Discovery of a Caffeic Acid Thioamide with Potent Activity against Pathogenic Fungi and Fluconazole Tolerance-Eliminating Effects in Candida albicans.

BACKGROUND: The increasing incidence of invasive fungal infections, coupled with the rising threat of antifungal resistance and tolerance, has created an urgent demand for novel antifungal agents with distinct mechanisms of action. METHODS: In this study, a series of caffeic acid amide derivatives (59 compounds) from our in‑house library was systematically screened for in vitro antifungal activity against clinically relevant pathogenic fungi. Structure-activity relationship (SAR) analysis was performed to identify key pharmacophoric features. The lead compound F4 was further characterized via checkerboard microdilution assays, biofilm formation inhibition assays, and spot assays to evaluate its combination effects, antibiofilm potential, and ability to eliminate fluconazole (FLC) tolerance. RESULTS: SAR studies revealed that the ortho‑dihydroxy moiety and thioamide substitution are critical determinants of antifungal activity. F4 demonstrated potent and broad‑spectrum activity against Cryptococcus neoformans and Candida glabrata (MIC50 = 0.5-16 μg/mL), and moderate activity against drug‑resistant Candida auris (MIC50 = 1-8 μg/mL), outperforming FLC against certain strains. Furthermore, F4 significantly inhibited biofilm formation in a dose‑dependent manner across multiple species, with strain‑dependent potency relative to FLC. Checkerboard assays indicated additive effects when F4 was combined with FLC against C. auris, C. glabrata, and C. neoformans. Importantly, although F4 showed no synergy and lacked direct growth inhibition in YPD medium (MIC > 64 μg/mL) against Candida albicans SC5314, spot assays demonstrated that F4 at concentrations ≥16 μg/mL effectively eliminated FLC tolerance at 37 °C, a temperature‑dependent effect not observed at 30 °C. CONCLUSION: Compound F4 emerges as a promising lead for antifungal drug development, distinguished by its broad‑spectrum activity, antibiofilm properties, and unique capacity to eliminate FLC tolerance. These findings warrant further mechanistic and in vivo investigations.