【摘要】 本文设计合成了一系列杜仲活性成分香豆素类与苯丁酸氮芥衍生物（B-1～B-4）,所有化合物均通过¹HNMR、¹³C-NMR、HRMS进行结构确认。通过CCK-8法筛选出新型衍生物B-2对A-253细胞具有较高的抗增殖作用,IC₅₀值为18.490±1.398μM,且对人正常细胞的毒性最低,IC₅₀值为80.9±10.432μM。采用流式细胞术、蛋白免疫印迹技术、吖啶橙AO染色等方法,研究了B-2对A-253细胞细胞凋亡、细胞周期及线粒体膜电位的影响。研究发现,随着B-2浓度的升高,A-253细胞处于G2/M期的比例升高,凋亡细胞逐渐增多,线粒体功能受损逐渐加重。B-2以浓度依赖的方式下调了细胞周期相关蛋白CDK2和Cyclin A2的表达,使A-253细胞周期停滞;上调细胞凋亡关键蛋白Cleaved caspase-3和Cleaved PARP而诱导A-253细胞发生凋亡;激活自噬相关标记物Beclin 1和LC3的表达,诱导A-253细胞发生自噬,进一步抑制细胞生长。更多还原

【Abstract】 In this paper,a series of novel derivatives of coumarins in eucommia ulmoides and chlorambucil（B-1～B-4） were designed and synthesized,and compounds’ structures were identified by ¹H-NMR,¹³C-NMR and HRMS.The new derivative B-2 was selected by CCK-8 method and showed stronger antiproliferative activity against A-253 cells with IC₅₀ value of 18.490±1.398μM,and with lower toxicity no normal cells with IC₅₀ 80.9±10.432 μM.The effects of B-2 on apoptosis,cell cycle and mitochondrial membrane potential of A-253 cells was examined by flow cytometry,western blotting and acridine orange AO staining.It was found that after treating A-253 cells with B-2,with the increase of B-2 concentration,the proportion of A-253 cells in G2/M phase,the early apoptotic cells and late apoptotic cells gradually increased,and the impairment of mitochondrial function was gradually aggravated.The B-2 could inhibit the proliferation of A-253 cells through a variety of molecular biological mechanisms,including down-regulation of CDK2 and Cyclin A2 expression to arrest the cell cycle,up-regulation expression of Cleaved Caspase-3 and Cleaved PARP to induce cell apoptosis,raising Beclinl and LC3 expression to induce autophagy.更多还原