Study On Synthesis, Structure, DNA Interaction, Protein Binding And Cytotoxicity Of Copper(Ⅱ) Complexes Based On Schiff Base

Copper is one of the essential microelements in human. It is widely distributed in organism and plays a role with metal enzymes. It has been confirmed that classic inorganic anticancer drug, cisplatin, flow into and out of cancer cell through copper transporter protein CTR1,ATP7 A, ATP7 B and so on, indicating that copper has potential anticancer activity. Copper element has valence alternation properties, generating ROS in the process of transition between Cu(I) and Cu(II) and causing cell damage and apoptosis. Copper complex is a hot topic in the field of inorganic cancer drug due to these special properties. Schiff base is a class of stable imine(-RC=N-)connected on the nitrogen atom with an alkyl or aryl, it could be synthesized by the condensation of an amine and an aldehyde or ketone. Schiff base and its metal complex have good anticancer, antibiosis, and it can interact with DNA as well. The synthetic condition of synthesis Schiff base is not harsh, and various derivatives of Schiff base ligands can be designed through modulating the substitution groups of amines or carbonyl compounds. The family of Schiff base shows structural diversity. Iron chelator which is derived from Schiff base acts on ribonucleotide reductase, which fully expresses p53 anticancer protein in some ways. To our knowledge, amino acid plays a key role in organisms. Owing to the presence of amino group, it has a great possibility of forming Schiff base. We design and synthesize Schiff base ligand and its copper complexes according to the structure features of iron chelator and amino acid.Object: Design and synthesize Schiff base ligand and its copper complexes. Investigate their structure characteristics, chemical nucleases activity, mode of the interaction between copper complex with HSA and cytotoxicity, to get copper complex with good biological activity.Methods: Several Schiff base ligands: N-methyl-2-(phenyl(pyridin-2-yl)methylene) hydrazine carbothioamide(L1) 、 2-(phenyl(pyridin-2-yl)methylene) hydrazine carbothioamide(L2) 、 5-chloro-2-hydroxybenzylidene-glycine(L3) 、 5-chloro-2-hydroxybenzylidene-alanine(L4) were syntheszed using phenyl(pyridin-2-yl) methanone, hydrazinecarbothioamide, 4-methyl-hydrazinecarbothioamide,5-chlorosalicylaldehyde, glycine and L-alanine. Then copper complexes can be synthesized with these ligands. All structures mentioned above were characterized by IR, 1H-NMR, element analysis and X-ray crystallography, respectively. Absorption spectroscopy, fluorescence spectroscopy and agarose gel electrophoresis were applied to assess the chemical nuclease activity and interaction between complexes and HSA. MTT assay was performed to test the cytotoxicity of Schiff base ligands and their copper complexes.Result: Seven copper Schiff base complexes, [Cu2(L1)2]Cl2(1)、[Cu2(L1)2]Br2(2)、[Cu(L2)]Cl(3) 、 [Cu(L2)]Br(4) 、 [CuII(L3)(H2O)2](5) 、 [CuII(L4)(H2O)](6) 、[CuII(L3)(bipy)]·0.5H2O(7) were synthesized. Their structures were characterized by IR, 1H-NMR, element analysis and X-ray crystallography, respectively. Complex 1 has one dimensional zigzag chains structural feature. In viscosity assay, along with the rising concentration of the ratio of complexes versus DNA, the viscosity of DNA solution was also rising. With the increasing concentrations of complexes 1-7, the fluorescence of Ethidium Bromide-DNA(EB-DNA) solution was quenched simutaneously. Calculated apparent binding constants(Kapp) of the complexes 1-7 were in the order of 105 magnitudes. With adding complexes 1-4, the fluorescence intensity of HSA decreased gradually, in addition, Ksv decreases with the rising temperature. When synchronous fluorescence spectra at Δλ = 60 nm, the spectral redshift is obtained. DNA cleavage experiments performed by agarose gel electrophoresis proved that in the presence of H2O2, Complexes 1-7 can effectively converted p UC19 DNA from Form I to Form II, and the higher concentration is, the more drastic the conversion degree is.Conclusion: Intercalative mode of copper complexes with CT-DNA was proved by viscosity measurement. The fluorescence spectra of EB-DNA shows that the seven copper complexes can effectively compete binding sites with EB. The DNA cleavage experiments performed by agarose gel electrophoresis proved that complexes 1-7 could effectively cleavage DNA. The mechanism is oxidative cleavage including hydroxyl radical, singlet oxygen-like species and hydrogen peroxide act as active species in the cleavage reaction. The HSA fluorescence quenching and UV absorption experiments determine that the quenching mechanism is static mechanism forcomplexes 1-4. The binding site is proved to be in tryptophan residues. IC50 values of complexes 1-4 were in the order of n M, suggesting superb anticancer activity in terms of cellular level.