Design, Synthesis, and Anti-Biofilm Activity of C-28 Modified Betulinic Acid Derivatives Targeting SarA in Drug-Resistant Staphylococcus aureus

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 <b>3b</b>, <b>3d</b>, <b>3e</b>, and <b>3o</b>, displayed notable antibacterial activity against Gram-positive bacteria, including <i>Staphylococcus aureus</i> and vancomycin-resistant <i>Staphylococcus aureus</i> (VRSA). The most active compound, <b>3d</b>, 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 <b>3d</b> 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 <b>3d</b> and SarA. These results highlight compound <b>3d</b> as a promising lead for the development of novel anti-biofilm agents targeting drug-resistant Gram-positive infections.