Expanding the Chemistry of Dihaloacetamides as Tunable Electrophiles for Reversible Covalent Targeting of Cysteines

Abstract

The choice of an appropriate electrophile is crucial in the design of targeted covalent inhibitors (TCIs). In this report, we systematically investigated the glutathione (GSH) reactivity of various haloacetamides and the aqueous stability of their thiol adducts. Our findings revealed that dihaloacetamides cover a broad range of GSH reactivity depending on the combination of the halogen atoms and the structure of the amine scaffold. Among the dihaloacetamides, dichloroacetamide (DCA) exhibited slightly lower GSH reactivity than chlorofluoroacetamide (CFA). The DCA−thiol adduct is readily hydrolyzed under aqueous conditions, but it can stably exist in the solvent-sequestered binding pocket of the protein. These reactivity profiles of DCA were successfully exploited in the design of TCIs targeting noncatalytic cysteines of KRAS G12C and EGFR L858R/T790M . These inhibitors exhibited strong antiproliferative activities against cancer cells. Our findings provide valuable insights for designing dihaloacetamide-based reversible covalent inhibitors.

Emerging and Re-emerging Warheads for Targeted Covalent Inhibitors: An Update

“…Another significant novelty since our previous Perspective was published, are double and triple α-halogenated acetamides, which can be used as tempered electrophiles for covalent ligands. Especially chloro­fluoro­acetamides (CFAs) and dichloro­acetamides (DCAs) have been investigated in much detail as part of the pioneering work of the Ojida group ( vide infra ). , Notably, the attenuating effect of an (additional) geminal halogen atom on α-haloesters had already been described in the 1950s, and DCAs have a long history in medicinal chemistry, albeit with little systematic exploration. For example, a DCA group is present in the approved antibiotic chloramphenicol, which has been known since the late 1940s, yet the primary mechanism of action of this compound, i.e., binding to the 50S ribosomal subunit to block peptidyl transferase activity, does not involve the formation of a covalent bond.…”

“…The broadest studies on di- and trihalogenated acetamides with respect to their TCI-like properties were published very recently by the Ojida group. , An initial report by Shindo et al focused on CFA as a novel reactive group for covalent ligands. They demonstrated the utility of this warhead as a weakly reactive and tunable electrophile toward cysteine residues, characterizing its properties in various in vitro and in vivo assays.…”

“…As mentioned, even more detailed investigations on various halo­acetamides were very recently published . First, the GSH reactivity (pH 7.2) of model compounds bearing acrylamide, CA, or CFA groups was assessed.…”

“…As a part of their most recent study on dihalo­acetamide warheads ( vide supra ), the Ojida group also tested DCA electrophiles systematically in the context of EGFR DM and KRAS G12C inhibition . To this end, various compounds derived from afatinib ( 1 , Figure ) and adagrasib ( 6 ) were synthesized.…”

Use of pyridazinediones for tuneable and reversible covalent cysteine modification applied to peptides, proteins and hydrogels

“…26 As an example, dihaloacetamides have been found to react with cysteines and, upon hydrolysis at neutral pH, the original cysteine may be released from the cysteine–haloacetamide conjugate, but these reagents are limited by being protein microenvironment specific and the dihaloacetamide is not re-formed post-hydrolysis. 27 Another area where reversible covalent modification has found application is the release of a drug from a surface/material to a local area or via a carrier protein (Fig. 1B).…”