HK40094658B - Heteroaryl heterocyclic compounds and uses thereof - Google Patents

Description

Heteroaryl heterocyclic compounds and their uses

Cross-references to related applications

This application claims priority to Chinese application 202010993583.8, filed on September 21, 2020; Chinese application 202110175357.3, filed on February 7, 2021; and Chinese application 202111077860.1, filed on September 15, 2021; the contents of which are incorporated herein by reference in their entirety.

Technical Field

This invention relates to heteroaryl heterocyclic compounds, pharmaceutical compositions comprising them, and methods for their preparation and use.

Background Technology

Bruton's tyrosine kinase (BTK) belongs to the Tec family of non-receptor tyrosine proteins (including BTK, LTK, TEC, BMX, and TXK). It is widely expressed in hematopoietic cells, except for T cells, NK cells, and differentiated plasma cells. BTK plays a crucial role in signal transduction via 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, signal transduction, and survival. It controls B cell development and differentiation by activating positive cell cycle regulators and differentiation factors, and also controls B cell survival and proliferation by regulating the expression of pro-apoptotic and anti-apoptotic proteins. BTK also plays an important role in the migration and adhesion of B lymphoma cells. In addition, BTK plays a role in many other hematopoietic cell signaling pathways, such as TNF-α production mediated by Toll-like receptor (TLR) and cytokine receptor in macrophages, signal transduction by IgE receptor (FceRI) in mast cells, inhibition of Fas/APO-1 apoptosis signaling in B-cell lymphoid cells, and collagen-stimulated platelet aggregation.

In humans, mutations in the BTK gene lead to X-linked agammaglobulinaemia (XLA), a hereditary immunodeficiency disease. Human XLA patients involve point mutations in the BTK gene, associated with extremely low BTK mRNA levels and BTK protein expression. Therefore, the lack of BTK kinase activity results in a near-complete deficiency of mature B cells and immunoglobulins, as well as a significant attenuation of persistent calcium signaling in response to BCR stimulation. The effect of BTK mutations is limited to the B cell population; no significant developmental defects in other immune cells have been observed in XLA patients. Spontaneous mutations in the BTK gene have also been found in X-linked immunodeficiency (xid) mice, showing a similar but less severe phenotype. In xid mice or mutation-induced BTK knockout mice, B cell differentiation is partially arrested, the number of mature B cells in circulation is reduced, and resistance to collagen-induced arthritis and staphylococcal-induced arthritis models is exhibited. Extensive 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 leukemia and lymphoma. Abnormal activation of the BCR signaling pathway has been confirmed in these autoimmune and B-cell-related diseases, and inhibition of B cells, the BCR signaling pathway, and BTK can all slow disease progression to varying degrees.

Based on the crucial 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. Various BTK inhibitors are being developed for clinical trials in hematological malignancies and autoimmune diseases. Small molecule BTK inhibitors (such as ibrutinib, acalabrutinib, zanubrutinib, PRN1008, and GDC-0853) have shown relatively effective therapeutic effects. For example, the irreversible BTK inhibitor ibrutinib has demonstrated high durable 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), in 2015 for the treatment of macroglobulinemia (WM), and in 2017 for the treatment of relapsed/refractory marginal zone lymphoma (MZL). In particular, its approval indications were expanded to include chronic graft-versus-host disease (GVHD) in 2017, demonstrating the mechanism of BTK in treating chronic autoimmune diseases. Furthermore, the irreversible BTK inhibitor acalabrutinib received approval for adult mesenteric lymphoma (MCL) in 2017 and for chronic lymphocytic lymphoma (CLL) in 2019; zanubrutinib received FDA approval for MCL in November 2019; and PRN1008 is currently undergoing a Phase 3 study for pemphigus. Several irreversible BTK inhibitors (tirabrutinib, spebrutinib, evobrutinib) and reversible BTK inhibitors (GDC-0853, ARQ-531, and LOXO-305) are under preclinical and clinical development.

Therefore, BTK inhibitors represent an attractive approach to developing treatments for related diseases, particularly autoimmune diseases, inflammatory diseases, or cancer.

Invention Summary

This invention provides a compound of formula (I):

Or a pharmaceutically acceptable salt thereof, or a solvate, racemic mixture, enantiomer, diastereomer, or tautomer thereof, wherein:

_X1 , _X2 and _X3 are independently CH or N;

U and V are independently N or CR ₉ ;

_Y1 and _Y2 are independently CR ₁₀ or N;

_R1 and _R2 are independently selected from hydrogen, deuterium, halogen, -CN, hydroxyl, _C1-6 alkyl, _C3-6 cyclic hydrocarbon, _C2-6 alkynyl, _C1-6 deuterated alkyl and _C1-6 haloalkyl; or, R1 and R2 together with the carbon atoms attached to them form a 3-6 membered cyclic hydrocarbon.

_R3 is hydrogen, deuterium, halogen, -CN, or _C1-6 haloalkyl;

_R4 is hydrogen, halogen, -CN, _C1-6 alkyl, _C2-6 alkynyl, -( _C1-3 alkyl)-OH, -( _C1-3 alkyl)-O-( _C1-3 alkyl), -O-( _C1-3 alkyl), -CHO, -C(O) _NH2 , -C(O) _NHCH3 , -C(O)N( _CH3 ) ₂ or 3-hydroxy-oxetane-3-yl, wherein the _C1-6 alkyl or _C1-3 alkyl is optionally substituted by one or more deuterium or halogens;

R ₅ is selected from hydrogen, _C1-6 alkyl and _C3-6 cyclic hydrocarbon groups, wherein the _C1-6 alkyl group is optionally substituted with one or more deuterium or halogens;

_Z1 , _Z2 , _Z3 and _Z4 are independently CH or N, provided that at least one of _Z1 , _Z2 , _Z3 and _Z4 is N;

_R6 and _R7 are each independently selected from _C1-6 alkyl groups;

_R8 is hydrogen, _C1-6 alkyl, _C3-6 cyclic hydrocarbon, or 4-8 membered heterocyclic group, wherein the _C1-6 alkyl, _C3-6 cyclic hydrocarbon, or 4-8 membered heterocyclic group is optionally substituted by one or more groups selected from: deuterium, halogen, _C1-6 alkyl, trifluoromethyl, -OH, _-NH2 , -O-( _C1-6 alkyl), -NH( _C1-6 alkyl) or -N( _C1-6 alkyl) ₂ ;

_R9 is hydrogen, deuterium, or halogen;

R ₁₀ is hydrogen, deuterium, halogen, CN, _C1-6 alkyl, or _C1-6 haloalkyl;

n is 0, 1, or 2; the condition is that when n is 1, _R3 is not hydrogen.

The compounds described above and the active compounds disclosed in the context of this invention (including compounds of general formula 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 this invention".

The present invention also provides a pharmaceutical composition comprising the compounds of the present invention and optionally comprising pharmaceutically acceptable excipients.

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.

The present invention also provides a method for treating or preventing diseases mediated by BTK or at least partially mediated by BTK, comprising administering an effective amount of the compound of the present invention to an individual in need of it.

The present invention also provides a method for treating or preventing autoimmune diseases, inflammatory diseases, or cancer, comprising administering an effective amount of the compound of the present invention to an individual in need of it.

The present invention also provides the use of the compounds of the present invention in the treatment or prevention of diseases mediated by BTK or at least partially mediated by BTK.

This invention also provides the use of the compounds of this invention in the treatment or prevention of autoimmune diseases, inflammatory diseases or cancer.

The present invention also provides the use of the compounds of the present invention in the preparation of medicaments for the treatment or prevention of diseases mediated by BTK or at least partially mediated by BTK.

The present invention also provides the use of the compounds of the present invention in the preparation of medicaments for the treatment or prevention of autoimmune diseases, inflammatory diseases or cancer.

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

The present invention also provides compounds of the present invention for use as pharmaceuticals.

The present invention also provides compounds of the present invention for use as medicines for treating or preventing diseases mediated by BTK or at least partially mediated by BTK, particularly for treating or preventing autoimmune diseases, inflammatory diseases or cancer.

The present invention also provides a pharmaceutical combination comprising the compounds of the present invention and at least one additional therapeutic agent, the therapeutic agent preferably selected from: anti-inflammatory agents, immunomodulators or antitumor agents, the antitumor agents including chemotherapeutic agents, immune checkpoint inhibitors or agonists, and targeted therapeutic agents.

The present invention also provides a kit for treating or preventing diseases mediated by BTK or at least partially mediated by BTK. The kit may contain a pharmaceutical composition of the present invention and instructions for use, wherein the pharmaceutical composition contains compounds of the present invention.

Attached Figure Description

Figure 1: The inhibitory effect of the compound of the present invention on the activation of B cells in whole blood of mice induced by anti-IgD antibody.

Figure 2: Effect of the compound of the present invention on paw volume in rats with CIA (collagen-induced arthritis) (hind paw volume was measured by a paw volume analyzer, and the data are expressed as mean ± standard deviation. The groups are the normal group, solvent control group, 0.25 mg/kg and 4 mg/kg GDC-0853 group, and different doses of compound 1QD group (normal group: n = 6, other groups: n = 8).

Figure 3: Effect of the compounds of this invention on platelet levels in peripheral blood of ITP (anti-mouse CD41 antibody-induced idiopathic thrombocytopenic purpura) mice. Platelet levels were measured using an automated blood analyzer. Data are expressed as mean ± standard deviation. The groups were divided into normal control group and model group (the solvent control group, the 40 mg/kg PRN1008 group, and the group with different doses of compound 1, respectively) (N = 8 for each group).

Invention Details

definition

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

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

As used herein, the term "alkyl" refers to a straight-chain or branched saturated hydrocarbon group having 1-18 carbon atoms (C _1-18 ), preferably 1-10 carbon atoms (C _1-10 ), particularly preferably 1-6 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" indicates an alkyl group having 1-6 carbon atoms, and "C _1-3 alkyl" indicates an alkyl group having 1-3 carbon atoms. Examples of _{C 1-6} 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, etc.

As used herein, the term "alkynyl" refers to a straight-chain or branched unsaturated hydrocarbon group containing one or more, for example, 1, 2, or 3 carbon-carbon triple bonds (C≡C), having 2-18 carbon atoms (C _2-18 ), preferably 2-10 carbon atoms (C _2-10 ), more preferably 2-6 carbon atoms (C _2-6 ), and even more preferably 2-4 carbon atoms (C _2-4 ). When the term "alkynyl" is prefixed with "C," it indicates the number of carbon atoms. For example, "C _2-6 alkynyl" indicates an alkynyl group having 2-6 carbon atoms, and "C _2-4 alkynyl" indicates an alkynyl group having 2-4 carbon atoms. Examples of _{C 2-6} alkynyl groups include, but are not limited to, ethynyl, propynyl (e.g., 2-propynyl), and butynyl (e.g., 2-butynyl). The alkynyl group may or may not be attached to a triple bond.

As used herein, the term "halogen" or "halogenated" refers to fluorine, chlorine, bromine, and iodine, preferably fluorine, chlorine, and bromine, and more preferably fluorine and chlorine.

As used herein, the term "haloalkyl" 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 each other. 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 each other. 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 each other. When the term "haloalkyl" is prefixed with "C", it indicates the number of carbon atoms. For example, "C _1-6 haloalkyl" indicates a haloalkyl group as defined above having 1-6 carbon atoms, and "C _1-4 haloalkyl" indicates a haloalkyl group as defined above having 1-4 carbon atoms. Examples of _C1-6 haloalkyl groups include, but are not limited to _{, -CF3} , _-CHF2 , _-CH2F , _-CH2CF3 , -CH( _CF3 ) ₂ , etc.

As used herein, the term "cyclic hydrocarbon group" refers to a saturated or partially unsaturated cyclic hydrocarbon group containing 3-12 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 )); 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 cyclic hydrocarbon group" or "3-6 membered cyclic hydrocarbon group" indicates a cyclic hydrocarbon group having 3-6 ring carbon atoms. Cyclic hydrocarbon groups may include fused or bridged rings and spirocyclic rings. The ring of a cyclic hydrocarbon group can be saturated, and its ring can contain one or more, for example, one or two double bonds (i.e., partially unsaturated), but it is not fully conjugated, nor is it an "aryl" group as defined in this invention. Examples of _C3-6 cyclic hydrocarbon groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, spiro[2.2]pentyl, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclopentadienyl, cyclohexenyl, etc.

As used herein, the terms "heterocyclic group" or "heterocycle" are used interchangeably and refer to a saturated or partially unsaturated ring having 3-12 ring atoms (e.g., 3-8, 4-8, 4-6, or 4-5 ring atoms), wherein the ring atoms comprise one or more (e.g., 1, 2, or 3, preferably 1 or 2) heteroatoms independently selected from N, O, and S, and the remaining ring atoms are carbon atoms; it may have one or more rings, such as 1, 2, or 3, preferably 1 or 2 rings. N and S may optionally be oxidized to various oxidation states. The junction of the heterocyclic group may be on an N heteroatom or a carbon atom. For example, "4-8 membered heterocyclic group" refers to a heterocyclic group having 4-8 (4, 5, 6, 7 or 8) ring atoms, comprising at least one, for example 1, 2 or 3, preferably 1 or 2 heteroatoms independently selected from N, O and S; "4-6 membered heterocyclic group" refers to a heterocyclic group having 4-6 (4, 5 or 6) ring atoms, comprising at least one, preferably 1 or 2 heteroatoms independently selected from N, O and S (preferably N and O, more preferably O), and is preferably a monocyclic group; "4-5 membered heterocyclic group" refers to a heterocyclic group having 4 or 5 ring atoms, comprising at least one, preferably 1 or 2 heteroatoms independently selected from N, O and S (preferably N and O, more preferably O), and is a monocyclic group. Heterocyclic groups may include fused or bridged rings and spirocyclic groups. The ring of the heterocyclic group can be saturated, and its ring can contain one or more, for example one or two double bonds (i.e., partially unsaturated), but it is not fully conjugated, nor is it a "heteroaryl" as defined in this invention. Examples of heterocyclic groups include, but are not limited to: 4-8-membered heterocyclic groups, 4-6-membered heterocyclic groups, 4-5-membered heterocyclic groups and 4-membered heterocyclic groups, such as oxetane (e.g., oxetane-3-yl), azirane, pyrrolidinyl, tetrahydrofuranyl, dioxopentane, tetrahydropyranyl, morpholinyl, thiomorpholinyl, piperidinyl, piperazinyl, tetrahydropyridinyl, pyrazinyl, pyrazolyl and oxaspiro[3.3]heptyl, preferably oxetane (e.g., oxetane-3-yl), azirane, tetrahydropyranyl, morpholinyl (e.g., morpholino), piperazinyl (e.g., piperazin-1-yl), tetrahydropyridinyl (e.g., 1,2,3,6-tetrahydropyridinyl).

The term "-OH" as used in this article refers to a hydroxyl group.

The term "-CN" used in this article refers to the cyano group.

The term "oxo" as used in this article refers to =O.

Any asymmetric atom (e.g., carbon) in a compound of formula (I) may be present in a racemic or enantiomer-rich form, for example, in (R)-, (S)-, or (RS)- configurations. In some embodiments, in the (R)- or (S) configuration, each asymmetric atom has an enantiomeric excess of at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100%.

As used herein, the terms “optional,” “optional,” or “optionally” mean that the event or condition described below may or may not occur, and the description includes both scenarios where the event or condition occurs and scenarios where it does not occur. For example, “optionally substituted by one or more…” includes both unsubstituted and substituted by one, two, three, or more of the described substituents. Those skilled in the art will understand that, for any group containing one or more substituents, the group does not include any substitution pattern that is spatially impractical, chemically incorrect, synthetically infeasible, and/or inherently unstable.

As used herein, the terms “substituted” or “replaced by” mean 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 selected from a given group of substituents or groups, provided that the replacement does not exceed the normal valence of the given atom, and the substituents may be the same as or different from each other. The terms “replaced by one or more groups selected from” or “replaced by one or more” as used herein mean that one or more hydrogen atoms on a given atom or group are independently replaced by one or more groups selected from a given group of substituents or groups, wherein the groups may be the same as or different from each other. Preferably, “replaced by one or more groups selected from” or “replaced 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 group of substituents or groups, wherein the groups may be the same as or different from each other. In some embodiments, when the substituent is oxo (i.e., =O), two hydrogen atoms on a single atom are replaced. Optional substituents can be various groups, provided that the combination of substituents and/or variables produces a chemically correct and stable compound. A chemically correct and stable compound means that the compound is stable enough to be isolated from the reaction mixture. Preferably, the substituents are those implemented in the example compounds of this application.

Unless otherwise stated, substituents are named within the core structure. For example, it should be understood that when a (cycloalkyl)alkyl group is listed as a possible substituent, it indicates that the substituent is attached to the core structure at the alkyl moiety.

Those skilled in the art will understand that some compounds of formula (I) may contain one or more chiral centers, and thus have two or more stereoisomers. Racemic mixtures of these isomers, mixtures of single isomers and one enantiomer-enriched mixture, and mixtures of diastereomers and specific diastereomer-enriched mixtures when there are two chiral centers are all within the scope of this invention. Those skilled in the art will also understand that this invention includes all single stereoisomers (e.g., enantiomers), racemic mixtures, or partially separated mixtures of compounds of formula (I), and, where appropriate, single tautomers thereof.

Racemic mixtures can be used in their original form or can be resolved into their individual isomers. Resolution yields stereochemically pure compounds or mixtures enriched with one or more isomers. Methods for isomer separation are well-known (see Allinger N.L. and Eliel E.L., "Topics in Stereochemistry", Vol. 6, Wiley Interscience, 1971), including physical methods such as chromatography using chiral adsorbents. Individual isomers in chiral forms can be prepared from chiral precursors. Alternatively, a single isomer can be obtained by chemical separation of the mixture as follows: forming a diastereomer salt with a chiral acid (e.g., a single enantiomer of 10-camphorsulfonate, camphorate, α-bromocamphorate, tartaric acid, diacetyltartaric acid, malic acid, pyrrolidone-5-carboxylic acid, etc.), fractionally crystallizing the salt, and then freeing one or two of the resolved bases. This process can optionally be repeated to obtain one or two isomers that substantially do not contain the other isomer, i.e., isomers with an optical purity >95%. Alternatively, a racemic compound can be covalently attached to a chiral compound (auxiliary compound) to obtain a diastereomer, which can be separated by chromatography or fractional crystallization, followed by chemical removal of the chiral auxiliary compound to obtain a pure enantiomer.

The term "tautomer" refers to a functional group isomer that arises from the rapid movement of an atom between two positions in a molecule. Tautomers can interconvert; for example, enols and ketones are typical tautomers.

"Pharmaceutical-acceptable salts" refer to salts of free acids or bases of formula (I) that are non-toxic, biologically tolerable, or otherwise biologically suitable for administration to an individual for treatment or prevention. For example, acid addition salts include, for instance, addition salts derived from inorganic and organic 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, etc. For a general description of pharmaceutically acceptable salts, see, for example, S.M. Berge et al., "Pharmaceutical Salts", J. Pharm. Sci., 1977, 66: 1-19, and Handbook of Pharmaceutical Salts, Properties, Selection, and Use, Stahl and Wermuth eds., Wiley-VCH and VHCA, Zurich, 2002.

Furthermore, if the compound described herein is obtained as an acid addition salt, its free base form can be obtained by alkalizing the 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 following the conventional procedure for preparing acid addition salts from basic compounds, by dissolving the free base in a suitable solvent and treating the solution with acid. Those skilled in the art can determine various synthetic methods for preparing non-toxic, pharmaceutically acceptable acid or base addition salts without extensive experimentation.

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

The term "solvent" refers to a solvation form containing stoichiometric or non-stoichiometric solvents. Some compounds have a tendency to engulf solvent molecules in a fixed molar ratio in the solid state, thus forming solvates. If the solvent is water, the formed solvate is a hydrate. When the solvent is ethanol, the formed solvate 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 state of _H₂O . Such combinations can form one or more hydrates, such as hemihydrates, monohydrates, and dihydrates.

The terms “group” and “base” used in this article are synonyms and are used to refer to functional groups or molecular segments that can be linked to other molecular segments.

The term "active ingredient" is used to refer to a chemical substance that has biological activity. In some implementations, the "active ingredient" is a chemical substance with pharmaceutical uses.

As used herein, the term "pharmaceutical combination" refers to a product obtained by mixing or combining two or more active ingredients, including fixed and non-fixed combinations of active ingredients, such as a pillbox or pharmaceutical composition. The term "fixed combination" means that two or more active ingredients (e.g., the compounds of the present invention and additional therapeutic agents) are administered simultaneously to a patient in the form of a single entity or dose. The term "non-fixed combination" means that two or more active ingredients (e.g., the compounds of the present invention and additional therapeutic agents) are administered simultaneously, in parallel, or sequentially to a patient in separate entities, wherein the administration to the patient provides a therapeutically effective level of the compound.

The terms "treatment," "treatment," or "prevention" refer to the administration of one or more pharmaceutical substances, particularly compounds of the present invention, to an individual suffering from, exhibiting symptoms of, or being susceptible to the disease or disorder, in order to cure, heal, alleviate, reduce, alter, treat, improve, enhance, or influence the disease or disorder, its symptoms, or the individual's susceptibility to it. In some embodiments, the disease or disorder is cancer, such as a solid tumor or a hematologic malignancy, including lymphoma, leukemia, and myeloma. In other embodiments, the disease or disorder is an inflammatory disease or an autoimmune disease.

When referring to chemical reactions, the terms “processing,” “contact,” and “reaction” mean the addition or mixing of two or more reagents under appropriate conditions to produce the shown and/or desired product. It should be understood that the reaction producing the shown and/or desired product may not necessarily originate directly from the combination of the two initially added reagents; that is, one or more intermediates may be present in the mixture that ultimately lead to the formation of the shown and/or desired product.

As used herein, the term "effective amount" refers to an amount or dose of a BTK inhibitor that is generally sufficient to produce a beneficial effect for a patient requiring treatment or prevention of a disease or disorder mediated by BTK activity or at least partially mediated by BTK. The effective amount or dose of the active ingredient in this invention can be determined by conventional methods (e.g., modeling, dose-escalation studies, or clinical trials) in conjunction with conventional influencing factors (e.g., the manner or route of administration, the pharmacokinetics of the drug component, the severity and duration of the disease or disorder, the individual's prior or ongoing treatment, the individual's health condition and response to the drug, and the judgment of the attending physician).

Typical dosage ranges are from about 0.0001 to about 200 mg of active ingredient per kilogram of individual body weight per day, for example, about 0.001 to 100 mg/kg/day, or about 0.01 to 35 mg/kg/day, or about 0.1 to 10 mg/kg, taken once daily or in divided doses (e.g., twice daily, three times daily, four times daily). For a person weighing 70 kg, an example range of appropriate dosage is about 0.05 to about 7 g/day, or about 0.2 to about 5 g/day. Once the patient's condition or impairment improves, the dosage can be adjusted to maintain the effect. For example, the dosage or frequency of administration can be adjusted, or the dosage and frequency of administration can be reduced, to maintain the desired therapeutic or preventative effect, depending on changes in symptoms. Of course, treatment can be discontinued if symptoms have improved to an appropriate level. However, for recurrence of symptoms, patients may require intermittent long-term treatment.

The term "inhibition" refers to a reduction in the baseline activity of a biological activity or process. The term "inhibition of BTK activity" is the actual pharmaceutical activity used for the purposes of this invention, referring to the reduction in BTK activity resulting from a direct or indirect response to the presence of the compounds of this invention, relative to the BTK activity in the absence of the compounds of this invention. The reduction in activity can be caused by a direct interaction between the compounds of this invention and BTK, or by the interaction of the compounds of this invention with one or more other factors that affect BTK activity. For example, the compounds of this invention can reduce BTK activity by directly binding to BTK, by directly or indirectly affecting another factor, or by directly or indirectly reducing the amount of BTK present in cells or the body.

As used herein, the terms “individual” or “patient” refer to both mammals and non-mammals. Mammals include any member of the mammalian class, including but not limited to: humans; non-human primates such as chimpanzees and other ape 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; etc. Examples of non-mammals include, but are not limited to, birds. The terms “individual” or “patient” do not limit a specific age or sex. In some embodiments, the individual or patient is a human being.

Generally, the term "about" is used in this article to adjust the given value to be 20% higher or lower than that value.

Undefined technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains.

All numerical ranges herein should be understood as disclosing every numerical value and subset thereof within that range, regardless of whether they are specifically disclosed otherwise. For example, reference to any numerical range should be considered as reference to every numerical value within that range, such as every integer within that range, for example, C _1-6 in this document represents 1, 2, 3, 4, 5, or 6 Cs. This invention relates to all values falling within these ranges, all smaller ranges, and upper or lower limits of numerical ranges.

Detailed Implementation

Implementation Scheme 1. Compound of Formula (I):

Or a pharmaceutically acceptable salt thereof, or a solvate, racemic mixture, enantiomer, diastereomer, or tautomer thereof, wherein:

_X1 , _X2 and _X3 are independently CH or N;

U and V are independently N or CR ₉ ;

_Y1 and _Y2 are independently CR ₁₀ or N;

_R1 and _R2 are independently selected from hydrogen, deuterium, halogen, -CN, hydroxyl, _C1-6 alkyl, 3-6 membered ring hydrocarbon, _C2-6 alkynyl, _C1-6 deuterated alkyl and _C1-6 haloalkyl; or, _R1 and _R2 together with the carbon atoms attached to them form a 3-6 membered ring hydrocarbon.

_R3 is hydrogen, deuterium, halogen, -CN, or _C1-6 haloalkyl;

_R4 is hydrogen, halogen, -CN, _C1-6 alkyl, _C2-6 alkynyl, -( _C1-3 alkyl)-OH, -( _C1-3 alkyl)-O-( _C1-3 alkyl), -O-( _C1-3 alkyl), -CHO, -C(O) _NH2 , -C(O) _NHCH3 , -C(O)N( _CH3 ) ₂ or 3-hydroxy-oxetane-3-yl, wherein the _C1-6 alkyl or _C1-3 alkyl is optionally substituted by one or more deuterium or halogens;

R ₅ is selected from hydrogen, _C1-6 alkyl and _C3-6 cyclic hydrocarbon groups, wherein the _C1-6 alkyl group is optionally substituted with one or more deuterium or halogens;

_Z1 , _Z2 , _Z3 and _Z4 are independently CH or N, provided that at least one of _Z1 , _Z2 , _Z3 and _Z4 is N;

_R6 and _R7 are each independently selected from _C1-6 alkyl groups;

_R8 is hydrogen, _C1-6 alkyl, _C3-6 cyclic hydrocarbon, or 4-8 membered heterocyclic group, wherein the _C1-6 alkyl, _C3-6 cyclic hydrocarbon, or 4-8 membered heterocyclic group is optionally substituted by one or more groups selected from: deuterium, halogen, _C1-6 alkyl, trifluoromethyl, -OH, _-NH2 , -O-( _C1-6 alkyl), -NH( _C1-6 alkyl) or -N( _C1-6 alkyl) ₂ ;

_R9 is hydrogen, deuterium, or halogen;

R ₁₀ is hydrogen, deuterium, halogen, CN, _C1-6 alkyl, or _C1-6 haloalkyl;

n is 0, 1, or 2; the condition is that when n is 1, _R3 is not hydrogen.

Implementation Scheme 2. The compound as described in Implementation Scheme 1, or a pharmaceutically acceptable salt thereof, or a solvate, racemic mixture, enantiomer, diastereomer, or tautomer thereof, wherein _R4 is a _C1-6 alkyl, -( _C1-3 alkyl)-OH, -( _C1-3 deuterated alkyl)-OH, -CHO, -C(O) _NH2 , -C(O) _NHCH3 , or -C(O)N( _CH3 ) ₂ ;

Preferably, _R4 is a _C1-6 alkyl, -( _C1-3 alkyl)-OH, -( _C1-3 deuterated alkyl)-OH, or -CHO.

Implementation Scheme 3. The compound as described in Implementation Scheme 1 or 2, or a pharmaceutically acceptable salt thereof, or a solvate, racemic mixture, enantiomer, diastereomer, or tautomer thereof, wherein:

_X1 , _X2 and _X3 are independently CH or N;

U and V are each independently CR ₉ ;

_Y1 and _Y2 are each independently CR ₁₀ ;

_R1 and _R2 are independently selected from hydrogen, deuterium, halogen, -CN, hydroxyl, _C1-6 alkyl, _C1-6 deuterated alkyl and _C1-6 haloalkyl;

_R3 is hydrogen, deuterium, halogen, -CN, or _C1-6 haloalkyl;

_R4 is -( _C1-3 alkyl)-OH, -C(O) _NH2 , -C(O) _NHCH3 or -C(O)N( _CH3 ) ₂ , wherein the _C1-3 alkyl is optionally substituted with one or more deuteriums;

_R5 is selected from hydrogen and _C1-6 alkyl, wherein the _C1-6 alkyl is optionally substituted with one or more deuterium;

_Z1 , _Z2 , _Z3 and _Z4 are independently CH or N, provided that at least one of _Z1 , _Z2 , _Z3 and _Z4 is N;

_R6 and _R7 are each independently selected from _C1-6 alkyl groups;

_R8 is hydrogen, _C1-6 alkyl, _C3-6 cyclic hydrocarbon or 4-8 membered heterocyclic group, wherein the _C1-6 alkyl, _C3-6 cyclic hydrocarbon or 4-8 membered heterocyclic group is optionally substituted by one or more groups selected from: deuterium, halogen, _C1-6 alkyl, trifluoromethyl, -OH or _-NH2 ;

_R9 is hydrogen or deuterium;

R ₁₀ is hydrogen or deuterium;

n is 0, 1, or 2; the condition is that when n is 1, _R3 is not hydrogen.

Implementation Scheme 4. The compound of any one of Implementation Schemes 1-3, or a pharmaceutically acceptable salt thereof, or a solvate, racemic mixture, enantiomer, diastereomer, or tautomer thereof, wherein:

_X1 , _X2 and _X3 are independently CH or N;

U and V are each independently CR ₉ ;

_Y1 and _Y2 are each independently CR ₁₀ ;

_R1 and _R2 are independently selected from hydrogen, deuterium, halogen, -CN, hydroxyl, _C1-6 alkyl, _C1-6 deuterated alkyl and _C1-6 haloalkyl;

_R3 is hydrogen, deuterium, halogen, -CN, or _C1-6 haloalkyl;

_R4 is -( _C1-3 alkyl)-OH, wherein the _C1-3 alkyl is optionally substituted with one or more deuterium;

_R5 is selected from _C1-6 alkyl groups, wherein the _C1-6 alkyl group is optionally substituted with one or more deuterium groups;

_Z1 , _Z2 , _Z3 and _Z4 are independently CH or N, provided that at least one of _Z1 , _Z2 , _Z3 and _Z4 is N;

_R6 and _R7 are each independently selected from _C1-6 alkyl groups;

_R8 is hydrogen, _C1-6 alkyl, _C3-6 cyclic hydrocarbon or 4-8 membered heterocyclic group, wherein the _C1-6 alkyl, _C3-6 cyclic hydrocarbon or 4-8 membered heterocyclic group is optionally substituted by one or more groups selected from: deuterium, halogen, _C1-6 alkyl, trifluoromethyl, -OH or _-NH2 ;

_R9 is hydrogen or deuterium;

R ₁₀ is hydrogen or deuterium;

n is 0, 1, or 2; the condition is that when n is 1, _R3 is not hydrogen.

Implementation Scheme 5. The compound of any one of Implementation Schemes 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.

Implementation Scheme 6. The compound of any one of Implementation Schemes 1-5, or a pharmaceutically acceptable salt thereof, or a solvate, racemic mixture, enantiomer, diastereomer or tautomer thereof, wherein _X3 is N.

Implementation Scheme 7. The compound of any one of Implementation Schemes 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.

Implementation Scheme 8. The compound of any one of Implementation Schemes 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 .

Implementation Scheme 9. The compound as described in Implementation Scheme 8, or a pharmaceutically acceptable salt thereof, or a solvate, racemic mixture, enantiomer, diastereomer or tautomer thereof, wherein R ₁₀ is hydrogen.

Implementation Scheme 10. The compound of any one of Implementation Schemes 1-9, or a pharmaceutically acceptable salt thereof, or a solvate, racemic mixture, enantiomer, diastereomer or tautomer thereof, wherein _R1 and _R2 are each independently selected from _C1-6 alkyl groups;

Preferably, _R1 and _R2 are each independently selected from _C1-3 alkyl groups;

More preferably, both _R1 and _R2 are methyl groups.

Implementation Scheme 11. The compound of any one of Implementation Schemes 1-10, or a pharmaceutically acceptable salt thereof, or a solvate, racemic mixture, enantiomer, diastereomer or tautomer thereof, wherein _R3 is hydrogen or a halogen;

Preferably, _R3 is hydrogen.

Scheme 12. The compound of any one of Schemes 1-11, or a pharmaceutically acceptable salt thereof, or a solvate, racemic mixture, enantiomer, diastereomer or tautomer thereof, wherein, preferably, _R4 is -( _C1-3 alkyl)-OH or -( _C1-3 deuterated alkyl)-OH;

Preferably, _R4 is a hydroxymethyl or a hydroxydeuterated methyl;

More preferably, _R4 is a hydroxymethyl group.

Implementation Scheme 13. The compound of any one of Implementation Schemes 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 -( _C1-3 alkyl)-OH.

Implementation Scheme 14. The compound of any one of Implementation Schemes 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.

Implementation Scheme 15. The compound of any one of Implementation Schemes 1-14, or a pharmaceutically acceptable salt thereof, or a solvate, racemic mixture, enantiomer, diastereomer or tautomer thereof, wherein: R ₅ is a C _1-6 alkyl group;

Preferably, _R5 is a _C1-3 alkyl group;

More preferably, _R5 is methyl.

Implementation Scheme 16. The compound of any one of Implementation Schemes 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.

Implementation Scheme 17. The compound of any one of Implementation Schemes 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.

Implementation Scheme 18. The compound of any one of Implementation Schemes 1-17, or a pharmaceutically acceptable salt thereof, or a solvate, racemic mixture, enantiomer, diastereomer, or tautomer thereof, wherein: R ₈ is hydrogen, C _1-6 alkyl, C _3-6 cyclic hydrocarbon, or 4-8 membered heterocyclic group, wherein the C _1-6 alkyl, C _3-6 cyclic hydrocarbon, or 4-5 membered heterocyclic group is optionally substituted by one or more groups selected from: deuterium, halogen, C _1-6 alkyl, trifluoromethyl, -OH, or -NH ₂ ;

Preferably, _R8 is hydrogen, _C1-6 alkyl or 4-5 membered heterocyclic group, wherein the _C1-6 alkyl or 4-5 membered heterocyclic group is optionally substituted by one or more groups selected from: deuterium, halogen, _C1-6 alkyl, trifluoromethyl, -OH or _-NH2 ;

Preferably, _R8 is a 4-5 membered heterocyclic group, optionally substituted by one or two groups selected from the following: deuterium, halogen, _C1-3 alkyl, trifluoromethyl, -OH or _-NH2 ;

Preferably, _R8 is a 4-5 member heterocyclic group;

More preferably, _R8 is a 4-membered heterocyclic group.

Implementation Scheme 19. The compound of any one of Implementation Schemes 1-18, or a pharmaceutically acceptable salt thereof, or a solvate, racemic mixture, enantiomer, diastereomer or tautomer thereof, wherein: R ₈ is an oxetane or tetrahydrofuranyl.

Implementation Scheme 20. The compound as described in Implementation Scheme 19, or a pharmaceutically acceptable salt thereof, or a solvate, racemic mixture, enantiomer, diastereomer, or tautomer thereof, wherein R ₈ is

Implementation Scheme 21. The compound as described in Implementation Scheme 1, or a pharmaceutically acceptable salt thereof, or a solvate, racemic mixture, enantiomer, diastereomer, or tautomer thereof,

It is selected from:

Implementation Scheme 22. A pharmaceutical composition comprising any of the compounds described in Implementation Schemes 1-21 and/or their pharmaceutically acceptable salts, and optionally comprising a pharmaceutically acceptable excipient.

Implementation Scheme 23. A method for inhibiting BTK activity in vivo or in vitro, comprising contacting BTK with an effective amount of any of the compounds described in Implementation Schemes 1-21 and/or their pharmaceutically acceptable salts.

Implementation Scheme 24. Use of any of the compounds described in Implementation Schemes 1-21 and/or their pharmaceutically acceptable salts in the preparation of a medicament for the treatment or prevention of diseases mediated by or at least partially mediated by BTK, preferably for the treatment or prevention of autoimmune diseases, inflammatory diseases, or cancer; wherein the inflammatory diseases or autoimmune diseases are preferably selected from: systemic and local inflammation, arthritis, rheumatoid arthritis, immunosuppression-related inflammation, organ transplant rejection, allergic diseases, ulcerative colitis, Crohn's disease, dermatitis, asthma, lupus erythematosus, Sjögren's syndrome, multiple sclerosis The cancers are: scleroderma, multiple sclerosis, osteoporosis, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, antineutrophil cytoplasmic antibody vasculitis, chronic obstructive pulmonary disease, psoriasis, Sjögren's syndrome, pemphigus vulgaris, kidney transplant-related diseases, thyroid autoimmune diseases, chronic lymphocytic thyroiditis, hyperthyroidism, pernicious anemia with chronic atrophic gastritis, pulmonary hemorrhage nephritis syndrome, bullous pemphigoid, primary biliary cirrhosis, acute idiopathic polyneuritis, systemic lupus erythematosus, and mixed connective tissue diseases; the cancers are preferably solid tumors or hematologic malignancies, including lymphoma, leukemia, and myeloma.

The cancers mentioned are 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, Waldenström macroglobulinemia, marginal zone lymphoma, Burkitt lymphoma, non-Burkkitt high-grade B-cell lymphoma, extranodal marginal zone B-cell lymphoma, small lymphocytic lymphoma (SLL), lymphoblastic lymphoma, and lymphocytes. Leukemia, myeloid leukemia, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), human acute monocytic leukemia, acute lymphoblastic 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 transplant-versus-host disease.

Implementation Scheme 25. A method for treating or preventing a disease in an individual, comprising administering to an individual in need an effective amount of any of the compounds described in Implementation Schemes 1-21 and/or their pharmaceutically acceptable salts, wherein the disease is BTK-mediated or at least partially BTK-mediated; the disease is preferably an autoimmune disease, an inflammatory disease, or cancer; the inflammatory disease or autoimmune disease is preferably selected from: systemic and local inflammation, arthritis, rheumatoid arthritis, immunosuppression-related inflammation, organ transplant rejection, allergic diseases, ulcerative joint diseases. Enteritis, Crohn's disease, dermatitis, asthma, lupus erythematosus, Sjögren's syndrome, multiple sclerosis, scleroderma, osteoporosis with multiple sclerosis, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, antineutrophil cytoplasmic antibody vasculitis, chronic obstructive pulmonary disease, psoriasis, Sjögren's syndrome, pemphigus vulgaris, kidney transplant-related diseases, thyroid autoimmune diseases, chronic lymphocytic thyroiditis, hyperthyroidism, pernicious anemia with chronic atrophic gastritis, pulmonary hemorrhage nephritis syndrome, bullous pemphigoid, primary biliary cirrhosis, acute idiopathic multiple sclerosis Neuritis, systemic lupus erythematosus, mixed connective tissue disease; the cancer is preferably a solid tumor or hematologic 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, Waldenström macroglobulinemia, marginal zone lymphoma, Burkitt lymphoma, non-Burkkitt 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 lymphoblastic 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 transplant-versus-host disease.

Implementation Scheme 26. Any of the compounds described in Implementation Schemes 1-21 and/or their pharmaceutically acceptable salts, which are used as medicines.

Implementation Scheme 27. Any compound described in Implementation Schemes 1-21 and/or a pharmaceutically acceptable salt thereof, for the treatment or prevention of diseases mediated by or at least partially mediated by BTK, preferably for the treatment or prevention of autoimmune diseases, inflammatory diseases, or cancer; wherein the inflammatory diseases or autoimmune diseases are preferably selected from: systemic and local inflammation, arthritis, rheumatoid arthritis, immunosuppression-related inflammation, organ transplant rejection, allergic diseases, ulcerative colitis, Crohn's disease, dermatitis, asthma, lupus erythematosus, Sjögren's syndrome, multiple sclerosis, scleroderma, osteoporosis with multiple sclerosis, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, antineutrophil cytoplasmic antibody vasculitis, chronic obstructive pulmonary disease, psoriasis, Sjögren's syndrome, pemphigus vulgaris, kidney transplant-related diseases, thyroid autoimmune diseases, chronic lymphocytic thyroiditis, hyperthyroidism, pernicious anemia with chronic atrophic gastritis, pulmonary hemorrhage and nephritis syndrome, bullous pemphigoid, primary biliary cirrhosis, acute idiopathic polyneuritis, systemic lupus erythematosus, mixed Connective tissue diseases; the cancer is preferably a solid tumor or hematologic 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, Waldenström macroglobulinemia, marginal zone lymphoma, Burkitt lymphoma, non-Burkkitt high-grade B-cell lymphoma, extranodal marginal zone B-cell lymphoma, and small lymphocytic lymphoma. SLL, lymphoblastic lymphoma, lymphocytic leukemia, myeloid leukemia, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), human acute monocytic leukemia, acute lymphoblastic 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 transplant-resistant host disease.

Implementation Scheme 28. A pharmaceutical combination comprising any of the compounds described in Implementation Schemes 1-21 and/or their pharmaceutically acceptable salts, and at least one additional therapeutic agent, said therapeutic agent preferably selected from: anti-inflammatory agents, immunomodulators or antitumor active agents, said antitumor active agents including chemotherapeutic agents, immune checkpoint inhibitors or agonists, and targeted therapeutic agents.

The various embodiments of the present invention (including the embodiments below) and the features of the various embodiments should be understood to be arbitrarily combined with each other, and all such combinations are included within the scope of the present invention, just as such combinations are specifically and individually listed herein, unless the context clearly indicates otherwise.

General Synthesis Method

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

Process 1:

Wherein: Hal represents halogen, and _R1 , _R2 , _R3 , _R4 , _R5 , R6, _R7 , _R8 , _X1 , _X2 , _X3 , _Z1 , _Z2 , _Z3 , _Z4 , U, V, _Y1 , _Y2 and _n are as defined in this paper.

As shown in process 1, the compound of formula 1-1 reacts with the dihaloaryl aldehyde compound of formula 1-2 under cuprous iodide catalysis to give the compound of formula 1-3. The cuprous iodide-catalyzed carbon-nitrogen coupling reaction is carried out under suitable conditions. The solvent used can be selected from polar solvents such as 1,4-dioxane, DMF, etc., and the base used can be selected _{from Cs₂CO₃} , _Na₂CO₃ , _K₃PO₄ , _etc. Formula 1-3 is reduced under suitable conditions to give the compound of formula 1-4 of this 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 mixture of methanol and dichloromethane. The acetyl group is added to the compound of formula 1-4 to give the compound of formula 1-5. The compound of formula 1-5 reacts with pinacol diborate under suitable conditions to give the boric acid or borate ester compound of formula 1-6. Compounds of formulas 1-6, under appropriate palladium reagent catalysis, undergo a Suzuki coupling reaction with halogenated derivatives of formulas 1-7 to yield compounds of formulas 1-8. The palladium-catalyzed carbon-carbon coupling reaction is carried out under suitable conditions. The solvent used can be a polar solvent such as 1,4 _- dioxane, DMF, THF, or a mixture of 1,4-dioxane and water. The base used can be _Cs₂CO₃ , _Na₂CO₃ , _{K₃PO₄ , etc.} , _and the catalyst can be Pd(dppf) _Cl₂ · _CH₂Cl₂ , Pd( _PPh₃ ) _₄ , Pd(OAc) _₂ , etc. Compounds of formulas 1-8 are deacetylated under appropriate basic conditions to yield compounds of formula (I-1) of this invention. The base used can be potassium carbonate, sodium carbonate, lithium hydroxide, etc., and the solvent used can be a polar solvent such as methanol, ethanol, or a mixture of methanol and water.

Process 2:

As shown in process 2, compounds of formula 1-3, under appropriate palladium reagent catalysis, undergo a Suzuki coupling reaction with boric acid or borate ester of formula 2-1 to obtain compound 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 mixture of 1,4- _dioxane and water, etc. 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. Compound 2-2 is then reduced under suitable conditions to obtain compound (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 mixture of methanol and dichloromethane, etc.

Process 3:

As shown in process 3, the compound of formula 3-1 undergoes a substitution reaction with bromoacetaldehyde diethanolate under suitable conditions to give the compound of formula 3-2. The base used can be selected from cesium carbonate, etc., and the solvent 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 give the compound of formula 3-3. The base used can be selected from lithium hydroxide, potassium carbonate, sodium carbonate, etc., and the solvent can be selected from polar solvents such as methanol, ethanol, or a mixture of methanol and water. The compound of formula 3-3 undergoes a condensation reaction with HATU and ammonia to give the compound of formula 3-4. The compound of formula 3-4 undergoes cyclization in acetic acid to give the compound of formula 3-5.

Process 4:

As shown in process 4, the compound of formula 3-1 undergoes a substitution reaction with hydrazine hydrate to give the compound of formula 4-1. The compound of formula 4-1 undergoes a cyclization reaction with triethyl orthoformate in DMF solution to give the compound of formula 4-2.

The substituents of the compounds obtained by the above methods can be further modified to obtain other desired compounds. For synthetic chemical transformation methods, see, for example: 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.

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

Pharmaceutical Compositions and Uses

The compounds of the present invention (such as those described in any of the examples herein) can be formulated into pharmaceutical compositions, alone or in combination with one or more other therapeutic agents. A pharmaceutical composition comprises: (a) an effective amount of the compound of the present invention; (b) a pharmaceutically acceptable excipient (e.g., one or more pharmaceutically acceptable carriers); and optionally (c) at least one other therapeutic agent.

Pharmaceutically acceptable excipients are those that are compatible with (and in some embodiments, stabilize) the active ingredient in the composition and are harmless to the individual being treated. For example, solubilizers such as cyclodextrins (which can form specific, more soluble complexes with the compounds of the present invention) can be used as pharmaceutical excipients to deliver the active ingredient. Other examples of excipients include colloidal silica, magnesium stearate, cellulose, sodium lauryl sulfate, and pigments such as D&C Yellow #10. Suitable pharmaceutically acceptable excipients are disclosed in standard references in the art (Remington's Pharmaceutical Sciences, A.Osol).

Pharmaceutical compositions comprising the compounds of the present invention can be administered in a variety of known manners, such as oral, topical, rectal, parenteral, inhalation, or implantation. The term "parenteral" as used herein includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intra-articular, intrasynovial, intrasternal, intravertebral, intra-affective, and intracranial injection or infusion.

The pharmaceutical compositions described herein may be prepared in the form of tablets, capsules, sachets, sugar-coated pills, powders, granules, lozenges, powder for injection, liquid preparations, or suppositories. In some embodiments, the pharmaceutical compositions comprising the compounds of the present invention may be formulated for intravenous infusion, topical administration, or oral administration.

Orally administered compositions can be in any orally acceptable dosage form, including but not limited to: tablets, capsules, emulsions, and aqueous suspensions, dispersants, and solutions. Common tablet carriers include lactose and corn starch. Lubricants such as magnesium stearate are also frequently incorporated into tablets. When administered orally in capsule form, useful diluents include lactose and dried corn starch. When administered orally in aqueous suspension or emulsion form, emulsifiers or suspending agents can be used to suspend or dissolve the active ingredient in the oil phase. If desired, certain sweeteners, flavoring agents, or colorings may be added.

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

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 non-toxic, parenteral-acceptable diluents or solvents, such as solutions in 1,3-butanediol. Pharmaceutically acceptable carriers and solvents, particularly mannitol, water, Ringer's solution, and physiological saline, are commonly used. Furthermore, sterile, non-volatile oils, such as synthetic mono- or diglycerides, are often used as solvents or suspension media. Fatty acids, such as oleic acid and its glyceride derivatives, and natural, pharmaceutically acceptable oils, such as olive oil or castor oil (especially in their polyoxyethylated forms), are commonly used as injectable media. These oil solutions or suspensions may also contain long-chain alcohol diluents or dispersants, or carboxymethyl cellulose or similar dispersants.

Inhalation compositions can be prepared using benzyl alcohol or other suitable preservatives, absorption enhancers that improve bioavailability, fluorocarbons and/or other solubilizers or dispersants known in the art, according to techniques well known in the pharmaceutical formulation field, or they can be prepared as solutions in saline.

Topical compositions can be formulated as oils, creams, lotions, ointments, etc. Suitable carriers for the compositions include vegetable or mineral oils, white petrolatum (white paraffin), branched-chain fatty acids or oils, animal fats, and high molecular weight alcohols (i.e., alcohols with more than 12 carbon atoms). In some embodiments, pharmaceutically acceptable carriers are those in which the active ingredient can dissolve. If desired, the composition may also contain emulsifiers, stabilizers, wetting agents, and antioxidants, as well as substances that impart color or fragrance. Furthermore, transdermal penetration enhancers may be added to the topical formulation. Examples of such enhancers can be found in U.S. Patent Nos. 3,989,816 and 4,444,762.

Creams can be formulated from a mixture of mineral oil, self-emulsifying beeswax, and water, with an active ingredient dissolved in a small amount of oil, such as almond oil, incorporated therein. An example of a cream contains approximately 40 parts by weight of water, approximately 20 parts by weight of beeswax, approximately 40 parts by weight of mineral oil, and approximately 1 part by weight of almond oil. Ointments can be formulated by mixing a solution of the active ingredient in a vegetable oil, such as almond oil, with warm paraffin wax and then cooling the mixture. An example of an ointment contains approximately 30% by weight of almond oil and approximately 70% by weight of white paraffin wax.

Suitable in vitro experiments can be used to evaluate the effect of the compounds of the present invention on inhibiting BTK activity. Further in vivo experiments can be conducted to detect additional effects of the compounds of the present invention in the prevention or treatment of cancer. For example, the compounds of the present invention can be administered to animals with cancer (e.g., mouse models), and their therapeutic effect can then be evaluated. If the results of preclinical trials are successful, the dosage range and route of administration in animals, such as humans, can also be predicted.

The compounds of the present invention show sufficient preclinical practical use to warrant clinical trials and are expected to demonstrate beneficial therapeutic or preventive effects, for example, in individuals with cancer.

As used herein, the term "cancer" refers to a cellular disorder characterized by uncontrolled or disordered cell proliferation, reduced cell differentiation, inappropriate invasion of surrounding tissues, and/or the ability to establish new growth sites in other locations. The term "cancer" includes, but is not limited to, solid tumors and hematologic malignancies (e.g., leukemia, lymphoma, or myeloma). 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, recurrent, and refractory cancers.

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 non-androgen-dependent 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), bronchioloalveolar carcinoma (BAC), and lung adenocarcinoma; ovarian cancer, including, for example, progressive epithelial carcinoma or primary peritoneal carcinoma; cervical cancer; endometrial cancer; and gastric cancer. Cancer; esophageal cancer; head and neck cancer, including, for example, squamous cell carcinoma of the head and neck; skin cancer, including, for example, melanoma and basal carcinoma; neuroendocrine carcinoma, including metastatic neuroendocrine tumors; brain tumors, including, for example, glioma, anaplastic oligodendroglioma, adult glioblastoma multiforme and adult anaplastic astrocytoma; bone cancer; sarcoma, including, for example, Kaposi's sarcoma; adrenal carcinoma; mesothelial endometrial carcinoma; choriocarcinoma; muscle cancer; connective tissue carcinoma; and thyroid cancer.

Non-limiting examples of hematologic malignancies include acute myeloid leukemia (AML); chronic myeloid leukemia (CML), including accelerated phase CML and CML blast crisis (CML-BP); acute lymphoblastic 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); Waldenström macroglobulinemia; myelodysplastic syndromes (MDS), including refractory anemia (RA), refractory anemia with ringed sideroblasts (RARS), refractory anemia with excessive budding cells (RAEB), and refractory anemia with excessive budding cells and acute transformation (RAEB-T); and myeloproliferative syndrome.

In some implementations, hematologic malignancies are 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, and relapsed or refractory multiple myeloma.

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

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

The term "autoimmune disease" refers to a disease or condition caused by damage to one's own tissues or organs due to an immune response to self-antigens. 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), osteoporosis with multiple sclerosis, 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, pemphigus vulgaris, kidney transplant-related diseases, and myeloproliferative disorders such as myelofibrosis. Autoimmune diseases include: rheumatoid arthritis (rhosis), post-polycythemia vera/essential thrombocytosis myelofibrosis (post-PV/ET myelofibrosis), thyroid autoimmune diseases, chronic lymphocytic thyroiditis, hyperthyroidism, pernicious anemia with chronic atrophic gastritis, goodpasture syndrome, bullous pemphigoid, primary biliary cirrhosis, acute idiopathic polyneuritis, systemic lupus erythematosus, and mixed connective tissue diseases. In some implementation plans, autoimmune diseases are selected from arthritis, such as rheumatoid arthritis and collagen-induced arthritis.

The term "inflammatory disease" or "inflammatory condition" refers to a pathological state that causes inflammation, particularly due to neutrophil chemotaxis. Non-limiting examples of inflammatory diseases include systemic and local inflammation, immunosuppression-related inflammation, organ transplant rejection, allergic diseases, inflammatory skin diseases (including psoriasis and atopic dermatitis); systemic scleroderma and sclerosis; reactions associated with inflammatory bowel diseases (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, post-cardiac reperfusion and abnormal contractile responses of coronary vessels after percutaneous coronary angioplasty, tissue reperfusion injury after stroke and abdominal aortic aneurysm surgery; stroke-related cerebral edema; head 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), Sjorgen's syndrome, vasculitis; diseases involving leukocyte exudation; sepsis or trauma-induced inflammatory diseases of the central nervous system (CNS), multiple organ injury syndrome; alcoholic hepatitis; bacterial pneumonia; antigen-antibody complex-mediated diseases, including glomerulonephritis; sepsis; sarcoidosis; immunopathological reactions following tissue/organ transplantation; lung inflammation, including pleurisy, alveolitis, vasculitis, pneumonia, chronic bronchitis, bronchiectasis, diffuse panbronchiolitis, allergic pneumonia, idiopathic pulmonary fibrosis (IPF), and cystic fibrosis, etc. Preferred indications include, but are not limited to, chronic inflammation, autoimmune diabetes, rheumatoid arthritis (RA), rheumatoid spondylitis, gouty arthritis and other joint diseases, 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, and any disease associated with inflammation and related conditions.

Furthermore, the compounds of the present invention (such as those described in any of the examples herein) can be used in combination with additional therapeutic agents to treat the diseases or conditions described herein, such as autoimmune diseases, inflammatory diseases, or cancer. The additional therapeutic agents may be administered separately from the compounds of the present invention or may be included in a pharmaceutical composition, such as a fixed-dose combination drug, in accordance with this disclosure. In some embodiments, the additional therapeutic agents are those known or found to be effective in treating BTK-mediated or at least partially BTK-mediated diseases, such as other BTK inhibitors or compounds that effectively antagonize other targets associated with that particular disease. Combination therapy can be used to improve efficacy (e.g., by including compounds that enhance the potency 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 compounds of the present invention.

In some embodiments, the compounds of the present invention (such as any of the compounds described herein) may be used in combination with other therapeutic agents, such as anti-inflammatory agents, immunomodulators, or antitumor agents, including chemotherapeutic agents, immune checkpoint inhibitors or agonists, and targeted therapeutic agents. As used herein, the term "antitumor agent" refers to any agent administered to a subject suffering from cancer for the purpose of treating cancer, including chemotherapeutic agents, immune checkpoint inhibitors or agonists, and targeted therapeutic agents.

Non-limiting examples of anti-inflammatory agents and immunomodulators include: immunosuppressants (e.g., tacrolimus, cyclosporine, rapamycin, methotrexate, cyclophosphamide, azathioprine, mercaptopurine, mycophenolate mofetil, 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, arylalkyl acids, 2-arylpropionic acid, N-aryl-o-aminobenzoic acid, oxacins, coxibi, or thiodianilide), and cyclooxygenase-2-specific inhibitors (e.g., vardicoxib, celecoxib, or...). Lofecoxib), leflunomide, aurthioglucose, aurthiomalic acid, orolimus, sulfasalazine, hydroxychloroquine, minocycline, TNF-α binding protein (e.g., infliximab, etanercept, or adalimumab), abatacept, anaerobicin, interferon, interferon-γ, interleukin-2, interleukin-6, interleukin-12/23, interleukin-17 antibody drugs, allergic vaccines, antihistamines, antileukotriene drugs, β-agonists, theophylline or anticholinergic drugs; JAK3 kinase inhibitors, including all known but not limited to: tofacitinib; IRAK4, RIPK1 inhibitors, etc.

Non-limiting examples of chemotherapeutic agents include topoisomerase I inhibitors (e.g., irinotecan, topotecan, camptothecin and its analogues or metabolites, and doxorubicin); topoisomerase II inhibitors (e.g., etoposide, teniposide, mitoxantrone, demethoxydaunorubicin, and donomycin); and alkylating agents (e.g., melphalan, chlorambucil, busulfan, thiotepa, ifosfamide, nitrosourea mustard, cyclohexanenitrosourea, methylcyclohexanenitrosourea, streptozotocin, aminoimide, methotrexate, and mitomycin). C and cyclophosphamide); DNA intercalating agents (e.g., cisplatin, oxaliplatin, and carboplatin); and free radical generating agents such as bleomycin; as well as nucleoside analogs (e.g., 5-fluorouracil, capecitabine, gemcitabine, fludarabine, cytarabine, azacitidine, mercaptopurine, thioguanine, pentostatin, and hydroxyurea), paclitaxel, taxanes, and related analogs; vincristine, vinblastine, and related analogs; sedatives and related analogs (e.g., CC-5013 and CC-4047).

Non-limiting examples of immune checkpoint inhibitors or agonists include PD-1 inhibitors, such as anti-PD-1 antibodies, such as pembrolizumab and nivolumab; PD-L1 inhibitors, such as anti-PD-L1 antibodies, such as 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, etc.

Targeted therapies include a variety of small or large molecule targeted therapies, non-limiting examples of which include: protein tyrosine kinase inhibitors (e.g., imatinib mesylate and gefitinib); proteasome inhibitors (e.g., 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 (e.g., rituximab, teimomab, tosimob), anti-Her2 monoclonal antibodies (trastuzumab), anti-EGFR antibodies (cetuximab), and anti-VEGFR antibodies (bevacizumab); anti-angiogenic drugs, such as lenalidomide; and other protein or enzyme inhibitors known to be upregulated, overexpressed, or activated in cancer, and whose inhibition can downregulate cell replication.

Example

The following examples are illustrative of the invention and do not limit the invention in any way. The data given (e.g., quantities, temperatures, etc.) are intended to be accurate; however, those skilled in the art should understand that some experimental errors and biases may occur. Unless otherwise stated, all parts are by weight, temperatures are in Celsius, and pressures are at or near atmospheric pressure. All mass spectrometry data were obtained using Agilent 6120 and 1100. All nuclear magnetic resonance data were obtained using a Varian 400MR. Except for the synthetic intermediates, all reagents and raw materials used in this invention were commercially available. The positive control GDC-0853 (fenebrutinib) was purchased from Shanghai Lingkai Pharmaceutical Technology Co., Ltd. All compound names, except for reagents, were generated using Chemdraw 16.0.

In any structural formula of this application, if there is a vacant valence on any atom, the vacant valence is actually for the sake of simplicity and is not specifically described as a hydrogen atom.

In this application, if both the name and structural formula of a compound are given, and the two are inconsistent, the structure of the compound shall prevail, unless the context indicates that the structure of the compound is incorrect while the name is correct.

The following list of abbreviations is used in the embodiments:

CD ₃ OD Deuterated Methanol

DCM (Dichloromethane)

DIEA N,N-Diisopropylethylamine

DMF (dimethylformamide)

DMSO (Dimethyl sulfoxide)

DMSO- _d6 -deuterated dimethyl sulfoxide

g gram

HATU 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylureonium hexafluorophosphate

HPMC (Hydroxypropyl Methylcellulose)

L Up

M Moles/L

mg

mL

mmol (millimoles)

mol

NBS N-bromosuccinimide

_Pd2 (dba) _3tris (dibenzylacetone)dipalladium

Pd( _dppf ) _{Cl₂CH₂Cl₂} [1,1' _- bis(diphenylphosphine)ferrocene]palladium dichloride dichloromethane complex

Xphos 2-Dicyclohexylphosphine-2',4',6'-triisopropylbiphenyl

Xant-phos 4,5-Bis(diphenylphosphine)-9,9-dimethyloxanthracene

Example 1: Synthesis of the compound

Intermediate I-1

3-((5-((2S,6S)-2,6-dimethyl-4-(oxecyclobutane-3-yl)piperazin-1-yl)pyridin-2-yl)amino)-1-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)pyridin-2(1H)-one

Step 1: (3S,5S)-3,5-dimethyl-4-(6-nitropyridin-3-yl)piperazine-1-carboxylic acid tert-butyl ester

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

Step 2: (3S,5S)-4-(6-aminopyridin-3-yl)-3,5-dimethylpiperazine-1-carboxylic acid tert-butyl ester

Hydrogen gas was bubbled into a mixture of (3S,5S)-3,5-dimethyl-4-(6-nitropyridin-3-yl)piperazine-1-carboxylic acid tert-butyl ester (4.5 g, 13.4 mmol) and 10% palladium-carbon (containing 50% water, 3.0 g) in methanol (100 mL) at room temperature, and the reaction was carried out at 40 °C for 3 hours. The reaction solution was filtered, and the filtrate was collected and concentrated under vacuum to obtain the target product (3.9 g, 95% yield), which was used directly in the next step of the reaction. [M+H] ⁺ 307.2

Step 3: (3S,5S)-4-(6-((5-bromo-1-methyl-2-oxo-1,2-dihydropyridin-3-yl)amino)pyridin-3-yl)-3,5-dimethylpiperazine-1-carboxylic acid tert-butyl ester

Under nitrogen protection, Xant-phos (433 mg, 0.75 mmol), Pd²⁻(dba)³⁻ (343 mg, 0.375 mmol), and cesium carbonate (4.9 g, 15.0 mmol) were added to a solution of (3S,5S)-4-(6-aminopyridin-3-yl)-3,5-dimethylpiperazin-1-carboxylic acid tert-butyl ester (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). The mixture was reacted at 90 °C for 12 hours, cooled to room temperature, filtered, and the filtrate was collected and concentrated. The residue was purified by silica gel column chromatography (methanol/dichloromethane) to give the target product (3.0 g, 81% yield). [M+H] ⁺ 492.1,494.1

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

Add concentrated hydrochloric acid (8 mL) to a methanol (15 mL) solution of (3S,5S)-4-(6-(((5-bromo-1-methyl-2-oxo-1,2-dihydropyridin-3-yl)amino)pyridin-3-yl)-3,5-dimethylpiperazine-1-carboxylic acid tert-butyl ester (3.0 g, 6.1 mmol) and stir at 50 °C for 30 minutes.

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

Step 5: 3-((5-((2S,6S)-2,6-dimethyl-4-(oxecyclobutane-3-yl)piperazin-1-yl)pyridin-2-yl)amino)-1-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)pyridin-2(1H)-one

Under nitrogen protection, Xphos (128 mg, 0.27 mmol), Pd₂(dba)₃ (247 mg, 0.27 mmol), and potassium acetate (784 mg, 8.0 mmol) were added to a solution of 5-bromo-3-((5-(((2S,6S)-2,6-dimethyl-4-(oxetane-3-yl)piperazin-1-yl)pyridin-2-yl)amino)-1-methylpyridin- ₂ (1H)-one (1.2 g, 2.68 mmol), pinacol diboronate ( _1.7 g, 6.7 mmol), and 1,4-dioxane (60 mL). The mixture was reacted at 65 °C for 6 hours, cooled to room temperature, filtered, and the filtrate was collected. The filtrate was concentrated under vacuum, and the residue was purified by silica gel column chromatography (methanol/dichloromethane) to give the target compound (650 mg, 50% purity, 24% yield).

[M+H] ⁺ 496.3

Following the preparation steps of intermediate I-1, and using the corresponding raw materials and reagents, the intermediates listed in the table below were prepared:

Intermediate I-3

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

Step 1: ethyl 1-(2,2-diethoxyethyl)-5,5-dimethyl-1,4,5,6-tetrahydrocyclopentadien[b]pyrrole-2-carboxylate

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

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

Lithium hydroxide monohydrate (14.2 g, 338 mmol) was added to a solution of ethyl 1-(2,2-diethoxyethyl)-5,5-dimethyl-1,4,5,6-tetrahydrocyclopentadieno[b]pyrrole-2-carboxylate (31.0 g, 96 mmol) in ethanol (150 mL) and water (150 mL), and the reaction was carried out at 80 °C for 12 h. Ethanol was removed under vacuum, and the pH was adjusted to 5–6 with concentrated hydrochloric acid under ice bath cooling. Water (200 mL) was added, and the mixture was extracted with ethyl acetate (200 mL x 3). The combined organic phases were collected, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to give the target product (26.7 g, 94% yield). [MH] ^- 294.1

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

Under nitrogen protection at 0–5 °C, triethylamine (25 mL, 181 mmol) was added to a DMF (150 mL) solution of 1-(2,2-diethoxyethyl)-5,5-dimethyl-1,4,5,6-tetrahydrocyclopentadieno[b]pyrrole-2-carboxylic acid (26.7 g, 90.5 mmol), followed by HATU (51.6 g, 136 mmol). The reaction was carried out at room temperature for 1 hour. The reaction solution was then poured into concentrated ammonia (800 mL) and stirred for 10 minutes. The solution was extracted with dichloromethane (300 mL x 2), and the organic phases were collected and combined. The product was concentrated to obtain the target product (26.6 g, 100% yield), which was used directly in the next reaction.

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

1-(2,2-diethoxyethyl)-5,5-dimethyl-1,4,5,6-tetrahydrocyclopentadien[b]pyrrole-2-carboxamide (26.6 g, 90.5 mmol) was dissolved in acetic acid (100 mL) and reacted at 100 °C for 4 h. Acetic acid was removed under vacuum, and the pH was adjusted to 8-9 with ammonia. Water (200 mL) was added, and the mixture was extracted with dichloromethane (200 mL x 3). The combined organic phases were collected and concentrated under vacuum. The residue was purified by silica gel column chromatography (dichloromethane/methanol) to give the target product (18.3 g, 100% yield). [M+H] ⁺ 203.1. ¹ H NMR (400MHz, DMSO-d ₆ )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).

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

Under nitrogen protection, 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) were added to a solution of 7,7-dimethyl-7,8-dihydro-2H-cyclopentadieno[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). The mixture was reacted at 80 °C for 16 hours, cooled to room temperature, filtered, and the filtrate was collected. The filtrate was concentrated under vacuum, and the residue was recrystallized from ethanol to give the target product (13.8 g, 58% yield). [M+H] ⁺ 342.1. ¹ HNMR (400MHz, CDCl ₃ )δ10.21(s,1H),8.53-8.43(m,1H),7.40-7.31(m,1H),7.10-7.02(m,1H),6.95(s,1H),6.89-6.81(m,1H),2.68-2.54(m,4H),1.27(s,6H).

Intermediate I-4

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

Hydrogen gas was bubbled 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 the reaction was carried out at room temperature for 8 hours. The reaction mixture was filtered, the filtrate was collected, and the pH was adjusted to 8 with concentrated ammonia. Extraction with ethyl acetate and concentration under vacuum were performed to give 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

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

Intermediate I-5

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

Step 1: 5,5-Dimethyl-1,4,5,6-tetrahydrocyclopentadiene[b]pyrrole-2-carboxylhydrazine

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

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

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

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

Under nitrogen protection, 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) were added to a solution of 7,7-dimethyl-7,8-dihydro-2H-cyclopentadieno[4,5]pyrrolo[1,2-d][1,2,4]triazine-1(6H)-one (3.25 g, 16.0 mmol), 2-bromo-4-chloronicotinaldehyde, and 1,4-dioxane (60 mL). The mixture was reacted at 80 °C for 4 hours and then cooled to room temperature. The mixture was concentrated under vacuum, and the residue was purified by silica gel column chromatography to give the target product (3.43 g, 63% yield). [M+H] ⁺ 343.1

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

Sodium borohydride (0.19 g, 5.0 mmol) was added to a solution of 4-chloro-2-(7,7-dimethyl-1-oxo-1,6,7,8-tetrahydro-2H-cyclopentadieno[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) at 0–5 °C under nitrogen protection, and the reaction was carried out for 10 min at this temperature. A saturated aqueous solution of ammonium chloride (10 mL) was added to the reaction mixture, and the mixture was extracted with dichloromethane (80 mL x 2). The combined organic phases were collected and concentrated under vacuum to obtain the target product (3.33 g, 97% yield), which was used directly in the next reaction. [M+H] ⁺ 345.1

Step 5: Acetic acid (4-chloro-2-(7,7-dimethyl-1-oxo-1,6,7,8-tetrahydro-2H-cyclopentadieno[4,5]pyrrolo[1,2-d][1,2,4]triazin-2-yl)pyridin-3-yl)methyl ester

Acetyl chloride (11.4 g, 145 mmol) was added to a solution of 2-(4-chloro-3-(hydroxymethyl)pyridin-2-yl)-7,7-dimethyl-7,8-dihydro-2H-cyclopentadieno[4,5]pyrrolo[1,2-d][1,2,4]triazine-1(6H)-one (3.33 g, 9.7 mmol) and triethylamine (3.91 g, 38.6 mmol) in dichloromethane (60 mL) at 0–5 °C under nitrogen protection, and the reaction was carried out at this temperature for 1 hour. Water (30 mL) and dichloromethane (80 mL) were added to the reaction solution, and the organic phases were collected and combined. The mixture was concentrated under vacuum, and the residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate) to give the target product (2.84 g, 76% yield). [M+H] ⁺ 387.1

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

Under nitrogen protection, Xphos (0.35 g, 0.73 mmol), Pd(dppf)Cl₂CH₂Cl₂ (0.60 g, 0.73 mmol), and potassium acetate (2.16 g, 22.0 mmol) were added to a solution of 4-chloro-2-(7,7-dimethyl-1-oxo-1,6,7,8-tetrahydro-2H-cyclopentadieno[4,5]pyrrolo[1,2-d][1,2,4]triazin- _{2-yl)pyridin-3 -} yl)methyl acetic acid (2.84 g, 7.3 mmol), _{pinacol diboronate} (5.59 g, 22.0 mmol), and 1,4-dioxane (200 mL). 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 target product (2.55 g, yield 88%). [M+H] ⁺ 397.1

Following steps 4-6 of the preparation of intermediate I-5, the intermediates listed in the table below were prepared using intermediate I-3 and the corresponding raw materials and reagents:

Compound 1

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

Step 1: 2'-(7,7-dimethyl-1-oxo-1,6,7,8-tetrahydro-2H-cyclopentadieno[4,5]pyrrolo[1,2-a]pyrazin-2-yl)-5-((5-((2S,6S)-2,6-dimethyl-4-(oxetane-3-yl)piperazin-1-yl)pyridin-2-yl)amino)-1-methyl-6-oxo-1,6-dihydro-[3,4'-bispyridine]-3'-carboxaldehyde

Under nitrogen protection, Xphos (9 mg, 0.02 mmol), Pd(dppf)Cl₂CH₂Cl₂ (16 mg, 0.02 mmol), and cesium carbonate (130 mg, 0.40 mmol) were added to a solution of intermediate I-1 (99 mg, 0.20 mmol) and intermediate I- ₃ (68 mg, 0.20 mmol) in 3 _mL of 1,4 _- dioxane and 0.2 mL of water. The mixture was reacted at 90 °C for 2 hours and then cooled to room temperature. The mixture was filtered, and the filtrate was collected and concentrated under vacuum to obtain the target product, which was used directly in the next reaction. [M+H] ⁺ 675.3

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

Sodium borohydride (7 mg, 0.20 mmol) was added to a methanol (0.5 mL) and dichloromethane (5 mL) solution of 2'-(7,7-dimethyl-1-oxo-1,6,7,8-tetrahydro-2H-cyclopentadieno[4,5]pyrrolo[1,2-a]pyrazin-2-yl)-5-((5-((2S,6S)-2,6-dimethyl-4-(oxetane-3-yl)piperazin-1-yl)pyridin-2-yl)amino)-1-methyl-6-oxo-1,6-dihydro-[3,4'-bispyridine]-3'-carboxaldehyde at 0–5 °C under nitrogen protection. The reaction was carried out at room temperature for 5 minutes. Water (0.5 mL) was added to the reaction solution, and the mixture was concentrated under vacuum. The residue was purified by silica gel column chromatography (methanol/water) and thin-layer chromatography (methanol/dichloromethane = 1/20) to obtain the target product (74 mg, yield 55%). [M+H] ⁺ 677.4. ¹ HNMR (400 MHz, CD ₃₎ OD): 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),4.6 4-4.57(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).

Following the preparation steps of compound 1, and using the corresponding intermediates and reagents, the compounds listed in the table below were prepared:

Compound 4

2-(5-((5-((2S,6S)-2,6-dimethyl-4-(oxetane-3-yl)piperazin-1-yl)pyridin-2-yl)amino)-3'-(hydroxymethyl)-1-methyl-6-oxo-1,6-dihydro-[3,4'-bispyridin]-2'-yl)-7,7-dimethyl-7,8-dihydro-2H-cyclopentadieno[4,5]pyrrolo[1,2-d][1,2,4]triazine-1(6H)-one

Under nitrogen protection, Xphos (31 mg, 0.066 mmol), Pd(dppf)Cl₂CH₂Cl₂ (27 mg, 0.033 mmol), and potassium phosphate trihydrate (264 mg, 0.99 mmol) were added to a solution of 5-bromo-3-((5-(((2S,6S)-2,6-dimethyl-4-(oxetane-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 in the preparation method 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). 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 residue was purified by silica gel column chromatography (methanol/water).

The obtained solid ([M+H] ⁺ 720.3) was dissolved in methanol (3 mL), and potassium carbonate (137 mg, 0.99 mmol) was added. The reaction mixture was reacted at room temperature for 2 hours. The reaction solution was concentrated under vacuum, and the residue was purified by silica gel column chromatography (methanol/water) and thin-layer chromatography (methanol/dichloromethane = 1/20) to obtain the target product (30 mg, yield 13%). [M+H] ⁺ 678.3. ^1H NMR (400 MHz, CD3 ₎ OD): 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),2.67-2.61(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).

Following the preparation steps of compound 4, and using the corresponding intermediates and reagents, the compounds listed in the table below were prepared:

Compound 5

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'-bispyridin]-2'-yl)-7,7-dimethyl-7,8-dihydro-2H-cyclopentadieno[4,5]pyrrolo[1,2-a]pyrazin-1(6H)-one

Compound 5a (500 mg, 0.69 mmol) was dissolved in trifluoroacetic acid (5 mL) and stirred at room temperature for 30 minutes. The solution was concentrated under vacuum, and the residue was dissolved in methanol (5 mL). Triethylamine (1 mL) was added, and the solution was concentrated again under vacuum. The residue was purified by silica gel column chromatography (methanol/water) to give the target product (340 mg, 79% yield). [M+H] ⁺ 621.4. ^1H NMR (400 MHz, CD3 ₎ OD)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),4.6 4-4.58(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).

Compound 6

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'-bispyridin]-2'-yl)-7,7-dimethyl-7,8-dihydro-2H-cyclopentadieno[4,5]pyrrolo[1,2-a]pyrazin-1(6H)-one

To a methanol (5 mL) solution of compound 5 (200 mg, 0.32 mmol), 1.2 mL of aqueous formaldehyde solution was added, and the mixture was stirred at room temperature for 5 minutes. Sodium borohydride (38 mg, 1.0 mmol) was then added, and the mixture was stirred at room temperature for 30 minutes. The reaction mixture was purified by silica gel column chromatography (methanol/water) to give the target product (78 mg, 38% yield). [M+H] ⁺ 635.3. ^1H NMR (400 MHz, CD3 ₎ OD)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).

Example 2: Determination of Biochemical BTK

1. Reagents and Materials

BTK recombinant protein: Invitrogen, catalog number PV3363;

Kinase Assay Kit - Tyrosine 1-peptide: Invitrogen, Catalog No. PV3190;

384-hole low flange black flat bottom polystyrene NBS microplate, no cap, no sterilization: Corning, item number 3575;

96-well polystyrene conical bottom MicroWell ^™ plate with cap: Thermo Scientific ^™ Nunc ^™ , part number 277143;

Envision Multimode Plate Reader: PerkinElmer;

Vibrating plate apparatus: Eppendorf;

TS-2102 Shaking Incubator: TENSUC;

2. Methods

Biochemical assays utilize fluorescence resonance energy transfer (FRET) technology, employing a dual-enzyme approach and different sensitivities to phosphorylated and non-phosphorylated short peptide protein cleavage. Two fluorescent groups are labeled at both ends of the short peptide substrate, forming a FRET pair. In the primary reaction (enzymatic reaction), the enzyme transfers a γ-phosphate group from ATP to a serine or threonine residue on the short peptide substrate. In the second step (chromogenic reaction), a site-specific protease (chromogenic reagent) recognizes and cleaves the non-phosphorylated short peptide. Phosphorylated short peptides inhibit this cleavage. Cleavage disrupts the donor (e.g., coumarin) and acceptor fluorescent groups (fluorescein) on the short peptide, while phosphorylated short peptides retain FRET. The ratio is calculated as follows: the ratio of the emission signals generated by the donor fluorescent group after excitation at 400 nm to the recipient. Emission signal ratio = Coumarin emission (445 nm) / Fluorescein emission (520 nm). If the FRET peptide is phosphorylated (e.g., without a kinase inhibitor), the emission ratio will remain low; if the FRET peptide is non-phosphorylated (e.g., with a kinase inhibitor), the emission ratio will be higher. This is used to differentiate the inhibitory effects of different compound inhibitors on BTK kinase activity.

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

Experimental Procedure: This experiment included three control groups, each with eight replicates: C1 100% inhibition group (no ATP), C2 0% inhibition group (ATP added), and C3 100% phosphorylation group. Add 2.5 μl of serially diluted compound to each well of a 384-well plate, in duplicate. Add 4% DMSO solution to wells C1, C2, and C3. Then, remove well C3, and add 2.5 μl of BTK enzyme solution to each of the remaining wells. Incubate at 4°C for 30 minutes. Next, remove well C3, and add 2.5 μl of short peptide solution to each well. Add 5 μl of phosphorylated short peptide solution to each well of C3. Add 2.5 μl of 1.33× kinase buffer to each of wells C1 and C3, and add 2.5 μl of ATP solution to each of the remaining wells. Centrifuge briefly at 1000 rpm for 30 seconds. Incubate the 384-well plate in a shaking incubator at room temperature for 1 hour, protected from light. After the enzyme reaction is complete, add 5 μl of colorimetric solution to each well, centrifuge briefly, shake the plate at 1000 rpm for 30 seconds, and centrifuge briefly again. Place the 384 plate in a shaking incubator in the dark and incubate at room temperature for 1 hour to complete the colorimetric reaction.

3. Testing

After color development, the 384-well plate was removed and read using an Envision multi-mode plate reader. The emission wavelength was 405nm, and the excitation wavelengths were 460nm/535nm. The optical signal was detected. The 460nm/535nm reading for each well was taken as the signal value for each well.

4. Calculation

The phosphorylation rate of short peptides in the presence of BTK kinase was calculated using the average signal value of C3 as 100% phosphorylation, the average signal value of C1 as 0% phosphorylation, and the average signal value of C2. The inhibition rate (%) of each compound concentration was calculated based on the signal value of each well, and the _IC50 value was obtained using the 205 model in XL-Fit 5.3 software (ID Business Solutions Limited).

The phosphorylation rate is calculated as follows:

Phosphorylation rate (%) = 100 - 100 × [(Emit signal ratio × F _100% ) - C _100% ] / {(C _0% - C _100% ) + [Emit signal ratio × (F _100% - F _0% )]}

Wherein, the emission signal ratio = coumarin emission signal (460nm) / fluorescein emission signal (535nm); _C100% = average coumarin emission signal of C3; C0 _% = average coumarin emission signal of C1; _F100% = average fluorescein emission signal of C3; F0 _% = average fluorescein emission signal of C1.

The inhibition rate is calculated as follows:

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

5. Test Results

Compound numbering <![CDATA[IC₅₀(μM)]]> 1 0.010 2 0.007 3 0.003 4 0.005 5 0.008 6 0.007

Example 3

Assay of phosphorylated BTK in Ramos cells

1. Reagents and Materials

Ramos cells: Ramos cells were purchased from the American Standard Biological Collection Center (ATCC) cell bank and cultured in PRMI 1640 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, with 10% fetal bovine serum (FBS) added. The cells were cultured normally in a cell culture incubator at 5% _CO2 and 37°C.

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

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

Hank's Balanced Salt Solution (HBSS): GIBCO, Catalog No. 14025-092;

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

3% hydrogen peroxide (3% _{H₂O₂ )} : Sigma, catalog number 88597-100ML-F;

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

Microplate reader: Envision, Perkin Elmer;

384-well plate, CulturPlate™ 384: Perkin Elmer, part number 6007680

96-well plate: Corning, part number 3799.

2. Methods

Ramos cells were starved for 2 hours in PRMI 1640 medium with 1% FBS. After starvation, the Ramos cells were diluted with Hank's balanced salt solution to 5.0 x ^10⁶ cells/ml and seeded into 96-well plates at 20 μL/well (1.0 x ^10⁵ cells/well). The cells were then cultured in a cell culture incubator at ₃₇ °C with 5% CO₂. After 1 hour of incubation, the test compounds were serially diluted 4-fold with Hank's balanced salt solution to the corresponding concentrations. Then, 5 μL/well of the diluted test compounds at different concentrations (final concentrations of 3.0, 0.75, 0.188, 0.047, 0.012, 0.0029, 0.0007, and 0.00018 μM, final DMSO concentration of 0.3%, double replicates) or 5 μL/well of control solution (1.5% DMSO, 8 replicates) was added to a 20 μL/well cell culture system and incubated for 1 hour. Then, 5 μL/well of a mixture of human immunoglobulin M diluted with Hank's balanced salt solution (final concentration of 10 μg/mL) and hydrogen peroxide (final concentration of 3.3 mM) was added to the test compound treatment wells and the anti-human immunoglobulin M control treatment wells, and 5 μL/well of Hank's balanced salt solution was added to the negative control treatment wells. The mixture was then incubated at 5% CO2 _. Incubate in a cell culture incubator at 37°C for 10 minutes.

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

3. Testing

The 384-well plate, incubated overnight in a constant temperature incubator, was removed and the light signal was detected using an Envision microplate reader at an emission wavelength of 320 nm and an excitation wavelength of 665 nm/615 nm. The signal value for each well was obtained by multiplying (665 nm reading/615 nm reading) by ^10⁴ .

4. Calculation

The average signal value of wells containing a mixture of human immunoglobulin M (final concentration 10 μg/mL) and hydrogen peroxide (final concentration 3.3 mM) but without the analyte compound was used as the high value, and the average signal value of wells without human immunoglobulin M stimulation and without the analyte compound was used as the low value. The inhibition rate (%) of each compound concentration was calculated based on the signal value of each well, and then the _IC50 value was obtained using the 205 model in XL-Fit 5.3 software (ID Business Solutions Limited).

The inhibition rate is calculated as follows:

Inhibition rate (%) = 100% - {(analyte treatment wells - negative control treatment wells) / (anti-human immunoglobulin M control treatment wells - negative control treatment wells)} × 100%, where,

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

Anti-human immunoglobulin M control wells: These represent the signal values of Ramos cells treated with anti-human immunoglobulin M and hydrogen peroxide but without the target compound.

Negative control wells: These represent the signal values of Ramos cells that were not stimulated with immunoglobulins and did not contain the test compound.

5. Test Results

Compound numbering <![CDATA[IC₅₀(μM)]]> 1 0.005 2 0.006 3 0.003 4 0.003 5 0.008 6 0.007

Example 4: Determination of B cell activity in whole blood of rats

1. Reagents and Materials

Peripheral whole blood from female Wistar rats;

Phosphate-buffered saline solution PBS: GIBCO, catalog number C20012500BT;

Anti-rat B220 PE antibody (PE anti rat B220): eBioscience, catalog number 12-0460-82;

Anti-rat CD86 FITC antibody: eBioscience, catalog number 11-0860-82;

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

Fixative (IC fixation buffer): Invitrogen, part number 00-8222-49;

96-hole U-shaped base plate: Nunc, part number 163320;

96-hole V-shaped base plate: Nunc, part number 49952;

Dimethyl sulfoxide (DMSO): Sigma-Aldrich, catalog number 34869-4L;

Anti-rat immunoglobulin D (Mouse anti-rat IgD): Bio-rad, catalog number MCA190;

Flow cytometer: BD FACS Canto II, BD.

2. Methods

For compound activity assays, 80 μL of rat peripheral whole blood was added to each well of a 96-well plate and incubated in a cell culture incubator at 37°C and 5% _CO2 . After half an hour, the test compound was diluted with PBS and serially diluted 3-fold to the corresponding concentrations. Then, 10 μL of the diluted test compound was added to each well of the rat whole blood culture system (final concentrations of test compound were 1.0, 0.33, 0.11, 0.037, 0.012, 0.0041, 0.0014, and 0.0005 μM, final concentration of DMSO was 0.3%, double replicates) or 10 μL of control solution (0.3% DMSO, 6 replicates) was added to the corresponding wells and incubated in a cell culture incubator for one hour. Then add 10 μL/well of anti-rat immunoglobulin D diluted with PBS (final concentration of 10 μg/mL) to the test compound treatment well and the anti-rat immunoglobulin D control treatment well, or 10 μL/well of PBS to the negative control treatment well, mix well, and continue to incubate in a cell culture incubator at 37°C with ₅ % CO2 for 18 hours.

The next day, the 96-well plate was removed, and a flow cytometry antibody mixture diluted with PBS (final concentration of anti-rat B220PE antibody 1 μg/mL and final concentration of anti-rat CD86 FITC antibody 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 the red blood cells. The mixture was shaken for 20 minutes, centrifuged to remove the supernatant, and then washed, fixed, and analyzed by flow cytometry.

3. Testing

The activation of B cells in the sample was determined by flow cytometry.

4. Calculation

The average proportion of activated B cells in wells containing anti-rat immunoglobulin D but without the test compound was used as the anti-rat immunoglobulin D control wells, and the average proportion of activated B cells in wells without immunoglobulin D stimulation and without the test compound was used as the negative control wells. The inhibition rate (%) for each concentration was calculated based on the B cell activation rate in each well, and then the _IC50 value was obtained using the 205 model in XL-Fit 5.3 software (ID Business Solutions Limited).

The inhibition rate is calculated as follows:

Inhibition rate (%) = 100% - {(analyte treatment wells - negative control treatment wells) / (anti-rat immunoglobulin D control treatment wells - negative control treatment wells)} × 100%, where,

Test compound treatment wells: indicate the activation rate of B cells in whole blood of rats treated with anti-rat immunoglobulin D and the test compound.

Anti-rat immunoglobulin D control wells: These indicate the activation rate of B cells in whole blood of rats treated with anti-rat immunoglobulin D but without the test compound.

Negative control wells: indicate the activation rate of B cells in whole blood of rats that were free of the test compound and not stimulated by immunoglobulins.

Based on the above tests, the compounds of the present invention exhibit strong inhibitory activity against B cell activation in rat whole blood. Specifically, compound 1 has an _IC50 value of 0.001 μM.

Example 5: Liver Microsome Stability Test

1. Experimental materials:

Mixed liver microsomes from male CD-1 mice and mixed liver microsomes from male SD rats were purchased from Bioreclamation IVT, Inc., USA.

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

2. Solution preparation:

10mM Stock Solution of Test Compound: Weigh a certain amount of the test compound, dissolve it in an appropriate volume of DMSO, and prepare a stock solution with a concentration of 10mM for later use.

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

3. Experimental methods:

The stock solution of the test compound is diluted to 0.1 mM (the concentration of the compound in the final reaction system is 1 μM) with an organic solvent (usually a mixture of acetonitrile, methanol and water in different proportions, depending on the solubility of the compound; if necessary, 1 N hydrochloric acid or 1 N sodium hydroxide is added to aid dissolution). The concentration of the organic solvent in the incubation system should not exceed 1% (of which the proportion of 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 preparation, it is pre-incubated in a 37°C water bath for 10 minutes and then placed on ice for later use as the NADPH regeneration solution. A 20 mg/mL liver microsome solution was mixed with 200 mM phosphate buffer and diluted with ultrapure water to obtain a liver microsome solution containing 2.5 mg/mL liver microsomes (final concentration in the reaction system was 0.5 mg/mL) and 50 mM phosphate buffer. The diluted liver microsome solution was mixed with 0.1 mM of the compound solution, and an appropriate volume of 100 mM EDTA, 300 mM _MgCl₂ solution, 200 mM phosphate buffer (final system was 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 started in a 37°C water bath for 30 minutes. The reaction was terminated by adding ice-cold acetonitrile containing an internal standard. The 0-minute sample was not incubated in a 37°C water bath, and unlike the 30-minute sample, the ice-cold acetonitrile containing an internal standard was added first, followed by the NADPH regeneration solution. After the sample containing the internal standard solution for terminating the reaction was vortexed and mixed, it was centrifuged at 4400 rpm for 10 minutes. The supernatant was then diluted tenfold with 50% methanol and analyzed by LC-MS/MS.

4. Analytical Methods:

The concentrations of compounds in the samples were determined using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The percentage of remaining compounds after 30 minutes of incubation was calculated, using the peak area ratio of the compound to the internal standard as an indicator, compared to the sample at 0 minutes, to evaluate the metabolic stability of the compounds.

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

Chromatographic column: Waters XSELECT Hss T3 C ₁₈ (2.5μm, 2.1×50mm) or CAPCELLPAK MG (5μm, 2.0×50mm)

Mobile phase:

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

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

The test results are shown in the table below:

Compound numbering 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%

*RLM, rat liver microsomes.

MLM stands for mouse liver microsomes.

Example 6: Pharmacodynamic evaluation of BTK target inhibition in vivo

Objective: To investigate the inhibitory effect of the compound of the present invention on B cell activation in vivo by inducing activation of B cells in mouse whole blood with anti-IgD antibody, thereby determining the in vivo BTK target inhibitory effect of the compound of the present invention.

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

Table 1. In vivo administration grouping information

After administration of the drug, animals in each group were anesthetized in _CO2 at a predetermined time, and blood was collected from the inner canthus of the eye, anticoagulated with heparin. 90 μL of whole blood from each mouse group was added to a 96-well culture plate, with anti-mouse IgD antibody (BIO-RAD, catalog number MCA4693) added to each well to a final concentration of 0.01 μg/μL (for each drug treatment group and the anti-IgD antibody-induced solvent group, respectively). Separately, 90 μL of whole blood from the solvent group mice was added to the same 96-well culture plate, with PBS (phosphate-buffered saline, GIBCO, catalog number C20012500BT) added to each well to a final concentration of 0.01 μg/μL (serving as the solvent control group). All groups were thoroughly mixed and incubated at 37°C/5% _CO2 for 4 hours. Blood from the drug treatment group mice was centrifuged to separate plasma for blood drug concentration analysis.

Add fluorescently labeled antibodies Anti-CD19-APC (BD Biosciences, catalog number 550992) and Anti-CD69-PE (BD Biosciences, catalog number 553237) to the cultured whole blood, mix well, and incubate at room temperature in the dark for 30 minutes. Transfer 50 μL of the sample to a 96-well deep V-type culture plate containing 380 μL of freshly prepared lysis buffer (BD Biosciences, catalog number 555899), vortex, and incubate at room temperature in the dark for 15 minutes to remove red blood cells. Cells were added to 400 μL of flow cytometry buffer (2% FBS//PBS, FBS: fetal bovine serum, GIBCO, catalog number 100100-147; PBS: GIBCO, catalog number C20012500BT), and centrifuged at 1200 rpm for 8 minutes at 4°C. The supernatant was discarded, and the cell clumps were washed twice with flow cytometry buffer and centrifuged again. The cells were then resuspended in 400 μL of flow cytometry buffer, and the expression of CD69+ in CD19+ positive cells (B cells) was detected and the data were analyzed using a BDFACS LSR Tortessa flow cytometer.

Calculation of B cell activation rate:

B cell activation rate = Percentage of CD69+CD19+ double-positive B cells / Percentage of CD19+ single-positive B cells

Calculation of inhibition rate:

Inhibition rate = (Percentage of B cell activation in the anti-IgD antibody-induced solvent group - Percentage of B cell activation in the drug-treated group) / (Percentage of B cell activation in the anti-IgD antibody-induced solvent group - Percentage of B cell activation in the solvent control group) × 100%

All data are expressed as mean ± standard error. Comparisons between each drug treatment group and the anti-IgD antibody-induced solvent group were performed using Graphpad Prism with one-way ANOVA and Dunnett's test to calculate p-values. Comparisons between drug treatment groups were performed using unpaired t-tests to calculate p-values.

Results: The experimental results are shown in Figure 1 and Table 2.

In this experiment, 16 hours after administration, GDC-0853 at 20 mg/kg showed a 9% inhibition rate of B cell activation; compound 2 of the present invention showed a 43% inhibition rate of B cell activation at a dose of 20 mg/kg. Compound 1 of the present invention showed a 60% inhibition rate of B cell activation at a dose of 5 mg/kg, which was statistically significant compared with the anti-IgD antibody-induced solvent group.

Table 2. Effects of in vivo administration of anti-IgD antibody-induced B cell activation in mouse whole blood.

#### indicates that compared with the solvent control group, p<0.0001;

* indicates p < 0.05 compared to the anti-IgD antibody-induced solvent group.

Example 7: Therapeutic effect of the compounds of the present invention on a rat model of type II collagen-induced arthritis.

1. Research Methods

Weigh an appropriate amount of bovine type II collagen (CII, Chondrex (Redmond, WA, USA), Cat#20021) and dissolve it in 0.1 mol acetic acid (SPGC Sinopharm Chemical Reagent Co., Ltd (Shanghai, P.R. China), Cat#:10000218.) to prepare a solution with a concentration of 6 mg/mL. Stir overnight at 4°C, then add an equal volume of Freund's incomplete adjuvant (Sigma-Aldrich. (St. Louis, MO, USA), Cat#:SLBW0366.) and emulsify thoroughly to prepare an emulsion with a CII concentration of 3 mg/mL.

Female Lewis rats were purchased from Vital River Laboratory Animal Technology Co., Ltd. (Certificate No. 20200928Aazz0619000579, initial weight 110-130 g). Six rats were randomly selected as the normal control group, and the remaining rats were used for immunization to establish the Lewis rat model. On day 0, the rats (excluding the normal control 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. A second challenge was performed on day 7, with the same intradermal injection of 0.2 mL of the emulsion. After the animals developed symptoms on day 10, their condition was closely monitored. After day 13, the average foot volume of the model animals was between 1.5 and 1.7 ml. The model animals were then randomly grouped and administered the drugs according to Table 3.

Table 3. Grouping information for drug administration during modeling.

After grouping, the normal group received no medication, while the other groups of rats were administered the control solvent, reference GDC-0853 at doses of 0.25 mg/kg and 4 mg/kg, and compound 1 orally once daily until the end of the experiment. The grouping and administration regimens are shown in Table 3.

Foot volume was measured starting on day 10 post-immunization, and the left and right hind foot volumes (V) were measured daily after an increase in foot volume was detected.

The foot volume of each animal's left and right hind limb joints was measured, and the average foot volume (APV) was calculated using the following formula:

Average Foot Volume (APV) = (V _left + V _right ) / 2

The effect of the drug on mean paw volume was analyzed using GraphPad with repeated measure ANOVA and Dunnett's multiple comparison test for significance. p-values were calculated, with ^### p < 0.001 indicating a highly significant difference from the normal group, *p < 0.05 indicating a statistically significant difference from the solvent control group, and **p < 0.01 indicating a highly significant difference from the solvent control group. The mean paw volume before administration was taken as baseline (or considered as 100% inflammation suppression). Average paw swelling (APS) was calculated for each animal using the following formula, where APV _d1 is the mean paw volume on day 1 after administration, and APV _dt is the mean paw volume on day t after administration:

The average volume change APS _dt = (APV _dt – APV _d1 )

The area under the curve (AUC) for the change in average foot volume is the area under the curve for the change in joint score calculated using the trapezoidal method. The formula is as follows:

AUC _APS = 1/2×(APS _d1 +APS _d2 )×(d ₂ -d ₁ )+1/2×(APS _d2 +APS _d3 )×(d ₃ -d ₂ )+…+1/2×(APS _dn +APS _d(n-1) )×(d _n -d _n-1 ).

Formula for calculating the area under the curve (IR _AUC ):

Inhibition rate (IR) _AUC % = (Average AUC _APS of model group - AUC _APS of drug-treated group) / (Average AUC _APS of model group - Average AUC _APS of normal group) × 100%

The ED ₅₀ was calculated using XLfit software based on the area under the curve (AUC) of the mean foot volume change, with the selected model being "log(inhibitor) vs. response-variable slope".

2. Results

Lewis rats developed the disease on day 10 after their first immunization with bovine type II collagen. As the disease progressed, paw volume gradually increased. A statistically significant difference in paw volume was observed between the solvent control group and the normal group ( ^p <0.001). GDC-0853-0.25 mg/kg did not improve paw volume growth in rats, while GDC-0853-4 mg/kg significantly reduced paw volume compared to the solvent control group (p<0.05). Once-daily oral administration of compound 1 solution at doses of 0.06, 0.25, and 4 mg/kg QD inhibited paw swelling in a dose-dependent manner, with inhibition rates (IR _AUC ) of 76.2%, 83.1%, and 200.2%, respectively; the lowest effective dose was 0.06 mg/kg/day. Compound 1 at 0.25 mg/kg (inhibition rate 83.1% AUC) showed statistically significant differences compared to the same dose of GDC-0853 (inhibition rate -6.4%), and compound 1 at 4 mg/kg (inhibition rate 200.2% AUC) showed statistically significant differences compared to the same dose of GDC-0853 (inhibition rate 144.7%), both significantly improving the sustained improvement in foot volume swelling (p<0.01, one-way repeated ANOVA, Graphpad test). Results are shown in Figure 2.

Example 8: The therapeutic effect of the compound of the present invention on idiopathic thrombocytopenic purpura induced by CD41 antibody.

1. Research Methods

Male C57BL/6 mice were purchased from Shanghai Lingchang Biotechnology Co., Ltd. (Certificate No. 20180003011079, initial weight 18-20 g) and randomly divided into groups of 8 mice each, according to Table 4. Before modeling, mice were administered single doses as shown in Table 4 at different time points: intraperitoneal injection of 2000 mg/kg IVIg of the positive control drug (Rongsheng, Lot#:201604B026), oral administration of 40 mg/kg PRN1008 (rilzabrutinib), and different doses of compound 1. At modeling time, each mouse was intraperitoneally injected with 200 μL of PBS solution containing 2 μg of anti-mouse CD41 antibody (BD, Cat#:553487, Lot#:7026765).

Table 4. Grouping information for drug administration during modeling

Eight hours and 24 hours after modeling, whole blood was collected and placed in centrifuge tubes pre-filled with 10% citrate-phosphate-dextrose-adenine (CPDA). Platelet levels (PLT) in the whole blood were measured using an XT-2000i (SYSMEX) automated blood analyzer from Shanghai Laboratory Animal Research Centre Sino-British SIPPR/B&K Lab Animal Ltd.

The mean platelet level in peripheral blood was statistically analyzed using Graphpad software using one-way ANOVA and Fisher's LSD multiple comparison test. p-values were calculated, with *p<0.05 indicating a statistically significant difference compared to the solvent control group, **p<0.01 indicating a highly statistically significant difference compared to the solvent control group, and ##p<0.01 indicating a highly statistically significant difference compared to the normal group.

The platelet recovery rate (RR) is calculated using the following formula:

RR%=(PLT _treatment –PLT _model )/(PLT _naive –PLT _model )×100%.

2. Results

Eight hours after intraperitoneal injection of anti-mouse CD41 antibody, platelet levels in peripheral blood of C57BL/6 mice were measured. The mean platelet levels of the solvent control group and the normal group were compared, showing a statistically significant difference (p<0.01). Intraperitoneal injection of 2 g/kg IVIg significantly restored platelet levels compared to the solvent control group (p<0.01), with a recovery rate of 54%. Oral administration of 40 mg/kg PRN1008 significantly restored platelet levels compared to the solvent control group (p<0.01), with a recovery rate of 40%. Single oral administration of compound 1 solution at doses of 0.004, 0.04, 0.4, and 4 mg/kg dose-dependently restored the anti-mouse CD41 antibody-induced decrease in platelet count, with recovery rates of 25%, 37%, 44%, and 51%, respectively; the lowest effective dose was 0.04 mg/kg.

Twenty-four hours after intraperitoneal injection of anti-mouse CD41 antibody, platelet levels in peripheral blood of C57BL/6 mice were measured. The mean platelet levels of the solvent control group and the normal group were compared, showing a statistically significant difference (##, p<0.01). Intraperitoneal injection of 2 g/kg IVIg significantly restored platelet levels compared to the solvent control group (**p<0.01), with a recovery rate of 53%. Oral administration of 40 mg/kg PRN1008 did not significantly restore mean platelet levels compared to the solvent control group, with a recovery rate of only 16%. Single oral administration of compound 1 solution at doses of 0.004, 0.04, 0.4, and 4 mg/kg dose-dependently restored the anti-mouse CD41 antibody-induced decrease in platelet count, with recovery rates of 5%, 21%, 29%, and 29%, respectively. Results are shown in Figure 3.

Claims (19)

1. Compound of formula (I): Or a pharmaceutically acceptable salt thereof, or a racemic mixture thereof, enantiomer or diastereomer, wherein: _X1 is CH or N; _X2 is CH; X ₃ is N; U and V are each independently CR ₉ ; _Y1 and _Y2 are each independently CR ₁₀ ; _R1 and _R2 are independently selected from _C1-6 alkyl and _C1-6 deuterated alkyl, respectively; _R3 is hydrogen or deuterium; _R4 is -( _C1-3 alkyl)-OH, wherein the _C1-3 alkyl is optionally substituted with one or more deuterium; _R5 is _C1-6 alkyl, wherein the _C1-6 alkyl is optionally substituted with one or more deuterium; _Z1 , _Z2 , _Z3 and _Z4 are independently CH or N, provided that one of _Z1 , _Z2 , _Z3 and _Z4 is N; _R6 and _R7 are each independently selected from _C1-6 alkyl groups; _R8 is hydrogen, _C1-6 alkyl or 4-6 membered heterocyclic group, wherein the _C1-6 alkyl or 4-6 membered heterocyclic group is optionally substituted with one or more deuterium; wherein the 4-6 membered heterocyclic group comprises one or two 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 of 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 of any one of claims 1-2, or a pharmaceutically acceptable salt thereof, or a racemic mixture, enantiomer or diastereomer thereof, wherein _Y1 and _Y2 are both _CR10 and _R10 is hydrogen. 4. The compound of any one of claims 1-2, 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 of any one of claims 1-2, or a pharmaceutically acceptable salt thereof, or a racemic mixture, enantiomer or diastereomer thereof, wherein _R3 is hydrogen. 6. The compound of any one of claims 1-2, or a pharmaceutically acceptable salt thereof, or a racemic mixture, enantiomer or diastereomer thereof, wherein _R4 is -( _C1-3 alkyl)-OH or -( _C1-3 deuterated alkyl)-OH. 7. The compound of any one of claims 1-2, or a pharmaceutically acceptable salt thereof, or a racemic mixture, enantiomer or diastereomer thereof, wherein _R3 is hydrogen and _R4 is -( _C1-3 alkyl)-OH. 8. The compound of any one of claims 1-2, or a pharmaceutically acceptable salt thereof, or a racemic mixture, enantiomer or diastereomer thereof, wherein U and V are both CH. 9. The compound of any one of claims 1-2, or a pharmaceutically acceptable salt thereof, or a racemic mixture, enantiomer or diastereomer thereof, wherein _R5 is a _C1-6 alkyl group. 10. The compound of any one of claims 1-2, 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 of any one of claims 1-2, or a pharmaceutically acceptable salt thereof, or a racemic mixture, enantiomer or diastereomer thereof, wherein _R6 and _R7 are both methyl. 12. The compound of any one of claims 1-2, or a pharmaceutically acceptable salt thereof, or a racemic mixture, enantiomer or diastereomer thereof, wherein R ₈ is an oxacyclobutane 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 any one of the compounds of claims 1-14 and/or a pharmaceutically acceptable salt thereof, and optionally comprising a pharmaceutically acceptable excipient. 16. A method for in vitro inhibition of BTK activity for non-therapeutic and non-diagnostic purposes, comprising contacting an effective amount of any of the compounds of claims 1-14 and/or a pharmaceutically acceptable salt thereof with BTK. 17. A pharmaceutical combination comprising any one of the compounds of claims 1-14 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: anti-inflammatory agents, immunomodulators, or antitumor active agents. 19. The pharmaceutical combination of claim 18, wherein the antitumor active agent is selected from chemotherapeutic agents, immune checkpoint inhibitors or agonists, and targeted therapeutic agents.