Insights into the Antiparasitic Activity of Pyrazole-benzimidazole against Trypanosoma cruzi

Background: Chagas disease (CD), a life-threatening disease caused by Trypanosoma cruzi, remains a significant global public health concern. The limited efficacy of the available drugs (nifurtimox and benznidazole), their severe adverse events, and the unsatisfactory outcomes of clinical trials drive the search for new, effective, and safe drugs. Objective: This study describes the synthesis, structural characterization, and in vitro antiparasitic activity of novel pyrazole-benzimidazole derivatives against mammalian developmental stages of T. cruzi. Methods: Phenotypic screening was used to assess the effect of pyrazole-benzimidazole derivatives against T. cruzi. Three-dimensional cardiac spheroids were employed to evaluate the toxic effect and drug efficacy. Molecular docking and cysteine protease activity were also performed. Results: Pyrazole-benzimidazole derivatives showed activity against both trypomastigotes and intracellular amastigotes. Compounds 1i (IC50 = 6.6 μM) and 1j (IC50 = 9.4 μM) demonstrated the most potent activity with a high selectivity index (SI > 45) against intracellular amastigotes. Both compounds exhibited high efficacy on 3D cardiac spheroids, effectively reducing the parasite load by over 80%. Molecular docking analysis revealed that both compounds target the catalytic domain of cruzain through pi-stacking and hydrogen bonding interactions and inhibit T. cruzi cysteine protease. These derivatives also showed an additive effect in combination with the reference drug (Bz). Conclusion: Our findings emphasize the significance of pyrazole-benzimidazole hybrids in the search for new anti-T. cruzi agents.