Synthesis,Characterization And Antitumor Activities Of Two Novel Pyrazine Anthrahydrazone Compounds And Their Metal Complexes

In this paper,two novel derivatives of anthrahydrazone were synthesized by replacing the pharmacophor of side chain of anthrahydrazone with pyrazine,based on the unique anti-tumor drug of anthrahydrazone,bisantrene.The ligands and their complexes were characterized by elemental analyses,IR,¹H-NMR,¹³C-NMR,UV-Visible,X-ray diffraction analysis.At the cellular level,8 compounds were screened for their antitumor activity by MTT assay.At the molecular level,by agarose gel electrophoresis and fluorescence spectrometry experiments of compound and DNA topoisomeraseⅠpreliminary research was conducted on the molecular mechanism of action.These works provide research experience and basis for the research of new anthracyclines anti-tumor drugs.The main contents are as follows:Two novel derivatives ligands of anthrahydrazone were synthesized,which were anthracene-9-pyrazine hydrazone（9-APZH）and anthracene-9,10-dipyrazine hydrazone（9,10-APZH）.A series of bioactive metal elements（Pt,Pd,Cu,Co,Ni,Zn）were selected to form a series of metal complexes with two ligands,namely,9-APZH-Cu,9-APZH-Co,9-APZH-Ni,9-APZH-Pd,9-APZH-Zn,9-APZH-Pt and 9,10-APZH-Cu.The structures of the nine compounds were characterized by ESI-MS,IR,elemental analysis and X-ray single crystal diffraction analysis.The nine compounds were stable at room temperature indicated by UV-Visible spectrophotometry.At the cellular level,MTT assay was used to test the antitumor activity of the compounds against 8 types of cells（human gastric cancer cell MGC-803,human cervical cancer cell He La229,human bladder cancer cell T-24,human liver cancer cell Hep-G2,human non-small lung cancer cell A549-DDP,human lung cancer cell NCl-H460）,and compared with human normal embryonic lung fibroblast WI-38 and human normal liver cell HL-7702,to explore its cytotoxic selectivity.Through the determination of the inhibition rate and IC₅₀ value,it can be seen that the cytotoxicity of the two pyrazine anthrahydrazone ligands is not significant in vitro,but from the preliminary screening inhibition rate,the activity of double pyrazine anthrahydrazone 9,10-APZH is generally higher than that of the single side chain 9-APZH.In terms of complexes,several metal complexes of 9-APZH showed no significant cytotoxicity,with IC₅₀ value of>20 M.Only 9,10-APZH-Cu showed a better inhibitory effect on the proliferation of Hep-G2,A549-DDP,NCl-H460 and He La229 cell lines in a concentration-dependent manner,and its activity was not only higher than that of 9,10-APZH ligand,but also higher than that of cisplatin,a clinical anticancer drug.It is worth noting,9,10-APZH-Cu complexes of normal embryonic lung fibroblast WI-38 and normal liver cells HL-7702 did not produce poisonous to the cells,showing the complexes have good selectivity,cytotoxicity have a thorough research and development as a new anticancer drug of potential prospects.Subsequently,the tumor cell cycle arrest and apoptosis induced by 9,10-APZH-Cu,Hep-G2,A549-DDP,NCI-H460 and He La229 were detected by flow cytometry.The results showed that the 9,10-APZH-Cu complex showed obvious periodic retardation in the induced periodic retardation experiment.In the experiment of inducing apoptosis,9,10-apzh-cu complexes can induce apoptosis of Hep-G2,NCI-H460 and He La229 cells to varying degrees,is in this paper,the synthesis of a series of new type of anthracene hydrazone derivatives of the most important discovery.At the molecular level,in view of typical targets:anthracycline-based drugs and DNA topoisomerase,application of competitive bonding and agarose gel electrophores is and fluorescence emission spectrum analysis method was studied more than nine compounds with DNA and topoisomeraseⅠmolecular mechanisms.The results showed that,on the whole,most of the compounds and DNA have some insertion effect,which is consistent with the molecular mechanism of the classical anthracycline anticancer drugs.In agarose gel electrophoresis experiment,this article expounds the compounds have to TopoⅠactivity has significantly inhibitory effect,are strong TopoⅠinhibitors.The subsequent discussion on the anti-tumor molecular mechanism of the target compound provided important data and references.From the perspective of structure-activity relationship,the 9-APZH ligands with substituents and the 9-APZH-Cu ligands with substituents at 9 and 10 positions did not show good biological activity in the two ligands synthesized with anthrahydrazone derivatives of pyrazines.But9,10-APZH-Cu antitumor activity is better.However,9,10-APZH-Cu,the only copper complex of 9,10-APZH-Cu,has better anti-tumor activity,which may be attributed to two reasons:（1）introducing into the pyrazine ring together at 9-and10-to generate a double pyrazine anthrahydrazone structure similar to the symmetrical structure of bisantrene,which not only expands the conjugate area of ligands,but also maintains a good aromatic planar property.（2）further to coordinate with the biological active ion,Cu（Ⅱ）,to achieve a positive synergistic effect.These merits have been proven to be beneficial for their DNA intercalation,thus greatly improved their inhibition activity towards DNA transcription by DNA topoisomerase,two enzymes closely related to both DNA and tumor cell growth.This is also consistent with the experimental results that 9,10-APZH-Cu showed a good inhibitory effect on the proliferation of tumor cells.Therefore,this research work provides scientific data and research basis for the mechanism of action of novel anti-tumor drugs based on anthrahydrazone structure.