CN112209933A - BTK inhibitors containing 4-azaspiroheptane - Google Patents

Abstract

The application belongs to the field of pharmaceutical chemistry, relates to a BTK inhibitor containing 4-azaspiroheptane, and particularly relates to a compound shown in a formula (I) or a 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-azaspiroheptane

Technical Field

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

Background

Bruton's Tyrosine Kinase (BTK) is mainly expressed in B cells, distributed in the lymphatic, hematopoietic and blood systems, is a member of the non-receptor type tyrosine kinase Tec family, which also includes Tec, ITK/TSK/EMT and BMX, and has a high structural homology. BTK plays a crucial role in B cell signaling pathways that link cell surface B cell receptor (B-cell receptor) stimulation to downstream intracellular responses, and is a key regulator of B cell development, activation, signaling, and survival. In recent years, researches on B cells, particularly B cell non-Hodgkin lymphoma and rheumatoid arthritis show that BTK is often abnormally expressed.

The BTK signal transduction pathway-based development of small molecule targeted drugs provides a brand-new approach for the treatment of B cell tumors such as leukemia and multiple myeloma and B cell immune diseases. Currently, irreversible inhibitors such as ibrutinib on the market often have mutations at BTK binding sites, which leads to reduced pharmaceutical activity and thus drug resistance, so that more BTK inhibitors are clinically needed and have higher selectivity for BTK, thereby avoiding toxic and side effects caused by off-target effect.

Detailed Description

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

wherein the content of the first and second substances,

ring A is selected from 5-10 membered heteroaryl or C_6-10An aryl group;

R¹independently selected from halogen, hydroxy, amino, cyano, C_1-6Alkoxy radical, C_1-6Alkyl, 5-10 membered heteroaryl, 5-10 membered heterocycloalkyl, C_6-10Aryl or C_6-10Cycloalkyl radical, said C_1-6Alkoxy radical, C_1-6Alkyl, 5-10 membered heteroaryl, 5-10 membered heterocycloalkyl, C_6-10Aryl or C_6-10Cycloalkyl optionally substituted by hydroxy, amino, cyano, halogen or halogeno C_1-6Alkyl 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)₂O-or-OS (O)₂-；

R²Independently selected from halogen, hydroxy, amino, cyano, C_1-6Alkyl or C_1-6Alkoxy radical, said C_1-6Alkyl or C_1-6Alkoxy is optionally substituted with halogen;

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

R³selected from hydrogen, R^aS(O)₂-、(R^aO)₂P (O) -or R^aC(O)-；

Wherein R is^aIndependently selected from C_2-6Alkynyl, C_2-6Alkenyl radical, C_1-6Alkyl radical, C_3-6Cycloalkyl group, (C)_1-6Alkyl) NH-, (C)_1-6Alkyl radical)₂N-, 3-6 membered heterocycloalkyl, 5-10 membered heteroaryl or C_6-10Aryl radical, the above R^aOptionally is (C)_1-6Alkyl radical)₂N-、(C_1-6Alkyl) 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 pyridyl.

In some embodiments, R¹Independently selected from halogen, cyano, C_1-3Alkoxy radical, C_1-3Alkyl, 5-6 membered heteroaryl, 5-6 membered heterocycloalkyl, C_6-10Aryl or C_6-10Cycloalkyl radicalsSaid C is_1-3Alkoxy radical, C_1-3Alkyl, 5-6 membered heteroaryl, 5-6 membered heterocycloalkyl, C_6-10Aryl or C_6-10Cycloalkyl optionally substituted by cyano, halogen or halogeno C_1-3Alkyl substitution; in some embodiments, R¹Independently selected from halogen, C_1-3Alkyl or C_6-10Aryl radical, said C_1-3Alkyl or C_6-10Aryl being optionally substituted by halogen or halogeno C_1-3Alkyl substitution; in some embodiments, R¹Independently selected from fluoro, chloro, bromo, halomethyl or phenyl optionally substituted with halo; in some embodiments, R¹Independently selected from trifluoromethyl or phenyl optionally substituted by 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 embodiments, R²Independently selected from halogen, hydroxy, amino, cyano, C_1-3Alkyl or C_1-3An alkoxy group; in some embodiments, R²Independently selected from fluorine, chlorine or bromine; in some embodiments, R²Independently 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, R³Is selected from R^aC(O)-。

In some embodiments, R^aIndependently selected from C_2-6Alkynyl, C_2-6Alkenyl radical, C_1-6Alkyl radical, C_3-6Cycloalkyl or 3-6 membered heterocycloalkyl; in some embodiments, R^aIndependently selected from C_2-3Alkynyl or C_2-3An alkenyl group; in some embodiments, R^aSelected from propynyl.

In some embodiments, R³Is selected from

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

wherein, ring A, L, R¹、R²、R³M and n are as defined above.

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

wherein R is¹、R²M and n are as defined above.

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

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

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

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

In another aspect, the present application relates to the above-mentioned compound or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof for preventing or treating BTK-related diseases.

In some embodiments, the BTK-related disease is selected from an autoimmune disease, an inflammatory disease, or a cancer.

Definition of

The following terms used in the present application have the following meanings, unless otherwise specified. A particular term should not be considered as ambiguous or unclear without special definition, but rather construed according to ordinary meaning in the art. When a trade name appears herein, it is intended to refer to its corresponding commodity or its active ingredient.

The term "substituted" means that any one or more hydrogen atoms on a particular atom is replaced with a substituent, so long as 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 aryl.

The terms "optionally" or "optionally" mean 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 is "optionally" substituted with halo, meaning that ethyl may be unsubstituted (CH)₂CH₃) Monosubstituted (e.g. CH)₂CH₂F) Polysubstituted (e.g. CHFCH)₂F、CH₂CHF₂Etc.) or completely substituted (CF)₂CF₃). It will be appreciated by those skilled in the art that any group containing one or more substituents will not incorporate any substitution or substitution pattern which is sterically impossible and/or cannot be synthesized.

Herein C_m-nThat the portion is in the given rangeAn integer number of carbon atoms. E.g. "C_1-6By "is meant that the group can 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 one time in the composition or structure of a compound, its definition in each case is independent. For example, if a group contains 2R, then each R has a separate option.

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

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

The term "amino" refers to the group-NH₂A group.

The term "cyano" refers to the group — CN.

The term "alkyl" refers to a group of formula C_nH_2n+1A hydrocarbon group of (1). The alkyl group may be linear or branched. For example, the term "C_1-6Alkyl "means an alkyl group having 1 to 6 carbon atoms (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, neopentyl, hexyl, 2-methylpentyl, and the like). Similarly, the alkyl portion (i.e., alkyl) of alkoxy, alkylamino, dialkylamino, alkylsulfonyl and alkylthio groups have the same definitions as above.

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

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, ethenyl, 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 having at least one triple bond composed of carbon atoms and hydrogen atoms. Non-limiting examples of alkynyl groups include, but are not limited to, ethynyl (-C ≡ CH), 1-propynyl (-C ≡ C-CH)₃) 2-propynyl (-CH)₂-C.ident.CH), 1, 3-butadiynyl (-C.ident.C-C.ident.CH), and the like.

The term "cycloalkyl" refers to a carbon ring that is fully saturated and may exist as a single ring, a bridged ring, or a 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, a bridged ring, or a spiro ring. Unless otherwise indicated, the heterocyclic ring 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 include, but are not limited to, oxiranyl, thietanyl, cycloazenyl, non-limiting examples of 4-membered heterocycloalkyl include, but are not limited to, azetidinyl, oxetanyl, thiabutinyl, examples of 5-membered heterocycloalkyl include, but are not limited to, tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, isoxazolidinyl, oxazolidinyl, isothiazolidinyl, thiazolidinyl, imidazolidinyl, examples of tetrahydropyrazolyl, 6-membered heterocycloalkyl include, but are not limited to, piperidinyl, tetrahydropyranyl, tetrahydrothiopyranyl, morpholinyl, piperazinyl, 1, 4-thialkyl, 1, 4-dioxanyl, thiomorpholinyl, 1, 3-dithianyl, 1, 4-dithianyl, and examples of 7-membered heterocycloalkyl include, but are not limited to, azepanyl, oxepanyl, thiepanyl. Monocyclic heterocycloalkyl groups having 5 or 6 ring atoms are preferred.

The term "aryl" refers to an all-carbon monocyclic or fused polycyclic aromatic ring group having a conjugated pi-electron system. For example, the aryl group can have 6 to 20 carbon atoms, 6 to 14 carbon atoms, or 6 to 12 carbon atoms. Non-limiting examples of aryl groups include, but are not limited to, phenyl, naphthyl, anthracenyl, 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 heteroaryls 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 "treating" means administering a compound or formulation described herein to ameliorate or eliminate a disease or one or more symptoms associated with the disease, and includes:

(i) inhibiting the disease or disease state, i.e., arresting its development;

(ii) alleviating the disease or condition, i.e., causing regression of the disease or condition.

The term "preventing" means administering a compound or formulation described herein to prevent a disease or one or more symptoms associated with the disease, including: prevention of a disease or condition occurs in a mammal, particularly when such mammal is susceptible to the disease condition, but has not yet been diagnosed as having the disease condition.

The term "therapeutically effective amount" means an amount of a compound of the present 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 a compound of the present application that constitutes a "therapeutically effective amount" varies 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 those skilled in the art with their own knowledge and this 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 the pharmaceutically acceptable salt, for example, a metal salt, an ammonium salt, a salt with an organic base, a salt with an inorganic acid, a salt with an organic acid, a salt with a basic or acidic amino acid, and the like can be mentioned.

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

The term "pharmaceutically acceptable adjuvants" refers to those adjuvants which do not have a significant irritating effect on the organism and do not impair the biological activity and properties of the active compound. Suitable adjuvants are well known to those 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 "comprise" and variations thereof such as "comprises" or "comprising," are to be understood in an open, non-exclusive sense, i.e., "including but not limited to.

The compounds and intermediates of the present application may also exist in different tautomeric forms, and all such forms are included within the scope of the present 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 referred to as proton transfer tautomers) include interconversion via proton migration, such as keto-enol and imine-enamine isomerizations. A specific example of a proton tautomer is an imidazole moiety, wherein the proton can migrate between two ring nitrogens. Valence tautomers include interconversion by recombination of some of the bonding electrons.

The present application also includes isotopically-labeled compounds of the present 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 mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into compounds of the present application include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine and chlorine, such as respectively²H、³H、¹¹C、¹³C、¹⁴C、¹³N、¹⁵N、¹⁵O、¹⁷O、¹⁸O、³¹P、³²P、³⁵S、¹⁸F、¹²³I、¹²⁵I and³⁶cl, and the like.

Certain isotopically-labelled compounds of the present application (e.g. with³H and¹⁴c-labeled ones) can be used in compound and/or substrate tissue distribution assays. Tritiated (i.e. by tritiation)³H) And carbon-14 (i.e.¹⁴C) Isotopes are particularly preferred for their ease of preparation and detectability. Positron emitting isotopes, such as¹⁵O、¹³N、¹¹C and¹⁸f 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 in the examples below, by substituting an isotopically labeled reagent for a non-isotopically labeled reagent.

In addition, heavier isotopes are used (such as deuterium (i.e., deuterium)²H) Substitution may provide certain therapeutic advantages resulting from greater 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 present application may be asymmetric, e.g., having one or more stereoisomers. Unless otherwise indicated, all stereoisomers include, for example, enantiomers and diastereomers. The compounds of the present application containing asymmetric carbon atoms can be isolated in optically active pure form or in racemic form. The optically active pure form can be resolved from a racemic mixture or synthesized by using chiral starting materials or chiral reagents.

The pharmaceutical compositions of the present application can be prepared by combining the compounds of the present application with suitable pharmaceutically acceptable excipients, for example, can be formulated into 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 a compound of the present application or a pharmaceutically acceptable salt thereof or a pharmaceutical composition 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 can be manufactured by methods well known in the art, such as conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, lyophilizing, and the like.

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

Solid oral compositions may be prepared by conventional mixing, filling or tableting methods. For example, it can be obtained by the following method: the active compounds are mixed with solid adjuvants, optionally the mixture obtained is milled, if desired with further suitable adjuvants, and the mixture is then processed to granules, to give tablets or dragee cores. 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 adapted for parenteral administration, as sterile solutions, suspensions or lyophilized products in suitable unit dosage forms.

In all methods of administration of the compounds of the general formula I described herein, the daily dose is from 0.01 to 200mg/kg body weight, in single or 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 listed below, embodiments formed by combinations thereof with other chemical synthetic methods, and equivalents thereof known to those skilled in the art, with preferred embodiments including, but not limited to, the examples of the present application.

The chemical reactions of the embodiments herein are carried out 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 based on the existing embodiments.

An important consideration in the art of synthetic route planning is the selection of suitable protecting Groups for reactive functional Groups (e.g., amino Groups as used herein), for example, see Greene's Protective Groups in Organic Synthesis (4th Ed.) Hoboken, New Jersey: John Wiley & Sons, Inc.

In some embodiments, the compounds of formula (II) herein may be prepared by methods known in the art by those skilled in the art of organic synthesis by the following routes:

wherein R is¹、R²M or n are as defined above, R⁴Is an amino protecting group selected from Cbz (benzyloxycarbonyl) or Boc (tert-butoxycarbonyl).

The following abbreviations are used in this application:

PE represents petroleum ether; EA represents ethyl acetate; DMSO represents dimethyl sulfoxide; DMF represents N, N-dimethylformamide; DCM represents dichloromethane; TBSCl represents tert-butyldimethylsilyl chloride; boc₂O represents di-tert-butyl dicarbonate; NBS represents N-bromosuccinimide; MeOH represents methanol; DTT represents dithiothreitol; EGTA stands for ethylene glycol bis (2-aminoethyl ether) tetraacetic acid; HATU stands for 2- (7-benzotriazol oxide) -N, N, N ', N' -tetramethyluronium hexafluorophosphate.

For clarity, the invention is further illustrated by examples, which do not limit the scope of the application. All reagents used herein were commercially available and used without further purification.

Detailed Description

Example 1: preparation of (S) -4- (8-amino-3- (4- (but-2-ynoyl) -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)

Intermediate 7-1(50g) was added to a reaction flask and dissolved in DCM (500mL), imidazole (59.1g) was added at 0 deg.C, TBSCl (2.21g) was added while maintaining the temperature, the reaction was continued at 0 deg.C, after completion of the reaction, the reaction solution was added to 500mL of saturated brine, methyl t-butyl ether was extracted, the organic phase was washed with a 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 (63.9g) is added into a reaction bottle, tetrahydrofuran (1000mL) is added, intermediate 7-2(100g) is added into the bottle dropwise at 0 ℃ under the protection of nitrogen, the temperature is maintained for reaction for about 1h, benzyl bromide (71mL) is added into the system for room temperature reaction, after the reaction is completed, the reaction solution is slowly added into 2L of ice water, methyl tert-butyl ether is extracted, an organic phase is washed by a saturated saline solution, dried by anhydrous sodium sulfate, filtered, and concentrated and purified by column chromatography (a developing agent: PE: EA: 100:50) to obtain compound 7-3(124.56 g).

¹H 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.

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

Adding tetrahydrofuran (1000mL) into a dry reaction bottle, adding ethyl magnesium bromide (310mL) under the protection of nitrogen, cooling at 70 ℃, dropwise adding tetraisopropyl titanate (92mL) into the bottle after the temperature is reached, maintaining the temperature for reaction for about 1h, adding 7-3(100g) into the system for room temperature reaction, slowly adding the reaction solution into 1L of ice water after the reaction is finished, performing suction filtration to remove black solid insoluble substances, performing extraction with methyl tert-butyl ether, washing an organic phase with saturated saline solution, drying with anhydrous sodium sulfate, filtering, and concentrating to obtain a crude compound 7-4 (110.52 g).

¹H 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

And 4, step 4: (S) - (4-benzyl-4-azaspiro [2.4] heptan-5-yl) methanol (intermediate 7-5)

The crude intermediate 7-4(100g) was charged into a reaction flask, dissolved in tetrahydrofuran (1000mL), and then a tetrahydrofuran solution of tetrabutylammonium fluoride (302mL,1mol/L) was added to react at 40 ℃, after completion of the reaction, the reaction system was concentrated, and then extracted with methyl tert-butyl ether and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to purify by column chromatography (developing solvent: DCM: MeOH 100:5) to obtain compound 7-5(16.18 g).

¹H 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.

And 5: (S) - (4-Azaspiro [2.4] hept-5-yl) methanol hydrochloride (intermediate 7-6)

Adding compound 7-5(15g) into a reaction flask, adding methanol (150mL) and concentrated hydrochloric acid (22.63mL), adding reduced palladium carbon (1.086g), replacing with hydrogen for 3-4 times, reacting at 30 deg.C with hydrogen balloon under hydrogen atmosphere, concentrating the reaction system, and removing water to obtain compound 7-6(12.51g)

¹H 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)

Compound 7-6(12g) was added to a reaction flask, dissolved in DCM (150mL) and cooled to 0 deg.C, triethylamine (29.2mL) was added to the system, followed by Boc₂O (17.65 mL). The temperature is maintained for reaction, and after the reaction is finished, methyl tert-butyl ether and saturated saline solution are used for extraction. Dried over anhydrous sodium sulfate, filtered and concentrated to give compound 7-7(19.86g)

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

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

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

Compound 7-8(17.56g) was added to a reaction flask, and (3-chloropyrazin-2-yl) methylamine hydrochloride (12.72g) was added, dissolved with DCM (200mL), and triethylamine (23.42mL) was added, followed by HATU (17g) and the mixture was reacted at room temperature. After the reaction, the reaction system was diluted with methyl t-butyl ether and 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 (developing solvent: PE: EA ═ 100:20) to give compound 7-9(12.78g)

¹H 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.

And 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 compound 7-9(12g) is added into a reaction bottle at 0 ℃ and N₂Ethyl acetate (120mL), DMF (7.46mL) was added under protection, and then phosphorus oxychloride (20.96mL) was added dropwise to the system to react at room temperature. After the reaction, the reaction system was slowly added to an ice water solution of ammonia water, the mixture was stirred to neutralize the reaction, and then methyl tert-butyl ether was used to extract the reaction system, the organic phase was dried over anhydrous sodium sulfate, and the mixture was concentrated to obtain compound 7-10(8.62g)

¹H 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, compounds 7-10(3.9g), DMF (40mL), NBS (2.69g), N were added in that order₂Under protection, the mixture is heated to 60 ℃ for reaction. After completion of the reaction, the reaction mixture was cooled to room temperature, and 200mL of ethyl acetate and 20mL of a saturated aqueous solution of sodium thiosulfate were added to the reaction mixture, followed by extraction with saturated brine, drying over anhydrous sodium sulfate, filtration, and purification by column chromatography (developer: PE: EA ═ 100:10) by concentration 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)

A pressure-tight reaction flask was charged with compound 7-11(1.5g), a solution of ammonia in isopropanol, (32.5ml,2mol/L)), and the mixture was heated to 120 ℃. After completion of the reaction, the reaction mixture was cooled to room temperature, concentrated and purified by column chromatography (developer: PE: EA: 100:10) to obtain compound 7-12(440mg)

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 compound 7-12(150mg), 3-fluoro-N- (4- (4-fluorophenyl) pyridin-2-yl) -4- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborane-2-yl) benzamide (210mg), potassium carbonate (177mg), [1,1' -bis (diphenylphosphino) ferrocene ] dichloropalladium (65.5mg), then 1, 4-dioxane (5mL), water (1mL), nitrogen substitution 3-4 times, heating to 80 ℃ for reaction, cooling to room temperature after the reaction was completed, adding a saturated saline solution to dilute the system, and extracting 2-3 times with DCM. The organic phase was dried over anhydrous sodium sulfate, concentrated and purified by column chromatography (developing solvent: DCM: MeOH: 100:3) to give compound 7-13(136 mg).

¹H 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-ynoyl) -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 compound 7-13(120mg) into methanol solution (20% -30%, 3mL) of hydrochloric acid, reacting at 50 deg.C for about 5-10min, concentrating, and drying to constant weight to obtain compound 7-14; to the reaction flask were added HATU (79mg), 2-butynoic acid (15.82mg), which was dissolved in DCM, and triethylamine (0.105mL) was added to react at room temperature. After the reaction, saturated sodium bicarbonate was added to dilute the reaction solution, followed by extraction with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate, concentrated and purified by column chromatography (developing solvent: DCM: MeOH ═ 100:3) to give compound I-1(125mg)

¹H NMR(500MHz,DMSO-d₆)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-ynoyl) -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 a reaction flask were added compounds 7-12(200mg), (2-fluoro-4- ((4-phenylpyridin-2-yl) carbamoyl) phenyl) boronic acid (232mg), potassium carbonate (136mg), [1,1' -bis (diphenylphosphino) ferrocene ] dichloropalladium (87mg) in this order, followed by addition of 1, 4-dioxane (5mL), water (1mL), nitrogen substitution 3-4 times, heating to 80 ℃ for reaction, cooling to room temperature after completion of the reaction, addition of a saturated saline solution to dilute the system, and extraction with DCM 2-3 times. The organic phase was dried over anhydrous sodium sulfate, concentrated and purified by column chromatography (developing solvent: DCM: MeOH: 100:3) to give compounds 8-13(136 mg).

¹H NMR(500MHz,DMSO-d₆)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-ynoyl) -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 compound 8-13(180mg) into methanol solution (20% -30%, 3mL) of hydrochloric acid, reacting at 50 deg.C for about 5-10min, concentrating, and drying to constant weight to obtain compound 8-14; to the reaction flask were added HATU (121mg) and 2-butynoic acid (24.42mg) successively, and after dissolving with DCM, triethylamine (118mg) was added and the reaction was carried out at room temperature. After the reaction, saturated sodium bicarbonate was added to dilute the reaction solution, followed by extraction with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate, concentrated and purified by column chromatography (developing solvent: DCM: MeOH ═ 100:3) to give compound I-2(105mg)

¹H NMR(500MHz,DMSO-d₆)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-ynoyl) -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 a reaction flask were added compounds 7-12(200mg), N- (pyridin-2-yl) -4- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborane-2-yl) benzamide (159mg), potassium carbonate (243mg), [1,1' -bis (diphenylphosphino) ferrocene ] dichloropalladium (35.8mg) in this order, followed by addition of 1, 4-dioxane (5mL), water (1mL), nitrogen substitution 3-4 times, reaction by heating to 80 ℃ and cooling to room temperature after completion of the reaction, addition of a saturated saline dilution system and extraction with DCM 2-3 times. The organic phase was dried over anhydrous sodium sulfate, concentrated and purified by column chromatography (developing solvent: DCM: MeOH: 100:5) to give compound 9-13(178 mg).

¹H 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-ynoyl) -4-azaspiro [2.4] heptan-5-yl) imidazo [1,5-a ] pyrazin-1-yl) -N- (pyridin-2-yl) benzamide (Compound I-3)

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

¹H 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-ynoyl) -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)

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)

The compound 7-12(200mg), 3-fluoro-4- (4,4,5, 5-tetramethyl-1, 3, 2-dioxolan-2-yl) -N- (4- (trifluoromethyl) pyridin-2-yl) benzamide (215mg), potassium carbonate (243mg), [1,1' -bis (diphenylphosphino) ferrocene ] dichloropalladium (35.4mg) were sequentially added to a reaction flask, followed by addition of 1, 4-dioxane (5mL), water (1mL), nitrogen substitution 3 to 4 times, heating to 80 ℃ for reaction, cooling to room temperature after the reaction was completed, addition of a saturated saline solution as a dilution system, and extraction with DCM 2 to 3 times. The organic phase was dried over anhydrous sodium sulfate, concentrated and purified by column chromatography (developing solvent: DCM: MeOH: 100:5) to give compound 10-13(248 mg).

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

¹H 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 inhibitory Activity screening

With kinase buffer (50mM HEPES, 10mM MgCl)₂2mM DTT, 1mM EGTA, 0.01% Tween 20) 350 ng/. mu.L of BTK (WT) stock was diluted, 6. mu.L of 1.67X 0.0334 ng/. mu.L working solution (final concentration of 0.02 ng/. mu.L) was added to each well, DMSO-dissolved different compounds were added to the wells using a nanoliter loading apparatus to give a final concentration of 1000nM-0.244nM, 4-fold gradient for 7 concentrations, blank control empty (no enzyme) and negative control wells (enzyme-containing, vehicle DMSO) were set up in 2 duplicate wells. After 30min of reaction of the enzyme with the compound or vehicle, 5 × 100 μ M ATP (final concentration of 20 μ M) and 5 × 0.5 μ M substrate (final concentration of 0.1 μ M, ULight-poly GT) in kinase buffer were mixed as described in 1:1 mixing, adding 4 mu L of the mixture into each hole; after the plate was sealed with a membrane, after reacting at room temperature for 2 hours, 5. mu.L of 4X 40mM EDTA (final concentration: 10mM) was added to each well for 5 minutes at room temperature, and 5. mu.L of 4X 8nM detection reagent (final concentration: 2nM, Ab) was added to each well and incubated at room temperature for 1 hour; and (3) reading the plate (excitation is 620nm, emission is 665nm) by using a PE Envision multifunctional microplate reader, and calculating IC50 by adopting four-parameter fitting.

1.2 BTK (C481S) inhibition Activity Screen

With kinase buffer (50mM HEPES, 10mM MgCl)₂2mM DTT, 1mM EGTA, 0.01% Tween 20) 50 ng/. mu.L of BTK (C481S) stock was diluted, 6. mu.l of 1.67 X0.25 ng/. mu.l working solution (final concentration of 0.15 ng/. mu.l) was added to each well, DMSO-dissolved different compounds were added to the wells using a nanoliter loader to give a final concentration of 1000nM to 0.244nM, 4-fold gradient, 7 concentrations, blank control empty (no enzyme) and negative control wells (enzyme-containing, vehicle DMSO) were set, and 2 duplicate wells were set. After the enzyme reacts with the compound or the solvent for 30min,5 X250. mu.M ATP (final concentration 50. mu.M) and 5 X0.5. mu.M substrate (final concentration 0.1. mu.M, ULight-poly GT) in kinase buffer were mixed in a ratio of 1:1 mixing, adding 4 mu L of the mixture into each hole; after the plate was sealed with a membrane, after reacting at room temperature for 2 hours, 5. mu.L of 4X 40mM EDTA (final concentration: 10mM) was added to each well for 5 minutes at room temperature, and 5. mu.L of 4X 8nM detection reagent (final concentration: 2nM, Ab) was added to each well and incubated at room temperature for 1 hour; and (3) reading the plate (excitation is 620nm, emission is 665nm) by using a PE Envision multifunctional microplate reader, and calculating IC50 by adopting four-parameter fitting.

1.3 EGFR (epidermal growth factor receptor) inhibitory Activity Screen

With kinase buffer (50mM HEPES, 10mM MgCl)₂2mM DTT, 1mM EGTA, 0.01% Tween 20) 50 ng/. mu.L of EGFR (WT) stock was diluted, 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, DMSO-dissolved different compounds were added to the wells using a nanoliter loading apparatus to give a final concentration of 1000nM-0.48nM, 4-fold gradient for 7 concentrations, blank control empty (no enzyme) and negative control wells (enzyme-containing, vehicle DMSO) were set up in 2 duplicate wells. After the enzyme reacts with the compound or the solvent for 10min, 5 × 25 μ M ATP (final concentration of 5 μ M) prepared with a kinase buffer solution and 5 × 0.5 μ M substrate (final concentration of 0.1 μ M, ULight-poly GT) are mixed in a ratio of 1:1 and added to the wells at 4 μ L per well; after the plate is sealed and the plate is covered with a membrane, after the reaction is carried out for 2 hours at room temperature, 5 mu L of 4 multiplied 40mM EDTA (10 mM final concentration) is added into each hole, the reaction is carried out for 5 minutes at room temperature, 5 mu L of 4 multiplied 8nM detection reagent (2 nM final concentration, Eu-anti-phosphorus-tyrosinane antibody) is added into each hole, the incubation is carried out for 1 hour at room temperature, a PE Envision multifunctional enzyme-linked immunosorbent assay is used for reading the plate (excitation 320nM, emission 665nM), and the IC50 is calculated by adopting four-parameter fitting.

1.4 TEC inhibition Activity screening

With kinase buffer (50mM HEPES, 10mM MgCl)₂2mM DTT, 1mM EGTA, 0.01% Tween 20) 50 ng/. mu.L of TEC stock solution was diluted, 6. mu.L of 1.67X 0.01336 g/. mu.L working solution (final concentration of 0.008 ng/. mu.L) was added to each well, and different compounds dissolved in DMSO were added to the wells using a nanoliter loading apparatus to give a final concentration of 1000nM to 0.24nM, 4-fold gradient, total of 7 concentrated solutionsBlank control wells (no enzyme) and negative control wells (enzyme, vehicle DMSO) were also set. After the enzyme reacts with the compound or the solvent for 30min, 5 × 50 μ M ATP (final concentration of 10 μ M) prepared by using a kinase buffer solution and 5 × 0.5 μ M substrate (final concentration of 0.1 μ M, ULight-poly GT) are mixed according to a ratio of 1:1 and added into the wells according to 4 μ L per well; after the plate is sealed and the plate is covered with a membrane, after reacting for 2h at room temperature, 5. mu.L of 4X 40mM EDTA (final concentration 10mM) is added to each well for 5min at room temperature, and 5. mu.L of 4X 8nM detection reagent (final concentration 2nM, Eu-anti-phosphorus-tyrosine antibody) is added to each well and incubated for 1h at room temperature; and (3) reading the plate (excitation is 320nm, emission is 665nm) by using a PE Envision multifunctional microplate reader, and calculating IC50 by adopting four-parameter fitting.

1.5 ITK (Interleukin-2-indole T-cell kinase) inhibition activity screening

With kinase buffer (50mM HEPES, 10mM MgCl)₂2mM DTT, 1mM EGTA, 0.01% Tween 20) 50 ng/. mu.L of ITK stock solution was diluted, 6. mu.L of 1.67X 0.0835 g/. mu.L working solution (final concentration of 0.05 ng/. mu.L) was added to each well, and DMSO-dissolved different compounds were added to the wells using a nanoliter applicator to give a final concentration of 1000nM to 0.24nM, 4-fold gradient, 7 concentrations, while blank control empty (no enzyme) and negative control wells (enzyme-containing, vehicle DMSO) were set. After the enzyme reacts with the compound or the solvent for 30min, 5 × 50 μ M ATP (final concentration of 10 μ M) prepared by using a kinase buffer solution and 5 × 0.5 μ M substrate (final concentration of 0.1 μ M, ULight-poly GT) are mixed according to a ratio of 1:1 and added into the wells according to 4 μ L per well; after the plate is sealed and the plate is covered with a membrane, after reacting for 2h at room temperature, 5. mu.L of 4X 40mM EDTA (final concentration 10mM) is added to each well for 5min at room temperature, and 5. mu.L of 4X 8nM detection reagent (final concentration 2nM, Eu-anti-phosphorus-tyrosine antibody) is added to each well and incubated for 1h at room temperature; and (3) reading the plate (excitation is 320nm, emission is 665nm) by using a PE Envision multifunctional microplate reader, and calculating IC50 by adopting four-parameter fitting.

1.6 proliferation inhibition of TMD-8 cells by Compounds

Collecting TMD-8 cells in exponential growth phase, collecting cells, centrifuging at 1500 rpm for 3min in a low-speed desktop centrifuge, discarding supernatant, and adding 2mL of plating medium (RPMI basic culture)Base + 5% FBS +0.05mM 2-mercaptoethanol) were used. Counting with a cell counter, taking the required amount of cells, adjusting the density to 5 × 10⁴one/mL of the cells were inoculated onto a 96-well plate using a discharge gun at 100. mu.L/well, and cultured in a cell culture chamber containing 5% CO2 at 37 ℃ and saturated humidity. After 24h of culture, compound loading is carried out by using a nano-lift loading instrument, 2 duplicate wells are arranged at each concentration, cells without the compound are used as negative control, CCK-8 and 10 mu L/well are added after 72 h, the absorbance value is detected at 450nm of an Envision enzyme-labeling instrument after 4h, four-parameter analysis is carried out, a dose-effect curve is fitted, and IC50 is calculated.

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

TABLE 1

Test example 2: in vivo pharmacokinetic experiments in mice

ICR mice with the weight of 18-22 g are randomly grouped after being adapted for 3-5 days, 9 mice in each group are separately gavaged with related compounds according to the dose of 10mg/kg, and the related compounds are separately injected statically at the dose of 1 mg/kg. The test animals (ICR mice) were fasted for 12h before administration and food for 4h after administration, and water was freely available before and after the experiment and during the experiment. About 0.1mL of blood is taken from the orbit at 0.25(15min), 0.5(30min), 1,2, 4, 6, 8, 10 and 24h after the gastric lavage, about 0.1mL of blood is taken from the orbit at 0.083(5min), 0.167(10min), 0.5(30min), 1,2, 6, 8, 10 and 24h after the intravenous injection, 3 to 4 time points are collected from each mouse, 3 mice are collected at each time point, the whole blood is placed in a centrifuge tube containing EDTA-K2 and sodium fluoride, the whole blood is transferred to 4 ℃ within 30min, and the plasma is separated by centrifugation at 4000rpm x 10 min. All plasma was collected and immediately stored at-20 ℃ for testing. Absorbing 20 mu L of plasma sample to be detected and a standard curve sample, adding 300 mu L of acetonitrile solution containing an internal standard (diazepam 20mg/mL), shaking and uniformly mixing for 5min, centrifuging at 13000rpm for 10min, taking 80 mu L of supernatant, adding 80 mu L of ultrapure water for dilution, uniformly mixing, absorbing 1 mu L of ultrapure water for LC/MS/MS determination, and recording a chromatogram. Oral, intravenous exposure of the compounds of the invention was evaluated by in vivo pharmacokinetic experiments in mice and the results are shown in table 2.

TABLE 2

Note: NA indicates no detection.

Test example 3: in vivo efficacy study

OCI-LY10 (human diffuse large B-cell lymphoma) mice subcutaneous transplantation tumor at a concentration of 1X 10⁸0.1 ml/mouse, inoculated under sterile conditions in the right axilla of NOD-SCID mice (the site of inoculation was shaved). After the subcutaneous transplantation tumor is inoculated, the tumor volume is up to 100-300mm³Left and right animals were grouped:

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

The vehicle or drug is administered by intragastric administration in a volume of 10ml/kg, 2 times daily for 23 days. Measuring the tumor volume 2-3 times per week, weighing the mice, and recording data; animal performance was observed daily. After all dosing was complete, the animals were sacrificed, and the tumors were stripped and weighed.

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

tumor Volume (TV) ═ length x width²)/2。

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

Therapeutic Effect of the Compounds of Table 3 on mouse OCI-LY10 transplantable tumors

Experimental materials:

SCID mouse, female, 6-8 weeks, Shanghai division of Experimental animals technology, Inc., Weitonglihua, Beijing, license number SCXK (Jing) 2016-: 1100111911008632.

diffuse large B-cell lymphoma cell line OCI-LY10 (Shanghai Baili Biotech Co., Ltd.).

OCI-LY10 was cultured in IMDM medium (GIBCO, USA) containing 20% fetal bovine serum FBS (GIBCO, USA). Cultured in an incubator containing 5% CO2 at room temperature.

Matrigel (BD company, usa).

Preparation of test compounds: anhydrous ethanol: tween 80: NS (V/V) is 10: 10: storing at 80 and 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 at ratio of 1:1, and regulating cell suspension concentration to 4 × 10⁷And/ml. SCID mice were inoculated subcutaneously on the right dorsal side with 4X 10 tumor cells⁶0.1 ml/mouse.

When the average tumor volume reaches about 167mm³Then selecting the tumor volume of 119.39-214.10mm³The animals were grouped by randomization based on the mean tumor volume. The Day of the grouping was Day 0 and dosing was started according to average body weight. Animal body weights and tumor sizes were determined twice a week during the experiment.

TABLE 4

The evaluation index of the anti-tumor activity is relative tumor proliferation rate T/C (%), and the calculation formula is as follows: T/C (%) ═ T_RTV/C_RTV*100％。(T_RTV: treatment group RTV; c_RTV: vehicle control group RTV);

relative Tumor Volume (RTV), the formula is: RTV is Vt/V0. Where V0 is the tumor volume measured at the time of caged administration (i.e., Day 0) and Vt is the tumor volume at each measurement.

The change (%) in body weight of tumor-bearing animals was calculated as follows: (weight at measurement-weight at time of group)/weight at time of group × 100%.

The results are shown in Table 5.

TABLE 5

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

Claims (10)

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

wherein the content of the first and second substances,

ring A is selected from 5-10 membered heteroaryl or C_6-10An aryl group;

R¹independently selected from halogen, hydroxy, amino, cyano, C_1-6Alkoxy radical, C_1-6Alkyl, 5-10 membered heteroaryl, 5-10 membered heterocycloalkyl, C_6-10Aryl or C_6-10Cycloalkyl radical, said C_1-6Alkoxy radical, C_1-6Alkyl, 5-10 membered heteroaryl, 5-10 membered heterocycloalkyl, C_6-10Aryl or C_6-10Cycloalkyl optionally substituted by hydroxy, amino, cyano, halogen or halogeno C_1-6Alkyl 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)₂O-or-OS (O)₂-；

R²Independently selected from halogen, hydroxy, amino, cyano, C_1-6Alkyl or C_1-6Alkoxy radical, said C_1-6Alkyl or C_1-6Alkoxy is optionally substituted with halogen;

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

R³selected from hydrogen, R^aS(O)₂-、(R^aO)₂P (O) -or R^aC(O)-；

Wherein R is^aIs selected from C_2-6Alkynyl, C_2-6Alkenyl radical, C_1-6Alkyl radical, C_3-6Cycloalkyl radical, C_1-6Alkyl NH-, (C)_1-6Alkyl radical)₂N-, 3-6 membered heterocycloalkyl, 5-10 membered heteroaryl or C_6-10Aryl radical, the above R^aOptionally is (C)_1-6Alkyl radical)₂N-、C_1-6Alkyl NH-, hydroxy, amino, halogen or cyano.

2. A compound of formula (I) or a pharmaceutically acceptable salt thereof as claimed in claim 1 wherein ring a is selected from phenyl or 5-6 membered heteroaryl; optionally, wherein ring a is selected from 6-membered heteroaryl; optionally, wherein ring a is selected from 6-membered nitrogen-containing heteroaryl; optionally wherein ring a is selected from pyridyl.

3. A compound of formula (I) or a pharmaceutically acceptable salt thereof as claimed in claims 1-2 wherein R¹Independently selected from halogen, cyano, C_1-3Alkoxy radical, C_1-3Alkyl, 5-6 membered heteroaryl, 5-6 membered heterocycloalkyl, C_6-10Aryl or C_6-10Cycloalkyl radical, said C_1-3Alkoxy radical, C_1-3Alkyl, 5-6 membered heteroaryl, 5-6 membered heterocycloalkyl, C_6-10Aryl or C_6-10Cycloalkyl optionally substituted by cyano, halogen or halogeno C_1-3Alkyl substitution; optionally, wherein R is¹Independently selected from halogen, C_1-3Alkyl or C_6-10Aryl radical, said C_1-3Alkyl or C_6-10Aryl being optionally substituted by halogen or halogeno C_1-3Alkyl substitution; optionally, wherein R is¹Independently selected from fluoro, chloro, bromo, halomethyl or phenyl optionally substituted with halo; optionally, wherein R is¹Independently selected from trifluoromethyl or phenyl optionally substituted by fluorine;

optionally, wherein m is selected from 0, 1,2,3 or 4; optionally, wherein m is selected from 0, 1 or 2; optionally, wherein m is selected from 0 or 1.

4. A compound of formula (I), or a pharmaceutically acceptable salt thereof, as claimed in any one of claims 1-3 wherein L is selected from-c (o) NH-or-nhc (o) -; optionally, wherein L is selected from-C (O) NH-.

5. A compound of formula (I) as claimed in any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof, which isIn R²Independently selected from halogen, hydroxy, amino, cyano, C_1-3Alkyl or C_1-3An alkoxy group; optionally, wherein R is²Independently selected from fluorine, chlorine or bromine; optionally, wherein R is²Independently selected from fluorine;

optionally, wherein n is selected from 0, 1 or 2; optionally, wherein n is selected from 0 or 1.

6. A compound of formula (I), or a pharmaceutically acceptable salt thereof, as claimed in any one of claims 1-5 wherein R³Is selected from R^aC(O)-；

Optionally, wherein R is^aIs selected from C_2-6Alkynyl, C_2-6Alkenyl radical, C_1-6Alkyl radical, C_3-6Cycloalkyl or 3-6 membered heterocycloalkyl; optionally, wherein R is^aIs selected from C_2-3Alkynyl or C_2-3An alkenyl group; optionally, wherein R is^aSelected from propynyl;

optionally, wherein R is³Is selected from

7. A compound of formula (I) according to claim 1, or a pharmaceutically acceptable salt thereof, selected from a compound of formula (I-1) or a pharmaceutically acceptable salt thereof,

8. a compound of formula (I) or a pharmaceutically acceptable salt thereof as claimed in claim 1 selected from a compound of formula (II) or formula (II-1) or a pharmaceutically acceptable salt thereof,

9. the following compounds or pharmaceutically acceptable salts thereof:

10. use of a compound of any one of claims 1-9, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the prevention or treatment of a BTK-related disease, optionally wherein the BTK-related disease is selected from an autoimmune disease, an inflammatory disease, or cancer.