2.1. In Vitro Anti-Trypanosomal Assay

The activity of the compounds against 427 Trypanosoma brucei brucei trypomastigotes was determined as previously described [37]. Briefly, parasites were incubated in 96-well plates with 20 µM or three-fold serial dilutions (100 µM starting concentration) of test compounds in Iscove’s modified Dulbecco’s medium (IMDM) (Lonza, Basel, Switzerland) supplemented with 10% fetal calf serum, HMI-9 supplement, hypoxanthine, and penicillin/streptomycin at 37 °C in a 5% CO₂ incubator. After 48 h, 20 µL of resazurin reagent (0.135 mg/mL resazurin in phosphate-buffered saline) was added to each well and the fluorescence (Ex₅₆₀/Em₅₉₀) read after 2–4 h in a Spectramax M3 plate reader (Molecular Devices, San Jose, CA, USA). The fluorescence readings were used to calculate % parasite viability relative to the readings obtained from the wells containing untreated control parasite cultures. The IC₅₀ values were determined by plotting % viability vs. log(compound) and performing a non-linear regression using a GraphPad Prism (v. 5.02) (San Diego, CA, USA).