Synthesis,Crystal Structure And Antitumor Activity Of Copper Complexes With 4-acyl Pyrazolone 2-thiophenecarboxylic Acid Hydrazide / Salicylhydrazide

The copper complexes with 4-acyl pyrazolone Schiff base have been proven to have good antitumor activity and have potential research value in the field of anticancer drugs.This paper takes 4-acyl pyrazolone and different hydrazides（2-thiophenecarboxylic acid hydrazide and salicylhydrazide）as the raw materials to form two kinds of Schiff bases,and then chooses planar diimine co-ligands including2,2’-bipyridine（bpy）,1,10-phenanthroline（phen）and dipyrido[3,2-d:2’,3’-f]quinoxaline（dpq）to form seven kinds of complexes with copper ion.The mixed ligand copper complexes derived from Schiff base have not been reported in the literature.The complexes were characterized by X-ray single crystal diffraction.The spectrophotometry were performed to study the bonding mode and strength of the complexes with herring sperm DNA（HS-DNA）.The in vitro cytotoxicities of these complexes against tumor cells（Hela and Eca-109）were further evaluated by MTT assay,and two representative complexes 4 and 7 with anticancer property were screened.The Hoechst 33258 and the Annexin V-FITC/PI double staining experiments were carried out to further study the mechanism of complex 4 and 7inhibiting Eca-109 cells proliferation.This work can provide valuable information for the design and research of metal antitumor drugs.The main research results are as follows:1.Synthesis and crystal structure of the complexes 1-7.N-（1-phenyl-3-methyl-4-benzoyl）-5-pyrazolone)-2-thiophenecarboxylic acid hydrazide（H₂L_a）and N-（1-phenyl-3-methyl-4-benzoyl）-5-pyrazolone)-2-salicylidene hydrazide（H₂L_b）were selected as two main ligands,the seven copper complexes were obtained by the reaction of two ligands with copper acetate and co-ligands（bpy,phen and dpq）,which are[Cu（L_a）]·CHCl₃（1）、[Cu（L_a）（bpy）]（2）、[Cu（L_a）（phen）]·（CH₃CN）·（Et OH）_0.5·（H₂O）_0.5（3）、[Cu（L_a）（dpq）]（4）、[Cu（L_b）（bpy）]（5）、[Cu（L_b）（phen）]（6）和[Cu（L_b）（dpq）]·（CHCl₃）₂（7）.These complexes were characterized by elemental analysis and X-ray single crystal diffraction.X-ray single crystal diffraction showed that the central Cu（II）of the seven copper complexes mainly adopt tetragonal pyramid geometry with five coordination.2.Study on the binding affinity of complexes 1-7 with HS-DNA.The binding of complexes 1-7 to HS-DNA was studied by UV-vis spectra and fluorescence quenching spectra.The binding and fluorescence quenching constants were calculated.The results showed that the complexes could bind with HS-DNA mainly by insertion.The complexes 4 and 7 have strongest binding abilities with HS-DNA in H₂L_a and H₂L_b series,respectively.The binding constants of complexes 4and 7 were 3.21×10⁵ M^-1 and 2.10×10⁵ M^-1,respectively,and the fluorescence quenching constants were 3.20×10⁵ M^-1 and 7.92×10⁴ M^-1,respectively.3.Study on the in vitro antitumor activities of complexes 1-7.The antiproliferative activities of complexes 1-7 against He La cells and Eca-109cells were examined by MTT assay.The results showed that complexes 1-7 exhibit excellent antitumor activity and superior to the clinical antitumor drug cisplatin.The overall trend of the antiproliferative effect of the complexes on two tumor cells follows:H₂L_a series copper complexes>H₂L_b series copper complexes,predicting that the anticancer activity is related to the hydrazide moiety of Schiff base,and the anticancer activity of thiophene hydrazide Schiff base is better than that of salicylhydrazone Schiff base.The anticancer activity of same series of complexes against Eca-109 is superior to He La.Comparing the copper complexes of the same series,it was found that the copper complexes containing the larger planar co-ligand showed better antitumor activity in vitro.The complexes 4 and 7 showed the highest cytotoxicity against both He La and Eca-109 cells in H₂L_a and H₂L_b series,respectively.The IC₅₀ values of the complex 4 on Eca-109 cells and He La cells were 1.16±0.21μM and 1.49±0.08μM,the IC₅₀ values of the complex 7 on Eca-109 cells and He La cells were 2.80±0.50μM and 3.02±0.11μM.The cell morphology of Eca-109treated with complexes 4 and 7 was detected by fluorescence microscope using Hoechst 33258 staining,which showed karyopyknosis and chromatin condensation,the results indicated that the cells were apoptotic.Annexin V-FITC/PI double staining experiments showed that complexes 4 and 7 could inhibit the proliferation of Eca-109cells by inducing apoptosis in a dose-dependent manner.