Unlocking Anti-Toxoplasma Activity: The Essential Role of a Cis-Configured Vinyl Linker.

Separation of cis/trans isomers is critical for SAR studies but challenging due to similar properties. To address this, we designed a rigid triple-connected heterocyclic scaffold locking stereochemical geometry, enabling reliable separation and unambiguous assignment. From a library of cis/trans oxadiazole derivatives, cis compound 6 emerged as the most potent anti-Toxoplasma agent, displaying superior selectivity, lower cytotoxicity, and enhanced intracellular parasite clearance compared to its trans counterpart and reference drug spiramycin. Mechanistic investigations revealed compound 6 induces pronounced oxidative stress, evidenced by elevated NO/ROS and GSH depletion. Complementary in silico studies, including docking and 100-ns MD simulations, supported a stereospecific binding mode within TgNTPDase, with cis-6 forming dual hydrogen bonds that the trans isomer cannot access; the simulation further demonstrated stable protein-ligand interactions with backbone RMSD 0.235 ± 0.043 nm and minimum distance 0.15-0.22 nm throughout. ADMET predictions indicated improved solubility, slower clearance, and lower hERG risk for cis-6, consistent with its better selectivity. Collectively, this work establishes the rigid tris-heterocyclic core as a versatile stereochemically predictable platform for isomer-resolved drug design, positioning compound 6 as a promising lead for anti-toxoplasmosis therapy.