CN112209934A - BTK inhibitors containing azaspiroheptanes - Google Patents

BTK inhibitors containing azaspiroheptanes Download PDF

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Publication number
CN112209934A
CN112209934A CN202010656864.4A CN202010656864A CN112209934A CN 112209934 A CN112209934 A CN 112209934A CN 202010656864 A CN202010656864 A CN 202010656864A CN 112209934 A CN112209934 A CN 112209934A
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optionally
compound
independently selected
ring
alkyl
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CN112209934B (en
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张寅生
任景
徐胜
王庆璘
赵天笑
施伟
王晓金
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Chia Tai Tianqing Pharmaceutical Group Co Ltd
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Chia Tai Tianqing Pharmaceutical Group Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators

Abstract

The application belongs to the field of pharmaceutical chemistry, relates to a BTK inhibitor containing 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 azaspiroheptanes
Technical Field
The present application relates to BTK inhibitors containing azaspiroheptanes, 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,
Figure BDA0002577067610000011
wherein the content of the first and second substances,
ring A is selected from 5-10 membered heteroaryl or C6-10An aryl group;
ring D is selected from 5-10 membered heteroaryl, C6-10Aryl or 5-10 membered heterocycloalkyl;
R1independently selected from halogen, hydroxy, amino, cyano, C1-6Alkoxy or C1-6Alkyl radical, said C1-6Alkoxy or C1-6Alkyl optionally substituted with halo;
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, C1-6Alkyl or C1-6Alkoxy radical, said C1-6Alkyl or C1-6Alkoxy is optionally substituted with halogen;
n is selected from 0, 1,2,3 or 4;
structure of the product
Figure BDA0002577067610000012
Is selected from
Figure BDA0002577067610000013
Wherein the content of the first and second substances,
R3independently selected from hydrogen, RaS(O)2-、(RaO)2P (O) -or RaC(O)-；
Wherein R isaIndependently selected from C2-6Alkynyl, C2-6Alkenyl radical, C1-6Alkyl radical, C3-6Cycloalkyl group, (C)1-6Alkyl) NH-, (C)1-6Alkyl radical)2N-, 3-6 membered heterocycloalkyl, 5-10 membered heteroaryl or C6-10Aryl radical, the above RaOptionally is (C)1-6Alkyl radical)2N-、(C1-6Alkyl) NH-, hydroxy, amino, halogen or cyano;
R4independently selected from ═ O, ═ S, C1-6Alkyl radical, C1-6Alkoxy, cyano or halogen;
q is selected from 0, 1,2,3 or 4.
In some embodiments, ring a is selected from phenyl or 5-6 membered heteroaryl; in some embodiments, ring a is selected from 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, ring D is selected from 5-6 membered heteroaryl, phenyl, or 5-6 membered heterocycloalkyl; in some embodiments, ring D is selected from 5-membered heteroaryl, 6-membered heteroaryl, phenyl, or 6-membered heterocycloalkyl; in some embodiments, ring D is selected from pyridyl, pyrimidinyl, oxazolyl, thiazolyl, imidazolyl, phenyl, piperidinyl, morpholinyl, piperazinyl, or thiophene; in some embodiments, ring D is selected from pyridyl, pyrimidinyl, oxazolyl, thiazolyl, imidazolyl, phenyl, piperidinyl, morpholinyl, or piperazinyl.
In some embodiments, R1Independently selected from halogen, cyano, C optionally substituted by halogen1-3Alkyl or C optionally substituted by halogen1-3An alkoxy group; in some embodiments, R1Independently selected from halogen, C optionally substituted by halogen1-3An alkyl group; in some embodiments, R1Independently selected from halogen or C1-3An alkyl group; in some embodiments, R1Independently selected from fluoro, methyl or ethyl.
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, 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, C1-3Alkyl or C1-3An alkoxy group; in some embodiments, R2Independently selected from halogen; 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, n is selected from 1.
In some embodiments, R3Independently selected from RaC(O)-。
In some embodiments, RaIndependently selected from C2-6Alkynyl, C2-6Alkenyl radical, C1-6Alkyl radical, C3-6Cycloalkyl or 3-6 membered heterocycloalkyl; in some embodiments, RaIndependently selected from C2-3Alkynyl or C3-4A cycloalkyl group; in some embodiments, RaIndependently selected from propynyl or cyclopropyl.
In some embodiments, R4Independently selected from ═ O, ═ S, C1-3Alkyl or C1-3An alkoxy group; in some embodiments, R4Independently selected from ═ O, ═ S, or methyl.
In some embodiments, q is selected from 0, 1 or 2.
In some embodiments, a structure
Figure BDA0002577067610000021
Is selected from
Figure BDA0002577067610000022
Wherein R is3、R4And q 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 a pharmaceutically acceptable salt thereof,
Figure BDA0002577067610000031
wherein, ring D, R1、R2、R3、R4M, n, q 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 (III) or a pharmaceutically acceptable salt thereof,
Figure BDA0002577067610000032
wherein, ring D, R1、R2、R3、R4M, n, q 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 (IV) or a pharmaceutically acceptable salt thereof,
Figure BDA0002577067610000033
wherein, ring D, R1、R2、R3、R4M, n, q 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:
Figure BDA0002577067610000041
Figure BDA0002577067610000051
in another aspect, the present application relates to a pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof, as described herein. 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 of formula (I) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.
In another aspect, the present application relates to the use of a compound of formula (I), 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 present application relates to the use of a compound of formula (I) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, in the prevention or treatment of a BTK-related disease.
In another aspect, the present application relates to a compound of formula (I) 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 Cm-nIt is the moiety that has an integer number of carbon atoms in the given range. E.g. "C1-6By "it is meant that the group may have1 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.
For the
Figure BDA0002577067610000052
When L is selected from-CO (NH) -, it represents
Figure BDA0002577067610000053
Is composed of
Figure BDA0002577067610000054
When a substituent's bond is cross-linked to two atoms on a ring, such substituent may be bonded to any atom on the ring.
For example,
Figure BDA0002577067610000061
when q is 1, it represents
Figure BDA0002577067610000062
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 CnH2n+1A hydrocarbon group of (1). The alkyl group may be linear or branched. For example, the term "C1-6Alkyl "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). Class ISimilarly, 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 a 5-to 6-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 prevent, ameliorate or eliminate a disease or one or more symptoms associated with the disease, and includes:
(i) preventing the occurrence of a disease or condition in a mammal, particularly when such mammal is susceptible to the disease condition, but has not yet been diagnosed as having the disease condition;
(ii) inhibiting the disease or disease state, i.e., arresting its development;
(iii) alleviating the disease or condition, i.e., causing regression of the disease or 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, with partial deuterium substitutionMeaning that at least one hydrogen is substituted by at least one deuterium, e.g.
Figure BDA0002577067610000081
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 (such as amino Groups in the present application), for example, reference may be made to Greene's Protective Groups in Organic Synthesis (4th Ed.) Hoboken, New Jersey: John Wiley & Sons, Inc. all references cited herein are incorporated herein in their entirety.
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:
Figure BDA0002577067610000091
wherein, ring D, R1、m、R2、n、R3、R4Or q is as defined above.
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; NBS represents N-bromosuccinimide; DIPEA stands for diisopropylethylamine; MeOH represents methanol; EDTA represents ethylene diamine tetraacetic acid; DTT represents dithiothreitol; EGTA stands for ethylene glycol bis (2-aminoethyl ether) tetraacetic acid; ATP represents adenosine triphosphate; HATU represents 2- (7-benzotriazol oxide) -N, N' -tetramethyluronium hexafluorophosphate; ACN represents acetonitrile; et (Et)3N represents triethylamine; cbz represents benzyloxycarbonyl; PdCl2(dppf)CH2Cl2Represents [1,1' -bis (diphenylphosphino) ferrocene]A palladium dichloride dichloromethane complex; NIS stands for N-iodosuccinimide.
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 (2-fluoro-4- ((4-phenylpyridin-2-yl) carbamoyl) phenyl) boronic acid (intermediate H)
Figure BDA0002577067610000092
Intermediate H1(3.74g) was added to a reaction flask and dissolved in DMF (25mL), intermediate H2(2.86g) and DIPEA (4.38g) were added at 0 ℃, after stirring for 10 minutes at 0 ℃, HATU (7.73g) was added, heated to 80 ℃ under nitrogen protection for 6 hours to complete the reaction, the reaction solution was poured into ice water of 2 to 3 times the volume of the reaction solution, stirred uniformly and filtered, and the obtained filter cake was purified by silica gel column chromatography (developer: PE: EA: 1, and then DCM: MeOH: 4:1) to obtain intermediate H (3.03 g).
1H NMR(500MHz,DMSO)δ10.978(s,1H),8.406(s,2H),7.792-7.765(m,3H),7.738-7.659(m,2H),7.581-7.494(m,4H),3.180(s,2H).MS(ESI,[M-H]+)m/z：335.3.
Example 2: preparation of intermediates 1 to 5
Figure BDA0002577067610000101
Step 1: (S) -6- (((3-Chloropyrazin-2-yl) methyl) carbamoyl) -5-azaspiro [2.4] heptane-5-carboxylic acid benzyl ester (1-2)
Adding (S) -5- ((benzyloxy) carbonyl) -5-azaspiro [2.4] into a reaction bottle]Heptane-6-carboxylic acid (8.41g) dissolved in DCM (150ml) and intermediate 1(5g) and Et were added3N (13.63mL), HATU (11.15g) was added under ice-water bath, after completion of the addition, the mixture was warmed to room temperature and stirred continuously until the reaction was completed, and the reaction mixture was successively treated with 2M concentrated hydrochloric acid and saturated NaHCO3Washing with water solution, saturated NaCl water solution, and washing the organic layer with anhydrous MgSO4The mixture was stirred, dried, filtered, and the filtrate was concentrated and purified by a silica gel column (developing solvent: PE: EA: 80:20 to 50:50) to obtain intermediate 1-2(8.24 g).
1H NMR(500MHz,DMSO-d6):δ8.62-8.54(dd,J1＝34.0Hz,J2＝2.0Hz,1H),8.47-8.40(d,J＝32.0Hz,1H),8.43(s,1H),7.38-7.30(m,5H),5.08-5.03(m,2H),4.56-4.39(m,3H),3.46-3.38(dd,J1＝30.5Hz,J2＝10.0Hz,1H),2.29-2.25(m,1H),2.03-1.97(m,1H),1.82-1.77(m,1H),0.58-0.48(m,4H).MS(ESI,[M+H]+)m/z：401.3.
Step 2: (S) -benzyl 6- (8-chloroimidazo [1,5-a ] pyrazin-3-yl) -5-azaspiro [2.4] heptane-5-carboxylate (1-3)
Intermediate 1-2(7.3g), ACN (75mL) and DMF (1.5mL) were added to a reaction flask and POCl was slowly added dropwise over an ice-water bath3(6.69ml), after the dropwise addition, the temperature was gradually raised to room temperature, and the stirring was continued for 1.5 hours to complete the reaction. Slowly adding the reaction solution into a mixed system of concentrated ammonia water and crushed ice, stirring until the crushed ice is melted, alkalifying the reaction solution until the pH value is about 9, extracting with EA for 2 times, and combining organic layersWashing with saturated salt water for 2 times, anhydrous MgSO4Drying, filtration and concentration gave intermediate 1-3(7.56 g).
1H NMR(500MHz,DMSO-d6):δ8.49-8.27(dd,J1＝107Hz,J2＝4.5Hz,1H),7.87-7.78(d,J＝40.5Hz,1H),7.43-7.30(m,3H),7.21-7.17(dd,J1＝15.0Hz,J2＝4.5Hz,1H),7.10-7.08(m,1H),6.67-6.66(d,J＝7.0Hz,1H),5.58-5.56(m,1H),5.03-4.93(m,1H),4.93-4.63(dd,J1＝138.5Hz,J2＝12.5Hz,1H),3.65-3.61(m,1H),3.49-3.41(dd,J1＝30Hz,J2＝10Hz,1H),2.26-2.24(m,2H),0.73-0.58(m,4H).MS(ESI,[M+H]+)m/z：383.3.
And step 3: (S) -benzyl 6- (1-bromo-8-chloroimidazo [1,5-a ] pyrazin-3-yl) -5-azaspiro [2.4] heptane-5-carboxylate (1-4)
Adding intermediate 1-3(7g) and DMF (70ml) into a reaction flask, stirring at room temperature to dissolve, adding NBS (3.32g) in ice water bath, reacting at room temperature for 1 hr after adding, adding water (700ml), extracting with EA for 2 times, mixing organic layers, washing with saturated salt water for 2 times, and anhydrous MgSO4Drying, filtration and concentration gave intermediate 1-4(7.21 g). MS (ESI, [ M + H ]]+)m/z：461.3.
And 4, step 4: (S) -benzyl 6- (8-amino-1-bromoimidazo [1,5-a ] pyrazin-3-yl) -5-azaspiro [2.4] heptane-5-carboxylate (intermediate 1-5)
Intermediate 1-4(7.1g) was dissolved in isopropanol (40ml), transferred to a pressure-resistant sealed tube, and Et was added3N (4.14mL) and an isopropanol solution of ammonia (149mL,2.0mmol/mL), sealing the reaction device, heating to 120 ℃, stirring for reacting for 20 hours until the reaction is complete, and cooling the reaction solution to room temperature. Concentrating the reaction solution, adding water to the concentrated residue, extracting with EA for 3 times, mixing the organic layers, washing with saturated salt water for 2 times, and mixing the organic layers with MgSO4Drying, filtering, concentrating the filtrate, and purifying by column chromatography (developing solvent: DCM: MeOH: 98: 2-96: 4) to obtain intermediate 1-5(4.86 g).
1H NMR(500MHz,DMSO-d6):δ7.73-7.60(dd,J＝60Hz,4.0Hz,1H),7.36-7.15(m,4H),7.01-6.90(dd,J＝53.5Hz,4.0Hz,1H),6.6-6.74(d,J＝6.5Hz,1H),6.62(s,2H),5.44-5.40(d,J＝20Hz,1H),5.02-4.69(m,2H),3.60-3.58(d,J＝10.5Hz,1H),3.46-3.39(m,1H),2.25-2.21(m,1H),2.13-2.10(m,1H),0.70(s,1H),0.69-0.64(d,J＝24Hz,1H),0.56-0.55(d,J＝6.0Hz,2H).MS(ESI,[M+H]+)m/z：442.5.
Example 3: (S) -4- (8-amino-3- (5- (but-2-ynoyl) -5-azaspiro [2.4] heptan-6-yl) imidazo [1,5-a ] pyrazin-1-yl) -3-fluoro-N- (4-phenylpyridin-2-yl) benzamide (Compound I-1)
Figure BDA0002577067610000111
Step 1: (S) -benzyl 6- (8-amino-1- (2-fluoro-4- ((4-phenylpyridin-2-yl) carbamoyl) phenyl) imidazo [1,5-a ] pyrazin-3-yl) -5-azaspiro [2.4] heptane-5-carboxylate (intermediate 2-6)
Intermediates 1 to 5(1.26g) and intermediate H (0.98g) were added to a sealed tube, dissolved in dioxane (10mL), potassium carbonate (1.58g) and water (1mL) were added, stirring was carried out for 1 minute, then [1,1' -bis (diphenylphosphino) ferrocene ] dichloropalladium (II) dichloromethane complex (0.58g) was added, nitrogen was bubbled through for 1 minute, and then the microwave reactor was heated to 80 ℃ under 150W for 40 minutes. After the reaction, the reaction mixture was dissolved in 40mL of water, extracted with EA, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography (developing solvent: DCM: MeOH: 100:1 to 40:1) to obtain intermediate 2-6(0.48 g).
1H NMR(500MHz,DMSO)δ11.07(s,1H),8.06-8.03(m,2H),7.81-7.66(m,4H),7.60-7.51(m,5H),7.33(s,3H),7.18-7.12(m,3H),6.78-6.76(d,J＝7Hz,1H),6.01(s,2H),5.76(s,1H),5.02-5.00(d,J＝11.5Hz,2H),3.62-3.60(m,2H),2.33-2.19(m,2H),0.71-0.55(m,4H).MS(ESI,[M+H]+)m/z：654.5.
Step 2: (S) -4- (8-amino-3- (5-azaspiro [2.4] heptan-6-yl) imidazo [1,5-a ] pyrazin-1-yl) -3-fluoro-N- (4-phenylpyridin-2-yl) benzamide (intermediate 2-7)
Intermediate 2-6(0.40g) was added to the reaction flask and dissolved in MeOH (30mL), ammonium formate (0.32g) and palladium on carbon (0.054g) were added and refluxed overnight under nitrogen. After the reaction, the reaction solution was filtered, the filtrate was concentrated and then added to 50mL of water, EA was extracted, the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography (developing solvent: DCM: MeOH: 20:1 to 10:1) to obtain intermediate 2 to 7(0.13 g).
1H NMR(500MHz,DMSO)δ11.06(s,1H),8.54-8.49(m,2H),8.06-8.03(m,2H),7.87-7.86(d,J＝5Hz,1H),7.81-7.79(m,2H),7.67-7.63(m,1H),7.59-7.52(m,4H),7.12-7.11(d,J＝4.5Hz,1H),6.03(s,2H),4.88(s,1H),2.96-2.86(m,2H)2.44-2.06(m,2H),0.66-0.58(m,4H).MS(ESI,[M+H]+)m/z：520.4.
And step 3: intermediate 2-7(0.060g) was charged to a reaction flask and dissolved in DCM (6mL), 2-butynoic acid (0.0092g) and triethylamine (0.047g) were added at 0 ℃ and after stirring for 5 minutes at 0 ℃, HATU (0.046g) was added and the reaction continued at 0 ℃, after completion of the reaction, the reaction was dissolved in 30mL of water, DCM was extracted, the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated and purified by preparative thin layer chromatography (developer: EA: MeOH 10:1) to give compound I-1(0.047 g).
1H NMR(500MHz,DMSO)δ11.06(s,1H),8.54-8.49(m,2H),8.06-8.03(m,2H),7.85-7.79(m,3H),7.64-7.52(m,5H),7.15-7.11(m,1H),6.10-6.06(d,J＝18Hz,2H),5.80-5.60(m,1H),3.80-3.52(m,2H),2.37-2.15(m,2H),2.00(s,3H),0.71-0.60(m,4H).HR-MS(ESI,[M+H]+)m/z：586.2335.
Example 4: (S) -4- (8-amino-3- (5- (cyclopropanecarbonyl) -5-azaspiro [2.4] hept-6-yl) imidazo [1,5-a ] pyrazin-1-yl) -3-fluoro-N- (4-phenylpyridin-2-yl) benzamide (Compound I-2)
Figure BDA0002577067610000121
Step 1: intermediate 2-7(0.060g) was charged to a reaction flask and dissolved in DCM (6mL), cyclopropanecarboxylic acid (0.0099g) and triethylamine (0.047g) were added at 0 ℃, after stirring for 5 minutes at 0 ℃, HATU (0.0461g) was added, the mixture was reacted at 0 ℃ for 1 hour, the reaction solution was dissolved in 30mL of water, DCM was extracted, the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by preparative thin layer chromatography (developer: EA: MeOH ═ 15:1) to give compound I-2(0.043 g).
1H NMR(500MHz,DMSO)δ11.06(s,1H),8.54-8.49(m,2H),8.05-8.03(m,2H),7.81-7.78(m,3H),7.65-7.52(m,5H),7.08-7.07(m,1H),6.09-6.06(d,J＝17.5Hz,2H),5.57-5.55(m,1H),3.89-3.74(m,2H),2.27-2.18(m,2H),1.34(s,1H)0.86-0.67(m,8H).HR-MS(ESI,[M+H]+)m/z：588.2484.
Example 5: preparation of (2-fluoro-4- ((4- (4-fluorophenyl) pyridin-2-yl) carbamoyl) phenyl) boronic acid (intermediate G)
Figure BDA0002577067610000122
Step 1: 4- (4-fluorophenyl) pyridin-2-amine (intermediate G3)
Intermediate G1(10G), intermediate G2(12.13G) and potassium carbonate (32G) were charged into a reaction flask, dissolved in dioxane (240mL), and [1,1' -bis (diphenylphosphino) ferrocene ] dichloropalladium (II) dichloromethane complex (4.72G) was added to the flask, followed by addition of water (40mL), nitrogen substitution 3 to 4 times and heating to 80 ℃ for 60 minutes. After the reaction, the reaction mixture was dissolved in 300mL of water, extracted with DCM, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography (developing solvent: DCM: MeOH: 100:2 to 100:4) to obtain intermediate G3 (9.94G). MS (ESI, [ M + H ] +) M/z: 189.4.
step 2: (2-fluoro-4- (4- (4- (4-fluorophenyl) pyridin-2-yl) carbamoyl) phenyl) boronic acid (intermediate G)
Intermediate H1(17.47G) was added to a reaction flask and dissolved in DMF (180mL), intermediate G3(8.94G) and DIPEA (18.41G) were added at 0 ℃ and after stirring at 0 ℃ for 10 minutes HATU (36.1G) was added and heated to 80 ℃ under nitrogen protection to react for 80 minutes, the reaction was completed, the reaction solution was poured into ice water of 2 to 3 times the volume of the reaction solution, stirred well and filtered, and the resulting filter cake was slurried with 0.5N diluted hydrochloric acid solution and filtered to give intermediate G (16.107G).
1H NMR(500MHz,DMSO)δ11.859(s,1H),8.603(s,1H),8.531-8.519(d,J＝6Hz,1H),7.966-7.875(m,4H),7.743-7.713(m,2H),7.467-7.433(m,2H).MS(ESI,[M-H]+)m/z：353.3.
Example 6: (S) -4- (8-amino-3- (5-but-2-ynoyl) -5-azaspiro [2.4] heptan-6-yl) imidazo [1,5-a ] pyrazin-1-yl) -3-fluoro-N- (4- (4-fluorophenyl) pyridin-2-yl) benzamide (Compound I-3)
Figure BDA0002577067610000131
Step 1: (S) -6- (8-amino-1- (2-fluoro-4- ((4- (4-fluorophenyl) pyridin-2-yl) carbamoyl) phenyl) imidazo [1,5-a ] pyrazin-3-yl) -5-azaspiro [2.4] heptane-5-carboxylic acid benzyl ester (intermediate 3-6)
Intermediate 1 to 5(7.2G), intermediate G (9.72G) and potassium carbonate (9G) were charged into a reaction flask, dissolved in dioxane (216mL), and [1,1' -bis (diphenylphosphino) ferrocene ] dichloropalladium (II) dichloromethane complex (3.32G) was added, water (36mL) was added, and after 3 to 4 nitrogen replacements, the reaction was carried out at 80 ℃ for 60 minutes. After the reaction, the reaction mixture was dissolved in 300mL of water, extracted with DCM, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography (developing solvent: DCM: MeOH: 100:3) to obtain intermediate 3-6(7.8 g). MS (ESI, [ M + H ] +) M/z: 672.5.
step 2: (S) -4 (8-amino-3- (5-azaspiro [2.4] heptan-6-yl) imidazo [1,5-a ] pyrazin-1-yl) -3-fluoro-N- (4- (4-fluorophenyl) pyridin-2-yl) benzamide (intermediate 3-7)
Step 2: intermediate 3-6(7g) was added to the reaction flask and dissolved in MeOH (400mL), ammonium formate (19.71g) and palladium on carbon (3.3g) were added and refluxed overnight under nitrogen. After completion of the reaction, the reaction mixture was filtered, the filtrate was concentrated, and the resulting product was purified by silica gel column chromatography (developing solvent: DCM: MeOH: 100:5 to 100:8) to obtain intermediate 3-7(3.18 g). MS (ESI, [ M + H ]]+)m/z：538.5.
And step 3: adding the intermediate 3-7(3g) into a reaction bottle, dissolving the intermediate in DCM (300mL), adding 2-butynoic acid (0.42g) and triethylamine (2.24g) at 0 ℃, stirring for 5 minutes at 0 ℃, adding HATU (2.21g), continuing to react at 0 ℃, dissolving the reaction solution in 300mL of water after the reaction is finished, extracting DCM, drying an organic phase anhydrous sodium sulfate, filtering, concentrating, and purifying by silica gel column chromatography to obtain the compound I-3(1.73 g).
1H NMR(500MHz,DMSO)δ11.08(s,1H),8.52-8.48(m,2H),8.06-8.03(m,2H),7.87-7.85(m,3H),7.79-7.78(d,J＝5Hz,1H),7.65-7.51(m,1H),7.42-7.39(m,2H),7.15-7.11(m,1H),6.10-6.04(m,2H),5.81-5.58(m,1H),3.82-3.50(m,2H),2.32-2.12(m,2H),2.00(s,2H),1.59(s,1H),0.76-0.59(m,4H).MS(ESI,[M+H]+)m/z：604.5.
Example 7: preparation of N- (4-bromopyridin-2-yl) -3-fluoro-4- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) benzamide (intermediate J)
Figure BDA0002577067610000132
Step 1: 3-fluoro-4- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) benzoic acid (24.54g), 4-bromopyridin-2-amine (13.3g) and triethylamine (31.1g) were added to a reaction flask, and dissolved in DCM (300mL), after stirring at room temperature for 20 minutes, HATU (35.1g) was added, and the mixture was heated to 70 ℃ to react for 6 hours, the reaction was completed, the reaction mixture was added to water (500mL) for liquid separation, the organic phase was evaporated and redissolved in DMF (50mL), 1M aqueous HCl (500mL) was added, the mixture was stirred uniformly and filtered, and the resulting cake was slurried with a little EA and dried to give intermediate J (17.277 g). MS ESI [ M + H- (2, 3-dimethylbutane)]+m/z: 339.3 (boric acid form molecular weight).
Example 8: preparation of (S) -4- (8-amino-3- (5-but-2-ynoyl) -5-azaspiro [2.4] heptan-6-yl) imidazo [1,5-a ] pyrazin-1-yl) -N- (4-bromopyridin-2-yl) -3-fluorobenzamide (intermediate K)
Figure BDA0002577067610000141
Step 1: (S) -benzyl 6- (1-iodo-8-chloroimidazo [1,5-a ] pyrazin-3-yl) -5-azaspiro [2.4] heptane-5-carboxylate (4-4)
Adding the intermediate 1-3(50.9g) and DMF (400ml) into a reaction flask, stirring at room temperature to dissolve, adding NIS (42.6g) into an ice water bath, reacting at 60 ℃ for overnight hours after the addition is finished, completely reacting, adding ice water (800ml) of saturated sodium thiosulfate, extracting with EA for 2 times, combining organic layers, washing with saturated salt water for 2 timesAnhydrous NaSO4Drying, filtration and concentration gave intermediate 4-4(62.81 g). MS (ESI, [ M + H ]]+)m/z：509.3.
Step 2: (S) -benzyl 6- (8-amino-1-iodoimidazo [1,5-a ] pyrazin-3-yl) -5-azaspiro [2.4] heptane-5-carboxylate (intermediate 4-5)
Dissolving the intermediate 4-4(62.8g) in isopropanol (180mL), transferring to a pressure-resistant sealed tube, adding an isopropanol solution of ammonia (1400mL,2.0mmol/mL), sealing the reaction device, heating to 120 ℃, stirring for reacting overnight, completely reacting, and cooling the reaction solution to room temperature. The reaction mixture was concentrated, and the concentrated residue was purified by column chromatography (developing solvent: PE: EA: 100:0 to 30:70) to give intermediate 4-5(41.0 g). 1H NMR (500MHz, DMSO) δ 7.79-7.66(dd,1H),7.36-7.16(M,4H),7.01-6.89(dd,1H),6.72-6.71(d, J ═ 6.5Hz,1H),6.56(s,2H),5.44-5.39(M,1H),5.03-4.70(M,2H),3.61-3.59(M,1H),3.45-3.38(M,1H),2.19-2.12(M,2H),0.70-0.55(M,4H). MS (ESI, [ M + H ], [ M + H-]+)m/z：490.3.
And step 3: (S) -benzyl 6- (8-amino-1- (4- ((4-bromopyridin-2-yl) carbamoyl) -2-fluorophenyl) imidazo [1,5-a ] pyrazin-3-yl) -5-azaspiro [2.4] heptane-5-carboxylate (intermediate 4-6)
And step 3: intermediate 4-5(16.5g), intermediate J (14.63g) and potassium carbonate (18.64g) were added to a reaction flask, dissolved in dioxane (300mL), and [1,1' -bis (diphenylphosphino) ferrocene was added]Palladium (II) dichloride dichloromethane complex (4.68g), water (50mL) was added, and after 3 to 4 nitrogen replacements, the reaction was carried out at 80 ℃ for 60 minutes. After completion of the reaction, the reaction mixture was dissolved in 300mL of water, extracted with DCM, dried over anhydrous sodium sulfate, filtered, concentrated and purified by silica gel column chromatography (developing solvent: DCM: MeOH: 97.5:2.5) to give intermediate 4 to 6(12.56 g). 1H NMR (500MHz, DMSO) δ 11.23(s,1H),8.50(s,1H),8.35-8.34(d, J ═ 5Hz,1H),8.04-8.00(M,2H),7.81-7.63(M,2H),7.53-7.47(M,2H),7.33(s,2H),7.17-7.00(M,2H),6.77-6.76(d, J ═ 6.5Hz,1H),6.08(s,2H),5.54(M,1H),5.03-4.74(M,2H),3.62-3.60(M,1H),3.51-3.43(M,1H),2.33-2.29(M,1H),2.19(s,1H),0.70-0.56(M,4H), [ M, ESI ] + M (M,1H), [ M,1H ], (M,1H) ], 0.56(M, 1H, M,1H, M]+)m/z：656.4.
And 4, step 4: (S) -4- (8-amino-3- (5-azaspiro [2.4] heptan-6-yl) imidazo [1,5-a ] pyrazin-1-yl) -N- (4-bromopyridin-2-yl) -3-fluorobenzamide (intermediate 4-7)
The intermediates 4 to 6(12g) and an acetic acid solution (150ml) of 33% HBr were put into a pressure resistant tube and reacted at room temperature for 2 hours. At the end of the reaction, the reaction was concentrated under reduced pressure to remove most of the solution, the concentrate was redissolved with water (600ml) and washed with DCM (200ml × 3), and the base was adjusted in an aqueous ice bath with DCM: MeOH ═ 10:1 mixed solvent extraction (2.5L), organic phase dried over anhydrous sodium sulfate, and dried by rotary drying under reduced pressure to give intermediate 4-7(7.85 g). 1H NMR (500MHz, DMSO) δ 11.22(s,1H),8.49(s,1H),8.34-8.33(d, J ═ 5Hz,1H),8.02-7.98(M,2H),7.86-7.85(d, J ═ 4.5Hz,1H),7.66-7.62(M,1H),7.48-7.47(d, J ═ 4.5Hz,1H),7.09-7.08(d, J ═ 4.5Hz,1H),6.01(s,2H),4.79-4.76(M,1H),2.88-2.80(M,2H),2.41-2.37(M,1H),2.05-2.01(M,1H),0.63-0.54(M,4H), MS (ESI, [ M + H ], (ESI, M + H),0.63-0.54(M,4H), and M (ESI) [ 1H ], (M,1H)]+)m/z：522.3.
And 5: (S) -4- (8-amino-3- (5-but-2-ynoyl) -5-azaspiro [2.4] heptan-6-yl) imidazo [1,5-a ] pyrazin-1-yl) -N- (4-bromopyridin-2-yl) -3-fluorobenzamide (intermediate K)
And 5: intermediate 4-7(7.85g), 2-butynoic acid (1.12g) and triethylamine (6.01g) were added to a reaction flask, and dissolved in DCM (500mL), and after stirring at room temperature for 5 minutes, HATU (5.93g) was added, and the reaction was completed at room temperature for 1 hour, and the reaction solution was added to water (400mL) for liquid separation, DCM was extracted (100mL × 3), and the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography (developing solvent: DCM: MeOH ═ 97:3), to give intermediate K. 1H NMR (500MHz, DMSO) δ 11.23(s,1H),8.49(s,1H),8.35-8.34(d, J ═ 5.5Hz,1H),8.02-7.99(M,2H),7.85-7.78(M,1H),7.65-7.61(M,1H),7.49-7.48(d, J ═ 5.5Hz,1H),7.15-7.10(M,1H),6.09-6.03(d, J ═ 28Hz,2H),5.81-5.58(M,1H),3.81-3.50(M,2H),2.39-2.12(M,2H),1.59-1.24(M,3H),0.76-0.56(M,4H), MS (ESI, [ M + H ], [ M,4H ], [ M + H ]]+)m/z：588.3.
Example 9: preparation of (S) -N- ([4, 4' -bipyridinyl ] -2-yl) -4- (8-amino-3- (5-but-2-ynoyl) -5-azaspiro [2.4] heptan-6-yl) imidazo [1,5-a ] pyrazin-1-yl) -3-fluorobenzamide (Compound I-4)
Figure BDA0002577067610000151
Step 1: intermediate K (0.3g), 4-pyridineboronic acid (0.0626g) and potassium carbonate (0.282g) were added to a reaction flask, dissolved in dioxane (6mL), and [1,1' -bis (diphenylphosphino) ferrocene was added]Palladium (II) dichloride dichloromethane complex (0.083g), water (1mL) was added, and after 3 to 4 nitrogen replacements, the reaction was carried out at 80 ℃ for 60 minutes. After the reaction, the reaction solution was dissolved in 100mL of water, extracted with DCM, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography (developing solvent: DCM: MeOH: 97:3) to obtain compound I-4.1H NMR(500MHz,DMSO)δ11.18(s,1H),8.77-8.76(d,J＝5.5Hz,2H),8.61(s,1H),8.58-8.57(d,J＝5Hz,1H),8.06-8.04(m,2H),7.86-7.79(m,3H),7.67-7.63(m,2H),7.15-7.11(m,1H),6.10-6.05(m,2H),5.80-5.60(m,1H),3.82-3.50(m,2H),2.39-2.12(m,2H),1.59-1.24(m,3H),0.76-0.56(m,4H).MS(ESI,[M+H]+)m/z：587.33.
Example 10: preparation of (S) -4- (8-amino-3- (5-but-2-ynoyl) -5-azaspiro [2.4] heptan-6-yl) imidazo [1,5-a ] pyrazin-1-yl) -N- (4- (2, 6-difluorophenyl) pyridin-2-yl) -3-fluorobenzamide (Compound I-5)
Figure BDA0002577067610000152
Step 1: intermediate K (0.5g), 2, 6-difluorophenylboronic acid (0.335g) and potassium carbonate (0.470g) were added to a reaction flask, dissolved in dioxane (6mL), and [1,1' -bis (diphenylphosphino) ferrocene was added]Palladium (II) dichloride dichloromethane complex (0.139g), water (1mL) was added, and after 3 to 4 nitrogen replacements, the reaction was carried out at 80 ℃ for 2 hours. After the reaction, the reaction mixture was dissolved in 100mL of water, extracted with DCM, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography (developing solvent: DCM: MeOH (containing 10% ammonia) ═ 97:3) to obtain compound I-5. 1H NMR (500MHz, DMSO) δ 11.17(s,1H),8.57-8.56(d, J ═ 5Hz,1H),8.35(s,1H),8.04-8.01(M,2H),7.85-7.78(M,1H),7.65-7.57(M,2H),7.34-7.30(M,3H),7.15-7.10(M,1H),6.08-6.02(M,2H),5.79-5.60(M,1H),3.82-3.50(M,2H),2.39-2.12(M,2H),1.59-1.24(M,3H),0.76-0.59(M,4H). MS (ESI, [ M +2H ], 2H, 1H, 1.5, 1H, and MS (ESI)]2+/2)m/z：311.80.
Example 11: preparation of (S) -4- (8-amino-3- (5-but-2-ynoyl) -5-azaspiro [2.4] heptan-6-yl) imidazo [1,5-a ] pyrazin-1-yl) -3-fluoro-N- (4- (pyrimidin-5-yl) pyridin-2-yl) benzamide (Compound I-6)
Figure BDA0002577067610000161
Step 1: intermediate K (0.3g), pyrimidine-5-boronic acid (0.0694g) and potassium carbonate (0.282g) were added to a reaction flask, dissolved in dioxane (6mL), and [1,1' -bis (diphenylphosphino) ferrocene was added]Palladium (II) dichloride dichloromethane complex (0.083g), water (1mL) was added, and after 3 to 4 nitrogen replacements, the reaction was carried out at 80 ℃ for 1.5 hours. After the reaction, the reaction solution was dissolved in 100mL of water, extracted with DCM, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography (developing solvent: DCM: MeOH ═ 94:6) to obtain compound I-6. 1H NMR (500MHz, DMSO). delta.11.19 (s,1H),9.33-9.25(M,3H),8.58(s,2H),8.05(s,2H),7.86-7.79(M,1H),7.67(s,2H),7.14-7.12(M,1H),6.11-6.07(M,2H),5.80-5.61(M,1H),3.82-3.50(M,2H),2.37-2.13(M,2H),1.59-1.24(M,3H),0.74-0.59(M,4H). MS (ESI, [ M +2H ]]2+/2)m/z：294.75.
Example 12: preparation of (S) -4- (8-amino-3- (5-but-2-ynoyl) -5-azaspiro [2.4] heptan-6-yl) imidazo [1,5-a ] pyrazin-1-yl) -3-fluoro-N- (4- (oxazol-2-yl) pyridin-2-yl) benzamide (Compound I-7)
Figure BDA0002577067610000162
Step 1: intermediate K (0.5g), tetrakistriphenylphosphine palladium (0.098g) and anhydrous DMF (10ml) were charged into a reaction flask, and after 3 to 4 nitrogen replacements, 2- (tri-n-butylstannyl) oxazole (0.335g) was injected and the reaction was allowed to proceed overnight at 110 ℃. After the reaction, the reaction mixture was dissolved in 200mL of water, EA extracted (150mL × 3), the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to prepare a plate for purification (developing solvent: DCM: MeOH: aqueous ammonia: 30:1:0.2) to obtain compound I-7. 1H NMR (500MHz, DMSO). delta.11.23 (s,1H),8.86(s,1H),8.60-8.59(d, J)＝5Hz,1H),8.40(s,1H),8.06-8.03(m,2H),7.85-7.72(m,2H),7.66-7.62(m,1H),7.54(s,1H),7.15-7.11(m,1H),6.09-6.03(m,2H),5.80-5.60(m,1H),3.82-3.70(m,2H),2.39-2.12(m,2H),1.59-1.360(m,3H),0.74-0.59(m,4H).MS(ESI,[M+2H]2+/2)m/z：289.19.
Example 13: preparation of (S) -4- (8-amino-3- (5- (but-2-ynoyl) -5-azaspiro [2.4] heptan-6-yl) imidazo [1,5-a ] pyrazin-1-yl) -N- (4- (2, 4-difluorophenyl) pyridin-2-yl) -3-fluorobenzamide (Compound I-8)
Figure BDA0002577067610000171
Step 1: to a reaction flask were added, in order, intermediate K (200mg), 2, 4-difluorophenylboronic acid (54.8mg), K2CO3(188mg) and solvent 1, 4-dioxane (6mL), water (1mL), and stirred, N2Bubbling for 1min, and adding a catalyst PdCl2(dppf)CH2Cl2(69.4mg), then N2Bubbling for 1min, and heating in oil bath at 80 deg.C for one hour. After the reaction, 10mL of water is added into the reaction liquid, DCM-MeOH (20:1) is used for extraction, the organic phase is washed by saturated saline solution, dried by anhydrous sodium sulfate and filtered and steamed in a rotary way, the crude product is obtained by silica gel column chromatography purification (developing agent: DCM-MeOH (99:1 to 95:5)), and the target compound I-8 is obtained after high pressure reverse phase preparative chromatography separation.
1H NMR(500MHz,DMSO)11.12(s,1H),8.52(d,J＝4.9Hz,1H),8.44(s,1H),8.03(t,J＝7.6Hz,2H),7.82(dd,J＝30.2,4.8Hz,1H),7.74(dd,J＝15.5,8.5Hz,1H),7.64(dd,J＝12.8,7.4Hz,1H),7.48(t,J＝10.1Hz,1H),7.39(d,J＝4.5Hz,1H),7.30(t,J＝7.8Hz,1H),7.13(dd,J＝16.3,4.6Hz,1H),6.15-5.96(m,2H),5.83-5.57(m,1H),3.85-3.47(m,2H),2.41-2.10(m,2H),2.07-1.55(m,3H),0.90–0.52(m,4H).MS(ESI,[M+H]+)m/z:622.2148.
Example 14: preparation of (S) -N- ([3,4 '-bipyridinyl ] -2' -yl) -4- (8-amino-3- (5- (but-2-ynoyl) -5-azaspiro [2.4] heptan-6-yl) imidazo [1,5-a ] pyrazin-1-yl) -3-fluorobenzamide (Compound I-9)
Figure BDA0002577067610000172
Step 1: to a reaction flask, intermediate K (300mg), pyridine-3-phenylboronic acid (81.5mg), K2CO3(282mg), and solvent 1, 4-dioxane (9mL), water (1.5mL) were added in this order, and the mixture was stirred, followed by N2Bubbling for 1min, and adding a catalyst PdCl2(dppf)CH2Cl2(104.1mg), then N2Bubbling for 1min, and heating in oil bath at 80 deg.C for two hours. After completion of the reaction, 10mL of water was added to the reaction mixture, the mixture was extracted with DCM-MeOH (20:1), the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered and rotary-evaporated, and purified by silica gel column chromatography (developing solvent: DCM-MeOH (99:1 to 96:4)) to obtain the objective compound I-9.
1H NMR(500MHz,DMSO)δ11.14(s,1H),9.02(s,1H),8.72(s,1H),8.56(s,2H),8.20(d,J＝5.1Hz,1H),8.05(s,2H),7.83(d,J＝28.1Hz,1H),7.62(d,J＝19.0Hz,2H),7.13(d,J＝13.8Hz,1H),6.09(s,2H),5.87–5.54(m,1H),3.85-3.45(m,2H),2.42-2.10(m,2H),2.06-1.53(m,3H),0.88–0.52(m,4H).MS(ESI,[M+H]+)m/z:587.2279.
Example 15: preparation of (S) -4- (8-amino-3- (5- (but-2-ynoyl) -5-azaspiro [2.4] heptan-6-yl) imidazo [1,5-a ] pyrazin-1-yl) -3-fluoro-N- (4- (thien-2-yl) pyridin-2-yl) benzamide (Compound I-10)
Figure BDA0002577067610000181
Step 1: to a reaction flask were added, in order, intermediate K (300mg), thiophene-2-boronic acid (85mg), K2CO3(282mg), and solvent 1, 4-dioxane (9mL), water (1.5mL), followed by stirring, and N2Bubbling for 1min, and adding a catalyst PdCl2(dppf)CH2Cl2(416mg), then N2Bubbling for 1min, and heating in oil bath at 80 deg.C for 2.5 hr. After completion of the reaction, 10mL of water was added to the reaction mixture, the mixture was extracted with DCM-MeOH (20:1), the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered and rotary-evaporated, and purified by silica gel column chromatography (developing solvent: DCM-MeOH (99:1 to 95:5)) to obtain a crude product. The crude product was purified on a preparative plate after being washed with DCM-MeOH (20:1) (developer: EA-MeOH-NH)3H2O (40:1:0.2)) to obtain the target compound I-10。
1H NMR(500MHz,CDCl3)δ8.77(d,J＝16.2Hz,1H),8.70(s,1H),8.31(d,J＝5.2Hz,1H),7.92-7.82(m,2H),7.79–7.70(m,2H),7.64(d,J＝3.3Hz,1H),7.47(d,J＝4.8Hz,1H),7.40-7.32(m,1H),7.22-7.12(m,2H),5.74-5.55(m,1H),5.13-4.91(m,2H),3.98–3.65(m,2H),2.81–2.17(m,2H),2.02-1.66(m,3H),0.94–0.64(m,4H).MS(ESI,[M+H]+)m/z:592.1919.
Example 16: preparation of (S) -4- (8-amino-3- (5- (but-2-ynoyl) -5-azaspiro [2.4] heptan-6-yl) imidazo [1,5-a ] pyrazin-1-yl) -3-fluoro-N- (4- (piperidin-1-yl) pyridin-2-yl) benzamide (Compound I-11)
Figure BDA0002577067610000182
Step 1: (S) -6- (8-amino-1- (2-fluoro-4- ((4- (piperidin-1-yl) pyridin-2-yl) carbamoyl) phenyl) imidazo [1,5-a ] pyrazin-3-yl) -5-azaspiro [2.4] heptane-5-carboxylic acid benzyl ester (intermediate 5-7)
Intermediate 4-6(1.0g), piperidine (10mL) was added to the reaction flask and reacted at 150 ℃ for 30 minutes in a microwave oven. After the reaction, the reaction mixture was dissolved in 10mL of water, extracted twice with 1mL of DCM, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography (developing solvent: DCM: MeOH: 100:3) to obtain intermediate 5-7(0.460 g). MS (ESI, [ M + H ] +) M/z: 661.7.
step 2: (S) -4- (8-amino-3- (5-azaspiro [2.4] hept-6-yl) imidazo [1,5-a ] pyrazin-1-yl) -3-fluoro-N- (4- (piperidin-1-yl) pyridin-2-yl) benzamide (intermediate 5-8)
The reaction flask was charged with 5-7(350mg) of compound and an aqueous solution of hydrobromic acid (5mL, 48%), the mouth of the flask was sealed, and after completion of the reaction, the reaction was carried out at 40 ℃. Diluting with saturated saline, extracting with methyl tert-butyl ether to remove organic phase, and retaining aqueous phase. The aqueous phase was adjusted to a slightly alkaline pH with saturated 10% sodium hydroxide solution. Then, the mixture was extracted with DCM 2-3 times and dried over anhydrous sodium sulfate. Concentration gave compound 5-8(300mg)
And step 3: (S) -4- (8-amino-3- (5- (but-2-ynoyl) -5-azaspiro [2.4] heptan-6-yl) imidazo [1,5-a ] pyrazin-1-yl) -3-fluoro-N- (4- (piperidin-1-yl) pyridin-2-yl) benzamide (Compound I-11)
The reaction flask was charged with Compound 5-8(300mg), HATU (222mg) and 2-butynoic acid (44.5mg) were dissolved in DCM (10mL), and triethylamine (0.29mL) was added to conduct 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: 100:5) to give compound I-11.
1H NMR(500MHz,DMSO-d6)δ10.58(s,1H),8.11-7.94(m,3H),7.91–7.70(m,2H),7.64-7.55(m,1H),7.16-7.07(m,1H),6.70-6.62(m,1H),6.16-5.90(m,2H),5.83-5.54(m,1H),3.88–3.66(m,1H),3.64–3.47(m,1H),3.38(s,4H),2.42-2.11(m,2H),2.00(s,2H),1.69–1.51(m,7H),0.82–0.49(m,4H).MS(ESI,[M+H]+)m/z：593.5.
Example 17: preparation of (S) -4- (8-amino-3- (5- (but-2-ynoyl) -5-azaspiro [2.4] heptan-6-yl) imidazo [1,5-a ] pyrazin-1-yl) -3-fluoro-N- (4-morpholinopyridin-2-yl) benzamide (Compound I-12)
Figure BDA0002577067610000191
Step 1: (S) -6- (8-amino-1- (2-fluoro-4- ((4-morpholinopyridin-2-yl) carbamoyl) phenyl) imidazo [1,5-a ] pyrazin-3-yl) -5-azaspiro [2.4] heptane-5-carboxylic acid benzyl ester (intermediate 6-7)
Intermediate 4-6(1.0g), morpholine (10mL) was added to the reaction flask and reacted for 30 minutes at 150 ℃ with microwave. After the reaction, the reaction mixture was dissolved in 10mL of water, extracted twice with 10mL of DCM, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography (developing solvent: DCM: MeOH: 100:3) to obtain intermediate 6-7(0.160 g). MS (ESI, [ M + H ] +) M/z: 663.35.
step 2: (S) -4- (8-amino-3- (5-azaspiro [2.4] heptan-6-yl) imidazo [1,5-a ] pyrazin-1-yl) -3-fluoro-N- (4-morpholinopyridin-2-yl) benzamide (intermediate 6-8)
The reaction flask was charged with compound 6-7(130mg) and hydrobromic acid in acetic acid (3mL, 33%), the flask was sealed, and the reaction was carried out at room temperature after completion of the reaction. Diluting with saturated saline, extracting with methyl tert-butyl ether to remove organic phase, and retaining aqueous phase. The aqueous phase was adjusted to a slightly alkaline pH with saturated sodium bicarbonate solution. Then, the mixture was extracted with DCM 2-3 times and dried over anhydrous sodium sulfate. Concentration gave compound 6-8(80mg)
Step 3 (S) -4- (8-amino-3- (5- (but-2-ynoyl) -5-azaspiro [2.4] hept-6-yl) imidazo [1,5-a ] pyrazin-1-yl) -3-fluoro-N- (4-morpholinopyridin-2-yl) benzamide (Compound I-12)
The reaction flask was charged with Compound 6-8(80mg), added with HATU (82mg) and 2-butynoic acid (14.84mg), dissolved in DCM (5mL), added with triethylamine (0.109mL), and reacted 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: 100:5) to give compound I-12.
1H NMR(500MHz,DMSO-d6)δ10.74(s,1H),8.06(d,J＝6.1Hz,1H),8.03–7.96(m,2H),7.88-7.78(m,1H),7.73(s,1H),7.65-7.58(m,1H),7.15-7.10(m,1H),6.78-6.72(m,1H),6.15(s,2H),5.83–5.56(m,1H),3.83–3.72(m,5H),3.60–3.48(m,1H),3.39–3.33(m,4H),2.41-2.26(m,1H),2.23-2.10(m,1H),1.99(s,2H),1.59(s,1H),0.79–0.49(m,4H).HR-MS(ESI,[M+H]+)m/z：595.2597.
Example 22: preparation of (S) -4- (8-amino-3- (5- (but-2-ynoyl) -5-azaspiro [2.4] hept-6-yl) imidazo [1,5-a ] pyrazin-1-yl) -3-fluoro-N- (4- (4-methylpiperazin-1-yl) pyridin-2-yl) benzamide (I-13)
Figure BDA0002577067610000201
Step 1: (S) -benzyl 6- (8-amino-1- (2-fluoro-4- ((4- (4-methylpiperazin-1-yl) pyridin-2-yl) carbamoyl) phenyl) imidazo [1,5- ] a ] pyrazin-3-yl) -5-azaspiro [2.4] heptane-5-carboxylate (intermediate 11-7)
Intermediate 4-6(1.0g), N-methylpiperazine (10mL) were added to a reaction flask and reacted at 150 ℃ for 30 minutes under microwave. After the reaction, the reaction mixture was dissolved in 10mL of water, extracted with DCM, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography (developing solvent: DCM: MeOH: 100:3) to obtain intermediate 11-7(0.611 g). MS (ESI, [ M + H ] +) M/z: 676.40.
step 2: (S) -4- (8-amino-3- (5-azaspiro [2.4] hept-6-yl) imidazo [1,5-a ] pyrazin-1-yl) -3-fluoro-N- (4- (4-methylpiperazin-1-yl) pyridin-2-yl) benzamide (intermediate 11-8)
To a reaction flask were added 11-7(578mg) and hydrobromic acid in acetic acid (6mL, 33%), the flask was sealed, and the reaction was carried out at room temperature after completion of the reaction. Dilute with saturated brine, extract with DCM to remove the organic phase and retain the aqueous phase. The aqueous phase was adjusted to a slightly alkaline pH with saturated sodium bicarbonate solution. Extracted three more times with DCM and dried over anhydrous sodium sulfate. Concentration gave compound 11-8(392 mg). MS (ESI, [ M + H ]]+)m/z：542.6.
And step 3: (S) -4- (8-amino-3- (5- (but-2-ynoyl) -5-azaspiro [2.4] hept-6-yl) imidazo [1,5-a ] pyrazin-1-yl) -3-fluoro-N- (4- (4-methylpiperazin-1-yl) pyridin-2-yl) benzamide (I-13)
Adding the intermediate 11-8(0.373g) into a reaction bottle, dissolving the intermediate in DCM (20mL), adding 2-butynoic acid (0.052g) and triethylamine (0.279g) at 0 ℃, stirring for 5 minutes at 0 ℃, adding HATU (0.275g), continuing to react at 0 ℃, dissolving the reaction solution in 50mL of water after the reaction is finished, extracting DCM, drying the organic phase with anhydrous sodium sulfate, filtering, concentrating, and purifying by silica gel column chromatography to obtain the compound I-13. HR-MS (ESI, [ M + H ]]+)m/z：608.2933.
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. Enzymes and compoundsAfter 30min reaction with the substrate or vehicle, 5 × 100 μ M ATP (final 20 μ M) in kinase buffer was mixed with 5 × 0.5 μ M substrate (final 0.1 μ M, ULight-poly GT) in a 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 30min of reaction of the enzyme with the compound or vehicle, 5 × 250 μ M ATP (final concentration 50 μ M) and 5 × 0.5 μ M substrate (final concentration 0.1 μ 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 solution 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, various compounds dissolved in DMSO were added to the wells using a nano-liter loading apparatus to give final concentrations of 1000nM to 0.48nM, 4-fold gradientIn total, 7 concentrations were set, and 2 duplicate wells were set with blank control empty (no enzyme) and negative control wells (enzyme-containing, vehicle DMSO). 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 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 DMSO-dissolved different compounds were added to the wells using a nanoliter loading apparatus 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-added) 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.
1.5 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, and 6. mu.L of 1.67X 0.0835 g/. mu.L of reagent was added per wellAs a working solution (final concentration 0.05 ng/. mu.L), DMSO-solubilized different compounds were added to the wells using a nanoliter load-meter to give a final concentration of compounds of 1000nM to 0.24nM, 4-fold gradient, for a total of 7 concentrations, while blank control wells (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
Taking TMD-8 cells in an exponential growth phase, collecting the cells to a centrifuge tube, centrifuging for 3min at 1500rpm by using a low-speed desktop centrifuge, discarding the supernatant, and adding 2mL of plating medium (RPMI basic medium + 5% FBS +0.05mM 2-mercaptoethanol) for cell resuspension. 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
Figure BDA0002577067610000221
Test example 2: in vivo pharmacokinetic experiments in mice
ICR mice, weight 18-22 g, after adapting for 3-5 days, were randomly grouped, 9 mice per group, and were individually gavaged with related compounds at a dose of 10 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. After the intragastric administration, about 0.1mL of blood is collected at orbital time of 0.25(15min), 0.5(30min), 1,2, 4, 6, 8, 10 and 24h, 3-4 time points are collected for each mouse, 3 mice are collected at each time point, whole blood is collected and placed in centrifuge tubes containing EDTA-K2 and sodium fluoride, the whole blood is transferred to 4 ℃ within 30min, and plasma is centrifugally separated under the condition of 4000rpm multiplied by 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 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
Parameter(s) Unit of I-7(ig-10mg/kg) I-9(ig-10mg/kg) I-10(ig-10mg/kg)
AUC(0-t) μg/L*h 63.1 11.3 48.4
AUC(0-∞) μg/L*h 63.1 20.1 52.5
MRT(0-t) h 2.59 0.857 0.691
t1/2z h 0.332 1.35 0.527
Tmax h 0.25 0.25 0.25
Cmax μg/L 35.1 11.3 56.3
。
Test example 2: cellular level BTK (Y223) phosphorylation inhibition Activity screening
mu.L of 30% hydrogen peroxide was added to 860. mu.L of double distilled water to prepare 200mM hydrogen peroxide. PV (sodium pervanadate): adding (200 mmol/sodium orthovanadate 10 μ L) and (200mmol/L hydrogen peroxide 10 μ L) into (80 μ L phenol red-free 1640 complete medium) at room temperature for reaction for 15min, adding phenol red-free 1640 complete medium, diluting to 6mM, and using as it is. Taking Ramos lymphoma cells growing in a logarithmic phase, centrifuging for 3min at 1500rpm by a low-speed desktop centrifuge, adding a proper amount of phenol-free red 1640, completely suspending the cells in a culture medium, counting, taking a proper amount of cell suspension, adding a proper amount of corresponding culture medium, and adjusting the cell density to be about 1-2 x 10E7 cells/mL. The cells were plated (384 wells) at the above cell density, 20. mu.L/well; adding 5 mu L of compound into each hole and incubating for 1 h; 20mM PV was diluted to 6mM (final concentration 1mM) in complete medium without phenol red 1640; then adding 5 mul PV into each hole according to the plate distribution, and incubating for 15min-20 min; the blank group was inoculated with cells without compound and without PV; control group, inoculated cells, no compound, added PV; add 10. mu.L of blocking buffer added lysate (4X) immediately and incubate with shaking at room temperature for 30 min. After mixing, 16 μ L of lysate was transferred to another 384 well small volume white plate. Add 4. mu.L of pre-mixed antibody (vol/vol) in assay buffer, cover plate, centrifuge to mix well, and incubate overnight at room temperature. The PE Envision multifunctional plate reader detects 665nm/620nm signal values, four-parameter fitting calculates IC50, and the results are shown in Table 3.
TABLE 3
Figure BDA0002577067610000231
Test example 3: effect on human diffuse large B-cell lymphoma OCI-LY10 SCID mouse xenograft tumor
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 Compound I-1: 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
Figure BDA0002577067610000232
The evaluation index of the anti-tumor activity is relative tumor proliferation rate T/C (%), and the calculation formula is as follows: T/C (%) ═ TRTV/CRTV*100％。(TRTV: treatment group RTV; cRTV: 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
Figure BDA0002577067610000241
***: p <0.001 compared to vehicle group.

Claims (10)

1. A compound of formula (I) or a pharmaceutically acceptable salt thereof,
Figure FDA0002577067600000011
wherein the content of the first and second substances,
ring A is selected from 5-10 membered heteroaryl or C6-10An aryl group;
ring D is selected from 5-10 membered heteroaryl, C6-10Aryl or 5-10 membered heterocycloalkyl;
R1independently selected from halogen, hydroxy, amino, cyano, C1-6Alkoxy or C1-6Alkyl radical, said C1-6Alkoxy or C1-6Alkyl optionally substituted with halo;
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, C1-6Alkyl or C1-6Alkoxy radical, said C1-6Alkyl or C1-6Alkoxy is optionally substituted with halogen;
n is selected from 0, 1,2,3 or 4;
structure of the product
Figure FDA0002577067600000012
Is selected from
Figure FDA0002577067600000013
Wherein the content of the first and second substances,
R3independently selected from hydrogen, RaS(O)2-、(RaO)2P (O) -or RaC(O)-；
Wherein R isaIndependently selected from C2-6Alkynyl, C2-6Alkenyl radical, C1-6Alkyl radical, C3-6CycloalkanesBase, (C)1-6Alkyl) NH-, (C)1-6Alkyl radical)2N-, 3-6 membered heterocycloalkyl, 5-10 membered heteroaryl or C6-10Aryl radical, the above RaOptionally is (C)1-6Alkyl radical)2N-、(C1-6Alkyl) NH-, hydroxy, amino, halogen or cyano;
R4independently selected from ═ O, ═ S, C1-6Alkyl radical, C1-6Alkoxy, cyano or halogen;
q is selected from 0, 1,2,3 or 4.
2. A compound of formula (I) as claimed in claim 1 wherein ring a is selected from phenyl or 5-6 membered heteroaryl; optionally, wherein ring a is selected from 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;
optionally, wherein ring D is selected from 5-6 membered heteroaryl, phenyl or 5-6 membered heterocycloalkyl; optionally, wherein ring D is selected from 5-membered heteroaryl, 6-membered heteroaryl, phenyl, or 6-membered heterocycloalkyl; optionally, wherein ring D is selected from pyridyl, pyrimidinyl, oxazolyl, thiazolyl, imidazolyl, phenyl, piperidinyl, morpholinyl, piperazinyl, or thiophene; optionally wherein ring D is selected from pyridyl, pyrimidinyl, oxazolyl, thiazolyl, imidazolyl, phenyl, piperidinyl, morpholinyl, or piperazinyl.
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 optionally substituted by halogen1-3Alkyl or C optionally substituted by halogen1-3An alkoxy group; optionally, wherein R is1Independently selected from halogen, C optionally substituted by halogen1-3An alkyl group; optionally, wherein R is1Independently selected from halogen or C1-3An alkyl group; optionally, wherein R is1Independently selected from fluoro, methyl or ethyl;
optionally, wherein m is selected from 0, 1,2,3 or 4; optionally, wherein m is selected from 0, 1 or 2.
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), or a pharmaceutically acceptable salt thereof, as claimed in any one of claims 1-4 wherein R2Independently selected from halogen, hydroxy, amino, cyano, C1-3Alkyl or C1-3An alkoxy group; optionally, wherein R is2Independently selected from halogen; 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; optionally, wherein n is selected from 1.
6. A compound of formula (I), or a pharmaceutically acceptable salt thereof, as claimed in any one of claims 1-5 wherein R3Independently selected from RaC(O)-；
Optionally, wherein R isaIndependently selected from C2-6Alkynyl, C2-6Alkenyl radical, C1-6Alkyl radical, C3-6Cycloalkyl or 3-6 membered heterocycloalkyl; optionally, wherein R isaIndependently selected from C2-3Alkynyl or C3-4A cycloalkyl group; optionally, wherein R isaIndependently selected from propynyl or cyclopropyl.
7. A compound of formula (I), or a pharmaceutically acceptable salt thereof, as claimed in any one of claims 1-6 wherein R4Independently selected from ═ O, ═ S, C1-3Alkyl or C1-3An alkoxy group; optionally, wherein R is4Independently selected from ═ O, ═ S, or methyl;
optionally, wherein q is selected from 0, 1 or 2.
8. A compound of formula (I) according to claim 1, wherein the compound of formula (I) is selected from a compound of formula (II), a compound of formula (III) or a compound of formula (IV),
Figure FDA0002577067600000021
Figure FDA0002577067600000031
9. the following compounds or pharmaceutically acceptable salts thereof:
Figure FDA0002577067600000032
Figure FDA0002577067600000041
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.
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106831787A (en) * 2017-01-20 2017-06-13 成都倍特药业有限公司 Compound as bruton's tyrosine kinase inhibitor and its preparation method and application
US20170362242A1 (en) * 2014-12-31 2017-12-21 Merck Sharp & Dohme Corp. Benzamide imidazopyrazine btk inhibitors

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20170362242A1 (en) * 2014-12-31 2017-12-21 Merck Sharp & Dohme Corp. Benzamide imidazopyrazine btk inhibitors
CN106831787A (en) * 2017-01-20 2017-06-13 成都倍特药业有限公司 Compound as bruton's tyrosine kinase inhibitor and its preparation method and application

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