A drug for treating tumors

Abstract

The present invention provides a compound represented by formula (I), which exhibits excellent inhibitory activity against TRK, ALK, ROS1 kinases and their mutants, especially against TRK kinase and its mutant forms; has a strong inhibitory effect on a variety of TRK-mutated cells (including TRK-mutated drug-resistant cells), and its anti-tumor activity in vitro and in vivo is superior to that of the existing control compounds. Moreover, the combined use of the compound of the present invention and a KRAS<supgt;G12C< / supgt; inhibitor can synergistically enhance the inhibitory effect of the drug on tumors with KRAS<supgt;G12C< / supgt> mutations. The compound of the present invention has good application prospects as a drug.

Description

[0001] This application claims the priority of a previous application titled "A Drug for Treating Tumors" with the patent application number 202111345327.9, which was filed with the China National Intellectual Property Administration on November 15, 2021. The full text of this application is incorporated into this application by reference. Technical Field

[0002] The present invention belongs to the field of medicine, and specifically relates to a drug for treating tumors. Background Art

[0003] 1. KRAS G12C and Tumors

[0004] RAS is a guanine nucleotide-binding protein that acts as a molecular switch in the cycle of interconversion between GTP / GDP near the cell membrane. The RAS gene mainly has three subtypes (HRAS, NRAS, or KRAS). Among them, KRAS is one of the most common oncogenes in solid tumors and can occur in tumors such as lung cancer, colorectal cancer, pancreatic cancer, uterine cancer, bladder cancer, esophageal cancer, gastric cancer, kidney cancer, breast cancer, skin cancer, prostate cancer, leukemia (such as acute myeloid leukemia, acute lymphoblastic leukemia), cervical cancer, liver cancer, ovarian cancer, and brain cancer. KRAS mutations occur in approximately 90% of pancreatic cancers, 50% of colorectal cancers, and 25% of lung cancers. From the overall mutation frequency of various tumors, the KRAS mutation rate is about 30%.

[0005] The three amino acids where KRAS is most likely to mutate are glycine 12 (G12), glycine 13 (G13), and glutamine 61 (Q61). G12 and G13 are around the nucleotide-binding pocket, and their mutations will produce a certain steric effect, preventing the formation of the conformation required for hydrolysis, thereby inhibiting the hydrolysis of GTP-RAS. KRAS G12C mutations account for about 21% of the mutations at codon 12. Compared with the wild type, KRAS G12X mutations generally have a lower affinity for RAF. Among them, the G12D, G12R, and G12V mutations have a low affinity for RAF, the G12A mutation has a slightly lower affinity, while the G12C mutation shows an affinity similar to that of the wild type. Therefore, KRAS G12C mutations are more likely to activate the RAF-mediated MEK-ERK signaling pathway, promoting the growth and proliferation of cancer cells. In non-small cell lung cancer, KRAS mutations account for about 25%, and among them, KRAS G12C mutations account for 14% of all non-small cell lung cancers. And compared with lung cancer patients without a smoking history, lung cancer patients with a smoking history have a higher incidence of KRAS G12CThe proportion of mutations increased significantly. Correlation analysis between ctDNA KRAS mutation detection and prognosis showed that KRAS mutation was associated with poor prognosis in pancreatic cancer patients, and G12C accounted for approximately 2% in pancreatic cancer patients with KRAS mutations. KRAS G12C Autophosphorylation was more significant in pancreatic cancer and colorectal cancer. In colorectal cancer, KRAS mutations accounted for approximately 45%, among which KRAS G12C mutations accounted for 5%.

[0006] AMG510 is an orally active KRAS G12C inhibitor developed by Amgen. The phase 1 clinical trial was initiated on August 27, 2018, and its NDA application was accepted by the FDA in February 2021. MRTX849 is a KRAS G12C inhibitor developed by Mirati. The phase 1 clinical trial was initiated in December 2018 and is currently in the phase 3 clinical stage. However, from the clinical data, due to tumor heterogeneity and differences in the sensitivity of clinical patients to KRAS G12C inhibitors, AMG510, MRTX849, etc. have limited effects on tumor patients with KRAS G12C mutations (objective response rate ORR ≤ 50%), and there are significant differences in the responses to different tumor types, limiting the clinical value of single use.

[0007]

[0008] Multiple combination trials have been conducted on both AMG510 and MRTX849. The main combination targets include: PD-1, EGFR, SHP2, MEK, etc.

Paper 1

Paper 2

[0009] 2. TRK and Tumors

[0010] Tropomyosin receptor kinase (TRK) belongs to the receptor tyrosine kinase family, and its family members include three proteins, TRKA, TRKB, and TRKC, encoded by the NTRK1, NTRK2, and NTRK3 genes, respectively. Research results in recent decades have shown that TRK kinases are closely related to the occurrence, metastasis, and deterioration of various tumors. For example, TRK kinases are overexpressed in non-small cell lung cancer, colorectal cancer, melanoma, gallbladder cancer, thyroid cancer, malignant glioma, etc., and this overexpression is closely related to the proliferation and migration of tumor cells. NTRK gene fusions, gene mutations, and high expression of TRK proteins can all cause constitutive phosphorylation of TRK tyrosine residues, continuously activating downstream signaling pathways such as PI3K, RAS / Raf / ERK, and PLC-γ, leading to the occurrence and development of tumors.

[0011] Cancers carrying NTRK gene fusions or mutations are divided into two major categories: (1) rare cancers with a relatively high proportion of NTRK fusion mutations. For example, the vast majority of infantile fibrosarcomas carry NTRK fusion mutations, but the disease itself is extremely rare; (2) common cancers, such as non-small cell lung cancer, with a relatively low proportion of NTRK fusion mutations, estimated to be only about 1%.

[0012] There are currently only two drugs on the market targeting TRK, both of which belong to the first-generation TRK inhibitors. In November 2018, Larotrectinib (trade name VITRAKVI, code name LOXO-101, 25mg / 100mg capsules, 20mg / mL oral solution) jointly developed by LoxoOncology and Bayer was approved for marketing, for the treatment of children and adult patients with advanced solid tumors carrying NTRK gene fusions. In August 2019, Entrectinib (trade name ROZLYTREK, code name RXDX-101, 100mg / 200mg capsules) developed by Roche was approved for marketing, with two indications, for the treatment of ROS1-positive non-small cell lung cancer, and children (over 12 years old) and adult patients with neurotrophic tyrosine receptor kinase (NTRK) gene fusions. According to literature reports, acquired resistance mutations of TRK inhibitors have been found during the clinical trial research process. Currently, second-generation TRK inhibitors such as LOXO-195 and TPX-0005 developed based on the resistance mutation sites of patients have also entered the clinical research stage.

[0013]

[0014]

Paper 3

[0015] However, not all TRK inhibitors have an inhibitory effect on the SRC / FAK / JAK2 targets. Except for TPX-0005 mentioned in [Paper 3], whether the combination of other TRK inhibitors and KRAS G12C inhibitors can synergistically enhance the effect is currently uncertain. Summary of the Invention

[0016] The present invention provides a novel class of TRK inhibitors that can effectively inhibit TRK kinases, prevent and / or treat diseases mediated by TRK; and simultaneously provides a combination treatment regimen of the TRK inhibitor and a KRAS G12C inhibitor to increase the efficacy against tumors (especially tumors with KRAS G12C mutations).

[0017] Specifically:

[0018] The present invention provides the use of Compound A in the preparation of a drug for treating tumors, and the structure of Compound A is shown in the following formula (I):

[0019]

[0020] Wherein, X is selected from: a bond, -O-, -S-, -NH- or -CH2-;

[0021] Y, Y1, Y2, Y3, Y4 are each independently selected from: -CH-, N or C;

[0022] X2 is selected from: a bond, -(CH2) p -, or -NH-, wherein p is 1, 2, 3 or 4; when X2 is a bond, the benzene ring is directly connected to Ring A;

[0023] Indicates: the presence or absence of a key;

[0024] R is selected from: C 5～12 aryl or heteroaryl, wherein each aryl or heteroaryl is unsubstituted or substituted with at least one substituent selected from R1;

[0025] Each occurrence of R1 is independently selected from: hydrogen, halogen, -OH, amino, -NHC 1～6 alkyl, -N(C 1～6 alkyl)2, cyano, unsubstituted or substituted with at least one R 1a substituted C 1～6 alkyl, unsubstituted or substituted with at least one R 1a substituted C 1～6 alkoxy, unsubstituted or substituted with at least one R 1a substituted C 3～6 cycloalkyl, unsubstituted or substituted with at least one R 1a substituted C 3～6 cycloalkoxy or -SC 1～6 alkyl;

[0026] R 1a Each occurrence is independently selected from: C 1-6 alkyl, C 1-6 alkoxy, nitro, halogen, -OH, amino, -NHC 1～6 alkyl, -N(C 1～6 alkyl)2 or cyano;

[0027] R2 is selected from: H, halogen, hydroxy, amino, or substituted or unsubstituted C 1～6 alkyl, wherein the substitution means being substituted with 1, 2 or 3 substituents selected from halogen or hydroxy;

[0028] R3 is selected from: H, halogen, -OH, amino, C 1～6 alkyl or C 1～6 alkoxy;

[0029] Ring A is selected from: cycloalkyl, heterocycloalkyl, bridged ring, heterobridged ring, fused ring, heterofused ring, spiro ring, heterospiro ring, wherein the heteroatoms in the heterocycloalkyl, heterobridged ring, heterofused ring and heterospiro ring are independently selected from O, S or N, and the number of heteroatoms is selected from 1, 2, 3 or 4;

[0030] R4 is located at any substitutable position on Ring A and is independently selected from: -H, -OH, halogen, -CN, oxo group, substituted or unsubstituted C 1～6 alkyl, -(CH2) m -OH, -(CH2) m -COOH, -(CH2)m -CO-NH2, -CO-(CH2) m -NH2, -CO-CR 4a R 4b -OH or -CO-R 4b ; wherein, the oxo group means that two Hs at the same substitution position are replaced by the same O to form a double bond; m is selected from 1, 2, 3 or 4; R 4a is selected from hydrogen, unsubstituted or substituted C 1～4 alkyl; R 4b is selected from H, unsubstituted or substituted C 1～6 alkyl or unsubstituted or substituted C 3～6 cycloalkyl, and the substituents of the substitution are independently selected from -OH, -NH2, halogen, and the number of substituents is selected from 1, 2 or 3;

[0031] n is selected from 1, 2, 3 or 4.

[0032] The present invention provides the use of compound A in combination with a KRAS G12C inhibitor in the preparation of a medicament for treating tumors, and the structure of compound A is shown in the above formula (I).

[0033] The present invention provides the use of compound A in the preparation of a medicament for improving the therapeutic effect of a KRAS G12C inhibitor on tumors, and the structure of compound A is shown in the above formula (I).

[0034] The present invention provides the use of a KRAS G12C inhibitor in the preparation of a medicament for improving the therapeutic effect of compound A on tumors, and the structure of compound A is shown in the above formula (I).

[0035] The present invention provides a medicament which comprises compound A, and the structure of compound A is shown in the above formula (I). Preferably, the medicament is used for treating tumors.

[0036] The present invention provides a medicament which comprises compound A and a KRAS G12C inhibitor, and the structure of compound A is shown in the above formula (I). Preferably, the medicament is used for treating tumors.

[0037] The present invention provides a medicament which comprises compound A, and the structure of compound A is shown in the above formula (I), and the medicament is used for improving the therapeutic effect of a KRAS G12C inhibitor on tumors.

[0038] The present invention provides a medicament which comprises a KRAS G12C inhibitor, and the medicament is used for improving the therapeutic effect of compound A on tumors, and the structure of compound A is shown in the above formula (I).

[0039] The present invention provides a method for treating tumors, comprising: administering to a patient or subject a therapeutically effective amount of compound A, the structure of compound A being as shown in the above formula (I).

[0040] The present invention provides a method for treating tumors, comprising: administering to a patient or subject a therapeutically effective amount of compound A and a KRAS G12C inhibitor, the structure of compound A being as shown in the above formula (I).

[0041] The present invention provides a method for improving the effect of a KRAS G12C inhibitor in treating tumors, comprising: administering to a patient or subject a therapeutically effective amount of compound A, the structure of compound A being as shown in the above formula (I).

[0042] The present invention provides a method for improving the effect of compound A in treating tumors, comprising: administering to a patient or subject a therapeutically effective amount of a KRAS G12C inhibitor, the structure of compound A being as shown in the above formula (I).

[0043] In the above technical solutions:

[0044] Preferably, for the said compound A, in one embodiment, R is selected from: aryl or heteroaryl of C 5～9 , wherein each aryl or heteroaryl is unsubstituted or substituted by at least one substituent selected from R1; or, R is selected from: aryl or heteroaryl of C 5～6 , wherein each aryl or heteroaryl is unsubstituted or substituted by at least one substituent selected from R1.

[0045] In one embodiment, the compound A has a structure as shown in formula (I-1) or (I-2):

[0046]

[0047] wherein, R1, R2, R3, R4, X, X2, Y, Y1, Y2, Y3, Y4, ring A and n have the same definitions as in the compound of formula (I) of the present invention, and X1 is selected from: -CH- or N.

[0048] In one embodiment, the compound A has a structure as shown in formula (I-A):

[0049]

[0050] wherein, R1, R2, R3, R4, X, X1, X2, Y, Y1, Y2, Y3, Y4, ring A and n have the same definitions as in the compound of formula (I-1) or (I-2) of the present invention.

[0051] In one embodiment, the compound A has a structure as shown in formula (I-A-1a), (I-A-1b), or (I-A-1c):

[0052]

[0053] Wherein, R1, R2, R3, R4, X, X1, X2, ring A, and n have the same definitions as in the compounds of formula (I-1) or (I-2) of the present invention.

[0054] In one embodiment, the compound A has a structure as shown in formula (I-B):

[0055]

[0056] Wherein, R1, R2, R3, R4, X1, X2, Y, Y1, Y2, Y3, Y4, ring A, and n have the same definitions as in the compounds of formula (I-1) or (I-2) of the present invention.

[0057] In one embodiment, the compound A has a structure as shown in formula (I-B-1a), (I-B-1b), or (I-B-1c):

[0058]

[0059] Wherein, R1, R2, R3, R4, X1, X2, ring A, and n have the same definitions as in the compounds of formula (I-1) or (I-2) of the present invention.

[0060] In one embodiment, the compound A has a structure as shown in formula (I-C):

[0061]

[0062] Wherein, R1, R2, R3, R4, X1, X2, Y, Y1, Y2, Y3, Y4, ring A, and n have the same definitions as in the compounds of formula (I-1) or (I-2) of the present invention.

[0063] In one embodiment, the compound A has a structure as shown in formula (I-C-1a), (I-C-1b), or (I-C-1c):

[0064]

[0065] Wherein, R1, R2, R3, R4, X1, X2, ring A, and n have the same definitions as in the compounds of formula (I-1) or (I-2) of the present invention.

[0066] In one embodiment, the compound A (including the compound of formula (I), the compound of formula (I-1), the compound of formula (I-2), the compound of formula (I-A), the compound of formula (I-A-1a), the compound of formula (I-A-1b), the compound of formula (I-A-1c), the compound of formula (I-B), the compound of formula (I-B-1a), the compound of formula (I-B-1b), the compound of formula (I-B-1c), the compound of formula (I-C), the compound of formula (I-C-1a), the compound of formula (I-C-1b), the compound of formula (I-C-1c)), wherein, each occurrence of R1 is independently selected from: hydrogen, halogen, -OH, amino, cyano, unsubstituted or substituted by at least one R 1a substituted C 1～6 alkyl, unsubstituted or substituted by at least one R 1a substituted C 1～6 alkoxy, unsubstituted or substituted by at least one R 1a substituted C 3～6 cycloalkyl or unsubstituted or substituted by at least one R 1a substituted C 3～6 cycloalkoxy; or, each occurrence of R1 is independently selected from: hydrogen, halogen, -OH, amino, cyano, unsubstituted or substituted by at least one R 1a substituted C 1～6 alkyl or unsubstituted or substituted by at least one R 1a substituted C 1～6 alkoxy; or, each occurrence of R1 is independently selected from: hydrogen, halogen, -OH, amino, C 1～6 alkyl or C 1～6 alkoxy; or, each occurrence of R1 is independently selected from: hydrogen, halogen, -OH, amino, C 1～3 alkyl or C 1～3 alkoxy; or, each occurrence of R1 is independently selected from: hydrogen, halogen, -OH, amino, methyl, ethyl, methoxy or ethoxy; or, each occurrence of R1 is independently selected from: hydrogen, F, Cl, -OH, methyl or methoxy; or, each occurrence of R1 is independently selected from: F, -OH, methyl or methoxy.

[0067] In one embodiment, R 1a each occurrence is independently selected from: C 1-3 alkyl, C 1-3 alkoxy, nitro, halogen, -OH, amino, -NHC 1～6 alkyl, -N(C 1～6 alkyl)2 or cyano; or, R 1a is selected from: halogen, -OH, amino, -NHC 1～3 alkyl, -N(C1～3 (alkyl)2 or cyano; or, R 1a is selected from: halogen, -OH, amino or cyano; or, R 1a is selected from: F, Cl or -OH.

[0068] In one embodiment, the compound A (including the compound of formula (I), the compound of formula (I-1), the compound of formula (I-2), the compound of formula (I-A), the compound of formula (I-A-1a), the compound of formula (I-A-1b), the compound of formula (I-A-1c), the compound of formula (I-B), the compound of formula (I-B-1a), the compound of formula (I-B-1b), the compound of formula (I-B-1c), the compound of formula (I-C), the compound of formula (I-C-1a), the compound of formula (I-C-1b), the compound of formula (I-C-1c)), wherein, R2 is selected from: H, F, OH or substituted or unsubstituted C 1～6 alkyl, the substitution means being substituted by 1, 2 or 3 substituents selected from halogen or hydroxyl; or, R2 is selected from H, F, -OH or C 1～6 alkyl; or, R2 is selected from H, F, -OH or C 1～3 alkyl; or, R2 is selected from H or F.

[0069] In one embodiment, the compound A (including the compound of formula (I), the compound of formula (I-1), the compound of formula (I-2), the compound of formula (I-A), the compound of formula (I-A-1a), the compound of formula (I-A-1b), the compound of formula (I-A-1c), the compound of formula (I-B), the compound of formula (I-B-1a), the compound of formula (I-B-1b), the compound of formula (I-B-1c), the compound of formula (I-C), the compound of formula (I-C-1a), the compound of formula (I-C-1b), the compound of formula (I-C-1c)), wherein, X1 is selected from -CH- or N, and R1 is selected from F or -OCH3.

[0070] In one embodiment, X1 is selected from -CH-, and R1 is selected from F; or, X1 is selected from N, and R1 is selected from -OCH3.

[0071] In one embodiment, the compound A has the structure shown in formula (I-D):

[0072]

[0073] wherein, R3, R4, X2, Y, Y1, Y2, Y3, Y4, ring A and n have the same definitions as in the compound of formula (I) of the present invention.

[0074] In one embodiment, Compound A has a structure represented by Formula (I-D-a), Formula (I-D-b), or Formula (I-D-c):

[0075]

[0076] Wherein, R3, R4, X2, Ring A, and n have the same definitions as in the compound of Formula (I) of the present invention.

[0077] In one embodiment, Compound A (including the compound of Formula (I), the compound of Formula (I-1), the compound of Formula (I-2), the compound of Formula (I-A), the compound of Formula (I-A-1a), the compound of Formula (I-A-1b), the compound of Formula (I-A-1c), the compound of Formula (I-B), the compound of Formula (I-B-1a), the compound of Formula (I-B-1b), the compound of Formula (I-B-1c), the compound of Formula (I-C), the compound of Formula (I-C-1a), the compound of Formula (I-C-1b), the compound of Formula (I-C-1c), the compound of Formula (I-D), the compound of Formula (I-D-a), the compound of Formula (I-D-b), the compound of Formula (I-D-c)), wherein, R3 is selected from: H, halogen, -OH, amino, C 1～3 alkyl or C 1～3 alkoxy; alternatively, R3 is selected from: H, F, Cl, -OH, methyl, or methoxy; alternatively, R3 is selected from: H or F.

[0078] In one embodiment, Compound A has a structure represented by Formula (I-D-1a), Formula (I-D-1b), or Formula (I-D-1c):

[0079]

[0080] Wherein, R4, X2, Ring A, and n have the same definitions as in the compound of Formula (I) of the present invention.

[0081] In one embodiment, Compound A has a structure represented by Formula (I-C-1a-1):

[0082]

[0083] Wherein, R4, X2, Ring A, and n have the same definitions as in Formula (I) of the present invention.

[0084] In one embodiment, Compound A has a structure represented by Formula (I-C-1a-2):

[0085]

[0086] Wherein, R4, X2, Ring A, and n have the same definitions as in the compound of Formula (I) of the present invention.

[0087] In one embodiment, the compound A (including the compounds of formula (I), formula (I-1), formula (I-2), formula (I-A), formula (I-A-1a), formula (I-A-1b), formula (I-A-1c), formula (I-B), formula (I-B-1a), formula (I-B-1b), formula (I-B-1c), formula (I-C), formula (I-C-1a), formula (I-C-1b), formula (I-C-1c), formula (I-D), formula (I-D-a), formula (I-D-b), formula (I-D-c), formula (I-D-1a), formula (I-D-1b), formula (I-D-1c), formula (I-C-1a-1), and formula (I-C-1a-2), the same hereinafter), wherein ring A is selected from: C 3-6 cycloalkyl, 4-6 membered heterocycloalkyl, C 6-8 bridged ring group, 6-8 membered heterobridged ring group, C 8-10 fused ring group, 8-10 membered heterofused ring group, C 7-12 monospiro ring group, 7-12 membered heteromonospiro ring group; or, ring A is selected from: 4-6 membered heterocycloalkyl, 6-8 membered heterobridged ring group, 8-10 membered heterofused ring group, 7-12 membered heteromonospiro ring group; wherein the heteroatoms in the heterocycloalkyl, heterobridged ring group, heterofused ring group and heteromonospiro ring group are independently selected from O, S or N, and the number of heteroatoms is selected from 1, 2, 3 or 4; or, ring A is selected from the following structures:

[0088]

[0089] Or, ring A is selected from the following structures:

[0090]

[0091] Or, ring A is selected from the following structures:

[0092]

[0093] Or, ring A is selected from the following structures:

[0094]

[0095] Or, ring A is selected from the following structures:

[0096]

[0097] In one embodiment, the compound A (including the compound of formula (I), the compound of formula (I-1), the compound of formula (I-2), the compound of formula (I-A), the compound of formula (I-A-1a), the compound of formula (I-A-1b), the compound of formula (I-A-1c), the compound of formula (I-B), the compound of formula (I-B-1a), the compound of formula (I-B-1b), the compound of formula (I-B-1c), the compound of formula (I-C), the compound of formula (I-C-1a), the compound of formula (I-C-1b), the compound of formula (I-C-1c), the compound of formula (I-D), the compound of formula (I-D-a), the compound of formula (I-D-b), the compound of formula (I-D-c), the compound of formula (I-D-1a), the compound of formula (I-D-1b), the compound of formula (I-D-1c), the compound of formula (I-C-1a-1), and the compound of formula (I-C-1a-2)), wherein, R4 is independently selected from: -H, -OH, halogen, -CN, oxo group, substituted or unsubstituted C 1～6 alkyl, -COOH, -CONH2, -CO-CR 4a R 4b -OH or -CO-R 4b ; or, R4 is independently selected from: -H, -OH, halogen, -CN, oxo group, substituted or unsubstituted C 1～6 alkyl, -CO-CR 4a R 4b -OH or -CO-R 4b ; or, R4 is independently selected from: -H, -OH, halogen, substituted or unsubstituted C 1～6 alkyl, -CO-CR 4a R 4b -OH or -CO-R 4b ; or, R4 is independently selected from: -H, -OH, halogen, substituted or unsubstituted C 1～3 alkyl, -CO-CR 4a R 4b -OH or -CO-R 4b ; or, R4 is independently selected from: -H, -OH, substituted or unsubstituted C 1～3 alkyl, -CO-CR 4a R 4b -OH or -CO-R 4b ; or, R4 is independently selected from: -CO-CR 4a R 4b -OH or -CO-R 4b ; wherein, the oxo group means that two H at the same substitution position are replaced by the same O to form a double bond; R 4a is selected from hydrogen, unsubstituted or substituted C 1～4 alkyl; R 4bSelected from unsubstituted or substituted C 1～6 alkyl or unsubstituted or substituted C 3～6 cycloalkyl, wherein the substituents of the substitution are independently selected from -OH, -NH2, or halogen, and the number of substituents is selected from 1, 2, or 3; alternatively, the substituents of the substitution are independently selected from -OH or F, and the number of substituents is selected from 1, 2, or 3.

[0098] In one embodiment, the compound A has the structure shown in formula (I-A-1a):

[0099]

[0100] Wherein, X is selected from: a bond, -O-, -S-, -NH-, or -CH2-;

[0101] X1 is selected from: -CH- or N;

[0102] X2 is selected from: a bond, -(CH2) p -, or -NH-, wherein p is 1, 2, 3, or 4;

[0103] R1 is selected from: H, halogen, -OH, amino, C 1～6 alkyl or C 1～6 alkoxy;

[0104] R2 is selected from: H, F, OH, or substituted or unsubstituted C 1～6 alkyl, and the substitution means being substituted by 1, 2, or 3 substituents selected from halogen or hydroxyl;

[0105] R3 is selected from: H, halogen, -OH, amino, C 1～6 alkyl or C 1～6 alkoxy;

[0106] Ring A is selected from: cycloalkyl, heterocycloalkyl, bridged ring group, heterobridged ring group, fused ring group, hetero-fused ring group, spiro ring group, heterospiro ring group, wherein the heteroatoms in the heterocycloalkyl, heterobridged ring group, hetero-fused ring group, and heterospiro ring group are independently selected from O, S, or N, and the number of heteroatoms is selected from 1, 2, 3, or 4;

[0107] R4 is located at any substitutable position on ring A and is independently selected from: -H, -OH, halogen, -CN, oxo group, substituted or unsubstituted C 1～6 alkyl, -(CH2) m -OH, -(CH2) m -COOH, -(CH2) m -CO-NH2, -CO-(CH2) m -NH2, -CO-CR 4a R 4b -OH or -CO-R4b ; wherein, the oxo group means that two Hs at the same substitution position are replaced by the same O to form a double bond; m is selected from 1, 2, 3 or 4; R 4a is selected from hydrogen, unsubstituted or substituted C 1～4 alkyl; R 4b is selected from H, unsubstituted or substituted C 1～6 alkyl or unsubstituted or substituted C 3～6 cycloalkyl, and the substituents of the substitution are independently selected from -OH, -NH2, halogen, and the number of substituents is selected from 1, 2 or 3;

[0108] n is selected from 1, 2, 3 or 4.

[0109] In one embodiment, the compound A has a structure shown in formula (I-B-1a):

[0110]

[0111] wherein, R1, R2, R3, R4, X1, X2, ring A and n have the same definitions as in the compound of formula (I-A-1a) of the present invention.

[0112] In one embodiment, the compound A has a structure shown in formula (I-D-1a):

[0113]

[0114] wherein, R1, R3, R4, X1, X2, ring A and n have the same definitions as in the compound of formula (I-A-1a) of the present invention.

[0115] In one embodiment, the compound A has a structure shown in formula (I-C-1a):

[0116]

[0117] wherein, R1, R3, R4, X1, X2, ring A and n have the same definitions as in the compound of formula (I-A-1a) of the present invention.

[0118] In one embodiment, the compound A has a structure shown in formula (I-C-1a-1):

[0119]

[0120] wherein, R4, X2, ring A and n have the same definitions as in the compound of formula (I-A-1a) of the present invention.

[0121] In one embodiment, the compound A has a structure shown in formula (I-C-1a-2):

[0122]

[0123] Among them, R4, X2, ring A and n have the same definitions as in the compound of formula (I-A-1a) of the present invention.

[0124] In one embodiment, in the compound A, ring A is selected from: C 3-6 cycloalkyl, 4- to 6-membered heterocycloalkyl, C 6-8 bridged ring group, 6- to 8-membered heterobridged ring group, C 8-10 fused ring group, 8- to 10-membered heterofused ring group, C 7-12 monospiro ring group, 7- to 12-membered heteromonospiro ring group, wherein the heteroatoms in the heterocycloalkyl, heterobridged ring group, heterofused ring group and heteromonospiro ring group are independently selected from O, S or N, and the number of heteroatoms is selected from 1, 2 or 3.

[0125] In one embodiment, in the compound A, ring A is selected from the following structures:

[0126]

[0127] In one embodiment, in the compound A, R4 is independently selected from: -H, -OH, halogen, -CN, oxo group, substituted or unsubstituted C 1～6 alkyl, -COOH, -CONH2, -CO-CR 4a R 4b -OH or -CO-R 4b , wherein the oxo group means that two Hs at the same substitution position are replaced by the same O to form a double bond; R 4a is selected from hydrogen, unsubstituted or substituted C 1～4 alkyl; R 4b is selected from unsubstituted or substituted C 1～6 alkyl or unsubstituted or substituted C 3～6 cycloalkyl, and the substituents of the substituted ones are independently selected from -OH, -NH2, halogen, and the number of substituents is selected from 1, 2 or 3.

[0128] In one embodiment, the compound A has the following structure:

[0129]

[0130]

[0131]

[0132]

[0133]

[0134]

[0135]

[0136] In one embodiment, the compound A has the structure shown in the following formula (4):

[0137]

[0138] In the above technical solutions,

[0139] Preferably, the KRAS G12C inhibitor is selected from AMG-510, MRTX849, JNJ-74699157 (ARS-3248), ARS-1620, MRTX1257, RM-007, ADT-007, preferably AMG510 and MRTX849, more preferably AMG510. The structures of AMG510 and MRTX849 are shown as follows respectively:

[0140]

[0141] Preferably, the tumor is selected from tumors with KRAS G12C mutation, and the tumors include lung cancer, colorectal cancer, pancreatic cancer, uterine cancer, bladder cancer, esophageal cancer, gastric cancer, kidney cancer, breast cancer, skin cancer, prostate cancer, leukemia, cervical cancer, liver cancer, ovarian cancer and brain cancer. The leukemia includes acute myeloid leukemia and acute lymphoblastic leukemia; preferably lung cancer, pancreatic cancer and colorectal cancer, and the lung cancer is preferably non-small cell lung cancer and lung adenocarcinoma.

[0142] In some embodiments, the drug of the present invention further comprises a pharmaceutically acceptable excipient or a pharmaceutically acceptable carrier.

[0143] Each preparation unit of the drug of the present invention contains 0.1-1000 mg of compound A, preferably 1-500 mg, and exemplary contents are 1000 mg, 900 mg, 800 mg, 700 mg, 600 mg, 500 mg, 400 mg, 300 mg, 250 mg, 200 mg, 190 mg, 180 mg, 170 mg, 160 mg, 150 mg, 140 mg, 130 mg, 120 mg, 110 mg, 100 mg, 90 mg, 80 mg, 70 mg, 60 mg, 50 mg, 40 mg, 30 mg, 20 mg, 15 mg, 10 mg, 5 mg, 3 mg, 2 mg, 1 mg and range values between any two of the above point values.

[0144] Each preparation unit of the drug of the present invention contains 0.1-1000 mg of KRAS G12CAn inhibitor, preferably 1 - 1000 mg, exemplary amounts being 1000 mg, 900 mg, 800 mg, 700 mg, 600 mg, 500 mg, 400 mg, 300 mg, 250 mg, 200 mg, 190 mg, 180 mg, 170 mg, 160 mg, 150 mg, 140 mg, 130 mg, 120 mg, 110 mg, 100 mg, 90 mg, 80 mg, 70 mg, 60 mg, 50 mg, 40 mg, 30 mg, 20 mg, 15 mg, 10 mg, 5 mg, 3 mg, 2 mg, 1 mg, and ranges between any two of these point values. In some embodiments, the KRAS G12C inhibitor is AMG510, and each dosage unit of the drug contains about 1 - 1000 mg of AMG510, preferably 1 - 500 mg, 1 - 400 mg, 1 - 300 mg, or 1 - 200 mg, for example, containing about 960 mg, 480 mg, 240 mg, 120 mg of AMG510. In some embodiments, the KRAS G12C inhibitor is MRTX849, and each dosage unit of the drug contains about 1 - 1000 mg of MRTX849, preferably 1 - 600 mg, 1 - 500 mg, 1 - 400 mg, 1 - 300 mg, or 1 - 200 mg, for example, containing about 600 mg, 300 mg, 200 mg, 150 mg, 100 mg of MRTX849.

[0145] Compound A and the KRAS G12C inhibitor can be included in the same dosage unit or in separate dosage units, and the drug can be formulated in the form of a combination product. The dosage forms are selected from oral dosage forms, injection dosage forms, topical dosage forms, or external use dosage forms, etc. Accordingly, compound A and the KRAS G12C inhibitor are administered by injection, orally, topically, or in vitro. Compound A and the KRAS G12C inhibitor can be administered simultaneously, or at intervals, or sequentially.

[0146] The drug of the present invention is administered to a patient or subject in a therapeutically effective amount to treat tumors. The therapeutically effective amount can relieve or eliminate the clinical symptoms, signs, pathological changes, etc. of the tumors of the patient or subject. Among them, the therapeutically effective amount of Compound A is 1 - 1000 mg, preferably 1 - 800 mg, 1 - 600 mg, 1 - 500 mg, 1 - 400 mg, 1 - 350 mg, 1 - 360 mg, 1 - 300 mg, 1 - 250 mg, 1 - 240 mg, 1 - 200 mg, 1 - 180 mg, 1 - 150 mg. Exemplary doses include 15 mg, 30 mg, 60 mg, 75 mg, 90 mg, 120 mg, 150 mg, 180 mg, 200 mg, 240 mg, 250 mg, 300 mg, 350 mg, 360 mg, 400 mg, 450 mg, 500 mg and the range values between any two of the above point values. The above therapeutically effective amount can be a single administration dose or a cumulative dose administered in 2 - 3 divided doses per day. For example, 15 mg, 30 mg, 60 mg, 90 mg, 120 mg or 150 mg each time, orally administered 1 or 2 times a day.

[0147] KRAS G12C The therapeutically effective amount of the inhibitor is 1 - 1500 mg, such as 1500 mg, 1200 mg, 1000 mg, 900 mg, 800 mg, 700 mg, 600 mg, 500 mg, 400 mg, 300 mg, 250 mg, 200 mg, 190 mg, 180 mg, 170 mg, 160 mg, 150 mg, 140 mg, 130 mg, 120 mg, 110 mg, 100 mg, 90 mg, 80 mg, 70 mg, 60 mg, 50 mg, 40 mg, 30 mg, 20 mg, 15 mg, 10 mg, 5 mg, 3 mg, 2 mg, 1 mg and the range values between any two of the above point values; the above therapeutically effective amount can be a single administration dose or a cumulative dose administered in 2 - 3 divided doses per day. In some embodiments, the G12C inhibitor is AMG510, and its therapeutically effective amount is about 1 - 1200 mg, preferably 100 - 1200 mg, 200 - 1200 mg, 300 - 1200 mg, 400 - 1100 mg, 500 - 1100 mg, 600 - 1100 mg, 700 - 1100 mg, 800 - 1000 mg or 900 - 1000 mg; the above therapeutically effective amount can be a single administration dose or a cumulative dose administered in 2 - 3 divided doses per day. In one embodiment, the therapeutically effective amount of AMG510 is 960 mg each time, orally administered 1 time a day. In some embodiments, the G12CThe inhibitor is MRTX849, and its therapeutically effective amount is 1 - 1500 mg, preferably 100 - 1500 mg, 200 - 1400 mg, 300 - 1300 mg, 400 - 1200 mg, 500 - 1200 mg, 600 - 1200 mg, 700 - 1200 mg,

[0148] 800 - 1200 mg, 900 - 1200 mg or 1000 - 1200 mg; the above - mentioned therapeutically effective amount can be a single - administration dose or an accumulated dose administered 2 - 3 times a day. In one embodiment, the effective therapeutic amount of MRTX849 is 600 mg each time, orally administered 2 times a day.

[0149] The above - mentioned embodiments represent exemplary embodiments of the present invention, but the present invention is not limited to the above - mentioned embodiments. In addition, the various technical features in the above - mentioned embodiments of the present invention can be combined with each other, thereby constituting one or more new technical solutions, and these new technical solutions also fall within the scope of the present invention as long as such new technical solutions are technically feasible.

[0150] The present invention also provides a method for preparing compound A, comprising the following steps:

[0151]

[0152] Compound Im reacts with compound R4 - OH to obtain the compound shown in formula I;

[0153] Wherein, R, R2, R3, R4, X, X2, Y, Y1, Y2, Y3, Y4, Ring A and n have the same definitions as above.

[0154] In one embodiment, the compound Im is prepared by the following method:

[0155]

[0156] Compound In reacts with compound Ip to obtain compound Im;

[0157] Wherein, R, R2, R3, R4, X, X2, Y, Y1, Y2, Y3, Y4, and ring A have the same definitions as above.

[0158] Definitions

[0159] Unless otherwise specified, the following terms referred to in the present invention have the following definitions.

[0160] In the present invention, for some substituents, the " " indicates the connection site.

[0161] The term "alkyl" refers to a monovalent saturated aliphatic hydrocarbon group, including straight-chain or branched-chain groups containing 1-20 carbon atoms, preferably containing 1-10 carbon atoms (i.e., C 1-10 alkyl), more preferably containing 1-8 carbon atoms (C 1-8 alkyl), still more preferably containing 1-6 carbon atoms (i.e., C 1-6 alkyl). For example, "C 1-6 alkyl" means that the group is an alkyl group and the number of carbon atoms in the carbon chain is between 1 and 6 (specifically 1, 2, 3, 4, 5, or 6). Examples include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, neopentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, n-heptyl, n-octyl, etc.

[0162] The term "carbocyclic group" or "carbocycle" denotes a monovalent or polyvalent saturated or partially unsaturated monocyclic, bicyclic or tricyclic system containing 3-12 carbon atoms, wherein the monocyclic, bicyclic or tricyclic system does not contain an aromatic ring. Carbobicyclic groups include bridged-ring groups, spiro-ring groups, fused-ring groups, etc. A bridged-ring group means that any two rings share two directly or indirectly connected atoms.

[0163] The term "cycloalkyl" refers to a monocyclic saturated aliphatic hydrocarbon group having a specific number of carbon atoms, preferably containing 3-12 carbon atoms (i.e., C 3-12 cycloalkyl), more preferably containing 3-10 carbon atoms (C 3-10 cycloalkyl), still more preferably 3-6 carbon atoms (C 3-6 cycloalkyl), 4-6 carbon atoms (C 4-6 cycloalkyl), 5-6 carbon atoms (C 5-6 cycloalkyl). Examples include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, methylcyclopropyl, 2-ethyl-cyclopentyl, dimethylcyclobutyl, etc.

[0164] The term "alkoxy" refers to -O-alkyl, where the alkyl is as defined above, i.e., containing 1-20 carbon atoms, preferably containing 1-10 carbon atoms, more preferably 1-8 carbon atoms, still more preferably 1-6 carbon atoms (specifically 1, 2, 3, 4, 5, or 6). Representative examples include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, butoxy, 1-methylpropoxy, 2-methylpropoxy, tert-butoxy, pentyloxy, 1-methylbutoxy, 2-methylbutoxy, 3-methylbutoxy, 1,1-dimethylpropoxy, 1,2-dimethylpropoxy, 2,2-dimethylpropoxy, 1-ethylpropoxy, etc.

[0165] The term "halogen" or "halo" means F, Cl, Br, I. The term "haloalkyl" means that one, two or more hydrogen atoms or all hydrogen atoms in the alkyl group as defined above are replaced by halogen. Representative examples of haloalkyl include CCl3, CF3, CHCl2, CH2Cl, CH2Br, CH2I, CH2CF3, CF2CF3, etc.

[0166] The term "heterocyclic group" refers to a saturated or partially unsaturated monocyclic, bicyclic or polycyclic cyclic hydrocarbon substituent, which is a non-aromatic structure, containing 3 - 20 ring atoms, wherein one, two, three or more ring atoms are selected from N, O or S, and the remaining ring atoms are C. Preferably, it contains 3 - 12 ring atoms (C 3-12 heterocyclic group), more preferably 3 - 10 ring atoms (C 3-10 heterocyclic group), or 3 - 8 ring atoms (C 3-8 heterocyclic group), or 3 - 6 ring atoms (C 3-6 heterocyclic group), or 4 - 6 ring atoms (C 4-6 heterocyclic group), or 5 - 6 ring atoms (C 5-6 heterocyclic group). The heteroatoms are preferably 1 - 4, more preferably 1 - 3 (i.e., 1, 2 or 3). Examples of monocyclic heterocyclic groups include pyrrolidinyl, imidazolidinyl, tetrahydrofuryl, dihydropyrrolyl, piperidinyl, piperazinyl, pyranyl, etc. Polycyclic heterocyclic groups include heterospirocyclic groups, heteroannular groups, heterofused ring groups and heterobridged ring groups, etc.

[0167] The term "heterocycloalkyl" means a saturated "heterocyclic group" as defined above, containing 3 - 20 ring atoms, wherein one, two, three or more ring atoms are selected from N, O or S, and the remaining ring atoms are C. Preferably, it contains 3 - 12 ring atoms (C 3-12 heterocycloalkyl), more preferably 3 - 10 ring atoms (C 3-10 heterocycloalkyl), or 3 - 8 ring atoms (C 3-8 heterocycloalkyl), or 3 - 7 ring atoms (C 3-7 heterocycloalkyl), or 3 - 6 ring atoms (C 3-6 heterocycloalkyl), or 4 - 6 ring atoms (C 4-6 heterocycloalkyl), or 5 - 6 ring atoms (C 5-6 heterocycloalkyl). The heteroatoms are preferably 1 - 4, more preferably 1 - 3 (i.e., 1, 2 or 3). Examples include aziridinyl, oxiranyl, thiiranyl, azetidinyl, oxetanyl, thietanyl, pyrrolidinyl, tetrahydrofuryl, oxane, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, dioxolanyl, dithiane, oxazolidinyl, thiazolidinyl, pyrazolidinyl, imidazolidine, etc.

[0168] The term "aryl" refers to a monocyclic, bicyclic, and tricyclic aromatic carbocyclic system containing 6 - 16 carbon atoms, or 6 - 14 carbon atoms, or 6 - 12 carbon atoms, or 6 - 10 carbon atoms, preferably 6 - 10 carbon atoms. The term "aryl" can be used interchangeably with the term "aromatic ring". Examples of aryl groups can include, but are not limited to, phenyl, naphthyl, anthracenyl, phenanthryl, pyrenyl, etc.

[0169] The term "heteroaryl" refers to an aromatic monocyclic or polycyclic ring system having a 5 - 12 membered structure, or preferably a 5 - 10 membered structure, a 5 - 8 membered structure, more preferably a 5 - 6 membered structure, wherein one, two, three or more ring atoms are heteroatoms and the remaining atoms are carbon. The heteroatoms are independently selected from O, N, or S, and the number of heteroatoms is preferably 1, 2, or 3. Examples of heteroaryl include, but are not limited to, furyl, thienyl, oxazolyl, thiazolyl, isoxazolyl, oxadiazolyl, thiadiazolyl, pyrrolyl, pyrazolyl, imidazolyl, triazolyl, tetrazolyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, thiodiazolyl, triazinyl, phthalazinyl, quinolinyl, isoquinolinyl, pteridinyl, purinyl, indolyl, isoindolyl, indazolyl, benzofuryl, benzothienyl, benzopyridyl, benzopyrimidinyl, benzopyrazinyl, benzimidazolyl, benzophthalazinyl, pyrrolo[2,3 - b]pyridyl, imidazo[1,2 - a]pyridyl, pyrazolo[1,5 - a]pyridyl, pyrazolo[1,5 - a]pyrimidinyl, imidazo[1,2 - b]pyridazinyl, [1,2,4]triazolo[4,3 - b]pyridazinyl, [1,2,4]triazolo[1,5 - a]pyrimidinyl, [1,2,4]triazolo[1,5 - a]pyridyl, etc.

[0170] The active compounds described in the present invention, such as compound A, AMG510, and MRTX849, etc., are interpreted to include the said compounds and their tautomers, stereoisomers, optical isomers, solvates, N - oxides, prodrugs, isotope derivatives, or pharmaceutically acceptable salts thereof. The tautomers, stereoisomers, optical isomers, solvates, N - oxides, prodrugs, isotope derivatives, or pharmaceutically acceptable salts of the said compounds are obtained by conventional technical means in the art and exert the same or similar effects in vivo and in vitro with substantially the same mechanism of action as the said compounds.

[0171] The term "pharmaceutically acceptable salt" or "medicinal salt" refers to a salt that is suitable for contact with mammalian tissues, especially human tissues, within the scope of reasonable medical judgment, without excessive toxicity, irritation, allergic reaction, etc., and is commensurate with a reasonable benefit / risk ratio. If the said compound is basic, the pharmaceutically acceptable salt includes salts prepared from inorganic acids and also includes salts prepared from organic acids. If the said compound is acidic, the pharmaceutically acceptable salt includes salts prepared from inorganic bases and / or organic bases.

[0172] The term "stereoisomer" refers to isomers resulting from different spatial arrangements of atoms in a molecule, including configurational isomers and conformational isomers. Among them, configurational isomers include geometric isomers (or cis-trans isomers) and optical isomers (including enantiomers and diastereomers).

[0173] Geometric isomers can exist in this compound. The compounds of the present invention may contain carbon-carbon double bonds or carbon-nitrogen double bonds in the E or Z configuration, where the term "E" represents higher-order substituents on the opposite sides of the carbon-carbon or carbon-nitrogen double bond, and the term "Z" represents higher-order substituents on the same side of the carbon-carbon or carbon-nitrogen double bond (determined using the Cahn-Ingold-Prelog priority rules). The compounds of the present invention may also exist in the form of a mixture of "E" and "Z" isomers. The substituents around the cycloalkyl or heterocycloalkyl are referred to as cis or trans configurations.

[0174] Optical isomers refer to substances with exactly the same molecular structure, similar physical and chemical properties, but different optical activities. The compounds of the present invention may contain asymmetrically substituted carbon atoms in the R or S configuration, where the terms "R" and "S" are defined as in IUPAC 1974 Recommendations for Section E, Fundamental Stereochemistry, Pure Appl. Chem. (1976) 45, 13-10. Compounds with asymmetrically substituted carbon atoms (with an equal number of R and S configurations) are racemic at those carbon atoms. Atoms with an excess of one configuration (relative to the other) result in a higher number of that configuration, preferably in an excess of about 85%-90%, more preferably in an excess of about 95%-99%, and even more preferably in an excess greater than about 99%. Accordingly, the present invention includes racemic mixtures, relative and absolute optical isomers, and mixtures of relative and absolute optical isomers.

[0175] The term "nitroxide" means that when a compound contains several amine functional groups, one or more nitrogen atoms can be oxidized to form N-oxides. Specific examples of N-oxides are N-oxides of tertiary amines or N-oxides on nitrogen atoms of nitrogen-containing heterocycles.

[0176] The term "solvate" refers to an association formed by one or more solvent molecules with the compounds of the present invention.

[0177] The term "tautomer" refers to structural isomers that have different energies and can interconvert via a low energy barrier. If tautomerization is possible (e.g., in solution), a chemical equilibrium of tautomers can be achieved. For example, prototropic tautomers (also known as proton-transfer tautomers) include interconversions that occur via proton migration, such as keto-enol isomerization and imine-enamine isomerization. Valence tautomers include interconversions that occur via the reorganization of some bonding electrons. Unless otherwise indicated, all tautomeric forms of the compounds of the present invention are within the scope of the present invention.

[0178] The term "isotope derivative" means that the compounds of the present invention can exist in isotopically labeled or enriched forms, containing one or more atoms whose atomic weights or mass numbers are different from those of the atoms found in the greatest amounts in nature. Isotopes can be radioactive or non-radioactive isotopes. Isotopes of atoms such as hydrogen, carbon, phosphorus, sulfur, fluorine, chlorine, and iodine include, but are not limited to: 2 H, 3 H, 13 C, 14 C, 15 N, 18 O, 32 P, 35 S, 18 F, 36 Cl and 125 I. Compounds containing these and / or other isotopes of atoms are within the scope of the present invention.

[0179] In another embodiment, the isotopically labeled compound contains deuterium ( 2 H), tritium ( 3 H), or 14 C isotopes. The isotopically labeled compounds of the present invention can be prepared using general methods well known to those of ordinary skill in the art. In this regard, relevant literature includes: Lizondo, J et al, Drugs Fut, 21(11), 1116(1996); Brickner, S J et al, J Med Chem, 39(3), 673(1996); Mallesham, B et al, Org Lett, 5(7), 963(2003).

[0180] Compounds containing isotopes have been used in pharmaceutical research to study the in vivo metabolic fate of compounds by evaluating the mechanism of action and metabolic pathways of the parent compound using non-isotopic tracers (Blake et al, J. Pharm. Sci. 64, 3, 367 - 391 (1975)). Such metabolic studies are important in the design of safe and effective therapeutic agents because the active compound in vivo administered to a patient or a metabolite produced from the parent compound has been shown to be toxic or carcinogenic (Kushner et al, Can. J. Physiol. Pharmacol., 77, 79 - 88 (1999); Foster et al, Advances in Drug Research Vol. 14, pp. 2 - 36, Academic press, London, 1985; Kato et al, J. Labelled Comp. Radiopharmaceut., 36(10):927 - 932 (1995)).

[0181] In addition, drugs containing non-radioactive active isotopes, such as deuterated drugs known as "heavy drugs", can be used to treat related diseases and disorders. Increasing the amount of the isotope present in the above compounds above its natural abundance is referred to as enrichment. Examples of amounts of enrichment include about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 16, 21, 25, 29, 33, 37, 42, 46, 50, 54, 58, 63, 67, 71, 75, 79, 84, 88, 92, 96 to about 100 mol%.

[0182] Any possible site in the molecular structure can be substituted with an isotope to obtain an isotopic derivative. For example, any possible site in the molecule can be substituted with deuterium ( 2 2H) to obtain a deuterated form of the derivative.

[0183] Drugs traced with stable isotopes can alter the physicochemical properties of the drug, such as pKa and lipid solubility. If the isotope substitution affects the region involved in ligand-receptor interaction, then these effects and alterations can affect the pharmacodynamic response of the drug molecule. Although some physical properties of the stable isotope-traced molecule are different from those of the non-traced molecule, the chemical and biological properties are the same. An important difference is that due to the increased mass of the heavy isotope, any bond involving the heavy isotope and another atom is stronger than the same bond between the light isotope and that atom. Accordingly, incorporating an isotope at the site of metabolism or enzyme-catalyzed transformation can potentially slow down the reaction and, relative to the non-isotopic compound, can alter the pharmacokinetic properties or effects.

[0184] The term "prodrug" or "precursor drug" is a derivative of a designed active drug that can improve certain defined, undesirable physical or biological properties. Physical properties are generally related to solubility (excessive or insufficient lipid or water solubility) or stability, while problematic biological characteristics include too rapid metabolism or poor bioavailability, which may itself be related to physicochemical properties.

[0185] Prodrugs are generally prepared as follows: a) forming esters, half-esters, carbonates, nitrates, amides, hydroxamic acids, carbamates, imines, Mannich bases, phosphates, phosphonates, and enamines of the active drug; b) functionalizing the drug with azo, glycoside, peptide, and ether functional groups; c) using acetalamine, hemiacetalamine, polymer, salt, complex, phosphoramide, acetal, hemiacetal, and ketal forms of the drug. See, e.g., Andrejus Korolkovas’s, “Essentials of Medicinal Chemistry”, John Wiley-Interscience Pulications, John Wiley and Sons, New York (1988), pp. 97-118, which is incorporated herein by reference in its entirety. Esters can be prepared from substrates containing hydroxyl or carboxyl groups by general methods known to those skilled in the art. Typical reactions of these compounds are substitutions in which one heteroatom is replaced by another atom. Amides can be prepared in a similar manner from substrates containing amino or carboxyl groups. Esters can also react with amines or ammonia to form amides. Another way to prepare amides is to heat carboxylic acids and amines together.

[0186] The term "pharmaceutically acceptable excipient" or "pharmaceutically acceptable carrier" includes, but is not limited to, any adjuvant, carrier, excipient, glidant, sweetening agent, diluent, preservative, dye, colorant, flavoring agent, surfactant, wetting agent, dispersing agent, suspending agent, stabilizing agent, isotonic agent, solvent, or emulsifying agent approved by the U.S. Food and Drug Administration, the China National Medical Products Administration, etc. for use in humans or domestic animals.

[0187] The term "tumor" includes benign tumors, malignant tumors, and borderline tumors, where malignant tumors are collectively referred to as cancers.

[0188] As used herein, the term "treatment" refers to alleviating, relieving, or improving the symptoms of a disease or disorder, improving symptoms caused by underlying metabolism, inhibiting a disease or disorder, e.g., preventing the development of a disease or disorder, alleviating a disease or disorder, causing regression of a disease or disorder, alleviating the condition caused by a disease or disorder, or preventing the symptoms of a disease or disorder.

[0189] The term "patient or subject" includes humans and other animals. In one embodiment, the methods described herein can be used in human clinical medicine, veterinary medicine, and animal testing. Thus, a "patient or subject" can be a human, or in the case of veterinary applications, can be a farm animal or pet, or in the course of animal testing, can be a laboratory animal such as a rodent (e.g., mouse, rat), dog, monkey, etc.

[0190] The term "KRAS" G12C "inhibitor" includes, but is not limited to: any compound or reagent known in the art that selectively inhibits the KRAS G12C gene or selectively inhibits the protein (K-ras G12C protein) encoded by the KRAS G12C gene, which is involved in the RAS / MAPK signaling pathway, wherein the K-ras protein product encoded by the KRAS gene has a missense mutation G12C. The terms KRAS gene, K-ras protein, and RAS / MAPK signaling pathway are known and understood by those skilled in the art. The K-ras G12C protein encoded by the mutant gene has a glycine-cysteine mutation at position 12 of the K-ras G12C protein). As the product of the KRAS G12C gene, the production of the K-ras G12C protein is the result of a mutation in the coding sequence, such as a guanine-to-thymine substitution at position 34 of the coding sequence. The KRAS G12C inhibitor can be any reagent known in the art that selectively targets the KRAS G12C gene, and includes those that can selectively inhibit or can selectively interfere with the transcription (and / or translation) of the KRAS G12C gene (or its corresponding messenger RNA) into the K-ras G12C protein, such as siRNA, oligonucleotides, ribonucleic acids, etc. In some embodiments, the KRAS G12C inhibitor can selectively inhibit or selectively interfere with the transcription / translation of KRAS G12C and / or the corresponding messenger RNA, and the KRAS G12C inhibitor can be a biological agent, such as siRNA, oligonucleotides, ribonucleic acids, etc. In some embodiments, the KRAS G12C inhibitor can selectively inhibit the K-ras G12C protein encoded by the KRAS G12C gene, and the KRAS G12C inhibitor can be a biological agent, such as an antibody (e.g., monoclonal antibody or mAb), small molecule drug / inhibitor, or targeting agent. "KRAS" G12CExamples of the "inhibitor" include, but are not limited to, AMG-510, MRTX849, JNJ-74699157 (i.e., ARS-3248), ARS-1620, MRTX1257, RM-007, ADT-007, etc. In some embodiments, the KRAS G12C inhibitor is a compound disclosed in U.S. Patent US20180334454, which is hereby incorporated by reference as an exemplary reference for the KRAS G12C inhibitor. In some embodiments, the KRAS G12C inhibitor is AMG510. In some embodiments, the KRAS G12C inhibitor is a compound disclosed in U.S. Patents US20190270743 and US20190144444, which are hereby incorporated by reference as exemplary references for the KRAS G12C inhibitor. In some embodiments, the KRAS G12C inhibitor is MRTX849. Among them, the structures of AMG510 and MRTX849 are shown as follows:

[0191]

[0192] For the sake of providing a more concise description, some quantitative data in this article do not use the term "about". It should be understood that whether the term "about" is explicitly used or not, each numerical value given here not only includes the actually given value (the given value), but also means including the approximate value of such given value reasonably inferred by those of ordinary skill in the art, including the equivalents and approximate values of such given value due to experimental and / or measurement conditions. The approximate value is preferably ±20%, ±15%, ±10%, ±8%, ±6%, ±5%, ±4%, ±3%, 2%, ±1% based on the given value.

[0193] All patents, patent applications and other disclosures mentioned herein are incorporated herein by reference in their entirety. If the definitions set forth in this article are contrary to or inconsistent with the definitions set forth in the patents, patent applications or other disclosures incorporated herein by reference, the definitions set forth in this article shall prevail over the definitions incorporated herein by reference.

[0194] In the present invention, the terms used are as described below:

[0195] DCM: Dichloromethane; DIPEA: Diisopropylethylamine; DMF: N,N-Dimethylformamide; EA: Ethyl acetate; NBS: N-Bromosuccinimide; PE: Petroleum ether; DMSO: Dimethyl sulfoxide; TBTU: O-Benzotriazol-1-yl-N,N,N',N'-tetramethyluronium tetrafluoroborate; BOP: Benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate; ATP: 5'-Adenosine triphosphate; DTT: 1,4-Dithiothreitol; MTT: 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide.

[0196] The beneficial effects of the present invention are as follows:

[0197] The present invention provides a class of compounds with novel structures. In vitro kinase activity inhibition assays show that the compounds of the present invention exhibit excellent inhibitory activities against TRK, ALK, ROS1 kinases and their mutants, especially against TRK and its mutant forms. In vitro cell inhibition activity assays show that the compounds of the present invention have strong inhibitory effects on various TRK-mutated cells (including drug-resistant cells with TRK mutations), and the IC 50 of the preferred compounds is below 10 nM, and preferably below 3 nM. The results of in vivo tumor inhibition assays show that compared with the control compounds, the compounds of the present invention have better in vivo anti-tumor effects, better tolerance, and higher potential for drug development. In vivo mechanism of action research assays show that the compounds of the present invention can inhibit TRK in tumor tissues, thereby effectively inhibiting the phosphorylation of PLCγ and AKT and inhibiting the growth of tumor tissues. The combined use of the compounds of the present invention and KRAS 50 inhibitors (such as AMG510) can synergistically enhance the inhibitory effects of the drugs on KRAS G12C mutated tumors (such as non-small cell lung cancer, pancreatic cancer), and have good application prospects. G12C BRIEF DESCRIPTION OF THE DRAWINGS

[0198] Attached Figure 1 Time-effect relationship western blot of the inhibitory effect of Compound 4 of the present invention on the phosphorylation of downstream target proteins in tumor tissues of TRKA-G595R mutant drug-resistant tumor-bearing mice. DETAILED DESCRIPTION OF THE INVENTION

[0199] The raw materials, reaction reagents, catalysts or solvents involved in the following detailed description can all be obtained through commercial channels or prepared by conventional methods of the prior art.

[0200] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are generally carried out under conventional conditions or according to the conditions recommended by the manufacturers. Unless otherwise defined, all professional and scientific terms used herein have the same meaning as those familiar to professionals in the field. In addition, any methods and materials similar or equivalent to the described content can be applied to the method of the present invention. The preferred implementation methods and materials shown in the text are only for demonstration purposes.

[0201] Preparation Example A: Preparation of Ethyl 6-chloroimidazo[1,2-b]pyridazine-3-carboxylate

[0202]

[0203] Cool a solution of ethyl (E)-3-ethoxyacrylate (5.00 g, 34.7 mmol, 1 eq) in 1,4-dioxane (50 mL) and water (50 mL) to -10 °C, and add NBS (6.80 g, 38.15 mmol, 1.1 eq) in portions. Allow the temperature to rise to room temperature naturally and react for 2 h. Add 6-chloro-3-aminopyridazine (4.5 g, 34.7 mmol, 1 eq), heat to 80 °C and react for 1.5 h, then cool to room temperature. Concentrate under reduced pressure. Add water (100 mL) and EA (100 mL) to the residue, stir, separate the layers, and extract the aqueous phase with EA (20 mL × 2). Combine the organic phases. Wash the organic phase with H2O (50 mL) and saturated brine (50 mL), concentrate under reduced pressure to remove the organic solvent, and separate the residue by silica gel column chromatography (eluent: n-hexane:EA = 5:1 to 1:1, v / v) to obtain ethyl 6-chloroimidazo[1,2-b]pyridazine-3-carboxylate (5.2 g, yield 59.75%), (ES, m / z): 225.91 [M+H] + 。

[0204] Intermediate Preparation Example 1: (R)-5-(2-(2,5-Difluorophenyl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylic acid (Intermediate A1)

[0205]

[0206] Step a: A mixed solution of (R)-2-(2,5-difluorophenyl)pyrrolidine (5.0 g, 27.292 mmol), ethyl 5-chloropyrazolo[1,5-a]pyrimidine-3-carboxylate (6.14 g, 27.292 mmol), n-butanol (70 mL) and diisopropylamine (6.9 g, 68.230 mmol) was refluxed and stirred at 100 °C for 4 h, concentrated under reduced pressure to obtain an orange viscous solid, anhydrous ether was added, a large amount of solid precipitated out after stirring, and the crude product of ethyl (R)-5-(2-(2,5-difluorophenyl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylate (6.224 g) was obtained by filtration. Without purification, it was directly used in the next reaction, (ES, m / z): 373.02 [M+H] + 。

[0207] Step b: The crude product of ethyl (R)-5-(2-(2,5-difluorophenyl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylate (6.224 g, 16.714 mmol) was dissolved in anhydrous ethanol (40 mL), stirred at 75 °C until the mixed solution was clear and transparent, and then an aqueous solution of LiOH (2.805 g, 66.856 mmol) (40 mL) was added, and the mixture was stirred at 75 °C for 3 h. After cooling to room temperature, it was concentrated under reduced pressure to remove anhydrous ethanol. 1N HCl aqueous solution was slowly added dropwise to adjust the pH to 3-4, a large amount of white solid precipitated out, stirred at room temperature for 30 min and then filtered, and the filter cake was washed with a small amount of purified water. The filter cake was collected, dried and weighed to obtain (R)-5-(2-(2,5-difluorophenyl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylic acid (5.64 g, 98%), (ES, m / z): 345.02 [M+H] + 。

[0208] Intermediate Preparation Example 2: 5-((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylic acid (Intermediate A2)

[0209]

[0210] Step a: A mixed solution of (2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidine (5.826 g, 28.958 mmol), ethyl 5-chloropyrazolo[1,5-a]pyrimidine-3-carboxylate (6.534 g, 28.958 mmol), n-butanol (50 mL) and diisopropylamine (8.790 g, 86.874 mmol) was reacted at 100 °C for 4 h, and concentrated under reduced pressure to obtain crude ethyl 5-((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylate. Without purification, it was directly used in the next step reaction, (ES, m / z): 391.05 [M+H] + .

[0211] Step b: The crude ethyl 5-((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylate was dissolved in anhydrous ethanol (50 mL), and stirred at 75 °C until the system was clear and transparent. Then an aqueous solution of LiOH (4.86 g, 115.832 mmol) (50 mL) was added, and the mixture was stirred at 75 °C for 5 h. After cooling to room temperature, it was concentrated under reduced pressure to remove anhydrous ethanol. 1N HCl aqueous solution was slowly added dropwise to adjust the pH to 3-4, and a large amount of white solid precipitated. After stirring at room temperature for 30 min, it was filtered by suction, and the filter cake was washed with a small amount of purified water. The filter cake was collected, dried and weighed to obtain a white powdery solid, 5-((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylic acid (9.9 g). The filtrate was extracted with EA (2×50 mL), the organic phases were combined, washed with water (2×50 mL) and saturated NaCl aqueous solution (50 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. Purification by column chromatography (PE:EA = 4:1 - 2:1, v / v), the product spot was collected, and concentrated under reduced pressure to obtain a white powdery solid, 5-((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylic acid (386 mg). A total of 10.286 g (98%) of pure 5-((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylic acid was obtained, (ES, m / z): 363.04 [M+H] + .

[0212] Intermediate Preparation Examples 1a - 2a:

[0213] Referring to the process steps of Intermediate Preparation Examples 1 and 2, the corresponding starting materials and preparation methods were used to prepare Intermediates A1a - A2a as follows:

[0214]

[0215]

[0216] Intermediate Preparation Example 3: 5-(3-(2,5-difluorophenyl)morpholino)pyrazolo[1,5-a]pyrimidine-3-carboxylic acid (Intermediate A3)

[0217]

[0218] Step a: Under nitrogen protection and at 0 °C, trimethylcyanosilane (39.66 g, 0.4 mol) was added dropwise to a 7 mol / L NH3 / CH3OH solution (30 mL) of 2,5-difluorobenzaldehyde (28.4 g, 0.2 mol). After the addition was complete, the reaction mixture was stirred overnight at room temperature. The reaction solution was concentrated under reduced pressure, and the residue was purified by column chromatography (CH2Cl2:CH3OH = 50:1, V / V) to obtain 2-amino-2-(2,5-difluorophenyl)acetonitrile (21.92 g, 65.2%), (ES, m / z): 169 [M+H] + 。

[0219] Step b: 2-Amino-2-(2,5-difluorophenyl)acetonitrile (21.92 g, 130 mmol) was added to a 2 mol / L NaOH aqueous solution (130 mL), and the reaction was carried out under reflux for 6 h. The temperature was lowered to 0 °C, and the pH was adjusted to 3 with concentrated hydrochloric acid. The solution was concentrated to dryness under reduced pressure. Tetrahydrofuran (200 mL) was added to the residue, and the mixture was stirred for 30 min, filtered, and the filtrate was dried overnight with anhydrous sodium sulfate, filtered, and concentrated to dryness under reduced pressure to obtain crude 2-amino-2-(2,5-difluorophenyl)acetic acid (20.78 g), which was used directly in the next step without purification, (ES, m / z): 186.02 [M-H] - 。

[0220] Step c: Under nitrogen protection, a solution of 2-amino-2-(2,5-difluorophenyl)acetic acid (20.78 g, 111 mmol) in tetrahydrofuran (600 mL) was cooled to -10 to -5 °C, and lithium aluminum hydride (10.53 g, 278 mmol) was added in portions. After the addition was complete, the reaction mixture was stirred overnight at room temperature. A saturated ammonium chloride solution (500 mL) was added to the reaction solution to quench the reaction. The mixture was filtered through diatomaceous earth, and the filtrate was separated into layers. The aqueous phase was extracted with EA (2 × 100 mL), and the organic phases were combined. The combined organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (CH2Cl2:CH3OH = 10:1, V / V) to obtain 2-amino-2-(2,5-difluorophenyl)ethanol (7.177 g, total yield in two steps 32%), (ES, m / z): 174.02 [M+H] + 。

[0221] Step d: Under nitrogen protection and at 0 °C, chloroacetyl chloride (5.62 g, 49.736 mmol) was added to a solution of 2-amino-2-(2,5-difluorophenyl)ethanol (7.177 g, 41.447 mmol) and triethylamine (8.388 g, 82.894 mmol) in tetrahydrofuran (200 ml). The mixture was stirred at a constant temperature for 30 min. 60% NaH (4.974 g, 124.341 mmol) was added to the reaction system in portions. After addition, the reaction was carried out at room temperature for 2 h. The reaction was quenched with saturated ammonium chloride (100 ml). The layers were separated, and the aqueous phase was extracted with EA (2 × 50 ml). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (CH2Cl2:CH3OH = 80:1 - 20:1, V / V) to obtain 5-(2,5-difluorophenyl)morpholin-3-one (4.532 g, 51.3%), (ES, m / z): 214.01 [M+H] + 。

[0222] Step e: Under nitrogen protection and at 0 °C, lithium aluminum hydride (3.229 g, 85.849 mmol) was added to a solution of 5-(2,5-difluorophenyl)morpholin-3-one (4.532 g, 21.271 mmol) in tetrahydrofuran (100 mL) in portions. After addition, the reaction was carried out at 50 °C for 2 h. The temperature was cooled to 0 °C, and the reaction was quenched with saturated ammonium chloride (90 mL). The mixture was concentrated under reduced pressure, and the aqueous phase was extracted with EA (3 × 50 mL). The organic phases were combined, washed with water (50 mL) and brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (CH2Cl2:CH3OH = 50:1 - 25:1, V / V) to obtain 3-(2,5-difluorophenyl)morpholine (3.40 g, 80.3%), (ES, m / z): 200.03 [M+H] + 。

[0223] Step f: A mixed solution of 3-(2,5-difluorophenyl)morpholine (3.40 g, 17.068 mmol), ethyl 5-chloropyrazolo[1,5-a]pyrimidine-3-carboxylate (3.851 g, 17.068 mmol), n-butanol (50 mL) and diisopropylamine (5.181 g, 51.204 mmol) was reacted overnight at 100 °C; concentrated under reduced pressure. EA (100 mL) and water (100 mL) were added to the residue, stirred, and separated. The aqueous phase was extracted with EA (2 × 50 mL), and the organic phases were combined; the combined organic phases were washed with water (100 mL) and brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure; the residue was purified by column chromatography (n-hexane:ethyl acetate = 50:1 to 10:1, V / V) to obtain ethyl 5-(3-(2,5-difluorophenyl)morpholino)pyrazolo[1,5-a]pyrimidine-3-carboxylate (6.066 g, 91.5%), (ES, m / z): 389.05 [M+H] + .

[0224] Step g: An aqueous solution of LiOH (2.622 g, 62.492 mmol) in water (60 mL) was added to a solution of ethyl 5-(3-(2,5-difluorophenyl)morpholino)pyrazolo[1,5-a]pyrimidine-3-carboxylate (6.066 g, 15.623 mmol) in ethanol (60 mL), and the mixture was heated to 75 °C and reacted overnight. After cooling to room temperature, it was concentrated under reduced pressure. The pH of the aqueous phase was adjusted to 2 - 3 with 1N hydrochloric acid aqueous solution, and extracted with EA (3 × 60 mL), and the organic phases were combined; the combined organic phases were washed with water (100 mL) and brine (50 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure, and purified by column chromatography (PE:EA = 4:1 to 1:1, v / v) to obtain 5-(3-(2,5-difluorophenyl)morpholino)pyrazolo[1,5-a]pyrimidine-3-carboxylic acid (5.324 g, 94.6%), (ES, m / z): 361.09 [M+H] + .

[0225] Intermediate Preparation Example 4: 5-(3-(5-Fluoro-2-methoxypyridin-3-yl)morpholino)pyrazolo[1,5-a]pyrimidine-3-carboxylic acid (Intermediate A4)

[0226]

[0227] Referring to the method of steps a - f in Intermediate Preparation Example 3, using 5-fluoro-2-methoxy-3-pyridinecarboxaldehyde as the raw material, ethyl 5-(3-(5-fluoro-2-methoxypyridin-3-yl)morpholino)pyrazolo[1,5-a]pyrimidine-3-carboxylate was prepared.

[0228] Ethyl 5-(3-(5-fluoro-2-methoxypyridin-3-yl)morpholino)pyrazolo[1,5-a]pyrimidine-3-carboxylate (5.00 g, 12.46 mmol) in anhydrous ethanol (50 mL) was added with an aqueous solution of LiOH (2.091 g, 49.827 mmol) (50 mL), and the reaction was carried out overnight at 75 °C. After cooling to room temperature, it was concentrated under reduced pressure. The aqueous phase was adjusted to pH 2 - 3 with 1N hydrochloric acid aqueous solution, extracted with EA (3 × 50 ml), and the organic phases were combined; the organic phase was washed with water (50 ml) and brine (50 ml), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure, and purified by column chromatography (PE:EA = 4:1 - 1:1, v / v) to obtain 5-(3-(5-fluoro-2-methoxypyridin-3-yl)morpholino)pyrazolo[1,5-a]pyrimidine-3-carboxylic acid (4.277 g, 92%), (ES, m / z): 374.02 [M+H] + .

[0229] Intermediate Preparation Example 5: (R)-5-(2-(2,5-difluorophenyl)pyrrolidin-1-yl)-N-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (Intermediate A5)

[0230]

[0231] Step a: 1-Boc-4-(4-aminophenyl)piperazine (850 mg, 3.067 mmol) was added to a solution of (R)-5-(2-(2,5-difluorophenyl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylic acid (880 mg, 2.556 mmol) and TBTU (985 mg, 3.067 mmol) in anhydrous DMF (10 mL), and then DIPEA (991 mg, 7.668 mmol) was added dropwise at 0 °C, and the reaction was carried out overnight at room temperature. The reaction solution was added with water (50 mL) and stirred, and a solid precipitated. The filter cake was obtained by suction filtration under reduced pressure and dried in a vacuum drying oven to obtain (R)-tert-butyl 4-(4-(5-(2-(2,5-difluorophenyl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamido)phenyl)piperazine-1-carboxylate (1.450 g, 94%), (ES, m / z): 604.52 [M+H] + .

[0232] Step b: Add DCM and CF3COOH (12 mL, 3 / 1, v / v) to (R)-tert-butyl 4-(4-(5-(2-(2,5-difluorophenyl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamido)phenyl)piperazine-1-carboxylate (1.45 g, 2.402 mmol), stir at room temperature for 2.5 h, concentrate the reaction solution under reduced pressure, add water (12 mL) and EA (6 mL) to the residue, adjust the pH to 9 with ammonia water, stir to precipitate a solid, obtain the filter cake by suction filtration under reduced pressure, wash the filter cake with a small amount of water, dry it and weigh to obtain (R)-5-(2-(2,5-difluorophenyl)pyrrolidin-1-yl)-N-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (999 mg), (ES, m / z): 504.13 [M+H] + 。

[0233] Intermediate Preparation Examples 5a and 6-9:

[0234] Refer to the process steps of Intermediate Preparation Example 5, and use the following corresponding starting materials and preparation methods to prepare Intermediates A5a and A6-A9:

[0235]

[0236] Intermediate Preparation Example 10: 5-((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidin-1-yl)-N-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (Intermediate A10)

[0237]

[0238] Step a: Add 1-Boc-4-(4-aminophenyl)piperazine (918 mg, 3.312 mmol) to an anhydrous DMF (10 mL) solution containing 5-((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylic acid (Intermediate A2, 1000 mg, 2.76 mmol) and TBTU (1063 mg, 3.312 mmol), then add DIPEA (1284 mg, 9.936 mmol) dropwise at 0 °C, and react overnight at room temperature. Add water (50 mL) to the reaction solution and stir, precipitate a solid, obtain the filter cake by suction filtration under reduced pressure, and dry it in a vacuum drying oven to obtain tert-butyl 4-(4-(5-((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamido)phenyl)piperazine-1-carboxylate (1320 mg, 77%). (ES, m / z): 622.09 [M+H] + 。

[0239] Step b: To tert-butyl 4-(4-(5-((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamido)phenyl)piperazine-1-carboxylate (1.320 g, 2.125 mmol), add DCM and CF3COOH (12 mL, 3 / 1, v / v), stir at room temperature for 4 h, concentrate the reaction solution under reduced pressure, add water (80 mL) and EA (10 mL) to the residue, adjust the pH to 9 with ammonia water, stir until a solid precipitates, obtain the filter cake by suction filtration under reduced pressure, wash the filter cake with a small amount of water, dry and weigh to obtain 5-((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidin-1-yl)-N-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (907 mg, 82%), (ES, m / z): 522.09 [M+H] + 。

[0240] Intermediate Preparation Example 10a:

[0241] Referring to the process steps of Intermediate Preparation Example 10, prepare Intermediate A10a using the following corresponding starting materials and preparation methods:

[0242]

[0243]

[0244] Intermediate Preparation Example 11: 5-((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidin-1-yl)-N-(4-(piperidin-4-yl)phenyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (Intermediate A11)

[0245]

[0246] Step a: 1-Boc-4-(4-aminophenyl)piperidine (458 mg, 1.656 mmol) was added to a solution of 5-((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylic acid (Intermediate A2, 500 mg, 1.380 mmol) and TBTU (532 mg, 1.656 mmol) in anhydrous DMF (5 mL). Then, DIPEA (535 mg, 4.140 mmol) was added dropwise at 0 °C, and the reaction was stirred overnight at room temperature for 18 h. The reaction mixture was added to water (50 mL) and stirred. A solid precipitated out. The solid was obtained by suction filtration under reduced pressure and dried in a vacuum drying oven to obtain tert-butyl 4-(4-(5-((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamido)phenyl)piperidine-1-carboxylate (627 mg, 73%). (ES, m / z): 621.15 [M+H] + .

[0247] Step b: To tert-butyl 4-(4-(5-((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamido)phenyl)piperidine-1-carboxylate (627 mg, 1.101 mmol) was added DCM and CF3COOH (8 mL, 3 / 1, v / v). The mixture was stirred at room temperature for 4 h. The reaction mixture was concentrated under reduced pressure. Water (80 mL) and EA (50 mL) were added to the residue. The pH was adjusted to 9 with ammonia water. The aqueous phase was extracted with EA (55 mL × 2), and the combined extracts were washed with H2O (20 mL) and brine (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was eluted on a silica gel column for column chromatography. First, impurities with relatively low polarity were washed off with 3% (v / v) MeOH-DCM, and then the elution was changed to 10% (v / v) MeOH-DCM. The product spot was collected and concentrated to obtain 5-((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidin-1-yl)-N-(4-(piperidin-4-yl)phenyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (437 mg, 76%). (ES, m / z): 521.14 [M+H] + .

[0248] Intermediate Preparation Examples 11a and 12:

[0249] Referring to the process steps of Intermediate Preparation Example 11, Intermediate A11a and A12 were prepared using the following corresponding starting materials and preparation methods:

[0250]

[0251] Intermediate Preparation Example 13: 5-(3-(2,5-Difluorophenyl)morpholino)-N-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (Intermediate A13)

[0252]

[0253] Step a: Under nitrogen protection, 1-Boc-4-(4-aminophenyl)piperazine (3.812 g, 13.756 mmol) was added to a solution of 5-(3-(2,5-difluorophenyl)morpholino)pyrazolo[1,5-a]pyrimidine-3-carboxylic acid (4.127 g, 11.46 mmol) and TBTU (4.417 g, 13.756 mmol) in anhydrous DMF (20 mL). Then, DIPEA (4.441 g, 34.362 mmol) was added dropwise at 0 °C, and the reaction was carried out overnight at room temperature. The reaction solution was added to a mixture of EA (100 ml) and water (100 mL), stirred, and separated. The aqueous phase was extracted with EA (2 × 50 ml), and the organic phases were combined. The combined organic phase was washed with water (2 × 50 mL) and brine (50 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. Purification by column chromatography (PE:EA = 80:1 - 60:1, v / v) gave tert-butyl 4-(4-(5-(3-(2,5-difluorophenyl)morpholino)pyrazolo[1,5-a]pyrimidine-3-carboxamido)phenyl)piperazine-1-carboxylate (3.924 g, 55.3%), (ES, m / z): 620.49 [M+H] + 。

[0254] Step b: Trifluoroacetic acid (10 ml) was added to a solution of tert-butyl 4-(4-(5-(3-(2,5-difluorophenyl)morpholino)pyrazolo[1,5-a]pyrimidine-3-carboxamido)phenyl)piperazine-1-carboxylate (3.924 g, 6.332 mmol) in DCM (30 ml), and the mixture was stirred at room temperature for 4 h. The reaction solution was concentrated under reduced pressure. Water (100 mL) and EA (100 ml) were added to the residue, and the pH was adjusted to 9 with ammonia. The layers were separated, and the aqueous phase was extracted with EA (2 × 50 ml). The organic phases were combined. The combined organic phase was washed with water (100 mL) and brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (CH2Cl2:CH3OH = 50:1 - 10:1, v / v) to give 5-(3-(2,5-difluorophenyl)morpholino)-N-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (2.978 g, 90.5%), (ES, m / z): 520.11 [M+H] + 。

[0255] Intermediate Preparation Example 14:

[0256] Referring to the process steps of Intermediate Preparation Example 13, Intermediate A14 was prepared using the following corresponding starting materials and preparation methods:

[0257]

[0258] Intermediate Preparation Example 15: 5-(3-(5-Fluoro-2-methoxypyridin-3-yl)morpholino)-N-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (Intermediate A15)

[0259]

[0260] Step a: Under nitrogen protection, 1-Boc-4-(4-aminophenyl)piperazine (3.813 g, 13.747 mmol) was added to a solution of 5-(3-(5-fluoro-2-methoxypyridin-3-yl)morpholino)pyrazolo[1,5-a]pyrimidine-3-carboxylic acid (4.277 g, 11.46 mmol) and TBTU (4.414 g, 13.747 mmol) in anhydrous DMF (20 mL). Then, DIPEA (4.441 g, 34.368 mmol) was added dropwise at 0 °C, and the reaction was carried out overnight at room temperature. The reaction solution was added to a mixture of EA (100 ml) and water (100 mL), stirred, and separated; the aqueous phase was extracted with EA (2 × 50 ml), and the organic phases were combined; the organic phase was washed with water (2 × 50 mL) and brine (50 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. Purification by column chromatography (PE:EA = 100:1 to 50:1, v / v) gave tert-butyl 4-(4-(5-(3-(5-fluoro-2-methoxypyridin-3-yl)morpholino)pyrazolo[1,5-a]pyrimidine-3-carboxamido)phenyl)piperazine-1-carboxylate (4.736 g, 65.3%), (ES, m / z): 633.09 [M+H] + 。

[0261] Step b: Add CF3COOH (10 ml) to a solution of tert-butyl 4-(4-(5-(3-(5-fluoro-2-methoxypyridin-3-yl)morpholino)pyrazolo[1,5-a]pyrimidine-3-carboxamido)phenyl)piperazine-1-carboxylate (4.736 g, 7.485 mmol) in DCM (30 ml). Stir at room temperature for 4 h. Concentrate the reaction solution under reduced pressure. Add water (100 mL) and EA (100 ml) to the residue. Adjust the pH to 9 with ammonia water. Separate the layers. Extract the aqueous phase with EA (2 × 50 ml). Combine the organic phases. Wash the organic phases with water (100 mL) and brine (50 mL). Dry over anhydrous sodium sulfate, filter, concentrate under reduced pressure, and purify the residue by column chromatography (CH2Cl2:CH3OH = 50:1 - 10:1, v / v) to obtain 5-(3-(5-fluoro-2-methoxypyridin-3-yl)morpholino)-N-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (3.685 g, 92.5%), (ES, m / z): 533.10 [M+H] + 。

[0262] Intermediate Preparation Example 16:

[0263] Referring to the process steps of Intermediate Preparation Example 15, prepare Intermediate A16 using the following corresponding starting materials and preparation methods:

[0264]

[0265] Intermediate Preparation Example 17: N1-(oxetan-3-yl)benzene-1,4-diamine (Intermediate B1)

[0266]

[0267] Step a: Add 3-oxetanamine (124 mg, 1.701 mmol) to a solution of p-fluoronitrobenzene (200 mg, 1.417 mmol) in DMSO (3 mL). Then add DIPEA (366 mg, 2.834 mmol). Stir and react at 120 °C for 5 h. Add water (20 mL) to the reaction solution. Stir until a large amount of solid precipitates. Filter under reduced pressure to obtain the filter cake N-(4-nitrophenyl)oxetan-3-amine (268 mg, 97%), (ES, m / z): 195.01 [M+H] + 。

[0268] Step b: Add the filter cake N-(4-nitrophenyl)oxetan-3-amine (268 mg, 1.380 mmol) and methanol (15 mL) obtained in the previous step into a reaction flask, add 10% Pd / C, replace with H2 and stir for 4 h. Filter out Pd / C by suction, and rotary evaporate the filtrate to obtain the solid product of N-1-(oxetan-3-yl)benzene-1,4-diamine (110 mg), (ES, m / z): 164.92 [M+H] + 。

[0269] Intermediate Preparation Examples 18 - 24:

[0270] Refer to the process steps of Intermediate Preparation Example 17, and use the following corresponding starting materials, p-fluoronitrobenzene and preparation methods to prepare Intermediates B2 - B8:

[0271]

[0272]

[0273] Intermediate Preparation Example 25: (R)-6-(2-(2,5-difluorophenyl)pyrrolidin-1-yl)-[1,2,4]triazolo[4,3-a]pyrazine-3-carboxylic acid (Intermediate C1)

[0274]

[0275] Steps a - c: Refer to the methods of the first to third steps in Example 1 of Patent CN108794484B to prepare (R)-6-(2-(2,5-difluorophenyl)pyrrolidin-1-yl)-[1,2,4]triazolo[4,3-a]pyrazine-3-carboxylic acid, (ES, m / z): 346.02 [M+H] + 。

[0276] Intermediate Preparation Example 26: 6-((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidin-1-yl)-[1,2,4]triazolo[4,3-a]pyrazine-3-carboxylic acid (Intermediate C2)

[0277]

[0278] Steps a - c: Refer to the process steps of Intermediate Preparation Example 25 to prepare 6-((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidin-1-yl)-[1,2,4]triazolo[4,3-a]pyrazine-3-carboxylic acid, (ES, m / z): 364.03 [M+H] + 。

[0279] Intermediate Preparation Examples 27 - 30:

[0280] Referring to the process steps of Intermediate Preparation Example 5, Intermediate C3 - C6 were prepared using the following corresponding starting materials and preparation methods:

[0281]

[0282]

[0283] The following are examples of the compounds of the present invention.

[0284] Example 1: (R)-5-(2-(2,5-Difluorophenyl)pyrrolidin-1-yl)-N-(4-(4-(2-hydroxyacetyl)piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (Compound 1)

[0285]

[0286] Glycolic acid (32 mg, 0.417 mmol) was added to a solution of anhydrous DMF (3 mL) containing (R)-5-(2-(2,5-difluorophenyl)pyrrolidin-1-yl)-N-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (Intermediate A5, 70 mg, 0.139 mmol) and BOP (74 mg, 0.167 mmol). Then, DIPEA (54 mg, 0.417 mmol) was added dropwise at 0 °C, and the mixture was stirred at room temperature for 4 h. The reaction solution was mixed with water (20 mL), and the mixture was extracted with EA (15 mL × 2). The combined organic phases were washed with H2O (20 mL) and brine (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was eluted on a silica gel column for column chromatography, first with 1% (v / v) MeOH-DCM and then with 2% (v / v) MeOH-DCM. The product spot was collected and concentrated to obtain (R)-5-(2-(2,5-difluorophenyl)pyrrolidin-1-yl)-N-(4-(4-(2-hydroxyacetyl)piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (44 mg, 56%), (ES, m / z): 562.13 [M+H] + 。

[0287] Examples 1a, 1b and 2 - 3:

[0288] Referring to the preparation process route and operation of Example 1, Compounds 1a, 1b and 2 - 3 were prepared using the following materials and Intermediate A5, A5a or C3 as starting materials.

[0289]

[0290] Example 4: 5-((2R,4S)-2-(2,5-Difluorophenyl)-4-fluoropyrrolidin-1-yl)-N-(4-(4-(2-hydroxyacetyl)piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (Compound 4)

[0291]

[0292] Glycolic acid (306 mg, 4.026 mmol) was added to a solution of 5-((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidin-1-yl)-N-(4-(piperazin-4-yl)phenyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (Intermediate A10, 700 mg, 1.342 mmol) and BOP (712 mg, 1.610 mmol) in anhydrous DMF (10 mL). Then, DIPEA (520 mg, 4.026 mmol) was added dropwise at 0 °C, and the reaction mixture was stirred at room temperature for 4 h. The reaction solution was mixed with water (80 mL), and the mixture was extracted with EA (55 mL × 2). The combined organic phases were washed with H2O (80 mL) and brine (80 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. Elution was carried out using a silica gel column for column chromatography, first with 1% (v / v) MeOH-DCM and then with 2% (v / v) MeOH-DCM. The product spot was collected and concentrated to obtain 5-((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidin-1-yl)-N-(4-(4-(2-hydroxyacetyl)piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (666 mg, 86%), (ES, m / z): 580.14 [M+H] + 。 1 1H NMR (600 MHz, DMSO-d6) δ 9.810 (s, 1H), 8.906 - 8.723 (m, 1H), 8.283 - 8.229 (m, 1H), 7.623 (s, 1H), 7.343 (s, 1H), 7.210 (s, 2H), 7.061 - 6.842 (m, 4H), 5.711 - 5.495 (m, 2H), 4.631 (t, J = 5.4 Hz, 1H), 4.556 - 4.548 (m, 1H), 4.318 - 4.225 (m, 1H), 4.150 (d, J = 5.4 Hz, 2H), 3.637 (s, 2H), 3.513 (s, 2H), 3.124 - 3.106 (m, 4H), 2.957 - 2.912 (m, 1H).

[0293] Examples 4a - 4b, 5a, 7a - 7b, and 5 - 9:

[0294] According to the preparation process route and operation of Example 4, using intermediate A10, A10a or C4 and the following materials as starting materials, compounds 4a - 4b, 5a, 7a - 7b and 5 - 9 are prepared.

[0295]

[0296]

[0297]

[0298] Example 10: 5 - ((2R,4S)-2-(2,5 - difluorophenyl)-4 - fluoropyrrolidin - 1 - yl)-N-(4-(1-(2 - hydroxyacetyl)piperidin - 4 - yl)phenyl)pyrazolo[1,5 - a]pyrimidine - 3 - carboxamide (Compound 10)

[0299]

[0300] Glycolic acid (438 mg, 5.763 mmol) was added to a solution of 5 - ((2R,4S)-2-(2,5 - difluorophenyl)-4 - fluoropyrrolidin - 1 - yl)-N-(4-(piperidin - 4 - yl)phenyl)pyrazolo[1,5 - a]pyrimidine - 3 - carboxamide (Intermediate A11, 1000 mg, 1.921 mmol) and BOP (1274 mg, 2.882 mmol) in anhydrous DMF (10 mL), and then DIPEA (745 mg, 5.763 mmol) was added dropwise at 0 °C, and the reaction was stirred overnight at room temperature. The reaction solution was mixed with water (100 mL), and the mixture was extracted with EA (80 mL × 2), and the combined organic phases were washed with H2O (100 mL) and brine (110 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a residue, which was eluted with a silica gel column for column chromatography. First, 1% (v / v) MeOH - DCM was used, and then 2% (v / v) MeOH - DCM was used for elution. The product spots were collected and concentrated to obtain 5 - ((2R,4S)-2-(2,5 - difluorophenyl)-4 - fluoropyrrolidin - 1 - yl)-N-(4-(1-(2 - hydroxyacetyl)piperidin - 4 - yl)phenyl)pyrazolo[1,5 - a]pyrimidine - 3 - carboxamide (820 mg, 74%), (ES, m / z): 579.14 [M + H] + . 1HNMR(600MHz, DMSO-d6) δ 9.952 (s, 1H), 9.022 - 8.753 (m, 1H), 8.355 - 8.152 (m, 2H), 7.663 (s, 1H), 7.343 - 7.042 (m, 6H), 5.773 - 5.552 (m, 2H), 4.529 - 4.481 (m, 2H), 4.358 - 4.115 (m, 3H), 3.803 - 3.779 (m, 1H), 3.640 - 3.603 (m, 1H), 3.090 - 3.049 (m, 1H), 2.968 - 2.957 (m, 1H), 2.792 - 2.752 (m, 1H), 2.722 - 2.682 (m, 1H), 2.241 - 2.212 (m, 1H), 1.808 (s, 2H), 1.608 - 1.589 (m, 1H), 1.492 - 1.476 (m, 1H).

[0301] Examples 10a - 10b, 11a - 11b and 11 - 17:

[0302] According to the preparation process route and operation of Example 10, using intermediate A11, A11a or C6 and the following materials as starting materials, compounds 10a - 10b, 11a - 11b and 11 - 17 were prepared.

[0303]

[0304]

[0305]

[0306] Example 18: 5 - ((2R,4S) - 2 - (2,5 - difluorophenyl) - 4 - fluoropyrrolidin - 1 - yl) - N - (4 - (4 - (2 - (2 - hydroxyacetyl)piperazin - 1 - yl)methyl)phenyl)pyrazolo[1,5 - a]pyrimidine - 3 - carboxamide (Compound 18)

[0307]

[0308] Glycolic acid (51 mg, 0.672 mmol) was added to a solution of 5-((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidin-1-yl)-N-(4-(piperazin-1-ylmethyl)phenyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (Intermediate A12, 120 mg, 0.224 mmol) and BOP reagent (149 mg, 0.336 mmol) in anhydrous DMF (12 mL). Then, DIPEA (87 mg, 0.672 mmol) was added dropwise at 0 °C, and the reaction mixture was stirred at room temperature for 4 h. The reaction solution was mixed with water (20 mL), and the mixture was extracted with EA (10 mL × 2). The combined organic phases were washed with H2O (10 mL) and brine (10 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a residue. The residue was eluted through a silica gel column for column chromatography, first with 1% (v / v) MeOH-DCM and then increasing to 2% (v / v) MeOH-DCM to obtain 5-((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidin-1-yl)-N-(4-(4-(2-(2-hydroxyacetyl)piperazin-1-yl)methyl)phenyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (30 mg, 22%), (ES, m / z): 594.12 [M+H] + .

[0309] Example 19: 5-((2R,4S)-2-(2,5-Difluorophenyl)-4-fluoropyrrolidin-1-yl)-N-(4-(oxetan-3-ylamino)phenyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (Compound 19)

[0310]

[0311] N1-(Oxetan-3-yl)benzene-1,4-diamine (105 mg, 0.640 mmol) was added to a solution of 5-((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidin-1-yl)-N-(1,2,3,4-tetrahydroisoquinolin-7-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (Intermediate A2, 193 mg, 0.534 mmol) and TBTU (205 mg, 0.640 mmol) in anhydrous DMF (5 mL). Then, DIPEA (205 mg, 1.602 mmol) was added dropwise at 0 °C, and the reaction mixture was stirred overnight at room temperature for 14 h. The reaction solution was mixed with water (25 mL), and the mixture was extracted with EA (15 mL × 2). The combined organic phases were washed with H2O (20 mL) and brine (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a residue. The residue was separated by preparative liquid chromatography column (X-Bridge C18 19*150 mm 5 um, elution system: gradient elution with 5-25% aqueous acetonitrile solution modified with 0.05% NH4HCO3) to obtain 5-((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidin-1-yl)-N-(4-(oxetan-3-ylamino)phenyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (60 mg, 22.1%), (ES, m / z): 509.12 [M+H] + .

[0312] Examples 19a - 19b and 20 - 24:

[0313] According to the preparation process and operation of Example 19, the following intermediates were used as starting materials to prepare Compounds 19a - 19b and 20 - 24:

[0314]

[0315]

[0316]

[0317] Example 25: 5-((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidin-1-yl)-N-(4-(6-(2-hydroxyacetyl)-3,6-diazabicyclo[3.1.1]hept-3-yl)phenyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (Compound 25)

[0318]

[0319] Step a: Add tert-butyl 3-(4-aminophenyl)-3,6-diazabicyclo[3.1.1]heptane-6-carboxylate (442 mg, 1.528 mmol) (Intermediate B5) to a solution of 5-((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylic acid (Intermediate A2, 460 mg, 1.270 mmol) and TBTU (491 mg, 1.528 mmol) in anhydrous DMF (5 mL). Then, add DIPEA (494 mg, 3.819 mmol) dropwise at 0 °C, and stir the reaction mixture overnight at room temperature for 14 h. Add water (100 mL) to the reaction solution and stir. A large amount of solid precipitates. Filter under reduced pressure and dry to obtain the crude product of tert-butyl 3-(4-(5-((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamido)phenyl)-3,6-diazabicyclo[3.1.1]heptane-6-carboxylate as a solid.

[0320] Step b: Dissolve the crude product of tert-butyl 3-(4-(5-((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamido)phenyl)-3,6-diazabicyclo[3.1.1]heptane-6-carboxylate obtained above in DCM (25 mL), add CF3COOH (5 mL), and stir at room temperature for 3 h. Rotate the reaction solution to dryness, dilute with water, adjust the pH to alkaline with ammonia water, and a solid precipitates. Filter and air-dry to obtain the solid product of N-(4-(3,6-diazabicyclo[3.1.1]heptan-3-yl)phenyl)-5-((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (450 mg).

[0321] Step c: Glycolic acid (35 mg, 0.450 mmol) was added to a solution of N-(4-(3,6-diazabicyclo[3.1.1]heptan-3-yl)phenyl)-5-((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (200 mg, 0.375 mmol) and TBTU (145 mg, 0.450 mmol) in anhydrous DMF (5 mL). Then, DIPEA (146 mg, 1.125 mmol) was added dropwise at 0 °C, and the reaction mixture was stirred at room temperature for 4 h. The reaction solution was mixed with water (30 mL), and the mixture was extracted with EA (20 mL × 2). The combined organic phases were washed with H2O (30 mL) and brine (40 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a residue. The residue was eluted on a silica gel column for column chromatography, first with 1% MeOH-DCM and then with 2% MeOH-DCM. The product spots were collected and concentrated to obtain 5-((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidin-1-yl)-N-(4-(6-(2-hydroxyacetyl)-3,6-diazabicyclo[3.1.1]hept-3-yl)phenyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (180 mg, 81%), (ES, m / z): 592.02 [M+H] + .

[0322] Example 26: 5-((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidin-1-yl)-N-(4-(6-(2-hydroxyacetyl)-2,6-diazaspiro[3.3]heptan-2-yl)phenyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (Compound 26)

[0323]

[0324] Step a: Add tert-butyl 6-(4-aminophenyl)-2,6-diazaspiro[3.3]heptane-2-carboxylate (178 mg, 0.607 mmol) (Intermediate B8) to a solution of 5-((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylic acid (Intermediate A2, 200 mg, 0.552 mmol) and TBTU (217 mg, 0.607 mmol) in anhydrous DMF (5 mL), then add DIPEA (217 mg, 1.656 mmol) dropwise at 0 °C, and stir the reaction mixture overnight at room temperature for 14 h. Add water (30 mL) to the reaction solution and stir. A large amount of solid precipitates. Filter under reduced pressure and dry to obtain crude tert-butyl 6-(4-(5-((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamido)phenyl)-2,6-diazaspiro[3.3]heptane-2-carboxylate. (ES, m / z): 534.12 [M+H] + .

[0325] Step b: Dissolve the above-obtained crude tert-butyl 6-(4-(5-((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamido)phenyl)-2,6-diazaspiro[3.3]heptane-2-carboxylate in DCM (6 mL), add CF3COOH (2 mL), and stir at room temperature for 3 h. Rotavap the reaction solution, dilute with water, adjust the pH to alkaline with ammonia water, and a solid precipitates. Filter and air-dry to obtain the solid product of N-(4-(2,6-diazaspiro[3.3]heptan-2-yl)phenyl)-5-((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (218 mg).

[0326] Step c: Glycolic acid (37 mg, 0.490 mmol) was added to a solution of N-(4-(2,6-diazaspiro[3.3]heptan-2-yl)phenyl)-5-((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (218 mg, 0.409 mmol) and TBTU (157 mg, 0.490 mmol) in anhydrous DMF (10 mL). Then, DIPEA (158 mg, 1.226 mmol) was added dropwise at 0 °C, and the reaction mixture was stirred at room temperature for 4 h. The reaction solution was mixed with water (30 mL), and the mixture was extracted with EA (20 mL × 2). The combined organic phases were washed with H2O (30 mL) and brine (40 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by preparative TLC (eluent: DCM:MeOH = 15:1, v / v) to obtain 5-((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidin-1-yl)-N-(4-(6-(2-hydroxyacetyl)-2,6-diazaspiro[3.3]heptan-2-yl)phenyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (51 mg, 94%), (ES, m / z): 592.12 [M+H] + .

[0327] Example 27: 5-(3-(2,5-Difluorophenyl)morpholinyl)-N-(4-(4-(2-hydroxyacetyl)piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (Compound 27)

[0328]

[0329] Glycolic acid (584 mg, 7.676 mmol) was added to a solution of 5-((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidin-1-yl)-N-(4-(piperidin-4-yl)phenyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (Intermediate A13, 997 mg, 1.919 mmol) and BOP (1274 mg, 2.878 mmol) in anhydrous DMF (10 mL). Then, DIPEA (744 mg, 5.756 mmol) was added dropwise at 0 °C, and the reaction mixture was stirred overnight at room temperature. The reaction solution was mixed with water (100 mL) and extracted with EA (80 mL × 2). The organic phases were combined, washed with H2O (100 mL) and brine (110 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was eluted on a silica gel column for column chromatography, and the eluent was (DCM:CH3OH = 100:1 - 50:1, v / v). The product spot was collected and concentrated to obtain 5-(3-(2,5-difluorophenyl)morpholinyl)-N-(4-(4-(2-hydroxyacetyl)piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (609.8 mg, 55%), (ES, m / z): 578.12 [M+H] + 。

[0330] Example 28

[0331] According to the preparation process route and operation of Example 27, using A14 and glycolic acid as starting materials, Compound 28 was prepared.

[0332]

[0333]

[0334] Example 29: 5-(3-(5-Fluoro-2-methoxypyridin-3-yl)morpholinyl)-N-(4-(4-(2-hydroxyacetyl)piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (Compound 29)

[0335]

[0336] Glycolic acid (511 mg, 6.723 mmol) was added to a solution of 5-(3-(5-fluoro-2-methoxypyridin-3-yl)morpholino)-N-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (Intermediate A15, 1.022 g, 1.92 mmol) and BOP (1274 mg, 2.878 mmol) in anhydrous DMF (10 mL). Then, DIPEA (744 mg, 5.763 mmol) was added dropwise at 0 °C, and the reaction mixture was stirred overnight at room temperature. The reaction solution was mixed with water (100 mL) and extracted with EA (80 mL × 2). The organic phases were combined, washed with H2O (100 mL) and brine (110 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was eluted on a silica gel column for column chromatography, and the eluent was (DCM:CH3OH = 100:1 to 50:1, v / v). The product spot was collected and concentrated to obtain 5-(3-(5-fluoro-2-methoxypyridin-3-yl)morpholino)-N-(4-(4-(2-hydroxyacetyl)piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (737 mg, 65.02%), (ES, m / z): 591.20 [M+H] + 。

[0337] Example 30

[0338] According to the preparation process route and operation of Example 29, using A16 and glycolic acid as starting materials, Compound 30 was prepared.

[0339]

[0340] Examples 31 - 47

[0341] With reference to the preparation process routes and operations of Intermediate Preparation Examples 1 - 30 and Examples 1 - 30, and referring to the method disclosed in CN111936500A, using (2R)-2-(2,5-difluorophenyl)-3-azabicyclo[3.1.0]hexane and each intermediate as raw materials, Compounds 31 - 47 were prepared.

[0342]

[0343]

[0344]

[0345] Reference Substance Preparation Examples 1 - 5:

[0346] In addition: With reference to the preparation process routes and operations in the patent documents WO2019029629A1 and WO2012034095A1, Compounds D1 - D5 were prepared.

[0347]

[0348]

[0349] Test Example 1: Kinase Inhibition Test of Compounds against TRK

[0350] 1. Operating Procedure:

[0351] 1.1 Kinase Reaction:

[0352] Add test compounds with a certain concentration gradient, enzyme solution (add kinase buffer (1X kinase buffer (Cisbio, Cat#62EZBFDD), pH 7.5; 5 mM MgCl2, 1 mM DTT) to the negative control wells) into the compound plate in sequence, and centrifuge at 1000 rpm for 30 seconds. Seal the plate and incubate it in a constant temperature incubator at 25°C for 30 minutes. Prepare substrate solutions of TK-Sub-biotin (Cisbio, Cat#61TKOBL) and ATP (Sigma, Cat#R0441), and add the substrate mixed solution into the 384-well plate, then centrifuge at 1000 rpm for 30 seconds. Seal the plate and incubate it in a constant temperature incubator at 25°C for 60 minutes.

[0353]

[0354] 1.2 Kinase Detection:

[0355] Dilute, mix and add the TK antibody and XL665 into the assay plate, then centrifuge at 1000 rpm for 30 seconds. Seal the plate and incubate it in a constant temperature incubator at 25°C for 60 minutes. Place the assay plate on the Envision machine for reading. (HTRF 665 / 615 ratio: 665 nm signal value / 615 nm signal value)

[0356] Inhibition Rate = (Ratio 阴性对照孔 - Ratio 化合物孔 ) / (Ratio 阴性对照孔 - Ratio 无酶对照孔 ) × 100%

[0357] 1.3 Data Analysis and Curve Fitting

[0358] Fit the data in the XLFit excel add-in version 5.4.0.8 to obtain the IC 50 value.

[0359] 1.4 QC Parameters

[0360] Reference compounds are included in each plate, and their IC 50Within 3 times each time.

[0361] 2. Test results: As shown in Table 1

[0362] Table 1 Kinase inhibitory activities of the compounds of the present invention and control compounds against TRK

[0363]

[0364] Note: The above RXDX-101, LOXO-195, and LOXO-101 are all publicly disclosed compounds and commercially available products (pharmaceutical or chemical grade products).

[0365] The results show that: The compounds of the present invention exhibit high kinase inhibitory activities among various kinases, and the activities are superior to or equivalent to those of RXDX-101, LOXO-195, and LOXO-101 in TRKA, TRKB, TRKC, and TRKC-G696A, while the inhibitory activities in various mutant drug-resistant kinases (G595R, G667C, G623R) are significantly superior to those of RXDX-101, LOXO-195, and LOXO-101.

[0366] Test Example 2: Kinase inhibitory test of compounds against ALK and ROS1

[0367] 1. Operating steps:

[0368] 1.1 Kinase reaction:

[0369] The compounds are diluted to a certain concentration with DMSO and serially diluted 4-fold. Add a certain concentration of the compound, enzyme solution, and DMSO to a 384-well plate and incubate at room temperature for 10 min; add a fluorescein-labeled peptide and incubate with ATP (sigma, Cat. No.: A7699-1G, Lot No.: 987-65-5) at 28 °C for a certain time; add a stop solution. Read the values.

[0370] Inhibition rate formula for a single concentration: Inhibition rate = (OD 阴性对照孔 - OD 化合物孔 ) / (OD 阴性对照孔 - OD 无酶对照孔 ) × 100%

[0371]

[0372] 1.2 Data analysis and curve fitting

[0373] Fit the data in the XLFit excel add-in version 4.3.1 to obtain the IC 50 value, and the results are shown in Table 2.

[0374] Table 2 Inhibitory activities of the compounds of the present invention and control compounds against ALK and ROS1 kinases

[0375]

[0376] The results showed that: Multiple compounds of the present invention exhibited strong inhibitory activities against ROS1 kinase, significantly superior to RXDX-101 and LOXO-101, and superior to LOXO-195; they also had good inhibitory activities against ALK kinase, significantly superior to LOXO-101 and LOXO-195.

[0377] Test Example 3: In vitro inhibitory test of compounds on cells

[0378] 1. Cell lines

[0379] Six test cell lines were sourced from Kangyuan Botech (Beijing) Co., Ltd.

[0380] Cell type: Murine B cells

[0381] Culture medium: RPMI-1640 + 10% FBS

[0382] 2. Test method

[0383] Harvest cells in the logarithmic growth phase and perform cell counting using a platelet counter. Pipette a cell suspension of a certain density evenly and inoculate it into a 96-well plate, 100 μL per well, shake to evenly disperse it into the wells; add 100 μl of a drug solution with a certain concentration gradient to each well, and set three replicates for each drug concentration; culture in a 37°C CO2 incubator for 72 hours; add MTT working solution (5 mg / ml), 20 μl per well; incubate at 37°C for 4 hours; centrifuge at 1000 rpm / min for 5 min using a plate centrifuge, aspirate 180 μl of the culture medium and then add 150 μl of DMSO, mix well using a microporous oscillator, wipe the bottom of the plate clean, and measure the optical density value (OD) at 550 nm using an enzyme-linked immunosorbent assay reader.

[0384] 3. Data analysis

[0385] Inhibition rate = (OD of control wells - OD of test wells) / (OD of control wells - OD of blank wells) * 100%, and according to the inhibition rates at each concentration, use SPSS software to calculate the half-maximal inhibitory concentration IC 50 value.

[0386] 4. Test results: The results are shown in Table 3 as follows:

[0387] Table 3 Inhibitory activities of the compounds of the present invention and control compounds against TRK mutant cell lines

[0388]

[0389] Note: —— Indicates not detected.

[0390] Table 4 Inhibitory Activity of Control Compounds against TRK Mutant Cell Lines

[0391]

[0392] The results showed that multiple compounds of the present invention exhibited good in vitro cell activity in a variety of wild-type and mutant drug-resistant cell lines, significantly superior to RXDX-101, LOXO-195, LOXO-101, and the compounds D1-D5 disclosed in the prior art.

[0393] Test Example 4: Study on the in Vivo Mechanism of Compounds

[0394] 1. Test Method

[0395] 1.1 Model Preparation:

[0396] Take mutant drug-resistant cells Ba / F3LMNA-NTRK1-G595R in the logarithmic growth phase, collect and resuspend them in serum-free medium to make the cell concentration 6×10 7 -10×10 7 cells / mL, and add an equal volume of Matrigel to the cell suspension to make the final cell concentration 3×10 7 -5×10 7 cells / mL. Subcutaneously inoculate 0.1 mL of the tumor cell suspension under the axilla of the forelimb of NuNu mice (Beijing Vital River, 4-6 weeks old, female), and the inoculation dose is 3×10 6 -5×10 6 cells / mouse to prepare an animal model.

[0397] 1.2 Test Grouping:

[0398] Use vernier calipers to measure the maximum and minimum tumor diameters of the xenografts in nude mice, and calculate the tumor volume: The calculation formula for the tumor volume (Tumor volume, TV) is: V = 1 / 2 × a × b 2 , where a and b represent the maximum and minimum diameters of the tumor mass respectively. 12-15 days after inoculation, select nude mice with a tumor volume of 200-300 mm 3 , and evenly divide the animals into 7 groups according to the tumor volume by the random number method, with 3 mice in each group.

[0399] 1.3 Administration

[0400] Administer the drug by gavage according to the animal body weight, and the administration volume is 10 ml / kg. Compound 4 is configured into the required administration concentration using "3% DMSO + 96% HP-β-CD (0.5 g / mL) + 1% HCl".

[0401] There were three rats in the control group. Tumor tissues were collected and frozen 4 h after the administration of the vehicle. In other groups, the compound of the present invention was administered at 4100 mg / kg, and tumor tissues were collected and frozen at 0.25 h, 1 h, 4 h, 8 h, 12 h, and 24 h, respectively.

[0402] 1.4 Protein extraction and quantification

[0403] A certain mass of tumor tissue was added to the corresponding volume of protein lysate (RIPA lysis buffer (ThermoFisher, catalog number 89900): protease inhibitor (cOmplete, Mini, EDTA-free, EASYpack; Roche, catalog number 04693159001): phosphatase inhibitor (PhosStop, EASY pack; Roche, catalog number 04906837001) = 8:1:1), homogenized, and lysed on ice bath for 30 min. After centrifugation at low temperature and high speed, the supernatant was taken for BCA protein quantification (operated according to the BCA protein quantification kit (Tiangen, catalog number: #PA115-01)). Finally, the protein concentration was adjusted to a uniform concentration with the lysis buffer, and then loading buffer was added, and boiled at 100 °C for 10 min.

[0404] 1.5 Western-blot

[0405] A 4-20% 10-well precast gel was used; the sample loading amount was 100 μg; electrophoresis was carried out at 140 V for 1-1.5 h; wet transfer was carried out at 300 mA for 1.5-2 h; blocked with 5% BSA for 2-3 h; incubated with primary antibodies at 4 °C overnight (Trk1:5000, p-Trk, PLCγ1, p-PLCγ1, AKT, p-AKT, actin1:1000); washed with 0.1% TBST for 4×5 min; incubated with secondary antibodies at room temperature for 2 h (1:5000), developed with ECL, and exposed.

[0406]

[0407]

[0408] 2. Test results: As shown in the Figure 1 appendix.

[0409] It can be seen from the test results that: as time prolongs, Figure 1 the levels of TRK, p-TRK, p-PLCγ1, and p-AKT in the

[0410] Experimental Example 5: In Vivo Pharmacodynamic Experiment of Compounds on NTRK Mutation-Resistant Tumor Model

[0411] Test Method

[0412] 1.1 Model Preparation

[0413] Cells in the logarithmic growth phase were collected, resuspended in serum-free medium, and adjusted to a cell concentration of 6×10 7 -10×10 7 cells / mL. An equal volume of Matrigel was added to the cell suspension to give a final cell concentration of 3×10 7 -5×10 7 cells / mL. 0.1 mL of the tumor cell suspension (inoculation dose: 3×10 6 -5×10 6 cells / mouse) was subcutaneously inoculated into the axilla of the forelimb of NuNu mice (Vital River Laboratories, Beijing, 4 - 6 weeks old, female) to prepare an animal model.

[0414] 1.2 Test Grouping

[0415] The maximum and minimum tumor diameters of the xenografts in nude mice were measured with vernier calipers, and the tumor volume was calculated using the formula: Tumor volume (TV) = V = 1 / 2×a×b 2 , where a and b represent the maximum and minimum diameters of the tumor mass, respectively. 7 - 10 days after inoculation, nude mice with a tumor volume of 100 - 200 mm 3 were selected and evenly grouped by tumor volume using the random number method, with 6 mice in each group.

[0416] 1.3 Observation Indicators

[0417] Gavage administration was started on the day of grouping according to the animal body weight. The administration volume was 10 ml / kg. LOXO-195 was formulated into the required administration solution using 0.5% CMC-Na, and Compounds 4 and 10 were formulated into the required administration solutions using "3% DMSO + 96% HP-β-CD (0.5 g / mL) + 1% HCl". The vehicle control group was given the vehicle "3% DMSO + 96% HP-β-CD (0.5 g / mL) + 1% HCl". The tumor diameter was measured twice a week, and the tumor volume was calculated. The specific indicators are as follows:

[0418] Animal body weight: The animals were weighed every morning before administration. A weight loss of more than 20% was defined as a toxic reaction to the drug (observed until the day after the last administration);

[0419] Tumor volume (TV) = V = 1 / 2×a×b 2, where a and b represent the maximum diameter and minimum diameter of the tumor mass (observed until the day after the last administration);

[0420] Relative tumor proliferation rate T / C(%) : T / C(%) = TRTV / CRTV×100% (TRTV: RTV of the drug administration group, CRTV: RTV of the control group);

[0421] Tumor growth inhibition rate (TGI) = [1 - (Ti - T0) / (Vi - V0)]×100%. (Where Ti represents the average tumor volume of a certain drug administration group on a certain day; T0 is the average tumor volume of this drug administration group at the start of drug administration; Vi is the average tumor volume of the vehicle control group on a certain day (the same day as Ti); V0 is the average tumor volume of the vehicle control group at the start of drug administration);

[0422] Tumor inhibition rate: At the end of the experiment, the animals were sacrificed by cervical dislocation, the tumor masses were dissected and weighed, photographed, and the tumor inhibition rate was calculated. Tumor inhibition rate = (average tumor weight of the control group - average tumor weight of the drug administration group) / average tumor weight of the control group×100%.

[0423] Test results

[0424] 2.1 Ba / F3 LMNA-NTRK1-G667C model

[0425] 2.1.1 Effect of the drug on the body weight of tumor-bearing mice

[0426] The body weights of each compound at each dose group showed an upward trend, and the upward trend was more obvious than that of the control group. The significant increase in the body weights of each compound at each dose group may be related to the compound, or it may be due to the inhibition of tumor growth, resulting in better condition of the mice and significant weight gain. The test results are shown in Table 5.

[0427] 2.1.2 Effect of the drug on the tumor weight and tumor inhibition rate of tumor-bearing mice

[0428] The data results showed that: at the same drug administration dose (100 mg / kg), compared with LOXO-195, Compounds 4 and 10 of the present invention had more significant inhibition on tumor growth; further, compared with the higher drug administration dose group of LOXO-195 (200 mg / kg), Compounds 4 and 10 (100 mg / kg) of the present invention also showed better tumor inhibition effect. The results are shown in Table 5.

[0429] Table 5 In vivo results of Ba / F3 LMNA-NTRK1-G667C model

[0430]

[0431] 2.2 Ba / F3 LMNA-NTRK1-G595R model

[0432] 2.2.1 Effects of drugs on the body weight of tumor-bearing mice

[0433] The body weights of each dose group of each compound showed an increasing trend, and the increasing trend was more obvious than that of the control group. The significant increase in body weight in each dose group of each compound may be related to the compound or may be due to the inhibition of tumor growth, resulting in a better state of the mice and significant weight gain. The test results are shown in Table 6.

[0434] 2.2.2 Effects of drugs on tumor weight and tumor inhibition rate of tumor-bearing mice

[0435] The data results showed that: compared with LOXO-195 (100 mg / kg), at a lower dosage (50 mg / kg), compounds 4 and 10 of the present invention could significantly inhibit the weight of tumor tissue, and the tumor weight inhibition rate > 90%. The test results are shown in Table 6.

[0436] Table 6 In vivo results of Ba / F3 LMNA-NTRK1-G595R model

[0437]

[0438] Test Example 6: Pharmacodynamic test of the combination of compounds and AMG510 on the Nu / Nu mouse human non-small cell lung cancer H358 xenograft tumor model

[0439] 1. Test method

[0440] 1.1 Preparation of H358 mouse xenograft tumor model

[0441] Cells in the logarithmic growth phase were collected and resuspended in serum-free medium to a cell concentration of 2×10 7 cells / mL, and an equal volume of Matrigel was added to the cell suspension to make the final cell concentration 1×10 7 cells / mL. 0.1 mL of the tumor cell suspension was subcutaneously inoculated into the axilla of the forelimb of NuNu mice (Beijing Vital River, 4 - 6 weeks old, female) at an inoculation dose of 1×10 6 cells / mouse to prepare an animal model.

[0442] 1.2 Test grouping

[0443] The maximum and minimum tumor diameters of the xenograft tumors in nude mice were measured with vernier calipers, and the tumor volume was calculated: the calculation formula for tumor volume (Tumor volume, TV) is: V = 1 / 2 × a × b 2 , where a and b represent the maximum diameter and minimum diameter of the tumor mass respectively. Four days after inoculation, nude mice with a tumor volume of 100 - 200 mm 3 were selected and randomly divided into 7 groups with 6 mice in each group according to the tumor volume by the random number method.

[0444] 1.3 Observation indicators

[0445] Gavage administration was started according to the animal body weight on the day of grouping. The total administration volume was 10 ml / kg. Compound 4 was prepared into the required administration solution using "3% DMSO + 96% HP-β-CD (0.5 g / mL) + 1% HCl". AMG510 was prepared into the required administration solution using "99.6% (0.5% MC) + 0.4% Tween-80". The vehicle control group used the vehicle "3% DMSO + 96% HP-β-CD (0.5 g / mL) + 1% HCl".

[0446] The tumor diameter was measured twice a week, and the tumor volume was calculated. The specific indicators are as follows:

[0447] Animal body weight: The animals were weighed before drug administration every day. A body weight reduction of more than 20% was defined as a toxic reaction to the drug (observed until the day after the last drug administration);

[0448] Tumor volume (TV) = V = 1 / 2 × a × b 2 , where a and b represent the maximum diameter and minimum diameter of the tumor mass respectively (observed until the day after the last drug administration);

[0449] Relative tumor proliferation rate T / C (%) : T / C (%) = TRTV / CRTV × 100% (TRTV: RTV of the drug administration group, CRTV: RTV of the control group);

[0450] Tumor growth inhibition rate (TGI) = [1 - (Ti - T0) / (Vi - V0)] × 100%. (where Ti represents the average tumor volume of a certain drug administration group on a certain day; T0 is the average tumor volume of this drug administration group at the start of drug administration; Vi is the average tumor volume of the vehicle control group on a certain day (the same day as Ti); V0 is the average tumor volume of the vehicle control group at the start of drug administration);

[0451] Synergy factor Q: Burgi correction formula: Q = E(a + b) / [E(a) + E(b) – E(a)*E(b)]. (E(a + b): Tumor volume inhibition rate after combined administration of drugs a and b; E(a): Tumor volume inhibition rate when a is administered alone; E(b): Tumor volume inhibition rate when b is administered alone)

[0452] Q < 0.85 Antagonism

[0453] 0.85 ≤ Q < 1.15 Additive

[0454] Q ≥ 1.15 Synergy

[0455] 2. Test results

[0456] 2.1 Effects of drugs on the body weight of tumor-bearing mice

[0457] The body weights of the groups treated with Compound 4 alone, AMG510 alone, and their combination all showed an upward trend. The upward trends in the body weights of the groups treated with Compound 4 alone and in combination were significant, and no obvious drug toxicity reactions were observed. The test results are shown in Table 7.

[0458] 2.2 Effects of the drug on the tumor inhibition rate in tumor-bearing mice

[0459] The results showed that: compared with the vehicle control group, both combination groups showed obvious tumor inhibition effects, with significant statistical differences. Compared with Compound 4 at 30 mg / kg, the combination group of Compound 4 at 30 mg / kg and AMG510 at 3 mg / kg showed significant statistical differences (P < 0.05) and obvious synergistic effects (Q = 1.429). The combination group of Compound 4 at 100 mg / kg and AMG510 at 6 mg / kg showed significant statistical differences (P < 0.05) compared with the corresponding single-treatment groups and obvious synergistic effects (Q = 1.257). The test results are shown in Table 7.

[0460] Table 7 In vivo pharmacodynamic results of the H358 model

[0461]

[0462]

[0463] Note: Compared with the vehicle control group, *P < 0.05; compared with Compound 4 at 30 mg / kg, #P < 0.05; compared with Compound 4 at 100 mg / kg, ▲P < 0.05; compared with AMG510 at 6 mg / kg, ●P < 0.05.

[0464] Experimental Example 7: Pharmacodynamic test of the combination of compound and AMG510 on the MIAPaCa2 xenograft model of human pancreatic cancer in Nu / Nu mice

[0465] 1. Test method

[0466] 1.1 Preparation of the MIA PaCa2 model

[0467] Cells in the logarithmic growth phase were collected and resuspended in serum-free medium to a cell concentration of 1×10 8 cells / mL. 0.1 mL of the tumor cell suspension was subcutaneously inoculated into the axilla of the forelimb of NuNu mice (Beijing Vital River, 4 - 6 weeks old, female) at an inoculation dose of 1×10 7 cells / mouse to prepare an animal model.

[0468] 1.2 Test grouping

[0469] Measure the maximum tumor diameter and minimum tumor diameter of the xenograft tumors in nude mice with a vernier caliper, and calculate the tumor volume: The calculation formula for the tumor volume (TV) is: V = 1 / 2 × a × b 2 , where a and b represent the maximum diameter and minimum diameter of the tumor mass, respectively. Seven days after inoculation, select nude mice with a tumor volume of 100 - 200 mm 3 , and evenly divide the animals into 4 groups according to the tumor volume by the random number method, with 6 animals in each group.

[0470] 1.3 Observation indicators

[0471] Start gavage administration according to the animal body weight on the day of grouping. The total administration volume is 10 ml / kg. Compound 4 is configured into the required administration solution using "3% DMSO + 96% HP-β-CD (0.5 g / mL) + 1% HCl". AMG510 is configured into the required administration solution using "99.6% (0.5% MC) + 0.4% Tween-80". The vehicle control group uses the vehicle "3% DMSO + 96% HP-β-CD (0.5 g / mL) + 1% HCl".

[0472] Measure the tumor diameter twice a week and calculate the tumor volume. The specific indicators are as follows:

[0473] Animal body weight: Weigh the animals before administration every day. A body weight reduction of more than 20% is defined as a toxic reaction of the drug (observed until the day after the last administration);

[0474] Tumor volume (TV) = V = 1 / 2 × a × b 2 , where a and b represent the maximum diameter and minimum diameter of the tumor mass, respectively (observed until the day after the last administration);

[0475] Relative tumor proliferation rate T / C (%) : T / C (%) = TRTV / CRTV × 100% (TRTV: RTV of the administration group, CRTV: RTV of the control group);

[0476] Tumor growth inhibition rate (TGI) = [1 - (Ti - T0) / (Vi - V0)] × 100%. (Where Ti represents the average tumor volume of a certain administration group on a certain day; T0 is the average tumor volume of this administration group at the start of administration; Vi is the average tumor volume of the vehicle control group on a certain day (the same day as Ti); V0 is the average tumor volume of the vehicle control group at the start of administration);

[0477] Synergistic factor Q: Burgi correction formula: Q = E(a + b) / [E(a) + E(b) – E(a)*E(b)]. (E(a + b): tumor volume inhibition rate after combined administration of drugs a and b; E(a): tumor volume inhibition rate when drug a is administered alone; E(b): tumor volume inhibition rate when drug b is administered alone)

[0478] Q < 0.85 antagonism

[0479] 0.85 ≤ Q < 1.15 additive

[0480] Q ≥ 1.15 synergy

[0481] 2. Test results

[0482] 2.1 Effects of drugs on the body weight of tumor-bearing mice

[0483] The body weights of the groups treated with compound 4 alone, AMG510 alone, and in combination showed an upward trend. The upward trend in the body weights of the groups treated with compound 4 alone and in combination was significant, and no obvious drug toxicity reactions were observed. The test results are shown in Table 8.

[0484] 2.2 Effects of drugs on the tumor inhibition rate of tumor-bearing mice

[0485] The results showed that: compared with the solvent control group, the combined treatment group showed obvious tumor inhibition effects, with significant statistical differences (P < 0.01); after calculation, the combined treatment group had obvious synergistic effects compared with the single-treatment groups (Q = 1.86). The test results are shown in Table 8.

[0486] Table 8 In vivo pharmacodynamic results of the MIA PaCa2 model

[0487]

[0488] Note: Compared with the solvent control group, *P < 0.05, **P < 0.01; compared with AMG510 at 10 mg / kg, ##P < 0.01; compared with compound 4 at 30 mg / kg, ▲▲▲P < 0.001.

Claims

1. Use of Compound A in combination with a KRAS G12C inhibitor in the preparation of a medicament for treating tumors, wherein the structure of Compound A is shown in the following formula (4): The KRAS G12C inhibitor is selected from AMG510: The tumor is non-small cell lung cancer or pancreatic cancer.

2. Use of Compound A in the preparation of a drug for improving the therapeutic effect of KRAS G12C inhibitor in treating tumors, wherein the structure of Compound A is shown in the following formula (4): The KRAS G12C inhibitor is selected from AMG510: The tumor is non-small cell lung cancer or pancreatic cancer.

3. KRAS G12C Use of an inhibitor in the preparation of a drug for improving the therapeutic effect of compound A on tumors, wherein the structure of compound A is shown in the following formula (4): The KRAS G12C inhibitor is selected from AMG510: The tumor is non-small cell lung cancer or pancreatic cancer.

4. The use according to any one of claims 1 to 3, characterized in that: The drug further comprises a pharmaceutically acceptable excipient or a pharmaceutically acceptable carrier.

5. The use according to any one of claims 1 to 3, characterized in that: Each preparation unit of the described drug contains 0.1 - 1000 mg of compound A; and / or, each preparation unit of the described drug contains G12C 0.1 - 1000 mg of a KRAS inhibitor.

6. The use according to any one of claims 1 to 3, characterized in that: Each preparation unit of the described drug contains 1 - 500 mg of compound A; and / or, each preparation unit of the described drug contains 1 - 1000 mg of KRAS G12C inhibitor 1.

7. The use according to claim 1 or 3, characterized in that: Each preparation unit of the drug contains 1 - 1000 mg of AMG510.

8. The use according to claim 1 or 3, characterized in that: Each preparation unit of the drug contains 1 - 500 mg of AMG510.

9. The use according to claim 1 or 3, characterized in that: Each preparation unit of the drug contains 1 - 400 mg of AMG510.

10. The use according to claim 1 or 3, characterized in that: Each preparation unit of the drug contains 1 - 300 mg of AMG510.

11. The use according to claim 1 or 3, characterized in that: Each preparation unit of the drug contains 1 - 200 mg of AMG510.

12. The use according to any one of claims 1-3, characterized in that: The compound A and KRAS G12C inhibitor are included in the same pharmaceutical unit, or are formulated into the drug in different pharmaceutical preparations respectively in the form of a combination product; the pharmaceutical preparation is selected from oral preparations, injection preparations, topical administration preparations or external use preparations.

13. A drug, characterized in that: Containing compound A and a KRAS G12C inhibitor, and the structure of the compound A is shown in the following formula (4): The KRAS G12C inhibitor is selected from AMG510: The drug is used for treating non-small cell lung cancer or pancreatic cancer.

14. The drug according to claim 13, characterized in that: The compound A and KRAS G12C inhibitors are included in the same dosage unit, or are respectively formulated in different formulations in the form of a combination product; the formulations are selected from oral formulations, injection formulations, topical administration formulations or external use formulations.

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