Preparative HPLC separation using a reverse phase Luna 5 µm phenyl-hexyl column (Phenomenex) provided high purity (>99%) samples of 1 and 2 as their TFA salts. CHIKV nsP2 protease inhibition was found to reside exclusively in 1 (IC50 = 60 nM) with the byproduct 2 devoid of activity in the enzyme assay at 100 µM . Using a combination of 1 H and 13 C NMR spectroscopy, the chemical structure of 2 was determined to be a dihydropyrazolo pyrazin-4(5H)-one (Figure ) arising from intramolecular cyclization of 1. 1 H NMR analysis of 1 showed characteristic olefinic protons corresponding to the (E)-vinyl sulfone at δ 6.69 ppm (dt, J = 15.3, 1.8 Hz, H14) and δ 6.82 ppm (dt, J = 15.3, 4.4 Hz, H13) (Figure ). These olefin resonances were absent in 2 (Figure ) and were replaced by a multiplet at δ 5.07 ppm (m, J = 8.9, 4.5 Hz, H9) and signals for two protons at δ 3.76-3.72 ppm (m, H11) and δ 3.98 ppm (dd, J = 14. . Other resonances consistent with the cyclic structure of 2 were the non-equivalent methylene protons at δ 3.70 ppm (td, J = 6.3, 3.5 Hz, H8) and δ 3.93 ppm (ddd, J = 13.3, 4.4, 2.3 Hz, H8) (Figure ), which appeared as a single multiplet at δ 4.11 ppm in acyclic 1 (Figure , H12). Additional evidence for the cyclic structure of 2 was provided by a 1 H-13 C heteronuclear multiple bond correlation (HMBC) experiment (Figure ) that indicated a three-bond correlation between atoms H9 and C7 in 2 that was absent in the acyclic 1. Formation of 2 is proposed to occur under the basic conditions of the amide coupling by a formal aza-Michael conjugate addition of the pyrazole N2 into the b-carbon of the vinyl sulfone (Scheme 2). The resulting dihydropyrazolo pyrazin-4(5H)-one, which masks the reactive vinyl sulfone warhead within its cyclic structure, was stable under normal laboratory conditions with no propensity to revert to the acyclic 1 upon storage as a solid or as a 10 mM DMSO stock solution. However, researchers are cautioned that commercial samples of 1 or other heterocyclic b-amidomethyl-vinyl sulfones (ChemSpace, Enamine) may contain undetermined quantities of the respective dihydropyrazolo pyrazin-4(5H)-ones unless careful quality control by 1 H NMR and UPLC analysis has been performed.