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

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 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.