Synthesis, Structure And Antitumor Activities Of Transition Metal Complexes Bearing Tropolone

In this paper,tropolone,which is also called cyclohexenate,was chosen as a ligand to react with different metal salts using solvothermal or routine solution methods in the absence or presence of different ancillary ligands of 2-methyl-8-hydroxyquinolin（HMQ）,8-hydroxyquinoline（HQ）,1,10-phenanthrene（phen）,2,2’-pyridine（2,2’-bpy）,5,7-dichloro-8-hydroxyquinoline（HQCl₂）,5-chloro-8-hydroxyquinoline（HQCl）,giving 14 new complexes which are divided into three series.In the first series of four Cu（II）complexes,the aim is to investigate the effects of different ancillary ligands on the antitumor activity of the Cu（II）-tropolone complexes.In the second series of six Pt（II）complexes,the effects of different ancillary ligands on the antitumor activity of the Pt（II）-tropolone complex were investigated.Four different transition metals complex bearing tropolone were involved in the third series,which were used to examine the effects of different metal ions on anti-tumor activities.Their structures were characterized and analyzed by infrared spectroscopy,ultraviolet-visible spectrophotometer,electrospray ionization mass spectrometry and single crystal X-ray diffractionanalyses.The in vitro antitumor activities of these complexes were screeened at cellular level by MTT assay,flow cytometry and cell morphology and the relevant cycle arrest and apoptosis of MGC80-3,T24 and Hela cells induced by these complexes were explored.The specific research contents are listed below.In this research,tropolone was chosen as a ligand to react with different metal salts using solvothermal or routine solution methods,giving 14 new complexes which are divided into three series.They have the formula of[Cu L₂]（1）,[Cu（phen）LCl]·0.5H₂O（2）,[Cu₂（MQ）₂L₂]（3）,[Cu（2,2’-bpy）LCl]·H₂O（4）,[Pt（Q）L]（5）,[Pt（MQ）L]（6）,[（QCl₂）Pt（DMSO）Cl]（7）,[（Phen）Cu LCl₃Pt（DMSO）]（8）,[Pt（QCl₂）₂]（9）,[Pt（QCl）₂Cl₂]（10）,[Mn（phen）L₂]（11）,[Co（phen）L₂]（12）,[Ni（phen）L₂]（13）,[Zn（phen）L₂]（14）.There are four Cu（II）complexes（1-4）in the first series,in which the Cu（II）ion of complex 1 is a four-coordinate in planar square geometry and the Cu（II）ions of complexes 2-4 are five-coordinated in square pyramidal configurations.The two Cu（II）ions in complex 3 are bridged by two hydroxyl oxygen atoms from two 2-methyl-8-hydroxyquinoline ligands to form a binuclear structure.Complexes 1,2,and 4 are all mononuclear Cu（II）complexes.The second series are Pt（II）metal complexes（5-10）,including five mononuclear Pt（II）complexes and one Pt-Cu heterometallic dinuclear complex.The Pt（II）ions in complexes 5 and 6 are coordinated by a tropolone ligand and a quinoline ligand to form a Pt O₃N planar square configuration.The Pt（II）ion in complex 7 is coordinated by a dehydrogenated 5,7-dichloro-8-hydroxyquinoline ligand,one DMSO and one Cl^-ligand to form a planar squre geometry.The planar square geometried Pt（II）ion and the tetrahedral Cu（II）ion in complex 8 are bridged by a Cl^-ion to form a heterometallic dinuclear structure.The Pt（II）ion of complex 9 is coordinated by two 5,7-dichloro-8-hydroxyquinoline ligands to form a Pt O₂N₂ planar square geometry.The Pt（II）ion in complex 10 is coordinated by two5-chloro-8-hydroxyquinoline ligands and two chloride ligands to form a six-coordinated octahedral geometry.The third series contains four isomorphous complexes（11-14）with different metal ions,in which the metal ions are six-coordianted in distorted octahedral geometries.According to the preliminary characterization of stabilities and solubilities of these complexes,complexes 1-4 in the first series,complexes 5 and 6 in the second series,and complexes 11-14 in the third series were selected for screening their antitumor activities.The proliferation-inhibiting abilities of complexes 1-6 and 11-14,and the associated ligands were evaluated by MTT method on the selected human tumor cells（T-24,NCI-H460,Hep-G2,SK-OV-3,MGC80-3,BEL-7402,Hela,A549）and human normal liver cell HL-7702.The experimental results showed that complexes 1-6 and 11-14 have certain inhibitory effects on different tumor cells.Complexes 1-4 show nice inhibitory effects on different tumor cells,in which complex 2 had the best inhibitory effect on MGC80-3 with a IC₅₀ value of 3.5±0.26μM.Complexes 5 and 6 have certain inhibitory activities on selected tumor cells.The IC₅₀ values of complex 5 on A549 cells,MGC80-3 cells,T24 cells and Hela cells are all below 10,in which complex 5 show a better anti-tumor effect on T24 than that for cisplatin with a IC₅₀ value of 3.6±0.63μM.Complexes 11-14 have a certain selectivity for their cytotoxicities on different tumor cells.Complex 11 has low IC₅₀ values for MGC80-3 and He La cells,and complex 14 has low IC₅₀ values for Hela and SK-OV-3 cells.Complex 12 and complex 13 have low toxicity against selected tumor cell lines with their IC₅₀ values above 30μM.It is possibly related to the different metal ions in the complexes of the third series.Among them,the IC₅₀ value of complex 11against Hela cells is 15.86±1.13μM,which is similar to that for cisplatin,however,with much lower toxicity to human HL-7702 normal cells.Based on the preliminary screening results of anti-tumor activity,complex 2 in the first series,complex 5 in the second series and complex 11 in the third series were selected for the further investigation on anti-tumor mechanisms.Flow cytometry was used to study the specific situation of cell cycle arrest and cell cycle regulators.The experimental results showed that complex 2 has no obvious cycle arrest on MGC80-3 cells.Complex 5 induces T24 cell cycle arrest at the G2 phase.Complex 11 induces hela cell cycle arrest at the G1 phase.The results for cell apoptosis induced by these complexes showed that the percentage of apoptosis induced by complex 2,5 and 11 are 33.72%,81.7%and 35.31%,respectively.The Western blotting study of the mechanism for complex 5 on tumor cell cycle arrest revealed it can activate P53 and P27proteins and inhibit the expression levels of Cdc25 A,Cyclin A,Cyclin B and CDK2 proteins,which results in the termination of DNA replication in tumor cell,and thereby leading to cell cycle arrest at the G2 phase.Based on the results of cellular uptake,Cell morphology,Hoechst 33258 staining,AO/EB double staining,measurement of ROS generation,Ca²⁺fluctuation,and mitochondrial membrane potential（JC-1）,caspase-3/9 activation assay and Western Blot,the anti-tumor mechanism of complexes 2,5 and 11 was studied.Autophagy was detected for complex 2 by MDC staining with the autophagy-related protein expression checked by Western Blot.The experimental results showed that complexes 2,5 and 11 can induce apoptosis of corresponding tumor cells by mitochondrial dysfunction and complex 2 can also induce autophagy of MGC80-3.Complexes 2,5,and 11 can decrease the mitochondrial membrane potential of MGC80-3 cells,T24 cells,and Hela cells,increase the level of intracellular reactive oxygen species（ROS）and release calcium ions(Ca²⁺),and activate the activities of Caspase-3 and Caspase-9,as well as up-regulate the expression of apoptosis-related proteins Cytochrime c,Bak,Bax and Apaf-1 and decrease the expression level of Bcl-2 protein.