7-Prenyloxycoumarins as Promising Antileishmanial Agents: In Vitro, In Vivo, and In Silico Evaluation Against Leishmania amazonensis

<b>Background/Objectives:</b> Leishmaniasis remains a major neglected tropical disease, and current chemotherapeutic options are limited by toxicity and resistance in <i>Leishmania</i> species, including <i>L. amazonensis</i>. Prenylated coumarins have emerged as promising bioactive scaffolds. Altissimacoumarin D and its analogues inhibit fungal efflux pumps associated with resistance. However, their antileishmanial potential and mechanisms of action remain unclear. Here, we evaluated the in vitro, in vivo, and in silico effects of altissimacoumarin D and seven analogues against <i>L. amazonensis</i>. <b>Methods:</b> In vitro assays were performed to identify active compounds and assess toxicity in keratinocytes. In vivo experiments in hamsters evaluated antileishmanial activity and renal and hepatic toxicity. In silico analyses were conducted to investigate the mechanism of action of the substances. <b>Results:</b> In vitro assays showed that ACS47, ACS48, and ACS51 were the most active and safe compounds. In a hamster infection model, daily administration of ACS47 and ACS48 (2.5 mg/kg) significantly reduced parasite burden and lesion size, while maintaining normal renal and hepatic biochemical parameters. Histological analysis correlated reduced lesion size with marked decreases in amastigote density. Based on in silico analysis, spermidine synthase was supported as a plausible molecular target. <b>Conclusions:</b> Collectively, these findings identify ACS47 and ACS48 as promising lead compounds for future antileishmanial drug development.