Characterization of a novel degradation product of 2,2 '-dithiobis[N-isoleucylbenzamide], an inhibitor of HIV nucleocapsid protein zinc fingers

Zinc finger motifs have been found to be important in a variety of protein structures including transcription factors and viral nucleocapsid proteins. Recently, it was demonstrated that various aromatic disulfides effectively remove the metal ion from the zinc finger, resulting in an alteration of t ertiary structure in this region of the protein, thereby inhibiting transcr iption. Among these compounds, 2,2'-dithiobis[N-isoleucylbenzamide] exhibit s activity against human immunodeficiency virus (HIV)-type 1 in vitro and h as been selected for preclinical development as an anti-HIV agent. Analysis of this agent by reversed-phase high-performance liquid chromatography (HP LC) indicated a significant quantity of two additional compounds. Identifyi ng the parent disulfide was accomplished by scanning eluting peaks with pos itive ion thermospray ionization (TSP) mass spectrometry (RIS). Solution-in duced disproportionation of the disulfide into its sulfydryl monomer was de monstrated by treating the drug with dithiothreitol (DTT) prior to HPLC ana lysis. TSP-MS analysis of the remaining chromatographic peak suggested a mo lecular weight of 265, which, with H-1-nuclear magnetic resonance (NMR) dat a of the isolated material, allowed us to elucidate the chemical structure as N-isoleucyl-benzisothiazolone. Contact with stainless steel, such as tha t employed in an HPLC system, was found to accelerate degradation of the pa rent disulfide to the benzisothiazolone. (C) Published by Elsevier Science B.V.