A compound that can penetrate the brain and act as a BTK or HER2 inhibitor, its preparation method and uses.

Abstract

A compound used as a BTK inhibitor or HER2 inhibitor, its preparation method, and its uses are disclosed. The compound has the structure shown in Formula II or its tautomers, mesosomes, racemates, enantiomers, diastereomers, or mixtures thereof, as well as pharmaceutically acceptable hydrates, solvates, or salts. The BTK protein kinase inhibitors of this invention exhibit strong inhibitory activity against both wild-type BTK and mutant BTK (C481S), with some compounds showing high brain penetration rates. The HER2 inhibitors of this invention exhibit good HER2 kinase inhibitory activity and high blood-brain barrier permeability. Furthermore, the compounds of this invention possess favorable pharmacokinetic properties and show promising application prospects.

Description

[0001] This application claims priority to Chinese Patent Application No. 202010679776.6, filed on July 15, 2020, entitled "A compound as a BTK inhibitor, its preparation method and use thereof", and also claims priority to Chinese Patent Application No. 202011337022.9, filed on November 25, 2020, entitled "A compound as a BTK inhibitor, its preparation method and use thereof", the entire contents of which are incorporated herein by reference. Technical Field

[0002] This invention relates to the field of pharmaceutical technology, and in particular to a compound with good brain penetration as a BTK or HER2 protein kinase inhibitor, its preparation method, and its uses. Background Technology

[0003] Bruton's tyrosine protein kinase (BTK) is a member of the Tec family of non-receptor protein tyrosine kinases, primarily expressed in various hematopoietic cell lines. The Tec family is the second largest family of human non-receptor kinases after the Src family, and its main members include BTK, BMX (etk), ITK, TEC, and TXK (RLK). In 1993, BTK was identified as a defective protein in human X-linked agammaglobulinemia (XLA). BTK is a key regulator of the B cell receptor (BCR) signaling pathway, playing a crucial role in B cell activation, proliferation, differentiation, and survival, and is closely associated with various B-cell tumors and autoimmune diseases.

[0004] The BTK structure contains five main domains: the PH domain (Pleckstrin homology), the TH domain (Tec homology), the SH3 domain (Src homology 3), the SH2 domain (Src homology 2), and the SH1 domain (Src homology 1). BTK activation (phosphorylation) initially occurs in the activation loop within the SH1 domain, with further activation occurring in the SH2 and SH3 domains, which contain the main autophosphorylation sites. These SH domains also contain the nuclear localization signal (NLS) and nuclear export sequence (NES) required for BTK nucleocytoplasmic shuttle.

[0005] BTK plays an irreplaceable role in B lymphocyte development. It controls B cell development and differentiation by activating positive cell cycle regulators and differentiation factors, and also controls B cell survival and proliferation by regulating the expression of pro-apoptotic and anti-apoptotic proteins. Sustained BTK activation is a prerequisite for the development of chronic lymphocytic leukemia (CLL); abnormal BCR-BTK signaling promotes the survival of activated B cell subtypes in diffuse large B-cell lymphoma (DLBCL). BTK gain-of-function mutations have also been confirmed in colorectal cancer, acute lymphoblastic leukemia (ALL), and chronic myeloid leukemia (CML). Therefore, abnormal activation of the BTK-dependent pathway has been shown to be closely related to the development and progression of various tumors.

[0006] Currently approved irreversible BTK inhibitors, such as ibrutinib, acalabrutinib, and zanubrutinib, selectively bind irreversibly to the cysteine ​​residue (Cys-481) of BTK, inhibiting BTK activity to treat related diseases. However, some cancer patients develop resistance to first-generation BTK inhibitors, creating unmet clinical needs. Studies have shown that BTK-C481S mutation is one of the main resistance mechanisms associated with this condition; therefore, drugs that target and inhibit BTK-C481S mutations hold promise for providing new treatment options. For example, ARQ-531 is an orally bioavailable, potent, and reversible dual inhibitor of wild-type and C481S-mutant BTK; early clinical results for ARQ-531 have demonstrated its effectiveness in patients with C481S-mutant BTK.

[0007] The annual incidence of primary central nervous system lymphoma (PCNSL) is approximately 0.47 per 100,000, with over 10,000 patients diagnosed annually in China. After decades of treatment development, the current first-line treatment regimen is based on high-dose methotrexate (HD-MTX) induction chemotherapy to achieve radiographic complete response (CR), followed by whole-brain radiotherapy (WBRT) as consolidation therapy. This regimen has a median overall survival (OS) of 30–50 months. However, this regimen can cause severe neurotoxicity, which increases with patient age and radiation dose, manifesting as memory loss, cognitive impairment, gait disturbances, and dementia, significantly impacting patients' quality of life. Studies have also explored the efficacy of autologous hematopoietic stem cell transplantation versus whole-brain radiotherapy in PCNSL consolidation therapy, showing no difference between the two. It should be noted that whole-brain radiotherapy causes irreversible damage to brain function. Regardless of the treatment regimen, the prognosis is poor.

[0008] Due to the presence of the blood-brain barrier (BBB), conventional immunochemotherapy is difficult to penetrate, resulting in poor efficacy. The efficacy of drugs treating primary or secondary CNS involvement depends on their distribution in the brain after crossing the BBB. Only lipophilic molecules can passively diffuse and easily cross the BBB. Conventional immunochemotherapy, such as rituximab, is a monoclonal antibody with a large molecular weight, making it difficult to penetrate the blood-brain barrier and thus unable to exert its full therapeutic effect.

[0009] As the pathogenesis of PCNSL becomes increasingly well understood, recent breakthroughs in molecularly specific targeted drugs against the BCR pathway have provided new treatment options for these patients. Bruton's tyrosine kinase (BTK) is a kinase that connects the BCR signaling pathway and the NF-κB signaling pathway, and is a fundamental target for the treatment of ABC-DLBCL inhibition. BTK inhibitors are selective drugs that can directly act on MYD88 and CD79B in the BCR pathway, and mutations in the BCR signaling pathway in central nervous system lymphomas are often predominantly MYD88 and CD79B. PCNSL is diagnosed according to the WHO 2008 classification and requires immunohistochemical results, with key markers including all B-cell markers (CD19, CD20, PAX5), BCL6, MUM1 / IRF4, and CD10. The NCCN guidelines for central nervous system malignancies (2020.V1) recommend BTKi for relapsed / refractory PCNSL.

[0010] In March 2020, the BTK inhibitor Velexbru (Tirabrutinib hydrochloride) was approved in Japan for the treatment of relapsed or refractory primary central nervous system lymphoma (PCNSL). Velexbru is the first BTK inhibitor approved globally for the treatment of relapsed or refractory PCNSL. Results showed that 52.9% of patients achieved a response after treatment with Velexbru. A phase I / II study of tirabrutinib in relapsed / refractory central nervous system lymphoma (CNSL) enrolled 44 patients. Phase I used a 3+3 dose-escalation design, with patients receiving 320 mg and 480 mg of tirabrutinib once daily (QD), and dose-limiting toxicities (DLT) assessed over 28 days. Phase II administered 480 mg of tirabrutinib QD under fasting conditions. Results showed that compared to 320 mg, patients receiving 480 mg had superior objective response rate (ORR) and progression-free survival (PFS). ORR: 320 mg vs 480 mg vs 480 mg (fasting conditions) = 60% vs 100% vs 52.9%, PFS: 320 mg vs 480 mg vs 480 mg (fasting conditions) = 2.1 vs 11.1 vs 5.8 months. However, the lower ORR in the 480 mg (fasting) group compared to the 320 mg group may be due to differences in patient characteristics. Plasma drug concentrations of tirabrutinib were comparable at all doses; at four different time points, the trough concentration of tirabrutinib in the 480mg group was higher than that in the 320mg group, and the cerebrospinal fluid concentration increased with the increase of plasma drug concentration; it can be seen that the efficacy of BTKi in treating central nervous system lymphoma is related to the drug concentration of BTKi in cerebrospinal fluid (CSF).

[0011] Treatment options for PCNSL are limited, and progression-free survival (PFS) and overall survival (OS) are far from meeting expectations. While a first-generation irreversible BTK inhibitor (Tirabrutinib) has been approved for the treatment of this disease abroad, this drug has weak activity against the BTK protein, requires excessively high clinical doses (480 mg once daily), has significant side effects, poor patient compliance, and carries the risk of developing resistance due to C481S mutations after a period of use.

[0012] Studies show that BTK inhibitors can also be used for autoimmune diseases such as rheumatoid arthritis, multiple sclerosis (MS), and psoriasis. The pathogenesis of MS is directly related to brain lesions. In 2019, the global market size for MS drugs was US$25 billion, and it is projected to reach US$40.66 billion by 2027, with a projected CAGR of 7.1%. Clinical and economic research data show that the annual treatment cost for MS is US$28,000. BTK, as a key kinase in the B-cell receptor signaling pathway, is crucial for the development and function of immune cells involved in the pathogenesis of MS, such as B lymphocytes, macrophages, and microglia. Therefore, BTK inhibitors hold promise for providing novel treatment options for autoimmune diseases such as MS.

[0013] HER2 (human epidermal growth factor receptor-2), also known as ERBB2, is located on chromosome 17 and is a proto-oncogene. Its encoded product, HER2 protein, is a transmembrane protein with tyrosine protein kinase activity and belongs to the EGFR family. It is overexpressed in various cancers, including breast cancer, ovarian cancer, and gastric cancer. HER2 mediates cell growth, differentiation, and survival, and can promote the invasive spread of cancer cells.

[0014] Approximately 15% to 20% of breast cancers are HER2-positive (HER2+). Compared to HER2-negative cancers, HER2+ tumors are more aggressive and are associated with shorter survival, poorer overall survival, a higher risk of recurrence, and central nervous system disorders (brain metastases). About 30% to 50% of HER2+ breast cancer patients will develop brain metastases over time.

[0015] According to the 2018 GLOBOCAN report, nearly 2.1 million new cases of breast cancer were diagnosed globally, with an incidence rate of 27.5 per 100,000, accounting for 11.6% of all cancer cases. Comparing new cases and deaths worldwide, breast cancer is the leading cause of cancer death among women globally, with the highest incidence and mortality rates. In my country, breast cancer has the highest incidence rate among female malignant tumors, reaching 43 per 100,000. In 2018, nearly 368,000 new cases of breast cancer were diagnosed in China, with breast cancer drug expenditures reaching 40 billion yuan. The growth rate of breast cancer incidence in China is twice the global average (an increase of over 300,000 cases annually), ranking first globally. There is still no effective treatment for HER2-positive breast cancer patients with brain metastases, and the mortality rate remains high (10 out of every 100,000 people die from breast cancer).

[0016] Tukysa (tucatinib) is a small-molecule oral tyrosine kinase inhibitor (TKI) with excellent targeting selectivity for HER2. It was granted Breakthrough Therapy Designation (BTD) by the FDA in 2019. In addition to this designation, it also received Fast Track and Orphan Drug designations in 2017. It is used in combination with trastuzumab and capecitabine to treat patients with locally advanced, unresectable, or metastatic (including those with brain metastases) HER2-positive breast cancer who have previously received trastuzumab, pertuzumab, or T-DM1 (ado-trastuzumab emtansine). Summary of the Invention

[0017] In view of this, this application provides a compound as a BTK inhibitor or HER2 inhibitor, a method for its preparation, and its uses. The compound provided by this invention can be used as a BTK protein kinase inhibitor or a HER2 protein kinase inhibitor and has the characteristics of high inhibitory activity.

[0018] This invention provides a compound having the structure shown in Formula I or a tautomer thereof, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, a pharmaceutically acceptable hydrate, solvate, or salt thereof:

[0019]

[0020] Among them: A1, A2, A3, A4, A5, and A6 are each independently selected from C-R5 or nitrogen (N); and at least one of A1, A2, A3, A4, A5, and A6 is N;

[0021] M is selected from substituted or unsubstituted saturated hydrocarbon groups or heterosaturated hydrocarbon groups, substituted or unsubstituted unsaturated cyclic groups or heterocyclic groups, and substituted or unsubstituted monocyclic, bicyclic, or tricyclic aryl or heteroaryl groups; wherein the substituted groups are each independently selected from aryl or heteroaryl groups substituted by any group, alkyl or heteroalkyl, cycloalkyl or heterocycloalkyl, unsaturated cyclic or heterocyclic groups, phenoxy, halogen, hydroxy, cyano, amino, ester, nitro, mercapto, amide, sulfonyl, phosphoryl, alkyloxyphosphoryl, alkylsulfone, and alkyl sulfoxide groups; further, the substituted groups are aryl or heteroaryl groups substituted by any group, more preferably phenyl groups substituted by any group;

[0022] Q is selected from CR 10 R 11 NR 12 Oxygen (O), Sulfur (S), S(O), S(O)2;

[0023] R1, R2, R3, R4, R5, R 10 R 11 R12 Each of the following groups is independently selected from hydrogen, deuterium, halogen, substituted or unsubstituted alkyl or heteroalkyl, substituted or unsubstituted cycloalkyl or heterocycloalkyl, substituted or unsubstituted unsaturated cycloalkyl or heterocycloalkyl, substituted or unsubstituted aryl or heteroaryl, hydroxyl, cyano, amino, ester, nitro, mercapto, amide, sulfonyl, phosphoryl, alkyloxyphosphoryl, alkylsulfone, alkyl sulfoxide; or R3, R4 together with the carbon atom attached to them to form a substituted or unsubstituted C3-C10 cycloalkyl or heterocycloalkyl; wherein the substituent is selected from halogen, hydroxyl, cyano, amino, mercapto, nitro, carboxyl, hydroxyamino, alkyl, cycloalkyl, heteroalkyl, heterocycloalkyl, aryl, heteroaryl, ester, acyl, amide, sulfonyl, phosphoryl;

[0024] m is selected from integers from 0 to 6; n is selected from integers from 0 to 3.

[0025] The compounds described in this invention are in any form having the structure of Formula I, including tautomers, meso compounds, racemates, enantiomers, diastereomers or mixtures thereof, pharmaceutically acceptable hydrates, solvates or salts, etc.

[0026] In this application, "selected from" generally indicates a parallel relationship of "or". In the structure shown in Formula I, three or four of A1, A2, A3, A4, A5, and A6 are preferably N; the position of R2 is not limited, but it is preferably in the para position of R1. In this application, the substitution can be monosubstituted or polysubstituted (e.g., disubstituted, trisubstituted), and the specific substitution position is not particularly limited. The unsubstituted saturated hydrocarbon group includes unsubstituted alkyl and unsubstituted cycloalkyl groups; the heterocyclic group, heteroaryl group, etc., means that one or more carbon atoms can be replaced by heteroatoms, which are atoms other than carbon (C), such as oxygen, sulfur, nitrogen, and phosphorus (P). In addition, the above-mentioned halogens include fluorine (F), chlorine (Cl), bromine (Br), etc., preferably fluorine or chlorine. The above-mentioned "C3-C10" refers to the number of carbon atoms selected from an integer from 3 to 10, and similar expressions will not be repeated below.

[0027] In this application, the bridging atom is chemically bonded to the ring system formed on the ring (as shown in the following formula). This means that the bridging atom can be connected to any connectable C atom on the ring, thus forming any spirocyclic or bridged ring compound. For example, the following formula indicates that the bridging atom Q can be connected to any connectable C atom on the six-membered ring, that is, connected to the same C atom to form a spirocyclic compound, such as all bridging atoms being connected to C atom number 2 or all being connected to C atom number 3, etc.; connected to different C atoms to form a bridged ring compound, such as the bridging atom being connected to C atoms number 1 and 4 or C atoms number 2 and 4, etc.

[0028]

[0029] Preferably, the compound has the structure shown in Formula II or its tautomers, meso compounds, racemates, enantiomers, diastereomers, or mixtures thereof, pharmaceutically acceptable hydrates, solvates, or salts.

[0030]

[0031] Wherein, R1 is selected from hydrogen, halogen, hydroxyl, cyano, amino, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted C1-C6 heteroalkyl, substituted or unsubstituted C3-C6 heterocycloalkyl; further, R1 is selected from hydrogen, amino, methyl, ethyl, methoxy, cyano, trifluoromethyl, isopropyl, cyclopropyl; even further, R1 is selected from hydrogen (H), amino (NH2), methyl (CH3);

[0032] R2 is selected from hydrogen, halogen, hydroxyl, cyano, amino, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted C1-C6 heteroalkyl, and substituted or unsubstituted C3-C6 heterocycloalkyl; further, R2 is selected from hydrogen, fluorine, chlorine, bromine, methyl, ethyl, methoxy, cyano, trifluoromethyl, isopropyl, and cyclopropyl; even further, R2 is selected from hydrogen, chlorine, and methyl.

[0033] R3 and R4 are selected from hydrogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted C1-C6 heteroalkyl, substituted or unsubstituted C3-C6 heterocycloalkyl; or R3 and R4 together with the carbon atom attached to them form a substituted or unsubstituted C3-C6 cycloalkyl or a heterocycloalkyl containing N or O atoms.

[0034] Furthermore, R3 and R4 are selected from hydrogen, methyl, ethyl, isopropyl, cyclopropyl, or R3 and R4 together with the carbon atom attached to them to form cyclopropyl, azirrobutyl, azirropentyl, azirrohexyl, oxacyclobutyl, oxacyclopentyl, and oxacyclohexyl.

[0035] R6 is selected from hydrogen, halogen, hydroxyl, cyano, amino, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted C1-C6 heteroalkyl, and substituted or unsubstituted C3-C6 heterocycloalkyl; further, R6 is selected from hydrogen, halogen, cyano, substituted or unsubstituted C1-C3 alkyl, and substituted or unsubstituted C1-C3 alkoxy; further, R6 is selected from hydrogen, fluorine, chlorine, bromine, trifluoromethyl, methyl, methoxy, trifluoromethoxy, and difluoromethoxy; even further, R6 is hydrogen or fluorine.

[0036] m is selected from 0, 1, 2, 3; n is selected from 0, 1, 2; n1 is selected from 0, 1, 2, 3, 4;

[0037] R7 is selected from substituted or unsubstituted aryl and substituted or unsubstituted pyridyl groups, wherein the substituents are independently selected from halogens, hydroxyl groups, amino groups, cyano groups, alkyl groups, heteroalkyl groups, cycloalkyl groups, and heterocycloalkyl groups; further, the substituents are independently selected from fluorine, chlorine, bromine, cyano, amino, C1-C3 alkyl, C1-C3 alkoxy, C3-C6 cycloalkyl, and C3-C6 heterocycloalkyl groups; even further, the substituents are independently selected from fluorine, chlorine, bromine, cyano, trifluoromethyl, trifluoromethoxy, difluoromethoxy, methoxy, deuterated methoxy, cyclopropyl, cyclopropylmethoxy, ethyl, isopropyl, and isobutyl groups; wherein the number of substituents is an integer between 0 and 5.

[0038] X is selected from An acceptable linking group. In some embodiments, X is... Among them, R9, R 13 Independently selected from hydrogen, halogen, hydroxyl, amino, cyano, C1-C3 alkyl, C1-C3 alkoxy, C3-C6 cycloalkyl, C3-C6 heterocycloalkyl; R9 and R 13 Together with the carbon atoms attached thereto, they form substituted or unsubstituted C3-C6 cycloalkyl groups or substituted or unsubstituted C3-C6 heterocycloalkyl groups containing N or O; further, they are independently selected from hydrogen, fluorine, chlorine, cyano, methyl, ethyl, isopropyl, cyclopropyl, trifluoromethyl, isobutyl, R9 and R 13 Together with the carbon atom it is attached to, they form a cyclopropyl group, which is further selected from hydrogen, fluorine, deuterium, chlorine, methyl, hydroxyl, and amino. Specifically, X can be: Where X is All of these compounds can act as brain-penetrating BTK inhibitors or HER2 inhibitors, with R9 and R2 being more preferred. 13 All are selected from fluorine.

[0039] In some embodiments of this application, the compound has the structure shown in Formula III or Formula IV, or in the form of tautomers, mesosomes, racemates, enantiomers, diastereomers, or mixtures thereof, pharmaceutically acceptable hydrates, solvates, or salts thereof.

[0040]

[0041] The structures of R1, R2, R3, R4, R6, and X are as described above; m, n, and n1 are also as described above; for example, X is... wait.

[0042] In Equations III-IV, n2 is selected from 0, 1, 2, 3, and 4;

[0043] R8 is independently selected from hydrogen, halogen, hydroxyl, amino, cyano, alkyl, heteroalkyl, cycloalkyl, and heterocycloalkyl; further, R8 is independently selected from hydrogen, fluorine, chlorine, bromine, cyano, amino, C1-C3 alkyl, C1-C3 alkoxy, C3-C6 cycloalkyl, and C3-C6 heterocycloalkyl; even further, the substituent is independently selected from hydrogen, fluorine, chlorine, bromine, cyano, trifluoromethyl, trifluoromethoxy, difluoromethoxy, methoxy, deuterated methoxy, cyclopropyl, cyclopropylmethoxy, ethyl, isopropyl, and isobutyl; wherein the number of substituents is an integer between 0 and 5 (including the endpoints); multiple substituents may be the same or different; in Formula IV, the connection position of the substituted or unsubstituted pyridinyl group is not limited and may be connected at the ortho position of N.

[0044] In some embodiments of this application, the N-fused ring in Formulas II-IV can be used. Alternatively, the single bonds at both ends are connecting bonds. Furthermore, in the X structure of formulas II-IV, single bonds with curved lines represent connecting bonds. The position of R6 is not limited; n1 is preferably 0, 1, or 2. When n = 0, it is a pentagonal ring; when n = 1, it is a hexagramal ring, and so on.

[0045] Preferably, in formulas II-IV, R1 is an amino group, R2 is hydrogen or chlorine, and R6 is hydrogen or a monosubstituted fluorine group; in formula II, R7 is a substituted or unsubstituted phenyl or pyridyl group; X is mainly an ether or amide structure, and the nitrogen atom of the amide is attached to R7. Preferably, n is 0 or 1, m is 0 or 2, and R3 and R4 are both hydrogen, methyl, or cyclopropyl with the attached carbon atom.

[0046] Specifically, the compound structure described in this application is selected from one of the following (wherein, the single-bonded form at one end is methyl, as shown in Formula 5 of compound 5); more preferably, the compounds shown in Formula 2, Formula 5, Formula 34, Formula 42, Formula 89, Formula 100, Formula 101, Formula 103, Formula 106, Formula 109, Formula 111, Formula 114, Formula 116, Formula 118, Formula 121, Formula 125, Formula 130, Formula 145, Formula 146, Formula 152, and Formula 155 have better performance:

[0047]

[0048]

[0049]

[0050]

[0051]

[0052]

[0053]

[0054] This invention provides a pharmaceutical composition in which the active ingredient is selected from one or more of the compounds described above or their stereoisomers, solvates, hydrates, pharmaceutically acceptable salts, or eutectics. Furthermore, this invention does not impose any particular limitations on the formulation type of the pharmaceutical composition.

[0055] This invention provides the use of the aforementioned compound or its stereoisomers, solvates, hydrates, pharmaceutically acceptable salts, or cocrystals in the preparation of protein kinase inhibitors; further, the kinase inhibitor is a BTK inhibitor or a HER2 inhibitor. Alternatively, this invention provides the use of the aforementioned compound or its stereoisomers, solvates, hydrates, pharmaceutically acceptable salts, or cocrystals in the preparation of medicaments for treating diseases caused by BTK kinase or HER2 kinase overexpression.

[0056] This invention provides the use of the compounds described above, or their stereoisomers, solvates, hydrates, pharmaceutically acceptable salts, or eutectics, in the preparation of medicaments for the treatment of any one or more of the following diseases: autoimmune diseases, inflammatory diseases, thromboembolic diseases, allergies, infectious diseases, proliferative disorders, and cancer.

[0057] Further, the diseases may be selected from: arthritis, rheumatoid arthritis, urticaria, vitiligo, organ transplant rejection, ulcerative colitis, Crohn's disease, dermatitis, asthma, Sjögren's syndrome, systemic lupus erythematosus, multiple sclerosis, idiopathic thrombocytopenic purpura, rash, anti-neutrophil cytoplasmic antibody vasculitis, pemphigus, pemphigus vulgaris, chronic obstructive pulmonary disease, psoriasis; breast cancer, mantle cell lymphoma, ovarian cancer, esophageal cancer, laryngeal cancer, glioblastoma, neuroblastoma, gastric cancer, hepatocellular carcinoma, gastric cancer, glioma, endometrial cancer, melanoma, kidney cancer, bladder cancer, melanoma, bladder cancer. Biliary tract cancer, kidney cancer, pancreatic cancer, lymphoma, pilocarcinoma, nasopharyngeal carcinoma, pharyngeal cancer, colorectal cancer, rectal cancer, brain and central nervous system cancers, cervical cancer, prostate cancer, testicular cancer, urogenital tract cancer, lung cancer, non-small cell lung cancer, small cell carcinoma, lung adenocarcinoma, bone cancer, colon cancer, adenoma, pancreatic cancer, adenocarcinoma, thyroid cancer, follicular carcinoma, Hodgkin's leukemia, bronchial cancer, thyroid cancer, endometrial cancer, cervical cancer, multiple myeloma, acute myeloid leukemia, chronic myeloid leukemia, lymphocytic leukemia, chronic lymphocytic leukemia, myeloid leukemia, non-Hodgkin's lymphoma, primary macroglobulinemia.

[0058] In existing technologies, the inhibitory activity of ARQ-531 still needs improvement. Its inhibitory activity against TMD8 and REC-1 cells is poor, leading to problems such as excessive clinical dosage and high side effects. Furthermore, ARQ-531 has poor selectivity, exhibiting high inhibitory activity against TEC and EGFR, which can easily cause side effects such as bleeding, diarrhea, and eczema. Moreover, its pharmacokinetics are not ideal; preclinical studies showed that its bioavailability was only 38% in canine pharmacokinetic experiments. In short, ARQ-531 has significant room for improvement in terms of inhibitory activity, selectivity, and pharmacokinetics.

[0059] Treatment options for PCNSL are limited, and progression-free survival (PFS) and overall survival (OS) are far from meeting expectations. While a first-generation irreversible BTK inhibitor (Tirabrutinib) has been approved for treatment abroad, this drug has weak activity against the BTK protein, requires excessively high clinical doses (480 mg once daily), has significant side effects, poor patient compliance, and carries the risk of developing resistance due to C481S mutations after a period of use. The high clinical dose is primarily due to two factors: the compound's relatively weak activity and, more importantly, its low blood-brain barrier permeability, requiring higher doses to achieve the effective concentration needed to cross the blood-brain barrier. There is significant room for improvement in Tirabrutinib's inhibitory activity, pharmacokinetics, and brain penetration.

[0060] Tucatinib is a small-molecule oral tyrosine kinase inhibitor (TKI) with excellent targeting selectivity for HER2. It was granted Breakthrough Therapy Designation (BTD) by the FDA in 2019. In addition to this designation, it also received Fast Track and Orphan Drug designations in 2017. Used in combination with trastuzumab and capecitabine for the treatment of patients with locally advanced, unresectable, or metastatic (including brain metastases) HER2-positive breast cancer who have received prior therapy with trastuzumab, pertuzumab, or T-DM1 (ado-trastuzumab emtansine), it was approved by the U.S. Food and Drug Administration (FDA) on April 17, 2020. The clinical dosage is high, 300 mg twice daily, with significant side effects. Literature indicates that the brain penetration rate of tucatinib in mice is less than 5%. There is significant room for improvement in the pharmacokinetics and brain penetration rate of tucatinib.

[0061] In the in vitro BTK inhibition and HER2 inhibition kinase activity assays of this invention, the compound powder was dissolved in 100% DMSO to prepare a 10mM stock solution; it was then frozen at -20°C protected from light. During the kinase reaction, the test concentration of the compound was 1μM, diluted 100 times to a final concentration in 100% DMSO solution in a 384 source plate, with 3-fold dilutions for 10 concentrations. Furthermore, this invention also used the compound to conduct experiments on liver microsomal stability, rat pharmacokinetics, rat brain penetration rate, and pharmacodynamic models. Compared with existing clinical drugs (ARQ-531), the compound of this invention, as a BTK protein kinase inhibitor, has advantages in BTK and BTK(C481S) inhibitory activity, liver microsomal stability, rat pharmacokinetics, and toxicity. Compared with the existing marketed drug Tirabrutinib, the compound of this invention, as a BTK protein kinase inhibitor, has advantages in BTK and BTK(C481S) inhibitory activity, cellular activity, liver microsomal stability, rat pharmacokinetics, and rat blood-brain barrier penetration rate. The HER2 inhibitor compound of the present invention is comparable to the existing marketed drug Tucatinib in terms of HER2 inhibitory activity, BT474 cell activity, and NCI-N87 cell activity; and is significantly superior to Tucatinib in terms of pharmacokinetics and blood-brain barrier permeability in rats.

[0062] This invention has designed and synthesized several target compounds. The specific preparation process is as follows: Intermediate A (also known as the boric acid or borate ester compound represented by Formula A) and intermediate B (the brominated derivative represented by Formula B) undergo a Suzuki reaction to synthesize intermediate C (the intermediate represented by Formula C), followed by deprotection to obtain the compound with the structure shown in Formula II. In a specific embodiment, commercially available boric acid A or a self-made borate ester A is coupled with a self-made brominated derivative B under palladium catalysis to obtain intermediate C. Deprotection of intermediate C yields the compound of the example. Compared to the phase II clinical drug ARQ-531, this compound of the present invention exhibits significant improvements in BTK and BTK(C481S) inhibitory activity, liver microsomal stability, and rat pharmacokinetics.

[0063] Furthermore, the following synthesis methods in the embodiments of the present invention are simple and have a high yield.

[0064]

[0065] This invention provides an intermediate compound for preparing the aforementioned BTK inhibitor or HER2 inhibitor, having the following structure:

[0066] The explanations for R1, R2, R3, R4, m, and n are as described above; for example:

[0067] In addition, other intermediate compounds also include wait.

[0068] This invention provides another intermediate compound for the preparation of the aforementioned BTK inhibitor or HER2 inhibitor, having the structure shown below; furthermore, the compound with the structure shown below can be used in the preparation of drugs that can cross the blood-brain barrier:

[0069] The explanations for R6, R8, n1, and n2 are as described above. Attached Figure Description

[0070] Figure 1 These are the pharmacodynamic model test results of some compounds in this invention, specifically TMD8.

[0071] Figure 2 These are the pharmacodynamic model test results of some compounds in this invention, specifically TMD8.

[0072] Figure 3 These are the results of pharmacodynamic model tests of some compounds of the present invention, DOHH-2-Luc, in a brain tumor.

[0073] Figure 4 These are fluorescence images showing the efficacy test results of DOHH-2-Luc, a compound from this invention, in a brain tumor pharmacological model. Detailed Implementation

[0074] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0075] To further understand this application, the following detailed description, in conjunction with embodiments, of the compounds provided in this application that can be used as BTK protein kinase inhibitors, their preparation methods, and uses.

[0076] In this embodiment of the invention, the structure of the compound is determined by mass spectrometry (MS) or nuclear magnetic resonance (NMR). 1 Determined by ¹H NMR equipment. The term "room temperature" refers to a temperature between 10°C and 25°C. Chemical abbreviations have the following meanings:

[0077] DMF: N,N-dimethylformamide; DIEA: N,N-diisopropylethylamine;

[0078] HATU: O-(7-azabenzotriazol-1-yl)-N,N,N′;-tetramethylurea hexafluorophosphate;

[0079] PdCl2(dppf): [1,1′-bis(diphenylphosphino)ferrocene]palladium dichloride;

[0080] DCM: Dichloromethane; TEA: Triethylamine; TBDPSCl: Lithium bis(trimethylsilylamine);

[0081] 9-BBN: 9-Boronbicyclo[3.3.1]nonane; Dess-Martin: Dess-Martin oxidant;

[0082] DME: Dimethyl ethylene glycol; TosMIC: p-Toluenesulfonylmethylisocyanate;

[0083] t-BuOK: Potassium tert-butoxide; Dibal-H: Diisobutylaluminum hydride; THF: Tetrahydrofuran;

[0084] NBS: N-bromosuccinimide; TBAF: Tetrabutylammonium fluoride; DMSO: Dimethyl sulfoxide;

[0085] LDA: Lithium diisopropylaminodimethylamine;

[0086] HBTU: Benzotriazole-N,N,N′,N′-Tetramethylurea hexafluorophosphate;

[0087] NMP: N-methylpyrrolidone; BAST: bis(2-methoxyethyl)aminosulfur trifluoride;

[0088] PMDTA: Pentamethyldiethylenetriamine; DMA: N,N-dimethylacetamide;

[0089] dppf: 1,1′-bis(diphenylphosphine)ferrocene; Pd2(dba)3: tris(dibenzylacetone)dipalladium;

[0090] TsCl: 4-Toluenesulfonyl chloride; DMAP: 4-Dimethylaminopyridine; PDC: Pyridine dichromate;

[0091] DIAD: Diisopropyl azodicarbonate; NCS: N-chlorosuccinimide.

[0092] Preparation of intermediate A-1:

[0093]

[0094] Compound A-1-1 (5.0 g, 53.1 mmol), DMF (50 mL), A-1-2 (11.6 g, 53.1 mmol), and DIEA (20.6 g, 159.3 mmol) were added to a reaction flask. The reaction mixture was purged with nitrogen and cooled to 0 °C. HATU (24.2 g, 63.7 mmol) was added in portions. The reaction mixture was slowly heated to room temperature and stirred overnight. TLC showed that the starting material had completely reacted. Water was added to the reaction system, and the mixture was extracted twice with ethyl acetate. The organic phases were combined, washed with water and saturated brine, dried over anhydrous Na₂SO₄, and purified by silica gel column chromatography after vacuum evaporation to give 11.9 g of product A-1-3, yield: 76%.

[0095] Compound A-1-3 (5.0 g, 16.9 mmol), dioxane (50 mL), bis-pinacol boronic acid ester (5.2 g, 20.3 mmol), and potassium acetate (2.5 g, 25.4 mmol) were added to the reaction flask. The reaction solution was purged with nitrogen. PdCl2 (dppf) (500 mg, 0.68 mmol) was added to the reaction solution, and the reaction solution was purged with nitrogen again. The reaction mixture was heated to 90 °C and stirred overnight. TLC showed that the starting materials reacted completely. After the reaction system cooled, silica gel was added directly and stirred. The mixture was then purified by silica gel column chromatography to obtain the crude product. The crude product was slurried with petroleum ether to give 3.4 g of product A-1, yield: 62%.

[0096] Preparation of intermediate A-2:

[0097]

[0098] Compound A-2-1 (2.0 g, 11.8 mmol) and dioxane (20 mL) were added to a reaction flask and cooled in an ice-water bath. Hydrogen peroxide (20 mL, 30%) was added dropwise to the reaction mixture, and the mixture was stirred overnight at room temperature to stop the reaction. Water was added to the reaction system, and the mixture was extracted four times with ethyl acetate. The organic phases were combined, washed with water and saturated brine, and purified by vacuum evaporation and silica gel column chromatography to give 1.6 g of product A-2-2 (yield: 96%).

[0099] Compound A-2-2 (1.00 g, 7.04 mmol), DMF (20 mL), p-bromoiodobenzene (1.99 g, 7.04 mmol), tetrabutylammonium bromide (230 mg, 0.704 mmol), potassium phosphate (2.99 g, 14.1 mmol), and cuprous iodide (140 mg, 0.704 mmol) were added to the reaction flask. The reaction mixture was purged with nitrogen, and then heated to 140 °C with stirring overnight. The reaction system was cooled to room temperature, water was added, and the mixture was extracted three times with ethyl acetate. The organic phases were combined, washed with water and saturated brine, and purified by vacuum evaporation and silica gel column chromatography to obtain 800 mg of product A-2-3, yield: 38%.

[0100] Compound A-2-3 (800 mg, 2.69 mmol), dioxane (16 mL), bis-pinacol boronic acid ester (821 mg, 3.23 mmol), and potassium acetate (528 mg, 5.38 mmol) were added to the reaction flask. The reaction solution was purged with nitrogen. PdCl2 (dppf) (80 mg, 0.109 mmol) was added to the reaction solution, and the reaction solution was purged with nitrogen again. The reaction mixture was heated to 80 °C and stirred for 16 hours. After the reaction system cooled, silica gel was added directly and stirred. Then, the product A-2 was purified by silica gel column chromatography to obtain 520 mg of product A-2, yield: 56%.

[0101] Preparation of intermediate A-3:

[0102]

[0103] Compound A-3-1 (1.28 g, 10.5 mmol), DCM (40 mL), A-3-2 (1.0 g, 5.2 mmol), Cu(OAc)2 (945 mg, 5.2 mmol), TEA (1.58 g, 15.6 mmol), and 4A molecular sieve (1.66 g) were added to a reaction flask. The reaction mixture was stirred overnight at room temperature. The reaction mixture was filtered, and the filtrate was mixed with silica gel and then purified by silica gel column chromatography to obtain 600 mg of product A-3-3, yield: 43%.

[0104] The method for synthesizing A-3 is the same as the method for synthesizing A-2 from A-2-3.

[0105] Preparation of intermediate A-4:

[0106]

[0107] Compound A-4-1 (1.06 g, 6.3 mmol), DMF (10 mL), A-3-2 (1.0 g, 5.2 mmol), and potassium carbonate (1.45 g, 10.5 mmol) were added to a reaction flask. The reaction mixture was heated to 100 °C and stirred overnight. TLC showed that the starting material reacted completely. Water was added to the reaction system, and the mixture was extracted twice with ethyl acetate. The organic phases were combined, washed with water and saturated brine, dried over anhydrous Na₂SO₄, and evaporated under vacuum to give 1.8 g of product A-4-2, yield: 100%. The product was used directly in the next step without further purification.

[0108] The synthesis method of A-4 is the same as that of A-2 synthesized from A-2-3.

[0109] The following compounds were synthesized using commercially available raw materials through the methods for preparing the above intermediates.

[0110] Table 1: Structure and Synthesis of Intermediates A-5 to A-12

[0111]

[0112]

[0113] Preparation of intermediate A-13:

[0114]

[0115] Compound A-13-1 (1.00 g, 5.55 mmol), THF (15 mL), and triisopropyl borate (1.25 g, 6.66 mmol) were added to a reaction flask. The reaction solution was cooled to -70 °C, and LDA (2 M, 3.3 mL, 6.6 mmol) was added dropwise. After the addition was complete, the reaction solution was slowly warmed to room temperature, and the reaction was quenched with dilute hydrochloric acid. The mixture was extracted twice with ethyl acetate, and the organic phases were combined, washed with saturated brine, evaporated to dryness, and purified by silica gel column chromatography to give 838 mg of product A-13-2, yield: 67%.

[0116] The synthesis method of A-13 is the same as that of A-2 synthesized from A-2-1.

[0117] Preparation of intermediate A-14:

[0118]

[0119] Compound A-14-1 (500 mg, 3.90 mmol), acetonitrile (5 mL), potassium carbonate (647 mg, 4.68 mmol), and deuterated iodomethane (566 mg, 3.90 mmol) were added to a reaction flask. The reaction mixture was heated to 50 °C and stirred overnight. The reaction mixture was poured into water, acidified with dilute hydrochloric acid, and extracted twice with ethyl acetate. The organic phases were combined, washed with saturated brine, evaporated to dryness, and purified by silica gel column chromatography to give 432 mg of product A-14-2, yield: 76%.

[0120] The synthesis method of A-14 is the same as that of A-2 synthesized from A-2-2.

[0121] Preparation of intermediate A-15:

[0122]

[0123] Compound A-2-3 (500 mg, 1.68 mmol) and dichloromethane (8 mL) were added to a reaction flask. The reaction solution was cooled to -70 °C, and a dichloromethane solution of boron tribromide (1 M, 5 mL, 5.0 mmol) was added dropwise. After the addition was complete, the reaction was maintained at this temperature for 2 hours. The reaction solution was quenched with water, extracted twice with dichloromethane, and the organic phases were combined, evaporated to dryness, and the residue was purified by silica gel column chromatography to give 390 mg of product A-15-1, yield: 82%.

[0124] Compound A-15-1 (200 mg, 0.706 mmol), DMF (2 mL), bromocyclopropane (171 mg, 1.41 mmol), cesium carbonate (276 mg, 0.847 mmol), and sodium iodide (53 mg, 0.353 mmol) were added to a reaction flask. The reaction mixture was heated to 150 °C and reacted for 15 hours. The reaction mixture was poured into water and extracted twice with ethyl acetate. The organic phases were combined, washed with water and saturated brine, evaporated to dryness, and purified by silica gel column chromatography to give 121 mg of product A-15-2, yield: 53%.

[0125] The synthesis method of A-15 is the same as that of A-2 synthesized from A-2-3.

[0126] Preparation of intermediate A-16:

[0127]

[0128] The synthesis method of A-16 is the same as that of A-13 synthesized from A-13-1.

[0129] Preparation of intermediate A-17:

[0130]

[0131] Compound A-17-1 (250 mg, 1.76 mmol), dichloromethane (5 mL), p-bromophenylboronic acid (706 mg, 3.52 mmol), copper acetate (320 mg, 1.76 mmol), pyridine (418 mg, 5.28 mmol), and 4A molecular sieve (powder, 500 mg) were added to the reaction flask. The reaction solution was stirred at room temperature under atmospheric conditions for two days. Silica gel was then added directly to the reaction solution and stirred. The solution was purified by silica gel column chromatography to obtain 367 mg of product A-17-2, yield: 70%.

[0132] The synthesis method of A-17 is the same as that of A-2 synthesized from A-2-3.

[0133] Preparation of intermediate A-18:

[0134]

[0135] Compound A-18-1 (500 mg, 2.44 mmol), toluene (10 mL), cyclopropylboronic acid (315 mg, 3.66 mmol), potassium phosphate (1036 mg, 4.88 mmol), tricyclohexylphosphine (68 mg, 0.244 mmol), palladium acetate (30 mg), and water (0.5 mL) were added to a reaction flask. The reaction mixture was purged with nitrogen and heated to 100 °C overnight. After cooling, silica gel was added directly and stirred until the mixture was purified by silica gel column chromatography to obtain 316 mg of product A-18-2, yield: 78%.

[0136] The method for synthesizing A-18-3 is the same as the method for synthesizing A-15-1 from A-2-3.

[0137] The synthesis method of A-18 is the same as that of A-2 synthesized from A-2-2.

[0138] The following compounds were synthesized using commercially available raw materials through the methods for preparing the above intermediates.

[0139] Table 2: Structure and Synthesis of Intermediates A-19 and A-20

[0140]

[0141] Preparation of intermediate A-21:

[0142]

[0143] Compound A-21-1 (500 mg, 1.91 mmol) and tetrahydrofuran (8 mL) were added to a reaction flask. The reaction solution was purged with nitrogen and cooled in an ice-salt bath. Methylmagnesium bromide (1 M tetrahydrofuran solution, 2.3 mL, 2.3 mmol) was added dropwise to the reaction solution. After the addition was complete, the mixture was allowed to rise to room temperature and react for 1 hour. The reaction solution was quenched with saturated ammonium chloride aqueous solution and extracted twice with ethyl acetate. The organic phases were combined, washed with water and saturated brine, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness to give 535 mg of product A-21-1, yield: 100%. The product was used directly in the next step without further purification.

[0144] The synthesis method of A-21 is the same as that of A-2 synthesized from A-2-3.

[0145] Preparation of intermediate A-22:

[0146]

[0147] The synthesis of A-2 is described in the Journal of Medicinal Chemistry, 2020, vol.63, #10, 5102-5118.

[0148] The following compounds were synthesized using commercially available raw materials through the methods for preparing the above intermediates.

[0149] Table 3: Structure and Synthesis of Intermediates A-23 to A-25

[0150]

[0151]

[0152] Preparation of intermediate A-26:

[0153]

[0154] The method for synthesizing A-26-2 is the same as the method for synthesizing A-18-2 from A-18-1.

[0155] Compound A-26-2 (500 mg, 2.57 mmol), methanol (8 mL), and sodium hydroxide aqueous solution (1 M, 5.1 mL, 5.1 mmol) were added to a reaction flask, and the reaction mixture was allowed to react overnight at room temperature. The reaction mixture was diluted with water, acidified with dilute hydrochloric acid, and extracted twice with ethyl acetate. The organic phases were combined, washed with water and saturated brine, dried over anhydrous sodium sulfate, and filtered under vacuum to obtain 440 mg of product A-26-3, yield: 95%. The product was used directly in the next step without further purification.

[0156] Compound A-26-3 (200 mg, 1.11 mmol), DMF (2 mL), pinacol 4-aminophenylboronic acid (268 mg, 1.22 mmol), and DIEA (430 mg, 3.33 mmol) were added to the reaction flask. HATU (633 mg, 1.67 mmol) was added to the reaction mixture in a single addition, and the mixture was allowed to react overnight at room temperature. The reaction mixture was quenched with water, extracted twice with ethyl acetate, and the organic phases were combined, washed with water and saturated brine, dried over anhydrous Na₂SO₄, and purified by silica gel column chromatography to obtain 219 mg of product A-26 (yield: 52%).

[0157] Preparation of intermediate A-27:

[0158]

[0159] Compound A-21-1 (1000 mg, 3.83 mmol), S-tert-butylsulfinamide (511 mg, 4.21 mmol), and 1,4-dioxane (10 mL) were added to the reaction flask. The reaction solution was purged with nitrogen, and tetraethyl titanate (2184 mg, 9.58 mmol) was added. The reaction solution was heated to 100 °C and stirred for 5 hours. The reaction solution was cooled, quenched with water, and extracted twice with ethyl acetate. The organic phases were combined, washed with water and saturated brine, and purified by silica gel column chromatography after vacuum evaporation to obtain 882 mg of product A-27-1, yield: 63%.

[0160] Compound A-27-1 (882 mg, 2.42 mmol) and tetrahydrofuran (14 mL) were added to a reaction flask. The reaction solution was purged with nitrogen and cooled in an ice-salt bath. Methyl magnesium bromide (1 M tetrahydrofuran solution, 2.9 mL, 2.9 mmol) was added dropwise to the reaction solution. After the addition was complete, the mixture was allowed to react at room temperature for 1 hour. The reaction solution was quenched with saturated ammonium chloride aqueous solution, extracted twice with ethyl acetate, and the organic phases were combined, washed with water and saturated brine, evaporated under vacuum, and purified by silica gel column chromatography to obtain 630 mg of product A-27-2, yield: 68%.

[0161] Compound A-27-2 (630 mg, 1.66 mmol) and methanol (10 mL) were added to a reaction flask, followed by a 1,4-dioxane solution of hydrogen chloride (4 M, 6 mL). The reaction mixture was reacted at room temperature for 1 hour, then concentrated to dryness under reduced pressure. The residue was diluted with water, adjusted to an alkaline solution with sodium hydroxide, and extracted twice with ethyl acetate. The organic phases were combined, washed with water and saturated brine, evaporated under vacuum, and purified by silica gel column chromatography to obtain 371 mg of product A-27-3, yield: 81%.

[0162] The synthesis method of A-27 is the same as that of A-2 synthesized from A-2-3.

[0163] Preparation of intermediate A-28:

[0164]

[0165] The synthesis method of A-28 is the same as that of A-2 synthesized from A-2-3.

[0166] Preparation of intermediate A-29:

[0167]

[0168] Compound A-29-1 (1000 mg, 5.17 mmol) and tetrahydrofuran (10 mL) were added to a reaction flask. The reaction mixture was purged with nitrogen and cooled in an ice-water bath. Isopropyl magnesium chloride (1 M tetrahydrofuran solution, 6.2 mL, 6.2 mmol) was added dropwise to the reaction mixture. After the addition was complete, the reaction mixture was brought to room temperature and stirred for 1 hour. Then, a tetrahydrofuran solution of p-fluorobenzaldehyde (770 mg, 6.20 mmol) (4 mL) was added dropwise. After the addition was complete, the reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was quenched with saturated ammonium chloride aqueous solution, extracted twice with ethyl acetate, and the organic phases were combined, washed with water and saturated brine, evaporated under vacuum, and purified by silica gel column chromatography to obtain 612 mg of product A-29-2 (yield: 50%).

[0169] Compound A-29-2 (612 mg, 2.56 mmol), dichloromethane (12 mL), and Dess-Martin oxidant (1632 mg, 3.85 mmol) were added to the reaction flask. The reaction was carried out at room temperature for 1 hour, and TLC showed that the reaction was complete. The reaction solution was then directly added to silica gel and stirred. Silica gel column chromatography was used for purification to give 495 mg of product A-29-3, yield: 82%.

[0170] The synthesis method of A-29 is the same as that of A-27 synthesized from A-21-1.

[0171] Preparation of intermediate A-30:

[0172]

[0173] Compound A-30-1 (500 mg, 2.92 mmol), acetonitrile (5 mL), p-bromophenol (607 mg, 3.51 mmol), and potassium carbonate (485 mg, 3.51 mmol) were added to a reaction flask. The reaction mixture was heated to 70 °C and stirred overnight. After cooling, the mixture was filtered, washed with ethyl acetate, and the filtrate was evaporated under vacuum and purified by silica gel column chromatography to obtain 742 mg of product A-30-2, yield: 97%.

[0174] The synthesis method of A-30 is the same as that of A-2 synthesized from A-2-3.

[0175] Preparation of intermediate A-31:

[0176]

[0177] Compound A-31-1 (500 mg, 2.82 mmol), 5-bromo-2-chloropyrimidine (546 mg, 2.82 mmol), and N-methylpyrrolidone (5 mL) were added to a reaction flask. The reaction mixture was heated to 150 °C and stirred for 2 hours. The reaction mixture was cooled, diluted with water, and extracted twice with ethyl acetate. The organic phases were combined, washed with water and saturated brine, and purified by vacuum evaporation and silica gel column chromatography to obtain 580 mg of product A-31-2, yield: 62%.

[0178] The synthesis method of A-31 is the same as that of A-2 synthesized from A-2-3.

[0179] The following compounds were synthesized using commercially available raw materials through the methods for preparing the above intermediates.

[0180] Table 4: Structure and Synthesis of Intermediates A-32 to A-33

[0181]

[0182] Preparation of intermediate A-34:

[0183]

[0184] Compound A-34-1 (500 mg, 2.94 mmol), DMF (5 mL), dimethylhydroxylamine hydrochloride (344 mg, 3.53 mmol), and DIEA (1520 mg, 11.8 mmol) were added to the reaction flask with stirring. HBTU (1449 mg, 3.82 mmol) was added to the reaction mixture in one batch. The reaction mixture was stirred overnight at room temperature. The mixture was then poured into water and extracted four times with ethyl acetate. The organic phases were combined, washed with saturated brine, and purified by silica gel column chromatography after vacuum evaporation to obtain 600 mg of product A-34-2, yield: 96%.

[0185] Add p-bromoiodobenzene (876 mg, 3.10 mmol) and tetrahydrofuran (10 mL) to the reaction flask. Purge the reaction solution with nitrogen and cool to -70°C in a dry ice / ethanol bath. Add n-butyllithium (2.5 M, 1.24 mL, 3.10 mmol) dropwise to the reaction solution. After stirring for 30 minutes, add a tetrahydrofuran solution of A-34-2 (600 mg, 2.81 mmol) (3 mL). After the addition is complete, slowly raise the reaction solution to room temperature and react for 1 hour. Quench the reaction solution with water, extract twice with ethyl acetate, combine the organic phases, wash with saturated brine, evaporate under vacuum, and purify by silica gel column chromatography to obtain 360 mg of product A-34-3, yield: 41%.

[0186] The synthesis method of A-34 is the same as that of A-2 synthesized from A-2-3.

[0187] Preparation of intermediate A-35:

[0188]

[0189] The synthesis method of A-35-2 is the same as that of A-34-3 synthesized from A-34-2.

[0190] The synthesis method of A-35-3 is the same as that of A-29-2.

[0191] The synthesis method of A-35 is the same as that of A-2 synthesized from A-2-3.

[0192] The following compounds were synthesized using commercially available raw materials through the methods for preparing the above intermediates.

[0193] Table 5: Structure and Synthesis of Intermediates A-36 to A-39

[0194]

[0195]

[0196] Preparation of intermediate A-40:

[0197]

[0198] Compound A-38 (100 mg, 0.309 mmol), DMF (1 mL), methyl iodide (48 mg, 0.340 mmol), and potassium carbonate (51 mg, 0.371 mmol) were added to a reaction flask. The reaction mixture was heated to 80 °C and reacted for 5 hours. The reaction mixture was cooled, poured into water, and extracted twice with ethyl acetate. The organic phases were combined, washed with water and saturated brine, dried over anhydrous sodium sulfate, and purified by vacuum evaporation using silica gel filtration to obtain 62 mg of product A-40, yield: 60%.

[0199] The following compounds were synthesized using commercially available raw materials through the methods for preparing the above intermediates.

[0200] Table 6: Structure and Synthesis of Intermediates A-41 to A-47

[0201]

[0202]

[0203] Preparation of intermediate A-48:

[0204]

[0205] Compound A-15-1 (200 mg, 0.706 mmol), DMF (4 mL), and sodium difluorochloroacetate (215 mg, 1.41 mmol) were added to a reaction flask. The reaction solution was purged with nitrogen and heated to 100 °C for 6 hours. The reaction solution was cooled, poured into water, and extracted twice with ethyl acetate. The organic phases were combined, washed with water and saturated brine, and purified by vacuum evaporation and silica gel column chromatography to obtain 128 mg of product A-48-1, yield: 54%.

[0206] The synthesis method of A-48 is the same as that of A-2 synthesized from A-2-3.

[0207] The following compounds were synthesized using commercially available raw materials through the methods for preparing the above intermediates.

[0208] Table 7: Structure and Synthesis of Intermediates A-49 to A-53

[0209]

[0210] Preparation of intermediate A-54:

[0211]

[0212] Compound A-54-1 (500 mg, 3.89 mmol), 5-bromo-2-chloropyrimidine (752 mg, 3.89 mmol), DMF (5 mL), and potassium carbonate (645 mg, 4.67 mmol) were added to a reaction flask. The reaction mixture was heated to 100 °C and reacted for 4 hours. The reaction mixture was cooled, poured into water, and extracted twice with ethyl acetate. The organic phases were combined, washed with water and saturated brine, and purified by vacuum evaporation and silica gel column chromatography to obtain 511 mg of product A-54-2, yield: 46%.

[0213] The synthesis method of A-54 is the same as that of A-2 synthesized from A-2-3.

[0214] Preparation of intermediate A-55:

[0215]

[0216] Compound A-55-1 (1.00 g, 7.35 mmol), Pd / C (10%, 200 mg), and methanol (25 mL) were added to the reaction flask. The reaction mixture was purged with hydrogen, and then stirred overnight under hydrogen pressure (balloon). The reaction mixture was filtered, and the filtrate was directly evaporated to dryness to give 1.00 g of product A-55-2, yield: 99%. The product was used directly in the next step without purification.

[0217] Compound A-55-2 (800 mg, 5.79 mmol) and tetrahydrofuran (10 mL) were added to a reaction flask. The reaction mixture was purged with nitrogen and cooled to -70°C in a dry ice / ethanol bath. Butyllithium (2.5 M, 2.8 mL, 6.95 mmol) was added dropwise to the reaction mixture. After stirring for 30 minutes, a solution of trimethyl borate (723 mg, 6.95 mmol) in tetrahydrofuran (3 mL) was added dropwise. After the addition was complete, the reaction mixture was slowly heated to room temperature and reacted for 30 minutes. The reaction mixture was quenched with dilute hydrochloric acid, extracted twice with ethyl acetate, and the organic phases were combined, washed with saturated brine, evaporated under vacuum, and purified by silica gel column chromatography to obtain 430 mg of product A-55-3, yield: 41%.

[0218] The synthesis method of A-55 is the same as that of A-2 synthesized from A-2-1.

[0219] Preparation of intermediate A-56:

[0220]

[0221] Compound A-56-1 (500 mg, 3.90 mmol), dibromomethane (1018 mg, 5.85 mmol), DMF (8 mL), and potassium carbonate (1348 mg, 9.75 mmol) were added to a reaction flask. The reaction mixture was heated to 100 °C and reacted for 4 hours. The reaction mixture was cooled, poured into water, and extracted twice with ethyl acetate. The organic phases were combined, washed with water and saturated brine, and purified by vacuum evaporation and silica gel column chromatography to obtain 320 mg of product A-56-2, yield: 59%.

[0222] The synthesis method of A-56 is the same as that of A-55 synthesized from A-55-2.

[0223] The following compounds were synthesized using commercially available raw materials through the methods for preparing the above intermediates.

[0224] Table 8: Structure and Synthesis of Intermediates A-57 to A-59

[0225]

[0226]

[0227] Preparation of intermediate A-60:

[0228]

[0229] Compound A-60-1 (500 mg, 3.31 mmol), p-bromophenol (685 mg, 3.96 mmol), NMP (10 mL), and cesium carbonate (3.20 g, 9.90 mmol) were added to a reaction flask. The reaction mixture was heated to 80 °C and reacted overnight. The reaction mixture was cooled, poured into water, and extracted twice with ethyl acetate. The organic phases were combined, washed with water and saturated brine, evaporated under vacuum, and purified by silica gel column chromatography to obtain 830 mg of product A-60-2, yield: 82%.

[0230] The synthesis method of A-60 is the same as that of A-2 synthesized from A-2-3.

[0231] The following compounds were synthesized using commercially available raw materials through the methods for preparing the above intermediates.

[0232] Table 9: Structure and Synthesis of Intermediates A-61 to A-63

[0233]

[0234] Preparation of intermediate A-64:

[0235]

[0236] Compound A-64-1 (1.192 g, 8.05 mmol), bromobenzene (10.1 g, 64.3 mmol), and aluminum trichloride (2.15 g, 16.1 mmol) were added to a reaction flask. The reaction mixture was purged with nitrogen and heated to 90 °C for 3 hours. The reaction mixture was cooled and poured into dilute hydrochloric acid. It was extracted three times with dichloromethane, and the organic phases were combined. The organic phase was extracted three times with an aqueous sodium carbonate solution. The aqueous phase was acidified to pH 3 with dilute hydrochloric acid. The precipitated solid was filtered, washed with water, and the filter cake was collected and dried under vacuum to give 2.0 g of product A-64-2, yield: 81%. The product was used directly in the next step without further purification.

[0237] The synthesis method of A-64-3 is the same as that of A-2 synthesized from A-2-3.

[0238] Compound A-64-3 (200 mg, 0.57 mmol), DMF (3 mL), ammonium chloride (152 mg, 2.85 mmol), and DIEA (220 mg, 1.71 mmol) were added to the reaction flask with stirring. HBTU (324 mg, 0.85 mmol) was added to the reaction mixture in one batch. The reaction mixture was stirred overnight at room temperature. The mixture was then poured into water and extracted three times with ethyl acetate. The organic phases were combined, washed with saturated brine, and purified by vacuum evaporation using silica gel ablation to obtain 70 mg of product A-64 (yield: 35%).

[0239] The following compounds were synthesized using commercially available raw materials through the methods for preparing the above intermediates.

[0240] Table 10: Structure and Synthesis of Intermediates A-65 to A-67

[0241]

[0242] Preparation of intermediate A-68:

[0243]

[0244] Compound A-68-1 (200 mg, 1.39 mmol), p-bromophenol (361 mg, 2.09 mmol), NMP (2 mL), and potassium carbonate (384 mg, 2.78 mmol) were added to a reaction flask. The reaction mixture was heated to 180 °C and reacted for 8 hours. The reaction mixture was cooled, poured into water, and extracted twice with ethyl acetate. The organic phases were combined, washed with water and saturated brine, dried over anhydrous sodium sulfate, and purified by vacuum evaporation using silica gel ablation to obtain 60 mg of product A-68-2 (yield: 15%).

[0245] The synthesis method of A-68 is the same as that of A-2 synthesized from A-2-3.

[0246] Preparation of intermediate A-69:

[0247]

[0248] The synthesis method of A-69 is the same as that of A-1 synthesized from A-1-1 and A-1-2.

[0249] Preparation of intermediate A-70:

[0250]

[0251] Compound A-21-1 (200 mg, 0.766 mmol), triethylsilane (267 mg, 2.31 mmol), dichloromethane (4 mL), and trifluoromethanesulfonic acid (35 mg, 0.231 mmol) were added to a reaction flask, and the reaction mixture was stirred overnight at room temperature. The reaction mixture was poured into water, extracted twice with ethyl acetate, and the organic phases were combined. After vacuum evaporation, the mixture was purified by silica gel column chromatography to give 190 mg of product A-70-1, yield: 100%.

[0252] The synthesis method of A-70 is the same as that of A-2 synthesized from A-2-3.

[0253] Preparation of intermediate A-71:

[0254]

[0255] The synthesis method of A-71 is the same as that of A-54 synthesized from A-54-1.

[0256] Preparation of intermediate A-72:

[0257]

[0258] Compound A-72-2 (2.00 g, 8.06 mmol), DMF (15 mL), A-72-1 (1.31 g, 8.06 mmol), DIEA (3.12 g, 24.2 mmol), and HATU (4.60 g, 12.1 mmol) were added to a reaction flask. The reaction mixture was heated to 60 °C and reacted overnight. The reaction mixture was cooled, poured into water, and extracted twice with ethyl acetate. The organic phases were combined, washed with water and saturated brine, evaporated under vacuum, and purified by silica gel column chromatography to give 1.56 g of product A-72, yield: 49%.

[0259] Preparation of intermediate A-73:

[0260]

[0261] Compound A-21-1 (300 mg, 1.15 mmol) and BAST (3 mL) were added to a reaction tube, which was then sealed and heated to 90 °C overnight. The reaction mixture was cooled, poured into water, and extracted twice with ethyl acetate. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and purified by vacuum evaporation using a silica gel preparative plate to obtain 270 mg of product A-73-1 (yield: 83%).

[0262] The synthesis method of A-73 is the same as that of A-2 synthesized from A-2-3.

[0263] Preparation of intermediate A-74:

[0264]

[0265] Compound A-74-1 (1.00 g, 7.93 mmol), PMDTA (1.44 g, 8.32 mmol), and tetrahydrofuran (10 mL) were added to the reaction flask. The reaction solution was purged with nitrogen and cooled to -70°C in a dry ice / ethanol bath. Butyllithium (2.5 M, 3.3 mL, 8.30 mmol) was added dropwise to the reaction solution. After stirring at this temperature for 2 hours, dry ice was carefully added to the reaction solution, and the solution was slowly brought to room temperature. The reaction solution was quenched with dilute hydrochloric acid, extracted three times with dichloromethane, and the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and evaporated under vacuum to give 1.00 g of product A-74-2 (yield: 74%). The product was used directly in the next step without further purification.

[0266] Compound A-74-2 (800 mg, 4.70 mmol) and dichloromethane (8 mL) were added to a reaction flask. The reaction solution was purged with nitrogen and cooled in an ice-water bath. A dichloromethane solution of boron tribromide (17%, 27.7 g, 18.8 mmol) was added dropwise to the reaction solution. After the addition was complete, the reaction solution was brought to room temperature and reacted for 30 minutes. The reaction solution was then cooled again in an ice-water bath, and the reaction was quenched by slow dropwise addition of methanol. The reaction solution was directly evaporated under vacuum and purified by silica gel column chromatography to obtain 560 mg of product A-74-3, yield: 76%.

[0267] Compound A-74-3 (560 mg, 3.59 mmol), DMF (5 mL), methylamine solution (30%, 557 mg, 5.38 mmol), DIEA (1392 mg, 10.8 mmol), and HATU (1775 mg, 4.67 mmol) were added to the reaction flask. The reaction mixture was stirred overnight at room temperature. The reaction mixture was poured into water and extracted six times with ethyl acetate. The organic phases were combined, evaporated under vacuum, and purified by silica gel column chromatography to give 254 mg of product A-74-4, yield: 42%.

[0268] The synthesis method of A-74 is the same as that of A-17 synthesized from A-17-1.

[0269] Preparation of intermediate A-75:

[0270]

[0271] The synthesis method of A-75-1 is the same as that of A-34-2 synthesized from A-34-1.

[0272] Compound A-75-1 (500 mg, 1.91 mmol) and tetrahydrofuran (8 mL) were added to the reaction flask. The reaction mixture was purged with nitrogen and cooled in an ice-salt bath. A tetrahydrofuran solution of magnesium phenyl bromide (1 M, 2.3 mL, 2.3 mmol) was added dropwise to the reaction mixture. After the addition was complete, the reaction mixture was slowly heated to room temperature and stirred for 1 hour. The reaction mixture was quenched with dilute hydrochloric acid, extracted twice with ethyl acetate, and the organic phases were combined, washed with water and saturated brine, dried over anhydrous sodium sulfate, and evaporated under vacuum to give 538 mg of product A-75-2, yield: 100%. The product was used directly in the next step without further purification.

[0273] The synthesis method of A-75 is the same as that of A-21-1 for synthesizing A-73.

[0274] Preparation of intermediate A-76:

[0275]

[0276] Compound A-76-1 (200 mg, 1.10 mmol), p-bromophenol (228 mg, 1.32 mmol), NMP (2 mL), and cesium carbonate (538 mg, 1.65 mmol) were added to a reaction flask. The reaction mixture was heated to 80 °C and reacted overnight. The reaction mixture was cooled, poured into water, and extracted twice with ethyl acetate. The organic phases were combined, washed with water and saturated brine, evaporated under vacuum, and purified by silica gel column chromatography to obtain 280 mg of product A-76-2, yield: 76%.

[0277] Compound A-76-2 (200 mg, 0.597 mmol), sodium methoxide (161 mg, 2.98 mmol), and NMP (2 mL) were added to a reaction flask. The reaction mixture was heated to 80 °C and reacted overnight. After cooling, the mixture was poured into water and extracted twice with ethyl acetate. The organic phases were combined, washed with water and saturated brine, and purified by vacuum evaporation and silica gel column chromatography to give 116 mg of product A-76-3, yield: 56%.

[0278] The synthesis method of A-76 is the same as that of A-2 synthesized from A-2-3.

[0279] Preparation of intermediate A-77:

[0280]

[0281] The method for synthesizing A-77-3 is the same as the method for synthesizing A-34-3 from A-34-1.

[0282] The synthesis method of A-77 is the same as that of A-21-1 to synthesize A-73.

[0283] Preparation of intermediate A-78:

[0284]

[0285] The method for synthesizing A-78-2 is the same as the method for synthesizing A-34-2 from A-34-1.

[0286] Compound A-78-2 (474 ​​mg, 1.73 mmol), zinc cyanide (305 mg, 2.59 mmol), DMA (5 mL), zinc powder (47 mg), dppf (94 mg), and Pd2(dba)3 (94 mg) were added to the reaction flask. The reaction solution was purged with nitrogen and heated to 110 °C overnight. The reaction solution was cooled, poured into water, and extracted twice with ethyl acetate. The organic phases were combined, washed with saturated brine, evaporated under vacuum, and purified by silica gel column chromatography to obtain 473 mg of product A-78-3, yield: 100%.

[0287] The synthesis method of A-78-4 is the same as the method for synthesizing A-34-3 from A-34-2.

[0288] The synthesis method of A-78 is the same as that of A-21-1 to synthesize A-73.

[0289] The following compounds were synthesized using commercially available raw materials through the methods for preparing the above intermediates.

[0290] Table 11: Structure and Synthesis of Intermediates A-79 to A-81

[0291]

[0292] Preparation of intermediate A-82:

[0293]

[0294] Add 5 mL of dichloromethane to the reaction flask, purge with nitrogen, cool to -40 °C in a dry ice / acetonitrile bath, add titanium tetrachloride (1453 mg, 7.66 mmol), then slowly add a toluene solution of dimethyl zinc (1 M, 7.7 mL, 7.7 mmol). After the addition is complete, maintain the reaction temperature for 30 minutes. Add 3 mL of dichloromethane solution of compound A-21-1 (500 mg, 1.91 mmol) to the reaction solution. After the addition is complete, maintain the reaction temperature for 1 hour, then slowly raise the temperature to room temperature and stir overnight. Quench the reaction solution with water, extract twice with dichloromethane, combine the organic phases, wash with water, evaporate under vacuum, and purify by silica gel column chromatography to obtain 326 mg of product A-82-1, yield: 62%.

[0295] The synthesis method of A-82 is the same as that of A-2 synthesized from A-2-3.

[0296] The following compounds were synthesized using commercially available raw materials through the methods for preparing the above intermediates.

[0297] Table 12: Structure and Synthesis of Intermediates A-83 to A-100

[0298]

[0299]

[0300]

[0301] Preparation of intermediate A-101:

[0302]

[0303] Add p-bromoiodobenzene (1.71 g, 6.03 mmol) and tetrahydrofuran (15 mL) to the reaction flask, purge with nitrogen, cool to -70 °C in a dry ice / ethanol bath, then slowly add n-butyllithium (2.5 M, 2.4 mL, 6.03 mmol). After the addition is complete, maintain the reaction temperature for 30 minutes. Weigh A-101-1 (1.00 g, 5.74 mmol) and dissolve it in tetrahydrofuran (5 mL), then add it dropwise to the reaction solution. After 10 minutes, slowly raise the reaction solution to room temperature. Quench the reaction solution with water, extract twice with ethyl acetate, combine the organic phases, wash with water, evaporate under vacuum, and purify by silica gel column chromatography to obtain 1.4 g of product A-101-2, yield: 73%.

[0304] The synthesis method of A-101 is the same as that of A-2 synthesized from A-2-3.

[0305] Preparation of intermediate A-102:

[0306]

[0307] Add A-101-2 (550 mg, 1.66 mmol) and dichloromethane (6 mL) to the reaction flask, purge with nitrogen, add DAST (402 mg, 2.49 mmol) under ice bath, and incubate for 2 hours. Quench the reaction solution with sodium bicarbonate aqueous solution, extract twice with dichloromethane, combine the organic phases, dry to anhydrous sodium sulfate, evaporate under vacuum, and purify by silica gel plate to obtain 410 mg of product A-102, yield: 74%.

[0308] The synthesis method of A-102 is the same as that of A-2 synthesized from A-2-3.

[0309] Preparation of intermediate A-103:

[0310]

[0311] Add A-103-1 (500 mg, 1.50 mmol) and hydrobromic acid aqueous solution (5 mL) to the reaction flask, cool in an ice bath, add sodium nitrite (645 mg, 9.35 mmol), and then incubate the reaction for 20 minutes. Add CuBr (2.69 g, 18.75 mmol) in hydrobromic acid aqueous solution (5 mL) to the reaction solution, and slowly raise the reaction solution to room temperature for 3 hours. Dilute the reaction solution with water, extract twice with ethyl acetate, combine the organic phases, wash with water, evaporate under vacuum, and purify by silica gel column chromatography to obtain 620 mg of product A-103-2, yield: 89%.

[0312] Add A-103-2 (200 mg, 0.43 mmol) and tetrahydrofuran (5 mL) to the reaction flask, purge with nitrogen, cool to -70°C in a dry ice / ethanol bath, then slowly add n-butyllithium (2.5 M, 0.17 mL, 0.43 mmol). After the addition is complete, maintain the reaction temperature for 1 hour. Quench the reaction solution with dilute hydrochloric acid, extract twice with ethyl acetate, combine the organic phases, dry over anhydrous sodium sulfate, and evaporate under vacuum to obtain 170 mg of crude A-103-3. The crude product was used directly in the next step without purification.

[0313] The synthesis method of A-103 is the same as that of A-2 synthesized from A-2-3.

[0314] Preparation of intermediate B-1:

[0315]

[0316] Compound B-1-1 (2.00 g, 17.5 mmol), imidazole (1.43 g, 21.0 mmol), and DMF solution (10 mL) were added to a reaction flask. The reaction mixture was purged with nitrogen, and TBDPSCl (5.30 g, 19.3 mmol) was added in an ice-water bath. After the addition was complete, the ice was removed, and the reaction mixture was stirred at room temperature for 16 h. TLC showed that the reaction was complete. The reaction mixture was poured into water, extracted twice with ethyl acetate, and the extracts were combined. The extracts were then washed twice with water, followed by washing with saturated NaCl solution. Finally, the extract was dried over anhydrous Na₂SO₄ and directly evaporated under vacuum to give 6.68 g of product B-1-2. This unpurified product was used directly in the next step.

[0317] A tetrahydrofuran solution (35 mL) of B-1-2 (6.68 g, 18.9 mmol) was added to the reaction flask. The reaction solution was purged with nitrogen, and 9-BBN (0.5 M, 91 mL) was added dropwise under an ice-water bath. After the addition was complete, the reaction mixture was stirred at room temperature for 16 h. TLC showed that the starting material reacted completely. The reaction solution was cooled again under an ice-water bath, and 24 mL of 10% NaOH solution and 12 mL of 30% H2O2 solution were slowly added. Stirring was continued for 1 h, and TLC showed that the intermediate reacted completely. The reaction solution was poured into water and extracted twice with ethyl acetate. The extracts were combined, washed twice with water, washed with saturated NaCl solution, and then directly added to silica gel for mixing. The product was then purified by silica gel column chromatography to obtain 6.49 g of product B-1-3. The two-step yield was 100%.

[0318] A 50 mL solution of compound B-1-3 (6.49 g, 17.5 mmol) in dichloromethane was added to the reaction flask. The reaction mixture was purged with nitrogen, and Dess-Martin oxidant (11.14 g, 26.3 mmol) was added to the flask in an ice-water bath. The mixture was stirred for 1.5 h, and TLC showed that the reaction was complete. Silica gel was added directly for mixing, and the mixture was then purified by silica gel column chromatography to obtain 6.45 g of product B-1-4, yield: 100%.

[0319] Compound B-1-4 (6.45 g, 17.5 mmol), ethanol (926 mg, 20.1 mmol), DME (60 mL), and TosMIC (3.92 g, 20.1 mmol) were added to the reaction flask, purged with nitrogen, and cooled in an ice-water bath. t-BuOK (3.83 g, 34.1 mmol) was added to the reaction mixture, and the mixture was stirred for 30 min, then slowly brought to room temperature and stirred for another 1.5 h. TLC showed that the starting material reacted completely. The reaction mixture was poured into a saturated NH4Cl solution (350 mL), extracted twice with ethyl acetate, the extracts were combined, silica gel was added and stirred, and then purified by silica gel column chromatography to obtain 2.27 g of product B-1-5 (two spots on TLC), yield: 34%.

[0320] A dichloromethane solution (35 mL) of B-1-5 (2.27 g, 5.97 mmol) was added to the reaction flask. The reaction solution was purged with nitrogen and cooled in a dry ice-ethanol bath. Dibal-H (1 M, 9 mL) was slowly added dropwise to the reaction solution. The mixture was stirred at this temperature for 1.5 h. TLC showed that the reaction was complete. The reaction solution was then added to cold dilute hydrochloric acid (1 M, 10 mL) and stirred until no bubbles were observed. DCM was then added and extracted twice. The organic phases were combined, washed with saturated NaCl solution, and finally dried over anhydrous Na₂SO₄. The reaction solution was directly evaporated under vacuum to give 2.22 g of product B-1-6. Yield: 97%.

[0321] Compound B-1-6 (2.22 g, 5.81 mmol), THF (60 mL), water (30 mL), and K₂CO₃ (4.82 g, 34.8 mmol) were added to a reaction flask, followed by the addition of KMnO₄ (3.67 g, 23.2 mmol) in portions. The reaction mixture was stirred at room temperature for 30 min. TLC showed that the starting material had reacted completely. The reaction mixture was acidified with dilute hydrochloric acid, and then NaHSO₃ solution was added until the reaction mixture became colorless. The mixture was extracted twice with ethyl acetate, and the organic phases were combined, washed with saturated NaCl solution, and finally dried over anhydrous Na₂SO₄. The reaction mixture was directly evaporated under vacuum to give 1.89 g of product B-1-7. Yield: 82%.

[0322] Compound B-1-7 (1.89 g, 4.74 mmol), 3-chloropyrazine-2-methylamine dihydrochloride (1.03 g, 4.74 mmol), and DMF (20 mL) were added to the reaction flask. The mixture was purged with nitrogen and cooled in an ice-water bath. HATU (2.16 g, 5.69 mmol) and DIEA (3.06 g, 23.7 mmol) were then added to the reaction solution. The mixture was stirred for 30 min, then the ice was removed, and stirring continued for another 30 min at room temperature. TLC showed complete reaction of the starting material. The reaction solution was poured into water and extracted twice with ethyl acetate. The extracts were combined, washed twice with water, and then washed with saturated NaCl solution. Silica gel was added and stirred, followed by purification by silica gel column chromatography to obtain 1.94 g of product B-1-8 (two spots on TLC). Yield: 78%.

[0323] Compound B-1-8 (790 mg, 1.51 mmol), DMF (0.8 mL), and ethyl acetate (8 mL) were added to a reaction flask. The mixture was purged with nitrogen, and phosphorus oxychloride (1.39 g, 9.08 mmol) was added dropwise under an ice-water bath. The reaction mixture was stirred for 1 h. TLC showed that the starting material reacted completely. The reaction mixture was quenched in NaHCO3 solution (4.5 g NaHCO3 / 30 mL H2O), extracted twice with ethyl acetate, and the extracts were combined. The extracts were then washed twice with water, followed by washing with saturated NaCl solution. Finally, the mixture was dried over anhydrous Na2SO4 and directly evaporated under vacuum to give 680 mg of product B-1-9 (two spots on TLC). Yield: 89.0%.

[0324] A 7 mL solution of B-1-9 (680 mg, 1.34 mmol) in DMF was added to the reaction flask. The reaction solution was purged with nitrogen, and NBS (287 mg, 1.61 mmol) was added under an ice-water bath. The mixture was stirred for 40 min, and TLC showed that the reaction was complete. The reaction solution was quenched in water, extracted twice with ethyl acetate, and the extracts were combined. The samples were then washed twice with water, followed by washing with saturated NaCl solution. Silica gel was added and the mixture was stirred. The solution was then purified by silica gel column chromatography to obtain 298 mg of product B-1-10-A (the less polar spot on TLC, which eluted first) and 339 mg of product B-1-10-B (the more polar spot on TLC, which eluted later). Overall yield: 81%.

[0325] Compound B-1-10-A (298 mg, 0.509 mmol), ammonia (6 mL), and n-butanol solution (3 mL) were added to a sealed flask. The flask was then heated to 95 °C and stirred for 16 h. The reaction mixture was cooled, evaporated to dryness under vacuum, and then purified by silica gel column chromatography to obtain 184 mg of product B-1-A, yield: 64%.

[0326] B-1-B was prepared by reacting B-1-10-B with ammonia water using the same method as the synthesis of B-1-A.

[0327] Preparation of intermediate B-2:

[0328]

[0329] Weigh out 8.56 g (357 mmol) of NaH and suspend it in 80 mL of anhydrous THF. Heat the solution to 40-45 °C and add a THF solution of compound B-2-1 (22.85 g, 149 mmol) dropwise. After the addition is complete, maintain the temperature and stir the reaction for 15 minutes. Add a THF solution of ethyl acrylate dropwise. After the addition is complete, continue the reaction for 15 minutes. Cool the reaction solution to room temperature and add it to ice water. Adjust the pH to 3 with concentrated HCl. Extract twice with ethyl acetate. Combine the organic phases, dry over anhydrous sodium sulfate, filter, concentrate under reduced pressure, and purify by column chromatography to obtain compound B-2-2 as a colorless oil (37.6 g, 83%).

[0330] Compound B-2-2 (37.6 g, 120 mmol) and sodium chloride (20.97 g, 359 mmol) were added to DMSO (170 mL) and H2O (5 mL) and reacted at 160 °C for 1.7 h. The reaction solution was cooled, added to ice water, extracted with ethyl acetate, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. Column chromatography purified compound B-2-3 as a colorless oil (21.6 g, 75%).

[0331] Compound B-2-3 (21.6 g, 89.2 mmol), ethylene glycol (6.64 g, 107 mmol), and p-toluenesulfonic acid hydrate (169 mg, 0.89 mmol) were weighed and added to toluene (180 mL). The mixture was refluxed at 120 °C for 4 h. The reaction solution was cooled and added to a saturated aqueous solution of NaHCO3. The solution was extracted with ethyl acetate, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by column chromatography to give compound B-2-4 as a pale yellow liquid (23.7 g, 93%).

[0332] Weigh LAH (6.47 g, 166 mmol) into a three-necked flask, add anhydrous THF (150 mL), purge with nitrogen, and add a THF solution of compound B-2-4 (23.7 g, 82.8 mmol) (100 mL) dropwise under ice-salt bath cooling. After the addition is complete, slowly raise the temperature to room temperature and react for 5 h. Add H2O / THF (1:1, 30 mL) dropwise to the reaction mixture under ice-water bath cooling, then add 5N sodium hydroxide aqueous solution (8 mL), and stir overnight at room temperature. Dilute the reaction solution with DCM / MeOH (5:1, 250 mL) in the reaction flask, filter, and rinse with DCM / MeOH (5:1). Add 50 g of silica gel to the filtrate, stir for 15 min, filter, and rinse. Concentrate the filtrate under reduced pressure to obtain compound B-2-5 (16.7 g, 99%).

[0333] Compound B-2-5 (16.7 g, 82.6 mmol) was weighed into a reaction flask, pyridine (100 mL) was added, and TsCl (34.6 g, 182 mmol) was added under ice-water bath. The mixture was stirred overnight at room temperature. The reaction solution was diluted with ethyl acetate, washed with 10% citric acid solution and saturated sodium chloride, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was slurried with ethanol to give compound B-2-6 as a white solid (35 g, 83%).

[0334] Compound B-2-6 (35 g, 68.5 mmol) was weighed into a reaction flask, and 260 mL of 1 N HCl solution and 300 mL of THF were added. The reaction mixture was reacted at 80 °C for 5 h. The reaction solution was extracted with ethyl acetate, the organic phase was washed with water, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The solution was purified by column chromatography to give compound B-2-7 (26.2 g, 82%).

[0335] Compound 1,3-dithiane (2.1 g, 17.4 mmol) and anhydrous THF (40 mL) were added to a reaction flask. The mixture was purged with nitrogen, and n-butyllithium (2.5 M, 8.5 mL) was added dropwise under dry ice-ethanol bath cooling. After the addition was complete, the temperature was raised to 0 °C and the reaction proceeded for 1 h. After further cooling in a dry ice-ethanol bath, a THF solution of compound B-2-7 (6.5 g, 13.9 mmol) was added dropwise. After the addition was complete, the temperature was raised to room temperature and the reaction proceeded for 1 h. The reaction solution was quenched in a saturated NH4Cl solution, extracted with ethyl acetate, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. Column chromatography was used to purify compound B-2-8 (6.77 g, 83%).

[0336] Compound B-2-8 (6.77 g, 11.5 mmol), NaOH (1.38 g, 34.6 mmol), and THF (170 mL) were added to the reaction flask and refluxed overnight at 70 °C. The reaction solution was cooled to room temperature, water was added, and the mixture was extracted with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. Column chromatography was used to purify compound B-2-9 (3.7 g, 77%).

[0337] Compound B-2-9 (3.7 g, 8.92 mmol), acetonitrile (50 mL), and water (12.5 mL) were added to a reaction flask. NBS (5.56 g, 31.2 mmol) was added under an ice-water bath. After the addition was complete, the mixture was allowed to react at room temperature for 3 h. The reaction solution was added to a saturated NaHCO3 solution, extracted with ethyl acetate, and the organic phases were combined. The organic phase was backwashed with saturated NaCl solution, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain crude compound B-2-10, which was not further purified.

[0338] The crude compound B-2-10 obtained above was dissolved in ethanol, and NaBH4 (508 mg, 13.4 mmol) was added under ice-water bath. The reaction was allowed to proceed at room temperature for 1 h. The reaction solution was quenched in saturated NH4Cl solution, extracted with ethyl acetate, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. Column chromatography was used to purify compound B-2-11 (2.66 g, 91% yield in two steps).

[0339] Compound B-2-11 (2.56 g, 7.84 mmol), DMAP (287 mg, 2.35 mmol), imidazole (1.06 g, 15.7 mmol), and DMF (15 mL) were added to a reaction flask, followed by TBDPSCl (2.59 g, 9.41 mmol). The reaction mixture was reacted at room temperature for 0.5 h. The reaction solution was added to water, extracted with ethyl acetate, and the organic phases were combined. The organic phase was backwashed with saturated NaCl solution, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. Column chromatography was used to purify compound B-2-12 (4.0 g, 90%).

[0340] Compound B-2-12 (4.0 g, 7.08 mmol) and methanol (120 mL) were added to a reaction flask. Mg (1.89 g, 77.9 mmol) was added with stirring at room temperature. After 30 minutes, the reaction proceeded exothermically with stirring overnight. The reaction mixture was then added to a saturated NH4Cl solution, extracted with ethyl acetate, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. Column chromatography was used to purify compound B-2-13 (2.4 g, 83%).

[0341] Compound B-2-13 (2.4 g, 5.85 mmol) and DMF (30 mL) were added to the reaction flask. PDC (6.6 g, 17.6 mmol) was added under ice-water bath conditions. After the addition was complete, the mixture was allowed to react at room temperature for 2 h. The reaction solution was diluted with ethyl acetate, extracted with water, and the organic phases were combined. The organic phase was backwashed with saturated NaCl solution, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. Column chromatography was used to purify compound B-2-14 (1.99 g, 80%).

[0342] Compound B-2-14 (1.99 g, 4.69 mmol), 3-chloropyrazine-2-methylamine dihydrochloride (1.02 g, 4.69 mmol), and DMF (10 mL) were added to the reaction flask. Then, HBTU (2.13 g, 5.62 mmol) and DIEA (2.42 g, 18.8 mmol) were added to the reaction solution, and the mixture was reacted at room temperature for 1 h. The reaction solution was then added to water and extracted twice with ethyl acetate. The combined organic phases were backwashed with saturated NaCl solution, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. Column chromatography was used to purify compound B-2-15 (1.99 g, 77%).

[0343] Compound B-2-15 (1.59 g, 2.89 mmol) was added to a reaction flask and dissolved in 30 mL of DCM. Under nitrogen protection, pyridine (1.83 g, 23.1 mmol) and trifluoromethanesulfonic anhydride (4.89 g, 17.3 mmol) were added in an ice-water bath. After the addition was complete, the mixture was allowed to react at room temperature for 4 h. The reaction solution was added to a saturated NaHCO3 solution, extracted with ethyl acetate, and the organic phases were combined. The organic phase was backwashed with saturated NaCl solution, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain crude compound B-2-16, which was not further purified.

[0344] The crude compound B-2-16 obtained above was dissolved in DMF (8 mL), and NBS (566 mg, 3.18 mmol) was added. The mixture was reacted at room temperature for 0.5 h. The reaction solution was added to NaHCO3 solution, extracted with ethyl acetate, and the organic phases were combined. The organic phase was backwashed with saturated NaCl solution, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. Column chromatography was used to purify compound B-2-17 (1.43 g, 81% yield in two steps).

[0345] Compound B-2-17 (1.43 g, 2.34 mmol), ammonia (20 mL), and n-butanol (8 mL) were added to a sealed container. The reaction system was heated to 95 °C and stirred for 16 h. The reaction solution was evaporated to dryness under vacuum and purified by column chromatography to obtain compound B-2 (1.2 g, 87%).

[0346] Preparation of intermediate B-3:

[0347]

[0348] B-3-1 was prepared according to the method described in the reference (Angew. Chem. Int. Ed. 2020, 59, 7161-7167).

[0349] Compound B-3-1 (1.25 g, 6.71 mmol), imidazole (548 mg, 8.06 mmol), and DMF (12 mL) were added to a reaction flask, followed by TBDPSCl (1.94 g, 7.05 mmol). The reaction mixture was reacted overnight at room temperature. The reaction solution was added to water and extracted twice with ethyl acetate. The combined organic phases were washed with water, washed with saturated NaCl solution, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give compound B-3-2 (2.85 g, 100%). No further purification was performed.

[0350] Compound B-3-2 (2.85 g, 6.71 mmol) was dissolved in ethanol (25 mL), and an aqueous solution of sodium hydroxide (403 mg, 10.1 mmol) (10 mL) was added. The reaction mixture was heated to 60 °C and reacted overnight. The reaction mixture was cooled, added to water, and acidified with dilute hydrochloric acid. The mixture was extracted twice with ethyl acetate, and the combined organic phases were washed with water, washed with saturated NaCl solution, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give compound B-3-3 (2.53 g, 95%). No further purification was performed.

[0351] The method for preparing B-3 from B-3-3 is the same as the method for preparing B-2 from B-2-14.

[0352] Preparation of intermediate B-4:

[0353]

[0354] Compounds B-4-1 (100 mg, 0.383 mmol), B-1-3 (170 mg, 0.460 mmol), triphenylphosphine (251 mg, 0.958 mmol), and THF (2 mL) were added to a reaction flask. The reaction solution was purged with nitrogen and heated to 60 °C. DIAD (194 mg, 0.958 mmol) was added dropwise to the reaction solution, and the reaction was maintained at this temperature overnight. The reaction solution was cooled, concentrated to dryness under reduced pressure, and purified by silica gel column chromatography to give compounds B-4-A (52 mg, low polarity) and B-4-B (140 mg, high polarity, containing the impurity triphenylphosphine). Overall yield: 82%.

[0355] Preparation of intermediate B-5:

[0356]

[0357] Compound B-1-3 (500 mg, 1.35 mmol), TEA (273 mg, 2.70 mmol), DCM (5 mL), and p-toluenesulfonyl chloride (309 mg, 1.62 mmol) were added to a reaction flask. The reaction mixture was stirred at room temperature for 2 h, and TLC showed almost no reaction. DMAP (198 mg, 1.62 mmol) was added to the reaction mixture, and the reaction was allowed to proceed overnight. The reaction mixture was then added to water, acidified with dilute hydrochloric acid, and extracted twice with ethyl acetate. The combined organic phases were washed with water and saturated NaCl solution, concentrated under reduced pressure to dryness, and purified by silica gel column chromatography to give compound B-5-1 (550 mg, 78%).

[0358] Compound B-5-1 (200 mg, 0.381 mmol), 3-bromo-4-chloro-1H-pyrazolo[4,3-C]pyridine (89 mg, 0.381 mmol), cesium carbonate (149 mg, 0.457 mmol), and DMA (2 mL) were added to a reaction flask. The reaction mixture was heated to 100 °C and stirred overnight. After cooling, the reaction mixture was added to water and extracted twice with ethyl acetate. The organic phases were combined, washed with water and saturated NaCl solution, concentrated under reduced pressure to dryness, and purified by silica gel plate to obtain compound B-5-2 (60 mg, 27%).

[0359] Compound B-5-2 (60 mg, 0.103 mmol), ammonia (2 mL), and n-butanol (1 mL) were added to a sealed tube. The reaction system was heated to 100 °C and stirred overnight. The reaction solution was cooled, evaporated under vacuum, and purified by silica gel stencil to obtain compound B-5 (38 mg, 66%).

[0360] Preparation of intermediate B-6:

[0361]

[0362] Compound B-1-10-B (200 mg, 0.342 mmol), THF (3 mL), and TBAF (1 M, 0.7 mL, 0.7 mmol) were added to the reaction flask. The reaction solution was stirred at room temperature for 4 h. The reaction solution was then purified directly by silica gel plate preparation to obtain compound B-6-1 (108 mg, 91%).

[0363] Compound B-6-1 was oxidized with PDC (see the synthesis of B-2-14 from B-2-13) to prepare B-6-2.

[0364] Compound B-6-2 (90 mg, 0.25 mmol), potassium carbonate (69 mg, 0.50 mmol), DMF (1 mL), and iodomethane (53 mg, 0.374 mmol) were added to a reaction flask, and the reaction mixture was stirred overnight at room temperature. The reaction mixture was then added to water, extracted twice with ethyl acetate, and the organic phases were combined, washed with water, washed with saturated NaCl solution, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure to dryness, and purified by silica gel filtration to obtain compound B-6-3 (80 mg, 85%).

[0365] Compound B-6-3 (80 mg, 0.21 mmol) and THF (2 mL) were added to the reaction flask, purged with nitrogen, and cooled in an ice-water bath. Tetraisopropyl titanate (28 mg, 0.10 mmol) was added to the reaction mixture, followed by slow dropwise addition of magnesium ethyl bromide (0.6 mL, 0.6 mmol, 1 M). After the addition was complete, the reaction mixture was brought to room temperature and stirred overnight. The reaction mixture was quenched in an aqueous ammonium chloride solution, extracted twice with ethyl acetate, the organic phases were combined, washed with saturated brine, concentrated under reduced pressure to dryness, and purified by silica gel filtration to give 22 mg of compound B-6-4, yield: 28%.

[0366] The method for preparing B-6 from B-6-4 is the same as the method for preparing B-2 from B-2-17.

[0367] Preparation of intermediate B-7:

[0368]

[0369] Compound B-6-3 (100 mg, 0.267 mmol) and THF (2 mL) were added to the reaction flask, purged with nitrogen, and cooled in an ice-water bath. Methylmagnesium bromide (0.8 mL, 0.8 mmol, 1 M) was added dropwise to the reaction mixture. After addition was complete, the reaction mixture was brought to room temperature and stirred overnight. The reaction mixture was quenched in an aqueous ammonium chloride solution, extracted twice with ethyl acetate, the organic phases were combined, washed with saturated brine, concentrated under reduced pressure to dryness, and purified by silica gel filtration to give 65 mg of compound B-7-1, yield: 65%.

[0370] The method for preparing B-7 from B-7-1 is the same as the method for preparing B-2 from B-2-17.

[0371] Preparation of intermediate B-8:

[0372]

[0373] Compound B-8-1 (CAS: 652-67-5, 1.00 g, 6.84 mmol), imidazole (559 mg, 8.21 mmol), and DMF (15 mL) were added to a reaction flask, followed by TBDPSCl (1.88 g, 6.84 mmol). The reaction mixture was allowed to react overnight at room temperature. The reaction solution was poured into water and extracted twice with ethyl acetate. The organic phases were combined, washed with water and saturated NaCl solution, concentrated under reduced pressure, and purified by silica gel column chromatography to obtain compound B-8-2 (1.63 g, 62%).

[0374] The method for preparing B-8 using B-8-2 is the same as the method for preparing B-1-A(B) from B-1-3.

[0375] Preparation of intermediate B-9:

[0376]

[0377] Compound B-9-1 (8.0 g, 76.1 mmol), dioxane (120 mL), and Raney Ni (approximately 1 g) were added to the reaction flask. The reaction mixture was purged with hydrogen, heated to 90 °C under hydrogen pressure, and stirred overnight. TLC showed that the starting materials had largely reacted completely. The reaction mixture was cooled, filtered, and the filtrate was evaporated to dryness under reduced pressure to give 8.3 g of product B-9-2, yield: 100%. The unpurified product was used directly in the next step.

[0378] B-9 is prepared by condensation, cyclization, and bromination of B-9-2 and B-1-7, with the specific method referring to the method for preparing B-1-10-A(B) from B-1-7.

[0379] Preparation of intermediate B-10:

[0380]

[0381] Compound B-1-9 (200 mg, 0.395 mmol) and tetrahydrofuran (3 mL) were added to the reaction flask. The reaction solution was purged with nitrogen and cooled to -70°C in a dry ice / ethanol bath. Butyllithium (2.5 M, 0.19 mL, 0.474 mmol) was added dropwise to the reaction solution. After the addition was complete, the reaction was maintained at this temperature for 30 minutes. Iodomethane (112 mg, 0.790 mmol) was added dropwise to the reaction solution. After the addition was complete, the reaction solution was slowly brought to room temperature. The reaction was quenched with ammonium chloride aqueous solution. The mixture was extracted twice with ethyl acetate, and the organic phases were combined, concentrated to dryness under reduced pressure, and purified by column chromatography to obtain B-10-1 (160 mg, 78%).

[0382] B-10-1 is prepared by bromine on NBS and then ammonolysis, with the specific method referring to the method for preparing B-1-A(B) from B-1-9.

[0383] Example 1: Preparation of Compound 1

[0384]

[0385] Compound B-1-B (205 mg, 0.362 mmol), A-1 (161 mg, 0.471 mmol), Na₂CO₃ (77 mg, 0.724 mmol), PdCl₂ (dppf) (20 mg), dioxane (6 mL), and water (2 mL) were added to a reaction flask. The mixture was purged with nitrogen and the temperature was raised to 95 °C for 2.5 h. TLC showed that the reaction was complete. The reaction solution was diluted with ethyl acetate, stirred directly into silica gel, and then purified by silica gel column chromatography to obtain 182 mg of product C-1-B, yield: 72%.

[0386] Compound C-1-B (182 mg, 0.260 mmol) and tetrahydrofuran (4 mL) were added to the reaction flask, followed by TBAF (1 M, 0.39 mL). The reaction mixture was stirred at room temperature for 1.5 h. TLC showed that the reaction was complete. The reaction solution was purified directly through a silica gel plate (DCM / MeOH = 15 / 1) to give 70 mg of product 1-B, yield: 58%.

[0387] The structure of the product was characterized by nuclear magnetic resonance and mass spectrometry, and the results are as follows:

[0388] 1 H NMR (400MHz, d6-DMSO) δ1.56-1.60 (1H, m), 1.94-2.07 (2H, m), 2.20-2.25 (1H, m), 3.28-3.31 (1H, m), 3.38-3 .42 (2H, m), 3.47-3.51 (1H, m), 3.77 (1H, dd, J = 11.8Hz, 3.2Hz), 4.11 (1H, dd, J = 11.8Hz, 1.8Hz), 4.59 (1H, t, J=5.8Hz), 6.03 (2H, brs), 7.07 (1H, d, J=5.0Hz), 7.20 (1H, ddd, J=7.4Hz, 4.9Hz, 1.0Hz), 7.63 (2H, dd, J=10. 4Hz, 5.4Hz), 7.85-7.90 (1H, m), 7.98-8.02 (2H, m), 8.21 (1H, d, J=8.4Hz), 8.41-8.43 (1H, m), 10.97 (1H, s).

[0389] MS(ESI)m / z(M+H) + :463.0.

[0390] 1-A was prepared by using A-1 and B-1-A in the method of synthesizing 1-B.

[0391] The structure of the product was characterized by nuclear magnetic resonance and mass spectrometry, and the results are as follows:

[0392] 1H NMR (400MHz, d6-DMSO) δ1.38-1.50 (1H, m), 1.73-1.75 (1H, m), 1.80-1.92 (1H, m), 2.12-2.15 (1H, m) ), 3.36-3.47 (3H, m), 3.62 (1H, t, J = 11.0Hz), 4.07-4.10 (1H, m), 4.69 (1H, t, J = 5.5Hz), 6.02 (2H, s) , 7.07 (1H, d, J = 5.0Hz), 7.20 (1H, dd, J = 6.9Hz, 5.2Hz), 7.61 (1H, t, J = 7.9Hz), 7.76 (1H, d, J = 5.0Hz) , 7.83-7.91 (1H, m), 7.95-8.04 (2H, m), 8.21 (1H, d, J = 8.4Hz), 8.42 (1H, d, J = 3.8Hz), 10.97 (1H, s).

[0393] MS(ESI)m / z(M+H) + :463.1.

[0394] Example 2: Preparation of Compound 2

[0395]

[0396] 2-A was prepared by reacting A-5 with B-1-A using the method for synthesizing 1-B.

[0397] The structure of the product was characterized by nuclear magnetic resonance and mass spectrometry, and the results are as follows:

[0398] 1 H NMR (400MHz, d6-DMSO) δ1.41-1.51 (1H, m), 1.50-1.78 (1H, m), 1.85-1.97 (1H, m), 2.13-2.15 (1H, m), 3.3 5-3.49 (4H, m), 3.65 (1H, t, J = 11.0Hz), 4.10 (1H, ddd, J = 11.0Hz, 3.6Hz, 1.6Hz), 4.69 (1H, t, J = 5.6Hz), 6 .13 (2H, brs), 7.09 (1H, d, J = 4.9Hz), 7.19 (1H, dd, J = 6.9Hz, 5.2Hz), 7.76 (3H, dd, J = 9.5Hz, 6.7Hz), 7.83 -7.90 (1H, m), 8.16 (2H, d, J = 8.4Hz), 8.23 ​​(1H, d, J = 8.4Hz), 8.41 (1H, dd, J = 4.8Hz, 1.0Hz), 10.84 (1H, s).

[0399] MS(ESI)m / z(M+H) + :445.2.

[0400] Compound 2-A was resolved by SFC to give 2-A-P1 (first peak) and 2-A-P2 (later peak).

[0401] SFC preparation conditions:

[0402] Instrument: SFC-80 (Thar, Waters)

[0403] Column: CHIRALCEL OJ (30*250mm 5μm) (Daicel)

[0404] Column temperature: 35℃

[0405] Mobile phase: A=CO2 Co-Solvent B=ETOH

[0406] Cycle Time: 12.5min Run Time: 21min

[0407]

[0408] 2-B was prepared by reacting A-5 with B-1-B using the method for synthesizing 1-B.

[0409] The structure of the product was characterized by nuclear magnetic resonance and mass spectrometry, and the results are as follows:

[0410] 1 H NMR (400MHz, d6-DMSO) δ1.58-1.63 (1H, m), 1.95-2.02 (1H, m), 2.08-2.17 (1H, m), 2.24-2.28 (1H, m), 3.39 -3.51 (4H, m), 3.78 (1H, dd, J = 11.7, 3.2Hz), 4.10 (1H, d, J = 10.1Hz), 4.60 (1H, t, J = 5.2Hz), 6.14 (2H, brs) , 7.09 (1H, d, J = 4.9Hz), 7.18 (1H, dd, J = 6.9Hz, 5.3Hz), 7.63 (1H, d, J = 5.0Hz), 7.76 (2H, d, J = 8.3Hz), 7.84 -7.88 (1H, m), 8.16 (2H, d, J = 8.3Hz), 8.23 ​​(1H, d, J = 8.3Hz), 8.41 (1H, dd, J = 4.8Hz, 1.0Hz), 10.84 (1H, s).

[0411] MS(ESI)m / z(M+H) + :445.2.

[0412] Example 3: Preparation of Compound 3

[0413]

[0414] Compound 2-B (50 mg, 0.113 mmol), NCS (16.5 mg, 0.124 mmol), and glacial acetic acid (1 mL) were added to a reaction flask. The reaction mixture was heated to 80 °C and reacted for 2 h. The reaction mixture was concentrated to dryness under reduced pressure, and an aqueous sodium bicarbonate solution was added. The mixture was extracted twice with ethyl acetate, and the organic phases were combined, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, concentrated to dryness under reduced pressure, and purified by silica gel plate preparation to give 28 mg of product 3, yield: 52%.

[0415] MS(ESI)m / z(M+H) + :479.2.

[0416] Examples 4-119: Preparation of compounds 4-119

[0417] Compounds 4–119 were obtained using different intermediates obtained through the same methods used to prepare compounds 1-B or 3. The intermediate numbers, structural formulas, MS data, and other parameters used are described below. 1 The H-NMR data are shown in Table 13.

[0418] Table 13: Structure, MS and... of Examples 4-119 1 H-NMR data

[0419]

[0420]

[0421]

[0422]

[0423]

[0424]

[0425]

[0426]

[0427]

[0428]

[0429]

[0430]

[0431]

[0432]

[0433] Example 120: Preparation of Compound 120

[0434]

[0435] Compound B-1-4 (3.10 g, 8.41 mmol), methanol (31 mL), hydroxylamine hydrochloride (1.17 g, 16.8 mmol), and sodium acetate (2.07 g, 25.2 mmol) were added to the reaction flask. The reaction mixture was stirred overnight at room temperature. The solution was poured into water and extracted twice with ethyl acetate. The organic phases were combined, washed with saturated brine, concentrated under reduced pressure to dryness, and then purified by silica gel column chromatography to give 1.90 g of product 120-1, yield: 59%.

[0436] Compound 120-1 (1.90 g, 4.95 mmol) and tetrahydrofuran (20 mL) were added to the reaction flask and cooled in an ice bath. Lithium aluminum hydride (376 mg, 9.91 mmol) was added in portions to the reaction solution. The reaction solution was brought to room temperature and reacted for 3 h. The reaction solution was then cooled in an ice bath again, and the reaction was quenched by slowly adding water (380 mg), 15% NaOH aqueous solution (380 mg), and water (1.14 g) dropwise. The resulting suspension was filtered, washed with DCM / MeOH (10 / 1), concentrated to dryness under reduced pressure, and then purified by silica gel column chromatography to obtain 200 mg of product 120-2, yield: 31%.

[0437] Compound 2,4-dichloro-3-nitropyridine (294 mg, 1.52 mmol), DMF (2 mL), 120-2 (200 mg, 1.52 mmol), and triethylamine (231 mg, 2.29 mmol) were added to a reaction flask. The reaction mixture was stirred at room temperature for 4 h. The reaction mixture was poured into water and extracted three times with ethyl acetate. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain 464 mg of product 120-3, yield: 100%. The product was not further purified.

[0438] Compound 120-3 (464 mg, 1.61 mmol), isopropanol (5 mL), bis-(4-methoxybenzyl)-amine (415 mg, 1.61 mmol), and triethylamine (212 mg, 2.10 mmol) were added to a reaction flask. The reaction mixture was heated to 95 °C and stirred for 4 h. After cooling, the mixture was concentrated to dryness under reduced pressure and then purified by silica gel column chromatography to obtain 540 mg of product 120-4, yield: 66%.

[0439] Compound 120-4 (388 mg, 0.76 mmol), DMF (4 mL), imidazole (78 mg, 1.14 mmol), DMAP (10 mg, 0.076 mmol), and tert-butyldiphenylchlorosilane (210 mg, 0.76 mmol) were added to the reaction flask. The reaction mixture was heated to 60 °C and stirred overnight. TLC showed that a large amount of starting material remained. Imidazole (150 mg), DMAP (40 mg), and tert-butyldiphenylchlorosilane (100 mg) were added to the reaction mixture, and the temperature was raised to 80 °C for 2 h. tert-butyldiphenylchlorosilane (200 mg) was added to the reaction mixture, and after 2 h, TLC showed that the reaction was complete. The reaction mixture was cooled, poured into water, extracted three times with ethyl acetate, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure to dryness, and then purified by silica gel column chromatography to obtain 650 mg of product 120-5, yield: 100%.

[0440] Compound 120-5 (650 mg, 0.87 mmol), methanol / glacial acetic acid (5 mL / 5 mL), and iron powder (486 mg, 8.7 mmol) were added to a reaction flask. The reaction mixture was stirred at room temperature for 4 h. The reaction mixture was slowly poured into an aqueous solution of NaHCO3, extracted twice with ethyl acetate, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give 612 mg of product 120-6, yield: 98%. The product was not further purified.

[0441] Compound 120-6 (612 mg, 0.85 mmol), acetonitrile (6 mL), and N,N′-carbonyldiimidazole (280 mg, 1.71 mmol) were added to a reaction flask. The reaction mixture was heated to 80 °C and stirred overnight. After cooling, the mixture was concentrated to dryness under reduced pressure and then purified by silica gel column chromatography to give 470 mg of product 120-7, yield: 74%.

[0442] Compound 120-7 (470 mg, 0.63 mmol), dichloromethane (15 mL), 4-phenoxyphenylboronic acid (271 mg, 1.27 mmol), ketone acetate (115 mg, 0.63 mmol), 4A molecular sieve (500 mg), and triethylamine (192 mg, 1.90 mmol) were added to the reaction flask. The reaction mixture was stirred at room temperature for 36 h. The reaction mixture was filtered through a diatomaceous earth filter, washed with ethyl acetate, and the filtrate was concentrated to dryness under reduced pressure. The filtrate was then purified by silica gel column chromatography to obtain 240 mg of product 120-8, yield: 41%.

[0443] Compound 120-8 (260 mg, 0.29 mmol), dichloromethane (4 mL), and trifluoroacetic acid (4 mL) were added to a reaction flask. The reaction mixture was heated to 50 °C and stirred for 3 h. The reaction mixture was cooled, concentrated to dryness under reduced pressure, and extracted twice with ethyl acetate by adding NaHCO3 aqueous solution. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was dissolved in tetrahydrofuran (2 mL), and TBAF (1 M, 0.2 mL) was added. The mixture was stirred at room temperature for 1 h. The reaction mixture was directly purified by silica gel ablation to give 40 mg of product 120, yield: 30%.

[0444] The structure of the product was characterized by nuclear magnetic resonance and mass spectrometry, and the results are as follows:

[0445] 1 H NMR (400MHz, d6-DMSO) δ1.37-1.53 ​​(1H, m), 1.56-1.71 (1H, m), 1.81-1.95 (1H, m), 2.12-2 .29(1H,m), 2.34-2.44(4H,m), 3.40-3.54(1.5H,m), 3.59-3.70(1H,m), 3.77-4.02(1H,m ), 4.17-4.39 (1H, m), 4.71 (0.5H, t, J = 5.7Hz), 4.76-4.83 (2H, m), 6.94 (0.5H, d, J = 5.6Hz) ), 7.13 (4H, t, J = 8.5Hz), 7.18-7.25 (1.5H, m), 7.40-7.48 (4H, m), 7.74 (1H, t, J = 5.6Hz).

[0446] MS(ESI)m / z(M+H)+ :433.2.

[0447] Examples 121-138: Preparation of compounds 121-138

[0448] Compounds 121–138 were obtained using different intermediates prepared by the same methods as compounds 1-B or 3. The intermediate numbers, structural formulas, MS data, and other parameters used are described below. 1 The H-NMR data are shown in Table 14.

[0449] Table 14: Structure, MS and... of Examples 121-138 1 H-NMR data

[0450]

[0451]

[0452]

[0453]

[0454]

[0455]

[0456]

[0457] Pharmacological trials

[0458] Experimental Example 1: In vitro BTK inhibitory kinase activity assay

[0459] 1: Compound preparation

[0460] Dissolve the compound powder in 100% DMSO to prepare a 10 mM stock solution. Store frozen at -20°C, protected from light.

[0461] 2: Kinase reaction process

[0462] (1) Prepare 1×Kinase buffer;

[0463] (2) Preparation of compound concentration gradients: The test concentration of the test compound was 1 μM. It was diluted 100 times to a final concentration of 100% DMSO solution in a 384source plate. The compound was diluted 3 times to obtain 10 concentrations. 250 nL of the 100-fold final concentration of the compound was transferred to the target plate OptiPlate-384F using an Echo 550 dispenser.

[0464] (3) Prepare a kinase solution with a final concentration of 2.5 times using 1×Kinase buffer;

[0465] (4) Add 10 μL of kinase solution at 2.5 times the final concentration to the compound well and the positive control well, respectively; add 10 μL of 1×Kinase buffer to the negative control well;

[0466] (5) Centrifuge at 1000 rpm for 30 seconds, shake the reaction plate to mix, and incubate at room temperature for 10 minutes;

[0467] (6) Prepare a mixed solution of ATP and Kinase substrate 2 at a final concentration of 5 / 3 times using 1×Kinase buffer;

[0468] (7) Add 15 μL of a mixture of ATP and substrate at 5 / 3 times the final concentration to initiate the reaction;

[0469] (8) Centrifuge the 384-well plate at 1000 rpm for 30 seconds, shake to mix, and incubate at room temperature for 10 minutes;

[0470] (9) Add 30 μL of the stop detection solution to stop the kinase reaction, centrifuge at 1000 rpm for 30 seconds, and vortex to mix.

[0471] (10) Read the conversion rate using Caliper EZ Reader.

[0472] 3: Data Analysis

[0473] Calculation formula:

[0474]

[0475] Where: Conversion%_sample is the conversion rate reading of the sample; Conversion%_min: the mean value of the negative control wells, representing the conversion rate reading of wells without enzyme activity; Conversion%_max: the mean value of the positive control wells, representing the conversion rate reading of wells without compound inhibition.

[0476] Fitting dose-response curve

[0477] Using the log value of concentration as the X-axis and the percentage inhibition rate as the Y-axis, the dose-response curve was fitted using the log(inhibitor) vs. response-variable slope of the analysis software GraphPad Prism 5 to obtain the IC50 value of each compound on enzyme activity.

[0478] The calculation formula is Y = Bottom + (Top - Bottom) / (1 + 10^((LogIC50 - X) * HillSlope)).

[0479] The inhibitory activities of the compounds of this invention against wild-type and mutant BTK C481S kinases are shown in Table 15:

[0480] IC50: A≤5nM; 5nM<B≤20nM; 20nM<C≤100nM; 100nM<D≤1000nM; E>1000nM.

[0481] Table 15: Inhibitory activity of the compounds of the present invention against BTK and BTK-C481S kinase

[0482]

[0483]

[0484]

[0485]

[0486] “\” indicates that this test was not performed.

[0487] Experimental Example 2: Liver Microsomal Stability Test

[0488] 1. Add 10 μL of test or control working solution and 80 μL of microsomal working solution (liver microsomal protein concentration of 0.5 mg / mL) to the T0, T5, T10, T20, T30, T60 and NCF60 sample wells. Add only microsomal working solution to the Blank60 well. Then place the Blank60, T5, T10, T20, T30 and T60 samples other than T0 and NCF60 in a 37°C water bath for pre-incubation for about 10 minutes.

[0489] 2: First, add 300 μL of stop solution (acetonitrile solution containing 200 ng / mL tolbutamide and 200 ng / mL labetalol) to the T0 sample, then add 10 μL of NADPH regeneration system working solution;

[0490] 3: After the pre-incubation of Blank60, T5, T10, T20, T30 and T60 incubation plates, add 10 μL of NADPH regeneration system working solution to each sample well to start the reaction. Add 10 μL of 100mM potassium phosphate buffer to the NCF60 sample well.

[0491] 4. After incubation for an appropriate time (e.g., 5, 10, 20, 30 and 60 minutes), add 300 μL of stop solution to each sample well of the test sample and the control sample well of the Blank60, T5, T10, T20, T30, T60 and NCF60 plates to terminate the reaction.

[0492] 5. Shake all sample plates well and centrifuge at 4000 rpm for 20 minutes. Take 100 μL of the supernatant of the test sample or reference sample and dilute it in 300 μL of pure water for LC-MS / MS analysis.

[0493] 6: Data analysis, calculating T based on first-order elimination kinetics. 1 / 2 and CL int(mic) (μL / min / mg) value, the first-order elimination kinetic equation is:

[0494]

[0495]

[0496]

[0497]

[0498]

[0499] The results of the stability test of human and rat liver microsomes are shown in Table 16:

[0500] Table 16: Results of liver microsomal stability tests of the compounds of this invention

[0501]

[0502]

[0503] Experimental Example 3: Pharmacokinetic Test

[0504] Pharmacokinetic studies were conducted on SD rats by single oral administration (10 mg / kg, 3 rats per group). The test compounds were dissolved in 5% DMSO + 10% solubilizer + 85% saline, vortexed for 1-2 min, and sonicated for 5-10 min to prepare a colorless, transparent, and clear solution. Animals were fasted overnight before oral administration and resumed feeding 4 hours later. Pharmacokinetic samples were collected from SD rats via orbital blood collection at the following time points: 0.25 h, 0.5 h, 1 h, 2 h, 2.5 h, 3 h, 4 h, 6 h, 8 h, and 10 h post-administration. Three whole blood samples (approximately 0.2–0.3 mL) were collected at each time point. Blood samples were immediately placed on ice and centrifuged within 15 minutes to separate the plasma (centrifugation conditions: 8000 rpm, 1 min, room temperature). Collected plasma was stored at -20°C before analysis. Take 20 μL of plasma sample into a 1.6 mL 96-well deep plate, add 200 μL of working internal standard solution (for blank, add the same volume of solvent without internal standard), vortex mix for 1 min, centrifuge at 5800 rpm for 10 min, take 100 μL of supernatant and add it to a 96-well sample plate, and analyze by LC-MS / MS.

[0505] The pharmacokinetic test results of some compounds of this invention are shown in Table 17 below:

[0506] Table 17 Pharmacokinetic Test Results of Some Compounds of the Invention

[0507]

[0508]

[0509] Experiment 4: In vitro cell proliferation inhibition activity test

[0510] 1: Cell Culture

[0511] Cells were cultured in 1640 medium with 10% inactivated FBS and 1% antibiotics, and cultured at 37°C and 5% CO2.

[0512] 2: Cell plating

[0513] (1) Culture the cells routinely until the cell saturation is 80%-90% and the required number is reached, then collect the cells.

[0514] (2) Resuspend the cells in the appropriate culture medium, count them, and prepare a cell suspension of appropriate density.

[0515] (3) Add the cell suspension to a 96-well plate, 100 μL per well.

[0516] (4) The cells were cultured overnight at 37°C in a 5% CO2 incubator.

[0517] 3: Preparation of Compounds

[0518] (1) The test compounds were diluted with DMSO to prepare a stock solution with a final concentration of 20 mM for later use.

[0519] (2) Dilute the mother liquor from 20mM to 2mM by 10 times with DMSO, and then dilute it by 3 times to 9 concentrations starting from 2mM.

[0520] (3) The blank control wells were filled with 0.5% DMSO and used as high-reading control wells.

[0521] (4) Cell-free wells containing only culture medium were used as low-read control wells.

[0522] 4: Compound treatment of cells

[0523] (1) After the cells are plated for 24 hours, the compound is used alone. 99 μL of growth medium is added to each well, and then 1 μL of the compound prepared in step 4.3a), b), and c) is added. Gently shake to ensure that the mixture is evenly mixed, and then place it in a 37°C, 5% CO2 incubator.

[0524] (2) Place the cell plate in an incubator for 72 hours.

[0525] 5: CTG method detection

[0526] (1) Place the cell test plate at room temperature for 30 minutes to equilibrate, and discard 100 μL of culture medium from each well.

[0527] (2) Add 100 μL of CTG reagent (CelltiterGlo kit) to each well, place on a shaker and shake for 2 minutes, then place at room temperature in the dark for 30 minutes.

[0528] (3) Use an Envision instrument to read the chemiluminescence signal value.

[0529] 6: Data Analysis

[0530] Calculate IC using GraphPad Prism 8 software 50 The IC of the compound is obtained using the following nonlinear fitting formula. 50 (Half-maximal inhibitory concentration), the results are shown in the table below:

[0531] Y=Bottom+(Top-Bottom) / (1+10^((LogIC 50 -X)*HillSlope))

[0532] X: Log value of compound concentration, Y: Inhibition rate (% inhibition)

[0533] Inhibition rate (% inhibition) = (High-value control reading - Compound well reading) / (High-value control reading - Low-value control reading) * 100

[0534] Table 18: Inhibitory activity of some compounds of the present invention against TMD8 cell proliferation

[0535]

[0536]

[0537] Table 19: Inhibitory activity of some compounds of the present invention on DOHH2 cell proliferation

[0538]

[0539] Table 20: Inhibitory activity of some compounds of the present invention on BT474 cell proliferation

[0540]

[0541] Table 21: Inhibitory activity of some compounds of the present invention on NCI-N87 cell proliferation

[0542]

[0543]

[0544] Experimental Example 5: HER2 Kinase Activity Assay

[0545] 1. Her2 kinase test procedure

[0546] 1) Preparation of 1× kinase reaction buffer: 1 volume of 5× kinase reaction buffer and 4 volumes of water, 1 mM dithiothreitol, 5 mM magnesium chloride, 1 mM manganese chloride, 12.5 mM SEB.

[0547] 2) Transfer 100 nmol of the diluted working solution of the compound to each well of the reaction plate (784075, Greiner) using an Echo 550. Seal the reaction plate with sealing film and centrifuge at 1000 g for 1 minute.

[0548] 3) Prepare a 1 ng / μL Her2 kinase solution using 1× kinase reaction buffer.

[0549] 4) Add 5 μL of the prepared kinase solution to each well of the reaction plate. Seal the plate with sealing film, centrifuge at 1000g for 1 minute, and incubate at room temperature for 10 minutes.

[0550] 5) Prepare a mixture of 2× kinase substrate and ATP using 1× kinase reaction buffer. The 2× Her2 kinase substrate consists of 2 μM MTK-substrate-biotin and 4 μM ATP.

[0551] 6) Add 5 μL of 2×TK-substrate-biotin and ATP mixture to the reaction plate, centrifuge at 1000g for 30 seconds, and start the reaction.

[0552] 7) Her2 kinase test: react at room temperature for 50 minutes.

[0553] 8) Prepare a mixture of Sa-XL 665 (125 nM) and TK-antibody-Cryptate using HTRF detection buffer.

[0554] 9) Add 10 μL of Sa-XL 665 and TK-antibody-Cryptate mixture to each well, centrifuge at 1000g for 30 seconds, and react at room temperature for 1 hour.

[0555] 10) Use Envision 2104 to read the fluorescence signals at 615nm (Cryptate) and 665nm (XL665).

[0556] 2. Data Analysis

[0557] 1) The inhibition percentage is calculated as follows:

[0558]

[0559] The average value of the Ratio 665 / 615nm for all positive control wells on the entire plate.

[0560] The average value of the Ratio 665 / 615nm for all negative control wells on the entire plate.

[0561] 2) Calculate IC50 and fit the dose-response curve of the compound:

[0562] Using GraphPad 6.0, the IC50 of the compound is obtained using the following nonlinear fitting formula.

[0563] Y=Bottom+(Top-Bottom) / (1+10^((LogIC50-X)*HillSlope))

[0564] X: Log value of compound concentration; Y: Percentage of compound inhibition

[0565] Example HER2 IC50(nm) 130 4.35 145 1.69 146 3.92 152 4.19 155 9.44 Tucatinib 3.00

[0566] Experimental Example 6: Blood-brain barrier permeability test

[0567] Pharmacokinetic studies of each test compound were conducted using SD rats after a single oral administration at a dose of 10 mg / kg, with 9 animals per group. The test compounds were dissolved in 5% DMSO + 10% solubilizer + 85% saline, vortexed for 1-2 min, and sonicated for 5-10 min to prepare a colorless, transparent, and clear solution. Animals were fasted overnight before administration. At 1 h, 2 h, and 4 h post-administration, approximately 0.2–0.3 mL of blood was collected from 3 rats each via the orbital sinus. Blood samples were immediately placed on ice and centrifuged within 15 minutes to separate the plasma (centrifugation conditions: 8000 rpm, 1 min, room temperature). The collected plasma was stored at -20°C before analysis. Cerebrospinal fluid and brain tissue were collected immediately after blood collection. Cerebrospinal fluid (CSF) was extracted via dural puncture using a microsyringe under direct vision. After anesthesia with chloral hydrate, the head was fixed, the back hair was clipped, and a 2cm transverse incision was made at the line connecting the bases of the ears. The muscle layers of the neck and skull base were bluntly scraped to expose the foramen magnum. Approximately 100μl of CSF was collected using a 100μl microsyringe and stored at -20℃ before analysis. The rats were then immediately euthanized, decapitated, and the brain tissue was dissected, the surface capillaries were removed, and the tissue was weighed. Three times the volume of ice-cold physiological saline was added, and the tissue was homogenized for 1 minute. The homogenate was then stored at -20℃ before analysis. Take 20 μL of plasma sample and brain homogenate sample respectively, add 200 μL of working internal standard solution (for blank samples, add the same volume of solvent without internal standard), vortex mix for 1 min, centrifuge at 13500 rpm for 10 min, and collect 100 μL of supernatant for LC-MS / MS analysis. Take 20 μL of cerebrospinal fluid sample, add 60 μL of working internal standard solution (for blank samples, add the same volume of solvent without internal standard), vortex mix for 1 min, centrifuge at 13500 rpm for 10 min, and collect 50 μL of supernatant for LC-MS / MS analysis.

[0568] Table 23: Blood-brain barrier permeability test results of some compounds of the present invention

[0569]

[0570]

[0571] Experimental Example 7: TMD8 Pharmacodynamic Model Test

[0572] Human diffuse large B-cell lymphoma TMD8 cells were cultured in vitro in monolayer under the following conditions: RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin, incubated at 37°C in a 5% CO2 incubator. Cells were passaged twice weekly using trypsin-EDTA digestion. When cell saturation reached 80%-90% and the desired number was achieved, cells were harvested, counted, and seeded. 0.2 ml (1 x 10⁻⁶ cells) of the medium was used for seeding. 7 TMD8 cells (with matrix gel, volume ratio 1:1) were subcutaneously inoculated into the right posterior dorsal region of each mouse, resulting in an average tumor volume of approximately 137 mm². 3 Dosing was initiated in groups at that time. Tumor diameter was measured twice weekly using calipers. The tumor volume was calculated using the formula: V = 0.5a × b 2 , where a and b represent the long and short diameters of the tumor, respectively.

[0573] See results Figure 1 and Figure 2 ;according to Figure 1 Based on the TMD8 mouse subcutaneous xenograft tumor efficacy model, under the same dosage of 10 mg / kg, compounds 118 and 89-P1 showed significantly better tumor-inhibiting effects than the clinical phase II drug ARQ-531 and the marketed drug ibrutinib. Figure 2 In the TMD8 mouse subcutaneous xenograft tumor model, under the same dosage of 20 mg / kg, Examples 111-P1 and 125 showed significantly better tumor inhibition effects than tirabrutinib. In particular, Example 111-P1, with a tumor inhibition rate of 93% (TGI), nearly twice that of tirabrutinib, almost completely controlled tumor growth, demonstrating a very significant efficacy advantage.

[0574] Experimental Example 8: DOHH-2-Luc Intratumoral Tumor Efficacy Model Testing

[0575] 1. Cell Culture

[0576] DOHH-2-luc tumor cells were cultured in vitro in RPMI 1640 medium containing 10% fetal bovine serum and 500 ng / mL puromycin at 37°C and 5% CO2. The medium was replenished or replaced every 2-3 days, and the number of passages was limited to 4-5. Tumor cells in the logarithmic growth phase were then used for in vivo tumor seeding.

[0577] 2. Inoculation and grouping of tumor cells

[0578] After anesthetizing the animal with a salbutamol injection, it was fixed prone on the operating table. The skin on the top of its head was disinfected with iodine and 75% alcohol. The skin was incised about 0.5 cm along the midline of the head to expose the coronal and sagittal lines. Using a brain locator, the location was determined approximately 0.5-1.0 mm above the coronal line and 2 mm to the right of the sagittal line. A hole was drilled with a 1 mL syringe needle at the location, and a microsyringe was inserted vertically to a depth of 3 mm. 3 × 10⁶ DOHH-2-luc tumor cells were slowly injected (over 1 minute). 5 A 2 μL suspension was administered and the needle was left in place for 1 minute. After needle removal, the puncture site was quickly sealed with bone wax, and the wound was sutured with a skin stapler. Approximately 7 days after tumor inoculation, the animals were randomly divided into 5 groups of 5 animals each, based on their body weight and the optical signal intensity of the tumor site.

[0579] 3. Imaging analysis

[0580] The small animal in vivo imaging system IVIS Lumina III (Perkin Elmer) was used to image mice 1-2 times per week according to their condition. The signal intensity of bioluminescence imaging (BLI, unit: photosns / s) at the tumor cell inoculation site was monitored as a key indicator for assessing tumor growth and drug efficacy. The specific operation is as follows:

[0581] Mice were intraperitoneally injected with D-luciferin (15 mg / mL, or 5 μL / g depending on the animal's body weight), and then anesthetized by inhalation with 1%-2% isoflurane. Ten minutes after the D-luciferin injection, the animals were imaged using IVIS Lumina III. The data were analyzed using Living Image software (Perkin Elmer) to calculate the optical signal intensity within the region of interest (ROI) for each animal.

[0582] See results Figure 3 , Figure 4 ;according to Figure 3 In a mouse brain DOHH2 tumor model study, under the same dosage of 30 mg / kg (BID), Examples 111-P1 and 125 showed significantly better tumor inhibition than Tirabrutinib, with a very obvious advantage in efficacy, and no side effects were found after 21 days of administration.

[0583] Figure 4The images show fluorescence images of all tested animals. The size of the tumor in the brain is represented by color and area size; the redder the color, the larger the tumor. As can be seen from the images, at the same dose, Examples 111-P1 and 125 showed excellent tumor inhibition, with almost no red areas, indicating that the tumors in these two groups were very small. In contrast, all animals in the model group and the Tirabrutinib group showed large red areas, indicating that the tumors were very large.

[0584] As can be seen from the above embodiments, the compounds of the present invention, as BTK protein kinase inhibitors, have the structure of Formula I, preferably the structure of Formula II; they exhibit strong inhibitory effects on both wild-type BTK and mutant BTK (C481S), and possess favorable pharmacokinetic properties, making them suitable for preparing drugs to treat diseases caused by BTK kinase overexpression. Some of these compounds showed significantly better efficacy than marketed BTK inhibitors Ibrutinib, Tirabrutinib, and ARQ-531 (currently in phase II clinical trials) in a TMD8 subcutaneous tumor pharmacodynamic model experiment.

[0585] The compounds of this invention exhibit significantly superior blood-brain barrier permeability, liver microsomal stability, and pharmacokinetics compared to the marketed drugs tirabrutinib and tucatinib. In the DOHH-2-Luc intracerebral pharmacodynamic model, some compounds demonstrate excellent efficacy, validating the brain permeability data. These compounds can be used to prepare drugs for treating diseases caused by BTK or HER2 kinase overexpression, particularly brain diseases.

[0586] The compounds or their stereoisomers, solvates, hydrates, pharmaceutically acceptable salts or cocrystals described above in this invention can be used to prepare medicaments for treating any one or more of the following diseases: autoimmune diseases, inflammatory diseases, thromboembolic diseases, allergies, infectious diseases, proliferative diseases and cancer, and are expected to provide new and effective treatment options.

[0587] The above description is merely a preferred embodiment of the present invention. It should be noted that those skilled in the art can make various modifications to these embodiments without departing from the technical principles of the present invention, and these modifications should also be considered within the scope of protection of the present invention.

Claims

1. A compound as a BTK inhibitor or a HER2 inhibitor, characterized in that, having the structure of Formula III, Formula IV, or a pharmaceutically acceptable salt thereof: Formula III; Formula IV; wherein R1 is amino; R2 is hydrogen; R3, R4 are both hydrogen; R6 is hydrogen or fluorine; m is selected from 0, 1, 2; n is selected from 0, 1; n1 is selected from 0, 1, 2, 3, 4; X is selected from wherein R9, R 13 are each selected from fluorine; n2 is selected from 0, 1, 2, 3, 4; R8 substituents are independently selected from fluorine, chlorine, bromine, cyano, trifluoromethyl, trifluoromethoxy, difluoromethoxy, methoxy, deuterated methoxy, cyclopropyl, cyclopropylmethoxy, ethyl, isopropyl, isobutyl; wherein the number of said substituents is an integer between 0-4.

2. A compound that is a BTK inhibitor or a HER2 inhibitor, characterized in that, said compound structure is selected from one of the following: 111、 113、 114、 121、 123、 124、 125、 126、 127、 129、 130、 132、 138、 139、 140、 141、 142、 143、 144、 145、 146、 147、 148、 149、 150、 151、 152、 153、 154、 155、 156。 3. A pharmaceutical composition comprising a compound of claim 1 or 2 and a pharmaceutically acceptable carrier. The active ingredient of the pharmaceutical composition is selected from one or a combination of two or more of the compounds or pharmaceutically acceptable salts thereof according to any one of claims 1-2.

4. Use of a compound or a pharmaceutically acceptable salt thereof according to any one of claims 1-2 in the manufacture of a medicament for the treatment of a disease selected from the group consisting of arthritis, urticaria, vitiligo, organ transplant rejection, ulcerative colitis, Crohn's disease, dermatitis, asthma, Sjogren's syndrome, systemic lupus erythematosus, multiple sclerosis, idiopathic thrombocytopenic purpura, skin rash, anti-neutrophil cytoplasmic antibody vasculitis, pemphigus, chronic obstructive pulmonary disease, psoriasis; esophageal cancer, laryngeal cancer, brain and central nervous system cancer, stomach cancer, hepatocellular carcinoma, melanoma, biliary tract cancer, lymphoma, hairy cell cancer, pharyngeal cancer, large intestine cancer, rectal cancer, urogenital tract cancer, lung cancer, small cell cancer, bone cancer, colon cancer, adenoma, follicular carcinoma, Hodgkin's leukemia, bronchial cancer, multiple myeloma, lymphocytic leukemia, myelogenous leukemia, primary macroglobulinemia.

5. Use according to claim 4, characterized in that, said disease is selected from the group consisting of ovarian cancer, kidney cancer, bladder cancer.

6. Use of a compound or a pharmaceutically acceptable salt thereof according to any one of claims 1-2 in the manufacture of a medicament for the treatment of a disease selected from the group consisting of rheumatoid arthritis, mantle cell lymphoma, non-Hodgkin's lymphoma, nasopharyngeal carcinoma, non-small cell lung cancer, glioblastoma, neuroblastoma.

7. Use of a compound or a pharmaceutically acceptable salt thereof according to any one of claims 1-2 in the manufacture of a medicament for the treatment of a disease selected from the group consisting of pemphigus vulgaris, prostate cancer, testicular cancer, endometrial cancer, uterine body cancer, uterine cervix cancer, adenocarcinoma, acute myeloid leukemia, chronic myeloid leukemia, chronic lymphoid leukemia.

8. Use of a compound or a pharmaceutically acceptable salt thereof according to any one of claims 1-2 in the manufacture of a medicament for the treatment of a disease selected from the group consisting of breast cancer, lung adenocarcinoma, thyroid cancer, pancreatic cancer, glioma.

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