WO2024175065A1 - 一类含有芳基取代的cdk12/13的降解剂，及其制备方法、药物组合物和应用 - Google Patents

一类含有芳基取代的cdk12/13的降解剂，及其制备方法、药物组合物和应用 Download PDF

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WO2024175065A1
WO2024175065A1 PCT/CN2024/078146 CN2024078146W WO2024175065A1 WO 2024175065 A1 WO2024175065 A1 WO 2024175065A1 CN 2024078146 W CN2024078146 W CN 2024078146W WO 2024175065 A1 WO2024175065 A1 WO 2024175065A1
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independently selected
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French (fr)
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丁克
钦奈延阿鲁
阳建章
常玉
周立成
周锴捷
王孝举
王震
黄维雪
周凤涛
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Shanghai Institute of Organic Chemistry of CAS
Livzon Pharmaceutical Group Inc
University of Michigan System
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Shanghai Institute of Organic Chemistry of CAS
Livzon Pharmaceutical Group Inc
University of Michigan System
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Priority claimed from CN202310175306.XA external-priority patent/CN118580220A/zh
Priority claimed from CN202311588550.5A external-priority patent/CN120040418A/zh
Priority to JP2025549667A priority Critical patent/JP2026507677A/ja
Application filed by Shanghai Institute of Organic Chemistry of CAS, Livzon Pharmaceutical Group Inc, University of Michigan System filed Critical Shanghai Institute of Organic Chemistry of CAS
Priority to EP24759739.6A priority patent/EP4671239A1/en
Priority to IL322876A priority patent/IL322876A/en
Priority to KR1020257031885A priority patent/KR20250159187A/ko
Priority to AU2024225659A priority patent/AU2024225659A1/en
Priority to CN202480014782.6A priority patent/CN121175298A/zh
Publication of WO2024175065A1 publication Critical patent/WO2024175065A1/zh
Priority to MX2025009857A priority patent/MX2025009857A/es
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    • C07—ORGANIC CHEMISTRY
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    • C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene or sparfloxacin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/517—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with carbocyclic ring systems, e.g. quinazoline, perimidine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
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    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/04—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/14—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing three or more hetero rings
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    • 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
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    • 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
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    • 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
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    • 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 belongs to the field of chemical medicine, and in particular relates to a degrader of cell cycle-dependent protein kinase 12/13 (CDK12/13), a pharmaceutical composition thereof and an application thereof.
  • CDK12/13 cell cycle-dependent protein kinase 12/13
  • Protein kinases are key regulators of cell function and constitute one of the largest and most diverse gene families. Protein kinases direct the activity, localization, and overall function of many proteins through phosphorylation of substrate proteins, and are involved in almost all cellular activities. Abnormalities in the expression, activation, and localization of protein kinases are closely related to the occurrence and development of a variety of diseases, and are important driving factors for a variety of diseases such as tumors and inflammation. As of December 31, 2020, the FDA has approved a total of 62 kinase inhibitor drugs for marketing. Studies have found that the non-kinase functions of proteins play an important role in diseases such as tumors.
  • PROTAC refers to a class of small molecule compounds that can specifically recognize and induce the degradation of target proteins; the molecular structure consists of three parts: target protein recognition ligand, Linker and E3 recognition ligand.
  • the advantage of PROTAC is that it not only effectively inhibits the kinase activity of the target protein, but also can quickly degrade and remove the target protein. In theory, only a catalytic amount of drug is needed to degrade almost all proteins in the cell (including membrane proteins), so it has high safety, drug resistance and broad application prospects.
  • degraders for target proteins such as ERR, ABL, BET, CDK4/6 have been successfully developed. The results show that it can not only treat the proliferation of gene-driven tumors, but also overcome inhibitor resistance.
  • CDK12/13 (Cyclin-dependent kinase 12/13) is a member of the cyclin-dependent kinase family (CDKs) of serine/threonine protein kinases, and forms a complex with Cyclin K to exert biological functions.
  • CDK12/13 contains 1490 and 1512 amino acids respectively, and shares 46% homology.
  • the kinase region consists of 300 amino acids with a homology of up to 92%.
  • CDK12/13 forms a complex with Cyclin K by phosphorylating the C-terminal domain (CTD) of RNA polymerase II (RNA Pol II).
  • CTD consists of a highly repetitive sequence of seven amino acids, YSPTSPS. In humans, CTD includes a 52-repeat unit.
  • CDK12/13 mainly phosphorylates Ser2 to regulate transcription and post-transcriptional mRNA processing. Genetic studies have shown that CDK12 promotes the transcription of full-length gene products by inhibiting the cleavage of intronic polyadenylation sites. Many homologous recombination repair genes (such as BRCA1/2, ATM, ATR, FANCD2, FANCI, etc.) contain more intronic polyadenylation sites. Therefore, the expression of these genes is more sensitive to the loss or inhibition of CDK12. Compared with other transcriptional CDKs, the N-terminus of CDK12/13 contains additional arginine/serine-rich (RS) motifs, which are commonly found in proteins involved in pre-mRNA splicing.
  • RS arginine/serine-rich
  • CDK12/13 may have non-kinase functions (such as protein-protein interactions) and play an important role in mRNA splicing and mRNA 3' end processing. Therefore, it is far from enough to use only CDK12/13 kinase inhibitors as probe tool molecules to study the biological functions of CDK12/13. Inducing CDK12/13 degradation and inhibiting its kinase and non-kinase functions simultaneously has great potential in studying the biological functions of CDK12/13 and disease treatment, and has attracted increasing interest among scientists in recent years.
  • the present invention provides a class of trans-1,4-cyclohexanediamine compounds and their use as CDK12/13 degraders, which can effectively and highly selectively degrade CDK12/13 protein kinases and inhibit the proliferation, migration and invasion of various tumor cells.
  • a compound having a structure of formula (I) is provided, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, or a prodrug molecule thereof:
  • Z is CH 2 or CO
  • V is selected from the following group:
  • X and Y are each independently selected from the following group: N, CH or CR 3 ; wherein R 3 is selected from the following group: halogen, cyano, hydroxyl, amino, C 1 -C 3 alkyl, halogenated C 1 -C 3 alkyl, C 1 -C 3 alkoxy, halogenated C 1 -C 3 alkoxy, C 3 -C 8 cycloalkyl, or 3-8 membered heterocyclyl;
  • B is selected from the following group: NH, O, CO, CH 2 ; wherein each U and W are independently selected from the following group: N or CH; each m, p, m', p' are independently: 0, 1, 2 or 3;
  • Ring A is selected from the group consisting of:
  • Q and W are each independently selected from: CH, N;
  • R' is independently selected from the group consisting of hydrogen, halogen, cyano, hydroxyl, substituted hydroxyl, amino, substituted amino, C 1 -C 5 alkyl, halogenated C 1 -C 5 alkyl, C 1 -C 5 alkoxy, halogenated C 1 -C 5 alkoxy, C 3 -C 8 cycloalkyl, and when Q and W are independently selected from CH, R' can be a substituent on Q and W;
  • D, E, F, G are each independently selected from the following group: CH, N, CR 6 ; wherein R 6 is selected from the following group: halogen, trifluoromethyl, hydroxyl, cyano, amino, methyl, methoxy, trifluoromethoxy;
  • R 1 is selected from the group consisting of H, -NHR 7 , -OR 7 , -(C(R 9 )R 8 )R 7 ;
  • R 7 is -R 10 , -CH 2 R 10 or -(CH 2 ) 2 R 10 ;
  • R 8 and R 9 are each independently selected from the group consisting of hydrogen, halogen, cyano, methyl, halomethyl, methoxy, halomethoxy, ethyl, haloethyl, ethoxy, haloethoxy, hydroxy, amino, a 3-8 membered heteroatom containing 1, 2 or 3 heteroatoms, Ring, and the heteroatom is selected from O, S or N;
  • R 8 , R 9 and the C atoms to which they are connected together form a 3-7 heterocyclic ring;
  • R10 is selected from the following group:
  • Q 1 , Q 2 , Q 3 , Q 4 , and Q 5 are each independently selected from: CH, N, or CR 11 ;
  • Each R 11 is independently selected from the group consisting of halogen, cyano, hydroxy, amino, nitro, C 1 -C 3 alkyl, halogenated C 1 -C 3 alkyl, C 1 -C 4 alkoxy, halogenated C 1 -C 4 alkoxy, C 3 -C 8 cycloalkyl;
  • R 2 is selected from the group consisting of H, C 1 -C 3 alkyl,
  • B is selected from the following group: NH, O, CO, CH 2 ; wherein each U and W are independently selected from the following group: N or CH; each m, p, m', p' are independently: 0, 1, 2 or 3;
  • R L6 is a ring and can be selected from the following structures:
  • p L1 , p L2 , p L3 , p L4 , p L5 and p L6 are independently selected from 0, 1, 2, 3, 4, 5, or 6.
  • each m, p, m', p' is independently: 0, 1, 2 or 3;
  • p L1 , p L2 , p L3 , p L4 , p L5 and p L6 are independently selected from 0, 1, 2, 3, 4, 5, or 6.
  • V is selected from the group consisting of:
  • X and Y are each independently selected from the following group: N, CH or CR 3 ;
  • R 3 is selected from the following group: halogen, cyano, hydroxyl, amino, C 1 -C 3 alkyl, halogenated C 1 -C 3 alkyl, C 1 -C 3 alkoxy, halogenated C 1 -C 3 alkoxy.
  • B is selected from the group consisting of: NH, O, CO;
  • each U and W are independently selected from the following group: N or CH; and each m, p, m', p' are independently 0, 1, 2 or 3.
  • Ring A is selected from:
  • W is selected from the following group: CH, N.
  • R' is optionally selected from: hydrogen, halogen, cyano, hydroxyl, amino, C 1 -C 5 alkyl, halogenated C 1 -C 5 alkyl, C 1 -C 5 alkoxy, halogenated C 1 -C 5 alkoxy, C 3 -C 8 cycloalkyl
  • D, E, F, and G are CH and CR 6 ; wherein R 6 is selected from the following group: halogen.
  • the R 1 is -NHR 7 ;
  • R 7 is -CH 2 R 10 ; R 10 is as defined above.
  • the R 10 is selected from the following group: cyano, C 1 -C 5 alkyl, halogenated C 1 -C 4 alkyl, C 1 -C 4 alkoxy, C 3 -C 10 cycloalkyl, 4-7 membered heterocyclic group, C 6 -C 10 aryl; wherein the aryl is substituted by one or more CR 11 ; R 11 is defined as above.
  • B is selected from the following group: NH, O, CO;
  • the Linker is selected from the following group:
  • linker is selected from the group consisting of chemical bonds, wherein each n is independently 0, 1, 2, 3, 4, 5 or 6; each r and m is independently 0, 1 or 2; each U and W is independently selected from the group consisting of N or CH.
  • the compound has a structure shown in formula (II):
  • B is selected from: wherein each U and W are independently selected from the following group: N or CH; each m, p, m', p' are independently 0, 1, 2 or 3;
  • R' is optionally selected from: hydrogen, halogen, cyano, hydroxyl, amino;
  • R 11 and R 6 are selected from: hydrogen, halogen;
  • X, Y are independently selected from: CH, N or CR 3 ; wherein, R 3 is selected from: halogen, cyano, hydroxyl, amino;
  • Z is selected from: CH 2 or CO
  • the compound is selected from the group consisting of:
  • Z is selected from the group consisting of: CH 2 or CO;
  • V is selected from the following group:
  • X and Y are selected from the following groups: N, CH or CR 3 ; wherein R 3 is selected from the following groups: halogen, cyano, hydroxyl, amino, C 1 -C 3 alkyl, halogenated C 1 -C 3 alkyl, C 1 -C 3 alkoxy, halogenated C 1 -C 3 alkoxy, C 3 -C 8 cycloalkyl, or 3-8 membered heterocyclyl;
  • B is selected from the following group: NH, O, CO, CH 2 ; wherein each U and W are independently selected from the following group: N or CH; each m and p are independently: 0, 1 or 2;
  • Ring A is selected from the group consisting of:
  • M and T are each independently selected from the following group: N, CR 4 ;
  • I, J, K are each independently selected from the group consisting of N, O, S, CR 4 and NR 4 ;
  • R 4 is selected from the group consisting of hydrogen, halogen, cyano, hydroxy, amino, C 1 -C 5 alkyl, halogenated C 1 -C 5 alkyl, C 1 -C 5 alkoxy, halogenated C 1 -C 5 alkoxy, C 3 -C 8 cycloalkyl, -CH 2 R 5 , -(CH 2 ) 2 R 5 , -(CH 2 ) 3 R 5 or 3-8 membered heterocyclyl;
  • R 5 is selected from the group consisting of cyano, hydroxy, amino, C 3 -C 8 cycloalkyl, or 3-8 membered heterocyclyl;
  • D, E, F, G are each independently selected from the following group: CH, N, CR 6 ; wherein R 6 is selected from the following group: halogen, trifluoromethyl, hydroxyl, cyano, amino, methyl, methoxy, trifluoromethoxy;
  • R 1 is selected from the group consisting of H, -NHR 7 , -OR 7 , -(C(R 9 )R 8 )R 7 ;
  • R 7 is -R 10 , -CH 2 R 10 or -(CH 2 ) 2 R 10 ;
  • R 8 and R 9 are each independently selected from the following group: hydrogen, halogen, cyano, methyl, halomethyl, methoxy, halomethoxy, ethyl, haloethyl, ethoxy, haloethoxy, hydroxyl, amino, a 3-8 membered heterocycle containing 1, 2 or 3 heteroatoms, wherein the heteroatoms are selected from O, S or N;
  • R 8 , R 9 and the C atoms to which they are connected together form a 3-7 heterocyclic ring;
  • R10 is selected from the following group:
  • Q 1 , Q 2 , Q 3 , Q 4 , and Q 5 are each independently selected from: CH, N, or CR 11 ;
  • Each R 11 is independently selected from the group consisting of halogen, cyano, hydroxy, amino, nitro, C 1 -C 3 alkyl, halogenated C 1 -C 3 alkyl, C 1 -C 4 alkoxy, halogenated C 1 -C 4 alkoxy, C 3 -C 8 cycloalkyl;
  • R 2 is selected from the group consisting of H, C 1 -C 3 alkyl,
  • substitution refers to substitution by one or more groups selected from the group consisting of hydrogen, deuterium, C 1 -C 18 alkyl, deuterated C 1 -C 18 alkyl, halogenated C 1 -C 18 alkyl, halogenated C 1 -C 18 alkylhydroxy, C 3 -C 20 cycloalkyl, C 1 -C 18 alkoxy, deuterium substituted C 1 -C 18 alkoxy, halogenated C 1 -C 18 alkoxy, C 6 -C 14 aryl, 5-14 membered heteroaryl, 4-20 membered heterocyclyl, halogen, oxo, nitro, hydroxy, cyano, ester, amine, amide, sulfonamide or urea;
  • Each of p L1 -p L6 is independently selected from 0, 1, 2, 3, 4, 5, or 6.
  • V is selected from the group consisting of:
  • X and Y are each independently selected from the following group: N, CH or CR 3 ;
  • R 3 is selected from the following group: halogen, cyano, hydroxyl, amino, C 1 -C 3 alkyl, halogenated C 1 -C 3 alkyl, C 1 -C 3 alkoxy, halogenated C 1 -C 3 alkoxy.
  • B is selected from the group consisting of: NH, O, CO;
  • each U and W are independently selected from the following group: N or CH; and each m and p are independently 0 or 1.
  • Ring A is selected from the group consisting of:
  • T is selected from the following group: CH, N.
  • R 4 is selected from the group consisting of hydrogen, halogen, cyano, hydroxyl, amino, C 1 -C 5 alkyl, halogenated C 1 -C 5 alkyl, C 1 -C 5 alkoxy, halogenated C 1 -C 5 alkoxy, C 3 -C 8 cycloalkyl, -CH 2 R 5 , -(CH 2 ) 2 R 5 , -(CH 2 ) 3 R 5 , or 3-8 membered heterocyclyl.
  • D, E, F, and G are CH.
  • the R 1 is -NHR 7 ;
  • R 7 is -CH 2 R 10 ; R 10 is as defined above.
  • the R 10 is selected from the following group: cyano, C 1 -C 5 alkyl, halogenated C 1 -C 4 alkyl, C 1 -C 4 alkoxy, C 3 -C 10 cycloalkyl, 4-7 membered heterocyclic group, C 6 -C 10 aryl; wherein the aryl is substituted by one or more CR 11 ; R 11 is defined as above.
  • the Linker is selected from the following group: wherein each n is independently 0, 1, 2, 3, 4, 5 or 6; each r and m is independently 0, 1 or 2; each U and W is independently selected from the group consisting of N or CH.
  • Linker is selected from the following group:
  • each n is independently 0, 1, 2, 3, 4, 5 or 6; each r and m is independently 0, 1 or 2; each U and W is independently selected from the group consisting of N or CH.
  • the compound has a structure shown in formula (II) or formula (III):
  • U and W are independently selected from: CH or N;
  • X, Y are independently selected from: CH, N or CR 3 ;
  • Z is selected from: CH 2 or CO
  • n, m are independently selected from: 0 or 1.
  • the compound is selected from the group consisting of:
  • a pharmaceutical composition characterized in that it comprises
  • Optional (2) a pharmaceutically acceptable carrier.
  • a compound as described in the first aspect or the second aspect of the present invention or a pharmaceutically acceptable salt or a stereoisomer or a prodrug molecule thereof, or an application of the pharmaceutical composition as described in the third aspect of the present invention, characterized in that it is used for the preparation of a CDK12/13 protein kinase degrader.
  • a compound as described in the first aspect or the second aspect of the present invention or a pharmaceutically acceptable salt or a stereoisomer or a prodrug molecule thereof, or a use of the pharmaceutical composition as described in the third aspect of the present invention, characterized in that it is used to prepare a drug for preventing and/or treating diseases mediated by CDK12/13 serine/threonine protein kinase.
  • the disease mediated by CDK12/13 serine/threonine protein kinase is selected from the following group: prostate cancer, breast cancer, uterine cancer, ovarian cancer, non-small cell lung cancer, small cell lung cancer, Ewing sarcoma, lung adenocarcinoma, lung squamous cell carcinoma, pancreatic cancer, liver cancer, skin cancer, epithelial cell carcinoma, gastrointestinal stromal tumor, leukemia, histiocytic lymphoma, nasopharyngeal carcinoma, head and neck tumors, colon cancer, rectal cancer, glioma.
  • Figure 1 shows (A) the WB results of CDK12 and CDK13 protein degradation after some compounds were treated with MDA-MB-231 cells at a concentration of 0.1 ⁇ M for 15 hours; (B) the WB results of CDK12 and CDK13 protein degradation after some compounds were treated with MDA-MB-231 cells at a concentration of 0.3 ⁇ M for 15 hours; (C) using the control group DMSO as a reference standard, the CDK12/13 level was quantified by the gray value of the band in the WB result image ( Figure A); (D) using the control group DMSO as a reference standard, the CDK12/13 level was quantified by the gray value of the band in the WB result image ( Figure B).
  • Figure 2 shows (A) time-dependent degradation of CDK12 and CDK13 proteins in MDA-MB-231 cells by representative compounds; (B) WB determination results of intracellular CDK12/13 protein levels after MDA-MB-231 cells were treated with representative compounds at different concentrations for 15 hours.
  • FIG3 is a result of measuring the inhibitory activity of the compounds on triple-negative breast cancer cells MDA-MB-231.
  • Figure 4 shows (A) WB results of CDK12 and CDK13 protein degradation after some compounds were treated with 500 nM concentration for 6 hours in 22RV1 cells.
  • B Compound YJZ9069 dose-dependently degraded CDK12 and CDK13 proteins in 22RV1 cells and dose-dependently inhibited RNA polymerase II (RNA Pol II) C-terminal 2-serine
  • C WB results of CDK12 and CDK13 protein degradation after treating LnCap cells with some compounds at 500 nM for 6 hours.
  • D Using the control group DMSO as the reference standard, the protein level was quantified by the gray value of the bands in the WB result images ( Figures A, C).
  • Figure 5 shows the WB measurement results of intracellular CDK12 and CDK13 protein levels after 22RV1 cells were treated with representative compounds YJZ9069, YJZ1201, YJZ1202, YJZ1203, YJZ1204, YJZ1097, YJZ1205 and YJZ1206 at different concentrations for 6 hours.
  • FIG6 is a result of measuring the proliferation inhibition activity of the compounds on the prostate cancer cell line VCap.
  • the "plurality” mentioned in the present invention refers to two or more than two.
  • “And/or” describes the association relationship of the associated objects, indicating that there may be three relationships.
  • a and/or B can represent: A exists alone, A and B exist at the same time, and B exists alone.
  • the character “three” generally indicates that the associated objects are in an "or” relationship.
  • any variable e.g., R 10 , R 11 , etc.
  • its definition at each occurrence is independent of its definition at every other occurrence.
  • combinations of substituents and variables are permitted so long as such combinations render the compound stable.
  • Lines drawn from substituents into the ring system indicate that the indicated bond may be attached to any substitutable ring atom. If the ring system is polycyclic, it means that such bonds are attached only to any suitable carbon atom of the adjacent ring.
  • substituents and substitution patterns for the compounds of the present invention can select substituents and substitution patterns for the compounds of the present invention to provide compounds that are chemically stable and that can be readily synthesized from readily available starting materials by techniques in the art and the methods set forth below. If a substituent is itself substituted with more than one group, it is to be understood that these groups may be on the same carbon atom or on different carbon atoms so long as the structure is stable.
  • alkyl is intended to include both branched and straight chain saturated aliphatic hydrocarbon groups having a specified number of carbon atoms.
  • C 1 -C 8 in “C 1 -C 8 alkyl” includes groups having 1, 2, 3, 4, 5, 6, 7 or 8 carbon atoms in a straight or branched arrangement.
  • cycloalkyl refers to a monocyclic saturated aliphatic hydrocarbon group having a specified number of carbon atoms.
  • cycloalkyl includes cyclopropyl, methyl-cyclopropyl, 2,2-dimethyl-cyclobutyl, 2-ethyl-cyclopentyl, cyclohexyl, etc.
  • alkenyl includes straight or branched alkenyl groups.
  • C2 - C6 alkenyl refers to straight or branched alkenyl groups having 2 to 6 carbon atoms, such as vinyl, allyl, 1-propenyl, isopropenyl, 1-butenyl, 2-butenyl, or the like.
  • alkynyl includes straight or branched chain alkynyl groups.
  • C2 - C6 alkynyl refers to a 2-6 carbon atom.
  • cycloalkyl refers to a cyclic saturated aliphatic hydrocarbon group having a specific number of carbon atoms.
  • C3 - C10 alkenyl refers to a cyclic saturated aliphatic hydrocarbon group having 3-10 carbon atoms. It can be a single ring, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or similar groups. It can also be a bicyclic form, such as a bridged ring or a spiro ring form.
  • heterocyclyl or “heterocycloalkyl” refers to a saturated or partially saturated cyclic group having a specific number of ring atoms (e.g., 3-10 ring atoms), wherein 1-3 atoms are heteroatoms selected from N, S and O. It can be a monocyclic, bicyclic or polycyclic form, such as a fused ring, a bridged ring or a spirocyclic form.
  • alkylamino refers to an amine group substituted by an alkyl group.
  • C 1 -C 6 alkylamino refers to an amine group substituted by a C 1 -C 6 alkyl group, which may be monosubstituted or disubstituted; for example, methylamino, ethylamino, propylamino, isopropylamino, butylamino, isobutylamino, tert-butylamino, dimethylamino, diethylamino, dipropylamino, diisopropylamino, dibutylamino, diisobutylamino, di-tert-butylamino, etc.
  • alkoxy refers to a group having an alkyl-oxy structure.
  • C 1 -C 6 alkoxy refers to a straight or branched alkoxy group having 1 to 6 carbon atoms, including methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, tert-butoxy, etc.
  • haloalkyl represents an alkyl group wherein one or more hydrogen atoms are replaced by halogen, wherein alkyl is as defined above.
  • haloalkoxy represents an alkoxy group having one or more hydrogen atoms replaced by a halogen, wherein alkoxy is as defined above.
  • halogen as used herein is meant to include F, Cl, Br and I. More preferably, the halogen or halogen atom is selected from F, Cl and Br. "Halogenated” means substituted with an atom selected from F, Cl, Br, and I.
  • the groups described in the present invention may be substituted by substituents selected from the following groups: halogen, nitrile, nitro, hydroxyl, amino, C 1 -C 6 alkyl-amino, C 1 -C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C 1 -C 6 alkoxy, halogenated C 1 -C 6 alkyl, halogenated C 2 -C 6 alkenyl, halogenated C 2 -C 6 alkynyl, halogenated C 1 -C 6 alkoxy, allyl, benzyl, C 6 -C 12 aryl, C 1 -C 6 alkoxy- C 1 -C 6 alkyl, C 1 -C 6 alkoxy - carbonyl, phenoxycarbonyl, C 2 -C 6 alkynyl-carbonyl, C 2 -
  • the present invention includes free forms of compounds of formula (I) (herein, formula (I) includes formula (II)), and also includes pharmaceutically acceptable salts thereof, stereoisomers thereof, and prodrug molecules thereof.
  • the term "free form” refers to a compound in a non-salt form.
  • the pharmaceutically acceptable salts included include not only the exemplary salts of the specific compounds described herein, but also typical pharmaceutically acceptable salts of the free forms of all compounds of formula (I) or formula (II).
  • the free form of a specific salt of the compound can be separated using techniques known in the art.
  • the free form can be regenerated by treating the salt with an appropriate dilute aqueous alkali solution, such as a dilute aqueous solution of NaOH, a dilute aqueous solution of potassium carbonate, a dilute aqueous ammonia, and a dilute aqueous solution of sodium bicarbonate.
  • an appropriate dilute aqueous alkali solution such as a dilute aqueous solution of NaOH, a dilute aqueous solution of potassium carbonate, a dilute aqueous ammonia, and a dilute aqueous solution of sodium bicarbonate.
  • the free form is somewhat different from its respective salt form in certain physical properties, such as solubility in polar solvents, but for the purpose of the invention, such acid salts and base salts are equivalent to their respective free forms in other pharmaceutical aspects.
  • salts of the present invention can be synthesized from compounds of the present invention containing a basic or acidic moiety by conventional chemical methods.
  • salts of basic compounds are prepared by ion exchange chromatography or by reacting a free base with a stoichiometric amount or excess of an inorganic or organic acid in the desired salt form in an appropriate solvent or combination of solvents.
  • salts of acidic compounds are formed by reacting with an appropriate inorganic or organic base.
  • the pharmaceutically acceptable salts of the compounds of the present invention include those prepared by reacting a basic compound of the present invention with an inorganic or organic
  • Conventional non-toxic salts of the compounds of the present invention formed by organic acid reaction include salts obtained from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, nitric acid, etc.
  • organic acids such as acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, pamoic acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, p-aminobenzenesulfonic acid, 2-acetoxy-benzoic acid, fumaric acid, benzenesulfonic acid, toluenesul
  • inorganic acids such as hydrochloric acid, hydro
  • suitable "pharmaceutically acceptable salts” refer to salts prepared from pharmaceutically acceptable non-toxic bases including inorganic bases and organic bases.
  • Salts derived from inorganic bases include aluminum salts, ammonium salts, calcium salts, copper salts, iron salts, ferrous salts, lithium salts, magnesium salts, manganic salts, manganous salts, potassium salts, sodium salts, zinc salts, etc. Ammonium salts, calcium salts, magnesium salts, potassium salts and sodium salts are particularly preferred.
  • Salts derived from pharmaceutically acceptable organic non-toxic bases including salts of primary, secondary and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins such as arginine, betaine, caffeine, choline, N,N'-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, aminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucosamine, glucosamine, histidine, hydroxocobalamin, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purine, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, and the like.
  • basic ion exchange resins such
  • the structural formulas described in the present invention are intended to include all isomeric forms (such as enantiomers, diastereomers and geometric isomers (or conformational isomers)): for example, R, S configurations containing asymmetric centers, (Z), (E) isomers of double bonds, etc. Therefore, single stereochemical isomers of the compounds of the present invention or mixtures of their enantiomers, diastereomers or geometric isomers (or conformational isomers) are all within the scope of the present invention.
  • tautomer means that structural isomers with different energies can interconvert across a low energy barrier.
  • proton tautomers i.e., prototropic
  • Valence tautomers include interconversions via reorganization of some bonding electrons.
  • solvate refers to a complex in which the compound of the present invention is coordinated with solvent molecules to form a specific ratio.
  • hydrate refers to a complex formed by coordination of a compound of the present invention with water.
  • the compound of the present invention can also be in the form of a prodrug.
  • prodrug refers to a compound that produces an active compound when metabolized (e.g., in vivo or in vitro).
  • the prodrug can be inactive, or have a lower activity than a free drug, but can provide a favorable treatment, administration or metabolic characteristics.
  • the exemplary prodrug moiety of the present invention can be connected to the free drug by the hydroxyl, amino, phosphate or thiophosphate backbone of a nucleotide, and can include esters, carbamates, carbonyls, thioesters, amides, isocyanates, ureas, thioureas or other physiologically acceptable metabolically unstable parts.
  • the prodrug is activated by enzymatic hydrolysis.
  • the present disclosure also includes isotopically labeled compounds, which are identical to compounds of formula (I) (including compounds of formula (II)), but in which one or more atoms are replaced by atoms having an atomic mass or mass number different from the atomic mass or mass number commonly found in nature.
  • isotopes suitable for inclusion in the compounds of the present invention are hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine and chlorine, such as but not limited to 2 H, 3 H, 13 C, 14 C, 15 N, 18 O, 31 P, 35 S, 18 F and 36 Cl.
  • Substitution with heavier isotopes may provide certain therapeutic advantages due to greater metabolic stability, such as greater in vivo half-life or lower dosage requirements, and may therefore be preferred in certain circumstances.
  • the compounds may be combined with positron emission tomography (PET) for medical imaging and positron emission tomography (PET) studies.
  • PET positron emission tomography
  • Suitable positron emitting isotopes that can be incorporated into compounds of formula (I) or (II) are 11 C, 13 N, 15 O and 18 F.
  • Isotopically labeled compounds of formula (I) or (II) can generally be prepared by conventional techniques known to those skilled in the art, or by methods analogous to those described herein, using an appropriate isotopically labeled reagent instead of a non-isotopically labeled reagent.
  • the compounds disclosed herein can exist in solvated and unsolvated forms with pharmaceutically acceptable solvents such as water, ethanol, etc., and the present invention is intended to cover solvated and unsolvated forms.
  • the compound is amorphous.
  • the compound is a single polymorph.
  • the compound is a mixture of polymorphs.
  • the compound is in crystalline form.
  • the compounds of the present invention are CDK12/13 protein degraders, the compounds and pharmaceutically acceptable salts thereof as well as other compound forms disclosed herein can be included in pharmaceutical compositions useful for treating, preventing and alleviating diseases associated with CDK12/13 activity.
  • the pharmaceutical composition of the present invention comprises an effective amount, such as a safe and effective amount of a compound of the present invention or a pharmacologically acceptable salt thereof and a pharmacologically acceptable excipient or carrier.
  • Effective amount refers to an amount sufficient to induce a desired biological response (e.g., to treat a condition).
  • Safe and effective amount means: the amount of the compound is sufficient to significantly improve the condition without causing serious side effects.
  • the pharmaceutical composition comprises 1 to 3000 mg (active dose range of 3 to 30 mg/kg) of the disclosed compound/dose, and more preferably comprises 10 to 2000 mg of the disclosed compound/dose.
  • "one dose” is a capsule or tablet.
  • “Pharmaceutically acceptable carrier” refers to: one or more compatible solid or liquid fillers or gel substances suitable for human use, which must have sufficient purity and sufficiently low toxicity. "Compatibility” herein means that the components of the composition can be mixed with the compounds of the present disclosure or with each other without significantly reducing the efficacy of the compounds.
  • Examples of pharmaceutically acceptable carrier parts include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose and cellulose acetate), gelatin, talc, solid lubricants (such as stearic acid and magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil), polyols (propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (Tween etc.), wetting agents (sodium lauryl sulfate, etc.), coloring agents, flavoring agents, stabilizers, antioxidants, preservatives and pyrogen-free water.
  • cellulose and its derivatives such as sodium carboxymethyl cellulose, sodium ethyl cellulose and cellulose acetate
  • gelatin such as talc
  • solid lubricants such as stearic acid and magnesium stearate
  • calcium sulfate such as soybean oil
  • compositions of the present invention include (but are not limited to): oral, intratumoral, rectal, parenteral (intravenous, intramuscular or subcutaneous) and topical administration.
  • Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules.
  • the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients: (a) fillers or bulking agents, such as starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) binders, such as hydroxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and gum arabic; (c) humectants, such as glycerol; (d) disintegrants, such as agar, calcium carbonate, potato starch or tapioca starch, alginic acid, some complex silicates and sodium carbonate; (e) retarding solvents, such as paraffin; (f) absorption promoters, such as quaternary ammonium compounds; (g) wetting agents, such as cetyl alcohol and gly
  • Solid dosage forms such as tablets, dragees, capsules, pills, and granules, may be prepared with coatings and shells, such as enteric coatings and other materials well known in the art. They may contain opacifying agents, and the active compound or compounds in such compositions that release in a portion of the digestive tract in a delayed manner. Examples of useful embedding components The active compound can also be in microencapsulated form with one or more of the above-mentioned excipients, if desired.
  • Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or elixirs.
  • the liquid dosage form may contain an inert diluent commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butylene glycol, dimethylformamide and oils, in particular cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil, or mixtures thereof.
  • an inert diluent commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butylene glycol, dimethylformamide and oils, in particular cottonseed oil
  • composition may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.
  • adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.
  • Suspensions in addition to the active compounds, may contain suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methanol and agar, or mixtures thereof.
  • suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methanol and agar, or mixtures thereof.
  • compositions for parenteral injection may include physiologically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions.
  • Suitable aqueous and nonaqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.
  • Topical dosage forms for the compounds of this invention include ointments, powders, patches, sprays and inhalants.
  • the active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers or propellants, if required.
  • the compounds of the present disclosure may be administered alone or in combination with other pharmaceutically acceptable compounds.
  • the safe and effective amount of the compound of the present invention is suitable for mammals (such as humans) in need of treatment, wherein the dosage is a dosage that is considered pharmaceutically effective when administered, and the daily dosage for a person weighing 60 kg is generally 1-2000 mg, preferably 6-600 mg.
  • the specific dosage should also be determined by comprehensively considering factors such as the route of administration and the health status of the patient, which are all within the capabilities of skilled physicians.
  • the compound of the present invention is a CDK12/13 protein degrader, so the compound or a composition comprising the compound can be used to treat, prevent and alleviate diseases associated with CDK12/13 activity or abnormal expression.
  • a compound of the present invention in the preparation of a medicament for preventing and/or treating a disease mediated by CDK12/13 serine/threonine protein kinase.
  • a compound of the present invention for preventing and/or treating a disease mediated by CDK12/13 serine/threonine protein kinase.
  • disclosed herein is a method for treating a disease mediated by CDK12/13 serine/threonine protein kinase in a subject in need thereof, comprising administering to the subject an effective amount of a compound of the present invention.
  • diseases mediated by CDK12/13 serine/threonine protein kinases include: prostate cancer, breast cancer, uterine cancer, ovarian cancer, non-small cell lung cancer, small cell lung cancer, Ewing sarcoma, lung adenocarcinoma, squamous cell lung cancer, pancreatic cancer, liver cancer, skin cancer, epithelial cell carcinoma, gastrointestinal stromal tumor, leukemia, histiocytic lymphoma, nasopharyngeal carcinoma, head and neck tumors, colon cancer, rectal cancer, glioma.
  • the disclosed compounds and compositions can be used in combination with other known therapies.
  • “combination" administration refers to the delivery of two (or more) different treatments to a subject while the subject is suffering from a condition, for example, two or more treatments are performed after the subject is diagnosed with a condition and before the condition is cured or eliminated, or the treatment is stopped for other reasons.
  • delivery of one treatment is still ongoing when delivery of the second treatment begins, so there is overlap in administration. This is sometimes referred to herein as “simultaneous" or “parallel delivery.”
  • delivery of one treatment is started before delivery of another treatment.
  • the combination of the first and second treatments may be administered in combination with the first treatment.
  • the second treatment may be administered in combination with the first treatment.
  • the second treatment may be administered in combination with the first treatment.
  • the combination of the first and second treatments ...
  • Compounds or compositions disclosed herein and at least one additional therapeutic agent can be administered simultaneously, in the same or separate compositions, or sequentially.
  • a compound as described herein can be administered first, and then additional agents can be administered, or the order of administration can be reversed.
  • the compounds described herein are administered in combination with other treatment modalities, including surgery, radiation, transplantation (e.g., stem cell transplantation, bone marrow transplantation), chemotherapy, immunotherapy, cryotherapy, and/or thermotherapy.
  • other treatment modalities including surgery, radiation, transplantation (e.g., stem cell transplantation, bone marrow transplantation), chemotherapy, immunotherapy, cryotherapy, and/or thermotherapy.
  • transplantation e.g., stem cell transplantation, bone marrow transplantation
  • chemotherapy e.g., immunotherapy, cryotherapy, and/or thermotherapy.
  • the compounds described herein are administered with at least one additional therapeutic agent, such as a chemotherapeutic agent.
  • the compounds described herein are administered in combination with one or more additional chemotherapeutic agents.
  • the chemotherapeutic agent can be a chemotherapeutic agent identified in the "A to Z List of Cancer Drugs" published by the National Cancer Institute.
  • the cell cycle-dependent protein kinase 12/13 (CDK12/13) degrader provided by the present invention can effectively degrade CDK12 and CDK13 protein kinases, and can be used to prepare drugs for preventing or treating diseases mediated by CDK12 and/or CDK13 protein kinases, such as prostate cancer, breast cancer, uterine cancer, ovarian cancer, non-small cell lung cancer, small cell lung cancer, Ewing sarcoma, lung adenocarcinoma, lung squamous cell carcinoma, pancreatic cancer, liver cancer, skin cancer, epithelial cell carcinoma, gastrointestinal stromal tumors, leukemia, histiocytic lymphoma, nasopharyngeal carcinoma, head and neck tumors, colon cancer, rectal cancer, glioma, etc.
  • diseases mediated by CDK12 and/or CDK13 protein kinases such as prostate cancer, breast cancer, uterine cancer, ovarian cancer, non-small cell lung cancer, small cell
  • the cell cycle-dependent protein kinase 12/13 (CDK12/13) degrading agent provided by the present invention has strong degradation activity.
  • the cell cycle-dependent protein kinase 12/13 (CDK12/13) degrading agent provided by the present invention has high protein kinase degradation selectivity.
  • trans-cyclohexane-1,4-diamine 2 (1.2 g, 10.5 mmol), potassium phosphate (1.3 g, 6 mmol), CuI (57 mg, 0.3 mmol), and D-proline (35 mg, 0.3 mmol) were added to a system of tert-butyl 4-(4-bromophenyl)piperazine-1-carboxylate (1.02 g, 3 mmol) in anhydrous DMSO (15 mL) in sequence. After completion, the gas was replaced with argon three times, and the temperature was raised to 100°C.
  • the reaction was moved to room temperature, filtered with diatomaceous earth, washed three times with DCM/MeOH (10:1) system, and the filtrate was concentrated under reduced pressure, wet-loaded, and purified on a silica gel normal phase chromatography column to obtain the target compound 3-86 (off-white solid, 480 mg, yield 43%).
  • benzyl isocyanate 4 (1.2 g, 9 mmol) and DIPEA (1.6 mL) were added to a DMF (3 mL) system of 4-39 (1.438 g, 3 mmol) in sequence. After completion, the temperature was raised to 95°C. After 6 h, the reaction was completed as monitored by TLC. The mixture was moved to room temperature, concentrated under reduced pressure, and purified by normal phase chromatography on silica gel to obtain the target intermediate 4-44 (white solid, 1.3 g, yield 72%).
  • HATU 137 mg, 0.36 mmol
  • DIPEA 0.1 mL, 0.6 mmol
  • 1-N-Boc-3-azetidinecarboxylic acid 67 mg, 0.33 mmol
  • the synthesis method is the same as step f of Example 1.
  • the target compound 4-64 (yellow solid 3.9 g, yield 72%) was obtained by column chromatography.
  • the intermediate 4-68 (3.7 g, 7.3 mmol) was dissolved in 5 mL DCM, and 2.5 mL trifluoroacetic acid (TFA) was added. After heating to 55°C and reflux for 6 h, the solvent was evaporated under reduced pressure and the target compound 4-69 (yellow solid 2.5 g, yield 70%) was obtained by column chromatography.
  • TFA trifluoroacetic acid
  • the synthesis method is the same as step f of Example 1.
  • Step 1 Preparation of tert-butyl 4-(4-(((1r,4r)-4-aminocyclohexyl)amino)phenyl)piperazine-1-carboxylate (Compound 3)
  • the reaction mixture was then heated and stirred at 100 ° C for 10 hours, filtered using diatomaceous earth, and the filter residue was washed with ethyl acetate 2-3 times. After evaporating the filtrate under reduced pressure and purifying it through a silica gel column, 12 g of the target compound was obtained, which was off-white. Solid (44% yield).
  • Step 2 Preparation of tert-butyl 4-(4-(((1r,4r)-4-(quinazolin-2-ylamino)cyclohexyl)amino)phenyl)piperazine-1-carboxylate (Compound 5)
  • Step 3 Preparation of tert-butyl 4-(4-(3-benzyl-1-((1r, 4r)-4-(quinazolin-2-ylamino)cyclohexyl)ureido)phenyl)piperazine-1-carboxylate (Compound 7)
  • Step 4 Preparation of 3-benzyl-1-(4-(piperazin-1-yl)phenyl)-1-(1r,4r)-4-(quinazolin-2-ylamino)cyclohexyl)urea (Compound 8)
  • Step 5 Preparation of 3-benzyl-1-(4-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)oxy)acetyl)piperazin-1-yl)phenyl)-1-((1r,4r)-4-(quinazolin-2-ylamino)cyclohexyl) (Compound YJZ9069)
  • Example 24 3-Benzyl-1-(4-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-5-yl)oxy)acetyl)piperazin-1-yl)phenyl)-1-((1r,4r)-4-(quinazolin-2-ylamino)cyclohexyl)urea (Compound YJZ1090)
  • the synthesis method is similar to that shown in Example 23.
  • Example 25 3-Benzyl-1-(4-(2-((2-(2,6-dioxopiperidin-3-yl)-3-oxoisoindol-5-yl)oxy)acetyl)piperazin-1-yl)phenyl)-1-((1r,4r)-4-(quinazolin-2-ylamino)cyclohexyl)urea (Compound YJZ1094)
  • the synthesis method is similar to that shown in Example 23.
  • the synthesis method is similar to that shown in Example 23.
  • Example 27 3-Benzyl-1-(4-(4-((2-(2,6-dioxopiperidin-3-yl)-3-oxoisoindol-5-yl)glyceryl)piperazin-1-yl)phenyl)-1-((1r,4r)-4-(quinazolin-2-ylamino)cyclohexyl)urea (Compound YJZ1095)
  • the synthesis method is similar to that shown in Example 23.
  • Example 28 3-Benzyl-1-(4-(4-(1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindole-5-yl)azetidine-3-carbonyl)piperazin-1-yl)phenyl)-1-((1r,4r)-4-(quinazolin-2-ylamino)cyclohexyl)urea (Compound YJZ1093)
  • the synthesis method is similar to that shown in Example 23.
  • the synthesis method is similar to that shown in Example 23.
  • the synthesis method is similar to that shown in Example 23.
  • the synthesis method is similar to that shown in Example 23.
  • the synthesis method is similar to that shown in Example 23.
  • Example 33 3-Benzyl-1-(4-(4-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)oxy)acetyl)piperazin-1-yl)phenyl)-1-((1r,4r)-4-((1-propyl-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)cyclohexyl)urea (Compound YJZ9108)
  • the synthesis method is similar to that shown in Example 23.
  • Example 34 N-(4-(4-(3-(3-benzyl-1-((1r,4r)-4-(quinazolin-2-ylamino)cyclohexyl)ureido)phenyl)piperazin-1-yl)butyl)-2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)oxy)acetamide (Compound YJZ9049)
  • the synthesis method is similar to that shown in Example 23.
  • Example 35 N-(5-(4-(3-(3-benzyl-1-((1r,4r)-4-(quinazolin-2-ylamino)cyclohexyl)ureido)phenyl)piperazin-1-yl)pentyl)-2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)oxy)acetamide (Compound YJZ9048)
  • the synthesis method is similar to that shown in Example 23.
  • Example 36 N-(6-(4-(3-(3-benzyl-1-((1r,4r)-4-(quinazolin-2-ylamino)cyclohexyl)ureido)phenyl)piperazin-1-yl)hexyl)-2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)oxy)acetamide (Compound YJZ9043)
  • the synthesis method is similar to that shown in Example 23.
  • Example 37 N-(7-(4-(3-(3-benzyl-1-((1r,4r)-4-(quinazolin-2-ylamino)cyclohexyl)ureido)phenyl)piperazin-1-yl)heptyl)-2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)oxy)acetamide (Compound YJZ9047)
  • the synthesis method is similar to that shown in Example 23.
  • Example 38 3-Benzyl-1-(3-(4-(4-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)amino)butanoyl)piperazin-1-yl)phenyl)-1-((1r,4r)-4-(quinazolin-2-ylamino)cyclohexyl)urea (Compound YJZ9058)
  • the synthesis method is similar to that shown in Example 23.
  • Example 39 3-Benzyl-1-(3-(4-(6-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)amino)hexyl)piperazin-1-yl)phenyl)-1-((1r,4r)-4-(quinazolin-2-ylamino)cyclohexyl)urea (Compound YJZ9052)
  • the synthesis method is similar to that shown in Example 23.
  • Example 40 3-Benzyl-1-(3-(4-(8-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)amino)octyl)piperazin-1-yl)phenyl)-1-((1r,4r)-4-(quinazolin-2-ylamino)cyclohexyl)urea (Compound YJZ9053)
  • the synthesis method is similar to that shown in Example 23.
  • Example 41 3-Benzyl-1-(3-(4-(10-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)amino)decanoyl)piperazin-1-yl)phenyl)-1-((1r,4r)-4-(quinazolin-2-ylamino)cyclohexyl)urea (Compound YJZ9055)
  • the synthesis method is similar to that shown in Example 23.
  • Example 42 N-(3-(3-benzyl-1-((1r,4r)-4-(quinazolin-2-ylamino)cyclohexyl)ureido)phenyl)-4-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)amino)butanamide (Compound YJZ9062)
  • the synthesis method is similar to that shown in Example 23.
  • the synthesis method is similar to that shown in Example 23.
  • Step 1 Preparation of tert-butyl 3-(4-(4-(3-benzyl-1-((1r,4r)-4-(quinazolin-2-ylamino)cyclohexyl)ureido)phenyl)piperazin-1-yl)methyl)azetidin-1-carboxylate (Compound 11)
  • Step 2 Preparation of 1-(4-(4-(azetidin-3-ylmethyl)piperazin-1-yl)phenyl)-3-benzyl-1-((1r,4r)-4-(quinazolin-2-ylamino)cyclohexyl)urea (Compound 12)
  • Step 3 3-Benzyl-1-(4-((1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-5-yl)azetidin-3-ylmethyl)piperazin-1-yl)phenyl)-1-((1r,4r)-4-(quinazolin-2-ylamino)cyclohexyl)urea (Compound YJZ1102)
  • Example 45 Preparation of 3-benzyl-1-(4-(4-(1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-5-yl)piperidin-4-yl)-piperazin-1-yl)phenyl)-1-((1r,4r)-4-(quinazolin-2-ylamino)cyclohexyl)urea (Compound YJZ1105)
  • the synthesis method is similar to that shown in Example 44.
  • Example 46 3-Benzyl-1-(4-(4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-5-yl)azetidin-3-yl(piperazin-1-yl)phenyl)-1-((1r,4r)-4-(quinazolin-2-ylamino)cyclohexyl)urea (Compound YJZ1103)
  • the synthesis method is similar to that shown in Example 44.
  • the synthesis method is similar to that shown in Example 44.
  • Example 48 3-Benzyl-1-(4-(4-((1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindole-5-yl)piperidin-4-yl)methyl)piperazin-1-yl)phenyl)-1-((1r,4r)-4-(quinazolin-2-ylamino)cyclohexyl)urea (YJZ1097)
  • the synthesis method is similar to that shown in Example 44.
  • the synthesis method is similar to that shown in Example 44.
  • Example 50 1-(4-(4-(1-(1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindole-5-yl)piperidin-4-yl)-piperazin-1-yl)phenyl)-3-(2-fluorobenzyl)-1-((1r,4r)-4-(quinazolin-2-ylamino)cyclohexyl)urea (YJZ1203)
  • the synthesis method is similar to that shown in Example 44.
  • Example 51 3-Benzyl-1-(6-(4-(1-(1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindole-5-yl)piperidin-4-yl)pyrazin-1-yl)pyridin-3-yl]-1-((1r,4r)-4-(quinazolin-2-ylamino)cyclohexyl)urea (YJZ1205)
  • the synthesis method is similar to that shown in Example 44.
  • Example 52 3-Benzyl-1-(6-(4-(1-(1-(2-(2-(2,6-dioxopiperidin-3-yl)-6-fluoro-1,3-dioxoisoindole-5-yl)piperidin-4-yl)pyrrolazin-1-yl)pyridin-3-yl]-1-((1r,4r)-4-(quinazolin-2-ylamino)cyclohexyl)urea (YJZ1206)
  • the synthesis method is similar to that shown in Example 44.
  • Step 1 Preparation of tert-butyl ((1r, 4r)-4-((4-bromophenyl)amino)cyclohexyl)carbamate (Compound 16)
  • Step 2 Preparation of tert-butyl ((1r, 4r)-4-(3-benzyl-1-(4-bromophenyl)ureido)cyclohexyl)carbamate (Compound 17)
  • Step 3 Preparation of 1-((1r, 4r)-4-aminocyclohexyl)-3-benzyl-1-(4-bromophenyl)urea (Compound 18)
  • Step 4 Preparation of 3-benzyl-1-(4-bromophenyl)-1-((1r,4r)-4-(quinazolin-2-ylamino)cyclohexyl)urea (Compound 19)
  • Step 5 Preparation of tert-butyl 4-(1-(4-(3-benzyl-1-((1r,4r)-4-(quinazolin-2-ylamino)cyclohexyl)ureido)phenyl)piperidin-4-yl)piperazine-1-carboxylate (Compound 21)
  • Step 6 Preparation of 3-benzyl-1-(4-(4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindole-5-yl)piperazin-1-yl)piperidin-1-ylphenyl)-1-((1r,4r)-4-(quinazolin-2-ylamino)cyclohexyl)urea (YJZ1202)
  • Example 54 3-Benzyl-1-(4-(4-(1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindole-5-yl)piperidin-4-yl)-piperazin-1-yl)-3-fluorophenyl)-1-((1r,4r)-4-(quinazolin-2-ylamino)cyclohexyl)urea (YJZ1204)
  • the synthesis method is similar to that shown in Example 53.
  • Example 55 Testing of representative compounds for CDK12/13 degradation activity in MDA-MB-231 cells
  • Cell line MDA-MB-231 was purchased from the American Type Culture Collection (ATCC). Conventional Western Blot was used for detection, as follows: MDA-MB-231 cells were seeded in a 12-well plate at a density of 2 ⁇ 10 5 /mL, 1mL per well, and cultured overnight in an incubator. Then, a certain concentration of compound was added for 15 hours, and the cells were lysed with lysis buffer to collect protein samples. An appropriate amount of sample was taken for SDS-PAGE electrophoresis. After the electrophoresis, the protein was transferred to a polyvinylidene fluoride (PVDF) membrane using a wet electrotransfer system.
  • PVDF polyvinylidene fluoride
  • the electrotransferred PVDF membrane was cut into several required bands, and then placed in a blocking solution (5% skim milk powder diluted in TBS containing 0.1% Tween 20) and blocked at room temperature for 1.5 hours.
  • a blocking solution 5% skim milk powder diluted in TBS containing 0.1% Tween 20
  • the primary antibody of the corresponding target protein was diluted in TBS containing 0.1% Tween 20 to complete the preparation of the primary antibody solution.
  • the blocked membrane was placed in the corresponding primary antibody solution and incubated at 4°C for 12-14 hours. Then it was washed three times with TBS containing 0.1% Tween 20, each time for 5 minutes.
  • the washed membrane was placed in the secondary antibody solution (horseradish peroxidase-labeled goat anti-rabbit IgG, diluted 1:2000 in TBS containing 0.1% Tween 20) and reacted at room temperature for 2 hours. After washing the membrane three times as above, use StarSignal plus reagent and Omni-ECL reagent Colorimetry and photography were performed using Amersham Imager 800 system.
  • the results in Figure 1 show that some compounds can effectively degrade the levels of CDK12 and CDK13 proteins in cells, such as compounds ZLC4-77, ZLC4-91, ZLC5-6, ZLC5-11, ZLC7-36, ZLC5-106, ZLC6-1, ZLC6-37, ZLC6-42 and ZLC6-103.
  • the results in Figure 2 show that compounds ZLC4-91 and ZLC5-11 can Time-dependent degradation of CDK12 and CDK13 proteins in MDA-MB-231 cells. After treating cells at a concentration of 1 ⁇ M for 15 hours, the proteins were almost completely degraded. In addition, the compound had a time-dependent degradation effect on the partner protein Cyclin K of CDK12 and CDK13. After 15 hours, the Cyclin K protein in the cells was almost completely degraded.
  • Figure 1 (A) WB results of CDK12 and CDK13 protein degradation after some compounds were treated with MDA-MB-231 cells at a concentration of 0.1 ⁇ M for 15 hours; (B) WB results of CDK12 and CDK13 protein degradation after some compounds were treated with MDA-MB-231 cells at a concentration of 0.3 ⁇ M for 15 hours; (C) Using the control group DMSO as a reference standard, the CDK12/13 level was quantified by the gray value of the band in the WB result image ( Figure A); (D) Using the control group DMSO as a reference standard, the CDK12/13 level was quantified by the gray value of the band in the WB result image ( Figure B).
  • FIG 2 (A) Representative compounds time-dependently degrade CDK12 and CDK13 proteins in MDA-MB-231 cells; (B) WB determination results of intracellular CDK12/13 protein levels after MDA-MB-231 cells were treated with representative compounds at different concentrations for 15 hours.
  • Example 56 Study on the proliferation inhibitory activity of compounds on triple-negative breast cancer cells MDA-MB-231
  • the cell proliferation inhibitory activity of the compound was determined using Cell Counting Kit-8 Cell Viability Assay (Selleck.cn).
  • the cells were seeded in 384-well plates ( PS 3701) and incubated in an incubator at 37°C with 5% CO 2. After overnight incubation, prepare a high concentration stock solution of each compound to be tested; use 650 Liquid Handler Compounds were added to a 384-well plate according to a certain concentration gradient. After administration, the cell plate was placed in an incubator at 37°C and 5% CO 2 for 5 days. (Thermo Scientific TM ) Add 5 ⁇ L of cck-8 reagent to each well of the 384-well plate and return the plate to the incubator for 1.5-2 hours.
  • Example 57 In vivo pharmacokinetic study of representative compounds zlc-4-91 and zlc-4-93 of cell cycle-dependent protein kinase 12/13 (CDK12/13) degraders
  • the oral bioavailability of the compound was calculated based on the area under the blood concentration-time curve (AUC) data.
  • the results are shown in Table 1.
  • the experimental results show (Table 1) that the representative compounds zlc-4-91 and zlc-4-93 have excellent oral pharmacokinetic properties.
  • Cmax refers to the maximum blood drug concentration
  • T1 /2 is the half-life
  • CL refers to the clearance rate
  • F refers to the bioavailability.
  • Example 58 Testing of representative compounds for CDK12/13 degradation activity in 22RV1 cells
  • Cell line 22RV1 cell line was purchased from the American Type Culture Collection (ATCC). Conventional Western Blot was used for detection, as follows: 22RV1 was seeded in a certain number in a 12-well plate, cultured overnight in an incubator, and then a certain concentration of compound was added for 6 hours, and the cells were lysed with a lysis buffer to collect samples. Then, an appropriate amount of sample was taken for SDS-PAGE electrophoresis. After the electrophoresis, the protein was transferred to a nitrocellulose membrane using a semi-dry electrotransfer system.
  • ATCC American Type Culture Collection
  • the nitrocellulose membrane was placed in a blocking solution (5% skim milk powder diluted in TBS containing 0.1% Tween 20) and blocked at room temperature for 2 hours, and then the membrane was placed in a primary antibody solution (1:1000 diluted in TBS containing 0.1% Tween 20) and incubated overnight at 4°C. Wash three times with TBS containing 0.1% Tween 20, each time for 15 minutes. The membrane was placed in a secondary antibody solution (horseradish peroxidase-labeled goat anti-rabbit IgG, diluted 1:1000 in TBS containing 0.1% Tween 20) for 1 hour at room temperature. After washing the membrane three times as above, the color was developed with ECL plus reagent and photographed with Amersham Imager 600 system.
  • a blocking solution 5% skim milk powder diluted in TBS containing 0.1% Tween 20
  • a primary antibody solution (1:1000 diluted in TBS containing 0.1% Tween 20) and incubated overnight at 4°C. Wash three times with TBS
  • Compound YJZ9069 can almost completely degrade intracellular CDK12 and CDK13 proteins after treating 22RV1 cells at a concentration of 0.5 ⁇ M for 15 hours, and dose-dependently inhibit the phosphorylation of serine 2 at the C-terminus of RNA polymerase II (RNA Pol II) (phoSer2).
  • FIG 4 (A) WB results of CDK12 and CDK13 protein degradation after some compounds were treated with 500nM concentration for 6 hours in 22RV1 cells.
  • B Compound YJZ9069 dose-dependently degraded CDK12 and CDK13 proteins in 22RV1 cells, and dose-dependently inhibited the phosphorylation of serine 2 at the C-terminus of RNA polymerase II (RNA Pol II) (phoSer2).
  • C WB results of CDK12 and CDK13 protein degradation after some compounds were treated with 500nM concentration in LnCap cells for 6 hours.
  • D Using the control group DMSO as the reference standard, the protein level was quantified by the gray value of the bands in the WB result images ( Figures A, C).
  • Example 59 Study on the proliferation inhibitory activity of compounds on prostate cancer VCap cells
  • the cell proliferation activity of the compound was determined using CellTiter-Glo Luminescent Cell Viability Assay (Promega, Madison, WI). The cells were seeded on 96-well plates in their respective culture media and incubated at 37°C in a 5% CO2 incubator. After overnight incubation, serial dilutions of the compound were prepared and added to the 96-well plates. A volume of CellTiter-Glo reagent equal to the volume of cell culture medium present in each well was added (e.g., 100 microliters of reagent was added to the culture medium of 100 microliters of 96-well plate cells). The contents were mixed on an orbital shaker for 2 minutes to induce cell lysis.
  • the 96-well plate was incubated at room temperature for 10 minutes to stabilize the luminescent signal. Luminescence was recorded: The luminescent signal of each well was obtained using an Infinite M1000Pro plate reader (Tecan, Switzerland, and the data was analyzed using GraphPad Prism software (GraphPad Software Inc, La Jolla, CA).
  • Example 60 In vivo pharmacokinetic experiments of representative compounds YJZ9069, YJZ1102, YJZ1105, YJZ1201, YJZ1202, YJZ1203, YJZ1204, YJZ1097, YJZ1205 and YJZ1206 of cell cycle-dependent protein kinase 12/13 (CDK12/13) degraders
  • the oral bioavailability of the compound was calculated based on the area under the blood concentration-time curve (AUC) data.
  • the results are shown in Table 2-6.
  • the experimental results show (Table 2-6) that the representative compounds YJZ1102, YJZ1105, YJZ1201, YJZ1202, YJZ1203, YJZ1204, YJZ1097, YJZ1205 and YJZ1206 have excellent oral pharmacokinetic properties.
  • Cmax refers to the maximum blood drug concentration
  • T1 /2 is the half-life
  • CL refers to the clearance rate
  • F refers to the bioavailability.
  • Cmax refers to the maximum blood drug concentration
  • T1 /2 is the half-life
  • CL refers to the clearance rate
  • F refers to the bioavailability.
  • Cmax refers to the maximum blood drug concentration
  • T1 /2 is the half-life
  • CL refers to the clearance rate
  • F refers to the bioavailability.
  • Cmax refers to the maximum blood drug concentration
  • T1 /2 is the half-life
  • CL refers to the clearance rate
  • F refers to the bioavailability.
  • Cmax refers to the maximum blood drug concentration
  • T1 /2 is the half-life
  • CL refers to the clearance rate
  • F refers to the bioavailability.

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Abstract

本发明涉及一类含有芳基取代的CDK12/13的降解剂，及其制备方法、药物组合物和应用。本发明的细胞周期依赖性蛋白激酶12/13（CDK12/13）的降解剂具有式（I）所示结构，该类化合物可作为蛋白激酶降解剂，能够有效地、高选择性地降解CDK12/13蛋白并且能抑制多种肿瘤细胞的增殖、迁移和侵袭。

Description

一类含有芳基取代的CDK12/13的降解剂，及其制备方法、药物组合物和应用 技术领域
本发明属于化学医药领域，特别是涉及细胞周期依赖性蛋白激酶12/13(CDK12/13)的降解剂及其药用组合物和应用。
背景技术
蛋白激酶是细胞功能的关键调节分子，构成了最大且功能最多样的基因家族之一。蛋白激酶通过底物蛋白的磷酸化，指导许多蛋白的活动、定位以及总体功能，并且参与几乎所有细胞活动。蛋白激酶的表达、活化、定位等异常，与多种疾病的发生、发展密切相关，是肿瘤、炎症等多种疾病的重要驱动因子。截止到2020年12月31日，FDA共计批准了62个激酶抑制剂药物上市。研宄发现，蛋白的非激酶功能在肿瘤等疾病中发挥重要作用。文献已经报道了CDK6、FAK、EGFR、PIPK3、PDK1、BRAF、CRAF、CHK2、ZAP70、AKT、Aurora A等激酶的非激酶功能。因此，单纯抑制激酶的活性无法完全抑制激酶的非激酶功能，从而带来疗效低、耐药等潜在不利影响。因此，开发蛋白降解剂有望全面抑制激酶的酶活和非激酶功能，发挥强效治疗作用。而基于PROTAC原理的蛋白降解剂是目前最为成功和成熟的蛋白降解策略，并成功用于多个激酶及其他靶标的降解剂开发。
PROTAC特指一类可以特异性识别并诱导降解靶蛋白的小分子化合物；分子结构由靶蛋白识别配体、Linker和E3识别配体三部分组成。PROTAC的优点是不但有效抑制靶蛋白的激酶活性，而且可以快速降解清除靶蛋白。理论上只需要催化量的药物，就可以降解细胞内几乎所有的蛋白质(包括膜蛋白)，故具有较高的安全性、耐药性和广阔的应用前景。目前已经开发成功了针对ERR、ABL、BET、CDK4/6等靶蛋白的降解剂，结果表明其不但能治疗基因驱动型肿瘤的增殖，也能克服抑制剂耐药。2018年，辉瑞公司宣布投资8.3亿美元进行基于PROTAC技术的蛋白降解剂药物研发。2019年3月，药明康德的合作伙伴Arinas公司宣布，其开发的针对雄激素受体的蛋白降解剂ARV-110进入临床研究；这是全球首个进入临床研究阶段的蛋白降解剂。
CDK12/13(Cyclin-dependent kinase 12/13)属于丝氨酸/苏氨酸蛋白激酶的细胞周期蛋白依赖性激酶家族(CDKs)的成员，与Cyclin K形成复合物发挥生物学功能。CDK12/13分别含有1490、1512个氨基酸，且享有46％的同源性，激酶区域由300个氨基酸组成其同源性高达92％。CDK12/13与Cyclin K形成复合物通过磷酸化RNA聚合酶II(RNA Pol II)C端结构域(CTD)，CTD由YSPTSPS七个氨基酸组成的高度重复序列，在人类中，CTD包括一个52重复单元。CDK12/13主要对Ser2进行磷酸化从而调节转录，及转录后的mRNA的加工。遗传研究表明，CDK12通过抑制内含子聚腺苷酸化位点的切割，促进转录出全长基因的产物，许多同源重组修复基因(如：BRCA1/2、ATM、ATR、FANCD2、FANCI等)包含更多的内含子多腺苷化位点，因此，这些基因的表达对CDK12的丢失或抑制更为敏感。与其他转录CDKs相比CDK12/13的N末端含有额外的精氨酸/富含丝氨酸(RS)基序，RS基序常见于参与前mRNA剪接的蛋白质中。Proline-rich motifs(PRIM)的基序也见于N-和C-末端，可能作为SH3，WW或含有肌动蛋白结合蛋白(profilin)结构域的蛋白质的结合位点。这些结构提示CDK12/13可能存在非激酶功能(如:蛋白-蛋白相互作用)在mRNA剪接，mRNA 3'末端加工发挥着重要作用。因此，仅使用CDK12/13的激酶抑制剂做为研究CDK12/13生物学功能的探针工具分子是远远不够的。利用蛋白水解靶向嵌合体(PROTACs) 诱导CDK12/13降解，同时抑制其激酶功能和非激酶功能，在研究CDK12/13的生物学功能和疾病治疗当中具有巨大的潜力，近年来引起了科学家越来越大的兴趣。
发明内容
基于此，本发明提供了一类反式-1,4-环己二胺类化合物及其作为CDK12/13降解剂的应用，该类化合物能够有效地、高选择性地降解CDK12/13蛋白激酶，且能抑制多种肿瘤细胞的增殖、迁移和侵袭。
在本发明的第一方面，提供了一种具有式(I)结构的化合物，或其药学上可接受的盐，或其药物组合物或其前药分子：
其中，Z为CH2或CO；
V选自下组：
X、Y各自独立地选自下组：N、CH或CR3；其中，R3选自下组：卤素、氰基、羟基、氨基、C1-C3烷基、卤代C1-C3烷基、C1-C3烷氧基、卤代C1-C3烷氧基、C3-C8环烷基，或3-8元杂环基；
B选自下组： NH、O、CO、CH2；其中，各个U、W各自独立选自下组：N或CH；各个m、p、m'、p'各自独立地为：0、1、2或3；
A环选自下组：
其中，Q、W各独立地选自：CH、N；
R'独立地选自：氢、卤素、氰基、羟基、取代羟基、氨基、取代氨基、C1-C5烷基、卤代C1-C5烷基、C1-C5烷氧基、卤代C1-C5烷氧基、C3-C8环烷基，且当Q、W独立地选自CH时，R'可以为Q、W上的取代基；
D、E、F、G各自独立选自下组：CH、N、CR6；其中，R6选自下组：卤素、三氟甲基、羟基、氰基、胺基、甲基、甲氧基、三氟甲氧基；
R1选自下组：H、-NHR7、-OR7、-(C(R9)R8)R7；
其中，R7为-R10、-CH2R10或-(CH2)2R10；
R8、R9各自独立地选自下组：氢、卤素、氰基、甲基、卤代甲基、甲氧基、卤代甲氧基、乙基、卤代乙基、乙氧基、卤代乙氧基、羟基、氨基、含有1、2或3个杂原子的3-8元杂 环，且所述的杂原子选自O、S或N；
或者R8、R9及其相连的C原子共同形成3-7杂环；
R10选自下组：
1)氰基、C1-C5烷基、卤代C1-C4烷基、C1-C4烷氧基、C3～C10环烷基、取代或未取代的5-12元芳环、取代或未取代的3-12元杂环；
2)
其中Q1、Q2、Q3、Q4、Q5各自独立地选自：CH、N或CR11；
各个R11各自独立地选自下组：卤素、氰基、羟基、氨基、硝基、C1-C3烷基、卤代C1-C3烷基、C1-C4烷氧基、卤代C1-C4烷氧基、C3-C8环烷基；
R2选自下组：H、C1-C3烷基、
当B选自下组： NH、O、CO、CH2；其中，各个U、W各自独立选自下组：N或CH；各个m、p、m'、p'各自独立地为：0、1、2或3；时
Linker为：
其中，
RL1、RL2、RL3、RL4和RL5相同或不同，且各自独立地选自取代或未取代的下组基团：化学键、CH2、CHD、CD2、C＝O、O、NH、SO、SO2、P＝O、NHCO、NHSO2、OCH2、OCH2CH2、CH2OCH2、NHCH2、NMeCH2、NHCH2CH2、NMeCH2CH2、CH2NHCO、NHCOCH2、

RL6为环，且任选自以下结构：


其中各个n各自独立地为0，1，2，3，4，5或6；各个r、m各自独立地为0，1或2；各个U、W各自独立选自下组：N或CH
pL1、pL2、pL3、pL4、pL5和pL6独立地选自0、1、2、3、4、5、或6。
当B为其中各个m、p、m'、p'各自独立地为：0、1、2或3；时
Linker为：
其中，
RL1、RL2、RL3、RL4、RL5和RL6相同或不同，且各自独立地选自取代或未取代的下组基团：化学键、CH2、CHD、CD2、C＝O、O、NH、SO、SO2、P＝O、NHCO、NHSO2、OCH2、OCH2CH2、CH2OCH2、NHCH2、NMeCH2、NHCH2CH2、NMeCH2CH2、CH2NHCO、NHCOCH2、



其中各个n各自独立地为0，1，2，3，4，5或6；各个r、m各自独立地为0，1或2；各个U、W各自独立选自下组：N或CH；
pL1、pL2、pL3、pL4、pL5和pL6独立地选自0、1、2、3、4、5、或6。
在部分实施方式中，V选自下组：
在部分实施方式中，X、Y各自独立选自下组：N、CH或CR3；
其中，R3选自下组：卤素、氰基、羟基、氨基、C1-C3烷基、卤代C1-C3烷基、C1-C3烷氧基、卤代C1-C3烷氧基。
在部分实施方式中，B选自下组： NH、O、CO；
其中，各个U、W各自独立选自下组：N或CH；各个m、p、m'、p'各自独立地为0、1、2或3。
在部分实施方式中，A环选自：
其中，W选自下组：CH、N。
R'任选自：氢、卤素、氰基、羟基、氨基、C1-C5烷基、卤代C1-C5烷基、C1-C5烷氧基、卤代C1-C5烷氧基、C3-C8环烷基
在部分实施方式中，所述的D、E、F、G为CH、CR6；其中，R6选自下组：卤素。
在部分实施方式中，所述的R1为-NHR7；
其中，R7为-CH2R10；R10的定义如前所述。
在另一优选例中，所述的R10选自下组：氰基、C1-C5烷基、卤代C1-C4烷基、C1-C4烷氧基、C3-C10环烷基、4-7元杂环基、C6-C10芳基；其中所述的芳基被一个或多个CR11取代；R11的定义如前所述。
在部分实施方式中，
当B选自下组：NH、O、CO；
时，所述的Linker选自下组：
且当B为时，所述的Linker选自下组：化学键、 其中各个n各自独立地为0，1，2，3，4，5或6；各个r、m各自独立地为0，1或2；各个U、W各自独立选自下组：N或CH。
在部分实施方式中，所述的化合物具有式(II)所示结构：
其中，B选自：其中，各个U、W各自独立选自下组：N或CH；各个m、p、m'、p'各自独立地为0、1、2或3；
R'任选自：氢、卤素、氰基、羟基、氨基；
R11、R6任选自：氢、卤素；
X，Y独立选自：CH、N或CR3；其中，R3任选自：卤素、氰基、羟基、氨基；
Z任选自：CH2或CO；
在部分实施方式中，所述化合物选自下组：



在本发明的第二方面，提供了一种具有式(I)结构的化合物，或其药学上可接受的盐，或其药物组合物或其前药分子：
其中，Z选自下组：CH2或CO；
V选自下组：
X、Y选自下组：N、CH或CR3；其中，R3选自下组：卤素、氰基、羟基、氨基、C1-C3烷基、卤代C1-C3烷基、C1-C3烷氧基、卤代C1-C3烷氧基、C3-C8环烷基，或3-8元杂环基；
B选自下组： NH、O、CO、CH2；其中，各个U、W各自独立选自下组：N或CH；各个m、p各自独立地为：0、1或2；
A环选自下组：
M、T各自独立选自下组：N、CR4；
I、J、K各自独立选自下组：N、O、S、CR4和NR4；
R4选自下组：氢、卤素、氰基、羟基、氨基、C1-C5烷基、卤代C1-C5烷基、C1-C5烷氧基、卤代C1-C5烷氧基、C3-C8环烷基、-CH2R5、-(CH2)2R5、-(CH2)3R5或3-8元杂环基；
R5选自下组：氰基、羟基、氨基、C3-C8环烷基、或3-8元杂环基；
D、E、F、G各自独立选自下组：CH、N、CR6；其中，R6选自下组：卤素、三氟甲基、羟基、氰基、胺基、甲基、甲氧基、三氟甲氧基；
R1选自下组：H、-NHR7、-OR7、-(C(R9)R8)R7；
其中，R7为-R10、-CH2R10或-(CH2)2R10；
R8、R9各自独立地选自下组：氢、卤素、氰基、甲基、卤代甲基、甲氧基、卤代甲氧基、乙基、卤代乙基、乙氧基、卤代乙氧基、羟基、氨基、含有1、2或3个杂原子的3-8元杂环，且所述的杂原子选自O、S或N；
或者R8、R9及其相连的C原子共同形成3-7杂环；
R10选自下组：
1)氰基、C1-C5烷基、卤代C1-C4烷基、C1-C4烷氧基、C3～C10环烷基、取代或未取代的5-12元芳环、取代或未取代的3-12元杂环；
2)
其中Q1、Q2、Q3、Q4、Q5各自独立地选自：CH、N或CR11；
各个R11各自独立地选自下组：卤素、氰基、羟基、氨基、硝基、C1-C3烷基、卤代C1-C3烷基、C1-C4烷氧基、卤代C1-C4烷氧基、C3-C8环烷基；
R2选自下组：H、C1-C3烷基、
Linker为：
其中，RL1-RL6相同或不同，且各自独立地选自取代或未取代的下组基团：键、CH2、CHD、CD2、C＝O、O、NH、SO、SO2、P＝O、NHCO、NHSO2、OCH2、OCH2CH2、CH2OCH2、NHCH2、NMeCH2、NHCH2CH2、NMeCH2CH2、CH2NHCO、NHCOCH2、


其中各个n各自独立地为0，1，2，3，4，5或6；各个r、m各自独立地为0，1或2；各个U、W各自独立选自下组：N或CH
所述取代是指被选自下组的一个或多个基团取代：氢、氘、C1-C18烷基、氘代C1-C18烷基、卤代C1-C18烷基、卤代C1-C18烷基羟基、C3-C20环烷基、C1-C18烷氧基、氘 代C1-C18烷氧基、卤代C1-C18烷氧基、C6-C14芳基、5-14元杂芳基、4-20元杂环基、卤素、氧代基、硝基、羟基、氰基、酯基、胺基、酰胺基、磺酰胺基或脲基；
各pL1-pL6独立地选自0、1、2、3、4、5、或6。
在部分实施方式中，V选自下组：
在部分实施方式中，X、Y各自独立选自下组：N、CH或CR3；
其中，R3选自下组：卤素、氰基、羟基、氨基、C1-C3烷基、卤代C1-C3烷基、C1-C3烷氧基、卤代C1-C3烷氧基。
在部分实施方式中，B选自下组：NH、O、CO；
其中，各个U、W各自独立选自下组：N或CH；各个m、p各自独立地为0或1。
在部分实施方式中，A环选自下组：
其中，T选自下组：CH、N。
在部分实施方式中，R4选自下组：氢、卤素、氰基、羟基、氨基、C1-C5烷基、卤代C1-C5烷基、C1-C5烷氧基、卤代C1-C5烷氧基、C3-C8环烷基、-CH2R5、-(CH2)2R5、-(CH2)3R5或3-8元杂环基。
在部分实施方式中，所述的D、E、F、G为CH。
在部分实施方式中，所述的R1为-NHR7；
其中，R7为-CH2R10；R10的定义如前所述。
在另一优选例中，所述的R10选自下组：氰基、C1-C5烷基、卤代C1-C4烷基、C1-C4烷氧基、C3-C10环烷基、4-7元杂环基、C6-C10芳基；其中所述的芳基被一个或多个CR11取代；R11的定义如前所述。
在部分实施方式中，Linker选自下组： 其中各个n各自独立地为0，1，2，3，4，5或6；各个r、m各自独立地为0，1或2；各个U、W各自独立选自下组：N或CH。
在另一优选例中，Linker选自下组：
其中各个n各自独立地为0，1，2，3，4，5或6；各个r、m各自独立地为0，1或2；各个U、W各自独立选自下组：N或CH。
在部分实施方式中，在部分实施方式中，所述的化合物具有式(II)或式(III)所示结构：
其中，U，W独立选自：CH或N；
X，Y独立选自：CH、N或CR3；
Z任选自：CH2或CO；
n，m独立选自：0或1。
在部分实施方式中，所述化合物选自下组：



在本发明的第三方面，提供了一种药物组合物，其特征在于，包含
(1)作为活性成分的如本发明第一方面或第二方面所述的化合物，或者药学上可接受的盐、其立体异构体或其前药分子；和
任选的(2)药学上可接受的载体。
在本发明的第四方面，提供了一种如本发明第一方面或第二方面所述的化合物，或者其药学上可接受的盐或者其立体异构体或者其前药分子，或者如本发明第三方面所述的药物组合物的应用，其特征在于，用于制备CDK12/13蛋白激酶降解剂。
在本发明的第五方面，提供了一种如本发明第一方面或第二方面所述的化合物，或者其药学上可接受的盐或者其立体异构体或者其前药分子，或者如本发明第三方面所述的药物组合物的应用，其特征在于，用于制备预防和/或治疗由CDK12/13丝氨酸/苏氨酸蛋白激酶介导的疾病的药物。
在部分实施方式中，所述由CDK12/13丝氨酸/苏氨酸蛋白激酶介导的疾病选自下组：前列腺癌、乳腺癌、子宫癌、卵巢癌、非小细胞肺癌、小细胞肺癌、尤文肉瘤、肺腺癌、肺鳞癌、胰腺癌、肝癌、皮肤癌、上皮细胞癌、胃肠间质瘤、白血病、组织细胞性淋巴癌、鼻咽癌、头颈部肿瘤、结肠癌、直肠癌、胶质瘤。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1为(A)部分化合物在0.1μΜ浓度下处理MDA-MB-231细胞15小时后对CDK12、CDK13蛋白降解的WB结果；(B)部分化合物在0.3μΜ浓度下处理MDA-MB-231细胞15小时后对CDK12、CDK13蛋白降解的WB结果；(C)以对照组DMSO为参照标准，通过对WB结果图像(图A)中条带的灰度值定量CDK12/13水平；(D)以对照组DMSO为参照标准，通过对WB结果图像(图B)中条带的灰度值定量CDK12/13水平。
图2为(A)代表性化合物时间依赖性降解MDA-MB-231细胞中的CDK12和CDK13蛋白；(B)代表性化合物在不同浓度下处理MDA-MB-231细胞15小时后，细胞内CDK12/13蛋白水平的WB测定结果。
图3为化合物对三阴性乳腺癌细胞MDA-MB-231的抑制活性的测定结果。
图4为(A)部分化合物在500nM浓度下处理22RV1细胞6小时后对CDK12和CDK13蛋白降解的WB结果。(B)化合物YJZ9069剂量依赖性地降解22RV1细胞中的CDK12和CDK13蛋白，及剂量依赖性地抑制RNA聚合酶II(RNA Pol II)C端2位丝氨 酸的磷酸化(phoSer2)。(C)部分化合物在500nM浓度下处理LnCap细胞6小时后对CDK12和CDK13蛋白降解的WB结果。(D)以对照组DMSO为参照标准，通过对WB结果图像(图A，C)中条带的灰度值来定量蛋白质水平。
图5为代表性化合物YJZ9069、YJZ1201、YJZ1202、YJZ1203、YJZ1204、YJZ1097、YJZ1205和YJZ1206在不同浓度下处理22RV1细胞6小时后，细胞内CDK12和CDK13蛋白水平的WB测定结果。
图6为化合物对前列腺癌细胞系VCap的增殖抑制活性测定结果。
具体实施方式
发明人经过广泛而深入的研究，出乎意料地发现一类反式-1,4-环己二胺类化合物，对其进行了一系列生物活性测试，从而发现其具有出色的CDK12/13抑制性能。在此基础上完成了本发明。
本发明下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。实施例中所用到的各种常用化学试剂，均为市售产品。
除非另有定义，本发明所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。本发明的说明书中所使用的术语只是为了描述具体的实施例的目的，不用于限制本发明。
本发明的术语“包括”和“具有”以及它们任何变形，意图在于覆盖不排他的包含。例如包含了一系列步骤的过程、方法、装置、产品或设备没有限定于已列出的步骤或模块，而是可选地还包括没有列出的步骤，或可选地还包括对于这些过程、方法、产品或设备固有的其它步骤。
在本发明中提及的“多个”是指两个或两个以上。“和/或”，描述关联对象的关联关系，表示可以存在三种关系，例如，A和/或B，可以表示：单独存在A，同时存在A和B，单独存在B这三种情况。字符“三种一般表示前后关联对象是一种“或”的关系。
本发明所述化合物中，当任何变量(例如R10、R11等)在任何组分中出现超过一次，则其每次出现的定义独立于其它每次出现的定义。同样，允许取代基及变量的组合，只要这种组合使化合物稳定。自取代基划入环系统的线表示所指的键可连接到任何能取代的环原子上。如果环系统为多环，其意味着这种键仅连接到邻近环的任何适当的碳原子上。要理解本领域普通技术人员可选择本发明化合物的取代基及取代型式而提供化学上稳定的并可通过本领域技术和下列提出的方法自可容易获得的原料容易合成的化合物。如果取代基自身被超过一个基团取代，应理解这些基团可在相同碳原子上或不同碳原子上，只要使结构稳定。
本文所用术语“烷基”意指包括具有特定碳原子数目的支链的和直链的饱和脂肪烃基。例如，“C1-C8烷基”中“C1-C8”的定义包括以直链或支链排列的具有1、2、3、4、5、6、7或8个碳原子的基团。术语“环烷基”指具有特定碳原子数目的单环饱和脂肪烃基。例如“环烷基”包括环丙基、甲基-环丙基、2,2-二甲基-环丁基、2-乙基-环戊基、环己基等。
如本文所用，术语“烯基”包括直链或支链的烯基。例如C2-C6烯基指具有2-6个碳原子的直链或支链的烯基，例如乙烯基、烯丙基、1-丙烯基、异丙烯基、1-丁烯基、2-丁烯基、或类似基团。
如本文所用，术语“炔基”包括直链或支链的炔基。例如C2-C6炔基是指具有2-6个碳 原子的直链或支链的炔基，例如乙炔基、丙炔基、丁炔基、或类似基团。
如本文所用，术语“环烷基”是指具有特定碳原子数目的环状饱和脂肪烃基。例如C3-C10烯基指具有3-10个碳原子的环状饱和脂肪烃基。其可以是单环，例如环丙基、环丁基、环戊基、环己基、或类似基团。也可以是双环形式，例如桥环或螺环形式。
如本文所用，术语“杂环基”或者“杂环烷基”是指具有特定的环原子数(如3-10个环原子)的，且其中1-3个原子为选自N、S和O的杂原子的饱和或部分饱和的环状基团。其可以是单环，也可以是双环或多环形式，例如稠环、桥环或螺环形式。具体的实例可以为氧杂环丁烷基、氮杂环丁烷基、四氢-2H-吡喃基、哌啶基、四氢呋喃基、吗啉基和吡咯烷基等。
如本文所用，术语“烷胺基”是指被烷基所取代的胺基。例如，“C1-C6烷胺基”是指被C1-C6烷基所取代的胺基，可以是单取代或双取代的；例如，甲胺基、乙胺基、丙胺基、异丙胺基、丁胺基、异丁胺基、叔丁胺基、二甲胺基、二乙胺基、二丙胺基、二异丙胺基、二丁胺基、二异丁胺基、二叔丁胺基等。
如本文所用，术语“烷氧基”是指具有烷基-氧基结构的基团。例如，“C1-C6烷氧基”是指具有1-6个碳原子的直链或支链的烷氧基，包括甲氧基、乙氧基、丙氧基、异丙氧基、丁氧基、异丁氧基、叔丁氧基等。
如本文所用，术语“卤代烷基”代表其中有一个或多个氢原子被卤素取代的烷基基团，其中烷基的定义如上所述。
如本文所用，术语“卤代烷氧基”代表有一个或多个氢原子被卤素取代的烷氧基基团，其中烷氧基的定义如上所述。
正如本领域技术人员所理解的，本文中所用“卤素”意指包括F、Cl、Br和I。更佳地，卤素或卤原子选自F、Cl和Br。“卤代的”是指被选自F、Cl、Br、和I的原子所取代。
本发明所述的基团除非特别说明是“取代的或未取代的”，否则本发明的基团均可被选自下组的取代基所取代：卤素、腈基、硝基、羟基、氨基、C1-C6烷基-胺基、C1-C6烷基、C2-C6烯基、C2-C6炔基、C1-C6烷氧基、卤代C1-C6烷基、卤代C2-C6烯基、卤代C2-C6炔基、卤代C1-C6烷氧基、烯丙基、苄基、C6-C12芳基、C1-C6烷氧基-C1-C6烷基、C1-C6烷氧基-羰基、苯氧羰基、C2-C6炔基-羰基、C2-C6烯基-羰基、C3-C6环烷基-羰基、C1-C6烷基-磺酰基等。
本发明包括式(I)(在此，式(I)包括式(II))化合物的游离形式，也包括其药学上可接受的盐、其立体异构体及其前药分子。术语“游离形式”指以非盐形式的化合物。包括在内的药学上可接受盐不仅包括本文所述特定化合物的示例性盐，也包括所有式(I)或式(II)化合物游离形式的典型的药学上可接受的盐。可使用本领域已知技术分离所述化合物特定盐的游离形式。例如，可通过用适当的碱稀水溶液例如NaOH稀水溶液、碳酸钾稀水溶液、稀氨水及碳酸氢钠稀水溶液处理该盐使游离形式再生。游离形式在某些物理性质例如在极性溶剂中溶解度上与其各自盐形式多少有些区别，但是为发明的目的这种酸盐及碱盐在其它药学方面与其各自游离形式相当。
可通过常规化学方法自含有碱性部分或酸性部分的本发明化合物合成本发明的药学上可接受的盐。通常，通过离子交换色谱或通过游离碱和化学计算量或过量的所需盐形式的无机或有机酸在适当溶剂或多种溶剂的组合中反应制备碱性化合物的盐。类似的，通过和适当的无机或有机碱反应形成酸性化合物的盐。
因此，本发明化合物的药学上可接受的盐包括通过碱性本发明化合物和无机或有 机酸反应形成的本发明化合物的常规无毒盐。例如，常规的无毒盐包括得自无机酸例如盐酸、氢溴酸、硫酸、氨基磺酸、磷酸、硝酸等制备的盐，也包括得自有机酸例如乙酸、丙酸、琥珀酸、乙醇酸、硬脂酸、乳酸、苹果酸、酒石酸、柠檬酸、抗坏血酸、扑酸、马来酸、羟基马来酸、苯乙酸、谷氨酸、苯甲酸、水杨酸、对氨基苯磺酸、2-乙酰氧基一苯甲酸、富马酸、苯磺酸、甲苯磺酸、甲磺酸、乙烷二磺酸、草酸、羟乙基磺酸、三氟乙酸等制备的盐。
如果本发明化合物为酸性的，则适当的“药学上可接受的盐”指通过药学上可接受的无毒碱包括无机碱及有机碱制备的盐。得自无机碱的盐包括铝盐、铵盐、钙盐、铜盐、铁盐、亚铁盐、锂盐、镁盐、锰盐、亚锰盐、钾盐、钠盐、锌盐等。特别优选铵盐、钙盐、镁盐、钾盐和钠盐。得自药学上可接受的有机无毒碱的盐，所述碱包括伯胺、仲胺和叔胺的盐，取代的胺包括天然存在的取代胺、环状胺及碱性离子交换树脂例如精氨酸、甜菜碱、咖啡因、胆碱、N,N'-二苄基乙二胺、二乙胺、2-二乙基氨基乙醇、2-二甲基氨基乙醇、氨基乙醇、乙醇胺、乙二胺、N-乙基吗啉、N-乙基哌啶、葡萄糖胺、氨基葡萄糖、组氨酸、羟钴胺、异丙基胺、赖氨酸、甲基葡萄糖胺、吗啉、哌嗪,哌啶、多胺树脂、普鲁卡因、嘌呤、可可碱、三乙胺、三甲胺、三丙胺、氨基丁三醇等。
Berg等，“Pharmaceutical Salts”J.Pharm.Sci.’1977：66：1-19更详细描述了上文所述药学上可接受的盐及其它典型的药学上可接受的盐的制备。
除非特别说明，本发明所描述的结构式意在包括所有的同分异构形式(如对映异构，非对映异构和几何异构体(或构象异构体))：例如含有不对称中心的R、S构型，双键的(Z)、(E)异构体等。因此，本发明化合物的单个立体化学异构体或其对映异构体、非对映异构体或几何异构体(或构象异构体)的混合物都属于本发明的范围。
如本文所用，术语“互变异构体”表示具有不同能量的结构同分异构体可以越过低能垒，从而互相转化。比如，质子互变异构体(即质子移变)包括通过质子迁移进行互变，如1H-吲唑与2H-吲唑。化合价互变异构体包括通过一些成键电子重组而进行互变。
如本文所用，术语“溶剂合物”是指本发明化合物与溶剂分子配位形成特定比例的配合物。
如本文所用，术语“水合物”是指本发明化合物与水进行配位形成的配合物。
本发明的化合物也可以为前药形式。如本文所用，术语“前药”是指当代谢(例如体内或体外)时产生活性化合物的化合物。在一些实施方式中，前药可以是无活性的，或具有比游离药物更低的活性，但可以提供有利的处理、给药或代谢特性。本发明的示例性前药部分可以通过核苷酸的羟基、氨基、磷酸酯或硫代磷酸酯主链与游离药物连接，并且可以包含酯、氨基甲酸酯、羰基、硫酯、酰胺、异氰酸酯、脲、硫脲或其他生理上可接受的代谢不稳定部分。在一些实施方式中，前药通过酶促水解被激活。
本公开还包括同位素标记的化合物，其与式(I)化合物(包括式(II)的化合物)相同，但其中一个或多个原子被原子质量或质量数与自然界中通常发现的原子质量或质量数不同的原子所取代。适合包括在本发明化合物中的同位素的实例是氢、碳、氮、氧、磷、硫、氟和氯，例如但不限于2H、3H、13C、14C、15N、18O、31P、35S、18F和36Cl。用较重的同位素(例如氘，即2H)替代可以提供某些治疗优势，这是由于更高的代谢稳定性，例如更高的体内半衰期或更低的剂量需求，因此在某些情况下可能是优选的。所述的化合物可以结合用于医学成像和正电子发射断层扫描(PET)研究的正电子发射同 位素，以确定受体的分布。可以并入式(I)或(II)化合物中的合适的正电子发射同位素是11C、13N、15O和18F。同位素标记的式(I)或(II)化合物通常可以通过本领域技术人员已知的常规技术，或者通过类似于本文所述的方法，使用适当的同位素标记的试剂代替非同位素标记的试剂制备。
本文公开的化合物可以与药学上可接受的溶剂例如水、乙醇等以溶剂化和非溶剂化形式存在，并且本发明旨在涵盖溶剂化和非溶剂化形式。在一个实施方式中，所述的化合物是无定形的。在一个实施方式中，所述的化合物是单一的多晶型物。在另一个实施方案中，所述的化合物是多晶型物的混合物。在另一个实施方式中，所述的化合物呈结晶形式。
药物组合物及给药方法
由于本发明化合物为CDK12/13蛋白降解剂，所述的化合物及其药学上可接受的盐以及本文公开的其他化合物形式可以包含在可用于治疗、预防和减轻与CDK12/13活性相关的疾病的药物组合物中。
本发明的药物组合物包含有效量的，例如安全有效量的本发明化合物或其药理学上可接受的盐和药理学上可接受的赋形剂或载体。“有效量”是指足以引发所需生物反应(例如，治疗病症)的量。“安全有效量”是指：化合物的量足以显着改善病情而不引起严重的副作用。通常，药物组合物包含1至3000mg(活性剂量范围为3至30mg/kg)的本公开的化合物/剂量，并且更优选地包含10至2000mg的本发明的化合物/剂量。优选地，“一剂”是胶囊剂或片剂。
“药学上可接受的载体”是指：一种或多种相容的适合人类使用的固体或液体填充剂或凝胶物质，其必须具有足够的纯度和足够低的毒性。本文中的“相容性”是指组合物的组分可以与本公开的化合物混合、彼此混合而不会显着降低化合物的功效。药学上可接受的载体部分的实例包括纤维素及其衍生物(例如羧甲基纤维素钠、乙基纤维素钠和醋酸纤维素)、明胶、滑石粉、固体润滑剂(例如硬脂酸和硬脂酸镁)、硫酸钙、植物油(例如大豆油、芝麻油、花生油、橄榄油)、多元醇(丙二醇、甘油、甘露醇、山梨糖醇等)、乳化剂(吐温等)、润湿剂(十二烷基硫酸钠等)、着色剂、调味剂、稳定剂、抗氧化剂、防腐剂和无热原水。
本发明的化合物或药物组合物的给药方式没有特别限制，代表性的给药方式包括(但不限于)：口服、瘤内、直肠、肠胃外(静脉内、肌内或皮下)和局部给药。
用于口服给药的固体剂型包括胶囊剂、片剂、丸剂、散剂和颗粒剂。在这些固体剂型中，活性化合物与至少一种常规惰性赋形剂(或载体)混合，例如柠檬酸钠或磷酸二钙，或与以下成分混合：(a)填充剂或填充剂，例如淀粉、乳糖、蔗糖、葡萄糖、甘露醇和硅酸；(b)粘合剂，例如羟甲基纤维素、海藻酸盐、明胶、聚乙烯吡咯烷酮、蔗糖和阿拉伯树胶；(c)保湿剂，例如甘油；(d)崩解剂，例如琼脂、碳酸钙、马铃薯淀粉或木薯淀粉、海藻酸、一些复合硅酸盐和碳酸钠；(e)缓凝溶剂，例如石蜡；(f)吸收促进剂，例如季胺化合物；(g)润湿剂，例如鲸蜡醇和单硬脂酸甘油酯；(h)吸附剂，例如高岭土；(i)润滑剂，例如滑石粉、硬脂酸钙、硬脂酸镁、固体聚乙二醇、月桂基硫酸钠或其混合物。在胶囊剂、片剂和丸剂中，剂型还可以包括缓冲剂。
固体剂型，例如片剂、糖衣丸、胶囊、丸剂和颗粒剂，可以由包衣和外壳材料制备，例如肠溶包衣和本领域熟知的其他材料。其可以包含遮光剂，和活性化合物或在此类组合物中以延迟方式在消化道的一部分中释放的化合物。可用的包埋组分的实例 是聚合物质和蜡。必要时，活性化合物还可以与一种或多种上述赋形剂形成微囊形式。
用于口服给药的液体剂型包括药学上可接受的乳剂、溶液剂、混悬剂、糖浆剂或酏剂。除活性化合物外，液体剂型可包含本领域常用的惰性稀释剂，例如水或其他溶剂，增溶剂和乳化剂，例如乙醇、异丙醇、碳酸乙酯、乙酸乙酯、丙二醇，1,3-丁二醇、二甲基甲酰胺和油，特别是棉籽油、花生油、玉米胚芽油、橄榄油、蓖麻油和芝麻油，或其混合物。
除了这些惰性稀释剂外，该组合物还可包含助剂，例如润湿剂、乳化剂和助悬剂、甜味剂、矫味剂和香料。
除活性化合物外，混悬剂还可包含助悬剂，例如乙氧基化异十八烷醇、聚氧乙烯山梨糖醇和失水山梨糖醇酯、微晶纤维素、甲醇铝和琼脂，或其混合物。
用于肠胃外注射的组合物可包含生理上可接受的无菌水溶液或无水溶液、分散体、悬浮液或乳剂，以及用于再溶解成无菌可注射溶液或分散体的无菌粉末。合适的水性和非水性载体、稀释剂、溶剂或赋形剂包括水、乙醇、多元醇及其合适的混合物。
本发明化合物的局部给药剂型包括软膏剂、粉剂、贴剂、喷雾剂和吸入剂。活性成分在无菌条件下与生理上可接受的载体和任何防腐剂、缓冲剂或抛射剂(如果需要)混合。
本公开的化合物可以单独给药或可以与其他药学上可接受的化合物组合给药。
当使用该药物组合物时，本发明化合物的安全有效量适用于需要治疗的哺乳动物(例如人)，其中剂量为给药时药学上认为有效的剂量，体重60kg的人每日给药剂量一般为1-2000mg，优选6-600mg。当然，具体的剂量还应综合考虑给药途径、患者的健康状况等因素来确定，这些都在熟练医师的能力范围内。
用途和治疗方法
如上所述，本发明的化合物是CDK12/13蛋白降解剂，因此该化合物或包含该化合物的组合物可用于治疗、预防和减轻与CDK12/13活性或异常表达相关的疾病。在一些实施方式中，本文公开了本发明化合物在制备用于预防和/或治疗由CDK12/13丝氨酸/苏氨酸蛋白激酶介导的疾病的药物中的用途。在一些实施方式中，本文公开了用于预防和/或治疗由CDK12/13丝氨酸/苏氨酸蛋白激酶介导的疾病的本发明化合物。在一些实施方案中，本文公开了在有需要的受试者中治疗由CDK12/13丝氨酸/苏氨酸蛋白激酶介导的疾病的方法，包括向受试者施用有效量的本发明化合物。在一些实施方式中，CDK12/13丝氨酸/苏氨酸蛋白激酶介导的疾病包括：前列腺癌、乳腺癌、子宫癌、卵巢癌、非小细胞肺癌、小细胞肺癌、尤文肉瘤、肺腺癌、鳞状细胞肺癌、胰腺癌、肝癌、皮肤癌、上皮细胞癌、胃肠道间质瘤、白血病、组织细胞淋巴瘤、鼻咽癌、头颈部肿瘤、结肠癌、直肠癌、神经胶质瘤。
当用于本文所公开的用途和方法时，所公开的化合物和组合物可以与其他已知疗法组合使用。如本文所用，“组合”施用是指在受试者患有病症的过程中向受试者递送两种(或更多)不同的治疗，例如，在受试者被诊断患有病症之后并且在病症被治愈或消除，或者治疗因其他原因停止之前进行两种或更多种治疗。在一些实施方式，当第二种治疗的递送开始时，一种治疗的递送仍在进行，因此在施用方面存在重叠。这有时在本文中称为“同时”或“并列递送”。在其他实施方式中，一种治疗的递送在另 一种治疗的递送开始之前结束。在任一种情况的一些实施方式中，由于组合施用，治疗更有效。例如，第二种治疗更有效，例如，与在没有第一次治疗的情况下进行第二次治疗相比，用较少的第二次治疗可以看到相同的效果，或者第二次治疗可以更大程度地减轻症状，或者对于第一次治疗时观察到类似情况。在一些实施方式中，递送使得症状或与病症相关的其他参数的减少大于在没有另一种治疗的情况下递送的一种治疗所观察到的减少。两种处理的效果可以部分相加、完全相加或大于相加。递送可以使得递送的第一种治疗的效果在递送第二种治疗时仍然是可检测的。
本文公开的化合物或组合物和至少一种另外的治疗剂可以同时施用、在相同或分开的组合物中施用、或依次施用。对于顺序施用，可首先施用本文所述的化合物，随后可施用另外的药剂，或可颠倒施用顺序。
在一些实施方式中，本文所述的化合物与其他治疗方式组合施用，包括手术、放射、移植(例如，干细胞移植、骨髓移植)、化学疗法、免疫疗法、冷冻疗法和/或温热疗法。这样的组合疗法可以允许较低剂量的施用药剂和/或其他药剂，从而避免与各种疗法相关的可能的毒性或并发症。
在一些实施方式中，本文所述的化合物与至少一种另外的治疗剂例如化学治疗剂一起施用。在特定实施方式中，本文所述的化合物与一种或多种另外的化学治疗剂联合施用。化学治疗剂可以是在由美国国家癌症研究所公布的“A到Z癌症药物清单(A to Z List of Cancer Drugs)”中鉴定的化学治疗剂。
本发明的主要优点在于：
1、本发明提供的细胞周期依赖性蛋白激酶12/13(CDK12/13)的降解剂，可以有效地降解CDK12和CDK13蛋白激酶，可以用于制备预防或者治疗由CDK12和/或CDK13蛋白激酶介导的疾病的药物，比如前列腺癌、乳腺癌、子宫癌、卵巢癌、非小细胞肺癌、小细胞肺癌、尤文肉瘤、肺腺癌、肺鳞癌、胰腺癌、肝癌、皮肤癌、上皮细胞癌、胃肠间质瘤、白血病、组织细胞性淋巴癌、鼻咽癌、头颈部肿瘤、结肠癌、直肠癌、胶质瘤等。
2、本发明提供的细胞周期依赖性蛋白激酶12/13(CDK12/13)的降解剂具有较强的降解活性。
3、本发明提供的细胞周期依赖性蛋白激酶12/13(CDK12/13)的降解剂具有较高的蛋白激酶降解选择性。
以下结合具体实施例对本发明作进一步详细的说明。
实施例1：化合物zlc-4-77
步骤1：中间体3-86的制备
三口圆底烧瓶中，向4-(4-溴苯基)哌嗪-1-羧酸叔丁酯(1.02g,3mmol)的无水DMSO(15mL)体系中依次加反式环己烷-1,4-二胺2(1.2g,10.5mmol)、磷酸钾(1.3g,6mmol)、CuI(57mg,0.3mmol)、D-脯氨酸(35mg,0.3mmol)，完毕，氩气置换气三次，升温至100℃，TLC监测反应完成后，移至室温，硅藻土过滤，DCM/MeOH(10:1)体系洗涤三次，滤液减压浓缩，湿法上样，硅胶正相色谱柱纯化，得到目标化合物3-86(灰白色固体,480mg,产率43％)。1H NMR(400MHz,DMSO-d6)δ6.74(d,J＝8.8Hz,2H),6.49(d,J＝8.9Hz,2H),4.88(d,J＝8.2Hz,1H),3.42(t,J＝5.1Hz,4H),3.03(s,1H),2.83(t,J＝5.1Hz,4H),2.76(s,1H),1.95(d,J＝12.8Hz,2H),1.85(d,J＝12.4Hz,2H),1.41(s,9H),1.26(q,J＝10.9Hz,2H),1.11(q,J＝11.6Hz,2H).HRMS(ESI)for C21H34N4O2[M+H]+,calcd:375.2755,found:375.2739.
步骤2：中间体4-39的制备
100mL圆底烧瓶中，向3-86(1.38g,3.7mmol)的DMF(7.5mL)体系中依次加入5-氰基-2-氟吡啶3(451mg,3.7mmol)、Cs2CO3(1.45g,4.44mol),完毕，室温反应过夜，TLC监测反应完成后，过滤，滤液减压浓缩。经硅胶柱层析正相色谱纯化，得到中间体4-39(白色固体,1.4g,80％)。1H NMR(400MHz,DMSO-d6)δ8.37(s,1H),7.63(d,J＝8.5Hz,1H),7.51(d,J＝1Hz,1H),6.75(d,J＝6.9Hz,2H),6.52(d,J＝8.3Hz,3H),4.92(s,1H),3.75(s,1H),3.42(s,4H),3.12(s,1H),2.83(s,4H),1.98(s,4H),1.41(s,9H),1.36–1.28(m,2H),1.25–1.17(m,2H).
步骤3：中间体4-44的制备
25mL圆底烧瓶中，向4-39(1.438g,3mmol)的DMF(3mL)体系中依次加入异氰酸苄酯4(1.2g,9mmol)、DIPEA(1.6mL),完毕，升温至95℃,6h后，TLC监测反应完成，移至室温，减压浓缩，硅胶正相色谱纯化，得到目标中间体4-44(白色固体，1.3g,产率72％)。1H NMR(400MHz,DMSO-d6)δ8.29(s,1H),7.60(d,J＝9.0Hz,1H),7.46(d,J＝6.6Hz,1H),7.28–7.24(m,2H),7.18–7.14(m,3H),7.03–6.98(m,4H),6.46(d,J＝8.7Hz,1H),5.58-5.56(m,1H),4.26(t,J＝11.9Hz,1H),4.14(d,J＝5.4Hz,2H),3.45(s,5H),3.16(s,4H),1.90(d,J＝11.1Hz,2H),1.76(d,J＝11.6Hz,2H),1.42(d,J＝1.6Hz,9H),1.27(m,2H),1.14–1.04(m,2H).
步骤4：中间体4-46的制备
25mL圆底烧瓶中，向4-44(1.83g,3mmol)的CH2Cl2(2mL)体系中滴加CF3COOH(1mL),室温搅拌过夜，反应完成后，减压浓缩，饱和碳酸氢钠-CH2Cl2/MeOH(10:1)体系萃取，无水硫酸钠干燥，经硅胶柱层析纯化，得到目标中间体4-46(白色固体，1.43g,94％)。1H NMR(500MHz,DMSO-d6)δ8.29(s,1H),7.60(d,J＝9.2Hz,1H),7.48(s,1H),7.28–7.25(m,2H),7.18–7.15(m,3H),7.00–6.95(m,4H),6.47(d,J＝8.9Hz,1H),5.56(s,1H),4.26(m,1H),4.14(d,J＝5.9Hz,2H),3.48(s,1H),3.37–3.34(m,1H),3.11–3.09(m,4H),2.85–2.83(m,4H),1.90(d,J＝11.4Hz,2H),1.76(d,J＝10.7Hz,2H),1.33–1.27(m,2H),1.13–1.06(m,2H).
步骤5：中间体4-65的制备
25mL圆底烧瓶中，向4-46(153mg,0.3mmol)的DMF(2mL)体系中依次碳酸钾(83mg,0.6mmol)、1-Boc-4-溴甲基哌啶(167mg,0.6mmol),完毕，升温至80℃，搅拌过夜，反应完成后，反应液减压浓缩，硅胶柱正相色谱纯化，得到目标中间体4-65(白色固体，110mg,52％)。1H NMR(500MHz,DMSO-d6)δ8.29(d,J＝2.1Hz,1H),7.60(d,J＝8.7Hz,1H),7.49(d,J＝6.6Hz,1H),7.28–7.26(m,2H),7.18–7.14(m,3H),7.03–6.96(m,4H),6.46(d,J＝8.8Hz,1H),5.59(t,J＝5.6Hz,1H),4.28–4.23(s,1H),4.14–4.13(d,J＝6.0Hz,2H),3.92(s,2H),3.50(s,1H),3.17(s,4H),2.47(s,4H),2.17(d,J＝6.6Hz,2H),1.90(d,J＝9.7Hz,2H),1.77–1.68(m,5H),1.39(s,9H),1.33–1.26(m,2H),1.13–1.08(m,2H),0.98–0.94(m,2H),0.84–0.81(m,2H).MS(ESI)for C41H55N8O3[M+H]+,calcd:707.4,found:707.5.
步骤6：化合物zlc-4-77的制备
25mL圆底烧瓶中，向4-65(79mg,0.112mmol)的CH2Cl2(1mL)体系中滴加CF3COOH(0.3mL)，室温搅拌过夜，反应完成后，溶剂减压浓缩后，依次加入DMF(2mL)、2-(2,6-二氧代-哌啶-3-基)-5-氟-异吲哚-1,3-二酮(31mg,0.112mmol)、DIPEA(0.023mL,0.135mmol),升温至105℃,监测反应完成后，移至室温，减压浓缩，硅胶正相色谱纯化，得到目标产物zlc-4-77(黄色固体,30mg,32％)。1H NMR(600MHz,DMSO-d6)δ11.08(s,1H),8.29(d,J＝2.1Hz,1H),7.65(d,J＝8.5Hz,1H),7.60(d,J＝8.4Hz,1H),7.47(s,1H),7.31(s,1H),7.28–7.25(m,2H),7.23(d,J＝8.6Hz,1H),7.18–7.15(m,3H),7.01–6.97(m,4H),6.51–6.44(m,1H),5.57(t,J＝5.2Hz,1H),5.06(dd,J＝12.8,5.4Hz,1H),4.26(t,J＝11.8Hz,1H),4.15(d,J＝5.7Hz,2H),4.05(d,J＝12.7Hz,2H),3.48(s,1H),3.19(s,4H),2.97(t,J＝12.0Hz,2H),2.93–2.84(m,1H),2.63–2.53(m,2H),2.52–2.50(m,4H),2.20(d,J＝6.1Hz,2H),2.04–1.99(m,1H),1.90(d,J＝10.3Hz,3H),1.82(d,J＝12.5Hz,2H),1.76(d,J＝10.6Hz,2H),1.35–1.28(m,2H),1.17–1.05(m,4H).13C NMR(151MHz,DMSO)δ172.81,170.12,167.64,166.97,159.25,156.80,155.01,153.10,150.15,141.33,134.05,131.50,128.15,128.02,126.69,126.23,125.01,119.12,117.58,117.35,115.34,107.74,93.99,63.71,53.21,52.91,48.73,47.70,47.27,43.47,32.49,31.31,30.98,30.20,29.64,22.20,14.09.HRMS(ESI)for C49H55N10O5[M+H]+,calcd:863.4351,found:863.4360.
实施例2：化合物zlc-4-79的制备
步骤1：中间体4-66的制备
25mL圆底烧瓶中，向4-46(153mg,0.3mmol)的DMF(2mL)体系中依次加入HATU(137mg,0.36mmol)、DIPEA(0.1mL,0.6mmol)和1-N-Boc-3-吖丁啶羧酸(67mg,0.33mmol)，完毕，室温搅拌反应，监测反应完成后，加水淬灭，析出固体，过滤，滤饼洗涤(CH2Cl2/MeOH＝10:1),无水硫酸钠干燥，硅胶正向色谱纯化，得到目标中间体4-66(白色固体,120mg,59％)。1H NMR(500MHz,DMSO-d6)δ8.29(d,J＝2.1Hz,1H),7.60(d,J＝9.2Hz,1H),7.48(s,1H),7.28–7.25(m,2H),7.18–7.14(m,3H),7.03–6.99(m,4H),6.46(d,J＝8.9Hz,2H),5.61–5.59(m,1H),4.28–4.23(m,1H),4.14(d,J＝5.8Hz,2H),3.72–3.68(m,1H),3.63–3.60(m,4H),3.40(s,2H),3.19–3.17(m,3H),3.16–3.12(m,4H),1.90(d,J＝10.9Hz,2H),1.76(d,J＝12.3Hz,2H),1.37(s,9H),1.32–1.27(m,2H),1.13–1.06(m,2H).MS(ESI)for C39H49N8O4[M+H]+,calcd:693.4,found:692.8.
步骤2：化合物zlc-4-79的制备
合成方法与实施例1步骤f。
1H NMR(600MHz,DMSO-d6)δ11.07(s,1H),8.29(d,J＝2.2Hz,1H),7.66(d,J＝8.3Hz,1H),7.60(d,J＝8.2Hz,1H),7.51–7.44(m,1H),7.28–7.26(m,2H),7.19–7.15(m,3H),7.05–7.01(m,4H),6.84(d,J＝1.8Hz,1H),6.70(dd,J＝8.4,2.0Hz,1H),6.47(d,J＝8.9Hz,1H),5.58(t,J＝5.5Hz,1H),5.06(dd,J＝12.8,5.4Hz,1H),4.28–4.25(m,3H),4.18–4.15(m,4H),4.00–3.97(m,1H),3.65(s,2H),3.49(s,3H),3.22(m,4H),2.91–2.84(m,1H),2.59–2.53(m,2H),2.03–2.00(m,1H),1.91(d,J＝10.3Hz,2H),1.77(d,J＝10.6Hz,2H),1.31(m,2H),1.10(m,2H).13C NMR(151MHz,DMSO)δ172.80,170.10,169.35,167.43,167.15,159.24,156.77,154.98,153.10,149.92,141.30,133.80,131.60,128.76,128.03,126.71,126.25,124.83,119.12,117.24,115.97,114.30,104.56,94.00,53.63,52.92,48.72,48.07,47.75,44.42,43.48,41.30,31.30,31.21,30.98,30.20,22.19,14.09.HRMS(ESI)for C47H49N10O6[M+H]+,calcd:849.3831,found:849.3839.
实施例3：化合物zlc-4-91的制备
步骤1：中间体4-64的制备
将化合物对溴碘苯(5.0g,17.7mmol)、N-BOC-反式-1,4-环己二胺(3.2g,14.7mmol)、Pd2(dba)3(1.37g,1.5mmol)、Xantphos(1.7g,2.94mmol)和tert-ButONa(2.8g,29.4mmol)混合溶于150mL甲苯中，用氩气置换三次后将反应液加热至100℃反应过夜。反应结束后用硅藻土过滤反应液，减压旋转浓缩反应液。经柱层析纯化得目标化合物4-64(黄色固体3.9g,产率72％)。1H NMR(500MHz,DMSO-d6)δ7.16–7.13(m,2H),6.79(d,J＝7.9Hz,1H),6.49(dd,J＝8.9,2.0Hz,2H),5.63(d,J＝8.0Hz,1H),3.21–3.19(m,1H),3.06–3.03(m,1H),1.93(d,J＝12.6Hz,2H),1.77(d,J＝12.5Hz,2H),1.28 –1.21(m,1H),1.16–1.11(m,2H).
步骤2：中间体4-68的制备
将中间体4-64(3.9g,10.6mmol)溶于4mL DMF中，加入苄基异氰酸酯(4.2g,31.7mmol)，DIPEA(1.59g,12.3mmol)，95℃反应5h。旋干溶剂，柱层析得目标化合物4-68(黄色固体3.7g,产率69％)。1H NMR(500MHz,DMSO-d6)δ7.64–7.62(m,2H),7.27–7.25(m,2H),7.19–7.15(m,3H),7.12–7.10(m,2H),6.69(d,J＝7.9Hz,1H),6.01(t,J＝6.1Hz,1H),4.24–4.16(m,1H),4.13(d,J＝6.1Hz,2H),2.94(m,1H),1.72(d,J＝11.7Hz,4H),1.34(s,9H),1.24–1.21(m,2H),1.03–0.96(d,J＝12.1Hz,2H).
步骤3：中间体4-69的制备
将中间体4-68(3.7g,7.3mmol)溶于5mL DCM中，加入2.5mL三氟乙酸(TFA)。加热至55℃回流6h后，减压旋干溶剂，柱层析得目标化合物4-69(黄色固体2.5g,产率70％)。1H NMR(500MHz,DMSO-d6)δ7.65–7.63(m,2H),7.28–7.25(m,2H),7.19–7.11(m,5H),6.04(m,1H),4.23–4.18(m,1H),4.12(d,J＝5.3Hz,2H),2.79(m,1H),1.89(d,J＝12.8Hz,2H),1.79(d,J＝12.2Hz,2H),1.38(m,2H),1.09–1.01(m,2H).
步骤4：中间体4-87的制备
将化合物4-69(2.5g,5.1mmol)溶于15mL DMF中，加入5-氰基-2-氟吡啶(744mg,6.1mol)，Cs2CO3(2.0g,6.1mol)，混合物在室温下搅拌15min后升至60℃反应40min。反应结束后减压旋干溶剂，柱层析纯化得目标化合物4-87(白色固体，1.9g,产率70％)。1H NMR(500MHz,DMSO-d6)δ8.30(d,J＝2.2Hz,1H),7.65–7.60(m,3H),7.48(d,J＝6.5Hz,1H),7.29–7.26(m,2H),7.19–7.13(m,5H),6.47(d,J＝8.9Hz,1H),6.03(t,J＝5.9Hz,1H),4.30–4.23(m,1H),4.14(d,J＝6.0Hz,2H),3.51(s,1H),1.91(d,J＝11.0Hz,1H),1.78(d,J＝11.4Hz,2H),1.31(m,2H),1.10(m,2H).
步骤5：中间体4-88的制备
25mL史莱克瓶中依次加入化合物4-87(1.51g,3mmol)、4-哌嗪-1-基哌啶-1-羧酸叔丁酯(986mg,3.6mmol)、Pd2(dba)3(274mg,0.3mmol)、Xantphos(347mg,0.6mmol)和tert-ButONa(576g,6mmol)，氩气置换三次，加甲苯(6mL)，升温至110至，反应过夜。完成后硅藻土过滤，减压浓缩，柱层析纯化得目标化合物4-88(黄色固体，1.27g,61％)。1H NMR(500MHz,DMSO-d6)δ8.29(d,J＝1.7Hz,1H),7.60(d,J＝8.5Hz,1H),7.48(s,1H),7.28–7.25(m,2H),7.18–7.14(m,3H),7.00–6.95(m,5H),6.46(d,J＝9.0Hz,1H),5.59(s,1H),4.28–4.23(m,1H),4.14(d,J＝5.9Hz,2H),3.95(s,2H),3.16(s,4H),2.73(s,1H),2.61(s,4H),2.40–2.37(m,1H),1.90(d,J＝10.8Hz,2H),1.77(m,4H),1.39(s,9H),1.33–1.23(m,6H),1.13–1.05(m,2H).
步骤6：化合物zlc-4-91的制备
合成方法与实施例1步骤f。
1H NMR(600MHz,DMSO-d6)δ11.11(s,1H),8.29(d,J＝2.2Hz,1H),7.71(d,J＝11.4Hz,1H),7.59(d,J＝8.4Hz,1H),7.45(m,2H),7.28–7.25(m,2H),7.18–7.15(m,3H),7.01–6.96(m,4H),6.46(d,J＝8.9Hz,1H),5.57(m,1H),5.11(dd,J＝12.9,5.4Hz,2H),4.28–4.26(m,1H),4.15(d,J＝5.7Hz,2H),3.67(d,J＝11.5Hz,2H),3.49(s,1H),3.19(s,4H),2.93–2.86(m,3H),2.66(s,4H),2.61–2.58(m,1H),2.54–2.52(d,J＝13.2Hz,1H),2.49–2.44(d,J＝15.7Hz,1H),2.04–2.02(m,1H),1.93–1.90(m,4H),1.76(d,J＝10.4Hz,2H),1.62–1.57(m,2H),1.33–1.27(m,2H),1.13–1.07(d,2H).13C NMR(151MHz,DMSO)δ172.76,169.91,166.69,166.21,159.25,158.09,156.81,156.41,153.09,150.15,145.47,141.34,131.50,128.77,128.14,128.01,126.69,126.22,122.98,119.11,115.29,113.72,112.00,111.83,93.99,59.76,52.92,49.33,49.05,48.77,48.02,43.48,31.31,30.96,30.20,27.88,22.09,14.09.HRMS(ESI)for C48H52N10O5[M+H]+,calcd:867.4101,found:867.4104.
实施例4：化合物zlc-4-93的制备
合成方法参考实施例1。
1H NMR(600MHz,DMSO-d6)δ11.08(s,1H),8.29(d,J＝2.2Hz,1H),7.66(d,J＝8.5Hz,1H),7.60(d,J＝8.2Hz,1H),7.47(s,1H),7.33(s,1H),7.27–7.25(m,3H),7.18–7.15(m,3H),7.00–6.95(m,4H),6.46(d,J＝8.9Hz,1H),5.58–5.56(m,1H),5.08–5.05(dd,J＝12.8,5.5Hz,1H),4.28–4.23(m,1H),4.14(d,J＝5.8Hz,2H),4.09(d,J＝12.5Hz,2H),3.48(s,1H),3.17(s,4H),3.01–2.97(m,2H),2.91–2.85(m,1H),2.63(s,4H),2.60–2.50(m,4H),2.03–2.00(m,1H),1.90(d,J＝10.3Hz,4H),1.76(d,J＝10.5Hz,2H),1.51–1.46(m,2H),1.33–1.27(m,2H),1.12–1.06(m,2H).13C NMR(151MHz,DMSO)δ172.81,170.11,167.61,166.96,159.24,156.80,154.75,153.09,150.14,141.34,134.04,131.49,128.14,128.01,126.69,126.22,125.01,119.12,117.70,115.30,107.82,93.98,59.76,52.90,48.74,48.00,46.61,43.47,31.30,30.98,30.19,27.21,22.19,14.09.HRMS(ESI)for C48H53N10O5[M+H]+,calcd:849.4195,found:849.4199.
实施例5：化合物zlc-5-6的制备
合成方法参考实施例1。
1H NMR(500MHz,DMSO-d6)δ11.07(s,1H),8.29(d,J＝2.2Hz,1H),7.64(d,J＝8.3Hz,1H),7.60(d,J＝10.2Hz,1H),7.47(d,J＝7.0Hz,1H),7.28–7.25(m,2H),7.18–7.15(m,3H),7.02–6.97(m,4H),6.78(d,J＝1.5Hz,1H),6.65(dd,J＝8.4,1.7Hz,1H),6.47(d,J＝8.8Hz,1H),5.58(m,1H),5.07–5.03(m,1H),4.29–4.24(m,1H),4.17–4.14(m,4H),3.73–3.70(m,2H),3.50(s,1H),3.50-3.46(m,1H),3.19(s,4H),3.05(s,1H),2.90–2.84(m,1H),2.66(d,J＝5.4Hz,2H),2.60–2.54(m,6H),2.02–2.00(m,1H),1.91(d,J＝9.7Hz,2H),1.77(d,J＝10.2Hz,2H),1.33–1.26(m,2H),1.13–1.06(m,2H).13C NMR(151MHz,DMSO)δ172.80,170.11,167.49,167.18,159.24,156.80,155.19,153.10,150.12,141.34,133.82,131.50,128.22,128.02,126.69,126.23,124.80,119.12,116.68,115.42,114.06,104.35,93.98,61.76,55.75,52.85,48.70,47.63,43.46,31.30,30.98,30.20,27.00,22.21.HRMS(ESI)for C47H51N10O5[M+H]+,calcd:835.4038,found:835.4043.
实施例6：化合物zlc-5-11的制备
合成方法参考实施例1。
1H NMR(500MHz,DMSO-d6)δ11.09(s,1H),8.31–8.28(m,1H),7.68(d,J＝8.7Hz,1H),7.60(d,J＝8.8Hz,1H),7.49(s,1H),7.34(s,1H),7.28–7.25(m,3H),7.18–7.15(m,3H),6.99(m,4H),6.46(d,J＝8.8Hz,1H),5.59–5.57(m,1H),5.07(dd,J＝12.8,5.4Hz,1H),4.28–4.14(m,1H),4.14(d,J＝5.6Hz,2H),3.79(d,J＝9.4Hz,2H),3.44(m,4H),2.92–2.85(m,1H),2.75–2.70(m,2H),2.65(m,4H),2.60–2.56(m,2H),2.41–2.36(m,1H),2.03–2.00(m,1H),1.91(s,4H),1.76(d,J＝11.0Hz,2H),1.53–1.51(m,2H),1.33–1.25(m,2H),1.13–1.06(m,2H).13C NMR(126MHz,DMSO)δ172.87,170.14,167.60,167.02,159.26,156.85,153.14,150.15,141.36,133.87,131.53,128.06,127.91,126.71,126.27,124.94,119.17,115.82,94.00,59.81,52.91,48.79,48.42,47.47,43.49,31.34,31.01,30.24,22.21,14.13.HRMS(ESI)for C48H53N10O5[M+H]+,calcd:849.4195,found:849.4192.
实施例7：化合物zlc-5-15的制备
合成方法参考实施例1。
1H NMR(500MHz,DMSO-d6)δ11.09(s,1H),8.29(d,J＝2.3Hz,1H),7.66(d,J＝8.2Hz,1H),7.60(d,J＝8.9Hz,1H),7.49(s,1H),7.28–7.25(m,2H),7.18–7.15(m,3H),7.03–6.98(m,4H),6.82(s,1H),6.67(d,J＝8.4Hz,1H),6.46(d,J＝8.9Hz,1H),5.62–5.60(m,1H),5.08–5.04(m,1H),4.28–4.23(m,1H),4.14(d,J＝5.2Hz,4H),3.92(s,2H),3.39(s,1H),3.23(s,4H),2.91–2.84(m,1H),2.64–2.51(m,6H),2.02–2.01(m,1H),1.90(d,J＝11.6Hz,2H),1.77–1.75(m,2H),1.35–1.22(m,3H),1.17–1.05(m,2H).13C NMR(126MHz,DMSO)δ172.87,170.17,167.51,167.20,159.26,156.83,154.94,153.14,150.06,141.37,133.86,131.57,128.05,126.72,126.27,124.91,119.17,116.92,115.45,114.27,104.56,94.00,55.06,54.19,52.91,49.16,48.74,47.40,43.49,31.33,31.01,30.23,22.23,14.13.HRMS(ESI)for C46H49N10O5[M+H]+,calcd:821.3882,found:821.3884.
实施例8：化合物zlc-5-20的制备
合成方法参考实施例1。
1H NMR(500MHz,DMSO-d6)δ11.08(s,1H),8.29(d,J＝1.8Hz,1H),7.65(d,J＝8.4Hz,1H),7.60(d,J＝8.6Hz,1H),7.48(d,J＝5.9Hz,1H),7.28–7.25(m,2H),7.19–7.15(m,3H),7.01–6.98(m,5H),6.86(d,J＝8.6Hz,1H),6.47(d,J＝8.9Hz,1H),5.59(t,J＝5.7Hz,1H),5.06(dd,J＝12.8,5.3Hz,1H),4.29–4.23(m,1H),4.15(d,J＝5.7Hz,2H),3.75–3.72(m,1H),3.61–3.58(m,1H),3.50–3.38(m,2H),3.29–3.22(m,5H),3.05–2.98(m,1H),2.91–2.85(m,1H),2.69–2.61(m,4H),2.61–2.52(m,2H),2.31–2.27(m,1H),2.02–2.00(m,1H),1.91–1.89(m,3H),1.77(d,J＝10.5Hz,2H),1.33–1.27(m,2H),1.13–1.06(m,2H).13C NMR(126MHz,DMSO)δ172.89,170.22,167.75,167.29,159.27,156.85,153.15,151.81,150.13,141.38,134.04,131.56,128.29,126.72,126.27,124.96,119.18,115.80,115.48,115.25,105.63,94.01,63.55,52.92,52.03,51.35,48.71,47.64,47.05,43.50,31.34,31.03,30.24,28.89,22.29.HRMS(ESI)for C47H51N10O5[M+H]+,calcd:835.4038,found:835.4036.
实施例9：化合物zlc-5-31的制备
合成方法参考实施例1。
1H NMR(500MHz,DMSO-d6)δ11.10(s,1H),8.30(d,J＝1.8Hz,1H),7.67(d,J＝8.5Hz,1H),7.60(d,J＝8.5Hz,1H),7.54–7.46(m,1H),7.34(s,1H),7.28–7.24(m,3H),7.19–7.15(m,3H),7.05–7.00(m,4H),6.47(d,J＝8.9Hz,1H),5.60–5.59(m,1H),5.07(dd,J＝12.8,5.3Hz,1H),4.29–4.24(m,1H),4.15(d,J＝5.5Hz,2H),4.08(d,J＝12.6Hz,2H),3.71(s,2H),3.61(s,2H),3.49(s,1H),3.23(s,2H),3.17(s,2H),3.11–3.02(m,3H),2.92–2.85(m,1H),2.60–2.54(m,2H),2.03–2.00(m,1H),1.90(d,J＝10.5Hz,2H),1.78–1.73(m,4H),1.67–1.60(m,2H),1.33–1.27(m,2H),1.13–1.06(m,2H).13C NMR(151MHz,DMSO)δ172.81,172.34,170.10,167.60,166.96,159.24,156.77,154.82,153.09,149.92,141.30,134.05,131.60,128.70,128.02,126.70,126.24,125.03,119.11,117.64,115.88,107.85,93.99,52.92,48.73,48.41,47.84,46.72,44.62,43.48,41.07,36.80,31.30,30.98,30.20,27.42,22.19,14.09.HRMS(ESI)for C49H53N10O6[M+H]+,calcd:877.4144,found:877.4139.
实施例10：化合物zlc-5-103的制备
合成方法参考实施例1。
1H NMR(600MHz,DMSO-d6)δ11.08(s,1H),8.30–8.28(m,1H),7.68(d,J＝8.5Hz,1H),7.60(d,J＝8.6Hz,1H),7.47(s,1H),7.36(s,1H),7.28–7.25(m,3H),7.18–7.15(m,3H),6.97(d,J＝8.6Hz,2H),6.49(d,J＝8.7Hz,2H),6.46(d,J＝9.0Hz,1H),5.51(t,J＝5.7Hz,1H),5.07(dd,J＝12.8,5.4Hz,1H),4.28–4.24(m,1H),4.15(d,J＝5.9Hz,2H),3.98(t,J＝7.1Hz,2H),3.70–3.68(m,2H),3.50–3.42(m,5H),3.38–3.37(m,2H),2.91–2.85(m,1H),2.60–2.52(m,3H),2.02–2.00(m,1H),1.90(d,J＝10.2Hz,2H),1.76(d,J＝10.6Hz,2H),1.33–1.23(m,4H),1.12–1.06(m,2H).13C NMR(151MHz,DMSO)δ173.28,170.55,168.03,167.45,159.72,157.38,155.70,153.58,151.37,141.79,134.31,131.92,128.50,127.19,127.15,126.71,125.37,119.59,118.93,118.37,112.19,108.49,94.46,56.10,54.60,53.37,49.25,49.10,47.17,43.94,31.78,31.45,30.67,22.65.HRMS(ESI)for C46H49N10O5[M+H]+,calcd:821.3882,found:821.3884.
实施例11：化合物zlc-5-104的制备
合成方法参考实施例1。
1H NMR(600MHz,DMSO-d6)δ11.07(s,1H),8.29(d,J＝2.3Hz,1H),7.63(d,J＝8.5Hz,1H),7.60(d,J＝8.3Hz,1H),7.48(s,1H),7.28–7.25(m,3H),7.21–7.15(m,4H),6.97(d,J＝8.5Hz,2H),6.67(d,J＝8.4Hz,2H),6.47(d,J＝8.9Hz,1H),5.53(s,1H),5.05(dd,J＝12.8,5.5Hz,1H),4.29–4.25(m,1H),4.15(d,J＝5.8Hz,2H),4.02(d,J＝12.3Hz,2H),3.41(m,3H),3.10(m,2H),2.96–2.84(m,5H),2.59–2.53(m,4H),2.43(s,1H),2.28–2.23(m,2H),2.01–1.97(m,2H),1.91–1.90(m,2H),1.79–1.75(m,5H),1.31–1.27(m,2H),1.24–1.23(m,2H),1.13–1.09(m,2H).HRMS(ESI)for C51H57N10O5[M+H]+,calcd:889.4508,found:889.4515.
实施例12：化合物zlc-5-105的制备
合成方法参考实施例1。
1H NMR(600MHz,DMSO-d6)δ11.08(s,1H),8.29(d,J＝2.3Hz,1H),7.64(d,J＝8.6Hz,1H),7.60(d,J＝8.0Hz,1H),7.48(s,1H),7.29–7.25(m,3H),7.21(dd,J＝8.7,2.1Hz,1H),7.18–7.14(m,3H),6.95(d,J＝8.6Hz,2H),6.47–6.44(m,3H),5.47(t,J＝5.5Hz,1H),5.06(dd,J＝12.8,5.5Hz,1H),4.27–4.23(m,1H),4.14(d,J＝5.9Hz,2H),4.01(d,J＝12.9Hz,2H),3.90(s,4H),3.30(s,4H),2.95–2.91(m,2H),2.91–2.85(m,1H),2.60–2.52(m,2H),2.27(s,1H),2.02–2.00(m,1H),1.90(d,J＝12.1Hz,2H),1.75(d,J＝11.8Hz,4H),1.30–1.24(m,4H),1.17–1.11(m,2H),1.08–1.06(m,2H).13C NMR(151MHz,DMSO)δ173.29,170.59,168.11,167.43,159.71,157.35,155.40,153.57,151.12,141.74,134.52,131.89,128.51,125.49,119.59,118.02,117.81,112.31,108.16,94.46,70.25,64.88,62.14,53.35,49.20,47.71,43.94,34.71,31.77,31.45,30.65,29.90,22.66.HRMS(ESI)for C50H55N10O5[M+H]+,calcd:875.4351,found:875.4354.
实施例13：化合物zlc-5-106的制备
合成方法参考实施例1。
1H NMR(600MHz,DMSO-d6)δ11.07(s,1H),8.29(d,J＝2.2Hz,1H),7.64(d,J＝8.4Hz,1H),7.60(d,J＝8.7Hz,1H),7.48(s,1H),7.28–7.25(m,2H),7.18–7.15(m,3H),7.02–6.97(m,4H),6.90(d,J＝1.8Hz,1H),6.81(dd,J＝8.6,2.1Hz,1H),6.47(d,J＝8.9Hz,1H),5.57(t,J＝5.6Hz,1H),5.05(dd,J＝12.7,5.5Hz,1H),4.28–4.23(m,1H),4.15(d,J＝5.9Hz,2H),3.59–3.57(m,1H),3.52–3.49(m,2H),3.43–3.37(m,2H),3.21(s,4H),3.17–3.14(m,1H),2.91–2.85(m,1H),2.68–2.63(m,1H),2.59–2.56(m,5H),2.41(d,J＝6.9Hz,2H),2.18–2.14(m,1H),2.02–1.99(m,1H),1.90(d,J＝10.4Hz,2H),1.81–1.76(m,3H),1.31–1.27(m,2H),1.13–1.07(m,2H).13C NMR(151MHz,DMSO)δ173.29,170.63,168.20,167.71,159.72,157.29,153.57,152.35,150.61,141.80,134.49,131.98,128.50,127.16,126.71,125.44,119.59,115.96,115.84,115.73,105.93,94.46,61.37,53.56,53.39,52.68,49.15,48.14,47.69,43.94,35.99,31.78,31.47,30.67,29.74,22.73.HRMS(ESI)for C48H53N10O5[M+H]+,calcd:849.4195,found:849.4200.
实施例14：化合物zlc-6-1的制备
合成方法参考实施例1。
1H NMR(600MHz,DMSO-d6)δ11.06(s,1H),8.29(d,J＝2.2Hz,1H),7.64(d,J＝8.4Hz,1H),7.60(d,J＝8.5Hz,1H),7.48(s,1H),7.28–7.25(m,2H),7.18–7.15(m,3H),7.02–6.97(m,4H),6.91(d,J＝1.7Hz,1H),6.82(dd,J＝8.6,2.0Hz,1H),6.47(d,J＝8.9Hz,1H),5.58–5.56(m,1H),5.05(dd,J＝12.7,5.5Hz,1H),4.28–4.24(m,1H),4.15(d,J＝5.8Hz,2H),3.60–3.57(m,1H),3.53–3.49(m,2H),3.43–3.39(m,2H),3.21(s,4H),3.17–3.15(m,1H),2.91–2.85(m,1H),2.67–2.64(m,1H),2.59–2.52(m,5H),2.42(d,J＝7.0Hz,2H),2.18–2.15(m,1H),2.02–1.96(m,1H),1.90(d,J＝10.3Hz,2H),1.81–1.76(m,3H),1.31–1.29(m,2H),1.13–1.07(m,2H).13C NMR(151MHz,DMSO)δ173.29,170.63,168.20,167.71,159.72,157.29,153.58,152.35,150.61,141.80,134.49,131.98,128.50,127.16,126.71,125.45,119.59,115.96,115.84,115.73,105.93,94.46,61.37,53.57,53.39,52.68,49.15,48.14,47.69,43.94,35.99,31.78,31.46,30.67,29.74,22.56.HRMS(ESI)for C48H53N10O5[M+H]+,calcd:849.4195,found:849.4191.
实施例15：化合物zlc-6-35的制备
合成方法参考实施例1。
1H NMR(600MHz,DMSO-d6)δ11.08(s,1H),8.29(d,J＝2.2Hz,1H),7.68(d,J＝8.5Hz,1H),7.60(d,J＝8.9Hz,1H),7.47(s,1H),7.36–7.34(m,1H),7.28–7.25(m,3H),7.18–7.14(m,3H),6.95(d,J＝8.5Hz,2H),6.47–6.45(m,3H),5.48–5.47(m,1H),5.07(dd,J＝12.8,5.4Hz,1H),4.27–4.23(m,1H),4.14(d,J＝5.9Hz,2H),3.97(m,2H),3.54–3.50(m,2H),3.45(s,4H),3.00–2.95(m,1H),2.64(d,J＝7.3Hz,2H),2.61–2.57(m,2H),2.54–2.52(m,5H),2.03–1.99(m,1H),1.90(d,J＝10.4Hz,2H),1.75(d,J＝11.6Hz,2H),1.31–1.27(m,2H),1.11–1.05(m,2H).13C NMR(151MHz,DMSO)δ172.82,170.09,167.57,166.99,159.25,156.92,155.24,153.11,150.90,141.29,133.87,131.44,128.05,126.70,126.49,124.91,119.13,118.35,117.85,111.48,107.93,94.00,56.11,52.87,52.34,48.78,46.88,43.48,31.30,30.98,30.20,29.03,27.22.HRMS(ESI)for C47H51N10O5[M+H]+,calcd:835.4038,found:835.4045.
实施例16：化合物zlc-6-37的制备
合成方法参考实施例1。
1H NMR(600MHz,DMSO-d6)δ11.07(s,1H),8.29(d,J＝2.2Hz,1H),7.64(d,J＝8.3Hz,1H),7.60(d,J＝7.8Hz,1H),7.47(s,1H),7.28–7.25(m,2H),7.18–7.14(m,3H),6.99–6.94(m,4H),6.79(s,1H),6.65–6.63(dd,J＝8.4,1.9Hz,1H),6.47–6.46(m,1H),5.54(t,J＝5.7Hz,1H),5.05(dd,J＝12.8,5.4Hz,1H),4.28–4.22(m,1H),4.14(d,J＝5.9Hz,2H),4.10(s,4H),3.71(d,J＝12.1Hz,2H),3.36(m,2H),2.90–2.84(m,1H),2.67–2.63(m,2H),2.59–2.56(m,1H),2.56–2.52(m,2H),2.48–2.46(m,2H),2.02–1.98(m,1H),1.90(d,J＝10.3Hz,2H),1.76(d,J＝11.9Hz,4H),1.45–1.38(m,1H),1.33–1.26(m,2H),1.24–1.17(m,4H),1.13–1.06(m,2H).13C NMR(151MHz,DMSO)δ173.28,170.57,167.96,167.64,159.72,157.30,155.34,153.57,151.04,141.79,134.26,131.92,128.50,128.23,127.15,126.71,125.28,119.59,117.46,116.27,114.92,105.17,94.46,64.77,61.69,53.38,49.19,48.61,43.94,34.69,31.77,31.45,30.67,22.67.HRMS(ESI)for C50H55N10O5[M+H]+,calcd:875.4351,found:875.4349.
实施例17：化合物zlc-6-38的制备
合成方法参考实施例1。
1H NMR(600MHz,DMSO-d6)δ11.14(s,1H),8.29(d,J＝2.2Hz,1H),7.92(d,J＝2.9Hz,3H),7.60(d,J＝8.9Hz,1H),7.47(s,1H),7.28–7.25(m,2H),7.18–7.15(m,3H),7.02–6.99(s,4H),6.46(d,J＝8.9Hz,1H),5.57(t,J＝5.8Hz,1H),5.16(dd,J＝12.9,5.4Hz,1H),4.28–4.23(m,1H),4.14(d,J＝6.0Hz,2H),3.68(s,2H),3.39–3.36(m,1H),3.27–3.22(m,4H),2.92–2.86(m,1H),2.72–2.69(m,4H),2.62–2.59(m,1H),2.56–2.53(m,1H),2.08–2.04(m,1H),1.93–1.87(m,2H),1.76(d,J＝11.4Hz,2H),1.32–1.26(m,2H),1.12–1.06(m,2H).13C NMR(151MHz,DMSO)δ172.75,169.76,166.50,166.42,156.80,153.11,150.14,141.33,137.58,131.83,131.52,130.28,128.77,128.32,128.03,126.70,126.24,125.74,123.83,119.13,115.55,93.99,90.49,83.72,52.91,51.46,49.12,47.62,46.75,43.47,31.31,30.92,30.20,21.93.HRMS(ESI)for C46H46N9O5[M+H]+,calcd:804.3616,found:804.3623.
实施例18：化合物zlc-6-42的制备
合成方法参考实施例1。
1H NMR(600MHz,DMSO-d6)δ11.08(s,1H),8.29(d,J＝2.4,1H),7.68(d,J＝8.5Hz,1H),7.60(d,J＝9.0Hz,1H),7.47(s,1H),7.34(d,J＝2.2Hz,1H),7.28–7.25(m,3H),7.18–7.15(m,3H),6.99–6.96(m,4H),6.47(d,J＝8.9Hz,1H),5.55(t,J＝6.0Hz,1H),5.07(dd,J＝12.8,5.4Hz,1H),4.28–4.23(m,1H),4.15(d,J＝6.1Hz,2H),3.74(d,J＝11.8Hz,2H),3.44(s,4H),3.42–3.36(m,4H),2.90–2.85(m,1H),2.72–2.68(m,2H),2.61–2.54(m,2H),2.23(d,J＝7.2Hz,2H),2.03–1.99(m,1H),1.90(d,J＝11.6Hz,2H),1.84(d,J＝12.1Hz,2H),1.77–1.72(m,3H),1.31–1.28(m,2H),1.25–1.20(m,4H),1.13–1.07(m,2H).13C NMR(151MHz,DMSO)δ172.83,170.10,167.58,167.00,159.26,156.85,155.27,153.11,150.62,141.33,133.87,131.46,128.04,127.74,126.70,126.25,124.92,119.14,118.32,117.78,115.80,107.89,94.00,63.74,52.91,52.76,48.78,48.12,46.96,43.48,32.44,31.32,30.99,30.32,30.21,22.19.HRMS(ESI)for C49H55N10O5[M+H]+,calcd:863.4351,found:863.4357.
实施例19：化合物zlc-6-101的制备
合成方法参考实施例1。
1H NMR(600MHz,DMSO-d6)δ11.07(s,1H),8.29(d,J＝2.1Hz,1H),7.66(d,J＝8.5Hz,1H),7.60(d,J＝8.7Hz,1H),7.48(s,1H),7.32(s,1H),7.28–7.23(m,3H),7.18–7.15(m,3H),6.99(s,4H),6.50–6.43(m,1H),5.54–5.52(m,1H),5.06(dd,J＝12.8,5.5Hz,1H),4.28–4.24(m,1H),4.15(d,J＝5.8Hz,2H),3.51(s,6H),3.22(s,4H),2.91–2.85(m,1H),2.60–2.57(m,2H),2.03–1.99(m,1H),1.91(d,J＝10.6Hz,2H),1.77(d,J＝10.4Hz,2H),1.63–1.59(m,8H),1.33–1.28(m,2H),1.14–1.07(m,2H).13C NMR(151MHz,DMSO)δ172.83,170.14,167.67,167.00,159.25,156.84,154.97,153.11,141.31,134.04,131.46,128.05,127.66,126.70,126.26,125.00,119.13,117.35,115.47,107.50,94.00,69.79,52.90,48.74,43.48,42.89,34.82,34.12,31.31,30.99,30.21,29.14,22.21.HRMS(ESI)for C48H52N9O5[M+H]+,calcd:834.4086,found:834.4073.
实施例20：化合物zlc-6-102的制备
合成方法参考实施例1。
1H NMR(600MHz,DMSO-d6)δ11.06(s,1H),8.30(d,J＝2.2Hz,1H),7.65(d,J＝8.4Hz,1H),7.60(d,J＝8.2Hz,1H),7.48–7.45(m,1H),7.28–7.26(m,2H),7.19–7.15(m,3H),7.02–6.99(m,4H),6.96(s,1H),6.83(dd,J＝8.6,1.6Hz,1H),6.47(d,J＝8.9Hz,1H),5.52(t,J＝5.6Hz,1H),5.07–5.04(m,1H),4.29–4.24(m,1H),4.15(d,J＝5.9Hz,2H),3.53–3.50(m,2H),3.37(m,4H),3.24–3.21(m,1H),2.91–2.85(m,1H),2.61–2.52(m,4H),2.02–1.98(m,1H),1.97–1.95(m,2H),1.91(d,J＝10.7Hz,2H),1.77(d,J＝10.3Hz,2H),1.73–1.66(m,4H),1.33–1.27(m,2H),1.13–1.07(m,2H).13C NMR(151MHz,DMSO)δ172.83,170.16,167.74,167.26,159.25,156.82,153.12,152.06,150.42,141.28,134.02,131.48,128.05,127.83,126.70,126.27,124.94,119.13,115.80,115.54,115.22,105.58,94.00,57.28,52.90,48.69,46.32,45.44,43.49,35.02,34.06,31.31,31.00,30.22,22.26.HRMS(ESI)for C47H50N9O5[M+H]+,calcd:820.3929,found:820.3933.
实施例21：化合物zlc-6-103的制备
合成方法参考实施例1。
1H NMR(600MHz,DMSO-d6)δ11.07(s,1H),8.30(d,J＝2.2Hz,1H),7.65(d,J＝8.3Hz,1H),7.60(d,J＝8.1Hz,1H),7.48(s,1H),7.28–7.26(m,2H),7.19–7.15(m,3H),7.06–7.03–6.99(m,4H),6.80(d,J＝1.8Hz,1H),6.67(dd,J＝8.4,2.0Hz,1H),6.47(d,J＝8.8Hz,1H),5.56(t,J＝5.6Hz,1H),5.05(dd,J＝12.8,5.5Hz,1H),4.29–4.23(m,1H),4.15(d,J＝5.8Hz,1H),3.83(m,4H),3.43–3.41(m,2H),3.22(m,3H),2.91–2.85(m,1H),2.59–2.53(m,2H),2.02–1.89(m,1H),1.91–1.89(m,6H),1.77(d,J＝10.1Hz,2H),1.33–1.27(m,2H),1.13–1.07(m,2H).13C NMR(151MHz,DMSO)δ172.83,170.12,167.53,167.21,159.26,156.83,155.20,153.12,150.21,141.33,133.86,131.53,128.04,126.71,126.26,124.87,122.51,119.14,116.69,115.95,114.18,104.42,94.00,60.78,52.92,48.73,45.32,43.49,34.65,34.21,31.32,30.99,30.21,22.23,20.45.HRMS(ESI)for C46H48N9O5[M+H]+,calcd:806.3773,found:806.3767.
实施例22：化合物zlc-7-36的制备
合成方法参考实施例1。
1H NMR(600MHz,DMSO-d6)δ11.08(s,1H),8.29(d,J＝2.2Hz,1H),7.60–7.58(m,2H),7.51–7.44(m,1H),7.29–7.25(m,2H),7.18–7.15(m,3H),7.01–6.97(m,4H),6.91(d,J＝7.6Hz,1H),6.46(d,J＝8.9Hz,1H),5.57(t,J＝5.6Hz,1H),5.06(dd,J＝12.9,5.4Hz,1H),4.30–4.24(m,3H),4.15(d,J＝5.8Hz,2H),3.87–3.85(m,2H),3.53–3.44(m,1H),3.19(s,4H),3.04–3000(m,1H),2.90–2.84(m,1H),2.67(d,J＝6.8Hz,2H),2.61–2.51(m,6H),2.03–1.99(m,1H),1.90(d,J＝10.5Hz,2H),1.76(d,J＝10.6Hz,2H),1.31–1.27(m,2H),1.12–1.07(m,2H).13C NMR(151MHz,DMSO)δ172.80,170.02,166.84,166.43,159.26,156.83,154.43,153.11,152.79,144.21,144.13,141.34,131.52,129.38,128.23,128.04,126.70,119.14,118.30,115.43,111.33,111.19,107.95,94.00,61.57,57.75,52.93,52.83,48.94,47.64,43.48,31.32,30.97,30.21,27.85,22.17,22.08,13.99.HRMS(ESI)for C47H50FN10O5[M+H]+,calcd:853.3944,found:853.3956.
实施例23：3-苄基-1-(4-(2-((2-(2,6-二氧代哌啶-3-基)-1,3-二氧代异吲哚-4-基)氧基)乙酰基)哌嗪-1-基)苯基)-1-((1r，4r)-4-(喹唑啉-2-基氨基)环己基)脲(YJZ9069)
步骤1：4-(4-(((1r,4r)-4-氨基环己基)氨基)苯基)哌嗪-1-羧酸叔丁酯(化合物3)的制备
将磷酸钾(31g,146mmol)、反式环己烷-1,4-二胺2(29.3g,256.4mmol)、4-(4-溴苯基)哌嗪-1-羧酸叔丁酯1(25g,73.26mmol)、CuI(1.39g 7.3mmol)和D-脯氨酸(843mg,7.3mmol)溶于无水DMSO(500mL)中。然后将所得悬浮氩气保护，置换三次。然后将反应混合物在100℃加热搅拌10小时后，使用硅藻土过滤，用乙酸乙酯洗涤滤渣2-3次。减压蒸发滤液后并通过硅胶柱纯化，得到目标化合物12g，为灰白色 固体(产率44％)。1H NMR(400MHz,DMSO-d6)δ6.74(d,J＝8.8Hz,2H),6.49(d,J＝8.9Hz,2H),4.88(d,J＝8.2Hz,1H),3.42(t,J＝5.1Hz,4H),3.03(s,1H),2.83(t,J＝5.1Hz,4H),2.76(s,1H),1.95(d,J＝12.8Hz,2H),1.85(d,J＝12.4Hz,2H),1.41(s,9H),1.26(q,J＝10.9Hz,2H),1.11(q,J＝11.6Hz,2H).HRMS(ESI)for C21H34N4O2[M+H]+,calcd:375.2755,found:375.2739.
步骤2：4-(4-(((1r,4r)-4-(喹唑啉-2-基氨基)环己基)氨基)苯基)哌嗪-1-羧酸叔丁酯(化合物5)的制备
将4-(4-(((1r,4r)-4-氨基环己基)氨基)苯基)哌嗪-1-羧酸叔丁酯3(9g,24mmol)溶于DMF(40mL)中的溶液中，加入2-氯喹唑啉4(4g,24mol)，Cs2CO3(9.4g,28.9mol)，混合物在室温下搅拌15min后升至60℃反应40min。然后过滤反应液，并减压旋蒸浓缩滤液。经柱层析纯化，得到黄白色固体11.2g(产率93％)。1H NMR(400MHz,DMSO-d6)δ9.09(s,1H),7.77(d,J＝8.0Hz,1H),7.67(t,J＝7.7Hz,1H),7.45(d,J＝8.5Hz,1H),7.29(d,J＝8.0Hz,1H),7.20(t,J＝7.4Hz,1H),6.76(d,J＝8.2Hz,2H),6.52(d,J＝8.3Hz,2H),4.95(s,1H),3.86(d,J＝9.8Hz,1H),3.43(t,J＝5.0Hz,4H),3.18–3.01(m,1H),2.84(s,4H),2.01(d,J＝11.6Hz,4H),1.50–1.32(m,11H),1.30–1.15(m,2H).
步骤3：4-(4-(3-苄基-1-((1r，4r)-4-(喹唑啉-2-基氨基)环己基)脲基)苯基)哌嗪-1-羧酸叔丁酯(化合物7)的制备
将4-(4-(((1r,4r)-4-(喹唑啉-2-基氨基)环己基)氨基)苯基)哌嗪-1-羧酸叔丁酯5(11.2g,22.25mmol)，DIPEA(8.6g,66.76mmol)，异氰酸苄酯6(8.9g,66.76mmol)溶于15mL DMF中。混合物在95℃搅拌4小时。减压除去溶剂，柱层析纯化得白色固体10.2g(产率72％)。1H NMR(400MHz,DMSO-d6)δ9.05(s,1H),7.74(d,J＝8.0Hz,1H),7.63(t,J＝7.8Hz,1H),7.40(d,J＝8.8Hz,1H),7.31–7.22(m,3H),7.21–7.13(m,4H),7.08–6.98(m,4H),5.58(t,J＝6.1Hz,1H),4.28(t,J＝12.3Hz,1H),4.16(d,J＝5.8Hz,2H),3.58(s,1H),3.47(t,J＝5.1Hz,4H),3.18(t,J＝5.2Hz,4H),1.96(d,J＝12.0Hz,2H),1.79(d,J＝12.3Hz,2H),1.50–1.33(m,12H),1.13(q,J＝13.5,12.5Hz,2H).
步骤4：3-苄基-1-(4-(哌嗪-1-基)苯基)-1-(1r，4r)-4-(喹唑啉-2-基氨基)环己基)脲(化合物8)的制备
4-(4-(3-苄基-1-((1r，4r)-4-(喹唑啉-2-基氨基)环己基)脲基)苯基)哌嗪-1-羧酸叔丁酯7(7.3g,11.5mmol)溶于DCM(20mL)中，加入TFA(10mL)，并在50℃下搅拌回流过夜。然后在减压下将反应混合物浓缩，并通过柱层析纯化所得透明油状物体4.3g(产率70％)。1H NMR(400MHz,DMSO-d6)δ9.05(s,1H),7.74(dd,J＝8.0,1.5Hz,1H),7.63(t,J＝8.5Hz,1H),7.41(d,J＝8.5Hz,1H),7.31–7.21(m,3H),7.21–7.13(m,4H),7.00(q,J＝9.1Hz,4H),5.56(t,J＝6.1Hz,1H),4.28(tt,J＝12.1,3.7Hz,1H),4.16(d,J＝6.0Hz,2H),3.65–3.53(m,1H),3.11(dd,J＝6.3,3.7Hz,4H),2.84(t,J＝5.0Hz,4H),1.96(d,J＝10.9Hz,2H),1.79(d,J＝10.8Hz,2H),1.41(q,J＝13.1Hz,2H),1.13(q,J＝13.0Hz,2H).
步骤5：3-苄基-1-(4-(2-((2-(2,6-二氧代哌啶-3-基)-1,3-二氧代异吲哚-4-基)氧基)乙酰基)哌嗪-1-基)苯基)-1-((1r，4r)-4-(喹唑啉-2-基氨基)环己基)(化合物YJZ9069)的制备
将2-(2,6-二氧代哌啶-3-基)-1,3-二氧异吲哚啉-5-羧酸9(39.9mg,0.12)、HATU(45.1mg,0.12mmol)、DIPEA(21.3mg,0.16mmol)和化合物8(59mg,0.11mmol)溶于DMF(6mL)中。混合物在室温下搅拌15分钟后，将反应液减压旋转蒸发，通过硅胶柱色谱纯化，得到目标化合物，为白色固体70mg(产率75％)。
1H NMR(400MHz,DMSO-d6)δ11.12(s,1H),9.05(s,1H),7.79(t,J＝7.9Hz,1H),7.74(d,J＝8.0Hz,1H),7.63(t,J＝7.8Hz,1H),7.46(d,J＝7.2Hz,1H),7.39(t,J＝8.1Hz,2H),7.27(q,J＝9.9,8.7Hz,3H),7.22–7.13(m,4H),7.11–7.02(m,4H),5.60(t,J＝6.0Hz,1H),5.26(s,2H),5.12(dd,J＝12.8,5.4Hz,1H),4.27(d,J＝12.4Hz,1H),4.17(d,J＝6.0Hz,2H),3.62(s,4H),3.31(s,1H),3.24(s,2H),2.96–2.82(m,1H),2.65–2.53(m,2H),2.10–2.00(m,1H),1.97(d,J＝10.8Hz,2H),1.80(d,J＝11.8Hz,2H),1.41(q,J＝12.5Hz,2H),1.13(q,J＝12.3Hz,2H).13C NMR(151MHz,DMSO-d6)δ173.31,170.43,167.29,165.76,165.61,162.50,157.33,156.06,150.34,141.73,137.09,134.48,133.55,132.13(2C),129.17,128.52(3C),128.32,127.17(4C),126.75,122.21,120.67,119.95,116.60,116.40(2C),116.00,66.57,53.54,49.23,49.13,48.29,48.16,44.33,43.94,41.69,40.043,31.75,31.40,30.87(2C),22.46.HRMS(ESI)for C47H47N9O7[M+H]+,calcd:850.36712,found:850.3646.HPLC analysis:MeOH-H2O(80:20),12.08min,97.4％purity.
实施例24：3-苄基-1-(4-(2-((2-(2,6-二氧代哌啶-3-基)-1,3-二氧代异吲哚-5-基)氧基)乙酰基)哌嗪-1-基)苯基)-1-((1r，4r)-4-(喹唑啉-2-基氨基)环己基)脲(化合物YJZ1090)
合成方法与实施例23所示方法相似。
1H NMR(400MHz,DMSO-d6)δ11.11(s,1H),9.05(s,1H),7.85(d,J＝8.3Hz,1H),7.74(d,J＝8.3Hz,1H),7.63(t,J＝7.9Hz,1H),7.48(d,J＝2.3Hz,1H),7.43–7.35(m,2H),7.32–7.24(m,3H),7.23(d,J＝8.2Hz,1H),7.21–7.13(m,4H),7.11–7.00(m,4H),5.60(t,J＝5.8Hz,1H),5.21(s,2H),5.12(dd,J＝12.9,5.4Hz,1H),4.27(t,J＝12.3Hz,1H),4.17(d,J＝6.0Hz,2H),3.70–3.52(m,5H),3.31(s,2H),3.24(s,2H),2.95–2.83(m,1H),2.65–2.54(m,2H),2.10–2.01(m,1H),1.97(d,J＝11.4Hz,2H),1.80(d,J＝11.9Hz,2H),1.41(q,J＝12.4Hz,2H),1.14(q,J＝12.0Hz,2H).13C NMR(151MHz,DMSO-d6)δ173.30,170.42,167.42,167.27,165.69,164.15,162.50,157.32,150.32,141.74,134.47,134.17,132.13(2C),129.19,128.52(3C),128.32,127.17(4C),126.74,125.65,123.70,122.21,121.69,119.95,116.39(2C),109.58,66.60,53.54,49.45,49.13,48.34,48.16,44.28,43.94,41.64,40.44,31.75,31.41,30.87(2C),22.52.HRMS(ESI)forC47H47N9O7[M+H]+,calcd:850.36712,found:850.3633.HPLC analysis:MeOH-H2O(75:25),6.96min,95.0％purity.
实施例25：3-苄基-1-(4-(2-((2-(2,6-二氧哌啶-3-基)-3-氧代异吲哚-5-基)氧基)乙酰基)哌嗪-1-基)苯基)-1-((1r，4r)-4-(喹唑啉-2-基氨基)环己基)脲(化合物YJZ1094)
合成方法与实施例23所示方法相似。
1H NMR(400MHz,DMSO-d6)δ10.98(s,1H),9.05(s,1H),7.74(d,J＝7.9Hz,1H),7.63(t,J＝7.7Hz,1H),7.51(d,J＝8.2Hz,1H),7.40(d,J＝8.5Hz,1H),7.31–7.21(m,5H),7.21–7.13(m,4H),7.08–7.00(m,4H),5.60(t,J＝5.9Hz,1H),5.11(dd,J＝13.2,5.1Hz,1H),5.03(s,2H),4.43–4.21(m,3H),4.16(d,J＝6.0Hz,2H),3.71–3.53(m,5H),3.30(s,2H),3.23(s,2H),2.96–2.84(m,1H),2.68–2.53(m,2H),2.04–1.90(s,3H),1.80(d,J＝12.1Hz,2H),1.41(q,J＝12.3Hz,2H),1.20–1.06(m,2H).13C NMR(151MHz,DMSO-d6)δ173.41,171.47,168.57,166.31,162.50,158.74,157.33,150.34,141.75,134.97,134.48,133.27,132.11(2C),129.16,128.52(3C),128.32,127.16(4C),126.74,124.92,122.21,120.65,119.95,116.38(2C),107.67,66.52,53.54,52.21(2C),48.45,48.18,47.28,44.44,43.93,41.63,40.43,31.74,31.66,30.86(2C),22.92.HRMS(ESI)forC47H49N9O6[M+H]+,calcd:836.38786,found:836.3843.HPLC analysis:MeOH-H2O(75:25),6.33min,95.6％purity.
实施例26：3-苄基-1-(4-(4-((2-(2,6-二氧代哌啶-3-基)-1,3-二氧代异吲哚-5-基)甘氨酸)哌嗪-1-基)苯基)-1-((1r，4r)-4-(喹唑啉-2-基氨基)环己基)脲(化合物YJZ1091)
合成方法与实施例23所示方法相似。
1H NMR(400MHz,DMSO-d6)δ11.06(s,1H),9.05(s,1H),7.74(d,J＝8.0Hz,1H),7.63(t,J＝7.5Hz,1H),7.59(d,J＝8.3Hz,1H),7.40(d,J＝8.5Hz,1H),7.31–7.25(dd,J＝8.5,6.5Hz,2H),7.23(d,J＝8.2Hz,1H),7.21–7.11(m,6H),7.09–9.00(m,5H),5.60(t,J＝5.6Hz,1H),5.05(dd,J＝12.9,5.5Hz,1H),4.27(t,J＝12.3Hz,1H),4.22(d,J＝5.1Hz,2H),4.16(d,J＝6.2Hz,2H),3.68(s,4H),3.58(s,1H),3.30(s,2H),3.24(s,2H),2.93–2.81(m,1H),2.62–2.53(m,2H),2.45–1.89(m,3H),1.80(d,J＝12.1Hz,2H),1.41(q,J＝12.2Hz,2H),1.14(d,J＝13.3Hz,2H).13C NMR(151MHz,DMSO-d6)δ173.34,170.66,168.23,167.70,167.35,162.50,159.29,157.33,154.52,152.04,150.36,141.74,134.47,132.12(2C),129.17,128.52(4C),128.32,127.17(4C),126.74,125.28,122.21,119.95,117.14,116.39(2C),53.54,49.11(2C),48.40,48.18,44.73,44.30,43.94,41.83,40.43,31.75,31.44,30.87(2C),22.69.HRMS(ESI)for C47H48N10O6[M+H]+,calcd:849.38311,found:849.3803.HPLC analysis:MeOH-H2O(75:25),7.47min,96.8％purity.
实施例27：3-苄基-1-(4-(4-((2-(2,6-二氧哌啶-3-基)-3-氧代异吲哚-5-基)甘油基)哌嗪-1-基)苯基)-1-((1r，4r)-4-(喹唑啉-2-基氨基)环己基)脲(化合物YJZ1095)
合成方法与实施例23所示方法相似。
1H NMR(400MHz,DMSO-d6)δ10.95(s,1H),9.05(s,1H),7.74(d,J＝7.3Hz,1H),7.63(t,J＝7.8Hz,1H),7.40(d,J＝8.5Hz,1H),7.28(t,J＝7.6Hz,3H),7.23(d,J＝8.0Hz,1H),7.18(t,J＝7.1Hz,4H),7.10–6.99(m,5H),6.94(d,J＝2.2Hz,1H),5.97(t,J＝5.1Hz,1H),5.60(t,J＝6.0Hz,1H),5.08(dd,J＝13.3,5.1Hz,1H),4.35–4.23(d,J＝16.5Hz,2H),4.19–4.12(m,3H),4.07(d,J＝5.1Hz,2H),3.69(d,J＝17.3Hz,4H),3.59(s,1H),3.31(s,2H),3.23(s,2H),2.96–2.84(m,1H),2.69–2.55(m,2H),2.43–2.30(m,1H),1.97(d,J＝11.7Hz,2H),1.80(d,J＝11.7Hz,2H),1.41(q,J＝12.6,12.2Hz,2H),1.14(q,J＝13.5,11.1Hz,2H).13C NMR(151MHz,DMSO-d6)δ173.41,171.47,168.57,166.31,162.50,158.74,157.33,150.34,141.75,134.97,134.48,133.27,132.11(2C),129.16,128.52(3C),128.32,127.16(4C),126.74,124.92,122.21,120.65,119.95,116.38(2C),107.67,66.52,53.54,52.21,49.11,48.45,48.18,47.28,44.44,43.93,41.63,40.43,31.75,31.66,30.86(2C),22.92.HRMS(ESI)for C47H50N10O5[M+H]+,calcd:835.40384,found:835.4012.HPLC analysis:MeOH-H2O(75:25),6.70min,95.8％purity.
实施例28：3-苄基-1-(4-(4-(1-(2-(2,6-二氧哌啶-3-基)-1,3-二氧异吲哚-5-基)氮杂环丁烷-3-羰基)哌嗪-1-基)苯基)-1-((1r，4r)-4-(喹唑啉-2-基氨基)环己基)脲(化合物YJZ1093)
合成方法与实施例23所示方法相似。
1H NMR(400MHz,DMSO-d6)δ11.07(s,1H),9.05(s,1H),7.74(d,J＝7.9Hz,1H),7.66(d,J＝8.3Hz,1H),7.62(t,J＝8.0Hz,1H),7.40(d,J＝8.5Hz,1H),7.31–7.25(m,2H),7.24(d,J＝8.2Hz,1H),7.21–7.13(m,4H),7.10–7.00(m,4H),6.85(d,J＝2.1Hz,1H),6.71(dd,J＝8.4,2.2Hz,1H),5.58(t,J＝6.2Hz,1H),5.06(dd,J＝12.9,5.5Hz,1H),4.34–4.22(m,3H),4.22–4.08(m,4H),4.05–3.94(m,1H),3.66(s,2H),3.58(s,1H),3.51(s,2H),3.29–3.16(m,4H),2.94–2.82(m,1H),2.70–2.52(m,2H),2.06–1.90(m,3H),1.80(d,J＝11.9Hz,2H),1.41(q,J＝12.3Hz,2H),1.19–1.06(m,2H).13C NMR(151MHz,DMSO-d6)δ173.34,170.59,169.88,167.92,167.65,162.50,157.33,155.46,150.39,141.70,134.49,134.25,132.11(2C),129.20,128.53(3C),128.32,127.17(4C),126.76,125.33,122.22,119.95,117.67,116.46(2C),114.80,105.02,54.09(2C),53.53,49.18(2C),48.53,48.20,44.89,43.93,41.79,40.40,31.74,31.68,31.42,30.87(2C),22.65.HRMS(ESI)for C49H50N10O6[M+H]+,calcd:875.39876,found:875.3958.HPLC analysis:MeOH-H2O(75:25),8.69min,97.7％purity.
实施例29：3-苄基-1-(4-(4-((E)-3-(2-(2,6-二氧代哌啶-3-基)-1,3-二氧代异吲哚-5-基)丙烯酰基)哌嗪-1-基)苯基)-1-((1r，4r)-4-(喹唑啉-2-基氨基)环己基)脲(化合物YJZ1114)
合成方法与实施例23所示方法相似。
1H NMR(400MHz,DMSO-d6)δ11.15(s,1H),9.05(s,1H),8.48(s,1H),8.19(d,J＝7.8Hz,1H),7.96(d,J＝7.7Hz,1H),7.74(d,J＝7.9Hz,1H),7.71(s,2H),7.63(t,J＝7.8Hz,1H),7.39(d,J＝8.6Hz,1H),7.31–7.21(m,3H),7.21–7.13(m,4H),7.07(s,4H),5.59(t,J＝6.4Hz,1H),5.19(dd,J＝13.0,5.4Hz,1H),4.27(t,J＝11.9Hz,1H),4.17(d,J＝5.8Hz,2H),3.95(s,2H),3.76(s,2H),3.58(s,1H),2.97–2.83(m,1H),2.69–2.56(m,2H),2.13–2.04(m,2H),1.96(d,J＝11.9Hz,2H),1.80(d,J＝11.9Hz,2H),1.41(q,J＝12.1Hz,2H),1.16(q,J＝12.1Hz,2H).13C NMR(151MHz,DMSO-d6)δ173.26,170.31,167.43,167.23,164.45,162.49,157.29,150.37,142.28,141.79,140.14,135.37,134.45,132.59,132.13(2C),131.66,129.21,128.51(3C),128.31,127.18(4C),126.72,124.38,122.87,122.63,122.18,119.95,116.38(2C),53.54,49.57(2C),48.90,48.27,45.38,43.95,42.19,40.50,31.76,31.41,30.88(2C),22.45.HRMS(ESI)for C48H47N9O6[M+H]+,calcd:846.37221,found:846.3687.HPLC analysis:MeOH-H2O(75:25),14.00min,99.3％purity.
实施例30：3-苄基-1-(4-(4-(3-(2-(2,6-二氧代哌啶-3-基)-1,3-二氧代异吲哚-5-基)苯甲酰基)哌嗪-1-基)苯基)-1-((1r，4r)-4-(喹唑啉-2-基氨基)环己基)脲(化合物YJZ1130)
合成方法与实施例23所示方法相似。
1H NMR(400MHz,DMSO-d6)δ11.15(s,1H),9.05(s,1H),8.30–8.21(m,2H),8.03(d,J＝7.7Hz,1H),7.97(d,J＝7.9Hz,1H),7.92(s,1H),7.75(d,J＝8.1Hz,1H),7.68–7.60(m,2H),7.55(d,J＝7.6Hz,1H),7.40(d,J＝8.5Hz,1H),7.31–7.23(m,3H),7.21–7.13(m,4H),7.11–6.98(m,4H),5.57(t,J＝6.8Hz,1H),5.19(dd,J＝12.6,5.3Hz,1H),4.28(t,J＝12.1Hz,1H),4.16(d,J＝5.7Hz,2H),3.82(s,1H),3.57(s,4H),3.25(s,4H),2.97–2.84(m,1H),2.69–2.58(m,2H),2.14–2.04(m,1H),1.96(d,J＝11.4Hz,2H),1.80(d,J＝12.0Hz,2H),1.41(q,J＝12.1Hz,2H),1.14(q,J＝12.1Hz,2H).13C NMR(151MHz,DMSO-d6)δ173.26,170.33,169.02,167.42,167.39,162.48,157.27,150.37,146.31,141.76,138.78,137.40,134.44,133.72,132.81,132.13(2C),130.67,129.99,129.25,128.96,128.51(3C),128.31,127.93,127.17(4C),126.72,126.26,124.58,122.21,122.18,119.96,116.46(2C),53.53,49.57(2C),49.12,48.28,47.52,43.95,40.51,31.75,31.42(2C),30.88(2C),22.47.HRMS(ESI)for C52H49N9O6[M+H]+,calcd:896.38786,found:896.3845.HPLC analysis:MeOH-H2O(75:25),19.82min,98.9％purity.
实施例31：3-苄基-1-(4-(4-(1-(2-(2,6-二氧哌啶-3-基)-1,3-二氧异吲哚-5-基)-1-吡唑-3-羰基)哌嗪-1-基)苯基)-1-((1r，4r)-4-(喹唑啉-2-基氨基)环己基)脲(化合物YJZ1131)
合成方法与实施例23所示方法相似。
1H NMR(400MHz,DMSO-d6)δ11.16(s,1H),9.19(s,1H),9.05(s,1H),8.49(s,1H),8.44(d,J＝8.3Hz,1H),8.16(s,1H),8.09(d,J＝8.2Hz,1H),7.74(d,J＝8.0Hz,1H),7.63(t,J＝7.8Hz,1H),7.39(d,J＝8.6Hz,1H),7.31–7.21(m,3H),7.21–7.13(m,4H),7.11–6.97(m,4H),5.60(t,J＝6.2Hz,1H),5.21(dd,J＝13.0,5.4Hz,1H),4.29(t,J＝12.2Hz,1H),4.17(d,J＝6.0Hz,2H),3.83(s,4H),3.58(s,1H),3.31(s,4H),2.98–2.85(m,1H),2.69–2.57(m,2H),2.15–2.04(m,1H),1.97(d,J＝12.6Hz,2H),1.80(d,J＝11.9Hz,2H),1.41(q,J＝12.9,12.4Hz,2H),1.14(q,J＝12.9,12.1Hz,2H).13C NMR(151MHz,DMSO-d6)δ173.24,170.29,166.93,166.88,162.48,162.33,157.29,150.33,144.34,143.30,141.78,134.44,133.77,132.17(2C),130.30,129.21,129.05,128.51(3C),128.31,127.18(4C),126.73,125.67,125.25,124.64,122.17,119.98,119.96,116.32(2C),113.73,53.55,49.66(2C),49.13,48.46,43.96(2C),40.51,31.76,31.41(2C),30.89(2C),22.45. HRMS(ESI)for C49H47N11O6[M+H]+,calcd:886.37835,found:886.3749.HPLC analysis:MeOH-H2O(75:25),14.54min,96.4％purity.
实施例32：3-苄基-1-(4-(2-((2-(1-甲基-2,6-二氧代哌啶-3-基)-1,3-二氧代异吲哚-4-基)氧基)乙酰基)哌嗪-1-基)苯基)-1-((1r，4r)-4-(喹唑啉-2-基氨基)环己基)脲(化合物YJZ1078)
合成方法与实施例23所示方法相似。
1H NMR(400MHz,DMSO-d6)δ9.05(s,1H),7.79(t,J＝7.8Hz,1H),7.74(d,J＝8.0Hz,1H),7.63(t,J＝7.8Hz,1H),7.47(d,J＝7.3Hz,1H),7.39(d,J＝8.4Hz,2H),7.31–7.21(m,3H),7.21–7.13(m,4H),7.10–6.99(s,4H),5.59(t,J＝6.0Hz,1H),5.25(s,2H),5.18(dd,J＝12.7,5.0Hz,1H),4.28(t,J＝10.4Hz,1H),4.17(d,J＝6.0Hz,2H),3.67–3.56(m,5H),3.32(s,2H),3.24(s,2H),3.02(s,3H),3.00–2.88(m,1H),2.81–2.70(m,1H),2.60–2.54(m,1H),2.06(d,J＝9.7Hz,1H),1.97(d,J＝11.6Hz,2H),1.80(d,J＝11.9Hz,2H),1.42(q,J＝12.8,12.4Hz,2H),1.14(q,J＝12.3,11.6Hz,2H).13C NMR(151MHz,DMSO-d6)δ172.28,170.18,167.28,165.76,165.60,162.50,157.33,156.07,150.34,141.72,137.14,134.48,133.54,132.12(2C),129.17,128.52(3C),128.32,127.17(4C),126.75,122.21,120.71,119.95,116.58,116.39(2C),116.03,66.61,53.54,49.81,49.13,48.31,48.15,44.34,43.94,41.70,40.43,31.75,31.55,30.87(2C),27.08,21.67.HRMS(ESI)for C48H49N9O7[M+H]+,calcd:864.38277,found:864.3804.HPLC analysis:MeOH-H2O(75:25),9.27min,97.5％purity.
实施例33：3-苄基-1-(4-(4-(2-((2-(2,6-二氧代哌啶-3-基)-1,3-二氧代异吲哚-4-基)氧基)乙酰基)哌嗪-1-基)苯基)-1-((1r，4r)-4-((1-丙基-1H-吡唑并[3,4-d]嘧啶-6-基)氨基)环己基)脲(化合物YJZ9108)
合成方法与实施例23所示方法相似。
1H NMR(400MHz,DMSO-d6)δ11.12(s,1H),8.73(s,1H),7.89(s,1H),7.79(dd,J＝8.5,7.3Hz,1H),7.46(d,J＝7.2Hz,1H),7.38(d,J＝8.6Hz,1H),7.28(dd,J＝8.4,6.4Hz,3H),7.21–7.13(m,3H),7.09–7.00(m,4H),5.57(s,1H),5.25(s,2H),5.11(dd,J＝12.9,5.4Hz,1H),4.26(s,1H),4.16(d,J＝6.0Hz,2H),4.09(t,J＝6.8Hz,2H),3.62(s,4H), 3.51(s,1H),3.30(s,2H),3.24(s,2H),2.96–2.82(m,1H),2.64–2.53(m,2H),2.09–1.92(m,3H),1.84–1.70(m,4H),1.40(q,J＝12.7Hz,2H),1.16–1.03(m,2H),0.76(s,3H).
实施例34：N-(4-(4-(3-(3-苄基-1-((1r，4r)-4-(喹唑啉-2-基氨基)环己基)脲基)苯基)哌嗪-1-基)丁基)-2-((2-(2,6-二氧代哌啶-3-基)-1,3-二氧代异吲哚-4-基)氧基)乙酰胺(化合物YJZ9049)
合成方法与实施例23所示方法相似。
1H NMR(400MHz,DMSO-d6)δ11.12(s,1H),9.05(s,1H),7.97(t,J＝4.9Hz 1H),7.82(t,J＝7.9Hz,1H),7.74(d,J＝8.1Hz,1H),7.63(t,J＝7.8Hz,1H),7.50(d,J＝7.3Hz,1H),7.40(d,J＝8.5Hz,2H),7.33–7.21m,4H),7.21–7.13(m,4H),6.97(d,J＝8.5Hz,1H),6.69(s,1H),6.61(d,J＝7.6Hz,1H),5.60(t,J＝6.2Hz,1H),5.12(dd,J＝13.0,5.4Hz,1H),4.78(s,2H),4.25(t,J＝12.4Hz,1H),4.18(d,J＝5.9Hz,2H),3.61(d,J＝9.3Hz,1H),3.25–3.08(m,6H),2.96–2.83(m,1H),2.63–2.53(m,2H),2.48–2.44(m,2H),2.32(d,J＝6.6Hz,2H),2.03(d,J＝12.5Hz,1H),1.97(d,J＝12.0Hz,2H),1.83(d,J＝11.8Hz,2H),1.48(s,4H),1.40(t,J＝12.4Hz,2H),1.27–1.13(m,4H).
实施例35：N-(5-(4-(3-(3-苄基-1-((1r，4r)-4-(喹唑啉-2-基氨基)环己基)脲基)苯基)哌嗪-1-基)戊基)-2-((2-(2,6-二氧代哌啶-3-基)-1,3-二氧代异吲哚-4-基)氧基)乙酰胺(化合物YJZ9048)
合成方法与实施例23所示方法相似。
1H NMR(400MHz,DMSO-d6)δ11.15(s,1H),9.05(s,1H),7.94(t,J＝5.8Hz,1H),7.81(t,J＝8.0Hz,1H),7.74(d,J＝8.0Hz,1H),7.63(t,J＝7.7Hz,1H),7.50(d,J＝7.2Hz,1H),7.40(d,J＝8.5Hz,2H),7.33–7.24(m,3H),7.22(d,J＝8.0Hz,1H),7.20–7.14(m,4H),6.97(d,J＝8.5Hz,1H),6.69(s,1H),6.61(d,J＝7.7Hz,1H),5.60(t,J＝6.1Hz,1H),5.12(dd,J＝12.9,5.3Hz,1H),4.77(s,2H),4.28(t,J＝12.3Hz,1H),4.17(d,J＝5.9Hz,2H),3.61(s,1H),3.20–3.11(m,6H),2.96–2.82(m,1H),2.65–2.53(m,2H),2.47(s,2H),2.29(t,J＝7.4Hz,2H),2.03(dd,J＝11.8,6.3Hz,1H),1.97(d,J＝11.6Hz,2H),1.83(d,J＝11.9Hz,2H),1.44(dd,J＝22.9,10.2Hz,6H),1.30(d,J＝7.3Hz,2H),1.26–1.14(m,2H).
实施例36：N-(6-(4-(3-(3-苄基-1-((1r，4r)-4-(喹唑啉-2-基氨基)环己基)脲基)苯基)哌嗪-1-基)己基)-2-((2-(2,6-二氧代哌啶-3-基)-1,3-二氧代异吲哚-4-基)氧基)乙酰胺(化合物YJZ9043)
合成方法与实施例23所示方法相似。
1H NMR(400MHz,DMSO-d6)δ11.16(s,1H),9.05(s,1H),7.93(t,J＝5.7Hz,1H),7.81(t,J＝7.9Hz,1H),7.74(d,J＝8.0Hz,1H),7.63(t,J＝7.7Hz,1H),7.50(d,J＝7.3Hz,1H),7.40(d,J＝8.5Hz,2H),7.33–7.24(m,3H),7.22(d,J＝7.8Hz,1H),7.20–7.14(m,4H),6.97(d,J＝8.6Hz,1H),6.69(s,1H),6.61(d,J＝7.6Hz,1H),5.59(t,J＝6.0Hz,1H),5.12(dd,J＝12.9,5.4Hz,1H),4.77(s,2H),4.27(t,J＝11.8Hz,1H),4.18(d,J＝6.0Hz,2H),3.62(s,1H),3.16(t,J＝6.2Hz,8H),2.95–2.84(m,1H),2.64–2.54(m,2H),2.49–2.45(m,2H),2.30(t,J＝7.5Hz,2H),2.08–2.00(m,1H),1.97(d,J＝12.1Hz,2H),1.83(d,J＝11.9Hz,2H),1.50–1.35(m,6H),1.33–1.26(m,4H),1.23–1.14(m,2H).
实施例37：N-(7-(4-(3-(3-苄基-1-((1r，4r)-4-(喹唑啉-2-基氨基)环己基)脲基)苯基)哌嗪-1-基)庚基)-2-(2-(2,6-二氧代哌啶-3-基)-1,3-二氧代异吲哚-4-基)氧基)乙酰胺(化合物YJZ9047)
合成方法与实施例23所示方法相似。
1H NMR(400MHz,DMSO-d6)δ11.15(s,1H),9.05(s,1H),7.92(t,J＝5.6Hz,1H),7.81(t,J＝7.9Hz,1H),7.74(d,J＝8.0Hz,1H),7.63(t,J＝7.7Hz,1H),7.50(d,J＝7.3Hz,1H),7.43–7.35(m,2H),7.28(q,J＝8.2,7.8Hz,3H),7.22(d,J＝7.8Hz,1H),7.21–7.15(m,4H),6.97(d,J＝8.5Hz,1H),6.69(s,1H),6.61(d,J＝7.6Hz,1H),5.76(s,1H),5.60(t,J＝6.1Hz,1H),5.12(dd,J＝13.0,5.4Hz,1H),4.77(s,2H),4.25(t,J＝12.6Hz,1H),4.18(d,J＝6.0Hz,2H),3.62(s,1H),3.16(dd,J＝8.9,5.0Hz,7H),2.95–2.84(m,1H),2.65–2.54(m,2H),2.48(s,2H),2.30(t,J＝7.5Hz,2H),2.08–2.01(m,1H),1.97(d,J＝12.3Hz,2H),1.83(d,J＝11.9Hz,2H),1.51–1.34(m,6H),1.33–1.13(m,10H).
实施例38：3-苄基-1-(3-(4-(4-((2-(2,6-二氧代哌啶-3-基)-1,3-二氧代异吲哚-4-基)氨基)丁酰基)哌嗪-1-基)苯基)-1-((1r，4r)-4-(喹唑啉-2-基氨基)环己基)脲(化合物YJZ9058)
合成方法与实施例23所示方法相似。
1H NMR(400MHz,DMSO-d6)δ11.08(s,1H),9.05(s,1H),7.74(d,J＝7.9Hz,1H),7.61(dt,J＝15.9,7.8Hz,2H),7.39(d,J＝8.5Hz,1H),7.35–7.24(m,3H),7.22(d,J＝7.9 Hz,1H),7.20–7.14(m,5H),7.03–6.97(m,2H),6.72(s,1H),6.70–6.61(m,2H),5.61(t,J＝6.1Hz,1H),5.05(dd,J＝12.9,5.4Hz,1H),4.26(t,J＝12.1Hz,1H),4.18(d,J＝6.0Hz,2H),3.68–3.54(m,5H),3.23–3.09(m,4H),2.93–2.82(m,1H),2.62–2.54(m,2H),2.46(t,J＝7.0Hz,2H),2.06–1.93(m,4H),1.89–1.78(m,4H),1.42(q,J＝12.4Hz,2H),1.18(q,J＝12.0Hz,2H).
实施例39：3-苄基-1-(3-(4-(6-((2-(2,6-二氧代哌啶-3-基)-1,3-二氧代异吲哚-4-基)氨基)己基)哌嗪-1-基)苯基)-1-((1r，4r)-4-(喹唑啉-2-基氨基)环己基)脲(化合物YJZ9052)
合成方法与实施例23所示方法相似。
1H NMR(400MHz,DMSO-d6)δ11.09(s,1H),9.05(s,1H),7.74(d,J＝7.7Hz,1H),7.63(t,J＝7.7Hz,1H),7.57(dd,J＝8.6,7.1Hz,1H),7.39(d,J＝8.5Hz,1H),7.35–7.25(m,3H),7.23(d,J＝7.8Hz,1H),7.21–7.13(m,4H),7.09(d,J＝8.6Hz,1H),7.03–6.97(m,2H),6.73(s,1H),6.65(d,J＝7.5Hz,1H),6.54(t,J＝5.9Hz,1H),5.62(t,J＝6.1Hz,1H),5.04(dd,J＝12.9,5.3Hz,1H),4.29(t,J＝12.0Hz,1H),4.18(d,J＝6.0Hz,2H),3.60(s,5H),3.32–3.26(m,1H),3.16(d,J＝19.2Hz,4H),2.93–2.81(m,1H),2.62–2.53(m,2H),2.37(t,J＝7.3Hz,2H),2.06–1.91(m,3H),1.83(d,J＝11.8Hz,2H),1.59(dp,J＝14.9,7.2Hz,4H),1.48–1.34(m,4H),1.18(q,J＝12.6Hz,2H).
实施例40：3-苄基-1-(3-(4-(8-(2-(2,6-二氧代哌啶-3-基)-1,3-二氧代异吲哚-4-基)氨基)辛基)哌嗪-1-基)苯基)-1-((1r，4r)-4-(喹唑啉-2-基氨基)环己基)脲(化合物YJZ9053)
合成方法与实施例23所示方法相似。
1H NMR(400MHz,DMSO-d6)δ11.09(s,1H),9.04(s,1H),7.74(d,J＝8.0Hz,1H),7.62(t,J＝7.7Hz,1H),7.59–7.54(m,1H),7.39(d,J＝8.5Hz,1H),7.35–7.25(m,3H),7.22(d,J＝8.0Hz,1H),7.20–7.14(m,4H),7.08(d,J＝8.6Hz,1H),7.04–6.98(m,2H),6.73(s,1H),6.65(d,J＝7.6Hz,1H),6.52(t,J＝6.0Hz,1H),5.62(t,J＝6.0Hz,1H),5.04(dd,J＝12.9,5.4Hz,1H),4.26(t,J＝12.1Hz,1H),4.17(d,J＝6.0Hz,2H),3.59(s,5H),3.31–3.24(m,1H),3.16(d,J＝20.5Hz,4H),2.93–2.81(m,1H),2.62–2.53(m,2H),2.35(t,J＝7.4Hz,2H),2.06–1.91(m,3H),1.83(d,J＝12.0Hz,2H),1.61–1.48(m,4H),1.41(q,J＝12.5Hz,2H),1.36–1.27(m,6H),1.18(q,J＝12.5Hz,2H).
实施例41：3-苄基-1-(3-(4-(10-(2-(2,6-二氧代哌啶-3-基)-1,3-二氧代异吲哚-4-基)氨基)癸酰基)哌嗪-1-基)苯基)-1-((1r，4r)-4-(喹唑啉-2-基氨基)环己基)脲(化合物YJZ9055)
合成方法与实施例23所示方法相似。
1H NMR(400MHz,DMSO-d6)δ11.09(s,1H),9.04(s,1H),7.74(d,J＝8.0Hz,1H),7.62(t,J＝7.7Hz,1H),7.57(dd,J＝8.6,7.1Hz,1H),7.39(d,J＝8.5Hz,1H),7.35–7.24(m,3H),7.23(d,J＝7.9Hz,1H),7.21–7.13(m,4H),7.07(d,J＝8.6Hz,1H),7.04–6.97(m,2H),6.73(s,1H),6.65(d,J＝7.6Hz,1H),6.51(t,J＝5.9Hz,1H),5.62(t,J＝5.9Hz,1H),5.05(dd,J＝12.9,5.4Hz,1H),4.27(t,J＝12.1Hz,1H),4.17(d,J＝6.0Hz,2H),3.59(s,5H),3.27(p,J＝6.6Hz,2H)3.16(d,J＝20.9Hz,4H),2.94–2.82(m,1H),2.63–2.55(m,2H),2.34(t,J＝7.4Hz,2H),2.06–1.90(m,3H),1.83(d,J＝11.9Hz,2H),1.61–1.48(m,4H),1.41(q,J＝12.3Hz,2H),1.36–1.12(m,12H).
实施例42：N-(3-(3-苄基-1-((1r，4r)-4-(喹唑啉-2-基氨基)环己基)脲基)苯基)-4-((2-(2,6-二氧代哌啶-3-基)-1,3-二氧代异吲哚-4-基)氨基)丁酰胺(化合物YJZ9062)
合成方法与实施例23所示方法相似。
1H NMR(400MHz,DMSO-d6)δ11.10(s,1H),10.13(s,1H),9.04(s,1H),7.72(dd,J＝11.5,7.9Hz,2H),7.66–7.55(m,2H),7.48(s,1H),7.43–7.35(m,2H),7.30–7.21(m,3H),7.21–7.12(m,5H),7.03(d,J＝7.0Hz,1H),6.87(d,J＝8.1Hz,1H),6.69(t,J＝6.2Hz,1H),5.79(t,J＝6.1Hz,1H),5.05(dd,J＝12.8,5.4Hz,1H),4.27(t,J＝11.8Hz,1H),4.16(d,J＝6.0Hz,2H),3.58(s,1H),2.94–2.81(m,1H),2.64–2.53(m,2H),2.45(t,J＝7.2Hz,2H),2.07–1.86(m,5H),1.80(d,J＝12.1Hz,2H),1.42(q,J＝12.2Hz,2H),1.28–1.11(m,4H).
实施例43：N-(3-(3-苄基-1-((1r，4r)-4-(喹唑啉-2-基氨基)环己基)脲基)苯基)-8-((2-(2,6-二氧哌啶-3-基)-1,3-二氧异吲哚-4-基)氨基)辛酰胺(化合物YJZ9059)
合成方法与实施例23所示方法相似。
1H NMR(400MHz,DMSO-d6)δ11.10(s,1H),10.03(s,1H),9.04(s,1H),7.74(d,J＝8.0Hz,1H),7.69(d,J＝8.4Hz,1H),7.65–7.52(m,2H),7.49(s,1H),7.43–7.34(m,2H),7.30–7.20(m,4H),7.20–7.14(m,4H),7.07(d,J＝8.6Hz,1H),7.01(d,J＝7.1Hz,1H), 6.86(d,J＝7.5Hz,1H),6.52(t,J＝5.9Hz,1H),5.79(t,J＝5.9Hz,1H),5.04(dd,J＝13.0,5.4Hz,1H),4.28(t,J＝12.1Hz,1H),4.16(d,J＝5.9Hz,2H),3.58(s,1H),3.29(p,J＝6.7Hz,2H)2.94–2.80(m,1H),2.63–2.54(m,2H),2.32(t,J＝7.3Hz,2H),2.06–1.91(m,3H),1.79(d,J＝12.0Hz,2H),1.66–1.51(s,4H),1.43(q,J＝12.3Hz,2H),1.37–1.31(m,4H),1.28–1.22(m,2H),1.21–1.10(m,2H).
实施例44：3-苄基-1-(4-((1-(2-(2,6-二氧代哌啶-3-基)-1,3-二氧代异吲哚-5-基)氮杂环丁烷-3-基甲基)哌嗪-1-基)苯基)-1-((1r，4r)-4-(喹唑啉-2-基氨基)环己基)脲(化合物YJZ1102)
步骤1：3-(4-(4-(3-苄基-1-((1r，4r)-4-(喹唑啉-2-基氨基)环己基)脲基)苯基)哌嗪-1-基)甲基)氮杂环丁酯-1-羧酸叔丁酯(化合物11)的制备
将KHCO3(345mg,2.5mmol)加入混有化合物8(620mg,1.2mmol)、3-(溴甲基)氮杂环丁烷-1-羧酸叔丁酯10(630mg,2.5mmol)的DMF(40mL)中。在80℃搅拌5小时后，过滤反应液。减压旋蒸浓缩滤液通过柱层析纯化得白色固体的560mg(产率65％)。1H NMR(400MHz,DMSO-d6)δ9.05(s,1H),7.74(d,J＝8.0Hz,1H),7.63(t,J＝7.8Hz,1H),7.40(d,J＝8.7Hz,1H),7.27(t,J＝7.5Hz,2H),7.23(d,J＝8.0Hz,1H),7.21–7.12(m,4H),7.01(q,J＝9.2Hz,4H),5.57(t,J＝6.1Hz,1H),4.26(t,J＝12.1Hz,1H),4.16(d,J＝6.0Hz,2H),3.92(s,2H),3.67–3.44(m,4H),3.31(s,2H),3.17(d,J＝6.2Hz,4H),2.89(s,1H),2.73(s,1H),2.57(d,J＝7.2Hz,2H),1.96(d,J＝11.3Hz,2H),1.79(d,J＝11.9Hz,2H),1.47–1.31(m,11H),1.13(q,J＝12.6Hz,2H).
步骤2：1-(4-(4-(氮杂环丁烷-3-基甲基)哌嗪-1-基)苯基)-3-苄基-1-((1r，4r)-4-(喹唑啉-2-基氨基)环己基)脲(化合物12)的制备
3-(4-(4-(3-苄基-1-((1r，4r)-4-(喹唑啉-2-基氨基)环己基)脲基)苯基)哌嗪-1-基)甲基)氮杂环丁酯-1-羧酸叔丁酯11(100mg,0.14mmol)溶于DCM(4mL)中，加入TFA(2mL)，并在50℃下搅拌回流过夜。然后在减压下将反应混合物浓缩，并通过柱层析纯化所得白色物体60mg(产率70％)。
步骤3：3-苄基-1-(4-((1-(2-(2,6-二氧代哌啶-3-基)-1,3-二氧代异吲哚-5-基)氮杂环丁烷-3-基甲基)哌嗪-1-基)苯基)-1-((1r，4r)-4-(喹唑啉-2-基氨基)环己基)脲(化合物YJZ1102)
将2-(2,6-二氧哌啶-3-基)-5-氟异吲哚啉-1,3-二酮13(42.9mg,0.16mmol)溶在6mL DMSO中，在室温下加入DIPEA(25.1mg,0.19mmol)和化合物12(84.5mg,0.13mmol)。将所得混合物在120℃搅拌8小时。反应结束后在真空下除去溶剂，通过柱色谱纯化得到黄色固体状化合物71mg(产率63％)。1H NMR(400MHz,DMSO-d6)δ11.07(s,1H),9.05(s,1H),7.74(d,J＝7.9Hz,1H),7.69–7.59(m,2H),7.40(d,J＝8.5Hz,1H),7.32–7.25(m,2H),7.23(d,J＝8.0Hz,1H),7.18(t,J＝7.5Hz,4H),7.09–6.95(m,4H),6.79(d,J＝2.1Hz,1H),6.66(dd,J＝8.4,2.1Hz,1H),5.58(t,J＝6.1Hz,1H),5.06(dd,J＝12.9,5.4Hz,1H),4.30(t,J＝12.4Hz,2H),4.22–4.09(m,4H),3.72(dd,J＝8.4,5.4Hz,2H),3.59(s,1H),3.21(t,J＝4.8Hz,4H),3.06(p,J＝6.9Hz,1H),2.95–2.81(m,1H),2.67(d,J＝7.2Hz,2H),2.63–2.53(m,6H),2.08–1.89(m,3H),1.79(d,J＝11.7Hz,2H),1.41(q,J＝12.3,11.6Hz,2H),1.13(q,J＝12.4Hz,2H).13C NMR(151MHz,DMSO-d6)δ173.33,170.61,167.99,167.68,162.49,157.35,155.66,150.60,141.76,134.46,134.28,132.02(2C),128.68,128.52(3C),128.31,127.16(4C),126.74,125.30,122.20,119.95,117.11,115.90(2C),114.55,104.80,62.22,56.21(2C),53.52,53.30(2C),49.17(2C),48.06(2C),43.93,40.43,31.75,31.44,30.87(2C),27.47,22.68.HRMS(ESI)for C49H52N10O5[M+H]+,calcd:861.41949,found:861.4163.HPLC analysis:MeOH-H2O(80:20),11.34min,98.3％purity.
实施例45：3-苄基-1-(4-(4-(1-(2-(2,6-二氧代哌啶-3-基)-1,3-二氧代异吲哚-5-基)哌啶-4-基)-哌嗪-1-基)苯基)-1-((1r，4r)-4-(喹唑啉-2-基氨基)环己基)脲(化合物YJZ1105)的制备
合成方法与实施例44所示方法相似。
1H NMR(400MHz,DMSO-d6)δ11.08(s,1H),9.05(s,1H),7.74(d,J＝8.0Hz,1H),7.67(d,J＝8.5Hz,1H),7.63(t,J＝7.5Hz,2H),7.40(d,J＝8.5Hz,1H),7.34(d,J＝2.2Hz,1H),7.30–7.24(m,3H),7.23(d,J＝8.1Hz,1H),7.20–7.12(m,4H),7.01(q,J＝8.8Hz,4H),5.57(t,J＝6.0Hz,1H),5.07(dd,J＝12.9,5.4Hz,1H),4.26(t,J＝12.1Hz,1H),4.16(d,J＝6.0Hz,2H),4.09(d,J＝10.1Hz,2H),3.65–3.53(m,1H),3.26–3.12(m,4H),3.00(t,J＝12.3Hz,2H),2.94–2.82(m,1H),2.65(t,J＝4.9Hz,4H),2.62–2.59(m,1H),2.59–2.53(m,2H),2.07–1.86(m,5H),1.78(d,J＝11.6Hz,2H),1.50(q,J＝11.1Hz,2H),1.41(q,J＝12.5Hz,4H),1.13(q,J＝12.5,11.9Hz,2H).13C NMR(151MHz,DMSO-d6)δ173.37,170.61,168.11,167.48,162.50,157.39,155.22,150.62,141.70,134.48,131.99(2C),128.56,128.52(3C),128.32,127.14(4C),126.75,125.53,125.24,122.23,119.94,118.17,118.01,115.79(2C),108.24,60.99,53.51,49.20(3C),48.38(2C),47.06(2C),43.91,40.73,40.36,31.75,31.42,30.85(2C),27.64(2C),22.65.HRMS(ESI)for C50H54N10O5[M+H]+,calcd:875.43514,found:875.4322.HPLC analysis:MeOH-H2O(80:20),11.36min,99.2％purity.
实施例46：3-苄基-1-(4-(4-(2-(2,6-二氧代哌啶-3-基)-1,3-二氧代异吲哚-5-基)氮杂环丁烷-3-基(哌嗪-1-基)苯基)-1-((1r，4r)-4-(喹唑啉-2-基氨基)环己基)脲(化合物YJZ1103)
合成方法与实施例44所示方法相似。
1H NMR(400MHz,DMSO-d6)δ11.08(s,1H),9.05(s,1H),7.74(d,J＝8.5Hz,1H),7.67(d,J＝8.3Hz,1H),7.63(t,J＝7.7Hz,1H),7.40(d,J＝8.5Hz,1H),7.31–7.25(m,2H),7.23(d,J＝7.9Hz,1H),7.21–7.14(m,4H),7.08–6.98(m,4H),6.82(d,J＝2.1Hz,1H),6.68(dd,J＝8.4,2.1Hz,1H),5.58(t,J＝6.3Hz,1H),5.06(dd,J＝12.9,5.4Hz,1H),4.34–4.23(m,1H),4.15(t,J＝7.6Hz,4H),3.94(dd,J＝8.8,4.9Hz,2H),3.66–3.51(m,1H),3.45–3.37(m,1H),3.26(t,J＝5.7Hz,4H),2.94–2.80(m,1H),2.64–2.53(m,6H),2.06–1.99(m,1H),1.96(d,J＝14.5Hz,2H),1.79(d,J＝11.7Hz,2H),1.41(q,J＝12.4Hz,2H),1.13(q,J＝12.8Hz,2H).13C NMR(151MHz,DMSO-d6)δ173.33,170.61,167.97,167.67,162.49,157.35,155.42,150.54,141.76,134.47,134.29,132.04(2C),128.76,128.52(3C),128.31,127.16(4C),126.73,125.35,122.20,119.95,117.33,115.94(2C),114.72,104.97,55.50(2C),54.62,53.51,49.59(2C),49.18(2C),47.83(2C),43.92, 40.43,31.74,31.43,30.86(2C),22.67.HRMS(ESI)for C48H50N10O5[M+H]+,calcd:847.40384,found:847.4000.HPLC analysis:MeOH-H2O(75:25),13.02min,97.9％purity.
实施例47：3-苄基-1-(4-(4-(1-(2-(2,6-二氧代哌啶-3-基)-1,3-二氧代异吲哚-5-基)吡咯烷-3-基(哌嗪-1-基)苯基)-1-((1r，4r)-4-(喹唑啉-2-基氨基)环己基)脲(化合物YJZ1096)
合成方法与实施例44所示方法相似。
1H NMR(400MHz,DMSO-d6)δ11.07(s,1H),9.05(s,1H),7.74(d,J＝8.0Hz,1H),7.68–7.59(m,2H),7.40(d,J＝8.4Hz,1H),7.28(t,J＝7.5Hz,2H),7.23(d,J＝8.1Hz,1H),7.18(t,J＝7.3Hz,4H),7.04(s,4H),6.99(s,1H),6.87(d,J＝9.0Hz,1H),5.58(d,J＝7.0Hz,1H),5.06(dd,J＝12.7,5.4Hz,1H),4.30(t,J＝12.4Hz,1H),4.16(d,J＝5.9Hz,2H),3.75(t,J＝8.4Hz,1H),3.66–3.53(m,2H),3.42(q,J＝8.8Hz,2H),3.31–3.27(m,1H),3.24(t,J＝6.2Hz,4H),3.02(p,J＝7.6Hz,1H),2.95–2.82(m,1H),2.67(s,4H),2.63–2.53(m,2H),2.34–2.26(m,1H),2.05–1.88(m,4H),1.79(d,J＝11.7Hz,2H),1.41(q,J＝12.4Hz,2H),1.13(q,J＝12.8Hz,2H).13C NMR(151MHz,DMSO-d6)δ173.35,170.65,168.20,167.74,162.49,157.36,152.27,150.57,141.76,134.46,132.03(2C),128.74,128.52(4C),128.31,127.15(4C),126.74,125.41,122.21,119.95,116.22,115.92(2C),115.71,106.05,63.97,53.52,52.42,51.76(2C),49.15(2C),48.05(2C),47.49,43.93,40.43,31.76,31.45,30.87(2C),29.28,22.71.HRMS(ESI)for C49H52N10O5[M+H]+,calcd:861.41949,found:861.4168.HPLC analysis:MeOH-H2O(75:25),15.48min,98.2％purity.
实施例48：3-苄基-1-(4-(4-((1-(2-(2,6-二氧哌啶-3-基)-1,3-二氧异吲哚-5-基)哌啶-4-基)甲基)哌嗪-1-基)苯基)-1-((1r，4r)-4-(喹唑啉-2-基氨基)环己基)脲(YJZ1097)
合成方法与实施例44所示方法相似。
1H NMR(600MHz,DMSO-d6)δ11.09(s,1H),9.05(s,1H),7.75(d,J＝7.9Hz,1H),7.66(d,J＝8.5Hz,1H),7.63(t,J＝7.6Hz,1H),7.41(d,J＝8.6Hz,1H),7.32(s,1H),7.30–7.26(m,2H),7.24(d,J＝8.8Hz,2H),7.21–7.13(m,4H),7.02(q,J＝8.7Hz,4H),5.58(t,J＝6.1Hz,1H),5.07(dd,J＝12.8,5.5Hz,1H),4.29(t,J＝11.7Hz,1H),4.16(d,J＝6.0Hz,2H),4.06(d,J＝12.8Hz,2H),3.65–3.54(m,1H),3.26–3.15(s,4H),2.99(t,J＝12.3Hz,2H),2.93–2.83(m,1H),2.64–2.54(m,2H),2.52(m,4H),2.21(d,J＝5.2Hz,2H),2.05–1.99(m,1H),1.99–1.93(m,2H),1.92–1.85(m,1H),1.85–1.75(m,4H),1.41(q,J＝12.4Hz,2H),1.20–1.09(m,4H).13C NMR(151MHz,DMSO-d6)δ173.30,170.60,168.12,167.45,162.48,157.32,155.48,150.62,141.81,134.52,134.42,132.02(2C), 128.67,128.51(3C),128.31,127.16(4C),126.71,125.49,125.28,122.17,119.96,118.06,117.80,115.83(2C),108.21,64.18,53.68(2C),53.52,49.20(2C),48.15,47.74(2C),43.94,40.51,32.94,31.76,31.45,30.88(2C),30.11(2C),29.45,22.67.HRMS(ESI)for C51H56N10O5[M+H]+,calcd:889.45079,found:889.4473.HPLC analysis:MeOH-H2O(80:20),15.05min,96.9％purity.
实施例49：3-苄基-1-(4-(4-(1-(2-(2-(2,6-二氧哌啶-3-基)-6-氟-1,3-二氧异吲哚-5-基)哌啶-4-基)-哌嗪-1-基)苯基)-1-((1r，4r)-4-(喹唑啉-2-基氨基)环己基)脲(YJZ1201)
合成方法与实施例44所示方法相似。
1H NMR(400MHz,DMSO-d6)δ11.11(s,1H),9.05(s,1H),7.77–7.68(m,2H),7.63(t,J＝7.9Hz,1H),7.47(d,J＝7.3Hz,1H),7.40(d,J＝8.5Hz,1H),7.31–7.24(m,2H),7.23(d,J＝7.0Hz,1H),7.21–7.13(m,4H),7.07–6.95(m,4H),5.57(t,J＝6.8Hz,1H),5.11(dd,J＝13.1,5.3Hz,1H),4.28(t,J＝11.4Hz,1H),4.16(d,J＝5.8Hz,2H),3.68(d,J＝11.7Hz,2H),3.58(s,1H),3.21(s,4H),2.99–2.82(m,3H),2.68(s,4H),2.64–2.54(m,3H),2.08–1.89(m,5H),1.78(d,J＝9.6Hz,2H),1.61(q,J＝12.2Hz,3H),1.41(q,J＝14.5,13.7Hz,2H),1.13(q,J＝13.1Hz,2H).13C NMR(151MHz,DMSO-d6)δ173.25,170.40,167.17,166.69,162.47,158.57(d,J＝253.3Hz,1C),157.32,150.63,146.01(d,J＝8.8Hz,1C),141.82,134.43,132.02(2C),129.26(d,J＝2.2Hz,1C),128.65,128.50(3C),128.30,127.16(4C),126.71,123.51(d,J＝9.6Hz,1C),122.17,119.96,115.79(2C),114.23(d,J＝4.8Hz,1C),112.49(d,J＝25.3Hz,1C),60.86,53.52,52.46,49.84,49.81,49.51,49.25(2C),48.49(2C),43.94,40.51,31.78,31.42,30.87(2C),28.35(2C),22.55.HRMS(ESI)for C50H53N10O5F[M+H]+,calcd:893.42572,found:893.4229.HPLC analysis:MeOH-H2O(80:20),11.79min,95.8％purity.
实施例50：1-(4-(4-(1-(1-(2-(2,6-二氧哌啶-3-基)-1,3-二氧异吲哚-5-基)哌啶-4-基)-哌嗪-1-基)苯基)-3-(2-氟苄基)-1-((1r，4r)-4-(喹唑啉-2-基氨基)环己基)脲(YJZ1203)
合成方法与实施例44所示方法相似。
1H NMR(400MHz,DMSO-d6)δ11.08(s,1H),9.05(s,1H),7.74(d,J＝8.0Hz,1H),7.67(d,J＝8.6Hz,1H),7.63(t,J＝7.7Hz,1H),7.40(d,J＝8.6Hz,1H),7.34(s,1H),7.29 –7.14(m,6H),7.14–7.08(m,1H),7.03(q,J＝8.6Hz,4H),5.59(s,1H),5.07(dd,J＝13.4,5.5Hz,1H),4.31–4.17(m,3H),4.10(d,J＝12.7Hz,2H),3.58(s,1H),3.20(s,4H),3.00(t,J＝12.3Hz,2H),2.95–2.82(m,1H),2.66(s,4H),2.63–2.53(m,3H),2.07–1.86(m,5H),1.79(d,J＝11.9Hz,2H),1.50(q,J＝11.5Hz,2H),1.44–1.33(m,2H),1.20–1.09(m,2H).13C NMR(151MHz,DMSO-d6)δ173.30,170.59,168.09,167.44,162.47,161.01(d,J＝243.4Hz,1C),157.23,155.23,150.66,134.51,134.43,131.98(2C),129.03(d,J＝4.7Hz,2C),128.58,128.55,128.53,128.44(d,J＝14.2Hz,1C),128.30,125.49,125.29,124.61(d,J＝3.3Hz,1C),122.17,119.96,118.18,118.10,115.81(2C),115.12(d,J＝21.1Hz,1C),108.29,61.02,53.57,49.25(2C),49.21,49.10,48.46(2C),47.09(2C),40.51,37.73,31.76,31.45,30.84(2C),27.67(2C),22.66.HRMS(ESI)for C50H53N10O5F[M+H]+,calcd:893.42572,found:893.4224.HPLC analysis:MeOH-H2O(80:20),9.19min,96.2％purity.
实施例51：3-苄基-1-(6-(4-(1-(1-(2-(2,6-二氧哌啶-3-基)-1,3-二氧异吲哚-5-基)哌啶-4-基)吡嗪-1-基)吡啶-3-基]-1-((1r，4r)-4-(喹唑啉-2-基氨基)环己基)脲(YJZ1205)
合成方法与实施例44所示方法相似。
1H NMR(400MHz,DMSO-d6)δ11.08(s,1H),9.05(s,1H),7.91(s,1H),7.74(d,J＝8.0Hz,1H),7.70–7.58(m,2H),7.40(d,J＝8.4Hz,1H),7.34(s,2H),7.31–7.21(m,4H),7.21–7.13(m,4H),6.88(d,J＝9.0Hz,1H),6.05(t,J＝6.6Hz,1H),5.07(dd,J＝12.8,5.3Hz,1H),4.26(t,J＝12.8Hz,1H),4.12(dd,J＝18.7,9.2Hz,4H),3.60(s,1H),3.52(s,4H),3.00(t,J＝12.4Hz,2H),2.94–2.81(m,1H),2.70–2.53(m,7H),2.07–1.93(m,3H),1.90(d,J＝12.2Hz,2H),1.78(d,J＝11.7Hz,2H),1.57–1.33(m,4H),1.09(q,J＝12.9Hz,2H).13C NMR(151MHz,DMSO-d6)δ173.30,170.59,168.09,167.44,162.47,158.32,157.45,155.22,149.85,141.82,140.60,134.51,134.43,128.48(3C),128.31,127.18(4C),126.68,125.49,124.43,122.17,119.97,118.17,118.09,108.28,107.40,61.12,55.39,53.49,49.21(2C),49.10(2C),47.10(2C),45.29,43.96,31.70,31.45,30.85(2C),27.66(2C),22.66.HRMS(ESI)for C49H53N11O5[M+H]+,calcd:876.43039,found:876.4276.HPLC analysis:MeOH-H2O(80:20),6.94min,97.8％purity.
实施例52：3-苄基-1-(6-(4-(1-(1-(2-(2-(2,6-二氧哌啶-3-基)-6-氟-1,3-二氧异吲哚-5-基)哌啶-4-基)吡咯嗪-1-基)吡啶-3-基]-1-((1r，4r)-4-(喹唑啉-2-基氨基)环己基)脲(YJZ1206)
合成方法与实施例44所示方法相似。
1H NMR(400MHz,DMSO-d6)δ11.11(s,1H),9.05(s,1H),7.92(s,1H),7.73(t,J＝9.7Hz,2H),7.63(t,J＝8.0Hz,1H),7.46(d,J＝7.4Hz,1H),7.41(d,J＝8.5Hz,1H),7.35(d,J＝9.1Hz,1H),7.31–7.25(m,2H),7.21(d,J＝7.5Hz,1H),7.21–7.13(m,3H),6.90(d,J＝9.0Hz,1H),6.05(t,J＝5.4Hz,1H),5.11(dd,J＝13.0,5.3Hz,1H),4.28(t,J＝12.3Hz,1H),4.15(d,J＝6.0Hz,2H),3.68(d,J＝11.9Hz,2H),3.63–3.48(m,5H),2.99–2.82(m,3H),2.64(s,4H),2.59–2.52(m,3H),2.08–1.87(m,5H),1.79(d,J＝11.7Hz,2H),1.61(d,J＝12.0Hz,2H),1.41(q,J＝13.6,13.0Hz,2H),1.10(d,J＝12.8Hz,3H).13C NMR(151MHz,DMSO-d6)δ173.25,170.40,167.17,166.69,162.47,158.33,157.45,156.89(d,J＝253.4Hz,1C),149.86,146.00(d,J＝8.49Hz,1C),141.83,140.60,134.42,129.26(d,J＝2.2Hz,1C),128.48(3C),128.30,127.19(4C),126.69,125.28,124.42,123.52(d,J＝9.9Hz,1C),122.17,119.97,114.24(d,J＝4.5Hz,1C),112.48(d,J＝25.5Hz,1C),107.40,60.96,55.39,53.50,49.84(2C),49.52(2C),49.11(2C),45.30,43.97,40.51,31.72,31.42,30.85(2C),28.32(2C),22.55.HRMS(ESI)for C49H52N11O5F[M+H]+,calcd:894.42097,found:894.4176.HPLC analysis:MeOH-H2O(80:20),8.79min,98.9％purity.
实施例53：3-苄基-1-(4-(4-(2-(2,6-二氧哌啶-3-基)-1,3-二氧异吲哚-5-基)哌嗪-1-基)哌啶-1-基苯基)-1-((1r，4r)-4-(喹唑啉-2-基氨基)环己基)脲(YJZ1202)
步骤1：((1r，4r)-4-((4-溴苯基)氨基)环己基)氨基甲酸叔丁酯(化合物16)的制备
将化合物14(5.0g,17.7mmol)、化合物15(3.2g,14.7mmol)、Pd2(dba)3(1.37mg,1.5mmol)、Xantphos(1.7g,2.94mmol)和tert-ButONa(2.8g,29.4mmol)混合溶于150mL甲苯中，用氩气置换三次后将反应液加热至100℃反应过夜。反应结束后用硅藻土过滤反应液，减压旋转浓缩反应液。经柱层析纯化得黄色固体3.9g(产率72％)。1H  NMR(400MHz,DMSO-d6)δ7.15(d,J＝6.8Hz,2H),6.76(d,J＝7.9Hz,1H),6.50(d,J＝8.9Hz,2H),5.60(d,J＝8.0Hz,1H),3.27–3.13(m,1H),3.12–2.99(m,1H),1.94(d,J＝12.7Hz,2H),1.79(d,J＝12.4Hz,2H),1.38(s,9H),1.26(q,J＝12.2,11.3Hz,2H),1.14(q,J＝12.2Hz,2H).
步骤2：((1r，4r)-4-(3-苄基-1-(4-溴苯基)脲基)环己基)氨基甲酸叔丁酯(化合物17)的制备
将化合物16(3.9g,10.6mmol)溶于4mL DMF中，加入苄基异氰酸酯6(4.2g,31.7mmol)，DIPEA(1.59g,12.3mmol)，95℃反应5h。旋干溶剂，柱层析得黄色固体3.7g(产率69％)。1H NMR(400MHz,DMSO-d6)δ7.63(d,J＝8.4Hz,2H),7.30–7.22(m,2H),7.20–7.14(m,3H),7.11(d,J＝7.5Hz,2H),6.66(d,J＝8.0Hz,1H),5.98(t,J＝6.2Hz,1H),4.22–4.07(m,3H),3.02–2.87(m,1H),1.73(d,J＝10.8Hz,4H),1.35(s,9H),1.24(q,J＝12.5Hz,2H),1.01(q,J＝12.5,12.0Hz,2H).
步骤3：1-((1r，4r)-4-氨基环己基)-3-苄基-1-(4-溴苯基)脲(化合物18)的制备
将化合物17(3.7g,7.3mmol)溶于5mL DCM中，加入2.5mL三氟乙酸(TFA)。加热至55℃回流6h后，减压旋干溶剂，柱层析得目标化合物黄色固体2.5g(产率70％)。1H NMR(400MHz,DMSO-d6)δ7.62(d,J＝8.0Hz,2H),7.30–7.22(m,2H),7.21–7.14(m,3H),7.11(d,J＝8.0Hz,2H),5.95(t,J＝6.1Hz,1H),4.20(t,J＝11.9Hz,1H),4.14(d,J＝6.1Hz,2H),2.26(t,J＝7.8Hz,1H),1.80–1.60(m,4H),1.10(q,J＝11.9,11.2Hz,2H),0.98(q,J＝12.5,12.1Hz,2H).
步骤4：3-苄基-1-(4-溴苯基)-1-((1r，4r)-4-(喹唑啉-2-基氨基)环己基)脲(化合物19)的制备
将化合物18(2.5g,5.1mmol)溶于15mL DMF中，加入2-氯喹唑啉4(1.0g,6.1mol)，Cs2CO3(2.0g,6.1mol)，混合物在室温下搅拌15min后升至60℃反应40min。反应结束后减压旋干溶剂，柱层析纯化得1.9g(产率70％)。1H NMR(400MHz,DMSO-d6)δ9.05(s,1H),7.74(d,J＝8.0Hz,1H),7.69–7.59(m,3H),7.40(d,J＝8.5Hz,1H),7.31–7.25(m,2H),7.23(d,J＝7.8Hz,1H),7.21–7.12(m,6H),6.02(t,J＝6.2Hz,1H),4.29(t,J＝12.5Hz,1H),4.16(d,J＝5.9Hz,2H),3.68–3.53m,1H),1.98(d,J＝13.6Hz,2H),1.81(d,J＝12.1Hz,2H),1.42(q,J＝12.4Hz,2H),1.13(q,J＝12.5Hz,2H).
步骤5：4-(1-(4-(3-苄基-1-((1r，4r)-4-(喹唑啉-2-基氨基)环己基)脲基)苯基)哌啶-4-基)哌嗪-1-甲酸叔丁酯(化合物21)的制备
将化合物19(1.9g,3.6mmol)、化合物20(1.25g,4.6mmol)、Pd2(dba)3(327mg,0.36mmol)、Xantphos(414mg,0.72mmol)和tert-ButONa(688mg,7.2mmol)混合溶于80mL甲苯中，用氩气置换三次后将反应液加热至100℃反应过夜。反应结束后用硅藻土过滤反应液，减压旋转浓缩反应液。经柱层析纯化得淡黄色固体1785mg(产率69％)。1H NMR(400MHz,DMSO-d6)δ9.05(s,1H),7.74(d,J＝8.2Hz,1H),7.66–7.60(m,1H),7.40(d,J＝9.1Hz,1H),7.30–7.24(m,2H),7.24–7.13(m,5H),7.04–6.95(m,4H),5.53(t,J＝7.4Hz,1H),4.29(t,J＝12.6Hz,1H),4.16(d,J＝6.0Hz,2H),3.79(d,J＝12.1Hz,2H),3.65–3.51(m,1H),3.30(s,4H),2.71(t,J＝12.1Hz,2H),2.46(s,4H),2.35(t,J＝11.5Hz,1H),2.01–1.91(m,2H),(d,J＝15.7Hz,2H),1.87(d,J＝11.8Hz,2H),1.78(d,J＝12.0Hz,2H),1.48(q,J＝12.0Hz,2H),1.44–1.32(m,11H),1.13(q,J＝12.5Hz,2H).将上述反应产物溶于5mL二氯甲烷中，加入2.5mL三氟乙酸，在55℃下回流4h，脱去Boc保护基后得1.1g用于下一步。
步骤6：3-苄基-1-(4-(4-(2-(2,6-二氧哌啶-3-基)-1,3-二氧异吲哚-5-基)哌嗪-1-基)哌啶-1-基苯基)-1-((1r，4r)-4-(喹唑啉-2-基氨基)环己基)脲(YJZ1202)的制备
将化合物21通过实施例44步骤3的类似方法获得YJZ1102。1H NMR(400MHz,DMSO-d6)δ11.08(s,1H),9.05(s,1H),7.74(d,J＝8.0Hz,1H),7.68(d,J＝8.4Hz,1H),7.63(t,J＝7.9Hz,1H),7.40(d,J＝8.5Hz,1H),7.34(s,1H),7.31–7.21(m,4H),7.21–7.13(m,4H),7.01(s,4H),5.55(t,J＝6.5Hz,1H),5.08(dd,J＝13.0,5.4Hz,1H),4.28(t,J＝12.3Hz,1H),4.16(d,J＝6.0Hz,2H),3.81(d,J＝11.7Hz,2H),3.59(s,1H),3.44(s,4H),2.95–2.83(m,1H),2.74(t,J＝11.8Hz,2H),2.66(s,4H),2.63–2.53(m,2H),2.40(t,J＝11.0Hz,1H),2.06–1.87(m,5H),1.79(d,J＝11.9Hz,2H),1.53(q,J＝11.9Hz,2H),1.41(q,J＝12.5Hz,2H),1.13(q,J＝12.6Hz,2H).13C NMR(151MHz,DMSO-d6)δ173.29,170.56,168.04,167.47,162.47,157.33,155.73,150.64,141.80,134.42,134.32,131.99(2C),128.51(3C),128.38,128.30,127.16(4C),126.71,125.36,122.17,119.96,118.76,118.21,116.24(2C),108.28,61.03,53.53,49.24,49.13,48.91(2C),47.94(2C),47.71(2C),43.94,40.51,31.77,31.45,30.88(2C),28.38(2C),22.65.HRMS(ESI)for C50H54N10O5[M+H]+,calcd:875.43514,found:875.4315.HPLC analysis:MeOH-H2O(80:20),9.43min,95.2％purity.
实施例54：3-苄基-1-(4-(4-(1-(2-(2,6-二氧哌啶-3-基)-1,3-二氧异吲哚-5-基)哌啶-4-基)-哌嗪-1-基)-3-氟苯基)-1-((1r，4r)-4-(喹唑啉-2-基氨基)环己基)脲(YJZ1204)
合成方法与实施例53所示方法相似。
1H NMR(400MHz,DMSO-d6)δ11.08(s,1H),9.05(s,1H),7.74(d,J＝8.0Hz,1H),7.69–7.59(m,2H),7.40(d,J＝8.5Hz,1H),7.34(s,1H),7.31–7.21(m,4H),7.21–7.14(m,4H),7.06(t,J＝9.2Hz,1H),7.02–6.91(m,2H),5.92(t,J＝6.4Hz,1H),5.07(dd,J＝13.2,5.4Hz,1H),4.24(t,J＝13.3Hz,1H),4.10(d,J＝12.7Hz,2H),3.67–3.54(m,1H),3.08(s,4H),3.00(t,J＝12.3Hz,2H),2.94–2.82(m,1H),2.67(s,4H),2.63–2.53(m,3H),2.07–1.93(m,3H),1.90(d,J＝12.4Hz,2H),1.81(d,J＝11.8Hz,2H),1.50(q,J＝11.8Hz,2H),1.40(q,J＝12.2Hz,2H),1.14(q,J＝11.9Hz,2H).13C NMR(151MHz,DMSO-d6)δ173.30,170.59,168.09,167.44,162.48,157.02,155.23,153.97(d,J＝246.4Hz,1C),141.77,139.82(d,J＝8.1Hz,1C),134.51,134.44,132.01(d,J＝9.2Hz,1C),128.49(3C),128.32,128.04,127.17(4C),126.70,125.49,122.18,119.97,119.43,119.30(d,J＝14.5Hz,1C),118.09(d,J＝14.1Hz,1C),108.29,61.06,53.80,50.74,49.27(2C),49.21(2C),49.06,47.10(2C),43.94,40.51,31.76,31.45,30.77(2C),27.64(2C),22.66.HRMS(ESI)for C50H53N10O5F[M+H]+,calcd:893.42572,found:893.4225.HPLC analysis:MeOH-H2O(80:20),10.01min,95.8％purity.
实施例55：代表性化合物对MDA-MB-231细胞中CDK12/13降解活性的测试
实验方法:细胞株MDA-MB-231购自美国标准生物品收藏中心(ATCC)。使用常规Western Blot(免疫印迹法)进行检测，具体如下：将MDA-MB-231细胞按2×105/mL密度种于12孔板，每孔种1mL，于培养箱内贴壁培养过夜。随后加入一定浓度的化合物作用15h，用裂解液裂解细胞，收取蛋白质样品。取适量样品进行SDS-PAGE电泳，电泳结束后用湿法电转移系统将蛋白转移至聚偏二氟乙烯(PVDF)膜，将电转完成的PVDF膜切割为所需的若干条带，随后将其置于封闭液(5％脱脂奶粉稀释于含0.1％Tween 20的TBS)中室温封闭1.5h。按照不同抗体的稀释要求如1:1000(CST)，1：3000(Proteintech，Bethyl)，1：200(Santacruz)将对应靶蛋白的一抗稀释于含0.1％Tween 20的TBS中完成一抗溶液的配制，将封闭完成后的膜被分别置于对应的一抗溶液中于4℃孵育12-14h。随后用含0.1％Tween 20的TBS洗涤三次，每次5min。将洗涤完成后的膜置于二抗溶液(辣根过氧化物酶标记羊抗兔的IgG，1:2000稀释于含0.1％Tween 20的TBS)中室温反应2h。同上洗膜三次后，用StarSignal plus试剂和Omni-ECL试剂发色，Amersham Imager 800system拍照。
图1结果表明，部分化合物能有效降解细胞内的CDK12和CDK13蛋白水平，如化合物ZLC4-77、ZLC4-91、ZLC5-6、ZLC5-11、ZLC7-36、ZLC5-106、ZLC6-1、ZLC6-37、ZLC6-42和ZLC6-103等。图2结果显示，化合物ZLC4-91和ZLC5-11可 时间依赖性降解MDA-MB-231细胞中的CDK12和CDK13蛋白，在1μΜ浓度下处理细胞15小时后蛋白已被几乎完全降解。此外，化合物对CDK12和CDK13的伴侣蛋白Cyclin K具有时间依赖性的降解效果，15小时后细胞内的Cyclin K蛋白已被几乎完全降解。
我们进一步测定了代表性化合物在不同浓度下对MDA-MB-231细胞中CDK12和CDK13蛋白的降解情况。实验结果表明这些代表性化合物能剂量依赖性地降解MDA-MB-231细胞中CDK12和CDK13蛋白，在1μM浓度下可将靶蛋白有效降解(图2)。
图1，(A)部分化合物在0.1μΜ浓度下处理MDA-MB-231细胞15小时后对CDK12、CDK13蛋白降解的WB结果；(B)部分化合物在0.3μΜ浓度下处理MDA-MB-231细胞15小时后对CDK12、CDK13蛋白降解的WB结果；(C)以对照组DMSO为参照标准，通过对WB结果图像(图A)中条带的灰度值定量CDK12/13水平；(D)以对照组DMSO为参照标准，通过对WB结果图像(图B)中条带的灰度值定量CDK12/13水平。
图2，(A)代表性化合物时间依赖性降解MDA-MB-231细胞中的CDK12和CDK13蛋白；(B)代表性化合物在不同浓度下处理MDA-MB-231细胞15小时后，细胞内CDK12/13蛋白水平的WB测定结果。
实施例56：化合物对三阴乳腺癌细胞MDA-MB-231的增殖抑制活性研究
采用Cell Counting Kit-8Cell Viability Assay(Selleck.cn)测定化合物的细胞增殖抑制活性。将细胞接种在含其对应培养基的384孔板(PS 3701)中，于37℃、含5％CO2的培养箱孵育。孵育过夜后，配制各待测化合物的高浓度母液；使用650 Liquid Handler将化合物按照一定浓度梯度添加到384孔板中。给药完成后将细胞板置于37℃、含5％CO2的培养箱孵育5天。随后使用(Thermo ScientificTM)向384孔板中每孔加入5μL cck-8试剂，将孔板重新放回培养箱中孵育1.5-2h。孵育完成后将孔板取出，离心除气泡后使用获取每个孔于450nm和650nm处的吸光度值，使用GraphPad Prism软件(GraphPad Software Inc)处理分析数据。
图3结果表明，能有效降解CDK12和CDK13蛋白水平的化合物如：ZLC4-77、ZLC4-91、ZLC5-6、ZLC5-31和ZLC6-1可有效抑制三阴性乳腺癌细胞MDA-MB-231的生长，对MDA-MB-231细胞系的IC50分别为610.4nM、622.5nM、497.9nM、389.0nM和288.6nM。
实施例57：细胞周期依赖性蛋白激酶12/13(CDK12/13)降解剂的代表性化合物zlc-4-91、zlc-4-93的体内药代动力学实验
大鼠药代动力学和生物利用度试验。SD大鼠，单次口服(10mg/kg)、静脉(2.5mg/kg)和腹腔注射(10mg/kg)给药，给药后在合适的时间点采集动物血样，肝素抗凝，8000转/分钟，离心6分钟，取上清，-20℃保存备HPLC-MS分析。血样采用乙腈沉淀蛋白，12000rpm*10min，上清液用于HPCL-MS分析。数据采用DAS2.0进行参数拟合，分别获得房室模型和非房室模型参数。根据血药浓度-时间曲线下面积(AUC)数据计算化合物的口服生物利用度。结果见表1。实验结果表明(表1)代表性化合物zlc-4-91、zlc-4-93具有较优异的口服药代动力学性质。
表1.化合物zlc-4-93的药代动力学实验结果
Cmax是指最大血药浓度，T1/2为半衰期，CL是指清除率，F是指生物利用度。
实施例58：代表性化合物对22RV1细胞中CDK12/13降解活性的测试
实验方法:细胞株:22RV1细胞株购自美国标准生物品收藏中心(ATCC)。使用常规Western Blot(免疫印迹法)进行检测，具体如下：将22RV1按一定数量种于12孔板，培养箱内贴壁培养过夜后，加入一定浓度的化合物作用6h，用裂解液裂解细胞收样。然后取适量样品进行SDS-PAGE电泳，电泳结束后用半干电转移系统将蛋白转移至硝酸纤维素膜，将硝酸纤维素膜置于封闭液(5％脱脂奶粉稀释于含0.1％Tween 20的TBS)中室温封闭2h，然后将膜分别置于一抗溶液(1:1000稀释于含0.1％Tween 20的TBS)中4℃孵育过夜。用含0.1％Tween 20的TBS洗涤三次，每次15min。将膜置于二抗溶液(辣根过氧化物酶标记羊抗兔的IgG，1:1000稀释于含0.1％Tween 20的TBS)中室温反应lh。同上洗膜三次后，用ECL plus试剂发色，Amersham Imager 600system拍照。
图4结果表明，部分化合物能有效降解细胞内的CDK12和CDK13蛋白水平，如化合物YJZ9069、YJZ1102、YJZ1105、YJZ1201、YJZ1202、YJZ1203、YJZ1204、YJZ1097、YJZ1205和YJZ1206等。其中化合物YJZ9069可以剂量依赖性降解22RV1细胞中的CDK12和CDK13蛋白，化合物YJZ9069在0.5μΜ浓度下处理22RV1细胞15小时后几乎完全降解细胞内的CDK12和CDK13蛋白，并且剂量依赖性地抑制RNA聚合酶II(RNA Pol II)C端2位丝氨酸的磷酸化(phoSer2)。
我们进一步测定了代表性化合物YJZ9069、YJZ1201、YJZ1202、YJZ1203、YJZ1204、YJZ1097、YJZ1205和YJZ1206在不同浓度下对22RV1细胞中CDK12和CDK13蛋白的降解情况(图2)。WB实验结果表明代表性化合物YJZ9069、YJZ1201、YJZ1202、YJZ1203、YJZ1204、YJZ1097、YJZ1205和YJZ1206能剂量依赖性地在降解22RV1细胞中CDK12和CDK13蛋白水平，并且在100nM浓度下几乎完全地降解细胞内的CDK12和CDK13蛋白(图5)
图4，(A)部分化合物在500nM浓度下处理22RV1细胞6小时后对CDK12和CDK13蛋白降解的WB结果。(B)化合物YJZ9069剂量依赖性地降解22RV1细胞中的CDK12和CDK13蛋白，及剂量依赖性地抑制RNA聚合酶II(RNA Pol II)C端2位丝氨酸的磷酸化(phoSer2)。(C)部分化合物在500nM浓度下处理LnCap细胞6小时后对CDK12和CDK13蛋白降解的WB结果。(D)以对照组DMSO为参照标准，通过对WB结果图像(图A，C)中条带的灰度值来定量蛋白质水平。
图5，代表性化合物YJZ9069、YJZ1201、YJZ1202、YJZ1203、YJZ1204、YJZ1097、YJZ1205和YJZ1206在不同浓度下处理22RV1细胞6小时后，细胞内CDK12和CDK13蛋白水平的WB测定结果。
实施例59：化合物对前列腺癌VCap细胞的增殖抑制活性研究
采用CellTiter-Glo Luminescent Cell Viability Assay(Promega,Madison,WI)测定化合物的细胞增殖活性。将细胞接种在其各自培养基中的96孔板上，37℃下在5％CO2的培养箱孵育。孵育过夜后，制备化合物的系列稀释液，并将其添加到96孔板中。加入等于每个孔中存在的细胞培养基体积的CellTiter-Glo试剂的体积(例如，向100微升96孔板细胞的培养基中添加100微升试剂)。在轨道振荡器上混合内容物2分钟以诱导细胞裂解。让96孔板在室温下孵育10分钟以稳定发光信号。记录发光：使用Infinite M1000Pro读板器(Tecan,Zürich,Switzerland)获取每个孔的发光信号，使用GraphPad Prism软件(GraphPad Software Inc,La Jolla,CA)分析数据。
图6结果表明，能有效降解CDK12和CDK13蛋白水平的化合物如：YJZ9069、YJZ1102、YJZ1105、YJZ1201、YJZ1202、YJZ1203、YJZ1204、YJZ1097、YJZ1205和YJZ1206可以强效地抑制前列腺癌Vcap细胞的生长，对VCap细胞系的IC50分别为22.90nM、16.31nM、15.11nM、64.4nM、17.46nM、18.78nM、23.31nM、3.93nM、和13.50nM。而不能有效诱导CDK12和CDK13蛋白降解的化合物如：YJZ9055和YJZ1078对VCap细胞系的IC50分别为2202nM和>5μM,说明该系列化合物有效抑制Vcap细胞的生长是由于降解CDK12和CDK13蛋白引起的。
实施例60：细胞周期依赖性蛋白激酶12/13(CDK12/13)降解剂的代表性化合物YJZ9069、YJZ1102、YJZ1105、YJZ1201、YJZ1202、YJZ1203、YJZ1204、YJZ1097、YJZ1205和YJZ1206的体内药代动力学实验
大鼠药代动力学和生物利用度试验。SD大鼠，单次口服(10mg/kg)、静脉(2.5mg/kg)和腹腔注射(10mg/kg)给药，给药后在合适的时间点采集动物血样，肝素抗凝，8000转/分钟，离心6分钟，取上清，-20℃保存备HPLC-MS分析。血样采用乙腈沉淀蛋白，12000rpm*10min，上清液用于HPCL-MS分析。数据采用DAS2.0进行参数拟合，分别获得房室模型和非房室模型参数。根据血药浓度-时间曲线下面积(AUC)数据计算化合物的口服生物利用度。结果见表2-6。实验结果表明(表2-6)代表性化合物YJZ1102、YJZ1105、YJZ1201、YJZ1202、YJZ1203、YJZ1204、YJZ1097、YJZ1205和YJZ1206具有较优异的口服药代动力学性质。
表2.化合物YJZ9069和YJZ1102的药代动力学实验结果

Cmax是指最大血药浓度，T1/2为半衰期，CL是指清除率，F是指生物利用度。
表3.化合物YJZ1105和YJZ1202的药代动力学实验结果
Cmax是指最大血药浓度，T1/2为半衰期，CL是指清除率，F是指生物利用度。
表4.化合物YJZ1203和YJZ1204的药代动力学实验结果
Cmax是指最大血药浓度，T1/2为半衰期，CL是指清除率，F是指生物利用度。
表5.化合物YJZ1205和YJZ1201的药代动力学实验结果
Cmax是指最大血药浓度，T1/2为半衰期，CL是指清除率，F是指生物利用度。
表6.化合物YJZ1206和YJZ1097的药代动力学实验结果
Cmax是指最大血药浓度，T1/2为半衰期，CL是指清除率，F是指生物利用度。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对发明专利范围的限制。以上所述实施例的各技术特征可以进行任意的组合，为使描述简洁，未对上述实施例中的各个技术特征所有可能的组合都进行描述，然而，只要这些技术特征的组合不存在矛盾，都应当认为是本说明书记载的范围。

Claims (22)

  1. 具有式(I)结构的化合物，或其药学上可接受的盐，或其药物组合物或其前药分子：
    其中，Z为：CH2或CO；
    V选自下组：
    X、Y各自独立地选自下组：N、CH或CR3；其中，R3选自下组：卤素、氰基、羟基、氨基、C1-C3烷基、卤代C1-C3烷基、C1-C3烷氧基、卤代C1-C3烷氧基、C3-C8环烷基，或3-8元杂环基；
    B选自下组： NH、O、CO、CH2；其中，各个U、W各自独立选自下组：N或CH；各个m、p、m'、p'各自独立地为：0、1、2或3；
    A环选自下组：
    其中，Q、W各自独立地选自：CH、N；
    R'独立地选自：氢、卤素、氰基、羟基、取代羟基、氨基、取代氨基、C1-C5烷基、卤代C1-C5烷基、C1-C5烷氧基、卤代C1-C5烷氧基、C3-C8环烷基，且当Q、W独立地选自CH时，R'可以为Q、W上的取代基；
    D、E、F、G各自独立选自下组：CH、N、CR6；其中，R6选自下组：卤素、三氟甲基、羟基、氰基、胺基、甲基、甲氧基、三氟甲氧基；且当D、E、F、G中任一为CH时，该原子可以作为与linker的连接位点，此时所述的D、E、F或G为C；
    R1选自下组：H、-NHR7、-OR7、-(C(R9)R8)R7；
    其中，R7为-R10、-CH2R10或-(CH2)2R10；
    R8、R9各自独立地选自下组：氢、卤素、氰基、甲基、卤代甲基、甲氧基、卤代甲氧基、乙基、卤代乙基、乙氧基、卤代乙氧基、羟基、氨基、含有1、2或3个杂原子的3-8元杂环，且所述的杂原子选自O、S或N；
    或者R8、R9及其相连的C原子共同形成3-7杂环；
    R10选自下组：
    1)氰基、C1-C5烷基、卤代C1-C4烷基、C1-C4烷氧基、C3～C10环烷基、取代或未取代的 5-12元芳环、取代或未取代的3-12元杂环；
    2)
    其中Q1、Q2、Q3、Q4、Q5各自独立地选自：CH、N或CR11；
    各个R11各自独立地选自下组：卤素、氰基、羟基、氨基、硝基、C1-C3烷基、卤代C1-C3烷基、C1-C4烷氧基、卤代C1-C4烷氧基、C3-C8环烷基；
    R2选自下组：H、C1-C3烷基、
    当B选自下组： NH、O、CO、CH2；其中，各个U、W各自独立选自下组：N或CH；各个m、p、m'、p'各自独立地为：0、1、2或3；时
    Linker为：
    其中，
    RL1、RL2、RL3、RL4和RL5相同或不同，且各自独立地选自取代或未取代的下组基团：化学键、CH2、CHD、CD2、C＝O、O、NH、SO、SO2、P＝O、NHCO、NHSO2、OCH2、OCH2CH2、CH2OCH2、NHCH2、NMeCH2、NHCH2CH2、NMeCH2CH2、CH2NHCO、NHCOCH2、

    RL6为环，且任选自以下结构：


    其中各个n各自独立地为0，1，2，3，4，5或6；各个r、m各自独立地为0，1或2；各个U、W各自独立选自下组：N或CH；
    pL1、pL2、pL3、pL4、pL5和pL6各自独立地选自0、1、2、3、4、5、或6；或
    当B为其中各个m、p、m'、p'各自独立地为：0、1、2或3；时
    Linker为：
    其中，
    RL1、RL2、RL3、RL4、RL5和RL6相同或不同，且各自独立地选自取代或未取代的下组基团：化学键、CH2、CHD、CD2、C＝O、O、NH、SO、SO2、P＝O、NHCO、NHSO2、OCH2、OCH2CH2、CH2OCH2、NHCH2、NMeCH2、NHCH2CH2、NMeCH2CH2、CH2NHCO、NHCOCH2、



    其中各个n各自独立地为0，1，2，3，4，5或6；各个r、m各自独立地为0，1或2；各个U、W各自独立选自下组：N或CH；
    pL1、pL2、pL3、pL4、pL5和pL6各自独立地选自0、1、2、3、4、5、或6。
  2. 如权利要求1所述的化合物，或者其药学上可接受的盐或者其立体异构体或者其前药分子，其特征在于，
    B选自下组：NH、O、CO；
    其中，各个U、W各自独立选自下组：N或CH；各个m、p、m'、p'各自独立地为0、1、2或3。
  3. 如权利要求1所述的化合物，或者其药学上可接受的盐或者其立体异构体或者其前药分子，其特征在于，
    A环选自下组：
    其中，W选自下组：CH、N；
    R'选自：氢、卤素、氰基、羟基、氨基、C1-C5烷基、卤代C1-C5烷基、C1-C5烷氧基、卤代C1-C5烷氧基、C3-C8环烷基。
  4. 如权利要求1所述的化合物，或者其药学上可接受的盐或者其立体异构体或者其前药分子，其特征在于，所述的D、E、F、G为CH、CR6；其中，R6选自下组：卤素。
  5. 如权利要求1所述的化合物，或者其药学上可接受的盐或者其立体异构体或者其前药分子，其特征在于，
    当B选自下组：NH、O、CO；时，所述的Linker选自下组：
    且当B为时，所述的Linker选自下组：化学键、
  6. 如权利要求1所述的化合物，或者其药学上可接受的盐或者其立体异构体或者其前药分子，其特征在于，具有式(II)所示结构：
    其中，B选自：
    其中，各个U、W各自独立选自下组：N或CH；各个m、p、m'、p'各自独立地为0、1、2或3；
    R'任选自：氢、卤素、氰基、羟基、氨基；
    R11、R6任选自：氢、卤素；
    X，Y独立选自：CH、N或CR3；
    其中，R3任选自：卤素、氰基、羟基、氨基；
    Z任选自：CH2或CO。
  7. 具有式(I)结构的化合物，或其药学上可接受的盐，或其药物组合物或其前药分子：
    其中，Z选自下组：CH2或CO；
    V选自下组：
    X、Y选自下组：N、CH或CR3；其中，R3选自下组：卤素、氰基、羟基、氨基、C1-C3烷基、卤代C1-C3烷基、C1-C3烷氧基、卤代C1-C3烷氧基、C3-C8环烷基，或3-8元杂环基；
    B选自下组： NH、O、CO、CH2；其中，各个U、W各自独立选自下组：N或CH；各个m、p各自独立地为：0、1或2；
    A环选自下组：
    M、T各自独立选自下组：N、CR4；
    I、J、K各自独立选自下组：N、O、S、CR4和NR4；
    R4选自下组：氢、卤素、氰基、羟基、氨基、C1-C5烷基、卤代C1-C5烷基、C1-C5烷氧基、卤代C1-C5烷氧基、C3-C8环烷基、-CH2R5、-(CH2)2R5、-(CH2)3R5或3-8元杂环基；
    R5选自下组：氰基、羟基、氨基、C3-C8环烷基、或3-8元杂环基；
    D、E、F、G各自独立选自下组：CH、N、CR6；其中，R6选自下组：卤素、三氟甲基、羟基、氰基、胺基、甲基、甲氧基、三氟甲氧基；
    R1选自下组：H、-NHR7、-OR7、-(C(R9)R8)R7；
    其中，R7为-R10、-CH2R10或-(CH2)2R10；
    R8、R9各自独立地选自下组：氢、卤素、氰基、甲基、卤代甲基、甲氧基、卤代甲氧基、乙基、卤代乙基、乙氧基、卤代乙氧基、羟基、氨基、含有1、2或3个杂原子的3-8元杂环，且所述的杂原子选自O、S或N；
    或者R8、R9及其相连的C原子共同形成3-7杂环；
    R10选自下组：
    1)氰基、C1-C5烷基、卤代C1-C4烷基、C1-C4烷氧基、C3～C10环烷基、取代或未取代的 5-12元芳环、取代或未取代的3-12元杂环；
    2)
    其中Q1、Q2、Q3、Q4、Q5各自独立地选自：CH、N或CR11；
    各个R11各自独立地选自下组：卤素、氰基、羟基、氨基、硝基、C1-C3烷基、卤代C1-C3烷基、C1-C4烷氧基、卤代C1-C4烷氧基、C3-C8环烷基；
    R2选自下组：H、C1-C3烷基、
    Linker为：
    其中，RL1-RL6相同或不同，且各自独立地选自取代或未取代的下组基团：键、CH2、CHD、CD2、C＝O、O、NH、SO、SO2、P＝O、NHCO、NHSO2、OCH2、OCH2CH2、CH2OCH2、NHCH2、NMeCH2、NHCH2CH2、NMeCH2CH2、CH2NHCO、NHCOCH2、
    其中各个n各自独立地为0，1，2，3，4，5或6；各个r、m各自独立地为0，1或2；各个U、W各自独立选自下组：N或CH
    所述取代是指被选自下组的一个或多个基团取代：氢、氘、C1-C18烷基、氘代C1- C18烷基、卤代C1-C18烷基、卤代C1-C18烷基羟基、C3-C20环烷基、C1-C18烷氧基、氘代C1-C18烷氧基、卤代C1-C18烷氧基、C6-C14芳基、5-14元杂芳基、4-20元杂环基、卤素、氧代基、硝基、羟基、氰基、酯基、胺基、酰胺基、磺酰胺基或脲基；
    各pL1-pL6独立地选自0、1、2、3、4、5、或6。
  8. 如权利要求1或7所述的化合物，或者其药学上可接受的盐或者其立体异构体或者其前药分子，其特征在于，
    V选自下组：
  9. 如权利要求1或7所述的化合物，或者其药学上可接受的盐或者其立体异构体或者其前药分子，其特征在于，
    X、Y各自独立选自下组：N、CH或CR3；
    其中，R3选自下组：卤素、氰基、羟基、氨基、C1-C3烷基、卤代C1-C3烷基、C1-C3烷氧基、卤代C1-C3烷氧基。
  10. 如权利要求7所述的化合物，或者其药学上可接受的盐或者其立体异构体或者其前药分子，其特征在于，
    B选自下组：NH、O、CO；
    其中，各个U、W各自独立选自下组：N或CH；各个m、p各自独立地为0或1。
  11. 如权利要求7所述的化合物，或者其药学上可接受的盐或者其立体异构体或者其前药分子，其特征在于，
    A环选自下组：
    其中，T选自下组：CH、N。
  12. 如权利要求7所述的化合物，或者其药学上可接受的盐或者其立体异构体或者其前药分子，其特征在于，
    R4选自下组：氢、卤素、氰基、羟基、氨基、C1-C5烷基、卤代C1-C5烷基、C1-C5烷氧基、卤代C1-C5烷氧基、C3-C8环烷基、-CH2R5、-(CH2)2R5、-(CH2)3R5或3-8元杂环基。
  13. 如权利要求7所述的化合物，或者其药学上可接受的盐或者其立体异构体或者其前药分子，其特征在于，所述的D、E、F、G为CH。
  14. 如权利要求7所述的化合物，或者其药学上可接受的盐或者其立体异构体或者其前药分子，其特征在于，所述的R1为-NHR7；
    其中，R7为-CH2R10；R10的定义如权利要求7中所述。
    在另一优选例中，所述的R10选自下组：氰基、C1-C5烷基、卤代C1-C4烷基、C1-C4烷氧基、C3-C10环烷基、4-7元杂环基、C6-C10芳基；其中所述的芳基被一个或多个CR11取代；R11的定义如权利要求7中所述。
  15. 如权利要求7所述的化合物，或者其药学上可接受的盐或者其立体异构体或者其前药分子，其特征在于，
    Linker选自下组： 其中各个n各自独立地为0，1，2，3，4，5或6；各个r、m各自独立地为0，1或2；各个U、W各自独立选自下组：N或CH。
    在另一优选例中，Linker选自下组：
    其中各个n各自独立地为0，1，2，3，4，5或6；各个r、m各自独立地为0，1或2；各个U、W各自独立选自下组：N或CH。
  16. 如权利要求7所述的化合物，或者其药学上可接受的盐或者其立体异构体或者其前药分子，其特征在于，具有式(II)或式(III)所示结构：
    其中，U，W独立选自：CH或N；
    X，Y独立选自：CH、N或CR3；
    Z任选自：CH2或CO；
    n，m独立选自：0或1。
  17. 如权利要求1所述的化合物，或者其药学上可接受的盐或者其立体异构体或者其前药分子，其特征在于，所述化合物选自下组：



  18. 如权利要求7所述的化合物，或者其药学上可接受的盐或者其立体异构体或 者其前药分子，其特征在于，所述化合物选自下组：


  19. 一种药物组合物，其特征在于，包含
    (1)作为活性成分的如权利要求1-18任一所述的化合物，或者药学上可接受的盐、其立体异构体或其前药分子；和
    任选的(2)药学上可接受的载体。
  20. 如权利要求1-18任一所述的化合物，或者其药学上可接受的盐或者其立体异构体或者其前药分子，或者如权利要求19所述的药物组合物的应用，其特征在于，用于制备CDK12/13蛋白激酶降解剂。
  21. 如权利要求1-18任一所述的化合物，或者其药学上可接受的盐或者其立体异构体或者其前药分子，或者如权利要求19所述的药物组合物的应用，其特征在于，用于制备预防和/或治疗由CDK12/13丝氨酸/苏氨酸蛋白激酶介导的疾病的药物。
  22. 如权利要求21所述的应用，其特征在于，所述由CDK12/13丝氨酸/苏氨酸蛋白激酶介导的疾病选自下组：前列腺癌、乳腺癌、子宫癌、卵巢癌、非小细胞肺癌、小细胞肺癌、尤文肉瘤、肺腺癌、肺鳞癌、胰腺癌、肝癌、皮肤癌、上皮细胞癌、胃肠间质瘤、白血病、组织细胞性淋巴癌、鼻咽癌、头颈部肿瘤、结肠癌、直肠癌、胶质瘤。
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WO2025176178A1 (zh) * 2024-02-20 2025-08-28 中国科学院上海有机化学研究所 一类并环结构取代的新型cdk12/13共价抑制剂或其药物组合物和应用

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