WO2021073593A1 - 作为cdk2/4/6三重抑制剂的氨基嘧啶类化合物 - Google Patents

作为cdk2/4/6三重抑制剂的氨基嘧啶类化合物 Download PDF

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WO2021073593A1
WO2021073593A1 PCT/CN2020/121390 CN2020121390W WO2021073593A1 WO 2021073593 A1 WO2021073593 A1 WO 2021073593A1 CN 2020121390 W CN2020121390 W CN 2020121390W WO 2021073593 A1 WO2021073593 A1 WO 2021073593A1
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compound
pharmaceutically acceptable
acceptable salt
group
alkyl
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PCT/CN2020/121390
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English (en)
French (fr)
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周明
徐招兵
李刚
胡利红
丁照中
江文
胡国平
黎健
陈曙辉
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南京明德新药研发有限公司
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Priority to CN202080072813.5A priority Critical patent/CN114555600B/zh
Priority to EP20877068.5A priority patent/EP4046999A4/en
Priority to JP2022523308A priority patent/JP2023501110A/ja
Publication of WO2021073593A1 publication Critical patent/WO2021073593A1/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
    • 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
    • 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
    • C07D471/10—Spiro-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
    • 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
    • C07D487/10—Spiro-condensed systems

Definitions

  • the present invention relates to new aminopyrimidine compounds as CDK2/4/6 inhibitors, in particular to compounds represented by formula (I) and pharmaceutically acceptable salts thereof, as well as compounds represented by formula (I) and pharmaceutically acceptable salts thereof
  • Malignant tumors are one of the main diseases that endanger human life.
  • humans have developed a variety of diagnosis and treatment methods, including the most commonly used chemotherapy, surgery, radiotherapy, and targeted therapy. These therapies delay the development of tumors to a certain extent and prolong the lives of patients.
  • the above therapies still cannot achieve the desired inhibitory effect.
  • the toxic and side effects of the above therapies are also key factors that limit their applications.
  • CDK cyclin-dependent kinases
  • CDK2 overexpression is associated with abnormal cell cycle regulation, and CDK2/Cyclin E participates in the regulation of cell cycle G1 to S phase.
  • the CDK2/Cyclin E complex can also catalyze the phosphorylation of Rb, thereby promoting the progression of the cell cycle from the G1 phase to the S phase; in the S phase, the CDK2/cyclin A complex can promote the DNA replication process.
  • CDK2 corresponding cyclin Cyclin E is common in tumors expression.
  • Cyclin E1 is related to the poor prognosis of ovarian cancer, gastric cancer, breast cancer and other tumors.
  • CCNE1 is related to poor survival and potential therapeutic target inovarian cancer, cancer(2010)116:2621-34; Etemadmoghadam et al.,Resistance to CDK2 selected inhibitors with polyassociated CCNE1 cell -Amplified Ovarian cancer,clin cancerres(2013)19:5960-71; Au-Yeung et al.,selective targeting of cyclin E1-Amplified high-grade serous ovarian cancer by cyclin-dependent kind 2 and AKT inhibition,Clin.Cancer res.(2017)30:297-303; Ooi et al., Gene amplification of CCNE1, CCND1, and CDK6 in gastric cancers detected by multiplex ligation-dependent probe amplification and fluorescence in situ
  • Cyclin E2 overexpression and Breast cancer is related to endocrine therapy resistance, and inhibition of CDK2 can make tamoxifen resistant and CCNE2 overexpressing cells are resusceptible to tamoxifen and CDK4/6 inhibitors.
  • Cyclin E2 overexpression is associated with endocrine resistance but not insensitivity to CDK2 inhibition in human breast cancer cells.Mol cancer Ther.
  • Cyclin E amplification is also related to the resistance of HER2-positive breast cancer to trastuzumab. (Scaltriti et al., Cyclin E amplification/overexpression is a mechanism of trastuzumab resistance in HER2+breast cancer patients, Proc Natl Acad Sci. (2011) 108:3761-6).
  • the small molecule CDK inhibitor dinaciclib inhibits CDK1, CDK2, CDK5 and CDK9 at the same time, and is currently undergoing clinical trials for breast cancer and hematoma. Seliciclib inhibits CDK2, CDK7 and CDK9 at the same time, and is currently undergoing clinical trials in combination with chemotherapy for the treatment of solid tumors.
  • the CDK2/4/6 inhibitor PF-06873600 (WO2018033815A1) developed by Pfizer has undergone clinical trials. Its CDK2 activity is high, but its selectivity to other CDK subtypes such as CDK9 is poor. So far, no CDK2 inhibitor has been approved for marketing. Small molecule drugs with CDK2 inhibitory activity and novel kinase inhibitory spectrum are still unmet clinical needs.
  • the present invention provides a compound represented by formula (I) or a pharmaceutically acceptable salt thereof,
  • T is N or CH
  • R 1 is C 4-6 cycloalkyl, wherein said C 4-6 cycloalkyl is substituted with two or three R a;
  • Each R a is independently F, Cl, Br, I, -CN, -OH, C 1-3 alkoxy, or C 1-3 alkyl, a C 1-3 alkoxy wherein said C 1-3 alkoxy and The alkyl group is optionally substituted with 1, 2 or 3 substituents independently selected from F, Cl, Br, -CN, -OH and -NH 2 ;
  • R 2 and R 3 are each independently H, F, Cl, Br, I, -CN, -OH, C 1-3 alkoxy or C 1-3 alkyl, wherein the C 1-3 alkoxy And C 1-3 alkyl is optionally substituted with 1, 2 or 3 substituents independently selected from F, Cl, Br, -CN, -OH and -NH 2 ;
  • R 2 and R 3 are joined together to form a C 3-5 cycloalkyl group together with the carbon atom to which they are connected, and the C 3-5 cycloalkyl group is optionally substituted by 1, 2 or 3 R b ;
  • Each R b is independently H, F, Cl, Br, I, -CN, -OH, C 1-3 alkoxy, C 1-3 alkyl, or C 1-3 haloalkyl;
  • R 4 and R 5 are each independently H, F, Cl, Br, I, -CN, -OH, C 1-3 alkoxy or C 1-3 alkyl, wherein the C 1-3 alkoxy And C 1-3 alkyl is optionally substituted with 1, 2 or 3 substituents independently selected from F, Cl, Br, -CN, -OH and -NH 2 ;
  • R 3 and R 4 are joined together to form a C 3-5 cycloalkyl group together with the carbon atom to which they are connected, and the C 3-5 cycloalkyl group is optionally substituted with 1, 2 or 3 R c ;
  • Each R c is independently H, F, Cl, Br, I, -CN, -OH, C 1-3 alkoxy, C 1-3 alkyl, or C 1-3 haloalkyl;
  • R 6 is H, F, Cl, Br, I, -CN, -OH, C 1-3 alkoxy, C 1-3 alkyl or C 1-3 haloalkyl;
  • R 7 is -NH 2 , C 1-3 alkylamino, C 1-6 alkyl, C 3-5 cycloalkyl, 4-6 heterocycloalkyl, 5-6 membered heteroaryl or phenyl, wherein The C 1-6 alkyl group, C 3-5 cycloalkyl group, 4-6 heterocycloalkyl group, 5-6 membered heteroaryl group and phenyl group are optionally substituted by 1, 2 or 3 R d ;
  • Each R d is independently H, F, Cl, Br, I, -CN, -OH, C 1-3 alkoxy or C 1-3 alkyl, wherein the C 1-3 alkoxy and C 1 -3 Alkyl is optionally substituted with 1, 2 or 3 substituents independently selected from F, Cl, Br, -CN, -OH and -NH 2 ;
  • n 0, 1 or 2;
  • the 4-6 membered heterocycloalkyl group and the 5-6 membered heteroaryl group respectively contain 1, 2, 3 or 4 heteroatoms independently selected from N, -O- and -S-.
  • the present invention also provides a compound represented by formula (I) or a pharmaceutically acceptable salt thereof,
  • T is N or CH
  • R 1 is C 4-6 cycloalkyl, wherein said C 4-6 cycloalkyl is optionally substituted with 1, 2 or 3 substituents R a;
  • Each R a is independently H, F, Cl, Br, I, -CN, -OH, C 1-3 alkoxy, or C 1-3 alkyl, wherein a C 1-3 alkoxy groups or C 1 -3 Alkyl is optionally substituted with 1, 2 or 3 substituents independently selected from F, Cl, Br, -CN, -OH and -NH 2 ;
  • R 2 and R 3 are each independently H, F, Cl, Br, I, -CN, -OH, C 1-3 alkoxy or C 1-3 alkyl, wherein the C 1-3 alkoxy And C 1-3 alkyl is optionally substituted with 1, 2 or 3 substituents independently selected from F, Cl, Br, -CN, -OH and -NH 2 ;
  • R 2 and R 3 are joined together to form a C 3-5 cycloalkyl group together with the carbon atom to which they are connected, and the C 3-5 cycloalkyl group is optionally substituted by 1, 2 or 3 R b ;
  • Each R b is independently H, F, Cl, Br, I, -CN, -OH, C 1-3 alkoxy, C 1-3 alkyl, or C 1-3 haloalkyl;
  • R 4 and R 5 are each independently H, F, Cl, Br, I, -CN, -OH, C 1-3 alkoxy or C 1-3 alkyl, wherein the C 1-3 alkoxy And C 1-3 alkyl is optionally substituted with 1, 2 or 3 substituents independently selected from F, Cl, Br, -CN, -OH and -NH 2 ;
  • R 3 and R 4 are joined together to form a C 3-5 cycloalkyl group together with the carbon atom to which they are connected, and the C 3-5 cycloalkyl group is optionally substituted with 1, 2 or 3 R c ;
  • Each R c is independently H, F, Cl, Br, I, -CN, -OH, C 1-3 alkoxy, C 1-3 alkyl, or C 1-3 haloalkyl;
  • R 6 is H, F, Cl, Br, I, -CN, -OH, C 1-3 alkoxy, C 1-3 alkyl or C 1-3 haloalkyl;
  • R 7 is -NH 2 , C 1-3 alkylamino, C 1-6 alkyl, C 3-5 cycloalkyl, 4-6 heterocycloalkyl, 5-6 membered heteroaryl or phenyl, wherein The C 1-6 alkyl group, C 3-5 cycloalkyl group, 4-6 heterocycloalkyl group, 5-6 membered heteroaryl group and phenyl group are optionally substituted by 1, 2 or 3 R d ;
  • Each R d is independently H, F, Cl, Br, I, -CN, -OH, C 1-3 alkoxy or C 1-3 alkyl, wherein the C 1-3 alkoxy and C 1 -3 Alkyl is optionally substituted with 1, 2 or 3 substituents independently selected from F, Cl, Br, -CN, -OH and -NH 2 ;
  • n 0, 1 or 2;
  • the 4-6 membered heterocycloalkyl group and the 5-6 membered heteroaryl group respectively contain 1, 2, 3 or 4 heteroatoms independently selected from N, -O- and -S-.
  • each of the foregoing Ra is independently H, F, Cl, Br, I, -CN, -OH, -OCH 3 , -CH 3 , -CF 3 or -CH 2 CH 3 , others
  • the variables are as defined in the present invention.
  • each of the foregoing Ra is independently F, Cl, Br, I, -CN, -OH, -OCH 3 , -CH 3 , -CF 3 or -CH 2 CH 3 , and other variables such as Defined by the present invention.
  • R 1 is Which said Optionally substituted with 1, 2 or 3 R a, R a and each of the other variables are as defined herein.
  • the above-mentioned R 1 is Which said Is substituted with two or three R a, R a and each of the other variables are as defined in the present invention.
  • R 1 is Each of R a and all other variables are as defined in the present invention.
  • the above-mentioned compound has a structure represented by formula (I-1):
  • R 2, R 3, R 4, R 5, R 6, R 7, R a and n are as defined in the present invention, p is 1, 2 or 3.
  • the above-mentioned compound has a structure represented by formula (I-1):
  • R 2, R 3, R 4, R 5, R 6, R 7, R a and n are as defined in the present invention, p is 1, 2 or 3.
  • the above-mentioned compound has a structure represented by formula (I-2):
  • R 2, R 3, R 4, R 5, R 6, R 7, R a and n are as defined in the present invention.
  • each of the foregoing R b is independently H, F, Cl, Br, I, -CN, -OH, -OCH 3 , -CH 3 , -CF 3 or -CH 2 CH 3 , and others
  • the variables are as defined in the present invention.
  • R 2 and R 3 are connected together to form a cyclopropyl group together with the carbon atom to which they are connected, and the cyclopropyl group is optionally substituted by 1, 2 or 3 R b , and each R b and other variables are as defined in the present invention.
  • R 4 and R 5 are each independently H, F, Cl, Br, I, -CN, -OH, -OCH 3 , -CH 3 , -CF 3 or -CH 2 CH 3.
  • Other variables are as defined in the present invention.
  • each of the foregoing R c is independently H, F, Cl, Br, I, -CN, -OH, -OCH 3 , -CH 3 , -CF 3 or -CH 2 CH 3 , others
  • the variables are as defined in the present invention.
  • R 3 and R 4 are connected together to form a cyclopropyl group together with the carbon atom to which they are connected, and the cyclopropyl group is optionally substituted with 1, 2 or 3 R c , and each R c and other variables are as defined in the present invention.
  • the above-mentioned compound has a structure represented by any one of formulas (I-3) to (I-7):
  • R a , R b , R c , T, R 6 , R 7 and p are as defined in the present invention, and q is 0, 1, 2 or 3.
  • the above-mentioned compound has a structure shown in any one of formulas (I-8) to (I-12):
  • R a, R b, R c, T, R 6, R 7, p and q are as defined in the present invention.
  • R 2 and R 3 are each independently H, F, Cl, Br, I, -CN, -OH, -OCH 3 , -CH 3 , -CF 3 or -CH 2 CH 3.
  • Other variables are as defined in the present invention.
  • R 6 is H, F, Cl, Br, I, -CN, -OH, -OCH 3 , -CH 3 or -CH 2 CH 3 , and other variables are as defined in the present invention.
  • each of the foregoing Rds is independently H, F, Cl, Br, I, -CN, -OH, -OCH 3 , -CH 3 , -CF 3 or -CH 2 CH 3 , others
  • the variables are as defined in the present invention.
  • each of the foregoing Rds is independently H, F, Cl, Br, I, -CN, -OH, -OCH 3 , -CH 3 , -CF 3 , -CH 2 CH 3 or- CH(CH 3 ) 2 , and other variables are as defined in the present invention.
  • the above-mentioned R 7 is -NH 2 , -NH(CH 3 ), -NH(CH 2 CH 3 ), -N(CH 3 ) 2 , -CH 3 , -CH 2 CH 3 , -CH 2 CH 2 CH 3 , -CH(CH 3 ) 2 , cyclopropyl, cyclopentyl, pyrrolidinyl, tetrahydrofuranyl, piperidinyl, pyrazolyl, pyridyl or phenyl, wherein the -CH 3 , -CH 2 CH 3 , -CH 2 CH 2 CH 3 , -CH(CH 3 ) 2 , cyclopropyl, cyclopentyl, pyrrolidinyl, tetrahydrofuranyl, piperidinyl, pyrazolyl, pyridyl and The phenyl group is optionally substituted with 1, 2 or 3 Rd , and each
  • R 7 is -NH 2 , -NH(CH 3 ), -NH(CH 2 CH 3 ), -N(CH 3 ) 2 , -CH 3 , -CH 2 CH 3 , -CH 2 CH 2 CH 3 , -CH(CH 3 ) 2 , cyclopropyl, cyclopentyl, azetidinyl, pyrrolidinyl, tetrahydrofuranyl, piperidinyl, pyrazolyl, pyridyl or phenyl , Wherein the -CH 3 , -CH 2 CH 3 , -CH 2 CH 2 CH 3 , -CH(CH 3 ) 2 , cyclopropyl, cyclopentyl, azetidinyl, pyrrolidinyl, tetrahydrofuranyl , Piperidinyl, pyrazolyl, pyridyl and phen
  • R 7 is -NH 2 , -NH(CH 3 ), -NH(CH 2 CH 3 ), -N(CH 3 ) 2 , -C(R d ) 3 , -CH 2 CH 2 R d ,
  • Rd and other variables are as defined in the present invention.
  • R 7 is -NH 2 , -NH(CH 3 ), -NH(CH 2 CH 3 ), -N(CH 3 ) 2 , -C(R d ) 3 , -CH 2 CH 2 R d ,
  • Rd and other variables are as defined in the present invention.
  • the above-mentioned R 7 is -NH 2 , -NH(CH 3 ), -NH(CH 2 CH 3 ), -N(CH 3 ) 2 , -C(R d ) 3 , -CH 2 CH 2 R d ⁇ -CH(CH 3 ) 2 ⁇
  • Rd and other variables are as defined in the present invention.
  • R 7 is -NH 2 , -NH(CH 3 ), -N(CH 3 ) 2 , -CH 3 , -CF 3 , -CH 2 CH 3 ,
  • Other variables are as defined in the present invention.
  • R 7 is -NH 2 , -NH(CH 3 ), -N(CH 3 ) 2 , -CH 3 , -CF 3 , -CH 2 CH 3 ,
  • Other variables are as defined in the present invention.
  • R 7 is -NH 2 , -NH(CH 3 ), -N(CH 3 ) 2 , -CH 3 , -CF 3 , -CH 2 CH 3 , -CH(CH 3 ) 2 , -CH 2 CH 2 OCH 3 ,
  • Other variables are as defined in the present invention.
  • the above-mentioned pharmaceutically acceptable salt is hydrochloride.
  • the present invention also provides a pharmaceutical composition, which contains a therapeutically effective amount of the above-mentioned compound or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.
  • the present invention also provides the application of the above-mentioned compound or its pharmaceutically acceptable salt and the above-mentioned pharmaceutical composition in the preparation of CDK2/4/6 inhibitor drugs.
  • the present invention also provides the application of the above-mentioned compound or its pharmaceutically acceptable salt and the above-mentioned pharmaceutical composition in the preparation of a medicine for treating solid tumors.
  • the aforementioned solid tumor is colorectal cancer or breast cancer.
  • the present invention provides a novel structure of CDK2/4/6 triple inhibitor.
  • This series of compounds has excellent inhibitory activity against CDK2/4/6 enzyme levels, and the selectivity for CDK9 is significantly better than PF-06873600, and the safety risk caused by off-target is small; for colorectal cancer cells with high Cyclin E expression levels
  • the proliferation of HCT116 and triple-negative breast cancer cells HCC1806 has obvious inhibitory activity, and the selectivity to Rb-negative triple-negative breast cancer MDA-MB-468 cells is significantly better than that of PF-06873600.
  • the compound of the present invention also has lower clearance rate, higher AUC, higher oral bioavailability, and better comprehensive pharmacokinetic properties.
  • 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 the 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, for example, 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
  • the pharmaceutically acceptable salt of the present invention can be synthesized from the parent compound containing acid or base by conventional chemical methods. In general, 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 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 their 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 groups. 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 freely rotate the double bond or the single bond of the ring-forming carbon atom.
  • diastereomer refers to a stereoisomer in which the molecule has two or more chiral centers and the relationship between the molecules is non-mirror mirror image.
  • 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 dashed 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 tautomer also called prototropic tautomer
  • 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 tautomerization 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 an appropriate 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 uses a chiral stationary phase and is optionally combined with chemical derivatization (for example, the formation of amino groups from amines). Formate).
  • the compound 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).
  • deuterium can be substituted for hydrogen to form deuterated drugs.
  • the bond formed by deuterium and carbon is stronger than the bond formed by ordinary hydrogen and carbon.
  • deuterated drugs can reduce toxic side effects and increase drug stability. , Enhance the efficacy, prolong 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 may 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 does 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 can 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 dashed 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 line in indicates 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 examples 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 indicate 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 that are 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 1-3 alkylamino refers to those alkyl groups containing 1 to 3 carbon atoms attached to the rest of the molecule through an amino group.
  • the C 1-3 alkylamino group includes C 1-2 , C 2 and C 3 alkylamino groups and the like.
  • 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(CH 3 ) 2 and so on.
  • halogen or halogen by itself or as part of another substituent means a fluorine, chlorine, bromine or iodine atom.
  • C 1-3 haloalkyl means monohaloalkyl and polyhaloalkyl containing 1 to 3 carbon atoms.
  • the C 1-3 haloalkyl group includes C 1-2 , C 2-3 , C 3 , C 2 and C 1 haloalkyl group and the like.
  • Examples of C 1-3 haloalkyl include, but are not limited to, trifluoromethyl, trichloromethyl, 2,2,2-trifluoroethyl, pentafluoroethyl, pentachloroethyl, 3-bromopropyl, and the like.
  • C 4-6 cycloalkyl means a saturated cyclic hydrocarbon group composed of 4 to 6 carbon atoms, which is a monocyclic and bicyclic ring system.
  • the C 4-6 cycloalkyl includes C 4-5 and C 5-6 cycloalkyl, etc.; it can be monovalent, divalent or multivalent.
  • Examples of C 4-6 cycloalkyl include, but are not limited to, 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 and 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.
  • 4-6 membered heterocycloalkyl by itself or in combination with other terms means a saturated cyclic group consisting of 4 to 6 ring atoms, with 1, 2, 3 or 4 ring atoms.
  • heteroatoms independently selected from O, S and N, and the rest are carbon atoms, wherein nitrogen atoms are optionally quaternized, and nitrogen and sulfur heteroatoms can be optionally oxidized (ie, NO and S(O) p , p Is 1 or 2). It includes monocyclic and bicyclic ring systems, where the bicyclic ring system includes spiro, fused, and bridged rings.
  • a heteroatom may occupy the connection position of the heterocycloalkyl group with the rest of the molecule.
  • the 4-6 membered heterocycloalkyl group includes 5-6 membered, 4-membered, 5-membered and 6-membered heterocycloalkyl groups and the like.
  • 4-6 membered heterocycloalkyl groups 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 and 4-morpholinyl, etc.), Dioxanyl, dithiazinyl, isoxazolidinyl, isothiazolidin
  • 5-6 membered heteroaryl ring and “5-6 membered heteroaryl group” can be used interchangeably in the present invention.
  • the term “5-6 membered heteroaryl group” means a ring consisting of 5 to 6 ring atoms. It is composed of a monocyclic group with a conjugated ⁇ -electron system, in which 1, 2, 3 or 4 ring atoms are heteroatoms independently selected from O, S and N, and the rest are carbon atoms. Where the nitrogen atom is optionally quaternized, the nitrogen and sulfur heteroatoms may optionally be oxidized (ie NO and S(O) p , p is 1 or 2).
  • the 5-6 membered heteroaryl group can be attached to the rest of the molecule through a heteroatom or a carbon atom.
  • the 5-6 membered heteroaryl group includes 5-membered and 6-membered heteroaryl groups.
  • Examples of the 5-6 membered heteroaryl include, but are not limited to, pyrrolyl (including N-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, etc.), pyrazolyl (including 2-pyrazolyl and 3-pyrrolyl, etc.) Azolyl, etc.), imidazolyl (including N-imidazolyl, 2-imidazolyl, 4-imidazolyl and 5-imidazolyl, etc.), oxazolyl (including 2-oxazolyl, 4-oxazolyl and 5- Oxazolyl, etc.), triazolyl (1H-1,2,3-triazolyl, 2H-1,2,3-triazolyl, 1H-1,2,4-triazolyl and 4H-1, 2,
  • C 6-10 aromatic ring and “C 6-10 aryl” can be used interchangeably in the present invention.
  • C 6-10 aromatic ring or “C 6-10 aryl” means that A cyclic hydrocarbon group with a conjugated ⁇ -electron system composed of 6 to 10 carbon atoms, which can be a monocyclic, fused bicyclic or fused tricyclic system, in which each ring is aromatic. It may be monovalent, divalent or multivalent, and C 6-10 aryl groups include C 6-9 , C 9 , C 10 and C 6 aryl groups and the like. Examples of C 6-10 aryl groups include, but are not limited to, phenyl, naphthyl (including 1-naphthyl, 2-naphthyl, etc.).
  • 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.; similarly, from n to n +m member means that the number of atoms in the ring is from 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 group, such as trimethylsilyl (TMS) and tert-butyldi
  • hydroxyl 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 (
  • 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 relates to the absolute configuration of the compound, the absolute configuration can be confirmed by conventional technical means in the art.
  • the single crystal X-ray diffraction method uses the Bruker D8 venture diffractometer to collect the diffraction intensity data of the cultured single crystal.
  • the light source is CuK ⁇ radiation
  • the scanning method After scanning and collecting relevant data, the direct method (Shelxs97) is further used to analyze the crystal structure to confirm the absolute configuration.
  • 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.
  • DIBAL-H stands for diisobutylaluminum hydride
  • DMSO dimethyl sulfoxide
  • DBU 1,8-diazabicycloundec-7-ene
  • EDTA stands for ethyl Diaminetetraacetic acid
  • HPMC stands for hydroxypropyl methylcellulose
  • LCMS liquid chromatography mass spectrometry
  • Rh(PPh 3 ) 3 Cl stands for tris(triphenylphosphine) rhodium chloride
  • SFC stands for supercritical fluid chromatography
  • TLC represents thin layer chromatography analysis
  • Pd(dppf)Cl 2 ⁇ CH 2 Cl 2 represents [1,1'-bis(diphenylphosphine)ferrocene] palladium dichloride dichloromethane
  • Psi represents pounds per inch
  • DMSO Represents dimethyl sulfoxide
  • ATP represents adenosine triphosphate
  • Example 1 Compound 1, Compound 1A and Compound 1B
  • the reaction solution was cooled to 20°C, water (1000 mL) was added and extracted with ethyl acetate (800 mL ⁇ 3), the organic phases were combined and washed with saturated brine (500 mL ⁇ 4), dried with anhydrous sodium sulfate, filtered, and the filtrate was evaporated to dryness .
  • the crude product was stirred with methyl tert-butyl ether (500 mL) at 20°C for 16 hours, and then petroleum ether (200 mL) was added to continue stirring for 3 hours, and compound 1-q was obtained by filtration.
  • Compound 1 was prepared by SFC (column: polyoxymethylene coated chiral stationary phase (250mm ⁇ 30mm ⁇ 10 ⁇ m); mobile phase: 45% [0.1% ammonia ethanol solution]; purified to obtain compound 1A (retention time 0.548 minutes) and compound 1B (Retention time 0.895 minutes).
  • reaction solution was quenched with saturated ammonium chloride (25mL) and extracted with ethyl acetate (10mL ⁇ 3).
  • organic phases were combined and washed with saturated brine (20mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was evaporated to dryness.
  • reaction solution was concentrated under reduced pressure and passed through preparative chromatography (column: Waters Xbridge 150 ⁇ 25mm ⁇ 5 ⁇ m; mobile phase: 0.05% aqueous ammonia solution as mobile phase A, acetonitrile as mobile phase B, B%: 18%-48%, Gradient time: 9min) Purification to obtain compound 6.
  • reaction solution was filtered through diatomaceous earth, and the filtrate was evaporated to dryness, and the crude product obtained by preparative chromatography (column: Waters Xbridge C18 250 ⁇ 50mm ⁇ 10 ⁇ m; mobile phase: 0.05% ammonia solution as mobile phase A, acetonitrile as mobile phase B, B%: 15%-30%, gradient time: 20 min) Purification to obtain compound 7.
  • reaction solution was evaporated to dryness under reduced pressure, and the obtained crude product was prepared by chromatography (column: Waters Xbridge C18 150 ⁇ 25mm ⁇ 5 ⁇ m; mobile phase: 10mM ammonium bicarbonate solution as mobile phase A, acetonitrile as mobile phase B, B% : 18%-48%, gradient time: 9 min) to obtain compound 8.
  • reaction solution was evaporated to dryness under reduced pressure, and the obtained crude product was prepared by chromatography (column: Waters Xbridge C18 150 ⁇ 25mm ⁇ 5 ⁇ m; mobile phase: 10mM ammonium bicarbonate solution as mobile phase A, acetonitrile as mobile phase B, B% : 22%-52%, gradient time: 9 min) to obtain compound 9.
  • reaction solution was evaporated to dryness under reduced pressure, and the obtained crude product was preparative chromatography (column: Waters Xbridge C18 150 ⁇ 30mm ⁇ 5 ⁇ m; mobile phase: 10mM ammonium bicarbonate solution as mobile phase A, acetonitrile as mobile phase B, B% : 16%-46%, gradient time: 11.5min) Purification to obtain compound 10.
  • Example 11 Compound 11, Compound 11A and Compound 11B
  • the compound 11 was chiral separated by SFC to obtain 11A and 11B.
  • Compound 11 was prepared by SFC (column: polyoxymethylene coated chiral stationary phase (250mm ⁇ 30mm ⁇ 10 ⁇ m); mobile phase: 65% [0.1% ammonia ethanol solution]; purified to obtain compound 11A (retention time 0.479 minutes) and compound 11B (Retention time 1.516 minutes).
  • CDK2/CyclinA2 kinase detection kit was purchased from Promega. Nivo multi-label analyzer (PerkinElmer).
  • Experimental method Use the kinase buffer in the kit to dilute the enzyme, substrate, adenosine triphosphate and inhibitor.
  • the compound to be tested was diluted 5 times with a discharge gun to the 8th concentration, that is, diluted from 50 ⁇ M to 0.65 nM, with a DMSO concentration of 5%, and a 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, integration time 0.5 seconds.
  • CDK2/CyclinE1 kinase detection kit was purchased from Promega. Nivo multi-label analyzer (PerkinElmer).
  • Experimental method Use the kinase buffer in the kit to dilute the enzyme, substrate, adenosine triphosphate and inhibitor.
  • the compound to be tested was diluted 5 times with a discharge gun to the 8th concentration, that is, diluted from 50 ⁇ M to 0.65 nM, with a DMSO concentration of 5%, and a 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. The integration time is 0.5. second.
  • CDK4/CyclinD1 kinase was purchased from Invitrogen, and the reaction substrates LANCE Ultra ULight TM- 4E-BP-1 (Thr37146) Peptide (peptide) and EU-ANTI-P-4EBP1 (THR37/46) were purchased from PerkinElmer. Nivo multi-label analyzer (PerkinElmer).
  • Kinase buffer preparation The components of the buffer include: pH 7.5 hydroxyethylpiperazine ethanesulfonic acid solution 50mM, ethylenediaminetetraacetic acid 1mM, magnesium chloride 10mM, 0.01% polyoxyethylene laureth (Brij-35) ), dithiothreitol 2mM diluted enzyme with kinase buffer, substrate LANCE Ultra ULight TM- 4E-BP-1 (Thr37146) Peptide (peptide), adenosine triphosphate and inhibitor.
  • the compound to be tested was diluted 5 times with a discharge gun to the 8th concentration, that is, diluted from 40 ⁇ M to 0.512 nM, the DMSO concentration was 4%, and the double-well experiment was set up.
  • the final concentration gradient of the compound is 10 ⁇ M diluted to 0.128 nM.
  • the reaction system was placed at 25°C for 120 minutes.
  • CDK6/CyclinD1 kinase was purchased from Carna.
  • the reaction substrates LANCE Ultra ULight TM- 4E-BP-1 (Thr37146) Peptide (peptide) and EU-ANTI-P-4EBP1 (THR37/46) were purchased from PerkinElmer. Nivo multi-label analyzer (PerkinElmer).
  • Kinase buffer preparation The components of the buffer include: pH 7.5 hydroxyethylpiperazine ethanesulfonic acid solution 50mM, ethylenediaminetetraacetic acid 1mM, magnesium chloride 10mM, 0.01% polyoxyethylene laureth (Brij-35) ), dithiothreitol 2mM diluted enzyme with kinase buffer, substrate LANCE Ultra ULight TM- 4E-BP-1 (Thr37146) Peptide (peptide), adenosine triphosphate and inhibitor.
  • the compound to be tested was diluted 5 times with a discharge gun to the 8th concentration, that is, diluted from 40 ⁇ M to 0.512 nM, the DMSO concentration was 4%, and the double-well experiment was set up.
  • the final concentration gradient of the compound is 10 ⁇ M diluted to 0.128 nM.
  • the reaction system was placed at 25°C for 120 minutes.
  • 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
  • Experimental method Use the kinase buffer in the kit to dilute the enzyme, substrate, adenosine triphosphate and inhibitor.
  • the compound to be tested was diluted 5 times with a discharge gun to the 8th concentration, that is, diluted from 50 ⁇ M to 0.65 nM, with a DMSO concentration of 5%, and a 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 activities of the compounds of the present invention on CDK2/CyclinA2, CDK2/CyclinE1, CDK4/CyclinD1, CDK6/CyclinD1, and CDK9/CyclinT1.
  • the compound of the present invention has significant inhibitory activity on CDK2, CDK4 and CDK6 kinase, and has high selectivity for CDK9.
  • McCoy'5a medium, fetal bovine serum, penicillin/streptomycin antibiotics were purchased from Vicente.
  • CellTiter-Glo (cell viability chemiluminescence detection reagent) reagent was purchased from Promega.
  • the HCT116 cell line was purchased from Nanjing Kebai Biotechnology Co., Ltd. Nivo multi-label analyzer (PerkinElmer).
  • Plant HCT116 cells in a white 96-well plate 80 ⁇ L of cell suspension per well, which contains 1000 HCT116 cells.
  • the cell plate was placed in a carbon dioxide incubator for overnight culture.
  • the compound to be tested was diluted 5-fold to the 9th concentration with a discharge gun, that is, diluted from 2mM to 5.12nM, and a double-well experiment was set up.
  • Add 78 ⁇ L of culture 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 concentration of the compound transferred to the cell plate ranges from 10 ⁇ M to 0.0256 nM.
  • the cell plate was placed in a carbon dioxide incubator for 4 days.
  • prepare a cell plate and read the signal value as the maximum value (Max value in the following equation) on the day of dosing to participate in data analysis.
  • Add 25 ⁇ L of cell viability chemiluminescence detection reagent to each well of this cell plate, and incubate for 10 minutes at room temperature to stabilize the luminescence signal. Using multi-marker analyzer readings.
  • Plant HCC1806 cells in a white 96-well plate 80 ⁇ L of cell suspension per well, which contains 3000 HCC1806 cells.
  • the cell plate was placed in a carbon dioxide incubator for overnight culture.
  • the compound to be tested was diluted 5-fold to the 9th concentration with a discharge gun, that is, diluted from 10 mM to 25.6 nM, and a double-well experiment was set up.
  • the concentration of the compound transferred to the cell plate ranges from 50 ⁇ M to 0.128 nM.
  • the cell plate was placed in a carbon dioxide incubator for 7 days. After reaching the culture time, remove the cell supernatant, put the cell plate in the -80°C refrigerator for 1 hour, then add 100 ⁇ L Cyquant reagent to each well, and use the multi-label analyzer to read. Another cell plate is prepared, and the signal value is read as the maximum value (Max value in the following equation) on the day of drug addition to participate in data analysis.
  • CellTiter-Glo (cell viability chemiluminescence detection reagent) reagent was purchased from Promega.
  • the MDA-MB-468 cell line was purchased from Nanjing Kebai Biotechnology Co., Ltd. Nivo multi-label analyzer (PerkinElmer).
  • Plant MDA-MB-468 cells in a white 96-well plate 80 ⁇ L of cell suspension per well, which contains 1000 MDA-MB-468 cells.
  • the cell plate was placed in a carbon dioxide incubator for overnight culture.
  • the compound to be tested was diluted 5-fold to the 9th concentration with a discharge gun, that is, diluted from 10 mM to 25.6 nM, and a double-well experiment was set up.
  • the concentration of the compound transferred to the cell plate ranges from 50 ⁇ M to 0.128 nM.
  • the cell plate was placed in a carbon dioxide incubator for 7 days.
  • prepare a cell plate and read the signal value as the maximum value (Max value in the following equation) on the day of dosing to participate in data analysis.
  • Add 25 ⁇ L of cell viability chemiluminescence detection reagent to each well of this cell plate, and incubate for 10 minutes at room temperature to stabilize the luminescence signal. Using multi-marker analyzer readings.
  • the compound of the present invention has significant inhibitory activity on the proliferation of HCT 116 and HCC1806 cells, and has poor activity on Rb-negative MDA-MB-468, and the selectivity is significantly better than PF-06873600.
  • the healthy adult female Balb/c mice used in this study were purchased from Shanghai Lingchang Biotechnology Co., Ltd.
  • the administration vehicle is 10% DMSO + 10% solutol + 80% water.
  • the intravenous injection group solution was filtered with a 2 ⁇ m filter membrane before administration.
  • mice Four female Balb/c mice were divided into two groups according to two in each group. In the first group, 2 mg/kg of the test compound was administered intravenously; in the second group, 10 mg/kg of the test compound was administered intragastrically.
  • the continuous blood sampling method is adopted, and the blood of two animals is collected at each time point.
  • 30 ⁇ L of whole blood was collected before and after administration at 0.0833, 0.25, 0.5, 1, 2 (only intragastric group), 4, 6, 8 (only intravenous group), and 24 hours after administration.
  • the whole blood was placed in an anticoagulation tube, centrifuged at 3200g for 10 minutes at 4°C, and plasma was prepared and stored at -80°C.
  • LC/MS-MS was used to determine the drug concentration in plasma.
  • the compound of the present invention has low clearance rate in mice, high exposure, good oral bioavailability, and excellent comprehensive pharmacokinetic properties.

Abstract

公开了作为CDK2/4/6抑制剂的氨基嘧啶类化合物，具体公开了式(I)所示化合物及其药学上可接受的盐，以及式(I)所示化合物及其药学上可接受的盐在制备治疗实体瘤药物中的应用。

Description

作为CDK2/4/6三重抑制剂的氨基嘧啶类化合物
本申请主张如下优先权：
CN201910988432.0，申请日2019年10月17日；
CN202010558823.1，申请日2020年06月18日。
技术领域
本发明涉及新的作为CDK2/4/6抑制剂的氨基嘧啶类化合物，具体涉及式(I)所示化合物及其药学上可接受的盐，以及式(I)所示化合物及其药学上可接受的盐在制备治疗实体瘤药物中的应用。
背景技术
恶性肿瘤是现如今危害人类生命安全的主要疾病之一。近百年来，人类为了对抗恶性肿瘤，发展出了多种包括最常用的化疗，手术，放疗以及靶向疗法在内的诊疗手段与治疗方法。这些疗法在一定程度上延缓肿瘤的发展，延长了患者生命。但是由于恶性肿瘤的无限制生长，浸润与转移的特性，以上疗法依旧无法达到理想的抑制效果。同时，以上的疗法的毒副作用也是限制其应用的关键因素。
细胞周期的调控主要受一系列丝氨酸/苏氨酸激酶的影响，这类激酶又被称作为细胞周期蛋白依赖性激酶(CDK),他们通过预期相对应的调节亚基周期素(cyclins)相结合，推动细胞周期的进行、遗传信息的转录和细胞的正常***增殖。CDK异常活化与肿瘤发展相关，CDK抑制剂已经被证明可以用于肿瘤的治疗。目前CDK4/6抑制剂Palbociclib，Abemaciclib和Ribociclib已被批准用于HR阳性/HER-2阴性乳腺癌的治疗。尽管CDK4/6抑制剂在HR阳性的转移性乳腺癌上展现出较好的临床药效，但其对其他CDK亚型的抑制活性较弱，容易发生原发性耐药和获得性耐药。
CDK2过表达与细胞周期调控异常相关，CDK2/Cyclin E参与细胞周期G1期到S期调控。G1期末期，CDK2/Cyclin E的复合物同样可以催化Rb磷酸化，从而促进细胞周期从G1期进展到S期；在S期，CDK2/cyclin A复合物可以促进DNA的复制过程。(Asghar et al.The history and future of targeting cyclin-dependent kinases in cancer therapy,Nat.Rev.Drug.Discov.2015；14(2):130-146)CDK2对应的周期素Cyclin E在肿瘤中普遍过表达。Cyclin E1的扩增和过表达与卵巢癌、胃癌和乳腺癌等肿瘤的不良预后相关。(Nakayama et al.,Gene amplification CCNE1 is related to poor survival and potential therapeutic target in ovarian cancer,cancer(2010)116:2621-34；Etemadmoghadam et al.,Resistance to CDK2 inhibitors is associated with selection of polyploidy cells in CCNE1-Amplified Ovarian cancer,clin cancer res(2013)19:5960-71；Au-Yeung et al.,selective targeting of cyclin E1-Amplified high-grade serous ovarian cancer by cyclin-dependent kinase 2 and AKT inhibition,Clin.Cancer res.(2017)30:297-303；Ooi et al.,Gene amplification of CCNE1,CCND1,and CDK6 in gastric cancers detected by multiplex ligation-dependent probe amplification and fluorescence in situ hybridization,Hum Pathol.(2017)61:58-67；Noske et al.,detection of CCNE1/UR/(19q12)amplification by in situ hybridization is common in high grade and type 2 endometrial cancer,oncotarget(2017)8:14794-14805).Cyclin E2过表达与乳腺癌内分泌疗法耐药相关，且抑制CDK2可以使他莫昔芬耐药且CCNE2过表达的细胞重新对他莫昔芬和CDK4/6抑制剂敏感。(caldon et al.,Cyclin E2 overexpression is associated with endocrine resistance but not insensitivity to CDK2 inhibition in human breast cancer cells.Mol cancer Ther.(2012)11:1488-99；Herrera-abreu et al., Early adaption and acquired resistance to CDK4/6 inhibition in Estrogen Receptor-positive breast cancer,cancer res.(2016)76:2301-2313).Cyclin E扩增同样与HER2阳性的乳腺癌对曲妥珠单抗耐药有关。(Scaltriti et al.,Cyclin E amplification/overexpression is a mechanism of trastuzumab resistance in HER2+breast cancer patients,Proc Natl Acad Sci.(2011)108:3761-6).
小分子CDK抑制剂dinaciclib同时抑制CDK1,CDK2,CDK5和CDK9，目前正在开展乳腺癌和血液瘤的临床实验。Seliciclib同时抑制CDK2,CDK7和CDK9，目前正在开展与化疗联用用于治疗实体瘤的临床实验。Pfizer研发的CDK2/4/6抑制剂PF-06873600(WO2018033815A1)已开展临床实验，其CDK2活性较高，但其对其他CDK亚型如CDK9的选择性较差。到目前为止，仍然没有CDK2抑制剂被批准上市。对CDK2有抑制活性且具有新颖的激酶抑制谱的小分子药物依然是临床未被满足的需求。
Figure PCTCN2020121390-appb-000001
发明内容
一方面，本发明提供了式(I)所示化合物或其药学上可接受的盐，
Figure PCTCN2020121390-appb-000002
其中，
T为N或CH；
R 1为C 4-6环烷基，其中所述C 4-6环烷基被1、2或3个R a所取代；
各R a独立地为F、Cl、Br、I、-CN、-OH、C 1-3烷氧基或C 1-3烷基，其中所述C 1-3烷氧基和C 1-3烷基任选被1、2或3个独立选自F、Cl、Br、-CN、-OH和-NH 2的取代基所取代；
R 2和R 3各自独立地为H、F、Cl、Br、I、-CN、-OH、C 1-3烷氧基或C 1-3烷基，其中所述C 1-3烷氧基和C 1-3烷基任选被1、2或3个独立选自F、Cl、Br、-CN、-OH和-NH 2的取代基所取代；
或者R 2和R 3连接在一起，与其相连的碳原子一起形成C 3-5环烷基，所述C 3-5环烷基任选被1、2或3个R b所取代；
各R b独立地为H、F、Cl、Br、I、-CN、-OH、C 1-3烷氧基、C 1-3烷基或C 1-3卤代烷基；
R 4和R 5各自独立地为H、F、Cl、Br、I、-CN、-OH、C 1-3烷氧基或C 1-3烷基，其中所述C 1-3烷氧基和C 1-3烷基任选被1、2或3个独立选自F、Cl、Br、-CN、-OH和-NH 2的取代基所取代；
或者R 3和R 4连接在一起，与其相连的碳原子一起形成C 3-5环烷基，所述C 3-5环烷基任选被1、2或3个R c所取代；
各R c独立地为H、F、Cl、Br、I、-CN、-OH、C 1-3烷氧基、C 1-3烷基或C 1-3卤代烷基；
R 6为H、F、Cl、Br、I、-CN、-OH、C 1-3烷氧基、C 1-3烷基或C 1-3卤代烷基；
R 7为-NH 2、C 1-3烷氨基、C 1-6烷基、C 3-5环烷基、4-6杂环烷基、5-6元杂芳基或苯基，其中所述C 1-6烷基、C 3-5环烷基、4-6杂环烷基、5-6元杂芳基和苯基任选被1、2或3个R d所取代；
各R d独立地为H、F、Cl、Br、I、-CN、-OH、C 1-3烷氧基或C 1-3烷基，其中所述C 1-3烷氧基和C 1-3烷基任选被1、2或3个独立选自F、Cl、Br、-CN、-OH和-NH 2的取代基所取代；
n为0、1或2；
所述4-6元杂环烷基和5-6元杂芳基分别包含1、2、3或4个独立选自N、-O-和-S-的杂原子。
本发明还提供了式(I)所示化合物或其药学上可接受的盐，
Figure PCTCN2020121390-appb-000003
其中，
T为N或CH；
R 1为C 4-6环烷基，其中所述C 4-6环烷基任选被1、2或3个R a所取代；
各R a独立地为H、F、Cl、Br、I、-CN、-OH、C 1-3烷氧基或C 1-3烷基，其中所述C 1-3烷氧基和C 1-3烷基任选被1、2或3个独立选自F、Cl、Br、-CN、-OH和-NH 2的取代基所取代；
R 2和R 3各自独立地为H、F、Cl、Br、I、-CN、-OH、C 1-3烷氧基或C 1-3烷基，其中所述C 1-3烷氧基和C 1-3烷基任选被1、2或3个独立选自F、Cl、Br、-CN、-OH和-NH 2的取代基所取代；
或者R 2和R 3连接在一起，与其相连的碳原子一起形成C 3-5环烷基，所述C 3-5环烷基任选被1、2或3个R b所取代；
各R b独立地为H、F、Cl、Br、I、-CN、-OH、C 1-3烷氧基、C 1-3烷基或C 1-3卤代烷基；
R 4和R 5各自独立地为H、F、Cl、Br、I、-CN、-OH、C 1-3烷氧基或C 1-3烷基，其中所述C 1-3烷氧基和C 1-3烷基任选被1、2或3个独立选自F、Cl、Br、-CN、-OH和-NH 2的取代基所取代；
或者R 3和R 4连接在一起，与其相连的碳原子一起形成C 3-5环烷基，所述C 3-5环烷基任选被1、2或3个R c所取代；
各R c独立地为H、F、Cl、Br、I、-CN、-OH、C 1-3烷氧基、C 1-3烷基或C 1-3卤代烷基；
R 6为H、F、Cl、Br、I、-CN、-OH、C 1-3烷氧基、C 1-3烷基或C 1-3卤代烷基；
R 7为-NH 2、C 1-3烷氨基、C 1-6烷基、C 3-5环烷基、4-6杂环烷基、5-6元杂芳基或苯基，其中所述C 1-6烷基、C 3-5环烷基、4-6杂环烷基、5-6元杂芳基和苯基任选被1、2或3个R d所取代；
各R d独立地为H、F、Cl、Br、I、-CN、-OH、C 1-3烷氧基或C 1-3烷基，其中所述C 1-3烷氧基和C 1-3烷基任选被1、2或3个独立选自F、Cl、Br、-CN、-OH和-NH 2的取代基所取代；
n为0、1或2；
所述4-6元杂环烷基和5-6元杂芳基分别包含1、2、3或4个独立选自N、-O-和-S-的杂原子。
在本发明的一些方案中，上述各R a独立地为H、F、Cl、Br、I、-CN、-OH、-OCH 3、-CH 3、-CF 3或-CH 2CH 3，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F、Cl、Br、I、-CN、-OH、-OCH 3、-CH 3、-CF 3或-CH 2CH 3，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
Figure PCTCN2020121390-appb-000004
其中所述
Figure PCTCN2020121390-appb-000005
任选被1、2或3个R a所取代，各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 1为
Figure PCTCN2020121390-appb-000006
其中所述
Figure PCTCN2020121390-appb-000007
被1、2或3个R a所取代，各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 1为
Figure PCTCN2020121390-appb-000008
各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 1为
Figure PCTCN2020121390-appb-000009
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
Figure PCTCN2020121390-appb-000010
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)所示结构：
Figure PCTCN2020121390-appb-000011
其中，R 2、R 3、R 4、R 5、R 6、R 7、R a和n如本发明所定义，p为0、1、2或3。
在本发明的一些方案中，上述化合物具有式(I-1)所示结构：
Figure PCTCN2020121390-appb-000012
其中，R 2、R 3、R 4、R 5、R 6、R 7、R a和n如本发明所定义，p为1、2或3。
在本发明的一些方案中，上述化合物具有式(I-2)所示结构：
Figure PCTCN2020121390-appb-000013
其中，R 2、R 3、R 4、R 5、R 6、R 7、R a和n如本发明所定义。
在本发明的一些方案中，上述各R b独立地为H、F、Cl、Br、I、-CN、-OH、-OCH 3、-CH 3、-CF 3或-CH 2CH 3，其他变量如本发明所定义。
在本发明的一些方案中，上述R 2和R 3连接在一起，与其相连的碳原子一起形成环丙基，所述环丙基任选被1、2或3个R b所取代，各R b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 4和R 5各自独立地为H、F、Cl、Br、I、-CN、-OH、-OCH 3、-CH 3、-CF 3或-CH 2CH 3，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 c独立地为H、F、Cl、Br、I、-CN、-OH、-OCH 3、-CH 3、-CF 3或-CH 2CH 3，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连接在一起，与其相连的碳原子一起形成环丙基，所述环丙基任选被1、2或3个R c所取代，各R c及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-3)～(I-7)任一结构式所示结构：
Figure PCTCN2020121390-appb-000014
Figure PCTCN2020121390-appb-000015
其中，R a、R b、R c、T、R 6、R 7和p如本发明所定义，q为0、1、2或3。
在本发明的一些方案中，上述化合物具有式(I-8)～(I-12)任一结构式所示结构：
Figure PCTCN2020121390-appb-000016
其中，R a、R b、R c、T、R 6、R 7、p和q如本发明所定义。
在本发明的一些方案中，上述R 2和R 3各自独立地为H、F、Cl、Br、I、-CN、-OH、-OCH 3、-CH 3、-CF 3或-CH 2CH 3，其他变量如本发明所定义。
在本发明的一些方案中，上述R 6为H、F、Cl、Br、I、-CN、-OH、-OCH 3、-CH 3或-CH 2CH 3，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 d独立地为H、F、Cl、Br、I、-CN、-OH、-OCH 3、-CH 3、-CF 3或-CH 2CH 3，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 d独立地为H、F、Cl、Br、I、-CN、-OH、-OCH 3、-CH 3、-CF 3、-CH 2CH 3或-CH(CH 3) 2，其他变量如本发明所定义。
在本发明的一些方案中，上述R 7为-NH 2、-NH(CH 3)、-NH(CH 2CH 3)、-N(CH 3) 2、-CH 3、-CH 2CH 3、-CH 2CH 2CH 3、-CH(CH 3) 2、环丙基、环戊基、吡咯烷基、四氢呋喃基、哌啶基、吡唑基、吡啶基或苯基，其中所述-CH 3、-CH 2CH 3、-CH 2CH 2CH 3、-CH(CH 3) 2、环丙基、环戊基、吡咯烷基、四氢呋喃基、 哌啶基、吡唑基、吡啶基和苯基任选被1、2或3个R d所取代，各R d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 7为-NH 2、-NH(CH 3)、-NH(CH 2CH 3)、-N(CH 3) 2、-CH 3、-CH 2CH 3、-CH 2CH 2CH 3、-CH(CH 3) 2、环丙基、环戊基、氮杂环丁基、吡咯烷基、四氢呋喃基、哌啶基、吡唑基、吡啶基或苯基，其中所述-CH 3、-CH 2CH 3、-CH 2CH 2CH 3、-CH(CH 3) 2、环丙基、环戊基、氮杂环丁基、吡咯烷基、四氢呋喃基、哌啶基、吡唑基、吡啶基和苯基任选被1、2或3个R d所取代，各R d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 7为-NH 2、-NH(CH 3)、-NH(CH 2CH 3)、-N(CH 3) 2、-C(R d) 3、-CH 2CH 2R d、
Figure PCTCN2020121390-appb-000017
各R d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 7为-NH 2、-NH(CH 3)、-NH(CH 2CH 3)、-N(CH 3) 2、-C(R d) 3、-CH 2CH 2R d、
Figure PCTCN2020121390-appb-000018
各R d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 7为-NH 2、-NH(CH 3)、-NH(CH 2CH 3)、-N(CH 3) 2、-C(R d) 3、-CH 2CH 2R d、-CH(CH 3) 2、
Figure PCTCN2020121390-appb-000019
各R d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 7为-NH 2、-NH(CH 3)、-N(CH 3) 2、-CH 3、-CF 3、-CH 2CH 3、
Figure PCTCN2020121390-appb-000020
Figure PCTCN2020121390-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R 7为-NH 2、-NH(CH 3)、-N(CH 3) 2、-CH 3、-CF 3、-CH 2CH 3、
Figure PCTCN2020121390-appb-000022
Figure PCTCN2020121390-appb-000023
Figure PCTCN2020121390-appb-000024
其他变量如本发明所定义。
在本发明的一些方案中，上述R 7为-NH 2、-NH(CH 3)、-N(CH 3) 2、-CH 3、-CF 3、-CH 2CH 3、-CH(CH 3) 2、-CH 2CH 2OCH 3、
Figure PCTCN2020121390-appb-000025
Figure PCTCN2020121390-appb-000026
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 PCTCN2020121390-appb-000027
在本发明的一些方案中，上述化合物为
Figure PCTCN2020121390-appb-000028
Figure PCTCN2020121390-appb-000029
在本发明的一些方案中，上述药学上可接受的盐为盐酸盐。
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或其药学上可接受的盐和药学上可接受的载体。
另一方面，本发明还提供了上述化合物或其药学上可接受的盐及上述药物组合物在制备CDK2/4/6抑制剂药物中的应用。
本发明还提供了上述化合物或其药学上可接受的盐及上述药物组合物在制备治疗实体瘤药物中的应用。在一些实施方案中，上述实体瘤为结直肠癌或乳腺癌。
技术效果
本发明提供一种新型结构的CDK2/4/6三重抑制剂。该系列的化合物对CDK2/4/6的酶水平抑制活性优异，且对CDK9的选择性显著优于PF-06873600，脱靶引起的安全性风险小；对Cyclin E表达水平较高的结直肠癌细胞HCT116及三阴性乳腺癌细胞HCC1806的增殖具有明显的抑制活性，且对Rb阴性的三阴性乳腺癌MDA-MB-468细胞具有显著优于PF-06873600的选择性。本发明化合物还具有较低的清除率，较高的AUC、口服生物利用度较高，综合药代动力学性质较优。
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 PCTCN2020121390-appb-000030
和楔形虚线键
Figure PCTCN2020121390-appb-000031
表示一个立体中心的绝对构型，用直形实线键
Figure PCTCN2020121390-appb-000032
和直形虚线键
Figure PCTCN2020121390-appb-000033
表示立体中心的相对构型，用波浪线
Figure PCTCN2020121390-appb-000034
表示楔形实线键
Figure PCTCN2020121390-appb-000035
或楔形虚线键
Figure PCTCN2020121390-appb-000036
或用波浪线
Figure PCTCN2020121390-appb-000037
表示直形实线键
Figure PCTCN2020121390-appb-000038
和直形虚线键
Figure PCTCN2020121390-appb-000039
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 PCTCN2020121390-appb-000040
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 PCTCN2020121390-appb-000041
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 PCTCN2020121390-appb-000042
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 PCTCN2020121390-appb-000043
直形虚线键
Figure PCTCN2020121390-appb-000044
或波浪线
Figure PCTCN2020121390-appb-000045
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 PCTCN2020121390-appb-000046
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 PCTCN2020121390-appb-000047
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，术语“C 1-3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3个碳原子的烷基基团。所述C 1-3烷氨基包括C 1-2、C 2和C 3烷氨基等。C 1-3烷氨基的实例包括但不限于-NHCH 3、-N(CH 3) 2、-NHCH 2CH 3、-N(CH 3)CH 2CH 3、-NHCH 2CH 2CH 3、-NHCH(CH 3) 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，术语“C 1-3卤代烷基”表示包含1至3个碳原子的单卤代烷基和多卤代烷基。所述C 1-3卤代烷基包括C 1-2、C 2-3、C 3、C 2和C 1卤代烷基等。C 1-3卤代烷基的实例包括但不限于三氟甲基、三氯甲基、2,2,2-三氟乙基、五氟乙基、五氯乙基、3-溴丙基等。
除非另有规定，“C 4-6环烷基”表示由4至6个碳原子组成的饱和环状碳氢基团，其为单环和双环体系，所述C 4-6环烷基包括C 4-5和C 5-6环烷基等；其可以是一价、二价或者多价。C 4-6环烷基的实例包括，但不限于，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和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，术语“4-6元杂环烷基”本身或者与其他术语联合分别表示由4至6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“4-6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4-6元杂环烷基包括5-6元、4元、5元和6元杂环烷基等。4-6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、六氢哒嗪基、高哌嗪基或高哌啶基等。
除非另有规定，本发明术语“5-6元杂芳环”和“5-6元杂芳基”可以互换使用，术语“5-6元杂芳基”表示由5至6个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)。5-6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5-6元杂芳基包括5元和6元杂芳基。所述5-6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)、***基(1H-1,2,3-***基、2H-1,2,3-***基、1H-1,2,4-***基和4H-1,2,4-***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)、吡啶基(包括2-吡啶基、3-吡啶基和4-吡啶基等)、吡嗪基或嘧啶基(包括2-嘧啶基和4-嘧啶基等)。
除非另有规定，本发明术语“C 6-10芳环”和“C 6-10芳基”可以互换使用，术语“C 6-10芳环”或“C 6-10芳基”表示由6至10个碳原子组成的具有共轭π电子体系的环状碳氢基团，它可以是单环、稠合双环或稠合三环体系，其中各个环均为芳香性的。其可以是一价、二价或者多价，C 6-10芳基包括C 6-9、C 9、C 10和C 6芳基等。C 6-10芳基的实例包括但不限于苯基、萘基(包括1-萘基和2-萘基等)。
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：
Figure PCTCN2020121390-appb-000048
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：DIBAL-H代表二异丁基氢化铝；DMSO代表二甲基亚砜；DBU代表1,8-二氮杂二环十一碳-7-烯；EDTA代表乙二胺四乙酸；HPMC代表羟丙基甲基纤维素；LCMS代表液质联用色谱；Rh(PPh 3) 3Cl代表三(三苯基膦)氯化铑；SFC代表超临界流体色谱；TLC代表薄层色谱分析；Pd(dppf)Cl 2·CH 2Cl 2代表[1,1'-双(二苯基膦)二茂铁]二氯化钯二氯甲烷；Psi代表磅/英寸；DMSO代表二甲基亚砜；ATP代表腺嘌呤核苷三磷酸；ADP-Glo代表一种试剂盒。
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。
实施例1：化合物1、化合物1A和化合物1B
Figure PCTCN2020121390-appb-000049
化合物1-n的合成：
将化合物1-l(100g,438.84mmol,56.18mL)加入乙腈(1L)中，然后加入三乙胺(66.61g,658.26mmol,91.62mL)，冷却到0℃，缓慢滴加化合物1-m(60.65g,526.61mmol)，保持反应体系温度为0-5℃，将反应液在0-5℃下搅拌3小时。向反应体系中加入水(500mL)，用乙酸乙酯(500mL×3)萃取，有机相合并用饱和食盐水(600mL×3)洗涤，用无水硫酸钠干燥，过滤，滤液蒸干。粗产品用石油醚：乙酸乙酯＝10:1(500mL)在20℃悬浮搅拌2小时，过滤得到化合物1-n。 1H NMR(400MHz,CDCl 3):δ8.19(s,1H),5.50(br d,J＝2.6Hz,1H),4.36(br s,1H),4.23(ddd,J＝5.7,8.1,10.2Hz,1H),2.36-2.28(m,1H),2.08-1.99(m,1H),1.91-1.75(m,3H),1.62-1.55(m,1H),1.17(s,3H)；LCMS(ESI):m/z:308.0(M+1)。
化合物1-o的合成：
将化合物1-n(110g,358.79mmol)和化合物1-k(115.56g,538.19mmol,HCl)加入二甲基亚砜(1100mL)中，然后加入碳酸钾(148.76g,1.08mol)，将混合物在120℃下搅拌16小时。将混合物冷却到20℃，加入水(1L)，用乙酸乙酯(500mL×3)萃取，有机相合并用饱和食盐水(800mL×3)洗涤，用无水硫酸钠干燥，过滤，滤液蒸干。得到的粗产品用乙酸乙酯：石油醚＝2:3(500mL)在70℃下搅拌30分钟，然 后冷却到20℃搅拌3小时。过滤得到化合物1-o。LCMS(ESI):m/z:450.0(M+1)。
化合物1-q的合成：
将化合物1-o(100g,218.77mmol,98.09％纯度)加入N,N-二甲基甲酰胺(1000mL)中加入丙烯酸乙酯(219.02g,2.19mol,237.81mL)，然后加入三乙胺(88.55g,875.07mmol,121.80mL)和Pd(dppf)Cl 2·CH 2Cl 2(17.87g,21.88mmol)，将混合物用氮气置换几次，加热到100℃氮气保护下搅拌16小时。将反应液冷却到20℃，加入水(1000mL)用乙酸乙酯(800mL×3)萃取，有机相合并用饱和食盐水(500mL×4)洗涤，用无水硫酸钠干燥，过滤，滤液蒸干。将粗产品用甲基叔丁基醚(500mL)在20℃搅拌16小时，然后加入石油醚(200mL)继续搅拌3小时，过滤得到化合物1-q。 1H NMR(400MHz,CD 3OD):δ7.98(s,1H),5.28(br t,J＝8.5Hz,1H),4.15(quin,J＝7.6Hz,1H),3.95-3.88(m,1H),3.74(br dd,J＝3.7,12.4Hz,2H),3.68(t,J＝8.5Hz,2H),3.46(t,J＝8.1Hz,2H),3.02(br t,J＝11.7Hz,2H),2.77-2.71(m,2H),2.65-2.56(m,4H),2.53-2.43(m,1H),2.24-2.04(m,4H),1.95-1.86(m,2H),1.79-1.72(m,1H),1.62-1.49(m,2H),1.18(s,3H),1.00(t,J＝7.2Hz,3H)；LCMS(ESI):m/z:468.1(M+1)。
化合物1-r的合成：
将化合物1-q(101g,210.30mmol,97.36％纯度)加入乙醇(1000mL)，在氮气保护下加入湿Pd/C(10％,10g)，反应体系用氢气置换几次。将反应液在氢气(15psi)80℃条件下搅拌32小时。将混合物冷却到20℃，过滤，滤液蒸干得到粗产品。将粗产品用甲基叔丁基醚(500mL)室温搅拌3小时，过滤得到化合物1-r。 1H NMR(400MHz,CD 3OD):δ7.56(s,1H),4.36(t,J＝8.3Hz,1H),4.13(q,J＝7.2Hz,2H),3.79(tt,J＝4.0,10.6Hz,1H),3.74-3.67(m,2H),2.95(dt,J＝2.6,11.9Hz,2H),2.87(s,3H),2.73-2.67(m,2H),2.58-2.52(m,2H),2.23-2.04(m,3H),1.90-1.72(m,5H),1.64-1.53(m,2H),1.24(t,J＝7.2Hz,3H),1.16(s,3H)；LCMS(ESI):m/z:470.3(M+1)。
化合物1A的合成：
将化合物1-r(7.8g,16.61mmol)加入二甲基亚砜(80mL)然后加入DBU(5.06g,33.22mmol,5.01mL)。将混合物加热到120℃搅拌10小时。将反应冷却到20℃，加入水(100mL)用乙酸乙酯(100mL×2)萃取，有机相合并用饱和食盐水(100mL×2)洗涤，用无水硫酸钠干燥，过滤，滤液减压浓缩。粗品通过制备色谱(色谱柱：Waters Xbridge BEH C18 50×50mm×10μm；流动相：以0.05％的氨水溶液为流动相A，乙腈为流动相B，B％：20％-42％，梯度时间：15min)纯化。得到化合物1A。 1H NMR(400MHz,DMSO-d 6):δ8.00(s,1H),5.24-5.11(m,1H),4.22(s,1H),3.90-3.73(m,1H),3.61-3.46(m,2H),2.92-2.78(m,5H),2.66-2.58(m,2H),2.56-2.51(m,2H),2.35-2.22(m,1H),2.09-1.85(m,5H),1.81-1.70(m,2H),1.65-1.43(m,3H),1.11-0.99(m,3H)；LCMS(ESI):m/z:424.2(M+1)。
Figure PCTCN2020121390-appb-000050
化合物1-B的合成：
将化合物1-a(100g,429.76mmol)加入四氢呋喃(1000mL)中，冷却到-78℃，氮气保护下缓慢滴加DIBAL-H(1M,859.52mL)，加完后将反应液升温到25℃，并在氮气保护下搅拌16小时。TLC(石油醚:乙酸乙酯＝3:1)显示化合物1-a有少量剩余，并有新产物生成。停止反应，用饱和氯化铵(800mL)淬灭反应，用乙酸乙酯(500mL×3)萃取，有机相合并用饱和食盐水(500mL×2)洗涤，用无水硫酸钠干燥，过滤，滤液减压浓缩，得到的粗产品于25℃下在石油醚:乙酸乙酯＝10:1(200mL)中搅拌16小时，得到化合物1-b。
1H NMR(400MHz,CDCl 3):8.57(s,1H),4.76(s,2H),2.59(s,3H)。
化合物1-d的合成：
将化合物1-b(20g,104.90mmol)和化合物1-c(12.69g,110.15mmol)加入乙腈(200mL)中，然后加入三乙胺(15.92g,157.36mmol,21.90mL)，将反应液在80℃下搅拌12小时。TLC(石油醚:乙酸乙酯＝3:1)显示化合物1-b反应完全，并有新产物生成。将反应液冷却到25℃，减压浓缩除去多余的乙腈。得到的粗产物，通过硅胶柱层析(石油醚/乙酸乙酯＝1/1)纯化，得到化合物1-d。 1H NMR(400MHz,CDCl 3):7.71(s,1H),4.25(m,1H),3.49(s,3H),2.50(s,3H),2.21(dt,J＝3.8,8.1Hz,1H),1.96(dt,J＝3.6,7.7Hz,1H),1.90-1.77(m,4H),1.76-1.68(m,1H),1.58-1.49(m,1H),1.11(s,3H)。
化合物1-e的合成：
将化合物1-d(20g,74.25mmol)加入乙酸乙酯(150mL)和甲醇(50mL)中，然后加入活性二氧化锰(64.55g,742.50mmol)，将反应液在50℃下搅拌4小时。TLC(石油醚:乙酸乙酯＝1:1)显示化合物1-d反应完全，并有新产物生成。将反应液冷却到25℃，过滤，滤饼用甲醇(50mL×2)洗涤，滤液减压浓缩，得到化合物1-e。 1H NMR(400MHz,CDCl 3):9.65(s,1H),8.27(s,1H),4.31(ddd,J＝6.5,8.2,9.5Hz,1H), 2.49(s,3H),2.26-2.15(m,1H),1.92-1.85(m,1H),1.81-1.73(m,2H),1.67-1.50(m,2H),1.09(s,3H)。
化合物1-g的合成：
将化合物1-e(5g,18.70mmol)加入二氯甲烷(100mL)中，然后加入1-f(6.84g,19.64mmol)，将反应液在25℃下搅拌2小时。TLC(石油醚:乙酸乙酯＝3:1)显示化合物1-e反应完全，并有新产物生成。将反应液减压浓缩除去多余的二氯甲烷。得到的粗产物，通过硅胶柱层析(石油醚/乙酸乙酯＝3/1)纯化，得到化合物1-g。 1H NMR(400MHz,CDCl 3):8.21(d,J＝0.6Hz,1H),7.52(d,J＝15.9Hz,1H),6.32(d,J＝15.8Hz,1H),5.06(br d,J＝5.1Hz,1H),4.32-4.29(m,2H),2.55(s,3H),2.31-2.21(m,1H),2.07-1.97(m,1H),1.93-1.71(m,3H),1.64-1.52(m,1H),1.37(t,J＝7.1Hz,3H),1.14(s,3H)。
化合物1-h的合成：
将化合物1-g(560mg,1.66mmol)加入四氢呋喃(10mL)中，然后在氮气保护下加入(PPh 3) 3RhCl(307.09mg,331.91μmol)，将反应液用氢气置换几次，将反应液在氢气压力50Psi，50℃下搅拌16小时。LCMS显示反应完全。将反应液冷却到25℃，减压浓缩，得到的粗产物通过硅胶柱层析(石油醚/乙酸乙酯＝3/1)纯化，得到化合物1-h。 1H NMR(400MHz,CDCl 3):7.74(s,1H),5.73(br d,J＝4.3Hz,1H),5.46(s,1H),4.19-4.12(m,1H),2.66-2.59(m,2H),2.55-2.48(m,2H),2.42(s,3H),2.17-2.07(m,1H),1.96-1.87(m,1H),1.81-1.70(m,2H),1.57-1.47(m,2H),1.21-1.18(m,3H),1.01(s,3H)。
化合物1-i的合成：
将化合物1-h(360mg,1.06mmol)加入N-甲基吡咯烷酮(5mL)中，然后加入DBU(322.90mg,2.12mmol,319.71μL)，将反应液在120℃下搅拌16小时。LCMS显示反应完全。将反应液冷却到25℃，用乙酸乙酯(10mL×3)萃取，有机相合并用饱和食盐水(20mL)洗涤，用无水硫酸钠干燥，过滤，滤液浓缩，得到的粗产物通过薄层层析法(石油醚/乙酸乙酯＝1/1)纯化，得到化合物1-i。LCMS(ESI):m/z:294.2(M+1)；
1H NMR(400MHz,CDCl 3):8.23(s,1H),5.00(t,J＝8.8Hz,1H),2.88-2.81(m,2H),2.75-2.65(m,4H),2.57(s,3H),2.05-1.90(m,4H),1.19(s,3H)。
化合物1-j的合成：
将化合物1-i(100mg,340.85μmol)加入2-甲基四氢呋喃(1mL)和水(0.2mL)中，然后加入过硫酸氢钾复合盐(523.86mg,852.13μmol)，在25℃下搅拌2小时。LCMS显示反应完全。将反应液用饱和亚硫酸钠(15mL)淬灭，用乙酸乙酯(10mL×3)萃取，有机相合并用饱和食盐水(15mL)洗涤，用无水硫酸钠干燥，过滤，滤液浓缩，得到化合物1-j。LCMS(ESI):m/z:307.8(M-18+1)；
1H NMR(400MHz,CDCl 3):8.45(s,1H),5.01(t,J＝8.7Hz,1H),3.25(s,3H),2.96-2.91(m,2H),2.76-2.69(m,2H),2.12-1.98(m,2H),1.96-1.88(m,2H),1.84-1.78(m,2H),1.09(s,3H)。
化合物1的合成：
将化合物1-j(80mg,245.86μmol)加入二甲基亚砜(1mL)中然后加入1-k(79.19mg,368.80μmol,盐酸盐)和二异丙基乙胺(158.88mg,1.23mmol,214.13μL)，将反应液在120℃搅拌16小时。LCMS显示反应完全。将反应液冷却到25℃，加入水(10mL)用乙酸乙酯(10mL×3)萃取，有机相合并用饱和食盐水(15mL×2)洗涤，用无水硫酸钠干燥，过滤，滤液浓缩，得到的粗品通过制备色谱(色谱柱：3_Phenomenex Luna C18 75×30mm×3μm；流动相：以0.05％的盐酸溶液为流动相A，乙腈为流动相B， B％：11％-31％，梯度时间：6.5min)纯化。纯化后的混合液用饱和碳酸氢钠调pH到7～8，然后用二氯甲烷(10mL×3)萃取，有机相合并用饱和食盐水(15mL×2)洗涤，用无水硫酸钠干燥，过滤，滤液蒸干，得到化合物1。LCMS(ESI)m/z:424.1(M+1)。
化合物1B的合成：
将化合物1经SFC手性分离得到1A和1B。
化合物1通过SFC制备(柱子：聚甲醛涂敷型手性固定相(250mm×30mm×10μm)；流动相：45％[0.1％氨水乙醇溶液]；纯化得到化合物1A(保留时间0.548分钟)和化合物1B(保留时间0.895分钟)。
化合物1B： 1H NMR(400MHz,MeOD)δ7.87(s,1H),5.17(br t,J＝8.6Hz,1H),3.85-3.76(m,1H),3.64-3.55(m,2H),2.89-2.81(m,2H),2.76(s,3H),2.66-2.59(m,2H),2.54-2.48(m,2H),2.40-2.31(m,1H),2.12-1.92(m,4H),1.82-1.73(m,2H),1.67-1.60(m,1H),1.57-1.46(m,2H),1.06(s,3H)。
实施例2：化合物2的盐酸盐
Figure PCTCN2020121390-appb-000051
化合物2-a的合成：
将化合物1-b(5g,26.23mmol)加入乙酸乙酯(50mL)中，然后加入活性二氧化锰(22.80g,262.26mmol)，将反应液在50℃下搅拌16小时。TLC(石油醚:乙酸乙酯＝3:1)显示化合物1-b反应完全，并有新产物 生成。将反应液冷却到25℃，过滤，滤饼用甲醇(30mL×3)洗涤，滤液蒸干，得到的粗产物通过硅胶柱层析(石油醚/乙酸乙酯＝3/1)纯化，得到化合物2-a。 1H NMR(400MHz,CDCl 3):δ10.33(s,1H),8.89(s,1H),2.66(s,3H)。
化合物2-b的合成：
将化合物2-a(3g,15.90mmol)加入二氯甲烷(20mL)中，然后加入化合物1-f(5.82g,16.70mmol)，将反应液在30℃下搅拌2小时。TLC(石油醚:乙酸乙酯＝10:1)显示化合物2-a反应完全，并有新产物生成。将反应液减压浓缩，得到的粗产物，通过硅胶柱层析(石油醚/乙酸乙酯＝3/1)纯化，得到化合物2-b。
1H NMR(400MHz,CDCl 3):δ8.66(s,1H),7.81(d,J＝16.1Hz,1H),6.51(d,J＝16.3Hz,1H),4.31(q,J＝7.2Hz,2H),2.61(s,3H),1.37(t,J＝7.1Hz,3H)。
化合物2-c的合成：
将化合物2-b(4.2g,16.23mmol)加入2-甲基四氢呋喃(20mL)中，然后加入Rh(PPh 3) 3Cl(3.00g,3.25mmol)，反应液用氢气置换几次，将反应液在氢气(15Psi)，80℃下搅拌16小时。LCMS显示反应完全。将反应液冷却到20℃，减压浓缩除去多余的2-甲基四氢呋喃，得到的粗产物，通过硅胶柱层析(石油醚/乙酸乙酯＝10/1)纯化，得到化合物2-c。LCMS(ESI):m/z:260.9(M+1)。
化合物2-d的合成：
将化合物2-c(1.9g,7.29mmol)加入四氢呋喃(20mL)中，冷却到-78℃，在氮气保护下缓慢加入二异丙基氨基锂(1M,18.22mL)，将反应液在-78℃下搅拌30分钟，然后加入碘甲烷(1.55g,10.93mmol,680.46μL)，将反应液升到25℃，并搅拌1.5小时。TLC(石油醚:乙酸乙酯＝10:1)显示化合物2-c反应完全，并有新产物生成。反应液用饱和氯化铵(25mL)淬灭，用乙酸乙酯(10mL×3)萃取，有机相合并用饱和食盐水(20mL)洗涤，用无水硫酸钠干燥，过滤，滤液蒸干，得到的粗产物，通过硅胶柱层析(石油醚/乙酸乙酯＝10/1)纯化，得到化合物2-d。
化合物2-e的合成：
将化合物2-d(1.2g,4.37mmol)和化合物1-c(1.01g,8.73mmol)加入到二甲基亚砜(20mL)中，然后加入二异丙基乙胺(1.69g,13.10mmol,2.28mL)，将反应液在110℃下搅拌16小时。LCMS显示反应完全。将反应液冷却到25℃，加入水(5mL)，用乙酸乙酯(10mL×3)，有机相合并用饱和食盐水(20mL)洗涤，用无水硫酸钠干燥，过滤，滤液蒸干，得到的粗产物，通过硅胶柱层析(石油醚/乙酸乙酯＝3/1)纯化，得到化合物2-e。LCMS(ESI):m/z:354.2(M+1)； 1H NMR(400MHz,CDCl 3):7.84-7.81(m,1H),4.17-4.11(m,2H),2.80(td,J＝8.0,15.0Hz,1H),2.66-2.59(m,1H),2.51(d,J＝0.6Hz,3H),2.49-2.41(m,1H),2.28-2.17(m,1H),2.00(dt,J＝3.5,7.5Hz,1H),1.90-1.81(m,2H),1.79-1.68(m,1H),1.61(s,5H),1.27-1.25(m,3H),1.11(d,J＝8.2Hz,3H)。
化合物2-f的合成：
将化合物2-e(1.1g,3.11mmol)加入N-甲基吡咯烷酮(15mL)中然后加入DBU(947.50mg,6.22mmol,938.12μL)，将反应液在120℃下搅拌12小时。LCMS显示反应完全。将反应液冷却到25℃，加入水(20mL)，用乙酸乙酯(20mL×3)，有机相合并用饱和食盐水(30mL×2)洗涤，用无水硫酸钠干燥，过滤，滤液蒸干，得到的粗产物，通过硅胶柱层析(石油醚/乙酸乙酯＝2/1)纯化，得到化合物2-f。LCMS (ESI):m/z:208.2(M+1)。
化合物2-g的合成：
将化合物2-f(1g,3.25mmol)加入2-甲基四氢呋喃(10mL)和水(2mL)中，然后加入过硫酸氢钾复盐(5.00g,8.13mmol)，将反应液在25℃下搅拌2小时。LCMS显示反应完全。将反应液用饱和亚硫酸钠(50mL)淬灭，用乙酸乙酯(20mL×3)萃取，有机相合并用饱和食盐水(30mL×3)洗涤，用无水硫酸钠干燥，过滤，滤液浓缩，得到化合物2-g。LCMS(ESI):m/z:322.1(M-18+1)； 1H NMR(400MHz,CDCl 3):8.46-8.42(m,1H),3.31(t,J＝7.1Hz,2H),3.25(s,3H),3.00(dt,J＝4.5,14.1Hz,1H),2.77(s,3H),2.28-2.28(m,1H),2.30(t,J＝8.1Hz,1H),1.81-1.77(m,1H),1.27-1.19(m,4H),1.10-1.06(m,3H)。
化合物2的合成：
将化合物2-g(500mg,1.47mmol)和化合物1-k(474.46mg,2.21mmol,盐酸盐)加入到二甲基亚砜(8mL)中，然后加入二异丙基乙胺(951.95mg,7.37mmol,1.28mL)，将反应液在140℃下搅拌16小时。LCMS显示反应完全。将反应液冷却到25℃，加入水(20mL)，用乙酸乙酯(10mL×3)，有机相合并用饱和食盐水(15mL×2)洗涤，用无水硫酸钠干燥，过滤，滤液蒸干，得到的粗产物，通过制备色谱(色谱柱：3_Phenomenex Luna C18 75×30mm×3μm；流动相：以0.05％的盐酸溶液为流动相A，乙腈为流动相B，B％：13％-33％，梯度时间：6.5min)纯化，得到化合物2的盐酸盐。LCMS(ESI):m/z:438.1(M+1)；
1H NMR(400MHz,MeOD)δ8.01(d,J＝2.4Hz,1H),3.83-3.75(m,1H),3.09-2.93(m,4H),2.90(s,3H),2.78-2.57(m,2H),2.53-2.25(m,2H),2.25-2.10(m,4H),1.97-1.89(m,2H),1.82-1.67(m,4H),1.28(dd,J＝6.7,11.4Hz,3H),1.17(d,J＝2.4Hz,3H)。
实施例3：化合物3
Figure PCTCN2020121390-appb-000052
Figure PCTCN2020121390-appb-000053
化合物3-b的合成：
将化合物3-a(5g,24.97mmol)和三乙胺(5.05g,49.93mmol)溶于二氯甲烷(50mL)后，所得混合物在15℃氮气保护下搅拌0.5小时，然后缓慢加入氯甲酸苄酯(6.39g,37.45mmol)，所得混合物在该温度下搅拌1.5小时。将反应液加水(80mL)稀释，二氯甲烷(30mL×4)萃取。所得有机相用饱和食盐水(60mL×1)洗涤，无水Na 2SO 4干燥，过滤，浓缩。得到的粗品，通过石油醚(50mL)在25℃下搅拌3小时纯化，得到化合物3-b。 1H NMR(400MHz,CD 3OD)δppm 1.24-1.37(m,2H)1.43(s,9H)1.83(br d,J＝10.51Hz,2H)2.95(br s,2H)3.44-3.57(m,1H)4.05(br d,J＝13.51Hz,2H)5.10(s,2H)7.23-7.40(m,5H)；LCMS(ESI):m/z:335.2(M+1)。
化合物3-c的合成：
将化合物3-b(7g,20.93mmol)溶于乙酸乙酯(100mL)，然后加入盐酸乙酸乙酯溶液(4M,52.33mL)，所得混合液在15℃下搅拌2小时，将反应液浓缩后得到化合物3-c。 1H NMR(400MHz,CD 3OD)δppm1.49(qd,J＝12.29,4.46Hz,2H)2.00(br d,J＝12.35Hz,2H)2.94(br s,2H)3.34(s,2H)4.22(dt,J＝13.97,2.25Hz,2H)5.12(s,2H)7.27-7.38(m,5H)；LCMS(ESI):m/z:235.2(M+1)。
化合物3-e的合成：
将化合物3-d(1.2g,3.69mmol)，化合物3-c(2.59g,11.06mmol)和二异丙基乙胺(3.81g,29.50mmol)溶于二甲基亚砜(20mL)后，所得混合物在110℃下搅拌12小时，将反应液加水(100mL)稀释，乙酸乙酯(60mL×3)萃取。所得有机相用饱和食盐水(100mL×1)洗涤，无水Na 2SO 4干燥，过滤，浓缩。得到粗品化合物通过薄层层析法(二氯甲烷：甲醇＝10:1)纯化得到化合物3-e。LCMS(ESI):m/z:480.3(M+1)。化合物3-f的合成：
将化合物3-e(830mg,1.73mmol)和湿钯碳(300mg,10％)溶于甲醇(15mL)后，所得混合液在15℃，15psi氢气下搅拌2小时，将反应液过滤，滤液浓缩后得到化合物3-f。
化合物3的合成：
将化合物3-f(100mg,289.49μmol)，3-g(61.05mg,434.23μmol)和三乙胺(43.94mg,434.23μmol)溶于二氯甲烷(2mL)，所得混合液在15℃下搅拌1小时。将反应液浓缩后通过制备色谱(色谱柱：Waters Xbridge 150×25mm×5μm；流动相：以10mM的碳酸氢铵溶液为流动相A，乙腈为流动相B，B％：20％-50％，梯度时间：9min)纯化得到化合物3。 1H NMR(400MHz,CD 3OD)δppm 1.01-1.07(m,4H)1.16(s,3H)1.53-1.66(m,2H)1.68-1.77(m,1H)1.83-1.92(m,2H)2.00-2.20(m,4H)2.42-2.53(m,2H)2.56-2.64(m,2H)2.68-2.78(m,2H)3.07(td,J＝12.10,2.08Hz,2H)3.74(br dd,J＝12.23,3.55Hz,2H)3.86-3.98(m,1H)5.27(br t,J＝8.44Hz,1H)7.97(s,1H).LCMS(ESI):m/z:450.3(M+1)。
实施例4：化合物4
Figure PCTCN2020121390-appb-000054
化合物4的合成：
将化合物3-f(100mg,289.49μmol)，4-a(55.83mg,434.23μmol)和三乙胺(43.94mg,434.23μmol)溶于二氯甲烷(2mL)，所得混合液在15℃下搅拌1小时。将反应液浓缩后通过制备色谱(色谱柱：Waters Xbridge 150×25mm×5μm；流动相：以10mM的碳酸氢铵溶液为流动相A，乙腈为流动相B，B％：15％-48％，梯度时间：10min)纯化得到化合物4。 1H NMR(400MHz,CD 3OD-d 4)δppm 1.17(s,3H)1.34(t,J＝7.40Hz,3H)1.52-1.65(m,2H)1.69-1.77(m,1H)1.83-1.94(m,2H)2.00-2.22(m,4H)2.42-2.52(m,1H)2.56-2.67(m,2H)2.68-2.77(m,2H)3.00-3.09(m,4H)3.70-3.78(m,2H)3.86-3.97(m,1H)5.27(br t,J＝8.62Hz,1H)7.98(s,1H)；LCMS(ESI):m/z:438.3(M+1)。
实施例5：化合物5
Figure PCTCN2020121390-appb-000055
Figure PCTCN2020121390-appb-000056
化合物5的合成：
将化合物3-f(100mg,289.49μmol)，5-a(61.92mg,434.23μmol)和三乙胺(43.94mg,434.23μmol)溶于二氯甲烷(2mL)，所得混合液在15℃下搅拌1小时。将反应液浓缩后通过制备色谱(色谱柱：Waters Xbridge 150×25mm×5μm；流动相：以10mM的碳酸氢铵溶液为流动相A，乙腈为流动相B，B％：21％-51％，梯度时间：9min)，纯化得到化合物5。 1H NMR(400MHz,METHANOL-d 4)δppm 1.16(s,3H)1.32(d,J＝6.72Hz,6H)1.49-1.60(m,2H)1.69-1.76(m,1H)1.83-1.93(m,2H)1.98-2.11(m,3H)2.17(td,J＝11.80,8.07Hz,1H)2.41-2.52(m,1H)2.57-2.64(m,2H)2.67-2.76(m,2H)3.10(br t,J＝11.37Hz,2H)3.73-3.82(m,2H)3.86-3.98(m,1H)5.26(br t,J＝8.56Hz,1H)7.96(s,1H)；LCMS(ESI):m/z:452.3(M+1)。
实施例6：化合物6
Figure PCTCN2020121390-appb-000057
化合物6的合成：
在0℃下向化合物3-f(5mg，14.47μmol)和三乙胺(2.93mg，28.95μmol，4.03μmL)的二氯甲烷(1mL)溶液中滴加6-a(2.30mg，14.47μmol)，反应液在15℃下搅拌2小时。LC-MS显示原料反应完全，并检测到目标产物已生成。反应液减压浓缩，通过制备色谱(色谱柱：Waters Xbridge 150×25mm×5μm；流动相：以0.05％的氨水溶液为流动相A，乙腈为流动相B，B％：18％-48％，梯度时间：9min)纯化，得到化合物6。 1H NMR(400MHz,CD 3OD)δ7.87(s,1H)，5.17(br s,1H)，3.84-3.73(m,1H)，3.68-3.56(m,4H)，3.28(s,3H)，3.20-3.15(m,2H)，2.92(br t,J＝11.3Hz,2H)，2.67-2.57(m,2H)，2.55-2.47(m,2H)，2.43-2.31(m,1H)，2.13-1.89(m,4H)，1.84-1.74(m,2H)，1.66-1.59(m,1H)，1.55-1.40(m,2H)，1.24-1.17(m,1H)，1.10-1.02(m,3H)。LCMS(ESI)m/z:468.4(M+1)。
实施例7：化合物7
Figure PCTCN2020121390-appb-000058
化合物7-b的合成：
将化合物7-a(7.5g,36.19mmol)和三乙胺(5.49g,54.29mmol,7.56mL)加入二氯甲烷(100mL)中，冷却到0℃，然后向反应液中滴加甲烷磺酰氯(4.98g,43.43mmol,3.36mL)，将反应温度升到15℃，并搅拌3小时。TLC(石油醚/乙酸乙酯＝1/1)显示反应完全，有新化合物生成。将反应液用1M盐酸溶液(50mL)淬灭，用二氯甲烷(50mL×3)萃取，有机相合并用饱和食盐水(100mL)洗涤，用无水硫酸钠干燥，过滤，滤液蒸干，得到化合物7-b。 1H NMR(400MHz,CDCl 3):δ7.32-7.23(m,5H),5.16(tt,J＝4.2,6.7Hz,1H),5.03(s,2H),4.32-4.26(m,2H),4.13-4.08(m,2H),2.99(s,3H)。
化合物7-d的合成：
将化合物7-b(5g,17.52mmol)加入N,N-二甲基甲酰胺(50mL)中，加入碳酸钾(3.63g,26.29mmol)，将反应液冷却到0℃，然后滴加7-c(2.00g,26.29mmol)。将反应液加热到80℃搅拌16小时。LCMS显示反应完全。将反应液冷却到20℃，加入水(100mL)，用乙酸乙酯(50mL×3)萃取，有机相合并用饱和食盐水(100)洗涤，用无水硫酸钠干燥，过滤，滤液蒸干，得到的粗产物通过硅胶柱层析(石油醚/乙酸乙酯＝5/1)纯化，得到化合物7-d。 1H NMR(400MHz,CDCl 3):δ7.41-7.30(m,5H),5.12(s,2H),4.48(t,J＝8.8Hz,2H),4.23(tt,J＝5.6,8.2Hz,1H),3.92(dd,J＝5.6,9.5Hz,2H),2.35(s,3H)；LCMS(ESI):m/z:266.0(M+1)。
化合物7-e的合成：
将化合物7-d(2.5g,9.42mmol)加入水(10mL)和二氯甲烷(20mL)中，冷却到0℃，然后向反应体系中通入氯气，将反应液在0-10℃下通氯气反应1小时。TLC(石油醚/乙酸乙酯＝5/1)显示反应完全，有新化合物生成。将反应液分液，有机相用水(50mL)，饱和碳酸钠(50mL×1)和饱和食盐水(50mL×1)洗涤，用无水硫酸钠干燥，过滤，滤液蒸干，得到的粗产物通过硅胶柱层析(石油醚/乙酸乙酯＝5/1)纯化，得到化合物7-e。 1H NMR(400MHz,CDCl 3):δ7.31-7.26(m,5H),5.06(s,2H),4.55-4.34(m,5H)。
化合物7-f的合成：
将化合物3-f(1g,2.89mmol)和三乙胺(585.86mg,5.79mmol,805.86μL)加入二氯甲烷(10mL)中，然后加入化合物7-e(838.75mg,2.89mmol)，将反应液在15℃下搅拌16小时。LCMS显示反应完全。反应液减压蒸干，得到的粗产物通过硅胶柱层析(石油醚/乙酸乙酯＝0/1)纯化，得到化合物7-f。 1H NMR(400MHz,CD 3OD):δ7.98(s,1H),7.38-7.29(m,5H),5.28(br t,J＝8.5Hz,1H),5.14-5.13(m,2H),4.34-4.22(m,5H),3.95-3.89(m,1H),3.81-3.75(m,2H),3.10-3.02(m,2H),2.99-2.88(m,1H),2.77-2.71(m,2H),2.65-2.59(m,2H),2.51-2.43(m,1H),2.24-2.16(m,1H),1.95-1.86(m,3H),1.82-1.72(m,2H),1.61-1.51(m,2H),1.17(s,3H)；LCMS(ESI):m/z:599.4(M+1)。
化合物7的合成：
将化合物7-f(1.1g,1.84mmol)加入甲醇(20mL)，在氮气保护下加入湿钯/碳(10％,200mg)，反应液用氢气置换几次。将反应液在氢气(15psi)保护15℃下搅拌10小时。LCMS显示反应完全。将反应液通过硅藻土过滤，滤液蒸干，得到的粗产物制备色谱(色谱柱：Waters Xbridge C18 250×50mm×10μm；流动相：以0.05％的氨水溶液为流动相A，乙腈为流动相B，B％：15％-30％，梯度时间：20min)纯化，得到化合物7。 1H NMR(400MHz,CDCl 3):δ7.98(s,1H),5.28(br t,J＝8.4Hz,1H),4.39(quin,J＝7.6Hz,1H),4.02-3.70(m,7H),3.02(br t,J＝11.6Hz,2H),2.77-2.70(m,2H),2.66-2.59(m,2H),2.51-2.41(m,1H),2.23-2.03(m,4H),1.94-1.86(m,2H),1.78-1.73(m,1H),1.55(dq,J＝8.1,11.6Hz,2H),1.18(s,3H)；LCMS(ESI):m/z:465.3(M+1)。
实施例8：化合物8
Figure PCTCN2020121390-appb-000059
化合物8的合成：
将化合物7(70mg,145.05μmol)和乙醛(31.95mg,725.27μmol,40.70μL)加入甲醇(2mL)，在15℃下搅拌30分钟，然后加入氰基硼氢化钠(18.23mg,290.11μmol)，在15℃下搅拌1小时。LCMS显示反应完全。将反应液减压蒸干，得到的粗产物制备色谱(色谱柱：Waters Xbridge C18 150×25mm×5μm；流动相：以10mM的碳酸氢铵溶液为流动相A，乙腈为流动相B，B％：18％-48％，梯度时间：9min)纯化，得到化合物8。 1H NMR(400MHz,CD 3OD):δ7.98(s,1H),5.28(br t,J＝8.5Hz,1H),4.15(quin,J＝7.6Hz,1H),3.95-3.88(m,1H),3.74(br dd,J＝3.7,12.4Hz,2H),3.68(t,J＝8.5Hz,2H),3.46(t,J＝8.1Hz,2H),3.02(br t,J＝11.7Hz,2H),2.77-2.71(m,2H),2.65-2.56(m,4H),2.53-2.43(m,1H),2.24-2.04(m,4H),1.95-1.86(m,2H),1.79-1.72(m,1H),1.62-1.49(m,2H),1.18(s,3H),1.00(t,J＝7.2Hz,3H)；LCMS(ESI):m/z:493.4(M+1)。
实施例9：化合物9
Figure PCTCN2020121390-appb-000060
将化合物7(70mg,145.05μmol)和丙酮(42.12mg,725.25μmol,53.32μL)加入甲醇(2mL)，在15℃下搅拌30分钟，然后加入氰基硼氢化钠(18.23mg,290.11μmol)，在15℃下搅拌1小时。LCMS显示反应完全。将反应液减压蒸干，得到的粗产物制备色谱(色谱柱：Waters Xbridge C18 150×25mm×5μm；流动相：以10mM的碳酸氢铵溶液为流动相A，乙腈为流动相B，B％：22％-52％，梯度时间：9min)纯化，得到化合物9。 1H NMR(400MHz,CD 3OD):δ7.98(s,1H),5.28(br t,J＝8.6Hz,1H),4.08(quin,J＝7.7Hz,1H),3.97-3.87(m,1H),3.74(br dd,J＝3.8,12.3Hz,2H),3.67(t,J＝8.4Hz,2H),3.46(t,J＝8.2Hz,2H),3.03(br t,J＝11.7Hz,2H),2.79-2.70(m,2H),2.67-2.58(m,2H),2.55-2.43(m,2H),2.24-2.03(m,4H),1.94-1.84(m,2H),1.79-1.72(m,1H),1.62-1.49(m,2H),1.18(s,3H),0.97(d,J＝6.2Hz,6H)；LCMS(ESI):m/z:507.4(M+1)。
实施例10：化合物10
Figure PCTCN2020121390-appb-000061
化合物10的合成：
将化合物7(90mg,145.05μmol)和甲醛(75.67mg,932.49μmol,69.42μL,37％纯度)加入甲醇(2mL)，在15℃下搅拌30分钟，然后加入氰基硼氢化钠(23.44mg,372.99μmol)，在15℃下搅拌1小时。LCMS显示反应完全。将反应液减压蒸干，得到的粗产物制备色谱(色谱柱：Waters Xbridge C18 150×30mm×5μm；流动相：以10mM的碳酸氢铵溶液为流动相A，乙腈为流动相B，B％：16％-46％， 梯度时间：11.5min)纯化得到化合物10。 1H NMR(400MHz,CD 3OD):δ7.99(s,1H),5.28(br t,J＝8.4Hz,1H),4.36-4.28(m,1H),4.20(t,J＝9.4Hz,2H),4.03(dd,J＝6.5,10.5Hz,2H),3.97-3.90(m,1H),3.80-3.74(m,2H),3.11-3.03(m,2H),2.78-2.70(m,5H),2.66-2.60(m,2H),2.53-2.42(m,1H),2.24-2.04(m,4H),1.95-1.85(m,2H),1.79-1.72(m,1H),1.63-1.52(m,2H),1.18(s,3H)；LCMS(ESI):m/z:479.3(M+1)。
实施例11：化合物11、化合物11A和化合物11B
Figure PCTCN2020121390-appb-000062
化合物11-b的合成：
将化合物11-a(4.2g,43.67mmol)加入二氯甲烷(150mL)，冷却到0℃，然后缓慢分批加入间氯过氧苯甲酸(13.30g,65.51mmol,85％纯度)，将反应液在15℃反应16小时。将混合物过滤，滤液用饱和亚硫酸钠溶液(50mL)淬灭。分液水相用二氯甲烷(20mL×2)萃取，有机相合并，用饱和碳酸氢钠溶液(50mL×2)和饱和食盐水(50mL)洗涤，用无水硫酸钠干燥，过滤，滤液在不高于15℃减压蒸干，得到化合物11-b。 1H NMR(400MHz,CDCl 3):δ2.07-1.32(m,9H),0.98(t,J＝7.5Hz,3H)。
化合物11-c的合成：
将化合物11-b(5.1g,45.47mmol)加入水(50mL)中，加入苄胺(4.38g,40.92mmol,4.46mL)，将反应液在100℃下搅拌16小时。LCMS显示反应完全。将反应液用冰水冷却，用浓盐酸调pH＝1，用乙酸乙酯(30mL×2)萃取。将水相用5M氢氧化钠调pH＝10。用乙酸乙酯(30mL×3)萃取，有机相合并用饱和食盐水(30mL×2)洗涤，用无水硫酸钠干燥，过滤，蒸干。得到的粗产物通过硅胶柱层析(石油醚/乙酸乙酯＝1/1)纯化，得到化合物11-c。 1H NMR(400MHz,CDCl 3):δ7.19-7.15(m,1H),7.30-7.11(m,4H),3.83-3.62(m,2H),2.81(t,J＝6.6Hz,1H),1.79-1.43(m,8H),0.93-0.86(m,3H)；LCMS(ESI):m/z:220.2(M+1)。
化合物11-d的合成：
将化合物11-c(2.6g,11.85mmol)加入异丙醇(30mL)中，在氮气保护下加入湿氢氧化钯/碳(0.5g,50％)。然后反应体系用氢气置换几次，将反应液在氢气(50psi)25℃下搅拌16小时。TLC(二氯甲烷:甲醇＝10:1)显示反应完全。将反应液通过硅藻土过滤，滤液蒸干，得到化合物11-d。 1H NMR(400MHz,CDCl 3):δ3.10(dd,J＝5.2,6.3Hz,1H),2.17(ddd,J＝2.6,6.6,13.1Hz,1H),1.79-1.72(m,3H),1.61-1.50(m,3H),1.41-1.30(m,1H),1.03-0.99(m,3H)。
化合物11-f的合成：
将化合物11-e(2.3g,12.06mmol)和化合物12-d(1.56g,12.06mmol)，然后加入乙腈(30mL)中，加入三乙胺(1.83g,18.10mmol,2.52mL)，将反应液在80℃下搅拌16小时。LCMS显示反应完全。将反应液冷却到25℃，减压浓缩除去乙腈，得到的粗产物通过硅胶柱层析(石油醚/乙酸乙酯＝1/1)纯化，得到化合物11-f。 1H NMR(400MHz,CDCl 3):δ7.78(s,1H),4.53(t,J＝6.7Hz,1H),4.49(s,2H),2.53(s,3H),2.37-2.27(m,1H),1.87-1.49(m,8H),0.96(t,J＝7.4Hz,3H)；LCMS(ESI):m/z:284.2(M+1)。
化合物11-g的合成：
将化合物11-f(2.38g,8.40mmol)加入乙酸乙酯(40mL)中，然后加入二氧化锰(7.30g,83.98mmol)，将混合物在60℃下反应2小时。TLC(石油醚/乙酸乙酯＝1/1)显示反应完全。将反应液冷却到25℃，过滤，滤饼用甲醇(20mL×3)洗涤，滤液蒸干，得到化合物11-g。 1H NMR(400MHz,CDCl 3):δ9.73(s,1H),8.66(br d,J＝4.8Hz,1H),8.35(s,1H),4.47(td,J＝7.6,8.7Hz,1H),2.58(s,3H),2.34-2.26(m,1H),1.95-1.70(m,5H),1.66-1.60(m,1H),1.41(q,J＝7.3Hz,2H),0.96(t,J＝7.3Hz,3H)。
化合物11-i的合成：
将化合物11-g(2.3g,8.17mmol)加入二氯甲烷(50mL)中，然后加入化合物11-h(2.99g,8.58mmol)，将反应液在20℃下搅拌2小时。TLC(石油醚/乙酸乙酯＝5/1)显示反应完全，有新化合物生成。将反应液减压蒸干，得到的粗产物通过硅胶柱层析(石油醚/乙酸乙酯＝5/1)纯化，得到化合物11-i。 1H NMR(400MHz,CDCl 3):δ8.11(s,1H),7.42(d,J＝15.8Hz,1H),6.22(d,J＝15.8Hz,1H),4.95(br d,J＝5.5Hz,1H), 4.29(ddd,J＝6.0,7.8,10.5Hz,2H),4.21(q,J＝7.1Hz,2H),2.46(s,3H),2.22-2.10(m,1H),1.84-1.71(m,3H),1.70-1.59(m,1H),1.53-1.42(m,1H),1.27(t,J＝7.2Hz,3H),0.88(t,J＝7.3Hz,3H)。
化合物11-j的合成：
将化合物11-i(2.7g,7.68mmol)加入2-甲基四氢呋喃(50mL)中，然后加入Rh(PPh 3) 3Cl(710.77mg,768.22μmol)，将反应液用氢气置换几次，在氢气(15Psi)保护，80℃下搅拌20小时。TLC(石油醚/乙酸乙酯＝3/1)显示反应完全，有新化合物生成。将反应液减压蒸干，得到的粗产物通过硅胶柱层析(石油醚/乙酸乙酯＝3/1)纯化，得到化合物11-j。 1H NMR(400MHz,CDCl 3):δ7.73(s,1H),5.66(br d,J＝5.3Hz,1H),4.21(ddd,J＝5.8,7.9,10.4Hz,1H),4.10-4.04(m,3H),2.64-2.60(m,2H),2.53-2.48(m,2H),2.42(s,3H),2.14-2.07(m,1H),1.84-1.71(m,3H),1.62-1.48(m,3H),1.20-1.18(m,3H),0.88-0.85(m,3H)。
化合物11-k的合成：
将化合物11-j(600mg,1.70mmol)加入N-甲基吡咯烷酮(10mL)中，然后加入DBU(516.82mg,3.39mmol,511.70μL)，将反应液在80℃下搅拌16小时。LCMS显示反应完全。将反应液冷却到25℃，加入水(10mL)，用乙酸乙酯(10mL×3)萃取，有机相合并用饱和食盐水(20mL×2)洗涤，用无水硫酸钠干燥，过滤，滤液蒸干，得到的粗产物通过硅胶柱层析(石油醚/乙酸乙酯＝1/1)纯化，得到化合物11-k。 1H NMR(400MHz,CDCl 3):δ8.13(s,1H),4.96(br t,J＝8.9Hz,1H),2.77-2.71(m,2H),2.64-2.60(m,2H),2.48(s,3H),1.96-1.73(m,6H),1.48-1.41(m,1H),1.22-1.18(m,2H),0.84(t,J＝7.4Hz,3H)；LCMS(ESI):m/z:308.1(M+1)。
化合物11-l的合成：
将化合物11-k(200mg,650.60μmol)加入2-甲基四氢呋喃(5mL)和水(1mL)中，然后加入过硫酸氢钾(999.91mg,1.63mmol)，将反应液在25℃下搅拌2小时。LCMS显示反应完全。将反应液用饱和亚硫酸钠溶液(20mL)淬灭，用乙酸乙酯(20mL×3)萃取，有机相合并用饱和食盐水(20mL)洗涤，用无水硫酸钠干燥，过滤，滤液蒸干，得到化合物11-l。 1H NMR(400MHz,CDCl 3):δ8.54(s,1H),5.11(br t,J＝7.4Hz,1H),3.33(s,3H),3.06-2.99(m,2H),2.87-2.78(m,2H),2.05-1.81(m,6H),1.30-1.23(m,2H),0.94(t,J＝7.4Hz,3H)；LCMS(ESI):m/z:322.1(M+1-18)。
化合物11的合成：
将化合物11-l(390mg,1.15mmol)和化合物1-k(1.23g,5.75mmol,盐酸盐)加入N-甲基吡咯烷酮(10mL)，然后加入二异丙基乙胺(1.04g,8.04mmol,1.40mL)，将反应液在140℃下搅拌16小时。LCMS显示反应完全。将反应液冷却到20℃，加入水(10mL)，用乙酸乙酯(15mL×3)萃取，有机相合并用饱和食盐水(20mL)洗涤，用无水硫酸钠干燥，过滤，滤液蒸干，得到的粗产物制备色谱(色谱柱：Waters Xbridge C18 150×50mm×10μm；流动相：以0.05％的氨水溶液为流动相A，乙腈为流动相B，B％：18％-48％，梯度时间：11.5min)纯化，得到化合物11。 1H NMR(400MHz,CD 3OD):δ7.99(s,1H),5.31(br t,J＝8.4Hz,1H),3.96-3.86(m,1H),3.72(br dd,J＝3.4,11.9Hz,2H),2.97(dt,J＝2.3,11.6Hz,2H),2.88(s,3H),2.77-2.70(m,2H),2.65-2.59(m,2H),2.49-2.38(m,1H),2.15-2.05(m,5H),1.96-1.85(m,2H),1.77(dd,J＝6.1,12.0Hz,1H),1.68-1.57(m,2H),1.52-1.41(m,2H),0.91(t,J＝7.4Hz,3H)；LCMS(ESI):m/z:438.3(M+1)。
化合物11的拆分：
将化合物11经SFC手性分离得到11A和11B。
化合物11过SFC制备(柱子：聚甲醛涂敷型手性固定相(250mm×30mm×10μm)；流动相：65％[0.1％氨水乙醇溶液]；纯化得到化合物11A(保留时间0.479分钟)和化合物11B(保留时间1.516分钟)。
化合物11A： 1H NMR(400MHz,CD 3OD):δ7.87(s,1H),5.19(br t,J＝8.4Hz,1H),3.84-3.76(m,1H),3.60(br dd,J＝3.5,11.9Hz,2H),2.85(dt,J＝2.4,11.6Hz,2H),2.76(s,3H),2.64-2.57(m,2H),2.52-2.46(m,2H),2.37-2.26(m,1H),2.05-1.95(m,4H),1.86-1.73(m,2H),1.65(dd,J＝6.0,12.1Hz,1H),1.55-1.45(m,2H),1.40-1.30(m,2H),0.79(t,J＝7.4Hz,3H)；LCMS(ESI):m/z:438.3(M+1)。
化合物11B： 1H NMR(400MHz,CD 3OD):δ7.87(s,1H),5.19(br t,J＝8.4Hz,1H),3.84-3.75(m,1H),3.64-3.56(m,2H),2.85(dt,J＝2.5,11.6Hz,2H),2.76(s,3H),2.66-2.56(m,2H),2.53-2.46(m,2H),2.38-2.26(m,1H),2.06-1.93(m,4H),1.86-1.72(m,2H),1.65(dd,J＝6.2,11.9Hz,1H),1.57-1.44(m,2H),1.39-1.28(m,2H),0.79(t,J＝7.4Hz,3H)；LCMS(ESI):m/z:438.3(M+1)。
活性测试
实验例1：酶活性测试
■CDK2/CyclinA2激酶活性测试
实验材料：CDK2/CyclinA2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/CyclinA2酶(1.6ng)，2μL底物和ATP的混合物(50μM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的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。
■CDK2/CyclinE1激酶活性测试
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。
■CDK4/CyclinD1激酶活性测试
实验材料：CDK4/CyclinD1激酶购自Invitrogen，反应底物LANCE Ultra ULight  TM-4E-BP-1(Thr37146)Peptide(肽)和EU-ANTI-P-4EBP1(THR37/46)购自PerkinElmer。Nivo多标记分析仪 (PerkinElmer)。
实验方法：激酶缓冲液配制：缓冲液的成分包括：PH 7.5的羟乙基哌嗪乙硫磺酸溶液50mM，乙二胺四乙酸1mM，氯化镁10mM，0.01％月桂醇聚氧乙烯醚(Brij-35)，二硫苏糖醇2mM用激酶缓冲液稀释酶，底物LANCE Ultra ULight  TM-4E-BP-1(Thr37146)Peptide(肽)，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40μM稀释至0.512nM，DMSO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，5μL CDK4/CyclinD1酶(0.5ng)，置于25℃反应60分钟后，再加入2.5μL底物和ATP的混合物(350μM三磷酸腺苷，12.5nM底物)，此时化合物终浓度梯度为10μM稀释至0.128nM。反应体系置于25℃反应120分钟。反应结束后，每孔加入5μL EDTA和2X LANCE TM检测缓冲液(Detection Buffer)(1:1)混合液，25℃反应5分钟，结束反应后每孔加入5μL的LANCE Ultra Eu-anti-P-4E-BP1(Thr37MS)(4nM)，25℃反应60分钟后，根据时间分辨荧光共振能量转移原理利用Nivo仪器检测反应信号。
■CDK6/CyclinD1激酶活性测试
实验材料：CDK6/CyclinD1激酶购自Carna。反应底物LANCE Ultra ULight  TM-4E-BP-1(Thr37146)Peptide(肽)和EU-ANTI-P-4EBP1(THR37/46)购自PerkinElmer。Nivo多标记分析仪(PerkinElmer)。
实验方法：激酶缓冲液配制：缓冲液的成分包括：PH 7.5的羟乙基哌嗪乙硫磺酸溶液50mM，乙二胺四乙酸1mM，氯化镁10mM，0.01％月桂醇聚氧乙烯醚(Brij-35)，二硫苏糖醇2mM用激酶缓冲液稀释酶，底物LANCE Ultra ULight  TM-4E-BP-1(Thr37146)Peptide(肽)，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40μM稀释至0.512nM，DMSO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，5μL CDK6/CyclinD1酶(0.5ng)，置于25℃反应60分钟后，再加入2.5μL底物和ATP的混合物(250μM三磷酸腺苷，12.5nM底物)，此时化合物终浓度梯度为10μM稀释至0.128nM。反应体系置于25℃反应120分钟。反应结束后，每孔加入5μL EDTA和2X LANCE TM检测缓冲液(Detection Buffer)(1:1)混合液，25℃反应5分钟，结束反应后每孔加入5μL的LANCE Ultra Eu-anti-P-4E-BP1(Thr37MS)(4nM)，25℃反应60分钟后，根据时间分辨荧光共振能量转移原理利用Nivo仪器检测反应信号。
■CDK9/CyclinT1激酶活性测试
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CDK2/CyclinA2，CDK2/CyclinE1，CDK4/CyclinD1，CDK6/CyclinD1，CDK9/CyclinT1酶学抑制活性。
表1本发明实施例化合物的酶活性数据(IC 50)
Figure PCTCN2020121390-appb-000063
实验结论：本发明化合物对CDK2、CDK4和CDK6激酶具有显著的抑制活性，且对CDK9具有较高的选择性。
实验例2：细胞实验
■HCT116细胞活性测试
实验材料：McCoy'5a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。HCT116细胞系购自南京科佰生物科技有限公司。Nivo多标记分析仪(PerkinElmer)。
实验方法：将HCT116细胞种于白色96孔板中，80μL细胞悬液每孔，其中包含1000个HCT116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9个浓度，即从2mM稀释至5.12nM，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。转移到细胞板中的化合物浓度范围是10μM至0.0256nM。细胞板置于二氧化碳培养箱中培养4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25μL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。
■HCC1806细胞活性测试
实验材料：RPMI-1640培养基，胎牛血清，盘尼西林/链霉素抗生素购自维森特。CyQUANT Cell Proliferation Assays(细胞增殖分析试剂盒)试剂购自ThermoFisher。HCC1806细胞系购自南京科佰生物科技有限公司。Nivo多标记分析仪(PerkinElmer)。
实验方法：将HCC1806细胞种于白色96孔板中，80μL细胞悬液每孔，其中包含3000个HCC1806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9个浓度，即从10mM稀释至25.6nM，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。转移到细胞板中的化合物浓度范围是50μM至0.128nM。细胞板置于二氧化碳培养箱中培养7天。到达培养时间后去掉细胞上清，将细胞板放入-80℃冰箱1小时，之后每孔加入100μL Cyquant试剂，采用多标记分析仪进行读数。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。
■MDA-MB-468细胞活性测试
实验材料：L15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。MDA-MB-468细胞系购自南京科佰生物科技有限公司。Nivo多标记分析仪(PerkinElmer)。
实验方法：将MDA-MB-468细胞种于白色96孔板中，80μL细胞悬液每孔，其中包含1000个MDA-MB-468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9个浓度，即从10mM稀释至25.6nM，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。转移到细胞板中的化合物浓度范围是50μM至0.128nM。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25μL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，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2提供了本发明的化合物对细胞增殖的抑制活性。
表2本发明实施例化合物对细胞的抗增殖活性(IC 50)
Figure PCTCN2020121390-appb-000064
实验结论：本发明化合物对HCT 116及HCC1806细胞增殖具有显著的抑制活性，且对Rb阴性的MDA-MB-468活性较差，选择性显著优于PF-06873600。
实验例3：本发明化合物的药代动力学评价
试验动物
本研究使用的健康的成年雌性Balb/c小鼠，均购自上海灵畅生物科技有限公司。
药物的配制
静脉注射组给药溶液的配制
精确称量1.11mg待测化合物，加入109.8μL的DMSO涡旋1分钟后，加入109.8μL的solutol涡旋1分钟得到澄清溶液，再加入878μL的水涡旋1分钟，得到终浓度为1mg/mL的澄清溶液，给药溶媒为10％DMSO+10％solutol+80％水。静脉注射组溶液在给药前用2μm的滤膜进行过滤。
口服给药组给药溶液的配制
精确称量1.61mg待测化合物，加入796μL的1％HPMC在45℃超声20分钟，再搅拌15分钟后得到均匀的混悬溶液，得到终浓度为2mg/mL的混悬溶液，给药溶媒为1％HPMC。
给药
4只雌性Balb/c小鼠，按照每组2只，分为2组。第一组，静脉注射给予2mg/kg的待测化合物；第二组，灌胃给予10mg/kg待测化合物。
样品采集
采用连续采血方式，每个时间点采集两只动物的血液。分别于给药前及给药后0.0833、0.25、0.5、1、2(仅灌胃组)、4、6、8(仅静脉组)、24小时采集30μL全血。将全血置于抗凝管中，在4℃条件下，3200g离心10分钟，制备血浆并保存于-80℃。应用LC/MS-MS测定血浆中的药物浓度。
表3本发明实施例化合物的药代动力学结果
Figure PCTCN2020121390-appb-000065
实验结论：本发明化合物在小鼠体内清除率低，暴露量高，口服生物利用度较好，综合药代动力学性质优异。

Claims (27)

  1. 式(I)所示化合物或其药学上可接受的盐，
    Figure PCTCN2020121390-appb-100001
    其中，
    T为N或CH；
    R 1为C 4-6环烷基，其中所述C 4-6环烷基被1、2或3个R a所取代；
    各R a独立地为F、Cl、Br、I、-CN、-OH、C 1-3烷氧基或C 1-3烷基，其中所述C 1-3烷氧基和C 1-3烷基任选被1、2或3个独立选自F、Cl、Br、-CN、-OH和-NH 2的取代基所取代；
    R 2和R 3各自独立地为H、F、Cl、Br、I、-CN、-OH、C 1-3烷氧基或C 1-3烷基，其中所述C 1-3烷氧基和C 1-3烷基任选被1、2或3个独立选自F、Cl、Br、-CN、-OH和-NH 2的取代基所取代；
    或者R 2和R 3连接在一起，与其相连的碳原子一起形成C 3-5环烷基，所述C 3-5环烷基任选被1、2或3个R b所取代；
    各R b独立地为H、F、Cl、Br、I、-CN、-OH、C 1-3烷氧基、C 1-3烷基或C 1-3卤代烷基；
    R 4和R 5各自独立地为H、F、Cl、Br、I、-CN、-OH、C 1-3烷氧基或C 1-3烷基，其中所述C 1-3烷氧基和C 1-3烷基任选被1、2或3个独立选自F、Cl、Br、-CN、-OH和-NH 2的取代基所取代；
    或者R 3和R 4连接在一起，与其相连的碳原子一起形成C 3-5环烷基，所述C 3-5环烷基任选被1、2或3个R c所取代；
    各R c独立地为H、F、Cl、Br、I、-CN、-OH、C 1-3烷氧基、C 1-3烷基或C 1-3卤代烷基；
    R 6为H、F、Cl、Br、I、-CN、-OH、C 1-3烷氧基、C 1-3烷基或C 1-3卤代烷基；
    R 7为-NH 2、C 1-3烷氨基、C 1-6烷基、C 3-5环烷基、4-6杂环烷基、5-6元杂芳基或苯基，其中所述C 1-6烷基、C 3-5环烷基、4-6杂环烷基、5-6元杂芳基和苯基任选被1、2或3个R d所取代；
    各R d独立地为H、F、Cl、Br、I、-CN、-OH、C 1-3烷氧基或C 1-3烷基，其中所述C 1-3烷氧基和C 1-3烷基任选被1、2或3个独立选自F、Cl、Br、-CN、-OH和-NH 2的取代基所取代；
    n为0、1或2；
    所述4-6元杂环烷基和5-6元杂芳基分别包含1、2、3或4个独立选自N、-O-和-S-的杂原子。
  2. 根据权利要求1所述化合物或其药学上可接受的盐，其中各R a独立地为F、Cl、Br、I、-CN、-OH、-OCH 3、-CH 3、-CF 3或-CH 2CH 3。
  3. 根据权利要求1或2所述化合物或其药学上可接受的盐，其中R 1为
    Figure PCTCN2020121390-appb-100002
    其中所述
    Figure PCTCN2020121390-appb-100003
    被1、2或3个R a所取代。
  4. 根据权利要求3所述化合物或其药学上可接受的盐，其中R 1为
    Figure PCTCN2020121390-appb-100004
  5. 根据权利要求4所述化合物或其药学上可接受的盐，其中R 1为
    Figure PCTCN2020121390-appb-100005
  6. 根据权利要求1所述化合物或其药学上可接受的盐，其中所述化合物具有式(I-1)所示结构：
    Figure PCTCN2020121390-appb-100006
    其中，R 2、R 3、R 4、R 5、R 6、R 7、R a和n如权利要求1所定义，p为1、2或3。
  7. 根据权利要求6所述化合物或其药学上可接受的盐，其中所述化合物具有式(I-2)所示结构：
    Figure PCTCN2020121390-appb-100007
    其中，R 2、R 3、R 4、R 5、R 6、R 7、R a和n如权利要求6所定义。
  8. 根据权利要求1所述化合物或其药学上可接受的盐，其中各R b独立地为H、F、Cl、Br、I、-CN、-OH、-OCH 3、-CH 3、-CF 3或-CH 2CH 3。
  9. 根据权利要求1或6～8任一项所述化合物或其药学上可接受的盐，其中R 2和R 3连接在一起，与其相连的碳原子一起形成环丙基，所述环丙基任选被1、2或3个R b所取代。
  10. 根据权利要求1、6或7任一项所述化合物或其药学上可接受的盐，其中R 4和R 5各自独立地为H、F、Cl、Br、I、-CN、-OH、-OCH 3、-CH 3、-CF 3或-CH 2CH 3。
  11. 根据权利要求1所述化合物或其药学上可接受的盐，其中各R c独立地为H、F、Cl、Br、I、-CN、-OH、-OCH 3、-CH 3、-CF 3或-CH 2CH 3。
  12. 根据权利要求1、6、7或11任一项所述化合物或其药学上可接受的盐，其中R 3和R 4连接在一起，与其相连的碳原子一起形成环丙基，所述环丙基任选被1、2或3个R c所取代。
  13. 根据权利要求6所述化合物或其药学上可接受的盐，其中所述化合物具有式(I-3)～(I-7)任一结构式所示结构：
    Figure PCTCN2020121390-appb-100008
    其中，R a、R b、R c、T、R 6、R 7和p如权利要求6所定义，q为0、1、2或3。
  14. 根据权利要求13所述化合物或其药学上可接受的盐，其中所述化合物具有式(I-8)～(I-12)任一结构式所示结构：
    Figure PCTCN2020121390-appb-100009
    Figure PCTCN2020121390-appb-100010
    其中，R a、R b、R c、T、R 6、R 7、p和q如权利要求13所定义。
  15. 根据权利要求1、6、7、13或14任一项所述化合物或其药学上可接受的盐，其中R 2和R 3各自独立地为H、F、Cl、Br、I、-CN、-OH、-OCH 3、-CH 3、-CF 3或-CH 2CH 3。
  16. 根据权利要求1、6、7、13或14任一项所述化合物或其药学上可接受的盐，其中R 6为H、F、Cl、Br、I、-CN、-OH、-OCH 3、-CH 3或-CH 2CH 3。
  17. 根据权利要求1所述化合物或其药学上可接受的盐，其中各R d独立地为H、F、Cl、Br、I、-CN、-OH、-OCH 3、-CH 3、-CF 3、-CH 2CH 3或-CH(CH 3) 2。
  18. 根据权利要求1、6、7、13、14或17任一项所述化合物或其药学上可接受的盐，其中R 7为-NH 2、-NH(CH 3)、-NH(CH 2CH 3)、-N(CH 3) 2、-CH 3、-CH 2CH 3、-CH 2CH 2CH 3、-CH(CH 3) 2、环丙基、环戊基、氮杂环丁基、吡咯烷基、四氢呋喃基、哌啶基、吡唑基、吡啶基或苯基，其中所述-CH 3、-CH 2CH 3、-CH 2CH 2CH 3、-CH(CH 3) 2、环丙基、环戊基、氮杂环丁基、吡咯烷基、四氢呋喃基、哌啶基、吡唑基、吡啶基和苯基任选被1、2或3个R d所取代。
  19. 根据权利要求18所述化合物或其药学上可接受的盐，其中R 7为-NH 2、-NH(CH 3)、-NH(CH 2CH 3)、-N(CH 3) 2、-C(R d) 3、-CH 2CH 2R d、-CH(CH 3) 2、
    Figure PCTCN2020121390-appb-100011
    Figure PCTCN2020121390-appb-100012
  20. 根据权利要求19所述化合物或其药学上可接受的盐，其中R 7为-NH 2、-NH(CH 3)、-N(CH 3) 2、-CH 3、-CF 3、-CH 2CH 3、-CH(CH 3) 2、-CH 2CH 2OCH 3、
    Figure PCTCN2020121390-appb-100013
    Figure PCTCN2020121390-appb-100014
  21. 下式化合物或其药学上可接受的盐：
    Figure PCTCN2020121390-appb-100015
    Figure PCTCN2020121390-appb-100016
  22. 下式化合物或其药学上可接受的盐：
    Figure PCTCN2020121390-appb-100017
    Figure PCTCN2020121390-appb-100018
    Figure PCTCN2020121390-appb-100019
  23. 根据权利要求1～22任一项所述化合物或其药学上可接受的盐，其中药学上可接受的盐为盐酸盐。
  24. 一种药物组合物，其含有治疗有效量的根据权利要求1～23任一项所述化合物或其药学上可接受的盐和药学上可接受的载体。
  25. 根据权利要求1～23任一项所述化合物或其药学上可接受的盐或根据权利要求24所述的药物组合物在制备CDK2/4/6抑制剂药物中的应用。
  26. 根据权利要求1～23任一项所述化合物或其药学上可接受的盐或根据权利要求24所述的药物组合物在制备治疗实体瘤药物中的应用。
  27. 根据权利要求26所述应用，其中实体瘤为结直肠癌或乳腺癌。
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