Studies On Synthesis,Antitumor Activity And Mechanism Of Schiff Base-Transition Metal Complexes

This treatise committed to design and reform series of Schiff base hydrazide and thiosemicarbazide ligands,and finally synthesized a series of new complexes by coordination with transition metals.The infrared spectroscopy,Nuclear Magnetic Resonance,Mass spectrometry,elemental analysis and X-ray single crystal diffractometer were employed to fully characterized the ligands and complexes.The inhibition rate of ligands and complexes on tumor cells was detected using MTT assay.Flow cytometry was used to analyze the cell cycle distribution and cell apoptosis inducing by the complexes.Inductively coupled plasma mass spectrometry(ICP-MS),Leica inverted fluorescence microscope,UV-visible Spectroscopy,Fluorescence spectroscopy,Western Blotting,agarose gel electrophoresis,TRAP assay,and molecular docking simulation were used to study the distribution of complexes in tumor cells,the interaction of complexes with DNA,the effects of complexes on cell cycle and apoptosis-related proteins and on telomerase activity,and the 3D cell sphere were also employed to simulate the tumor body in vitro to study the effects of complexes on tumors.Finally,some new Schiff base metal complexes with significant antitumor activities were obtained.1.To screen Schiff base-transition metal complexes with higher anti-tumor activity,this thesis optimized the structure based on pyridine benzoyl hydrazide(L1),quinoline benzoyl hydrazide(L2),naphthalene formaldehyde benzoyl hydrazide(L8-12)and salicylaldehyde benzoyl hydrazide(L13-17)were synthesized,and the pyridine thiosemicarbazide ligands were also synthesized by the design of carbonyl groups being substituted by sulfhydryl groups on Acyl hydrazine(L8-12).The complexes C1-C21 were synthesized by chelating the above ligands with transition metal ions through solution method,the crystals of the complexes are obtained by solvent evaporation method or solvent diffusion method,X-ray techniques were used to characterize the structure of the complexes,the results showed that all complexes are monomuclear molecules,and the central copper(?)ions in the complexes C1 and C4 are tridentate-coordinated with one Ligand-1 or Ligand-2 vis the N atoms,heterocyclic N atoms,and carbonyl O atom.The central copper(?)ion of complexes C7-C11 are tridentate coordinated with a thiosemicarbazide Ligand 3-7 via the N atom,the heterocyclic N atom and the sulfhydryl S atom,and the central copper(?)ions of the complexes C10 and C11 are also coordinated with a methanol molecule to form five-coordinated mode.The central Pt(?)ion of the complexes C2 and C5 is bidentate coordinated with two N atoms on the L1 and L2,and then coordinated with two chloride ions to form a four-coordinated mode which similar to the cisplatin molecule.The central Ru(?)ion of the complexes C3 and C6 are coordinated with Ligand 1 and Ligand 2 via the N atom and the carbonyl O atom on the ligands to form a bidentate,and then coordinated with two chloride ions and two dimethyl groups via the sulfone molecule to form a hexacoordinate mode;the central Pt(?)ion of the complexes C12 and C13 are coordinated with Ligand 8 and Ligand 9 via the N atom and the carbonyl O atom on the ligands to form bidentate,and a chloride ion and a sulfoxide atom on dimethyl are coordinated with the central Pt(?)ion to form a planar quadrilateral.The central Pt(?)ion of the complexes C14,C15 and C16 are bidentate coordinated with L10,L11 and L12 via the N atom and the dehydrogenated O atom on the benzene ring,and a chloride ion and the dimethyl sulfoxide molecules are also coordinated to the central Pt(?)ion to form a planar quadrilateral;The central Pt(?)ion of the complexes C17,C18,C19,and C20 is bidentate coordinated to the N atom and the carbonyl O atom on the L13-16,in addition to a chloride ion and a dimethyl sulfoxide molecule,coupling to form a planar quadrilateral.The center Pt(?)ion of complex C21 is a four-coordinated with the N atom,the dehydrogenated O atom on the benzene ring,a chloride ion and a dimethyl sulfoxide molecule to form a planar quadrilateral.These complexes have some differences in coordination mode and molecular structure,resulting in differences in their biological activities.2.The inhibition rate of ligands and complexes against tumor cells was detected by MTT assay.Excepting the thiosemicarbazone L4-7 with a higher inhibition rate(52.27%-76.49%),other ligands have a lower inhibition rate against tumor cells(<40%).The inhibition rate was greatly enhanced by modified N-4 of the thiosemicarbazide Ligand3 to form Ligand4-7.The complexes C1-C6 are obtained via L1 and L2 coordinating with three different metal ions,respectively.They have a significant difference in the inhibition rate against tumor cells.The complexes C1 and C4 showed relatively higher activity,the IC50 values were(22.02 ± 1.83)?M and(15.68 ± 1.29)?M against to MGC80-3 cells,respectively,while the IC50 values were more than 40?M for the Pt(?)complexes C2 and C5 against to MGC80-3 cells,and the IC50 values were more than 50 ?M for the Ru(?)complexes against MGC80-3 cells.ICP-MS was used to detect the total amount of three metals in the cells after the complexes C1-C6 were used to treatment 1×106 MGC80-3 cells.The results showed that the total amounts of copper in the cells were(3.87 ± 0.22 nmol Cu/106cells)(C1)and(7.32 ± 0.22 nmol Cu/106cells)(C4),respectively.The total amounts of Pt in the cells were(2.98 ± 0.32 nmol Pt/106cells)(C2)and(4.68 ± 0.42 nmol Pt/106cells)(C5),respectively.The total amount of Ru in the cells was(0.042 ± 0.012 nmol Ru/106cells)(C3)and(0.044 ± 0.003 nmol Ru/106cells)(C6),respectively.The Ru complexes were less absorbed into the cells relative to the Cu and Pt complexes,which may be the reason for the complexes C3 and C6 to display a lower activity.The 2-pyridylthiosemicarbazide ligands and the complexes showed a higher inhibitory rate against to MGC80-3 cells and SK-OV-3 cells,and the IC50 values of L3 and the corresponding complex C7 were(19.04 ± 0.43)?M and(12.11± 0.82)?M against to MGC80-3 cells,respectively.By introducing a benzene ring(C8)at the N-4,forming a piperidine ring(C9)or a pyrrolidine ring(C11),the amount of complexes absorbed into tumor cells increased significantly,the activities of the complexes were increased up several times against the MGC80-3 cells.The cytotoxicity of the complex C10 was more than14 times higher than that of C7 after introducing two methyl groups at N-4.The results showed that the modification of N-4 could improve the cytotoxicity of the ligands and their complexes.The complexes C12-C16 were obtained by using hydroxyl,hydrocarbyl or halogen substituted benzoyl hydrazine ring,the inhibition rates were higher against to MGC80-3 cells than of the other tumor cells,and the IC50 were(7.18±0.58)?M,(10.64 ± 0.87)?M,(5.22 ± 0.70)?M,(4.38± 0.38)?M and(13.07 ± 0.31)?M,respectively,which were slightly lower(17.87 ± 0.35)?M than cisplatin.The complex C15 with propyl para-substituted has shown the best antitumor activity.The complex C18-C21 were synthesized by substituting a hydrogen atom on the salicylaldehyde benzene ring with a halogen or a methyl group,and the complexes showed good activity against to tumor cells,the IC50 values were in the range of 5.67-186.46?M,which were higher than the cytotoxicity of the un-modified complex C17(IC50 value was in the range of15.41-27.76?M and the complexes C17-C21 showed the same activity on A549 and A549 cis R cells.Among them,the complex C19 which substituted with bromide ion had the best activity against these two cells,IC50 values were(8.03 ± 0.26)?M and(8.07 ± 0.28)?M,respectively,which was superior to cisplatin against A549 and A549 cis R cells with IC50 values of(20.25 ±0.74)?M and(49.24 ± 0.57)?M,respectively,indicating that the complex C19 is not cross-resistant to cisplatin.The above researches showed that after structural modification and modification,the amount of complexes by absorbing into tumor cells could be increased and enhance their activity.3.Flow cytometry was used to detect the apoptosis of tumor cells induced by complexes.The results showed that the complexes C7 and C10 could induce late apoptosis of MGC80-3cells with the percentages of apoptosis of 22.76% and 32.75%,respectively.Complexes C12-C16 induced early apoptosis of MGC80-3 cells significantly,the percentages of apoptosis were 36.66%,30.90%,42.85%,71.45%,and 22.79%,respectively.Complexes C17 and C19 could induce A549 cis R cell early apoptosis with the percentages of 38.97% and 45.07%,respectively.In addition,the flow cytometry detected that the cell cycle of MGC80-3 was blocked in G1 phase by the complex C7,while the cell cycle of MGC80-3 was blocked in S phase by the complex C10.The complexes C17 and C19 blocked the A549 cis R cell cycle in the S phase,and the complexes caused a decrease in intracellular mitochondrial membrane potential(??m),and then led to apoptosis of tumor cells.The complexes above could cause the production of intracellular reactive oxygen species(ROS).The morphology of A549 cis R cells can be altered and cell migration can be blocked by complexes C17 and C19.In addition,the growth of 3D tumor cell spheres can be inhibited in vitro by complexes C17 and C19.4.The anti-tumor mechanisms of the complexes were studied by molecular docking simulation,ultraviolet-visible spectroscopy,fluorescence spectroscopy,agarose gel electrophoresis,Western Blot,flow cytometry,and TRAP silver staining.The results show that the complexes could induce apoptosis through multiple pathways.(1)Ultraviolet-visible spectroscopy experiments showed that the absorption peaks of the complexes were reduced with warying degrees by adding CT-DNA to the solutions of complexes C10,C15,C17,and C19,which confirmed that the complexes were bound to the CT-DNA helix by insertion.Fluorescence spectroscopy experiments show that the complexes C10,C15,C17,and C19 could compete and substituted EB in EB-DNA to cause a decrease in the fluorescence intensity of the solution.The results of agarose gel electrophoresis show that the Pt(?)complexes of C12,C15,C17,and C19 have a stronger effect on DNA than the Cu(?)complexes of C7 and C10.Only the high dose(250 ?M)of the complex C10 showed a significant effect on DNA,while the Pt(?)complexes of C12,C15,C17,and C19 can obviously effect on DNA with a lower dose(10 ?M).Simulated molecular docking results showed that the complexes could be embedded in DNA molecules,interacting with DNA by hydrogen bonds,resulting in DNA damage,then caused cell cycle block,which coinciding to the results of the effects on the cell cycle detected by flow cytometry.The results confirmed by western blot showed that the complexes C7 and C10 could significantly inhibit the expression of Cyclin A,cyclin-dependent protein kinase-2(CDK2)and the regulatory factor Cdc25 A,and significantly up-regulate the expression of p21 and p27 in a concentration-dependent.The complexes C12-C16 could inhibit the expression of Cyclin A and CDK2 in varying degrees,and up-regulate the expression of p27,complexes C13-C16 could significantly up-regulate the expression of tumor suppressor gene p21;complexes C17 and C19 inhibit the expression of Cyclin A and CDK2 with dose-dependent,significantly inhibiting at high doses,which could also significantly up-regulate the expression of p21.The above experiments have confirmed that complexes can interacted with DNA and affect the cell cycle by regulating the expression of cycle-related proteins.(2)Mitochondria is one of the targets of most metal complexes.The experiments showed that the mitochondrial membrane potential was significantly loosed.The effect of complexes on apoptosis-related proteins was detected by Western blot.The results showed that the expression of Bcl-2 was significantly inhibited by the complex C10,the expression of proapoptotic proteins Bad and Bax were up-regulated,the expression of cytochrome C and apoptotic activating factor Apaf-1 were also up-regulated.The expression of Bcl-2 and Bax were effected in different level by complexes C12-C16,in which the complexes C12-C15 could significantly inhibit the expression of Bcl-2,and the complexes C14-C16 significantly up-regulated the expression of the pro-apoptotic protein Bax and led to the release of cytochrome C.The expression of Bcl-2 and Bcl-x L were significantly inhibited by the complexes C17 and C19 at high doses,and the expression of Bax was up-regulated,the expression of cytochrome C was increased at high dose.The expression of Caspase-3/9 was detected by flow cytometry.The results showed that complexes C7,C10,C12-C16,C17,and C19 can activate the expression of Caspase-9 to different degrees and cause Caspase-3 to be activated.The experiment confirmed that complexes can regulate mitochondrial-related proteins,inducing the loss of mitochondrial membrane potential,and activate apoptosis executive factors to cause apoptosis.(3)Western blot analysis showed that the complexes C7,C10,C12-C16,C17,and C19 inhibited the expression of protooncogene c-myc and telomerase reverse transcription h TERT significantly,and led to the decrease of telomerase activity in cells.The results of telomerase activity assay showed that the complexes C7,C10,C12-C16,C17,and C19 inhibited the telomerase activity in tumor cells to varying degrees.Inhibition of telomerase activity in tumor cells is also one of the pathways to induce its apoptosis.