Design,Synthesis And Mechanism Of Action Of Anthraquinone Derivatives As Potential Anticancer Drugs

Anthraquinone anti-cancer drugs are widely used and highly effective chemotherapy drugs,which are mainly used clinically in the treatment of hematological malignancies and solid tumors such as lymphoma and ovarian cancer.Studies have shown that the rigid ring structure of anthraquinones,which consists of a planar tricyclic aromatic anthracene system,can be intercalated in DNA base pairs and has the ability to bind to the active site of Topoisomerase II（Topo II）,thus interfering with normal DNA replication and transcription as well as catalyzing DNA double-strand breaks（DSB）to kill tumor cells.However,in the current clinical application,the problems of drug resistance and cardiotoxicity of traditional anthraquinone anti-cancer drugs greatly limit their application prospects,so the design and development of novel anthraquinone anticancer drugs is very necessary.In this work,two novel imidazotetrazine-anthraquinone derivatives C-1 and C-9 with dual functions of DNA intercalation and methylation were designed,screened and synthesized.Their mechanism of action was investigated using electrochemical,molecular docking,electrophoresis and spectroscopic methods.We first designed a series of bifunctional antitumor candidates by introducing the imidazotetrazine structure,which can cause alkylation damage to DNA,into the parent nucleus of anthraquinone to achieve the goal of introducing alkylation damage while intercalating DNA to catalyze DNA double-strand breakage.Subsequently,two small molecules with high affinity for DNA and Topo II（C-1 and C-9）,were screened by molecular docking with MOE software to simulate the interaction of the candidate compounds with DNA and Topo II,using binding energy as the evaluation criterion.The synthetic route was rationally designed and two small molecule compounds were successfully synthesized.The redox mechanisms were investigated by cyclic voltammetry（CV）,square wave voltammetry（SWV）,and differential pulse voltammetry（DPV）.Both reduction processes were found to be controlled by diffusion.The redox processes of C-1 and C-9 were similar,involving reversible redox processes on the anthraquinone ring and irreversible oxidation and reduction processes on the imidazolotetrazine ring.Then we modeled the three-dimensional interaction of two small molecules with DNA and Topo II by molecular docking,and found that the two compounds could not only bind to DNA by intercalating in DNA base pairs,but also interact with Topo II by hydrogen bonding with amino acid residues of Topo II.The interaction of two small molecule compounds with DNA was tested in DNA solution using differential pulse voltammetry（DPV）,and both were found to be intercalated in the DNA strand and to cause oxidative damage to DNA.DNA electrochemical biosensors were established to study the interaction of two small molecule compounds with DNA,and the results were in general agreement with the relevant tests in DNA solution.DNA electrochemical biosensors were established to study the interaction of two small molecule compounds with DNA,and the results were in general agreement with the relevant tests in DNA solution.The interaction of C-1 and C-9 with DNA was further validated using UV-Vis absorption titration experiments and fluorescence emission titration experiments,demonstrating that both interact with DNA in an intercalated and spontaneous manner.The binding constants of C-1 and C-9 were 9.31×10⁵ M^-1 and 12.74×10⁵ M^-1,respectively.Agarose gel electrophoresis studies showed that two small molecule compounds can produce deconvolution and cleavage damage to DNA at the micromolar level.The results of in vitro anti-tumor activity assay showed that C-1 and C-9 had strong anti-proliferative effects on human non-small cell lung cancer cells（A549）and mouse glioblastoma cells（GL261）.For the A549 cell line,the IC₅₀ values for both compounds were 6.31μM（C-1）and 9.71μM（C-9）,respectively.For the GL261 cell line,the IC₅₀ values for both compounds were 7.40μM（C-1）and10.86μM（C-9）,respectively.This study provides new ideas for the molecular design and development of novel anthraquinone-based anticancer drugs,and also provides a template for the pre-study of lead compounds.