Heteroaryl heterocyclic compounds and uses thereof

Abstract

Provided herein are heteroarylheterocyclic compounds of formula (I), pharmaceutical compositions containing them, methods for their preparation, and uses thereof, wherein each variable is as defined in the specification. The compound is a Btk inhibitor. #imgabs0#

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Chinese application No. 202010993583.8 filed on September 21, 2020, Chinese application No. 202110175357.3 filed on February 7, 2021, and Chinese application No. 202111077860.1 filed on September 15, 2021; the contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] The present invention relates to heteroaryl heterocyclic compounds, pharmaceutical compositions containing the same, and preparation methods and uses thereof. Background Art

[0004] Bruton's tyrosine kinase (BTK), a member of the Tec family of non-receptor tyrosine proteins (including BTK, LTK, TEC, BMX, and TXK), is widely expressed in hematopoietic cells, in addition to T cells, NK cells, and differentiated plasma cells. BTK plays a crucial role in signal transduction through the B cell antigen receptor (BCR) and Fcγ receptor (FcγR) in B cells and myeloid cells. It is a key regulator of B cell development, activation, signaling, and survival. It controls B cell development and differentiation by activating positive cell cycle regulators and differentiation factors, and controls B cell survival and proliferation by regulating the expression of pro-apoptotic and anti-apoptotic proteins. BTK also plays a crucial role in the migration and adhesion of B lymphoma cells. In addition, BTK plays a role in numerous other hematopoietic cell signaling pathways, such as Toll-like receptor (TLR) and cytokine receptor-mediated TNF-α production in macrophages, IgE receptor (FceRI) signaling in mast cells, inhibition of Fas / API-1 apoptotic signaling in B-lineage lymphoid cells, and collagen-stimulated platelet aggregation.

[0005] In humans, mutations in the BTK gene can cause the inherited immunodeficiency disease X-linked agammaglobulinaemia (XLA). Human XLA patients have point mutations in the BTK gene, which are associated with extremely low BTK mRNA levels and BTK protein expression. As a result, the loss of BTK kinase activity leads to an almost complete lack of mature B cells and immunoglobulins, as well as a significant attenuation of sustained calcium signals in response to BCR stimulation. The effects of BTK mutations are limited to the B cell population, and no significant developmental defects of other immune cells have been found in XLA patients. Spontaneous mutations in the BTK gene have also been found in X-linked immunodeficiency (xid) mice, which display a similar but less severe phenotype. In xid mice or mutation-induced BTK gene knockout mice, B cell differentiation is partially blocked, the number of mature B cells in the blood circulation is reduced, and they show resistance to collagen-induced arthritis and staphylococcal-induced arthritis models. A wealth of evidence shows that BTK is highly expressed in peripheral B cells of patients with autoimmune diseases such as rheumatoid arthritis (RA), primary Sjögren's syndrome (pSS), and systemic lupus erythematosus (SLE), as well as in B-cell leukemias and lymphomas. Abnormal activation of BCR signaling has been demonstrated in these autoimmune and B-cell-related diseases, and inhibition of B cells, the BCR signaling pathway, and BTK can mitigate disease progression to varying degrees.

[0006] Based on the key role of BTK in B cell development and function, BTK is considered a potential target for the treatment of B cell malignancies and autoimmune diseases. A variety of BTK inhibitors are being developed for clinical studies of hematological malignancies and autoimmune diseases. Small molecule BTK inhibitors (such as ibrutinib, acalabrutinib, zanubrutinib, PRN1008, GDC-0853) have shown relatively effective therapeutic effects. For example, the irreversible BTK inhibitor ibrutinib has shown high lasting efficacy and low toxicity in clinical studies. It was approved by the U.S. Food and Drug Administration (FDA) in 2013 for the treatment of relapsed mantle cell lymphoma (MCL), in 2014 for chronic lymphocytic leukemia (CLL), and in 2015 for It was approved in 2017 for the treatment of WM and relapsed / refractory marginal zone lymphoma (MZL). In particular, its 2017 expansion of its approved indication to include chronic graft-versus-host disease (GVHD) demonstrated the mechanistic role of BTK in the treatment of chronic autoimmune diseases. Furthermore, the irreversible BTK inhibitor acalabrutinib was approved for adult MCL in 2017 and CLL in 2019; zanubrutinib received FDA approval for MCL in November 2019; and PRN1008 is undergoing a Phase 3 study for pemphigus vulgaris. Several irreversible BTK inhibitors (tirabrutinib, spebrutinib, evobrutinib) and reversible BTK inhibitors (GDC-0853, ARQ-531, and LOXO-305) are undergoing preclinical and clinical development.

[0007] Therefore, BTK inhibitors represent an attractive approach for development in the treatment of related diseases, in particular autoimmune diseases, inflammatory diseases or cancer.

[0008] Summary of the Invention

[0009] The present invention provides a compound of formula (I):

[0010]

[0011] or a pharmaceutically acceptable salt thereof, or a solvate, racemic mixture, enantiomer, diastereomer or tautomer thereof, wherein:

[0012] X1, X2 and X3 are each independently CH or N;

[0013] U and V are each independently N or CR9;

[0014] Y1 and Y2 are each independently CR 10 or N;

[0015] R1 and R2 are independently selected from hydrogen, deuterium, halogen, -CN, hydroxyl, C 1-6 Alkyl, C 3-6 Cycloalkyl, C 2-6 Alkynyl, C 1-6 Deuterated alkyl and C 1-6 Haloalkyl; or, R1, R2 together with the carbon atom to which they are attached form a 3-6 membered cyclic hydrocarbon group;

[0016] R3 is hydrogen, deuterium, halogen, -CN or C 1-6 alkyl halide;

[0017] R4 is hydrogen, halogen, -CN, C1-6 Alkyl, C 2-6 Alkynyl, -(C 1-3 Alkyl)-OH, -(C 1-3 alkyl)-O-(C 1-3 alkyl), -O-(C 1-3 alkyl), -CHO, -C(O)NH2, -C(O)NHCH3, -C(O)N(CH3)2 or 3-hydroxy-oxetane-3-yl, wherein the C 1-6 Alkyl or C 1-3 The alkyl groups are each optionally substituted with one or more deuterium or halogen;

[0018] R5 is selected from hydrogen, C 1-6 Alkyl and C 3-6 Cycloalkyl, wherein the C 1-6 The alkyl group is optionally substituted with one or more deuterium or halogen;

[0019] Z1, Z2, Z3 and Z4 are each independently CH or N, provided that at least one of Z1, Z2, Z3 and Z4 is N;

[0020] R6 and R7 are independently selected from C 1-6 alkyl;

[0021] R8 is hydrogen, C 1-6 Alkyl, C 3-6 Cycloalkyl or 4-8 membered heterocyclic group, wherein the C 1-6 Alkyl, C 3-6 The cycloalkyl or 4-8 membered heterocyclyl is optionally substituted by one or more groups selected from the group consisting of deuterium, halogen, C 1-6 Alkyl, trifluoromethyl, -OH, -NH2, -O-(C 1-6 Alkyl), -NH(C 1-6 alkyl) or -N(C 1-6 Alkyl)2;

[0022] R9 is hydrogen, deuterium or halogen;

[0023] R 10 For hydrogen, deuterium, halogen, CN, C 1-6 Alkyl or C 1-6 alkyl halide;

[0024] n is 0, 1 or 2; provided that, when n is 1, R3 is not hydrogen.

[0025] The above-mentioned compounds and the active compounds disclosed in the context of the present invention (including general compounds and specific compounds), as well as pharmaceutically acceptable salts thereof, or their solvates, racemic mixtures, enantiomers, diastereomers or tautomers, are referred to herein as "compounds of the present invention".

[0026] The present invention also provides a pharmaceutical composition comprising a compound of the present invention and optionally a pharmaceutically acceptable excipient.

[0027] The present invention also provides a method for inhibiting BTK activity in vivo or in vitro, comprising contacting an effective amount of the compound of the present invention with BTK.

[0028] The present invention also provides a method for treating or preventing a disease mediated or at least partially mediated by BTK, comprising administering to a subject in need thereof an effective amount of a compound of the present invention.

[0029] The present invention also provides a method for treating or preventing autoimmune diseases, inflammatory diseases or cancer, which comprises administering an effective amount of a compound of the present invention to a subject in need thereof.

[0030] The present invention also provides the use of the compounds of the present invention in treating or preventing diseases mediated or at least partially mediated by BTK.

[0031] The present invention also provides use of the compound of the present invention in treating or preventing autoimmune diseases, inflammatory diseases or cancer.

[0032] The present invention also provides use of the compound of the present invention in the preparation of a medicament for treating or preventing a disease mediated or at least partially mediated by BTK.

[0033] The present invention also provides use of the compound of the present invention in preparing a medicament for treating or preventing autoimmune diseases, inflammatory diseases or cancer.

[0034] The present invention also provides compounds of the present invention for use in inhibiting BTK activity in vivo or in vitro.

[0035] The present invention also provides a compound of the invention for use as a medicament.

[0036] The present invention also provides a compound of the invention for use as a medicament in the treatment or prevention of a disease mediated or at least in part by BTK, in particular for the treatment or prevention of an autoimmune disease, an inflammatory disease or cancer.

[0037] The present invention also provides a pharmaceutical combination comprising a compound of the present invention and at least one additional therapeutic agent, wherein the therapeutic agent is preferably selected from: an anti-inflammatory agent, an immunomodulatory agent or an anti-tumor active agent, wherein the anti-tumor active agent includes a chemotherapeutic agent, an immune checkpoint inhibitor or agonist, and a targeted therapeutic agent.

[0038] The present invention also provides a kit for treating or preventing a disease mediated or at least partially mediated by BTK. The kit may comprise a pharmaceutical composition of the present invention and instructions for use, wherein the pharmaceutical composition comprises a compound of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 : The inhibitory effect of the compounds of the present invention on B cell activation in mouse whole blood induced by anti-IgD antibodies.

[0040] Figure 2 Effects of the compounds of the present invention on the joint paw volume of CIA (collagen-induced arthritis) rats (the hind paw volume was measured using a paw volume meter, and the data were expressed as mean ± standard deviation. The groups were normal group, vehicle control group, 0.25 mg / kg and 4 mg / kg GDC-0853 groups, and different doses of compound 1 QD groups (normal group: n=6, other groups: n=8).

[0041] Figure 3 Effects of the compounds of the present invention on peripheral blood platelet levels in mice with ITP (idiopathic thrombocytopenic purpura induced by anti-mouse CD41 antibodies). Platelet levels were measured using an automated hematology analyzer. Data are presented as mean ± standard deviation. The groups included a normal control group and a modeling group (respectively, a vehicle control group, a 40 mg / kg PRN1008 group, and groups receiving different doses of Compound 1) (N = 8 per group). Detailed Description of the Invention

[0043] definition

[0044] The following words, phrases and symbols used in this application have the meanings described below, unless the context indicates otherwise.

[0045] A hyphen ("-") that is not between two letters or symbols indicates the point of attachment of a substituent. For example, -OR 3 Refers to R 3 It is connected to the rest of the molecule through an oxygen atom.

[0046] The term "alkyl" as used herein refers to a group having 1 to 18 carbon atoms (C 1-18 ), preferably 1-10 carbon atoms (C 1-10 ), particularly preferably 1 to 6 carbon atoms (C 1-6), more preferably 1-4 carbon atoms (C 1-4 ) or 1-3 carbon atoms (C 1-3 ). When the term "alkyl" is prefixed with "C", it indicates the number of carbon atoms. For example, "C 1-6 "Alkyl" means an alkyl group having 1 to 6 carbon atoms, "C 1-3 "Alkyl" means an alkyl group having 1 to 3 carbon atoms. 1-6 Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl (e.g., n-propyl, isopropyl), butyl (e.g., n-butyl, isobutyl, sec-butyl, and tert-butyl), pentyl (e.g., n-pentyl, isopentyl, neopentyl), hexyl, and the like.

[0047] The term "alkynyl" as used herein refers to a group having 2 to 18 carbon atoms (C≡C) containing one or more, for example 1, 2 or 3, carbon-carbon triple bonds (C≡C). 2-18 ), preferably 2-10 carbon atoms (C 2-10 ), more preferably 2-6 carbon atoms (C 2-6 ), more preferably 2-4 carbon atoms (C 2-4 ) is a straight or branched unsaturated hydrocarbon group. When the term "alkynyl" is prefixed with "C", it indicates the number of carbon atoms. For example, "C 2-6 "Alkynyl" means an alkynyl group having 2 to 6 carbon atoms, "C 2-4 "Alkynyl" means an alkynyl group having 2 to 4 carbon atoms. 2-6 Examples of alkynyl groups include, but are not limited to, ethynyl, propynyl (e.g., 2-propynyl), butynyl (e.g., 2-butynyl), etc. The point of attachment of the alkynyl group may or may not be on a triple bond.

[0048] As used herein, the term "halogen" or "halo" refers to fluorine, chlorine, bromine and iodine, preferably fluorine, chlorine and bromine, more preferably fluorine and chlorine.

[0049] The term "haloalkyl" as used herein refers to an alkyl group as defined herein in which one or more hydrogen atoms, such as 1, 2, 3, 4 or 5 hydrogen atoms, are replaced by halogen atoms, and when more than one hydrogen atom is replaced by a halogen atom, the halogen atoms may be the same as or different from one another. In one embodiment, the term "haloalkyl" as used herein refers to an alkyl group as defined herein in which two or more hydrogen atoms, such as 2, 3, 4 or 5 hydrogen atoms, are replaced by halogen atoms, wherein the halogen atoms are the same as one another. In another embodiment, the term "haloalkyl" as used herein refers to an alkyl group as defined herein in which two or more hydrogen atoms, such as 2, 3, 4 or 5 hydrogen atoms, are replaced by halogen atoms, wherein the halogen atoms are different from one another. When the term "haloalkyl" is prefixed with "C", it indicates the number of carbon atoms. For example, "C 1-6"Haloalkyl" means a haloalkyl group as defined above having 1 to 6 carbon atoms, "C 1-4 "Haloalkyl" means a haloalkyl group as defined above having 1 to 4 carbon atoms. 1-6 Examples of haloalkyl include, but are not limited to, -CF3, -CHF2, -CH2F, -CH2CF3, -CH(CF3)2, and the like.

[0050] The term "cycloalkyl" as used herein refers to a group containing 3 to 12 ring carbon atoms (C 3-12 )(e.g. 3-8 ring carbon atoms (C 3-8 ), 5-7 ring carbon atoms (C 5-7 ), 4-7 ring carbon atoms (C 4-7 ) or 3-6 ring carbon atoms (C 3-6 )) is a saturated or partially unsaturated cyclic hydrocarbon group; it may have one or more rings, such as 1, 2 or 3, preferably 1 or 2 rings. When the term "cyclic hydrocarbon group" is prefixed with "C", it indicates the number of carbon atoms. For example, "C 3-6 "Cycloalkyl" or "3-6 membered cycloalkyl" means a cycloalkyl having 3-6 ring carbon atoms. The cycloalkyl may include fused or bridged rings and spiro rings. The ring of the cycloalkyl may be saturated, and may contain one or more, for example one or two, double bonds (i.e., partially unsaturated), but it is not completely conjugated and is not an "aryl" as defined in the present invention. 3-6 Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, spiro[2.2]pentanyl, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclopentadienyl, cyclohexenyl, and the like.

[0051] As used herein, the terms "heterocyclyl" or "heterocycle" are used interchangeably and refer to a saturated or partially unsaturated ring having 3-12 ring atoms (e.g., 3-8 ring atoms, 4-8 ring atoms, 4-6 ring atoms, or 4-5 ring atoms), wherein the ring atoms include one or more (e.g., 1, 2, or 3, preferably 1 or 2) heteroatoms independently selected from N, O, and S, with the remaining ring atoms being carbon atoms; which may have one or more rings, e.g., 1, 2, or 3, preferably 1 or 2 rings. Wherein, N and S may optionally be oxidized to various oxidation states. The point of attachment of the heterocyclyl group may be on the N heteroatom or on a carbon atom. For example, "4-8 membered heterocyclyl" means a heterocyclyl having 4-8 (4, 5, 6, 7 or 8) ring atoms, which contains at least one, for example 1, 2 or 3, preferably 1 or 2 heteroatoms independently selected from N, O and S; "4-6 membered heterocyclyl" means a heterocyclyl having 4-6 (4, 5 or 6) ring atoms, which contains at least one, preferably 1 or 2 heteroatoms independently selected from N, O and S (preferably N and O, more preferably O), which is preferably a monocycle; "4-5 membered heterocyclyl" means a heterocyclyl having 4 or 5 ring atoms, which contains at least one, preferably 1 or 2 heteroatoms independently selected from N, O and S (preferably N and O, more preferably O), which is a monocycle. Heterocyclyls may include fused or bridged rings as well as spirocycles. The ring of the heterocyclic group may be saturated, and may contain one or more, for example one or two, double bonds (ie, partially unsaturated), but it is not completely conjugated and is not a "heteroaryl" as defined in the present invention. Examples of heterocyclic groups include, but are not limited to, 4- to 8-membered heterocyclic groups, 4- to 6-membered heterocyclic groups, 4- to 5-membered heterocyclic groups, and 4-membered heterocyclic groups, such as oxetanyl (e.g., oxetan-3-yl), azetidinyl, pyrrolidinyl, tetrahydrofuranyl, dioxolanyl, tetrahydropyranyl, morpholinyl, thiomorpholinyl, piperidinyl, piperazinyl, tetrahydropyridinyl, pyrazinyl, pyrazolidinyl, and oxaspiro[3.3]heptanyl, preferably oxetanyl (e.g., oxetan-3-yl), azetidinyl, tetrahydropyranyl, morpholinyl (e.g., morpholino), piperazinyl (e.g., piperazin-1-yl), tetrahydropyridinyl (e.g., 1,2,3,6-tetrahydropyridinyl).

[0052] The term "-OH" as used herein refers to a hydroxyl group.

[0053] As used herein, the term "-CN" refers to a cyano group.

[0054] The term "oxo" as used herein refers to =0.

[0055] Any asymmetric atom (e.g., carbon, etc.) of the compound of formula (I) may be in racemic or enantiomerically enriched form, for example, in (R)-, (S)-, or (RS)-configuration. In some embodiments, the asymmetric atom in the (R)- or (S)-configuration each has at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% enantiomeric excess.

[0056] As used herein, the terms "optional," "optionally," or "optionally" mean that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not. For example, "optionally substituted with one or more..." includes unsubstituted and substituted with 1, 2, 3, or more of the described substituents. It will be understood by those skilled in the art that for any group containing one or more substituents, the group does not include any substitution pattern that is sterically impractical, chemically incorrect, synthetically infeasible, and / or inherently unstable.

[0057] As used herein, the term "substituted" or "substituted by..." means that one or more (e.g., 1, 2, 3, or 4) hydrogen atoms on a given atom or group are replaced by one or more (e.g., 1, 2, 3, or 4) substituents, preferably substituents selected from a given substituent group or group group, provided that the normal valence of the given atom is not exceeded, and the substituents may be the same or different from each other. As used herein, the term "substituted by one or more groups selected from..." or "substituted by one or more..." means that one or more hydrogen atoms on a given atom or group are independently replaced by one or more groups selected from a given substituent group or group group, wherein the groups may be the same or different from each other. Preferably, "substituted by one or more groups selected from..." or "substituted by one or more..." means that a given atom or group is replaced by 1, 2, 3, or 4 groups independently selected from a given substituent group or group group, wherein the groups may be the same or different from each other. In some embodiments, when the substituent is oxo (i.e., =O), then two hydrogen atoms on a single atom are replaced. The optional substituents may be various groups, provided that the combination of substituents and / or variables produces a chemically correct and stable compound. Chemically correct and stable compounds mean compounds that are sufficiently stable to be isolated from a reaction mixture. Preferably, the substituents are those exemplified in the example compounds of this application.

[0058] Unless otherwise indicated, substituents are named into the core structure. For example, it is understood that when (cycloalkyl)alkyl is listed as a possible substituent, it means that the point of attachment of the substituent to the core structure is at the alkyl portion.

[0059] It will be appreciated by those skilled in the art that some compounds of formula (I) may contain one or more chiral centers and therefore have two or more stereoisomers. Racemic mixtures of these isomers, single isomers, and mixtures enriched in one enantiomer, as well as diastereomers and mixtures partially enriched in specific diastereomers when there are two chiral centers are all within the scope of the present invention. It will also be appreciated by those skilled in the art that the present invention includes all single stereoisomers (e.g., enantiomers), racemic mixtures, or partially resolved mixtures of compounds of formula (I), and, where appropriate, individual tautomers thereof.

[0060] Racemic mixtures can be used as such or resolved into their individual isomers. Resolution can yield stereochemically pure compounds or mixtures enriched in one or more isomers. Methods for separating isomers are well known (see Allinger NL and Eliel EL, "Topics in Stereochemistry," Vol. 6, Wiley Interscience, 1971) and include physical methods such as chromatography using chiral adsorbents. Individual isomers can be prepared in chiral form from chiral precursors. Alternatively, the individual isomers can be chemically separated from the mixture by forming diastereomeric salts with chiral acids (e.g., 10-camphorsulfonate, camphorate, α-bromocamphorate, tartaric acid, diacetyltartaric acid, malic acid, pyrrolidone-5-carboxylic acid, etc.), fractionally crystallizing the salts, then liberating one or both of the resolved bases, and optionally repeating this process to obtain one or two isomers that are substantially free of the other isomer, i.e., isomers with an optical purity of >95%. Alternatively, the racemate can be covalently linked to a chiral compound (auxiliary) to obtain diastereoisomers, which can be separated by chromatography or fractional crystallization, followed by chemical removal of the chiral auxiliary to obtain the pure enantiomers.

[0061] The term "tautomer" refers to functional group isomers resulting from the rapid shift of an atom between two positions in a molecule. Tautomers can be interconverted, for example, the enol and keto forms are typical tautomers.

[0062] "Pharmaceutically acceptable salts" refer to salts of free acids or bases of compounds of formula (I) that are non-toxic, biologically tolerable, or otherwise biologically suitable for administration to treat or prevent a subject. For example, acid addition salts include, for example, those derived from inorganic acids, such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, and nitric acid, and organic acids, such as p-toluenesulfonic acid, salicylic acid, methanesulfonic acid, oxalic acid, succinic acid, citric acid, malic acid, lactic acid, fumaric acid, and the like. For a general description of pharmaceutically acceptable salts, see, for example, SM Berge et al., "Pharmaceutical Salts," J. Pharm. Sci., 1977, 66: 1-19, and Handbook of Pharmaceutical Salts, Properties, Selection, and Use, ed. Stahl and Wermuth, Wiley-VCH and VHCA, Zurich, 2002.

[0063] Furthermore, if the compounds described herein are obtained in the form of acid addition salts, their free base forms can be obtained by basifying a solution of the acid addition salt. Conversely, if the product is in the form of a free base, its acid addition salt, particularly a pharmaceutically acceptable acid addition salt, can be obtained by dissolving the free base in a suitable solvent and treating the solution with an acid according to conventional procedures for preparing acid addition salts from basic compounds. Those skilled in the art can readily identify various synthetic methods for preparing non-toxic pharmaceutically acceptable acid addition salts or base addition salts without undue experimentation.

[0064] The term "deuterated compound" refers to a compound in which one or more hydrogen atoms, for example 1, 2, 3, 4 or 5 hydrogen atoms, are replaced by deuterium atoms (D).

[0065] The term "solvate" refers to a solvent addition form containing either stoichiometric or non-stoichiometric amounts of a solvent. Some compounds have a tendency to entrain fixed molar ratios of solvent molecules in the solid state, thereby forming a solvate. If the solvent is water, the solvate formed is a hydrate. When the solvent is ethanol, the solvate formed is an ethanolate. Hydrates are formed by one or more or fewer molecules of water with one molecule of the substance, wherein the water retains its molecular form of HO. Such a combination can form one or more hydrates, such as hemihydrates, monohydrates, and dihydrates.

[0066] As used herein, the terms "group" and "radical" are synonymous and are used to denote a functional group or a molecular fragment that can be attached to other molecular fragments.

[0067] The term "active ingredient" is used to refer to a chemical substance that has biological activity. In some embodiments, the "active ingredient" is a chemical substance that has pharmaceutical uses.

[0068] As used herein, the term "pharmaceutical combination" means a product obtained by mixing or combining two or more active ingredients, including fixed and non-fixed combinations of the active ingredients, such as kits and pharmaceutical compositions. The term "fixed combination" means that two or more active ingredients (e.g., a compound of the invention and an additional therapeutic agent) are administered to a patient simultaneously in the form of a single entity or dosage. The term "non-fixed combination" means that two or more active ingredients (e.g., a compound of the invention and an additional therapeutic agent) are administered to a patient simultaneously, concurrently or sequentially as separate entities, wherein the administration provides therapeutically effective levels of the compounds in the patient.

[0069] The terms "treating" or "treatment" or "preventing" a disease or disorder refer to the administration of one or more pharmaceutical substances, particularly compounds of the present invention, to a subject suffering from the disease or disorder, or having symptoms of the disease or disorder, or having a predisposition to the disease or disorder, in order to cure, heal, alleviate, alleviate, alter, cure, improve, ameliorate, or affect the disease or disorder, the symptoms of the disease or disorder, or the predisposition to the disease or disorder. In some embodiments, the disease or disorder is cancer, such as a solid tumor or a hematological malignancy, including lymphoma, leukemia, and myeloma. In other embodiments, the disease or disorder is an inflammatory disease or an autoimmune disease.

[0070] When referring to a chemical reaction, the terms "treating," "contacting," and "reacting" mean adding or mixing two or more reagents under appropriate conditions to produce the indicated and / or desired product. It should be understood that the reaction that produces the indicated and / or desired product may not necessarily result directly from the combination of the two reagents initially added, that is, there may be one or more intermediates generated in the mixture that ultimately lead to the formation of the indicated and / or desired product.

[0071] The term "effective amount" as used herein refers to an amount or dosage of a BTK inhibitor that is generally sufficient to produce a beneficial effect in a patient who needs treatment or prevention of a disease or disorder mediated by BTK activity or at least in part by BTK. The effective amount or dosage of the active ingredient in the present invention can be determined by conventional methods (e.g., modeling, dose escalation studies, or clinical trials) in combination with conventional influencing factors (e.g., the mode or route of administration or administration, the pharmacokinetics of the pharmaceutical ingredient, the severity and course of the disease or disorder, the individual's previous or ongoing treatment, the individual's health status and response to the drug, and the judgment of the attending physician).

[0072] Typical dosage range is from about 0.0001 to about 200 milligrams of active ingredient every kilogram of individual body weight every day, for example, be about 0.001 to 100 mg / kg / day, or be about 0.01 to 35 mg / kg / day, or be about 0.1 to 10 mg / kg, once a day or divided dose unit taking (for example, twice a day, three times a day, four times a day).For 70 kilograms of people, the example range of suitable dose is about 0.05 to about 7 grams / day, or be about 0.2 to about 5 grams / day.Once the patient's disease or obstacle appear to improve, dosage can be adjusted to maintain effect.For example, according to the variation of symptoms, dosage or administration frequency or dosage and administration frequency can be reduced to the level of maintenance desired treatment or preventive effect.Certainly, if the symptoms are alleviated to appropriate level, treatment can be stopped.But, for the recurrence of symptoms, the patient may need intermittent long-term treatment.

[0073] The term "inhibit" refers to a reduction in the baseline activity of a biological activity or process. The term "inhibit BTK activity" is the actual drug activity for the purposes of this invention and refers to a reduction in BTK activity that results, directly or indirectly, in response to the presence of a compound of the invention, relative to the activity of BTK in the absence of the compound of the invention. The reduction in activity can be caused by a direct interaction between the compound of the invention and BTK, or by an interaction between the compound of the invention and one or more other factors that affect BTK activity. For example, the compounds of the invention can reduce the activity of BTK by directly binding to BTK, can reduce the activity of BTK by directly or indirectly affecting another factor, or can reduce the activity of BTK by directly or indirectly reducing the amount of BTK present in a cell or organism.

[0074] As used herein, the term "subject" or "patient" refers to both mammals and non-mammals. Mammals refer to any member of the mammalian family, including but not limited to humans; non-human primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, and pigs; livestock such as rabbits, dogs, and cats; laboratory animals, including rodents such as rats, mice, and guinea pigs; and the like. Examples of non-mammals include but are not limited to birds. The term "subject" or "patient" does not limit the subject to a particular age or sex. In some embodiments, the subject or patient is a human.

[0075] In general, the term "about" is used herein to modify a stated numerical value by 20% above or below that value.

[0076] Technical and scientific terms used herein without specific definition have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0077] All numerical ranges herein should be understood to disclose every value and subset of values ​​within the range, regardless of whether they are specifically disclosed otherwise. For example, when any numerical range is mentioned, it should be considered to have mentioned every value within the numerical range, such as every integer within the numerical range, such as C herein. 1-6 It means including 1, 2, 3, 4, 5 or 6 C. The present invention relates to all values ​​falling within these ranges, all smaller ranges and upper or lower limits of numerical ranges. DETAILED DESCRIPTION

[0078] Embodiment 1. Compound of formula (I):

[0079]

[0080] or a pharmaceutically acceptable salt thereof, or a solvate, racemic mixture, enantiomer, diastereomer or tautomer thereof, wherein:

[0081] X1, X2 and X3 are each independently CH or N;

[0082] U and V are each independently N or CR9;

[0083] Y1 and Y2 are each independently CR 10 or N;

[0084] R1 and R2 are independently selected from hydrogen, deuterium, halogen, -CN, hydroxyl, C 1-6 Alkyl, 3-6 membered cyclic hydrocarbon, C 2-6 Alkynyl, C 1-6 Deuterated alkyl and C 1-6 Haloalkyl; or, R1, R2 together with the carbon atom to which they are attached form a 3-6 membered cyclic hydrocarbon group;

[0085] R3 is hydrogen, deuterium, halogen, -CN or C 1-6 alkyl halide;

[0086] R4 is hydrogen, halogen, -CN, C 1-6 Alkyl, C 2-6 Alkynyl, -(C 1-3 Alkyl)-OH, -(C 1-3 alkyl)-O-(C 1-3 alkyl), -O-(C 1-3 alkyl), -CHO, -C(O)NH2, -C(O)NHCH3, -C(O)N(CH3)2 or 3-hydroxy-oxetane-3-yl, wherein the C 1-6 Alkyl or C 1-3 The alkyl groups are each optionally substituted with one or more deuterium or halogen;

[0087] R5 is selected from hydrogen, C 1-6 Alkyl and C 3-6 Cycloalkyl, wherein the C 1-6 The alkyl group is optionally substituted with one or more deuterium or halogen;

[0088] Z1, Z2, Z3 and Z4 are each independently CH or N, provided that at least one of Z1, Z2, Z3 and Z4 is N;

[0089] R6 and R7 are independently selected from C 1-6 alkyl;

[0090] R8 is hydrogen, C 1-6 Alkyl, C 3-6 Cycloalkyl or 4-8 membered heterocyclic group, wherein the C 1-6 Alkyl, C 3-6 The cycloalkyl or 4-8 membered heterocyclyl is optionally substituted by one or more groups selected from the group consisting of deuterium, halogen, C 1-6 Alkyl, trifluoromethyl, -OH, -NH2, -O-(C 1-6 Alkyl), -NH(C 1-6 alkyl) or -N(C 1-6 Alkyl)2;

[0091] R9 is hydrogen, deuterium or halogen;

[0092] R 10 For hydrogen, deuterium, halogen, CN, C 1-6 Alkyl or C 1-6 alkyl halide;

[0093] n is 0, 1 or 2; provided that, when n is 1, R3 is not hydrogen.

[0094] Embodiment 2. The compound according to embodiment 1, or a pharmaceutically acceptable salt thereof, or a solvate, racemic mixture, enantiomer, diastereomer or tautomer thereof, wherein R4 is C 1-6 Alkyl, -(C 1-3 Alkyl)-OH, -(C 1-3 deuterated alkyl) -OH, -CHO, -C(O)NH2, -C(O)NHCH3 or -C(O)N(CH3)2;

[0095] Preferably, R4 is C 1-6 Alkyl, -(C 1-3 Alkyl)-OH, -(C 1-3 deuterated alkyl) -OH or -CHO.

[0096] Embodiment 3. The compound of Embodiment 1 or 2, or a pharmaceutically acceptable salt thereof, or a solvate, racemic mixture, enantiomer, diastereomer or tautomer thereof, wherein:

[0097] X1, X2 and X3 are each independently CH or N;

[0098] U and V are each independently CR9;

[0099] Y1 and Y2 are each independently CR 10 ;

[0100] R1 and R2 are independently selected from hydrogen, deuterium, halogen, -CN, hydroxyl, C 1-6 Alkyl, C 1-6 Deuterated alkyl and C 1-6 alkyl halide;

[0101] R3 is hydrogen, deuterium, halogen, -CN or C 1-6 alkyl halide;

[0102] R4 is -(C 1-3 alkyl)-OH, -C(O)NH2, -C(O)NHCH3 or -C(O)N(CH3)2, wherein the C 1-3 The alkyl group is optionally substituted with one or more deuterium groups;

[0103] R5 is selected from hydrogen and C 1-6 Alkyl, wherein the C 1-6 The alkyl group is optionally substituted with one or more deuterium groups;

[0104] Z1, Z2, Z3 and Z4 are each independently CH or N, provided that at least one of Z1, Z2, Z3 and Z4 is N;

[0105] R6 and R7 are independently selected from C 1-6 alkyl;

[0106] R8 is hydrogen, C 1-6 Alkyl, C 3-6 Cycloalkyl or 4-8 membered heterocyclic group, wherein the C 1-6 Alkyl, C 3-6 The cycloalkyl or 4-8 membered heterocyclyl is optionally substituted by one or more groups selected from the group consisting of deuterium, halogen, C 1-6 Alkyl, trifluoromethyl, -OH or -NH2;

[0107] R9 is hydrogen or deuterium;

[0108] R 10 is hydrogen or deuterium;

[0109] n is 0, 1 or 2; provided that, when n is 1, R3 is not hydrogen.

[0110] Embodiment 4. The compound of any one of Embodiments 1-3, or a pharmaceutically acceptable salt thereof, or a solvate, racemic mixture, enantiomer, diastereomer or tautomer thereof, wherein:

[0111] X1, X2 and X3 are each independently CH or N;

[0112] U and V are each independently CR9;

[0113] Y1 and Y2 are each independently CR 10 ;

[0114] R1 and R2 are independently selected from hydrogen, deuterium, halogen, -CN, hydroxyl, C 1-6 Alkyl, C 1-6 Deuterated alkyl and C 1-6 alkyl halide;

[0115] R3 is hydrogen, deuterium, halogen, -CN or C 1-6 alkyl halide;

[0116] R4 is -(C 1-3 Alkyl)-OH, wherein the C 1-3 The alkyl group is optionally substituted with one or more deuterium groups;

[0117] R5 is selected from C 1-6 Alkyl, wherein the C 1-6 The alkyl group is optionally substituted with one or more deuterium groups;

[0118] Z1, Z2, Z3 and Z4 are each independently CH or N, provided that at least one of Z1, Z2, Z3 and Z4 is N;

[0119] R6 and R7 are independently selected from C 1-6 alkyl;

[0120] R8 is hydrogen, C 1-6 Alkyl, C 3-6 Cycloalkyl or 4-8 membered heterocyclic group, wherein the C 1-6 Alkyl, C 3-6 The cycloalkyl or 4-8 membered heterocyclyl is optionally substituted by one or more groups selected from the group consisting of deuterium, halogen, C 1-6 Alkyl, trifluoromethyl, -OH or -NH2;

[0121] R9 is hydrogen or deuterium;

[0122] R 10 is hydrogen or deuterium;

[0123] n is 0, 1 or 2; provided that, when n is 1, R3 is not hydrogen.

[0124] Embodiment 5. The compound of any one of Embodiments 1-4, or a pharmaceutically acceptable salt thereof, or a solvate, racemic mixture, enantiomer, diastereomer or tautomer thereof, wherein X1 is CH or N, X2 is CH, and X3 is N.

[0125] Embodiment 6. The compound of any one of Embodiments 1-5, or a pharmaceutically acceptable salt thereof, or a solvate, racemic mixture, enantiomer, diastereomer or tautomer thereof, wherein X3 is N.

[0126] Embodiment 7. The compound of any one of Embodiments 1-6, or a pharmaceutically acceptable salt thereof, or a solvate, racemic mixture, enantiomer, diastereomer or tautomer thereof, wherein X1 and X2 are both CH.

[0127] Embodiment 8. The compound of any one of Embodiments 1-7, or a pharmaceutically acceptable salt thereof, or a solvate, racemic mixture, enantiomer, diastereomer or tautomer thereof, wherein Y1 and Y2 are both CR 10 .

[0128] Embodiment 9. The compound of embodiment 8, or a pharmaceutically acceptable salt thereof, or a solvate, racemic mixture, enantiomer, diastereomer or tautomer thereof, wherein R 10 For hydrogen.

[0129] Embodiment 10. The compound of any one of Embodiments 1-9, or a pharmaceutically acceptable salt thereof, or a solvate, racemic mixture, enantiomer, diastereomer or tautomer thereof, wherein R1 and R2 are independently selected from C 1-6 alkyl;

[0130] Preferably, R1 and R2 are independently selected from C 1-3 alkyl;

[0131] More preferably, R1 and R2 are both methyl.

[0132] Embodiment 11. The compound of any one of Embodiments 1-10, or a pharmaceutically acceptable salt thereof, or a solvate, racemic mixture, enantiomer, diastereomer or tautomer thereof, wherein R3 is hydrogen or halogen;

[0133] Preferably, R3 is hydrogen.

[0134] Embodiment 12. The compound of any one of Embodiments 1-11, or a pharmaceutically acceptable salt thereof, or a solvate, racemic mixture, enantiomer, diastereomer or tautomer thereof, wherein, preferably, R4 is -(C 1-3 Alkyl)-OH or -(C 1-3 deuterated alkyl)-OH;

[0135] Preferably, R4 is hydroxymethyl or hydroxydeuterated methyl;

[0136] More preferably, R4 is hydroxymethyl.

[0137] Embodiment 13. The compound of any one of Embodiments 1-12, or a pharmaceutically acceptable salt thereof, or a solvate, racemic mixture, enantiomer, diastereomer or tautomer thereof, wherein R3 is hydrogen, and R4 is -(C 1-3 alkyl)-OH.

[0138] Embodiment 14. The compound of any one of Embodiments 1-13, or a pharmaceutically acceptable salt thereof, or a solvate, racemic mixture, enantiomer, diastereomer or tautomer thereof, wherein U and V are both CH.

[0139] Embodiment 15. The compound of any one of Embodiments 1-14, or a pharmaceutically acceptable salt thereof, or a solvate, racemic mixture, enantiomer, diastereomer or tautomer thereof, wherein: R5 is C 1-6 alkyl;

[0140] Preferably, R5 is C 1-3 alkyl;

[0141] More preferably, R5 is methyl.

[0142] Embodiment 16. A compound as described in any one of Embodiments 1-15, or a pharmaceutically acceptable salt thereof, or a solvate, racemic mixture, enantiomer, diastereomer or tautomer thereof, wherein: Z1 is N, and Z2, Z3 and Z4 are all CH.

[0143] Embodiment 17. The compound of any one of Embodiments 1-16, or a pharmaceutically acceptable salt thereof, or a solvate, racemic mixture, enantiomer, diastereomer or tautomer thereof, wherein R6 and R7 are both methyl.

[0144] Embodiment 18. A compound as described in any one of Embodiments 1-17, or a pharmaceutically acceptable salt thereof, or a solvate, racemic mixture, enantiomer, diastereomer or tautomer thereof, wherein: R8 is hydrogen, C 1-6 Alkyl, C 3-6 Cycloalkyl or 4-8 membered heterocyclic group, wherein the C 1-6 Alkyl, C 3-6 The cycloalkyl or 4-5 membered heterocyclic group is optionally substituted by one or more groups selected from the group consisting of deuterium, halogen, C 1-6 Alkyl, trifluoromethyl, -OH or -NH2;

[0145] Preferably, R8 is hydrogen, C 1-6 Alkyl or 4-5 membered heterocyclic group, wherein the C 1-6 The alkyl or 4-5 membered heterocyclic group is optionally substituted by one or more groups selected from the group consisting of deuterium, halogen, C 1-6 Alkyl, trifluoromethyl, -OH or -NH2;

[0146] Preferably, R8 is a 4-5 membered heterocyclic group, optionally substituted by 1 or 2 groups selected from the group consisting of deuterium, halogen, C 1-3 Alkyl, trifluoromethyl, -OH or -NH2;

[0147] Preferably, R8 is a 4-5 membered heterocyclic group;

[0148] More preferably, R8 is a 4-membered heterocyclic group.

[0149] Embodiment 19. A compound as described in any one of Embodiments 1-18, or a pharmaceutically acceptable salt thereof, or a solvate, racemic mixture, enantiomer, diastereomer or tautomer thereof, wherein: R8 is oxetanyl or tetrahydrofuranyl.

[0150] Embodiment 20. The compound of embodiment 19, or a pharmaceutically acceptable salt thereof, or a solvate, racemic mixture, enantiomer, diastereomer or tautomer thereof, wherein R8 is

[0151] Embodiment 21. The compound of embodiment 1, or a pharmaceutically acceptable salt thereof, or a solvate, racemic mixture, enantiomer, diastereomer or tautomer thereof,

[0152] It is selected from:

[0153]

[0154] Embodiment 22. A pharmaceutical composition comprising the compound of any one of Embodiments 1-21 and / or a pharmaceutically acceptable salt thereof, and optionally comprising a pharmaceutically acceptable excipient.

[0155] Embodiment 23. A method for inhibiting BTK activity in vivo or in vitro, comprising contacting BTK with an effective amount of the compound of any one of Embodiments 1-21 and / or a pharmaceutically acceptable salt thereof.

[0156] Embodiment 24. Use of the compound of any one of Embodiments 1-21 and / or its pharmaceutically acceptable salt in the preparation of a medicament for treating or preventing a disease mediated or at least partially mediated by BTK, wherein the medicament is preferably used to treat or prevent an autoimmune disease, an inflammatory disease or cancer; wherein the inflammatory disease or autoimmune disease is preferably selected from the group consisting of systemic inflammation and local inflammation, arthritis, rheumatoid arthritis, inflammation associated with immunosuppression, organ transplant rejection, allergic disease, ulcerative colitis, Crohn's disease, dermatitis, asthma, lupus erythematosus, Sjögren's syndrome, multiple sclerosis chemosis, scleroderma, multiple sclerosis osteoporosis, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, antineutrophil cytoplasmic antibody vasculitis, chronic obstructive pulmonary disease, psoriasis, Sjögren's syndrome, herpes vulgaris, diseases related to kidney transplantation, thyroid autoimmune disease, chronic lymphocytic thyroiditis, hyperthyroidism, pernicious anemia with chronic atrophic gastritis, Goodpasture's syndrome, pemphigoid, primary biliary cirrhosis, acute idiopathic polyneuritis, systemic lupus erythematosus, mixed connective tissue disease; the cancer is preferably a solid tumor or a hematological malignancy, including lymphoma, leukemia and myeloma;

[0157] The cancer is more preferably selected from B cell malignancies, diffuse large B cell lymphoma (DLBCL), large B cell lymphoma (LBCL), B cell lymphoma, mantle cell lymphoma, follicular lymphoma, non-Hodgkin lymphoma, Hodgkin lymphoma, Waldenstrom's macroglobulinemia, marginal zone lymphoma, Burkitt lymphoma, non-Burkitt high grade B cell lymphoma, extranodal marginal zone B cell lymphoma, small lymphocytic lymphoma (SLL), lymphoblastic lymphoma, lymphocytes Leukemia, myeloid leukemia, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), human acute monocytic leukemia, acute lymphocytic leukemia (ALL), B-cell acute lymphocytic leukemia (B-ALL), hairy cell leukemia, chronic lymphocytic leukemia (CLL) (e.g., high-risk CLL), myelodysplastic syndrome, acute lymphoblastic leukemia, myeloma (e.g., multiple myeloma), or graft-versus-host disease.

[0158] Embodiment 25. A method for treating or preventing a disease in an individual, comprising administering to an individual in need thereof an effective amount of a compound and / or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-21, wherein the disease is mediated by BTK or at least partially mediated by BTK; the disease is preferably an autoimmune disease, an inflammatory disease or cancer; the inflammatory disease or autoimmune disease is preferably selected from the group consisting of systemic inflammation and local inflammation, arthritis, rheumatoid arthritis, inflammation associated with immunosuppression, organ transplant rejection, allergic disease, ulcerative colitis, leukemia ... Enteritis, Crohn's disease, dermatitis, asthma, lupus erythematosus, Sjögren's syndrome, multiple sclerosis, scleroderma, multiple sclerosis osteoporosis, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, antineutrophil cytoplasmic antibody vasculitis, chronic obstructive pulmonary disease, psoriasis, Sjögren's syndrome, pityriasis vulgaris, diseases related to kidney transplantation, thyroid autoimmune disease, chronic lymphocytic thyroiditis, hyperthyroidism, pernicious anemia with chronic atrophic gastritis, Goodpasture's syndrome, pemphigoid, primary biliary cirrhosis, acute idiopathic multiple sclerosis Neuroitis, systemic lupus erythematosus, mixed connective tissue disease; the cancer is preferably a solid tumor or a hematological malignancy, including lymphoma, leukemia and myeloma; the cancer is more preferably selected from B cell malignancies, diffuse large B cell lymphoma (DLBCL), large B cell lymphoma (LBCL), B cell lymphoma, mantle cell lymphoma, follicular lymphoma, non-Hodgkin lymphoma, Hodgkin lymphoma, Waldenstrom's macroglobulinemia, marginal zone lymphoma, Burkitt lymphoma, non-Burkitt high-grade B cell lymphoma, extranodal marginal zone B cell lymphoma, Small lymphocytic lymphoma (SLL), lymphoblastic lymphoma, lymphocytic leukemia, myeloid leukemia, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), human acute monocytic leukemia, acute lymphocytic leukemia (ALL), B-cell acute lymphocytic leukemia (B-ALL), hairy cell leukemia, chronic lymphocytic leukemia (CLL) (e.g., high-risk CLL), myelodysplastic syndrome, acute lymphoblastic leukemia, myeloma (e.g., multiple myeloma), or graft-versus-host disease.

[0159] Embodiment 26. A compound according to any one of Embodiments 1 to 21 and / or a pharmaceutically acceptable salt thereof for use as a medicament.

[0160] Embodiment 27. The compound of any one of Embodiments 1-21 and / or its pharmaceutically acceptable salt, for use in treating or preventing a disease mediated or at least partially mediated by BTK, preferably for treating or preventing an autoimmune disease, an inflammatory disease or cancer; the inflammatory disease or autoimmune disease is preferably selected from the group consisting of systemic inflammation and local inflammation, arthritis, rheumatoid arthritis, inflammation associated with immunosuppression, organ transplant rejection, allergic diseases, ulcerative colitis, Crohn's disease, dermatitis, asthma, lupus erythematosus, Sjögren's syndrome, multiple sclerosis, scleroderma, multiple sclerosis osteoporosis, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, antineutrophil cytoplasmic antibody vasculitis, chronic obstructive pulmonary disease, psoriasis, Sjögren's syndrome, herpes vulgaris, diseases related to kidney transplantation, thyroid autoimmune disease, chronic lymphocytic thyroiditis, hyperthyroidism, pernicious anemia with chronic atrophic gastritis, Goodpasture's syndrome, pemphigoid, primary biliary cirrhosis, acute idiopathic polyneuritis, systemic lupus erythematosus, mixed Connective tissue disease; the cancer is preferably a solid tumor or a hematological malignancy, including lymphoma, leukemia and myeloma; the cancer is more preferably selected from B cell malignancies, diffuse large B cell lymphoma (DLBCL), large B cell lymphoma (LBCL), B cell lymphoma, mantle cell lymphoma, follicular lymphoma, non-Hodgkin lymphoma, Hodgkin lymphoma, Waldenstrom's macroglobulinemia, marginal zone lymphoma, Burkitt lymphoma, non-Burkitt high-grade B cell lymphoma, extranodal marginal zone B cell lymphoma, small lymphocytic lymphoma The present invention relates to a type of leukemia or other related disease, wherein the present invention includes but is not limited to single-cell lymphoma (SLL), lymphoblastic lymphoma, lymphocytic leukemia, myeloid leukemia, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), human acute monocytic leukemia, acute lymphocytic leukemia (ALL), B-cell acute lymphoblastic leukemia (B-ALL), hairy cell leukemia, chronic lymphocytic leukemia (CLL) (e.g., high-risk CLL), myelodysplastic syndrome, acute lymphoblastic leukemia, myeloma (e.g., multiple myeloma), or graft-versus-host disease.

[0161] Embodiment 28. A pharmaceutical combination comprising a compound according to any one of Embodiments 1-21 and / or a pharmaceutically acceptable salt thereof, and at least one additional therapeutic agent, preferably selected from: an anti-inflammatory agent, an immunomodulatory agent, or an anti-tumor agent, wherein the anti-tumor agent includes a chemotherapeutic agent, an immune checkpoint inhibitor or agonist, and a targeted therapeutic agent.

[0162] The various embodiments described in the present invention (including the examples below) and the features in the various embodiments should be understood to be able to be combined with each other in any way, and the various solutions obtained by these combinations are included in the scope of the present invention, just as the solutions obtained by these combinations are specifically and one by one listed in this document, unless the context clearly indicates otherwise.

[0163] General synthetic method

[0164] The compounds of formula (I) described herein and / or pharmaceutically acceptable salts thereof can be synthesized using commercially available starting materials, by methods known in the art, or by methods disclosed in this patent application. The synthetic routes shown in Schemes 1-2 illustrate general synthetic methods for the compounds of the present invention, and the synthetic routes shown in Schemes 3-6 illustrate general synthetic methods for the starting material 1-1 used in Schemes 1-2.

[0165] Process 1:

[0166]

[0167] wherein: Hal represents halogen, R1, R2, R3, R4, R5, R6, R7, R8, X1, X2, X3, Z1, Z2, Z3, Z4, U, V, Y1, Y2 and n are as defined herein.

[0168] As shown in Scheme 1, a compound of Formula 1-1 reacts with a dihaloarylaldehyde compound of Formula 1-2 under the catalysis of cuprous iodide to produce a compound of Formula 1-3. The cuprous iodide-catalyzed carbon-nitrogen coupling reaction is carried out under suitable conditions. The solvent used can be a polar solvent such as 1,4-dioxane or DMF, and the base used can be selected from Cs2CO3, Na2CO3, K3PO4, etc. Formula 1-3 is reduced under suitable conditions to produce a compound of Formula 1-4 of the present invention. The reducing agent used can be selected from sodium borohydride, potassium borohydride, lithium borohydride, etc., and the solvent used can be a polar solvent such as methanol, ethanol, or a mixture of methanol and dichloromethane. An acetyl group is added to the compound of Formula 1-4 to produce a compound of Formula 1-5. The compound of Formula 1-5 reacts with biboronic acid pinacol ester under suitable conditions to produce a boronic acid or boric acid ester compound of Formula 1-6. The compound of Formula 1-6 is reacted with a halide of Formula 1-7 under the catalysis of an appropriate palladium reagent via a Suzuki coupling reaction to obtain a compound of Formula 1-8. The palladium-catalyzed carbon-carbon coupling reaction is carried out under suitable conditions. The solvent used can be selected from polar solvents such as 1,4-dioxane, DMF, THF, or a mixed solvent of 1,4-dioxane and water. The base used can be selected from Cs2CO3, Na2CO3, K3PO4, etc. The catalyst used can be selected from Pd(dppf)Cl2·CH2Cl2, Pd(PPh3)4, Pd(OAc)2, etc. The compound of Formula 1-8 is deacetylated under appropriate alkaline conditions to obtain the compound of Formula (I-1) of the present invention. The base used can be selected from potassium carbonate, sodium carbonate, lithium hydroxide, etc. The solvent used can be selected from polar solvents such as methanol, ethanol, or a mixed solvent of methanol and water.

[0169] Process 2:

[0170]

[0171] As shown in Scheme 2, the compound of Formula 1-3 is reacted with a boronic acid or boronic ester of Formula 2-1 via a Suzuki coupling reaction under the catalysis of an appropriate palladium reagent to obtain a compound of Formula 2-2. The palladium-catalyzed carbon-carbon coupling reaction is carried out under suitable conditions. The solvent used can be selected from polar solvents such as 1,4-dioxane, DMF, THF, or a mixed solvent of 1,4-dioxane and water. The base used can be selected from Cs2CO3, Na2CO3, K3PO4, etc., and the catalyst used can be selected from Pd(dppf)Cl2·CH2Cl2, Pd(PPh3)4, Pd(OAc)2, etc. The compound of Formula 2-2 is reduced under suitable conditions to obtain the compound of Formula (I-1) of the present invention. The reducing agent used can be selected from sodium borohydride, potassium borohydride, lithium borohydride, etc., and the solvent used can be selected from polar solvents such as methanol, ethanol, or a mixed solvent of methanol and dichloromethane.

[0172] Process 3:

[0173]

[0174] As shown in Scheme 3, the compound of Formula 3-1 undergoes a substitution reaction with bromoacetaldehyde diethyl acetal under appropriate conditions to obtain a compound of Formula 3-2. The base used can be selected from cesium carbonate, etc., and the solvent used can be selected from polar solvents such as DMF and 1,4-dioxane. The compound of Formula 3-2 is hydrolyzed in an alkaline solution to obtain a compound of Formula 3-3. The base used can be selected from lithium hydroxide, potassium carbonate, sodium carbonate, etc., and the solvent used can be selected from polar solvents such as methanol, ethanol, or a mixed solvent of methanol and water. The compound of Formula 3-3 undergoes a condensation reaction with HATU and ammonia water to obtain a compound of Formula 3-4. The compound of Formula 3-4 is ring-closed in acetic acid to obtain a compound of Formula 3-5.

[0175] Process 4:

[0176]

[0177] As shown in Scheme 4, the compound of Formula 3-1 undergoes a substitution reaction with hydrazine hydrate to obtain a compound of Formula 4-1. The compound of Formula 4-1 undergoes a ring-closure reaction with triethyl orthoformate in a DMF solution to obtain a compound of Formula 4-2.

[0178] The substituents of the compounds obtained by the above methods can be further modified to obtain other desired compounds. Synthetic chemical transformation methods can be referenced, for example, in: R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995) and subsequent editions.

[0179] Before use, the compounds of the present invention can be purified by column chromatography, high performance liquid chromatography, crystallization or other appropriate methods.

[0180] Pharmaceutical compositions and uses

[0181] The compounds of the present invention (e.g., any of the compounds of the Examples herein) can be formulated into pharmaceutical compositions, alone or in combination with one or more additional therapeutic agents. Pharmaceutical compositions include: (a) an effective amount of a compound of the present invention; (b) a pharmaceutically acceptable excipient (e.g., one or more pharmaceutically acceptable carriers); and optionally (c) at least one additional therapeutic agent.

[0182] A pharmaceutically acceptable excipient is an excipient that is compatible with the active ingredient in the composition (in some embodiments, stabilizes the active ingredient) and is not harmful to the individual being treated. For example, solubilizers such as cyclodextrins (which form specific, more soluble complexes with the compounds of the invention) can be used as pharmaceutical excipients to deliver the active ingredient. Examples of other excipients include colloidal silicon dioxide, magnesium stearate, cellulose, sodium lauryl sulfate, and pigments such as D&C Yellow #10. Suitable pharmaceutically acceptable excipients are disclosed in the standard reference text in this field (Remington's Pharmaceutical Sciences, A. Osol).

[0183] The pharmaceutical composition comprising the compound of the present invention can be administered in various known ways, such as orally, topically, rectally, parenterally, by inhalation or implantation. The term "parenteral" as used herein includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intraspinal, intralesional and intracranial injection or infusion.

[0184] The pharmaceutical compositions described herein can be prepared in the form of tablets, capsules, bagged granules, dragees, powders, granules, lozenges, powder injections, liquid preparations or suppositories. In some embodiments, the pharmaceutical compositions comprising the compounds of the present invention can be formulated for intravenous infusion, topical administration or oral administration.

[0185] Compositions for oral administration can be in any orally acceptable dosage form, including but not limited to tablets, capsules, emulsions, and aqueous suspensions, dispersions, and solutions. Common tablet carriers include lactose and corn starch. Lubricants such as magnesium stearate are also commonly added to tablets. When administered orally in capsule form, useful diluents include lactose and dried corn starch. When administered orally in the form of an aqueous suspension or emulsion, an emulsifier or suspending agent can be used to suspend or dissolve the active ingredient in the oil phase. If desired, certain sweeteners, flavorings, or pigments can also be added.

[0186] In some embodiments, the amount of the compound of the present invention in a tablet can be 1, 5, 10, 15, 20, 25, 50, 75, 80, 85, 90, 95, 100, 125, 150, 200, 250, 300, 400 and 500 mg. In some embodiments, the amount of the compound of the present invention in a capsule can be 1, 5, 10, 15, 20, 25, 50, 75, 80, 85, 90, 95, 100, 125, 150, 200, 250, 300, 400 and 500 mg.

[0187] Sterile injectable compositions (such as aqueous or oily suspensions) can be formulated using suitable dispersants or wetting agents (e.g., Tween 80) and suspending agents according to techniques known in the art. Sterile injectable compositions can also be sterile injectable solutions or suspensions in a nontoxic, parenterally acceptable diluent or solvent, such as a solution in 1,3-butanediol. Pharmaceutically acceptable carriers and solvents are particularly useful, including mannitol, water, Ringer's solution, and normal saline. In addition, sterile, nonvolatile oils such as synthetic mono- or diglycerides are commonly used as solvents or suspending media. Fatty acids such as oleic acid and its glyceride derivatives, as well as natural pharmaceutically acceptable oils such as olive oil or castor oil (especially their polyoxyethylated forms), are commonly used as injectable media. These oil solutions or suspensions may also contain long-chain alcohol diluents or dispersants, or carboxymethylcellulose or similar dispersants.

[0188] The inhalation composition can be prepared according to well-known techniques in the field of pharmaceutical formulation using benzyl alcohol or other suitable preservatives, absorption promoters to improve bioavailability, fluorocarbons and / or other solubilizers or dispersants known in the art, or it can be prepared as a solution in saline.

[0189] Topical compositions can be formulated into forms such as oils, creams, lotions, ointments, etc. Suitable carriers for the compositions include vegetable oils or mineral oils, white petrolatum (white soft paraffin), branched fats or oils, animal fats, and high molecular weight alcohols (i.e., alcohols having a carbon number greater than 12). In some embodiments, a pharmaceutically acceptable carrier is a carrier in which the active ingredient can be dissolved. If necessary, the composition may also include an emulsifier, a stabilizer, a wetting agent, and an antioxidant, as well as a substance that imparts color or fragrance. In addition, a transdermal penetration enhancer may also be added to the topical preparation. Examples of such enhancers are found in U.S. Patent Nos. 3,989,816 and 4,444,762.

[0190] Creams can be formulated from a mixture of mineral oil, self-emulsifying beeswax, and water, into which the active ingredient dissolved in a small amount of oil, such as almond oil, is mixed. One example of a cream comprises, by weight, about 40 parts water, about 20 parts beeswax, about 40 parts mineral oil, and about 1 part almond oil. Ointments can be prepared by mixing a solution of the active ingredient in a vegetable oil, such as almond oil, with warm soft paraffin and cooling the mixture. One example of an ointment comprises, by weight, about 30% almond oil and about 70% white soft paraffin.

[0191] Suitable in vitro experiments can be used to evaluate the effect of the compounds of the present invention on inhibiting BTK activity. The other effects of the compounds of the present invention in preventing or treating cancer can be further detected by in vivo tests. For example, the compounds of the present invention can be administered to animals (such as mouse models) suffering from cancer, and then their therapeutic effects are evaluated. If the results of the preclinical trials are successful, the dosage range and route of administration for animals such as humans can also be predicted.

[0192] The compounds of the invention show sufficient preclinical utility to warrant clinical trials and are expected to show beneficial therapeutic or prophylactic effects, for example, in individuals suffering from cancer.

[0193] As used herein, the term "cancer" refers to a cellular disorder characterized by uncontrolled or dysregulated cell proliferation, decreased cell differentiation, inappropriate ability to invade surrounding tissues, and / or the ability to establish new growth foci at other sites. The term "cancer" includes, but is not limited to, solid tumors and hematological malignancies (e.g., leukemias, lymphomas, or myelomas). The term "cancer" includes cancers of the skin, tissues, organs, bones, cartilage, blood, and blood vessels. The term "cancer" includes both primary cancers and metastatic cancers, recurrent cancers, and refractory cancers.

[0194] Non-limiting examples of solid tumors include pancreatic cancer; bladder cancer; colorectal cancer; breast cancer, including metastatic breast cancer; prostate cancer, including androgen-dependent and androgen-independent prostate cancer; testicular cancer; kidney cancer, including, for example, metastatic renal cell carcinoma; urothelial carcinoma; liver cancer; hepatocellular carcinoma; lung cancer, including, for example, non-small cell lung cancer (NSCLC), bronchioalveolar carcinoma (BAC), and lung adenocarcinoma; ovarian cancer, including, for example, progressive epithelial carcinoma or primary peritoneal cancer; cervical cancer; endometrial cancer; gastric cancer; esophageal cancer; head and neck cancer, including, for example, head and neck squamous cell carcinoma; skin cancer, including, for example, melanoma and basal cell carcinoma; neuroendocrine cancer, including metastatic neuroendocrine tumors; brain tumors, including, for example, gliomas, anaplastic oligodendrogliomas, adult glioblastoma multiforme, and adult anaplastic astrocytomas; bone cancer; sarcomas, including, for example, Kaposi's sarcoma (Kaposi's sarcoma). sarcoma); adrenal cancer; mesothelial carcinoma; choriocarcinoma; muscle cancer; connective tissue cancer; and thyroid cancer.

[0195] Non-limiting examples of hematological malignancies include acute myeloid leukemia (AML); chronic myeloid leukemia (CML), including accelerated phase CML and CML blast crisis (CML-BP); acute lymphocytic leukemia (ALL); chronic lymphocytic leukemia (CLL), including high-risk CLL; human acute monocytic leukemia (M(5)); hairy cell leukemia; lymphocytic leukemia; chronic lymphoid leukemia; myeloid leukemia; myelodysplastic syndrome or acute lymphoblastic leukemia; small lymphocytic lymphoma (SLL), lymphoblastic lymphoma, Hodgkin lymphoma; non-Hodgkin lymphoma (NHL); follicular lymphoma; mantle cell lymphoma (MCL); B-cell lymphoma; T-cell lymphoma; diffuse large B-cell lymphoma (DLBCL); large B-cell lymphoma (LBCL); follicular lymphoma, marginal zone lymphoma, Burkitt lymphoma, non-Burkitt high-grade B-cell lymphoma, extranodal marginal zone B-cell lymphoma; multiple myeloma (MM); Waldenstrom's macroglobulinemia; myelodysplastic syndromes (MDS), including refractory anemia (RA), refractory anemia with ringed sideroblasts (RARS), refractory anemia with excess blasts (RAEB), and refractory anemia with excess blasts acutely transformed (RAEB-T); and myeloproliferative syndrome.

[0196] In some embodiments, the hematological malignancy is relapsed or refractory diffuse large B-cell lymphoma (DLBCL), relapsed or refractory mantle cell lymphoma, relapsed or refractory follicular lymphoma, relapsed or refractory CLL, relapsed or refractory SLL, relapsed or refractory multiple myeloma.

[0197] The compounds of the present invention can be used to achieve beneficial therapeutic or prophylactic effects, for example, in individuals suffering from cancer.

[0198] The compounds of the invention can be used to achieve beneficial therapeutic or prophylactic effects, for example, in individuals suffering from autoimmune diseases or in individuals suffering from inflammatory diseases.

[0199] The term "autoimmune disease" refers to a disease or condition caused by an immune response to self-antigens that results in damage to one's own tissues or organs. Examples of autoimmune diseases include, but are not limited to, chronic obstructive pulmonary disease (COPD), allergic rhinitis, lupus erythematosus, myasthenia gravis, Sjögren's syndrome, multiple sclerosis (MS), scleroderma (also known as systemic sclerosis), multiple sclerosis osteoporosis, arthritis (e.g., rheumatoid arthritis (RA), collagen-induced arthritis), psoriasis, inflammatory bowel disease (e.g., ulcerative colitis, Crohn's disease), asthma, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, antineutrophil cytoplasmic antibody vasculitis, Sjögren's syndrome, pyroptosis vulgaris, diseases associated with kidney transplantation, and myeloproliferative disorders such as myelofibrosis, post-polycythemia vera / essential thrombocytosis myelofibrosis (post-PV / ET). myelofibrosis), thyroid autoimmune disease, chronic lymphocytic thyroiditis, hyperthyroidism, pernicious anemia with chronic atrophic gastritis, Goodpasture syndrome, pemphigoid, primary biliary cirrhosis, acute idiopathic polyneuritis, systemic lupus erythematosus, mixed connective tissue disease, etc. In some embodiments, the autoimmune disease is selected from arthritis, such as rheumatoid arthritis, collagen-induced arthritis, etc.

[0200] The term "inflammatory disease" or "inflammatory condition" refers to a pathological state that results in inflammation, particularly due to neutrophil chemotaxis. Non-limiting examples of inflammatory diseases include systemic and local inflammation, inflammation associated with immunosuppression, organ transplant rejection, allergic diseases, inflammatory skin diseases (including psoriasis and atopic dermatitis); systemic scleroderma and sclerosis; reactions associated with inflammatory bowel disease (IBD, such as Crohn's disease and ulcerative colitis); ischemia-reperfusion injury, including surgically induced tissue reperfusion injury, myocardial ischemia such as myocardial infarction, cardiac arrest, postoperative reperfusion after cardiac surgery, and abnormal contractile responses of coronary vessels after percutaneous transluminal coronary angioplasty, stroke, and abdominal aortic aneurysm surgery; cerebral edema secondary to stroke; cranial trauma, hemorrhagic shock; asphyxia; adult respiratory distress syndrome; acute lung injury; Behcet's disease; dermatomyositis; polymyositis; multiple sclerosis (MS) S); dermatitis; meningitis; encephalitis; uveitis; osteoarthritis; lupus nephritis; autoimmune diseases such as rheumatoid arthritis (RA), Sjögren's syndrome, and vasculitis; diseases involving leukocytic infiltration; central nervous system (CNS) inflammatory diseases secondary to sepsis or trauma, and multiple organ injury syndrome; alcoholic hepatitis; bacterial pneumonia; antigen-antibody complex-mediated diseases, including glomerulonephritis; sepsis; sarcoidosis; immunopathological reactions caused by tissue / organ transplantation; lung inflammation, including pleurisy, alveolitis, vasculitis, pneumonia, chronic bronchitis, bronchiectasis, diffuse panbronchiolitis, hypersensitivity pneumonitis, idiopathic pulmonary fibrosis (IPF), and cystic fibrosis. Preferred indications include, but are not limited to, chronic inflammation, autoimmune diabetes, rheumatoid arthritis (RA), rheumatoid spondylitis, gouty arthritis and other joint disorders, multiple sclerosis (MS), asthma, systemic lupus erythematosus, adult respiratory distress syndrome, Behcet's disease, psoriasis, chronic inflammatory lung disease, graft-versus-host disease, Crohn's disease, ulcerative colitis, inflammatory bowel disease (IBD), Alzheimer's disease and pyresis, as well as any disease associated with inflammation and related disorders.

[0201] In addition, the compounds of the present invention (e.g., any of the example compounds herein) can be used in combination with additional therapeutic agents for the treatment of diseases or conditions described herein, such as autoimmune diseases, inflammatory diseases, or cancer. The additional therapeutic agent can be administered separately from the compounds of the present invention, or it can be included in a pharmaceutical composition according to the present disclosure, such as a fixed-dose combination drug. In some embodiments, the additional therapeutic agent is an ingredient that is known or has been found to be effective for treating diseases mediated by BTK or at least partially mediated by BTK, such as other BTK inhibitors or compounds that can effectively antagonize other targets associated with the specific disease. Combination therapy can be used to improve the efficacy (e.g., by including a compound that can enhance the efficacy or effectiveness of the compounds of the present invention in the combination therapy), reduce one or more side effects, or reduce the required dose of the compound of the present invention.

[0202] In some embodiments, the compounds of the present invention (e.g., any compound herein) can be used in combination with another therapeutic agent, such as an anti-inflammatory agent, an immunomodulator, or an anti-tumor agent, including a chemotherapeutic agent, an immune checkpoint inhibitor or agonist, and a targeted therapeutic agent. The term "anti-tumor agent" as used herein refers to any agent administered to a subject with cancer for the purpose of treating cancer, such as a chemotherapeutic agent, an immune checkpoint inhibitor or agonist, and a targeted therapeutic agent.

[0203] Non-limiting examples of anti-inflammatory agents and immunomodulators include: immunosuppressants (e.g., tacrolimus, cyclosporine, rapamycin, methotrexate, cyclophosphamide, azathioprine, mercaptopurine, mycophenolate or FTY720), glucocorticoids (e.g., prednisone, cortisone acetate, prednisolone, methylprednisolone, dexamethasone, betamethasone, triamcinolone, hydrocortisone, beclomethasone, fludrocortisone acetate, deoxycorticosterone acetate, aldosterone), nonsteroidal anti-inflammatory drugs (e.g., salicylates, arylalkanoic acids, 2-arylpropionic acids, N-arylanthranilic acids, oxicams, coxibs or sulfanilides), cyclooxygenase-2-specific inhibitors (e.g., valdecoxib, celecoxib or rofecoxib), leflunomide, gold thioglucose, gold thiomalate, orolis, sulfasalazine, hydroxychloroquine, minocycline, TNF-α binding protein (such as infliximab, etanercept or adalimumab), abatacept, anakinra, interferon, interferon-γ, interleukin-2, interleukin-6, interleukin-12 / 23, interleukin-17 antibody drugs, allergy vaccines, antihistamines, antileukotrienes, beta-agonists, theophylline or anticholinergic drugs; JAK3 kinase inhibitors, including all known but not limited to tofacitinib; IRAK4, RIPK1 inhibitors, etc.

[0204] Non-limiting examples of chemotherapeutic agents include topoisomerase I inhibitors (e.g., irinotecan, topotecan, camptothecin and its analogs or metabolites, and doxorubicin); topoisomerase II inhibitors (e.g., etoposide, teniposide, mitoxantrone, daunorubicin, and daunomycin); alkylating agents (e.g., melphalan, chlorambucil, busulfan, thiotepa, ifosfamide, carbamazepine, lomustine, methyllomustine, streptozotocin, decarbazine, methotrexate, mitomycin, chlorambucil, thiotepa, ifosfamide, nitrosocarbamide, cyclohexane, methyllomustine, streptozotocin, decarbazine, methotrexate, mitomycin, chlorambucil, thiotepa, ifosfamide, nitrosocarbamide, lomustine, methyllomustine, streptozotocin, decameron, methotrexate, mitomycin, nitroglycerin, chlorambucil ...chlorambucil, methyllomustine, streptozotocin, decameron, methotrexate, mitomycin, nitroglycerin, chloram C and cyclophosphamide); DNA intercalators (e.g., cisplatin, oxaliplatin, and carboplatin); and free radical generators such as bleomycin; as well as nucleoside analogs (e.g., 5-fluorouracil, capecitabine, gemcitabine, fludarabine, cytarabine, azacitidine, mercaptopurine, thioguanine, pentostatin, and hydroxyurea), paclitaxel, docetaxel, and related analogs; vincristine, vinblastine, and related analogs; sedatives and related analogs (e.g., CC-5013 and CC-4047).

[0205] Non-limiting examples of immune checkpoint inhibitors or agonists include PD-1 inhibitors, such as anti-PD-1 antibodies, e.g., pembrolizumab and nivolumab; PD-L1 inhibitors, such as anti-PD-L1 antibodies, e.g., atezolizumab, durvalumab, and avelumab; CTLA-4 inhibitors, such as ipilimumab; and BTLA inhibitors, LAG-3 inhibitors, TIM3 inhibitors, TIGIT inhibitors, VISTA inhibitors, OX-40 agonists, and the like.

[0206] Targeted therapeutics include various small molecule or large molecule targeted therapeutics, non-limiting examples of which include: protein tyrosine kinase inhibitors (such as imatinib mesylate and gefitinib); proteasome inhibitors (such as bortezomib); NF-κB inhibitors, including IκB kinase inhibitors; PI3Kδ inhibitors; SYK inhibitors; Bcl2 inhibitors; antibodies that bind to proteins overexpressed in cancer and thereby downregulate cell replication, such as anti-CD20 antibodies (such as rituximab, ibritumomab tiuxetan, tositumomab), anti-Her2 monoclonal antibodies (trastuzumab), anti-EGFR antibodies (cetuximab) and anti-VEGFR antibodies (bevacizumab); anti-angiogenic drugs, such as lenalidomide, etc.; and other protein or enzyme inhibitors, which are known to be upregulated, overexpressed or activated in cancer, and whose inhibition can downregulate cell replication.

[0207] Example

[0208] The following examples are illustrative of the present invention and are not intended to limit the present invention in any way. The data provided (e.g., amount, temperature, etc.) strive to ensure their accuracy, but those skilled in the art will appreciate that there will be some experimental errors and deviations. Unless otherwise stated, all parts are by weight, temperatures are degrees Celsius, and pressures are atmospheric pressure or near atmospheric pressure. All mass spectrometry data were measured by Agilent 6120 and 1100. All nuclear magnetic resonance data were measured by Varian 400MR. Except for the synthetic intermediates, all reagents and raw materials used in the present invention were obtained from commercial sources. The positive control GDC-0853 (fenebrutinib) was purchased from Shanghai Lingkai Pharmaceutical Technology Co., Ltd. The names of all compounds except the reagents were generated by Chemdraw 16.0.

[0209] In any structural formula herein, if there are vacant valencies on any atom, the vacant valencies are actually hydrogen atoms that are not specifically depicted for simplicity.

[0210] In this application, if both the name and structural formula of a compound are given, in the event of a discrepancy between the two, the structure of the compound shall prevail unless the context indicates that the structure of the compound is incorrect and the name is correct.

[0211] The following is a list of abbreviations used in the examples:

[0212] CD3OD deuterated methanol

[0213] DCM dichloromethane

[0214] DIEA N,N-Diisopropylethylamine

[0215] DMF N,N-dimethylformamide

[0216] DMSO dimethyl sulfoxide

[0217] DMSO-d6 deuterated dimethyl sulfoxide

[0218] g grams

[0219] HATU 2-(7-Azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate

[0220] HPMC Hydroxypropyl Methylcellulose

[0221] L liter

[0222] M moles / liter

[0223] mg milligrams

[0224] mL milliliters

[0225] mmol millimole

[0226] mol mole

[0227] NBS N-bromosuccinimide

[0228] Pd2(dba)3 tris(dibenzylideneacetone)dipalladium

[0229] Pd(dppf)Cl2 CH2Cl2 [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride dichloromethane complex

[0230] Xphos 2-dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl

[0231] Xant-phos 4,5-bis(diphenylphosphine)-9,9-dimethylxanthene

[0232] Example 1 Synthesis of Compounds

[0233] Intermediate I-1

[0234] 3-((5-((2S,6S)-2,6-dimethyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-yl)amino)-1-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2(1H)-one

[0235]

[0236] Step 1: tert-Butyl (3S,5S)-3,5-dimethyl-4-(6-nitropyridin-3-yl)piperazine-1-carboxylate

[0237] Under nitrogen, DIEA (40 mL) was added to a DMSO (40 mL) solution of 5-fluoro-2-nitropyridine (4.5 g, 31.7 mmol) and tert-butyl (3S,5S)-3,5-dimethylpiperazine-1-carboxylate (5.0 g, 23.3 mmol). The mixture was reacted at 120°C for 24 hours, then cooled to room temperature and concentrated under vacuum. The resulting residue was purified by silica gel column chromatography (dichloromethane / ethyl acetate) to yield the desired product (6.0 g, 77% yield). [M+H] + 337.1

[0238] Step 2: tert-Butyl (3S,5S)-4-(6-aminopyridin-3-yl)-3,5-dimethylpiperazine-1-carboxylate

[0239] At room temperature, hydrogen was introduced into a mixture of tert-butyl (3S,5S)-3,5-dimethyl-4-(6-nitropyridin-3-yl)piperazine-1-carboxylate (4.5 g, 13.4 mmol) and 10% palladium-carbon (containing 50% water, 3.0 g) in methanol (100 mL). The mixture was reacted at 40°C for 3 hours. The reaction mixture was filtered, the filtrate collected, and concentrated under vacuum to afford the desired product (3.9 g, 95% yield), which was used directly in the next reaction. [M+H] + 307.2

[0240] Step 3: (3S,5S)-tert-Butyl 4-(6-((5-bromo-1-methyl-2-oxo-1,2-dihydropyridin-3-yl)amino)pyridin-3-yl)-3,5-dimethylpiperazine-1-carboxylate

[0241] Under nitrogen, to a solution of tert-butyl (3S,5S)-4-(6-aminopyridin-3-yl)-3,5-dimethylpiperazine-1-carboxylate (3.0 g, 9.8 mmol) and 3,5-dibromo-1-methylpyridin-2(1H)-one (2.0 g, 7.5 mmol) in 1,4-dioxane (100 mL) were added Xant-phos (433 mg, 0.75 mmol), Pd2(dba)3 (343 mg, 0.375 mmol), and cesium carbonate (4.9 g, 15.0 mmol). The mixture was reacted at 90°C for 12 hours, then cooled to room temperature, filtered, and the filtrate was collected and concentrated. The resulting residue was purified by silica gel column chromatography (methanol / dichloromethane) to obtain the desired product (3.0 g, 81% yield). [M+H] + 492.1,494.1

[0242] Step 4: 5-Bromo-3-((5-((2S,6S)-2,6-dimethyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-yl)amino)-1-methylpyridin-2(1H)-one

[0243] To a solution of tert-butyl (3S,5S)-4-(6-((5-bromo-1-methyl-2-oxo-1,2-dihydropyridin-3-yl)amino)pyridin-3-yl)-3,5-dimethylpiperazine-1-carboxylate (3.0 g, 6.1 mmol) in methanol (15 mL) was added concentrated hydrochloric acid (8 mL), followed by stirring at 50°C for 30 minutes.

[0244] The mixture was concentrated under reduced pressure in vacuo, and a suspension of zinc chloride (2.5 g, 18.3 mmol) and sodium cyanoborohydride (2.3 g, 36.6 mmol) in methanol (50 mL) was added to a solution of the resulting residue in methanol (30 mL). The reaction was stirred at 50°C for 4 hours, concentrated under reduced pressure in vacuo, and the resulting residue was purified by silica gel column chromatography (methanol / water) to yield the desired product (2.0 g, 73% yield). [M+H] + 448.1,450.1

[0245] Step 5: 3-((5-((2S,6S)-2,6-dimethyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-yl)amino)-1-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2(1H)-one

[0246] Under nitrogen, to a solution of 5-bromo-3-((5-((2S,6S)-2,6-dimethyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-yl)amino)-1-methylpyridin-2(1H)-one (1.2 g, 2.68 mmol) and pinacol diboron (1.7 g, 6.7 mmol) in 1,4-dioxane (60 mL) were added Xphos (128 mg, 0.27 mmol), Pd2(dba)3 (247 mg, 0.27 mmol), and potassium acetate (784 mg, 8.0 mmol). The mixture was reacted at 65°C for 6 hours and then cooled to room temperature. The filtrate was filtered and concentrated under vacuum. The residue was purified by silica gel column chromatography (methanol / dichloromethane) to give the title compound (650 mg, 50% purity, 24% yield).

[0247] [M+H] + 496.3

[0248] Referring to the preparation steps of intermediate I-1, the intermediates in the following table were prepared using corresponding raw materials and reagents:

[0249]

[0250]

[0251] Intermediate I-3

[0252] 4-Chloro-2-(7,7-dimethyl-1-oxo-1,6,7,8-tetrahydro-2H-cyclopenta[4,5]pyrrolo[1,2-a]pyrazin-2-yl)nicotinaldehyde

[0253]

[0254] Step 1: Ethyl 1-(2,2-diethoxyethyl)-5,5-dimethyl-1,4,5,6-tetrahydrocyclopenta[b]pyrrole-2-carboxylate

[0255] To a solution of ethyl 5,5-dimethyl-1,4,5,6-tetrahydrocyclopenta[b]pyrrole-2-carboxylate (20.0 g, 96 mmol) in DMF (120 mL) was added cesium carbonate (80.0 g, 245 mmol) and bromoacetaldehyde diethyl acetal (40.0 g, 203 mmol), and the mixture was reacted at 100°C for 16 hours. Water (200 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate (200 mL x 2). The organic phases were combined and concentrated under vacuum. The resulting residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate) to obtain the desired product (31.0 g, 100% yield). [M+Na] + 324.1

[0256] Step 2: 1-(2,2-diethoxyethyl)-5,5-dimethyl-1,4,5,6-tetrahydrocyclopenta[b]pyrrole-2-carboxylic acid

[0257] To a solution of ethyl 1-(2,2-diethoxyethyl)-5,5-dimethyl-1,4,5,6-tetrahydrocyclopenta[b]pyrrole-2-carboxylate (31.0 g, 96 mmol) in ethanol (150 mL) and water (150 mL) was added lithium hydroxide monohydrate (14.2 g, 338 mmol) and the mixture was allowed to react at 80°C for 12 hours. The ethanol was removed under vacuum, and the pH was adjusted to 5-6 with concentrated hydrochloric acid under ice-cooling. Water (200 mL) was added, and the mixture was extracted with ethyl acetate (200 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain the desired product (26.7 g, 94% yield). [MH] - 294.1

[0258] Step 3: 1-(2,2-diethoxyethyl)-5,5-dimethyl-1,4,5,6-tetrahydrocyclopenta[b]pyrrole-2-carboxamide

[0259] To a solution of 1-(2,2-diethoxyethyl)-5,5-dimethyl-1,4,5,6-tetrahydrocyclopenta[b]pyrrole-2-carboxylic acid (26.7 g, 90.5 mmol) in DMF (150 mL) at 0-5°C under nitrogen was added triethylamine (25 mL, 181 mmol) and then HATU (51.6 g, 136 mmol). The reaction was allowed to react at room temperature for 1 hour. The reaction solution was poured into concentrated aqueous ammonia (800 mL) and stirred for 10 minutes. The mixture was extracted with dichloromethane (300 mL x 2). The organic phases were combined and concentrated to afford the desired product (26.6 g, 100% yield), which was used directly in the next reaction.

[0260] Step 4: 7,7-Dimethyl-7,8-dihydro-2H-cyclopenta[4,5]pyrrolo[1,2-a]pyrazin-1(6H)-one

[0261] Dissolve 1-(2,2-diethoxyethyl)-5,5-dimethyl-1,4,5,6-tetrahydrocyclopenta[b]pyrrole-2-carboxamide (26.6 g, 90.5 mmol) in acetic acid (100 mL) and react at 100°C for 4 hours. Remove the acetic acid under vacuum, adjust the pH to 8-9 with aqueous ammonia, add water (200 mL), and extract with dichloromethane (200 mL x 3). Collect and combine the organic phases, concentrate under vacuum, and purify the resulting residue by silica gel column chromatography (dichloromethane / methanol) to obtain the desired product (18.3 g, 100% yield). [M+H] + 203.1. 1 H NMR (400MHz, DMSO-d6) δ10.21 (s, 1H), 6.98 (d, J = 5.6Hz, 1H), 6.58 (s, 1H), 6. 48(t,J=5.6Hz,1H),2.62-2.60(m,2H),2.47-2.46(m,2H),1.19-1.17(m,6H).

[0262] Step 5: 4-chloro-2-(7,7-dimethyl-1-oxo-1,6,7,8-tetrahydro-2H-cyclopenta[4,5]pyrrolo[1,2-a]pyrazin-2-yl)nicotinaldehyde

[0263] Under nitrogen, to a solution of 7,7-dimethyl-7,8-dihydro-2H-cyclopenta[4,5]pyrrolo[1,2-a]pyrazin-1(6H)-one (14.2 g, 70.2 mmol) and 2-bromo-4-chloronicotinaldehyde (30.9 g, 141 mmol) in 1,4-dioxane (500 mL) were added cuprous iodide (13.6 g, 70.2 mmol), 4,7-dimethoxy-1,10-phenanthroline (1.18 g, 49.2 mmol), and cesium carbonate (68.6 g, 211 mmol). The mixture was reacted at 80°C for 16 hours, then cooled to room temperature, filtered, and the filtrate was collected and concentrated under vacuum. The resulting residue was recrystallized from ethanol to obtain the desired product (13.8 g, 58% yield). [M+H] + 342.1. 1HNMR(400MHz, CDCl3)δ10.21(s,1H),8.53-8.43(m,1H),7.40-7.31(m,1H),7.1 0-7.02(m,1H),6.95(s,1H),6.89-6.81(m,1H),2.68-2.54(m,4H),1.27(s,6H).

[0264] Intermediate I-4

[0265] 4-Chloro-2-(10-fluoro-1-oxo-6,7,8,9-tetrahydropyrazino[1,2-a]indol-2(1H)-yl)nicotinaldehyde

[0266]

[0267] Hydrogen was introduced into a mixture of ethyl 3-fluoro-1H-indole-2-carboxylate (10.5 g, 13.4 mmol) and platinum dioxide (1.57 g, 6.9 mmol) in acetic acid (210 mL) and allowed to react at room temperature for 8 hours. The reaction mixture was filtered, the filtrate collected, and the pH was adjusted to 8 with concentrated aqueous ammonia. Extraction with ethyl acetate and concentration under vacuum afforded ethyl 3-fluoro-4,5,6,7-tetrahydro-1H-indole-2-carboxylate (10.5 g, 98% yield), which was used directly in the next reaction. [M+H] + 212.0

[0268] Referring to the preparation steps 1-5 of intermediate I-3, 3-fluoro-4,5,6,7-tetrahydro-1H-indole-2-carboxylic acid ethyl ester and corresponding raw materials and reagents were used to prepare the target product intermediate I-4. [M+H] + 346.1

[0269] Intermediate I-5

[0270] (3-(Acetoxymethyl)-2-(7,7-dimethyl-1-oxo-1,6,7,8-tetrahydro-2H-cyclopenta[4,5]pyrrolo[1,2-d][1,2,4]triazin-2-yl)pyridin-4-yl)boronic acid

[0271]

[0272] Step 1: 5,5-Dimethyl-1,4,5,6-tetrahydrocyclopenta[b]pyrrole-2-carboxylic acid hydrazide

[0273] To a solution of ethyl 5,5-dimethyl-1,4,5,6-tetrahydrocyclopenta[b]pyrrole-2-carboxylate (6.50 g, 31.4 mmol) in ethanol (15 mL) was added aqueous hydrazine hydrate (45 mL, 36.0 mmol) and reacted in a microwave reactor at 150°C for 2 hours. The mixture was cooled to room temperature, filtered, and the filter cake was washed with water. The filter cake was collected and dried under vacuum to obtain the desired product (5.60 g, 92% yield), which was used directly in the next reaction. [M+H] + 194.1

[0274] Step 2: 7,7-Dimethyl-7,8-dihydro-2H-cyclopenta[4,5]pyrrolo[1,2-d][1,2,4]triazin-1(6H)-one

[0275] Under nitrogen, triethyl orthoformate (3.11 g, 21.0 mmol) was added to a solution of 5,5-dimethyl-1,4,5,6-tetrahydrocyclopenta[b]pyrrole-2-carbohydrazide (5.60 g, 29.0 mmol) in DMF (16 mL) and reacted at 160°C for 16 hours. The mixture was cooled to room temperature, filtered, and the filter cake was washed with a small amount of methanol. The filter cake was collected and dried under vacuum to obtain the desired product (3.25 g, 55% yield), which was used directly in the next reaction. [M+H] + 204.1

[0276] Step 3: 4-chloro-2-(7,7-dimethyl-1-oxo-1,6,7,8-tetrahydro-2H-cyclopenta[4,5]pyrrolo[1,2-d][1,2,4]triazin-2-yl)nicotinaldehyde

[0277] Under nitrogen, to a solution of 7,7-dimethyl-7,8-dihydro-2H-cyclopenta[4,5]pyrrolo[1,2-d][1,2,4]triazine-1(6H)-one (3.25 g, 16.0 mmol) and 2-bromo-4-chloronicotinaldehyde in 1,4-dioxane (60 mL) were added cuprous iodide (1.52 g, 8.0 mmol), 4,7-dimethoxy-1,10-phenanthroline (1.35 g, 5.6 mmol), and cesium carbonate (10.4 g, 32.0 mmol). The mixture was reacted at 80°C for 4 hours and then cooled to room temperature. The product was concentrated under vacuum, and the resulting residue was purified by silica gel column chromatography to yield the desired product (3.43 g, 63% yield). [M+H] + 343.1

[0278] Step 4: 2-(4-chloro-3-(hydroxymethyl)pyridin-2-yl)-7,7-dimethyl-7,8-dihydro-2H-cyclopenta[4,5]pyrrolo[1,2-d][1,2,4]triazin-1(6H)-one

[0279] To a solution of 4-chloro-2-(7,7-dimethyl-1-oxo-1,6,7,8-tetrahydro-2H-cyclopenta[4,5]pyrrolo[1,2-d][1,2,4]triazin-2-yl)nicotinaldehyde (3.43 g, 10.0 mmol) in methanol (10 mL) and dichloromethane (30 mL) was added sodium borohydride (0.19 g, 5.0 mmol) at 0-5°C under nitrogen. The mixture was reacted for 10 minutes at this temperature. Saturated aqueous ammonium chloride (10 mL) was added to the reaction solution, and the mixture was extracted with dichloromethane (80 mL x 2). The organic phases were combined and concentrated under vacuum to afford the desired product (3.33 g, 97% yield), which was used directly in the next reaction. [M+H] + 345.1

[0280] Step 5: 4-chloro-2-(7,7-dimethyl-1-oxo-1,6,7,8-tetrahydro-2H-cyclopenta[4,5]pyrrolo[1,2-d][1,2,4]triazin-2-yl)pyridin-3-yl)methyl acetate

[0281] To a solution of 2-(4-chloro-3-(hydroxymethyl)pyridin-2-yl)-7,7-dimethyl-7,8-dihydro-2H-cyclopenta[4,5]pyrrolo[1,2-d][1,2,4]triazin-1(6H)-one (3.33 g, 9.7 mmol) and triethylamine (3.91 g, 38.6 mmol) in dichloromethane (60 mL) was added acetyl chloride (11.4 g, 145 mmol) at 0-5°C under nitrogen. The mixture was allowed to react for 1 hour. Water (30 mL) and dichloromethane (80 mL) were added to the reaction solution, and the organic phases were combined and concentrated under vacuum. The resulting residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate) to afford the desired product (2.84 g, 76% yield). [M+H] + 387.1

[0282] Step 6: (3-(Acetoxymethyl)-2-(7,7-dimethyl-1-oxo-1,6,7,8-tetrahydro-2H-cyclopenta[4,5]pyrrolo[1,2-d][1,2,4]triazin-2-yl)pyridin-4-yl)boronic acid

[0283] Under nitrogen, to a solution of 4-chloro-2-(7,7-dimethyl-1-oxo-1,6,7,8-tetrahydro-2H-cyclopenta[4,5]pyrrolo[1,2-d][1,2,4]triazin-2-yl)pyridin-3-yl)methyl acetate (2.84 g, 7.3 mmol) and pinacol diboron (5.59 g, 22.0 mmol) in 1,4-dioxane (200 mL) were added Xphos (0.35 g, 0.73 mmol), Pd(dppf)Cl₂ CH₂Cl₂ (0.60 g, 0.73 mmol), and potassium acetate (2.16 g, 22.0 mmol). The mixture was reacted at 100°C for 16 hours and then cooled to room temperature. The reaction solution was concentrated under vacuum, and the resulting residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate) to obtain the desired product (2.55 g, 88% yield). [M+H] + 397.1

[0284] Referring to steps 4-6 of the preparation of intermediate I-5, intermediate I-3 and corresponding raw materials and reagents were used to prepare the intermediates in the following table:

[0285]

[0286] Compound 1

[0287] 2-(5-((5-((2S,6S)-2,6-dimethyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-yl)amino)-3'-(hydroxymethyl)-1-methyl-6-oxo-1,6-dihydro-[3,4'-bipyridinyl]-2'-yl)-7,7-dimethyl-7,8-dihydro-2H-cyclopenta[4,5]pyrrolo[1,2-a]pyrazin-1(6H)-one

[0288]

[0289] Step 1: 2'-(7,7-Dimethyl-1-oxo-1,6,7,8-tetrahydro-2H-cyclopenta[4,5]pyrrolo[1,2-a]pyrazin-2-yl)-5-((5-((2S,6S)-2,6-dimethyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-yl)amino)-1-methyl-6-oxo-1,6-dihydro-[3,4'-bipyridine]-3'-carbaldehyde

[0290] Under nitrogen, to a solution of intermediate I-1 (99 mg, 0.20 mmol) and intermediate I-3 (68 mg, 0.20 mmol) in 1,4-dioxane (3 mL) and water (0.2 mL) were added Xphos (9 mg, 0.02 mmol), Pd(dppf)Cl2 CH2Cl2 (16 mg, 0.02 mmol), and cesium carbonate (130 mg, 0.40 mmol). The mixture was reacted at 90°C for 2 hours and then cooled to room temperature. The filtrate was collected by filtration and concentrated under reduced pressure to obtain the desired product, which was used directly in the next reaction. [M+H] + 675.3

[0291] Step 2: 2-(5-((5-((2S,6S)-2,6-dimethyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-yl)amino)-3'-(hydroxymethyl)-1-methyl-6-oxo-1,6-dihydro-[3,4'-bipyridinyl]-2'-yl)-7,7-dimethyl-7,8-dihydro-2H-cyclopenta[4,5]pyrrolo[1,2-a]pyrazin-1(6H)-one

[0292] To a solution of 2'-(7,7-dimethyl-1-oxo-1,6,7,8-tetrahydro-2H-cyclopenta[4,5]pyrrolo[1,2-a]pyrazin-2-yl)-5-((5-((2S,6S)-2,6-dimethyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-yl)amino)-1-methyl-6-oxo-1,6-dihydro-[3,4'-bipyridine]-3'-carbaldehyde obtained in step 1 in methanol (0.5 mL) and dichloromethane (5 mL) was added sodium borohydride (7 mg, 0.20 mmol) at 0-5°C under nitrogen protection, and the mixture was reacted at room temperature for 5 minutes. Water (0.5 mL) was added to the reaction solution, and the mixture was concentrated under vacuum. The resulting residue was purified by silica gel column chromatography (methanol / water) and thin layer chromatography (methanol / dichloromethane = 1 / 20) to obtain the desired product (74 mg, 55% yield). [M+H] + 677.4. 1HNMR (400MHz, CD3OD): δ8.74-8.69(m,1H),8.56-8.51(m,1H),7.95-7.91(m,1H),7.60-7.57(m,1H),7.54-7.51(m ,1H),7.40-7.36(m,1H),7.23-7.19(m,1H),7.03-7.00(m,1H),6.95-6.90(m,1H),6.80-6.75(m,1H),4.70-4.65(m ,2H),4.64-4.57(m,2H),4.56-4.52(m,1H),4.50-4.45(m,1H),3.69(s,3H),3.54-3.46(m,2H),3.46-3.39(m,1H), 2.78-2.68(m,2H),2.65-2.58(m,2H),2.57-2.52(m,2H),2.22-2.13(m,2H),1.30-1.26(m,6H),0.98-0.94(m,6H).

[0293] Referring to the preparation steps of compound 1, the compounds in the following table were prepared using the corresponding intermediates and reagents:

[0294]

[0295]

[0296] Compound 4

[0297] 2-(5-((5-((2S,6S)-2,6-dimethyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-yl)amino)-3'-(hydroxymethyl)-1-methyl-6-oxo-1,6-dihydro-[3,4'-bipyridinyl]-2'-yl)-7,7-dimethyl-7,8-dihydro-2H-cyclopenta[4,5]pyrrolo[1,2-d][1,2,4]triazin-1(6H)-one

[0298]

[0299] Under nitrogen, to a solution of 5-bromo-3-((5-((2S,6S)-2,6-dimethyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-yl)amino)-1-methylpyridin-2(1H)-one (148 mg, 0.33 mmol) (i.e., the product of step 4 of the preparation of intermediate I-1) and intermediate I-5 (130 mg, 0.33 mmol) in 1,4-dioxane (5.0 mL) and water (0.5 mL) were added Xphos (31 mg, 0.066 mmol), Pd(dppf)Cl2 CHCl2 (27 mg, 0.033 mmol), and potassium phosphate trihydrate (264 mg, 0.99 mmol). The mixture was reacted at 100°C for 4 hours and then cooled to room temperature. The reaction solution was concentrated under vacuum, and the resulting residue was purified by silica gel column chromatography (methanol / water).

[0300] The obtained solid ([M+H] + 720.3) was dissolved in methanol (3 mL), and potassium carbonate (137 mg, 0.99 mmol) was added. The mixture was allowed to react at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure in vacuo, and the resulting residue was purified by silica gel column chromatography (methanol / water) and thin layer chromatography (methanol / dichloromethane = 1 / 20) to obtain the desired product (30 mg, 13% yield). [M+H] + 678.3. 1 H NMR (400MHz, CD3OD): δ8.72-8.86(m,1H),8.58-8.52(m,1H),8.43-8.38(m,1H),7.97 -7.92(m,1H),7.63-7.58(m,1H),7.53-7.48(m,1H),7.42-7.36(m,1H),7.08-6.99(m ,2H),4.70-4.52(m,6H),3.70(s,3H),3.55-3.41(m,3H),2.87-280(m,2H),2.67-2.6 1(m,2H),2.60-2.51(m,2H),2.24-2.12(m,2H),1.32-1.29(m,6H),1.00-0.94(m,6H).

[0301] Referring to the preparation steps of compound 4, the compounds in the following table were prepared using the corresponding intermediates and reagents:

[0302]

[0303] Compound 5

[0304] 2-(5-((5-((2S,6S)-2,6-dimethylpiperazin-1-yl)pyridin-2-yl)amino)-3'-(hydroxymethyl)-1-methyl-6-oxo-1,6-dihydro-[3,4'-bipyridinyl]-2'-yl)-7,7-dimethyl-7,8-dihydro-2H-cyclopenta[4,5]pyrrolo[1,2-a]pyrazin-1(6H)-one

[0305]

[0306] Compound 5a (500 mg, 0.69 mmol) was dissolved in trifluoroacetic acid (5 mL) and stirred at room temperature for 30 minutes. The mixture was concentrated under vacuum, and the resulting residue was dissolved in methanol (5 mL). Triethylamine (1 mL) was added and the mixture was concentrated under vacuum again. The resulting residue was purified by silica gel column chromatography (methanol / water) to obtain the desired product (340 mg, 79% yield). [M+H] + 621.4. 1 H NMR(400MHz,CD3OD)δ8.78(s,1H),8.60-8.51(m,1H),8.00(s,1H),7.61-7.56(m,1H),7.56-7. 51(m,1H),7.48-7.38(m,1H),7.27-7.18(m,1H),7.09-7.01(m,1H),6.93(s,1H),6.81-6.75(m, 1H),4.64-4.58(m,1H),4.53-4.46(m,1H),3.70(s,3H),3.69-3.61(m,2H),3.44-3.37(m,2H), 3.11-3.02(m,2H),2.79-2.68(m,2H),2.66-2.56(m,2H),1.30-1.26(m,6H),1.09-0.99(m,6H).

[0307] Compound 6

[0308] 2-(3'-(Hydroxymethyl)-1-methyl-6-oxo-5-((5-((2S,6S)-2,4,6-trimethylpiperazin-1-yl)pyridin-2-yl)amino)-1,6-dihydro-[3,4'-bipyridinyl]-2'-yl)-7,7-dimethyl-7,8-dihydro-2H-cyclopenta[4,5]pyrrolo[1,2-a]pyrazin-1(6H)-one

[0309]

[0310] To a solution of compound 5 (200 mg, 0.32 mmol) in methanol (5 mL) was added aqueous formaldehyde solution (1.2 mL) and stirred at room temperature for 5 minutes. Sodium borohydride (38 mg, 1.0 mmol) was added and stirred at room temperature for 30 minutes. The reaction solution was purified by silica gel column chromatography (methanol / water) to obtain the desired product (78 mg, 38% yield). [M+H] + 635.3. 1 H NMR (400MHz, CD3OD) δ8.74-8.69(m,1H),8.58-8.51(m,1H),7.97-7.90(m,1H),7.62-7.56(m, 1H),7.55-7.50(m,1H),7.41-7.34(m,1H),7.25-7.19(m,1H),7.05-6.99(m,1H),6.93(s,1H) ,6.81-6.75(m,1H),4.62-4.58(m,1H),4.51-4.46(m,1H),3.70(s,3H),3.53-3.46(m,2H),2. 78-2.69(m,2H),2.69-2.58(m,4H),2.36-2.18(m,5H),1.29-1.27(m,6H),0.98-0.90(m,6H).

[0311] Example 2 Biochemical BTK Determination

[0312] 1. Reagents and Materials

[0313] BTK recombinant protein: Invitrogen, catalog number PV3363;

[0314] Kinase assay kit-tyrosine 1 peptide: Invitrogen, cat. no. PV3190;

[0315] 384-well low-flange black flat-bottom polystyrene NBS microplate, without lid, non-sterile: Corning, Cat. No. 3575;

[0316] 96-well polystyrene conical bottom MicroWell TM Plate, with lid closure: Thermo Scientific TM Nunc TM , item number 277143;

[0317] Envision multimode plate reader: PerkinElmer;

[0318] Shaking plate instrument: Eppendorf;

[0319] TS-2102 shaking incubator: TENSUC;

[0320] 2. Methods

[0321] This biochemical assay utilizes fluorescence resonance energy transfer (FRET) technology, a dual-enzyme format, and differential sensitivity to protein cleavage between phosphorylated and non-phosphorylated peptides. Two fluorophores are labeled at each end of the peptide substrate, forming a FRET pair. In the primary reaction (enzymatic reaction), the enzyme transfers the gamma-phosphate group from ATP to a serine or threonine residue in the peptide substrate. In the secondary reaction (chromogenic reaction), a site-specific protease (chromogenic reagent) recognizes and cleaves the non-phosphorylated peptide. Phosphorylated peptides inhibit this cleavage. Peptide cleavage disrupts the donor (e.g., coumarin) and acceptor fluorophores (fluorescein) on the peptide, while phosphorylated peptides maintain FRET. The ratio is calculated by calculating the ratio of the emission signals from the donor fluorophore to the acceptor upon excitation at 400 nm. Emission signal ratio = coumarin emission (445 nm) / fluorescein emission (520 nm). If the FRET peptide is phosphorylated (e.g., no kinase inhibitor), the emission ratio will remain low; if the FRET peptide is non-phosphorylated (e.g., kinase inhibition), the emission ratio will be higher. This allows us to distinguish the inhibitory effects of different compound inhibitors on BTK kinase activity.

[0322] Reference Kinase Assay Kit - Tyrosine 1 Peptide Instructions for Experimental Procedures. Reagent Preparation: 1.33× Kinase Buffer: Dilute 5× Kinase Buffer with water to 1.33× Kinase Buffer; Enzyme Solution: Dissolve the kinase in 1.33× Kinase Buffer to a final working concentration of 3.32nM; Short Peptide Solution: Dissolve the short peptide stock solution (1mM dissolved in DMSO) in 1.33× Kinase Buffer to a final working concentration of 2μM; Z′-LYTE Tyr01 Phosphorylated Short Peptide Solution: Dissolve 0.6μl of the stock solution (1mM dissolved in DMSO) in 149.4μl 1.33× kinase buffer; ATP solution, dissolve ATP stock solution (10mM aqueous solution) in 1.33× kinase buffer to a final working concentration of 32μM; chromogenic solution, dissolve chromogenic solution B in chromogenic buffer to a final working concentration of 1× chromogenic solution; 4× compound configuration, dilute the compound in a 3-fold gradient concentration to finally obtain a 4% DMSO aqueous solution containing different concentrations of the compound, with final working concentrations of 3000, 1000, 333.33, 111.11, 37.04, 12.35, 4.12, and 1.37nM, a total of 8 concentration points.

[0323] Experimental Procedure: This experiment included three control groups, each with eight replicates: C1 100% inhibition group (no ATP), C2 0% inhibition group (with ATP), and C3 100% phosphorylation group. 2.5 μl of serially diluted compound was added to each well of a 384-well plate in duplicate, with 4% DMSO added to wells C1, C2, and C3. Then, well C3 was removed, and 2.5 μl of BTK enzyme solution was added to each remaining well. The plate was incubated at 4°C for 30 minutes. Then, well C3 was removed, and 2.5 μl of the peptide solution was added to each well, while 5 μl of the phosphorylated peptide solution was added to each well of C3. 2.5 μl of 1.33× kinase buffer was added to each well of C1 and C3, and 2.5 μl of ATP solution was added to each remaining well. The plate was centrifuged briefly, shaken at 1000 rpm for 30 seconds, and then centrifuged briefly. The 384-well plate was placed in a shaking incubator, protected from light, and incubated at room temperature for 1 hour. After the enzyme reaction is complete, add 5 μl of color development solution to each well, centrifuge briefly, shake the plate at 1000 rpm for 30 seconds, and centrifuge briefly. Place the 384-well plate in a shaking incubator away from light and incubate at room temperature for 1 hour to complete the color development reaction.

[0324] 3. Detection

[0325] After color development is complete, remove the 384-well plate and read it using an Envision multi-mode plate reader at an emission wavelength of 405 nm and excitation wavelengths of 460 nm / 535 nm. The light signal is measured by combining the reading at 460 nm and the reading at 535 nm for each well.

[0326] 4. Calculation

[0327] The average signal value of C3 was taken as 100% phosphorylation, the average signal value of C1 was taken as 0% phosphorylation, and the average signal value of C2 was taken as the phosphorylation rate of the short peptide in the presence of BTK kinase. The inhibition rate (%) of each concentration of the compound was calculated based on the signal value of each well, and the IC was obtained using the 205 model in XL-Fit 5.3 software (ID Business Solutions Limited). 50 value.

[0328] The phosphorylation rate is calculated as follows:

[0329] Phosphorylation rate (%) = 100-100 × [(emission signal ratio × F 100％ )-C 100％ ] / {(C 0％ -C 100％ )+[emission signal ratio×(F 100％ -F 0％ )]}

[0330] Wherein, emission signal ratio = coumarin emission signal (460 nm) / fluorescein emission signal (535 nm); C100％ = average coumarin emission signal of C3; C 0％ = average value of coumarin emission signal of C1; F 100％ = average fluorescence emission signal of C3; F 0％ = the average value of the fluorescein emission signal of C1.

[0331] The inhibition rate was calculated as follows:

[0332] Inhibition rate (%) = 100 × (C2 phosphorylation rate - detection well phosphorylation rate) / C2 phosphorylation rate

[0333] 5. Test Results

[0334] Compound number <![CDATA[IC 50 (μM)]]> 1 0.010 2 0.007 3 0.003 4 0.005 5 0.008 6 0.007

[0335] Example 3

[0336] Determination of phosphorylated BTK in Ramos cells

[0337] 1. Reagents and Materials

[0338] Ramos cells: Ramos cells were purchased from the American Type Culture Collection (ATCC) and cultured in PRMI1640 medium containing L-glutamine, 1.5 g / L sodium bicarbonate, 2.383 g / L HEPES solution, 0.11 g / L sodium pyruvate, and 4.5 g / L glucose, supplemented with 10% fetal bovine serum (FBS) in a 5% CO2, 37°C cell culture incubator.

[0339] PRMI 1640 medium: GIBCO, catalog number A10491-01;

[0340] Fetal bovine serum (FBS): GIBCO, catalog number 100100-147;

[0341] Hank's balanced salt solution (HBSS): GIBCO, product number 14025-092;

[0342] Immunoglobulin M (IgM): Jackson Immuno, catalog number 109-006-129;

[0343] 3% hydrogen peroxide (3% H2O2): Sigma, product number 88597-100ML-F;

[0344] BTK phospho-Y223 HTRF kit: Cisbio, catalog number 63ADK017PEH;

[0345] Microplate reader: Envision, Perkin Elmer;

[0346] 384-well plate CulturPlateTM384: Perkin Elmer, catalog number 6007680

[0347] 96-well plate: Corning, cat. no. 3799.

[0348] 2. Methods

[0349] Ramos cells were starved for 2 hours with PRMI 1640 medium containing 1% FBS. The starved Ramos cells were diluted to 5.0 x 10 6 cells / ml, and seeded into 96-well plates at 20 μL / well, i.e. 1.0x10 5 Cells / well were cultured in a cell culture incubator at 5% CO2 and 37°C. After culturing for 1 hour, the test compound was diluted 4-fold with Hank's balanced salt solution to the corresponding concentration, and then 5 μL / well of the diluted test compound of different concentrations (the final concentration of the test compound was 3.0, 0.75, 0.188, 0.047, 0.012, 0.0029, 0.0007 and 0.00018 μM, the final concentration of DMSO was 0.3%, duplicate wells) or 5 μL / well of control solution (1.5% DMSO, 8 replicates) were added. Add 20 μL / well of the cell culture system and incubate for one hour. Then, add 5 μL / well of a mixture of human immunoglobulin M (final concentration of 10 μg / mL) and hydrogen peroxide (final concentration of 3.3 mM) diluted with Hank's balanced salt solution to the test compound-treated wells and anti-human immunoglobulin M control-treated wells, and 5 μL / well of Hank's balanced salt solution to the negative control-treated wells. Incubate in a cell culture incubator at 5% CO2 and 37°C for 10 minutes.

[0350] Add 10 μL / well of cell lysis buffer to each well of a 96-well plate, mix well, and lyse at room temperature for 30 minutes. Pipette 16 μL / well of lysate into a new 384-well plate, then add 4 μL / well of phosphorylated BTK antibody. Centrifuge (1000 rpm) for 1 minute, shake for 1 minute, and centrifuge again (1000 rpm) for 1 minute. Finally, incubate in a constant temperature incubator overnight. Detect the cells the next day.

[0351] 3. Detection

[0352] The 384-well plate incubated overnight in the constant temperature incubator was taken out and the light signal was detected using an Envision microplate reader with an emission wavelength of 320 nm and an excitation wavelength of 665 nm / 615 nm. The value of (the reading at 665 nm / the reading at 615 nm of each well) was multiplied by 10. 4as the signal value for each well.

[0353] 4. Calculation

[0354] The average signal value of the wells containing a mixture of human immunoglobulin M (final concentration of 10 μg / mL) and hydrogen peroxide (final concentration of 3.3 mM) but without the test compound was taken as the high value, and the average signal value of the wells without human immunoglobulin M stimulation and without the test compound was taken as the low value. The inhibition rate (%) of each compound concentration was calculated based on the signal value of each well, and then the 205 model in XL-Fit 5.3 software (ID Business Solutions Limited) was used to calculate the IC 50 value.

[0355] The inhibition rate was calculated as follows:

[0356] Inhibition rate (%) = 100% - {(test compound treated well - negative control treated well) / (anti-human immunoglobulin M control treated well - negative control treated well)} × 100%, where,

[0357] Test compound-treated wells: represent the signal values ​​of Ramos cells treated with anti-human immunoglobulin M, hydrogen peroxide and the test compound.

[0358] Anti-human immunoglobulin M control-treated wells: indicate the signal value of Ramos cells treated with anti-human immunoglobulin M and hydrogen peroxide but without the test compound.

[0359] Negative control treatment wells: represent the signal value of Ramos cells without test compound and without immunoglobulin stimulation.

[0360] 5. Test Results

[0361] Compound number <![CDATA[IC 50 (μM)]]> 1 0.005 2 0.006 3 0.003 4 0.003 5 0.008 6 0.007

[0362] Example 4 Determination of B cell activity in rat whole blood

[0363] 1. Reagents and Materials

[0364] Peripheral whole blood of female Wistar rats;

[0365] Phosphate buffered saline (PBS): GIBCO, product number C20012500BT;

[0366] PE anti rat B220: eBioscience, catalog number 12-0460-82;

[0367] FITC anti rat CD86 antibody: eBioscience, catalog number 11-0860-82;

[0368] 10× lysis buffer: BD Biosciences, catalog number 555899;

[0369] IC fixation buffer: Invitrogen, cat. no. 00-8222-49;

[0370] 96-well U-bottom plate: Nunc, catalog number 163320;

[0371] 96-well V-bottom plate: Nunc, catalog number 49952;

[0372] Dimethyl sulfoxide (DMSO): Sigma-Aldrich, product number 34869-4L;

[0373] Mouse anti-rat IgD: Bio-rad, catalog number MCA190;

[0374] Flow cytometer: BD FACS Canto II, BD.

[0375] 2. Methods

[0376] For compound activity assays, rat peripheral whole blood was collected and added to a 96-well plate at 80 μL / well and incubated in a cell culture incubator at 5% CO2 and 37°C. Half an hour later, the test compound was diluted with PBS and serially diluted 3-fold to the corresponding concentration. Then, 10 μL / well of the diluted test compound at different concentrations was added to the rat whole blood culture system (final concentrations of the test compound were 1.0, 0.33, 0.11, 0.037, 0.012, 0.0041, 0.0014, and 0.0005 μM, final DMSO concentration was 0.3%, in duplicate). Alternatively, 10 μL / well of control solution (0.3% DMSO, in six replicates) was added to the corresponding wells, and the cells were incubated in a cell culture incubator for one hour. Then, add 10 μL / well of anti-rat immunoglobulin D diluted in PBS (final concentration of 10 μg / mL) to the test compound-treated wells and anti-rat immunoglobulin D control-treated wells, or 10 μL / well of PBS to the negative control-treated wells, mix well, and continue culturing in a cell culture incubator with 5% CO2 and 37°C for 18 hours.

[0377] On the next day, the 96-well plate was removed and a flow cytometry antibody mixture diluted with PBS (the final concentration of anti-rat B220PE antibody was 1 μg / mL and the final concentration of anti-rat CD86 FITC antibody was 1 μg / mL) was added to each well. After incubation in the dark for 30 minutes, 50 μL of blood was aspirated from each well and added to 500 μL of freshly prepared lysis buffer to lyse red blood cells. The plate was shaken for 20 minutes, centrifuged to remove the supernatant, and then washed, fixed, and detected by flow cytometry.

[0378] 3. Detection

[0379] The activation of B cells in the samples was determined by flow cytometry.

[0380] 4. Calculation

[0381] The average value of the activated B cell ratio in the wells treated with anti-rat immunoglobulin D but without the test compound was used as the anti-rat immunoglobulin D control wells, and the average value of the activated B cell ratio in the wells without immunoglobulin D stimulation and without the test compound was used as the negative control wells. The inhibition rate (%) of each concentration was calculated based on the B cell activation ratio in each well, and then the 205 model in XL-Fit 5.3 software (ID Business Solutions Limited) was used to calculate the IC 50 value.

[0382] The inhibition rate was calculated as follows:

[0383] Inhibition rate (%) = 100% - {(test compound treated well - negative control treated well) / (anti-rat immunoglobulin D control treated well - negative control treated well)} × 100%, where,

[0384] Test compound-treated wells: indicate the activation ratio of B cells in rat whole blood treated with anti-rat immunoglobulin D and the test compound.

[0385] Anti-rat IgG control-treated wells: indicate the activation ratio of B cells in rat whole blood treated with anti-rat IgG but without the test compound.

[0386] Negative control treatment wells: represent the activation ratio of B cells in rat whole blood without test compound and immunoglobulin stimulation.

[0387] The above tests show that the compounds of the present invention have a strong ability to inhibit the activation of B cells in rat whole blood. 50 The value is 0.001 μM.

[0388] Example 5 Liver microsome stability test

[0389] 1. Experimental Materials:

[0390] Male CD-1 mouse mixed liver microsomes and male SD rat mixed liver microsomes were purchased from Bioreclamation IVT, USA.

[0391] Phenacetin, glucose-6-phosphate dehydrogenase (G-6-PDH), and nicotinamide adenine dinucleotide phosphate (NADP) were purchased from Sigma-Aldrich, USA. Glucose-6-phosphate (G-6-P) was purchased from Shanghai Libo Chemical Technology Co., Ltd. and Carbosynth China Limit.

[0392] 2. Solution preparation:

[0393] 10 mM stock solution of the test compound: Weigh a certain amount of the test compound and dissolve it in an appropriate volume of DMSO to prepare a 10 mM stock solution for later use.

[0394] Reaction stop solution: Dissolve an appropriate amount of the internal standard compound phenacetin in acetonitrile to prepare a reaction stop solution with a concentration of 1000 ng / mL and keep it at room temperature for use.

[0395] 3. Experimental methods:

[0396] The stock solution of the test compound is diluted to 0.1 mM (the final concentration of the compound in the reaction system is 1 μM) with an organic solvent (usually a mixture of acetonitrile, methanol, and water in varying proportions, depending on the solubility of the compound; if necessary, 1N hydrochloric acid or 1N sodium hydroxide is added to aid solubilization). The organic solvent concentration in the incubation system should not exceed 1% (DMSO should not exceed 0.1%). Appropriate amounts of 100 mM NADP, 500 mM G-6-P, and 100 Unit / mL G-6-PDH are mixed and diluted with ultrapure water (the final system contains 1 mM NADP, 5 mM G-6-P, and 1 Unit / mL G-6-PDH). After pre-incubation in a 37°C water bath for 10 minutes, place on ice until ready to use as the NADPH regeneration solution. A 20 mg / mL liver microsomal solution was mixed with 200 mM phosphate buffer and diluted with ultrapure water to yield a solution containing 2.5 mg / mL liver microsomes (final reaction concentration 0.5 mg / mL) and 50 mM phosphate buffer. The diluted liver microsomal solution was mixed with a 0.1 mM compound solution, and an appropriate volume of a mixture of 100 mM EDTA, 300 mM MgCl₂ solution, 200 mM phosphate buffer (final reaction system: 3 mM MgCl₂, 1 mM EDTA, and 50 mM phosphate buffer), and water was added. Finally, NADPH regeneration solution was added, and the reaction was initiated in a 37°C water bath for 30 minutes. The reaction was terminated by adding glacial acetonitrile reaction stop solution containing an internal standard. The 0-minute sample was not incubated in a 37°C water bath. Unlike the 30-minute sample, glacial acetonitrile reaction stop solution containing an internal standard was added first, followed by the addition of NADPH regeneration solution. The sample to which the internal standard solution was added was vortexed and mixed, and then centrifuged at 4400 rpm for 10 minutes. The supernatant was diluted ten-fold with 50% methanol and then analyzed by LC-MS / MS.

[0397] 4. Analytical methods:

[0398] Liquid chromatography-tandem mass spectrometry (LC-MS / MS) was used to determine the concentration of the compound in the sample. The metabolic stability of the compound was evaluated by calculating the percentage of compound remaining after 30 minutes of incubation compared to the 0-minute sample, using the peak area ratio of the compound to the internal standard as an indicator.

[0399] Instruments: API4500, API4000 or LTQ mass spectrometer; liquid phase is UHPLC system (Shimadzu LC-30AD, model Nexra X2) including liquid delivery unit, column oven, detector and autosampler; or Agilent 1200 dual pump series HPLC and CTC autosampler.

[0400] Column: Waters XSELECT Hss T3 C18 (2.5μm, 2.1×50mm) or CAPCELLPAK MG (5μm, 2.0×50mm)

[0401] Mobile phase:

[0402] A: Water containing 0.1% FA (formic acid) (with or without 0.1% ACN (acetonitrile))

[0403] B: Acetonitrile containing 0.1% FA (formic acid).

[0404] The test results are shown in the following table:

[0405] Compound number RLM* MLM GDC-0853 81.0％ 76.3％ 1 87.9％ 91.6％ 2 85.7％ 92.6％ 4 97.4％ 90.9％ 5 95.4％ 67.5％

[0406] *RLM, rat liver microsomes.

[0407] **MLM, mouse liver microsomes.

[0408] Example 6 Evaluation of the efficacy of BTK target inhibition in vivo

[0409] Objective: To study the inhibitory effect of the compounds of the present invention on B cell activation in vivo by inducing activation of B cells in mouse whole blood with anti-IgD antibodies, thereby determining the BTK target inhibitory effect of the compounds of the present invention in vivo.

[0410] Methods: C57BL / 6 mice (female, 18-20 g, purchased from Shanghai Lingchang Biotechnology Co., Ltd.) were grouped according to Table 1.

[0411] Table 1 In vivo drug administration group information

[0412]

[0413] After dosing, animals in each group were anesthetized with CO2 at the designated time. Blood was collected from the medial canthus of the orbit and anticoagulated with heparin. 90 μL of whole blood from each mouse was added to a 96-well plate, and anti-mouse IgD antibody (BIO-RAD, Catalog No. MCA4693) was added to each well to a final concentration of 0.01 μg / μL (respectively, for each drug-treated group and the anti-IgD antibody-induced vehicle group). Separately, 90 μL of whole blood from mice in the vehicle group was added to the same 96-well plate, and PBS (phosphate-buffered saline, GIBCO, Catalog No. C20012500BT) was added to each well to a final concentration of 0.01 μg / μL (respectively, for the vehicle control group). All groups were mixed thoroughly and incubated in a 37°C / 5% CO2 incubator for 4 hours. Blood from mice in the drug-treated groups was centrifuged to separate plasma for analysis of drug concentrations.

[0414] Fluorescently labeled antibodies Anti-CD19-APC (BD Biosciences, Catalog No. 550992) and Anti-CD69-PE (BD Biosciences, Catalog No. 553237) were added to the cultured whole blood, mixed evenly, and incubated at room temperature in the dark for 30 minutes; 50 μL of sample was transferred to a 96-well deep V-shaped culture plate containing 380 μL of freshly prepared lysis buffer (BD Biosciences, Catalog No. 555899), shaken, and placed at room temperature in the dark for 15 minutes to remove red blood cells; 400 μL of flow cytometry buffer (2% FBS / / PBS, FBS: fetal bovine serum, GIBCO, Catalog No. 100100-147; PBS: GIBCO, Catalog No. C20012500BT) was added, and the plate was centrifuged at 1200 rpm and 4°C for 8 minutes; the supernatant was removed, and the cell pellet was washed twice with flow cytometry buffer and centrifuged; the cells were resuspended in 400 μL of flow cytometry buffer and analyzed by BD FACS. LSRFortessa flow cytometer was used to detect the expression of CD69+ in CD19+ positive cells (B cells) and analyze the data.

[0415] Calculation of B cell activation rate:

[0416] B cell activation rate = percentage of CD69+CD19+ double-positive B cells / percentage of CD19+ single-positive B cells

[0417] Calculation of inhibition rate:

[0418] Inhibition rate = (B cell activation rate percentage of anti-IgD antibody-induced vehicle group - B cell activation rate percentage of drug-treated group) / (B cell activation rate percentage of anti-IgD antibody-induced vehicle group - B cell activation rate percentage of vehicle control group) × 100%

[0419] Data are presented as mean ± standard error. The p-values ​​were calculated by one-way analysis of variance and Dunnett's test for comparison between each drug-treated group and the anti-IgD antibody-induced vehicle group using Graphpad Prism. The p-values ​​were calculated by unpaired t test for comparison between drug-treated groups.

[0420] Results: The experimental results are as follows Figure 1 As shown in Table 2.

[0421] In this experiment, 16 hours after administration, GDC-0853 at 20 mg / kg inhibited B cell activation by 9%, while Compound 2 of the present invention inhibited B cell activation by 43% at a 20 mg / kg dose. Compound 1 of the present invention inhibited B cell activation by 60% at a 5 mg / kg dose, demonstrating statistically significant differences compared to the anti-IgD antibody-induced vehicle group.

[0422] Table 2 Effects of in vivo administration on B cell activation in mouse whole blood induced by anti-IgD antibodies

[0423]

[0424] #### indicates p < 0.0001 compared with the vehicle control group;

[0425] *Indicates p < 0.05 compared with the anti-IgD antibody-induced vehicle group.

[0426] Example 7 Therapeutic effects of the compounds of the present invention on type II collagen-induced arthritis model in rats

[0427] 1. Research Methods

[0428] An appropriate amount of bovine type II collagen (CII, Chondrex (Redmond, WA, USA), Cat# 20021) was weighed and dissolved in 0.1 M acetic acid (SPGC Sinopharm Chemical Reagent Co., Ltd (Shanghai, PR China), Cat# 10000218) to prepare a 6 mg / mL solution. The solution was stirred overnight at 4°C, and an equal volume of incomplete Freund's adjuvant (Sigma-Aldrich. (St. Louis, MO, USA), Cat# SLBW0366) was added and fully emulsified to prepare an emulsion with a CII concentration of 3 mg / mL.

[0429] Female Lewis rats were purchased from Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China) (certificate number 20200928Aazz0619000579, initial weight 110-130 g). Six rats were randomly selected as the normal group, and all remaining rats were immunized for modeling. On day 0, for the initial immunization, all rats except the normal group were anesthetized with isoflurane (Hebei Yipin Pharmaceutical Co., Ltd., Lot: C002170601). After disinfection with 75% alcohol, 0.2 mL of the emulsion was injected intradermally at the base of the tail. On day 7, a secondary challenge was performed with 0.2 mL of the emulsion injected intradermally using the same method. After the onset of disease on day 10, the animals were closely observed for disease progression. After day 13, the average paw volume of the modeled animals was between 1.5 and 1.7 ml. The modeled animals were randomly divided and dosed according to Table 3.

[0430] Table 3 Modeling and drug administration group information

[0431]

[0432] After grouping, the normal group received no medication, while the other groups received control vehicle, reference GDC-0853 0.25 mg / kg, 4 mg / kg, and Compound 1 orally once daily for the duration of the experiment. Grouping and dosing schedules are shown in Table 3.

[0433] Paw volume was measured starting from day 10 after immunization, and the volume of the left and right hind paws (V) was measured every day after the increase in paw volume was detected.

[0434] The paw volume of the left and right hind limb joints of each animal was measured, and the average paw volume (APV) was calculated according to the following formula:

[0435] Average foot volume APV=(V left +V right ) / 2

[0436] The effects of drugs on mean paw volume were analyzed by repeated measure ANOVA with Dunnett's multiple comparison test in GraphPad, and the p value was calculated. ### p<0.001 indicates a statistically significant difference from the normal group, *p<0.05 indicates a statistically significant difference from the vehicle control group, and **p<0.01 indicates a statistically significant difference from the vehicle control group. The average paw volume of each animal before administration was the baseline (or 100% inhibition of inflammation was considered). The average paw volume change (APS) of each animal was calculated according to the following formula, where APV d1 APV is the average paw volume on day 1 of medication. dt is the average foot volume administered on day t:

[0437] Average foot volume change (APS) dt =(APV dt –APV d1 )

[0438] The area under the curve (AUC) of the mean foot volume change is the area under the curve of the joint score change calculated by the trapezoidal method, and the calculation formula is:

[0439] AUC APS =1 / 2×(APS d1 +APS d2 )×(d2-d1)+1 / 2×(APS d2 +APS d3 )×(d3-d2)+……+1 / 2×(APS dn +APSd(n-1) )×(d n -d n-1 ).

[0440] The inhibition rate (IR) of the area under the curve AUC )Calculation formula:

[0441] Inhibition rate IR AUC % = (mean AUC of the model group APS -AUC in drug-treated groups APS ) / (Average AUC of the model group APS -Average AUC of normal group APS )×100%

[0442] ED 50 The inhibition rate was calculated using the area under the curve (AUC) of mean foot volume change using XLfit software, using the "log (inhibitor) vs. response-Variable slope" model:

[0443]

[0444] 2. Results

[0445] Lewis rats began to develop the disease on the 10th day after the first immunization with bovine type II collagen. As the disease progressed, the foot volume of the hind limbs gradually increased. The foot volume growth of the vehicle control group was compared with that of the normal group, and there was a statistically significant difference ( ### P < 0.001). GDC-0853-0.25 mg / kg had no effect on the increase of paw volume in rats, while GDC-0853-4 mg / kg significantly reduced the paw volume compared with the vehicle control group (p < 0.05). Once-daily oral administration of compound 1 solution at 0.06, 0.25 and 4 mg / kg QD inhibited paw swelling in a dose-dependent manner, with the inhibition rate under the curve (IR AUC ) were 76.2%, 83.1% and 200.2% respectively; the lowest effective dose was 0.06 mg / kg / day. 0.25 mg / kg of compound 1 (inhibition rate of area under the curve 83.1%) was compared with the same dose of GDC-0853 (inhibition rate of area under the curve -6.4%), and 4 mg / kg of compound 1 (inhibition rate of area under the curve 200.2%) was compared with the same dose of GDC-0853 (inhibition rate of area under the curve 144.7%). There were statistically significant differences, both of which could significantly improve the sustained improvement of foot volume swelling (p < 0.01, one-way repeated variance analysis, tested by Graphpad). The results are shown in Figure 2 .

[0446] Example 8 Therapeutic Effects of the Compounds of the Present Invention on Idiopathic Thrombocytopenic Purpura Induced by Anti-CD41 Antibodies

[0447] 1. Research Methods

[0448] Male C57BL / 6 mice were purchased from Shanghai Lingchang Biotechnology Co., Ltd. (certificate number 20180003011079, initial weight 18-20 g) and randomly divided into groups of 8 mice per group according to Table 4. At various times before model establishment, mice were administered a single dose as shown in Table 4: intraperitoneal injection of the active agent 2000 mg / kg IVIg (Rongsheng, Lot#: 201604B026), oral administration of 40 mg / kg PRN1008 (rilzabrutinib), and various doses of Compound 1. During model establishment, each mouse received an intraperitoneal injection of 200 μL of PBS containing 2 μg of anti-mouse CD41 antibody (BD, Cat#: 553487, Lot#: 7026765).

[0449] Table 4 Modeling and medication grouping information

[0450]

[0451] 8 hours and 24 hours after modeling, whole blood was collected and placed in a centrifuge tube pre-filled with 10% citrate-phosphate-dextrose-adenine (CPDA), and the platelet level (PLT) in the whole blood was detected using the Shanghai Laboratory Animal Research Centre Sino-British SIPPR / B&K Lab Animal Ltd XT-2000i (SYSMEX) automatic hematology analyzer.

[0452] The mean platelet level in peripheral blood was statistically analyzed using Graphpad statistical software with one-way ANOVA followed by Fisher LSD multiple comparison test. The p value was calculated. *p<0.05 indicates a statistically significant difference compared with the vehicle control group, **p<0.01 indicates a statistically extremely significant difference compared with the vehicle control group, and ##p<0.01 indicates a statistically extremely significant difference compared with the normal group.

[0453] The platelet level recovery rate (RR) was calculated according to the following formula:

[0454] RR%=(PLT treatment –PLTmodel ) / (PLT naive –PLT model )×100%.

[0455] 2. Results

[0456] Eight hours after intraperitoneal injection of anti-mouse CD41 antibody, platelet levels in the peripheral blood of C57BL / 6 mice were measured. Comparison of mean platelet levels between the vehicle control group and the normal control group revealed a highly statistically significant difference (##p<0.01). Intraperitoneal administration of 2g / kg IVIg significantly restored platelet levels compared to the vehicle control group (**p<0.01), with a platelet recovery rate of 54%. Oral administration of 40mg / kg PRN1008 also significantly restored platelet levels compared to the vehicle control group (**p<0.01), with a platelet recovery rate of 40%. A single oral dose of Compound 1 solution at 0.004, 0.04, 0.4, and 4mg / kg dose-dependently restored anti-mouse CD41 antibody-induced platelet reduction, with platelet recovery rates of 25%, 37%, 44%, and 51%, respectively; the minimum effective dose was 0.04mg / kg.

[0457] 24 hours after intraperitoneal injection of anti-mouse CD41 antibody, the platelet level in the peripheral blood of C57BL / 6 mice was detected, and the average platelet level of the vehicle control group and the normal group was compared, which showed a statistically significant difference (##, p<0.01). Intraperitoneal injection of 2g / kg IVIg significantly restored platelet levels compared with the vehicle control group (**p<0.01), and the platelet recovery rate was 53%. Oral administration of 40mg / kg PRN1008 did not significantly restore the average platelet level compared with the vehicle control group, and the platelet recovery rate was only 16%. A single oral administration of compound 1 solution at 0.004, 0.04, 0.4 and 4mg / kg dose-dependently restored the decrease in platelets induced by anti-mouse CD41 antibody, with platelet recovery rates of 5%, 21%, 29% and 29%, respectively. The results are shown in Figure 3 .

Claims

1. Compound of formula (I): or a pharmaceutically acceptable salt thereof, or a racemic mixture, enantiomer or diastereomer thereof, wherein: X1 is CH or N; X2 is CH; X3 is N; U and V are each independently CR9; Y1 and Y2 are each independently CR 10 ; R1 and R2 are independently selected from C 1-6 Alkyl and C 1-6 deuterated alkyl; R3 is hydrogen or deuterium; R4 is -(C 1-3 Alkyl)-OH, wherein the C 1-3 Alkyl is optionally substituted with one or more deuterium; R5 is C 1-6 Alkyl, wherein the C 1-6 The alkyl group is optionally substituted with one or more deuterium groups; Z1, Z2, Z3 and Z4 are each independently CH or N, provided that one of Z1, Z2, Z3 and Z4 is N; R6 and R7 are independently selected from C 1-6 alkyl; R8 is hydrogen, C 1-6 Alkyl or 4-6 membered heterocyclic group, wherein the C 1-6 The alkyl or 4-6 membered heterocyclyl is optionally substituted with one or more deuterium; wherein the 4-6 membered heterocyclyl contains 1 or 2 heteroatoms independently selected from N, O and S; R9 is hydrogen or deuterium; R 10 is hydrogen or deuterium; n is 0.

2. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, or a racemic mixture, enantiomer or diastereomer thereof, wherein: X1 and X2 are both CH.

3. The compound according to any one of claims 1 to 2, or a pharmaceutically acceptable salt thereof, or a racemic mixture, enantiomer or diastereomer thereof, wherein: Y1 and Y2 are both CR 10 , and R 10 For hydrogen.

4. The compound according to any one of claims 1 to 2, or a pharmaceutically acceptable salt thereof, or a racemic mixture, enantiomer or diastereomer thereof, wherein: R1 and R2 are each independently selected from methyl.

5. The compound according to any one of claims 1 to 2, or a pharmaceutically acceptable salt thereof, or a racemic mixture, enantiomer or diastereomer thereof, wherein: R3 is hydrogen.

6. The compound according to any one of claims 1 to 2, or a pharmaceutically acceptable salt thereof, or a racemic mixture, enantiomer or diastereomer thereof, wherein: R4 is -(C 1-3 Alkyl)-OH or -(C 1-3 deuterated alkyl)-OH.

7. The compound according to any one of claims 1 to 2, or a pharmaceutically acceptable salt thereof, or a racemic mixture, enantiomer or diastereomer thereof, wherein: R3 is hydrogen, and R4 is -(C 1-3 alkyl)-OH.

8. The compound according to any one of claims 1 to 2, or a pharmaceutically acceptable salt thereof, or a racemic mixture, enantiomer or diastereomer thereof, wherein: U and V are both CH.

9. The compound according to any one of claims 1 to 2, or a pharmaceutically acceptable salt thereof, or a racemic mixture, enantiomer or diastereomer thereof, wherein: R5 is C 1-6 alkyl.

10. The compound according to any one of claims 1 to 2, or a pharmaceutically acceptable salt thereof, or a racemic mixture, enantiomer or diastereomer thereof, wherein: Z1 is N, and Z2, Z3 and Z4 are all CH.

11. The compound according to any one of claims 1 to 2, or a pharmaceutically acceptable salt thereof, or a racemic mixture, enantiomer or diastereomer thereof, wherein: R6 and R7 are both methyl groups.

12. The compound according to any one of claims 1 to 2, or a pharmaceutically acceptable salt thereof, or a racemic mixture, enantiomer or diastereomer thereof, wherein: R8 is oxetanyl or tetrahydrofuranyl.

13. The compound of claim 12, or a pharmaceutically acceptable salt thereof, or a racemic mixture, enantiomer or diastereomer thereof, wherein: R8 is 14. The compound of claim 1, or a pharmaceutically acceptable salt thereof, or a racemic mixture, enantiomer or diastereomer thereof, selected from:

15. A pharmaceutical composition comprising the compound according to any one of claims 1 to 14 and / or a pharmaceutically acceptable salt thereof, and optionally comprising a pharmaceutically acceptable excipient.

16. A method for inhibiting BTK activity in vitro for non-therapeutic and non-diagnostic purposes, comprising contacting BTK with an effective amount of the compound according to any one of claims 1 to 14 and / or a pharmaceutically acceptable salt thereof.

17. A pharmaceutical combination comprising a compound according to any one of claims 1 to 14 and / or a pharmaceutically acceptable salt thereof, and at least one additional therapeutic agent.

18. The pharmaceutical combination of claim 17, wherein the therapeutic agent is selected from the group consisting of an anti-inflammatory agent, an immunomodulatory agent, or an anti-tumor active agent.

19. The pharmaceutical combination of claim 18, wherein the anti-tumor active agent is selected from chemotherapeutic agents, immune checkpoint inhibitors or agonists, and targeted therapeutic agents.

Images