Substituted pyrazole compounds, compositions containing the same and uses thereof

Abstract

The present invention provides a substituted pyrazole compound and a composition comprising the compound and its use, wherein the compound is a compound represented by formula (I) or its tautomer, stereoisomer, prodrug, crystal form, pharmaceutically acceptable salt, hydrate or solvent compound. The compound of formula (I) can be used as a reversible Bruton's tyrosine kinase (BTK) inhibitor for treating diseases related to BTK, and has high selectivity and good pharmacokinetic properties.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and in particular, relates to a substituted pyrazole compound and a composition comprising the compound and its use. More specifically, the present invention relates to certain deuterium-substituted 5-amino-3-(4-(((5-fluoro-2-methoxybenzoyl)amino)methyl)phenyl)-1-(1,1,1-trifluoropropane-2-yl)-1H-pyrazole-4-carboxamide and its derivatives and their tautomers, stereoisomers, prodrugs, crystal forms, pharmaceutically acceptable salts, hydrates or solvates. These deuterium-substituted compounds and their compositions can be used as reversible Bruton's tyrosine kinase (BTK) inhibitors for the treatment of diseases associated with BTK, with high selectivity and good pharmacokinetic properties. Background Art

[0002] BTK is a member of the Src-related Tec family of cytoplasmic tyrosine kinases. BTK is a key kinase in the BCR signaling pathway. When the BCR signaling pathway is activated, the signal is transmitted through immunoglobulin λ (lgλ), inducing the phosphorylation of Src kinase family members, catalyzing the dual phosphorylation of Tyr551 and Tyr223 in BTK to activate BTK. Activated BTK promotes the phosphorylation of downstream phospholipase Cγ (phospholipase C gamma, PLCγ), and phosphorylated PLCγ hydrolyzes phosphatidylinositol 4,5-bisphosphate (phosphatidylinositol4,5-bisphosphate, PIP2) to generate inositol triphosphate (inositol triphosphate, IP3) and diacylglycerol (diacyl glycerol, DAG). IP3 promotes the release of intracellular calcium, and DAG cooperates with calcium ions to activate signaling pathways such as protein kinases C (MAPKs) and mammalian target of rapamycin (mTOR), thereby regulating the expression of genes and cytokines. On the other hand, BTK can activate IκB kinase, thereby promoting the phosphorylation of IκB and inducing its separation from nuclear factor kappa-B (NF-κB). NF-κB separated from IκB translocates into the nucleus due to the exposure of its nuclear localization sequence, and specifically binds to the NF-κB site on DNA to regulate cell function.

[0003] BTK is an essential component of normal and malignant B-cell receptor signaling. BTK plays a key role in the B-cell antigen receptor signaling pathway and is required for the development, activation, and survival of B cells. BTK is a validated molecular target found in many B-cell leukemias and lymphomas, including chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), Waldenstrom's macroglobulinemia (WM), and marginal zone lymphoma (MZL).

[0004] Some covalent BTK inhibitors, including ibrutinib, acalabrutinib, and zanubrutinib, all irreversibly bind to C481 in the kinase domain of the BTK protein through covalent binding, thereby inhibiting the activity of the BTK enzyme. After long-term use of these drugs, the amino acid residue C481 on BTK may mutate, making it impossible for the original inhibitor to form a covalent bond with BTK, greatly weakening the inhibitory activity on BTK, and thus causing the body to develop resistance to covalent BTK inhibitors.

[0005] Pirtobrutinib (chemical name: (S)-5-amino-3-(4-(((5-fluoro-2-methoxybenzoyl)amino)methyl)phenyl)-1-(1,1,1-trifluoropropan-2-yl)-1H-pyrazole-4-carboxamide, having the following structural formula) is an investigational, oral, highly selective, non-covalent BTK inhibitor that provides sustained high target coverage regardless of BTK turnover rate, maintains activity in the presence of C481 acquired resistance mutations, and avoids complications caused by kinase off-targets of covalent and investigational non-covalent BTK inhibitors.

[0006]

[0007] Poor absorption, distribution, metabolism and / or excretion (ADME) properties are known to be the main reason for the failure of many candidate drugs in clinical trials. Many drugs currently on the market also limit their scope of application due to poor ADME properties. Rapid drug metabolism will cause many drugs that could have been highly effective in treating diseases to be difficult to make into medicines due to excessively rapid metabolism and clearance from the body. Although frequent or high-dose medication may solve the problem of rapid drug clearance, this method will bring about problems such as poor patient compliance, side effects caused by high-dose medication, and rising treatment costs. In addition, rapidly metabolized drugs may also expose patients to adverse toxic or reactive metabolites.

[0008] It is still a challenging task to find novel and effective non-covalent BTK inhibitors with good oral bioavailability and druggability. Therefore, there is still a need in the art to develop compounds suitable for the next generation of non-covalent BTK inhibitors with selective inhibitory activity and / or better pharmacodynamics / pharmacokinetics, and the present invention provides such compounds. Summary of the invention

[0009] In view of the above technical problems, the present invention discloses a novel deuterium-substituted pyrazole compound as an effective next-generation reversible non-covalent BTK inhibitor, whose inhibitory activity against BTK and C481 mutant BTK reaches the nanomolar level, which is significantly higher than other kinases (such as EGFR), which ensures that the compound of the present invention has high activity and high selectivity against BTK. In addition, the compound of the present invention also shows better metabolic stability and / or pharmacokinetic properties.

[0010] To this end, the present invention adopts the following technical solutions:

[0011] The first aspect of the present invention provides a compound of formula (I):

[0012]

[0013] in,

[0014] Y1, Y2, Y3, Y4, Y5, Y6 and Y7 are each independently selected from hydrogen, deuterium, halogen or trifluoromethyl;

[0015] R1, R2 and R3 are each independently selected from hydrogen or deuterium;

[0016] X1 and X2 are each independently selected from CH3, CD3, CHD2 or CH2D;

[0017] Provided that the above compound contains at least one deuterium atom;

[0018] or a tautomer, stereoisomer, prodrug, crystal form, pharmaceutically acceptable salt, hydrate or solvate thereof.

[0019] On the other hand, the present invention provides a pharmaceutical composition containing a compound of the present invention or its tautomer, stereoisomer, prodrug, crystal form, pharmaceutically acceptable salt, hydrate or solvent compound and a pharmaceutically acceptable excipient. In a specific embodiment, the compound of the present invention is provided in the pharmaceutical composition in an effective amount. In a specific embodiment, the compound of the present invention is provided in a therapeutically effective amount. In a specific embodiment, the compound of the present invention is provided in a preventive effective amount. In a specific embodiment, the pharmaceutical composition also includes another therapeutic agent.

[0020] On the other hand, the present invention provides a method for preparing the pharmaceutical composition as described above, comprising the following steps: mixing a pharmaceutically acceptable excipient with the compound of the present invention or its tautomer, stereoisomer, prodrug, crystalline form, pharmaceutically acceptable salt, hydrate or solvate to form a pharmaceutical composition.

[0021] In another aspect, the present invention provides the use of a compound of the present invention or its tautomer, stereoisomer, prodrug, crystal form, pharmaceutically acceptable salt, hydrate or solvate, or the above-mentioned pharmaceutical composition in the preparation of a medicament for treating and / or preventing a disease associated with BTK.

[0022] In another aspect, the present invention further provides a method for treating and / or preventing a disease associated with BTK, which comprises administering to a subject in need thereof a therapeutically effective amount of a compound of the present invention or its tautomer, stereoisomer, prodrug, crystalline form, pharmaceutically acceptable salt, hydrate or solvate, or a pharmaceutical composition of the present invention.

[0023] In a specific embodiment, the BTK includes wild-type BTK and mutant BTK; preferably, the mutant BTK is selected from BTK C481S, BTK C481F, BTK C481Y, BTK C481R, BTK C481T, BTK C481G or BTK C481W; more preferably, the mutant BTK is selected from BTK C481S.

[0024] In a specific embodiment, the BTK-related disease is selected from cancer, inflammatory conditions, immune diseases or fibrosis. In a specific embodiment, the cancer is selected from hematological cancer. In a specific embodiment, hematological cancer is selected from acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), acute promyelocytic leukemia (APL), chronic lymphocytic lymphoma (CLL), chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML), chronic neutrophilic leukemia (CNL), acute undifferentiated leukemia (AUL), anaplastic large cell lymphoma (ALCL), prolymphocytic leukemia (PML), juvenile myelomonocytic leukemia (JMML), adult T-cell leukemia (ALL), acute myeloid leukemia ( In some embodiments, the cancer is selected from glioma, leukemia, myelodysplastic syndrome, myeloproliferative disease, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone lymphoma (e.g., splenic marginal zone lymphoma, extranodal marginal zone B-cell lymphoma), Burkitt's lymphoma, Waldenstrom's macroglobulinemia (lymphoplasmacytic lymphoma), primary central nervous system lymphoma, small lymphocytic lymphoma, precursor B-cell lymphoblastic leukemia, hairy cell leukemia, mucosa-associated lymphoid tissue lymphoma, plasma cell myeloma, plasmacytoma, and multiple myeloma. In a specific embodiment, the cancer is selected from glioma. In a specific embodiment, the immune disease is selected from arthritis, multiple sclerosis or osteoporosis.

[0025] Other objects and advantages of the present invention will be apparent to those skilled in the art from the following detailed description, examples and claims.

[0026] definition

[0027] Herein, unless otherwise specified, "deuterated" means that one or more hydrogen atoms in a compound or group are replaced by deuterium; deuterated can be monosubstituted, disubstituted, polysubstituted or fully substituted. The term "one or more deuterated" and "one or more deuterated" can be used interchangeably.

[0028] As used herein, unless otherwise specified, a "non-deuterated compound" refers to a compound containing deuterium atoms at a ratio not higher than the natural deuterium isotope content (0.015%).

[0029] As used herein, the term "subject" includes, but is not limited to, humans (i.e., males or females of any age group, e.g., pediatric subjects (e.g., infants, children, adolescents) or adult subjects (e.g., young adults, middle-aged adults, or older adults)) and / or non-human animals, e.g., mammals, e.g., primates (e.g., cynomolgus monkeys, rhesus monkeys), cattle, pigs, horses, sheep, goats, rodents, cats, and / or dogs. In some embodiments, the subject is a human. In other embodiments, the subject is a non-human animal.

[0030] "Disease," "disorder," and "condition" are used interchangeably herein.

[0031] As used herein, and unless otherwise indicated, the terms "treat," "treatment," "treating ...

[0032] Generally, an "effective amount" of a compound refers to an amount sufficient to cause a target biological response. As will be appreciated by those of ordinary skill in the art, the effective amount of the compounds of the invention may vary depending on factors such as the biological target, the pharmacokinetics of the compound, the disease being treated, the mode of administration, and the age, health, and symptoms of the subject. An effective amount includes both therapeutic and prophylactic treatment effective amounts.

[0033] Unless otherwise specified, a "therapeutically effective amount" of a compound as used herein is an amount sufficient to provide a therapeutic benefit in the treatment of a disease, disorder, or condition, or to delay or minimize one or more symptoms associated with the disease, disorder, or condition. A therapeutically effective amount of a compound refers to an amount of a therapeutic agent, used alone or in combination with other therapies, that provides a therapeutic benefit in the treatment of a disease, disorder, or condition. The term "therapeutically effective amount" may include an amount that improves overall treatment, reduces or avoids symptoms or causes of a disease or condition, or enhances the therapeutic efficacy of other therapeutic agents.

[0034] Unless otherwise specified, a "prophylactically effective amount" of a compound as used herein is an amount sufficient to prevent a disease, disorder, or condition, or an amount sufficient to prevent one or more symptoms associated with a disease, disorder, or condition, or an amount to prevent the recurrence of a disease, disorder, or condition. A prophylactically effective amount of a compound refers to an amount of a therapeutic agent used alone or in combination with other agents that provides a prophylactic benefit in the process of preventing a disease, disorder, or condition. The term "prophylactically effective amount" may include an amount that improves overall prevention, or an amount that enhances the prophylactic efficacy of other prophylactic agents.

[0035] "Combination" and related terms refer to simultaneous or sequential administration of therapeutic agents of the invention. For example, a compound of the invention can be administered simultaneously or sequentially with another therapeutic agent in separate unit dosage forms, or simultaneously with another therapeutic agent in a single unit dosage form. DETAILED DESCRIPTION

[0036] Compound

[0037] As used herein, "compounds of the present invention" refers to compounds of the following formula (I)-(II) and subsets thereof (e.g., compounds of formula (IA), (IB)), or tautomers, stereoisomers, prodrugs, crystal forms, pharmaceutically acceptable salts, hydrates or solvates thereof.

[0038] In one embodiment, the present invention relates to compounds of formula (I):

[0039]

[0040] in,

[0041] Y1, Y2, Y3, Y4, Y5, Y6 and Y7 are each independently selected from hydrogen, deuterium, halogen or trifluoromethyl;

[0042] R1, R2 and R3 are each independently selected from hydrogen or deuterium;

[0043] X1 and X2 are each independently selected from CH3, CD3, CHD2 or CH2D;

[0044] Provided that the above compound contains at least one deuterium atom;

[0045] or a tautomer, stereoisomer, prodrug, crystal form, pharmaceutically acceptable salt, hydrate or solvate thereof.

[0046] In a specific embodiment, the deuterium isotope content of deuterium at the deuterated position is at least 0.015% greater than the natural deuterium isotope content, preferably greater than 30%, more preferably greater than 50%, more preferably greater than 55%, more preferably greater than 60%, more preferably greater than 65%, more preferably greater than 70%, more preferably greater than 75%, more preferably greater than 80%, more preferably greater than 85%, more preferably greater than 90%, more preferably greater than 95%, and more preferably greater than 99%.

[0047] Specifically, in the present invention, the deuterium isotope content of each deuterated position of Y1, Y2, Y3, Y4, Y5, Y6, Y7, R1, R2, R3, X1 and X2 is at least greater than the natural isotope content by 0.015%, more preferably greater than 1%, more preferably greater than 5%, more preferably greater than 10%, more preferably greater than 15%, more preferably greater than 20%, more preferably greater than 25%, more preferably greater than 30%, more preferably greater than 35%, more preferably greater than 40%, more preferably greater than 45%, more preferably greater than 50%, more preferably greater than 55%, more preferably greater than 60%, more preferably greater than 65%, more preferably greater than 70%, more preferably greater than 75%, more preferably greater than 80%, more preferably greater than 85%, more preferably greater than 90%, more preferably greater than 95%, more preferably greater than 99%.

[0048] In another specific embodiment, the compound of the present invention contains at least one deuterium atom, more preferably two deuterium atoms, more preferably three deuterium atoms, more preferably four deuterium atoms, more preferably five deuterium atoms, more preferably six deuterium atoms, more preferably seven deuterium atoms, more preferably eight deuterium atoms, more preferably nine deuterium atoms, more preferably ten deuterium atoms, more preferably eleven deuterium atoms, more preferably twelve deuterium atoms, more preferably thirteen deuterium atoms, more preferably fourteen deuterium atoms, more preferably fifteen deuterium atoms, more preferably sixteen deuterium atoms.

[0049] In another specific embodiment, "Y1, Y2, Y3, Y4, Y5, Y6 and Y7 are each independently selected from hydrogen, deuterium, halogen or trifluoromethyl" includes the technical scheme that Y1 is selected from hydrogen, deuterium, halogen or trifluoromethyl, Y2 is selected from hydrogen, deuterium, halogen or trifluoromethyl, Y3 is selected from hydrogen, deuterium, halogen or trifluoromethyl, and so on, until Y7 is selected from hydrogen, deuterium, halogen or trifluoromethyl. More specifically, it includes the technical scheme that Y1 is hydrogen, Y1 is deuterium, Y1 is halogen (F, Cl, Br or I) or Y1 is trifluoromethyl, Y2 is hydrogen, Y2 is deuterium, Y2 is halogen (F, Cl, Br or I) or Y2 is trifluoromethyl, Y3 is hydrogen, Y3 is deuterium, Y3 is halogen (F, Cl, Br or I) or Y3 is trifluoromethyl, and so on, until Y7 is hydrogen, Y7 is deuterium, Y7 is halogen (F, Cl, Br or I) or Y7 is trifluoromethyl.

[0050] In another specific embodiment, "R1, R2 and R3 are each independently selected from hydrogen or deuterium" includes the technical solution that R1 is selected from hydrogen or deuterium, R2 is selected from hydrogen or deuterium, and R3 is selected from hydrogen or deuterium. More specifically, it includes the technical solution that R1 is hydrogen or R1 is deuterium, R2 is hydrogen or R2 is deuterium, and R3 is hydrogen or R3 is deuterium.

[0051] In another specific embodiment, "X1 and X2 are each independently selected from CH3, CD3, CHD2 or CH2D" includes the technical scheme that X1 is selected from CH3, CD3, CHD2 or CH2D, and X2 is selected from CH3, CD3, CHD2 or CH2D. More specifically, it includes the technical scheme that X1 is CH3, X1 is CD3, X1 is CHD2 or X1 is CH2D, and X2 is CH3, X2 is CD3, X2 is CHD2 or X2 is CH2D.

[0052] In another embodiment, the present invention is directed to compounds of formula (IA):

[0053]

[0054] in,

[0055] Y1, Y2, Y3, Y4, Y5, Y6 and Y7 are each independently selected from hydrogen, deuterium, halogen or trifluoromethyl;

[0056] R1, R2 and R3 are each independently selected from hydrogen or deuterium;

[0057] X1 and X2 are each independently selected from CH3, CD3, CHD2 or CH2D;

[0058] Provided that the above compound contains at least one deuterium atom;

[0059] or a tautomer, stereoisomer, prodrug, crystal form, pharmaceutically acceptable salt, hydrate or solvate thereof.

[0060] In another embodiment, the present invention is directed to compounds of formula (IB):

[0061]

[0062] in,

[0063] Y1, Y2, Y3, Y4, Y5, Y6 and Y7 are each independently selected from hydrogen, deuterium, halogen or trifluoromethyl;

[0064] R1, R2 and R3 are each independently selected from hydrogen or deuterium;

[0065] X1 and X2 are each independently selected from CH3, CD3, CHD2 or CH2D;

[0066] Provided that the above compound contains at least one deuterium atom;

[0067] or a tautomer, stereoisomer, prodrug, crystal form, pharmaceutically acceptable salt, hydrate or solvate thereof.

[0068] In some embodiments of general formula (I), formula (IA) and formula (IB), preferably, the present invention relates to the above-mentioned compounds, or their tautomers, stereoisomers, prodrugs, crystal forms, pharmaceutically acceptable salts, hydrates or solvates, wherein X1 is CD3, and Y1, Y2, Y3, Y4, Y5, Y6, Y7, R1, R2, R3 and X2 are as defined above.

[0069] In some embodiments of general formula (I), formula (IA) and formula (IB), preferably, the present invention relates to the above-mentioned compounds, or their tautomers, stereoisomers, prodrugs, crystal forms, pharmaceutically acceptable salts, hydrates or solvates, wherein X2 is CD3, Y1, Y2, Y3, Y4, Y5, Y6, Y7, R1, R2, R3 and X1 are as defined above.

[0070] In some embodiments of general formula (I), formula (IA) and formula (IB), preferably, the present invention relates to the above-mentioned compounds, or their tautomers, stereoisomers, prodrugs, crystal forms, pharmaceutically acceptable salts, hydrates or solvates, wherein X1 and X2 are CD3, and Y1, Y2, Y3, Y4, Y5, Y6, Y7, R1, R2 and R3 are as defined above.

[0071] In some embodiments of general formula (I), formula (IA) and formula (IB), preferably, the present invention relates to the above-mentioned compounds, or their tautomers, stereoisomers, prodrugs, crystal forms, pharmaceutically acceptable salts, hydrates or solvates, wherein R1 is deuterium, and Y1, Y2, Y3, Y4, Y5, Y6, Y7, R2, R3, X1 and X2 are as defined above.

[0072] In some embodiments of general formula (I), formula (IA) and formula (IB), preferably, the present invention relates to the above-mentioned compounds, or their tautomers, stereoisomers, prodrugs, crystal forms, pharmaceutically acceptable salts, hydrates or solvates, wherein R1 is deuterium, X1 is CD3, and Y1, Y2, Y3, Y4, Y5, Y6, Y7, R2, R3 and X2 are as defined above.

[0073] In some embodiments of general formula (I), formula (IA) and formula (IB), preferably, the present invention relates to the above-mentioned compounds, or their tautomers, stereoisomers, prodrugs, crystal forms, pharmaceutically acceptable salts, hydrates or solvates, wherein R2 is deuterium, and Y1, Y2, Y3, Y4, Y5, Y6, Y7, R1, R3, X1 and X2 are as defined above.

[0074] In some embodiments of general formula (I), formula (IA) and formula (IB), preferably, the present invention relates to the above-mentioned compounds, or their tautomers, stereoisomers, prodrugs, crystal forms, pharmaceutically acceptable salts, hydrates or solvates, wherein R3 is deuterium, and Y1, Y2, Y3, Y4, Y5, Y6, Y7, R1, R2, X1 and X2 are as defined above.

[0075] In some embodiments of general formula (I), formula (IA) and formula (IB), preferably, the present invention relates to the above-mentioned compounds, or their tautomers, stereoisomers, prodrugs, crystal forms, pharmaceutically acceptable salts, hydrates or solvates, wherein R2 and R3 are deuterium, and Y1, Y2, Y3, Y4, Y5, Y6, Y7, R1, X1 and X2 are as defined above.

[0076] In another embodiment, the present invention relates to compounds of formula (II):

[0077]

[0078] in,

[0079] R1, R2 and R3 are each independently selected from hydrogen or deuterium;

[0080] X1 and X2 are each independently selected from CH3, CD3, CHD2 or CH2D;

[0081] Provided that the above compound contains at least one deuterium atom;

[0082] or a tautomer, stereoisomer, prodrug, crystal form, pharmaceutically acceptable salt, hydrate or solvate thereof.

[0083] In another embodiment, the present invention is directed to compounds of formula (IIA):

[0084]

[0085] in,

[0086] R1, R2 and R3 are each independently selected from hydrogen or deuterium;

[0087] X1 and X2 are each independently selected from CH3, CD3, CHD2 or CH2D;

[0088] Provided that the above compound contains at least one deuterium atom;

[0089] or a tautomer, stereoisomer, prodrug, crystal form, pharmaceutically acceptable salt, hydrate or solvate thereof.

[0090] In another embodiment, the present invention is directed to compounds of formula (IIB):

[0091]

[0092] in,

[0093] R1, R2 and R3 are each independently selected from hydrogen or deuterium;

[0094] X1 and X2 are each independently selected from CH3, CD3, CHD2 or CH2D;

[0095] Provided that the above compound contains at least one deuterium atom;

[0096] or a tautomer, stereoisomer, prodrug, crystal form, pharmaceutically acceptable salt, hydrate or solvate thereof.

[0097] In some embodiments of general formula (II), formula (IIA) and formula (IIB), preferably, the present invention relates to the above-mentioned compounds, or their tautomers, stereoisomers, prodrugs, crystal forms, pharmaceutically acceptable salts, hydrates or solvates, wherein X1 is CD3, R1, R2, R3 and X2 are as defined above.

[0098] In some embodiments of general formula (II), formula (IIA) and formula (IIB), preferably, the present invention relates to the above-mentioned compounds, or their tautomers, stereoisomers, prodrugs, crystal forms, pharmaceutically acceptable salts, hydrates or solvates, wherein X2 is CD3, R1, R2, R3 and X1 are as defined above.

[0099] In some embodiments of general formula (II), formula (IIA) and formula (IIB), preferably, the present invention relates to the above-mentioned compounds, or their tautomers, stereoisomers, prodrugs, crystal forms, pharmaceutically acceptable salts, hydrates or solvates, wherein X1 and X2 are CD3, and R1, R2 and R3 are as defined above.

[0100] In some embodiments of general formula (II), formula (IIA) and formula (IIB), preferably, the present invention relates to the above-mentioned compounds, or their tautomers, stereoisomers, prodrugs, crystal forms, pharmaceutically acceptable salts, hydrates or solvates, wherein R1 is deuterium, and R2, R3, X1 and X2 are as defined above.

[0101] In some embodiments of general formula (II), formula (IIA) and formula (IIB), preferably, the present invention relates to the above-mentioned compounds, or their tautomers, stereoisomers, prodrugs, crystal forms, pharmaceutically acceptable salts, hydrates or solvates, wherein R1 is deuterium, X1 is CD3, and R2, R3 and X2 are as defined above.

[0102] In some embodiments of general formula (II), formula (IIA) and formula (IIB), preferably, the present invention relates to the above-mentioned compounds, or their tautomers, stereoisomers, prodrugs, crystal forms, pharmaceutically acceptable salts, hydrates or solvates, wherein R1 is deuterium, X2 is CD3, and R2, R3 and X1 are as defined above.

[0103] In some embodiments of general formula (II), formula (IIA) and formula (IIB), preferably, the present invention relates to the above-mentioned compounds, or their tautomers, stereoisomers, prodrugs, crystal forms, pharmaceutically acceptable salts, hydrates or solvates, wherein R1 is deuterium, X1 and X2 are CD3, and R2 and R3 are as defined above.

[0104] In some embodiments of general formula (II), formula (IIA) and formula (IIB), preferably, the present invention relates to the above-mentioned compounds, or their tautomers, stereoisomers, prodrugs, crystal forms, pharmaceutically acceptable salts, hydrates or solvates, wherein R2 is deuterium, and R1, R3, X1 and X2 are as defined above.

[0105] In some embodiments of general formula (II), formula (IIA) and formula (IIB), preferably, the present invention relates to the above-mentioned compounds, or their tautomers, stereoisomers, prodrugs, crystal forms, pharmaceutically acceptable salts, hydrates or solvates, wherein R3 is deuterium, and R1, R2, X1 and X2 are as defined above.

[0106] In some embodiments of general formula (II), formula (IIA) and formula (IIB), preferably, the present invention relates to the above-mentioned compounds, or their tautomers, stereoisomers, prodrugs, crystal forms, pharmaceutically acceptable salts, hydrates or solvates, wherein R2 and R3 are deuterium, and R1, X1 and X2 are as defined above.

[0107] In some embodiments of general formula (II), formula (IIA) and formula (IIB), preferably, the present invention relates to the above-mentioned compounds, or their tautomers, stereoisomers, prodrugs, crystal forms, pharmaceutically acceptable salts, hydrates or solvates, wherein X1 is CD3, R2 and R3 are deuterium, and R1 and X2 are as defined above.

[0108] In some embodiments of general formula (II), formula (IIA) and formula (IIB), preferably, the present invention relates to the above-mentioned compounds, or their tautomers, stereoisomers, prodrugs, crystal forms, pharmaceutically acceptable salts, hydrates or solvates, wherein X2 is CD3, R2 and R3 are deuterium, and R1 and X1 are as defined above.

[0109] As a preferred embodiment of the present invention, the compound, or its tautomer, stereoisomer, prodrug, crystal form, pharmaceutically acceptable salt, hydrate or solvate, is selected from any of the following compounds:

[0110]

[0111]

[0112]

[0113]

[0114]

[0115]

[0116]

[0117]

[0118]

[0119]

[0120]

[0121] The compounds of the present invention may include one or more asymmetric centers and may therefore exist in a variety of stereoisomeric forms, for example, enantiomers and / or diastereoisomeric forms. For example, the compounds of the present invention may be individual enantiomers, diastereomers or geometric isomers (e.g., cis and trans isomers), or may be in the form of a mixture of stereoisomers, including racemic mixtures and mixtures enriched in one or more stereoisomers. Isomers may be separated from the mixture by methods known to those skilled in the art, including chiral high pressure liquid chromatography (HPLC) and the formation and crystallization of chiral salts; or preferred isomers may be prepared by asymmetric synthesis.

[0122] Those skilled in the art will appreciate that organic compounds can form complexes with solvents in which they react or from which they precipitate or crystallize. These complexes are referred to as "solvates". When the solvent is water, the complex is referred to as a "hydrate". The present invention encompasses all solvates of the compounds of the present invention.

[0123] The term "solvate" refers to a form of a compound or a salt thereof that is combined with a solvent, usually formed by a solvolysis reaction. This physical association may include hydrogen bonding. Conventional solvents include water, methanol, ethanol, acetic acid, DMSO, THF, ether, etc. The compounds described herein can be prepared, for example, in crystalline form and can be solvated. Suitable solvates include pharmaceutically acceptable solvates and further include stoichiometric solvates and non-stoichiometric solvates. In some cases, the solvate will be able to separate, for example, when one or more solvent molecules are incorporated into the crystal lattice of the crystalline solid. "Solvate" includes solvates in solution and separable solvates. Representative solvates include hydrates, ethanolates and methanolates.

[0124] The term "hydrate" refers to a compound that is combined with water. Generally, the ratio of the number of water molecules contained in the hydrate of a compound to the number of molecules of the compound in the hydrate is determined. Therefore, the hydrate of a compound can be represented by, for example, the general formula R·xH2O, where R is the compound, and x is a number greater than 0. A given compound can form more than one type of hydrate, including, for example, a monohydrate (x is 1), a lower hydrate (x is a number greater than 0 and less than 1, for example, a hemihydrate (R·0.5H2O)) and a polyhydrate (x is a number greater than 1, for example, a dihydrate (R·2H2O) and a hexahydrate (R·6H2O)).

[0125] The compounds of the present invention can be in amorphous or crystalline form (polymorph). In addition, the compounds of the present invention can exist in one or more crystalline forms. Therefore, the present invention includes all amorphous or crystalline forms of the compounds of the present invention within its scope. The term "polymorph" refers to the crystalline form (or its salt, hydrate or solvate) of a compound with a specific crystal packing arrangement. All polymorphs have the same elemental composition. Different crystalline forms usually have different X-ray diffraction patterns, infrared spectra, melting points, densities, hardnesses, crystal shapes, photoelectric properties, stability and solubility. Recrystallization solvents, crystallization rates, storage temperatures and other factors can lead to a dominant crystalline form. The various polymorphs of a compound can be prepared by crystallization under different conditions.

[0126] The present invention also includes isotopically labeled compounds which are identical to those of the present invention but for which one or more atoms have been replaced by atoms having an atomic mass or mass number different from the atomic mass or mass number normally found in nature. Examples of isotopes that can be introduced into the present compounds include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine and chlorine, e.g. 2 H. 3 H. 13 C. 11 C. 14 C. 15 N.18 O. 17 O. 31 P. 32 P. 35 S. 18 F and 36 Cl. Compounds of the present invention containing the above isotopes and / or other isotopes of other atoms, their prodrugs and pharmaceutically acceptable salts of the compounds or prodrugs are within the scope of the present invention. Certain isotopically labeled compounds of the present invention, such as those in which a radioactive isotope (e.g. 3 H and 14 C) can be used for drug and / or substrate tissue distribution assays. 3 H and carbon-14, i.e. 14 C isotopes are particularly preferred because they are easy to prepare and detect. 2 H, because higher metabolic stability can provide therapeutic benefits, such as prolonged half-life in vivo or reduced dosage requirements, and thus may be preferred in some cases. Isotope-labeled compounds of formula (I) of the present invention and their prodrugs can generally be prepared by replacing non-isotope-labeled reagents with readily available isotope-labeled reagents when carrying out the processes disclosed in the following schemes and / or Examples and Preparation Examples.

[0127] In addition, prodrugs are also included in the context of the present invention. The term "prodrug" as used herein refers to a compound that is converted into its active form having a medical effect in vivo by, for example, hydrolysis in the blood. Pharmaceutically acceptable prodrugs are described in T. Higuchi and V. Stella, Prodrugs as Novel Delivery Systems, Vol. 14 of the ACSSymposium Series, Edward B. Roche, ed., Bioreversible Carriers in Drug Design, American Pharmaceutical Association and Pergamon Press, 1987, and D. Fleisher, S. Ramon and H. Barbra "Improved oral drug delivery: solubility limitations overcome by the use of prodrugs", Advanced Drug Delivery Reviews (1996) 19 (2) 115-130, each of which is incorporated herein by reference.

[0128] Prodrugs are any covalently bonded compounds of the present invention that release the parent compound in vivo when such prodrugs are administered to a patient. Prodrugs are usually prepared by modifying functional groups, and the modification is carried out in a manner that allows the modification to be cleaved to produce the parent compound by conventional manipulation or in vivo. Prodrugs include, for example, compounds of the present invention in which hydroxyl, amino or sulfhydryl groups are bonded to any group, which can be cleaved to form hydroxyl, amino or sulfhydryl groups when administered to a patient. Therefore, representative examples of prodrugs include, but are not limited to, acetate / amide, formate / amide and benzoate / amide derivatives of hydroxyl, sulfhydryl and amino functional groups of compounds of formula (I). In addition, in the case of carboxylic acid (-COOH), esters such as methyl esters, ethyl esters, etc. can be used. Esters themselves can be active and / or can be hydrolyzed under human body conditions. Suitable pharmaceutically acceptable in vivo hydrolyzable ester groups include those groups that are easily decomposed in the human body to release the parent acid or its salt.

[0129] Methods for preparing the compounds of the present invention

[0130] The compounds of the present invention (including salts thereof) can be prepared using known organic synthesis techniques and can be synthesized according to any of a variety of possible synthetic pathways (such as those in the scheme below). The reaction for preparing the compounds of the present invention can be carried out in a suitable solvent, and a technician in the field of organic synthesis can easily select a solvent. A suitable solvent can be substantially unreactive with a starting material (reactant), an intermediate or a product at a temperature for carrying out the reaction (e.g., a temperature within the range of the solvent freezing temperature to the solvent boiling temperature). A given reaction can be carried out in a solvent or a mixture of more than one solvent. A technician can select a solvent for a specific reaction step according to the specific reaction step.

[0131] The preparation of the compounds of the present invention may involve the protection and removal of different chemical groups. One skilled in the art can easily determine whether protection and removal of protection and the selection of appropriate protecting groups are required. The chemical properties of protecting groups can be found, for example, in Wuts and Greene, Protective Groups in Organic Synthesis, 4th Edition, John Wiley & Sons: New Jersey, (2006), which is incorporated herein by reference in its entirety.

[0132] The compounds of the present invention can be reacted with an optically active resolving agent to form a pair of diastereomeric compounds, separate diastereoisomers and recycle the enantiomers of optical purity to prepare their single stereoisomers. The diastereomeric derivatives of the compounds of the present invention can be used to carry out the separation of enantiomers, preferably dissociable complexes (e.g., crystalline diastereomeric salts). Diastereomers have significantly different physical properties (e.g., melting point, boiling point, solubility, reactivity, etc.), and can be easily separated by the advantages of these dissimilarity. Diastereomers can be separated by chromatography, preferably by separation / resolving techniques based on differences in solubility. Then, by any practical means that will not racemize, the optically pure enantiomer is recovered, together with the resolving agent. A more detailed description of techniques suitable for resolving stereoisomers of compounds starting from racemic mixtures can be found in Jean Jacques, Andre Collet, Samuel H. Wilen, "Enantiomers, Racemates and Resolutions", John Wiley And Sons, Inc., 1981.

[0133] The reaction can be monitored by any suitable method known in the art. For example, the reaction can be monitored by spectroscopic means such as nuclear magnetic resonance (NMR) spectroscopy (e.g. 1 H or 13 C), infrared (IR) spectroscopy, spectrophotometry (e.g., UV-visible), mass spectrometry (MS), or by chromatographic methods such as high performance liquid chromatography (HPLC) or thin layer chromatography (TLC).

[0134] Pharmaceutical compositions, preparations and kits

[0135] In another aspect, the present invention provides a pharmaceutical composition comprising a compound of the present invention (also referred to as an "active ingredient") and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises an effective amount of the active ingredient. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of the active ingredient. In some embodiments, the pharmaceutical composition comprises a preventively effective amount of the active ingredient.

[0136] The pharmaceutically acceptable excipient used in the present invention refers to a non-toxic carrier, adjuvant or medium that does not destroy the pharmacological activity of the compound formulated together. The pharmaceutically acceptable carrier, adjuvant or medium that can be used in the composition of the present invention include, but are not limited to, ion exchangers, aluminum oxide, aluminum stearate, lecithin, serum proteins (such as human serum albumin), buffer substances (such as phosphates), glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes (such as protamine sulfate), disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, silica gel, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based materials, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and lanolin.

[0137] The present invention also includes a kit (e.g., a pharmaceutical package). The kit provided may include a compound of the invention, other therapeutic agents, and a first and second container (e.g., a vial, an ampoule, a bottle, a syringe, and / or a dispersible package or other suitable container) containing the compound of the invention and other therapeutic agents. In some embodiments, the kit provided may also optionally include a third container containing a pharmaceutical excipient for diluting or suspending the compound of the invention and / or other therapeutic agents. In some embodiments, the compound of the invention and other therapeutic agents provided in the first container and the second container are combined to form a unit dosage form.

[0138] Pharmaceutical composition provided by the invention can be administered by many ways, including but not limited to: oral administration, parenteral administration, inhalation administration, topical administration, rectal administration, nasal administration, oral administration, vaginal administration, administration by implant or other modes of administration. For example, parenteral administration used herein includes subcutaneous administration, intradermal administration, intravenous administration, intramuscular administration, intraarticular administration, intraarterial administration, intrasynovial cavity administration, intrasternal administration, intrathecal administration, intralesional administration and intracranial injection or infusion technology.

[0139] Generally, an effective amount of the compounds provided herein is administered. The amount of the compound actually administered can be determined by a physician according to the relevant circumstances, including the condition being treated, the route of administration selected, the compound actually administered, the age, weight and response of the individual patient, the severity of the patient's symptoms, and the like.

[0140] When used to prevent the conditions described herein, the compounds provided herein are administered to a subject at risk of developing the condition, typically based on the advice of a physician and under the supervision of a physician, at dosage levels as described above. Subjects at risk of developing a specific condition typically include those with a family history of the condition, or those identified by genetic testing or screening as being particularly susceptible to developing the condition.

[0141] The pharmaceutical compositions provided herein can also be administered over a long period of time ("chronic administration"). Chronic administration refers to administration of a compound or a pharmaceutical composition thereof over a long period of time, e.g., 3 months, 6 months, 1 year, 2 years, 3 years, 5 years, etc., or administration may continue indefinitely, e.g., for the rest of the subject's life. In some embodiments, chronic administration is intended to provide a constant level of the compound in the blood over a long period of time, e.g., within a therapeutic window.

[0142] Various methods of administration can be used to further deliver the pharmaceutical composition of the present invention. For example, in some embodiments, the pharmaceutical composition can be administered by push injection, for example, in order to quickly increase the concentration of the compound in the blood to an effective level. The push dose depends on the target systemic level of the active component, for example, the intramuscular or subcutaneous push dose slowly releases the active component, and the push delivered directly to the vein (for example, by IV intravenous drip) can be delivered more quickly, so that the concentration of the active component in the blood is quickly increased to an effective level. In other embodiments, the pharmaceutical composition can be given in a continuous infusion form, for example, by IV intravenous drip, so as to provide a steady-state concentration of the active component in the subject's body. In addition, in other embodiments, the pharmaceutical composition of the push dose can be first given, and then continuous infusion.

[0143] Oral compositions can be in the form of bulk liquid solutions or suspensions or bulk powders. However, more generally, in order to facilitate accurate dosing, the composition is provided in unit dosage form. The term "unit dosage form" refers to a physical discrete unit suitable as a unit dose for human patients and other mammals, each unit containing a predetermined amount of active substances suitable for producing the desired therapeutic effect and a suitable pharmaceutical excipient. Typical unit dosage forms include pre-filled, pre-measured ampoules or syringes of liquid compositions, or pills, tablets, capsules, etc. in the case of solid compositions. In such compositions, the compound is usually a relatively small component (about 0.1 to about 50% by weight, or preferably about 1 to about 40% by weight), and the remainder is various carriers or excipients and processing aids useful for forming the desired administration form.

[0144] For oral dosage, a representative regimen is one to five oral doses per day, especially two to four oral doses, typically three oral doses. Using these dosage modes, each dose provides about 0.01 to about 20 mg / kg of the compound of the invention, and preferred doses each provide about 0.1 to about 10 mg / kg, especially about 1 to about 5 mg / kg.

[0145] In order to provide blood levels similar to those using an injectable dose, or lower blood levels than using an injectable dose, a transdermal dose is generally selected in an amount of about 0.01 to about 20% by weight, preferably about 0.1 to about 20% by weight, preferably about 0.1 to about 10% by weight, and more preferably about 0.5 to about 15% by weight.

[0146] From about 1 to about 120 hours, especially 24 to 96 hours, the injected dosage level is in the range of about 0.1 mg / kg / hour to at least 10 mg / kg / hour. In order to obtain sufficient steady state levels, a preload bolus of about 0.1 mg / kg to about 10 mg / kg or more may also be given. For a 40 to 80 kg human patient, the maximum total dose cannot exceed about 2 g / day.

[0147] Liquid forms suitable for oral administration may include a suitable aqueous or non-aqueous carrier and buffers, suspending and dispersing agents, colorants, flavoring agents, etc. Solid forms may include, for example, any of the following components, or compounds of a similar nature: binders, such as microcrystalline cellulose, tragacanth, or gelatin; excipients, such as starch or lactose, disintegrants, such as alginic acid, Primogel, or corn starch; lubricants, such as magnesium stearate; glidants, such as colloidal silicon dioxide; sweeteners, such as sucrose or saccharin; or flavoring agents, such as peppermint, methyl salicylate, or orange flavoring.

[0148] Injectable compositions are typically based on injectable sterile saline or phosphate buffered saline, or other injectable excipients known in the art. As previously mentioned, in such compositions, the active compound is typically a minor component, often about 0.05 to 10% by weight, and the remainder is an injectable excipient, etc.

[0149] Typically, transdermal compositions are formulated as topical ointments or creams containing active ingredients. When formulated as an ointment, the active ingredient is typically combined with a paraffin or water-miscible ointment base. Alternatively, the active ingredient can be formulated as an ointment together with, for example, an oil-in-water cream base. Such transdermal formulations are well known in the art and typically include other components for enhancing the stable skin penetration of the active ingredient or formulation. All such known transdermal formulations and components are included within the scope provided by the invention.

[0150] The compounds of the invention may also be administered by transdermal means. Thus, transdermal administration may be achieved using patches of the reservoir or porous membrane type, or a variety of solid matrices.

[0151] The above components for oral administration, injection or topical administration are representative only. Other materials and processing techniques are described in Remington's Pharmaceutical Sciences, 17th edition, 1985, Mack Publishing Company, Easton, Pennsylvania, Part 8, which is incorporated herein by reference.

[0152] The compounds of the invention may also be administered in sustained release form or from a sustained release delivery system. Descriptions of representative sustained release materials can be found in Remington's Pharmaceutical Sciences.

[0153] The present invention also relates to pharmaceutically acceptable formulations of the compounds of the present invention. In one embodiment, the formulation comprises water. In another embodiment, the formulation comprises a cyclodextrin derivative. The most common cyclodextrins are α-, β- and γ-cyclodextrins consisting of 6, 7 and 8 α-1,4-linked glucose units, respectively, which optionally include one or more substituents on the linked sugar moiety, including but not limited to: methylated, hydroxyalkylated, acylated and sulfoalkyl ether substitutions. In some embodiments, the cyclodextrin is a sulfoalkyl ether β-cyclodextrin, for example, sulfobutyl ether β-cyclodextrin, also known as Captisol. See, for example, US5,376,645. In some embodiments, the formulation includes hexapropyl-β-cyclodextrin (e.g., in water, 10-50%).

[0154] Indications

[0155] In another aspect, provided is the use of the compound of formula (I) disclosed in the present invention (including all individual embodiments and generic subsets disclosed herein) or its tautomer, stereoisomer, prodrug, crystal form, pharmaceutically acceptable salt, hydrate or solvate as a medicine.

[0156] The compounds of the present invention exhibit potent and selective BTK inhibition. For example, the compounds of the present invention exhibit nanomolar potency against wild-type BTK and BTK kinases encoded by BTK genes that include BTK kinase inhibitor resistance mutations, including, for example, C481S, C481F, C481Y, C481R, C481T, C481G or C481W, preferably, the mutation is C481S. In some embodiments, the inhibition of C481S is similar to the inhibition observed for wild-type BTK.

[0157] In some embodiments, the compounds of the invention selectively target BTK kinase. For example, relative to another kinase or non-kinase target, the compounds of the invention can selectively target BTK kinase. In some embodiments, relative to one or more kinases in BRK, CSK, ERBB4, FYN, MEK1, MEK2, TEC, TXK, YES1, BMX, BLK, EGFR, ITK, SRC, JAK1, JAK2 and JAK3, the compounds of the invention can selectively target BTK kinase.

[0158] In some embodiments, relative to another kinase, the compounds of the present invention exhibit at least 30 times selectivity for BTK kinase. For example, relative to another kinase, the compounds of the present invention exhibit at least 40 times selectivity for BTK kinase; at least 50 times selectivity; at least 60 times selectivity; at least 70 times selectivity; at least 80 times selectivity; at least 90 times selectivity; at least 100 times selectivity; at least 200 times selectivity; at least 300 times selectivity; at least 400 times selectivity; at least 500 times selectivity; at least 600 times selectivity; at least 700 times selectivity; at least 800 times selectivity; at least 900 times selectivity; or at least 1000 times selectivity. In some embodiments, relative to another kinase, the compounds of the present invention exhibit at least 100 times selectivity for BTK kinase. In some embodiments, the selectivity for BTK kinase relative to another kinase is measured in a cell assay (e.g., a cell assay provided herein).

[0159] The compounds of the invention are useful for treating diseases and conditions that are treated with BTK kinase inhibitors, such as diseases and conditions associated with BTK, such as proliferative diseases, such as cancer, including hematological cancers and solid tumors, and inflammatory conditions, immune diseases or fibrosis.

[0160] In some embodiments of any of the methods or uses described herein, the cancer (e.g., a cancer associated with BTK) is a hematological cancer. In some embodiments of any of the methods or uses described herein, the cancer (e.g., a cancer associated with BTK) is a solid tumor. In some embodiments of any of the methods or uses described herein, the cancer (e.g., a cancer associated with BTK) is a B cell malignancy. In some embodiments of any of the methods or uses described herein, the cancer (e.g., a BTK-related cancer) is Hodgkin lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone lymphoma (e.g., splenic marginal zone lymphoma, extranodal marginal zone B-cell lymphoma), Burkitt's lymphoma, Waldenstrom's macroglobulinemia (lymphoplasmacytic lymphoma), primary central nervous system lymphoma, small lymphocytic lymphoma, chronic lymphocytic lymphoma, acute lymphocytic leukemia, B-cell prolymphocytic leukemia, precursor B-cell lymphoblastic leukemia, hairy cell leukemia, acute myeloid leukemia, chronic myeloid leukemia, multiple myeloma, plasma cell myeloma, plasmacytoma, bone cancer, bone metastasis, breast cancer, gastro-esophageal cancer, pancreatic cancer, ovarian cancer, prostate cancer, lung cancer, colon cancer, head and neck cancer, or glioma.

[0161] In some embodiments, the hematological cancer is selected from leukemia, non-Hodgkin's lymphoma, Hodgkin's lymphoma or myeloma, for example, acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), acute promyelocytic leukemia (APL), chronic lymphocytic lymphoma (CLL), chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML), chronic neutrophilic leukemia (CNL), acute undifferentiated leukemia (AUL), anaplastic large cell lymphoma (ALCL), prolymphocytic leukemia (PML), juvenile myelomonocytic leukemia (JMML), adult T-cell leukemia (ALL), trilineage myeloproliferation Acute myeloid leukemia (AML / TMDs), mixed lineage leukemia (MLL), myelodysplastic syndrome (MDS), myeloproliferative disorders (MPDs), diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone lymphoma (e.g., splenic marginal zone lymphoma, extranodal marginal zone B-cell lymphoma), Burkitt's lymphoma, Waldenstrom's macroglobulinemia (lymphoplasmacytic lymphoma), primary central nervous system lymphoma, small lymphocytic lymphoma, precursor B-cell lymphoblastic leukemia, hairy cell leukemia, mucosa-associated lymphoid tissue lymphoma, plasma cell myeloma, plasmacytoma, and multiple myeloma. Other examples of hematological cancers include myelodysplastic disorders (MPDs), such as polycythemia vera (PV), essential thrombocytopenia (ET), and idiopathic primary myelofibrosis (IMF / IPF / PMF). In some embodiments, the hematological cancer is mantle cell lymphoma, chronic lymphocytic lymphoma, small lymphocytic lymphoma, Waldenstrom's macroglobulinemia, or marginal zone lymphoma.

[0162] In some embodiments, the solid tumor is selected from bone cancer, bone metastasis, breast cancer, gastro-esophageal cancer, pancreatic cancer, ovarian cancer, prostate cancer, lung cancer, colon cancer, head and neck cancer.

[0163] In some embodiments, the immune disease is selected from arthritis, multiple sclerosis, osteoporosis, irritable bowel syndrome, inflammatory bowel disease, Crohn's disease, lupus, rheumatoid arthritis, psoriatic arthritis, osteoarthritis, Still's disease, juvenile arthritis, diabetes, myasthenia gravis, Hashimoto's thyroiditis, Ord's thyroiditis, Graves' disease, Sjögren's syndrome, Guillain-Barré syndrome, acute disseminated encephalomyelitis, Addison's disease, opsoclonus syndrome, obsessive compulsive disorder, Spondylitis, antiphospholipid antibody syndrome, aplastic anemia, autoimmune hepatitis, celiac disease, Goodpasture's syndrome, idiopathic thrombocytopenic purpura, optic neuritis, scleroderma, primary biliary cirrhosis, Reiter's syndrome, Takayasu's arteritis, warm autoimmune hemolytic anemia, Wegener's granulomatosis, psoriasis, alopecia universalis, Behcet's disease, chronic fatigue, dysautonomia, endometriosis, interstitial cystitis, neuromyotonia, Scleroderma and vulvar pain, asthma, appendicitis, blepharitis, bronchiolitis, bronchitis, bursitis, cervicitis, cholangitis, cholecystitis, colitis, conjunctivitis, colitis, cystitis, lacrimal gland inflammation, dermatitis, dermatomyositis, encephalitis, endocarditis, endometritis, enteritis, enterocolitis, epicondylitis, epididymitis, fasciitis, fibrositis, gastritis, gastroenteritis, hepatitis, inflammatory enteritis, suppurative inflammation, laryngitis, mastitis, meningitis, myelitis, myocarditis, myositis, nephritis inflammation, oophoritis, orchitis, osteitis, otitis, pancreatitis, mumps, pericarditis, peritonitis, pharyngitis, pleurisy, phlebitis, pneumonia, pneumonitis, proctitis, prostatitis, pyelonephritis, rhinitis, salpingitis, sinusitis, stomatitis, synovitis, tendinitis, tonsillitis, uveitis, vaginitis, vasculitis, vulvitis, graft-versus-host disease, transplantation, transfusion, anaphylactic reaction, allergic reaction, type I hypersensitivity reaction, allergic conjunctivitis, allergic rhinitis, and atopic dermatitis.

[0164] In some embodiments, the inflammatory disease is selected from arthritis, asthma, appendicitis, blepharitis, bronchiolitis, bronchitis, bursitis, cervicitis, cholangitis, cholecystitis, colitis, conjunctivitis, cystitis, dacryoadenitis, dermatitis, dermatomyositis, encephalitis, endocarditis, endometritis, enteritis, enterocolitis, epicondylitis, epididymitis, fasciitis fibrositis, gastritis, gastroenteritis, hepatitis, hidradenitis suppurativa, laryngitis, mastitis, meningitis, osteomyelitis myocarditis, myositis, nephritis, oophoritis, orchitis, osteitis, otitis, pancreatitis, mumps, pericarditis, peritonitis, pharyngitis, pleurisy, phlebitis, pneumonia, lung disease, proctitis, prostatitis, pyelonephritis, rhinitis, salpingitis, sinusitis, stomatitis, synovitis, tendonitis, tonsillitis, uveitis, vaginitis, vasculitis, and vulvitis.

[0165] In some embodiments, the autoimmune disease is selected from lupus and Sjögren's syndrome, rheumatoid arthritis, psoriatic arthritis, osteoarthritis, Still's disease, juvenile arthritis, diabetes, myasthenia gravis, Hashimoto's thyroiditis, Ord's thyroiditis, Graves' disease, Sjögren's syndrome, Guillain-Barre syndrome, acute disseminated encephalomyelitis, Addison's disease, opsoclonus-myoclonus syndrome, ankylosing spondylitis, antiphospholipid antibody syndrome, aplastic anemia, autoimmune hepatitis, celiac disease, Goodpasture's syndrome, idiopathic thrombocytopenic purpura, optic neuritis, scleroderma, primary biliary cirrhosis, Reiter's syndrome, Takayasu's arteritis, hemolytic anemia, Wegener's granulomatosis, psoriasis, alopecia universalis, Behcet's disease, chronic fatigue, dysautonomia, endometriosis, interstitial cystitis, neuromyotonia, scleroderma, and vulvodynia.

[0166] In some embodiments, the heteroimmune disease is selected from the group consisting of graft versus host disease, transplantation, transfusion, anaphylaxis, allergic reaction, type I hypersensitivity reaction, allergic conjunctivitis, allergic rhinitis, and atopic dermatitis.

[0167] In some embodiments, fibrosis is selected from pulmonary fibrosis, idiopathic pulmonary fibrosis (IPF), usual interstitial pneumonia (UIP), interstitial lung disease, cryptogenic fibrosing alveolitis (CFA), bronchiolitis obliterans, bronchiectasis, fatty liver disease, steatosis (e.g., non-alcoholic steatohepatitis (NASH), cholestatic liver disease (e.g., primary biliary cirrhosis (PBC)), cirrhosis, alcohol-induced liver fibrosis, bile duct damage, biliary fibrosis, cholestasis, or bile duct disease.

[0168] Example

[0169] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only intended to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods for the following examples without specifying specific conditions are usually based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, parts and percentages are by weight and weight percentage.

[0170] Abbreviations:

[0171] Tol: Toluene

[0172] THF: Tetrahydrofuran

[0173] HCl: hydrochloric acid

[0174] MeOH: Methanol

[0175] TsOMe: methyl p-toluenesulfonate

[0176] K2CO3: Potassium carbonate

[0177] LiOH: lithium hydroxide

[0178] DMF: N,N-dimethylformamide

[0179] DCM: dichloromethane

[0180] KHMDS: Potassium bis(trimethylsilyl)amide

[0181] DIPEA: N,N-diisopropylethylamine

[0182] Pd(OAc)2: Palladium acetate

[0183] Xphos: 2-Dicyclohexylphosphino-2′,4′,6′-triisopropylbiphenyl

[0184] Cs2CO3: Cesium carbonate

[0185] Preparation of intermediate A-1 (1,1,1-trifluoropropan-2-yl) hydrazine hydrochloride

[0186]

[0187] The following synthetic route was used

[0188]

[0189] Step 1 Synthesis of compound N'-(1,1,1-trifluoropropan-2-yl)benzoylhydrazide

[0190] Add benzoyl hydrazide (13.6 g, 100 mmol) and toluene (200 mL) to a 500 mL sealed tube equipped with a magnetic rod, stir to dissolve, add 1,1,1-trifluoropropan-2-one (16.8 g, 150 mmol), seal, place in an oil bath, heat to 110°C, keep warm and stir to react overnight. Cool to room temperature, evaporate the solvent under reduced pressure, and pass the residue through a silica gel column to obtain 16.7 g of a white solid with a yield of 72.6%. LC-MS (APCI): m / z=231.0 (M+1) + .

[0191] Step 2 Synthesis of compound N'-(1,1,1-trifluoropropan-2-yl)benzoylhydrazide

[0192] Add N'-(1,1,1-trifluoropropane-2-yl)benzoylhydrazide (9.2g, 40mmol) and anhydrous THF (100mL) to a 500mL three-necked flask equipped with a magnetic stirrer and a condenser, stir to dissolve, evacuate and replace with nitrogen 3 times, cool in an ice-water bath, slowly drop BH3-THF (1M, 80mL, 80mmol), remove the ice bath, stir and react at room temperature under nitrogen atmosphere overnight. Under ice-water bath cooling, add methanol (50mL) to quench the reaction, remove the ice bath, concentrate to dryness under reduced pressure, add dichloromethane (150mL), stir for 30 minutes, filter out insoluble solids, wash with saturated ammonium chloride solution (50mL), dry with anhydrous sodium sulfate, filter, concentrate to dryness, add petroleum ether (50mL), stir for 30 minutes under ice-water bath cooling, a large amount of white solid precipitates, filter, and air dry to obtain 6.5g, yield 70.0%. LC-MS (APCI): m / z = 233.0 (M+1) + .

[0193] Step 3 Synthesis of Intermediate A-1

[0194] Add N'-(1,1,1-trifluoropropane-2-yl)benzoylhydrazide (4.6 g, 20 mmol) and methanol (15 mL) to a 100 mL single-necked flask equipped with a magnetic stirrer and a condenser, stir to dissolve, add concentrated hydrochloric acid (27 mL), heat to 80 ° C under a nitrogen atmosphere, keep stirring and react overnight. Cool to room temperature, concentrate to dryness under reduced pressure, add ethyl acetate (30 mL), stir for 10 minutes, filter, and dry to obtain 1.9 g of white solid, with a yield of 58.0%.

[0195] Intermediate A-2 (1,1,1-trifluoropropan-2-yl-3,3,3-d 3 ) Preparation of hydrazine hydrochloride

[0196]

[0197] The following synthetic route was used

[0198]

[0199] Step 1 Synthesis of compound N'-(1,1,1-trifluoropropan-2-yl-3,3,3-d3)benzoylhydrazide

[0200] Add N'-(1,1,1-trifluoropropane-2-yl)benzoylhydrazide (4.6 g, 20 mmol), deuterated methanol (15 mL) and heavy water (15 mL) to a 100 mL single-necked flask equipped with a magnetic stirrer, stir to dissolve, add a heavy water solution of sodium deuteride (4.0 g, 40 mmol, 40% heavy water solution), heat to 60 ° C under a nitrogen atmosphere, and stir overnight. LC-MS shows complete conversion, cool to room temperature, evaporate the organic solvent under reduced pressure, extract with dichloromethane (20 mL x 2), combine the organic phases, dry over anhydrous sodium sulfate, filter, concentrate and pass through a silica gel column to obtain 3.8 g of a white solid, with a yield of 81.5%. LC-MS (APCI): m / z = 234.1 (M+1) + .

[0201] Step 2 Synthesis of compound N'-(1,1,1-trifluoropropan-2-yl-3,3,3-d3)benzoylhydrazide

[0202] To a 250mL three-necked flask equipped with a magnetic stirrer and a condenser, add N'-(1,1,1-trifluoropropan-2-yl-3,3,3-d3)benzohydrazide (3.5 g, 15 mmol) and anhydrous THF (40 mL), stir to dissolve, evacuate and replace with nitrogen three times, cool in an ice-water bath, slowly add BH3-THF (1M, 30 mL, 30 mmol) dropwise, remove the ice bath, and react with stirring at room temperature under nitrogen atmosphere overnight. Under ice-water bath cooling, methanol (30 mL) was added to quench the reaction, the ice bath was removed, the mixture was concentrated to dryness under reduced pressure, dichloromethane (60 mL) was added, stirred for 30 minutes, insoluble solids were filtered out, washed with saturated ammonium chloride solution (30 mL), dried over anhydrous sodium sulfate, filtered, concentrated to dryness, petroleum ether (30 mL) was added, stirred for 30 minutes under ice-water bath cooling, a large amount of white solids precipitated, filtered, and air-dried to obtain 2.6 g, with a yield of 73.7%. LC-MS (APCI): m / z=236.0 (M+1) + .

[0203] Step 3 Synthesis of intermediate A-2

[0204] Add N'-(1,1,1-trifluoropropane-2-yl-3,3,3-d3)benzoylhydrazide (2.6 g, 11 mmol) and methanol (8 mL) to a 100 mL single-necked flask equipped with a magnetic stirrer and a condenser, stir to dissolve, add concentrated hydrochloric acid (15 mL), heat to 80 ° C under a nitrogen atmosphere, keep stirring and react overnight. Cool to room temperature, concentrate to dryness under reduced pressure, add ethyl acetate (30 mL), stir for 10 minutes, filter, and dry to obtain 1.2 g of white solid, with a yield of 65.3%.

[0205] Preparation of intermediate B-1 (((5-fluoro-2-methoxybenzoyl)amino)methyl) potassium trifluoroborate

[0206]

[0207] The following synthetic route was used

[0208]

[0209] Step 1 Synthesis of compound 5-fluoro-2-methoxybenzoyl chloride

[0210] Add 5-fluoro-2-methoxybenzoic acid (2.6 g, 15 mmol) and anhydrous dichloromethane (30 mL) to a 100 mL three-necked flask equipped with a magnetic stirrer, stir until dissolved, cool in an ice-water bath, dropwise add oxalyl chloride (3.8 g, 30 mmol) and anhydrous DMF (110 mgm, 1.5 mmol) under a nitrogen atmosphere, remove the ice bath, stir and react at room temperature overnight. Evaporate the solvent and unreacted oxalyl chloride under reduced pressure for standby use.

[0211] Step 2 Synthesis of intermediate B-1

[0212] To a 100 mL three-necked flask equipped with a magnetic stirrer, (4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)methanamine (3.63 g, 16.5 mmol) and anhydrous THF (30 mL) were added, cooled to -78 °C under a nitrogen atmosphere, KHMDS (16.5 mL, 1 M THF solution) was added dropwise, and the mixture was stirred for 30 minutes at room temperature. The mixture was heated to room temperature and stirred for 30 minutes. Anhydrous methanol (2.11 g, 66 mmol) was added, and the mixture was stirred at room temperature for 1 hour. The insoluble solid was filtered off, and the solvent of the filtrate was evaporated at no more than 30 °C. Anhydrous THF was taken twice (20 mL), dissolved in anhydrous THF (20 mL), and the above-mentioned THF solution of 5-fluoro-2-methoxybenzoyl chloride (20 mL) was slowly added dropwise. After the dropwise addition, the mixture was stirred at room temperature under a nitrogen atmosphere overnight. The solvent was evaporated, dissolved in methanol (30 mL), potassium bifluoride aqueous solution (6.4 g, 82.5 mmol, 20 mL) was added, and the reaction was stirred at room temperature overnight. The solvent was evaporated under reduced pressure, toluene was added with water twice (50 mL), the residue was washed with MTBE, filtered, the filter cake was washed with hot methanol / acetone (1 / 3, 100 mL) solution, the filtrate was concentrated to dryness, MTBE was added for pulping, the precipitated solid was filtered, and dried to obtain 3.5 g of white solid, with a yield of 80.7%. 1 H NMR (400MHz, DMSO-D6) δ (ppm): 7.75 (br s, 1H), 7.65-7.62 (m, 1H), 7.30-7.25 (m, 1H), 7.17-7.14 (m, 1H), 3.87 (s, 3H), 2.13-2.10 (m, 2H).

[0213] Intermediate B-2(((5-fluoro-2-(methoxy-d 3 Preparation of potassium trifluoroborate (2-( ...

[0214]

[0215] The following synthetic route was used

[0216]

[0217] Step 1 Synthesis of Compound 5-fluoro-2-(methoxy-d3)benzoic acid methyl ester

[0218] Add 5-fluoro-2-hydroxybenzoic acid methyl ester (5.1 g, 30 mmol) and acetonitrile (80 mL) to a 250 mL single-necked flask equipped with a magnetic stirrer and a condenser, stir to dissolve, add potassium carbonate (8.28 g, 60 mmol) and TsOMe-d3 (7.37 g, 39 mmol), heat to 80 ° C under a nitrogen atmosphere, and stir to react overnight. Cool to room temperature, add ethyl acetate (100 mL), filter out insoluble solids, concentrate the filtrate and pass it through a silica gel column to obtain 4.2 g of white solid, with a yield of 74.8%. LC-MS (APCI): m / z=188.0 (M+1) + .

[0219] Step 2 Synthesis of Compound 5-Fluoro-2-(Methoxy-d3)benzoic Acid

[0220] Add 5-fluoro-2-(methoxy-d3)benzoic acid methyl ester (3.74 g, 20 mmol), THF (20 mL) and water (20 mL) to a 100 mL single-necked flask equipped with a magnetic stirrer, stir to dissolve, add lithium hydroxide (0.96 g, 40 mmol), and stir at room temperature for 3 hours under nitrogen atmosphere. The organic solvent is evaporated under reduced pressure, and solid citric acid (2.56 g, 13.3 mmol) is slowly added under ice-water cooling to precipitate a large amount of white solid, which is filtered, washed with a small amount of ice water, and vacuum dried to obtain 3.0 g of the white solid, with a yield of 86.6%. LC-MS (APCI): m / z=174.0 (M+1) + .1H NMR (400MHz, DMSO-D6) δ (ppm): 12.92 (s, 1H), 7.43-7.34 (m, 2H), 7.16-7.12 (m, 1H).

[0221] Step 3 Synthesis of compound 5-fluoro-2-(methoxy-d3)benzoyl chloride

[0222] Add 5-fluoro-2-(methoxy-d3)benzoic acid (2.6 g, 15 mmol) and anhydrous dichloromethane (30 mL) to a 100 mL three-necked flask equipped with a magnetic stirrer, stir until dissolved, cool in an ice-water bath, dropwise add oxalyl chloride (3.8 g, 30 mmol) and anhydrous DMF (110 mgm, 1.5 mmol) under a nitrogen atmosphere, remove the ice bath, stir and react at room temperature overnight. Evaporate the solvent and unreacted oxalyl chloride under reduced pressure for standby use.

[0223] Step 4 Synthesis of intermediate B-2

[0224] To a 100mL three-necked flask equipped with a magnetic stirrer, add (4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)methanamine (3.63 g, 16.5 mmol) and anhydrous THF (30 mL), cool to -78°C under a nitrogen atmosphere, add KHMDS (16.5 mL, 1M THF solution) dropwise, keep stirring for 30 minutes, warm to room temperature and stir to react for 30 minutes, add anhydrous methanol (2.11 g, 66 mmol), stir at room temperature for 1 hour, filter out insoluble solids, evaporate the filtrate solvent at no more than 30°C, add anhydrous THF twice (20 mL), dissolve in anhydrous THF (20 mL), slowly add the above-mentioned THF solution (20 mL) of 5-fluoro-2-(methoxy-d3)benzoyl chloride dropwise, and after the dropwise addition, stir and react at room temperature under a nitrogen atmosphere overnight. The solvent was evaporated, dissolved in methanol (30 mL), potassium bifluoride aqueous solution (6.4 g, 82.5 mmol, 20 mL) was added, and the reaction was stirred at room temperature overnight. The solvent was evaporated under reduced pressure, toluene was added with water twice (50 mL), the residue was washed with MTBE, filtered, the filter cake was washed with hot methanol / acetone (1 / 3, 100 mL) solution, the filtrate was concentrated to dryness, MTBE was added for pulping, the precipitated solid was filtered, and dried to obtain 3.1 g of white solid, with a yield of 70.7%. 1 H NMR (400MHz, DMSO-D6) δ (ppm): 7.75 (br s, 1H), 7.65-7.62 (m, 1H), 7.30-7.25 (m, 1H), 7.17-7.14 (m, 1H), 2.13-2.10 (m, 2H).

[0225] Example 1 5-amino-3-(4-((5-fluoro-2-(methoxy-d 3 )benzoylamino)methyl)phenyl)-1-(1, 1,1-trifluoropropan-2-yl)-1H-pyrazole-4-carboxamide (Compound T-1);

[0226] (S)-5-amino-3-(4-((5-fluoro-2-(methoxy-d 3 )benzoylamino)methyl)phenyl)-1-(1,1,1- (trifluoropropan-2-yl)-1H-pyrazole-4-carboxamide (compound T-1-S);

[0227] (R)-5-amino-3-(4-((5-fluoro-2-(methoxy-d 3 )benzoylamino)methyl)phenyl)-1-(1,1,1- Preparation of trifluoropropan-2-yl)-1H-pyrazole-4-carboxamide (Compound T-1-R)

[0228]

[0229]

[0230] The following synthetic route was used

[0231]

[0232] Step 1 Synthesis of compound 2-((4-bromophenyl)(hydroxy)methylene)malononitrile

[0233] Toluene (100 mL), THF (20 mL) and malononitrile (6.3 mL, 100.3 mmol) were added to a 250 mL single-necked flask equipped with a magnetic stirrer in sequence, and the mixture was stirred to dissolve. The mixture was cooled to -10 °C, 4-bromobenzoyl chloride (20 g, 91.1 mmol) was added, and the mixture was stirred for 10 minutes. DIPEA (31.8 mL, 182.3 mmol) was slowly added dropwise. The mixture was added dropwise within half an hour, and the internal temperature was controlled not to exceed -10 °C. After the addition was completed, the mixture was slowly heated to room temperature and stirred for 2 hours. Ethyl acetate (200 mL) and 1M hydrochloric acid aqueous solution (200 mL) were added, stirred for 10 minutes, the organic phase was separated, washed with 1M hydrochloric acid (100 mL), washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness. The residue was added to petroleum ether / ethyl acetate (100 mL, 20 / 1), stirred for 20 minutes, filtered, washed with petroleum ether, and air-dried to obtain 20 g of gray solid, with a yield of 88.1%. LC-MS (APCI): m / z=249.0 (M+1) + .

[0234] Step 2 Synthesis of compound 2-((4-bromophenyl)(methoxy)methylene)malononitrile

[0235] Sodium hydride (2.48 g, 61.84 mmol, 60% by mass dispersed in silicone oil) was added to a 250 mL three-necked flask equipped with a magnetic stirrer and a condenser, and the mixture was evacuated and replaced with nitrogen three times. Anhydrous THF (40 mL) was added under ice-water cooling, and the mixture was stirred and dispersed. A solution of 2-((4-bromophenyl)(hydroxy)methylene)malononitrile (14 g, 56.2 mmol) in anhydrous THF (40 mL) was added dropwise, and the mixture was stirred and reacted for 30 minutes under ice-water bath. Dimethyl sulfate (16.0 mL, 168.6 mmol) was added dropwise. After the addition was completed, the mixture was heated to 80°C, and the mixture was stirred and reacted overnight. The mixture was cooled to room temperature, and saturated brine (80 mL) was added to quench the reaction, the organic phase was separated, and the aqueous phase was extracted with ethyl acetate (50 mL x 2). The organic phases were combined, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, concentrated, and passed through a silica gel column to obtain 7.3 g of a white solid with a yield of 49.4%. LC-MS (APCI): m / z = 231.0 (M+1)+ .

[0236] Step 3 Compound 5-amino-3-(4-bromophenyl)-1-(1,1,1-trifluoropropan-2-yl)-1H-pyrazole-4-carbonitrile

[0237] Add intermediate A-1 (1.6 g, 10 mmol) and anhydrous ethanol (60 mL) to a 100 mL single-necked flask equipped with a magnetic stirrer and a condenser. Add triethylamine (4.04 g, 40 mmol) and 2-((4-bromophenyl)(methoxy)methylene)malononitrile (1.75 g, 6.67 mmol) under stirring, heat to 80°C under a nitrogen atmosphere, and stir and react overnight. Cool to room temperature, evaporate the solvent under reduced pressure, add saturated brine (20 mL) and ethyl acetate (30 mL), stir for 10 minutes, separate the organic phase, extract the aqueous phase with ethyl acetate (30 mL x 2), combine the organic phases, dry over anhydrous sodium sulfate, filter, concentrate and pass through a silica gel column to obtain 2.2 g of a white solid, with a yield of 91.8%. LC-MS (APCI): m / z=359.0 (M+1) + . 1 H NMR (400MHz, DMSO-D6) δ (ppm): 7.74-7.67 (m, 4H), 7.15 (br s, 2H), 5.34-5.26 (m, 1H), 1.63 (d, J = 6.4Hz, 3H).

[0238] Step 4 Compound N-(4-(5-amino-4-cyano-1-(1,1,1-trifluoropropan-2-yl)-1H-pyrazol-3-yl)phenyl)-5-fluoro-2-(methoxy-d3)benzamide

[0239] Add intermediate B-2 (0.29 g, 1.0 mmol), 5-amino-3-(4-bromophenyl)-1-(1,1,1-trifluoropropane-2-yl)-1H-pyrazole-4-carbonitrile (0.36 g, 1.0 mmol), Cs2CO3 (0.98 g, 3.0 mmol), Xphos (48 mg, 0.1 mmol), Pd(OAc)2 (12 mg, 0.05 mmol), THF (10 mL) and water (1 mL) to a 50 mL single-necked flask equipped with a magnetic stirrer and a condenser, evacuate and replace with nitrogen three times, heat to 85 ° C and stir to react overnight. Cool to room temperature, evaporate the solvent under reduced pressure, add saturated brine (5 mL) and ethyl acetate (20 mL), separate the organic phase, extract the aqueous phase with ethyl acetate (20 mL x 2), combine the organic phases, dry over anhydrous sodium sulfate, concentrate and pass through a silica gel column to obtain 0.38 g of a white solid, with a yield of 81.8%. LC-MS (APCI): m / z = 465.2 (M+1) + .

[0240] Step 4 Synthesis of Compound T-1

[0241] N-(4-(5-amino-4-cyano-1-(1,1,1-trifluoropropane-2-yl)-1H-pyrazol-3-yl)phenyl)-5-fluoro-2-(methoxy-d3)benzamide (0.38 g, 0.82 mmol) and methanesulfonic acid (3 mL) were added to a 50 mL single-necked flask equipped with a magnetic stirrer and a condenser, and the temperature was raised to 80°C. The mixture was stirred and reacted for 1 hour under a nitrogen atmosphere. After cooling to room temperature, saturated brine (20 mL) and ethyl acetate (20 mL) were added, and the pH was adjusted to 8 with ammonia water. The organic phase was separated, and the aqueous phase was extracted with ethyl acetate (20 mL x 2). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated and passed through a silica gel column to obtain 0.31 g of a white solid with a yield of 78.5%. LC-MS (APCI): m / z=483.2 (M+1) + .

[0242] Step 5 Synthesis of compounds T-1-S and T-1-R

[0243] The racemic compound T-1 (0.31 g) was dissolved in methanol (10 mL) and subjected to chiral preparation by IC column using the following conditions: flow rate: 4 ml / min; mobile phase: MTBE:MeOH:EtOH=91:2:7 (0.1% triethylamine);

[0244] The corresponding fractions were collected and dried by spin drying to obtain 120 mg of compound T-1-S with a corresponding retention time of 26.358 min and 120 mg of compound T-1-R with a corresponding retention time of 31.922 min.

[0245] Example 2 5-amino-3-(4-((5-fluoro-2-methoxybenzoylamino)methyl)phenyl)-1-(1,1,1- Trifluoropropan-2-yl-3,3,3-d3)-1H-pyrazole-4-carboxamide (compound T-2);

[0246] (S)-5-amino-3-(4-((5-fluoro-2-methoxybenzoylamino)methyl)phenyl)-1-(1,1,1-trifluoro Propan-2-yl-3,3,3-d3)-1H-pyrazole-4-carboxamide (compound T-2-S);

[0247] (R)-5-amino-3-(4-((5-fluoro-2-methoxybenzoylamino)methyl)phenyl)-1-(1,1,1-trifluoro Preparation of propan-2-yl-3,3,3-d3)-1H-pyrazole-4-carboxamide (compound T-2-R)

[0248]

[0249] The following synthetic route was used

[0250]

[0251] Step 1 Synthesis of compound 5-amino-3-(4-bromophenyl)-1-(1,1,1-trifluoropropan-2-yl-3,3,3-d3)-1H-pyrazole-4-carbonitrile

[0252] Add intermediate A-2 (1.2 g, 7.2 mmol) and anhydrous ethanol (40 mL) to a 100 mL single-necked flask equipped with a magnetic stirrer and a condenser. Add triethylamine (2.9 g, 28.7 mmol) and 2-((4-bromophenyl)(methoxy)methylene)malononitrile (1.26 g, 4.8 mmol) under stirring. Heat to 80°C under a nitrogen atmosphere and stir overnight. Cool to room temperature, evaporate the solvent under reduced pressure, add saturated brine (20 mL) and ethyl acetate (30 mL), stir for 10 minutes, separate the organic phase, extract the aqueous phase with ethyl acetate (30 mL x 2), combine the organic phases, dry over anhydrous sodium sulfate, filter, concentrate and pass through a silica gel column to obtain 1.6 g of a white solid with a yield of 92.0%. LC-MS (APCI): m / z=362.0 (M+1) + . 1 H NMR (400MHz, DMSO-D6) δ (ppm): 7.74-7.67 (m, 4H), 7.15 (br s, 2H), 5.31 (s, 1H).

[0253] Step 2 Synthesis of compound N-(4-(5-amino-4-cyano-1-(1,1,1-trifluoropropan-2-yl-3,3,3-d3)-1H-pyrazol-3-yl)phenyl)-5-fluoro-2-methoxybenzamide

[0254] To a 50 mL single-necked flask equipped with a magnetic stirrer and a condenser, intermediate B-1 (0.29 g, 1.0 mmol), 5-amino-3-(4-bromophenyl)-1-(1,1,1-trifluoropropan-2-yl-3,3,3-d3)-1H-pyrazole-4-carbonitrile (0.36 g, 1.0 mmol), Cs2CO3 (0.98 g, 3.0 mmol), Xphos (48 mg, 0.1 mmol), Pd(OAc)2 (12 mg, 0.05 mmol), THF (10 mL) and water (1 mL) were added. The mixture was evacuated and replaced with nitrogen three times. The temperature was raised to 85°C and the reaction was stirred and kept warm overnight. After cooling to room temperature, the solvent was evaporated under reduced pressure, saturated brine (5 mL) and ethyl acetate (20 mL) were added, the organic phase was separated, the aqueous phase was extracted with ethyl acetate (20 mL x 2), the organic phases were combined, dried over anhydrous sodium sulfate, concentrated and passed through a silica gel column to obtain 0.36 g of a white solid, with a yield of 77.5%. LC-MS (APCI): m / z = 465.2 (M+1) + .

[0255] Step 3 Synthesis of Compound T-2

[0256] Add N-(4-(5-amino-4-cyano-1-(1,1,1-trifluoropropan-2-yl-3,3,3-d3)-1H-pyrazol-3-yl)phenyl)-5-fluoro-2-methoxybenzamide (0.36 g, 0.82 mmol) and methanesulfonic acid (3 mL) to a 50 mL single-necked flask equipped with a magnetic stirrer and a condenser, heat to 80°C, and stir under a nitrogen atmosphere for 1 hour. Cool to room temperature, add saturated brine (20 mL) and ethyl acetate (20 mL), adjust pH to 8 with ammonia water, separate the organic phase, extract the aqueous phase with ethyl acetate (20 mL x 2), combine the organic phases, dry over anhydrous sodium sulfate, filter, concentrate and pass through a silica gel column to obtain 0.30 g of a white solid, with a yield of 75.8%. LC-MS (APCI): m / z=483.2 (M+1) + .

[0257] Step 4 Synthesis of Compound T-2-S and Compound T-2-R

[0258] The racemic compound T-2 (0.30 g) was dissolved in methanol (10 mL) and subjected to chiral preparation by IC column using the following conditions: flow rate: 4 ml / min; mobile phase: MTBE:MeOH:EtOH=91:2:7 (0.1% triethylamine);

[0259] The corresponding fractions were collected and dried by spin drying to obtain 120 mg of compound T-2-S with a corresponding retention time of 24.483 min and 120 mg of compound T-2-R with a corresponding retention time of 29.807 min.

[0260] Example 3 5-amino-3-(4-((5-fluoro-2-methoxybenzoylamino)methyl)phenyl)-1-(1,1,1- Trifluoropropan-2-yl-3,3,3-d3)-1H-pyrazole-4-carboxamide (compound T-3);

[0261] (S)-5-amino-3-(4-((5-fluoro-2-methoxybenzoylamino)methyl)phenyl)-1-(1,1,1-trifluoro Propan-2-yl-3,3,3-d3)-1H-pyrazole-4-carboxamide (compound T-3-S);

[0262] (R)-5-amino-3-(4-((5-fluoro-2-methoxybenzoylamino)methyl)phenyl)-1-(1,1,1-trifluoro Preparation of propan-2-yl-3,3,3-d3)-1H-pyrazole-4-carboxamide (compound T-3-R)

[0263]

[0264]

[0265] The following synthetic route was used

[0266]

[0267] Step 1 Synthesis of compound N-(4-(5-amino-4-cyano-1-(1,1,1-trifluoropropan-2-yl-3,3,3-d3)-1H-pyrazol-3-yl)phenyl)-5-fluoro-2-(methoxy-d3)benzamide

[0268] To a 50 mL single-necked flask equipped with a magnetic stirrer and a condenser, add intermediate B-2 (0.29 g, 1.0 mmol), 5-amino-3-(4-bromophenyl)-1-(1,1,1-trifluoropropan-2-yl-3,3,3-d3)-1H-pyrazole-4-carbonitrile (0.36 g, 1.0 mmol), Cs2CO3 (0.98 g, 3.0 mmol), Xphos (48 mg, 0.1 mmol), Pd(OAc)2 (12 mg, 0.05 mmol), THF (10 mL) and water (1 mL), evacuate the mixture and replace with nitrogen three times, raise the temperature to 85°C and stir to react overnight. After cooling to room temperature, the solvent was evaporated under reduced pressure, saturated brine (5 mL) and ethyl acetate (20 mL) were added, the organic phase was separated, the aqueous phase was extracted with ethyl acetate (20 mL x 2), the organic phases were combined, dried over anhydrous sodium sulfate, concentrated and passed through a silica gel column to obtain 0.35 g of a white solid, with a yield of 75.0%. LC-MS (APCI): m / z = 468.2 (M+1) + .

[0269] Step 2 Synthesis of Compound T-3

[0270] Add N-(4-(5-amino-4-cyano-1-(1,1,1-trifluoropropan-2-yl-3,3,3-d3)-1H-pyrazol-3-yl)phenyl)-5-fluoro-2-(methoxy-d3)benzamide (0.35 g, 0.75 mmol) and methanesulfonic acid (3 mL) to a 50 mL single-necked flask equipped with a magnetic stirrer and a condenser, heat to 80°C, and stir under a nitrogen atmosphere for 1 hour. Cool to room temperature, add saturated brine (20 mL) and ethyl acetate (20 mL), adjust pH to 8 with ammonia water, separate the organic phase, extract the aqueous phase with ethyl acetate (20 mL x 2), combine the organic phases, dry over anhydrous sodium sulfate, filter, concentrate and pass through a silica gel column to obtain 0.30 g of a white solid with a yield of 82.4%. LC-MS (APCI): m / z=486.2 (M+1) + .

[0271] Step 3 Synthesis of compounds T-3-S and T-3-R

[0272] The racemic compound T-3 (0.30 g) was dissolved in methanol (10 mL) and subjected to chiral preparation by IC column using the following conditions: flow rate: 4 ml / min; mobile phase: MTBE:MeOH:EtOH=91:2:7 (0.1% triethylamine);

[0273] The corresponding fractions were collected and dried by spin drying to obtain 120 mg of compound T-3-S with a corresponding retention time of 25.942 min and 120 mg of compound T-3-R with a corresponding retention time of 31.415 min.

[0274] Biological activity testing.

[0275] (1) Kinase activity inhibition test

[0276] This experiment uses the HTRF TK Kit (Catalog No. 62TK0PEC) provided by Cisbio to detect the inhibitory activity of the compound against BTK kinase (Invitrogen, Catalog No. PV3587) and BTK C481S kinase (Carna Biosciences, Catalog No. 08-547) and the selectivity relative to EGFR, ITK and TEC kinase. After optimizing the detection conditions and confirming the detection conditions according to the kit instructions, prepare 10uL reaction system on ice, add the compound, substrate, ATP, and kinase to the 384-well plate in sequence, and use the kinase-free well as a negative control, i.e., a blank well; and use the compound-free well as a positive control. After reacting at room temperature for 30-60min, add the detection reagent, and after reacting at room temperature for 1h, use the microplate reader (BioTek, Synergy Neo2) to read the values ​​of 620nm (Cryptate) and 665nm (XL665), calculate the ratio of 665 / 620 for each well, and export the data. Calculate IC with GraphPadPrism7.0 software 50 .

[0277] Reagents and consumables: BTK kinase (Invitrogen, catalog number PV3587), BTK C481S (Carna, catalog number 08-547), ITK (Carna, catalog number 08-181), EGFR (Carna, catalog number 08-115), TEC (Carna, catalog number 08-182), HTRF-TK kit (Cisbio, catalog number 62TK0PEC), MgCl2 (Sigma, catalog number M1028), MnCl2 (Sigma, catalog number M1787), DTT (Sigma, catalog number D0632), ATP (Sigma, catalog number A7699-1g), DMSO (Sigma, catalog number D2650), 384-well plate (PE, catalog number 6008280).

[0278] Kinase activity analysis: Prepare the required compounds, and the final concentration of DMSO in the kinase detection system should not exceed 1%. The entire operation steps are carried out on ice, and the total reaction volume is 10μL. Add the compounds (4μL), substrates and ATP (4μL), and kinases (2μL) to the 384-well plate in sequence, shake for 30 seconds, react at room temperature for an appropriate time, add the detection reagent, react at room temperature for 1 hour, and detect with an enzyme reader. The specific steps are as follows:

[0279] a) Calculate the 1× kinase buffer required for kinase assay according to the amount of test compound and prepare as needed;

[0280] b) Prepare a 2.5× working concentration of the compound, dilute the 100-fold compound gradient concentration solution 40× to the required concentration, mix well, and add 4 μL to each well;

[0281] c) Prepare 5× working concentration of substrate and ATP, mix 1:1 before adding to 384-well plate, and add 4 μL to each well;

[0282] d) Prepare 5× working concentration of kinase and add 2 μL to each well;

[0283] e) After shaking on a microplate shaker for 30 seconds, seal the plate with a sealing film and react at room temperature for an appropriate time;

[0284] f) Prepare 4× working concentrations of streptavidin XL-665 and TK antibody-cryptate, mix 1:1 before adding to the 384-well plate, and add 10 μL to each well;

[0285] g) After 1 hour of reaction at room temperature, read the plate;

[0286] h) The blank control wells were those in which only substrate, ATP and 1% DMSO were added but no kinase was added, and the positive control wells were those in which only substrate, ATP, 1% DMSO and kinase were added.

[0287] The IC of the compound was obtained using the following nonlinear fitting formula: 50 (half inhibitory concentration):

[0288] Y=Bottom+(Top-Bottom) / (1+10^((LogIC50-X)*HillSlope))

[0289] X: log value of compound concentration

[0290] Y: Inhibition rate (%inhibition)

[0291] The calculation formula of kinase inhibition rate Inhibition (inhibition rate %) is:

[0292] Inhi. (%) = 1-(Ratio of test drug-Ratio of negative control) / (Ratio of positive control-Ratio of negative control)×100%.

[0293] The compounds of the present invention were tested in the above kinase inhibition experiment, and the experimental results showed that compared with the non-deuterated compounds, the compounds of the present invention had more potent activity against both wild-type BTK kinase and drug-resistant mutant BTK C481S kinase and excellent selectivity over EGFR, ITK and TEC kinase. The results of the representative example compounds are summarized in Table 1 below, where A represents IC 50 ≤10nM, B represents IC50 10-50nM, C represents IC 50 50-100nM, D represents IC 50 100-1000nM, E represents IC 50 1000-10000nM, F represents IC 50 >10000nM.

[0294] Table 1:

[0295]

[0296]

[0297] (2) Test of cell growth inhibitory activity

[0298] This experiment used the Promega Luminescence cell viability detection kit is a homogenous cell viability detection method that measures the cell viability of cultured cells (TMD-8 cells: human diffuse large B lymphoma cell line, REC1 cells: human mantle cell lymphoma cells, DOHH2 cells: human follicular lymphoma cells, Pfeiffer cells: human diffuse large cell lymphoma cells, Raji cells: human Burkitt lymphoma cells, RPMI-8226 cells: human multiple myeloma cells) by quantitative ATP. The specific experimental steps are as follows:

[0299] a) Collect cells in the logarithmic growth phase, prepare a cell suspension, count the cells, and calculate the cell concentration.

[0300] b) Dilute the cell suspension to the desired concentration and add it to the middle well of the 96-well plate. Add PBS to the edge wells. The wells without cells but with culture medium are used as blank controls.

[0301] c) In addition to the cell plate used for compound analysis, prepare another 96-well plate as the T0 plate, add 3-6 blank wells and 3-6 cell wells, and detect cell viability by CTG the next day as the T0 viability value of the cells.

[0302] d) Place the cell plate in a 37°C, 5% CO2 incubator and culture overnight.

[0303] e) The next day, the compound was diluted in various concentrations and the cell plate was placed in a 37°C, 5% CO2 incubator for 72 hours. The wells without compound but with 0.5% DMSO were used as positive controls for cell growth.

[0304] f) Add CTG reagent (CellTiter-Glo kit) according to the manufacturer's instructions to detect cell viability.

[0305] g) Read the plate using Biotek cytation3.0 microplate reader and export the data.

[0306] h) IC50 was calculated using GraphPad Prism 7.0 software.

[0307] The IC of the compound was obtained using the following nonlinear fitting formula: 50 (half inhibitory concentration):

[0308] Y=Bottom+(Top-Bottom) / (1+10^((LogIC 50 -X)*HillSlope))

[0309] X: log value of compound concentration

[0310] Y: Inhibition rate (%inhibition)

[0311] The calculation formula of cell inhibition rate Inhibition (inhibition rate %) is:

[0312] Inhi. (%) = 1-(Lum test drug-Lum vehicle control) / (Lum cell control-Lum vehicle control)×100%.

[0313] The above experimental results show that: compared with non-deuterated compounds, the compounds of the present invention have more potent activity against TMD8 cells, REC1 cells, DOHH2 cells, Pfeiffer cells, Raji cells and RPMI-8226 cells.

[0314] (3) Phosphorylation experiment

[0315] Using the Western Blot method, this experiment detected the target inhibitory effect of the compound of the present invention on BTK and BTK C481S in NIH / 3T3-FL-BTK and NIH / 3T3-FL-BTK-C481S cells by using a BCA protein concentration assay kit (brand beyotime, catalog number P0012).

[0316] Instruments and equipment: high-speed refrigerated centrifuge (Thermo Fisher / Sorvall Legend Micro 17R), vortexer (Scilogex / MX-S), microplate reader (Molecular Devices / SpectraMax Paradigm), electrophoresis instrument (Bio-rad / 1658001), membrane transfer instrument (Bio-rad / 1703935), automatic chemiluminescence image analysis system (Tanon / 5200), cell counter (Countstar / IC1000), TS-100 decolorization shaker (Qilin Bell / TS-100)

[0317] Cells and culture conditions:

[0318] cell characteristic Culture medium Remark NIH / 3T3-FL-BTK Adherent cells DMEM+10%FBS+1%PS 37℃, 5% CO2 NIH / 3T3-FL-BTK-C481S Adherent cells DMEM+10%FBS+1%PS 37℃, 5% CO2

[0319] Culture conditions: NIH / 3T3-FL-BTK and NIH / 3T3-FL-BTK-C481S were cultured in DMEM medium (Corning, USA) + 10% fetal bovine serum (Fetal Bovine Serum, Excell Bio) + 1% Penicillin Streptomycin solution (Coring, USA). After cell recovery, they were cultured for two generations before testing.

[0320] Experimental steps:

[0321] 1) Cell plating

[0322] One day in advance, take cells in logarithmic growth phase and count them with a cell counter. The cell viability is required to be >90%. Inoculate the cells into a sterile 6-well plate with 0.8×106 cells per well. Supplement DMEM complete medium to 2000 μL. Place the 6-well plate in a constant temperature incubator at 37°C and 5% CO2 to allow the cells to adhere to the wall.

[0323] 2) Compound administration

[0324] On the next day, the 6-well plate was taken out and the cells were observed under a microscope to verify that they were normal. In the clean bench, fresh DMEM complete medium was replaced with 1998 μL per well. 2 μL of the prepared gradient diluted 1000× stock solution (ARQ-531) was taken and added to the 6-well plate inoculated with cells. The final concentrations of the compounds were 1000 nM, 300 nM, 100 nM, 30 nM, 10 nM, 1 nM, and 0 nM, respectively. The 6-well plate was gently shaken to ensure even distribution of the compounds.

[0325] 3) Incubation

[0326] The 6-well plate was incubated in a constant temperature incubator at 37°C and 5% CO2 for 4 h;

[0327] 4) Sample collection

[0328] After the cells were cultured for 4 hours, the culture medium in the culture dish was removed, the cells were washed with PBS, and then digested with trypsin. The cells were centrifuged at 8000 rpm for 3 minutes, and the cells were collected in a 1.5 mL EP tube. The supernatant was discarded and the total protein was extracted.

[0329] 5) Cell lysis

[0330] Collect cells from each group into a 1.5 ml EP tube, and then add a certain volume (determined by the amount of cells, about 66 μL for every 0.8×106 cells) of Lysis Buffer (RIPA: protease / phosphatase inhibitor = 100:1). After adding Lysis Buffer, quickly place the EP tube on ice and use a vortexer to vortex the EP tube to fully lyse the cells. Be careful not to vortex for too long, and quickly place the sample tube on ice for lysis, shaking once every 10 minutes, for a total of three times. After the lysis time is over, place the EP tube in a precooled 4°C centrifuge and centrifuge at 12000rpm for 10 minutes. After centrifugation, transfer the supernatant to a new 1.5 ml EP tube and mark it.

[0331] 6) BCA protein concentration determination

[0332] Use the BCA protein concentration determination kit to quantify the protein in the sample lysate supernatant according to the kit instructions. After the protein concentration is determined, the lysate supernatant with the lowest concentration is used as the benchmark, and the remaining lysate supernatants are diluted to this concentration using Lysis Buffer after calculation according to the BCA standard curve. Add the corresponding volume of 5× loading Buffer to the diluted lysate supernatant, heat it in a 95℃ metal bath for 10 minutes, then cool it on ice, and store the sample in a -20℃ refrigerator.

[0333] 7) Glue making

[0334] Wash the glass plates used for making gel, align the bottoms of the two glass plates, put them into the bracket, and fix them with clamps. Prepare the separation gel according to the 10% separation gel formula, mix it well and add it to the glass plate, stop when it is about 3 / 4 of the glass plate, and press the gel with 75% ethanol. After 90 minutes, the separation gel solidifies, pour out the 75% ethanol, use filter paper to absorb the residual ethanol solution on the glass plate, prepare 5% concentrated gel, mix it well and add it to the glass plate, then insert the sample comb, and wait for 60 minutes to make the gel.

[0335] 8) Protein loading and electrophoresis

[0336] Place the gel plate in the electrophoresis tank and fix it. Fill the inner tank with 1× electrophoresis solution and the outer tank exceeds the metal wire. Remove the sample comb, load 25μg of protein samples of NIH / 3T3-FL-BTK and NIH / 3T3-FL-BTK-C481S cells, and then load the protein ladder on each gel. Turn on the power supply, run the electrophoresis at a constant voltage of 70V for 30min, and then continue the electrophoresis at a constant voltage of 86V for 1h20min until the bromophenol blue indicator band reaches the bottom, and then stop the electrophoresis.

[0337] 9) Protein electroporation

[0338] Soak the PVDF membrane in methanol for 30 seconds to wake up the membrane, then put it into the transfer box together with 4 filter papers and 2 sponges. Pour the pre-prepared 1× electrotransfer solution into the transfer box. Take out the gel, cut it into the required size, and place it in the transfer folder in the order of sponge, filter paper, gel, PVDF membrane, filter paper, and sponge. Remove bubbles, place the membrane on the anode side and the gel on the cathode side (black gel and white film), insert it into the electrophoresis tank, pour in the transfer buffer, put in ice bags, and place ice bags around the electrophoresis tank to cool it down. Constant voltage 100V electrophoresis transfer for 90 minutes.

[0339] 10) Blocking and incubation with primary antibody

[0340] After electroporation, place the PVDF membrane in a blocking solution of 5% skim milk, place it on a shaker and shake it slowly, and block it at room temperature for 1 hour. Wash the blocked PVDF membrane slowly in TBST for 3 times, 10 minutes each time. After washing, place the PVDF membrane in the antibody box of the primary antibody, dilute the primary antibody with the primary antibody diluent at a ratio of 1:1000, and place the antibody box in a 4°C gentle shaker for incubation (10-16 hours).

[0341] 11) Incubate with secondary antibody and develop color

[0342] Remove the PVDF membrane from the antibody box after overnight incubation and recover the primary antibody. Wash the PVDF membrane slowly in TBST for 3 times, 10 minutes each time. Place the washed PVDF membrane in the dilution solution of the corresponding species (1:3000) (containing 5% skim milk) and incubate it on a gentle shaker at room temperature for 1 hour. After the secondary antibody incubation, remove the PVDF membrane and wash it slowly in TBST for 3 times, 10 minutes each time. Mix an equal volume of A solution and B solution in the ECL kit, evenly add it to the surface of the membrane (protein side), place it in a Tanon 5200 luminescence imager (turn on precooling 5 minutes in advance) for exposure and development, and take pictures to save the pictures.

[0343] 12) Reagent formula:

[0344] 1x electrophoresis buffer formula: Tris 3.02g, glycine 18.8g, SDS 1.0g;

[0345] 10x electroporation buffer formula: glycine 151.1g, Tris 30.3g;

[0346] 1x electrotransfer buffer recipe: 200 ml of 75% ethanol, 100 ml of 10x electrotransfer buffer, 700 ml of water, and dilute to 1 L;

[0347] 1x TPST formula: NaCl 8.7g, Tris 2.4g, pH 7.2-7.4, Tween 20 1ml;

[0348] 13) Western Blot band gray value analysis

[0349] Image J2X software was used to analyze the gray value of the p-BTKY223 and GAPDH bands in the Western Blot result graph. The gray value results were displayed in curve graphs, with the abscissa being the compound concentration and the ordinate being the value of the drug concentration inhibition rate. The ordinate value was calculated using the following formula: Inhibition (%) = 100% - Gray compound / Gray dmso * 100%, and was fitted using GraphPad Prism software, with the fitting method being log (inhibitor) vs. response -- Variable slope (four parameters).

[0350] The above experimental results show that compared with non-deuterated compounds, the compounds of the present invention have better BTK and BTK C481S target inhibitory activity on NIH / 3T3-FL-BTK and NIH / 3T3-FL-BTK-C481S cells.

[0351] (4) Metabolic stability evaluation

[0352] Metabolic stability is generally used to describe the speed and extent of a compound's metabolism, and is one of the main factors affecting pharmacokinetic properties. Many compounds are substrates for CYP450 enzymes and other drug-metabolizing enzymes, and liver microsomes are a system rich in CYP450. The purpose of this experiment is to study the in vitro metabolic stability of the compound by incubating it with human and SD rat liver microsomes and using LC-MS / MS to detect the remaining proportion of the compound.

[0353] ① Preparation of solution

[0354] Phosphate buffered saline (PBS): Mix 150 mL of pre-prepared KH2PO4 (0.5 M) solution and 700 mL of K2HPO4 (0.5 M) solution, and adjust the pH value of the mixture to 7.4 with K2HPO4 (0.5 M) solution to prepare 5-fold PBS, and store at 4°C for later use. Dilute 5-fold with ultrapure water before use, and add 3.3 mM magnesium chloride to obtain phosphate buffered saline (PBS) (100 mM).

[0355] NADPH regeneration system solution: Use 5 mL of PBS to prepare an NADPH solution containing 6.5 mM NADP, 16.5 mM G-6-P, and 3 U / mL G-6-PD.

[0356] Internal standard stop solution: Prepare 50 ng / mL propranolol hydrochloride and 200 ng / mL tolbutamide in acetonitrile as internal standard working solution.

[0357] Human liver microsome solution: Take 0.31 mL of human liver microsomes (25 mg / mL) and add them to 0.961 mL of PBS (pH 7.4) and mix well to obtain a human liver microsome dilution solution with a protein concentration of 0.625 mg / mL.

[0358] SD rat liver microsome solution: Take 0.31 mL of SD rat liver microsome (25 mg / mL) and add it to 0.961 mL of PBS (pH 7.4) and mix well to obtain a SD rat liver microsome dilution solution with a protein concentration of 0.625 mg / mL.

[0359] Sample working solution: The compound of the present invention and non-deuterated compound powder, positive control dextromethorphan powder and omeprazole powder were prepared with DMSO to 10 mM as sample stock solution, and then diluted with 70% acetonitrile-water to obtain 0.25 mM sample working solution.

[0360] ②Sample incubation

[0361] 398 μL of human liver microsome dilution was added to a 96-well incubation plate (N=2), and 2 μL of 0.25 mM test compound and dextromethorphan were added respectively and mixed.

[0362] 398 μL of SD rat liver microsome dilution was added to a 96-well incubation plate (N=2), and 2 μL of 0.25 mM test compound and omeprazole were added respectively and mixed.

[0363] Add 300 μL of pre-cooled stop solution to each well of a 96-well deep-well plate and place on ice to serve as a stop plate.

[0364] Place the 96-well incubation plate and NADPH regeneration system in a 37°C water bath, shake at 100 rpm, and pre-incubate for 5 minutes. Take out 80 μL of incubation solution from each well of the incubation plate and add it to the termination plate, mix well, and add 20 μL of NADPH regeneration system solution as the 0min sample. Add 80 μL of NADPH regeneration system solution to each well of the incubation plate, start the reaction, and start timing. The reaction concentration of the test compound is 1 μM, and the protein concentration is 0.5 mg / mL.

[0365] At 10, 30, and 90 min of reaction, 100 μL of the reaction solution was taken and added to the stop plate, and vortexed for 3 min to terminate the reaction.

[0366] The stop plate was centrifuged at 5000 rpm and 4°C for 15 min. 200 μL of supernatant was taken into a 96-well plate pre-added with 200 μL of ultrapure water, mixed well, and sampled by LC-MS / MS, injecting 10 uL.

[0367] ③Sample analysis method

[0368] In this experiment, the LC-MS / MS system was used to detect the peak areas of the test compound, dextromethorphan, omeprazole and internal standard, and the peak area ratio of the compound to the internal standard was calculated.

[0369] ④Data processing

[0370] The peak areas of the samples and internal standards were obtained by mass spectrometer and Analyst software. The residual amount of the compound (R%) and time were plotted using the single exponential degradation model of Graphpad prism7.0 software to obtain the substrate elimination rate constant K.

[0371] Ct / C0=exp(-K*t)

[0372] The half-life T is calculated according to the following formula: 1 / 2 and intrinsic clearance CL int , where V / M is equal to 1 / C(protein).

[0373] t 1 / 2 (min); CL int (μL / min / mg).

[0374] Experimental results: The compounds of the present invention and their non-deuterated compounds were tested and compared to evaluate their metabolic stability in human and SD rat liver microsomes. Compared with the non-deuterated compounds, the compounds of the present invention have a longer half-life T 1 / 2 and lower clearance CL int The results of representative example compounds are summarized in Table 2 below.

[0375] Table 2:

[0376]

[0377] (5) Pharmacokinetic experiments in rats

[0378] Six male Sprague-Dawley rats, 7-8 weeks old, weighing about 210 g, were divided into two groups, 3 rats in each group, and a single dose of the compound (10 mg / kg orally) was administered intravenously or orally to compare the pharmacokinetic differences.

[0379] Rats were fed with standard diet and given water. Fasting began 16 hours before the experiment. Drugs were dissolved with PEG400 and dimethyl sulfoxide. Blood was collected from the orbit at 0.083 hours, 0.25 hours, 0.5 hours, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, and 24 hours after administration.

[0380] The rats were briefly anesthetized by inhalation of ether, and 300 μL of blood was collected from the orbits into test tubes. The test tubes contained 30 μL of 1% heparin saline solution. Before use, the test tubes were dried at 60°C overnight. After the last time point of blood sampling, the rats were killed under ether anesthesia.

[0381] After blood sample collection, gently invert the test tube at least 5 times to ensure sufficient mixing and place on ice. The blood sample is centrifuged at 4°C 5000rpm for 5 minutes to separate plasma from red blood cells. Use a pipette to aspirate 100μL of plasma into a clean plastic centrifuge tube and mark the name of the compound and time point. The plasma is stored at -80°C before analysis. The concentration of the compound of the present invention in plasma is determined by LC-MS / MS. Pharmacokinetic parameters are calculated based on the blood drug concentration of each animal at different time points.

[0382] The experiments show that the compounds of the present invention have better pharmacokinetic properties in animals, and therefore have better pharmacodynamics and therapeutic effects. The results of the representative example compounds are summarized in Table 3 below.

[0383] Table 3:

[0384]

[0385]

[0386] The above contents are further detailed descriptions of the present invention in combination with specific preferred embodiments, and it cannot be determined that the specific implementation of the present invention is limited to these descriptions. For ordinary technicians in the technical field to which the present invention belongs, several simple deductions or substitutions can be made without departing from the concept of the present invention, which should be regarded as falling within the protection scope of the present invention.

Claims

1. Compound of formula (II): in, R1, R2 and R3 are hydrogen; X1 and X2 are each independently selected from CH3 or CD3; Provided that the above compound contains at least three deuterium atoms; or a pharmaceutically acceptable salt thereof.

2. The compound according to claim 1, which is a compound of formula (IIA): in, R1, R2 and R3 are hydrogen; X1 and X2 are each independently selected from CH3 or CD3; Provided that the above compound contains at least three deuterium atoms; or a pharmaceutically acceptable salt thereof.

3. The compound according to claim 1, which is a compound of formula (IIB): in, R1, R2 and R3 are hydrogen; X1 and X2 are each independently selected from CH3 or CD3; Provided that the above compound contains at least three deuterium atoms; or a pharmaceutically acceptable salt thereof.

4. The compound according to any one of claims 1 to 3, wherein X1 is CD3.

5. The compound according to any one of claims 1 to 3, wherein X2 is CD3.

6. The compound according to claim 4, wherein X2 is CD3.

7. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein The compound is selected from:

8. A pharmaceutical composition comprising a pharmaceutically acceptable excipient and the compound or pharmaceutically acceptable salt according to any one of claims 1 to 7.

9. Use of the compound or pharmaceutically acceptable salt according to any one of claims 1 to 7, or the pharmaceutical composition according to claim 8, in the preparation of a medicament for treating and / or preventing a disease associated with BTK.

10. The use according to claim 9, wherein The BTK is selected from wild-type BTK or mutant BTK.

11. The use according to claim 10, wherein The mutant BTK is selected from BTK C481S, BTK C481F, BTK C481Y, BTK C481R, BTK C481T, BTK C481G or BTK C481W.

12. The use according to claim 11, wherein the mutant BTK is selected from BTK C481S.

13. The use of claim 9, wherein The BTK-related disease is selected from cancer, inflammatory disorder, immune disease or fibrosis.

14. The use according to claim 13, wherein The cancer is selected from hematological cancers.

15. The use according to claim 14, wherein The hematological cancer is selected from the group consisting of acute lymphocytic leukemia, acute myeloid leukemia, acute promyelocytic leukemia, chronic lymphocytic lymphoma, chronic myeloid leukemia, chronic myelomonocytic leukemia, chronic neutrophilic leukemia, acute undifferentiated leukemia, anaplastic large cell lymphoma, prolymphocytic leukemia, juvenile myelomonocytic leukemia, adult T-cell leukemia, acute myeloid leukemia with trilineage myelodysplasia, Mixed lineage leukemia, myelodysplastic syndrome, myeloproliferative disorders, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone lymphoma, Burkitt lymphoma, Waldenstrom's macroglobulinemia, primary central nervous system lymphoma, small lymphocytic lymphoma, precursor B-cell lymphoblastic leukemia, hairy cell leukemia, mucosa-associated lymphoid tissue lymphoma, plasma cell myeloma, plasmacytoma, and multiple myeloma.

16. The use of claim 13, wherein The cancer is selected from glioma.

17. The use of claim 13, wherein The immune disease is selected from arthritis, multiple sclerosis or osteoporosis.

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