Amino-containing macrocyclic compounds as protein kinase regulators

Abstract

The present invention relates to compounds that can be used as RET and / or BTK inhibitors and their use in treating RET and / or BTK-mediated diseases#imgabs0#

Description

Technical Field

[0001] The present invention relates to compounds or pharmaceutically acceptable salts thereof and their use as RET and / or BTK inhibitors. More specifically, the present invention provides novel RET and / or BTK inhibitors and their use in treating RET and / or BTK-mediated diseases. Background Art

[0002] RET (rearranged during transfection) is a proto-oncogene located on chromosome 10. The RET protein encoded by the RET gene is a cell membrane receptor tyrosine kinase (RTK) that belongs to the cadherin superfamily. The RET gene plays an important role in the development of the kidney and enteric nervous system during embryonic development. It is also crucial for the homeostasis of multiple tissues, including neurons, neuroendocrine tissue, hematopoietic tissue, and male germ cells. While classical RTK activation requires ligand-receptor interaction, RET activation requires interaction between its ligand (glial cell line-derived neurotrophic factor family ligand, GFLS) and a coreceptor (GFLS family receptor-α). The resulting GFLs-GFRα complex binds to the extracellular domain of RET, leading to phosphorylation of the intracellular tyrosine kinase domain, recruitment of associated adaptor proteins, and activation of a signaling cascade that promotes cell proliferation, thereby activating several pathways, including MAPK, PI3K, JAK-STAT, PKA, and PKC.

[0003] There are two primary mechanisms for RET oncogenic activation. First, chromosomal rearrangements generate new fusion proteins, typically a fusion of the RET kinase domain and a protein containing the self-dimerization domain. Second, point mutations in the RET gene may encode a RET protein with abnormal activity, which can transmit aberrant signals and affect multiple aspects of cell growth, survival, invasion, and metastasis. Sustained signaling can lead to excessive cell proliferation and induce various cancers.

[0004] RET rearrangements occur in 1%-2% of NSCLC patients and 5%-10% of papillary thyroid cancer patients, and RET point mutations occur in 60% of medullary thyroid cancer. The most common RET fusion types are KIF5B-RET and CCDC6-RET, followed by NCOA4-RET and TRIM33-RET. ZNF477P-RET, ERCC1-RET, HTR4-RET, and CLIP1-RET have also been reported.

[0005] Currently, several multi-target tyrosine kinase inhibitors have been used in clinical studies for patients with RET gene mutations, such as vandetanib (primarily indicated for the treatment of unresectable, locally advanced, or metastatic, symptomatic, or progressive medullary thyroid cancer) and sorafenib (for liver cancer, kidney cancer, and locally recurrent or metastatic, progressive, or radioactive iodine-refractory differentiated thyroid cancer). While broad anticancer efficacy may also result in toxic side effects, the most common adverse drug reactions (>20%) with vandetanib are diarrhea, rash, acne, nausea, hypertension, headache, fatigue, decreased appetite, and abdominal pain (Vandetanib product insert, FDA); the most common drug-related adverse events with sorafenib are rash (38%), diarrhea (37%), hand-foot skin reaction (35%), and fatigue (33%) (Sorafenib product insert, FDA). Summary of the Invention

[0006] One or more embodiments of the present application provide a compound or stereoisomer, pharmaceutically acceptable salt, solvate, or tautomer of formula (I):

[0007]

[0008] in:

[0009] M is CH or N;

[0010] R 1 is H, C1-C4 alkyl or halogenated C1-C4 alkyl;

[0011] R 2 is CN, C2-C6 alkenyl, 6-8 membered aryl, 5-6 membered heteroaryl or 8-10 membered fused heteroaryl;

[0012] The C2-C6 alkenyl group is optionally substituted with a substituent selected from halogen, halogenated C1-C3 alkyl, C1-C6 alkyl and C1-C6 alkoxy;

[0013] The 6-8 membered aryl or 5-6 membered heteroaryl is optionally selected from halogenated C1-C3 alkyl, halogen, C1-C6 alkyl, C1-C6 alkoxy, 3-5 membered cycloalkyl, cyano, (CH2) n OR a 、NHR a 、NHC(=O)NHR a 、NHC(=O)OR a 、NHC(=O)Cyc 1 、NHCyc 2 、NHC(=O)(CH2) n OR a 、NHS(=O)NHRa 、NHC(=O)C(=O)NHR a 、C(=O)NHR a 、C(=O)OR a and OC(=O)NHR a Substituents substituted; Cyc 1 is a four-membered ring containing one N heteroatom and is optionally substituted by a substituent selected from halogen, amino, C1-C6 alkyl and C1-C6 alkoxy; Cyc 2 A four-membered ring containing one or more oxo groups, and optionally substituted by a substituent selected from halogen, amino, C1-C6 alkyl and C1-C6 alkoxy; the 5-6 membered heteroaryl group has a heteroatom selected from N, O and S; n is 1, 2 or 3;

[0014] wherein R a is hydrogen or C1-C4 alkyl, wherein the C1-C4 alkyl is optionally substituted by a substituent selected from halogen, amino and hydroxy;

[0015] The 8-10 membered fused heteroaryl group contains one or more heteroatoms, and is optionally substituted by a substituent selected from halogen, amino and C1-C4 alkyl, wherein the heteroatoms are selected from N, S and O;

[0016] The conditions are:

[0017] And R 1 When selected from H, the R 2 Not the following structures:

[0018]

[0019]

[0020] In one or more embodiments, wherein R2 is a C2-C6 alkenyl group selected from the following substituted or unsubstituted structures: Its substituents are selected from the group consisting of halogen, halogenated C1-C3 alkyl, C1-C4 alkyl and C1-C4 alkoxy.

[0021] In one or more embodiments, wherein R2 is a C2-C6 alkenyl group selected from the following substituted or unsubstituted structures: Its substituents are selected from the group consisting of: F, CF3, CF2, and CH3; R1 is H or CH3.

[0022] In one or more embodiments, the substituent of the 6-8 membered aryl or 5-6 membered heteroaryl is NHC(=O)Cyc 1 、NHCyc 2 When Cyc 1is a four-membered ring containing one N heteroatom and optionally substituted by a substituent selected from halogen, amino, C1-C4 alkyl and C1-C4 alkoxy; wherein Cyc 2 A four-membered ring containing two oxo groups, and optionally substituted by a substituent selected from halogen, amino, C1-C6 alkyl and C1-C4 alkoxy.

[0023] In one or more embodiments, wherein Cyc 1 for Cyc 2 for

[0024] In one or more embodiments, wherein R 2 is phenyl, wherein the phenyl group is arbitrarily selected from halogenated C1-C3 alkyl, halogen, C1-C6 alkyl, C1-C6 alkoxy, 3-5 membered cycloalkyl, cyano, (CH2) n OR a 、NHR a 、NHC(=O)NHR a 、NHC(=O)OR a 、NHC(=O)Cyc 1 、NHCyc 2 、NHC(=O)(CH2) n OR a 、NHS(=O)NHR a 、NHC(=O)C(=O)NHR a 、C(=O)NHR a 、C(=O)OR a , and OC(=O)NHR a Substituents substituted; Cyc 1 for Cyc 2 for wherein R e Each is independently hydrogen or C1-C4 alkyl, wherein the C1-C4 alkyl is optionally substituted with a substituent selected from halogen, amino and hydroxy.

[0025] In one or more embodiments, wherein said R2 is a 5-6 membered heteroaryl group selected from the following substituted or unsubstituted structures: The substituents are as defined above.

[0026] In one or more embodiments, wherein said R2 is a 5-6 membered heteroaryl group selected from the following substituted or substituted structures: Its substituents are selected from CH3, C2H5, OCH3, CH2OCH3, CH2CH2OCH3, C(=O)NHCH3, C(=O)OCH3, CN and CF2; R1 is H, CH3, C2H5 or C3H7.

[0027] In one or more embodiments, the 8-10 membered fused heteroaryl group is the following structure: The structure is optionally substituted with substituents selected from the group consisting of halogen, amino, and C1-C4 alkyl.

[0028] In one or more embodiments, wherein M is N.

[0029] In one or more embodiments, wherein R1 is H, CH3, C2H5 or CH(CH3)2.

[0030] In one or more embodiments, wherein R 2 Substituted with halogen, amino, methyl or ethyl Phenyl.

[0031] In one or more embodiments, wherein R 2 for

[0032] In one or more embodiments, wherein R1 is CH3 or C2H5, CH(CH3)2; M is N; R 2 for

[0033] One or more embodiments of the present application provide a compound or stereoisomer, pharmaceutically acceptable salt, solvate, or tautomer of formula (I'):

[0034]

[0035] in:

[0036] M is CH or N;

[0037] R 1 is H, C1-C4 alkyl or halogenated C1-C4 alkyl;

[0038] R 2 is CN, C2-C6 alkenyl, 6-8 membered aryl, 5-6 membered heteroaryl, 8-10 membered fused heteroaryl, or 5-membered heterocycloalkyl containing an oxygen atom or a nitrogen atom;

[0039] The C2-C6 alkenyl group is optionally substituted by halogen, halogenated C1-C3 alkyl, C1-C6 alkyl, or C1-C6 alkoxy;

[0040] The 6-8 membered aryl, 5-6 membered heteroaryl or 5 membered heterocycloalkyl containing nitrogen atoms is optionally selected from oxo, halogenated C1-C3 alkyl, halogen, C1-C6 alkyl, C1-C6 alkoxy, 3-5 membered cycloalkyl, C1-C3 alkyl, halogen-substituted oxygen or nitrogen-containing 4-5 membered heterocyclic group, cyano, (CH2) n OR a 、NHR a 、NHC(=O)NHR a 、NHC(=O)OR a 、NHC(=O)Cyc 1 、NHCyc 2 、NHC(=O)(CH2) n OR a 、NHS(=O)NHR a 、NHC(=O)C(=O)NHR a 、C(=O)NHR a 、C(=O)OR a , and OC(=O)NHR a Substituents substituted; Cyc 1 is a four-membered ring containing one N heteroatom and is optionally substituted by a substituent selected from halogen, amino, C1-C6 alkyl and C1-C6 alkoxy; Cyc 2 is a four-membered ring containing one or more oxo groups, and is optionally substituted by a substituent selected from halogen, amino, C1-C6 alkyl and C1-C6 alkoxy; the heteroatom of the 5-6 membered heteroaryl group is N, O or S; n is 1, 2 or 3;

[0041] wherein R a is hydrogen or C1-C4 alkyl, wherein the C1-C4 alkyl is optionally substituted by a substituent selected from halogen, amino and hydroxy;

[0042] The 8-10 membered fused heteroaryl group contains one or more heteroatoms, and is optionally substituted by a substituent selected from halogen, amino and C1-C4 alkyl, wherein the heteroatom is N, S or O;

[0043] The conditions are:

[0044] And R 1 When H, the R 2 Not the following structures:

[0045]

[0046] In one or more embodiments, wherein R2 is a C2-C6 alkenyl group, the C2-C6 alkenyl group is optionally substituted with a substituent selected from halogen, halogenated C1-C3 alkyl, C1-C4 alkyl and C1-C4 alkoxy:

[0047] In one or more embodiments, wherein R2 is a C2-C6 alkenyl group, the C2-C6 alkenyl group is the following structure optionally substituted with a substituent selected from F, CF3, CF2 and CH3: R1 is H or CH3.

[0048] In one or more embodiments, the substituent of the 6-8 membered aryl or 5-6 membered heteroaryl is NHC(=O)Cyc 1 、NHCyc 2 When Cyc 1 is a four-membered ring containing one N heteroatom and optionally substituted by a substituent selected from halogen, amino, C1-C4 alkyl and C1-C4 alkoxy; wherein Cyc 2 A four-membered ring containing two oxo groups, and optionally substituted by a substituent selected from halogen, amino, C1-C6 alkyl and C1-C4 alkoxy.

[0049] In one or more embodiments, wherein Cyc 1 for Cyc 2 for

[0050] In one or more embodiments, wherein R 2 is phenyl, wherein the phenyl group is optionally selected from halogenated C1-C3 alkyl, halogen, C1-C6 alkyl, C1-C6 alkoxy, 3-5 membered cycloalkyl, oxygen- or nitrogen-containing 4-5 membered heterocyclic group substituted by oxo, C1-C3 alkyl, halogen, cyano, (CH2) n OR a 、NHR a 、NHC(=O)NHR a 、NHC(=O)OR a 、NHC(=O)Cyc 1 、NHCyc 2 、NHC(=O)(CH2) n OR a 、NHS(=O)NHR a 、NHC(=O)C(=O)NHR a 、C(=O)NHR a 、C(=O)OR a 、OC(=O)NHR a Substituents substituted; Cyc 1 for Cyc 2 for wherein R aEach is independently hydrogen or C1-C4 alkyl, wherein the C1-C4 alkyl is optionally substituted with a substituent selected from halogen, amino and hydroxy.

[0051] In one or more embodiments, wherein said R2 is a 5-6 membered heteroaryl group, which is optionally substituted by a substituent selected from the group defined above:

[0052] In one or more embodiments, wherein the R2 is a 5-6 membered heteroaryl, the 5-6 membered heteroaryl is optionally substituted by a substituent selected from CH3, C2H5, OCH3, CHOCH3, CH2CH2OCH3, C(=O)NHCH3, C(=O)OCH3, CN and CF2: R1 is H, CH3, C2H5 or C3H7.

[0053] In one or more embodiments, the 8-10 membered fused heteroaryl group is the following structure: The structure is optionally substituted with substituents selected from the group consisting of halogen, amino, C1-C4 alkyl, and C1-C4 alkoxy.

[0054] In one or more embodiments, wherein M is N.

[0055] In one or more embodiments, wherein R1 is H, CH3, C2H5 or CH(CH3)2.

[0056] In one or more embodiments, wherein R 2 Substituted with halogen, amino, methyl or ethyl or phenyl.

[0057] In one or more embodiments, R 2 for

[0058] In one or more embodiments, wherein R1 is CH3, C2H5 or CH(CH3)2; M is N; R 2 for

[0059] In one or more embodiments, the compounds of the present application have the following structure:

[0060]

[0061]

[0062]

[0063] One or more embodiments of the present application provide a pharmaceutical composition comprising the compound of the present application or a stereoisomer, a pharmaceutically acceptable salt, a solvate, or a tautomer thereof, and a pharmaceutically acceptable excipient.

[0064] One or more embodiments of the present application provide use of the compound of the present application or its stereoisomer, pharmaceutically acceptable salt, solvate, or tautomer, or the pharmaceutical composition of the present application in the preparation of a medicament for treating cancer.

[0065] One or more embodiments of the present application provide a compound or pharmaceutical composition of the present application for use as a medicament.

[0066] One or more embodiments of the present application provide the compound or pharmaceutical composition of the present application for treating and / or preventing cancer.

[0067] One or more embodiments of the present application provide a method for treating and / or preventing cancer, comprising administering an effective amount of the compound or pharmaceutical composition of the present application to a subject in need thereof.

[0068] In one or more embodiments, the cancer is lung cancer, papillary thyroid cancer, medullary thyroid cancer, differentiated thyroid cancer, recurrent thyroid cancer, refractory differentiated thyroid cancer, multiple endocrine neoplasia type 2A or 2B (MEN2A or MEN2B, respectively), pheochromocytoma, parathyroid hyperplasia, breast cancer, colorectal cancer, papillary renal cell carcinoma, gastrointestinal ganglioneuroma, or cervical cancer.

[0069] In one or more embodiments, the cancer is a RET or mutant RET mediated cancer.

[0070] In one or more embodiments, the cancer is medullary thyroid carcinoma, non-small cell lung cancer, metastatic solid tumor or advanced solid tumor with RET gene mutation or fusion.

[0071] One or more embodiments of the present application provide uses of the compound of the present application or its stereoisomer, pharmaceutically acceptable salt, solvate, or tautomer, or the pharmaceutical composition of the present application in the preparation of a medicament for treating BTK-mediated diseases.

[0072] One or more embodiments of the present application provide the compound or pharmaceutical composition of the present application for treating BTK-mediated diseases.

[0073] One or more embodiments of the present application provide a method for treating and / or preventing a BTK-mediated disease, comprising administering an effective amount of a compound or pharmaceutical composition of the present application to a subject in need thereof.

[0074] In one or more embodiments, the BTK-mediated disease is cancer, autoimmune disease or allergic disease.

[0075] In one or more embodiments, the cancer is one or more of subtype diffuse large B-cell lymphoma, mantle cell lymphoma, chronic lymphocytic lymphoma, extranodal marginal zone B-cell lymphoma, B-cell chronic lymphocytic leukemia, B-cell prolymphocytic leukemia, mature B-cell acute lymphoblastic leukemia, 17p deleted chronic lymphocytic leukemia, Waldenstrom macroglobulinemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasma cell myeloma, plasmacytoma, nodal marginal zone B-cell lymphoma, mantle cell lymphoma, intravascular large B-cell lymphoma and primary effusion lymphoma; the autoimmune disease is systemic lupus erythematosus, rheumatoid arthritis, Sjögren's syndrome, multiple sclerosis, inflammatory bowel disease, Inflammation such as Crohn's disease and ulcerative colitis, urticaria, immune thrombocytopenia, IgA nephropathy, hidradenitis suppurativa, psoriasis, vitiligo, neutrophilic dermatosis, autoimmune blistering disease such as pemphigus and pemphigoid, IgG4-related disease, autoimmune hemolytic anemia, rheumatic fever, antiphospholipid syndrome, systemic sclerosis / scleroderma, autoimmune hepatitis, primary sclerosing cholangitis, primary biliary cirrhosis, Henoch-Schonlein purpura, Churg-Strauss syndrome / allergic granulomatosis with polyangiitis, Behçet's disease / Behçet's disease, ANCA-associated small vessel vasculitis, and one or more of dermatitis herpetiformis; the allergic disease is one or more of allergic conjunctivitis, allergic rhinitis, allergic asthma, atopic dermatitis, and chronic asthma. BRIEF DESCRIPTION OF THE DRAWINGS

[0076] Figure 1 It shows the changes in tumor volume after administration of diffuse large B-type transformed lymphoma DOHH2 model control group (vehicle), ARQ-531 (30 mg / kg, QD), compound 29 (10 mg / kg, 30 mg / kg, 60 mg / kg, BID), and compound 58 (30 mg / kg, 60 mg / kg, BID).

[0077] Figure 2 The absolute body weight changes after administration of diffuse large B-cell transformed lymphoma DOHH2 model control group (vehicle), ARQ-531 (30 mg / kg, QD), compound 29 (10 mg / kg 30 mg / kg, 60 mg / kg, BID), and compound 58 (30 mg / kg, 60 mg / kg, BID).

[0078] Figure 3The relative body weight changes of diffuse large B-cell transformed lymphoma TMD8 model control group (vehicle), LOXO-305 (10 mg / kg, BID), compound 29 (10 mg / kg, BID), and compound 29 (30 mg / kg, BID) after administration.

[0079] Figure 4 It shows the changes in tumor volume of diffuse large B-type transformed lymphoma TMD8 model control group (vehicle), LOXO-305 (10 mg / kg, BID), compound 29 (10 mg / kg, BID), and compound 29 (30 mg / kg, BID) after administration.

[0080] Figure 5 The fluorescence values of tumor cells in the MiniPDX control group (vehicle, vehicle-D7), ibrutinib (10 mg / kg), LOXO-305 (50 mg / kg), ARQ-531 (50 mg / kg), compound 29 (50 mg / kg), and compound 23 (50 mg / kg), a tumor model of diffuse large B-cell transformation of chronic lymphocytic leukemia, were shown.

[0081] Figure 6 The T / C (%) after administration of MiniPDX control group (vehicle, vehicle-D7), ibrutinib (10 mg / kg), LOXO-305 (50 mg / kg), ARQ-531 (50 mg / kg), compound 29 (50 mg / kg), and compound 23 (50 mg / kg), a tumor model of diffuse large B-cell transformation of chronic lymphocytic leukemia.

[0082] Figure 7 The relative body weight changes of MiniPDX control group (vehicle, vehicle-D7), ibrutinib (10 mg / kg), LOXO-305 (50 mg / kg), ARQ-531 (50 mg / kg), compound 29 (50 mg / kg), and compound 23 (50 mg / kg), a tumor model of diffuse large B-cell transformation of chronic lymphocytic leukemia, were shown after administration. DETAILED DESCRIPTION

[0083] Before further describing the present invention, it should be understood that the present invention is not limited to the specific embodiments described. The terms used herein are for the purpose of describing the specific embodiments only and are not intended to be limiting. In addition, the scope of the present invention is limited only by the appended claims and the specification. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0084] Chemical definition

[0085] As used herein, the term "alkyl" refers to an aliphatic hydrocarbon group, including saturated hydrocarbon groups. The alkyl portion may be a straight-chain or branched chain alkyl group. For example, C1-6 alkyl, C1-4 alkyl, or C1-3 alkyl. C1-6 alkyl refers to an alkyl group having 1 to 6 carbon atoms, such as 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, or 6 carbon atoms. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, tert-pentyl, hexyl, isohexyl, and groups equivalent to any of the foregoing examples, as considered by those skilled in the art and the teachings provided herein. The alkyl group may be unsubstituted or substituted with one or more substituents, including, but not limited to, alkyl, alkoxy, cyano, hydroxy, carbonyl, carboxyl, aryl, heteroaryl, amine, halogen, sulfonyl, sulfinyl, phosphono, and the like.

[0086] As used herein, the term "alkenyl" refers to a straight or branched hydrocarbon group having from 2 to 12 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12) carbon atoms in the chain and having one or more double bonds. An alkenyl group can be unsubstituted or substituted as described for an alkyl group or as described in the various examples provided herein. The term includes cis- and trans-isomers and mixtures thereof.

[0087] As used herein, the term "alkynyl" refers to a straight or branched hydrocarbon group having 2 to 12 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12) carbon atoms in the chain and having one or more triple bonds. Alkynyl groups can be unsubstituted or substituted as described for alkyl groups or as described in the various examples provided herein.

[0088] In the term, "ring" refers to any covalently closed structure, including, for example, carbocyclic rings (e.g., aryl or cycloalkyl), heterocyclic rings (e.g., heteroaryl or heterocycloalkyl), aromatic groups (e.g., aryl or heteroaryl), non-aromatic groups (e.g., cycloalkyl or heterocycloalkyl). The ring can be optionally substituted and can be a monocyclic or polycyclic ring. Typical polycyclic rings generally include bicyclic and tricyclic rings. The ring of the present application generally has 1-20 ring atoms, for example, 1 ring atom, 2 ring atoms, 3 ring atoms, 4 ring atoms, 5 ring atoms, 6 ring atoms, 7 ring atoms, 8 ring atoms, 9 ring atoms, 10 ring atoms, 11 ring atoms, 12 ring atoms, 13 ring atoms, 14 ring atoms, 15 ring atoms, 16 ring atoms, 17 ring atoms, 18 ring atoms, 19 ring atoms or 20 ring atoms.

[0089] The term "membered" refers to the number of atoms that make up the ring. Typical five-membered rings include cyclopentyl, pyrrole, imidazole, thiazole, furan, and thiophene; typical six-membered rings include cyclohexyl, pyridine, pyran, pyrazine, thiopyran, pyridazine, pyrimidine, and benzene. A ring containing heteroatoms among its backbone atoms is a heterocycle; an aromatic group containing heteroatoms is a heteroaryl group; and a non-aromatic group containing heteroatoms is a heterocycloalkyl group, which includes heterocycloalkyls.

[0090] The term "heteroatom" refers to an atom other than carbon or hydrogen. The one or more heteroatoms in the heterocycle of the present application may be independently selected from O, S, N, Si and P, but is not limited thereto.

[0091] The term "oxo" refers to the replacement of a hydrogen on a carbon by =0.

[0092] The term "halogen" refers to fluorine, chlorine, bromine or iodine.

[0093] The term "haloalkyl" refers to an alkyl group in which at least one hydrogen atom is replaced by a halogen atom, such as CF3.

[0094] The term "cycloalkyl" refers to a saturated or partially unsaturated (containing one or more double bonds, but no ring has a completely conjugated π electron system) cyclic hydrocarbon substituent containing 1-3 (e.g., 1, 2, or 3) rings, including monocyclic alkyl, bicyclic alkyl, and tricyclic alkyl, which contains 3-20 (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) carbon atoms that can form a ring, preferably 3-10 carbon atoms (i.e., 3-10 membered cycloalkyl, also referred to as C3-C10 cycloalkyl), such as 3 to 8, 3 to 7, 3 to 6, or 5 to 6 carbon atoms. Preferably, the cycloalkyl is selected from a monovalent cycloalkyl derived from the following rings:

[0095]

[0096] Preferred are cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclopentenyl or cyclohexenyl.

[0097] "Heterocycloalkyl" and "cycloheteroalkyl" are used interchangeably and refer to saturated, non-aromatic, monocyclic, fused, bridged, and spirocyclic rings containing one or more (e.g., 1, 2, 3, or 4) heteroatoms, wherein the heteroatoms may be N, O, or S. Heterocycloalkyl may be a 3- to 10-membered (e.g., 3-, 4-, 5-, 6-, 7-, 8-, 9-, or 10-membered, i.e., containing 3, 4, 5, 6, 7, 8, 9, or 10 ring atoms) monocyclic, bicyclic, or tricyclic group. The ring structure may optionally contain up to two oxo groups on carbon or sulfur ring members.

[0098] Typical heterocycloalkyl groups include, but are not limited to, monovalent groups derived from the following rings:

[0099]

[0100] These heterocycloalkyl groups can also be represented by commonly understood structural formulas, such as

[0101] It should be understood that when a heterocycloalkyl group is connected to two groups according to the structure or context, the heterocycloalkyl group is a divalent group, i.e., it has two attachment sites. In this case, it can also be referred to as a heterocycloalkylene group. Examples of heterocycloalkylene groups include, but are not limited to, divalent groups formed from the above groups, such as:

[0102]

[0103] The term "aryl" refers to a monocyclic or fused polycyclic (i.e., rings that share adjacent pairs of carbon atoms) group having 6 to 14 carbon atoms (6 to 14 members) (e.g., 6, 7, 8, 9, 10, 11, 12, 13, or 14) with a conjugated π electron system. Exemplary aryl groups include, but are not limited to, phenyl, naphthyl, and anthracenyl. Aryl groups can be unsubstituted or substituted as described for alkyl groups or as described in the various examples provided herein.

[0104] The term "heteroaryl" refers to a heteroaromatic system containing 1 to 4 (e.g., 1, 2, 3, or 4) heteroatoms, 5 to 14 ring atoms (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14), wherein the heteroatoms are selected from O, S, and N. Illustrative examples of heteroaryl groups include the following entities in the form of appropriate bonding moieties:

[0105]

[0106] The heteroaryl ring may be fused to an aryl, heterocycloalkyl, cycloalkyl ring or another heteroaryl to form a fused heteroaryl. The fused heteroaryl is preferably an 8-10 membered fused heteroaryl, wherein the heteroatoms are selected from O, S and N, and may be optionally substituted with halogen, oxo, amino or C1-C6 alkyl.

[0107] "Substitution" means that one or more hydrogen atoms, preferably up to 5 (e.g., 1, 2, 3, 4, 5), and more preferably 1 to 3 hydrogen atoms, in a group can be replaced independently of one another by a corresponding number of substituents. It goes without saying that the substituents are only in their possible chemical positions, and a person skilled in the art can determine (by experiment or theory) without undue effort which substitutions are possible or impossible. For example, an amino or hydroxyl group having free hydrogen may be unstable when combined with a carbon atom having an unsaturated (e.g., olefinic) bond.

[0108] "Optionally" and "may" mean that the subsequently described event or circumstance can but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0109] The term "substituted or unsubstituted" herein refers to any group that is monosubstituted or polysubstituted by a specified substituent to the extent that such monosubstituted or polysubstituted (including multiple substitutions on the same moiety) is chemically permitted, and each substituent can be located at any available position on the group and can be attached through any available atom on the substituent. "Any available position" refers to any position on the group that is chemically accessible by methods known in the art or methods taught herein and does not produce an overly unstable molecule. When there are two or more substituents on any group, each substituent is defined independently of any other substituent and can therefore be the same or different.

[0110] The term "compounds of the invention" as used herein is intended to encompass compounds of formula (I) and (I') as defined herein, or any preferred or specific embodiment thereof, or pharmaceutically acceptable salts, tautomers or solvates thereof.

[0111] The term "pharmaceutically acceptable" as used herein refers to molecular entities and compositions that are or are approvable by relevant agencies in various countries, or are listed in the generally recognized pharmacopoeia for use in animals, and more particularly humans, or that do not produce adverse, allergic or other untoward reactions when administered in appropriate amounts to animals, such as humans.

[0112] The term "pharmaceutically acceptable salt" as used herein refers to a salt of a compound of the present invention that is pharmaceutically acceptable and possesses the desired pharmacological activity of the parent compound. Specifically, such salts are non-toxic and can be inorganic acid addition salts or organic acid addition salts and base addition salts.

[0113] The "pharmaceutical composition" of the present invention refers to a composition comprising one or more compounds of formula (I) and (I') or stereoisomers, tautomers, pharmaceutically acceptable salts or solvates thereof, and a carrier or excipient generally accepted in the art for delivering biologically active compounds to an organism (e.g., a human).

[0114] It should be understood that the structures and groups of the compounds of the present invention conform to the rules of chemical valence bonds. When writing certain groups or structures, their connecting bonds are omitted. For example, in some cases, it is recorded that M in formula (I) is selected from N, and based on the general structure, M is =N-. Whether it is written as M selected from N or M selected from =N-, it is understood by those skilled in the art. In addition, in the present invention, substituents such as NHR a Based on the general structure, we know that NHR a -NH-R a Other groups can also be understood and explained similarly.

[0115] Obviously, based on the above contents of the present invention, according to the common technical knowledge and means in this field, without departing from the above basic technical ideas of the present invention, other various forms of modifications, replacements or changes can be made.

[0116] One or more embodiments of the present application provide compounds having the structural characteristics of the above general formulas (I) and (I'). Studies have found that such compounds can effectively inhibit RET and / or BTK-mediated, or mutated RET and / or BTK-mediated diseases, thereby preventing or treating RET and / or related diseases.

[0117] In one or more embodiments, the compounds of the present application have one or more of the following technical effects:

[0118] High RET and / or BTK kinase inhibitory activity Preferred compounds of the present invention show an IC50 of 0.1 nM to 1 μM, such as 0.1 nM to 0.1 μM in assays; and / or

[0119] It has high activity against mutant RET and / or BTK, and thus can be used to treat related diseases that have developed drug resistance due to mutations; and / or it has high kinase selectivity, and thus has reduced side effects.

[0120] The compounds of one or more embodiments of the present application have advantages such as good bioavailability and metabolic stability, and low cardiotoxicity.

[0121] The compounds of one or more embodiments of the present application have better solubility, stability, hygroscopicity, pharmacokinetic properties, bioavailability, etc.

[0122] Based on the beneficial effects of the above compounds of the present invention, the present invention also provides the following technical solutions in various aspects.

[0123] The pharmaceutical composition of the present invention can be prepared by techniques known to those skilled in the art. The pharmaceutical composition of the present invention can be prepared by mixing the compound of the present invention or a pharmaceutically acceptable salt thereof with one or more pharmaceutically acceptable excipients.

[0124] Examples of pharmaceutically acceptable excipients in the present invention include adjuvants, diluents, carriers, pH regulators, buffers, sweeteners, fillers, stabilizers, surfactants, wetting agents, lubricants, emulsifiers, suspending agents, preservatives, antioxidants, opacifiers, glidants, processing aids, colorants, flavorings, flavorings, diluents, binders and other known additives.

[0125] The compounds of the present invention or pharmaceutically acceptable salts thereof can be formulated into solutions, emulsions, suspensions or dispersions in suitable pharmaceutical solvents or carriers according to conventional methods known in the art for preparing various dosage forms, or formulated together with pharmaceutically acceptable excipients into tablets, capsules, syrups, powders, granules, aqueous or oily solutions or suspensions, (lipid) emulsions, dispersible powders, suppositories, ointments, creams, drops, aerosols, dry powder formulations and sterile injectable aqueous or oily solutions or suspensions. The pharmaceutical compositions of the present specification can be delivered by a suitable mode of administration, for example, oral, intravenous, rectal, parenteral, topical, transdermal, ophthalmic, nasal, buccal or pulmonary (inhalation) administration, wherein parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration.

[0126] This patent also provides a method for synthesizing the above-mentioned compounds. The method of the invention is mainly based on the preparation methods reported in chemical literature or on the relevant synthesis using commercially available chemical reagents as starting materials.

[0127] In the synthetic examples of the present application:

[0128] Cs2CO3 represents cesium carbonate;

[0129] K2CO3 represents potassium carbonate;

[0130] Na2CO3 represents sodium carbonate;

[0131] DMF stands for N,N-dimethylformamide;

[0132] DCM means dichloromethane;

[0133] EtOH indicates ethanol;

[0134] THF stands for tetrahydrofuran;

[0135] TEA stands for triethylamine;

[0136] EA represents ethyl acetate;

[0137] 1,4-dioxane means 1,4-dioxane;

[0138] DIPEA stands for N,N-diisopropylethylamine;

[0139] DIAD stands for diisopropyl azodicarboxylate;

[0140] PPh3 represents triphenylphosphine

[0141] LiOH represents lithium hydroxide;

[0142] HCl stands for hydrogen chloride;

[0143] POCl3 represents phosphorus oxychloride;

[0144] FDPP stands for pentafluorophenyl diphenyl phosphate;

[0145] PPh3 represents triphenylphosphine;

[0146] NBS denotes N-bromosuccinimide;

[0147] CDI stands for N,N'-carbonyldiimidazole;

[0148] RuPhos Pd G3 represents methanesulfonic acid (2-dicyclohexylphosphino-2′,6′-diisopropoxy-1,1′-biphenyl)(2-amino-1,1′-biphenyl-2-yl) palladium(II);

[0149] RuPhos represents 2-bicyclohexylphosphino-2',6'-diisopropoxybiphenyl;

[0150] Xphos Pd G3 represents methanesulfonic acid (2-dicyclohexylphosphino-2',4',6'-tri-isopropyl-1,1'-biphenyl

[0151] yl)(2'-amino-1,1'-biphenyl-2-yl)palladium(II);

[0152] K3PO4 represents anhydrous potassium phosphate;

[0153] Pd(dppf)cl2 represents [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride;

[0154] Pd(PPh3)4 represents tetrakistriphenylphosphine palladium;

[0155] RuCl3·H2O represents hydrated ruthenium trichloride;

[0156] HATU represents 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate;

[0157] NaHMDS stands for sodium hexamethyldisilazide;

[0158] Xphos represents 2-dicyclohexylphosphino-2,4,6-triisopropylbiphenyl;

[0159] TBAF represents tetrabutylammonium fluoride;

[0160] TMSOTf stands for trimethylsilyl trifluoromethanesulfonate.

[0161] Example 1: (S, 1 3 E,1 4 E)-17-amino-4 5 -Fluoro-6-methyl-1 6The preparation and synthesis steps of -(4-(trifluoromethyl)phenyl)-5-oxa-2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidin-4(3,2)-pyridine heterocycle nonacyclopentane-9-one (Compound 1) are as follows:

[0162]

[0163] Step 1: Preparation of 2-((5-fluoro-2-methoxypyridin-3-yl)methyl)isoindoline-1,3-dione (Compound 1A)

[0164]

[0165] Under argon protection, (5-fluoro-2-methoxypyridin-3-yl)methanol (3.14 g, 20 mmol) and TEA (2.83 g, 28 mmol) were dissolved in DCM (100 mL) in a 250 mL single-necked flask. Methanesulfonyl chloride (2.4 g, 21 mmol) was added dropwise to the above solution at 0°C, and the reaction system was stirred at room temperature for 2 h, quenched with water, extracted with EA, and the organic layer was washed with water and saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered and concentrated. The residue was dissolved in DMF (50 mL), and potassium phthalimide (5.55 g, 30 mmol) was subsequently added at 0°C. The mixture was then stirred at room temperature overnight, quenched with water, extracted with EA, and the organic layer was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered and concentrated to obtain compound 1A.

[0166] MS (ESI) m / z 287.0 (M+H) +

[0167] Step 2: Preparation of 2-((5-fluoro-2-hydroxypyridin-3-yl)methyl)isoindoline-1,3-dione (Compound 1B)

[0168]

[0169] 2-((5-Fluoro-2-methoxypyridin-3-yl)methyl)isoindoline-1,3-dione (2 g, 6.99 mmol) was dissolved in 30 mL of ethanol, and a 4N HCl solution in dioxane (50 mL, 209.7 mmol) was added. The system was stirred at 80°C overnight. After the reaction was complete, the reaction solution was concentrated, and a small amount of water was added to the residue. The residue was filtered, and the resulting solid was rinsed with water and dried in vacuo to obtain Compound 1B.

[0170] MS (ESI) m / z 273.1 (M+H) + .

[0171] Step 3: Preparation of (S)-tert-butyl(2-((3-(((1,3-dioxoisoindol-2-yl)methyl)-5-fluoropyridin-2-yl)oxy)propyl)carbamate (Compound 1C)

[0172]

[0173] Under argon protection, 2-((5-fluoro-2-hydroxypyridin-3-yl)methyl)isoindoline-1,3-dione (600 mg, 2.21 mmol), (R)-tert-butyl(2-hydroxypropyl)carbamate (772 mg, 4.41 mmol), and PPh3 (1.16 g, 4.41 mmol) were dissolved in DCM (30 mL). DIAD (891 mg, 4.41 mmol) was added dropwise at 0°C and allowed to react at room temperature for 2 h. After the reaction was complete, the reaction solution was concentrated to dryness, and the residue was purified by column chromatography to obtain compound 1C. MS (ESI) m / z 430.2 (M+H) + .

[0174] Step 4: Preparation of (S)-tert-butyl(2-((3-(aminomethyl)-5-fluoropyridin-2-yl)oxy)propyl)carbamate (Compound 1D)

[0175]

[0176] Tert-butyl (S)-(2-((3-((1,3-dioxoisoindol-2-yl)methyl)-5-fluoropyridin-2-yl)oxy)propyl)carbamate (0.9 g, 2.10 mmol) was dissolved in 50 mL of ethanol, followed by the addition of hydrazine hydrate (0.262 g, 4.20 mmol, 80%) and stirring at 80°C for 2 h. After completion of the reaction, the mixture was filtered, the filtrate collected, and concentrated. The crude product was purified via a C-18 flash column to afford compound 1D.

[0177] MS (ESI) m / z 300.2 (M+H) + .

[0178] Step 5: Preparation of methyl 5,7-dihydroxypyrazolo[1,5-a]pyrimidine-3-carboxylate (Compound 1E)

[0179]

[0180] Methyl 5-amino-1H-pyrazole-4-carboxylate (28.2 g, 200.0 mmol) was dissolved in methanol. Diethyl malonate (64.0 g, 400.0 mmol) and sodium methoxide (32.4 g, 600.0 mmol) were added sequentially in an ice-water bath. The reaction mixture was heated to 90°C and stirred for 16 h. After completion of the reaction, the reaction system was cooled to 0°C, filtered, and the filter cake was rinsed with ethanol. The solid was dissolved in water, and hydrochloric acid was added to adjust the pH to 1. A large amount of solid precipitated, which was then filtered, the filter cake was rinsed with water, and dried to obtain compound 1E.

[0181] MS (ESI) m / z 210.1 (M+H) + .

[0182] Step 6: Preparation of methyl 5,7-dichloropyrazolo[1,5-a]pyrimidine-3-carboxylate (Compound 1F)

[0183]

[0184] To a reaction flask containing methyl 5,7-dihydroxypyrazolo[1,5-a]pyrimidine-3-carboxylate (10.45 g, 50.0 mmol) at 0°C was added POCl3 (100 mL), followed by the slow dropwise addition of N,N-dimethylaniline (15.1 g, 125.0 mmol). After the addition was complete, the reaction mixture was heated to 80°C and allowed to react for 16 h. Upon completion, the reaction solution was slowly poured into ice water to quench the reaction. The mixture was extracted twice with DCM, and the combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography to obtain compound 1F.

[0185] MS (ESI) m / z 246.0 (M+H) + .

[0186] Step 7: Preparation of methyl 5-chloro-7-(dibenzylamino)pyrazolo[1,5-a]pyrimidine-3-carboxylate (Compound 1G)

[0187]

[0188] Methyl 5,7-dichloropyrazolo[1,5-a]pyrimidine-3-carboxylate (7.35 g, 30.0 mmol) was dissolved in a mixture of 1,4-dioxane and DCM. DIPEA (7.7 g, 60.0 mmol) and dibenzylamine (7.1 g, 36.0 mmol) were then added sequentially. The reaction system was stirred at room temperature for 3 h. After completion of the reaction, the reaction solution was concentrated to remove excess POCl3, and the residue was purified by column chromatography to obtain compound 1G.

[0189] MS (ESI) m / z 407.1 (M+H) + .

[0190] Step 8: Preparation of methyl 6-bromo-5-chloro-7-(dibenzylamino)pyrazolo[1,5-a]pyrimidine-3-carboxylate (Compound 1H)

[0191]

[0192] Methyl 5-chloro-7-(dibenzylamino)pyrazolo[1,5-a]pyrimidine-3-carboxylate (8.2 g, 20.0 mmol) was dissolved in DCM, followed by the addition of NBS (4.3 g, 24.0 mmol). The reaction was stirred at room temperature for 16 h. After completion of the reaction, the mixture was concentrated under reduced pressure, and the residue was purified by column chromatography to yield compound 1H.

[0193] MS (ESI) m / z 485.0 (M+H) + .

[0194] Step 9: Preparation of (S)-methyl 6-bromo-5-((((2-((1-(((tert-butoxycarbonyl)amino)propan-2-yl)oxy)-5-fluoropyridin-3-yl)methyl)amino)-7-(dibenzylamino)pyrazolo[1,5-a]pyrimidine-3-carboxylate (Compound II)

[0195]

[0196] Methyl 6-bromo-5-chloro-7-(dibenzylamino)pyrazolo[1,5-a]pyrimidine-3-carboxylate (7.26 g, 15.0 mmol) was dissolved in ethanol, followed by the addition of tert-butyl (S)-(2-((3-(aminomethyl)-5-fluoropyridin-2-yloxy)propyl)carbamate (6.78 g, 22.5 mmol) and DIPEA (5.8 g, 45.0 mmol). The reaction system was heated to 80°C and stirred for 16 h. After completion of the reaction, the mixture was concentrated under reduced pressure, and the residue was purified by column chromatography to obtain compound 1I.

[0197] MS (ESI) m / z 748.7 (M+H) +

[0198] Step 10: Preparation of (S)-methyl 5-((2-((1-(((tert-butoxycarbonyl)amino)propan-2-yl)oxy)-5-fluoropyridin-3-yl)methyl)amino)-7-(dibenzylamino)-6-(4-(trifluoromethyl)phenyl)pyrazolo[1,5-a]pyrimidine-3-carboxylate (Compound 1J)

[0199]

[0200] Under nitrogen protection, (S)-6-bromo-5-((((2-((1-(((tert-butoxycarbonyl)amino)propan-2-yl)oxy)-5-fluoropyridin-3-yl)methyl)amino)-7-(dibenzylamino)pyrazolo[1,5-a]pyrimidine-3-carboxylic acid methyl ester (1.0 g, 1.34 mmol) and 4-trifluoromethylphenylboronic acid (509 mg, 2.68 mmol) were added to a flask containing a mixed solvent (1,4-diol). The mixture was added to a reaction flask of 4-nitro-1,4-dioxane / H2O, 4 / 1, 35 mL, and then XPhos (63.8 mg, 0.13 mmol), XPhos-Pd-G3 (113 mg, 0.13 mmol) and K3PO4 (568 mg, 2.68 mmol). The reaction solution was stirred at 80°C for 3 h. After the reaction was complete, the reaction system was concentrated under reduced pressure. The residue was separated and purified by silica gel column chromatography to obtain compound 1J. MS (ESI) m / z 814.0 (M+H) +

[0201] Step 11: (S,1 3 E,1 4 E)-1 7 -(dibenzylamino)-4-(dibenzylamino)- 5 -Fluoro-6-methyl-1 6 Preparation of -(4-(trifluoromethyl)phenyl)-5-oxa-2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidin-4(3,2)-pyridine heterocycle 9-one (Compound 1K)

[0202]

[0203] (S)-5-((2-((1-(((tert-Butoxycarbonyl)amino)propan-2-yl)oxy)-5-fluoropyridin-3-yl)methyl)amino)-7-(dibenzylamino)-6-(4-(trifluoromethyl)phenyl)pyrazolo[1,5-a]pyrimidine-3-carboxylate (8.13 g, 10.0 mmol) was dissolved in a mixed solution of methanol and THF, followed by the addition of LiOH (4.8 g, 200.0 mmol) aqueous solution. After the addition was complete, the reaction system was heated to 60 ° C and stirred for 16 h. The reaction system was then cooled to 0°C, the pH adjusted to 2-3 with 2N hydrochloric acid, and extracted three times with DCM. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. A solution of hydrochloric acid in 1,4-dioxane was added, and the mixture was stirred at room temperature for 1 hour. After the reaction was complete, the reaction system was concentrated to dryness, and DCM and DMF were added, followed by DIPEA (19.32 g, 150.0 mmol) and FDPP (19.2 g, 50.0 mmol). After the additions were complete, the mixture was stirred at room temperature for 16 hours. After the reaction was complete, the reaction system was concentrated to dryness under reduced pressure, and the residue was purified by silica gel column chromatography to obtain compound 1K.

[0204] MS (ESI) m / z 682.2 (M+H) +

[0205] Step 12: (S,1 3 E,1 4 E)-1 7 -Amino-4 5 -Fluoro-6-methyl-1 6 Preparation of -(4-(trifluoromethyl)phenyl)-5-oxa-2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidin-4(3,2)-pyridine heterocycle 9-one (Compound 1)

[0206]

[0207] (S,1 3 E,1 4 E)-1 7 -(dibenzylamino)-4-(dibenzylamino)- 5 -Fluoro-6-methyl-1 6 -(4-(Trifluoromethyl)phenyl)-5-oxa-2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidine-4(3,2)-pyridineheterocyclic 9-one (68.1 mg, 0.1 mmol) was dissolved in DCM (10.0 mL), and trifluoromethanesulfonic acid (150.0 mg, 1.0 mmol) was added at 0°C, and the reaction was maintained at 0°C for 2 h. After the reaction was complete, TEA was slowly added at 0°C to adjust the pH to 7-8. The reaction solution was concentrated under reduced pressure, and the residue was separated by high pressure preparative separation to obtain Compound 1 of Example 1.

[0208] MS (ESI) m / z 502.2 (M+H) +

[0209] 1 H NMR (400MHz, DMSO) δ9.86(d,J=8.7Hz,1H),8.08(s,1H),8.00(d,J=3.0Hz,1H),7.91(t,J=14.0Hz,2H),7.67(dd,J=8.8,2.9Hz,1H),7.58(s,2H),7 .07(t,J=6.0Hz,1H),6.93(d,J=46.4Hz,2H),5.11–4.88(m,2H),4.01(m, 1H), 3.83 (m, 1H), 3.12 (dd, J=12.3, 10.6Hz, 1H), 1.43 (t, J=16.6Hz, 3H).

[0210] Example 2: (S, 1 3 E,1 4E)-1 7 -Amino-1 6 -(1-ethyl-1H-pyrazol-4-yl)-4 5 The preparation and synthesis steps of -fluoro-6-methyl-5-oxa-2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidine-4(3,2)-pyridine heterocycle 9-one (Compound 2) are as follows:

[0211]

[0212] Step 1: Preparation of (S)-methyl 5-((2-((1-(((tert-butoxycarbonyl)amino)propan-2-yl)oxy)-5-fluoropyridin-3-yl)methyl)amino)-7-(dibenzylamino)-6-(1-ethyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylate (Compound 2A)

[0213]

[0214] Under nitrogen, (S)-6-bromo-5-((((2-((1-(((tert-Butoxycarbonyl)amino)propan-2-yl)oxy)-5-fluoropyridin-3-yl)methyl)amino)-7-(dibenzylamino)pyrazolo[1,5-a]pyrimidine-3-carboxylic acid methyl ester (800 mg, 1.07 mmol) and (1-ethyl-1H-pyrazol-4-yl)boronic acid (165 mg, 1.18 mmol) were added to a reaction flask containing a mixed solvent (1,4-dioxane / H2O, 4 / 1, 35 mL). Pd(dppf)Cl2 (78 mg, 0.11 mol) and K3PO4 (443 mg, 3.21 mol) were then added. The reaction solution was stirred at 80°C for 16 h. After the reaction was complete, the reaction system was concentrated under reduced pressure. The residue was separated and purified by column chromatography to obtain compound 2A. MS (ESI) m / z 764.2(M+H) +

[0215] Step 2: (S, 1 3 E,1 4 E)-1 7 -(dibenzylamino)-1 6 -(1-ethyl-1H-pyrazol-4-yl)-4 5 Preparation of 6-fluoro-5-methyl-5-oxa-2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidin-4(3,2)-pyridine heterocycle 9-one (Compound 2B)

[0216]

[0217] (S)-5-((2-((1-(((tert-Butoxycarbonyl)amino)propan-2-yl)oxy)-5-fluoropyridin-3-yl)methyl)amino)-7-(dibenzylamino)-6-(1-ethyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylate (330 mg, 0.43 mmol) was dissolved in a mixed solution of methanol and THF, and then LiOH (208 mg, 8.66 mmol)) aqueous solution was added. After the addition was complete, the reaction system was heated to 60°C and stirred for 16 h. After the reaction was complete, the reaction system was cooled to 0°C, the pH was adjusted to 2-3 with 2N hydrochloric acid, and the mixture was extracted three times with DCM. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. A solution of hydrochloric acid in 1,4-dioxane was added, and the mixture was stirred at room temperature for 1 hour. After the reaction was complete, the reaction system was concentrated to dryness, and DCM and DMF were added, followed by DIPEA (2.23 g, 17.32 mmol) and FDPP (334 mg, 0.87 mmol). After the additions were complete, the mixture was stirred at room temperature for 16 hours. After the reaction was complete, the reaction system was concentrated to dryness under reduced pressure, and the residue was purified by silica gel column chromatography to obtain compound 2B.

[0218] MS (ESI) m / z 632.3 (M+H) +

[0219] Step 3: (S, 1 3 E,1 4 E)-1 7 -Amino-4 5 -Fluoro-6-methyl-1 6 Preparation of -(1-ethyl-1H-pyrazol-4-yl)-5-oxa-2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidin-4(3,2)-pyridine heterocycle 9-one (Compound 2)

[0220]

[0221] (S,1 3 E,1 4 E)-1 7 -(dibenzylamino)-1 6 -(1-ethyl-1H-pyrazol-4-yl)-4 56-Fluoro-6-methyl-5-oxa-2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidine-4(3,2)-pyridineheterocyclic 9-one (63.1 mg, 0.1 mmol) was dissolved in DCM (10.0 mL), and trifluoromethanesulfonic acid (150.0 mg, 1.0 mmol) was added at 0°C, and the reaction was maintained at 0°C for 2 h. After the reaction was complete, TEA was slowly added at 0°C to adjust the pH to 7-8. The reaction solution was concentrated under reduced pressure, and the residue was subjected to high pressure preparative separation to obtain Compound 2 of Example 2.

[0222] MS (ESI) m / z 452.2 (M+H) +

[0223] 1 H NMR(400MHz,DMSO)δ9.88(d,J=8.8Hz,1H),8.04(s,1H),8.00(d,J=3.0Hz,1H),7.87 (s,1H),7.76(dd,J=8.8,2.9Hz,1H),7.51(s,1H),7.21(t,J=6.1Hz,1H),6.88(s,2H ),5.06–4.97(m,1H),4.93(dd,J=14.5,5.2Hz,1H),4.22(q,J=7.3Hz,2H),3.99(m,1 H),3.91(dd,J=14.5,5.6Hz,1H),3.11(dd,J=12.3,10.6Hz,1H),1.55–1.42(m,6H).

[0224] Example 3: (S, 1 3 E,1 4 E)-1 7 -Amino-4 5 -Fluoro-6-methyl-1 6 Preparation of -(1-(2-methoxyethyl)-1H-pyrazol-4-yl)-5-oxa-2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidin-4(3,2)-pyridine heterocycle 9-one (Compound 3)

[0225]

[0226] Using (1-(2-methoxyethyl)-1H-pyrazol-4-yl)boronic acid instead of (1-ethyl-1H-pyrazol-4-yl)boronic acid in step 1 of Example 2, the same preparation method as Example 2 was used to obtain Compound 3 of Example 3. MS (ESI) m / z 482.3 (M+H) +

[0227] 1H NMR (400MHz, DMSO) δ9.87(d,J=8.8Hz,1H),8.03(d,J=12.0Hz,1H),8.00(d,J=3.0Hz,1H),7.86 (s,1H),7.76(dd,J=8.8,2.9Hz,1H),7.54(s,1H),7.22(t,J=6.0Hz,1H),6.86(s,2H),5.00(m, 1H),4.93(dd,J=14.4,5.7Hz,1H),4.35(t,J=5.7Hz,2H),3.99(m,1H),3.92(dd,J=14.5,5.5Hz ,1H),3.81(t,J=5.8Hz,2H),3.33(s,3H),3.11(dd,J=12.3,10.8Hz,1H),1.46(d,J=6.1Hz,3H).

[0228] Example 4: (S, 1 3 E,1 4 E)-1 7 -Amino-4 5 -Fluoro-6-methyl-1 6 Preparation of -(1-methyl-1H-pyrazol-3-yl)-5-oxa-2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidin-4(3,2)-pyridine heterocycle 9-one (Compound 4)

[0229]

[0230] Substituting 1-methylpyrazol-3-yl-boronic acid pinacol ester for (1-ethyl-1H-pyrazol-4-yl)boric acid in step 1 of Example 2, the same preparation method as in Example 2 was used to obtain compound 4 of Example 4.

[0231] MS (ESI) m / z 438.1 (M+H) +

[0232] 1 H NMR (400MHz, DMSO) δ9.78(d,J=8.7Hz,1H),8.08(s,1H),8.00(d,J=3.0Hz,1H),7.96(d,J=2.1Hz,1H),7.82(m,2H),7.43( s,2H),6.60(d,J=2.2Hz,1H),5.09–4.93(m,2H),4.10–3.91(m,5H),3.12(dd,J=12.2,10.6Hz,1H),1.46(d,J=6.1Hz,3H).

[0233] Example 5: (S, 13 E,1 4 E)-1 7 -Amino-4 5 -Fluoro-6-methyl-1 6 Preparation of -(1-methyl-1H-pyrrol-3-yl)-5-oxa-2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidin-4(3,2)-pyridine heterocycle 9-one (Compound 5)

[0234]

[0235] Substituting 1-methylpyrrol-3-yl-boronic acid pinacol ester for (1-ethyl-1H-pyrazol-4-yl)boric acid in step 1 of Example 2, the same preparation method as in Example 2 was used to obtain Compound 5 of Example 5.

[0236] MS (ESI) m / z 437.1 (M+H) +

[0237] 1 H NMR (400MHz, DMSO) δ9.89 (d, J=8.8Hz, 1H), 8.02 (dd, J=17.1, 6.7Hz, 2H), 7.78 (d d,J=8.9,2.9Hz,1H),7.60(dd,J=15.7,7.1Hz,1H),7.18(dd,J=11.4,5.0Hz,1H), 6.95(dd,J=10.8,5.4Hz,1H),6.89(s,1H),6.59(s,1H),6.11–6.01(m,1H),5.07– 4.89(m,2H),3.97(m,2H),3.73(s,3H),3.15–3.06(m,1H),1.46(d,J=6.1Hz,3H).

[0238] Example 6: (S, 1 3 E,1 4 E)-1 7 -Amino-4 5 -Fluoro-6-methyl-1 6 Preparation of -(4-ethylphenyl)-5-oxa-2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidin-4(3,2)-pyridine heterocycle 9-one (Compound 6)

[0239]

[0240] Substituting 4-ethylphenylboronic acid pinacol ester for (1-ethyl-1H-pyrazol-4-yl)boronic acid in step 1 of Example 2, the same preparation method as in Example 2 was used to obtain compound 6 of Example 6.

[0241] MS (ESI) m / z 462.2 (M+H) +

[0242] 1 H NMR(400MHz, DMSO-d6)δ9.87(d,J=8.9Hz,1H),8.06(s,1H),8.00(d,J=3.1Hz,1H) ,7.71-7.69(m,1H),7.44(d,J=7.7Hz,2H),7.25(d,J=16.2Hz,2H),6.98(t,J=6.1 Hz,1H),6.66(s,2H),5.07–4.90(m,2H),4.04–4.00(m,1H),3.98–3.86(m,1H),3. 15–3.07(m,1H),2.75–2.70(m,2H),1.45(d,J=6.1Hz,3H),1.30(t,J=7.6Hz,3H).

[0243] Example 7: (S, 1 3 E,1 4 E)-1 7 -Amino-4 5 -Fluoro-6-methyl-1 6 Preparation of -(2-methoxypyridin-4-yl)-5-oxa-2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidine-4(3,2)-pyridine heterocycle 9-one (Compound 7)

[0244]

[0245] Substituting (2-methoxypyridin-4-yl)boronic acid for (1-ethyl-1H-pyrazol-4-yl)boronic acid in step 1 of Example 2, the same preparation method as in Example 2 was used to obtain Compound 7 of Example 7.

[0246] MS (ESI) m / z 465.17 (M+H) +

[0247] 1H NMR(400MHz,DMSO-d6)δ9.83(d,J=8.9Hz,1H),8.33(d,J=5.2Hz,1H),8.06(s, 1H), 7.99 (d, J = 3.1Hz, 1H), 7.67 (d, J = 7.8Hz, 1H), 7.17 (t, J = 6.1Hz, 1H), 7.03 (s,2H),6.93(d,J=5.2Hz,1H),6.80(s,1H),5.04–4.88(m,2H),4.02–4.02(m, 1H), 3.93 (s, 3H), 3.87–3.83 (m, 1H), 3.12–3.08 (m, 1H), 1.45 (d, J = 6.1Hz, 3H).

[0248] Example 8: (S, 1 3 E,1 4 E)-1 7 -Amino-1 6 -(4-cyclopropylphenyl)-4 5 Preparation of 6-fluoro-5-methyl-5-oxa-2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidine-4(3,2)-pyridine heterocycle 9-one (Compound 8)

[0249]

[0250] Substituting 2-(4-cyclopropylphenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane for (1-ethyl-1H-pyrazol-4-yl)boronic acid in step 1 of Example 2, the same preparation method as in Example 2 was used to obtain Compound 8 of Example 8.

[0251] MS (ESI) m / z 474.2 (M+H) +

[0252] 1 H NMR(400MHz,DMSO-d6)δ9.86(br s,1H),8.07(s,1H),8.00(s,1H),7.84–7.46(m,2H),7.48–7.08(m,4H),7.08–6.49(m,2H),5 .09–4.83(m,2H),4.07–3.88(m,2H),3.12(t,J=11.9Hz,1H),1.59–0.96(m,5H),1.45(s,3H).

[0253] Example 9: (S, 1 3 E,1 4 E)-1 7 -Amino-1 6-(4-chloro-3-fluorophenyl)-4 5 Preparation of 6-fluoro-5-methyl-5-oxa-2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidine-4(3,2)-pyridine heterocycle 9-one (Compound 9)

[0254]

[0255] Substituting 4-chloro-3-fluorophenylboronic acid for (1-ethyl-1H-pyrazol-4-yl)boronic acid in step 1 of Example 2, the same preparation method as in Example 2 was used to obtain Compound 9 of Example 9.

[0256] MS (ESI) m / z 486.2 (M+H) +

[0257] 1H NMR (400MHz, DMSO) δ9.86(d,J=8.8Hz,1H),8.06(s,1H),8.00(d,J=3.0Hz,1H),7.76(t,J=8.0Hz,1H),7.65(s,1H),7.42(s,1H),7.18(s,1H ),7.11(t,J=5.8Hz,1H),7.02(s,2H),5.06–4.89(m,2H),4.02–3.97(m,1H),3.86–63.81(m,1H),3.17–3.04(m,1H),1.45(d,J=6.1Hz,3H).

[0258] Example 10: (S, 1 3 E,1 4 E)-1 7 -Amino-1 6 -(4-chloro-3-fluorophenyl)-4 5 The preparation and synthesis steps of 1-fluoro-6-methyl-5-oxa-2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidine-4(3,2)-pyridine heterocycle 9-one (Compound 10) are as follows:

[0259]

[0260]

[0261] Step 1: Preparation of (S)-methyl 5-(((2-((1-(((tert-Butoxycarbonyl)amino)propan-2-yl)oxy)-5-fluoropyridin-3-yl)methyl)amino)-7-(dibenzylamino)-6-((4-fluorophenyl)amino)pyrazolo[1,5-a]pyrimidine-3-carboxylate (Compound 10A)

[0262]

[0263] Under nitrogen, (S)-methyl 6-bromo-5-((((2-((1-(((tert-butoxycarbonyl)amino)propan-2-yl)oxy)-5-fluoropyridin-3-yl)methyl)amino)-7-(dibenzylamino)pyrazolo[1,5-a]pyrimidine-3-carboxylate (1.5 g, 2.0 mmol) and 4-fluoroaniline (333.0 mg, 3.0 mmol) were added to a reaction flask containing tert-butanol (50 mL). Subsequently, Ruphos Pd G3 (167.4 mg, 0.2 mmol), Ruphos (186.2 mg, 0.4 mmol) and Cs2CO3 (1.9 g, 6.0 mmol) were added. The reaction solution was stirred at 100°C for 3 h. After the reaction was complete, the reaction system was concentrated under reduced pressure. The residue was separated and purified by silica gel column chromatography to obtain compound 10A. MS (ESI) m / z 779.3 (M+H) +

[0264] Step 2: (S, 1 3 E,1 4 E)-1 7 -(dibenzylamino)-4-(dibenzylamino)- 5 -Fluoro-6-methyl-1 6 Preparation of -((4-fluorophenyl)amino)-5-oxa-2,8-diaza-1(5,3)-pyrazolin[1,5-a]pyrimidine 4(3,2)-pyridine heterocycle 9-one (Compound 10B)

[0265]

[0266] (S)-5-(((2-((1-(((tert-Butoxycarbonyl)amino)propan-2-yl)oxy)-5-fluoropyridin-3-yl)methyl)amino)-7-(dibenzylamino)-6-((4-fluorophenyl)amino)pyrazolo[1,5-a]pyrimidine-3-carboxylate (780 mg, 1.0 mol) was dissolved in a mixed solution of methanol and THF, followed by addition of LiOH (480 mg, 20.0 mmol) aqueous solution. After addition, the reaction system was heated to 60°C and stirred for 16 h. After the reaction was complete, the reaction system was cooled to 0°C. The pH was adjusted to 2-3 with 2N hydrochloric acid, extracted three times with DCM, and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. A solution of hydrochloric acid in 1,4-dioxane was added and stirred at room temperature for 1 hour. After the reaction was complete, the reaction system was concentrated to dryness, and then DCM and DMF were added, followed by DIPEA (1.9 g, 15.0 mmol) and FDPP (1.92 g, 5.0 mmol). After the addition was complete, the mixture was stirred at room temperature for 16 hours. After the reaction was complete, the reaction system was concentrated to dryness under reduced pressure, and the residue was separated by silica gel column chromatography to obtain compound 10B.

[0267] MS (ESI) m / z 647.2 (M+H) +

[0268] Step 3: (S, 1 3 E,1 4 E)-1 7 -Amino-4 5 -Fluoro-6-methyl-1 6 Preparation of -((4-fluorophenyl)amino)-5-oxa-2,8-diaza-1(5,3)-pyrazolin[1,5-a]pyrimidine 4(3,2)-pyridine heterocycle 9-one (Compound 10)

[0269]

[0270] (S,1 3 E,1 4 E)-1 7 -(dibenzylamino)-1 6 -(1-ethyl-1H-pyrazol-4-yl)-4 5 6-Fluoro-6-methyl-5-oxa-2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidine-4(3,2)-pyridineheterocyclyl-9-one (64.6 mg, 0.1 mmol) was dissolved in DCM (10.0 mL), and trifluoromethanesulfonic acid (150.0 mg, 1.0 mmol) was added at 0°C, and the reaction was maintained at 0°C for 2 h. After the reaction was complete, TEA was slowly added at 0°C to adjust the pH to 7-8. The reaction solution was concentrated under reduced pressure, and the residue was subjected to high pressure preparative separation to obtain Compound 10 of Example 10.

[0271] MS (ESI) m / z 467.1 (M+H) +

[0272] 1 H NMR (400MHz, DMSO) δ9.99(d,J=9.1Hz,1H),8.05(s,1H),8.00(d,J=3.0Hz,1H),7.70(dd,J=17.4,11.2Hz,2H),7.20(s,2H),6. 95(t,J=8.8Hz,2H),6.77(s,1H),6.49(s,2H),5.02-4.91(m,2H),4.07–3.88(m,2H),3.19–3.03(m,1H),1.46(d,J=6.1Hz,3H).

[0273] Example 11: (S, 1 3 E,1 4 E)-1 7-Amino-1 6 -(thiazol-2-yl)-4-nitropropene 5 The preparation and synthesis steps of 1-fluoro-6-methyl-5-oxa-2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidine-4(3,2)-pyridine heterocycle 9-one (Compound 11) are as follows:

[0274]

[0275] Step 1: Preparation of (S)-methyl 5-(((((2-((1-(((tert-Butoxycarbonyl)amino)propan-2-yl)oxy)-5-fluoropyridin-3-yl)methyl)amino)-7-(dibenzylamino)-6-(thiazol-2-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylate (Compound 11A)

[0276]

[0277] (S)-6-bromo-5-((((2-((1-(((tert-Butoxycarbonyl)amino)propan-2-yl)oxy)-5-fluoropyridin-3-yl)methyl)amino)-7-(dibenzylamino)pyrazolo[1,5-a]pyrimidine-3-carboxylic acid methyl ester (1.5 g, 2.0 mmol) was added to a reaction bottle containing dry toluene (50 mL), and then Pd(PPh3)4 (310 mg, 0.27 mmol) was added at room temperature. The system was purged with nitrogen three times, and then 2-(tributylstannyl)thiazole (842 μL, 2.68 mmol) was added to the reaction solution. Finally, the reaction solution was stirred at 110°C for 16 h. After the reaction was complete, potassium fluoride aqueous solution was added to quench the reaction. The reaction system was concentrated under reduced pressure, and the residue was separated and purified by column chromatography to obtain compound 11A.

[0278] MS (ESI) m / z 753.2 (M+H) +

[0279] Step 2: (S, 1 3 E,1 4 E)-1 7 -(dibenzylamino)-4-(dibenzylamino)- 5 -Fluoro-6-methyl-1 6 Preparation of -(thiazol-2-yl)-5-oxa-2,8-diaza-1(5,3)-pyrazolin[1,5-a]pyrimidine 4(3,2)-pyridine heterocycle 9-one (Compound 11B)

[0280]

[0281] (S)-5-(((((2-((1-(((tert-Butoxycarbonyl)amino)propan-2-yl)oxy)-5-fluoropyridin-3-yl)methyl)amino)-7-(dibenzylamino)-6-(thiazol-2-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylate (753.0 mg, 1.0 mol) was dissolved in a mixed solution of methanol and THF, followed by addition of LiOH (480 mg, 20.0 mmol) aqueous solution. After addition, the reaction system was heated to 60°C and stirred for 16 h. After the reaction was complete, the reaction system was then The temperature was lowered to 0°C, the pH was adjusted to 2-3 with 2N hydrochloric acid, and the mixture was extracted three times with DCM. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. A solution of hydrochloric acid in 1,4-dioxane was added and stirred at room temperature for 1 hour. After the reaction was complete, the reaction solution was concentrated to dryness, and DCM and DMF were added, followed by DIPEA (1.9 g, 15.0 mmol) and FDPP (1.92 g, 5.0 mmol). After the addition was complete, the mixture was stirred at room temperature for 16 hours. The reaction solution was concentrated to dryness under reduced pressure, and the residue was separated and purified by silica gel column chromatography to obtain compound 11B.

[0282] MS (ESI) m / z 621.2 (M+H) +

[0283] Step 3: (S, 1 3 E,1 4 E)-1 7 -Amino-4 5 -Fluoro-6-methyl-1 6 Preparation of -(thiazol-2-yl)-5-oxa-2,8-diaza-1(5,3)-pyrazolin[1,5-a]pyrimidine 4(3,2)-pyridine heterocycle 9-one (Compound 11)

[0284]

[0285] (S,1 3 E,1 4 E)-1 7 -(dibenzylamino)-1 6 -(1-ethyl-1H-pyrazol-4-yl)-4 5 6-Fluoro-6-methyl-5-oxa-2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidine-4(3,2)-pyridineheterocyclyl-9-one (62.0 mg, 0.1 mmol) was dissolved in DCM (10.0 mL), and trifluoromethanesulfonic acid (150.0 mg, 1.0 mmol) was added at 0°C, and the reaction was maintained at 0°C for 2 h. After completion of the reaction, TEA was slowly added at 0°C to adjust the pH to 7-8. The reaction solution was concentrated under reduced pressure, and the residue was subjected to high pressure preparative separation to obtain Compound 11 of Example 11.

[0286] MS (ESI) m / z 441.2 (M+H) +

[0287] 1 H NMR (400MHz, DMSO-d6) δ9.68(d,J=8.9Hz,1H),8.38(t,J=5.9Hz,1H),8.21(s,2H),8.15(s,1H),8.02(dd,J=7.3,3.2Hz,2H),7.94( d,J=3.4Hz,1H),7.82(dd,J=8.9,3.1Hz,1H),5.04(m,2H),4.17–3.94(m,2H),3.13(dd,J=13.2,10.1Hz,1H),1.47(d,J=6.1Hz,3H).

[0288] Example 12: (S, 1 3 E,1 4 E)-1 7 -Amino-1 6 -(4-(ethylamino)phenyl)-4 5 Preparation of 6-fluoro-5-methyl-5-oxa-2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidine-4(3,2)-pyridine heterocycle 9-one (Compound 12)

[0289]

[0290] Substituting tert-butyl ethyl(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)carbamate for (1-ethyl-1H-pyrazol-4-yl)boric acid in step 1 of Example 2, the same preparation method as in Example 2 was used to obtain compound 12 of Example 12.

[0291] MS (ESI) m / z 476.9 (M+H) +

[0292] 1H NMR (400MHz, DMSO) δ9.91(d,J=8.8Hz,1H),8.00(dd,J=15.4,12.6Hz,2H),7.72(dd,J=8.9,2 .8Hz,1H),7.12–6.93(m,3H),6.75(d,J=7.1Hz,2H),6.50(s,2H),5.88(t,J=5.0Hz,1H),5.0 8–4.96(m,1H),4.92(dd,J=14.3,6.6Hz,1H),4.01(dd,J=15.7,6.5Hz,1H),3.89(dd,J=14.5 ,5.5Hz,1H),3.29–3.18(m,1H),3.20–3.03(m,3H),1.46(d,J=6.0Hz,2H),1.27–1.15(m,3H).

[0293] Example 13: 1-(4-((S,1 3 E,1 4 E)-1 7 -Amino-4 5 -Fluoro-6-methyl-9-oxo-5-oxa-2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidine-4(3,2)-pyridine heterocycle Jiufan-1 6 The preparation synthesis route of 3-propylurea (compound 13) is as follows:

[0294]

[0295] Step 1: Preparation of (S)-methyl 6-((4-((tert-butoxycarbonyl)amino)phenyl)-5-(((2-((1-((tert-butoxycarbonyl)amino)propan-2-yl)oxy)-5)-fluoropyridin-3-yl)methyl)amino)-7-(dibenzylamino)pyrazolo[1,5-a]pyrimidine-3-carboxylate (Compound 13A)

[0296]

[0297] Under nitrogen protection, (S)-6-bromo-5-((((2-((1-(((tert-butoxycarbonyl)amino)propan-2-yl)oxy)-5-fluoropyridin-3-yl)methyl)amino)-7-(dibenzylamino)pyrazolo[1,5-a]pyrimidine-3-carboxylic acid methyl ester (1.0 g, 1.34 mmol) and 4-tert-butoxycarbonylaminophenylboronic acid pinacol ester (858 mg, 2.68 mmol) were added to a mixture containing a mixed solvent (1,4- dioxane / H2O, 4 / 1, 35 mL) was added to a reaction flask, followed by the addition of XPhos (63.8 mg, 0.13 mmol), XPhos-Pd-G3 (113 mg, 0.13 mmol) and K3PO4 (568 mg, 2.68 mmol). The reaction solution was stirred at 80°C for 3 h. After the reaction was complete, the reaction system was concentrated under reduced pressure, and the residue was separated and purified by column chromatography to obtain compound 13A.

[0298] MS (ESI) m / z 862.1 (M+H) +

[0299] Step 2: (S, 1 3 E,1 4 E)-1 6 -(4-amino)phenyl)-1 7 -(dibenzylamino)-4-(dibenzylamino)- 5 -Fluorine-1 12 Preparation of 6-dimethyl-5-oxa-2,8-diaza-1(5,3)-pyrazolino[1,5-a]pyrimidin-4(3,2)-pyridine heterocycle 9-one (Compound 13B)

[0300]

[0301] (S)-6-((4-((tert-Butoxycarbonyl)amino)phenyl)-5-(((2-((1-((tert-Butoxycarbonyl)amino)propan-2-yl)oxy)-5)-fluoropyridin-3-yl)methyl)amino)-7-(dibenzylamino)pyrazolo[1,5-a]pyrimidine-3-carboxylate (860 mg, 1.0 mol) was dissolved in a mixed solution of methanol and THF, followed by the addition of LiOH (480 mg, 20.0 mmol) aqueous solution. After the addition was complete, the reaction system was heated to 60 °C and stirred for 16 h. The reaction system was then cooled to 0°C, the pH adjusted to 2-3 with 2N hydrochloric acid, and extracted three times with DCM. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. A solution of hydrochloric acid in 1,4-dioxane was added, and the mixture was stirred at room temperature for 1 hour. The reaction system was concentrated to dryness, and DCM and DMF were added, followed by DIPEA (1.93 g, 15.0 mmol) and FDPP (1.92 g, 5.0 mmol). After the additions were complete, the mixture was stirred at room temperature for 16 hours. After completion of the reaction, the reaction system was concentrated to dryness under reduced pressure, and the residue was purified by silica gel column chromatography to obtain compound 13B.

[0302] MS (ESI) m / z 629.8 (M+H) +

[0303] Step 3: 1-(4-((S,1 3 E,1 4 E)-1 7 -(dibenzylamino)-4-(dibenzylamino)- 5 -Fluoro-6-methyl-9-oxo-5-oxa-2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidine-4(3,2)-pyridine heterocycle Jiufan-1 6 Preparation of 2-amino-3-methyl-phenyl-3-propylurea (Compound 13C)

[0304]

[0305] (S,1 3 E,1 4 E)-1 6 -(4-amino)phenyl)-1 7 -(dibenzylamino)-4-(dibenzylamino)- 5 -Fluorine-1 126-Dimethyl-5-oxa-2,8-diaza-1(5,3)-pyrazolino[1,5-a]pyrimidin-4(3,2)-pyridineheterocyclic cyclopentadien-9-one (300.0 mg, 0.48 mmol) was dissolved in dichloromethane (10.0 mL). DIPEA (186.0 mg, 1.44 mmol) and CDI (233.0 mg, 1.44 mmol) were then added at 0°C. The reaction system was stirred at 40°C for 16 hours. After the reaction was complete, n-propylamine (58.0 mg, 0.96 mmol) was slowly added to the reaction system at room temperature, and the system was stirred at 40°C for 3 hours. After the reaction was complete, the reaction solution was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to obtain compound 13C.

[0306] MS (ESI) m / z 714.3 (M+H) +

[0307] Step 4: 1-(4-((S,1 3 E,1 4 E)-1 7 -Amino-4 5 -Fluoro-6-methyl-9-oxo-5-oxa-2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidine-4(3,2)-pyridine heterocycle Jiufan-1 6 Preparation of -yl)-phenyl)-3-propylurea (Compound 13)

[0308]

[0309] 1-(4-((S,1 3 E,1 4 E)-1 7 -(dibenzylamino)-4-(dibenzylamino)- 5 -Fluoro-6-methyl-9-oxo-5-oxa-2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidine-4(3,2)-pyridine heterocycle Jiufan-1 6 3-Propyl-1-(2-amino-4-phenyl)-3-propylurea (72.0 mg, 0.1 mmol) was dissolved in DCM (10.0 mL), and trifluoromethanesulfonic acid (150.0 mg, 1.0 mmol) was added at 0°C, and the reaction was maintained at 0°C for 2 h. After completion of the reaction, TEA was slowly added at 0°C to adjust the pH to 7-8. The reaction solution was concentrated under reduced pressure, and the residue was isolated by high pressure preparative separation to obtain Compound 13 of Example 13.

[0310] MS (ESI) m / z 533.9 (M+H) +

[0311] 1H NMR(400MHz,DMSO)δ9.90(d,J=8.9Hz,1H),8.63(s,1H),8.11–8.03(m,1H),8.00(d,J=3.0Hz,1 H),7.70(dd,J=8.9,2.9Hz,1H),7.61(s,2H),7.17(d,J=17.5Hz,2H),6.99(t,J=6.1Hz,1H),6. 66(s,2H),6.20(t,J=5.7Hz,1H),5.05-4.98(m,1H),4.93(dd,J=14.3,5.7Hz,1H),4.04-3.97( m,1H),3.87(dd,J=14.5,5.5Hz,1H),3.19–3.00(m,3H),1.53–1.38(m,5H),0.94–0.81(m,3H).

[0312] Example 14: (4-((S,1 3 E,1 4 E)-1 7 -Amino-4 5 -Fluoro-6-methyl-9-oxo-5-oxa-2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidine-4(1,2)-phenyl ring heterocyclic nine-fan-1 6 The preparation synthesis route of ethyl carbamate (compound 14) is as follows:

[0313]

[0314]

[0315] Step 1: (4-((S,1 3 E,1 4 E)-1 7 -(dibenzylamino)-4-(dibenzylamino)- 5 -Fluoro-6-methyl-9-oxo-5-oxa-2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidine-4(1,2)-benzoheterocyclic-1-(6-fluoro-9-methyl-5-oxa-2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidine-4(1,2)-benzoheterocyclic-1-(6-fluoro-9- 6 Preparation of ethyl)phenyl)urethane (Compound 14A):

[0316]

[0317] (S,1 3 E,1 4 E)-1 6 -(4-aminophenyl)-1 7 -(dibenzylamino)-4-(dibenzylamino)- 56-Fluoro-6-methyl-5-oxa-2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidin-4(3,2)-pyridineheterocyclyl-9-one (800 mg, 1.27 mmol) and DIPEA (492.0 mg, 3.81 mmol) were dissolved in THF (12 mL). The reaction mixture was cooled to 0°C and ethyl chloroformate (183 μL, 1.91 mmol) was added dropwise. Stirring was continued for 3.0 h. After the reaction was complete, the reaction mixture was concentrated under reduced pressure, and the residue was purified by column chromatography to obtain compound 14A.

[0318] MS (ESI) m / z 701.2 (M+H) +

[0319] Step 2: (4-((S,1 3 E,1 4 E)-1 7 -Amino-4 5 -Fluoro-6-methyl-9-oxo-5-oxa-2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidine-4(1,2)-phenyl ring heterocyclic nine-fan-1 6 Preparation of ethyl)phenyl)urethane (Compound 14):

[0320]

[0321] (4-((S,1 3 E,1 4 E)-1 7 -(dibenzylamino)-4-(dibenzylamino)- 5 -Fluoro-6-methyl-9-oxo-5-oxa-2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidine-4(1,2)-phenyl ring heterocyclic nine-fan-1 6 Ethyl (2-amino)phenyl)carbamate (70.0 mg, 0.1 mmol) was dissolved in DCM (10.0 mL), and trifluoromethanesulfonic acid (150.0 mg, 1.0 mmol) was added at 0°C. The reaction was maintained at 0°C for 2 h. After completion of the reaction, TEA was slowly added at 0°C to adjust the pH to 7-8. The reaction solution was concentrated under reduced pressure, and the residue was isolated by high pressure preparative separation to obtain Compound 14 of Example 14.

[0322] MS (ESI) m / z 520.2 (M+H) +

[0323] 1H NMR (400MHz, DMSO) δ10.04(d,J=7.3Hz,1H),9.85(s,1H),8.04(s,1H),7.68(d,J=8. 3Hz,2H),7.33–7.12(m,3H),7.07–6.90(m,3H),6.65(s,2H),5.07(dd,J=14.2,6.3H z,1H),4.43(s,1H),4.18(q,J=7.1Hz,2H),3.93(dd,J=11.4,6.7Hz,1H),3.74(dd,J =14.4,5.7Hz,1H),3.17–3.06(m,1H),1.45(d,J=6.0Hz,3H),1.28(t,J=7.1Hz,3H).

[0324] Example 15: N-(4-((S,1 3 E,1 4 E)-1 7 -Amino-4 5 -Fluoro-6-methyl-9-oxo-5-oxa-2,8-diaza-1(5,3)-pyrazolin[1,5-a]pyrimidine-4(3,2)-pyridine heterocycle Jiufan-1 6 Preparation of -yl)-phenyl)-2-methoxyacetamide (Compound 15)

[0325]

[0326] Substituting methoxyacetyl chloride for ethyl chloroformate in step 1 of Example 14, the same preparation method as in Example 14 was used to obtain Compound 15 of Example 15.

[0327] MS (ESI) m / z 521.5 (M+H) +

[0328] 1H NMR (400MHz, DMSO) δ9.99(s,1H),9.88(d,J=8.8Hz,1H),8.06(d,J=8.2Hz,1H),7.99(d,J=3.0H z,1H),7.91(d,J=8.5Hz,2H),7.68(dd,J=8.9,3.0Hz,1H),7.25(s,2H),6.99(t,J=6.1Hz,1H),6 .71(s,2H),5.05-4.98(m,1H),4.93(dd,J=14.4,5.1Hz,1H),4.06(s,2H),4.03-3.97(m,1H),3 .86(dd,J=14.6,5.6Hz,1H),3.40(s,3H),3.11(dd,J=12.2,10.5Hz,1H),1.45(d,J=6.1Hz,3H).

[0329] Example 16: N-(4-((S,1 3 E,1 4 E)-1 7 -Amino-4 5 -Fluoro-6-methyl-9-oxo-5-oxa-2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidine-4(3,2)-pyridine heterocycle Jiufan-1 6 Preparation of azetidine-1-carboxamide (Compound 16)

[0330]

[0331] Substituting azetidine for n-propylamine in step 3 of Example 13, the same preparation method as in Example 13 was used to obtain compound 16 of Example 16.

[0332] MS (ESI) m / z 532.5 (M+H) +

[0333] 1H NMR (400MHz, DMSO) δ9.88 (d, J=8.7Hz, 1H), 8.58 (s, 1H), 8.04 (s, 1H), 7.99 (d, J= 3.0Hz,1H),7.79–7.65(m,3H),7.17(dd,J=31.2,19.8Hz,2H),6.98(dd,J=7.9,4 .3Hz,1H),6.64(s,2H),5.03-4.96(m,1H),4.95-4.90(m,1H),4.06–3.92(m,5H) ,3.92–3.77(m,1H),3.13-3.06(m,1H),2.28–2.14(m,2H),1.45(d,J=6.1Hz,3H).

[0334] Example 17: 1-(4-((S,1 3 E,1 4 E)-1 7 -Amino-4 5 -Fluoro-6-methyl-9-oxo-5-oxa-2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidine-4(3,2)-pyridine heterocycle Jiufan-1 6 The preparation synthesis route of 2-ethyl-3-thiourea (compound 17) is as follows:

[0335]

[0336]

[0337] Step 1: 1-(4-((S,1 3 E,1 4 E)-1 7 -(dibenzylamino)-4-(dibenzylamino)- 5 -Fluoro-6-methyl-9-oxo-5-oxa-2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidine-4(3,2)-pyridine heterocycle Jiufan-1 6 Preparation of -yl)-phenyl)-3-ethylthiourea (Compound 17A):

[0338]

[0339] (S,1 3 E,1 4 E)-1 6 -(4-amino)phenyl)-1 7 -(dibenzylamino)-4-(dibenzylamino)- 5 -Fluorine-1 126-Dimethyl-5-oxa-2,8-diaza-1(5,3)-pyrazolino[1,5-a]pyrimidin-4(3,2)-pyridineheterocyclic 9-one (400.0 mg, 0.637 mmol) was dissolved in anhydrous pyridine (5.0 mL), and ethyl isothiocyanate (110.0 mg, 1.28 mmol) was added. The reaction system was stirred at 80°C for 16 hours. After completion of the reaction, the reaction solution was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to obtain compound 17A.

[0340] MS (ESI) m / z 716.3 (M+H) +

[0341] Step 2: 1-(4-((S,1 3 E,1 4 E)-1 7 -Amino-4 5 -Fluoro-6-methyl-9-oxo-5-oxa-2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidine-4(3,2)-pyridine heterocycle Jiufan-1 6 Preparation of -yl)-phenyl)-3-ethylthiourea (Compound 17):

[0342]

[0343] 1-(4-((S,1 3 E,1 4 E)-1 7 -(dibenzylamino)-4-(dibenzylamino)- 5 -Fluoro-6-methyl-9-oxo-5-oxa-2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidine-4(3,2)-pyridine heterocycle Jiufan-1 6 1-(2-(4-(2-amino-3-phenyl)-3-ethylthiourea) (72.0 mg, 0.1 mmol) was dissolved in DCM (10.0 mL), and trifluoromethanesulfonic acid (150.0 mg, 1.0 mmol) was added at 0°C. The reaction was maintained at 0°C for 2 h. After the reaction was complete, TEA was slowly added at 0°C to adjust the pH to 7-8. The reaction solution was concentrated under reduced pressure, and the residue was purified by high pressure preparative separation to obtain Compound 17 of Example 17. MS (ESI) m / z 536.6 (M+H) +

[0344] 1H NMR(400MHz,DMSO)δ9.88(d,J=8.8Hz,1H),9.81(s,1H),8.09–8.03(m,1H),7.9 9(d,J=3.0Hz,1H),7.79–7.56(m,4H),7.27(d,J=8.8Hz,2H),7.07(t,J=6.1Hz,1 H),6.70(s,2H),5.08–4.88(m,2H),4.03-3.97(m,1H),3.88(dd,J=14.6,5.6Hz ,1H),3.58–3.47(m,2H),3.18–3.01(m,1H),1.45(d,J=6.1Hz,3H),1.17(m,3H).

[0345] Example 18: 1-(4-((S,1 3 E,1 4 E)-1 7 -Amino-4 5 -Fluoro-6-methyl-9-oxo-5-oxa-2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidine-4(3,2)-pyridine heterocycle Jiufan-1 6 Preparation of 2-fluorophenyl-3-yl)-2-fluorophenyl-3-ethylurea (Compound 18)

[0346]

[0347] Substituting tert-butyl (2-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)carbamate for 4-tert-butoxycarbonylaminophenylboronic acid pinacol ester in step 1 of Example 13, and substituting ethylamine for n-propylamine in step 3 of Example 13, the same preparation method as in Example 13 was used to obtain compound 18 of Example 18.

[0348] MS (ESI) m / z 538.2 (M+H) +

[0349] 1H NMR(400MHz, DMSO-d6)δ9.89(d,J=8.9Hz,1H),8.43(d,J=2.8Hz,1H),8.36(t,J= 8.6Hz,1H),8.05(s,1H),8.00(d,J=3.1Hz,1H),7.68(s,1H),7.08(t,J=16.3Hz,3 H),6.84(s,2H),6.65(t,J=5.5Hz,1H),5.03–4.90(m,2H),3.95–4.05(m,1H),3. 84–3.88(m,1H),3.20–3.07(m,3H),1.45(d,J=6.1Hz,3H),1.08(t,J=7.2Hz,3H).

[0350] Example 19: 1-(4-((S,1 3 E,1 4 E)-1 7 -Amino-4 5 -Fluoro-6-methyl-9-oxo-5-oxa-2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidine-4(3,2)-pyridine heterocycle Jiufan-1 6 Preparation of 2-chlorophenyl)-3-ethylurea (Compound 19)

[0351]

[0352] Substituting tert-butyl (2-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)carbamate in Example 18 was effected using the same preparation method as in Example 18 to obtain Compound 19 of Example 19.

[0353] MS (ESI) m / z 554.2 (M+H) +

[0354] 1H NMR(400MHz,DMSO-d6)δ9.89(d,J=9.0Hz,1H),8.40(d,J=8.5Hz,1H),8.12(s ,1H),8.06(s,1H),8.00(d,J=3.0Hz,1H),7.68(s,1H),7.40–7.23(m,1H),7.2 4–7.05(m,3H),6.86(s,2H),5.07–4.87(m,2H),4.01(m,1H),3.86(dd,J=14.7 ,5.7Hz,1H),3.24–3.07(m,3H),1.46(d,J=6.1Hz,3H),1.10(t,J=7.2Hz,3H).

[0355] Example 20: 3-(4-((S,1 3 E,1 4 E)-1 7 -(dibenzylamino)-4-(dibenzylamino)- 5 -Fluoro-6-methyl-9-oxo-5-oxa-2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidine-4(3,2)-pyridine heterocycle Jiufan-1 6 The preparation synthesis route of (4-ethoxycyclobut-3-ene-1,2-dione) (compound 20) is as follows:

[0356]

[0357] Step 1: 3-(4-((S,1 3 E,1 4 E)-1 7 -(dibenzylamino)-4-(dibenzylamino)- 5 -Fluoro-6-methyl-9-oxo-5-oxa-2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidine-4(3,2)-pyridine heterocycle Jiufan-1 6 Preparation of 4-ethoxycyclobutane-1,2-dione (Compound 20A):

[0358]

[0359] (S,1 3 E,1 4 E)-1 6 -(4-amino)phenyl)-1 7 -(dibenzylamino)-4-(dibenzylamino)- 5 -Fluorine-1 126-Dimethyl-5-oxa-2,8-diaza-1(5,3)-pyrazolino[1,5-a]pyrimidine-4(3,2)-pyridineheterocyclic cyclopentane-9-one (400.0 mg, 0.637 mmol) was dissolved in anhydrous ethanol (5.0 mL), and 3,4-diethoxycyclobutane-1,2-dione (140.0 mg, 0.81 mmol) was added. The reaction mixture was stirred at room temperature for 16 hours. After the reaction was complete, the reaction solution was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to obtain Compound 20A.

[0360] MS (ESI) m / z 755.8 (M+H) +

[0361] Step 2: 3-(4-((S,1 3 E,1 4 E)-1 7 -(dibenzylamino)-4-(dibenzylamino)- 5 -Fluoro-6-methyl-9-oxo-5-oxa-2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidine-4(3,2)-pyridine heterocycle Jiufan-1 6 Preparation of 4-ethoxycyclobut-3-ene-1,2-dione (Compound 20):

[0362]

[0363] 3-(4-((S,1 3 E,1 4 E)-1 7 -(dibenzylamino)-4-(dibenzylamino)- 5 -Fluoro-6-methyl-9-oxo-5-oxa-2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidine-4(3,2)-pyridine heterocycle Jiufan-1 6 (-yl)-phenyl)amino)-4-ethoxycyclobutane-1,2-dione (76.0 mg, 0.1 mmol) was dissolved in DCM (10.0 mL), and trifluoromethanesulfonic acid (150.0 mg, 1.0 mmol) was added at 0°C, and the reaction was maintained at 0°C for 2 h. After the reaction was complete, TEA was slowly added at 0°C, and the pH was adjusted to 7-8. The reaction solution was concentrated under reduced pressure, and the residue was purified by high pressure preparative separation to obtain Compound 20 of Example 20.

[0364] MS (ESI) m / z 573.5 (M+H) +

[0365] 1H NMR (400 MHz, DMSO-d6) δ 10.96 (s, 1H), 9.94–9.84 (m, 1H), 8.06 (s, 1H), 8.00 (d, J = 3.1 Hz, 1H), 7.70 (dd, J = 9.0, 3.1 Hz, 1H), 7.66–7.57 (m, 2H), 7.32 (s, 2H), 7.01 (t, J = 6.2 Hz, 1H), 6.74 (s, 2H), 5.02 (m, 1H), 4.94 (m, 1H), 4.84 (m, 2H), 4.00 (m, 1H), 3.87 (dd, J = 14.7, 5.7 Hz, 1H), 3.12 (m, 1H), 1.52–1.42 (m, 6H). Example 21: 3-(4-((S,1 3 E,1 4 E)-1 7 -(dibenzylamino)-4-(dibenzylamino)- 5 -Fluoro-6-methyl-9-oxo-5-oxa-2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidine-4(3,2)-pyridine heterocycle Jiufan-1 6 The preparation synthesis route of (4-ethoxy)-cyclobut-3-ene-1,2-dione (compound 21) is as follows:

[0366]

[0367] Step 1: Preparation of (R)-3-(1-aminoethyl)-5-fluoropyridin-2-ol (Compound 21A):

[0368]

[0369] (R)-1-(5-Fluoro-2-methoxypyridin-3-yl)ethan-1-amine (1.00 g, 2.62 mmol), ethanol (10 mL), and 1,4-dioxane hydrochloride (10 mL) were added to a reaction flask, and the system was stirred at 80°C for 4 hours. After the reaction was complete, the reaction solution was concentrated under reduced pressure to obtain compound 21A.

[0370] MS (ESI) m / z 157.2 (M+H) + .

[0371] Step 2: Preparation of (R)-benzyl(1-(5-fluoro-2-hydroxypyridin-3-yl)ethyl)carbamate (Compound 21B):

[0372]

[0373] (R)-3-(1-aminoethyl)-5-fluoropyridin-2-ol (1.00 g, 2.71 mmol), ethanol (20 mL), and water (20 mL) were added to a reaction flask. KCO (1.12 g, 8.13 mmol) was added portionwise. N-benzylsuccinimidyl carbonate (1.35 g, 5.42 mmol) was added at 0°C and allowed to react for 2 hours at room temperature. After the reaction was complete, the mixture was poured into water and extracted with ethyl acetate. The combined organic phases were dried over sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography to yield compound 21B.

[0374] MS (ESI) m / z 291.2 (M+H) + .

[0375] Step 3: Preparation of tert-butyl ((S)-2-((3-((R)-1-(((benzyloxy)carbonyl)amino)ethyl)-5-fluoropyridin-2-yl)oxy)propyl)carbamate (Compound 21C):

[0376]

[0377] Under argon, benzyl (R)-(1-(5-fluoro-2-hydroxypyridin-3-yl)ethyl)carbamate (0.70 g, 2.41 mmol), tert-butyl (1-(hydroxymethyl)cyclopropyl)carbamate (0.63 g, 3.61 mmol), and PPh3 (1.26 g, 4.82 mmol) were dissolved in THF (50 mL). DIAD (0.97 g, 4.82 mmol) was added dropwise at 0°C. The reaction system was allowed to warm to room temperature and stirred overnight. After completion of the reaction, the reaction solution was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to yield compound 21C.

[0378] MS (ESI) m / z 348.1 (M+H) + .

[0379] Step 4: Preparation of tert-butyl ((S)-2-((3-((R)-1-aminoethyl)-5-fluoropyridin-2-yl)oxy)propyl)carbamate (Compound 21D):

[0380]

[0381] Tert-butyl ((S)-2-((3-((R)-1-(((benzyloxy)carbonyl)amino)ethyl)-5-fluoropyridin-2-yl)oxy)propyl)carbamate (0.89 g, 6.12 mmol) was dissolved in methanol (20 mL). Palladium on carbon (200 mg) was then added to the reaction flask to displace the hydrogen atmosphere. The reaction was allowed to proceed at room temperature for 5 hours. After the reaction was complete, the filtrate was filtered, collected, and concentrated to yield compound 21D.

[0382] MS (ESI) m / z 314.2 (M+H) + .

[0383] Step 5: Preparation of methyl 6-bromo-5-(((R)-1-(2-((S)-1-((tert-butoxycarbonyl)amino)propan-2-yl)oxy)-5-fluoropyridin-3-yl)ethyl)amino)-7-(dibenzylamino)pyrazolo[1,5-a]pyrimidine-3-carboxylate (Compound 21E):

[0384]

[0385] Tert-butyl ((S)-2-((3-((R)-1-aminoethyl)-5-fluoropyridin-2-yl)oxy)propyl)carbamate (0.60 g, 1.92 mmol), methyl 6-bromo-5-chloro-7-(dibenzylamino)pyrazolo[1,5-a]pyrimidine-3-carboxylate (0.87 g, 1.74 mmol), and DIPEA (0.67 g, 5.22 mmol) were dissolved in ethanol (40 mL) and reacted at 60°C overnight. After the reaction was complete, the reaction solution was concentrated under reduced pressure, and the residue was separated and purified by silica gel column chromatography to obtain compound 21E. MS (ESI) m / z 776.2 (M+H) + .

[0386] Step 6: Preparation of methyl 6-(4-((tert-butoxycarbonyl)amino)phenyl)-5-((R)-1-(2-((S)-1-((tert-butoxycarbonyl)amino)propan-2-yl)oxy)-5-fluoropyridin-3-yl)ethyl)amino)-7-(dibenzylamino)pyrazolo[1,5-a]pyrimidine-3-carboxylate (Compound 21F):

[0387]

[0388] Under argon protection, methyl 6-bromo-5-(((R)-1-(2-((S)-1-((tert-butoxycarbonyl)amino)propan-2-yl)oxy)-5-fluoropyridin-3-yl)ethyl)amino)-7-(dibenzylamino)pyrazolo[1,5-a]pyrimidine-3-carboxylate (0.90 g, 1.16 mmol), N-Boc-4-aminophenylboronic acid pinacol ester (0.48 g, 1.51 mmol), Pd2(dba)3 (109 mg, 0.12 mmol), K2CO3 (0.48 g, 3.48 mmol), 1,4-dioxane (20 mL) and water (4 mL) were added to the reaction flask and reacted at 80°C overnight. After the reaction was complete, the reaction solution was poured into water and extracted with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated, and the residue was separated and purified by silica gel column chromatography to obtain compound 21F.

[0389] MS (ESI) m / z 889.2 (M+H) +

[0390] Step 7: (1 3 E,1 4 E,3R,6S)-1 6 -(4-aminophenyl)-1 7 -(dibenzylamino)-4-(dibenzylamino)- 5 Preparation of 2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidine-4(3,2)-pyridine heterocycle 9-one (Compound 21G):

[0391]

[0392] Ethyl 6-(4-((tert-butoxycarbonyl)amino)phenyl)-5-((R)-1-(2-((S)-1-((tert-butoxycarbonyl)amino)propan-2-yl)oxy)-5-fluoropyridin-3-yl)ethyl)amino)-7-(dibenzylamino)pyrazolo[1,5-a]pyrimidine-3-carboxylate (0.59 g, 0.66 mmol) was dissolved in a mixture of methanol (18 mL), tetrahydrofuran (6 mL), and water (6 mL). Lithium hydroxide (318 mg, 13.28 mmol) was then added. After complete addition, the reaction system was heated to 60°C and stirred overnight. After completion of the reaction, the reaction system was cooled to 0°C, the pH was adjusted to 2-3 with 2N hydrochloric acid, and the mixture was extracted with EA. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated. A solution of DCM and hydrochloric acid in 1,4-dioxane (10 mL) was added and the mixture was allowed to react at room temperature for 3 hours. The reaction mixture was concentrated under reduced pressure, and the resulting crude product was dissolved in DMF (70 mL) and DCM (140 mL). DIPEA (3.42 g, 26.56 mmol) and FDPP (0.51 g, 1.33 mmol) were then added. The system was stirred at room temperature for 16 hours. After completion of the reaction, NaCO solution was added to quench the reaction, followed by extraction with DCM. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography to yield compound 21G.

[0393] MS (ESI) m / z 643.2 (M+H) +

[0394] Step 8: (4-((1 3 E,1 4 E,3R,6S)-1 7 -(dibenzylamino)-4-(dibenzylamino)- 5 -Fluoro-3,6-dimethyl-9-oxo-5-oxo-2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidine-4(3,2)-pyridine heterocycle Jiufan-16 Preparation of ethyl)amino)phenyl)carbamate (Compound 21H):

[0395]

[0396] (1 3 E,1 4 E,3R,6S)-1 6 -(4-aminophenyl)-1 7 -(dibenzylamino)-4-(dibenzylamino)- 5 3-Fluoro-3,6-dimethyl-5-oxo-2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidin-4(3,2)-pyridineheterocyclyl-9-one (340 mg, 0.53 mmol), DIPEA (137 mg, 1.06 mmol), and DCM (15 mL) were added to a reaction flask. Ethyl chloroformate (115 mg, 1.06 mmol) was added dropwise at 0°C and allowed to react overnight at room temperature. After completion of the reaction, the reaction solution was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to obtain compound 21H.

[0397] MS (ESI) m / z 715.2 (M+H) +

[0398] Step 9: (4-((1 3 E,1 4 E,3R,6S)-1 7 -Amino-4 5 -Fluoro-3,6-dimethyl-9-oxo-5-oxo-2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidine-4(3,2)-pyridine heterocycle Jiufan-1 6 Preparation of ethyl)amino)phenyl)carbamate (Compound 21):

[0399]

[0400] (4-((1 3 E,1 4 E,3R,6S)-1 7 -(dibenzylamino)-4-(dibenzylamino)- 5 -Fluoro-3,6-dimethyl-9-oxo-5-oxo-2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidine-4(3,2)-pyridine heterocycle Jiufan-1 6Ethyl (2-amino)phenyl)carbamate (72.0 mg, 0.1 mmol) was dissolved in DCM (10.0 mL), and trifluoromethanesulfonic acid (150.0 mg, 1.0 mmol) was added at 0°C, and the reaction was maintained at 0°C for 2 h. After the reaction was complete, TEA was slowly added at 0°C to adjust the pH to 7-8. The reaction solution was concentrated under reduced pressure, and the residue was purified by high-pressure preparative separation to obtain Compound 21 of Example 21.

[0401] MS (ESI) m / z 535.3 (M+H) +

[0402] 1 H NMR (400MHz, DMSO) δ9.83(d,J=11.7Hz,1H),9.79(d,J=8.7Hz,1H),8.05(s,1H),7. 96(d,J=3.0Hz,1H),7.91(dd,J=9.1,3.0Hz,1H),7.68(d,J=8.7Hz,2H),7.24(s,2H) ,6.67(s,2H),6.40(d,J=7.4Hz,1H),5.53–5.43(m,1H),5.02(m,1H),4.17(m,2H),3 .99(m,1H),3.11(m,1H),1.46(d,J=6.1Hz,3H),1.37(d,J=7.1Hz,3H),1.27(m,3H).

[0403] Example 22: (1 3 E,1 4 E,3R,6S)-1 7 -Amino-1 6 -(4-methylphenyl)4 5 The preparation and synthesis route of 2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidine-4(3,2)-pyridine heterocycle 9-one (Compound 22) is as follows:

[0404]

[0405]

[0406] Step 1: Preparation of methyl 5-(((R)-1-(2-((S)-1-((tert-butoxycarbonyl)amino)propan-2-yl)oxy)-5-fluoropyridin-3-yl)ethyl)amino)-7-(dibenzylamino)-6-(p-tolyl)pyrazolo[1,5-a]pyrimidine-3-carboxylate (Compound 22A):

[0407]

[0408] Under argon, methyl 6-bromo-5-(((R)-1-(2-((S)-1-((tert-butoxycarbonyl)amino)propan-2-yl)oxy)-5-fluoropyridin-3-yl)ethyl)amino)-7-(dibenzylamino)pyrazolo[1,5-a]pyrimidine-3-carboxylate (650 mg, 0.84 mmol), p-methylphenylboronic acid (170 mg, 1.26 mmol), K3PO4 (530 mg, 2.52 mol), Xphos Pd G3 (35.5 mg, 0.04 mol), 1,4-dioxane (16 mL), and water (4 mL) were added. The reaction system was stirred at 80°C overnight. After completion of the reaction, the reaction solution was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to obtain Compound 22A.

[0409] MS (ESI) m / z 788.2 (M+H) +

[0410] Step 2: (1 3 E,1 4 E,3R,6S)-1 6 -(4-methylphenyl)-1 7 -(dibenzylamino)-4-(dibenzylamino)- 5 Preparation of 2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidine-4(3,2)-pyridine heterocycle 9-one (Compound 22B):

[0411]

[0412] Methyl 5-(((R)-1-(2-((S)-1-((tert-Butoxycarbonyl)amino)propan-2-yl)oxy)-5-fluoropyridin-3-yl)ethyl)amino)-7-(dibenzylamino)-6-(p-tolyl)pyrazolo[1,5-a]pyrimidine-3-carboxylate (788 mg, 1.0 mol) was dissolved in a mixed solution of MeOH and THF, followed by the addition of LiOH (480 mg, 20.0 mmol) aqueous solution. After the addition, the reaction system was heated to 60°C and stirred for 16 h. The reaction system was then cooled to 0°C, the pH adjusted to 2-3 with 2N hydrochloric acid, and extracted three times with DCM. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. A solution of hydrochloric acid in 1,4-dioxane was added, and the mixture was stirred at room temperature for 1 hour. The reaction system was concentrated to dryness, and DCM and DMF were added, followed by DIPEA (1.93 g, 15.0 mmol) and FDPP (1.92 g, 5.0 mmol). After the additions were complete, the mixture was stirred at room temperature for 16 hours. After the reaction was complete, the reaction system was concentrated to dryness under reduced pressure, and the residue was purified by silica gel column chromatography to obtain compound 22B.

[0413] MS (ESI) m / z 642.1 (M+H) +

[0414] Step 3: (1 3 E,1 4 E,3R,6S)-1 7 -Amino-1 6 -(4-methylphenyl)4 5 Preparation of -fluoro-3,6-dimethyl-5-oxo-2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidine-4(3,2)-pyridine heterocycle 9-one (Compound 22):

[0415]

[0416] (1 3 E,1 4 E,3R,6S)-1 6 -(4-methylphenyl)-1 7 -(dibenzylamino)-4-(dibenzylamino)- 5 1-Fluoro-3,6-dimethyl-5-oxo-2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidine-4(3,2)-pyridineheterocyclic 9-one (64.0 mg, 0.1 mmol) was dissolved in DCM (10.0 mL), and trifluoromethanesulfonic acid (150.0 mg, 1.0 mmol) was added at 0°C, and the reaction was maintained at 0°C for 2 h. After the reaction was complete, TEA was slowly added at 0°C to adjust the pH to 7-8. The reaction solution was concentrated under reduced pressure, and the residue was isolated by high pressure preparative separation to obtain Compound 22 of Example 22.

[0417] MS (ESI) m / z 462.1 (M+H) +

[0418] 1 H NMR(400MHz,DMSO)δ9.79(d,J=8.7Hz,1H),8.05(s,1H),7.95(t,J=6.2Hz,1H), 7.97–7.90(dd,J=9.1,2.9Hz,1H),7.40(d,J=7.9Hz,2H),7.23(s,2H),6.64(s,2 H),6.39(d,J=7.4Hz,1H),5.54–5.45(m,1H),5.04–4.99(m,1H),4.02–3.96(m,1 H),3.09–3.15m,1H),2.42(s,3H),1.46(d,J=6.1Hz,3H),1.37(d,J=7.1Hz,3H).

[0419] Example 23: (13 E,1 4 E,3R,6S)-1 7 -Amino-1 6 -(1-methyl-1H-pyrrol-3-yl)-4 5 Preparation of -fluoro-3,6-dimethyl-5-oxo-2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidine-4(3,2)-pyridine heterocycle 9-one (Compound 23):

[0420]

[0421] Substituting N-methylpyrrole-3-boronic acid pinacol ester for p-methylphenylboronic acid in step 1 of Example 22, the same preparation method as in Example 22 was used to obtain compound 23 of Example 23.

[0422] MS (ESI) m / z 451.2 (M+H) +

[0423] 1H NMR (400MHz, DMSO) δ9.80(d,J=8.3Hz,1H),8.03(d,J=4.7Hz,1H),7.99(m,2H),6.97(m,1H),6.91(t,J=1.8Hz,1H),6.63(s,2H),6.50(d,J=7. 2Hz,1H),6.12–6.09(m,1H),5.46(m,1H),5.04(m,1H),3.98(m,1H),3.73(s,3H),3.12(dd,J=12.1,10.2Hz,1H),1.45(dd,J=11.6,6.6Hz,6H).

[0424] Example 24: 4-((1 3 E,1 4 E,3R,6S)-1 7 -Amino-4 5 -Fluoro-3,6-dimethyl-9-oxa-5-oxa-2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidine-4(3,2)-pyridine heterocycle Jiufan-1 6 Preparation of -yl)-1-methyl-1H-pyrrole-2-carbonitrile (Compound 24):

[0425]

[0426] Substituting 1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrole-2-carbonitrile for p-methylphenylboronic acid in step 1 of Example 22, the same preparation method as in Example 22 was used to obtain Compound 24 of Example 24.

[0427] MS (ESI) m / z 476.19 (M+H) +

[0428] 1 H NMR (400MHz, DMSO-d6) δ9.88–9.71(m,1H),8.04(s,1H),8.02–7.90(m,2H),7.29(d,J=2.0Hz,1H),7.01(d,J=2.0Hz,1H),6.93(s,2H), 6.65(d,J=7.6Hz,1H),5.47(m,1H),5.01(m,1H),3.98(m,1H),3.83(s,3H),3.10(m,1H),1.46(d,J=6.0Hz,3H),1.41(d,J=7.2Hz,3H).

[0429] Example 25: (1 3 E,1 4 E,3R,6S)-1 7 -Amino-1 6 -(pyridin-3-yl)-4 5 Preparation of 2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidine-4(3,2)-pyridine heterocycle 9-one (Compound 25):

[0430]

[0431] Substituting 3-pyridineboronic acid for p-methylphenylboronic acid in step 1 of Example 22, the same preparation method as in Example 22 was used to obtain Compound 25 of Example 25.

[0432] MS (ESI) m / z 449.18 (M+H) +

[0433] 1H NMR(400MHz,DMSO-d6)δ9.84–9.71(m,1H),8.70(dd,J=4.8,1.6Hz,1H),8.50( s,1H),8.07(s,1H),7.97(d,J=3.2Hz,1H),7.86(d,J=9.1Hz,1H),7.75(d,J=8. 0Hz,1H),7.58(m,1H),6.97(s,2H),6.62(d,J=7.6Hz,1H),5.51(m,1H),5.02( m,1H),3.99(m,1H),3.12(m,1H),1.46(d,J=6.0Hz,3H),1.36(d,J=7.2Hz,3H).

[0434] Example 26: (1 3 E,1 4 E,3R,6S)-1 7 -Amino-1 6 -(3,5-difluoro-4-methylphenyl)-4 5 Preparation of -fluoro-3,6-dimethyl-5-oxo-2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidine-4(3,2)-pyridine heterocycle 9-one (Compound 26):

[0435]

[0436] Substituting (3,5-difluoro-4-methylphenyl)boronic acid for p-methylphenylboronic acid in step 1 of Example 22, the same preparation method as in Example 22 was used to obtain Compound 26 of Example 26.

[0437] MS (ESI) m / z 498.3 (M+H) +

[0438] 1 H NMR (400MHz, DMSO) δ9.78(d,J=8.6Hz,1H),8.06(s,1H),7.98(d,J=3.0Hz,1H),7.86(dd,J=9.1,3.0Hz,1H),7.04(d,J=8.7Hz,2H),6.98(s,2H),6 .63(d,J=7.5Hz,1H),5.55–5.45(m,1H),5.06–4.97(m,1H),3.99(m,1H) ,3.12(m,1H),2.26(s,3H),1.47(d,J=6.1Hz,3H),1.39(d,J=7.1Hz,3H).

[0439] Example 27: (1 3 E,1 4 E,3R,6S)-1 7 -Amino-1 6 -(4-(difluoromethyl)phenyl)-4 5 Preparation of -fluoro-3,6-dimethyl-5-oxo-2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidine-4(3,2)-pyridine heterocycle 9-one (Compound 27):

[0440]

[0441] Substituting 4-(difluoromethyl)phenylboronic acid pinacol ester for p-tolueneboronic acid in step 1 of Example 22, the same preparation method as in Example 22 was used to obtain Compound 27 of Example 27.

[0442] MS (ESI) m / z 498.3 (M+H) +

[0443] 1 H NMR(400MHz,DMSO)δ9.77(d,J=8.7Hz,1H),8.07(d,J=1.0Hz,1H),8.03–7.88(m ,2H),7.78(d,J=7.7Hz,2H),7.51(s,2H),7.16(t,J=55.9Hz,1H),6.84(s,2H),6 .52(d,J=7.3Hz,1H),5.59–5.46(m,1H),5.13–4.94(m,1H),4.00(ddd,J=12.8,9 .1,3.7Hz,1H),3.22–3.07(m,1H),1.46(t,J=8.8Hz,3H),1.37(d,J=6.9Hz,3H).

[0444] Example 28: (1 3 E,1 4 E,3R,6S)-1 7 -Amino-1 6 -(3-fluoro-4-methylphenyl)-4 5 Preparation of -fluoro-3,6-dimethyl-5-oxo-2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidine-4(3,2)-pyridine heterocycle 9-one (Compound 28):

[0445]

[0446] Substituting (3-fluoro-4-methylphenyl)boronic acid for p-tolueneboronic acid in step 1 of Example 22, the same preparation method as in Example 22 was used to obtain Compound 28 of Example 28.

[0447] MS (ESI) m / z 480.19 (M+H) +

[0448] 1H NMR(400MHz,DMSO-d6)δ9.84–9.76(m,1H),8.06(s,1H),7.97(d,J=3.0Hz,1H) ,7.90(s,1H),7.48(t,J=8.0Hz,1H),7.13–7.06(m,2H),6.82(s,2H),6.52(d,J =7.5Hz,1H),5.55–5.47(m,1H),5.06–4.98(m,1H),4.03–3.96(m,1H),3.15–3. 09(m,1H),2.35(d,J=1.8Hz,3H),1.47(d,J=6.2Hz,3H),1.38(d,J=7.1Hz,3H).

[0449] Example 29: (1 3 E,1 4 E,3R,6S)-1 7 -Amino-1 6 -(4-chlorophenyl)-4 5 Preparation of -fluoro-3,6-dimethyl-5-oxo-2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidine-4(3,2)-pyridine heterocycle 9-one (Compound 29):

[0450]

[0451] Substituting 4-chlorophenylboronic acid for p-methylphenylboronic acid in step 1 of Example 22, the same preparation method as in Example 22 was used to obtain compound 29 of Example 29.

[0452] MS (ESI) m / z 482.14 (M+H) +

[0453] 1 H NMR(400MHz,DMSO)δ9.79(d,J=8.8Hz,1H),8.06(s,1H),7.97(d,J=3.0Hz,1H ),7.88–7.85(dd,J=9.1,3.0Hz,1H),7.62(d,J=8.6Hz,2H),7.35(s,2H),6.8 5(s,2H),6.53(d,J=7.5Hz,1H),5.55–5.44(m,1H),5.05–4.94(m,1H),4.02– 3.96(m,1H),3.18–3.05(m,1H),1.46(d,J=6.1Hz,3H),1.37(d,J=7.1Hz,3H).

[0454] Example 30: (S, 1 3 E,14 E)-1 6 -(1-ethyl-1H-pyrrol-3-yl)-1 7 -Amino-4 5 The preparation and synthesis route of 1-fluoro-6-methyl-5-oxa-2,8-diaza-1(5,3)-pyrazolino[1,5-a]pyrimidine-4(3,2)-pyridine heterocycle 9-one (Compound 30) is as follows:

[0455]

[0456] Step 1: (S, 1 3 E,1 4 E)-1 6 -Br-1 7 -(dibenzylamino)-4-(dibenzylamino)- 5 -Fluorine-1 12 Preparation of 6-dimethyl-5-oxa-2,8-diaza-1(5,3)-pyrazolino[1,5-a]pyrimidine-4(3,2)-pyridine heterocycle 9-one (Compound 30A):

[0457]

[0458] (S)-6-bromo-5-((((2-((1-(((tert-Butoxycarbonyl)amino)propan-2-yl)oxy)-5-fluoropyridin-3-yl)methyl)amino)-7-(dibenzylamino)pyrazolo[1,5-a]pyrimidine-3-carboxylic acid methyl ester (7.47 g, 10.0 mol) was dissolved in a mixed solution of methanol and THF, followed by the addition of LiOH (4.8 g, 200.0 mmol) aqueous solution. After the addition was complete, the reaction system was heated to 60°C and stirred for 16 h. The reaction system was then cooled to 0°C and adjusted with 2N hydrochloric acid. The pH was adjusted to 2-3, and the mixture was extracted three times with DCM. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. A solution of hydrochloric acid in 1,4-dioxane was added, and the mixture was stirred at room temperature for 1 hour. The reaction system was concentrated to dryness, and then DCM and DMF were added, followed by DIPEA (19.32 g, 150.0 mmol) and FDPP (19.2 g, 50.0 mmol). After the additions were complete, the mixture was stirred at room temperature for 16 hours. After the reaction was complete, the reaction system was concentrated to dryness under reduced pressure, and the residue was isolated by C-18 reverse preparative separation to obtain compound 30A.

[0459] MS (ESI) m / z 630.2 (M+H) +

[0460] Step 2: (S, 1 3 E,1 4 E)-1 6 -(1-ethyl-1H-pyrrol-3-yl)-17 -(Benzhydrylamino)-4-(Benzhydrylamino)- 5 Preparation of 6-fluoro-5-methyl-5-oxa-2,8-diaza-1(5,3)-pyrazolin[1,5-a]pyrimidine-4(3,2)-pyridine heterocycle 9-one (Compound 30B):

[0461]

[0462] Under nitrogen protection, (S,1 3 E,1 4 E)-1 6 -Br-1 7 -(Benzhydrylamino)-4-(Benzhydrylamino)- 5 6-Fluoro-6-methyl-5-oxa-2,8-diaza-1(5,3)-pyrazolino[1,5-a]pyrimidin-4(3,2)-pyridineheterocyclopentadien-9-one (250.0 mg, 0.40 mmol), 1-ethyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrole (107.6 mg, 0.49 mmol), and K2CO3 (112.1 mg, 0.81 mmol) were dissolved in a mixture of dioxane and water (4:1, 5.0 mL), followed by the addition of Pd(PPh3)4 (46.9 mg, 0.04 mmol). The reaction system was stirred at 80°C for 16 hours. After completion of the reaction, the reaction system was concentrated to dryness under reduced pressure, and the residue was purified by silica gel column chromatography to obtain compound 30B.

[0463] MS (ESI) m / z 631.6 (M+H) +

[0464] Step 3: (S, 1 3 E,1 4 E)-1 6 -(1-ethyl-1H-pyrrol-3-yl)-1 7 -Amino-4 5 Preparation of 6-fluoro-5-methyl-5-oxa-2,8-diaza-1(5,3)-pyrazolin[1,5-a]pyrimidine-4(3,2)-pyridine heterocycle 9-one (Compound 30):

[0465]

[0466] (S,1 3 E,1 4 E)-1 6 -(1-ethyl-1H-pyrrol-3-yl)-1 7 -(Benzhydrylamino)-4-(Benzhydrylamino)- 56-Fluoro-6-methyl-5-oxa-2,8-diaza-1(5,3)-pyrazolino[1,5-a]pyrimidine-4(3,2)-pyridineheterocyclic 9-one (63.0 mg, 0.1 mmol) was dissolved in DCM (10.0 mL), and trifluoromethanesulfonic acid (150.0 mg, 1.0 mmol) was added at 0°C, and the reaction was maintained at 0°C for 2 h. After the reaction was complete, TEA was slowly added at 0°C to adjust the pH to 7-8. The reaction solution was concentrated under reduced pressure, and the residue was isolated by high pressure preparative separation to obtain Compound 30 of Example 30.

[0467] MS (ESI) m / z 451.3 (M+H)+

[0468] 1H NMR (400MHz, DMSO-d6) δ9.87(d,J=8.8Hz,1H),8.03(s,1H),8.02-7.94(m,2H),7.78(m,1H),7.14(t,J=6.0Hz,2H),7.03(s,1H),6.9 5(s,1H),6.57(s,2H),6.08(s,1H),5.11-4.96(m,1H),4.99-4.87(m,1H),4.09-3.90(m,4H),3.17-3.04(m,1H),1.49-1.39(m,6H).

[0469] Example 31: 4-((S,1 3 E,1 4 E)-1 7 -Amino-4 5 -Fluoro-6-methyl-9-oxo-5-oxa-2,8-diaza-1(5,3)-pyrazolin[1,5-a]pyrimidine 4(3,2)-pyridine heterocycle Jiufan-1 6 Preparation of -yl)-N-methyl-1H-pyrrole-2-carboxamide (Compound 31):

[0470]

[0471] 4-Borate-N,1-dimethyl-1H-pyrrole-2-carboxamide was used to replace 1-ethyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrole in step 2 of Example 30, and the same preparation method as Example 30 was used to obtain Compound 31 of Example 31.

[0472] MS (ESI) m / z 494.3 (M+H) +

[0473] 1H NMR (400MHz, DMSO-d6) δ9.87(d,J=9.1Hz,1H),8.04-8.00(m,2H),7.99(s,1H),7.78(d,J=3.0Hz,1H),7.23(m,2H),7.05(s ,1H),6.78(s,3H),5.04-4.91(m,2H),4.05-3.85(m,5H),3.15-3.05(m,1H),2.80(d,J=6.3Hz,3H),1.46(d,J=6.1Hz,3H).

[0474] Example 32: 4-((S,1 3 E,1 4 E)-1 7 -Amino-4 5 -Fluoro-6-methyl-9-oxo-5-oxa-2,8-diaza-1(5,3)-pyrazolin[1,5-a]pyrimidine 4(3,2)-pyridine heterocycle Jiufan-1 6 -yl)-N-methyl-1H-2-pyrrolecarboxamide (Compound 32):

[0475]

[0476] 1-Benzyl-N-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrole-2-carboxamide was used to replace 1-ethyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrole in step 2 of Example 30, and the same preparation method as in Example 30 was used to obtain compound 32 of Example 32.

[0477] MS (ESI) m / z 480.2 (M+H) + .

[0478] 1 H NMR (400MHz, DMSO-d6) δ11.84(s,1H),9.88(d,J=10.6Hz,1H),8.07(s,1H),8.03(s,1H),7.99(s,1H),7.75(d,J=11.8Hz,1H),7.16(s,1H),6 .94(s,1H),6.77(s,1H),6.67(s,2H),5.00(m,1H),4.92(d,J=17.9Hz ,1H),4.04-3.87(m,2H),3.15-3.06(m,1H),2.78(s,3H),1.45(s,3H).

[0479] Example 33: 4-((S,1 3 E,1 4 E)-1 7 -Amino-4 5 -Fluoro-6-methyl-9-oxo-5-oxa-2,8-diaza-1(5,3)-pyrazolin[1,5-a]pyrimidine 4(3,2)-pyridine heterocycle Jiufan-1 6 Preparation of methyl-1H-pyrrole-2-carboxylate (compound 33):

[0480]

[0481] Substituting 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrole-2-carboxylic acid methyl ester for 1-ethyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrole in step 2 of Example 30, the same preparation method as in Example 30 was used to obtain compound 33 of Example 33.

[0482] MS (ESI) m / z 481.2 (M+H) + .

[0483] 1 H NMR (400MHz, DMSO-d6) δ12.24(s,1H),9.85(d,J=8.5Hz,1H),7.99(s,1H),7.95(d,J=3.1Hz,1H),7.68(dd,J=8.9,3.0Hz,1H),7.10(dd,J=11.9,5.6 Hz,2H),6.77(s,1H),6.69(s,2H),4.96(m,1H),4.88(m,1H),3.95(m,1H), 3.88-3.82(m,1H),3.78(s,3H),3.10-3.02(m,1H),1.41(d,J=6.1Hz,3H).

[0484] Example 34: (S, 1 3 E,1 4 E)-1 6 -(1H-pyrrol-3-yl)-1 7 -Amino-4 5 Preparation of 6-fluoro-5-methyl-5-oxa-2,8-diaza-1(5,3)-pyrazolin[1,5-a]pyrimidine-4(3,2)-pyridine heterocycle 9-one (Compound 34):

[0485]

[0486] Substituting 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrole for 1-ethyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrole in step 2 of Example 30, the same preparation method as in Example 30 was used to obtain compound 34 of Example 34.

[0487] MS (ESI) m / z 423.2 (M+H) +

[0488] 1 H NMR (400MHz, DMSO-d6) δ11.22(s,1H),9.89(d,J=8.9Hz,1H),8.02(s,1H),8.00–7.97(m,1H),7.81–7.70(m,1H),7.12(t,J=6.1Hz,1H),7.02(m, 1H),6.92(m,1H),6.49(m,2H),6.11(m,1H),5.08–4.96(m,1H),4.98–4. 83(m,1H),4.10–3.83(m,2H),3.20–3.03(m,1H),1.45(d,J=5.9Hz,3H).

[0489] Example 35: 4-((S,1 3 E,1 4 E)-1 7 -Amino-4 5 -Fluoro-6-methyl-9-oxo-5-oxa-2,8-diaza-1(5,3)-pyrazolin[1,5-a]pyrimidine 4(3,2)-pyridine heterocycle Jiufan-1 6 Preparation of methyl-N-methyl-1H-pyrrole-2-carboxylate (Compound 35):

[0490]

[0491] 1-Methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrole-2-carboxylic acid methyl ester was used to replace 1-ethyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrole in step 2 of Example 30, and the same preparation method as Example 30 was used to obtain Compound 35 of Example 35.

[0492] MS (ESI) m / z 495.2 (M+H) + .

[0493] 1H NMR (400MHz, DMSO-d6) δ9.84(d,J=8.9Hz,1H),7.99(s,1H),7.95(d,J=2.4Hz,1H),7. 70(dd,J=9.2,2.6Hz,1H),7.20(d,J=1.9Hz,1H),7.18-7.14(m,1H),6.83(d,J=1.7Hz ,1H),6.79(s,2H),5.01-4.93(m,1H),4.93-4.84(m,1H),3.99-3.93(m,1H),3.92-3. 90(m,3H),3.89-3.82(m,1H),3.76(s,3H),3.11-3.03(m,1H),1.41(d,J=6.1Hz,3H).

[0494] Example 36: (S, 1 3 E,1 4 E)-1 7 -Amino-4 5 -Fluoro-6-methyl-1 6 Preparation of -(1-methyl-1H-4-imidazolyl)-5-oxa-2,8-diaza-1(5,3)-pyrazolin[1,5-a]pyrimidine 4(3,2)-pyridine heterocycle 9-one (Compound 36):

[0495]

[0496] Substituting 1-methyl-4-(tributylstannyl)-1H-imidazole for 2-(tributylstannyl)thiazole in step 1 of Example 11, the same preparation method as Example 11 was used to obtain Compound 36 of Example 36.

[0497] MS (ESI) m / z 438.2 (M+H) +

[0498] 1 H NMR(400MHz, DMSO-d6)δ9.75(d,J=6.6Hz,1H),8.09(s,1H),8.06(s,1H),7.99(s,1H),7.89(s,1H),7.86-7.81(m,1H), 7.65(s,2H),7.58(s,1H),5.02(d,J=16.3Hz,2H),4.11-3.94(m,2H),3.78(s,3H),3.12(t,J=12.1Hz,1H),1.46(s,3H).

[0499] Example 37: N 1 -(4-((S,1 3 E,14 E)-1 7 -(Benzhydrylamino)-4-(Benzhydrylamino)- 5 -Fluoro-6-methyl-9-oxo-5-oxa-2,8-diaza-1(5,3)-pyrazolin[1,5-a]pyrimidine 4(3,2)-pyridine heterocycle Jiufan-1 6 -yl)phenyl)-N 2 The preparation synthesis route of -methyloxalamide (Compound 37) is as follows:

[0500]

[0501] Step 1: N 1 -(4-((S,1 3 E,1 4 E)-1 7 -(Benzhydrylamino)-4-(Benzhydrylamino)- 5 -Fluoro-6-methyl-9-oxo-5-oxa-2,8-diaza-1(5,3)-pyrazolin[1,5-a]pyrimidine 4(3,2)-pyridine heterocycle Jiufan-1 6 -yl)phenyl)-N 2 Preparation of -methyloxalamide (Compound 37A):

[0502]

[0503] The compound (S,1 3 E,1 4 E)-1 6 -(4-aminophenyl)-1 7 -Benzhydrylamino-4-(2-nitrophenyl)amino)- 5 6-Fluoro-6-methyl-5-oxa-2,8-diaza-1(5,3)-pyrazolino[1,5-a]pyrimidine-4(3,2)-pyridine heterocycle-9-one (340 mg, 0.54 mmol), 2-(methylamino)-2-oxoacetic acid (61 mg, 0.59 mmol), HATU (267 mg, 0.70 mmol), and DIPEA (209 mg, 1.62 mmol) were dissolved in DMF (100 mL). The mixture was stirred at room temperature for 2 hours. After the reaction was complete, the reaction system was concentrated to dryness under reduced pressure, and the residue was separated and purified by silica gel column chromatography to obtain compound 37A. MS (ESI) m / z 714.8 (M+H) +

[0504] Step 2: N 1 -(4-((S,1 3 E,1 4 E)-1 7 -(Benzhydrylamino)-4-(Benzhydrylamino)- 5-Fluoro-6-methyl-9-oxo-5-oxa-2,8-diaza-1(5,3)-pyrazolin[1,5-a]pyrimidine 4(3,2)-pyridine heterocycle Jiufan-1 6 -yl)phenyl)-N 2 Preparation of -methyloxalamide (Compound 37):

[0505]

[0506] N 1 -(4-((S,1 3 E,1 4 E)-1 7 -(Benzhydrylamino)-4-(Benzhydrylamino)- 5 -Fluoro-6-methyl-9-oxo-5-oxa-2,8-diaza-1(5,3)-pyrazolin[1,5-a]pyrimidine 4(3,2)-pyridine heterocycle Jiufan-1 6 -yl)phenyl)-N 2 Methyloxalamide (63.0 mg, 0.1 mmol) was dissolved in DCM (10.0 mL), and trifluoromethanesulfonic acid (150.0 mg, 1.0 mmol) was added at 0°C, and the reaction was maintained at 0°C for 2 h. After the reaction was completed, TEA was slowly added at 0°C to adjust the pH to 7-8. The reaction solution was concentrated under reduced pressure, and the residue was isolated by high pressure preparative separation to obtain Compound 37 of Example 37.

[0507] MS (ESI) m / z 534.3 (M+H) +

[0508] 1 H NMR (400MHz, DMSO-d6) δ10.81(s,1H),9.84(d,J=8.8Hz,1H),8.98(q,J=4.8Hz,1H),8.05 (d,J=8.8Hz,2H),8.01(s,1H),8.0-7.96(m,1H),7.66(dd,J=9.1,3.0Hz,1H),7.27(s,2H ),6.97(t,J=6.2Hz,1H),6.70(s,2H),5.01-4.87(m,2H),3.99-3.93(m,1H),3.82(dd,J= 14.6,5.8Hz,1H),3.07(dd,J=12.0,10.4Hz,1H),2.74-2.69(m,3H),1.41(d,J=6.0Hz,3H)

[0509] Example 38: (1 3 E,1 4 E,3R,6S)-1 7 -amino-3-ethyl-45 -Fluoro-6-methyl-1 6 The preparation and synthesis route of -(p-tolyl)-5-oxo-2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidine-4(3,2)-pyridine heterocycle 9-one (Compound 38) is as follows:

[0510]

[0511] Step 1: Preparation of (R,E)-N-((5-fluoro-2-methoxypyridin-3-yl)methylene)-2-methylpropane-2-sulfenamide (Compound 38A)

[0512]

[0513] 5-Fluoro-2-methoxypyridine-3-carboxaldehyde (15.5 g, 100.0 mmol) was dissolved in DCM (10 mL). Cs2CO3 (65.6 g, 200.0 mmol) and (R)-2-methylpropane-2-sulfenamide (14.5 g, 120.0 mmol) were added sequentially. The mixture was allowed to react at 30°C for 16 hours. After completion of the reaction, the mixture was filtered and the filtrate was concentrated under reduced pressure to obtain compound 38A.

[0514] MS (ESI) m / z 259.3 (M+H) + .

[0515] Step 2: Preparation of (R)-N-((R)-1-(5-fluoro-2-methoxypyridin-3-yl)propyl)propane-2-sulfenamide (Compound 38B)

[0516]

[0517] Under nitrogen, (R,E)-N-((5-fluoro-2-methoxypyridin-3-yl)methylene)-2-methylpropane-2-sulfenamide (18.0 g, 70.0 mmol) was dissolved in anhydrous THF (260 mL). Ethylmagnesium chloride (2.0 M in THF) (88.0 mL, 175.0 mmol) was added at 0°C and allowed to react at room temperature for 6 hours. After the reaction was complete, saturated ammonium chloride solution was slowly added at 0°C to quench the reaction. After stirring for half an hour, the mixture was extracted with ethyl acetate, the combined organic phases were dried over sodium sulfate, filtered, and concentrated, and the residue was purified by silica gel column chromatography to obtain compound 38B. MS (ESI) m / z 275.3 (M+H) + .

[0518] Step 3: Preparation of (R)-1-(5-fluoro-2-methoxypyridin-3-yl)propan-1-amine (Compound 38C)

[0519]

[0520] Dissolve (R)-N-((R)-1-(5-fluoro-2-methoxypyridin-3-yl)propyl)propane-2-sulfenamide (9.6 g, 35.0 mmol) in DCM (80.0 mL). Add hydrochloric acid in dioxane (35.0 mL, 70.0 mmol) at room temperature. Stir the mixture at room temperature for 4 hours. After completion of the reaction, concentrate the reaction mixture under reduced pressure to obtain the hydrochloride salt of compound 38C.

[0521] MS (ESI) m / z 185.2 (M+H) + .

[0522] Step 4: Preparation of (R)-3-(1-aminopropyl)-5-fluoropyridin-2-ol (Compound 38D)

[0523]

[0524] (R)-1-(5-Fluoro-2-methoxypyridin-3-yl)propan-1-amine (5.2 g, 28.3 mmol) was dissolved in ethanol (80.0 mL). A solution of hydrochloric acid in dioxane (13.0 mL, 57.0 mmol) was added at room temperature. The mixture was then reacted at 80°C overnight. After completion of the reaction, the reaction mixture was concentrated under reduced pressure to yield compound 38D.

[0525] MS (ESI) m / z 171.2 (M+H) + .

[0526] Step 5: Preparation of (R)-methyl 6-bromo-7-(dibenzylamino)-5-((1-(5-fluoro-2-hydroxypyridin-3-yl)propyl)amino)pyrazolo[1,5-a]pyrimidine-3-carboxylate (Compound 38E)

[0527]

[0528] (R)-3-(1-Aminopropyl)-5-fluoropyridin-2-ol (3.60 g, 20.6 mmol), methyl 6-bromo-5-chloro-7-(dibenzylamino)pyrazolo[1,5-a]pyrimidine-3-carboxylate (10.3 g, 20.6 mmol), DIPEA (8.0 g, 61.8 mmol), and ethanol (100 mL) were added to a reaction flask and reacted at 80°C overnight. After completion of the reaction, the reaction solution was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to obtain compound 38E.

[0529] MS (ESI) m / z 619.2 (M+H) + .

[0530] Step 6: Preparation of methyl 6-bromo-5-(((R)-1-(2-(((S)-1-((tert-butoxycarbonyl)amino)propan-2-yl)oxy)-5-fluoropyridin-3-yl))propyl)amino)-7-(dibenzylamino)pyrazolo[1,5-a]pyrimidine-3-carboxylate (Compound 38F)

[0531]

[0532] Under nitrogen, (R)-methyl 6-bromo-7-(dibenzylamino)-5-((1-(5-fluoro-2-hydroxypyridin-3-yl)propyl)amino)pyrazolo[1,5-a]pyrimidine-3-carboxylate (6.3 g, 10.0 mmol) was dissolved in anhydrous THF. Tert-butyl (S)-(2-hydroxypropyl)carbamate (1.75 g, 11.0 mmol) and PPh3 (4.0 g, 15.0 mmol) were then added. After the addition, the reaction system was cooled to 0°C, and DIAD (4.1 g, 20.0 mmol) was slowly added dropwise. After the addition, the system was stirred at room temperature for 16 hours. After completion of the reaction, the reaction solution was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to obtain compound 38F.

[0533] MS (ESI) m / z 776.2 (M+H) +

[0534] Step 7: Preparation of methyl 5-(((R)-1-(2-(((S)-1-((tert-butoxycarbonyl)amino)propan-2-yl)oxy)-5-fluoropyridin-3-yl)propyl)amino)-7-(dibenzylamino)-6-(p-tolyl)pyrazolo[1,5-a]pyrimidine-3-carboxylate (Compound 38G)

[0535]

[0536] Under argon protection, 6-bromo-5-(((R)-1-(2-(((S)-1-((tert-butoxycarbonyl)amino)propan-2-yl)oxy)-5-fluoropyridin-3-yl))propyl)amino)-7-(dibenzylamino)pyrazolo[1,5-a]pyrimidine-3-carboxylic acid methyl ester (4.0 g, 5.1 mmol), p-methylphenylboronic acid (1.1 g, 7.65 mmol), XPhos (242.6 mg, 0.51 mmol), XPhos-Pd-G3 (447.0 mg, 0.51 mmol) and phosphorus K3PO4 (3.23 g, 15.3 mmol), 1,4-dioxane (50 mL) and water (10.0 mL) were added to the reaction flask and reacted at 80°C overnight. After the reaction was completed, the reaction solution was concentrated under reduced pressure, and the residue was separated and purified by silica gel column chromatography to obtain compound 38G.

[0537] MS (ESI) m / z 788.3 (M+H) +

[0538] Step 8: (1 3 E,1 4 E,3R,6S)-1 7 -(dibenzylamino)-3-ethyl-4- 5 -Fluoro-6-methyl-1 6 Preparation of -(p-Tolyl)-5-oxo-2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidin-4(3,2)-pyridine heterocycle 9-one (Compound 38H)

[0539]

[0540] Methyl 5-(((R)-1-(2-(((S)-1-((tert-butoxycarbonyl)amino)propan-2-yl)oxy)-5-fluoropyridin-3-yl)propyl)amino)-7-(dibenzylamino)-6-(p-tolyl)pyrazolo[1,5-a]pyrimidine-3-carboxylate (788.0 mg, 1.0 mmol) was dissolved in a mixed solution of methanol and THF, followed by the addition of LiOH (480 mg, 20.0 mmol) aqueous solution. After the addition, the reaction system was heated to 60°C and stirred for 16 h. The reaction system was then cooled to 0°C, the pH adjusted to 2-3 with 2N hydrochloric acid, and extracted three times with DCM. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. A solution of hydrochloric acid in 1,4-dioxane was added, and the mixture was stirred at room temperature for 1 hour. The reaction system was concentrated to dryness, and DCM and DMF were added, followed by DIPEA (1.93 g, 15.0 mmol) and FDPP (1.92 g, 5.0 mmol). After the additions were complete, the mixture was stirred at room temperature for 16 hours. After completion of the reaction, the reaction system was concentrated to dryness under reduced pressure, and the residue was purified by silica gel column chromatography to obtain compound 38H.

[0541] MS (ESI) m / z 656.7 (M+H) +

[0542] Step 9: (1 3 E,1 4 E,3R,6S)-1 7 -amino-3-ethyl-4 5 -Fluoro-6-methyl-1 6 Preparation of -(p-Tolyl)-5-oxo-2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidine-4(3,2)-pyridine heterocycle Jiufan-9-one (Compound 38)

[0543]

[0544] (1 3 E,1 4 E,3R,6S)-1 7 -(dibenzylamino)-3-ethyl-4- 5 -Fluoro-6-methyl-1 6 1-(p-Tolyl)-5-oxo-2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidin-4(3,2)-pyridinecyclononane-9-one (66.0 mg, 0.1 mmol) was dissolved in DCM (10.0 mL), and trifluoromethanesulfonic acid (150.0 mg, 1.0 mmol) was added at 0°C. The reaction was maintained at 0°C for 2 h. After the reaction was complete, TEA was slowly added at 0°C to adjust the pH to 7-8. The reaction solution was concentrated under reduced pressure, and the residue was isolated by high pressure preparative separation to obtain Compound 38 of Example 38.

[0545] MS (ESI) m / z 476.3 (M+H) +

[0546] 1 H NMR(400MHz,DMSO-d6)δ9.81(dd,J=9.0,1.5Hz,1H),8.06(s,1H),7.97(d,J=3.0Hz,1H ),7.88(dd,J=9.2,3.1Hz,1H),7.45–7.38(m,2H),7.25(d,J=7.6Hz,2H),6.63(s,2H), 6.38(d,J=7.6Hz,1H),5.31–5.23(m,1H),5.03(m,1H),3.99(m,1H),3.12(m,1H),2.43 (s,3H),1.88(m,1H),1.69–1.56(m,1H),1.46(d,J=6.1Hz,3H),0.84(t,J=7.2Hz,3H).

[0547] Example 39: (S, 1 3 E,1 4 E)-1 7 -Amino-4 5 -Fluoro-6-methyl-1 6 Preparation of -(2-oxo-1,4-dihydro-2H-benzo[d][1,3]oxazin-6-yl)-5-oxo-2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidin-4(3,2)-pyridine heterocycle 9-one (Compound 39)

[0548]

[0549] Using 6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1,4-dihydro-2H-benzo[d][1,3]oxazin-2-one instead of 1-ethyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrole in step 2 of Example 30, the same preparation method as Example 30 was used to obtain Compound 39 of Example 39. MS (ESI) m / z 505.3 (M+H) +

[0550] 1 H NMR (400MHz, DMSO) δ10.35(d,J=7.3Hz,1H),9.87(s,1H),8.05(s,1H),7.99(d,J=3.1Hz,1H),7.73–7.61(m,1H),7.26–7.12(m,2H),7.10–6.97(m,2 H),6.80(s,2H),5.40–5.29(m,2H),5.01(s,1H),4.95–4.90(m,1H),4.00– 3.96(m,1H),3.87–3.82(m,1H),3.13–3.08(m,1H),1.45(d,J=6.1Hz,3H).

[0551] Example 40: (S, 1 3 E,1 4 E)-1 7 -Amino-4 5 -Fluoro-6-methyl-1 6 Preparation of -(prop-1-en-2-yl)-5-oxa-2,8-diaza-1(5,3)-pyrazolin[1,5-a]pyrimidine-4(3,2)-pyridine heterocycle 9-one (Compound 40)

[0552]

[0553] Substituting isopropenylboronic acid for (1-ethyl-1H-pyrazol-4-yl)boronic acid in step 1 of Example 2, the same preparation method as in Example 2 was used to obtain Compound 40 of Example 40.

[0554] MS (ESI) m / z 398.2 (M+H) +

[0555] 1H NMR (400MHz, DMSO-d6) δ9.91–9.84(m,1H),8.03(s,1H),7.99(d,J=3.1Hz,1H),7.78(dd,J=8.9,3.1Hz,1H),7.46(t,J=6.2Hz,1H),7 .00(s,2H),5.62–5.55(m,1H),5.18–5.11(m,1H),4.97(m,2H),4.04–3.92(m,2H),3.10(m,1H),1.97(s,3H),1.45(d,J=6.1Hz,3H).

[0556] Example 41: (S, 1 3 E,1 4 E)-1 7 -Amino-1 6 -(4-(dimethylamino)-3-fluorophenyl)-4 5 Preparation of 6-fluoro-5-methyl-5-oxa-2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidine-4(3,2)-pyridine heterocycle 9-one (Compound 41)

[0557]

[0558] Substituting 2-fluoro-N,N-dimethyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline for (1-ethyl-1H-pyrazol-4-yl)boronic acid in step 1 of Example 2, the same preparation method as in Example 2 was used to obtain compound 41 of Example 41.

[0559] MS (ESI) m / z 495.2 (M+H) +

[0560] 1 H NMR(400MHz, DMSO-d6)δ9.88(d,J=8.7Hz,1H),8.05(s,1H),8.00(d,J=3.0Hz,1H),7.71(s,1H),7.21–6.95(m,4H),6.79(s,2H),5.06–4.88(m,2H) ,4.03–3.97(m,4.0Hz,1H),3.88(dd,J=14.6,5.6Hz,1H),3.14–3.08(ddd,J=13.5,10.1,1.5Hz,1H),2.89(d,J=0.8Hz,6H),1.46(d,J=6.1Hz,3H).

[0561] Example 42: (1 3 E,1 4E,3R,6S)-1 7 -amino-3-ethyl-4 5 -Fluoro-6-methyl-1 6 Preparation of -(p-chlorophenyl)-5-oxo-2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidine-4(3,2)-pyridine heterocycle 9-one (Compound 42)

[0562]

[0563] Substituting p-chlorophenylboronic acid for p-tolueneboronic acid in step 7 of Example 38, the same preparation method as in Example 38 was used to obtain compound 42 of Example 42.

[0564] MS (ESI) m / z 497.3 (M+H) +

[0565] 1 H NMR(400MHz,DMSO-d6)δ9.81(d,J=8.9Hz,1H),8.07(d,J=1.2Hz,1H),8.00-7.94(m,1H) ,7.85-7.82(m,1H),7.67-7.57(m,2H),7.36(d,J=7.9Hz,2H),6.85(s,2H),6.51(d,J=7 .5Hz,1H),5.27(q,J=7.9Hz,1H),5.11–4.92(m,1H),4.02-3.96(m,1H),3.20-3.06(m,1 H),1.92-1.81(m,1H),1.68-1.57(m,1H),1.46(d,J=6.0Hz,3H),0.84(t,J=7.2Hz,3H).

[0566] Example 43: (1 3 E,1 4 E,3R,6S)-1 7 -amino-3-ethyl-4 5 -Fluoro-6-methyl-1 6 Preparation of -(p-chlorophenyl)-5-oxo-2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidine-4(3,2)-pyridine heterocycle 9-one (Compound 43)

[0567]

[0568] Substituting 1-methylpyrrole-3-pinacol boronate for p-methylphenylboronic acid in step 7 of Example 38, the same preparation method as in Example 38 was used to obtain compound 43 of Example 43.

[0569] MS (ESI) m / z 465.1 (M+H)

[0570] 1 H NMR(400MHz,DMSO-d6)δ9.82(d,J=8.7Hz,1H),8.03(d,J=1.2Hz,1H),8.00-7.91(m,2H),6.97(q ,J=2.0Hz,1H),6.90(q,J=1.8Hz,1H),6.63(s,2H),6.51(d,J=7.4Hz,1H),6.10(q,J=1.9Hz,1H), 5.24(q,J=7.8Hz,1H),5.08-5.00(m,1H),4.01-3.98(m,1H),3.72(d,J=1.2Hz,3H),3.18-3.04( m,1H),2.00-1.89(m,1H),1.76-1.62(m,1H),1.45(dd,J=6.0,1.2Hz,3H),0.86(t,J=7.2Hz,3H).

[0571] Example 44: (1 3 E,1 4 E,3R,6S)-1 7 -amino-3-ethyl-4 5 -Fluoro-6-methyl-1 6 The preparation and synthesis route of -(p-chlorophenyl)-5-oxo-2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidine-4(3,2)-pyridine heterocycle 9-one (Compound 44) is as follows:

[0572]

[0573] Step 1: Preparation of (R)-N-((R)-1-(5-fluoro-2-methoxypyridin-3-yl)-2-methylpropyl)-2-methylpropane-2-sulfenamide (Compound 44A)

[0574]

[0575] Under nitrogen, (R,E)-N-((5-fluoro-2-methoxypyridin-3-yl)methylene)-2-methylpropane-2-sulfenamide (8.0 g, 30.9 mmol) was dissolved in anhydrous THF (260 mL). The reaction temperature was lowered to -78°C, followed by the addition of isopropylmagnesium bromide (1.0 M in THF) (93.0 mL, 92.9 mmol). The mixture was allowed to react at room temperature for 6 hours. After completion of the reaction, saturated ammonium chloride solution was slowly added at 0°C to quench the reaction. After stirring for half an hour, the mixture was extracted with ethyl acetate. The combined organic phases were dried over sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography to obtain Compound 44A.

[0576] MS (ESI) m / z 303.2 (M+H) + .

[0577] Step 2: Preparation of (R)-1-(5-fluoro-2-methoxypyridin-3-yl)-2-methylpropan-1-amine (Compound 44B)

[0578]

[0579] Dissolve (R)-N-((R)-1-(5-fluoro-2-methoxypyridin-3-yl)-2-methylpropyl)-2-methylpropane-2-sulfenamide (6.0 g, 19.8 mmol) in DCM (80.0 mL). Add a solution of hydrochloric acid in dioxane (10.0 mL, 40.0 mmol) at room temperature. Stir the mixture at room temperature for 1 hour. After completion of the reaction, concentrate the reaction mixture under reduced pressure to obtain the hydrochloride salt of compound 44B.

[0580] MS (ESI) m / z 199.2 (M+H) + .

[0581] Step 3: Preparation of (R)-3-(1-amino-2-methylpropyl)-5-fluoropyridin-2-ol (Compound 44C)

[0582]

[0583] (R)-1-(5-Fluoro-2-methoxypyridin-3-yl)-2-methylpropan-1-amine (6.0 g, 19.8 mmol) was dissolved in ethanol (80.0 mL). A solution of hydrochloric acid in dioxane (13.0 mL, 57.0 mmol) was added at room temperature. The mixture was then reacted at 80°C overnight. After completion of the reaction, the reaction mixture was concentrated under reduced pressure to yield compound 44C.

[0584] MS (ESI) m / z 185.2 (M+H) + .

[0585] Step 4: Preparation of (R)-methyl 6-bromo-7-(dibenzylamino)-5-((1-(5-fluoro-2-hydroxypyridin-3-yl)-2-methylpropyl)amino)pyrazolo[1,5-a]pyrimidine-3-carboxylate (Compound 44D)

[0586]

[0587] (R)-3-(1-amino-2-methylpropyl)-5-fluoropyridin-2-ol (3.0 g, 16.3 mmol), methyl 6-bromo-5-chloro-7-(dibenzylamino)pyrazolo[1,5-a]pyrimidine-3-carboxylate (8.7 g, 17.9 mmol), DIPEA (6.26 g, 48.9 mmol), and ethanol (100 mL) were added to a reaction flask and reacted at 80°C overnight. After completion of the reaction, the reaction solution was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to obtain compound 44D.

[0588] MS (ESI) m / z 647.2 (M+H) + .

[0589] Step 5: Preparation of methyl 6-bromo-5-(((R)-1-(2-(((S)-1-((tert-butoxycarbonyl)amino)propan-2-yl)oxy)-5-fluoropyridin-3-yl))-2-methylpropyl)amino)-7-(dibenzylamino)pyrazolo[1,5-a]pyrimidine-3-carboxylate (Compound 44E)

[0590]

[0591] Under nitrogen, (R)-methyl 6-bromo-7-(dibenzylamino)-5-((1-(5-fluoro-2-hydroxypyridin-3-yl)-2-methylpropyl)amino)pyrazolo[1,5-a]pyrimidine-3-carboxylate (6.4 g, 10.0 mmol) was dissolved in anhydrous THF. Tert-butyl (S)-(2-hydroxypropyl)carbamate (1.75 g, 11.0 mmol) and PPh3 (4.0 g, 15.0 mmol) were then added. After the addition, the reaction system was cooled to 0°C, and DIAD (4.1 g, 20.0 mmol) was slowly added dropwise. After the addition, the system was stirred at room temperature for 16 hours. After completion of the reaction, the reaction solution was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to obtain compound 44E.

[0592] MS (ESI) m / z 790.2 (M+H) +

[0593] Step 6: Preparation of methyl 5-(((R)-1-(2-(((S)-1-((tert-butoxycarbonyl)amino)propan-2-yl)oxy)-5-fluoropyridin-3-yl)-2-methylpropyl)amino)-7-(dibenzylamino)-6-(1-methyl-1H-pyrrol-3-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylate (Compound 44F)

[0594]

[0595] Under argon protection, 6-bromo-5-(((R)-1-(2-(((S)-1-((tert-butoxycarbonyl)amino)propan-2-yl)oxy)-5-fluoropyridin-3-yl))-2-methylpropyl)amino)-7-(dibenzylamino)pyrazolo[1,5-a]pyrimidine-3-carboxylate (4.1 g, 5.1 mmol), p-methylphenylboronic acid (1.1 g, 7.65 mmol), XPhos (242.6 mg, 0.51 mmol), XPhos-Pd-G3 (447.0 mg, 0.51 mmol) and phosphorus K3PO4 (3.23 g, 15.3 mmol), 1,4-dioxane (50 mL) and water (10.0 mL) were added to the reaction flask and reacted at 80°C overnight. After the reaction was completed, the reaction solution was concentrated under reduced pressure, and the residue was separated and purified by silica gel column chromatography to obtain compound 44F.

[0596] MS (ESI) m / z 791.4 (M+H) +

[0597] Step 7: (1 3 E,1 4 E,3R,6S)-1 7 -(dibenzylamino)-4-(dibenzylamino)- 5 -fluoro-3-isopropyl-6-methyl-1 6 Preparation of -(1-methyl-1H-pyrrol-3-yl)-5-oxa-2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidin-4(3,2)-pyridine heterocycle 9-one (Compound 44G)

[0598]

[0599] Methyl 5-(((R)-1-(2-(((S)-1-((tert-Butoxycarbonyl)amino)propan-2-yl)oxy)-5-fluoropyridin-3-yl)-2-methylpropyl)amino)-7-(dibenzylamino)-6-(1-methyl-1H-pyrrol-3-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylate (790.0 mg, 1.0 mmol) was dissolved in a mixed solution of methanol and THF, followed by the addition of LiOH (480 mg, 20.0 mmol) aqueous solution. After the addition was complete, the reaction system was heated to 60°C and stirred for 16 h. The reaction system was then cooled to 0°C, the pH adjusted to 2-3 with 2N hydrochloric acid, and extracted three times with DCM. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. A solution of hydrochloric acid in 1,4-dioxane was added, and the mixture was stirred at room temperature for 1 hour. The reaction system was concentrated to dryness, and DCM and DMF were added, followed by DIPEA (1.93 g, 15.0 mmol) and FDPP (1.92 g, 5.0 mmol). After the additions were complete, the mixture was stirred at room temperature for 16 hours. After completion of the reaction, the reaction system was concentrated to dryness under reduced pressure, and the residue was purified by silica gel column chromatography to obtain compound 44G.

[0600] MS (ESI) m / z 659.2 (M+H) +

[0601] Step 8: (1 3 E,1 4 E,3R,6S)-1 7 -Amino-4 5 -fluoro-3-isopropyl-6-methyl-1 6 Preparation of -(1-methyl-1H-pyrrol-3-yl)-5-oxa-2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidin-4(3,2)-pyridine heterocycle 9-one (Compound 44)

[0602]

[0603] (1 3 E,1 4 E,3R,6S)-1 7 -(dibenzylamino)-4-(dibenzylamino)- 5 -fluoro-3-isopropyl-6-methyl-1 6-(1-Methyl-1H-pyrrol-3-yl)-5-oxa-2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidin-4(3,2)-pyridineheterocyclyl-9-one (66.0 mg, 0.1 mmol) was dissolved in DCM (10.0 mL), and trifluoromethanesulfonic acid (150.0 mg, 1.0 mmol) was added at 0°C, and the reaction was maintained at 0°C for 2 h. After the reaction was complete, TEA was slowly added at 0°C to adjust the pH to 7-8. The reaction solution was concentrated under reduced pressure, and the residue was isolated by high pressure preparative separation to obtain Compound 44 of Example 44.

[0604] MS (ESI) m / z 479.22 (M+H) +

[0605] 1 H NMR(400MHz,DMSO-d6)δ9.81(d,J=8.8Hz,1H),8.02(s,1H),7.95–7.93(m,2H),6.9 7(t,J=2.4Hz,1H),6.88(t,J=2.0Hz,1H),6.67–6.51(m,3H),6.10–6.05(m,1H),5.0 8–4.99(m,1H),4.94(s,1H),4.00–3.94(m,1H),3.72(s,3H),3.15–3.07(m,1H),2.1 9–2.10(m,1H),1.43(d,J=6.1Hz,3H), 1.06(d,J=6.4Hz,3H),0.67(d,J=6.6Hz,3H).

[0606] Example 45: 4-((1 3 E,1 4 E,3R,6S)-1 7 -Amino-4 5 -Fluoro-3,6-dimethyl-9-oxo-5-oxa-2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidine-4(3,2)-pyridine heterocycle Jiufan-1 6 -yl)-1H-pyrrole-2-carbonitrile (Compound 45) is prepared as follows:

[0607]

[0608]

[0609] Step 1: (1 3 E,1 4 E,3R,6S)-1 6 -Br-1 7 -(dibenzylamino)-4-(dibenzylamino)- 5Preparation of 3,6-dimethyl-5-oxa-2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidin-4(3,2)-pyridine heterocycle 9-one (Compound 45A)

[0610]

[0611] Methyl 5-(((R)-1-(2-(((S)-1-((tert-Butoxycarbonyl)amino)propan-2-yl)oxy)-5-fluoropyridin-3-yl)-2-methylpropyl)amino)-7-(dibenzylamino)-6-(1-methyl-1H-pyrrol-3-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylate (2.5 g, 3.22 mmol) was dissolved in a mixed solution of methanol and THF, followed by the addition of LiOH (246.0 mg, 10.2 mmol) aqueous solution. After the addition was complete, the reaction system was heated to 60°C and stirred for 16 h. The reaction system was then cooled to 0°C, the pH was adjusted to 2-3 with 2N hydrochloric acid, and the mixture was extracted three times with DCM. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The crude product was dissolved in DCM, and TMSOTf (3 mL) was slowly added dropwise at -15°C and stirred for half an hour. After the reaction was complete, DIPEA was added to adjust the pH of the system to a weak base. The solvent was then removed by distillation under reduced pressure. DCM and DMF were then added, followed by DIPEA (1.93 g, 15.0 mmol) and FDPP (1.92 g, 5.0 mmol). After addition, the mixture was stirred at room temperature for 16 h. After the reaction was complete, the reaction system was concentrated to dryness under reduced pressure, and the residue was separated and purified by silica gel column chromatography to obtain compound 45A.

[0612] MS (ESI) m / z 630.2 (M+H) +

[0613] Step 2: 4-((1 3 E,1 4 E,3R,6S)-1 7 -(dibenzylamino)-4-(dibenzylamino)- 5 -Fluoro-3,6-dimethyl-9-oxo-5-oxa-2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidine-4(3,2)-pyridinecyclononane-1 6 Preparation of 1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrole-2-carbonitrile (Compound 45B)

[0614]

[0615] (1 3 E,1 4 E,3R,6S)-1 6 -Br-1 7-(dibenzylamino)-4-(dibenzylamino)- 5 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrole-2-carbonitrile (995 mg, 2.86 mmol), Pd(dppf)Cl2 (139 mg, 0.190 mmol), cesium carbonate (1.86 g, 5.73 mmol), 1,4-dioxane (18 mL), and water (4.5 mL) were added to a reaction flask and reacted overnight at 110°C. After completion of the reaction, the reaction solution was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to obtain compound 45B.

[0616] MS (ESI) m / z 772.35 (M+H) +

[0617] Step 3:4-((1 3 E,1 4 E,3R,6S)-1 7 -(dibenzylamino)-4-(dibenzylamino)- 5 -Fluoro-3,6-dimethyl-9-oxo-5-oxa-2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidine-4(3,2)-pyridinecyclononane-1 6 Preparation of -yl)-1H-pyrrole-2-carbonitrile (Compound 45C)

[0618]

[0619] 4-((1 3 E,1 4 E,3R,6S)-1 7 -(dibenzylamino)-4-(dibenzylamino)- 5 -Fluoro-3,6-dimethyl-9-oxo-5-oxa-2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidine-4(3,2)-pyridinecyclononane-1 6 45C was obtained by dissolving 1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrole-2-carbonitrile (340 mg, 0.44 mmol) in tetrahydrofuran and adding TBAF (1 M in THF, 0.88 mL, 0.88 mmol). The mixture was refluxed for three hours. After completion of the reaction, the reaction solution was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to obtain compound 45C.

[0620] MS (ESI) m / z 642.2 (M+H) +

[0621] Step 4: 4-((1 3 E,1 4 E,3R,6S)-1 7 -Amino-4 5 -Fluoro-3,6-dimethyl-9-oxo-5-oxa-2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidine-4(3,2)-pyridinecyclononane-1 6 Preparation of -yl)-1H-pyrrole-2-carbonitrile (Compound 45)

[0622]

[0623] 4-((1 3 E,1 4 E,3R,6S)-1 7 -(dibenzylamino)-4-(dibenzylamino)- 5 -Fluoro-3,6-dimethyl-9-oxo-5-oxa-2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidine-4(3,2)-pyridinecyclononane-1 6 -yl)-1H-pyrrole-2-carbonitrile (229 mg, 0.36 mmol) was dissolved in DCM (10.0 mL), and trifluoromethanesulfonic acid (150.0 mg, 1.0 mmol) was added at 0°C, and the reaction was maintained at 0°C for 2 h. After the reaction was complete, TEA was slowly added at 0°C to adjust the pH to 7-8. The reaction solution was concentrated under reduced pressure, and the residue was isolated by high pressure preparative separation to obtain Compound 45 of Example 45.

[0624] MS (ESI) m / z 462.17 (M+H) +

[0625] 1 H NMR (400MHz, DMSO-d6) δ12.59 (s, 1H), 9.79 (d, J = 8.8Hz, 1H), 8.03 (s, 1H), 8.01 –7.90(m,2H),7.69–7.52(m,1H),7.26(t,J=1.2Hz,1H),6.99(t,J=1.2Hz,1H), 6.85(s,2H),6.58(d,J=7.2Hz,1H),5.59–5.37(m,1H),5.04–4.98(m,1H),4.01 –3.95(m,1H),3.14–3.08(m,1H),1.46(d,J=6.0Hz,3H),1.40(d,J=7.2Hz,3H).

[0626] Example 46: (1 3 E,1 4E,3R,6S)-1 7 -Amino-4 5 -fluoro-3,6-dimethyl-1 6 The preparation route of -(1-methyl-1H-pyrazol-4-yl)-5-oxa-2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidine-4(3,2)-pyridine heterocycle 9-one (Compound 46) is as follows:

[0627]

[0628] Step 1: (1 3 E,1 4 E,3R,6S)-1 7 -(dibenzylamino)-4-(dibenzylamino)- 5 -fluoro-3,6-dimethyl-1 6 Preparation of -(1-methyl-1H-pyrazol-4-yl)-5-oxa-2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidin-4(3,2)-pyridine heterocycle 9-one (Compound 46A)

[0629]

[0630] (1 3 E,1 4 E,3R,6S)-1 6 -Br-1 7 -(dibenzylamino)-4-(dibenzylamino)- 5 1-Fluoro-3,6-dimethyl-5-oxa-2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidin-4(3,2)-pyridineheterocycle-9-one (230 mg, 0.36 mmol), (1-methyl-1H-pyrazol-4-yl)boronic acid (68.9 mg, 0.55 mmol), anhydrous cesium carbonate (352 mg, 1.08 mmol), tetrakistriphenylphosphine palladium (41.6 mg, 0.036 mmol), dioxane (5 mL), and water (1.25 mL) were placed in a reaction flask and heated to 105°C under argon for 16 hours. After the reaction was complete, the reaction solution was concentrated under reduced pressure, and the residue was separated and purified by silica gel column chromatography to obtain compound 46A.

[0631] MS (ESI) m / z 632.29 (M+H) +

[0632] Step 2: (1 3 E,1 4 E,3R,6S)-1 7 -Amino-4 5 -fluoro-3,6-dimethyl-1 6Preparation of -(1-methyl-1H-pyrazol-4-yl)-5-oxa-2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidine-4(3,2)-pyridine heterocycle 9-one (Compound 46)

[0633]

[0634] (1 3 E,1 4 E,3R,6S)-1 7 -(dibenzylamino)-4-(dibenzylamino)- 5 -fluoro-3,6-dimethyl-1 6 -(1-Methyl-1H-pyrazol-4-yl)-5-oxa-2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidine-4(3,2)-pyridineheterocyclyl-9-one (190 mg, 0.3 mmol) was dissolved in DCM (6.0 mL), and trifluoromethanesulfonic acid (150.0 mg, 1.0 mmol) was added at -20°C. The reaction was maintained at -20°C for 2 h. After the reaction was complete, TEA was slowly added at 0°C to adjust the pH to 7-8. The reaction solution was concentrated under reduced pressure, and the residue was isolated by high pressure preparative separation to obtain Compound 46 of Example 46.

[0635] MS (ESI) m / z 452.20 (M+H) +

[0636] 1 H NMR (400MHz, DMSO-d6) δ9.80(d,J=8.8Hz,1H),8.03(s,1H),7.97–7.94(m,2H),7.82(s,1H),7.49(s,1H),6.87(s,2H),6.66(d,J=7.6Hz,1 H),5.49–5.45(m,1H),5.04–4.98(m,1H),4.02–3.96(m,1H),3.94(s,3H),3.19–3.05(m,1H),1.45(d,J=6.1Hz,3H),1.41(d,J=7.1Hz,3H).

[0637] Example 47: (1 3 E,1 4 E,3R,6S)-1 7 -Amino-4 5 -fluoro-3,6-dimethyl-1 6Preparation of -(4-(1-methyl-5-oxo-1,5-dihydro-4H-1,2,4-triazol-4-yl)phenyl)-5-oxa-2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidin-4(3,2)-pyridine heterocycle 9-one (Compound 47)

[0638]

[0639] Substituting 2-methyl-4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-2,4-dihydro-3H-1,2,4-triazol-3-one for (1-methyl-1H-pyrazol-4-yl)boric acid in step 1 of Example 46, the same preparation method as in Example 46 was used to obtain compound 47 of Example 47.

[0640] MS (ESI) m / z 545.2 (M+H) +

[0641] 1 H NMR(400MHz,DMSO-d6)δ9.85–9.74(m,1H),8.61(s,1H),8.07(s,1H),7.99–7 .96(m,2H),7.95(brs,1H),7.93–7.90(m,1H),7.49(s,2H),6.83(s,2H),6.5 8(d,J=7.6Hz,1H),5.53–5.49(m,1H),5.02–4.99(m,1H),4.03–3.96(m,1H), 3.45(s,3H),3.20–3.02(m,1H),1.46(d,J=6.0Hz,3H),1.38(d,J=7.2Hz,3H).

[0642] Example 48: (1 3 E,1 4 E,3R,6S)-1 7 -Amino-1 6 -(2-ethoxy-1H-benzo[d]imidazol-5-yl)-4-nitropropene 5 Preparation of 3,6-dimethyl-5-oxa-2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidine-4(3,2)-pyridine heterocycle 9-one (Compound 48)

[0643]

[0644] Substituting tert-butyl 2-ethoxy-5-(4,4,5,5-tetramethyl-1,3,2-dioxolane)-1H-benzo[d]imidazole-1-carboxylate for (1-methyl-1H-pyrazol-4-yl)boronic acid in step 1 of Example 46, the same preparation method as in Example 46 was used to obtain Compound 48 of Example 48.

[0645] MS (ESI) m / z 532.2 (M+H) +

[0646] 1 H NMR (400MHz, DMSO-d6) δ11.98(d,J=5.7Hz,1H),9.86(d,J=8.9Hz,1H),8.06(s,1H),7.98(d,J= 2.8Hz,1H),7.95–7.80(m,1H),7.56(t,J=6.8Hz,1H),7.46–7.33(m,1H),7.05–6.90(m,1H),6.6 3(s,1H),6.60–6.54(m,1H),6.46–6.35(m,1H),5.56–5.48(m,1H),5.06–4.97(m,1H),4.56–4.5 1(m,2H),4.04–3.97(m,1H),3.18–3.07(m,1H),1.50–1.37(m,6H),1.36(dd,J=7.4,4.1Hz,3H).

[0647] Example 49: (1 3 E,1 4 E,3R,6S)-1 7 -Amino-4 5 -fluoro-3,6-dimethyl-1 6 Preparation of -(4-(3-methyl-5-oxo-1,2,4-oxadiazol-4(5H)-yl)phenyl)-5-oxa-2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidin-4(3,2)-pyridine heterocycle 9-one (Compound 49)

[0648]

[0649] Substituting 3-methyl-4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-1,2,4-oxadiazol-5(4H)-one for (1-methyl-1H-pyrazol-4-yl)boronic acid in step 1 of Example 46, the same preparation method as in Example 46 was used to obtain Compound 49 of Example 49.

[0650] MS (ESI) m / z 546.2 (M+H)+

[0651] 1 H NMR (400MHz, DMSO-d6) δ9.75(d,J=8.8Hz,1H),8.08(s,1H),7.97(m,2H),7.73(d,J=8.4Hz,2H),7.53(d,J=8.4Hz,2H),6.91(brs,2H),6.58(d,J=7 .2Hz,1H),5.54–5.49(m,1H),5.07–5.02(m,1H),4.03–3.96(m,1H),3.20 –3.05(m,1H),2.28(s,3H),1.47(d,J=6.0Hz,3H),1.39(d,J=7.2Hz,3H).

[0652] Example 50: (1 3 E,1 4 E,3R,6S)-1 7 -Amino-1 6 -(4-(dimethylamino)-3,5-difluorophenyl)-4 5 Preparation of 3,6-dimethyl-5-oxa-2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidine-4(3,2)-pyridine heterocycle 9-one (Compound 50)

[0653]

[0654] Substituting 2,6-difluoro-N,N-dimethyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline for (1-methyl-1H-pyrazol-4-yl)boronic acid in step 1 of Example 46, the same preparation method as in Example 46 was used to obtain Compound 50 of Example 50.

[0655] MS (ESI) m / z 527.2 (M+H) +

[0656] 1H NMR (400MHz, DMSO-d6) δ9.78(d,J=8.9Hz,1H),8.06(s,1H),7.98(d,J=3.0Hz,1H),7.91(dd,J=9.1,3.1Hz,1H),6.98(d,J=9.7Hz,4H),6.69(d, J=7.5Hz,1H),5.52–5.49(m,1H),5.03–4.98(m,1H),4.02–3.96(m,1H),3.14–3.09(m,1H),2.92(d,J=1.7Hz,6H),1.43(dd,J=25.2,6.6Hz,6H).

[0657] Example 51: (1 3 E,1 4 E,3R,6S)-1 7 -Amino-1 6 -(2-ethoxy-1-methyl-1H-benzo[d]imidazol-5-yl)-4-nitropropene 5 Preparation of 3,6-dimethyl-5-oxa-2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidine-4(3,2)-pyridine heterocycle 9-one (Compound 51)

[0658]

[0659] Substituting 2-ethoxy-1-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-benzo[d]imidazole for (1-methyl-1H-pyrazol-4-yl)boronic acid in step 1 of Example 46, the same preparation method as in Example 46 was used to obtain Compound 51 of Example 51.

[0660] MS (ESI) m / z 546.2 (M+H) +

[0661] 1H NMR (400MHz, DMSO-d6) δ9.86(d,J=9.0Hz,1H),8.06(s,1H),7.97(d,J=3.0Hz,1H),7.95–7. 81(m,1H),7.57–7.54(m,1H),7.38–7.30(m,1H),7.09–6.98(m,1H),6.59(s,2H),6.36(t,J =7.7Hz,1H),5.52(d,J=6.9Hz,1H),5.02(q,J=5.5Hz,1H),4.62–4.56(m,2H),4.04–3.97(m ,1H),3.63(s,3H),3.13(dd,J=13.1,10.2Hz,1H),1.48–1.43(m,6H),1.35(t,J=6.4Hz,3H).

[0662] Example 52: (1 3 E,1 4 E,3R,6S)-1 7 -Amino-1 6 -(2,4-difluorophenyl)-4 5 The preparation route of 1-fluoro-3,6-dimethyl-5-oxa-2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidine-4(3,2)-pyridine heterocycle 9-one (Compound 52) is as follows:

[0663]

[0664] Step 1: Preparation of ethyl 2-(2,4-difluorophenyl)acetate (Compound 52A)

[0665]

[0666] 2-(2,4-Difluorophenyl)acetic acid (24.00 g, 0.14 mol), ethanol (200 mL), and concentrated sulfuric acid (1.5 mL) were added to a reaction flask and reacted at 80°C overnight. After the reaction was complete, the reaction solution was concentrated under reduced pressure, and the residue was separated and purified by silica gel column chromatography to obtain compound 52A.

[0667] MS (ESI) m / z 201.2 (M+H) + .

[0668] Step 2: Preparation of diethyl 2-(2,4-difluorophenyl)malonate (Compound 52B)

[0669]

[0670] Ethyl 2-(2,4-difluorophenyl)acetate (23.0 g, 0.115 mol) was dissolved in tetrahydrofuran at room temperature. Sodium hydride (9.2 g, 0.23 mol) was added portionwise at 0°C and stirred for 15 minutes. Diethyl carbonate (27.2 g, 0.23 mol) was then slowly added dropwise. After the addition was complete, the system was heated to 60°C and allowed to react overnight. After completion of the reaction, the mixture was quenched with water and extracted with ethyl acetate. The organic phase was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to yield Compound 52B.

[0671] MS (ESI) m / z 273.2 (M+H) + .

[0672] Step 3: Preparation of ethyl 6-(2,4-difluorophenyl)-5,7-dihydroxypyrazolo[1,5-a]pyrimidine-3-carboxylate (Compound 52C)

[0673]

[0674] Diethyl 2-(2,4-difluorophenyl)malonate (18.6 g, 68.32 mmol), ethyl 5-amino-1H-pyrazole-4-carboxylate (11.6 g, 75.15 mmol), and tri-n-butylamine (15.2 g, 82.0 mmol) were added to a reaction flask and reacted at 180°C for 10 hours. After the reaction was complete, the organic phase was concentrated under reduced pressure to obtain crude compound 52C, which was directly used for the next step without purification.

[0675] MS (ESI) m / z 336.2 (M+H) + .

[0676] Step 4: Preparation of ethyl 5,7-dichloro-6-(2,4-difluorophenyl)pyrazolo[1,5-a]pyrimidine-3-carboxylate (Compound 52D)

[0677]

[0678] Ethyl 6-(2,4-difluorophenyl)-5,7-dihydroxypyrazolo[1,5-a]pyrimidine-3-carboxylate (21.0 g, 62.64 mmol), phosphorus pentachloride (14.35 g, 68.9 mmol), and phosphorus oxychloride (200 mL) were added to a reaction flask and reacted at 100°C overnight. After the reaction was complete, the organic phase was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to obtain Compound 52D.

[0679] MS (ESI) m / z 373.1 (M+H) +

[0680] Step 5: Preparation of ethyl 5-chloro-6-(2,4-difluorophenyl)-7-(dibenzylamino)pyrazolo[1,5-a]pyrimidine-3-carboxylate (Compound 52E)

[0681]

[0682] Ethyl 5,7-dichloro-6-(4-chloro-2-fluorophenyl)pyrazolo[1,5-a]pyrimidine-3-carboxylate (6.4 g, 17.2 mmol) and N,N-diisopropylethylamine (6.67 g, 51.6 mmol) were dissolved in DCM. Dibenzylamine (6.79 g, 34.4 mmol) was added dropwise. After the addition was complete, the mixture was heated to 30°C and stirred overnight. After the reaction was complete, the organic phase was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to obtain Compound 52E.

[0683] MS (ESI) m / z 533.1 (M+H) + .

[0684] Step 6: Preparation of ethyl 5-(((R)-1-(2-(((S)-1-((tert-butoxycarbonyl)amino)propan-2-yl)oxy)-5-fluoropyridin-3-yl)ethyl)amino)-7-(dibenzylamino)-6-(2,4-difluorophenyl)pyrazolo[1,5-a]pyrimidine-3-carboxylate (Compound 52F)

[0685]

[0686] Ethyl 5-chloro-6-(2,4-difluorophenyl)-7-(dibenzylamino)pyrazolo[1,5-a]pyrimidine-3-carboxylate (4.0 g, 7.51 mmol), tert-butyl ((S)-2-((3-((R)-1-aminoethyl)-5-fluoropyridin-2-yl)oxy)propyl)carbamate (2.82 g, 9.01 mmol), n-butanol (20.0 mL), and N,N-diisopropylethylamine (2.91 g, 22.53 mmol) were added to a reaction flask and heated to 120°C for 24 hours. After the reaction was complete, the organic phase was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to obtain Compound 52F.

[0687] MS (ESI) m / z 810.1 (M+H) + .

[0688] Step 7: (1 3 E,1 4 E,3R,6S)-1 7 -(dibenzylamino)-1 6 -(2,4-difluorophenyl)-4 5Preparation of 3,6-dimethyl-5-oxa-2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidine-4(3,2)-pyridine heterocycle 9-one (Compound 52G)

[0689]

[0690] Ethyl 5-(((R)-1-(2-(((S)-1-((tert-butoxycarbonyl)amino)propan-2-yl)oxy)-5-fluoropyridin-3-yl)ethyl)amino)-7-(dibenzylamino)-6-(2,4-difluorophenyl)pyrazolo[1,5-a]pyrimidine-3-carboxylate (810.0 mg, 1.0 mmol) was dissolved in a mixed solution of methanol and THF, followed by the addition of LiOH (480 mg, 20.0 mmol) aqueous solution. After the addition was complete, the reaction system was heated to 60°C and stirred for 16 h. The reaction system was then cooled to 0°C, the pH adjusted to 2-3 with 2N hydrochloric acid, and extracted three times with DCM. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. A solution of hydrochloric acid in 1,4-dioxane was added, and the mixture was stirred at room temperature for 1 hour. The reaction system was concentrated to dryness, and DCM and DMF were added, followed by DIPEA (1.93 g, 15.0 mmol) and FDPP (1.92 g, 5.0 mmol). After the additions were complete, the mixture was stirred at room temperature for 16 hours. After completion of the reaction, the reaction system was concentrated to dryness under reduced pressure, and the residue was purified by silica gel column chromatography to obtain Compound 52G.

[0691] MS (ESI) m / z 664.2 (M+H) +

[0692] Step 8: (1 3 E,1 4 E,3R,6S)-1 7 -Amino-1 6 -(2,4-difluorophenyl)-4 5 Preparation of 3,6-dimethyl-5-oxa-2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidine-4(3,2)-pyridine heterocycle 9-one (Compound 52)

[0693]

[0694] (1 3 E,1 4 E,3R,6S)-1 7 -(dibenzylamino)-1 6 -(2,4-difluorophenyl)-4 51-Fluoro-3,6-dimethyl-5-oxa-2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidine-4(3,2)-pyridineheterocyclyl-9-one (200.0 mg, 0.3 mmol) was dissolved in DCM (6.0 mL), and trifluoromethanesulfonic acid (150.0 mg, 1.0 mmol) was added at -20°C. The reaction was maintained at -20°C for 2 h. After the reaction was complete, TEA was slowly added at 0°C to adjust the pH to 7-8. The reaction solution was concentrated under reduced pressure, and the residue was isolated by high pressure preparative separation to obtain Compound 52 of Example 52.

[0695] MS (ESI) m / z 484.4 (M+H) +

[0696] 1 H NMR (400MHz, DMSO-d6) δ9.84-9.75 (m, 1H), 8.09 (s, 1H), 7.98 (dd, J = 3.1, 1. 9Hz,1H),7.82(m,1H),7.51-7.35(m,2H),7.35-7.21(m,1H),7.07(d,J=3.8H z,2H),6.71(dd,J=12.2,7.5Hz,1H),5.52(m,1H),5.08-4.95(m,1H),4.09-3 .95(m,1H),3.12(m,1H),1.47(dd,J=6.1,1.3Hz,3H),1.38(d,J=7.1Hz,3H).

[0697] Example 53: (1 3 E,1 4 E,3R,6S)-1 7 -Amino-1 6 -(4-chloro-2-fluorophenyl)-4 5 Preparation of 3,6-dimethyl-5-oxa-2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidine-4(3,2)-pyridine heterocycle 9-one (Compound 53)

[0698]

[0699] Substituting 2-(4-chloro-2-fluorophenyl)acetic acid for 2-(2,4-difluorophenyl)acetic acid in step 1 of Example 52, the same preparation method as in Example 52 was used to obtain Compound 53 of Example 53.

[0700] MS (ESI) m / z 500.2 (M+H) +

[0701] 1H NMR (400MHz, DMSO) δ9.77(d,J=9.1Hz,1H),8.07(s,1H),7.97(dd,J=2.9,2.1Hz,1 H),7.83–7.75(m,1H),7.69–7.60(m,1H),7.49–7.32(m,2H),7.11(d,J=4.8Hz,2H ),6.75(dd,J=11.4,7.5Hz,1H),5.50(dd,J=6.9,5.0Hz,1H),5.05–4.94(m,1H),4 .06–3.92(m,1H),3.16–3.04(m,1H),1.46(d,J=6.1Hz,3H),1.36(d,J=7.1Hz,3H).

[0702] Example 54: (1 3 E,1 4 E,3R,6S)-1 7 -Amino-4 5 -fluoro-3,6-dimethyl-1 6 Preparation of -(pyrrolidin-1-yl)-5-oxa-2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidin-4(3,2)-pyridine heterocycle 9-one (Compound 54)

[0703]

[0704] Using diethyl 2-(pyrrolidin-1-yl)malonate instead of diethyl 2-(2,4-difluorophenyl)malonate in step 3 of Example 52, the same preparation method as Example 52 was used to obtain Compound 54 of Example 54. MS (ESI) m / z 441.22 (M+H) +

[0705] 1 H NMR (400MHz, DMSO-d6) δ9.87(d,J=9.2Hz,1H),8.04(dd,J=9.2,2.8Hz,1H),8.00(s,1H),7.96(d,J=2.8Hz,1H),7.05(s,2H),6.95(d,J=7.6Hz ,1H),5.55–5.36(m,1H),5.00–4.95(m,1H),3.99–3.93(m,1H),3.09–3 .00(m,5H),2.02(s,4H),1.53(d,J=7.2Hz,3H),1.45(d,J=6.0Hz,3H).

[0706] Example 55: (1 3 E,1 4E,3R,6S)-1 7 -Amino-4 5 -fluoro-3,6-dimethyl-1 6 Preparation of -(3-methylpyrrolidin-1-yl)-5-oxa-2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidin-4(3,2)-pyridine heterocycle 9-one (Compound 55)

[0707]

[0708] Substituting diethyl 2-(3-methylpyrrolidin-1-yl)malonate for diethyl 2-(2,4-difluorophenyl)malonate in step 3 of Example 52, the same preparation method as in Example 52 was used to obtain Compound 55 of Example 55.

[0709] MS (ESI) m / z 455.2 (M+H) +

[0710] 1 H NMR(400MHz,DMSO-d6)δ9.91–9.82(m,2H),8.06(m,2H),8.01(s,2H),7.97(d,J=2.8Hz,2H),7 .01(d,J=6.0Hz,4H),6.89–6.83(m,2H),5.50–5.38(m,2H),5.01–4.97(m,2H),3.99–3.92(m, 2H),3.17–3.00(m,8H),2.84–2.80(m,1H),2.73(t,J=7.8Hz,1H),2.63–2.51(m,2H),2.15–2. 00(m,2H),1.79–1.70(m,2H),1.53(d,J=7.2Hz,6H),1.45(d,J=6.0Hz,6H),1.18–1.03(m,6H).

[0711] Example 56: (1 3 E,1 4 E,3R,6S)-1 7 -Amino-4 5 -fluoro-3,6-dimethyl-1 6 Preparation of -(1H-pyrrol-1-yl)-5-oxa-2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidin-4(3,2)-pyridine heterocycle 9-one (Compound 56)

[0712]

[0713] Substituting diethyl 2-(1H-pyrrol-1-yl)malonate for diethyl 2-(2,4-difluorophenyl)malonate in step 3 of Example 52, the same preparation method as in Example 52 was used to obtain Compound 56 of Example 56.

[0714] MS (ESI) m / z 437.2 (M+H) +

[0715] 1 H NMR (400MHz, DMSO-d6) δ9.77(d,J=8.8Hz,1H),8.11(d,J=2.0Hz,1H),7.98(d,J=2 .8Hz,1H),7.92(dd,J=9.2,2.4Hz,1H),6.89(s,2H),6.80(d,J=10.0Hz,2H),6.49( d,J=7.2Hz,1H),6.39(s,2H),5.48(t,J=7.2Hz,1H),5.10–4.96(m,1H),3.98(t,J= 10.8Hz, 1H), 3.12 (t, J = 12.0Hz, 1H), 1.46 (d, J = 6.4Hz, 3H), 1.40 (d, J = 7.2Hz, 3H).

[0716] Example 57: (1 3 E,1 4 E,3R,6S)-1 7 -Amino-4 5 -fluoro-3,6-dimethyl-1 6 The preparation route of -(1H-pyrrol-1-yl)-5-oxa-2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidine-4(3,2)-pyridine heterocycle 9-one (Compound 57) is as follows:

[0717]

[0718] Step 1: Preparation of methyl 5-((R)-1-(2-((S)-1-((tert-Butoxycarbonyl)amino)propan-2-yloxy)-5-fluoropyridin-3-yl)ethyl)amino)-7-(dibenzylamino)-6-(1,2,5-trimethyl-1H-pyrrol-3-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylate (Compound 57A)

[0719]

[0720] Under argon protection, methyl 6-bromo-5-(((R)-1-(2-(((S)-1-((tert-butoxycarbonyl)amino)propan-2-yl)oxy)-5-fluoropyridin-3-yl))-2-methylpropyl)amino)-7-(dibenzylamino)pyrazolo[1,5-a]pyrimidine-3-carboxylate (1.0 g, 1.29 mmol), 1,2,5-trimethyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrole (360 mg, 1.54 mmol), potassium phosphate (820 mg, 3.86 mol), Pd(dppf)Cl2 (94 mg, 0.13 mol), 1,4-dioxane (40 mL), and water (8 mL) were added to a reaction flask and reacted at 80°C overnight. After the reaction was completed, the reaction solution was concentrated under reduced pressure, and the residue was separated and purified by silica gel column chromatography to obtain compound 57A.

[0721] MS (ESI) m / z 791.4 (M+H) +

[0722] Step 2: (1 3 E,1 4 E,3R,6S)-1 7 -(dibenzylamino)-4-(dibenzylamino)- 5 -fluoro-3,6-dimethyl-1 6 Preparation of -(1,2,5-trimethyl-1H-pyrrol-3-yl)-5-oxa-2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidin-4(3,2)-pyridine heterocycle 9-one (Compound 57B)

[0723]

[0724] 5-((R)-1-(2-((S)-1-((tert-Butoxycarbonyl)amino)propan-2-yloxy)-5-fluoropyridin-3-yl)ethyl)amino)-7-(dibenzylamino)-6-(1,2,5-trimethyl-1H-pyrrol-3-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylic acid methyl ester (790.0 mg, 1.0 mmol) was dissolved in a mixed solution of methanol and THF, followed by the addition of LiOH (480 mg, 20.0 mmol) aqueous solution. After the addition, the reaction system was heated to 60°C and stirred for 16 h. The reaction system was then cooled to 0°C, the pH adjusted to 2-3 with 2N hydrochloric acid, and extracted three times with DCM. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. A solution of hydrochloric acid in 1,4-dioxane was added, and the mixture was stirred at room temperature for 1 hour. The reaction system was concentrated to dryness, and DCM and DMF were added, followed by DIPEA (1.93 g, 15.0 mmol) and FDPP (1.92 g, 5.0 mmol). After the additions were complete, the mixture was stirred at room temperature for 16 hours. After completion of the reaction, the reaction system was concentrated to dryness under reduced pressure, and the residue was purified by silica gel column chromatography to obtain compound 57B.

[0725] MS (ESI) m / z 659.2 (M+H) +

[0726] Step 3: (1 3 E,1 4 E,3R,6S)-1 7 -Amino-4 5 -fluoro-3,6-dimethyl-1 6 Preparation of -(1,2,5-trimethyl-1H-pyrrol-3-yl)-5-oxa-2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidin-4(3,2)-pyridine heterocycle 9-one (Compound 57)

[0727]

[0728] (1 3 E,1 4 E,3R,6S)-1 7 -(dibenzylamino)-4-(dibenzylamino)- 5 -fluoro-3,6-dimethyl-1 6-(1,2,5-Trimethyl-1H-pyrrol-3-yl)-5-oxa-2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidin-4(3,2)-pyridineheterocycle-9-one (197.0 mg, 0.3 mmol) was dissolved in DCM (6.0 mL), and trifluoromethanesulfonic acid (150.0 mg, 1.0 mmol) was added at -20°C. The reaction was maintained at -20°C for 2 h. After the reaction was complete, TEA was slowly added at 0°C to adjust the pH to 7-8. The reaction solution was concentrated under reduced pressure, and the residue was isolated by high pressure preparative separation to obtain Compound 57 of Example 57.

[0729] MS (ESI) m / z 479.1 (M+H) +

[0730] 1 H NMR (400MHz, DMSO-d6) δ9.90–9.83(m,1H),8.05–8.01(m,1H),8.01–7.91(m,2H),6.47(d,J=16.1Hz,2H),6.42–6.36(m,1H),5.49(dd,J=9.8,4.4 Hz,1H),5.02(s,1H),3.99(s,1H),3.47(s,3H),3.12(t,J=11.6Hz,1H), 2.36–2.18(m,3H),1.97(d,J=37.0Hz,3H),1.45(dd,J=13.3,6.6Hz,6H).

[0731] Example 58: (1 3 E,1 4 E,3R,6S)-1 7 -Amino-1 6 -(4-Fluorophenyl)-4 5 Preparation of -fluoro-3,6-dimethyl-5-oxa-2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidine-4(3,2)-pyridine heterocycle 9-one (Compound 58):

[0732]

[0733] Substituting ethyl 2-(4-fluorophenyl)acetate for ethyl 2-(2,4-difluorophenyl)acetate in step 2 of Example 52, the same preparation method as in Example 52 was used to obtain Compound 58 of Example 58.

[0734] MS (ESI) m / z 466.2 (M+H) +

[0735] 1H NMR (400MHz, DMSO) δ9.81(d,J=8.7Hz,1H),8.07(s,1H),7.97(d,J=3.0Hz,1H),7.88(dd,J=9.1,3.0Hz,1H),7.40(d,J=8.1Hz,4H),6.81(s,2H),6. 48(d,J=7.5Hz,1H),5.50(dd,J=13.7,6.5Hz,1H),5.06–4.95(m,1H),4.0 3–3.97(m,1H),2.89(s,1H),1.47(d,J=6.1Hz,3H),1.37(d,J=7.1Hz,3H).

[0736] Biological test data

[0737] Unless otherwise specified, the experimental materials, reagents, procedures, and methods used in the following active examples can be obtained from commercial sources or easily known or prepared based on existing technologies. The structure of LOX305 is as follows: It is commercially available and has the following structure: It can be purchased commercially.

[0738] Experimental Example 1: In vitro kinase activity test of the compounds of the present invention

[0739] Purpose of the experiment

[0740] IC of the compound 50 The half-inhibitory concentration (CI) value was used as an indicator to evaluate the inhibitory effect of the compound on RET wild-type, RET mutant, BTK and / or mutant BTK kinases.

[0741] Experimental methods

[0742] Mobility shift assays were used to test the inhibitory activity of compounds against the following kinases: wild-type RET, mutant RET, BTK, and / or mutant BTK. Compounds were tested at a starting concentration of 1000 nM, with 3-fold dilutions and 10 concentrations in a single-well assay.

[0743] Reagents and consumables:

[0744] Reagent name Availability Item No. batch number R is called ET Carna 08-15 13CBS-0134F RETG810R Proqinase 1724-0000-1 002 RET V804M signalchem R02-12GG Y985- BTK Carna 08-18 14CBS-0619Q BTK C481S Carna 08-54 14CBS-0633H Kinase substrate 2 GL 19086 P200807-YS1908 Kinase substrate 4 GL 11239 P171211-XY1123 Kinase substrate 22 GL 11239 P200403-CL1123 DMS Sigm D8418-1L SHBG3288V 384-well plate Cornin 3573 12619003 384-well plate Cornin 3575 BC 31316039 MgCl2 Sigma M1028 / ATP Prome V910B / DTT Sigma D0632 /

[0745] instrument:

[0746] Centrifuge (manufacturer: Eppendorf, model: 5430 )

[0747] Microplate reader (Manufacturer: Perkin Elmer, Model: Caliper EZ Reader )

[0748] Echo 550 (Manufacturer: Labcyte, Model: Echo 550)

[0749] Preparation of kinase reaction buffer:

[0750] 20 mM hydroxyethylpiperazine ethanesulfonic acid (Hepes) (pH 7.5) buffer, 10 mM MgCl2, 1 mM ethylene glycol bisaminoethyl ether tetraacetic acid (EGTA), 0.02% polyoxyethylene lauryl ether (Brij35), 0.02 mg / ml N, O-bis(trimethylsilyl)acetamide (BSA), 0.1 mM Na3VO4, 2 mM dithiothreitol (DTT), 1% DMSO.

[0751] Compound:

[0752] The test compound was dissolved in 100% dimethyl sulfoxide (DMSO) system, prepared to 10 mM and stored in a nitrogen cabinet in the dark.

[0753] Reaction conditions:

[0754] ATP (μM) Reaction time RET 16 60min RET G810R 201 4h RET V804M 5.4 60min BTK 71 30min BTK C481S 90 30min

[0755] Kinase reaction process:

[0756] (1) Prepare 1× kinase reaction buffer.

[0757] (2) Preparation of compound concentration gradient: The starting concentration of the compound to be tested was 1000 nM. The compound was diluted to 100 times the final concentration of 100% dimethyl sulfoxide (DMSO) solution in a 384-well plate. The compound was diluted accurately 3-fold in kinase buffer, 10 times the final concentration, to a final concentration of 0.0508 nM. The compound was diluted to 100 times the final concentration of 100% DMSO solution in a 384 source plate. 250 nL of the compound at 100 times the final concentration was transferred to the destination 384-well plate using an Echo 550 dispenser. 250 nL of DMSO was added to the positive and negative control wells.

[0758] (3) Prepare a kinase solution with a final concentration of 2.5 times using 1× kinase buffer.

[0759] (4) Add 10 μL of kinase solution at 2.5 times the final concentration to the compound wells and positive control wells respectively; add 10 μL of 1× kinase buffer to the negative control wells.

[0760] (5) Centrifuge at 1000 rpm for 30 seconds, shake the reaction plate to mix, and incubate at room temperature for 10 minutes.

[0761] (6) Prepare a mixed solution of adenosine triphosphate (ATP) and kinase substrate with a final concentration of 25 / 15 using 1× kinase buffer.

[0762] (7) Add 15 μL of a mixed solution of adenosine triphosphate (ATP) and substrate at 25 / 15 times the final concentration to start the reaction.

[0763] (8) Centrifuge the 384-well plate at 1000 rpm for 30 seconds, shake to mix, and incubate at room temperature for 30-240 minutes.

[0764] (9) After stopping the kinase reaction, centrifuge at 1000 rpm for 30 seconds and vortex to mix.

[0765] (10) Read the conversion rate using Caliper EZ Reader.

[0766] Data Analysis

[0767] Calculation formula

[0768]

[0769] Where: Conversion%_sample is the conversion rate reading of the sample; Conversion%_min is the mean value of the negative control wells, representing the conversion rate reading of the wells without enzyme activity; Conversion%_max is the mean value of the positive control wells, representing the conversion rate reading of the wells without compound inhibition.

[0770] Fitting dose-effect curve

[0771] The logarithmic value of the concentration was used as the X-axis and the percentage inhibition rate was used as the Y-axis. Graphpad 6.0 analysis software was used to fit the dose-effect curve to obtain the IC value of each compound on the enzyme activity. 50 value.

[0772] The experimental results are shown in Table 1:

[0773] Table 1

[0774]

[0775]

[0776] “-” means not detected.

[0777] Example 2 Cell Proliferation Inhibition

[0778] 2.1 TMD 8, Ba / F3-KIF5B-RET, Ba / F3-KIF5B-RET-V804M, and Ba / F3-KIF5B-RET-G810R cell proliferation inhibition

[0779] Experimental methods:

[0780] (1) Day 0: Cell plating

[0781] Count cells using an automated counter and dilute to the desired concentration based on the seeding density. TMD 8 cells were seeded at a density of 3000 cells per well, while Ba / F3-KIF5B-RET, Ba / F3-KIF5B-RET-V804M, and Ba / F3-KIF5B-RET-G810R cells were seeded at a density of 2000 cells per well. 100 μL of cell suspension was added to each well of a 96-well plate. Culture medium was used as a background control (min). The plates were incubated in a 37°C, 5% CO2 incubator overnight.

[0782] (2) Day 1: Drug administration

[0783] Ten dosing dose groups were set for each compound, with an initial concentration of 10 μmol / L and 3-fold dilution.

[0784] Prepare a 200x stock solution by dissolving the compound in dimethyl sulfoxide (DMSO). Upon use, dilute the stock solution to 3x the final concentration with culture medium: add 3 μl of the 200x stock solution to 197 μl of culture medium. Add 50 μl of this diluted mixture to a 96-well plate and incubate in a 37°C, 5% CO2 incubator for 72 hours.

[0785] (3) Day 4: Data Testing

[0786] Before the test, place the 96-well plate at room temperature and add 40 μl of CellTiter-Glo to each well. @ Reagents; mix on a plate shaker for 2 minutes to lyse cells. Incubate at room temperature for 60 minutes and measure the fluorescence signal after it stabilizes.

[0787] (4) Data Analysis

[0788] GraphPad Prism 5 software was used to draw sigmoidal dose-response curves and calculate IC50 values.

[0789] The inhibition rate (IR) of the test compound was calculated using the following formula:

[0790] IR (%)=(1−(RLU compound−RLU blank control) / (RLU vehicle control−RLU blank control)×100%.

[0791] The inhibition rates of compounds at different concentrations were calculated in Excel, and then GraphPad Prism software was used to draw inhibition curves and calculate relevant parameters, including minimum inhibition rate, maximum inhibition rate and IC50.

[0792] RLU: relative light unit, that is, fluorescence value.

[0793] 2.2 Inhibition of DoHH-2 Cell Proliferation

[0794] Experimental methods:

[0795] (1) Day 1: Cell plating and test article addition

[0796] a) 96-well plate, DoHH-2 cells seeded at a density of 1.5 x 10 4 cells per well. Add 100 μl of culture medium to each well;

[0797] b) First, the stock solution of the compound was diluted from 10 mM to 1000, 300, 100, 30, 10, 3, 1, 0.3, and 0.1 μM. The stock solution of panobinostat was diluted from 100 μM to 10, 3, 1, 0.3, 0.1, 0.03, 0.01, 0.003, and 0.001 μM, with a DMSO content of 100%; c) After thorough mixing, 4.6 μL was removed with an electric dispenser and added to 225.4 μL of culture medium, resulting in a 50-fold dilution and a DMSO content of 2%;

[0798] d) Add 100 μL of (c) to each of the two replicate wells (a) to give final compound concentrations of 10, 3, 1, 0.3, 0.1, 0.03, 0.01, 0.003, and 0.001 μM, respectively. The final concentrations of panobinastat were 100, 30, 10, 3, 1, 0.3, 0.1, 0.03, and 0.01 nmol / L, in a total of 200 μL of culture medium containing 1% DMSO.

[0799] e) The cells were incubated in a 37°C, 5% CO2 incubator for 3 days before detection.

[0800] (2) Day 4: Testing

[0801] a) Leave 50 μl of culture medium in each well and add 50 μl of CTG reagent;

[0802] b) Incubate on a shaker for 5 minutes and allow to equilibrate for 5 minutes;

[0803] c) Detection using a multifunctional microplate reader.

[0804] (3) Data Analysis

[0805] a) Cell viability

[0806] Cell viability = As / Ac × 100%

[0807] As: test wells (culture medium containing cells, CTG, test compounds)

[0808] Ac: control well (containing cell culture medium, CTG, no test compound)

[0809] b)IC 50

[0810] IC 50 Fitting was performed using GraphPad Prism.

[0811] See Table 2 for specific data

[0812] Table 2

[0813]

[0814] Example 3 CYP liver microsome test

[0815] (1) Preparation, addition, incubation and detection of test samples

[0816] a) First, dilute the stock solution of the positive compound 4-fold and set the final concentration:

[0817] Furafylline (0.6, 0.2, 0.06 micromolar), Quinidine (200, 60, 20 nanomolar), Ketoconazole (0.06, 0.02, 0.006 micromolar), Sulfaphenazole (0.2, 0.06, 0.02 micromolar), Ticlopidine HCl (0.6, 0.2, 0.06 micromolar)

[0818] b) The test compound was diluted to 10, 3, and 1 μmol / L (final concentration).

[0819] c) Prepare the reaction substrate according to the protocol of each kit, aliquot 5 μL of the solution into a 384-well plate, then add 5 μL of a 4-fold diluted compound (so that the dimethyl sulfoxide content in the system is 0.2%), mix thoroughly to avoid bubble formation, and incubate at 25°C for 10 minutes;

[0820] d) Add 15 μL of the prepared reduced nicotinamide adenine dinucleotide phosphate solution and incubate at 25°C for 30 min / 45 min / 30 min / 30 min / 30 min (1A2 / 2D6 / 3A4 / 2C9 / 2C19);

[0821] e) In another white 384-well plate, add 15 μl of detection buffer. Mix 15 μl of the solution from step d with the buffer, avoiding bubbles. Incubate at 25°C in the dark for 20 minutes.

[0822] g) Detection using a multifunctional microplate reader.

[0823] (2) Data Analysis

[0824] a) Inhibition rate

[0825] Inhibition rate = [1-(Fs-Fb) / (Fc-Fb)] × 100%

[0826] Fs: test well (containing enzyme, substrate, and test compound)

[0827] Fc: Control well (contains enzyme, substrate, and no test compound)

[0828] Fb: Blank well (contains enzyme, membrane, and no test compound)

[0829] b)IC 50

[0830] IC 50 Sigma Plot 11 was used for fitting.

[0831] The results are shown in Table 3:

[0832] Table 3

[0833]

[0834] Example 4 hERG test

[0835] (1) Experimental methods

[0836] Prepare a gradient of test compounds and the positive compound E-4031 at concentrations of 30, 10, and 1 μM. Dilute hERG Tracer Red to 1 nmol / L. Add 2.5 μL of compound, 5 μL of hERG Membrane, and 2.5 μL of hERG Tracer Red to each well of a 384-well plate and incubate at 25°C for 4 hours. Detect using a multi-function microplate reader, collect data, and process them. For detailed procedures, refer to the Thermo Fisher Scientific Predictor™ hERGFluorescence Polarization Assay Kit instructions.

[0837] E-4031 was used as the positive compound to calculate the relative inhibition rate of the compound. GraphPad Prism 6 was used to process the data and the IC was calculated after the curve was drawn. 50 The results showed that compounds 22, 23, and 29 had no hERG inhibition (IC 50 >30 μM).

[0838] Example 5 Mouse PK test

[0839] a) Experimental animals

[0840] Male CD1 mice were used for the experiment, with 3 mice in each group. The animals were fasted overnight before administration and had free access to water. They were given feed 4 hours after administration.

[0841] b) Drug delivery design

[0842] The dosing schedule was: 1 mg / kg for the intravenous injection group and 5 mg / kg for the oral administration group. The dosing volume was 5 ml / kg, and the actual dosing volume was calculated based on animal body weight. PK data were collected at 0.083, 0.25, 0.5, 1, 2, 4, 8, and 24 hours after dosing.

[0843] c) In vivo evaluation

[0844] Evaluation criteria: Cage-side observation (twice daily), detailed clinical observation before randomization and on the day of dosing, and body weight recorded before dosing. If necessary, unplanned observations should be conducted and recorded.

[0845] d) Blood sample collection and processing process

[0846] At the time points specified in the experimental design, approximately 30 μL of whole blood was collected from the orbital vein of each animal and placed in an anticoagulant tube containing EDTA-K2 and mixed thoroughly by inversion several times. The collected blood sample was centrifuged at 4000 g for 5 minutes at 4°C to obtain plasma. Plasma samples were aliquoted into clean polyethylene microcentrifuge tubes and stored at -75 ± 15°C until analysis.

[0847] e) Sample analysis

[0848] All samples were analyzed by LC-MS / MS, and the concentration of the drug in the sample was determined by the standard curve method. TM ,) or other similar software to calculate pharmacokinetic parameters. If applicable plasma drug concentration-time data are available, calculate the following pharmacokinetic parameters:

[0849] CL (clearance rate)

[0850] Vd (apparent volume of distribution)

[0851] T 1 / 2 (elimination half-life)

[0852] C0 (initial concentration)

[0853] C max (peak concentration)

[0854] Tmax (Peak Time)

[0855] AUC (area under the plasma concentration-time curve)

[0856] MRT (mean residence time)

[0857] F(bioavailability)

[0858] Pharmacokinetic data were presented using descriptive statistics such as mean, standard deviation, and sample size. Calculations were performed using Microsoft Excel 2007. The results showed that both compounds 23 and 29 exhibited good exposure and oral bioavailability in mice. The PK profile of compounds 29 and 58 in mice was linear.

[0859]

[0860] Example 6 Rat PK test

[0861] a) Experimental animals

[0862] Male SD rats were used for the experiment, with 3 rats in each group. The animals were fasted overnight before administration and had free access to water. They were given feed 4 hours after administration.

[0863] b) Drug delivery design

[0864] The dosing schedule was: 1 mg / kg for intravenous administration and 10 mg / kg for oral administration. The dosing volume was 0.2 mg / mL for intravenous administration and 1 mg / mL for oral administration. The actual dosing volume was calculated based on animal body weight. PK data were collected at 0.083, 0.25, 0.5, 0.75, 1, 2, 4, 8, and 24 hours after dosing.

[0865] c) In vivo evaluation

[0866] Evaluation criteria: Cage-side observation (twice daily), detailed clinical observation before randomization and on the day of dosing, and body weight recorded before dosing. If necessary, unplanned observations should be conducted and recorded.

[0867] d) Blood sample collection and processing process

[0868] At the time points specified in the experimental design, approximately 30 μL of whole blood was collected from the orbital vein of each animal and placed in an anticoagulant tube containing EDTA-K2 and mixed thoroughly by inversion several times. The collected blood sample was centrifuged at 4000 g for 5 minutes at 4°C to obtain plasma. Plasma samples were aliquoted into clean polyethylene microcentrifuge tubes and stored at -75 ± 15°C until analysis.

[0869] e) Sample analysis

[0870] All samples were analyzed by LC-MS / MS, and the concentration of the drug in the sample was determined by the standard curve method. TM ,) or other similar software to calculate pharmacokinetic parameters. If applicable plasma drug concentration-time data are available, calculate the following pharmacokinetic parameters:

[0871] CL (clearance rate)

[0872] Vd (apparent volume of distribution)

[0873] T 1 / 2 (elimination half-life)

[0874] C0 (initial concentration)

[0875] C max (peak concentration)

[0876] T max (Peak Time)

[0877] AUC (area under the plasma concentration-time curve)

[0878] MRT (mean residence time)

[0879] F(bioavailability)

[0880] Pharmacokinetic data were described using descriptive statistics such as mean, standard deviation, and sample size. Calculations were performed using Microsoft Excel 2007. The results showed that compound 58 exhibited good exposure and oral bioavailability in rats.

[0881]

[0882] Example 7 In vivo efficacy data

[0883] 7.1 Drug Efficacy Testing in DOHH2 Xenograft Model

[0884] (1) Cell culture

[0885] DoHH-2 cells were cultured in RPMI1640 medium containing 10% fetal bovine serum. DoHH-2 cells in the exponential growth phase were collected and resuspended in PBS to a suitable concentration for subcutaneous tumor inoculation in nude mice.

[0886] (2) Animal modeling

[0887] CB17 / SCID mice were subcutaneously inoculated with 5×10 6 DoHH-2 cells were resuspended in 1:1 PBS and Matrigel (0.1 ml / cell) and the tumor growth was observed regularly.

[0888] (3) Random grouping

[0889] When the tumor grows to an average volume of 100-150 mm 3 Animals were randomly divided into groups according to tumor size for drug administration, with 5 animals in each group. The day of tumor cell inoculation was defined as day 0.

[0890] (4) Dosage method, experimental observation and data collection

[0891] Compounds 29 and 58 were orally administered to mice twice daily for 21 consecutive days. ARQ531 mice were orally administered with test compounds once daily for 21 consecutive days.

[0892] After tumor cell inoculation, routine monitoring includes tumor growth and the effect of treatment on the normal behavior of the animals, including the activity of the experimental animals, food and water intake, weight gain or loss, eyes, fur and other abnormalities. Clinical symptoms observed during the experiment are recorded in the original data. After the start of drug administration, the weight and tumor size of the mice are measured twice a week. Tumor volume calculation formula: Tumor volume (mm 3 )=1 / 2×(a×b 2 )(where a represents the major diameter and b represents the minor diameter).

[0893] The relative tumor growth rate, T / C (%), is the percentage of the tumor volume or weight of the treatment group and the control group at a certain time point. The calculation formula is as follows:

[0894] T / C (%) = TRTV / CRTV × 100% (TRTV: average RTV of the treatment group; CRTV: average RTV of the vehicle control group; RTV = Vt / V0, V0 is the tumor volume of the animal at the time of grouping, and Vt is the tumor volume of the animal after treatment);

[0895] Or T / C (%) = TTW / CTW x 100% (TTW: average tumor weight at the end of the experiment in the treatment group; CTW: average tumor weight at the end of the experiment in the vehicle control group).

[0896] The relative tumor inhibition rate (TGI) was calculated as follows: TGI% = (1-T / C) × 100%. (T and C are the relative tumor volume (RTV) or tumor weight (TW) of the treatment and control groups at a specific time point, respectively.)

[0897] At the end of the experiment (21 days after administration), no mice died or lost weight in the control group, ARQ-531 (30 mg / kg, QD*21 days), compound 29 (10 mg / kg, 30 mg / kg, 60 mg / kg BID*21 days), or compound 58 (30 mg / kg, 60 mg / kg BID*21 days), and there was no obvious drug toxicity. All mice were well tolerated during the treatment period.

[0898] The results are as follows Figure 1 and Figure 2 shown.

[0899] 7.2 Efficacy Testing in TMD8 Xenograft Model

[0900] (1) Cell culture and inoculation

[0901] TMD8 cells were cultured in RPMI1640 medium at 37°C and 5% CO2, and passaged 2-3 times a week. When the cell saturation reached 80%-90%, cells were harvested, counted, and plated.

[0902] When the cells in the logarithmic growth phase reached the required number for the experiment, the cells were collected and centrifuged at 1000 rpm for 5 min to remove the supernatant. The cells were resuspended in culture medium and counted using a cell counter. Based on the counting results, the original solution was diluted to a viable cell concentration of 1×10 8 The cell suspension was prepared at a cell viability of 90.28% at passage 31. The diluted cell suspension was diluted with Matrigel at a ratio of 1:1. After mixing, the suspension was placed on ice. A 1ml sterile insulin syringe was used to draw up the suspension. 0.2ml of the cell suspension was inoculated subcutaneously in the right armpit of each mouse. Each mouse was inoculated with 1×10 TMD8 cells. 7 indivual.

[0903] (2) Grouping method

[0904] After the inoculation, the tumor growth status was observed daily, and the average tumor volume reached about 112.28 mm 3 The mice were randomly divided into 7 groups according to tumor volume.

[0905] (3) Dosage method

[0906] Dosage is based on mouse body weight: 10 μl / g. If body weight loss exceeds 20%, the dosing regimen should be adjusted accordingly.

[0907] (4) Observation

[0908] The development and any modifications of this experimental protocol have been evaluated and approved by the Experimental Animal Welfare Ethics Committee of Hefei Zhongke Puruisheng Biotechnology Co., Ltd. The health status and mortality of the animals were monitored daily. Routine examinations included tumor growth, activity, diet, weight, eyes, hair, and other abnormal behaviors. Tumor volume and weight were measured twice a week (Monday and Thursday). Tumor volume was measured with a vernier caliper using the formula TV = 0.5a × b 2 , where a is the long diameter of the tumor and b is the short diameter of the tumor.

[0909] (5) Experimental endpoint

[0910] The experimental indicator examined whether tumor growth was inhibited, delayed, or cured. The compound's anti-tumor efficacy was evaluated using the relative tumor growth rate (T / C%). In principle, the evaluation criteria were: T / C (%) > 40% for ineffectiveness; T / C (%) ≤ 40% with p < 0.05 for significant difference.

[0911] The vehicle group and compound 29 did not show significant weight loss, morbidity or mortality.

[0912] The results are as follows Figure 3 and Figure 4 shown.

[0913] Tumor volume in tumor-bearing mice in the vehicle control group increased significantly and rapidly. T / C (%) values for the LOXO305 30 mg / kg and Compound 29 30 mg / kg groups were 0% and 20% on day 21, respectively, with T / C values below 40%. T / C (%) values for the Compound 29 60 mg / kg group were 0% and below 40% on day 35, respectively. P < 0.05 compared to the control group. LOXO305 30 mg / kg, Compound 29 60 mg / kg, and Compound 29 30 mg / kg groups demonstrated significant inhibitory effects in the TMD8 xenograft tumor model. Compound 29 (10 mg / kg) demonstrated a modest antitumor effect in this model, with a dose-dependent inhibitory effect.

[0914] 7.3miniPDX

[0915] (1) MiniPDX preparation and grouping

[0916] The tumor model of diffuse large B-cell transformed chronic lymphocytic leukemia required for the resuscitation experiment was grown to 500-800mm. 3 The tumor tissues were surgically removed aseptically, and non-tumor tissue and necrotic tissue were removed in a biosafety cabinet; the tumor tissues were cut into 1-3 mm 3For small tumor pieces, digest the tumor in digestion buffer at 37°C for 1-2 hours. Then, pass the cell suspension through a 70μm sieve, centrifuge at 1200 rpm for 3 minutes, remove the supernatant, resuspend the cells in 10mL of PBS containing 1% FBS, and count on a hemocytometer. After removing the mouse cells, centrifuge at 1200 rpm for 3 minutes, remove the supernatant, resuspend the cells in BIO-MPM-1 cell culture medium, count on a hemocytometer, and adjust the cell density. The cell suspension was then filled into MiniPDX capsules. Mice were randomly divided into groups according to body weight and inoculated subcutaneously with MiniPDX capsules on the back of the mice. Day 0 was designated as day 0, and the experiment lasted for 7 days.

[0917] In the MiniPDX model of chronic lymphocytic leukemia (CLL) diffuse large B-cell transformed lymphoma, the relative proliferation rates of tumor cells in the ibrutinib (10 mg / kg), LOXO305 (50 mg / kg), ARQ531 (50 mg / kg), compound 29 (50 mg / kg), and compound 23 (50 mg / kg) treatment groups were 32%, 61%, 27%, 41%, and 90%, respectively, on day 7 after dosing. Compared with the control group, all treatment groups showed varying degrees of reduction in fluorescence (RLU), with significant reductions in fluorescence (RLU) in the ibrutinib (10 mg / kg), ARQ531 (50 mg / kg), and compound 29 (50 mg / kg) treatment groups (p<0.001), demonstrating significant tumor inhibition.

[0918] The results are as follows Figure 5 、 Figure 6 、 Figure 7 shown.

[0919] Example 8 Solubility

[0920] (1) Solubility determination method

[0921] 15 μL of a 10 mM stock solution of compound 58 was placed into the corresponding 96-well plates in order. 485 μL of either pH 2.0 or pH 7.4 PBS was added, respectively. The experiment was performed in duplicate. The samples were then transferred to an Eppendorf Thermomixer Comfort shaker at 25°C, 1100 rpm, for 2 hours. The samples were filtered, and 5 μL of the filtrate and 5 μL of DMSO were added to 490 μL of a mixture of water and acetonitrile containing the internal standard. A certain proportion of ultrapure water was used for dilution based on the peak shape of the solution. The dilution factor was adjusted based on the solubility value and LC-MS signal response.

[0922] (2) Preparation of 300 μM standard solution (STD)

[0923] Transfer 6 μL from the 10 mM DMSO STD plate to the remaining empty plate, then add 194 μL of DMSO to this plate for an STD concentration of 300 μM. From the 300 μM DMSO STD plate, transfer 5 μL of DMSO STD and 5 μL of PBS (pH 2.0 or pH 7.4) to the remaining empty plate, then add 490 μL of a 1:1 water-acetonitrile mixture containing the internal standard to this plate for a final STD concentration of 3 μM. Use a certain ratio of ultrapure water to dilute the solution based on the peak shape. Adjust the concentration of the standard sample based on the LC-MS signal response.

[0924]

[0925]

[0926] Those skilled in the art will appreciate that the foregoing description is exemplary and illustrative in nature and is intended to illustrate the present invention and its preferred embodiments. Through routine experimentation, the skilled artisan will appreciate that obvious modifications and variations can be made without departing from the spirit of the present invention. All such modifications within the scope of the appended claims are intended to be included therein. It is therefore intended that the present invention be defined not by the foregoing description but by the scope of the following claims and their equivalents. All publications cited in this specification are incorporated herein by reference.

Claims

1. A compound of formula (I) or a pharmaceutically acceptable salt thereof: in: M is selected from N; R 1 Selected from C1-C4 alkyl; R 2 is selected from 6-8 membered aryl or 5-6 membered heteroaryl; The 6-8 membered aryl or 5-6 membered heteroaryl is selected from: halogenated C1-C3 alkyl, halogen, C1-C6 alkyl, NHC(=O)NHR a 、NHC(=O)OR a The 5-6 membered heteroaryl group has a heteroatom selected from N; wherein R a Selected from C1-C4 alkyl.

2. The compound of formula (I) or pharmaceutically acceptable salt according to claim 1, wherein R 2 is selected from phenyl, wherein the phenyl is selected from: halogenated C1-C3 alkyl, halogen, C1-C6 alkyl, NHC(=O)NHR a , and NHC(=O)OR a substituted by a substituent.

3. The compound of formula (I) or pharmaceutically acceptable salt according to claim 1, wherein R 2 is a 5-6 membered heteroaryl group selected from the following substituted structures: The substituents are as defined in claim 1.

4. The compound of formula (I) or pharmaceutically acceptable salt according to claim 3, wherein R 2 is a 5-6 membered heteroaryl group selected from the following substituted structures: The substituent is selected from: CH3, C2H5, or CHF2; R 1 It is CH3, C2H5 or C3H7.

5. A compound of formula (I) or a pharmaceutically acceptable salt according to any one of claims 1 to 4, wherein R 1 It is CH3, C2H5, or CH(CH3)2.

6. A compound of formula (I) or a pharmaceutically acceptable salt according to any one of claims 1 to 4, wherein R 2 Methyl or ethyl substituted or a phenyl group substituted with a methyl group or an ethyl group.

7. A compound or pharmaceutically acceptable salt of the general formula (I) according to any one of claims 1 to 4, wherein R 2 for 8. A compound of formula (I) or a pharmaceutically acceptable salt according to any one of claims 1 to 4, wherein R 1 is CH3, C2H5, or CH(CH3)2; R 2 for 9. A compound of formula (I') or a pharmaceutically acceptable salt thereof: in: M is N; R 1 is a C1-C4 alkyl group; R 2 is a 6-8 membered aryl group, a 5-6 membered heteroaryl group or a 5 membered heterocycloalkyl group containing a nitrogen atom; The 6-8 membered aryl, 5-6 membered heteroaryl or 5 membered heterocycloalkyl containing nitrogen atom is selected from halogenated C1-C3 alkyl, halogen, C1-C6 alkyl, NHC(=O)NHR a 、NHC(=O)OR a The 5-6 membered heteroaryl group has a heteroatom of N; wherein R a It is a C1-C4 alkyl group.

10. The compound according to claim 9 or a pharmaceutically acceptable salt thereof, wherein R 2 is phenyl, wherein the phenyl is selected from halogenated C1-C3 alkyl, halogen, C1-C6 alkyl, NHC(=O)NHR a , and NHC(=O)OR a substituted by a substituent.

11. The compound according to claim 9 or a pharmaceutically acceptable salt thereof, wherein R 2 is a 5-6 membered heteroaryl group, which is substituted by a substituent selected from the group defined in claim 9:

12. The compound according to claim 11 or a pharmaceutically acceptable salt thereof, wherein R 2 is a 5-6 membered heteroaryl group, wherein the 5-6 membered heteroaryl group is the following structure substituted by a substituent selected from CH3, C2H5, and CHF2: R 1 It is CH3, C2H5 or C3H7.

13. The compound according to any one of claims 9 to 12, or a pharmaceutically acceptable salt thereof, wherein R 1 It is CH3, C2H5 or CH(CH3)2.

14. The compound according to any one of claims 9 to 12, or a pharmaceutically acceptable salt thereof, wherein R 2 Methyl or ethyl substituted or a phenyl group substituted with a methyl group or an ethyl group.

15. The compound or pharmaceutically acceptable salt according to any one of claims 9 to 12, wherein R 2 for 16. The compound according to any one of claims 9 to 12, or a pharmaceutically acceptable salt thereof, wherein R 1 is CH3, C2H5 or CH(CH3)2; R 2 for 17. A compound having the following structure or a pharmaceutically acceptable salt thereof:

18. A pharmaceutical composition comprising the compound according to any one of claims 1 to 17 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

19. Use of the compound according to any one of claims 1 to 17 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to claim 18 in the preparation of a medicament for treating cancer.

20. The use according to claim 19, wherein the cancer is lung cancer, papillary thyroid cancer, medullary thyroid cancer, differentiated thyroid cancer, recurrent thyroid cancer, refractory differentiated thyroid cancer, multiple endocrine carcinoma type 2A or 2B, pheochromocytoma, parathyroid hyperplasia, breast cancer, colorectal cancer, papillary renal cell carcinoma, gastrointestinal mucosal ganglioneuroma or cervical cancer.

21. The use according to claim 19, wherein the cancer is a RET or mutant RET-mediated cancer.

22. The use according to claim 20 or 21, wherein the cancer is medullary thyroid carcinoma, non-small cell lung cancer, metastatic solid tumor or advanced solid tumor with RET gene mutation or fusion.

23. Use of the compound according to any one of claims 1 to 17 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to claim 18, in the preparation of a medicament for treating BTK-mediated diseases. The use according to claim 23 , wherein the BTK-mediated disease is cancer, autoimmune disease or allergic disease.

25. The use according to claim 24, wherein the cancer is one or more of subtype diffuse large B-cell lymphoma, mantle cell lymphoma, chronic lymphocytic lymphoma, extranodal marginal zone B-cell lymphoma, B-cell chronic lymphocytic leukemia, B-cell prolymphocytic leukemia, mature B-cell acute lymphoblastic leukemia, 17p deleted chronic lymphocytic leukemia, Waldenstrom macroglobulinemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasma cell myeloma, plasmacytoma, nodal marginal zone B-cell lymphoma, mantle cell lymphoma, intravascular large B-cell lymphoma and primary effusion lymphoma; the autoimmune disease is systemic lupus erythematosus, rheumatoid arthritis, Sjögren's syndrome, multiple sclerosis, inflammatory bowel disease, The present invention relates to one or more of the following diseases: enteritis such as Crohn's disease and ulcerative colitis, urticaria, immune thrombocytopenia, IgA nephropathy, hidradenitis suppurativa, psoriasis, vitiligo, neutrophilic dermatosis, autoimmune blistering disease such as pemphigus and pemphigoid, IgG4-related disease, autoimmune hemolytic anemia, rheumatic fever, antiphospholipid syndrome, systemic sclerosis / scleroderma, autoimmune hepatitis, primary sclerosing cholangitis, primary biliary cirrhosis, Henoch-Schonlein purpura, Churg-Strauss syndrome / allergic granulomatosis with polyangiitis, Behçet's disease / Behçet's disease, ANCA-associated small vessel vasculitis, and dermatitis herpetiformis; the allergic disease is one or more of allergic conjunctivitis, allergic rhinitis, allergic asthma, atopic dermatitis, and chronic asthma.

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