4- O -Allyl Derivative of Natural Licarin A Exhibits Selective In Vitro Activity against Toxoplasma gondii Ta

Abstract

Toxoplasma gondii is an obligate intracellular protozoan responsible for toxoplasmosis, a widespread parasitic disease that poses a serious health threat, especially to immunocompromised patients and pregnant women. Current therapeutic options remain unsatisfactory because they are associated with adverse effects and do not eliminate latent infections, emphasizing the need for new antitoxoplasmic agents. In the present study, the natural neolignan (−)-licarin A (1), isolated from the Brazilian species Nectandra oppositifolia, together with three semisynthetic derivatives, 4-O-methyl (1a), 4-O-acetyl (1b), and 4-O-allyl (1c), were investigated for their in vitro activity against T. gondii tachyzoites. Antiparasitic activity (EC50) was determined using a β-galactosidase-based assay, while cytotoxicity (CC50) toward Human Foreskin Fibroblasts (HFF) was assessed by the resazurin assay. Among the tested compounds, derivative 1c exhibited the highest potency (EC50 = 3.49 μM) and lowest cytotoxicity (CC50 = 120.10 μM), resulting in the highest selectivity index (SI = 34.41). In silico ADMET analysis predicted favorable gastrointestinal absorption and blood–brain barrier permeability for all licarin derivatives. Mechanistic assays demonstrated that licarin A derivatives 1a–1c did not compromise parasite plasma membrane integrity but significantly reduced mitochondrial membrane potential, supporting an intracellular mode of action. Further functional analyses revealed that compound 1c inhibited intracellular parasite proliferation in a concentration-dependent manner and maintained significant antiparasitic activity after drug removal only at the highest concentration tested, without affecting parasite invasion or intracellular replication following pretreatment of extracellular tachyzoites. Overall, these findings identify the 4-O-allyl derivative of licarin A as the most selective compound of this series and provide additional evidence that semisynthetic modification of the licarin scaffold represents a promising strategy for discovering new therapeutic candidates against T. gondii.