SYNTHESIS AND STRUCTURAL ELUCIDATION OF GLUTATHIONE AND N-ACETYL-CYSTEINE CONJUGATES OF 5-AMINOSALICYLIC ACID

The ability of 5-aminosalicylic acid (5-ASA) to be oxidized to a quino ne monoimine compound capable of conjugating with nucleophilic compoun ds such as N-acetyl-cysteine (NAC) and glutathione (GSH) has been inve stigated in vitro. Three isomeric conjugates of 5-ASA and NAC as well as three isomeric conjugates of 5-ASA and GSH were found to be formed. 5-ASA was initially oxidized by PbO2 in a solution of TRIS-HCl buffer pH 9.3 followed by the in situ addition of N-acetyl-cysteine or gluta thione to the oxidized 5-ASA at pH 7.5. The resulting conjugates were N-acetylated at the aromatic amino group in order to avoid autooxidati on of the products formed. The N-acetylated conjugates were isolated b y preparative HPLC and the structures were characterized by nuclear ma gnetic resonance (H-1-NMR and C-13-NMR) spectroscopy as well as by mas s spectrometry (MS) and the data obtained confirmed the formation of t hioether linkages between 5-ASA and the SH compounds. The chemical nat ure of the reactive intermediate capable of adding SH compounds was ve rified to be the 2-carboxy-quinone monoimine by H-1-NMR spectroscopy. The N-acetylated conjugates of 5-ASA and NAC were used as reference st andards in order to investigate whether such conjugates are excreted i n the urine from persons treated with 5-ASA. The N-acetyl-cysteine con jugates could be detected by fluorescense, which resulted in low detec tion limits ranging from 0.02 mug to 0.06 mug per ml corresponding to the transformation of about 0.003% of the daily dose of 5-ASA into mer capturic acids of 5-ASA, when 1 g of 5-ASA was ingested. In spite of t he low detection limits, none of the mercapturic acid conjugates was d etected in the urine from persons treated with 5-ASA.