteroaryl group in ring C are independently 6-membered heteroaryl groups, with heteroatoms of 1, 2 or 3, and N can be pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl or 1,3,5-triazinyl.

[0060] In some schemes, the heteroatom in the 5-6 member heteroaryl group of the ring C is one or more of N, O and S, and the number is 1 or 2, which can be furanyl, thiophene, pyrrole, 1H-pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl or isothiazolyl.

[0061] In some schemes, the 5-6 membered heterocyclic alkyl group in the ring C is a 5-6 membered monocyclic heterocyclic alkyl group with heteroatoms of N and / or O, and the number is 1 or 2, which can be tetrahydrofuranyl, pyrrolidinyl or piperidinyl.

[0062] In some schemes, R a R b R c R1, R2, R3, R4, R5, each of R6, each of R7, and each of R R-2 In the context, C 1-6 Alkyl group is C 1-3 Alkyl groups, such as methyl, ethyl, n-propyl, or isopropyl, and again, methyl or ethyl.

[0063] In some schemes, R a R b R1, R2, R3, R4, R5 and each R R-2 In the context, C 1-6 The alkoxy group is C 1-3 Alkoxy groups, such as methoxy, ethoxy, n-propoxy, or isopropoxy, and again, methoxy or ethoxy.

[0064] In some schemes, each R R-1 Each R R-2 Each R R-3 Each R R-4 R3, R4, R5, Ra and R b In the above, the halogen is independently F, Cl, Br or I, preferably Cl or F.

[0065] In some schemes, in R1 and R2, when the C 1-6 Alkyl groups are formed by one or more R groups. R-1 When replacing, the C 1-6 Alkyl group is C 1-3 Alkyl, R R-1 The C is a hydroxyl group; 1-6 Alkyl groups can be

[0066] In some schemes, in R3, R4, and R5, when the aforementioned C 1-6 Alkyl groups are formed by one or more R groups. R-2 When replacing, the C 1-6 Alkyl group is C 1-3 Alkyl groups, each R R-2 Independently hydroxyl, deuterium, amino, halogen, -S(=O)2-C 1-6 Alkyl or C 1-6 Alkoxy; the C 1-6 Alkyl groups can be -CD3, -CH2F, -CHF2, -CF3,

[0067] In some schemes, R3, R4, and R5, when the aforementioned C 1-6 Alkyl groups are formed by one or more R groups. R-2 When replacing, the C 1-6 The alkoxy group is C 1-3 Alkoxy groups, each R R-2 It is an amino group; the C is... 1-6 Alkoxy groups can be

[0068] In some schemes, R3, R4, and R5, the aforementioned C 2-6 The alkynyl group is either ethynyl or propynyl, for example, ethynyl.

[0069] In some schemes, R3, R4, and R5, the -C 1-6 C in alkylene-NR6R7 1-6 Alkylene is C 1-3 Alkylenes, such as methylene, ethylene, n-propylene, or isopropylene.

[0070] In some schemes, each R R-2 In the context, -S(=O)2-C 1-6 C in alkyl 1-6 Alkyl group is C 1-3 Alkyl groups, such as methyl, ethyl, n-propyl, or isopropyl, and methyl, for example.

[0071] In some schemes, in each R6 and each R7, when the C 1-6 Alkyl groups are formed by one or more R groups.R-3 When replacing, the C 1-6 Alkyl group is C 1-3 Alkyl groups, each R R-3 Independent halogen or hydroxyl group; the C 1-6 Alkyl groups can be

[0072] In some schemes, in M4, the C mentioned 1-6 Alkylene is C 1-3 Alkylenes, such as methylene, ethylene, n-propylene, or isopropylene, such as methylene.

[0073] In some schemes, in M4, the C mentioned 2-6 The ethynyl group is either ethynyl or propynyl, for example, ethynyl.

[0074] In some schemes, the 3-10 membered heterocyclic alkyl group in ring B is a 3-6 membered monocyclic heterocyclic alkyl group or a 6-8 membered bicyclic spirocyclic heterocyclic alkyl group, the heteroatom is one or more of N, O or S, the number is 1 or 2, and it may also be aza-butyl, pyrrolidinyl, 1,3-dioxolanecycloyl, piperidinyl, oxacyclobutyl, tetrahydrofuranyl, tetrahydropyranyl, piperazinyl, morpholinyl, thiomorpholinyl, isoxazolyl, oxazolyl, 2-oxa-6-azaspiro[3.4]octyl or 2-oxa-6-azaspiro[3.3]heptyl.

[0075] In some schemes, the 3-6 membered heterocyclic alkenyl group in ring B is a 5-6 membered heterocyclic alkenyl group, the heteroatom is one or more of N, O or S, the number is 1 or 2, and it contains 1 or 2 double bonds, which may be 3,6-dihydro-2H-pyranyl.

[0076] In some schemes, in ring B, the heteroatom in the 5-6 membered heteroaryl group is one or more of N, O or S, and the number is 1 or 2, which can be furanyl, thiophene, pyrrole, 1H-pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, or isothiazolyl.

[0077] In some schemes, R a In the context, C 1-6 The alkyl halide is C 1-3 Halogenated alkyl groups.

[0078] In some schemes, R c It is hydrogen.

[0079] In some schemes, X is S or O.

[0080] In some schemes, one group in R1 and R2 is hydrogen, and the other group is C. 1-3 Alkyl, the C 1-3The alkyl group may optionally be substituted with one or more hydroxyl groups;

[0081] In some schemes, one group in R1 and R2 is hydrogen, and the other group is methyl or...

[0082] In some schemes, for For example

[0083] In some schemes, for For example

[0084] In some schemes, ring A is a 6-membered heteroaryl, a 6-membered heteroaryl-5-membered heteroaryl, a 6-membered heteroaryl-5-membered heterocycloalkyl, or a 6-membered heteroaryl-6-membered heterocycloalkyl; for example, ring A is a 6-membered heteroaryl or a 6-membered heteroaryl-5-membered heterocycloalkyl.

[0085] In some schemes, ring A is

[0086]

[0087] In some schemes, ring B is C. 3-6 Cycloalkyl, 3-6 membered monocyclic heterocyclic alkyl, 6-8 membered bicyclic spirocyclic heterocyclic alkyl, 5-6 membered heterocyclic alkenyl or 5-6 membered heteroaryl.

[0088] In some schemes, ring B is

[0089] In some schemes, R3 is hydrogen, halogen, or C. 1-6 Alkyl, C 1-6 Alkyl, -NR6R7 or The C mentioned 1-6 Alkyl groups may optionally be surrounded by one or more R groups. R-2 Substitution; ring B is C 3-6 Cycloalkyl, 3-10 membered heterocycloalkyl, phenyl or 5-6 membered heteroaryl; each R R-2 Independently hydroxyl, deuterium, amino, halogen, C 1-6 Alkyl or C 1-6 Alkoxy; R b Halogen, C 1-6 Alkyl or C 1-6 Alkoxy;

[0090]

[0091] 1 or more R R-2 Replace; each R R-2 Independently hydroxyl, deuterium, amino, halogen, C 1-3 Alkyl or C 1-3 Alkoxy groups; each R6 and each R7 is independently hydrogen or C. 1-3 Alkyl; ring B is a 5-6 membered monocyclic heterocyclic alkyl or a 5-6 membered heteroaryl; R b Hydroxyl group, halogen, oxo group (=O), C 1-6 Alkyl or C 1-6 Alkoxy, R b Halogen, C 1-3 Alkyl or C 1-3 Alkoxy;

[0092] In some schemes, R3 is hydrogen, Cl, -CH3, -N(CH3)2,

[0093] In some schemes, R4 and R5 are independently hydrogen, halogen, cyano, or C. 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 Alkyne group, -C(=O)-NR6R7, -C(=O)-OR6, -C(=O)-R6, -R6R7 or The C mentioned 1-6 Alkyl groups and the C 1-6 The alkoxy group may optionally be surrounded by one or more R groups. R-2 Substitution: hydroxyl, deuterium, amino, halogen, -S(=O)2-C 1-6 Alkyl, C 1-6 Alkyl or C 1-6 Alkyl group; M4 is a linking bond, -C(=O)-, O, NH, C 1-6 Alkylene or C 2-6 etymynyl; ring B is C 3-6 Cycloalkyl, 3-10-membered heterocycloalkyl, 3-6-membered heterocyclic alkenyl or 5-6-membered heteroaryl; each R6 and each R7 is independently hydrogen or C. 1-3 alkyl;

[0094] Preferably, R4 and R5 are independently hydrogen, halogen, cyano, or C. 1-3 Alkyl, C 1-3 Alkoxy, C 2-6 Alkyne group, -C(=O)-NR6R7, -C(=O)-OR6, -C(=O)-R6, -R6R7 or The C mentioned 1-3 Alkyl groups and the C 1-3 The alkoxy group may optionally be surrounded by one or more R groups. R-2Substitution: hydroxyl, deuterium, amino, halogen, -S(=O)2-C 1-3 Alkyl, C 1-3 Alkyl or C 1-3 Alkyl group; M4 is a linking bond, -C(=O)-, C 1-3 Alkylene or C 2-3 Alynyl group; each R6 and each R7 is independently hydrogen or C. 1-3 Alkyl, the C 1-3 Alkyl groups may optionally be surrounded by one or more R groups. R-3 replace;

[0095] Ring B is C 3-6 Cycloalkyl, 3-6 membered monocyclic heterocyclic alkyl, 6-8 membered bicyclic spirocyclic heterocyclic alkyl, 5-6 membered heterocyclic alkenyl or 5-6 membered heteroaryl.

[0096] In some schemes, R4 and R5 are independently hydrogen, F, Cl, -CN, -CH3, -CD3, -CHF, -CHF2, -CF3, -OCH3, -OCH2CH3, -N(CH3)2、

[0097]

[0098]

[0099] In some schemes, For any of the following situations:

[0100] Case 1: Ring A is a 6-membered heteroaryl group, and R3, R4, and R5 are independently hydrogen, halogen, cyano, or C. 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 Alkyne group, -C(=O)-NR6R7, -C(=O)-OR6, -C(=O)-R6, -NR6R7 or -C 1-6 Alkylene-NR6R7; the C 1-6 Alkyl groups and the C 1-6 The alkoxy group may optionally be surrounded by one or more R groups. R-2 replace:

[0101] Case 2: Ring A is a 6-membered heteroaryl group, and one of R3, R4, and R5 is... The definitions of the other two groups are the same as those in case 1;

[0102] Case 3: Ring A is a 6-membered heteroaryl 5-6-membered heteroaryl or a 6-membered heteroaryl 5-6-membered heterocycloalkyl, and the definitions of R3, R4 and R5 are the same as those in Case 1.

[0103] Better place,

[0104] Case 1: Ring A is a 6-membered heteroaryl group, and R3 is H, halogen, or C. 1-6 Alkyl, the C 1-6 Alkyl groups may optionally be surrounded by one or more R groups. R-2 replace;

[0105] R4 and R5 are independently hydrogen, halogen, cyano, or C. 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 Alkyne group, -C(=O)-NR6R7, -C(=O)-OR6, -C(=O)-R6, -NR6R7 or -C 1-6 Alkylene-NR6R7, the C 1-6 Alkyl groups and the C 1-6 The alkoxy group may optionally be surrounded by one or more R groups. R-2 replace;

[0106] Case 2: Ring A is a 6-membered heteroaryl group, R3 is a halogen, one of R4 and R5 is H, and the other group is... M4 is a connector, -C = O-, O, C 1-6 Alkylene or C 2-6 Ethyne group;

[0107] Case 3: Ring A is a 6-membered heteroaryl-5-6-membered heteroaryl or a 6-membered heteroaryl-5-6-membered heterocycloalkyl, R3 is a halogen, and R4 and R5 are independently H or C. 1-6 alkyl.

[0108] Better,

[0109] Case 1: Ring A is a pyrimidine group, and R3 is a halogen (such as Cl);

[0110] R4 and R5 are independently hydrogen, halogen, cyano, or C. 1-3 Alkyl, C 1-3 Alkoxy, C 2-6 Alkyne group, -C(=O)-NR6R7, -C(=O)-OR6 or -C(=O)-R6; the C 1-6 Alkyl groups and the C 1-6 The alkoxy group may optionally be surrounded by one or more R groups. R-2 Replace; each R R-2 Independently hydroxyl, deuterium, amino, halogen, -S(=O)2-C 1-3 Alkyl or C 1-3 Alkoxy;

[0111] R6 and each of the R7s are independently either hydrogen or C. 1-3 Alkyl, the C 1-3Alkyl groups may optionally be surrounded by one or more R groups. R-3 replace;

[0112] Case 2: When ring A is a pyrimidine group, R3 is a halogen (such as Cl), one of R4 and R5 is H or a halogen, and the other group is... M4 is a connector, -C = O-, C 1-3 Alkylene or C 2-3 etymynyl group, ring B is C 3-6 Cycloalkyl, 3-6 membered monocyclic heterocyclic alkyl, 6-8 membered bicyclic spirocyclic heterocyclic alkyl, 5-6 membered heterocyclic alkenyl or 5-6 membered heteroaryl; n is 0, 1, 2 or 3; R b Hydroxyl, halogen, oxo group, C 1-3 Alkyl or C 1-3 Alkoxy, the C 1-3 Alkyl groups may optionally be surrounded by one or more R groups. R-4 Replace; each R R-4 Independently selected as hydroxyl, amino, or halogen;

[0113] Case 3: When ring A is 5,7-dihydrofurano[3,4-d]pyrimidinyl, 5,6,7,8-tetrahydropyrido[4,3-d]pyrimidinyl, or pyrrolo[3,4-d]pyrimidinyl, R3 is a halogen (such as Cl), and R4 and R5 are independently H or C. 1-3 alkyl.

[0114] Better, for

[0115]

[0116]

[0117] In some embodiments, the compound represented by Formula I is any one of the following compounds, its corresponding isomer, or a mixture of both:

[0118]

[0119]

[0120]

[0121]

[0122]

[0123]

[0124] Typical compounds of Formula I or their pharmaceutically acceptable salts include, but are not limited to:

[0125]

[0126]

[0127]

[0128]

[0129] It also includes:

[0130]

[0131]

[0132] The present invention also provides a method for preparing the compound of Formula I or a pharmaceutically acceptable salt thereof, comprising the following steps: in the presence of a base (e.g., cesium carbonate), carrying out an aromatic nucleophilic substitution reaction between the compound of Formula III and the compound of Formula IV in a solvent (N,N-dimethylformamide) to obtain the compound of Formula I;

[0133]

[0134] Where L is a leaving group (e.g., halogens, such as fluorine, chlorine), and rings A, X, M1, M2, M3, R a R c The definitions of R1, R2, R3, R4 and R5 are the same as those described above.

[0135] The present invention also provides a pharmaceutical composition comprising the compound represented by Formula I above or a pharmaceutically acceptable salt thereof (therapeutic amount), and pharmaceutical excipients.

[0136] The present invention also includes the use of the compound of Formula I above, or a pharmaceutically acceptable salt thereof (therapeutic amount), in the preparation of an MK2 inhibitor.

[0137] The present invention also includes the use of the compound of Formula I above, or a pharmaceutically acceptable salt thereof (in an effective therapeutic amount), in the preparation of a medicament for treating or preventing diseases associated with protein HSP27 phosphorylation.

[0138] The present invention also includes a compound III, V, or VI (which can be used to prepare a compound represented by formula I):

[0139]

[0140] Among them, R8 and R 11 Independently for C 1-6Alkyl groups; R9 and R8 are independently amino protecting groups (e.g., THP, Boc), and the other groups are defined as described above.

[0141] Compound III, V, or VI is preferably one of the following compounds:

[0142]

[0143] Unless otherwise specified, the terms used in this invention have the following meanings:

[0144] The term "pharmaceuticalally acceptable salt" refers to a salt obtained by reacting a compound with a pharmaceutically acceptable acid or base. When a compound contains a relatively acidic functional group, a base addition salt can be obtained by contacting the compound with a sufficient amount of a pharmaceutically acceptable base in a suitable inert solvent. Pharmaceutically acceptable base addition salts include, but are not limited to, sodium, potassium, calcium, aluminum, magnesium, bismuth, and ammonium salts. When a compound contains a relatively basic functional group, an acid addition salt can be obtained by contacting the compound with a sufficient amount of a pharmaceutically acceptable acid in a suitable inert solvent. Pharmaceutically acceptable acid addition salts include, but are not limited to, hydrochloride, sulfate, and methanesulfonate salts. For details, see Handbook of Pharmaceutical Salts: Properties, Selection, and Use (P. Heinrich Stahl, Camille G. Wermuth, 2011, 2nd Revised Edition).

[0145] In structural fragments This refers to the structural segment being connected to the rest of the molecule through this site. For example, It refers to cyclohexyl.

[0146] The "-" at the end of a group indicates that the group is attached to the rest of the molecule through that site. For example, CH3-C(=O)- refers to an acetyl group.

[0147] The term "one or more" refers to one, two, or three.

[0148] The term "halogen" refers to fluorine, chlorine, bromine, or iodine.

[0149] The term "oxo" refers to the =O group, where an oxygen atom replaces two hydrogen atoms on the same carbon atom; that is, a carbonyl group replaces a methylene group.

[0150] The term "alkyl" refers to a straight-chain or branched, saturated monovalent hydrocarbon group having a specified number of carbon atoms (e.g., C1-C6). Alkyl groups include, but are not limited to: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, n-hexyl, etc.

[0151] The term "alkylene" refers to a divalent group that is attached to the rest of the molecule by two single bonds, and the rest of the definition is the same as that of the term "alkyl".

[0152] The term "alkoxy" refers to the group R. X -O-,R X The definition is the same as the term "alkyl". Alkoxy groups include, but are not limited to: methoxy, ethoxy, n-propoxy, isopropoxy, etc.

[0153] The term "alkenyl" refers to a straight-chain or branched, unsaturated monovalent hydrocarbon group having a specified number of carbon atoms (e.g., C2-C6) and having one or more (e.g., 1, 2, or 3) carbon-carbon sp groups. 2 Double bond. Alkenyl groups include, but are not limited to: vinyl groups, wait.

[0154] The term "alkynyl" refers to a straight-chain or branched, unsaturated monovalent hydrocarbon group having a specified number of carbon atoms (e.g., C2-C6) and having one or more (e.g., 1, 2, or 3) carbon-carbon sp groups. 3 Triple bond. Alkynyl groups include, but are not limited to: ethynyl, wait.

[0155] The term "ynynyl" refers to a divalent group that is connected to the rest of the molecule by two single bonds, and the rest of the definition is the same as that of the term "ynyl".

[0156] The term "cycloalkyl" refers to a cyclic, saturated monovalent hydrocarbon group having a specified number of carbon atoms (e.g., C3-C6), which is monocyclic. (Monocyclic)cycloalkyl groups include, but are not limited to: wait.

[0157] The term "aryl" refers to an aryl group having a specified number of carbon atoms (e.g., C6-C). 10 A cyclic, unsaturated monovalent hydrocarbon group, which is a monocyclic aryl group including but not limited to: phenyl, etc.

[0158] The term "aromatic ring" is defined as follows, with the rest being the same as the term "aryl": 1. It is connected to the rest of the molecule by two or more single bonds; 2. It shares two atoms and one bond with the rest of the molecule.

[0159] The term "heterocyclic alkyl" refers to a cyclic, saturated monovalent group having a specified number of ring atoms (e.g., 3-10), a specified number of heteroatoms (e.g., 1, 2, or 3), and a specified type of heteroatom (one or more of N, O, and S), which is a monocyclic or polycyclic (2- or 3-cyclic) spirocyclic group. Heterocyclic alkyl groups are attached to the rest of the molecule via carbon atoms or heteroatoms. Heterocyclic alkyl groups include, but are not limited to: azirrocyclic butyl, pyrrolidinyl, piperidinyl, piperazineyl, etc.

[0160] The term “heterocyclic” is defined as follows, with the remaining definitions being the same as the term “heterocyclic alkyl”: 1. It is connected to the rest of the molecule by two or more single bonds; 2. It shares two atoms and one bond with the rest of the molecule.

[0161] The term "heterocyclic alkenyl" refers to a cyclic, unsaturated monovalent hydrocarbon group having a specified number of ring atoms (e.g., 5 to 10), a specified number of heteroatoms (e.g., 1, 2, or 3), and a specified type of heteroatom (one or more of N, O, and S), having one or more (e.g., 1, 2, or 3) carbon-carbon sp groups. 2 A double bond, which is a monocyclic compound, is non-aromatic. Heterocyclic alkenyl groups are attached to the rest of the molecule via carbon atoms or heteroatoms. (Monocyclic) heterocyclic alkenyl groups include, but are not limited to: wait.

[0162] The term "heteroaryl" refers to a cyclic, unsaturated monovalent group having a specified number of ring atoms (e.g., 5-10), a specified number of heteroatoms (e.g., 1, 2, or 3), and a specified type of heteroatom (one or more of N, O, and S), and is monocyclic. The heteroaryl group is attached to the rest of the molecule via a carbon atom or heteroatom; heteroaryls include, but are not limited to: wait.

[0163] The term “heteroaryl ring” meets any of the following conditions, and the rest of the definition is the same as that of the term “heteroaryl group”: 1. It is connected to the rest of the molecule by two or more single bonds; 2. It shares two atoms and one bond with the rest of the molecule.

[0164] The term "therapeutic effective dose" refers to the amount given to a patient that is sufficient to effectively treat the disease. Therapeutic effective doses will vary depending on the type of compound, the type of disease, the severity of the disease, the patient's age, etc., but may be adjusted as appropriate by those skilled in the art.

[0165] The term "pharmaceutical excipients" refers to all substances contained in pharmaceutical preparations other than the active pharmaceutical ingredient, and are generally divided into two main categories: excipients and additives. For details, please refer to the *Pharmacopoeia of the People's Republic of China (2020 Edition)* and *Handbook of Pharmaceutical Excipients* (Paul J Sheskey, Bruno C Hancock, Gary P Moss, David J Goldfarb, 2020, 9th Edition).

[0166] The term "treatment" refers to eliminating the cause of an illness or relieving symptoms.

[0167] The term "prevention" refers to reducing the risk of developing a disease.

[0168] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0169] The reagents and raw materials used in this invention are all commercially available.

[0170] The positive and progressive effects of this invention are as follows: the compounds of this invention have excellent inhibitory activity against phosphorylation of MK2 enzyme and HSP27, the main downstream protein of MK2. Attached Figure Description

[0171] Figure 1 The image shows the MicroED Fourier difference plot of compound 1A. Detailed Implementation

[0172] The following examples are illustrative of the invention and do not limit the invention in any way. Experimental methods in the examples of this invention that do not specify specific conditions are generally performed under conventional conditions or as recommended by the raw material or product manufacturer. Except for synthetic intermediates, all reagents used in this invention are commercially available.

[0173] The structure of the compounds was determined by nuclear magnetic resonance (NMR) and / or mass spectrometry (MS). NMR chemical shifts (δ) are given in ppm. NMR measurements were performed using a Bruker AVANCE-400 NMR spectrometer with deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl3), or deuterated methanol (Methanol-d4) as the solvent and tetramethylsilane (TMS) as the internal standard.

[0174] The HPLC determination was performed using a Shimadzu LC-20ADXR Waters SunFire C18 5μm, 150x4.6 mm anode.

[0175] Preparative HPLC conditions: Waters 3767 / Qda; Column: SunFire C18, 19*250mm, 10μm

[0176] MS measurements were performed using a Shimadzu LCMS-2020 Waters SunFire C18 5μm 50*4.6mm sensor, scanned in positive / negative ion mode, with a mass scan range of 80-1200.

[0177] The known starting materials disclosed herein can be synthesized using or in accordance with methods known in the art, or can be purchased from companies such as Shanghai Titan Technology, Shaoyuan Chemical Technology, and Bid Pharmaceutical.

[0178] Unless otherwise stated, all fractions are by weight, temperatures are in Celsius, and pressures are in atmospheres or close to atmospheres.

[0179] Use the following abbreviations:

[0180]

[0181]

[0182] Preparation of intermediate Int-1:

[0183]

[0184] Step 1: Synthesis of 3-amino-2-methylbenzonitrile

[0185]

[0186] 3-Bromo-2-methylaniline (10.0 g, 53.8 mmol) was dissolved in 80 mL of N,N-dimethylformamide, and zinc cyanide (12.6 g, 10.76 mmol) and tetrakis(triphenylphosphine)palladium (6.22 g, 5.38 mmol) were added. The reaction was carried out at 100 °C for 16 h under nitrogen protection, cooled to room temperature, filtered, diluted with water, and extracted with ethyl acetate (500 mL x 2). The organic phase was washed with brine (100 mL x 4). The combined organic solvents were concentrated, and the residue was purified by kerogen column chromatography (100% ethyl acetate / petroleum ether) to give 3-amino-2-methylbenzonitrile (5.81 g, 71% yield). LCMS: m / z (ESI) = 133.1 [M+H] + .

[0187] Step 2: Synthesis of 3-amino-6-bromo-2-methylbenzonitrile

[0188]

[0189] 3-Amino-2-methylbenzonitrile (5.81 g, 44 mmol) was dissolved in methanol (120 mL) and acetic acid (60 mL), and liquid bromine (1.32 g, 44 mmol) was added dropwise at 0 °C. The mixture was stirred at 0 °C for 2 hours, quenched with sodium thiosulfate solution (100 mL) and an aqueous solution of sodium carbonate (100 mL), and extracted with ethyl acetate (400 mL x 2). The organic phase was concentrated, and the residue was purified by ketene column chromatography (0-50% ethyl acetate / petroleum ether) to give 3-amino-6-bromo-2-methylbenzonitrile (7.8 g, yield 83%). LCMS: m / z (ESI) = 212.9 [M+H] + .

[0190] Step 3: Synthesis of 1-acetyl-5-bromo-1H-indazole-4-formonitrile

[0191]

[0192] 3-Amino-6-bromo-2-methylbenzonitrile (6.6 g, 31.28 mmol) was dissolved in toluene (140 mL), heated to 90 °C, and acetic anhydride (10.6 g, 103.22 mmol) was added. The mixture was then stirred at 90 °C for 2 hours. Tert-butyl nitrite (4.58 g, 44.42 mmol) was added at 90 °C, and the mixture was stirred at 115 °C for 3 hours. The mixture was concentrated to dryness to obtain a crude product, which was then slurried with methanol to give 1-acetyl-5-bromo-1H-indazole-4-carboxynitrile (5.7 g, 69% yield). LCMS: m / z (ESI) = 265.8 [M+H] + .

[0193] Step 4: Synthesis of 5-bromo-1H-indazole-4-formonitrile

[0194]

[0195] 1-Acetyl-5-bromo-1H-indazole-4-carboxynitrile (4 g, 15.2 mmol) was dissolved in methanol (400 mL) and water (80 mL), and potassium carbonate (4.2 g, 30.4 mmol) was added. The mixture was stirred at room temperature for 16 hours and concentrated to dryness. The residue was purified by ketene column chromatography (0-50% ethyl acetate / petroleum ether) to give 5-bromo-1H-indazole-4-carboxynitrile (3.3 g, 98% yield). LCMS: m / z (ESI) = 223.0 [M+H] + .

[0196] Step 5: Synthesis of 5-bromo-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole-4-carboxynitrile

[0197]

[0198] 5-Bromo-1H-indazole-4-carboxynitrile (3.3 g, 14.86 mmol) was dissolved in DCM (120 mL), and 3,4-dihydro-2H-pyran (3.75 g, 44.59 mmol) and p-toluenesulfonic acid (283 mg, 1.49 mmol) were added. The mixture was stirred at room temperature for 16 hours, concentrated to dryness, and the residue was purified by ketene column chromatography (0-25% ethyl acetate / petroleum ether) to give 5-bromo-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole-4-carboxynitrile (4.3 g, 96% yield). LCMS: m / z (ESI) = 223.0 [M+H] + .

[0199] Step 6: Synthesis of methyl 8-amino-3-(tetrahydro-2H-pyran-2-yl)-3H-thieno[3,2-e]indazole-7-carboxylic acid ester

[0200]

[0201] 5-Bromo-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole-4-carboxynitrile (4.3 g, 14.05 mmol) was dissolved in dimethyl sulfoxide (80 mL). Methyl 2-mercaptoacetate (2.98 g, 28.10 mmol) and sodium methoxide (1.52 g, 28.1 mmol) were added at room temperature. The mixture was heated to 75 °C and stirred for 16 hours. The mixture was quenched with water and extracted with ethyl acetate (100 m x 3). The organic phases were combined and concentrated to dryness. The residue was purified by kerogen column chromatography (0-50% ethyl acetate / petroleum ether) to give methyl 8-amino-3-(tetrahydro-2H-pyran-2-yl)-3H-thieno[3,2-e]indazole-7-carboxylic acid ester (3.82 g, 92% yield). LCMS: m / z (ESI) = 332.0 [M+H] + .

[0202] Step 7: Synthesis of methyl 8-((R)-2-(tert-butoxycarbonylamino)propylamino)-3-(tetrahydro-2H-pyran-2-yl)-3H-thieno[3,2-e]indazole-7-carboxylic acid ester

[0203]

[0204] Methyl 8-amino-3-(tetrahydro-2H-pyran-2-yl)-3H-thieno[3,2-e]indazole-7-carboxylic acid ester (3.6 g, 10.8 mmol) was dissolved in 32 mL of N,N-dimethylformamide. Sodium hydrogen (522 mg, 13.0 mmol) was added at 0 °C, and the mixture was stirred for 30 minutes. Then (R)-4-methyl-1,2,3-oxathiazolidin-3-carboxylic acid tert-butyl ester 2,2-dioxide (2.83 g, 11.9 mmol) was added, and the mixture was stirred at 0 °C for 2 hours. The mixture was quenched with water, extracted with ethyl acetate (100 mL x 2), concentrated to dryness, and the residue was purified by pyrogallol column chromatography (0-33% ethyl acetate / petroleum ether) to give methyl 8-((R)-2-(tert-butoxycarbonylamino)propylamino)-3-(tetrahydro-2H-pyran-2-yl)-3H-thieno[3,2-e]indazole-7-carboxylic acid ester (5.3 g, 99% yield). LCMS: m / z (ESI) = 489.1 [M+H] + .

[0205] Step 8: Synthesis of methyl 8-((R)-2-(tert-butoxycarbonylamino)propylamino)-3-(tetrahydro-2H-pyran-2-yl)-3H-thieno[3,2-e]indazole-7-carboxylic acid ester

[0206]

[0207] A methanol (10 mL) solution of methyl 8-((R)-2-(tert-butoxycarbonylamino)propylamino)-3-(tetrahydro-2H-pyran-2-yl)-3H-thieno[3,2-e]indazole-7-carboxylate (1.36 g, 2.66 mmol) was mixed with hydrochloric acid / 1,4-dioxane (4 mol / L, 10 mL), and the mixture was stirred at room temperature for 3 hours. The reaction mixture was concentrated to dryness to give crude methyl 8-((R)-2-(tert-butoxycarbonylamino)propylamino)-3-(tetrahydro-2H-pyran-2-yl)-3H-thieno[3,2-e]indazole-7-carboxylate (1.1 g), which was used directly for the next step. LCMS: m / z (ESI) = 305.0 [M+H] + .

[0208] Step 9: Synthesis of (R)-9-methyl-8,9,10,11-tetrahydro-[1,4]diazo-heptan[5',6':4,5]thieno[3,2-e]indazole-7(3H)-one

[0209]

[0210] Methyl 8-((R)-2-(tert-butoxycarbonylamino)propylamino)-3-(tetrahydro-2H-pyran-2-yl)-3H-thieno[3,2-e]indazole-7-carboxylic acid ester (3 g, 9.8 mmol) was dissolved in methanol (60 mL), and 1,8-diazobisspiro[5.4.0]undec-7-ene (7.5 mL, 49.8 mmol) was added. The reaction mixture was stirred at 70 °C for 16 hours. After the reaction was complete, the reaction mixture was concentrated to dryness, and the residue was purified by preparative liquid chromatography to give (R)-9-methyl-8,9,10,11-tetrahydro-[1,4]diazo-heptan[5′,6′:4,5]thieno[3,2-e]indazole-7(3H)-one (Int-1) (1.9 g, yield 71%). LCMS: m / z(ESI) = 272.9 [M+H] + . 1 H NMR (400MHz, DMSO-d6): δ13.44(s,1H),8.74(s,1H),7.86(d,J=4.0Hz,1H),7.67(d,J=8 .0Hz,2H),6.84(s,1H),3.63-3.61(m,1H),3.48(d,J=4.0Hz,2H),1.20(d,J=6.8Hz,3H).

[0211] Example 1: Synthesis of compounds 1A, 1B, 1C and 1D

[0212]

[0213] (R)-9-methyl-8,9,10,11-tetrahydro-[1,4]diazolypeptido[5',6':4,5]thieno[3,2-e]indazole-7(3H)-one (Int-1) (250 mg, 0.92 mmol) was dissolved in 10 mL of N,N-dimethylformamide, and cesium carbonate (898 mg, 2.76 mmol) and 2,4-dichloro-6-methylpyrimidine (372 mg, 2.30 mmol) were added. The reaction mixture was reacted at 50 °C for 2 hours. The reaction solution was filtered, and the filtrate was concentrated to obtain the crude product. Then, it was purified by preparative high performance liquid chromatography (solvent A: water + 0.1% formic acid; solvent B: acetonitrile; gradient: 45-55%) to obtain:

[0214] 1A:(R)-2-(2-chloro-6-methylpyrimidin-4-yl)-9-methyl-8,9,10,11-tetrahydro-[1,4]diazoylheptan[5',6':4,5]thieno[3,2-e]indazole-7(2H)-one (51.83 mg, yield 14.17%) was a pale yellow solid. LC-MS: m / z (ESI) = 399.0 [M+H] + ;1 H NMR (400MHz, DMSO-d6): δ9.86(s,1H),8.16(s,1H),7.93(d,J=4.0Hz,1H),7.77(d,J=8.0Hz,1H),7.70(d ,J=8.0Hz,1H),7.12(brs,1H),3.63-3.61(m,1H),3.51-3.47(m,2H),2.62(s,3H),1.19(d,J=8.0Hz,3H).

[0215] 1B:(R)-3-(2-chloro-6-methylpyrimidin-4-yl)-9-methyl-8,9,10,11-tetrahydro-[1,4]diazoylheptan[5',6':4,5]thieno[3,2-e]indazole-7(3H)-one (25.61 mg, yield 7.0%) was a pale yellow solid. LC-MS: m / z (ESI) = 399.0 [M+H] + ; 1 H NMR (400MHz, DMSO-d6): δ9.24(s,1H),8.78(d,J=8.0Hz,1H),8.08(d,J=8.0Hz,1H),8.03-8. 01(m,2H),7.02(brs,1H),3.65(brs,1H),3.49(brs,2H),2.58(s,3H),1.21(d,J=8.0Hz,3H).

[0216] 1C:(R)-2-(4-chloro-6-methylpyrimidin-2-yl)-9-methyl-8,9,10,11-tetrahydro-[1,4]diazoylheptan[5',6':4,5]thieno[3,2-e]indazole-7(2H)-one (20.64 mg, yield 5.64%) was a pale yellow solid. LC-MS: m / z (ESI) = 399.0 [M+H] + ; 1 H NMR (400MHz, DMSO-d6): δ9.86 (s, 1H), 7.91 (d, J = 4.0Hz, 1H), 7.73 -7.71(m,3H),7.10-7.07(m,1H),3.63-3.62(m,1H),3.48-3.46(m,2H),2.64(s,3H),1.19(d,J=8.0Hz,3H).

[0217] 1D:(R)-3-(4-chloro-6-methylpyrimidin-2-yl)-9-methyl-8,9,10,11-tetrahydro-[1,4]diazoylheptan[5',6':4,5]thieno[3,2-e]indazole-7(3H)-one (10.02 mg, yield 2.74%) was a pale yellow solid. LC-MS: m / z (ESI) = 399.0 [M+H] + ; 1 H NMR (400MHz, DMSO-d6): δ9.17 (s, 1H), 8.80 (d, J = 8.0Hz, 1H), 8.02-7.99 (m, 2H), 7.59 (s, 1H), 7.04-7.02(m,1H),3.65-3.63(m,1H),3.49-3.46(m,2H),2.64(s,3H),1.21(d,J=8.0Hz,3H).

[0218] Structural identification: Five portions of compound 1A, each 0.16 mg, were weighed and placed in five separate glass vials, and dissolved in ethyl acetate, 1,4-dioxane, acetonitrile, methanol, and acetone, respectively. After evaporation at room temperature in a fume hood for two days, needle-like crystals were obtained under all conditions. A small amount of the needle-like crystals was placed on a clean glass slide, and the presence of microcrystals was confirmed using a polarizing microscope. Micro-crystal electron diffraction (MicroED) analysis revealed that the sample molecule had an indazole N2-substituted framework (excluding 1B and 1D). The MicroED Fourier difference plot based on the ball-and-stick model of the molecular stereostructure of the smallest asymmetric unit (e.g., ...) is shown below. Figure 1 The residual potential indicates the position of hydrogen atoms to distinguish the structures of compounds 1A and 1C.

[0219] Based on the above conclusions and the NMR data of the four products, the structures of the corresponding compounds can be obtained.

[0220] Example 2: Synthesis of compounds 2A and 2B

[0221]

[0222] Step 1: Synthesis of 6-(difluoromethyl)pyrimidine-2,4-diol

[0223]

[0224] Ethyl 4,4-difluoro-3-carbonylbutyrate (2 g, 12 mmol) and carbodiamine (1.44 g, 24 mmol) were dissolved in ethanol (50 mL). Sodium ethoxide (8.16 g, 24 mmol, 20% ethanol solution) was added at room temperature, and the mixture was reacted at 90 °C for 12 hours. The reaction solution was evaporated to dryness, water (50 mL) was added, the pH was adjusted to neutral, and the mixture was extracted with ethyl acetate (100 mL). The organic phase was washed with saturated brine, dried over sodium sulfate, and concentrated to obtain the crude product. The crude product was then purified by pyrogallol column chromatography (petroleum ether:ethyl acetate (V / V) = 1:1) to give 6-(difluoromethyl)pyrimidine-2,4-diol (400 mg, yield 20%) as a brown solid. LC-MS: m / z (ESI) = 163.0 [M+H] + ;

[0225] Step 2: Synthesis of 2,4-dichloro-6-(difluoromethyl)pyrimidine

[0226]

[0227] 6-(difluoromethyl)pyrimidine-2,4-diol (400 mg, 2.45 mmol) was dissolved in phosphorus oxychloride (5 mL) and reacted at 100 °C for 12 hours. The reaction solution was evaporated to dryness, dissolved in dichloromethane (50 mL), slowly added to ice, and the pH was adjusted to neutral. Extraction was then performed with dichloromethane (50 mL), the organic phase was washed with saturated brine (50 mL), dried over sodium sulfate, and concentrated to obtain the crude product. Purification was then achieved by pyrogallol column chromatography (petroleum ether:ethyl acetate (V / V) = 5:1) to give 2,4-dichloro-6-(difluoromethyl)pyrimidine (120 mg, yield 24%) as an off-white solid. LC-MS: m / z (ESI) = 199.0 [M+H] + ;

[0228] Step 3:

[0229] (R)-9-methyl-8,9,10,11-tetrahydro-[1,4]diazolypeptido[5′,6′:4,5]thieno[3,2-e]indazole-7(2H)-one (73 mg, 0.26 mmol) was dissolved in (4 mL) N,N-dimethylformamide, followed by the addition of cesium carbonate (172 mg, 0.53 mmol) and 2,4-dichloro-6-(difluoromethyl)pyrimidine (100 mg, 0.5 mmol). The reaction mixture was reacted at room temperature for 2 hours. The reaction mixture was filtered, and the filtrate was concentrated to obtain the crude product. The crude product was then purified by preparative high-performance liquid chromatography (HPLC) (solvent A: water + 0.1% formic acid; solvent B: acetonitrile; gradient: 35–46%) to obtain the final product.

[0230] Compound 2A:(R)-2-(2-chloro-6-(difluoromethyl)pyrimidin-4-yl)-9-methyl-8,9,10,11-tetrahydro-[1,4]diazoylheptan[5',6':4,5]thieno[3,2-e]indazole-7(2H)-one (7.99 mg, 7% yield) is a yellow solid.

[0231] LC-MS: m / z (ESI) = 435.1 [M+H] + ;

[0232] 1 H NMR (400MHz, DMSO-d6): δ9.93(s,1H),8.38(s,1H),7.95(d,J=4.0Hz,1H),7.80(d,J=8.0Hz,1H),7. 73(d,J=8.0Hz,1H),7.30-7.02(m,2H),3.67-3.60(m,1H),3.51-3.44(m,2H),1.19(d,J=8.0Hz,3H).

[0233] Compound 2B: (R)-3-(2-chloro-6-(difluoromethyl)pyrimidin-4-yl)-9-methyl-8,9,10,11-tetrahydro-[1,4]diazoylheptan[5',6':4,5]thieno[3,2-e]indazole-7(3H)-one (11.74 mg, 10% yield) is a yellow solid.

[0234] LC-MS: m / z (ESI) = 435.1 [M+H] + ;

[0235] 1 H NMR (400MHz, DMSO-d6): δ9.32(s,1H),8.78(d,J=8.0Hz,1H),8.27(s,1H),8.14(d,J=8.0Hz,1H),8. 05(d,J=4.0Hz,1H),7.26-6.99(m,2H),3.68-3.62(m,1H),3.53-3.47(m,2H),1.21(d,J=8.0Hz,3H).

[0236] Example 3: Synthesis of compounds 3A, 3B, 3C and 3D

[0237]

[0238] Step 1: Synthesis of 2,4-dichloro-6-(methyl-d3)pyrimidine

[0239]

[0240] 2,4,6-Trichloropyrimidine (2000 mg, 10.90 mmol) was dissolved in tetrahydrofuran (15 mL), and then cuprous iodide (208 mg, 1.09 mmol) was added. Under nitrogen protection, deuterated methyl magnesium bromide (1330 mg, 10.9 mmol) was added dropwise at 0 °C for 2 hours. Water (100 mL) was added to the reaction mixture, followed by extraction with ethyl acetate (100 mL). The organic phase was washed with saturated brine (100 mL), dried over sodium sulfate, and concentrated to obtain the crude product. The crude product was then purified by pyrogallol column chromatography (petroleum ether:ethyl acetate (V / V) = 8:1) to give 2,4-dichloro-6-(methyl-d3)pyrimidine (200 mg, yield 11.05%) as an off-white solid. LC-MS: m / z (ESI) = 166.1 [M+H] + ;

[0241] Step Two:

[0242] (R)-9-methyl-8,9,10,11-tetrahydro-[1,4]diazolypeptido[5′,6′:4,5]thieno[3,2-e]indazole-7(2H)-one (109 mg, 0.402 mmol) was dissolved in N,N-dimethylformamide (5 mL), followed by the addition of cesium carbonate (393 mg, 1.20 mmol) and 2,4-dichloro-6-(methyl-d3)pyrimidine (200 mg, 1.2 mmol). The reaction mixture was reacted at 50 °C for 5 hours. The reaction mixture was filtered, and the filtrate was concentrated to obtain the crude product. The crude product was then purified by preparative high-performance liquid chromatography (HPLC) (solvent A: water + 0.1% formic acid; solvent B: acetonitrile; gradient: 51–61%) to obtain:

[0243] 3A:(R)-2-(2-chloro-6-(methyl-d3)pyrimidin-4-yl)-9-methyl-8,9,10,11-tetrahydro-[1,4]diazoylheptan[5',6':4,5]thieno[3,2-e]indazole-7(2H)-one (7.36 mg, yield 4.56%) was a pale yellow solid. LC-MS: m / z (ESI) = 402.0 [M+H] + ; 1 HNMR (400MHz, DMSO-d6): δ9.86(s,1H),8.16(s,1H),7.92(d,J=4.0Hz,1H),7.77(d,J=8.0Hz,1H),7.6 9(d,J=8.0Hz,1H),7.12(t,J=4.0Hz,1H),3.63-3.61(m,1H),3.49-3.46(m,2H),1.18(d,J=8.0Hz,3H).

[0244] 3B:(R)-2-(2-chloro-6-(methyl-d3)pyrimidin-4-yl)-9-methyl-8,9,10,11-tetrahydro-[1,4]diazoylheptan[5',6':4,5]thieno[3,2-e]indazole-7(2H)-one (8.03 mg, yield 4.98%) was a pale yellow solid. LC-MS: m / z (ESI) = 401.9 [M+H] + ; 1 HNMR (400MHz, DMSO-d6): δ9.24(s,1H),8.78(d,J=8.0Hz,1H),8.08(d,J=8.0Hz,1H),8.02( s,2H),7.03(t,J=4.0Hz,1H),3.64-3.62(m,1H),3.49-3.46(m,2H),1.20(d,J=8.0Hz,3H).

[0245] 3C:(R)-2-(2-chloro-6-(methyl-d3)pyrimidin-4-yl)-9-methyl-8,9,10,11-tetrahydro-[1,4]diazoylheptan[5',6':4,5]thieno[3,2-e]indazole-7(2H)-one (0.76 mg, yield 0.47%) was a pale yellow solid. LC-MS: m / z (ESI) = 402.0 [M+H] + ; 1 HNMR (400MHz, DMSO-d6): δ9.85(s,1H),7.89(s,1H),7.77-7.75(m,3H),7.08( t,J=4.0Hz,1H),3.63-3.61(m,1H),3.47-3.45(m,2H),1.18(d,J=8.0Hz,3H).

[0246] 3D:(R)-2-(2-chloro-6-(methyl-d3)pyrimidin-4-yl)-9-methyl-8,9,10,11-tetrahydro-[1,4]diazoylheptanzo[5',6':4,5]thieno[3,2-e]indazole-7(2H)-one (1.95 mg, yield 1.21%) was a pale yellow solid. LC-MS: m / z (ESI) = 401.9 [M+H] + ; 1 HNMR (400MHz, DMSO-d6): δ9.18 (s, 1H), 8.81 (d, J = 8.0Hz, 1H), 8.04-8.00 (m, 2H), 7.60 ( s,1H),7.04(t,J=4.0Hz,1H),3.65(brs,1H),3.50-3.47(m,2H),1.22(d,J=4.0Hz,3H).

[0247] Example 4: Synthesis of compounds 4A and 4B

[0248]

[0249] Step 1: Synthesis of 2,4-dichloro-6-cyclopropyl-5-fluoropyrimidine

[0250]

[0251] 2,4-Dichloro-5-fluoropyrimidine (0.5 g, 3.0 mmol) was dissolved in 3 mL of tetrahydrofuran, and cyclopropylmagnesium bromide (3 mL, 3.0 mmol) was added. The reaction was carried out at 0 °C for 1 h. A solution of iodine (0.75 g, 3 mmol) and triethylamine (303 mg, 3 mmol) in tetrahydrofuran (10 mL) was added dropwise, and the reaction was allowed to proceed at room temperature for 12 h after the addition was complete. After the reaction was complete, the solution was diluted with saturated ammonium chloride solution and extracted with ethyl acetate. The resulting organic phase was dried over anhydrous sodium sulfate and concentrated to obtain a crude product. The product was purified by silica gel column chromatography (elution with petroleum ether / ethyl acetate = 1 / 1). The product, 2,4-dichloro-6-cyclopropyl-5-fluoropyrimidine (0.1 g, yield 16.1%), was a yellow solid. LC-MS: m / z (ESI) = 207.0 [M+H] +

[0252] Step Two:

[0253] 2,4-Dichloro-6-cyclopropyl-5-fluoropyrimidine (0.1 g, 0.48 mmol) was dissolved in 5 mL of N,N-dimethylformamide, and cesium carbonate (475.4 mg, 1.45 mmol) and (R)-9-methyl-8,9,10,11-tetrahydro-[1,4]diazoylheptan[5′,6':4,5]thieno[3,2-e]indazole-7(2H)-one (130.6 mg, 0.48 mmol) were added. The mixture was stirred at 25 °C for 1 hour. After the reaction was complete, the product was obtained by preparative high performance liquid chromatography.

[0254] 4A:(R)-2-(2-chloro-6-cyclopropyl-5-fluoropyrimidin-4-yl)-9-methyl-8,9,10,11-tetrahydro-[1,4]diazoylheptan[5′,6':4,5]thieno[3,2-e]indazole-7(2H)-one (12.14 mg, yield 5.7%), as a yellow solid. LC-MS: m / z (ESI) = 443.0 [M+H] + ; 1H NMR (400MHz, DMSO-d6): δ9.79(s,1H),7.92(d,J=4.0Hz,1H),7.78-7.72(m,2H),7.10(brs,1H),3.62(br ,1H),3.47-3.45(m,2H),2.54-2.52(m,1H),1.34-1.31(m,2H),1.23-1.21(m,2H),1.19(d,J=8.0Hz,3H).

[0255] 4B:(R)-3-(2-chloro-6-cyclopropyl-5-fluoropyrimidin-4-yl)-9-methyl-8,9,10,11-tetrahydro-[1,4]diazoylheptan[5',6':4,5]thieno[3,2-e]indazole-7(3H)-one (18.33 mg, yield 8.6%), product as a yellow solid. LC-MS: m / z (ESI) = 443.0 [M+H] + ; 1 HNMR (400MHz, DMSO-d6): δ9.25(s,1H),8.46(d,J=8.0Hz,1H),8.05(d,J=8.0Hz,1H),8.02(d,J=8.0Hz,1H),7.03(t,J=4.0Hz ,1H),3.68-3.64(m,1H),3.51-3.48(m,2H),1.56-1.54(m,1H),1.32-1.27(m,2H),1.21(d,J=8.0Hz,3H),1.19-1.16(m,2H).

[0256] Example 5: Synthesis of compounds 5A and 5B

[0257]

[0258] Step 1: Synthesis of 2,4-dichloro-5-fluoro-6-(methoxymethyl)pyrimidine

[0259]

[0260] 2,4-Dichloro-5-fluoropyrimidine (1 g, 6 mmol) was dissolved in dry tetrahydrofuran (20 mL). Magnesium bromide (9 mL, 9 mmol, 1 M tetrahydrofuran solution) was added dropwise at 0 °C under nitrogen protection, and the reaction was allowed to proceed at room temperature for 1 hour. The temperature was lowered to 0 °C, and a tetrahydrofuran solution of iodine (1.5 g, 6 mmol) and triethylamine (605 mg, 6 mmol) was added dropwise. The reaction was allowed to proceed at room temperature for 12 hours after the addition was complete. Water (50 mL) was added to the reaction mixture, followed by extraction with ethyl acetate (100 mL). The organic phase was washed with saturated brine, dried over sodium sulfate, and concentrated to obtain the crude product. The crude product was then purified by kerogen column chromatography (petroleum ether:ethyl acetate (V / V) = 10:1) to obtain 2,4-dichloro-5-fluoro-6-(methoxymethyl)pyrimidine (400 mg, yield 31%) as an oily liquid. LC-MS: m / z(ESI) = 211[M+H] + ;

[0261] Step Two:

[0262] (R)-9-methyl-8,9,10,11-tetrahydro-[1,4]diazo-heptan[5′,6′:4,5]thieno[3,2-e]indazole-7(2H)-one (50 mg, 0.18 mmol) was dissolved in (3 mL) N,N-dimethylformamide, and then cesium carbonate (117 mg, 0.36 mmol) and 2,4-dichloro-5-fluoro-6-(methoxymethyl)pyrimidine (72 mg, 0.34 mmol) were added. The reaction solution was reacted at 50 °C for 4 hours. The reaction solution was filtered, and the filtrate was concentrated to obtain the crude product. Then, it was purified by preparative high performance liquid chromatography (solvent A: water + 0.1% formic acid; solvent B: acetonitrile; gradient: 42-58%) to obtain:

[0263] 5A:(R)-2-(2-chloro-5-fluoro-6-(methoxymethyl)pyrimidin-4-yl)-9-methyl-8,9,10,11-tetrahydro-[1,4]diazoylheptan[5',6':4,5]thieno[3,2-e]indazole-7(2H)-one (7 mg, yield 8.6%) was a yellow solid. LC-MS: m / z (ESI) = 447.0 [M+H] + ; 1 H NMR (400MHz, DMSO-d6): δ9.83 (s, 1H), 7.93 (d, J = 4.0Hz, 1H), 7.78-7.71 (m, 2H), 7.11 (brs, 1H), 4.71 (s, 2H), 3.64-3.58 (m, 1H), 3.52-3.45 (m, 2H), 3.43 (s, 3H), 1.19 (d, J = 8.0Hz, 3H).

[0264] 5B:(R)-3-(2-chloro-5-fluoro-6-(methoxymethyl)pyrimidin-4-yl)-9-methyl-8,9,10,11-tetrahydro-[1,4]diazoylheptan[5′,6′:4,5]thieno[3,2-e]indazole-7(3H)-one (11.19 mg, yield 14%) was a yellow solid. 1 H NMR (400MHz, DMSO-d6): δ9.27(s,1H),8.52(d,J=8.0Hz,1H),8.08(d,J=8.0Hz,1H),8.03(d,J=4.0Hz,1H),7 .03(t,J=4.0Hz,1H),4.68(s,2H),3.67-3.63(m,1H),3.49-3.45(m,2H),3.42(s,3H),1.21(d,J=8.0Hz,3H).

[0265] Example 6: Synthesis of compounds 6A and 6B

[0266]

[0267] Step 1: Synthesis of 2,4-dichloro-6-(oxobutylcyclo-3-yl)pyrimidine

[0268]

[0269] Under nitrogen protection, a solution of nickel dichloro(dimethoxyethane) (6 mg, 0.03 mmol) and 4,4′-di-tert-butyl-2,2′-dipyridine (7 mg, 0.03 mmol) in 1,2-dimethoxyethane (2 mL) was added to 2,4,6-trichloropyrimidine (500 mg, 2.73 mmol), 3-bromooxidine (374 mg, 2.73 mmol), and (4,4′-di-tert-butyl-2,2′-bipyridine). The reaction mixture was reacted in a solution of bis[3,5-difluoro-2-[5-(trifluoromethyl)-2-pyridyl]phenyl]iridium(III) hexafluorophosphate (31 mg, 0.03 mmol), tris(trimethylsilyl)silane (2.03 g, 8.2 mmol), and sodium carbonate (579 mg, 5.46 mmol) in 1,2-dimethoxyethane (10 mL) at room temperature under blue LED light (420 nm, 40 W) for 12 h. Water (20 mL) was added to the reaction mixture, followed by extraction with ethyl acetate (50 mL). The organic phase was washed with saturated brine (50 mL), dried over sodium sulfate, and concentrated to obtain the crude product. The crude product was then purified by pyrogallol column chromatography (petroleum ether:ethyl acetate (V / V) = 10:1) to give 2,4-dichloro-6-(oxadicyclo-3-yl)pyrimidine (100 mg, yield 18%) as a white solid. LC-MS: m / z(ESI) = 205[M+H] + ;

[0270] Step Two:

[0271] (R)-9-methyl-8,9,10,11-tetrahydro-[1,4]diazolypeptido[5′,6′:4,5]thieno[3,2-e]indazole-7(2H)-one (50 mg, 0.18 mmol) was dissolved in (3 mL) N,N-dimethylformamide, and then cesium carbonate (117 mg, 0.36 mmol) and 2,4-dichloro-6-(oxobutylcyclo-3-yl)pyrimidine (70 mg, 0.34 mmol) were added. The reaction mixture was reacted at room temperature for 2 hours. The reaction mixture was filtered, and the filtrate was concentrated to obtain the crude product. Then, it was purified by preparative high performance liquid chromatography (solvent A: water + 0.1% formic acid; solvent B: acetonitrile; gradient: 41–54%) to obtain:

[0272] 6A:(R)-2-(2-chloro-6-(oxadiazon-3-yl)pyrimidin-4-yl)-9-methyl-8,9,10,11-tetrahydro-[1,4]diazoylheptan[5′,6′:4,5]thieno[3,2-e]indazole-7(2H)-one (5.66 mg, 7% yield) was a yellow solid.

[0273] LC-MS: m / z (ESI) = 441.2 [M+H] + ; 1 H NMR (400MHz, DMSO-d6): δ9.89(s,1H),8.19(s,1H),7.94

[0274] (d,J=4.0Hz,1H),7.78(d,J=8.0Hz,1H),7.71(d,J=8.0Hz,1H),7.14(t,J=4.0Hz,1H),4.96-4.92(m,2H) ,4.82(t,J=8.0Hz,2H),4.66-4.57(m,1H),3.66-3.60(m,1H),3.52-3.45(m,2H),1.19(d,J=8.0Hz,3H).

[0275] 6B:(R)-3-(2-chloro-6-(oxobutylcyclo-3-yl)pyrimidin-4-yl)-9-methyl-8,9,10,11-tetrahydro-[1,4]diazoylheptan[5′,6′:4,5]thieno[3,2-e]indazole-7(3H)-one (13.61 mg, 17% yield) was a yellow solid. 1H NMR (400MHz, DMSO-d6): δ9.25(s,1H),8.78(d,J=8.0Hz,1H),8.09(d,J=8.0Hz,1H),8.03(s,2H),7.02(t,J=4.0Hz,1H),4 .94-4.91(m,2H),4.81(t,J=4.0Hz,2H),4.59-4.55(m,1H),3.68-3.61(m,1H),3.52-3.46(m,2H),1.21(d,J=8.0Hz,3H).

[0276] Example 7: Synthesis of Compound 7A

[0277]

[0278] Step 1: Synthesis of 2,4-dichloro-6-((trimethylsilyl)ethynyl)pyrimidine

[0279]

[0280] 2,4,6-Trichloropyrimidine (500 mg, 2.73 mmol), cuprous iodide (16 mg, 0.08 mmol), triethylamine (552 mg, 5.46 mmol), and palladium dichloride bis(triphenylphosphine) (173 mg, 0.25 mmol) were dissolved in tetrahydrofuran (20 mL). Ethynyltrimethylsilane (268 mg, 2.73 mmol) was added under nitrogen protection, and the reaction was allowed to proceed at room temperature for 12 hours after the addition was complete. Water (50 mL) was added to the reaction mixture, followed by extraction with ethyl acetate (100 mL). The organic phase was washed with saturated brine, dried over sodium sulfate, and concentrated to obtain the crude product. The crude product was then purified by pyrogallol column chromatography (petroleum ether:ethyl acetate (V / V) = 5:1) to give 2,4-dichloro-6-((trimethylsilyl)ethynyl)pyrimidine (300 mg, yield 44.8%) as a white solid. LC-MS: m / z (ESI) = 245 [M+H] + ;

[0281] Step 2: Synthesis of (R)-2-(2-chloro-6-ethynylpyrimidin-4-yl)-9-methyl-8,9,10,11-tetrahydro-[1,4]diazoylheptan[5′,6′:4,5]thieno[3,2-e]indazole-7(2H)-one

[0282] (R)-9-methyl-8,9,10,11-tetrahydro-[1,4]diazolypton[5′,6′:4,5]thieno[3,2-e]indazole-7(2H)-one (100 mg, 0.37 mmol) was dissolved in (5 mL) N,N-dimethylformamide, followed by the addition of potassium carbonate (101 mg, 0.74 mmol) and 2,4-dichloro-6-((trimethylsilyl)ethynyl)pyrimidine (180 mg, 0.74 mmol). The reaction mixture was reacted at 50 °C for 4 hours. The reaction mixture was filtered, and the filtrate was concentrated to obtain the crude product. The solution was then purified by preparative high-performance liquid chromatography (HPLC) (solvent A: water + 0.1% formic acid; solvent B: acetonitrile; gradient: 40–53%) to give (R)-2-(2-chloro-6-ethynylpyrimidin-4-yl)-9-methyl-8,9,10,11-tetrahydro-[1,4]diazoylheptanzo[5′,6′:4,5]thieno[3,2-e]indazole-7(2H)-one (7A) (9.73 mg, yield 6.4%) as a yellow solid. LC-MS: m / z (ESI) = 409.0 [M+H] + ; 1 HNMR (400MHz, DMSO-d6): δ9.92(s,1H),8.24(s,1H),7.95(d,J=8.0Hz,1H),7.78(d,J=8.0Hz,1H),7.70(d,J= 8.0Hz,1H),7.16(t,J=4.0Hz,1H),5.13(s,1H),3.64-3.59(m,1H),3.50-3.46(m,2H),1.19(d,J=4.0Hz,3H).

[0283] Example 8: Synthesis of compounds 8A, 8B, 8C and 8D

[0284]

[0285] Step 1: Synthesis of 2,4-dichloro-6-(1-methyl-1H-pyrazol-3-yl)pyrimidine

[0286]

[0287] 2,4,6-Trichloropyrimidine (200 mg, 1.09 mmol) was dissolved in (5 mL) tetrahydrofuran and (1 mL) water. Then, 1-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxoboropentane-2-yl)-1H-pyrazole (227 mg, 1.09 mmol), sodium bicarbonate (275 mg, 3.27 mmol), and tetrakis(triphenylphosphine)palladium (126 mg, 0.109 mmol) were added. The reaction mixture was reacted at 80 °C for 6 hours. Water (100 mL) was added to the reaction mixture, and the mixture was extracted with ethyl acetate (100 mL). The organic phase was washed with saturated brine (100 mL), dried over sodium sulfate, and concentrated to obtain the crude product. The product was then purified by stannin column chromatography (petroleum ether:ethyl acetate (V / V) = 2:1) to give 2,4-dichloro-6-(1-methyl-1H-pyrazol-3-yl)pyrimidine (70 mg, yield 28.03%) as an off-white solid. LC-MS: m / z (ESI) = 229.0 [M+H] + ;

[0288] Step Two:

[0289] (R)-9-methyl-8,9,10,11-tetrahydro-[1,4]diazolypeptido[5′,6′:4,5]thieno[3,2-e]indazole-7(2H)-one (42 mg, 0.153 mmol) was dissolved in (3 mL) N,N-dimethylformamide, and then cesium carbonate (149 mg, 0.458 mmol) and 2,4-dichloro-6-(1-methyl-1H-pyrazol-3-yl)pyrimidine (70 mg, 0.306 mmol) were added. The reaction solution was reacted at 50 °C for 5 hours. The reaction solution was filtered, and the filtrate was concentrated to obtain the crude product. Then, it was purified by preparative high performance liquid chromatography (solvent A: water + 0.1% formic acid; solvent B: acetonitrile; gradient: 61-71%) to obtain:

[0290] 8A:(R)-2-(2-chloro-6-(1-methyl-1H-pyrazol-3-yl)pyrimidin-4-yl)-9-methyl-8,9,10,11-tetrahydro-[1,4]diazoylheptan[5′,6′:4,5]thieno[3,2-e]indazole-7(2H)-one (6.64 mg, yield 9.35%) was a pale yellow solid. LC-MS: m / z (ESI) = 465.0 [M+H] + ; 1H NMR (400MHz, DMSO-d6): δ9.93(s,1H),7.99(d,J=4.0Hz,1H),7.97(s,1H),7.90(d,J=4.0Hz,1H),7.77-7.72(m ,2H),7.28(d,J=4.0Hz,1H),7.09(brs,1H),4.02(s,3H),3.63(brs,1H),3.50(brs,2H),1.21(d,J=8.0Hz,3H).

[0291] 8B:(R)-2-(2-chloro-6-(1-methyl-1H-pyrazol-3-yl)pyrimidin-4-yl)-9-methyl-8,9,10,11-tetrahydro-[1,4]diazoylheptan[5′,6′:4,5]thieno[3,2-e]indazole-7(2H)-one (8.58 mg, yield 12.08%) was a pale yellow solid. LC-MS: m / z (ESI) = 465.0 [M+H] + ; 1 H NMR (400MHz, DMSO-d6) δ9.27(s,1H),8.81(d,J=8.0Hz,1H),8.42(s,1H),8.10(d,J=8.0Hz,1H),8.03(d,J=4.0Hz, 1H),7.93(brs,1H),7.05(s,1H),6.99(s,1H),4.01(s,3H),3.65(brs,1H),3.49(brs,2H),1.21(d,J=4.0Hz,3H).

[0292] 8C:(R)-2-(2-chloro-6-(1-methyl-1H-pyrazol-3-yl)pyrimidin-4-yl)-9-methyl-8,9,10,11-tetrahydro-[1,4]diazoylheptan[5′,6′:4,5]thieno[3,2-e]indazole-7(2H)-one (2.16 mg, yield 3.04%) was a pale yellow solid. LC-MS: m / z (ESI) = 465.0 [M+H] + ; 1 H NMR (400MHz, DMSO-d6): δ9.89 (s, 1H), 8.55 (s, 1H), 7.96-7.92 (m, 2H), 7.77 (d, J = 4.0Hz, 2H), 7 .14(brs,1H),7.02(s,1H),4.03(s,3H),3.64(brs,1H),3.48(brs,2H),1.19(d,J=8.0Hz,3H).

[0293] 8D:(R)-2-(2-chloro-6-(1-methyl-1H-pyrazol-3-yl)pyrimidin-4-yl)-9-methyl-8,9,10,11-tetrahydro-[1,4]diazoylheptan[5′,6′:4,5]thieno[3,2-e]indazole-7(2H)-one (2.35 mg, yield 3.31%) was a pale yellow solid. LC-MS: m / z (ESI) = 465.0 [M+H] + ; 1 H NMR (400MHz, DMSO-d6): δ9.19(s,1H),8.90(d,J=8.0Hz,1H),8.06(d,J=8.0Hz,1H),8.00(d,J=4.0Hz,2H),7 .88(s,1H),7.18(s,1H),7.04(brs,1H),4.02(s,3H),3.65(brs,1H),3.50(brs,2H),1.21(d,J=8.0Hz,3H).

[0294] Example 9: Synthesis of compounds 9A and 9B

[0295]

[0296] Step 1: Synthesis of tert-butoxycarbonyl ((2,4-dichloropyrimidin-5-yl)methyl)carbamate

[0297]

[0298] 2,4-Dichloro-5-(chloromethyl)pyrimidine (300 mg, 1.52 mmol) was dissolved in 10 mL of tetrahydrofuran, followed by the addition of potassium di(tert-butoxycarbonyl)amide (621 mg, 2.43 mmol) and sodium iodide (456 mg, 3.04 mmol). The reaction mixture was reacted at 30 °C for 2 hours. Water (100 mL) was added to the reaction mixture, followed by extraction with ethyl acetate (100 mL). The organic phase was washed with saturated brine (100 mL), dried over sodium sulfate, and concentrated to obtain the crude product. The crude product was then purified by kerogen column chromatography (petroleum ether:ethyl acetate (V / V) = 2:1) to give tert-butoxycarbonyl((2,4-dichloropyrimidine-5-yl)methyl)carbamate (300 mg, yield 52.20%) as an off-white solid. LC-MS: m / z (ESI) = 378.0 [M+H] + ;

[0299] Step Two:

[0300] (R)-9-methyl-8,9,10,11-tetrahydro-[1,4]diazo-heptan[5′,6′:4,5]thieno[3,2-e]indazole-7(2H)-one (53 mg, 0.194 mmol) was dissolved in N,N-dimethylformamide (3 mL), followed by the addition of cesium carbonate (190 mg, 0.582 mmol) and tert-butoxycarbonyl((2,4-dichloropyrimidin-5-yl)methyl)carbamate (110 mg, 0.291 mmol). The reaction mixture was reacted at 50 °C for 5 hours. The reaction mixture was filtered, and the filtrate was diluted with water and filtered again to obtain product mixtures 9-3A and 9-3B (80 mg, yield 67.19%), as pale yellow solids. LC-MS: m / z (ESI) = 614.0 [M+H] + ;

[0301] Step 3:

[0302] The above mixture (80 mg, 0.130 mmol) was dissolved in hydrochloric acid / dioxane (5 mL). The reaction solution was reacted at 25 °C for 2 hours. The reaction solution was concentrated to dryness to obtain the crude product. Then, it was purified by preparative high performance liquid chromatography (solvent A: water + 0.1% formic acid; solvent B: acetonitrile; gradient: 15–25%) to obtain:

[0303] 9A:(R)-2-(5-(aminomethyl)-2-chloropyrimidin-4-yl)-9-methyl-8,9,10,11-tetrahydro-[1,4]diazoylheptan[5′,6′:4,5]thieno[3,2-e]indazole-7(2H)-one (1.35 mg, yield 2.50%) was a pale yellow solid. LC-MS: m / z (ESI) = 414.0 [M+H] + ; 1 H NMR (400MHz, DMSO-d6): δ9.89(s,1H),9.06(s,1H),8.21(s,1H),7.93(d,J=4.0Hz,1H),7.79-7.72( m,2H),7.12(t,J=4.0Hz,1H),4.27(s,2H),3.63(brs,1H),3.49-3.46(m,2H),1.20(d,J=8.0Hz,3H).

[0304] 9B:(R)-2-(5-(aminomethyl)-2-chloropyrimidin-4-yl)-9-methyl-8,9,10,11-tetrahydro-[1,4]diazoylheptan[5′,6′:4,5]thieno[3,2-e]indazole-7(2H)-one (1.99 mg, yield 3.69%) was a pale yellow solid. LC-MS: m / z (ESI) = 414.0 [M+H] + ; 1H NMR (400MHz, DMSO-d6): δ9.23(s,1H),8.98(s,1H),8.52(d,J=8.0Hz,1H),8.23(s,1H),8.06-8.01(m, 2H),7.02(t,J=4.0Hz,1H),4.13(s,2H),3.65-3.62(m,1H),3.49-3.47(m,2H),1.20(d,J=8.0Hz,3H).

[0305] Example 10: Synthesis of compounds 10A and 10B

[0306]

[0307] Step 1: Synthesis of 5-((3-methoxyacin-1-yl)methyl)pyrimidine-2,4-diol

[0308]

[0309] 3-Methoxyacridine (0.54 g, 6.25 mmol) was dissolved in dichloromethane, and triethylamine (1.9 g, 18.75 mmol) and 5-(chloromethyl)pyrimidine-2,4-diol (1 g, 6.25 mmol) were added. The mixture was reacted at 25 °C for 1 hour. After the reaction was complete, the solution was diluted with saturated sodium chloride aqueous solution and extracted with dichloromethane to obtain a crude product. The crude product was then purified by preparative high-performance liquid chromatography (HPLC) to obtain the product 5-((3-methoxyacridine-1-yl)methyl)pyrimidine-2,4-diol (0.2 g, yield 15%) as a white solid. LC-MS: m / z (ESI) = 211.2 [M+H] + .

[0310] Step 2: Synthesis of 2,4-dichloro-5-((3-methoxyacryl-1-yl)methyl)pyrimidine

[0311]

[0312] 5-((3-methoxyacrimidin-1-yl)methyl)pyrimidine-2,4-diol (0.2 g, 0.95 mmol) was dissolved in 5 mL of phosphorus oxychloride and stirred at 100 °C for 1 hour. After the reaction was complete, the reaction solution was concentrated to dryness, diluted with saturated sodium chloride aqueous solution, and extracted with dichloromethane to obtain the crude product. The crude product was then purified by preparative high-performance liquid chromatography (HPLC) to obtain the product 2,4-dichloro-5-((3-methoxyacrimidin-1-yl)methyl)pyrimidine (0.1 g, yield 42.4%) as a yellow solid. LC-MS: m / z (ESI) = 248.1 [M+H] + .

[0313] Step 3:

[0314] 2,4-Dichloro-5-((3-methoxyacinidin-1-yl)methyl)pyrimidine (0.10 g, 0.40 mmol) was dissolved in 5 mL of N,N-dimethylformamide, and 8,9,10,11-tetrahydro-[1,4]diazolyptono[5′,6′:4,5]thieno[3,2-e]indazole-7(2H)-one (0.1 g, 0.4 mmol) and cesium carbonate (394 mg, 1.2 mmol) were added. The mixture was stirred at 25 °C for 1 hour. After the reaction was complete, the reaction solution was concentrated to dryness under vacuum to obtain the crude product, which was then purified by preparative high performance liquid chromatography.

[0315] 10A:(R)-2-(2-chloro-5-((3-methoxyacinidin-1-yl)methyl)pyrimidin-4-yl)-9-methyl-8,9,10,11-tetrahydro-[1,4]diazoylheptan[5′,6′:4,5]thieno[3,2-e]indazole-7(2H)-one (20.0 mg, yield 10.3%), as a yellow solid. LC-MS: m / z (ESI) = 484.0 [M+H] + ; 1 H NMR (400MHz, DMSO-d): δ9.98 (s, 1H), 9.14 (brs, 1H), 7.96 (d, J = 4.0Hz, 1H), 7.87-7.84 (m, 2H), 7.17-7.14 (m, 1H), 4.96-4 .89(m,2H),4.64-4.60(m,2H),4.42-4.35(m,3H),3.63(brs,1H),3.49-3.46(m,2H),3.28(s,3H),1.19(d,J=4.0Hz,3H).

[0316] 10B:(R)-3-(2-chloro-5-((3-methoxyacinidin-1-yl)methyl)pyrimidin-4-yl)-9-methyl-8,9,10,11-tetrahydro-[1,4]diazoylheptan[5′,6′:4,5]thieno[3,2-e]indazole-7(3H)-one (10.0 mg, yield 5.2%), is a yellow solid. LC-MS: m / z (ESI) = 484.0 [M+H] + ; 1H NMR (400MHz, DMSO-d6): δ9.36(s,1H),9.04(brs,1H),8.68(d,J=8.0Hz,1H),8.15(d,J=8.0Hz,1H),8.07(d,J=4.0Hz,1H),7. 10(s,1H),4.87-4.85(m,2H),4.48-4.45(s,2H),4.26-4.22(m,3H),3.65(br,2H),3.29-3.26(m,4H),1.20(d,J=4.0Hz,3H).

[0317] Example 11: Synthesis of compounds 11A and 11B

[0318]

[0319] Step 1: Synthesis of tert-butyl (2-((2,4-dichloropyrimidin-5-yl)oxy)ethyl)carbamate

[0320]

[0321] 2,4-Dichloropyrimidin-5-phenol (200 mg, 1.21 mmol) was dissolved in tetrahydrofuran (5 mL), and then tert-butyl(2-hydroxyethyl)carbamate (391 mg, 2.42 mmol), triphenylphosphine (636 mg, 2.42 mmol), and di-tert-butyl azodicarbonate (558 mg, 2.42 mmol) were added. The reaction mixture was reacted at 25 °C for 6 hours. Water (100 mL) was added to the reaction mixture, and the mixture was extracted with ethyl acetate (100 mL). The organic phase was washed with saturated brine (100 mL), dried over sodium sulfate, and concentrated to obtain the crude product. The crude product was then purified by kerogen column chromatography (petroleum ether:ethyl acetate (V / V) = 2:1) to obtain (2-((2,4-dichloropyrimidin-5-yl)oxy)ethyl)carbamate tert-butyl ester (350 mg, yield 93.69%) as an off-white solid. LC-MS: m / z (ESI) = 251.9 [M+H] + ;

[0322] Step Two:

[0323] (R)-9-methyl-8,9,10,11-tetrahydro-[1,4]diazolypeptido[5′,6′:4,5]thieno[3,2-e]indazole-7(2H)-one (66 mg, 0.243 mmol) was dissolved in N,N-dimethylformamide (3 mL), followed by the addition of sodium hydroxide (12 mg, 0.486 mmol). The reaction mixture was reacted at 0 °C for 1 hour. Then, tert-butyl (2-((2,4-dichloropyrimidin-5-yl)oxy)ethyl)carbamate (150 mg, 0.487 mmol) was added, and the reaction mixture was reacted at 25 °C for 5 hours. Water (10 mL) was added to the reaction mixture, followed by extraction with ethyl acetate (10 mL). The organic phase was washed with saturated brine (10 mL), dried over sodium sulfate, and concentrated to obtain the crude product. The mixture was then purified by pyrite column chromatography (petroleum ether:ethyl acetate (V / V) = 3:1) to give mixtures 11-3A and 11-3B (50 mg, yield 37.76%) as pale yellow solids. LC-MS: m / z (ESI) = 544.2 [M+H] + ;

[0324] Step 3:

[0325] The above mixture (0.050 g, 0.092 mmol) was dissolved in 3 mL of dichloromethane, and trifluoroacetic acid (3 mL) was added dropwise with stirring. The reaction mixture was stirred at 25 °C for 2 hours. The solution was concentrated to dryness under vacuum, and the residue was diluted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness to obtain the crude product. This crude product was then purified by preparative high-performance liquid chromatography (solvent A: water + 0.1% trifluoroacetic acid; solvent B: acetonitrile; gradient: 22–32%) to obtain:

[0326] 11A:(R)-2-(5-(2-aminoethoxy)-2-chloropyrimidin-4-yl)-9-methyl-8,9,10,11-tetrahydro-[1,4]diazoylheptan[5′,6′:4,5]thieno[3,2-e]indazole-7(2H)-one (12.22 mg, yield 29.95%), product was a pale yellow solid. LC-MS: m / z (ESI) = 444.0 [M+H] + ; 1 H NMR (400MHz, DMSO-d6): δ9.77(s,1H),9.01(s,1H),8.04(brs,3H),7.94(d,J=4.0Hz,1H),7.82-7.75(m,2H),7 .01(s,1H),4.57(t,J=8.0Hz,2H),3.63-3.59(m,1H),3.47(brs,2H),3.38-3.36(m,2H),1.19(d,J=4.0Hz,3H).

[0327] 11B:(R)-2-(5-(2-aminoethoxy)-2-chloropyrimidin-4-yl)-9-methyl-8,9,10,11-tetrahydro-[1,4]diazoylheptan[5′,6′:4,5]thieno[3,2-e]indazole-7(2H)-one (23.17 mg, yield 56.79%), product was a pale yellow solid. LC-MS: m / z (ESI) = 444.0 [M+H] + ; 1 H NMR (400MHz, DMSO-d6): δ9.21(s,1H),8.94(s,1H),8.19(d,J=8.0Hz,1H),8.03-7.99(m,2H),7.90(brs,3H ),7.04(brs,1H),4.47-4.45(m,2H),3.65(brs,1H),3.49(brs,2H),3.24(brs,2H),1.21(d,J=4.0Hz,3H).

[0328] Example 12: Synthesis of compounds 12A and 12B

[0329]

[0330] Step 1: Synthesis of 2,6-dichloro-N-(2,2-difluoroethyl)pyrimidine-4-carboxamide

[0331]

[0332] 2,6-Dichloropyrimidine-4-carbonyl chloride (1 g, 4.74 mmol) was dissolved in dichloromethane (10 mL) and added dropwise under ice bath to a dichloromethane (20 mL) solution of 2,2-difluoroethane-1-amine (384 mg, 4.74 mmol) and triethylamine (957 mg, 9.48 mmol). The reaction was carried out at room temperature for 4 hours. Water (50 mL) was added to the reaction solution, followed by extraction with dichloromethane (100 mL). The organic phase was washed with saturated brine, dried over sodium sulfate, and concentrated to obtain the crude product. The crude product was then purified by pyrogallol column chromatography (petroleum ether:ethyl acetate (V / V) = 2:1) to give 2,6-dichloro-N-(2,2-difluoroethyl)pyrimidine-4-carboxamide (500 mg, yield 41%) as an off-white solid. LC-MS: m / z (ESI) = 256 [M+H] + ;

[0333] Step Two:

[0334] (R)-9-methyl-8,9,10,11-tetrahydro-[1,4]diazolypeptido[5′,6′:4,5]thieno[3,2-e]indazole-7(2H)-one (50 mg, 0.18 mmol) was dissolved in 3 mL of N,N-dimethylformamide, followed by the addition of cesium carbonate (117 mg, 0.36 mmol) and 2,6-dichloro-N-methylpyrimidine-4-carboxamide (89 mg, 0.34 mmol). The reaction mixture was reacted at 50 °C for 4 hours. The reaction mixture was filtered, and the filtrate was concentrated to obtain the crude product. The crude product was then purified by preparative high-performance liquid chromatography (HPLC) (solvent A: water + 0.1% formic acid; solvent B: acetonitrile; gradient: 40–55%) to obtain:

[0335] 12A: (R)-2-chloro-N-(2,2-difluoroethyl)-6-(9-methyl-7-carbonyl-8,9,10,11-tetrahydro-[1,4]diazoylheptan[5′,6′:4,5]thieno[3,2-e]indazole-2(7H)-yl)pyrimidin-4-carboxamide (17.59 mg, yield 19.8%) was a yellow solid. LC-MS: m / z (ESI) = 492.0 [M+H] + ; 1 H NMR (400MHz, DMSO-d6): δ9.93(s,1H),9.37(t,J=8.0Hz,1H),8.58(s,1H),7.94(d,J=4.0Hz,1H),7.81-7.74(m,2H),7. 14(t,J=4.0Hz,1H),6.38-6.03(m,1H),3.82-3.77(m,2H),3.66-3.63(m,1H),3.52-3.48(m,2H),1.19(d,J=8.0Hz,3H).

[0336] 12B:(R)-2-chloro-N-(2,2-difluoroethyl)-6-(9-methyl-7-carbonyl-8,9,10,11-tetrahydro-[1,4]diazoylheptan[5′,6′:4,5]thieno[3,2-e]indazole-3(7H)-yl)pyrimidin-4-carboxamide (4.99 mg, yield 5.6%) was a yellow solid. LC-MS: m / z (ESI) = 499.9 [M+H] + ; 1H NMR (400MHz, DMSO-d6): δ9.39(s,1H),8.79(d,J=8.0Hz,1H),8.48(s,1H),8.13(d,J=8.0Hz,1H),8.05(d,J=4.0Hz,1H),7.75-7.72 (m,1H),7.15(t,J=4.0Hz,1H),6.35-6.05(m,1H),3.77-3.71(m,2H),3.66-3.63(m,1H),3.51-3.49(m,2H),1.19(d,J=8.0Hz,3H).

[0337] Example 13: Synthesis of compounds 13A and 13B

[0338]

[0339] Step 1: Synthesis of (2,6-dichloropyrimidin-4-yl)(isoxazolidine-2-yl)methyl ketone

[0340]

[0341] 2,6-Dichloropyrimidin-4-carbonyl chloride (400 mg, 1.89 mmol) was dissolved in dichloromethane (10 mL), followed by the addition of isoxazolidine hydrochloride (207 mg, 1.89 mmol) and N,N-diisopropylethylamine (0.734 g, 5.68 mmol). The reaction mixture was reacted at 30 °C for 10 hours. Water (100 mL) was added to the reaction mixture, followed by extraction with ethyl acetate (100 mL). The organic phase was washed with saturated brine (100 mL), dried over sodium sulfate, and concentrated to obtain the crude product. The crude product was then purified by pyrogallol column chromatography (petroleum ether:ethyl acetate (V / V) = 1:1) to give (2,6-dichloropyrimidin-4-yl)(isooxazolidine-2-yl) methyl ketone (100 mg, yield 21.31%) as an off-white solid. LC-MS: m / z (ESI) = 247.9 [M+H] + ;

[0342] Step Two:

[0343] (R)-9-methyl-8,9,10,11-tetrahydro-[1,4]diazolypeptido[5′,6′:4,5]thieno[3,2-e]indazole-7(2H)-one (36 mg, 0.134 mmol) was dissolved in N,N-dimethylformamide (3 mL), followed by the addition of cesium carbonate (131 mg, 0.403 mmol) and (2,6-dichloropyrimidin-4-yl)(isoxazolidine-2-yl)methyl ketone (100 mg, 0.403 mmol). The reaction mixture was reacted at 50 °C for 5 hours. The reaction mixture was concentrated to dryness to obtain a crude product. This crude product was then purified by preparative high-performance liquid chromatography (HPLC) (solvent A: water + 0.1% formic acid; solvent B: acetonitrile; gradient: 45–55%) to obtain:

[0344] 13A:(R)-2-(2-chloro-6-(isoxazolidin-2-carbonyl)pyrimidin-4-yl)-9-methyl-8,9,10,11-tetrahydro-[1,4]diazoylheptan[5′,6′:4,5]thieno[3,2-e]indazole-7(2H)-one (3.93 mg, yield 6.04%) was a pale yellow solid. LC-MS: m / z (ESI) = 484.0 [M+H] + ; 1 H NMR (400MHz, DMSO-d6): δ9.91 (s, 1H), 8.38-8.35 (m, 1H), 7.94 (d, J = 4Hz, 1H), 7.80-7.73 (m, 2H), 7.14 (brs,1H),4.05(br,4H),3.64-3.62(m,1H),3.47(brs,2H),2.41-2.32(m,2H),1.18(d,J=4.0Hz,3H).

[0345] 13B:(R)-2-(2-chloro-6-(isoxazolidin-2-carbonyl)pyrimidin-4-yl)-9-methyl-8,9,10,11-tetrahydro-[1,4]diazoylheptan[5′,6′:4,5]thieno[3,2-e]indazole-7(2H)-one (6.62 mg, yield 10.18%) was a pale yellow solid. LC-MS: m / z (ESI) = 484.0 [M+H] + ; 1 H NMR (400MHz, DMSO-d6): δ9.30(s,1H),8.78(d,J=8.0Hz,1H),8.29(br,1H),8.13(d,J=8.0Hz,1H),8.06(s, 1H),7.05(brs,1H),4.05(br,4H),3.66(brs,1H),3.49(brs,2H),2.39-2.33(m,2H),1.21(d,J=8.0Hz,3H).

[0346] Example 14: Synthesis of compounds 14A and 14B

[0347]

[0348] Step 1: Synthesis of methyl N-(tert-butoxycarbonyl)-O-(tert-butyldimethylsilyl)-D-serine ester

[0349]

[0350] N-(tert-butoxycarbonyl)-D-serine methyl ester (15 g, 68.5 mmol) and imidazole (13.9 g, 205 mmol) were dissolved in dichloromethane (200 mL). Tert-butyldimethylchlorosilane (11.3 g, 75.3 mmol) was added under ice bath conditions, and the reaction was allowed to proceed for 12 hours at room temperature. Water (100 mL) was added to the reaction mixture, followed by extraction with dichloromethane. The organic phase was washed with saturated brine (100 mL), dried over sodium sulfate, and concentrated to obtain the crude product. Purification was then achieved by kerogen column chromatography (petroleum ether:ethyl acetate (V / V) = 20:1) to give methyl N-(tert-butoxycarbonyl)-O-(tert-butyldimethylsilyl)-D-serine ester (20 g, 87% yield) as an oily liquid. LC-MS: m / z (ESI) = 334 [M+H] + ;

[0351] Step 2: Synthesis of tert-butyl(S)-(1-((tert-butyldimethylsilyl)oxo)-3-hydroxypropane-2-yl)carbamate

[0352]

[0353] Methyl N-(tert-butoxycarbonyl)-O-(tert-butyldimethylsilyl)-D-serine ester (20 g, 60 mmol) was dissolved in dry tetrahydrofuran (100 mL), and lithium aluminum hydride (60 mL, 60 mmol, 1 M tetrahydrofuran solution) was added dropwise at -15 °C under nitrogen protection. After the addition was complete, the reaction was carried out at room temperature for 1 hour. The reaction was quenched at -15 °C with saturated ammonium chloride aqueous solution, extracted with ethyl acetate, washed with saturated brine, dried over sodium sulfate, and concentrated to obtain the crude product. The crude product was then purified by kerogen column chromatography (petroleum ether:ethyl acetate (V / V) = 10:1) to give tert-butyl(S)-(1-((tert-butyldimethylsilyl)oxo)-3-hydroxypropane-2-yl)carbamate (10 g, yield 54%) as an oily liquid. LC-MS: m / z (ESI) = 306 [M+H] + ;

[0354] Step 3: Synthesis of 2-oxidation of tert-butyl(4R)-4-(((tert-butyldimethylsilyl)oxo)methyl)-1,2,3-oxathiazolidin-3-carboxylic acid ester

[0355]

[0356] Imidazole (6.69 g, 98.4 mmol) was dissolved in dichloromethane (100 mL), and thionyl chloride (7.8 g, 65.6 mmol) in dichloromethane (10 mL) was slowly added at 0 °C. The reaction was allowed to proceed at room temperature for 1 hour after the addition was complete. The temperature was lowered to 0 °C, and tert-butyl(S)-(1-((tert-butyldimethylsilyl)oxo)-3-hydroxypropane-2-yl)carbamate (10 g, 32.8 mmol) in dichloromethane (20 mL) was added dropwise. The reaction was allowed to proceed at room temperature for 1 hour after the addition was complete, and the reaction was quenched with ice water. The mixture was extracted with dichloromethane, washed with saturated brine, dried over sodium sulfate, and concentrated to obtain the crude product. The product was then purified by pyrite column chromatography (dichloromethane:methanol (V / V) = 20:1) to give tert-butyl(4R)-4-(((tert-butyldimethylsilyl)oxo)methyl)-1,2,3-oxathiazolidin-3-carboxylic acid ester 2-oxidation (5 g, yield 43%) as a yellow oily liquid. LC-MS: m / z (ESI) = 352 [M+H] + ;

[0357] Step 4: Synthesis of tert-butyl(R)-4-(((tert-butyldimethylsilyl)oxo)methyl)-1,2,3-oxathiazolidin-3-carboxylic acid ester 2,2-dioxide

[0358]

[0359] 2-Oxytosiazole-3-carboxylic acid ester tert-butyl(4R)-4-(((tert-butyldimethylsilyl)oxo)methyl)-1,2,3-oxathiazolidin-3-carboxylic acid ester 2-oxide (5 g, 14.25 mmol) was dissolved in acetonitrile (50 mL) and water (50 mL). Ruthenium trichloride monohydrate (296 mg, 1.42 mmol) and sodium periodate (6.1 g, 28.49 mmol) were added at 0 °C. After the addition was complete, the reaction was carried out at room temperature for 12 hours. After the reaction was complete, the solution was diluted with ice water, extracted with dichloromethane, washed with saturated brine, dried over sodium sulfate, and concentrated to obtain the crude product. The product was then purified by slag column chromatography (dichloromethane:methanol (V / V) = 10:1) to give tert-butyl(R)-4-(((tert-butyldimethylsilyl)oxo)methyl)-1,2,3-oxathiazolidin-3-carboxylic acid ester 2,2-dioxide (2 g, yield 38%) as a white solid. LC-MS: m / z (ESI) = 368.1 [M+H] + ;

[0360] Step 5: Synthesis of methyl 8-(((S)-2-((tert-butoxycarbonyl)amino)-3-((tert-butyldimethylsilyl)oxo)propyl)amino)-3-(tetrahydro-2H-pyran-2-yl)-3H-thieno[3,2-e]indazole-7-carboxylic acid ester

[0361]

[0362] Methyl 8-amino-3-(tetrahydro-2H-pyran-2-yl)-3H-thieno[3,2-e]indazole-7-carboxylic acid ester (1.6 g, 4.83 mmol) was dissolved in N,N-dimethylformamide (10 mL), and sodium hydride (232 mg, 5.8 mmol) was added at 0 °C under nitrogen. After the addition was complete, the mixture was reacted at 0 °C for 30 minutes. Then, tert-butyl(R)-4-(((tert-butyldimethylsilyl)oxo)methyl)-1,2,3-oxathiazolidin-3-carboxylic acid ester 2,2-dioxide (1.95 g, 5.32 mmol) was added, and the mixture was reacted at 0 °C for 2 hours. After the reaction was complete, the mixture was quenched with water, extracted with ethyl acetate, washed with saturated brine, dried over sodium sulfate, and concentrated to obtain the crude product. The product was then purified by pyrogallol column chromatography (dichloromethane:methanol (V / V) = 10:1) to give methyl 8-(((S)-2-((tert-butoxycarbonyl)amino)-3-((tert-butyldimethylsilyl)oxo)propyl)amino)-3-(tetrahydro-2H-pyran-2-yl)-3H-thieno[3,2-e]indazole-7-carboxylic acid ester (1 g, yield 33%) as an off-white solid. LC-MS: m / z (ESI) = 619.0 [M+H] + ;

[0363] Step Six: Synthesis of methyl(S)-8-((2-amino-3-hydroxypropyl)amino)-3H-thieno[3,2-e]indazole-7-carboxylic acid ester

[0364]

[0365] Methyl 8-(((S)-2-((tert-butoxycarbonyl)amino)-3-((tert-butyldimethylsilyl)oxo)propyl)amino)-3-(tetrahydro-2H-pyran-2-yl)-3H-thieno[3,2-e]indazole-7-carboxylic acid ester (1 g, 1.62 mmol) was dissolved in methanol (10 mL), and a solution of dioxane (5 mL, 20 mmol) of hydrogen chloride was added. The reaction was carried out at room temperature for 3 hours. After the reaction was completed, the solution was concentrated to give crude methyl (S)-8-((2-amino-3-hydroxypropyl)amino)-3H-thieno[3,2-e]indazole-7-carboxylic acid ester (500 mg, 96% yield) as a brown solid. LC-MS: m / z (ESI) = 321.0 [M+H] + ;

[0366] Step 7: (S)-9-(hydroxymethyl)-8,9,10,11-tetrahydro-[1,4]diazo-heptan[5′,6′:4,5]thieno[3,2-e]indazole-7(3H)-one

[0367] Methyl (S)-8-((2-amino-3-hydroxypropyl)amino)-3H-thieno[3,2-e]indazole-7-carboxylic acid ester (500 mg, 1.56 mmol) was dissolved in methanol (10 mL), and 1,8-diazabicyclo[5,4,0]undec-7-ene (1.18 g, 7.81 mmol) was added. The mixture was heated to 70 °C and reacted for 12 hours. After the reaction was complete, the crude product was concentrated, and dichloromethane was added. The solid precipitated, filtered, and the filter cake was dried to give (S)-9-(hydroxymethyl)-8,9,10,11-tetrahydro-[1,4]diazoylheptan[5′,6′:4,5]thieno[3,2-e]indazole-7(3H)-one (250 mg, 50% yield) as a brown solid. LC-MS: m / z (ESI) = 289.0 [M+H] + ;

[0368] Step 8:

[0369] (S)-9-(hydroxymethyl)-8,9,10,11-tetrahydro-[1,4]diazo-heptan[5′,6′:4,5]thieno[3,2-e]indazole-7(3H)-one (50 mg, 0.17 mmol) was dissolved in (3 mL) N,N-dimethylformamide, and then cesium carbonate (170 mg, 0.52 mmol) and 2,4-dichloro-5-fluoro-6-methylpyrimidine (47 mg, 0.26 mmol) were added. The reaction was carried out at 50 °C for 4 hours. The reaction solution was filtered, and the filtrate was concentrated to obtain the crude product. Then, it was purified by preparative high performance liquid chromatography (solvent A: water + 0.1% formic acid; solvent B: acetonitrile; gradient: 43-58%) to obtain:

[0370] 14A:(S)-2-(2-chloro-5-fluoro-6-methylpyrimidin-4-yl)-9-(hydroxymethyl)-8,9,10,11-tetrahydro-[1,4]diazoylheptan[5′,6′:4,5]thieno[3,2-e]indazole-7(2H)-one (2 mg, yield 2.7%) is a yellow solid.

[0371] LC-MS: m / z (ESI) = 433.0 [M+H] + ;

[0372] 1H NMR (400MHz, DMSO-d6): δ9.84 (s, 1H), 7.78-7.72 (m, 3H), 7.09 (t, J = 4.0Hz, 1H), 5.03 -5.01(m,1H),3.62-3.59(m,1H),3.53-3.47(m,2H),3.44-3.40(m,2H),2.61(s,3H).

[0373] 14B:(S)-3-(2-chloro-5-fluoro-6-methylpyrimidin-4-yl)-9-(hydroxymethyl)-8,9,10,11-tetrahydro-[1,4]diazoylheptan[5′,6′:4,5]thieno[3,2-e]indazole-7(3H)-one (3.08 mg, 4% yield) was a yellow solid.

[0374] 1 H NMR (400MHz, DMSO-d6): δ9.28(s,1H),8.49(d,J=8.0Hz,1H),8.07(d,J=12.0Hz,1H),7.85(d,J=4.0Hz,1H),7. 01(t,J=4.0Hz,1H),5.05-5.02(m,1H),3.67-3.62(m,1H),3.60-3.52(m,2H),3.48-3.44(m,2H),2.58(s,3H).

[0375] Example 15: Synthesis of Compound 15

[0376]

[0377] Step 1: Methyl 3-aminothiopheno[3,2-b]pyridine-2-carboxylate (15-2)

[0378]

[0379] 3-Chloro-2-cyanopyridine (15-1) (10 g, 72.2 mmol, 1.0 eq.), methyl mercaptoacetate (7.1 mL, 79.4 mmol, 1.1 eq.) and potassium carbonate (10.1 g, 79.4 mmol, 1.1 eq.) were added to N,N-dimethylformamide (100 mL) and water (10 mL) and stirred at 40 °C for 3 hours.

[0380] LC-MS analysis of the reaction solution showed that the reaction was complete. The reaction solution was added to water (1000 mL), filtered, and the filter cake was washed with water (500 mL). The filter cake was dissolved in ethyl acetate (500 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give a yellow solid product, methyl 3-aminothiopheno[3,2-b]pyridine-2-carboxylic acid ester (15-2) (11 g, yield 73%).

[0381] LC-MS: m / z (ESI) = 208.0 [M+H] + ;

[0382] Step 2: Methyl(S)-3-((2-((tert-butoxycarbonyl)amino)propyl)amino)thieno[3,2-b]pyridine-2-carboxylate (15-3)

[0383]

[0384] Methyl 3-aminothiopheno[3,2-b]pyridine-2-carboxylate (15-2) (1 g, 4.8 mmol, 1.0 eq.) and sodium hydride (380 mg, 9.6 mmol, 2.0 eq.) were added to N,N-dimethylformamide (20 mL) and reacted at 0 °C for 1 hour. Then, tert-butyl(R)-4-methyl-1,2,3-oxathiazolidin-3-carboxylate 2,2-dioxide (1.1 g, 4.8 mmol, 1.0 eq.) was added and reacted at 0 °C for 1 hour. LC-MS analysis of the reaction solution showed that the reaction was complete. The reaction solution was quenched with saturated ammonium chloride solution (100 mL) and extracted three times with ethyl acetate (100 mL). The organic phases were combined, dried with anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a yellow solid product, methyl(S)-3-((2-((tert-butoxycarbonyl)amino)propyl)amino)thiopheno[3,2-b]pyridine-2-carboxylic acid ester (15-3) (1.7 g).

[0385] LC-MS: m / z (ESI) = 365.0 [M+H] +

[0386] Step 3: Methyl(S)-3-((2-aminopropyl)amino)thieno[3,2-b]pyridine-2-carboxylate (15-4)

[0387]

[0388] Methyl(S)-3-((2-((tert-butoxycarbonyl)amino)propyl)amino)thieno[3,2-b]pyridine-2-carboxylate (15-3) (1.7 g, 4.65 mmol, 1.0 eq.) was added to dichloromethane (20 mL), followed by slow addition of 4 M hydrochloric acid / dioxane solution (20 mL). The reaction was carried out at 20 °C for 16 hours. LC-MS analysis of the reaction solution showed that the reaction was complete. The reaction solution was directly concentrated under reduced pressure to obtain a yellow solid crude product, methyl(S)-3-((2-aminopropyl)amino)thieno[3,2-b]pyridine-2-carboxylate (15-4) (2 g).

[0389] LC-MS: m / z (ESI) = 265.0 [M+H] +

[0390] Step 4: (R)-3-methyl-1,2,3,4-tetrahydro-5H-pyrido[2′,3′:4,5]thieno[3,2-e][1,4]diazo-heptan-5-one (15-5)

[0391]

[0392] Methyl (S)-3-((2-aminopropyl)amino)thieno[3,2-b]pyridine-2-carboxylic acid ester (15-4) (2 g, 7.54 mmol, 1.0 eq.) and DBU (5 mL, 37.3 mmol, 5.0 eq.) were added to methanol (20 mL), and the reaction was carried out at 70 °C for 16 hours. LC-MS analysis of the reaction solution showed that the reaction was complete. The reaction solution was concentrated under reduced pressure, and the residue was purified by reverse-phase column chromatography (C18, acetonitrile / water (trifluoroacetic acid)) (acetonitrile:water = 70%) to obtain the yellow solid product (R)-3-methyl-1,2,3,4-tetrahydro-5H-pyrido[2′,3′:4,5]thieno[3,2-e][1,4]diazoylheptan-5-one (15-5) (1.7 g, yield 46%).

[0393] LC-MS: m / z (ESI) = 233.1 [M+H] +

[0394] Step 5: Di-tert-butyl(R)-3-methyl-5-carbonyl-2,3-dihydro-1H-pyrido[2',3′:4,5]thieno[3,2-e][1,4]diazazo-1,4(5H)-dicarboxylic acid ester (15-6)

[0395]

[0396] (R)-3-methyl-1,2,3,4-tetrahydro-5H-pyrido[2′,3′:4,5]thieno[3,2-e][1,4]diazo-heptan-5-one (15-5) (1.7 g, 7.3 mmol, 1.0 eq.), ditert-butyl dicarbonate (7 mL, 29.2 mmol, 4.0 eq.), 4-dimethylaminopyridine (150 mg, 1.5 mmol, 0.2 eq.), and triethylamine (3 mL, 22 mmol, 3.0 eq.) were added to dichloromethane (20 mL) and reacted at room temperature for 16 hours. LC-MS analysis of the reaction solution showed that the reaction was complete. The reaction solution was concentrated under reduced pressure, and the residue was purified by column chromatography (dichloromethane) to give a yellow solid product, di-tert-butyl(R)-3-methyl-5-carbonyl-2,3-dihydro-1H-pyrido[2′,3′:4,5]thieno[3,2-e][1,4]diazapheno-1,4(5H)-dicarboxylic acid ester (15-6) (1.7 g, yield 56%).

[0397] LC-MS: m / z (ESI) = 433.0 [M+H] +

[0398] Step 6: (R)-1,4-Di(tert-butoxycarbonyl)-3-methyl-5-carbonyl-2,3,4,5-tetrahydro-1H-pyrido[2′,3′:4,5]thiopheno[3,2-e][1,4]diazazo-10-N-oxide (15-7)

[0399]

[0400] Di-tert-butyl(R)-3-methyl-5-carbonyl-2,3-dihydro-1H-pyrido[2′,3′:4,5]thieno[3,2-e][1,4]diazaphen-1,4(5H)-dicarboxylic acid ester (15-6) (1.5 g, 3.5 mmol, 1.0 eq.) was added to dichloromethane (20 mL), followed by the addition of m-chloroperoxybenzoic acid (750 mg, 3.7 mmol, 1.5 eq.), and the mixture was stirred at room temperature for 2 hours. LC-MS analysis of the reaction solution showed that the reaction was complete. The reaction solution was quenched in sodium thiosulfate solution (100 mL), and extracted three times with dichloromethane (50 mL * 3). The organic phases were combined, concentrated under reduced pressure, and the residue was purified by column chromatography (petroleum ether: ethyl acetate = 1:2) to give a yellow solid product (R)-1,4-di(tert-butoxycarbonyl)-3-methyl-5-carbonyl-2,3,4,5-tetrahydro-1H-pyrido[2′,3′:4,5]thieno[3,2-e][1,4]diazaphene 10-N-oxide (15-7) (100 mg, yield 6%).

[0401] LC-MS: m / z (ESI) = 449.3 [M+H] +

[0402] Step 7: Di-tert-butyl(R)-9-amino-3-methyl-5-carbonyl-2,3-dihydro-1H-pyrido[2′,3′:4,5]thieno[3,2-e][1,4]diazazo-1,4(5H)-dicarboxylic acid ester (15-8)

[0403]

[0404] (R)-1,4-di(tert-butoxycarbonyl)-3-methyl-5-carbonyl-2,3,4,5-tetrahydro-1H-pyrido[2′,3′:4,5]thieno[3,2-e][1,4]diazaphene-10-oxide(15-7) (110 mg, 0.24 mmol, 1.0 eq.) and p-toluenesulfonic anhydride (14 mg, 0.48 mmol, 2.0 eq.) were added to ammonia (0.5 mL) and dichloromethane (2 mL). The mixture was reacted at 45 °C for 1 hour, and then ammonia (0.5 mL) and p-toluenesulfonic anhydride (14 mg, 0.48 mmol, 2.0 eq.) were added as supplemental solutions. 8 mmol (2.0 eq.) was added, and the mixture was stirred at 45 °C for 1 hour. LC-MS analysis of the reaction solution showed that the reaction was complete. The reaction solution was concentrated under reduced pressure, and the residue was purified by reverse-phase column chromatography (C18, acetonitrile / water (trifluoroacetic acid)) (acetonitrile:water = 50%) to obtain a yellow solid product, di-tert-butyl(R)-9-amino-3-methyl-5-carbonyl-2,3-dihydro-1H-pyrido[2′,3′:4,5]thieno[3,2-e][1,4]diazaphen-1,4(5H)-dicarboxylic acid ester (15-8) (50 mg, yield 45%).

[0405] LC-MS: m / z (ESI) = 448.2 [M+H] +

[0406] Step 8: 2-Bromo-1-(2-chloropyrimidin-4-yl)ethane-1-one (15-10)

[0407]

[0408] 1-(2-chloropyrimidin-4-yl)ethane-1-one (15-9) (50 mg, 0.32 mmol, 1.0 eq.), hydrobromic acid (58 μL, 0.32 mmol, 1.0 eq.), and liquid bromine (16 μL, 0.32 mmol, 1.0 eq.) were added to acetic acid (1 mL), and the reaction was carried out at 20 °C for 16 hours. LC-MS analysis of the reaction mixture showed that the reaction was complete. The reaction mixture was directly filtered, and the filter cake was washed with acetic acid (0.5 mL). The filter cake was then transferred and dried to give a white solid product, 2-bromo-1-(2-chloropyrimidin-4-yl)ethane-1-one (15-10) (70 mg, 93% yield).

[0409] LC-MS: m / z (ESI) = 235 [M+H] +

[0410] Step Nine: (R)-2-(2-chloropyrimidin-4-yl)-9-methyl-8,9,10,11-tetrahydro-7H-imidazo[1”,2”:1′,6′]pyrido[2′,3′:4,5]thieno[3,2-e][1,4]diazo-heptan-7-one (15)

[0411] Di-tert-butyl(R)-9-amino-3-methyl-5-carbonyl-2,3-dihydro-1H-pyrido[2′,3':4,5]thieno[3,2-e][1,4]diazapheno-1,4(5H)-dicarboxylic acid ester (15-8) (40 mg, 0.09 mmol, 1.0 eq.) and 2-bromo-1-(2-chloropyrimidin-4-yl)ethane-1-one (15-9) (40 mg, 0.1 mmol, 1.1 eq.) were added to N,N-dimethylacetamide (3 mL) and reacted at 120 °C for 24 hours. LC-MS analysis of the reaction solution showed that the reaction... The reaction mixture was concentrated under reduced pressure. The residue was purified by reverse-phase column chromatography (C18, acetonitrile / water (trifluoroacetic acid)) (acetonitrile:water = 40%). The product was concentrated under reduced pressure, and the residue was then purified by column chromatography (dichloromethane:methanol = 10:1) to obtain a yellow solid product (R)-2-(2-chloropyrimidin-4-yl)-9-methyl-8,9,10,11-tetrahydro-7H-imidazo[1”,2”:1′,6′]pyrido[2',3':4,5]thieno[3,2-e][1,4]diazoylheptan-7-one (15) (4 mg, yield 11%).

[0412] LC-MS: m / z (ESI) = 385.0 [M+H] +1H NMR (400MHz, DMSO-d6) δ9.38 (s, 1H), 8.82 (d, J = 5.0Hz, 1H), 8.23-8.20 (m, 1H), 8.14 (d, J = 5. 2Hz,1H),7.85(d,J=9.3Hz,1H),7.71(d,J=8.8Hz,1H),2.03-1.97(m,4H),0.88-0.83(m,3H).

[0413] Example 16: Synthesis of compounds 16A and 16B

[0414]

[0415] Step 1: 4-Bromo-1H-indazole-5-ol (16-2)

[0416]

[0417] 1H-indazole-5-ol (16-1) (25 g, 186 mmol, 1.0 eq.) and N-bromosuccinimide (33 g, 186 mmol, 1.0 eq.) were added to tetrahydrofuran (200 mL), and the reaction was carried out overnight at 0 °C to 25 °C for 16 h. The reaction was monitored by LC-MS to confirm completion. The reaction solution was added to H₂O (1 L), and the mixture was extracted three times with ethyl acetate (1 L * 3). The combined organic phases were dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain a crude brown solid product. The crude product was added to dichloromethane (100 mL), stirred at 25 °C for 1 h, filtered, and the filter cake was washed with dichloromethane (10 mL * 3). The filter cake was transferred and dried to obtain a brown solid product, 4-bromo-1H-indazole-5-ol (16-2) (35 g, yield 89%).

[0418] LC-MS: m / z = 214 [M+H] +

[0419] Step 2: Ethyl 2-((4-bromo-1H-indazol-5-yl)oxo)acetate (16-3)

[0420]

[0421] 4-Bromo-1H-indazole-5-ol (16-2) (2 g, 9.4 mmol, 1.0 eq.) and ethyl bromoacetate (1 mL, 10 mmol, 1.1 eq.) were added to acetone (20 mL), followed by slow addition of potassium carbonate (2.6 g, 20 mmol, 2.0 eq.). The reaction mixture was stirred at 55 °C for 16 h. LC-MS analysis confirmed the reaction was complete. The reaction mixture was then added to water (100 mL), extracted three times with ethyl acetate (100 mL x 3), and then three times with dichloromethane (100 mL x 3). The organic phases were combined and concentrated under reduced pressure. The residue was purified by column chromatography (petroleum ether:ethyl acetate = 2:1) to obtain a yellow oily liquid product, ethyl 2-((4-bromo-1H-indazole-5-yl)oxo)acetate (16-3) (1.3 g, yield 46%).

[0422] LC-MS: m / z = 298,300 [M+H] +

[0423] Step 3: Ethyl 2-((4-bromo-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-5-yl)oxo)acetate (16-4)

[0424]

[0425] Ethyl 2-((4-bromo-1H-indazol-5-yl)oxo)acetate (16-3) (1.3 g, 4.3 mmol, 1.0 eq.) and p-toluenesulfonic acid (74 mg, 0.4 mmol, 0.1 eq.) were added to dichloromethane (10 mL), followed by slow addition of dihydropyran (1 mL). The reaction mixture was stirred at 25 °C for 2 hours. The reaction was confirmed to be complete by LC-MS. The reaction mixture was concentrated under reduced pressure, and the residue was purified by column chromatography (petroleum ether: ethyl acetate = 2:1) to give the colorless liquid product ethyl 2-((4-bromo-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-5-yl)oxo)acetate (16-4) (0.8 g, yield 48%).

[0426] LC-MS: m / z = 298 [M+H] +

[0427] Step 4: Ethyl 2-((4-cyano-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-5-yl)oxo)acetate (16-5)

[0428]

[0429] Ethyl 2-((4-bromo-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-5-yl)oxo)acetate (16-4) (0.8 g, 2.1 mmol, 1.0 eq.), zinc cyanide (488 mg, 4.2 mmol, 2.0 eq.) and tetraphenylphosphine palladium (240 mg, 0.2 mmol, 0.2 eq.) were added to DMF (10 mL), and the reaction mixture was stirred at 100 °C for 16 hours. The reaction solution was detected as complete by LC-MS. The reaction solution was added to water (100 mL), filtered, and the filter cake was washed three times with water (10 mL * 3). The filter cake was dissolved in ethyl acetate (100 mL), dried over anhydrous sodium sulfate, and the organic phase was concentrated under reduced pressure to obtain a white solid crude product: ethyl 2-((4-cyano-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole-5-yl)oxo)acetate (16-5) (680 mg, crude product).

[0430] LC-MS: m / z = 245 [M+H] +

[0431] Step 5: Ethyl 8-amino-3-(tetrahydro-2H-pyran-2-yl)-3H-furano[3,2-e]indazole-7-carboxylic acid ester (16-6)

[0432]

[0433] Ethyl 2-((4-cyano-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole-5-yl)oxo)acetate (16-5) (680 mg, 2.0 mmol, 1.0 eq.) was added to tetrahydrofuran (10 mL), followed by potassium tert-butoxide (230 mg, 2.0 mmol, 2.0 eq.). The reaction mixture was stirred at 0 °C for 1 hour. The reaction was confirmed to be complete by LC-MS. The reaction mixture was then slowly added to saturated ammonium chloride (100 mL), and extracted three times with ethyl acetate (100 mL * 3). The combined organic phases were concentrated under reduced pressure to obtain a yellow solid crude product: ethyl 8-amino-3-(tetrahydro-2H-pyran-2-yl)-3H-furano[3,2-e]indazole-7-carboxylic acid ester (16-6) (250 mg, crude).

[0434] LC-MS: m / z = 329 [M+H] +

[0435] Step 6: Ethyl 8-(((R)-2-((tert-butoxycarbonyl)amino)propyl)amino)-3-(tetrahydro-2H-pyran-2-yl)-3H-furano[3,2-e]indazole-7-carboxylic acid ester (16-7)

[0436]

[0437] Ethyl 8-amino-3-(tetrahydro-2H-pyran-2-yl)-3H-furano[3,2-e]indazole-7-carboxylic acid ester (16-6) (250 mg, 0.76 mmol, 1.0 eq.), (R)-4-methyl-1,2,3-oxathiazolidin-3-carboxylic acid tert-butyl ester 2,2-dioxide (180 mg, 0.76 mmol, 1.0 eq.) were added to N,N-dimethylformamide (5 mL), followed by slow addition of sodium hydride (36 mg, 0.9 mmol, 1.2 eq.). The reaction mixture was stirred at room temperature for 1 hour. LC-MS analysis of the reaction solution showed that the reaction was complete. The reaction solution was added to 0.5M hydrochloric acid (aq.) (50 mL) and extracted three times with ethyl acetate (50 mL * 3). The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain crude yellow solid product ethyl 8-(((R)-2-((tert-butoxycarbonyl)amino)propyl)amino)-3-(tetrahydro-2H-pyran-2-yl)-3H-furano[3,2-e]indazole-7-carboxylic acid ester (16-7) (350 mg, crude product).

[0438] Step 7: Ethyl(R)-8-((2-aminopropyl)amino)-3H-furano[3,2-e]indazole-7-carboxylic acid ester (16-8)

[0439]

[0440] Ethyl(R)-2-((tert-butoxycarbonyl)amino)propyl)amino)-3-(tetrahydro-2H-pyran-2-yl)-3H-furano[3,2-e]indazole-7-carboxylic acid ester (16-7) (350 mg, 0.72 mmol, 1.0 eq.) was added to dichloromethane (100 mL), followed by 4M hydrochloric acid / dioxane (5 mL). The reaction mixture was stirred at 25 °C for 2 hours. LC-MS analysis showed that the reaction was complete. The reaction mixture was concentrated under reduced pressure to obtain a yellow solid crude product, ethyl(R)-8-((2-aminopropyl)amino)-3H-furano[3,2-e]indazole-7-carboxylic acid ester (16-8) (300 mg, crude product).

[0441] LC-MS: m / z = 302[M+H] +

[0442] Step 8: (R)-9-methyl-8,9,10,11-tetrahydro-[1,4]diazo-heptan[5',6':4,5]furano[3,2-e]indazole-7(3H)-one (16-9)

[0443]

[0444] Ethyl(R)-8-((2-aminopropyl)amino)-3H-furano[3,2-e]indazole-7-carboxylic acid ester (16-8) (300 mg, 1.0 mmol, 1.0 eq.) was added to methanol (10 mL), followed by DBU (1.5 mL, 5.0 mmol, 5.0 eq.). The reaction mixture was stirred at 70 °C for 16 hours. LC-MS analysis of the reaction mixture showed that the reaction was complete. The reaction mixture was concentrated under reduced pressure, and the residue was purified by reverse-phase column chromatography (C18, acetonitrile / water (trifluoroacetic acid)) (acetonitrile:water = 20%) to obtain a black oily product (R)-9-methyl-8,9,10,11-tetrahydro-[1,4]diazoylheptanzo[5′,6′:4,5]furano[3,2-e]indazole-7(3H)-one (180 mg, yield 75%).

[0445] LC-MS: m / z = 256 [M+H] +

[0446] Step Nine:

[0447] (R)-9-methyl-8,9,10,11-tetrahydro-[1,4]diazolypeptido[5',6':4,5]furano[3,2-e]indazole-7(2H)-one (100 mg, 0.39 mmol) was dissolved in 3 mL of N,N-dimethylformamide, and cesium carbonate (382 mg, 1.17 mmol) and 2,4-dichloro-6-methylpyrimidine (127 mg, 0.78 mmol) were added. The reaction mixture was reacted at 50 °C for 1 hour. The reaction solution was filtered, and the filtrate was concentrated to obtain the crude product. Then, it was purified by preparative high performance liquid chromatography (solvent A: water + 0.1% formic acid; solvent B: acetonitrile; gradient: 35-45%) to obtain:

[0448] 16A:(R)-2-(2-chloro-6-methylpyrimidin-4-yl)-9-methyl-8,9,10,11-tetrahydro-[1,4]diazoylheptanzo[5',6′:4,5]furano[3,2-e]indazole-7(2H)-one (2.20 mg, yield 1.5%) was a pale yellow solid. LC-MS: m / z (ESI) = 383.1 [M+H] + ; 1 H NMR (400MHz, DMSO-d6): δ9.83(s,1H),8.16(s,1H),7.73(d,J=8.0Hz,1H),7.64(d,J=8.0Hz,1H),7.58( d,J=4.0Hz,1H),7.17(brs,1H),3.58(brs,1H),3.35-3.32(m,2H),2.62(s,3H),1.18(d,J=8.0Hz,3H).

[0449] 16B:(R)-3-(4-chloro-6-methylpyrimidin-2-yl)-9-methyl-8,9,10,11-tetrahydro-[1,4]diazoylheptan[5′,6′:4,5]furano[3,2-e]indazole-7(3H)-one (2.14 mg, yield 1.43%) was a pale yellow solid. LC-MS: m / z (ESI) = 383.1 [M+H] + ; 1 H NMR (400MHz, DMSO-d6): δ9.16(s,1H),8.78(d,J=8.0Hz,1H),8.00(s,1H),7.88(d,J=8.0Hz,1H),7.73( d,J=4.0Hz,1H),7.03(brs,1H),3.61(brs,1H),3.38-3.36(m,2H),2.57(s,3H),1.20(d,J=8.0Hz,3H).

[0450] Example 17: Synthesis of compounds 17A and 17B

[0451]

[0452] Step 1: Synthesis of tert-butyl(R)-2-chloro-4-(9-methyl-7-carbonyl-8,9,10,11-tetrahydro-[1,4]diazo-heptan[5',6':4,5]thieno[3,2-e]indazole-2(7H)-yl)-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carboxylic acid ester

[0453] (R)-9-methyl-8,9,10,11-tetrahydro-[1,4]diazolypeptido[5',6':4,5]thieno[3,2-e]indazole-7(2H)-one (100 mg, 0.367 mmol) was dissolved in (5 mL) 1,4-dioxane, followed by the addition of cesium carbonate (359 mg, 1.10 mmol) and tert-butyl-2,4-dichloro-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carboxylic acid ester (17-1, 167 mg, 0.551 mmol). The reaction mixture was reacted at 100 °C under nitrogen protection for 16 hours. Water (100 mL) was added to the reaction mixture, followed by extraction with ethyl acetate (100 mL). The organic phase was washed with saturated brine (100 mL), dried over sodium sulfate, and concentrated to obtain the crude product. The product was then purified by stannin column chromatography (petroleum ether:ethyl acetate (V / V) = 1:1) to give tert-butyl(R)-2-chloro-4-(9-methyl-7-carbonyl-8,9,10,11-tetrahydro-[1,4]diazoylheptan[5′,6':4,5]thieno[3,2-e]indazole-2(7H)-yl)-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carboxylic acid ester (17-2, 120 mg, yield 60.51%) as an off-white solid. LC-MS: m / z (ESI) = 538.2 [MH] - ;

[0454] Step 2: Synthesis of (R)-2-(2-chloro-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-4-yl)-9-methyl-8,9,10,11-tetrahydro-[1,4]diazoylheptan[5',6':4,5]thieno[3,2-e]indazole-7(2H)-one

[0455] tert-Butyl(R)-2-chloro-4-(9-methyl-7-carbonyl-8,9,10,11-tetrahydro-[1,4]diazo-heptan[5′,6':4,5]thieno[3,2-e]indazole-2(7H)-yl)-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carboxylic acid ester (17-2) (50 mg, 0.093 mmol) was dissolved in hydrochloric acid / dioxane (5 mL). The reaction solution was reacted at 25 °C for 2 hours. The reaction solution was concentrated to dryness to obtain a crude product. Then, it was purified by preparative high performance liquid chromatography (solvent A: water + 0.1% formic acid; solvent B: acetonitrile; gradient: 48–58%) to obtain:

[0456] 17A:(R)-2-(2-chloro-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-4-yl)-9-methyl-8,9,10,11-tetrahydro-[1,4]diazoylheptanzo[5′,6′:4,5]thieno[3,2-e]indazole-7(2H)-one (13.67 mg, yield 33.56%) was a pale yellow solid. LC-MS: m / z (ESI) = 440.0 [M+H] + ; 1 H NMR (400MHz, DMSO-d6): δ9.95(s,1H),9.20(s,2H),7.96(d,J=4.0Hz,1H),7.81(d,J=8.0Hz,1H),7.69(d ,J=8.0Hz,1H),7.13(brs,1H),4.91(s,2H),3.63-3.56(m,5H),3.25-3.22(m,2H),1.19(d,J=8.0Hz,3H).

[0457] 17B:(R)-2-(2-chloro-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-4-yl)-9-methyl-8,9,10,11-tetrahydro-[1,4]diazoylheptanzo[5′,6′:4,5]thieno[3,2-e]indazole-7(2H)-one (3.75 mg, yield 9.21%) was a pale yellow solid. LC-MS: m / z (ESI) = 440.0 [M+H] + ; 1 H NMR (400MHz, DMSO-d6): δ9.29(s,1H),9.16(brs,2H),8.58(d,J=8.0Hz,1H),8.10(d,J=8.0Hz,1H),8.05(d,J=4.0Hz,1H),7. 03(brs,1H),4.75(s,2H),3.66-3.64(m,1H),3.59-3.56(m,2H),3.50-3.48(m,2H),3.24-3.21(m,2H),1.21(d,J=8.0Hz,3H).

[0458] Example 18: Synthesis of compounds 18A and 18B

[0459]

[0460] Step 1: Synthesis of 2,4-dichloro-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidine

[0461]

[0462] 0.350 g (1.15 mmol) of tert-butyl-2,4-dichloro-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carboxylic acid ester was dissolved in 3 mL of dichloromethane, and trifluoroacetic acid (3 mL) was added dropwise with stirring. The reaction mixture was stirred at 25 °C for 2 hours. The solution was concentrated to dryness under vacuum, and the residue was diluted with saturated sodium carbonate aqueous solution, extracted with ethyl acetate, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness to obtain the crude product. The crude product, 2,4-dichloro-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidine (200 mg, yield 85.18%), was a pale yellow solid. LC-MS: m / z (ESI) = 204.0 [M+H] + ;

[0463] Step 2: Synthesis of 2,4-dichloro-6-methyl-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidine

[0464]

[0465] 2,4-Dichloro-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidine (200 mg, 0.98 mmol) was dissolved in 5 mL of dichloromethane, followed by the addition of 3 mL of formaldehyde aqueous solution and then sodium cyanoborohydride (123 mg, 1.96 mmol). The reaction mixture was reacted at 25 °C for 6 hours. Water (10 mL) was added to the reaction mixture, followed by extraction with ethyl acetate (10 mL). The organic phase was washed with saturated brine (10 mL), dried over sodium sulfate, and concentrated to obtain the crude product. The crude product was then purified by pyrogallol column chromatography (petroleum ether:ethyl acetate (V / V) = 2:1) to give 2,4-dichloro-6-methyl-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidine (200 mg, yield 93.57%) as an off-white solid. LC-MS: m / z (ESI) = 219.7 [M+H] + ;

[0466] Step 3: Synthesis of (R)-2-(2-chloro-6-methyl-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-4-yl)-9-methyl-8,9,10,11-tetrahydro-[1,4]diazoylheptan[5′,6′:4,5]thieno[3,2-e]indazole-7(2H)-one

[0467] 2,4-Dichloro-6-methyl-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidine (200 mg, 0.917 mmol) was dissolved in (3 mL) N,N-dimethylformamide, followed by the addition of cesium carbonate (299 mg, 0.917 mmol) and 2,4-dichloro-6-(1-methyl-1H-pyrazol-3-yl)pyrimidine (83 mg, 0.306 mmol). The reaction mixture was reacted at 50 °C for 5 hours. The reaction mixture was filtered, and the filtrate was concentrated to obtain the crude product. The crude product was then purified by preparative high-performance liquid chromatography (HPLC) (solvent A: water + 0.1% formic acid; solvent B: acetonitrile; gradient: 19–29%) to obtain:

[0468] 18A:(R)-2-(2-chloro-6-methyl-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-4-yl)-9-methyl-8,9,10,11-tetrahydro-[1,4]diazoylheptanzo[5′,6′:4,5]thieno[3,2-e]indazole-7(2H)-one (10.06 mg, yield 7.25%), product was a pale yellow solid. LC-MS: m / z (ESI) = 454.1 [M+H] + ; 1 H NMR (400MHz, DMSO-d6): δ9.94(s,1H),7.96(d,J=4.0Hz,1H),7.81(d,J=12.0Hz,1H),7.71(d,J=8.0Hz,1H) ,7.13(s,1H),3.64-3.62(m,1H),3.48-3.44(m,4H),3.33-3.30(m,4H),3.06(s,3H),1.19(d,J=4.0Hz,3H).

[0469] 18B:(R)-3-(2-chloro-6-methyl-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-4-yl)-9-methyl-8,9,10,11-tetrahydro-[1,4]diazoylheptanzo[5′,6′:4,5]thieno[3,2-e]indazole-7(3H)-one (6.70 mg, yield 4.83%), product was a pale yellow solid. LC-MS: m / z (ESI) = 454.1 [M+H] + ; 1H NMR (400MHz, DMSO-d6): δ9.29(s,1H),8.55(d,J=8.0Hz,1H),8.10(d,J=8.0Hz,1H),8.05(d,J=4.0Hz,1H) ,7.03(brs,1H),3.65-3.61(m,3H),3.50(br,3H),3.32-3.29(m,3H),3.02(s,3H),1.20(d,J=8.0Hz,3H).

[0470] Example 19: Synthesis of compounds 19A and 19B

[0471]

[0472] Step 1: 2-(2,6-Dichloropyrimidin-4-yl)oxazole

[0473] 2,4,6-Trichloropyrimidine (1200 mg, 6.59 mmol) and 2-(tributyltinyl)oxazole (2124 mg, 5.93 mmol) were dissolved in 20 mL of N,N-dimethylformamide, and palladium dichloride bis(triphenylphosphine) (462 mg, 0.66 mmol) was added. The reaction mixture was reacted at 90 °C for 12 h under nitrogen protection. The reaction solution was filtered, and the filtrate was diluted with water and ethyl acetate. The organic phase was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 3 / 1 elution) to give 2-(2,6-dichloropyrimidine-4-yl)oxazole (200 mg, yield 14.10%) as a pale yellow solid. LC-MS: m / z (ESI) = 216.0 [M+H] + ;

[0474] Step 2: (R)-2-(2-chloro-6-(oxazol-2-yl)pyrimidin-4-yl)-9-methyl-8,9,10,11-tetrahydro-[1,4]diazo-heptan[5',6':4,5]thieno[3,2-e]indazole-7(2H)-one

[0475] (R)-9-methyl-8,9,10,11-tetrahydro-[1,4]diazolypeptido[5′,6′:4,5]thieno[3,2-e]indazole-7(2H)-one (40 mg, 0.15 mmol) was dissolved in 5 mL of N,N-dimethylformamide, followed by the addition of cesium carbonate (144 mg, 0.44 mmol) and 2-(2,6-dichloropyrimidin-4-yl)oxazole (47 mg, 0.22 mmol). The reaction mixture was reacted at 25 °C for 2 hours. The reaction solution was filtered, and the filtrate was concentrated to obtain the crude product. The crude product was then purified by preparative high-performance liquid chromatography (HPLC) (solvent A: water + 0.1% formic acid; solvent B: acetonitrile; gradient: 43-53%) to obtain:

[0476] 19A:(R)-2-(2-chloro-6-(oxazol-2-yl)pyrimidin-4-yl)-9-methyl-8,9,10,11-tetrahydro-[1,4]diazoylheptan[5',6':4,5]thieno[3,2-e]indazole-7(2H)-one (2.39 mg, yield 3.60%) was a pale yellow solid. LC-MS: m / z (ESI) = 452.0 [M+H] + ; 1 HNMR (400MHz, DMSO-d6): δ9.93(s,1H),8.64(s,1H),8.55(s,1H),7.94(d,J=4.0Hz,1H),7.81-7.77( m,2H),7.71(s,1H),7.16-7.14(m,1H),3.64-7.62(m,1H),3.50-3.48(d,2H),1.20(d,J=8.0Hz,3H).

[0477] 19B:(R)-3-(2-chloro-6-(oxazol-2-yl)pyrimidin-4-yl)-9-methyl-8,9,10,11-tetrahydro-[1,4]diazoylheptan[5',6':4,5]thieno[3,2-e]indazole-7(3H)-one (3.99 mg, yield 6.02%) was a pale yellow solid. LC-MS: m / z (ESI) = 452.0 [M+H] + ; 1 HNMR (400MHz, DMSO-d6): δ9.31(s,1H),8.77(d,J=12.0Hz,1H),8.51(d,J=4.0Hz,2H),8.11(d,J=8.0Hz,1H ),8.03(d,J=4.0Hz,1H),7.67(s,1H),7.05(brs,1H),3.65(brs,1H),3.49(brs,2H),1.21(d,J=8.0Hz,3H).

[0478] Example 20: Synthesis of compounds 20A and 20B

[0479]

[0480] Step 1: Ethyl 4-fluoro-3-carbonylbutyrate

[0481]

[0482] Ethyl acetate (20 g, 227.27 mmol) was dissolved in 200 mL of tetrahydrofuran. The temperature of the mixture was lowered to -60 °C, and diisopropylaminolithium (125 mL, 250.0 mmol) was slowly added under nitrogen protection. After the addition was complete, the mixture was maintained at -60 °C for 1 hour. Then, ethyl 2-fluoroacetate (21.7 g, 204.55 mmol) was added, and the temperature of the mixture was slowly raised to 25 °C. The reaction was carried out at room temperature for 12 hours. New spots were observed on a TLC plate. The reaction solution was concentrated to obtain the crude product. Then, it was purified by ketene column chromatography (petroleum ether:ethyl acetate (V / V) = 1:1) to obtain ethyl 4-fluoro-3-carbonylbutyrate (27 g, yield 80.31%) as a colorless liquid.

[0483] Step 2: 6-(fluoromethyl)pyrimidine-2,4-diol

[0484]

[0485] Ethyl 4-fluoro-3-carbonylbutyrate (15 g, 101.35 mmol) was dissolved in 150 mL of methanol, and urea (12.2 g, 202.70 mmol) and a methanol solution of sodium methoxide (61 mL, 304.05 mmol) were added. The reaction mixture was reacted at 65 °C for 12 hours. The reaction solution was concentrated to obtain a crude product. The crude product was dissolved in water, and the pH was adjusted to neutral with 4N hydrochloric acid. The product was extracted with ethyl acetate, and the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness under vacuum to obtain a crude product. The crude product was purified by silica gel column chromatography and then by ketene column chromatography (dichloromethane:methanol (V / V) = 10:1) to give 6-(fluoromethyl)pyrimidine-2,4-diol (200 mg, yield 1.37%) as a white solid. LC-MS: m / z (ESI) = 145.0 [M+H] + ;

[0486] Step 3: 2,4-Dichloro-6-(fluoromethyl)pyrimidine

[0487]

[0488] 6-(fluoromethyl)pyrimidine-2,4-diol (200 mg, 1.39 mmol) was dissolved in 10 mL of phosphorus oxychloride and reacted at 100 °C for 12 hours. The reaction solution was concentrated to obtain a crude product. The crude product was dissolved in 2 mL of dichloromethane, quenched dropwise in 10 mL of ice water, extracted with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness under vacuum to obtain a crude product. The crude product was purified by silica gel column chromatography followed by ketene column chromatography (petroleum ether:ethyl acetate (V / V) = 5:1) to give 2,4-dichloro-6-(fluoromethyl)pyrimidine (140 mg, yield 56.0%) as a white solid. LC-MS: m / z (ESI) = 181.0 [M+H] + ;

[0489] Step 4: (R)-2-(2-chloro-6-(fluoromethyl)pyrimidin-4-yl)-9-methyl-8,9,10,11-tetrahydro-[1,4]diazo-heptan[5',6':4,5]thieno[3,2-e]indazole-7(2H)-one

[0490] (R)-9-methyl-8,9,10,11-tetrahydro-[1,4]diazolypeptido[5',6′:4,5]thieno[3,2-e]indazole-7(2H)-one (50 mg, 0.18 mmol) was dissolved in 5 mL of N,N-dimethylformamide, followed by the addition of cesium carbonate (180 mg, 0.55 mmol) and 2,4-dichloro-6-(fluoromethyl)pyrimidine (50 mg, 0.27 mmol). The reaction mixture was reacted at 25 °C for 30 min. The reaction solution was filtered, and the filtrate was concentrated to obtain the crude product. The crude product was then purified by preparative high-performance liquid chromatography (HPLC) (solvent A: water + 0.1% formic acid; solvent B: acetonitrile; gradient: 37-47%) to obtain:

[0491] 20A: (R)-2-(2-chloro-6-(fluoromethyl)pyrimidin-4-yl)-9-methyl-8,9,10,11-tetrahydro-[1,4]diazoylheptan[5′,6′:4,5]thieno[3,2-e]indazole-7(2H)-one (4.21 mg, yield 5.51%) was a yellow solid. LC-MS: m / z (ESI) = 417.0 [M+H] + ; 1H NMR (400MHz, DMSO-d6): δ9.90(s,1H),8.18(s,1H),7.94(d,J=4.0Hz,1H),7.75(dd,J=24.0,12.0Hz,2H ),7.14-7.12(m,1H),5.67(d,J=48Hz,2H),3.65-3.63(m,1H),3.50-3.44(m,2H),1.19(d,J=8.0Hz,3H).

[0492] 20B:(R)-3-(2-chloro-6-(fluoromethyl)pyrimidin-4-yl)-9-methyl-8,9,10,11-tetrahydro-[1,4]diazoylheptan[5′,6′:4,5]thieno[3,2-e]indazole-7(3H)-one (12.55 mg, yield 16.41%) was a pale yellow solid. LC-MS: m / z (ESI) = 417.0 [M+H] + ; 1 HNMR (400MHz, DMSO-d6): δ9.27(s,1H),8.77(d,J=8.0Hz,1H),8.10(d,J=12.0Hz,1H),8.05-8.03(m,2H ),7.05-7.02(m,1H),5.63(d,J=44Hz,2H),3.66-3.64(m,1H),3.49-3.46(m,2H),1.21(d,J=8.0Hz,3H).

[0493] Example 21: Synthesis of compounds 21A and 21B

[0494]

[0495] Step 1: Synthesis of (R)-2-(6-fluoropyrimidin-4-yl)-9-methyl-8,9,10,11-tetrahydro-[1,4]diazo-heptan[5′,6′:4,5]thieno[3,2-e]indazole-7(2H)-one

[0496] (R)-9-methyl-8,9,10,11-tetrahydro-[1,4]diazolypeptido[5′,6′:4,5]thieno[3,2-e]indazole-7(2H)-one (50 mg, 0.18 mmol) was dissolved in (3 mL) N,N-dimethylformamide, followed by the addition of cesium carbonate (120 mg, 0.37 mmol) and 4,6-difluoropyrimidine (43 mg, 0.37 mmol). The reaction mixture was reacted at 50 °C for 4 hours. The reaction mixture was filtered, and the filtrate was concentrated to obtain the crude product. The crude product was then purified by preparative high-performance liquid chromatography (HPLC) (solvent A: water + 0.1% formic acid; solvent B: acetonitrile; gradient: 44–58%) to obtain:

[0497] 21A:(R)-2-(6-fluoropyrimidin-4-yl)-9-methyl-8,9,10,11-tetrahydro-[1,4]diazoylheptan[5′,6′:4,5]thieno[3,2-e]indazole-7(2H)-one (8 mg, 12% yield) was a yellow solid. LC-MS: m / z (ESI) = 369.1 [M+H] + ; 1 H NMR (400MHz, DMSO-d6): δ10.00(s,1H),9.07(s,1H),7.96(s,1H),7.93(d,J=4.0Hz,1H),7.75(dd,J= 24.0,8.0Hz,2H),7.10(t,J=4.0Hz,1H),3.66-3.59(m,1H),3.52-3.44(m,2H),1.18(d,J=4.0Hz,3H).

[0498] 21B:(R)-3-(6-fluoropyrimidin-4-yl)-9-methyl-8,9,10,11-tetrahydro-[1,4]diazoylheptan[5′,6′:4,5]thieno[3,2-e]indazole-7(3H)-one (17.83 mg, yield 26.8%) was a yellow solid. LC-MS: m / z (ESI) = 369.1 [M+H] + ; 1 H NMR (400MHz, DMSO-d6): δ9.26(s,1H),9.03(d,J=4.0Hz,1H),8.94(d,J=12.0Hz,1H),8.07(d,J=8.0Hz,1H),8.03( d,J=4.0Hz,1H),7.81(s,1H),7.03(t,J=4.0Hz,1H),3.66-3.61(m,1H),3.53-3.45(m,2H),1.21(d,J=8.0Hz,3H).

[0499] Example 22: Synthesis of compounds 22A and 22B

[0500]

[0501] Step 1: Synthesis of (R)-4-(2,6-dichloropyrimidin-4-yl)-3-methylmorpholine

[0502]

[0503] 2,4,6-Trichloropyrimidine (600 mg, 3.27 mmol) was dissolved in (10 mL) dichloromethane, and then (R)-3-methylmorpholine (298 mg, 2.94 mmol) and triethylamine (1.66 g, 16.36 mmol) were added. The reaction mixture was reacted at 30 °C for 2 hours. Water (100 mL) was added to the reaction mixture, followed by extraction with ethyl acetate (100 mL). The organic phase was washed with saturated brine (100 mL), dried over sodium sulfate, and concentrated to obtain the crude product. The crude product was then purified by pyrogallol column chromatography (petroleum ether:ethyl acetate (V / V) = 2:1) to give (R)-4-(2,6-dichloropyrimidin-4-yl)-3-methylmorpholine (380 mg, yield 46.82%) as an off-white solid. LC-MS: m / z (ESI) = 248.1 [M+H] + ;

[0504] Step 2: Synthesis of (R)-2-(2-chloro-6-((R)-3-methylmorpholino)pyrimidin-4-yl)-9-methyl-8,9,10,11-tetrahydro-[1,4]diazoylheptan[5′,6′:4,5]thieno[3,2-e]indazole-7(2H)-one

[0505] (R)-4-(2,6-dichloropyrimidin-4-yl)-3-methylmorpholine (115 mg, 0.464 mmol) was dissolved in (3 mL) N,N-dimethylformamide, followed by the addition of cesium carbonate (151 mg, 0.464 mmol) and (R)-9-methyl-8,9,10,11-tetrahydro-[1,4]diazoylheptan[5′,6′:4,5]thieno[3,2-e]indazole-7(2H)-one (42 mg, 0.155 mmol). The reaction mixture was reacted at 50 °C for 5 hours. The reaction mixture was filtered, and the filtrate was concentrated to obtain the crude product. The crude product was then purified by preparative high-performance liquid chromatography (HPLC) (solvent A: water + 0.1% formic acid; solvent B: acetonitrile; gradient: 45–55%) to obtain:

[0506] 22A: (R)-2-(2-chloro-6-((R)-3-methylmorpholino)pyrimidin-4-yl)-9-methyl-8,9,10,11-tetrahydro-[1,4]diazoylheptan[5′,6′:4,5]thieno[3,2-e]indazole-7(2H)-one (28.19 mg, yield 37.70%) was a pale yellow solid. LC-MS: m / z (ESI) = 484.0 [M+H] + ; 1H NMR (400MHz, DMSO-d6): δ9.72 (s, 1H), 7.88 (d, J = 4.4Hz, 1H), 7.74-7.67 (m, 2H), 7.04-7.02 (m, 2H), 4.01-3.99 (m, 1H), 3.79-3. 76(m,1H),3.67-3.63(m,2H),3.55-3.49(m,1H),3.47-3.44(m,2H),3.35(s,3H),1.28(d,J=8.0Hz,3H),1.19(d,J=8.0Hz,3H).

[0507] 22B: (R)-3-(2-chloro-6-((R)-3-methylmorpholino)pyrimidin-4-yl)-9-methyl-8,9,10,11-tetrahydro-[1,4]diazoylheptan[5′,6′:4,5]thieno[3,2-e]indazole-7(3H)-one (14.45 mg, yield 19.32%) was a pale yellow solid. LC-MS: m / z (ESI) = 484.0 [M+H] + ; 1 H NMR (400MHz, DMSO): δ9.07(s,1H),8.64(d,J=12.0Hz,1H),7.98-7.95(m,2H),6.99(t,J=4.0Hz,1H),6.93(s,1H),4.02-3.96(m,1H),3.79-3.76(m ,1H),3.68-3.64(m,2H),3.54-3.48(m,3H),3.36-3.34(m,1H),3.32-3.3 0(m,1H),3.28-3.26(m,1H),1.30(d,J=4.0Hz,3H),1.20(d,J=8.0Hz,3H).

[0508] Example 23: Synthesis of compounds 23A and 23B

[0509]

[0510] Step 1: Synthesis of 3-bromo-4-methyl-5-nitropyridine-2-phenol

[0511]

[0512] 4-Methyl-5-nitropyridine-2-phenol (5 g, 32.67 mmol) was dissolved in water (100 mL), heated to 40 °C, and liquid bromine (5.75 g, 35.71 mmol) was added dropwise. After the addition was complete, the reaction was carried out for 5 hours, and a solid precipitated. The mixture was cooled to room temperature, filtered, washed with water, and dried to give 3-bromo-4-methyl-5-nitropyridine-2-phenol (5 g, yield 65.6%) as a yellow solid. LC-MS: m / z (ESI) = 233 [M+H] + ;

[0513] Step 2: Synthesis of 3-bromo-2-chloro-4-methyl-5-nitropyridine

[0514]

[0515] 3-Bromo-4-methyl-5-nitropyridine-2-phenol (5 g, 21.64 mmol) was dissolved in phosphorus oxychloride (20 mL), heated to 100 °C, and reacted for 12 hours. The reaction mixture was placed on ice, extracted with ethyl acetate, washed with saturated brine, dried over sodium sulfate, and concentrated to obtain the crude product. The crude product was then purified by pyrite column chromatography (petroleum ether:ethyl acetate (V / V) = 10:1) to give 3-bromo-2-chloro-4-methyl-5-nitropyridine (5 g, yield 92%) as a yellow solid. LC-MS: m / z (ESI) = 251 [M+H] + ;

[0516] Step 3: Synthesis of 5-bromo-6-chloro-4-methylpyridine-3-amine

[0517]

[0518] 3-Bromo-2-chloro-4-methyl-5-nitropyridine (5 g, 19.92 mmol) was dissolved in ethanol (50 mL), and iron powder (5.5 g, 99.6 mmol) and saturated ammonium chloride aqueous solution (20 mL) were added. The mixture was heated to 70 °C and reacted for 12 hours. The iron powder was filtered off, and the mixture was extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over sodium sulfate, and concentrated to obtain the crude product. The crude product was then purified by pyrite column chromatography (petroleum ether:ethyl acetate (V / V) = 5:1) to give 5-bromo-6-chloro-4-methylpyridine-3-amine (4 g, 91% yield) as a yellow solid. LC-MS: m / z (ESI) = 221 [M+H] + ;

[0519] Step 4: Synthesis of 4-bromo-5-chloro-1H-pyrazolo[3,4-c]pyridine

[0520]

[0521] 5-Bromo-6-chloro-4-methylpyridin-3-amine (4 g, 18.1 mmol) was dissolved in water (20 mL) and acetic acid (20 mL). Sodium nitrite (1.25 g, 18.1 mmol) was added under ice bath conditions. After the addition was complete, the reaction was carried out at room temperature for 12 hours. The mixture was extracted with ethyl acetate, washed with saturated brine, dried over sodium sulfate, and concentrated to obtain the crude product. The crude product was then purified by pyrazolite column chromatography (petroleum ether:ethyl acetate (V / V) = 5:1) to give 4-bromo-5-chloro-1H-pyrazolo[3,4-c]pyridine (2 g, 47% yield) as a yellow solid. LC-MS: m / z (ESI) = 232 [M+H] + ;

[0522] Step 5: Synthesis of 4-bromo-5-chloro-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-c]pyridine

[0523]

[0524] 4-Bromo-5-chloro-1H-pyrazolo[3,4-c]pyridine (2 g, 8.62 mmol) was dissolved in dichloromethane (20 mL), and 3,4-dihydro-2H-pyran (2.16 g, 25.86 mmol) and p-toluenesulfonic acid (150 mg, 0.86 mmol) were added. The mixture was stirred at room temperature for 16 hours and concentrated to obtain a crude product. The crude product was then purified by pyrazolite column chromatography (petroleum ether:ethyl acetate (V / V) = 10:1) to give 4-bromo-5-chloro-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-c]pyridine (2 g, 73% yield) as a yellow solid. LC-MS: m / z (ESI) = 316 [M+H] + ;

[0525] Step Six: Synthesis of 5-chloro-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-c]pyridine-4-carboxylonitrile

[0526]

[0527] 4-Bromo-5-chloro-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-c]pyridine (2 g, 6.31 mmol) was dissolved in 20 mL of N,N-dimethylformamide, and zinc cyanide (740 mg, 6.31 mmol) and tetra(triphenylphosphine)palladium (720 mg, 0.63 mmol) were added. The reaction was carried out at 100 °C for 16 h under nitrogen protection, cooled to room temperature, filtered, diluted with water, extracted with ethyl acetate, washed with saturated brine, dried over sodium sulfate, and concentrated to obtain the crude product. The crude product was then purified by kerogen column chromatography (petroleum ether:ethyl acetate (V / V) = 10:1) to give 5-chloro-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-c]pyridine-4-carboxynitrile (650 mg, yield 39%) as a yellow solid. LCMS: m / z(ESI) = 263[M+H] + .

[0528] Step 7: Synthesis of methyl 8-amino-3-(tetrahydro-2H-pyran-2-yl)-3H-pyrazolo[4,3-d]thieno[2,3-b]pyridine-7-carboxylic acid ester

[0529]

[0530] 5-Chloro-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-c]pyridine-4-carboxylonitrile (650 mg, 2.47 mmol) was dissolved in methanol (10 mL). Methyl 2-mercaptoacetate (523 mg, 4.94 mmol) and sodium methoxide (267 mg, 4.94 mmol) were added at room temperature. The mixture was heated to 75 °C and stirred for 16 hours. The mixture was quenched with water, extracted with ethyl acetate, and the organic phases were combined and concentrated to dryness. The residue was purified by kerogen column chromatography (0-50% ethyl acetate / petroleum ether) to give methyl 8-amino-3-(tetrahydro-2H-pyran-2-yl)-3H-pyrazolo[4,3-d]thieno[2,3-b]pyridine-7-carboxylic acid ester (400 mg, 48% yield). LCMS: m / z (ESI) = 333.0 [M+H] + .

[0531] Step 8: Synthesis of methyl 8-(((R)-2-((tert-butoxycarbonyl)amino)propyl)amino)-3-(tetrahydro-2H-pyran-2-yl)-3H-pyrazolo[4,3-d]thieno[2,3-b]pyridine-7-carboxylic acid ester

[0532]

[0533] Methyl 8-amino-3-(tetrahydro-2H-pyran-2-yl)-3H-pyrazolo[4,3-d]thieno[2,3-b]pyridine-7-carboxylic acid ester (400 mg, 1.2 mmol) was dissolved in N,N-dimethylformamide (10 mL). Sodium hydride (57 mg, 1.44 mmol) was added at 0 °C under nitrogen atmosphere. After the addition was complete, the mixture was reacted at 0 °C for 30 minutes. Then, tert-butyl(R)-4-(((tert-butyldimethylsilyl)oxo)methyl)-1,2,3-oxathiazolidin-3-carboxylic acid ester 2,2-dioxide (52 g, 1.43 mmol) was added. The mixture was reacted at 0 °C for 2 hours. After the reaction was complete, the mixture was quenched with water, extracted with ethyl acetate, washed with saturated brine, dried over sodium sulfate, and concentrated to obtain the crude product. The product was then purified by pyrazolite column chromatography (dichloromethane:methanol (V / V) = 10:1) to give methyl 8-(((R)-2-((tert-butoxycarbonyl)amino)propyl)amino)-3-(tetrahydro-2H-pyran-2-yl)-3H-pyrazolo[4,3-d]thieno[2,3-b]pyridine-7-carboxylic acid ester (250 mg, yield 42%) as an off-white solid. LC-MS: m / z (ESI) = 490 [M+H] + ;

[0534] Step Nine: Synthesis of methyl(R)-8-((2-aminopropyl)amino)-3H-pyrazolo[4,3-d]thieno[2,3-b]pyridine-7-carboxylic acid ester

[0535]

[0536] Methyl(R)-2-((tert-butoxycarbonyl)amino)propyl)amino)-3-(tetrahydro-2H-pyran-2-yl)-3H-pyrazolo[4,3-d]thieno[2,3-b]pyridine-7-carboxylic acid ester (250 mg, 0.51 mmol) was dissolved in methanol (5 mL), and a solution of dioxane (2 mL, 8 mmol) of hydrogen chloride was added. The reaction was carried out at room temperature for 3 hours. After the reaction was completed, the solution was concentrated to give methyl(R)-8-((2-aminopropyl)amino)-3H-pyrazolo[4,3-d]thieno[2,3-b]pyridine-7-carboxylic acid ester (200 mg, 100% yield) as a crude brown solid. LC-MS: m / z (ESI) = 306 [M+H] + ;

[0537] Step 10: Synthesis of (R)-9-methyl-8,9,10,11-tetrahydropyrazolo[4”,3”:4′,5′]pyrido[3′,2′:4,5]thiopheno[3,2-e][1,4]diazo-heptan-7(3H)-one

[0538]

[0539] Methyl(R)-8-((2-aminopropyl)amino)-3H-pyrazolo[4,3-d]thieno[2,3-b]pyridine-7-carboxylic acid ester (200 mg, 0.65 mmol) was dissolved in methanol (5 mL), and 1,8-diazabicyclo[5,4,0]undec-7-ene (476 mg, 3.15 mmol) was added. The mixture was heated to 70 °C and reacted for 12 hours. After the reaction was completed, the reaction mixture was concentrated to dryness, and the residue was purified by preparative liquid chromatography to obtain (R)-9-methyl-8,9,10,11-tetrahydropyrazolo[4”,3”:4',5']pyridolo[3',2':4,5]thieno[3,2-e][1,4]diazo-heptan-7(3H)-one (70 mg, 39% yield) as a brown solid. LC-MS: m / z (ESI) = 274.0 [M+H] + ;

[0540] Step 11: Synthesis of (R)-2-(2-chloro-6-methylpyrimidin-4-yl)-9-methyl-8,9,10,11-tetrahydropyrazolo[4”,3”:4',5']pyridolo[3',2':4,5]thieno[3,2-e][1,4]diazo-heptan-7(2H)-one

[0541] (R)-9-methyl-8,9,10,11-tetrahydropyrazolo[4”,3”:4',5']pyrido[3',2':4,5]thieno[3,2-e][1,4]diazo-heptan-7(3H)-one (50 mg, 0.17 mmol) was dissolved in (3 mL) N,N-dimethylformamide, and then cesium carbonate (170 mg, 0.52 mmol) and 2,4-dichloro-6-methylpyrimidine (43 mg, 0.26 mmol) were added. The reaction was carried out at room temperature for 4 hours. The reaction solution was filtered, and the filtrate was concentrated to obtain the crude product. Then, it was purified by preparative high performance liquid chromatography (solvent A: water + 0.1% formic acid; solvent B: acetonitrile; gradient: 43-58%) to obtain:

[0542] 23A:(R)-2-(2-chloro-6-methylpyrimidin-4-yl)-9-methyl-8,9,10,11-tetrahydropyrazolo[4”,3”:4′,5’]pyridolo[3’,2′:4,5]thieno[3,2-e][1,4]diazoylheptan-7(2H)-one (5.41 mg, yield 7.9%) was a yellow solid. LC-MS: m / z (ESI) = 400.0 [M+H] + ;

[0543] 1H NMR (400MHz, DMSO-d6) δ10.02(s,1H),9.30(s,1H),8.26(s,1H),8.01(d,J=4.0Hz,1H),7.30 (t,J=4.0Hz,1H),3.66-3.60(m,1H),3.54-3.43(m,2H),2.66(s,3H),1.19(d,J=8.0Hz,3H).

[0544] 23B:(R)-3-(2-chloro-6-methylpyrimidin-4-yl)-9-methyl-8,9,10,11-tetrahydropyrazolo[4”,3”:4′,5']pyridolo[3',2′:4,5]thieno[3,2-e][1,4]diazo-heptan-7(3H)-one (5.49 mg, 8% yield) is a yellow solid.

[0545] LC-MS: m / z (ESI) = 400.0 [M+H] + ;

[0546] 1H NMR(400MHz,DMSO-d6)δ10.01(s,1H),9.34(s,1H),8.12(d,J=4.0Hz,1H),8.04(s,1H),7.21 (t,J=4.0Hz,1H),3.67-3.63(m,1H),3.53-3.47(m,2H),2.60(s,3H),1.21(d,J=8.0Hz,3H).

[0547] Referring to the preparation methods in the above embodiments, the following compounds were prepared by SNAr reaction with different chloroaromatic heterocycles and (R)-9-methyl-8,9,10,11-tetrahydro-[1,4]diazo-heptan[5′,6′:4,5]thieno[3,2-e]indazole-7(3H)-one (Int-1):

[0548]

[0549]

[0550]

[0551]

[0552]

[0553]

[0554]

[0555]

[0556]

[0557]

[0558]

[0559]

[0560]

[0561]

[0562]

[0563]

[0564] Biological evaluation

[0565] Test Example 1: In vitro MK2 enzyme activity detection experiment

[0566] 1. Experimental reagents and instruments

[0567]

[0568] Instruments and equipment supplier Item number or model Plate shaker Thermo 4625-ECN / THZQ Centrifuge Eppendorf 5810R Envision 2104multi-label Reader PerkinElmer 2104 Echo Labcyte 655

[0569] 2. Experimental Procedure

[0570] CC-99677 structural formula:

[0571] In vitro MK2 kinase activity was measured via ADP-Glo TM The kinase assay was used for testing. In the experiment, the initial concentration for testing the inhibitory effect of the test compound on MK2 activity was 3000 nM, diluted 3-fold, for a total of 10 concentrations, and tested in replicates. Compound CC-99677 was used as a standard control, and DMSO was also included as a control.

[0572] Add the compound dilution buffer to a 384 assay plate (784075, Greiner) using an Echo 550; seal the plate and centrifuge at 1000g for 1 minute; prepare a 2× enzyme solution by adding an appropriate amount of MK2 kinase to 1× kinase buffer; add 2.5 μl of the 2× kinase solution to the assay plate; centrifuge at 1000g for 30 seconds and incubate at room temperature for 10 minutes; prepare a 2× substrate and ATP mixture by adding an appropriate amount of substrate and ATP to 1× kinase buffer; add 2.5 μl of the 2× substrate and ATP mixture to the 384 assay plate to start the reaction; centrifuge at 1000g for 30 seconds, seal the assay plate, and incubate at room temperature for 2 hours; add 4 μl of ADP-Glo ​​reagent and incubate at room temperature for 1 hour; add 8 μl of kinase assay reagent and incubate at room temperature for 1 hour; read the luminescence signal of each well on the plate using an Envision 2104 plate reader.

[0573] Inhibition rate calculation formula:

[0574] %inhibition=100-(Signalcmpd-SignalAve_PC) / (SignalAve_VC-SignalAve_PC)×100.

[0575] 3. Statistical methods:

[0576] Calculate IC50 and plot the compound effect-dose curve: Use GraphPad 6.0 to fit the % inhibition value and the logarithm of the compound concentration into a nonlinear regression (dose response-variable slope) to calculate IC50.

[0577] Y = Bottom + (Top - Bottom) / (1 + 10^((LogIC50 - X) * HillSlope)), where X is the log value of the compound's inhibitory concentration, and Y is the inhibition rate.

[0578] Table 1: The effect of the compounds of the present invention on MK2 inhibition IC 50 value

[0579]

[0580]

[0581]

[0582]

[0583] Conclusion: The compounds disclosed herein exhibit strong inhibitory activity against MK2 enzyme, and the inhibitory activity of some compounds against MK2 enzyme is significantly superior to that of CC-99677 (IC50). 50 =156.3nM).

[0584] Test Example 2: Detection of pHSP27 in THP-1 cells using the HCS method

[0585] 1. Experimental reagents and instruments

[0586]

[0587]

[0588] 2. Experimental Procedure

[0589] The fluorescence value of pHSP27 in THP-1 cells was detected using the High Content Screening (HCS) method. In the experiment, the initial concentration of the test compound for inhibiting pHSP27 was 10000 nM, diluted 3-fold, for a total of 10 concentrations, and tested in replicates. Compound CC-99677 was used as a standard control, and DMSO was also included as a control.

[0590] PMA-treated THP-1 cells were seeded in 96-well cell culture plates and cultured at 37°C and 5% CO2 for 72 hours. LPS (50 ng / ml) or culture medium was added, and the plates were incubated at 37°C and 5% CO2 for 30 minutes. Paraformaldehyde was added to each well, and the plates were incubated at room temperature for 20 minutes. 0.1% Trintonx-100 was added to the plates for cell perforation. Odessey blocking buffer was added, and the plates were incubated at room temperature for 1 hour. After washing, primary antibody (Anti-Hsp27 (phospho S78) antibody, ab32501) was added to the plates, and the plates were incubated overnight at 4°C. The plates were washed with PBST, and secondary antibody (Alexa Fluor 488 antibody, A11008) and Hoechst 33342 were added to the plates, and the plates were incubated at room temperature for 1 hour. The plates were washed with PBST, and images were acquired using the Operetta CLS high-content imaging system.

[0591] Inhibition rate calculation formula:

[0592]

[0593] 3. Statistical methods:

[0594] Computing IC 50 And plot the compound effect dose curve: calculate IC using nonlinear regression (dose response - variable slope) in Graphpad 8.0. 50 .

[0595] Y = Bottom + (Top - Bottom) / (1 + 10^((LogIC50 - X) * HillSlope)), where X is the log value of the compound's inhibitory concentration, and Y is the inhibition rate.

[0596] Table 2: Inhibition IC50 of the compounds of this invention on pHSP27 in THP-1 cells 50 value

[0597]

[0598]

[0599] Conclusion: The compounds disclosed herein exhibit strong inhibitory activity against the phosphorylation of HSP27, the major downstream protein of MK2, in the THP-1 mononuclear cell line. Some compounds show pHSP27 inhibitory activity comparable to that of CC-99677.

[0600] Test Example 3: Rat Pharmacokinetic Evaluation of Compounds

[0601] In this study, SD rats (SPF grade) were used as test compounds. The plasma drug concentrations of the test and reference compounds administered intravenously or orally to rats at different time points were quantitatively determined using LCMS / MS to evaluate the pharmacokinetic characteristics of the test drugs in rats.

[0602] A clear or suspension solution of the test compound was injected into rats via the tail vein (without fasting) or administered via gavage (rats were fasted overnight before administration and fed 4 hours after administration). Blood samples were collected via jugular vein at 0 h (before administration) and at 0.083 h, 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, and 24 h after intravenous or oral administration. Approximately 0.25 mL of blood was collected per sample, anticoagulated with EDTA-K2, and placed on moist ice after collection. Plasma was centrifuged within 1 hour (centrifugation conditions: 6000 g, 3 minutes, 2-8 °C). Plasma samples were stored at -80 °C before analysis. Blood drug concentrations were determined using LC-MS / MS. Pharmacokinetic parameters were calculated using Phoenix WinNonlin 8.2.0 based on blood drug concentration data at different time points. The results provided parameters such as AUC0-t, AUC0-∞, MRT0-∞, Cmax, Tmax, and T1 / 2, along with their mean and standard deviation.

[0603] Table 3. Pharmacokinetic parameters of the compounds of the present invention in rats

[0604]

[0605] Conclusion: The compound of this invention exhibits superior pharmacokinetics in rats compared to CC-99677 and demonstrates good drug-like properties.

Claims

1. A compound of formula I or a pharmaceutically acceptable salt thereof, in: R c It is hydrogen or C 1-6 alkyl; R1 and R2 are each independently hydrogen and C. 1-6 Alkyl or C 1-6 Alkoxy, the C 1-6 Alkyl groups and the C 1-6 The alkoxy group may optionally be surrounded by one or more R groups. R-1 replace; Each R R-1 Independently hydroxyl, amino, or halogen; X is S, O, CH2, or NH; M1, M2 and M3 are each independently N, NH, C or CH; Ring A is phenyl, 5-6 heteroaryl cyclic C, and ring C is 5-6-membered heteroaryl or 5-6-membered heterocyclic alkyl. The heteroatom in the heteroaryl or heterocyclic alkyl is one or more of N, O or S, and the number is 1, 2, 3 or 4. R3, R4, and R5 are independently hydrogen, halogen, cyano, or C. 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 Alkyne group, -C(=O)-NR6R7, -C(=O)-OR6, -C(=O)-R6, -NR6R7, -C 1-6 Alkylene-NR6R7 or The C mentioned 1-6 Alkyl groups and the C 1-6 The alkoxy group may optionally be surrounded by one or more R groups. R-2 replace: Each R R-2 Independently hydroxyl, deuterium, amino, halogen, oxo group (=O), -S (=O)2-C 1-6 Alkyl, C 1-6 Alkyl or C 1-6 Alkoxy; Each R6 and each R7 is independently either hydrogen or C. 1-6 Alkyl, the C 1-6 Alkyl groups may optionally be surrounded by one or more R groups. R-3 replace; Each R R-3 Independently selected as hydroxyl, amino, or halogen; M4 is a connecting bond, -C = O-, O, NH, C 1-6 Alkylene or C 2-6 Ethyne group; n is 0, 1, 2, or 3; Ring B is C 3-6 Cycloalkyl, 3-10-membered heterocycloalkyl, 3-6-membered heterocyclic alkenyl, phenyl or 5-6-membered heteroaryl; The heteroatoms in the 3-6 heterocyclic alkyl group, the 3-6 heterocyclic alkenyl group, and the 5-6 heteroaryl group are independently one or more of N, O, or S, and the number is 1, 2, 3, or 4. R b Hydroxyl, halogen, oxo group, C 1-6 Alkyl or C 1-6 Alkoxy, the C 1-6 Alkyl groups may optionally be surrounded by one or more R groups. R-4 replace; Each R R-4 Independently selected as hydroxyl, amino, or halogen; R a Halogen, cyano, C 1-6 Alkyl, C 1-6 Halogenated alkyl or C 1-6 Alkoxy; m can be 0, 1, or 2.

2. The compound of formula I as claimed in claim 1, or a pharmaceutically acceptable salt thereof, characterized in that, for And / or, for R1 is C 1-6 Alkyl or C 1-6 Alkoxy, the C 1-6 Alkyl groups and the C 1-6 The alkoxy group may optionally be surrounded by one or more R groups. R-1 replace.

3. The compound of formula I as claimed in claim 1, characterized in that, The compound represented by Formula I is a compound represented by the following general formulas II-1, II-2, or II-3:

4. The compound of formula I as claimed in claim 3, or a pharmaceutically acceptable salt thereof, characterized in that, The general formula II-1 is general formula II-1a, II-1b, II-1c, II-1d, II-1e, II-1f or II-1g: Preferably, in general formula II-1a, R3 is a halogen; one of the groups in R4 and R5 is... The other group is hydrogen, halogen, C 1-6 Alkyl or C 1-6 Alkoxy; M4 is the connecting key, and ring B is C. 3-6 Cycloalkyl or 3-10 membered heterocycloalkyl, where n is 0, 1, 2 or 3, R b Hydroxyl, halogen, C 1-6 Alkyl or C 1-6 Alkoxy; The general formula II-2 is general formula II-2a, II-2b, II-2c, II-2d, II-2e, II-2f or II-2g: The general formula II-3 is general formula II-3a, II-3b, II-3c, II-3d, II-3e, II-3f or II-3g:

5. The compound of formula I as claimed in any one of claims 1-3, or a pharmaceutically acceptable salt thereof, characterized in that, It meets one or more of the following conditions: (1) In ring A, the 5-6 member heteroaryl and the 5-6 heteroaryl in ring C are independently 6 member heteroaryl, the heteroatom is N, and the number is 1, 2 or 3; (2) In ring C, the heteroatom in the 5-6 member heteroaryl group is one or more of N, O and S, and the number is 1 or 2; (3) In the ring C, the 5-6 membered heterocyclic alkyl group is a 5-6 membered monocyclic heterocyclic alkyl group, the heteroatom is N and / or O, the number is 1 or 2, and it can be tetrahydrofuranyl, pyrrolidinyl or piperidinyl; (4)R a R b R c R1, R2, R3, R4, R5, each of R6, each of R7, and each of R R-2 In the context, C 1-6 Alkyl group is C 1-3 alkyl; (5)R a R b R1, R2, R3, R4, R5 and each R R-2 In the context, C 1-6 The alkoxy group is C 1-3 Alkoxy; (6) Each R R-1 Each R R-2 Each R R-3 Each R R-4 R3, R4, R5, R a and R b In this context, the halogen is independently F, Cl, Br, or I, for example, Cl or F; (7) In R1 and R2, when the aforementioned C 1-6 Alkyl groups are formed by one or more R groups. R-1 When replacing, the C 1-6 Alkyl group is C 1-3 Alkyl, R R-1 It is a hydroxyl group; (8) Among R3, R4 and R5, when the aforementioned C 1-6 Alkyl groups are formed by one or more R groups. R-2 When replacing, the C 1-6 Alkyl group is C 1-3 Alkyl groups, each R R-2 Independently hydroxyl, deuterium, amino, halogen, -S(=O)2-C 1-6 Alkyl or C 1-6 Alkoxy; (9) Among R3, R4 and R5, when the aforementioned C 1-6 Alkyl groups are formed by one or more R groups. R-2 When replacing, the C 1-6 The alkoxy group is C 1-3 Alkoxy groups, each R R-2 It is an amino group; (10) Among R3, R4 and R5, the C 2-6 The alkynyl group is either ethynyl or propynyl; (11) Among R3, R4 and R5, the -C 1-6 C in alkylene-NR6R7 1-6 Alkylene is C 1-3 Alkylene; (12) Each R R-2 In the context, -S(=O)2-C 1-6 C in alkyl 1-6 Alkyl group is C 1-3 alkyl; (13) Among each R6 and each R7, when the aforementioned C 1-6 Alkyl groups are formed by one or more R groups. R-3 When replacing, the C 1-6 Alkyl group is C 1-3 Alkyl groups, each R R-3 Independently halogenated or hydroxyl; (14) In M4, the C 1-6 Alkylene is C 1-3 Alkylene; (15) In M4, the C 2-6 The ynyl group is either ethynyl or propynyl; (16) In ring B, the 3-10 member heterocyclic alkyl group is a 3-6 member monocyclic heterocyclic alkyl group or a 6-8 member bicyclic spirocyclic heterocyclic alkyl group, and the heteroatom is one or more of N, O or S, and the number is 1 or 2. (17) In ring B, the 3-6 membered heterocyclic alkenyl group is a 5-6 membered heterocyclic alkenyl group, the heteroatom is one or more of N, O or S, the number is 1 or 2, and it contains 1 or 2 double bonds; (18) In ring B, the 5-6 membered heteroaryl group is a 5 membered heteroaryl group, and the heteroatom is one or more of N, O and S, and the number is 1 or 2; and (19)R a In the context, C 1-6 Haloalkyl groups can be C10-3 ... 1-3 Halogenated alkyl groups.

6. The compound of formula I as claimed in any one of claims 1-3, or a pharmaceutically acceptable salt thereof, characterized in that, It meets one or more of the following conditions: (1) In ring A, the 6-membered heteroaryl group is pyridinyl, pyrimidinyl, pyridazinyl, pyrazinyl or 1,3,5-triazinyl; (2) In ring C, the 5-6 member heteroaryl group is furanyl, thiophene, pyrrole, 1H-pyrazolyl, imidazole, oxazolyl, isoxazolyl, thiazolyl or isothiazolyl; (3) In ring C, the 5-6 member monocyclic heterocyclic alkyl group is tetrahydrofuranyl, pyrrolidinyl or piperidinyl; (4)R a R b R c R1, R2, R3, R4, R5, each of R6, each of R7, and each of R R-2 In the context, C 1-3 The alkyl group is methyl or ethyl; (5)R a R b R1, R2, R3, R4, R5 and each R R-2 In the context, C 1-3 The alkoxy group is either methoxy or ethoxy; (6) Each R R-1 Each R R-2 Each R R-3 Each R R-4 R3, R4, R5, R a and R b In this context, the halogen is independently either Cl or F; (7) In R1 and R2, when the aforementioned C 1-6 Alkyl groups are formed by one or more R groups. R-1 When replacing, the C 1-6 Alkyl is (8) Among R3, R4 and R5, when the aforementioned C 1-6 Alkyl groups are formed by one or more R groups. R-2 When replaced, the C 1-6 Alkyl groups are -CD3, -CH2F, -CHF2, -CF3, (9) Among R3, R4 and R5, when the aforementioned C 1-6 Alkoxyl-containing compounds are covered by one or more R R-2 When replaced, the C 1-6 alkoxy group is (10) Among R3, R4 and R5, the C 2-6 The alkynyl group is an ethynyl group; (11) Each R R-2 In the context, -S(=O)2-C 1-3 C in alkyl 1-3 The alkyl group is methyl; (12) Among each R6 and each R7, when the aforementioned C 1-6 Alkyl groups are formed by one or more R groups. R-3 When replacing, the C 1-6 The alkyl group is -CH2CH3, (13) In M4, the C 1-3 The alkylene group is methylene; (14) In M4, the C 2-6 The ethynyl group is an ethynyl group; (15) In ring B, the 3-6 member monocyclic heterocyclic alkyl group is azahexacyclic butyl, pyrrolidinyl, 1,3-dioxolanecycloyl, piperidinyl, oxacyclobutyl, tetrahydrofuranyl, tetrahydropyranyl, piperazinyl, morpholinyl, thiomorpholinyl, isoxazolyl or oxazolyl. (16) In ring B, the heterocyclic alkyl group of the 6-8 member bicyclic spirocyclic ring is 2-oxa-6-azaspiro[3.4]octyl or 2-oxa-6-azaspiro[3.3]heptyl; (17) In ring B, the 5-6 membered heterocyclic alkenyl group is 3,6-dihydro-2H-pyranyl; (18) In ring B, the 5-membered heteroaryl group is furanyl, thiophene, pyrrole, 1H-pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl or isothiazolyl.

7. The compound of formula I as claimed in any one of claims 1-3, or a pharmaceutically acceptable salt thereof, characterized in that, It meets one or more of the following conditions: (1)R c It is hydrogen; (2) X is S or O; (3) One of the groups in R1 and R2 is hydrogen, and the other group is C. 1-3 alkyl or hydroxy substituted C 1-3 alkyl; Preferably, one of the groups in R1 and R2 is hydrogen, and the other group is methyl or... (4) Ring A is a 6-membered heteroaryl, a 6-membered heteroaryl-5-membered heteroaryl, a 6-membered heteroaryl-5-membered heterocycloalkyl or a 6-membered heteroaryl-6-membered heterocycloalkyl; Preferably, ring A is (5) R3 is hydrogen, halogen, or C. 1-3 Alkyl, -NR6R7 or The C mentioned 1-3 Alkyl groups may optionally be surrounded by one or more R groups. R-2 Replace; each R R-2 Independently hydroxyl, deuterium, amino, halogen, C 1-3 Alkyl or C 1-3 Alkoxy groups; each R6 and each R7 is independently hydrogen or C. 1-3 Alkyl; ring B is a 5-6 membered monocyclic heterocyclic alkyl or a 5-6 membered heteroaryl; R b Hydroxyl group, halogen, oxo group (=O), C 1-6 Alkyl or C 1-6 Alkoxy, R b Halogen, C 1-3 Alkyl or C 1-3 Alkoxy Preferably, R3 is hydrogen, Cl, -CH3, -N(CH3)2, (6) R4 and R5 are independently hydrogen, halogen, cyano, C 1-3 Alkyl, C 1-3 Alkoxy, C 2-6 Alkyne group, -C(=O)-NR6R7, -C(=O)-OR6, -C(=O)-R6, -R6R7 or The C mentioned 1-3 Alkyl groups and the C 1-3 The alkoxy group may optionally be surrounded by one or more R groups. R-2 Substitution: hydroxyl, deuterium, amino, halogen, -S(=O)2-C 1-3 Alkyl, C 1-3 Alkyl or C 1-3 Alkyl group; M4 is a linking bond, -C(=O)-, C 1-3 Alkylene or C 2-3 Alynyl group; each R6 and each R7 is independently hydrogen or C. 1-3 Alkyl, the C 1-3 Alkyl groups may optionally be surrounded by one or more R groups. R-3 replace; Ring B is C 3-6 Cycloalkyl, 3-6 membered monocyclic heterocyclic alkyl, 6-8 membered bicyclic spirocyclic heterocyclic alkyl, 5-6 membered heterocyclic alkenyl or 5-6 membered heteroaryl; Preferably, R4 and R5 are independently hydrogen, F, Cl, -CN, -CH3, -CH2CH3, -CD3, -CHF, -CHF2, -CF3, -OCH3, -OCH2CH3, -N(CH3)2、 8. The compound of formula I as claimed in claim 1, characterized in that, It meets one or more of the following conditions: (1) for (2) for and (3) For any of the following situations: Case 1: Ring A is a 6-membered heteroaryl group, and R3, R4, and R5 are independently hydrogen, halogen, cyano, or C. 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 Alkyne group, -C(=O)-NR6R7, -C(=O)-OR6, -C(=O)-R6, -NR6R7 or -C 1-6 Alkylene-NR6R7; the C 1-6 Alkyl groups and the C 1-6 The alkoxy group may optionally be surrounded by one or more R groups. R-2 replace: Case 2: Ring A is a 6-membered heteroaryl group, and one of R3, R4, and R5 is... The definitions of the other two groups are the same as those in case 1; Case 3: Ring A is a 6-membered heteroaryl 5-6-membered heteroaryl or a 6-membered heteroaryl 5-6-membered heterocycloalkyl, and the definitions of R3, R4 and R5 are the same as those in Case 1; Better place, Case 1: When ring A is a 6-membered heteroaryl group, R3 is H, halogen, or C. 1-6 Alkyl, the C 1-6 Alkyl groups may optionally be surrounded by one or more R groups. R-2 replace; R4 and R5 are independently hydrogen, halogen, cyano, or C. 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 Alkyne group, -C(=O)-NR6R7, -C(=O)-OR6, -C(=O)-R6, -NR6R7 or -C 1-6 Alkylene-NR6R7, the C 1-6 Alkyl groups and the C 1-6 The alkoxy group may optionally be surrounded by one or more R groups. R-2 replace; Case 2: Ring A is a 6-membered heteroaryl group, R3 is a halogen, one of R4 and R5 is H or F, and the other group is... M4 is a connector, -C = O-, O, C 1-6 Alkylene or C 2-6 Ethyne group; Case 3: Ring A is a 6-membered heteroaryl-5-6-membered heteroaryl or a 6-membered heteroaryl-5-6-membered heterocycloalkyl, R3 is a halogen, and R4 and R5 are independently H or C. 1-6 alkyl; Better, Case 1: Ring A is a pyrimidinyl group, and R3 is a halogen; R4 and R5 are independently hydrogen, halogen, cyano, or C. 1-3 Alkyl, C 1-3 Alkoxy, C 2-6 Alkyne group, -C(=O)-NR6R7, -C(=O)-OR6 or -C(=O)-R6; the C 1-6 Alkyl groups and the C 1-6 The alkoxy group may optionally be surrounded by one or more R groups. R-2 Replace; each R R-2 Independently hydroxyl, deuterium, amino, halogen, -S(=O)2-C 1-3 Alkyl or C 1-3 Alkoxy; R6 and each of the R7s are independently either hydrogen or C. 1-3 Alkyl, the C 1-3 Alkyl groups may optionally be surrounded by one or more R groups. R-3 replace; Case 2: When ring A is a pyrimidinyl group, R3 is a halogen, one of R4 and R5 is H or a halogen, and the other group is... M4 is a connector, -C = O-, C 1-3 Alkylene or C 2-3 etymynyl group, ring B is C 3-6 Cycloalkyl, 3-6 membered monocyclic heterocyclic alkyl, 6-8 membered bicyclic spirocyclic heterocyclic alkyl, 5-6 membered heterocyclic alkenyl or 5-6 membered heteroaryl; n is 0, 1, 2 or 3; R b Hydroxyl, halogen, oxo group, C 1-3 Alkyl or C 1-3 Alkoxy, the C 1-3 Alkyl groups may optionally be surrounded by one or more R groups. R-4 Replace; each R R-4 Independently selected as hydroxyl, amino, or halogen; Case 3: When ring A is 5,7-dihydrofurano[3,4-d]pyrimidinyl, 5,6,7,8-tetrahydropyrido[4,3-d]pyrimidinyl, or pyrrolo[3,4-d]pyrimidinyl, R3 is halogen, and R4 and R5 are independently H or C. 1-3 alkyl; Better, for 9. The compound of formula I as claimed in claim 1, characterized in that, The compound represented by Formula I is any one of the following compounds, its corresponding isomer, or a mixture of both:

10. A method for preparing a compound of formula I as described in any one of claims 1-9, or a pharmaceutically acceptable salt thereof, characterized in that, It includes the following steps: in the presence of a base, the compound of formula III and the compound of formula IV are subjected to an aromatic nucleophilic substitution reaction in a solvent to obtain the compound shown in formula I; Where L is the leaving group, and rings A, X, M1, M2, M3, and R are also present. a R c The definitions of R1, R2, R3, R4 and R5 are as described in any one of claims 1-9.

11. A pharmaceutical composition, characterized in that, It includes the compound of formula I as described in any one of claims 1-9 or a pharmaceutically acceptable salt thereof, and pharmaceutical excipients.

12. The use of a compound of Formula I as described in any one of claims 1-9, or a pharmaceutically acceptable salt thereof, in the preparation of a MK2 inhibitor or a medicament for the treatment or prevention of diseases associated with protein HSP27 phosphorylation.

13. A compound III, V, or VI: in, R8 and R 11 Independently for C 1-6 Alkyl groups; R9 and R 10 Independently protected by amino groups, X, M1, M2, M3, R a and R c The definition is as described in any one of claims 1-9; Compound III, V, or VI is preferably one of the following compounds:

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