Hydrogen/Deuterium Exchange for Chiral Stability Assessment in Acidic Methine-Containing Compounds

Acidic methine-containing compounds are prone to racemization driven by keto-enol tautomerization, which can lead to products with distinct pharmacological and toxicological properties. Glutarimide, a core structural component of Cereblon binders, and thiazolidinediones, a class of antidiabetic agents, are particularly susceptible to this instability. In this study, we developed an H/D exchange method to investigate the racemization or epimerization kinetics of 28 compounds, including 3 thiazolidinediones, as well as 13 Molecular Glues and 12 proteolysis targeting chimeras (PROTACs), both containing the glutarimide scaffold. In phosphate buffer (0.1 M, D₂O, pD 7.8) at 37 °C, the racemization half-lives of glutarimide-based Molecular Glues ranged from 3.0 to 7.3 h, while thiazolidinediones exhibited substantially faster racemization with half-lives of 0.8 to 1.0 h. The correlation of H/D exchange and racemization rates were confirmed using chiral supercritical fluid chromatography. Reverse (D/H) exchange demonstrated a 3.6- to 8.5-fold improvement in chiral stability for methine-deuterated compounds, attributed to a primary kinetic isotope effect. Moreover, several PROTACs demonstrated substantially greater chiral stability than Molecular Glues, highlighting their distinct chiral behavior. Human serum albumin was found to influence chiral stability, accelerating racemization in some compounds while stabilizing Others. Finally, conformational analysis revealed that PROTACs can adopt three distinct conformations; however, a clear correlation between enhanced chiral stability and specific conformers has yet to be established. These findings highlight the utility of the H/D exchange method as a robust and streamlined technique for assessing racemization and epimerization kinetics in various biologically relevant media. This approach enables rapid evaluation of chiral stability and highlights the potential of deuteration as a strategy to enhance chiral stability and optimize therapeutic agents within these compound classes.