Mono-N-alkylation of Amphotericin B and Nystatin A1 and Its Amides: Effect on the In Vitro Activity, Cytotoxicity and Permeabilization of Model Membranes

<b>Objectives</b>: In 2022, the World Health Organization highlighted the necessity for the development of new antifungal agents. Polyene antibiotics are characterized by a low risk of drug resistance; however, their use is limited by low solubility and severe side effects. <b>Methods</b>: A series of <i>N</i>-alkylated derivatives of amphotericin B and nystatin A₁ as well as their <i>N</i>-(2-hydroxyethyl)amides were synthesized. Their antifungal activity was evaluated against various <i>Candida</i> strains and <i>Aspergillus fumigatus</i> using the broth microdilution method. Cytotoxicity was assessed using an MTT assay on human embryonic kidney cells HEK293 and human skin fibroblast cells hFB-hTERT6, as well as a hemolysis assay on erythrocytes. Membrane activity was analyzed by fluorimetric measurement of calcein leakage from model liposomes. <b>Results</b>: Derivatives containing the <i>N</i>-(hydroxyethyl)amino)ethyl fragment (compounds <b>3</b> and <b>4</b>) exhibited relatively high antifungal activity, as did <i>N</i>-(2-hydroxyethyl)amides <b>5</b> and <b>9</b>. Bis-modified compounds <b>6</b> and <b>10</b> did not outperform their mono-modified analogues in terms of activity or cytotoxicity. The mono-<i>N</i>-alkylated compound <b>3</b> showed the highest activity/toxicity ratio, which correlated well with its selectivity for ergosterol-containing model membranes. <b>Discussion</b>: Combining two successful modifications does not necessarily improve the activity/toxicity ratio of polyenes. Further studies can be performed for the optimization of carboxyl group of <b>3</b>.