Identification of glutathione conjugates of acetylene-containing positive allosteric modulators of metabotropic glutamate receptor subtype 5

Abstract

A recent medicinal chemistry campaign to identify positive allosteric modulators (PAMs) of metabotropic glutamate receptor subtype 5 (mGluR5) led to the discovery of potent compounds featuring an oxazolidinone structural core flanked by biaryl acetylene and haloaryl moieties. However, biotransformation studies of some of these mGluR5 PAMs demonstrated the formation of glutathione (GSH) conjugates. The conjugates in question were formed independently of NADPH as the main products in liver microsomes and liver cytosol (rat and human) and exhibited masses that were 307 u greater than their respective substrates, indicating the involvement of a reductive step in the formation of these metabolites. To further characterize the relevant metabolic sequences, GSH conjugates of (4R,5R)-5-(3-fluorophenyl)-4-(5-(pyrazin-2-ylethynyl)pyridin-3-yl)oxazolidin-2-one and (4R,5R)-5-(4-fluorophenyl)-4-(6-((3-fluoropyridin-2-yl)ethynyl)pyridin-2-yl)oxazolidin-2-one were biosynthesized and isolated. Subsequent analysis by NMR showed that GSH had reacted with the acetylene carbon atoms of these mGluR5 PAMs, suggesting a conjugate addition mechanism and implicating cytosolic and microsomal GSH S-transferases (GSTs) in catalysis. Interestingly, five closely related mGluR5 PAMs were not similarly prone to the formation of GSH conjugates in vitro. These compounds also featured acetylenes, but were flanked by either phenyl or cyclohexyl rings, which indicated that the formation of GSH conjugates was influenced by proximal functional groups that modulated the electron density of the triple bond and/or differences in enzyme-substrate specificity. These results informed an ongoing drug-discovery effort to identify mGluR5 PAMs with drug-like properties and a low risk of reactivity with endogenous thiols.