Syntheses And Antitumor Activities Of Diphyllin Glycosides Targeting Tumor Acidic Microenvironment

ObjectiveTo synthesize two natural antitumor-active diphyllin glycosides(Cleistanthin A and Cleistanthoside A), modify their structures and study on the structure-activity relationships, as well as their molecular targets.Methods1) Chemistry: A five-step process, including Adol and Diels-Alder reactions, was applied to obtain diphyllin from 6-bromovertraldehyde with overall 42.5% yield. Then, two natural diphyllin glycosides and twelve analogues were synthesized from diphyllin and D-xylose in twelve steps involving phase transfer catalysis(PTC) and Schmidt glycosylations. The chemical structures of these glycosides were confirmed by 1H NMR, 13 C NMR and HRMS.2) Antitumor activities: These glycosides were evaluated for their anti-proliferative activities against A549、HCT116、HepG2 and RM tumor cell lines by MTT assay.3) Antitumor mechanism: These glycosides were investigated for V-ATPase inhibition by V-ATPase activity assay kit and evaluated for inhibitory activities of lysosomal acidity in HepG2 cells by cell fluorescence technique.Results1) The total syntheses of two natural diphyllin glycosides(Cleistanthin A and Cleistanthoside A) have been accomplished, then twelve glycosides analogues were achieved by structure modification. Their structures were confirmed by 1H NMR, 13 C NMR and HRMS.2) The IC50 values of these glycosides on four tumor cell lines A549、HCT116、HepG2 and RM were obtained by MTT assay. All diphyllin glycosides displayed strong anti-proliferative activities against tumor cell lines, especially HCT116 and HepG2, with IC50 values of 0.02-3.8 μM.3) Diphyllin glycosides showed potent V-ATPase inhibitory activity. The V-ATPase activities dropped to below 20% after HepG2 was treated with diphyllin glycosides at 100 nM.4) Lysosomal acidity was completely reversed after HepG2 was treated with diphyllin glycosides at 100 nM.ConclusionsTwo natural diphyllin glycosides(Cleistanthin A and Cleistanthoside A) and twelve glycosides analogues were synthesized successfully. The majority of diphyllin glycosides displayed strong anti-proliferative activities. Diphyllin glycosides exert their antitumor activities mainly through inhibiting V-ATPase to reverse tumor acidic microenvironment.