Application of 19F-n.m.r. spectroscopy to the identification of dog urinary metabolites of imirestat, a spirohydantoin aldose reductase inhibitor.
1. Urine from a dog dosed orally at 20 mg/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 beta-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.
Gilbert
P J
PJ
Department of Drug Metabolism, Hoechst Pharmaceutical Research Laboratories, Hoechst UK Ltd., Walton, Milton Keynes, UK.
Hartley
T E
TE
Troke
J A
JA
Turcan
R G
RG
Vose
C W
CW
Watson
K V
KV
D000449:Aldehyde Reductase / Q000037:antagonists & inhibitors*; D000818:Animals; D002250:Carbon Radioisotopes; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004285:Dogs; D005449:Fluorenes / Q000032:analysis / Q000652:urine*; D005461:Fluorine; D005965:Glucuronates / Q000302:isolation & purification / Q000652:urine; D006827:Hydantoins / Q000032:analysis / Q000378:metabolism / Q000652:urine*; D006868:Hydrolysis; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008297:Male
D002250:Carbon Radioisotopes; D005449:Fluorenes; D005965:Glucuronates; D006827:Hydantoins; C052241:imirestat; D005461:Fluorine; D000449:Aldehyde Reductase