Antifungal Activity of Polyalthic Acid Analogs Against <i>Cryptococcus neoformans</i> Involves Production of Reactive Oxygen Species (ROS) and Disruption of Cell Membrane Integrity

Invasive fungal infections have a high mortality rate, and the current antifungal arsenal is limited by toxicity, resistance, and the small number of available drugs. Polyalthic acid is a natural product with modest antifungal activity and low toxicity. Therefore, it is a suitable candidate for structural modification to enhance its antifungal activity. Six polyalthic acid analogs were synthesized, and their activity was tested against Candida albicans and Cryptococcus neoformans. The best compound was 2b with MICs of 21.8 and 12.3 µg/mL, respectively. We further investigated the mechanism of action of 2b by assessing its effects on ROS production and plasma membrane integrity in C. neoformans. Intracellular ROS levels were measured using H₂DCFH-DA, while membrane integrity was evaluated using DAPI staining. A significant increase in ROS generation was detected at concentrations ≥ 4 µg/mL as early as 1 h after treatment. DAPI intensity and focal staining pattern were observed at all concentrations tested after 7.5 h of treatment. These findings indicated that the antifungal activity of 2b involves reactive oxygen species production and disruption of membrane integrity. The current study supports 2b as a potential lead compound for the development of new antifungal agents to address the limitations of current therapies.