Time-of-addition study of compound 1. The compound was added to each well (doses ranging from 100 μM to 0.2 μM) at designated time points immediately before (−0.5 h) or after (0, 2, 4, 6, and 8 h) inoculation of C. albicans DAY185 into microplates. Following 48 h of incubation, the wells were washed twice with PBS and the extent of biofilm formation determined by XTT assay. The 50% inhibitory concentration (IC50) value for biofilm inhibition was calculated using GraphPad Prism software. The experiment was replicated three times with each treatment tested in triplicate during each trial. The effects of single doses of compound 1 at these time points are shown in the Supporting Information, Figures S42 and S43. The difference in the relative potency of 1 at the −0.5 and 0 h addition time points is due to the delay in solubilization of 1 in aqueous medium at room temperature (25 °C).