WO2023284765A1

WO2023284765A1 - Substituted pyrazole compound, composition containing same and use thereof

Info

Publication number: WO2023284765A1
Application number: PCT/CN2022/105358
Authority: WO
Inventors: 王义汉; 李焕银
Original assignee: 深圳市塔吉瑞生物医药有限公司
Priority date: 2021-07-16
Filing date: 2022-07-13
Publication date: 2023-01-19
Also published as: CN115611810A

Abstract

A substituted pyrazole compound, a composition containing the compound, and a use thereof, the compound being a compound represented by formula (I) or a tautomer, stereoisomer, prodrug, crystal form, pharmaceutically acceptable salt, hydrate or solvate thereof. The compound of formula (I) can be used as a reversible Bruton's tyrosine kinase (BTK) inhibitor for the treatment of BTK-related diseases, and has high selectivity and good pharmacokinetic properties.

Description

Substituted pyrazole compounds and compositions comprising the same and uses thereof

technical field

The invention belongs to the technical field of medicine, and in particular relates to a substituted pyrazole compound, a composition containing the compound and its application. More specifically, the present invention relates to certain deuterium-substituted 5-amino-3-(4-(((5-fluoro-2-methoxybenzoyl)amino)methyl)phenyl)-1-( Compounds of 1,1,1-trifluoropropan-2-yl)-1H-pyrazole-4-carboxamide and its derivatives and their tautomers, stereoisomers, prodrugs, crystal forms, pharmacy acceptable salts, hydrates or solvates. These deuterium-substituted compounds and compositions thereof can be used as reversible Bruton's tyrosine kinase (Bruton's tyrosine kinase, BTK) inhibitors for the treatment of BTK-related diseases with high selectivity and good pharmacokinetics academic characteristics.

Background technique

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 transduced by immunoglobulin λ (immunoglobulin, lgλ), which induces the phosphorylation of members of the Src kinase family, and catalyzes the double phosphorylation of Tyr551 and Tyr223 in BTK to activate BTK. Activated BTK promotes phosphorylation of downstream phospholipase C gamma (PLC gamma), and phosphorylated PLC gamma hydrolyzes 4,5-bisphosphate phosphatidylinositol (phosphatidylinositol4,5-bisphosphate, PIP2) to generate inositol triphosphate ( inositol triphosphate, IP3) and diacyl glycerol (DAG). IP3 promotes intracellular calcium release, and DAG cooperates with calcium ion to activate protein kinases C (protein kinases, MAPKs), mammalian target of rapamycin (mTOR) and other signaling pathways to activate, 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 NF-κB sites on DNA to play a role in regulating cell functions.

BTK is an essential component of receptor signaling in normal and malignant B cells. 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 Macroglobulinemia (waldenstrom's macroglobulinemia, WM) and marginal zone lymphoma (marginal zone lymphoma, MZL).

Some covalent bond BTK inhibitors, including ibrutinib, acatinib, zanubrutinib, etc., all irreversibly bind to C481 in the BTK protein kinase domain through covalent bonding, thereby inhibiting BTK enzyme activity the goal of. After long-term use of these drugs, the amino acid residue C481 on BTK may be mutated, so that the original inhibitor can no longer form a covalent bond with BTK, and the inhibitory activity on BTK is greatly weakened, which in turn leads to the development of covalent BTK inhibitors in the human body. drug resistance.

Pirtobrutinib (chemical name is (S)-5-amino-3-(4-(((5-fluoro-2-methoxybenzoyl)amino)methyl)phenyl)-1-(1,1, 1-trifluoropropan-2-yl)-1H-pyrazole-4-carboxamide, which has the following structural formula) is an investigational, oral, highly selective non-covalent BTK inhibitor regardless of BTK turnover rate Either way, it provides sustained high on-target coverage, maintains activity in the presence of the C481 acquired resistance mutation, and avoids complications caused by off-target kinases of covalent and non-covalent BTK inhibitors under development.

Poor absorption, distribution, metabolism and/or excretion (ADME) properties are known to be the main reason for the failure of many drug candidates in clinical trials. Many currently marketed drugs also limit their range of applications due to poor ADME properties. The rapid metabolism of drugs will cause many drugs that can treat diseases with high efficiency to be eliminated from the body too quickly, making it difficult to become drugs. Although frequent or high doses of drugs may solve the problem of rapid drug clearance, this method will bring problems such as poor patient compliance, side effects caused by high doses of drugs, and increased treatment costs. In addition, rapidly metabolizing drugs may also expose patients to undesirable toxic or reactive metabolites.

Discovering new and effective non-covalent BTK inhibitors with good oral bioavailability and druggability is still a challenging task. Therefore, there is still a need in the art to develop compounds with selective inhibitory activity and/or better pharmacodynamics/pharmacokinetics for use as next-generation non-covalent BTK inhibitors, and the present invention provides such compounds.

Contents of the invention

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 bond BTK inhibitor, whose inhibitory activity on BTK and C481 mutant BTK reaches the nanomolar level, Significantly higher than other kinases (such as EGFR), which ensures that the compound of the present invention has high activity and high selectivity for BTK. In addition, the compounds of the present invention also show better metabolic stability and/or pharmacokinetic properties.

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

The first aspect of the present invention provides formula (I) compound:

in,

Y ₁ , Y ₂ , Y ₃ , Y ₄ , Y ₅ , Y ₆ and Y ₇ are each independently selected from hydrogen, deuterium, halogen or trifluoromethyl;

R ₁ , R ₂ and R ₃ are each independently selected from hydrogen or deuterium;

X ₁ and X ₂ are each independently selected from CH ₃ , CD ₃ , CHD ₂ or CH ₂ D;

The additional condition is that the above compounds contain at least one deuterium atom;

Or a tautomer, stereoisomer, prodrug, crystal form, pharmaceutically acceptable salt, hydrate or solvate thereof.

In another aspect, the present invention provides compounds containing the present invention or tautomers, stereoisomers, prodrugs, crystal forms, pharmaceutically acceptable salts, hydrates or solvates and pharmaceutically acceptable Excipients for pharmaceutical compositions. In a specific embodiment, the compound of the invention is provided in said pharmaceutical composition in an effective amount. In specific embodiments, the compounds of the invention are provided in a therapeutically effective amount. In specific embodiments, the compounds of the invention are provided in a prophylactically effective amount. In specific embodiments, the pharmaceutical composition further comprises an additional therapeutic agent.

In another aspect, the present invention provides a method for preparing the above-mentioned pharmaceutical composition, comprising the following steps: mixing a pharmaceutically acceptable excipient with the compound of the present invention or its tautomer, stereoisomer body, prodrug, crystal form, pharmaceutically acceptable salt, hydrate or solvate are mixed to form a pharmaceutical composition.

In another aspect, the present invention provides the compound of the present invention or its tautomer, stereoisomer, prodrug, crystal form, pharmaceutically acceptable salt, hydrate or solvate, or the above pharmaceutical composition in Use in the preparation of medicines for treating and/or preventing diseases related to BTK.

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

In specific embodiments, 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.

In specific embodiments, said BTK-associated disease is selected from cancer, inflammatory disorders, immune diseases or fibrosis. In a specific embodiment, the cancer is selected from hematological cancers. In particular embodiments, the hematological cancer is selected from acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), acute promyelocytic leukemia (APL), chronic lymphocytic lymphoma (CLL), chronic myeloid Leukemia (CML), Chronic Myelomonocytic Leukemia (CMML), Chronic Neutrophil 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 with trilineage myelodysplastic disorder (AML/TMDS), mixed lineage leukemia (MLL), myelodysplastic syndrome syndrome (MDS), myeloproliferative disorder (MPD), diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone lymphoma (eg, splenic marginal zone lymphoma, extranodal marginal zone B lymphoma), Burkitt's lymphoma, Waldenstrom's macroglobulinemia (lymphoplasmacytic lymphoma), primary central nervous system lymphoma, small lymphocytic lymphoma, precursor B 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 glioblastoma. In a specific embodiment, said immune disease is selected from arthritis, multiple sclerosis, osteoporosis.

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

definition

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

Herein, unless otherwise specified, "non-deuterated compound" refers to a compound containing deuterium atoms in a proportion not higher than the natural deuterium isotope content (0.015%).

As used herein, the term "subject" includes, but is not limited to: human (i.e., male or female of any age group, e.g., pediatric subjects (e.g., infants, children, adolescents) or adult subjects (e.g., Young, middle-aged, or older adults)) and/or non-human animals, e.g., mammals, e.g., primates (e.g., cynomolgus monkeys, rhesus monkeys), cows, 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.

"Disease", "disorder" and "condition" are used interchangeably herein.

As used herein, unless otherwise specified, the term "treating" includes an effect on a subject suffering from a particular disease, disorder or condition, which reduces the severity of the disease, disorder or condition, or delays or slows down the disease, disorder or the development of a condition ("therapeutic treatment"), and also includes effects that occur before a subject begins to suffer from a particular disease, disorder or disease ("prophylactic treatment").

In general, an "effective amount" of a compound refers to an amount sufficient to elicit a desired biological response. As will be appreciated by those of ordinary skill in the art, the effective amount of a compound of the present invention may vary depending on factors such as, for example, the biological target, the pharmacokinetics of the compound, the disease being treated, the mode of administration, and the age of the subject. Health conditions and symptoms. An effective amount includes therapeutically and prophylactically effective amounts.

As used herein, unless otherwise specified, a "therapeutically effective amount" of a compound is an amount sufficient to provide a therapeutic benefit in the treatment of a disease, disorder, or condition, or to induce one or more effects associated with the disease, disorder, or condition. Symptoms are delayed or minimized. A therapeutically effective amount of a compound refers to that amount of the therapeutic agent, alone or in combination with other therapies, which provides a therapeutic benefit in the treatment of a disease, disorder or condition. The term "therapeutically effective amount" can include an amount that improves overall therapy, reduces or avoids symptoms or causes of a disease or disorder, or enhances the therapeutic efficacy of other therapeutic agents.

As used herein, unless otherwise specified, a "prophylactically effective amount" of a compound 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 to prevent a disease , the number of recurrences of the disorder or condition. A prophylactically effective amount of a compound refers to that amount of the therapeutic agent, alone or in combination with other agents, which provides a prophylactic benefit in the prevention of a disease, disorder or condition. The term "prophylactically effective amount" can include amounts that improve overall prophylaxis, or that enhance the prophylactic efficacy of other prophylactic agents.

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

detailed description

compound

Herein, "the compound of the present invention" refers to the following formula (I)-formula (II) compound and its subset (for example, the compound of formula (IA), formula (IB)), or its tautomer, Stereoisomers, prodrugs, crystal forms, pharmaceutically acceptable salts, hydrates or solvates.

In one embodiment, the invention relates to compounds of formula (I):

in,

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 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%.

Specifically, in the present invention, deuterium at each deuterated position of Y ₁ , Y ₂ , Y ₃ , Y ₄ , Y ₅ , Y ₆ , Y ₇ , R ₁ , R ₂ , R ₃ , X ₁ and X ₂ The isotopic content is at least 0.015% greater than the natural isotopic content, 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%.

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 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 Preferably contain eleven deuterium atoms, more preferably contain twelve deuterium atoms, more preferably contain thirteen deuterium atoms, more preferably contain fourteen deuterium atoms, more preferably contain fifteen deuterium atoms, more preferably Preferably contains sixteen deuterium atoms.

In another specific embodiment, "Y ₁ , Y ₂ , Y ₃ , Y ₄ , Y ₅ , Y ₆ and Y ₇ are each independently selected from hydrogen, deuterium, halogen or trifluoromethyl" includes Y ₁ 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 The technical solution of hydrogen, deuterium, halogen or trifluoromethyl. More specifically, including Y ₁ is hydrogen, Y ₁ is deuterium, Y ₁ is halogen (F, Cl, Br or I) or Y ₁ is trifluoromethyl, Y ₂ is hydrogen, Y ₂ is deuterium, Y ₂ 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 Y ₇ is hydrogen, Y ₇ is deuterium, Y ₇ is a halogen (F, Cl, Br or I) or Y ₇ is a technical scheme of trifluoromethyl.

In another specific embodiment, "R ₁ , R ₂ and R ₃ are each independently selected from hydrogen or deuterium" includes R ₁ is selected from hydrogen or deuterium, R ₂ is selected from hydrogen or deuterium, and R ₃ is selected from hydrogen or deuterium Deuterium technical solution. More specifically, including R ₁ is hydrogen or R ₁ is deuterium, R ₂ is hydrogen or R ₂ is deuterium, and R ₃ is hydrogen or R ₃ is deuterium technical scheme.

In another specific embodiment, "X ₁ and X ₂ are each independently selected from CH ₃ , CD ₃ , CHD ₂ or CH ₂ D" includes that X ₁ is selected from CH ₃ , CD ₃ , CHD ₂ or CH ₂ D, and X ₂ are selected from the technical scheme of CH ₃ , CD ₃ , CHD ₂ or CH ₂ D. More specifically, including X ₁ is CH ₃ , X ₁ is CD ₃ , X ₁ is CHD ₂ or X ₁ is CH ₂ D, and X ₂ is CH ₃ , X ₂ is CD ₃ , X ₂ is CHD ₂ or X ₂ is the technical scheme of CH ₂ D.

In another embodiment, the present invention relates to compounds of formula (IA):

in,

In another embodiment, the present invention relates to compounds of formula (IB):

in,

In some embodiments of general formula (I), formula (IA) and formula (IB), preferably, the present invention relates to the above compounds, or their tautomers, stereoisomers, prodrugs, crystal forms, pharmaceutical Acceptable salts, hydrates or solvates above, wherein X ₁ is CD ₃ , Y ₁ , Y ₂ , Y ₃ , Y ₄ , Y ₅ , Y ₆ , Y ₇ , R ₁ , R ₂ , R ₃ and _X2 is as defined above.

In some embodiments of general formula (I), formula (IA) and formula (IB), preferably, the present invention relates to the above compounds, or their tautomers, stereoisomers, prodrugs, crystal forms, pharmaceutical Acceptable salts, hydrates or solvates above, wherein X ₂ is CD ₃ , Y ₁ , Y ₂ , Y ₃ , Y ₄ , Y ₅ , Y ₆ , Y ₇ , R ₁ , R ₂ , R ₃ and _X1 is as defined above.

In some embodiments of general formula (I), formula (IA) and formula (IB), preferably, the present invention relates to the above compounds, or their tautomers, stereoisomers, prodrugs, crystal forms, pharmaceutical Acceptable salts, hydrates or solvates above, wherein X ₁ and X ₂ are CD ₃ , Y ₁ , Y ₂ , Y ₃ , Y ₄ , Y ₅ , Y ₆ , Y ₇ , R ₁ , R ₂ and _R3 is as defined above.

In some embodiments of general formula (I), formula (IA) and formula (IB), preferably, the present invention relates to the above compounds, or their tautomers, stereoisomers, prodrugs, crystal forms, pharmaceutical Acceptable salts, hydrates or solvates above, wherein, R ₁ is deuterium, Y ₁ , Y ₂ , Y ₃ , Y ₄ , Y ₅ , Y ₆ , Y ₇ , R ₂ , R ₃ , X ₁ and X ₂ as defined above.

In some embodiments of general formula (I), formula (IA) and formula (IB), preferably, the present invention relates to the above compounds, or their tautomers, stereoisomers, prodrugs, crystal forms, pharmaceutical Acceptable salts, hydrates or solvates above, wherein, R ₁ is deuterium, X ₁ is CD ₃ , Y ₁ , Y ₂ , Y ₃ , Y ₄ , Y ₅ , Y ₆ , Y ₇ , R ₂ , R ₃ and _X2 are as defined above.

In some embodiments of general formula (I), formula (IA) and formula (IB), preferably, the present invention relates to the above compounds, or their tautomers, stereoisomers, prodrugs, crystal forms, pharmaceutical Acceptable salts, hydrates or solvates above, wherein R ₂ is deuterium, Y ₁ , Y ₂ , Y ₃ , Y ₄ , Y ₅ , Y ₆ , Y ₇ , R ₁ , R ₃ , X ₁ and X ₂ as defined above.

In some embodiments of general formula (I), formula (IA) and formula (IB), preferably, the present invention relates to the above compounds, or their tautomers, stereoisomers, prodrugs, crystal forms, pharmaceutical Acceptable salts, hydrates or solvates above, wherein, R ₃ is deuterium, Y ₁ , Y ₂ , Y ₃ , Y ₄ , Y ₅ , Y ₆ , Y ₇ , R ₁ , R ₂ , X ₁ and X ₂ as defined above.

In some embodiments of general formula (I), formula (IA) and formula (IB), preferably, the present invention relates to the above compounds, or their tautomers, stereoisomers, prodrugs, crystal forms, pharmaceutical Acceptable salts, hydrates or solvates above, wherein R ₂ and R ₃ are deuterium, Y ₁ , Y ₂ , Y ₃ , Y ₄ , Y ₅ , Y ₆ , Y ₇ , R ₁ , X ₁ and X ₂ as defined above.

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

in,

In another embodiment, the present invention relates to compounds of formula (IIA):

in,

In another embodiment, the present invention relates to compounds of formula (IIB):

in,

In some embodiments of general formula (II), formula (IIA) and formula (IIB), preferably, the present invention relates to the above compounds, or their tautomers, stereoisomers, prodrugs, crystal forms, pharmaceutical An acceptable salt, hydrate or solvate of the above, wherein X ₁ is CD ₃ , and R ₁ , R ₂ , R ₃ and X ₂ are as defined above.

In some embodiments of general formula (II), formula (IIA) and formula (IIB), preferably, the present invention relates to the above compounds, or their tautomers, stereoisomers, prodrugs, crystal forms, pharmaceutical An acceptable salt, hydrate or solvate of the above, wherein X ₂ is CD ₃ , and R ₁ , R ₂ , R ₃ and X ₁ are as defined above.

In some embodiments of general formula (II), formula (IIA) and formula (IIB), preferably, the present invention relates to the above compounds, or their tautomers, stereoisomers, prodrugs, crystal forms, pharmaceutical An acceptable salt, hydrate or solvate of the above, wherein X ₁ and X ₂ are CD ₃ , and R ₁ , R ₂ and R ₃ are as defined above.

In some embodiments of general formula (II), formula (IIA) and formula (IIB), preferably, the present invention relates to the above compounds, or their tautomers, stereoisomers, prodrugs, crystal forms, pharmaceutical An acceptable salt, hydrate or solvate of the above, wherein R ₁ is deuterium, and R ₂ , R ₃ , X ₁ and X ₂ are as defined above.

In some embodiments of general formula (II), formula (IIA) and formula (IIB), preferably, the present invention relates to the above compounds, or their tautomers, stereoisomers, prodrugs, crystal forms, pharmaceutical The above acceptable salt, hydrate or solvate, wherein, R ₁ is deuterium, X ₁ is CD ₃ , R ₂ , R ₃ and X ₂ are as defined above.

In some embodiments of general formula (II), formula (IIA) and formula (IIB), preferably, the present invention relates to the above compounds, or their tautomers, stereoisomers, prodrugs, crystal forms, pharmaceutical The above acceptable salt, hydrate or solvate, wherein, R ₁ is deuterium, X ₂ is CD ₃ , R ₂ , R ₃ and X ₁ are as defined above.

In some embodiments of general formula (II), formula (IIA) and formula (IIB), preferably, the present invention relates to the above compounds, or their tautomers, stereoisomers, prodrugs, crystal forms, pharmaceutical The above acceptable salt, hydrate or solvate, wherein, R ₁ is deuterium, X ₁ and X ₂ are CD ₃ , R ₂ and R ₃ are as defined above.

In some embodiments of general formula (II), formula (IIA) and formula (IIB), preferably, the present invention relates to the above compounds, or their tautomers, stereoisomers, prodrugs, crystal forms, pharmaceutical An acceptable salt, hydrate or solvate of the above, wherein R ₂ is deuterium, and R ₁ , R ₃ , X ₁ and X ₂ are as defined above.

In some embodiments of general formula (II), formula (IIA) and formula (IIB), preferably, the present invention relates to the above compounds, or their tautomers, stereoisomers, prodrugs, crystal forms, pharmaceutical An acceptable salt, hydrate or solvate of the above, wherein R ₃ is deuterium, and R ₁ , R ₂ , X ₁ and X ₂ are as defined above.

In some embodiments of general formula (II), formula (IIA) and formula (IIB), preferably, the present invention relates to the above compounds, or their tautomers, stereoisomers, prodrugs, crystal forms, pharmaceutical An acceptable salt, hydrate or solvate of the above, wherein R ₂ and R ₃ are deuterium, and R ₁ , X ₁ and X ₂ are as defined above.

In some embodiments of general formula (II), formula (IIA) and formula (IIB), preferably, the present invention relates to the above compounds, or their tautomers, stereoisomers, prodrugs, crystal forms, pharmaceutical An acceptable salt, hydrate or solvate of the above, wherein X ₁ is CD ₃ , R ₂ and R ₃ are deuterium, and R ₁ and X ₂ are as defined above.

In some embodiments of general formula (II), formula (IIA) and formula (IIB), preferably, the present invention relates to the above compounds, or their tautomers, stereoisomers, prodrugs, crystal forms, pharmaceutical An acceptable salt, hydrate or solvate of the above, wherein X ₂ is CD ₃ , R ₂ and R ₃ are deuterium, and R ₁ and X ₁ are as defined above.

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 :

The compounds of the present invention may include one or more asymmetric centers, and thus may exist in various stereoisomeric forms, eg, enantiomeric and/or diastereomeric forms. For example, the compounds of the invention may be individual enantiomers, diastereoisomers or geometric isomers (eg cis and trans isomers), or may be in the form of a mixture of stereoisomers, Racemic mixtures and mixtures enriched in one or more stereoisomers are included. Isomers can be separated from mixtures by methods known to those skilled in the art, including: chiral high pressure liquid chromatography (HPLC) and formation and crystallization of chiral salts; or preferred isomers can be obtained by prepared by asymmetric synthesis.

Those skilled in the art will appreciate that organic compounds may form complexes with solvents in which they react or from which they are precipitated or crystallized. These complexes are known as "solvates". When the solvent is water, the complex is called a "hydrate". The invention covers all solvates of the compounds of the invention.

The term "solvate" refers to a form of a compound, or a salt thereof, which is associated with a solvent, usually formed by a solvolysis reaction. This physical association may include hydrogen bonding. Common solvents include water, methanol, ethanol, acetic acid, DMSO, THF, diethyl ether, and the like. The compounds described herein can be prepared, for example, in crystalline forms, and can be solvated. Suitable solvates include pharmaceutically acceptable solvates and further include stoichiometric solvates and non-stoichiometric solvates. In some instances, such solvates will be capable of isolation, for example, when one or more solvent molecules are incorporated into the crystal lattice of the crystalline solid. "Solvate" includes both solution state solvates and isolatable solvates. Representative solvates include hydrates, ethanolates and methanolates.

The term "hydrate" refers to a compound that combines with water. Generally, the ratio of the number of water molecules contained in a hydrate of a compound to the number of molecules of the compound in the hydrate is determined. Thus, a hydrate of a compound can be represented, for example, by the general formula R.x H ₂ O, where R is the compound, and x is a number greater than zero. A given compound may form more than one hydrate type, including, for example, monohydrates (x is 1), lower hydrates (x is a number greater than 0 and less than 1, for example, hemihydrates (R 0.5 _H2 O)) and polyhydrates (x is a number greater than 1, eg, dihydrate (R·2 H ₂ O) and hexahydrate (R·6 H ₂ O)).

The compounds of the invention may be in amorphous or crystalline form (polymorphs). Furthermore, the compounds of the invention may exist in one or more crystalline forms. Accordingly, the present invention includes within its scope all amorphous or crystalline forms of the compounds of the invention. The term "polymorph" refers to a crystalline form of a compound (or a salt, hydrate or solvate thereof) in a particular crystal packing arrangement. All polymorphs have the same elemental composition. Different crystalline forms generally have different X-ray diffraction patterns, infrared spectra, melting points, densities, hardness, crystal shapes, optoelectronic properties, stability and solubility. Recrystallization solvent, crystallization rate, storage temperature, and other factors can cause one crystalline form to predominate. Various polymorphs of a compound can be prepared by crystallization under different conditions.

The present invention also includes isotopically labeled compounds which are identical to those of the present invention but wherein one or more atoms are 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 may be incorporated into the compounds of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine and chlorine, such as 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. The compounds of the present invention, their prodrugs and pharmaceutically acceptable salts of the compounds or the prodrugs containing the above-mentioned isotopes and/or other isotopes of other atoms all belong to the scope of the present invention. Certain isotopically-labeled compounds of the invention, eg, those incorporating radioactive isotopes (eg, ^3H ^and14C ), are useful in drug and/or substrate tissue distribution assays. Tritium, ^ie3H , and carbon- ¹⁴ , ie14C isotopes are particularly preferred because of their ease of preparation and detection. Furthermore, substitution with heavier isotopes, such as deuterium, ^ie2H , may be preferred in some circumstances since greater metabolic stability may afford therapeutic benefits, such as increased in vivo half-life or reduced dosage requirements. The isotope-labeled compound of formula (I) of the present invention and its prodrug can generally be prepared in this way. When carrying out the processes disclosed in the following schemes and/or examples and preparation examples, replace non-isotope-labeled reagents with readily available isotope-labeled reagents Labeled reagents.

Furthermore, prodrugs are also included within the context of the present invention. The term "prodrug" as used herein refers to a compound that is converted in vivo to its active form having a medical effect, for example by hydrolysis in blood. Pharmaceutically acceptable prodrugs are described in T. Higuchi and V. Stella, Prodrugs as Novel Delivery Systems, Vol. 14 of A.C.S. Symposium 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, per intro This article is for reference.

A prodrug is any covalently bonded compound of the invention which, when administered to a patient, releases the parent compound in vivo. Prodrugs are generally prepared by modifying functional groups in such a way that the modification can be cleaved by routine manipulation or in vivo to yield the parent compound. Prodrugs include, for example, compounds of the invention wherein a hydroxy, amino, or thiol group is bonded to any group that, when administered to a patient, cleaves to form the hydroxy, amino, or thiol group. Thus, representative examples of prodrugs include, but are not limited to, acetate/amide, formate/amide and benzoate/amide derivatives of the hydroxy, sulfhydryl and amino functional groups of the compounds of formula (I). In addition, in the case of carboxylic acid (-COOH), esters such as methyl ester, ethyl ester and the like can be used. The esters themselves may be reactive and/or hydrolyzable under human in vivo conditions. Suitable pharmaceutically acceptable in vivo hydrolyzable ester groups include those which break down readily in the human body to release the parent acid or a salt thereof.

Processes for the preparation of compounds of the invention

Compounds of the invention, including salts thereof, can be prepared using known organic synthesis techniques and can be synthesized according to any of a number of possible synthetic routes, such as those in the schemes below. The reactions used to prepare the compounds of the present invention can be carried out in suitable solvents, which can be readily selected by those skilled in the art of organic synthesis. Suitable solvents may be substantially nonreactive with the starting materials (reactants), intermediates or products at the temperatures at which the reactions are carried out (eg, temperatures ranging from the solvent's freezing temperature to the solvent's boiling temperature). A given reaction can be carried out in one solvent or a mixture of more than one solvent. A skilled person can select a solvent for a specific reaction step according to the specific reaction step.

The preparation of the compounds of the present invention may involve the protection and deprotection of various chemical groups. The need for protection and deprotection and selection of appropriate protecting groups can be readily determined by those skilled in the art. The chemistry of protecting groups can be found in, eg, Wuts and Greene, Protective Groups in Organic Synthesis, 4th ed., John Wiley & Sons: New Jersey, (2006), which is hereby incorporated by reference in its entirety.

The compounds of the present invention can be prepared as their individual stereoisomers by reacting a racemic mixture of the compound with an optically active resolving agent to form a pair of diastereoisomeric compounds, separating the diastereomers and recovering the optically pure enantiomers. isomer. Enantiomeric resolution may be performed using diastereomeric derivatives of the compounds of the invention, preferentially dissociable complexes (eg, crystalline diastereomeric salts). Diastereomers have markedly different physical properties (eg, melting points, boiling points, solubilities, reactivities, etc.) and can be readily separated by taking advantage of these dissimilarities. Diastereomers can be separated by chromatography, preferably by separation/resolution techniques based on differences in solubility. The optically pure enantiomer is then recovered, along with the resolving reagents, by any practical means that will not result in racemization. A more detailed description of techniques applicable to the resolution of stereoisomers of compounds starting from racemic mixtures can be found in Jean Jacques, Andre Collet, Samue1 H. Wilen, "Enantiomers, Racemates and Resolution" ("Enantiomers , Racemates and Resolutions"), John Wiley And Sons, Inc., 1981.

The reaction can be monitored according to any suitable method known in the art. For example, 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 chromatography Product formation is monitored by methods such as high performance liquid chromatography (HPLC) or thin layer chromatography (TLC).

Pharmaceutical compositions, preparations and kits

In another aspect, the invention provides pharmaceutical compositions comprising a compound of the invention (also referred to as "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 prophylactically effective amount of the active ingredient.

A pharmaceutically acceptable excipient used in the present invention refers to a non-toxic carrier, adjuvant or vehicle that does not destroy the pharmacological activity of the compound with which it is formulated. Pharmaceutically acceptable carriers, adjuvants or vehicles that may be used in the compositions 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 phosphate), glycine, sorbic acid, potassium sorbate, partial glyceride mixture of saturated vegetable fatty acids, water, salt or electrolyte (such as protamine sulfate), disodium hydrogen phosphate, potassium hydrogen phosphate, Sodium chloride, zinc salts, silica gel, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-embedded segmented polymers, polyethylene glycol, and lanolin.

The invention also includes kits (eg, pharmaceutical packs). Provided kits can include a compound of the invention, another therapeutic agent, and first and second containers (e.g., vials, ampoules, bottles, syringes, and/or dispersible packs or other suitable container). In some embodiments, provided kits can also optionally include a third container containing a pharmaceutically acceptable excipient for diluting or suspending a compound of the invention and/or other therapeutic agent. In some embodiments, a compound of the invention and other therapeutic agent provided in a first container and a second container are combined to form a unit dosage form.

The pharmaceutical composition provided by the present invention can be administered by many routes, including but not limited to: oral administration, parenteral administration, inhalation administration, topical administration, rectal administration, nasal cavity administration, buccal administration, vaginal administration Drugs, by implants, or by other means of administration. For example, parenteral administration as used herein includes subcutaneous administration, intradermal administration, intravenous administration, intramuscular administration, intraarticular administration, intraarterial administration, intrasynovial administration, intrasternal administration , intracerebrospinal administration, intralesional administration, and intracranial injection or infusion techniques.

Typically, an effective amount of a compound provided herein is administered. The amount of the compound actually administered can be determined by the physician according to the circumstances, including the condition being treated, the route of administration chosen, the compound actually administered, the age, weight and response of the individual patient, the severity of the patient's symptoms, etc. .

When used to prevent a condition described herein, the compounds provided herein are administered to a subject at risk of developing the condition, typically on the advice and supervision of a physician, at dosage levels as described above. Subjects at risk of developing a particular condition generally include those with a family history of the condition, or those determined by genetic testing or screening to be particularly susceptible to developing the condition.

Long-term administration of the pharmaceutical compositions provided herein ("chronic administration") can also be used. Long-term administration refers to administering a compound or a pharmaceutical composition thereof for a long period of time, for example, 3 months, 6 months, 1 year, 2 years, 3 years, 5 years, etc., or may continue administration indefinitely, For example, 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 an extended period of time, eg, within the therapeutic window.

Various methods of administration may be used to further deliver the pharmaceutical compositions of the present invention. For example, in some embodiments, pharmaceutical compositions may be administered as a bolus injection, eg, in order to rapidly increase the blood concentration of the compound to effective levels. The bolus dose depends on the target systemic level of the active ingredient, for example, an intramuscular or subcutaneous bolus dose provides slow release of the active ingredient, while a bolus delivered directly into a vein (e.g., by IV infusion) can be more effective. The rapid delivery results in a rapid rise in blood concentration of the active ingredient to effective levels. In other embodiments, the pharmaceutical compositions may be administered as a continuous infusion, eg, by IV infusion, to provide a steady state concentration of the active ingredient in the subject's body. Additionally, in other embodiments, a bolus dose of the pharmaceutical composition may be administered first, followed by a continuous infusion.

Oral compositions may take the form of bulk liquid solutions or suspensions or bulk powders. More usually, however, the compositions will be presented in unit dosage form for ease of precise dosing. The term "unit dosage form" refers to physically discrete units suitable as unitary dosages for human patients and other mammals, each unit containing a predetermined quantity of active material suitable to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient. Typical unit dosage forms include prefilled, premeasured ampoules or syringes for liquid compositions, or pills, tablets, capsules and the like in the case of solid compositions. In such compositions, the compound will generally be a minor component (from about 0.1 to about 50% by weight, or preferably from about 1 to about 40% by weight), with the remainder being various components useful for forming the desired administration form. Carriers or excipients and processing aids.

For oral dosages, a typical regimen is one to five oral dosages per day, especially two to four oral dosages, typically three oral dosages. Using these dosing patterns, each dose provides from about 0.01 to about 20 mg/kg of the compound of the invention, with preferred doses each providing from about 0.1 to about 10 mg/kg, especially about 1 to about 5 mg/kg.

In order to provide blood levels similar to, or lower than, the injected dose, the 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 from about 0.5 to about 15% by weight.

Injection dosage levels range from about 0.1 mg/kg/hour to at least 10 mg/kg/hour from about 1 to about 120 hours, especially 24 to 96 hours. A preload bolus of about 0.1 mg/kg to about 10 mg/kg or more may also be given in order to achieve adequate steady state levels. For a human patient of 40 to 80 kg, the maximum total dose should not exceed approximately 2 g/day.

Liquid forms suitable for oral administration may include suitable aqueous or non-aqueous carriers as well as buffering, suspending and dispersing agents, coloring agents, flavoring agents, and the like. The solid form may comprise, for example, any of the following components, or compounds of similar nature: binders, such as microcrystalline cellulose, tragacanth, or gelatin; excipients, such as starch or lactose, disintegrants, For example, alginic acid, Primogel, or corn starch; lubricants, for example, magnesium stearate; glidants, for example, colloidal silicon dioxide; sweeteners, for example, sucrose or saccharin; or flavoring agents, for example, peppermint, water Methyl sylate or orange flavoring.

Injectable compositions are typically based on injectable sterile saline or phosphate buffered saline, or other injectable excipients known in the art. In such compositions, as previously mentioned, the active compound is typically a minor component, often from about 0.05 to 10% by weight, the remainder being injectable excipients and the like.

Transdermal compositions are typically formulated as topical ointments or creams containing the active ingredient. When formulated in an ointment, the active ingredients are typically combined with a paraffinic or a water-miscible ointment base. Alternatively, the active ingredients may be formulated in a cream, with, for example, an oil-in-water cream base. Such transdermal formulations are well known in the art, and generally include other ingredients for enhancing the stable skin penetration of the active ingredient or formulation. All such known transdermal formulations and compositions are included within the scope of the present invention.

The compounds of the invention may also be administered by transdermal devices. Thus, transdermal administration can be achieved using patches of the reservoir or porous membrane type, or various solid matrices.

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

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.

The invention also relates to pharmaceutically acceptable formulations of the compounds of the 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 composed of 6, 7, and 8 α-1,4-linked glucose units, respectively, optionally including a or multiple substituents including, but not limited to, methylated, hydroxyalkylated, acylated, and sulfoalkyl ether substitutions. In some embodiments, the cyclodextrin is a sulfoalkyl ether β-cyclodextrin, eg, sulfobutyl ether β-cyclodextrin, also known as Captisol. See, eg, U.S. 5,376,645. In some embodiments, the formulation includes hexapropyl-β-cyclodextrin (eg, 10-50% in water).

Indications

In another aspect, there is provided a compound of formula (I) disclosed herein (including all individual embodiments and generic subsets disclosed herein) or tautomers, stereoisomers, prodrugs, crystal forms thereof , the use of a pharmaceutically acceptable salt, hydrate or solvate as a medicine.

Compounds of the invention exhibit potent and selective BTK inhibition. For example, the compounds of the present invention exhibit nanomolar potency against wild-type BTK and BTK kinase encoded by the BTK gene comprising BTK kinase inhibitor resistance mutations such as C481S, C481F, C481Y, C481R, C481T, C481G or C481W, preferably Accordingly, the mutation is C481S. In some embodiments, the inhibition of C481S is similar to that observed for wild-type BTK.

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

In some embodiments, the compounds of the invention exhibit at least 30-fold selectivity for BTK kinase relative to another kinase. For example, compounds of the invention exhibit at least 40-fold selectivity for BTK kinase; at least 50-fold selectivity; at least 60-fold selectivity; at least 70-fold selectivity; at least 80-fold selectivity for BTK kinase relative to another kinase at least 90-fold selectivity; at least 100-fold selectivity; at least 200-fold selectivity; at least 300-fold selectivity; at least 400-fold selectivity; at least 500-fold selectivity; at least 600-fold selectivity at least 700-fold selectivity; at least 800-fold selectivity; at least 900-fold selectivity; or at least 1000-fold selectivity. In some embodiments, compounds of the invention exhibit at least 100-fold selectivity for BTK kinase relative to another kinase. In some embodiments, selectivity for a BTK kinase over another kinase is measured in a cellular assay (eg, a cellular assay provided herein).

The compounds of the invention are useful in the treatment of diseases and conditions treated with BTK kinase inhibitors, such as diseases and conditions associated with BTK, such as proliferative diseases, such as cancers, including hematological cancers and solid tumors, and inflammatory conditions, immune diseases or fibrosis.

In some embodiments of any of the methods or uses described herein, the cancer (eg, a BTK-related cancer) is a hematological cancer. In some embodiments of any of the methods or uses described herein, the cancer (eg, a BTK-related cancer) is a solid tumor. In some embodiments of any of the methods or uses described herein, the cancer (eg, a BTK-related cancer) is a B-cell malignancy. In some embodiments of any of the methods or uses described herein, the cancer (e.g., a cancer associated with BTK) is Hodgkin's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, Marginal zone lymphoma (eg, 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 lymphoblastic leukemia, B-cell prolymphocytic leukemia, precursor B-cell lymphoblastic leukemia, hairy cell leukemia, acute myeloid Leukemia, chronic myeloid leukemia, multiple myeloma, plasma cell myeloma, plasma cell tumor, bone cancer, bone metastases, breast cancer, gastroesophageal cancer, pancreatic cancer, ovarian cancer, prostate cancer, lung cancer, colon cancer, head and neck cancer or glioma.

In some embodiments, the hematological cancer is selected from leukemia, non-Hodgkin's lymphoma, Hodgkin's lymphoma or myeloma, for example, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), acute early Myeloid leukemia (APL), chronic lymphocytic lymphoma (CLL), chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML), chronic neutrophil leukemia (CNL), acute undifferentiated leukemia (AUL), anaplastic large cell lymphoma (ALCL), prolymphocytic leukemia (PML), juvenile myelomonocytic leukemia (JMML), adult T-cell leukemia (ALL), acute myelodysplastic disorder with trilineage Myeloid leukemia (AML/TMDS), mixed lineage leukemia (MLL), myelodysplastic syndrome (MDS), myeloproliferative disorder (MPD), diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma lymphoma, marginal zone lymphoma (eg, 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.

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

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, silver Psoriatic arthritis, osteoarthritis, Still's disease, juvenile arthritis, diabetes, myasthenia gravis, Hashimoto's thyroiditis, Oder's thyroiditis, Graves' disease, Sjogren's syndrome, Guillain-Barre syndrome, acute disseminated encephalomyelitis, Addison's disease, oculoclonus syndrome, ankylosing spondylitis, antiphospholipid antibody syndrome, aplastic anemia, autoimmune hepatitis, celiac disease, Goodpaschu Syndrome, Idiopathic Thrombocytopenic Purpura, Optic Neuritis, Scleroderma, Primary Biliary Cirrhosis, Reiter's Syndrome, Taurin's Arteritis, Warm Autoimmune Hemolytic Anemia, Wegener's Granulomatosis , psoriasis, alopecia universalis, Behcet's disease, chronic fatigue, dysautonomia, endometriosis, interstitial cystitis, neuromyotonia, scleroderma and vulvodynia, asthma, Appendicitis, blepharitis, bronchiolitis, bronchitis, bursitis, cervicitis, cholangitis, cholecystitis, colitis, conjunctivitis, colitis, cystitis, lacrimal glanditis, dermatitis, dermatomyositis, encephalitis, Endocarditis, endometritis, enteritis, enterocolitis, epicondylitis, epididymitis, fasciitis, fibrous inflammation, gastritis, gastroenteritis, hepatitis, inflammatory bowel disease, suppurative inflammation, laryngitis, Mastitis, meningitis, myelitis, 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, vulvitis, graft-versus-host disease, transplantation, Blood transfusion, anaphylaxis, anaphylaxis, type I hypersensitivity, allergic conjunctivitis, allergic rhinitis, and atopic dermatitis.

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

In some embodiments, the autoimmune disease is selected from lupus and Sjogren's syndrome, rheumatoid arthritis, psoriatic arthritis, osteoarthritis, Still's disease, juvenile arthritis, diabetes, myasthenia gravis, Hashimoto's thyroiditis, Oder's thyroiditis, Graves' disease, Sjögren's syndrome, Guillain-Barré syndrome, acute disseminated encephalomyelitis, Addison's disease, oculoclonus-myoclonus syndrome syndrome, ankylosing spondylitis, antiphospholipid antibody syndrome, aplastic anemia, autoimmune hepatitis, celiac disease, Goodpasture syndrome, idiopathic thrombocytopenic purpura, optic neuritis, scleroderma, Primary biliary cirrhosis, Reiter's syndrome, Taurus arteritis, hemolytic anemia, Wegener's granulomatosis, psoriasis, alopecia pervasive, Behcet's disease, chronic fatigue, autonomic disturbances , endometriosis, interstitial cystitis, neuromyotonia, scleroderma and vulvodynia.

In some embodiments, the heteroimmune disease is selected from graft versus host disease, transplantation, transfusion, allergy, allergy, type I hypersensitivity, allergic conjunctivitis, allergic rhinitis, and atopic dermatitis.

In some embodiments, the fibrosis is selected from pulmonary fibrosis, idiopathic pulmonary fibrosis (IPF), common interstitial pneumonia (UIP), interstitial lung disease, cryptogenic fibrotic alveolitis (CFA), occlusive bronchiolitis, bronchiectasis, fatty liver disease, steatosis (eg, nonalcoholic steatohepatitis (NASH), cholestatic liver disease (eg, primary biliary cirrhosis (PBC)), cirrhosis, alcohol Caused by liver fibrosis, bile duct injury, bile fibrosis, cholestasis or bile duct disease.

Example

Below in conjunction with specific embodiment, further elaborate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. For the experimental methods without specific conditions indicated in the following examples, the conventional conditions or the conditions suggested by the manufacturer are usually followed. Parts and percentages are by weight unless otherwise indicated.

Abbreviations:

Tol: toluene

THF: Tetrahydrofuran

HCl: hydrochloric acid

MeOH: Methanol

TsOMe: methyl p-toluenesulfonate

K ₂ CO ₃ : potassium carbonate

LiOH: lithium hydroxide

DMF: N,N-Dimethylformamide

DCM: dichloromethane

KHMDS: potassium bis(trimethylsilyl)amide

DIPEA: N,N-Diisopropylethylamine

Pd(OAc) ₂ : palladium acetate

Xphos: 2-Dicyclohexylphosphonium-2′,4′,6′-triisopropylbiphenyl

Cs ₂ CO ₃ : cesium carbonate

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

Using the following synthetic route

Step 1 Synthesis of compound N'-(1,1,1-trifluoropropan-2-ylidene)benzohydrazide

Add benzohydrazide (13.6g, 100mmol) and toluene (200mL) to a 500mL sealed tube equipped with a magnet, stir to dissolve, add 1,1,1-trifluoropropan-2-one (16.8g, 150mmol ), sealed, placed in an oil bath sink, heated up to 110°C, kept warm and stirred overnight. After cooling to room temperature, the solvent was evaporated under reduced pressure, and the residue was passed 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) ⁺ .

Step 2 Synthesis of compound N'-(1,1,1-trifluoroprop-2-yl)benzohydrazide

Add N'-(1,1,1-trifluoropropan-2-ylidene)benzohydrazide (9.2 g, 40 mmol) and anhydrous THF (100 mL ), stirred and dissolved, vacuumed and replaced with nitrogen three times, cooled in an ice-water bath, slowly added BH ₃ -THF (1M, 80mL, 80mmol) dropwise, after the drop was complete, removed the ice bath, and stirred at room temperature overnight under a nitrogen atmosphere. Under cooling in an ice-water bath, add methanol (50 mL) to quench the reaction, remove the ice bath, concentrate to dryness under reduced pressure, add dichloromethane (150 mL), stir for 30 minutes, filter out insoluble solids, and wash with saturated ammonium chloride ( 50 mL), dried over anhydrous sodium sulfate, filtered, concentrated to dryness, added petroleum ether (50 mL), stirred for 30 minutes under cooling in an ice-water bath, a large amount of white solid precipitated, filtered, and air-dried to obtain 6.5 g, yield 70.0%. LC-MS(APCI): m/z＝233.0(M+1) ⁺ .

Step 3 Synthesis of Intermediate A-1

Add N'-(1,1,1-trifluoropropan-2-yl)benzohydrazide (4.6g, 20mmol) and methanol (15mL) to a 100mL single-necked flask equipped with magnetic stirring and a condenser, and stir to dissolve After clearing, concentrated hydrochloric acid (27 mL) was added, and the temperature was raised to 80° C. under a nitrogen atmosphere, and the reaction was carried out overnight with stirring. 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, yield 58.0%.

Preparation of intermediate A-2 (1,1,1-trifluoropropan-2-yl-3,3,3-d ₃ ) hydrazine hydrochloride

Using the following synthetic route

Step 1 Synthesis of compound N'-(1,1,1-trifluoropropan-2-ylidene-3,3,3-d ₃ )benzohydrazide

Add N'-(1,1,1-trifluoropropan-2-ylidene)benzohydrazide (4.6g, 20mmol), deuterated methanol (15mL) and Heavy water (15mL) was dissolved by stirring, and a heavy aqueous solution of sodium deuterium oxide (4.0g, 40mmol, 40% heavy aqueous solution) was added, and the temperature was raised to 60°C under a nitrogen atmosphere, and kept stirring overnight. LC-MS showed that the conversion was complete, cooled to room temperature, evaporated the organic solvent under reduced pressure, extracted with dichloromethane (20mL x 2), combined the organic phases, dried over anhydrous sodium sulfate, filtered, concentrated and passed through a silica gel column to obtain a white solid 3.8 g, yield 81.5%. LC-MS(APCI): m/z＝234.1(M+1) ⁺ .

Step 2 Synthesis of compound N'-(1,1,1-trifluoropropan-2-yl-3,3,3-d ₃ )benzohydrazide

Add N'-(1,1,1-trifluoropropan-2-ylidene-3,3,3-d ₃ )benzohydrazide (3.5g , 15mmol) and anhydrous THF (40mL), stir to dissolve, vacuumize and replace with nitrogen three times, cool in an ice-water bath, slowly add BH ₃ -THF (1M, 30mL, 30mmol) dropwise, after the drop is complete, remove the ice bath, The reaction was stirred overnight at room temperature under a nitrogen atmosphere. Under cooling in an ice-water bath, add methanol (30 mL) to quench the reaction, remove the ice bath, concentrate to dryness under reduced pressure, add dichloromethane (60 mL), stir for 30 minutes, filter out insoluble solids, wash with saturated ammonium chloride ( 30 mL), dried over anhydrous sodium sulfate, filtered, concentrated to dryness, added petroleum ether (30 mL), stirred for 30 minutes under cooling in an ice-water bath, a large amount of white solid precipitated, filtered, and air-dried to obtain 2.6 g, yield 73.7%. LC-MS(APCI): m/z＝236.0(M+1) ⁺ .

Step 3 Synthesis of Intermediate A-2

Add N'-(1,1,1-trifluoropropan-2-yl-3,3,3-d ₃ )benzohydrazide (2.6g, 11mmol ) and methanol (8mL), stirred to dissolve, added concentrated hydrochloric acid (15mL), heated to 80°C under a nitrogen atmosphere, kept stirring and reacted 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, yield 65.3%.

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

Using the following synthetic route

Synthesis of step 1 compound 5-fluoro-2-methoxybenzoyl chloride

Add 5-fluoro-2-methoxybenzoic acid (2.6g, 15mmol) and anhydrous dichloromethane (30mL) into a 100mL three-neck flask equipped with magnetic stirring, stir to dissolve, cool in an ice-water bath, drop under a nitrogen atmosphere Oxalyl chloride (3.8g, 30mmol) and anhydrous DMF (110mgm, 1.5mmol) were added, and after the drop was completed, the ice bath was removed, and the reaction was stirred overnight at room temperature. The solvent and unreacted oxalyl chloride were evaporated under reduced pressure for use.

Step 2 Synthesis of Intermediate B-1

Add (4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)methanamine (3.63g, 16.5mmol ) and anhydrous THF (30mL), cooled to -78°C under a nitrogen atmosphere, added KHMDS (16.5mL, 1M THF solution) dropwise, kept stirring for 30 minutes, warmed up to room temperature and stirred for 30 minutes, added anhydrous methanol (2.11 g, 66mmol), stirred at room temperature for 1 hour, filtered off insoluble solids, distilled off the filtrate solvent at no more than 30°C, took two times in anhydrous THF (20mL), dissolved in anhydrous THF (20mL), slowly added dropwise the above 5- A THF solution of fluoro-2-methoxybenzoyl chloride (20 mL) was added dropwise, and stirred at room temperature under a nitrogen atmosphere to react overnight. The solvent was evaporated, dissolved in methanol (30 mL), and an aqueous solution of potassium hydrogen fluoride (6.4 g, 82.5 mmol, 20 mL) was added, and the reaction was stirred overnight at room temperature. Evaporate the solvent under reduced pressure, take toluene with water twice (50mL), wash the residue with MTBE, filter, wash the filter cake with a hot methanol/acetone (1/3, 100mL) solution, concentrate the filtrate to dryness, add MTBE for beating, and precipitate a solid It was filtered and dried to obtain 3.5 g of white solid with a yield of 80.7%. ¹ H NMR(400MHz,DMSO-D ₆ )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).

Preparation of intermediate B-2(((5-fluoro-2-(methoxy-d ₃ )benzoyl)amino)methyl)potassium trifluoroborate

Using the following synthetic route

Step 1 Synthesis of compound 5-fluoro-2-(methoxy-d ₃ )methyl benzoate

Add methyl 5-fluoro-2-hydroxybenzoate (5.1g, 30mmol) and acetonitrile (80mL) to a 250mL single-necked flask equipped with magnetic stirring and a condenser, stir to dissolve, add potassium carbonate (8.28g, 60mmol) and TsOMe-d ₃ (7.37g, 39mmol), the temperature was raised to 80°C under a nitrogen atmosphere, and the mixture was incubated and stirred overnight. After cooling to room temperature, ethyl acetate (100 mL) was added, and the insoluble solid was filtered off. The filtrate was concentrated and passed through a silica gel column to obtain 4.2 g of a white solid, with a yield of 74.8%. LC-MS(APCI): m/z＝188.0(M+1) ⁺ .

Step 2 Synthesis of compound 5-fluoro-2-(methoxy-d ₃ )benzoic acid

Add methyl 5-fluoro-2-(methoxy-d ₃ )benzoate (3.74g, 20mmol), THF (20mL) and water (20mL) into a 100mL single-necked flask equipped with magnetic stirring, stir to dissolve, Lithium hydroxide (0.96 g, 40 mmol) was added, and the reaction was stirred at room temperature for 3 hours under nitrogen atmosphere. The organic solvent was evaporated under reduced pressure, and solid citric acid (2.56 g, 13.3 mmol) was slowly added under cooling in an ice-water bath. A large amount of white solid precipitated, filtered, washed with a small amount of ice water, and dried in vacuo to obtain 3.0 g of the white solid, with a yield of 86.6%. LC-MS(APCI):m/z＝174.0(M+1) ⁺ . ¹ H NMR(400MHz,DMSO-D ₆ )δ(ppm):12.92(s,1H),7.43-7.34(m,2H) ,7.16-7.12(m,1H).

Step 3 Synthesis of compound 5-fluoro-2-(methoxy-d ₃ )benzoyl chloride

Add 5-fluoro-2-(methoxy-d ₃ )benzoic acid (2.6g, 15mmol) and anhydrous dichloromethane (30mL) into a 100mL three-necked flask equipped with magnetic stirring, stir to dissolve, and cool in an ice-water bath , under nitrogen atmosphere, oxalyl chloride (3.8g, 30mmol) and anhydrous DMF (110mgm, 1.5mmol) were added dropwise. The solvent and unreacted oxalyl chloride were evaporated under reduced pressure for use.

Step 4 Synthesis of Intermediate B-2

Add (4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)methanamine (3.63g, 16.5mmol ) and anhydrous THF (30mL), cooled to -78°C under a nitrogen atmosphere, added KHMDS (16.5mL, 1M THF solution) dropwise, kept stirring for 30 minutes, warmed up to room temperature and stirred for 30 minutes, added anhydrous methanol (2.11 g, 66mmol), stirred at room temperature for 1 hour, filtered off insoluble solids, distilled off the filtrate solvent at no more than 30°C, took two times in anhydrous THF (20mL), dissolved in anhydrous THF (20mL), slowly added dropwise the above 5- A THF solution of fluoro-2-(methoxy-d ₃ )benzoyl chloride (20 mL) was added dropwise, and the reaction was stirred overnight at room temperature under a nitrogen atmosphere. The solvent was evaporated, dissolved in methanol (30 mL), and an aqueous solution of potassium hydrogen fluoride (6.4 g, 82.5 mmol, 20 mL) was added, and the reaction was stirred overnight at room temperature. Evaporate the solvent under reduced pressure, take toluene with water twice (50mL), wash the residue with MTBE, filter, wash the filter cake with a hot methanol/acetone (1/3, 100mL) solution, concentrate the filtrate to dryness, add MTBE for beating, and precipitate a solid It was filtered and dried to obtain 3.1 g of white solid with a yield of 70.7%. ¹ H NMR(400MHz,DMSO-D ₆ )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).

Example 1 5-amino-3-(4-((5-fluoro-2-(methoxy-d ₃ )benzoylamino)methyl)phenyl)-1-(1,1,1-tri fluorine Propan-2-yl)-1H-pyrazole-4-carboxamide (compound T-1);

(S)-5-amino-3-(4-((5-fluoro-2-(methoxy-d ₃ )benzoylamino)methyl)phenyl)-1-(1,1,1- Trifluoropropane-2- Base)-1H-pyrazole-4-carboxamide (compound T-1-S);

(R)-5-amino-3-(4-((5-fluoro-2-(methoxy-d ₃ )benzoylamino)methyl)phenyl)-1-(1,1,1- Trifluoropropane-2- base)-1H-pyrazole-4-carboxamide (compound T-1-R) preparation

Using the following synthetic route

Synthesis of step 1 compound 2-((4-bromophenyl)(hydroxyl)methylene)malononitrile

Add toluene (100mL), THF (20mL) and malononitrile (6.3mL, 100.3mmol) to a 250mL single-necked flask equipped with magnetic stirring in sequence, stir to dissolve, cool to -10°C, add 4-bromobenzoyl chloride ( 20g, 91.1mmol), stirred for 10 minutes, slowly added DIPEA (31.8mL, 182.3mmol) dropwise, and finished dropping in half an hour. Add ethyl acetate (200mL) and 1M aqueous hydrochloric acid solution (200mL), stir for 10 minutes, separate the organic phase, wash with 1M hydrochloric acid (100mL), wash with saturated brine (100mL), dry over anhydrous sodium sulfate, filter, and concentrate the filtrate to After drying, 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 a gray solid with a yield of 88.1%. LC-MS(APCI): m/z＝249.0(M+1) ⁺ .

Synthesis of step 2 compound 2-((4-bromophenyl)(methoxy)methylene)malononitrile

Sodium hydride (2.48g, 61.84mmol, 60% mass fraction dispersed in silicone oil) was added to a 250mL three-necked flask equipped with magnetic stirring and a condenser, vacuumized and replaced with nitrogen for 3 times, and anhydrous THF ( 40mL), stirred and dispersed, added dropwise 2-((4-bromophenyl)(hydroxyl)methylene)malononitrile (14g, 56.2mmol) in anhydrous THF (40mL) solution, stirred and reacted under ice-water bath for 30 minutes , dropwise added dimethyl sulfate (16.0mL, 168.6mmol), after the dropwise completion, the temperature was raised to 80° C., and the reaction was carried out overnight with insulation and stirring. Cool to room temperature, add saturated brine (80mL) to quench the reaction, separate the organic phase, extract the aqueous phase with ethyl acetate (50mLx2), combine the organic phases, wash with saturated brine (50mL), dry over anhydrous sodium sulfate, filter, Concentrate and pass through a silica gel column to obtain 7.3 g of white solid with a yield of 49.4%. LC-MS(APCI): m/z＝231.0(M+1) ⁺ .

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

Add intermediate A-1 (1.6g, 10mmol) and absolute ethanol (60mL) in the 100mL one-necked flask that is equipped with magnetic stirring and condenser tube, under stirring, add triethylamine (4.04g, 40mmol) and 2-( (4-Bromophenyl)(methoxy)methylene)malononitrile (1.75 g, 6.67 mmol), heated to 80° C. under nitrogen atmosphere, kept stirring and reacted overnight. Cool to room temperature, distill off the solvent under reduced pressure, add saturated brine (20mL) and ethyl acetate (30mL), stir for 10 minutes, separate the organic phase, extract the aqueous phase with ethyl acetate (30mLx2), combine the organic phases, anhydrous It was dried over sodium sulfate, filtered, concentrated and passed through a silica gel column to obtain 2.2 g of white solid with a yield of 91.8%. LC-MS (APCI): m/z＝359.0(M+1) ⁺ . ¹ H NMR (400MHz, DMSO-D ₆ ) 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).

Step 4 Compound N-(4-(5-amino-4-cyano-1-(1,1,1-trifluoroprop-2-yl)-1H-pyrazol-3-yl)phenyl)-5 -Fluoro-2-(methoxy-d ₃ )benzamide

Add intermediate B-2 (0.29 g, 1.0 mmol), 5-amino-3-(4-bromophenyl)-1-(1,1,1-tri Fluoropropan-2-yl)-1H-pyrazole-4-carbonitrile (0.36g, 1.0mmol), _Cs2CO3 (0.98g, _3.0mmol ), Xphos (48mg, 0.1mmol), Pd(OAc) ₂ (12mg, 0.05mmol), THF (10mL) and water (1mL), vacuumize and replace with nitrogen three times, heat up to 85°C and keep stirring overnight. Cool to room temperature, evaporate the solvent under reduced pressure, add saturated brine (5mL) and ethyl acetate (20mL), separate the organic phase, extract the aqueous phase with ethyl acetate (20mLx2), combine the organic phases, and 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) ⁺ .

Synthesis of step 4 compound T-1

Add N-(4-(5-amino-4-cyano-1-(1,1,1-trifluoropropan-2-yl)-1H-pyrazole -3-yl)phenyl)-5-fluoro-2-(methoxy-d ₃ )benzamide (0.38g, 0.82mmol) and methanesulfonic acid (3mL), heated to 80°C, kept under nitrogen atmosphere The reaction was stirred for 1 hour. Cool to room temperature, add saturated brine (20mL) and ethyl acetate (20mL), adjust the pH to 8 with ammonia water, separate the organic phase, extract the aqueous phase with ethyl acetate (20mL X 2), combine the organic phases, anhydrous sodium sulfate It was dried, filtered, concentrated and passed through a silica gel column to obtain 0.31 g of white solid with a yield of 78.5%. LC-MS (APCI): m/z = 483.2 (M+1) ⁺ .

Step 5 Synthesis of compounds T-1-S and T-1-R

The racemate compound T-1 (0.31g) was dissolved in methanol (10mL), and was prepared chirally through an IC column using the following conditions: flow rate: 4ml/min; mobile phase: MTBE:MeOH:EtOH=91:2:7 (0.1% triethylamine);

The corresponding fractions were collected and spin-dried to obtain 120 mg of compound T-1-S, corresponding to a retention time of 26.358 min; and 120 mg of compound T-1-R, corresponding to a retention time of 31.922 min.

Example 2 5-amino-3-(4-((5-fluoro-2-methoxybenzoylamino)methyl)phenyl)-1-(1,1,1-trifluoropropane-2- Base-3,3,3-d ₃ )-1H-pyrazole-4-carboxamide (compound T-2);

(S)-5-amino-3-(4-((5-fluoro-2-methoxybenzoylamino)methyl)phenyl)-1-(1,1,1-trifluoropropane-2 -base -3,3,3-d ₃ )-1H-pyrazole-4-carboxamide (compound T-2-S);

(R)-5-amino-3-(4-((5-fluoro-2-methoxybenzoylamino)methyl)phenyl)-1-(1,1,1-trifluoropropane-2 -base Preparation of -3,3,3-d ₃ )-1H-pyrazole-4-carboxamide (compound T-2-R)

Using the following synthetic route

Step 1 Compound 5-amino-3-(4-bromophenyl)-1-(1,1,1-trifluoroprop-2-yl-3,3,3-d ₃ )-1H-pyrazole-4 -Synthesis of forminonitrile

Add intermediate A-2 (1.2g, 7.2mmol) and dehydrated ethanol (40mL) to the 100mL single-necked flask equipped with magnetic stirring and condenser tube, under stirring, add triethylamine (2.9g, 28.7mmol) and 2 -((4-Bromophenyl)(methoxy)methylene)malononitrile (1.26g, 4.8mmol), heated to 80°C under nitrogen atmosphere, kept stirring and reacted overnight. Cool to room temperature, distill off the solvent under reduced pressure, add saturated brine (20mL) and ethyl acetate (30mL), stir for 10 minutes, separate the organic phase, extract the aqueous phase with ethyl acetate (30mLx2), combine the organic phases, anhydrous It was dried over sodium sulfate, filtered, concentrated and passed through a silica gel column to obtain 1.6 g of white solid with a yield of 92.0%. LC-MS (APCI): m/z＝362.0(M+1) ⁺ . ¹ H NMR (400MHz, DMSO-D ₆ ) δ (ppm): 7.74-7.67 (m, 4H), 7.15 (br s, 2H ),5.31(s,1H).

Step 2 Compound N-(4-(5-amino-4-cyano-1-(1,1,1-trifluoroprop-2-yl-3,3,3-d ₃ )-1H-pyrazole- Synthesis of 3-yl)phenyl)-5-fluoro-2-methoxybenzamide

Add intermediate B-1 (0.29 g, 1.0 mmol), 5-amino-3-(4-bromophenyl)-1-(1,1,1-tri Fluoroprop-2-yl-3,3,3-d ₃ )-1H-pyrazole-4-carbonitrile (0.36g, 1.0mmol), Cs ₂ CO ₃ (0.98g, 3.0mmol), Xphos (48mg, 0.1mmol), Pd(OAc) ₂ (12mg, 0.05mmol), THF (10mL) and water (1mL), vacuumize and nitrogen replacement 3 times, heat up to 85°C and keep stirring overnight. Cool to room temperature, evaporate the solvent under reduced pressure, add saturated brine (5mL) and ethyl acetate (20mL), separate the organic phase, extract the aqueous phase with ethyl acetate (20mLx2), combine the organic phases, and dry over anhydrous sodium sulfate. Concentrate and pass through a silica gel column to obtain 0.36 g of white solid with a yield of 77.5%. LC-MS (APCI): m/z = 465.2 (M+1) ⁺ .

Step 3 Synthesis of compound T-2

Add N-(4-(5-amino-4-cyano-1-(1,1,1-trifluoropropan-2-yl-3,3,3 -d ₃ )-1H-pyrazol-3-yl)phenyl)-5-fluoro-2-methoxybenzamide (0.36g, 0.82mmol) and methanesulfonic acid (3mL), heated to 80°C, Under a nitrogen atmosphere, the mixture was incubated and stirred for 1 hour. Cool to room temperature, add saturated brine (20mL) and ethyl acetate (20mL), adjust the pH to 8 with ammonia water, separate the organic phase, extract the aqueous phase with ethyl acetate (20mL x 2), combine the organic phases, anhydrous sodium sulfate Dry, filter, concentrate and pass through a silica gel column to obtain 0.30 g of white solid, yield 75.8%. LC-MS (APCI): m/z = 483.2 (M+1) ⁺ .

Step 4 Synthesis of compound T-2-S and compound T-2-R

The racemic compound T-2 (0.30g) was dissolved in methanol (10mL), and was chirally prepared through an IC column using the following conditions: flow rate: 4ml/min; mobile phase: MTBE:MeOH:EtOH=91:2:7 (0.1% triethylamine);

The corresponding fractions were collected and spin-dried to obtain 120 mg of compound T-2-S, corresponding to a retention time of 24.483 min; and 120 mg of compound T-2-R, corresponding to a retention time of 29.807 min.

Example 3 5-amino-3-(4-((5-fluoro-2-methoxybenzoylamino)methyl)phenyl)-1-(1,1,1-trifluoropropane-2- Base-3,3,3-d ₃ )-1H-pyrazole-4-carboxamide (compound T-3);

(S)-5-amino-3-(4-((5-fluoro-2-methoxybenzoylamino)methyl)phenyl)-1-(1,1,1-trifluoropropane-2 -base -3,3,3-d ₃ )-1H-pyrazole-4-carboxamide (compound T-3-S);

(R)-5-amino-3-(4-((5-fluoro-2-methoxybenzoylamino)methyl)phenyl)-1-(1,1,1-trifluoropropane-2 -base Preparation of -3,3,3-d ₃ )-1H-pyrazole-4-carboxamide (compound T-3-R)

Using the following synthetic route

Step 1 Compound N-(4-(5-amino-4-cyano-1-(1,1,1-trifluoroprop-2-yl-3,3,3-d ₃ )-1H-pyrazole- Synthesis of 3-yl)phenyl)-5-fluoro-2-(methoxy-d ₃ )benzamide

Add intermediate B-2 (0.29 g, 1.0 mmol), 5-amino-3-(4-bromophenyl)-1-(1,1,1-tri Fluoroprop-2-yl-3,3,3-d ₃ )-1H-pyrazole-4-carbonitrile (0.36g, 1.0mmol), Cs ₂ CO ₃ (0.98g, 3.0mmol), Xphos (48mg, 0.1mmol), Pd(OAc) ₂ (12mg, 0.05mmol), THF (10mL) and water (1mL), vacuumize and nitrogen replacement 3 times, heat up to 85°C and keep stirring overnight. Cool to room temperature, evaporate the solvent under reduced pressure, add saturated brine (5mL) and ethyl acetate (20mL), separate the organic phase, extract the aqueous phase with ethyl acetate (20mLx2), combine the organic phases, and dry over anhydrous sodium sulfate. Concentrate and pass 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) ⁺ .

Synthesis of step 2 compound T-3

Add N-(4-(5-amino-4-cyano-1-(1,1,1-trifluoropropan-2-yl-3,3,3 -d ₃ )-1H-pyrazol-3-yl)phenyl)-5-fluoro-2-(methoxy-d ₃ )benzamide (0.35 g, 0.75 mmol) and methanesulfonic acid (3 mL), The temperature was raised to 80° C., and the mixture was incubated and stirred for 1 hour under a nitrogen atmosphere. Cool to room temperature, add saturated brine (20mL) and ethyl acetate (20mL), adjust the pH to 8 with ammonia water, separate the organic phase, extract the aqueous phase with ethyl acetate (20mLx2), combine the organic phases, and dry over anhydrous sodium sulfate. After filtering, concentrating and passing through a silica gel column, 0.30 g of a white solid was obtained, with a yield of 82.4%. LC-MS (APCI): m/z = 486.2 (M+1) ⁺ .

Step 3 Synthesis of compounds T-3-S and T-3-R

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

The corresponding fractions were collected and spin-dried to obtain 120 mg of compound T-3-S, corresponding to a retention time of 25.942 min; and 120 mg of compound T-3-R, corresponding to a retention time of 31.415 min.

Biological activity test.

(1) Kinase activity inhibition test

In this experiment, the HTRF TK Kit (catalog number 62TK0PEC) kit provided by Cisbio was used to detect the inhibitory activity of the compound on BTK kinase (Invitrogen, catalog number PV3587) and BTK C481S kinase (Carna Biosciences, catalog number 08-547) and relative to Selectivity of EGFR, ITK and TEC kinases. After optimizing the detection conditions and confirming the detection conditions according to the kit instructions, prepare a 10uL reaction system on ice, add compounds, substrates, ATP, and kinases to the 384-well plate in sequence, and use no kinase wells as negative controls, that is, blank wells; Wells without compound were used as positive control. After reacting at room temperature for 30-60 minutes, add the detection reagent, and react at room temperature for 1 hour, read the values of 620nm (Cryptate) and 665nm (XL665) with a microplate reader (BioTek, Synergy Neo2), calculate the ratio of 665/620 in each well, and export the data . IC ₅₀ was calculated with GraphPad Prism7.0 software.

Reagents and consumables: BTK Kinase (Invitrogen, Cat. No. PV3587), BTK C481S (Carna, Cat. No. 08-547), ITK (Carna, Cat. No. 08-181), EGFR (Carna, Cat. No. 08-115), TEC (Carna, Cat. No. 08-182), HTRF-TK kit (Cisbio, product number 62TK0PEC), MgCl ₂ (Sigma, product number M1028), MnCl ₂ (Sigma, product number M1787), DTT (Sigma, product number D0632), ATP (Sigma, product number A7699-1g ), DMSO (Sigma, Cat. No. D2650), 384-well plate (PE, Cat. No. 6008280).

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 were carried out on ice, with a total reaction volume of 10 μL. Add compounds (4 μL), substrates and ATP (4 μL), and kinases (2 μL) into the 384-well plate in sequence, shake for 30 seconds, and react appropriately at room temperature. After a certain time, the detection reagent was added, reacted at room temperature for 1 hour, and detected by a microplate reader. Specific steps are as follows:

a) Calculate the 1×kinase buffer required for kinase detection according to the amount of the detection compound, and prepare as needed;

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

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

d) Prepare a kinase with a working concentration of 5×, and add 2 μL to each well;

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

f) Prepare streptavidin XL-665 and TK antibody-cryptate at a working concentration of 4×, mix 1:1 before adding to a 384-well plate, and add 10 μL to each well;

g) Read the plate after reacting at room temperature for 1 hour;

h) Add only substrate, ATP, 1% DMSO, and no kinase as blank control wells, and only add substrate, ATP, 1% DMSO and kinase as positive control wells.

The _IC50 (half maximal inhibitory concentration) of the compound was obtained using the following non-linear fitting formula:

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

X: log value of compound concentration

Y: Inhibition rate (%inhibition)

Kinase inhibition rate Inhibition (inhibition rate %) calculation formula is:

Inhi.(%)=1-(Ratio drug to be tested-Ratio negative control)/(Ratio positive control-Ratio negative control)×100%.

The compound of the present invention was tested in the above-mentioned kinase inhibition experiment, and the experimental results showed that: compared with non-deuterated compounds, the compound of the present invention has more potent activity and superiority to wild-type BTK kinase and drug-resistant mutant BTK C481S kinase. Excellent selectivity for EGFR, ITK and TEC kinases. The results of representative example compounds are summarized in the following table 1, wherein, A represents IC ₅₀ ≤ 10nM, B represents IC ₅₀ of 10-50nM, C represents IC ₅₀ of 50-100nM, D represents IC ₅₀ of 100-1000nM, E indicates IC ₅₀ is 1000-10000nM, F indicates IC ₅₀ >10000nM.

Table 1:

Example compound number

BTK

BTK C481S

EGFR

ITK

TEC

PirtobrutinibPirtobrutinib	AA	AA	BB	Ff	DD.
T-1T-1	AA	AA	DD.	Ff	DD.
T-1-ST-1-S	AA	AA	CC	Ff	DD.
T-1-RT-1-R	AA	AA	DD.	Ff	EE.
T-2T-2	AA	AA	DD.	Ff	DD.
T-2-RT-2-R	AA	AA	DD.	Ff	DD.
T-3T-3	AA	AA	DD.	Ff	DD.
T-3-ST-3-S	AA	AA	CC	Ff	DD.
T-3-RT-3-R	AA	AA	DD.	Ff	EE.

(2) Test of growth inhibitory activity of cells

This experiment uses the Promega company to provide

Luminescence method cell viability detection kit, which is a homogeneous cell viability detection method, can be used to measure cultured cells by quantifying ATP (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). The specific experimental steps are as follows:

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

b) After diluting the cell suspension to the desired concentration, add it to the middle well of a 96-well plate, and add PBS to the edge wells. The wells where no cells were added and only culture medium were added were used as blank controls.

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

d) Place the cell plate in a 37° C., 5% CO ₂ incubator for overnight culture.

e) The next day, the compounds of various concentrations were added in gradient dilution, and the cell plate was placed in a 37° C., 5% CO ₂ incubator for 72 hours. The wells where no compound was added and only 0.5% DMSO were added were used as positive controls for cell growth.

f) According to the manufacturer's instructions, CTG reagent (CellTiter-Glo kit) was added to detect the cell viability value.

g) Read the plate with Biotek cytation3.0 microplate reader and export the data.

h) Calculate IC50 with GraphPad Prism7.0 software.

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

X: log value of compound concentration

Y: Inhibition rate (%inhibition)

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

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

The above experimental results show that: compared with non-deuterated compounds, the compound of the present invention has more potent activity on TMD8 cells, REC1 cells, DOHH2 cells, Pfeiffer cells, Raji cells and RPMI-8226 cells.

(3) Phosphorylation experiment

Adopt Western Blot method, this experiment detects the compound of the present invention in NIH/3T3-FL-BTK and NIH/3T3-FL-BTK-C481S cells by BCA protein concentration assay kit (brand beyotime, catalog number P0012), to BTK and On-target inhibition of BTK C481S.

Equipment: high-speed refrigerated centrifuge (Thermo Fisher/Sorvall Legend Micro 17R), vortex analyzer (Scilogex/MX-S), microplate reader (Molecular Devices/SpectraMax Paradigm), electrophoresis apparatus (Bio-rad/1658001), spin Membrane instrument (Bio-rad/1703935), automatic chemiluminescence image analysis system (Tanon/5200), cell counter (Countstar/IC1000), TS-100 decolorization shaker (Qilin Bell/TS-100)

Cells and culture conditions:

细胞cell	特性characteristic	培养基culture medium	备注Remark
NIH/3T3-FL-BTKNIH/3T3-FL-BTK	贴壁细胞adherent cells	DMEM+10％FBS+1％PSDMEM+10%FBS+1%PS	37℃，5％CO2培养37°C, 5% CO2 cultivation
NIH/3T3-FL-BTK-C481SNIH/3T3-FL-BTK-C481S	贴壁细胞adherent cells	DMEM+10％FBS+1％PSDMEM+10%FBS+1%PS	37℃，5％CO2培养37°C, 5% CO2 cultivation

Culture conditions: NIH/3T3-FL-BTK and NIH/3T3-FL-BTK-C481S adopt DMEM medium (Corning, USA) + 10% fetal bovine serum (Fetal Bovine Serum, Excell Bio) + 1% double antibody (Penicillin Streptomycin solution, Coring, USA) were cultured, and the cells were cultured for two generations after recovery, to be tested.

Experimental steps:

1) Cell plating

One day in advance, take the cells in the logarithmic growth phase and count them with a cell counter. The cell viability must be more than 90%. Inoculate the cells into a sterile 6-well plate, with 0.8×106 cells per well, and supplement DMEM complete medium to 2000 μL, 6-well plate was placed in a 37°C, 5% CO ₂ constant temperature incubator for static culture to allow the cells to adhere to the wall.

2) Compound administration

The next day, take out the 6-well plate and observe under the microscope that the cells are normal. In the ultra-clean workbench, replace with fresh DMEM complete medium, 1998 μL per well, and take 2 μL of the prepared serially diluted 1000× mother solution (ARQ-531) , were added to the 6-well plates inoculated with cells, the final concentrations of the compounds were 1000nM, 300nM, 100nM, 30nM, 10nM, 1nM, 0nM, and the 6-well plates were gently shaken to distribute the compounds evenly.

3) Incubation

Incubate the 6-well plate in a constant temperature incubator at 37°C and 5% CO ₂ for 4 hours;

4) Sample collection

After the cells were cultured for 4 hours, remove the medium in the culture dish, wash the cells with PBS, digest the cells with trypsin, centrifuge at 8000rpm for 3 minutes, collect the cells in a 1.5mL EP tube, and discard the supernatant , to extract total protein;

5) Cell Lysis

Collect the cells of each group into 1.5ml EP tubes, and then add a certain volume (determined according to the amount of cells, about 66 μL per 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 vortex to vortex the EP tube to fully lyse the cells. Note that the vortex time should not be too long, quickly place the sample tube on ice to lyse, shake once every 10 minutes, a total of three times. After the lysis time is over, put the EP tube into a centrifuge pre-cooled to 4°C and centrifuge at 12000rpm for 10min. After centrifugation, transfer the supernatant to a new 1.5ml EP tube and label it.

6) Determination of BCA protein concentration

Using the BCA protein concentration assay kit, according to the kit instructions, protein quantification was performed on the lysed supernatant of the sample. After completing the determination of protein concentration, the lysed supernatant with the lowest concentration was used as the benchmark, and the remaining lysed supernatants were calculated according to the BCA standard curve and then diluted to this concentration with Lysis Buffer. Add a corresponding volume of 5×loading Buffer to the diluted lysed supernatant, heat in a warm metal bath at 95°C for 10 minutes, then place it on ice to cool, and store the sample in a -20°C refrigerator.

7) Glue making

Clean the glass plates used for glue making, align the bottoms of the two glass plates and put them into the bracket, and fix them with clips. Prepare the separating gel according to the 10% separating gel formula, mix well and add to the glass plate, add about 3/4 of the glass plate to stop, and press the glue with 75% ethanol. After 90 minutes, the separation gel solidified, poured out 75% ethanol, blotted the remaining ethanol solution on the glass plate with filter paper, prepared 5% concentrated gel, mixed it and added it to the glass plate, then inserted the sample comb, and waited for 60 minutes , which is made into glue.

8) Protein loading and electrophoresis

Put the gel plate in the electrophoresis tank and fix it, fill the inner tank with 1× electrophoresis solution, and the outer tank exceeds the wire. Unplug the sample comb, load 25 μg of protein samples from 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 electrophoresis at a constant voltage of 70V for 30 minutes, then continue electrophoresis at a constant voltage of 86V for 1 hour and 20 minutes until the bromophenol blue indicator band reaches the bottom, and then stop the electrophoresis.

9) Protein electroporation

Soak the PVDF membrane in methanol for 30s to wake up the membrane, and then put it into the transfer membrane box together with 4 pieces of filter paper and 2 pieces of sponge. Pour the pre-configured 1× electrotransfer solution into the transfer box. Take out the gel, cut it into the required size, put it in the transfer film folder in the order of sponge, filter paper, gel, PVDF membrane, filter paper, and sponge, remove air bubbles, place the membrane on the anode side, and the gel on the cathode side (Black glue and white film), insert into the electrophoresis tank, pour the transfer buffer, put in ice packs, and place ice packs around the electrophoresis tank to cool down. Constant voltage 100V electrophoretic transfer for 90min.

10) Block and incubate primary antibody

After the electroporation was completed, the PVDF membrane was put into the blocking solution of 5% skimmed milk, shaken slowly on a shaker, and blocked at room temperature for 1 hour. The blocked PVDF membrane was slowly washed 3 times in TBST, 10 min each time. After washing, put the PVDF membrane into the antibody box of the primary antibody. The primary antibody was diluted with the primary antibody diluent at a ratio of 1:1000, and the antibody box was incubated at 4°C on a gentle shaker (10-16h).

11) Incubate secondary antibody and develop color

Remove the overnight incubated PVDF membrane from the antibody box and recover the primary antibody. The PVDF membrane was slowly washed 3 times in TBST, 10 min each time. Put the washed PVDF membrane into the corresponding species of secondary antibody (1:3000) dilution solution (containing 5% skimmed milk), and incubate for 1 h at room temperature on a gentle shaker. After the secondary antibody incubation, the PVDF membrane was taken out and washed slowly in TBST three times, 10 min each time. Mix medium volumes of solution A and solution B in the ECL kit, add evenly on the surface of the membrane (protein surface), put it into a Tanon 5200 luminescence imager (turn on pre-cooling 5 minutes in advance) for exposure and development, take pictures and save the pictures.

12) Reagent formula:

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

10x Electroporation Buffer Formula: Glycine 151.1g, Tris 30.3g;

1x electrotransfer buffer formula: take 200ml of 75% ethanol, 100ml of 10x electrotransfer solution, 700ml of water, and dilute to 1L;

1x TPST formula: Nacl 8.7g, Tris 2.4g, PH 7.2-7.4, Tween 20 1ml;

13) Western Blot strip gray value analysis

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

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.

(4) Evaluation of metabolic stability

Metabolic stability is generally used to describe the speed and extent of a compound being metabolized, and is one of the main factors affecting pharmacokinetic properties. Many compounds are substrates of CYP450 enzymes and other drug-metabolizing enzymes, and liver microsomes are a system rich in CYP450. The purpose of this experiment is to incubate the compounds of the present invention with human and SD rat liver microsomes and use LC-MS/MS was used to detect the remaining proportion of the compound to study the in vitro stability of the compound.

①Preparation of solution

Phosphate buffer saline (PBS): Mix 150mL of pre-prepared KH ₂ PO ₄ (0.5M) solution and 700mL of K ₂ HPO ₄ (0.5M) solution, then adjust the mixture with K ₂ HPO ₄ (0.5M) solution When the pH value reaches 7.4, it is used as 5-fold concentration PBS and stored at 4°C for later use. Before use, it was diluted 5 times with ultrapure water, and 3.3 mM magnesium chloride was added to obtain phosphate buffered saline PBS (100 mM).

NADPH regeneration system solution: use 5mL of PBS to prepare NADPH solution containing 6.5mM NADP, 16.5mM G-6-P, 3U/mL G-6-PD.

Internal standard stop solution: use acetonitrile to prepare 50ng/mL propranolol hydrochloride and 200ng/mL tolbutamide as internal standard working solution.

Human liver microsome solution: Add 0.31mL human liver microsome (25mg/mL) into 0.961mL PBS (pH7.4) and mix well to obtain a dilution of human liver microsome with a protein concentration of 0.625mg/mL.

SD rat liver microsomes solution: take 0.31mL SD rat liver microsomes (25mg/mL) and add them into 0.961mL PBS (pH7.4) and mix well to obtain a dilution of SD rat liver microsomes with a protein concentration of 0.625mg/mL liquid.

Sample working solution: Prepare the compound of the present invention and non-deuterated compound powder, positive control dextromethorphan powder and omeprazole powder to 10 mM with DMSO as the sample stock solution. Then dilute with 70% acetonitrile-water to obtain 0.25mM sample working solution.

② Sample incubation

Take 398 μL of human liver microsome dilution and add it to a 96-well incubation plate (N=2), add 2 μL of 0.25 mM test compound and dextromethorphan respectively, and mix well.

Take 398 μL of SD rat liver microsome dilution and add it to a 96-well incubation plate (N=2), add 2 μL of 0.25 mM test compound and omeprazole respectively, and mix well.

Add 300 μL of pre-cooled stop solution to each well into a 96-well deep-well plate and place it on ice as a stop plate.

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

When reacting for 10, 30, and 90 minutes, respectively, take 100 μL of the reaction solution, add it to the stop plate, and vortex for 3 minutes to stop the reaction.

The termination plate was centrifuged at 5000 rpm at 4°C for 15 min. Take 200 μL of supernatant to a 96-well plate pre-added with 200 μL of ultrapure water, mix well, use LC-MS/MS for sample analysis, and inject 10 μL.

③ Sample analysis method

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

④Data processing

The peak area of the sample and the internal standard is obtained by the mass spectrometer and Analyst software, and the substrate elimination rate constant K can be obtained by plotting the remaining amount of the compound (R%) and time using the single exponential degradation model of the Graphpad prism7.0 software

Ct/C0=exp(-K*t)

And calculate the half-life T _1/2 and the internal clearance CL _int according to the following formula, where V/M is equal to 1/C (protein).

t _1/2 (min); CL _int (μL/min/mg).

Experimental results: The compounds of the present invention and their non-deuterated compounds were tested and compared simultaneously, and their metabolic stability in human and SD rat liver microsomes was evaluated. Compared with non-deuterated compounds, the compound of the present invention has longer half-life T _1/2 and lower clearance rate CL _int , and can obviously improve metabolic stability. Results for representative example compounds are summarized in Table 2 below.

Table 2:

(5) Rat pharmacokinetic experiment

6 male Sprague-Dawley rats, 7-8 weeks old, weighing about 210g, were divided into 2 groups, 3 rats in each group, and a single dose of the compound (10 mg/kg orally) was administered intravenously or orally, and the pharmacokinetic differences were compared .

Rats were fed with standard diet and given water. Fasting started 16 hours before the test. Drugs were dissolved with PEG400 and DMSO. Orbital blood was collected 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.

Rats were briefly anesthetized after inhalation of ether, and 300 μL blood samples were collected from the orbits in test tubes. There is 30 μL of 1% heparin saline solution in the test tube. The test tubes were dried overnight at 60°C before use. After blood sampling at the last time point, the rats were anesthetized with ether and sacrificed.

Immediately after blood sample collection, gently invert the tube at least 5 times to ensure thorough mixing and place on ice. Blood samples were centrifuged at 5000rpm at 4°C for 5 minutes to separate plasma from red blood cells. Aspirate 100 μL of plasma with a pipette into a clean plastic centrifuge tube, labeling the name and time point of the compound. Plasma was stored at -80°C until analysis. Concentrations of compounds of the invention in plasma were determined by LC-MS/MS. Pharmacokinetic parameters were calculated based on the plasma concentrations of each animal at different time points.

Experiments show that the compound of the present invention has better pharmacokinetic properties in animals, and therefore has better pharmacodynamics and therapeutic effects. Results for representative example compounds are summarized in Table 3 below.

the	PirtobrutinibPirtobrutinib	T-1-ST-1-S	T-3-ST-3-S
给药方式/剂量Administration/Dosage	IV(0.5mg/kg)IV (0.5mg/kg)	IV(0.5mg/kg)IV (0.5mg/kg)	IV(0.5mg/kg)IV (0.5mg/kg)

C _max(ng/mL) C _max (ng/mL)	363363	22462246	659659
AUC _last(hng/mL) AUC _last (hng/mL)	293293	18071807	414414
AUC _{INF_pred}(hng/mL) AUC _{INF_pred} (hng/mL)	297297	18211821	418418
MRT _last(h) MRT _last (h)	1.271.27	1.491.49	1.271.27
Vz _{_pred}(L/kg) _{Vz_pred} (L/kg)	4.824.82	2.792.79	2.982.98
Cl _{_pred}(L/h/kg) _{Cl_pred} (L/h/kg)	29.129.1	9.259.25	20.520.5
T _1/2(h) T _1/2 (h)	2.022.02	3.493.49	1.71.7

The above content is a further detailed description of the present invention in conjunction with specific preferred embodiments, and it cannot be assumed that the specific implementation of the present invention is limited to these descriptions. For those of ordinary skill in the technical field of the present invention, without departing from the concept of the present invention, some simple deduction or replacement can be made, which should be regarded as belonging to the protection scope of the present invention.

Claims

Compound of formula (I), or its tautomer, stereoisomer, prodrug, crystal form, pharmaceutically acceptable salt, hydrate or solvate:

in,

Y 1 , Y 2 , Y 3 , Y 4 , Y 5 , Y 6 and Y 7 are each independently selected from hydrogen, deuterium, halogen or trifluoromethyl;

R 1 , R 2 and R 3 are each independently selected from hydrogen or deuterium;

X 1 and X 2 are each independently selected from CH 3 , CD 3 , CHD 2 or CH 2 D;

The additional proviso is that the above compounds contain at least one deuterium atom.
The compound according to claim 1, which is a compound of formula (IA):

in,

Y 1 , Y 2 , Y 3 , Y 4 , Y 5 , Y 6 and Y 7 are each independently selected from hydrogen, deuterium, halogen or trifluoromethyl;

R 1 , R 2 and R 3 are each independently selected from hydrogen or deuterium;

X 1 and X 2 are each independently selected from CH 3 , CD 3 , CHD 2 or CH 2 D;

The additional condition is that the above compounds contain at least one deuterium atom;

Or a tautomer, stereoisomer, prodrug, crystal form, pharmaceutically acceptable salt, hydrate or solvate thereof.
The compound according to claim 1, which is a compound of formula (IB):

in,

Y 1 , Y 2 , Y 3 , Y 4 , Y 5 , Y 6 and Y 7 are each independently selected from hydrogen, deuterium, halogen or trifluoromethyl;

R 1 , R 2 and R 3 are each independently selected from hydrogen or deuterium;

X 1 and X 2 are each independently selected from CH 3 , CD 3 , CHD 2 or CH 2 D;

The additional condition is that the above compounds contain at least one deuterium atom;

Or a tautomer, stereoisomer, prodrug, crystal form, pharmaceutically acceptable salt, hydrate or solvate thereof.
The compound according to any one of claims 1-3, wherein X 1 is CD 3 .
The compound according to any one of claims 1-4, wherein X 2 is CD 3 .
The compound according to any one of claims 1-5, wherein R 1 is deuterium.
The compound according to any one of claims 1-6, wherein R 2 is deuterium.
The compound according to any one of claims 1-7, wherein R 3 is deuterium.
The compound according to claim 1, which is a compound of formula (II):

in,

R 1 , R 2 and R 3 are each independently selected from hydrogen or deuterium;

X 1 and X 2 are each independently selected from CH 3 , CD 3 , CHD 2 or CH 2 D;

The additional condition is that the above compounds contain at least one deuterium atom;

Or a tautomer, stereoisomer, prodrug, crystal form, pharmaceutically acceptable salt, hydrate or solvate thereof.
The compound according to claim 9, which is a compound of formula (IIA):

in,

R 1 , R 2 and R 3 are each independently selected from hydrogen or deuterium;

X 1 and X 2 are each independently selected from CH 3 , CD 3 , CHD 2 or CH 2 D;

The additional condition is that the above compounds contain at least one deuterium atom;

Or a tautomer, stereoisomer, prodrug, crystal form, pharmaceutically acceptable salt, hydrate or solvate thereof.
The compound according to claim 9, which is a compound of formula (IIB):

in,

R 1 , R 2 and R 3 are each independently selected from hydrogen or deuterium;

X 1 and X 2 are each independently selected from CH 3 , CD 3 , CHD 2 or CH 2 D;

The additional condition is that the above compounds contain at least one deuterium atom;

Or a tautomer, stereoisomer, prodrug, crystal form, pharmaceutically acceptable salt, hydrate or solvate thereof.
The compound according to any one of claims 9-11, wherein X 1 is CD 3 .
The compound according to any one of claims 9-12, wherein X 2 is CD 3 .
The compound according to any one of claims 9-13, wherein R 1 is deuterium.
The compound according to any one of claims 9-14, wherein R 2 is deuterium.
The compound according to any one of claims 9-15, wherein R 3 is deuterium.
The compound according to claim 1, or its tautomer, stereoisomer, prodrug, crystal form, pharmaceutically acceptable salt, hydrate or solvate, wherein the compound is selected from:
A pharmaceutical composition, which contains a pharmaceutically acceptable excipient and the compound described in any one of claims 1-17, or its tautomer, stereoisomer, prodrug, crystal form, A pharmaceutically acceptable salt, hydrate or solvate.
The compound according to any one of claims 1-17, or its tautomer, stereoisomer, prodrug, crystal form, pharmaceutically acceptable salt, hydrate or solvate, or claim 18 Use of the pharmaceutical composition in preparing medicines for treating and/or preventing diseases related to BTK.
The use according to claim 19, wherein the BTK is selected from wild-type BTK or mutant BTK.
The use according to claim 20, wherein the mutant BTK is selected from BTK C481S, BTK C481F, BTK C481Y, BTK C481R, BTK C481T, BTK C481G or BTK C481W; preferably, the mutant BTK is selected from BTK C481S.
The use according to claim 19, wherein the BTK-related diseases are selected from cancer, inflammatory disorders, immune diseases or fibrosis.
The use according to claim 22, wherein the cancer is selected from hematological cancers.
The use according to claim 23, wherein the hematological cancer is selected from acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), acute promyelocytic leukemia (APL), chronic lymphocytic lymphoma (CLL), Chronic Myeloid Leukemia (CML), Chronic Myelomonocytic Leukemia (CMML), Chronic Neutrophil 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 with trilineage myelodysplastic disorder (AML/TMDS), mixed lineage leukemia ( MLL), myelodysplastic syndrome (MDS), myeloproliferative disorder (MPD), diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone lymphoma (eg, splenic marginal zone lymphoma lymphoma, extranodal marginal zone B-cell lymphoma), Burkitt lymphoma, Waldenstrom's macroglobulinemia (lymphoplasmacytic lymphoma), primary central nervous system lymphoma, small lymphoma Cell lymphoma, precursor B-cell lymphoblastic leukemia, hairy cell leukemia, mucosa-associated lymphoid tissue lymphoma, plasma cell myeloma, plasmacytoma, and multiple myeloma.
The use according to claim 22, wherein the cancer is selected from glioblastoma.
The use according to claim 22, wherein the immune disease is selected from arthritis, multiple sclerosis and osteoporosis.