Kinetic decay curves of nitroxides (2 mM in a PBS buffer/DMSO solution 1:1 v/v) in the presence of the reducing agent sodium ascorbate (16 equivalents) were recorded by CW-EPR spectroscopy at X-band frequencies, by observing the decreasing peak intensity of the characteristic triplet spectrum over time (Figure ). At these relatively high concentrations the measurements exhibited a rapid decay within the initial seconds. Such apparent deviation from pseudo-first-order behaviour is consistent with the complex mechanism of ascorbate degradation, especially under aerobic conditions. Consequently, determining the absolute rate constants is not possible. Nevertheless, a comparison of the reduction profiles remains a valid indication of the relative kinetic stability of these nitroxides. The stability of exo-methylene-substituted nitroxides 7 and 8 was benchmarked against the established monosubstituted pyrrolinyl and pyrrolidinyl nitroxides 5 and 9, respectively (Figure ). Additionally, 7 and 8 were compared with the disubstituted pyrrolidinyl nitroxide 10 (as a mixture of diastereomers) and the most stable species from our previous research, tetraethylsubstituted piperidinyl nitroxide 11 (see the Supporting Information for synthesis). The tetraethyl-substituted piperidinyl nitroxide 11 remains the most stable also in this set of nitroxides, exhibiting 50% remaining radical after exposure to an excess of reducing agent for 40 minutes. By comparison, all the spirocyclohexyl-substituted nitroxides were more rapidly reduced, presumably due to the less effective steric shielding provided by the spirocyclohexyl moieties. The difference in stability between pyrrolinyl nitroxide 5 (10% remaining radical) and pyrrolidinyl nitroxide 9 (25% remaining radical) is evident. This finding is in contrast with previous studies that indicate a negligible difference in reduction stability between spirocyclohexyl-substituted pyrrolinyl and pyrrolidinyl nitroxides, which has been ascribed to subtle differences in the relative population of open and semi-open conformations in these scaffolds. Pyrrolidinyl nitroxides, which are inherently more stable towards reduction, prefer open scaffold conformations that leave the radical centre more exposed and thereby facilitate reaction with ascorbate. By contrast, pyrrolinyl nitroxides favour semi-open conformations that partially shield the nitroxide group, thereby compensating for the effect of the intrinsically less stable unsaturated ring. These apparently contradictory results can, however, be explained by differences in the conditions and the functional groups present in the nitroxides between this previous study and our work, both of which can have a significant impact on kinetic rates. Interestingly, the novel disubstituted pyrrolidinyl nitroxide 10 (11% remaining radical) has a comparable stability to pyrrolinyl nitroxide 5. Here, it is possible that the stability expected for a pyrrolidinyl nitroxide (e.g. 9) is counterbalanced by the electronic effects of the nitro functionality.