DNA/HSA Binding Interactions And In Vitro Cytotoxicity Studies Of Ternary Copper(?) Complexes

Since cisplatin was widely applied to clinical as chemotherapy drug,studies on metal drugs have drawn great attentions of researchers.Most metal drugs used for clinic are platinum and ruthenium drugs,which are easily synthesized and powerful anticancer abilities compared with natural products such as paclitaxel.However,platinum or ruthenium drugs have serious side effects and causes cross-resistance.Hence,researches and developments of metal anti-cancer drugs with fewer side effects,high selectivity and good water-solubility are of great significance.Copper,one of the essential trace elements for human body,is important constituent of some enzymes in human.Hence,copper complexes may have fewer side effects.N,N-arylamines are a group of compounds with planar structures and hydrophobicity,which are similar to DNA bases and can interact with DN A/protein efficiently.N,N-arylamine compounds possess lots of biological activities,and metal complexes containing N,N-arylamine exert enhanced activities.Amino acids and oligopeptides are endogenous micromolecules,which helps metal complexes recognize biomacromolecules and increase their solubilities.Therefore,copper complexes of N,N-arylamine and amino acid are promising anticancer drugs.This paper aims at exploiting copper???complexes that have potential anticancer activities,and studying DNA/HSA binding properties.In this paper,two kinds of opper???complexes of N,N-arylamines and amino acids have been studied and the main results are listed as follows:In part I,four copper???complexes of 2,4-diamido-6-?2'-pyridyl?-1,3,5-triazine?PyTA?and amino acid,[Cu?PyTA??L-Phe?C l]·2H₂O?1??[C u?PyTA??L-Met?C l]·H₂O?2??[Cu?PyTA??L-Thr??C lO₄?]·0.75H₂O?3??[Cu?PyTA??L-Ser??C lO₄?]·H₂O?4??where L-Phe=L-Phenylalanine,L-Met=L-Methionine,L-Thr=L-Threonine,L-Ser=L-Serine?,were prepared by slow evaporation and characterized by elemental analysis,IR,UV-vis spectroscopy,molar conductivity meter and ESI-MS.The crystal structures of 1³ were gained by using X-crystal diffraction spectrometer,indicating that the copper atoms of the comlexes were located in distorted tetragonal pyramids.The DNA binding interactions of1⁴ were studied by multi-spectroscopic methods,hydromechanical method and molecular docking technique.The results indicated that these complexes bound to DNA in minor groove with similar binding intensity.The gel electrophoresis experiments showed that the complexes can effectively cleave pBR 322 DN A in the presence of reductant,involving in the generation of reactive oxygen species.The HSA binding interactions of 1⁴ were studied by multi-spectroscopy,isotherm titration microcalorimetry and molecular docking,showing the complexes had strong affinity with HSA.Electronic absorption spectra and CD spectra results showed that the secondary structures and hydrophobic microenvironment were changed in the binding processes.Site-marker competitive binding experiments and molecular docking proved that complexes 1 and 2 bond to HSA in site I near to Trp-214.The cytotoxicity tests showed that complexes 1 and 2 had perfect anticancer abilitiy towards Eca-109 and A549 cell lines(IC₅₀=22²8?M).Complexes 3⁴ showed inhibiting effects on Bel-7402 cell line(IC₅₀=42.1?M for 3,IC₅₀=38.6?M for 4).In addition,the cytotoxicities towards He La cell line of 1 and 2 were enhanced by 2 times through binding to HSA,but the cytotoxicities against normal cells had not been changed.The apoptosis assays towards Eca-109 cells showed that complexes 1 and 2 inhibited cell proliferation by inducing apoptosis pathway.In part ?,two copper???complexes of 5-methyl-2-?2'-pyridyl?benzimidazole?HPBM?and amino acid,[C u?HPBM??Gly??H₂O?]·0.5H₂O·ClO₄?5??[C u?HPBM??L-Phe??H₂O?]·C lO₄?6??where Gly=Glycine,L-Phe==L-Phenylalanine?,were prepared by slow solvent evaporation and characterized by elemental analysis,IR,UV-vis spectroscopy,molar conductivity meter,ESI-MS.The DNA binding interactions of 5 and 6 were studied by multi-spectroscopic and hydromechanical methods,indicating that the binding constants of 5 and 6 were 7.26×10⁴ and 7.38×10⁴ M^-1,respectively.The lowest-binding energies of 5and 6 abtained by molecular docking method were-8.82 and-9.59 kcal/mol,respectively.The gel electrophoresis experiments showed that the complexes can effectively cleave pBR322 DNA in the presence of reductant,involving in the generation of singlet oxygen ¹O₂and superoxide anion O₂^-.The cytotoxic tests showed that complexes 5 and 6 have good anticancer abilitiy towards Eca-109,He La and A549 cell lines(IC₅₀<8?M),and inhibited Eca-109 proliferation by inducing apoptosis pathway.Comet assay showed that the complexes had concentration-dependent DNA damages in Eca-109 cells.The complexes caused a decrease of ROS level in Eca-109 cells and promoted the death of cells.Mitochondrial membrane potential detection suggested that the complexes could decrease the membrane potential in cells.The cell cycle distribution revealed that complex 5 arrested the cell growth at S and G2/M phase,while complex 5 arrested the cell growth at S phase.