Development of 6-methyl-4,5-dihydropyrazolo[3,4-d]pyrimidin-4-one derivatives as lead sigma-1 receptor inhibitor: synthesis and biological evaluation via MSI.

The present study describes the design, synthesis, and biological evaluation of a series of pyrazolo[3,4-d]pyrimidin-4-one derivatives (n = 27) as σ1 receptor antagonists. Among these, compound 39 showed the highest binding affinity for the σ1 receptor, with a Kiσ1 value of 7.48 ± 1.11 nM. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) analysis indicated that compound 39 binds predominantly to the β-barrel and α5 regions of the σ1 receptor, in a manner comparable to classical antagonists such as haloperidol. In vivo mass spectrometry imaging (MSI) demonstrated that compound 39 effectively crosses the blood-brain barrier. In a formalin-induced mouse nociceptive pain model, compound 39 produced dose-dependent antinociceptive effects, and it showed no observable cytotoxicity in vitro and was well tolerated in vivo in the assays performed. Taken together, these results indicate that compound 39 represents a preliminary lead with potential for further preclinical development.