Metabolite profile of Nectandra oppositifolia Nees & Mart. and assessment of antitrypanosomal activity of bioactive compounds through efficiency analyses

EtOH extracts from the leaves and twigs of Nectandra oppositifolia Nees & Mart. shown activity against amastigote forms of Trypanosoma cruzi. These extracts were subjected to successive liquid-liquid partitioning to afford bioactive CH2Cl2 fractions. UHPLC-TOF-HRMS/MS and molecular networking were used to obtain an overview of the phytochemical composition of these active fractions. Aiming to isolate the active compounds, both CH2Cl2 fractions were subjected to fractionation using medium pressure chromatography combined with semi-preparative HPLC-UV. Using this approach, twelve compounds (1-12) were isolated and identified by NMR and HRMS analysis. Several isolated compounds displayed activity against the amastigote forms of T. cruzi, especially ethyl protocatechuate (7) with EC50 value of 18.1 μM, similar to positive control benznidazole (18.7 μM). Considering the potential of compound 7, protocatechuic acid and its respective methyl (7a), n-propyl (7b), n-butyl (7c), n-pentyl (7d), and n-hexyl (7e) esters were tested. Regarding antitrypanosomal activity, protocatechuic acid and compound 7a were inactive, while 7b-7e exhibited EC50 values from 20.4 to 11.7 μM, without cytotoxicity to mammalian cells. These results suggest that lipophilicity and molecular complexity play an important role in the activity while efficiency analysis indicates that the natural compound 7 is a promising prototype for further modifications to obtain compounds effective against the intracellular forms of T. cruzi

Improving the drug-likeness of inspiring natural products - evaluation of the antiparasitic activity against Trypanosoma cruzi through semi-synthetic and simplified analogues of licarin A

Neolignan licarin A (1) was isolated from leaves of Nectandra oppositifolia (Lauraceae) and displayed activity against trypomastigote forms of the etiologic agent of American trypanosomiasis, Trypanosoma cruzi. Aiming for the establishment of SAR, five different compounds (1a - 1e) were prepared and tested against T. cruzi. The 2-allyl derivative of licarin A (1d) exhibited higher activity against trypomastigotes of T. cruzi (IC50 = 5.0 μM and SI = 9.0), while its heterocyclic derivative 1e displayed IC50 of 10.5 μM and reduced toxicity against NCTC cells (SI > 19.0). However, these compounds presented limited oral bioavailability estimation ( 200 μM). In addition, these simplified analogues showed a better permeability profile (Papp > 1.0 × 10-6 cm/s) on PAMPA models, resulting in improved drug-likeness. Vanillyl alcohol acetyl derivative (3b) and isoeugenol methyl derivative (4a) displayed activity against the extracellular forms of T. cruzi (trypomastigotes) with IC50 values of 5.1 and 8.8 μM respectively. Based on these results, compounds with higher selectivity index against extracellular forms of the parasite (1d, 1e, 3d, and 4a) were selected for a mechanism of action study. After a short incubation period (1 h) all compounds increased the reactive oxygen species (ROS) levels of trypomastigotes, suggesting cellular oxidative stress. The ATP levels were increased after two hours of incubation, possibly involving a high energy expenditure of the parasite to control the homeostasis. Except for compound 4a, all compounds induced hyperpolarization of mitochondrial membrane potential, demonstrating a mitochondrial imbalance. Considering the unique mitochondria apparatus of T. cruzi and the lethal alterations induced by structurally based on licarin A, these compounds are interesting hits for future drug discovery studies in Chagas disease