CN108191871B

CN108191871B - Novel Bruton's tyrosine kinase inhibitor and preparation method and application thereof

Info

Publication number: CN108191871B
Application number: CN201810002152.3A
Authority: CN
Inventors: 李英富; 黄浩喜; 刘冠锋; 陈垌珲; 任俊峰; 易守兵; 苏忠海
Original assignee: Chengdu Haibo Rui Pharmaceutical Co Ltd; CHENGDU BRILLIANT PHARMACEUTICAL CO LTD
Current assignee: Chengdu Beite Pharmaceutical Co ltd; Scinnohub Pharmaceutical Co Ltd
Priority date: 2018-01-02
Filing date: 2018-01-02
Publication date: 2020-02-18
Anticipated expiration: 2038-01-02
Also published as: CN108191871A

Abstract

The invention relates to a reversible Bruton's tyrosine kinase inhibitor, which comprises a compound shown as a formula (I) and a stereoisomer, a hydrate, a solvate, a pharmaceutically acceptable salt, a eutectic crystal or a prodrug thereof, a preparation method of the compound, and a method and application for inhibiting BTK kinase activity and mutant BTK kinase activity by using the novel compound.

Description

Novel Bruton's tyrosine kinase inhibitor and preparation method and application thereof

Technical Field

The invention belongs to the field of pharmaceutical chemistry, and particularly relates to a novel reversible inhibitor compound of Bruton's tyrosine kinase, a preparation method of the compound, and a method and application for inhibiting BTK kinase activity and mutant BTK kinase activity by using the novel compound.

Background

Btk is a non-receptor Tec family cytoplasmic tyrosine kinase expressed in hematopoietic cells (including myeloid cells and B cells, but not T cells), and plays an important role in B cell maturation and activation, and is highly expressed in B cell malignancies.Btk inhibitor ibrutinib has demonstrated clinical efficacy in a range of B cell malignancies, including Chronic Lymphocytic Leukemia (CLL), relapsed or refractory Mantle Cell Lymphoma (MCL), macroglobulinemia (WM), etc. BTK inhibitors can inhibit the production of B cell autoantibodies and cytokines in addition to B cell lymphomas and leukemias, in autoimmune diseases, B cells present autoantigens, promote the activation and secretion of inflammatory factors by T cells to cause tissue damage, while activating B cells to produce large amounts of antibodies, triggering autoimmune responses.T and B cells interact to form positive feedback regulatory chains, leading to an exacerbation of autoimmune responses, histopathological damage, studies have shown that regulatory B cells in vivo secrete interleukin 10(IL-10) or interleukin 10(β) to inhibit systemic inflammatory responses such as systemic lupus erythematosus, Systemic Lupus Erythematosus (SLE) inflammatory disease, systemic lupus erythematosus, and other immune response-mediated by systemic inflammatory diseases (SLE) drugs such as SLE 1-mediated by TGF-mediated immune response.

Although the covalent irreversible binding mechanism of btk (ibrutinib) is clinically effective, it also results in a series of toxic side effects, with atrial fibrillation, diarrhea, rash, joint pain and bleeding in the clinic, and clinical data show that some patients are susceptible to Progressive Disease (PD) after drug withdrawal, such as Richter Syndrome (RS). In addition, as ibrutinib is widely used, drug resistance of patients also appears, and the generation reason is mainly that cysteine at the site of BTK481 is mutated into serine (C481S), and the mutation of Cys481 blocks the covalent irreversible binding reaction of Btk with the ibrutinib and other inhibitors, so that the inhibition effect of BTK inhibitors (ibrutinib) on mutant BTK is greatly reduced, and the drug resistance is generated.

Although ibrutinib treatment is effective, a considerable part of patients with clinical B-cell lymphoma are not sensitive to treatment except for a part of patients who develop resistance at a later stage, for example, about 1/3 patients in MCL do not respond to treatment, and the response rate in DLBCL is not high, and doctors and patients need additional treatment options in view of the above problems.

Therefore, the invention provides a selective non-covalent reversible BTK inhibitor different from ibrutinib, and the non-covalent structure can inhibit BTK C481S mutation and wild type BTK inhibition. The invention reports a preferable non-covalent BTK inhibitor, and aims to provide a BTK inhibitor with good curative effect, good tolerance or low toxic and side effect, and application thereof in preparing medicines for autoimmune diseases, inflammatory diseases and cancers. The inflammatory diseases include rheumatoid arthritis, atopic dermatitis, etc., and the autoimmune diseases include systemic lupus erythematosus. The cancer is leukemia or lymphoma, etc.

There are currently reports of non-covalently reversible BTK inhibitors:

WO-2017103611 discloses a reversible Btk inhibitor having the general structure:

the description in this patent is not considered part of this patent, since the general structure is quite different from that of the present invention.

WO-2017046604 discloses a reversible Btk inhibitor having the general structure:

Disclosure of Invention

The invention relates to a reversible Bruton's tyrosine kinase inhibitor, which has the following structure:

a reversible bruton's tyrosine kinase inhibitor comprising a compound of formula (i) and stereoisomers, hydrates, solvates, pharmaceutically acceptable salts, co-crystals or prodrugs thereof:

a is optionally selected from the group consisting of

Is absent; wherein

Wherein the N-terminal is connected to Q and the C-terminal is connected to a pyrazinoimidazole group;

wherein the 4-carbon atom is attached to the pyrazinoimidazole group and the 2-carbon atom is attached to Q;

wherein N is attached to Q and C is attached to a pyrazinoimidazole group;

q is selected from R₁,-COR₂,-SOR₃,-SO₂R₄,-NHCONH(R₅)，-NHCOR₆，-(CH₂)nCONHR₇Wherein R is₁，R₂，R₃,R₄，R₅，R₆，R₇Is substituted or unsubstituted C₁～C₆Alkyl radical, C₃～C₆Cycloalkyl, 3-to 6-membered heterocycloalkyl, C₅～C₆Aryl, 5-to 6-membered heteroaryl; the substituent is 1-4C₁～C₆Alkyl radical, C₃～C₆Cycloalkyl, 3-to 6-membered heterocycloalkyl, hydroxy-substituted C₁～C₆Alkyl, hydroxy, amino, cyano, nitro, isocyano, halogen, ═ O, trifluoromethyl; the heterocycloalkyl or heteroaryl group contains 0-3 heteroatoms N, S or O;

n＝1～3；

when A is absent, Q is-COR₂；

R₈Is hydrogen, substituted or unsubstituted C₁～C₆Alkyl radical, C₃～C₆Cycloalkyl, 3-to 6-membered heterocycloalkyl, C₅～C₆Aryl, 5-to 6-membered heteroaryl; the substituent is 1-4C₁～C₆Alkyl radical, C₃～C₆Cycloalkyl, 3-to 6-membered heterocycloalkyl, hydroxy-substituted C₁～C₆Alkyl, hydroxy, amino, cyano, nitro, isocyano, halogen, ═ O, trifluoromethyl; the heterocycloalkyl or heteroaryl group contains 0-3 heteroatoms N, S or O;

alternatively, when R₈And Q is ortho, and R₈Is selected from C₁～C₆Alkyl groups, optionally together with the carbon atom to which they are attached, and the phenyl ring form a five-or six-membered ring substituted by carbonyl groups;

the B ring is selected from a substituted or unsubstituted 5-6-membered aromatic ring or a heteroaromatic ring, the heteroaromatic group contains 0-3 heteroatoms N, S or O, and the substituent is methyl, ethyl, isopropyl, hydroxyl, amino, cyano, nitro, isocyano, halogen or trifluoromethyl;

l is selected from O, S, CONH or CH₂；

The D ring is selected from a substituted or unsubstituted 5-6-membered aromatic ring or a heteroaromatic ring, the heteroaromatic group contains 0-3 heteroatoms N, S or O, and the substituent is methyl, ethyl, isopropyl, hydroxyl, amino, cyano, nitro, isocyano, halogen or trifluoromethyl;

in a preferred embodiment of the present invention, a compound represented by the general formula (II) or a stereoisomer, hydrate, solvate, pharmaceutically acceptable salt, co-crystal or prodrug thereof, wherein the reversible bruton's tyrosine kinase inhibitor according to claim 1 is characterized by the following structure:

wherein X is C-H or N;

in a preferred embodiment of the present invention, a compound represented by the general formula (II) or a stereoisomer, hydrate, solvate, pharmaceutically acceptable salt, co-crystal or prodrug thereof, wherein L is O or CONH, and NH is linked to the compound when L is CONHTo the carbon atom ortho to X;

the invention discloses a compound shown in a general formula (II) or a stereoisomer, a hydrate, a solvate, a pharmaceutically acceptable salt, a eutectic crystal or a prodrug thereof, wherein when X is C-H, L is O; when X is N, L is CONH; NH is attached to when L is CONHOn a carbon atom ortho to the middle pyridine;

the invention provides a compound shown in general formulas (III), (IV), (V), (VI) and (VII) or a stereoisomer, a hydrate, a metabolite, a solvate, a pharmaceutically acceptable salt, a eutectic crystal or a prodrug thereof:

wherein Q₁Is selected from R₁，-COR₂,-SOR₃,-SO₂R₄,；

Q₂Is selected from R₁,-COR₂,-NHCONH(R₅)，-NHCOR₆，-(CH₂)nCONHR₇；

Q₃，Q₄,Q₅Is selected from-COR₂；

R₁，R₂，R₃，R₄，R₅，R₆，R₇Is substituted or unsubstituted C₁～C₆Alkyl radical, C₃～C₆Cycloalkyl, 3-to 6-membered heterocycloalkyl, C₅～C₆Aryl, 5-to 6-membered heteroaryl; the substituent is 1-4C₁～C₆Alkyl radical, C₃～C₆Cycloalkyl, 3-to 6-membered heterocycloalkyl, hydroxy-substituted C₁～C₆Alkyl, hydroxy, amino, cyano, nitro, isocyano, halogen, ═ O, trifluoromethyl; the heterocycloalkyl or heteroaryl group contains 0-3 heteroatoms N, S or O;

n＝1～3；

in a preferred embodiment of the present invention, the present invention provides a compound represented by the general formula (III), (IV), (V), (VI), (VII) or a stereoisomer, hydrate, solvate, pharmaceutically acceptable salt, co-crystal or prodrug thereof:

wherein Q₁Is selected from R₁，-COR₂；

Q₃，Q₄，Q₅Is selected from-COR₂；

Wherein R is₁，R₂，R₃，R₄Is substituted or unsubstituted C₁～C₆Alkyl radical, C₃～C₆Cycloalkyl, 3-to 6-membered heterocycloalkyl, C₅～C₆Aryl, 5-to 6-membered heteroaryl; the substituent is 1-4C₁～C₆Alkyl radical, C₃～C₆Cycloalkyl, 3-6 membered heterocycloalkyl, hydroxy-substituted C₁～C₆Alkyl, hydroxy, amino, cyano, nitro, isocyano, halogen, ═ O, trifluoromethyl; the heterocycloalkyl or heteroaryl group contains 0-3 heteroatoms N, S or O;

n＝1；

R₅selected from substituted or unsubstituted aryl, heteroaryl, said substituent being C₁～C₆Alkyl, hydroxy, amino, cyano, halo, ═ O, trifluoromethyl; the heterocycloalkyl or heteroaryl group contains 0-3 heteroatoms N, S or O;

R₆selected from substituted or unsubstituted aryl, heteroaryl, said heteroaryl comprising 0-3 heteroatoms N, S or O; the substituent is 1-3C₁～C₆Alkyl radical, C₃～C₆Cycloalkyl, hydroxy, amino, cyano, nitro, isocyano, halogen, ═ O, trifluoromethyl;

R₇selected from substituted aryl, the substituent being C₁～C₆Alkyl radical, C₃～C₆Cycloalkyl, hydroxy, amino, cyano, nitro, isocyano, halogen, ═ O, trifluoromethyl;

R₈selected from hydrogen, substituted or unsubstituted C₁～C₆Alkyl radical, C₃～C₆A cycloalkyl group; the substituent is 1-4C₁～C₆Alkyl radical, C₃～C₆Cycloalkyl, 3-to 6-membered heterocycloalkyl, hydroxy-substituted C₁～C₆Alkyl, hydroxy, amino, cyano, nitro, isocyano, halogen, ═ O, trifluoromethyl;

alternatively, when R₈And Q is ortho, and R₈Is selected from C₁～C₆When alkyl, the carbon atoms to which they are attached may be selected to form, together with the phenyl ring, an oxo-substituted benzo five-or six-membered ring;

in a preferred embodiment of the invention, the invention relates to a compound selected from, but not limited to:

according to a specific embodiment of the present invention, the compound of the present invention or its stereoisomer, hydrate, solvate, pharmaceutically acceptable salt, co-crystal or prodrug is selected from hydrochloride, hydrobromide, sulfate, phosphate, acetate, trifluoroacetate, thiocyanate, maleate, hydroxymaleate, glutarate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, benzoate, salicylate, phenylacetate, cinnamate, lactate, malonate, pivalate, succinate, fumarate, malate, mandelate, tartrate, gallate, gluconate, laurate, palmitate, pectate, picrate, citrate or a combination thereof, preferably, the salt is selected from hydrochloride, hydrobromide, sulfate, or a combination thereof, preferably, the salt is selected from hydrochloride, hydrobromide, sulfate, or a combination thereof, Phosphate, acetate, maleate, mesylate, besylate, p-toluenesulfonate, benzoate, salicylate, cinnamate, lactate, malonate, succinate, fumarate, malate, tartrate, citrate, or combinations thereof

The invention also provides application of the compound or a stereoisomer, a hydrate, a solvate, a pharmaceutically acceptable salt, a eutectic crystal or a prodrug thereof in preparation of pharmaceutical preparations for treating inhibition of Bruton's tyrosine kinase, in particular application in preparation of pharmaceutical preparations for treating and/or preventing hyperproliferative diseases

The invention also provides the use of the compound or a stereoisomer, hydrate, solvate, pharmaceutically acceptable salt, cocrystal or prodrug thereof in the preparation of medicaments for treating autoimmune diseases, inflammatory diseases and cancers.

The invention also provides the application of the compound or the stereoisomer, the hydrate, the solvate, the pharmaceutically acceptable salt, the eutectic crystal or the prodrug thereof in rheumatoid arthritis.

Preferably, the invention also provides the use of the compound or the stereoisomer, the hydrate, the solvate, the pharmaceutically acceptable salt, the cocrystal or the prodrug thereof in the treatment of the systemic lupus erythematosus.

The invention also provides the use of the compound or the stereoisomer, the hydrate, the solvate, the pharmaceutically acceptable salt, the eutectic crystal or the prodrug thereof in atopic dermatitis.

Preferably, the invention also provides the use of the compound or the stereoisomer, hydrate, solvate, pharmaceutically acceptable salt, eutectic crystal or prodrug thereof in leukemia or lymphoma.

Unless stated to the contrary, the terms used in the specification and claims have the following meanings.

Carbon, hydrogen, oxygen, sulfur, nitrogen or halogen referred to in the groups and compounds of the present invention all include isotopes thereof, and carbon, hydrogen, oxygen, sulfur or nitrogen referred to in the groups and compounds of the present invention are optionally further replaced by one or more of their corresponding isotopes, wherein isotopes of carbon include 12C, 13C and 14C, isotopes of hydrogen include protium (H), deuterium (D, also called deuterium), tritium (T, also called deuterium), isotopes of oxygen include 16O, 17O and 18O, isotopes of sulfur include 32S, 33S, 34S and 36S, isotopes of nitrogen include 14N and 15N, isotopes of fluorine include 17F and 19F, isotopes of chlorine include 35Cl and 37Cl, and isotopes of bromine include 79Br and 81 Br.

"alkyl" means a straight or branched chain saturated aliphatic hydrocarbon group containing 1 to 20 carbon atoms, preferably an alkyl group of 1 to 8 carbon atoms, more preferably an alkyl group of 1 to 6 carbon atoms, non-limiting examples of which include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, n-pentyl, 2-pentyl, 3-pentyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 3-methyl-3-pentyl, 2, 3-dimethyl-2-butyl, 3-dimethyl-2-butyl, n-heptyl, n-octyl, and various branched chain isomers thereof; the alkyl group can be optionally further substituted by 0 to 4 groups selected from F, Cl, Br, I, hydroxyl, nitro, cyano, isocyano, hydroxyalkyl, carbocyclyl, heterocyclic group, substituted or unsubstituted 5-to 6-membered aromatic or heteroaromatic ring, wherein the heteroaromatic group contains 0 to 3 heteroatoms N, S or O, and the substituent is methyl, ethyl, isopropyl, hydroxyl, amino, cyano, halogen or trifluoromethyl.

"heterocyclyl" means a substituted or unsubstituted saturated or unsaturated aromatic or non-aromatic ring which may be a 3 to 8 membered monocyclic, 4 to 12 membered bicyclic or 10 to 15 membered tricyclic ring system and contain 1 to 3 heteroatoms selected from N, O or S, preferably a 3 to 6 membered heterocyclyl, the optionally substituted N, S in the ring of the heterocyclyl may be oxidized to various oxidation states. The substituent is 0-4C₁～C₆Alkyl radical, C₃～C₆Cycloalkyl, 3-6 membered heterocycloalkyl, hydroxy-substituted C₁～C₆Alkyl, hydroxy, amino, cyano, halo, ═ O, trifluoromethyl; non-limiting examples include the following structures

"carbocyclyl" refers to a saturated or unsaturated aromatic or non-aromatic ring which may be a 3-to 10-membered monocyclic, 4-to 12-membered bicyclic, or 10-to 15-membered tricyclic ring system, preferably a 3-to 6-membered ring system

The term "halogen" means F, Cl, Br or I;

as used herein, the term "oxo" means an oxygen bonded to a carbon atom with a double bond, thereby forming a carbonyl group

By "pharmaceutically acceptable salt" or "pharmaceutically acceptable salt thereof" is meant a salt of a compound of the invention that retains the biological effectiveness and properties of the free acid or free base obtained by reaction with a non-toxic inorganic or organic base, and the free base obtained by reaction with a non-toxic inorganic or organic acid. Generally, acids suitable for pharmaceutically forming salts include, but are not limited to: inorganic acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, phosphoric acid, etc., organic acids such as formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, methanesulfonic acid, phenylmethanesulfonic acid, benzenesulfonic acid, etc.; and acidic amino acids such as aspartic acid and glutamic acid.

By "prodrug" is meant a compound of the invention that is metabolically convertible in vivo to a biologically active compound. Prodrugs of the invention are prepared by modifying hydroxy groups in compounds of the invention, which modifications may be removed by routine manipulation or in vivo, to yield the parent compound. When a prodrug of the present invention is administered to a mammalian subject, the prodrug is cleaved to form a free hydroxyl group.

"cocrystal" refers to a crystal of an Active Pharmaceutical Ingredient (API) and a cocrystal former (CCF) bound by hydrogen bonding or other non-covalent bonds, wherein the API and CCF are both solid in their pure state at room temperature and a fixed stoichiometric ratio exists between the components. A co-crystal is a multi-component crystal that contains both a binary co-crystal formed between two neutral solids and a multicomponent co-crystal formed between a neutral solid and a salt or solvate.

"stereoisomers" refers to isomers resulting from the different arrangement of atoms in a molecule, including cis, trans isomers, enantiomers and conformational isomers.

"optional" or "optionally" or "selective" or "selectively" means that the subsequently described event or circumstance may, but need not, occur, and that the description includes instances where the event or circumstance occurs and instances where it does not. For example, "a heterocyclic group optionally substituted with an alkyl group" means that the alkyl group may, but need not, be present, and the description includes the case where the heterocyclic group is substituted with an alkyl group, and the case where the heterocyclic group is not substituted with an alkyl group.

The active ingredients of the invention are prepared into pharmaceutical preparations, and different preparation forms, such as solution, solid preparation and the like, can be prepared according to the physicochemical properties and the actual medication requirements of the active ingredients.

One aspect of the present invention relates to the use of a compound having the general structure (I), (II), (III), (IV), (V), (VI) or (VII) for the manufacture of a medicament for the treatment of a disorder responsive to inhibition of bruton's tyrosine kinase;

further relates to the use of compounds of formula (I), (II), (III), (IV), (V), (VI) or (VII) for the manufacture of a medicament for the treatment of autoimmune, inflammatory and cancer disorders; further relates to the use of compounds of general structure (I), (II), (III), (IV), (V), (VI) or (VII) for the preparation of a medicament for the treatment of rheumatoid arthritis, systemic lupus erythematosus, atopic dermatitis;

in some embodiments, Btk inhibitors are useful for treating diseases and disorders that can be alleviated by inhibiting (i.e., decreasing) Btk enzymatic activity. "disease" means a disease or disease symptom. Thus, the present invention provides methods of treating autoimmune disorders, inflammatory disorders, and cancer in a subject in need thereof. Such methods comprise administering to the subject a therapeutically effective amount of a Btk inhibitor.

The term "autoimmune disorder" includes diseases or disorders involving inappropriate immune responses to natural antigens, such as Acute Disseminated Encephalomyelitis (ADEM), Addison's disease, alopecia areata, antiphospholipid antibody syndrome (APS), autoimmune hemolytic anemia, autoimmune hepatitis, Bullous Pemphigoid (BP), celiac disease, dermatomyositis, type 1 diabetes, Goodpasture's syndrome, Graves 'disease, Guillain-barre syndrome (GBS), Hashimoto's disease, idiopathic thrombocytopenic purpura, lupus erythematosus, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, malignant anemia, multiple myositis, primary biliary cirrhosis, scholar syndrome (syndrom), Temporal arteritis and Wegener's granulomatosis. The term "inflammatory disorder" includes diseases or disorders involving acute or chronic inflammation, such as allergy, asthma, prostatitis, glomerulonephritis, Pelvic Inflammatory Disease (PID), inflammatory bowel disease (IBD, e.g. crohn's disease, ulcerative colitis), reperfusion injury, rheumatoid arthritis, graft rejection and vasculitis. In some embodiments, the invention provides a method of treating rheumatoid arthritis or lupus.

The term "cancer" includes diseases or disorders involving abnormal cell growth and/or proliferation, such as glioma, thyroid cancer, breast cancer, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer), gastric cancer, gastrointestinal stromal tumor, pancreatic cancer, cholangiocarcinoma, ovarian cancer, endometrial cancer, prostate cancer, renal cell carcinoma, lymphoma (e.g., anaplastic large cell lymphoma), leukemia (e.g., acute myelogenous leukemia, T-cell leukemia, chronic lymphocytic leukemia), multiple myeloma, malignant mesothelioma, malignant melanoma, and colon cancer (e.g., high microsatellite instability colorectal cancer). In some embodiments, the invention provides a method of treating leukemia or lymphoma.

In certain embodiments, the compounds of the present invention are used in medicine. In some embodiments, the compounds of the invention

The compounds of the invention are useful as kinase inhibitors. In certain embodiments, the compounds of the invention are selective inhibitors of Btk wild-type Btk kinase and mutant Btk-C481S.

In some embodiments the IC50 of the Btk inhibitor for the wild-type Btk kinase is less than 400nM, in some embodiments the IC50 of the Btk inhibitor for the mutant Btk-C481S is less than 400 nM. In some embodiments, the IC50 of the Btk inhibitor for the wild-type Btk kinase is less than 10 nM. In some embodiments, the IC50 of the Btk inhibitor against mutant Btk-C481S is 10nM to 10 uM.

In order to develop a suitable BTK inhibitor, an in vitro assay for inhibition of kinase activity by BTK-C481S can be identified. The activity of the inhibitor compounds can be determined using methods known in the art and/or those provided herein.

The Bruton tyrosine kinase inhibitor provided by the invention has stronger and almost equivalent inhibitory activity to wild BTK and C481S mutant BTK, and has extremely important significance for solving the drug resistance of the existing BTK inhibitor.

Detailed Description

The following detailed description is provided for the purpose of illustrating the embodiments and the advantageous effects thereof, and is not intended to limit the scope of the present disclosure.

The following abbreviations have the meanings indicated below:

DMF means N, N-dimethylformamide;

NBS represents N-bromosuccinimide;

DCM represents dichloromethane;

TEA represents triethylamine;

TFA represents trifluoroacetic acid;

THF represents tetrahydrofuran;

EA represents ethyl acetate;

PE represents petroleum ether;

MeOH for methanol;

TBSCl represents tert-butyldimethylsilyl chloride;

HBTU represents benzotriazole-N, N, N ', N' -tetramethyluronium hexafluorophosphate;

TLC indicated spot silica gel plate;

KOAc represents potassium acetate;

Ac₂o represents acetic anhydride

BPO represents benzoyl peroxide;

Pd(pph₃)₄represents palladium triphenylphosphine;

Pd(dppf)Cl₂represents [1,1' -bis (diphenylphosphino) ferrocene]Palladium dichloride;

EDCI represents 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride;

HOBT represents 1-hydroxybenzotriazole;

STAB represents sodium triacetoxyborohydride;

FAM represents carboxyfluorescein;

ATP represents adenosine triphosphate.

Example 1: preparation of 4- (8-amino-3- ((1R,3S,4S) -2- (2-chloropyrimidine-4-carbonyl) -2-azabicyclo [2.2.1] heptan-3-yl) imidazo [1,5-a ] pyrazin-1-yl) -N- (pyridin-2-yl) benzamide

The synthesis steps are as follows:

step 1: preparation of tert-butyl (1R,3R,4S) -3- (((3-chloropyrazin-2-yl) methyl) carbamoyl) -2-azabicyclo [2.2.1] heptane-2-carboxylate

To a reaction vessel containing (3-chloropyrazin-2-yl) methylamine (3.43g, 24mmol), (1R,3R,4S) -2- (tert-butoxycarbonyl) -2-azabicyclo [2.2.1] under nitrogen protection]Heptane-3-carboxylic acid (5.80g, 20mmol), HOBt (4.21g,31.2mmol) and TEA (4.37g, 43.2mmol) in 30mL DMF (0 ℃) were added portionwise EDCI (5.97g, 31.2mmol), the reaction mixture was stirred at room temperature overnight, TLC showed completion of the reaction of the starting material, water was added to quench the reaction, EA was extracted (50mL × 3), the organic phase was back-washed with saturated saline, anhydrous Na₂SO₄After drying thoroughly, evaporation in vacuo and purification by column chromatography (PE/EA 5/1-3/1) 8.0g of the title compound was obtained as a brown solid.

Step 2: preparation of tert-butyl (1R,3S,4S) -3- (8-chloroimidazo [1,5-a ] pyrazin-3-yl) -2-azabicyclo [2.2.1] heptane-2-carboxylate

Charging (1R,3R,4S) -3- (((3-chloropyrazin-2-yl) methyl) carbamoyl) -2-azabicyclo [2.2.1] under ice salt bath]A mixed solution of tert-butyl heptane-2-carboxylate (6.3g, 17.18mmol) in DMF/EA (7.5mL/50mL) was slowly added dropwise POCl₃(12.6mL, 103.08mmol), after addition, the reaction mixture was stirred at room temperature for 2h and TLC showed complete reaction of starting materials, Na was slowly added to the reaction mixture₂CO₃(6mol/L), keeping pH above 8, separating organic phase, extracting aqueous phase with EA (20mL x 3), combining the above organic phases with anhydrous Na₂SO₄Drying was carried out thoroughly, evaporated in vacuo and purified by column chromatography (PE/EA-3/1) to give 5.6g of the title compound as a yellow solid.

And step 3: preparation of tert-butyl (1R,3S,4S) -3- (1-bromo-8-chloroimidazo [1,5-a ] pyrazin-3-yl) -2-azabicyclo [2.2.1] heptane-2-carboxylate

Charging (1R,3S,4S) -3- (8-chloroimidazo [1, 5-a) in ice salt bath]Pyrazin-3-yl) -2-azabicyclo [2.2.1]NBS (2.66g, 14.9mmol) was added in portions to a solution of tert-butyl heptane-2-carboxylate (4.95g, 14.19mmol) in 50mL DMF and the reaction mixture was stirred for 1h in an ice salt bath, after TLC showed completion of the starting material reaction, the reaction was slowly added to NaHCO₃(1mol/L) quenching the reaction, extraction with EA (20 mL. times.3), washing the organic phase with saturated NaCl, anhydrous Na₂SO₄Drying thoroughly, evaporation in vacuo and purification by column chromatography (PE/EA-5/1) gave 5.2g of the title compound as a pale yellow solid.

And 4, step 4: preparation of tert-butyl (1R,3S,4S) -3- (8-amino-1-bromoimidazo [1,5-a ] pyrazin-3-yl) -2-azabicyclo [2.2.1] heptane-2-carboxylate

15mL of 2-BuOH and 30mL of aqueous ammonia were added to an autoclave charged with tert-butyl (1R,3S,4S) -3- (1-bromo-8-chloroimidazo [1,5-a ] pyrazin-3-yl) -2-azabicyclo [2.2.1] heptane-2-carboxylate (4.4g, 10.29mmol) at room temperature, the reaction mixture was stirred at 90 ℃ for 15h, TLC showed that the starting materials were completely reacted, the reaction solution was concentrated in vacuo to give a crude solid, which was slurried with EA/PE (5/1) to give 3.2g of the title compound as a pale yellow solid.

And 5: preparation of (1R,3S,4S) -3- (8-amino-1- (4- (pyridin-2-ylcarbamoyl) phenyl) imidazo [1,5-a ] pyrazin-3-yl) -2-azabicyclo [2.2.1] heptane-2-carboxylic acid tert-butyl ester

Under the protection of nitrogen, the mixture is charged with (1R,3S,4S) -3- (8-amino-1-bromoimidazo [1,5-a ]]Pyrazin-3-yl) -2-azabicyclo [2.2.1]Heptane-2-carboxylic acid tert-butyl ester (3.5g, 8.57mmol), N- (pyridin-2-yl) -4- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) benzamide (3.33g, 10.28mmol), Na₂CO₃(1.82g, 17.14mmol) of a mixed dioxane/EtOH/water solution (36mL/12mL/12mL), Pd (PPh) was added₃)₄(496.89mg, 0.43mmol), the reaction mixture was stirred at 90 ℃ overnight, TLC showed complete reaction of the starting materials, water was added to the reaction solution to quench, EA (40 mL. times.3) was used for extraction, the organic phase was back-washed with saturated brine, and anhydrous Na₂SO₄Drying, evaporation in vacuo and column chromatography (DCM/MeOH-60/1) afforded 2.8g of the title compound as a pale yellow solid.

Step 6: preparation of 4- (8-amino-3- ((1R,3S,4S) -2-azabicyclo [2.2.1] heptan-3-yl) imidazo [1,5-a ] pyrazin-1-yl) -N- (pyridin-2-yl) benzamide

To the reaction vessel is charged with (1R,3S,4S) -3- (8-amino-1- (4- (pyridin-2-ylcarbamoyl) phenyl) imidazo [1,5-a]Pyrazin-3-yl) -2-azabicyclo [2.2.1]To a solution of tert-butyl heptane-2-carboxylate (2.98g, 5.68mmol) in DCM (20mL) was added TFA (3.5mL), the reaction mixture was stirred at room temperature overnight, and after TLC showed complete reaction of the starting materials, the reaction was concentratedBy using Na₂CO₃(3mol/L) to pH 8, extracting with DCM/MeOH (10/1), and separating the organic phase with anhydrous Na₂SO₄Drying, evaporation in vacuo and purification by column chromatography (DCM/MeOH 60/1-10/1) afforded 2.0g of the title compound as a white solid.

And 7: preparation of 4- (8-amino-3- ((1R,3S,4S) -2- (2-chloropyrimidine-4-carbonyl) -2-azabicyclo [2.2.1] heptan-3-yl) imidazo [1,5-a ] pyrazin-1-yl) -N- (pyridin-2-yl) benzamide

To the flask was charged 2-chloro-4-pyrimidinecarboxylic acid (16mg, 0.10mmol), 4- (8-amino-3- ((1R,3S,4S) -2-azabicyclo [2.2.1] under nitrogen protection]Heptane-3-yl) imidazo [1,5-a]To a solution of pyrazin-1-yl) -N- (pyridin-2-yl) benzamide (43mg, 0.10mmol) and TEA (20mg, 0.20mmol) in 1mL THF was added HBTU (57mg, 0.15mmol), the reaction mixture was stirred at room temperature overnight, TLC showed completion of the starting material reaction, water was added to quench the reaction, EA was extracted (5 mL. times.3), the organic phase was back-washed with saturated saline, anhydrous Na₂SO₄Dry well, evaporate in vacuo and purify on silica gel plate (DCM/EA ═ 1/1) to give 21mg of the title compound as a yellow solid.

The product structure was characterized by nuclear magnetic resonance and mass spectrometry with the following results:

¹H NMR(400MHz,d₆-DMSO)δ1.43-1.51(1H,m),1.78-1.83(3.5H,m),1.97-2.00(0.5H,m),2.31-2.34(0.5H,m),2.66-2.69(1H,m),2.75-2.77(0.5H,m),4.78(0.5H,s),4.92(0.5H,s),5.22(0.5H,s),5.47(0.5H,s),6.08-6.17(2H,m),7.09-7.20(2H,m),7.54-7.58(1.5H,m),7.70-7.76(2H,m),7.84-7.89(1H,m),7.98(0.5H,d,J＝4.8Hz),8.11-8.17(2.5H,m),8.21-8.24(0.5H,m),8.41(1H,d,J＝4.0Hz),8.66(0.5H,d,J＝4.8Hz),8.94(0.5H,d,J＝4.8Hz),10.85(1H,s).

EM (calculated): 565.2; MS (ESI) M/e (M +1H)⁺：566.2。

It can be seen that the compounds prepared herein are structurally identical to the compounds in the above reaction schemes.

EXAMPLE 2 preparation of 4- (8-amino-3- ((1R,3S,4S) -2- (2, 3-dihydroxypropyl) -2-azabicyclo [2.2.1] hept-3-yl) imidazo [1,5- α ] pyrazin-1-yl) -N- (pyridin-2-yl) benzamide

To a solution of 4- (8-amino-3- ((1R,3S,4S) -2-azabicyclo [2.2.1] heptan-3-yl) imidazo [1,5-a ] pyrazin-1-yl) -N- (pyridin-2-yl) benzamide (43mg, 0.10mmol) in 1mL of methanol under nitrogen was added glycidol (15mg, 0.20mmol), the reaction mixture was stirred at 70 ℃ for 5h, the reaction was directly spun dry and then purified by silica gel plate (DCM/MeOH ═ 20/1) to give 13mg of the title compound as a yellow solid.

¹H NMR(400MHz,d₆-DMSO)δ1.24-1.26(1H,m),1.42-1.50(1H,m),1.66-1.68(2H,m),1.86-1.96(2H,m),2.38-2.50(2.3H,m),2.87-2.92(0.7H,m),3.27-3.32(2H,m),3.42-3.48(2H,m),3.72(1H,s),4.50-4.52(1H,m),4.89-4.91(1H,m),6.13-6.18(2H,m),7.08-7.12(1H,m),7.17-7.20(1H,m),7.67-7.78(3H,m),7.84-7.88(1H,m),8.14(2H,d,J＝8.4Hz),8.22(1H,d,J＝8.4Hz),8.41(1H,dd,J＝4.8Hz,1.2Hz),10.83(1H,s).

EM (calculated): 499.2, respectively; MS (ESI) M/e (M +1H)⁺：500.2。

EXAMPLE 3 preparation of 4- (-amino-3- ((1R,3S,4S) -2- (oxetan-3-yl) -2-azabicyclo [2.2.1] heptan-3-yl) imidazo [1,5- α ] pyrazin-1-yl) -N- (pyridin-2-yl) benzamide

Charged with 3-oxetanone (14mg, 0.20mmol) and 4- (8-amino-3- ((1R,3S,4S) -2-azabicyclo [2.2.1]]Heptane-3-yl) imidazo [1,5-a]Pyrazin-1-yl) -N-, (Pyridine-2-yl) benzamide (43mg, 0.10mmol) in 1mL 1, 2-dichloroethane was stirred at room temperature for 1h, STAB (42mg, 0.20mmol) was added to the reaction mixture, the reaction mixture was stirred at room temperature for 2h, TLC showed complete reaction of the starting materials, water was added to quench the reaction, EA was extracted (5 mL. times.3), the organic phase was back-washed with saturated saline, anhydrous Na₂SO₄Dry well, evaporate in vacuo and purify on silica gel plate (DCM/EA ═ 2/1) to give 27mg of the title compound as a pale yellow solid.

¹H NMR(400MHz,d₆-DMSO)δ1.29(1H,d,J＝9.2Hz),1.39-1.42(1H,m),1.56-1.65(3H,m),2.15(1H,d,J＝9.2Hz),2.42(1H,s),3.49(1H,s),3.95(1H,s),4.14-4.17(1H,m),4.28-4.32(2H,m),4.48-4.54(2H,m),6.13(2H,brs),7.10(1H,d,J＝4.8Hz),7.17-7.20(1H,m),7.76(2H,d,J＝8.0Hz),7.85-7.89(1H,m),8.00(1H,d,J＝5.2Hz),8.17(2H,d,J＝8.0Hz),8.23(1H,d,J＝8.4Hz),8.41(1H,d,J＝4.4Hz),10.87(1H,s).

EM (calculated): 481.2, respectively; MS (ESI) M/e (M +1H)⁺：482.2。

Examples 4 to 16

The following compounds are prepared by the preparation method of example 1 or example 3 by using the following compounds as raw materials, the structures and nuclear magnetic characterization data of the compounds are shown in table 1, and table 1 is a summary of the structures and structural analysis data of the compounds prepared in examples 4 to 16 of the present application.

The above reagents are all obtained by direct purchase

TABLE 1 Structure and structural analysis data for compounds prepared in examples 4-16

Example 17: preparation of 1- ((1R,3S,4S) -3- (8-amino-1- (4-phenoxyphenyl) imidazo [1,5-a ] pyrazin-3-yl) -2-azabicyclo [2.2.1] heptanepyridin-2-yl) propane-1, 2-dione

The synthesis steps are as follows:

step 1: preparation of tert-butyl (1R,3S,4S) -3- (8-amino-1- (4-phenoxyphenyl) imidazo [1,5-a ] pyrazin-3-yl) -2-azabicyclo [2.2.1] heptane-2-carboxylate

Under the protection of nitrogen, the mixture is charged with (1R,3S,4S) -3- (8-amino-1-bromoimidazo [1,5-a ]]Pyrazin-3-yl) -2-azabicyclo [2.2.1]Heptane-2-carboxylic acid tert-butyl ester (4.08g, 10.00mmol), 4,5, 5-tetramethyl-2- (4-phenoxyphenyl) -1,3, 2-dioxolane (4.44g, 15.00mmol), Na₂CO₃(2.12g, 20.00mmol) of a mixed solution of dioxane/EtOH/water (36mL/12mL/12mL), Pd (PPh) was added₃)₄(0.58g, 0.50mmol), stirring the reaction mixture at 90 deg.C overnight, quenching the reaction solution with water, extracting with EA (50 mL. times.3), backwashing the organic phase with saturated brine, anhydrous Na₂SO₄Drying, evaporation in vacuo and purification by column chromatography (PE/EA ═ 2/1) gave 3.81g of the title compound as a pale yellow solid.

Step 2: preparation of 3- ((1R,3S,4S) -2-azabicyclo [2.2.1] hept-3-yl) -1- (4-phenoxyphenyl) imidazo [1,5-a ] pyrazin-8-amine

To the reaction vessel is charged with (1R,3S,4S) -3- (8-amino-1- (4-phenoxyphenyl) imidazo [1,5-a]Pyrazin-3-yl) -2-azabicyclo [2.2.1]To a solution of tert-butyl heptane-2-carboxylate (3.8g, 7.64mmol) in DCM (50mL) was added TFA (10mL), the reaction mixture was stirred at room temperature overnight, after TLC showed complete reaction of the starting materials, the reaction was concentrated and Na was used₂CO₃(3mol/L) to pH 8, extracting with DCM/MeOH (10/1), and separating the organic phase with anhydrous Na₂SO₄Drying, evaporation in vacuo and purification by column chromatography (DCM/MeOH ═ 20/1) afforded 2.8g of the title compound as a pale yellow solid.

And step 3: preparation of 1- ((1R,3S,4S) -3- (8-amino-1- (4-phenoxyphenyl) imidazo [1,5-a ] pyrazin-3-yl) -2-azabicyclo [2.2.1] heptanepyridin-2-yl) propane-1, 2-dione

Charging pyruvic acid (10mg, 0.11mmol), 3- ((1R,3S,4S) -2-azabicyclo [2.2.1] under nitrogen protection]Hept-3-yl) -1- (4-phenoxyphenyl) imidazo [1,5-a]HBTU (57mg, 0.15mmol) was added to a solution of pyrazin-8-amine (40mg, 0.10mmol) and TEA (20mg, 0.20mmol) in 1mL THF, the reaction mixture was stirred at room temperature overnight, TLC showed the starting materials reacted completely, water was added to quench the reaction, EA was extracted (5 mL. times.3), the organic phase was back-washed with saturated saline, anhydrous Na₂SO₄Dry well, evaporate in vacuo and purify on silica gel plate (DCM/EA-1/1) to give 33mg of the title compound as an off-white solid.

¹H NMR(400MHz,d₆-DMSO)δ1.34-1.36(0.5H,m),1.46-1.48(0.5H,m),1.60-1.62(0.5H,m),1.75-1.79(3H,m),1.80-2.00(2H,m),2.31(1.5H,s),2.55-2.59(1H,m),2.67-2.68(1H,m),4.57(0.5H,s),4.73(0.5H,s),5.03(0.5H,s),5.24(0.5H,s),6.02-6.11(2H,m),7.08-7.13(5H,m),7.15-7.19(1H,m),7.41-7.44(2H,m),7.54-7.59(2H,m),7.73(0.5H,d,J＝5.2Hz),7.86(0.5H,d,J＝5.2Hz).

EM (calculated): 467.2, respectively; MS (ESI) M/e (M +1H)⁺：468.2。

Example 18: preparation of 1- ((1R,3S,4S) -3- (8-amino-1- (4-phenoxyphenyl) imidazo [1,5-a ] pyrazin-3-yl) -2-azabicyclo [2.2.1] heptanepyridin-2-yl) propane-1, 3-diol

Charged with 1, 3-dihydroxyacetone (18mg, 0.20mmol) and 3- ((1R,3S,4S) -2-azabicyclo [ 2.2.1)]Hept-3-yl) -1- (4-phenoxyphenyl) imidazo [1,5-a]A solution of pyrazin-8-amine (40mg, 0.10mmol) in 1mL of 1, 2-dichloroethane was stirred at room temperature for 1h, then STAB (42mg, 0.20mmol) was added to the reaction mixture, and the reaction mixture was stirred at room temperature overnight. The reaction was quenched with water, extracted with EA (5 mL. times.3), the organic phase was back-washed with saturated brine, anhydrous Na₂SO₄And (5) fully drying. Evaporation in vacuo followed by purification on silica gel plate (DCM/EA ═ 2/1) afforded 5mg of the title compound as a yellow solid.

¹H NMR(400MHz,d₆-DMSO)δ1.44-1.46(1H,m),1.63-1.66(2H,m),1.71-1.73(1H,m),1.91-1.94(1H,m),2.35(1H,s),2.55-2.56(2H,m),3.07-3.10(1H,m),3.47-3.57(4H,m),3.83(1H,s),4.47(1H,dd,J＝8.8Hz,3.2Hz),4.59-4.62(1H,m),6.07(2H,brs),7.04(1H,d,J＝5.2Hz),7.11-7.13(4H,m),7.17-7.20(1H,m),7.41-7.45(2H,m),7.60(2H,d,J＝8.8Hz),7.78(1H,d,J＝4.8Hz).

EM (calculated): 471.2; MS (ESI) M/e (M +1H)⁺：472.2。

Example 19: preparation of 1- ((1R,3S,4S) -3- (8-amino-1- (4-phenoxyphenyl) imidazo [1,5-a ] pyrazin-3-yl) -2-azabicyclo [2.2.1] heptanepyridin-2-yl) propan-2-one

Under the protection of nitrogen, the mixture is charged with 3- ((1R,3S,4S) -2-azabicyclo [2.2.1]Hept-3-yl) -1- (4-phenoxyphenyl) imidazo [1,5-a]1mL CH of pyrazin-8-amine (40mg, 0.10mmol) and bromoacetone (14mg, 0.10mmol)₃Adding K into CN solution₂CO₃(28mg, 0.2mmol), the reaction mixture was stirred at 60 ℃ for one day, TLC showed the starting material to react completely, the system was filtered, the filtrate was evaporated in vacuo and purified by silica gel plate (DCM/MeOH ═ 15/1) to give 30mg of the title compound as a white solid.

¹H NMR(400MHz,d₆-DMSO)δ1.31-1.34(1H,m),1.40-1.43(1H,m),1.59-1.66(2H,m),1.87-1.91(1H,m),1.97(3H,s),2.12-2.18(1H,m),2.33-2.38(1H,m),3.36(0.4H,s),3.41(0.6H,s),3.52(1H,s),3.56(0.6H,s),3.60(0.4H,s),3.71(1H,s),5.97(2H,brs),6.95(1H,d,J＝4.8Hz),7.08-7.12(4H,m),7.15-7.21(1H,m),7.40-7.46(2H,m),7.57-7.62(2H,m),7.96-8.02(1H,m).

EM (calculated): 453.2; MS (ESI) M/e (M +1H)⁺：454.2。

Examples 20 to 23

The following compounds are prepared by the preparation method of example 17 or example 19 using the following compounds as raw materials, and the structures and nuclear magnetic characterization data of the compounds are shown in table 2, and table 2 is a summary of the structures and structural analysis data of the compounds prepared in examples 20 to 23 of the present application.

The above reagents are all obtained by direct purchase

TABLE 2 Structure and structural analysis data for the compounds prepared in examples 20-23

Example 24: preparation of 4- (3- (3-acetylphenyl) -8-aminoimidazo [1,5-a ] pyrazin-1-yl) -N- (pyridin-2-yl) benzamide

The synthesis steps are as follows:

step 1: preparation of 1- (3- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) phenyl) ethanone

Pd (dppf) Cl is added to a 300mL solution of dioxane containing 3' -bromoacetophenone (19.9g, 100.0mmol), pinacol diboron ester (25.4g, 100.0mmol) and AcOK (19.6g, 200.0mmol) under nitrogen₂(3.7g, 5.0 mmol). The reaction mixture was stirred at 80 ℃ for 4 h. The reaction solution was quenched by adding water, extracted with EA (200 mL. times.3), and the organic phase was back-washed with saturated brine and anhydrous Na₂SO₄Drying, evaporation in vacuo and purification by column chromatography (PE/EA-10/1) gave 20.6g of the title compound as an off-white solid.

Step 2: preparation of N- ((3-chloropyrazin-2-yl) methyl) formamide

A mixed solution of acetic anhydride (40mL) and formic acid (80mL) was stirred at 60 ℃ for 2h, and then 3-chloropyrazine-2-methylamine dihydrochloride (21.6g, 100.0mmol) was added to the reaction system. After the addition, the reaction mixture was stirred at 60 ℃ for 2 hours, TLC showed complete reaction of the starting materials, the reaction was concentrated to an oil, and Na was slowly added thereto₂CO₃(6mol/L), the pH is maintained above 8. Extract with EA (200 mL. times.3), combine the organic phases and use anhydrous Na₂SO₄Drying was carried out thoroughly and evaporation in vacuo gave 15.6g of the title compound as a brown solid.

And step 3: preparation of 8-chloroimidazo [1,5-a ] pyrazines

POCl was slowly dropped into a DMF/EA (25mL/150mL) mixed solution containing N- ((3-chloropyrazin-2-yl) methyl) formamide (15.0g, 87.7mmol) under a salt bath₃(26.8g, 175.4mmol), after addition, the reaction mixture was stirred at room temperature for 2h, TLC showed complete reaction of starting materials, then Na was slowly added to the reaction mixture₂CO₃(6mol/L), keeping pH above 8, separating organic phase, extracting aqueous phase with EA (100mL x 3), combining the above organic phases with anhydrous Na₂SO₄Drying was carried out thoroughly and evaporation in vacuo gave 11.0g of the title compound as a yellow solid.

And 4, step 4: preparation of 1, 3-dibromo-8-chloroimidazo [1,5-a ] pyrazine

Charging 8-chloroimidazo [1,5-a ] in ice salt bath]NBS (23.2g, 130.6mmol) was added portionwise to pyrazine (10.0g, 65.3mmol) in 100mL DMF and the reaction mixture was stirred at room temperature overnight, TLC showed the starting material reaction was completeThereafter, the reaction solution was slowly added to NaHCO₃(1mol/L) quenching the reaction, extraction with EA (100 mL. times.3), washing the organic phase with saturated NaCl, anhydrous Na₂SO₄Drying was carried out thoroughly, evaporated in vacuo and purified by column chromatography (PE/EA-5/1) to give 16.4g of the title compound as a yellow solid.

And 5: preparation of 1, 3-dibromoimidazo [1,5-a ] pyrazin-8-amine

At room temperature, 100mL of 2-BuOH and 200mL of ammonia water are added into a high-pressure reaction kettle filled with 1, 3-dibromo-8-chloroimidazo [1,5-a ] pyrazine (15g, 48.2mmol), the reaction mixture is stirred and reacted for 15h at 90 ℃, after TLC shows that the raw materials are completely reacted, the reaction liquid is concentrated in vacuum to obtain a solid crude product, and EA/PE (5/1) is used for pulping to obtain a pure product 11.7g of a target compound which is a light yellow solid.

Step 6: preparation of 1- (3- (8-amino-1-bromoimidazo [1,5-a ] pyrazin-3-yl) -phenyl) ethanone

Under the protection of nitrogen, 1, 3-dibromo imidazo [1,5-a ] is charged]Pyrazin-8-amine (292mg, 1.0mmol), 1- (3- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) phenyl) ethanone (246mg, 1.0mmol) and Na₂CO₃(212mg, 2.0mmol) of a mixed solution of dioxane/EtOH/water (3mL/1mL/1mL), Pd (dppf) Cl was added₂(73mg, 0.1 mmol). The reaction mixture was stirred at 90 ℃ for 4 h. TLC showed the reaction of the starting materials was completed, the reaction solution was quenched by adding water, extracted with EA (5 mL. times.3), and the organic phase was back-washed with saturated brine and anhydrous Na₂SO₄Drying, evaporation in vacuo gave a crude solid which was slurried with EA/PE (1/2) to give 280mg of the title compound as a brown solid.

And 7: preparation of 4- (3- (3-acetylphenyl) -8-aminoimidazo [1,5-a ] pyrazin-1-yl) -N- (pyridin-2-yl) benzamide

Charging 1- (3- (8-amino-1-bromoimidazo [1,5-a ] under the protection of nitrogen]Pyrazin-3-yl) -phenyl) ethanone (280mg, 0.85mmol), N- (pyridin-2-yl) -4- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) benzamide (247mg, 0.76mmol) and Na₂CO₃(212mg, 2.0mmol) of a mixed solution of dioxane/EtOH/water (3mL/1mL/1mL), Pd (dppf) Cl was added₂(73mg, 0.1 mmol). The reaction mixture was stirred at 90 ℃ for 4 h. TLC showed the reaction of the starting materials was completed, the reaction solution was quenched by adding water, extracted with EA (5 mL. times.3), and the organic phase was back-washed with saturated brine and anhydrous Na₂SO₄Drying, evaporation in vacuo and purification on silica gel plate (DCM/EA-1/1) afforded 135mg of the title compound as an off-white solid.

¹H NMR(400MHz,d₆-DMSO)δ2.69(3H,s),6.33(2H,brs),7.18-7.21(2H,m),7.74-7.78(1H,m),7.84-7.89(4H,m),8.09-8.15(2H,m),8.19-8.25(3H,m),8.39-8.43(2H,m),10.90(1H,s).

EM (calculated): 448.2, respectively; MS (ESI) M/e (M +1H)⁺：449.2。

Example 25: preparation of 4- (8-amino-3- (3- (1-hydroxyethyl) phenyl) imidazo [1,5-a ] pyrazin-1-yl) -N- (pyridin-2-yl) benzamide

To the mixture is charged with 4- (3- (3-acetylphenyl) -8-aminoimidazo [1,5-a]Pyrazin-1-yl) -N- (pyridin-2-yl) benzamide (45mg, 0.10mmol) in 2mL methanol was added NaBH₄(15mg, 0.4mmol), the reaction mixture was stirred at room temperature for 0.5h, TLC showed complete reaction of starting material, water was added to quench the reaction, EA extraction (5)mL. times.3), the organic phase was back-washed with saturated brine and anhydrous Na₂SO₄Dry well, evaporate in vacuo and purify on silica gel plate (DCM/MeOH ═ 20/1) to give 21mg of the title compound as a white solid.

¹H NMR(400MHz,d₆-DMSO)δ1.40(3H,d,J＝6.4Hz),4.84-4.87(1H,m),5.34(1H,d,J＝4.0Hz),6.29(2H,brs),7.17-7.21(2H,m),7.49-7.57(2H,m),7.72-7.90(6H,m),8.19-8.29(3H,m),8.42-8.43(1H,m),10.95(1H,s).

EM (calculated): 450.2 of the total weight of the mixture; MS (ESI) M/e (M +1H)⁺：451.2。

Examples 26 to 38

The following compounds were prepared by the preparation methods of example 24 or example 25 using the following compounds as raw materials, and the structures and nuclear magnetic characterization data of the compounds are shown in table 3, and table 3 is a summary of the structures and structural analysis data of the compounds prepared in examples 26 to 38 of the present application.

The above reagents are obtained by direct purchase or custom synthesis

TABLE 3 Structure and structural analysis data for compounds prepared in examples 26-38

Example 39: preparation of 1- (3- (8-amino-1- (4-phenoxyphenyl) imidazo [1,5-a ] pyrazin-3-yl) -2- (hydroxymethyl) phenyl) -3- (p-tolyl) urea

The synthetic procedure is shown below

Step 1: preparation of 1-bromo-2- (bromomethyl) -3-nitrobenzene

To a solution of 2-bromo-6-nitrotoluene (21.6g, 100.0mmol) and BPO (2.4g, 10.0mmol) in 30mL of CH at room temperature₃NBS (17.8g, 100.0mmol) was added to CN solution, the reaction mixture was stirred at room temperature for 4h, TLC showed complete reaction of the starting materials, water was added to quench the reaction, EA was extracted (30 mL. times.3), the organic phase was back-washed with saturated brine, anhydrous Na₂SO₄Drying was carried out thoroughly, evaporated in vacuo and purified by column chromatography (PE/EA-10/1) to give 24.0g of the title compound as a yellow solid.

Step 2: preparation of 2-bromo-6-nitrobenzyl acetate

A solution of 1-bromo-2- (bromomethyl) -3-nitrobenzene (20.0g, 67.8mmol) and AcOK (13.3g, 135.6mmol) in 200mL DMF was stirred at 80 ℃ for 4 h. TLC showed complete reaction of starting material, water was added to quench the reaction, EA was extracted (100 mL. times.3), and the organic phase was back-washed with saturated brine and anhydrous Na₂SO₄Drying was carried out thoroughly and evaporation in vacuo gave 14.5g of the title compound as a yellow solid.

And step 3: preparation of 2-bromo-6-nitrobenzol

A mixed solution containing 2-bromo-6-nitrobenzyl acetate (12.0g, 43.8mmol), aqueous sodium hydroxide (3M, 50mL) and 50mL of ethanol was stirred at room temperature overnight. TLC showed complete reaction of the starting materials, diluted with water, extracted with EA (50 mL. times.3), and the organic phase back-washed with saturated brine, anhydrous Na₂SO₄After thorough drying and evaporation in vacuo, 9.5g of the title compound are obtained as a yellow solid.

And 4, step 4: preparation of ((2-bromo-6-nitrobenzyl) oxy) (tert-butyl) dimethylsilane

TBSCl (5.8g, 38.5mmol) was added dropwise to a solution of 2-bromo-6-nitrobenzol (9.0g, 38.8mmol) and imidazole (5.3g, 77.6mmol) in 100mL of EDCM at room temperature, and the reaction mixture was stirred at room temperature for 5 h. TLC showed complete reaction of starting material, water was added to quench the reaction, DCM was extracted (50 mL. times.3), the organic phase was back-washed with saturated brine, anhydrous Na₂SO₄Drying was carried out thoroughly and evaporation in vacuo gave 11.4g of the title compound as a yellow solid.

And 5: preparation of 3-bromo-2- (((tert-butyldimethylsilyl) oxy) methyl) aniline

To the flask was charged ((2-bromo-6-nitrobenzyl) oxy) (tert-butyl) dimethylsilane (11.0g, 31.8mmol) and NH₄Iron powder (8.9g, 159.0mmol) was added to a solution of Cl (8.5g, 159.0mmol) in EtOH/water (100mL/20mL) and the system was stirred at 80 ℃ for 4 h. TLC showed complete reaction of the starting materials, diluted with water, extracted with EA (100 mL. times.3), and the organic phase was back-washed with saturated brine and anhydrous Na₂SO₄After thorough drying and evaporation in vacuo, 9.5g of the title compound are obtained as a yellow solid.

Step 6: preparation of 1- (3-bromo-2- (((tert-butyldimethylsilyl) oxy) methyl) phenyl) -3- (p-tolyl) urea

100mL of CH charged with 3-bromo-2- (((tert-butyldimethylsilyl) oxy) methyl) aniline (9.0g, 28.5mmol) and p-tolylene isocyanate (3.8g, 28.5mmol)₃The CN solution was stirred at room temperature overnight. TLC showed complete reaction of starting material, water was added to quench the reaction, EA was extracted (100 mL. times.4), and the organic phase was back-washed with saturated brine and anhydrous Na₂SO₄Dried well, evaporated in vacuo and purified by column chromatography (DCM/MeOH ═ 20/1) to give 6.9g of the title compound as a pale yellow solid.

And 7: preparation of 1- (2- (((tert-butyldimethylsilyl) oxy) methyl) -3- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) phenyl) -3- (p-tolyl) urea

To a solution of 1- (3-bromo-2- (((tert-butyldimethylsilyl) oxy) methyl) phenyl) -3- (p-tolyl) urea (4.5g, 10.0mmol), pinacol diboron (2.5g, 10.0mmol), AcOK (2.0g, 20.4mmol) in 50mL of dioxane under nitrogen protection was added Pd (dppf) Cl₂(0.4g, 0.5 mmol). The reaction mixture was stirred at 80 ℃ for 4 h. The reaction solution was quenched by adding water, extracted with EA (200 mL. times.3), and the organic phase was back-washed with saturated brine and anhydrous Na₂SO₄Drying, vacuum evaporation and pulping the crude product with PE to give 4.5g of the title compound as a brown solid.

And 8: preparation of 1- (3- (8-amino-1-bromoimidazo [1,5-a ] pyrazin-3-yl) -2- (((tert-butyldimethylsilyl) oxy) methyl) phenyl) -3- (p-tolyl) urea

To a mixture of 1- (2- (((tert-butyldimethylsilyl) oxy) methyl) -3- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) phenyl) -3- (p-tolyl) urea (4.0g, 8.1mmol), 1, 3-dibromoimidazo [1,5-a ] under nitrogen]Pyrazin-8-amine (2.4g, 8.2mmol) and Na₂CO₃(1.7g, 16.2mmol) of a dioxane/water (40mL/15mL) mixed solution, Pd (dppf) Cl was added₂(0.3g, 0.4 mmol). The reaction mixture was stirred at 90 ℃ for 4 h. TLC showed the reaction of the starting materials was completed, the reaction solution was quenched by adding water, extracted with EA (50 mL. times.3), and the organic phase was back-washed with saturated brine and anhydrous Na₂SO₄Drying, evaporation in vacuo and purification by column chromatography (DCM/MeOH ═ 20/1) gave 200mg of the title compound as a yellow solid.

And step 9: preparation of 1- (3- (8-amino-1- (4-phenoxyphenyl) imidazo [1,5-a ] pyrazin-3-yl) -2- (((tert-butyldimethylsilyl) oxy) methyl) phenyl) -3- (p-tolyl) urea

Charging 1- (3- (8-amino-1-bromoimidazo [1,5-a ] under the protection of nitrogen]Pyrazin-3-yl) -2- (((tert-butyldimethylsilyl) oxy) methyl) phenyl) -3- (p-tolyl) urea (190mg, 0.33mmol), 4,4,5, 5-tetramethyl-2- (4-phenoxyphenyl) -1,3, 2-dioxaborolan (97mg, 0.33mmol) and Na₂CO₃(69mg, 0.65mmol) of a dioxane/water (4mL/1.5mL) mixed solution, Pd (dppf) Cl was added₂(15mg, 0.02 mmol). The reaction mixture was stirred at 90 ℃ for 4 h. TLC showed the reaction of the starting materials was completed, the reaction solution was quenched by adding water, extracted with EA (5 mL. times.3), and the organic phase was back-washed with saturated brine and anhydrous Na₂SO₄Drying, evaporation in vacuo and purification on silica gel plate (DCM/MeOH ═ 20/1) afforded 25mg of the title compound as a yellow solid.

Step 10: preparation of 1- (3- (8-amino-1- (4-phenoxyphenyl) imidazo [1,5-a ] pyrazin-3-yl) -2- (hydroxymethyl) phenyl) -3- (p-tolyl) urea

1- (3- (8-amino-1- (4-phenoxyphenyl) imidazo [1, 5-a)]A solution of pyrazin-3-yl) -2- (((tert-butyldimethylsilyl) oxy) methyl) phenyl) -3- (p-tolyl) urea (20mg, 0.03mmol) in HCl/EA (4M, 4mL) was stirred at room temperature overnight. TLC showed the starting material was completely reacted, diluted with water and Na₂CO₃Adjusting the system to be alkalescent, extracting with DCM (5 mL. times.5), backwashing the organic phase with saturated saline solution and anhydrous Na₂SO₄Dry well, evaporate in vacuo and purify on a silica gel plate (DCM/MeOH ═ 10/1) to give 10mg of the title compound as a pale yellow solid.

¹H NMR(400MHz,d₆-DMSO)δ2.25(3H,s),4.46(2H,d,J＝4.8Hz),5.44(1H,t,J＝5.0Hz),6.21(2H,brs),7.05-710(3H,m),7.13-7.21(6H,m),7.28(1H,d,J＝4.8Hz),7.37-7.46(5H,m),7.69(2H,d,J＝8.8Hz),8.09(1H,d,J＝8.4Hz),8.53(1H,s),9.43(1H,s).

EM (calculated): 556.2, respectively; MS (ESI) M/e (M +1H)⁺：557.2。

Example 40: preparation of 1- (3- (8-amino-1- (4-phenoxyphenyl) imidazo [1,5-a ] pyrazin-3-yl) -2- (hydroxymethyl) phenyl) -3- (p-tolyl) urea

Starting with 3-bromo-acetophenone, the preparation was carried out as described in example 39, Steps 7,8,9

¹H NMR(400MHz,d₆-DMSO)δ2.68(3H,s),6.24(2H,brs),7.13-7.21(6H,m),7.43-7.47(2H,m),7.69-7.77(3H,m),7.82(1H,d,J＝5.2Hz),8.08-8.13(2H,m),8.37(1H,s).

EM (calculated): 420.2; MS (ESI) M/e (M +1H)⁺：421.2。

Example 41: preparation of 2- (3- (8-amino-1- (4-phenoxyphenyl) imidazo [1,5-a ] pyrazin-3-yl) -2-methylphenyl) -N- (p-tolyl) acetamide

Prepared by the method of steps 7,8 and 9 of example 39, using custom-made synthetic 2- (3-bromo-2-methylphenyl) -N- (p-tolyl) acetamide as a starting material

¹H NMR(400MHz,d₆-DMSO)δ2.13(3H,s),2.24(3H,s),3.82(2H,s),6.20(2H,brs),7.05-7.20(9H,m),7.34-7.37(2H,m),7.42-7.50(5H,m),7.70(2H,d,J＝8.8Hz),10.12(1H,s).

EM (calculated): 539.2, respectively; MS (ESI) M/e (M +1H)⁺：590.2。

Example 42: preparation of 4- (8-amino-3- (cyclopropanecarbonyl) imidazo [1,5-a ] pyrazin-1-yl) -N- (pyridin-2-yl) benzamide

The synthetic procedure is shown below

Step 1: preparation of (1-bromo-8-chloroimidazo [1,5-a ] pyrazin-3-yl) (cyclopropyl) methanone

Under the protection of nitrogen, 1, 3-dibromo-8-chloroimidazo [1,5-a ] is reacted]Pyrazine (311mg, 1.0mmol) in THF (5mL) was cooled to-70 deg.C, then N-butyllithium (2.5M in THF, 0.4mL, 0.1mmol) was added dropwise at this temperature, stirring was continued for 0.5h after the addition was complete, then N-methoxy-N-methylcyclopropanecarboxamide (129mg, 1.0mmol) in THF (1mL) was added dropwise to the reaction, and the reaction mixture was continued stirring at-70 deg.C for 1h after the addition was complete. Saturated NH is poured into the reaction system₄In aqueous Cl solution, EA was extracted (10 mL. times.3), the organic phase was back-washed with saturated brine and anhydrous Na₂SO₄Dried thoroughly, evaporated in vacuo and purified by column chromatography (PE/EA-2/1) to give 180mg of the title compound as a brown solid.

Step 2: preparation of (8-amino-1-bromoimidazo [1,5-a ] pyrazin-3-yl) (cyclopropyl) methanone

Under the condition of room temperature, 2mL of 2-BuOH and 4mL of ammonia water are added into an autoclave containing (1-bromo-8-chloroimidazo [1,5-a ] pyrazin-3-yl) (cyclopropyl) methanone (150mg, 0.5mmol), the reaction mixture is stirred at 90 ℃ for reaction overnight, TLC shows that the raw materials are completely reacted, the reaction solution is concentrated in vacuum to obtain a solid crude product, and EA/PE (3/1) is used for pulping to obtain a pure product 105mg of a target compound which is a light yellow solid.

And step 3: preparation of 4- (8-amino-3- (cyclopropanecarbonyl) imidazo [1,5-a ] pyrazin-1-yl) -N- (pyridin-2-yl) benzamide

Under the protection of nitrogen, filling (8-amino-1-bromoimidazo [1,5-a ]]Pyrazin-3-yl) (cyclopropyl) methanone (56mg, 0.2mmol), N- (pyridin-2-yl) -4- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) benzamide (65mg, 0.2mmol) and Na₂CO₃(42mg, 0.4mmol) of a mixed solution of dioxane/EtOH/water (1.5mL/0.5mL/0.5mL), Pd (dppf) Cl was added₂(7mg, 0.01 mmol). The reaction mixture was stirred at 90 ℃ for 4 h. TLC showed the reaction of the starting materials was completed, the reaction solution was quenched by adding water, extracted with EA (5 mL. times.3), and the organic phase was back-washed with saturated brine and anhydrous Na₂SO₄Drying, evaporation in vacuo and purification on silica gel plate (DCM/EA-1/1) afforded 29mg of the title compound as a white solid.

¹H NMR(400MHz,d₆-DMSO)δ1.20(4H,d,J＝6.0Hz),1.30(1H,s),6.55(2H,brs),7.26(1H,dd,J＝5.4Hz,7.0Hz),7.57(1H,d,J＝4.4Hz),7.90-7.96(3H,m),8.27-8.31(3H,m),8.49(1H,d,J＝4.0Hz),8.80(1H,d,J＝4.8Hz),10.99(1H,s).

EM (calculated): 398.1; MS (ESI) M/e (M +1H)⁺：399.2。

Example 43: preparation of 4- (8-amino-3- ((1S) -2- (2-chloropyrimidine-4-carbonyl) octahydrocyclopenta [ c ] pyrrol-1-yl) imidazo [1,5-a ] pyrazin-1-yl) -N- (pyridin-2-yl) benzamide

Prepared by the method of example 1 using (1S) -2- (tert-butoxycarbonyl) octahydrocyclopenta [ c ] pyrrole-1-carboxylic acid and the corresponding compound of example 1 as starting materials

¹H NMR(400MHz,d₆-DMSO)δ1.45-2.02(7H,m),2.79-2.90(1H,m),3.05-3.10(1H,m),3.57-3.69(1H,m),3.98-4.18(1H,m),5.46-5.64(1H,m),6.07-6.18(3H,m),7.07-7.20(2H,m),7.40-7.49(0.5H,m),7.67(0.5H,d,J＝8.4Hz),7.76-7.79(2H,m),7.84-7.89(1H,m),7.95(0.5H,d,J＝5.2Hz),8.13-8.17(2H,m),8.22(0.5H,dd,J＝8.4Hz,0.8Hz),8.40-8.42(1H,m),8.68(0.5H,d,J＝5.2Hz),8.94(0.5H,d,J＝5.2Hz).

EM (calculated):579.2；MS(ESI)m/e(M+1H)⁺：580.2。

Test example 1: in vitro wild-type BTK inhibition kinase Activity assay

1: the test principle is as follows:

a Mobility detection technology (Mobility-Shift Assay) of a microfluidic chip technology applies the basic concept of capillary electrophoresis to a microfluidic environment, a substrate for experiments is polypeptide with fluorescent labels, the substrate is converted into a product under the action of enzyme in a reaction system, and charges carried by the product are correspondingly changed. 2: the test method comprises the following steps:

(1) preparing a sample to be tested: diluting the reaction solution with 100% DMSO to 50 times of the final reaction concentration, namely 25 umol/L;

(2) diluting: 25umol/L is used as the initial concentration, and then diluted by 4 times of concentration, and 10 concentration gradients are diluted;

(3) adding 100% DMSO into the positive control well and the negative control well respectively;

(4) diluting the prepared compounds with 10 concentrations by 10 times with 1 time of kinase buffer solution respectively; wherein the kinase buffer solution contains 50mmol/L of hydroxyethyl piperazine ethanethiosulfonic acid with pH of 7.5, 0.01% of dodecyl polyglycol ether, 10mmol/L of magnesium chloride, and 2mmol/L of dithiothreitol;

(5) preparing 2.5 times of enzyme solution: adding kinase into 1 time of kinase buffer solution to form 2.5 times of enzyme solution;

(6) preparing 2.5 times of substrate solution: adding FAM-labeled polypeptide and ATP into 1-time kinase buffer solution to form 2.5-time substrate solution;

(7) add enzyme solution to 384-well plates: 5 mul of 5-fold compound dissolved in 10% DMSO was added to 384-well reaction plates, then 10 mul of 2.5-fold enzyme solution was added, and incubation was carried out for 10 minutes at room temperature;

(8) add substrate solution to 384-well plate: add 10. mu.l of 2.5 fold substrate solution to 384 well reaction plates;

(9) kinase reaction and termination: incubating for 1h at 28 ℃, and then adding 25 mu l of stop solution to stop the reaction; wherein the termination solution comprises hydroxyethyl piperazine ethanethiosulfonic acid with the concentration of 100mmol/L and the pH value of 7.5, 0.015 percent of dodecyl polyglycol ether, 0.2 percent of No. 3 surface reagent and 20mmol/L of ethylenediamine tetraacetic acid;

(10) reading conversion rate data on the Caliper reading data;

(11) inhibition calculations conversion data were replicated from Caliper.

The conversion was converted to inhibition data, where max refers to the conversion of the DMSO control and min is the conversion of the no enzyme live control.

Percent inhibition＝(max-conversion)/(max-min)*100.

Test example 2: in vitro mutant BTK-C481S kinase activity inhibition assay

1: the test method comprises the following steps:

1) dissolving a compound to be tested in DMSO, taking 1mM as an initial concentration, then diluting by 3-fold concentration, and carrying out 10 concentration gradients; DMSO-dissolved test compound, BTK-C481S protein, substrate Ploy E₄Y₁And ATP were diluted to 2.5-fold reaction solutions with 1 XBuffer, 1 XBuffer: 40mM Tris, 7.5; 20mM MgCl₂；0.1mg/ml BSA；2mM MnCl₂,50μM DTT

2) Adding 1.25 muL of a 2.5-time test compound containing 4% DMSO and 1.25 muL of a 2.5-time enzyme solution into a 384-well plate respectively, and incubating for 30 minutes at room temperature; then 2.5. mu.L of PolyE was added₄Y₁ATP reaction solution, and incubation is carried out for 60 minutes at room temperature;

3) add 5. mu.L ADP-Glo Reagent to 384 well plates and incubate for 40 min at room temperature;

4) add 10. mu.L of Kinase Detection Reagent to 384 well plates and incubate for 30 minutes at room temperature;

5) envision Plate-Reader reading

2: and (3) experimental verification:

the vehicle group (containing kinase, ATP, polypeptide substrate and 1% DMSO) was used as 100% phosphorinone control in the experiment; containing kinase, ATP and 1% DMSO reaction groups as 0% phosphorylation control (without polypeptide substrate); the reference compound PCI32765 was used in the experiments from Haoyuan Chemexpress co.

3: as a result:

the Envision Plate-reader reading gave the corresponding per-well chemiluminescence RLU. The original data of the test compound is RLU_DrugAs control data, RLU was used as 100% phosphorylation control₁₀₀RLU was used as the control group of 0% phosphorinone₀(ii) a For RLU_DrugPerforming a pre-treatment, i.e. deducting background values RLU₀Then, normalization is performed:

％Enzyme Activity＝(RLU_Drug-RLU₀)/(RLU₁₀₀-RLU₀)*100％

adopting Graph Pad Prism version 5.0 to perform nonlinear regression curve fitting on BTK-C481S kinase relative residual Activity (% Enzyme Activity) value of single concentration of the compound to be tested to obtain IC₅₀Value of

TABLE 4 results of the inhibitory activity of the compounds of the examples on wild-type BTK kinase and mutant BTK-C481S

As can be seen from the data in the table, examples 15, 17, 19, 23, 24 and 36 have higher inhibition effects on the kinase activities of wild-type BTK and mutant BTK-C481S. Among them, the performances of the embodiments 19 and 23 are particularly prominent: the wild-type BTK kinase inhibition of both compounds was approximately equivalent to the positive control ibrutinib; meanwhile, the mutant BTK-C481S kinase inhibition effect is better than that of the positive control GDC-0853. Therefore, the compound designed by the invention can be used as a BTK inhibitor, and can simultaneously inhibit wild BTK and mutant BTK-C481S, thereby overcoming drug resistance caused by mutation. Has wide application prospect in resisting malignant tumor.

Claims

1. A reversible bruton's tyrosine kinase inhibitor comprising a compound of formula (III):

wherein X is C-H or N, L is O or CONH, wherein when X is C-H, L is O; when X is N, L is CONH; NH is attached to when L is CONH

On a carbon atom ortho to the middle pyridine;

wherein Q₁Is selected from R₁，-COR₂；

Wherein R is₁，R₂Is substituted or unsubstituted C₁～C₆Alkyl radical, C₃～C₆Cycloalkyl, 3-to 6-membered heterocycloalkyl, C₅～C₆Aryl, 5-to 6-membered heteroaryl; the substituent is 1-4C₁～C₆Alkyl, hydroxy substituted C₁～C₆Alkyl, hydroxy, amino, cyano, nitro, isocyano, halogen, ═ O, trifluoromethyl; the heterocycloalkyl or heteroaryl group contains 0-3 heteroatoms N or O.

2. The reversible bruton's tyrosine kinase inhibitor according to claim 1, characterized by the following structure:

3. use of a compound according to any one of claims 1 or 2 in the manufacture of a medicament for the treatment of a condition responsive to inhibition of bruton's tyrosine kinase.

4. Use of a compound according to any one of claims 1 or 2 in the manufacture of a medicament for the treatment of autoimmune disorders, inflammatory disorders and cancer.

5. Use according to claim 4, selected from rheumatoid arthritis, systemic lupus erythematosus, atopic dermatitis, leukemia or lymphoma.