新型三氟甲氧查尔酮类衍生物的设计合成及体外抗宫颈癌活性研究

Design Synthesis and in Vitro Anti-cervical Cancer Activity of New Trifluoromethoxy Chalcone Derivatives

目的以天然甘草查尔酮母核为先导化合物骨架,设计、合成三氟甲氧基查尔酮类衍生物,并对其进行体外抗宫癌活性研究。方法通过Claisen-Schmidt羟醛缩合反应,合成全新三氟甲氧基查尔酮类衍生物,结构经^(1)H-NMR、^(13)C-NMR及HR-ESI-MS进行确证。四唑盐(MTT)法测定目标化合物对HeLa、SiHa、C-33A三种宫颈癌细胞及正常宫颈上皮永生化H8细胞的毒性,并做构效关系分析,选定候选化合物。Transwell法和流式细胞仪技术测定候选化合物3o对HeLa细胞的侵袭迁移,促凋亡及对细胞周期的影响;分子对接法将候选化合物与鼠双微粒2(MDM2)蛋白靶点进行分子对接,测定化合物3o与靶蛋白分子的结合能力及结合特点;Western blot法测定候选化合物3o对MDM2和p53蛋白的调控作用。结果合成20个全新三氟甲氧基查尔酮类目标化合物,候选化合物3o对宫颈癌细胞的毒性最强(IC_(50)=4.60±0.40μmol·L^(-1)),明显优于阳性药顺铂(IC_(50)=17.16±0.93μmol·L^(-1));候选化合物3o可有效抑制HeLa细胞的侵袭迁移,对HeLa细胞有显著的促凋亡作用并让细胞周期停留在G_(0)/G_(1)期;候选化合物3o与MDM2蛋白p53结合口袋的关键氨基酸有效结合(结合能-37.62 kcal·mol^(-1));候选化合物3o对MDM2蛋白有明显下调作用,对p53蛋白有明显上调作用。结论本研究结果为筛选有效低毒的新型靶向查尔酮类抗肿瘤候选药物提供一定实验基础。

OBJECTIVE To design and synthesize trifluoromethoxy chalcone derivatives based on the natural licorice chalcone parent as the lead compound backbone,and to study their anti-cervical cancer activity in vitro.METHODS New trifluoromethoxy chalcone derivatives were synthesized by Claisen-Schmidt aldol condensation.The structures were confirmed by^(1)H-NMR,^(13)C-NMR and HR-ESI-MS.The cytotoxic activity of the target compounds on cervical cancer cell lines HeLa,SiHa,C-33A and normal cervical epithelial immortalized H8 cells were determined by MTT assay,and the structure activity relationship(SAR)was analyzed and the candidate compounds were selected.The effects of candidate compound 3o on invasion,migration,apoptosis and cell cycle of HeLa cells were determined by Transwell and flow cytometry.The candidate compound 3o was docked to the MDM2 and protein target by molecular docking method,and the binding ability and binding characteristics of the compound to the target protein molecules were determined.The regulatory effects of the candidate compounds on MDM2 and p53 proteins were assessed using Western Blot analysis.RESULTS Twenty new trifluoromethoxy chalcones were synthesized.Candidate compound 3o showed the strongest inhibitory activity against cervical cancer cells(IC_(50)=4.60±0.40μmol·L^(-1)),which was significantly better than that of positive drug cisplatin(IC_(50)=17.16±0.93μmol·L^(-1)).The candidate compound 3o could effectively inhibit the invasion and migration of HeLa cells,induce apoptosis and arrest the cell cycle at G_(0)/G_(1)phase.Candidate compound 3o binds to key amino acids in p53 binding pocket of MDM2 protein(binding energy-37.62 kcal·mol^(-1)).The compounds significantly downregulated MDM2 protein expression while upregulating p53 protein levels.CONCLUSION The research results provide experimental evidence for screening new chalcone derivatives as targeted,effective,and low-toxicity anti-tumor candidates against cervical cancer.