small, it could not be isolated for further spectral analysis. Therefore, the positions of hydroxy and methoxy substitution on the aromatic system were assigned, based on the following information: (a) the presence of the 163 and 164 pair in the mass spectrum precluded the possibility of any substitution located ortho to the diphenyl ether oxygen; ( b ) the product of enzymatic hydroxylation of para-substituted monohydric phenols is catechol, which undergoes 0-methylation yielding para-substituted orthomethoxy phenol (6); and (c) catechol 0-methyltransferase does not 0-methylate resorcinol derivatives (7).
N-Methyl-N'-p-(4-hydroxy-3-methoxyphenoxy)phenylsulfa-
mide (VII1)-Compound VIII is the N-demethylation product of VII. It was found in the urine of all three species in conjugated form. The amount was very small but was separated from the crude urine extract by repeated TLC. The mass spectrum of VIII of the trimethylsilyl derivative showed that the molecular ion was at m/e 540. Loss of the N-trimethylsilyl-N-methylsulfamoyl group ( M -166) and the appearance of the 4-hydroxy-3-methoxy-N-methylphenoxyaniline ion a t 316 ( M -224) indicated a monomethyl sulfamide analog. Loss of 30 or 31 mass units suggested it was a methoxy phenol. Presence of the 163 and 164 pair indicated that the hydroxyl and methoxy substitutions were not ortho to the phenoxy oxygen.
The NMR spectrum of VIII was difficult to interpret because of the overlapping of aromatic protons. However, a set of AA'BB' multiplets indicated one phenyl ring had para-para symmetric disubstitution. This ruled out the possibility that the aniline ring was modified. Based on the same argument as for VII, VIII was assigned the structure of the N-demethyl-4-hydroxy-3-methoxy analog of the parent drug.
In conclusion, the results of these experiments indicated that the metabolic fate of the parent drug was different in these three species. Rats can only demethylate the drug a t reduced efficiency, as shown by the presence of only trace amounts of I1 (the N -demethylated product) and the presence of VII in rat urine. The lack of IV and VI (ortho-hydroxylated metabolites) in human urine may indicate that humans can only hydroxylate I in the paraposition. However, since the human subjects in this study were only administered a single dose of the drug while the animals were administered multiple doses, this apparent species variation may be caused by enzyme induction in animals.