The C-3-substituted and C-5-substituted 20-deoxyingenol monoesters are important active components in Euphorbiaceae plants. Nonetheless, their similar physical properties make them difficult to distinguish. The present study developed fast and efficient rules for identifying the esterification sites of 20-deoxyingenol based on a series of chemical syntheses of monoesters and literature research, utilizing NMR spectroscopy, optical rotation analysis, and chromatographic retention behavior. In addition, a series of 20-deoxyingenol ether derivatives, including 1,3,4-oxadiazole derivatives, were synthesized. The cytotoxic activities of all 20-deoxyingenol derivatives were evaluated on A549 and HepG2 cell lines. Notably, 20-deoxyingenol 1,3,4-oxadiazole derivative 22 (IC 50 = 8.8 μM) exhibited significant anticancer activity against HepG2 cells with low toxicity to normal cells (IC 50 > 50 μM), making it a promising compound. We investigated the potential anticancer mechanism of compound 22 by examining protein expression changes in HepG2 cells using quantitative proteomics. Our findings indicated that 22 induced G1/S phase cell cycle arrest and, In a dose-dependent manner, inhibited CDK4 and CyclinD1 expression while upregulating P21. Moreover, 22 promoted the accumulation of autophagosomes and the proteins LC3 and PINK1, enhancing autophagy and mitophagy in HepG2 cells. Collectively, compound 22 might serve as a novel autophagy inhibitor with anticancer properties.

Structural characteristics-guided investigation of Ailanthus altissima (Mill.) Swingle resulted in the isolation and identification of seven undescribed potential Michael reaction acceptors (1–7). Ailanlactone A (1) possesses an unusual 1,7-epoxy-11,12-seco quassinoid core. Ailanterpene B (6) was a rare guaianolide-type sesquiterpene with a 5/6/6/6-fused skeleton. Their structures were determined through extensive analysis of physiochemical and spectroscopic data, quantum chemical calculations, and single crystal X-ray crystallo-graphic technology using Cu Kα radiation. The cytotoxic activities against HepG2 and Hep3B cells of isolates were determined. En-couragingly, ailanaltiolide K (4) showed significant cytotoxicity against Hep3B cells with IC50 values of 1.41 ± 0.21 μM, whose covalent binding mode was uncovered in silico.