WO2021008441A1 - 含有5-氮杂螺庚烷的btk抑制剂 - Google Patents

含有5-氮杂螺庚烷的btk抑制剂 Download PDF

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Publication number
WO2021008441A1
WO2021008441A1 PCT/CN2020/101200 CN2020101200W WO2021008441A1 WO 2021008441 A1 WO2021008441 A1 WO 2021008441A1 CN 2020101200 W CN2020101200 W CN 2020101200W WO 2021008441 A1 WO2021008441 A1 WO 2021008441A1
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Prior art keywords
compound
pharmaceutically acceptable
formula
alkyl
acceptable salt
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PCT/CN2020/101200
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English (en)
French (fr)
Inventor
张寅生
任景
徐胜
王庆璘
邓力
刘戌时
施伟
宋伟
张旻澄
张颖
徐宏江
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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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Priority to CN202080043175.4A priority Critical patent/CN113993871B/zh
Publication of WO2021008441A1 publication Critical patent/WO2021008441A1/zh
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/4985—Pyrazines or piperazines ortho- or peri-condensed with heterocyclic ring systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • 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
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • 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
    • 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

Definitions

  • This application relates to a compound containing 5-azaspiroheptane as a BTK inhibitor, a preparation method thereof, a pharmaceutical composition containing the compound, and its use in the treatment of BTK-related diseases.
  • BTK Bruton's tyrosine kinase
  • B-cell tumors such as leukemia, multiple myeloma and B-cell immune diseases.
  • the irreversible inhibitors currently on the market, such as ibrutinib often have mutations in the BTK binding site, resulting in decreased drug activity and drug resistance. Therefore, more BTK inhibitors are needed clinically, and they have a higher resistance to BTK. Selectivity to avoid the side effects caused by off-target effects.
  • the application provides a compound of formula (I), its stereoisomers or pharmaceutically acceptable salts thereof,
  • Ring A is selected from 5-10 membered heteroaryl or C 6-10 aryl
  • R 1 is independently selected from halogen, hydroxy, amino, cyano, C 1-6 alkoxy or C 1-6 alkyl, the C 1-6 alkoxy or C 1-6 alkyl is optionally Halogen substitution;
  • n is selected from 0, 1, 2, 3, 4, 5 or 6;
  • R 2 is selected from hydrogen, R a S (O) 2 -, (R a O) 2 P (O) - , or R a C (O) -;
  • R a is independently selected from a C 2-6 alkynyl group, C 2-6 alkenyl, C 1-6 alkyl, C 3-6 cycloalkyl, (C 1-6 alkyl) NH -, (C 1 -6 alkyl) 2 N-, 3-6 membered heterocycloalkyl, 5-10 membered heteroaryl, or C 6-10 aryl group, the above R a is optionally substituted (C 1-6 alkyl) 2 N -, (C 1-6 alkyl)NH-, hydroxyl, amino, halogen or cyano substitution.
  • the present application provides a pharmaceutical composition, which comprises the compound of formula (I) described in the present application, its stereoisomer or a pharmaceutically acceptable salt thereof.
  • the present application provides a method for preventing or treating BTK-related diseases in mammals, including administering a therapeutically effective amount of the compound of formula (I) described in the present application, and its stereoisomers to mammals in need of such prevention or treatment Or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition described in this application.
  • this application provides that the compound of formula (I), its stereoisomers or pharmaceutically acceptable salts thereof, or the pharmaceutical composition described in this application are prepared for prevention or treatment with BTK Use in medicine for related diseases.
  • this application provides that the compound of formula (I) described in this application, its stereoisomers or pharmaceutically acceptable salts thereof, or the pharmaceutical composition described in this application are used in the prevention or treatment of diseases related to BTK the use of.
  • this application provides the compound of formula (I) described in this application, its stereoisomers or pharmaceutically acceptable salts thereof, or the drug combination described in this application for preventing or treating diseases related to BTK Things.
  • This application relates to a compound of formula (I), its stereoisomers or pharmaceutically acceptable salts thereof,
  • Ring A is selected from 5-10 membered heteroaryl or C 6-10 aryl
  • R 1 is independently selected from halogen, hydroxy, amino, cyano, C 1-6 alkoxy or C 1-6 alkyl, the C 1-6 alkoxy or C 1-6 alkyl is optionally Halogen substitution;
  • n is selected from 0, 1, 2, 3, 4, 5 or 6;
  • R 2 is selected from hydrogen, R a S (O) 2 -, (R a O) 2 P (O) - , or R a C (O) -;
  • R a is independently selected from a C 2-6 alkynyl group, C 2-6 alkenyl, C 1-6 alkyl, C 3-6 cycloalkyl, (C 1-6 alkyl) NH -, (C 1 -6 alkyl) 2 N-, 3-6 membered heterocycloalkyl, 5-10 membered heteroaryl, or C 6-10 aryl group, the above R a is optionally substituted (C 1-6 alkyl) 2 N -, (C 1-6 alkyl)NH-, hydroxyl, amino, halogen or cyano substitution.
  • 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 A is pyridin-2-yl. In some embodiments, ring A is phenyl.
  • R 1 is independently selected from halogen, cyano, C 1-3 alkyl or C 1-3 alkoxy, the C 1-3 alkyl or C 1-3 alkoxy optionally Ground is substituted by halogen (e.g., fluorine).
  • halogen e.g., fluorine
  • R 1 is independently selected from halogen, cyano, C 1-3 alkoxy, or C 1-3 alkyl optionally substituted with halogen; in some embodiments, R 1 is independently Is selected from fluorine, chlorine, bromine, iodine, cyano, methoxy, or methyl optionally substituted by halogen; in some embodiments, R 1 is independently selected from chlorine, bromine, cyano, -CHF 2 or methyl Oxy; In some embodiments, R 1 is independently selected from bromo.
  • n is selected from 0, 1, 2, 3, or 4; in some embodiments, m is selected from 0, 1, or 2; in some embodiments, m is selected from 1.
  • R 2 is selected from R a C (O) -.
  • R a is selected from C 2-6 alkynyl group, C 2-6 alkenyl, C 1-6 alkyl, C 3-6 cycloalkyl, or 3-6 membered heterocycloalkyl; in some embodiments, R a is selected from C 2-3 alkynyl or C 3-4 cycloalkyl; in some embodiments, R a is selected from propynyl.
  • R 2 is selected from R a C (O) -, wherein R a is selected from C 2-6 alkynyl group, C 2-6 alkenyl, C 1-6 alkyl, C 3-6 cycloalkyl R a is selected from C 2-3 alkynyl or C 3-4 cycloalkyl; or R a is propynyl.
  • R 2 is selected from
  • the compound of formula (I), its stereoisomer, or pharmaceutically acceptable salt thereof of the present application is selected from the group of formula (I-1), its stereoisomer or its pharmaceutically acceptable salt salt,
  • ring A, R 1 , R 2 , and m are as defined above.
  • the compound of formula (I) of the present application its stereoisomers or pharmaceutically acceptable salts thereof are selected from compounds of formula (II) or formula (II-1) or formula (II-2), Or a stereoisomer or a pharmaceutically acceptable salt thereof,
  • R 1 and m are as defined above.
  • the compound of formula (I) of the present application is selected from the following compounds or their pharmaceutically acceptable salts:
  • the present application relates to a pharmaceutical composition, which comprises the compound of formula (I) of the present application, its stereoisomer or a pharmaceutically acceptable salt thereof.
  • the pharmaceutical composition of the present application further includes pharmaceutically acceptable excipients.
  • the present application relates to a method for preventing or treating BTK-related diseases in mammals, including administering a therapeutically effective amount of a compound of formula (I), its stereoisomers or a mammal in need of such prevention or treatment, preferably a human Its pharmaceutically acceptable salt, or its pharmaceutical composition.
  • this application relates to the use of a compound of formula (I), its stereoisomers or pharmaceutically acceptable salts, or pharmaceutical compositions thereof in the preparation of drugs for preventing or treating BTK-related diseases.
  • the present application relates to the use of a compound of formula (I), its stereoisomer or its pharmaceutically acceptable salt, or its pharmaceutical composition in the prevention or treatment of BTK-related diseases.
  • the present application relates to a compound of formula (I), its stereoisomer or its pharmaceutically acceptable salt, or its pharmaceutical composition for preventing or treating BTK-related diseases.
  • the aforementioned BTK-related diseases are BTK-mediated diseases.
  • the above-mentioned BTK-related diseases are selected from autoimmune diseases, inflammatory diseases or cancer.
  • the above-mentioned BTK-related diseases are selected from B lymphoma. In some specific embodiments, the above-mentioned BTK-related diseases are selected from diffuse large B-cell lymphoma.
  • substituted means that any one or more hydrogen atoms on a specific group are replaced by a substituent, as long as the valence of the specific group is normal and the compound after substitution is stable.
  • it means that two hydrogen atoms are replaced, and the oxo will not occur on the aromatic group.
  • the term “optional” or “optionally” means that the event or situation described later can occur or not occur, and the description includes occurrence of said event or situation and non-occurrence of said event or situation.
  • the ethyl group is "optionally" substituted by halogen, meaning that the ethyl group can be unsubstituted (CH 2 CH 3 ), monosubstituted (such as CH 2 CH 2 F), or polysubstituted (such as CHFCH 2 F, CH 2 CHF 2 etc.) or completely substituted (CF 2 CF 3 ).
  • CH 2 CH 3 unsubstituted
  • monosubstituted such as CH 2 CH 2 F
  • polysubstituted such as CHFCH 2 F, CH 2 CHF 2 etc.
  • CF 2 CF 3 completely substituted
  • C mn in this context means that the part has an integer number of carbon atoms in a given range.
  • C 1-6 means that the group can have 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, or 6 carbon atoms.
  • any variable such as R
  • its definition in each case is independent. For example, if a group contains 2 Rs, each R has independent options.
  • halo or halogen refers to fluorine, chlorine, bromine and iodine.
  • hydroxy refers to the -OH group.
  • amino refers to the -NH 2 group.
  • cyano refers to the -CN group.
  • alkyl refers to a hydrocarbon of the formula C n H 2n + 1, for example, C 1 - 6 alkyl, C 1 - 3 alkyl.
  • the alkyl group may be linear or branched.
  • C 1 - 6 alkyl refers to (e.g., methyl, ethyl, n-propyl, isopropyl, alkyl containing 1 to 6 carbon atoms, n-butyl, isobutyl, sec-butyl, Tert-butyl, n-pentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, neopentyl, hexyl, 2-methylpentyl, etc.).
  • the alkyl moiety (ie, alkyl) of alkoxy, alkylamino, dialkylamino, alkylsulfonyl and alkylthio have the same definition as above.
  • alkoxy refers to -O- alkyl, e.g., -OC 1 - 6 alkyl, -OC 1 - 3 alkyl.
  • alkenyl refers to an unsaturated aliphatic hydrocarbon group having at least one double bond of carbon atoms and hydrogen atoms, straight-chain or branched-chain, e.g., C 2 - 6 alkenyl, C 2 - 3 alkenyl group.
  • alkenyl include, but are not limited to, vinyl, 1-propenyl, 2-propenyl, 1-butenyl, isobutenyl, 1,3-butadienyl, and the like.
  • alkynyl refers to an unsaturated aliphatic hydrocarbon group having at least one triple bond in a straight or branched chain of carbon atoms and hydrogen atoms, e.g., C 2 - 6 alkynyl, C 2 - 3 alkynyl group.
  • alkynyl groups include, but are not limited to, ethynyl (-C ⁇ CH), 1-propynyl (-C ⁇ C-CH 3 ), 2-propynyl (-CH 2 -C ⁇ CH), 1,3-Butadiynyl (-C ⁇ CC ⁇ CH) and so on.
  • cycloalkyl refers to a carbocyclic ring that is fully saturated and may exist as a monocyclic, bridged, or spiro ring. Unless otherwise indicated, the carbocyclic ring is usually a 3 to 10 membered ring, or a 3 to 6 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, adamantane Alkyl and so on.
  • heterocycloalkyl refers to a cyclic group that is fully saturated and may exist as a monocyclic, bridged, or spiro ring. Unless otherwise indicated, the heterocyclic ring is generally a 3 to 7 membered ring containing 1 to 3 heteroatoms (preferably 1 or 2 heteroatoms) independently selected from sulfur, oxygen and/or nitrogen.
  • the heterocycloalkyl group may be a 3 to 6 membered ring containing 1 or 2 heteroatoms independently selected from oxygen and/or nitrogen. Examples of 3-membered heterocycloalkyl groups include, but are not limited to, oxirane, sulfiethane, and azathione groups.
  • Non-limiting examples of 4-membered heterocycloalkyl include, but are not limited to, azetidinyl, oxetane
  • Examples of cyclic groups, thiabutanyl, and 5-membered heterocycloalkyl groups include, but are not limited to, tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, isoxazolidinyl, oxazolidinyl, isothiazolidinyl, thiazolidine
  • 6-membered heterocycloalkyl include but are not limited to piperidinyl, tetrahydropyranyl, tetrahydrothiopyranyl, morpholinyl, piperazinyl, 1, Examples of 4-thiaxanyl, 1,4-dioxanyl, thiomorpholinyl, 1,3-dithianyl, 1,4-dithianyl, and 7-membered heterocycloalkyl include But
  • aryl refers to an all-carbon monocyclic or fused polycyclic aromatic ring group with a conjugated ⁇ -electron system.
  • the aryl group may have 6-20 carbon atoms, 6-14 carbon atoms, or 6-12 carbon atoms, or 6-10 carbon atoms.
  • Non-limiting examples of aryl groups include, but are not limited to, phenyl, naphthyl, anthracenyl, 1,2,3,4-tetralin and the like.
  • heteroaryl refers to a monocyclic or fused polycyclic ring system containing at least one (for example, 1, 2 or 3) ring atoms selected from N, O and/or S, and the remaining ring atoms are C, And has at least one aromatic ring.
  • Preferred heteroaryl groups have a single 4- to 8-membered ring, or 5- to 8-membered ring, or 6-membered ring (for example, a 6-membered heteroaryl containing 1 or 2 N and/or O), or 6 to 14 One, especially multiple fused polycyclic rings of 6 to 10 ring atoms.
  • heteroaryl groups include, but are not limited to, pyrrolyl, furyl, thienyl, imidazolyl, oxazolyl, pyrazolyl, pyridyl, pyrimidinyl, pyrazinyl, quinolinyl, isoquinolinyl , Tetrazolyl, triazolyl, triazinyl, benzofuranyl, benzothienyl, indolyl, isoindolyl, etc.
  • treatment means administering the compound or formulation described in this application to ameliorate or eliminate a disease or one or more symptoms related to the disease, and includes:
  • prevention means administering the compound or formulation described in this application to prevent a disease or one or more symptoms related to the disease, including: preventing the occurrence of a disease or disease state in a mammal, especially when this When mammals are susceptible to this disease state, but have not been diagnosed as having this disease state.
  • terapéuticaally effective amount means (i) treating or preventing a specific disease, condition or disorder, (ii) reducing, ameliorating or eliminating one or more symptoms of a specific disease, condition or disorder, or (iii) preventing or delaying The amount of the compound of the present application for the onset of one or more symptoms of a specific disease, condition, or disorder described herein.
  • the amount of the 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 it can be routinely determined by those skilled in the art. Determined by its own knowledge and this disclosure.
  • pharmaceutically acceptable refers to those compounds, materials, compositions and/or dosage forms that are within the scope of reliable medical judgment and are suitable for use in contact with human and animal tissues, but not Many toxicity, irritation, allergic reactions or other problems or complications are commensurate with a reasonable benefit/risk ratio.
  • Examples of pharmaceutically acceptable salts include metal salts, ammonium salts, salts with organic bases, salts with inorganic acids, salts with organic acids, salts with basic or acidic amino acids, and the like.
  • pharmaceutical composition refers to a mixture of one or more of the compounds of the application or their salts and pharmaceutically acceptable excipients.
  • the purpose of the pharmaceutical composition is to facilitate the administration of the compound of the present application to the organism.
  • pharmaceutically acceptable excipients refers to those excipients that have no obvious stimulating effect on the organism and will not damage the biological activity and performance of the active compound.
  • Suitable auxiliary materials 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.
  • tautomer or "tautomeric form” refers to structural isomers of different energy that can interconvert via a low energy barrier.
  • proton tautomers also called proton transfer tautomers
  • proton migration such as keto-enol and imine-enamine isomerization.
  • a specific example of a proton tautomer is the imidazole moiety, where protons can migrate between two ring nitrogens.
  • Valence tautomers include interconversion through the recombination of some bonding electrons.
  • the present application also includes compounds of the present application that are the same as those described herein, but with one or more atoms replaced by an isotope-labeled atom having an atomic weight or mass number different from those generally found in nature.
  • isotopes that can be incorporated into the compounds of the present application include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine, such as 2 H, 3 H, 11 C, 13 C, 14 C, 13 N, 15 N, 15 O, 17 O, 18 O, 31 P, 32 P, 35 S, 18 F, 123 I, 125 I and 36 Cl, etc.
  • Certain isotope-labeled compounds of the application can be used in the analysis of compound and/or substrate tissue distribution. Tritiated (ie 3 H) and carbon-14 (ie 14 C) isotopes are especially preferred due to their ease of preparation and detectability.
  • Positron emission isotopes, such as 15 O, 13 N, 11 C, and 18 F can be used in positron emission tomography (PET) studies to determine substrate occupancy.
  • PET positron emission tomography
  • the isotope-labeled compounds of the present application can generally be prepared by the following procedures similar to those disclosed in the schemes and/or examples below, by replacing non-isotopically-labeled reagents with isotope-labeled reagents.
  • substitution with heavier isotopes can provide certain therapeutic advantages resulting from higher metabolic stability (for example, increased in vivo half-life or reduced dosage requirements), and therefore in certain situations
  • deuterium substitution can be partial or complete
  • partial deuterium substitution refers to the substitution of at least one hydrogen with at least one deuterium.
  • the compounds of the application may be asymmetric, for example, have one or more stereoisomers. Unless otherwise specified, all stereoisomers include, for example, enantiomers and diastereomers.
  • the compound containing asymmetric carbon atoms of the present application can be isolated in an optically pure form or a racemic form. The optically active pure form can be resolved from the racemic mixture or synthesized by using chiral starting materials or chiral reagents.
  • the pharmaceutical composition of the application can be prepared by combining the compound of the application with suitable pharmaceutically acceptable excipients, for example, can be formulated into solid, semi-solid, liquid or gaseous preparations, such as tablets, pills, capsules, powders , Granules, ointments, emulsions, suspensions, suppositories, injections, inhalants, gels, microspheres and aerosols.
  • suitable pharmaceutically acceptable excipients for example, can be formulated into solid, semi-solid, liquid or gaseous preparations, such as tablets, pills, capsules, powders , Granules, ointments, emulsions, suspensions, suppositories, injections, inhalants, gels, microspheres and aerosols.
  • Typical routes for administering the compound of the application or a pharmaceutically acceptable salt or pharmaceutical composition thereof include, but are not limited to, oral, rectal, topical, inhalation, parenteral, sublingual, intravaginal, intranasal, intraocular, intraperitoneal, Intramuscular, subcutaneous, and intravenous administration.
  • the pharmaceutical composition of the present application can be manufactured by methods well known in the art, such as conventional mixing method, dissolution method, granulation method, sugar-coated pill method, grinding method, emulsification method, freeze-drying method, etc.
  • the pharmaceutical composition is in oral form.
  • the pharmaceutical composition can be formulated by mixing the active compound with pharmaceutically acceptable excipients well known in the art. These auxiliary materials enable the compound of the present application to be formulated into tablets, pills, lozenges, sugar-coated agents, capsules, liquids, gels, slurries, suspensions, etc., for oral administration to patients.
  • the solid oral composition can be prepared by conventional mixing, filling or tabletting methods. For example, it can be obtained by the following method: mixing the active compound with solid excipients, optionally grinding the resulting mixture, adding other suitable excipients if necessary, and processing the mixture into granules to obtain tablets Or the core of the dragee.
  • suitable excipients include but are not limited to: binders, diluents, disintegrants, lubricants, glidants, sweeteners, or flavoring agents.
  • the pharmaceutical composition may also be suitable for parenteral administration, such as a sterile solution, suspension or lyophilized product in a suitable unit dosage form.
  • the daily dose is 0.01 to 200 mg/kg body weight, in the form of single or divided doses.
  • the compound of the present application can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, the embodiments formed by combining them with other chemical synthesis methods, and those well known to those skilled in the art Equivalent replacement manners, preferred implementation manners include but are not limited to the embodiments of the present application.
  • PE petroleum ether
  • EA ethyl acetate
  • DMSO dimethyl sulfoxide
  • DMF N,N-dimethylformamide
  • DCM dichloromethane
  • MeOH stands for methanol
  • EDTA ethylenediaminetetraacetic acid
  • DTT Represents dithiothreitol
  • EGTA represents ethylene glycol bis(2-aminoethyl ether) tetraacetic acid
  • ATP represents adenosine triphosphate
  • HATU 2-(7-oxybenzotriazole)-N,N, N',N'-tetramethylurea hexafluorophosphate
  • ACN stands for acetonitrile
  • Et 3 N stands for triethylamine
  • NIS stands for N-iodosuccinimide
  • Cbz- stands for benzyloxycarbonyl
  • Boc- stands for tertiary Butoxycarbonyl
  • Step 1 (S)-6-(((3-chloropyrazin-2-yl)methyl)carbamoyl)-5-azaspiro[2.4]heptane-5-carboxylic acid benzyl ester (1- 2)
  • Step 2 (S)-6-(8-chloroimidazo[1,5-a]pyrazin-3-yl)-5-azaspiro[2.4]heptane-5-carboxylic acid benzyl ester (1- 3)
  • Step 1 Add 3-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoic acid (24.54 g), 4-bromopyridin-2-amine (13.3g) and triethylamine (31.1g), dissolved in DCM (300mL), stirred at room temperature for 20 minutes, added HATU (35.1g), heated to 70 After reacting at °C for 6 hours, the reaction is complete. The reaction solution is added to water (500ml) for liquid separation. The organic phase is spin-dried under reduced pressure and re-dissolved in DMF (50ml). Add 1M HCl aqueous solution (500ml), stir well and filter.
  • Step 1 (S)-6-(1-iodo-8-chloroimidazo[1,5-a]pyrazin-3-yl)-5-azaspiro[2.4]heptane-5-carboxylic acid benzyl Esters (1-4)
  • Step 2 (S)-6-(8-Amino-1-iodoimidazo[1,5-a]pyrazin-3-yl)-5-azaspiro[2.4]heptane-5-carboxylic acid benzyl Ester (Intermediate 1-5)
  • Step 3 (S)-6-(8-amino-1-(4-((4-bromopyridin-2-yl)carbamoyl)-2-fluorophenyl)imidazole[1,5-a]pyridine (Azin-3-yl)-5-azaspirocyclo[2.4]heptane-5-carboxylic acid benzyl ester (Intermediate 1-6)
  • Step 4 (S)-4-(8-amino-3-(5-azaspiro[2.4]heptane-6-yl)imidazo[1,5-a]pyrazin-1-yl)-N -(4-Bromopyridin-2-yl)-3-fluorobenzamide (Intermediate 1-7)
  • Step 5 (S)-4-(8-amino-3-(5-but-2-ynyl)-5-azaspiro[2.4]heptane-6-yl)imidazo[1,5-a ]Pyrazin-1-yl)-N-(4-bromopyridin-2-yl)-3-fluorobenzamide (Compound I-1)
  • Step 1 (S)-6-(((3-chloropyrazin-2-yl)methyl)carbamoyl)-5-azaspiro[2.4]heptane-5-carboxylic acid tert-butyl ester (2 -2)
  • Step 2 (S)-6-(8-chloroimidazo[1,5-a]pyrazin-3-yl)-5-azaspiro[2.4]heptane-5--carboxylic acid tert-butyl ester ( 2-3)
  • Step 1 (S)-6-(1-iodo-8-chloroimidazo[1,5-a]pyrazin-3-yl)-5-azaspiro[2.4]heptane-5-carboxylic acid tert Butyl ester (2-4)
  • Step 2 (S)-6-(8-Amino-1-iodoimidazo[1,5-a]pyrazin-3-yl)-5-azaspiro[2.4]heptane-5-carboxylic acid tert Butyl ester (Intermediate 2-5)
  • Step 3 (S)-6-(8-amino-1-(4-((4-chloropyridin-2-yl)carbamoyl)-2-fluorophenyl)imidazole[1,5-a]pyridine (Azin-3-yl)-5-azaspiro[2.4]heptane-5-carboxylic acid tert-butyl ester (Intermediate 2-6)
  • Step 4 (S)-4-(8-amino-3-(5-but-2-ynyl)-5-azaspiro[2.4]heptane-6-yl)imidazo[1,5-a ]Pyrazin-1-yl)-N-(4-chloropyridin-2-yl)-3-fluorobenzamide (Compound I-2)
  • Step 1 Add 3-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoic acid (5g ), 4-cyanopyridin-2-amine (2.69g) and triethylamine (5.7g), and dissolved in DMF (20mL), add HATU (7.86g), heat to 60°C and react for 6 hours, the reaction is complete The reaction solution was added to saturated brine (200 mL), ethyl acetate (200 mL) was added for extraction, the organic phase was dried over anhydrous sodium sulfate, and spin-dried under reduced pressure to obtain Intermediate L (5.12 g).
  • Step 1 (S)-6-(8-amino-1-(4-((4-cyanopyridin-2-yl)carbamoyl)-2-fluorophenyl)imidazole [1,5-a] Pyrazin-3-yl)-5-azaspiro[2.4]heptane-5-carboxylic acid tert-butyl ester (Intermediate 3-6)
  • Step 2 (S)-4-(8-amino-3-(5-but-2-ynyl)-5-azaspiro[2.4]heptane-6-yl)imidazo[1,5-a ]Pyrazin-1-yl)-N-(4-cyanopyridin-2-yl)-3-fluorobenzamide (Compound I-3)
  • Step 1 Add 3-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoic acid (5g ), 4-difluoromethylpyridin-2-amine (2.84g) and triethylamine (5.7g), dissolved in DMF (20mL), HATU (7.86g) was added, heated to 60°C for 6 hours, After the reaction was completed, the reaction solution was added to saturated brine (200 mL), ethyl acetate (200 mL) was added for extraction, the organic phase was dried over anhydrous sodium sulfate, and spin-dried under reduced pressure to obtain Intermediate M (4.45 g).
  • Step 1 (S)-6-(8-amino-1-(4-((4-difluoromethylpyridin-2-yl)carbamoyl)-2-fluorophenyl)imidazole [1,5- a]Pyrazin-3-yl)-5-azaspiro[2.4]heptane-5-carboxylic acid tert-butyl ester (Intermediate 4-6)
  • Step 2 (S)-4-(8-amino-3-(5-but-2-ynyl)-5-azaspiro[2.4]heptane-6-yl)imidazo[1,5-a ]Pyrazin-1-yl)-N-(4-difluoromethylpyridin-2-yl)-3-fluorobenzamide (Compound I-4)
  • Step 1 Add 3-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoic acid (5.14 g), 4-methoxypyridine-2-amine (2g) and triethylamine (4.89g), dissolved in DMF (30mL), stirred at room temperature for 20 minutes, added HATU (7.35g), heated to The reaction was reacted at 60°C for 3 hours. The reaction was completed. The reaction solution was extracted with water (200ml) and ethyl acetate. The organic phase was washed with water and saturated aqueous sodium chloride solution, then dried over anhydrous sodium sulfate, filtered, and concentrated intermediate N (2.8g) ).
  • Step 1 (S)-6-(8-amino-1-(4-((4-methoxypyridin-2-yl)carbamoyl)-2-fluorophenyl)imidazole [1,5-a ]Pyrazin-3-yl)-5-azaspiro[2.4]heptane-5-carboxylic acid tert-butyl ester (Intermediate 6-6)
  • Step 2 (S)-4-(8-amino-3-(5-but-2-ynyl)-5-azaspiro[2.4]heptane-6-yl)imidazo[1,5-a ]Pyrazin-1-yl)-N-(4-methoxypyridin-2-yl)-3-fluorobenzamide (Compound I-5)
  • Test Example 1 In vitro activity
  • ⁇ 8nM detection reagent (final concentration 2nM, Ab), incubated for 1 hour at room temperature; PE Envision multifunctional microplate reader reads the plate (excitation 620nm, emission 665nm), using four-parameter fitting to calculate IC50.
  • ⁇ 8nM detection reagent final concentration of 2nM, Eu-anti-phospho-tyrosine antibody
  • PE Envision multi-functional microplate reader reads the plate (excitation 320nm, emission 665nm), using four-parameter fitting, Calculate IC50.
  • TEC stock solution Dilute the 50ng/ ⁇ L TEC stock solution with kinase buffer (50mM HEPES, 10mM MgCl 2 , 2mM DTT, 1mM EGTA, 0.01% Tween 20), and add 6 ⁇ L 1.67 ⁇ 0.01336g/ ⁇ L working solution (final The concentration is 0.008ng/ ⁇ L), the different compounds dissolved in DMSO are added to the wells with a nanoliter sampler to make the final compound concentration of 1000nM-0.24nM, a 4-fold gradient, a total of 7 concentrations, and a blank control empty ( Without enzyme) and negative control wells (containing enzyme, plus solvent DMSO).
  • kinase buffer 50mM HEPES, 10mM MgCl 2 , 2mM DTT, 1mM EGTA, 0.01% Tween 20
  • 6 ⁇ L 1.67 ⁇ 0.01336g/ ⁇ L working solution final The concentration is 0.008ng/ ⁇ L
  • ⁇ 8nM detection reagent final concentration of 2nM, Eu-anti-phospho-tyrosine antibody, incubate at room temperature for 1 hour; PE Envision multi-functional microplate reader reads the plate (excitation 320nm, emission 665nm), using four-parameter fitting, Calculate IC50.
  • ITK Interleukin-2-inducible T-cellkinase inhibitory activity screening
  • kinase buffer 50mM HEPES, 10mM MgCl 2, 2mM DTT, 1mM EGTA, 0.01% Tween 20
  • 50ng / ⁇ L of diluted mother liquor ITK is added per well of fluid 6 ⁇ L 1.67 ⁇ 0.0835g / ⁇ L (final The concentration is 0.05ng/ ⁇ L)
  • the different compounds dissolved in DMSO are added to the wells with a nanoliter sampler to make the final compound concentration of 1000nM-0.24nM, a 4-fold gradient, a total of 7 concentrations, and a blank control empty ( Without enzyme) and negative control wells (containing enzyme, plus solvent DMSO).
  • ⁇ 8nM detection reagent final concentration of 2nM, Eu-anti-phospho-tyrosine antibody, incubate at room temperature for 1 hour; PE Envision multi-functional microplate reader reads the plate (excitation 320nm, emission 665nm), using four-parameter fitting, Calculate IC50.
  • PV sodium pervanadate
  • the base is diluted to 6mM, ready to use.
  • TMD-8 cells in good exponential growth phase, collect the cells into a centrifuge tube, low-speed benchtop centrifuge, 1500 rpm, centrifuge for 3 minutes, discard the supernatant, and add 2mL seed plate medium (RPMI basic medium + 5% FBS+0.05mM 2-mercaptoethanol) to resuspend the cells.
  • 2mL seed plate medium RPMI basic medium + 5% FBS+0.05mM 2-mercaptoethanol
  • Use a cell counter to count take the required amount of cells to adjust the density to 5 ⁇ 10 4 cells/mL, use a row gun to inoculate the 96-well plate, 100 ⁇ L/well, and place the cells at 37°C and 5% CO 2 saturated humidity Cultivate in an incubator. After culturing for 24 hours, use a nanoliter sampler to add compound samples. Set 2 replicate wells for each concentration.
  • ICR mice weighing 18-22g, were adapted for 3 to 5 days, and then randomly divided into groups, 9 mice in each group, and the related compounds were intragastrically administered at a dose of 10 mg/kg, and the related compounds were injected intravenously at a dose of 1 mg/kg.
  • the test animals (ICR mice) were fasted for 12 hours before administration, and were given food 4 hours after administration. They were free to drink water before and after the experiment and during the experiment.
  • each mouse collected 3 to 4 time points, each time point 3 mice, collect whole blood In a centrifuge tube containing EDTA-K2 and sodium fluoride, transfer to 4°C within 30 minutes and centrifuge at 4000 rpm ⁇ 10 minutes to separate plasma. After collecting all plasma, store it at -20°C for testing.
  • ig gavage
  • iv intravenous injection
  • MRT mean residence time
  • Vz apparent volume of distribution
  • CLz clearance rate
  • Model group vehicle 6; compound I-1: 50mg/kg, bid, i.g 6.
  • the vehicle or drug was administered by intragastric administration at a volume of 10ml/kg, twice a day, for 23 consecutive days. Measure the tumor volume 2-3 times a week, weigh the mouse at the same time, and record the data; observe the animal performance daily. After all the administration was finished, the animals were sacrificed and the tumors were stripped and weighed.
  • Tumor volume (TV) (length ⁇ width 2) / 2.
  • Tumor growth inhibition (1-tumor weight in the treatment group / tumor weight in the model group) ⁇ 100%.

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Abstract

含有5-氮杂螺庚烷的BTK抑制剂，具体涉及式(I)化合物、其立体异构体或其药物上可接受的盐、其制备方法、含有该化合物的药物组合物，及其在治疗BTK相关疾病中的用途。

Description

含有5-氮杂螺庚烷的BTK抑制剂
相关申请的交叉引用
本申请要求于2019年07月12日向中国国家知识产权局提交的第201910629943.3号中国专利申请以及于2019年09月10日向中国国家知识产权局提交的第201910851109.9号中国专利申请的优先权和权益，所述申请公开的内容通过引用整体并入本文中。
技术领域
本申请涉及作为BTK抑制剂的含有5-氮杂螺庚烷的化合物、其制备方法、含有该化合物的药物组合物、以及其在治疗BTK相关疾病中的用途。
背景技术
布鲁顿酪氨酸激酶(Bruton's tyrosine kinase，BTK)主要在B细胞中表达，分布于淋巴系统、造血及血液系统，是非受体型酪氨酸激酶Tec家族中一员，该家族成员还包括TEC、ITK/TSK/EMT和BMX，在结构上具有高度同源性。BTK在连接细胞表面B细胞受体(B-cell receptor)刺激至下游细胞内应答的B细胞信号传导途径中起至关重要的作用，是B细胞发育、激活、信号传导和存活的关键调节物。近年来有关B细胞特别是针对B细胞非霍奇金性淋巴癌和类风湿关节炎的研究发现，BTK往往会出现异常表达。
基于BTK信号传导通路开发小分子靶向药物，为B细胞类肿瘤如白血病、多发性骨髓瘤及B细胞类免疫疾病的治疗提供一条全新的途径。目前上市的不可逆抑制剂，比如依鲁替尼，其BTK结合位点往往会发生突变，导致药物活性下降从而产生耐药性，因此临床上需要更多的BTK抑制剂，且对BTK具有较高的选择性，从而避免脱靶效应所带来的毒副作用。
发明概述
一方面，本申请提供式(I)化合物、其立体异构体或其药学上可接受的盐，
Figure PCTCN2020101200-appb-000001
其中，
环A选自5-10元杂芳基或C 6-10芳基；
R 1独立地选自卤素、羟基、氨基、氰基、C 1-6烷氧基或C 1-6烷基，所述C 1-6烷氧基或C 1-6烷基任选地被卤素取代；
m选自0、1、2、3、4、5或6；
R 2选自氢、R aS(O) 2-、(R aO) 2P(O)-或R aC(O)-；
其中R a独立地选自C 2-6炔基、C 2-6烯基、C 1-6烷基、C 3-6环烷基、(C 1-6烷基)NH-、(C 1-6烷基) 2N-、3-6元杂环烷基、5-10元杂芳基或C 6-10芳基，上述R a任选地被(C 1-6烷基) 2N-、(C 1-6烷基)NH-、羟基、氨基、卤素或氰基取代。
另一方面，本申请提供药物组合物，其包含本申请所述的式(I)化合物、其立体异构体或其药学上可接受的盐。
另一方面，本申请提供预防或治疗哺乳动物与BTK相关疾病的方法，包括对需要该预防或治疗的哺乳动物给予治疗有效量的本申请所述的式(I)化合物、其立体异构体或其药学上可接受的盐、或本申请所述的药物组合物。
另一方面，本申请提供本申请所述的式(I)化合物、其立体异构体或其药学上可接受的盐、或本申请所述的药物组合物在制备用于预防或治疗与BTK相关疾病的药物中的用途。
另一方面，本申请提供本申请所述的式(I)化合物、其立体异构体或其药学上可接受的盐、或本申请所述的药物组合物在预防或治疗与BTK相关疾病中的用途。
另一方面，本申请提供用于预防或治疗与BTK相关疾病的本申请所述的式(I)化合物、其立体异构体或其药学上可接受的盐、或本申请所述的药物组合物。
发明详述
本申请涉及式(I)化合物、其立体异构体或其药学上可接受的盐，
Figure PCTCN2020101200-appb-000002
其中，
环A选自5-10元杂芳基或C 6-10芳基；
R 1独立地选自卤素、羟基、氨基、氰基、C 1-6烷氧基或C 1-6烷基，所述C 1-6烷氧基或C 1-6烷基任选地被卤素取代；
m选自0、1、2、3、4、5或6；
R 2选自氢、R aS(O) 2-、(R aO) 2P(O)-或R aC(O)-；
其中R a独立地选自C 2-6炔基、C 2-6烯基、C 1-6烷基、C 3-6环烷基、(C 1-6烷基)NH-、(C 1-6烷基) 2N-、3-6元杂环烷基、5-10元杂芳基或C 6-10芳基，上述R a任选地被(C 1-6烷基) 2N-、(C 1-6烷基)NH-、羟基、氨基、卤素或氰基取代。
在一些实施方案中，环A选自5-6元杂芳基；在一些实施方案中，环A选自6元杂芳基；在一些实施方案中，环A选自6元含氮杂芳基；在一些实施方案中，环A选自吡啶基。在一些实施方案中，环A为吡啶-2-基。在一些实施方案中，环A为苯基。
在一些实施方案中，R 1独立地选自卤素、氰基、C 1-3烷基或C 1-3烷氧基，所述C 1-3烷基或C 1-3烷氧基任选地被卤素(例如，氟)取代。在一些实施方案中，R 1独立地选自卤素、氰基、C 1-3烷氧基、或任选地被卤素取代的C 1-3烷基；在一些实施方案中，R 1独立地选自氟、氯、溴、碘、氰基、甲氧基或任选被卤素取代的甲基；在一些实施方案中，R 1独立地选自氯、溴、氰基、-CHF 2或甲氧基；在一些实施方案中，R 1独立地选自溴。
在一些实施方案中，m选自0、1、2、3或4；在一些实施方案中，m选自0、1或2；在一些实施方案中，m选自1。
在一些实施方案中，R 2选自R aC(O)-。
在一些实施方案中，R a选自C 2-6炔基、C 2-6烯基、C 1-6烷基、C 3-6环烷基或3-6元杂环烷基；在一些实施方案中，R a选自C 2-3炔基或C 3-4环烷基；在一些实施方案中，R a选自丙炔基。
在一些实施方案中，R 2选自R aC(O)-，其中R a选自C 2-6炔基、C 2-6烯基、C 1-6烷基、C 3-6环烷基或3-6元杂环烷基；或者R a选自C 2-3炔基或C 3-4环烷基；或者R a为丙炔基。
在一些实施方案中，R 2选自
Figure PCTCN2020101200-appb-000003
在一些实施方案中，本申请的式(I)化合物、其立体异构体或其药学上可接受的盐选自式(I-1)化合物、其立体异构体或其药学上可接受的盐，
Figure PCTCN2020101200-appb-000004
其中，环A、R 1、R 2、m如上定义。
在一些实施方案中，本申请的式(I)化合物、其立体异构体或其药学上可接受的盐选自式(II)或式(II-1)或式(II-2)化合物、或其立体异构体或其药学上可接受的盐，
Figure PCTCN2020101200-appb-000005
其中，R 1、m如上定义。
在一些实施方案中，本申请的式(I)化合物、其立体异构体或其药学上可接受的盐选自以下化合物或其药学上可接受的盐：
Figure PCTCN2020101200-appb-000006
Figure PCTCN2020101200-appb-000007
另一方面，本申请涉及药物组合物，其包含本申请的式(I)化合物、其立体异构体或其药学上可接受的盐。在一些实施方案中，本申请的药物组合物还包括药学上可接受的辅料。
另一方面，本申请涉及预防或治疗哺乳动物与BTK相关疾病的方法，包括对需要该预防或治疗的哺乳动物，优选人类，给予治疗有效量的式(I)化合物、其立体异构体或其药学上可接受的盐、或其药物组合物。
另一方面，本申请涉及式(I)化合物、其立体异构体或其药学上可接受的盐、或其药物组合物在制备预防或治疗与BTK相关疾病的药物中的用途。
另一方面，本申请涉及式(I)化合物、其立体异构体或其药学上可接受的盐、或其药物组合物在预防或治疗与BTK相关疾病中的用途。
另一方面，本申请涉及预防或治疗与BTK相关疾病的式(I)化合物、其立体异构体或其药学上可接受的盐、或其药物组合物。
在一些具体实施方案中，上述BTK相关疾病为BTK介导的疾病。
在一些具体实施方案中，上述BTK相关疾病选自自身免疫性疾病、炎症疾病或癌症。
在一些具体实施方案中，上述BTK相关疾病选自B淋巴细胞瘤。在一些具体实施方案中，上述BTK相关疾病选自弥漫性大B细胞淋巴瘤。
定义
除非另有说明，本申请中所用的下列术语具有下列含义。一个特定的术语在没有特别定义的情况下不应该被认为是不确定的或不清楚的，而应该按照本领域普通的含义去理解。当本文中出现商品名时，意在指代其对应的商品或其活性成分。
术语“被取代”是指特定基团上的任意一个或多个氢原子被取代基取代，只要特定基团的价态是正常的并且取代后的化合物是稳定的。当取代基为氧代(即＝O)时，意味着两个氢原子被取代，氧代不会发生在芳香基上。
术语“任选”或“任选地”是指随后描述的事件或情况可以发生或不发生，该描述包括发生所述事件或情 况和不发生所述事件或情况。例如，乙基“任选”被卤素取代，指乙基可以是未被取代的(CH 2CH 3)、单取代的(如CH 2CH 2F)、多取代的(如CHFCH 2F、CH 2CHF 2等)或完全被取代的(CF 2CF 3)。本领域技术人员可理解，对于包含一个或多个取代基的任何基团，不会引入任何在空间上不可能存在和/或不能合成的取代或取代模式。
本文中的C m-n，是该部分具有给定范围中的整数个碳原子。例如“C 1-6”是指该基团可具有1个碳原子、2个碳原子、3个碳原子、4个碳原子、5个碳原子或6个碳原子。
当任何变量(例如R)在化合物的组成或结构中出现一次以上时，其在每一种情况下的定义都是独立的。例如，如果一个基团含2个R，则每个R都有独立的选项。
术语“卤”或“卤素”是指氟、氯、溴和碘。
术语“羟基”指-OH基团。
术语“氨基”指-NH 2基团。
术语“氰基”指-CN基团。
术语“烷基”是指通式为C nH 2n+1的烃基，例如，C 1- 6烷基、C 1- 3烷基。该烷基可以是直链或支链的。例如，术语“C 1- 6烷基”指含有1至6个碳原子的烷基(例如甲基、乙基、正丙基、异丙基、正丁基、异丁基、仲丁基、叔丁基、正戊基、1-甲基丁基、2-甲基丁基、3-甲基丁基、新戊基、己基、2-甲基戊基等)。类似地，烷氧基、烷基氨基、二烷基氨基、烷基磺酰基和烷硫基的烷基部分(即烷基)具有上述相同定义。
术语“烷氧基”指-O-烷基，例如，-O-C 1- 6烷基、-O-C 1- 3烷基。
术语“烯基”是指由碳原子和氢原子组成的直链或支链的具有至少一个双键的不饱和脂肪族烃基，例如，C 2- 6烯基、C 2- 3烯基。烯基的非限制性实例包括但不限于乙烯基、1-丙烯基、2-丙烯基、1-丁烯基、异丁烯基、1,3-丁二烯基等。
术语“炔基”是指由碳原子和氢原子组成的直链或支链的具有至少一个三键的不饱和脂肪族烃基，例如，C 2- 6炔基、C 2- 3炔基。炔基的非限制性实例包括但不限于乙炔基(-C≡CH)、1-丙炔基(-C≡C-CH 3)、2-丙炔基(-CH 2-C≡CH)、1,3-丁二炔基(-C≡C-C≡CH)等。
术语“环烷基”指完全饱和的并且可以以呈单环、桥环或螺环存在的碳环。除非另有指示，该碳环通常为3至10元环，或3至6元环。环烷基非限制性实例包括但不限于环丙基、环丁基、环戊基、环己基、降冰片基(双环[2.2.1]庚基)、双环[2.2.2]辛基、金刚烷基等。
术语“杂环烷基”是指完全饱和的并且可以以单环、桥环或螺环存在的环状基团。除非另有指示，该杂环通常为含有1至3个独立地选自硫、氧和/或氮的杂原子(优选1或2个杂原子)的3至7元环。杂环烷基可以为含有1或2个独立地选自氧和/或氮的杂原子的3至6元环。3元杂环烷基的实例包括但不限于环氧乙烷基、环硫乙烷基、环氮乙烷基，4元杂环烷基的非限制性实例包括但不限于吖丁啶基、噁丁环基、噻丁环基，5元杂环烷基的实例包括但不限于四氢呋喃基、四氢噻吩基、吡咯烷基、异噁唑烷基、噁唑烷 基、异噻唑烷基、噻唑烷基、咪唑烷基、四氢吡唑基，6元杂环烷基的实例包括但不限于哌啶基、四氢吡喃基、四氢噻喃基、吗啉基、哌嗪基、1,4-噻噁烷基、1,4-二氧六环基、硫代吗啉基、1,3-二噻烷基、1,4-二噻烷基，7元杂环烷基的实例包括但不限于氮杂环庚烷基、氧杂环庚烷基、硫杂环庚烷基。优选为具有5或6个环原子的单环杂环烷基。
术语“芳基”是指具有共轭的π电子体系的全碳单环或稠合多环的芳香环基团。例如，芳基可以具有6-20个碳原子，6-14个碳原子或6-12个碳原子，或者6-10个碳原子。芳基的非限制性实例包括但不限于苯基、萘基、蒽基和1,2,3,4-四氢化萘等。
术语“杂芳基”是指单环或稠合多环体系，其中含有至少一个(例如，1、2或3个)选自N、O和/或S的环原子，其余环原子为C，并且具有至少一个芳香环。优选的杂芳基具有单个4至8元环，或者5至8元环，或者6元环(例如，含有1或2个N和/或O的6元杂芳基)，或包含6至14个，尤其是6至10个环原子的多个稠合多环。杂芳基的非限制性实例包括但不限于吡咯基、呋喃基、噻吩基、咪唑基、噁唑基、吡唑基、吡啶基、嘧啶基、吡嗪基、喹啉基、异喹啉基、四唑基、三唑基、三嗪基、苯并呋喃基、苯并噻吩基、吲哚基、异吲哚基等。
术语“治疗”意为将本申请所述化合物或制剂进行给药以改善或消除疾病或与所述疾病相关的一个或多个症状，且包括：
(i)抑制疾病或疾病状态，即遏制其发展；
(ii)缓解疾病或疾病状态，即使该疾病或疾病状态消退。
术语“预防”意为将本申请所述化合物或制剂进行给药以预防疾病或与所述疾病相关的一个或多个症状，包括：预防疾病或疾病状态在哺乳动物中出现，特别是当这类哺乳动物易患有该疾病状态，但尚未被诊断为已患有该疾病状态时。
术语“治疗有效量”意指(i)治疗或预防特定疾病、病况或障碍，(ii)减轻、改善或消除特定疾病、病况或障碍的一种或多种症状，或(iii)预防或延迟本文中所述的特定疾病、病况或障碍的一种或多种症状发作的本申请化合物的用量。构成“治疗有效量”的本申请化合物的量取决于该化合物、疾病状态及其严重性、给药方式以及待被治疗的哺乳动物的年龄而改变，但可例行性地由本领域技术人员根据其自身的知识及本公开内容而确定。
术语“药学上可接受的”，是针对那些化合物、材料、组合物和/或剂型而言，它们在可靠的医学判断的范围之内，适用于与人类和动物的组织接触使用，而没有过多的毒性、刺激性、过敏性反应或其它问题或并发症，与合理的利益/风险比相称。
作为药学上可接受的盐，例如，可以包括金属盐、铵盐、与有机碱形成的盐、与无机酸形成的盐、与有机酸形成的盐、与碱性或者酸性氨基酸形成的盐等。
术语“药物组合物”是指一种或多种本申请的化合物或其盐与药学上可接受的辅料组成的混合物。药物 组合物的目的是有利于对有机体给予本申请的化合物。
术语“药学上可接受的辅料”是指对有机体无明显刺激作用，而且不会损害该活性化合物的生物活性及性能的那些辅料。合适的辅料是本领域技术人员熟知的，例如碳水化合物、蜡、水溶性和/或水可膨胀的聚合物、亲水性或疏水性材料、明胶、油、溶剂、水等。
词语“包括(comprise)”或“包含(comprise)”及其英文变体例如comprises或comprising应理解为开放的、非排他性的意义，即“包括但不限于”。
本申请的化合物和中间体还可以以不同的互变异构体形式存在，并且所有这样的形式包含于本申请的范围内。术语“互变异构体”或“互变异构体形式”是指可经由低能垒互变的不同能量的结构异构体。例如，质子互变异构体(也称为质子转移互变异构体)包括经由质子迁移的互变，如酮-烯醇及亚胺-烯胺异构化。质子互变异构体的具体实例是咪唑部分，其中质子可在两个环氮间迁移。价互变异构体包括通过一些成键电子的重组的互变。
本申请还包括与本文中记载的那些相同的，但一个或多个原子被原子量或质量数不同于自然中通常发现的原子量或质量数的原子置换的同位素标记的本申请化合物。可结合到本申请化合物的同位素的实例包括氢、碳、氮、氧、磷、硫、氟、碘和氯的同位素，诸如分别为 2H、 3H、 11C、 13C、 14C、 13N、 15N、 15O、 17O、 18O、 31P、 32P、 35S、 18F、 123I、 125I和 36Cl等。
某些同位素标记的本申请化合物(例如用 3H及 14C标记的那些)可用于化合物和/或底物组织分布分析中。氚化(即 3H)和碳-14(即 14C)同位素对于由于它们易于制备和可检测性是尤其优选的。正电子发射同位素，诸如 15O、 13N、 11C和 18F可用于正电子发射断层扫描(PET)研究以测定底物占有率。通常可以通过与公开于下文的方案和/或实施例中的那些类似的下列程序，通过同位素标记试剂取代未经同位素标记的试剂来制备同位素标记的本申请化合物。
此外，用较重同位素(诸如氘(即 2H))取代可以提供某些由更高的代谢稳定性产生的治疗优点(例如增加的体内半衰期或降低的剂量需求)，并且因此在某些情形下可能是优选的，其中氘取代可以是部分或完全的，部分氘取代是指至少一个氢被至少一个氘取代。
本申请化合物可以是不对称的，例如，具有一个或多个立体异构体。除非另有说明，所有立体异构体都包括，如对映异构体和非对映异构体。本申请的含有不对称碳原子的化合物可以以光学活性纯的形式或外消旋形式被分离出来。光学活性纯的形式可以从外消旋混合物拆分，或通过使用手性原料或手性试剂合成。
本申请的药物组合物可通过将本申请的化合物与适宜的药学上可接受的辅料组合而制备，例如可配制成固态、半固态、液态或气态制剂，如片剂、丸剂、胶囊剂、粉剂、颗粒剂、膏剂、乳剂、悬浮剂、栓剂、注射剂、吸入剂、凝胶剂、微球及气溶胶等。
给予本申请化合物或其药学上可接受的盐或其药物组合物的典型途径包括但不限于口服、直肠、局部、 吸入、肠胃外、舌下、阴道内、鼻内、眼内、腹膜内、肌内、皮下、静脉内给药。
本申请的药物组合物可以采用本领域众所周知的方法制造，如常规的混合法、溶解法、制粒法、制糖衣药丸法、磨细法、乳化法、冷冻干燥法等。
在一些实施方案中，药物组合物是口服形式。对于口服给药，可以通过将活性化合物与本领域熟知的药学上可接受的辅料混合，来配制该药物组合物。这些辅料能使本申请的化合物被配制成片剂、丸剂、锭剂、糖衣剂、胶囊剂、液体、凝胶剂、浆剂、悬浮剂等，用于对患者的口服给药。
可以通过常规的混合、填充或压片方法来制备固体口服组合物。例如，可通过下述方法获得：将所述的活性化合物与固体辅料混合，任选地碾磨所得的混合物，如果需要则加入其它合适的辅料，然后将该混合物加工成颗粒，得到了片剂或糖衣剂的核心。适合的辅料包括但不限于：粘合剂、稀释剂、崩解剂、润滑剂、助流剂、甜味剂或矫味剂等。
药物组合物还可适用于肠胃外给药，如合适的单位剂型的无菌溶液剂、混悬剂或冻干产品。
本文所述的通式Ⅰ化合物的所有施用方法中，每天给药的剂量为0.01到200mg/kg体重，以单独或分开剂量的形式。
本申请的化合物可以通过本领域技术人员所熟知的多种合成方法来制备，包括下面列举的具体实施方式、其与其他化学合成方法的结合所形成的实施方式以及本领域技术上人员所熟知的等同替换方式，优选的实施方式包括但不限于本申请的实施例。
本申请具体实施方式的化学反应是在合适的溶剂中完成的，所述的溶剂须适合于本申请的化学变化及其所需的试剂和物料。为了获得本申请的化合物，有时需要本领域技术人员在已有实施方式的基础上对合成步骤或者反应流程进行修改或选择。
本领域合成路线规划中的一个重要考量因素是为反应性官能团(如本申请中的氨基)选择合适的保护基，例如，可参考Greene's Protective Groups in Organic Synthesis(4th Ed).Hoboken,New Jersey:John Wiley&Sons,Inc.。
本申请采用下述缩略词：
PE代表石油醚；EA代表乙酸乙酯；DMSO代表二甲基亚砜；DMF代表N，N-二甲基甲酰胺；DCM代表二氯甲烷；MeOH代表甲醇；EDTA代表乙二胺四乙酸；DTT代表二硫苏糖醇；EGTA代表乙二醇双(2-氨基乙基醚)四乙酸；ATP代表腺苷三磷酸；HATU代表2-(7-氧化苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯；ACN代表乙腈；Et 3N代表三乙胺；NIS代表N-碘代丁二酰亚胺；Cbz-代表苄氧羰基；Boc-代表叔丁氧羰基；HEPES代表4-羟乙基哌嗪乙磺酸；BTK代表布鲁顿酪氨酸激酶；EGFR代表epidermal growth factor receptor，表皮生长因子受体；TEC代表tyrosine kinase expressed in hepatocellular carcinoma，酪氨酸激酶在肝细胞癌中的表达；ULight-poly GT代表LANCE Ultra ULight-Poly GT；WT代表野生型。
为清楚起见，进一步用实施例来阐述本发明，但是实施例并非限制本申请的范围。本申请所使用的所 有试剂是市售的，无需进一步纯化即可使用。
实施例1中间体1-3的制备
Figure PCTCN2020101200-appb-000008
步骤1：(S)-6-(((3-氯吡嗪-2-基)甲基)氨基甲酰)-5-氮杂螺[2.4]庚烷-5-羧酸苄酯(1-2)
在反应瓶中加入(S)-5-((苄氧基)羰基)-5-氮杂螺[2.4]庚烷-6-羧酸(8.41g)并溶解于DCM(150ml)中，再加入中间体1(5g)和Et 3N(13.63mL)，在冰水浴下加入HATU(11.15g)，加完升至室温继续搅拌，反应完全，反应液依次用2M浓盐酸、饱和NaHCO 3水溶液、饱和NaCl水溶液洗，有机层用无水MgSO 4搅拌干燥，过滤，滤液浓缩经硅胶柱纯化(展开剂：PE：EA＝80:20～50:50)，得中间体1-2(8.24g)。
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.
步骤2：(S)-6-(8-氯咪唑并[1,5-a]吡嗪-3-基)-5-氮杂螺[2.4]庚烷-5-羧酸苄酯(1-3)
在反应瓶中加入中间体1-2(7.3g)、ACN(75ml)及DMF(1.5mL)，冰水浴下缓慢滴加POCl 3(6.69ml)，滴加完毕，逐渐升温到室温，继续搅拌1.5小时，反应完成。将反应液缓慢滴加到浓氨水与碎冰混合体系中，滴加完毕，继续搅拌至碎冰融化，碱化反应液至约pH＝9，用EA萃取2次，合并有机层，用饱和食盐水洗2次，无水MgSO 4干燥，过滤，浓缩干得中间体1-3(7.56g)。
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.
实施例2：N-(4-溴吡啶-2-基)-3-氟-4-(4,4,5,5-四甲基-1,3,2-二氧杂硼杂戊环-2-基)苯甲酰胺(中间体J)的制备
Figure PCTCN2020101200-appb-000009
步骤1：向反应瓶中加入3-氟-4-(4,4,5,5-四甲基-1,3,2-二氧杂硼杂环戊环-2-基)苯甲酸(24.54g)，4-溴吡啶-2-胺(13.3g)和三乙胺(31.1g)，并溶于DCM(300mL)中，室温下搅拌20分钟后，加入HATU(35.1g)，加热至70℃反应6小时，反应完成，将反应液加入水(500ml)中分液，有机相减压旋干后重新溶解于DMF(50ml)，加入1M HCl水溶液(500ml)中，搅拌均匀后过滤，所得滤饼用少许EA打浆后烘干，得中间体J(17.277g)。 1H NMR(500MHz,DMSO-d6)δ11.22(d,J＝36.6Hz,1H),8.44(dd,J＝7.1,1.5Hz,1H),8.32(d,J＝5.3Hz,1H),7.83(ddd,J＝16.5,7.7,1.2Hz,1H),7.78(d,J＝5.4Hz,1H),7.76–7.69(m,1H),7.50–7.43(m,1H),1.31(d,J＝4.9Hz,12H).MS:ESI[M+H-(2,3-二甲基丁烷)] +m/z：339.3(硼酸形式).
实施例3：(S)-4-(8-氨基-3-(5-丁-2-炔酰基)-5-氮螺环[2.4]庚烷-6-基)咪唑[1,5-a]吡嗪-1-基)-N-(4-溴吡啶-2-基)-3-氟苯甲酰胺(化合物I-1)的制备
Figure PCTCN2020101200-appb-000010
步骤1：(S)-6-(1-碘-8-氯咪唑并[1,5-a]吡嗪-3-基)-5-氮杂螺[2.4]庚烷-5-羧酸苄酯(1-4)
在反应瓶中加入中间体1-3(50.9g)和DMF(400ml)，室温搅拌溶清后冰水浴加入NIS(42.6g)，加毕，在60℃下反应过夜小时，反应完全，加饱和硫代硫酸钠的冰水(800ml)，EA萃取2次，有机层合并，饱和食盐水洗2次，无水NaSO 4干燥，后过滤浓缩得中间体1-4(62.81g)。MS(ESI,[M+H]+)m/z：509.3.
步骤2：(S)-6-(8-氨基-1-碘咪唑并[1,5-a]吡嗪-3-基)-5-氮杂螺[2.4]庚烷-5-羧酸苄酯(中间体1-5)
将中间体1-4(62.8g)溶于异丙醇(180ml)中，转移至耐压密封管中，加入氨的异丙醇溶液(1400ml,2.0mmol/mL)，密闭反应装置，加热120℃，搅拌反应过夜，反应完全，反应液冷却至室温。反应液浓缩，浓缩残余物柱层析纯化(展开剂：PE：EA＝100:0～30:70)得中间体1-5(41.0g)。 1H NMR(500MHz,DMSO)δ7.79-7.66(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/z：490.3.
步骤3：(S)-6-(8-氨基-1-(4-((4-溴吡啶-2-基)氨甲酰)-2-氟苯基)咪唑[1,5-a]吡嗪-3-基)-5-氮杂螺环[2.4]庚烷-5-羧酸苄酯(中间体1-6)
向反应瓶中加入中间体1-5(16.5g)，中间体J(14.63g)与碳酸钾(18.64g)，溶于二氧六环(300mL)中，加入[1,1’-双(二苯基膦基)二茂铁]二氯化钯(II)二氯甲烷络合物(4.68g)，加入水(50mL)，氮气置换3-4次后，80℃反应60分钟。反应结束，将反应液溶于300mL水中，DCM萃取，有机相无水硫酸钠干燥，过滤，浓缩，硅胶柱层析纯化(展开剂：DCM：MeOH＝97.5:2.5)，得中间体1-6(12.56g)。 1H NMR(500MHz,DMSO)δ11.23(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).MS(ESI,[M+H] +)m/z：656.4.
步骤4：(S)-4-(8-氨基-3-(5-氮螺环[2.4]庚烷-6-基)咪唑并[1,5-a]吡嗪-1-基)-N-(4-溴吡啶-2-基)-3-氟苯甲酰胺(中间体1-7)
在耐压管中加入中间体1-6(12g)与33％HBr的醋酸溶液(150ml)，室温反应2小时。反应结束，将反应液减压浓缩除去大部分溶液，浓缩物用水(600ml)重溶后DCM洗涤(200ml*3)，水相冰浴下调碱，DCM：MeOH＝10:1混合溶剂萃取(2.5L)，有机相无水硫酸钠干燥，减压旋干，得中间体1-7(7.85g)。 1H NMR(500MHz,DMSO)δ11.22(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] +)m/z：522.3.
步骤5：(S)-4-(8-氨基-3-(5-丁-2-炔酰基)-5-氮螺环[2.4]庚烷-6-基)咪唑并[1,5-a]吡嗪-1-基)-N-(4-溴吡啶-2-基)-3-氟苯甲酰胺(化合物I-1)
向反应瓶中加入中间体1-7(7.85g)，2-丁炔酸(1.12g)和三乙胺(6.01g)，并溶于DCM(500mL)中，室温下搅拌5分钟后，加入HATU(5.93g)，室温反应1小时，反应完成，将反应液加入水(400ml)中分液，DCM萃取(100ml*3)，合并有机相，饱和食盐水洗涤后无水硫酸钠干燥，过滤，浓缩，硅胶柱层析纯化(展开剂：DCM：MeOH＝97:3)，得化合物I-1(7.72g)。 1H NMR(500MHz,DMSO)δ11.23(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/z：588.3.
实施例4：中间体2-3的制备
Figure PCTCN2020101200-appb-000011
步骤1：(S)-6-(((3-氯吡嗪-2-基)甲基)氨基甲酰)-5-氮杂螺[2.4]庚烷-5-羧酸叔丁酯(2-2)
在反应瓶中加入(S)-5-(叔丁氧羰基)-5-氮杂螺[2.4]庚烷-6-羧酸(200g)并溶解于DCM(2500ml)中，再加入中间体1(149g)和Et 3N(347mL)，在冰水浴下加入HATU(347g)，加完升至室温继续搅拌，反应完全，反应液依次用0.5M浓盐酸、饱和NaHCO 3水溶液、饱和NaCl水溶液洗涤，有机层用无水MgSO 4搅拌干燥，过滤，浓缩，得中间体2-2(300g)。
1H NMR(500MHz,DMSO-d6)δ8.61(d,J＝2.5Hz,1H),8.48–8.30(m,2H),4.61–4.57(m,1H),4.49–4.43(m,1H),4.31–4.24(m,1H),3.35–3.15(m,2H),2.23–2.14(m,1H),1.79–1.71(m,1H),1.36(s,3H),1.32(s,6H),0.65–0.38(m,4H).MS(ESI,[M+Na] +)m/z：389.4。
步骤2：(S)-6-(8-氯咪唑并[1,5-a]吡嗪-3-基)-5-氮杂螺[2.4]庚烷-5--羧酸叔丁酯(2-3)
在反应瓶中加入中间体2-2(300g)、EA(2500ml)及DMF(190mL)，冰水浴下缓慢滴加POCl 3(267ml)，滴加完毕，逐渐升温到室温搅拌过夜，反应完成。将反应液缓慢滴加到浓氨水与碎冰混合体系中，滴加完毕，继续搅拌至碎冰融化，碱化反应液至约pH＝9，用EA萃取2次，合并有机层，用饱和食盐水洗2次，无水MgSO 4干燥，过滤，浓缩干得中间体2-3(338g)。
1H NMR(500MHz,DMSO-d6)δ8.46(d,J＝5.0Hz,1H),8.00–7.83(m,1H),7.43(d,J＝5.0Hz,1H),5.49–5.43(m,1H),3.63–3.28(m,2H),2.34–2.04(m,2H),1.32(s,3H),0.91(s,6H),0.77–0.40(m,4H).MS(ESI,[M+Na] +)m/z：371.4。
实施例5：N-(4-氯吡啶-2-基)-3-氟-4-(4,4,5,5-四甲基-1,3,2-二氧杂硼杂戊环-2-基)苯甲酰胺(中间体K)的制备
Figure PCTCN2020101200-appb-000012
步骤1：向反应瓶中加入3-氟-4-(4,4,5,5-四甲基-1,3,2-二氧杂硼杂环戊环-2-基)苯甲酸(5g)，4-氯吡啶-2-胺(2.5g)和三乙胺(5.7g)，并溶于DMF(20mL)中，加入HATU(7.86g)，加热至60℃反应6小时，反应完成，将反应液加入饱和食盐水(200mL)，加入乙酸乙酯(200mL)萃取，有机相用无水硫酸钠干燥，减压旋干后得中间体K(5.3g)， 1H NMR(500MHz,DMSO-d6)δ11.18(d,J＝34.0Hz,1H),8.39(d,J＝5.3Hz,1H),8.29(dt,J＝7.2,1.3Hz,1H),7.89–7.83(m,2H),7.81–7.73(m,2H),1.32(s,12H).MS:ESI[M+H-(2,3- 二甲基丁烷)] +m/z：295.1(硼酸形式)。
实施例6：(S)-4-(8-氨基-3-(5-丁-2-炔酰基)-5-氮螺环[2.4]庚烷-6-基)咪唑[1,5-a]吡嗪-1-基)-N-(4-氯吡啶-2-基)-3-氟苯甲酰胺(化合物I-2)的制备
Figure PCTCN2020101200-appb-000013
步骤1：(S)-6-(1-碘-8-氯咪唑并[1,5-a]吡嗪-3-基)-5-氮杂螺[2.4]庚烷-5-羧酸叔丁酯(2-4)
在反应瓶中加入中间体2-3(60g)和DMF(400ml)，室温搅拌溶清后冰水浴加入NIS(58g)，加毕，在60℃下反应过夜，反应完全，加饱和硫代硫酸钠的冰水(1500ml)，EA萃取2次，有机层合并，饱和食盐水洗2次，无水NaSO 4干燥，后过滤浓缩得中间体2-4(79.86g)。 1H NMR(500MHz,DMSO-d6)δ8.53(d,J＝5.1Hz,1H),7.42(d,J＝5.0Hz,1H),5.57–5.33(m,1H),3.72–3.45(m,1H),3.32–3.16(m,1H),2.32–2.05(m,2H),1.33(s,4H),0.93(s,5H),0.84–0.31(m,4H).MS(ESI,[M+H]+)m/z：475.4.
步骤2：(S)-6-(8-氨基-1-碘咪唑并[1,5-a]吡嗪-3-基)-5-氮杂螺[2.4]庚烷-5-羧酸叔丁酯(中间体2-5)
将中间体2-4(80g)转移至耐压密封管中，加入仲丁醇(100mL)，氨水(200mL)密闭反应装置，加热120℃，搅拌反应16h，反应完全，反应液冷却至室温。加入乙酸乙酯萃取，有机相浓缩后得到中间体2-5(75.5g)。MS(ESI,[M+H] +)m/z：456.4。
步骤3：(S)-6-(8-氨基-1-(4-((4-氯吡啶-2-基)氨甲酰)-2-氟苯基)咪唑[1,5-a]吡嗪-3-基)-5-氮杂螺环[2.4]庚烷-5-羧酸叔丁酯(中间体2-6)
向反应瓶中加入中间体2-5(250mg)，中间体K(372mg)与碳酸钾(219mg)，溶于二氧六环(5mL)中，加入[1,1’-双(二苯基膦基)二茂铁]二氯化钯(II)二氯甲烷络合物(43mg)，加入水(1mL)，氮气置换3-4次后，80℃反应120分钟。反应结束，将反应液溶于200mL水中，DCM萃取，有机相无水硫酸钠干燥，过滤，浓缩，硅胶柱层析纯化(展开剂：DCM：MeOH＝97:3)，得中间体2-6(206mg)。
1H NMR(500MHz,DMSO-d6)δ11.24(s,1H),8.42(d,J＝5.4Hz,1H),8.33(d,J＝1.9Hz,1H),8.04–7.97(m,2H),7.82–7.52(m,2H),7.35(dd,J＝5.4,1.9Hz,1H),7.09(d,J＝5.0Hz,1H),6.01(s,2H),5.48–5.25(m,1H),3.63(d,J＝10.5Hz,1H),3.26(d,J＝10.4Hz,1H),2.36–1.93(m,2H),1.23(s,3H),0.98(s,6H),0.79–0.41(m,4H).MS(ESI,[M+H] +)m/z：578.6。
步骤4：(S)-4-(8-氨基-3-(5-丁-2-炔酰基)-5-氮螺环[2.4]庚烷-6-基)咪唑并[1,5-a]吡嗪-1-基)-N-(4-氯吡啶-2-基)-3-氟苯甲酰胺(化合物I-2)
反应瓶中加入中间体2-6(190mg),加入1,4-二氧六环(5mL)，加入4M的盐酸二氧六环溶液(4mL)室温反应1小时。反应结束，将反应液减压旋干，得中间体2-7(185mg)。
向反应瓶中加入中间体2-7(185mg)，2-丁炔酸(26.3mg)，溶于DCM(5mL)，加入三乙胺(133mg)，室温下搅拌5分钟后，加入HATU(125mg)，室温反应1小时，反应完成，将反应液加入饱和食盐水(200mL)中分液，EA萃取(200mL)，有机相用无水硫酸钠干燥，过滤，浓缩，硅胶柱层析纯化(展开剂：DCM：MeOH＝97:3)，得化合物I-2(55mg)。
1H NMR(500MHz,DMSO-d6)δ11.25(s,1H),8.42(d,J＝5.4Hz,1H),8.33(d,J＝1.9Hz,1H),8.09–7.93(m,2H),7.81(dd,J＝30.7,5.0Hz,1H),7.68–7.56(m,1H),7.35(dd,J＝5.4,1.9Hz,1H),7.12(dd,J＝16.7,5.0Hz,1H),6.05(d,J＝30.6Hz,2H),5.86–5.51(m,1H),3.84–3.66(m,1H),3.62–3.47(m,1H),2.34(ddd,J＝29.5,12.6,7.7Hz,1H),2.17(ddd,J＝33.8,12.6,5.9Hz,1H),1.99(s,2H),1.59(s,1H),0.79–0.51(m,4H).MS(ESI,[M+H] +)m/z：544.3。
实施例7：N-(4-氰基吡啶-2-基)-3-氟-4-(4,4,5,5-四甲基-1,3,2-二氧杂硼杂戊环-2-基)苯甲酰胺(中间体L)的制备
Figure PCTCN2020101200-appb-000014
步骤1：向反应瓶中加入3-氟-4-(4,4,5,5-四甲基-1,3,2-二氧杂硼杂环戊环-2-基)苯甲酸(5g)，4-氰基吡啶-2-胺(2.69g)和三乙胺(5.7g)，并溶于DMF(20mL)中，加入HATU(7.86g)，加热至60℃反应6小时，反应完成，将反应液加入饱和食盐水(200mL)，加入乙酸乙酯(200mL)萃取，有机相用无水硫酸钠干燥，减压旋干后得中间体L(5.12g)。 1H NMR(500MHz,DMSO-d6)δ11.42(s,1H),8.67(dd,J＝5.0,0.9Hz,1H),8.49(t,J＝1.1Hz,1H),7.87(dd,J＝7.7,1.5Hz,1H),7.82–7.74(m,2H),7.65(dd,J＝5.0,1.4Hz,1H),1.33(s,12H).MS(ESI,[M-H] -)m/z：366.5。
实施例8：(S)-4-(8-氨基-3-(5-丁-2-炔酰基)-5-氮螺环[2.4]庚烷-6-基)咪唑[1,5-a]吡嗪-1-基)-N-(4-氰基吡啶-2-基)-3-氟苯甲酰胺(化合物I-3)的制备
Figure PCTCN2020101200-appb-000015
步骤1：(S)-6-(8-氨基-1-(4-((4-氰基吡啶-2-基)氨甲酰)-2-氟苯基)咪唑[1,5-a]吡嗪-3-基)-5-氮杂螺环[2.4]庚烷-5-羧酸叔丁酯(中间体3-6)
向反应瓶中加入中间体2-5(250mg)，中间体L(332mg)与碳酸钾(219mg)，溶于二氧六环(5mL)中，加入[1,1’-双(二苯基膦基)二茂铁]二氯化钯(II)二氯甲烷络合物(43mg)，加入水(1mL)，氮气置换3-4次后，80℃反应120分钟。反应结束，将反应液溶于200mL水中，DCM萃取，有机相无水硫酸钠干燥，过滤，浓缩，硅胶柱层析纯化(展开剂：DCM：MeOH＝97:3)，得中间体3-6(260mg)。
1H NMR(500MHz,DMSO-d6)δ11.42(s,1H),8.68(d,J＝4.9Hz,1H),8.59–8.50(m,1H),8.11–7.97(m,2H),7.80–7.57(m,3H),7.13–7.05(m,1H),6.01(s,2H),5.53–5.27(m,1H),3.63(d,J＝10.4Hz,1H),3.26(d,J＝10.4Hz,1H),2.37–1.88(m,2H),1.41–1.21(m,4H),0.98(s,5H),0.78–0.44(m,4H).MS(ESI,[M+H]+)m/z：569.6。
步骤2：(S)-4-(8-氨基-3-(5-丁-2-炔酰基)-5-氮螺环[2.4]庚烷-6-基)咪唑并[1,5-a]吡嗪-1-基)-N-(4-氰基吡啶-2-基)-3-氟苯甲酰胺(化合物I-3)
反应瓶中加入中间体3-6(200mg),加入1,4-二氧六环(5mL)，加入4M的盐酸二氧六环溶液(4mL)室温反应1小时。反应结束，将反应液减压旋干，得中间体3-7(198mg)。
向反应瓶中加入中间体3-7(198mg)，2-丁炔酸(21.56mg)，溶于DCM(5mL)，加入三乙胺(115mg)，室温下搅拌5分钟后，加入HATU(108mg)，室温反应1小时，反应完成，将反应液加入饱和食盐水(200mL)中分液，EA萃取(200mL)，有机相用无水硫酸钠干燥，过滤，浓缩，硅胶柱层析纯化(展开剂：DCM：MeOH＝97:3)，得化合物I-3(50mg)。 1H NMR(500MHz,DMSO-d6)δ11.43(s,1H),8.69(d,J＝5.2Hz,1H),8.54(s,1H),8.09–7.97(m,2H),7.82(dd,J＝31.1,5.0Hz,1H),7.71–7.55(m,2H),7.13(dd,J＝16.6,5.0Hz,1H),6.05(d,J＝30.9Hz,2H),5.84–5.50(m,1H),3.85–3.66(m,1H),3.60–3.46(m,1H),2.34(ddd,J＝29.6,12.6,7.8Hz,1H),2.17(ddd,J＝34.1,12.6,5.8Hz,1H),1.99(s,2H),1.59(s,1H),0.79–0.50(m,4H).MS(ESI,[M+H]+)m/z：535.5。
实施例9：N-(4-二氟甲基吡啶-2-基)-3-氟-4-(4,4,5,5-四甲基-1,3,2-二氧杂硼杂戊环-2-基)苯甲酰胺(中间体M)的制备
Figure PCTCN2020101200-appb-000016
步骤1：向反应瓶中加入3-氟-4-(4,4,5,5-四甲基-1,3,2-二氧杂硼杂环戊环-2-基)苯甲酸(5g)，4-二氟甲基吡啶-2-胺(2.84g)和三乙胺(5.7g)，并溶于DMF(20mL)中，加入HATU(7.86g)，加热至60℃反应6小时，反应完成，将反应液加入饱和食盐水(200mL)，加入乙酸乙酯(200mL)萃取，有机相用无水硫酸钠干燥，减压旋干后得中间体M(4.45g)。 1H NMR(500MHz,DMSO-d6)δ11.25(s,1H),8.58(d,J＝5.0Hz,1H),8.40(d,J＝6.5Hz,1H),7.88(dd,J＝7.7,1.5Hz,1H),7.82–7.73(m,2H),7.37(dd,J＝5.1,1.4Hz,1H),7.17(t,J＝55.2Hz,1H),1.33(s,12H).MS(ESI,[M+H] +)m/z：393.5。
实施例10：(S)-4-(8-氨基-3-(5-丁-2-炔酰基)-5-氮螺环[2.4]庚烷-6-基)咪唑[1,5-a]吡嗪-1-基)-N-(4-二氟甲基吡啶-2-基)-3-氟苯甲酰胺(化合物I-4)的制备
Figure PCTCN2020101200-appb-000017
步骤1：(S)-6-(8-氨基-1-(4-((4-二氟甲基吡啶-2-基)氨甲酰)-2-氟苯基)咪唑[1,5-a]吡嗪-3-基)-5-氮杂螺环[2.4]庚烷-5-羧酸叔丁酯(中间体4-6)
向反应瓶中加入中间体2-5(250mg)，中间体M(294mg)与碳酸钾(219mg)，溶于二氧六环(5mL)中，加入[1,1’-双(二苯基膦基)二茂铁]二氯化钯(II)二氯甲烷络合物(43mg)，加入水(1mL)，氮气置换3-4次后，80℃反应120分钟。反应结束，将反应液溶于200mL水中，DCM萃取，有机相无水硫酸钠干燥，过滤，浓缩，硅胶柱层析纯化(展开剂：DCM：MeOH＝97:3)，得中间体4-6(280mg)。
1H NMR(500MHz,DMSO-d6)δ11.25(s,1H),8.59(d,J＝5.1Hz,1H),8.44(s,1H),8.11–7.98(m,2H),7.81–7.53(m,2H),7.38(d,J＝5.0Hz,1H),7.30–7.03(m,2H),6.03(s,2H),5.49–5.27(m,1H),3.64(d,J＝10.4Hz,1H),3.26(d,J＝10.4Hz,1H),2.36–1.97(m,2H),1.36(s,3H),0.98(s,6H),0.78–0.49(m,4H).MS(ESI,[M+H]+)m/z：594.5。
步骤2：(S)-4-(8-氨基-3-(5-丁-2-炔酰基)-5-氮螺环[2.4]庚烷-6-基)咪唑并[1,5-a]吡嗪-1-基)-N-(4-二氟甲基吡啶-2-基)-3-氟苯甲酰胺(化合物I-4)
反应瓶中加入中间体4-6(220mg),加入1,4-二氧六环(5mL)，加入4M的盐酸二氧六环溶液(4mL) 室温反应1小时。反应结束，将反应液减压旋干，得中间体4-7(225mg)。
向反应瓶中加入中间体4-7(225mg)，2-丁炔酸(26.6mg)，溶于DCM(5mL)，加入三乙胺(135mg)，室温下搅拌5分钟后，加入HATU(127mg)，室温反应1小时，反应完成，将反应液加入饱和食盐水(200mL)中分液，EA萃取(200mL)，有机相用无水硫酸钠干燥，过滤，浓缩，硅胶柱层析纯化(展开剂：DCM：MeOH＝97:3)，得化合物I-4(90mg)。 1H NMR(500MHz,DMSO-d6)δ11.25(s,1H),8.60(d,J＝5.0Hz,1H),8.44(s,1H),8.10–7.97(m,2H),7.82(dd,J＝30.4,5.1Hz,1H),7.70–7.58(m,1H),7.38(dd,J＝5.1,1.3Hz,1H),7.32–7.03(m,2H),6.05(d,J＝30.6Hz,2H),5.86–5.53(m,1H),3.86–3.67(m,1H),3.63–3.47(m,1H),2.34(ddd,J＝29.4,12.6,7.8Hz,1H),2.17(ddd,J＝33.9,12.6,5.8Hz,1H),1.99(s,2H),1.59(s,1H),0.79–0.49(m,4H).MS(ESI,[M+H]+)m/z：560.5。
实施例11：N-(4-甲氧基吡啶-2-基)-3-氟-4-(4,4,5,5-四甲基-1,3,2-二氧杂硼杂戊环-2-基)苯甲酰胺(中间体N)的制备
Figure PCTCN2020101200-appb-000018
步骤1：向反应瓶中加入3-氟-4-(4,4,5,5-四甲基-1,3,2-二氧杂硼杂环戊环-2-基)苯甲酸(5.14g)，4-甲氧基吡啶-2-胺(2g)和三乙胺(4.89g)，并溶于DMF(30mL)中，室温下搅拌20分钟后，加入HATU(7.35g)，加热至60℃反应3小时，反应完成，反应液加入水(200ml)和乙酸乙酯萃取，有机相用水和饱和氯化钠水溶液洗涤，然后用无水硫酸钠干燥，过滤，浓缩中间体N(2.8g)。
1H NMR(500MHz,DMSO-d6)δ10.95(s,1H),8.21(d,J＝5.8Hz,1H),7.85–7.84(m,1H),7.82(d,J＝2.3Hz,1H),7.79–7.76(m,1H),7.75–7.72(m,1H),6.81–6.79(m,1H),3.85(s,3H),1.31(s,12H).MS:ESI[M+H-(2,3-二甲基丁烷)] +m/z：291.2(硼酸形式)。
实施例12：(S)-4-(8-氨基-3-(5-丁-2-炔酰基)-5-氮螺环[2.4]庚烷-6-基)咪唑[1,5-a]吡嗪-1-基)-N-(4-甲氧基吡啶-2-基)-3-氟苯甲酰胺(化合物I-5)的制备
Figure PCTCN2020101200-appb-000019
步骤1：(S)-6-(8-氨基-1-(4-((4-甲氧基吡啶-2-基)氨甲酰)-2-氟苯基)咪唑[1,5-a]吡嗪-3-基)-5-氮杂螺环[2.4]庚烷-5-羧酸叔丁酯(中间体6-6)
向反应瓶中加入中间体2-5(250mg)，中间体N(290mg)与碳酸钾(148mg)，溶于二氧六环(5mL)中，加入[1,1’-双(二苯基膦基)二茂铁]二氯化钯(II)二氯甲烷络合物(41.1mg)，加入水(1mL)，氮气置换3-4次后，80℃反应60分钟。反应结束，将反应液溶于300mL水中，DCM萃取，有机相无水硫酸钠干燥，过滤，浓缩，硅胶柱层析纯化(展开剂：DCM：MeOH＝97:3)，得中间体6-6(214mg)。MS(ESI,[M+H] +)m/z：574.6。
步骤2：(S)-4-(8-氨基-3-(5-丁-2-炔酰基)-5-氮螺环[2.4]庚烷-6-基)咪唑并[1,5-a]吡嗪-1-基)-N-(4-甲氧基吡啶-2-基)-3-氟苯甲酰胺(化合物I-5)
反应瓶中加入中间体6-6(204mg),加入1,4-二氧六环(5mL)，加入4M的盐酸二氧六环溶液(4mL)室温反应1小时。反应结束，将反应液减压旋干，得中间体6-7(140mg)。
向反应瓶中加入中间体6-7(140mg)，2-丁炔酸(22.4mg)，溶于DCM(5mL)，加入三乙胺(90mg)，室温下搅拌5分钟后，加入HATU(118mg)，室温反应1小时，反应完成，将反应液加入饱和食盐水(200mL)中分液，EA萃取(200mL)，有机相用无水硫酸钠干燥，过滤，浓缩，硅胶柱层析纯化(展开剂：DCM：MeOH＝97:3)，得化合物I-5(118mg)。
1H NMR(500MHz,DMSO-d6)δ10.91(s,1H),8.23(d,J＝5.8Hz,1H),8.02–7.99(m,1H),7.86(d,J＝2.3Hz,1H),7.81(dd,J＝30.6,5.1Hz,1H),7.63–7.59(m,1H),7.13–7.09(m,1H),6.82–6.80(m,1H),6.04(d,J＝26.9Hz,2H),5.80–5.57(m,1H),3.87(s,3H),3.81–3.71(m,1H),3.60–3.47(m,1H),2.39–2.15(m,2H),1.99(s,3H),0.71–0.49(m,4H).MS(ESI,[M+H] +)m/z：540.5。
试验例1：体外活性
1.1 BTK抑制活性筛选
用激酶缓冲液(50mM HEPES、10mM MgCl 2、2mM DTT、1mM EGTA、0.01％Tween 20)将350ng/μL的BTK(WT)母液进行稀释，按每孔加入6μL 1.67×的0.0334ng/μL的工作液(终浓度为0.02ng/μL)，用纳升加样仪将DMSO溶解的不同化合物加入到孔中，使化合物终浓度为1000nM-0.244nM，4倍梯度，共7个浓度，同时设空白对照空(不含酶)与阴性对照孔(含酶，加溶媒DMSO)，设2个复孔。酶与化合物或溶媒反应30min后，将用激酶缓冲液配制好的5×的100μM ATP(终浓度为20μM)与5×的0.5μM底物(终浓度为0.1μM，ULight-poly GT)，按1：1混合，按每孔4μL加入孔中；封板膜封板以后，室温反应2h后，每孔加入5μL 4×的40mM EDTA(终浓度为10mM)，室温5min，再每孔加入5μL 4×的8nM检测试剂(终浓度为2nM，Ab)，室温孵育1小时；PE Envision多功能酶标仪进行读板(激发620nm，发射665nm)，采用四参数拟合，计算IC50。
1.2 BTK(C481S)抑制活性筛选
用激酶缓冲液(50mM HEPES、10mM MgCl 2、2mM DTT、1mM EGTA、0.01％Tween 20)将50ng/μL的BTK(C481S)母液进行稀释，按每孔加入6μl 1.67×的0.25ng/μl的工作液(终浓度为0.15ng/μl)，用纳升加样仪将DMSO溶解的不同化合物加入到孔中，使化合物终浓度为1000nM-0.244nM，4倍梯度，共7个浓度，同时设空白对照空(不含酶)与阴性对照孔(含酶，加溶媒DMSO)，设2个复孔。酶与化合物或溶媒反应30min后，将用激酶缓冲液配制好的5×的250μM ATP(终浓度为50μM)与5×的0.5μM底物(终浓度为0.1μM，ULight-poly GT)，按1：1混合，按每孔4μL加入孔中；封板膜封板以后，室温反应2h后，每孔加入5μL4×的40mM EDTA(终浓度为10mM)，室温5min，再每孔加入5μL 4×的8nM检测试剂(终浓度为2nM，Ab)，室温孵育1小时；PE Envision多功能酶标仪进行读板(激发620nm，发射665nm)，采用四参数拟合，计算IC50。
1.3 EGFR(epidermal growth factor receptor)抑制活性筛选
用激酶缓冲液(50mM HEPES、10mM MgCl 2、2mM DTT、1mM EGTA、0.01％Tween 20)将50ng/μL的EGFR(WT)母液进行稀释，按每孔加入6μL 1.67×的0.01336ng/μL的工作液(终浓度为0.008ng/μL)，用纳升加样仪将DMSO溶解的不同化合物加入到孔中，使化合物终浓度为1000nM-0.48nM，4倍梯度，共7个浓度，同时设空白对照空(不含酶)与阴性对照孔(含酶，加溶媒DMSO)，设2个复孔。酶与化合物或溶媒反应10min后，将用激酶缓冲液配制好的5×的25μM ATP(终浓度为5μM)与5×的0.5μM底物(终浓度为0.1μM，ULight-poly GT)，按1:1混合后按每孔4μL加入孔中；封板膜封板以后，室温反应2h后，每孔加入5μL 4×的40mM EDTA(终浓度为10mM)，室温5min，再每孔加入5μL 4×的8nM检测试剂(终浓度为2nM，Eu-anti-phospho-tyrosine antibody)，室温孵育1小时，PE Envision多功能酶标仪进行读板(激发320nm，发射665nm)，采用四参数拟合，计算IC50。
1.4 TEC抑制活性筛选
用激酶缓冲液(50mM HEPES、10mM MgCl 2、2mM DTT、1mM EGTA、0.01％Tween 20)将50ng/μL的TEC母液进行稀释，按每孔加入6μL 1.67×的0.01336g/μL的工作液(终浓度为0.008ng/μL)，用纳升加样仪将DMSO溶解的不同化合物加入到孔中，使化合物终浓度为1000nM-0.24nM，4倍梯度，共7个浓度，同时设空白对照空(不含酶)与阴性对照孔(含酶，加溶媒DMSO)。酶与化合物或溶媒反应30min后，将用激酶缓冲液配制好的5×的50μM ATP(终浓度为10μM)与5×的0.5μM底物(终浓度为0.1μM，ULight-poly GT)，按1:1混合后按每孔4μL加入孔中；封板膜封板以后，室温反应2h后，每孔加入5μL 4×的40mM EDTA(终浓度为10mM)，室温5min，再每孔加入5μL 4×的8nM检测试剂(终浓度为2nM，Eu-anti-phospho-tyrosine antibody)，室温孵育1小时；PE Envision多功能酶标仪进行读板(激发320nm，发射665nm)，采用四参数拟合，计算IC50。
1.5 ITK(Interleukin-2-inducible T-cell kinase)抑制活性筛选
用激酶缓冲液(50mM HEPES、10mM MgCl 2、2mM DTT、1mM EGTA、0.01％Tween 20)将50ng/μL的ITK母液进行稀释，按每孔加入6μL 1.67×的0.0835g/μL的工作液(终浓度为0.05ng/μL)，用纳升加样仪将DMSO溶解的不同化合物加入到孔中，使化合物终浓度为1000nM-0.24nM，4倍梯度，共7个浓度，同时设空白对照空(不含酶)与阴性对照孔(含酶，加溶媒DMSO)。酶与化合物或溶媒反应30min后，将用激酶缓冲液配制好的5×的50μM ATP(终浓度为10μM)与5×的0.5μM底物(终浓度为0.1μM，ULight-poly GT)，按1:1混合后按每孔4μL加入孔中；封板膜封板以后，室温反应2h后，每孔加入5μL 4×的40mM EDTA(终浓度为10mM)，室温5min，再每孔加入5μL 4×的8nM检测试剂(终浓度为2nM，Eu-anti-phospho-tyrosine antibody)，室温孵育1小时；PE Envision多功能酶标仪进行读板(激发320nm，发射665nm)，采用四参数拟合，计算IC50。
上述测试结果见表1。
表1
Figure PCTCN2020101200-appb-000020
NA：未测定。
试验例2：细胞活性
2.1细胞水平BTK(Y223)磷酸化抑制活性筛选
取20μL的30％的过氧化氢，加入860μL双蒸水，配制成200mM的过氧化氢。PV(过钒酸钠)：取(200mmol/原钒酸钠10μL)添加(200mmol/L过氧化氢10μL)添加(80μL无酚红1640完全培养基)室温反应15min，加无酚红1640完全培养基稀释至6mM，现配现用。取对数期生长的Ramos淋巴瘤细胞，低速台式离心机，1500rpm离心3min，加入适量无酚红1640完全培养基重悬后计数，取适量细胞悬液加入适量相应的培养基进行细胞密度调整，调整细胞密度约为1-2*10E7个/mL。细胞按照上述细胞密度进行种板(384孔)，20μL/孔；每孔加入5μL化合物孵育1h；取20mM的PV采用无酚红1640完全培养基稀释至6mM(终浓度1mM)；随后根据板分布每孔加入5μL PV，孵育15min-20min；空白组接种细胞，不加化合物，不加PV；对照组，接种细胞，不加化合物，加PV；立即加入10μL添加封闭液的裂解液(4X)，并在室温下摇动孵育30min。混匀后，将16μL裂解物转移到另一个384孔小体积白板。加入4μL检测缓冲液配制的预先混合的抗体(vol/vol)，盖板，离心使混合均匀，室温孵育过夜。PE Envision多功能读板仪进行检测665nm/620nm信号值，四参数拟合计算IC 50。测试结果见表2。
表2
Figure PCTCN2020101200-appb-000021
2.2化合物对TMD-8细胞的增殖抑制作用
取处于指数生长期状态良好的TMD-8细胞，收集细胞至离心管，低速台式离心机，1500转/min，离心3min，弃上清，加入2mL种板培养基(RPMI基础培养基+5％FBS+0.05mM 2-巯基乙醇)进行细胞重悬。使用细胞计数仪计数，取所需量的细胞调整密度至5×10 4个/mL，使用排枪接种于96孔板上，100μL/孔，置于37℃、含5％CO 2饱和湿度的细胞培养箱中培养。培养24h后，使用纳升加样仪进行化合物加样，每一浓度设置2个复孔，以不加化合物的细胞作为阴性对照，72小时后加CCK-8，10μL/孔，4小时后，Envision酶标仪450nm处检测其吸光值，四参数分析，拟合量效曲线，计算IC50，结果见表3。
表3
Figure PCTCN2020101200-appb-000022
2.3化合物对OCI-LY10细胞的增殖抑制作用
取处于指数生长期状态良好的OCI-LY10细胞，收集细胞至离心管，取20μL计数，根据计数结果取实验所需细胞量，低速台式离心机，1500转/min，离心3min，弃上清，加入合适量种板培养基(RPMI基础培养基+10％FBS)进行细胞重悬，调整细胞密度至1×10 5个/mL，使用排枪接种于96孔板上，100μL/孔，置于37℃、含5％CO 2饱和湿度的细胞培养箱中培养。培养24h后，使用纳升加样仪进行化合物加样，每一浓度设置2个复孔，以不加化合物的细胞作为阴性对照，72小时后加CCK-8，10μL/孔，4小时后，Envision酶标仪450nm处检测其吸光值，四参数分析，拟合量效曲线，计算IC50。
Figure PCTCN2020101200-appb-000023
试验例3：小鼠体内药代动力学实验
ICR小鼠，体重18～22g，适应3～5天后，随机分组，每组9只，按10mg/kg剂量分别灌胃相关化合物，1mg/kg剂量分别静注相关化合物。受试动物(ICR小鼠)给药前禁食12h，给药后4h给食物，实验前后和实验过程中均自由饮水。灌胃给药后于0.25(15min)、0.5(30min)、1、2、4、6、8、10、24h眼眶取血约0.1mL，静注给药后于0.083(5min)、0.167(10min)、0.5(30min)、1、2、6、8、10、24h眼眶取血约0.1mL，每只小鼠采集3～4个时间点，每个时间点3只小鼠，采集全血置于含EDTA-K2和氟化钠离心管中，30min内转移到4℃，4000rpm×10min条件下离心分离血浆。收集全部血浆后立即于-20℃保存待测。吸取20μL待测血浆样品和标曲样品，加入300μL含内标(地西泮20mg/mL)的乙腈溶液，振荡混匀5min，13000rpm离心10min，取上清80μL，加入80μL超纯水稀释，混匀，吸取1μL用于LC/MS/MS测定，记录色谱图。通过小鼠体内药物动力学实验评估本发明化合物的口服、静注暴露量，结果见表4。
表4
Figure PCTCN2020101200-appb-000024
注：ig：灌胃；iv：静脉注射；MRT：平均驻留时间；Vz：表观分布容积；CLz：清除率。
试验例4：体内药效研究
OCI-LY10小鼠皮下移植瘤，浓度1×10 8/ml*0.1ml/只，在无菌条件下，接种于NOD-SCID小鼠右侧腋窝下(接种时接种部位剃毛)。皮下移植瘤接种后待肿瘤瘤体积至100-300mm 3左右将动物分组：
模型组：溶媒6只；化合物I-1：50mg/kg，bid，i.g 6只。
按10ml/kg的体积分别灌胃给予溶媒或药物，每日2次，连续给药23天。每周测2-3次瘤体积，同时称鼠重，记录数据；每日观察动物表现。待全部给药结束后，处死动物，剥瘤称重。
使用下面公式计算肿瘤体积和抑瘤率：
肿瘤体积(TV)＝(长×宽 2)/2。
抑瘤率(tumor growth inhibition,TGI)＝(1-治疗组瘤重量/模型组瘤重量)×100％。
表5化合物对小鼠OCI-LY10移植瘤的疗效
Figure PCTCN2020101200-appb-000025

Claims (10)

  1. 式(I)化合物、其立体异构体或其药学上可接受的盐，
    Figure PCTCN2020101200-appb-100001
    其中，
    环A选自5-10元杂芳基或C 6-10芳基；
    R 1独立地选自卤素、羟基、氨基、氰基、C 1-6烷氧基或C 1-6烷基，所述C 1-6烷氧基或C 1-6烷基任选地被卤素取代；
    m选自0、1、2、3、4、5或6；
    R 2选自氢、R aS(O) 2-、(R aO) 2P(O)-或R aC(O)-；
    其中R a独立地选自C 2-6炔基、C 2-6烯基、C 1-6烷基、C 3-6环烷基、(C 1-6烷基)NH-、(C 1-6烷基) 2N-、3-6元杂环烷基、5-10元杂芳基或C 6-10芳基，所述R a任选地被(C 1-6烷基) 2N-、(C 1-6烷基)NH-、羟基、氨基、卤素或氰基取代。
  2. 权利要求1所述的式(I)化合物、其立体异构体或其药学上可接受的盐，其中环A选自5-6元杂芳基；或者，环A选自6元杂芳基；或者，环A选自6元含氮杂芳基；或者，环A选自吡啶基。
  3. 权利要求1或2所述的式(I)化合物、其立体异构体或其药学上可接受的盐，其中R 1独立地选自卤素、氰基、C 1-3烷氧基、或任选地被卤素取代的C 1-3烷基；或者，R 1独立地选自氟、氯、溴、碘、氰基、甲氧基或任选被卤素取代的甲基；或者，R 1独立地选自氯、溴、氰基、-CHF 2或甲氧基；或者，R 1独立地为溴。
  4. 权利要求1-3中任一项所述的式(I)化合物、其立体异构体或其药学上可接受的盐，其中m选自0、1、2、3或4；或者，m选自0、1或2；或者，m为1。
  5. 权利要求1-4中任一项所述的式(I)化合物、其立体异构体或其药学上可接受的盐，其中R 2选自R aC(O)-；或者，R 2选自
    Figure PCTCN2020101200-appb-100002
  6. 权利要求1-5中任一项所述的式(I)化合物、其立体异构体或其药学上可接受的盐，其中R a选自C 2-6 炔基、C 2-6烯基、C 1-6烷基、C 3-6环烷基或3-6元杂环烷基；或者，R a选自C 2-3炔基或C 3-4环烷基；或者，R a为丙炔基。
  7. 权利要求1所述的式(I)化合物、其立体异构体或其药学上可接受的盐，选自式(I-1)、式(II)、式(II-1)或式(II-2)化合物或其药学上可接受的盐，
    Figure PCTCN2020101200-appb-100003
    其中环A、R 1、R 2和m如权利要求1所定义。
  8. 以下化合物、其立体异构体或其药学上可接受的盐：
    Figure PCTCN2020101200-appb-100004
    Figure PCTCN2020101200-appb-100005
  9. 一种药物组合物，其包含权利要求1-8任一项所述的化合物、其立体异构体或其药学上可接受的盐，还包括药学上可接受的辅料。
  10. 用于预防或治疗与BTK相关疾病的权利要求1-8任一项所述的化合物、其立体异构体或其药学上可接受的盐、或权利要求9所述的药物组合物。
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