Overcoming the Hydrolytic Susceptibility of Thiol-Reactive Reversible Warheads through the Development of Substituted...

The ascent of α-cyanoacrylamide covalent warheads for the reversible engagement of cysteine thiols has led to the development of a novel class of selective inhibitors. As part of our efforts to develop peptide-based hydrogel materials that incorporate these warheads, we observed that α-cyanoacrylamides undergo undesired retro-Knoevenagel hydrolysis under physiological conditions. This instability extends to small molecule kinase inhibitors as well, possibly affecting their pharmacokinetics. To gain an understanding of this hydrolytic activity, we performed a series of reactivity studies employing BTK and sickle cell inhibitors as well as model peptides. During these studies, we unexpectedly discovered that an α-cyanoacrylamide-functionalized derivative of the sickle cell inhibitor, voxelotor, was exceptionally stable toward hydrolysis. Subsequent structure-activity studies coupled with molecular truncation afforded a small, hydrolytically stable dimethoxyphenyl cyanoacrylamide (DiMe) warhead. This compact unit demonstrates negligible hydrolysis for over a week under physiological conditions while still being prone to reversible thiol addition. Extensive quantum chemistry calculations provide an electronic and structural understanding of this selective reactivity. The utility of the DiMe warhead was demonstrated in the context of small molecule BTK inhibition and should find broad use as a hydrolytically stable yet thiol-reactive reversible warhead in other applications.