Synthesis And Cytotoxic Evaluation Of Halogenated ?-exo-methylene-lactones

Objective:Alpha-exo-methylene-lactone is a natural product which possesses various bioactivity,and is important in drug discovery.This research developed a simple and efficient method to synthesize a series of halogenated?-exo-methylene-lactones by using dienoic acids as substrates.The cytotoxic activities of these compounds against non-small cell lung cancer cells?NSCLC?are tested.Methods:A collection of halogenated?-exo-methylene-?-butyrolactones were synthesized by fluorination,bromination,chlorination and iodization of dienoic acid substrates.Based on the?-exo-methylene-lactone moiety,study of the lactone ring was broadened to six-member ring and seven-member ring?-exo-methylene-lactones which were synthesized by using the substrates with extended chain length,and study was furthered in?-exo-methylene-lactones with two chiral centers and spiro?-exo-methylene-lactones which were synthesized by using the substrates with different functional groups substituted the double bond away from the carbonyl.The compounds were subjected to in vitro cytotoxic evaluation against NSCLC cells.¹H-NMR was used to perform mechanism study for in vitro reaction of?-exo-methylene-lactone with GSH.Results:1.By using dienoic acid as substrates,32?-exo-methylene-lactones were successfully synthesized in efficiency with yield up to 70%-99%?except 7 and 16?,among which2a-2k,?R?-3a,?S?-3a,4,5a,5b,6,7 were?-exo-methylene-?-butyrolactones,enantiomers of 9a were?-exo-methylene-?-valerolactones,11 was seven-member ring?-exo-methylene-lactone,13,15a,15b,16 were?-exo-methylene-?-butyrolactones with two chiral centers,18 and 20a-20c were spiro?-exo-methylene-?-butyrolactones.2.Bioassay results showed that compound 2a-2j had significant cytotoxic activity on H1975 NSCLC cells bearing L858R/T709M mutant EGFR and A431 wild type cells in vitro.The structure-activity relationship suggested more potent antiproliferative effect be found on both cells when the benzene ring was substituted in para-position,and the cytotoxic activity was enhanced when the benzene ring was substituted by electron-withdrawing functional groups,but was decreased by electron-donating functional groups.3.A stronger activity was observed in bromo-?-exo-methylene-?-butyrolactones than in fluoro-and chloro-ones.In addition,iodo-?-exo-methylene-?-butyrolactones exhibited potent antiproliferative activity against mutant H1795 cells,which was three-fold efficacious than that against the wild type A431 cells.4.The cytotoxic activity of bromo-?-exo-methylene-?-butyrolactone was stronger than bromo-?-exo-methylene-?-valerolactone,and seven-member ring?-exo-methylene-lactone led to a decreased cytotoxic activity.5.For?-exo-methylene-?-butyrolactone bearing two chiral centers,compound 15a selectively inhibited mutant H1975 cells with a lower IC₅₀ value,and spiro?-exo-methylene-?-butyrolactone 20b and 20c showed comparable pharmacological effect compared with Parthenolide.6.Although compound 2h,5a,5b,15a exhibited comparable cytotoxic activity on both HCC827 and A546 cells,they were observed with low selectivity on normal HL7702cells,and showed unpromising IC₅₀values against EGFR?L858R/T790M?and wild type enzymes.7.GSH conjugation assay was performed to investigate the intrinsic reactivity mechanism,the results illustrated that the reactivity of double-bond warhead of 5a with GSH by Michael addition was more reactive than that of the iodide group with GSH by S_N2 reaction,suggesting a weaker reactivity of iodide group than that of the?-exo-methylene-lactone moiety.Conclusion:In this study,a series of halogenated?-exo-methylene-lactones were synthesized.Their cytotoxic activity against non-small cell lung cancer cells were tevaluated in vitro.The brominated?-exo-methylene-?-butyrolactone structure was the most potent core for the anticancer effect.In addition,phenyl groups substituted in chiral centers were the essential moiety for its bioactivity.For example,compound 15a was able to selectively inhibit mutant non-small cell lung cancer cells.However,this series of compounds inhibited normal liver HL7702 cells with no selection while exhibiting cytotoxic activities against HCC827 and A549 NSCLC cells,and had low inhibition against EGFR?L858R/T790?and EGFR?WT?enzyme activities as well.GSH conjugation assay suggested an irreversible covalent binding formed between the double bond warhead in lactone moiety and thiol-containing nucleophiles by Michael addition led to cytotoxicity against tumor cells.