A compound as a BTK inhibitor and its preparation method and application

Abstract

The present invention provides a compound as a BTK inhibitor, its preparation method, and use. The compound has the structure represented by Formula I, or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable hydrate, solvate, or salt thereof. R1 is selected from a substituted or unsubstituted C1-C8 alkyl group, a substituted or unsubstituted C3-C10 cycloalkyl group, a substituted or unsubstituted C1-C8 heteroalkyl group, a substituted or unsubstituted 3- to 10-membered heterocycloalkyl group, or a substituted or unsubstituted aryl or heteroaryl group; R3 is selected from a substituted or unsubstituted aryl group, or a substituted or unsubstituted pyridyl group; A and B are independently selected from C, O, N, amide, or S; n is 0, 1, 2, 3, or 4; and m is 0, 1, or 2. The compound can be used as a BTK protein kinase inhibitor, exhibits strong inhibitory activity against BTK, and has good pharmacokinetic properties and blood-brain barrier permeability, showing promising application prospects.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and more specifically, to a compound serving as a BTK inhibitor, a preparation method thereof, and an application thereof. Background Art

[0002] Bruton tyrosine kinase (BTK), a member of the Tec family of non-receptor tyrosine kinases, is a key kinase in the B cell receptor (BCR) signaling pathway. Excessive BTK expression leads to abnormal activation of the BCR pathway, which can affect B cell proliferation, differentiation, and apoptosis, thereby triggering various malignant lymphomas, primarily B cell lymphomas within the non-Hodgkin lymphoma category. Furthermore, excessive BTK expression and abnormal activation of the BCR signaling pathway can cause B cell dysfunction, altered immune tolerance, and the transformation of B cells into autoreactive B cells, which secrete large amounts of autoantibodies and induce autoimmune diseases.

[0003] Based on the above mechanism, BTK inhibitors have been increasingly studied in autoimmune diseases (including rheumatoid arthritis) in recent years. Although only ibrutinib received FDA approval in 2017 for the treatment of an autoimmune disease (chronic graft-versus-host disease (cGVHD)), more than a dozen small molecule inhibitors have entered clinical trials. BTK has become the most widely used target for clinical drugs in the field of autoimmune diseases, alongside TNF (tumor necrosis factor) and CD20 (B lymphocyte antigen CD20), and is expected to become a new target for the treatment of autoimmune diseases in the future.

[0004] Currently, five BTK inhibitors have been approved for marketing: ibrutinib, acalabrutinib, zanubrutinib, tilarabrutinib, and obeticholic acid. These five BTK inhibitors are all irreversible BTK inhibitors with the same mechanism of action. Irreversible BTK inhibitors, such as ibrutinib, have developed significant resistance, with resistance rates exceeding 50% after three years and over 80% after five years. Therefore, the development of reversible BTK inhibitors to combat resistance is urgently needed. Irreversible BTK inhibitors bind irreversibly to Cys481, the active site of the target protein BTK, thereby inhibiting BTK autophosphorylation and thereby suppressing the BTK signaling pathway. Once BTK develops the C481S mutation, irreversible BTK inhibitors lose their binding site and, consequently, their inhibitory activity. The C481S mutation is a key cause of drug resistance in B-cell malignancies. Studies have shown that nearly 80% of patients who develop resistance to irreversible BTK inhibitors such as ibrutinib have the C481S mutation. Off-target effects of irreversible BTK inhibitors are a major cause of intolerance in some patients. In addition to irreversibly binding to Cys481, the active site of the target protein BTK, irreversible BTK inhibitors also irreversibly bind to Cys (cysteine) residues on other proteins in the body, resulting in off-target effects. Reversible BTK inhibitors can effectively address issues such as resistance and off-target effects associated with irreversible BTK inhibitors and are currently a major research and development focus, with active international clinical trials.

[0005] There are about 100 different types of autoimmune diseases in the world that affect the human body, including common ones such as rheumatoid arthritis, ankylosing spondylitis, psoriasis, systemic lupus erythematosus, Sjögren's syndrome, pemphigus, Crohn's disease, etc.

[0006] Clinical research on BTK inhibitors for chronic inflammatory and autoimmune diseases is very active. Over 10 BTK inhibitors have entered clinical trials, with indications including rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), Sjögren's syndrome (SS), multiple sclerosis (MS), asthma, atopic dermatitis, chronic spontaneous urticaria, febrile autoimmune hemolytic anemia, and pemphigus. These diseases are characterized by altered B cell selection leading to the production of autoreactive antibodies and proinflammatory cytokines.

[0007] Central nervous system lymphomas include primary CNS lymphomas and secondary lymphomas that arise from systemic lymphomas invading the CNS. The incidence of this disease is low, accounting for 1% to 3% of CNS tumors. However, with the use of immunosuppressants, the incidence has been increasing in recent years. Primary CNS lymphomas account for approximately 8% of cases, and approximately 50% of intracranial lymphoma cases are accompanied by systemic lymphomas. Multiple sclerosis is one of the most common idiopathic inflammatory demyelinating diseases of the CNS. Lesions are often distributed in the optic nerves, periventricular white matter, corpus callosum, brainstem, cerebellum, and cervical spinal cord white matter. Progression can ultimately lead to loss of muscle coordination, vision impairment, and functional impairment. Due to the blood-brain barrier, most drugs have difficulty entering the brain, making them ineffective for treating conditions within the brain. The development of a brain-penetrating BTK inhibitor would be of great significance for the treatment of CNS lymphoma and multiple sclerosis. Summary of the Invention

[0008] In view of this, the object of the present invention is to provide a compound as a BTK inhibitor, a preparation method and application thereof. The compound as a BTK inhibitor is a class of compounds containing a 5-aminopyrazole-4-carboxamide structure, which can be used as a BTK protein kinase inhibitor, has high inhibitory activity and other characteristics, and can be used for the treatment of tumors and autoimmune diseases.

[0009] The present invention provides a compound as a BTK inhibitor having the structure shown in Formula I, or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, a pharmaceutically acceptable hydrate, solvate, or salt thereof:

[0010]

[0011] In Formula I, R1 is selected from substituted or unsubstituted C1-C8 alkyl, substituted or unsubstituted C3-C10 cycloalkyl, substituted or unsubstituted C1-C8 heteroalkyl, substituted or unsubstituted 3-10 membered heterocycloalkyl, or substituted or unsubstituted aryl or heteroaryl;

[0012] R2 is selected from hydrogen, halogen, hydroxy, cyano, amino, amide, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted C1-C6 heteroalkyl, substituted or unsubstituted 3- to 8-membered heterocycloalkyl, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted C3-C6 cycloalkyloxy, or substituted or unsubstituted 3- to 8-membered heterocycloalkoxy;

[0013] R3 is selected from substituted or unsubstituted aryl, or substituted or unsubstituted pyridyl;

[0014] wherein the substituted substituents are independently selected from halogen, hydroxy, amino, cyano, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkoxy, cycloalkyloxy, heterocycloalkyloxy, amide, sulfonyl, phosphino, alkyloxyphosphino, alkylsulfone or alkylsulfoxide; further, the substituted substituents are independently selected from halogen, cyano, amide, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3-C6 cycloalkyl containing at least one or more N, O, S, substituted or unsubstituted C1-C6 heteroalkyl containing at least one or more N, O, S, substituted or unsubstituted 3 to 8-membered heterocycloalkyl containing at least one or more N, O, S, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted C1-C6 trifluoroalkyl, substituted or unsubstituted C3-C6 cycloalkyloxy, or substituted or unsubstituted 3 to 8-membered heterocycloalkoxy;

[0015] A and B are independently selected from C, O, N, amide or S;

[0016] n is 0, 1, 2, 3 or 4;

[0017] m is 0, 1 or 2.

[0018] In the present invention, "selected from" is generally a parallel relationship of or, and the substitution can be monosubstituted or polysubstituted (for example, disubstituted, trisubstituted, tetrasubstituted), and the specific substitution position is not particularly limited. In the present invention, the unsubstituted saturated hydrocarbon group includes unsubstituted alkyl and unsubstituted cycloalkyl; the heterocyclic group, heteroaryl and other groups are groups in which one or more carbon atoms can be replaced by heteroatoms, and heteroatoms are atoms such as oxygen, sulfur, nitrogen, phosphorus, etc. other than carbon. In addition, the above-mentioned halogen includes fluorine, chlorine, bromine, etc., preferably fluorine or chlorine. The above-mentioned "C3-C10" is an integer with a carbon atom number selected from 3 to 10, and similar expressions are not repeated below.

[0019] Preferably, in the structure shown in Formula I, when m is 0, the compound as a BTK inhibitor has the structure shown in Formula II, or its tautomers, mesomers, racemates, enantiomers, diastereomers or mixtures thereof, pharmaceutically acceptable hydrates, solvates or salts:

[0020]

[0021] In formula II, the structures of R1, R2, and R3 are as described above;

[0022] n is 0, 1, 2, 3 or 4, preferably 0 or 1.

[0023] In a preferred embodiment of the present invention, the compound as a BTK inhibitor has a structure represented by Formula III, or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, a pharmaceutically acceptable hydrate, solvate, or salt thereof:

[0024]

[0025] In formula III, R1 is selected from substituted or unsubstituted C1-C8 alkyl, substituted or unsubstituted C3-C10 cycloalkyl, substituted or unsubstituted C1-C8 heteroalkyl, substituted or unsubstituted 3-10 membered heterocycloalkyl, or substituted or unsubstituted aryl or heteroaryl; further, R1 is selected from substituted or unsubstituted C1-C3 alkyl, substituted or unsubstituted C3-C6 cycloalkyl, or substituted or unsubstituted 3-6 membered heterocycloalkyl;

[0026] R4 is selected from hydrogen, halogen, hydroxy, cyano, amino, amide, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted C1-C6 heteroalkyl, substituted or unsubstituted 3- to 8-membered heterocycloalkyl, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted C3-C6 cycloalkyloxy, or substituted or unsubstituted 3- to 8-membered heterocycloalkoxy;

[0027] n is 0, 1, 2, 3 or 4.

[0028] In a preferred embodiment of the present invention, the compound as a BTK inhibitor has a structure shown in the following formula:

[0029]

[0030]

[0031]

[0032]

[0033] The present invention also provides a pharmaceutical composition, wherein the active ingredient of the pharmaceutical composition is selected from one or a combination of two or more of the compounds described in the above technical solutions, or their stereoisomers, solvates, hydrates, pharmaceutically acceptable salts, or cocrystals. Furthermore, the present invention has no particular limitations on the formulation type of the pharmaceutical composition.

[0034] The present invention also provides use of the compound described in the above technical solution or its stereoisomers, solvates, hydrates, pharmaceutically acceptable salts or cocrystals in the preparation of protein kinase inhibitors; further, the protein kinase inhibitor is a BTK inhibitor.

[0035] The present invention also provides use of the compound described in the above technical solution or its stereoisomers, solvates, hydrates, pharmaceutically acceptable salts or cocrystals in the preparation of drugs for treating diseases caused by overexpression of BTK kinase.

[0036] The present invention also provides the use of the compound described in the above technical solution or its stereoisomers, solvates, hydrates, pharmaceutically acceptable salts or cocrystals in the preparation of drugs for treating any one or more of autoimmune diseases, inflammatory diseases, thromboembolic diseases, allergies, infectious diseases, proliferative disorders and cancer.

[0037] Further, the disease can 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 vulgaris, chronic obstructive pulmonary disease, psoriasis, anxiety, post-traumatic stress disorder (PTSD), thrombocytopenia, autoimmune hemolytic anemia, atopic dermatitis, myasthenia gravis, lupus nephritis, lung disease and new coronavirus pneumonia, primary membranous nephropathy, cardiac thrombotic inflammatory disease, 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, hairy cell carcinoma, nasopharyngeal cancer, pharyngeal cancer, colorectal cancer, rectal cancer, brain and central nervous system cancer, cervical cancer, prostate cancer, testicular cancer, genitourinary 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, uterine corpus cancer, cervical cancer, multiple myeloma, acute myeloid leukemia, chronic myeloid leukemia, lymphocytic leukemia, chronic lymphoid leukemia, myeloid leukemia, non-Hodgkin's lymphoma, essential macroglobulinemia.

[0038] In the in vitro BTK kinase inhibition activity assay described in the examples of the present invention, the compound powder was dissolved in 100% DMSO to prepare a 10 mM stock solution. During the kinase reaction, the test compound was tested at a starting concentration of 1 μM, with the compound diluted 3-fold over 10 concentrations. The compounds described in the examples of the present invention were also tested for rat brain penetration. The compounds of the present invention, as BTK protein kinase inhibitors, exhibited excellent BTK kinase inhibitory activity and high rat blood-brain barrier penetration.

[0039] The present invention also provides a method for preparing the compound as a BTK inhibitor described in the above technical solution, comprising the following steps:

[0040] The starting material A of the following formula undergoes a substitution reaction with a halide R1X to obtain an intermediate B, and then the intermediate B is subjected to a Suzuki coupling reaction with a borate compound intermediate C to obtain compound D;

[0041]

[0042] The present invention designs and synthesizes multiple target compounds. The specific preparation process is shown in the above formula: a commercially available starting material A is reacted with a halide (R1X) to produce an intermediate B, which is then subjected to a Suzuki coupling reaction with a borate compound intermediate C to produce compound D (Example). The synthesis method of the present invention is simple and has a high yield.

[0043] The present invention provides a compound as a BTK inhibitor, a preparation method and use thereof; the compound as a BTK inhibitor has the structure shown in Formula I, or its tautomers, mesomers, racemates, enantiomers, diastereomers or mixtures thereof, pharmaceutically acceptable hydrates, solvates or salts; wherein R1 is selected from substituted or unsubstituted C1-C8 alkyl, substituted or unsubstituted C3-C10 cycloalkyl, substituted or unsubstituted C1-C8 heteroalkyl, substituted or unsubstituted 3 to 10 membered heterocycloalkyl, or substituted or unsubstituted aryl or heterocyclic aryl; R2 is selected from hydrogen, halogen, hydroxyl, cyano, amino, amide, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3- C6 cycloalkyl, substituted or unsubstituted C1-C6 heteroalkyl, substituted or unsubstituted 3- to 8-membered heterocycloalkyl, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted C3-C6 cycloalkyloxy, or substituted or unsubstituted 3- to 8-membered heterocycloalkoxy; R3 is selected from substituted or unsubstituted aryl, or substituted or unsubstituted pyridyl; wherein the substituted substituents are independently selected from halogen, hydroxy, amino, cyano, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkoxy, cycloalkyloxy, heterocycloalkoxy, amide, sulfonyl, phosphino, alkyloxyphosphino, alkylsulfone or alkylsulfoxide; A and B are independently selected from C, O, N, amide or S; n is 0, 1, 2, 3 or 4; m is 0, 1 or 2. The compounds provided by the present invention as BTK inhibitors are a class of compounds containing a 5-aminopyrazole-4-carboxamide structure, which can be used as BTK protein kinase inhibitors, have strong inhibitory activity against BTK, and have good pharmacokinetic properties and blood-brain barrier permeability, and have good application prospects. DETAILED DESCRIPTION

[0044] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0045] To further illustrate the present invention, the following examples provide a detailed description of the compounds provided by the present invention that can be used as BTK protein kinase inhibitors, their preparation methods, and uses. In the following examples of the present invention, the structures of the compounds are determined by mass spectrometry (MS) or nuclear magnetic resonance (NMR) 1 The term "room temperature" refers to 10°C to 30°C. Chemical abbreviations have the following meanings:

[0046] NBS: N-bromosuccinimide; DMF: N,N-dimethylformamide; PE: petroleum ether; EA: ethyl acetate; TFA: trifluoroacetic acid; TEA: triethylamine; DCE: 1,2-dichloroethane; TsCl: 4-toluenesulfonyl chloride; DCM: dichloromethane; MeOH: methanol; BAST: bis(2-methoxyethyl)aminosulfur trifluoride; DIEA: N,N-diisopropylethylamine; HATU: O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate; DMSO: dimethyl sulfoxide; PdCl2(dppf): [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride.

[0047] Preparation of intermediate A:

[0048]

[0049] A-1 (23.0 g, 213 mmol) and DMF (230 mL) were added to a reaction flask. The reaction solution was cooled in an ice bath and NBS (41.6 g, 234 mmol) was added portionwise. After the addition was complete, the reaction solution was warmed to room temperature and stirred overnight. The reaction solution was poured into water, and a small amount of sodium thiosulfate was added to remove the color. The solution was extracted three times with ethyl acetate. The organic phases were combined, evaporated in vacuo, and purified on a silica gel column (PE / EA = 1 / 1 to 1 / 2) to obtain 7.5 g of product A-2 as a light yellow solid. The yield was 19%.

[0050] A-2 (7.5 g, 40.1 mmol) and TFA (183 g, 1604 mmol) were added to a reaction flask. Concentrated sulfuric acid (39.3 g, 401 mmol) was added dropwise to the reaction solution, which was then heated to 50°C and stirred overnight. The reaction solution was evaporated in vacuo to remove TFA, and the residue was added dropwise to ice water and extracted with ethyl acetate (100 ml x 50). The organic phases were combined and evaporated in vacuo to obtain 7.6 g of product A as a light yellow solid (yield: 93%).

[0051] Preparation of intermediate B-1:

[0052]

[0053] B-1-1 (5.0 g, 43.8 mmol), TEA (5.3 g, 52.6 mmol), and DCE (50 mL) were added to the reaction flask. TsCl (9.2 g, 48.2 mmol) was added portionwise to the reaction solution. After the addition was complete, the reaction solution was heated to 60°C and stirred overnight. The reaction system was cooled to room temperature and directly added to silica gel for mixing. After vacuum evaporation, the sample was purified on a silica gel column (PE / EA = 50 / 1) to obtain 6.0 g of the product B-1-2 as a colorless oil, with a yield of 51%.

[0054] To the reaction flask were added B-1-2 (1.3 g, 4.88 mmol), A (500 mg, 2.44 mmol), K2CO3 (674 mg, 4.88 mmol), KI (486 mg, 2.93 mmol), and DMF (5 mL). The reaction solution was heated to 130°C and stirred for 24 hours. The reaction system was cooled to room temperature, filtered, and the filtrate was evaporated in vacuo to remove DMF. The residue was directly purified on a silica gel preparation plate (DCM / MeOH = 10 / 1) to give 180 mg of the product B-1 as a light yellow crude product in a yield of 25%.

[0055] Preparation of intermediate B-2:

[0056]

[0057] To a reaction flask were added B-2-1 (1215 mg, 7.32 mmol), A (1000 mg, 4.88 mmol), K2CO3 (1348 mg, 9.76 mmol), 18-crown-6 (1418 mg, 5.37 mmol), and DMF (10 mL). The reaction solution was heated to 100°C and stirred overnight. B-2-1 (800 mg, 4.82 mmol) and K2CO3 (800 mg, 5.79 mmol) were further added to the reaction solution, and the reaction was continued at 100°C for 7 hours. The reaction system was cooled, poured into water, and extracted six times with ethyl acetate. The organic phases were combined, dried over anhydrous Na2SO4, evaporated in vacuo, and purified on a silica gel column (PE / EA = 1 / 1 to 1 / 2) to give 640 mg of product B-2 as a light yellow oil in a yield of 53%.

[0058] The following compounds were synthesized by the method for preparing intermediate B-2, using commercially available corresponding raw materials instead of B-2-1, as shown in Table 1.

[0059] Table 1 Structure and synthesis of intermediates B-3 to B-8

[0060]

[0061] Preparation of intermediate B-9:

[0062]

[0063] The synthesis method of B-9 refers to the method for synthesizing B-1 from B-1-1.

[0064] Preparation of intermediate C-1:

[0065]

[0066] C-1-1 (10.0 g, 38.3 mmol) and BAST (35 mL) were added to a reaction tube, sealed, and heated to 90°C overnight. The reaction solution was cooled, poured into water, and extracted twice with ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, evaporated in vacuo, and purified on a silica gel column (PE) to obtain 9.9 g of the product C-1-2 as a colorless oil (yield: 91%).

[0067] C-1-2 (9.9 g, 34.97 mmol), dioxane (100 mL), bis-pinacol boronate (13.32 g, 52.45 mmol), and potassium acetate (10.3 g, 104.9 mmol) were added to a reaction flask. The reaction mixture was purged with nitrogen. PdCl2(dppf) (1.28 g, 1.75 mmol) was added to the reaction mixture, and the reaction mixture was purged with nitrogen again. The reaction mixture was heated to 90°C and stirred overnight. The reaction mixture was evaporated in vacuo to remove the dioxane, and dichloromethane was added to dissolve it. Silica gel was then added directly to the sample and the sample was purified on a silica gel column (PE-PE / EA = 70 / 1) to obtain 6.1 g of product C-1, with a yield of 53%.

[0068] Preparation of intermediate C-2:

[0069]

[0070] To a reaction flask, C-2-1 (1.00 g, 5.73 mmol), DMF (10 mL), dimethylhydroxylamine hydrochloride (671 mg, 6.87 mmol), and DIEA (2.96 g, 22.92 mmol) were added. HATU (3.27 g, 8.59 mmol) was added to the reaction mixture in one portion while stirring. The reaction mixture was stirred at room temperature overnight, poured into water, and extracted three times with ethyl acetate. The organic phases were combined, washed with saturated brine, evaporated in vacuo, and purified on a silica gel column (PE / EA = 2 / 1) to give 1.19 g of product C-2-2 as a yellow liquid in a 95% yield.

[0071] To the reaction flask, p-bromoiodobenzene (1.62 g, 5.74 mmol) and tetrahydrofuran (15 mL) were added, the reaction solution was nitrogen-purged, and the dry ice / ethanol bath was cooled to -70 ° C. n-Butyl lithium (2.5 M, 2.4 mL, 6.02 mmol) was added dropwise to the reaction solution. After stirring for 30 minutes, a solution of C-2-2 (1.19 g, 5.47 mmol) in tetrahydrofuran (3 mL) was added dropwise to the reaction solution. After the addition was complete, the reaction solution was slowly warmed to room temperature and reacted for 1 hour. The reaction solution was quenched with saturated aqueous ammonium chloride solution, extracted twice with ethyl acetate, the organic phases were combined, washed with saturated brine, evaporated in vacuo, and purified on a silica gel column (PE / EA=50 / 1) to give 1.2 g of product C-2-3 as a light yellow solid in a yield of 71%.

[0072] The synthesis method of C-2 refers to the method for synthesizing C-1 from C-1-1.

[0073] Preparation of intermediate C-3:

[0074]

[0075] C-3-1 (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 the reaction flask. The reaction solution was heated to 70°C and stirred overnight. The reaction solution was cooled, filtered, and washed with ethyl acetate. The filtrate was evaporated in vacuo and purified on a silica gel column to give 742 mg of product C-3-2 in a yield of 97%.

[0076] The synthesis method of C-3 refers to the method for synthesizing C-1 from C-1-2.

[0077] Preparation of intermediate C-4:

[0078]

[0079] The synthesis method of C-4-2 refers to the method for synthesizing C-2-2 from C-2-1.

[0080] C-4-2 (1.05 g, 3.83 mmol) and tetrahydrofuran (10 mL) were added to the reaction flask, the reaction solution was replaced with nitrogen, and the reaction solution was cooled in an ice-salt bath. Phenylmagnesium bromide in tetrahydrofuran (1 M, 4.6 mL, 4.6 mmol) was added dropwise to the reaction solution. After the addition was complete, the reaction solution was slowly warmed to room temperature and stirred for 1 hour. The reaction solution was quenched with saturated aqueous ammonium chloride solution and extracted twice with ethyl acetate. The organic phases were combined, washed with saturated brine, evaporated in vacuo, and purified on a silica gel column (PE / EA=20 / 1) to give 960 mg of the product C-4-3 as a white solid in a yield of 86%.

[0081] The synthesis method of C-4 refers to the method for synthesizing C-1 from C-1-1.

[0082] Preparation of intermediate C-5:

[0083]

[0084] To a reaction flask, C-5-1 (5.0 g, 24.87 mmol), cyclopropylboronic acid (4.3 g, 49.75 mmol), n-butyldi(1-adamantyl)phosphine (892 mg, 2.49 mmol), sodium carbonate (5.3 g, 49.75 mmol), and 1,4-dioxane / water (40 / 10 mL) were added and the atmosphere was purged with nitrogen three times. Palladium acetate (280 mg, 1.24 mmol) was added and the atmosphere was purged with nitrogen three more times. The reaction mixture was heated to 90°C and stirred overnight. The reaction mixture was cooled, diluted with water, and the acidity was adjusted with 1N HCl. The mixture was extracted three times with ethyl acetate. The organic phases were combined, washed with saturated brine, evaporated in vacuo, and purified on a silica gel column (PE / EA = 10 / 1) to give 3.7 g of product C-5-2 in a 92% yield.

[0085] The synthesis method of C-5 refers to the method for synthesizing C-2 from C-2-1.

[0086] Preparation of intermediate C-6:

[0087]

[0088] The synthesis method of C-6-4 refers to the method for synthesizing C-2-4 from C-2-1.

[0089] C-6-4 (120 mg, 0.39 mmol), potassium carbonate (81 mg, 0.58 mmol) and DMSO / water (2 / 0.2 mL) were added to the reaction flask, and hydrogen peroxide (30%, 88 mg, 0.78 mmol) was added with stirring. The reaction solution was stirred at room temperature for 2 hours and then added to water. A solid precipitated, which was filtered and dried to obtain 115 mg of product C-6-5 (yield: 90%).

[0090] The synthesis method of C-6 refers to the method for synthesizing C-1 from C-1-2.

[0091] Preparation of intermediate C-7:

[0092]

[0093] The synthesis method of C-7-2 refers to the method for synthesizing C-2-2 from C-2-1.

[0094] To the reaction flask, o-bromoiodobenzene (2.0 g, 7.07 mmol) and tetrahydrofuran (20 mL) were added, the reaction solution was nitrogen-purged, and the dry ice / ethanol bath was cooled to -70 ° C. n-Butyl lithium (2.5 M, 3 mL, 7.5 mmol) was added dropwise to the reaction solution. After stirring for 1 hour, a solution of dihydro-3 (2H) -furanone (608 mg, 7.07 mmol) in tetrahydrofuran (3 mL) was added dropwise to the reaction solution. After the addition was complete, the reaction solution was slowly warmed to room temperature and reacted for 1 hour. The reaction solution was quenched with saturated aqueous ammonium chloride solution and extracted twice with ethyl acetate. The organic phases were combined, washed with saturated brine, evaporated in vacuo, and purified on a silica gel column (PE / EA = 3 / 1) to obtain 1.2 g of product C-7-4 as a colorless solid in a yield of 71%.

[0095] C-7-4 (600 mg, 2.5 mmol), triethylsilane (2.3 g, 20 mmol), trifluoroacetic acid (3.1 g, 27.5 mmol) and dichloromethane (6 mL) were added to the reaction flask, replaced with nitrogen three times, cooled in an ice bath, and added with boron trifluoride etherate (3.5 mL). The reaction solution was slowly warmed to room temperature and stirred overnight. The reaction solution was poured into an aqueous sodium carbonate solution to quench the reaction, extracted three times with ethyl acetate, the organic phases were combined, washed with saturated brine, evaporated in vacuo, and purified on a silica gel column (PE / EA=50 / 1) to give 340 mg of product C-7-5, yield: 61%.

[0096] C-7-5 (340 mg, 1.53 mmol) and tetrahydrofuran (4 mL) were added to the reaction flask, the reaction solution was replaced with nitrogen, and the dry ice / ethanol bath was cooled to -70 ° C. n-Butyl lithium (2.5 M, 0.64 mL, 1.6 mmol) was added dropwise to the reaction solution. After stirring for 40 minutes, a solution of C-7-2 (373 mg, 1.53 mmol) in tetrahydrofuran (1.5 mL) was added dropwise to the reaction solution. After the addition was completed, the reaction solution was slowly warmed to room temperature and reacted for 1 hour. The reaction solution was quenched with saturated aqueous ammonium chloride solution and extracted twice with ethyl acetate. The organic phases were combined, washed with saturated brine, evaporated in vacuo, and purified on a silica gel column (PE / EA = 10 / 1) to give 300 mg of the product C-7-6 as a colorless solid in a yield of 57%.

[0097] The synthesis method of C-7 refers to the method for synthesizing C-1 from C-1-1.

[0098] Preparation of intermediate C-8:

[0099]

[0100] C-8-1 (1.0 g, 6.57 mmol), cyclopropane bromide (7.95 g, 65.7 mmol), cesium carbonate (6.41 g, 19.7 mmol), potassium iodide (1.1 g, 6.57 mmol) and DMF (100 mL) were added to the reaction flask, sealed, and heated to 130 ° C for 48 hours. The reaction solution was cooled, diluted with water, and washed with ethyl acetate. The aqueous phase was adjusted to acidity with dilute hydrochloric acid and extracted twice with ethyl acetate. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and evaporated in vacuo to obtain 1.23 g of product C-8-2 as a brown oil. The crude product was used directly in the next step without purification. It mainly contained o-hydroxybenzoic acid as an impurity.

[0101] The synthesis method of C-8 refers to the method for synthesizing C-2 from C-2-1.

[0102] Preparation of intermediate C-9:

[0103]

[0104] To a reaction flask, C-9-1 (400 mg, 1.86 mmol), potassium cyclopentane trifluoroborate (360 mg, 2.05 mmol), cesium carbonate (1.2 g, 3.72 mmol), n-butyldi(1-adamantyl)phosphine (67 mg, 0.186 mmol), palladium acetate (21 mg, 0.093 mmol), and toluene / water (5 / 1 mL) were added. The atmosphere was replaced with nitrogen and heated to 100°C for overnight reaction. The reaction solution was cooled, and silica gel was added directly to the sample, which was then dried and purified on a silica gel column (PE / EA = 30 / 1) to give 310 mg of the product C-9-2 as a yellow oil in an 82% yield.

[0105] C-9-2 (310 mg, 1.52 mmol) and methanol (3 mL) were added to a reaction flask, followed by a solution of sodium hydroxide (182 mg, 4.55 mmol) in water (3 mL). The mixture was heated to 50°C and reacted for 2 hours. The reaction solution was evaporated in vacuo to remove methanol, diluted with water, acidified with dilute hydrochloric acid, and extracted twice with ethyl acetate. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and evaporated in vacuo to give 290 mg of product C-9-3 as a brown solid. Yield: 100%.

[0106] The synthesis method of C-9 refers to the method for synthesizing C-2 from C-2-1.

[0107] Preparation of intermediate C-10:

[0108]

[0109] To a reaction flask, add C-8-1 (1.0 g, 6.57 mmol), bromomethylcyclopropane (1.07 g, 7.89 mmol), potassium carbonate (2.72 g, 19.7 mmol), potassium iodide (1.1 g, 6.57 mmol), and acetonitrile (10 mL). Heat to 80°C and react overnight. The reaction solution is cooled, diluted with water, and extracted twice with ethyl acetate. The organic phases are combined, washed with saturated brine, dried over anhydrous sodium sulfate, and evaporated in vacuo to give 1.34 g of product C-10-1 as a yellow-green oil (yield: 99%).

[0110] The synthesis method of C-10-2 refers to the method for synthesizing C-9-3 from C-9-2.

[0111] The synthesis method of C-10 refers to the method for synthesizing C-2 from C-2-1.

[0112] Preparation of intermediate C-11:

[0113]

[0114] C-11-1 (1.0 g, 5.78 mmol), 2-iodopropane (1.18 g, 6.57 mmol), potassium carbonate (1.04 g, 7.23 mmol), and DMF (10 mL) were added to a reaction flask and heated to 55°C overnight. The reaction solution was cooled, diluted with water, and extracted twice with ethyl acetate. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, evaporated in vacuo, and purified on a silica gel column to give 778 mg of the product C-11-2 as a colorless liquid in a yield of 63%.

[0115] The synthesis method of C-11 refers to the method for synthesizing C-7 from C-7-5.

[0116] The following compounds were synthesized by the methods for preparing the above intermediates using commercially available corresponding raw materials, as shown in Table 2.

[0117] Table 2 Structure and synthesis of intermediates C-12 to C-38

[0118]

[0119]

[0120]

[0121]

[0122] Example: Preparation of Compound 1

[0123] To a reaction flask were added B-1 (30 mg, 0.10 mmol), C-1 (50 mg, 0.15 mmol), NaCO (21 mg, 0.20 mmol), PdCl(dppf) (4 mg), dioxane (1 mL), and water (0.22 mL). The atmosphere was replaced with nitrogen and the temperature was raised to 90°C for overnight reaction. The reaction solution was directly purified by preparative silica gel (PE / EA = 1 / 3) to afford 11 mg of product 1, in a yield of 26%.

[0124] The product structure was characterized by nuclear magnetic resonance and mass spectrometry, and the results are as follows:

[0125] 1 H NMR (400MHz, d6-DMSO) δ1.61(3H,d,J=6.8Hz),5.25-5.36(1H,m),6.61(2H,s),7.48-7.53(3H,m),7.54-7.57(2H,m),7.61(1H,dd,J=19.2,8.5Hz).

[0126] MS (ESI) m / z (M+H)+ :425.1.

[0127] Examples 2-54: Preparation of Compounds 2-54

[0128] Compounds 2 to 54 were prepared by using different intermediates using the method for preparing compound 1. The structural formula, intermediate number, MS and 1 The H-NMR data are shown in Table 3.

[0129] Table 3 Structural formula, MS and 1H-NMR data of Examples 2 to 54

[0130]

[0131]

[0132]

[0133]

[0134]

[0135]

[0136]

[0137]

[0138] ND: Not tested.

[0139] Efficacy trials

[0140] Test Example 1: In vitro BTK inhibition kinase activity test

[0141] 1: Kinase reaction experimental steps

[0142] 1) Dilute the test compound (stock solution 10 mM) 10-fold with 100% DMSO, perform 3-fold equal dilutions in a 96-well dilution plate, add 1 μL of the compound to 39 μL of kinase reaction buffer, and shake on a microplate shaker for 20 minutes.

[0143] 2) Transfer 2 μL of kinase to a 384-well reaction plate, add 1 μL of the test compound to the 384-well reaction plate (Greiner, 784075), centrifuge at 1000 rpm / min for 1 min, and incubate at 25°C for 10 min.

[0144] 3) Transfer 2 μL of substrate mixture to a 384-well plate, centrifuge at 1000 rpm / min for 1 min, and incubate at 25°C for 60 min. Final concentrations of the test compounds in the reaction system were 1000, 333, 111, 37, 12, 4, 1, 0.5, 0.15, and 0.005 nM. The final DMSO concentration was 0.5%.

[0145] 4) Prepare a 2X Sa-XL 665 / TK-antibody-Cryptate mixture using HTRF detection buffer.

[0146] 5) Add 5 μL of Sa-XL 665 / TK-antibody-Cryptate to each well, centrifuge at 1000 rpm / min for 30 seconds, and react at room temperature for 1 hour.

[0147] 6) The fluorescence signals at 615 nm (Cryptate) and 665 nm (XL665) were read using Biotek.

[0148] 2: Experimental data processing method

[0149] Compound inhibition rate (%inh) = (negative control - compound) / (negative control - positive control) × 100%;

[0150] Negative control: DMSO;

[0151] Positive control: 1000 nM Ibrutinib;

[0152] The IC50 (half maximal inhibitory concentration) of the compound was obtained using the following nonlinear fitting formula:

[0153] Y=Bottom+(Top-Bottom) / [1+10^((LogIC 50 -X)×HillSlope)];

[0154] X: log value of compound concentration;

[0155] Y: compound inhibition rate (% inh).

[0156] The inhibitory activity of the compounds of the present invention on BTK kinase is shown in Table 4.

[0157] Table 4 Inhibitory activity of the compounds of the present invention on BTK kinase

[0158]

[0159]

[0160] Test Example 2: Blood-brain barrier permeability test

[0161] Pharmacokinetic studies were conducted on SD rats undergoing a single oral administration of each test compound at a dose of 10 mg / kg, with nine animals per group. The test compound was dissolved in 5% DMSO + 10% solutol + 85% saline, vortexed for 1-2 minutes, and sonicated for 5-10 minutes to prepare a colorless, transparent, and clear dosing solution. Animals were fasted overnight prior to dosing. Approximately 0.2-0.3 mL of blood was collected from three SD rats via orbital sampling 1, 2, and 4 hours after dosing. Blood samples were immediately placed on ice and centrifuged within 15 minutes to separate plasma (centrifugation conditions: 8000 rpm, 1 minute, room temperature). Collected plasma was stored at -20°C prior to analysis. Cerebrospinal fluid and brain tissue were immediately obtained following blood collection. Cerebrospinal fluid (CSF) was obtained by puncturing the dura mater under direct vision using a microinjector. After chloral hydrate anesthesia, the skull was immobilized, the dorsal hair was trimmed, and a 2-cm transverse incision was made along the line connecting the two ear roots. The muscle layer of the neck and skull base was bluntly scraped to expose the foramen magnum. Approximately 100 μl of CSF was collected using a 100 μl microinjector and stored at −20°C until analysis. The rats were immediately sacrificed and decapitated. The brain tissue was dissected, the surface capillaries stripped, and the tissue was weighed. Three times the volume of ice-cold saline was added and homogenized in a homogenizer for 1 min. The tissue was then stored at −20°C until analysis. Take 20 μl of plasma sample and brain homogenate sample respectively, add 200 μl of working internal standard solution (blank without internal standard and add the same volume of solvent), vortex mix for 1 min, centrifuge at 13500 rpm for 10 min, take 100 μl of supernatant, and analyze by LC-MS / MS injection. Take 20 μl of cerebrospinal fluid sample, add 60 μl of working internal standard solution (blank without internal standard and add the same volume of solvent), vortex mix for 1 min, centrifuge at 13500 rpm for 10 min, take 50 μl of supernatant, and analyze by LC-MS / MS injection. The blood-brain barrier permeability test results of some compounds of the present invention are shown in Table 5.

[0162] Table 5 Blood-brain barrier permeability test results of some compounds of the present invention

[0163]

[0164]

[0165] As shown in the above examples, the compounds of the present invention, as BTK protein kinase inhibitors, have a structure of Formula I, preferably Formula II or III; exhibit potent inhibitory effects against wild-type BTK; and demonstrate significantly higher plasma and brain tissue drug concentrations and blood-brain barrier permeability in rats than LOXO-305, a drug currently in Phase III clinical trials. These compounds can be used to prepare therapeutics for diseases caused by BTK kinase overexpression, including brain diseases.

[0166] The compounds described above in the present invention, or their stereoisomers, solvates, hydrates, pharmaceutically acceptable salts or cocrystals, can be used to prepare drugs for treating any one or more of autoimmune diseases, inflammatory diseases, thromboembolic diseases, allergies, infectious diseases, proliferative disorders and cancers, and are expected to provide new and effective treatment options.

[0167] The above description of the disclosed embodiments will enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is to be construed in the widest manner consistent with the principles and novel features disclosed herein.

Claims

1. A compound as a BTK inhibitor, characterized in that Having the structure shown in Formula III, or its tautomer, mesomer, racemate, enantiomer, diastereomer or mixture thereof, or pharmaceutically acceptable salt: Formula III; The formula III is selected from the following structures: 。 2. A compound as a BTK inhibitor, characterized in that Having the following structure, or a tautomer, mesomer, racemate, enantiomer, diastereomer or mixture thereof, or a pharmaceutically acceptable salt thereof: 。 3. A method for preparing the compound as a BTK inhibitor according to claim 1, characterized in that: The following steps are involved: The starting material A of the following formula undergoes a substitution reaction with a halide R1X to obtain an intermediate B, and then the intermediate B is subjected to a Suzuki coupling reaction with a borate compound intermediate C to obtain compound D; 。 4. A pharmaceutical composition, characterized in that The active ingredient of the pharmaceutical composition is selected from one or a combination of two or more of the compound or its stereoisomers and pharmaceutically acceptable salts according to claim 1 or 2.

5. Use of the compound according to claim 1 or 2, or a stereoisomer or pharmaceutically acceptable salt thereof, in the preparation of a protein kinase inhibitor; the protein kinase inhibitor is a BTK inhibitor.

6. Use of the compound according to claim 1 or 2, or a stereoisomer or pharmaceutically acceptable salt thereof, in the preparation of a medicament for treating diseases caused by overexpression of BTK kinase.

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