Synthesis Of Mitochondria-targeted Coumarin Formamide-Cinnamic Acid Hybrids As Anticancer Agents

Organelle Mitochondria are acting as the power houses of cells and the control center for programmed cell death.Compared to normal cellular mitochondria,cancer cells have extensive variations in mitochondria,such as higher membrane potential（Δψ_m）,damaged metabolic pathways of oxidative phosphorylation,excess production of reactive oxygen species（ROS）,and decreased response to pro-apoptotic stimulate.Therefore,mitochondria have been recognized as one of the most important targets for new drug design in cancer.The natural product cinnamic acids have a broad spectrum of pharmacological activities,such as anti-tumor,anti-rheumatic,anti-inflammatory,anti-inflammatory,anti-aging dementia and et al.Previously,our research group has synthesized the mitochondria-targeted cinnamic acids by covalently tying the triphenylphosphonium cation（TPP）as a targeting carrier to cinnamic acids by alkyl chain,which displayed the enhanced cytotoxicity against human hepatoblastoma HepG2 cells and lower toxicity to the normal liver cells LO2.However,the MitoHCAs without fluorescence labeling cannot be used for the mitochondrial visualization and track the therapeutic effect simultaneously.Recently,thecumulatingevidencehasdemonstratedthatthe fluorophore7-（Diethylamino）coumarin-3-carboxamide can deliver the drugs specifically to mitochondria.Hence,we designed and synthesized of a series of fluorescent cinnamides by the employment of mitochondria-targeted fluorophore coumarin-3-carboxamide.And the research contents cover the mitochondrial localization,cytotoxicity screening and mechanism investigation.The main findings are as following:（1）Design and synthesis of the hybrids:we employed cinnamic acid（β-phenylacrylic acid）as the bioactive fraction whose structural modification involved the variations in the number and position of the methoxyl group on aryl ring,the introductions of substituted aryl group into theα-position of the olefinic bond,and reducing the olefinic bond to a single bond.In addition,piperazine,ethylenediamine,butanediamine,and hexamethylenediamine were used as the linkers,and 21 target compounds were produced by the amide reaction.All the compounds were identified by ¹H NMR,¹³C NMR and HRMS,including the purities determined by HPLC（≥95%）.（2）Cytotoxic activity screening and structure-activity relationship analysis:by using the MTT method,the cytotoxic activities were measured.The cell lines used are:Hela（human cervical cancer cells）,HepG2（human liver cancer cells）,MCF-7（human breast cancer cells）,HL-60（human acute promyelocytic leukemia cells）and human normal cell PBMC（human peripheral blood mononuclear cells）.The results showed that the hybrids exhibited high cytotoxic activity against HL-60 cells.Among them,the 48 h-IC₅₀ of 18c toward HL-60 cells was 9.10μM,while 18c showed no toxicity to normal cells PBMC at a dose of 50μM after 48 h treatment.Besides,the structure-cytotoxicity activity relationships are obtained.（3）Cytotoxicity mechanism of 18c:by laser confocal microscopy and flow cytometry analysis,we found that 18c could rapidly enter HL-60 cells and specifically localize in mitochondria.Besides,18c induced the burst of ROS,depletion of Δψ_m,occurrence of apoptosis and cell cycle arrest.（4）A thioether addition reaction of 18c with cysteamine:the presence of a thioether addition reaction was confirmed by UV-Vis spectroscopy and HRMS analysis.These results suggested that 18c might react with the sulfhydryl within mitochondria,which would improve its retention ability in mitochondria.Taken together,we found the highly bioactive compound 18c as anti-leukemia agent,and explored its primary anti-cancer mechanism,which might shed the light on the further studying.