Antitrypanosomal activity and evaluation of the mechanism of action of dehydrodieugenol isolated from Nectandra leucantha (Lauraceae) and its methylated derivative against Trypanosoma cruzi. BACKGROUND:  From a previous screening of Brazilian biodiversity for antiprotozoal activity, the hexane extract from leaves of Nectandra leucantha (Nees & Mart.) (Lauraceae) demonstrated activity against Trypanosoma cruzi. Chromatographic separation of this extract afforded bioactive dehydrodieugenol (1). Furthermore, methylated derivative 2 (dehydrodieugenol dimethyl ether) was prepared and also tested against T. cruzi. PURPOSE:  To examine the therapeutical potential of compounds 1 and 2 against T. cruzi as well as to elucidate the mechanism of action of bioactive compound 1 against T. cruzi. METHODS/STUDY DESIGN:  Crude hexane extract from leaves was subjected to chromatographic steps to afford bioactive compound 1. In order to analyze the effect of additional methyl group in the antiparasitic activity of 1, derivative 2 was prepared (both are no pan-assay interference compounds - PAINS). These compounds were evaluated in vitro against T. cruzi (trypomastigote and amastigote forms) and analyzed for the potential effect in host cells through the production of nitric oxide and reactive oxygen species. Finally, the plasma membrane effect of the most potent compound 1 was investigated in T. cruzi trypomastigotes. RESULTS:  Compounds 1 and 2 displayed activity against amastigotes of T. cruzi. Although both compounds promoted activity against intracellular amastigotes, the production of nitric oxide and reactive oxygen species of host cells were unaltered, suggesting an antiparasitic activity other than host cell activation. Considering 1 the most effective compound against T. cruzi, the interference in the plasma membrane of the trypomastigotes was investigated using the fluorescent probe SYTOX® Green. After a short-term incubation, the fluidity and integrity of the plasma membrane was completely altered, suggesting it as a primary target for compound 1 in T. cruzi. CONCLUSION:  Compounds 1 and 2 selectively eliminated the intracellular parasites without host cell activation and could be important scaffolds for the search of new hit compounds.