Studies On The Nuclease And Antitumor Activities Of Thiosemicarbazones And Their Derivatives Copper/Platinum Complexes

Cisplatin is used widely in the clinical and kill the tumor cell effectively.It can kill tumor cells and normal cells at the same time.It is a hot topic on how to reduce the toxicity and platinum drug resistance.The research and development of artificial nuclease provides valuable information for new DNA-target chemotherapy drugs.Topoisomerase inhibitor can suppress tumor cells effectively.Utilizing high topoisomerase expression characteristics in tumor cells,platinum and topoisomerase inhibitors are combined for improving the platinum drug toxicity at the molecular level.Object:New thiosemicarbazone and amino thiazolone and their complexes were designed and synthesized.All these aim to provide valuable information for developing novel metal antitumor drugs.In order to provide valuable information for the research and development of new metal antitumor drugs,it can be screened out with good nucleic acid reagents or antitumor agents.Methods:Using a potential thiosemicarbazones Topo-II inhibitor（Dp44m T）,"cyclization thiosemicarbazones ligands"（amino thiazolone）were obtained.The corresponding copper/platinum complexes were obtained by the two ligands.These were characterized by IR,element analyses and X-ray crystallography.The nuclease activities of copper and platinum complexes were explored by gel electrophoresis,UV and fluorescent spectra.We screened the complexes with good nuclease properties further to explore their cytotoxic activity.The antitumor activity and mechanism of the complexes were investigated by MTT assay,Hoechst 33342staining assay,Annexin V/PI analysis,comet assay and cell cycle arrest experiments.The protein binding abilities to platinum complexes were monitored using SA as model protein.Result:We got thirty-five compounds including nine thiosemicarbazone ligands,nine unreported amino thiazolone ligand and seventeen new copper/platinum complexes.The main contributions in this work are as follows:1.Nine thiosemicarbazones ligands were synthesized.Two new copper complexes,[Cu（MM-TSC）Cl]（1）and[Cu（NM-TSC）（OAc）]·H₂O（2）were obtained.The nuclease activities of 1 and 2 were showed without any external agents,1<2.The DNA cleavage mechanism indicated the involvement of singlet oxygen,this implied that DNA cleavage could be attributed to oxidation mechanism.In order to study the antitumor activities of two copper complexes,the MTT assay was performed in He La,Hep G-2 and MCF-7.The results indicated that two complexes exhibited significant cytotoxic activity,IC₅₀:1<2.In Hep G-2 cells,complex 2 were superior to cisplatin against the same cell lines.The antitumor mechanisms of 2 based on Hep G-2 cells were investigated by Hoechst 33342 staining assay,Annexin V/PI analysis,cell cycle arrest,ROS generation experiments.It was observed that 2induced cells apoptosis via ROS pathway.2.A novel tetranuclear"well"platinum complex,[Pt₄（NM-TSC）₄]·16.25H₂O（3）,was synthesized with NM-TSC ligand.3 could bind CT-DNA by an efficient intercalation mode.The nuclease activity of 3 was micromolar levels.DNA was degraded under high concentration,which indicated that it can be used as an effective nucleic acid enzyme reagent in vitro.The MTT assay was performed,but 3 exhibited a poor cytotoxic activity.3.In order to avoid the bridging coordination of sulfur atom of NM-TSC ligand,we designed a class of amino thiazolone compounds.Twelve copper（II）complexes were synthesized with nine new amino thiazolone ligands.Complex 11,[Cu（MB-TZO）]Cl₂,exhibited prominent DNA cleavage activity without any external agents,which varied as 11>6-10,12>13>5,4>14,15.In the presence of H₂O₂,the DNA cleavage activity of 11 was still the best of them and the oxidation mechanisms of 11 were discussed.MTT assay was performed to test the antitumor activities of 4-15,and all of them exhibited excellent cytotoxic activities except 12and 13.The structure-activity relationships of twelve copper complexes were preliminarily discussed,the cytotoxic activity of 8 was the highest among them.The antitumor activities for 4,11,13 and 15 were also investigated using comet assay.4.Two new complexes[Pt（MH-TZO）]Cl（16）and[Pt（ME-TZO）]Cl₂（17）,were synthesized with MH-TZO and ME-TZO ligands.They could bind CT-DNA by an intercalation mode.Gel electrophoresis experiments was applied to study DNA helicase activity of 16 and 17,giving 16<17.MTT assay was performed to test the cytotoxic activity of 16 and 17,showing that 16 had more obvious cytotoxic activity than 17.It was concluded that DNA helicase activity of complexes might not directly associate with the antitumor activity.Conclusion:The nuclease activities of 1 and 2 were showed without the H₂O₂,1<2.It was observed that 2 induced cells apoptosis via ROS pathway.The nuclease activity of 3was micromolar levels.DNA was degraded under high concentration.The DNA cleavage activity of 11 was the best of them and the oxidation mechanisms of 11 were discussed.MTT assay was performed to test the antitumor activities of 4-15,and all of them exhibited excellent cytotoxic activities except 12 and 13.Gel electrophoresis experiments was applied to study DNA helicase activity of 16 and 17,giving 16<17.MTT assay was performed to test the cytotoxic activity of 16 and 17,showing that 16had more obvious cytotoxic activity than 17.It was concluded that DNA helicase activity of complexes might not directly associate with the antitumor activity.