Studies On The Synthesis,Structural Characterization And In Vitro Antitumour Activity Of Complexes With Liriodenine Derivatives

This dissertation mainly focused on a typical oxoaporphine alkaloid,liriodenine derivatives(La,Lb)and its transition metal complexes.Five new trantion metal complexes have been synthesized and characterized by elemental analysis,IR,ESI-MS and single crystal X-ray diffraction analysis.In the cellular level,the antitumor activity of liriodenine metal complexes against a series of tumor cell lines were screened by MTT assay and their cell cycle,cell apoptosis induction were examined by flow cytometry(FCM)and Western Blot.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 follows:1.Five transition metal complexes with liriodenine derivatives(La,Lb)were synthesized,which were[CuLb(NO3)2](1);[Zn(La)2(NO3)2](2);[Co(La)2(N03)2];(3)[Ni(La)2(NO3)2](4);[La:H][FeCl4](5).All of the complexes were structurally characterized by IR,EMI-MS and X-ray diffraction analysis.Complex 1 adopt an six-coordinated structure,in which the central Cu(Ⅱ)was coordinated by two bi-dentated Lb ligands,two bi-dentated nitrate ligands,respectively,and formed a octahedronal geometry;Complexes 2～4 had the similar coordination mode,in which the central metal ion is six-coordinated by two bi-dentated La ligands via the 6-N atom and 7-O atom,two bi-dentated nitrate ligands via two O atoms.respectively,to form a octahedronal geometry;The structure of complex 5 was special and iron ion has not coordinated with La.Heterocyclic N atom of La was protonated by H+ of reaction solution,forming a[La:H]+cations.Then[La:H]+cations reacted with[FeCl4]-anions forms a new type of ionic compound.2.The antitumor activities of these complexes against a series of human tumor cell lines(NCI-H460,MGC80-3,T-24,Hep-G2,HeLa229,SK-OV-3,A549)and human normal liver cell line HL-7702 were screened by MTT assay,and the IC50 values were also evaluated.It was found that the T-24 tumor cell line was the most sensitive to these complexes.Complexes 1 and 2 showed the higher cytotoxicity towards these tumor cell lines than the other complexes,especially to the T-24 tumor cell lines,with IC50 values lower than 10 μM.3.Both complexes 1 and 2 were examined for their cell cycle arrest in the T-24 cells using flow cytometry and Western blot.The cell cycle experimental results showed that the complexe 1 arrested the cell cycle of T-24 cells in S phase;Complex 2 arrested the cell cycle of T-24 cells in G2/M phase.By Western blot assay,the presence of complexes 1 was found to effectively inhibit the expression of CyclinA2,Cdk2,p-Cdk2(the key proteins of S phase);enhance the expression of p21,p53,cyclinE1;complexes 2 was found to effectively inhibit the expression of Cdc25C,CDK1,CyclinB1(the key proteins of G2/M phase).The results showed that the cell cycle arrest of complex 1 was relate to CyclinA2,Cdk2,p-Cdk2,p21,p53,CyclinE1;Complex 2 was relate to Cdc25C,CDK1,CyclinB1.The distribution of drugs on T-24 cells were measured by ICP-MS,the results have shown that a large amount of metal accumulated in mitochondria after treatment with complexes 1 and complex 2 was mainly located on cytomembrance fraction and cytosolic fraction.It found that the mechanism that complex 1 reactes with T-24 cell was different from complex 2.4.The apoptotic mechanism in T-24 cells induced by complexes 1 and 2 was further studied by cellular biological method,flow cytometry and Western blot assay.By cellular morphology observation using different probes,both complexes 1 and 2 were found to significantly induce cell apoptosis in T-24 cells.Using the Hoechst33342 staining and DID staining,the typical apoptotic events could be observed,and the loss of mitochondrial membrane potential was also detected by JC-1 staining.The ROS level was detected to be enhanced in tumor cells in the presence of complexes 1 and 2,along with the increasement 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 1 and 2 was found to effectively inhibit the expression of the anti-apoptotic gene,bcl-2,bcl-xl and to enhance the expression of the pro-apoptotic gene,Bax,Bak and cytochrome c.It strongly suggested that both complexes 1 and 2 might induce the cell apoptosis via the mitochondrial pathway.