Advances in Azole Antifungal Agents: From Structural Foundations to Resistance Challenges

Azoles are the most widely used antifungal agents in clinical practice, with important applications in human medicine, agriculture, and veterinary science. This review systematically summarizes the structural features, classification, six-decade development history, mechanisms of action, antifungal spectra, and resistance mechanisms of azole antifungals. Based on the number of nitrogen atoms in the azole ring, these compounds are divided into imidazoles (two nitrogens) and triazoles (three nitrogens), which differ markedly in target enzyme selectivity, pharmacokinetic properties, and safety. Tetrazoles, as a new generation of drugs, exhibit lower basicity and reduced inhibition of human CYP450 enzymes, and have demonstrated improved selectivity and better safety profiles in preclinical studies; however, their clinical advantages still require further validation. Since the introduction of the first clinically used imidazole clotrimazole in 1969, azoles have evolved from topical formulations to orally or intravenously administrable triazoles across four generations, each iteration broadening the antifungal spectrum and improving safety and pharmacokinetics. Azoles act by inhibiting fungal sterol 14α-demethylase (CYP51), thereby blocking ergosterol biosynthesis and disrupting fungal cell membrane integrity. Their spectrum covers most clinically relevant fungi, but significant inter-generational differences exist. However, the widespread emergence of resistance mechanisms—including efflux pump overexpression, target enzyme alterations, biofilm formation, and other mechanisms—combined with cross-resistance risks between agricultural azole fungicides and clinical azoles, severely compromises their clinical utility. This review also discusses future directions, including novel tetrazoles, dual/multi-target inhibitors, AI-assisted drug design, and cross-sectoral resistance control within the One Health framework.