含吡唑酮基团的喹唑啉衍生物的合成及其作为EGFR/VEGFR-2双靶标酪氨酸激酶抑制剂

Synthesis of Quinazoline Derivatives Possessing Pyrazolone groups as Dual EGFR/VEGFR-2 Receptor Tyrosine Kinase Inhibitors

双靶标酪氨酸激酶抑制剂在克服药物抗性和减少药物毒副作用方面具有重要作用,本文设计并合成了含有吡唑酮基团的喹唑啉衍生物作为EGFR/VEGFR-2双靶标酪氨酸激酶抑制剂。目标化合物由喹唑啉中间体和吡唑酮中间体通过亲核取代反应合成。喹唑啉中间体以2,3,4-三羟基苯甲酸为原料,通过酯化、硝化、还原、氯化和环化等反应合成;吡唑酮中间体以4-取代苯基肼盐酸盐为原料,通过甲基化和氧化等反应合成。目标化合物通过^(1)H NMR、^(13)C NMR和HR-MS进行结构鉴定。分别采用ADP-Glo激酶活性检测方法和CCK-8法测定了目标化合物对EGFR和VEGFR-2的抑制活性以及对Hela细胞、A549细胞、HUVEC细胞的抗增殖活性,其对EGFR和VEGFR-2抑制活性IC_(50)值为10~899 nM,15~712 nM;对部分在分子水平测定表现出较高活性的化合物进行了抗增殖活性测定,所选定的化合物对人肺癌A549细胞的半抑制浓度IC_(50)值为10~267 nM,对人脐静脉内皮细胞HUVEC的半抑制浓度IC_(50)值为11~433 nM,对人宫颈癌细胞Hela细胞几乎没有表现出抑制活性。对在细胞和分子水平测试均表现出良好活性的化合物5l通过分子对接研究发现其能够很好地结合在EGFR激酶和VEGFR-2激酶的活性口袋中。本研究为发现EGFR和VEGFR-2双靶标小分子酪氨酸激酶抑制剂奠定了良好的基础。

Quinazoline derivatives containing pyrazolone groups were designed and synthesized as EGFR/VEGFR-2 dual-target tyrosine kinase inhibitors.The target compounds were synthesized from intermediate quinazoline and intermediate pyrazolone by nucleophilic substitution reaction.Quinazoline intermediates were synthesized from reagent 2,3,4-trihydroxybenzoic acid by esterification,nitration,reduction,chlorination and cyclization.Pyrazolone intermediates were synthesized from 4-substituted phenylhydrazine hydrochloride by methylation and oxidation.The structure of all target compound was identified by ^(1)H NMR,^(13)C NMR and HR-MS.ADP-Glo kinase activity assay was used to determine the inhibitory activity of the target compounds on EGFR and VEGFR-2.CCK-8 assay were used to evaluate anti-proliferation activity of the target compounds on Hela cells,A549 cells and HUVEC cells.The IC_(50) values of the target compounds against EGFR and VEGFR-2 ranged from 10-899 nM and 15-712 nM,respectively.The compounds that showed high activity at molecular level were assayed antiproliferative activity on human lung adenocarcinoma cell A549 with IC_(50) of 10-30 nM and human umbilical vein endothelial cells HUVEC with IC_(50) of 17-57 nM and on the human Hela cells with little inhibitory activity.Compounds that showed good activity at both cellular and molecular levels were found to be able to enter the pockets of EGFR kinase and VEGFR-2 kinase by molecular docking studies.This work established the foundation for the forthcoming research and practical application on the development of dual-target small molecule tyrosine kinase inhibitors for EGFR and VEGFR-2.