Thiol-induced nitric oxide release from 3-halogeno-3,4-dihydrodiazete 1,2-dioxides

In this work we studied the mechanism of nitric oxide (NO) release underlying the vasorelaxant and antiaggregant effect of 3,4-dihydrodiazete 1,2-dioxides (DD). Six derivatives were included in the investigations, namely, 3-bromo- and 3-chloro-3,4,4-trimethyl-DD (1a,b), 3-bromo- and 3-chloro-4-methyl-3,4-hexamethylene-DD (2a,b), 3,3,4,4-tetramethyl-DD (3), and 3-methyl-3,4-hexamethylene-DD (4), and their reactivity toward thiols was analyzed. The 3-bromo- and 3-chloro-DD derivatives were found to react with thiols; this reaction can lead to NO formation, DD 2a being the most reactive compound. 2-(Hydroxyamino)-2-methylbutan-3-one oxime (5a) and 2-hydroxy-2-methylbutan-3-one oxime (6) were the main products isolated from the reaction of 1a with cysteine. Reaction rates of DD with thiols were dependent upon pH and concentration of the reagents. Maximum rates of NO release corresponded to thiol concentrations in the range of 1 mM. Consistent with reaction kinetics data and products isolated, a reaction mechanism was proposed. Addition of 2a to bovine aortic endothelial cells led to strong NO release indicating a reaction with endogenous thiols. In rat mesenterial arteries, the vasorelaxant action of 2a was only slightly influenced by addition of thiol to the incubation medium. For the most reactive DD derivatives, cytotoxic effects were observed at concentrations roughly 2 orders of magnitude higher than those inducing vasorelaxation.