Studies On The Synthesis,in Vitro Antitumour Activity And Action Mechanism Of The Rare Earth Metal Complexes With Oxonantenine

This dissertation mainly focused on a typical oxoaporphine alkaloid,oxonantenine(ONT),and its rare earth metal complexes.Eight new rare metal complexes have been synthesized and their structures have been characterized by elemental analysis,IR,ESI-MS and single crystal X-ray diffraction analysis.On the cellular level,the antitumor activity of ONT metal complexes against a series of tumor cell lines were screened by MTT assay and their cell apoptosis induction were examined by flow cytometry(FCM).On the molecular level,the interaction of ONT and its complexes with DNA were investigated by UV-vis,fluorescence,CD spectroscopy,agarose gel electrophoresis assay and Western Blot assay.These researches will be beneficial to the research and development of new metal-based antitumor agents derived from the natural active ligands.The main content are as the follow:1.Eight rare metal complexes with oxonantenine(ONT)were synthesized,which were[Ce(ONT)2(H2O)(NO3)3](1);[Nd(ONT)2(NO3)3](2);[Gd(ONT)2(NO3)3](3);[Tb(ONT)2(NO3)3](4);[Dy(ONT)2(NO3)3](5);[Ho(ONT)2(NO3)3](6);[Tm(ONT)2(NO3)3](7)and[Lu(ONT)2(NO3)3](8).All of the complexes were structurally characterized by IR、EMI-MS and X-ray diffraction analysis.Complexes 2～8 had the similar coordination mode,in which the central metal ion is ten-coordinated by two bi-dentated ONT ligands via the 7-N atom and 6-O atom,three bi-dentated nitrate ligands via two O atoms,respectively,to form a bicapped tetragonal antiprism geometry.As for complex 1,it adopts an unusual eleven-coordinated mode,in which the central Ce(Ⅲ)was coordinated by two bi-dentated ONT ligands,three bi-dentated nitrate ligands and one H2O molecule,respectively,to form a one-capped pentagonal antiprism geometry.2.The antitumor activities of these complexes against a series of human tumor cell lines(NCI-H460、MGC80-3、BEL-7404、T-24、Hep-G2、HeLa229、SK-OV-3、A549)and human normal liver cell line HL-7702 were screeened by MTT assay,and the IC50 values were also evaluated.It was found that the BEL-7404 tumor cell line was the most sensitive to these complexes.Complexes 3 and 4 showed the higher cytotoxicity towards these tumor cell lines than the other complexes,especially to the BEL-7404 tumor cell lines,with IC50 values lower than 10 μM.Both complexes 3 and 4 were examined for their cell cycle arrest and cell apoptosis induction in the BEL-7404 cells using flow cytometry.The cell cycle experimental results showed that both the complexes arrested the cell cycle of BEL-7404 cells in the S phase.While by cellular morphology observation using different probes,both complexes 3 and 4 were found to significantly induce cell apoptosis in BEL-7404 cells.Using the Hoechst33258 staining and AO/EB staining,the typical apoptotic events could be observed,and the loss of mitochondrial membrane potential was also detected by Rh123 staining.3.The apoptotic mechanism in BEL-7404 cells induced by complexes 3 and 4 was further studied by cellular biological method,flow cytometry and Western blot assay.The ROS level was detected to be enhanced in tumor cells in the presence of complexes 3 and 4,along with the increment on the[Ca2+].By flow cytometry,the key factors for the caspase cascade,caspase-3 and caspase-9,were found to be activated.By Western blot assay,the presence of complexes 3 and 4 was found to effectively inhibit the expression of the anti-apoptotic gene,bcl-2,and to enhance the expression of the pro-apoptotic gene,Bax,cytochrome c and Apaf-1.It strongly suggested that both complexes 3 and 4 might induce the cell apoptosis via the mitochondrial pathway.4.Through a variety of spectroscopic analytic methods as well as the agarose gel electrophoresis assay,the possible interaction mechanism between the ONT complexes and DNA was discussed on the molecular level,since DNA was generally regarded as the primary intracellular target.The results showed that ONT interacted with ct-DNA in an intercalative binding mode due to its good planarity and extended cyclic conjugated system.Most of the metal complexes of ONT could also intercalatively bind with DNA,with higher binding intensities than ONT ligand,which could be ascribed to the central lanthanide(Ⅲ)who acting the key role to promote the DNA binding.From the results of the agarose gel electrophoresis experiment,the metal complexes of ONT could more effectively reduce the shift mobility of supercoiled DNA than ONT,which confirmed their intercalative binding mode with DNA.While quite some of these metal complexes also showed the ability of DNA cleavage at higher concentration than 80 μM,which most probably due to the redox activity of the central lanthanide(Ⅲ).