Design, Synthesis, and Anti-ischemic Stroke Activity Evaluation of Novel GSNOR Inhibitors.

S-nitrosoglutathione reductase (GSNOR), a key regulator of protein S-nitrosation, is a promising therapeutic target for cerebral ischemia. We report the design, synthesis, and evaluation of indole-based GSNOR inhibitors with potent anti-ischemic activity. Molecular docking and enzymatic assays identified lead compound 19, and structural optimization yielded compound 45, the most potent inhibitor (IC50 = 44.12 ± 8.33 nM). To enhance drug-like properties, we developed compound 50, a methyl ester prodrug of 45, which demonstrated improved neuroprotection in the OGD/R cell model. In a rat ischemic stroke model, compound 50 exhibited favorable pharmacokinetics, good brain penetration, and significantly reduced infarct volume while improving neurological deficits. Nitroso-proteomics and transcriptomic analyses suggest that compound 50 may exert neuroprotection by regulating calcium signaling and synaptic function via Clstn1 S-nitrosation and by inhibiting neuronal apoptosis. These results highlight compound 50 as a promising therapeutic candidate for ischemic stroke with enhanced neuroprotective efficacy.