Synthesis of Novel Isoxazoline Derivatives with Selective Inhibition of the Drug Efflux Pump Mdr1 to Reverse Drug Resistance of Candida albicans In Vitro and In Vivo.

The transcriptional upregulation of drug efflux pumps constitutes a primary mechanism of azole resistance acquired by diverse fungal pathogens. This overexpression diminishes intracellular azole accumulation, thereby compromising drug efficacy and contributing to therapeutic failure. The coadministration of azole antifungals with efflux pump inhibitors presents a promising therapeutic strategy to combat resistant fungal infections. In the search for novel antifungal drugs, we obtained 49 isoxazoline derivatives through structural modification of the isoxazolin-3-one skeleton. Among them, 6ao showed potent synergistic activity with fluconazole (FLC) against azole-resistant Candida albicans (FICI = 0.094). In the Galleria mellonella larvae and mice model, 6ao exhibited potent in vivo therapeutic efficacy. Interestingly, mechanistic investigations revealed that 6ao, when combined with FLC, reduced the expression of MDR1, an efflux pump gene, in the multidrug-resistant clinical isolate CA632. Taken together, these results identify 6ao as a promising lead candidate for combating azole-resistant Candida infections.