A kinetic study of thiol addition to N-phenylchloroacetamide.

Irreversible enzyme inhibitors bind covalently to their target and permanently limit its function. The redox-sensitive thiol group on the side chain of cysteine (Cys) residues is often the nucleophilic group that is targeted for reaction with the electrophilic warhead of irreversible inhibitors. While the acrylamide group is the warhead applied most frequently currently in the design of inhibitors with therapeutic potential, the chloroacetamide group offers a comparable reactivity profile. In that context, we have studied the details of the mechanism of thiol addition to N-phenylchloroacetamide (NPC). A kinetic assay was developed to accurately monitor the reaction progress between NPC and a small library of thiols with varying pKa values. From these data, a Brønsted-type plot was constructed, from which a ßnucRS- value of 0.22 ± 0.07 was derived, indicative of a relatively early transition state with respect to attack by the thiolate. The halide leaving group was also varied, for the reaction with one thiol, providing rate constants consistent with a transition state that is early with respect to leaving group departure. The effects of temperature and ionic strength were also studied, and all data are consistent with an early transition state for a concerted SN2 mechanism of addition. Molecular modelling was also performed, and these calculations confirm the concerted transition state and relative reactivity of the haloacetamides. Finally, this study allows a detailed comparison of the reactivity and reaction mechanisms of the chloroacetamide group with the benchmark acrylamides used in many irreversible inhibitor drugs.

A mechanistic study of thiol addition to N-acryloylpiperidine.

Nucleophilic cysteine (Cys) residues are present in many enzyme active sites and represent the target of many different irreversible enzyme inhibitors. Given its fine balance between aqueous stability and thiolate reactivity, the acrylamide group is a particularly popular warhead pharmacophore among inhibitors designed for biological and therapeutic application. The acrylamide group is well known to undergo thiol addition, but the precise mechanism of this addition reaction has not been studied in as much detail. In this work we have focussed on the reaction of N-acryloylpiperidine (AcrPip), which represents a motif found in many targeted covalent inhibitor drugs. Using a precise HPLC-based assay, we measured the second order rate constants for the reaction of AcrPip with a panel of thiols possessing different pKa values. This allowed construction of a Brønsted-type plot that reveals the relative insensitivity of the reaction to the nucleophilicity of the thiolate. By studying temperature effects, we were able to construct an Eyring plot from which the enthalpy and entropy of activation were calculated. Ionic strength and solvent kinetic isotope effects were also studied, informing on charge dispersal and proton transfer in the transition state. DFT calculations were also performed, providing information on the potential structure of the activated complex. Taken together, these data strongly support one cohesive addition mechanism that is the microscopic reverse of the E1cb elimination, and highly relevant to the intrinsic thiol selectivity of AcrPip inhibitors and their subsequent design.

A mechanistic study of thiol addition to N-phenylacrylamide.

Cysteine (Cys) residues contain a redox-sensitive thiol and are commonly found in enzyme active sites. In recent years, the presence of a reactive thiolate group on a protein has been exploited in the development of irreversible enzyme inhibitors as therapeutic agents. Many targeted covalent inhibitors (TCIs) are designed to covalently react with a specific Cys residue on a target protein active site, irreversibly modifying the target and inhibiting its normal function. The electrophilic warhead most commonly used in this way is the acrylamide functional group. Although the acrylamide group is well known for its ability to undergo thiol-addition reactions, very few studies have been conducted to elucidate the detailed mechanism of this reaction, which inspired us to conduct a thorough kinetic investigation. First, we developed a robust kinetic assay to accurately monitor reaction progress between N-phenylacrylamide (NPA) and a small library of alkyl thiols having widely varying pKa values. This allowed us to construct a Brønsted-type plot for the thiol addition reaction, revealing a ßnucRS- value of 0.07 ± 0.04. We also studied the solvent kinetic isotope effects (SKIEs), pH dependence, and temperature dependence of the reaction, which showed that reaction has a relatively large negative ΔS‡, and a small ΔH‡. Computational studies provided a structure for the transition state that is consistent with the experimental data. All of these data are consistent with rate-limiting nucleophilic attack, followed by rapid protonation of the enolate, corresponding to the microscopic reverse of the E1revcb elimination mechanism.