To assess if cyclization of 1 into 2 could occur during the assessment of cellular antiviral activity, the stability of 1 in neutral pH phosphate buffer was investigated. To achieve this, a stock solution of 1 was prepared in DMSO-d6 and diluted into pH 7.4 phosphate buffer (with 10% D2O) to a final concentration of 2 mM, in the presence of maleic acid as an internal standard. The solution was analyzed by 1 H NMR spectroscopy over 48 h at room temperature (Figure ). At t = 1 h, vinyl sulfone 1 was present in solution at the expected 2 mM concentration. After 24 h, a reduction in the abundance of 1 by 10% and the appearance of approximately 9% of its cyclic isomer 2 was observed. After 48 h, 81% of 1 remained with approximately 19% of 2 present in the phosphate buffer. These results demonstrated that partial cyclization of 1 into 2 might occur under standard cell culture conditions. Whether this decrease in the effective concentration of 1 would influence its efficacy as an antiviral nsP2 inhibitor would depend on the frequency of dosing and time course of the bioassay.