Synthesis And Antitumor Activity Studies Of Novel Oxovanadium(?) Complexes With 1,10-phenanthroline Derivative Ligands

Oxovanadium complexes are very important types of high-value transition metal complexes,with a variety of biological activities such as anti-tumor,anti-bacterial,anti-fungal,anti-viral,anti-parasitic effects.In addition,it has many biological activities such as catalyst,reduction of hyperlipemia and hypertension,and insulin enhancement.Recent studies have shown that oxovanadium complexes have low IC₅₀??M range?effect on tumor cells,and have pro-apoptotic and anti-proliferative effects.Therefore,in-depth studies of the anti-tumor properties and mechanisms of such complexes is of great significance and can provide an important experimental basis for further clinical applications.In terms of anti-tumor mechanism,although several mechanisms have been proposed to explain the inhibition of tumor cell cycle and induction of apoptosis by oxovanadium complexes,the specific mechanism remains unclear.In order to further understand the biological activity and mechanism of oxovanadium complexes,a series of novel oxovanadium???complexes with 1,10-phenanthroline derivative ligands were designed and synthesized,and they were tested by biological experiments and their antitumor activity and mechanism were explored.The main research work of this paper is as follows:1.Four novel Oxovanadium???complexes with pyridoxal thiosemicarbazone Schiff base as the main ligand and 1,10-phenanthroline as ancillary ligands were designed and synthesized,namely[VO?Hthpy??phen?]?1?,[VO?Hpthpy??phen?]?2?,[VO?Hmthpy??phen?]?3?and[VO?Hethpy??phen?]?4?.The structures are characterized by elemental analysis,UV-Vis,IR,ESI-MS and nuclear magnetic resonance.2.ThreenovelOxovanadium???complexeswith o-hydroxyacetophenone thiosemicarbazone Schiff base as the main ligand and1,10-phenanthroline as ancillary ligands were designed and synthesized,namely[VO?HoHAT??phen?]?5?,[VO?HoHMT??phen?]?6?and[VO?HoHET??phen?]?7?.The structures are characterized by elemental analysis,UV-Vis,IR,ESI-MS and nuclear magnetic resonance.3.Three novel Oxovanadium???complexes with pyridoxal isonicotanoyl Schiff base as the main ligand and 1,10-phenanthroline imidazole derivatives as ancillary ligands were designed and synthesized,namely[VO?PIH??FPIP?]?8?,[VO?PIH??ClPIP?]?9?and[VO?PIH??OHPIP?]?10?.The structures are characterized by elemental analysis,UV-Vis,IR,ESI-MS and nuclear magnetic resonance.4.The interaction of the above novel complexes with DNA was studied by UV-Vis spectral titration,fluorescence spectrometry and agarose gel electrophoresis.The experiments confirmed that these complexes bind to DNA by insertional binding mode.The plasmid DNA can be efficiently cleaved under physiological conditions to exhibit chemical nuclease activity.The interaction of the complexes with BSA were studied by fluorescence spectroscopy.The results showed that such complexes have a good tendency to bind to proteins.5.The antitumor activities of these novel complexes and the possible of their anti-tumor mechanisms were studied by using a series of in vitro experiments,respectively.?1?The antitumor activity of the complex against lung cancer cells?A549?,cervical cancer cells?Hela?and liver cancer cells?HepG2?in vitro was evaluated by MTT assay.The results showed that the growth of selected cancer cells was significantly inhibited,and the inhibition rate of the new complexes on tumor cells was comparable to that of the clinical drug cisplatin,and some compounds were even higher than cisplatin.?2?It was observed by AO/EB and Hoechst 33342 staining experiments that the tumor cells after the action of the complex showed a distinct apoptotic state.The cell cycle and apoptosis of tumor cells were determined by flow cytometry.It was found that the complex can block the cell cycle and the cell cycle arrests at a certain stage and can induce apoptosis of tumor cells.?3?Qualitative and quantitative analysis of intracellular reactive oxygen species and mitochondrial membrane potential using fluorescence microscopy and flow cytometry,and the results showed that the measured complexes significantly increased the level of reactive oxygen species in the tumor cells,while the mitochondrial membrane potential decreased significantly.These results indicated that the mechanism of complex led to tumor cell apoptosis possibly due to the increase of reactive oxygen species?ROS?.Simultaneously,the oxidative damage of the mitochondrial membrane and the permeability of the membrane are changed,which could induced the decrease in mitochondrial membrane potential.Eventually leads to apoptosis.?4?In order to further explore the mechanism of action of anti-tumor activity of the complex,four genes related to cell cycle arrest and apoptosis were selected as targets,and Q-PCR was used to verify the gene level.The expression level of the proapoptotic gene Bax and the tumor suppressor gene p57^Kip2ip2 is increased,and the expression levels of the anti-apoptotic genes Bcl-2 and C-myc are decreased.?5?Finally,the related protein levels were tested by using Western Blot technique.The results showed that these complexes could up-regulate the expression of Bax and p57^Kip2,and down-regulate the expression of Bcl-2protein.Based on the above experimental results,it could be suggested that these novel oxyvanadium complexes have very good antitumor activities.The mechanism by which they induce tumor cell apoptosis may be as follows:the oxyvanadium complexes could induce apoptosis of tumor cells and inhibit tumor invasion through ROS-mediated mitochondrial pathway.When the complex interacted with tumor cells,it will stimulated an increase of intracellular ROS,thereby the expression of the pro-protein Bax is activated.Meanwhile,the expression of the anti-apoptotic protein Bcl-2 is inhibited,and ion channels were formed on the mitochondrial membranes,which caused the change in mitochondrial permeability.Accordingly,the released of apoptosis-related active substances in mitochondria,ultimately leading to apoptosis.On the other hand,the intervention of the complex activated the expression of the tumor suppressor factor p57^Kip2,thus inhibited the effect of cyclin-dependent kinase?CDK?,arresting the cell cycle in the G₁ phase,but could not smoothly enter the S phase,cells proliferation were inhibited,and finally the inhibition of tumor invasion is achieved.