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.