An NMR study of the metabolic fate of 2-, 3- and 4-fluorobenzyl alcohols in the rat: detection of N-acetylcysteinyl conjugates as minor metabolites in urine.

Following the administration of 2-, 3- and 4-fluorobenzyl alcohols, the major metabolites detected in urine corresponded to the glycine conjugates of the corresponding benzoic acids. Little, or no, unchanged parent compound was detected in the samples. In addition to glycine-conjugated benzoic acids, a small proportion of the urinary metabolites for each of the fluorobenzyl alcohols was found to correspond to N-acetylcysteinyl conjugate. These were probably formed as the result of the production of a reactive sulphate ester during metabolism. The overall urinary recoveries of metabolites for the 2- and 3-fluorobenzyl alcohols were lower than that observed for the corresponding benzoic acids whilst that for 4-fluorobenzyl alcohol was similar.

Application of (1)H- and (19)F-NMR spectroscopy in the investigation of the urinary and biliary excretion of 3,5-, 2,4-ditrifluoromethylbenzoic and pentafluorobenzoic acids in rat.

1. The metabolism and excretion of 2,4-, 3,5-ditrifluoromethyl- and pentafluorobenzoic acids were studied in the bile-cannulated rat using (1)H- and (19)F-NMR spectroscopy following intraperitoneal administration at 50 mg kg(-1). 2. Pentafluorobenzoic acid was excreted in the urine entirely unchanged. No detectable compound or metabolites were eliminated in the bile. A total of 63.5 +/- 6.7% of the dose was recovered in the 24-h collection period. 3. In the case of 2,4-ditrifluromethyl benzoic acid, 83.9 +/- 5.2% of the dose was recovered in the 24h after administration, with about 52% being excreted in the urine and 32% in the bile. The majority of the material present in the urine was unchanged parent compound. In bile, some 60% of the compound-related material excreted was present as transacylated ester glucuronide conjugates. 4. For 3,5-ditrifluoromethylbenzoic acid, 49.6 +/- 5.3% of the dose was recovered in the 24-h collection period, with about 22% being excreted in the urine and 28% in the bile. The material excreted in both the urine and bile was a mixture of the parent acid and transacylated ester glucuronides. 5. Urinary excretion in bile-cannulated animals was similar to that found in studies using non-cannulated animals dosed at 100mg kg(-1).

The metabolism and excretion of [14c] 2- and 4-chlorobenzoic acids in the rat.

The metabolic fate of [14C]-labelled 2 and 4-chlorobenzoic acids (2- and 4-CBA) has been determined in the rat following intraperitoneal (i.p.) administration at 100 mg/kg to male rats. The major route of elimination for both 2-and 4-CBA was urine with > 80%, of the dose recovered in the initial 0-24 h after administration. Glycine conjugation was found to be the dominant metabolic fate for both [14C] 2- and 4-CBA however, the position of chloro substitution had a clear effect on the extent of metabolism via this route with ortho substitution reducing the extent of metabolism via this pathway.

1H and 19F-nmr spectroscopic studies on the metabolism and urinary excretion of mono- and disubstituted phenols in the rat.

1. 1H and 19F-nmr spectroscopy was used to investigate quantitatively the urinary excretion of the metabolites of 15 substituted phenols in the rat. The compounds studied were: 2-, 3-, and 4-fluorophenols; 2-, 3-, and 4-trifluoromethylphenol; 2,4-, 2,6- and 3,4-difluorophenol; 2-fluoro-5-trifluoromethylphenol, 3-fluoro-5-trifluoromethylphenol, 2-trifluoromethyl-4-fluorophenol; 3-chloro-4-fluorophenol, 3-fluoro-4-chlorophenol, and 3-methyl-4-fluorophenol. All compounds were dosed to the Sprague-Dawley rat (10 mg/kg i.p.) and urine was collected over the periods 0-8, 8-24 and 24-48 h post-dosing and analyzed using nmr spectroscopy. 2. The compounds were excreted in the urine mainly as glucuronide or sulphate conjugates or as the unchanged parent compound. There was considerable variation in the urinary excretion of the compounds over 48 h ranging from 22.1 to 93.6% of the dose. There was no apparent relationship between the molecular weight of compounds or their metabolites and the percentage molar recovery of each in the urine. 3. Ortho-substituted phenols in general showed a greater propensity for glucuronidation than did either meta- or para-substituted compounds, irrespective of the substituent group. The molar glucuronide-to-sulphate ratio for ortho-substituted compounds was found to be 2.2 +/- 0.9 whereas the ratio for both meta- and para-substituted compounds was 0.8 +/- 0.2 (p < 0.0001). 4. There were characteristic substituent effects of phenolic glucuronidation or sulphation on the 19F-nmr chemical shifts for both F- and CF3-substituted phemols and these substituent effects were a useful aid to metabolite signal assignment. 5. These studies show that nmr spectroscopy provides a rapid and convenient approach to the construction of metabolic databases of simple xenobiotics for the investigation of structure-metabolism relationships.

Prediction of urinary sulphate and glucuronide conjugate excretion for substituted phenols in the rat using quantitative structure-metabolism relationships.

1. The quantitative urinary excretion of the sulphate and glucuronide metabolites of 15 substituted phenols dosed to rat has been determined using high resolution 19F-nmr spectroscopy. 2. The urinary metabolic fate of each of the compounds was related to a series of calculated physicochemical properties for each compound to produce quantitative structure-metabolism relationships (QSMRs). Using these calculated molecular properties it was possible to predict the urinary recovery of xenobiotic material as a percentage of the administered dose, to classify the compounds according to their 'dominant' metabolite pattern and to predict quantitatively the proportions of glucuronide and sulphate conjugates in the urine by the use of multiple linear regression. 3. The quantitative predictions were tested by cross-validation and good prediction of total xenobiotic urinary recovery as a percentage of the administered dose was achieved based on an equation involving the electrophilic superdelocalizability at C4 (para to the hydroxyl function), the smallest principal ellipsoid axis dimension and the heat of formation. The largest moment of inertia and the electrophilic superdelocalizability at C3 were found to be the most significant factors for the prediction of the percentage glucuronide in the urine, and the urinary excretion of sulphate conjugates as a percentage of total urinary recovery was negatively correlated with the glucuronide excretion as little parent compound was excreted.

Quantitative structure-metabolism relationships for substituted benzoic acids in the rat. Computational chemistry, NMR spectroscopy and pattern recognition studies.

An extensive set of computed molecular properties, both steric and electronic, have been calculated using molecular orbital and empirical methods for benzoic acid (1) and a congeneric series of substituted benzoic acids, i.e. 2-, 3- and 4-fluorobenzoic acids (2-4), 2-, 3- and 4-trifluoromethyl benzoic acids (5-7), 2-, 3- and 4-methylbenzoic acids (8-10), 4-amino benzoic acid (11), 2-fluoro-4-trifluoromethyl benzoic acid (12), 4-fluoro-2-trifluoromethyl benzoic acid (13), 3-trifluoromethyl-4-fluorobenzoic acid (14). We have monitored the urinary excretion profiles and determined the metabolic fate of compounds 2-7, 12-14 in the rat using high resolution 1H and 19F NMR spectroscopy. Corresponding data for compounds 1,8-11 are taken from the literature. In all cases phase II glucuronidation or glycine conjugation reactions dominated the metabolism of these compounds. Compounds 5, 7, 12, 13 have ester glucuronides as their major metabolites; the rest primarily form glycine conjugates. Compounds (1-12) have been classified according to their calculated physicochemical properties using pattern recognition methods and principal components maps have been used as a novel type of structure-metabolism diagram. The maps of compounds in the physicochemical property space served to separate the compounds into the two major classes which related to their principal metabolic fate in vivo, namely glucuronidation versus glycine conjugation. Compounds 13 and 14 were used as further probes of the property space, and dominant metabolic fates of glucuronidation and glycine conjugation, respectively, were predicted from the previous "training set map". The metabolic fate of compounds 1-14 can thus be classified according to a simple set of physicochemical rules. Investigation of the physicochemical properties which are important in distinguishing the metabolic fate of the compounds may give insight into key features of the drug-metabolizing enzyme active sites and hence provide information on basic mechanisms of benzoate metabolism.