Application of ¹⁹F.n.m.r. spectroscopy to the identification of dog urinary metabolites of imirestat, a spirohydantoin aldose reductase inhibitor

Abstract

1. Urine from a dog dosed orally at 20mg/kg with ¹⁴C-imirestat, a spirohydantoin aldose reductase inhibitor, contained 17.7 and 12.5% of the administered radioactivity at 0-48 and 48-72 h respectively.

2. Radio-h.p.l.c. of the 0-48 h urine revealed a complex mixture of metabolites and a small proportion of parent drug (1.6% of dose). Direct ¹⁹F-n.m.r. spectroscopy of this urine showed the fluoride ion, numerous metabolites which were predominantly glucuronide conjugates and, as a minor component, the parent drug.

3. After incubation with β-glucuronidase the 0-48 h urine gave a ¹⁹F-n.m.r. spectrum showing fewer signals. This finding is consistent with aromatic ring hydroxylation followed by glucuronidation being the major metabolite pathways.

4. Deconjugated urine was analysed by proton-coupled ¹⁹F-n.m.r. and two-dimensional ¹⁹F-¹⁹F correlated spectroscopy. Results indicate that major components included three monohydroxy metabolites, a diphenol with both phenolic functions in the same ring, and a phenolic metabolite containing only one fluorine atom.

5. Semi-preparative h.p.l.c. of 0-48 h dog urine gave individual glucuronides isolated as mixtures of C-9 epimers. These fractions were hydrolysed and purified a second time by h.p.l.c. to give aglycones which were analysed by multi-nuclear n.m.r. and g.l.c.-mass spectrometry. The 3- and 4-hydroxy derivatives of imirestat were identified, as was the 2-hydroxy product obtained during or following defluorination. The other major aglycone was postulated to be the 3-fluoro-2-hydroxy metabolite. This represents a novel ‘NIH-shift’ type pathway for the metabolism of fluorobenzenes.