WO2024051727A1 - 吡唑类衍生物、药物组合物及应用 - Google Patents

吡唑类衍生物、药物组合物及应用 Download PDF

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WO2024051727A1
WO2024051727A1 PCT/CN2023/117196 CN2023117196W WO2024051727A1 WO 2024051727 A1 WO2024051727 A1 WO 2024051727A1 CN 2023117196 W CN2023117196 W CN 2023117196W WO 2024051727 A1 WO2024051727 A1 WO 2024051727A1
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alkyl
compound
pharmaceutically acceptable
stereoisomer
acceptable salt
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PCT/CN2023/117196
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English (en)
French (fr)
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胡璞
陆居权
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楚浦创制（武汉）医药科技有限公司
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Publication of WO2024051727A1 publication Critical patent/WO2024051727A1/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
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/415—1,2-Diazoles
    • 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
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/415—1,2-Diazoles
    • A61K31/4162—1,2-Diazoles condensed with heterocyclic ring systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
    • 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
    • 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

  • the present invention relates to the field of medical technology, and in particular to a pyrazole derivative, pharmaceutical composition and application.
  • CDKs Cell cycle-dependent kinases
  • CDKs belong to the serine/threonine kinase family. They participate in physiological processes such as cell proliferation and transcription. According to the different functions of CDKs, they can be divided into two categories: 1) One type of CDK is involved in cell cycle regulation, mainly including CDK1, CDK2, CDK4, CDK6, etc.; 2) The other type of CDK is involved in transcriptional regulation, mainly including CDK7, CDK8, CDK9, CDK12, CDK13, etc.
  • CDK2 plays a key role in promoting G1/S transition and S phase progression.
  • CDK2 forms a complex with Cyclin E and phosphorylates retinoblastoma family members (pRb, etc.), leading to the release and activation of E2F transcription factors, promoting the transition of the cell cycle from G1 phase to S phase, thereby causing CDK2/ Cyclin A activation promotes cell cycle DNA synthesis, replication and other processes.
  • Cyclin E1 Increased copy number and overexpression of Cyclin E1 have been identified in ovarian cancer, gastric cancer, endometrial cancer, breast cancer and other cancers, and are positively correlated with poor prognosis of the corresponding tumors.
  • high expression of Cyclin E2 is often accompanied by resistance to hormone therapy (Mol. Cancer Ther., 2012, 11, 1488-1499), and amplification or overexpression of Cyclin E is associated with poor prognosis of breast cancer. There is a close relationship (N.Engl.J.Med, 2002, 347, 1566-1575).
  • Cyclin E amplification has also been reported to contribute to trastuzumab resistance (Proc. Natl. Acad.
  • the object of the present invention is to provide a pyrazole derivative with good selectivity for inhibiting the activity of CDK2.
  • the first aspect of the present invention provides a compound represented by formula (I), or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof:
  • R 1 is selected from H, halogen, C 1-6 alkyl, halogenated C 1-6 alkyl, C 3-7 cycloalkyl, 3 to 7 membered heterocycloalkyl, nitro, isocyanyl, -( CH 2 ) n R 5 ;
  • R 5 is selected from -CN, -OR 6 , -NR 6 R 7 , -C(O)OR 6 , -C(O)R 6 , -S(O) 2 R 6 , -P(O)(OR 6 ) 2 , -O-alicyclic group, -O-aliphatic heterocyclic group, -O-aryl, -O-heteroaryl or -C(O)-heteroaryl group, wherein the alicyclic group, alicyclic group Cyclic group, aryl group and heteroaryl group are unsubstituted or substituted by one or more R 0 ;
  • R 0 is selected from halogen, C 1-6 alkyl, halogenated C 1-6 alkyl, C 3-7 cycloalkyl, 3-7 membered heterocycloalkyl, cyano, amino, nitro or hydroxyl;
  • R 6 and R 7 are each independently selected from H, C 1-6 alkyl, halogen, halogenated C 1-6 alkyl, C 3-7 cycloalkyl, 3-7 membered heterocycloalkyl, aryl or heteroaryl;
  • n 0, 1, 2, 3 or 4;
  • Ring A is selected from
  • X is each independently N or CR 6 ;
  • R 8 is selected from H, hydroxyl, C 1-6 alkyl, halogenated C 1-6 alkyl, C 3-7 cycloalkyl or 3-7 membered heterocycloalkyl;
  • R 2 and R 3 are each independently selected from H, C 1-6 alkyl, halo C 1-6 alkyl, C 3-7 cycloalkyl or 3-7 membered heterocycloalkyl, wherein said C 3 -7 cycloalkyl and 3-7 membered heterocycloalkyl are unsubstituted or substituted by one or more R 9 ;
  • R 9 is selected from halogen, C 1-6 alkyl, halogenated C 1-6 alkyl, cyano, amino, nitro or hydroxyl;
  • R 4 is selected from H, halogen, amino, hydroxyl or C 1 -C 6 alkyl
  • one or more hydrogen atoms are substituted or unsubstituted by deuterium atoms;
  • the compound does not have the structure shown below:
  • the R 1 when in the structure shown in formula (I), the R 1 is selected from -CH 2 -O-CH 3 and ring A is selected from
  • R 2 is selected from H
  • R 3 is selected from -CH(CH 3 ) 2
  • R 4 is selected from H
  • one or more hydrogen atoms in the structure represented by formula (I) are replaced by deuterium atoms.
  • R 1 , ring A, R 2 , R 3 and R 4 are selected from other groups in the structure represented by formula (I), one or more hydrogens in the structure represented by formula (I) Atoms are substituted or unsubstituted with deuterium atoms.
  • the R 1 is selected from -CH 2 R 5 and the R 5 is selected from -OR 61 , -NR 6 R 7 , -CN, -C(O)OR 6 , -C(O) R 6 , -S(O) 2 R 6 , -P(O)(OR 6 ) 2 , -O-alicyclic group, -O-alicyclic heterocyclic group, -O-aryl, -O-heteroaryl group or -C(O)-heteroaryl, wherein the alicyclic group, alicyclic heterocyclic group, aryl group, heteroaryl group is unsubstituted or substituted with one or more R 0 ;
  • the R 61 is selected from H, C 1-6 alkyl in which one or more hydrogen atoms are replaced by deuterium atoms, halogenated C 1 -C 6 alkyl, C 3-7 cycloalkyl, 3-7 membered heterocycle Alkyl, aryl or heteroaryl.
  • R 1 is selected from -CH 2 R 5
  • R 5 is selected from -OR 61 , -NR 6 R 7 or -CN.
  • the R 1 is selected from -CH 2 R 5 and the R 5 is selected from -OR 61 or -NR 6 R 7 ;
  • the R 61 is selected from H, a C 1-6 alkyl group in which one or more hydrogen atoms are replaced by deuterium atoms or a halogenated C 1 -C 6 alkyl group;
  • the R 6 and R 7 are each independently selected from H, C 1-6 alkyl or halo C 1-6 alkyl.
  • said R 1 is -OH.
  • R 2 is H
  • R 3 is selected from C 1-6 alkyl or halo C 1 -C 6 alkyl.
  • the R 3 is -C(CH 3 ) 3 .
  • the R 1 is selected from -(CH 2 ) n R 5 , and one or more hydrogen atoms in the R 5 are replaced by deuterium atoms. Further preferably, R 5 is selected from -OR 6 .
  • the structure represented by formula (I) is selected from the structure represented by any one of formulas (I-1) to (I-3):
  • the structure represented by formula (I) is selected from the structure represented by formula (I-4):
  • X is selected from CR6 .
  • R 6 is selected from H, C 1-6 alkyl, halogen or halogenated C 1-6 alkyl.
  • R 1 is selected from H, halogen, C 3-7 cycloalkyl, 3 to 7 membered heterocycloalkyl, nitro, isocyanate, -(CH 2 ) n R 5 ;
  • n is 1, and R 1 is -CH 2 R 5 .
  • the structure represented by formula (I) is selected from the structure represented by formula (I-5):
  • X is selected from CR6 .
  • R 6 is selected from H, C 1-6 alkyl, halogen or halogenated C 1-6 alkyl.
  • the R 5 is selected from -CN, -OR 6 or -NR 6 R 7 ;
  • the R 6 and R 7 are each independently selected from H, C 1-6 alkyl or halo C 1-6 alkyl.
  • R5 is selected from -CN, -OC 1-3 alkyl, -N(H)C 1-3 alkyl, -N(C 1-3 alkyl)(C 1-3 alkyl ).
  • the R 2 is selected from H, C 1 -C 6 alkyl or halo C 1 -C 6 alkyl;
  • the R 3 is selected from H, C 1 -C 6 alkyl, halogenated C 1 -C 6 alkyl, C 3-7 cycloalkyl or 3-7 membered heterocycloalkyl, wherein the C 3-7 Cycloalkyl and 3-7 membered heterocycloalkyl are unsubstituted or substituted with one or more R 9 .
  • the R 2 is selected from H;
  • the R 3 is selected from H, C 1 -C 6 alkyl, halogenated C 1 -C 6 alkyl, unsubstituted or 5-membered heterocycloalkyl substituted by one R 9 .
  • the R 9 is selected from halogen, C 1-6 alkyl or halogenated C 1-6 alkyl; the heteroatom of the 5-membered heterocycloalkyl is an oxygen atom.
  • R4 is selected from H, F or methyl. Further preferably, R4 is selected from H.
  • R 0 is selected from C 3-7 cycloalkyl, 3-7 membered heterocycloalkyl, cyano, amino, nitro or hydroxyl.
  • R 8 is selected from H, C 1-6 alkyl, halo C 1-6 alkyl, C 3-7 cycloalkyl or 3-7 membered heterocycloalkyl.
  • the compounds include, but are not limited to, the following structures:
  • the pharmaceutically acceptable salt is an alkyl salt. Further, the pharmaceutically acceptable salt is formate.
  • a second aspect of the present invention provides a pharmaceutical composition, which includes the compound described in the first aspect of the present invention, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, and a pharmaceutically acceptable carrier.
  • the third aspect of the present invention provides the compound described in the first aspect of the present invention, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, and the pharmaceutical composition described in the third aspect of the present invention in the preparation of treatment or prevention and Applications in medicines for diseases related to or mediated by CDK2 activity.
  • the disease associated with or mediated by CDK2 activity is cancer.
  • the inventor unexpectedly discovered a compound represented by formula (I).
  • the compound has unexpected inhibitory activity on CDK2 and can be used to treat various cancers with high Cyclin E expression, especially for cancer patients with resistance to CDK4/6 inhibitors, with excellent therapeutic effects. Based on the above findings, the inventor completed the present invention.
  • pharmaceutically acceptable refers to those compounds, materials, compositions and/or dosage forms which, within the scope of sound medical judgment, are suitable for use in contact with human and animal tissue without multiple toxicity, irritation, allergic reactions, or other problems or complications, commensurate with a reasonable benefit/risk ratio.
  • pharmaceutically acceptable salts refers to salts of compounds of the present invention prepared from compounds having specific substituents found in the present invention and relatively non-toxic acids or bases.
  • base addition salts can be obtained by contacting such compounds with a sufficient amount of base in pure solution or in a suitable inert solvent.
  • Pharmacy Acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amine or magnesium salts or similar salts.
  • acid addition salts can be obtained by contacting such compounds with a sufficient amount of acid in neat solution or in 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, bicarbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, Hydrogen sulfate, hydriodic acid, phosphorous acid, etc.; and organic acid salts, including formic acid, acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, octanoic acid, etc.
  • inorganic acid salts including, for example, hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, Hydrogen sulfate, hydriodic acid, phosphorous acid, etc.
  • organic acid salts including formic acid, acetic acid, propionic acid, is
  • Acid fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, methanesulfonic acid and other similar acids; also includes amino acids (such as arginine, etc.) salts, as well as salts of organic acids such as glucuronic acid. Certain specific compounds of the present invention contain both basic and acidic functional groups and thus can be converted into either base or acid addition salts.
  • the pharmaceutically acceptable salts of the present invention can be synthesized by conventional chemical methods from parent compounds containing acid groups or bases.
  • such salts are prepared by reacting the free acid or base form of these compounds with a stoichiometric amount of the appropriate base or acid in water or an organic solvent or a mixture of the two.
  • 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, diastereoisomers isomer, the (D)-isomer, the (L)-isomer, as well as their racemic mixtures and other mixtures, such as enantiomeric or diastereomerically enriched mixtures, all of which belong to the present invention. within the scope of the invention. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All such isomers, as well as mixtures thereof, are included within the scope of the present invention.
  • enantiomers or “optical isomers” refer to stereoisomers that are mirror images of each other.
  • cis-trans isomers or “geometric isomers” refers to the inability of the double bonds or single bonds of the carbon atoms in the ring to rotate freely.
  • diastereomer refers to stereoisomers whose molecules have two or more chiral centers and are in a non-mirror image relationship between the molecules.
  • use wedge-shaped solid line keys and wedge-shaped dotted keys Represents the absolute configuration of a three-dimensional center
  • using straight solid line keys and straight dotted keys Represent the relative configuration of the three-dimensional center with a wavy line
  • wedge-shaped solid line key or wedge-shaped dotted key or use tilde Represents a straight solid line key and straight dotted keys
  • formula (A) It means that the compound exists in the form of a single isomer of formula (A-1) or formula (A-2) or in the form of a mixture of two isomers of formula (A-1) and formula (A-2); below Formula (B) indicates that the compound exists as a single isomer of formula (B-1) or formula (B-2) or as a mixture of two isomers of formula (B-1) and formula (B-2). Form exists.
  • the following formula (C) indicates that the compound exists in the form of a single isomer of formula (C-1) or formula (C-2) or in the form of two isomers of formula (C-1) and formula (C-2). Exists in mixture form.
  • tautomer or “tautomeric form” means that at room temperature, isomers with different functional groups are in dynamic equilibrium and can quickly convert into each other.
  • proton tautomers also known as proton transfer tautomers
  • proton migration such as keto-enol isomerization and imine-enol isomerization.
  • Valence tautomers involve interconversions through the reorganization of some bonding electrons.
  • keto-enol tautomerization is the tautomerization between pentane-2,4-dione and 4-hydroxypent-3-en-2-one.
  • the terms “enriched in an isomer,” “enantiomerically enriched,” “enriched in an enantiomer,” or “enantiomerically enriched” refer to one of the isomers or enantiomers.
  • 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 greater than or equal to 96%, or greater than or equal to 97%, or greater than or equal to 98%, or greater than or equal to 99%, or greater than or equal to 99.5%, or greater than or equal to 99.6%, or greater than or equal to 99.7%, or greater than or equal to 99.8%, or greater than or equal to 99.9%.
  • the term “isomer excess” or “enantiomeric excess” refers to the relative percentages of two isomers or two enantiomers. The difference between the numbers. For example, if the content of one isomer or enantiomer is 90% and the content of the other isomer or enantiomer is 10%, then the isomer or enantiomeric excess (ee value) is 80% .
  • optically active (R)- and (S)-isomers as well as the D and L isomers can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If one enantiomer of a compound of the invention is desired, it can be prepared by asymmetric synthesis or derivatization with chiral auxiliaries, in which the resulting diastereomeric mixture is separated and the auxiliary group is cleaved to provide pure desired enantiomer.
  • a diastereomeric salt is formed with a suitable optically active acid or base, and then the salt is formed by conventional methods known in the art. Diastereomeric resolution is performed and the pure enantiomers are recovered. Furthermore, the separation of enantiomers and diastereomers is usually accomplished by the use of chromatography using chiral stationary phases, optionally combined with chemical derivatization methods (e.g., generation 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 that make up the compound.
  • compounds can be labeled with radioactive isotopes, such as tritium ( 3 H), iodine-125 ( 125 I), or C-14 ( 14 C).
  • deuterated drugs can be replaced by heavy hydrogen to form deuterated drugs. The bond between deuterium and carbon is stronger than the bond between ordinary hydrogen and carbon. Compared with non-deuterated drugs, deuterated drugs can reduce side effects and increase drug stability. , enhance efficacy, extend drug biological half-life and other advantages. All variations in the isotopic composition of the compounds of the invention, whether radioactive or not, are included within the scope of the invention.
  • substitution means that any one or more hydrogen atoms on a specific atom are replaced by a substituent, which can include deuterium and hydrogen variants, as long as the valence state of the specific atom is normal and the substituted compound is stable of.
  • the type and number of substituents can be arbitrary on the basis of chemical achievability.
  • any variable eg, R1
  • its definition in each instance is independent.
  • R1 When any variable (eg, R1 ) occurs more than once in the composition or structure of a compound, its definition in each instance is independent.
  • R1 When any variable (eg, R1 ) occurs more than once in the composition or structure of a compound, its definition in each instance is independent.
  • R1 When any variable (eg, R1 ) occurs more than once in the composition or structure of a compound, its definition in each instance is independent.
  • R1 e.g, R1
  • R1 When any variable (eg, R1 ) occurs more than once in the composition or structure of a compound, its definition in each instance is independent.
  • a group 0-2 R1 , then said group may optionally be substituted by up to two R1 , with independent options for R1 in each case.
  • substituents and/or variants thereof are permitted only 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.
  • substituent When a substituent is vacant, it means that the substituent does not exist. For example, when X in AX is vacant, it means that the structure is actually A. When a listed substituent does not indicate through which atom it is connected to the substituted group, this Each substituent can be bonded through any atom thereof. For example, a pyridyl group as a substituent can be bonded to the substituted group through any carbon atom on the pyridine ring.
  • the direction of connection is arbitrary, for example, The middle linking group L is -MW-.
  • -MW- can be connected to ring A and ring B in the same direction as the reading order from left to right. You can also connect ring A and ring B in the opposite direction to the reading order from left to right.
  • any one or more sites of the group can be connected to other groups through chemical bonds.
  • connection mode of the chemical bond is non-positioned and there are H atoms at the connectable site, when the chemical bond is connected, the number of H atoms at the site will be reduced correspondingly with the number of connected chemical bonds and become the corresponding valence. group.
  • the chemical bond connecting the site to other groups can be a straight solid line bond straight dashed key or wavy lines express.
  • the straight solid line bond in -OCH 3 means that it is connected to other groups through the oxygen atom in the group;
  • the straight dotted bond in means that it is connected to other groups through both ends of the nitrogen atoms in the group;
  • the wavy lines in indicate that the phenyl group is connected to other groups through the 1 and 2 carbon atoms in the phenyl group;
  • the number of atoms on a ring is usually defined as the number of ring members.
  • a "3-7 membered ring” refers to a “ring” with 3-7 atoms arranged around it.
  • C 1-6 alkyl is used to mean a straight or branched chain saturated hydrocarbon group consisting of 1 to 6 carbon atoms.
  • a C 1-4 alkyl group which may be monovalent (such as methyl), divalent (such as methylene) or multivalent (such as methine).
  • Examples of C 1-3 alkyl groups include, but are not limited to, methyl (Me), ethyl (Et), propyl (including n-propyl and isopropyl), butyl (including Including n-butyl, isobutyl, tert-butyl and sec-butyl).
  • C 1-6 alkoxy means those alkyl groups containing 1 to 6 carbon atoms that are attached to the remainder of the molecule through an oxygen atom. Preferred is C 1-3 alkoxy. Examples of C 1-3 alkoxy include, but are not limited to, methoxy, ethoxy, propoxy (including n-propoxy and isopropoxy), and the like.
  • C 1-6 alkylamino means those alkyl groups containing 1 to 6 carbon atoms attached to the remainder of the molecule through an amino group. Preferred is C 1-3 alkylamino. Examples of C 1-3 alkylamino groups include, but are not limited to, -NHCH 3 , -N(CH 3 ) 2 , -NHCH 2 CH 3 , -N(CH 3 )CH 2 CH 3 , -NHCH 2 CH 2 CH 3 , - NHCH 2 (CH 3 ) 2 etc.
  • halogen or halogen by itself or as part of another substituent means a fluorine, chlorine, bromine or iodine atom.
  • 5-membered heteroaryl ring and “5-membered heteroaryl” in the present invention can be used interchangeably.
  • the term “5-membered heteroaryl” refers to a single ring consisting of 5 ring atoms and having a conjugated ⁇ electron system.
  • the nitrogen atoms are optionally quaternized, and the nitrogen and sulfur heteroatoms are optionally oxidized (i.e., NO and S(O) p , p is 1 or 2).
  • a 5-membered heteroaryl group can be attached to the rest of the molecule through a heteroatom or a carbon atom.
  • the 5-membered heteroaryl include but are not limited to pyrrolyl (including N-pyrrolyl, 2-pyrrolyl and 3-pyrrolyl, etc.), pyrazolyl (including 2-pyrazolyl and 3-pyrazolyl) etc.), imidazolyl (including N-imidazolyl, 2-imidazolyl, 4-imidazolyl and 5-imidazolyl, etc.), oxazolyl (including 2-oxazolyl, 4-oxazolyl and 5-oxazolyl) base, etc.), triazolyl (1H-1,2,3-triazolyl, 2H-1,2,3-triazolyl, 1H-1,2,4-triazolyl and 4H-1,2, 4-triazolyl, etc.), tetrazolyl, isoxazolyl (3-isoxazolyl, 4-isoxazoly
  • 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 , also include 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.; similarly, n yuan to n The +m member indicates that the number of atoms in the ring is n to n+m.
  • a 3-12 membered ring includes a 3-membered ring, a 4-membered ring, a 5-membered ring, a 6-membered ring, a 7-membered ring, an 8-membered ring, and a 9-membered ring.
  • 3-membered ring includes 3-6-membered ring, 3-9-membered ring, 5-6-membered ring ring, 5-7 membered ring, 6-7 membered ring, 6-8 membered ring, and 6-10 membered ring, etc.
  • C 3-7 cycloalkyl means a saturated cyclic hydrocarbon group composed of 3 to 7 carbon atoms, which includes monocyclic and bicyclic systems, where the bicyclic system includes spirocyclic, paracyclic and Bridge ring.
  • the C 3-7 cycloalkyl group includes C 3-6 , C 4-6 , C 4-5 , C 5-7 or C 5-6 cycloalkyl group, etc.; it can be monovalent, divalent or multivalent.
  • Examples of C 3-7 cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and the like.
  • the term "3-7 membered heterocycloalkyl" by itself or in combination with other terms means a group consisting of 3 to 7 ring atoms, respectively.
  • a heteroatom may occupy the attachment position of the heterocycloalkyl to the rest of the molecule.
  • the 3-7-membered heterocycloalkyl group includes 5-7-membered, 3-membered, 4-membered, 5-membered, 6-membered and 7-membered heterocycloalkyl groups, etc.
  • 3-7 membered heterocycloalkyl examples include, but are not limited to, azetidinyl, oxetanyl, thietanyl, pyrrolidinyl, pyrazolidinyl, imidazolidinyl, tetrahydrothienyl ( Including tetrahydrothiophen-2-yl and tetrahydrothiophen-3-yl, etc.), tetrahydrofuranyl (including tetrahydrofuran-2-yl, etc.), tetrahydropyranyl, piperidinyl (including 1-piperidinyl, 2- Piperidinyl and 3-piperidinyl, etc.), piperazinyl (including 1-piperazinyl and 2-piperazinyl, etc.), morpholinyl (including 3-morpholinyl, 4-morpholinyl, etc.), Dioxanyl, dithianyl, isoxazolidinyl, isothiazolidinyl
  • 3-7 membered nitrogen-containing heterocycloalkyl means a 3-7 membered heterocycloalkyl group containing at least one N atom.
  • Alicyclic group refers to a saturated or partially unsaturated all-carbon ring system. Where “partially unsaturated” refers to a ring portion that includes at least one double or triple bond, “partially unsaturated” is intended to encompass rings having multiple sites of unsaturation, but is not intended to include aryl groups as defined herein or Heteroaryl moieties.
  • Non-limiting examples include cyclopropyl ring, cyclobutyl ring, cyclopentyl ring, cyclopentenyl ring, cyclohexyl ring, cyclohexenyl ring, cyclohexadienyl ring, cycloheptyl ring, cycloheptyl tri Alkenyl ring, cyclopentanone ring, cyclopentane-1,3-dione ring, etc.
  • Aliphatic heterocyclic group refers to a saturated or partially unsaturated alicyclic group in which 1, 2 or 3 ring carbon atoms are selected from nitrogen, oxygen or S(O) t (where t is an integer from 0 to 2). Substituted, but not including the ring portion of -OO-, -OS- or -SS-, the remaining ring atoms are carbon.
  • Non-limiting examples include propylene oxide ring, azetidine ring, oxetane ring, tetrahydrofuran ring, tetrahydrothiophene ring, tetrahydropyrrole ring, piperidine ring, pyrroline ring, oxazolidine ring , piperazine ring, dioxolane, dioxane, morpholine ring, thiomorpholine ring, thiomorpholine-1,1-dioxide, tetrahydropyran ring, azetidine- 2-keto ring, oxetane-2-one ring, pyrrolidine-2-one ring, pyrrolidine-2,5-dione ring, piperidin-2-one ring, dihydrofuran-2(3H )-keto ring, dihydrofuran-2,5-dione ring, tetrahydro-2H-pyran-2-one
  • Non-limiting examples of partially unsaturated monoheterocycles include 1,2-dihydroazetidine ring, 1,2-dihydrooxetadiene ring, 2,5-dihydro-1H- Pyrrole ring, 2,5-dihydrofuran ring, 2,3-dihydrofuran ring, 2,3-dihydro-1H-pyrrole ring, 3,4-dihydro-2H-pyran ring, 1,2, 3,4-tetrahydropyridine ring, 3,6-dihydro-2H-pyran ring, 1,2,3,6-tetrahydropyridine ring, 4,5-dihydro-1H-imidazole ring, 1,4 ,5,6-tetrahydropyrimidine ring, 3,4,7,8-tetrahydro-2H-1,4,6-oxadiazolin ring, 1,6-dihydropyrimidine ring, 4,5,6, 7-tetrahydro-1H-1,3-d
  • Aryl and “aromatic ring” are used interchangeably and both refer to an all-carbon monocyclic or fused polycyclic (that is, a ring that shares adjacent pairs of carbon atoms) group with a conjugated ⁇ electron system. Can be fused with a cycloalkyl ring, heterocycloalkyl ring, cycloalkenyl ring, heterocycloalkenyl ring or heteroaryl group.
  • C 6-10 aryl refers to a monocyclic or bicyclic aryl group having 6 to 10 carbon atoms, a non-limiting embodiment of aryl Examples include phenyl, naphthyl, etc.
  • Heteroaryl and “heteroaryl ring” are used interchangeably and both refer to a monocyclic, bicyclic or polycyclic 4n+2 aromatic ring system having ring carbon atoms and ring heteroatoms (e.g., having a Arrange shared 6 or 10 ⁇ electrons) groups in which each heteroatom is independently selected from nitrogen, oxygen and sulfur.
  • heteroaryl also includes ring systems in which the above-mentioned heteroaryl ring is fused with one or more cycloalkyl rings, heterocycloalkyl rings, cycloalkenyl rings, heterocycloalkenyl rings or aromatic rings. Heteroaryl rings may be optionally substituted.
  • 5- to 10-membered heteroaryl refers to a monocyclic or bicyclic heteroaryl group having 5 to 10 ring atoms, of which 1, 2, 3 or 4 ring atoms are heteroatoms.
  • “5- to 6-membered heteroaryl” refers to a monocyclic heteroaryl group with 5 to 6 ring atoms, of which 1, 2, 3 or 4 ring atoms are heteroatoms.
  • Non-limiting examples include thienyl, furan base, thiazolyl, isothiazolyl, imidazolyl, oxazolyl, pyrrolyl, pyrazolyl, triazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,2 ,5-triazolyl, 1,3,4-triazolyl, tetrazolyl, isoxazolyl, oxadiazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl Azolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, tetrazine base.
  • 8- to 10-membered heteroaryl refers to a bicyclic heteroaryl group having 8 to 10 ring atoms, in which 1, 2, 3 or 4 ring atoms are heteroatoms.
  • Non-limiting examples include indolyl, iso- Indolyl, indazolyl, benzotriazolyl, benzothienyl, isobenzothienyl, benzofuranyl, benzisofuranyl, benzimidazolyl, benzoxazolyl, benzoiso Oxazolyl, benzoxadiazolyl, benzothiazolyl, benzisothiazolyl, benzothiadiazolyl, indanazinyl, purinyl, pyrido[3,2-d]pyrimidinyl, pyrido [2,3-d]pyrimidinyl, pyrido[3,4-d]pyrimidinyl, pyrido[4,3-d]pyrimidinyl, 1,8-naph
  • Heteroatom means nitrogen, oxygen or sulfur. In heteroaryl groups containing one or more nitrogen atoms, the point of attachment may be a carbon or nitrogen atom as long as the valency permits. Heteroaryl bicyclic systems may include one or more heteroatoms in one or both rings.
  • leaving group refers to a functional group or atom that can be replaced by another functional group or atom through a substitution reaction, such as a nucleophilic substitution reaction.
  • representative leaving groups include triflate; chlorine, bromine, iodine; sulfonate groups such as mesylate, tosylate, p-bromobenzenesulfonate, p-toluenesulfonate Ester, etc.; acyloxy group, such as acetoxy group, trifluoroacetoxy group, etc.
  • 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 nitrogen position of an amino group.
  • 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) ; Arylmethoxycarbonyl, such as benzyloxycarbonyl (Cbz) and 9-fluorenylmethoxycarbonyl (Fmoc); Arylmethyl, such as benzyl (Bn), trityl (Tr), 1,1-di -(4'-methoxyphenyl)methyl; silyl groups, such as trimethylsilyl (TMS) and tert-butyldimethylsilyl (TBS) and so on.
  • acyl such as alkanoyl (such as acetyl, trichloroacetyl or trifluoroacetyl); alkoxycarbonyl, such as
  • hydroxyl protecting group refers to a protecting group suitable for preventing hydroxyl side reactions.
  • Representative hydroxyl protecting groups include, but are not limited to: alkyl groups, such as methyl, ethyl, and tert-butyl; acyl groups, such as alkanoyl (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-butyldimethylsilyl (TBS), etc.
  • alkyl groups such as methyl, ethyl, and tert-butyl
  • acyl groups such as alkanoyl (such as acetyl)
  • arylmethyl groups such as benzyl (Bn), p-methyl Oxybenzyl (PMB),
  • substituted substituents independently selected from mean that when more than one hydrogen on the group is replaced by a substituent, the types of the substituents may be the same or different, so The substituents selected from are of their own independent species.
  • the compounds of the present invention, or pharmaceutically acceptable salts thereof, or stereoisomers thereof can be combined with one or more pharmaceutical carriers to form a suitable dosage form for administration.
  • dosage forms are suitable for oral, rectal, topical, intraoral, and other parenteral administration (e.g., subcutaneous, intramuscular, intravenous, etc.).
  • dosage forms suitable for oral administration include capsules, tablets, granules, syrups, etc.
  • the compounds of the present invention contained in these preparations can be solid powder or granules; solutions or suspensions in aqueous or non-aqueous liquids; water-in-oil or oil-in-water emulsions, etc.
  • the above dosage forms can be prepared from the active compound and one or more carriers or excipients through common pharmaceutical methods.
  • the above carriers need to be compatible with the active compound or other excipients.
  • commonly used non-toxic carriers include but are not limited to mannitol, lactose, starch, magnesium stearate, cellulose, glucose, sucrose, etc.
  • Carriers used for liquid preparations include water, physiological saline, glucose aqueous solution, ethylene glycol, polyethylene glycol, etc.
  • the active compounds can form solutions or suspensions with the above-mentioned carriers.
  • compositions of the present invention are formulated, dosed, and administered in a manner consistent with good medical practice.
  • the "therapeutically effective amount" of a compound administered is determined by factors such as the specific condition to be treated, the individual being treated, the cause of the condition, the target of the drug, and the mode of administration.
  • a “therapeutically effective amount” refers to an amount of a compound of the invention that will cause a biological or medical response in an individual, such as reducing or inhibiting enzyme or protein activity or ameliorating symptoms, alleviating a condition, slowing or delaying the progression of a disease, or preventing a disease, etc.
  • the pharmaceutical composition of the present invention or the therapeutically effective amount of the compound of the present invention or a pharmaceutically acceptable salt thereof or a stereoisomer thereof contained in the pharmaceutical composition is preferably 0.1 mg-5g/kg (body weight ).
  • Patient means an animal, preferably a mammal, more preferably a human being.
  • mammal refers to warm-blooded vertebrate mammals, including, for example, cats, dogs, rabbits, bears, foxes, wolves, monkeys, deer, rats, pigs, and humans.
  • Treatment means to alleviate, delay the progression, attenuate, prevent, or maintain an existing disease or condition (eg, cancer). Treatment also includes curing, preventing the progression of, or alleviating to some degree one or more symptoms of a disease or condition.
  • 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, embodiments formed by combining them with other chemical synthesis methods, and methods well known to those skilled in the art. Equivalent alternatives and preferred embodiments include, but are not limited to, embodiments of the present invention.
  • the structure of the compound of the present invention can be confirmed by conventional methods well known to those skilled in the art. If the present invention involves the absolute configuration of the compound, the absolute configuration can be confirmed by conventional technical means in the art.
  • single crystal X-ray diffraction uses a Bruker D8 venture diffractometer to collect diffraction intensity data on the cultured single crystal.
  • the light source is CuK ⁇ radiation.
  • the scanning method is: After scanning and collecting relevant data, the direct method (Shelxs97) is further used to analyze the crystal structure, and the absolute configuration can be confirmed.
  • the solvent used in the present invention is commercially available.
  • the present invention uses the following abbreviations: Pd/C represents palladium on carbon; H 2 represents hydrogen; N 2 represents nitrogen; mL represents milliliter; MeNH 2 represents dimethylamine; BBr 3 represents boron tribromide.
  • step 1
  • the reaction solution was slowly poured into saturated ammonium chloride solution (3.00mL), and extracted three times with ethyl acetate (3.00mL*3).
  • the organic phases were combined, washed once with saturated brine (3.00 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain a crude product.
  • the crude product was purified using preparative HPLC (preparative column: 3_Phenomenex Luna C18 75*30mm*3um; mobile phase: [water (TFA)-ACN]; B%: 22%-52%, 7mins).
  • step 1
  • Dissolve compound 2-3 (6.20g, 18.4mmol, 1.00eq) in tetrahydrofuran (100mL) at room temperature, cool to -20°C, and add lithium bis(trimethylsilyl)amide and 1M n-hexane solution under N2 protection. (1.00M, 55.1mL, 3.00eq), stirred at -20°C for 1 hour, dissolved compound 2-1 (3.43g, 17.8mmol, 94.1% purity, 0.970eq) in tetrahydrofuran (20.0mL) and added. Use N 2 to replace the air three times. Under N 2 conditions, monitor after reacting at -20°C for 1 hour.
  • Compound 2-4 (9.00g, 16.4mmol, 91.0% purity, 1.00eq) was dissolved in formic acid (200g, 4.34mol, 164mL, 264eq) at room temperature. Use N2 to replace the air 3 times, stir under N2 conditions at 20°C for 2 hours. The mixture was concentrated to remove most of the formic acid, methanol (160 mL) and water (40.0 mL) were added to the residue and stirred to dissolve, and lithium hydroxide monohydrate (6.89 g, 164 mmol, 10.0 eq) was added under N protection. Use N2 to replace the air three times, and monitor after reacting at 20°C for 1 hour.
  • step 1
  • Triethylamine (79.0mg, 780umol, 109uL, 3.00eq), N,N-dimethylformamide (1.00mL) and compound 3-1 (193mg, 1.56mmol, 6.00eq) were added in sequence.
  • step 1
  • step 1
  • Dissolve compound 1 (700mg, 1.69mmol, 1.00eq) in dichloromethane (7mL) at room temperature, cool to -70°C, and add boron tribromide (5.20g, 20.8mmol, 2.00mL, 12.3eq) dropwise. .
  • Dissolve compound 1 (700mg, 1.69mmol, 1.00eq) in dichloromethane (7mL) at room temperature, cool to -70°C, and add boron tribromide (5.20g, 20.8mmol, 2.00mL, 12.3eq) dropwise. .
  • step 1
  • Dissolve compound 1-8 (100mg, 324umol, 1.00eq) in tetrahydrofuran (2.00mL) at room temperature, control the temperature to -20°C, and add lithium bis(trimethylsilyl)amide and 1.00M tetrahydrofuran solution under N2 protection. (1.00M, 972uL, 3.00eq), stir at -20°C Mix for 1 hour.
  • Compound 9-2 (59.7mg, 324umol, 1.00eq) was dissolved in tetrahydrofuran (1.00mL) and added. Use N2 to replace the air three times. Under N2 conditions, monitor after reacting at -20°C for 1 hour. LCMS compound 1-8 has been completely consumed.
  • step 1
  • CDK2/CyclinE 1 was purchased from Xingenocon.
  • Ulight-4E-BP1 polypeptide, Eu-anti-phospho-tyrosine antibody, and 1X detection buffer were purchased from PerkinElmer.
  • High-purity ATP was purchased from Promega Company.
  • EDTA was purchased from Sigma. Nivo multi-label analyzer (PerkinElmer).
  • Kinase buffer contains 50mM HEPES, 1mM EDTA, 10mM MgCl2 , 0.01% Brij-35, pH7.4. Add 2.38g HEPES, 58mg EDTA, 406mg MgCl2 , 20mg Brij-35 to 200ml buffer, adjust PH to 7.4.
  • Dilute enzyme, Ulight-4E-BP1 peptide, ATP and inhibitors using kinase buffer Dilute the Eu-anti-phospho-tyrosine antibody to a concentration of 8 nM/L using detection buffer.
  • the compound to be tested was diluted 5 times to the 8th concentration using a volley gun, that is, diluted from 4 ⁇ M to 0.0512 nM.
  • the final concentration of DMSO was 4%, and a double well experiment was set up. Add 2.5 ⁇ L of each concentration gradient of inhibitor, 5 ⁇ L of CDK2/CyclinE 1 enzyme (10 ng), and 2.5 ⁇ L of substrate and ATP mixture to the microplate. (4mMATP, 100nM Ulight-4E-BP1 polypeptide).
  • the final concentration gradient of the compound is 1 ⁇ M diluted to 0.0128Nm, and the final concentrations of ATP and substrate are 1mM and 25nM.
  • the reaction system was placed at 25 degrees for 120 minutes. After the reaction, add 5 ⁇ L of stop solution to each well, and continue the reaction at 25°C for 5 minutes. After the reaction, add 5 ⁇ L of Eu-anti-phospho-tyrosine antibody diluent to each well, react at 25°C for 60 minutes, and use the PerkinElmerNivo multi-label analyzer TR.
  • -FRET mode for data collection excitation wavelength is 320nm and emission wavelengths are 615nm and 665nm).
  • IC 50 can be obtained by curve fitting with four parameters (log(inhibitor) vs. response in GraphPad Prism --Variable slope mode derived).
  • Table 1 provides the CDK2/CyclinE 1 enzymatic inhibitory activity of the compounds of the present invention.
  • CDK1/CyclinB 1 was purchased from CARNA.
  • Ulight-4E-BP1 polypeptide, Eu-anti-phospho-tyrosine antibody, and 1X detection buffer were purchased from PerkinElmer.
  • High-purity ATP was purchased from Promega Company.
  • EDTA was purchased from Sigma. Nivo multi-label analyzer (PerkinElmer).
  • Kinase buffer contains 50mM HEPES, 1mM EDTA, 10mM MgCl2, 0.01% Brij-35, pH7.4. Add 2.38g HEPES, 58mg EDTA, 406mg MgCl2, 20mg Brij-35 to 200ml buffer, and adjust the pH to 7.4.
  • the compound to be tested was diluted 5 times to the 8th concentration using a volley gun, that is, diluted from 4 ⁇ M to 0.0512 nM.
  • the final concentration of DMSO was 4%, and a double well experiment was set up.
  • the final concentration gradient of the compound is 1 ⁇ M was diluted to 0.0128nM, giving final concentrations of ATP and substrate of 1mM and 50nM.
  • the reaction system was placed at 25 degrees for 60 minutes.
  • IC 50 can be obtained by curve fitting with four parameters (log(inhibitor) vs. response in GraphPad Prism --Variable slope mode derived).
  • Table 1 provides the CDK1/CyclinB 1 enzymatic inhibitory activity of the compounds of the present invention.
  • GSK3 ⁇ Active was purchased from SignalChem; GSK3 Substrate was purchased from SignalChem; ADP-Glo Kinase Assay was purchased from Promega; Kinase assay buffer III was purchased from SignalChem; Nivo multi-label analyzer (PerkinElmer).
  • the compound to be tested was diluted with 100% DMSO to 100 ⁇ M as the first concentration, and then diluted 5 times to the eighth concentration using a volley gun, that is, diluted from 100 ⁇ M to 0.0013 ⁇ M.
  • Use 1X kinasebuffer to dilute each concentration point of the compound 20 times to prepare a compound working solution containing 5% DMSO.
  • the final concentration gradient of the compound is 1 ⁇ M and diluted to 0.013nM.
  • the final concentration of ATP and substrate is 25 ⁇ M and 0.2 ⁇ g/ ⁇ l.
  • the reaction system was placed at 25 degrees for 60 minutes. After the reaction, add 5 ⁇ l ADP-Glo reagent to each well and continue the reaction for 40 minutes at 25°C. After the reaction, add 10uL kinase detection reagent to each well. After reacting for 30 minutes at 25°C, use the PerkinElmer Nivo multi-label analyzer to read the chemiluminescence, with an integration time of 0.5 Second.
  • Example-Min Use the equation (Sample-Min)/(Max-Min)*100% to convert the original data into an inhibition rate.
  • the value of IC50 can be obtained by curve fitting with four parameters (log(inhibitor) vs. response in GraphPad Prism- -Variable slope mode).
  • Table 1 provides the GSK3 ⁇ enzymatic inhibitory activity of the compounds of the present invention.
  • Min well The negative control well reads the well containing 1% DMSO solvent.
  • 1640 medium, fetal calf serum, and penicillin/streptomycin antibiotics were purchased from Vicente.
  • CellTiter-Glo (cell viability chemiluminescence detection reagent) reagent was purchased from Promega.
  • the OVCAR3 cell line was purchased from Nanjing Kebai Biotechnology Co., Ltd. Envision multi-label analyzer (PerkinElmer).
  • OVCAR3 cells were seeded in a white 384-well plate, with 40 ⁇ L of cell suspension per well containing 300 OVCAR3 cells. cell. The cell plate was cultured overnight in a carbon dioxide incubator.
  • Example-Min Use the equation (Sample-Min)/(Max-Min)*100% to convert the original data into an inhibition rate.
  • the value of IC 50 can be obtained by curve fitting with four parameters ("log(inhibitor) vs.” in GraphPad Prism. response--Variable slope” mode).
  • Table 1 provides the inhibitory activity of the compounds of the present invention on OVCAR3 cell proliferation.
  • 1640 medium, fetal calf serum, and penicillin/streptomycin antibiotics were purchased from Vicente.
  • CellTiter-Glo (cell viability chemiluminescence detection reagent) reagent was purchased from Promega.
  • the T-47D cell line was purchased from Nanjing Kebai Biotechnology Co., Ltd. Envision multi-label analyzer (PerkinElmer).
  • T-47D cells were seeded in a white 384-well plate, and each well of 40 ⁇ L cell suspension contained 300 T-47D cells. The cell plate was cultured overnight in a carbon dioxide incubator.
  • Example-Min Use the equation (Sample-Min)/(Max-Min)*100% to convert the original data into inhibition rate.
  • the value of IC 50 can be used Obtained by curve fitting with four parameters (obtained by "log(inhibitor) vs. response--Variable slope" mode in GraphPad Prism).
  • Table 1 provides the inhibitory activity of the compounds of the present invention on T-47D cell proliferation.

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Abstract

本发明涉及一种吡唑类衍生物、药物组合物及应用。所述吡唑类衍生物具有式(I)所示的结构。所述吡唑类衍生物对CDK2的活性抑制选择性好。

Description

吡唑类衍生物、药物组合物及应用 技术领域
本发明涉及医药技术领域，特别涉及一种吡唑类衍生物、药物组合物及应用。
背景技术
细胞周期依赖性激酶(CDK)属于丝氨酸/苏氨酸激酶家族。它们参与细胞的增殖，转录等生理过程。根据CDK功能的不同，可以将其分为两大类：1)一类CDK参与细胞周期调控，主要包括CDK1、CDK2、CDK4、CDK6等；2)另一类CDK参与转录调节，主要包括CDK7、CDK8、CDK9、CDK12、CDK13等。
CDK2活性失调经常发生在多种人类癌症中，故CDK2令科研人员感兴趣。CDK2在促进G1/S转换及S期进程中起关键作用。CDK2与细胞周期蛋白E(Cyclin E)形成复合物，磷酸化视网膜母细胞瘤家族成员(pRb等)，导致E2F转录因子释放并活化，推动细胞周期自G1期至S期转换，进而使得CDK2/Cyclin A活化，促进细胞周期DNA合成、复制等过程。
Cyclin E1拷贝数增加及过表达已经在卵巢癌、胃癌、子宫内膜癌、乳腺癌及其他癌症中得到鉴定，且与对应的肿瘤不良预后正相关。在ER+乳腺癌细胞中，Cyclin E2的高表达常伴随着激素治疗耐药(Mol.Cancer Ther.,2012,11,1488-1499)，且Cyclin E的扩增或过表达与乳腺癌的不良预后有着密切关系(N.Engl.J.Med,2002,347,1566-1575)。在HER2+乳腺癌中，Cyclin E的扩增也被报道出对于曲妥珠单抗的耐药有一定的贡献(Proc.Natl.Acad.Sci.,2011,108,3671-3676)。更有报告称，Cyclin E的过表达在三阴性乳腺癌(Breast Care,2011,6,273-278)或发炎性乳腺癌(Oncotarget,2017,8,14897-14911)进展中也起到重要作用。因此，CDK2可能成为一个重要抗肿瘤靶点。
当前有少数具有CDK2活性的小分子抑制剂处于临床研究阶段，例如辉瑞的PF-3600，结构式为：其能够抑制CDK2、CDK4和CDK6，但其对其它CDK亚型也有较强的抑制活性，这样难免带来一些脱靶副作用。另外，到目前为止，还没有任何CDK2抑制剂批准，因此迫切需要开发新颖的、安全有效的、能够治疗多种癌症的CDK2抑制剂，特别是选择性靶向CDK2的小分子抑制剂，可能会具有更好的安全性。
发明内容
本发明的目的是提供一种对CDK2的活性抑制选择性好的吡唑类衍生物。
本发明第一方面提供了一种式(I)所示的化合物、或其药学上可接受的盐、或其立体异构体：
其中，
R1选自H，卤素，C1-6烷基，卤代C1-6烷基，C3-7环烷基，3至7元杂环烷基，硝基，异氰基，-(CH2)nR5；
R5选自-CN，-OR6，-NR6R7，-C(O)OR6，-C(O)R6，-S(O)2R6，-P(O)(OR6)2，-O-脂环基，-O-脂杂环基，-O-芳基，-O-杂芳基或-C(O)-杂芳基，其中所述脂环基，脂杂环基，芳基，杂芳基为未取代或经一个或多个R0取代；
R0选自卤素，C1-6烷基，卤代C1-6烷基，C3-7环烷基，3-7元杂环烷基，氰基，氨基，硝基或羟基；
R6和R7各自独立地选自H，C1-6烷基，卤素，卤代C1-6烷基，C3-7环烷基，3-7元杂环烷基，芳基或杂芳基；
n为0、1、2、3或4；
环A选自
X各自独立地为N或CR6；
R8选自H，羟基，C1-6烷基，卤代C1-6烷基，C3-7环烷基或3-7元杂环烷基；
R2和R3各自独立地选自H，C1-6烷基，卤代C1-6烷基，C3-7环烷基或3-7元杂环烷基，其中所述C3-7环烷基和3-7元杂环烷基为未取代或经一个或多个R9取代；
R9选自卤素，C1-6烷基，卤代C1-6烷基，氰基，氨基，硝基或羟基；
R4选自H，卤素，氨基，羟基或C1-C6烷基；
式(I)所示的结构中一个或多个氢原子被氘原子取代或未取代；
所述化合物不具有如下所示结构：
在一些实施例中，当式(I)所示的结构中，所述R1选自-CH2-O-CH3，环A选自 R2选自H，R3选自-CH(CH3)2，R4选自H时，式(I)所示的结构中一个或多个氢原子被氘原子取代。
可以理解地，当R1、环A、R2、R3和R4选自式(I)所示的结构中的其它基团时，式(I)所示的结构中一个或多个氢原子被氘原子取代或未取代。
在一些实施例中，所述R1选自-CH2R5，所述R5选自-OR61，-NR6R7，-CN，-C(O)OR6，-C(O)R6，-S(O)2R6，-P(O)(OR6)2，-O-脂环基，-O-脂杂环基，-O-芳基，-O-杂芳基或-C(O)-杂芳基，其中所述脂环基，脂杂环基，芳基，杂芳基为未取代或经一个或多个R0取代；
所述R61选自H，一个或多个氢原子被氘原子取代的C1-6烷基，卤代C1-C6烷基，C3-7环烷基，3-7元杂环烷基，芳基或杂芳基。
进一步地，所述R1选自-CH2R5，所述R5选自-OR61，-NR6R7或-CN。
在一些优选的实施例中，所述R1选自-CH2R5，所述R5选自-OR61或-NR6R7；
所述R61选自H，一个或多个氢原子被氘原子取代的C1-6烷基或卤代C1-C6烷基；
所述R6和R7各自独立地选自H，C1-6烷基或卤代C1-6烷基。
进一步优选地，所述R1为-OH。
在一些优选的实施例中，所述R2为H，R3选自C1-6烷基或卤代C1-C6烷基。
进一步优选地，所述R3为-C(CH3)3。
在一些优选的实施例中，所述R1选自-(CH2)nR5，所述R5中一个或多个氢原子被氘原子取代。进一步优选地，R5选自-OR6。
在一些优选的实施例中，式(I)所示的结构选自式(I-1)～(I-3)任一式所示的结构：

进一步优选地，式(I)所示的结构选自式(I-4)所示的结构：
在一些优选的实施例中，X选自CR6。进一步优选地，R6选自H，C1-6烷基，卤素或卤代C1-6烷基。
在一些优选的实施例中，R1选自H，卤素，C3-7环烷基，3至7元杂环烷基，硝基，异氰基，-(CH2)nR5；
进一步优选地，n为1，所述R1为-CH2R5，此时，式(I)所示的结构选自式(I-5)所示的结构：
在一些优选的实施例中，X选自CR6。进一步优选地，R6选自H，C1-6烷基，卤素或卤代C1-6烷基。
进一步优选地，所述R5选自-CN，-OR6或-NR6R7；
所述R6和R7各自独立地选自H，C1-6烷基或卤代C1-6烷基。
在一些实施例中，R5选自-CN，-OC1-3烷基，-N(H)C1-3烷基，-N(C1-3烷基)(C1-3烷基)。
在一些优选的实施例中，所述R2选自H，C1-C6烷基或卤代C1-C6烷基；
所述R3选自H，C1-C6烷基，卤代C1-C6烷基，C3-7环烷基或3-7元杂环烷基，其中所述C3-7环烷基和3-7元杂环烷基为未取代或经一个或多个R9取代。
进一步优选地，所述R2选自H；
所述R3选自H，C1-C6烷基，卤代C1-C6烷基，未取代或经一个R9取代的5元杂环烷基。
进一步优选地，所述R9选自卤素，C1-6烷基或卤代C1-6烷基；所述5元杂环烷基的杂原子为氧原子。
进一步优选地，所述式(I)中选自如下结构中的一种：
在一些优选的实施例中，R4选自H，F或甲基。进一步优选地，R4选自H。
在一些优选的实施例中，R0选自C3-7环烷基，3-7元杂环烷基，氰基，氨基，硝基或羟基。
在一些优选的实施例中，R8选自H，C1-6烷基，卤代C1-6烷基，C3-7环烷基或3-7元杂环烷基。
在一些实施例中，所述化合物包括的结构但不限于以下结构：
在一些实施例中，所述药学上可接受的盐为烷基酸盐。进一步地，所述药学上可接受的盐为甲酸盐。
本发明第二方面提供了一种药物组合物，其包括本发明第一方面所述的化合物、或其药学上可接受的盐、或其立体异构体，以及药学可接受的载体。
本发明第三方面提供了本发明第一方面所述的化合物、或其药学上可接受的盐、或其立体异构体以及本发明第三方面所述的药物组合物在制备治疗或预防与CDK2活性相关的或由CDK2活性介导的疾病的药物中的应用。
在某些实施方案中，CDK2活性相关的或由CDK2活性介导的疾病是癌症。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
本发明人经过长期而深入的研究，意外地发现了一种如式(I)所示的化合物。所述的化合物对CDK2具有出乎意料的抑制活性，可用于治疗Cyclin E高表达的各类癌症，特别是CDK4/6抑制剂耐药的癌症病人具有优异的治疗效果。基于上述发现，发明人完成了本发明。
具体实施方式
术语定义
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。
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，所述有机酸包括如甲酸、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，用楔形实线键和楔形虚线键表示一个立体中心的绝对构型，用直形实线键和直形虚线键表示立体中心的相对构型，用波浪线表示楔形实线键或楔形虚线键或用波浪线表示直形实线键和直形虚线键
除非另有说明，当化合物中存在双键结构，如碳碳双键、碳氮双键和氮氮双键，且双键上的各个原子均连接有两个不同的取代基时(包含氮原子的双键中，氮原子上的一对孤对电子视为其连接的一个取代基)，如果该化合物中双键上的原子与其取代基之间用波浪线连接，则表示该化合物的(Z)型异构体、(E)型异构体或两种异构体的混合物。例如下式(A) 表示该化合物以式(A-1)或式(A-2)的单一异构体形式存在或以式(A-1)和式(A-2)两种异构体的混合物形式存在；下式(B)表示该化合物以式(B-1)或式(B-2)的单一异构体形式存在或以式(B-1)和式(B-2)两种异构体的混合物形式存在。下式(C)表示该化合物以式(C-1)或式(C-2)的单一异构体形式存在或以式(C-1)和式(C-2)两种异构体的混合物形式存在。
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。例如本发明中， 为互变异构体。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。
术语“取代”是指特定原子上的任意一个或多个氢原子被取代基取代，取代基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。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。
当任何变量(例如R1)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R1所取代，则所述基团可以任选地至多被两个R1所取代，并且每种情况下的R1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，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。当该化学键的连接方式是不定位的，且可连接位点存在H原子时，则连接化学键时，该位点的H原子的个数会随所连接化学键的个数而对应减少变成相应价数的基团。所述位点与其他基团连接的化学键可以用直形实线键直形虚线键或波浪线表示。例如-OCH3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；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；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；表示该哌啶基上的任意可连接位点可以通过1个化学键与其他基团相连，至少包括 这4种连接方式，即使-N-上画出了H原子，但是仍包括这种连接方式的基团，只是在连接1个化学键时，该位点的的H会对应减少1个变成相应的一价哌啶基。
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，“3-7元环”是指环绕排列3-7个原子的“环”。
除非另有规定，术语“C1-6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。优选为C1-4烷基，其可以是一价(如甲基)、二价(如亚甲基)或者多价(如次甲基)。C1-3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)、丁基(包 括正丁基、异丁基、叔丁基和仲丁基)。
除非另有规定，术语“C1-6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6个碳原子的烷基基团。优选为C1-3烷氧基。C1-3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，术语“C1-6烷氨基”表示通过氨基连接到分子的其余部分的那些包含1至6个碳原子的烷基基团。优选为C1-3烷氨基。C1-3烷氨基的实例包括但不限于-NHCH3、-N(CH3)2、-NHCH2CH3、-N(CH3)CH2CH3、-NHCH2CH2CH3、-NHCH2(CH3)2等。
除非另有规定，术语“卤代素”或“卤素”本身或作为另一取代基的一部分表示氟、氯、溴或碘原子。
除非另有规定，本发明术语“5杂芳环”和“5元杂芳基”可以互换使用，术语“5元杂芳基”表示由5环原子组成的具有共轭π电子体系的单环基团，其1、2、3或4个环原子为独立选自O、S和N的杂原子，其余为碳原子。其中氮原子任选地被季铵化，氮和硫杂原子可任选被氧化(即NO和S(O)p，p是1或2)。5元杂芳基可通过杂原子或碳原子连接到分子的其余部分。所述5元杂芳基的实例包括但不限于吡咯基(包括N-吡咯基、2-吡咯基和3-吡咯基等)、吡唑基(包括2-吡唑基和3-吡唑基等)、咪唑基(包括N-咪唑基、2-咪唑基、4-咪唑基和5-咪唑基等)、噁唑基(包括2-噁唑基、4-噁唑基和5-噁唑基等)、三唑基(1H-1,2,3-三唑基、2H-1,2,3-三唑基、1H-1,2,4-三唑基和4H-1,2,4-三唑基等)、四唑基、异噁唑基(3-异噁唑基、4-异噁唑基和5-异噁唑基等)、噻唑基(包括2-噻唑基、4-噻唑基和5-噻唑基等)、呋喃基(包括2-呋喃基和3-呋喃基等)、噻吩基(包括2-噻吩基和3-噻吩基等)。
除非另有规定，Cn-n+m或Cn-Cn+m包括n至n+m个碳的任何一种具体情况，例如C1-12包括C1、C2、C3、C4、C5、C6、C7、C8、C9、C10、C11、和C12，也包括n至n+m中的任何一个范围，例如C1-12包括C1-3、C1-6、C1-9、C3-6、C3-9、C3-12、C6-9、C6-12、和C9-12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。
除非另有规定，“C3-7环烷基”表示由3至7个碳原子组成的饱和环状碳氢基团，其包括单环和双环体系，其中双环体系包括螺环、并环和桥环。所述C3-7环烷基包括C3-6、C4-6、C4-5、C5-7或C5-6环烷基等；其可以是一价、二价或者多价。C3-7环烷基的实例包括，但不限于，环丙基、环丁基、环戊基、环己基、环庚基等。
除非另有规定，术语“3-7元杂环烷基”本身或者与其他术语联合分别表示由3至7个环原 子组成的饱和环状基团，其1、2、3或4个环原子为独立选自O、S和N的杂原子，其余为碳原子，其中氮原子任选地被季铵化，氮和硫杂原子可任选被氧化(即NO和S(O)p，p是1或2)。其包括单环和双环体系，其中双环体系包括螺环、并环和桥环。此外，就该“3-7元杂环烷基”而言，杂原子可以占据杂环烷基与分子其余部分的连接位置。所述3-7元杂环烷基包括5-7元、3元、4元、5元、6元和7元杂环烷基等。3-7元杂环烷基的实例包括但不限于氮杂环丁基、氧杂环丁基、硫杂环丁基、吡咯烷基、吡唑烷基、咪唑烷基、四氢噻吩基(包括四氢噻吩-2-基和四氢噻吩-3-基等)、四氢呋喃基(包括四氢呋喃-2-基等)、四氢吡喃基、哌啶基(包括1-哌啶基、2-哌啶基和3-哌啶基等)、哌嗪基(包括1-哌嗪基和2-哌嗪基等)、吗啉基(包括3-吗啉基和4-吗啉基等)、二噁烷基、二噻烷基、异噁唑烷基、异噻唑烷基、1,2-噁嗪基、1,2-噻嗪基或六氢哒嗪基等。
除非另有规定，术语“3-7元含氮杂环烷基”表示为至少含有一个N原子的3-7元杂环烷基。
脂环基是指饱和或部分不饱和的全碳环系统。其中“部分不饱和”是指包括至少一个双键或三键的环部分，“部分不饱和”意图涵盖具有多个不饱和位点的环，但并不意图包括如本文所定义的芳基或杂芳基部分。非限制性实施例包括环丙基环、环丁基环、环戊基环、环戊烯基环、环己基环、环己烯基环、环己二烯基环、环庚基环、环庚三烯基环、环戊酮环、环戊烷-1,3-二酮环等。
脂杂环基是指饱和或部分不饱和脂环基中的1、2或3个环碳原子被选自氮、氧或S(O)t(其中t是整数0至2)的杂原子所取代，但不包括-O-O-、-O-S-或-S-S-的环部分，其余环原子为碳。非限制性实施例包括环氧丙烷环、氮杂环丁烷环、氧杂环丁烷环、四氢呋喃环、四氢噻吩环、四氢吡咯环、哌啶环、吡咯啉环、噁唑烷环、哌嗪环、二氧戊环、二氧六环、吗啉环、硫代吗啉环、硫代吗啉-1,1-二氧化物、四氢吡喃环、氮杂环丁烷-2-酮环、氧杂环丁烷-2-酮环、吡咯烷-2-酮环、吡咯烷-2,5-二酮环、哌啶-2-酮环、二氢呋喃-2(3H)-酮环、二氢呋喃-2,5-二酮环、四氢-2H-吡喃-2-酮环、哌嗪-2-酮环、吗啉-3-酮环。部分不饱和单杂环的非限制性实施例包括1,2-二氢氮杂环丁二烯环、1,2-二氢氧杂环丁二烯环、2,5-二氢-1H-吡咯环、2,5-二氢呋喃环、2,3-二氢呋喃环、2,3-二氢-1H-吡咯环、3,4-二氢-2H-吡喃环、1,2,3,4-四氢吡啶环、3,6-二氢-2H-吡喃环、1,2,3,6-四氢吡啶环、4,5-二氢-1H-咪唑环、1,4,5,6-四氢嘧啶环、3,4,7,8-四氢-2H-1,4,6-噁二唑嗪环、1,6-二氢嘧啶环、4,5,6,7-四氢-1H-1,3-二氮杂环、2,5,6,7-四氢-1,3,5-噁二氮杂环等。
“芳基”和“芳环”可互换使用，均指具有共轭的π电子体系的全碳单环或稠合多环(也就是共享毗邻碳原子对的环)基团，该基团可以与环烷基环、杂环烷基环、环烯基环、杂环烯基环或杂芳基稠合。“C6-10芳基”指具有6至10个碳原子的单环或双环芳基，芳基的非限制性实施 例包括苯基、萘基等。
“杂芳基”和“杂芳基环”可互换使用，均指具有环碳原子和环杂原子的单环、双环或多环的4n+2芳族环体系(例如，具有以环状排列共享的6或10个π电子)的基团，其中每个杂原子独立地选自氮、氧和硫。本发明中，杂芳基还包括其中上述杂芳基环与一个或多个环烷基环、杂环烷基环、环烯基环、杂环烯基环或芳环稠合的环系统。杂芳基环可以任选地被取代。“5至10元杂芳基”是指具有5至10个环原子，其中1、2、3或4个环原子为杂原子的单环或双环杂芳基。“5至6元杂芳基”是指具有5至6个环原子，其中1、2、3或4个环原子为杂原子的单环杂芳基，非限制性实施例包括噻吩基、呋喃基、噻唑基、异噻唑基、咪唑基、噁唑基、吡咯基、吡唑基、三唑基、1,2,3-三唑基、1,2,4-三唑基、1,2,5-三唑基、1,3,4-三唑基、四唑基、异噁唑基、噁二唑基、1,2,3-噁二唑基、1,2,4-噁二唑基、1,2,5-噁二唑基、1,3,4-恶二唑基、噻二唑基、吡啶基、哒嗪基、嘧啶基、吡嗪基、三嗪基、四嗪基。“8至10元杂芳基”是指具有8至10个环原子，其中1、2、3或4个环原子为杂原子的双环杂芳基，非限制性实施例包括吲哚基、异吲哚基、吲唑基、苯并三唑基、苯并噻吩基、异苯并噻吩基、苯并呋喃基、苯并异呋喃基、苯并咪唑基、苯并噁唑基、苯并异噁唑基、苯并噁二唑基、苯并噻唑基、苯并异噻唑基、苯并噻二唑基、茚嗪基、嘌呤基、吡啶并[3,2-d]嘧啶基、吡啶并[2,3-d]嘧啶基、吡啶并[3,4-d]嘧啶基、吡啶并[4,3-d]嘧啶基、1,8-萘啶基、1,7-萘啶基、1,6-萘啶基、1,5-萘啶基、喋啶基、喹啉基、异喹啉基、噌琳基、喹喔啉基、酞嗪基和喹唑啉基。“杂原子”是指氮、氧或硫。在含有一个或多个氮原子的杂芳基中，只要化合价允许，连接点可以是碳或氮原子。杂芳基双环系统在一个或两个环中可以包括一个或多个杂原子。
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(例如亲核取代反应)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。
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。
除非另有定义，本发明所述“各自独立地选自……的取代基”是指当基团上的一个以上的氢被取代基取代时，所述的取代基种类可以相同或不同，所选自的取代基为各自独立的种类。
通常本发明化合物或其药学可接受的盐、或其立体异构体可以与一种或多种药用载体形成适合的剂型施用。这些剂型适用于口服、直肠给药、局部给药、口内给药以及其他非胃肠道施用(例如，皮下、肌肉、静脉等)。例如，适合口服给药的剂型包括胶囊、片剂、颗粒剂以及糖浆等。这些制剂中包含的本发明的化合物可以是固体粉末或颗粒；水性或非水性液体中的溶液或是混悬液；油包水或水包油的乳剂等。上述剂型可由活性化合物与一种或多种载体或辅料经由通用的药剂学方法制成。上述的载体需要与活性化合物或其他辅料兼容。对于固体制剂，常用的无毒载体包括但不限于甘露醇、乳糖、淀粉、硬脂酸镁、纤维素、葡萄糖、蔗糖等。用于液体制剂的载体包括水、生理盐水、葡萄糖水溶液、乙二醇和聚乙二醇等。活性化合物可与上述载体形成溶液或是混悬液。
本发明的组合物以符合医学实践规范的方式配制，定量和给药。给予化合物的“治疗有效量”由要治疗的具体病症、治疗的个体、病症的起因、药物的靶点以及给药方式等因素决定。
“治疗有效量”是指将引起个体的生物学或医学响应，例如降低或抑制酶或蛋白质活性或改善症状、缓解病症、减缓或延迟疾病进程或预防疾病等的本发明化合物的量。
本发明的所述药物组合物或所述药用组合物中含有的本发明化合物或其药学上可接受的盐、或其立体异构体的治疗有效量优选为0.1mg-5g/kg(体重)。
“患者”是指一种动物，最好为哺乳动物，更好的为人。术语“哺乳动物”是指温血脊椎类哺乳动物，包括如猫、狗、兔、熊、狐狸、狼、猴子、鹿、鼠、猪和人类。
“治疗”是指减轻、延缓进展、衰减、预防，或维持现有疾病或病症(例如癌症)。治疗还包括将疾病或病症的一个或多个症状治愈、预防其发展或减轻到某种程度。
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。
本发明的化合物可以通过本领域技术人员所熟知的常规方法来确认结构，如果本发明涉及化合物的绝对构型，则该绝对构型可以通过本领域常规技术手段予以确证。例如单晶X射线衍射法(SXRD)，把培养出的单晶用Bruker D8 venture衍射仪收集衍射强度数据，光源为CuKα辐射，扫描方式：扫描，收集相关数据后，进一步采用直接法(Shelxs97)解析晶体结构，便可以确证绝对构型。
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：Pd/C代表钯碳；H2代表氢气；N2代表氮气；mL代表毫升；MeNH2代表二甲胺；BBr3代表三溴化硼。
化合物依据本领域常规命名原则或者使用软件命名，市售化合物采用供应商目录名称。
下面经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。
化合物1
第1步：
将化合物1-1溶于四氢呋喃(2mL)，使用氮气置换空气3次。将体系降温至0-10℃，氮气环境下缓慢加二异丁基氢化铝(1M,4.43mL,5.00eq)。20-30℃反应3小时后监测。LCMS显示化合物1-1消耗完全，监测到一个目标产物峰(m/z＝183.9)。冰水浴条件下向反应液中以 此加入水(0.70mL)、15％氢氧化钠水溶液(0.70mL)和水(2.00mL)。反应搅拌10分钟后加入无水硫酸钠干燥，过滤，浓缩滤液得到粗产品化合物1-2。LCMS:m/z(M+H)+＝183.9。1H NMR:(400MHz,DMSO-d6)δ8.78(d,J＝4.6Hz,1H),7.72(d,J＝4.8Hz,1H),6.92(s,1H),5.51(t,J＝5.8Hz,1H),4.70(d,J＝5.8Hz,2H)。
第2步：
将化合物1-2溶于四氢呋喃(2.00mL)，使用氮气置换空气3次，降温至0-10℃。氮气环境下，向反应液加入氢化钠(28.3mg,708umol,60％纯度,1.30eq)；反应30分钟后，加入碘甲烷(116mg,817umol,50.9uL,1.50eq)。10-20℃反应1小时后监测。LCMS显示化合物1-2消耗完全，监测到一个目标产物峰(m/z＝198.5)。将反应液缓慢倒入饱和氯化铵溶液(3.00mL)，乙酸乙酯(3.00mL*3)萃取三次。合并有机相，用饱和食盐水(3.00mL)洗涤一次，用无水硫酸钠干燥，过滤，浓缩滤液得到粗产品。粗产品使用制备HPLC(制备柱:3_Phenomenex Luna C18 75*30mm*3um；mobile phase:[water(TFA)-ACN]；B％:22％-52％,7mins)纯化。使用碳酸钠调节制备液pH值至7-8，用乙酸乙酯(10.0mL*2)萃取三次，用无水硫酸钠干燥，过滤，浓缩滤液得到化合物1-3。LCMS:m/z(M+H)+＝198.5.1H NMR:(400MHz,DMSO-d6)δ8.81(dd,J＝4.71,0.92Hz,1H),7.76(d,J＝4.77Hz,1H),6.99(s,1H),4.64(s,2H),3.34(s,3H)。
第3步：
氮气保护下将化合物1-4(300mg,839umol,1.00eq)和对硝基苯甲酰氯(219mg,1.09mmol,1.30eq)溶于二氯甲烷(3.00mL)。将反应体系降温至0℃，依次加入吡啶(199mg,2.52mmol,203uL,3.00eq)和4-二甲氨基吡啶(10.3mg,83.9umol,0.10eq)。反应2小时后监测，TLC(石油醚:乙酸乙酯＝2:1)显示化合物1-4(Rf＝0.20)消耗完全并且有一个主点(Rf＝0.70)生成。将反应液浓缩，得到化合物1-6。LCMS:m/z(M+H)+＝523.3。
第4步：
将化合物1-6(600mg,1.15mmol,1.00eq)溶于二氯甲烷(6.00mL)，依次加入N,N-二异丙基乙胺(594mg,4.59mmol,800uL,4.00eq)和异丙胺(204mg,3.44mmol,296uL,3.00eq)。25℃反应1小时后监测，TLC(石油醚:乙酸乙酯＝2:1)显示化合物1-6(Rf＝0.70)消耗完全，一个主要新点(Rf＝0.30)生成。将反应液缓慢导入水中(3.00mL)，用二氯甲烷(3.00mL*2)萃取2次。合并有机相，饱和食盐水(3.00mL*2)洗涤两次，无水硫酸钠干燥，过滤，浓缩滤液得到粗产品。粗产品使用柱层析纯化(300-400目硅胶,石油醚:乙酸乙酯＝100:1至2:1,产物:Rf＝0.3)得到化合物1-7。LCMS:m/z(M+H)+＝443.3。
第5步：
将化合物1-7(300mg,678umol,1.00eq)溶于无水四氢呋喃(3.00mL)，使用氮气置换三次 空气，加入湿钯碳(30.0mg,50％纯度)。使用氢气(15psi)置换氮气三次。25℃和氢气(15psi)条件下反应2小时后监测。TLC(石油醚:乙酸乙酯＝1:1)显示，化合物1-7(Rf＝0.70)消耗完全，一个主要新点(Rf＝0.40)生成。过滤反应液，浓缩滤液得到化合物1-8。LCMS:m/z(M+H)+＝309.3。
第6步：
将化合物1-8(312.mg,1.01mmol,2.50eq),化合物1-3(0.08g,404umol,1.00eq)溶于二氧六环(2.00mL),依次加入三(二亚苄基丙酮)二钯(37.1mg,40.5umol,0.10eq),碳酸钠(85.8mg,810umol,2.00eq)和4,5-双二苯基膦-9,9-二甲基氧杂氧杂蒽杂蒽(46.9mg,81.0umol,0.20eq)。使用氮气置换空气3次，50-60℃反应12小时后监测。LCMS显示化合物1-8消耗完全，监测到一个目标产物峰(m/z＝470.3)。将反应液过滤，浓缩滤液得到粗产品。粗产品使用柱层析纯化(300-400目硅胶,石油醚:乙酸乙酯＝10:1至1:1)得到化合物1-9。LCMS:m/z(M+H)+＝470.4。
第7步：
将化合物1-9(60.0mg,128umol,1.00eq)溶于无水甲酸(3.00mL)。100℃反应16小时厚监测。TLC(石油醚:乙酸乙酯＝1:2)显示化合物1-9(Rf＝0.60)消耗完全，一个新点(Rf＝0.10)生成。将反应液浓缩去除甲酸，加入二氯甲烷(10.0mL)稀释，使用饱和碳酸氢钠水溶液洗涤两次和饱和食盐水洗涤一次，使用无水硫酸钠干燥，过滤，浓缩滤液得到粗产品。粗产品使用柱层析纯化(300-400目硅胶,石油醚:乙酸乙酯＝5:1至0:1,产物:石油醚:乙酸乙酯＝1:2,Rf＝0.10)得到化合物1。LCMS:m/z(M+H)+＝414.3。1H NMR:(400MHz,CDCl3)δ7.86(m,1H)7.36-7.51(m,1H)6.71-6.87(m,1H)6.48-6.69(m,1H)5.09-5.29(m,1H)4.71(s,1H)4.65(s,2H)3.66-3.90(m,1H)3.44(s,3H)3.13-3.28(m,1H)2.43-2.61(m,1H)2.11-2.22(m,1H)1.87-1.98(m,4H)1.09-1.16(m,6H)。
化合物2

第1步：
室温下，将化合物1-3(5.00g,25.3mmol,100％purity,1.00eq)和氟化钾(7.35g,127mmol,2.96mL,5.00eq)溶于N,N-二甲基甲酰胺(50.0mL)。使用N2置换空气3次，在N2条件下，140℃反应16小时后监测，LCMS显示化合物1-3消耗完全并且有一个产物峰(m/z＝182.0(M+H)+)生成。在反应液中加入水(50.0mL)，用乙酸乙酯(100mL*3)萃取3次。合并有机相，饱和氯化钠水溶液(100mL*3)洗涤3次，无水硫酸钠干燥，过滤，浓缩滤液得到粗产品。使用柱层析纯化(SiO2,石油醚:乙酸乙酯＝5:1至2:1,产物:Rf＝0.60)得到将化合物2-1。LCMS:m/z＝181.9(M+H)+。
第2步：
室温下，将化合物1-4(8.00g,22.4mmol,1.00eq)和咪唑(2.29g,33.6mmol,1.50eq)溶于N,N-二甲基甲酰胺(80.0mL)，再加入二甲基叔丁基氯硅烷(5.06g,33.6mmol,4.11mL,1.50eq)。置换N2三次后，N2条件下20℃反应6小时后监测，LCMS显示化合物1-4消耗完全并且监测到一个目标产物峰(m/z＝472.1(M+H)+)。将反应液缓慢倒入水中(80.0mL)，用乙酸乙酯(80.0mL*3)萃取3次。合并有机相，用饱和氯化钠水溶液(50.0mL*3)洗涤3次，无水硫酸钠干燥，过滤，浓缩滤液得到粗产品。粗产品使用柱层析纯化(SiO2,石油醚:乙酸乙酯＝5:1至2:1,产物:Rf＝0.50)得到化合物2-2。LCMS:m/z＝472.3(M+H)+。
第3步：
室温下，将四氢呋喃(100mL)和乙酸乙酯(100mL)加入500ml三颈烧瓶，在N2保护下依次加入湿钯碳(1.89g,1.78mmol,1.91mL,10.0％purity)和化合物2-2(9.50g,20.0mmol,99.5％purity,1.00eq)。置换氢气三次。在氢气(15.0Psi)条件下，20℃反应3小时后监测，LCMS显示化合物2-2消耗完全并且监测到一个目标产物峰(m/z＝338.0(M+H)+)。过滤，浓缩滤液得到化合物2-3。LCMS:m/z＝338.0(M+H)+。
第4步：
室温下，将化合物2-3(6.20g,18.4mmol,1.00eq)溶于四氢呋喃(100mL)，降温至-20℃，N2保护下加入二(三甲基硅)氨基锂，1M正己烷溶液(1.00M,55.1mL,3.00eq)，-20℃下搅拌1小时，将化合物2-1(3.43g,17.8mmol,94.1％purity,0.970eq)溶于四氢呋喃(20.0mL)后加入。使用N2置换空气3次，在N2条件下，-20℃反应1小时后监测，LCMS显示化合物3消耗完全并且监测到一个目标产物峰(m/z＝499.2(M+H)+)。将反应液缓慢倒入饱和氯化铵水溶液中(300mL)，用乙酸乙酯(120mL*3)萃取3次。合并有机相，饱和氯化钠水溶液(120mL*2)洗涤2次，无水硫酸钠干燥，过滤，浓缩滤液得到粗产品。粗产品使用柱层析纯化(SiO2,石油醚:乙酸乙酯＝5:1至1:1,产物:Rf＝0.50)得到化合物2-4。LCMS:m/z＝499.2(M+H)+1H NMR:(400MHz,DMSO-d6)δ9.00(s,1H),8.04(d,J＝4.4Hz,1H),7.30(d,J＝4.4Hz,1H),6.85-6.77(m,1H),6.01(s,1H),4.56(s,2H),4.37-4.25(m,1H),3.33-3.31(m,3H),3.04-2.91(m,1H),2.31-2.21(m,1H),1.96-1.86(m,1H),1.85-1.72(m,2H),1.65-1.59(m,1H),1.57-1.52(m,1H),1.49(s,9H),0.83(s,9H),0.05-0.02(m,6H)。
第5步：
室温下，将化合物2-4(9.00g,16.4mmol,91.0％purity,1.00eq)溶于甲酸(200g,4.34mol,164mL,264eq)。使用N2置换空气3次，N2条件下，20℃搅拌2小时。浓缩混合物除去大部分甲酸，在残留物中加入甲醇(160mL)和水(40.0mL)搅拌使其溶解，在N2保护下加入一水合氢氧化锂(6.89g,164mmol,10.0eq)。使用N2置换空气3次，20℃反应1小时后监测。LCMS显示化合物2-4消耗完全，监测到一个目标产物峰(m/z＝385.1(M+H)+)。将反应液缓慢倒入水中(450mL)，用乙酸乙酯(160mL*3)萃取3次。合并有机相，无水硫酸钠干燥，过滤，浓缩滤液得到粗产品。使用柱层析纯化(SiO2,石油醚:乙酸乙酯＝50:1至0:1,产物:Rf＝0.50)得到化合物2-5。LCMS:m/z＝385.1(M+H)+。
第6步：
室温下，将化合物2-5(101mg,260umol,98.8％purity,1.00eq)溶于2-甲基四氢呋喃(1.00mL)，依次加入1,1-羰基二咪唑(127mg,780umol,3.00eq)和4-二甲胺基吡啶(6.36mg,52.0umol,0.200eq)。使用N2置换空气3次，在N2条件下，20℃搅拌1小时。依次加入化合物2-6(237mg,2.34mmol,9.00eq)和三乙胺(263mg,2.60mmol,362uL,10.0eq)。使用N2置换空 气3次，在N2条件下，20℃反应24小时后监测，LCMS显示化合物2-5消耗完全并且监测到一个目标产物峰(m/z＝512.1(M+H)+)。将反应液缓慢倒入水中(5.00mL)，用乙酸乙酯(10.0mL*3)萃取3次。合并有机相，饱和氯化钠水溶液(10.0mL)洗涤1次，无水硫酸钠干燥，过滤，浓缩滤液得到化合物2-7。LCMS:m/z＝512.1(M+H)+。
第7步：
室温下，将化合物2-7(159mg,311umol,1.00eq)溶于甲酸(3.55g,77.1mmol,2.91mL,248eq)。使用N2置换空气3次，在N2条件下，80℃反应1小时后监测，LCMS显示化合物2-7消耗完全，监测到一个目标产物峰(m/z＝456.1(M+H)+)。浓缩混合物除去大部分甲酸，在残留物中加入二氯甲烷(10.0mL)使其全部溶解后，用饱和碳酸氢钠水溶液(20.0mL*3)和饱和氯化钠水溶液(20.0mL*3)分别萃取3次，无水硫酸钠干燥，过滤，浓缩滤液得到粗产品。粗产品使用HPLC(制备柱:Phenomenex luna C18 150*25mm*10um；mobile phase:[water(FA)-ACN]；B％:9％-39％,10min)制备纯化冻干，得到化合物2。LCMS:m/z＝456.1(M+H)+.1H NMR:(400MHz,MeOD)δ7.93(d,J＝4.4Hz,1H),7.39(s,1H),7.07(s,1H),6.30(s,1H),5.09(s,1H),4.64(s,2H),3.98-3.91(m,1H),3.90-3.82(m,2H),3.57(d,J＝8.8Hz,1H),3.43(s,3H),3.24-3.12(m,1H),2.61-2.50(m,1H),2.27-2.08(m,2H),2.01-1.81(m,5H),1.42(s,3H)。
化合物3
第1步：
室温下，将化合物2-5(101mg,260umol,98.8％purity,1.00eq)溶于2-甲基四氢呋喃(1.00mL)。依次加入1,1-羰基二咪唑(127mg,780umol,3.00eq)和4-二甲胺基吡啶(6.36mg,52.0umol,0.200eq)。使用N2置换空气3次，在N2条件下，20℃反应1小时。再依次加入三乙胺(79.0mg,780umol,109uL,3.00eq)，N,N-二甲基甲酰胺(1.00mL)和化合物3-1(193mg,1.56mmol,6.00eq)。使用N2置换空气3次，在N2条件下，80℃反应16小时监测，TLC(石油醚:乙酸乙酯＝0:1)显示化合物2-5(Rf＝0.50)消耗完全并且有一个主点(Rf＝0.70)生成。将反应液倒入水中(6.00mL)，用乙酸乙酯(10.0mL*3)萃取3次。合并有机相，饱和氯化钠水溶液(10.0 mL)洗涤1次，无水硫酸钠干燥，过滤，浓缩滤液得到化合物3-2。
第2步：
室温下，将化合物3-2(180mg,362umol,1.00eq)溶于甲酸(4.13g,89.7mmol,3.38mL,248eq)。使用N2置换空气3次，在N2条件下，80℃反应1小时后监测，LCMS显示化合物9消耗完全，监测到一个目标产物峰(m/z＝442.1(M+H)+)。浓缩混合物除去大部分甲酸，在残留物中加入二氯甲烷(10.0mL)使其全部溶解后，用饱和碳酸氢钠水溶液(20.0mL*3)和饱和氯化钠水溶液(20.0mL*3)分别萃取3次，无水硫酸钠干燥，过滤，浓缩滤液得到粗产品。粗产品使用HPLC(制备柱:Phenomenex luna C18 150*25mm*10um；mobile phase:[water(FA)-ACN]；B％:6％-36％,10min)制备纯化冻干，得到化合物3。LCMS:m/z＝442.2(M+H)+.1H NMR:(400MHz,DMSO)δ12.26-12.03(m,1H),10.07-9.89(m,1H),8.01(d,J＝4.8Hz,1H),7.41-7.32(m,2H),7.27(s,1H),6.70-6.46(m,1H),5.12-4.96(m,1H),4.56(s,2H),4.10-3.95(m,1H),3.73(dd,J＝6.0,8.8Hz,2H),3.64(d,J＝7.2Hz,1H),3.42(dd,J＝4.0,8.4Hz,1H),3.32(s,3H),3.19-3.02(m,1H),2.12-1.60(m,8H)。
化合物4
第1步：
室温下，将化合物2-5(170mg,437umol,98.8％purity,1.00eq)溶于二氯甲烷(2.00mL)，降温至0℃。依次加入吡啶(104mg,1.31mmol,106uL,3.00eq),4-二甲氨基吡啶(5.34mg,43.7umol,0.100eq)和4-硝基苯酚氯甲酸酯(114mg,568umol,1.30eq)。使用N2置换空气3次，在N2条件下，20℃反应0.5小时。再加入化合物4-1(639mg,8.74mmol,918uL,20.0eq)，用N2置换空气3次，在N2条件下，20℃反应2小时后监测，LCMS显示化合物5仍有残留(m/z＝385.0(M+H)+)，并且有一个产物峰(m/z＝484.2(M+H)+)生成。将反应液倒入水中(5.00mL)，用乙酸乙酯(10.0mL*3)萃取3次。合并有机相，饱和氯化钠水溶液(10.0mL)洗涤1次，无水 硫酸钠干燥，过滤，浓缩滤液得到粗产品。使用柱层析纯化(SiO2,石油醚:乙酸乙酯＝5:1至1:1,产物:Rf＝0.50)得打化合物4-2。LCMS:m/z＝484.2(M+H)+.
第2步：
室温下，将化合物4-2(180mg,372umol,100％purity,1.00eq)溶于甲酸(4.20g,91.2mmol,3.44mL,245eq)。使用N2置换空气3次，在N2条件下，80℃反应1小时后监测，LCMS显示化合物4-2消耗完全，监测到一个目标产物峰(m/z＝428.0(M+H)+)。浓缩混合物除去大部分甲酸，在残留物中加入二氯甲烷(10.0mL)使其全部溶解后，用饱和碳酸氢钠水溶液(20.0mL*3)和饱和氯化钠水溶液(20.0mL*3)分别萃取3次，无水硫酸钠干燥，过滤，浓缩滤液得到粗产品化合物4-3。LCMS:m/z＝428.0(M+H)+.
第3步：
室温下，将化合物4-3(100mg,234umol,1.00eq)溶于二氯甲烷(1mL)，降温至-78℃，逐滴加入BBr3(117mg,468umol,45.1uL,2.00eq)。使用N2置换空气3次，在N2条件下，0℃反应1小时后监测，LCMS显示化合物4-3仍有残留(m/z＝428.1(M+H)+)，有一个目标产物峰(m/z＝414.1(M+H)+)。将反应液倒入0～5℃冰水中(15.0mL)，用饱和碳酸氢钠水溶液调pH至6～8，用二氯甲烷(20.0mL*3)萃取3次。合并有机相，饱和氯化钠水溶液(30.0mL*2)洗涤2次，无水硫酸钠干燥，过滤，浓缩滤液得到粗产品。粗产品使用HPLC(制备柱:Phenomenex luna C18 150*25mm*10um；mobile phase:[water(FA)-ACN]；B％:10％-40％,10min)制备纯化冻干，得到化合物4。LCMS:m/z＝414.1(M+H)+.1H NMR:(400MHz,MeOD)δ7.95(d,J＝4.8Hz,1H),7.39(d,J＝4.8Hz,1H),7.13(s,1H),6.29(s,1H),5.07(d,J＝3.2Hz,1H),4.80(s,2H),3.26-3.12(m,1H),2.64-2.50(m,1H),2.22-2.08(m,1H),2.06-1.76(m,5H),1.29(s,9H)。
化合物5
第1步：
室温下，将化合物1(700mg,1.69mmol,1.00eq)溶于二氯甲烷(7mL)，降温至-70℃，逐滴加入三溴化硼(5.20g,20.8mmol,2.00mL,12.3eq)。使用N2置换空气3次，在N2条件下， -70～-50℃反应1小时后监测，LCMS显示化合物1消耗完全，监测到目标产物峰(m/z＝463.9(M+H)+)。将反应液倒入0～5℃冰水中(50.0mL)，用饱和碳酸氢钠水溶液调pH至6～8，用二氯甲烷(50.0mL*3)萃取3次。合并有机相，饱和氯化钠水溶液(50.0mL*2)洗涤2次，无水硫酸钠干燥，过滤，浓缩滤液得到化合物5-1。LCMS:m/z＝463.9(M+H)+.
第2步：
室温下，将化合物5-1(100mg,216mmol,1.00eq)溶于四氢呋喃(7mL)。使用N2置换空气3次，在N2条件下，缓慢滴加MeNH2(2M,541uL,5.00eq)溶液，20℃反应1小时后监测，LCMS显示化合物5-1消耗完全，监测到目标产物峰(m/z＝413.2(M+H)+)。将反应液缓慢倒入1M盐酸水溶液中(保持溶液pH＝7-8)，用二氯甲烷(15.0mL*3)萃取3次。合并有机相，饱和氯化钠水溶液(15.0mL*2)洗涤2次，无水硫酸钠干燥，过滤，浓缩滤液得到粗产品。粗产品使用HPLC(制备柱:column:Phenomenex luna C18 150*25mm*10um；mobile phase:[water(FA)-ACN]；B％:1％-31％,10min)制备纯化冻干，得到化合物5的甲酸盐。LCMS:m/z＝413.2(M+H)+.1H NMR:(400MHz,MeOD)δ8.50(s,1H),7.97(d,J＝4.8Hz,1H),7.44(d,J＝4.4Hz,1H),7.11(s,1H),6.41-6.38(m,1H),5.13-5.08(m,1H),4.38(s,2H),3.73-3.67(m,1H),3.19-3.13(m,1H),2.77(s,3H),2.60-2.54(m,1H),2.15-1.83(m,5H),1.13-1.10(m,6H)。
化合物6
室温下，将化合物1(700mg,1.69mmol,1.00eq)溶于二氯甲烷(7mL)，降温至-70℃，逐滴加入三溴化硼(5.20g,20.8mmol,2.00mL,12.3eq)。使用N2置换空气3次，在N2条件下，-70～-50℃反应1小时后监测，LCMS显示化合物1消耗完全，监测到目标产物峰(m/z＝400.1(M+H)+)。将反应液倒入0～5℃冰水中(50.0mL)，用饱和碳酸氢钠水溶液调pH至6～8，用二氯甲烷(50.0mL*3)萃取3次。合并有机相，饱和氯化钠水溶液(50.0mL*2)洗涤2次，无水硫酸钠干燥，过滤，浓缩滤液得到粗产品。粗产品使用HPLC(制备柱:column:YMC Triart C18 250*50mm*7um；mobile phase:[water(FA)-ACN]；B％:30％-60％,20min)制备纯化冻干，得到化合物6。LCMS:m/z＝400.1(M+H)+.1H NMR:(400MHz,MeOD)δ7.91(s,1H),7.37(d,J＝4.8Hz,1H),7.05(s,1H),6.38-6.25(m,1H),5.16-5.09(m,1H),4.79(s,2H),3.72-3.63(m,1H),3.13(d,J＝0.8Hz,1H),2.59-2.52(m,1H),2.14-1.82(m,5H),1.11(d,J＝6.4Hz,6H)。
化合物7
室温下，将化合物5-1(0.24g,519umol,1.00eq)溶于四氢呋喃(3mL),加入氰化钾(70.0mg,1.08mmol,46.1uL,2.07eq),二甲基亚砜(0.5mL)，室温反应2小时后,加入水(0.5mL),十八冠醚-6(206mg,779umol,1.50eq)。70℃反应16小时后监测，LCMS显示化合物5-1消耗完全，监测到目标产物峰(m/z＝409.2(M+H)+)。将反应液缓慢倒入水中(10mL)，用乙酸乙酯(10mL*3)萃取3次，饱和氯化钠水溶液(10mL*3)洗涤3次，合并有机相，无水硫酸钠干燥，过滤，浓缩滤液得到粗产品。粗产品使用HPLC(制备柱:column:Phenomenex luna C18 150*25mm*10um；mobile phase:[water(HCl)-ACN]；B％:11％-41％,10min)制备纯化冻干，得到化合物7。LCMS:m/z＝409.2(M+H)+.1H NMR:(400MHz,MeOD)δ8.23(d,J＝6.0Hz,1H),7.53-7.50(m,2H),6.20(s,1H),5.11(d,J＝2.8Hz,1H),4.25(s,2H),3.73-3.68(m,1H),3.26-3.24(m,1H),2.66-2.61(m,1H),2.19-2.03(m,1H),2.02-1.79(m,4H),1.12(d,J＝6.4Hz,6H)。
化合物8
室温下，将化合物5-1(100mg,216umol,1.00eq)溶于四氢呋喃(1mL),缓慢滴加二甲胺四氢呋喃溶液(1M,1.08mL,5.00eq)，使用N2置换空气3次，在N2条件下，室温反应1小时后监测，LCMS显示化合物5-1消耗完全，监测到目标产物峰(RT＝0.385min,m/z＝427.1(M+H)+)。将反应液过滤，浓缩滤液得到粗产品。粗产品使用HPLC(制备柱:column:Phenomenex luna C18 150*25mm*10um；mobile phase:[water(HCl)-ACN]；B％:0％-30％,10min)制备纯化冻干，得到化合物8。LCMS:m/z＝427.1(M+H)+.1H NMR:(400MHz,MeOD)δ8.32(d,J＝5.6Hz,1H),7.91(s,1H),7.60(d,J＝5.6Hz,1H),6.32(s,1H),5.13(s,1H),4.69(s,2H),3.63-3.58(m,1H),3.29-3.22(m,1H),2.99(s,6H),2.70-2.55(m,1H),2.20-2.18(m,4H),2.04-1.90(m,4H),1.20-1.12(m,6H)。
化合物9

第1步：
将化合物1-2(1.00g,5.45mmol,1.00eq)溶于四氢呋喃(10.0mL)，使用氮气置换空气3次，降温至-5℃。氮气环境下，向反应液依次加入氘代碘甲烷(4.64g,32.7mmol,1.99mL,6.00eq)，氢化钠(261mg,6.54mmol,109uL,60.0％purity,1.20eq)，-5℃反应1小时后监测。LCMS显示化合物1-2消耗完全。将反应液缓慢倒入饱和氯化铵溶液(10.0mL)，乙酸乙酯(10.0mL x3)萃取三次。合并有机相，用饱和食盐水(10.0mL)洗涤一次，用无水硫酸钠干燥，过滤，浓缩滤液得到化合物9-1。LCMS:MS(ESI)m/z＝201.0[M+H]+。1H NMR:EC6456-215-P1N1,DMSO-d6,400MHzδ＝8.28(d,J＝4.8Hz,1H),7.64(d,J＝4.4Hz,1H),6.89(s,1H),4.71(s,2H)。
第2步：
室温下，将四丁基氟化氨(2.66g,10.1mmol,4.00eq)溶于N-甲基吡咯烷酮(5.00mL)，升温至130℃反应1小时，N2保护下加入化合物9-1(0.51g,2.54mmol,1.00eq)，130℃下搅拌3小时后监测。LCMS显示化合物9-1已完全消耗。将反应液倒入冰水(20.0mL)中，用乙酸乙酯(30.0mL x3)萃取3次。合并有机相，饱和食盐水(10.0mL x 2)洗涤两次，无水硫酸钠干燥，过滤，浓缩滤液得到粗产品。粗产品使用柱层析纯化(100-200目硅胶,石油醚:乙酸乙酯＝100:1至1:100,产物:Rf＝0.33)得到目标产物9-2。LCMS:MS(ESI)m/z＝184.9[M+H]+。1H NMR:EC6456-218-P1N1,CDCl3,400MHzδppm 8.24(d,J＝4.8Hz,1H),7.41(d,J＝4.4Hz,1H),6.87(s,1H),4.72(s,2H)。
第3步：
室温下，将化合物1-8(100mg,324umol,1.00eq)溶于四氢呋喃(2.00mL)，控温至-20℃，N2保护下加入二(三甲基硅)氨基锂,1.00M四氢呋喃溶液(1.00M,972uL,3.00eq)，-20℃下搅 拌1小时。将化合物9-2(59.7mg,324umol,1.00eq)溶于四氢呋喃(1.00mL)后加入。使用N2置换空气3次，在N2条件下，-20℃反应1小时后监测，LCMS化合物1-8已完全消耗。将反应液倒入饱和氯化铵水溶液(5.00mL)中，用乙酸乙酯(5.00mL x3)萃取3次。合并有机相，饱和食盐水(5.00mL x 2)洗涤两次，无水硫酸钠干燥，过滤，浓缩滤液得到粗产品9-3。LCMS:MS(ESI)m/z＝473.4[M+H]+。1H NMR:EC6456-219-P1N2,DMSO-d6,400MHzδ＝9.00(s,1H),8.03(d,J＝4.8Hz,1H),7.29(d,J＝4.8Hz,1H),6.91(d,J＝7.6Hz,1H),6.84(s,1H),6.02(s,1H),5.05-4.92(m,1H),4.59-4.51(m,2H),3.64-3.50(m,2H),3.05-2.95(m,1H),1.92-1.63(m,6H),1.49(s,9H),1.00(d,J＝6.4Hz,6H)。
第4步：
将化合物9-3(100mg,211umol,1.00eq)溶于无水甲酸(1.00mL)。100℃反应10分钟后监测。LCMS显示化合物9-3已完全消耗。将反应液浓缩去除甲酸得到粗产品。粗产品用反相高效液相色谱法(甲酸条件)纯化得到化合物9的甲酸盐。LCMS:MS(ESI)m/z＝417.1[M+H]+。1H NMR:EC6456-222-P1N1,DMSO-d6,400MHzδ＝9.97(s,1H),8.01(d,J＝4.80Hz,1H),7.37(d,J＝4.80Hz,1H),7.27(s,1H),6.96(d,J＝7.60Hz,1H),6.58(s,1H),5.01(d,J＝4.00Hz,1H),4.55(s,2H),3.57(tt,J＝12.0,6.80Hz,2H),3.03-3.14(m,1H),1.58-2.11(m,6H),1.00-1.06(m,6H)。
化合物10

第1步：
室温下，将化合物9-2(200mg,1.09mmol,1.00eq)和N-碘代丁二酰亚胺(256mg,1.14mmol,1.05eq)溶于乙腈(2.00mL)，25℃下搅拌20小时。将混合液倒入水(5.00mL)中，用乙酸乙酯(3.00mL x3)萃取3次。合并有机相，饱和食盐水(2.00mL x 2)洗涤两次，无水硫酸钠干燥，过滤，浓缩滤液得到粗产品。粗产品使用常规反相分离(中性方法)得到目标产物10-1。LCMS:MS(ESI)m/z＝310.8[M+H]+。1H NMR:EC6456-221-P1N2,CDCl3,400MHz
δ＝8.30(dd,J＝4.80,2.00Hz,1H),7.44(dd,J＝4.80,1.20Hz,1H),4.68(s,2H)。
第2步：
将化合物10-1(66.0mg,212umol,1.00eq)溶于二氧六环(2.00mL)，依次将三甲基环硼氧烷50％四氢呋喃(608uL,10.0eq)，碳酸钾(88.2mg,638umol,3.00eq)加入到体系中，使用氮气置换空气3次，氮气环境下加入(7.85mg,21.2umol,0.10eq)。90℃反应10小时后监测。将混合液倒入水(5.00mL)中，用乙酸乙酯(3.00mL x 3)萃取3次。合并有机相，饱和食盐水(2.00mL x 2)洗涤两次，无水硫酸钠干燥，过滤，浓缩滤液得到粗产品。粗产品使用常规反相分离(中性方法)得到得到化合物10-2。LCMS:MS(ESI)m/z＝199.0[M+H]+。1H NMR:EC6456-243-P1N1,CDCl3,400MHzδ＝8.14(d,J＝4.80Hz,2.40Hz,1H),7.30(d,J＝4.80Hz,2.00Hz,1H),4.68(s,2H),2.48(s,3H)。
第3步：
室温下，将化合物1-8(38.0mg,123umol,1.00eq)溶于四氢呋喃(2.00mL)，控温至-20℃，N2保护下二(三甲基硅)氨基锂(1.00M,369uL,3.00eq)，-20℃下搅拌1小时。将化合物10-2(24.4 mg,123umol,1.00eq)溶于四氢呋喃(1.00mL)后加入。使用N2置换空气3次，在N2条件下，-20℃反应1小时后监测，LCMS化合物10-2已完全消耗。将反应液倒入饱和氯化铵水溶液(5.00mL)中，用乙酸乙酯(5.00mL x3)萃取3次。合并有机相，饱和食盐水(5.00mL x 2)洗涤两次，无水硫酸钠干燥，过滤，浓缩滤液得到粗产品。粗产品使用常规反相分离(甲酸方法)得到目标产物10-3。LCMS:MS(ESI)m/z＝487.4[M+H]+。
第4步：
将化合物10-3(20.0mg,41.1umol,1.00eq)溶于无水甲酸(1.00mL)。100℃反应10分钟后监测。LCMS显示化合物3已完全消耗。将反应液浓缩去除甲酸得到粗产品。粗产品用反相高效液相色谱法(甲酸条件)纯化得到化合物10的甲酸盐。LCMS:MS(ESI)m/z＝431.3[M+H]+。1H NMR:EC6456-253-P1N2,DMSO-d6,400MHzδppm 12.18(s,1H),7.98(d,J＝3.20Hz,1H),7.18-7.37(m,1H),6.95(s,1H)6.43(d,J＝2.80Hz,1H),5.88(s,1H),5.00(s,2H),4.44-4.53(m,3H),3.55-3.60(m,1H),3.02-3.15(m,1H),2.03-2.07(m,1H),1.85–1.95(m,2H),1.64–1.79(m,4H),1.03(d,J＝5.20Hz,6H)。
体外活性测试
实验例一：体外CDK2/CyclinE酶活性测试
实验材料：
CDK2/CyclinE 1购自新格诺康。Ulight-4E-BP1多肽，Eu-anti-phospho-tyrosine抗体，1X检测缓冲液购自PerkinElmer公司。高纯度ATP购自Promega公司。EDTA购自Sigma。Nivo多标记分析仪(PerkinElmer)。
实验方法：
激酶缓冲液配制：激酶缓冲液包含50mM HEPES，1mM EDTA，10mM MgCl2，0.01％Brij-35，PH7.4 200ml缓冲液中加入2.38g HEPES，58mg EDTA，406mg MgCl2，20mg Brij-35，调整PH到7.4。
终止液配制：
使用100μL1MEDTA原液加上0.625uL的1X检测缓冲液与1725uL蒸馏水混合配置成终止液。
使用激酶缓冲液稀释酶，Ulight-4E-BP1多肽，ATP和抑制剂。使用检测缓冲液稀释Eu-anti-phospho-tyrosine抗体稀释至8nM/L浓度。将待测化合物用排枪进行5倍稀释至第8个浓度，即从4μM稀释至0.0512nM，DMSO终浓度为4％，设置双复孔实验。向微孔板中加入2.5μL抑制剂各浓度梯度，5μLCDK2/CyclinE 1酶(10ng)，2.5μL底物和ATP的混合物 (4mMATP，100nM Ulight-4E-BP1多肽)，此时化合物终浓度梯度为1μM稀释至0.0128Nm,ATP和底物终浓度为1mM和25nM。反应体系置于25度反应120分钟。反应结束后，每孔加入5μL终止液，25度继续反应5分钟，结束反应后每孔加入5uL的Eu-anti-phospho-tyrosine抗体稀释液，25度反应60分钟后采用PerkinElmerNivo多标记分析仪TR-FRET模式进行数据采集(激发波长为320nm发射波长为615nm和665nm)。
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，IC50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/CyclinE 1酶学抑制活性。
实验例二：体外CDK1/CyclinB1酶活性测试
实验材料：
CDK1/CyclinB 1购自CARNA。Ulight-4E-BP1多肽，Eu-anti-phospho-tyrosine抗体，1X检测缓冲液购自PerkinElmer公司。高纯度ATP购自Promega公司。EDTA购自Sigma。Nivo多标记分析仪(PerkinElmer)。
实验方法：
激酶缓冲液配制：激酶缓冲液包含50mM HEPES，1mM EDTA，10mM MgCl2，0.01％Brij-35，PH7.4 200ml缓冲液中加入2.38g HEPES，58mg EDTA，406mg MgCl2，20mg Brij-35，调整PH到7.4。
终止液配制：
使用100μL 1MEDTA原液加上0.625uL的1X检测缓冲液与1725uL蒸馏水混合配置成终止液。
使用激酶缓冲液稀释酶，Ulight-4E-BP1多肽，ATP和抑制剂。
使用检测缓冲液稀释Eu-anti-phospho-tyrosine抗体稀释至8nM/L浓度。
将待测化合物用排枪进行5倍稀释至第8个浓度，即从4μM稀释至0.0512nM，DMSO终浓度为4％，设置双复孔实验。向微孔板中加入2.5μL抑制剂各浓度梯度，5μLCDK1/CyclinB1酶(0.5ng)，2.5μL底物和ATP的混合物(4mMATP，200nM Ulight-4E-BP1多肽)，此时化合物终浓度梯度为1μM稀释至0.0128nM，ATP和底物终浓度为1mM和50nM。反应体系置于25度反应60分钟。反应结束后，每孔加入5μL终止液，25度继续反应5分钟，结束反应后每孔加入5uL的Eu-anti-phospho-tyrosine抗体稀释液，25度反应60分钟后采用PerkinElmerNivo多标记分析仪TR-FRET模式进行数据采集(激发波长为320nm发射波长为615nm和665nm)。
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，IC50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/CyclinB 1酶学抑制活性。
实验例三：体外GSK3β酶活性测试
实验材料：
GSK3βActive购自SignalChem；GSK3 Substrate购自SignalChem；ADP-Glo Kinase Assay购自Promega；Kinase assay buffer III购自SignalChem；Nivo多标记分析仪(PerkinElmer)。
实验方法：
将待测化合物用100％DMSO稀释到100μM作为第一个浓度，然后再用排枪进行5倍稀释至第8个浓度，即从100μM稀释至0.0013μM。用1X kinasebuffer将化合物各浓度点进行20倍稀释，配制成含有5％DMSO的化合物工作液，向微孔板中加入1μL化合物各浓度梯度工作液，设置双复孔。向微孔板中加入2μl GSK3β酶(1ng)，2μl底物和ATP的混合物(62.5μMATP，0.5μg/μlGSK3Substrate)，此时化合物终浓度梯度为1μM稀释至0.013nM，ATP和底物终浓度为25μM和0.2μg/μl。反应体系置于25度反应60分钟。反应结束后，每孔加入5μlADP-Glo试剂，25度继续反应40分钟，结束反应后每孔加入10uL的kinasedetection试剂，25度反应30分钟后采用PerkinElmer Nivo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，IC50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GSK3β酶学抑制活性。
Max孔:阳性对照孔读值无酶空白孔
Min孔：阴性对照孔读值为含有1％DMSO溶剂孔
实验例四：体外OVCAR3细胞活性测试
实验材料：
1640培养基，胎牛血清，盘尼西林/链霉素抗生素购自维森特。CellTiter-Glo(细胞活率化学发光检测试剂)试剂购自Promega。OVCAR3细胞系购自南京科佰生物科技有限公司。Envision多标记分析仪(PerkinElmer)。
实验方法：
将OVCAR3细胞种于白色384孔板中，40μL细胞悬液每孔，其中包含300个OVCAR3 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2000μM稀释至0.00512μM，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10μL每孔到细胞板中。转移到细胞板中的化合物浓度范围是10μM至0.026nM。细胞板置于二氧化碳培养箱中培养7天。另准备一块细胞板，在加药当天读取信号值作为最大值(下面方程式中Max值)参与数据分析。向此细胞板每孔加入10μL细胞活率化学发光检测试剂，室温孵育10分钟使发光信号稳定。采用多标记分析仪读数。
向细胞板中加入每孔10μL的细胞活率化学发光检测试剂，室温孵育10分钟使发光信号稳定。采用多标记分析仪读数。
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，IC50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OVCAR3细胞增殖的抑制活性。
实验例五：体外T47D细胞活性测试
实验材料：
1640培养基，胎牛血清，盘尼西林/链霉素抗生素购自维森特。CellTiter-Glo(细胞活率化学发光检测试剂)试剂购自Promega。T-47D细胞系购自南京科佰生物科技有限公司。Envision多标记分析仪(PerkinElmer)。
实验方法：
将T-47D细胞种于白色384孔板中，40μL细胞悬液每孔，其中包含300个T-47D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2000μM稀释至0.00512μM，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10μL每孔到细胞板中。转移到细胞板中的化合物浓度范围是10μM至0.026nM。细胞板置于二氧化碳培养箱中培养7天。另准备一块细胞板，在加药当天读取信号值作为最大值(下面方程式中Max值)参与数据分析。向此细胞板每孔加入10μL细胞活率化学发光检测试剂，室温孵育10分钟使发光信号稳定。采用多标记分析仪读数。
向细胞板中加入每孔10μL的细胞活率化学发光检测试剂，室温孵育10分钟使发光信号稳定。采用多标记分析仪读数。
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，IC50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T-47D细胞增殖的抑制活性。
表1
以上所述实施例的各技术特征可以进行任意的组合，为使描述简洁，未对上述实施例中的各个技术特征所有可能的组合都进行描述，然而，只要这些技术特征的组合不存在矛盾，都应当认为是本说明书记载的范围。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。

Claims (21)

  1. 一种具有式(I)所示的结构的化合物、或其药学上可接受的盐、或其立体异构体：
    其中，
    R1选自H，卤素，C1-6烷基，卤代C1-6烷基，C3-7环烷基，3至7元杂环烷基，硝基，异氰基或-(CH2)nR5；
    R5选自-CN，-OR6，-NR6R7，-C(O)OR6，-C(O)R6，-S(O)2R6，-P(O)(OR6)2，-O-脂环基，-O-脂杂环基，-O-芳基，-O-杂芳基或-C(O)-杂芳基，其中所述脂环基，脂杂环基，芳基，杂芳基为未取代或经一个或多个R0取代；
    R0选自卤素，C1-6烷基，卤代C1-6烷基，C3-7环烷基，3-7元杂环烷基，氰基，氨基，硝基或羟基；
    R6和R7各自独立地选自H，C1-6烷基，卤素，卤代C1-6烷基，C3-7环烷基，3-7元杂环烷基，芳基或杂芳基；
    n为0、1、2、3或4；
    环A选自
    X各自独立地为N或CR6；
    R8选自H，羟基，C1-6烷基，卤代C1-6烷基，C3-7环烷基或3-7元杂环烷基；
    R2和R3各自独立地选自H，C1-6烷基，卤代C1-6烷基，C3-7环烷基或3-7元杂环烷基，其中所述C3-7环烷基和3-7元杂环烷基为未取代或经一个或多个R9取代；
    R9选自卤素，C1-6烷基，卤代C1-6烷基，氰基，氨基，硝基或羟基；
    R4选自H，卤素，氨基，羟基或C1-C6烷基；
    式(I)所示的结构中一个或多个氢原子被氘原子取代或未取代；
    所述化合物不具有如下所示结构：

  2. 如权利要求1所述的化合物、或其药学上可接受的盐、或其立体异构体，其特征在于，当式(I)所示的结构中，所述R1选自-CH2-O-CH3，环A选自R2选自H，R3选自-CH(CH3)2，R4选自H时，式(I)所示的结构中一个或多个氢原子被氘原子取代。
  3. 如权利要求1所述的化合物、或其药学上可接受的盐、或其立体异构体，其特征在于，所述R1选自-CH2R5，所述R5选自-OR61，-NR6R7，-CN，-C(O)OR6，-C(O)R6，-S(O)2R6，-P(O)(OR6)2，-O-脂环基，-O-脂杂环基，-O-芳基，-O-杂芳基或-C(O)-杂芳基，其中所述脂环 基，脂杂环基，芳基，杂芳基为未取代或经一个或多个R0取代；
    所述R61选自H，一个或多个氢原子被氘原子取代的C1-6烷基，卤代C1-C6烷基，C3-7环烷基，3-7元杂环烷基，芳基或杂芳基。
  4. 如权利要求3所述的化合物、或其药学上可接受的盐、或其立体异构体，其特征在于，所述R1选自-CH2R5，所述R5选自-OR61或-NR6R7；
    所述R61选自H，一个或多个氢原子被氘原子取代的C1-6烷基或卤代C1-C6烷基；
    所述R6和R7各自独立地选自H，C1-6烷基或卤代C1-6烷基。
  5. 如权利要求4所述的化合物、或其药学上可接受的盐、或其立体异构体，其特征在于，所述R1为-OH。
  6. 如权利要求1所述的化合物、或其药学上可接受的盐、或其立体异构体，其特征在于，所述R2为H，R3选自C1-6烷基或卤代C1-C6烷基。
  7. 如权利要求6所述的化合物、或其药学上可接受的盐、或其立体异构体，其特征在于，所述R3为-C(CH3)3。
  8. 如权利要求1所述的化合物、或其药学上可接受的盐、或其立体异构体，其特征在于，所述R1选自-(CH2)nR5，所述R5中一个或多个氢原子被氘原子取代。
  9. 如权利要求1所述的化合物、或其药学上可接受的盐、或其立体异构体，其特征在于，式(I)所示的结构选自式(I-1)～(I-3)任一式所示的结构：
  10. 如权利要求9所述的化合物、或其药学上可接受的盐、或其立体异构体，其特征在于，式(I)所示的结构选自式(I-4)所示的结构：
  11. 如权利要求10所述的化合物、或其药学上可接受的盐、或其立体异构体，其特征在于，X选自CR6。
  12. 如权利要求11所述的化合物、或其药学上可接受的盐、或其立体异构体，其特征在于，R6选自H，C1-6烷基，卤素或卤代C1-6烷基。
  13. 如权利要求1所述的化合物、或其药学上可接受的盐、或其立体异构体，其特征在于，所述R1为-CH2R5。
  14. 如权利要求13所述的化合物、或其药学上可接受的盐、或其立体异构体，其特征在于，所述R5选自-CN，-OR6或-NR6R7；
    所述R6和R7各自独立地选自H，C1-6烷基或卤代C1-6烷基。
  15. 如权利要求1所述的化合物、或其药学上可接受的盐、或其立体异构体，其特征在于，所述R2选自H，C1-C6烷基或卤代C1-C6烷基；
    所述R3选自H，C1-C6烷基，卤代C1-C6烷基，C3-7环烷基或3-7元杂环烷基，其中所述C3-7环烷基和3-7元杂环烷基为未取代或经一个或多个R9取代。
  16. 如权利要求15所述的化合物、或其药学上可接受的盐、或其立体异构体，其特征在于，所述R2选自H；
    所述R3选自H，C1-C6烷基，卤代C1-C6烷基，未取代或经一个R9取代的5元杂环烷基。
  17. 如权利要求16所述的化合物、或其药学上可接受的盐、或其立体异构体，其特征在于，所述R9选自卤素，C1-6烷基或卤代C1-6烷基；所述5元杂环烷基的杂原子为氧原子。
  18. 如权利要求17所述的化合物、或其药学上可接受的盐、或其立体异构体，其特征在于，所述式(I)中选自如下结构中的一种：
  19. 如权利要求1～18任一项所述的化合物、或其药学上可接受的盐、或其立体异构体，其特征在于，所述化合物具有如下结构中的一种：
  20. 一种药物组合物，其特征在于，包括权利要求1-19中任一项所述的化合物、或其药学上可接受的盐、或其立体异构体；以及药学可接受的载体。
  21. 根据权利要求1-19任一所述的化合物、或其药学上可接受的盐、或其立体异构体，或权利要求20所述的药物组合物在制备治疗或预防与CDK2活性相关的或由CDK2活性介导的疾病的药物中的应用。
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