WO2020259556A1 - 作为cdk9抑制剂的氮杂吲哚连吡唑类化合物 - Google Patents

作为cdk9抑制剂的氮杂吲哚连吡唑类化合物 Download PDF

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WO2020259556A1
WO2020259556A1 PCT/CN2020/098002 CN2020098002W WO2020259556A1 WO 2020259556 A1 WO2020259556 A1 WO 2020259556A1 CN 2020098002 W CN2020098002 W CN 2020098002W WO 2020259556 A1 WO2020259556 A1 WO 2020259556A1
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compound
pharmaceutically acceptable
isomer
acceptable salt
reaction
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English (en)
French (fr)
Inventor
刘迎春
徐招兵
胡利红
丁照中
江文
黎健
陈曙辉
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Medshine Discovery Inc
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Medshine Discovery Inc
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Priority to CN202080046613.2A priority Critical patent/CN114008046B/zh
Priority to US17/622,570 priority patent/US20220267321A1/en
Publication of WO2020259556A1 publication Critical patent/WO2020259556A1/zh
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    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D519/00—Heterocyclic compounds containing more than one system of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring system not provided for in groups C07D453/00 or C07D455/00

Definitions

  • the present invention relates to new azaindolepyrazole compounds as CDK9 inhibitors, and specifically relates to compounds represented by formula (I), pharmaceutically acceptable salts and isomers thereof, and compounds represented by formula (I) Application of the compound, its pharmaceutically acceptable salt and its isomers, and the pharmaceutical composition containing them in the preparation of cancer drugs.
  • CDK Cell Dependent Kinase
  • CDK9 is a member of the CDK family and is mainly involved in the process of transcription regulation.
  • the heterodimer composed of CDK9 and cyclin (T1, T2a, T2b, K) participates in the formation of positive transcription elongation factor (p-TEFb), of which there are approximately 80% of CDK9 binds to cyclinT1.
  • P-TEFb phosphorylates the carboxy-terminal domain of RNA polymerase II, mainly Ser-2 phosphorylation, to regulate transcription elongation.
  • the inhibition of CDK9 and transcriptional repression leads to the rapid consumption of short-lived mRNA transcripts and related proteins (including Myc and Mcl-1), leading to the death of cancer cells that are highly dependent on these anti-apoptotic proteins. Therefore, targeting CDK9 represents a treatment strategy for tumor types that are highly dependent on these anti-apoptotic proteins.
  • CDK9 small molecule inhibitors have entered the clinical research stage for cancer treatment, namely Bayer's BAY1251152 and AstraZeneca's AZD4573. These patents include WO2012160034, WO2014076091, WO2009047359, WO2011110612, US2016376287.
  • the present invention provides a compound represented by formula (I), a pharmaceutically acceptable salt or isomer thereof,
  • T 1 is N or CR
  • R is H or Cl
  • T 2 is N or CH
  • R 1 is H or C 1-6 alkyl, wherein the C 1-6 alkyl is optionally selected by 1, 2 or 3 independently selected from F, Cl, -OH, -NH 2 and C 1-3 alkoxy Substitution of the group;
  • R 2 is H, F or Cl
  • R 3 and R 4 are each independently H, F, Cl or C 1-3 alkyl
  • R 5 is H, C 3-6 cycloalkyl or phenyl, wherein the C 3-6 cycloalkyl and phenyl optionally substituted with 1, 2, or 3 R a;
  • Each R a is independently H, F, Cl, C 1-3 alkyl or a C 1-3 alkoxy group.
  • the above-mentioned compound has a structure represented by formula (I-1) or (I-2):
  • R, T 2 , R 1 , R 2 , R 3 , R 4 and R 5 are as defined in the present invention.
  • the above-mentioned compound has a structure represented by formula (I-1-a) or (I-1-b):
  • R, R 1 , R 2 , R 3 , R 4 and R 5 are as defined in the present invention.
  • R 3 and R 4 are each independently H, F or Other variables are as defined in the present invention.
  • R 3 and R 4 are each independently H, and other variables are as defined in the present invention.
  • the above-mentioned compound has a structure represented by formula (I-1-c) or (I-1-d):
  • R, R 1 , R 2 and R 5 are as defined in the present invention.
  • each of the above Ra is independently H, F, Cl or Other variables are as defined in the present invention.
  • each of the above Ra is independently Cl, and other variables are as defined in the present invention.
  • R 5 is H, Which said Optionally substituted with 1, 2 or 3 R a, R a, and the other variables are as defined in the present invention.
  • R 5 is H
  • R a and the other variables are as defined in the present invention.
  • R 5 is H
  • Other variables are as defined in the present invention.
  • R 5 is H
  • Other variables are as defined in the present invention.
  • the above-mentioned compound has a structure represented by formula (I-1-e), (I-1-f), (I-1-g) or (I-1-h):
  • R, R 1, R 2 and R a are as defined in the present invention.
  • R 1 is H, Which said Optionally substituted by 1, 2 or 3 substituents independently selected from F, Cl, -OH, -NH 2 and -OCH 3 , and other variables are as defined in the present invention.
  • R 1 is H
  • Other variables are as defined in the present invention.
  • the above-mentioned compound is:
  • the present invention also provides a pharmaceutical composition, which contains a therapeutically effective amount of the above-mentioned compound, its isomer or its pharmaceutically acceptable salt, and a pharmaceutically acceptable carrier.
  • the present invention also provides the application of the above-mentioned compound, its pharmaceutically acceptable salt or its isomer and the above-mentioned pharmaceutical composition in the preparation of CDK9 inhibitor drugs.
  • the present invention also provides the application of the above-mentioned compound, its pharmaceutically acceptable salt or its isomers, and the above-mentioned pharmaceutical composition in the preparation of cancer drugs.
  • the compound of the present invention designs the mother nucleus as an azaindole structure, which forms a strong double hydrogen bond with the hinge region of the CDK9 structure, and the nitrogen on the pyrazole forms a hydrogen bond with Lys48 of CDK9.
  • the piperidine in the solvent region of the compound of the present invention is very basic. In CDK9, it can form a salt bridge with Asp109 and maintain high activity. In other subtypes of CDK such as CDK2, the positively charged piperidine interacts with the cavity mouth. The positive charge of Lys89 has electrostatic repulsion, so the molecule has better selectivity for CDK2 and other kinase subtypes.
  • the compound of the present invention has excellent anti-tumor activity and safety. Has a very good prospect of medicine.
  • 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 , Without excessive toxicity, irritation, allergic reactions or other problems or complications, commensurate with a reasonable benefit/risk ratio.
  • pharmaceutically acceptable salt refers to a salt of the compound of the present invention, which is prepared from a compound with specific substituents discovered in the present invention and a relatively non-toxic acid or base.
  • a base addition salt can be obtained by contacting the compound with a sufficient amount of base in a pure solution or a suitable inert solvent.
  • Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amine or magnesium salt or similar salts.
  • the acid addition salt can be obtained by contacting the compound with a sufficient amount of acid in a pure solution or a suitable inert solvent.
  • Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts including, for example, hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, hydrogen carbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, Hydrogen sulfate, hydroiodic acid, phosphorous acid, etc.; and organic acid salts, the organic acid includes such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, Similar acids such as fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid and methanesulfonic acid; also include salts of amino acids (such as arginine, etc.) , And salts of organic acids such as glucuronic acid. Certain specific compounds of the present invention contain basic and acidic
  • the pharmaceutically acceptable salt of the present invention can be synthesized from the parent compound containing acid or base by conventional chemical methods. Generally, such salts are prepared by reacting these compounds in free acid or base form with a stoichiometric amount of appropriate base or acid in water or an organic solvent or a mixture of both.
  • the compounds of the present invention may exist in specific geometric or stereoisomeric forms.
  • the present invention contemplates all such compounds, including cis and trans isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers Isomers, (D)-isomers, (L)-isomers, and racemic mixtures and other mixtures, such as enantiomers or diastereomer-enriched mixtures, all of these mixtures belong to this Within the scope of the invention.
  • Additional asymmetric carbon atoms may be present in substituents such as alkyl. All these isomers and their mixtures are included in the scope of the present invention.
  • enantiomer or “optical isomer” refers to stereoisomers that are mirror images of each other.
  • cis-trans isomer or “geometric isomer” is caused by the inability to rotate freely because of double bonds or single bonds of ring-forming carbon atoms.
  • diastereomer refers to a stereoisomer in which a molecule has two or more chiral centers and the relationship between the molecules is not mirror images.
  • wedge-shaped solid line keys And wedge-shaped dashed key Represents the absolute configuration of a solid center, with a straight solid line key And straight dashed key Indicates the relative configuration of the three-dimensional center, using wavy lines Represents a wedge-shaped solid line key Or wedge-shaped dotted key Or use wavy lines Represents a straight solid line key And straight dashed key
  • the compound of the present invention may be specific.
  • tautomer or “tautomeric form” means that at room temperature, the isomers of different functional groups are in dynamic equilibrium and can be transformed into each other quickly. If tautomers are possible (such as in solution), the chemical equilibrium of tautomers can be reached.
  • proton tautomers also called prototropic tautomers
  • proton migration such as keto-enol isomerization and imine-ene Amine isomerization.
  • Valence isomers include some recombination of bonding electrons to carry out mutual transformation.
  • keto-enol tautomerization is the tautomerism between two tautomers of pentane-2,4-dione and 4-hydroxypent-3-en-2-one.
  • the terms “enriched in one isomer”, “enriched in isomers”, “enriched in one enantiomer” or “enriched in enantiomers” refer to one of the isomers or pairs of
  • the content of the enantiomer is less than 100%, and the content of the isomer or enantiomer is greater than or equal to 60%, or greater than or equal to 70%, or greater than or equal to 80%, or greater than or equal to 90%, or greater than or equal to 95%, or 96% or greater, or 97% or greater, or 98% or greater, or 99% or greater, or 99.5% or greater, or 99.6% or greater, or 99.7% or greater, or 99.8% or greater, or greater than or equal 99.9%.
  • the term “isomer excess” or “enantiomeric excess” refers to the difference between the relative percentages of two isomers or two enantiomers. For example, if the content of one isomer or enantiomer is 90%, and the content of the other isomer or enantiomer is 10%, the isomer or enantiomer excess (ee value) is 80% .
  • optically active (R)- and (S)-isomers and D and L isomers can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If you want to obtain an enantiomer of a compound of the present invention, it can be prepared by asymmetric synthesis or derivatization with chiral auxiliary agents, in which the resulting diastereomeric mixture is separated and the auxiliary group is cleaved to provide pure The desired enantiomer.
  • the molecule when the molecule contains a basic functional group (such as an amino group) or an acidic functional group (such as a carboxyl group), it forms a diastereomeric salt with a suitable optically active acid or base, and then passes through a conventional method known in the art The diastereoisomers are resolved, and then the pure enantiomers are recovered.
  • the separation of enantiomers and diastereomers is usually accomplished through the use of chromatography, which employs a chiral stationary phase and is optionally combined with chemical derivatization (for example, the formation of amino groups from amines). Formate).
  • the compounds of the present invention may contain unnatural proportions of atomic isotopes on one or more of the atoms constituting the compound.
  • compounds can be labeled with radioisotopes, such as tritium ( 3 H), iodine-125 ( 125 I), or C-14 ( 14 C).
  • deuterated drugs can be formed by replacing hydrogen with heavy hydrogen. The bond formed by deuterium and carbon is stronger than the bond formed by ordinary hydrogen and carbon. Compared with undeuterated drugs, deuterated drugs have reduced toxic side effects and increased drug stability. , Enhance the efficacy, extend the biological half-life of drugs and other advantages. All changes in the isotopic composition of the compounds of the present invention, whether radioactive or not, are included in the scope of the present invention. "Optional" or “optionally” means that the event or condition described later may but not necessarily occur, and the description includes a situation in which the event or condition occurs and a situation in which the event or condition does not occur.
  • the term "effective amount” or “therapeutically effective amount” refers to a sufficient amount of a drug or agent that is non-toxic but can achieve the desired effect.
  • the "effective amount” of one active substance in the composition refers to the amount required to achieve the desired effect when combined with another active substance in the composition.
  • the determination of the effective amount varies from person to person, and depends on the age and general conditions of the recipient, as well as the specific active substance. The appropriate effective amount in a case can be determined by those skilled in the art according to routine experiments.
  • substituted means that any one or more hydrogen atoms on a specific atom are replaced by substituents, and can include deuterium and hydrogen variants, as long as the valence of the specific atom is normal and the substituted compound is stable of.
  • oxygen it means that two hydrogen atoms are replaced. Oxygen substitution will not occur on aromatic groups.
  • optionally substituted means that it can be substituted or unsubstituted. Unless otherwise specified, the type and number of substituents can be arbitrary on the basis that they can be chemically realized.
  • any variable such as R
  • its definition in each case is independent.
  • the group may optionally be substituted with up to two Rs, and R has independent options in each case.
  • combinations of substituents and/or variants thereof are only permitted if such combinations result in stable compounds.
  • linking group When the number of a linking group is 0, such as -(CRR) 0 -, it means that the linking group is a single bond.
  • substituents When a substituent is vacant, it means that the substituent is absent. For example, when X in A-X is vacant, it means that the structure is actually A.
  • substituents do not indicate which atom is connected to the substituted group, such substituents can be bonded via any atom.
  • a pyridyl group can pass through any one of the pyridine ring as a substituent. The carbon atom is attached to the substituted group.
  • the middle linking group L is -MW-, at this time -MW- can be formed by connecting ring A and ring B in the same direction as the reading order from left to right It can also be formed by connecting ring A and ring B in the direction opposite to the reading order from left to right Combinations of the linking groups, substituents, and/or variants thereof are only permitted if such combinations result in stable compounds.
  • any one or more sites of the group can be connected to other groups through chemical bonds.
  • the chemical bond between the site and other groups can be a straight solid bond Straight dotted key Or wavy line Said.
  • the straight solid bond in -OCH 3 means that it is connected to other groups through the oxygen atom in the group;
  • the straight dashed bond in indicates that the two ends of the nitrogen atom in the group are connected to other groups;
  • the wavy lines in indicate that the phenyl group is connected to other groups through the 1 and 2 carbon atoms.
  • the number of atoms in a ring is generally defined as the number of ring members.
  • “5-7 membered ring” refers to a “ring” in which 5-7 atoms are arranged around.
  • C 1-6 alkyl is used to indicate a linear or branched saturated hydrocarbon group composed of 1 to 6 carbon atoms.
  • the C 1-6 alkyl group includes C 1-5 , C 1-4 , C 1-3 , C 1-2 , C 2-6 , C 2-4 , C 6 and C 5 alkyl groups, etc.; it may Is monovalent (such as methyl), divalent (such as methylene) or multivalent (such as methine).
  • C 1-6 alkyl groups include, but are not limited to, methyl (Me), ethyl (Et), propyl (including n-propyl and isopropyl), butyl (including n-butyl, isobutyl) , S-butyl and t-butyl), pentyl (including n-pentyl, isopentyl and neopentyl), hexyl, etc.
  • C 1-4 alkyl is used to denote a linear or branched saturated hydrocarbon group composed of 1 to 4 carbon atoms.
  • the C 1-4 alkyl group includes C 1-2 , C 1-3 and C 2-3 alkyl groups, etc.; it can be monovalent (such as methyl), divalent (such as methylene) or multivalent ( Such as methine).
  • Examples of C 1-4 alkyl groups include, but are not limited to, methyl (Me), ethyl (Et), propyl (including n-propyl and isopropyl), butyl (including n-butyl, isobutyl) , S-butyl and t-butyl) and so on.
  • C 1-3 alkyl is used to indicate a linear or branched saturated hydrocarbon group composed of 1 to 3 carbon atoms.
  • the C 1-3 alkyl group includes C 1-2 and C 2-3 alkyl groups, etc.; it can be monovalent (such as methyl), divalent (such as methylene) or multivalent (such as methine) .
  • Examples of C 1-3 alkyl include, but are not limited to, methyl (Me), ethyl (Et), propyl (including n-propyl and isopropyl), and the like.
  • C 1-3 alkoxy refers to those alkyl groups containing 1 to 3 carbon atoms attached to the rest of the molecule through an oxygen atom.
  • the C 1-3 alkoxy group includes C 1-2 , C 2-3 , C 3 and C 2 alkoxy groups and the like.
  • Examples of C 1-3 alkoxy include but are not limited to methoxy, ethoxy, propoxy (including n-propoxy and isopropoxy) and the like.
  • C 3-6 cycloalkyl means a saturated cyclic hydrocarbon group composed of 3 to 6 carbon atoms, which is a monocyclic ring system, and the C 3-6 cycloalkyl includes C 3 -5 , C 3-4 or C 4-5 cycloalkyl, etc.; it can be monovalent, divalent or multivalent.
  • Examples of C 3-6 cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and the like.
  • C 3-5 cycloalkyl means a saturated cyclic hydrocarbon group composed of 3 to 5 carbon atoms, which is a monocyclic ring system, and the C 3-5 cycloalkyl includes C 3 -4 or C 4-5 cycloalkyl, etc.; it can be monovalent, divalent or multivalent.
  • Examples of C 3-5 cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl and the like.
  • C n-n+m or C n -C n+m includes any specific case of n to n+m carbons, for example, C 1-12 includes C 1 , C 2 , C 3 , C 4 , C 5 , C 6 , C 7 , C 8 , C 9 , C 10 , C 11 , and C 12 , including any range from n to n+m, for example, C 1-12 includes C 1-3 , C 1-6 , C 1-9 , C 3-6 , C 3-9 , C 3-12 , C 6-9 , C 6-12 , and C 9-12, etc.; in the same way, from n to n +m means the number of atoms in the ring is n to n+m, for example, 3-12 membered ring includes 3-membered ring, 4-membered ring, 5-membered ring, 6-membered ring, 7-membered ring, 8-membered ring, 9-membered
  • leaving group refers to a functional group or atom that can be replaced by another functional group or atom through a substitution reaction (for example, an affinity substitution reaction).
  • representative leaving groups include triflate; chlorine, bromine, iodine; sulfonate groups, such as mesylate, tosylate, p-bromobenzenesulfonate, p-toluenesulfonic acid Esters, etc.; acyloxy groups, such as acetoxy, trifluoroacetoxy and the like.
  • protecting group includes but is not limited to "amino protecting group", “hydroxy protecting group” or “thiol protecting group”.
  • amino protecting group refers to a protecting group suitable for preventing side reactions at the amino nitrogen position.
  • Representative amino protecting groups include, but are not limited to: formyl; acyl, such as alkanoyl (such as acetyl, trichloroacetyl or trifluoroacetyl); alkoxycarbonyl, such as tert-butoxycarbonyl (Boc) ; Arylmethyloxycarbonyl, such as benzyloxycarbonyl (Cbz) and 9-fluorenylmethyloxycarbonyl (Fmoc); arylmethyl, such as benzyl (Bn), trityl (Tr), 1,1-di -(4'-Methoxyphenyl)methyl; silyl groups, such as trimethylsilyl (TMS) and tert-butyldimethyls
  • hydroxy protecting group refers to a protecting group suitable for preventing side reactions of the hydroxyl group.
  • Representative hydroxy protecting groups include but are not limited to: alkyl groups, such as methyl, ethyl, and tert-butyl; acyl groups, such as alkanoyl groups (such as acetyl); arylmethyl groups, such as benzyl (Bn), p-methyl Oxybenzyl (PMB), 9-fluorenylmethyl (Fm) and diphenylmethyl (diphenylmethyl, DPM); silyl groups such as trimethylsilyl (TMS) and tert-butyl Dimethylsilyl (TBS) and so on.
  • alkyl groups such as methyl, ethyl, and tert-butyl
  • acyl groups such as alkanoyl groups (such as acetyl)
  • arylmethyl groups such as benzyl (Bn), p-methyl Oxybenzyl (P
  • the compounds of the present invention 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 alternatives, preferred implementations include but are not limited to the embodiments of the present invention.
  • the solvent used in the present invention is commercially available.
  • DMF stands for N,N-dimethylformamide
  • Cs 2 CO 3 stands for cesium carbonate
  • EtOAc stands for ethyl acetate
  • EA stands for ethyl acetate
  • THF stands for tetrahydrofuran
  • MeOH stands for methanol
  • DCM Stands for dichloromethane
  • PE stands for petroleum ether
  • EtOH stands for ethanol
  • CuI stands for cuprous iodide
  • NCS stands for N-chlorosuccinimide
  • NBS stands for N-bromosuccinimide
  • ICl stands for monochlorination Iodine
  • Pd(dppf)Cl 2 represents 1,1'-bis(diphenylphosphorus)ferrocene palladium chloride
  • Pd(PPh 3 ) 4 represents palladium tetrakistriphenylphosphine
  • ACN represents acetonitrile
  • FA represents formic acid
  • Reaction scheme 1 prepares the compound represented by formula (I-1-a),
  • R, R 1 , R 2 , R 3 , R 4 and R 5 are as defined in the present invention.
  • Compound (E) can be Sonigashira coupling between compound (C) and compound (D).
  • the reaction requires a suitable catalyst (such as palladium tetraphenylphosphine, cuprous iodide), a suitable base (such as triethylamine), and a suitable solvent (such as toluene, acetonitrile).
  • a suitable catalyst such as palladium tetraphenylphosphine, cuprous iodide
  • a suitable base such as triethylamine
  • a suitable solvent such as toluene, acetonitrile
  • reaction is more preferred to be carried out at high temperature and requires a suitable base (such as potassium tert-butoxide) and a suitable solvent (such as DMF).
  • a suitable base such as potassium tert-butoxide
  • a suitable solvent such as DMF.
  • Compound (L) can be prepared by substitution reaction between compound (J) and bromide (K). This reaction is more preferred to proceed at high temperature and requires a suitable base (such as cesium carbonate) and a suitable solvent (such as DMF).
  • Compound (F) and compound (L) are coupled under palladium catalyzed conditions (such as Pd(dppf)Cl 2 ) to obtain compound (G).
  • Compound (G) is deprotected under acidic conditions (such as hydrogen chloride/ethyl acetate solution) to obtain compound (H), and compound (H) undergoes substitution reaction or reductive amination reaction with corresponding raw materials to obtain formula (I-1-a) The compound shown.
  • the reaction solution was concentrated under reduced pressure, dissolved in water (10 ml), the pH of the aqueous phase was adjusted to 9 with saturated sodium carbonate, extracted with ethyl acetate (20 ml*2), the organic phase was washed with brine (20 ml), and concentrated under reduced pressure
  • the obtained residue was purified by preparative HPLC (column: Phenomenex Synergi C18 150*25mm*10 ⁇ m; formic acid, eluent: [water (0.225% formic acid)-acetonitrile], gradient: acetonitrile 9%-39%) to obtain the compound 7.
  • the compounds of the present invention are CDK9 inhibitors.
  • IC 50 used herein refers to the maximum concentration producing 50% inhibition of the use of a reagent should be reagent.
  • CDK9/CyclinT1 kinase was purchased from Carna
  • ADP-Glo detection kit was purchased from Promega
  • PKDTide substrate and kinase reaction buffer were purchased from Signalchem. Nivo multi-label analyzer (PerkinElmer).
  • kinase buffer in the kit to dilute the enzyme, substrate, adenosine triphosphate and inhibitor.
  • the compound to be tested was diluted 5-fold to the 8th concentration with a discharge gun, that is, diluted from 50 ⁇ M to 0.65 nM, the DMSO concentration was 5%, and the double-well experiment was set up.
  • the reaction system was placed at 25°C for 120 minutes. After the reaction, add 5 ⁇ L of ADP-Glo reagent to each well, and continue the reaction at 25°C for 40 minutes. After the reaction, add 10 ⁇ L of kinase detection reagent to each well. After reacting at 25°C for 30 minutes, use a multi-label analyzer to read chemiluminescence.
  • the integration time is 0.5. second
  • the IC 50 value can be obtained by curve fitting with four parameters (log(inhibitor) vs.response in GraphPad Prism --Variable slope mode).
  • Table 1 provides the enzymatic inhibitory activity of the compounds of the present invention on CDK9/CyclinT1.
  • the compound of the invention has good activity on CDK9 kinase.
  • the activity is similar to the reference compounds BAY1251152 and AZD4573.
  • CDK1/CyclinB1 kinase detection kit was purchased from Promega. Nivo multi-label analyzer (PerkinElmer).
  • kinase buffer in the kit to dilute the enzyme, substrate, adenosine triphosphate and inhibitor.
  • the compound to be tested was diluted 5-fold to the 8th concentration with a discharge gun, that is, diluted from 50 ⁇ M to 0.65 nM, the DMSO concentration was 5%, and the double-well experiment was set up.
  • the reaction system was placed at 25°C for 120 minutes. After the reaction, add 5 ⁇ L of ADP-Glo reagent to each well, and continue the reaction at 25°C for 40 minutes. After the reaction, add 10 ⁇ L of kinase detection reagent to each well. After reacting at 25°C for 30 minutes, use a multi-label analyzer to read chemiluminescence. second.
  • the IC 50 value can be obtained by curve fitting with four parameters (log(inhibitor) vs.response in GraphPad Prism --Variable slope mode).
  • Table 1 provides the inhibitory activity of the compounds of the present invention on CDK1/CyclinB1 enzymes.
  • the compound of the present invention does not have strong inhibitory activity on CDK1 kinase. Therefore, the compound of the present invention shows a better selectivity for CDK1 than BAY1251152 and AZD4573.
  • CDK2/CyclinE1 kinase detection kit was purchased from Promega. Nivo multi-label analyzer (PerkinElmer).
  • kinase buffer in the kit to dilute the enzyme, substrate, adenosine triphosphate and inhibitor.
  • the compound to be tested was diluted 5-fold to the 8th concentration with a discharge gun, that is, diluted from 50 ⁇ M to 0.65 nM, the DMSO concentration was 5%, and the double-well experiment was set up.
  • the reaction system was placed at 25°C for 60 minutes. After the reaction, add 5 ⁇ L of ADP-Glo reagent to each well, and continue the reaction at 25°C for 40 minutes. After the reaction, add 10 ⁇ L of kinase detection reagent to each well. After reacting at 25°C for 30 minutes, use a multi-label analyzer to read chemiluminescence. second.
  • the IC 50 value can be obtained by curve fitting with four parameters (log(inhibitor) vs.response in GraphPad Prism --Variable slope mode).
  • Table 1 provides the inhibitory activity of the compounds of the present invention on CDK2/CyclinE1 enzyme.
  • the compound of the present invention does not have strong inhibitory activity on CDK2 kinase. Therefore, the compound of the present invention shows a better selectivity for CDK2 than BAY1251152 and AZD4573.
  • IMDM medium fetal bovine serum, penicillin/streptomycin antibiotics were purchased from Promega (Madison, WI).
  • MV-4-11 cell line was purchased from the Cell Bank of the Chinese Academy of Sciences. Nivo multi-label analyzer (PerkinElmer).
  • Plant MV-4-11 cells in a white 96-well plate 80 ⁇ L of cell suspension per well, which contains 6000 MV-4-11 cells.
  • the cell plate was cultured overnight in a carbon dioxide incubator.
  • the compound to be tested was diluted 5-fold to the 8th concentration with a discharge gun, that is, from 2mM to 26nM, and a double-well experiment was set up. Add 78 ⁇ L of medium to the middle plate, and then transfer 2 ⁇ L of each well of the gradient dilution compound to the middle plate according to the corresponding position. After mixing, transfer 20 ⁇ L of each well to the cell plate. The final concentration of the compound is 10 ⁇ M to 0.13 nM. The cell plate was placed in a carbon dioxide incubator for 3 days.
  • the IC 50 value can be obtained by four-parameter curve fitting ("log(inhibitor) vs. GraphPad Prism" response--Variable slope” mode).
  • Table 1 provides the inhibitory activity of the compounds of the present invention on the proliferation of MV-4-11 cells.
  • the compound of the invention has good cell anti-proliferation activity against MV4-11.
  • CDX human-derived tumor cell line-based xenotransplantation
  • mice Female, 6-8 weeks old, weighing about 18-22 grams.
  • the mice are kept in a special pathogen-free environment and in a single ventilated cage (3 mice per cage). All cages, bedding and water are disinfected before use. All animals have free access to standard certified commercial laboratory food.
  • a total of 30 mice purchased from Shanghai Lingchang biological science and technology Co., LTD. were used for research.
  • Each mouse was implanted with tumor cells (10 ⁇ 10 6 in 0.2 ml of phosphate buffer) subcutaneously in the right flank for tumor growth. The administration was started when the average tumor volume reached approximately 110 cubic millimeters.
  • the test compound was injected weekly at a dose of 15 mg/kg.
  • the anti-tumor efficacy is determined by dividing the average tumor increase volume of animals treated with the compound by the average tumor increase volume of untreated animals.
  • the compound of the present invention exhibits good pharmacodynamics and safety.
  • CDX human-derived tumor cell line-based xenotransplantation
  • mice Female, 6-8 weeks old, weighing about 18-22 grams.
  • the mice are kept in a special pathogen-free environment and in a single ventilated cage (3 mice per cage). All cages, bedding and water are disinfected before use. All animals have free access to standard certified commercial laboratory food.
  • a total of 36 mice purchased from Shanghai Lingchang biological science and technology Co., LTD. were used for research.
  • Each mouse was implanted with tumor cells (10 ⁇ 10 6 in 0.2 ml of phosphate buffer) subcutaneously in the right flank for tumor growth. The administration was started when the average tumor volume reached approximately 121 cubic millimeters.
  • the test compound was injected weekly at a dose of 10 mg/kg.
  • the anti-tumor efficacy is determined by dividing the average tumor increase volume of animals treated with the compound by the average tumor increase volume of untreated animals.
  • the compound of the present invention exhibits good pharmacodynamics and safety.

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Abstract

一类作为CDK9抑制剂的氮杂吲哚连吡唑类化合物，具体涉及式(I)所示化合物、其药学上可接受的盐及其异构体，以及式(I)所示化合物、其药学上可接受的盐及其异构体以及包含它们的药用组合物在制备治疗癌症药物中的应用。

Description

作为CDK9抑制剂的氮杂吲哚连吡唑类化合物
本申请主张如下优先权：
申请号：CN201910566823.3，申请日：2019年06月27日。
技术领域
本发明涉及新的作为CDK9抑制剂的氮杂吲哚连吡唑类化合物，具体涉及式(I)所示化合物、其药学上可接受的盐及其异构体，以及式(I)所示化合物、其药学上可接受的盐及其异构体以及包含它们的药用组合物在制备治疗癌症药物中的应用。
背景技术
肿瘤的发生往往伴随着细胞的过度活化和持续性增殖，而CDK(细胞周期依赖性激酶)在细胞内外信号的调节下对细胞周期和转录过程发挥着重要的调控作用。在癌细胞中，CDK-cyclin(周期素)的活性往往是失调的，可能的原因包含：信号传导通路的过度激活、cyclin的过度表达、CDK的异常扩增、内源性抑制因子的失活或缺失，这些启发着人们通过不断寻找新型的CDK抑制剂来发展肿瘤治疗技术。
CDK9是CDK家族成员之一，主要参与转录调控过程，由CDK9和cyclin(T1、T2a、T2b、K)组成的异源二聚体参与组成正性转录延长因子(p-TEFb)，其中约有80％的CDK9与cyclinT1结合。P-TEFb通过使RNA聚合酶II的羧基端结构域磷酸化，主要是Ser-2磷酸化，调节转录延长。CDK9的抑制和转录阻遏导致快速消耗短寿命的mRNA转录物和相关蛋白(包括Myc和Mcl-1)，从而导致高度依赖这些抗凋亡蛋白的癌细胞死亡。因此靶向CDK9代表一种高度依赖这些抗凋亡蛋白的肿瘤类型的治疗策略。
目前，已经有CDK9小分子抑制剂进入临床研究阶段用于癌症的治疗，即拜耳的BAY1251152和阿斯利康的AZD4573。这些专利包括WO2012160034，WO2014076091，WO2009047359，WO2011110612，US2016376287。
Figure PCTCN2020098002-appb-000001
虽然在开发用于癌症和其他疾病治疗的CDK9抑制剂的道路上已经做了很多努力，但是到目前为止还没有针对该靶点的药物上市。在开展临床研究的这些药物中，BAY1251152的临床上最主要的3/4级和剂量限制性不良副作用为嗜中性白血球减少症，而AZD4573的激酶选择性和代谢不好，限制其发挥更好的药效。因此仍然迫切需要开发新颖的、更加安全有效的、能够治疗多种癌症(包括白血病和淋巴癌)的CDK9抑制剂。
发明内容
一方面，本发明提供了式(I)所示化合物、其药学上可接受的盐或其异构体，
Figure PCTCN2020098002-appb-000002
其中，T 1为N或CR；
R为H或Cl；
T 2为N或CH；
R 1为H或C 1-6烷基，其中所述C 1-6烷基任选被1、2或3个独立选自F、Cl、-OH、-NH 2和C 1-3烷氧基的取代基所取代；
R 2为H、F或Cl；
R 3和R 4各自独立地为H、F、Cl或C 1-3烷基；
R 5为H、C 3-6环烷基或苯基，其中所述C 3-6环烷基和苯基任选被1、2或3个R a所取代；
各R a独立地为H、F、Cl、C 1-3烷基或C 1-3烷氧基。
在本发明的一些方案中，上述化合物具有式(I-1)或(I-2)所示结构：
Figure PCTCN2020098002-appb-000003
其中，R、T 2、R 1、R 2、R 3、R 4和R 5如本发明所定义。
在本发明的一些方案中，上述化合物具有式(I-1-a)或(I-1-b)所示结构：
Figure PCTCN2020098002-appb-000004
其中，R、R 1、R 2、R 3、R 4和R 5如本发明所定义。
在本发明的一些方案中，上述R 3和R 4各自独立地为H、F或
Figure PCTCN2020098002-appb-000005
其他变量如本发明所定义。
在本发明的一些方案中，上述R 3和R 4各自独立地为H，其他变量如本发明所定义。
在本发明的一些方案中，上述化合物具有式(I-1-c)或(I-1-d)所示结构：
Figure PCTCN2020098002-appb-000006
其中，R、R 1、R 2和R 5如本发明所定义。
在本发明的一些方案中，上述各R a独立地为H、F、Cl或
Figure PCTCN2020098002-appb-000007
其他变量如本发明所定义。
在本发明的一些方案中，上述各R a独立地为Cl，其他变量如本发明所定义。
在本发明的一些方案中，上述R 5为H、
Figure PCTCN2020098002-appb-000008
其中所述
Figure PCTCN2020098002-appb-000009
Figure PCTCN2020098002-appb-000010
任选被1、2或3个R a所取代，R a及其他变量如本发明所定义。
在本发明的一些方案中，上述R 5为H、
Figure PCTCN2020098002-appb-000011
Figure PCTCN2020098002-appb-000012
R a及其他变量如本发明所定义。
在本发明的一些方案中，上述R 5为H、
Figure PCTCN2020098002-appb-000013
Figure PCTCN2020098002-appb-000014
其他变量如本发明所定义。
在本发明的一些方案中，上述R 5为H、
Figure PCTCN2020098002-appb-000015
Figure PCTCN2020098002-appb-000016
其他变量如本发明所定义。
在本发明的一些方案中，上述化合物具有式(I-1-e)、(I-1-f)、(I-1-g)或(I-1-h)所示结构：
Figure PCTCN2020098002-appb-000017
其中，R、R 1、R 2和R a如本发明所定义。
在本发明的一些方案中，上述R 1为H、
Figure PCTCN2020098002-appb-000018
其中所述
Figure PCTCN2020098002-appb-000019
Figure PCTCN2020098002-appb-000020
任选被1、2或3个独立选自F、Cl、-OH、-NH 2和-OCH 3的取代基所取代，其他变量如本发明所定义。
在本发明的一些方案中，上述R 1为H、
Figure PCTCN2020098002-appb-000021
其他变量如本发明所定义。
在本发明的一些方案中，上述结构单元
Figure PCTCN2020098002-appb-000022
为
Figure PCTCN2020098002-appb-000023
Figure PCTCN2020098002-appb-000024
其他变量如本发明所定义。
本发明还有一些方案是由上述变量任意组合而来。
在本发明的一些方案中，上述化合物为：
Figure PCTCN2020098002-appb-000025
Figure PCTCN2020098002-appb-000026
本发明还提供了一种药物组合物，其含有治疗有效量的上述化合物、其异构体或其药学上可接受的盐和药学上可接受的载体。
本发明还提供了上述化合物、其药学上可接受的盐或其异构体以及上述药物组合物在制备CDK9抑制剂药物中的应用。
本发明还提供了上述化合物、其药学上可接受的盐或其异构体以及上述药物组合物在制备治疗癌症药物中的应用。
技术效果
本发明化合物将母核设计为氮杂吲哚结构，与CDK9结构的铰链区形成强的双氢键作用，吡唑上的氮与CDK9的Lys48形成氢键作用。另外，本发明的化合物溶剂区的哌啶碱性很强，在CDK9中，能与Asp109形成盐桥，保持了高活性，在CDK其它亚型如CDK2中，带正电的哌啶与腔口的正电Lys89有静电斥力，故该分子对CDK2等激酶亚型的选择性较好。在小鼠体内肿瘤模型中，本发明化合物具有非常优秀的抗肿瘤活性，同时安全性也非常好。具有非常好的成药前景。
定义和说明
除非另有说明，本文所用的下列术语和短语旨在具有下列含义。一个特定的术语或短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。
这里所采用的术语“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。
术语“药学上可接受的盐”是指本发明化合物的盐，由本发明发现的具有特定取代基的化合物与相对无毒的酸或碱制备。当本发明的化合物中含有相对酸性的功能团时，可以通过在纯的溶液或合适的惰性溶剂中用足够量的碱与这类化合物接触的方式获得碱加成盐。药学上可接受的碱加成盐包括钠、钾、钙、铵、有机胺或镁盐或类似的盐。当本发明的化合物中含有相对碱性的官能团时，可以通过在纯的溶液或合适的惰性溶剂中用足够量的酸与这类化合物接触的方式获得酸加成盐。药学上可接受的酸加成盐的实例包括无机酸盐，所述无机酸包括例如盐酸、氢溴酸、硝酸、碳酸，碳酸氢根，磷酸、磷酸一氢根、磷酸二氢根、硫酸、硫酸氢根、氢碘酸、亚磷酸等；以及有机酸盐，所述有机酸包括如乙酸、丙酸、异丁酸、马来酸、丙二酸、苯甲酸、琥珀酸、辛二酸、反丁烯二酸、乳酸、扁桃酸、邻苯二甲酸、苯磺酸、对甲苯磺酸、柠檬酸、酒石酸和甲磺酸等类似的酸；还包括氨基酸(如精氨酸等)的盐，以及如葡糖醛酸等有机酸的盐。本发明的某些特定的化合物含有碱性和酸性的官能团，从而可以被转换成任一碱或酸加成盐。
本发明的药学上可接受的盐可由含有酸根或碱基的母体化合物通过常规化学方法合成。一般情况下，这样的盐的制备方法是：在水或有机溶剂或两者的混合物中，经由游离酸或碱形式的这些化合物与化学计量的适当的碱或酸反应来制备。
本发明的化合物可以存在特定的几何或立体异构体形式。本发明设想所有的这类化合物，包括顺式和反式异构体、(-)-和(+)-对映体、(R)-和(S)-对映体、非对映异构体、(D)-异构体、(L)-异构体，及其外消旋混合物和其他混合物，例如对映异构体或非对映体富集的混合物，所有这些混合物都属于本发明的范围之内。烷基等取代基中可存在另外的不对称碳原子。所有这些异构体以及它们的混合物，均包括在本发明的范围之内。
除非另有说明，术语“对映异构体”或者“旋光异构体”是指互为镜像关系的立体异构体。
除非另有说明，术语“顺反异构体”或者“几何异构体”系由因双键或者成环碳原子单键不能自由旋转而引起。
除非另有说明，术语“非对映异构体”是指分子具有两个或多个手性中心，并且分子间为非镜像的关系的立体异构体。
除非另有说明，“(+)”表示右旋，“(-)”表示左旋，“(±)”表示外消旋。
除非另有说明，用楔形实线键
Figure PCTCN2020098002-appb-000027
和楔形虚线键
Figure PCTCN2020098002-appb-000028
表示一个立体中心的绝对构型，用直形实线键
Figure PCTCN2020098002-appb-000029
和直形虚线键
Figure PCTCN2020098002-appb-000030
表示立体中心的相对构型，用波浪线
Figure PCTCN2020098002-appb-000031
表示楔形实线键
Figure PCTCN2020098002-appb-000032
或楔形虚线键
Figure PCTCN2020098002-appb-000033
或用波浪线
Figure PCTCN2020098002-appb-000034
表示直形实线键
Figure PCTCN2020098002-appb-000035
和直形虚线键
Figure PCTCN2020098002-appb-000036
本发明的化合物可以存在特定的。除非另有说明，术语“互变异构体”或“互变异构体形式”是指在室温下，不同官能团异构体处于动态平衡，并能很快的相互转化。若互变异构体是可能的(如在溶液中)，则可以达到互变异构体的化学平衡。例如，质子互变异构体(proton tautomer)(也称质子转移互变异构体(prototropic tautomer))包括通过质子迁移来进行的互相转化，如酮-烯醇异构化和亚胺-烯胺异构化。价键 异构体(valence tautomer)包括一些成键电子的重组来进行的相互转化。其中酮-烯醇互变异构化的具体实例是戊烷-2,4-二酮与4-羟基戊-3-烯-2-酮两个互变异构体之间的互变。
除非另有说明，术语“富含一种异构体”、“异构体富集”、“富含一种对映体”或者“对映体富集”指其中一种异构体或对映体的含量小于100％，并且，该异构体或对映体的含量大于等于60％，或者大于等于70％，或者大于等于80％，或者大于等于90％，或者大于等于95％，或者大于等于96％，或者大于等于97％，或者大于等于98％，或者大于等于99％，或者大于等于99.5％，或者大于等于99.6％，或者大于等于99.7％，或者大于等于99.8％，或者大于等于99.9％。
除非另有说明，术语“异构体过量”或“对映体过量”指两种异构体或两种对映体相对百分数之间的差值。例如，其中一种异构体或对映体的含量为90％，另一种异构体或对映体的含量为10％，则异构体或对映体过量(ee值)为80％。
可以通过的手性合成或手性试剂或者其他常规技术制备光学活性的(R)-和(S)-异构体以及D和L异构体。如果想得到本发明某化合物的一种对映体，可以通过不对称合成或者具有手性助剂的衍生作用来制备，其中将所得非对映体混合物分离，并且辅助基团裂开以提供纯的所需对映异构体。或者，当分子中含有碱性官能团(如氨基)或酸性官能团(如羧基)时，与适当的光学活性的酸或碱形成非对映异构体的盐，然后通过本领域所公知的常规方法进行非对映异构体拆分，然后回收得到纯的对映体。此外，对映异构体和非对映异构体的分离通常是通过使用色谱法完成的，所述色谱法采用手性固定相，并任选地与化学衍生法相结合(例如由胺生成氨基甲酸盐)。本发明的化合物可以在一个或多个构成该化合物的原子上包含非天然比例的原子同位素。例如，可用放射性同位素标记化合物，比如氚( 3H)，碘-125( 125I)或C-14( 14C)。又例如，可用重氢取代氢形成氘代药物，氘与碳构成的键比普通氢与碳构成的键更坚固，相比于未氘化药物，氘代药物有降低毒副作用、增加药物稳定性、增强疗效、延长药物生物半衰期等优势。本发明的化合物的所有同位素组成的变换，无论放射性与否，都包括在本发明的范围之内。“任选”或“任选地”指的是随后描述的事件或状况可能但不是必需出现的，并且该描述包括其中所述事件或状况发生的情况以及所述事件或状况不发生的情况。
针对药物或药理学活性剂而言，术语“有效量”或“治疗有效量”是指无毒的但能达到预期效果的药物或药剂的足够用量。对于本发明中的口服剂型，组合物中一种活性物质的“有效量”是指与该组合物中另一种活性物质联用时为了达到预期效果所需要的用量。有效量的确定因人而异，取决于受体的年龄和一般情况，也取决于具体的活性物质，个案中合适的有效量可以由本领域技术人员根据常规试验确定。
术语“被取代的”是指特定原子上的任意一个或多个氢原子被取代基取代，可以包括重氢和氢的变体，只要特定原子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(即＝O)时，意味着两个氢原子被取代。氧取代不会发生在芳香基上。术语“任选被取代的”是指可以被取代，也可以不被取代，除非另有规定，取代基的种类和数目在化学上可以实现的基础上可以是任意的。
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。因此，例如，如果一个基团被0-2个R所取代，则所述基团可以任选地至多被两个R所取代，并且每种情况下的R都有独立的选项。此外，取代基和/或其变体的组合只有在这样的组合会产生稳定的化合物的情况下才是被允许的。
当一个连接基团的数量为0时，比如-(CRR) 0-，表示该连接基团为单键。
当其中一个变量选自单键时，表示其连接的两个基团直接相连，比如A-L-Z中L代表单键时表示该结构实际上是A-Z。
当一个取代基为空缺时，表示该取代基是不存在的，比如A-X中X为空缺时表示该结构实际上是A。当所列举的取代基中没有指明其通过哪一个原子连接到被取代的基团上时，这种取代基可以通过其任何原子相键合，例如，吡啶基作为取代基可以通过吡啶环上任意一个碳原子连接到被取代的基团上。
当所列举的连接基团没有指明其连接方向，其连接方向是任意的，例如，
Figure PCTCN2020098002-appb-000037
中连接基团L为-M-W-，此时-M-W-既可以按与从左往右的读取顺序相同的方向连接环A和环B构成
Figure PCTCN2020098002-appb-000038
也可以按照与从左往右的读取顺序相反的方向连接环A和环B构成
Figure PCTCN2020098002-appb-000039
所述连接基团、取代基和/或其变体的组合只有在这样的组合会产生稳定的化合物的情况下才是被允许的。
除非另有规定，当某一基团具有一个或多个可连接位点时，该基团的任意一个或多个位点可以通过化学键与其他基团相连。所述位点与其他基团连接的化学键可以用直形实线键
Figure PCTCN2020098002-appb-000040
直形虚线键
Figure PCTCN2020098002-appb-000041
或波浪线
Figure PCTCN2020098002-appb-000042
表示。例如-OCH 3中的直形实线键表示通过该基团中的氧原子与其他基团相连；
Figure PCTCN2020098002-appb-000043
中的直形虚线键表示通过该基团中的氮原子的两端与其他基团相连；
Figure PCTCN2020098002-appb-000044
中的波浪线表示通过该苯基基团中的1和2位碳原子与其他基团相连。
除非另有规定，环上原子的数目通常被定义为环的元数，例如，“5-7元环”是指环绕排列5-7个原子的“环”。
除非另有规定，术语“C 1-6烷基”用于表示直链或支链的由1至6个碳原子组成的饱和碳氢基团。所述C 1-6烷基包括C 1-5、C 1-4、C 1-3、C 1-2、C 2-6、C 2-4、C 6和C 5烷基等；其可以是一价(如甲基)、二价(如亚甲基)或者多价(如次甲基)。C 1-6烷基的实例包括但不限于甲基(Me)、乙基(Et)、丙基(包括n-丙基和异丙基)、丁基(包括n-丁基，异丁基，s-丁基和t-丁基)、戊基(包括n-戊基，异戊基和新戊基)、己基等。
除非另有规定，术语“C 1-4烷基”用于表示直链或支链的由1至4个碳原子组成的饱和碳氢基团。所述C 1-4烷基包括C 1-2、C 1-3和C 2-3烷基等；其可以是一价(如甲基)、二价(如亚甲基)或者多价(如次甲基)。C 1-4烷基的实例包括但不限于甲基(Me)、乙基(Et)、丙基(包括n-丙基和异丙基)、丁基(包括n-丁基，异丁基，s-丁基和t-丁基)等。
除非另有规定，术语“C 1-3烷基”用于表示直链或支链的由1至3个碳原子组成的饱和碳氢基团。所述C 1-3烷基包括C 1-2和C 2-3烷基等；其可以是一价(如甲基)、二价(如亚甲基)或者多价(如次甲基)。C 1-3烷基的实例包括但不限于甲基(Me)、乙基(Et)、丙基(包括n-丙基和异丙基)等。
除非另有规定，术语“C 1-3烷氧基”表示通过一个氧原子连接到分子的其余部分的那些包含1至3个碳原子的烷基基团。所述C 1-3烷氧基包括C 1-2、C 2-3、C 3和C 2烷氧基等。C 1-3烷氧基的实例包括但不限于甲氧基、乙氧基、丙氧基(包括正丙氧基和异丙氧基)等。
除非另有规定，“C 3-6环烷基”表示由3至6个碳原子组成的饱和环状碳氢基团，其为单环体系，所述C 3-6环烷基包括C 3-5、C 3-4或C 4-5环烷基等；其可以是一价、二价或者多价。C 3-6环烷基的实例包括，但不限于，环丙基、环丁基、环戊基、环己基等。
除非另有规定，“C 3-5环烷基”表示由3至5个碳原子组成的饱和环状碳氢基团，其为单环体系，所述C 3-5环烷基包括C 3-4或C 4-5环烷基等；其可以是一价、二价或者多价。C 3-5环烷基的实例包括，但不限于，环丙基、环丁基、环戊基等。
除非另有规定，C n-n+m或C n-C n+m包括n至n+m个碳的任何一种具体情况，例如C 1-12包括C 1、C 2、C 3、C 4、C 5、C 6、C 7、C 8、C 9、C 10、C 11、和C 12，也包括n至n+m中的任何一个范围，例如C 1-12包括C 1-3、C 1-6、C 1-9、C 3-6、C 3-9、C 3-12、C 6-9、C 6-12、和C 9-12等；同理，n元至n+m元表示环上原子数为n至n+m个，例如3-12元环包括3元环、4元环、5元环、6元环、7元环、8元环、9元环、10元环、11元环、和12元环，也包括n至n+m中的任何一个范围，例如3-12元环包括3-6元环、3-9元环、5-6元环、5-7元环、6-7元环、6-8元环、和6-10元环等。
术语“离去基团”是指可以被另一种官能团或原子通过取代反应(例如亲和取代反应)所取代的官能团或原子。例如，代表性的离去基团包括三氟甲磺酸酯；氯、溴、碘；磺酸酯基，如甲磺酸酯、甲苯磺酸酯、对溴苯磺酸酯、对甲苯磺酸酯等；酰氧基，如乙酰氧基、三氟乙酰氧基等等。
术语“保护基”包括但不限于“氨基保护基”、“羟基保护基”或“巯基保护基”。术语“氨基保护基”是指适合用于阻止氨基氮位上副反应的保护基团。代表性的氨基保护基包括但不限于：甲酰基；酰基，例如链烷酰基(如乙酰基、三氯乙酰基或三氟乙酰基)；烷氧基羰基，如叔丁氧基羰基(Boc)；芳基甲氧羰基，如苄氧羰基(Cbz)和9-芴甲氧羰基(Fmoc)；芳基甲基，如苄基(Bn)、三苯甲基(Tr)、1,1-二-(4'-甲氧基苯基)甲基；甲硅烷基，如三甲基甲硅烷基(TMS)和叔丁基二甲基甲硅烷基(TBS)等等。术语“羟基保护基”是指适合用于阻止羟基副反应的保护基。代表性羟基保护基包括但不限于：烷基，如甲基、乙基和叔丁基；酰基，例如链烷酰基(如乙酰基)；芳基甲基，如苄基(Bn)，对甲氧基苄基(PMB)、9-芴基甲基(Fm)和二苯基甲基(二苯甲基，DPM)；甲硅烷基，如三甲基甲硅烷基(TMS)和叔丁基二甲基甲硅烷基(TBS)等等。
本发明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发明的实施例。
本发明所使用的溶剂可经市售获得。
本发明采用下述缩略词：DMF代表N,N-二甲基甲酰胺；Cs 2CO 3代表碳酸铯；EtOAc代表乙酸乙酯；EA代表乙酸乙酯；THF代表四氢呋喃；MeOH代表甲醇；DCM代表二氯甲烷；PE代表石油醚；EtOH代表乙醇；CuI代表碘化亚铜；NCS代表N-氯代丁二酰亚胺；NBS代表N-溴代丁二酰亚胺；ICl代表一氯化碘；Pd(dppf)Cl 2代表1,1'-双(二苯基磷)二茂铁氯化钯；Pd(PPh 3) 4代表四三苯基膦钯；ACN代表乙腈；FA 代表甲酸；NH 3·H 2O代表氨水；TEA代表三乙胺；Boc 2O代表二碳酸二叔丁酯；代表Boc代表叔丁氧羰基，是氨基的一种保护基；CDI代表N,N'-羰基二咪唑；LCMS代表液质联用色谱；HPLC代表液相色谱；TLC代表薄层层析。
化合物依据本领域常规命名原则或者使用
Figure PCTCN2020098002-appb-000045
软件命名，市售化合物采用供应商目录名称。
具体实施方式
下面通过实施例对本发明进行详细描述，但并不意味着对本发明任何不利限制。本文已经详细地描述了本发明，其中也公开了其具体实施例方式，对本领域的技术人员而言，在不脱离本发明精神和范围的情况下针对本发明具体实施方式进行各种变化和改进将是显而易见的。本发明化合物的盐酸盐或甲酸盐，加入饱和碳酸氢钠溶液调节pH到中性，经过高效液相色谱法分离(中性，碳酸氢铵体系)得到化合物的游离碱。
反应流程1 制备式(I-1-a)所示化合物，
Figure PCTCN2020098002-appb-000046
在反应流程1所示的反应中，R、R 1、R 2、R 3、R 4和R 5如本发明所定义，化合物(E)可以由化合物(C)与化合物(D)发生Sonigashira偶联反应制备，该反应需要合适的催化剂(比如四三苯基膦钯、碘化亚铜)，合适的碱(比如三乙胺)，合适的溶剂(比如甲苯、乙腈)。根据反应流程1，化合物(F)可以 由化合物(E)关环制备，该反应更偏好于在高温下进行，需要合适的碱(比如叔丁醇钾)，合适的溶剂(比如DMF)。化合物(L)可以由化合物(J)与溴代物(K)发生取代反应制备，该反应更偏好于在高温下进行，需要合适的碱(比如碳酸铯)，合适的溶剂(比如DMF)。
化合物(F)与化合物(L)在钯催化条件(比如Pd(dppf)Cl 2)下偶联得到化合物(G)。化合物(G)在酸性条件下(比如氯化氢/乙酸乙酯溶液)脱保护得到化合物(H)，化合物(H)与相应的原料发生取代反应或者还原胺化反应得到式(I-1-a)所示化合物。
实施例1
Figure PCTCN2020098002-appb-000047
第一步：
Figure PCTCN2020098002-appb-000048
在零下20℃，向化合物1-1(30.0克，173.40毫摩尔，1.0当量)的N,N-二甲基甲酰胺(300毫升)溶液中加入N-氯代丁二酰亚胺(27.79克，208.08毫摩尔，1.2当量)。混合物在25℃反应1小时。TLC(石油醚：乙酸乙酯＝3:1)显示原料反应完全。反应液倒入氢氧化钠(w％＝10％，500毫升)水溶液中，乙酸乙酯(300毫升*2)萃取，合并有机相，饱和食盐水(300毫升)洗涤，分出有机相，无水硫酸钠干燥， 过滤，滤液旋干得到残余物。残余物用硅胶柱(洗脱剂：石油醚：乙酸乙酯＝40:1到10:1)纯化得到化合物1-2。LCMS(ESI)m/z:208.9(M+1)。
第二步：
Figure PCTCN2020098002-appb-000049
在40℃，向化合物1-2(10.0克，48.20毫摩尔，1.0当量)的N,N-二甲基甲酰胺(200毫升)溶液中加入一氯化碘(11.74克，72.30毫摩尔，3.69毫升，1.5当量)，混合物在40℃反应3小时。TLC(石油醚：乙酸乙酯＝3:1)显示原料未反应完全。反应液在40℃继续反应12小时。TLC(石油醚：乙酸乙酯＝3:1)显示原料反应完全。反应液倒入水(600毫升)中，二氯甲烷(500毫升)萃取，有机相依次用亚硫酸钠(300毫升*2)、饱和食盐水(300毫升)洗涤。有机相浓缩得到残余物。残余物用硅胶柱(洗脱剂：石油醚：乙酸乙酯＝40:1到10:1)纯化得到化合物1-3。
第三步：
Figure PCTCN2020098002-appb-000050
向化合物1-3(5.0克，15.00毫摩尔，1.0当量)的三乙胺(100毫升)溶液中加入化合物1-4(4.71克，22.50毫摩尔，1.50当量)、二氯二(三苯基膦)钯(2.11克，3.00毫摩尔，0.20当量)和碘化亚酮(2.86克，15.00毫摩尔，1.0当量)，反应液用氮气置换三次后，110℃反应12小时。LCMS显示原料未反应完全。反应液冷却至30℃，硅藻土过滤，滤饼用乙酸乙酯(50毫升)洗涤。滤液减压浓缩，得到的残余物通过硅胶柱(石油醚：乙酸乙酯＝20：1到5：1)纯化，得到化合物1-5。LCMS(ESI)m/z:415.5(M+1)。
第四步：
Figure PCTCN2020098002-appb-000051
向化合物1-5(2.0克，4.82毫摩尔，1.0当量)的N,N-二甲基甲酰胺(100毫升)溶液中加入叔丁醇钾(1.62克，14.47毫摩尔，3.0当量)，反应液用氮气置换三次后，110℃反应2小时。TLC(石油醚：乙酸乙酯＝3：1)显示反应完全。反应液冷却至室温，过滤，滤饼用乙酸乙酯(50毫升)洗涤。滤液减压浓缩，得到的残余物通过硅胶柱层析(石油醚：乙酸乙酯＝10：1到3：1)纯化，得到化合物1-6。LCMS(ESI)m/z:415.8(M+1)。
第五步：
Figure PCTCN2020098002-appb-000052
向化合物1-9(2.0克，10.31毫摩尔，1.0当量)的N,N-二甲基甲酰胺(100毫升)溶液中加入溴甲基环丙烷(1-10，1.67克，12.37毫摩尔，1.18毫升，1.2当量)和碳酸铯(10.07克，30.92毫摩尔，3.0当量)。混合物在80℃反应5小时，TLC(石油醚：乙酸乙酯＝3:1)显示原料反应完全。反应液冷却至20℃，倒入水(60毫升)中，用乙酸乙酯(60毫升)萃取，有机相用饱和食盐水(60毫升)洗涤一次。分出有机相，浓缩得到残余物。残余物用硅胶柱(石油醚：乙酸乙酯＝10：1到3：1)纯化，得到化合物1-7。LCMS(ESI)m/z:249.2(M+1)。
第六步：
Figure PCTCN2020098002-appb-000053
向化合物1-6(200毫克，482.25微摩尔，1.0当量)的1,4-二氧六环(20毫升)和水(5毫升)溶液中加入化合物1-7(358.98毫克，1.45毫摩尔，3.0当量)、Pd(dppf)Cl 2(7.06毫克，9.64微摩尔，0.2当量)和碳酸铯(471.38毫克，1.45毫摩尔，3.0当量)。反应液用氮气置换三次后，100℃反应12小时。LCMS显示原料反应完全，反应液冷却至20℃，减压浓缩得到残余物。残余物通过薄层析板(石油醚：乙酸乙酯＝1:1)纯化得到化合物1-8。LCMS(ESI)m/z:456.2(M+1)。
第七步：
Figure PCTCN2020098002-appb-000054
向化合物1-8(120毫克，263.17微摩尔，1.0当量)的乙酸乙酯(5毫升)溶液中加入氯化氢/乙酸乙酯(4摩尔/升，5毫升，76.0当量)。混合物在20℃反应12小时。LCMS显示原料反应完全，反应液浓缩得到残余物。残余物通过制备HPLC(色谱柱:Phenomenex Synergi C18 150*25mm*10μm，盐酸，流动相：水(0.05％盐酸)-乙腈，梯度：乙腈13％-33％)纯化得到化合物1的盐酸盐。 1H NMR(400MHz,DMSO-d 6)δ12.03(s,1H),9.33(br d,J＝10.0Hz,1H),9.11(br d,J＝9.8Hz,1H),8.38(s,1H),8.20(s,1H),7.99(s,1H),6.35(d,J＝1.3Hz,1H),4.10(d,J＝7.2Hz,2H),3.33(br d,J＝12.5Hz,2H),3.13-2.95(m,3H),2.24(br d,J＝12.5Hz,2H),1.97-1.85(m,2H),1.38-1.26(m,1H),0.59-0.52(m,2H),0.45-0.39(m,2H)；LCMS(ESI)m/z:356.2(M+1)。
实施例2
Figure PCTCN2020098002-appb-000055
在20℃下，向化合物1(70毫克，152.46微摩尔，1.0当量)的甲醇(2.0毫升)溶液中加入37％的甲醛(218毫克，2.69毫摩尔，0.2毫升，17.85当量)溶液和氰基硼氢化钠(94.55毫克，1.50毫摩尔，10.0当量)，混合物在20℃下搅拌1小时。LCMS显示原料反应完全。将反应混合物的pH用1摩尔/升的盐酸溶液调至7，减压浓缩，得到残余物用制备HPLC(色谱柱：Phenomenex Synergi C18 150*25mm*10μm；甲酸，流动相：[水(0.225％甲酸)-乙腈]，梯度：乙腈22％-52％)纯化得到化合物2的甲酸盐。 1H NMR(400MHz,DMSO-d 6)δ11.82(br s,1H),8.35(s,1H),8.28(br s,1H),8.16(s,1H),7.98(s,1H),6.33(s,1H),4.09(d,J＝7.09Hz,2H),3.04(br d,J＝10.88Hz,2H),2.78(br t,J＝11.00Hz,1H),2.39-2.28(m,5H),2.05(br d,J＝11.98Hz,2H),1.82(q,J＝11.41Hz,2H),1.37-1.28(m,1H),0.60-0.53(m,2H),0.46-0.40(m,2H)；LCMS(ESI)m/z:370.0(M+1)。
实施例3
Figure PCTCN2020098002-appb-000056
向化合物1(150毫克，228.30微摩尔，1.0当量)的甲醇(5毫升)溶液中加入乙醛(125.71毫克，1.14毫摩尔，160.14微升，5.0当量)和氰基硼氢化钠(71.73毫克，1.14毫摩尔，5.0当量)。混合物在15℃反应2小时。LCMS显示原料反应完全，反应液浓缩得到残余物。向残余物中加入水(10毫升)和乙酸乙酯(20毫升)，分离有机相，浓缩得到残余物。残余物通过制备HPLC(色谱柱：Phenomenex Synergi C18150*25mm*10μm；盐酸，流动相：[水(0.05％盐酸)-乙腈]，梯度：乙腈10％-40％)纯化，得到化合物3的盐酸盐。 1H NMR(400MHz,DMSO-d 6)δ12.13-11.86(m,1H),10.68(br s,1H),8.44-8.34(m,1H),8.20(s,1H),8.06-7.92(m,1H),6.53-6.29(m,1H),4.10(d,J＝7.2Hz,2H),3.55(br d,J＝11.4Hz,2H),3.21-2.94(m,5H),2.38-2.25(m,2H),2.18-1.94(m,2H),1.40-1.20(m,4H),0.63-0.51(m,2H),0.48-0.37(m,2H)；LCMS(ESI)m/z:384.2(M+1)。
实施例4
Figure PCTCN2020098002-appb-000057
在20℃下，向化合物1(100毫克，152.20微摩尔，1.0当量)的甲醇(2毫升)溶液中加入丙酮(2.63 克，45.34毫摩尔，3.33毫升，297.90当量)和氰基硼氢化钠(95.64毫克，1.52毫摩尔，10.0当量)，混合物在20℃下搅拌2小时。LCMS显示原料剩余。继续加入丙酮(2.63克，45.34毫摩尔，3.33毫升，297.90当量)和氰基硼氢化钠(95.64毫克，1.52毫摩尔，10当量)，混合物在20℃下搅拌12小时。LCMS显示原料反应完全。反应混合物减压浓缩，溶解在水中(10毫升)，用乙酸乙酯(20毫升*2)萃取，有机相用无水硫酸钠干燥，减压浓缩，得到粗品。粗品用制备HPLC(色谱柱：Phenomenex Synergi C18 150*25mm*10μm；盐酸，流动相：水(0.05％盐酸)-乙腈，梯度：乙腈11％-41％)纯化得到化合物4的盐酸盐。 1H NMR(400MHz,DMSO-d 6)δ12.08-11.92(m,1H),10.54(br s,1H),8.42-8.34(m,1H),8.21(s,1H),7.98(s,1H),6.33(d,J＝1.10Hz,1H),4.10(d,J＝7.21Hz,2H),3.45(br d,J＝11.13Hz,3H),3.16-3.03(m,3H),2.34(br d,J＝11.13Hz,2H),2.21-2.02(m,2H),1.36-1.23(m,7H),0.62-0.53(m,2H),0.62-0.53(m,2H)；LCMS(ESI)m/z:398.2(M+1)。
实施例5
Figure PCTCN2020098002-appb-000058
向化合物1(200毫克，304.40微摩尔，1.0当量)的N,N-二甲基甲酰胺(10毫升)溶液中加入1-溴-2-甲氧基乙烷(211.54毫克，1.52毫摩尔，142.93微升，5.0当量)和三乙胺(154.01毫克，1.52毫摩尔，211.84微升，5.0当量)。混合物在15℃反应12小时。LCMS显示原料反应完全，反应液浓缩得到残余物。残余物通过制备HPLC(色谱柱：Phenomenex Synergi C18 150*25mm*10μm；盐酸，流动相：水(0.05％盐酸)-乙腈，梯度：乙腈10％-40％)纯化，得到化合物5的盐酸盐。 1H NMR(400MHz,DMSO-d 6)δ12.31-12.08(m,1H),10.95(br s,1H),8.48-8.35(m,1H),8.23(s,1H),8.07-7.96(m,1H),6.66-6.26(m,1H),4.10(d,J＝7.2Hz,2H),3.85-3.68(m,2H),3.63-3.38(m,2H),3.33-3.23(m,5H),3.22-2.98(m,3H),2.30(br d,J＝13.8Hz,2H),2.21-1.98(m,2H),1.41-1.22(m,1H),0.59-0.50(m,2H),0.46-0.37(m,2H)。LCMS(ESI)m/z:414.2(M+1)。
实施例6
Figure PCTCN2020098002-appb-000059
在20℃下，向化合物1(200毫克，304.40微摩尔，1.0当量)的甲醇(10毫升)溶液中加入甲氧基丙酮(268.19毫克，3.04毫摩尔，282.30微升，10当量)和氰基硼氢化钠(191.29毫克，3.04毫摩尔，10当量)，混合物在45℃下搅拌4小时。LCMS显示原料剩余。混合物在45℃下继续搅拌16小时。LCMS显示原料反应完全。用1摩尔/升的盐酸溶液将反应液的pH调到7，减压浓缩，得到残余物用制备HPLC(色谱柱：Phenomenex Synergi C18 150*25mm*10μm；甲酸，[洗脱剂：水(0.225％甲酸)-乙腈]，梯度：已腈:25％-55％)纯化得到化合物6的甲酸盐。 1H NMR(400MHz,DMSO-d 6)δ11.79(s,1H),8.35(s,1H),8.23 (s,1H),8.15(s,1H),7.98(s,1H),6.32(d,J＝1.22Hz,1H),4.09(d,J＝7.21Hz,2H),3.51-3.44(m,1H),3.33(dd,J＝9.66,5.14Hz,1H),3.29-3.22(m,3H),3.06-2.92(m,3H),2.82-2.71(m,1H),2.64-2.54(m,2H),2.04(br d,J＝11.98Hz,2H),1.85-1.70(m,1H),1.39-1.28(m,1H),1.04(d,J＝6.60Hz,3H),0.59-0.51(m,2H),0.45-0.38(m,2H)；LCMS(ESI)m/z:428.1(M+1)。
实施例7
Figure PCTCN2020098002-appb-000060
第一步：
Figure PCTCN2020098002-appb-000061
在25℃下，向化合物1-8(300毫克，657.92微摩尔，1.0当量)的N,N-二甲基甲酰胺(10毫升)溶液中加入N-氯代丁二酰亚胺(92.25毫克，690.82微摩尔，282.30微升，1.05当量)，混合物在25℃下搅拌2小时。LCMS显示原料剩余，混合物在25℃下继续搅拌1小时。LCMS显示原料反应完全。反应液倒入水(30毫升)中，过滤，滤饼用水(30毫升)洗涤，用乙酸乙酯(30毫升)溶解，有机相用盐水(20毫升)洗涤，减压浓缩，得到粗品。粗品用TLC(石油醚：乙酸乙酯＝2:1)板纯化，得到化合物7-1。LCMS(ESI)m/z:490.1(M+1)。
第二步：
Figure PCTCN2020098002-appb-000062
向化合物7-1(90毫克，183.51微摩尔，1.0当量)的乙酸乙酯(5毫升)溶液中加入氯化氢/乙酸乙酯溶液(4.0摩尔/升，5毫升，108.98当量)，混合物在15℃下反应1小时。LCMS显示原料反应完全。反应 液减压浓缩，用水(10毫升)溶解，水相pH用饱和的碳酸钠调到9，用乙酸乙酯(20毫升*2)萃取，有机相用盐水(20毫升)洗涤，减压浓缩，得到的残余物用制备HPLC(色谱柱：Phenomenex Synergi C18 150*25mm*10μm；甲酸，洗脱剂：[水(0.225％甲酸)-乙腈]，梯度：乙腈9％-39％)纯化得到化合物7。 1H NMR(400MHz,DMSO-d 6)δ8.39(s,1H),8.29(s,1H),7.99(s,1H),7.60(s,1H),4.07(d,J＝7.0Hz,2H),3.29-3.15(m,2H),3.31-3.14(m,1H),3.31-3.14(m,1H),2.94-2.84(m,2H),2.14-2.03(m,2H),1.82(br d,J＝12.9Hz,2H),1.34-1.25(m,1H),0.57-0.51(m,2H),0.42-0.37(m,2H)；LCMS(ESI)m/z:390.0(M+1)。
实施例8
Figure PCTCN2020098002-appb-000063
第一步：
Figure PCTCN2020098002-appb-000064
在25℃下，向1-9(200毫克，1.03毫摩尔，1.0当量)和8-1(201.68毫克，1.24毫摩尔，1.2当量)的N,N-二甲基甲酰胺(2毫升)溶液中加入碳酸铯(1.01克，3.09毫摩尔，3当量)，混合物在100℃下搅拌12小时。LCMS显示原料反应完全。反应液倒入水(10毫升)中，用乙酸乙酯(20*2毫升)萃取，有机相减压浓缩，得到的残余物通过硅胶柱(石油醚：乙酸乙酯＝1：1)纯化，得到化合物8-2。LCMS(ESI)m/z:277.1(M+1)。
第二步：
Figure PCTCN2020098002-appb-000065
在25℃，氮气保护下，向1-6(100毫克，241.12微摩尔，1.0当量)和8-2(98.89毫克，361.69微摩尔，1.5当量)的二氧六环(2毫升)和水(0.5毫升)混合液中加入Pd(dppf)Cl 2(17.64毫克，24.11微摩尔，0.1当量)和碳酸铯(235.69毫克，723.37微摩尔，3当量)，混合物在100℃下搅拌12小时。LCMS 显示原料反应完全。反应液用硅藻土过滤，滤饼用乙酸乙酯(30毫升)洗涤，滤液减压浓缩，得到的残余物用TLC板(石油醚：乙酸乙酯＝1：1)纯化，得到化合物8-3。LCMS(ESI)m/z:484.3(M+1)。
第三步：
Figure PCTCN2020098002-appb-000066
向化合物8-3(80毫克，165.28微摩尔，1.0当量)的乙酸乙酯(5毫升)溶液中加入氯化氢/乙酸乙酯(4.0摩尔/升，5毫升，121.02当量)，混合物在15℃下反应1小时。LCMS显示原料剩余，混合物在15℃下继续反应1小时，LCMS显示原料反应完全。反应液减压浓缩，用水(10毫升)溶解，水相pH用饱和的碳酸氢钠调到8，用乙酸乙酯(20毫升*2)萃取，有机相减压浓缩，得到的残余物用制备HPLC(色谱柱：Boston Green ODS 150*30mm*5μm；甲酸，流动相：[水(0.225％甲酸)-乙腈]，梯度：乙腈15％-45％)纯化得到化合物8的甲酸盐。 1H NMR(400MHz,DMSO-d 6)δ11.94(br s,1H),8.44(s,1H),8.46-8.39(m,1H),8.34(s,1H),8.17(s,1H),7.97(s,1H),6.31(s,1H),4.15(s,2H),3.26(br d,J＝11.1Hz,2H),3.06-2.82(m,3H),2.46-2.37(m,1H),2.13(br d,J＝12.6Hz,2H),1.83(q,J＝11.3Hz,2H),1.69-1.47(m,6H),1.37-1.22(m,2H)；LCMS(ESI)m/z:384.2(M+1)。
实施例9
Figure PCTCN2020098002-appb-000067
第一步：
Figure PCTCN2020098002-appb-000068
向化合物1-9(1.0克，5.15毫摩尔，1.0当量)的N,N-二甲基甲酰胺(30毫升)溶液中加入化合物9-1(1.06克，6.18毫摩尔，734.02微升，1.2当量)和碳酸铯(2.52克，7.73毫摩尔，1.5当量)。混合物在100℃反应12小时。LCMS显示原料反应完全。反应液冷却至15℃后，倒入水(100毫升)中，混合物用乙酸乙酯(50毫升*2)萃取，合并有机相，减压浓缩得到残余物。残余物用硅胶柱层析纯化(洗脱剂：石油醚：乙酸乙酯＝1:0到5:1)得到化合物9-2。LCMS(ESI)m/z:284.9(M+1)。
第二步：
Figure PCTCN2020098002-appb-000069
向化合物1-6(300毫克，723.37微摩尔，1.0当量)的1,4-二氧六环(20毫升)和水(10毫升)溶液中加入化合物9-2(308.33毫克，1.09毫摩尔，1.5当量)、Pd(dppf)Cl 2(105.86毫克，144.67微摩尔，0.2当量)和碳酸铯(471.38毫克，1.45毫摩尔，2.0当量)。混合物氮气置换三次后，100℃反应2小时。LCMS显示原料反应完全。反应液冷却至15℃后，反应液浓缩得到残余物，残余物通过硅胶板(石油醚：乙酸乙酯＝1:1)纯化，得到化合物9-3。LCMS(ESI)m/z:492.2(M+1)。
第三步：
Figure PCTCN2020098002-appb-000070
向化合物9-3(170毫克，320.23微摩尔，1.0当量)的乙酸乙酯(10毫升)溶液中加入氯化氢/乙酸乙酯溶液(4摩尔/升，10.0毫升，124.91当量)。混合物在15℃继续反应2小时，LCMS显示原料反应完全。反应液浓缩得到残余物，残余物溶于水(20毫升)中，用碳酸氢钠(w％＝10％)水溶液调节PH～8，混合物用二氯甲烷/甲醇＝10:1(50毫升)萃取，有机相减压浓缩得到残余物。残余物用制备HPLC(色谱柱：Phenomenex Synergi C18 150*25mm*10μm；甲酸，流动相：[水(0.225％甲酸)-乙腈]，梯度：乙腈11％-41％)纯化，得到化合物9的甲酸盐。 1H NMR(400MHz,DMSO-d 6)δ11.91(br s,1H),8.49(s,1H),8.42(s,1H),8.17(s,1H),8.01(s,1H),7.44-7.26(m,5H),6.31(s,1H),5.46(s,2H),3.24(br d,J＝12.3Hz,2H),3.05-2.80(m,3H), 2.12(br d,J＝12.2Hz,2H),1.91-1.72(m,2H)；LCMS(ESI)m/z:392.1(M+1)。
实施例10
Figure PCTCN2020098002-appb-000071
化合物10的甲酸盐的合成参考化合物9的甲酸盐。 1H NMR(400MHz,DMSO-d 6)δ11.97(br s,1H),9.14(br s,1H),8.88(br d,J＝9.2Hz,1H),8.50(s,1H),8.20(s,1H),8.05(s,1H),7.52(br d,J＝5.1Hz,1H),7.37(br d,J＝4.8Hz,2H),7.14(br d,J＝4.6Hz,1H),6.33(br s,1H),5.58(br s,2H),3.34(br d,J＝11.4Hz,2H),3.16-2.92(m,3H),2.24(br d,J＝13.4Hz,2H),1.87(q,J＝12.5Hz,2H)；LCMS(ESI)m/z:426.1(M+1)。
实施例11
Figure PCTCN2020098002-appb-000072
化合物11的甲酸盐的合成参考化合物9的甲酸盐。 1H NMR(400MHz,DMSO-d 6)δ11.98(br s,1H),9.19(br d,J＝8.4Hz,1H),8.93(br d,J＝8.8Hz,1H),8.56(s,1H),8.20(s,1H),8.04(s,1H),7.40(br s,3H),7.30(br d,J＝6.9Hz,1H),6.34(br s,1H),5.49(br s,2H),3.33(br d,J＝11.7Hz,2H),3.18-2.91(m,3H),2.24(br d,J＝13.4Hz,2H),1.88(q,J＝12.3Hz,2H)；LCMS(ESI)m/z:426.1(M+1)。
实施例12
Figure PCTCN2020098002-appb-000073
化合物12的合成参考化合物9。 1H NMR(400MHz,CD 3OD)δ8.57-8.48(m,1H),8.45-8.35(m,1H),8.19-8.10(m,1H),7.44-7.38(m,2H),7.37-7.30(m,2H),6.65-6.56(m,1H),5.50(s,2H),3.54(br d,J＝13.0Hz,2H),3.27-3.11(m,3H),2.36(br d,J＝12.5Hz,2H),2.09-1.93(m,2H)；LCMS(ESI)m/z:426.1(M+1)。
实施例13
Figure PCTCN2020098002-appb-000074
Figure PCTCN2020098002-appb-000075
第一步：
Figure PCTCN2020098002-appb-000076
在-50℃，氮气保护下，向二异丙胺(926.22毫克，9.15毫摩尔，1.29毫升，2..5当量)的四氢呋喃(5毫升)溶液中加入正丁基锂(2.5摩尔/升，3.66毫升，2.5当量)溶液，混合物在0℃下搅拌0.5小时。在-78℃，氮气保护下，将混合液滴加到13-1(1克，3.65毫摩尔，1.0当量)的四氢呋喃(5毫升)中，混合物在-78℃搅拌1小时。然后将溶解在四氢呋喃(2毫升)中的碘(1.02克，4.03毫摩尔，1.1当量)滴加到反应混合物中，半小时内升到15℃。TLC(石油醚：乙酸乙酯＝5：1)显示原料剩余。反应混合物倒入饱和氯化铵(30毫升)溶液中，用乙酸乙酯(30*2毫升)萃取，有机相用盐水(30毫升)洗涤，然后减压浓缩，得到的残余物通过硅胶柱(石油醚：乙酸乙酯＝5：1)纯化，得到化合物13-2。
第二步：
Figure PCTCN2020098002-appb-000077
向化合物13-2(400毫克，1.00毫摩尔，1.0当量)的乙酸乙酯(10毫升)溶液中加入氯化氢/乙酸乙酯(4.0摩尔/升，10毫升，39.90当量)，混合物在15℃下反应0.5小时。TLC(石油醚：乙酸乙酯＝5：1)板显示原料剩余，LCMS显示产物生成。混合物在15℃下继续反应0.5小时，TLC(石油醚：乙酸乙酯＝5：1)显示原料反应完全。反应混合物减压浓缩，得到粗品13-3直接用于下一步。LCMS(ESI)m/z:300.8(M+1)。
第三步：
Figure PCTCN2020098002-appb-000078
在氮气保护下，向13-3(0.37克，1.10毫摩尔，1.0当量)和1-4(230.90毫克，1.10毫摩尔，1.0当量)的三乙胺(10毫升)溶液中加入二(三苯基膦)二氯化钯(154.88毫克，220.65微摩尔，0.2当量)和 碘化亚铜(230.90毫克，1.10毫摩尔，1.0当量)，混合物在100℃下搅拌12小时。LCMS显示原料反应完全。反应液用硅藻土过滤，滤饼用乙酸乙酯(30毫升)洗涤，滤液减压浓缩，得到的残余物通过硅胶柱(石油醚：乙酸乙酯＝1：1)纯化，得到化合物13-4。LCMS(ESI)m/z:382.1(M+1)。
第四步：
Figure PCTCN2020098002-appb-000079
在15℃下，向13-4(150毫克，394.45微摩尔，1.0当量)的N,N-二甲基甲酰胺(5毫升)溶液中加入叔丁醇钾(132.79毫克，1.18毫摩尔，3当量)，混合物在100℃下搅拌12小时。TLC(石油醚：乙酸乙酯＝1：1)显示原料反应完全。反应液倒入水(20毫升)中，用乙酸乙酯(20毫升*2)萃取，有机相减压浓缩，得到粗品13-5直接用于下一步。
第五步：
Figure PCTCN2020098002-appb-000080
在25℃，氮气保护下，向13-5(130毫克，341.85微摩尔，1.0当量)和1-7(254.47毫克，1.03毫摩尔，3.0当量)的二氧六环(8毫升)和水(2毫升)混合液中加入Pd(dppf)Cl 2(50.03毫克，68.37微摩尔，0.2当量)和碳酸铯(334.05毫克，1.03毫摩尔，3当量)，混合物在100℃下搅拌12小时。TLC(石油醚：乙酸乙酯＝1：1)显示原料反应完全。反应液用硅藻土过滤，滤饼用乙酸乙酯(30毫升)洗涤，滤液减压浓缩，得到的残余物用硅胶柱(石油醚：乙酸乙酯＝1：1)纯化，得到化合物13-6。LCMS(ESI)m/z:422.2(M+1)。
第六步：
Figure PCTCN2020098002-appb-000081
向化合物13-6(150毫克，313.36微摩尔，1.0当量)的乙酸乙酯(5毫升)溶液中加入氯化氢/乙酸乙酯(4.0摩尔/升，5毫升，63.83当量)，混合物在15℃下反应15分钟。LCMS显示原料反应完全。反应混合物减压浓缩，残余物用水(15毫升)溶解，水相pH用饱和的碳酸钠溶液调到9，乙酸乙酯(20毫升*2)萃取，有机相减压浓缩，得到的残余物用制备HPLC(色谱柱：Shim-pack C18 150*25mm*10μm；甲酸，流动相：[水(0.225％甲酸)-乙腈]，梯度：乙腈0％-26％)纯化得到化合物13的甲酸盐。 1H NMR(400MHz,DMSO-d 6)δ11.64(br s,1H),8.49-8.45(m,1H),8.47(s,1H),8.35(br s,1H),8.11-8.06(m,2H),7.22(d,J＝5.0Hz,1H),6.51(s,1H),4.06(d,J＝7.2Hz,2H),3.32(br d,J＝12.4Hz,2H),3.08-2.90(m,1H),3.08-2.90(m,3H), 2.21(br d,J＝12.4Hz,2H),1.95-1.79(m,2H),1.38-1.24(m,1H),0.60-0.52(m,2H),0.45-0.35(m,2H)；LCMS(ESI)m/z:322.1(M+1)。
实施例14
Figure PCTCN2020098002-appb-000082
第一步：
Figure PCTCN2020098002-appb-000083
在0℃下，向化合物1-2(5.0克，24.10毫摩尔，1.0当量)的浓硫酸(60毫升)溶液中分批加入硝酸钾(3.66克，36.15毫摩尔，1.5当量)。混合物在15℃反应12小时，TLC(石油醚：乙酸乙酯＝2:1)显示原料反应完全。反应液缓慢倒入冰水(80毫升)中，混合物过滤，滤饼减压干燥得到化合物14-1。
第二步：
Figure PCTCN2020098002-appb-000084
向化合物14-1(1.0克，3.96毫摩尔，1.0当量)的乙醇(40毫升)和水(20毫升)溶液中加入氯化铵(1.06克，19.81毫摩尔，5.0当量)和铁粉(66.63毫克，11.88毫摩尔，3.0当量)。混合物在80℃反应2小时，LCMS显示原料反应完全。反应液冷却至15℃，过滤，滤饼用乙醇(50毫升)洗涤。滤液减压浓缩得到化合物14-2。LCMS(ESI)m/z:224.0(M+1)。
第三步：
Figure PCTCN2020098002-appb-000085
向化合物14-2(400毫克，1.80毫摩尔，1.0当量)的N,N-二甲基甲酰胺(20毫升)溶液中加入化合物14-3(618.34毫克，2.70毫摩尔，1.5当量)、羰基二咪唑(437.31毫克，2.70毫摩尔，1.5当量)和吡啶(426.66毫克，5.39毫摩尔，435.37微升，3.0当量)。混合物在氮气保护下，100℃反应2小时，LCMS显示原料未反应完全。向反应液中加入碳酸钾(585.82毫克，1.80毫摩尔，1.0当量)，混合物在氮气保护下，100℃继续反应10小时，LCMS显示原料反应完全。反应液倒入水(50毫升)中，乙酸乙酯(50毫升*2)萃取，合并有机相，饱和食盐水(50毫升)洗涤一次后，分出有机相，降压浓缩得到残余物。残余物用硅胶板分离(石油醚：乙酸乙酯＝1:1)，得到化合物14-4。LCMS(ESI)m/z:417.0(M+1)。
第四步：
Figure PCTCN2020098002-appb-000086
向化合物14-4(180毫克，383.37微摩尔，1.0当量)的1,4-二氧六环(10毫升)和水(5毫升)溶液中加入化合物1-7(190.25毫克，766.74微摩尔，2.0当量)、Pd(dppf)Cl 2(62.62毫克，76.67微摩尔，0.2当量)和碳酸铯(374.73毫克，1.15毫摩尔，3.0当量)。混合物用氮气置换三次后，110℃继续反应2小时。LCMS显示原料反应完全。反应液冷却至15℃，减压浓缩得到残余物。残余物用乙酸乙酯(50毫升*2)萃取，合并有机相，饱和食盐水(100毫升)洗涤。分出有机相，降压浓缩，得到残余物。残余物硅胶板分离(石油醚：乙酸乙酯＝1:1)，得到化合物14-5。LCMS(ESI)m/z:457.2(M+1)。
第五步：
Figure PCTCN2020098002-appb-000087
向化合物14-5(100毫克，212.27微摩尔，1.0当量)的乙酸乙酯(5毫升)溶液中加入氯化氢/乙酸乙酯溶液(4摩尔/升，5.0毫升，94.22当量)。混合物在15℃继续反应0.5小时，LCMS显示原料反应完全。反应液浓缩得到残余物，残余物溶于水(20毫升)中，用碳酸氢钠(w％＝10％)水溶液调节PH～8，混合物用乙酸乙酯(20毫升*2)萃取，合并有机相，减压浓缩得到残余物。残余物用制备HPLC(色谱柱：Boston Green ODS 150*30mm*5μm；甲酸，流动相：水(0.225％甲酸)-乙腈，梯度：乙腈10％-40％)纯化，得到化合物14。 1H NMR(400MHz,CD 3OD)δ8.66(s,1H),8.51(s,1H),8.45(s,1H),8.30(s,1H),4.13(d,J＝7.2Hz,2H),3.56(td,J＝3.7,12.9Hz,2H),3.40-3.32(m,1H),3.26-3.15(m,2H),2.42-2.31(m,2H),2.28-2.14(m,2H), 1.45-1.32(m,1H),0.71-0.64(m,2H),0.51-0.44(m,2H)；LCMS(ESI)m/z:357.2(M+1)。
实施例15
Figure PCTCN2020098002-appb-000088
第一步：
Figure PCTCN2020098002-appb-000089
向化合物1-6(0.5克，1.21毫摩尔，1当量)和化合物15-1(376.27毫克，1.81毫摩尔，1.5当量)的二氧六环(20毫升)和水(5毫升)溶液中加入Pd(dppf)Cl 2·CH 2Cl 2(196.91毫克，241.12微摩尔，0.2当量)、碳酸铯(1.18克，3.62毫摩尔，3当量)。混合物在100℃反应12小时。TLC(石油醚：乙酸乙酯＝1:1)显示原料反应完全，反应液浓缩得到残余物。残余物经柱层析(二氧化硅，洗脱剂：石油醚：乙酸乙酯＝1:1)纯化得到化合物15-2。LCMS(ESI)m/z:416.2(M+1)。
第二步：
Figure PCTCN2020098002-appb-000090
向化合物15-2(0.4克，961.73毫摩尔，1当量)的乙酸乙酯(10毫升)溶液中加入氯化氢/乙酸乙酯(4摩尔/升，10毫升，41.59当量)。反应液在15℃反应15分钟，LCMS显示原料反应完全。反应液浓缩得到残余物，残余物通过制备HPLC[色谱柱：Phenomenex luna C18 150*40mm*15μm；水(0.05％盐酸)-乙腈：7％-37％，10分钟]纯化得到化合物15的盐酸盐。 1H NMR(400MHz,DMSO-d 6)δ11.96(s,1H),9.21(br d,J＝8.8Hz,1H),8.97(br d,J＝10.2Hz,1H),8.31(s,1H),8.19(s,1H),7.96(s,1H),6.34(d,J＝1.4Hz,1H),3.96(s,3H),3.33(br d,J＝12.4Hz,2H),3.13-2.94(m,3H),2.24(br d,J＝12.2Hz,2H),1.98-1.81(m,2H)；LCMS(ESI)m/z:316.2(M+1)。
体外活性测试
本发明涉及的化合物是CDK9抑制剂。以下实验结果证实本专利列举的化合物确实是CDK9抑制剂并且可作为潜在的抗癌药。文中用到的IC 50是指使用某种试剂产生50％最大抑制时对应该试剂的浓度。
实验例一：体外CDK9/CyclinT1酶活性测试
实验材料：
CDK9/CyclinT1激酶购自Carna，ADP-Glo检测试剂盒购自Promega，PKDTide底物及激酶反应缓冲液购自Signalchem。Nivo多标记分析仪(PerkinElmer)。
实验方法：
使用试剂盒里的激酶缓冲液稀释酶，底物，三磷酸腺苷和抑制剂。
将待测化合物用排枪进行5倍稀释至第8个浓度，即从50μM稀释至0.65nM，DMSO浓度为5％，设置双复孔实验。向微孔板中加入1μL抑制剂各浓度梯度，2μL CDK9/CyclinT1酶(4ng)，2μL底物和ATP的混合物(100μM三磷酸腺苷，0.2μg/μL底物)，此时化合物终浓度梯度为10μM稀释至0.13nM。反应体系置于25℃反应120分钟。反应结束后，每孔加入5μL ADP-Glo试剂，25℃继续反应40分钟，结束反应后每孔加入10μL的激酶检测试剂，25℃反应30分钟后采用多标记分析仪读数化学发光，积分时间0.5秒。
数据分析：
利用方程式(Sample-Min)/(Max-Min)*100％将原始数据换算成抑制率，IC 50的值即可通过四参数进行曲线拟合得出(GraphPad Prism中log(inhibitor)vs.response--Variable slope模式得出)。表1提供了本发明的化合物对CDK9/CyclinT1酶学抑制活性。
实验结论：
本发明化合物对CDK9激酶具有良好的活性。和参照化合物BAY1251152和AZD4573活性类似。
实验例二：体外CDK1/CyclinB1酶活性测试
实验材料：
CDK1/CyclinB1激酶检测试剂盒购自Promega。Nivo多标记分析仪(PerkinElmer)。
实验方法：
使用试剂盒里的激酶缓冲液稀释酶，底物，三磷酸腺苷和抑制剂。
将待测化合物用排枪进行5倍稀释至第8个浓度，即从50μM稀释至0.65nM，DMSO浓度为5％，设置双复孔实验。向微孔板中加入1μL抑制剂各浓度梯度，2μL CDK1/CyclinB1酶(12.5ng)，2μL底物和ATP的混合物(25μM三磷酸腺苷，0.2μg/μL底物)，此时化合物终浓度梯度为10μM稀释至0.13nM。反应体系置于25℃反应120分钟。反应结束后，每孔加入5μL ADP-Glo试剂，25℃继续反应40分钟，结束反应后每孔加入10μL的激酶检测试剂，25℃反应30分钟后采用多标记分析仪读数化学发光，积分时间0.5秒。
数据分析：
利用方程式(Sample-Min)/(Max-Min)*100％将原始数据换算成抑制率，IC 50的值即可通过四参数进行曲线拟合得出(GraphPad Prism中log(inhibitor)vs.response--Variable slope模式得出)。表1提供了本发明的化合物对CDK1/CyclinB1酶学抑制活性。
实验结论：
本发明化合物对CDK1激酶的抑制活性不强，所以，本发明化合物表现出对CDK1的选择性好于BAY1251152和AZD4573。
实验例三：体外CDK2/CyclinE1酶活性测试
实验材料：
CDK2/CyclinE1激酶检测试剂盒购自Promega。Nivo多标记分析仪(PerkinElmer)。
实验方法：
使用试剂盒里的激酶缓冲液稀释酶，底物，三磷酸腺苷和抑制剂。
将待测化合物用排枪进行5倍稀释至第8个浓度，即从50μM稀释至0.65nM，DMSO浓度为5％，设置双复孔实验。向微孔板中加入1μL抑制剂各浓度梯度，2μL CDK2/CyclinE1酶(2ng)，2μL底物和ATP的混合物(150μM三磷酸腺苷，0.1μg/μL底物)，此时化合物终浓度梯度为10μM稀释至0.13nM。反应体系置于25℃反应60分钟。反应结束后，每孔加入5μL ADP-Glo试剂，25℃继续反应40分钟，结束反应后每孔加入10μL的激酶检测试剂，25℃反应30分钟后采用多标记分析仪读数化学发光，积分时间0.5秒。
数据分析：
利用方程式(Sample-Min)/(Max-Min)*100％将原始数据换算成抑制率，IC 50的值即可通过四参数进行曲线拟合得出(GraphPad Prism中log(inhibitor)vs.response--Variable slope模式得出)。表1提供了本发明的化合物对CDK2/CyclinE1酶学抑制活性。
实验结论：
本发明化合物对CDK2激酶的抑制活性不强，所以，本发明化合物表现出对CDK2的选择性好于BAY1251152和AZD4573。
实验例四：体外细胞活性测试
实验材料：
IMDM培养基，胎牛血清，盘尼西林/链霉素抗生素购自Promega(Madison,WI)。MV-4-11细胞系购自中国科学院细胞库。Nivo多标记分析仪(PerkinElmer)。
实验方法：
将MV-4-11细胞种于白色96孔板中，80μL细胞悬液每孔，其中包含6000个MV-4-11细胞。细胞板置于二氧化碳培养箱中过夜培养。
将待测化合物用排枪进行5倍稀释至第8个浓度，即从2mM稀释至26nM，设置双复孔实验。向中间板中加入78μL培养基，再按照对应位置，转移2μL每孔的梯度稀释化合物至中间板，混匀后转移20μL每孔到细胞板中。化合物终浓度为10μM至0.13nM。细胞板置于二氧化碳培养箱中培养3天。
向细胞板中加入每孔25μL的Promega CellTiter-Glo试剂，室温孵育10分钟使发光信号稳定。采用PerkinElmer Nivo多标记分析仪读数。
数据分析：
利用方程式(Sample-Min)/(Max-Min)*100％将原始数据换算成抑制率，IC 50的值即可通过四参数进行曲线拟合得出(GraphPad Prism中"log(inhibitor)vs.response--Variable slope"模式得出)。表1提供了本发明的化合物对MV-4-11细胞增殖的抑制活性。
实验结论：
本发明化合物对MV4-11具有良好的细胞抗增殖活性。
表1
Figure PCTCN2020098002-appb-000091
实验例五：体内药效研究(一)
在皮下植入MV4-11急性髓系白血病患者来源的基于人源肿瘤细胞系的异种移植(CDX)BALB/c裸小鼠上进行体内药效实验。
实验操作：
BALB/c裸鼠，雌性，6-8周，体重约18-22克，将小鼠保持在一个特殊的无病原体的环境中，且在单个通风笼中(3只小鼠每笼)。所有的笼子，铺垫和水在使用前进行消毒。所有的动物都可以自由获取标准认证的商业实验室饮食。共有30只购于上海灵畅生物科技有限公司(Shanghai Lingchang biological science and technology Co.,LTD.)的小鼠用于研究。每只小鼠在右胁腹皮下植入肿瘤细胞(10×10 6在0.2毫升磷酸盐缓冲液中)，用于肿瘤的生长。当平均肿瘤体积达到约110立方毫米时开始给药。将试验化合物每周注射给药，给药剂量为15毫克/公斤。肿瘤体积每周2次用二维卡尺测量，体积以立方毫米计量，通过以下公式计算：V＝0.5a×b 2，其中a和b分别是肿瘤的长径和短径。抗肿瘤药效是通过用化合物处理过的动物的平均肿瘤增加体积除以未处理过动物的平均肿瘤增加体积来确定。
实验结论：
在MV4-11急性髓系白血病CDX体内药效模型中，本发明化合物展现良好的药效和安全性。
表2
Figure PCTCN2020098002-appb-000092
Figure PCTCN2020098002-appb-000093
实验例六：体内药效研究(二)
在皮下植入MV4-11急性髓系白血病患者来源的基于人源肿瘤细胞系的异种移植(CDX)BALB/c裸小鼠上进行体内药效实验。
实验操作：
BALB/c裸鼠，雌性，6-8周，体重约18-22克，将小鼠保持在一个特殊的无病原体的环境中，且在单个通风笼中(3只小鼠每笼)。所有的笼子，铺垫和水在使用前进行消毒。所有的动物都可以自由获取标准认证的商业实验室饮食。共有36只购于上海灵畅生物科技有限公司(Shanghai Lingchang biological science and technology Co.,LTD.)的小鼠用于研究。每只小鼠在右胁腹皮下植入肿瘤细胞(10×10 6在0.2毫升磷酸盐缓冲液中)，用于肿瘤的生长。当平均肿瘤体积达到约121立方毫米时开始给药。将试验化合物每周注射给药，给药剂量为10毫克/公斤。肿瘤体积每周2次用二维卡尺测量，体积以立方毫米计量，通过以下公式计算：V＝0.5a×b 2，其中a和b分别是肿瘤的长径和短径。抗肿瘤药效是通过用化合物处理过的动物的平均肿瘤增加体积除以未处理过动物的平均肿瘤增加体积来确定。
实验结论：
在MV4-11急性髓系白血病CDX体内药效模型中，本发明化合物展现良好的药效和安全性。
表3
Figure PCTCN2020098002-appb-000094

Claims (17)

  1. 式(I)所示化合物、其药学上可接受的盐或其异构体，
    Figure PCTCN2020098002-appb-100001
    其中，T 1为N或CR；
    R为H或Cl；
    T 2为N或CH；
    R 1为H或C 1-6烷基，其中所述C 1-6烷基任选被1、2或3个独立选自F、Cl、-OH、-NH 2和C 1-3烷氧基的取代基所取代；
    R 2为H、F或Cl；
    R 3和R 4各自独立地为H、F、Cl或C 1-3烷基；
    R 5为H、C 3-6环烷基或苯基，其中所述C 3-6环烷基和苯基任选被1、2或3个R a所取代；
    各R a独立地为H、F、Cl、C 1-3烷基或C 1-3烷氧基。
  2. 根据权利要求1所述的化合物、其药学上可接受的盐或其异构体，其化合物具有式(I-1)或(I-2)所示结构：
    Figure PCTCN2020098002-appb-100002
    其中，R、T 2、R 1、R 2、R 3、R 4和R 5如权利要求1所定义。
  3. 根据权利要求2所述的化合物、其药学上可接受的盐或其异构体，其化合物具有式(I-1-a)或(I-1-b)所示结构：
    Figure PCTCN2020098002-appb-100003
    其中，R、R 1、R 2、R 3、R 4和R 5如权利要求2所定义。
  4. 根据权利要求1～3任一项所述的化合物、其药学上可接受的盐或其异构体，其中R 3和R 4各自独立地为H、F或
    Figure PCTCN2020098002-appb-100004
  5. 根据权利要求2所述的化合物、其药学上可接受的盐或其异构体，其化合物具有式(I-1-c)或(I-1-d)所示结构：
    Figure PCTCN2020098002-appb-100005
    其中，R、R 1、R 2和R 5如权利要求2所定义。
  6. 根据权利要求1所述的化合物、其药学上可接受的盐或其异构体，其中各R a独立地为H、F、Cl或
    Figure PCTCN2020098002-appb-100006
  7. 根据权利要求1～3、5或6任一项所述的化合物、其药学上可接受的盐或其异构体，其中R 5为H、
    Figure PCTCN2020098002-appb-100007
    Figure PCTCN2020098002-appb-100008
    其中所述
    Figure PCTCN2020098002-appb-100009
    任选被1、2或3个R a所取代。
  8. 根据权利要求7所述的化合物、其药学上可接受的盐或其异构体，其中R 5为H、
    Figure PCTCN2020098002-appb-100010
    Figure PCTCN2020098002-appb-100011
  9. 根据权利要求8所述的化合物、其药学上可接受的盐或其异构体，其中R 5为H、
    Figure PCTCN2020098002-appb-100012
    Figure PCTCN2020098002-appb-100013
  10. 根据权利要求7所述的化合物、其药学上可接受的盐或其异构体，其化合物具有式(I-1-e)、(I-1-f)、(I-1-g)或(I-1-h)所示结构：
    Figure PCTCN2020098002-appb-100014
    其中，R、R 1、R 2和R a如权利要求7所定义。
  11. 根据权利要求1～3、5或8～10任一项所述的化合物、其药学上可接受的盐或其异构体，其中R 1为H、
    Figure PCTCN2020098002-appb-100015
    其中所述
    Figure PCTCN2020098002-appb-100016
    任选被1、2或3个独立选自F、Cl、-OH、-NH 2和-OCH 3的取代基所取代。
  12. 根据权利要求11所述的化合物、其药学上可接受的盐或其异构体，其中R 1为H、
    Figure PCTCN2020098002-appb-100017
    Figure PCTCN2020098002-appb-100018
  13. 根据权利要求1～3任一项所述的化合物、其药学上可接受的盐或其异构体，其中结构单元
    Figure PCTCN2020098002-appb-100019
    为
    Figure PCTCN2020098002-appb-100020
  14. 下式化合物、其药学上可接受的盐或其异构体：
    Figure PCTCN2020098002-appb-100021
    Figure PCTCN2020098002-appb-100022
  15. 一种药物组合物，其含有治疗有效量的根据权利要求1～14任一项所述的化合物、其异构体或其药学上可接受的盐和药学上可接受的载体。
  16. 根据权利要求1～14任一项所述的化合物、其药学上可接受的盐或其异构体或根据权利要求15所述的药物组合物在制备CDK9抑制剂药物中的应用。
  17. 根据权利要求1～14任一项所述的化合物、其药学上可接受的盐或其异构体或根据权利要求15所述的药物组合物在制备治疗癌症药物中的应用。
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113666934A (zh) * 2021-07-28 2021-11-19 北京深度制耀科技有限公司 Cdk9激酶抑制剂
WO2022135580A1 (zh) * 2020-12-25 2022-06-30 南京明德新药研发有限公司 吡啶并吡咯类化合物的晶型、制备方法及其应用

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2025076115A1 (en) * 2023-10-02 2025-04-10 Prelude Therapeutics Incorporated Cdk inhibitors and their use as pharmaceuticals

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101389324A (zh) * 2005-12-23 2009-03-18 史密丝克莱恩比彻姆公司 Aurora激酶的氮杂吲哚抑制剂
WO2016040858A1 (en) * 2014-09-12 2016-03-17 G1 Therapeutics, Inc. Combinations and dosing regimes to treat rb-positive tumors
WO2017103670A1 (en) * 2015-12-14 2017-06-22 Zenith Epigenetics Lid. 1h-imidazo[4,5-b]pyridinyl and 2-oxo-2,3-dihydro-1h-imidazo[4,5-b]pyridinyl heterocyclic bet bromodomain inhibitors
WO2018192273A1 (zh) * 2017-04-19 2018-10-25 上海炯烁医药科技有限公司 一种新型细胞周期蛋白依赖性激酶cdk9抑制剂
WO2019058132A1 (en) * 2017-09-22 2019-03-28 The University Of Nottingham HETEROCYCLYL SUBSTITUTED PYRROLOPYRIDINES AS INHIBITORS OF KINASE CDK12

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
SG11201607973XA (en) * 2014-03-24 2016-11-29 Guangdong Zhongsheng Pharmaceutical Co Ltd Quinoline derivatives as smo inhibitors
ES2728356T3 (es) * 2015-06-29 2019-10-23 Astrazeneca Ab Derivados de amidas policíclicas como inhibidores de CDK9
EP3434676B1 (en) * 2016-03-25 2022-03-09 Chia Tai Tianqing Pharmaceutical Group Co., Ltd. Substituted pyrrolopyrimidine cdk inhibitor, pharmaceutical composition containing same and use thereof

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101389324A (zh) * 2005-12-23 2009-03-18 史密丝克莱恩比彻姆公司 Aurora激酶的氮杂吲哚抑制剂
WO2016040858A1 (en) * 2014-09-12 2016-03-17 G1 Therapeutics, Inc. Combinations and dosing regimes to treat rb-positive tumors
WO2017103670A1 (en) * 2015-12-14 2017-06-22 Zenith Epigenetics Lid. 1h-imidazo[4,5-b]pyridinyl and 2-oxo-2,3-dihydro-1h-imidazo[4,5-b]pyridinyl heterocyclic bet bromodomain inhibitors
WO2018192273A1 (zh) * 2017-04-19 2018-10-25 上海炯烁医药科技有限公司 一种新型细胞周期蛋白依赖性激酶cdk9抑制剂
WO2019058132A1 (en) * 2017-09-22 2019-03-28 The University Of Nottingham HETEROCYCLYL SUBSTITUTED PYRROLOPYRIDINES AS INHIBITORS OF KINASE CDK12

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
LAN, PING, ET AL.: "3D-QSAR and molecular docking studies of azaindole derivatives as Aurora B kinase inhibitors", 《JOURNAL OF MOLECULAR MODELING》, vol. 17, 11 August 2010 (2010-08-11), XP019903361, DOI: 10.1007/s00894-010-0820-7 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022135580A1 (zh) * 2020-12-25 2022-06-30 南京明德新药研发有限公司 吡啶并吡咯类化合物的晶型、制备方法及其应用
CN113666934A (zh) * 2021-07-28 2021-11-19 北京深度制耀科技有限公司 Cdk9激酶抑制剂
CN113666934B (zh) * 2021-07-28 2023-06-23 北京深度制耀科技有限公司 Cdk9激酶抑制剂

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