Tunable Methacrylamides for Covalent Ligand Directed Release Chemistry

Targeted covalent inhibitors are an important class of drugs and chemical probes. However, relatively few electrophiles meet the criteria for successful covalent inhibitor design. Here we describe alpha-substituted methacrylamides as a new class of electrophiles suitable for targeted covalent inhibitors. While typically alpha-substitutions inactivate acrylamides, we show that hetero alpha-substituted methacrylamides have higher thiol reactivity and undergo a conjugated addition-elimination reaction ultimately releasing the substituent. Their reactivity toward thiols is tunable and correlates with the pK(a)/pK(b) of the leaving group. In the context of the BTK inhibitor ibrutinib, these electrophiles showed lower intrinsic thiol reactivity than the unsubstituted ibrutinib acrylamide. This translated to comparable potency in protein labeling, in vitro kinase assays, and functional cellular assays, with improved selectivity. The conjugate addition-elimination reaction upon covalent binding to their target cysteine allows functionalizing alpha-substituted methacrylamides as turn-on probes. To demonstrate this, we prepared covalent ligand directed release (CoLDR) turn-on fluorescent probes for BTK, EGFR, and K-Ras(G12C). We further demonstrate a BTK CoLDR chemiluminescent probe that enabled a high-throughput screen for BTK inhibitors. Altogether we show that alpha-substituted methacrylamides represent a new and versatile addition to the toolbox of targeted covalent inhibitor design.

Automated summary

This study introduces a new class of alpha-substituted methacrylamides as potential electrophiles for targeted covalent inhibitors, showing higher thiol reactivity and undergoing a conjugated addition-elimination reaction. These electrophiles demonstrated comparable potency and improved selectivity in various assays, making them a versatile addition to the toolbox of targeted covalent inhibitor design.