However, how the substitution on the amidine groups affects the anti-trypanosomal properties of bisbenzofurans has not been explained. Thus in light of their reduced cytotoxicity compared to pentamidine these molecules require further investigation to study the influence of the type of cationic substituents and the distance between aromatic moieties on uptake and intracellular distribution of dicationic bisbenzofurans in order to understand better the mode of action and thus improve the efficacy of aromatic diamidines. Thus promising in vitro, anti-trypanosomal activity, excellent potency in the acute mouse model of trypanosomiasis and the reduced cytotoxicity (1.9 µM) of compound 1 compared to pentamidine warrants further pre-clinical and clinical trials of this molecule. While compound 32 and 20 can serve as lead compounds for further SAR optimization to derive congener with enhanced activity and lesser cytotoxicity. 