Designing Curcumin-inspired Anticancer Agents By Improving Its Stability,Eletrophilicity And Targeting To Mitochondrion

Increased therapeutic activity and selectivity is highly desirable for cancer chemotherapy.An effective strategy is to target an aspect of cancer cells that is not shared by normal cells.Compared with normal cells,cancer cells are generally characterized by elevated levels of reactive oxygen species?ROS?and energy metabolism to meet their malignant phenotypes.Mitochondria are considered as the main source of cellular ROS and also the cells'powerhouse.Additionally,multiple hallmarks of cancer including altered ATP aynthesis,increased ROS levels and mitochondrial membrane potential,have been linked to mitochondria dysfunctions.These unique biochemical properties of cancer cells make them vulnerable to further production of ROS by using prooxidants,and have opened a window for developing prooxidative anticancer agents?PPAs?,especially mitochondrial-targeted PPAs.Curcumin?Cur?,a polyphenolic compound isolated from the rhizome of Curcuma longa,stands out as a promising multi-target anticancer molecule in terms of its Michael acceptor-dependent covalent interaction with nucleophilic centers in proteins.However,its poor stability and low bioavailability restrict the clinical application.This study was thereby aimed at designing curcumin-inspired anticancer agents by improving its stability,eletrophilicity and targeting to mitochondrion.The main contents are summarized as follows:?1?We designed a curcumin analog?Cur-1?to improve its stability by introducing the geminal dimethyl substituents on the active methylene group,and further designed a mitochondria-targeted curcumin analog by introducing triphenylphosphonium cations?TPP⁺?to afford MitoCur-1.Compared with Cur and Cur-1,MitoCur-1 was identified as a stronger cytotoxic agent,and manifested more than 6-fold selectivity toward human hepatoma HepG2 cells over human normal liver L-02 cells with the IC₅₀ values being 1.5 and 9.7?M for HepG2 and L02 cells,respectively.Mechanistic study uncovers that due to its effective accumulate in the mitochondria of Hep G2 cells,MitoCur-1 interferes effectively with energy metabolism including inhibition of mitochondrial oxidative phosphorylation?OXPHOS?,anaerobic glycolysis and ATP production,as well as induces significantly Michael acceptor-dependent intracellular ROS(H₂O₂ and O₂^-·)generation.Higher ROS generation is responsible for more effective G0/G1 arrest and mitochondria-mediated apoptosis by MitoCur-1 than Cur and Cur-1.In contrast,MitoCur-1 was relatively powerless to change the above pathways in L02 cells.This work provides a case of successfully designing natural product-inspired and mitochondria-targeted prooxidative anticancer agents.?2?Activation of nuclear factor erythroid-2-related factor 2?Nrf2?is one of the most important cellular defence mechanisms against oxidative stress,and represents an effective means to prevent the development of cancer.We designed a series of curcumin analogs by introducing the geminal dimethyl substituents on the active methylene group to improve the stability and find more potent Nrf2 activators.And on this basis we also designed a catechol-type Cur-3 as a proelectrophile.Importantly,introduction of the geminal dimethyl substituents but also the catechol moieties improves the stability.Compound 3 with a combination of the geminal dimethyl substituents and the catechol moieties in one compound,was identified as a promising lead molecule in terms of its increased stability and cytoprotective activity against the tert-butyl hydroperoxide?t-BHP?-induced death of HepG2 cells.Mechanism studies indicate that its cytoprotective effects are mediated by activating the Nrf2 signaling pathway in the Michael acceptor-and catechol-dependent manners.Additionally,we verified by using copper and iron ion chelators that the two metal ion-mediated oxidations of Cur-3 to its corresponding electrophilic o-quinone,contribute significantly to its Nrf2-dependent cytoprotection.This work provides an example of successfully designing natural curcumin-directed Nrf2 activators by a stability-increasing and proelectrophilic strategy.?3?We selected the molecules mentioned in the second part to probe their cytotoxic mechanisms in SW620 cells.Among the test molecules,Cur-1,surfaced as a promising lead molecule in terms of its increased stability,electrophilicity and cytotoxicity.Mechanism studies suggest that it induces G2/M cell cycle arrest in SW620 cells by upregulating Cdc25C and Cdk1,down-regulating p21 and cyclin B1as well as inhibiting microtubule polymerization,in a Michael acceptor dependent manner.Interestingly,Cur-1 does induces an obvious ROS(H₂O₂ and O₂^-·)accumulation,but the accumulation is only an effect rather than a cause of its cytotoxicity.?4?We synthesized a battery of curcumin analogs by replacing its active methylene with a pyridine ring to improve its stability and eletrophilicity.Additionally,due to the important role of trifluoromethyl group in improving cellular membrane permeability and metabolism stability,introduction of this group was also applied.Compared with the parent Cur,compound B1 bearing two ortho trifluoromethyl groups on the aromatic rings exhibited increased stability and eletrophilicity and cytotoxicity against human lung adenocarcinoma A549 cells.This molecule was also identified as a stronger Michael acceptor-type ROS inducer to trigger apoptosis of A549 cells through the classical mitochondria pathway.?5?Last,we designed a suite of 4-arylidene curcumin analogues via Knoevenagel condensation to eliminate the active methylene.It was found that Cur-k9,was the most active in inducing death of A549 cells in a Michael acceptor-and ROS-dependent fashion.