Host-guest complexation of chlorzoxazone and its synthetic impurity 2-Amino-4-Chlorophenol by cationic water-soluble Pillar[5]arene

Chlorzoxazone is a centrally acting muscle relaxant, synthesized from 2-amino-4-chlorophenol, a toxic impurity whose concentration in chlorzoxazone formulations is strictly regulated to 0.5%. We investigate the host-guest interactions of chlorzoxazone, 2-amino-4-chlorophenol, and its protonated form with the cationic, water-soluble pillar[5]arene using ¹H NMR spectroscopy, molecular dynamics (MD) simulations, MM-PBSA binding free-energy calculations, and density functional theory (DFT). 1 H NMR spectra indicate that chlorzoxazone and 2-amino-4-chlorophenol are probably only partially included within the central cavity and mainly encapsulated in the pocket formed by the alkyl ammonium chains of the host. MD simulations indicate that all three guests form binary complexes with the host, while only chlorzoxazone forms ternary complexes. For the binary complexes, the oxazole ring of chlorzoxazone is partially inserted into the cavity, 2-amino-4-chlorophenol remains mostly above the central cavity and shows higher flexibility, and the ammonium group of the protonated form of 2-amino-4-chlorophenol is oriented inside the cavity where it engages in cation-π interactions. The results of the Molecular Mechanics Poisson-Boltzmann Surface Area reveal a strong preference for chlorzoxazone over 2-amino-4-chlorophenol in both binary and ternary complexes. Density Functional calculations support the binding preferences observed for the binary complexes and indicate that complexation is enthalpy-driven. This selectivity provides a potential strategy for impurity control in pharmaceutical formulations by preferentially capturing the target drug.