Cyclization of 1 to 2 was favored under basic conditions and 2 but occurred slowly at physiological pH. We were eager to determine if the cyclization was reversible and whether dihydropyrazolo The stability of the dihydropyrazolo pyrazin-4(5H)-one 2 under standard laboratory conditions suggested that equilibrium with the acyclic b-amidomethyl-vinyl sulfone 1 lay predominantly toward the cyclic form. For example, 1 was below the limits of UPLC detection in 10 mM DMSO stock solutions of 2 even after prolonged storage over several months. Since low levels of the electrophilic b-amidomethyl-vinyl sulfone could theoretically still be present, we decided to test whether 1 could be captured using glutathione (GSH). Incubation of 1 with a 100-fold excess of GSH in phosphate buffer at 30 °C led to complete conversion to GSH adduct 9 within 8 h as monitored by LCMS (Table and). In contrast, incubation of dihydropyrazolo pyrazin-4(5H)-one 2 with excess GSH did not result in formation the GSH adduct 9 even after 24 h. The GSH capture data demonstrated that no detectable level of the acyclic b-amidomethyl-vinyl sulfone 1 was present in phosphate buffer, making it extremely unlikely that dihydropyrazolo pyrazin-4(5H)-one 2, despite its improved pharmacokinetic properties, would be useful as a prodrug for 1.