Second Generation of 2,2′-Di(indol-3-yl)ethanamines as Antileishmanial Agents: Phenotypic Hit Identification and Pharmacophore Studies

: The urgent need for new antileishmanial drugs persists due to resistance and toxicity associated with current treatments. Although bisindoles represent a promising chemotype, our first-generation 2,2'-di(indol-3-yl)ethanamines, inspired by a natural marine alkaloid, displayed significant cytotoxicity despite potent activity against L. infantum. To address this limitation, we designed and synthesized a second-generation series in which the indole bromine substituents were removed, and N-methylation was prioritized as a strategy to mitigate cytotoxicity. Compounds 1e, 2b, 2f, 3d, and 3e emerged as promising hits, exhibiting potent activity against L. infantum promastigotes (IC50 = 0.6-3 μM) and markedly improved selectivity indices (SI = 11-66) in THP-1 cells. These compounds retained efficacy in an in vitro infection model, with 1e, 2b, and 2f displaying IC50 values below 10 μM against intracellular amastigotes. A pharmacophore model derived from URB1483 and 2f was generated to explore plausible mechanisms of action underlying the antileishmanial activity, with trypanothione reductase (TR) selected as a possible biological target. While compound 2f showed only weak inhibition (IC50 = 71 μM), this could provide a starting point for the structure-based design of future antileishmanial agents. Preliminary ADME-related evaluations of 1e and 2f revealed improved aqueous solubility, alongside favorable chemical and metabolic stability specifically for 1e. Consequently, while 2f could be a new hit for a still underexploited target, compound 1e emerges as the most balanced lead candidate for future development of antileishmanial derivatives.