Investigation of Glutarimide <i>N</i>-Alkylated Derivatives of Lenalidomide

Lenalidomide is a thalidomide derivative that engages the E3 ligase substrate receptor cereblon (CRBN) to promote targeted protein degradation. Lenalidomide possesses a glutarimide moiety, which is responsible for CRBN engagement, and an isoindoline moiety, which promotes neosubstrate recruitment. Modification of the glutarimide is a generalizable prodrug strategy to inhibit CRBN binding for the selective activation of CRBN-dependent activity, yet these compounds may possess CRBN-independent effects. We prepared six <i>N</i>-alkylated glutarimide derivatives and found CRBN-independent effects on TNFα inhibition and selective effects in the cell viability profiles. Evaluation of selected compounds by global proteomics in KG1a cells reveals that the downregulation of Rab28 is CRBN-independent and mediated by autophagy. Finally, we developed a representative prodrug to demonstrate the enzymatic release of lenalidomide. Collectively, although some CRBN-independent properties are observed, modification of glutarimide is a generally viable strategy to prevent CRBN engagement in a prodrug strategy.