Novel 4-(4-substituted Amidobenzyl) Furan-2-(5H)-one Derivatives As Antitumor Agents Based On Topoisomerase Ⅰ Target

Objective: Cancer is one of the global health problems worldwide along with an increasing number of patients every year.Currently the design and synthesis of new drugs for the treatment of cancer is a crucial area of research in medicinal chemistry.Topoisomerase I(TopoⅠ)is a ubiquitous nuclear enzyme that catalyzes transient cleavage and reconnection in DNA,which are associated with DNA replication,transcription,recombination and chromatin remodeling.Evidence suggests that DNA topoisomerase I is an effective target for the discovery of antitumor drugs as a result of its high over-expression in cancer cells.Compounds bearing α,β-unsaturated lactones or 2-furanone have been reported to play a broad spectrum of biological roles,including antitumor,antibacterial,and anti-inflammatory activities,which suggests that the α,β-unsaturated lactone was a promising pharmacophore for antitumor drug design.In addition,Securinine bearing an α,β-unsaturated lactone shows potent antiproliferative activity as a TopoⅠ inhibitor,therefore we simplified the structure of securinine and designed a series of compounds whose structure can be easily synthesized,and investigated their antitumor action to target topoisomerase I.Methods:(1)The design of target compounds: the target compounds were designed by simplifying Securinine and the scaffold hopping.In series I,C-4 of the furanone ring is a linear hydrocarbon,while in series II,C4 of the furanone ring was modified to produce an exocyclic double bond,which has been revealed that exocyclic double bondsare an essential structural feature which increases antitumor activity.(2)The synthesis of target compounds: Series I: 3-Phenylpropanalwas reacted with glyoxylic acid monohydrate to afford(Z)-3-benzyl-4-oxobut-2-enoic acid(1-1).Compound 1-1 was treated with nitrosonitric acid to obtainthep-nitrophenyl compound 1-2,which was reduced to the hydroxyl compound(1-3)with sodium borohydride.Compound 1-3 with N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride(EDCI)afforded 4-(4-nitrobenzyl)furan-2-(5H)-one(1-4),which was reduced to 4-(4-aminobenzyl)-furan-2-(5H)-one(1-5)by iron.This compound(1-5)was treatedwith acyl chloride to afford the A-1~A-7.Series II: Reaction of 1-(bromomethyl)-4-nitrobenzene with 2,4-pentanedione gave 4-(4-nitrophenyl)butan-2-one(2-1),which was treated with glyoxylic acid monohydrate to obtain compound 2-2.This was reacted with p-toluenesulfonic acid to afford 5-methylene-4-(4-nitrobenzyl)furan-2-(5H)-one(2-3).Compound 2-3 was reduced by iron to obtain the amine 2-4,which was treated with the appropriate acyl chloride to afford the target compounds B-1~B-15.(3)Biological activity:(A)TopoⅠ inhibitory activity: TopoⅠ inhibitory assay was used to investigate the TopoⅠ inhibition of the target compounds.(B)Cytotoxicity: The MTT assay was used to evaluate antitumor activity by testing the cytotoxicity against three human cancer cells lines,MCF-7(human breast adenocarcinoma cell line),HeLa(human cervix tumor cell line),A549(adenocarcinomic human alveolar basal epithelial cell line).(C)TopoⅠ inhibitory mechanism: DNA insertting assay,DNA-cleavage assay and electrophoretic mobility shift assay were conducted to investigate TopoⅠ inhibitory mechanism.(4)Molecular docking: In order to determine if the exocyclic double bond is essential to the TopoⅠ inhibitory activity and investigate the interaction between the active compounds and TopoⅠ,a molecular docking study was performed with Sybyl-8 and AMBER software.Results:(1)Chemistry: 22 target compounds were obtained,whose structures were identfied by 1H NMR,13 C NMR,MS and ESI-HRMS.(2)Biological activity:(A)TopoⅠ inhibitory activity: Compounds of series II with the exocyclic double bond generally showed higher inhibitory activity than series I.B-15 was the most active compound among series I and II.(B)Cytotoxicity: B-15 with the highest inhibitory activity,is the most active compound against all three human cancer cell lines.Consistent with the TopoⅠ inhibitory results,compounds of series I,lacking the exocyclic double bond,also generally showed poor inhibitory activity.(C)TopoⅠ inhibitory mechanism: Synthetic compounds can not only stabilize the drug-enzyme-DNA covalent ternary complex as well as camptothecin,but also interfere with the binding between TopoⅠ and DNA.(D)Molecular docking: Compounds in series II could interact with the active pocket of TopoⅠ by p-p stacking or the p-alkyl stacking,which were not observed between compounds in series I and the active pocket of TopoⅠ.The most active compound B-15 fitted well into the active pocket by forming hydrogen bonds.Conclusion:In this thesis,two series of novel 4-(4-substituted amidobenzyl)furan-2-(5H)-one derivatives bearing an α,β-unsaturated lactone moiety,a pharmacophore with potential antitumor activity,were designed,synthesized and evaluated for TopoⅠ inhibitory activity and cytotoxicity against three human cancer cell lines.The rusults of topo I inhibitory activit y and cytotoxicity suggested that introduction of the exocyclic double bond into the α,β-unsaturated lactones 2-furanone moiety was essential to develop 4-(4-substituted amidobenzyl)furan-2-(5H)-one derivatives as TopoⅠ-based antitumor agents.In series II,several compounds showing promising activity as TopoⅠ inhibitors and cytotoxicity,especially B-15,were identified.A series of mechanism studies indicate that our compounds showed dual mechanisms.These results may be useful in the future to guide the design and modification of new furanone derivatives as TopoⅠ inhibitors.