Halogenated Naphthoquinones Inhibit Biofilm Formation and Virulence in Drug-Resistant Candida with ROS-Associated Antifungal Activity

Antifungal resistance in Candida species, particularly in biofilm-associated infections, poses a major clinical challenge. We evaluated a series of halogenated 1,4-naphthoquinones for their antifungal, antibiofilm, and antivirulence activities against drug-resistant Candida species. Among the derivatives tested, 2,3-dichloro-5,8-dihydroxy-1,4-naphthoquinone (compound #10) emerged as the most potent, exhibiting strong activity against both C. albicans and non-albicans Candida strains. Notably, #10 at 1 μg/mL showed rapid fungicidal activity, with no viable cells detected within 15 min, and inhibited biofilm formation at the same concentration. Cell aggregation and hyphal development were also reduced at the sub-MIC of 0.5 μg/mL. Structure–activity comparisons suggest that hydroxyl substitution further enhances activity beyond halogenation alone. Mechanistically, #10 induced intracellular reactive oxygen species (ROS) production at low concentrations and was associated with rapid fungicidal activity and suppression of virulence-associated gene expression, including those involved in adhesion, hyphal morphogenesis, and biofilm regulation. Toxicity was further evaluated across plant, invertebrate, mammalian cell, and rat models. These findings identify #10 as a promising lead candidate for the development of antifungal agents targeting biofilm-associated and drug-resistant Candida infections.