Finally, all synthesized compounds were checked for PAINS filters A, B and C, undesirable substructure moieties, covalent inhibition, and compliance with the rule-offive with the FAF-Drugs4 webserver (fafdrugs4.mti.univ-paris-diderot.fr/) by inputing SMILES strings for the compounds. In vitro biological evaluation against T. brucei and L. infantum intramacrophage amastigotes. The efficacy against T. brucei brucei Lister 427 bloodstream forms was evaluated in a modified resazurin-based assay as previously described. Cells were grown at 37°C and 5% CO2 in a complete HMI-9 medium supplemented with 10% fetal calf serum (FCS) and 100 UI/mL of penicillin/streptomycin. The HMI-9 medium was selected due to its high folic acid content (9 µm), resulting in efficient parasite growth and enabling process standardization. The same medium was used in our previous experiments and allowed maintenance of similar relative folate metabolism levels between models studied in high and low throughput systems, namely, between Trypanosoma cells and mice models, and between mice models and human plasma. Cultures were then diluted to a cell density of 2×10 6 /mL.