Discovery and Structure‐Activity Relationship Study of Pyrazolyl Indolenine Derivatives as <i>Staphylococcus aureus</i> Biofilm Inhibitors

Staphylococcus aureus-associated infections pose a significant clinical challenge due to biofilm formation, which contributes to antibiotic resistance and persistent infections. The prevalence of methicillin-resistant S. aureus (MRSA) further exacerbates this issue, underscoring the urgent need for effective therapeutic strategies. In this study, we report a potent and selective second-generation MRSA biofilm inhibitor (4ad), which showed a minimum biofilm inhibition concentration (MBIC₅₀) of 0.78 µg/mL and demonstrated ≥ 128-fold selectivity for biofilm inhibition over planktonic growth. Through structural optimisation and fragmentation, we further identified a truncated analogue (5a) that effectively eradicated a preformed MRSA biofilm, with a minimum biofilm eradication concentration (MBEC₅₀) of 0.78 µg/mL-outperforming both the first-generation hit (4e) and the clinically used antibiotics oxacillin and vancomycin. Notably, both 4ad and 5a exhibited no significant impact on planktonic bacterial viability or Vero cell cytotoxicity. Given the broad-spectrum antibiofilm activity of 4e against S. aureus, we investigated its combinatorial effects with antibiotics. 4e demonstrated additive effects in combination with vancomycin, erythromycin, and amoxicillin. Importantly, both structure-activity relationship (SAR) and quantitative structure-activity relationship (QSAR) analyses identified key structural features associated with antibiofilm activity. Collectively, these findings support further development of pyrazolyl indolenine derivatives and mechanistic studies aimed at combating antibiotic-resistant S. aureus biofilm.