Omicron and its variant viruses are responsible for serious contagious respiratory illnesses in humans and bringing about global epidemics. Previously, we identified a 3-O-β-chacotriosyl OA saponin derivative H-1, which displayed broad-spectrum potency against SARS-CoV-2 and its variants tested by targeting the S-mediated entry process. This paper reports the structure optimization of H-1 based on a scaffold-hopping strategy, which resulted in the identification of OA amide derivatives featuring a N, O-disubstituted butterfly-shaped l-hydroxyproline scaffold at the C-3 position of OA with more potent inhibitory activity against Omicron entry. Initial screening using a pseudotype viral system and validation assays using authentic Omicron virus illustrated this set of OA benzyl amide derivatives could block Omicron entry into host cells, thus displaying good antiviral potency. Subsequent investigations confirmed the mechanism of action of the lead compound A28 through the SPR analysis, a Co-IP assay, a cell-cell fusion assay coupled with docking study, in which the N, O-disubstituted trans-l-hydroxyproline fragment as a "chacotrioside mimic" played an important role in displaying the success of this evolution strategy. These findings provide a core hopping-inspired novel SARS-CoV-2 fusion inhibitor for COVID-19 treatment.
