Designing Piperlongumine-and Curcumin-inspired Glutathione S-transferase Inhibitors And Anticancer Agents By An Electrophilicity-based Strategy

Compared with normal cells,cancer cells harbor a plethora of different biochemical properties,such as overexpression of glutathione S-transferases（GSTs）and increased levels of reactive oxygen species（ROS）.Cancer cells can develop drug resistance by overexpressing GSTs.Therefore,developing GST inhibitors as sensitizers has been proposed as a potential approach to overcome multiple drug resistance.On the other hand,cancer cells exploit increased levels of ROS to maintain their malignant phenotypes,and are more dependent on a “redox adaptation” mechanism to keep levels of ROS within the range that allows them to escape death under the ROS stress.As such,they are more vulnerable to further ROS generation.The contradictory role of ROS in cancer cells is called as “live by the sword,die by the sword”.This difference reinforces the idea that contrary to a traditional antioxidant therapy,further ROS generation induced by prooxidants could selectively kill cancer cells.The concepts of electrophilicity and nucleophilicity constitute basic principles of organic chemistry.The attack of a nucleophile at an electrophilic center leads to the formation of a new covalent bond.In nature,however,nucleophile and electrophile appear to be well split up.Binding of biologically active electrophilic natural products to a nucleophilic center in proteins or nucleic acids,allows nature to regulate various cellular processes.Piperlongumine and curcumin,characterized by the presence of two electrophilic Michael acceptor units,have been identified as potent prooxidative anticancer agents.Autophagy,the type II programmed cell death,has become one of the hottest research areas of life science following apoptosis.However,information on designing natural product-inspired GST inhibitors,prooxidative anticancer agents and autophagic activators is rather sparse.Taken together,the current conditions beg the following question: whether is it feasible to design piperlongumine-and curcumin-inspired GST inhibitors,prooxidative anticancer agents and autophagic activators by an electrophilicity-based strategy? Therefore,in this work,we constructed a series of piperlongumine-and curcumin-directed electrophiles and evaluated their GST-inhibitory（in the second and fourth parts）and prooxidative anticancer activities against cancer cells（in the third,fifth and sixth parts）.The main contents are summarized as follows:（1）We designed and synthesized thirteen analogs of piperlongumine by an electrophilicity-based strategy,and evaluated their GST-inhibitory activity.The results indicate that the electrophilicity plays a vital role in the GST-inhibitory activity.Among which,PL-13,characterized by the presence of a para-trifluoromethyl group and an α-chlorine on the aromatic and lactam ring,respectively,surfaced as a novel GST inhibitor to effectively overcome cisplatin resistance by virtue of its Michael acceptor units in lung cancer A549 cells.This work emphasizes a natural product-inspired and electrophilicity-based strategy to develop GST inhibitors.This strategy is exemplified by piperlongumine and its designed analogs.（2）We selected the molecules mentioned in the second part and U2 OS cells as a model,to investigate the feasibility in developing piperlongumine-inspired prooxidative autophagic activators.Compared with piperlongumine,PL-13 exhibits significantly increased cytotoxicity.Mechanistic study uncovers that this molecule promotes H₂O₂ generation by virtue of its Michael acceptor units,resulting in MAPK-mediated autophagic cell death.In this work,we not only clarified the ROS generation induced by PL-13 being upstream event of death of the cells,but also elucidated the type of ROS to be H₂O₂ by partial and complete inhibition of VE and CAT against the above signals,respectively.（3）We synthesized three series of mono-carbonyl curcumin analogs by introducing various substituents（trifluoromethyl,methoxy,and hydroxyl）on the aromatic rings by an electrophilictiy-based strategy and placing the 5-carbon linker（acetone,cyclopentanone or cyclohexanone）to omit β-diketone moiety of curcumin,then investigated their GST-inhibitory activity.Among which,NC-3,characterized by the presence of two meta-trifluoromethyl groups on the aromatic rings and one acetone spacer,surfaced as a lead.This molecule could sensitize cisplatin-resistant lung cancer A549 cells by inhibiting GSTs followed by activation of ASK1-JNK/p38 signaling pathway.（4）Piperidone-containing curcumin analogs favour solubility in aqueous media and potential to cross cellular membranes due to the presence of both a protonated and neutral form in the nitrogen-containing heterocycles.Therefore,we designed and synthesized three series of mono-carbonyl curcumin analogs by placing the 5-carbon linkers（cyclohexanone,piperidone and methylpiperidone）and introducing various substituents（trifluoromethyl and methoxy）on the aromatic rings by an electrophilictiy-based strategy.It was found that compared with curcumin,most of them exhibited significantly increased cytotoxicity against NCI-H460 cells.Especially,CNH-3,bearing two meta-trifluoromethyl groups on the aromatic rings and one piperidone spacer,exhibited the strongest cytotoxicity.Mechanistic study uncovers that this molecule induced intracellular generation of ROS（H₂O₂）by virtue of its Michael acceptor units,resulting in mitochondria-mediated apoptosis and p38-activated autophagic cell death.This work highlights the feasibility in designing curcumin-inspired apoptotic inducers and autophagic activators by an electrophilicity-based strategy.（5）We selected the molecules mentioned in the fourth part and NCI-H460 cells as a model,to investigate the feasibility in developing curcumin-inspired prooxidative cell cycle arrest agents.Some of them were more active than curcumin.Especially CP-5,bearing two ortho-methoxy groups on the aromatic rings and one cyclopentanone spacer,exhibited the strongest cytotoxicity among the test molecules.Mechanistic study uncovers this molecule could induce a Michael acceptor-and ROS-dependent G2/M cell cycle arrest by decreasing the Cdk1 expression via up-regulation of p53 and down-regulation of Cdc25 C.