Compounds as dhx9 inhibitors and uses thereof

Abstract

This invention belongs to the field of pharmaceutical chemistry and relates to a class of compounds as DHX9 inhibitors and their applications. Specifically, this invention provides compounds of formula (I) or isomers thereof, pharmaceutically acceptable salts, solvates, crystals or prodrugs, methods for their preparation, pharmaceutical compositions containing these compounds, and the use of these compounds or compositions for treating DHX9-mediated diseases.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical chemistry, specifically relating to compounds or isomers thereof, pharmaceutically acceptable salts, solvates, crystals or prodrugs as DHX9 inhibitors, methods for their preparation, pharmaceutical compositions containing these compounds, and the use of these compounds or compositions for the treatment of DHX9-mediated diseases. Background Technology

[0002] The DEAD-box protein family is a large class of ATP-dependent RNA helicases that promote DNA or RNA folding and / or conformational rearrangement by binding to and interacting with RNA, and occasionally participate in protein-nucleic acid interactions. These enzymes are involved in almost all aspects of nucleic acid metabolism, including replication, repair, recombination, transcription, splicing, chromosome segregation, and telomere maintenance. To support enhanced proliferation and meet the demands of accelerated nucleic acid metabolism, helicases are frequently overexpressed in cancer cells. Meanwhile, naturally occurring loss-of-function (LOF) mutations in helicases are associated with many diseases, including cancer (C. Gulliver et al, Futurescience OA, 7(2020) Fso650.).

[0003] DHX9 possesses the ability to unwind DNA, RNA, and complex polynucleotide structures, including DNA-RNA hybrids (R-Loops), DNA and RNA G-quadruplexes, and circular RNA. Dysregulation of DHX9 activity can lead to altered cell growth and subsequent tumorigenesis, and it is overexpressed in many cancer types, such as colorectal and lung cancer. Currently reported mechanisms of DHX9's tumor-suppressive effects mainly involve inducing apoptosis due to DNA replication stress and a "viral mimicry" response induced by dsRNA and R-Loop. 1. DHX9 participates in the unwinding of R-Loops or DNA / RNA hybrid structures. Excessive R-Loop formation can trigger genomic instability and DNA replication stress by impairing replication fork progression and inducing DSB (diverse DNA-RNA hybridization). Studies have shown that DHX9 deficiency leads to upregulation of DNA damage-related genes and downregulation of DNA replication-related genes in tumor cells, a significant increase in R-Loop structure, an increased proportion of DNA stall forks, and induces DNA replication stress damage (p-CHK1, p-CHK2, p-H2AX), which in turn leads to cellular genomic instability and activation of apoptosis-related pathways (Cleaved PARP). 2. Studies have shown that inhibiting DHX9 can increase intracellular immunogenicity induced by double-stranded RNA (dsRNA), triggering an immune response in tumors and enhancing the efficacy of immunotherapy. This mechanism is also known as a "viral mimicry" response. This study clarifies that DHX9 is a factor that induces viral mimicry, involving the inhibition of double-stranded RNA and R-Loop, and proposes DHX9 as a new target for enhancing anti-tumor immunity (T. Murayama et al, Cancer Discov., 14(2024) 468-491; R. Chen et al, Cancer Discov., 11(2021) 2707-2725.).

[0004] Preclinical studies have shown that microsatellite instability (MSI) tumors with mismatch repair deficiency (dMMR) and tumors lacking BRCA function (BRCA LOF) exhibit a strong dependence on DHX9, making this helicase an attractive target for therapeutic drugs targeting these tumors. Summary of the Invention

[0005] One object of the present invention is to provide a class of compounds, isomers thereof, pharmaceutically acceptable salts, solvates, crystals or prodrugs having DHX9 inhibitory activity as shown in formulas (I), (Ia), (Ib) and (Ic).

[0006] Another object of the present invention is to provide a method for preparing compounds of general formulas (I), (Ia), (Ib), and (Ic) of the present invention, or isomers thereof, pharmaceutically acceptable salts, solvates, crystals, or prodrugs.

[0007] Another object of the present invention is to provide compositions comprising compounds of general formulas (I), (Ia), (Ib), and (Ic) of the present invention, or isomers thereof, pharmaceutically acceptable salts, solvates, crystals or prodrugs, and pharmaceutically acceptable carriers, as well as compositions comprising compounds of general formulas (I), (Ia), (Ib), and (Ic) of the present invention, or isomers thereof, pharmaceutically acceptable salts, solvates, crystals or prodrugs, and one or more other pharmaceutical products.

[0008] Another object of the present invention is to provide a method for treating DHX9-mediated diseases using compounds of general formulas (I), (Ia), (Ib), and (Ic), or their isomers, pharmaceutically acceptable salts, solvates, crystals, or prodrugs of the present invention, and the use of compounds of general formulas (I), (Ia), (Ib), and (Ic), or their isomers, pharmaceutically acceptable salts, solvates, crystals, or prodrugs of the present invention in the preparation of medicaments for treating DHX9-mediated diseases.

[0009] To achieve the aforementioned objectives, the present invention provides the following technical solution:

[0010] In a first aspect, the present invention provides compounds of general formula (I) or isomers thereof, pharmaceutically acceptable salts, solvates, crystals, or prodrugs.

[0011]

[0012] in,

[0013] Ring A is selected from C 6-12 Aryl, 5-15 quinone heteroaryl, C 3-12 Cycloalkyl and 3-12 membered heterocyclic groups;

[0014] Ring B is selected from monocyclic, fused, or spirocyclic bicyclic or tricyclic aryl, heteroaryl, cycloalkyl, and heterocyclic groups;

[0015] R 1 R 2 R 3 and R 4 Each group is independently selected from hydrogen, halogen, hydroxyl, alkyl, haloalkyl, hydroxyalkyl, alkoxy, haloalkoxy, hydroxyalkoxy, nitro, carboxyl, cyano, amino, monoalkylamino, alkylacylamino, alkylacyl, aminoacyl, alkylaminoacyl, oxo group and dialkylamino;

[0016] L does not exist or is selected from C. 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 1-6 Alkoxy, C 2-6 alkenyloxy group, C 2-6 The alkynyl, acyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, and heterocyclic groups are optionally substituted by one or more groups selected from halogen, hydroxyl, alkyl, haloalkyl, hydroxyalkyl, alkoxy, haloalkoxy, hydroxyalkoxy, amino, monoalkylamino, alkylacylamino, alkylacyl, alkylsulfonyl, aminoacyl, alkylaminoacyl, dialkylamino, alkenyl, alkynyl, haloalkylacyl, hydroxyalkylacyl, and oxo groups.

[0017] R 5 Selected from hydrogen, halogen, hydroxyl, C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 Alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, and heterocyclic groups, optionally substituted by one or more groups selected from halogen, hydroxyl, alkyl, haloalkyl, hydroxyalkyl, alkoxy, haloalkoxy, hydroxyalkoxy, amino, monoalkylamino, alkylacylamino, alkylacyl, alkylsulfonyl, aminoacyl, alkylaminoacyl, dialkylamino, alkenyl, alkynyl, haloalkylacyl, hydroxyalkylacyl, cycloalkyl, heterocyclic, aryl, heteroaryl, halocycloalkyl, and oxo groups; and

[0018] m, n, and p are each independently selected from 0, 1, 2, and 3.

[0019] In some embodiments, the compound of the present invention is a compound of general formula (I) or an isomer thereof, a pharmaceutically acceptable salt, a solvate, a crystal, or a prodrug, wherein ring B is selected from monocyclic, fused, or spirocyclic bicyclic or tricyclic rings. 6-12 Aryl, 5-15 quinone heteroaryl, C 3-12 Cycloalkyl groups and 3-12 membered heterocyclic groups, wherein the heteroatom is selected from one or more N, O, S, P, R 4 Selected from hydrogen, halogens, C 1-6 Alkyl and oxo groups, p is selected from 0, 1, 2 and 3.

[0020] In some preferred embodiments, the compounds of the present invention are compounds of general formula (I) or isomers thereof, pharmaceutically acceptable salts, solvates, crystals, or prodrugs, wherein ring B is selected from...

[0021] In some embodiments, the compound of the present invention is a compound of general formula (I) or an isomer thereof, a pharmaceutically acceptable salt, a solvate, a crystal, or a prodrug, wherein R 5Selected from hydrogen, halogen, hydroxyl, C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 Alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, and heterocyclic groups, optionally composed of one or more groups selected from halogen, hydroxyl, C 1-6 Alkyl, Halogenated C 1-6 Alkyl, hydroxyl C 1-6 Alkyl, C 1-6 Alkoxy, halogenated C 1-6 Alkoxy, hydroxy C 1-6 Alkoxy, amino, mono-C 1-6 Alkylamino, C 1-6 Alkyl acylamino, C 1-6 Alkyl acyl, C 1-6 alkylsulfonyl, aminoacyl, C 1-6 Alkylaminoacyl, bis(C) 1-6 Alkylamino, C 2-6 alkenyl, C 2-6 alkynyl, halogenated C 1-6 Alkyl acyl, hydroxy C 1-6 Alkyl acyl, C 3-12 Cycloalkyl, 3-12 membered heterocyclic groups, C 6-12 Aryl, 5-15 quinone heteroaryl, halogenated C 3-12 The heteroatom is selected from one or more N, O, S, and P groups, and is substituted with cycloalkyl or oxo groups.

[0022] In some preferred embodiments, the compound of the present invention is a compound of general formula (I) or an isomer thereof, a pharmaceutically acceptable salt, a solvate, a crystal, or a prodrug, wherein R 5 Selected from hydrogen, fluorine, chlorine, bromine, iodine, hydroxyl, methyl, ethyl, vinyl, ethynyl, propynyl, cyclopropyl, cyclobutyl, oxetyl, pyridyl, cyclopentenyl, It may be optionally oxidized by one or more elements selected from fluorine, chlorine, bromine, iodine, hydroxyl, C 1-3 Alkyl, Halogenated C 1-3 Alkyl, hydroxyl C 1-3 Alkyl, C 1-3 Alkoxy, halogenated C 1- Alkoxy, hydroxy C 1-3 Alkoxy groups, amino groups, mono-C 1-3 Alkylamino, C 1-3 Alkyl acylamino, C 1-3 Alkyl acyl, C 1-3 alkylsulfonyl, aminoacyl, C 1-3 Alkylaminoacyl, bis(C) 1-3 Alkylamino, C 2-4 alkenyl, C2-4 alkynyl, halogenated C 1-3 Alkyl acyl, hydroxy C 1-3 Alkyl acyl, C 3-8 Cycloalkyl, 3-8 membered heterocyclic groups, C 6-8 Aryl, 5-8 quinone heteroaryl, halogenated C 3-8 The heteroatom is selected from one or more N, O, S, and P groups, and is substituted with cycloalkyl or oxo groups.

[0023] In some embodiments, the compounds of the present invention are compounds of general formula (I) or isomers thereof, pharmaceutically acceptable salts, solvates, crystals, or prodrugs having the structure of general formula (Ia).

[0024]

[0025] in,

[0026] Ring B is selected from fused or spirocyclic tricyclic heteroaryl and heterocyclic groups; and

[0027] R 1 R 2 R 3 R 4 m, n and p have the definitions described in the above general formula (I).

[0028] In some preferred embodiments, the compounds of the present invention are compounds of general formula (Ia) or isomers thereof, pharmaceutically acceptable salts, solvates, crystals, or prodrugs, wherein ring B is selected from fused or spirocyclic tricyclic 5-15-membered heteroaryl and 3-12-membered heterocyclic groups, and the heteroatom is selected from one or more N, O, S, P, R 4 Selected from hydrogen, halogens, C 1-6 Alkyl and oxo groups, p is selected from 0, 1, 2 and 3.

[0029] In some preferred embodiments, the compounds of the present invention are compounds of general formula (Ia) or isomers thereof, pharmaceutically acceptable salts, solvates, crystals, or prodrugs, wherein ring B is selected from...

[0030] In some embodiments, the compounds of the present invention are compounds of general formula (I) or isomers thereof, pharmaceutically acceptable salts, solvates, crystals, or prodrugs having the structure of the following general formula (Ib).

[0031]

[0032] L is selected from C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 1-6 Alkoxy, C2-6 alkenyloxy group, C 2-6 Acyl group;

[0033] The ring C is selected from cycloalkyl, heterocyclic, fused, or spirocyclic bicyclic or tricyclic heteroaryl groups, optionally substituted by one or more groups selected from halogen, hydroxyl, alkyl, haloalkyl, hydroxyalkyl, alkoxy, haloalkoxy, hydroxyalkoxy, amino, monoalkylamino, alkylacylamino, alkylacyl, alkylsulfonyl, aminoacyl, alkylaminoacyl, dialkylamino, alkenyl, alkynyl, haloalkylacyl, hydroxyalkylacyl, and oxo groups; and

[0034] Rings A and R 2 R 3 R 4 , n and p have the definitions described in the above general formula (I).

[0035] In some embodiments, the compounds of the present invention are compounds of general formula (Ib) or isomers thereof, pharmaceutically acceptable salts, solvates, crystals, or prodrugs, wherein L is selected from C 1-3 Alkyl, C 2-4 alkenyl, C 2-5 alkynyl group, C 1-3 Alkoxy, C 2-4 alkenyloxy group, C 2-5 Acyl group, alkynyl group.

[0036] In some specific embodiments, the compounds of the present invention are compounds of general formulas (I) and (Ib) or isomers thereof, pharmaceutically acceptable salts, solvates, crystals, or prodrugs, wherein L is selected from methyl, methoxy, ethoxy, Acyl group.

[0037] In some embodiments, the compounds of the present invention are compounds of general formula (Ib) or isomers thereof, pharmaceutically acceptable salts, solvates, crystals, or prodrugs, wherein the ring C is selected from C14. 3-12 Cycloalkyl, 3-12 heterocyclic, fused or spirocyclic bicyclic or tricyclic 5-15-membered heteroaryl groups, wherein the heteroatom is selected from one or more N, O, S, P, and optionally is surrounded by one or more halogens, hydroxyl groups, C 1-6 Alkyl, Halogenated C 1-6 Alkyl, hydroxyl C 1-6 Alkyl, C 1-6 Alkoxy, halogenated C 1-6 Alkoxy, hydroxy C 1-6 Alkoxy, amino, mono-C 1-6 Alkylamino, C 1-6 Alkyl acylamino, C 1-6 Alkyl acyl, C 1-6 alkylsulfonyl, aminoacyl, C 1-6Alkylaminoacyl, bis(C) 1-6 Alkylamino, C 2-4 alkenyl, C 2-4 alkynyl, halogenated C 1-6 Alkyl acyl, hydroxy C 1-6 Group substitution of alkyl acyl groups and oxo groups.

[0038] In some preferred embodiments, the compounds of the present invention are compounds of general formula (Ib) or isomers thereof, pharmaceutically acceptable salts, solvates, crystals, or prodrugs, wherein the ring C is selected from cyclopropyl, oxetyl, pyridyl, ... It may be optionally oxidized by one or more elements selected from fluorine, chlorine, bromine, iodine, hydroxyl, C 1-3 Alkyl, Halogenated C 1-3 Alkyl, hydroxyl C 1-3 Alkyl, C 1-3 Alkoxy, halogenated C 1- Alkoxy, hydroxy C 1-3 Alkoxy groups, amino groups, mono-C 1-3 Alkylamino, C 1-3 Alkyl acylamino, C 1-3 Alkyl acyl, C 1-3 alkylsulfonyl, aminoacyl, C 1-3 Alkylaminoacyl, bis(C) 1-3 Alkylamino, C 2-4 alkenyl, C 2-4 alkynyl, halogenated C 1-3 Alkyl acyl, hydroxy C 1-3 The heteroatom is selected from one or more N, O, S, and P groups, and is substituted with alkyl acyl or oxo groups.

[0039] In some embodiments, the compounds of the present invention are compounds of general formula (I) or isomers thereof, pharmaceutically acceptable salts, solvates, crystals, or prodrugs having the structure of the following general formula (Ic).

[0040]

[0041] in,

[0042] Cycle C and cycle D are each independently selected from aryl, heteroaryl, cycloalkyl, cycloalkenyl, and heterocyclic groups, and are optionally substituted by one or more groups selected from halogen, hydroxyl, alkyl, haloalkyl, hydroxyalkyl, alkoxy, haloalkoxy, hydroxyalkoxy, amino, monoalkylamino, alkylacylamino, alkylacyl, alkylsulfonyl, aminoacyl, alkylaminoacyl, dialkylamino, alkenyl, alkynyl, haloalkylacyl, hydroxyalkylacyl, and oxo groups;

[0043] When ring C is selected from monoheterocyclic and monoheteroaryl groups, its connection position with ring D is not N; and

[0044] Rings A and R 2 R 3 R 4 , n and p have the definitions described in the above general formula (I).

[0045] In some embodiments, the compounds of the present invention are compounds of general formula (Ic) or isomers thereof, pharmaceutically acceptable salts, solvates, crystals, or prodrugs, wherein ring C and ring D are each independently selected from C 6-12 Aryl, 5-15 quinone heteroaryl, C 3-12 cycloalkyl, C 3-12 Cycloalkenyl and 3-12 membered heterocyclic groups, wherein the heteroatom is selected from one or more N, O, S, P, and optionally is selected from one or more halogens, hydroxyl groups, C 1-6 Alkyl, Halogenated C 1-6 Alkyl, hydroxyl C 1-6 Alkyl, C 1-6 Alkoxy, halogenated C 1-6 Alkoxy, hydroxy C 1-6 Alkoxy, amino, mono-C 1-6 Alkylamino, C 1-6 Alkyl acylamino, C 1-6 Alkyl acyl, C 1-6 alkylsulfonyl, aminoacyl, C 1-6 Alkylaminoacyl, bis(C) 1-6 Alkylamino, C 2-4 alkenyl, C 2-4 alkynyl, halogenated C 1-6 Alkyl acyl, hydroxy C 1-6 Group substitution of alkyl acyl groups and oxo groups.

[0046] In some embodiments, the compounds of the present invention are compounds of general formulas (I), (Ia), (Ib), and (Ic), or isomers thereof, pharmaceutically acceptable salts, solvates, crystals, or prodrugs, wherein ring A is selected from C 6-8 aryl, 5-8-membered heteroaryl, wherein the heteroatom is selected from one or more N, O, S, P, R 3 Selected from hydrogen, C 1-6 Alkyl and Halogenated C 1-6 Alkyl group, n is selected from 0, 1 and 2.

[0047] In some specific embodiments, the compounds of the present invention are compounds of general formula (I), general formula (Ib), general formula (Ic) or isomers thereof, pharmaceutically acceptable salts, solvates, crystals or prodrugs, wherein ring A is selected from...

[0048] In some embodiments, the compounds of the present invention are compounds of general formulas (I), (Ia), (Ib), and (Ic), or isomers thereof, pharmaceutically acceptable salts, solvates, crystals, or prodrugs, wherein R 2 R 3 and R 4 Each is independently selected from hydrogen, fluorine, chlorine, bromine, iodine, hydroxyl, and C. 1-6 Alkyl, Halogenated C 1-6 Alkyl, hydroxyl C 1-6 Alkyl, C 1-6 Alkoxy, halogenated C 1-6 Alkoxy, hydroxy C 1-6 Alkoxy, nitro, carboxyl, cyano, amino, C 1-6 Alkylamino, C 1-6 Alkyl acylamino, C 1-6 Alkyl acyl, amino acyl, C 1-6 Alkylaminoacyl, oxo group and double C 1-6 Alkylamino.

[0049] In some preferred embodiments, the compounds of the present invention are compounds of general formulas (I), (Ia), (Ib), and (Ic), or isomers thereof, pharmaceutically acceptable salts, solvates, crystals, or prodrugs, wherein R 2 Selected from methyl,

[0050] This invention provides the following specific compounds or isomers thereof, pharmaceutically acceptable salts, solvates, crystals, or prodrugs:

[0051]

[0052]

[0053]

[0054]

[0055] In a second aspect, the present invention provides pharmaceutical compositions comprising the compounds of the present invention or isomers thereof, pharmaceutically acceptable salts, solvates, crystals, or prodrugs.

[0056] In some embodiments, the present invention provides compounds or isomers thereof, pharmaceutically acceptable salts, solvates, crystals or prodrugs of the present invention, and pharmaceutical compositions comprising compounds or isomers thereof, pharmaceutically acceptable salts, solvates, crystals or prodrugs of the present invention for treating DHX9-mediated diseases.

[0057] In some embodiments, the present invention provides pharmaceutical compositions comprising the compound of the present invention or an isomer thereof, a pharmaceutically acceptable salt, a solvate, a crystal or prodrug, and a pharmaceutically acceptable carrier.

[0058] The compounds of the present invention or their isomers, pharmaceutically acceptable salts, solvates, crystals, or prodrugs can be mixed with pharmaceutically acceptable carriers, diluents, or excipients to prepare pharmaceutical formulations suitable for oral or parenteral administration. Administration methods include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, and oral routes. The formulations can be administered via any route, such as by infusion or bolus, or by absorption through the epithelium or mucous membranes (e.g., oral mucosa or rectum). Administration can be systemic or local. Examples of oral formulations include solid or liquid dosage forms, specifically including tablets, pills, granules, powders, capsules, syrups, emulsions, suspensions, etc. The formulations can be prepared by methods known in the art and contain carriers, diluents, or excipients conventionally used in the field of pharmaceutical formulations.

[0059] Thirdly, the present invention provides a method for using compounds or isomers thereof, pharmaceutically acceptable salts, solvates, crystals or prodrugs, or pharmaceutical compositions comprising the present invention (I), (Ia), (Ib), (Ic) for treating DHX9-mediated diseases, and their use in the preparation of medicaments for treating DHX9-mediated diseases.

[0060] In some preferred embodiments, the present invention provides methods for using compounds or isomers thereof, pharmaceutically acceptable salts, solvates, crystals or prodrugs, or pharmaceutical compositions comprising thereof, represented by formulas (I), (Ia), (Ib), and (Ic) of the present invention, or pharmaceutical compositions thereof, for treating DHX9-mediated diseases, and uses in the preparation of medicaments for treating DHX9-mediated diseases, wherein the DHX9-mediated diseases include, but are not limited to, proliferative diseases, metabolic diseases, or hematologic disorders. In some embodiments, the DHX9-mediated diseases of the present invention are cancer.

[0061] In some embodiments, the DHX9-mediated diseases described in this invention include, but are not limited to, acoustic neuroma, adenocarcinoma, adrenal carcinoma, anal cancer, angiosarcoma (e.g., lymphangiosarcoma, lymphangioendothelial sarcoma, angiosarcoma), adnexal cancer, benign monoclonal gammopathy, biliary cancer (e.g., cholangiocarcinoma), bladder cancer, breast cancer (e.g., breast adenocarcinoma, papillary breast carcinoma, breast cancer, medullary breast carcinoma, triple-negative breast cancer), brain cancer (e.g., meningioma; glioma, such as astrocytoma, oligodendroglioma; medulloblastoma), and mycoplasma. Tracheal cancer, carcinoid tumor, cervical cancer (e.g., cervical adenocarcinoma), chordoma, craniopharyngioma, colorectal cancer (e.g., colon cancer, rectal cancer, colorectal adenocarcinoma), epithelial cancer, ependymoma, endothelial sarcoma (e.g., Kaposi's sarcoma, multiple idiopathic hemorrhagic sarcomas), endometrial cancer (e.g., uterine cancer, uterine sarcoma), esophageal cancer (e.g., esophageal adenocarcinoma, Barrett's adenocarcinoma), Ewing sarcoma. Cancers including sarcoma, ocular cancer (e.g., intraocular melanoma, retinoblastoma), familial eosinophilia, gallbladder cancer, gastric cancer (e.g., gastric adenocarcinoma), gastrointestinal stromal tumors (GIST), head and neck cancers (e.g., head and neck squamous cell carcinoma, oral cancer (e.g., oral squamous cell carcinoma (OSCC), pharyngeal cancer (e.g., laryngeal cancer, pharyngeal cancer, nasopharyngeal cancer, oropharyngeal cancer)), hematopoietic system cancers (e.g., leukemia such as acute lymphoblastic leukemia (ALL) (e.g., B-cell ALL, T-cell ALL), acute myeloid leukemia (AML) (e.g., B-cell AML, T-cell AML), chronic myeloid leukemia (CML) (e.g., B-cell CML, T-cell CML), and chronic lymphocytic leukemia (CLL) (e.g., B-cell CML). L-cell and T-cell CLL); lymphomas such as Hodgkin lymphoma (HL) (e.g., B-cell HL, T-cell HL) and non-Hodgkin lymphoma (NHL) (e.g., B-cell NHL such as diffuse large cell lymphoma (DLCL) (e.g., diffuse large B-cell lymphoma (DLBCL)), follicular lymphoma, chronic lymphocytic leukemia / small lymphocytic lymphoma (CLL / SLL), mantle cell lymphoma (MCL), marginal zone B-cell lymphoma (e.g., mucosa-associated lymphoid tissue (MALT) lymphoma, nodular marginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma), primary mediastinal B-cell lymphoma, Burkitt lymphoma, lymphoplasmacytic lymphoma (i.e., "Woldanstrom macroglobulinemia") macroglobulinemia, hairy cell leukemia (HCL), immunoblastic large cell lymphoma, precursor B-cell lymphoblastic lymphoma, and primary central nervous system (CNS) lymphoma; and T-cell NHL such as precursor T-cell lymphoblastic lymphoma / leukemia, peripheral T-cell lymphoma (PTCL) (e.g., cutaneous T-cell lymphoma (CTCL) (e.g., mycosis fungiodes, Sezary syndrome), angioimmunoblastic T-cell lymphoma, extranodular natural killer T-cell lymphoma, enteropathy-type T-cell lymphoma, subcutaneous panniculitis-like T-cell lymphoma, anaplastic large cell lymphoma); and mixtures of one or more leukemias / lymphomas as described above;And multiple myeloma (MM), heavy chain diseases (e.g., alpha chain disease, gamma chain disease, μ chain disease), angioblastoma, inflammatory myofibroblastoma, immune cell amyloidosis, renal cell carcinoma (e.g., nephroblastoma, also known as Wilms' tumor), hepatocellular carcinoma (e.g., hepatocellular carcinoma (HCC), malignant hepatocellular carcinoma), lung cancer (e.g., bronchial carcinoma, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), lung adenocarcinoma), leiomyosarcoma (LMS), mastocytosis (e.g., generalized mastocytosis), myelodyplastic syndrome (MDS), mesothelioma, myeloproliferative disorders (MPD) (e.g., polycythemia vera (PV), idiopathic thrombocythemia (ET), idiopathic extramedullary metaplasia (AMM)). This includes myelofibrosis (MF), chronic idiopathic myelofibrosis, chronic myeloid leukemia (CML), chronic neutrophilic leukemia (CNL), eosinophilic leukocytosis (HES), neuroblastoma, neurofibroma (e.g., type 1 or 2 multiple neurofibroma (NF), Schwannoma), neuroendocrine carcinoma (e.g., gastrointestinal pancreatic neuroendocrine tumor (GEP-NET), carcinoid tumor), osteosarcoma, ovarian cancer (e.g., cystadenocarcinoma, ovarian embryonal carcinoma, ovarian adenocarcinoma, ovarian clear cell carcinoma, ovarian serous cystadenocarcinoma), papillary adenocarcinoma, pancreatic cancer (e.g., pancreatic adenocarcinoma, intraductal papillary myxoma (IPMN), islet cell tumor), and penile cancer (e.g., Paget's disease of the penis and scrotum). Diseases including pineal gland tumors, primary neuroectodermal tumors (PNT), prostate cancer (e.g., prostate adenocarcinoma), rectal cancer, rhabdomyosarcoma, salivary duct cancer, skin cancer (e.g., squamous cell carcinoma (SCC), keratoacanthoma (KA), melanoma, basal cell carcinoma (BCC)), small bowel cancer (e.g., adnexal cancer), soft tissue sarcomas (e.g., malignant fibrous histiocytoma (MFH), liposarcoma, malignant peripheral nerve sheath tumor (MPNST), chondrosarcoma, fibrosarcoma, myxosarcoma), sebaceous gland cancer, sweat gland cancer, synovial tumors, testicular cancer (e.g., seminoma, embryonal testicular carcinoma), thyroid cancer (e.g., papillary thyroid carcinoma, papillary thyroid carcinoma (PTC), medullary thyroid carcinoma), urethral cancer, vaginal cancer, and vulvar cancer (e.g., vulvar Paget's disease), medulloblastoma, adenoid cystic carcinoma, melanoma, and glioblastoma.

[0062] In some preferred embodiments, the present invention provides methods for treating DHX9-mediated diseases using compounds or isomers thereof, pharmaceutically acceptable salts, solvates, crystals or prodrugs, or pharmaceutical compositions comprising thereof, represented by general formulas (I), (Ia), (Ib), and (Ic), as well as their use in the preparation of medicaments for treating DHX9-mediated diseases, wherein the DHX9-mediated diseases include, but are not limited to: breast cancer, esophageal cancer, bladder cancer, lung cancer, hematopoietic system cancers, lymphoma, medulloblastoma, medulloblastoma, rectal adenocarcinoma, colon cancer, gastric cancer, pancreatic cancer, liver cancer, adenoid cystic carcinoma, prostate cancer, lung cancer, head and neck squamous cell carcinoma, brain cancer, hepatocellular carcinoma, melanoma, oligodendroglioma, glioblastoma, testicular cancer, ovarian clear cell carcinoma, ovarian serous cystadenocarcinoma, thyroid cancer, multiple myeloma (AML), renal cell carcinoma, mantle cell lymphoma, triple-negative breast cancer, non-small cell lung cancer, hemoglobinopathies, diabetes, and obesity.

[0063] Terminology Definition

[0064] Unless otherwise stated, the terms used in the specification and claims have the following meanings.

[0065] In the compounds of this invention, "hydrogen," "carbon," and "oxygen" include all their isotopes. Isotopes should be understood to include those atoms having the same number of atoms but different mass numbers. For example, isotopes of hydrogen include protium, tritium, and deuterium, and isotopes of carbon include... 12 C 13 C and 14 C, oxygen isotopes include 16 O and 18 O etc.

[0066] In this invention, "isomers" refers to molecules with the same atomic composition and bonding but different three-dimensional spatial arrangements, including but not limited to diastereomers, enantiomers, cis-trans isomers, and mixtures thereof, such as racemic mixtures. Many organic compounds exist in optically active forms, meaning they are capable of rotating the plane of polarized light. In describing optically active compounds, the prefixes D, L, or R, S are used to indicate the absolute configuration of the chiral center of the molecule. The prefixes D, L, or (+), (-) are used to name the symbols for the plane polarization rotation of the compound; (-) or L indicates that the compound is levorotatory, and the prefix (+) or D indicates that the compound is dextrorotatory. These stereoisomers have the same chemical structure but different stereostructures. Specific stereoisomers can be enantiomers, and mixtures of isomers are usually called enantiomer mixtures. A 50:50 enantiomer mixture is called a racemic mixture or racemate, which may result in a lack of stereoselectivity or stereodirection during chemical reactions. The terms “racemic mixture” and “racemate” refer to a mixture of two equimolar enantiomers that lack optical activity.

[0067] Depending on the choice of starting materials and methods, the compounds of this invention can exist as one or a mixture of possible isomers, such as racemic mixtures and mixtures of non-corresponding isomers (depending on the number of asymmetric carbon atoms). Optically active (R)- or (S)- isomers can be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques.

[0068] Any mixture of stereoisomers obtained can be separated into pure or substantially pure geometric isomers, enantiomers, and diastereomers based on differences in the physicochemical properties of the components, for example, by chromatography and / or fractional crystallization.

[0069] In this invention, "halogen" refers to fluorine, chlorine, bromine, or iodine. "Halogenated" in this invention means substituted with fluorine, chlorine, bromine, or iodine.

[0070] In this invention, "alkyl" refers to a straight-chain or branched saturated aliphatic hydrocarbon group, preferably a straight-chain or branched group containing 1 to 6 carbon atoms, and more preferably a straight-chain or branched group containing 1 to 3 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, etc. The alkyl group can be substituted or unsubstituted, and when substituted, the substituent can be at any usable connection point.

[0071] In this invention, "carbonyl" and "acyl" both refer to -C(O)-.

[0072] In this invention, "sulfonyl" refers to -S(O)2-.

[0073] In this invention, "sulfonamide group" refers to -S(O)2NH-.

[0074] In this invention, "halogenated alkyl" refers to an alkyl group that is substituted with at least one halogen.

[0075] In this invention, "hydroxyalkyl" refers to an alkyl group that is substituted with at least one hydroxyl group.

[0076] In this invention, "alkoxy" refers to -O-alkyl. Non-limiting examples of alkoxy groups include: methoxy, ethoxy, propoxy, n-propoxy, isopropoxy, isobutoxy, sec-butoxy, etc. Alkoxy groups can be optionally substituted or unsubstituted, and when substituted, the substituent can be at any usable connection point.

[0077] In this invention, "cycloalkyl" refers to a cyclic saturated hydrocarbon group. Suitable cycloalkyl groups can be substituted or unsubstituted monocyclic groups having 3-12 carbon atoms, or fused, bridged, or spirocyclic bicyclic or tricyclic saturated hydrocarbon groups, such as cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0078] The term "heterocyclic group" in this invention refers to a group ("3- to 12-membered non-aromatic ring system") having 1 to 4 ring heteroatoms (each heteroatom being independently selected from nitrogen, oxygen, sulfur, boron, phosphorus, and silicon). In heterocyclic groups containing one or more nitrogen atoms, the connecting point can be a carbon or nitrogen atom, provided the valence permits. Heterocyclic groups can be monocyclic ("monocyclic heterocyclic group") or fused, bridged, or spirocyclic ring systems (e.g., bicyclic systems (also known as "bicyclic heterocyclic groups")), and can be saturated or partially unsaturated. Suitable heterocyclic groups include, but are not limited to, piperidinyl, aziridine, aziridine propane, tetrahydropyrroleyl, piperazineyl, dihydroquinazolinyl, oxacyclopropyl, oxacyclobutyl, tetrahydrofuranyl, tetrahydropyranyl, 6-aziridine[3.4]octyl, Each instance of a heterocyclic group can be optionally substituted or unsubstituted, and when substituted, the substituent can be at any usable junction.

[0079] In this invention, "aryl" refers to an aromatic system that may comprise a monocyclic or fused polycyclic ring, preferably a monocyclic or fused bicyclic aromatic system, containing 6 to 12 carbon atoms, more preferably about 6 to about 10 carbon atoms. Suitable aryl groups include, but are not limited to, phenyl, naphthyl, anthraceneyl, fluorenyl, and indanyl. The aryl group may be optionally substituted or unsubstituted, and when substituted, the substituent may be at any usable connection point.

[0080] In this invention, "heteroaryl" refers to an aryl group in which at least one carbon atom is replaced by a heteroatom. The heteroaryl group may have a monocyclic or fused, bridged, or spirocyclic polycyclic system, wherein at least one ring is aromatic. Preferably, it consists of 5-15 atoms (5-15-membered heteroaryl), more preferably 5-10 atoms (5-10-membered heteroaryl), wherein the heteroatom is O, S, or N. The heteroaryl groups include, but are not limited to, imidazolyl, pyrrolyl, furanyl, thiophene, pyrazolyl, oxazolyl, thiazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl, tetrazolyl, indoleyl, pyridyl, pyrimidinyl, pyrazinyl, triazinyl, isoindoleyl, benzopyrazolyl, benzimidazolyl, benzofuranyl, benzopyranyl, benzothiophene, benzooxazolyl, benzothiazolyl, benzothiazolyl, benzoisooxazolyl, benzoisothiazolyl, quinolinyl, isoquinolinyl, quinazolinyl, cenolinyl, quinoxazinyl, benzothiazolyl, imidazopyridyl, pyrimidinylpyrazolyl, pyrimidinylimidazole, benzo[d]thiazolyl, spiro[indoleyl] [Dolin-3,4'-piperidin]-2-one, 5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazinyl, 1,2,3,4-tetrahydroquinolinyl, 4,5,6,7-tetrahydro-1H-pyrrolo[3,2-c]pyridinyl, 2,4,5,6-tetrahydropyrrolo[3,4-c]pyrazolyl, 1,2,3,4-tetrahydroisoquinolinyl, 5,6,7,8-tetrahydroimidazo[1,2-a]pyrazinyl, 5,6,7,8-tetrahydro-[1,2,4]triazolo[1,5-a]pyrazinyl, 4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridinyl, etc. The heteroaryl group can be optionally substituted or unsubstituted; when substituted, the substituent can be at any usable linking point.

[0081] The term "pharmaceutically acceptable salt" in this invention refers to salts of the compounds of this invention that are safe and effective when used in mammals and possess the intended biological activity.

[0082] In the conventional sense, the term "solvent" in this invention refers to a complex formed by a combination of a solute (such as an active compound or a salt of an active compound) and a solvent (such as water). The solvent refers to a solvent known or readily identifiable to those skilled in the art. If water is present, the solvate is typically referred to as a hydrate, such as a hemihydrate, monohydrate, dihydrate, trihydrate, or a substitute thereof.

[0083] The in vivo effects of compounds having chemical formulas (I), (Ia), (Ib), and (Ic) can be partially exerted by one or more metabolites formed in the human or animal body after administration of the compounds having chemical formulas (I), (Ia), (Ib), and (Ic). As described above, the in vivo effects of compounds having chemical formulas (I), (Ia), (Ib), and (Ic) can also be exerted via the metabolism of a prodrug ("prodrug"). The "prodrug" of this invention refers to a compound that, under physiological conditions in an organism, is converted into the compound of this invention through reaction with enzymes, gastric acid, etc., i.e., a compound converted into the compound of this invention through enzymatic oxidation, reduction, hydrolysis, etc., and / or through hydrolytic reactions such as gastric acid, etc.

[0084] The “crystallization” of this invention refers to a solid whose internal structure is formed by the regular repetition of atoms (or groups thereof) in three dimensions, which is different from amorphous solids that do not have such a regular internal structure.

[0085] The term "pharmaceutical composition" as used in this invention refers to a mixture comprising any of the compounds described herein, including corresponding isomers, prodrugs, solvates, pharmaceutically acceptable salts or their chemically protected forms, and one or more pharmaceutically acceptable carriers and / or mixtures of other one or more drugs. The purpose of a pharmaceutical composition is to facilitate the administration of the compound to a living organism. Such compositions are typically used in the preparation of medicaments for the treatment and / or prevention of diseases mediated by one or more kinases.

[0086] The "pharmaceutical-grade carrier" of this invention refers to a carrier that does not cause significant irritation to the organism and does not interfere with the biological activity and properties of the administered compound. This includes all solvents, diluents or other excipients, dispersants, surfactants, isotonic agents, thickeners or emulsifiers, preservatives, solid binders, lubricants, etc., unless any conventional carrier medium is incompatible with the compounds of this invention. Some examples of pharmaceutically acceptable carriers include, but are not limited to, sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, as well as cellulose and cellulose acetate; malt, gelatin, etc.

[0087] In this invention, "excipient" refers to an inert substance added to a pharmaceutical composition to further promote the delivery of the compound. Excipients may include calcium carbonate, calcium phosphate, various sugars and various types of starch, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycol. Detailed Implementation

[0088] The present invention will be further described in detail below with reference to the embodiments, but the present invention is not limited to these embodiments. Unless otherwise specified, all materials used in the following embodiments are commercially available.

[0089] Intermediate 1: N-(3-chloro-5-(methylsulfonamido)phenyl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)thiophene-2-carboxamide

[0090]

[0091] Step 1: Preparation of N-(3-chloro-5-nitrophenyl)methanesulfonamide

[0092]

[0093] 3-Chloro-5-fluoronitrobenzene (3.5 g, 20 mmol), methanesulfonamide (2.8 g, 30 mmol), and cesium carbonate (19 g, 60 mmol) were dissolved in DMSO and reacted at 60 °C for 3 h. After the reaction was completed, monitored by LCMs, the reaction solution was extracted with saturated brine and ethyl acetate. The organic phase was collected, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography to obtain 3 g of the target compound. ESI-MS m / z: 250.9 [M+H] + .

[0094] Step 2: Preparation of N-(3-amino-5-chlorophenyl)methanesulfonamide

[0095]

[0096] N-(3-chloro-5-nitrophenyl)methanesulfonamide (3 g, 12 mmol), iron powder (6.7 g, 120 mmol), and NH4Cl (6.3 g, 120 mmol) were dissolved in 50 mL of ethanol and 30 mL of water, and stirred at 90 °C for 2 hours. After the reaction was complete as monitored by LCMs, the resulting mixture was filtered, and the filter cake was washed with methanol. The filtrate was concentrated under reduced pressure. The residue was subjected to column chromatography. The combined solutions yielded 1.7 g of the target product. ESI-MS m / z: 220.9 [M+H] + .

[0097] Step 3: Preparation of 4-bromo-N-(3-chloro-5-(methylsulfonamido)phenyl)thiophene-2-carboxamide

[0098]

[0099] 4-Bromothiophene-2-carboxylic acid (455 mg, 2.2 mmol), N-methylimidazolium (361 mg, 4.4 mmol), and N,N,N',N'-tetramethylchloromethanesulfonamide hexafluorophosphate (924 mg, 3.3 mmol) were dissolved in 10 mL of acetonitrile. Then, N-(3-amino-5-chlorophenyl)methanesulfonamide (398 mg, 1.8 mmol) was added, and the mixture was stirred at room temperature for 2 hours. After the reaction was complete as monitored by LCMs, the reaction solution was concentrated under reduced pressure and column chromatography was performed to obtain 491 mg of the target product. ESI-MS m / z: 408.7 [M+H] + .

[0100] Step 4: Preparation of N-(3-chloro-5-(methylsulfonamido)phenyl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)thiophene-2-carboxamide

[0101]

[0102] 4-Bromo-N-(3-chloro-5-(methylsulfonamido)phenyl)thiophene-2-carboxamide (491 mg, 1.2 mmol), pinacol diboronate (610 mg, 2.4 mmol), DPPF palladium dichloride (88 mg, 0.12 mmol), and potassium acetate (353 mg, 3.6 mmol) were dissolved in 20 mL of 1,4-dioxane. The mixture was heated to 80 °C and stirred for 1.5 hours under argon protection. After the reaction was completed as monitored by LCMs, the reaction solution was cooled to room temperature, extracted with ethyl acetate and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure by column chromatography to obtain 274 mg of the target product. ESI-MS m / z: 456.8 [M+H] + .

[0103] Intermediate 2: N-(3-chloro-5-(methylsulfonamido)phenyl)-5-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)thiophene-2-carboxamide

[0104]

[0105] Step 1: Preparation of 4-bromo-5-methylthiophene-2-carboxylic acid

[0106]

[0107] 10 g (70.33 mmol) of 5-methylthiophene-2-carboxylic acid and 12.365 g (77.37 mmol) of bromine and 100 mL of acetic acid were added to a 200 mL reaction flask. The mixture was stirred at 60 °C for 2 hours. After cooling to room temperature, 50 mL of water was added at 0 °C to precipitate the solid. The solid was filtered and washed with water. The filter cake was dried and proceeded directly to the next step.

[0108] Step 2: Preparation of 4-bromo-N-(3-chloro-5-(methylsulfonamido)phenyl)-5-methylthiophene-2-carboxamide

[0109]

[0110] Add 4-bromo-5-methylthiophene-2-carboxylic acid (884 mg, 4 mmol), TCFH (1683 mg, 6 mmol), and NMI (983 mg, 12 mmol) to a 50 mL round-bottom flask. Add 30 mL of solvent and stir for 5 minutes. Finally, add N-(3-amino-5-chlorophenyl)methanesulfonamide (880 mg, 4 mmol). Stir at room temperature for 1 hour. Quench the reaction with water, extract three times with ethyl acetate, combine the organic phases, and dry with saturated brine and anhydrous sodium sulfate. Column chromatography yields the product. ESI-MS m / z: 422.6 [M+H] + .

[0111] Step 3: Preparation of N-(3-chloro-5-(methylsulfonamido)phenyl)-5-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)thiophene-2-carboxamide

[0112]

[0113] To a 50 mL round-bottom flask, add 4-bromo-N-(3-chloro-5-(methylsulfonamido)phenyl)-5-methylthiophene-2-carboxamide (1 g, 2.36 mmol), diboron dipinacol (1.2 g, 4.72 mmol), Pd(dppf)Cl2 (86 mg, 0.118 mmol), potassium acetate (0.695 g, 7.08 mmol), and 20 mL of 1,4-dioxane. Under argon protection, stir at 90 °C for 1 hour. Filter directly, wash with ethyl acetate, evaporate directly to dryness, and obtain the product by column chromatography. ESI-MS m / z: 470.9 [M+H] + .

[0114] Example 1: N-(3-chloro-5-(methylsulfonamido)phenyl)-5-methyl-4-(3'H-spiro[cyclobutane-1,1'-furano[3,4-c]pyridine]-6'-yl)thiophene-2-carboxamide

[0115]

[0116] Step 1: Preparation of 6'-chloro-3'H-spiro[cyclobutane-1,1'-furano[3,4-c]pyridine]-3'′-one

[0117]

[0118] 2,2,6,6-Tetramethylpiperidine (2.7 g, 19.05 mmol) was dissolved in tetrahydrofuran (25 mL) and added to a 250 mL double-necked flask. Under argon protection, the mixture was cooled to -78 °C with liquid nitrogen and added. n-BuLi (2.5 M, 10 mL) was added dropwise, and the mixture was stirred for 1 hour. 6-Chloronicotinic acid (1 g, 6.35 mmol) was added, and the mixture was stirred for 1 hour. Cyclobutanone (0.89 g, 12.7 mmol) was then added, and the mixture was stirred for 1 hour. After the reaction was complete as monitored by LC-MS, the reaction solution was quenched with water, and the pH was adjusted to acidity with 6 M dilute hydrochloric acid. Ethyl acetate was added for extraction, and the organic phase was collected. During the concentration of the organic phase, a solid precipitated. The solid was filtered and washed to obtain the target product. ESI-MS m / z: 209.9 [M+H] + .

[0119] Step 2: Preparation of 1-(2-chloro-5-(hydroxymethyl)pyridin-4-yl)cyclobut-1-ol

[0120]

[0121] 6'-Chloro-3'H-spiro[cyclobutane-1,1'-furano[3,4-c]pyridine]-3'-one (1 g, 4.78 mmol) was added to a 100 mL single-necked flask and dissolved in 15 mL of THF. The mixture was cooled to 0 °C, and then lithium borohydride (2 M, 7.2 mL) was added. The mixture was stirred in an ice-water bath for 1 hour. After the reaction was completed, the reaction solution was diluted with water, extracted twice with ethyl acetate, and the organic phase was collected. The organic phase was washed twice with water, once with saturated brine, dried over anhydrous sodium sulfate, concentrated, and then subjected to column chromatography to obtain the product. The target product was obtained by column chromatography. ESI-MS m / z: 214.0 [M+H] +

[0122] Step 3: Preparation of 6'-chloro-3'H-spiro[cyclobutane-1,1'-furano[3,4-c]pyridine]

[0123]

[0124] 1-(2-chloro-5-(hydroxymethyl)pyridin-4-yl)cyclobutanol (0.1 g, 0.469 mmol) was added to a 100 mL single-necked flask, followed by 10 mL of THF and 1 mL of KHMDS (1 M, 1 mL). The mixture was cooled to 0 °C, and then p-toluenesulfonyl chloride (0.685 g, 5.96 mmol) was added. The reaction was carried out in an ice-water bath for 50 minutes. After the reaction was completed, the reaction solution was diluted with water, extracted twice with ethyl acetate, and the organic phase was collected. The organic phase was washed twice with water, once with saturated brine, dried over anhydrous sodium sulfate, concentrated, and then subjected to column chromatography to obtain the product. The target product was obtained by column chromatography. ESI-MS m / z: 196.0 [M+H]+

[0125] Step 4: Preparation of methyl 5-methyl-4-(3'H-spiro[cyclobutane-1,1'-furano[3,4-c]pyridin]-6'-yl)thiophene-2-carboxylate

[0126]

[0127] 6'-chloro-3'H-spiro[cyclobutane-1,1'-furano[3,4-c]pyridine] (0.056 g, 0.28 mmol) was added to a 100 mL single-necked flask, followed by methyl 5-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaboron-2-yl)thiophene-2-carboxylate (0.05 g, 0.176 mmol), Pd(OAc)2 (0.004 g, 0.0176 mmol), X-PHOS (0.008 g, 0.0176 mmol), K3PO4 (0.056 g, 0.264 mmol), and then 10 mL of dioxane. After the reaction was monitored by LC-MS, the reaction solution was diluted with water, extracted twice with ethyl acetate, and the organic phase was collected. The organic phase was washed twice with water, once with saturated brine, dried over anhydrous sodium sulfate, concentrated, and subjected to column chromatography to obtain the target product. ESI-MS m / z: 315.9 [M+H] +

[0128] Step 5: Preparation of 5-methyl-4-(3'H-spiro[cyclobutane-1,1'-furano[3,4-c]pyridin]-6'-yl)thiophene-2-carboxylic acid

[0129]

[0130] Weigh 0.16 g (0.508 mmol) of methyl 5-methyl-4-(3'H-spiro[cyclobutane-1,1'-furano[3,4-c]pyridin]-6'-yl)thiophene-2-carboxylate into a 100 mL single-necked flask, add 5 mL of dioxane and 5 mL of water, then add KOH (0.569 g, 10.16 mmol). Heat to 80 °C and react for 1 hour. After the reaction is complete as monitored by LC-MS, dilute the reaction solution with water, adjust the pH to 1-3 with 6 M dilute hydrochloric acid solution, extract twice with ethyl acetate, collect the organic phase, wash twice with water, wash once with saturated brine, dry over anhydrous sodium sulfate, concentrate, and precipitate by column chromatography to obtain the product. ESI-MS m / z: 301.9 [M+H] +

[0131] Step 6: Preparation of N-(3-chloro-5-(methylsulfonamido)phenyl)-5-methyl-4-(3'H-spiro[cyclobutane-1,1'-furano[3,4-c]pyridin]-6'-yl)thiophene-2-carboxamide

[0132]

[0133] 5-Methyl-4-(3'H-spiro[cyclobutane-1,1'-furano[3,4-c]pyridin]-6'-yl)thiophene-2-carboxylic acid (0.12 g, 0.399 mmol) was added to 5 mL of DMF, followed by TCFH (0.168 g, 0.599 mmol), NMI (0.049 g, 1.19 mmol), and then N-(3-amino-5-chlorophenyl)methanesulfonamide (0.097 g, 0.439 mmol). The mixture was reacted at 40 °C for 1 hour. After the reaction was complete as monitored by LC-MS, ethyl acetate was added for extraction, the organic phase was concentrated, and the product was obtained by column chromatography. ESI-MS m / z: 503.8 [M+H] + .

[0134] 1 H NMR (400MHz, DMSO) δ10.47(s,1H),10.07(s,1H),8.61(s,1H),8.42(s,1H),7.86(s,1H),7.70(t,J=1.8Hz,1H),7.65( t,J=1.8Hz,1H),6.95(t,J=1.9Hz,1H),5.07(s,2H),3.07(s,3H),2.73(s,3H),2.45-2.37(m,2H),2.12-1.88(m,4H).

[0135] Example 2: N-(3-chloro-5-(methylsulfonamido)phenyl)-5-methyl-4-(1'-methyl-2'-oxo-1',2'-dihydrospiro[cyclobutane-1,3'-pyrrolo[2,3-c]pyridine]-5'-yl)thiophene-2-carboxamide

[0136]

[0137] Step 1: Preparation of 5'-chlorospiro[cyclobutane-1,3'-pyrrolo[2,3-c]pyridine]-2'(1'H)-one

[0138]

[0139] Weigh 1.0 g (5.9 mmol) of 5-chloro-1,3-dihydro-2H-pyrrolo[2,3-c]pyridin-2-one into a two-necked flask, dissolve it in 5 mL of N,N-dimethylformamide, purge with argon, cool to -10 °C, and slowly add 10 mL of N,N-dimethylformamide solution containing 0.71 g (17.8 mmol) of sodium hydroxide. After addition, stir at -10 °C for 5 min, then add 10 mL of N,N-dimethylformamide solution containing 1.2 g (5.9 mmol) of 1,3-dibromopropane. After addition, stir at -10 °C for 2 h. After the reaction is complete, quench with water, extract with ethyl acetate, combine the organic phases, dry to anhydrous sodium sulfate, and perform silica gel column chromatography. The target product elutes at 15%-20% (ethyl acetate / petroleum ether), collect, and dry under vacuum to obtain 0.18 g of product. ESI-MS m / z: 209.0 [M+H] + .

[0140] Step 2: Preparation of 5'-chloro-1'-methylspiro[cyclobutane-1,3'-pyrrolo[2,3-c]pyridine]-2'(1'H)-one

[0141]

[0142] Weigh 0.18 g (0.86 mmol) of 5'-chlorospiro[cyclobutane-1,3'-pyrrolo[2,3-c]pyridine]-2'(1'H)-one into a reaction flask, dissolve in tetrahydrofuran (5 mL), cool to 0 °C in an ice bath, add sodium hydride (0.11 g, 2.64 mmol), stir for 5 min in an ice bath, add iodomethane (0.13 g, 0.86 mmol), react for 1.5 h in an ice bath. After the reaction is complete, quench with water, extract with ethyl acetate, dry under anhydrous sodium sulfate, and vacuum dry to give 0.15 g of product. ESI-MS m / z: 223.0 [M+H] +

[0143] Step 3: Preparation of methyl 5-methyl-4-(1'-methyl-2'-oxo-1',2'-dihydrospiro[cyclobutane-1,3'-pyrrolo[2,3-c]pyridine]-5'-yl)thiophene-2-carboxylate

[0144]

[0145] Weigh out 0.14 g (0.63 mmol) of 5'-chloro-1'-methylspiro[cyclobutane-1,3'-pyrrolo[2,3-c]pyridine]-2'(1'H)-one, methyl 5-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborin-2-yl)thiophene-2-carboxylate, 0.20 g (0.69 mmol) of palladium acetate, 0.014 g (0.63 mmol) of 2-dicyclohexylphosphine-2',4',6 '-Triisopropylbiphenyl (0.028 g, mmol) and potassium phosphate (0.42 g, 1.89 mmol) were added to a two-necked flask, followed by 1,4-dioxane (15 mL) and water (2.5 mL). The mixture was purged with argon and reacted in an oil bath at 70 °C for 8 h. After the reaction was complete, the mixture was filtered, extracted with ethyl acetate, and the organic phases were combined, dried over anhydrous sodium sulfate, and subjected to silica gel column chromatography. The target product was eluted at 15% (ethyl acetate / petroleum ether), collected, and dried under vacuum to give 0.17 g of the product. ESI-MS m / z: 342.9 [M+H] +

[0146] Step 4: Preparation of N-(3-chloro-5-(methylsulfonamido)phenyl)-5-methyl-4-(1'-methyl-2'-oxo-1',2'-dihydrospiro[cyclobutane-1,3'-pyrrolo[2,3-c]pyridine]-5'-yl)thiophene-2-carboxamide

[0147]

[0148] Weigh methyl 5-methyl-4-(1'-methyl-2'-oxo-1',2'-dihydrospiro[cyclobutane-1,3'-pyrrolo[2,3-c]pyridine]-5'-yl)thiophene-2-carboxylate (0.11 g, 0.32 mmol) and N-(3-amino-5-chlorophenyl)methanesulfonamide (0.088 g, 0.38 mmol) into a two-necked flask, dissolve in toluene (5 mL), purge with argon, cool to 0 °C in an ice bath, slowly add 2 mol / L trimethylaluminum toluene solution (0.48 mL, 0.96 mmol), stir for 5 min in an ice bath after addition, transfer to an oil bath, heat to 70 °C, react for 5 h, after the reaction is complete, quench with saturated sodium carbonate solution, extract with ethyl acetate, combine the organic phases, dry with anhydrous sodium sulfate, vacuum dry, and transfer to the separation platform to obtain 0.023 g of product. ESI-MS m / z: 530.8 [M+H] + . 1H NMR(400MHz,DMSO)δ10.46(s,1H),8.63(s,1H),8.39(s,1H),8.31(s,1H),7.72-7.65(m,2H),7.52(s,1H),6 .95(s,1H),4.48-4.42(m,2H),3.64(s,3H),3.07(s,3H),2.70(s,3H),2.69-2.64(m,2H),2.09-1.99(m,2H).

[0149] Example 3: N-(3-chloro-5-(methylsulfonamido)phenyl)-4-(5,6-dihydroimidazo[1,2-d]pyrido[4,3-f][1,4]oxazine) -10-yl)-5-methylthiophene-2-carboxamide

[0150]

[0151] Step 1: Preparation of 2-chloro-5-methoxypyridine

[0152]

[0153] 2-Chloro-5-hydroxypyridine (1.5 g, 11.6 mmol) was dissolved in dimethyl sulfoxide (10 mL), followed by the addition of potassium carbonate (4.8 g, 34.8 mmol) and methyl iodoform (1.0 mL, 17.4 mmol). The reaction was carried out at 120 °C for 3 hours. After the reaction was complete, the solution was diluted with water, extracted with ethyl acetate, washed with water and saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography to obtain 1.8 g of an oily substance. ESI-MS m / z: 144.1 [M+H] +

[0154] Step 2: Preparation of 2-chloro-5-methoxyisoniamaldehyde

[0155]

[0156] 2-Chloro-5-methoxypyridine (5 g, 34.7 mmol) was weighed into a 200 mL reaction flask and placed at -78 °C. It was dissolved in tetrahydrofuran (60 mL), and diisopropylaminolithium (35 mL, 70 mmol) was slowly added. The reaction was carried out at -78 °C for 1 h. Then, N,N-dimethylformamide (3.8 g, 53 mmol) was slowly added, and the reaction was carried out at -78 °C for 2 h. After the reaction was complete, the solution was diluted with water, extracted with ethyl acetate, washed with water and saturated brine, dried over anhydrous sodium sulfate, and separated by column chromatography to obtain 5.5 g. ESI-MS m / z: 172.1 [M+H] +

[0157] Step 3: Preparation of 2-chloro-4-(1H-imidazol-2-yl)-5-methoxypyridine

[0158]

[0159] 2-Chloro-5-methoxyisoninaldehyde (5.5 g, 32.2 mmol) was dissolved in ethanol (15 mL), and 40% glyoxal aqueous solution (10 mmol) and 25% ammonia solution (35 mL) were added. The reaction was allowed to proceed overnight at room temperature. After the reaction was complete, the reaction was quenched with water, the pH was adjusted to 2 with hydrochloric acid, and the extract was extracted with ethyl acetate and discarded. The pH was then adjusted to alkaline with sodium bicarbonate aqueous solution, and the extract was extracted with ethyl acetate. The ethyl acetate phase was collected, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 1.5 g. ESI-MS m / z: 210.1 [M+H] +

[0160] Step 4: Preparation of 6-chloro-4-(1H-imidazol-2-yl)pyridin-3-ol

[0161]

[0162] 1.5 g (7.1 mmol) of 2-chloro-4-(1H-imidazol-2-yl)-5-methoxypyridine was added to 30 mL of 40% hydrobromic acid aqueous solution and reacted overnight at 120 °C. After the reaction was complete, the solution was concentrated under reduced pressure to give 2.0 g of an oil. ESI-MS m / z: 196.1 [M+H] +

[0163] Step 5: 10-Chloro-5,6-dihydroimidazo[1,2-d]pyrido[4,3-f][1,4]oxazine Preparation

[0164]

[0165] 6-Chloro-4-(1H-imidazol-2-yl)pyridin-3-ol (2.0 g, 10.2 mmol) was added to N,N-dimethylformamide (20 mL), followed by dibromoethane (2 mL). The mixture was reacted at 90 °C for 3 hours. After the reaction was complete, the mixture was filtered, the filtrate was collected, concentrated under reduced pressure, and separated by column chromatography to obtain 150 mg. ESI-MS m / z: 222.1 [M+H] +

[0166] Step 6: N-(3-chloro-5-(methylsulfonamido)phenyl)-4-(5,6-dihydroimidazo[1,2-d]pyrido[4,3-f][1,4]oxazine) Preparation of 10-yl)-5-methylthiophene-2-carboxamide

[0167]

[0168] Weigh out 10-chloro-5,6-dihydroimidazo[1,2-d]pyrido[4,3-f][1,4]oxazine 0.15 g (0.67 mmol) and N-(3-chloro-5-(methylsulfonamido)phenyl)-5-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)thiophene-2-carboxamide (0.18 g, 0.38 mmol) were added to a reaction flask, dissolved in 10 mL of 1,4-dioxane, and 1,1-bis(diphenylphosphine)diberberine palladium dichloride (50 mg, 0.067 mmol) and cesium carbonate (653 mg, 2.01 mm) were added. The mixture was reacted at 90 °C for 1 h under argon protection. After the reaction was complete, the mixture was extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to obtain the target product. ESI-MS m / z: 529.9 [M+H] + . 1 H NMR (400MHz, DMSO) δ10.57(s,1H),8.58(s,1H),8.44(s,2H),7.65(d,J=21.2Hz,2 H),7.51(s,1H),7.20(s,1H),6.94(s,1H),4.56(s,4H),3.06(s,3H),2.72(s,3H).

[0169] Example 4: N-(3-chloro-5-(methylsulfonamido)phenyl)-5-methyl-4-(1'-methyl-2'-oxo-1',2'-dihydrospiro[cyclobutane-1,3'-pyrrolo[3,2-c]pyridine]-6'-yl)thiophene-2-carboxamide

[0170]

[0171] Step 1: Preparation of 6'-chlorospiro[cyclobutane-1,3'-pyrrolo[3,2-c]pyridine]-2'(1'H)-one

[0172]

[0173] Weigh 1.0 g (5.9 mmol) of 6-chloro-1,3-dihydro-2H-pyrrolo[3,2-c]pyridin-2-one into a two-necked flask, dissolve it in 5 mL of N,N-dimethylformamide, purge with argon, cool to -10 °C, and slowly add 10 mL of N,N-dimethylformamide solution containing 0.71 g (17.8 mmol) of sodium hydroxide. After addition, stir at -10 °C for 5 min, then add 10 mL of N,N-dimethylformamide solution containing 1.2 g (5.9 mmol) of 1,3-dibromopropane. After addition, stir at -10 °C for 2 h. After the reaction is complete, quench with water, extract with ethyl acetate, combine the organic phases, dry to anhydrous sodium sulfate, and perform silica gel column chromatography. The target product elutes at 15%-20% (ethyl acetate / petroleum ether), collect, and dry under vacuum to obtain 0.18 g of product. ESI-MS m / z: 209.0 [M+H] + .

[0174] Step 2: Preparation of 6'-chloro-1'-methylspiro[cyclobutane-1,3'-pyrrolo[3,2-c]pyridine]-2'(1'H)-one

[0175]

[0176] Weigh 0.18 g (0.86 mmol) of 6'-chlorospiro[cyclobutane-1,3'-pyrrolo[3,2-c]pyridine]-2'(1'H)-one into a reaction flask, dissolve in tetrahydrofuran (5 mL), cool to 0 °C in an ice bath, add sodium hydride (0.11 g, 2.64 mmol), stir for 5 min in an ice bath, add iodomethane (0.13 g, 0.86 mmol), react for 1.5 h in an ice bath. After the reaction is complete, quench with water, extract with ethyl acetate, dry under anhydrous sodium sulfate, and vacuum dry to give 0.15 g of product, yield 78.9%. ESI-MS m / z: 223.0 [M+H] +

[0177] Step 3: Preparation of methyl 5-methyl-4-(1'-methyl-2'-oxo-1',2'-dihydrospiro[cyclobutane-1,3'-pyrrolo[3,2-c]pyridine]-6'-yl)thiophene-2-carboxylate

[0178]

[0179] Weigh out 0.14 g (0.63 mmol) of 6'-chloro-1'-methylspiro[cyclobutane-1,3'-pyrrolo[3,2-c]pyridine]-2'(1'H)-one, methyl 5-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborin-2-yl)thiophene-2-carboxylate, 0.20 g (0.69 mmol) of palladium acetate, 0.014 g (0.63 mmol) of 2-dicyclohexylphosphine-2',4',6 '-Triisopropylbiphenyl (0.028 g, mmol) and potassium phosphate (0.42 g, 1.89 mmol) were added to a two-necked flask, followed by 1,4-dioxane (15 mL) and water (2.5 mL). The mixture was purged with argon and reacted in an oil bath at 70 °C for 8 h. After the reaction was complete, the mixture was filtered, extracted with ethyl acetate, and the organic phases were combined, dried over anhydrous sodium sulfate, and subjected to silica gel column chromatography. The target product was eluted at 15% (ethyl acetate / petroleum ether), collected, and dried under vacuum to give 0.17 g of the product. ESI-MS m / z: 342.9 [M+H] +

[0180] Step 4: Preparation of N-(3-chloro-5-(methylsulfonamido)phenyl)-5-methyl-4-(1'-methyl-2'-oxo-1',2'-dihydrospiro[cyclobutane-1,3'-pyrrolo[3,2-c]pyridine]-6'-yl)thiophene-2-carboxamide

[0181]

[0182] Weigh methyl 5-methyl-4-(1'-methyl-2'-oxo-1',2'-dihydrospiro[cyclobutane-1,3'-pyrrolo[3,2-c]pyridin]-6'-yl)thiophene-2-carboxylate (0.11 g, 0.32 mmol) and N-(3-amino-5-chlorophenyl)methanesulfonamide (0.088 g, 0.38 mmol) into a two-necked flask, dissolve in toluene (5 mL), purge with argon, cool to 0°C in an ice bath, slowly add 2 mol / L trimethylaluminum toluene solution (0.48 mL, 0.96 mmol), stir for 5 min in an ice bath after addition, transfer to an oil bath, heat to 70°C, react for 5 h. After the reaction is complete, quench with saturated sodium carbonate solution, extract with ethyl acetate, combine the organic phases, dry with anhydrous sodium sulfate, vacuum dry, and transfer to the separation platform to obtain 0.032 g of product. ESI-MS m / z: 530.8 [M+H] + . 1H NMR (400MHz, DMSO) δ10.49(s,1H),8.77(s,1H),8.37(s,1H),7.69(t,1H),7.64(t,J=1.8Hz,1H),7.30(s, 1H), 6.94 (t, J = 1.9Hz, 1H), 3.20 (s, 3H), 3.07 (s, 3H), 2.73 (s, 3H), 2.46-2.34 (m, 4H), 2.33-2.20 (m, 2H).

[0183] Example 5: (R)-N-(3-chloro-5-(methylsulfonamido)phenyl)-5-methyl-4-(9-oxo-6a,7,8,9-tetrahydro-6H-pyrido[3,4-b]pyrrolo[1,2-d][1,4]oxazin-2-yl)thiophene-2-carboxamide

[0184]

[0185] Step 1: Preparation of 4-bromo-6-chloropyridine-3-ol

[0186]

[0187] 4-Bromo-2-chloro-5-methoxypyridine (2.21 g, 10 mmol) was added to a reaction flask, purged three times with Ar gas, then DCM (20 mL) was added. BBr3 (2.89 mL, 30 mmol) was then slowly added dropwise at 0 °C, and the reaction mixture was stirred overnight at room temperature. The reaction was monitored by LC-MS. After the reaction was complete, the reaction solution was quenched with methanol, and methanol was added dropwise until a solid precipitated. The mixture was filtered, the filter cake was washed with a small amount of methanol, and then dried to give 1.98 g (95.69%) of a white solid. ESI-MS m / z: 207.8 [M+H] + .

[0188] Step 2: Preparation of (R)-5-(((4-bromo-6-chloropyridin-3-yl)oxy)methyl)pyrrolidine-2-one

[0189]

[0190] 4-Bromo-6-chloropyridin-3-ol (2.07 g, 10 mmol) and (S)-5-(bromomethyl)pyrrolidone-2-one (1.95 g, 11 mmol) were dissolved in DMF (30 mL), and then Cs₂CO₃ (9.77 mg, 30 mmol) was added. The reaction mixture was stirred overnight at room temperature. The reaction was monitored by LCMS. After the reaction was complete, the solid was filtered off, and the reaction mixture was evaporated to dryness. The mixture was extracted three times with EA, and the organic phases were combined, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give 2.696 g (88.70%) of crude yellow solid. ESI-MS m / z: 304.8 [M+H] + .

[0191] Step 3: Preparation of (R)-2-chloro-6,6a,7,8-tetrahydro-9H-pyrido[3,4-b]pyrrolo[1,2-d][1,4]oxazin-9-one

[0192]

[0193] (R)-5-(((4-bromo-6-chloropyridin-3-yl)oxy)methyl)pyrrolidone-2-one (2.619 g, 8.62 mmol), Pd2(dba)3 (394.7 mg, 0.431 mmol), Xantphos (498.8 mg, 0.862 mmol), and Cs2CO3 (8.4 g, 25.86 mmol) were added to a reaction flask, purged three times with Ar gas, and then Dioxane (60 mL) was added. The reaction system was stirred at 100 °C for 2 h. The reaction was monitored by LCMS. After the reaction was completed, the solid was filtered off, and the reaction solution was evaporated to dryness. The solution was extracted three times with EA, the organic phases were combined, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and column chromatography was performed to obtain 1.75 g (90.63%) of a yellow-green solid.

[0194] ESI-MS m / z: 225.0 [M+H] + .

[0195] Step 4: Preparation of (R)-N-(3-chloro-5-(methylsulfonamido)phenyl)-5-methyl-4-(9-oxo-6a,7,8,9-tetrahydro-6H-pyrido[3,4-b]pyrrolo[1,2-d][1,4]oxazin-2-yl)thiophene-2-carboxamide

[0196]

[0197] (R)-2-chloro-6,6a,7,8-tetrahydro-9H-pyrido[3,4-b]pyrrolo[1,2-d][1,4]oxazin-9-one (89.6 mg, 0.4 mmol), N-(3-chloro-5-(methylsulfonamido)phenyl)-5-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)thiophene-2-carboxamide (376.1 mg, 0.8 mmol), Pd(OAc)2 (9.1 mg, 0.04 mmol), X-phos (38.1 mg, 0.08 mmol), and K3PO4 (254.7 mg, 1.2 mmol) were added to the reaction flask, purged three times with Ar gas, and then Dioxane (10 mL) and H2O (2 mL) were added. The reaction system was stirred at 100 °C for 2 h. The reaction was monitored by LCMS. After the reaction was complete, the reaction mixture was evaporated to dryness. The solution was extracted three times with EA, the organic phases were combined, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain 25 mg of a white solid. ESI-MS m / z: 532.8 [M+H] + . 1 H NMR(400MHz,DMSO)δ10.53(s,1H),10.11(s,1H),8.62(s,1H),8.35(s,1H),8.32(s, 1H),7.68(s,1H),7.63(s,1H),6.94(s,1H),4.65(dd,J=10.7,3.0Hz,1H),4.13(qd, J=9.7,3.1Hz,1H),3.86(t,J=10.3Hz,1H),3.06(s,3H),2.75-2.67(m,1H),2.65(s, 3H), 2.41 (dd, J=17.0, 9.1Hz, 1H), 2.30-2.22 (m, 1H), 1.75 (dt, J=21.4, 10.6Hz, 1H).

[0198] Example 6: N-(3-chloro-5-(methylsulfonamido)phenyl)-4-(5H-imidazo[1,2-c]pyrido[4,3-e][1,3]oxazin-9-yl)-5-methylthiophene-2-carboxamide

[0199]

[0200] Step 1: Preparation of 9-chloro-5H-imidazolium[1,2-c]pyridine[4,3-e][1,3]oxazine

[0201]

[0202] 2.0 g (10.2 mmol) of 6-chloro-4-(1H-imidazol-2-yl)pyridin-3-ol was added to 20 mL of N,N-dimethylformamide, followed by 2 mL of diiodomethane. The mixture was reacted at 90 °C for 0.5 h. After the reaction was complete, the mixture was filtered, extracted with ethyl acetate, and the ethyl acetate phase was collected, dried over anhydrous sodium sulfate, and separated by column chromatography to obtain 130 mg. ESI-MS m / z: 207.9 [M+H] +

[0203] Step 2: Preparation of N-(3-chloro-5-(methylsulfonamido)phenyl)-4-(5H-imidazo[1,2-c]pyrido[4,3-e][1,3]oxazin-9-yl)-5-methylthiophene-2-carboxamide

[0204]

[0205] Weigh 0.13 g (0.628 mmol) of 9-chloro-5H-imidazolium[1,2-c]pyridine[4,3-e][1,3]oxazine and 0.3 g (0.6 mmol) of N-(3-chloro-5-(methylsulfonamido)phenyl)-5-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)thiophene-2-carboxamide into a reaction flask. Add 10 mL of 1,4-dioxane to dissolve 2 mL of water. Add 50 mg (0.067 mmol) of 1,1-bis(diphenylphosphine)diberberine palladium dichloride and 653 mg (2.01 mm) of cesium carbonate. Under argon protection, react at 90 °C for 1 h. After the reaction is complete, extract with ethyl acetate, wash with saturated brine, dry with anhydrous sodium sulfate, and concentrate to obtain the target product. ESI-MS m / z: 515.8 [M+H] + . 1 H NMR (400MHz, DMSO) δ10.49(s,1H),10.09(s,1H),8.56(s,1H),8.48(s,1H),8.00(s,1H),7.69(t,J=1.8Hz,1H),7.65(t,J =1.8Hz,1H),7.47(d,J=1.1Hz,1H),7.28(d,J=1.1Hz,1H),6.96(t,J=1.9Hz,1H),6.15(s,2H),3.07(s,3H),2.75(s,3H).

[0206] Example 7: (R)-N-(3-chloro-5-(methylsulfonamido)phenyl)-5-methyl-4-(9-oxo-6a,7,8,9-tetrahydro-6H-pyrido[4,3-b]pyrrolo[1,2-d][1,4]oxazin-3-yl)thiophene-2-carboxamide

[0207]

[0208] Step 1: Preparation of (S)-5-(((5-bromo-2-chloropyridin-4-yl)oxy)methyl)pyrrolidine-2-one

[0209]

[0210] 5-Bromo-2-chloropyridin-4-ol (1.557 g, 7.47 mmol) and (R)-5-(hydroxymethyl)pyrrolidone-2-one (1.032 g, 8.96 mmol) were added to a reaction flask, purged three times with Ar gas, and then THF (20 mL) and PPh3 (2.351 g, 8.96 mmol) were added. DEAD (1.411 mL, 8.96 mmol) was slowly added dropwise at 0 °C, and the mixture was stirred overnight at room temperature. After the reaction was complete, the reaction solution was evaporated to dryness. Water (30 mL) was added to the reaction system, and the mixture was extracted three times with EA. The organic phases were combined, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and column chromatography was performed to give 2.05 g (90.31%) of a yellow solid. ESI-MS m / z: 304.9 [M+H] + .

[0211] Step 2: Preparation of (R)-3-chloro-6,6a,7,8-tetrahydro-9H-pyrido[4,3-b]pyrrolo[1,2-d][1,4]oxazin-9-one

[0212]

[0213] (S)-5-(((5-bromo-2-chloropyridin-4-yl)oxy)methyl)pyrrolidone-2-one (2.371 g, 7.8 mmol), Pd2(dba)3 (714.2 mg, 0.78 mmol), Xantphos (902.6 mg, 1.56 mmol), and Cs2CO3 (7.624 g, 23.4 mmol) were added to a reaction tube, purged three times with Ar gas, and then Dioxane (40 mL) was added. The reaction system was stirred at 100 °C for 2.0 h. The reaction was monitored by TLC and LCMS. After the reaction was completed, the reaction solution was evaporated to dryness. Water (40 mL) was added to the reaction system, and the mixture was extracted three times with EA. The organic phases were combined, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and column chromatography was performed to obtain 0.9 g (51.52%) of a yellow-green solid.

[0214] ESI-MS m / z: 225.0 [M+H] + .

[0215] Step 3: Preparation of (R)-5-methyl-4-(9-oxo-6a,7,8,9-tetrahydro-6H-pyrido[4,3-b]pyrrolo[1,2-d][1,4]oxazin-3-yl)thiophene-2-carboxylic acid methyl ester

[0216]

[0217] (R)-3-chloro-6,6a,7,8-tetrahydro-9H-pyrido[4,3-b]pyrrolo[1,2-d][1,4]oxazin-9-one (900 mg, 4 mmol), methyl 5-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)thiophene-2-carboxylate (1.242 mg, 4.4 mmol), Pd(OAc)2 (90.6 mg, 0.4 mmol), X-phos (381.4 mg, 0.8 mmol), and K3PO4 (2.547 g, 12 mmol) were added to the reaction tube. The mixture was purged three times with Ar gas, and then Dioxane (30 mL) and H2O (4 mL) were added. The reaction system was stirred at 80 °C for 4.0 h. The reaction was monitored by TLC and LCMS. After the reaction was completed, the reaction solution was evaporated to dryness. Water (40 mL) was added to the reaction system, and the mixture was extracted three times with EA. The organic phases were combined, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by column chromatography to give 457 mg (33.21%) of a pale yellow solid. ESI-MS m / z: 344.9 [M+H] + .

[0218] Step 4: Preparation of (R)-5-methyl-4-(9-oxo-6a,7,8,9-tetrahydro-6H-pyrido[4,3-b]pyrrolo[1,2-d][1,4]oxazin-3-yl)thiophene-2-carboxylic acid

[0219]

[0220] (R)-5-methyl-4-(9-oxo-6a,7,8,9-tetrahydro-6H-pyrido[4,3-b]pyrrolo[1,2-d][1,4]oxazin-3-yl)thiophene-2-carboxylate (457 mg, 1.33 mmol) was dissolved in MeOH (10 mL) and H₂O (3 mL), and LiOH (159.6 mg, 6.65 mmol) was added. The reaction mixture was stirred at 50 °C for 2.0 h. The reaction was monitored by TLC and LCMS. After the reaction was complete, the pH of the reaction solution was adjusted to 5-6, and a solid precipitated out. The solid was filtered and dried to give 400 mg (91.17%) of white solid. ESI-MS m / z: 330.9 [M+H] + .

[0221] Step 5: Preparation of (R)-N-(3-bromo-5-chlorophenyl)-5-methyl-4-(9-oxo-6a,7,8,9-tetrahydro-6H-pyrido[4,3-b]pyrrolo[1,2-d][1,4]oxazin-3-yl)thiophene-2-carboxamide

[0222]

[0223] (R)-5-methyl-4-(9-oxo-6a,7,8,9-tetrahydro-6H-pyrido[4,3-b]pyrrolo[1,2-d][1,4]oxazin-3-yl)thiophene-2-carboxylic acid (369 mg, 1.12 mmol) and 3-bromo-5-chloroaniline (275.4 mg, 1.344 mmol) were dissolved in DMF (10 mL), NMI (309 μL, 3.92 mmol) was added, and the mixture was stirred for 5 min. Then, TCFH (377.1 mg, 1.344 mmol) was added, and the reaction mixture was stirred at room temperature for 4.0 h. The reaction was monitored by TLC and LCMS. After the reaction was completed, the reaction solution was evaporated to dryness. Water (40 mL) was added to the reaction system, and the mixture was extracted three times with EA. The organic phases were combined, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by column chromatography to give 429 mg (74.09%) of a yellow solid. ESI-MS m / z: 517.7 [M+H] + .

[0224] Step 6: Preparation of (R)-N-(3-chloro-5-(methylsulfonamido)phenyl)-5-methyl-4-(9-oxo-6a,7,8,9-tetrahydro-6H-pyrido[4,3-b]pyrrolo[1,2-d][1,4]oxazin-3-yl)thiophene-2-carboxamide

[0225]

[0226] (R)-N-(3-bromo-5-chlorophenyl)-5-methyl-4-(9-oxo-6a,7,8,9-tetrahydro-6H-pyrido[4,3-b]pyrrolo[1,2-d][1,4]oxazin-3-yl)thiophene-2-carboxamide (155 mg, 0.3 mmol), methanesulfonamide (57 mg, 0.6 mmol), CuI (28.6 mg, 0.15 mmol), N,N'-dimethyl-1,2-cyclohexanediamine (47.3 μL, 0.3 mmol), and K3PO4 (191.1 mg, 0.9 mmol) were added to a reaction tube. The mixture was purged three times with Ar gas, and then Dioxane (6 mL) was added. The reaction system was stirred at 100 °C for 8.0 h. The reaction was monitored by TLC and LCMS. After the reaction was complete, the solid was filtered off, and the reaction solution was evaporated to dryness. Column chromatography yielded 55 mg (34.41%) of a yellow product. After preparation and separation, 24 mg of a white solid was obtained. ESI-MS m / z: 532.8 [M+H] + . 1 H NMR(400MHz,DMSO)δ10.42(s,1H),9.51(s,1H),8.37(s,1H),7.66(s,1H),7.64(s,1H ),7.28(s,1H),6.94(s,1H),4.68(dd,J=10.6,2.8Hz,1H),4.09(td,J=9.7,2.9Hz,1H) ,3.96(t,J=10.3Hz,1H),3.06(s,3H),2.68(s,3H),2.63(dd,J=13.7,6.7Hz,1H),2.38 (dd,J=17.4,9.3Hz,1H),2.28(dt,J=11.6,8.9Hz,1H),1.76(dt,J=20.8,10.2Hz,1H).

[0227] Example 8: 4-(benzofurano[3,2-c]pyridin-3-yl)-N-(3-chloro-5-(methylsulfonamido)phenyl)thiophene-2-carboxamide

[0228]

[0229] Step 1: Preparation of 2-chloro-5-(2-methoxyphenyl)pyridine-4-amine

[0230]

[0231] Potassium phosphate (7.32 g, 31.44 mmol), triphenylphosphine (0.42 g, 1.57 mmol), 2-chloro-5-iodo-4-pyridinamine (4.00 g, 15.72 mmol), 2-methoxyphenylboronic acid (3.38 g, 22.32 mmol), and palladium acetate (0.18 g, 0.786 mmol) were added sequentially to a 250 mL three-necked flask. Acetonitrile and water were added, and the mixture was purged three times with argon. The mixture was stirred at 75 °C for 2 h under argon protection. After the reaction was completed as monitored by LC-MS, the reaction solution was filtered to remove the solid. The filtrate was extracted twice with ethyl acetate, and the organic phase was washed three times with water and saturated brine. The solution was dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The crude product was subjected to column chromatography with petroleum ether / ethyl acetate. The product was purified under reduced pressure at 21% concentration to obtain 3.8 g of a brownish-red solid. ESI-MS m / z: 235.58 [M+H]+.

[0232] Step 2: Preparation of 3-chlorobenzofurano[3,2-c]pyridine

[0233]

[0234] 2-Chloro-5-(2-methoxyphenyl)pyridine-4-amine was weighed into a 100 mL single-necked flask and dissolved in acetic acid (42 mL) and concentrated sulfuric acid (0.2 mL). Tert-butyl nitrite (2.00 g, 20.25 mmol) was dissolved in acetic acid (2.1 mL) and added to the reaction flask with stirring. After stirring for 30 min and monitoring the reaction at TCL until complete, acetic acid was removed directly under reduced pressure. The mixture was then extracted with dichloromethane. The organic phase was washed twice with water and saturated sodium bicarbonate, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The crude product was subjected to column chromatography with petroleum ether / ethyl acetate. The product was purified at 5% concentration, and the solvent was removed under reduced pressure to give 900 mg of a white solid. ESI-MS m / z: 204.5 [M+H]+.

[0235] Step 3: Preparation of 4-(benzofurano[3,2-c]pyridin-3-yl)-N-(3-chloro-5-(methylsulfonamido)phenyl)thiophene-2-carboxamide

[0236]

[0237] N-(3-chloro-5-(methylsulfonamido)phenyl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)thiophene-2-carboxamide (90 mg, 0.45 mmol), [1,1'-bis(diphenylphosphine)ferrocene]palladium dichloride (16 mg, 0.02 mmol), and potassium carbonate (186 mg, 1.35 mmol) were added sequentially to a 50 ml double-necked flask. 1,4-dioxane was added to dissolve the carboxylic acid (5 ml), followed by the addition of 3-chlorobenzofuran. [3,2-c]pyridine (205 mg, 0.45 mmol), water (1 ml), purged three times with argon, stirred at 90 °C for 2 hours under argon protection. After the reaction was complete as monitored by LC-MS, the mixture was extracted with ethyl acetate. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The crude product was subjected to column chromatography in a petroleum ether / ethyl acetate system. 200 mg of the brown solid obtained after solvent removal under reduced pressure at 50-60% was sent to a preparative separation platform for purification. After freeze-drying, 50 mg of a white solid was obtained. ESI-MS m / z: 497.8 [M+H] + . 1 H NMR (400MHz, DMSO) δ10.68(s,1H),10.15(s,1H),9.47(s,1H),8.87(d,J=1.1Hz,1H),8.66(d,J=1.1Hz,1H),8.33(s,1H),8.29(d,J= 7.3Hz,1H),7.83(d,J=8.2Hz,1H),7.75-7.72(m,2H),7.65-7.61(m,1H),7.52(t,J=7.5Hz,1H),6.99(t,J=1.9Hz,1H),3.11(s,3H).

[0238] Example 9: N-(3-chloro-5-(methylsulfonamido)phenyl)-4-(6-methyl-6,7,8,9-tetrahydro-5H-pyrrolo[2,3-b:4,5-c']bipyridin-2-yl)thiophene-2-carboxamide

[0239]

[0240] Step 1: Preparation of 2-bromo-6-(2-(1-methylpiperidin-4-yl)hydrazino)pyridine

[0241]

[0242] 2-Bromo-6-hydrazinopyridine (5 g, 27 mmol) was dissolved in ethanol (15 mL), and 1-methylpiperidin-4-one (3 g, 27 mmol) was added. The reaction was carried out at 50 °C for 2 hours. After the reaction was completed, the solution was concentrated under reduced pressure to give 7 g of a brown powdery solid. ESI-MS m / z: 282.9 [M+H] +

[0243] Step 2: Preparation of 2-bromo-6-methyl-6,7,8,9-tetrahydro-5H-pyrrolo[2,3-b:4,5-c']bipyridine

[0244]

[0245] Weigh 5 g (23 mmol) of 2-bromo-6-(2-(1-methylpiperidin-4-yl)hydrazino)pyridine and add it to polyphosphoric acid pre-stirred at 180 °C. Stir overnight at this temperature. After the reaction is complete, quench the reaction by slowly adding 300 mL of water at 90 °C, and adjust the pH to 8 with sodium hydroxide. A large amount of brown solid precipitates. Filter the mixture, wash the filter cake with plenty of water, collect the cake, and dry it under reduced pressure to obtain 3 g of powdery brown solid, which is directly added to the next step. ESI-MS m / z: 268.9 [M+H] +

[0246] Step 3: Preparation of N-(3-chloro-5-(methylsulfonamido)phenyl)-4-(6-methyl-6,7,8,9-tetrahydro-5H-pyrrolo[2,3-b:4,5-c'-]bipyridin-2-yl)thiophene-2-carboxamide

[0247]

[0248] Weigh 0.12 g (0.45 mmol) of 2-bromo-6-methyl-6,7,8,9-tetrahydro-5H-pyrrolo[2,3-b:4,5-c'-]bipyridine and 0.2 g (0.45 mmol) of N-(3-chloro-5-(methylsulfonamido)phenyl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)thiophene-2-carboxamide into a reaction flask. Add 5 mL of 1,4-dioxane to dissolve 1 mL of water. Add 320 mg (0.04 mmol) of 1,1-bis(diphenylphosphine)diferro-palladium dichloride and 286 mg (1.35 mmol) of cesium carbonate. Under argon protection, react at 90 °C for 1 h. After the reaction is complete, extract with ethyl acetate, wash with saturated brine, dry with anhydrous sodium sulfate, concentrate, and separate by column chromatography to obtain the target product. ESI-MS m / z: 515.8 [M+H] + . 1H NMR (400MHz, DMSO) δ11.37(s,1H),10.69(s,1H),8.68(d,J=1.2Hz,1H),8.33(d,J=1.2Hz,1H),7.83(d,J=8.1Hz,1H),7.73(t,J=1.8Hz,1H),7. 68(t,J=1.8Hz,1H),7.55(d,J=8.1Hz,1H),6.96(t,J=1.9Hz,1H),3.56( s,2H),3.09(s,3H),2.88-2.81(m,2H),2.81-2.74(m,2H),2.45(s,3H).

[0249] Example 10: N-(3-chloro-5-(methylsulfonamido)phenyl)-4-(7,8-dihydro-6H-pyrrolo[1',2':1,2]imidazo[4,5-c]pyridin-3-yl)-5-methylthiophene-2-carboxamide

[0250]

[0251] Step 1: Preparation of tert-butyl (6-chloro-4-(2-oxopyrrolidone-1-yl)pyridin-3-yl)carbamate

[0252]

[0253] Weigh (1.35 g, 3.8 mmol) of (6-chloro-4-iodopyridin-3-yl)carbamate tert-butyl ester ketone (0.33 g, 3.8 mmol), pyrrolidone-2-one (0.15 g, 0.76 mmol), cuprous iodide (0.15 g, 0.76 mmol), potassium carbonate (1.58 g, 8.4 mmol), and N,N'-dimethyl-1,2-cyclohexanediamine (0.22 g, 1.56 mmol) into a two-necked flask. Dissolve dioxane (10 mL), purge with argon, heat to 100 °C, and stir for 2 h. After the reaction is complete, filter, extract with ethyl acetate, combine the organic phases, dry to anhydrous sodium sulfate, and perform silica gel column chromatography. Elute with 15% (ethyl acetate / petroleum ether), collect, and dry under vacuum to give 360 ​​mg of product. ESI-MS m / z: 312.9 [M+H] + .

[0254] Step 2: Preparation of 1-(5-amino-2-chloropyridin-4-yl)pyrrolidine-2-one

[0255]

[0256] 0.36 g (1.2 mmol) of (6-chloro-4-(2-oxopyrrolidone-1-yl)pyridin-3-yl)carbamate tert-butyl ester was weighed into a reaction flask, dissolved in dichloromethane (5 mL), and trifluoroacetic acid (2 mL) was added. The mixture was stirred at room temperature for 2 h. After the reaction was complete, saturated sodium carbonate solution was added to quench the reaction. The mixture was extracted with ethyl acetate, and the organic phases were combined, dried over anhydrous sodium sulfate, and then dried under vacuum to give 0.23 g of the product. ESI-MS m / z: 211.6 [M+H] +

[0257] Step 3: Preparation of 3-chloro-7,8-dihydro-6H-pyrrolo[1',2':1,2]imidazo[4,5-c]pyridine

[0258]

[0259] Weigh 0.23 g (1.1 mmol) of 1-(5-amino-2-chloropyridin-4-yl)pyrrolidine-2-one into a reaction flask, dissolve in ethyl acetate (10 mL), add polyphosphoric acid (0.46 g), heat to 80 °C, stir for 3 h, and after the reaction is complete, quench with water while hot, adjust the pH to 7-8 with saturated sodium carbonate solution, extract with ethyl acetate, combine the organic phases, dry under anhydrous sodium sulfate, and vacuum dry to give 0.20 g of product. ESI-MS m / z: 194.5 [M+H] +

[0260] Step 4: Preparation of methyl 4-(7,8-dihydro-6H-pyrrolo[1',2':1,2]imidazo[4,5-c]pyridin-3-yl)-5-methylthiophene-2-carboxylate

[0261]

[0262] Weigh out 0.20 g (1.0 mmol) of 3-chloro-7,8-dihydro-6H-pyrrolo[1',2':1,2]imidazo[4,5-c]pyridine, 0.32 g (1.1 mmol) of methyl 5-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)thiophene-2-carboxylate, 0.025 g (0.1 mmol) of palladium acetate, and 2-dicyclohexylphosphine-2',4',6'-triisocyanate. Propylbiphenyl (0.10 g, 0.2 mmol) and potassium phosphate (0.64 g, 3.0 mmol) were added to a two-necked flask, along with 1,4-dioxane (15 mL) and water (1.5 mL). The mixture was purged with argon and reacted in an oil bath at 70 °C for 4 h. After the reaction was complete, the mixture was filtered, extracted with ethyl acetate, and the organic phases were combined, dried over anhydrous sodium sulfate, and subjected to silica gel column chromatography. The target product eluted at 15% (ethyl acetate / petroleum ether), collected, and dried under vacuum to give 0.30 g of the product. ESI-MS m / z: 314.8 [M+H]+

[0263] Step 5: Preparation of N-(3-chloro-5-(methylsulfonamido)phenyl)-4-(7,8-dihydro-6H-pyrrolo[1',2':1,2]imidazo[4,5-c]pyridin-3-yl)-5-methylthiophene-2-carboxamide

[0264]

[0265] Weigh methyl 4-(7,8-dihydro-6H-pyrrolo[1',2':1,2]imidazo[4,5-c]pyridin-3-yl)-5-methylthiophene-2-carboxylate (0.18 g, 0.57 mmol) and N-(3-amino-5-chlorophenyl)methanesulfonamide (0.13 g, 0.57 mmol) into a two-necked flask, dissolve in toluene (5 mL), purge with argon, cool to 0 °C in an ice bath, slowly add 2 mol / L trimethylaluminum toluene solution (0.80 mL, 1.71 mmol), stir for 5 min in an ice bath after addition, transfer to an oil bath, heat to 70 °C, react for 5 h, after the reaction is complete, quench with saturated sodium carbonate solution, add ethyl acetate, filter, wash the filter cake three times with water (5 mL), wash three times with ethyl acetate (5 mL), dissolve in methanol, and dry under vacuum to obtain 0.017 g of product. ESI-MS m / z: 501.8 [M+H] + . 1 H NMR (400MHz, DMSO) δ10.33(s,1H),8.88(d,J=0.6Hz,1H),8.53(s,1H),7.84(s,1H),7.38(s,1H),7.31(s,1H), 6.74(s,1H),4.19(t,J=7.1Hz,2H),3.06-2.88(m,2H),2.78(s,3H),2.72(s,3H),2.68(t,J=14.6,7.5Hz,2H).

[0266] Example 11: N-(3-chloro-5-(methylsulfonamido)phenyl)-4-(5-(cyclopropylethynyl)-3-methylpyridin-2-yl)thiophene-2-carboxamide

[0267]

[0268] Step 1: Preparation of 2-bromo-5-(cyclopropylethynyl)-3-methylpyridine

[0269]

[0270] 2-Bromo-5-iodo-3-methylpyridine (500 mg, 1.68 mmol), Pd(PPh3)2Cl2 (117 mg, 0.168 mmol), CuI (64 mg, 0.336 mmol), and Et3N (849 mg, 8.39 mmol) were mixed in 10 mL of tetrahydrofuran. After adding acetylenecyclopropane (89 mg, 1.34 mmol), the reaction was carried out at room temperature for 2.5 h under Ar protection. After the reaction was monitored by LC-MS until complete, the mixture was extracted with EA, dried to dryness, and subjected to column chromatography to obtain the target product. ESI-MS m / z: 235.09 [M+H] + .

[0271] Step 2: Preparation of N-(3-chloro-5-(methylsulfonamido)phenyl)-4-(5-(cyclopropylethynyl)-3-methylpyridin-2-yl)thiophene-2-carboxamide

[0272]

[0273] 2-Bromo-5-(cyclopropylethynyl)-3-methylpyridine (65 mg, 0.275 mmol), N-(3-chloro-5-(methylsulfonamido)phenyl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)thiophene-2-carboxamide (151 mg, 0.330 mmol), Pd(dppf)Cl2 (20 mg, 0.027 mmol), and K3PO4 (175 mg, 0.826 mmol) were mixed in 3 mL of dioxane, and 0.6 mL of water was added. The reaction was carried out at 90 °C for 2 h under Ar protection. After the reaction was monitored by LC-MS until complete, the mixture was extracted with EA, and the organic phase was dried and evaporated to dryness. After preliminary purification by column chromatography, it was sent to the preparation. ESI-MS m / z: 485.8 [M+H] + . 1 H NMR (400MHz, DMSO) δ10.58(s,1H),10.19(s,1H),8.53(d,J=1.2Hz,1H),8.48(d,J=1.9Hz,1H),8.23(d,J=1.2Hz,1H),7.76(d,J=1.5Hz,1H),7.69( t,J=1.7Hz,1H),7.66(t,J=1.8Hz,1H),6.95(t,J=1.9Hz,1H),3.07(s,3H ),2.48(s,3H),1.64-1.58(m,1H),0.96-0.91(m,2H),0.80-0.76(m,2H).

[0274] Example 12: N-(3-chloro-5-(methanesulfonylamino)phenyl)-4-(3-methyl-5-(oxetane-3-ylethynyl)pyridin-2-yl)thiophene-2-carboxamide

[0275]

[0276] Step 1: Preparation of 2-bromo-3-methyl-5-(oxetane-3-ylethynyl)pyridine

[0277]

[0278] 2-Bromo-5-iodo-3-methylpyridine (594 mg, 2 mmol), 3-ethynyloxetane (82.1 mg, 1 mmol), Pd(PPh3)Cl2 (140.38 mg, 0.2 mmol), CuI (76.18 mg, 0.4 mmol), and Et3N (1.01 g, 10 mmol) were added to THF (8 mL) and stirred at room temperature for 3 h. After the reaction was complete as monitored by LC-MS, saturated brine was added, and the mixture was extracted three times with ethyl acetate. The organic phase was dried under reduced pressure and the product was obtained by column chromatography. ESI-MS m / z: 251.9 [M+H] +

[0279] Step 2: Preparation of N-(3-chloro-5-(methanesulfonylamino)phenyl)-4-(3-methyl-5-(oxetane-3-ylethynyl)pyridin-2-yl)thiophene-2-carboxamide

[0280]

[0281] 2-Bromo-3-methyl-5-(oxetane-3-ylethynyl)pyridine (50 mg, 0.2 mmol), N-(3-chloro-5-(methylsulfonamido)phenyl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)thiophene-2-carboxamide (105.4 mg, 0.24 mmol), PdCl2 (dppf) (14.6 mg, 0.02 mmol), and K3PO4 (127.2 mg, 0.6 mmol) were added to a mixed solvent of dioxane and water (4 mL: 0.8 mL). The reaction was carried out at 90 °C for 3 hours under argon protection. After the reaction was completed as monitored by LC-MS, the reaction solution was quenched with water, extracted three times with ethyl acetate, and the organic phase was dried by rotary evaporation to obtain the target product. ESI-MS m / z: 501.8 [M+H] + . 1H NMR (400MHz, DMSO) δ10.60(s,1H),10.14(s,1H),8.64-8.51(m,2H),8.26(s,1H),7.86(s,1H),7.68(d,J=12.0Hz ,2H),6.96(s,1H),4.83(dd,J=8.4,5.5Hz,2H),4.68-4.63(m,2H),4.28-4.16(m,1H),3.07(s,3H),2.51(s,3H).

[0282] Example 13: N-(3-chloro-5-(methylsulfonamido)phenyl)-4-(5-(3-hydroxy-3-methylbut-1-yn-1-yl)-3-methylpyridin-2-yl)thiophene-2-carboxamide

[0283]

[0284] Step 1: Preparation of 4-(6-bromo-5-methylpyridin-3-yl)-2-methylbut-3-yn-2-ol

[0285]

[0286] 2-Bromo-5-iodo-3-methylpyridine (3 g, 10 mmol), palladium dichloride (701 mg, 1 mmol), and cuprous iodide (190 mg, 1 mmol) were dissolved in 10 mL of triethylamine, followed by the addition of 2-methyl-3-butyn-2-ol (840 mg, 10 mmol). The reaction was carried out at room temperature for 0.5 h under argon protection. After the reaction was completed as monitored by LC-MS, saturated ammonium chloride solution was added to quench the reaction. The mixture was extracted with ethyl acetate and saturated ammonium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure by column chromatography to give 2 g of the compound. ESI-MS m / z: 253.9 [M+H] + .

[0287] Step 2: Preparation of N-(3-chloro-5-(methylsulfonamido)phenyl)-4-(5-(3-hydroxy-3-methylbut-1-yn-1-yl)-3-methylpyridin-2-yl)thiophene-2-carboxamide

[0288]

[0289] N-(3-chloro-5-(methanesulfonamido)phenyl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaboran-2-yl)thiophene-2-carboxamide (100 mg, 0.22 mmol), 4-(6-bromo-5-methylpyridin-3-yl)-2-methylbut-3-yn-2-ol (127 mg, 0.5 mmol), DPPF palladium dichloride (14 mg, 0.02 mmol), and potassium carbonate (83 mg, 0.6 mmol) were dissolved in 2.5 mL of 1,4-dioxane and 0.5 mL of water. The mixture was heated to 80 °C and stirred for 2 hours under argon protection. After the reaction was complete as monitored by LCMs, the reaction solution was cooled to room temperature, extracted with ethyl acetate and saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and column chromatography to obtain a crude product, which was then used to prepare 8 mg of a white solid. ESI-MS m / z: 503.9 [M+H] + . 1 H NMR (400MHz, DMSO) δ10.60(s,1H),8.54(s,1H),8.51(d,J=1.7Hz,1H),8.26(d,J=0.9Hz,1H),7.81(d,J=1. 3Hz,1H),7.69(s,1H),7.66(s,1H),6.96(s,1H),5.59(s,1H),3.07(s,3H),2.50-2.48(m,3H),1.50(s,6H).

[0290] Example 14: N-(3-chloro-5-(methylsulfonamido)phenyl)-1-(5-(cyclopent-1-en-1-yl)pyridin-2-yl)-5-methyl-1H-pyrrole-3-carboxamide

[0291]

[0292] Step 1: Preparation of 2-bromo-5-(cyclopent-1-en-1-yl)pyridine

[0293]

[0294] Methyl 4-bromothiophene-2-carboxylate (1 g, 3.52 mmol) and cyclopent-1-en-1-ylboronic acid (0.38 g, 1.96 mmol) were dissolved in dioxane (15 mL). Pd(dppf)Cl2 (0.14 g, 0.192 mmol) and Cs2CO3 (1.911 g, 5.88 mmol) were added. The mixture was heated to 100 °C and stirred for 6 hours. After the reaction was complete as monitored by LC-MS, water was added to quench the reaction, followed by extraction with ethyl acetate. The organic phase was concentrated, and the product was obtained by column chromatography. ESI-MS m / z: 223.9 [M+H] + .

[0295] Step 2: Preparation of ethyl 1-(5-(cyclopent-1-en-1-yl)pyridin-2-yl)-5-methyl-1H-pyrrole-3-carboxylate

[0296]

[0297] Ethyl 5-methyl-1H-pyrrole-3-carboxylate (0.05 g, 0.36 mmol) was dissolved in 5 mL of toluene. Then, 2-bromo-5-(cyclopent-1-en-1-yl)pyridine (0.096 g, 0.432 mmol), CuI (6.9 mg, 0.036 mmol), DMEDA (6.3 mg, 0.072 mmol), and K3PO4 (0.206 g, 0.972 mmol) were added. The mixture was stirred at 120 °C for 24 hours. After the reaction was complete as monitored by LC-MS, the mixture was quenched with water, extracted with ethyl acetate, and the organic phase was concentrated. The product was obtained by column chromatography. ESI-MS m / z: 297.0 [M+H] + .

[0298] Step 3: Preparation of 1-(5-(cyclopent-1-en-1-yl)pyridin-2-yl)-5-methyl-1H-pyrrole-3-carboxylic acid

[0299]

[0300] Ethyl 1-(5-(cyclopent-1-en-1-yl)pyridin-2-yl)-5-methyl-1H-pyrrole-3-carboxylate (0.2 g, 0.673 mmol) was added to 5 mL of dioxane and 5 mL of water, followed by the addition of KOH (0.753 g, 13.46 mmol). The mixture was stirred at 80 °C for 2 hours. After the reaction was completed as monitored by LC-MS, the pH was adjusted to 5–6, and the mixture was extracted with ethyl acetate. The organic phase was then concentrated to obtain the target product. ESI-MS m / z: 269.0 [M+H] +

[0301] Step 4: Preparation of N-(3-chloro-5-(methylsulfonamido)phenyl)-1-(5-(cyclopent-1-en-1-yl)pyridin-2-yl)-5-methyl-1H-pyrrole-3-carboxamide

[0302]

[0303] 1-(5-(cyclopent-1-en-1-yl)pyridin-2-yl)-5-methyl-1H-pyrrole-3-carboxylic acid (0.1 g, 0.373 mmol) was added to 5 mL of DMF, followed by TCFH (0.157 g, 0.560 mmol), NMI (0.092 g, 0.112 mmol), and then N-(3-amino-5-chlorophenyl)methanesulfonamide (0.09 g, 0.410 mmol). The mixture was reacted at 50 °C for 2 hours. After the reaction was complete as monitored by LC-MS, ethyl acetate was added for extraction, the organic phase was concentrated, and the product was obtained by column chromatography. ESI-MS m / z: 470.9 [M+H] + . 1 H NMR (400MHz, DMSO) δ10.04(s,1H),9.86(s,1H),8.66(d,J=2.1Hz,1H),8.08(dd,J=8.4,2.2Hz,1H),7.98(d,J=1.8Hz,1H),7.71(s,1H),7.63(s,1H ),7.59(d,J=8.5Hz,1H),6.89(s,1H),6.58(s,1H),6.53(s,1H),3.06(s, 3H),2.73(t,J=6.4Hz,2H),2.54(s,2H),2.39(s,3H),2.04-1.95(m,2H).

[0304] Example 15: N-(3-chloro-5-(methylsulfonamido)phenyl)-1-(5-(4-(difluoromethylene)piperidin-1-yl)pyridin-2-yl)-5-methyl-1H-pyrrole-3-carboxamide

[0305]

[0306] Step 1: Preparation of 2-bromo-5-(4-(difluoromethylene)piperidin-1-yl)pyridine

[0307]

[0308] 2-Bromo-5-iodopyridine (1 g, 3.5 mmol), 4-(difluoromethylene)piperidine (388 mg, 2.92 mmol), Pd2(dba)3 (107 mg, 0.117 mmol), Xantphos (118 mg, 0.204 mmol), and sodium tert-butoxide (842 mg, 8.76 mmol) were added to toluene (20 mL). Under argon protection, the mixture was heated and stirred at 100 °C for 2 h. After the reaction was complete as monitored by LC-MS, toluene was removed by rotary evaporation, and the product was dissolved in ethyl acetate. Insoluble impurities were removed by filtration. The filtrate was washed three times with saturated brine, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography to obtain the target product. ESI-MS m / z: 289.0 [M+H] + .

[0309] Step 2: Preparation of ethyl 1-(5-(4-(difluoromethylene)piperidin-1-yl)pyridin-2-yl)-5-methyl-1H-pyrrole-3-carboxylate

[0310]

[0311] 2-Bromo-5-(4-(difluoromethylene)piperidin-1-yl)pyridine (69 mg, 0.24 mmol), ethyl 5-methyl-1H-pyrrole-3-carboxylate (30 mg, 0.2 mmol), CuI (7.6 mg, 0.04 mmol), K₂CO₃ (83 mg, 0.6 mmol), and N,N'-dimethyl-1,2-cyclohexanediamine (11.38 mg, 0.08 mmol) were added to acetonitrile (2 mL). Under argon protection, the mixture was stirred at 90 °C for two hours. After the reaction was completed as monitored by LC-MS, the acetonitrile was removed by rotary evaporation, dissolved in ethyl ester, washed three times with saturated brine, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography to obtain the target product. ESI-MS m / z: 362.168 [M+H] + .

[0312] Step 3: Preparation of 1-(5-(4-(difluoromethylene)piperidin-1-yl)pyridin-2-yl)-5-methyl-1H-pyrrole-3-carboxylic acid

[0313]

[0314] Ethyl 1-(5-(4-(difluoromethylene)piperidin-1-yl)pyridin-2-yl)-5-methyl-1H-pyrrole-3-carboxylate (240 mg, 0.66 mmol) and KOH (1.49 g, 26.5 mmol) were added to a mixed solvent of 1,4-dioxane and water (4 mL: 4 mL). The mixture was heated and stirred overnight at 90 °C. After the reaction was completed as monitored by LC-MS, 6 mol / L hydrochloric acid solution was slowly added dropwise to the reaction solution to adjust the pH to 4–5. A white solid precipitated out. The solid was filtered, washed with water, and dried to obtain the target product. ESI-MS m / z: 334.1367 [M+H] + .

[0315] Step 4: Preparation of N-(3-chloro-5-(methylsulfonamido)phenyl)-1-(5-(4-(difluoromethylene)piperidin-1-yl)pyridin-2-yl)-5-methyl-1H-pyrrole-3-carboxamide

[0316]

[0317] 1-(5-(4-(difluoromethylene)piperidin-1-yl)pyridin-2-yl)-5-methyl-1H-pyrrole-3-carboxylic acid (125 mg, 0.38 mmol), N-(3-amino-5-chlorophenyl)methanesulfonamide (100 mg, 0.456 mmol), TCFH (160 mg, 0.57 mmol), and NMI (93.6 mg, 1.14 mmol) were added to DMF (2 mL) and stirred overnight at 50 °C. After the reaction was completed as monitored by LC-MS, the product was obtained by column chromatography, yielding 60 mg. ESI-MS m / z: 536.1 [M+H] + . 1 H NMR (400MHz, DMSO) δ10.00(s,1H),9.80(s,1H),8.26(d,J=3.0Hz,1H),7.82(d,J=2.0Hz,1H),7.71(t,J=1.8Hz,1H),7.63(t,J=1.8Hz,1H),7.57 (dd,J=8.9,3.1Hz,1H),7.43(d,J=8.9Hz,1H),6.88(t,J=1.9Hz,1H),6.53(d,J=0.7Hz,1H),3.41-3.37(m,4H),3.06(s,3H),2.31-2.26(m,7H).

[0318] Example 16: N-(3-chloro-5-(methylsulfonamido)phenyl)-4-(5-(3,4-dihydro-1H-pyrrolo[2,1-c][1,4]oxazin-7-yl)pyridin-2-yl)-5-methylthiophene-2-carboxamide

[0319]

[0320] Step 1: Preparation of 1-(2-hydroxyethyl)-4-iodo-1H-pyrrole-2-carboxaldehyde

[0321]

[0322] 4-Iodo-1H-pyrrole-2-carboxaldehyde (5 g, 22.62 mmol) was dissolved in dioxane (50 mL), followed by the addition of KOH (3.81 g, 67.87 mmol) and bromoethanol (5.65 g, 45.25 mmol). The mixture was heated to 60 °C and stirred for 8 hours. LC-MS was used to monitor the reaction until the product growth ceased. The reaction was then stopped, and the pH was adjusted to near neutral with hydrochloric acid. The mixture was extracted with EA, and the organic phase was dried under reduced pressure and subjected to column chromatography to obtain the target product. ESI-MS m / z: 265.8 [M+H] + .

[0323] Step 2: Preparation of ethyl 2-(2-formyl-4-iodo-1H-pyrrole-1-yl)4-methylbenzenesulfonate

[0324]

[0325] 1-(2-hydroxyethyl)-4-iodo-1H-pyrrole-2-carboxaldehyde (2.5 g, 9.43 mmol) was dissolved in 60 mL of dichloromethane. TEA (2.86 g, 28.3 mmol) and p-toluenesulfonyl chloride (2.16 g, 11.32 mmol) were added, and the mixture was stirred at room temperature for 4 hours. After the reaction was complete as monitored by LC-MS, EA was added for extraction and separation. The organic phase was dried under reduced pressure, and column chromatography was used to obtain the target product. ESI-MS m / z: 419.7 [M+H] + .

[0326] Step 3: Preparation of ethyl 2-(2-(hydroxymethyl)-4-iodo-1H-pyrrole-1-yl)-4-methylbenzenesulfonate

[0327]

[0328] Ethyl 2-(2-formyl-4-iodo-1H-pyrrolo-1-yl)4-methylbenzenesulfonate (3.44 g, 8.21 mmol) was placed in a 100 mL reaction flask, and 40 mL of ethanol was added. Sodium borohydride (0.155 g, 4.10 mmol) was added under ice bath conditions. The reaction was carried out at 0 °C for 10 min, then the ice bath was removed, and the reaction was allowed to proceed at room temperature for 1 h. After the reaction was completed by TLC monitoring, a small amount of water was added to quench the reaction. The mixture was extracted with EA, and the organic phase was dried and evaporated to dryness to obtain the target product. ESI-MS m / z: 421.8 [M+H] +

[0329] Step 4: Preparation of 7-iodo-3,4-dihydro-1H-pyrrolo[2,1-c][1,4]oxazine

[0330]

[0331] Ethyl 2-(2-(hydroxymethyl)-4-iodo-1H-pyrrolo-1-yl)-4-methylbenzenesulfonate (3.45, 8.19 mmol) was added to 80 mL of tetrahydrofuran. NaH (328 mg, 8.19 mmol) with a concentration of 60% was added under ice bath conditions. The reaction was carried out at room temperature for 12 h. After the reaction was completed as monitored by LC-MS, saturated saline solution was added for dilution. The organic phase was extracted with EA, dried, and purified by silica gel column chromatography to obtain the target product. ESI-MS m / z: 249.9 [M+H] +

[0332] Step 5: Preparation of 7-(6-chloropyridin-3-yl)-3,4-dihydro-1H-pyrrolo[2,1-c][1,4]oxazine

[0333]

[0334] 7-Iodo-3,4-dihydro-1H-pyrrolo[2,1-c][1,4]oxazine (180 mg, 0.723 mmol), (6-chloropyridin-3-yl)boronic acid (170 mg,

[0335] 1.08 mmol), Pd(dppf)Cl2 (26 mg, 0.036 mmol), and potassium carbonate (300 mg, 2.17 mmol) were added to dioxane (8 mL), followed by 0.8 mL of water. The mixture was then reacted at 95 °C for 2 h under Ar protection. After the reaction was complete, the mixture was filtered to remove solid impurities, and the filtrate was evaporated to dryness and then subjected to column chromatography to obtain the target product. ESI-MS m / z: 234.8 [M+H] +

[0336] Step 6: Preparation of methyl 4-(5-(3,4-dihydro-1H-pyrrolo[2,1-c][1,4]oxazin-7-yl)pyridin-2-yl)-5-methylthiophene-2-carboxylic acid

[0337]

[0338] 7-(6-chloropyridin-3-yl)-3,4-dihydro-1H-pyrrolo[2,1-c][1,4]oxazine (165 mg, 0.703 mmol), methyl 5-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxoboran-2-yl)thiophene-2-carboxylate (297 mg, 1.05 mmol), Pd(dppf)Cl2.CH2Cl2 (57 mg, 0.070 mmol), and potassium carbonate (291 mg, 2.11 mmol) were suspended in 6 mL of dioxane and 0.6 mL of water. The reaction was carried out at 100 °C for 3 h under Ar atmosphere. After the reaction was completed, saturated saline was added for dilution, and the mixture was extracted with EA. The organic phase was dried and evaporated to dryness before column chromatography to obtain the target product. ESI-MS m / z: 354.9 [M+H]+.

[0339] Step 7: Preparation of 4-(5-(3,4-dihydro-1H-pyrrolo[2,1-c][1,4]oxazin-7-yl)pyridin-2-yl)-5-methylthiophene-2-carboxylic acid

[0340]

[0341] Methyl 4-(5-(3,4-dihydro-1H-pyrrolo[2,1-c][1,4]oxazin-7-yl)pyridin-2-yl)-5-methylthiophene-2-carboxylic acid (30 mg, 0.085 mmol) and NaOH (20.3 mg, 0.507 mmol) were added to a flask, along with 0.6 mL of tetrahydrofuran and 0.6 mL of water. The reaction was carried out at room temperature for 3 h. After the reaction was complete as monitored by LC-MS, the pH was adjusted to approximately 4 with 2N hydrochloric acid, resulting in the precipitation of an orange-yellow solid. The solid was filtered and dried to obtain the target product. ESI-MS m / z: 340.9 [M+H]+.

[0342] Step 8: Preparation of N-(3-chloro-5-(methylsulfonamido)phenyl)-4-(5-(3,4-dihydro-1H-pyrrolo[2,1-c][1,4]oxazin-7-yl)pyridin-2-yl)-5-methylthiophene-2-carboxamide

[0343]

[0344] 4-(5-(3,4-dihydro-1H-pyrrolo[2,1-c][1,4]oxazin-7-yl)pyridin-2-yl)-5-methylthiophene-2-carboxylic acid (26 mg, 0.076 mmol), TCFH (32 mg, 0.144 mmol), and NMI (19 mg, 0.229 mmol) were mixed in 1.5 mL of acetonitrile. N-(3-amino-5-chlorophenyl)methanesulfonamide (20 mg, 0.091 mmol) was added, and the reaction was carried out at room temperature for 1.5 h. After the reaction was monitored by LC-MS until complete, the mixture was diluted with saturated brine, extracted with EA, and the organic phase was dried under evaporation. A yellow solid was obtained. ESI-MS m / z: 542.8 [M+H] + . 1 H NMR (400MHz, DMSO) δ10.44(s,1H),10.07(s,1H),8.85(d,J=2.0Hz,1H),8.40(s,1H),8.04-7.98(m,1H),7.69-7. 62(m,3H),7.37(d,J=1.4Hz,1H),6.95(s,1H),6.33(s,1H),4.76(s,2H),3.99(s,4H),3.07(s,3H),2.72(s,3H).

[0345] Example 17: N-(3-chloro-5-(methylsulfonamido)phenyl)-4-(5-(5,6-dihydroimidazo[1,2-a]pyrazin-7(8H)-yl)pyridin-2-yl)-5-methylthiophene-2-carboxamide

[0346]

[0347] Step 1: Preparation of 7-(6-bromopyridin-3-yl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine

[0348]

[0349] 2-Bromo-5-iodopyridine (1 g, 3.5 mmol), 5,6,7,8-tetrahydroimidazole[1,2-a]pyrazine (360 mg, 2.92 mmol), Pd2(dba)3 (107 mg, 0.117 mmol), Xantphos (118 mg, 0.204 mmol), and sodium tert-butoxide (842 mg, 8.76 mmol) were added to toluene (25 mL). Under argon protection, the mixture was heated and stirred at 100 °C for 3 h. After the reaction was complete as monitored by LC-MS, toluene was removed by rotary evaporation. The product was then dissolved in ethyl acetate, filtered to remove insoluble impurities, washed three times with saturated brine, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography to obtain the target product. ESI-MS m / z: 279.0 [M+H] + .

[0350] Step 2: Preparation of N-(3-chloro-5-(methylsulfonamido)phenyl)-4-(5-(5,6-dihydroimidazo[1,2-a]pyrazin-7(8H)-yl)pyridin-2-yl)-5-methylthiophene-2-carboxamide

[0351]

[0352] Add 7-(6-bromopyridin-3-yl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine (84 mg, 0.3 mmol), N-(3-chloro-5-(methylsulfonamido)phenyl)-5-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)thiophene-2-carboxamide (156 mg, 0.33 mmol), Pd(dppf)Cl2·CH2Cl2 (24 mg, 0.03 mmol), and K3PO4 (191 mg, 0.9 mmol) to a 50 mL round-bottom flask, followed by 3 mL of 1,4-dioxane and 0.6 mL of water. Heat at 90 °C for two hours under argon protection. Quench the reaction with water, extract three times with ethyl acetate, combine the organic phases, and dry with saturated brine and anhydrous sodium sulfate. Column chromatography yields the product. ESI-MS m / z: 542.9 [M+H] + . 1H NMR (400MHz, DMSO) δ10.42(s,1H),8.51(d,J=2.2Hz,1H),8.34(s,1H),8.17(s,1H),7.67(t,J=1.7Hz,1H),7.64(t,J=1.7Hz,1H),7.62-7.50( m,2H),7.13(s,1H),6.94(t,J=1.9Hz,1H),6.91(s,1H),4.53(s,2H),4.14(t,J=5.3Hz,2H),3.84(t,J=5.4Hz,2H),3.06(s,3H),2.67(s,3H).

[0353] Example 18: N-(3-chloro-5-(methylsulfonamido)phenyl)-5-methyl-4-(5-(2-(trifluoromethyl)-5,6-dihydro-[1,2,4]triazolo[1,5-a]pyrazin-7(8H)-yl)pyridin-2-yl)thiophene-2-carboxamide

[0354]

[0355] Step 1: Preparation of 7-(6-bromopyridin-3-yl)-2-(trifluoromethyl)-5,6,7,8-tetrahydro-[1,2,4]triazolo[1,5-a]pyrazine

[0356]

[0357] 2-Bromo-5-iodopyridine (1.36 g, 4.8 mmol), 2-(trifluoromethyl)-5,6,7,8-tetrahydro-[1,2,4]triazolo[1,5-a]pyrazine (768.6 mg, 4.0 mmol), Pd2(dba)3 (146 mg, 0.16 mmol), Xantphos (162 mg, 0.28 mmol), and sodium tert-butoxide (1.15 g, 12.0 mmol) were added to toluene (30 mL). Under argon protection, the mixture was heated and stirred at 100 °C for 3 h. After the reaction was completed as monitored by LC-MS, toluene was removed by rotary evaporation. The product was then dissolved in ethyl acetate and water, filtered to remove insoluble impurities, washed three times with saturated brine, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography to obtain the target product. ESI-MS m / z: 349.8 [M+H] + .

[0358] Step 2: Preparation of N-(3-chloro-5-(methylsulfonamido)phenyl)-5-methyl-4-(5-(2-(trifluoromethyl)-5,6-dihydro-[1,2,4]triazolo[1,5-a]pyrazin-7(8H)-yl)pyridin-2-yl)-5-methylthiophene-2-carboxamide

[0359]

[0360] Add 7-(6-bromopyridin-3-yl)-2-(trifluoromethyl)-5,6,7,8-tetrahydro-[1,2,4]triazolo[1,5-a]pyrazine (104 mg, 0.3 mmol), N-(3-chloro-5-(methylsulfonamido)phenyl)-5-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)thiophene-2-carboxamide (156 mg, 0.33 mmol), Pd(dppf)Cl2·CH2Cl2 (24 mg, 0.03 mmol), and K3PO4 (191 mg, 0.9 mmol) to a 50 mL round-bottom flask, followed by 3 mL of 1,4-dioxane and 0.6 mL of water. Heat at 90 °C for two hours under argon protection. The reaction was quenched with water, and the mixture was extracted three times with ethyl acetate. The combined organic phases were dried over saturated brine and anhydrous sodium sulfate. Column chromatography yielded the target product. ESI-MS m / z: 611.8 [M+H] + . 1 H NMR (400MHz, DMSO) δ10.43(s,1H),10.10(s,1H),8.57(d,J=2.8Hz,1H),8.35(s,1H),7.70-7.63(m,3H),7.60(d,J=8. 8Hz,1H),6.94(t,J=1.9Hz,1H),4.79(s,2H),4.43(t,J=5.2Hz,2H),4.00(t,J=5.3Hz,2H),3.07(s,3H),2.68(s,3H).

[0361] Example 19: N-(3-chloro-5-(methylsulfonamido)phenyl)-4-(3-((2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl)methoxy)pyridin-2-yl)-5-methylthiophene-2-carboxamide

[0362]

[0363] Step 1: Preparation of (2S,7aR)-7a-(((2-bromopyridin-3-yl)oxy)methyl)-2-fluorohexahydro-1H-pyrrolizine

[0364]

[0365] Weigh 0.735 g (4.22 mmol) of ((2S,7aR)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl)methanol into a 250 mL three-necked flask, add 20 mL of tetrahydrofuran to dissolve it, then add 1.0 g (5.49 mmol) of 2-bromo-3-hydroxypyridine. After the addition is complete, stir for 5 minutes in an ice bath. During stirring, add 55 mL of tetrahydrofuran. After 5 minutes, add 1.44 g (5.49 mmol) of triphenylphosphine. l), after stirring in an ice bath for 5 minutes, added diisopropyl azodicarbonate (1.1 g, 5.49 mmol) dropwise. After the addition was complete, the ice bath was removed, and the mixture was stirred at room temperature for 2 hours. After the reaction was completed as monitored by LC-MS, the reaction solution was extracted with ethyl acetate, the organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The product was then subjected to dry column chromatography in a petroleum ether and ethyl acetate system with a polarity of 40-50%. The solvent was removed under reduced pressure to give 1.3 g of a white solid. ESI-MS m / z: 314.8 [M+H] + .

[0366] Step 2: Preparation of N-(3-chloro-5-(methylsulfonamido)phenyl)-4-(3-((2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl)methoxy)pyridin-2-yl)-5-methylthiophene-2-carboxamide

[0367]

[0368] N-(3-chloro-5-(methylsulfonamido)phenyl)-5-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)thiophene-2-carboxamide (99 mg, 0.21 mmol), [1,1'-bis(diphenylphosphine)ferrocene]palladium dichloride (8 mg, 0.01 mmol), and potassium carbonate (83 mg, 0.6 mmol) were added sequentially to a 50 ml double-necked flask. 1,4-dioxane was added to dissolve the carboxylic acid (6 ml), and then... (2S,7aR)-7a-((((2-bromopyridin-3-yl)oxy)methyl)-2-fluorohexahydro-1H-pyrrolizidine (63 mg, 0.20 mmol) and water (1 ml) were added. After purging three times with argon, the mixture was stirred at 90 °C for 2 hours under argon protection. After the reaction was completed as monitored by LC-MS, the mixture was extracted with ethyl acetate. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The mixture was then purified using a preparative separation platform and freeze-dried to give 40 mg of a pale yellow solid. ESI-MS m / z: 578.8 [M+H] + . 1H NMR (400MHz, DMSO) δ10.39(s,1H),8.41(dd,J=4.6,1.2Hz,1H),8.15(s,1H),7.69-7.63(m,3H),7.38( dd,J=8.3,4.6Hz,1H),6.94(t,J=1.9Hz,1H),4.74(d,J=53.1Hz,1H),3.15(dd,J=16.2,4.1Hz,1H),3. 06(s,2H),3.04-2.80(m,2H),2.72-2.60(m,2H),2.57(s,2H),2.45(s,3H),2.26(dd,J=62.5,7.9Hz,2 H),2.11-1.89(m,2H),1.72(dd,J=12.3,5.6Hz,1H),1.58-1.49(m,1H),1.34(dd,J=13.1,6.2Hz,1H).

[0369] Example 20: N-(3-chloro-5-(methylsulfonamido)phenyl)-4-(5-((2S,7aR)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl)methoxy)pyridin-2-yl)-5-methylthiophene-2-carboxamide

[0370]

[0371] Step 1: Preparation of (2S,7aR)-7a-((2-bromopyridin-5-yl)oxy)methyl)-2-fluorohexahydro-1H-pyrrolizine

[0372]

[0373] Weigh 0.735 g (4.22 mmol) of ((2S,7aR)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl)methanol into a 250 mL three-necked flask, add 20 mL of tetrahydrofuran to dissolve it, then add 1.0 g (5.49 mmol) of 2-bromo-5-hydroxypyridine. After the addition is complete, stir for 5 minutes in an ice bath. During stirring, add 55 mL of tetrahydrofuran. After 5 minutes, add 1.44 g (5.49 mmol) of triphenylphosphine. l), after stirring in an ice bath for 5 minutes, added diisopropyl azodicarbonate (1.1 g, 5.49 mmol) dropwise. After the addition was complete, the ice bath was removed, and the mixture was stirred at room temperature for 2 hours. After the reaction was completed as monitored by LC-MS, the reaction solution was extracted with ethyl acetate, the organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The product was then subjected to dry column chromatography in a petroleum ether and ethyl acetate system with a polarity of 40-50%. The solvent was removed under reduced pressure to give 1.3 g of a white solid. ESI-MS m / z: 314.8 [M+H] + .

[0374] Step 2: Preparation of N-(3-chloro-5-(methylsulfonamido)phenyl)-4-(5-((2S,7aR)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl)methoxy)pyridin-2-yl)-5-methylthiophene-2-carboxamide

[0375]

[0376] N-(3-chloro-5-(methylsulfonamido)phenyl)-5-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)thiophene-2-carboxamide (99 mg, 0.21 mmol), [1,1'-bis(diphenylphosphine)ferrocene]palladium dichloride (8 mg, 0.01 mmol), and potassium carbonate (83 mg, 0.6 mmol) were added sequentially to a 50 ml double-necked flask. 1,4-dioxane was added to dissolve the carboxylic acid (6 ml), and then... (2S,7aR)-7a-((((2-bromopyridin-5-yl)oxy)methyl)-2-fluorohexahydro-1H-pyrrolizidine (63 mg, 0.20 mmol) and water (1 ml) were added. After purging three times with argon, the mixture was stirred at 90 °C for 2 hours under argon protection. After the reaction was completed as monitored by LC-MS, the mixture was extracted with ethyl acetate. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The mixture was then purified using a preparative separation platform and freeze-dried to give 30 mg of a white solid. ESI-MS m / z: 578.8 [M+H] + . 1 H NMR(400MHz,D2O)δ8.36(d,J=2.3Hz,1H),8.31(s,1H),7.67(d,J=1.6Hz,1H),7.66-7.6 3(m,2H),7.60(dd,J=8.6,2.5Hz,1H),6.97(t,J=1.8Hz,1H),4.91(dt,J=51.9,4.2Hz,1 H),3.35-3.16(m,1H),3.11-2.99(m,4H),2.92-2.79(m,4H),2.72-2.63(m,3H),2.45-2 .35(m,1H),2.34-2.23(m,2H),1.99-1.91(m,1H),1.86-1.75(m,1H),1.59-1.47(m,1H).

[0377] Example 21: N-(3-chloro-5-(methylsulfonamido)phenyl)-5-methyl-4-(3-(4,6,7,7a-tetrahydro-5H-imidazo[4,5-c]pyridin-5-yl)pyridin-2-yl)thiophene-2-carboxamide

[0378]

[0379] Step 1: Preparation of 7-(2-bromopyridin-3-yl)-5,6,7,8-tetrahydroimidazolium[1,2-a]pyrazine

[0380]

[0381] 2-Bromo-3-iodopyridine (1.36 g, 4.8 mmol), 5,6,7,8-tetrahydroimidazole[1,2-a]pyrazine (768.6 mg, 4.0 mmol), Pd2(dba)3 (146 mg, 0.16 mmol), Xantphos (162 mg, 0.28 mmol), and sodium tert-butoxide (1.15 g, 12.0 mmol) were added to toluene (30 mL). Under argon protection, the mixture was heated and stirred at 130 °C for 6 h. After the reaction was completed as monitored by LC-MS, toluene was removed by rotary evaporation. The product was then dissolved in ethyl acetate and water, filtered to remove insoluble impurities, and extracted by liquid-liquid extraction. The organic phase was washed three times with saturated brine, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography to obtain 750 mg of the target product as an orange solid. ESI-MS m / z: 278.9 [M+H] + .

[0382] Step 2: Preparation of N-(3-chloro-5-(methylsulfonamido)phenyl)-5-methyl-4-(3-(4,6,7,7a-tetrahydro-5H-imidazo[4,5-c]pyridin-5-yl)pyridin-2-yl)thiophene-2-carboxamide

[0383]

[0384] Add 7-(2-bromopyridin-3-yl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine (84 mg, 0.3 mmol), N-(3-chloro-5-(methylsulfonamido)phenyl)-5-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)thiophene-2-carboxamide (156 mg, 0.33 mmol), Pd(dppf)Cl2·CH2Cl2 (24 mg, 0.03 mmol), and K3PO4 (191 mg, 0.9 mmol) to a 50 mL round-bottom flask, followed by 3 mL of 1,4-dioxane and 0.6 mL of water. Heat at 90 °C for two hours under argon protection. Quench the reaction with water, extract three times with ethyl acetate, combine the organic phases, and dry with saturated brine and anhydrous sodium sulfate. Column chromatography yields the product. ESI-MS m / z: 542.8 [M+H] + . 1H NMR (400MHz, DMSO) δ10.36(s,1H),10.08(s,1H),8.34(d,J=3.9Hz,1H),8.16(s,1H),7.69-7.62(m,2H),7.60(d,J=1.7Hz,1H),7.39(dd,J =8.2,4.6Hz,1H),7.06(s,1H),6.93(t,J=1.8Hz,1H),6.85(s,1H),4.17(s,2H),3.78(s,2H),3.24-3.18(m,2H),3.05(s,3H),2.36(s,3H).

[0385] Example 22: N-(3-chloro-5-(methylsulfonamido)phenyl)-4-(3-(3,4-dihydro-1H-pyrrolo[2,1-c][1,4]oxazin-7-yl)pyridin-2-yl)-5-methylthiophene-2-carboxamide

[0386]

[0387] Step 1: Preparation of 7-(2-chloropyridin-3-yl)-3,4-dihydro-1H-pyrrolo[2,1-c][1,4]oxazine

[0388]

[0389] 7-Iodo-3,4-dihydro-1H-pyrrolo[2,1-c][1,4]oxazine (105 mg, 0.421 mmol), (2-chloropyridin-3-yl)boronic acid (99 mg, 0.632 mmol), Pd(dppf)Cl2 (15 mg, 0.021 mmol), and K2CO3 (174 mg, 1.26 mmol) were added to a flask, along with 3 mL of Diaxane and 0.6 mL of H2O. The mixture was reacted at 90°C for 2–3 h under Ar atmosphere protection. After the reaction was complete as monitored by LC-MS, saturated saline solution was added for dilution, and the mixture was extracted with EA. The organic phase was dried under rotary evaporation, and the product was purified by column chromatography. ESI-MS m / z: 235.0 [M+H] +

[0390] Step 2: Preparation of N-(3-chloro-5-(methylsulfonamido)phenyl)-4-(3-(3,4-dihydro-1H-pyrrolo[2,1-c][1,4]oxazin-7-yl)pyridin-2-yl)-5-methylthiophene-2-carboxamide

[0391]

[0392] The following starting materials were added sequentially to a flask: 7-(2-chloropyridin-3-yl)-3,4-dihydro-1H-pyrrolo[2,1-c][1,4]oxazine (25 mg, 0.106 mmol), N-(3-chloro-5-(methylsulfonamido)phenyl)-5-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)thiophene-2-carboxamide (60 mg, 0.128 mmol), Pd(OAc)2 (3 mg, 0.010 mmol), X-phos (5 mg, 0.010 mmol), and K3PO4 (68 mg, 0.319 mmol). Then, 2 mL of Dioxane was added as the reaction solvent, followed by 0.4 mL of water. The reaction was carried out at 70 °C under Ar protection for 3 minutes. After the reaction was complete as monitored by LC-MS, saturated saline solution was added for dilution, and the mixture was extracted with EA. The organic phase was dried and evaporated to dryness. After initial purification by silica gel column chromatography, the product was further purified by HPLC and then lyophilized to obtain the target product. ESI-MS m / z: 542.8 [M+H] + . 1 H NMR (400MHz, DMSO) δ10.31 (s, 1H), 8.44 (dd, J = 4.6, 1.5Hz, 1H), 7.96 (s, 1H) ,7.92(dd,J=7.9,1.6Hz,1H),7.67(s,1H),7.60(s,1H),7.39(dd,J=7.9,4.7 Hz,1H),6.92(t,J=1.8Hz,1H),6.54(d,J=1.7Hz,1H),5.63(s,1H),4.62(s, 2H),3.89(d,J=5.2Hz,2H),3.86(d,J=5.2Hz,2H),3.04(s,3H),2.07(s,3H).

[0393] Example 23: 4-(3-(2-(2-oxa-6-azaspiro[3.3]heptane-6-yl)ethoxy)pyridin-2-yl)-N-(3-chloro-5-(methylsulfonamido)phenyl)-5-methylthiophene-2-carboxamide

[0394]

[0395] Step 1: Preparation of 2-bromo-3-(2-bromoethoxy)pyridine

[0396]

[0397] 2-Bromopyridin-3-ol (1.73 g, 10 mmol) was dissolved in CAN (30 mL), and 1,2-dibromoethane (1.28 mL, 15 mmol) and K₂CO₃ (4.15 g, 30 mmol) were added. The reaction mixture was stirred at 80 °C for 2 h. The reaction was monitored by LCMS. After the reaction was complete, the solid was filtered off, and the reaction mixture was evaporated to dryness. The mixture was extracted three times with EA, and the organic phases were combined, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give 2.1 g (75.30%) of crude yellow solid. ESI-MS m / z: 279.7 [M+H] + .

[0398] Step 2: Preparation of 6-(2-((2-bromopyridin-3-yl)oxy)ethyl)-2-oxa-6-azaspiro[3.3]heptane

[0399]

[0400] 2-Bromo-3-(2-bromoethoxy)pyridine (557.78 g, 2 mmol) and 2-oxa-6-azaspiro[3.3]heptane (216.2 μL, 2.4 mmol) were dissolved in ACN (20 mL), and then K2CO3 (829.2 mg, 6 mmol) was added. The reaction mixture was stirred overnight at room temperature. The reaction was monitored by LCMS. After the reaction was complete, the solid was filtered off, and the reaction mixture was evaporated to dryness. The mixture was extracted three times with EA, the organic phases were combined, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and column chromatography was performed to give 550 mg (92.28%) of yellow solid. ESI-MS m / z: 298.9 [M+H] + Step 3: Preparation of 4-(3-(2-(2-oxa-6-aza[3.3]heptane-6-yl)ethoxy)pyridin-2-yl)-N-(3-chloro-5-(methylsulfonamido)phenyl)-5-methylthiophene-2-carboxamide

[0401]

[0402] 6-(2-((2-bromopyridin-3-yl)oxy)ethyl)-2-oxa-6-azaspiro[3.3]heptane (89.4 mg, 0.3 mmol), N-(3-chloro-5-(methylsulfonamido)phenyl)-5-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaboron-2-yl)thiophene-2-carboxamide (155.13 mg, 0.33 mmol), Pd(dppf)Cl2 (21.95 mg, 0.03 mmol), and Cs2CO3 (293.2 mg, 0.9 mmol) were added to the reaction flask, purged three times with Ar gas, and then Dioxane (6 mL) and H2O (1.5 mL) were added. The reaction system was stirred at 100 °C for 2 h. The reaction was monitored by LCMS. After the reaction was completed, the reaction solution was evaporated to dryness. Extracted three times with EA, the organic phases were combined, washed with saturated sodium chloride, dried with anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and column chromatography to obtain 120 mg of dark brown solid (71.17%), which was then prepared to give 25 mg of white solid.

[0403] ESI-MS m / z: 562.8 [M+H] + . 1 H NMR (400MHz, DMSO) δ10.42(s,1H),8.25(d,J=4.4Hz,1H),8.21(s,1H),8.14(s,1H),7.70(s,1H),7.66(s,1H),7.56(d,J=8.3Hz ,1H),7.39(dd,J=8.2,4.6Hz,1H),6.94(s,1H),4.46(s,4H),4.02(s,2H),3.31(s,4H),3.06(s,3H),2.78(s,2H),2.44(s,3H).

[0404] Example 24: N-(3-chloro-5-(methylsulfonamido)phenyl)-5-methyl-4-(5-(5,6,7,8-tetrahydroimidazo[1,2-a]pyrazin-7-carbonyl)pyridin-2-yl)thiophene-2-carboxamide

[0405]

[0406] Step 1: Preparation of (6-bromopyridin-3-yl)(5,6-dihydroimidazo[1,2-a]pyrazin-7(8H)-yl) methyl ketone

[0407]

[0408] To a 50 mL round-bottom flask, 6-bromonicotinic acid (162 mg, 0.8 mmol), HATU (365 mg, 0.96 mmol), and DIPEA (310 mg, 2.4 mmol) were added sequentially. After stirring for 5 min, 5,6,7,8-tetrahydroimidazole[1,2-a]pyrazine (99 mg, 0.5 mmol) was added, and the mixture was stirred overnight at room temperature. The reaction was quenched with water, and the organic phase was extracted three times with ethyl acetate. The combined organic phases were washed twice with saturated brine, dehydrated with anhydrous sodium sulfate, concentrated under reduced pressure, and subjected to rapid column chromatography (15:1 = DCM:MeOH) to give 180 mg of a yellow solid. ESI-MS m / z: 370.8

[0409] [M+H] + .

[0410] Step 2: Preparation of N-(3-chloro-5-(methylsulfonamido)phenyl)-5-methyl-4-(5-(5,6,7,8-tetrahydroimidazo[1,2-a]pyrazin-7-carbonyl)pyridin-2-yl)thiophene-2-carboxamide

[0411]

[0412] Add (6-bromopyridin-3-yl)(5,6-dihydroimidazo[1,2-a]pyrazin-7(8H)-yl) methyl ketone (123 mg, 0.4 mmol), N-(3-chloro-5-(methylsulfonamido)phenyl)-5-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)thiophene-2-carboxamide (189 mg, 0.4 mmol), Pd(dppf)Cl2·CH2Cl2 (32 mg, 0.04 mmol), and K3PO4 (255 mg, 1.2 mmol) to a 50 mL round-bottom flask, followed by 4 mL of 1,4-dioxane and 0.8 mL of water. Heat at 90 °C for two hours under argon protection. Quench the reaction with water, extract three times with ethyl acetate, combine the organic phases, and dry with anhydrous sodium sulfate in saturated brine. Column chromatography (DCM:MeOH = 15:1) yielded 100 mg of a white product. ESI-MS m / z: 570.8 [M+H] + . 1H NMR (400MHz, DMSO) δ10.50(s,1H),8.82(s,1H),8.48(s,1H),8.08(d,J=7.3Hz,1H),7.81(d,J=8.2Hz,1H),7.69(t,J=1.8Hz,1H), 7.65(t,J=1.8Hz,1H),7.17(s,1H),6.95(dd,J=8.2,6.3Hz,2H),4.80(s,2H),4.12(s,2H),3.84(s,2H),3.07(s,3H),2.76(s,3H).

[0413] Example 25: N-(3-chloro-5-(methylsulfonamido)phenyl)-4-(5-((5,6-dihydroimidazo[1,2-a]pyrazin-7(8H)-yl)methyl)pyridin-2-yl)-5-methylthiophene-2-carboxamide

[0414]

[0415] Step 1: Preparation of 7-((6-chloropyridin-3-yl)methyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine

[0416]

[0417] 2-Chloro-5-(chloromethyl)pyridine (50 mg, 0.308 mmol) and 5,6,7,8-tetrahydroimidazolo[1,2-a]pyrazine (46 mg, 0.370 mmol) were mixed in 2.5 mL of DMSO and heated at 80 °C for 3 h. After the reaction was complete as monitored by LC-MS, EA was added for dilution, and the mixture was washed with saturated NaCl solution. The aqueous phase was then adjusted to strongly alkaline pH with NaOH solid. After three extractions with EA, the organic phases were combined and washed again with saturated NaCl solution to remove DMSO. The organic phase was then dried and evaporated to dryness to obtain the product. ESI-MS m / z: 249.0 [M+H] + .

[0418] Step 2: Preparation of N-(3-chloro-5-(methylsulfonamido)phenyl)-4-(5-((5,6-dihydroimidazo[1,2-a]pyrazin-7(8H)-yl)methyl)pyridin-2-yl)-5-methylthiophene-2-carboxamide

[0419]

[0420] 7-((6-chloropyridin-3-yl)methyl)-5,6,7,8-tetrahydroimidazole[1,2-a]pyrazine (60 mg, 0.241 mmol), N-(3-chloro-5-(methylsulfonamido)phenyl)-5-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)thiophene-2-carboxamide (113 mg, 0.241 mmol), Pd(dppf)Cl2.CH2Cl2 (16 mg, 0.019 mmol), and K2CO3 (100 mg, 0.724 mmol) were mixed in 2.5 mL of Dioxane. After adding 0.5 mL of water, the mixture was reacted at 90 °C for 2 h under Ar protection. After the reaction was complete as monitored by LC-MC, saturated brine was added for dilution, and the mixture was extracted with EA. The organic phase was dried and evaporated to dryness. After preliminary purification by silica gel column chromatography, the product was further purified to obtain the target product. ESI-MS m / z: 556.8 [M+H] + . 1 H NMR (400MHz, DMSO) δ10.47(s,1H),8.65(d,J=1.6Hz,1H),8.42(s,1H),7.91(dd,J=8.1,2.1Hz,1H),7.72-7.62(m,3H),7.06(s,1H),6 .94(t,J=1.9Hz,1H),6.84(s,1H),3.98(t,J=5.3Hz,2H),3.79(s,2H),3.61(s,2H),3.06(s,3H),2.88(t,J=5.4Hz,2H),2.72(s,3H).

[0421] Example 26: N-(3-chloro-5-(methylsulfonamido)phenyl)-4-(3-methyl-5-(2-methyl-2,7-diazaspiro[3.5]nonane-7-carbonyl)pyridin-2-yl)thiophene-2-carboxamide

[0422]

[0423] Step 1: Preparation of tert-butyl 7-(6-chloro-5-methylnicotinyl)-2,7-dihexerospiro[3.5]nonane-2-carboxylic acid

[0424]

[0425] 6-Chloro-5-methylnicotinic acid (150 mg, 0.874 mmol), TCFH (294 mg, 1.05 mmol), NMI (215 mg, 2.62 mmol), and 4 mL of acetonitrile were added to a 50 mL round-bottom flask. The mixture was stirred at room temperature for 30 min, then tert-butyl 2,7-diazaspiro[3.5]nonane-2-carboxylic acid (0.208 mg, 0.918 mmol) was added. After reacting for 1.5 h, half of the starting material remained. The reaction was continued overnight at room temperature. The mixture was diluted with saturated brine, extracted with ethyl acetate, and the organic phase was dried under evaporation. The product was obtained by column chromatography. ESI-MS m / z: 379.96 [M+H] + Step 2: Preparation of (6-chloro-5-methylpyridin-3-yl)(2,7-diazaspiro[3.5]non-7-yl)methyl ketone hydrochloride

[0426]

[0427] 7-(6-chloro-5-methylnicotinyl)-2,7-dihexaspiro[3.5]nonane-2-carboxylic acid ester (200 mg, 0.526 mmol) was added to a 50 mL round-bottom flask, dissolved in 1,4-dioxane, and then 1 mL of 1,4-dioxane hydrochloride solution was added. The mixture was stirred at room temperature for 3 h. After the reaction was complete as monitored by LC-MS, the solution was evaporated to dryness to obtain the target product. ESI-MS m / z: 280.16 [M+H]+.

[0428] Step 3: Preparation of (6-chloro-5-methylpyridin-3-yl)(2-methyl-2,7-diazaspiro[3.5]non-7-yl) methyl ketone

[0429]

[0430] (6-chloro-5-methylpyridin-3-yl)(2,7-diazaspiro[3.5]non-7-yl)methyl ketone hydrochloride (150 mg, 0.474 mmol) was added to a 50 mL round-bottom flask. 2.5 mL of LCM and 0.5 mL of MeOH were added as solvents. A 37% aqueous formaldehyde solution (77 mg, 0.949 mmol) was added, and the mixture was stirred at room temperature for 1 h. Then, sodium acetate (78 mg, 0.949 mmol) and sodium triacetylborohydride (201 mg, 0.949 mmol) were added sequentially, and the reaction was continued for 2 h. LC-MS was used to monitor the reaction until complete. After removing a small amount of solid impurities by filtration, the filtrate was evaporated to dryness to obtain the target product. ESI-MS m / z: 294.0 [M+H]+.

[0431] Step 4: Preparation of N-(3-chloro-5-(methylsulfonamido)phenyl)-4-(3-methyl-5-(2-methyl-2,7-diazaspiro[3.5]nonane-7-carbonyl)pyridin-2-yl)thiophene-2-carboxamide

[0432]

[0433] To a 50 mL round-bottom flask, add (6-chloro-5-methylpyridin-3-yl)(2-methyl-2,7-diazaspiro[3.5]non-7-yl) ketone (95 mg, 0.323 mmol), N-(3-chloro-5-(methylsulfonamido)phenyl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)thiophene-2-carboxamide (155 mg, 0.339 mmol), Pd(dppf)Cl2.CH2Cl2 (26 mg, 0.032 mmol), and K2CO3 (134 mg, 0.970 mmol). Then add 3 mL of 1,4-Dioxane and 0.5 mL of water. React at 90 °C for 3 h under Ar protection. After the reaction is complete as monitored by LC-MS, dilute with saturated brine, extract with ethyl acetate, separate the liquid from the liquid, dry the organic phase, and send to a preparative purification line to obtain the target product. ESI-MS m / z: 587.9 [M+H] + . 1 H NMR (400MHz, DMSO) δ10.59(s,1H),8.55(d,J=1.1Hz,1H),8.50(d,J=1.8Hz,1H),8.27(d,J=1.2Hz,1H),7.77(d,J=1.6Hz,1H),7.69(t,J=1.8Hz,1H), 7.66(t,J=1.8Hz,1H),6.96(t,J=1.9Hz,1H),3.57(s,2H),3.28(s,2H),3. 10(s,4H),3.07(s,3H),2.54(s,3H),2.32(s,3H),1.72(d,J=16.5Hz,4H).

[0434] Example 27: N-(3-chloro-5-(methylsulfonamido)phenyl)-5-methyl-1-(5-(4-(oxecyclobutane-3-yl)piperazin-1-yl)pyridin-2-yl)-1H-pyrrole-3-carboxamide

[0435]

[0436] Step 1: Preparation of 1-(6-bromopyridin-3-yl)-4-(oxetane-3-yl)piperazine

[0437]

[0438] 2-Bromo-5-iodopyridine (1 g, 3.5 mmol), 1-(3-oxetanebutyl)piperazine (449.3 mg, 2.92 mmol), Pd2(dba)3 (107 mg, 0.117 mmol), Xantphos (118 mg, 0.204 mmol), and sodium tert-butoxide (842 mg, 8.76 mmol) were added to toluene (25 mL). Under argon protection, the mixture was heated and stirred at 100 °C for 3 h. After the reaction was complete as monitored by LC-MS, toluene was removed by rotary evaporation. The product was then dissolved in ethyl acetate, filtered to remove insoluble impurities, washed three times with saturated brine, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography to obtain the target product. ESI-MS m / z: 298.0 [M+H] + .

[0439] Step 2: Preparation of ethyl 5-methyl-1-(5-(4-(oxecyclobutan-3-yl)piperazin-1-yl)pyridin-2-yl)-1H-pyrrole-3-carboxylate

[0440]

[0441] 1-(6-bromopyridin-3-yl)-4-(oxetane-3-yl)piperazine (357.5 mg, 1.2 mmol), ethyl 5-methyl-1H-pyrrole-3-carboxylate (153 mg, 1.0 mmol), CuI (38 mg, 0.2 mmol), K₂CO₃ (420 mg, 3.0 mmol), and N,N'-dimethyl-1,2-cyclohexanediamine (56.9 mg, 0.08 mmol) were added to acetonitrile (10 mL). Under argon protection, the mixture was stirred at 90 °C for 5 hours. After the reaction was completed as monitored by LC-MS, the reaction was quenched with water, and the mixture was extracted three times with ethyl ester. The combined organic phases were washed once with saturated brine, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography to obtain the target product. ESI-MS m / z: 372.16 [M+H] + .

[0442] Step 3: Preparation of 5-methyl-1-(5-(4-(oxecyclobutan-3-yl)piperazin-1-yl)pyridin-2-yl)-1H-pyrrole-3-carboxylic acid

[0443]

[0444] Ethyl 5-methyl-1-(5-(4-(oxecyclobutan-3-yl)piperazin-1-yl)pyridin-2-yl)-1H-pyrrole-3-carboxylate (297 mg, 0.8 mmol) and KOH (1.8 g, 32 mmol) were added to a mixed solvent of 1,4-dioxane and water (4 mL: 4 mL). The mixture was heated and stirred overnight at 90 °C. After the reaction was completed as monitored by LC-MS, 6 mol / L hydrochloric acid solution was slowly added dropwise to the reaction solution to adjust the pH to 4–5. The solution was then evaporated to dryness, and the target product was obtained by column chromatography. ESI-MS m / z: 343.0 [M+H] + .

[0445] Step 4: Preparation of N-(3-chloro-5-(methylsulfonamido)phenyl)-5-methyl-1-(5-(4-(oxecyclobutan-3-yl)piperazin-1-yl)pyridin-2-yl)-1H-pyrrole-3-carboxamide

[0446]

[0447] 5-Methyl-1-(5-(4-(oxecyclobutan-3-yl)piperazin-1-yl)pyridin-2-yl)-1H-pyrrolo-3-carboxylic acid (171 mg, 0.5 mmol), N-(3-amino-5-chlorophenyl)methanesulfonamide (132 mg, 0.6 mmol), TCFH (210 mg, 0.75 mmol), and NMI (123 mg, 1.5 mmol) were added to DMF (4 mL) and stirred overnight at 50 °C. After the reaction was completed as monitored by LC-MS, column chromatography yielded 120 mg of product, thus preparing 30 mg of the target product. ESI-MS m / z: 544.9 [M+H] + . 1H NMR (400MHz, DMSO) δ10.00(s,1H),9.81(s,1H),8.25(d,J=2.9Hz,1H),7.83(d,J=2.0Hz,1H),7.72(t,J=1.8Hz, 1H),7.64(t,J=1.8Hz,1H),7.56(dd,J=9.0,3.0Hz,1H),7.43(d,J=8.9Hz,1H),6.89(t,J=1.9Hz,1H),6.54(s,1H ), 4.59 (t, J = 6.5 Hz, 2H), 4.49 (t, J = 6.0 Hz, 2H), 3.47 (dt, J = 12.5, 6.3 Hz, 1H), 3.31 (s, 6H), 3.06 (s, 2H), 2.47-2.39 (m, 3H), 2.30 (s, 3H). Example 28: N-(3-chloro-5-(methylsulfonamido)phenyl)-4-(5-(4-(3,3-difluorocyclobutyl)piperazin-1-yl)-3-methylpyridin-2-yl)thiophene-2-carboxamide

[0448]

[0449] Step 1: Preparation of tert-butyl 4-(6-bromo-5-methylpyridin-3-yl)piperazine-1-carboxylate

[0450]

[0451] 2-Bromo-5-iodo-3-methylpyridine (4.5 g, 15 mmol), N-tert-butyloxycarbonyl-piperazine (5.6 g, 30 mmol), cesium carbonate (15 g, 45 mmol), tris(dibenzylacetone)dipalladium (686 mg, 0.75 mmol), and 4,5-bis(diphenylphosphine-9,9-dimethyloxanthracene) (433 mg, 0.75 mmol) were dissolved in 50 mL of 1,4-dioxane. The mixture was heated to 100 °C and stirred overnight under argon protection. After the reaction was completed as monitored by LC-MS, the reaction solution was cooled to room temperature, extracted with ethyl acetate and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give 4.6 g of crude product. ESI-MS m / z: 355.9 [M+H] + .

[0452] Step 2: Preparation of 1-(6-bromo-5-methylpyridin-3-yl)piperazine

[0453]

[0454] The crude product from the previous step, tert-butyl 4-(6-bromo-5-methylpyridin-3-yl)piperazine-1-carboxylate (1.4 g, 4 mmol), was dissolved in 5 mL of dichloromethane, and 5 mL of trifluoroacetic acid was added. The reaction mixture was reacted for 3 h. After the reaction was completed, saturated sodium bicarbonate solution was added to the reaction solution to adjust the pH to alkaline. The mixture was extracted with dichloromethane and saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography to obtain 0.8 g of the target compound. ESI-MS m / z: 255.9 [M+H] + .

[0455] Step 3: Preparation of 1-(6-bromo-5-methylpyridin-3-yl)-4-(3,3-difluorocyclobutyl)piperazine

[0456]

[0457] 1-(6-bromo-5-methylpyridin-3-yl)piperazine (0.8 g, 3.1 mmol) was dissolved in 20 mL of dichloromethane and cooled to -20 °C. 3,3-difluorocyclobutanone (328 mg, 3.1 mmol) was added and stirred for 2 h, followed by the addition of sodium cyanoborohydride (391 mg, 6.2 mmol). The mixture was heated to room temperature and stirred for 1 h. After the reaction was complete as monitored by LCMs, a saturated aqueous sodium bicarbonate solution was added to the reaction mixture. The mixture was extracted with dichloromethane and saturated brine. The organic phase was collected, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and column chromatography yielded 173 mg of the target compound as a yellow solid. ESI-MS m / z: 345.9 [M+H] + .

[0458] Step 4: Preparation of N-(3-chloro-5-(methylsulfonamido)phenyl)-4-(5-(4-(3,3-difluorocyclobutyl)piperazin-1-yl)-3-methylpyridin-2-yl)thiophene-2-carboxamide

[0459]

[0460] N-(3-chloro-5-(methanesulfonamido)phenyl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)thiophene-2-carboxamide (274 mg, 0.6 mmol), 1-(6-bromo-5-methylpyridin-3-yl)-4-(3,3-difluorocyclobutyl)piperazine (173 mg, 0.5 mmol), DPPF palladium dichloride (37 mg, 0.05 mmol), and potassium carbonate (248 mg, 1.8 mmol) were dissolved in 5 mL of 1,4-dioxane and 1 mL of water. The mixture was heated to 35 °C and stirred for 2 hours under argon protection. After the reaction was complete as monitored by LCMs, the reaction solution was cooled to room temperature, extracted with ethyl acetate and saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and column chromatography to obtain a crude product, which was then sent for further preparation. 20 mg of the target product was obtained as a white solid. ESI-MS m / z: 595.8 [M+H] + . 1 H NMR (400MHz, DMSO) δ10.55(s,1H),10.06(s,1H),8.46(d,J=1.2Hz,1H),8.22(d,J=2.7 Hz,1H),8.01(d,J=1.1Hz,1H),7.68(dt,J=9.1,1.8Hz,2H),7.28(d,J=2.5Hz,1H),6.95 (t, J = 1.9 Hz, 1H), 3.29-3.24 (m, 4H), 3.07 (s, 3H), 2.71 (dd, J = 14.6, 8.9 Hz, 3H), 2.44 (q, J = 7.0 Hz, 9H). Example 29: N-(3-chloro-5-((N,N-dimethylaminosulfonyl)amino)phenyl)-4-(3-methylpyridin-2-yl)thiophene-2-carboxamide

[0461]

[0462] Step 1: Preparation of N-(3-chloro-5-nitrophenyl)-N,N-dimethylaminosulfonamide

[0463]

[0464] 1-Chloro-3-fluoro-5-nitrobenzene (5 g, 28.5 mmol) and N,N-dimethylaminosulfonamide (2.71 g, 28.5 mmol) were dissolved in 60 mL of DMSO. Then, Cs₂CO₃ (27.8 g, 85.5 mmol) was added to the stirred solution, and the mixture was stirred at 60 °C for 2 h. After the reaction was complete as monitored by LC-MS, Cs₂CO₃ was removed by filtration. The filtrate was then quenched with a large amount of water and extracted with ethyl acetate. The extract was dried over anhydrous sodium sulfate, and the resulting solid was slurried with dichloromethane and petroleum ether for 1 h. After filtration, washing, and drying yielded the target product as a yellow solid. ESI-MS m / z: 277.8 [MH] - .

[0465] Step 2: Preparation of N-(3-chloro-5-((N,N-dimethylaminosulfonyl)amino)phenyl)-4-(3-methylpyridin-2-yl)thiophene-2-carboxamide

[0466]

[0467] 4-(3-methylpyridin-2-yl)thiophene-2-carboxylic acid (87 mg, 0.40 mmol) and TCFH (170 mg, 0.60 mmol) were added sequentially to a 50 mL double-necked flask. 5 mL of acetonitrile was added to dissolve the acid, followed by the addition of NMI (99 mg, 1.20 mmol). The mixture was stirred at room temperature for 5 minutes, then N-(3-chloro-5-aminophenyl)-N,N-dimethylaminosulfonamide (100 mg, 0.40 mmol) was added. The mixture was stirred at room temperature for 2 hours. After the reaction was complete as monitored by LC-MS, the reaction solution was diluted with water, extracted three times with ethyl acetate, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified using a preparative separation platform to obtain the target product as a white solid. ESI-MS m / z: 450.9 [M+H] + . 1 H NMR (400MHz, DMSO) δ10.58(s,1H),10.25(s,1H),8.60(d,J=4.4Hz,1H),8.51(d,J=1.1Hz,1H),8.28(s,1H),7.94(d,J=7.3 Hz,1H),7.71(t,J=1.7Hz,1H),7.64(t,J=1.7Hz,1H),7.50-7.44(m,1H),6.97(t,J=1.8Hz,1H),2.78(s,6H),2.54(s,3H).

[0468] Example 30: N-(3-chloro-5-((N-methylaminosulfonyl)amino)phenyl)-4-(3-methylpyridin-2-yl)thiophene-2-carboxamide

[0469]

[0470] Step 1: Preparation of N-(3-chloro-5-nitrophenyl)-N-methylaminosulfonamide

[0471]

[0472] 1-Chloro-3-fluoro-5-nitrobenzene (795 mg, 4.53 mmol) and N-methylaminosulfonamide (500 mg, 4.53 mmol) were dissolved in 40 mL of DMSO. Then, Cs₂CO₃ (1.477 g, 13.59 mmol) was added to the stirred solution, and the mixture was stirred overnight at 100 °C. After the reaction was complete as monitored by LC-MS, Cs₂CO₃ was removed by filtration. The filtrate was then quenched with a large amount of water and extracted eight times with ethyl acetate. The organic phase was washed eight times with water / saturated brine and dried over anhydrous sodium sulfate. The crude product was obtained by rotary evaporation and purified by petroleum ether / ethyl acetate column chromatography. The product was approximately 30% concentrated, and 270 mg of a yellow solid was obtained by rotary evaporation. ESI-MS m / z: 263.8 [MH] - .

[0473] Step 2: Preparation of N-(3-chloro-5-((N-methylaminosulfonyl)amino)phenyl)-4-(3-methylpyridin-2-yl)thiophene-2-carboxamide

[0474]

[0475] 4-(3-methylpyridin-2-yl)thiophene-2-carboxylic acid (233 mg, 1.06 mmol) and TCFH (446 mg, 1.59 mmol) were added sequentially to a 50 mL single-necked flask. 5 mL of acetonitrile was added to dissolve the precipitate, followed by NMI (261 mg, 3.18 mmol). The mixture was stirred at room temperature for 2 minutes, then N-(3-chloro-5-aminophenyl)-N-methylaminosulfonamide (233 mg, 1.06 mmol) was added. The mixture was stirred at room temperature for 2 hours. After the reaction was complete as monitored by LC-MS, the reaction solution was diluted with water, extracted three times with ethyl acetate, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified using a preparative separation platform to obtain 100 mg of the target product as a white solid. ESI-MS m / z: 436.8 [M+H] + . 1H NMR (400MHz, DMSO) δ10.52(s,1H),10.02(s,1H),8.50(dd,J=6.3,1.2Hz,2H),8.18(d,J=1.3Hz,1H),7.75(dd,J=7.7,0.8Hz,1H ),7.56(dt,J=9.5,1.8Hz,2H),7.44(q,J=4.9Hz,1H),7.30(dd,J=7.7,4.7Hz,1H),6.99(t,J=1.9Hz,1H),2.47(d,J=4.9Hz,6H).

[0476] Example 31: (R)-N-(3-chloro-5-(methylsulfonamido)phenyl)-5-methyl-4-(6a,7,8,9-tetrahydro-6H-pyrido[4,3-b]pyrrolo[1,2-d][1,4]oxazin-3-yl)thiophene-2-carboxamide

[0477]

[0478] Step 1: Preparation of (R)-2-(((5-bromo-2-chloropyridin-4-yl)oxy)methyl)pyrrolidine-1-carboxylic acid tert-butyl ester

[0479]

[0480] 5-Bromo-2-chloro-4-hydroxypyridine (2.1 g, 10 mmol), Boc-D-prolyl (3 g, 15 mmol), and PPh3 (3.9 g, 15 mmol) were dissolved in 30 mL of tetrahydrofuran. After cooling to 0 °C under Ar protection, DEAD (2.6 g, 15 mmol) was slowly added dropwise. The mixture was then stirred overnight at room temperature. After the reaction was complete as monitored by LCMs, the solution was concentrated under reduced pressure and column chromatography was performed to give 3.4 g of the compound. ESI-MS m / z: 390.8 [M+H] + .

[0481] Step 2: Preparation of (R)-5-bromo-2-chloro-4-(pyrrolidine-2-ylmethoxy)pyridine

[0482]

[0483] (R)-2-(((5-bromo-2-chloropyridin-4-yl)oxy)methyl)pyrrolidine-1-carboxylic acid tert-butyl ester (3.4 g, 8.7 mmol) was dissolved in 10 mL of dichloromethane, and 10 mL of trifluoroacetic acid was added. The mixture was then stirred for 2 hours. After the reaction was completed, the pH was adjusted to weakly basic using saturated sodium bicarbonate as monitored by LCMs. The mixture was then extracted with saturated brine and dichloromethane, and the organic phase was collected, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give 2.3 g of crude compound, which was used directly in the next step. ESI-MS m / z: 290.8 [M+H] + .

[0484] Step 3: Preparation of ((R)-3-chloro-6a,7,8,9-tetrahydro-6H-pyrido[4,3-b]pyrrolo[1,2-d][1,4]oxazine

[0485]

[0486] (R)-5-bromo-2-chloro-4-(pyrrolidine-2-ylmethoxy)pyridine (2.3 g, 8 mmol), Pd2(dba)3 (732 mg, 0.8 mmol), Xantphos (462 mg, 0.8 mmol), and Cs2CO3 (5.2 g, 16 mmol) were dissolved in 30 mL of 1,4-dioxane and heated to 100 °C with stirring for 2 h under Ar protection. After the reaction was complete as monitored by LCMs, the reaction mixture was cooled to room temperature. The mixture was then extracted with saturated sodium chloride solution and ethyl acetate. The organic phase was collected, dried over anhydrous sodium sulfate, and concentrated under reduced pressure by column chromatography to give 893 mg of the compound as a yellow solid. ESI-MS m / z: 211.0 [M+H] + Step 4: Preparation of (R)-N-(3-chloro-5-(methylsulfonamido)phenyl)-5-methyl-4-(6a,7,8,9-tetrahydro-6H-pyrido[4,3-b]pyrrolo[1,2-d][1,4]oxazin-3-yl)thiophene-2-carboxamide

[0487]

[0488] N-(3-chloro-5-(methylsulfonamido)phenyl)-5-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)thiophene-2-carboxamide (188 mg, 0.4 mmol), ((R)-3-chloro-6a,7,8,9-tetrahydro-6H-pyrido[4,3-b]pyrrolo[1,2-d][1,4]oxazine (126 mg, 0.6 mmol), DPPF palladium dichloride (29 mg, 0.04 mmol) and potassium carbonate (110 mg, 0.8 mmol) were dissolved in 2.5 mL of 1,4-dioxane and 0.5 mL of water. The mixture was heated to 100 °C and stirred for 2 hours under argon protection. After the reaction was complete as monitored by LCMs, the reaction solution was cooled to room temperature, extracted with ethyl acetate and saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated by column chromatography under reduced pressure to obtain a crude product. This crude product was then used to prepare a white solid of 40 mg. ESI-MS m / z: 518.8 [M+H] + . 1 H NMR (400MHz, DMSO) δ10.38(s,1H),8.30(s,1H),7.95(s,1H),7.66(dd,J=5.1,1.8Hz,2H),7.09(s,1H),6.95(t,J=1.8Hz,1H),4.63- 4.55(m,1H),3.57-3.47(m,3H),3.24(dd,J=16.2,8.9Hz,1H),3.07(s,3H),2.65(s,3H),2.14-1.90(m,3H),1.47(t,J=19.4Hz,1H).

[0489] Example 32: N-(3-chloro-5-(methylsulfonamido)phenyl)-4-(4,4-difluoro-1'-methyl-2'-oxo-1',2'-dihydrospiro[cyclohexane-1,3'-pyrrolo[3,2-b]pyridine]-5'-yl)-5-methylthiophene-2-carboxamide

[0490]

[0491] Step 1: Preparation of N-(2-bromo-6-chloropyridin-3-yl)-4,4-difluorocyclohexane-1-carboxamide

[0492]

[0493] 4,4-Difluorocyclohexane-1-carboxylic acid (2.40 g, 14.40 mmol), TCFH (5.05 g, 18.00 mmol), and NMI (3.90 g, 48 mmol) were added sequentially to a 250 mL single-necked flask. 50 mL of acetonitrile was added, and the mixture was stirred for 3 min. Then, 2-bromo-6-chloropyridin-3-amine (2.5 g, 12.00 mmol) was added. After the addition was complete, the mixture was stirred overnight at 80 °C. The reaction was monitored by LC-MS until complete. The reaction was then quenched with water. The reaction solution was extracted with ethyl acetate. The organic phase was washed three times with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. Dry column chromatography was performed using a system of petroleum ether and ethyl acetate. The product was purified under 18% polarity. The solvent was removed under reduced pressure to give a white solid. ESI-MS m / z: 352.8 [M+H] + .

[0494] Step 2: Preparation of N-(2-bromo-6-chloropyridin-3-yl)-4,4-difluoro-N-methylcyclohexane-1-carboxamide

[0495]

[0496] N-(2-bromo-6-chloropyridin-3-yl)-4,4-difluorocyclohexane-1-carboxamide was added to a 100 mL single-necked flask, dissolved in 40 mL of tetrahydrofuran. Sodium hydride (0.48 g, 12 mmol) was added while stirring at 0 °C. After stirring for 10 min, iodomethane (2.13 g, 15.00 mmol) was added. The mixture was stirred at 50 °C for 2 h after the addition was complete. The reaction was monitored by LC-MS until complete. The mixture was then quenched with water, and the tetrahydrofuran was removed under reduced pressure. The residue was extracted three times with ethyl acetate, and the organic phase was washed three times with water and dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure. The crude product was precipitated by column chromatography in a petroleum ether / ethyl acetate system to give a white solid. ESI-MS m / z: 366.8 [M+H] + .

[0497] Step 3: Preparation of 5'-chloro-4,4-difluoro-1'-methylspiro[cyclohexane-1,3'-pyrrolo[3,2-b]pyridine]-2'(1'H)-one

[0498]

[0499] N-(2-bromo-6-chloropyridin-3-yl)-4,4-difluoro-N-methylcyclohexane-1-carboxamide was added to a 50 mL double-necked flask, followed by 15 mL of toluene. Then, PEPPSI-IPr (0.11 g, 0.16 mmol) and sodium tert-butoxide (0.31 g, 3.26 mmol) were added sequentially. After the addition was complete, the mixture was purged with argon three times and stirred for 4 h under argon protection at 100 °C. After the reaction was completed by LC-MS monitoring, the toluene was evaporated to dryness, and the residue was extracted with ethyl acetate. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, concentrated, and the crude product was analyzed by column chromatography using a petroleum ether / ethyl acetate system. The product was concentrated into a brown solid of 170 mg at 8-10%. ESI-MS m / z: 286.9 [M+H]+.

[0500] Step 4: Preparation of N-(3-chloro-5-(methylsulfonamido)phenyl)-4-(4,4-difluoro-1'-methyl-2'-oxo-1',2'-dihydrospiro[cyclohexane-1,3'-pyrrolo[3,2-b]pyridine]-5'-yl)-5-methylthiophene-2-carboxamide

[0501]

[0502] The following medications were added: 5'-chloro-4,4-difluoro-1'-methylspiro[cyclohexane-1,3'-pyrrolo[3,2-b]pyridine]-2'(1'H)-one (140 mg, 0.49 mmol), N-(3-chloro-5-(methylsulfonamido)phenyl)-5-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaboron-2-yl)thiophene-2-carboxamide (460 mg, 0.98 mmol), and [1,1'-bis(diphenylphosphine)ferrocene]palladium dichloride (37 mg, ...). 0.05 mmol of potassium phosphate (312 mg, 1.47 mmol) was added sequentially to a 50 mL double-necked flask. 5 mL of 1,4-dioxane dissolved in potassium phosphate and 1 mL of water were added. After purging three times with argon, the mixture was stirred at 90 °C for 10 hours under argon protection. After the reaction was complete as monitored by LC-MS, the mixture was extracted with ethyl acetate. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The crude product was purified by petroleum ether / ethyl acetate column chromatography. At 48%, the product was concentrated into a white solid of 90 mg. This was then sent to a preparative separation platform for purification and lyophilized to give 20 mg of a white solid. ESI-MS m / z: 594.8 [M+H] + . 1¹H NMR (400MHz, DMSO) δ 10.42 (s, 1H), 10.11 (s, 1H), 8.31 (s, 1H), 7.72–7.56 (m, 4H), 6.96 (s, 1H), 3.22 (s, 3H), 3.07 (s, 3H), 2.71 (s, 3H), 2.30 (d, J = 10.3 Hz, 2H), 2.08 (s, 1H), 1.95 (dd, J = 13.9, 4.2 Hz, 4H), 1.23 (s, 1H). Example 33: N-(3-chloro-5-(methylsulfonamido)phenyl)-4-(5-((3-(5-fluoropyridin-3-yl)prop-2-yn-1-yl)oxy)pyridin-2-yl)-5-methylthiophene-2-carboxamide

[0503]

[0504] Step 1: Preparation of 3-fluoro-5-(3-(tetrahydro-2H-pyran-2-yl)oxy)prop-1-yn-1-yl)pyridine

[0505]

[0506] Piperanone (3.00 g, 17.05 mmol) was weighed into a 250 mL single-necked flask and dissolved in 600 mL of N,N-dimethylformamide. Then, 5-bromo-3-fluoropyridine (7.16 g, 51.14 mmol), tetrakis(triphenylphosphine)palladium (1.00 g, 0.85 mmol), cuprous iodide (648 mg, 3.41 mmol), and triethylamine (5.175 g, 51.14 mmol) were added sequentially. After the addition was complete, the mixture was stirred at 85 °C for 1.5 h. The reaction was monitored by LC-MS until complete. The reaction was then quenched with water. The reaction solution was extracted with ethyl acetate. The organic phase was washed 4-5 times with water and saturated brine. After drying with anhydrous sodium sulfate, the solvent was removed under reduced pressure. Dry column chromatography was performed using petroleum ether and ethyl acetate. The product was purified under 5% polarity. The solvent was removed under reduced pressure to give 3.8 g of a brownish-yellow oil. ESI-MS m / z: 236.0 [M+H] + .

[0507] Step 2: Preparation of 3-(5-fluoropyridin-3-yl)prop-2-yn-1-ol

[0508]

[0509] Add 30 ml of acetic acid, 15 ml of tetrahydrofuran, and 7.5 ml of water sequentially to a 100 ml single-necked flask. While stirring, add 3-fluoro-5-(3-(tetrahydro-2H-pyran-2-yl)oxy)prop-1-yn-1-yl)pyridine (3.80 g, 16.17 mmol). Stir at 45 °C for 20 hours. After the reaction is complete as monitored by LC-MS, concentrate under reduced pressure to remove acetic acid. Extract the residue 4-5 times with ethyl acetate. Wash the organic phase 3 times with water and dry to anhydrous sodium sulfate. Remove the solvent under reduced pressure. Analyze the crude product using a petroleum ether / ethyl acetate column chromatography system. Load the sample dry and remove the product at 20%-30%. Remove the solvent under reduced pressure to give 1.48 g of a yellow solid. ESI-MS m / z: 152.0 [M+H] + .

[0510] Step 3: Preparation of 2-bromo-5-((3-(5-fluoropyridin-3-yl)prop-2-yn-1-yl)oxy)pyridine

[0511]

[0512] 3-(5-fluoropyridin-3-yl)prop-2-yn-1-ol (1.40 g, 9.27 mmol) and 2-bromo-5-hydroxypyridine (1.63 g, 9.27 mmol) were added sequentially to a 250 mL single-necked flask. Tetrahydrofuran (90 mL) was added to dissolve the flask, and the mixture was stirred at 0 °C for 5 min. Triphenylphosphine (3.17 g, 12.05 mmol) was added, and the mixture was stirred at 0 °C for 5 min. Diisopropyl azodicarbonate (2.43 g, 12.05 mmol) was added dropwise. After the addition was complete, the ice bath was removed, and the mixture was stirred at 25 °C for 2 h. After the reaction was completed by LC-MS monitoring, the mixture was extracted three times with ethyl acetate. The organic phase was washed three times with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated. The crude product was dry-mounted and precipitated by petroleum ether / ethyl acetate column chromatography. The product was a concentrated pale yellow solid at approximately 15%. ESI-MS m / z: 306.8 [M+H]+.

[0513] Step 4: Preparation of N-(3-chloro-5-(methylsulfonamido)phenyl)-4-(5-((3-(5-fluoropyridin-3-yl)prop-2-yn-1-yl)oxy)pyridin-2-yl)-5-methylthiophene-2-carboxamide

[0514]

[0515] The following compounds were added: 2-bromo-5-((3-(5-fluoropyridin-3-yl)prop-2-yn-1-yl)oxy)pyridine (380 mg, 1.24 mmol), N-(3-chloro-5-(methylsulfonamido)phenyl)-5-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)thiophene-2-carboxamide (583 mg, 1.24 mmol), and [1,1'-bis(diphenylphosphine)ferrocene]palladium dichloride·dichloromethane complex (41 mg, 0.05 mmol). 1,4-dioxane (10 ml) and potassium carbonate (207 mg, 1.49 mmol) were added sequentially to a 50 ml double-necked flask. Then, 1,4-dioxane (10 ml) and water (2 ml) were added. After purging three times with argon, the mixture was stirred at 90 °C for 10 hours under argon protection. After the reaction was complete as monitored by LC-MS, the mixture was extracted with ethyl acetate. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The crude product was analyzed by column chromatography using a petroleum ether / ethyl acetate system. The product was concentrated at 48% to give 90 mg of a pale yellow oil. This was then purified using a preparative separation platform and freeze-dried to give 20 mg of the target product, a white cotton-like solid. ESI-MS m / z: 570.8

[0516] [M+H]+. 1 H NMR (400MHz, DMSO) δ10.44(s,1H),10.11(s,1H),8.65(d,J=2.7Hz,1H),8.56(s,1H),8.49(d,J=2.5Hz,1H),8. 36(s,1H),8.00-7.91(m,1H),7.74-7.58(m,4H),6.94(t,J=1.8Hz,1H),5.27(s,2H),3.07(s,3H),2.68(s,3H).

[0517] Example 34: N-(3-chloro-5-(methylsulfonamido)phenyl)-4-(5-(imidazol[1,2-b]pyrimidazine-3-ylethynyl)pyridin-2-yl)-5-methylthiophene-2-carboxamide

[0518]

[0519] Step 1: 3-((trimethylsilyl)ethynyl)imidazo[1,2-b]pyridazine

[0520]

[0521] 3-Bromoimidazolo[1,2-b]pyridazine (100 mg, 0.505 mmol), Pd(PPh3)2Cl2 (35 mg, 0.050 mmol), CuI (10 mg, 0.050 mmol), and Et3N (153 mg, 1.51 mmol) were added to a 50 mL flask, followed by the addition of 4 mL LDM. After purging with Ar gas, ethynyltrimethylsilane (99 mg, 1.01 mmol) was added, and the mixture was heated at 80 °C for 3 h. After the reaction was monitored by LC-MS to ensure complete reaction, the mixture was diluted with EA and washed three times with saturated NaCl. The organic phase was dried under reduced pressure and then subjected to column chromatography to obtain the product. ESI-MS m / z: 216.0 [M+H] +

[0522] Step 2: 3-ethynylimidazo[1,2-b]pyridazine

[0523]

[0524] 3-((trimethylsilyl)ethynyl)imidazo[1,2-b]pyridazine (105 mg, 0.488 mmol) was dissolved in 3 mL of methanol, and 1.5 mL of saturated KF aqueous solution was added. The mixture was reacted at room temperature for 30 min. After removing solid impurities by filtration, the filtrate was evaporated to dryness and subjected to column chromatography to obtain the product. ESI-MS m / z: 144.1 [M+H] +

[0525] Step 3: 3-((6-bromopyridin-3-yl)ethynyl)imidazo[1,2-b]pyridazine

[0526]

[0527] 3-ethynylimidazo[1,2-b]pyridazine (43 mg, 0.300 mmol), 2-bromo-5-iodopyridine (85 mg, 0.300 mmol), Pd(PPh3)2Cl2 (21 mg, 0.030 mmol), CuI (5.7 mg, 0.030 mmol), Et3N (91 mg, 0.901 mmol), and 3 mL THF were added. The reaction was carried out at room temperature for 2.5 h under Ar protection. After the reaction was complete as monitored by LC-MS, saturated NaCl solution was added, and the mixture was extracted with EA. The organic phase was dried and evaporated to dryness. Column chromatography yielded the target product. ESI-MS m / z: 298.9, 300.9 [M+H] +

[0528] Step 4: N-(3-chloro-5-(methylsulfonamido)phenyl)-4-(5-(imidazo[1,2-b]pyridazin-3-ylethynyl)pyridin-2-yl)-5-methylthiophene-2-carboxamide

[0529]

[0530] 3-((6-bromopyridin-3-yl)ethynyl)imidazo[1,2-b]pyridazine (60 mg, 0.200 mmol), N-(3-chloro-5-(methylsulfonamido)phenyl)-5-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)thiophene-2-carboxamide (94 mg, 0.200 mmol), Pd(dppf)Cl2.CH2Cl2 (60 mg, 0.200 mmol), and K2CO3 (83 mg, 0.602 mmol) were added sequentially to a 50 mL reaction flask. 2.5 mL of Dioxane and 0.5 mL of water were added, and the mixture was heated to 90 °C for 3 h under Ar protection. After the reaction was complete as monitored by LC-MS, saturated brine was added, and the mixture was extracted with EA. The organic phase was dried by rotary evaporation, preliminarily purified by silica gel column chromatography, and then sent for preparative purification. The final product was obtained by lyophilization. ESI-MS m / z: 562.8 [M+H] + . 1 H NMR (400MHz, DMSO) δ10.49(s,1H),8.90(d,J=1.7Hz,1H),8.76-8.72(m,1H),8.49(s,1H),8.31-8.25(m,2H),8.17(dd,J=8.3, 2.2Hz,1H),7.81(d,J=8.4Hz,1H),7.64(d,J=19.6Hz,2H),7.42(dd,J=9.2,4.4Hz,1H),6.93(s,1H),3.05(s,3H),2.76(s,3H).

[0531] Example 35: N-(3-chloro-5-(methylsulfonamido)phenyl)-4-(4,4-difluoro-3'H-spiro[cyclohexane-1,1'-furan[3,4-c]pyridine]-6'-yl)-5-methylthiophene-2-carboxamide

[0532]

[0533] Step 1: Preparation of 6'-chloro-4,4-difluoro-3'H-spiro[cyclohexane-1,1'-furan[3,4-c]pyridine]-3'-one

[0534]

[0535] 2,2,6,6-Tetramethylpiperidine (2.7 g, 19.13 mmol) was dissolved in 25 mL of THF 1. The solution was cooled to -78 °C under Ar protection. 2.5 M n-BuLi solution (9.94 mL, 24.84 mmol) was added, and the mixture was stirred for 1 h. Then, 6-chloronicotinic acid (1.0 g, 6.37 mmol) was added, and the mixture was stirred for another 1 h. Next, 4,4-difluorocyclohexane-1-one (1.7 g, 12.7 mmol) was added, and the reaction was allowed to proceed for 1 h. The reaction was then quenched with water, and the pH was adjusted to acidic with 6N hydrochloric acid. The mixture was extracted with EA, and the combined organic phases were dried and evaporated to dryness before column chromatography to obtain the product. ESI-MS m / z: 273.9 [M+H] + .

[0536] Step 2: Preparation of 1-(2-chloro-5-(hydroxymethyl)pyridin-4-yl)-4,4-difluorocyclohexane-1-ol

[0537]

[0538] 1.08 g (4 mmol) of 6'-chloro-4,4-difluoro-3'H-spiro[cyclohexane-1,1'-furan[3,4-c]pyridine]-3'-one was dissolved in 20 mL of THF, and 0.12 g of MeOH was added. The mixture was cooled to 0°C, and 6 mL (12 mmol) of 2 M LiBH4 solution was added. The reaction was allowed to proceed for 1 h. After the reaction was completed by LC-MS monitoring, water was added to quench the reaction, and the mixture was extracted three times with EA. The synthesized organic phase was washed twice with water, dried by rotary evaporation, and then subjected to column chromatography to obtain the product. ESI-MS m / z: 277.9 [M+H] +

[0539] Step 3: Preparation of 6'-chloro-4,4-difluoro-3'H-spiro[cyclohexane-1,1'-furan[3,4-c]pyridine]

[0540]

[0541] 1-(2-chloro-5-(hydroxymethyl)pyridin-4-yl)-4,4-difluorocyclohexane-1-ol (50 mg, 0.18 mmol) was dissolved in 2.5 mL of THF, and 0.4 mL of 1 M KHMDS solution (0.4 mmol) was added. After cooling to 0 °C, p-toluenesulfonyl chloride (41 mg, 0.216 mmol) was added, and the reaction was carried out in an ice bath for 1 h. After the reaction was completed by TLC, the mixture was diluted with saturated brine, extracted twice with EA, and the combined organic phases were washed once with saturated brine. After drying to dryness with anhydrous sodium sulfate, the product was obtained by column chromatography. ESI-MS m / z: 260.06 [M+H] +

[0542] Step 4: Preparation of N-(3-chloro-5-(methylsulfonamido)phenyl)-4-(4,4-difluoro-3'H-spiro[cyclohexane-1,1'-furan[3,4-c]pyridine]-6'-yl)-5-methylthiophene-2-carboxamide

[0543]

[0544] The following ingredients were added: 6'-chloro-4,4-difluoro-3'H-spiro[cyclohexane-1,1'-furan[3,4-c]pyridine] (70 mg, 0.270 mmol), N-(3-chloro-5-(methylsulfonamido)phenyl)-5-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)thiophene-2-carboxamide (127 mg, 0.270 mmol), Pd(dppf)Cl2.CH2Cl2 22 mg (0.027 mmol) and K₂CO₃ (112 mg, 0.809 mmol) were added sequentially to a 50 mL reaction flask, followed by 2.5 mL of Dioxane and 0.5 mL of water. The mixture was heated at 90 °C for 3 h under Ar atmosphere protection. After the reaction was complete as monitored by LC-MS, saturated saline solution was added, and the mixture was extracted with EA. The organic phase was dried under rotary evaporation, preliminarily purified by silica gel column chromatography, and then sent for preparative purification. The final product was obtained by lyophilization. ESI-MS m / z: 567.8 [M+H] + . 1 HNMR (400MHz, DMSO) δ10.44(s,1H),10.12(s,1H),8.65(s,1H),8.37(s,1H),7.72(s,1H),7.69(t,J=1.8Hz,1H),7.63(t ,J=1.8Hz,1H),6.94(t,J=1.9Hz,1H),5.14(s,2H),3.06(s,3H),2.72(s,3H),2.19-2.05(m,6H),1.87(d,J=9.5Hz,2H).

[0545] Referring to the preparation methods of the above embodiments of the present invention, compounds of Examples 36-56 in Table 1 were prepared using different raw materials.

[0546] Table 1

[0547]

[0548]

[0549]

[0550]

[0551] Example 1: Evaluation of the in vitro kinase activity of the compound

[0552] 1. Experimental Materials

[0553] Compounds: The compounds of the present invention prepared in the above examples were each prepared into 10 mM by DMSO and then successively diluted to 5000 nM, 1250 nM, 312.5 nM, 78.125 nM, 19.53125 nM, 4.8828125 nM, 1.220703125 nM, 0.305175781 nM, 0.076293945 nM, and 0.019073486 nM.

[0554] Reagent: ADP-Glo TM Kinase Assay, purchased from Promega, V9103; DHX9-IN-2, purchased from Targetmol, T82568; SB-RNA, purchased from ICE, ICE-DRU-102; DHX9 (150-1150), purchased from ICE, H2309T-H01HF; 384-well plate, purchased from Perkin Elmer, 6007299;

[0555] instrument: 655 SYSTEM, purchased from LABCYTE; Centrifuge, purchased from CENCE; Microplate Reader, purchased from BMG;

[0556] 2. Experimental Methods

[0557] 2.1 Take 40 μL of each compound solution into a 384-well plate and dilute the compound with DMSO in a 1:4 gradient. Transfer 0.10 μL of the diluted compound solution in each row to a 384 detection plate, with two replicates for each concentration column.

[0558] 2.2 Add 5 μL of enzyme working solution to each well of the 384 detection plate, centrifuge at 1000 rpm for 1 min, and incubate at 25°C for 10 min.

[0559] 2.3 Add 5 μL of substrate and ATP working solution to each well of the 384 assay plate to initiate the reaction; incubate at 25°C for 60 minutes.

[0560] 2.4 Add 5 μL of ADP-Glo ​​Reagent to each well of the 384 detection plate, centrifuge at 1000 rpm for 1 min, and incubate at 25 °C for 40 min.

[0561] 2.5 Add 10 μL of detection solution to each well of the 384 detection plate, centrifuge at 1000 rpm for 1 min, and incubate at 25 °C for 40 min.

[0562] 2.6 The luminescence signal was read using BMG, and the inhibition rate (I%) was calculated using the formula: I% = 100 × (ave High control - cpd well) / (ave High control - ave Low control), where ave High control is DMSO and enzyme, and ave Low control is DMSO and analysis buffer. The nonlinear regression equation of the compound was fitted using XLfit 5.5.0 to obtain the IC50 value of the compound. 50 The values ​​represent the compound concentrations that, compared to the untreated group, correspond to 50% inhibition of enzyme activity. IC50 values ​​for some compounds are also shown. 50 The results are shown in Table 2.

[0563] Table 2. Inhibition of DHX9 enzymatic activity by the compounds of this invention

[0564]

[0565]

[0566] Experimental results show that the compounds of this invention have good inhibitory activity against DHX9.

[0567] Example 2: Evaluation of the in vitro cell activity of the compound

[0568] 1. Experimental Materials

[0569] Test compounds: The compounds of the present invention prepared in the above examples were each prepared in 20 mM solution with DMSO and then successively diluted 3-fold to 5 μM, 1.666666667 μM, 0.555555556 μM, 0.185185185 μM, 0.061728395 μM, 0.020576132 μM, 0.006858711 μM, 0.002286237 μM, and 0.000762079 μM.

[0570] The colorectal cancer cell lines LS411N and HCT116 were purchased from Nanjing Kebai Biotechnology Co., Ltd. and Wuhan Pronosai Life Technology Co., Ltd., respectively.

[0571] Reagents: RPMI-1640, purchased from Invitrogen, USA; FBS, purchased from Invitrogen, USA; EDTA, purchased from Sigma, USA; CellTiter- The Luminescent Cell Viability Assay Kit was purchased from Progema, Inc., USA.

[0572] 2. Experimental Methods

[0573] 2.1 Cell Culture:

[0574] Cell resuscitation: Dissolve the cells in a 37°C water bath, then transfer them to 15 mL of preheated culture medium, centrifuge at 1000 rpm for 5 minutes, discard the culture medium, resuspend the cells in 1 mL of fresh culture medium, transfer them to a T25 culture flask containing 7 mL of fresh culture medium, and incubate at 37°C with 5% CO2.

[0575] Cell passage: When cells reach 80-90% confluence, discard the original complete culture medium. Add 2 mL of PBS to wash away any remaining medium, then discard the PBS. Add 1 mL of 0.25% trypsin digestion solution and digest until, under a microscope, the pseudopodia retract and become rounded, but the cells have not yet detached in sheets. At this point, stop digestion with 2 mL of complete culture medium. Gently pipette and collect the cell suspension, transfer it to a 15 mL centrifuge tube, and centrifuge at 1000 rpm for 5 min. Discard the culture medium, resuspend the cells in 1 mL of fresh culture medium, and seed them at the desired density in culture dishes. Transfer the seeded cells to T25 culture flasks containing 7 mL of fresh culture medium and incubate at 37°C in a 5% CO2 incubator. Change the culture medium or passage the cells every 2-3 days depending on cell growth.

[0576] 2.2 Experimental Procedure:

[0577] Cell plating: T25 cells were grown in culture flasks until confluence reached 80-90%. Following cell passage methods, the cell suspension was collected and counted. The resuspended cells were adjusted to a concentration of 1.5 × 10⁻⁶ cells / mL. 4 Cells / mL. Add the cell suspension of the above concentration to a 96-well cell culture plate, adding 100 μL (1.5 × 10⁻⁶ cells / mL) to each well. 3 (cells / well); the outermost 36 wells of the 96-well plate were filled with 250 μL of PBS to prevent the culture medium at the edge from evaporating too quickly and causing too large differences in culture conditions between the inner wells; and then incubated in a 5% CO2 incubator at 37°C for 24 h.

[0578] Cellular drug administration: Add (2×) 100μL of drug to the original culture medium (100μL). The compound was prepared as follows: Weigh 1-2mg of the compound in advance and prepare a 20mM stock solution using DMSO. Dilute the drug with complete culture medium, starting at a maximum concentration of 5μM, and then dilute 3-fold to nine concentration gradients: 5μM, 1.666666667μM, 0.555555556μM, 0.185185185μM, 0.061728395μM, 0.020576132μM, 0.006858711μM, 0.002286237μM, and 0.000762079μM.

[0579] Assay: After 6 days of compound treatment, the culture medium in the wells was discarded. The CellTiter-Glo Luminescent Cell Viabillity Assay was removed 30 minutes before the scheduled incubation period and allowed to equilibrate to room temperature. Celltiter-Glo reagent was diluted 1:1 (equal volume) with complete culture medium, and 150 μL of the diluted Celltiter-Glo reagent was added to each well. The mixture was shaken at room temperature for 2 minutes. After incubation at room temperature for another 10 minutes, the chemiluminescence signal was detected by shaking. The read detection condition was 500 ms.

[0580] Results Analysis: Based on the AU values ​​exported from the microplate reader, the inhibition rate of each well relative to the solvent control well was calculated: Inhibition (%) = 100 - (AU experimental wells - AU blank wells) / (AU solvent control wells - AU blank wells) × 100. Based on different compound concentrations and their corresponding inhibition rates, IC50 curves were plotted using GraghPad 6.0 software. The data were analyzed to obtain the final IC50 values. The experimental results are shown in Table 3.

[0581] Table 3. Inhibitory activity of the compounds of this invention against HCT116 cell proliferation

[0582]

[0583]

[0584] Experimental results show that the compound of the present invention has good inhibitory activity against the proliferation of human colon cancer cells HCT116.

[0585] Although the present invention has been described in detail above, those skilled in the art will understand that various modifications and changes can be made to the invention without departing from its spirit and scope. The scope of the invention is not limited to the detailed description above, but should be attributed to the claims.

Claims

1. A compound of general formula (I) or an isomer thereof, a pharmaceutically acceptable salt, a solvate, a crystal, or a prodrug, in, Ring A is selected from C 6-12 Aryl, 5-15 quinone heteroaryl, C 3-12 Cycloalkyl and 3-12 membered heterocyclic groups; Ring B is selected from monocyclic, fused, or spirocyclic bicyclic or tricyclic aryl, heteroaryl, cycloalkyl, and heterocyclic groups; R 1 R 2 R 3 and R 4 Each group is independently selected from hydrogen, halogen, hydroxyl, alkyl, haloalkyl, hydroxyalkyl, alkoxy, haloalkoxy, hydroxyalkoxy, nitro, carboxyl, cyano, amino, monoalkylamino, alkylacylamino, alkylacyl, aminoacyl, alkylaminoacyl, oxo group and dialkylamino; L does not exist or is selected from C. 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 1-6 Alkoxy, C 2-6 alkenyloxy group, C 2-6 The alkynyl, acyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, and heterocyclic groups are optionally substituted by one or more groups selected from halogen, hydroxyl, alkyl, haloalkyl, hydroxyalkyl, alkoxy, haloalkoxy, hydroxyalkoxy, amino, monoalkylamino, alkylacylamino, alkylacyl, alkylsulfonyl, aminoacyl, alkylaminoacyl, dialkylamino, alkenyl, alkynyl, haloalkylacyl, hydroxyalkylacyl, and oxo groups. R 5 Selected from hydrogen, halogen, hydroxyl, C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 Alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, and heterocyclic groups, optionally substituted by one or more groups selected from halogen, hydroxyl, alkyl, haloalkyl, hydroxyalkyl, alkoxy, haloalkoxy, hydroxyalkoxy, amino, monoalkylamino, alkylacylamino, alkylacyl, alkylsulfonyl, aminoacyl, alkylaminoacyl, dialkylamino, alkenyl, alkynyl, haloalkylacyl, hydroxyalkylacyl, cycloalkyl, heterocyclic, aryl, heteroaryl, halocycloalkyl, and oxo groups; and m, n, and p are each independently selected from 0, 1, 2, and 3.

2. The compound or isomer thereof, pharmaceutically acceptable salt, solvate, crystal or prodrug according to claim 1, wherein general formula (I) has the structure of general formula (Ia). in, Ring B is selected from fused or spirocyclic tricyclic heteroaryl and heterocyclic groups; and R 1 R 2 R 3 R 4 m, n and p have the definitions described in general formula (I) of claim 1.

3. The compound or isomer thereof, pharmaceutically acceptable salt, solvate, crystal or prodrug according to claim 1, wherein general formula (I) has the structure of general formula (Ib). in, L is selected from C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 1-6 Alkoxy, C 2-6 alkenyloxy group, C 2-6 Acyl group; The ring C is selected from cycloalkyl, heterocyclic, fused, or spirocyclic bicyclic or tricyclic heteroaryl groups, optionally substituted by one or more groups selected from halogen, hydroxyl, alkyl, haloalkyl, hydroxyalkyl, alkoxy, haloalkoxy, hydroxyalkoxy, amino, monoalkylamino, alkylacylamino, alkylacyl, alkylsulfonyl, aminoacyl, alkylaminoacyl, dialkylamino, alkenyl, alkynyl, haloalkylacyl, hydroxyalkylacyl, and oxo groups; and Rings A and R 2 R 3 R 4 n and p have the definitions described in general formula (I) of claim 1.

4. The compound or isomer thereof, pharmaceutically acceptable salt, solvate, crystal or prodrug according to claim 1, wherein general formula (I) has the structure of general formula (Ic). in, Cycle C and cycle D are each independently selected from aryl, heteroaryl, cycloalkyl, cycloalkenyl, and heterocyclic groups, and are optionally substituted by one or more groups selected from halogen, hydroxyl, alkyl, haloalkyl, hydroxyalkyl, alkoxy, haloalkoxy, hydroxyalkoxy, amino, monoalkylamino, alkylacylamino, alkylacyl, alkylsulfonyl, aminoacyl, alkylaminoacyl, dialkylamino, alkenyl, alkynyl, haloalkylacyl, hydroxyalkylacyl, and oxo groups; When ring C is selected from monoheterocyclic and monoheteroaryl groups, its connection position with ring D is not N; and Rings A and R 2 R 3 R 4 n and p have the definitions described in general formula (I) of claim 1.

5. The compound or isomer thereof, pharmaceutically acceptable salt, solvate, crystal or prodrug according to any one of claims 1-4, wherein R 2 R 3 and R 4 Each is independently selected from hydrogen, fluorine, chlorine, bromine, iodine, hydroxyl, and C. 1-6 Alkyl, Halogenated C 1-6 Alkyl, hydroxyl C 1-6 Alkyl, C 1-6 Alkoxy, halogenated C 1-6 Alkoxy, hydroxy C 1-6 Alkoxy, nitro, carboxyl, cyano, amino, C 1-6 Alkylamino, C 1-6 Alkyl acylamino, C 1-6 Alkyl acyl, amino acyl, C 1-6 Alkylaminoacyl, oxo group and double C 1-6 Alkylamino.

6. The compound or isomer thereof, pharmaceutically acceptable salt, solvate, crystal or prodrug according to claim 1, wherein ring B is selected from...

7. The compound or isomer thereof, pharmaceutically acceptable salt, solvate, crystal or prodrug according to claim 1, wherein R 5 Selected from hydrogen, fluorine, chlorine, bromine, iodine, hydroxyl, methyl, ethyl, vinyl, ethynyl, propynyl, cyclopropyl, cyclobutyl, oxetyl, pyridyl, cyclopentenyl, It may be optionally oxidized by one or more elements selected from fluorine, chlorine, bromine, iodine, hydroxyl, C 1-3 Alkyl, Halogenated C 1-3 Alkyl, hydroxyl C 1-3 Alkyl, C 1-3 Alkoxy, halogenated C 1- Alkoxy, hydroxy C 1-3 Alkoxy, amino, mono-C 1-3 Alkylamino, C 1-3 Alkyl acylamino, C 1-3 Alkyl acyl, C 1-3 alkylsulfonyl, aminoacyl, C 1-3 Alkylaminoacyl, bis(C) 1-3 Alkylamino, C 2-4 alkenyl, C 2-4 alkynyl, halogenated C 1-3 Alkyl acyl, hydroxy C 1-3 Alkyl acyl, C 3-8 Cycloalkyl, 3-8 membered heterocyclic groups, C 6-8 Aryl, 5-8 quinone heteroaryl, halogenated C 3-8 Group substitution with cycloalkyl or oxo groups.

8. The compound or isomer thereof, pharmaceutically acceptable salt, solvate, crystal or prodrug according to claim 1, wherein the compound is selected from the group consisting of:

9. A pharmaceutical composition comprising a compound or an isomer thereof as claimed in any one of claims 1-8, a pharmaceutically acceptable salt, a solvate, a crystal or prodrug, and a pharmaceutically acceptable carrier.

10. The use of the compound or isomer thereof, pharmaceutically acceptable salt, solvate, crystal or prodrug, or pharmaceutical composition of any one of claims 1-8 in the preparation of a medicament for treating DHX9-mediated diseases.

Images