Amphiphilic cationic 2-morpholinoethanol analogues synthesis and study of N-alkylation role in self-assembly and...

The annual incidence of Candida albicans infection and accompanying mortality has risen due to the ineffectiveness of the existing antifungal drugs. Herein, we synthesized a series of six N-alkylammonium salts derived from 2-morpholinoethanol and assessed their antifungal efficacy against C. albicans. The examined 4-alkyl-4-(2-hydroxyethyl)morpholin-4-ium bromide (NAMEtOH) varies by N-alkyl chain length, which ranges from 8 to 18 carbon atoms. The critical micellar concentration (CMC) was used to determine the self-assembly properties of the molecules. The CMC decreases as the length of the N-alkyl chain increases, indicating that the hydrophobic forces aid in their self-assembly. The antifungal activity deduced from minimum inhibitory concentration (MIC) reveals that the compounds with alkyl chains in N-C₁₈H₃₅ (N-octadecyl) have the lowest MIC (15.63 µM). Conversely, N-C₈H₁₇ (N-octyl) had the highest MIC (1000 µM), indicating that molecular lipophilicity plays a role in the antifungal activity. The mechanistic study showed that 4-octadecyl-4-(2-hydroxyethyl)morpholin-4-ium bromide (N18MEtOH) showed higher membrane permeability, as evidenced by elevating the uptake of propidium iodide. Using the 2',7'-dichlorodihydrofluorescein diacetate assay, we confirmed that the accumulation of reactive oxygen species (ROS) increased following N18MEtOH treatment. Molecular docking of NAMEtOH with lanosterol 14α-demethylase, CYP51, a key enzyme in maintaining membrane integrity, reveals that N18MEtOH has higher binding affinity, primarily through hydrophobic interactions. Molecular dynamics simulation confirms that N18MEtOH-CYP51 forms a stable complex, preferentially in the presence of a cofactor, suggesting that N18MEtOH may influence the activity of important enzymes involved in maintaining fungal structures. The hemolytic assay revealed that NAMEtOH does not damage the human red cells, indicating that it is biocompatible. In summary, the findings shed light on the role of molecular lipophilicity in NAMEtOH antifungal activity, which might be considered for devising safer formulations for treating C. albicans infections.