In parallel, the team developed libraries of benzylMD analogues with improved physicochemical properties by incorporating structurally diverse, polar amino-chains in place of the 4-(trifluoromethyl) substituent present in the antimalarial plasmodione. By screening the libraries in a broad panel of parasitic assays, we identified an anti-T. cruzi early hit compound (4a) functionalized by a piperazine, among the 3-benzylMD series, and confirmed its potent and highly specific anti-T. cruzi amastigote activity (Table ). The activity in vivo was also investigated in the bioluminescent chronic Chagas disease model without obvious signs of toxicity in the murine model (Figure ). Furthermore, the anti-T. cruzi activity of the early hit 4a was tested for sterile cidality in high content imaging assays using microscopic counting of intracellular amastigotes in vitro and was validated under the conditions for causing no relapse of intra-macrophagic amastigotes after drug wash-out, identifying the potential for sterile cure in vivo. To extend these preliminary results, it has been necessary to better understand which pharmacophore parameters of the proposed scaffold are essential for the anti-T. cruzi activity of the molecule by making slight modifications to the hit's backbone. In the present work, we report the synthetic routes to prepare a library of analogues of this early hit, a detailed structure-activity relationship study including calculated pharmacokinetic properties, experimental characterization of the anti-T. cruzi activity (IC50 values) and cytotoxicity (CC50) of the new benzylMD derivatives. This has allowed us to identify a new early lead 11b, henceforth called cruzidione (CZ), to develop further. In addition, an exploratory study in yeast to investigate the mode of action of the most specific anti-T. cruzi early lead CZ (11b), was performed and revealed that the NADH-dehydrogenase contributed to the redox-cycling of 11b through its benzoylMD metabolite 25b, the cruzidione oxide (CZO).