WO2020253711A1 - 一种吡咯并吡啶酮类化合物、其制备方法、其组合物和用途 - Google Patents

一种吡咯并吡啶酮类化合物、其制备方法、其组合物和用途 Download PDF

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WO2020253711A1
WO2020253711A1 PCT/CN2020/096521 CN2020096521W WO2020253711A1 WO 2020253711 A1 WO2020253711 A1 WO 2020253711A1 CN 2020096521 W CN2020096521 W CN 2020096521W WO 2020253711 A1 WO2020253711 A1 WO 2020253711A1
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胡有洪
耿美玉
陈菁菁
沈爱军
谢志铖
刘红椿
曾丽敏
李亚磊
彭润泽
张敏敏
张婕
丁健
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中国科学院上海药物研究所
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/04—Ortho-condensed systems
    • 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
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/437—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a five-membered ring having nitrogen as a ring hetero atom, e.g. indolizine, beta-carboline
    • 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
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/444—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a six-membered ring with nitrogen as a ring heteroatom, e.g. amrinone
    • 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
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/4523—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems
    • A61K31/4545—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems containing a six-membered ring with nitrogen as a ring hetero atom, e.g. pipamperone, anabasine
    • 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
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
    • 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
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/538—1,4-Oxazines, e.g. morpholine ortho- or peri-condensed with carbocyclic ring systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the present invention relates to a pyrrolopyridone compound or a pharmaceutically acceptable salt thereof, a preparation method thereof, and a pharmaceutical use of the composition for treating tumor diseases related to the target.
  • acetylation of histone lysine residues is considered to be a marker of transcriptionally active genes (see Non-Patent Document 1).
  • lysine acetylation can neutralize its positive charge, resulting in a decrease in the affinity of histones for negatively charged DNA or loosening the structure of the nucleosome, ultimately forming an open and accessible chromatin structure , Can recruit transcription factors.
  • acetylated lysine provides a binding site for the protein recognition module.
  • the acetylation and deacetylation of histone lysine residues are mainly controlled by histone acetylase (HATs) and histone deacetylases (HDACs).
  • acetylation on histones can be specifically recognized by bromodomains (BRDs), recruiting chromatin regulation related proteins, transcription factors, chromatin remodeling factors, etc., thereby regulating gene transcription and chromatin remodeling Play an important role (see Non-Patent Document 2).
  • Bromodomains can be divided into 8 families according to structural and sequence similarity. Among them, the bromodomain and extra-terminal domain (BET) family belong to the second category in the BRD family, including BRD2, BRD3, BRD4, and BRDT. BET protein can recognize acetylated histones, recruit transcription factors and transcription elongation factors, etc., regulate cell transcription and cell cycle, and play an important role in a variety of diseases, especially tumors and inflammation (see Non-Patent Document 3). As the main member of this family, BRD4 is closely related to transcriptional regulation and tumorigenesis (see Non-Patent Document 4).
  • BRD4 Oncogenes that regulate cancer cell proliferation, anti-apoptosis and invasiveness are regulated by BRD4.
  • c-Myc is the first reported oncogene regulated by BRD4 in hematoma and solid tumors, and it is a BET protein inhibitor.
  • the development provides a theoretical basis (see Non-Patent Documents 5 and 6).
  • Some other oncogenes such as FOSL1 (FRA-1), BCL-2, RUNX2, c-KIT, etc. are regulated by BRD4 (see Non-Patent Documents 7-10).
  • BRD4 has been shown to regulate the molecular mechanisms related to the repair of damaged DNA and is related to abnormal telomere regulation in cancer, which highlights the functional diversity of this protein in the process of cancer (see Non-Patent Literature 11, 12).
  • BET inhibitors with multiple structural types have been developed, such as I-BET 762, JQ1, I-BET 151, OTX-015, TEN-010, ZEN003694, GS-5829, CPI-0610, ABBV-075 , ABBV-744, etc., are widely used in the research of hematoma, solid tumor and cardiovascular disease.
  • I-BET 762, JQ1, I-BET 151, OTX-015, TEN-010, ZEN003694, GS-5829, CPI-0610, ABBV-075 , ABBV-744, etc. are widely used in the research of hematoma, solid tumor and cardiovascular disease.
  • toxic side effects such as severe thrombocytopenia, fatigue, nausea, vomiting and diarrhea and other gastrointestinal side effects.
  • HDAC deacetylates histones, which makes the binding of histones and DNA closer, thereby inhibiting gene expression.
  • Current studies have shown that HDAC can affect the cell cycle, cell apoptosis, response to DNA damage, cancer cell metastasis, angiogenesis, autophagy and other cellular processes, and play a vital role in tumor progression (see Non-Patent Document 14).
  • Inhibition of HDAC can cause changes in the overall acetylation level of histones, especially the lack of acetylation or low-density acetylation regions (see Non-Patent Documents 15-17).
  • HDAC and BET protein may have some direct or indirect interactions through the genome acetylation profile (see Non-Patent Document 18).
  • the super enhancer in the growth center of cancer cells depends on the BRD4 protein, so BET inhibitors can preferentially inhibit cancer cell-dependent oncogene transcription (see Non-Patent Document 4); while HDAC inhibitors can increase the acetylation of histones and other transcriptional regulators.
  • BRD4 protein has the function of histone molecular chaperones, which can assist RNA polymerase II through acetylation of nucleosomes, while HDAC removes the acetylation of histones or other proteins, and changes BRD4 in histones in nucleosomes and staining. Binding of mass-related protein complexes (see Non-Patent Document 19).
  • HDAC and BRD4 inhibitors can exhibit synergistic effects in a variety of tumor cells, and the combination of this drug can achieve higher cytotoxicity at lower doses, which may avoid the problem of superimposed toxicity .
  • BET inhibitors can partially sensitize c-Myc overexpressing lymphoma cells induced by HDAC-silencing genes, while the combination of BET inhibitor RVX2135 and HDAC inhibitor SAHA has an effect on lymphoma.
  • the progression-free survival of tumor transplanted mice has a synergistic effect (see Non-Patent Document 20).
  • the combination therapy of pabirestat and JQ1 can induce apoptosis and growth inhibition more effectively than the single drug, while the treatment has no significant effect on normal cells and can Prevent tumor growth and improve the survival rate of transplanted tumor mice (see Non-Patent Documents 21 and 22).
  • JQ1 and SAHA can produce effective and sustained anti-tumor effects in both in vivo and in vitro models of pancreatic ductal carcinoma (PDAC) (see Non-Patent Document 23); in urothelial cancer cell lines, JQ1 and Romi
  • PDAC pancreatic ductal carcinoma
  • the combined use of Desine showed a synergistic effect (see Non-Patent Document 24);
  • the combined use of Pabirestat and I-BET151 can strongly induce apoptosis and cell cycle arrest in melanoma cell lines and BRAFi-resistant melanoma patients Hysteresis (see Non-Patent Document 25);
  • panobinostat and OTX-015 can inhibit the proliferation of glioblastoma cells, enhance caspase-mediated apoptosis, especially inhibit phosphorylation and glycolysis, leading to energy crisis and serious Stress response (see Non-Patent Document 26).
  • Non-Patent Document 1 Hewings DS, Rooney TPC, Jennings LE, et al. Progress in the Development and Application of Small Molecule Inhibitors of Bromodomain-Acetyl-lysine Interactions. Journal of Medicinal Chemistry 2012; 55:9393-413.
  • Non-Patent Document 2 Arrowsmith CH, Bountra C, Fish PV, Lee K, Schapira M. Epigenetic protein families: a new frontier for drug discovery. Nat Rev Drug Discov 2012; 11: 384-400.
  • Non-Patent Document 3 Liu Z, Wang P, Chen H, et al. Drug Discovery Targeting Bromodomain-Containing Protein 4.J Med Chem 2017; 60:4533-58.
  • Non-Patent Document 4 Donati B, Lorenzini E, Ciarrocchi A. BRD4 and Cancer: going beyond transcriptional regulation. Mol Cancer 2018; 17:164.
  • Non-Patent Document 5 Delmore JE, Issa GC, Lemieux ME, et al. BET bromodomain inhibition as a therapeutic strategy to target c-Myc.Cell 2011; 146:904-17.
  • Non-Patent Document 6 McCleland ML, Mesh K, Lorenzana E, et al.
  • CCAT1 is an enhancer-templated RNA that predicts BET sensitivity in colorectal cancer. J Clin Invest2016; 126:639-52.
  • Non-Patent Document 7 Wyce A, Ganji G, Smithman KN, et al. BET inhibition silences expression of MYCN and BCL2 and induces cytotoxicity in neuroblastoma tumor models.PLoS One 2013; 8:e72967.
  • Non-Patent Document 8 Zhao Y, Liu Q, Acharya P, et al. High-Resolution Mapping of RNA Polymerases Identifies Mechanisms of Sensitivity and Resistance to BET Inhibitors in t(8; 21) AML. Cell Reports 2016; 16: 2003- 16.
  • Non-Patent Document 9 Sancisi V, Manzotti G, Gugnoni M, et al. RUNX2 expression in thyroid and breast cancer requires the cooperation of three non-redundant enhancers under the control of BRD4 and c-JUN.Nucleic 2017; -67.
  • Non-Patent Document 10 Lockwood WW, Zejnullahu K, Bradner JE, Varmus H. Sensitivity of human lung adenocarcinoma cell lines to targeted inhibition of BET epigenetic signaling proteins: Proceedings of the 2012 National Academy 109;
  • Non-Patent Document 11 Wang S, Pike AM, Lee SS, Strong MA, Connelly CJ, Greider CW. BRD4 inhibitors block telomere elongation. Nucleic Acids Research 2017; 45: 8403-10.
  • Non-Patent Document 12 Li X, Baek G, Ramand SG, et al. BRD4 Promotes DNA Repair and Mediates the Formation of TMPRSS2-ERG Gene Rearrangements in Prostate Cancer Cell Reports 2018; 22:796-808.
  • Non-Patent Document 13 Doroshow DB, Eder JP, LoRusso PM. BET inhibitors: a novel epigenetic approach. Ann Oncol 2017; 28: 1776-87.
  • Non-Patent Literature 14 Roche J, Bertrand P. Inside HDACs with more selective HDAC inhibitors. European Journal of Medicinal Chemistry 2016; 121:451-83.
  • Non-Patent Document 15 Chou C-W, Wu M-S, Huang W-C, Chen C-C. HDAC Inhibition Decreases the Expression of EGFR in Colorectal Cancer Cells. PLOS ONE 2011; 6: e18087.
  • Non-Patent Document 16 Kim YJ, Greer CB, Cecchini KR, Harris LN, Tuck DP, Kim TH. HDAC inhibitors Induce transcriptional repression of high copy number genes in breast cancer through elongation blockade. Oncogene. 2013; 32: 2828
  • Non-Patent Document 17 Wang Z, Zang C, Cui K, et al. Genome-wide Mapping of HATs and HDACs Reveals Distinct Functions in Active and Inactive Genes. Cell 2009; 138: 1019-31.
  • Non-Patent Document 18 Manzotti G, Ciarrocchi A, Sancisi V. Inhibition of BET Proteins and Histone Deacetylase (HDACs): Crossing Roads in Cancer Therapy.Cancers(Basel) 2019; 11.
  • Non-Patent Literature 19 Kanno T, Kanno Y, LeRoy G, et al. BRD4 assists elongation of both coding and enhancer RNAs by interacting with acetylated histones. Nature Structural &Amp; Molecular Biology 2014; 21: 1047.
  • Non-Patent Document 20 Bhadury J, Nilsson LM, Muralidharan SV, et al. BET and HDAC inhibitors induce similar genes and biological effects and synergize to kill in Myc-induced Murine Science A 272; 2014 Acad 1; -30.
  • Non-Patent Document 21 Shahbazi J, Liu PY, Atmadibrata B, et al.
  • the Bromodomain Inhibitor JQ1 and the Histone Deacetylase Inhibitor Panobinostat Synergistically Reduce N-Myc Expression and Induce AnticancerlinEffects.
  • Non-Patent Document 22 Fiskus W, Sharma S, Qi J, et al. Highly active combination of BRD4 antagonist and hisstone deacetylase inhibitor against human acute myelogenous leukemia cells. Mol Cancer Ther 2014; 13:1142-54.
  • Non-Patent Document 23 Mazur PK, Herner A, Mello SS, et al. Combined inhibition of BET family proteins and histone deacetylases as a potential epigenetics-based therapy for pancreatic ductal adenocarcinoma. Nat 3-71. 2015; 21: 116
  • Non-Patent Document 24 Holscher AS, Schulz WA, Pinkerneil M, Niegisch G, Hoffmann MJ. Combined Inhibition of BET proteins and class I HDACs Synergistically Induces Apoptosis in urothelial genecarcinoma: Celllines. Clinoma 2018
  • Non-Patent Literature 25 Anja Heinemann1,3,Carleen Cullinane4,Ricardo De Paoli-Iseppi2,5,James S.,Wilmott2,Dilini Gunatilake1,2,3,Jason Madore2,5,Dario Strbenac6,Jean Y.YangG6,Kavishan1 ,Jessamy C.Tiffen1,2,3,Rab K.Prinjha7,Nicholas Smithers7,,Grant A.McArthur4PH,2,3,Stuart J.Gallagher1,2,3.Combining BET and HDAC inhibitors synerganistically induces up AK and YAP signaling. Oncotarget 2015; 6.
  • Non-Patent Document 26 Zhang Y, Ishida CT, Ishida W, et al. Combined HDAC and Bromodomain Protein Inhibition Reprograms Tumor Cell Metabolism and Elicits Synthetic Legality in Glioblastoma. Clin. Cancer Res 41-5 2018; 24: 24:
  • Non-Patent Document 27 Zhou B, Hu J, Xu F, et al. Discovery of a Small-Molecule Degrader of Bromodomain and Extra-Terminal (BET)Proteins with Picolar Cellular Potencies and Capable of AchievingJTumor 2018; 61:462-81.
  • Non-Patent Document 28 Toure M, Crews CM. Small-Molecule PROTACS: New Approaches to Protein Degradation. Angew Chem Int Ed Engl 2016; 55: 1966-73.
  • the present invention designs and synthesizes new compounds based on pyrrolopyridones as the core, which has HDAC and BRD4 dual target inhibitory activity, or has dual functions of inhibiting BRD4 and ubiquitination and degradation of BRD4 protein, and has clear cell biology Function, can be applied to anti-tumor and other diseases.
  • R 1 is selected from
  • R a represents one or more substituents, each independently selected from hydrogen, halogen, hydroxy, cyano, alkyl, optionally substituted alkylamino, haloalkyl, alkoxy,
  • X is selected from alkylene, heteroalkylene, alkenylene, heteroarylene, (alkylene) 0 or 1 -arylene-(alkylene) 0 or 1 , alkylene-CONH-alkylene Alkyl, cycloalkylene, cycloalkenylene, heterocyclylene, heterocycloalkenylene; the arylene, heteroarylene, (alkylene) 0 or 1 -arylene-(ethylene Alkyl) 0 or 1 , cycloalkylene, cycloalkenylene, heterocyclylene or heterocycloalkenylene is optionally cycloalkenyl, heterocyclic, heterocycloalkenyl, hydroxy, nitro, cyano , Amino groups, alkoxy groups, alkyl groups, alkenyl groups, aryl groups, heteroaryl groups, cycloalkyl groups, and heterochain hydrocarbon groups optionally substituted by alkyl groups,
  • A is selected from aryl and heteroaryl
  • the "halogen" is selected from fluorine, chlorine, bromine or iodine.
  • alkyl “amino substituted by alkyl”, “haloalkyl”, “alkoxy”, “hydroxyalkyl”, “heterocyclylalkyl”, “cycloalkylalkyl”
  • the alkyl groups in “oxy” are each independently a C 1 -C 20 linear or branched alkyl group, optionally a C 1 -C 15 linear or branched alkyl group, optionally a C 1 -C 10 Linear or branched alkyl, optionally C 1 -C 7 linear or branched alkyl, optionally C 1 -C 6 linear or branched alkyl, optionally C 1 -C 5 Straight-chain or branched alkyl, optionally C 1 -C 4 straight or branched alkyl, optionally selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, iso Butyl, tert-butyl, sec-butyl, n-
  • alkylene “(alkylene) 0 or 1 -arylene-(alkylene) 0 or 1 ” and “alkylene-CONH-alkylene”
  • the alkyl groups are each independently a C 1 -C 20 linear or branched alkylene group, optionally a C 1 -C 15 linear or branched alkylene group, optionally a C 1 -C 10 linear or Branched alkylene, optionally C 1 -C 8 linear or branched alkylene, optionally C 1 -C 6 linear or branched alkylene, optionally C 1 -C 4 linear or branched alkylene group, optionally C 1 -C 3 straight or branched chain alkylene group, optionally C 3 -C 6 straight or branched chain alkylene group.
  • the “heteroalkylene group” is a C 1 -C 20 linear or branched alkylene group containing one or more heteroatoms selected from O, S or N, optionally containing 1 -6 C 1 -C 20 linear or branched alkylene groups selected from O, S or N heteroatoms, optionally C 1 containing 1-4 heteroatoms selected from O, S or N -C 20 linear or branched alkylene, optionally containing 1-6 C 1 -C 20 linear or branched alkylene selected from O or S, optionally containing 1-4 O or S is selected from C 1 -C 20 straight or branched chain alkylene group optionally containing O atoms in the C 1-6 1 -C 20 linear or branched alkylene group, optionally C 1 -C 20 linear or branched alkylene containing 1-4 O atoms; optionally 1-15 carbon atoms, optionally 1-10, optionally 1-8
  • One, optionally 1-6; heteroalkylene is for example –(CH 2
  • the arylene group in the "arylene”, “(alkylene) 0 or 1 -arylene-(alkylene) 0 or 1 " is a 6-10 membered arylene group; Select phenylene or naphthylene.
  • the “aryl group” is a 6-10 membered aryl group; it may be a phenyl group or a naphthyl group.
  • aromatic ring is a 6-10 membered aromatic ring; it may be a benzene ring or a naphthalene ring.
  • the "heteroarylene” is a 5-10 membered heteroarylene group containing 1-3 heteroatoms selected from N, O and S; alternatively, it is a heteroarylene group containing 1-2 5-10 membered heteroarylene groups from heteroatoms in N, O, and S; optionally, the heteroarylene group is selected from pyridylene, pyrrolylene, pyrimidinylene, pyrazinylene, Pyridazinyl, thienylene, furanylene.
  • the ring in the "heteroaromatic ring” and “heteroaryl” is a 5-10 membered heteroaromatic ring containing 1-3 heteroatoms selected from N, O and S; optionally, Is a 5-10 membered heteroaromatic ring containing 1-2 heteroatoms selected from N, O and S; optionally, the ring is selected from pyridine ring, pyrrole ring, pyrimidine ring, pyrazine ring, pyridazine Ring, thiophene ring, furan ring.
  • the “(alkylene) 0 or 1 -arylene-(alkylene) 0 or 1 ” is preferably a phenylene group, a benzylidene group, an ethylenephenyl group, or a tolylene group.
  • the "cycloalkylene” is a C 3 -C 10 monocyclic, bicyclic, tricyclic, or tetracyclic cycloalkylene; optionally, the cycloalkylene is selected from cyclopropylene , Cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene and cyclooctylene.
  • the cycloalkane of the "cycloalkane” and “cycloalkyl” is a C 3 -C 10 monocyclic, bicyclic, tricyclic, or tetracyclic cycloalkane; optionally, the cycloalkane is selected from the group consisting of Propane, cyclobutane, cyclopentane, cyclohexane, cycloheptane and cyclooctane.
  • the “cycloalkenylene group” is a partially unsaturated monocyclic, bicyclic, tricyclic, or tetracyclic C 3 -C 12 cycloalkenylene group containing one or more double bonds; Cyclopropenyl, cyclobutenylene, cyclopentenylene, cyclohexenylene, cycloheptenylene, or cyclooctenylene.
  • the "cycloalkenyl group” is a partially unsaturated monocyclic, bicyclic, tricyclic, or tetracyclic C 3 -C 12 cycloalkenyl group containing one or more double bonds; alternatively, it is a cyclopropenyl group , Cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl or cyclooctenyl.
  • the heterocycle in the "heterocycle”, “heterocyclyl”, and “heterocyclylene” is a ring containing 1, 2, or 3 heteroatoms selected from N, O, S A 3-10 membered non-aromatic heterocyclic ring, optionally, the heterocyclic ring is a 3-6 membered non-aromatic ring containing one or two heteroatoms selected from N and O on the ring.
  • the "heterocycloalkenylene” is a 3-10 membered non-cyclic ring containing 1, 2, or 3 heteroatoms selected from N, O, and S, and containing 1 or more double bonds.
  • the aromatic heterocycloalkenylene group is, optionally, a 3-6 membered non-aromatic heterocycloalkenylene group containing one or two heteroatoms selected from N and O on the ring.
  • heterocycloalkenyl group is a 3-10 membered non-aromatic ring containing 1, 2, or 3 heteroatoms selected from N, O, S and 1 or more double bonds
  • the heterocycloalkenyl group is optionally a 3-6 membered non-aromatic heterocycloalkenyl group containing one or two heteroatoms selected from N and O on the ring.
  • the "hetero-chain hydrocarbon group” is a straight-chain or branched saturated or unsaturated C 1 -C 20 hetero-chain hydrocarbon structure containing 1-6 heteroatoms selected from N, O, S on the chain; Optionally, it is a linear or branched saturated or unsaturated C 1 -C 20 heterochain hydrocarbon structure containing 1, 2, 3 or 4 heteroatoms selected from N and O in the chain, optionally, It is a straight-chain or branched saturated or unsaturated C 1 -C 20 heterochain hydrocarbon structure containing 1, 2, 3 or 4 O's in the chain, optionally, it is a chain containing 1, 2, 3, 3 or 4 O straight or branched saturated or unsaturated C 1 -C 10 heterochain hydrocarbon structure.
  • R a represents one or more substituents, each independently selected from hydrogen or halogen
  • X is selected from C 1 -C 10 alkylene, C 1 -C 10 alkylene -CONH-C 1 -C 10 alkylene, --(CH 2 CH 2 O) n --, --(OCH 2 CH 2 ) m –, –(CH 2 CH 2 O) l CH 2 CH 2 –, –(OCH 2 ) k –, –(CH 2 O) i –, –(CH 2 O) j CH 2 –, C 1 -C 10 alkyleneoxy, (C 1 -C 10 alkylene) 0 or 1 -arylene-(C 1 -C 10 alkylene) 0 or 1 , wherein n, m, l, k, i, j Each independently is an integer of 1-6;
  • A is selected from phenyl
  • the compound of formula I is selected from the following compounds:
  • the pharmaceutically acceptable salt includes an anionic salt and a cationic salt of the compound of formula I;
  • the pharmaceutically acceptable salt includes the alkali metal salt, alkaline earth metal salt, and ammonium salt of the compound of formula I; preferably, the alkali metal includes sodium, potassium, lithium, cesium, and the alkaline earth Metals include magnesium, calcium and strontium;
  • the pharmaceutically acceptable salt includes the salt formed by the compound of formula I and an organic base; preferably, the organic base includes trialkylamine, pyridine, quinoline, piperidine, imidazole, and picoline , Dimethylaminopyridine, dimethylaniline, N-alkylmorpholine, 1,5-diazabicyclo[4.3.0]nonene-5(DBN), 1,8-diazabicyclo[5.4.
  • the organic base includes trialkylamine, pyridine, quinoline, piperidine, imidazole, and picoline , Dimethylaminopyridine, dimethylaniline, N-alkylmorpholine, 1,5-diazabicyclo[4.3.0]nonene-5(DBN), 1,8-diazabicyclo[5.4.
  • the trialkylamine includes trimethylamine, triethylamine, N- Ethyl diisopropylamine; preferably, the N-alkylmorpholine includes N-methylmorpholine;
  • the pharmaceutically acceptable salt includes the salt formed by the compound of formula I and an acid; preferably, the acid includes an inorganic acid, an organic acid; preferably, the inorganic acid includes hydrochloric acid, hydrobromic acid, Hydroiodic acid, sulfuric acid, nitric acid, phosphoric acid, carbonic acid; preferably, the organic acid includes formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, lemon Acid, citric acid, tartaric acid, carbonic acid, picric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, glutamic acid, pamoic acid.
  • the acid includes an inorganic acid, an organic acid
  • the inorganic acid includes hydrochloric acid, hydrobromic acid, Hydroiodic acid, sulfuric acid, nitric acid, phosphoric acid, carbonic acid
  • P is hydrogen or a protecting group
  • Q is hydrogen or C 1 -C 10 alkyl
  • a and R 2 are as defined above.
  • protecting group When a multifunctional organic compound reacts, in order to make the reaction only occur at the desired functional group and avoid other functional groups from being affected, the other functional groups should be protected before the reaction and restored after the reaction is complete.
  • Protecting group is a general term for such groups that can provide protection to the other functional groups.
  • the protecting group here can be, for example, benzyl, p-toluenesulfonyl, (trimethylsilyl)ethoxy)methyl).
  • R 1 is a compound having the same in formula I, R 1 is a compound having the same in formula I, R 1 is a compound having the same in formula I, R 1 is a compound having the same in formula I, R 1 is a compound having the same in formula I, R 1 is a compound having the same in formula I, R 1 is a compound having the same in formula I, R 1 is a compound having the same in formula I, R 1 is a compound having the
  • A, R 2 and X are as defined above, and M is hydrogen or C 1 -C 10 alkyl;
  • R 01 is a hydroxyl group
  • R a is as defined above;
  • a and R 2 are as defined above; M is hydrogen or C 1 -C 10 alkyl; X 1 is alkylene;
  • R 02 is an alkylene group-R 1 , and R 1 is as defined above.
  • the preparation method of the compound of formula III or compound of formula IV includes:
  • the preparation method of the compound of formula II includes: Suzuki coupling reaction of compound of formula V with aryl boronic acid to obtain compound of formula II,
  • the compound of formula V is subjected to a Suzuki coupling reaction to obtain the aryl borate compound of formula VI, and then the aryl borate compound of formula VI is subjected to a Suzuki coupling reaction with the bromide to obtain the compound of formula II.
  • PG represents a protecting group (for example, benzyl, p-toluenesulfonyl, (trimethylsilyl)ethoxy) methyl), Y represents halogen, and Q represents hydrogen or C 1 -C 10 alkyl ;
  • the aryl boronic acid is a compound of formula VII:
  • the bromide is a compound of formula VIII:
  • the preparation method of the compound of formula V includes:
  • the compound of formula XIV is reacted with N,N-dimethylformamide dimethyl acetal to obtain the compound of formula XIII.
  • the obtained compound of formula XIII is reduced under acidic conditions to obtain the compound of formula XII.
  • the compound of formula XII is combined with a halide under strong alkali conditions.
  • the compound of formula XI is obtained by the reaction, and the compound of formula XI is reacted with alkyl chloroformate under strong base conditions to obtain the compound of formula X.
  • the compound of formula X is treated with acid to obtain the compound of formula IX.
  • the compound of formula IX is methylated by a chemical reagent to obtain a compound of formula V;
  • the condensation reaction is carried out in a solvent under alkaline conditions and in the presence of a condensing agent; preferably, the condensing agent includes: N,N'-carbonyldiimidazole, bicyclic Hexylcarbodiimide, O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea, N-hydroxy-7-azobenzotriazide Azole, 2-(1H-benzotrisazo L-1-yl)-1,1,3,3-tetramethylurea tetrafluoroborate or 1-hydroxybenzotriazole; preferably, the The base used in the alkaline conditions includes one or more of triethylamine, diisopropylethylamine, and DMAP; the solvent includes: tetrahydrofuran, dichloromethane or N,N-dimethylformamide.
  • the condensing agent includes: N,N'-carbonyldiimidazo
  • the Suzuki coupling reaction is carried out in a solvent under alkaline conditions in the presence of a palladium catalyst, optionally in the presence of a ligand; preferably, the Suzuki coupling reaction is carried out at 60°C to 150°C.
  • the reaction is promoted by microwave radiation;
  • the alkali used in the alkaline conditions includes CsF, Cs 2 CO 3 , K 2 CO 3 , triethylamine, diisopropyl ethyl One or two or more of amine and DMAP;
  • the palladium catalyst includes: Pd 2 (dba) 3 , Pd(OAc) 2 or Pd(PPh 3 ) 4 ;
  • the ligand includes: 1,3,5,7-Tetramethyl-6-phenyl-2,4,8-trioxa-6-phosphoryladamantane, 2-dicyclohexylphosphorus-2',4',6'- Triisopropylbiphenyl, 1,1'-bis(diphenylphosphonyl)ferrocene;
  • the solvent includes: methanol, dimethoxyethane, N,N-dimethylformaldehyde Amide, di
  • the reaction of the compound of formula XIV with N,N-dimethylformamide dimethyl acetal is carried out at a temperature of 60°C to 100°C; preferably, it is carried out in a solvent, and the solvent includes: N,N- dimethylformamide;
  • the acid includes: ammonium chloride or acetic acid;
  • the reducing agent used in the reduction reaction includes reduced iron powder;
  • the strong base includes sodium hydride
  • the strong base includes lithium diisopropylamide
  • the step of treating the compound of formula X with an acid is carried out in a solvent at a temperature of 40°C to 100°C.
  • the acid includes hydrochloric acid and hydrobromic acid; and the solvent includes dioxane, water;
  • the methylation reaction of the compound of formula IX is carried out in a solvent at a temperature of 40° C. to 100° C.
  • the methylation reagent is methyl iodide; preferably, the base used in the basic conditions includes hydrogenation.
  • the solvent includes N,N-dimethylformamide or dimethylsulfoxide.
  • composition comprising the compound of formula I or a pharmaceutically acceptable salt thereof and pharmaceutically acceptable excipients.
  • a method for preventing or treating tumors which comprises administering to a patient in need a preventive or therapeutically effective amount of the compound of the above formula I or a pharmaceutically acceptable salt thereof, or the above pharmaceutical composition.
  • the tumor includes non-small cell lung cancer, breast cancer, thyroid cancer (medullary thyroid cancer, papillary thyroid cancer), stomach cancer, bladder cancer, endometrial cancer, prostate cancer, cervical cancer, colon cancer, esophageal cancer , Keratoblastoma, myeloma, rhabdomyosarcoma, acute leukemia, liver cancer, adenocarcinoma or pancreatic cancer.
  • thyroid cancer medullary thyroid cancer, papillary thyroid cancer
  • stomach cancer bladder cancer
  • endometrial cancer prostate cancer
  • cervical cancer colon cancer
  • esophageal cancer Keratoblastoma
  • myeloma myeloma
  • rhabdomyosarcoma acute leukemia
  • liver cancer adenocarcinoma or pancreatic cancer.
  • a bifunctional inhibitor having the effect of inhibiting BRD4 and HDAC is obtained, or, according to a specific embodiment of another aspect of the present invention, by introducing R Ubiquitination in 1 recruits pharmacophores, and a bifunctional compound with BRD4 inhibitory activity and ubiquitination to degrade BRD4 protein is obtained.
  • the test compound showed strong resistance in acute monocytic leukemia cell MV-4-11, myelodysplastic syndrome cell line SKM-1 and human pancreatic cancer cell line MIA Paca2.
  • the inhibitory activity of cell proliferation was assessed for the following conditions:
  • the test compound shows significant cell proliferation inhibitory activity in acute monocytic leukemia cells MV-4-11.
  • Figure 1 is a Western-Blotting diagram of the influence of the compounds of Examples 7 and 10 on the HDAC and BRD4 signaling pathways in SKM-1 cells in Experiment 2, where Ac-tub refers to acetylated tublin protein, and Ac-H3 refers to acetylated H3 protein.
  • Figure 2 is a Western-Blotting diagram of the effects of the compounds of Examples 7 and 10 on the MV-4-11HDAC and BRD4 signaling pathways in Experiment 2, where Ac-tub refers to acetylated tublin protein, and Ac-H3 refers to acetylated H3 protein.
  • Fig. 3 is a Western-Blotting diagram of the degradation function of the compound of Example 105 in Experiment 4 on the MV-4-11 cell line for the degradation of BRD4 protein.
  • the obtained solid was dissolved in acetic acid (400mL), and reduced iron powder (40g, 708.36mmol) was added under stirring, and the reaction was heated and refluxed in an oil bath for 4 hours; after the reaction was cooled down, the insoluble solid was filtered out with Celite, and washed with hot ethanol The residue was filtered until no more product, and the solvent was evaporated under reduced pressure.
  • the obtained solid was dissolved in ethyl acetate (300 mL), and the organic phase was washed with water (100 mL), saturated sodium carbonate aqueous solution (100 mL), saturated sodium chloride aqueous solution (100 mL) successively, and the organic phases were combined and dried with anhydrous sodium sulfate.
  • Example 2 The reaction was quenched by adding water, extracted with ethyl acetate and washed with water and saturated sodium chloride solution. The organic phases were combined and dried over anhydrous sodium sulfate, filtered and concentrated. The compound of Example 2 was isolated by flash chromatography (silica gel 0-7% dichloromethane/methanol gradient).
  • the synthesis method is the same as in Example 1, except that methyl 5-aminovalerate is used instead of methyl 4-aminobutyrate.
  • the synthesis method is as in Example 2 except that methyl 5-aminovalerate is used instead of methyl 4-aminobutyrate.
  • the synthesis method is the same as in Example 1, except that methyl 6-aminocaproate is used instead of methyl 4-aminobutyrate.
  • the synthesis method is the same as in Example 2 except that methyl 6-aminocaproate is used instead of methyl 4-aminobutyrate.
  • the synthesis method is the same as in Example 1, except that methyl 7-aminoheptanoate is used instead of methyl 4-aminobutyrate.
  • the synthesis method is the same as in Example 2 except that methyl 7-aminoheptanoate is used instead of methyl 4-aminobutyrate.
  • the synthesis method is the same as in Example 1, except that methyl 4-(aminomethyl)benzoate is used instead of methyl 4-aminobutyrate.
  • the synthesis method is the same as in Example 2 except that methyl 4-(aminomethyl)benzoate is used instead of methyl 4-aminobutyrate.
  • the synthesis method is as in Example 9 except that phenylboronic acid is used instead of (2-phenoxyphenyl)boronic acid.
  • the synthesis method was as in Example 10 except that phenylboronic acid was used instead of (2-phenoxyphenyl)boronic acid.
  • the synthesis method is as in Example 9 except that (2-isopropoxyphenyl)boronic acid is used instead of (2-phenoxyphenyl)boronic acid.
  • the synthesis method is as in Example 10 except that (2-isopropoxyphenyl)boronic acid is used instead of (2-phenoxyphenyl)boronic acid.
  • the synthesis method is as in Example 9 except that 2-fluorophenylboronic acid is used instead of (2-phenoxyphenyl)boronic acid.
  • the synthesis method is as in Example 10 except that 2-fluorophenylboronic acid is used instead of (2-phenoxyphenyl)boronic acid.
  • the synthesis method is as in Example 9 except that 2-chlorobenzeneboronic acid is used instead of (2-phenoxyphenyl)boronic acid.
  • the synthesis method is as in Example 10 except that 2-chlorophenylboronic acid is used instead of (2-phenoxyphenyl)boronic acid.
  • the synthesis method is as in Example 9 except that 2-cyanophenylboronic acid is used instead of (2-phenoxyphenyl)boronic acid.
  • the synthesis method is as in Example 10 except that 2-cyanophenylboronic acid is used instead of (2-phenoxyphenyl)boronic acid.
  • the synthesis method is as in Example 9 except that 2-trifluoromethylphenylboronic acid is used instead of (2-phenoxyphenyl)boronic acid.
  • the synthesis method is as in Example 10 except that 2-trifluoromethylphenylboronic acid is used instead of (2-phenoxyphenyl)boronic acid.
  • the synthesis method is as in Example 9 except that 2-methylphenylboronic acid is used instead of (2-phenoxyphenyl)boronic acid.
  • the synthesis method is as in Example 10 except that 2-methylphenylboronic acid is used instead of (2-phenoxyphenyl)boronic acid.
  • the synthesis method is as in Example 9 except that 2-isopropylbenzeneboronic acid is used instead of (2-phenoxyphenyl)boronic acid.
  • the synthesis method is as in Example 10 except that 2-isopropylphenylboronic acid is used instead of (2-phenoxyphenyl)boronic acid.
  • the synthesis method is the same as in Example 9 except that 2-methoxyphenylboronic acid is used instead of (2-phenoxyphenyl)boronic acid.
  • the synthesis method is as in Example 10 except that 2-methoxyphenylboronic acid is used instead of (2-phenoxyphenyl)boronic acid.
  • the synthesis method is the same as in Example 9, except that (2-cyclopropoxyphenyl)boronic acid is used instead of (2-phenoxyphenyl)boronic acid.
  • the synthesis method is the same as in Example 10 except that (2-cyclopropoxyphenyl)boronic acid is used instead of (2-phenoxyphenyl)boronic acid.
  • the synthesis method is as in Example 9 except that 2-(4-fluoro-2,6-dimethylphenoxy)phenyl)boronic acid is used instead of (2-phenoxyphenyl)boronic acid.
  • the synthesis method is the same as in Example 10, except that 2-(4-fluoro-2,6-dimethylphenoxy)phenyl)boronic acid is used instead of (2-phenoxyphenyl)boronic acid.
  • the synthesis method is as in Example 9 except that (2-(2,4-difluorophenoxy)phenyl)boronic acid is used instead of (2-phenoxyphenyl)boronic acid.
  • the synthesis method is as in Example 10 except that (2-(2,4-difluorophenoxy)phenyl)boronic acid is used instead of (2-phenoxyphenyl)boronic acid.
  • the synthesis method is the same as in Example 9 except that (3-(ethylsulfonamido)phenyl)boronic acid is used instead of (2-phenoxyphenyl)boronic acid.
  • the synthesis method is as in Example 10 except that (3-(ethylsulfonamido)phenyl)boronic acid is used instead of (2-phenoxyphenyl)boronic acid.
  • the synthesis method is as in Example 9 except that 3-methanesulfonylphenylboronic acid is used instead of (2-phenoxyphenyl)boronic acid.
  • the synthesis method is as in Example 10 except that 3-methanesulfonylphenylboronic acid is used instead of (2-phenoxyphenyl)boronic acid.
  • compound 39a (0.3g, 0.617mmol), compound 39b (0.110g, 0.514mmol), cesium fluoride (0.156g, 1.03mmol), three (dibenzylideneacetone) two palladium (0 ) (0.047g, 0.0514mmol), 1,3,5,7-tetramethyl-6-phenyl-2,4,8-trioxa-6-phosphoryladamantane (0.029g, 0.103mmol)
  • ethylene glycol dimethyl ether (2 mL) and water (0.16 mL)
  • the mixture was stirred for 2 hours at 60°C in an oil bath until the reaction was complete.
  • the synthesis method is as in Example 9 except that 3-(1-hydroxyethyl)phenylboronic acid is used instead of (2-phenoxyphenyl)boronic acid.
  • the synthesis method is as in Example 10, except that 3-(1-hydroxyethyl)phenylboronic acid is used instead of (2-phenoxyphenyl)boronic acid.
  • the synthesis method is as in Example 9 except that 3-methoxycarbonylphenylboronic acid is used instead of (2-phenoxyphenyl)boronic acid.
  • the synthesis method is as in Example 10 except that 3-methoxycarbonylbenzeneboronic acid is used instead of (2-phenoxyphenyl)boronic acid.
  • the synthesis method is the same as in Example 9 except that 3-hydroxyphenylboronic acid is used instead of (2-phenoxyphenyl)boronic acid.
  • the synthesis method is as in Example 10 except that 3-hydroxyphenylboronic acid is used instead of (2-phenoxyphenyl)boronic acid.
  • the synthesis method is as in Example 9 except that 3-methoxyphenylboronic acid is used instead of (2-phenoxyphenyl)boronic acid.
  • the synthesis method is as in Example 10 except that 3-methoxyphenylboronic acid is used instead of (2-phenoxyphenyl)boronic acid.
  • the synthesis method is as in Example 9 except that 3-methylphenylboronic acid is used instead of (2-phenoxyphenyl)boronic acid.
  • the synthesis method is as in Example 10 except that 3-methylphenylboronic acid is used instead of (2-phenoxyphenyl)boronic acid.
  • the synthesis method is as in Example 9 except that 3-trifluoromethylphenylboronic acid is used instead of (2-phenoxyphenyl)boronic acid.
  • the synthesis method is as in Example 10 except that 3-trifluoromethylphenylboronic acid is used instead of (2-phenoxyphenyl)boronic acid.
  • the synthesis method is the same as in Example 9 except that 3-fluorophenylboronic acid is used instead of (2-phenoxyphenyl)boronic acid.
  • the synthesis method is as in Example 10 except that 3-fluorophenylboronic acid is used instead of (2-phenoxyphenyl)boronic acid.
  • the synthesis method is as in Example 9 except that 3-chlorobenzeneboronic acid is used instead of (2-phenoxyphenyl)boronic acid.
  • the synthesis method is as in Example 10 except that 3-chlorophenylboronic acid is used instead of (2-phenoxyphenyl)boronic acid.
  • the synthesis method is as in Example 9 except that 3-cyanophenylboronic acid is used instead of (2-phenoxyphenyl)boronic acid.
  • the synthesis method is as in Example 10 except that 3-cyanophenylboronic acid is used instead of (2-phenoxyphenyl)boronic acid.
  • the synthesis method is as in Example 9 except that 4-methylphenylboronic acid is used instead of (2-phenoxyphenyl)boronic acid.
  • the synthesis method is as in Example 10 except that 4-methylphenylboronic acid is used instead of (2-phenoxyphenyl)boronic acid.
  • the synthesis method is as in Example 9 except that 4-trifluoromethylphenylboronic acid is used instead of (2-phenoxyphenyl)boronic acid.
  • the synthesis method is as in Example 10 except that 4-trifluoromethylphenylboronic acid is used instead of (2-phenoxyphenyl)boronic acid.
  • the synthesis method is as in Example 9 except that 4-methoxyphenylboronic acid is used instead of (2-phenoxyphenyl)boronic acid.
  • the synthesis method is as in Example 10 except that 4-methoxyphenylboronic acid is used instead of (2-phenoxyphenyl)boronic acid.
  • the synthesis method is as in Example 9 except that 4-fluorophenylboronic acid is used instead of (2-phenoxyphenyl)boronic acid.
  • the synthesis method is as in Example 10 except that 4-fluorophenylboronic acid is used instead of (2-phenoxyphenyl)boronic acid.
  • the synthesis method is as in Example 9 except that 4-chlorobenzeneboronic acid is used instead of (2-phenoxyphenyl)boronic acid.
  • the synthesis method is as in Example 10 except that 4-chlorophenylboronic acid is used instead of (2-phenoxyphenyl)boronic acid.
  • the synthesis method is as in Example 9 except that 4-cyanophenylboronic acid is used instead of (2-phenoxyphenyl)boronic acid.
  • the synthesis method is as in Example 10 except that 4-cyanophenylboronic acid is used instead of (2-phenoxyphenyl)boronic acid.
  • the synthesis method is as in Example 9 except that (4-(dimethylamino)methyl)phenyl)boronic acid is used instead of (2-phenoxyphenyl)boronic acid.
  • the synthesis method is the same as in Example 10 except that (4-(dimethylamino)methyl)phenyl)boronic acid is used instead of (2-phenoxyphenyl)boronic acid.
  • the synthesis method is the same as in Example 9 except that (4-(morpholinomethyl)phenyl)boronic acid is used instead of (2-phenoxyphenyl)boronic acid.
  • the synthesis method is as in Example 10 except that (4-(morpholinomethyl)phenyl)boronic acid is used instead of (2-phenoxyphenyl)boronic acid.
  • Example 39 Except that 2-bromo-1-(2,4-difluorophenoxy)-4-(methylsulfonyl)benzene was used instead of compound 39b, the synthesis method of Example 77 was referred to Example 39.
  • Example 40 Except that 2-bromo-1-(2,4-difluorophenoxy)-4-(methylsulfonyl)benzene was used instead of compound 39b, the synthesis method of Example 78 was referred to Example 40.
  • compound 39a (0.3g, 0.617mmol), compound 79b (0.176g, 0.514mmol), cesium fluoride (0.156g, 1.03mmol), three (dibenzylideneacetone) two palladium (0 ) (0.047g, 0.0514mmol), 1,3,5,7-tetramethyl-6-phenyl-2,4,8-trioxa-6-phosphoryladamantane (0.029g, 0.103mmol)
  • ethylene glycol dimethyl ether (2 mL) and water (0.16 mL)
  • the mixture was stirred for 2 hours at 60°C in an oil bath until the reaction was complete.
  • Example 80a 4-((4-(2-(2,4-Difluorophenoxy)-5-(2-hydroxypropan-2-yl)phenyl)-6-methyl-7-oxo- 6,7-Dihydro-1H-pyrrolo[2,1,3-c]pyridine-2-carboxamido)methyl)benzoic acid
  • Example 81a 4-(2-(4-Fluoro-2,6-dimethylphenoxy)-5-(2-hydroxypropan-2-yl)phenyl)-6-methyl-7-oxo -6,7-Dihydro-1H-pyrrolo[2,1,3-c]pyridine-2-carboxylic acid
  • Example 81a Except that 4-fluoro-2,6-dimethylphenol was used instead of 2,4-difluorophenol, the synthesis method of Example 81a was the same as that of Example 79d.
  • Example 81 The synthesis method of Example 81 is the same as that of Example 79 except that the compound of Example 81a is used instead of the compound of Example 79d.
  • Example 91 The synthesis method of Example 91 is the same as that of Example 79 except that methyl 3-bromo-4-methoxybenzoate is used instead of compound 79a.
  • Example 92a was the same as that of Example 79e.
  • Example 92b is the same as that of Example 80a.
  • Example 92 The synthesis method of Example 92 is the same as that of Example 80 except that 92b is used instead of 80a.
  • Example 93 The synthesis method of Example 93 is the same as that of Example 85 except that methyl 6-aminocaproate is used instead of methyl 4-(aminomethyl)benzoate.
  • Example 94 The synthesis method of Example 94 is the same as that of Example 85 except that ethyl 7-aminoheptanoate is used instead of methyl 4-(aminomethyl)benzoate.
  • Example 95 The synthesis method of Example 95 is the same as that of Example 83 except that methyl 6-aminocaproate is used instead of methyl 4-(aminomethyl)benzoate.
  • Example 96 The synthesis method of Example 96 is the same as that of Example 83 except that ethyl 7-aminoheptanoate is used instead of methyl 4-(aminomethyl)benzoate.
  • Example 97 The synthesis method of Example 97 was the same as that of Example 79 except that methyl 6-aminocaproate was used instead of methyl 4-(aminomethyl)benzoate.
  • Example 98 The synthesis method of Example 98 is the same as that of Example 79 except that ethyl 7-aminoheptanoate is used instead of methyl 4-(aminomethyl)benzoate.
  • Example 99 The synthesis method of Example 99 is the same as that of Example 81 except that methyl 6-aminocaproate is used instead of methyl 4-(aminomethyl)benzoate.
  • Example 100 The synthesis method of Example 100 is the same as that of Example 81 except that ethyl 7-aminoheptanoate is used instead of methyl 4-(aminomethyl)benzoate.
  • Example 101 In addition to 8-bromo-2,2,4-trimethyl-6-(methylsulfonyl)-2-H-benzo[b][1,4]oxazine-3(4-H)-one instead of compound 39b, the synthesis method of Example 101 is the same as that of Example 39.
  • Example 102 In addition to 8-bromo-2,2,4-trimethyl-6-(methylsulfonyl)-2-H-benzo[b][1,4]oxazine-3(4-H)-one instead of compound 39b, the synthesis method of Example 102 was the same as that of Example 40.
  • Example 80 Except that 4-fluoro-1,2-phenylenediamine was used instead of o-phenylenediamine, the synthesis method of Example 103 was referred to Example 80.
  • Example 104 Except that 4-fluoro-1,2-phenylenediamine was used instead of o-phenylenediamine, the synthesis method of Example 104 was referred to Example 76.
  • Example 10 The compound of Example 10 (25mg, 0.043mmol) was dissolved in dichloromethane (2mL) and methanol (1mL), 4M HCl in dioxane solution (0.021mL, 0.086mmol) was added, and the reaction was at room temperature for 3 hours and then evaporated under reduced pressure The solvent was removed, and the resultant was recrystallized from methyl tert-butyl ether to obtain the compound of Example 105.
  • Example 106a Except that 2-isopropoxypyridine-3-boronic acid is used instead of (2-phenoxyphenyl)boronic acid, the synthesis method of Example 106a is the same as that of Example 1g;
  • Example 106 The synthesis method of Example 106 is the same as that of Example 10 except that the compound of Example 106a is used instead of the compound of Example 1g.
  • the reaction was quenched by adding water, extracted with ethyl acetate and washed with water and saturated sodium chloride solution. The organic phases were combined and dried over anhydrous sodium sulfate, filtered and concentrated.
  • the product compound 107 was isolated by flash chromatography (silica gel 0-7% dichloromethane / petrol methanol gradient).
  • Example 107 Except that N-tert-butoxycarbonyl-1,4-butanediamine was replaced by the compound N-tert-butoxycarbonyl-1,5-pentanediamine, the synthesis method of Example 108 was referred to Example 107.
  • Example 109 Except that the compound N-tert-butoxycarbonyl-1,6-hexamethylenediamine is used instead of N-tert-butoxycarbonyl-1,4-butanediamine, the synthesis method of Example 109 refers to Example 107.
  • reaction was quenched by adding water, extracted with ethyl acetate and washed with water and saturated sodium chloride solution. The organic phases were combined and dried over anhydrous sodium sulfate, filtered and concentrated. The product was separated by flash chromatography (silica gel 0-7% dichloromethane/methanol gradient), and the resulting product was dissolved in dioxane solution (2mL), and 2M sodium hydroxide solution (0.2mL) was added at 90°C After 4 hours of reaction, the solid was acidified with 2M hydrochloric acid to precipitate a solid, which was filtered off with suction and washed with water and dried to obtain compound 110a.
  • the reaction was quenched by adding water, extracted with ethyl acetate and washed with water and saturated sodium chloride solution. The organic phases were combined and dried over anhydrous sodium sulfate, filtered and concentrated.
  • the product 110 was isolated by flash chromatography (silica gel 0-7% dichloromethane/hydrocarbon gradient).
  • the synthesis method is the same as in Example 110 except that methyl 5-aminohexanoate is used instead of methyl 5-aminovalerate.
  • the synthesis method is the same as in Example 110 except that methyl 5-aminoheptanoate is used instead of methyl 5-aminovalerate.
  • the synthesis method is the same as in Example 110 except that methyl 4-aminomethylbenzoate is used instead of methyl 5-aminovalerate.
  • the reaction was quenched by adding water, extracted with ethyl acetate and washed with water and saturated sodium chloride solution. The organic phases were combined and dried over anhydrous sodium sulfate, filtered and concentrated. The product was isolated by flash chromatography (silica gel 0-7% dichloromethane/methanol gradient). The obtained product was dissolved in a 4M HCl dioxane solution (5 mL), reacted at 60° C. for 4 hours, and concentrated under reduced pressure to remove the solvent to obtain compound 117a.
  • the synthesis method is the same as in Example 117 except that 12-amino-4,7,10-trioxa dodecanoate tert-butyl ester is used instead of 9-amino-4,7-dioxonanoate tert-butyl ester.
  • the synthesis method is the same as in Example 117 except that 15-amino-4,7,10,13-tetraoxapentadecanoic acid tert-butyl ester is substituted for 9-amino-4,7-dioxonanoate tert-butyl ester.
  • the synthesis method is the same as that in Example 118 except that it is used instead of pomalidomide.
  • the synthesis method was the same as that of Example 115 except that the compound of Example 81a was used instead of the compound of Example 1g.
  • the synthesis method was the same as that in Example 121 except that it was used instead of pomalidomide.
  • the synthesis method is the same as that of Example 115 except that the compound of Example 106a is used instead of the compound of Example 1g.
  • the synthesis method is the same as that of Example 119 except that the compound of Example 81a is used instead of the compound of Example 1g.
  • the synthesis method is the same as that in Example 124 except that nasalidomide is used instead of pomalidomide.
  • the synthesis method is the same as 115 except that compound 92b is used instead of compound 110a.
  • Test 1 Inhibition experiment of molecular level enzyme
  • the compound was prepared into a 10 mM stock solution with DMSO, and then diluted 10 times to obtain a 1 mM stock solution for later use. Then use Binding Domain diluent buffer (#62DLBDDF, Cisbio) to dilute it 10 times (the DMSO concentration in the final system is 0.1%), according to the initial concentration required by the experiment, 1:5 gradient dilution, a total of 5 concentrations.
  • Binding Domain diluent buffer #62DLBDDF, Cisbio
  • Binding Domain diluent buffer #62DLBDDF, Cisbio
  • BRD4 control (4 ⁇ LBRD4(1) or BRD4(2)+4 ⁇ L prepared Lys(5,8,12,16)Ac]H4(1-21)-biotin+2 ⁇ L Binding Domain diluent buffer), and no BRD4 control (4 ⁇ L prepared Lys(5,8,12,16)Ac]H4(1-21)-biotin+6 ⁇ L Binding Domaindiluent buffer).
  • BRD4 control (4 ⁇ LBRD4(1) or BRD4(2)+4 ⁇ L prepared Lys(5,8,12,16)Ac]H4(1-21)-biotin+2 ⁇ L Binding Domain diluent buffer)
  • no BRD4 control (4 ⁇ L prepared Lys(5,8,12,16)Ac]H4(1-21)-biotin+6 ⁇ L Binding Domaindiluent buffer).
  • the experimental ratio value 665nm fluorescence value/615nm fluorescence value.
  • the IC 50 value is obtained by using Prism software by three-parameter regression.
  • the compound powder was prepared into a 10mM mother liquor with DMSO, and then diluted 10 times to obtain a 1mM stock solution for later use. Then use 1 times Enzymatic buffer (50mM Tris-HCl pH 8.0, 137mM NaCl, 2.7mM KCl, 1mM MgCl 2 , 0.01% Tween20) diluted 10 times (the DMSO concentration in the final system is 0.1%), according to the initial requirements of the experiment Concentration, 1:5 gradient dilution, a total of 5 concentrations.
  • Enzymatic buffer 50mM Tris-HCl pH 8.0, 137mM NaCl, 2.7mM KCl, 1mM MgCl 2 , 0.01% Tween20
  • HDAC1 (#31504, Active Motif)/HDAC6 (#31943, Active Motif) with 1 times Enzymatic buffer to 5 times the final concentration for use.
  • HDAC control (2 ⁇ L HDAC1 or HDAC6+4 ⁇ L prepared Histone H3(1-21) lysine 9 acetylated biotinylated peptide+4 ⁇ L Enzymatic buffer)
  • HDAC control (4 ⁇ L prepared Histone H3) (1-21)lysine 9 acetylated biotinylated peptide+6 ⁇ L Enzymatic buffer).
  • the experimental ratio value 665nm fluorescence value/615nm fluorescence value.
  • the IC 50 value is obtained by using Prism software by three-parameter regression.
  • SAHA HDAC pan inhibitor
  • MS275 HDAC1 selective inhibitor
  • OTX015 BRD4 (1/2) pan inhibitor
  • the enzyme activity data of drug ABBV-744 (BRD4(2) selective inhibitor) against BRD4(1)/(2) and HDAC1/6 are shown in Table 1 to Table 2.
  • Example 1-106 of the present invention has a certain HDAC inhibitory activity while having BRD4 activity, while the corresponding BRD4 inhibitors ABBV-744 and OTX-015 as positive controls do not have HDAC inhibitory activity .
  • Test Example 2 Western Blot to detect the effect of compounds on HDAC and BRD4 signaling pathways in MV-4-11 cells
  • MV-4-11 cells in the logarithmic growth phase were seeded in a 12-well culture plate. After the cells adhered overnight, compounds (10, 100, 1000 nM) were added to treat the cells for 24 hours. After washing 3 times with pre-cooled PBS (containing 1mM sodium vanadate), add 1xSDS gel loading buffer (formulation: 50mM Tris-Cl (pH6.8), 100mM DTT, 2% SDS, 10% (v/ v) Glycerol, 0.1% (w/v) bromophenol blue) lyse cells. The cell lysate was heated in a boiling water bath for 15 minutes and stored at -20°C.
  • 1xSDS gel loading buffer formulation: 50mM Tris-Cl (pH6.8), 100mM DTT, 2% SDS, 10% (v/ v) Glycerol, 0.1% (w/v) bromophenol blue
  • the protein sample is placed in a density SDS-polyacrylamide gel, and electrophoresed in Tris-glycine-SDS electrophoresis buffer [25mmol/L Tris, 250mmol/L glycine (pH8.3), 0.1% SDS] at 80V for about 20 minutes Compress, then switch to 120V electrophoresis for about 2h for separation.
  • Tris-glycine-SDS electrophoresis buffer [25mmol/L Tris, 250mmol/L glycine (pH8.3), 0.1% SDS] at 80V for about 20 minutes Compress, then switch to 120V electrophoresis for about 2h for separation.
  • the protein is transferred from the gel to the nitrocellulose filter by semi-dry blotting.
  • the transfer buffer formula is 192mmol/L glycine, 25mmol/L Tris, 20% methanol, and transfer about 1h according to the required protein molecular weight. Use Ponceau S staining to determine the transfer status and the
  • the luminescent reagents include ECL Plus western blotting detection system and Advance ECL western blotting detection system, and Super Signal Western Pico Chemiluminescent Substrate.
  • the compounds of Examples 84 and 80 can up-regulate the acetylation level of histone H3 in a dose-dependent manner, but have little effect on the acetylation level of tubulin, and their effect is equivalent to that of the HDAC1 selective inhibitor MS275, indicating this
  • the compound is a selective inhibitor of HDAC1; at the same time, the compound can down-regulate the expression of c-Myc in a dose-dependent manner, and its effect is similar to the BRD4 inhibitor ABBV-744.
  • the compound of Example 96 can simultaneously dose-dependently up-regulate the acetylation levels of histone H3 and tubulin, and has a similar effect to the pan-HDAC inhibitor SAHA, indicating that the compound has pan-HDAC inhibitory activity.
  • the compound can down-regulate the expression of c-Myc in a dose-dependent manner, and its effect is similar to the BRD4 inhibitor ABBV-744.
  • the compound of Example 100 can increase the acetylation level of tubulin in a dose-dependent manner, but has little effect on the acetylation level of histone H3. Its effect is similar to that of the selective HDAC6 inhibitor ACY1215, indicating that the compound has a certain HDAC6 selectivity Inhibit activity. At the same time, the compound can down-regulate the expression of c-Myc in a dose-dependent manner, and its effect is similar to the BRD4 inhibitor ABBV-744.
  • Example 84 and 80 are selective HDAC1 and BRD4 dual-target inhibitors
  • the compound of Example 96 is a pan-HDAC and BRD4 dual-target inhibitor
  • the compound of Example 100 is a selective dual-target inhibitor of HDAC6 and BRD4.
  • Test example 3 Cell level inhibition test
  • the CCK-8 method was used to detect cell growth inhibition.
  • the compound inhibition rate and IC 50 value were obtained by four-parameter regression using the software included with the microplate reader.
  • N.T. means not determined.
  • Example 7 and Example 96 of the pan-HDAC and BRD4 dual-target inhibitor are in the human acute monocytic leukemia cell line MV-4-11 and the human myelodysplastic syndrome cell line SKM-1 Shows strong inhibitory activity, better than the positive control HDAC inhibitors SAHA and ABBV-744.
  • HDAC1 and BRD4 dual-target inhibitors of Example 10, Example 80 and Example 84 compounds well inhibit the proliferation of human acute monocytic leukemia cell line MV-4-11 and human myelodysplastic syndrome cell line SKM-1 At the same time, it showed better inhibitory activity on the human pancreatic cancer MIA Paca2 cell line, which was stronger than the positive compounds ABBV-744 and MS275.
  • Example 115 The compounds of Example 115, Example 127 and Example 128 that targeted ubiquitination to degrade BRD4 protein showed strong cell proliferation inhibitory activity on the MV-4-11 cell line, which was stronger than the BET inhibitor ABBV-744, and The positive control d-BET6 is equivalent.
  • Test Example 4 Western Blot hybridization (Western Blot) detection of compound BRD4 protein degradation
  • the logarithmic growth phase MV-4-11 cells were seeded in a 12-well culture plate, and different concentrations of compounds (10 nM, 100 nM, 1000 nM) were added to treat the cells for 3 h. After blowing the cells in each well, collect them in a 1.5ml centrifuge tube, mark them, centrifuge at 450g for 5 minutes, and discard the supernatant. Add 1ml of pre-cooled PBS (containing 1mM sodium vanadate) to wash three times, and centrifuge each time to discard the supernatant.
  • pre-cooled PBS containing 1mM sodium vanadate
  • 1xSDS gel loading buffer (the formula is: 50mM Tris-Cl (pH6.8), 100mM DTT, 2% SDS, 10% (v/v) glycerol, 0.1% (w/v) bromophenol blue ) Lyse cells.
  • the cell lysate was heated in a boiling water bath for 10 minutes. Take the above-mentioned cell lysate for SDS-PAGE electrophoresis. After the electrophoresis, the protein is transferred to the nitrocellulose membrane with a semi-dry electrotransfer system. After the transfer, stain with Ponceau S to determine the transfer and the position of the protein band on the nitrocellulose membrane.
  • Time-dependent degradation experimental protocol inoculate logarithmic growth phase MV-4-11 cells in a 6-well culture plate, and add compound to treat the cells 1h, 3h, 6h, 12h, and 24h before the sampling time point.
  • the cells in each well are blown well and collected in a 2ml centrifuge tube, labeled, centrifuged at 450g for 5 minutes, and the supernatant is discarded.
  • Add 1ml of pre-cooled PBS (containing 1mM sodium vanadate) to wash three times, and centrifuge each time to discard the supernatant.
  • 1xSDS gel loading buffer (the formula is: 50mM Tris-Cl (pH6.8), 100mM DTT, 2% SDS, 10% (v/v) glycerol, 0.1% (w/v) bromophenol blue) Lyse the cells.
  • the cell lysate was heated in a boiling water bath for 15 minutes and stored at -20°C.
  • SDS-PAGE electrophoresis After the electrophoresis, the protein is transferred to the nitrocellulose membrane with a semi-dry electrotransfer system. After the transfer, stain with Ponceau S to determine the transfer and the position of the protein band on the nitrocellulose membrane.
  • Figure 3 shows the degradation effect of the compound on BRD4 at different time points under the condition of 100 nM concentration. It can be seen from the figure that the degradation effect of the compound of Example 115 on BRD4 protein gradually increased with time, and it was almost completely degraded in 24 hours; similar to the positive control dBET6, the compound of Example 128 appeared to have an effect on BRD4-Short after completely degrading BRD4 in 6 hours. The rebound. With the increase of time, the compounds of Example 115 and Example 128 showed continuous down-regulation of its downstream protein c-Myc.

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Abstract

一种吡咯并吡啶酮类化合物或其药学上可接受的盐，以及它们的制备方法和用途。所述吡咯并吡啶酮类化合物具有I所示的结构。该化合物具有BRD4和HDAC双靶抑制活性的特点和/或具有靶向泛素化降解BRD4蛋白功能的特点，可应用于与该靶点相关的抗肿瘤等相关疾病。

Description

一种吡咯并吡啶酮类化合物、其制备方法、其组合物和用途 技术领域
本发明涉及一种吡咯并吡啶酮类化合物或其药学上可接受的盐，其制备方法、及其组合物用于治疗与该靶点相关的肿瘤疾病等的药物用途。
背景技术
组蛋白赖氨酸残基的乙酰化被认为是转录活性基因的标志(参见非专利文献1)。一方面，赖氨酸乙酰化可以中和其正电荷，导致组蛋白对带负电荷的DNA的亲和力降低或使核小体的结构变得松散，最终形成一个开放的、可进入的染色质结构，能够招募转录因子。另一方面，乙酰化赖氨酸为蛋白识别模块提供了结合位点。组蛋白赖氨酸残基的乙酰化和去乙酰化主要由组蛋白乙酰化酶(histone acetylase，HATs)和组蛋白去乙酰化酶(histone deacetylases,HDACs)控制。而组蛋白上的乙酰化能够特异的被溴结构域蛋白(bromodomains，BRDs)识别，招募染色质调节相关蛋白、转录因子、染色质重塑因子等，从而在调控基因转录和染色质重塑中发挥重要作用(参见非专利文献2)。
溴结构域蛋白(bromodomains，BRDs)按照结构和序列相似性可以被分为8个家族。其中，溴结构域和超末端结构蛋白(bromodomain and extra-terminal Domain,BET)家族属于BRD家族中的第二类，包括BRD2、BRD3、BRD4以及BRDT。BET蛋白能够识别乙酰化的组蛋白，招募转录因子和转录延长因子等，调控细胞的转录和细胞周期，在多种疾病尤其是肿瘤、炎症中扮演着重要的角色(参见非专利文献3)。BRD4作为该家族中主要的成员，与转录调控和肿瘤的发生等密切相关(参见非专利文献4)。许多调节癌细胞增殖、抗凋亡和侵袭性的癌基因都受BRD4调控，其中c-Myc作为第一个被报道的在血液瘤和实体瘤中受BRD4调控的致癌基因，为BET蛋白抑制剂的开发提供了理论依据(参见非专利文献5、6)。还有一些其他的致癌基因例如FOSL1(FRA-1)、BCL-2、RUNX2、c-KIT等受BRD4调控(参见非专利文献7-10)。此外，BRD4已被证明可以调控与修复受损DNA相关的分子机制，并与癌症中异常的端粒调控有关，这突出了这种蛋白在癌症发生过程中的功能多样性作用(参见非专利文献11、12)。
目前，已经发展出了有多种结构类型的BET抑制剂，例如I-BET 762,JQ1,I-BET 151,OTX-015,TEN-010,ZEN003694,GS-5829,CPI-0610,ABBV-075,ABBV-744等，被广泛应用于血液瘤、实体瘤和心血管疾病等的研究。该类抑制剂的早期临床研究虽然能够有取 得短暂的治疗效果，但是也存在一些毒副作用，例如严重的血小板减少、疲劳、恶心、呕吐和腹泻等胃肠道副作用等(参见非专利文献13)。
HDACs的作用使组蛋白去乙酰化，令组蛋白和DNA的结合更为紧密，从而抑制基因的表达。目前研究表明，HDAC能够影响细胞周期、细胞凋亡、DNA损伤相应、癌细胞转移、血管生成、自噬和其他细胞进程，在肿瘤进程中扮演至关重要的角色(参见非专利文献14)。HDAC的抑制会引起组蛋白总体乙酰化水平的改变，尤其是缺乏乙酰化或者低密度乙酰化区域(参见非专利文献15-17)。而整个基因组中乙酰化的重新分布能够影响BET蛋白的募集，使其从调控区域转移到新的乙酰化位点，使BET蛋白的促进转录功能作用降低。因此，HDAC和BET蛋白通过基因组乙酰化谱可能存在着一些直接或者间接的相互作用(参见非专利文献18)。例如癌细胞生长中心超级增强子依赖于BRD4蛋白，因此BET抑制剂能够优先抑制癌细胞依赖的癌基因转录(参见非专利文献4)；而HDAC抑制剂能使组蛋白和其他转录调节因子的乙酰化水平升高，这能够诱导RNA聚合酶Ⅱ的延长暂停，从而优先抑制高表达的致癌扩增子(参见非专利文献16)。此外BRD4蛋白具有组蛋白分子伴侣的功能，能够协助RNA聚合酶Ⅱ通过乙酰化的核小体，而HDAC通过去除组蛋白或者其他蛋白上的乙酰化，改变BRD4在组蛋白在核小体和染色质相关蛋白复合物的结合(参见非专利文献19)。
许多临床前研究表明，HDAC和BRD4抑制剂能够在多种肿瘤细胞中表现出协同作用，而且这种药物联用能够在较低的剂量下达到较高的细胞毒性，从而可能避免叠加毒性的问题。例如在c-Myc诱导的淋巴瘤小鼠中，BET抑制剂能够部分敏化由HDAC沉默基因诱导的c-Myc过表达淋巴瘤细胞，而BET抑制剂RVX2135和HDAC抑制泛剂SAHA联用对淋巴瘤移植小鼠的无进展生存期具有协同作用(参见非专利文献20)。在成神经细胞瘤和极性骨髓性白血病细胞系中，帕比司他和JQ1联合治疗相比单独用药能够更有效的诱导细胞凋亡和生长抑制，而治疗对正常细胞没有显著影响，且能够阻止肿瘤生长，提高移植瘤小鼠的存活率(参见非专利文献21、22)。同样，JQ1与SAHA联用在胰腺导管癌(PDAC)的体内外模型中都能够产生有效且持续的抗肿瘤效果(参见非专利文献23)；在尿路上皮癌细胞系中，JQ1和罗米地辛联用显示出协同作用(参见非专利文献24)；帕比司他和I-BET151联合使用可强烈诱导黑色素瘤细胞系和对BRAFi耐药的黑色素瘤患者的细胞凋亡和细胞周期阻滞(参见非专利文献25)；panobinostat和OTX-015能够抑制胶质母细胞瘤细胞的增殖，增强caspase介导的凋亡，尤其是抑制磷酸化和糖酵解，导致细胞出现能量危机和严重的应激反应(参见非专利文献26)。
这些研究证明了HDAC和BRD4抑制剂联合治疗的有效性，而将HDAC和BRD4抑制剂活性基团结合到同一个分子中，可开发出一类新型更为有效的双靶小分子药物，同时能够表观遗传机制研究提供新型的工具分子，目前尚没有吡咯并吡啶酮类HDAC和BRD4双靶抑制剂小分子的报道。
近年来还发展出一种利用蛋白水解靶向嵌合分(Proteolysis Targeting Chimeras,PROTACs)技术设计合成的双功能小分子，能够将BRD4蛋白和细胞内的E3拉近，从而导致目标蛋白的降解。和传统的抑制剂相比，该类双功能小分子能够更加有效的抑制肿瘤细胞的生长，促进其凋亡(参见非专利文献27)。发展能够靶向泛素化降解BRD4蛋白的双功能小分子在研究BRD4蛋白的功能以及新型抗肿瘤药物的研发中具有重要意义(参见非专利文献28)，目前上没有基于吡咯并吡啶酮类结构母核的BRD4泛素化降解小分子的报道。
现有技术文献
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发明内容
本发明基于吡咯并吡啶酮类为母核，设计合成出新化合物，其具有HDAC和BRD4双靶抑制活性，或者具有能够抑制BRD4和泛素化降解BRD4蛋白的双功能，具有明确的细胞生物学功能，可应用于抗肿瘤等疾病。
鉴于此，提供一种式I化合物或其药学上可接受的盐：
Figure PCTCN2020096521-appb-000001
其中，
R 1选自
Figure PCTCN2020096521-appb-000002
Figure PCTCN2020096521-appb-000003
其中，R a表示一个或多个取代基，各自独立地选自氢、卤素、羟基、氰基、烷基、任选被烷基取代的氨基、卤代烷基、烷氧基，
X选自亚烷基、杂亚烷基、亚烯基、亚杂芳基、(亚烷基) 0或1-亚芳基-(亚烷基) 0 或1、亚烷基-CONH-亚烷基、亚环烷基、亚环烯基、亚杂环基、亚杂环烯基；所述亚芳基、亚杂芳基、(亚烷基) 0或1-亚芳基-(亚烷基) 0或1、亚环烷基、亚环烯基、亚杂环基或亚杂环烯基任选被环烯基、杂环基、杂环烯基、羟基、硝基、氰基、任选被烷基取代的氨基、烷氧基、烷基，烯基、芳基、杂芳基、环烷基、杂链烃基取代，
A选自芳基、杂芳基，
R 2表示一个或多个取代基，各自独立地选自氢、＝O、卤素、羟基、氰基、烷基、卤代烷基、烷氧基、环烷基氧基、芳氧基、羟烷基、-(R b)NS(O) 2R c、-S(O) 2R d、R eO(C＝O)-、R fR gN-C 1-10烷基、杂环基烷基、环烷基烷氧基，其中，R b、R c、R d、R e、R f和R g各自独立地为氢、烷基或卤代烷基；其中所述烷氧基、环烷基氧基、芳氧基、杂环基烷基和环烷基烷氧基任选各自独立地被1、2、3、4或5个选自＝O、卤素、羟基、氰基、烷基、卤代烷基、环烷基的基团取代；
或者，R 2表示两个或两个以上取代基，其中两个相邻的取代基连接，并与所连接的A上的碳原子一起形成环烷、杂环、芳环、杂芳环；所述环烷、杂环、芳环、杂芳环任选被卤素、＝O、羟基、氰基、烷基、卤代烷基、烷氧基、胺基、磺酰基、烷基磺酰基、烷基磺酰氨基取代。
可选地，所述“卤素”选自氟、氯、溴或碘。
可选地，所述“烷基”、“被烷基取代的氨基”、“卤代烷基”、“烷氧基”、“羟烷基”、“杂环基烷基”、“环烷基烷氧基”中的烷基各自独立地为C 1-C 20直链或支链烷基，可选地为C 1-C 15直链或支链烷基，可选地为C 1-C 10直链或支链烷基，可选地为C 1-C 7直链或支链烷基，可选地为C 1-C 6直链或支链烷基，可选地为C 1-C 5直链或支链烷基，可选地C 1-C 4直链或支链烷基，可选地，选自甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、仲丁基、正戊基，1-甲基丁基、2-甲基丁基、3-甲基丁基、异戊基、1-乙基丙基、新戊基、正己基、1-甲基戊基、2-甲基戊基、3-甲基戊基、异己基、1,1-二甲基丁基、2,2-二甲基丁基、3,3-二甲基丁基、1,2-二甲基丁基、1,3-二甲基丁基、2,3-二甲基丁基、2-乙基丁基、正庚基、2-甲基己基、3-甲基己基、2,2-二甲基戊基、3,3-二甲基戊基、2,3-二甲基戊基、2,4-二甲基戊基、3-乙基戊基或2,2,3-三甲基丁基；可选地，选自甲基、乙基、丙基、异丙基、丁基或异丁基。
可选地，所述“亚烷基”、“(亚烷基) 0或1-亚芳基-(亚烷基) 0或1”和“亚烷基-CONH-亚烷基”中的亚烷基各自独立地为C 1-C 20直链或支链亚烷基，可选地为C 1-C 15直链或支链亚烷基，可选地为C 1-C 10直链或支链亚烷基，可选地为C 1-C 8直链或支链亚烷基，可选地为C 1-C 6直链或支链亚烷基，可选地为C 1-C 4直链或支链亚烷基，可选地为C 1-C 3直链或支链亚烷基，可选地为C 3-C 6直链或支链亚烷基。
可选地，所述“杂亚烷基”为含有1个或多个选自O、S或N的杂原子的C 1-C 20直链或支链亚烷基，可选地为含有1-6个选自O、S或N的杂原子的C 1-C 20直链或支链亚烷基，可选地为含有1-4个选自O、S或N的杂原子的C 1-C 20直链或支链亚烷基，可选地为含有1-6个选自O或S的C 1-C 20直链或支链亚烷基，可选地为含有1-4个选自O或S的C 1-C 20直链或支链亚烷基，可选地为含有1-6个O原子的C 1-C 20直链或支链亚烷基，可选地为含有1-4个O原子的C 1-C 20直链或支链亚烷基；可选地碳原子数为1-15个，可选地为1-10个，可选地为1-8个，可选地为1-6个；杂亚烷基为例如–(CH 2CH 2O) n–、–(OCH 2CH 2) m–、–(CH 2CH 2O) lCH 2CH 2–、–(OCH 2) k–、–(CH 2O) i–、–(CH 2O) jCH 2–、C 1-C 20亚烷氧基，其中n、m、l、k、i、j各自独立地为1-6的整数，可选地为1-4的整数，可 选地为1-3的整数，可选地为1或2；可选地杂亚烷基为例如–(CH 2CH 2O) 4CH 2CH 2–、–(CH 2CH 2O) 3CH 2CH 2–、–(CH 2CH 2O) 2CH 2CH 2–或C 1-C 10亚烷氧基，或者C 1-C 8亚烷氧基，或者C 1-C 6亚烷氧基，或者C 1-C 4亚烷氧基。
可选地，所述“亚烯基”为C 2-C 20直链或支链亚烯基，可选地为C 2-C 15直链或支链亚烯基，可选地为C 2-C 10直链或支链亚烯基，可选地为C 2-C 8直链或支链亚烯基，可选地为C 2-C 6直链或支链亚烯基，可选地为C 2-C 4直链或支链亚烯基，可选地所述亚烯基为-CH＝CH-、-CH＝CHCH 2-、-CH 2CH＝CH-或-CH 2CH＝CHCH 2-。
可选地，所述“烯基”为C 2-C 20直链或支链烯基，可选地为C 2-C 15直链或支链烯基，可选地为C 2-C 10直链或支链烯基，可选地为C 2-C 8直链或支链烯基，可选地为C 2-C 6直链或支链烯基，可选地为C 2-C 4直链或支链烯基，可选地所述烯基为CH 2＝CH-、CH 2＝CHCH 2-、CH 3CH＝CH-或CH 3CH＝CHCH 2-。
可选地，所述“亚芳基”、“(亚烷基) 0或1-亚芳基-(亚烷基) 0或1”中的亚芳基为6-10元亚芳基；可选为亚苯基或亚萘基。
可选地，所述“芳基”为6-10元芳基；可选为苯基或萘基。
可选地，所述“芳环”为6-10元芳环；可选为苯环或萘环。
可选地，所述“亚杂芳基”为含有1-3个选自N、O和S中的杂原子的5-10元亚杂芳基；可选地，为含有1-2个选自N、O和S中的杂原子的5-10元亚杂芳基；可选地，所述亚杂芳基选自亚吡啶基、亚吡咯基、亚嘧啶基、亚吡嗪基、亚哒嗪基、亚噻吩基、亚呋喃基。
可选地，所述“杂芳环”、“杂芳基”中的环为含有1-3个选自N、O和S中的杂原子的5-10元杂芳环；可选地，为含有1-2个选自N、O和S中的杂原子的5-10元杂芳环；可选地，所述环选自吡啶环、吡咯环、嘧啶环、吡嗪环、哒嗪环、噻吩环、呋喃环。
可选地，所述“(亚烷基) 0或1-亚芳基-(亚烷基) 0或1”优选为亚苯乙基、亚苄基、亚乙苯基、亚甲苯基。
可选地，所述“亚环烷基”为C 3-C 10单环、双环、三环、或四环亚环烷基；可选地，所述亚环烷基选自亚环丙基、亚环丁基、亚环戊基、亚环己基、亚环庚基及亚环辛基。
可选地，所述“环烷”、“环烷基”的环烷为C 3-C 10单环、双环、三环、或四环环烷；可选地，所述环烷选自环丙烷、环丁烷、环戊烷、环己烷、环庚烷及环辛烷。
可选地，所述“亚环烯基”为含一或多个双键的部分不饱和单环、双环、三环、或四环C 3-C 12亚环烯基；可选地为亚环丙烯基、亚环丁烯基、亚环戊烯基、亚环己烯基、 亚环庚烯基或亚环辛烯基。
可选地，所述“环烯基”为含一或多个双键的部分不饱和单环、双环、三环、或四环C 3-C 12环烯基；可选地为环丙烯基、环丁烯基、环戊烯基、环己烯基、环庚烯基或环辛烯基。
可选地，所述“杂环”、“杂环基”、“亚杂环基”中的杂环为环上含有1个、2个或3个选自N、O、S的杂原子的3-10元非芳香杂环，可选地，所述杂环为环上含有1个或2个选自N、O的杂原子的3-6元非芳香环。
可选地，所述“亚杂环烯基”为环上含有1个、2个或3个选自N、O、S的杂原子且含有1个或多个双键的3-10元非芳香亚杂环烯基，可选地，为环上含有1个或2个选自N、O的杂原子的3-6元非芳香亚杂环烯基。
可选地，所述“杂环烯基”为环上含有1个、2个或3个选自N、O、S的杂原子且含有1个或多个双键的3-10元非芳香杂环烯基，可选地，为环上含有1个或2个选自N、O的杂原子的3-6元非芳香杂环烯基。
可选地，所述“杂链烃基”为链上含1-6个选自N、O、S的杂原子的直链或支链饱和或不饱和C 1-C 20杂链烃结构；可选地，为链上含1个、2个、3个或4个选自N、O的杂原子的直链或支链饱和或不饱和C 1-C 20杂链烃结构，可选地，为链上含1个、2个、3个或4个O的直链或支链饱和或不饱和C 1-C 20杂链烃结构，可选地，为链上含1个、2个、3个或4个O的直链或支链饱和或不饱和C 1-C 10杂链烃结构。
可选地，所述的式I化合物或其药学上可接受的盐中，R a表示一个或多个取代基，各自独立地选自氢或卤素，
X选自C 1-C 10亚烷基、C 1-C 10亚烷基-CONH-C 1-C 10亚烷基、–(CH 2CH 2O) n–、–(OCH 2CH 2) m–、–(CH 2CH 2O) lCH 2CH 2–、–(OCH 2) k–、–(CH 2O) i–、–(CH 2O) jCH 2–、C 1-C 10亚烷氧基、(C 1-C 10亚烷基) 0或1-亚芳基-(C 1-C 10亚烷基) 0或1，其中n、m、l、k、i、j各自独立地为1-6的整数；
A选自苯基，
R 2表示1、2、3、4或5个取代基，各自独立地选自氢、＝O、卤素、羟基、氰基、C 1-6烷基、卤代C 1-6烷基、C 1-6烷氧基、C 3-6环烷基氧基、C 6-10芳氧基、羟基C 1-6烷基、-(R b)NS(O) 2R c、-S(O) 2R d、R eO(C＝O)-、R fR gN-C 1-10烷基、C 5-10杂环基C 1-6烷基、C 3-6环烷基C 1-6烷氧基，其中，R b、R c、R d、R e、R f和R g各自独立地为氢、C 1-6烷基或卤代C 1-6烷基；其中所述C 1-6烷氧基、C 3-6环烷基氧基、C 6-10芳氧基、C 5-10杂环基C 1-6烷基和 C 3-6环烷基C 1-6烷氧基任选各自独立地被1、2、3、4或5个选自卤素、羟基、氰基、C 1-6烷基、卤代C 1-6烷基、C 3-6环烷基的基团取代；
或者，R 2表示两个或两个以上取代基，其中两个相邻的取代基连接，并与所连接的A环上的碳原子一起形成杂环，所述杂环优选为[1,4]恶嗪环；所述杂环任选被＝O、烷基、磺酰基、烷基磺酰基取代。
可选地，上述式I化合物或其药学上可接受的盐中，所述式I化合物选自下列化合物：
Figure PCTCN2020096521-appb-000004
Figure PCTCN2020096521-appb-000005
Figure PCTCN2020096521-appb-000006
Figure PCTCN2020096521-appb-000007
Figure PCTCN2020096521-appb-000008
Figure PCTCN2020096521-appb-000009
Figure PCTCN2020096521-appb-000010
Figure PCTCN2020096521-appb-000011
Figure PCTCN2020096521-appb-000012
Figure PCTCN2020096521-appb-000013
Figure PCTCN2020096521-appb-000014
Figure PCTCN2020096521-appb-000015
Figure PCTCN2020096521-appb-000016
Figure PCTCN2020096521-appb-000017
可选地，上述式I化合物或其药学上可接受的盐中，所述药学上可接受的盐包括所述式I化合物的阴离子盐和阳离子盐；
优选地，所述药学上可接受的盐包括所述式I化合物的碱金属的盐、碱土金属的盐、铵盐；优选地，所述碱金属包括钠、钾、锂、铯，所述碱土金属包括镁、钙、锶；
优选地，所述药学上可接受的盐包括所述式I化合物与有机碱形成的盐；优选地，所述有机碱包括三烷基胺、吡啶、喹啉、哌啶、咪唑、甲基吡啶、二甲氨基吡啶、二甲基苯胺、N-烷基吗啉、1,5-二氮杂双环[4.3.0]壬烯-5(DBN)、1,8-二氮杂双环[5.4.0]十一碳烯-7(DBU)、1,4-二氮杂双环[2.2.2]辛烷(DABCO)；优选地，所述三烷基胺包括三甲胺、三乙胺、N-乙基二异丙胺；优选地，所述N-烷基吗啉包括N-甲基吗啉；
优选地，所述药学上可接受的盐包括所述式I化合物与酸形成的盐；优选地，所述酸包括无机酸、有机酸；优选地，所述无机酸包括盐酸、氢溴酸、氢碘酸、硫酸、硝酸、磷酸、碳酸；优选地，所述有机酸包括甲酸、乙酸、丙酸、草酸、丙二酸、琥珀酸、富马酸、马来酸、乳酸、苹果酸、柠檬酸、枸橼酸、酒石酸、碳酸、苦味酸、甲磺酸、乙 磺酸、对甲苯磺酸、谷氨酸、双羟萘酸。
另一方面，还提供制备上述式I化合物或其药学上可接受的盐的方法，
Figure PCTCN2020096521-appb-000018
其特征在于，包括：
将式II化合物与NH 2X-R 1发生缩合反应，得到式I化合物，
Figure PCTCN2020096521-appb-000019
其中，式I中，A、R 2、X、R 1如上所定义；
式II中，P为氢或保护基团，Q为氢或C 1-C 10烷基；A和R 2如上所定义。
关于保护基团：当多官能团有机化合物进行反应时，为使反应只发生在所希望的官能团处，而避免其他官能团遭受影响，在反应前将其他官能团先加以保护，当反应完成后再恢复。保护基团是这种能够对所述其他官能团提供保护的一类基团的总称。此处保护基团可以为例如苄基、对甲苯磺酰基、(三甲基甲硅烷基)乙氧基)甲基)。
式NH 2X-R 1中，X、R 1如上所定义。
或者，其特征在于，包括：
将式III化合物与式NH 2R 01发生缩合反应，得到式I化合物，
Figure PCTCN2020096521-appb-000020
其中，
式I中，R 1为
Figure PCTCN2020096521-appb-000021
A、R 2、X、R a如上所定义。
式III中，A、R 2、X如上所定义，M为氢或C 1-C 10烷基；
式NH 2R 01中，R 01为羟基、
Figure PCTCN2020096521-appb-000022
R a如上所定义；
或者，其特征在于，包括：将式IV化合物与NH 2R 02发生缩合反应，得到式I化合物，
Figure PCTCN2020096521-appb-000023
其中，式I中，A、R 2、R 1如上所定义；X为亚烷基-CONH-亚烷基，
式IV中，A、R 2如上所定义；M为氢或C 1-C 10烷基；X 1为亚烷基；
式NH 2R 02中，R 02为亚烷基-R 1，R 1如上所定义。
优选地，所述式III化合物或式IV化合物的制备方法包括：
将式II化合物与式H 2NXCOOM化合物或式H 2NX 1COOM化合物发生缩合反应，得到式III化合物或式IV化合物；
式H 2NXCOOM中，X定义同式III中的定义；
式H 2NX 1COOM中，X 1定义同式IV中的定义；
优选地，式II化合物的制备方法包括：将式V化合物与芳基硼酸发生Suzuki偶联反应得到式II化合物，
或者，
将式V化合物通过Suzuki偶联反应得到式VI的芳基硼酸酯化合物，然后将式VI的芳基硼酸酯化合物与溴代物发生Suzuki偶联反应得到式II化合物，
Figure PCTCN2020096521-appb-000024
式V中，PG表示保护基团(例如苄基、对甲苯磺酰基、(三甲基甲硅烷基)乙氧基)甲基)，Y表示卤素，Q为氢或C 1-C 10烷基；
式VI中，PG和Q的定义与式V中相同；
所述芳基硼酸为式VII化合物：
Figure PCTCN2020096521-appb-000025
其中，A和R 2的定义与式II中的相同；
所述溴代物为式VIII化合物：
Figure PCTCN2020096521-appb-000026
其中，A和R 2的定义与式II中的相同。
优选地，所述式V化合物的制备方法包括：
将式XIV化合物与N,N-二甲基甲酰胺二甲基缩醛反应得到式XIII化合物，所得式XIII化合物在酸性条件下还原得到式XII化合物，式XII化合物在强碱条件下和卤化物反应得到式XI化合物，所得式XI化合物在强碱条件下与氯甲酸烷基酯反应得到式X化合物，对所得式X化合物用酸处理得到式IX化合物，在碱性条件和溶剂中用甲基化试剂将式IX化合物甲基化得到式V化合物；
Figure PCTCN2020096521-appb-000027
式IX至式XIV中，Y、PG和Q的定义与式V中相同。
优选地，在上述制备方法中，所述缩合反应在碱性条件下和缩合剂的存在下，在溶剂中进行；优选地，所述缩合剂包括：N,N'-羰基二咪唑、二环己基碳二亚胺、O-(7-氮杂苯并三唑-1-基)-N,N,N',N'-四甲基脲、N-羟基-7-偶氮苯并三氮唑、2-(1H-苯并三偶氮L-1-基)-1,1,3,3-四甲基脲四氟硼酸酯或1-羟基苯并三唑；优选地，所述碱性条件所用的碱包括三乙胺、二异丙基乙胺、DMAP中的一种或者两种以上；所述溶剂包括：四氢呋喃、二氯甲烷或N,N-二甲基甲酰胺。
优选的，所述Suzuki偶联反应在钯催化剂的存在下在碱性条件下于溶剂中进行，任选地在配体的存在下进行；优选地，所述Suzuki偶联反应在60℃至150℃的温度下进行，优选地，该反应通过微波辐射促发；优选地，所述碱性条件所用的碱包括CsF、Cs 2CO 3、K 2CO 3、三乙胺、二异丙基乙胺、DMAP中的一种或者两种以上；优选地，所述钯催化剂包括：Pd 2(dba) 3、Pd(OAc) 2或Pd(PPh 3) 4；优选地，所述配体包括：1,3,5,7-四甲基-6-苯基-2,4,8-三氧杂-6-磷酰金刚烷、2-二环己基磷-2’,4’,6’-三异丙基联苯、1,1’-双(二苯基磷烷基)二茂铁；优选地，所述溶剂包括：甲醇、二甲氧基乙烷、N,N-二甲基甲酰胺、二甲亚砜、二氧杂环己烷、四氢呋喃和水，或其混合物；
优选地，式XIV化合物与N,N-二甲基甲酰胺二甲基缩醛的反应60℃至100℃的温度下)进行；优选地在溶剂中进行，所述溶剂包括：N,N-二甲基甲酰胺；
优选地，所述式XIII化合物在酸性条件下的还原反应中，所述酸包括：氯化铵或乙酸；还原反应所用的还原剂包括还原铁粉；
优选地，所述式XII化合物在强碱条件下和卤化物的反应中，所述强碱包括氢化钠；
优选地，所得式XI化合物在强碱条件下与氯甲酸烷基酯的反应中，所述强碱包括二 异丙基氨基锂；
优选地，所述式X化合物用酸处理步骤中，在溶剂中于40℃至100℃的温度下进行，优选的，所述酸包括盐酸、氢溴酸；所述溶剂包括二氧六环、水；
优选地，在式IX化合物甲基化反应，在溶剂中于40℃至100℃的温度下进行，优选地，甲基化试剂为碘甲烷；优选的，所述碱性条件所用的碱包括氢化钠、碳酸铯或碳酸钾；所述溶剂包括N，N-二甲基甲酰胺或二甲亚砜。
另一方面，还提供一种药物组合物，包括上述式I化合物或其药学上可接受的盐和药学上可接受的辅料。
另一方面，还提供上述式I化合物或其药学上可接受的盐或上述药物组合物在制备用于预防或治疗肿瘤的药物中的用途。
另一方面，还提供一种预防或治疗肿瘤的方法，包括给予需要的患者预防或治疗有效量的上述式I化合物或其药学上可接受的盐，或上述药物组合物。
优选地，所述肿瘤包括非小细胞肺癌、乳腺癌、甲状腺癌(甲状腺髓样癌、乳头状甲状腺癌)、胃癌、膀胱癌、子宫内膜癌、前列腺癌、宫颈癌、结肠癌、食管癌、角质母细胞瘤、骨髓瘤、横纹肌肉瘤、急性白血病、肝癌、腺癌或胰腺癌。
根据一方面的具体实施方式，通过引入R 1中的HDAC关键药效团，获得了具有抑制BRD4和HDAC作用的双功能抑制剂，或者，根据本发明另一方面的具体实施方式，通过引入R 1中的泛素化募集药效团，获得了具有BRD4抑制活性以及泛素化降解BRD4蛋白的双功能化合物。
根据本发明另一方面的具体实施方式，测试化合物在急性单核细胞白血病细胞MV-4-11、骨髓增生异常综合征细胞株SKM-1及人胰腺癌细胞株MIA Paca2均显示出了很强的抑制细胞增殖活性。
根据本发明另一方面的具体实施方式，测试化合物在急性单核细胞白血病细胞MV-4-11显示出了明显的抑制细胞增殖活性。
附图说明
图1为实验二中实施例7和10化合物在SKM-1细胞中HDAC和BRD4信号通路影响的Western-Blotting图，其中Ac-tub是指乙酰化的tublin蛋白，Ac-H3是指乙酰化的H3蛋白。
图2为实验二中实施例7和10化合物在MV-4-11HDAC和BRD4信号通路影响的Western-Blotting图，其中Ac-tub是指乙酰化的tublin蛋白，Ac-H3是指乙酰化的H3蛋白。
图3是实验四中实施例105化合物在MV-4-11细胞株上对BRD4蛋白降解功能的Western-Blotting图。
具体实施方式
以下对本发明的具体实施方式进行详细说明。应当理解的是，此处所描述的具体实施方式仅用于示例性地对本发明进行说明，并不用于限制本发明。
实施例
实施例1
Figure PCTCN2020096521-appb-000028
实施例1a 4-溴-7-甲氧基-1-氢-吡咯并[2,3-c]吡啶(化合物1a)
Figure PCTCN2020096521-appb-000029
将5-溴-2-甲氧基-4-甲基-3-硝基吡啶(25g,101.19mmol)溶于N,N-二甲基甲酰胺(50mL)，并加入N,N-二甲基甲酰胺二甲基缩醛(34mL,253mmol)，用油浴加热80℃下反应12小时；反应结束后冷却至室温，减压蒸干溶剂，得到红色固体。将所得固体溶于乙酸(400mL)，搅拌状态下加入还原铁粉(40g,708.36mmol)，用油浴加热回流反应4小时；反应结束冷却后用硅藻土滤除不溶固体，用热乙醇洗涤滤渣至不再有产物，减压蒸干溶剂。将所得固体溶于乙酸乙酯(300mL)，依次用水(100mL)、饱和碳酸钠水溶液(100mL)、饱和氯化钠水溶液(100mL)洗有机相，合并有机相后用无水硫酸钠干燥，减压蒸干溶剂后用石油醚和乙酸乙酯20:1溶液重结晶，得到产物4-溴-7-甲氧基-1-氢-吡咯并[2,3-c]吡啶20g，(产率：87％)。
实施例1b 4-溴-7-甲氧基-1-甲苯磺酰基吡咯并[2,3-c]吡啶(化合物1b)
Figure PCTCN2020096521-appb-000030
在室温条件下将化合物1a(10g,44.04mmol)溶于N,N-二甲基甲酰胺(100mL)。在冰浴条件下向该溶液中介入氢化钠(2.11g，1.27g的60％矿物油分散液，52.85mmol)，搅拌10分钟后分批加入对甲苯磺酰氯(10.08g,52.85mmol)，冰浴条件下氮气保护搅拌2小时至反应完全。在冰浴条件下用饱和氯化铵水溶液淬灭反应后，抽滤得米白色不溶固体并用水洗涤。收集固体并在50℃真空烘箱中干燥得到产物4-溴-7-甲氧基-1-甲苯磺酰基-1-氢吡咯并[2,3-c]吡啶16g(产率：95％)。
实施例1c 4-溴-7-甲氧基-1-甲苯磺酰基-1-氢-吡咯并[2,3-c]吡啶-2-甲酸甲酯(化合物1c)
Figure PCTCN2020096521-appb-000031
向干燥的三口圆底烧瓶中加入化合物1b(10g,26.23mmol)，室温条件下溶于干燥的四氢呋喃(80mL)后，在-78℃条件下滴加2M的二异丙基氨基锂的四氢呋喃溶液(20mL,40mmmol)。逐渐升温至-50℃反应30分钟后，滴加氯甲酸甲酯(3.05mL,39.34mmol)，继续反应45分钟后，用饱和氯化铵溶液淬灭反应，乙酸乙酯萃取后用饱和食盐水洗有机相，无水硫酸钠干燥后，减压蒸干溶剂后柱层析，得到产物4-溴-7-甲氧基-1-甲苯磺酰基-1-氢-吡咯并[2,3-c]吡啶-2-甲酸甲酯8g(产率:70％)。
实施例1d 4-溴-7-氧代-1-甲苯磺酰基-6,7-二氢-1H-吡咯并[2,3-c]吡啶-2-羧酸甲酯(化合物1d)
Figure PCTCN2020096521-appb-000032
将化合物1c(3g,6.83mmol)溶于4M的氯化氢(24mL,136.60mmol)的二氧六环溶液中，在60℃条件氩气保护反应2小时至反应完全，减压蒸干溶剂得到产物4-溴-7-氧代-1-甲苯磺酰基-6,7-二氢-1H-吡咯并[2,3-c]吡啶-2-羧酸甲酯2.9g(产率：100％)。
实施例1e 4-溴-6-甲基-7-氧代-1-甲苯磺酰基-6,7-二氢-1H-吡咯并[2,3-c]吡啶-2-羧酸甲酯(化合物1e)
Figure PCTCN2020096521-appb-000033
在室温条件下将化合物1d(2.9g,6.83mmol)溶于N,N-二甲基甲酰胺(30mL)。在冰浴条件下向该溶液中加入氢化钠(0.327g，0.106g的60％矿物油分散液，8.18mmol)，搅拌10分钟后加入碘甲烷(0.509mL,8.18mmol)。冰浴条件下氮气保护搅拌2小时至反应完全。在冰浴条件下用饱和氯化铵水溶液淬灭反应后，用乙酸乙酯萃取。然后用水和饱和氯化钠溶液洗涤，合并有机相经无水硫酸钠干燥，过滤浓缩后将所得固体用石油醚：乙酸乙酯10:1重结晶，得到产物4-溴-6-甲基-7-氧代-1-甲苯磺酰基-6,7-二氢-1H-吡咯并[2,3-c]吡啶-2-羧酸甲酯2.5g(产率：83％)。
实施例1f 6-甲基-7-氧代-4-(2-苯氧基苯基)-1-甲苯磺酰基-6,7-二氢-1H-吡咯并[2,3-c]吡啶-2-羧酸甲酯(化合物1f)
Figure PCTCN2020096521-appb-000034
在氩气保护条件下将化合物1e(2g,4.55mmol)、(2-苯氧基苯基)硼酸(1.46g,6.83mmol)、氟化铯(1.38g,9.10mmol)、三(二亚苄基丙酮)二钯(0)(0.417g,0.46mmol)、1,3,5,7-四甲基-6-苯基-2,4,8-三氧杂-6-磷酰金刚烷(0.20g,0.91mmol)溶于乙二醇二甲醚(2mL)和甲醇(0.4mL)混合溶液中，60℃油浴条件下搅拌2小时至反应完全。用硅藻土滤除不溶物后乙酸乙酯萃取。然后用氯化钠溶液洗涤，合并有机相经无水硫酸钠干燥，过滤并浓缩。通过快速层析法(硅胶2-50％乙酸乙酯/石油醚梯度)分离出产物6-甲基-7-氧代-4-(2-苯氧基苯基)-1-甲苯磺酰基-6,7-二氢-1H-吡咯并[2,3-c]吡啶-2-羧酸甲酯2g(产率：83％)。
实施例1g 6-甲基-7-氧代-4-(2-苯氧基苯基)-6,7-二氢-1H-吡咯并[2,3-c]吡啶-2-羧酸(化合物1g)
Figure PCTCN2020096521-appb-000035
将化合物1f(0.2g,0.378mmol)溶于二氧六环(2mL)后加入4M的氢氧化钠溶液(0.4mL,1.6mmol)，在氩气保护条件下90℃搅拌2小时至反应完全。用1N盐酸溶液调节体系pH至1-3，将析出的白色固体抽滤、干燥，得到产物6-甲基-7-氧代-4-(2-苯氧基苯基)-6,7-二氢-1H-吡咯并[2,3-c]吡啶-2-羧酸0.11g(产率：81％)。
实施例1h 4-(6-甲基-7-氧代-4-(2-苯氧基苯基)-6,7-二氢-1H-吡咯并[2,3-c]吡啶-2-甲酰胺基)丁酸甲酯(化合物1h)
Figure PCTCN2020096521-appb-000036
将化合物1g(0.11g,0.305mmol)溶于N,N-二甲基甲酰胺(1.5mL)后加入N,N-二异丙基乙胺(0.079mL,0.610mmol)、N-羟基-7-偶氮苯并三氮唑(0.062g,0.458mmol)和1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(0.088g,0.458mmol)，室温反应20分钟后加入4-氨基丁酸甲酯(0.054g,0.458mmol)。在室温下搅拌4小时至反应完全。加入水淬灭反应，乙酸乙酯萃取后用水和饱和氯化钠溶液洗涤，合并有机相并经无水硫酸钠干燥，过滤并浓缩。通过快速层析法(硅胶0-5％二氯甲烷/石甲醇梯度)分离出产物4-(6-甲基-7-氧代-4-(2-苯氧基苯基)-6,7-二氢-1H-吡咯并[2,3-c]吡啶-2-甲酰胺基)丁酸甲酯0.1g(产率：71％)
将化合物1h(0.1g,0.217mmol)溶于二氯甲烷(0.2mL)和甲醇(1mL)，冰浴条件下加入50％羟胺水溶液(0.286mL,4.35mmol)后加入氢氧化钠固体(0.174g,4.35mmol)，继续搅拌2小时至反应完全。用3N盐酸溶液调节体系pH至1-3，将析出的固体抽滤后用水洗，干燥，得到实施例1化合物，0.07g(产率：70％)。
1H NMR(400MHz,DMSO)δ12.21(s,1H),10.38(s,1H),8.71(s,1H),8.38(t,J＝5.4Hz,1H),7.52(dd,J＝7.6,1.7Hz,1H),7.45–7.39(m,1H),7.33–7.25(m,4H),7.07–7.00(m,2H),6.89–6.83(m,3H),3.51(s,3H),3.23(dd,J＝12.7,6.8Hz,2H),2.02(t,J＝7.5Hz, 2H),1.78-1.68(m,2H).LR-MS(ESI)m/z 461(M+1)
实施例2
Figure PCTCN2020096521-appb-000037
实施例2a 4-(6-甲基-7-氧代-4-(2-苯氧基苯基)-6,7-二氢-1H-吡咯并[2,3-c]吡啶-2-甲酰胺基)丁酸(化合物2a)
Figure PCTCN2020096521-appb-000038
将化合物1h(0.1g,0.218mmol)溶于二氧六环(2mL)后加入2M的氢氧化钠溶液0.436mL,0.872mmol)，在氩气保护条件下90℃搅拌2小时至反应完全。用1N盐酸溶液调节体系pH至1-3，将析出的白色固体抽滤干燥得到产物化合物2a。
将化合物2a(0.08g,0.180mmol)溶于N,N-二甲基甲酰胺(1mL)后加入N,N-二异丙基乙胺(0.089mL,0.540mmol)、N-羟基-7-偶氮苯并三氮唑(0.034g,0.252mmol)和1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(0.048g,mmol)，室温反应20分钟后加入邻苯二胺(0.029g,0.269mmol)。在室温下搅拌4小时至反应完全。加入水淬灭反应，乙酸乙酯萃取后用水和饱和氯化钠溶液洗涤，合并有机相并经无水硫酸钠干燥，过滤并浓缩。通过快速层析法(硅胶0-7％二氯甲烷/甲醇梯度)分离出实施例2化合物.
1H NMR(400MHz,DMSO)δ12.24(s,1H),9.13(s,1H),8.37(s,1H),7.52(s,1H),7.41(dd,J＝15.0,3.0Hz,1H),7.36–7.26(m,4H),6.68(ddd,J＝14.9,13.4,3.1Hz,2H),6.58(ddd,J＝15.0,9.7,6.9Hz,1H),6.53–6.43(m,3H),6.40(dd,J＝15.1,3.0Hz,1H),6.25(td,J＝14.8,3.0Hz,1H),5.83(s,1H),4.63(s,2H)3.48(s,3H),3.28(t,J＝9.8Hz,2H),3.20(dd,J＝15.8,5.4Hz,2H),1.49–1.35(m,2H)LR-MS(ESI)m/z 536(M+1).
实施例3
Figure PCTCN2020096521-appb-000039
除了用5-氨基戊酸甲酯代替4-氨基丁酸甲酯以外，合成方法如实施例1。
1H NMR(400MHz,DMSO)δ12.20(s,1H),10.35(s,1H),8.76–8.59(m,1H),8.35(t,J＝5.4Hz,1H),7.52(dd,J＝7.5,1.7Hz,1H),7.41(td,J＝7.8,1.7Hz,1H),7.32–7.24(m,4H),7.05–6.99(m,2H),6.88–6.83(m,3H),3.50(s,3H),3.22(dd,J＝12.3,6.2Hz,2H),1.97(t,J＝7.0Hz,2H),1.58–1.44(m,4H).LR-MS(ESI)m/z 475(M+1).
实施例4
Figure PCTCN2020096521-appb-000040
除了用5-氨基戊酸甲酯代替4-氨基丁酸甲酯以外，合成方法如实施例2。
1H NMR(400MHz,DMSO)δ12.24(s,1H),9.12(s,1H),8.40(t,J＝5.4Hz,1H),7.52(dd,J＝7.5,1.2Hz,1H),7.41(td,J＝7.9,1.4Hz,1H),7.34–7.24(m,4H),7.15(d,J＝7.4Hz,1H),7.06–6.98(m,2H),6.92–6.83(m,4H),6.71(d,J＝7.2Hz,1H),6.53(t,J＝7.6Hz,1H),4.83(s,2H),3.50(s,3H),3.28(dd,J＝11.0,5.1Hz,2H),2.35(t,J＝7.3Hz,2H),1.71–1.60(m,2H),1.60–1.50(m,2H).LR-MS(ESI)m/z 550(M+1)
实施例5
Figure PCTCN2020096521-appb-000041
除了用6-氨基己酸甲酯代替4-氨基丁酸甲酯以外，合成方法如实施例1。
1H NMR(400MHz,DMSO)δ12.22(s,1H),10.34(s,1H),8.67(s,1lH),8.34(s,1H),7.52(d,J＝7.6Hz,1H),7.42(t,J＝7.9Hz,1H),7.25–7.33(m,6.8Hz,4H),7.06–7.00(m, 2H),6.90–6.84(d,J＝5.6Hz,3H),3.51(s,3H),3.23(dd,J＝12.8,6.7Hz,2H),1.95(t,J＝6.7Hz,2H),1.58–1.43(m,4H),1.33–1.26(m,2H).LR-MS(ESI)m/z 489(M+1).
实施例6
Figure PCTCN2020096521-appb-000042
除了用6-氨基己酸甲酯代替4-氨基丁酸甲酯以外，合成方法如实施例2。
1H NMR(400MHz,DMSO)δ12.24(s,1H),9.13(s,1H),8.38(t,J＝5.4Hz,1H),7.52(dd,J＝7.6,1.5Hz,1H),7.42(td,J＝8.0,1.7Hz,1H),7.33–7.24(m,3H),7.14(dd,J＝7.9,1.1Hz,1H),7.06–7.00(m,2H),6.93–6.83(m,4H),6.72(dd,J＝8.0,1.1Hz,1H),6.53(t,J＝7.6Hz,1H),4.97(s,2H),3.51(s,3H),3.30–3.22(m,2H),2.32(t,J＝7.5Hz,2H),1.68–1.60(m,2H),1.59–1.51(m,3H),1.42–1.32(m,2H).LR-MS(ESI)m/z 564(M+1).
实施例7
Figure PCTCN2020096521-appb-000043
除了用7-氨基庚酸甲酯代替4-氨基丁酸甲酯以外，合成方法如实施例1。
1H NMR(600MHz,DMSO)δ12.20(s,1H),10.33(s,1H),8.66(s,1H),8.33(t,J＝5.4Hz,1H),7.52(dd,J＝7.6,1.6Hz,1H),7.42(td,J＝8.1,1.7Hz,1H),7.32–7.25(m,4H),7.05–7.01(m,2H),6.88–6.84(m,3H),3.51(s,3H),3.23(dd,J＝12.7,6.7Hz,2H),1.94(t,J＝7.4Hz,2H),1.53–1.46(m,4H),1.32–1.25(m,4H).LR-MS(ESI)m/z 503(M+1).
实施例8
除了用7-氨基庚酸甲酯代替4-氨基丁酸甲酯以外，合成方法如实施例2。
Figure PCTCN2020096521-appb-000044
1H NMR(400MHz,DMSO)δ12.22(s,1H),9.10(s,1H),8.35(t,J＝5.6Hz,1H),7.52(dd,J＝7.5,1.6Hz,1H),7.44–7.39(m,1H),7.33–7.24(m,4H),7.14(d,J＝6.9Hz,1H),7.05–7.00(m,2H),6.84–6.91(m,4H),6.70(d,J＝6.8Hz,1H),6.53(td,J＝7.9,1.4Hz,1H),4.82(s,2H),3.50(s,3H),3.24(dd,J＝11.5,5.9Hz,2H),2.31(t,J＝7.4Hz,2H),1.65–1.56(m,2H),1.55–1.47(m,2H),1.42–1.30(m,4H).LR-MS(ESI)m/z 578(M+1).
实施例9
Figure PCTCN2020096521-appb-000045
除了用4-(氨基甲基)苯甲酸甲酯代替4-氨基丁酸甲酯以外，合成方法如实施例1。
1H NMR(400MHz,DMSO)δ12.29(s,1H),11.19(s,1H),8.95(t,J＝5.6Hz,1H),7.72(d,J＝8.1Hz,2H),7.52(d,J＝6.5Hz,1H),7.45–7.35(dd,J＝17.4,7.5Hz,3H),7.33–7.25(m,4H),7.08–6.98(m,2H),6.95(s,1H),6.87(d,J＝7.5Hz,2H),4.49(d,J＝5.7Hz,2H),3.50(s,3H).LR-MS(ESI)m/z 509(M+1).
实施例10
Figure PCTCN2020096521-appb-000046
除了用4-(氨基甲基)苯甲酸甲酯代替4-氨基丁酸甲酯以外，合成方法如实施例2。
1H NMR(600MHz,DMSO)δ12.30(s,1H),9.64(s,1H),9.00(t,J＝5.5Hz,1H),7.95(t,J＝9.9Hz,2H),7.53(d,J＝7.5Hz,1H),7.49–7.37(m,3H),7.31(s,1H),7.24–7.30(m,3H),7.17(d,J＝7.5Hz,1H),7.03(d,J＝7.7Hz,2H),7.00-6.94(m,2H),6.87(d,J＝8.0Hz,2H),6.78(d,J＝7.9Hz,1H),6.60(t,J＝7.4Hz,1H),4.89(s,2H),4.53(d,J＝5.6Hz,2H),3.51(s,3H)LR-MS(ESI)m/z 584(M+1).
实施例11
Figure PCTCN2020096521-appb-000047
除了用苯硼酸代替(2-苯氧基苯基)硼酸以外，合成方法如实施例9。
1H NMR(600MHz,DMSO)δ12.40(s,1H),11.17(s,1H),10.14(s,1H),8.99(t,J＝5.8Hz,1H),7.71(d,J＝8.2Hz,2H),7.59(d,J＝7.2Hz,2H),7.47(t,J＝7.7Hz,2H),7.42(s,1H),7.38(d,J＝8.2Hz,2H),7.35(t,J＝7.4Hz,1H),7.13(d,J＝2.2Hz,1H),4.49(d,J＝5.7Hz,2H),3.57(s,3H).LR-MS(ESI)m/z 417(M+1)
实施例12
Figure PCTCN2020096521-appb-000048
除了用苯硼酸代替(2-苯氧基苯基)硼酸以外，合成方法如实施例10。
1H NMR(500MHz,DMSO)δ12.43(s,1H),9.63(s,1H),9.05(t,J＝5.8Hz,1H),7.97(d,J＝8.0Hz,1H),7.62(d,J＝7.3Hz,2H),7.50(t,J＝7.7Hz,2H),7.47(d,J＝8.2Hz,2H),7.45(s,1H),7.38(t,J＝7.4Hz,1H),7.20-7.4(m,2H),6.97(t,J＝7.0Hz,1H),6.79(d,J＝8.0Hz,1H),6.60(t,J＝7.2Hz,1H),4.88(s,2H),4.57(d,J＝5.7Hz,2H),3.61(s,3H).LR-MS(ESI)m/z 492(M+1)
实施例13
Figure PCTCN2020096521-appb-000049
除了用(2-异丙氧基苯基)硼酸代替(2-苯氧基苯基)硼酸以外，合成方法如实施例9。
1H NMR(600MHz,DMSO)δ12.21(s,1H),11.18(s,1H),8.99(s,1H),8.93(t,J＝5.4Hz,1H),7.71(d,J＝7.9Hz,2H),7.37(d,J＝7.9Hz,2H),7.33(d,J＝7.1Hz,2H),7.24(s,1H),7.11(d,J＝8.5Hz,1H),7.00(t,J＝7.3Hz,1H),6.82(s,1H),4.55(dt,J＝11.9,5.8Hz,1H),4.48(d,J＝5.4Hz,2H),3.56(s,3H),1.16(s,3H),1.15(s,3H).LR-MS(ESI)m/z 475(M+1)
实施例14
Figure PCTCN2020096521-appb-000050
除了用(2-异丙氧基苯基)硼酸代替(2-苯氧基苯基)硼酸以外，合成方法如实施例10。
1H NMR(600MHz,DMSO)δ12.21(s,1H),9.61(s,1H),8.95(t,J＝5.9Hz,1H),7.93(d,J＝8.0Hz,1H),7.42(d,J＝8.2Hz,2H),7.34-7.30(m,2H),7.23(s,1H),7.14(d,J＝7.6Hz,1H),7.10(d,J＝8.4Hz,1H),6.99(td,J＝7.4,0.9Hz,1H),6.97–6.93(m,1H),6.82(d,J＝1.6Hz,1H),6.76(dd,J＝8.0,1.2Hz,1H),6.57(dd,J＝11.0,4.1Hz,1H),4.54(dt,J＝12.2,6.1Hz,1H),4.86(s,2H),4.51(d,J＝5.8Hz,2H),3.55(s,3H),1.15(s,3H),1.14(s,3H).LR-MS(ESI)m/z 550(M+1).
实施例15
Figure PCTCN2020096521-appb-000051
除了用2-氟苯硼酸替代(2-苯氧基苯基)硼酸以外，合成方法如实施例9。
1H NMR(400MHz,DMSO)δ12.48(s,1H),11.24(s,1H),10.24(s,1H),9.04(s,1H),7.76(d,J＝7.9Hz,2H),7.58(t,J＝7.2Hz,1H),7.53-7.32(m,6H),6.91(s,1H),4.53(d,J＝5.0Hz,2H),3.62(s,3H).LR-MS(ESI)m/z 435(M+1).
实施例16
Figure PCTCN2020096521-appb-000052
除了用2-氟苯硼酸替代(2-苯氧基苯基)硼酸以外，合成方法如实施例10。
1H NMR(400MHz,DMSO)δ12.47(s,1H),9.65(s,1H),9.04(s,1H),7.96(d,J＝8.1Hz,2H),7.55(t,J＝6.8Hz,1H),7.50–7.41(m,4H),7.40–7.30(m,2H),7.16(d,J＝7.3Hz,1H),6.97(t,J＝7.6Hz,1H),6.89(s,1H),6.78(d,J＝8.0Hz,1H),6.60(t,J＝7.5Hz,1H),4.90(s,2H),4.54(d,J＝5.7Hz,2H),3.59(s,3H).LR-MS(ESI)m/z 510(M+1).
实施例17
Figure PCTCN2020096521-appb-000053
除了用2-氯苯硼酸替代(2-苯氧基苯基)硼酸以外，合成方法如实施例9。
1H NMR(400MHz,DMSO)δ12.48(s,1H),11.24(s,1H),10.24(s,1H),9.04(t,J＝5.6Hz,1H),8.22(s,1H),8.02–7.80(m,2H),7.57–7.49(m,1H),7.38–7.31(m,2H),7.30–7.20(m,2H),7.13–6.99(m,1H),6.94(s,1H),4.52(d,J＝5.6Hz,2H),3.67(s,3H)LR-MS(ESI)m/z 451(M+1).
实施例18
Figure PCTCN2020096521-appb-000054
除了用2-氯苯硼酸替代(2-苯氧基苯基)硼酸以外，合成方法如实施例10。
1H NMR(400MHz,DMSO)δ12.48(s,1H),9.64(s,1H),9.04(t,J＝5.6Hz,1H),7.97–7.85(m,2H),7.64(dd,J＝14.9,2.8Hz,1H),7.54–7.48(m,2H),7.43–7.34(m,2H),7.34–7.29(m,2H),7.28–7.17(m,2H),7.09–7.00(m,1H),6.98(dd,J＝15.1,(m,1H),6.94(s,1H),4.90(s,2H),4.54(d,J＝5.6Hz,2H),3.60(s,3H).LR-MS(ESI)m/z 526(M+1).
实施例19
Figure PCTCN2020096521-appb-000055
除了用2-氰基苯硼酸替代(2-苯氧基苯基)硼酸以外，合成方法如实施例9。
1H NMR(400MHz,DMSO)δ12.48(s,1H),11.23(s,1H),10.22(s,1H),9.04(t,J＝5.6Hz,1H),8.21(s,1H),7.99–7.87(m,2H),7.71–7.60(m,1H),7.60–7.45(m,3H),7.40–7.28(m,2H),6.90(s,1H),4.55(d,J＝5.6Hz,2H),3.60(s,3H)LR-MS(ESI)m/z 442(M+1).
实施例20
Figure PCTCN2020096521-appb-000056
除了用2-氰基苯硼酸替代(2-苯氧基苯基)硼酸以外，合成方法如实施例10。
1H NMR(400MHz,DMSO)δ12.48(s,1H),9.64(s,1H),9.05(t,J＝5.6Hz,1H),8.21(s,1H),8.01–7.86(m,2H),7.71–7.60(m,2H),7.59–7.52(m,2H),7.51–7.46(m,1H),7.45–7.36(m,1H),7.36–7.30(m,2H),7.00(dd,J＝14.9,3.1Hz,1H),6.93(s,1H),6.85(td,J＝14.9,3.1Hz,1H),4.91(s,2H),4.55(d,J＝5.6Hz,2H),3.60(s,3H).LR-MS(ESI)m/z 517 (M+1).
实施例21
Figure PCTCN2020096521-appb-000057
除了用2-三氟甲基苯硼酸替代(2-苯氧基苯基)硼酸以外，合成方法如实施例9。
1H NMR(400MHz,DMSO)δ12.21(s,1H),11.17(s,1H),9.00(s,1H),8.94(t,J＝5.4Hz,1H),8.19(s,1H),8.06–7.84(m,2H),7.46–7.39(m,1H),7.39–7.30(m,3H),7.27(dd,J＝14.9,3.6Hz,1H),7.10(td,J＝14.7,3.5Hz,1H),6.91(s,1H),4.54(d,J＝5.6Hz,2H),3.57(s,3H).LR-MS(ESI)m/z 485(M+1).
实施例22
Figure PCTCN2020096521-appb-000058
除了用2-三氟甲基苯硼酸替代(2-苯氧基苯基)硼酸以外，合成方法如实施例10。
1H NMR(400MHz,DMSO)δ12.48(s,1H),9.65(s,1H),9.06(t,J＝5.6Hz,1H),8.20(s,1H),8.08–7.84(m,2H),7.66(dd,J＝15.0,2.9Hz,1H),7.46–7.39(m,2H),7.39–7.31(m,3H),7.27(dd,J＝14.9,3.6Hz,1H),7.10(td,J＝14.7,3.5Hz,1H),7.00(dd,J＝15.1,3.0Hz,1H),6.93(s,1H),6.85(td,J＝14.9,3.1Hz,1H),4.91(s,2H),4.55(d,J＝5.6Hz,2H),3.61(s,3H).LR-MS(ESI)m/z 560(M+1).
实施例23
Figure PCTCN2020096521-appb-000059
除了用2-甲基苯硼酸替代(2-苯氧基苯基)硼酸以外，合成方法如实施例9。
1H NMR(400MHz,DMSO)δ12.21(s,1H),11.17(s,1H),9.00(s,1H),8.95(t,J＝5.4 Hz,1H),8.19(s,1H),8.06–7.82(m,2H),7.38–7.31(m,2H),7.31–7.21(m,2H),7.05–6.90(m,3H),4.65(s,1H),4.49(d,J＝5.6Hz,2H),3.56(s,3H),2.45(s,3H).LR-MS(ESI)m/z 431(M+1).
实施例24
Figure PCTCN2020096521-appb-000060
除了用2-甲基苯硼酸替代(2-苯氧基苯基)硼酸以外，合成方法如实施例10。
1H NMR(400MHz,DMSO)δ12.47(s,1H),9.65(s,1H),9.07(t,J＝5.6Hz,1H),8.13(s,1H),7.95–7.80(m,2H),7.64(dd,J＝14.9,2.9Hz,1H),7.44–7.34(m,1H),7.34–7.29(m,2H),7.28–7.19(m,2H),7.05–6.89(m,4H),6.83(td,J＝14.9,3.0Hz,1H),4.93(s,2H),4.50(d,J＝5.6Hz,2H),3.61(s,3H),2.44(s,3H).LR-MS(ESI)m/z 506(M+1).
实施例25
Figure PCTCN2020096521-appb-000061
除了用2-异丙基苯硼酸替代(2-苯氧基苯基)硼酸以外，合成方法如实施例9。
1H NMR(400MHz,DMSO)δ12.21(s,1H),11.17(s,1H),8.99(s,1H),8.95(t,J＝5.4Hz,1H),8.19(s,1H),8.07–7.84(m,2H),7.39–7.31(m,3H),7.31–7.27(m,1H),7.27–7.23(m,1H),7.23–7.11(m,1H),6.95(s,1H),4.50(d,J＝5.6Hz,2H),3.57(s,3H),3.06–2.69(m,1H),1.17(d,J＝12.8Hz,6H).LR-MS(ESI)m/z 459(M+1).
实施例26
Figure PCTCN2020096521-appb-000062
除了用2-异丙基苯硼酸替代(2-苯氧基苯基)硼酸以外，合成方法如实施例10。
1H NMR(400MHz,DMSO)δ12.48(s,1H),9.65(s,1H),9.06(t,J＝5.6Hz,1H),8.15(s,1H),7.98–7.84(m,2H),7.65(dd,J＝14.9,3.1Hz,1H),7.46–7.36(m,1H),7.36–7.30(m,3H),7.30–7.21(m,2H),7.21–7.11(m,1H),6.99(dd,J＝15.1,3.0Hz,1H),6.94(s,1H),6.84(td,J＝14.9,3.1Hz,1H),4.93(s,2H),4.50(d,J＝5.6Hz,2H),3.61(s,3H),3.14–2.66(m,1H),1.17(d,J＝12.7Hz,6H).LR-MS(ESI)m/z 534(M+1).
实施例27
Figure PCTCN2020096521-appb-000063
除了用2-甲氧基苯硼酸替代(2-苯氧基苯基)硼酸以外，合成方法如实施例9。
1H NMR(400MHz,DMSO)δ12.21(s,1H),11.17(s,1H),9.00(s,1H),8.96(t,J＝5.4Hz,1H),8.20(s,1H),7.97–7.86(m,2H),7.38–7.30(m,3H),7.29–7.18(m,1H),7.10–6.99(m,2H),6.99(s,1H),4.49(d,J＝5.6Hz,2H),3.57(s,3H),3.67(s,3H).LR-MS(ESI)m/z 447(M+1).
实施例28
Figure PCTCN2020096521-appb-000064
除了用2-甲氧基苯硼酸替代(2-苯氧基苯基)硼酸以外，合成方法如实施例10。
1H NMR(400MHz,DMSO)δ12.48(s,1H),9.65(s,1H),9.07(t,J＝5.6Hz,1H),8.23(s,1H),8.04–7.84(m,2H),7.66(dd,J＝14.9,3.1Hz,1H),7.47–7.36(m,1H),7.36–7.30(m,3H),7.29–7.17(m,1H),7.10–6.96(m,4H),6.85(td,J＝14.9,3.1Hz,1H),4.93(s,2H),4.51(d,J＝5.6Hz,2H),3.90(s,3H),3.61(s,3H).LR-MS(ESI)m/z 522(M+1).
实施例29
Figure PCTCN2020096521-appb-000065
除了用(2-环丙氧基苯基)硼酸替代(2-苯氧基苯基)硼酸以外，合成方法如实施例9。
1H NMR(400MHz,DMSO)δ12.22(s,1H),11.18(s,1H),9.00(s,1H),8.95(t,J＝5.4Hz,1H),8.19(s,1H),7.92(d,J＝7.4Hz,2H),7.34(d,J＝7.4Hz,3H),7.23(td,J＝7.5,1.5Hz,1H),7.04(ddd,J＝15.7,7.6,1.5Hz,2H),6.90(s,1H),4.49(d,J＝5.6Hz,2H),3.57(s,3H),3.12(p,J＝8.3Hz,1H),0.66–0.34(m,2H),0.25–0.02(m,2H).LR-MS(ESI)m/z 473(M+1).
实施例30
Figure PCTCN2020096521-appb-000066
除了用(2-环丙氧基苯基)硼酸替代(2-苯氧基苯基)硼酸以外，合成方法如实施例10。
1H NMR(400MHz,DMSO)δ12.49(s,1H),9.65(s,1H),9.07(t,J＝5.6Hz,1H),8.36(s,1H),7.99–7.87(m,2H),7.66(dd,J＝14.9,3.1Hz,1H),7.46–7.39(m,2H),7.38–7.30(m,3H),7.28–7.18(m,1H),7.10–6.96(m,3H),6.85(td,J＝14.9,3.1Hz,1H),4.92(s,2H),4.51(d,J＝5.6Hz,2H),3.60(s,3H),3.33(p,J＝16.4Hz,1H),0.57–0.31(m,2H),0.25–0.03(m,2H).LR-MS(ESI)m/z 548(M+1).
实施例31
Figure PCTCN2020096521-appb-000067
除了用2-(4-氟-2,6-二甲基苯氧基)苯基)硼酸代替(2-苯氧基苯基)硼酸以外，合成方法如实施例9。
1H NMR(400MHz,DMSO)δ12.29(s,1H),11.19(s,1H),9.00(s,1H),8.95(t,J＝5.6Hz,1H),8.28(s,1H),7.98–7.82(m,2H),7.46(td,J＝14.8,3.1Hz,1H),7.38–7.23(m,4H),7.14–7.05(m,1H),7.04(s,1H),6.86(s,1H),6.82(s,1H),4.50(d,J＝5.6Hz,2H),3.57(s,3H),2.15(s,6H).LR-MS(ESI)m/z 555(M+1).
实施例32
Figure PCTCN2020096521-appb-000068
除了用2-(4-氟-2,6-二甲基苯氧基)苯基)硼酸代替(2-苯氧基苯基)硼酸以外，合成方法如实施例10。
1H NMR(400MHz,DMSO)δ12.49(s,1H),9.65(s,1H),9.07(t,J＝5.6Hz,1H),8.27(s,1H),7.97–7.88(m,2H),7.66(dd,J＝15.0,2.9Hz,1H),7.52–7.35(m,3H),7.34–7.28(m,2H),7.27(dd,J＝12.7,2.3Hz,1H),7.09(td,J＝14.9,3.3Hz,1H),7.04–6.96(m,2H),6.92–6.78(m,3H),4.94(s,2H),4.51(d,J＝5.6Hz,2H),3.63(s,3H),2.15(s,6H).LR-MS(ESI)m/z 630(M+1).
实施例33
Figure PCTCN2020096521-appb-000069
除了用(2-(2,4-二氟苯氧基)苯基)硼酸代替(2-苯氧基苯基)硼酸以外，合成方法如实施例9。
1H NMR(400MHz,DMSO)δ12.29(s,1H),11.19(s,1H),9.00(s,1H),8.95(t,J＝5.6Hz,1H)8.06(s,1H),7.98–7.85(m,2H),7.46(td,J＝14.8,3.2Hz,1H),7.38–7.32(m,2H),7.32–7.23(m,2H),7.15(s,1H),7.09(td,J＝14.9,3.3Hz,1H),7.02–6.85(m,2H),6.74(td,J＝16.0,2.5Hz,1H),,4.50(d,J＝5.6Hz,2H),3.58(s,3H).LR-MS(ESI)m/z 545(M+1).
实施例34
Figure PCTCN2020096521-appb-000070
除了用(2-(2,4-二氟苯氧基)苯基)硼酸代替(2-苯氧基苯基)硼酸以外，合成方法如实施例10。
1H NMR(400MHz,DMSO)δ12.49(s,1H),9.65(s,1H),9.07(t,J＝5.6Hz,1H),8.36(s,1H),7.97–7.84(m,2H),7.64(dd,J＝14.9,3.1Hz,1H),7.49–7.42(m,1H),7.42–7.33(m,2H),7.33–7.27(m,2H),7.25(dd,J＝12.6,2.3Hz,1H),7.17(s,1H),7.07(td,J＝14.8,3.2Hz,1H),6.98(dd,J＝15.1,3.0Hz,1H),6.96–6.86(m,2H),6.87–6.80(m,1H),6.79–6.70(m,1H),5.00(s,2H),4.51(d,J＝5.6Hz,2H),3.63(s,3H).LR-MS(ESI)m/z 620(M+1).
实施例35
Figure PCTCN2020096521-appb-000071
除了用(3-(乙基磺酰胺基)苯基)硼酸代替(2-苯氧基苯基)硼酸以外，合成方法如实施例9。
1H NMR(400MHz,DMSO)δ12.52(s,1H),9.95(s,1H),9.66(s,1H),9.05(s,1H),7.97(d,J＝8.0Hz,2H),7.51–7.37(m,5H),7.32(d,J＝7.3Hz,1H),7.23(d,J＝7.2Hz,1H),7.20–7.11(m,2H),6.97(t,J＝7.7Hz,1H),6.78(d,J＝8.0Hz,1H),6.60(t,J＝7.5Hz,1H),4.91(s,2H),4.56(d,J＝5.4Hz,2H),3.60(s,3H),3.17–3.11(m,2H),1.22(t,J＝7.3Hz,3H).LR-MS(ESI)m/z 524(M+1).
实施例36
Figure PCTCN2020096521-appb-000072
除了用(3-(乙基磺酰胺基)苯基)硼酸代替(2-苯氧基苯基)硼酸以外，合成方法如实施例10。
1H NMR(400MHz,DMSO)δ12.49(s,1H),11.21(s,1H),9.94(s,1H),9.02(s,1H),7.73(d,J＝7.7Hz,2H),7.50–7.36(m,5H),7.31(d,J＝6.7Hz,1H),7.23(d,J＝7.5Hz,1H),7.14(s,1H),4.52(s,2H),3.60(s,3H),3.19–3.07(m,2H),1.22(t,J＝7.0Hz,3H).LR-MS (ESI)m/z 599(M+1).
实施例37
Figure PCTCN2020096521-appb-000073
除了用3-甲磺酰基苯硼酸代替(2-苯氧基苯基)硼酸以外，合成方法如实施例9。
1H NMR(400MHz,DMSO)δ12.57(s,1H),11.22(s,1H),10.26(s,1H),9.08(t,J＝5.7Hz,1H),8.09(s,1H),7.98(d,J＝7.7Hz,1H),7.91(d,J＝8.1Hz,1H),7.77(t,J＝7.8Hz,1H),7.72(d,J＝8.1Hz,2H),7.63(s,1H),7.40(d,J＝8.1Hz,2H),7.14(d,J＝1.7Hz,1H),4.52(d,J＝5.5Hz,2H),3.61(s,3H),3.30(s,3H).LR-MS(ESI)m/z 495(M+1).
实施例38
Figure PCTCN2020096521-appb-000074
除了用3-甲磺酰基苯硼酸代替(2-苯氧基苯基)硼酸以外，合成方法如实施例10。
1H NMR(400MHz,DMSO)δ12.59(s,1H),9.65(s,1H),9.07(s,1H),8.09(s,1H),8.01–7.88(m,4H),7.78(t,J＝7.8Hz,1H),7.63(s,1H),7.46(d,J＝8.2Hz,2H),7.15(d,J＝6.9Hz,2H),6.97(s,1H),6.77(d,J＝6.8Hz,1H),6.59(s,1H),4.90(s,2H),4.56(d,J＝5.2Hz,2H),3.62(s,3H),3.30(s,3H).LR-MS(ESI)m/z 570(M+1).
实施例39
Figure PCTCN2020096521-appb-000075
实施例39a 甲基-6-甲基-7-氧代-4-(4,4,5,5-四甲基-1,3,2-二氧杂硼杂环戊烷-2-基)-1-甲苯磺酰基-6,7-二氢-1H-吡咯并[2]，3-c]吡啶-2-羧酸乙酯(化合物39a)
Figure PCTCN2020096521-appb-000076
将化合物1e(0.500g,1.14mmol)，连硼酸频哪醇酯(0.347g,1.37mmol)和醋酸钾(0.223g,2.28mmol)溶于二氧六环溶液(2mL)中，Ar气保护条件下加入Pd 2(dba) 3(0.132g,0.114mmol)和X-Phos(0.137g,0.228mmol)，在90℃油浴条件下反应3小时后停止加热，冷却至室温后滤除不溶物后用EA萃取后合并有机相，用饱和食盐水洗涤有机相后，用无水硫酸钠干燥。减压蒸干溶剂后，柱层析得到目标产物化合物39a(0.450g，92％)。
实施例39b 2-(3-溴苯基)丙-2-醇
Figure PCTCN2020096521-appb-000077
将3-溴苯甲酸甲酯(2g,9.30mmol)溶于无水四氢呋喃(30mL)，在冰浴条件下滴加甲基溴化镁(1M in THF,11.6mL，11.6mmol)，然后将反应室温条件下反应两个小时后用饱和氯化铵溶液灭反应。用乙酸乙酯萃取后合并有机相，用饱和食盐水洗涤有机相后，用无水硫酸钠干燥。减压蒸干溶剂后柱层析得到目标产物化合物39b(0.2g，10％)。
实施例39c 4-(3-(2-羟基丙烷-2-基)苯基)-6-甲基-7-氧代-1-甲苯磺酰基-6,7-二氢-1H-吡咯并[2,3-c]吡啶-2-甲基羧酸
Figure PCTCN2020096521-appb-000078
在氩气保护条件下将化合物39a(0.3g,0.617mmol)、化合物39b(0.110g,0.514 mmol)、氟化铯(0.156g,1.03mmol)、三(二亚苄基丙酮)二钯(0)(0.047g,0.0514mmol)、1,3,5,7-四甲基-6-苯基-2,4,8-三氧杂-6-磷酰金刚烷(0.029g,0.103mmol)溶于乙二醇二甲醚(2mL)和水(0.16mL)混合溶液中，60℃油浴条件下搅拌2小时至反应完全。用硅藻土滤除不溶物后乙酸乙酯萃取。然后用氯化钠溶液洗涤，合并有机相经无水硫酸钠干燥，过滤并浓缩。通过快速层析法(硅胶2-50％乙酸乙酯/石油醚梯度)分离出产物4-(3-(2-羟基丙烷-2-基)苯基)-6-甲基-7-氧代-1-甲苯磺酰基-6,7-二氢-1H-吡咯并[2,3-c]吡啶-2-甲基羧酸0.2g，(产率：79％)。
实施例39d 4-(3-(2-羟基丙烷-2-基)苯基)-6-甲基-7-氧代-6,7-二氢-1H-吡咯并[2,3-c]吡啶-2-羧酸
Figure PCTCN2020096521-appb-000079
将化合物39c(0.2g,0.404mmol)溶于二氧六环(2mL)后加入4M的氢氧化钠溶液(0.4mL,1.6mmol)，在氩气保护条件下90℃搅拌2小时至反应完全。用1N盐酸溶液调节体系pH至1-3，将析出的白色固体抽滤、干燥，得到产物4-(3-(2-羟基丙烷-2-基)苯基)-6-甲基-7-氧代-6,7-二氢-1H-吡咯并[2,3-c]吡啶-2-羧酸0.11g(产率：83％)。
实施例39e 4-((4-(3-(2-羟基丙烷-2-基)苯基)-6-甲基-7-氧代-6,7-二氢-1H-吡咯并[2,3-c]吡啶-2-甲基甲酰氨基)甲基)苯甲酸甲酯
Figure PCTCN2020096521-appb-000080
将化合物39d(0.10g,0.306mmol)溶于N,N-二甲基甲酰胺(1.5mL)后加入N,N-二异丙基乙胺(0.080mL,0.612mmol)、N-羟基-7-偶氮苯并三氮唑(0.062g,0.458mmol)和1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(0.088g,0.458mmol)，室温反应20分钟 后加入4-(氨基甲基)苯甲酸甲酯(0.076g,0.459mmol)。在室温下搅拌4小时至反应完全。加入水淬灭反应，乙酸乙酯萃取后用水和饱和氯化钠溶液洗涤，合并有机相并经无水硫酸钠干燥，过滤并浓缩。通过快速层析法(硅胶0-5％二氯甲烷/石甲醇梯度)分离出产物4-((4-(3-(2-羟基丙烷-2-基)苯基)-6-甲基-7-氧代-6,7-二氢-1H-吡咯并[2,3-c]吡啶-2-甲基甲酰氨基)甲基)苯甲酸甲酯0.1g(产率：69％)
将化合物39e(0.1g,0.213mmol)溶于二氯甲烷(0.2mL)和甲醇(1mL)，冰浴条件下加入50％羟胺水溶液(0.286mL,4.35mmol)后加入氢氧化钠固体(0.174g,4.35mmol)，继续搅拌2小时至反应完全。用3N盐酸溶液调节体系pH至3，将析出的固体抽滤后用水洗，干燥，得到实施例39化合物，0.07g(产率：70％)。
1H NMR(400MHz,DMSO)δ12.46(s,1H),11.23(s,1H),10.29(s,1H),9.09(s,1H),7.77-7.65(m,4H),7.45-7.37(m,5H),7.14(s,1H),4.52(d,J＝5.3Hz,2H),1.49(s,6H).LC-MS(ESI)m/z＝475(M+1)
实施例40
Figure PCTCN2020096521-appb-000081
实施例41a 4-((4-(3-(2-羟基丙烷-2-基)苯基)-6-甲基-7-氧代-6,7-二氢-1H-吡咯并[2,3-c]吡啶-2-甲酰氨基)甲基)苯甲酸
Figure PCTCN2020096521-appb-000082
将化合物39e(0.1g,0.211mmol)溶于二氧六环(2mL)后加入2M的氢氧化钠溶液0.436mL,0.872mmol)，在氩气保护条件下90℃搅拌2小时至反应完全。用1N盐酸溶液调节体系pH至3，将析出的白色固体抽滤干燥得到产物4-((4-(3-(2-羟基丙烷-2-基)苯基)-6-甲基-7-氧代-6,7-二氢-1H-吡咯并[2,3-c]吡啶-2-甲酰氨基)甲基)苯甲酸0.08g(产率：82％)。
将化合物40a(0.08g,0.174mmol)溶于N,N-二甲基甲酰胺(1mL)后加入N,N-二异丙基乙胺(0.089mL,0.540mmol)、N-羟基-7-偶氮苯并三氮唑(0.034g,0.252mmol)和1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(0.048g,mmol)，室温反应20分钟后加入邻苯二胺(0.023g,0.208mmol)。在室温下搅拌4小时至反应完全。加入水淬灭反应，乙酸乙酯萃取后用水和饱和氯化钠溶液洗涤，合并有机相并经无水硫酸钠干燥，过滤并浓缩。通过快速层析法(硅胶0-7％二氯甲烷/甲醇梯度)分离出产物实施例40化合物0.06g(产率：63％)。
1H NMR(400MHz,DMSO)δ12.48(s,1H),9.67(s,1H),9.06(s,1H),7.97(d,J＝7.7Hz,2H),7.69(s,1H),7.45(dd,J＝18.1,7.5Hz,6H),7.18(d,J＝7.5Hz,1H),7.13(s,1H),6.98(t,J＝7.6Hz,1H),6.79(d,J＝8.0Hz,1H),6.61(t,J＝7.5Hz,1H),4.93(s,2H),4.57(d,J＝5.4Hz,2H),4.13(s,1H),3.62(s,3H),1.50(s,6H).LR-MS(ESI)m/z 550(M+1).
实施例41
Figure PCTCN2020096521-appb-000083
除了用3-(1-羟基乙基)苯硼酸替代(2-苯氧基苯基)硼酸以外，合成方法如实施例9。
1H NMR(400MHz,DMSO)δ12.29(s,1H),11.19(s,1H),9.00(s,1H),8.95(t,J＝5.6Hz,1H),8.23(s,1H),8.04–7.75(m,2H),7.39–7.31(m,2H),7.31–7.25(m,2H),7.25–7.15(m,2H),6.99(s,1H),5.17(s,1H),4.71(q,J＝11.3Hz,1H),4.51(d,J＝5.6Hz,2H),3.57(s,3H),1.57(d,J＝11.3Hz,3H).LR-MS(ESI)m/z 461(M+1).
实施例42
Figure PCTCN2020096521-appb-000084
除了用3-(1-羟基乙基)苯硼酸替代(2-苯氧基苯基)硼酸以外，合成方法如实施例 10。
1H NMR(400MHz,DMSO)δ12.49(s,1H),9.65(s,1H),9.07(t,J＝5.6Hz,1H),8.23(s,1H),7.99–7.83(m,2H),7.66(dd,J＝15.0,2.9Hz,1H),7.45–7.36(m,1H),7.36–7.27(m,4H),7.26–7.16(m,2H),7.04(s,1H),7.00(dd,J＝15.1,3.0Hz,1H),6.85(td,J＝14.9,3.1Hz,1H),5.15(d,J＝20.0Hz,3H),4.71(q,J＝11.3Hz,1H),4.61(d,J＝5.6Hz,2H),3.57(s,3H),1.57(d,J＝11.4Hz,3H).LR-MS(ESI)m/z 536(M+1).
实施例43
Figure PCTCN2020096521-appb-000085
除了用3-甲氧基羰基苯硼酸替代(2-苯氧基苯基)硼酸以外，合成方法如实施例9。
1H NMR(400MHz,DMSO)δ12.29(s,1H),11.19(s,1H),9.00(s,1H),8.95(t,J＝5.6Hz,1H),8.27(s,1H),7.96–7.84(m,3H),7.55(dt,J＝15.2,3.2Hz,1H),7.40(t,J＝13.5Hz,1H),7.36–7.30(m,3H),7.01(s,1H),4.50(d,J＝5.6Hz,2H),3.90(s,3H),3.57(s,3H).LR-MS(ESI)m/z 475(M+1).
实施例44
Figure PCTCN2020096521-appb-000086
除了用3-甲氧基羰基苯硼酸替代(2-苯氧基苯基)硼酸以外，合成方法如实施例10。
1H NMR(400MHz,DMSO)δ12.49(s,1H),9.67(s,1H),9.07(t,J＝5.6Hz,1H),8.28(s,1H),7.97–7.83(m,3H),7.66(dd,J＝15.0,2.9Hz,1H),7.55(dt,J＝15.2,3.2Hz,1H),7.46–7.36(m,2H),7.36–7.30(m,3H),7.04(s,1H),7.00(dd,J＝15.1,3.0Hz,1H),6.85(td,J＝14.9,3.1Hz,1H),5.00(s,2H),4.51(d,J＝5.6Hz,2H),3.90(s,3H),3.57(s,3H).LR-MS(ESI)m/z 550(M+1).
实施例45
Figure PCTCN2020096521-appb-000087
除了用3-羟基苯硼酸替代(2-苯氧基苯基)硼酸以外，合成方法如实施例9。
1H NMR(400MHz,DMSO)δ12.39(s,1H),11.19(s,1H),8.99(s,1H),8.95(t,J＝5.6Hz,1H),8.40(s,1H),8.26(s,1H),8.06–7.84(m,2H),7.38–7.25(m,3H),7.04(s,1H),6.90(dt,J＝14.9,2.9Hz,1H),6.77–6.66(m,2H),4.51(d,J＝5.6Hz,2H),3.58(s,3H).LR-MS(ESI)m/z 433(M+1).
实施例46
Figure PCTCN2020096521-appb-000088
除了用3-羟基苯硼酸替代(2-苯氧基苯基)硼酸以外，合成方法如实施例10。
1H NMR(400MHz,DMSO)δ12.50(s,1H),9.65(s,1H),9.40(s,1H),9.07(t,J＝5.6Hz,1H),8.25(s,1H),7.98–7.83(m,2H),7.66(dd,J＝15.0,2.9Hz,1H),7.47–7.36(m,1H),7.36–7.24(m,3H),7.04(s,1H),7.00(dd,J＝15.1,3.0Hz,1H),6.94–6.79(m,2H),6.71(m,2H),5.00(s,2H),4.50(d,J＝5.6Hz,2H),3.57(s,3H).LC-MS(ESI)m/z＝508(M+1)
实施例47
Figure PCTCN2020096521-appb-000089
除了用3-甲氧基苯硼酸替代(2-苯氧基苯基)硼酸以外，合成方法如实施例9。
1H NMR(400MHz,DMSO)δ12.45(s,1H),11.20(s,1H),10.02(d,J＝73.6Hz,1H),9.04(t,J＝5.8Hz,2H),7.72(d,J＝8.1Hz,2H),7.47(s,1H),7.39(d,J＝6.7Hz,3H),7.16(t,J＝12.7Hz,3H),6.94(d,J＝6.4Hz,1H),4.51(d,J＝5.5Hz,2H),3.82(s,3H),3.58(s,3H).LR-MS(ESI)m/z 447(M+1).
实施例48
Figure PCTCN2020096521-appb-000090
除了用3-甲氧基苯硼酸替代(2-苯氧基苯基)硼酸以外，合成方法如实施例10。
1H NMR(400MHz,DMSO)δ12.46(s,1H),9.65(s,1H),9.07(d,J＝6.0Hz,1H),7.95(d,J＝8.3Hz,2H),7.50–7.43(m,3H),7.40(t,J＝8.0Hz,1H),7.20–7.10(m,4H),7.00–6.91(m,2H),6.77(d,J＝6.8Hz,1H),6.59(t,J＝7.5Hz,1H),4.91(s,2H),4.55(d,J＝5.9Hz,2H),3.83(s,3H),3.59(s,3H).LR-MS(ESI)m/z 522(M+1).
实施例49
Figure PCTCN2020096521-appb-000091
除了用3-甲基苯硼酸替代(2-苯氧基苯基)硼酸以外，合成方法如实施例9。
1H NMR(400MHz,DMSO)δ12.44(s,1H),11.21(s,1H),10.05(d,J＝73.6Hz,1H),9.03(s,1H),7.73(d,J＝8.2Hz,2H),7.45–7.34(m,6H),7.19(d,J＝6.9Hz,1H),7.14(d,J＝2.0Hz,1H),4.52(d,J＝5.3Hz,2H),3.59(s,3H),2.39(s,3H).LR-MS(ESI)m/z 431(M+1).
实施例50
Figure PCTCN2020096521-appb-000092
除了用3-甲基苯硼酸替代(2-苯氧基苯基)硼酸以外，合成方法如实施例10。
1H NMR(400MHz,DMSO)δ12.45(s,1H),9.64(s,1H),9.06(t,J＝5.8Hz,1H),7.96(d,J＝7.8Hz,2H),7.46(d,J＝8.0Hz,2H),7.43–7.32(m,4H),7.18(d,J＝7.1Hz,2H),7.14(s,1H),6.97(t,J＝7.6Hz,1H),6.77(d,J＝7.8Hz,1H),6.59(t,J＝7.3Hz,1H),4.89(s, 2H),4.56(d,J＝5.7Hz,2H),3.59(s,3H),2.39(s,3H).LR-MS(ESI)m/z 506(M+1).
实施例51
Figure PCTCN2020096521-appb-000093
除了用3-三氟甲基苯硼酸替代(2-苯氧基苯基)硼酸以外，合成方法如实施例9。
1H NMR(400MHz,DMSO)δ12.56(s,1H),11.22(s,1H),10.24(s,1H),9.07(t,J＝5.8Hz,1H),7.95(s,1H),7.89(s,1H),7.76–7.70(m,4H),7.62(s,1H),7.40(d,J＝8.2Hz,2H),7.13(d,J＝2.0Hz,1H),4.52(d,J＝5.5Hz,2H),3.61(s,3H).LR-MS(ESI)m/z 485(M+1).
实施例52
Figure PCTCN2020096521-appb-000094
除了用3-三氟甲基苯硼酸替代(2-苯氧基苯基)硼酸以外，合成方法如实施例10。
1H NMR(400MHz,DMSO)δ12.56(s,1H),9.64(s,1H),9.07(s,1H),7.95(d,J＝8.0Hz,3H),7.89(s,1H),7.73(s,2H),7.62(s,1H),7.46(d,J＝8.0Hz,2H),7.15(d,J＝9.9Hz,2H),6.96(t,J＝7.6Hz,1H),6.77(d,J＝8.0Hz,1H),6.59(t,J＝7.8Hz,1H),4.89(s,2H),4.56(d,J＝5.5Hz,2H),3.60(s,3H).LR-MS(ESI)m/z 560(M+1).
实施例53
Figure PCTCN2020096521-appb-000095
除了用3-氟苯硼酸替代(2-苯氧基苯基)硼酸以外，合成方法如实施例9。
1H NMR(400MHz,DMSO)δ12.45(s,1H),11.21(s,1H),10.05(d,J＝73.6Hz,1H),9.03(s,1H),8.23(s,1H),8.02–7.78(m,2H),7.39–7.30(m,2H),7.30–7.19(m,1H),7.11 (dt,J＝14.9,3.1Hz,1H),7.05–7.00(m,1H),7.00–6.91(m,2H),4.51(d,J＝5.6Hz,2H),3.66(s,3H).LR-MS(ESI)m/z 435(M+1).
实施例54
Figure PCTCN2020096521-appb-000096
除了用3-氟苯硼酸替代(2-苯氧基苯基)硼酸以外，合成方法如实施例10。
1H NMR(400MHz,DMSO)δ12.52(s,1H),9.65(s,1H),9.08(t,J＝6.0Hz,1H),7.96(d,J＝8.0Hz,2H),7.57–7.50(m,2H),7.45(dd,J＝17.9,9.7Hz,4H),7.24–7.12(m,3H),6.97(t,J＝7.6Hz,1H),6.78(d,J＝7.8Hz,1H),6.59(t,J＝7.4Hz,1H),4.90(s,2H),4.56(d,J＝5.5Hz,2H),3.59(s,3H).LR-MS(ESI)m/z 510(M+1).
实施例55
Figure PCTCN2020096521-appb-000097
除了用3-氯苯硼酸替代(2-苯氧基苯基)硼酸以外，合成方法如实施例9。
1H NMR(400MHz,DMSO)δ12.56(s,1H),11.21(s,1H),10.24(s,1H),9.07(t,J＝5.8Hz,1H),8.26(s,1H),7.98–7.88(m,2H),7.40–7.30(m,4H),7.30–7.26(m,1H),7.22(ddd,J＝12.7,5.3,3.3Hz,1H),7.02(s,1H),4.51(d,J＝5.6Hz,2H),3.57(s,3H).LR-MS(ESI)m/z 451(M+1).
实施例56
Figure PCTCN2020096521-appb-000098
除了用3-氯苯硼酸替代(2-苯氧基苯基)硼酸以外，合成方法如实施例10。
1H NMR(400MHz,DMSO)δ12.53(s,1H),9.65(s,1H),9.08(t,J＝6.0Hz,1H),7.96(d,J＝8.1Hz,2H),7.64(s,1H),7.60(d,J＝7.8Hz,1H),7.56(s,1H),7.52(t,J＝7.9Hz,1H),7.45(t,J＝9.1Hz,3H),7.16(d,J＝6.8Hz,2H),6.97(t,J＝7.5Hz,1H),6.78(d,J＝8.0Hz,1H),6.60(t,J＝7.7Hz,1H),4.94(s,2H),4.56(d,J＝5.7Hz,2H),3.59(s,3H).LR-MS(ESI)m/z 526(M+1).
实施例57
Figure PCTCN2020096521-appb-000099
除了用3-氰基苯硼酸替代(2-苯氧基苯基)硼酸以外，合成方法如实施例9。
1H NMR(400MHz,DMSO)δ12.21(s,1H),11.17(s,1H),9.00(s,1H),8.95(t,J＝5.4Hz,1H),8.23(s,1H),7.97–7.87(m,2H),7.85–7.82(m,1H),7.82–7.75(m,1H),7.66–7.58(m,2H),7.40–7.27(m,2H),6.98(s,1H),4.51(d,J＝5.6Hz,2H),3.57(s,3H)LR-MS(ESI)m/z 442(M+1).
实施例58
Figure PCTCN2020096521-appb-000100
除了用3-氰基苯硼酸替代(2-苯氧基苯基)硼酸以外，合成方法如实施例10。
1H NMR(400MHz,DMSO)δ12.51(s,1H),9.68(s,1H),9.03(t,J＝5.6Hz,1H),8.01(s,1H),7.96–7.85(m,2H),7.84–7.72(m,2H),7.68–7.53(m,3H),7.44–7.34(m,1H),7.34–7.26(m,2H),7.02–6.93(m,2H),6.83(td,J＝14.9,3.1Hz,1H),5.00(s,2H),4.50(d,J＝5.6Hz,2H),3.60(s,3H).LR-MS(ESI)m/z 517(M+1).
实施例59
Figure PCTCN2020096521-appb-000101
除了用4-甲基苯硼酸替代(2-苯氧基苯基)硼酸以外，合成方法如实施例9。
1H NMR(400MHz,DMSO)δ12.21(s,1H),11.17(s,1H),9.00(s,1H),8.95(t,J＝5.4Hz,1H),8.22(s,1H),7.96–7.88(m,2H),7.38–7.32(m,2H),7.32–7.25(m,2H),7.16–7.07(m,2H),7.01(s,1H),4.50(d,J＝5.7Hz,2H),3.57(s,3H),2.41(s,3H).LR-MS(ESI)m/z 431(M+1).
实施例60
Figure PCTCN2020096521-appb-000102
除了用4-甲基苯硼酸替代(2-苯氧基苯基)硼酸以外，合成方法如实施例10。
1H NMR(400MHz,DMSO)δ12.49(s,1H),9.66(s,1H),9.06(t,J＝5.7Hz,1H),8.23(s,1H),7.97–7.87(m,2H),7.66(dd,J＝15.0,3.0Hz,1H),7.46–7.36(m,1H),7.36–7.31(m,2H),7.31–7.25(m,2H),7.16–7.07(m,2H),7.03(s,1H),7.00(dd,J＝15.0,3.0Hz,1H),6.85(td,J＝14.9,3.1Hz,1H),4.93(s,2H),4.51(d,J＝5.6Hz,2H),3.61(s,3H),2.41(s,3H).LR-MS(ESI)m/z 506(M+1).
实施例61
Figure PCTCN2020096521-appb-000103
除了用4-三氟甲基苯硼酸替代(2-苯氧基苯基)硼酸以外，合成方法如实施例9。
1H NMR(400MHz,DMSO)δ12.20(s,1H),11.17(s,1H),9.01(s,1H),8.95(t,J＝5.4 Hz,1H),8.21(s,1H),7.97–7.87(m,2H),7.46–7.38(m,2H),7.38–7.30(m,2H),7.30–7.23(m,2H),6.97(s,1H),4.51(d,J＝5.6Hz,2H),3.60(s,3H).LR-MS(ESI)m/z 485(M+1).
实施例62
Figure PCTCN2020096521-appb-000104
除了用4-三氟甲基苯硼酸替代(2-苯氧基苯基)硼酸以外，合成方法如实施例10。
1H NMR(400MHz,DMSO)δ12.45(s,1H),9.65(s,1H),9.06(t,J＝5.7Hz,1H),8.22(s,1H),8.05–7.82(m,2H),7.66(dd,J＝14.9,3.1Hz,1H),7.45–7.34(m,4H),7.33–7.31(m,1H),7.30–7.22(m,2H),7.04–6.95(m,2H),6.85(td,J＝14.9,3.1Hz,1H),4.90(s,2H),4.50(d,J＝5.6Hz,2H),3.57(s,3H).LR-MS(ESI)m/z 560(M+1).
实施例63
Figure PCTCN2020096521-appb-000105
除了用4-甲氧基苯硼酸替代(2-苯氧基苯基)硼酸以外，合成方法如实施例9。
1H NMR(400MHz,DMSO)δ12.21(s,1H),11.17(s,1H),9.00(s,1H),8.95(t,J＝5.4Hz,1H),8.22(s,1H),8.01–7.83(m,2H),7.42–7.26(m,4H),7.01(s,1H),6.95–6.81(m,2H),4.55(d,J＝5.7Hz,2H),3.81(s,3H),3.60(s,3H).LR-MS(ESI)m/z 447(M+1).
实施例64
Figure PCTCN2020096521-appb-000106
除了用4-甲氧基苯硼酸替代(2-苯氧基苯基)硼酸以外，合成方法如实施例10。
1H NMR(400MHz,DMSO)δ12.43(s,1H),9.63(s,1H),9.06(t,J＝5.7Hz,1H),8.21(s,1H),7.98–7.87(m,2H),7.66(dd,J＝15.0,2.9Hz,1H),7.46–7.29(m,5H),7.04–6.96(m,2H),6.93–6.80(m,3H),4.88(s,2H),4.55(d,J＝5.6Hz,2H),3.81(s,3H),3.57(s,3H).LR-MS(ESI)m/z 526(M+1).
实施例65
Figure PCTCN2020096521-appb-000107
除了用4-氟苯硼酸替代(2-苯氧基苯基)硼酸以外，合成方法如实施例9。
1H NMR(400MHz,DMSO)δ12.21(s,1H),11.17(s,1H),9.00(s,1H),8.95(t,J＝5.4Hz,1H),8.21(s,1H),7.98–7.86(m,2H),7.38–7.28(m,4H),7.24(ddd,J＝15.4,8.9,2.7Hz,2H),7.00(s,1H),4.53(d,J＝5.7Hz,2H),3.55(s,3H).LR-MS(ESI)m/z 435(M+1)
实施例66
Figure PCTCN2020096521-appb-000108
除了用4-氟苯硼酸替代(2-苯氧基苯基)硼酸以外，合成方法如实施例10。
1H NMR(400MHz,DMSO)δ12.45(s,1H),9.61(s,1H),9.06(t,J＝5.7Hz,1H),8.22(s,1H),8.05–7.84(m,2H),7.66(dd,J＝15.0,3.0Hz,1H),7.45–7.36(m,1H),7.36–7.28(m,4H),7.28–7.18(m,2H),7.03(s,1H),7.00(dd,J＝15.2,3.1Hz,1H),6.91–6.79(m,1H),4.85(s,2H),4.51(d,J＝5.7Hz,2H),3.57(s,3H).LR-MS(ESI)m/z 510(M+1).
实施例67
Figure PCTCN2020096521-appb-000109
除了用4-氯苯硼酸替代(2-苯氧基苯基)硼酸以外，合成方法如实施例9。
1H NMR(400MHz,DMSO)δ12.21(s,1H),11.17(s,1H),8.99(s,1H),8.95(t,J＝5.4Hz,1H),8.22(s,1H),7.98–7.85(m,2H),7.49–7.39(m,2H),7.38–7.31(m,2H),7.31–7.23(m,2H),7.01(s,1H),4.45(d,J＝5.7Hz,2H),3.43(s,3H).LR-MS(ESI)m/z 451(M+1).
实施例68
Figure PCTCN2020096521-appb-000110
除了用4-氯苯硼酸替代(2-苯氧基苯基)硼酸以外，合成方法如实施例10。
1H NMR(400MHz,DMSO)δ12.45(s,1H),9.60(s,1H),9.06(t,J＝5.7Hz,1H),8.21(s,1H),7.97–7.86(m,2H),7.66(dd,J＝15.0,2.9Hz,1H),7.48–7.36(m,3H),7.36–7.31(m,2H),7.31–7.24(m,2H),7.04–6.96(m,2H),6.85(td,J＝14.9,3.1Hz,1H),4.85(s,2H),4.55(d,J＝5.7Hz,2H),3.57(s,3H).LR-MS(ESI)m/z 527(M+1).
实施例69
Figure PCTCN2020096521-appb-000111
除了用4-氰基苯硼酸替代(2-苯氧基苯基)硼酸以外，合成方法如实施例9。
1H NMR(400MHz,DMSO)δ12.31(s,1H),11.37(s,1H),9.10(s,1H),8.95(t,J＝5.4Hz,1H),8.22(s,1H),7.97–7.90(m,2H),7.90–7.83(m,2H),7.57–7.47(m,2H),7.40– 7.28(m,2H),6.98(s,1H),4.40(d,J＝5.7Hz,2H),3.47(s,3H).LR-MS(ESI)m/z 442(M+1).
实施例70
Figure PCTCN2020096521-appb-000112
除了用4-氰基苯硼酸替代(2-苯氧基苯基)硼酸以外，合成方法如实施例10。
1H NMR(400MHz,DMSO)δ12.45(s,1H),9.60(s,1H),9.06(t,J＝5.7Hz,1H),8.22(s,1H),7.97–7.89(m,2H),7.90–7.83(m,2H),7.66(dd,J＝15.0,2.9Hz,1H),7.58–7.47(m,2H),7.46–7.36(m,1H),7.37–7.28(m,2H),7.04–6.96(m,2H),6.85(td,J＝14.9,3.1Hz,1H),4.81(s,2H),4.55(d,J＝5.7Hz,2H),3.57(s,3H).LR-MS(ESI)m/z 517(M+1).
实施例71
除了用(4-(二甲胺基)甲基)苯基)硼酸替代(2-苯氧基苯基)硼酸以外，合成方法如实施例9。
Figure PCTCN2020096521-appb-000113
1H NMR(400MHz,DMSO)δ12.21(s,1H),11.17(s,1H),9.00(s,1H),8.95(t,J＝5.4Hz,1H),8.20(s,1H),7.97–7.85(m,2H),7.42–7.28(m,4H),7.16–7.04(m,2H),6.94(s,1H),4.51(d,J＝5.7Hz,2H),3.87(s,2H),3.55(s,3H),2.16(s,6H)LR-MS(ESI)m/z 474(M+1).
实施例72
Figure PCTCN2020096521-appb-000114
除了用(4-(二甲胺基)甲基)苯基)硼酸替代(2-苯氧基苯基)硼酸以外，合成方法如实施例10。
1H NMR(400MHz,DMSO)δ12.45(s,1H),9.60(s,1H),9.06(t,J＝5.7Hz,1H),8.23(s,1H),8.08–7.80(m,2H),7.66(dd,J＝14.9,3.1Hz,1H),7.46–7.36(m,2H),7.36–7.30(m,3H),7.15–7.07(m,2H),7.03(s,1H),7.00(dd,J＝15.2,3.1Hz,1H),6.85(td,J＝14.9,3.1Hz,1H),4.83(s,2H),4.51(d,J＝5.7Hz,2H),3.87(s,2H),3.55(s,3H),2.16(s,6H).LR-MS(ESI)m/z 549(M+1).
实施例73
Figure PCTCN2020096521-appb-000115
除了用(4-(吗啉代甲基)苯基)硼酸替代(2-苯氧基苯基)硼酸以外，合成方法如实施例9。
1H NMR(400MHz,DMSO)δ12.31(s,1H),11.15(s,1H),8.99(s,1H),8.95(t,J＝5.4Hz,1H),8.22(s,1H),8.00–7.80(m,2H),7.49–7.25(m,4H),7.14–7.06(m,2H),6.96(s,1H),4.51(d,J＝5.7Hz,2H),3.76(s,2H),3.55(s,3H),3.57(t,J＝9.3Hz,4H),2.42(t,J＝9.4Hz,4H).LR-MS(ESI)m/z 516(M+1).
实施例74
Figure PCTCN2020096521-appb-000116
除了用(4-(吗啉代甲基)苯基)硼酸替代(2-苯氧基苯基)硼酸以外，合成方法如实施例10。
1H NMR(400MHz,DMSO)δ12.45(s,1H),9.61(s,1H),9.06(t,J＝5.7Hz,1H),8.21(s,1H),8.01–7.84(m,2H),7.66(dd,J＝14.9,3.1Hz,1H),7.42(dd,J＝15.0,3.0Hz,1H),7.39 –7.30(m,4H),7.16–7.07(m,2H),7.04–6.96(m,2H),6.85(td,J＝14.9,3.1Hz,1H),4.83(s,2H),4.55(d,J＝5.7Hz,2H),3.87(s,2H),3.56(s,3H),3.57(t,J＝9.4Hz,4H),2.42(t,J＝9.4Hz,4H).LR-MS(ESI)m/z 591(M+1).
实施例75
Figure PCTCN2020096521-appb-000117
除用N-(3-溴-4-(2,4-二氟苯氧基)苯基)乙磺酰胺替代化合物39b以外，实施例75化合物的合成方法参照实施例39。
1H NMR(400MHz,DMSO)δ12.40(s,1H),11.21(s,1H),9.86(s,1H),8.95(t,J＝5.6Hz,1H),7.73(d,J＝8.2Hz,2H),7.44–7.35(m,4H),7.34(d,J＝2.6Hz,1H),7.24(dd,J＝8.8,2.6Hz,1H),7.08(td,J＝9.0,5.6Hz,1H),7.01(t,J＝8.5Hz,1H),6.95(d,J＝8.8Hz,1H),6.92(d,J＝2.1Hz,1H),4.50(d,J＝5.4Hz,2H),3.54(s,3H),3.11(q,J＝7.3Hz,2H),1.23(t,J＝7.3Hz,3H).LR-MS(ESI)m/z 652(M+1).
实施例76
Figure PCTCN2020096521-appb-000118
除用N-(3-溴-4-(2,4-二氟苯氧基)苯基)乙磺酰胺替代化合物39b以外，实施例76化合物的合成方法参照实施例40。
1H NMR(400MHz,DMSO)δ12.41(s,1H),9.86(s,1H),9.67(s,1H),8.99(s,1H),7.96(d,J＝8.3Hz,2H),7.45(d,J＝8.0Hz,2H),7.40–7.33(m,3H),7.26–7.22(m,1H),7.17(d,J＝8.2Hz,1H),7.13–7.05(m,1H),7.03–6.97(m,2H),6.96(s,1H),6.93(s,1H),6.79(d,J＝7.9Hz,1H),6.61(t,J＝7.6Hz,1H),4.96(s,2H),4.54(d,J＝5.5Hz,2H),3.55(s,3H),3.12(q,J＝7.5Hz,2H),1.23(t,J＝7.3Hz,4H)LR-MS(ESI)m/z 727(M+1).
实施例77
Figure PCTCN2020096521-appb-000119
除用2-溴-1-(2,4-二氟苯氧基)-4-(甲基磺酰基)苯替代化合物39b，实施例77的合成方法参照实施例39。
1H NMR(400MHz,DMSO))δ12.31(s,1H),11.15(s,1H),8.99(s,1H),8.95(t,J＝5.4Hz,1H),8.30(s,1H),8.01–7.78(m,2H),7.72(d,J＝3.1Hz,1H),7.62(dd,J＝14.9,2.8Hz,1H),7.38–7.26(m,4H),7.03–6.85(m,2H),6.56(td,J＝15.9,2.7Hz,1H),4.51(d,J＝5.7Hz,2H),3.57(s,3H),3.31(s,3H).LR-MS(ESI)m/z 623(M+1).
实施例78
Figure PCTCN2020096521-appb-000120
除用2-溴-1-(2,4-二氟苯氧基)-4-(甲基磺酰基)苯替代化合物39b，实施例78的合成方法参照实施例40。
1H NMR(400MHz,DMSO)δ12.42(s,1H),9.60(s,1H),9.06(t,J＝5.7Hz,1H),8.25(s,1H),7.96–7.84(m,2H),7.70(d,J＝3.0Hz,1H),7.67–7.55(m,2H),7.44–7.35(m,1H),7.35–7.25(m,3H),6.98(dd,J＝15.0,3.1Hz,1H),6.96–6.88(m,2H),6.88–6.78(m,2H),6.70(td,J＝15.8,2.5Hz,1H),4.88(s,2H),4.50(d,J＝5.7Hz,2H),3.56(s,3H),3.30(s,3H).LR-MS(ESI)m/z 442(M+1).LR-MS(ESI)m/z 698(M+1).
实施例79
Figure PCTCN2020096521-appb-000121
实施例79a 1-(3-溴-4-(2,4-二氟苯氧基)苯基)乙烷-1-酮
Figure PCTCN2020096521-appb-000122
将3-溴-4-氟苯甲酮(2g,9.22mmol)、2,4-二氟苯酚(1.44g,11.06mmol)和碳酸铯(4.5g,13.8 2mmol)溶于二甲亚砜(20mL)，在90℃油浴加热条件下反应3小时，冷却至室温后用乙酸乙酯萃取反应，合并有机相后用水和饱和氯化钠溶液洗有机相，然后用无水硫酸钠干燥。减(压蒸发除去溶剂后柱层析得到化合物1-(3-溴-4-(2,4-二氟苯氧基)苯基)乙烷-1-酮2.5g(产率：83％)。
实施例79b 2-(3-溴-4-(2,4-二氟苯氧基)苯基)丙-2-醇
Figure PCTCN2020096521-appb-000123
将化合物79a(2g,6.11mmol)溶于无水四氢呋喃(30mL)，在冰浴条件下滴加甲基溴化镁(1M in THF,7.33mL，7.33mmol)，然后将反应室温条件下反应两个小时后用饱和氯化铵溶液灭反应。用乙酸乙酯萃取后合并有机相，用饱和食盐水洗涤有机相后，用无水硫酸钠干燥。减压蒸干溶剂后柱层析得到目标产物化合物2-(3-溴-4-(2,4-二氟苯氧基)苯基)丙-2-醇(0.8g，38％)。
实施例79c 4-(2-(2,4-二氟苯氧基)-5-(2-羟基丙烷-2-基)苯基)-6-甲基-7-氧代-1-甲苯磺酰基-6,7-二氢-1H-吡咯并[2,3-c]吡啶-2-羧酸乙酯
Figure PCTCN2020096521-appb-000124
在氩气保护条件下将化合物39a(0.3g,0.617mmol)、化合物79b(0.176g,0.514mmol)、氟化铯(0.156g,1.03mmol)、三(二亚苄基丙酮)二钯(0)(0.047g,0.0514mmol)、1,3,5,7-四甲基-6-苯基-2,4,8-三氧杂-6-磷酰金刚烷(0.029g,0.103mmol)溶于乙二醇二甲醚(2mL)和水(0.16mL)混合溶液中，60℃油浴条件下搅拌2小时至反应完全。用硅 藻土滤除不溶物后乙酸乙酯萃取。然后用氯化钠溶液洗涤，合并有机相经无水硫酸钠干燥，过滤并浓缩。通过快速层析法(硅胶2-50％乙酸乙酯/石油醚梯度)分离出产物44-(2-(2,4-二氟苯氧基)-5-(2-羟基丙烷-2-基)苯基)-6-甲基-7-氧代-1-甲苯磺酰基-6,7-二氢-1H-吡咯并[2,3-c]吡啶-2-羧酸乙酯0.2g，(产率：62％)。
实施例79d 4-(2-(2,4-二氟苯氧基)-5-(2-羟基丙烷-2-基)苯基)-6-甲基-7-氧代-6,7-二氢-1H-吡咯并[2,3-C]吡啶-2-羧酸
Figure PCTCN2020096521-appb-000125
将化合物79c(0.2g,0.321mmol)溶于二氧六环(2mL)后加入4M的氢氧化钠溶液(0.4mL,1.6mmol)，在氩气保护条件下90℃搅拌2小时至反应完全。用1N盐酸溶液调节体系pH至1-3，将析出的白色固体抽滤、干燥，得到产物4-(2-(2,4-二氟苯氧基)-5-(2-羟基丙烷-2-基)苯基)-6-甲基-7-氧代-6,7-二氢-1H-吡咯并[2,3-C]吡啶-2-羧酸0.11g(产率：75％)。
实施例79e 4-((4-(2-(2,4-二氟苯氧基)-5-(2-羟基丙烷-2-基)苯基)-6-甲基-7-氧代-6,7-二氢-1H-吡咯并[2,3-c]吡啶-2-甲酰氨基)甲基)苯甲酸甲酯
Figure PCTCN2020096521-appb-000126
将化合物79d(0.10g,0.220mmol)溶于N,N-二甲基甲酰胺(1.5mL)后加入N,N-二异丙基乙胺(0.080mL,0.612mmol)、N-羟基-7-偶氮苯并三氮唑(0.062g,0.458mmol)和1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(0.088g,0.458mmol)，室温反应20分钟后加入4-(氨基甲基)苯甲酸甲酯(0.054g,0.330mmol)。在室温下搅拌4小时至反应完全。加入水淬灭反应，乙酸乙酯萃取后用水和饱和氯化钠溶液洗涤，合并有机相并经 无水硫酸钠干燥，过滤并浓缩。通过快速层析法(硅胶0-5％二氯甲烷/石甲醇梯度)分离出产物4-((4-(2-(2,4-二氟苯氧基)-5-(2-羟基丙烷-2-基)苯基)-6-甲基-7-氧代-6,7-二氢-1H-吡咯并[2,3-c]吡啶-2-甲酰氨基)甲基)苯甲酸甲酯0.1g(产率：76％)
将化合物79e(0.1g,0.166mmol)溶于二氯甲烷(0.2mL)和甲醇(1mL)，冰浴条件下加入50％羟胺水溶液(0.286mL,4.35mmol)后加入氢氧化钠固体(0.174g,4.35mmol)，继续搅拌2小时至反应完全。用3N盐酸溶液调节体系pH至3，将析出的固体抽滤后用水洗，干燥，得到实施例39化合物，0.07g(产率：70％)。
1H NMR(400MHz,DMSO)δ12.46(s,1H),11.23(s,1H),10.29(s,1H),9.09(s,1H),8.34(s,1H),8.11–7.78(m,2H),7.39–7.32(m,2H),7.32–7.21(m,2H),7.02–6.85(m,3H),6.78–6.65(m,2H),5.52(s,1H),4.51(d,J＝5.7Hz,2H),3.66(s,3H),1.49(s,6H).LR-MS(ESI)m/z 603(M+1).
实施例80
Figure PCTCN2020096521-appb-000127
实施例80a 4-((4-(2-(2,4-二氟苯氧基)-5-(2-羟基丙烷-2-基)苯基)-6-甲基-7-氧代-6,7-二氢-1H-吡咯并[2,1，3-c]吡啶-2-甲酰胺基)甲基)苯甲酸
Figure PCTCN2020096521-appb-000128
将化合物79e(0.1g,0.166mmol)溶于二氧六环(2mL)后加入2M的氢氧化钠溶液0.436mL,0.872mmol)，在氩气保护条件下90℃搅拌2小时至反应完全。用1N盐酸溶液调节体系pH至3，将析出的白色固体抽滤干燥得到产物4-((4-(2-(2,4-二氟苯氧基)-5-(2-羟基丙烷-2-基)苯基)-6-甲基-7-氧代-6,7-二氢-1H-吡咯并[2,1，3-c]吡啶-2-甲酰胺基)甲基)苯甲酸0.08g(产率：81％)。
将化合物80a(0.08g,0.136mmol)溶于N,N-二甲基甲酰胺(1mL)后加入N,N- 二异丙基乙胺(0.089mL,0.540mmol)、N-羟基-7-偶氮苯并三氮唑(0.034g,0.252mmol)和1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(0.048g,mmol)，室温反应20分钟后加入邻苯二胺(0.017g,0.163mmol)。在室温下搅拌4小时至反应完全。加入水淬灭反应，乙酸乙酯萃取后用水和饱和氯化钠溶液洗涤，合并有机相并经无水硫酸钠干燥，过滤并浓缩。通过快速层析法(硅胶0-7％二氯甲烷/甲醇梯度)分离出产物实施例80化合物0.05g(产率：54％)。
1H NMR(400MHz,DMSO)δ12.36(s,1H),9.65(s,1H),8.98(t,J＝5.0Hz,1H),7.96(d,J＝8.2Hz,2H),7.56(s,1H),7.45(d,J＝7.9Hz,3H),7.39(t,J＝8.6Hz,1H),7.33(s,1H),7.16(d,J＝8.0Hz,1H),7.14–7.08(m,1H),7.03(t,J＝9.8Hz,1H),6.97(t,J＝7.8Hz,1H),6.92(s,1H),6.83(d,J＝8.6Hz,1H),6.78(d,J＝7.7Hz,1H),6.60(t,J＝7.6Hz,1H),5.10(s,1H),4.90(s,2H),4.54(d,J＝5.5Hz,2H),3.56(s,3H),1.47(s,6H).LR-MS(ESI)m/z 678(M+1).
实施例81
Figure PCTCN2020096521-appb-000129
实施例81a 4-(2-(4-氟-2,6-二甲基苯氧基)-5-(2-羟基丙烷-2-基)苯基)-6-甲基-7-氧代-6,7-二氢-1H-吡咯并[2,1，3-c]吡啶-2-羧酸
Figure PCTCN2020096521-appb-000130
除用4-氟-2,6-二甲基苯酚替代2,4-二氟苯酚外，实施例81a的合成方法如实施例79d。
除用实施例81a化合物替代实施例79d化合物以外，实施例81的合成方法如实施例 79。
1H NMR(400MHz,DMSO)δ12.31(s,1H),11.15(s,1H),8.99(s,1H),8.95(t,J＝5.4Hz,1H),8.32(s,1H),7.99–7.84(m,2H),7.38–7.32(m,2H),7.29(dd,J＝14.9,2.8Hz,1H),7.26–7.22(m,2H),6.98(d,J＝14.9Hz,1H),6.85(d,J＝15.8Hz,2H),5.52(s,1H),4.51(d,J＝5.7Hz,2H),3.57(s,3H),2.15(s,6H),1.35(s,6H).LR-MS(ESI)m/z 613(M+1).
实施例82
Figure PCTCN2020096521-appb-000131
除用4-氟-2,6-二甲基苯酚替代2,4-二氟苯酚，实施例的合成方法如实施例80。
1H NMR(400MHz,DMSO)δ12.45(s,1H),9.61(s,1H),9.06(t,J＝5.7Hz,1H),8.33(s,1H),8.04–7.84(m,2H),7.66(dd,J＝15.0,2.9Hz,1H),7.45–7.36(m,1H),7.36–7.32(m,2H),7.29(dd,J＝14.9,2.8Hz,1H),7.26(s,1H),7.24(d,J＝2.9Hz,1H),7.09–6.93(m,2H),6.92–6.79(m,3H),5.52(s,1H),4.88(s,2H),4.51(d,J＝5.7Hz,2H),3.60(s,3H),2.15(s,6H),1.35(s,6H).LR-MS(ESI)m/z 688(M+1).
实施例83
Figure PCTCN2020096521-appb-000132
除用异丙醇替代2,4-二氟苯酚，实施例的合成方法如实施例79。
1H NMR(400MHz,DMSO)δ12.31(s,1H),11.15(s,1H),8.99(s,1H),8.95(t,J＝5.4Hz,1H),8.33(d,J＝18.9Hz,2H),8.06–7.73(m,2H),7.47–7.30(m,2H),7.28(d,J＝2.9Hz,1H),7.08(s,1H),6.94(d,J＝14.9Hz,1H),5.52(s,1H),4.91–4.47(m,1H),4.45(d,J＝5.6Hz,2H),3.66(s,3H),1.35(s,6H),1.31(d,J＝11.2Hz,6H).LR-MS(ESI)m/z 533(M+1).
实施例84
Figure PCTCN2020096521-appb-000133
除用异丙醇替代2,4-二氟苯酚，实施例的合成方法如实施例80。
1H NMR(400MHz,DMSO)δ12.43(s,1H),9.58(s,1H),9.03(t,J＝5.6Hz,1H),8.24(s,1H),8.04–7.82(m,2H),7.63(dd,J＝14.9,3.1Hz,1H),7.43–7.34(m,1H),7.34–7.28(m,3H),7.25(d,J＝3.1Hz,1H),7.04(s,1H),6.97(dd,J＝15.0,3.0Hz,1H),6.91(d,J＝14.8Hz,1H),6.82(td,J＝14.9,3.0Hz,1H),5.50(s,1H),5.10(s,2H),4.67(hept,J＝11.2Hz,1H),4.50(d,J＝5.6Hz,2H),3.56(s,3H),1.35(s,6H),1.31(d,J＝11.1Hz,6H).LR-MS(ESI)m/z 608(M+1).
实施例85
Figure PCTCN2020096521-appb-000134
除用环丙醇替代2,4-二氟苯酚，实施例的合成方法如实施例79。
1H NMR(400MHz,DMSO)δ12.31(s,1H),11.15(s,1H),8.99(s,1H),8.95(t,J＝5.4Hz,1H),8.33(s,1H),8.00–7.85(m,2H),7.38–7.30(m,3H),7.28(d,J＝2.9Hz,1H),7.04(s,1H),6.94(d,J＝14.9Hz,1H),5.52(s,1H),4.51(d,J＝5.6Hz,2H),3.60(s,3H),1.35(s,6H),1.28–0.92(m,1H),0.42–0.06(m,4H).LR-MS(ESI)m/z 531(M+1).
实施例86
Figure PCTCN2020096521-appb-000135
除用环丙醇替代2,4-二氟苯酚，实施例的合成方法如实施例80。
1H NMR(400MHz,DMSO)δ12.44(s,1H),9.60(s,1H),9.05(t,J＝5.6Hz,1H),8.35(s,1H),7.96–7.86(m,2H),7.65(dd,J＝14.9,3.1Hz,1H),7.46(s,1H),7.44–7.36(m,1H), 7.36–7.25(m,4H),6.99(dd,J＝14.9,3.1Hz,1H),6.93(d,J＝14.8Hz,1H),6.84(td,J＝15.0,3.0Hz,1H),5.52(s,1H),4.88(s,2H),4.51(d,J＝5.6Hz,2H),3.67(s,3H),3.33(p,J＝16.6Hz,1H),1.35(s,6H),0.75–0.02(m,4H).LR-MS(ESI)m/z 606(M+1).
实施例87
Figure PCTCN2020096521-appb-000136
除用羟甲基环丙烷替代2,4-二氟苯酚，实施例的合成方法如实施例79。
1H NMR(400MHz,DMSO)δ12.31(s,1H),11.15(s,1H),8.99(s,1H),8.95(t,J＝5.4Hz,1H),8.33(s,1H),8.00–7.85(m,2H),7.38–7.30(m,3H),7.28(d,J＝2.9Hz,1H),7.04(s,1H),6.94(d,J＝14.9Hz,1H),5.52(s,1H),4.51(d,J＝5.6Hz,2H),3.85(d,J＝14.7Hz,2H),3.66(s,3H),1.35(s,6H),1.28–0.92(m,1H),0.42–0.06(m,4H).LR-MS(ESI)m/z545(M+1).
实施例88
Figure PCTCN2020096521-appb-000137
除用羟甲基环丙烷替代2,4-二氟苯酚，实施例的合成方法如实施例80。
1H NMR(400MHz,DMSO)δ12.45(s,1H),9.61(s,1H),9.06(t,J＝5.6Hz,1H),8.15(s,1H),7.97–7.88(m,2H),7.66(dd,J＝15.0,2.9Hz,1H),7.47–7.37(m,1H),7.36–7.30(m,3H),7.28(d,J＝2.9Hz,1H),7.00(dd,J＝15.1,3.0Hz,1H),6.94(t,J＝7.5Hz,2H),6.85(td,J＝14.9,3.1Hz,1H),5.52(s,1H),4.90(s,2H),4.51(d,J＝5.6Hz,2H),3.58(d,J＝16.0Hz,2H),3.67(s,3H),1.35(s,6H),1.28–0.94(m,1H),0.54–0.20(m,4H).LR-MS(ESI)m/z 620(M+1).
实施例89
Figure PCTCN2020096521-appb-000138
除用环己醇替代2,4-二氟苯酚，实施例的合成如参照实施例79。
1H NMR(400MHz,DMSO)δ12.31(s,1H),11.15(s,1H),8.99(s,1H),8.95(t,J＝5.4Hz,1H),8.26(s,1H),8.06–7.83(m,2H),7.37–7.25(m,4H),6.99(s,1H),6.94(d,J＝14.9Hz,1H),5.52(s,1H),4.51(d,J＝5.6Hz,2H),3.85–3.69(m,1H),3.57(s,3H),2.03–1.85(m,2H),1.82–1.67(m,1H),1.68–1.53(m,2H),1.35(s,6H),1.30–1.07(m,5H).LR-MS(ESI)m/z 573(M+1).
实施例90
Figure PCTCN2020096521-appb-000139
除用环己醇替代2,4-二氟苯酚，实施例的合成方法如实施例80。
1H NMR(400MHz,DMSO)δ12.45(s,1H),9.61(s,1H),9.06(t,J＝5.6Hz,1H),8.19(s,1H),7.97–7.87(m,2H),7.66(dd,J＝14.9,3.1Hz,1H),7.45–7.36(m,1H),7.36–7.30(m,3H),7.28(d,J＝2.9Hz,1H),7.00(dd,J＝14.9,3.1Hz,1H),6.94(d,J＝14.8Hz,1H),6.89–6.76(m,2H),5.52(s,1H),5.12(s,2H),4.51(d,J＝5.6Hz,2H),3.57(s,3H),3.65–3.54(m,1H),2.00–1.83(m,2H),1.81–1.54(m,3H),1.35(s,6H),1.32–1.10(m,5H).LR-MS(ESI)m/z 678(M+1).
实施例91
Figure PCTCN2020096521-appb-000140
除用3-溴-4-甲氧基苯甲酸甲酯替代化合物79a以外，实施例91的合成方法如实施例79。
1H NMR(400MHz,DMSO-d 6)δ12.38(s,1H),11.19(s,1H),9.09–8.92(m,2H),7.73(d,J＝8.2Hz,2H),7.68(s,1H),7.48–7.38(m,6H),7.10(s,1H),5.08(s,1H),4.52(d,J＝5.8Hz,2H),3.81(s,3H),3.53(s,3H),1.49(s,6H).LR-MS(ESI)m/z 505(M+1).
实施例92
Figure PCTCN2020096521-appb-000141
实施例92a：
Figure PCTCN2020096521-appb-000142
除用3-溴-4-甲氧基苯甲酸甲酯替代化合物79a，实施例92a的合成方法如实施例79e。
实施例92b:
Figure PCTCN2020096521-appb-000143
除用92a替代79e，实施例92b的合成方法如实施例80a。
除用92b替代80a，实施例92的合成方法如实施例80。
1H NMR(600MHz,DMSO)δ12.24(d,J＝1.8Hz,1H),9.66(s,1H),8.95(t,J＝5.9Hz,1H),7.95(d,J＝8.0Hz,2H),7.44(dd,J＝8.5,2.0Hz,3H),7.38(d,J＝2.4Hz,1H),7.19(s,1H),7.17(d,J＝7.3Hz,1H),7.05(d,J＝8.7Hz,1H),6.98(t,J＝7.0Hz,1H),6.80(d,J＝7.8Hz,1H),6.71(d,J＝2.3Hz,1H),6.63(t,J＝7.3Hz,1H),5.09(s,1H),4.95(s,2H),4.52(d,J＝5.8Hz,2H),3.71(s,3H),3.57(s,3H),1.44(s,6H).LR-MS(ESI)m/z 490(M+1).
实施例93
Figure PCTCN2020096521-appb-000144
除用6-氨基己酸甲酯替代4-(氨基甲基)苯甲酸甲酯，实施例93的合成方法如实施例85。
1H NMR(400MHz,DMSO-d 6)δ12.15(s,1H),10.34(s,1H),8.67(s,1H),8.29(t,J＝5.4Hz,1H),7.45(d,J＝8.6Hz,1H),7.38(s,1H),7.33(d,J＝8.5Hz,1H),7.16(s,1H),6.59(d,J＝1.9Hz,1H),4.97(s,1H),3.83–3.72(m,1H),3.56(s,3H),3.22(q,J＝6.1Hz,2H),1.95(t,J＝7.4Hz,2H),1.56–1.46(m,4H),1.45(s,6H),1.34–1.25(m,2H),0.76–0.68(m,2H),0.60–0.53(m,2H).LR-MS(ESI)m/z 511(M+1).
实施例94
Figure PCTCN2020096521-appb-000145
除用7-氨基庚酸乙酯替代4-(氨基甲基)苯甲酸甲酯，实施例94的合成方法如实施例85。
1H NMR(400MHz,DMSO-d 6)δ12.15(s,1H),10.34(s,1H),8.67(s,1H),8.28(s,1H),7.45(d,J＝10.3Hz,1H),7.38(s,1H),7.33(d,J＝8.5Hz,1H),7.16(s,1H),6.60(s,1H),4.97(s,1H),3.85–3.74(m,1H),3.56(s,3H),3.22(q,J＝6.4,6.0Hz,1H),1.94(t,J＝7.4Hz,2H),1.53–1.47(m,4H),1.45(s,6H),1.34–1.25(m,4H),0.75–0.66(m,2H),0.61–0.53(m,2H).LR-MS(ESI)m/z 525(M+1).
实施例95
Figure PCTCN2020096521-appb-000146
除用6-氨基己酸甲酯替代4-(氨基甲基)苯甲酸甲酯，实施例95的合成方法如实施例83。
1H NMR(400MHz,DMSO-d 6)δ12.14(s,1H),10.34(s,1H),8.67(s,1H),8.31(t,J＝5.3Hz,1H),7.43–7.36(m,2H),7.20(d,J＝1.2Hz,1H),7.02(d,J＝9.1Hz,1H),6.72(s,1H),4.96 (s,1H),4.50(p,J＝6.0Hz,1H),3.57(s,3H),3.22(q,J＝6.1Hz,2H),1.94(t,J＝7.3Hz,2H),1.50(q,J＝7.6Hz,4H),1.44(s,6H),1.35–1.22(m,2H),1.13(d,J＝6.1Hz,6H).LR-MS(ESI)m/z 513(M+1).
实施例96
Figure PCTCN2020096521-appb-000147
除用7-氨基庚酸乙酯替代4-(氨基甲基)苯甲酸甲酯，实施例96的合成方法如实施例83。
1H NMR(400MHz,DMSO-d 6)δ12.13(s,1H),10.34(s,1H),8.67(s,1H),8.30(t,J＝5.4Hz,1H),7.42–7.36(m,2H),7.20(s,1H),7.02(d,J＝9.4Hz,1H),6.72(s,1H),4.95(s,1H),4.50(p,J＝6.0Hz,1H),3.57(s,3H),3.22(q,J＝6.5Hz,2H),1.94(t,J＝7.3Hz,2H),1.50(q,J＝7.3Hz,4H),1.44(s,6H),1.37–1.25(m,4H),1.13(d,J＝6.0Hz,6H).LR-MS(ESI)m/z 527(M+1).
实施例97
Figure PCTCN2020096521-appb-000148
除用6-氨基己酸甲酯替代4-(氨基甲基)苯甲酸甲酯，实施例97的合成方法如实施例79。
1H NMR(400MHz,DMSO-d 6)δ12.23(s,1H),10.35(s,1H),8.68(s,1H),8.32(t,J＝5.4Hz,1H),7.55(d,J＝2.4Hz,1H),7.45(dd,J＝8.6,2.4Hz,1H),7.39(td,J＝11.5,8.8,2.9Hz,1H),7.31(s,1H),7.14–7.06(m,1H),7.02(td,J＝8.5,2.5Hz,1H),6.82(d,J＝7.4Hz,2H),5.10(s,1H),3.56(s,3H),3.22(q,J＝6.1Hz,2H),1.94(t,J＝7.3Hz,2H),1.50(q,J＝7.6Hz,4H),1.44(s,6H),1.35–1.22(m,2H).LR-MS(ESI)m/z 583(M+1).
实施例98
Figure PCTCN2020096521-appb-000149
除用7-氨基庚酸乙酯替代4-(氨基甲基)苯甲酸甲酯，实施例98的合成方法如实施例 79。
1H NMR(400MHz,DMSO-d 6)δ12.23(s,1H),10.35(s,1H),8.68(s,1H),8.32(t,J＝5.4Hz,1H),7.55(d,J＝2.4Hz,1H),7.45(dd,J＝8.6,2.4Hz,1H),7.39(td,J＝11.5,8.8,2.9Hz,1H),7.31(s,1H),7.14–7.06(m,1H),7.02(td,J＝8.5,2.5Hz,1H),6.82(d,J＝7.4Hz,2H),5.10(s,1H),3.56(s,3H),3.22(q,J＝6.5Hz,2H),1.94(t,J＝7.3Hz,2H),1.55–1.39(m,10H),1.34–1.24(m,4H).LR-MS(ESI)m/z 597(M+1).
实施例99
Figure PCTCN2020096521-appb-000150
除用6-氨基己酸甲酯替代4-(氨基甲基)苯甲酸甲酯，实施例99的合成方法如实施例81。
1H NMR(400MHz,DMSO-d 6)δ12.27(s,1H),10.35(s,1H),8.68(s,1H),8.34(t,J＝5.4Hz,1H),7.52(d,J＝2.4Hz,1H),7.35(s,1H),7.32(dd,J＝8.6,2.4Hz,1H),7.00(d,J＝9.1Hz,2H),6.85(s,1H),6.31(d,J＝8.6Hz,1H),5.02(s,1H),3.60(s,3H),3.22(q,J＝6.5Hz,2H),2.02(s,6H),1.95(t,J＝7.4Hz,2H),1.58–1.47(m,4H),1.44(s,6H),1.29(d,J＝7.3Hz,2H).LR-MS(ESI)m/z 593(M+1).
实施例100
Figure PCTCN2020096521-appb-000151
除用7-氨基庚酸乙酯替代4-(氨基甲基)苯甲酸甲酯，实施例100的合成方法如实施例81。
1H NMR(400MHz,DMSO-d 6)δ12.25(s,1H),10.34(s,1H),8.67(s,1H),8.33(t,J＝5.5Hz,1H),7.52(d,J＝2.4Hz,1H),7.35(s,1H),7.33(dd,J＝8.7,2.6Hz,1H),7.00(d,J＝9.1Hz,2H),6.85(s,1H),6.31(d,J＝8.5Hz,1H),5.01(s,1H),3.60(s,3H),3.23(q,J＝6.5Hz,2H),2.02(s,6H),1.94(t,J＝7.3Hz,2H),1.53–1.47(m,4H),1.44(s,6H),1.33–1.24(m,4H).LR-MS(ESI)m/z 607(M+1).
实施例101
Figure PCTCN2020096521-appb-000152
除用8-溴-2,2,4-三甲基-6-(甲基磺酰基)-2-H-苯并[b][1,4]恶嗪-3(4-H)-酮替代化合物39b，实施例101的合成方法如实施例39。
1H NMR(400MHz,DMSO)δ12.31(s,1H),11.15(s,1H),8.99(s,1H),8.95(t,J＝5.4Hz,1H),8.27(s,1H),7.98–7.86(m,2H),7.74(d,J＝3.1Hz,1H),7.50(d,J＝3.1Hz,1H),7.40–7.25(m,2H),7.07(s,1H),4.51(d,J＝5.6Hz,2H),3.57(s,3H),3.36(s,3H),3.31(s,3H),1.55(s,6H).LR-MS(ESI)m/z 608(M+1).
实施例102
Figure PCTCN2020096521-appb-000153
除用8-溴-2,2,4-三甲基-6-(甲基磺酰基)-2-H-苯并[b][1,4]恶嗪-3(4-H)-酮替代化合物39b，实施例102的合成方法如实施例40。
1H NMR(400MHz,DMSO)δ12.44(s,1H),9.60(s,1H),9.05(t,J＝5.6Hz,1H),8.38(s,1H),7.98–7.87(m,2H),7.73(d,J＝3.1Hz,1H),7.65(dd,J＝15.0,2.9Hz,1H),7.49(d,J＝2.8Hz,1H),7.45–7.36(m,1H),7.36–7.29(m,2H),6.99(dd,J＝15.0,3.0Hz,1H),6.84(td,J＝14.9,3.1Hz,1H),6.65(s,1H),4.88(s,2H),4.51(d,J＝5.6Hz,2H),3.66(s,3H),3.36(s,3H),3.31(s,3H),1.55(s,6H).LR-MS(ESI)m/z 683(M+1).
实施例103
Figure PCTCN2020096521-appb-000154
除用4-氟-1,2-苯二胺替代邻苯二胺外，实施例103的合成方法参照实施例80。
1H NMR(400MHz,DMSO)δ12.31(s,1H),11.15(s,1H),8.99(s,1H),8.95(t,J＝5.4Hz,1H),8.28(s,1H),7.95–7.82(m,2H),7.44(dd,J＝14.9,9.9Hz,1H),7.35–7.28(m,2H),7.25(dd,J＝14.9,3.1Hz,1H),7.20(d,J＝2.9Hz,1H),7.00–6.93(m,2H),6.93–6.82(m,2H),6.79–6.67(m,2H),6.62(ddd,J＝16.0,15.0,3.1Hz,1H),5.49(s,1H),4.91(s,1H),4.59(d,J＝5.6Hz,2H),3.55(s,3H),1.34(s,6H).LR-MS(ESI)m/z 696(M+1).
实施例104
Figure PCTCN2020096521-appb-000155
除用4-氟-1,2-苯二胺替代邻苯二胺外，实施例104的合成方法参照实施例76。
1H NMR(400MHz,DMSO)δ12.43(s,1H),9.60(s,1H),9.05(t,J＝5.6Hz,1H),8.34(s,1H),7.97–7.84(m,2H),7.47(dd,J＝15.0,10.0Hz,1H),7.36(s,1H),7.35–7.29(m,2H),7.03–6.97(m,1H),6.97–6.93(m,1H),6.92–6.84(m,1H),6.83–6.74(m,3H),6.70–6.59(m,2H),6.57(s,1H),4.93(s,2H),4.51(d,J＝5.6Hz,2H),3.57(s,3H),3.45(q,J＝13.8Hz,2H),1.27(t,J＝13.7Hz,3H).LR-MS(ESI)m/z 745(M+1).
实施例105
Figure PCTCN2020096521-appb-000156
将实施例10化合物(25mg,0.043mmol)溶于二氯甲烷(2mL)和甲醇(1mL)后加入4M的HCl的二氧六环溶液(0.021mL,0.086mmol)，室温反应3小时候减压蒸发除去溶剂，将所得用甲基叔丁基醚重结晶得到实施例105化合物。
1H NMR(400MHz,DMSO)δ12.35(s,1H),10.59(s,1H),9.10(t,J＝5.7Hz,1H),8.10(d,J＝8.1Hz,2H),7.60(d,J＝7.8Hz,1H),7.56–7.46(m,4H),7.46–7.34(m,3H),7.30(m,4H),7.08–7.01(m,2H),6.98(s,1H),6.88(d,J＝8.2Hz,2H),4.56(d,J＝5.7Hz,2H),3.92(s,3H),3.51(s,3H).LR-MS(ESI)m/z 621(M+1).
实施例106
Figure PCTCN2020096521-appb-000157
实施例106a
Figure PCTCN2020096521-appb-000158
除用2-异丙氧基吡啶-3-硼酸替代(2-苯氧基苯基)硼酸外，实施例106a的合成方法同实施例1g；
除用实施例106a化合物替代实施例1g化合物外，实施例106的合成方法同实施例10。
1H NMR(400MHz,DMSO)δ12.21(s,1H),9.61(s,1H),8.95(t,J＝5.9Hz,1H),δ7.92(d,J＝15.1Hz,2H),7.70–7.57(m,3H),7.41–7.28(m,4H),7.09(dd,J＝15.1,3.0Hz,1H), 6.94–6.79(m,2H),6.59(t,J＝15.0Hz,1H).4.86(s,2H),4.55(dt,J＝11.9,5.8Hz,1H),4.48(d,J＝5.4Hz,2H),3.56(s,3H),1.16(s,3H),1.15(s,3H).LR-MS(ESI)m/z 551(M+1).
实施例107
Figure PCTCN2020096521-appb-000159
实施例107a
Figure PCTCN2020096521-appb-000160
将3-氟酞酐(10g,60.20mmol)和3-氨基-2,6-哌啶二酮(0.26g,72.24mmol)溶于乙酸(70mL)，在90℃油浴加热条件下反应过夜后，有大量不溶固体析出，抽滤后用水和饱和碳酸钠溶液洗涤固体，干燥后得到产物107a(12g,72％)。
实施例107b
Figure PCTCN2020096521-appb-000161
将化合物107a(0.2g,0.724mmol)溶于N,N-二甲基甲酰胺(3mL)后，加入N-叔丁氧羰基-1,4-丁二胺(0.151g,0.869mmol)和碳酸铯(0.220g,1.45mmol)。在室温下搅拌4小时至反应完全。加入水淬灭反应，乙酸乙酯萃取后用水和饱和氯化钠溶液洗涤，合并有机相并经无水硫酸钠干燥，过滤并浓缩。通过快速层析法(硅胶0-7％二氯甲烷/石甲醇梯度)分离出产物后将所得产物溶于三氟乙酸，室温搅拌15分钟后加压蒸发除去溶剂，得到化合物107b(0.2g,84％)。
将化合物1g(0.1g,0.277mmol)溶于N,N-二甲基甲酰胺(1mL)后，加入N,N- 二异丙基乙胺(0.098mL,0.555mmol)、N-羟基-7-偶氮苯并三氮唑(0.053g,0.388mmol)和1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(0.074g,0.388mmol)，室温反应20分钟后加入107b(0.128g,0.388mmol)。在室温下搅拌4小时至反应完全。加入水淬灭反应，乙酸乙酯萃取后用水和饱和氯化钠溶液洗涤，合并有机相并经无水硫酸钠干燥，过滤并浓缩。通过快速层析法(硅胶0-7％二氯甲烷/石甲醇梯度)分离出产物化合物107。
1H NMR(400MHz,CDCl3)δ11.54(s,1H),9.44(s,1H),7.52(d,J＝6.3Hz,1H),7.47–7.41(m,1H),7.38–7.30(m,2H),7.23(dd,J＝11.0,4.9Hz,3H),7.12–7.04(m,3H),6.99(t,J＝7.4Hz,1H),6.96(s,1H),6.86(dd,J＝8.0,5.3Hz,3H),6.26(t,J＝5.4Hz,1H),4.97(dd,J＝11.8,5.0Hz,1H),3.62(s,3H),3.45(s,2H),3.30(s,2H),2.88–2.70(m,3H),2.09(d,J＝7.4Hz,1H),1.70(s,4H).LR-MS(ESI)m/z 687(M+1).
实施例108
Figure PCTCN2020096521-appb-000162
除用化合物N-叔丁氧羰基-1,5-戊二胺替代N-叔丁氧羰基-1,4-丁二胺外，实施例108的合成方法参照实施例107。
1H NMR(400MHz,DMSO)δ12.85(s,1H),11.06(s,1H),8.98(t,J＝5.6Hz,1H),8.32(s,1H),7.59(t,J＝14.9Hz,1H),7.51–7.37(m,3H),7.30(dd,J＝12.6,2.3Hz,1H),7.28–7.22(m,2H),7.22–7.14(m,1H),7.14–7.04(m,3H),7.02–6.94(m,2H),6.79(t,J＝5.8Hz,1H),4.52(t,J＝14.7Hz,1H),3.67(s,3H),3.30(t,J＝10.1Hz,2H),3.06(t,J＝15.0Hz,2H),2.89–2.66(m,1H),2.58–2.35(m,2H),2.34–2.15(m,1H),1.76–1.56(m,2H),1.55–1.41(m,2H),1.39–1.17(m,2H).LR-MS(ESI)m/z 701(M+1).
实施例109
Figure PCTCN2020096521-appb-000163
除用化合物N-叔丁氧羰基-1,6-己二胺替代N-叔丁氧羰基-1,4-丁二胺外，实施例109的合成方法参照实施例107。
1H NMR(400MHz,DMSO)δ12.85(s,1H),11.06(s,1H),8.98(t,J＝5.7Hz,1H),8.08(s,1H),7.59(t,J＝14.9Hz,1H),7.51–7.37(m,3H),7.30(dd,J＝12.6,2.3Hz,1H),7.28–7.15(m,4H),7.15–7.04(m,3H),6.98(dd,J＝14.9,3.1Hz,1H),6.79(t,J＝5.6Hz,1H),4.52(t,J＝14.8Hz,1H),3.67(s,3H),3.30(t,J＝13.8Hz,2H),3.06(t,J＝14.9Hz,2H),2.89–2.66(m,1H),2.56–2.34(m,2H),2.33–2.11(m,1H),1.72–1.16(m,8H).LR-MS(ESI)m/z715(M+1).
实施例110
Figure PCTCN2020096521-appb-000164
实施例110a
Figure PCTCN2020096521-appb-000165
将化合物1g(0.2g,0.555mmol)溶于N,N-二甲基甲酰胺(2mL)后加入N,N-二异丙基乙胺(0.197mL,1.11mmol)、N-羟基-7-偶氮苯并三氮唑(0.106g,0.776mmol)和1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(0.148g,0.776mmol)，室温反应20分钟后加入5-氨基戊酸甲酯(0.44g,0.832mmol)。在室温下搅拌4小时至反应完全。加入水淬灭反应，乙酸乙酯萃取后用水和饱和氯化钠溶液洗涤，合并有机相并经无水硫酸钠干燥，过滤并浓缩。通过快速层析法(硅胶0-7％二氯甲烷/甲醇梯度)分离出产物后将所得产物溶于二氧六环溶液(2mL)，加入2M的氢氧化钠溶液(0.2mL)，90℃反应4小时后用2M的盐酸酸化析出固体，抽滤后水洗干燥得到化合物110a。
将化合物110a(0.1g,0.218mmol)溶于N,N-二甲基甲酰胺(1mL)后加入N,N-二异丙基乙胺(0.076mL,0.435mmol)、N-羟基-7-偶氮苯并三氮唑(0.041g,0.305mmol) 和1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(0.058g,0.305mmol)，室温反应20分钟后加入97b(0.090g,0.261mmol)。在室温下搅拌4小时至反应完全。加入水淬灭反应，乙酸乙酯萃取后用水和饱和氯化钠溶液洗涤，合并有机相并经无水硫酸钠干燥，过滤并浓缩。通过快速层析法(硅胶0-7％二氯甲烷/石甲醇梯度)分离出产物110。
1H NMR(400MHz,CDCl 3)δ11.51(d,J＝19.4Hz,1H),9.54(d,J＝19.6Hz,1H),7.50(d,J＝7.6Hz,1H),7.45–7.39(m,1H),7.33(t,J＝7.7Hz,1H),7.22(q,J＝8.7Hz,3H),7.08–6.94(m,5H),6.85(d,J＝8.5Hz,2H),6.81(d,J＝8.6Hz,1H),6.34(d,J＝21.5Hz,1H),6.22(t,J＝5.4Hz,1H),4.98(dd,J＝12.0,5.5Hz,1H),3.60(s,3H),3.50(s,1H),3.38(d,J＝5.9Hz,2H),3.23(d,J＝8.2Hz,4H),2.91–2.71(m,3H),2.21(t,J＝6.9Hz,2H),1.75–1.49(m,9H).LR-MS(ESI)m/z 786(M+1).
实施例111
Figure PCTCN2020096521-appb-000166
除用5-氨基己酸甲酯替代5-氨基戊酸甲酯外，合成方法同实施例110。
1H NMR(400MHz,CDCl 3)δ11.43(s,1H),9.62(s,1H),7.51(d,J＝7.5Hz,1H),7.48–7.42(m,1H),7.36–7.31(m,1H),7.27–7.18(m,3H),7.06(d,J＝6.2Hz,3H),7.00(t,J＝7.4Hz,1H),6.95(s,1H),6.85(dd,J＝8.2,4.1Hz,3H),6.27(s,1H),6.10(s,1H),5.00(dd,J＝11.8,5.4Hz,1H),3.59(s,3H),3.42–3.15(m,6H),2.92–2.74(m,3H),2.15(dd,J＝14.5,7.6Hz,3H),1.70–1.44(m,9H),1.41–1.21(m,2H).LR-MS(ESI)m/z 800(M+1).
实施例112
Figure PCTCN2020096521-appb-000167
除用5-氨基庚酸甲酯替代5-氨基戊酸甲酯外，合成方法同实施例110。
1H NMR(600MHz,CDCl 3)δ11.23(s,1H),9.93(s,1H),8.02(s,1H),7.52–7.46(m,2H),7.34(td,J＝8.1,1.6Hz,1H),7.22(dd,J＝8.3,7.6Hz,4H),7.06(dd,J＝14.0,6.7Hz, 3H),6.99(t,J＝7.4Hz,1H),6.92(d,J＝8.6Hz,2H),6.88(s,1H),6.83(d,J＝7.8Hz,2H),6.28(t,J＝5.8Hz,1H),5.96(t,J＝5.8Hz,1H),4.97(dd,J＝12.3,5.4Hz,1H),3.61(s,3H),3.42–3.35(m,1H),3.34–3.30(m,1H),3.27(dt,J＝19.6,6.4Hz,3H),3.22–3.15(m,1H),2.82–2.71(m,2H),2.14(dt,J＝7.6,6.0Hz,4H),1.68–1.54(m,6H),1.29–1.18(m,4H).LR-MS(ESI)m/z 814(M+1).
实施例113
Figure PCTCN2020096521-appb-000168
除用4-氨甲基苯甲酸甲酯替代5-氨基戊酸甲酯，以及N-叔丁氧羰基-1,3-丙二胺替代N-叔丁氧羰基-1,4-丁二胺外，合成方法同实施例110。
1H NMR(400MHz,DMSO)δ12.85(s,1H),11.06(s,1H),9.26(t,J＝5.6Hz,1H),8.44(s,1H),8.32(s,1H),7.98–7.87(m,2H),7.59(t,J＝14.9Hz,1H),7.51–7.41(m,2H),7.41–7.30(m,3H),7.30–7.26(m,2H),7.26–7.22(m,1H),7.22–7.13(m,1H),7.13–7.04(m,4H),6.98(dd,J＝14.9,3.1Hz,1H),6.79(s,1H),5.01–4.81(m,1H),4.11(d,J＝5.6Hz,2H),3.67(s,3H),3.42(t,J＝10.9Hz,2H),3.35(t,J＝15.4Hz,2H),2.61–2.19(m,4H),1.99–1.78(m,2H).LR-MS(ESI)m/z 806(M+1).
实施例114
Figure PCTCN2020096521-appb-000169
除用4-氨甲基苯甲酸甲酯替代5-氨基戊酸甲酯，以及N-叔丁氧羰基-1,5-戊二胺替代N-叔丁氧羰基-1,4-丁二胺外，合成方法同实施例110。
1H NMR(400MHz,DMSO)δ12.85(s,1H),11.06(s,1H),9.26(t,J＝5.6Hz,1H),8.64(s,1H),8.31(s,1H),7.98–7.85(m,2H),7.59(t,J＝15.1Hz,1H),7.51–7.45(m,1H),7.44–7.34(m,3H),7.33–7.26(m,3H),7.26–7.22(m,1H),7.22–7.13(m,1H),7.13–7.03(m,3H),7.02–6.94(m,2H),6.79(s,1H),5.13–4.73(m,1H),4.11(d,J＝5.6Hz,2H),3.67(s,3H),3.43–3.10(m,4H),2.64–2.20(m,4H),1.73–1.45(m,4H),1.39–1.19(m,2H). LR-MS(ESI)m/z 834(M+1).
实施例115
Figure PCTCN2020096521-appb-000170
除用4-氨甲基苯甲酸甲酯替代5-氨基戊酸甲酯外，合成方法同实施例110。
1H NMR(400MHz,CDCl3)δ11.92(s,1H),9.33(s,1H),8.23(s,1H),7.60(s,2H),7.47(d,J＝6.8Hz,1H),7.43–7.35(m,1H),7.31(d,J＝7.2Hz,1H),7.21(t,J＝7.9Hz,4H),7.08(s,1H),7.00(t,J＝7.2Hz,4H),6.89(s,1H),6.84(d,J＝8.0Hz,2H),6.79(d,J＝8.6Hz,1H),6.20(t,J＝5.3Hz,1H),4.91(dd,J＝11.5,5.4Hz,1H),4.58(s,2H),3.43(s,3H),3.23(s,2H),2.84–2.62(m,3H),2.05(s,1H),1.77(s,2H),1.66(s,4H).LR-MS(ESI)m/z 820(M+1).
实施例116
Figure PCTCN2020096521-appb-000171
除用4-氨甲基苯甲酸甲酯替代5-氨基戊酸甲酯，以及N-叔丁氧羰基-1,6-己二胺替代N-叔丁氧羰基-1,4-丁二胺外，合成方法同实施例110。
1H NMR(400MHz,DMSO)δ12.85(s,1H),11.06(s,1H),9.26(t,J＝5.6Hz,1H),8.64(s,1H),7.96(s,1H),7.95–7.89(m,2H),7.59(t,J＝14.9Hz,1H),7.51–7.44(m,1H),7.44–7.37(m,2H),7.37–7.32(m,2H),7.31–7.26(m,2H),7.24(dd,J＝7.7,3.1Hz,1H),7.22–7.13(m,1H),7.13–7.04(m,4H),6.98(dd,J＝14.9,3.1Hz,1H),6.79(s,1H),5.07–4.80(m,1H),4.11(d,J＝5.6Hz,2H),3.67(s,3H),3.30(t,J＝14.5Hz,4H),2.63–2.21(m,4H),1.68–1.47(m,4H),1.46–1.19(m,4H).LR-MS(ESI)m/z 848(M+1).
实施例117
Figure PCTCN2020096521-appb-000172
实施例117a
Figure PCTCN2020096521-appb-000173
将化合物1g(0.2g,0.555mmol)溶于N,N-二甲基甲酰胺(2mL)后，加入N,N-二异丙基乙胺(0.197mL,1.11mmol)、N-羟基-7-偶氮苯并三氮唑(0.106g,0.776mmol)和1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(0.148g,0.776mmol)，室温反应20分钟后加入9-氨基-4,7-二氧杂壬酸叔丁酯(0.144g,0.832mmol)。在室温下搅拌4小时至反应完全。加入水淬灭反应，乙酸乙酯萃取后用水和饱和氯化钠溶液洗涤，合并有机相并经无水硫酸钠干燥，过滤并浓缩。通过快速层析法(硅胶0-7％二氯甲烷/甲醇梯度)分离出产物。将所得产物溶于4M的HCl的二氧六环溶液(5mL)中，60℃反应4小时后减压浓缩除去溶剂得到化合物117a。
将化合物117a(0.1g,0.192mmol)于N,N-二甲基甲酰胺(1mL)，然后滴加草酰氯(0.029g,0.231mmol)的二氯甲烷溶液(1mL)，室温反应4个小时后加入泊马度胺(0.105g,0.385mmol)，继续反应过夜后淬灭反应，乙酸乙酯萃取后用水和饱和氯化钠溶液洗涤，合并有机相并经无水硫酸钠干燥，过滤并浓缩。通过快速层析法(硅胶0-7％二氯甲烷/甲醇梯度)分离出产物117。
1H NMR(400MHz,DMSO)δ11.06(s,1H),10.01(s,1H),8.98(t,J＝7.4Hz,1H),8.31(s,1H),8.05(dd,J＝14.9,3.1Hz,1H),7.79(t,J＝14.9Hz,1H),7.48(dd,J＝14.8,3.0Hz,1H),7.45–7.37(m,3H),7.28(m,2H),7.23–7.13(m,1H),7.13–7.04(m,4H),4.60(t,J＝14.9Hz,1H),3.77–3.63(m,7H),3.52(s,4H),3.03(td,J＝21.9,7.4Hz,2H),2.87–2.65(m,1H),2.59–2.41(m,4H),2.38–2.15(m,1H).LR-MS(ESI)m/z 775(M+1).
实施例118
Figure PCTCN2020096521-appb-000174
除用12-氨基-4,7,10-三氧杂十二烷酸叔丁酯替代9-氨基-4,7-二氧杂壬酸叔丁酯外，合成方法同实施例117。
1H NMR(400MHz,DMSO)δ12.85(s,1H),11.06(s,1H),10.01(s,1H),8.98(t,J＝7.3Hz,1H),8.29(s,1H),8.05(dd,J＝14.9,3.1Hz,1H),7.79(t,J＝14.9Hz,1H),7.48(dd,J＝14.8,3.1Hz,1H),7.45–7.37(m,3H),7.30(dd,J＝12.7,2.3Hz,1H),7.27(dd,J＝12.6,2.3Hz,1H),7.23–7.13(m,1H),7.13–7.04(m,3H),6.42(s,1H),4.57(t,J＝15.0Hz,1H),3.78–3.62(m,7H),3.55–3.39(m,8H),3.03(td,J＝21.6,7.3Hz,2H),2.77–2.12(m,6H).LR-MS(ESI)m/z 819(M+1).
实施例119
除15-氨基-4,7,10,13-四氧杂十五烷酸叔丁酯替代9-氨基-4,7-二氧杂壬酸叔丁酯外，合成方法同实施例117。
Figure PCTCN2020096521-appb-000175
1H NMR(400MHz,DMSO)δ12.85(s,1H),11.06(s,1H),10.01(s,1H),8.98(t,J＝7.3Hz,1H),8.31(s,1H),8.05(dd,J＝14.9,3.1Hz,1H),7.79(t,J＝14.9Hz,1H),7.51–7.39(m,3H),7.39–7.28(m,2H),7.26(dd,J＝12.6,2.3Hz,1H),7.23–7.13(m,1H),7.13–7.02(m,4H),5.08–4.78(m,1H),3.77–3.62(m,7H),3.54–3.21(m,12H),3.12–2.96(m,2H),2.67–2.22(m,6H).LR-MS(ESI)m/z 863(M+1).
实施例120
Figure PCTCN2020096521-appb-000176
除用来那度胺替代泊马度胺外，合成方法同实施例118。
1H NMR(400MHz,DMSO)δ12.85(s,1H),11.06(s,1H),9.41(s,1H),8.98(t,J＝7.3Hz,1H),7.86(s,1H),7.68(dd,J＝13.8,4.2Hz,1H),7.62–7.49(m,2H),7.48–7.36(m,3H),7.30(dd,J＝12.6,2.3Hz,1H),7.27(dd,J＝12.7,2.3Hz,1H),7.23–7.16(m,2H),7.15–7.03(m,3H),5.15–4.77(m,1H),4.39(s,2H),3.77–3.59(m,7H),3.52(s,8H),3.04(t,J＝8.6Hz,2H),2.66–2.17(m,6H).LR-MS(ESI)m/z 805(M+1).
实施例121
Figure PCTCN2020096521-appb-000177
除用实施例81a化合物替代实施例1g化合物外，合成方法同实施例115。
1H NMR(400MHz,DMSO)δ12.83(s,1H),11.05(s,1H),9.25(t,J＝5.6Hz,1H),8.63(s,1H),8.29(s,1H),8.06–7.84(m,2H),7.58(t,J＝14.9Hz,1H),7.37–7.31(m,2H),7.31–7.21(m,3H),7.03–6.92(m,2H),6.88(s,1H),6.84(d,J＝16.0Hz,2H),6.78(t,J＝7.5Hz,1H),5.58–5.52(m,1H),5.51(s,1H),4.10(d,J＝5.6Hz,2H),3.67(s,3H),3.30(t,J＝9.9Hz,4H),2.71–2.43(m,1H),2.38–2.17(m,3H),2.15(s,6H),1.68–1.39(m,4H),1.35(s,6H).LR-MS(ESI)m/z 925(M+1).
实施例122
Figure PCTCN2020096521-appb-000178
除用来那度胺替代泊马度胺外，合成方法同实施例121。
1H NMR(400MHz,DMSO)δ12.85(s,1H),11.06(s,1H),9.26(t,J＝5.6Hz,1H),8.64(s,1H),8.28(s,1H),8.15–7.69(m,2H),7.38–7.30(m,3H),7.29–7.22(m,3H),6.98(t,J＝7.5Hz,2H),6.94(s,1H),6.90(s,1H),6.90–6.85(m,1H),6.79(s,1H),5.52(s,1H),5.15–4.75(m,1H),4.39(s,2H),4.11(d,J＝5.6Hz,2H),3.67(s,3H),3.42–3.21(m,4H),2.59–2.29(m,3H),2.15(s,6H),2.13–1.95(m,1H),1.71–1.38(m,4H),1.35(s,6H).LR-MS(ESI)m/z 911(M+1).
实施例123
Figure PCTCN2020096521-appb-000179
除用实施例106a化合物替代实施例1g化合物外，合成方法同实施例115。
1H NMR(400MHz,DMSO)δ12.85(s,1H),11.07(s,1H),10.01(s,1H),8.78(s,1H),7.96–7.87(m,2H),7.67(dd,J＝14.8,3.0Hz,1H),7.63–7.53(m,2H),7.40(s,1H),7.37–7.31(m,2H),7.25(dd,J＝15.1,3.0Hz,1H),7.09(dd,J＝15.0,2.9Hz,1H),6.98(dd,J＝15.0,3.1Hz,1H),6.29(t,J＝15.0Hz,1H),4.70(dt,J＝22.5,11.1Hz,1H),4.45(dd,J＝21.2,7.9Hz,1H),4.11(s,2H),3.67(s,3H),3.60(s,1H),3.36–3.24(m,4H),2.38–1.90(m,4H),1.66–1.40(m,4H),1.34(s,3H),1.33(s,3H).LR-MS(ESI)m/z 787(M+1).
实施例124
Figure PCTCN2020096521-appb-000180
除用实施例81a化合物替代实施例1g化合物外，合成方法同实施例119。
1H NMR(400MHz,DMSO)δ12.85(s,1H),11.06(s,1H),10.01(s,1H),8.98(t,J＝7.5Hz,1H),8.31(s,1H),8.05(dd,J＝14.9,3.1Hz,1H),7.79(t,J＝14.9Hz,1H),7.40(dd,J＝15.1,3.0Hz,1H),7.29(dd,J＝14.9,2.8Hz,1H),7.24(d,J＝2.9Hz,1H),7.11(s,1H),6.98(d,J＝14.9Hz,1H),6.84(d,J＝16.0Hz,2H),5.52(s,1H),5.09–4.62(m,1H),3.78–3.62(m,7H),3.54–3.20(m,12H),3.11–2.96(m,2H),2.71–2.23(m,6H),2.15(s,6H),1.35(s,6H)LR-MS(ESI)m/z 967(M+1).
实施例125
Figure PCTCN2020096521-appb-000181
除用来那度胺替代泊马度胺外，合成方法同实施例124。
1H NMR(400MHz,DMSO)δ12.85(s,1H),11.06(s,1H),10.01(s,1H),8.98(t,J＝7.5Hz,1H),8.20(s,1H),7.77(dd,J＝14.9,3.1Hz,1H),7.62(t,J＝14.9Hz,1H),7.31(t,J＝3.0Hz,1H),7.28(t,J＝3.0Hz,1H),7.24(d,J＝2.9Hz,1H),6.98(d,J＝15.6Hz,2H),6.79(d,J＝15.8Hz,2H),5.52(s,1H),5.37–5.20(m,1H),4.39(s,2H),3.78–3.63(m,7H),3.56–3.18(m,12H),3.04(t,J＝7.1Hz,2H),2.62–2.28(m,5H),2.15(s,6H),2.12–1.89(m,1H),1.35(s,6H).LR-MS(ESI)m/z 954(M+1).
实施例126
Figure PCTCN2020096521-appb-000182
除了用异丙醇替代4-氟-2,6-二甲基苯酚，合成方法同121.
1H NMR(400MHz,DMSO-d 6)δ12.21(s,1H),11.10(s,1H),8.92(t,J＝6.0Hz,1H),8.46(t,J＝5.7Hz,1H),7.80(d,J＝8.2Hz,2H),7.56(dd,J＝8.6,7.1Hz,1H),7.44–7.34(m,4H),7.21(s,1H),7.11(d,J＝8.6Hz,1H),7.06–6.98(m,2H),6.80(s,1H),6.59(t,J＝6.0Hz,1H),5.05(dd,J＝12.8,5.4Hz,1H),4.95(s,1H),4.54–4.44(m,3H),3.57(s,3H),3.33–3.25(m,3H),2.94–2.82(m,1H),2.71–2.54(m,2H),2.08–1.97(m,1H),1.65-1.56(m,4H),1.44(s,6H),1.13(d,J＝6.0Hz,6H).
实施例127
Figure PCTCN2020096521-appb-000183
除了用环丙醇替代4-氟-2,6-二甲基苯酚，合成方法同121.
1H NMR(400MHz,DMSO-d 6)δ12.24(s,1H),11.11(s,1H),8.91(t,J＝5.9Hz,1H),8.46(t,J＝5.7Hz,1H),7.80(d,J＝7.9Hz,2H),7.56(t,J＝7.8Hz,1H),7.45(d,J＝8.1Hz,1H),7.41–7.36(m,3H),7.33(d,J＝8.5Hz,1H),7.17(s,1H),7.11(d,J＝8.6Hz,1H),7.01(d,J＝7.0Hz,1H),6.68(s,1H),6.59(t,J＝6.1Hz,1H),5.05(dd,J＝12.8,5.4Hz,1H),4.97(s,1H),4.49(d,J＝5.7Hz,2H),3.83–3.72(m,1H),3.56(s,3H),3.32–3.25(m,3H),2.95–2.80(m,1H),2.70–2.53(m,2H),2.08–1.97(m,1H),1.70–1.55(m,4H),1.44(s,6H),0.78–0.70(m,2H),0.61–0.56(m,2H).
实施例128
Figure PCTCN2020096521-appb-000184
除了用苯酚替代4-氟-2,6-二甲基苯酚，合成方法同121.
1H NMR(400MHz,DMSO-d 6)δ12.30(s,1H),11.11(s,1H),8.95(t,J＝6.1Hz,1H),8.47(t,J＝5.2Hz,0H),7.81(d,J＝7.9Hz,2H),7.62–7.53(m,2H),7.48(dd,J＝8.6,2.4Hz,1H),7.38(d,J＝8.1Hz,2H),7.32–7.22(m,3H),7.12(d,J＝8.6Hz,1H),7.01(d,J＝7.3Hz,2H),6.97(d,J＝8.7Hz,1H),6.93(d,J＝2.1Hz,1H),6.84(d,J＝8.3Hz,2H),6.60(t,J＝5.7Hz,1H),5.11(s,1H),5.05(dd,J＝12.8,5.3Hz,1H),4.50(d,J＝5.7Hz,2H),3.52(s,3H),3.32–3.24(m,3H),2.95–2.81(m,1H),2.70–2.55(m,2H),2.07–2.00(m,1H),1.66–1.55(m,4H),1.48(s,6H).
实施例129
Figure PCTCN2020096521-appb-000185
除了用2，4-二氟苯酚替代4-氟-2,6-二甲基苯酚，合成方法同121.
1H NMR(400MHz,DMSO-d 6)δ12.32(s,1H),11.10(s,1H),8.93(t,J＝5.8Hz,1H),8.46(t,J＝5.7Hz,1H),7.81(d,J＝8.3Hz,2H),7.60–7.53(m,2H),7.45(dd,J＝8.6,2.4Hz,1H),7.42–7.34(m,3H),7.32(s,1H),7.14–7.07(m,2H),7.05–6.97(m,2H),6.90(s,1H),6.82(d,J＝8.5Hz,1H),6.59(t,J＝6.0Hz,1H),5.09(s,1H),5.05(dd,J＝12.8,5.4Hz,1H),4.50(d,J＝5.8Hz,2H),3.56(s,3H),3.33–3.26(m,3H),2.95–2.81(m,1H),2.64–2.53(m,2H),2.06–1.97(m,1H),1.61(s,4H),1.47(s,6H).
实施例130
Figure PCTCN2020096521-appb-000186
除了用化合物92b替代化合物110a以外，合成方法同115。
1H NMR(400MHz,DMSO-d 6)δ12.25(s,1H),11.11(s,1H),8.90(t,J＝5.9Hz,1H),8.46(t,J＝5.7Hz,1H),7.80(d,J＝7.9Hz,2H),7.56(t,J＝7.8Hz,1H),7.45(d,J＝8.1Hz,1H),7.41–7.36(m,3H),7.33(d,J＝8.5Hz,1H),7.17(s,1H),7.11(d,J＝8.6Hz,1H),7.01 (d,J＝7.0Hz,1H),6.68(s,1H),6.54(t,J＝6.1Hz,1H),5.05(dd,J＝12.8,5.4Hz,1H),4.97(s,1H),4.49(d,J＝5.7Hz,2H),3.70(s,3H),3.56(s,3H),3.32–3.26(m,3H),2.95–2.80(m,1H),2.71–2.53(m,2H),2.07–1.97(m,1H),1.70–1.55(m,4H),1.44(s,6H)
生物活性检测
试验一：分子水平酶的抑制实验
BRD4(1)/(2)分子水平抑制实验方法：
将化合物用DMSO配制成10mM母液，再稀释10倍得到1mM储液备用。然后用Binding Domain diluent buffer(#62DLBDDF，Cisbio)稀释10倍后(终体系中DMSO浓度为0.1％)，依据实验要求的起始浓度，1：5梯度稀释，共5个浓度。
用Binding Domain diluent buffer(#62DLBDDF，Cisbio)稀释BRD4(1)/(2)至5倍终浓度备用。用Binding Domain diluent buffer(#62DLBDDF，Cisbio)稀释[Lys(5,8,12,16)Ac]H4(1-21)-biotin(#64989，Cisbio)至5倍终浓度备用。
在384-well ProxiPlate(#6008280，PE)中加入4μL配制好的BRD4(1)/BRD(2)，然后转移2μL化合物到反应板中，最后在反应板中加4μL配制好的[Lys(5,8,12,16)Ac]H4(1-21)-biotin(#64989，Cisbio)。同时设置两组对照孔，分别为有BRD4对照(4μLBRD4(1)或者BRD4(2)+4μL配制好的Lys(5,8,12,16)Ac]H4(1-21)-biotin+2μL Binding Domain diluent buffer)，以及无BRD4对照(4μL配制好的Lys(5,8,12,16)Ac]H4(1-21)-biotin+6μL Binding Domain diluent buffer)。贴上贴膜，37℃孵育30min。
用Detection buffer配制SA-XL665(#610SAXLA，Cisbio)和MAb Anti-6HIS-Eu cryptate Gold(#61HI2KLB，Cisbio)或者MAb Anti FLAG M2-Eu cryptate(#61FG2KLA，Cisbio)的检测混合物，每孔加入10μL检测混合物，室温孵育3h，使用多功能酶标仪Envision读取mp值，参数设置如下：
Top mirror LANCE/DELFIA Dual/Bias(446)
Exc.Filter UV2(TRF)320
Ems.Filter APC 665
2nd Ems.Filter Europium 615
实验所得的ratio值＝665nm荧光值/615nm荧光值。
Figure PCTCN2020096521-appb-000187
IC 50值采用Prism软件以三参数法回归求得。
HDAC 1/6分子水平抑制实验方法
化合物粉末用DMSO配制成10mM母液，再稀释10倍得到1mM储液备用。然后用1倍Enzymatic buffer(50mM Tris-HCl pH 8.0,137mM NaCl,2.7mM KCl,1mM MgCl 2,0.01％Tween20)稀释10倍后(终体系中DMSO浓度为0.1％)，依据实验要求的起始浓度，1：5梯度稀释，共5个浓度。
用1倍Enzymatic buffer稀释HDAC1(#31504，Active Motif)/HDAC6(#31943，Active Motif)至5倍终浓度备用。用1倍Enzymatic buffer稀释Histone H3(1-21)lysine 9 acetylated biotinylated peptide(#AS-64361，AnaSpec)至2.5倍终浓度备用。
在384-well ProxiPlate(#6008280，PE)中加入2μL配制好的5倍HDAC1/HDAC6，然后加入4μL化合物到反应板中，最后在反应板中加4μL2.5倍Histone H3(1-21)lysine 9 acetylated biotinylated peptide(#AS-64361，AnaSpec)。同时设置两组对照孔，分别为有HDAC对照(2μLHDAC1或者HDAC6+4μL配制好的Histone H3(1-21)lysine 9 acetylated biotinylated peptide+4μL Enzymatic buffer)，以及无HDAC对照(4μL配制好的Histone H3(1-21)lysine 9 acetylated biotinylated peptide+6μL Enzymatic buffer)。贴上贴膜，37℃孵育1h。
用Detection buffer(10μM SAHA in KinEASEdetection buffer)配制SA-XL665(#610SAXLA，Cisbio)和anti-H3K9me0-Eu(K)(#61KB0KAD，Cisbio)的检测混合物，每孔加入10μL检测混合物，室温孵育0.5h，使用多功能酶标仪Envision读取mp值，参数设置如下：
Top mirror LANCE/DELFIA Dual/Bias(446)
Exc.Filter UV2(TRF)320
Ems.Filter APC 665
2nd Ems.Filter Europium 615
实验所得的ratio值＝665nm荧光值/615nm荧光值。
Figure PCTCN2020096521-appb-000188
IC 50值采用Prism软件以三参数法回归求得。
本发明实施例中制备的化合物以及阳性对照药SAHA(HDAC泛抑制剂)、阳性对照药 MS275(HDAC1选择性抑制剂)、阳性对照药OTX015(BRD4(1/2)泛抑制剂)、阳性对照药ABBV-744(BRD4(2)选择性抑制剂)对BRD4(1)/(2)以及HDAC1/6酶活数据见下表1至表2。
表1 对BRD4(1)/(2)酶活IC 50(nM)
Figure PCTCN2020096521-appb-000189
Figure PCTCN2020096521-appb-000190
表2 对HDAC(1)/(6)酶活IC 50(nM)
Figure PCTCN2020096521-appb-000191
Figure PCTCN2020096521-appb-000192
Figure PCTCN2020096521-appb-000193
实验结论：在生物活性评价中，由表1可知，本发明实施例的HDAC和BRD4双靶吡咯并吡啶酮类化合物(实施例1-106化合物)都具有较好的BRD4活性。部分化合物表现出对BRD4的两个作用区域BD1和BD2的选择性，不同亚型选择性的HDAC抑制剂SAHA,MS275和ACY1215均不具有BRD4抑制活性。
由表2可知，本发明的实施例1-106化合物在具有BRD4活性的同时也具有一定的HDAC抑制活性，而相应的作为阳性对照的BRD4抑制剂ABBV-744和OTX-015不具有HDAC抑制活性。
试验例二：蛋白质免疫印迹(Western Blot)检测化合物对MV-4-11细胞中HDAC和BRD4信号通路的影响
实验方法
将对数生长期MV-4-11细胞接种于12孔培养板中。待细胞贴壁过夜后，分别加入化合物(10，100，1000nM)处理细胞24h。用预冷PBS(含1mM钒酸钠)冲洗3遍后，加入1xSDS凝胶上样缓冲液(配方为：50mM Tris-Cl(pH6.8)，100mM DTT，2％SDS，10％(v/v)甘油，0.1％(w/v)溴酚蓝)裂解细胞。细胞裂解物于沸水浴中加热15min放入-20℃保存。
蛋白样品置于密度SDS-聚丙烯酰胺凝胶中，在Tris-甘氨酸-SDS电泳缓冲液[25mmol/L Tris，250mmol/L甘氨酸(pH8.3)，0.1％SDS]中以80V电泳约20min进行压缩，随后改用120V电泳约2h进行分离。电泳结束后用半干印迹法将蛋白从凝胶转移至硝酸纤维素滤膜，转移缓冲液配方为192mmol/L甘氨酸、25mmol/L Tris、20％甲醇，按所需蛋白分子量大小转移约1h。用丽春红(Ponceau S)染色确定转移情况和蛋白条带位置，依据蛋白Marker分子量裁剪相应目的条带，然后用封闭液(含3％BSA的TBST)室温封闭90min，与相应的抗体于4℃孵育过夜。次日，用TBST洗涤液[20mmol/L Tris-HCl(pH 7.2-7.4，室温)，150mmol/L NaCl，0.1％(v/v)Tween20]室温洗涤3次，每次10min。加入用封闭液稀释的辣根过氧化物酶标记的二抗(1：2000)，室温孵育1h。 然后用TBST漂洗三次，每次10min。根据曝光强度选择合适的发光试剂显色，发光试剂分别有ECL Plus western blotting detection system和Advance ECL western blotting detection system，以及Super Signal West Pico Chemiluminescent Substrate。
实验结论：实施例84和80，实施例96和81以及实施例9和100化合物在MV-4-11细胞株上对HDAC和BRD4信号通路的影响见图1。
由图可知，实施例84和80化合物能够剂量依赖的上调组蛋白H3的乙酰化水平，但是对微管蛋白的乙酰化水平影响不大，其作用效果和HDAC1选择性抑制剂MS275相当，说明该类化合物是HDAC1选择性抑制剂；同时化合物能够剂量依赖的下调c-Myc的表达，作用效果和BRD4抑制剂ABBV-744类似。
实施例96化合物能够同时剂量依赖的上调组蛋白H3和微管蛋白的乙酰化水平，与泛HDAC抑制剂SAHA作用效果类似，说明该化合物具有泛HDAC抑制活性。同时该化合物能够剂量依赖的下调c-Myc的表达，作用效果和BRD4抑制剂ABBV-744类似。
实施例100化合物能够剂量依赖的上调微管蛋白的乙酰化水平，但是对组蛋白H3乙酰化水平影响不大，其作用效果和选择性HDAC6抑制剂ACY1215类似，说明该化合物具有一定的HDAC6选择性抑制活性。同时该化合物能够剂量依赖的下调c-Myc的表达，作用效果和BRD4抑制剂ABBV-744类似。
综上所述，通过在MV-4-11细胞株上免疫印迹实验，证明实施例84和80化合物是选择性HDAC1和BRD4双靶抑制剂，实施例96化合物是泛HDAC和BRD4双靶抑制剂，实施例100化合物是选择性HDAC6和BRD4双靶抑制剂。
试验例三：细胞水平抑制试验
实验方法
细胞的生长抑制检测采用CCK-8方法。
处于对数生长期的细胞按合适密度接种至96孔培养板(#3599，Corning)，每孔90μL培养过夜。
10mM的待测化合物用DMSO以1：3梯度稀释后，吸取2μl加入到198μl的无菌PBS中稀释100倍待用。将稀释好的化合物吸取10μl加入到细胞板作用72h，每个浓度设三复孔，并设相应浓度的溶媒对照及无细胞调零孔。作用结束后，每孔加入10μL CCK-8，培养箱中孵育4h后，SpectraMax 190酶标仪测定450nm波长下的光密度(OD值)。
化合物抑制率和IC 50值采用酶标仪随机附带软件以四参数法回归求得。
实施例化合物以及阳性对照药SAHA、阳性对照药MS275、阳性对照药OTX015、阳性对照药ABBV-744对不同肿瘤细胞株抑制活性见表3：
表3：对不同细胞株细胞抑制IC 50(μM)
Figure PCTCN2020096521-appb-000194
N.T.是指未测定。
实验结论：由表3可知，泛HDAC和BRD4双靶抑制剂实施例7和实施例96化合物在人急性单核细胞白血病细胞株MV-4-11及人骨髓增生异常综合征细胞株SKM-1显示出了强抑制活性，优于阳性对照HDAC抑制剂SAHA和ABBV-744。
HDAC1和BRD4双靶抑制剂实施例10，实施例80和实施例84化合物较好地抑制人急性单核细胞白血病细胞株MV-4-11及人骨髓增生异常综合征细胞株SKM-1细胞增殖，同时在人胰腺癌MIA Paca2细胞株上表现出较好的抑制活性，强于阳性化合物ABBV-744和MS275。
靶向泛素化降解BRD4蛋白的实施例115，实施例127和实施例128化合物在MV-4-11细胞株上表现出较强的抑制细胞增殖活性，强于BET抑制剂ABBV-744，和阳性对照d-BET6相当。
试验例四：免疫印迹杂交(Western Blot)检测化合物BRD4蛋白降解作用
实验方法
将对数生长期MV-4-11细胞接种于12孔培养板中，分别加入不同浓度化合物(10nM，100nM，1000nM)处理细胞3h。将每孔细胞吹匀后收集到1.5ml离心管中，做好标记，450g离心5分钟，弃上清。加入1ml预冷PBS(含1mM钒酸钠)清洗三遍，每次均离心弃上清。，最后加入1xSDS凝胶上样缓冲液(配方为：50mM Tris-Cl(pH6.8)，100mM DTT，2％SDS，10％(v/v)甘油，0.1％(w/v)溴酚蓝)裂解细胞。细胞裂解物于沸水浴中加热10min。取上述细胞裂解液进行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上。转移结束后，用丽春红(Ponceau S)染色确定转移情况和蛋白条带在硝酸纤维素膜上的位置，标记后用含3％BSA的封闭液[5％BSA，20mM Tris-HCl pH7.2-7.4，150mM NaCl，0.1％Tween-20]于摇床室温封闭60min。然后，将膜置于抗体稀释液(3％BSA)稀释的一抗中4℃过夜。用洗液[100mM Tris-HCl pH7.2-7.4，0.9％NaCl，0.2％Tween-20]室温洗涤三次，每次10min。加入辣根过氧化物酶标记的二抗，室温置于摇床上平缓摇动1h。再用洗液洗涤三次后，根据曝光强度选择合适的发光试剂显色。
时间依赖降解的实验方案：将对数生长期MV-4-11细胞接种于6孔培养板中，分别于收样时间点前1h,3h,6h,12h,24h加入化合物处理细胞。收样时，将每孔细胞吹匀后收集到2ml离心管中，做好标记，450g离心5分钟，弃上清。加入1ml预冷PBS(含1mM钒酸钠)清洗三遍，每次均离心弃上清。最后加入1xSDS凝胶上样缓冲液(配方为：50mM Tris-Cl(pH6.8)，100mM DTT，2％SDS，10％(v/v)甘油，0.1％(w/v)溴酚蓝)裂解细胞。细胞裂解物于沸水浴中加热15min放入-20℃保存。取上述细胞裂解液进行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上。转移结束后，用丽春红(Ponceau S)染色确定转移情况和蛋白条带在硝酸纤维素膜上的位置，标记后用含3％BSA的封闭液[5％BSA，20mM Tris-HCl pH7.2-7.4，150mM NaCl，0.1％Tween-20]于摇床室温封闭60min。然后，将膜置于抗体稀释液(3％BSA)稀释的一抗中4℃过夜。用洗液[100mM Tris-HCl pH7.2-7.4，0.9％NaCl，0.2％Tween-20]室温洗涤三次，每次10min。加入辣根过氧化物酶标记的二抗，室温置于摇床上平缓摇动1h。 再用洗液洗涤三次后，根据曝光强度选择合适的发光试剂显色。
实验结论：不同浓度的(10nM，100nM，1000nM)代表化合物3h时对BRD4蛋白降解结果见图2。由图可知，实施例111，112，115，127和128化合物在3h时能够起到降解BRD4蛋白的作用，其中实施例115化合物在3h时对BRD4-Short的降解作用不明显。
化合物在100nM浓度条件下在不同时间点对BRD4的降解作用结果见图3。由图可知，实施例115化合物对BRD4蛋白的降解作用随着时间的延长而逐渐增加，24h基本完全降解；和阳性对照dBET6类似，实施例128化合物在6h完全降解了BRD4后出现对BRD4-Short的反弹。随着时间的增加，实施例115和实施例128化合物对其下游蛋白c-Myc出现持续的下调作用。
以上结合附图详细描述了本发明的优选实施方式，但是，本发明并不限于上述实施方式中的具体细节，在本发明的技术构思范围内，可以对本发明的技术方案进行多种简单变型，这种简单变型均属于本发明的保护范围。

Claims (10)

  1. 一种式I化合物或其药学上可接受的盐：
    Figure PCTCN2020096521-appb-100001
    其中，
    R 1选自
    Figure PCTCN2020096521-appb-100002
    Figure PCTCN2020096521-appb-100003
    其中，R a表示一个或多个取代基，各自独立地选自氢、卤素、羟基、氰基、烷基、任选被烷基取代的氨基、卤代烷基、烷氧基，
    X选自亚烷基、杂亚烷基、亚烯基、亚杂芳基、(亚烷基) 0或1-亚芳基-(亚烷基) 0 或1、亚烷基-CONH-亚烷基、亚环烷基、亚环烯基、亚杂环基、亚杂环烯基；所述亚芳基、亚杂芳基、(亚烷基) 0或1-亚芳基-(亚烷基) 0或1、亚环烷基、亚环烯基、亚杂环基或亚杂环烯基任选被环烯基、杂环基、杂环烯基、羟基、硝基、氰基、任选被烷基取代的氨基、烷氧基、烷基，烯基、芳基、杂芳基、环烷基、杂链烃基取代，
    A选自芳基、杂芳基；
    R 2表示一个或多个取代基，各自独立地选自氢、＝O、卤素、羟基、氰基、烷基、卤代烷基、烷氧基、环烷基氧基、芳氧基、羟烷基、-(R b)NS(O) 2R c、-S(O) 2R d、R eO(C＝O)-、R fR gN-C 1-10烷基、杂环基烷基、环烷基烷氧基，其中，R b、R c、R d、R e、R f和R g各自独立地为氢、烷基或卤代烷基；其中所述烷氧基、环烷基氧基、芳氧基、杂环基烷基和环烷基烷氧基任选各自独立地被1、2、3、4或5个选自＝O、卤素、羟基、氰基、烷基、卤代烷基、环烷基的基团取代；
    或者，R 2表示两个或两个以上取代基，其中两个相邻的取代基连接，并与所连接的A上的碳原子一起形成环烷、杂环、芳环、杂芳环；所述环烷、杂环、芳环、杂芳环任选被卤素、＝O、羟基、氰基、烷基、卤代烷基、烷氧基、胺基、磺酰基、烷基磺酰基、 烷基磺酰氨基取代。
  2. 根据权利要求1所述的式I化合物或其药学上可接受的盐，其中，所述“卤素”选自氟、氯、溴或碘；
    所述“烷基”、“被烷基取代的氨基”、“卤代烷基”、“烷氧基”、“羟烷基”、“杂环基烷基”、“环烷基烷氧基”中的烷基各自独立地为C 1-C 20直链或支链烷基，可选地为C 1-C 15直链或支链烷基，可选地为C 1-C 10直链或支链烷基，可选地为C 1-C 7直链或支链烷基，可选地为C 1-C 6直链或支链烷基，可选地为C 1-C 5直链或支链烷基，可选地C 1-C 4直链或支链烷基，可选地，选自甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、仲丁基、正戊基，1-甲基丁基、2-甲基丁基、3-甲基丁基、异戊基、1-乙基丙基、新戊基、正己基、1-甲基戊基、2-甲基戊基、3-甲基戊基、异己基、1,1-二甲基丁基、2,2-二甲基丁基、3,3-二甲基丁基、1,2-二甲基丁基、1,3-二甲基丁基、2,3-二甲基丁基、2-乙基丁基、正庚基、2-甲基己基、3-甲基己基、2,2-二甲基戊基、3,3-二甲基戊基、2,3-二甲基戊基、2,4-二甲基戊基、3-乙基戊基或2,2,3-三甲基丁基；可选地，选自甲基、乙基、丙基、异丙基、丁基或异丁基；
    所述“亚烷基”、“(亚烷基) 0或1-亚芳基-(亚烷基) 0或1”和“亚烷基-CONH-亚烷基”中的亚烷基各自独立地为C 1-C 20直链或支链亚烷基，可选地为C 1-C 15直链或支链亚烷基，可选地为C 1-C 10直链或支链亚烷基，可选地为C 1-C 8直链或支链亚烷基，可选地为C 1-C 6直链或支链亚烷基，可选地为C 1-C 4直链或支链亚烷基，可选地为C 1-C 3直链或支链亚烷基，可选地为C 3-C 6直链或支链亚烷基；
    所述“杂亚烷基”为含有1个或多个选自O、S或N的杂原子的C 1-C 20直链或支链亚烷基，可选地为含有1-6个选自O、S或N的杂原子的C 1-C 20直链或支链亚烷基，可选地为含有1-4个选自O、S或N的杂原子的C 1-C 20直链或支链亚烷基，可选地为含有1-6个选自O或S的C 1-C 20直链或支链亚烷基，可选地为含有1-4个选自O或S的C 1-C 20直链或支链亚烷基，可选地为含有1-6个O原子的C 1-C 20直链或支链亚烷基，可选地为含有1-4个O原子的C 1-C 20直链或支链亚烷基；可选地碳原子数为1-15个，可选地为1-10个，可选地为1-8个，可选地为1-6个；杂亚烷基为例如–(CH 2CH 2O) n–、–(OCH 2CH 2) m–、–(CH 2CH 2O) lCH 2CH 2–、–(OCH 2) k–、–(CH 2O) i–、–(CH 2O) jCH 2–、C 1-C 20亚烷氧基，其中n、m、l、k、i、j各自独立地为1-6的整数，可选地为1-4的整数，可选地为1-3的整数，可选地为1或2；可选地杂亚烷基为例如–(CH 2CH 2O) 4CH 2CH 2–、–(CH 2CH 2O) 3CH 2CH 2–、–(CH 2CH 2O) 2CH 2CH 2–或C 1-C 10亚烷氧基，或者C 1-C 8亚烷氧基， 或者C 1-C 6亚烷氧基，或者C 1-C 4亚烷氧基；
    所述“亚烯基”为C 2-C 20直链或支链亚烯基，可选地为C 2-C 15直链或支链亚烯基，可选地为C 2-C 10直链或支链亚烯基，可选地为C 2-C 8直链或支链亚烯基，可选地为C 2-C 6直链或支链亚烯基，可选地为C 2-C 4直链或支链亚烯基，可选地所述亚烯基为-CH＝CH-、-CH＝CHCH 2-、-CH 2CH＝CH-或-CH 2CH＝CHCH 2-；
    所述“烯基”为C 2-C 20直链或支链烯基，可选地为C 2-C 15直链或支链烯基，可选地为C 2-C 10直链或支链烯基，可选地为C 2-C 8直链或支链烯基，可选地为C 2-C 6直链或支链烯基，可选地为C 2-C 4直链或支链烯基，可选地所述烯基为CH 2＝CH-、CH 2＝CHCH 2-、CH 3CH＝CH-或CH 3CH＝CHCH 2-；
    所述“亚芳基”、“(亚烷基) 0或1-亚芳基-(亚烷基) 0或1”中的亚芳基为6-10元亚芳基；可选为亚苯基或亚萘基；
    所述“芳基”为6-10元芳基；可选为苯基或萘基；
    所述“芳环”为6-10元芳环；可选为苯环或萘环；
    所述“亚杂芳基”为含有1-3个选自N、O和S中的杂原子的5-10元亚杂芳基；可选地，为含有1-2个选自N、O和S中的杂原子的5-10元亚杂芳基；可选地，所述亚杂芳基选自亚吡啶基、亚吡咯基、亚嘧啶基、亚吡嗪基、亚哒嗪基、亚噻吩基、亚呋喃基；
    所述“杂芳环”、“杂芳基”中的环为含有1-3个选自N、O和S中的杂原子的5-10元杂芳环；可选地，为含有1-2个选自N、O和S中的杂原子的5-10元杂芳环；可选地，所述环选自吡啶环、吡咯环、嘧啶环、吡嗪环、哒嗪环、噻吩环、呋喃环；
    所述“(亚烷基) 0或1-亚芳基-(亚烷基) 0或1”优选为亚苯乙基、亚苄基、亚乙苯基、亚甲苯基；
    所述“亚环烷基”为C 3-C 10单环、双环、三环、或四环亚环烷基；可选地，所述亚环烷基选自亚环丙基、亚环丁基、亚环戊基、亚环己基、亚环庚基及亚环辛基；
    所述“环烷”、“环烷基”的环烷为C 3-C 10单环、双环、三环、或四环环烷；可选地，所述环烷选自环丙烷、环丁烷、环戊烷、环己烷、环庚烷及环辛烷；
    所述“亚环烯基”为含一或多个双键的部分不饱和单环、双环、三环、或四环C 3-C 12亚环烯基；可选地为亚环丙烯基、亚环丁烯基、亚环戊烯基、亚环己烯基、亚环庚烯基或亚环辛烯基；
    所述“环烯基”为含一或多个双键的部分不饱和单环、双环、三环、或四环C 3-C 12环烯基；可选地为环丙烯基、环丁烯基、环戊烯基、环己烯基、环庚烯基或环辛烯基；
    所述“杂环”、“杂环基”、“亚杂环基”中的杂环为环上含有1个、2个或3个选自N、O、S的杂原子的3-10元非芳香杂环，可选地，所述杂环为环上含有1个或2个选自N、O的杂原子的3-6元非芳香环；
    所述“亚杂环烯基”为环上含有1个、2个或3个选自N、O、S的杂原子且含有1个或多个双键的3-10元非芳香亚杂环烯基，可选地，为环上含有1个或2个选自N、O的杂原子的3-6元非芳香亚杂环烯基；
    所述“杂环烯基”为环上含有1个、2个或3个选自N、O、S的杂原子且含有1个或多个双键的3-10元非芳香杂环烯基，可选地，为环上含有1个或2个选自N、O的杂原子的3-6元非芳香杂环烯基；
    所述“杂链烃基”为链上含1-6个选自N、O、S的杂原子的直链或支链饱和或不饱和C 1-C 20杂链烃结构；可选地，为链上含1个、2个、3个或4个选自N、O的杂原子的直链或支链饱和或不饱和C 1-C 20杂链烃结构，可选地，为链上含1个、2个、3个或4个O的直链或支链饱和或不饱和C 1-C 20杂链烃结构，可选地，为链上含1个、2个、3个或4个O的直链或支链饱和或不饱和C 1-C 10杂链烃结构。
  3. 根据权利要求1或2所述的式I化合物或其药学上可接受的盐，其中，
    R a表示一个或多个取代基，各自独立地选自氢或卤素，
    X选自C 1-C 10亚烷基、C 1-C 10亚烷基-CONH-C 1-C 10亚烷基、–(CH 2CH 2O) n–、–(OCH 2CH 2) m–、–(CH 2CH 2O) lCH 2CH 2–、–(OCH 2) k–、–(CH 2O) i–、–(CH 2O) jCH 2–、C 1-C 10亚烷氧基、(C 1-C 10亚烷基) 0或1-亚芳基-(C 1-C 10亚烷基) 0或1，其中n、m、l、k、i、j各自独立地为1-6的整数；
    A选自苯基，
    R 2表示1、2、3、4或5个取代基，各自独立地选自氢、＝O、卤素、羟基、氰基、C 1-6烷基、卤代C 1-6烷基、C 1-6烷氧基、C 3-6环烷基氧基、C 6-10芳氧基、羟基C 1-6烷基、-(R b)NS(O) 2R c、-S(O) 2R d、R eO(C＝O)-、R fR gN-C 1-10烷基、C 5-10杂环基C 1-6烷基、C 3-6环烷基C 1-6烷氧基，其中，R b、R c、R d、R e、R f和R g各自独立地为氢、C 1-6烷基或卤代C 1-6烷基；其中所述C 1-6烷氧基、C 3-6环烷基氧基、C 6-10芳氧基、C 5-10杂环基C 1-6烷基和C 3-6环烷基C 1-6烷氧基任选各自独立地被1、2、3、4或5个选自卤素、羟基、氰基、C 1-6烷基、卤代C 1-6烷基、C 3-6环烷基的基团取代；
    或者，R 2表示两个或两个以上取代基，其中两个相邻的取代基连接，并与所连接的A环上的碳原子一起形成杂环，所述杂环优选为[1,4]恶嗪环；所述杂环任选被＝O、烷基、 磺酰基、烷基磺酰基取代。
  4. 根据权利要求1所述的式I化合物或其药学上可接受的盐，其中，所述式I化合物选自以下化合物：
    Figure PCTCN2020096521-appb-100004
    Figure PCTCN2020096521-appb-100005
    Figure PCTCN2020096521-appb-100006
    Figure PCTCN2020096521-appb-100007
    Figure PCTCN2020096521-appb-100008
    Figure PCTCN2020096521-appb-100009
    Figure PCTCN2020096521-appb-100010
    Figure PCTCN2020096521-appb-100011
    Figure PCTCN2020096521-appb-100012
    Figure PCTCN2020096521-appb-100013
    Figure PCTCN2020096521-appb-100014
    Figure PCTCN2020096521-appb-100015
    Figure PCTCN2020096521-appb-100016
    Figure PCTCN2020096521-appb-100017
  5. 根据权利要求1-4任一项所述的式I化合物或其药学上可接受的盐，其中，所述药学上可接受的盐包括所述式I化合物的阴离子盐和阳离子盐；
    优选地，所述药学上可接受的盐包括所述式I化合物的碱金属的盐、碱土金属的盐、铵盐；优选地，所述碱金属包括钠、钾、锂、铯，所述碱土金属包括镁、钙、锶；
    优选地，所述药学上可接受的盐包括所述式I化合物与有机碱形成的盐；优选地，所述有机碱包括三烷基胺、吡啶、喹啉、哌啶、咪唑、甲基吡啶、二甲氨基吡啶、二甲基苯胺、N-烷基吗啉、1,5-二氮杂双环[4.3.0]壬烯-5(DBN)、1,8-二氮杂双环[5.4.0]十一碳烯-7(DBU)、1,4-二氮杂双环[2.2.2]辛烷(DABCO)；优选地，所述三烷基胺包括三甲胺、三乙胺、N-乙基二异丙胺；优选地，所述N-烷基吗啉包括N-甲基吗啉；
    优选地，所述药学上可接受的盐包括所述式I化合物与酸形成的盐；优选地，所述酸包括无机酸、有机酸；优选地，所述无机酸包括盐酸、氢溴酸、氢碘酸、硫酸、硝酸、磷酸、碳酸；优选地，所述有机酸包括甲酸、乙酸、丙酸、草酸、丙二酸、琥珀酸、富马酸、马来酸、乳酸、苹果酸、柠檬酸、枸橼酸、酒石酸、碳酸、苦味酸、甲磺酸、乙磺酸、对甲苯磺酸、谷氨酸、双羟萘酸。
  6. 制备权利要求1-5任一项所述的式I化合物或其药学上可接受的盐的方法，
    Figure PCTCN2020096521-appb-100018
    其特征在于，包括：
    将式II化合物与NH 2X-R 1发生缩合反应，得到式I化合物，
    Figure PCTCN2020096521-appb-100019
    其中，式I中，A、R 2、X、R 1如权利要求1-5任一项中所定义；
    式II中，P为氢或保护基团(例如苄基、对甲苯磺酰基、(三甲基甲硅烷基)乙氧基)甲基)，Q为氢或C 1-C 10烷基；A和R 2如权利要求1-5任一项中所定义；
    式NH 2X-R 1中，X、R 1如权利要求1-5任一项中所定义；
    或者，其特征在于，包括：
    将式III化合物与式NH 2R 01发生缩合反应，得到式I化合物，
    Figure PCTCN2020096521-appb-100020
    其中，
    式I中，R 1为
    Figure PCTCN2020096521-appb-100021
    A、R 2、X、R a如权利要求1-5任一项中所定义；
    式III中，A、R 2、X如权利要求1-5任一项中所定义，M为氢或C 1-C 10烷基； 式NH 2R 01中，R 01为羟基、
    Figure PCTCN2020096521-appb-100022
    R a如权利要求1-5任一项中所定义；
    或者，其特征在于，包括：将式IV化合物与NH 2R 02发生缩合反应，得到式I化合物，
    Figure PCTCN2020096521-appb-100023
    其中，式I中，A、R 2、R 1如权利要求1-5任一项中所定义；X为亚烷基-CONH-亚烷基，
    式IV中，A、R 2如权利要求1-5任一项中所定义；M为氢或C 1-C 10烷基；X 1为亚烷基；
    式NH 2R 02中，R 02为亚烷基-R 1，R 1如权利要求1-5任一项中所定义；
    优选地，所述式III化合物或式IV化合物的制备方法包括：
    将式II化合物与式H 2NXCOOM化合物或式H 2NX 1COOM化合物发生缩合反应，得到式III化合物或式IV化合物；
    式H 2NXCOOM中，X定义同式III中的定义；
    式H 2NX 1COOM中，X 1定义同式IV中的定义；
    优选地，式II化合物的制备方法包括：将式V化合物与芳基硼酸发生Suzuki偶联反应得到式II化合物，
    或者，
    将式V化合物通过Suzuki偶联反应得到式VI的芳基硼酸酯化合物，然后将式VI的芳基硼酸酯化合物与溴代物发生Suzuki偶联反应得到式II化合物，
    Figure PCTCN2020096521-appb-100024
    式V中，PG表示保护基团(例如苄基、对甲苯磺酰基、(三甲基甲硅烷基)乙氧基)甲基)，Y表示卤素，Q为氢或C 1-C 10烷基；
    式VI中，PG和Q的定义与式V中相同；
    所述芳基硼酸为式VII化合物：
    Figure PCTCN2020096521-appb-100025
    其中，A和R 2的定义与式II中的相同；
    所述溴代物为式VIII化合物：
    Figure PCTCN2020096521-appb-100026
    其中，A和R 2的定义与式II中的相同；
    优选地，所述式V化合物的制备方法包括：
    将式XIV化合物与N,N-二甲基甲酰胺二甲基缩醛反应得到式XIII化合物，所得式XIII化合物在酸性条件下还原得到式XII化合物，式XII化合物在强碱条件下和卤化物反应得到式XI化合物，所得式XI化合物在强碱条件下与氯甲酸烷基酯反应得到式X化合物，对所得式X化合物用酸处理得到式IX化合物，在碱性条件和溶剂中用甲基化试剂将式IX化合物甲基化得到式V化合物；
    Figure PCTCN2020096521-appb-100027
    式IX至式XIV中，Y、PG和Q的定义与式V中相同。
  7. 根据权利要求6所述的方法，其中，所述缩合反应在碱性条件下和缩合剂的存在下，在溶剂中进行；优选地，所述缩合剂包括：N,N'-羰基二咪唑、二环己基碳二亚胺、O-(7-氮杂苯并三唑-1-基)-N,N,N',N'-四甲基脲、N-羟基-7-偶氮苯并三氮唑、2-(1H-苯并三偶氮L-1-基)-1,1,3,3-四甲基脲四氟硼酸酯或1-羟基苯并三唑；优选地，所述碱性条件所用的碱包括三乙胺、二异丙基乙胺、DMAP中的一种或者两种以上；所述溶剂包括：四氢呋喃、二氯甲烷或N,N-二甲基甲酰胺；
    优选的，所述Suzuki偶联反应在钯催化剂的存在下在碱性条件下于溶剂中进行，任选地在配体的存在下进行；优选地，所述Suzuki偶联反应在60℃至150℃的温度下进行，优选地，该反应通过微波辐射促发；优选地，所述碱性条件所用的碱包括CsF、Cs 2CO 3、K 2CO 3、三乙胺、二异丙基乙胺、DMAP中的一种或者两种以上；优选地，所述钯催化剂包括：Pd 2(dba) 3、Pd(OAc) 2或Pd(PPh 3) 4；优选地，所述配体包括：1,3,5,7-四甲基-6-苯基-2,4,8-三氧杂-6-磷酰金刚烷、2-二环己基磷-2’,4’,6’-三异丙基联苯、1,1’-双(二苯基磷烷基)二茂铁；优选地，所述溶剂包括：甲醇、二甲氧基乙烷、N,N-二甲基甲酰胺、二甲亚砜、二氧杂环己烷、四氢呋喃和水，或其混合物；
    优选地，式XIV化合物与N,N-二甲基甲酰胺二甲基缩醛的反应60℃至100℃的温度下)进行；优选地在溶剂中进行，所述溶剂包括：N,N-二甲基甲酰胺；
    优选地，所述式XIII化合物在酸性条件下的还原反应中，所述酸包括：氯化铵或乙酸；还原反应所用的还原剂包括还原铁粉；
    优选地，所述式XII化合物在强碱条件下和卤化物的反应中，所述强碱包括氢化钠；
    优选地，所得式XI化合物在强碱条件下与氯甲酸烷基酯的反应中，所述强碱包括二 异丙基氨基锂；
    优选地，所述式X化合物用酸处理步骤中，在溶剂中于40℃至100℃的温度下进行，优选的，所述酸包括盐酸、氢溴酸；所述溶剂包括二氧六环、水；
    优选地，在式IX化合物甲基化反应，在溶剂中于40℃至100℃的温度下进行，优选地，甲基化试剂为碘甲烷；优选的，所述碱性条件所用的碱包括氢化钠、碳酸铯或碳酸钾；所述溶剂包括N，N-二甲基甲酰胺或二甲亚砜。
  8. 一种药物组合物，包括权利要求1-5任一项所述的式I化合物或其药学上可接受的盐和药学上可接受的辅料。
  9. 权利要求1-5任一项所述的式I化合物或其药学上可接受的盐在制备用于预防或治疗肿瘤的药物中的用途。
  10. 根据权利要求9所述的用途，其特征在于，所述肿瘤包括非小细胞肺癌、乳腺癌、甲状腺癌(甲状腺髓样癌、乳头状甲状腺癌)、胃癌、膀胱癌、子宫内膜癌、前列腺癌、宫颈癌、结肠癌、食管癌、角质母细胞瘤、骨髓瘤、横纹肌肉瘤、急性白血病、肝癌、腺癌或胰腺癌。
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