To address the urgent challenge of antimicrobial resistance, a series of twenty novel C-28 modified betulinic acid derivatives was designed and synthesized. Several derivatives, particularly 3b, 3d, 3e, and 3o, displayed notable antibacterial activity against Gram-positive bacteria, including Staphylococcus aureus and vancomycin-resistant Staphylococcus aureus (VRSA). The most active compound, 3d, was subjected to further mechanistic evaluation: it produced concentration-dependent inhibition zones in Oxford cup assays, exhibited bactericidal kinetics in time-kill studies, and significantly suppressed biofilm formation. Molecular docking suggested that the anti-biofilm activity of 3d may be mediated through binding to the staphylococcal accessory regulator A (SarA), a key transcriptional regulator of biofilm formation. The molecular dynamics study provided additional confirmation of the effective binding between 3d and SarA. These results highlight compound 3d as a promising lead for the development of novel anti-biofilm agents targeting drug-resistant Gram-positive infections.

The escalating crisis of bacterial antimicrobial resistance necessitates the discovery of novel antibacterial agents with distinct mechanisms of action. Ursolic acid (UA), a naturally abundant pentacyclic triterpenoid, serves as a promising scaffold for structural modification. In this study, a series of 26 novel UA derivatives (A1–A7 and B1–B19) were designed and synthesized by introducing various substituents at the C-28 carboxyl group via a chloroacetyl chloride linker. Their antibacterial activities were evaluated against S. aureus, S. epidermidis, E. coli, and P. aeruginosa using the microbroth dilution method. Among them, compound B1 exhibited the most potent activity, with a minimum inhibitory concentration (MIC) of 37.5 μg/mL against S. aureus and 75 μg/mL against E. coli. Antibacterial kinetic and time-kill curve assays confirmed the sustained bactericidal effect of B1. Furthermore, B1 significantly inhibited biofilm formation in both S. aureus and E. coli in a time-dependent manner. Molecular docking studies revealed that B1 binds spontaneously to the S. aureus SarA protein through three hydrogen bonds and π-π stacking interactions, providing a structural basis for its antibacterial and anti-biofilm activities. This study demonstrates that piperazine-modified UA derivative B1 is a promising antibacterial candidate and offers a new strategy for the structural optimization of ursolic acid.