CN112209933B - BTK inhibitors containing 4-azacycloheptane - Google Patents

Abstract

The application belongs to the field of pharmaceutical chemistry, and relates to a BTK inhibitor containing 4-azacycloheptane, in particular to a compound shown in a formula (I) or pharmaceutically acceptable salt thereof, a preparation method thereof, a pharmaceutical composition containing the compound and application thereof in treating BTK related diseases.

Description

BTK inhibitors containing 4-azacycloheptane

Technical Field

The present application relates to BTK inhibitors containing 4-azaspiroheptane, to processes for their preparation, to pharmaceutical compositions containing them, and to their use in the treatment of BTK related diseases.

Background

Bruton's tyrosine kinase, BTK) is expressed predominantly in B cells and is distributed throughout the lymphatic, hematopoietic and blood systems, a member of the Tec family of non-receptor tyrosine kinases, which also include Tec, ITK/TSK/EMT and BMX, and is highly structurally homologous. BTK plays a critical role in connecting B cell signaling pathways of cell surface B cell receptor (B-cell receptor) stimulation to downstream intracellular responses, a key regulator of B cell development, activation, signaling and survival. Recent studies on B cells, particularly on B cell non-hodgkin's lymphoma and rheumatoid arthritis, have found that BTK tends to be aberrantly expressed.

The development of small molecule targeted drugs based on BTK signaling pathways provides a novel approach for the treatment of B cell tumors such as leukemia, multiple myeloma and B cell immune diseases. Irreversible inhibitors such as ibrutinib on the market at present have mutation on the BTK binding site, so that the drug activity is reduced to generate drug resistance, more BTK inhibitors are clinically needed, and the irreversible inhibitors have higher selectivity on BTK, so that toxic and side effects caused by off-target effects are avoided.

Detailed Description

The present application relates to compounds of formula (I) or a pharmaceutically acceptable salt thereof,

wherein, the liquid crystal display device comprises a liquid crystal display device,

ring A is selected from 5-10 membered heteroaryl or C _6-10 An aryl group;

R ¹ independently selected from halogen, hydroxy, amino, cyano, C _1-6 Alkoxy, C _1-6 Alkyl, 5-10 membered heteroaryl, 5-10 membered heterocycloalkyl, C _6-10 Aryl or C _6-10 Cycloalkyl group, the C _1-6 Alkoxy, C _1-6 Alkyl, 5-10 membered heteroaryl, 5-10 membered heterocycloalkyl, C _6-10 Aryl or C _6-10 Cycloalkyl optionally substituted by hydroxy, amino, cyano, halogen or halo C _1-6 Alkyl substitution;

m is selected from 0, 1, 2, 3, 4, 5 or 6;

l is selected from the group consisting of-C (O) NH-, -NHC (O) -, -O-, -NH-, -S-, -C (O) O-, -OC (O) -, -S (O) ₂ O-or-OS (O) ₂ -；

R ² Independently selected from halogen, hydroxy, amino, cyano, C _1-6 Alkyl or C _1-6 Alkoxy group, the C _1-6 Alkyl or C _1-6 Alkoxy is optionally substituted with halogen;

n is selected from 0, 1, 2, 3 or 4;

R ³ selected from hydrogen, R ^a S(O) ₂ -、(R ^a O) ₂ P (O) -or R ^a C(O)-；

Wherein R is ^a Independently selected from C _2-6 Alkynyl, C _2-6 Alkenyl, C _1-6 Alkyl, C _3-6 Cycloalkyl, (C) _1-6 Alkyl) NH- (C _1-6 Alkyl group ₂ N-, 3-6 membered heterocycloalkyl, 5-10 membered heteroaryl or C _6-10 Aryl, R is as above ^a Optionally quilt (C) _1-6 Alkyl group ₂ N-、(C _1-6 Alkyl) NH-, hydroxy, amino, halogen or cyano.

In some embodiments, ring a is selected from phenyl or 5-6 membered heteroaryl; in some embodiments, ring a is selected from 6 membered heteroaryl; in some embodiments, ring a is selected from 6 membered nitrogen containing heteroaryl; in some embodiments, ring a is selected from pyridinyl.

In some embodiments, R ¹ Independently selected from halogen, cyano, C _1-3 Alkoxy, C _1-3 Alkyl, 5-6 membered heteroaryl, 5-6 membered heterocycloalkyl, C _6-10 Aryl or C _6-10 Cycloalkyl group, the C _1-3 Alkoxy, C _1-3 Alkyl, 5-6 membered heteroaryl, 5-6 membered heterocycloalkyl, C _6-10 Aryl or C _6-10 Cycloalkyl optionally substituted by cyano, halogen or halo C _1-3 Alkyl substitution; in some embodiments, R ¹ Independently selected from halogen, C _1-3 Alkyl or C _6-10 Aryl, said C _1-3 Alkyl or C _6-10 Aryl optionally substituted by halogen or halo C _1-3 Alkyl substitution; in some embodiments, R ¹ Independently selected from fluorine, chlorine, bromine, halomethyl or phenyl optionally substituted with halogen; in some embodiments, R ¹ Independently selected from trifluoromethyl or phenyl optionally substituted with fluorine.

In some embodiments, m is selected from 0, 1, 2, 3, or 4; in some embodiments, m is selected from 0, 1, or 2; in some embodiments, m is selected from 0 or 1.

In some embodiments, L is selected from-C (O) NH-or-NHC (O) -; in some embodiments, L is selected from-C (O) NH-.

In some casesIn embodiments, R ² Independently selected from halogen, hydroxy, amino, cyano, C _1-3 Alkyl or C _1-3 An alkoxy group; in some embodiments, R ² Independently selected from fluorine, chlorine or bromine; in some embodiments, R ² Independently selected from fluorine.

In some embodiments, n is selected from 0, 1, or 2; in some embodiments, n is selected from 0 or 1.

In some embodiments, R ³ Selected from R ^a C(O)-。

In some embodiments, R ^a Independently selected from C _2-6 Alkynyl, C _2-6 Alkenyl, C _1-6 Alkyl, C _3-6 Cycloalkyl or 3-6 membered heterocycloalkyl; in some embodiments, R ^a Independently selected from C _2-3 Alkynyl or C _2-3 Alkenyl groups; in some embodiments, R ^a Selected from propynyl.

In some embodiments, R ³ Selected from the group consisting of

In some embodiments, the compound of formula (I) or a pharmaceutically acceptable salt thereof of the present application is selected from the group consisting of compounds of formula (I-1) or a pharmaceutically acceptable salt thereof,

wherein the ring A, L, R ¹ 、R ² 、R ³ And m and n are defined as above.

In some embodiments, the compound of formula (I) or a pharmaceutically acceptable salt thereof of the present application is selected from the group consisting of compounds of formula (II) or formula (II-1) or a pharmaceutically acceptable salt thereof,

wherein R is ¹ 、R ² And m and n are defined as above.

In some embodiments, the compound of formula (I) of the present application or a pharmaceutically acceptable salt thereof is selected from the following compounds or pharmaceutically acceptable salts thereof:

in another aspect, the application relates to a pharmaceutical composition comprising a compound of the application described above or a pharmaceutically acceptable salt thereof. In some embodiments, the pharmaceutical compositions of the present application further comprise pharmaceutically acceptable excipients.

In another aspect, the application relates to a method of treating a BTK-related disorder in a mammal, comprising administering to a mammal, preferably a human, in need of such treatment a therapeutically effective amount of a compound as described above, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

In another aspect, the application relates to the use of the above compound or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, in the manufacture of a medicament for the prevention or treatment of a BTK-related disease.

In another aspect, the application relates to the use of the above compound or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, for the prevention or treatment of a BTK-related disease.

In another aspect, the present application relates to the above-described compounds or pharmaceutically acceptable salts thereof, or pharmaceutical compositions thereof, for the prevention or treatment of BTK-related diseases.

In some embodiments, the BTK-related disease described above is selected from autoimmune diseases, inflammatory diseases, or cancer.

Definition of the definition

The following terms used in the present application have the following meanings unless otherwise indicated. A particular term, unless otherwise defined, shall not be construed as being ambiguous or otherwise unclear, but shall be construed in accordance with the ordinary meaning in the art. When trade names are presented herein, it is intended to refer to their corresponding commercial products or active ingredients thereof.

The term "substituted" means that any one or more hydrogen atoms on a particular atom is substituted with a substituent, provided that the valence of the particular atom is normal and the substituted compound is stable. When the substituent is oxo (i.e., =o), meaning that two hydrogen atoms are substituted, oxo does not occur on the aromatic group.

The term "optionally" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not. For example, ethyl "optionally" substituted with halogen means that ethyl may be unsubstituted (CH ₂ CH ₃ ) Monosubstituted (e.g. CH ₂ CH ₂ F) Polysubstituted (e.g. CHFCH ₂ F、CH ₂ CHF ₂ Etc.) or fully substituted (CF) ₂ CF ₃ ). It will be appreciated by those skilled in the art that for any group comprising one or more substituents, no substitution or pattern of substitution is introduced that is sterically impossible and/or synthetic.

C herein _m-n It is that the moiety has an integer number of carbon atoms in the given range. For example "C _1-6 By "is meant that the group may have 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, or 6 carbon atoms.

When any variable (e.g., R) occurs more than once in the composition or structure of a compound, its definition in each case is independent. For example, if a group contains 2R's, then each R has an independent option.

The term "halogen" or "halo" refers to fluorine, chlorine, bromine and iodine.

The term "hydroxy" refers to an-OH group.

The term "amino" refers to-NH ₂ A group.

The term "cyano" refers to a-CN group.

The term "alkyl" refers to a compound of the formula C _n H _2n+1 Is a hydrocarbon group of (a). The alkyl group may be linear or branched. For example, the term "C _1-6 Alkyl "means an alkyl group having 1 to 6 carbon atoms (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl) Group, sec-butyl, tert-butyl, n-pentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, neopentyl, hexyl, 2-methylpentyl, etc.). Similarly, the alkyl portion (i.e., alkyl) of alkoxy, alkylamino, dialkylamino, alkylsulfonyl, and alkylthio have the same definition as above.

The term "alkoxy" refers to an-O-alkyl group.

The term "alkenyl" refers to a straight or branched chain unsaturated aliphatic hydrocarbon group having at least one double bond consisting of carbon atoms and hydrogen atoms. Non-limiting examples of alkenyl groups include, but are not limited to, vinyl, 1-propenyl, 2-propenyl, 1-butenyl, isobutenyl, 1, 3-butadienyl, and the like.

The term "alkynyl" refers to a straight or branched chain unsaturated aliphatic hydrocarbon group consisting of carbon and hydrogen atoms having at least one triple bond. Non-limiting examples of alkynyl groups include, but are not limited to, ethynyl (-C.ident.CH), 1-propynyl (-C.ident.C-CH) ₃ ) 2-propynyl (-CH) ₂ -C.ident.CH), 1, 3-butadienyl (-C.ident.C-C.ident.CH), and the like.

The term "cycloalkyl" refers to a carbocycle that is fully saturated and may exist as a single ring, bridged ring, or spiro ring. Unless otherwise indicated, the carbocycle is typically a 3 to 10 membered ring. Non-limiting examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, norbornyl (bicyclo [2.2.1] heptyl), bicyclo [2.2.2] octyl, adamantyl, and the like.

The term "heterocycloalkyl" refers to a cyclic group that is fully saturated and may exist as a single ring, bridged ring, or spiro ring. Unless otherwise indicated, the heterocycle is typically a 3 to 7 membered ring containing 1 to 3 heteroatoms (preferably 1 or 2 heteroatoms) independently selected from sulfur, oxygen and/or nitrogen. Examples of 3-membered heterocycloalkyl groups include, but are not limited to, ethylene oxide, ethylene nitride, non-limiting examples of 4-membered heterocycloalkyl groups include, but are not limited to, azetidinyl, oxetanyl, thietanyl, examples of 5-membered heterocycloalkyl groups include, but are not limited to, tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, isoxazolidinyl, oxazolidinyl, isothiazolidinyl, thiazolidinyl, imidazolidinyl, tetrahydropyrazolyl, examples of 6-membered heterocycloalkyl groups include, but are not limited to, piperidinyl, tetrahydropyranyl, tetrahydrothiopyranyl, morpholinyl, piperazinyl, 1, 4-thiaalkyl, 1, 4-dioxanyl, thiomorpholinyl, 1, 3-dithianyl, 1, 4-dithianyl, examples of 7-membered heterocycloalkyl groups include, but are not limited to, azepanyl, oxepinyl, thiepanyl. Preferred are monocyclic heterocycloalkyl groups having 5 or 6 ring atoms.

The term "aryl" refers to an all-carbon monocyclic or fused-polycyclic aromatic ring radical having a conjugated pi-electron system. For example, an aryl group may have 6-20 carbon atoms, 6-14 carbon atoms, or 6-12 carbon atoms. Non-limiting examples of aryl groups include, but are not limited to, phenyl, naphthyl, anthryl, and 1,2,3, 4-tetrahydronaphthalene, and the like.

The term "heteroaryl" refers to a monocyclic or fused polycyclic ring system containing at least one ring atom selected from N, O, S, the remaining ring atoms being C and having at least one aromatic ring. Preferred heteroaryl groups have a single 4 to 8 membered ring, especially a 5 to 8 membered ring, or multiple fused rings containing 6 to 14, especially 6 to 10 ring atoms. Non-limiting examples of heteroaryl groups include, but are not limited to, pyrrolyl, furanyl, thienyl, imidazolyl, oxazolyl, pyrazolyl, pyridyl, pyrimidinyl, pyrazinyl, quinolinyl, isoquinolinyl, tetrazolyl, triazolyl, triazinyl, benzofuranyl, benzothienyl, indolyl, isoindolyl, and the like.

The term "treatment" means administration of a compound or formulation of the application to ameliorate or eliminate a disease or one or more symptoms associated with the disease, and includes:

(i) Inhibiting a disease or disease state, i.e., inhibiting its progression;

(ii) The disease or condition is alleviated, even if the disease or condition subsides.

The term "preventing" means that the compound or formulation of the application is administered to prevent a disease or one or more symptoms associated with the disease, including: preventing a disease or a disease state from occurring in a mammal, particularly when such mammal is susceptible to the disease state, but has not been diagnosed as having the disease state.

The term "therapeutically effective amount" means an amount of a compound of the application that (i) treats or prevents a particular disease, condition, or disorder, (ii) alleviates, ameliorates, or eliminates one or more symptoms of a particular disease, condition, or disorder, or (iii) prevents or delays the onset of one or more symptoms of a particular disease, condition, or disorder described herein. The amount of the compound of the present application that constitutes a "therapeutically effective amount" will vary depending on the compound, the disease state and its severity, the mode of administration, and the age of the mammal to be treated, but can be routinely determined by one of ordinary skill in the art based on his own knowledge and disclosure.

The term "pharmaceutically acceptable" is intended to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

As pharmaceutically acceptable salts, for example, metal salts, ammonium salts, salts with organic bases, salts with inorganic acids, salts with organic acids, salts with basic or acidic amino acids, and the like can be mentioned.

The term "pharmaceutical composition" refers to a mixture of one or more compounds of the application or salts thereof and pharmaceutically acceptable excipients. The purpose of the pharmaceutical composition is to facilitate the administration of the compounds of the application to an organism.

The term "pharmaceutically acceptable excipients" refers to those excipients which do not significantly stimulate the organism and which do not impair the biological activity and properties of the active compound. Suitable excipients are well known to the person skilled in the art, such as carbohydrates, waxes, water soluble and/or water swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water and the like.

The words "comprise" or "include" and variations thereof such as "comprises" or "comprising" are to be interpreted in an open, non-exclusive sense, i.e. "including but not limited to.

The compounds and intermediates of the application may also exist in different tautomeric forms and all such forms are included within the scope of the application. The term "tautomer" or "tautomeric form" refers to structural isomers of different energies that can interconvert via a low energy barrier. For example, proton tautomers (also known as proton transfer tautomers) include tautomers via proton transfer, such as keto-enol and imine-enamine isomerisation. A specific example of a proton tautomer is an imidazole moiety, where a proton can migrate between two ring nitrogens. Valence tautomers include tautomers by recombination of some bond-forming electrons.

The application also includes isotopically-labeled compounds of the application which are identical to those recited herein, but for the fact that one or more atoms are replaced by an atom having an atomic weight or mass number different from the atomic weight or mass number usually found in nature. Examples of isotopes that can be incorporated into compounds of the application include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine, such as, respectively ² H、 ³ H、 ¹¹ C、 ¹³ C、 ¹⁴ C、 ¹³ N、 ¹⁵ N、 ¹⁵ O、 ¹⁷ O、 ¹⁸ O、 ³¹ P、 ³² P、 ³⁵ S、 ¹⁸ F、 ¹²³ I、 ¹²⁵ I and ³⁶ cl, and the like.

Certain isotopically-labeled compounds of the application (e.g., with ³ H is H ¹⁴ C-labeled) can be used in compound and/or substrate tissue distribution analysis. Tritiation (i.e ³ H) And carbon-14 (i.e ¹⁴ C) Isotopes are particularly preferred for their ease of preparation and detectability. Positron emitting isotopes, such as ¹⁵ O、 ¹³ N、 ¹¹ C and C ¹⁸ F can be used in Positron Emission Tomography (PET) studies to determine substrate occupancy. Isotopically-labeled compounds of the present application can generally be prepared by following procedures analogous to those disclosed in the schemes and/or examples below by substituting an isotopically-labeled reagent for an non-isotopically-labeled reagent.

In addition, with heavier isotopes (such asDeuterium (i.e ² H) Substitution may provide certain therapeutic advantages resulting from higher metabolic stability (e.g., increased in vivo half-life or reduced dosage requirements), and thus may be preferred in certain circumstances, where deuterium substitution may be partial or complete, partial deuterium substitution meaning that at least one hydrogen is substituted with at least one deuterium.

The compounds of the application may be asymmetric, e.g., have one or more stereoisomers. Unless otherwise indicated, all stereoisomers include, for example, enantiomers and diastereomers. The compounds of the application containing asymmetric carbon atoms can be isolated in optically pure or racemic form. Optically pure forms can be resolved from the racemic mixture or synthesized by using chiral starting materials or chiral reagents.

The pharmaceutical compositions of the present application may be prepared by combining the compounds of the present application with suitable pharmaceutically acceptable excipients, for example, in solid, semi-solid, liquid or gaseous formulations such as tablets, pills, capsules, powders, granules, ointments, emulsions, suspensions, suppositories, injections, inhalants, gels, microspheres, aerosols and the like.

Typical routes of administration of the compounds of the application or pharmaceutically acceptable salts thereof or pharmaceutical compositions thereof include, but are not limited to, oral, rectal, topical, inhalation, parenteral, sublingual, intravaginal, intranasal, intraocular, intraperitoneal, intramuscular, subcutaneous, intravenous administration.

The pharmaceutical compositions of the present application may be manufactured by methods well known in the art, such as conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, freeze-drying, and the like.

In some embodiments, the pharmaceutical composition is in oral form. For oral administration, the pharmaceutical compositions may be formulated by mixing the active compound with pharmaceutically acceptable excipients well known in the art. These excipients enable the compounds of the present application to be formulated into tablets, pills, troches, dragees, capsules, liquids, gels, slurries, suspensions and the like for oral administration to a patient.

The solid oral compositions may be prepared by conventional mixing, filling or tabletting methods. For example, it can be obtained by the following method: the active compound is mixed with solid auxiliary materials, the resulting mixture is optionally milled, if desired with other suitable auxiliary materials, and the mixture is then processed to granules, giving a tablet or dragee core. Suitable excipients include, but are not limited to: binders, diluents, disintegrants, lubricants, glidants, sweeteners or flavoring agents, and the like.

The pharmaceutical compositions may also be suitable for parenteral administration, such as sterile solutions, suspensions or lyophilized products in suitable unit dosage forms.

In all methods of administration of the compounds of formula I described herein, the daily dosage is from 0.01 to 200mg/kg body weight, either alone or in divided doses.

The compounds of the present application may be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments set forth below, embodiments formed by combining with other chemical synthetic methods, and equivalent alternatives well known to those skilled in the art, preferred embodiments including but not limited to the examples of the present application.

The chemical reactions of the embodiments of the present application are accomplished in a suitable solvent that is compatible with the chemical changes of the present application and the reagents and materials required therefor. In order to obtain the compounds of the present application, it is sometimes necessary for a person skilled in the art to modify or select the synthesis steps or reaction schemes on the basis of the embodiments already present.

An important consideration in the art of synthetic route planning is the selection of suitable protecting groups for reactive functionalities (such as amino groups in the present application), for example, reference may be made to Greene's Protective Groups in Organic Synthesis (4 th Ed.) Hoboken, new Jersey: john Wiley & Sons, inc.

In some embodiments, the compounds of formula (II) of the present application can be prepared by those skilled in the art of organic synthesis by the following routes using existing methods in the art:

Wherein R is ¹ 、R ² R is as defined above for m or n ⁴ Is an amino protecting group selected from Cbz (benzyloxycarbonyl) or Boc (tert-butoxycarbonyl).

The application adopts the following abbreviations:

PE represents petroleum ether; EA represents ethyl acetate; DMSO represents dimethyl sulfoxide; DMF represents N, N-dimethylformamide; DCM represents dichloromethane; TBSCl represents tert-butyldimethylchlorosilane; boc ₂ O represents di-tert-butyl dicarbonate; NBS represents N-bromosuccinimide; meOH represents methanol; DTT represents dithiothreitol; EGTA represents ethylene glycol bis (2-aminoethylether) tetraacetic acid; HATU stands for 2- (7-oxybenzotriazol) -N, N' -tetramethylurea hexafluorophosphate.

The application is further illustrated by examples, which are not intended to limit the scope of the application, for clarity. All reagents used in the present application are commercially available and can be used without further purification.

Detailed Description

Example 1: preparation of (S) -4- (8-amino-3- (4- (but-2-ynyl) -4-azaspiro [2.4] heptan-5-yl) imidazo [1,5-a ] pyrazin-1-yl) -3-fluoro-N- (4- (4-fluorophenyl) pyridin-2-yl) benzamide (Compound I-1)

Step 1 (S) -5- (((tert-butyldimethylsilyl) oxy) methyl) pyrrolidin-2-one (intermediate 7-2)

To the reaction flask was added intermediate 7-1 (50 g) and dissolved in DCM (500 mL), imidazole (59.1 g) was added at 0 ℃, TBSCl (2.21 g) was added at maintained temperature, the reaction was continued at 0 ℃, after completion of the reaction, the reaction solution was added to 500mL of saturated brine, methyl tert-butyl ether was extracted, the organic phase was washed with 5% citric acid solution, dried over anhydrous sodium sulfate, filtered, and concentrated without further purification to give compound 7-2 (106.2 g). MS (ESI, [ M+H ] +) M/z:230.5.

step 2: (S) -1-benzyl-5- (((tert-butyldimethylsilyl) oxy) methyl) pyrrolidin-2-one (intermediate 7-3)

Sodium hydrogen (63.9 g) was added to the reaction flask, tetrahydrofuran (1000 mL) was then added thereto, intermediate 7-2 (100 g) was added dropwise to the flask under nitrogen protection at 0℃and maintained at the temperature for about 1 hour, benzyl bromide (71 mL) was then added to the system for room temperature reaction, after the completion of the reaction, the reaction solution was slowly added to 2L of ice water, methyl tert-butyl ether was extracted, the organic phase was washed with saturated saline solution, dried over anhydrous sodium sulfate, filtered, and purified by column chromatography (developer: PE: EA=100:50) to give compound 7-3 (124.56 g).

¹ H NMR(500MHz,DMSO-d6)δ7.51–6.97(m,5H),4.71(d,J＝15.2Hz,1H),4.12(d,J＝15.3Hz,1H),3.78–3.62(m,1H),3.61–3.51(m,1H),3.49(dd,J＝8.7,4.0Hz,1H),2.36(dt,J＝17.6,9.1Hz,1H),2.27–2.14(m,1H),2.08–1.95(m,1H),1.77(m,1H),0.84(s,9H),-0.01(d,J＝7.3Hz,6H).MS(ESI,[M+H]+)m/z：320.2.

Step 3: (S) -4-benzyl-5- (((tert-butyldimethylsilyl) oxy) methyl) -4-azaspiro [2.4] heptane (intermediate 7-4)

Tetrahydrofuran (1000 mL) is added into a dry reaction bottle, ethyl magnesium bromide (310 mL) is added under the protection of nitrogen, the temperature is reduced to 70 ℃, tetraisopropyl titanate (92 mL) is dropwise added into the bottle after the temperature is reached, the reaction is carried out for about 1h at the maintained temperature, 7-3 (100 g) room temperature is added into the system for reaction, after the reaction is completed, the reaction solution is slowly added into 1L ice water, black solid insoluble matters are removed by suction filtration, methyl tertiary butyl ether is extracted, an organic phase is washed by saturated saline solution, dried by anhydrous sodium sulfate, filtered and concentrated to obtain a crude product (110.52 g) of the compound 7-4.

¹ H NMR(500MHz,DMSO-d6)δ7.41–7.13(m,5H),3.68(d,J＝13.9Hz,1H),3.50(d,J＝13.9Hz,1H),3.39–3.32(m,2H),2.92(m,1H),2.01(m,1H),1.91(m,1H),1.66(m,1H),1.52(m,1H),0.86–0.82(m,1H),0.79(s,9H),0.54–0.33(m,3H),-0.10(d,J＝10.5Hz,6H).

HR-MS(ESI,[M+H] ⁺ )m/z：332.2377

Step 4: (S) - (4-benzyl-4-azaspiro [2.4] heptane-5-yl) methanol (intermediate 7-5)

The crude intermediate 7-4 (100 g) was added to a reaction flask, dissolved in tetrahydrofuran (1000 mL), and then reacted with a tetrahydrofuran solution of tetrabutylammonium fluoride (302 mL,1 mol/L) at 40℃to give, after completion of the reaction, a reaction system was concentrated, followed by extraction with methyl tert-butyl ether and saturated brine, drying over anhydrous sodium sulfate, filtration, and purification by column chromatography (developer: DCM: meOH=100:5) to give compound 7-5 (16.18 g).

¹ H NMR(500MHz,DMSO-d6)δ7.46–7.08(m,5H),4.13(s,1H),3.69(dd,J＝14.1,3.9Hz,1H),3.52(dd,J＝14.1,3.7Hz,1H),3.31–3.22(m,1H),3.16(t,J＝9.5Hz,1H),2.92(q,J＝6.6Hz,1H),1.97(m,2H),1.67(tt,J＝11.7,5.2Hz,1H),1.50(t,J＝9.9Hz,1H),0.81(h,J＝10.7,10.2Hz,1H),0.46(h,J＝6.1,5.4Hz,2H),0.42–0.32(m,1H).MS(ESI,[M+H]+)m/z：218.5.

Step 5: (S) - (4-azaspiro [2.4] hept-5-yl) methanolic hydrochloride (intermediate 7-6)

Adding the compound 7-5 (15 g) into a reaction bottle, adding methanol (150 mL), concentrated hydrochloric acid (22.63 mL), adding reduced palladium carbon (1.086 g), replacing hydrogen for 3-4 times, maintaining the hydrogen atmosphere through a hydrogen balloon, reacting at 30 ℃, concentrating the reaction system after the reaction is completed, and removing dry water to obtain the compound 7-6 (12.51 g)

¹ H NMR(500MHz,DMSO-d6)δ9.84(s,1H),9.07(s,1H),3.79–3.46(m,3H),2.08(h,J＝6.3Hz,1H),1.93(m,2H),1.81(m,1H),1.17(m,2H),0.76(s,2H).

Step 6: (S) -5- (hydroxymethyl) -4-azaspiro [2.4] heptane-4-carboxylic acid tert-butyl ester (intermediate 7-7)

Compounds 7-6 (12 g) were added to the reaction flask, then dissolved with DCM (150 mL) and cooled to 0deg.C, triethylamine (29.2 mL) was added to the system, and Boc was added ₂ O (17.65 mL). The reaction was carried out while maintaining the temperature, and after the completion of the reaction, the mixture was extracted with methyl t-butyl ether and saturated brine. Drying over anhydrous sodium sulfate, filtering and concentrating to obtain the compound 7-7 (19.86 g)

Step 7: (S) -4- (tert-Butoxycarbonyl) -4-azaspiro [2.4] heptane-5-carboxylic acid (intermediate 7-8)

To the reaction flask was added compound 7-7 (19.86 g), followed by acetonitrile (40 mL), carbon tetrachloride (40 mL) was dissolved, followed by aqueous sodium periodate (47.4 g,60 mL), the reaction temperature was maintained at 0℃and then ruthenium trichloride monohydrate (833 mg) was added, and the reaction was maintained at room temperature. After completion of the reaction, 500mL of saturated brine was added thereto, the mixture was extracted 3 times with DCM, the organic phases were combined, dried over anhydrous sodium sulfate and concentrated by filtration to give Compound 7-8 (17.56 g)

Step 8: (S) -4-benzyl-N- ((3-chloropyrazin-2-yl) methyl) -4-azaspiro [2.4] heptane-5-carboxamide (intermediate 7-9)

Compounds 7-8 (17.56 g) were added to the reaction flask, and (3-chloropyrazin-2-yl) methylamine hydrochloride (12.72 g) was added, dissolved with DCM (200 mL) and triethylamine (23.42 mL) was added followed by HATU (17 g) to react the mixture at room temperature. At the end of the reaction, methyl tert-butyl ether was added to the reaction system to dilute it, and the mixture was washed twice with 500ml of a saturated solution of sodium hydrogencarbonate. The organic phase was dried over anhydrous sodium sulfate, concentrated and purified by column chromatography (developer: PE: ea=100:20) to give compound 7-9 (12.78 g)

¹ H NMR(500MHz,DMSO-d6)δ8.62(d,J＝2.4Hz,1H),8.44(d,J＝2.4Hz,1H),8.32(t,J＝5.4Hz,1H),4.61(dd,J＝16.4,5.8Hz,1H),4.48(dd,J＝16.4,5.1Hz,1H),4.35(dd,J＝8.7,3.5Hz,1H),2.24–2.09(m,1H),2.02(m,1H),1.84(m,1H),1.67(m,1H),1.58(d,J＝10.4Hz,1H),1.42(d,J＝22.1Hz,1H),1.29(s,9H),0.46(d,J＝7.2Hz,2H).MS(ESI,[M+Na]+)m/z：389.11.

Step 9: (S) -5- (8-chloroimidazo [1,5-a ] pyrazin-3-yl) -4-azaspiro [2.4] heptane-4-carboxylic acid tert-butyl ester (intermediate 7-10)

The reaction flask was charged with compound 7-9 (12 g) at 0deg.C, N ₂ Ethyl acetate (120 mL), DMF (7.46 mL) and phosphorus oxychloride (20.96 mL) were added dropwise to the system under protection and reacted at room temperature. After the reaction, the reaction system was slowly added to an aqueous ammonia ice-water solution, followed by neutralization under stirring, the reaction system was extracted with methyl tert-butyl ether, and the organic phase was dried over anhydrous sodium sulfate and concentrated to give Compound 7-10 (8.62 g)

¹ H NMR(500MHz,DMSO-d6)δ8.43(d,J＝5.0Hz,1H),7.88(s,1H),7.44(d,J＝5.1Hz,1H),5.53(dd,J＝7.5,4.3Hz,1H),2.34(m,2H),2.07–1.78(m,2H),1.58(s,2H),1.16(s,9H),0.72–0.47(m,2H).MS(ESI,[M+H]+)m/z：349.14.

Step 10: (S) -5- (1-bromo-8-chloroimidazo [1,5-a ] pyrazin-3-yl) -4-azaspiro [2.4] heptane-4-carboxylic acid tert-butyl ester (intermediate 7-11)

In a reaction flask, compound 7-10 (3.9 g), DMF (40 mL) and NBS (2.69 g), N were added in this order ₂ The mixture was heated to 60℃under protection. After the completion of the reaction, the reaction mixture was cooled to room temperature, 200mL of ethyl acetate and 20mL of a saturated aqueous sodium thiosulfate solution were added to the reaction mixture, which was then extracted with saturated brine, dried over anhydrous sodium sulfate, filtered, and purified by column chromatography (developer: PE: ea=100:10) to give compound 7-11 (1.51 g). MS (ESI, [ M+Na)] ⁺ )m/z：451.03

Step 11: (S) -5- (8-amino-1-bromoimidazo [1,5-a ] pyrazin-3-yl) -4-azaspiro [2.4] heptane-4-carboxylic acid tert-butyl ester (intermediate 7-12)

Compounds 7-11 (1.5 g), ammonia in isopropanol (32.5 ml,2 mol/L)) were added to the pressure-tight reaction flask and the mixture was heated to 120 ℃. After completion of the reaction, the mixture was cooled to room temperature, and purified by column chromatography (developer: PE: ea=100:10) to give compounds 7 to 12 (440 mg)

MS(ESI,[M+H] ⁺ )m/z：408.16。

Step 12: (S) -5- (8-amino-1- (2-fluoro-4- ((4- (4-fluorophenyl) pyridin-2-yl) carbamoyl) phenyl) imidazo [1,5-a ] pyrazin-3-yl) -4-azaspiro [2.4] heptane-4-carboxylic acid tert-butyl ester (intermediate 7-13)

The reaction flask was charged with the compound 7-12 (150 mg), 3-fluoro-N- (4- (4-fluorophenyl) pyridin-2-yl) -4- (4, 5-tetramethyl-1, 3, 2-dioxaborane-2-yl) benzamide (210 mg), potassium carbonate (177 mg), [1,1' -bis (diphenylphosphino) ferrocene ] palladium dichloride (65.5 mg), then 1, 4-dioxane (5 mL), water (1 mL), nitrogen gas was substituted 3-4 times, heated to 80 ℃ for reaction, cooled to room temperature after the reaction was completed, a saturated saline dilution system was added, and 2-3 times of extraction was performed with DCM. The organic phase was dried over anhydrous sodium sulfate, concentrated and purified by column chromatography (developer: DCM: meoh=100:3) to give compounds 7-13 (136 mg).

¹ H NMR(500MHz,DMSO-d6)δ11.08(s,1H),8.52(s,1H),8.49(d,J＝5.2Hz,1H),8.05(d,J＝4.6Hz,1H),8.03(s,1H),7.86(dd,J＝8.4,5.3Hz,2H),7.76(d,J＝5.1Hz,1H),7.62(t,J＝7.8Hz,1H),7.53(d,J＝5.3Hz,1H),7.41(t,J＝8.6Hz,2H),7.11(d,J＝5.1Hz,1H),6.20(s,2H),5.46(m,1H),2.34(m,1H),2.20(m,1H),1.99(m,2H),1.59(s,2H),1.04(s,9H),0.63(d,J＝10.4Hz,1H),0.54(d,J＝10.1Hz,1H).MS(ESI,[M+H] ⁺ )m/z：638.56

Step 13: (S) -4- (8-amino-3- (4- (but-2-ynyl) -4-azaspiro [2.4] heptan-5-yl) imidazo [1,5-a ] pyrazin-1-yl) -3-fluoro-N- (4- (4-fluorophenyl) pyridin-2-yl) benzamide (Compound I-1)

Adding 7-13 (120 mg) of compound into a reaction bottle, adding a methanol solution (20% -30% and 3 mL) of hydrochloric acid, reacting at 50 ℃ for about 5-10min, concentrating, and drying to constant weight to obtain 7-14 of compound; to the reaction flask was added HATU (79 mg), 2-butynoic acid (15.82 mg), dissolved in DCM, and triethylamine (0.105 mL) was added and reacted at room temperature. After the reaction, saturated sodium hydrogencarbonate was added to dilute the mixture, followed by extraction with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate, concentrated and purified by column chromatography (developer: DCM: meoh=100:3) to give compound I-1 (125 mg)

¹ H NMR(500MHz,DMSO-d ₆ )δ11.09(s,1H),8.52(s,1H),8.49(d,J＝5.2Hz,1H),8.10–8.01(m,2H),7.92–7.80(m,3H),7.65(t,J＝7.8Hz,1H),7.53(d,J＝5.3,1H),7.41(t,J＝8.7Hz,2H),7.22–7.09(m,1H),6.21-6.00(m,2H),5.93–5.68(m,1H),2.49–2.31(m,2H),2.07–1.93(m,2H),1.92-1.81(m,2H),1.81-1.74(m,1H),1.59(s,2.4H),0.72-0.55(m,1.6H).HR-MS(ESI,[M+H] ⁺ )m/z：604.2297.

Example 2: preparation of (S) -4- (8-amino-3- (4- (but-2-ynyl) -4-azaspiro [2.4] heptan-5-yl) imidazo [1,5-a ] pyrazin-1-yl) -3-fluoro-N- (4-phenylpyridin-2-yl) benzamide (Compound I-2)

Step 1: (S) -5- (8-amino-1- (2-fluoro-4- ((4-phenylpyridin-2-yl) carbamoyl) phenyl) imidazo [1,5-a ] pyrazin-3-yl) -4-azaspiro [2.4] heptane-4-carboxylic acid tert-butyl ester (intermediate 8-13)

To the reaction flask was added the compound 7-12 (200 mg), (2-fluoro-4- ((4-phenylpyridin-2-yl) carbamoyl) phenyl) boronic acid (232 mg), potassium carbonate (136 mg), [1,1' -bis (diphenylphosphino) ferrocene ] palladium dichloride (87 mg), then 1, 4-dioxane (5 mL), water (1 mL), nitrogen substitution 3-4 times, heated to 80 ℃ for reaction, cooled to room temperature after the reaction was completed, added with a saturated saline dilution system, and extracted 2-3 times with DCM. The organic phase was dried over anhydrous sodium sulfate, concentrated and purified by column chromatography (developer: DCM: meoh=100:3) to give compounds 8-13 (136 mg).

¹ H NMR(500MHz,DMSO-d ₆ )δ11.07(s,1H),8.54(s,1H),8.50(d,J＝5.2Hz,1H),8.08–8.00(m,2H),7.80(d,J＝7.6Hz,2H),7.74(d,J＝5.0Hz,1H),7.66–7.56(m,4H),7.55–7.46(m,2H),7.10(d,J＝5.0Hz,1H),6.03(s,2H),5.49-5.41(m,1H),2.41–2.10(m,2H),2.07-1.90(m,2H),1.60(s,2H),1.03(s,9H),0.68-0.49(m,2H).MS(ESI,[M+H] ⁺ )m/z：620.34.

Step 2: (S) -4- (8-amino-3- (4- (but-2-ynyl) -4-azaspiro [2.4] heptan-5-yl) imidazo [1,5-a ] pyrazin-1-yl) -3-fluoro-N- (4-phenylpyridin-2-yl) benzamide (Compound I-2)

Adding 8-13 (180 mg) of compound into a reaction bottle, adding a methanol solution (20% -30% and 3 mL) of hydrochloric acid, reacting for 5-10min at 50 ℃, concentrating the acid, and drying to constant weight to obtain 8-14 of compound; to the flask was added HATU (121 mg), 2-butynoic acid (24.42 mg), dissolved in DCM, and triethylamine (118 mg) was added thereto to react at room temperature. After the reaction, saturated sodium hydrogencarbonate was added to dilute the mixture, followed by extraction with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate, concentrated and purified by column chromatography (developer: DCM: meoh=100:3) to give compound I-2 (105 mg)

¹ H NMR(500MHz,DMSO-d ₆ )δ11.08(s,1H),8.55(s,1H),8.50(d,J＝5.2Hz,1H),8.10-8.00(m,2H),7.87(d,J＝5.0Hz,1H),7.83–7.77(m,2H),7.65(t,J＝7.8Hz,1H),7.60-7.54(m,2H),7.55–7.50(m,2H),7.25–7.09(m,1H),6.07(d,J＝24.7Hz,2H),5.95–5.68(m,1H),2.48–2.30(m,2H),2.05-1.93(m,2H),1.94–1.82(m,2H),1.81-1.74(m,1H),1.59(s,2.4H),0.73–0.54(m,1.6H).HR-MS(ESI,[M+H] ⁺ )m/z：586.2384.

Example 3: preparation of (S) -4- (8-amino-3- (4- (but-2-ynyl) -4-azaspiro [2.4] heptan-5-yl) imidazo [1,5-a ] pyrazin-1-yl) -N- (pyridin-2-yl) benzamide (Compound I-3)

Step 1: (S) -5- (8-amino-1- (4- (pyridin-2-ylcarbamoyl) phenyl) imidazo [1,5-a ] pyrazin-3-yl) -4-azaspiro [2.4] heptane-4-carboxylic acid tert-butyl ester (intermediate 9-13)

To the flask was added the compound 7-12 (200 mg), N- (pyridin-2-yl) -4- (4, 5-tetramethyl-1, 3, 2-dioxaborane-2-yl) benzamide (159 mg), potassium carbonate (243 mg), 1' -bis (diphenylphosphino) ferrocene ] palladium dichloride (35.8 mg), then 1, 4-dioxane (5 mL), water (1 mL), nitrogen substitution 3-4 times, heated to 80 ℃ for reaction, cooled to room temperature after the end of the reaction, diluted with saturated saline solution and extracted 2-3 times with DCM. The organic phase was dried over anhydrous sodium sulfate, concentrated and purified by column chromatography (developer: DCM: meoh=100:5) to give compounds 9-13 (178 mg).

¹ H NMR(500MHz,DMSO-d6)δ10.82(s,1H),8.41(d,J＝4.8Hz,1H),8.23(d,J＝8.4Hz,1H),8.20–8.08(m,2H),7.87(t,J＝7.9Hz,1H),7.73(d,J＝7.4Hz,3H),7.19(t,J＝6.2Hz,1H),7.12(dd,J＝5.1,1.6Hz,1H),6.13(s,2H),5.45(s,1H),2.43–2.11(m,2H),2.08–1.86(m,2H),1.61(s,2H),1.03(s,9H),0.73–0.47(m,2H).MS(ESI,[M+H] ⁺ )m/z：526.5.

Step 2: (S) -4- (8-amino-3- (4- (but-2-ynyl) -4-azaspiro [2.4] heptane-5-yl) imidazo [1,5-a ] pyrazin-1-yl) -N- (pyridin-2-yl) benzamide (Compound I-3)

Adding 9-13 (166 mg) into a reaction bottle, adding methanol (5 mL), adding dioxane solution (4M, 4 mL) of hydrochloric acid, reacting at 50 ℃ for about 1h, concentrating, and drying to constant weight to obtain 9-14; HATU (126 mg) and 2-butynoic acid (25.4 mg) were added to the reaction flask, dissolved in DCM, and triethylamine (0.169 ml) was added thereto to react at room temperature. After the reaction, saturated sodium hydrogencarbonate was added to dilute the mixture, followed by extraction with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate, concentrated and purified by column chromatography (developer: DCM: meoh=100:3) to give compound I-3 (86 mg)

¹ H NMR(500MHz,DMSO-d6)δ10.83(s,1H),8.51–8.37(m,1H),8.23(d,J＝8.4Hz,1H),8.18(d,J＝7.9Hz,2H),7.94–7.84(m,2H),7.77(d,J＝8.3Hz,2H),7.26–7.07(m,2H),6.36–6.01(m,2H),5.99–5.64(m,1H),2.48–2.32(m,2H),2.05–1.95(m,2H),1.95–1.89(m,1H),1.90–1.73(m,2H),1.60(s,2H),0.75–0.54(m,2H).MS(ESI,[M+H] ⁺ )m/z：492.4.

Example 4: preparation of (S) -4- (8-amino-3- (4- (but-2-ynyl) -4-azaspiro [2.4] heptan-5-yl) imidazo [1,5-a ] pyrazin-1-yl) -3-fluoro-N- (4- (trifluoromethyl) pyridin-2-yl) benzamide (Compound I-4)

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Step 1: (S) -5- (8-amino-1- (2-fluoro-4- ((4- (trifluoromethyl) pyridin-2-yl) carbamoyl) phenyl) imidazo [1,5-a ] pyrazin-3-yl) -4-azaspiro [2.4] heptane-4-carboxylic acid tert-butyl ester (intermediate 10-13)

To the reaction flask was added the compound 7-12 (200 mg), 3-fluoro-4- (4, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) -N- (4- (trifluoromethyl) pyridin-2-yl) benzamide (215 mg), potassium carbonate (243 mg), [1,1' -bis (diphenylphosphino) ferrocene ] palladium dichloride (35.4 mg), then 1, 4-dioxane (5 mL), water (1 mL), nitrogen substitution 3-4 times, heated to 80 ℃ for reaction, cooled to room temperature after the reaction was completed, added a saturated saline dilution system, and extracted 2-3 times with DCM. The organic phase was dried over anhydrous sodium sulfate, concentrated and purified by column chromatography (developer: DCM: meoh=100:5) to give compound 10-13 (248 mg).

¹ H NMR(500MHz,DMSO-d6)δ11.44(s,1H),8.72(d,J＝5.1Hz,1H),8.57(s,1H),8.09–7.94(m,2H),7.73(d,J＝5.0Hz,1H),7.62(t,J＝7.7Hz,1H),7.58(d,J＝5.1Hz,1H),7.11(d,J＝4.9Hz,1H),6.02(s,2H),5.55–5.27(m,1H),2.43–2.08(m,2H),2.07–1.82(m,2H),1.59(s,2H),1.03(s,9H),0.67–0.45(m,2H).MS(ESI,[M+H] ⁺ )m/z：612.5.

Step 2: (S) -4- (8-amino-3- (4- (but-2-ynyl) -4-azaspiro [2.4] heptan-5-yl) imidazo [1,5-a ] pyrazin-1-yl) -3-fluoro-N- (4- (trifluoromethyl) pyridin-2-yl) benzamide (Compound I-4)

Adding 10-13 (230 mg) of compound into a reaction bottle, adding methanol (5 mL), adding dioxane solution (4M, 4.7 mL) of hydrochloric acid, reacting at 50 ℃ for about 1h, concentrating, and drying to constant weight to obtain 10-14 of compound; HATU (148 mg) and 2-butynoic acid (29.7 mg) were added to the reaction flask, and after dissolution in DCM, triethylamine (0.197 ml) was added thereto, followed by reaction at room temperature. After the reaction, saturated sodium hydrogencarbonate was added to dilute the mixture, followed by extraction with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate, concentrated and purified by column chromatography (eluent: DCM: meoh=100:3) to give compound I-4 (110 mg)

¹ H NMR(500MHz,DMSO-d6)δ11.46(s,1H),8.72(d,J＝5.0Hz,1H),8.57(s,1H),8.09–7.98(m,2H),7.89–7.76(m,1H),7.66(t,J＝7.9Hz,1H),7.62–7.52(m,1H),7.20–7.09(m,1H),6.19–5.95(m,2H),5.92–5.68(m,1H),2.48–2.29(m,2H),2.07–1.93(m,2H),1.93–1.70(m,3H),1.59(s,2H),0.73–0.53(m,2H).MS(ESI,[M+H] ⁺ )m/z：578.4.

Test example 1: in vitro Activity

1.1 BTK inhibition activity screening

With kinase buffer (50 mM HEPES, 10mM MgCl) ₂ 2mM DTT, 1mM EGTA, 0.01% Tween 20) was diluted with 350 ng/. Mu.L of BTK (WT) stock solution, 6. Mu.L of 1.67X 0.0334 ng/. Mu.L working solution (final concentration 0.02 ng/. Mu.L) was added to each well, and the various compounds dissolved in DMSO were added to the wells using a nanoliter-type applicator to give a final concentration of 1000nM-0.244nM, a 4-fold gradient for 7 total concentrations, and a blank (without enzyme) and a negative control well (containing enzyme, vehicle DMSO) were set, and 2 multiplex wells were set. After 30min of reaction of the enzyme with the compound or vehicle, 5X of the enzyme is prepared by using a kinase buffer 100. Mu.M ATP (final concentration of 20. Mu.M) with 5X 0.5. Mu.M substrate (final concentration of 0.1. Mu.M, ulight-poly GT) at a ratio of 1:1 mixing, adding 4 mu L of the mixture into each hole; after sealing the plates, the plates were allowed to react at room temperature for 2 hours, 5. Mu.L of 4X 40mM EDTA (final concentration of 10 mM) was added to each well, and the plates were incubated at room temperature for 5 minutes, followed by 5. Mu.L of 4X 8nM detection reagent (final concentration of 2nM, ab) to each well, and incubated at room temperature for 1 hour; the PE Envision multifunctional microplate reader reads the plate (excitation 620nm, emission 665 nm), and IC50 was calculated using four parameter fitting.

1.2 BTK (C481S) inhibitory activity screening

With kinase buffer (50 mM HEPES, 10mM MgCl) ₂ 50 ng/. Mu.L of BTK (C481S) stock was diluted with 2mM DTT, 1mM EGTA, 0.01% Tween 20, 6. Mu.L of 1.67X of 0.25 ng/. Mu.L of working solution (final concentration 0.15 ng/. Mu.L) was added to each well, and the various compounds dissolved in DMSO were added to the wells using a nanoliter-type applicator to give a final concentration of 1000nM-0.244nM, a 4-fold gradient for 7 total concentrations, and a blank (containing no enzyme) and a negative control well (containing enzyme, vehicle DMSO) were simultaneously set, and 2 multiplex wells were set. After 30min of reaction of the enzyme with the compound or vehicle, 5X 250. Mu.M ATP (final concentration of 50. Mu.M) and 5X 0.5. Mu.M substrate (final concentration of 0.1. Mu.M, ULIght-poly GT) in kinase buffer were mixed according to a 1:1 mixing, adding 4 mu L of the mixture into each hole; after sealing the plates, the plates were allowed to react at room temperature for 2 hours, 5. Mu.L of 4X 40mM EDTA (final concentration of 10 mM) was added to each well, and the plates were incubated at room temperature for 5 minutes, followed by 5. Mu.L of 4X 8nM detection reagent (final concentration of 2nM, ab) to each well, and incubated at room temperature for 1 hour; the PE Envision multifunctional microplate reader reads the plate (excitation 620nm, emission 665 nm), and IC50 was calculated using four parameter fitting.

1.3 EGFR (epidermal growth factor receptor) inhibitory Activity Screen

With kinase buffer (50 mM HEPES, 10mM MgCl) ₂ 50 ng/. Mu.L of EGFR (WT) stock solution was diluted with 2mM DTT, 1mM EGTA, 0.01% Tween 20, 6. Mu.L of 1.67X 0.01336 ng/. Mu.L working solution (final concentration of 0.008 ng/. Mu.L) was added to each well, and the various DMSO-dissolved compounds were added to the wells using a nanoliter-type applicator to give a final concentration of 1000nM-0.48nM, a 4-fold gradient for 7 concentrations, and a blank (without enzyme) and negative pair were setThe wells (containing enzyme, vehicle DMSO) were plated with 2 duplicate wells. After 10min of reaction of the enzyme with the compound or vehicle, 5. Mu.M ATP (final concentration of 5. Mu.M) prepared with kinase buffer was mixed with 5X 0.5. Mu.M substrate (final concentration of 0.1. Mu.M, ULIght-poly GT) at 1:1 and added to the wells at 4. Mu.L per well; after sealing the plate, after 2h reaction at room temperature, 5. Mu.L of 4X 40mM EDTA (final concentration: 10 mM) was added to each well, and 5min at room temperature, and then 5. Mu.L of 4X 8nM detection reagent (final concentration: 2nM, eu-anti-phospho-tyrosine antibody) was added to each well, and incubated at room temperature for 1 h, and a PE Envision multifunctional microplate reader was used to read plates (excitation 320nM, emission 665 nM) and IC50 was calculated using four-parameter fitting.

1.4 TEC inhibition Activity Screen

With kinase buffer (50 mM HEPES, 10mM MgCl) ₂ 50 ng/. Mu.L of TEC mother liquor was diluted with 2mM DTT, 1mM EGTA, 0.01% Tween 20, 6. Mu.L of 1.67X 0.01336 g/. Mu.L of working solution (final concentration of 0.008 ng/. Mu.L) was added to each well, and the different DMSO-dissolved compounds were added to the wells using a nanoliter-type loading apparatus to give a final concentration of 1000nM-0.24nM, 4-fold gradient for 7 concentrations, and blank control wells (containing enzyme, vehicle DMSO) were simultaneously set. After 30min of reaction of the enzyme with the compound or vehicle, 5. Mu.M ATP (final concentration of 10. Mu.M) prepared with kinase buffer was mixed with 5X 0.5. Mu.M substrate (final concentration of 0.1. Mu.M, ULIght-poly GT) at 1:1 and added to the wells at 4. Mu.L per well; after sealing the plate, the plate was sealed at room temperature for 2 hours, 5. Mu.L of 4X 40mM EDTA (final concentration: 10 mM) was added to each well, and the plate was incubated at room temperature for 5 minutes, and then 5. Mu.L of 4X 8nM detection reagent (final concentration: 2nM, eu-anti-phospho-tyrosine antibody) was added to each well, and the plate was incubated at room temperature for 1 hour; the PE Envision multifunctional microplate reader reads the plate (excitation 320nm, emission 665 nm), and IC50 was calculated using four parameter fitting.

1.5 ITK (Interlukin-2-subducible T-cell kinase) inhibitory activity screening

With kinase buffer (50 mM HEPES, 10mM MgCl) ₂ 50 ng/. Mu.L of ITK stock was diluted with 2mM DTT, 1mM EGTA, 0.01% Tween 20, 6. Mu.L of 1.67X 0.0835 g/. Mu.L of working solution (final concentration: 0.05 ng/. Mu.L) was added to each well, and DMSO was dissolved in a nanoliter-type loading deviceTo the wells, the final concentration of compound was made to be 1000nM-0.24nM, 4-fold gradient, 7 total, with blank control wells (enzyme-free) and negative control wells (enzyme-containing, vehicle DMSO). After 30min of reaction of the enzyme with the compound or vehicle, 5. Mu.M ATP (final concentration of 10. Mu.M) prepared with kinase buffer was mixed with 5X 0.5. Mu.M substrate (final concentration of 0.1. Mu.M, ULIght-poly GT) at 1:1 and added to the wells at 4. Mu.L per well; after sealing the plate, the plate was sealed at room temperature for 2 hours, 5. Mu.L of 4X 40mM EDTA (final concentration: 10 mM) was added to each well, and the plate was incubated at room temperature for 5 minutes, and then 5. Mu.L of 4X 8nM detection reagent (final concentration: 2nM, eu-anti-phospho-tyrosine antibody) was added to each well, and the plate was incubated at room temperature for 1 hour; the PE Envision multifunctional microplate reader reads the plate (excitation 320nm, emission 665 nm), and IC50 was calculated using four parameter fitting.

1.6 proliferation inhibition of TMD-8 cells by Compounds

Collecting TMD-8 cells in exponential growth phase, collecting cells into a centrifuge tube, centrifuging at low speed with a table centrifuge at 1500 rpm for 3min, discarding supernatant, and adding 2mL of plate culture medium (RPMI basal medium+5% FBS+0.05mM 2-mercaptoethanol) for cell resuspension. Counting with a cytometer, and adjusting the density of cells to 5×10 ⁴ Each mL was inoculated onto a 96-well plate using a row gun, 100. Mu.L/well, and cultured in a cell incubator at 37℃and 5% CO2 saturated humidity. After 24h incubation, compound addition was performed using a nanoliter addition apparatus, 2 duplicate wells were set for each concentration, and cells without compound were used as negative controls, after 72 hours CCK-8, 10 μl/well was added, after 4 hours, absorbance was measured at 450nm using an Envision microplate reader, four-parameter analysis, and the IC50 was calculated as a fit curve.

The results of the above test are shown in Table 1.

TABLE 1

Test example 2: in vivo pharmacokinetic experiments in mice

ICR mice weighing 18-22 g, after 3-5 days of adaptation, randomly grouping 9 mice in each group, and respectively injecting gastric-related compounds according to a dosage of 10mg/kg, and respectively injecting gastric-related compounds according to a dosage of 1 mg/kg. The animals (ICR mice) were fasted for 12h before dosing, and food was given 4h after dosing, with free water before, after and during the experiment. About 0.1mL of blood was collected from the orbits of 0.25 (15 min), 0.5 (30 min), 1, 2, 4, 6, 8, 10 and 24h after the administration of the gastric lavage, about 0.1mL of blood was collected from the orbits of 0.083 (5 min), 0.167 (10 min), 0.5 (30 min), 1, 2, 6, 8, 10 and 24h after the administration of the intravenous injection, 3 to 4 time points were collected from each mouse, 3 mice were collected from each time point, whole blood was placed in a centrifuge tube containing EDTA-K2 and sodium fluoride, and plasma was centrifuged at 4000rpm×10min after transferring to 4 ℃ for 30 min. All plasma was collected and stored at-20℃immediately for testing. Sucking 20 mu L of plasma sample to be detected and standard yeast sample, adding 300 mu L of acetonitrile solution containing internal standard (diazepam 20 mg/mL), shaking and mixing for 5min, centrifuging at 13000rpm for 10min, taking 80 mu L of supernatant, adding 80 mu L of ultrapure water for dilution, mixing, sucking 1 mu L for LC/MS/MS measurement, and recording a chromatogram. Oral, intravenous exposure of the compounds of the invention was assessed by in vivo pharmacokinetic experiments in mice and the results are shown in table 2.

TABLE 2

Note that: NA indicates no detection.

Test example 3: in vivo efficacy study

OCI-LY10 (human diffuse large B cell lymphoma) mice were subcutaneously transplanted with 1X 10 concentration ⁸ Per ml 0.1 ml/mouse, inoculated under aseptic conditions under the right armpit of NOD-SCID mice (inoculated sites shaved at the time of inoculation). Tumor volume after inoculation of subcutaneous transplanted tumor is 100-300mm ³ Animals were grouped right and left:

model group: 6 solvents; compound I-3:50mg/kg, bid, i.g. 6.

The vehicle or drug was administered by gavage at a volume of 10ml/kg, 2 times daily for 23 consecutive days. Tumor volume is measured for 2-3 times every week, meanwhile, the mice are weighed, and data are recorded; animal performance was observed daily. After all dosing was completed, animals were sacrificed and tumor-removed and weighed.

Tumor volume and tumor inhibition rate were calculated using the following formulas:

tumor Volume (TV) = (length x width ² )/2。

Tumor inhibition rate (tumor growth inhibition, TGI) = (1-treatment group tumor weight/model group tumor weight) ×100%.

Therapeutic effects of the compounds of Table 3 on mouse OCI-LY10 transplants

Experimental materials:

SCID mice, female, 6-8 weeks, beijing verdeli laboratory animal technologies limited, shanghai, license number SCXK (jing) 2016-0006, animal eligibility number: 1100111911008632.

Diffuse large B-cell lymphoma cell line OCI-LY10 (shanghai bayer biotechnology limited).

OCI-LY10 was grown in IMDM medium (GIBCO, USA) with 20% fetal bovine serum FBS (GIBCO, USA). Incubated in an incubator containing 5% CO 2.

Matrigel (BD company, usa).

Preparation of test compounds: absolute ethyl alcohol: tween 80: NS (V/V) is 10:10: stored at 80,4 ℃.

Establishing a human diffuse large B cell lymphoma OCI-LY10 SCID mouse subcutaneous transplantation tumor model:

collecting tumor cells in logarithmic growth phase, counting, re-suspending in IMDM culture medium, adding Matrigel 1:1, and adjusting cell suspension concentration to 4X10 ⁷ /ml. Subcutaneous inoculation of tumor cells, 4X 10, on the right dorsal aspect of SCID mice ⁶ 0.1 ml/mouse.

Until the average tumor volume reached about 167mm ³ When the tumor volume is 119.39-214.10mm ³ Animals were grouped by randomization based on tumor mean volume. The group diary was Day 0 and administration was started according to the average body weight. Animal body weight and tumor size were measured twice a week during the experiment.

TABLE 4 Table 4

The evaluation index of the anti-tumor activity is relative tumor increment rate T/C (%), and the calculation formula is as follows: T/C (%) =t _RTV /C _RTV *100％。(T _RTV : treatment group RTV; c (C) _RTV : RTV in vehicle control group);

Relative tumor volume (relative tumor volume, RTV), the calculation formula is: rtv=vt/V0. Where V0 is the tumor volume measured at the time of divided cage administration (i.e., day 0), and Vt is the tumor volume at each measurement.

The change in body weight (%) of the tumor-bearing animals was calculated as follows: (weight at measurement-weight at grouping)/weight at grouping x 100%.

The experimental results are shown in Table 5.

TABLE 5

* **: p <0.001 compared to vehicle group.

Claims (28)

1. A compound of formula (I) or a pharmaceutically acceptable salt thereof,

wherein, the liquid crystal display device comprises a liquid crystal display device,

ring a is selected from phenyl or 5-6 membered heteroaryl;

R ¹ independently selected from halogen, hydroxy, amino, cyano, C _1-6 Alkoxy, C _1-6 Alkyl, 5-6 membered heteroaryl, 5-6 membered heterocycloalkyl, C ₆ Aryl or C ₆ Cycloalkyl group, the C _1-6 Alkoxy, C _1-6 Alkyl, 5-6 membered heteroaryl, 5-6 membered heterocycloalkyl, C ₆ Aryl or C ₆ Cycloalkyl groups optionallyBy hydroxy, amino, cyano, halogen or halo C _1-6 Alkyl substitution;

m is selected from 0, 1, 2 or 3;

l is selected from-C (O) NH-, or-O-;

R ² independently selected from halogen, hydroxy, amino, cyano, C _1-6 Alkyl or C _1-6 Alkoxy group, the C _1-6 Alkyl or C _1-6 Alkoxy is optionally substituted with halogen;

n is selected from 0, 1, 2 or 3;

R ³ selected from hydrogen, or R ^a C(O)-；

Wherein R is ^a Selected from C _2-6 Alkynyl, or C _2-6 Alkenyl, R is as defined above ^a Optionally quilt (C) _1-6 Alkyl group ₂ N-、C _1-6 Alkyl NH-, hydroxy, amino, halogen or cyano.

2. A compound of formula (I) as defined in claim 1, or a pharmaceutically acceptable salt thereof, wherein ring a is selected from 6 membered heteroaryl.

3. A compound of formula (I) as defined in claim 1, or a pharmaceutically acceptable salt thereof, wherein ring a is selected from 6 membered nitrogen containing heteroaryl.

4. A compound of formula (I) as defined in claim 1, or a pharmaceutically acceptable salt thereof, wherein ring a is selected from pyridinyl.

5. A compound of formula (I) as defined in claim 1, or a pharmaceutically acceptable salt thereof, wherein R ¹ Independently selected from halogen, cyano, C _1-3 Alkoxy, C _1-3 Alkyl, 5-6 membered heteroaryl, 5-6 membered heterocycloalkyl, C ₆ Aryl or C ₆ Cycloalkyl group, the C _1-3 Alkoxy, C _1-3 Alkyl, 5-6 membered heteroaryl, 5-6 membered heterocycloalkyl, C ₆ Aryl or C ₆ Cycloalkyl optionally substituted by cyano, halogen or halo C _1-3 Alkyl substitution.

6. As claimed inThe compound of formula (I) or a pharmaceutically acceptable salt thereof as claimed in claim 1, wherein R ¹ Independently selected from halogen, C _1-3 Alkyl or C ₆ Aryl, said C _1-3 Alkyl or C ₆ Aryl optionally substituted by halogen or halo C _1-3 Alkyl substitution.

7. A compound of formula (I) as defined in claim 1, or a pharmaceutically acceptable salt thereof, wherein R ¹ Independently selected from fluorine, chlorine, bromine, halomethyl or phenyl optionally substituted with halogen.

8. A compound of formula (I) as defined in claim 1, or a pharmaceutically acceptable salt thereof, wherein R ¹ Independently selected from trifluoromethyl or phenyl optionally substituted with fluorine.

9. A compound of formula (I) as defined in claim 1, or a pharmaceutically acceptable salt thereof, wherein m is selected from 0, 1 or 2.

10. A compound of formula (I) as defined in claim 1, or a pharmaceutically acceptable salt thereof, wherein m is selected from 0 or 1.

11. A compound of formula (I) as defined in claim 1, or a pharmaceutically acceptable salt thereof, wherein L is selected from-C (O) NH-.

12. A compound of formula (I) as defined in claim 1, or a pharmaceutically acceptable salt thereof, wherein R ² Independently selected from halogen, hydroxy, amino, cyano, C _1-3 Alkyl or C _1-3 An alkoxy group.

13. A compound of formula (I) as defined in claim 1, or a pharmaceutically acceptable salt thereof, wherein R ² Independently selected from fluorine, chlorine or bromine.

14. A compound of formula (I) as defined in claim 1, or a pharmaceutically acceptable salt thereof, wherein R ² Independently selected from fluorine.

15. A compound of formula (I) as defined in claim 1, or a pharmaceutically acceptable salt thereof, wherein n is selected from 0, 1 or 2.

16. A compound of formula (I) as defined in claim 1, or a pharmaceutically acceptable salt thereof, wherein n is selected from 0 or 1.

17. A compound of formula (I) as defined in claim 1, or a pharmaceutically acceptable salt thereof, wherein R ³ Selected from R ^a C(O)-。

18. A compound of formula (I) as defined in claim 1, or a pharmaceutically acceptable salt thereof, wherein R ^a Selected from C _2-6 Alkynyl, or C _2-6 Alkenyl groups.

19. A compound of formula (I) as defined in claim 1, or a pharmaceutically acceptable salt thereof, wherein R ^a Selected from C _2-3 Alkynyl or C _2-3 Alkenyl groups.

20. A compound of formula (I) as defined in claim 1, or a pharmaceutically acceptable salt thereof, wherein R ^a Selected from propynyl.

21. A compound of formula (I) as defined in claim 1, or a pharmaceutically acceptable salt thereof, wherein R ³ Selected from the group consisting of

22. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 1, selected from the group consisting of a compound of formula (I-1) or a pharmaceutically acceptable salt thereof,

23. the compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 1, selected from the group consisting of a compound of formula (II) or a pharmaceutically acceptable salt thereof,

24. the compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 1, selected from the group consisting of a compound of formula (II-1) or a pharmaceutically acceptable salt thereof,

25. the following compounds or pharmaceutically acceptable salts thereof:

26. a pharmaceutical composition comprising a compound of any one of claims 1-25, or a pharmaceutically acceptable salt thereof.

27. Use of a compound according to any one of claims 1-25, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 26, in the manufacture of a medicament for the prevention or treatment of a BTK-related disease.

28. The use of claim 27, wherein the BTK-related disease is selected from an autoimmune disease, an inflammatory disease, or cancer.