Synthesis Of Mitochondria-Targeted Coumarin 3-Formamide Derivatives As Anti-Breast Cancer Agents

Breast cancer?BC?is a worldwide public health dilemma with the highest rates of morbidity and mortality among women.Among BC subtypes,triple negative breast cancer?TNBC?is generally characterized by aggressive features,high recurrence and mortality rate.Since there lake of expression the three types of receptor?ER,PR,HER-2?,the therapies of targeting endocrine or human epidermal growth factor receptor are invalid.Hence,lacking of specific molecular targets in the treatment of TNBC has been a huge barrier.It has acknowledged that mitochondrial alterations,including increased oxidative stress,aberrant apoptotic machinery,increased membrane potential and deleted or mutated mtDNA have detected in BC.Undoubtedly,mitochondria play an important role in the occurrence and development of TNBC,which can be used as a therapeutic targets for breast cancer.Coumarins widely distributed in nature and have a broad spectrum of activities,such as anti-hypertension,anti-coagulation,anti-bacterial,anti-viral,anti-cancer,anti-inflammatory and analgesics.Additionally,they also have very rare nephrotoxicity,hepatotoxicity,cardiotoxicity,skin toxicity and other side effects.Therefore,they have attracted the attentions of researchers and been considered as the privilege structure.In the previous work,we synthesized a series of derivatives with high selectivity for cancer cells by linking the cinnamic acid and the targeted-mitochondrial carrier triphenylphosphine?TPP?through the alkyl chain.Subsequently,we hybridized 7-diethylaminocoumarin-3-carboxamide of targeted-mitochondrial fluorescent carrier with the pharmacophore cinnamic acid to obtain a series of mitochondria targeted derivative,which can be monitored at the cellular level in real time.Further,in order to obtain coumarin derivatives with higher selectivity and higher activity for breast cancer cells,7-diethylaminocoumarin-3-carboxamide was used as a pharmacophore to hybridize with TPP.The research results have be obtained as following:?1?Design of mitochondria-targeted compounds:7-diethyl-amino-coumarin formamide was chosen as the pharmacophore to carry out structural transformation,we synthesized 8a-d derivatives of coumarin-3-formyl-n-butyl quaternary phosphine salt.?2?Evaluation of anti-proliferation activity and analysis of structure-cytotoxicity relationship:we evaluate the anti-proliferative activity of the title compounds against MCF-7 cells?ER-positive breast cancer cells?,MDA-MB-231 cells?triple negative breast?,SK-BR-3 cells?human breast cancer cells with high HER2 expression?,and normal cell line MCF-10A by MTT assay.The results revealed that 8a and 8b exhibited high anti-proliferative activity on three tumor cells,and had no significant toxicity to normal cell line?MCF-10A?.Compound 8b displayed the best activity(IC₅₀=3.03?M)against MDA-MB-231 cells.It can be inferred that when the C-3 position of coumarin is an amide ester,the antitumor activity is strongest.?3?Compound 8b induced mitochondrial dysfunction and apoptosis:Laser confocal microscopy revealed that 8b mainly distributed in the mitochondria.Flow cytometer analysis revealed that 8b entered the cells in a concentration-dependent manner.Subsequently,it induced burst of ROS,depolarization of??_m,and apoptosis.?4?The addition reaction of 8b with glutathione:By UV-Vis spectroscopy and HRMS analysis,we found that 8b had an addition reaction with glutathione;Subsequently,with the depletion of cellular GSH by agent BSO,the abilities of 8b to suppress the cell growth and depolarize??_m were weakened in MDA-MB-231 cells,suggesting that cytotoxic activity of 8b is closely related to GSH.?5?Inhibition of TrxR2:Through the detection of TrxR activity of the whole cell and the extracted mitochondria,it was found that 8b inhibited TrxR activity significantly in the mitochondria extracts,and with less effect on the total cell TrxR activity.This discovery was further confirmed by detection of protein expression level.Obviously,8b significantly inhibited the expression of TrxR2 protein in mitochondria,but had little effect on TrxR1 protein in cytoplasm.Besides,the results of molecular docking experiments showed that there was a hydrogen bond between 8b and the SER-24 residue of mitochondrial TrxR2 protein,and the binding energy was-49.76 kal/mol.Conclusively,we found the mitochondria-targeted coumarin derivative 8b displayed a high anti-TNBC activity and acted on TrxR2,which might shed the light on the further study of mitochondria-targeted coumarin derivatives.