Syntheses and Characterization of the Acyl Glucuronide and Hydroxy Metabolites of Diclofenac

Abstract

In humans, metabolism of the commonly used nonsteroidal antiinflammatory drug diclofenac 1 yields principally the 4'-hydroxy 2, 5-hydroxy 3, and acyl glucuronide 4 metabolites. All three metabolites have been implicated in rare idiosyncratic adverse reactions associated with this widely used drug. Therefore, for mechanistic toxicological studies of 1, substantial quantities of 2-4 are required and their syntheses and characterization are described here. Key steps were a convenient two-step preparation of aniline 5 from phenol, efficient and selective 6-iodination of amide 18, and high-yielding Ullmann couplings to generate diarylamines 11 and 21. The acyl glucuronide 4 was obtained by Mitsunobu reaction of 1 (free acid) with allyl glucuronate 23 followed by Pd(0) deprotection, using a modification of a published procedure. We report full characterization of 4 and note that this important metabolite has been made available pure and in quantity for the first time. We report also the metabolic fates of the synthetic metabolites: 2 and 3 were glucuronidated in rats, but only 3 formed glutathione adducts in vivo and by enzymatic synthesis via a quinoneimine intermediate. A previously undescribed glutathione adduct of 3 was obtained by enzymatic synthesis. Compound 4 formed an imine-linked protein conjugate as evinced by sodium cyanoborohydride trapping.

An Improved Synthesis of 4'-Hydroxydiclofenac

“…The synthesis of 2 has been well described in two previous studies by Waterhouse et al 8 and Kenny et al 9 but these synthetic methods were not entirely satisfactory for scale-up reaction due to low overall yield and harsh reaction conditions. Waterhouse and colleagues synthesized 2 from 3,5-dichlorophenol using N- (2,6-dichloro-4-methoxyphenyl)acetamide as the key intermediate, but the yield in the cyclization step of the key intermediate was only 12%, which resulted in a very low overall yield (3.63%).…”

Multidrug Resistance-Associated Protein 3 Plays an Important Role in Protection against Acute Toxicity of Diclofenac

“…b-Anomers spontaneously isomerize into b-glucuronidase-resistant 2-, 3-, and 4-O-acyl isomers (Sallustio et al, 2000). DCF-AG undergoes these rearrangements as the pH changes from acidic to physiologic conditions such as those that occur in the GI tract (Ebner et al, 1999;Kenny et al, 2004). DCF-AG can form adducts with multiple proteins in the liver and GI tract, and dipeptidyl peptidase IV was identified as a DCF-AG target in rat liver, where adduction resulted in decreased activity (Hargus et al, 1995).…”

Feasibility of Biomonitoring of Exposure to Permethrin Through Analysis of Long-Lived (Metabolite) Adducts to Proteins

“…The yields of the first synthesis method (Kenny et al, 2004) was very low. In this case, a mixture of α and β-anomers resulted, having the correct mass that could be separated after deprotection, i.e., removal of the allyl function The desired β-anomer could be obtained in mg amount and could be fully characterized.…”