Formation of unusual glutamate conjugates of 1-[3(aminomethyl)phenyl]-N-[3-fluoro-2 '-(methylsulfonyl)-[1,1 '-biphenyl]-4-yl]-3-(trifluoromethyl)-1H-pyrazole-5-carboxamide (DPC 423) and its analogs: The role of gamma-glutamyltranspeptidase in the biotransformation of benzylamines

The role of gamma -glutamyltranspeptidase (GGT) in transferring glutamate f rom endogenous glutathione (GSH) to the benzylamine moiety of a compound, s uch as 1-[3-(aminomethyl)phenyl]-N-[3-fluoro-2'-(methylsulfonyl)-[1,1'-biph enyl]-4-yl]-3-(trifluoromethyl)- iH-pyrazole-5-carboxamide (DPC 423), is de scribed. Studies were performed with structurally related analogs of DPC 42 3 to demonstrate that this type of reaction was common to compounds possess ing a benzylamine group. Synthesizing appropriate standards and confirming by liquid chromatography (LC)/mass spectroscopy and LC/NMR made unambiguous assignments of the structures of glutamate conjugates of DPC 423. The use of stable isotope-labeled GSH for metabolism studies has not been described before. In the present study, we report the novel use of deuterated GSH in conjunction with mass spectral analysis to demonstrate the glutamate trans fer to the benzylamines in the presence of GGT. To further demonstrate that the a protons on the benzylamines and glutamate (as part of glutathione) w ere unaffected during the transpeptidation, these protons were replaced wit h deuterium. Acivicin (AT-125), a potent and selective inhibitor of GGT, wa s used to abolish the formation of the glutamate conjugates of DPC 423 in v itro and in vivo. This provided further evidence of the role of GGT in form ing the glutamate conjugates of benzylamines. This study demonstrated concl usively that GGT was responsible for mediating the transfer of glutamic aci d from GSH to the benzylamine moiety of a series of structurally related co mpounds.