Exploring 6-Hydroxy-3-Aryl/Heteroarylcoumarins as Promising Candidates Against Trypanosoma cruzi

Chagas disease urgently requires the development of new, safe, and effective therapies. We evaluated a series of 6-hydroxy-3-aryl/heteroarylcoumarin derivatives against Trypanosoma cruzi to identify candidates with multistage activity. Our screening revealed that derivatives 1c (2-pyridyl) and 1f (2,3-dihydrobenzo[b][1,4]dioxine) are highly selective chemotypes toward trypomastigotes over Vero cells (SI = 549.45 and > 625, respectively), demonstrating submicromolar potency (IC₅₀ = 0.91 and 0.80 μM, respectively), an activity up to 23-fold higher than benznidazole (Bz). The derivative 1f, like Bz, also exhibited dual-stage activity, showing intracellular amastigote activity (IC₅₀ ∼ 10 μM). Mechanistic analysis using a washout assay revealed that, rather than eliminating intracellular forms, 1f prevents the release of new trypomastigotes. Our findings suggest that 1f targets the amastigote-to-trypomastigote differentiation pathway. The retention of a high intracellular parasitic load and the absence of trypomastigote release during the washout period strongly suggest that 1f acts as a selective inhibitor of metacyclogenesis. This insight positions the coumarin scaffold bearing a 2,3-dihydrobenzo[b][1,4]dioxine substituent at position 3 as a promising starting point for developing improved multi-stage therapeutic candidates against Trypanosoma cruzi.