Discovery of novel anti-Toxoplasma gondii agent derived from thiazolidinone-isatin hybrids: synthesis, biological assessment, and computational modeling.

Toxoplasmosis is a globally prevalent parasitic infection that disproportionately impacts immunocompromised individuals and remains a significant public health challenge. The limitations of current standard-of-care therapies, marked by severe adverse effects and an inability to eradicate persistent cysts, necessitate the urgent development of novel, safer, and more efficacious anti-Toxoplasma agents. Employing a rational molecular hybridization strategy, we designed and synthesized a novel series of seventeen thiazolidinone-isatin hybrid derivatives to evaluate their in vitro inhibitory effects against the Toxoplasma gondii RH strain. The phenotypic screening revealed that most of the synthesized hybrids exhibited significant antiparasitic activity, with compounds 4a, 7g, and 7j emerging as highly promising leads. Notably, the unsubstituted isatin derivative 4a was the most potent agent of the series (IC50 = 0.21 µg mL-1). Crucially, 4a exhibited an exceptional safety profile and a remarkably broad therapeutic window, achieving a selectivity index (SI) in Hs27 host cells of >1171, vastly outperforming pyrimethamine, sulfadiazine and trimethoprim. Subsequent mechanistic assays confirmed that 4a profoundly impedes the intracellular proliferation of T. gondii tachyzoites in a rapid, dose-dependent manner. In order to elucidate the putative mechanism of action at the molecular level, comprehensive in silico investigations were conducted. In silico molecular docking and 100 ns molecular dynamics simulations suggested TgCDPK1 as a potential target and revealed a highly stable binding complex with 4a. Overall, this study validates the thiazolidinone-isatin hybrid scaffold as a highly effective new chemotype for antitoxoplasmic drug discovery and highlights 4a as a compelling candidate for further in vivo preclinical evaluation.