Anti-Toxoplasma gondii activity of two novel synthetic compounds based on pyridine, benzimidazole and benzoxazole scaffolds in human maternal-fetal interface models.

Congenital toxoplasmosis remains a major public health concern, and current therapy with sulfadiazine and pyrimethamine is limited by toxicity and efficacy, highlighting the need for safer alternatives. This study evaluated the anti-Toxoplasma gondii activity of two novel synthetic compounds, RVI 30 and RVI 33, containing pyridine, benzimidazole, and benzoxazole scaffolds, using BeWo cells and human third-trimester villous explants as models of the maternal-fetal interface. Cell and tissue viability were assessed by MTT; parasite proliferation and invasion by β-galactosidase assay; ultrastructural alterations by transmission electron microscopy; adhesion and invasion by differential immunofluorescence; and cytokine production by ELISA. Both compounds reduced T. gondii proliferation at non-cytotoxic concentrations, showing favorable selectivity indices. The antiparasitic effect was dose-dependent and partially irreversible. Our results also showed that the compounds exhibited the best binding to T. gondii dihydrofolate reductase-thymidylate synthase (TgDHFR). Ultrastructural analysis revealed tachyzoite damage, including organelle disruption and membrane alterations, consistent with direct parasiticidal activity. Pretreatment of tachyzoites or host cells reduced invasion, indicating effects on both parasite and host. In infected BeWo cells, both compounds upregulated macrophage migration inhibitory factor (MIF), while RVI 30 also downregulated IL-8, suggesting immunomodulatory mechanisms. Importantly, neither compound affected the viability of explants, but both reduced parasite proliferation with greater efficacy than sulfadiazine plus pyrimethamine and no disruption of local cytokine homeostasis. These findings demonstrate that RVI 30 and RVI 33 exert potent anti-T. gondii activity at the maternal-fetal interface through direct parasite damage and modulation of invasion and inflammatory pathways, supporting their potential as therapeutic candidates.