Studies of intramolecular rearrangements of acyl-linked glucuronides using salicyclic acid, flufenamic acid, and (S)- and (R)-benoxaprofrn and confirmation of isomerization in acyl-linked .DELTA.9-11-carboxytetrahydrocannabinol glucuronide

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Abstract
NMR and HPLC have been used to investigate the rearrangements of 1-O-acylglucuronides in vitro and the occurrence of rearranged isomers in urine. Glucuronides of flufenamic acid, (s)- and (R)-benoxaprofen, salicylic acid, and .DELTA.9-11-carboxytetrahydrocannabinol were synthesized, by use of immobilized enzymes, or purified from urine. Ester-linked isomers of these glucuronides were characterized, and isomers derived from flufenamic acid, (S)-benoxaprofen, and salicylic acid were purified for further study by NMR and HPLC. The positions of the new ester linkages could be identified by two-dimensional NMR. Shifts not only in the resonance of the proton adjacent to the esterified hydroxyl group but also in the resonance of the anomeric proton on carbon 1 of the glucuronic acid moiety could be correlated with the position of eachisomeric ester bond. HPLC elution times also correlated with ester position in this small set of samples. The sequences of isomer formation were studied in situ by NMR and also at pH 8 by HPLC. These studies indicate that, for the three cases examined, the C-2 ester is formed first, followed by formation of C-3 and C-4 esters. The purified isomeric esters were found not to re-form the high-energy 1-O-acyl bond. All other rearrangement steps are reversible. In contrast to other glycosides and glycerol esters, no evidence could be found for rearrangements beyond nearest-neighbor hydroxyl groups in glucuronic acid. The sequence of formation and reversibility is consistent with an ortho ester intermediate, as has been proposed for rearrangements of other glycosides. Half-lives for the glucuronides studied fall in the middle of the range summarized by Rachmel, Hazelton, Yergey, and Liberato [(1985) Drug Metab. Dispos. 13, 705-710]. The diastereomeric glucuronides of (S)- and (R)-benoxaprofen were found to undergo hydrolysis and rearrangement at different rates, although the C-2 ester was the most stable in both diastereomeric series. The C-3 isomer of flufenamic acid was found to be the most stable. A single rearranged isomer of the 1-O-acylglucuronide of salicylic acid was observed, which had relatively high stability and a different mechanism of formation. HPLC, TLC color tests, and also 1H NMR were applied to urine samples expected to contain 1-O-acylglucuronides and their isomers formed from flufenamic acid, benoxaprofen, salicylic acid, and .DELTA.9-11-carboxytetrahydrocannabinol, in order to evaluate both the suitability of the techniques and also the occurrence or not of rearranged isomers in urines from rabbits, rhesus monkeys, and humans.