Triheterocyclic derivatives, pharmaceutical compositions thereof and uses thereof

Abstract

The application discloses a triheterocyclic derivative, a pharmaceutical composition thereof and application. The triheterocyclic derivative (I), a stereoisomer or a pharmaceutically acceptable salt thereof has the following structure. The triheterocyclic derivative has a good inhibitory effect on ATR in vivo and in vitro, and the triheterocyclic derivative can further be used for treating diseases caused by abnormal ATR, for example, cancer.

Description

Technical Field

[0001] This invention relates to a trihexacyclic derivative, pharmaceutical compositions thereof, and their use as therapeutic agents, particularly as cancer therapeutic agents. Background Technology

[0002] Human cells suffer hundreds of thousands of DNA damages every day. Causes of DNA damage include normal cellular functions (e.g., oxidative metabolites), DNA metabolites (e.g., spontaneous errors during transcription and replication), and environmental factors (e.g., ultraviolet radiation, ionizing radiation, genotoxins). If these damages are not properly repaired, cells or organisms may lose activity. Accumulated DNA damage can also affect the stability and integrity of the genome and promote cancer formation. DNA damage can occur through DNA base oxidation or alkylation, DNA base mismatches and dimers, breaks and discontinuities in the DNA backbone, intra- and inter-strand DNA crosslinks, and overall changes in DNA structure. To ensure the stability and integrity of the cellular genome, cells have a complex DNA damage response (DDR) mechanism that can recognize and process specific types of DNA damage in specific parts of the cell cycle to maintain genome integrity and cell viability. Studies have found that healthy cells possess multiple DDR mechanisms, and these repair mechanisms can compensate for each other during DNA repair (Jackson SP, Nature, 2009, 461(7267), 1071-1078). Many cancer cells have defects in multiple DNA repair pathways, thus exhibiting a greater dependence on undamaged DNA repair pathways.

[0003] The ataxia telangiectasia mutant gene and Rad3-related kinase ATR (also known as FRAP-Related Protein 1; FRP1; MEC1; SCK1; SECKL1) is a member of the phosphatidylinositol-3 kinase-associated kinase (PIKK) protein family. ATR is an important kinase that activates cellular responses after DNA damage, thereby arresting cell cycle progression, stabilizing replication forks, and repairing DNA, thus preventing apoptosis (Cimprich KA, Nature Rev. Mol. Cell Biol., 2008, 9:616-627). ATR functions by stabilizing arrested replication forks, regulating the activation of cell cycle checkpoints and DNA damage repair. Once activated, ATR activates three signal transduction pathways by regulating its downstream regulators (mainly Chk1, WRN, and FANCI) to arrest cell cycle progression, promote DNA repair, and stabilize replication forks. Although the presence of RPA-coated single-stranded DNA is a common feature of ATR activation, ATR can also be activated in some cases without DNA helicase polymerase uncoupling, such as by UV radiation, platinum chemotherapy, or alkylating agents.

[0004] Because DNA repair in tumor cells can be defective due to various mutations, it exhibits a greater dependence on undamaged DNA repair pathways. Therefore, the synthetic lethal theory can be used to kill specific tumor cells while preserving healthy cells. Current cancer treatments, including chemotherapy and ionizing radiation, can induce DNA damage and replication fork arrest, thereby activating cell cycle checkpoints and leading to cell cycle arrest. This mechanism is crucial for cancer cell survival during treatment. Broken double-stranded DNA or replication stress can rapidly activate the ATR (Advanced Metabolic Reproductive Tract), which in turn initiates a series of downstream targets such as Chk1 (an ATR substrate), p53, and DNA topoisomerase 2 binding protein (TopBP1), leading to DNA repair and cell cycle arrest. The ATR gene is rarely mutated and is therefore easily activated during cancer chemotherapy. Furthermore, inhibiting the ATR can generate several synthetic lethal interactions, particularly with the ATM / p53 pathway. p53 is the most common tumor suppressor gene mutation, and DNA repair in cells with ATM / p53 gene defects or mutations is more dependent on ATR activation (Reaper, PM, Nat. Chem. Biol., 2011, 7, 428-430).

[0005] Studies have shown that the loss of specific DNA repair proteins, such as X-ray cross-complementation repair gene 1 and mismatch excision cross-complementation repair gene 1, can also make tumor cells more sensitive to ATR inhibition (Sultana R, PLoS One, 2013, 8(2):e57098). In addition, hypoxic tumor cells may cause replication stress, making them more sensitive to ATR inhibition. Inhibiting ATR can selectively increase the sensitivity of tumor cells to ionizing radiation and chemotherapy, and increase their sensitivity to replication stress by many times compared to normal cells (Lecona E, Exp Cell Res, 2014, 329(1):26-34). In addition, since ATR is crucial for maintaining telomere homologous recombination, tumor cells that rely on telomere replacement extension pathways for DNA damage repair are also more sensitive to ATR inhibition.

[0006] The ATR pathway, as a DNA damage response mechanism, plays a crucial role in the survival of tumor cells. Inhibition of its key factor, ATR, can induce the death of ATR-dependent malignant tumor cells with minimal impact on normal cells, making it an ideal target for developing low-toxicity and highly effective targeted drugs. Currently, two small molecule entities, VX970 and AZD6738, have entered Phase II clinical trials, and several patents targeting the ATR pathway have been published: WO2015 / 084384, WO2017 / 180723, WO2016 / 061097, WO2014 / 140644, WO2007 / 015632, WO2017 / 123588, and WO2007 / 046426. However, no corresponding drugs have yet been marketed. The tri-heterocyclic derivative of this invention provides a new approach for the development of ATR inhibitors. Summary of the Invention

[0007] The technical problem to be solved by this invention is to provide a novel tri-heterocyclic derivative, its pharmaceutical composition, and its application. The tri-heterocyclic derivative of this invention has good ATR inhibitory activity and can effectively treat and / or alleviate various ATR-mediated related diseases, such as malignant tumors.

[0008] The present invention provides a compound of formula (I), its stereoisomer or pharmaceutically acceptable salt;

[0009]

[0010] in,

[0011] X is CR3 or NR5; X1 is CR 3a CR 3a R 4a or NR 5a X2 is CR3b CR 3b R 4b or NR 5b X3 is the connection key, CR 3c CR 3c R 4c or NR 5c ;

[0012] U is either N or CH;

[0013] U1 and U2 are each independently N or C; and U1 and U2 are not simultaneously N.

[0014] V is NR6 or CR7; V1 is N or NR. 6a or CR 7a V2 represents N and NR. 6b or CR 7b V3 is the connector key, N, NR 6c or CR 7c ;

[0015] R1 is hydrogen or C. 1-6 alkyl;

[0016] R2 is a methyl group;

[0017] R3, R 3a R 3b and R 3c Each can be independently represented by hydrogen, halogen, cyano, nitro, or C. 1-6 Alkyl, C 2-6 alkynyl group, C 2-6 alkenyl, C 6-10 Aryl, C 3-8 Cycloalkyl, 3-8 membered heterocycloalkyl, 5-6 membered heteroaryl, C 6-10 Aryl C 1-6 Alkyl, C 3-8 cycloalkyl C 1-6 Alkyl, 3-8 membered heterocyclic alkyl C 1-6 Alkyl, 5-6 membered heteroaryl C 1-6 Alkyl, -SR a -OR a -OC(O)R a -OC(O)OR a -OC(O)NR a R b -C(O)OR a -C(O)R a -C(O)NR a R b -C(O)N(R) b OR a -C(O)NR bS(O)2R a -C(=NH)R a -NR a R b -NR b C(O)R a -N(R) b )C(O)OR a -N(R) b )C(O)NR a R b -NR b S(O)2R a -NR b C(=NH)R a -NR b C(=NH)NR b R a -S(O) 1-2 R a -S(O)2NR a R b -S(O)(=NCN)R a -S(O)(=NR) b )R a or -NR b S(O)2NR a R b ; wherein, the C 1-6 Alkyl, C 2-6 alkynyl group, C 2-6 alkenyl, C 6-10 Aryl, C 3-8 Cycloalkyl, 3-8 membered heterocycloalkyl, 5-6 membered heteroaryl, C 6-10 Aryl C 1-6 Alkyl, C 3-8 cycloalkyl C 1-6 Alkyl, 3-8 membered heterocyclic alkyl C 1-6 Alkyl or 5-6-membered heteroaryl C 1-6 The alkyl group is unsubstituted or selectively replaced by 1 to 3 groups selected from halogen, cyano, nitro, -SR. a -OR a -OC(O)R a -OC(O)OR a -OC(O)NR a R b -C(O)OR a -C(O)R a -C(O)NR a R b -C(O)NR b S(O)2R a -NR aR b -NR b C(O)R a -N(R) b )C(O)OR a -N(R) b )C(O)NR a R b -NR b C(=NH)R a -NR b C(=NH)NR a R b -NR b S(O)2R a -NR b S(O)2NR a R b -S(O) 1-2 R a -S(O)2NR a R b -S(O)(=NCN)R a and -S(O)(=NR b )R a The substituents can be substituted at any position;

[0018] R 4a R 4b and R 4c Independently, they are hydrogen, halogen, and C. 1-6 Alkyl, C 1-6 Alkoxy, halogenated C 1-6 Alkyl or halogenated C 1-6 Alkoxy;

[0019] R5, R 5a R 5b and R 5c Independently, they are hydrogen and C respectively. 1-6 Alkyl, C 2-6 alkynyl group, C 2-6 alkenyl, C 6-10 Aryl, C 3-8 Cycloalkyl, 3-8 membered heterocycloalkyl, 5-6 membered heteroaryl, C 6-10 Aryl C 1-6 Alkyl, C 3-8 cycloalkyl C 1-6 Alkyl, 3-8 membered heterocyclic alkyl C 1-6 Alkyl, 5-6 membered heteroaryl C 1-6 Alkyl, -SR a -OR a -C(O)OR a -C(O)R a -C(O)NRa R b -C(O)N(R) b OR a -C(O)NR b S(O)2R a -C(=NH)R a -S(O) 1-2 R a -S(O)2NR a R b -S(O)(=NCN)R a or -S(O)(=NR) b )R a ; wherein, the C 1-6 Alkyl, C 2-6 alkynyl group, C 2-6 alkenyl, C 6-10 Aryl, C 3-8 Cycloalkyl, 3-8 membered heterocycloalkyl, 5-6 membered heteroaryl, C 6-10 Aryl C 1-6 Alkyl, C 3-8 cycloalkyl C 1-6 Alkyl, 3-8 membered heterocyclic alkyl C 1-6 Alkyl or 5-6-membered heteroaryl C 1-6 The alkyl group is unsubstituted or selectively replaced by 1 to 3 groups selected from halogen, cyano, nitro, -SR. a -OR a -OC(O)R a -OC(O)OR a -OC(O)NR a R b -C(O)OR a -C(O)R a -C(O)NR a R b -C(O)NR b S(O)2R a -NR a R b -NR b C(O)R a -N(R) b )C(O)OR a -N(R) b )C(O)NR a R b -NR b C(=NH)R a -NR b C(=NH)NR a R b -NR bS(O)2R a -NR b S(O)2NR a R b -S(O) 1-2 R a -S(O)2NR a R b -S(O)(=NCN)R a and -S(O)(=NR b )R a The substituents can be substituted at any position;

[0020] R6, R 6a R 6b and R 6c Independently, they are hydrogen and C respectively. 1-6 Alkyl, C 1-6 Alkoxy, halogenated C 1-6 Alkyl, Halogenated C 1-6 Alkoxy, C 3-8 Cycloalkyl, 3-8 membered heterocycloalkyl, C 6-10 Aryl, 5-10 heteroaryl, C 3-8 cycloalkyl C 1-6 Alkyl or 3-8 membered heterocyclic alkyl C 1-6 Alkyl, wherein the C 6-10 The aryl or 5-10 heteroaryl groups are unsubstituted or selectively selected from halogens, cyano groups, and -R groups. c -OR c -NR c R d -N(CN)R c -N(OR) d )R c -S(O) 0-2 R c -C(O)R c -C(O)OR c -C(O)NR c R d -C(NH)NR c R d -NR d C(O)R c -NR d C(O)NR c R d -NR d S(O)2R c and -OC(O)R c The substituents can be substituted at any position;

[0021] R7, R 7a R7b and R 7c Each is independently hydrogen, halogen, cyano, or C. 1-6 Alkyl, C 1-6 Alkoxy, halogenated C 1-6 Alkyl, Halogenated C 1-6 Alkoxy, C 3-8 Cycloalkyl, 3-8 membered heterocycloalkyl, C 6-10 Aryl, 5-10 heteroaryl, C 3-8 cycloalkyl C 1-6 Alkyl or 3-8 membered heterocyclic alkyl C 1-6 Alkyl, wherein the C 6-10 The aryl or 5-10 heteroaryl groups are unsubstituted or selectively selected from halogens, cyano groups, and -R groups. c -OR c -NR c R d -N(CN)R c -N(OR) d )R c -S(O) 0-2 R c -C(O)R c -C(O)OR c -C(O)NR c R d -C(NH)NR c R d -NR d C(O)R c -NR d C(O)NR c R d -NR d S(O)2R c and -OC(O)R c The substituents can be substituted at any position;

[0022] Each R a R b R c and R d Independently, they are hydrogen and C respectively. 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-8 Cycloalkyl, 3-8 membered heterocycloalkyl, C 6-10 Aryl, 5-6 quinone heteroaryl, C 3-8 cycloalkyl C 1-6 Alkyl, 3-8 membered heterocyclic alkyl C 1-6 Alkyl, phenyl C 1-6 Alkyl or 5-6-membered heteroaryl C 1-6 Alkyl; the Ra R b R c and R d For unsubstituted or selectively substituted groups selected from 1 to 3 groups chosen from halogen, hydroxyl, amino, carboxyl, C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Alkylamino, Halogenated C 1-6 Alkyl, Halogenated C 1-6 Alkoxy, C 2-6 alkenyl and C 2-6 The substituents of the alkynyl group can be substituted at any position.

[0023] All embodiments described below as in Formula (I), and any combination of embodiments, are included within the scope of the structural formula shown in Formula (I) of this invention.

[0024] In some implementations, R1 is hydrogen or methyl.

[0025] In some implementations, R2 is hydrogen or methyl.

[0026] In some implementations, R1 is hydrogen and R2 is methyl.

[0027] In some implementations, each R a R b R c and R d Independently, they are hydrogen and C respectively. 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-8 Cycloalkyl, 3-8 membered heterocycloalkyl, C 6-10 Aryl, 5-6 quinone heteroaryl, C 3-8 cycloalkyl C 1-6 Alkyl, 3-8 membered heterocyclic alkyl C 1-6 Alkyl, phenyl C 1-6 Alkyl or 5-6-membered heteroaryl C 1-6 Alkyl; the R a R b R c and R d For being unsubstituted or selectively substituted by one or more groups selected from halogen, hydroxyl, amino, carboxyl, C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Alkylamino, Halogenated C 1-6 Alkyl, Halogenated C 1-6 Alkoxy, C 2-6 alkenyl and C 2-6 The substituents of the alkynyl group can be substituted at any position.

[0028] In some implementation schemes, R a and R b Together with the N atoms they are connected to, they form 3-8 membered heterocyclic alkyl groups.

[0029] In some implementation schemes, R c and R d Together with the N atoms they are connected to, they form 3-8 membered heterocyclic alkyl groups.

[0030] In some implementations, each R a Independently hydrogen, C 1-6 Alkyl, C 3-8 Cycloalkyl or 3-8 membered heterocycloalkyl; the R a For unsubstituted or selectively substituted by 1 to 3 molecules selected from halogen, hydroxyl, amino, C 1-6 Alkoxy, C 1-6 Alkylamino, Halogenated C 1-6 Alkyl and Halogenated C 1-6 The alkoxy group can be substituted at any position.

[0031] In some implementations, each R a Independently hydrogen or C 1-6 Alkyl; the C 1-6 The alkyl group is unsubstituted or selectively surrounded by 1 to 3 groups selected from halogen, hydroxyl, amino, C. 1-6 Alkoxy, C 1-6 Alkylamino, Halogenated C 1-6 Alkyl and Halogenated C 1-6 The alkoxy group can be substituted at any position.

[0032] In some implementations, each R b Independently hydrogen or C 1-6 alkyl.

[0033] In some implementations, each R c Independently hydrogen or C 1-6 Alkyl; the C 1-6 The alkyl group is unsubstituted or selectively surrounded by 1 to 3 groups selected from halogen, hydroxyl, amino, C. 1-6 Alkoxy, C 1-6 Alkylamino, Halogenated C 1-6 Alkyl and Halogenated C 1-6 The alkoxy group can be substituted at any position.

[0034] In some implementations, each R d Independently hydrogen or C 1-6 alkyl.

[0035] In some implementations, R3 is C 1-6 Alkyl, phenyl, C3-8 Cycloalkyl, 3-8 membered heterocycloalkyl, 5-6 membered heteroaryl, C 3-8 cycloalkyl C 1-6 Alkyl, 3-8 membered heterocyclic alkyl C 1-6 Alkyl, 5-6 membered heteroaryl C 1-6 Alkyl, -NR b S(O)2R a -S(O) 1-2 R a -S(O)2NR a R b -S(O)(=NCN)R a or -S(O)(=NR) b )R a ; wherein, the C 1-6 Alkyl, phenyl, C 3-8 Cycloalkyl, 3-8 membered heterocycloalkyl, 5-6 membered heteroaryl, C 3-8 cycloalkyl C 1-6 Alkyl or 3-8 membered heterocyclic alkyl C 1-6 The alkyl group is unsubstituted or selectively replaced by 1 to 3 groups selected from halogens, -CN, -SR. a -OR a -C(O)OR a -C(O)R a -C(O)NR a R b -NR a R b -NR b C(O)R a -NR b S(O)2R a -S(O) 1-2 R a -S(O)2NR a R b -S(O)(=NCN)R a and -S(O)(=NR b )R a The substituents can be substituted at any position.

[0036] In some implementation schemes, R 3a R 3b and R 3c Each is independently hydrogen, halogen, cyano, or C. 1-6 Alkyl, Halogenated C 1-6 Alkyl or halogenated C 1-6 Alkyl group.

[0037] In some implementation schemes, R 3a R 3b and R3c Each is hydrogen, independently.

[0038] In some implementation schemes, R 4a R 4b and R 4c Each independently can be either hydrogen or C. 1-6 alkyl.

[0039] In some implementation schemes, R 4a R 4b and R 4c Each is hydrogen, independently.

[0040] In some implementations, R5 is C 1-6 Alkyl, phenyl, C 3-8 Cycloalkyl, 3-8 membered heterocycloalkyl, 5-6 membered heteroaryl, C 3-8 cycloalkyl C 1-6 Alkyl, 3-8 membered heterocyclic alkyl C 1-6 Alkyl, 5-6 membered heteroaryl C 1-6 Alkyl group, -S(O) 1-2 R a -S(O)2NR a R b -S(O)(=NCN)R a or -S(O)(=NR) b )R a ; wherein, the C 1-6 Alkyl, phenyl, C 3-8 Cycloalkyl, 3-8 membered heterocycloalkyl, 5-6 membered heteroaryl, C 3-8 cycloalkyl C 1-6 Alkyl or 3-8 membered heterocyclic alkyl C 1-6 The alkyl group is unsubstituted or selectively replaced by 1 to 3 groups selected from halogens, -CN, -SR. a -OR a -C(O)OR a -C(O)R a -C(O)NR a R b -NR a R b -NR b C(O)R a -NR b S(O)2R a -S(O) 1-2 R e -S(O)2NR a R b -S(O)(=NCN)R a and -S(O)(=NR b )Ra The substituents can be substituted at any position.

[0041] In some implementation schemes, R 5a R 5b and R 5c Independently, they are hydrogen and C respectively. 1-6 Alkyl, Halogenated C 1-6 Alkyl or C 3-8 Cycloalkyl.

[0042] In some implementation schemes, R 5a R 5b and R 5c Each is hydrogen, independently.

[0043] In some embodiments, R6 and R7 are each independently a 5-6 membered heteroaryl group; the 5-6 membered heteroaryl group is unsubstituted or selectively replaced by 1 to 3 groups selected from halogen, cyano, -R c -OR c -NR c R d -N(CN)R c -N(OR) d )R c -S(O) 0- 2R c -C(O)R c -C(O)OR c -C(O)NR c R d -C(NH)NR c R d -NR d C(O)R c -NR d C(O)NR c R d -NR d S(O)2R c and -OC(O)R c The substituents can be substituted at any position.

[0044] In some embodiments, R6 and R7 are independently pyrrolo, pyrazol, or isoxazol; the pyrrolo, pyrazol, or isoxazol is unsubstituted or selectively substituted with 1 to 3 groups selected from halogen, cyano, or -R. c -OR c -NR c R d -N(CN)R c -N(OR) d )R c -S(O) 0-2 Rc -C(O)R c -C(O)OR c -C(O)NR c R d -C(NH)NR c R d -NR d C(O)R c -NR d C(O)NR c R d -NR d S(O)2R c and -OC(O)R c The substituents can be substituted at any position.

[0045] In some embodiments, R6 and R7 are independently pyrrole, pyrazol, or isoxazolyl.

[0046] In some embodiments, R6 and R7 are each independently a pyrazolyl group.

[0047] In some implementations, the R 6a R 6b and R 6c Independently, they are hydrogen and C respectively. 1-6 Alkyl or halogenated C 1-6 alkyl.

[0048] In some implementations, the R 6a R 6b and R 6c Each is hydrogen, independently.

[0049] In some implementations, the R 7a R 7b and R 7c Each is independently hydrogen, halogen, cyano, or C. 1-6 Alkyl, C 1-6 Alkoxy, halogenated C 1-6 Alkyl or halogenated C 1-6 Alkyl group.

[0050] In some implementations, the R 7a R 7b and R 7c Each is hydrogen, independently.

[0051] In some implementations, X is CR3, N, O, S, SO2, S(O)(NH), CR3R4, or NR5; X1 is CR 3a ,N,O,S,SO2,S(O)(NH),CR 3a R 4aor NR 5a X2 is CR 3b ,N,O,S,SO2,S(O)(NH),CR 3b R 4b or NR 5b X3 is the connection key, CR 3c ,N,O,S,SO2,S(O)(NH),CR 3c R 4c or NR 5c R4 represents hydrogen, halogen, or C. 1-6 Alkyl, C 1-6 Alkoxy, halogenated C 1-6 Alkyl or halogenated C 1-6 Alkyl group.

[0052] In some implementations, X is CR3, N, O, S, CR3R4, or NR5; X1 is CR 3a N, O, S, CR 3a R 4a or NR 5a X2 is CR 3b N, O, S, CR 3b R 4b or NR 5b X3 is the connection key, CR 3c N, O, S, CR 3c R 4c or NR 5c R4 represents hydrogen, halogen, or C. 1-6 Alkyl, C 1-6 Alkoxy, halogenated C 1-6 Alkyl or halogenated C 1-6 Alkyl group.

[0053] In some implementations, X is NR5; X1 is CR 3a CR 3a R 4a or NR 5a X2 is CR 3b CR 3b R 4b or NR 5b X3 is the connection key.

[0054] In some implementations, V is N, NR6, or CR7; V1 is N or NR 6a or CR 7a V2 represents N and NR. 6b or CR 7b V3 is the connector key, N, NR 6c or CR 7c .

[0055] In some implementations, V is NR6 or CR7; V1 is N or CR 7a V2 is N or CR 7b V3 is a connection key, N or CR 7c .

[0056] In some implementations, U is N.

[0057] In some implementations, U1 and U2 are C.

[0058] In some embodiments, the definitions of certain groups in the compound of formula (I), its stereoisomers, or pharmaceutically acceptable salts may be as follows, and undescribed groups may be as described in any of the above embodiments:

[0059] Among them, the group It can be any of the following structures:

[0060]

[0061] In some embodiments, the compound shown in formula (I), its stereoisomer, or a pharmaceutically acceptable salt is the compound shown in formula (II), its stereoisomer, or a pharmaceutically acceptable salt:

[0062]

[0063] Where U1 and U2 are C respectively; V is NR6; V1 is N or CR 7a V2 is N or CR 7b ;

[0064] Alternatively, U1 is C; U2 is N; V is CR7; V1 is N or CR 7a V2 is N or CR 7b ;

[0065] Alternatively, U1 is N; U2 is C; V is CR7; V1 is N or CR 7a V2 is N or CR 7b ;

[0066] R1, R2, U, X, X1, X2, R6, R7, R 7a and R 7b The definition is as described above.

[0067] In some implementations, U1 and U2 are C; V is NR6; and V1 is N or CR. 7a V2 is N or CR 7b .

[0068] U is N; R6 is pyrrole, pyrazol, or isoxazol; the pyrrole, pyrazol, or isoxazol is unsubstituted or selectively substituted by 1 to 3 groups selected from halogen, cyano, -R c -OR c -NR c R d -N(CN)R c -N(OR) d )R c -S(O) 0-2 R c -C(O)R c -C(O)OR c -C(O)NR c R d -C(NH)NR c R d -NR d C(O)R c -NR d C(O)NR c R d -NR d S(O)2R c and -OC(O)R c The substituents can be substituted at any position;

[0069] R 7a and R 7b Each is independently hydrogen;

[0070] R c and R d Each independently can be either hydrogen or C. 1-6 alkyl.

[0071] In some embodiments, the compound represented by formula (I), its stereoisomer, or a pharmaceutically acceptable salt is the compound represented by formula (IIA), its stereoisomer, or a pharmaceutically acceptable salt:

[0072]

[0073] in, It can be a double bond or a single bond;

[0074] The definitions of X1, X2, V1, V2 and R5 are as described above.

[0075] In some implementations, X1 and X2 are independently N or CH.

[0076] In some implementations, X1 and X2 are independently CH2.

[0077] In some implementations, V1 and V2 are independently N or CH.

[0078] In some implementations, R5 is C 1-6 Alkyl, phenyl, C 3-8 Cycloalkyl, 3-8 membered heterocycloalkyl, 5-6 membered heteroaryl, C 3-8 cycloalkyl C 1-6 Alkyl, 3-8 membered heterocyclic alkyl C 1-6 Alkyl, 5-6 membered heteroaryl C 1-6 Alkyl group, -S(O) 1-2 R a -S(O)2NR a R b -S(O)(=NCN)R a or -S(O)(=NR) b )R a ; wherein, the C 1-6 Alkyl, phenyl, C 3-8 Cycloalkyl, 3-8 membered heterocycloalkyl, 5-6 membered heteroaryl, C 3-8 cycloalkyl C 1-6 Alkyl or 3-8 membered heterocyclic alkyl C 1-6 The alkyl group is unsubstituted or selectively replaced by 1 to 3 groups selected from halogens, -CN, -SR. a -OR a -C(O)OR a -C(O)R a -C(O)NR a R b -NR a R b -NR b C(O)R a -NR b S(O)2R a -S(O) 1-2 R a -S(O)2NR a R b -S(O)(=NCN)R a and -S(O)(=NR b )R a The substituents can be substituted at any position;

[0079] Each R a Independently hydrogen or C 1-6 Alkyl; the C 1-6 The alkyl group is unsubstituted or selectively surrounded by 1 to 3 groups selected from halogen, hydroxyl, amino, C. 1-6 Alkoxy, C 1-6 Alkylamino, Halogenated C 1-6 Alkyl and Halogenated C 1-6The alkoxy group can be substituted at any position;

[0080] Each R b Independently hydrogen or C 1-6 alkyl.

[0081] In some embodiments, the compound represented by formula (I), its stereoisomer, or a pharmaceutically acceptable salt is the compound represented by formula (III), its stereoisomer, or a pharmaceutically acceptable salt:

[0082]

[0083] Where U1 and U2 are each independently C; V is CR7; V1 is N or CR 7a V2 is N or CR 7b V3 is N or CR 7c ;

[0084] R1, R2, U, X, X1, X2, R7, R 7a R 7b and R 7c The definition is as described above.

[0085] In some embodiments, U is N; R7 is pyrrole, pyrazol, or isoxazol; said pyrrole, pyrazol, or isoxazol is unsubstituted or selectively substituted with 1 to 3 groups selected from halogen, cyano, -R c -OR c -NR c R d -N(CN)R c -N(OR) d )R c -S(O) 0-2 R c -C(O)R c -C(O)OR c -C(O)NR c R d -C(NH)NR c R d -NR d C(O)R c -NR d C(O)NR c R d -NR d S(O)2R c and -OC(O)R c The substituents can be substituted at any position;

[0086] R 7a R 7b and R 7cEach is independently hydrogen;

[0087] R c and R d Each independently can be either hydrogen or C. 1-6 alkyl.

[0088] In some embodiments, the compound represented by formula (I), its stereoisomer, or a pharmaceutically acceptable salt is the compound represented by formula (IIIA), its stereoisomer, or a pharmaceutically acceptable salt:

[0089]

[0090] in, It can be a double bond or a single bond;

[0091] The definitions of X1, X2, V1, V2, V3 and R5 are as described above.

[0092] In some implementations, X1 and X2 are independently N or CH.

[0093] In some implementations, X1 and X2 are independently CH2.

[0094] In some implementations, V1, V2, and V3 are each independently N or CH.

[0095] In some implementations, V1 is N; V2 and V3 are independently CH.

[0096] In some implementations, R5 is C 1-6 Alkyl, phenyl, C 3-8 Cycloalkyl, 3-8 membered heterocycloalkyl, 5-6 membered heteroaryl, C 3-8 cycloalkyl C 1-6 Alkyl, 3-8 membered heterocyclic alkyl C 1-6 Alkyl, 5-6 membered heteroaryl C 1-6 Alkyl group, -S(O) 1-2 R a -S(O)2NR a R b -S(O)(=NCN)R a or -S(O)(=NR) b )R a ; wherein, the C 1-6 Alkyl, phenyl, C 3-8 Cycloalkyl, 3-8 membered heterocycloalkyl, 5-6 membered heteroaryl, C 3-8 cycloalkyl C 1-6 Alkyl or 3-8 membered heterocyclic alkyl C 1-6 The alkyl group is unsubstituted or selectively replaced by 1 to 3 groups selected from halogens, -CN, -SR. a -ORa -C(O)OR a -C(O)R a -C(O)NR a R b -NR a R b -NR b C(O)R a -NR b S(O)2R a -S(O) 1-2 R a -S(O)2NR a R b -S(O)(=NCN)R a and -S(O)(=NR b )R a The substituents can be substituted at any position;

[0097] Each R a Independently hydrogen or C 1-6 Alkyl; the C 1-6 The alkyl group is unsubstituted or selectively surrounded by 1 to 3 groups selected from halogen, hydroxyl, amino, C. 1-6 Alkoxy, C 1-6 Alkylamino, Halogenated C 1-6 Alkyl and Halogenated C 1-6 The alkoxy group can be substituted at any position;

[0098] Each R b Independently hydrogen or C 1-6 alkyl.

[0099] In some embodiments, the compound represented by formula (I), its stereoisomers, or pharmaceutically acceptable salts may optionally be the following compounds:

[0100]

[0101] Or its pharmaceutically acceptable salt.

[0102] The present invention also provides a method for preparing the compound shown in formula (I), its stereoisomers, or pharmaceutically acceptable salts, which is any of the following methods:

[0103] Method 1:

[0104]

[0105] In Method 1, X, X1, X2, X3, R2, and R6 are defined as previously stated. Step 1: In a solvent (e.g., N,N-dimethylformamide), IV-1 reacts with phosphorus oxychloride to give IV-2; or IV-1 reacts with hexamethylenetetramine / trifluoroacetic acid to give IV-2. Step 2: In a solvent (e.g., ethanol), IV-2 reacts with a suitable organohydrazine (e.g., heteroarylhydrazine) to give IV-3. Step 3: In a solvent (e.g., N-methylpyrrolidone), IV-3 undergoes cyclization at high temperature to give the compound shown as Formula IV.

[0106] Method 2:

[0107]

[0108] In Method 2, Lev is a leaving group, preferably a halogen, more preferably chlorine and bromine; X, X1, X2, X3, V1, V2, V3, R2, and R7 are defined as previously stated. In a solvent (e.g., 1,4-dioxane / water), under alkaline conditions (e.g., potassium carbonate, sodium carbonate, or cesium carbonate), in the presence of a catalyst (e.g., 1,1'-bis(diphenylphosphine)ferrocene]palladium dichloride), V-1 undergoes a coupling reaction to yield the compound shown in Formula V.

[0109] Method 3:

[0110]

[0111] In Method 3, the definitions of X1, X2, V, V1, V2, V3, U, U1, U2, R2, and R5 are as described above.

[0112] 1) When R5 is a substituted or unsubstituted C 1-6 Alkyl, substituted or unsubstituted C 3-8 cycloalkyl C 1-6 Alkyl, substituted or unsubstituted 3-8 membered heterocyclic alkyl C 1-6 Alkyl, substituted or unsubstituted 5-6 membered heteroaryl C 1-6 Alkyl group, -S(O) 1-2 R a or -S(O)2NR a R b When D is a halogen (preferably chlorine, bromine, or iodine); VI-1 and R5-D under alkaline conditions undergo a nucleophilic substitution reaction to obtain the structure shown in formula VI.

[0113] 2) When R5 is a substituted or unsubstituted phenyl or a substituted or unsubstituted 5-6-membered heteroaryl group, D is a halogen (preferably chlorine or bromine); under alkaline conditions (e.g., potassium carbonate or cesium carbonate), in the presence of a catalyst (e.g., methanesulfonic acid (2-dicyclohexylphosphino-2',6'-diisopropoxy-1,1'-biphenyl)(2-amino-1,1'-biphenyl-2-yl)palladium(II)), VI-1 and R5-D undergo a coupling reaction to give the compound shown in formula VI.

[0114] 3) When R5 is a substituted or unsubstituted C 3-8 When the cycloalkyl or substituted or unsubstituted 3-8 membered heterocyclic alkyl group is present, D is a borate group or borate ester group; under basic conditions (e.g., sodium carbonate or potassium carbonate) in the presence of a catalyst (e.g., copper acetate), VI-1 and R5-D undergo a coupling reaction to give the compound shown in formula VI.

[0115] In methods 1, 2, or 3 above, when an amino group, hydroxyl group, or carboxyl group is present in X, X1, X2, X3, V, V1, V2, V3, -R5, -R6, or -R7, this amino group, hydroxyl group, or carboxyl group can be protected by a protecting group to prevent any side reactions from occurring. If the above-mentioned amino protecting group, hydroxyl protecting group, or carboxyl protecting group is present, a subsequent deprotection step is required to obtain the compound shown in formula IV, V, or VI. Any suitable amino protecting group, such as a tert-butoxycarbonyl (Boc) group or a benzyloxycarbonyl (Cbz) group, can be used to protect the amino group. If Boc is used as the protecting group, the subsequent deprotection reaction can be carried out under standard conditions, such as p-toluenesulfonic acid / methanol system, dichloromethane / trifluoroacetic acid system, organic solution system of hydrogen chloride (organic solutions include but are not limited to: diethyl ether solution, 1,4-dioxane solution, methanol solution, ethanol solution, isopropanol solution) or trimethylsilyl trifluoromethanesulfonate / 2,6-dimethylpyridine / dichloromethane system; the Cbz protecting group can be deprotected using palladium on carbon / hydrogen system. Any suitable hydroxyl protecting group, such as benzyl, methoxymethyl (MOM), 2-tetrahydropyranyl (THP), (trimethylsilyl)ethoxymethyl (SEM), or organosilicon groups (including but not limited to tert-butyldimethylsilyl and trimethylsilyl), can be used as a hydroxyl protecting group. Subsequent deprotection reactions can be performed under standard conditions. For example, benzyl can be deprotected using a palladium on carbon / hydrogen system; MOM protecting groups can be deprotected using an organic solution system of hydrogen chloride (organic solutions include but are not limited to: diethyl ether solution, 1,4-dioxane solution, methanol solution, ethanol solution, and isopropanol solution); THP and SEM protecting groups can be deprotected using a trifluoroacetic acid / dichloromethane system; and organosilicon groups can be deprotected using a tetrabutylammonium fluoride / tetrahydrofuran system. Any suitable carboxyl protecting group, such as those forming carboxylic acid ester groups (e.g., methyl carboxylate, ethyl carboxylate), can be used to protect the carboxyl group. Subsequent deprotection reactions can be performed under standard conditions, such as sodium hydroxide, potassium hydroxide, or lithium hydroxide in tetrahydrofuran, water, and / or methanol solvents. The above deprotection reaction is preferably carried out in the last step.

[0116] Pharmaceutically acceptable salts of the tri-heterocyclic derivative (I) can be synthesized by conventional chemical methods.

[0117] Generally, salts can be prepared by reacting a free base or acid with an equisional or excess amount of an acid (inorganic or organic) or a base (inorganic or organic) in a suitable solvent or solvent combination.

[0118] The present invention also provides a pharmaceutical composition comprising a therapeutically effective amount of an active ingredient and pharmaceutically acceptable excipients; said active ingredient comprising one or more of a triherocyclic derivative (I), its stereoisomers, or a pharmaceutically acceptable salt.

[0119] In the pharmaceutical composition, the active ingredient may also include other therapeutic agents for diseases related to ATR level abnormalities.

[0120] In the pharmaceutical composition, the pharmaceutically acceptable excipients may include pharmaceutically acceptable carriers, diluents, and / or excipients.

[0121] Depending on the therapeutic purpose, the pharmaceutical composition can be formulated into various types of dosage forms, such as tablets, pills, powders, liquids, suspensions, emulsions, granules, capsules, suppositories, and injections (solutions and suspensions), with liquids, suspensions, emulsions, suppositories, and injections (solutions and suspensions) being preferred.

[0122] To form a pharmaceutical composition in tablet form, any excipient known and widely used in the art may be used. Examples of carriers include lactose, white sugar, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, crystalline cellulose, and silica; binders include water, ethanol, propanol, common syrup, glucose solution, starch solution, gelatin solution, carboxymethyl cellulose, shellac, methyl cellulose, potassium phosphate, and polyvinylpyrrolidone; disintegrants include dry starch, sodium alginate, agar powder, and kelp powder, sodium bicarbonate, calcium carbonate, fatty acid esters of polyvinyl sorbitol, dodecyl sodium 2SO4, monoglyceride stearate, starch, and lactose; disintegration inhibitors include white sugar, glyceryl tristearate, coconut oil, and hydrogenated oil; adsorption promoters include quaternary ammonium base and dodecyl sodium 2SO4; wetting agents include glycerin and starch; adsorbents include starch, lactose, kaolin, bentonite, and colloidal silica; and lubricants include pure talc, stearates, boric acid powder, and polyethylene glycol. It can also be made into sugar-coated tablets, gelatin-coated tablets, sausage-coated tablets, coated tablets, double-layered tablets and multilayered tablets by selecting common coating materials as needed.

[0123] To form a pharmaceutical composition in pellet form, any excipient known and widely used in the art may be used, such as carriers, like lactose, starch, coconut oil, hardened vegetable oil, kaolin, and talc; binders, like gum arabic, tragacanth, gelatin, and ethanol; and disintegrants, like agar and kelp powder.

[0124] To form a pharmaceutical composition in suppository form, any excipient known and widely used in the art may be used, such as polyethylene glycol, coconut oil, higher alcohols, esters of higher alcohols, gelatin, and semi-synthetic glycerides.

[0125] To prepare a pharmaceutical composition for injection, the solution or suspension can be sterilized (preferably with the addition of appropriate amounts of sodium chloride, glucose, or glycerol) to create an injection with isotonicity similar to blood. Any commonly used carrier in the art can be used in the preparation of the injection. Examples include water, ethanol, propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, and fatty acid esters of polyvinyl sorbitol. Furthermore, common solvents, buffers, and analgesics can be added.

[0126] In this invention, the content of the composition in the pharmaceutical composition is not particularly limited and can be selected within a wide range, typically 5% to 95% by mass, and preferably 30% to 80% by mass.

[0127] In this invention, there are no particular limitations on the method of administration of the pharmaceutical composition. Various dosage forms can be selected for administration based on the patient's age, gender, other conditions, and symptoms. For example, tablets, pills, solutions, suspensions, emulsions, granules, or capsules can be administered orally; injections can be administered alone or mixed with an injection delivery solution (such as glucose solution and amino acid solution) for intravenous injection; suppositories are administered rectally.

[0128] The present invention also provides the use of the aforementioned triherocyclic derivative (I), its stereoisomers, or pharmaceutically acceptable salts, or the pharmaceutical composition thereof, in the preparation of an ATR inhibitor. The ATR inhibitor refers to one that can inhibit the activity or expression of ATR (including aberrant activity or overexpression of ATR).

[0129] The tri-heterocyclic derivative (I), its stereoisomers, or pharmaceutically acceptable salts provided by this invention, or the pharmaceutical composition thereof, have the effects of resisting tumor cell proliferation, promoting tumor cell apoptosis, and / or resisting tumor cell invasion. The effect of promoting tumor cell apoptosis is achieved by inhibiting ATR activity.

[0130] The present invention also provides the use of the triherocyclic derivative (I), its stereoisomers or pharmaceutically acceptable salts, or the pharmaceutical composition thereof in the preparation of medicaments for treating, alleviating and / or preventing ATR-mediated related diseases.

[0131] The present invention also provides the use of the triherocyclic derivative (I), its stereoisomers or pharmaceutically acceptable salts, or the pharmaceutical composition thereof in the preparation of therapeutic and / or alleviating cancer drugs.

[0132] The present invention also provides the use of the tri-heterocyclic derivative (I), its stereoisomers or pharmaceutically acceptable salts, or the pharmaceutical composition thereof in the preparation of a medicament having an antiproliferative effect in mammals.

[0133] The present invention also provides the use of the tri-heterocyclic derivative (I), its stereoisomers or pharmaceutically acceptable salts, or the pharmaceutical composition thereof in the preparation of a drug having a pro-apoptotic effect in mammals.

[0134] The present invention also provides the use of the tri-heterocyclic derivative (I), its stereoisomers or pharmaceutically acceptable salts, or the pharmaceutical composition thereof in the preparation of a medicament that has an effect against cancer cell invasion in mammals.

[0135] The present invention also provides the use of the tri-heterocyclic derivative (I), its stereoisomers or pharmaceutically acceptable salts, or the pharmaceutical composition thereof in the treatment and / or relief of cancer, comprising a compound of formula (I), its stereoisomers or pharmaceutically acceptable salts, or a pharmaceutical composition comprising a compound of formula (I), its stereoisomers or pharmaceutically acceptable salts, administered in a therapeutically effective dose to mammals.

[0136] The present invention also provides the triherocyclic derivative (I), its stereoisomers or pharmaceutically acceptable salts, or the pharmaceutical composition thereof in combination with one or more other kinds of therapeutic agents and / or treatment methods for treating, alleviating and / or preventing ATR-mediated diseases.

[0137] In this invention, the ATR-mediated related diseases are those caused by abnormal ATR levels, preferably proliferative diseases, and more preferably cancer.

[0138] In this invention, the other therapeutic agents for ATR-mediated related diseases are preferably other types of therapeutic agents used to treat cancer.

[0139] In this invention, the other types of therapeutic agents for treating cancer can be formulated into single-dose therapeutic formulations with the tri-heterocyclic derivative (I), or into therapeutic formulations that are administered sequentially.

[0140] In this invention, the other types of therapeutic agents for cancer treatment may include, but are not limited to: alkylating agents, topoisease I / II inhibitors, antimitotic agents, antimetabolites, hormones and hormone analogs, antitumor antibiotics, small molecule kinase inhibitors, small molecule immunomodulators, interferon, aromatase inhibitors, PARP inhibitors, antitumor vaccines, cytokines, chimeric antigen receptor T cells (CAR-T), monoclonal antibodies, and radiotherapy, or one or more of these.

[0141] In this invention, the alkylating agent may be selected from, but is not limited to, one or more of, the following: cisplatin, carboplatin, oxaliplatin, nedaplatin, nitrogen mustard, N-nitrogen mustard hydrochloride, cyclobutyrate nitrogen mustard, uracil nitrogen mustard, cyclophosphamide, ifosfamide, thiotepa, carboquinone, triaminoquinone, inprofentoxin tosylate, mannosulfan, triosulane, busulfan, nimustine hydrochloride, dibromomannitol, melphalan, dacarbazine, ramustine, carmustine, lomustine, streptozotocin, temozolomide, procarbazine, ethyleneimine derivatives, methanesulfonates, nitrosoureas, and triazines.

[0142] In this invention, the topozyme I / II inhibitor may be selected from, but is not limited to, one or more of: doxorubicin, daunorubicin, epirubicin, edabricin, irinotecan, topotecan, rubitecan, belotecone, etoposide, tiniposide, doxorubicin, dexrazoxane, and camptothecin.

[0143] In this invention, the antimitotic agents include, but are not limited to, one or more of the following: paclitaxel, docetaxel, polyglutamic acid paclitaxel, isovincalcin, vincristine, vinblastine, vinorelbine, etoposide, teniposide, ixaprilone, lalotaxetine, ortataxel, tesetaxel, tocosal, and ispinax.

[0144] In this invention, the antimetabolite drugs may be selected from, but are not limited to, folic acid antagonists, pyrimidine analogs, purine analogs, and adenosine deaminase inhibitors, such as methotrexate, 5-fluorouracil, fluorouracil, cytarabine, 6-mercaptopurine, 6-thioguanine, fludarabine phosphate, pentostatin, and gemcitabine.

[0145] In this invention, the hormone therapy agent may be selected from, but is not limited to, one or more of, the following: phosphostel, diethylstilbestrol, chlorestrol, medroxyprogesterone acetate, megestrol acetate, chlormedrone acetate, cyproterone acetate, danazol, dinogest, allylestradiol, gestrinone, nomenoprogesterone, ureaprol, mepertricin, raloxifene, olmexifen, zomeroxifene, anluminate, testosterone, anti-estrogens, LH-RH derivatives, aromatase inhibitors, anti-androgens, adrenocortical hormones, androgen synthesis inhibitors, retinoic acid, and drugs that delay retinoic acid metabolism.

[0146] In this invention, the antitumor antibiotics include, but are not limited to, one or more of the following: actinomycin D, doxorubicin, daunorubicin, bleomycin, pepromycin, mitomycin C, arubicin, pirarubicin, epirubicin, fenestrate, idarubicin, sirolimus, and pentorubicin.

[0147] In this invention, the small molecule kinase inhibitors include, but are not limited to: erlotinib, imatinib, apatinib, nilotinib, crizotinib, dasatinib, pazopanib, regorafenib, ruxolitinib, sorafenib, sunitinib, vandetanib, vemurafenib, bosutinib, gefitinib, afatinib, axitinib, dabrafenib, dacomitinib, nintedanib, lenvatinib, masatitinib, midotutolin, neratinib, panatinib, raditinib, trametinib, and alanine. One or more of the following: linibuprofen, sildenafil, cabozantinib malate, ibrutinib, icotinib, cipatinib, cobitinib, ederalipinib, ponatinib, alisertib, dinaciclib, linsitinib, orantinib, rigosertib, tipifarnib, tivozanib, pimasertib, buparlisib, and fedratinib.

[0148] In this invention, the anti-tumor vaccine includes, but is not limited to, synthetic peptides, DNA vaccines, and recombinant viruses.

[0149] In this invention, the cytokine therapy includes, but is not limited to, IL2 and GM-CSF.

[0150] In this invention, the monoclonal antibodies include, but are not limited to, one or more of the following: alenumab, bentoximab, cetuximab, rituximab, denosumab, ipilimumab, oflavin, panitumumab, tosimomab, trastuzumab, bevacizumab, pertuzumab, caputuzumab, elotoximab, ipraximab, nexituzumab, nimotoximab, tocilizumab, matoximab, zalumumab, atocuzumab, ramucirumab, nivolumab, mogamulizumab, ocaratuzumab, oregovomab, dalotuzumab, and onartuzumab.

[0151] In this invention, the small molecule immunomodulators include, but are not limited to, one or more of the following: TLR7 agonists, TLR8 agonists, TLR9 agonists, IDO inhibitors, CD73 inhibitors, STING inhibitors, and A2AR antagonists.

[0152] In this invention, the interferons used for cancer treatment include, but are not limited to: interferon α, interferon α-2a, interferon α-2b, interferon β, interferon γ-1a, or interferon γ-n1, etc.

[0153] In this invention, the aromatase inhibitors include, but are not limited to, one or more of anatozol, aminoglutethimide, exemestane, fazodazole, and letrozol.

[0154] In this invention, the PARP inhibitors include, but are not limited to, one or more of the following: Olaparib, Niraparib, Rucaparib, Veliparib, and SC10914.

[0155] In this invention, the cancer includes metastatic and non-metastatic cancers, as well as familial and incidental cancers, and may also include solid tumors and non-solid tumors.

[0156] In this invention, specific examples of solid tumors may include, but are not limited to: tumors of the eye, bone, lung, stomach, pancreas, breast, prostate, brain (including glioblastoma and medulloblastoma), ovary (including stromal cells, germ cells and stromal cells arising from epithelial cells), bladder, testis, spinal cord, kidney (including adenocarcinoma and nephroblastoma), mouth, lips, pharynx, oral cavity (including squamous cell carcinoma), nasal cavity, small intestine, colon, rectum, parathyroid gland, gallbladder, bile duct, cervix, heart, subpharyngeal gland, bronchus, liver, ureter, vagina, anus, laryngeal gland, thyroid gland (including thyroid cancer and medullary carcinoma), esophagus, nasopharyngeal gland, pituitary gland, salivary gland, adrenal gland, head and neck intraepithelial neoplasia (including Bowen's disease and Paget's disease), sarcoma (including leiomyosarcoma, rhabdomyosarcoma, liposarcoma, fibrosarcoma, osteosarcoma), skin (including melanoma, Kaposi's sarcoma, basocellular carcinoma and squamous cell carcinoma), and related tumors.

[0157] In this invention, the solid tumor is preferably one or more of the following: eye cancer, bone cancer, stomach cancer, pancreatic cancer, breast cancer, prostate cancer, brain cancer (including but not limited to malignant glioma and medulloblastoma), ovarian cancer, bladder cancer, cervical cancer, testicular cancer, kidney cancer (including but not limited to adenocarcinoma and nephroblastoma), oral cancer (including squamous cell carcinoma), tongue cancer, laryngeal cancer, nasopharyngeal cancer, head and neck cancer, colon cancer, small intestine cancer, rectal cancer, parathyroid cancer, thyroid cancer, esophageal cancer, gallbladder cancer, bile duct cancer, cervical cancer, liver cancer, lung cancer (including but not limited to small cell lung cancer and non-small cell lung cancer), choriocarcinoma, osteosarcoma, Ewing's tumor, soft tissue sarcoma, and skin cancer.

[0158] In this invention, specific examples of non-solid tumors (including hematologic tumors) may include, but are not limited to, one or more of the following: lymphocytic leukemia (including lymphoblastic leukemia, lymphoma, myeloma, chronic lymphocytic leukemia (T-cell chronic lymphocytic leukemia, B-cell chronic lymphocytic leukemia), Hodgkin lymphoma, and non-Hodgkin lymphoma), myeloid-associated leukemia (including acute myeloid leukemia and chronic myeloid leukemia), and AIDs-associated leukemia.

[0159] In this invention, the cancer is preferably one or more of the following: non-small cell lung cancer, small cell lung cancer, gastric cancer, esophageal cancer, melanoma, colon cancer, pancreatic cancer, breast cancer, uterine cancer, ovarian cancer, prostate cancer, brain cancer, bladder cancer, kidney cancer, myeloma, liver cancer, acute myeloid leukemia, chronic myeloid leukemia, lymphoblastic leukemia, chronic lymphocytic leukemia, and lymphoma.

[0160] In this invention, the mammal is preferably a human.

[0161] In this invention, "tumor" and "cancer" have the same meaning.

[0162] In this invention, unless otherwise stated, the term "substituted at any position by one or more groups" means that any one or more hydrogen atoms of one or more atoms specified on the group are substituted by the specified group, provided that the substitution does not exceed the normal valence of the specified atom. All such substitutions are reasonable substitutions commonly found in the art. For example: R a Selective substitution of 1 to 3 groups at any position refers to R a It can be reasonably substituted at any position by one, two, or three identical or different substituents.

[0163] In this invention, any combination of variables is permitted only if such a combination produces a stable compound; for example, V is NR6 or CR7; V1 is N or NR. 6a or CR 7a V2 represents N and NR. 6b or CR 7b V3 is the connector key, N, NR 6c or CR 7c At that time, V, V1, V2, and V3 include any of the following stable combinations: 1) V is NR6, V1 is N or CR. 7a V2 is N or CR 7b 1) V3 is the connection key; 2) V is CR7, V1 is N or CR 7a V2 is N or CR 7b 1) V3 is the connection key; 2) V is CR7, V1 is N or CR 7a V2 is N or CR 7b V3 is N or CR 7c ;4) V is CR7, V1 is N or CR 7a V2 is NR 6b V3 is the connection key; or 5) V is CR7, V1 is NR. 6a V2 is N or CR 7b V3 is the connection key.

[0164] In this invention, when any variable appears more than once in the composition or structure of a compound, its definition is independent in each case.

[0165] In this invention, unless otherwise specified, the cyclic group contains This indicates that the cyclic group is an aromatic ring or a non-aromatic ring; the cyclic group contains This indicates that the cyclic group is an aromatic ring. For example: [group name missing] It is a 5-6 membered aromatic ring or a 5-6 membered non-aromatic ring; the definitions of X, X1, X2, and X3 are as described above. (Group) It consists of 5-6 membered aromatic rings; the definitions of X, X1, X2 and X3 are as described above.

[0166] Unless otherwise stated, the following terms appearing in this specification and claims have the following meanings:

[0167] The term "alkyl" refers to a saturated straight-chain or branched hydrocarbon group containing 1 to 20 carbon atoms, preferably 1 to 10 carbon atoms, more preferably 1 to 8, 1 to 6, or 1 to 4 carbon atoms. Representative examples of alkyl groups include, but are not limited to: methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, isobutyl, n-pentyl, n-hexyl, n-heptyl, octyl, nonyl, decyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1 1-Dimethylbutyl, 1,2-Dimethylbutyl, 2,2-Dimethylbutyl, 1,3-Dimethylbutyl, 2,3-Dimethylbutyl, 2-Ethylbutyl, 2-Methylpentyl, 3-Methylpentyl, 4-Methylpentyl, 4,4-Dimethylpentyl, 2-Methylhexyl, 3-Methylhexyl, 4-Methylhexyl, 5-Methylhexyl, 2,3-Dimethylpentyl, 2,4-Dimethylpentyl, 2,2-Dimethylpentyl, 3,3-Dimethylpentyl, 2-Ethylpentyl, 3-Ethylpentyl, 2,2,4-Trimethylpentyl, Undecyl, Dodecyl, and their various isomers, etc.

[0168] The term "cycloalkyl" refers to a monocyclic or fused cyclic group containing 3-20 carbon atoms, either saturated or partially unsaturated (containing one or two double bonds). "Monocyclic cycloalkyl" is preferably a 3-10 member monocyclic cycloalkyl, more preferably a 3-8 or 3-6 member monocyclic cycloalkyl. Examples of cycloalkyl groups include, but are not limited to: cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclodecyl, cyclododecyl, cyclohexenyl, 2,3-dihydro-1-H-indenyl, decahydronaphthalene, etc. The cycloalkyl group can be linked to the parent molecule via any carbon atom on the ring.

[0169] The term "heterocyclic alkyl" refers to a 3-20 member non-aromatic cyclic group consisting of a carbon atom and heteroatoms selected from nitrogen, oxygen, or sulfur, and is either saturated or partially unsaturated (containing one or two double bonds). This cyclic group can be monocyclic or fused. In this invention, the number of heteroatoms in the heterocyclic alkyl group is preferably 1, 2, 3, or 4. The nitrogen, carbon, or sulfur atoms in the heterocyclic alkyl group may optionally be oxidized. The nitrogen atom may optionally be further substituted by other groups to form a tertiary amine or quaternary ammonium salt. The heterocyclic alkyl group is preferably a 3-10 member monocyclic heterocyclic alkyl group, more preferably a 3-6 member monocyclic heterocyclic alkyl group. Examples of heterocyclic alkyl groups include, but are not limited to: aziridinyl, tetrahydrofuran-2-yl, morpholin-4-yl, thiomorpholin-4-yl, thiomorpholin-S-oxide-4-yl, piperidin-1-yl, N-alkylpiperidin-4-yl, pyrrolidine-1-yl, N-alkylpyrrolidine-2-yl, piperazine-1-yl, 4-alkylpiperazine-1-yl, etc. The heterocyclic alkyl groups can be linked to the parent molecule via any ring atom. The aforementioned ring atoms specifically refer to the carbon and / or nitrogen atoms that form the ring skeleton.

[0170] The terms “non-aromatic group” or “non-aromatic ring” refer to “cycloalkyl” and / or “heterocyclic alkyl”, including the above definitions of cycloalkyl and / or heterocyclic alkyl.

[0171] The term "cycloalkylalkyl" refers to a cycloalkyl group that is linked to the parent structure via an alkyl group. Therefore, "cycloalkylalkyl" encompasses the definitions of alkyl and cycloalkyl groups described above.

[0172] The term "heterocyclic alkyl alkyl" refers to a heterocyclic alkyl group that is linked to the parent core structure via an alkyl group. Therefore, "heterocyclic alkyl alkyl" encompasses the definitions of alkyl and heterocyclic alkyl groups described above.

[0173] The term "alkoxy" refers to a cyclic or acyclic alkyl group having the stated number of carbon atoms connected by an oxygen bridge, including alkyloxy, cycloalkyloxy, and heterocyclic alkyloxy groups. Thus, "alkoxy" encompasses the definitions of alkyl, heterocyclic alkyl, and cycloalkyl groups described above.

[0174] The term "alkenyl" refers to a straight-chain, branched, or cyclic non-aromatic hydrocarbon group containing at least one carbon-carbon double bond. It may contain 1-3 carbon-carbon double bonds, preferably 1. The term "C"... 2-4 "Alkenyl" refers to an alkenyl group having 2-4 carbon atoms; the term "C" is used to indicate this. 2-6 "Alkenyl" refers to an alkenyl group having 2-6 carbon atoms, including vinyl, propenyl, butenyl, 2-methylbutenyl, and cyclohexenyl.

[0175] The term "alkynyl" refers to a straight-chain, branched, or cyclic hydrocarbon group containing at least one carbon-carbon triple bond. It may contain 1-3 carbon-carbon triple bonds, preferably 1. The term "C"... 2-6"Alynyl" refers to an alkynyl group with 2-6 carbon atoms, including ethynyl, propynyl, butynyl and 3-methylbutynyl.

[0176] The term "aryl" refers to any stable 6-10 member monocyclic or fused aromatic group, wherein at least one ring of the fused aromatic group is a benzene ring, and the remaining rings may be benzene rings, monocyclic cycloalkyl groups, or monocyclic heterocyclic alkyl groups. The aryl groups include, but are not limited to: phenyl, naphthyl, tetrahydronaphthyl, 2,3-dihydroindene, biphenyl, benzo[d][1,3]dioxolane, indolinyl, isoindolinyl, 2,3-dihydrobenzofuranyl, 2,3-dihydrobenzo[b]thiophene, benzopyranyl, 1,2,3,4-tetrahydroquinolinyl, 1,2,3,4-tetrahydroisoquinolinyl, 2,2-dioxide-1,3-dihydrobenzo[c]isothiazolyl, 1,1-dioxide-2,3-dihydrobenzo[b]thiophene, 1-imino-1-oxo-2,3-dihydrobenzo[b]thiophene, and 2-oxo-2,3-dihydro-1H-benzo[d]imidazolyl.

[0177] The term "heteroaryl" refers to an aromatic ring group formed by replacing at least one carbon atom in a ring with a heteroatom selected from nitrogen, oxygen, or sulfur. This group can be a 5-7 membered monocyclic structure or a 7-12 membered fused ring structure, wherein at least one ring in the fused ring structure is a heteroaryl group, and the remaining rings can optionally be aromatic rings, heteroaromatic rings, cycloalkyl groups, or heterocycloalkyl groups. In this invention, the number of heteroatoms is preferably 1, 2, 3, or 4, and the nitrogen atom in the heteroaryl group can optionally be oxidized. The heteroaryl group is preferably a 5-10 membered heteroaryl group, including but not limited to: pyridinyl, pyrimidinyl, pyrazinyl, pyridazin-3(2H)-keto, furanyl, thiopheneyl, thiazolyl, pyrroleyl, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, 1,2,5-oxadiazolyl, 1,2,4-oxadiazolyl, 1H-1,2,4-triazolyl, 4H-1,2,4-triazolyl, 1H-1,2,3-triazolyl, 1H-tetrazole, 1H-indazoleyl, 1H-pyrazolo[3,4-b]pyridinyl, 1H-pyrazolo[3,4-c]pyridinyl, 1H-pyrazolo[4,3-c]pyridinyl Pyridyl, 1H-indolyl, 1H-benzimidazolyl, 1H-benzofuranyl, benzothiophenyl, benzothiazolyl, benzooxazolyl, quinolinyl, isoquinolinyl, quinazolinyl, 1H-pyrrolo[3,2-c]pyridyl, 1H-pyrrolo[2,3-c]pyridyl, 1H-pyrrolo[2,3-b]pyridyl, 2,3-dihydro-1H-pyrrolo[2,3-c]pyridyl, 2,3-dihydro-1H-pyrrolo[2,3-b]pyridyl, 7H-pyrrolo[2,3-d]pyrimidinyl or 7-oxo-6,7-dihydro-1H-pyrrolo[2,3-c]pyridyl.

[0178] The term “aromatic group” or “aromatic ring” refers to “aryl” and / or “heteroaryl”, encompassing the above definitions of aryl and / or heteroaryl.

[0179] The term "arylalkyl" refers to an aryl group that is linked to the parent structure via an alkyl group. Therefore, "arylalkyl" encompasses the definitions of alkyl and aryl groups mentioned above.

[0180] The term "heteroarylalkyl" refers to a heterocyclic alkyl group that is linked to the parent nucleus via an alkyl group. Therefore, "heteroarylalkyl" encompasses the definitions of alkyl and heteroaryl groups mentioned above.

[0181] The term "halogen" refers to fluorine, chlorine, bromine, or iodine.

[0182] The term "halogenated alkyl" refers to an alkyl group that has been arbitrarily substituted with a halogen. Thus, "halogenated alkyl" encompasses the definitions of halogen and alkyl group mentioned above.

[0183] The term "haloalkoxy" refers to an alkoxy group that has been substituted with a halogen in any way. Therefore, "haloalkoxy" encompasses the definitions of halogen and alkoxy group mentioned above.

[0184] The term "amino" refers to -NH2, and the term "alkamino" refers to an amino group in which at least one hydrogen atom is replaced by an alkyl group, including but not limited to: -NHCH3, -N(CH3)2, -NHCH2CH3, -N(CH2CH3)2, and -N(CH3)(CH2CH3). Thus, "alkamino" encompasses the definitions of alkyl and amino groups mentioned above.

[0185] The term "nitro" refers to -NO2.

[0186] The term "cyano" refers to -CN.

[0187] The term "carboxyl group" refers to -C(O)OH.

[0188] The symbol “=" represents a double bond.

[0189] The "room temperature" mentioned in this invention refers to 15-30℃.

[0190] The “pharmaceutically acceptable salts” described in this invention are discussed in Berge, et al., “Pharmaceutically Acceptable Salts”, J. Pharm. Sci., 66, 1-19 (1977), and are obvious to medicinal chemists that the salts are substantially nontoxic and provide the desired pharmacokinetic properties, palatability, absorption, distribution, metabolism, or excretion. The compounds described in this invention may have acidic, basic, or amphoteric groups. Typical pharmaceutically acceptable salts include those prepared by reacting the compounds of this invention with acids, such as: hydrochloride, hydrobromide, sulfate, pyrosulfate, hydrogen sulfate, sulfite, bisulfite, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate, nitrate, acetate, propionate, decanoate, octanoate, formate, acrylate, isobutyrate, hexanoate, heptanoate, oxalate, malonate, succinate, octanoate, benzoate, methylbenzoate, phthalate, maleate, methanesulfonate, p-toluenesulfonate, (D,L)-tartaric acid, citric acid, maleic acid, (D,L)-malic acid, fumaric acid, succinic acid, succinate, lactate, trifluoromethanesulfonate, naphthalene-1-sulfonate, mandelate, pyruvate, stearate, ascorbate, and salicylate. When the compounds of the present invention contain acidic groups, their pharmaceutically acceptable salts may also include: alkali metal salts, such as sodium or potassium salts; alkaline earth metal salts, such as calcium or magnesium salts; organic base salts, such as salts formed with ammonia, alkylamines, hydroxyalkylamines, amino acids (lysine, arginine), N-methylglucosamine, etc.

[0191] The term "isomer" as used in this invention refers to compounds of formula (I) that can have asymmetric centers and racemic mixtures, racemic mixtures, and single diastereomers, all of which include stereoisomers, geometric isomers, and transisomers. In this invention, when a compound of formula (I) or a salt thereof exists in a stereoisomeric form (e.g., containing one or more asymmetric carbon atoms), individual stereoisomers (enantiomers and diastereomers) and mixtures thereof are included within the scope of this invention. This invention also includes individual isomers of compounds or salts represented by formula (I), and mixtures of isomers with inverted chiral centers. The scope of this invention includes mixtures of stereoisomers, and purified enantiomers or mixtures enriched with enantiomers / diastereomers. This invention includes mixtures of stereoisomers of all possible different combinations of all enantiomers and diastereomers. This invention includes all combinations and subsets of stereoisomers of all specific groups defined above. The present invention also includes geometric isomers of the compound of formula (I) or salts thereof, said geometric isomers including cis-trans isomers.

[0192] Without violating common sense in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0193] The reagents and raw materials used in this invention are all commercially available. Attached Figure Description

[0194] Appendix Figure 1 The curves show the tumor volume changes of compound 2 (5 mg / kg, 10 mg / kg, 20 mg / kg, po) and the positive control AZD6738 (20 mg / kg, po) in the OCI-LY19 human B-cell lymphoma mouse subcutaneous xenograft model. Detailed Implementation

[0195] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0196] The abbreviations used in this embodiment of the invention have the following meanings:

[0197] The structures of all compounds in this invention can be determined by nuclear magnetic resonance (NMR). 1 Identification by 1H NMR and / or mass spectrometry (MS).

[0198] 1 10 H NMR chemical shifts (δ) were recorded in pPM (10 -6 NMR was performed using a Bruker AVANCE-400 spectrometer. Suitable solvents were deuterated chloroform (CDCl3), deuterated methanol (CD3OD), and deuterated dimethyl sulfoxide (DMSO-d6), with tetramethylsilane as an internal standard (TMS).

[0199] Low-resolution mass spectrometry (MS) was performed using an Ultimate 3000 HPLC-MSQ Plus MS mass spectrometer with a Kinetex 2.6u C18 100A (50 × 4.6 mm) LCMS-02-001 ESI source. Gradient elution conditions were used: 95% solvent A and 5% solvent B (less than 1.5 min or more than 3 min), followed by 5% solvent A and 95% solvent B (1.5 min to 3 min). Percentages represent the volume percentage of a particular solvent in the total solvent volume. Solvent A: 10 mM NH4HCO3 (aq); Solvent B: acetonitrile.

[0200] The compounds and intermediates of this invention can be purified using conventional preparative silica gel stencils or rapid separators. The elution system can be an EtOAc / PE system or a DCM / MeOH system. Alternatively, preparative HPLC can be used for separation.

[0201] High-performance liquid chromatography (prep-HPLC) was performed using a SHIMADZU LC-20 preparative HPLC system with a Waters Xbridge Pre C18 column (10 μm, 19 × 260 mm). Alkaline elution gradient was used with mobile phase B: 15–70% (v / v%), elution time 20 min, mobile phase A: 10 mM NH4HCO3 (aq), mobile phase B: acetonitrile. Acidic gradient elution was used with mobile phase B: 15%–55% (v / v%), elution time 20 min, mobile phase A: 0.1% trifluoroacetic acid aqueous solution, mobile phase B: acetonitrile. Detection wavelengths were 214 nm, and / or 254 nm, and / or 262 nm; flow rate was 10.0 mL / min.

[0202] The microwave reaction described in this embodiment of the invention uses The initiator+Microwave System EU(356006) type microwave reactor. Unless otherwise specified, all reactions in the embodiments of this invention are carried out under a nitrogen or argon atmosphere.

[0203] The thin-layer silicone sheet (prep-TLC) is either Yantai Huanghai HSGF254 or Qingdao GF254 silicone sheet.

[0204] Rapid separation machine (flash column chromatography) (flash system / Cheetah) TM The system used was Agela Technologies MP200, and the matching separation column was Flash column Silica-CS (80g), Cat No. CS140080-0.

[0205] The hydrogen atmosphere of the present invention can be achieved by the following methods: 1) connecting a hydrogen balloon with a volume of about 1L to the reaction system; 2) continuously introducing hydrogen directly into the reaction system under normal pressure; 3) replacing the hydrogen with a sealing tube and then sealing it.

[0206] Unless otherwise specified, all reactions in this invention are carried out under nitrogen or argon protection.

[0207] Example 1: Synthesis of (R)-3-methyl-4-(1-(methanesulfonyl)-6-(1H-pyrazol-3-yl)-1,6-dihydropyrazolo[3,4-b]pyrrolo[2,3-d]pyridin-4-yl)morpholine trifluoroacetate (compound 1)

[0208]

[0209]

[0210] Step 1: At -70°C, a tetrahydrofuran solution of lithium diisopropylamino in 2.2 mL (2.0 M, 4.46 mmol) was added dropwise to a tetrahydrofuran solution of 10 mL of 2,6-difluoro-4-iodopyridine (1.0 g, 4.15 mmol). The reaction system was stirred at this temperature for 30 minutes. Methyl formate (412 mg, 5.58 mmol) was added to the above reaction system, and stirring was continued for 1 hour. The reaction was quenched with water, and the aqueous phase was extracted with ethyl acetate. The organic phase was separated and concentrated under reduced pressure. The residue was purified by Flash column chromatography (ethyl acetate / petroleum ether = 0–1 / 4) to give compound 1.1 (300 mg, yield: 27%) as a yellow solid.

[0211] Step 2: Add 3-hydrazino-1H-pyrazole (91 mg, 0.93 mmol) to a 95% ethanol (5 mL) solution of compound 1.1 (250 mg, 0.93 mmol), and stir the reaction mixture at room temperature for 2 hours. Quench the reaction by slowly adding a saturated sodium bicarbonate aqueous solution to the reaction system under ice-water bath conditions. Extract the aqueous phase with ethyl acetate, separate the organic phase, and concentrate under reduced pressure to obtain compound 1.2 (130 mg, yield: 40%) as a yellow solid.

[0212] Step 3: A solution of compound 1.2 (130 mg, 0.37 mmol) in 2 mL of N-methylpyrrolidone was microwaved at 200 °C for 15 minutes. The reaction solution was then poured directly into water and filtered. The filter cake was dried under vacuum until compound 1.3 (150 mg, crude product) was obtained as a yellow solid. m / z: [M+H] + 330.0.

[0213] Step 4: Add (R)-3-methylmorpholine (48 mg, 0.48 mmol) to a dimethyl sulfoxide (DMSO) solution of compound 1.3 (80 mg, 0.24 mmol). Stir the reaction mixture at 145 °C for 1 hour. Then pour the reaction mixture into water and filter. After drying the filter cake, compound 1.4 (70 mg, yield: 71%) is a yellow solid. m / z: [M+H] + 411.0.

[0214] Step 5: Under ice bath conditions, sodium hydroxide (60%, 14 mg, 0.35 mmol) was added to a tetrahydrofuran (3 mL) solution of compound 1.4 (130 mg, 0.32 mmol). The reaction solution was stirred at 0 °C for 30 minutes. 2-(trimethylsilyl)ethoxymethyl chloride (73 mg, 0.44 mmol) was added to the above reaction solution and stirred at room temperature for 2 hours. The reaction was quenched with water. The aqueous phase was extracted with ethyl acetate, the organic phase was separated and concentrated under reduced pressure, and the residue was purified by Flash column chromatography (ethyl acetate / petroleum ether = 0–1 / 3) to give compound 1.5 (90 mg, yield: 52%) as a yellow oil. m / z: [M+H] + 541.3.

[0215] Step 6: To a solution of compound 1.5 (26 mg, 0.05 mmol) in 1,4-dioxane (3 mL), aminoacetaldehyde dimethyl acetal (26 mg, 0.25 mmol), tris(dibenzylacetone)dipalladium (9 mg, 0.01 mmol), S-(+)-1,1'-binaphthyl-2,2'-bis(diphenylphosphine) (6 mg, 0.01 mmol), and cesium carbonate (32.5 mg, 0.1 mmol) were added sequentially. The reaction system was purged with nitrogen and stirred at 120 °C for 3 hours under a nitrogen atmosphere. The reaction solution was directly concentrated under reduced pressure, and the residue was purified by Flash column chromatography (ethyl acetate / petroleum ether = 0–1 / 1) to give compound 1.6 (40 mg, yield: 78%) as a yellow oil. m / z: [M+H] + 518.2.

[0216] Step 7: Under ice bath conditions, boron trifluoride diethyl ether (62 mg, 0.44 mmol) was added to a dichloromethane (3 mL) solution of compound 1.6 (150 mg, 0.29 mmol). The reaction mixture was stirred at 0 °C for 30 minutes, then quenched with saturated sodium bicarbonate aqueous solution. The aqueous phase was extracted with dichloromethane, and the organic phases were combined and concentrated under reduced pressure. The residue was purified by Flash column chromatography (ethyl acetate / petroleum ether = 1 / 3) to give compound 1.7 (17 mg, yield: 13%) as a yellow oil. m / z: [M+H] + 454.2.

[0217] Step 8: Under ice bath conditions, sodium hydroxide (60%, 2.4 mg, 0.06 mmol) was added to a tetrahydrofuran (2 mL) solution of compound 1.7 (17 mg, 0.04 mmol). The reaction mixture was stirred at 0 °C for 30 minutes. Methanesulfonyl chloride (6.8 mg, 0.06 mmol) was then added to the reaction mixture, and the mixture was stirred at room temperature for 2 hours. The reaction was quenched with water, and the aqueous phase was extracted with ethyl acetate. The combined organic phases were concentrated under reduced pressure to give compound 1.8 (20 mg, crude product) as a yellow oil. m / z: [M+H]+ 532.2.

[0218] Step 9: Under ice bath conditions, triethylsilane (33 mg, 0.29 mmol) and trifluoroacetic acid (0.5 mL) were added to a solution of compound 1.8 (20 mg, crude) in dichloromethane (0.7 mL). The reaction mixture was stirred at room temperature for 1 hour, then concentrated under reduced pressure. The residue was purified by prep-HPLC (acidic conditions) to give compound 1 (1.16 mg, two-step yield: 6%) as a gray solid. m / z: [M+H] + 402.1; 1 H NMR (400MHz, CDCl3): δ8.32(s,1H),7.86(d,J=2.4Hz,1H),7.56(d,J=3.6Hz,1H),7.12(d,J=4.0Hz,1H) ,6.86(d,J=2.4Hz,1H),4.56-4.54(m,1H),3.98-3.95(m,2H),3.81-3.53(m,7H),1.26(d,J=6.8Hz,3H).

[0219] Example 2: Synthesis of (R)-3-methyl-4-(1-(methanesulfonyl)-6-(1H-pyrazol-3-yl)-1,2,3,6-tetrahydropyrazolo[3,4-b]pyrrolo[2,3-d]pyridin-4-yl)morpholine trifluoroacetate (compound 2)

[0220]

[0221] Step 1: A solution of 2,6-difluoro-4-iodopyridine (4.0 g, 16.6 mmol) and (R)-3-methylmorpholine (1.68 g, 16.6 mmol) in dimethyl sulfoxide (30 mL) was stirred at 100 °C for 5 hours. After cooling to room temperature, the reaction was quenched with water. The aqueous phase was extracted with ethyl acetate. The organic phases were combined and washed successively with water and saturated brine. The organic phase was separated and dried over anhydrous sodium sulfate. The mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by Flash column chromatography (ethyl acetate / petroleum ether = 0–3 / 2) to give compound 2.1 (4.4 g, yield: 82%) as a colorless oil. m / z: [M+H] + 323.0.

[0222] Step 2: A mixture of compound 2.1 (4.4 g, 13.6 mmol), aminoacetaldehyde dimethyl acetal (7.2 g, 68.0 mmol), tris(dibenzylacetone)palladium (576 mg, 0.68 mmol), S-(+)-1,1'-binaphthyl-2,2'-diphenylphosphine (396 mg, 0.63 mmol), cesium carbonate (5.9 g, 18.2 mmol), and 1,4-dioxane (30 mL) was purged three times with nitrogen. The reaction mixture was stirred at 100 °C for 5 hours under nitrogen atmosphere. After cooling to room temperature, the mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by Flash column chromatography (ethyl acetate / petroleum ether = 0–1 / 1) to give compound 2.2 (3.0 g, yield: 74%) as a yellow oil. m / z: [M+H] + 300.2.

[0223] Step 3: At -10°C, a solution of compound 2.2 (3.0 g, 10.0 mmol) in dichloromethane (10 mL) was added to a suspension of aluminum trichloride (5.3 g, 40.0 mmol) in dichloromethane (40 mL), and the mixture was stirred at -10°C for 20 minutes. The reaction was quenched with water, and the aqueous phase was extracted with dichloromethane. The combined organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by Flash column chromatography (ethyl acetate / petroleum ether = 0–1 / 3) to give compound 2.3 (1.56 g, yield: 66%) as a yellow solid. m / z: [M+H] + 236.2.

[0224] Step 4: Sodium cyanoborohydride (1.49 g, 23.7 mmol) was added to a solution of compound 2.3 (2.8 g, 11.8 mmol) in acetic acid (25 mL). The resulting mixture was stirred at room temperature for 9 hours. The reaction solution was slowly poured into a saturated aqueous sodium bicarbonate solution. The aqueous phase was extracted with ethyl acetate. The combined organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by Flash column chromatography (ethyl acetate / petroleum ether = 0–1 / 4) to give compound 2.4 (1.65 g, yield: 59%) as a yellow oil. m / z: [M+H] + 238.2.

[0225] Step 5: Under ice bath conditions, methanesulfonyl chloride (1.0 mL) was added to a pyridine (5.0 mL) solution of compound 2.4 (1.6 g, 6.72 mmol). The reaction mixture was stirred at 0 °C for 1 hour. The reaction was quenched with water, and the aqueous phase was extracted with ethyl acetate. The combined organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by Flash column chromatography (ethyl acetate / petroleum ether = 0–1 / 2) to give compound 2.5 (1.7 g, yield: 80%) as a yellow oil. m / z: [M+H]+ 316.2.

[0226] Step 6: Hexamethylenetetramine (3.3 g, 23.5 mmol) was added to a trifluoroacetic acid (20 mL) solution of compound 2.5 (1.86 g, 5.9 mmol). The mixture was stirred at 70 °C for 1 hour. The reaction was quenched with water, and the aqueous phase was extracted with ethyl acetate. The combined organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by Flash column chromatography (ethyl acetate / petroleum ether = 0–1 / 2) to give compound 2.6 (0.24 g, yield: 12%) as a yellow solid. m / z: [M+H] + 344.2.

[0227] Step 7: Add 3-hydrazino-1H-pyrazole (0.34 g, 3.49 mmol) to a 95% ethanol (5 mL) solution of compound 2.6 (0.24 g, 0.7 mmol). Stir the reaction mixture at room temperature for 20 minutes. Quench the reaction with water, extract the aqueous phase with ethyl acetate, combine the organic phases, dry with anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure to give compound 2.7 (0.3 g, crude product) as a yellow solid. m / z: [M+H] + 424.2.

[0228] Step 8: A solution of compound 2.7 (0.3 g, crude) in N-methylpyrrolidone (4 mL) was microwaved at 180 °C for 20 min. The reaction was quenched with water, the aqueous phase was extracted with ethyl acetate, the combined organic phases were dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the residue was purified by prep-HPLC (acidic conditions) to give compound 2 (136 mg, two-step yield: 38%) as a yellow solid. m / z: [M+H] + 404.2; 1 H NMR (400MHz, DMSO-d6): δ8.24(s,1H),7.84(d,J=4.0Hz,1H),6.83(d,J=4.0Hz,1H),4.20-4.02(m,3H ),3.94-3.88(m,1H),3.76-3.66(m,3H),3.41-3.28(m,2H),3.24-3.10(m,5H),1.18(d,J=8.0Hz,3H).

[0229] Example 3: Synthesis of (R)-N,N-dimethyl-4-(3-methylmorpholine)-6-(1H-pyrazol-3-yl)pyrazolo[3,4-b]pyrrolo[2,3-d]pyridine-1(6H)-sulfonamide trifluoroacetate (compound 3)

[0230]

[0231] Using the synthetic method for compound 1, replacing the methanesulfonyl chloride in step 8 with dimethylaminosulfonyl chloride yields compound 3, a grayish-white solid. m / z: [M+H] + 431.2; 1 H NMR (400MHz, CDCl3): δ8.55(s,1H),7.81(s,1H),7.60-7.56(m,1H),7.04-7.02(m,2H),4.65(m,1 H), 4.05-4.03 (m, 2H), 3.95-3.91 (m, 1H), 3.81-3.78 (m, 4H), 2.89 (s, 6H), 1.41 (d, J = 6.4Hz, 3H).

[0232] Example 4: Synthesis of (R)-N,N-dimethyl-4-(3-methylmorpholine)-6-(1H-pyrazol-3-yl)-2,3-dihydropyrazolo[3,4-b]pyrrolo[2,3-d]pyridine-1(6H)-sulfonamide trifluoroacetate (compound 4)

[0233]

[0234] Step 1: Under ice bath conditions, sodium hydrogen (136 mg, 3.4 mmol) was added to a tetrahydrofuran (2 mL) solution of compound 2.3 (0.4 g, 1.7 mmol), and the reaction mixture was stirred at 0 °C for 1 hour. Then, dimethylaminosulfonyl chloride (0.4 g, 3.4 mmol) was added to the above reaction mixture, and the mixture was stirred at room temperature for 2 hours. The reaction was quenched by adding saturated ammonium chloride aqueous solution, and the aqueous phase was extracted with ethyl acetate. The combined organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by Flash column chromatography (ethyl acetate / petroleum ether = 0–1 / 1) to give compound 4.1 (0.45 g, yield: 77%) as a yellow oil. m / z: [M+H] + 343.2.

[0235] Step 2: Compound 4.1 (0.45 g, 1.3 mmol) was added to a boranetetrahydrofuran complex (5 mL, 5.0 mmol), and the mixture was stirred at 80 °C for 1 hour. After cooling to room temperature, methanol (5 mL) was added, and the reaction mixture was refluxed and stirred for 48 hours. The reaction solution was concentrated under reduced pressure, and the residue was purified by Flash column chromatography (ethyl acetate / petroleum ether = 0–2 / 1) to give compound 4.2 (0.1 g, yield: 22%) as a yellow solid. m / z: [M+H] + 345.2.

[0236] Step 3: Under ice bath conditions, phosphorus oxychloride (0.5 mL) was slowly added dropwise to N,N-dimethylformamide (2.0 mL). The reaction mixture was stirred for 30 minutes. Then, compound 4.2 (80 mg, 0.23 mmol) was added to the above reaction mixture, and the reaction system was stirred at 80 °C for 2 hours. The reaction was quenched with water, and the aqueous phase was extracted with ethyl acetate. The combined organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give compound 4.3 (39 mg, yield: 44%) as a yellow solid. m / z: [M+H] + 373.2.

[0237] Step 4: Add 50 mg (0.51 mmol) of 3-hydrazino-1H-pyrazole to a 3 mL (95%) ethanol solution of compound 4.3 (38 mg, 0.1 mmol). Stir the reaction mixture at room temperature for 20 minutes. Quench the reaction with water, extract the aqueous phase with ethyl acetate, combine the organic phases, dry with anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure to give compound 4.4 (38 mg, crude product) as a yellow solid. m / z: [M+H] + 453.2.

[0238] Step 5: A solution of compound 4.4 (38 mg, crude) in N-methylpyrrolidone (2 mL) was microwaved at 180 °C for 20 min. The reaction was quenched with water, the aqueous phase was extracted with ethyl acetate, the combined organic phases were dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the residue was purified by prep-HPLC (acidic conditions) to give compound 4 (5.2 mg, two-step yield: 9%) as a pale yellow solid. m / z: [M+H] + 433.2; 1 H NMR (400MHz, DMSO-d6): δ8.39(s,1H),7.69(s,1H),6.89(s,1H),4.25-3.97(m, 4H), 3.75-3.55 (m, 5H), 3.32-3.12 (m, 2H), 2.96 (s, 6H), 1.26 (d, J = 8.0Hz, 3H).

[0239] Example 5: Synthesis of (R)-4-(3-methylmorpholine)-6-(1H-pyrazol-3-yl)-2,3-dihydropyrazolo[3,4-b]pyrrolo[2,3-d]pyridine-1(6H)-sulfonamide (compound 5)

[0240]

[0241]

[0242] Step 1: Under ice bath conditions, sodium hydroxide (80 mg, 2.0 mmol) was added to a tetrahydrofuran (10 mL) solution of compound 2.4 (238 mg, 1.0 mmol). The reaction system was stirred at this temperature for 0.5 hours, then benzyl chloroformate (340 mg, 2.0 mmol) was added. The reaction solution was stirred overnight at room temperature, and the reaction was quenched with water. The aqueous phase was extracted with ethyl acetate, the organic phases were combined and dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by Flash column chromatography (ethyl acetate / petroleum ether = 0–1 / 2) to give compound 5.1 (250 mg, yield: 67%) as a yellow solid. m / z: [M+H] + 372.2.

[0243] Step 2: Phosphorus oxychloride (0.5 mL) was slowly added dropwise to N,N-dimethylformamide (2 mL) of compound 5.1 (150 mg, 0.4 mmol). The reaction mixture was stirred overnight at 80 °C. The reaction was quenched with water, and the aqueous phase was extracted with ethyl acetate. The organic phases were combined and dried over anhydrous sodium sulfate. The mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by Flash column chromatography (ethyl acetate / petroleum ether = 0–1 / 2) to give compound 5.2 (100 mg, yield: 63%) as a yellow oil. m / z: [M+H] + 400.2.

[0244] Step 3: 3-Hydroxy-1H-pyrazole (75 mg, 0.75 mmol) was added to a mixed solution of compound 5.2 (100 mg, 0.25 mmol) in dichloromethane (1 mL) and ethanol (1 mL). The reaction mixture was stirred at room temperature for 1 hour. The reaction was quenched with saturated sodium bicarbonate solution. The aqueous phase was extracted with ethyl acetate. The combined organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give compound 5.3 (167 mg, crude product) as a yellow solid. m / z: [M+H] + 480.2.

[0245] Step 4: A solution of compound 5.3 (167 mg, crude) in N-methylpyrrolidone (3 mL) was microwaved at 180 °C for 2 hours. The reaction was quenched with water, the aqueous phase was extracted with ethyl acetate, the organic phases were combined and dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the residue was purified by Flash column chromatography (methanol / dichloromethane = 0–3 / 100) to give compound 5.4 (60 mg, two-step yield: 52%) as a yellow oil. m / z: [M+H] + 460.2.

[0246] Step 5: N,N-diisopropylethylamine (52 mg, 0.4 mmol), (Boc)₂O (44 mg, 0.2 mmol), and 4-dimethylaminopyridine (3 mg) were added to a 10 mL solution of compound 5.4 (60 mg, 0.13 mmol) in dichloromethane. The reaction mixture was stirred at room temperature for 5 hours and then concentrated under reduced pressure. The residue was purified by Flash column chromatography (ethyl acetate / petroleum ether = 0–1 / 2) to give compound 5.5 (40 mg, yield: 55%) as a yellow solid. m / z: [M+H] + 560.2.

[0247] Step 6: Add Pd / C (10%, 40 mg) to a methanol (4 mL) solution of compound 5.5 (40 mg, 0.07 mmol). After purging the reaction system with hydrogen, stir at room temperature for 2 hours under a hydrogen atmosphere. Filter the reaction solution, and concentrate the filtrate under reduced pressure to obtain compound 5.6 (30 mg, yield: 100%) as a yellow solid. m / z: [M+H] + 426.2.

[0248] Step 7: N-(tert-butoxycarbonyl)sulfonyl chloride (27 mg, 0.12 mmol) was added to a pyridine (10 mL) solution of compound 5.6 (60 mg, 0.14 mmol). The reaction mixture was stirred at room temperature for 2 hours. The reaction was quenched with saturated sodium bicarbonate solution. The aqueous phase was extracted with ethyl acetate. The organic phases were combined and dried over anhydrous sodium sulfate. The mixture was filtered, and the filtrate was concentrated under reduced pressure to give compound 5.7 (20 mg, yield: 24%) as a yellow oil. m / z: [M+H] + 605.2.

[0249] Step 8: Add 0.5 mL of trifluoroacetic acid to a solution of compound 5.7 (20 mg, 0.03 mmol) in dichloromethane (0.5 mL). Stir the reaction mixture at room temperature for 3 hours, then concentrate under reduced pressure. The residue was purified by prep-HPLC (alkaline conditions) to give compound 5 (2 mg, yield: 16%) as a gray solid. m / z: [M+H] + 405.2; 1 H NMR (400MHz, DMSO-d6): δ13.18-12.44(br.s,1H),8.28(s,1H),7.82(d,J=2.4Hz,1H),6.82(d,J=2.4Hz, 1H), 4.10-3.85 (m, 5H), 3.74-3.70 (m, 1H), 3.63-3.54 (m, 4H), 3.22-3.07 (m, 3H), 1.15 (d, J = 6.4Hz, 3H).

[0250] Example 6: Synthesis of (R)-4-(6-(1H-pyrazol-3-yl)-1-((trifluoromethyl)sulfonyl)-1,2,3,6-tetrahydropyrazolo[3,4-b]pyrrolo[2,3-d]pyridin-4-yl)-3-methylmorpholine trifluoroacetic acid (compound 6)

[0251]

[0252] Step 1: Under ice bath conditions, sodium hydrogen (94 mg, 2.35 mmol) was added to a tetrahydrofuran (6 mL) solution of compound 5.4 (0.54 g, 1.17 mmol). The reaction mixture was stirred at 0 °C for 0.5 h. 2-(trimethylsilyl)ethoxymethyl chloride (0.39 g, 2.35 mmol) was added to the above reaction solution, and the resulting mixture was stirred at room temperature for 0.5 h. The reaction was quenched with water, and the aqueous phase was extracted with ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by Flash column chromatography (ethyl acetate / petroleum ether = 0–1 / 1) to give compound 6.1 (360 mg, yield: 52%) as a yellow solid. m / z: [M+H] + 590.2.

[0253] Step 2: Pd / C (10%, 120 mg) was added to a methanol (5 mL) solution of compound 6.1 (360 mg, 0.61 mmol). The reaction system was purged with hydrogen and stirred at room temperature for 2 hours under a hydrogen atmosphere. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure to give compound 6.2 (280 mg, yield: 100%) as a yellow oil. m / z: [M+H] + 456.2.

[0254] Step 3: Add trifluoromethanesulfonyl chloride (25.2 mg, 0.15 mmol) to a pyridine (2 mL) solution of compound 6.2 (34 mg, 0.08 mmol). Stir the reaction mixture at room temperature for 1 hour. Quench the reaction with water, extract the aqueous phase with ethyl acetate, combine the organic phases, dry them over anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure to give compound 6.3 (55 mg, crude product) as a yellow oil. m / z: [M+H] + 588.2.

[0255] Step 4: Triethylsilane (73.1 mg, 0.63 mmol) was added to a mixed solution of compound 6.3 (55 mg, crude) in trifluoroacetic acid (1 mL) and dichloromethane (1 mL). The resulting reaction solution was stirred at room temperature for 1 hour, then concentrated under reduced pressure. The residue was purified by prep-HPLC (acidic conditions) to give compound 6 (12 mg, two-step yield: 26%) as a pale yellow solid. m / z: [M+H] + 458.2;1 H NMR (400MHz, DMSO-d6): δ12.97(s,1H),8.12(s,1H),7.90(s,1H),6.85(s,1H),4.50-4.3 2(m,2H),4.24-4.18(m,1H),4.02-3.86(m,1H),3.76-3.52(m,7H),1.34(d,J=8.0Hz,3H).

[0256] Example 7: Synthesis of (R)-4-(3-methylmorpholino)-1-(methanesulfonyl)-6-(1H-pyrazol-3-yl)-1,2,3,6-tetrahydropyrazolo[3,4-b]pyrrolo[2,3-d]pyridine-8-nitrile (compound 7)

[0257]

[0258] Step 1: A mixture of compound 2.6 (0.51 g, 1.48 mmol), hydrazine hydrate (3 mL), and ethylene glycol dimethyl ether (5 mL) was stirred at room temperature for 5 hours, and then the reaction mixture was directly concentrated under reduced pressure. The residue was purified by Flash column chromatography (ethyl acetate / petroleum ether = 0–3 / 2) to give compound 7.1 (270 mg, yield: 54%) as a yellow solid. m / z: [M+H] + 338.2.

[0259] Step 2: Under nitrogen protection, N-iodosuccinimide (0.27 g, 1.2 mmol) was added to a solution of compound 7.1 (0.27 g, 0.8 mmol) in N,N-dimethylformamide (2.5 mL). The reaction mixture was stirred at 40 °C for 16 hours. The reaction was quenched with water, and the aqueous phase was extracted with ethyl acetate. The organic phases were combined and washed with water. The organic phase was separated, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by Flash column chromatography (ethyl acetate / petroleum ether = 0–2 / 1) to give compound 7.2 (120 mg, yield: 32%) as a yellow solid. m / z: [M+H] + 464.2.

[0260] Step 3: A mixture of compound 7.2 (100 mg, 0.22 mmol), 3-fluoro-N,N-dimethyl-1H-pyrazole-1-sulfonamide (84 mg, 0.44 mmol), cesium carbonate (215 mg, 0.66 mmol), and N,N-dimethylformamide (5 mL) was stirred at 100 °C for 10 hours. The reaction was quenched with water. The aqueous phase was extracted with ethyl acetate. The combined organic phases were washed with water, separated, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by Flash column chromatography (ethyl acetate / petroleum ether = 0–3 / 2) to give compound 7.3 (30 mg, yield: 21%) as a yellow solid. m / z: [M+H] + 637.2.

[0261] Step 4: Under nitrogen protection, a mixture of compound 7.3 (30 mg, 47 μmol), zinc powder (1.6 mg, 24 μmol), zinc cyanide (16.5 mg, 0.14 mmol), cuprous iodide (9 mg, 47 μmol), and 1,1'-bis(diphenylphosphine)ferrocene palladium dichloride (7 mg, 0.01 mmol) in N,N-dimethylformamide (2.5 mL) was purged with nitrogen and then microwaved at 120 °C for 5 hours. The reaction mixture was cooled to room temperature, the reaction was quenched with water, and the aqueous phase was extracted with ethyl acetate. The combined organic phases were washed with water, separated, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by Flash column chromatography (ethyl acetate / petroleum ether = 0–3 / 2) to give compound 7.4 (15 mg, yield: 60%) as a yellow oil. m / z: [M+H] + 536.2.

[0262] Step 5: A solution of compound 7.4 (15 mg, 28 μmol) in a mixture of trifluoroacetic acid (0.2 mL) and dichloromethane (1 mL) was stirred at room temperature for 1 hour. The reaction solution was concentrated under reduced pressure, and the residue was purified by prep-HPLC (alkaline conditions) to give compound 7 (1.55 mg, yield: 13%) as a pale yellow solid. m / z: [M+H] + 429.2.

[0263] Example 8: Synthesis of (R)-3-methyl-4-(1-(methanesulfonyl)-6-(1H-pyrazol-3-yl)-1H-pyrrolo[3,2-c][1,7]naphthidin-4-yl)morpholine (compound 8)

[0264]

[0265]

[0266] Step 1: N,N-diisopropylethylamine (5.5 g, 42.8 mmol) and N-phenylbis(trifluoromethanesulfonylimide) (9.2 g, 25.7 mmol) were added to a solution of (R)-8-chloro-2-(3-methylmorpholino)-1,7-naphthidine-4-ol (6 g, 21.4 mmol) in dichloromethane (200 mL). The reaction mixture was stirred overnight at room temperature and then concentrated under reduced pressure. The residue was purified by Flash column chromatography (ethyl acetate / petroleum ether = 0–2 / 3) to give compound 8.1 (8 g, yield: 91%) as a yellow solid. m / z: [M+H] + 412.2.

[0267] Step 2: To a solution of compound 8.1 (3.6 g, 8.6 mmol) in 1,4-dioxane (100 mL), 2,2-dimethoxyethylamine (1.2 g, 10.3 mmol), tris(dibenzylindeneacetone)dipalladium (394 mg, 0.43 mmol), Xantphos (249 mg, 0.43 mmol), and potassium phosphate (3.6 g, 17.2 mmol) were added. The reaction mixture was purged with nitrogen three times, and then stirred at 110 °C under nitrogen protection for 2 hours. After cooling to room temperature, the mixture was concentrated under reduced pressure. The residue was purified by Flash column chromatography (ethyl acetate / petroleum ether = 0–2 / 3) to give compound 8.2 (2.2 g, yield: 70%) as a yellow solid. m / z: [M+H] + 367.2.

[0268] Step 3: Add boron trifluoride diethyl ether complex (2 g, 14.2 mmol) to a solution of compound 8.2 (2.1 g, 5.7 mmol) in acetonitrile (30 mL). Stir the reaction mixture overnight at room temperature. Quench the reaction with saturated sodium bicarbonate solution. Extract the aqueous phase with dichloromethane. Combine the organic phases and dry them over anhydrous sodium sulfate. Filter, concentrate the filtrate under reduced pressure, and purify the residue by Flash column chromatography (ethyl acetate / petroleum ether = 0–7 / 10) to give compound 8.3 (1 g, yield: 58%) as a yellow solid. m / z: [M+H] + 303.2.

[0269] Step 4: Under ice bath conditions, sodium hydrogen (36 mg, 0.9 mmol) was added to a tetrahydrofuran (3 mL) solution of compound 8.3 (100 mg, 0.3 mmol), and the reaction system was stirred at 0 °C for 0.5 hours. Then, 2-(trimethylsilyl)ethoxymethyl chloride (140 mg, 0.9 mmol) was added, and the reaction solution was stirred overnight at room temperature. The reaction was then quenched with saturated sodium bicarbonate aqueous solution. The aqueous phase was extracted with ethyl acetate, and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by Flash column chromatography (ethyl acetate / petroleum ether = 0–2 / 3) to give compound 8.4 (120 mg, yield: 92%) as a yellow solid. m / z: [M+H] + 433.2.

[0270] Step 5: To a mixed solution of compound 8.4 (100 mg, 0.24 mmol) in 1,4-dioxane (4 mL) and water (1 mL), 1-(2-tetrahydropyranyl)-1H-pyrazole-5-boronic acid pinacol ester (68 mg, 0.34 mmol), 1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride (16 mg, 0.02 mmol), and cesium carbonate (79 mg, 0.24 mmol) were added sequentially. After purging the reaction system with nitrogen, the reaction was microwaved at 120 °C for 0.5 hours. The reaction system was cooled to room temperature and concentrated under reduced pressure. The residue was purified by Flash column chromatography (methanol / dichloromethane = 0–3 / 100) to give compound 8.5 (60 mg, yield: 46%) as a yellow oil. m / z: [M+H] + 549.2.

[0271] Step 6: Add a tetrahydrofuran solution (1M, 0.8mL) of tetrabutylamine fluoride to a tetrahydrofuran solution (2mL) of compound 8.5 (60mg, 0.1mmol). Stir the reaction mixture at 50°C for 6 hours. Quench the reaction with water. Extract the aqueous phase with dichloromethane. Combine the organic phases, dry them with anhydrous sodium sulfate, filter, concentrate the filtrate under reduced pressure, and purify the residue by Flash column chromatography (dichloromethane / methanol = 0–3 / 100) to give compound 8.6 (40mg, yield: 95%) as a yellow oil. m / z: [M+H] + 419.2.

[0272] Step 7: Add sodium hydroxide (12 mg, 0.3 mmol) to a tetrahydrofuran (6 mL) solution of compound 8.6 (40 mg, 0.1 mmol) in an ice-water bath. Stir the reaction mixture at 0 °C for 0.5 hours. Then add methanesulfonyl chloride (34 mg, 0.3 mmol). Stir the reaction mixture at room temperature for 3 hours. Quench the reaction with a saturated sodium bicarbonate solution. Extract the aqueous phase with dichloromethane. Combine the organic phases, dry them with anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure to give compound 8.7 (30 mg, yield: 60%) as a yellow oil. m / z: [M+H] + 497.2.

[0273] Step 8: Add trifluoroacetic acid (1 mL) to a 2 mL solution of compound 8.7 (30 mg, 0.06 mmol) in dichloromethane. After stirring the reaction mixture at room temperature for 1 hour, quench the reaction with a saturated sodium bicarbonate solution. Extract the aqueous phase with dichloromethane, combine the organic phases, dry them over anhydrous sodium sulfate, filter, concentrate the filtrate under reduced pressure, and purify the residue by prep-HPLC (alkaline conditions) to give compound 8 (17 mg, yield: 68%) as a yellow solid. m / z: [M+H] + 413.2; 1 H NMR (400MHz, DMSO-d6): δ13.41(s,1H),8.56-8.51(m,2H),7.96(d,J=3.6Hz,1H),7.63(s,1H),7.41(s,1H),7.21(d,J=3. 6Hz,1H),4.63-4.55(m,1H),4.06-4.03(m,1H),3.95-3.86(m,2H),3.81(s,3H),3.76-3.62(m,3H),1.27(d,J=6.8Hz,3H).

[0274] Example 9: Synthesis of (R)-3-methyl-4-(1-(methanesulfonyl)-7-(1H-pyrazol-3-yl)-2,3,4,7-tetrahydro-1H-pyrazolo[3,4-h][1,6]naphthidin-5-yl)morpholine trifluoroacetate (compound 9)

[0275]

[0276] Step 1: N,N-diisopropylethylamine (8.57 g, 66.3 mmol) and (R)-3-methylmorpholine (2.46 g, 24.3 mmol) were added to a solution of 5,7-dichloro-1,6-naphthidine (4.4 g, 22.1 mmol) in dimethyl sulfoxide (73 mL). The reaction mixture was stirred overnight at 110 °C. The reaction mixture was then cooled to room temperature, quenched with ice water, and the aqueous phase was extracted with ethyl acetate. The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by Flash column chromatography (ethyl acetate / petroleum ether = 0–1 / 1) to give compound 9.1 (5.1 g, yield: 87%) as a yellow solid. m / z: [M+H] + 264.2.

[0277] Step 2: Compound 9.1 (1.5 g, 5.69 mmol), dimethyl sulfoxide (20 mL), and cesium fluoride (1.73 g, 11.4 mmol) were sequentially added to a sealed tube. The reaction mixture was stirred at 145 °C for 3 days. The reaction mixture was then cooled to room temperature and quenched with ice water. The aqueous phase was extracted with ethyl acetate. The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by Flash column chromatography (ethyl acetate / petroleum ether = 0–1 / 1) to give compound 9.2 (0.9 g, yield: 64%) as a yellow solid. m / z: [M+H] + 248.2.

[0278] Step 3: Palladium on carbon (10%, 0.85 g) was added to a methanol (50 mL) solution of compound 9.2 (0.85 g, 3.44 mmol). The reaction system was purged with hydrogen and stirred overnight at room temperature under a hydrogen atmosphere. The reaction mixture was then filtered through diatomaceous earth, the filter cake was washed with methanol, the filtrates were combined and concentrated under reduced pressure, and the residue was purified by Flash column chromatography (ethyl acetate / petroleum ether = 0–1 / 1) to give compound 9.3 (0.64 g, yield: 74%) as a colorless oil. m / z: [M+H] + 252.2.

[0279] Step 4: Under ice bath conditions, methanesulfonyl chloride (2.8 mL) was added to an anhydrous pyridine (7 mL) solution of compound 9.3 (0.53 g, 2.11 mmol). The reaction mixture was stirred at 40 °C for 2 hours in a sealed tube, and then concentrated directly under reduced pressure. The residue was poured into water, and the aqueous phase was extracted with ethyl acetate. The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by Flash column chromatography (ethyl acetate / petroleum ether = 0–1 / 1) to give compound 9.4 (0.33 g, yield: 48%) as a yellow oil. m / z: [M+H] + 330.2.

[0280] Step 5: Under nitrogen protection, phosphorus oxychloride (0.38 g, 2.5 mmol) was added dropwise to a solution of compound 9.4 (0.33 g, 1 mmol) in N,N-dimethylformamide (5 mL). The reaction system was stirred at 80 °C for 4 hours. The reaction was then quenched with water and stirred for 1 hour. The aqueous phase was adjusted to pH 7–8 with saturated sodium bicarbonate solution and extracted with ethyl acetate. The combined organic phases were washed with saturated brine, separated, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by Flash column chromatography (ethyl acetate / petroleum ether = 0–2 / 1) to give compound 9.5 (221 mg, yield: 62%) as a yellow solid. m / z: [M+H] + 358.2.

[0281] Step 6: Add 3-hydrazino-1H-pyrazole (0.27 g, 2.8 mmol) to a 95% ethanol (5 mL) solution of compound 9.5 (0.25 g, 0.7 mmol). Stir the reaction mixture at room temperature for 20 minutes. Then concentrate the reaction mixture under reduced pressure. Add water (5 mL) to the residue. Adjust the pH of the aqueous phase to 7-8 with saturated sodium bicarbonate solution. Extract the aqueous phase with ethyl acetate. Combine the organic phases and wash with saturated brine. Separate the organic phase, dry it with anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure to give compound 9.6 (0.3 g, yield: 98%) as a yellow solid. m / z: [M+H] + 438.2.

[0282] Step 7: Compound 9.6 (0.3 g, 0.69 mmol) was dissolved in N-methylpyrrolidone (3 mL). The reaction system was purged with nitrogen three times and then microwaved at 180 °C for 20 min. The reaction mixture was then cooled to room temperature, the reaction was quenched with water, and the aqueous phase was extracted with ethyl acetate. The combined organic phases were concentrated under reduced pressure, and the residue was purified by prep-HPLC (acidic conditions) to give compound 9 (103 mg, yield: 28%) as an off-white solid. m / z: [M+H] + 418.2; 1H NMR (400MHz, DMSO-d6): δ8.13(s,1H),7.86(d,J=2.4Hz,1H),6.81(d,J=2.4Hz,1H),3.84-3.70(m,6H),3.68-3.62(m,1H),3.49-3.42(m,1H),3.34 -3.27(m,1H),3.26(s,3H),3.03-2.93(m,1H),2.88-2.77(m,1H),2.71-2 .61(m,1H),2.12-2.00(m,1H),1.88-1.75(m,1H),1.02(d,J=6.4Hz,3H).

[0283] Example 10: Synthesis of (R)-3-methyl-4-(9-(methanesulfonyl)-3-(1H-pyrazol-3-yl)-3H-pyrazolo[3,4-c]isoquinoline-5-yl)morpholine trifluoroacetate (compound 10)

[0284]

[0285] Step 1: Concentrated sulfuric acid (10 mL) and N-bromosuccinimide (10.8 g, 60.6 mmol) were added separately to a solution of 1,3-dichloroisoquinoline (10 g, 50.5 mmol) in acetonitrile (250 mL). The reaction mixture was stirred at room temperature for 3 days. The mixture was filtered, and the filter cake was dried under vacuum to give compound 10.1 (7.4 g, yield: 53%) as a white solid. m / z: [M+H] + 275.8.

[0286] Step 2: N,N-diisopropylethylamine (8.95 g, 69.2 mmol) and (R)-3-methylmorpholine (3.27 g, 32.3 mmol) were added to a dimethyl sulfoxide (DMSO) solution of compound 10.1 (6.39 g, 23.1 mmol) in 96 mL of water. The reaction mixture was stirred overnight at 110 °C. The reaction mixture was then cooled to room temperature and quenched with ice water. The aqueous phase was extracted with ethyl acetate. The combined organic phases were washed with saturated brine, separated, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure, and the residue was purified by Flash column chromatography (petroleum ether / ethyl acetate = 4 / 1) to give compound 10.2 (6.13 g, yield: 78%) as a yellow solid. m / z: [M+H] + 341.0.

[0287] Step 3: Sodium methanesulfinate (3.59 g, 35.1 mmol) and cuprous iodide (6.69 g, 35.1 mmol) were added to a 75 mL solution of compound 10.2 (3 g, 8.78 mmol). The reaction mixture was stirred at 120 °C for 6 hours under nitrogen protection. The reaction mixture was then cooled to room temperature and poured into a saturated aqueous solution of ammonium chloride. The aqueous phase was extracted with ethyl acetate, and the organic phases were combined and washed with saturated brine. The organic phase was separated, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure, and the residue was purified by Flash column chromatography (petroleum ether / ethyl acetate = 1 / 2) to give compound 10.3 (1.8 g, yield: 60%) as a yellow solid. m / z: [M+H] + 341.0.

[0288] Step 4: Compound 10.3 (1.8 g, 5.29 mmol), dimethyl sulfoxide (25 mL), and cesium fluoride (2.41 g, 15.9 mmol) were sequentially added to a sealed tube. The reaction mixture was stirred at 150 °C for 5 hours. The reaction mixture was then cooled to room temperature, quenched with ice water, and the aqueous phase was extracted with ethyl acetate. The combined organic phases were washed with saturated brine, the organic phase was separated, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by Flash column chromatography (petroleum ether / ethyl acetate = 0–1 / 2) to give compound 10.4 (1.03 g, yield: 60%) as a yellow solid. m / z: [M+H] + 325.0.

[0289] Step 5: A solution of phosphorus oxychloride (0.5 mL) in N,N-dimethylformamide (5 mL) was stirred at room temperature for 10 minutes. Then, compound 10.4 (100 mg, 0.31 mmol) was added to the reaction solution. The reaction mixture was stirred at 80 °C for 3 hours, cooled to room temperature, and poured into ice water (20 mL). The mixture was stirred for 1 hour. Ethyl acetate (10 mL) was added, and the pH was adjusted to 8 with saturated sodium carbonate aqueous solution. 3-Hydroxy-1H-pyrazole (100 mg, 1.02 mmol) was added. The resulting mixture was stirred overnight at room temperature, then water (10 mL) was added. The aqueous phase was extracted with ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by prep-TLC (dichloromethane / methanol = 10 / 1) to give compound 10.5 (70 mg, yield: 52%) as a yellow oil. m / z: [M+H] + 433.2.

[0290] Step 6: A solution of compound 10.5 (70 mg, 0.16 mmol) in 2.1 mL of N-methylpyrrolidone was microwaved at 180 °C for 20 min in a sealed tube. The reaction solution was cooled to room temperature, and the reaction was quenched with water (10 mL). The aqueous phase was extracted with ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by prep-HPLC (acidic conditions) to give compound 10 (10 mg, yield: 15%) as a yellow solid. m / z: [M+H] + 413.2; 1 H NMR (400MHz, CDCl3) δ9.05-9.07(m,1H),8.61-8.68(m,2H),7.76-7.78(m,1H),7.67-7.71(m,1H),7.10-7.12(m, 1H),4.05-4.08(m,3H),3.91-3.95(m,1H),3.68-3.72(m,2H),3.35-3.38(m,1H),3.31(s,3H),1.22-1.24(m,3H).

[0291] Example 11: Synthesis of (R)-4-(1-cyclopropyl-6-(1H-pyrazol-3-yl)-1,2,3,6-tetrahydropyrazolo[3,4-b]pyrrolo[2,3-d]pyridin-4-yl)-3-methylmorpholine (compound 11)

[0292]

[0293] Step 1: Under nitrogen protection, cyclopropylboronic acid (180 mg, 2.1 mmol), copper acetate (191 mg, 1.05 mmol), and sodium carbonate (223 mg, 2.1 mmol) were added sequentially to a 6 mL solution of compound 2.4 (250 mg, 1.05 mmol) in acetonitrile. The reaction system was stirred at 70 °C for 5 hours. After cooling the reaction solution to room temperature, the reaction was quenched with water. The aqueous phase was extracted with ethyl acetate, and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by Flash column chromatography (ethyl acetate / petroleum ether = 0–1 / 2) to give compound 11.1 (220 mg, yield: 75%) as a pale yellow solid. m / z: [M+H] + 278.2.

[0294] Step 2: Under nitrogen protection, phosphorus oxychloride (0.3 g, 2.01 mmol) was added to a solution of compound 11.1 (220 mg, 0.8 mmol) in N,N-dimethylformamide (4 mL). The reaction system was stirred at 80 °C for 4 hours. After cooling to room temperature, the reaction was quenched with ice water and stirring continued for 2 hours. The aqueous phase was extracted with ethyl acetate, the organic phases were combined and washed with water, the organic phase was separated, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by Flash column chromatography (ethyl acetate / petroleum ether = 0–1 / 1) to give compound 11.2 (230 mg, yield: 94%) as a pale yellow solid. m / z: [M+H] + 306.2.

[0295] Step 3: Add 3-hydrazino-1H-pyrazole (0.2 g, 2.0 mmol) to an ethanol (95%, 6 mL) solution of compound 11.2 (0.15 g, 0.49 mmol). Stir the reaction system at room temperature for 0.5 hours. Quench the reaction with water. Adjust the pH of the aqueous phase to 7-8 with saturated sodium bicarbonate solution. Extract the aqueous phase with ethyl acetate. Combine the organic phases and wash with saturated brine. Separate the organic phase and dry it with anhydrous sodium sulfate. Filter the solution and concentrate the filtrate under reduced pressure to obtain compound 11.3 (0.18 g, crude product) as a yellow solid.

[0296] Step 4: The N-methylpyrrolidone solution of compound 11.3 (0.18 g, crude) was purged with nitrogen three times and then microwaved at 180°C for 1 hour. The reaction mixture was then cooled to room temperature, the reaction was quenched with water, and the aqueous phase was extracted with ethyl acetate. The combined organic phases were concentrated under reduced pressure, and the residue was purified by prep-HPLC (alkaline conditions) to give compound 11 (22.3 mg, yield: 12%) as a pale yellow solid. m / z: [M+H] + 366.2; 1 H NMR (400MHz, CDCl3): δ8.19(s,1H),7.60(d,J=4.0Hz,1H),6.71(s,1H),4.20-3.98(m,2H),3.80-3.76(m ,1H),3.70-3.48(m,6H),3.16-2.98(m,2H),2.68-2.60(m,1H),1.34(d,J=8.0Hz,3H),0.92-0.80(m,4H).

[0297] Example 12: Synthesis of (R)-3-methyl-4-(1-methyl-6-(1H-pyrazol-3-yl)-tetrahydropyrazolo[3,4-b]pyrrolo[2,3-d]pyridin-4-yl)morpholine (compound 12)

[0298]

[0299] Step 1: Under ice bath conditions, sodium hydrogen (67.4 mg, 1.7 mmol) was added to a tetrahydrofuran (3 mL) solution of compound 2.4 (200 mg, 0.84 mmol). The reaction system was stirred at 0 °C for 0.5 hours, and then iodomethane (470 mg, 3.4 mmol) was added. The reaction solution was stirred at room temperature for 0.5 hours, and the reaction was quenched with water. The aqueous phase was extracted with ethyl acetate, and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by Flash column chromatography (ethyl acetate / petroleum ether = 0–1 / 2) to give compound 12.1 (170 mg, yield: 81%) as a pale yellow solid. m / z: [M+H] + 252.2.

[0300] Steps 2-4: Following the synthetic method described in steps 2-4 for compound 11, compound 12 was obtained from compound 12.1 as a pale yellow solid. m / z: [M+H] + 340.2; 1 H NMR (400MHz, CDCl3): δ8.02(s,1H),7.60(d,J=4.0Hz,1H),6.72(s,1H),4.14-3.98(m ,2H),3.80-3.76(m,1H),3.70-3.48(m,6H),3.16-2.98(m,5H),1.34(d,J=8.0Hz,3H).

[0301] Example 13: Synthesis of (R)-4-(6-(1H-pyrazol-3-yl)-1-(pyridin-3-yl)-1,2,3,6-tetrahydropyrazolo[3,4-b]pyrrolo[2,3-d]pyridin-4-yl)-3-methylmorpholine trifluoroacetate (compound 13)

[0302]

[0303] Step 1: To a 1,4-dioxane (5 mL) solution of compound 2.4 (300 mg, 1.26 mmol), 3-iodopyridine (310 mg, 1.51 mmol), methanesulfonic acid (2-dicyclohexylphosphino-2',6'-diisopropoxy-1,1'-biphenyl)(2-amino-1,1'-biphenyl-2-yl)palladium(II) (158 mg, 0.19 mmol), and cesium carbonate (820 mg, 2.52 mmol) were added sequentially. The reaction system was purged with nitrogen three times, and then stirred at 110 °C for 16 hours under a nitrogen atmosphere. After cooling the reaction system to room temperature, it was directly concentrated under reduced pressure. The residue was purified by Flash column chromatography (ethyl acetate / petroleum ether = 0–2 / 3) to give compound 13.1 (190 mg, yield: 48%) as a yellow solid. m / z: [M+H]+ 314.8.

[0304] Steps 2-4: Following the synthetic method described in steps 2-4 for compound 11, compound 13 was obtained from compound 13.1 as a pale yellow solid. m / z: [M+H] + 402.8; 1 H NMR (400MHz, DMSO-d6): δ8.81(d,J=2.4Hz,1H),8.58-8.54(m,1H),8.10-8.05(m,1H),7.92-7.88(m,1H),7.78-7.72(m,1H),7.51 (s,1H),6.91(d,J=2.4Hz,1H),4.35-4.15(m,4H),4.01-3.94(m,1H),3.82-3.78(m,2H),3.74-3.63(m,4H),1.26(d,J=6.4Hz,3H).

[0305] Example 14: Synthesis of (R)-2-(4-(3-methylmorpholino)-6-(1H-pyrazol-3-yl)-2,3-dihydropyrazolo[3,4-b]pyrrolo[2,3-d]pyridin-1(6H)-yl)ethanol trifluoroacetate (compound 14)

[0306]

[0307] Step 1: Triethylamine (66 mg, 0.65 mmol), (Boc)₂O (95.2 mg, 0.44 mmol), and 4-dimethylaminopyridine (2.4 mg, 0.02 mmol) were added sequentially to a dichloromethane (2 mL) solution of compound 5.4 (0.20 g, 0.44 mmol). The reaction mixture was stirred at room temperature for 2 hours. The reaction was quenched with water, and the aqueous phase was extracted with ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by Flash column chromatography (ethyl acetate / petroleum ether = 1 / 2) to give compound 14.1 (150 mg, yield: 61%) as a yellow solid.

[0308] Step 2: Pd / C (10%, 120 mg) was added to a methanol (5 mL) solution of compound 14.1 (400 mg, 0.94 mmol). The reaction system was purged with hydrogen and stirred at room temperature for 4 hours under a hydrogen atmosphere. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure to obtain compound 14.2 (300 mg, yield: 71%) as a pale yellow foamy solid.

[0309] Step 3: Add sodium hydrogen (56.4 mg, 1.4 mmol, 60%) to a tetrahydrofuran (3 mL) solution of compound 14.2 (300 mg, 0.71 mmol), and stir the reaction mixture at room temperature for 0.5 hours. Add 2-(2-bromoethoxy)tetrahydro-2H-pyran (295 mg, 1.4 mmol) to the above reaction system, and continue stirring the resulting mixture at room temperature for 6 hours. Quench the reaction with water, extract the aqueous phase with ethyl acetate, combine the organic phases, dry them with anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure to give compound 14.3 (200 mg, yield: 51%) as a yellow oil.

[0310] Step 4: A mixed solution of compound 14.3 (50 mg, 0.09 mmol) in trifluoroacetic acid (1 mL) and dichloromethane (2 mL) was stirred at room temperature for 1 hour, then concentrated under reduced pressure. The residue was purified by prep-HPLC (acidic conditions) to give compound 14 (1.7 mg, yield: 4%) as a pale yellow solid. m / z: [M+H] + 370.2; 1 H NMR (400MHz, CD3OD): δ8.07(s,1H),7.81(s,1H),6.69(s,1H),4.32(t,J=4.0Hz,2H),3.95-4 .00(m,6H),3.76(d,J=12.0Hz,1H),3.34-3.74(m,4H),3.26-3.30(m,1H),1.28-1.38(m,3H).

[0311] Biological Examples

[0312] Example 1: ATR Enzymatic Test

[0313] In this experiment, the phosphorylation level of substrate protein P53 (Eurofins, 14-952) was detected using HTRF technology to determine the activity of ATR / ATRIP (Eurofins, 14-953) kinase. Reaction buffer (25 mM HEPES pH 8.0, 0.01% Brij-35, 1% Glycerol, 5 mM DTT, 1 mg / mL BSA), stop buffer (12.5 mM HEPES pH 8.0, 0.005% Brij-35, 0.5% Glycerol, 250 mM EDTA), and assay buffer (50 mM HEPES pH 7.0, 150 mM NaCl, 267 mM KF, 0.1% sodium cholate, 0.01% Tween 20) were prepared in advance. Dilute ATR / ATRIP to a working solution of 2 ng / μL with reaction buffer, dilute the substrate protein P53 to a working solution of 80 nM with reaction buffer, and prepare a 4 nM ATP (Sigma, A2383) working solution (containing 40 mM MnCl2) with reaction buffer. Dilute the compounds 3-fold serially with DMSO, then dilute with reaction buffer to prepare working solutions. Add 2.5 μL / well to a 384-well plate and centrifuge at 1500 rpm for 40 s. Then, add 2.5 μL each of the ATR / ATRIP, P53, and ATP working solutions sequentially to the 384-well plate, centrifuge at 1500 rpm for 40 s, and react at room temperature for 30 min. After the reaction is complete, add 5 μL of stop buffer to each well and centrifuge at 1500 rpm for 40 s. Antibody working solution containing 0.083 μg / mL anti-phospho-p53(Ser15)-K (CisBio, cat.61P08KAE) and 5 μg / mL anti-GST-d2 (CisBio, cat.61GSTDLA) was prepared using detection buffer. 5 μL was added to each well of a 384-well plate, centrifuged at 1500 rpm for 40 s, and allowed to react overnight at room temperature. TR-FRET was detected using a microplate reader (Tecan, Infinite M1000 Pro). Data were analyzed using Graphpad software, and a four-parameter equation was used to fit the curve and calculate the inhibitor's IC50. 50 Values ​​(Table 1).

[0314] Table 1:

[0315] Compound numbering <![CDATA[IC 50 (nM)]]> 1 1.2 2 0.69 3 0.51 4 1.8 5 6.5 6 4.8 7 46 8 40 11 5.9 12 14 13 7.5

[0316] Example 2: Cell proliferation assay

[0317] In this invention, cell assays were used to evaluate the bioactivity of compounds. LOVO (Nanjing Kebai), a human colon cancer cell line, was seeded in 96-well plates using Dulbecco's Modified Eagle's medium, supplemented with 10% fetal bovine serum and 1% P / S, and cultured at 37°C with 5% CO2. The compound concentration range was 4.5 nM to 30 μM. The stock solution of the test compound was dissolved in DMSO and added to the medium at the indicated concentration, and incubated for 72 hours. Negative control cells were treated with vehicle only. In some assays, a known ATR inhibitor was added as a positive control. Cell viability was evaluated using the Cell titer glo kit (CTG, Promega) as directed in the product instructions. Data were analyzed using Graphpad software, and IC50 was obtained. 50 Values ​​and compound fitting curves (Table 2).

[0318] Table 2:

[0319] Compound numbering <![CDATA[IC 50 (nM)]]> 1 29.62 2 37.34 5 261.7 6 79.57 7 405.8 8 32.28 11 71.95 12 93.65

[0320] Example 3: Cytochrome oxidase P450 inhibition test

[0321] The inhibitory effects of the compounds on the CYP2C19, 2D6, and 3A4 isoforms were evaluated using LC-MS / MS. In this method, the test compounds were mixed with a solution of human liver microsomes containing CYP model substrates and co-incubated under NADPH. The inhibitory IC50 values ​​of the compounds against CYP2C19, 2D6, and 3A4 were calculated by measuring the amount of metabolites of the model substrates in the reaction solution. 50 .

[0322] The specific experimental method is as follows:

[0323] The test compound was prepared as a 10 mM stock solution using DMSO, and then diluted to 4 mM with acetonitrile. Simultaneously, a corresponding reference inhibitor solution was prepared for each CYP subtype, for example, Ketoconazole. Both solutions were prepared separately (8 mL DMSO stock solution + 12 mL acetonitrile). The samples prepared under these conditions were at a 400X concentration. The above solutions were then serially diluted 3-fold with a DMSO / acetonitrile mixture (v / v: 40 / 60) to prepare the final test solutions. Seven concentration points were set for each test compound, with an initial and final concentration of 10 μM. NADPH, CYP enzyme model substrate, and human liver microsome solution were diluted to appropriate concentrations using preheated potassium phosphate buffer (0.1 M, pH 7.4). The human liver microsome solution was purchased from BD Gentest (20 mg / mL, Corning, catalog number #452161).

[0324] Add 400 mL of human liver microsome solution (0.2 mg / mL) to each well of the test compound in a 96-well plate, followed by 2 mL of the serially diluted final test sample of the aforementioned test compound. For each well corresponding to the reference inhibitor, add 200 mL of human liver microsome solution (0.2 mg / mL) and 1 mL of the final test sample. Aliquot 15 mL of the prepared corresponding model substrate into each well of a 96-well plate. After mixing the microsome solution, transfer 30 mL of the test compound / reference inhibitor-human liver microsome mixture to the 96-well plate containing the substrate, mix well, and preheat at 37°C for 5 minutes. Then add 15 mL of 8 mM NADPH solution preheated at 37°C to begin the reaction. Each test includes a duplicate control and a blank control without the test substance. A 96-well plate containing 60 mL of reaction solution was incubated at 37 °C. After incubation, 120 μL of cold acetonitrile solution containing an internal standard was added to each well to terminate the reaction. The 96-well plate was then shaken on a microplate shaker for 5 minutes (600 rpm / min) and centrifuged at 6000 rpm, 4 °C for 20 minutes. 40 μL of supernatant was then transferred from each well to another 96-well plate, and 80 μL of ultrapure water was added to each well. The plate was then shaken for 5 minutes (600 rpm / min) and centrifuged at 6000 rpm, 4 °C for 20 minutes. LC-MS / MS analysis was then performed. The inhibition rate was determined by comparing the amount of model substrate metabolite at each test concentration and in the case without the test substance. In GraphPad Prism 5.0 software, a nonlinear regression (Sigmoidal (non-linear) dose-response model) analysis was performed with the logarithm of the test concentration on the x-axis and the inhibition rate on the y-axis to obtain the IC50 of the test compound. 50 Values. The results are shown in Table 3 below:

[0325] Table 3:

[0326]

[0327] Example 4: Cardiac Safety Evaluation - hERG Test

[0328] This experiment used a CHO cell line stably transfected with hERG cDNA and expressing the p15 hERG channel. Cells were cultured at 37°C in a humidified incubator containing 5% CO2 in medium (Ham's F12, 10% v / v FBS, 100 μg / mL hygromycin B, 100 μg / mL genimycin) (from Invitrogen). Cells were allowed to grow and reach approximately 80-90% confluence under these conditions. Cells were then treated with Detachin (Genlantis) for 3-5 minutes. The cells were titrated 15-20 times with medium at 37°C, and then resuspended in CHO-S-SFM II medium (serum-free medium, Invitrogen) buffered with HEPES (25 mM). Cells used in QPatch studies must meet the following criteria: under microscopic examination, most suspended cells should be single and isolated; viability should be greater than 95%; and the cell density in the final suspension should be in the range of 3–8 × 10⁶ cells / mL before application to the QPatch mixing chamber. Cells meeting the above criteria can be used for recording within 4 hours post-harvest.

[0329] Prepare a 10 mM DMSO stock solution of the test compound. Six dosages (30, 10, 3, 1, 0.3, and 0.1 μM) were selected to obtain the fitting curve and IC50. 50 The final DMSO concentration was 0.1% or lower. The IC50 of the positive control cisapride was... 50 The evaluation doses were 3, 1, 0.3, 0.1, 0.03, and 0.01 μM. The internal electrophysiological recording solution composition was: CaCl2 2 mM, MgCl2 1 mM, KCl 4 mM, NaCl 145 mM, Glucose 10 mM, HEPES 10 mM, pH 7.4 (NaOH). The external recording solution composition was: CaCl2 374 mM, MgCl2 1.75 mM, KCl 120 mM, HEPES 10 mM, EGTA 5 mM, Na-ATP 4 mM, pH 7.25 (KOH) (all reagents used were from Sigma).

[0330] Whole-cell recording was performed using an automated QPatch (Sophion Biosciences, Denmark). Cells were recorded for 120 seconds to assess current stability. The voltage was then applied to the cells every 15 seconds throughout the process. Only stable cells with recorded parameters above the threshold were allowed to proceed to the drug assay procedure. All experiments were performed at approximately 25°C. An external solution containing 0.1% DMSO (carrier) was applied to the cells to establish a baseline. After the current stabilized for 3 minutes, the test compound was tested. The compound solution was added, and the cells were held in the test solution until the compound's effect reached a stable state, for a maximum of 4 minutes. For dose-response assays, the compound was cumulatively applied to the cells from low to high concentrations. After compound testing, the cells were rinsed with the external solution.

[0331] The data were analyzed using Sophion Assay software (assay software V5.0), Microsoft Excel, and Graphpad Prism 5.0 to obtain the IC50 of the compound. 50 The results are shown in Table 4 below:

[0332] Table 4:

[0333] Compound numbering <![CDATA[IC 50 (μM)]]> 1 ﹥30 2 ﹥30 3 ﹥30

[0334] Example 5: In vivo efficacy experiment of OCI-LY19 human B-cell lymphoma mouse subcutaneous xenograft tumor model

[0335] Cell culture: Human B-cell lymphoma OCI-LY19 cells were maintained as a monolayer in MEM-α medium containing 10% fetal bovine serum at 37°C in a constant temperature incubator containing 5% CO2. Tumor cells were passaged twice a week. Cells in the exponential growth phase were harvested and counted for seeding.

[0336] Laboratory animals: BALB / c nude mice, 6-8 weeks old, 19-22g, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0337] Six experimental groups were set up for Vehicle, positive control (AZD6738, CAS No.: 1352226-88-0) and compound 2, as shown in Table 5 below:

[0338] Table 5:

[0339]

[0340] Note: po: oral administration

[0341] Experimental methods: OCI-LY19 cell line (3.0×10⁻⁶) 6(0.1 mL / mouse) was injected subcutaneously into the right back of each experimental mouse. The tumor growth was observed regularly until it grew to approximately 100 mm. 3 Mice were randomly grouped according to tumor size and body weight, and administered medication according to the dosing schedule shown in Table 5. Throughout the experiment, mouse body weight and tumor size were measured twice a week.

[0342] Formula for calculating tumor size: Tumor volume (mm) 3 = 0.5 × (tumor long diameter × tumor short diameter) 2 ).

[0343] The experimental results are shown in Table 6 and Appendix. Figure 1 :

[0344] Table 6:

[0345]

[0346]

[0347] The results showed that, compared with the positive control AZD6738, the compound of the present invention exhibited better efficacy in the OCI-LY19 human B-cell lymphoma mouse subcutaneous xenograft model.

Claims

1. A compound or a pharmaceutically acceptable salt thereof, characterized in that, The compound is a compound as shown in formula (II) or a compound as shown in formula (III); or ; In the compound shown in formula (II), V is NR6; in the compound shown in formula (III), V is CR7; V3 is CR 7c ; In the compounds shown in formula (II) and formula (III), X is NR5; X1 is CR. 3a or CR 3a R 4a X2 is CR 3b or CR 3b R 4b ; U is N; U1 and U2 are each independently C; V1 is N; V2 is CR 7b ; R1 is hydrogen or C. 1-6 alkyl; R2 is a methyl group; R 3a and R 3b Each can be independently represented by hydrogen, halogen, cyano, nitro, or C. 1-6 Alkyl, C 3-8 cycloalkyl, -OR a or -NR a R b ; wherein, the C 1-6 Alkyl or C 3-8 The cycloalkyl group is unsubstituted or selectively replaced by 1 to 3 groups selected from halogen, cyano, nitro, -OR. a and -NR a R b The substituents can be substituted at any position; R 4a and R 4b Independently, they are hydrogen, halogen, and C. 1-6 Alkyl, C 1-6 Alkoxy, halogenated C 1-6 Alkyl or halogenated C 1-6 Alkoxy; R5 is hydrogen, C 1-6 Alkyl, C 3-8 Cycloalkyl, 5-6 membered heteroaryl, -S(O) 1-2 R a -S(O)2NR a R b -S(O)(=NCN)R a or -S(O)(=NR b )R a ; wherein, the C 1-6 Alkyl, C 3-8 The cycloalkyl or 5-6-membered heteroaryl group is unsubstituted or selectively replaced by 1 to 3 groups selected from halogen, cyano, nitro, -OR a and -NR a R b The substituents can be substituted at any position; R6 and R7 are each independently a 5-6 membered heteroaryl group, wherein the 5-6 membered heteroaryl group is unsubstituted or selectively composed of 1 to 3 groups selected from halogen, cyano, -R c -OR c and -NR c R d The substituents can be substituted at any position; R 7b and R 7c Each is independently hydrogen, halogen, cyano, or C. 1-6 Alkyl, C 1-6 Alkoxy, halogenated C 1-6 Alkyl, Halogenated C 1-6 Alkoxy or C 3-8 cycloalkyl; Each R a R b R c and R d Selected independently from hydrogen and C 1-6 Alkyl or C 3-8 cycloalkyl; the R a R b R c and R d It is either unsubstituted or selectively substituted at any position by 1 to 3 substituents selected from halogens.

2. The compound of claim 1 or a pharmaceutically acceptable salt thereof, characterized in that, R1 is hydrogen; R2 is methyl.

3. The compound of claim 1 or a pharmaceutically acceptable salt thereof, characterized in that, R 3a and R 3b Each is independently hydrogen, halogen, cyano, or C. 1-6 Alkyl, Halogenated C 1-6 Alkyl or halogenated C 1-6 Alkoxy; And / or, R 4a and R 4b Each independently can be either hydrogen or C. 1-6 alkyl; And / or, R5 is C 1-6 Alkyl, C 3-8 Cycloalkyl, 5-6 membered heteroaryl, -S(O) 1-2 R a -S(O)2NR a R b -S(O)(=NCN)R a or -S(O)(=NR b )R a ; wherein, the C 1-6 Alkyl, C 3-8 The cycloalkyl or 5-6-membered heteroaryl group is unsubstituted or selectively replaced by 1 to 3 groups selected from halogen, -CN, -OR. a and -NR a R b The substituents can be substituted at any position; And / or, R 7b and R 7c Each is independently hydrogen, halogen, cyano, or C. 1-6 Alkyl, C 1-6 Alkoxy, halogenated C 1-6 Alkyl or halogenated C 1-6 Alkoxy; And / or, each R b Independently hydrogen or C 1-6 alkyl; And / or, each R c Independently hydrogen or C 1-6 Alkyl; the C 1-6 The alkyl group is either unsubstituted or selectively substituted at any position by 1 to 3 substituents selected from halogens; And / or, each R d Independently hydrogen or C 1-6 alkyl.

4. The compound of claim 1 or a pharmaceutically acceptable salt thereof, characterized in that, R6 and R7 are independently pyrrole, pyrazol, or isoxazolyl, respectively; the pyrrole, pyrazol, or isoxazolyl groups are unsubstituted or selectively represented by 1 to 3 groups selected from halogen, cyano, -R c -OR c and -NR c R d The substituents can be substituted at any position.

5. The compound according to any one of claims 1 to 4, or a pharmaceutically acceptable salt thereof, characterized in that, In the compound shown in formula (II), the group It can be any of the following structures: or ; In the compound shown in formula (III), the group for .

6. The compound of claim 5 or a pharmaceutically acceptable salt thereof, characterized in that, R 3a R 3b R 4a and R 4b Each is independently hydrogen; And / or, R 7b It is hydrogen; And / or, R6 and R7 are independently pyrrole, pyrazol, or isoxazolyl, respectively.

7. The compound of claim 1 or a pharmaceutically acceptable salt thereof, characterized in that, In the compound shown in formula (II), R6 is pyrrolo, pyrazol, or isoxazol; the pyrrolo, pyrazol, or isoxazol is unsubstituted or selectively substituted by 1 to 3 groups selected from halogen, cyano, -R c -OR c and -NR c R d The substituents can be substituted at any position; R 7b It is hydrogen; R c and R d Each independently can be either hydrogen or C. 1-6 alkyl; In the compound shown in formula (III), R7 is pyrrolo, pyrazol, or isoxazol; the pyrrolo, pyrazol, or isoxazol is unsubstituted or selectively substituted by 1 to 3 groups selected from halogen, cyano, -R c -OR c and -NR c R d The substituents can be substituted at any position; R 7b and R 7c Each is independently hydrogen; R c and R d Each independently can be either hydrogen or C. 1-6 alkyl.

8. The compound or a pharmaceutically acceptable salt thereof as claimed in any one of claims 1 to 4 and 7, characterized in that, The compound is a compound as shown in formula (IIA). ； in, It can be a double bond or a single bond; Alternatively, the compound may be a compound of formula (IIIA). ； in, It can be a double bond or a single bond.

9. The compound of claim 8 or a pharmaceutically acceptable salt thereof, characterized in that, X1 and X2 are each independently CH; Alternatively, X1 and X2 can be CH2 independently.

10. The compound of claim 8 or a pharmaceutically acceptable salt thereof, characterized in that, R5 is C 1-6 Alkyl, C 3-8 Cycloalkyl, 5-6 membered heteroaryl, -S(O) 1-2 R a -S(O)2NR a R b or -S(O)(=NR b )R a ; wherein, the C 1-6 Alkyl, C 3-8 The cycloalkyl or 5-6-membered heteroaryl group is unsubstituted or selectively replaced by 1 to 3 groups selected from halogen, -CN, -OR. a and -NR a R b The substituents can be substituted at any position; Each R a Independently hydrogen or C 1-6 Alkyl; the C 1-6 The alkyl group is either unsubstituted or selectively substituted at any position by 1 to 3 substituents selected from halogens; Each R b Independently hydrogen or C 1-6 alkyl.

11. The compound of claim 8 or a pharmaceutically acceptable salt thereof, characterized in that, The compound is the compound shown in formula (IIA), wherein V1 is N and V2 is CH.

12. The compound of claim 8 or a pharmaceutically acceptable salt thereof, characterized in that, The compound is the compound shown in formula (IIIA), wherein V1 is N; V2 and V3 are independently CH.

13. The compound of claim 1 or a pharmaceutically acceptable salt thereof has any of the following structures: , , , , , , , , , , , , , , , , , or or its pharmaceutically acceptable salt.

14. A pharmaceutical composition comprising a therapeutically effective amount of an active ingredient and a pharmaceutically acceptable excipient; said active ingredient comprising a compound as claimed in any one of claims 1 to 13 or a pharmaceutically acceptable salt thereof.

15. The use of the compound of any one of claims 1 to 13 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 14, in the preparation of an ATR inhibitor medicament.

16. The use of the compound of any one of claims 1 to 13 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 14, in the preparation of a medicament for treating and / or alleviating diseases related to ATR level abnormalities.

17. The application as described in claim 16, characterized in that, The disease associated with abnormal ATR levels is cancer.

18. The use of the compound of any one of claims 1 to 13 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 14, in the preparation of a medicament for treating cancer.

19. The use of the compound of any one of claims 1 to 13 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 14, in the preparation of a medicament for treating cancer, wherein, The compound or a pharmaceutically acceptable salt thereof or a pharmaceutical composition thereof is used in combination with one or more other types of therapeutic agents and / or treatments for cancer treatment.

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