CN116063325A

CN116063325A - Macrocyclic compounds with BTK modulating effect and uses thereof

Info

Publication number: CN116063325A
Application number: CN202211331479.8A
Authority: CN
Inventors: 王静; 赵树春; 李建宗; 韦学振; 黄婷婷; 邵涛; 周瑞捷; 陈泠颖; 曾绍梅; 张晓东; 唐军
Original assignee: Scinnohub Pharmaceutical Co Ltd
Current assignee: Scinnohub Pharmaceutical Co Ltd
Priority date: 2021-11-02
Filing date: 2022-10-28
Publication date: 2023-05-05

Abstract

The present application provides compounds of formula (I) or stereoisomers, pharmaceutically acceptable salts, solvates, or tautomers thereof having BTK kinase activity and the use thereof in medicine.

Description

Macrocyclic compounds with BTK modulating effect and uses thereof

Technical Field

The present invention relates to compounds and stereoisomers, pharmaceutically acceptable salts, solvates, or tautomers thereof and their use as BTK kinase inhibitors. More specifically, the present invention provides novel compounds that are BTK inhibitors and stereoisomers thereof and their use in the treatment of BTK mediated related diseases. The compound of the present invention has particularly excellent inhibitory activity against BTK and BTK C481S.

Background

After binding of the antigen to the B cell antigen receptor (BCR) at the plasma membrane BCR, PLCG2 is phosphorylated at several specific sites, and then downstream signaling pathways are triggered by calcium mobilization, finally activating Protein Kinase C (PKC) family members. PLCG2 phosphorylation is closely related to the adaptor B cell adaptor protein BLNK, which serves as a platform, aggregates multiple signaling proteins, and is involved in cytokine receptor signaling pathways. And BTK plays an important role in the function of innate immune cells and adaptive immunity as a component of the Toll-like receptor (TLR) pathway. The B Cell Receptor (BCR) dependence of BTK induces activation signal pathways, principally the pool transcription factor NF-. Kappa.B and the nuclear factor of activated T cells (NFAT). Both of these conditions are mediated by Protein Kinase C (PKC).

BTK kinases are involved in the transduction of a variety of important signals in the body, and their activation has a significant impact on a variety of cellular processes. BTK disorders can lead to severe immunodeficiency, affecting the developmental maturation of B cells. When the organism generates immune response, BTK induces gene expression by mediating B cell signal activation, thereby regulating proliferation and apoptosis of B cells. Over-expression of BTK in normal human monocytes will promote TNF- α production, whereas those with abnormal BTK genes will have reduced TNF- α production, resulting in BTK activation to induce macrophage production of pro-inflammatory factors. The structure and the functional activation mechanism of the BTK are integrated [4], so that the BTK becomes a target point with wide target diseases, such as B cell malignant tumor, asthma, rheumatic arthritis, systemic lupus erythematosus and the like.

Btk is a key molecule in B cell antigen receptor (BCR) coupled signaling, whose activity is regulated by Lyn and Syk. And studies have shown that Src family kinases act upstream of Btk, activated by non-phosphorylating mediated mechanisms (Ronen Gabizon, j.med. Chem.2020,63, 5100-51011). BTK inhibitors inhibit proliferation, chemotaxis, and adhesion of B-cell lymphoma cells. Is mainly used for B cell malignant tumors (IMBRUVICA, SUmmary Review, FDA@drugs) such as Mantle Cell Lymphoma (MCL), chronic Lymphocytic Leukemia (CLL), primary macroglobulinemia (WM) and the like. The mechanism of action of BTK inhibitors is to bind to Cys-481 at the BTK (active) site, preventing BTK activation. There is an urgent need to develop drugs effective in patients with BTK C481S resistance (Lian Xu, blood,.2017May 4;129 (18): 2519-2525).

Disclosure of Invention

In one aspect the invention provides a compound or stereoisomer, pharmaceutically acceptable salt, solvate, or tautomer of formula (I):

m is selected from N, CH, CR ₆ ；

X ₁ Selected from (CRaRb) m; wherein Ra, rb are each independently selected from hydrogen, halogen, C _1-4 Alkyl, carbonyl, amino, C _1-4 Alkoxy, halo C _1-4 An alkyl group; wherein m is selected from 0, 1, 2;

R ₁ selected from hydrogen or C _1-4 An alkyl group;

R ₂ selected from hydrogen, C _1-4 Alkyl, cyano, amino, nitro, halogen, halogenated C _1-4 An alkyl group;

R ₃ 、R ₄ each independently selected from hydrogen, C _1-4 Alkyl, halogenated C _1-4 Alkyl, cyano, amino, nitro, halogen; the C is _1-4 Alkyl, halogenated C _1-4 Alkyl optionally being cyano, amino, nitro, halogen, C _1-4 Substituents for alkoxy, 3-6 membered cycloalkyl, 3-6 membered heterocycloalkyl, phenyl, 5-6 membered heteroaryl; the 3-6 membered heterocycloalkyl, 5-6 membered heteroaryl may be substituted by halogen, amino, hydroxy, C _1-4 Alkyl, C _1-4 Alkoxy is substituted, and the heteroatom is optionally selected from 1 to 3N, O, S;

R ₅ selected from hydrogen or C _1-4 An alkyl group;

R ₆ selected from hydrogen, fluorine, chlorine, bromine, C _1-4 An alkyl group;

R ₇ selected from hydrogen or amino;

a is selected from 3-6 membered cycloalkyl, 3-6 membered heterocycloalkyl, and C _2-6 Olefins or C _2-6 Alkynes, 3-6 membered unsaturated cycloalkyl, 3-6 membered unsaturated heterocycloalkyl, 6-8 membered aryl, 5-10 membered heteroaryl, said 3-6 membered cycloalkyl, 3-6 membered heterocycloalkyl, C _2-6 Olefins or C _2-6 Alkynes, 3-6 membered unsaturated cycloalkyl, 3-6 membered unsaturated heterocycloalkyl, 6-8 membered aryl, 5-10 membered heteroaryl groups may be substituted by halogen, C1-4 alkyl, cyano, hydroxy, nitroSubstituted with a group, -NRcRd, -NHRc, - (CH 2) nNRcRd, -NHC (O) ORc, -NHC (O) NHRc, -NHC (O) Rc, -ORc, -OC (O) Rc, -C (O) NHRc, - (CH 2) nC (O) NHRc, -C (O) NRcRd;

n is selected from 1, 2 and 3.

Further, the present invention provides a compound, or a stereoisomer, pharmaceutically acceptable salt, solvate, or tautomer thereof, wherein a is selected from the group consisting of a benzene ring, a 5-6 membered heteroaryl; the benzene ring, 5-6 membered heteroaryl group may be substituted with halogen, C _1-4 Alkyl, cyano, hydroxy, nitro, -NRcRd, -NHRc, - (CH) ₂ )nNRcRd、-NHC(O)ORc、-NHC(O)NHRc、-NHC(O)Rc、-ORc、-OC(O)ORc、-OC(O)Rc、-C(O)Rc、-C(O)NHRc、-(CH ₂ ) nC (O) NHRc, -C (O) NRcRd; rc, rd are each independently selected from C _1-4 Alkyl, 3-6 membered cycloalkyl or 3-6 membered heterocycloalkyl, said C _1-4 Alkyl groups may be substituted by halogen, amino, hydroxy, C _1-4 Alkoxy substituted; the 3-6 membered cycloalkyl or 3-6 membered heterocycloalkyl can be substituted by halogen, amino, hydroxy, C _1-4 Alkyl, C _1-4 Alkoxy groups are substituted.

Further A is selected from benzene ring, pyridine ring,

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Ring A may be substituted with halogen, C _1-4 Alkyl, cyano, hydroxy, nitro, -NRcRd, -NHRc, - (CH) ₂ )nNRcRd、-NHC(O)ORc、-NHC(O)NHRc、-NHC(O)Rc、-ORc、-OC(O)ORc、-OC(O)Rc、-C(O)Rc、-C(O)NHRc、-(CH ₂ ) nC (O) NHRc, -C (O) NRcRd; rc, rd are each independently selected from C _1-4 An alkyl group; the C is _1-4 Alkyl groups may be substituted by halogen, amino, hydroxy, C _1-4 Alkoxy groups are substituted.

Further A is selected from benzene rings which may be substituted by halogen, C _1-4 Alkyl, cyano, hydroxy, nitro, -NRcRd, -NHRc, - (CH) ₂ )nNRcRd、-NHC(O)ORc、-NHC(O)NHRc、-NHC(O)Rc、-ORc、-OC(O)ORc、-OC(O)Rc、-C(O)Rc、-C(O)NHRc、-(CH ₂ ) nC (O) NHRc, -C (O) NRcRd; rc, rd are each independently selected from C _1-4 An alkyl group; the C is _1-4 Alkyl groups may be substituted by halogen, amino, hydroxy, C _1-4 Alkoxy groups are substituted.

In other embodiments, the invention provides a compound, or a stereoisomer, pharmaceutically acceptable salt, solvate, or tautomer thereof, wherein R ₃ 、R ₄ Each independently selected from hydrogen, C _1-4 Alkyl, halogenated C _1-4 Alkyl, cyano, amino, nitro, halogen; the C is _1-4 Alkyl, halogenated C _1-4 Alkyl optionally being cyano, amino, nitro, halogen, C _1-4 Alkoxy, 6 membered cycloalkyl, 6 membered heterocycloalkyl; the 6-membered cycloalkyl, 6-membered heterocycloalkyl may be substituted by halogen, amino, hydroxy, C _1-4 Alkyl, C _1-4 Alkoxy substituted; the 6-membered heterocycloalkyl is optionally selected from 1 to 3N; preferably, the 6-membered ring is methylpiperidinyl.

In other embodiments, the invention provides a compound, or a stereoisomer, pharmaceutically acceptable salt, solvate, or tautomer thereof, R ₅ Selected from hydrogen;

in other embodiments, the invention provides a compound, or a stereoisomer, pharmaceutically acceptable salt, solvate, or tautomer thereof, R ₆ Selected from hydrogen, fluorine, chlorine, bromine;

in some embodiments the compounds of the invention, or stereoisomers, pharmaceutically acceptable salts, solvates, or tautomers thereof, have the structure of formula (II):

wherein X is ₁ 、R ₁ 、R ₂ 、R ₃ 、R ₄ 、R ₅ As defined above.

In some embodiments the compounds of the invention, or stereoisomers, pharmaceutically acceptable salts, solvates, or tautomers thereof, have the structure of formula (III):

wherein R is ₁ 、R ₂ 、R ₃ 、R ₄ 、R ₅ As defined above.

The compounds provided by the invention or stereoisomers, pharmaceutically acceptable salts, solvates, or tautomers thereof have the following structure:

in another aspect the invention provides a pharmaceutical composition comprising a compound as described above or a stereoisomer, pharmaceutically acceptable salt, solvate, or tautomer thereof and a pharmaceutically acceptable excipient.

In another aspect, the invention provides the use of a compound as described above, or a stereoisomer, pharmaceutically acceptable salt, solvate, or tautomer thereof, in the manufacture of a medicament for the treatment of a BTK mediated disease.

Further the BTK mediated disease is selected from cancer, autoimmune disease or allergic disease.

Still further the cancer is selected from one or more of a diffuse large B-cell lymphoma, mantle cell lymphoma, chronic lymphocytic lymphoma, extranodal marginal zone B-cell lymphoma, B-cell chronic lymphocytic leukemia, B-cell prolymphocytic leukemia, mature B-cell acute lymphoblastic leukemia, 17 p-deleted chronic lymphocytic leukemia, waldenstrom macroglobulinemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasmacytic myeloma, plasmacytoma, intranodal marginal zone B-cell lymphoma, mantle cell lymphoma, intravascular large B-cell lymphoma, and primary exudative lymphoma; the autoimmune disease is selected from one or more of systemic lupus erythematosus, rheumatoid arthritis, sjogren's syndrome, multiple sclerosis, inflammatory enteritis such as Crohn's disease and ulcerative colitis, urticaria, immune thrombocytopenia, igA nephropathy, hidradenitis suppurativa, psoriasis, vitiligo, neutrophilic dermatoses, autoimmune vesicular diseases such as pemphigus and pemphigoid, igG 4-related diseases, autoimmune hemolytic anemia, rheumatic fever, antiphospholipid syndrome, systemic sclerosis/scleroderma, autoimmune hepatitis, primary sclerocholangitis, primary biliary cirrhosis, allergic purpura, churg-Strauss syndrome/allergic granulomatosis vasculitis, behcet's disease, ANCA-related small vessel inflammation, dermatitis herpetiformis; the allergic diseases are selected from one or more of allergic conjunctivitis, allergic rhinitis, allergic asthma, atopic dermatitis, and chronic asthma.

Detailed Description

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

"alkyl" refers to an aliphatic hydrocarbon group, and to a saturated hydrocarbon group. The alkyl moiety may be a straight chain alkyl group or a branched alkyl group. For example, C1-6 alkyl, C1-4 alkyl or C1-3 alkyl. C1-6 alkyl refers to an alkyl group having 1 to 6 carbon atoms, for example, an alkyl group having 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, 6 carbon atoms. Non-limiting examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, neopentyl, n-hexyl and the like. The alkyl group may be unsubstituted or substituted with one or more substituents including, but not limited to, alkyl, alkoxy, cyano, hydroxy, carbonyl, carboxyl, aryl, heteroaryl, amino, halogen, sulfonyl, sulfinyl, phosphonyl, and the like.

"Ring" refers to any covalently closed structure, including, for example, carbocycles (e.g., aryl or cycloalkyl), heterocycles (e.g., heteroaryl or heterocycloalkyl), aromatic groups (e.g., aryl or heteroaryl), non-aromatic groups (e.g., cycloalkyl or heterocycloalkyl). The ring may be optionally substituted and may be monocyclic or polycyclic. Typical polycyclic rings generally include bicyclic and tricyclic rings. The ring of the present application typically has 1 to 20 ring atoms, for example 1 ring atom, 2 ring atoms, 3 ring atoms, 4 ring atoms, 5 ring atoms, 6 ring atoms, 7 ring atoms, 8 ring atoms, 9 ring atoms, 10 ring atoms, 11 ring atoms, 12 ring atoms, 13 ring atoms, 14 ring atoms, 15 ring atoms, 16 ring atoms, 17 ring atoms, 18 ring atoms, 19 ring atoms, or 20 ring atoms.

"Yuan" means the number of skeleton atoms constituting a ring. Typical 5-membered rings include, for example, cyclopentyl, pyrrole, imidazole, thiazole, furan, thiophene, and the like; typical 6-membered rings include, for example, cyclohexyl, pyridine, pyran, pyrazine, thiopyran, pyridazine, pyrimidine, benzene, and the like. Wherein, the ring containing hetero atoms in the skeleton atom is a heterocycle; the heteroatom-containing aryl is heteroaryl; the non-aromatic group containing a heteroatom is a heterocycloalkyl group, which includes heterocycloalkyl groups.

"heteroatom" refers to an atom other than carbon or hydrogen. One or more heteroatoms in the heterocycles of the present application may be independently selected from O, S, N, si and P, but are not limited thereto.

"aryl" refers to a monocyclic or fused polycyclic (i.e., rings sharing adjacent pairs of carbon atoms) group of 6 to 14 carbon atoms (6 to 14 members) with a conjugated pi-electron system, preferably 6 to 10 atoms, such as phenyl and naphthyl. More preferably phenyl.

The term "heteroaryl" refers to a heteroaromatic system comprising 1 to 4 (e.g., 1, 2,3, or 4) heteroatoms, 5 to 14 ring atoms (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14), wherein the heteroatoms are selected from oxygen, sulfur, and nitrogen. Heteroaryl groups are preferably 5 to 10 membered, containing 1 to 3 heteroatoms; more preferably 5 or 6 membered, containing 1 to 2 heteroatoms; preferably, for example, imidazolyl, furyl, thienyl, thiazolyl, pyrazolyl, oxazolyl, pyrrolyl, tetrazolyl, pyridyl, pyrimidinyl, thiadiazole, pyrazinyl, and the like, preferably imidazolyl, thiazolyl, pyrazolyl or pyrimidinyl, thiazolyl; more preferably pyrazolyl such as 1H-pyrazol-4-yl or thiazolyl. The heteroaryl ring may be fused to an aryl, heterocycloalkyl, cycloalkyl ring, or another heteroaryl group, thereby forming a fused heteroaryl group. The fused heteroaryl group is preferably an 8-10 membered fused heteroaryl group including, but not limited to: indolyl such as 1H-indol-5-yl, 2-oxo-2, 3-dihydro-1H-benzo [ d ] imidazolyl such as 2-oxo-2, 3-dihydro-1H-benzo [ d ] imidazol-5-yl, or 1H-benzo [ d ] imidazolyl such as 1H-benzo [ d ] imidazol-6-yl.

"cycloalkyl" refers to a cyclic hydrocarbon substituent comprising 1-3 rings, saturated or partially unsaturated (containing one or more double bonds, but no ring has a fully conjugated pi electron system), which includes monocycloalkyl, bicycloalkyl, and tricycloalkyl groups containing 3-20 ring-formable carbon atoms, preferably 3-10 carbon atoms (i.e., 3-10 membered cycloalkyl groups, which may also be referred to as C3-C10 cycloalkyl groups), such as 3 to 8, 3 to 7, 3 to 6, 5 to 6 carbon atoms. Preferably, the cycloalkyl is selected from monovalent cycloalkyl groups obtained from the following rings:

preferred are cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclopentenyl or cyclohexenyl.

It will be appreciated that when a cycloalkyl group is attached to two groups, for example where the groups are cycloalkyl groups, depending on the structure or context, the cycloalkyl groups are divalent, i.e. there are two attachment sites. In this case, it may be called a cycloalkylene group. Examples of preferred cycloalkylene groups include, but are not limited to, monocyclic structures such as cyclopropyl, cyclobutyl, cyclopentyl (e.g., cyclopentyl-1, 2-diyl, cyclopentyl-1, 3-diyl), cyclohexyl (e.g., cyclohexyl-1, 2-diyl, cyclohexyl-1, 3-diyl, cyclohexyl-1, 4-diyl), cycloheptylene, cyclooctyl, and the like.

"heterocycloalkyl" and "cycloheteroalkyl" are used interchangeably to refer to saturated, non-aromatic, monocyclic, fused, bridged, and spiro rings containing one or more (e.g., 1, 2, 3, or 4) heteroatoms. Wherein the heteroatom may be N, O, S or SO ₂ N, O and/or S are preferred. The heterocycloalkyl group can be 3-10 membered (e.g., 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, i.e., containing 3, 4, 5, 6, 7, 8)9 or 10 ring atoms). Typical heterocycloalkyl groups include, but are not limited to, monovalent groups derived from the following rings:

these heterocycloalkyl groups can also be represented by the commonly understood structural formulae, e.g

It is understood that when a heterocycloalkyl group is attached to two groups, depending on structure or context, the heterocycloalkyl group is a divalent group, i.e., there are two attachment sites. In this case, it may be also called a heterocycloalkylene group.

"oxo" refers to the substitution of hydrogen on carbon with =o.

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

"haloalkyl" means that at least one hydrogen in the alkyl group is replaced with a halogen atom, such as CF3.

"substituted" means that one or more hydrogen atoms, preferably up to 5 (e.g., 1, 2, 3, 4, 5), more preferably 1 to 3 hydrogen atoms in the group may be substituted independently of each other with a corresponding number of substituents. It goes without saying that substituents are only in their possible chemical positions, and that the person skilled in the art is able to determine (by experiment or theory) possible or impossible substitutions without undue effort. For example, amino or hydroxyl groups having free hydrogen may be unstable when bound to carbon atoms having unsaturated (e.g., olefinic) bonds.

"inhibitor" refers to a substance that decreases the activity of an enzyme.

"optionally" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs or does not.

The term "substituted or unsubstituted" as used herein means that any group is mono-or polysubstituted by the indicated substituents to the extent chemically permitted by such mono-or polysubstituted (including polysubstituted at the same moiety), each substituent being able to be located at any available position on the group and being able to be attached by any available atom on said substituent. By "any available position" is meant any position on the group that is chemically available by methods known in the art or taught herein and that does not result in an unduly labile molecule. When there are two or more substituents on any group, each substituent is defined independently of any other substituent and thus may be the same or different.

"stereoisomers" as used herein, refers to "stereoisomers" that when a compound of the invention contains one or more asymmetric centers, it may exist as racemates and racemic mixtures, single enantiomers, diastereomeric mixtures, and individual diastereomers. The compounds of the invention may have asymmetric centers and thus result in the presence of two optical isomers. The scope of the present invention includes all possible optical isomers and mixtures thereof. If the compounds of the present invention contain olefinic double bonds, the scope of the present invention includes cis-isomers and trans-isomers unless specified otherwise. The compounds of the invention may exist in tautomeric (one of the functional group isomers) forms which have different points of attachment of hydrogen through one or more double bond shifts, for example, the keto and his enol forms are keto-enol tautomers. Each tautomer and mixtures thereof are within the scope of the present invention. Enantiomers of all compounds. Diastereomers, racemates, meso, cis-trans isomers, tautomers, geometric isomers, epimers, mixtures thereof, and the like are within the scope of the present invention.

The term "compound of the invention" as used herein is intended to encompass compounds of the general formula (I) as defined herein or any preferred or specific embodiment thereof, including compounds of the formulae (I), (II), (III) and the like, and example compounds, stereoisomers, pharmaceutically acceptable salts, tautomers or solvates thereof.

The term "pharmaceutically acceptable" as used herein means molecular entities and compositions approved by or by the corresponding agency of the respective country or listed in the generally recognized pharmacopoeia for animals, and more particularly humans, or which do not produce adverse, allergic or other untoward reactions when administered in appropriate amounts to animals, such as humans.

The term "pharmaceutically acceptable salt" as used herein means a salt of a compound of the invention which is pharmaceutically acceptable and which has the desired pharmacological activity of the parent compound. In particular, such salts are non-toxic and may be inorganic acid addition salts or organic acid addition salts and base addition salts.

The term "individual" as used herein includes humans or non-human animals. Exemplary human individuals include human individuals (referred to as patients) or normal individuals suffering from a disease (e.g., a disease described herein). "non-human animals" in the context of the present invention include all vertebrates, such as non-mammals (e.g., birds, amphibians, reptiles) and mammals, such as non-human primates, domestic animals and/or domesticated animals (e.g., sheep, dogs, cats, cows, pigs, etc.).

The term "pharmaceutical composition" as used herein refers to a composition comprising one or more compounds of formula (I) or stereoisomers, tautomers, pharmaceutically acceptable salts or solvates thereof, and a carrier or excipient commonly accepted in the art for delivering a biologically active compound to an organism (e.g., a human).

The term "pharmaceutical combination" as used herein means that the compounds of the present invention may be combined with other active agents for the purpose of the present invention. The other active agent may be one or more additional compounds of the present invention, or may be a second or additional (e.g., third) compound that is compatible with, i.e., does not adversely affect each other, or has complementary activity to, the compounds of the present invention. Such agents are suitably present in combination in an amount effective to achieve the intended purpose. The other active agents may be co-administered with the compounds of the present invention in a single pharmaceutical composition or may be administered separately in separate discrete units from the compounds of the present invention, either simultaneously or sequentially when administered separately. The successive administrations may be close or distant in time.

It is to be understood that the structures, groups, etc. of the compounds of the present invention conform to the chemical valence rules. Some groups or structures have their linkages omitted when written. For example, in some cases, it is described that M in formula I is selected from N, and M is=N-based on the general structure. Whether written M is selected from N or M is selected from = N-, is understood by those skilled in the art. Other groups may be similarly understood and interpreted.

It should be apparent that, in light of the foregoing, various modifications, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.

Advantageous effects of the invention

The invention provides a BTK and/or BTK C481S inhibitor with novel structure and high activity.

Pharmaceutical composition and administration thereof:

the pharmaceutical compositions of the invention may be formulated by techniques known to those skilled in the art, such as those disclosed in Remington's Pharmaceutical Sciences, 20 th edition. For example, the pharmaceutical compositions of the invention described above may be prepared by mixing a compound of the invention with one or more pharmaceutically acceptable excipients. The preparation may further comprise the step of mixing one or more additional active ingredients with the compound of the invention and one or more pharmaceutically acceptable excipients.

The choice of excipients included in a particular composition will depend on a variety of factors, such as the mode of administration and the form of the composition provided. Suitable pharmaceutically acceptable excipients are well known to those skilled in the art and are described, for example, in Ansel, howard C., et al, ansel's Pharmaceutical Dosage Forms and Drug Delivery systems, philadelphia: lippincott, williams & Wilkins,2004, including, for example, adjuvants, diluents, carriers, pH modifying agents, buffers, sweeteners, fillers, stabilizers, surfactants, wetting agents, lubricants, emulsifiers, suspending agents, preservatives, antioxidants, opacifiers, glidants, processing aids, colorants, flavoring agents, other known additives.

The pharmaceutical compositions of the present invention may be administered in a standard manner. For example, suitable modes of administration include oral, intravenous, rectal, parenteral, topical, transdermal, ocular, nasal, buccal, or pulmonary (inhalation), wherein parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. For these purposes, the compounds of the present invention may be formulated by methods known in the art into the form of, for example, tablets, capsules, syrups, powders, granules, aqueous or oily solutions or suspensions, (lipid) emulsions, dispersible powders, suppositories, ointments, creams, drops, aerosols, dry powder formulations and sterile injectable aqueous or oily solutions or suspensions.

The size of the prophylactic or therapeutic dose of a compound of the invention will vary depending on a number of factors, including the severity of the individual, disorder or condition being treated, the rate of administration, the disposition of the compound and the discretion of the prescribing physician. Generally, an effective dose is about 0.0001 to about 5000mg, e.g., about 0.01 to about 1000 mg/kg/day per kg body weight per day (single or divided administration). For a 70kg person, this amounts to about 0.007 mg/day to about 7000 mg/day, for example about 0.7 mg/day to about 1500 mg/day. Depending on the mode of administration, the compounds of the invention may be present in the pharmaceutical composition in an amount of about 0.01mg to about 1000mg, suitably 0.1 to 500mg, preferably 0.5 to 300mg, more preferably 1 to 150mg, particularly preferably 1 to 50mg, for example 1.5mg, 2mg, 4mg, 10mg, 25mg, etc.; accordingly, the pharmaceutical composition of the invention will comprise from 0.05 to 99% w/w (weight percent), such as from 0.05 to 80% w/w, such as from 0.10 to 70% w/w, such as from 0.10 to 50% w/w of the compound of the invention, all weight percentages being based on the total composition. It will be appreciated that it may be necessary in some circumstances to use doses beyond these limits.

Detailed Description

The following abbreviations have the meanings indicated below:

DMF means N, N-dimethylformamide;

DCM represents dichloromethane;

EtOH represents ethanol;

MeOH represents methanol;

THF represents tetrahydrofuran;

TEA represents triethylamine;

DIPEA represents N, N-diisopropylethylamine;

DIAD means diisopropyl azodicarboxylate

PPh ₃ Representing triphenylphosphine

LiOH represents lithium hydroxide

HCl represents hydrogen chloride

POCl ₃ Represents phosphorus oxychloride

FDPP represents pentafluorophenyl diphenyl phosphate

EDCI stands for 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride;

HOBT represents 1-hydroxybenzotriazole.

The patent also provides a synthesis method of the compound, and the synthesis method of the invention mainly comprises a preparation method reported in chemical literature or related synthesis by taking a commercial chemical reagent as a starting material.

Example 1: n- (((1) ³ E,1 ⁴ E)-1 ⁷ -amino-4 ⁵ -fluoro-9-oxo-1 ⁶ -phenyl-5-oxa-2, 8-diaza-1 (5, 3) -pyrazoline [1,5-a]The preparation and synthesis steps of pyrimidine-4 (3, 2) -pyridine heterocycle nine-6 yl) methyl acrylic amine (compound 1) are as follows:

step 1: preparation of 2- ((5-fluoro-2-methoxypyridin-3-yl) methyl) isoindoline-1, 3-dione (Compound 1A)

(5-fluoro-2-methoxypyridin-3-yl) methanol (3.14 g,20 mmol) and TEA (2.83 g,28 mmol) were dissolved in DCM (100 mL) under argon, methanesulfonyl chloride (2.4 g,21 mmol) was added dropwise at 0deg.C, and the reaction was stirred at room temperature for 2h. After the reaction is completed, adding water to quench After extraction with ethyl acetate, washing the organic layer with water, saturated sodium chloride solution, drying over anhydrous sodium sulfate, concentrating by filtration, dissolving the residue in DMF (50 mL), followed by addition of potassium phthalimide (5.55 g,30 mmol) at 0deg.C, and then stirring the mixture overnight at room temperature. After the reaction was completed, water was added to quench, extraction was performed with ethyl acetate, the organic layer was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered and concentrated to obtain compound 1A. MS (ESI) M/z 287.0 (M+H) ⁺

Step 2: preparation of 2- ((5-fluoro-2-hydroxypyridin-3-yl) methyl) isoindoline-1, 3-dione (Compound 1B)

2- ((5-fluoro-2-methoxypyridin-3-yl) methyl) isoindoline-1, 3-dione (2 g,6.99 mmol) was dissolved in EtOH (30 mL), 4N HCl dioxane solution (50 mL,209.7 mmol) was added and the system stirred at 80℃overnight. After the reaction was completed, the reaction solution was concentrated, a small amount of water was added to the residue, and the resultant solid was filtered, rinsed with water, and dried in vacuo to obtain compound 1B.

MS(ESI)m/z 273.1(M+H) ⁺ 。

Step 3: preparation of ethyl 5, 7-dihydroxy-6-phenylpyrazolo [1,5-a ] pyrimidine-3-carboxylate (Compound 1C)

Ethyl 5-amino-1H-pyrazole-4-carboxylate (15.0 g,96.77 mmol) was dissolved in EtOH (96 mL) and diethyl phenylmalonate (45.6 g,193.50 mmol) and sodium ethoxide (13.2 g,193.50 mmol) were added at room temperature. After the reaction is heated to 80 ℃, stirring is carried out for 20 hours, after the reaction is completed, cooling to room temperature, filtering, washing with ethanol, and finally obtaining the compound 1C. MS (ESI) M/z 300.2 (M+H) ⁺

Step 4: preparation of ethyl 5, 7-dichloro-6-phenylpyrazolo [1,5-a ] pyrimidine-3-carboxylate (Compound 1D)

5, 7-dihydroxy-6-phenylpyrazolo [1,5-a ]]Pyrimidine-3-carboxylic acid ethyl ester (14.5 g,42.27 mmol) was dissolved in POCl ₃ (70 mL) followed by N, N-dimethylaniline (4.0 mL,31.70 mmol) at room temperature. The reaction was stirred for 12 hours after rising to 110 ℃. After the reaction was completed, the reaction solution was concentrated, the residue was poured into ice water, DCM was extracted 3 times, the organic phase was concentrated, and the crude product was separated and purified by silica gel column chromatography to obtain compound 1D.

MS(ESI)m/z 336.2(M+H) ⁺

Step 5: preparation of 5-chloro-7-dibenzylamino-6-phenylpyrazolo [1,5-a ] pyrimidine-3-carboxylic acid ethyl ester (Compound 1E)

Ethyl 5, 7-dichloro-6-phenylpyrazolo [1,5-a ] pyrimidine-3-carboxylate (2.0 g,6.0 mmol) was dissolved in DCM (12 mL) and DIPEA (2.0 mL,12.0 mmol) and dibenzylamine (2.3 mL,12.0 mmol) were added at room temperature and the reaction stirred for 13 h. After the reaction was completed, the reaction solution was washed with saturated aqueous citric acid, extracted 3 times with DCM, and the concentrated organic phase was separated and purified by silica gel column chromatography to obtain compound 1E.

MS(ESI)m/z 497.2(M+H) ⁺

Step 6: preparation of benzyl tert-butyl (2-hydroxypropane-1, 3-diyl) dicarbamate (Compound 1F)

Tert-butyl (3-amino-2-hydroxypropyl) carbamate (3.8 g,21.1 mmol) was dissolved in DCM (128 mL) under nitrogen and DIPEA (3.07 g,17.5 mmol) was then added. The reaction solution was cooled to 0℃and benzyloxycarbonyl chloroformate (3.4 mL,24.0 mmol) was then added to the system, stirred for 30 minutes after the addition was completed, and stirred for a further 12 hours after the temperature was raised to room temperature. After the reaction was completed, a proper amount of water was added to dilute the reaction, DCM was extracted 3 times, the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered to concentrate, and the crude product was separated and purified by silica gel column chromatography to obtain compound 1F.

MS(ESI)m/z 325.2(M+H) ⁺

Step 7: preparation of benzyl tert-butyl (2- ((3- ((1, 3-dioxoisoindolin-2-yl) methyl) -5-fluoropyridin-2-yl) oxy) propane-1, 3-diyl) dicarbamate (Compound 1G)

Benzyl tert-butyl (2-hydroxypropane-1, 3-diyl) dicarbamate (3.0 g,11.0 mmol) was dissolved in dry THF (30 mL) under nitrogen, and 2- ((5-fluoro-2-hydroxypyridin-3-yl) methyl) isoindoline-1, 3-dione (5.0 g,15.0 mmol), PPh were added sequentially at 0deg.C ₃ (5.0 g,19.08 mmol) and DIAD (3.7 mL,19.08 mmol). After the addition was complete, the reaction was warmed to room temperature and stirring was continued for 3 hours. After the reaction was completed, a proper amount of water was added for dilution, DCM was extracted 3 times, the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated, and the obtained crude product was separated and purified by silica gel column chromatography to obtain compound 1G.

MS(ESI)m/z 579.2(M+H) ⁺

Step 8: preparation of benzyl tert-butyl (2- ((3- (aminomethyl) -5-fluoropyridin-2-yl) oxy) propane-1, 3-diyl) dicarbamate (Compound 1H)

Benzyl tert-butyl (2- ((3- ((1, 3-dioxoisoindolin-2-yl) methyl) -5-fluoropyridin-2-yl) oxy) propane-1, 3-diyl) dicarbamate was dissolved in EtOH (50 mL) and hydrazine hydrate (683. Mu.L, 22.0 mmol) was added. After the addition was completed, the reaction system was heated to 80℃and stirred for 12 hours. After the reaction is completed, the reaction solution is concentrated, and the obtained crude product is separated and purified by silica gel column chromatography to obtain the compound 1H.

MS(ESI)m/z 449.2(M+H) ⁺

Step 9: preparation of ethyl 7- (dibenzylamino-5- ((2- ((11, 11-dimethyl-3, 9-dioxo-1-phenyl-2, 10-dioxa-4, 8-diazadodecan-6-yl) oxy) -5-fluoropyridin-3-yl) methyl) amino) -6-phenylpyrazolo [1,5-a ] pyrimidine-3-carboxylate (Compound 1I)

Ethyl 5-chloro-7- (dibenzylamino) -6-phenylpyrazolo [1,5-a ] pyrimidine-3-carboxylate (4.8 g,9.78 mmol) was dissolved in n-butanol (65 mL) followed by the sequential addition of benzyl tert-butyl (2- ((3- (aminomethyl) -5-fluoropyridin-2-yl) oxy) propane-1, 3-diyl) dicarbamate (2.92 g,6.5 mmol) and DIPEA (1.6 mL,9.78 mmol). After the addition, the temperature was raised to 100℃and stirred for 11 hours. After the reaction is completed, the crude product is obtained by reduced pressure distillation and concentration, and the obtained crude product is separated and purified by silica gel column chromatography to obtain the compound 1I.

MS(ESI)m/z 909.4(M+H) ⁺

Step 10: preparation of 5- ((((2- ((1-amino-3- ((tert-butoxycarbonyl) amino) propan-2-yl) oxy) -5-fluoropyridin-3-yl) methyl) amino) -7- (dibenzylamino) -6-phenylpyrazolo [1,5-a ] pyrimidine-3-carboxylic acid (compound 1J)

5- ((((2- ((1-amino-3- ((tert-butoxycarbonyl) amino) propan-2-yl) oxy) -5-fluoropyridin-3-yl) methyl) amino) -7- (dibenzylamino) -6-phenylpyrazolo [1, 5-a)]Pyrimidine-3-carboxylic acid ethyl ester (2.3 g,2.53 mmol) and sodium hydroxide (2.0 g,50.7 mmol) were dissolved in THF/MeOH/H ₂ O mixed solvent (3:1:1, 25 mL). After the addition was completed, the reaction was warmed to 70℃and stirred for 24 hours. After completion of the reaction, the pH of the reaction mixture was adjusted to acidity by adding 1N aqueous hydrochloric acid, then extracted 3 times with DCM/MeOH (9/1), and the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated to give Compound 1J which was used directly in the next reaction.

MS(ESI)m/z 747.3(M+H) ⁺

Step 11: (((1 ³ E,1 ⁴ E)-1 ⁷ - (dibenzylamino) -4 ⁵ -fluoro-9-oxo-1 ⁶ -phenyl-5-oxa-2, 8-diaza-1 (5, 3) -pyrazolo [1,5-a]Preparation of tert-butyl pyrimidin-4 (3, 2) -pyridineheterocycloalkyi-6-yl) methyl carbamate (compound 1K)

5- ((((2- ((1-amino-3- ((tert-butoxycarbonyl) amino) propan-2-yl) oxy) -5-fluoropyridin-3-yl) methyl) amino) -7- (dibenzylamino) -6-phenylpyrazolo [1,5-a ] pyrimidine-3-carboxylic acid was dissolved in DCM (400 mL) and DMF (200 mL), then the reaction solution was cooled to 0 ℃, DIPEA (6.1 mL,36.9 mmol) and FDPP (3.6 g,9.4 mmol) were added to the reaction solution and stirred at room temperature for 12 hours after completion of the reaction, saturated aqueous sodium carbonate solution was added to quench the reaction solution, DCM was extracted 3 times, the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated to give crude compound 1K which was isolated and purified by silica gel column chromatography.

MS(ESI)m/z 729.3(M+H) ⁺

Step 12: (1 ³ E,1 ⁴ E) -6- (aminomethyl) -1 ⁷ - (dibenzylamino) -4 ⁵ -fluoro-1 ⁶ -phenyl-5-oxa-2, 8-diaza-1 (5, 3) -pyrazolo [1,5-a]Preparation of pyrimidine-4 (3, 2) -pyridineheterocycloamphan-9-one (Compound 1L)

Will (((1) ³ E,1 ⁴ E)-1 ⁷ - (dibenzylamino) -4 ⁵ Fluoro-9-oxo-1 ⁶ -phenyl-5-oxa-2, 8-diaza-1 (5, 3) -pyrazolo [1,5-a]Tert-butyl pyrimidin-4 (3, 2) -pyridylazepin-6-yl) methyl carbamate (330 mg,0.45 mmol) was dissolved in DCM (10.0 mL) and HCl (4M in 1,4-dioxane,5.0 mL) was then added slowly to the reaction and stirred at that temperature for 30 min. ReactionAfter completion, TEA was slowly added at 0℃to adjust pH to 7-8, concentrated under reduced pressure, and the obtained crude product was separated and purified by silica gel column chromatography to give Compound 1L.

MS(ESI)m/z 629.2(M+H) ⁺

Step 13: n- (((1) ³ E,1 ⁴ E)-1 ⁷ - (benzhydrylamino) -4 ⁵ -fluoro-9-oxo-1 ⁶ -phenyl-5-oxa-2, 8-diaza-1 (5, 3) -pyrazoline [1,5-a]Preparation of pyrimidine-4 (3, 2) -pyridinium heterocycloamine-6 yl) methyl acrylate (Compound 1M)

Will (1) ³ E,1 ⁴ E) -6- (aminomethyl) -1 ⁷ - (benzhydrylamino) -4 ⁵ -fluoro-1 ⁶ -phenyl-5-oxa-2, 8-diaza-1 (5, 3) -pyrazoline [1,5-a]Pyrimidine-4 (3, 2) -pyridine heterocycle nonatomato-9-one (300 mg,0.477 mmol) was dissolved in DCM (100 mL), cooled to 0deg.C, TEA (96.57 mg,0.95 mmol) and acrylic anhydride (60.18 mg,0.477 mmol) were added sequentially and the reaction stirred at room temperature for 2 hours. After completion of the reaction, the reaction solution was poured into water, extracted 3 times with DCM, the organic phases were combined and washed 2 times with saturated brine, the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated, and the crude product was purified by silica gel column chromatography to give compound 1M.

MS(ESI)m/z 683.28(M+H) ⁺

Step 14: n- (((1) ³ E,1 ⁴ E)-1 ⁷ -amino-4 ⁵ -fluoro-9-oxo-1 ⁶ -phenyl-5-oxa-2, 8-diaza-1 (5, 3) -pyrazoline [1,5-a]Preparation of pyrimidine-4 (3, 2) -pyridinium heterocycloamine-6 yl) methyl acrylate (Compound 1)

N- (((1) ³ E,1 ⁴ E)-1 ⁷ - (benzhydrylamino) -4 ⁵ -fluoro-9-oxo-1 ⁶ -phenyl-5-oxa-2, 8-diaza-1 (5, 3) -pyrazoline [1,5-a]pyrimidine-4 (3, 2) -pyridine heterocycle nonatomato-6 yl) methyl acrylic acid amine (48 mg,0.07 mmol) was dissolved in DCM (4.5 mL), the reaction solution was cooled to 0℃and trifluoromethanesulfonic acid (225. Mu.L) was added dropwise to the reaction solution and stirred at this temperature for 15 minutes, after the reaction was completed, TEA was slowly added at 0℃to adjust pH to 7-8, and concentrated under reduced pressure, and the crude product obtained was isolated by high-pressure preparation to give compound 1 of example 1.

MS(ESI)m/z 503.19(M+H) ⁺

¹ H-NMR(400MHz,DMSO-d ₆ )δ9.72(d,J＝7.1Hz,1H),8.34(t,J＝5.6Hz,1H),8.02(s,1H),7.95(d,J＝3.0Hz,1H),7.65(dd,J＝8.8,3.0Hz,1H),7.56(t,J＝7.3Hz,2H),7.47(t,J＝7.4Hz,1H),7.31(s,2H),6.93(t,J＝6.0Hz,1H),6.67(s,2H),6.24(dd,J＝17.0,10.2Hz,1H),6.08(dd,J＝17.0,2.2Hz,1H),5.59(dd,J＝9.9,2.2Hz,1H),5.06(m,1H),4.89(dd,J＝14.3,4.7Hz,1H),3.96-3.90(m,1H),3.85(dd,J＝14.6,5.8Hz,1H),3.66-3.55(m,2H),3.19-3.14(m,1H).

Example 2: n- (((1) ³ E,1 ⁴ E)-1 ⁷ - (amino) -4 ⁵ -fluoro-9-oxo-1 ⁶ -phenyl-5-oxa-2, 8-diaza-1 (5, 3) -pyrazolo [1,5-a]Preparation of pyrimidin-4 (3, 2) -pyridine heterocyclo nona-6-yl) methyl methacrylate amine (compound 2)

The same preparation method as in example 1 was used to obtain compound 2 of example 2 using 2-methacrylic anhydride instead of acrylic anhydride in example 1.

MS(ESI)m/z 517.53(M+H) ⁺

¹ H-NMR(400MHz,DMSO-d ₆ )δ9.73(d,J＝7.1Hz,1H),8.15(t,J＝5.8Hz,1H),8.02(s,1H),7.94(d,J＝3.0Hz,1H),7.65(dd,J＝8.8,3.0Hz,1H),7.56(t,J＝7.0Hz,2H),7.47(t,J＝7.3Hz,1H),7.32(s,2H),6.94(t,J＝5.9Hz,1H),6.68(s,2H),5.64(s,1H),5.32(s,1H),5.15(m,1H),4.89(dd,J＝14.4,5.1Hz,1H),3.95-3.82(m,2H),3.61-3.40(m,2H),3.22-3.13(m,1H),1.99-1.83(m,3H).

Implementation of the embodimentsExample 3: (E) -N- (((1) ³ E,1 ⁴ E)-1 ⁷ -amino-4 ⁵ -fluoro-9-oxo-1 ⁶ -phenyl-5-oxa-2, 8-diaza-1 (5, 3) -pyrazolo [1,5-a ]Preparation of pyrimidin-4 (3, 2) -pyridine heterocyclo nonatomato-6-yl) methyl) -2-cyano-4, 4-dimethyl pent-2-enamide (compound 3):

the synthesis steps are as follows:

/>

step 1: (1 ³ E,1 ⁴ E)-1 ⁷ -amino-6- (aminomethyl) -4 ⁵ -fluoro-1 ⁶ -phenyl-5-oxa-2, 8-diaza-1 (5, 3) -pyrazolo [1,5-a]Preparation of pyrimidine-4 (3, 2) -pyridineheterocycloamphan-9-one (Compound 3A)

Will (((1) ³ E,1 ⁴ E)-1 ⁷ - (dibenzylamino) -4 ⁵ -fluoro-9-oxo-1 ⁶ -phenyl-5-oxa-2, 8-diaza-1 (5, 3) -pyrazolo [1,5-a]Pyrimidine-4 (3, 2) -pyridineheterocycloalpan-6-yl) methyl) carbamic acid tert-butyl ester (220 mg,0.302 mmol) was dissolved in DCM (5 mL), and trifluoromethanesulfonic acid (1 mL) was added dropwise at 0deg.C and stirred for 20 min. After completion of the reaction, the reaction was quenched by addition of TEA (1 mL), extracted with DCM, the organic phases combined, dried over anhydrous sodium sulfate, filtered and concentrated to give compound 3A. MS (ESI) M/z 449.5 (M+H) ⁺

Step 2: (E) -N- (((1) ³ E,1 ⁴ E)-1 ⁷ -amino-4 ⁵ -fluoro-9-oxo-1 ⁶ -phenyl-5-oxa-2, 8-diaza-1 (5, 3) -pyrazolo [1,5-a]Preparation of pyrimidine-4 (3, 2) -pyridinium heterocycloalkyi-6-yl) methyl) -2-cyano-4, 4-dimethylpent-2-enamide (Compound 3)

Will (1) ³ E,1 ⁴ E)-1 ⁷ -amino-6- (aminomethyl) -4 ⁵ -fluoro-1 ⁶ -phenyl-5-oxa-2, 8-diaza-1 (5, 3) -pyrazolo [1,5-a]Pyrimidine-4 (3, 2) -pyridineheterocycloamphen-9-one (130 mg,0.268 mg), EDCI (111 mg,0.579 mmol), HOBT (78 mg,0.579 mmol) and DIPEA (173 mg,1.34 mmol) were dissolved in DCM/DMF (15:1, 6 mL) mixed solution and stirred at room temperature for 1 hour, followed by addition of 2-cyano-4, 4-dimethyl-2-pentenoic acid (80 mg,0.522 mmol) and stirring overnight at room temperature. After the reaction is completed, the reaction solution is concentrated, and the residue is separated and purified by high-pressure preparation to obtain the compound 3.MS (ESI) M/z 584.2 (M+H) ⁺

¹ H NMR(400MHz,DMSO-d ₆ )δ9.77(d,J＝8.0Hz,1H),8.58(t,J＝5.6Hz,1H),8.06(s,1H),7.96(d,J＝3.0Hz,1H),7.69(dd,J＝8.8,3.2Hz,1H),7.55(m,3H),7.35(d,J＝6.4Hz,3H),6.98(t,J＝6.0Hz,1H),6.72(s,2H),5.21(m,1H),4.93(m,1H),3.98(m,1H),3.88(m,1H),3.72–3.54(m,2H),3.27–3.16(m,1H),1.21(s,9H).

Biological test data:

unless otherwise indicated, the experimental materials, reagents, procedures and methods used in the following active examples are all available from commercial sources or are readily known or prepared based on the prior art.

Experimental example 1: in vitro kinase Activity test of the Compounds of the invention

Purpose of experiment

IC with compounds ₅₀ (half inhibitory concentration) values are used as indicators to evaluate the inhibition of BTK and/or mutated BTK kinase by the compounds.

Experimental method

Using the method of Mobility shift assay, compounds were tested for their inhibitory activity against the following kinases: BTK and/or mutated BTK. The initial concentration of the compound used was 1000nM, 3-fold dilution, 10 concentrations, single well assay.

Reagent and consumable:

reagent name	Suppliers of goods	Goods number	Lot number
				BTK	Carna	08-180	14CBS-0619Q
BTK C481S	Carna	08-547	14CBS-0633H
				Kinase substrate 2	GL	190861	P200807-YS190861
Kinase substrate 4	GL	112395	P171211-XY112395
				Kinase substrate 22	GL	112393	P200403-CL112393
DMSO	Sigma	D8418-1L	SHBG3288V
				384-wellplate	Corning	3573	12619003
384-wellplate	Corning	3575BC	31316039
				MgCl2	Sigma	M1028	/
ATP	Promeg	V910B	/
				DTT	Sigma	D0632	/

Instrument:

centrifuge (manufacturer: eppendorf type 5430)

Enzyme label instrument (manufacturer: perkin Elmer model Caliper EZ Reader)

Echo 550 (manufacturer: labcyte, model: echo 550)

Preparation of kinase reaction buffer:

20mM hydroxyethylpiperazine ethylsulfuric acid (Hepes) (pH 7.5) buffer, 10mM MgCl ₂ 1mM ethylene glycol bis (aminoethyl) ether tetraacetic acid (EGTA), 0.02% polyoxyethylene lauryl ether (Brij 35), 0.02mg/ml N, O-bis (trimethylsilyl) acetamide (BSA), 0.1mM Na ₃ VO ₄ 2mM Dithiothreitol (DTT), 1% DMSO.

A compound:

the compound to be tested is dissolved in a 100% dimethyl sulfoxide (DMSO) system and is prepared to be 10mM for later use, and is stored in a nitrogen cabinet in a dark place.

Reaction conditions:

	ATP(μM)	Reaction time
			BTK	71	30min
BTK C481S	90	30min

kinase reaction process:

(1) 1 Xkinase reaction buffer was prepared.

(2) Preparing a compound concentration gradient: the initial concentration of the compound was 1000nM, diluted to 100% dimethyl sulfoxide (DMSO) in 384 well plates at 100-fold final concentration, and the compound was diluted 3-fold precisely with kinase buffer at 10 concentrations at 0.0508nM. 100% DMSO solutions were diluted to 100-fold final concentration in 384source plates. 250nl of 100-fold final concentration of compound was transferred to the destination plate 384-well plate using a dispenser Echo 550. Positive and negative control wells were added with 250nl DMSO.

(3) A2.5-fold final concentration of kinase solution was prepared with 1 Xkinase buffer.

(4) Adding 10 mu L of kinase solution with 2.5 times of final concentration to each of the compound well and the positive control well; mu.L of 1 Xkinase buffer was added to the negative control wells.

(5) Centrifugation at 1000rpm for 30 seconds, the reaction plate was shaken and mixed well and incubated at room temperature for 10 minutes.

(6) A25/15-fold final concentration of a mixed solution of Adenosine Triphosphate (ATP) and kinase substrate was prepared with 1 Xkinase buffer.

(7) The reaction was initiated by adding 15. Mu.L of a 25/15-fold final concentration of a mixed solution of Adenosine Triphosphate (ATP) and substrate.

(8) The 384-well plate is centrifuged at 1000rpm for 30 seconds, and is incubated for 30-240 minutes at room temperature after shaking and mixing.

(9) After stopping the kinase reaction, the mixture was centrifuged at 1000rpm for 30 seconds and mixed with shaking.

(10) The conversion was read with Caliper EZ Reader.

Data analysis

Calculation formula

Wherein: conversion% _sample is a Conversion reading of the sample; convertion% _min: negative control Kong Junzhi, representing conversion reading without enzyme wells; convesion% _max: positive control Kong Junzhi, represents a conversion reading without compound inhibition wells.

Fitting dose-response curve

The log value of the concentration is taken as an X axis, the percent inhibition rate is taken as a Y axis, and the graph pad 6.0 analysis software is adopted to simulate a quantitative response curve, so that the IC of each compound on the enzyme activity is obtained ₅₀ Values.

The experimental results are shown in table 1:

TABLE 1

"-" represents undetected.

It will be appreciated by those skilled in the art that the foregoing description is exemplary and illustrative in nature and is intended to illustrate the invention and its preferred embodiments. Through routine experimentation, those skilled in the art will appreciate that obvious modifications and variations can be made without departing from the spirit of the invention. All such modifications are intended to be included within the scope of the following claims. Accordingly, it is intended that the invention be defined not by the above description but by the scope of the following claims and their equivalents.

All publications cited in this specification are herein incorporated by reference.

Claims

1. A compound or stereoisomer, pharmaceutically acceptable salt, solvate, or tautomer of formula (I):

m is selected from N, CH, CR ₆ ；

R ₁ selected from hydrogen or C _1-4 An alkyl group;

R ₃ 、R ₄ each independently selected from hydrogen, C _1-4 Alkyl, halogenated C _1-4 Alkyl, cyano, amino, nitro, halogen; the C is _1-4 Alkyl, halogenated C _1-4 Alkyl groupOptionally can be cyano, amino, nitro, halogen, C _1-4 Substituents for alkoxy, 3-6 membered cycloalkyl, 3-6 membered heterocycloalkyl, phenyl, 5-6 membered heteroaryl; the 3-6 membered heterocycloalkyl, 5-6 membered heteroaryl may be substituted by halogen, amino, hydroxy, C _1-4 Alkyl, C _1-4 Alkoxy is substituted, and the heteroatom is optionally selected from 1 to 3N, O, S;

R ₅ selected from hydrogen or C _1-4 An alkyl group;

R ₇ selected from hydrogen or amino;

a is selected from 3-6 membered cycloalkyl, 3-6 membered heterocycloalkyl, and C _2-6 Olefins or C _2-6 Alkynes, 3-6 membered unsaturated cycloalkyl, 3-6 membered unsaturated heterocycloalkyl, 6-8 membered aryl, 5-10 membered heteroaryl, said 3-6 membered cycloalkyl, 3-6 membered heterocycloalkyl, C _2-6 Olefins or C _2-6 Alkynes, 3-6 membered unsaturated cycloalkyl, 3-6 membered unsaturated heterocycloalkyl, 6-8 membered aryl, 5-10 membered heteroaryl may be substituted with halo, C1-4 alkyl, cyano, hydroxy, nitro, -NRcRd, -NHRc, - (CH 2) nNRcRd, -NHC (O) ORc, -NHC (O) NHRc, -NHC (O) Rc, -ORc, -OC (O) ORc, -C (O) Rc, -C (O) NHRc, - (CH 2) nC (O) NHRc, -C (O) NRcRd;

n is selected from 1, 2 and 3.

2. The compound of claim 1, or a stereoisomer, pharmaceutically acceptable salt, solvate, or tautomer thereof, wherein a is selected from the group consisting of a benzene ring, a 5-6 membered heteroaryl; the benzene ring, 5-6 membered heteroaryl group may be substituted with halogen, C _1-4 Alkyl, cyano, hydroxy, nitro, -NRcRd, -NHRc, - (CH) ₂ )nNRcRd、-NHC(O)ORc、-NHC(O)NHRc、-NHC(O)Rc、-ORc、-OC(O)ORc、-OC(O)Rc、-C(O)Rc、-C(O)NHRc、-(CH ₂ ) nC (O) NHRc, -C (O) NRcRd; rc, rd are each independently selected from C _1-4 Alkyl, 3-6 membered cycloalkyl or 3-6 membered heterocycloalkyl, said C _1-4 Alkyl groups may be substituted by halogen, amino, hydroxy, C _1-4 Alkoxy substituted; the 3-6 membered cycloalkyl or 3-6 membered heterocycloalkyl group may beTo be covered by halogen, amino, hydroxy, C _1-4 Alkyl, C _1-4 Alkoxy groups are substituted.

3. The compound of claim 2, or a stereoisomer, pharmaceutically acceptable salt, solvate, or tautomer thereof, wherein

A is selected from benzene ring, pyridine ring,

4. A compound according to claim 3, or a stereoisomer, pharmaceutically acceptable salt, solvate, or tautomer thereof, wherein a is selected from the group consisting of benzene rings which may be substituted with halogen, C _1-4 Alkyl, cyano, hydroxy, nitro, -NRcRd, -NHRc, - (CH) ₂ )nNRcRd、-NHC(O)ORc、-NHC(O)NHRc、-NHC(O)Rc、-ORc、-OC(O)ORc、-OC(O)Rc、-C(O)Rc、-C(O)NHRc、-(CH ₂ ) nC (O) NHRc, -C (O) NRcRd; rc, rd are each independently selected from C _1-4 An alkyl group; the C is _1-4 Alkyl groups may be substituted by halogen, amino, hydroxy, C _1-4 Alkoxy groups are substituted.

5. The compound of claim 1, or a stereoisomer, pharmaceutically acceptable salt, solvate, or tautomer thereof, wherein R ₃ 、R ₄ Each independently selected from hydrogen, C _1-4 Alkyl, halogenated C _1-4 Alkyl, cyano, amino, nitro, halogen; the C is _1-4 Alkyl, halogenated C _1-4 Alkyl groups optionally being substituted by cyano, amino, nitro, halogenElement, C _1-4 Alkoxy, 6 membered cycloalkyl, 6 membered heterocycloalkyl; the 6-membered cycloalkyl, 6-membered heterocycloalkyl may be substituted by halogen, amino, hydroxy, C _1-4 Alkyl, C _1-4 Alkoxy substituted; the 6-membered heterocycloalkyl is optionally selected from 1 to 3N; preferably, the 6-membered ring is methylpiperidinyl.

6. A compound according to claim 1, or a stereoisomer, pharmaceutically acceptable salt, solvate, or tautomer thereof, R ₅ Selected from hydrogen.

7. A compound according to claim 1, or a stereoisomer, pharmaceutically acceptable salt, solvate, or tautomer thereof, R ₆ Selected from hydrogen, fluorine, chlorine, bromine.

8. The compound of claim 1, or a stereoisomer, pharmaceutically acceptable salt, solvate, or tautomer thereof, having the structure of formula (II):

wherein X is ₁ 、R ₁ 、R ₂ 、R ₃ 、R ₄ 、R ₅ As defined in any one of claims 1 to 6.

9. The compound of claim 1, or a stereoisomer, pharmaceutically acceptable salt, solvate, or tautomer thereof, having the structure of formula (III):

wherein R is ₁ 、R ₂ 、R ₃ 、R ₄ 、R ₅ As defined in any one of claims 1 to 6.

10. The compound of claim 1, or a stereoisomer, pharmaceutically acceptable salt, solvate, or tautomer thereof, having the structure:

11. A pharmaceutical composition comprising a compound according to any one of claims 1-10, or a stereoisomer, pharmaceutically acceptable salt, solvate, or tautomer thereof, and a pharmaceutically acceptable excipient.

12. Use of a compound according to any one of claims 1-10, or a stereoisomer, pharmaceutically acceptable salt, solvate, or tautomer thereof, in the manufacture of a medicament for the treatment of a BTK mediated disease.

13. The use of claim 12, wherein the BTK-mediated disease is selected from cancer, autoimmune disease or allergic disease.

14. The use of claim 13, wherein the cancer is selected from one or more of a diffuse large B-cell lymphoma, mantle cell lymphoma, chronic lymphocytic lymphoma, extranodal marginal zone B-cell lymphoma, B-cell chronic lymphocytic leukemia, B-cell prolymphocytic leukemia, acute lymphoblastic leukemia of mature B-cells, 17 p-deleted chronic lymphocytic leukemia, waldenstrom macroglobulinemia, lymphoplasmacytomegaloma, splenic marginal zone lymphoma, plasmacytomenoma, intranodal marginal zone B-cell lymphoma, mantle cell lymphoma, intravascular large B-cell lymphoma, and primary exudative lymphoma; the autoimmune disease is selected from one or more of systemic lupus erythematosus, rheumatoid arthritis, sjogren's syndrome, multiple sclerosis, inflammatory enteritis such as Crohn's disease and ulcerative colitis, urticaria, immune thrombocytopenia, igA nephropathy, hidradenitis suppurativa, psoriasis, vitiligo, neutrophilic dermatoses, autoimmune vesicular diseases such as pemphigus and pemphigoid, igG 4-related diseases, autoimmune hemolytic anemia, rheumatic fever, antiphospholipid syndrome, systemic sclerosis/scleroderma, autoimmune hepatitis, primary sclerocholangitis, primary biliary cirrhosis, allergic purpura, churg-Strauss syndrome/allergic granulomatosis vasculitis, behcet's disease, ANCA-related small vessel inflammation, dermatitis herpetiformis; the allergic diseases are selected from one or more of allergic conjunctivitis, allergic rhinitis, allergic asthma, atopic dermatitis, and chronic asthma.