Discovery of novel azole derivatives with benzanilide-containing hydrophobic side chains for the treatment of drug-resistant fungal infections

As fungal resistance to existing antifungal drugs continues to rise, there is an urgent need for new drugs with anti-resistance activity. In this study, a series of newly designed and synthesized benzanilide-containing azoles exhibited promising antifungal activity against fluconazole-sensitive <i>Candida albicans</i>. Importantly, the newly synthesized compounds also displayed potent activity against azole-resistant strains, surpassing the performance of the positive control fluconazole. This suggests that these compounds may have the potential to combat drug-resistant fungal infections. Subsequent studies on the antifungal mechanisms revealed that the compound can inhibit fungal CYP51, thereby blocking ergosterol biosynthesis. Morphological observations of fungal cells further confirmed CYP51 as the target of action. Resistance mechanisms elucidated that these compounds can inhibit biofilm formation and the expression of resistance-related genes ERG11 and efflux pump gene CDR1, thereby reversing resistance. Meanwhile, the most potent compound A11 demonstrated the ability to stimulate reactive oxygen species, thereby exhibiting potent fungicidal activity. Furthermore, the compound A11 also showed good stability in liver microsomes and plasma metabolism. Cytotoxicity studies demonstrated low toxicity of the compounds against MRC-5 cells, indicating their potential safety for therapeutic use. <i>In vivo</i> experimental results indicated that the representative compound A11 significantly inhibited fungal infections caused by resistant strains. Molecular docking studies further supported the efficacy of compound A11, showing its ability to bind to <i>Candida albicans</i> CYP51. These findings highlight the promising antifungal activity and minimal cytotoxicity of the benzanilide-containing azoles, making them potential candidates for the treatment of drug-resistant fungal infections.