Chagas disease, caused by the protozoan Trypanosoma cruzi, afflicts millions in Latin America. Benznidazole is the only available drug in Brazil and very limited due to side effects.1,2 In this context, natural products offer a substantial variety of compounds that can be used for development of novel therapeutics3. Thus, looking to isolate further such compounds from Porcelia macrocarpa, bioactivity-guided fractionation against trypomastigotes of T. cruzi of the MeOH extract resulted in the characterization of a fraction containing two compounds. Antitrypanosomal activity of Fraction E was performed against cell-derived trypomastigotes and amastigotes. Cytotoxicity was evaluated against NCTC cells. In addiction the activity in the cell membrane permeability and calcium levels were evaluated. Part of this material (1 g) was subjected to column chromatography over Sephadex LH-20 and eight groups (A – H) were obtained. The 1H NMR spectrum of biologically active fraction D (18 mg) showed characteristic signals of a lactone ring as well as signals suggesting the presence of a triple bond similar to those previously isolated from seeds of P. macrocarpa4. However, additional peaks were indicative of an enyne conjugated system suggesting the presence of an internal double bond. Analysis of 13C and DEPT NMR spectra confirmed the proposal due to the observation of signals referring to the carbonyl carbon in the lactone ring, the internal double bond and the terminal double bond. Additionally, signals referring to two quaternary carbons were observed. Finally, by means of HPLC/ESI-HRMS it was possible to infer that bioactive fraction D is composed of a mixture of related compounds 1 and 2 (Figure 1), whose structures were determined for the first time in this work. Fraction E displayed EC50 of 4.9 µg/mL and 2.4 µg/mL against trypomastigote and amastigote forms of the parasite, respectively, with no cytotoxicity observed at the highest concentration tested (CC50 > 200 µg/mL). The selectivity index against amastigotes for this fraction (SI = 83.3) was higher than for the standard drug benznidazole (EC50 = 1.43 µg/mL and CC50 = 52.5 µg/mL, SI = 36.4). A SI higher than 10 is in agreement with hit criteria for Chagas disease treatment, established by the Drugs for Neglected Diseases iniciative (DNDi)2. Mechanism of action studies with trypomastigote forms showed that fraction E does not alter the parasite cell membrane but does affect intracellular calcium levels. Further studies will be done to determine if alterations are caused in T. cruzi mitochondria and/or acidocalsisomes (both organelles sources of calcium, important for cellular homeostasis in trypanosomatids), as well as analyses of the isolated compounds. While enyne γ-lactones have been isolated from other species of Annonaceae5, this is the first description of such conjugated compounds in P. macrocarpa.