Mechanistic Exploration of N,N′-Disubstituted Diamines as Promising Chagas Disease Treatments

<b>Introduction:</b> Chagas disease, caused by the protozoan <i>Trypanosoma cruzi</i>, remains a major public health concern due to the limited effectiveness of current treatments, especially in the chronic stage. <b>Objective:</b> Here, we wanted to advance a library of 30 <i>N</i>,<i>N'</i>-disubstituted diamines as promising antichagasic agents and gain insight into the mechanism of action. <b>Methods:</b> The library was evaluated for activity against the <i>T. cruzi</i> amastigote stage and trypanocidal efficacy. In addition, selected compounds were tested as potential polyamine transport inhibitors, and a fluorescent analog was employed to investigate compound internalization. <b>Results</b>: Five compounds exhibited potent activity (pIC₅₀ > 6.0), particularly those with short aliphatic linkers (3-6 carbon atoms), suggesting a structure-activity relationship favouring shorter chains. Mechanistic studies showed that compound <b>3c</b> strongly inhibited polyamine transport, a vital pathway in <i>T. cruzi</i>, though this was not a universal mechanism among active hits, indicating the potential for multiple targets. A fluorescent analog confirmed intracellular uptake in amastigotes but lacked antiparasitic activity, likely due to disrupted pharmacophoric features. Importantly, none of the compounds demonstrated trypanocidal activity in long-term assays, and some showed cytotoxicity, particularly in the benzyloxy-substituted series. <b>Conclusions</b>: These findings position <i>N</i>,<i>N'</i>-disubstituted diamines as a viable scaffold for Chagas disease drug discovery. However, further optimization is required to enhance selectivity, achieve trypanocidal effects, and better understand the underlying mechanisms of action.