UBC Theses and Dissertations
The metabolic profiling of clobazam in rats Borel, Anthony Gerard Francis
Clobazam (CLBZ) is a 1,5-benzodiazepine with potent anticonvulsant activity. The metabolism of this drug was investigated in the rat and in vitro with the aid of stable isotope-labelled analogues and gas chromatography-mass spectroscopy (GCMS). Pentadeuteriophenyl CLBZ [²H₅]CLBZ was synthesized in essentially quantitative isotopic purity, and characterized by ¹H-NMR and GCMS. Of the five steps involved in the synthesis of [²H₅]CLBZ, the most susceptible to deuterium exchange was the nucleophilic substitution of 2,4-dichloronitrobenzene by aniline-d₇ to form N-(5-chloro-2-nitrophenyl)pentadeuteriophenylamine 18. In this step, the isotopic impurity aniline-2,3,4,5,-d₅ introduced protons from nitrogen into the ortho and para positions of the deuteriophenyl ring of 18. Phenol and catechol metabolites of CLBZ and N-desmethylclobazam (DMC) were synthesized according to the method used for [²H₅]CLBZ using 4-methoxyaniline and 3,4-dimethoxyaniline as starting materials. The methyl ether protecting group was well-suited for the synthetic procedure and was cleanly removed under mild conditions with BBr₃ to afford the phenols and catechols in good yield. The 0-methylated catechols of CLBZ and DMC were enzymatically synthesized from the catechol analogues of CLBZ and DMC using rat liver cytosol as a source of catechol O-methyltransferase (COMT) and S-adenosyl-L-methionine as the methyl donor. A meta/para O-methylation ratio of 2 was obtained from the CLBZ catechol. Condensation of formaldehyde with DMC catalyzed by K₂CO₃ produced a compound whose GCMS (EI) spectral properties were consistent with the carbinolamide, however, this compound could not be isolated because of its marginal stability. A major product for this reaction was 3-hydroxymethyl DMC which was characterized by ¹H-NMR and LCMS. Formaldehyde condensation catalyzed by KOH also afforded 3-hydroxymethyl DMC. Whether formaldehyde addition at the 3-position occurs as a kinetic product or whether it arises as a result of an equilibrium process subsequent to N-hydroxymethylation remains to be resolved. Rats were administered CLBZ:[²H₅]CLBZ as an approximate 50:50 mixture. Isotope shifts detected by GCMS allowed the following CLBZ metabolites to be identified. In bile, decreasing levels of 4'-hydroxy CLBZ, 4'-hydroxy DMC, O-methylated CLBZ catechols, 4'-hydroxy-3'-methoxy DMC and 3',4'-dihydroxy CLBZ appeared as both glucuronide and sulfate conjugates. In urine decreasing levels of the following sulfate conjugates were observed: 4'-hydroxy-3'-methoxy CLBZ, 4'-hydroxy CLBZ, 4'-hydroxy DMC and 3',4'-dihydroxy CLBZ. In bile, the glucuronides were predominant over the sulfates, whereas in urine only sulfate conjugates were detected. There was no detectable evidence of unconjugated CLBZ metabolites in bile or urine. As the biliary glucuronide and urinary sulfate, 3'-hydroxy-4'-methoxy CLBZ constituted ≤2 % of the O-methylated CLBZ catechols, whereas in bile, the sulfate conjugate of this metabolite constituted 30 %. One possible explanation for the higher levels of this metabolite as a biliary sulfate could be the low regiospecific methylation of dihydroxy CLBZ by COMT combined with arylsulfatase activity present in the kidney. Neither the CLBZ carbinolamide nor the N-hydroxy DMC (hydroxamic acid) was present in detectable levels in the conjugated or unconjugated fractions of bile or urine. Isotope effects in the metabolism of CLBZ and DMC in the rat were marginal for phenyl ring oxidation as measured for phenol metabolites (kH/kD =1.103 ± .090) and for catechols (kH/kD. = 1.088 ± .207). The metabolism of CLBZ and DMC in vitro was examined using control and phenobarbital-induced rat liver microsomes. CLBZ metabolism resulted in N-demethylation to DMC and phenyl ring hydroxylation at the 4'-position. There was no detectable evidence for the carbinolamide intermediate. Induction appeared to increase N-demethylation compared to phenyl ring hydroxylation. DMC was metabolized to 4'-hydroxy DMC with no detectable production of the hydroxamic acid. The isotope effect associated with CLBZ N-demethylation was determined by incubating an approximate 50:50 mixture of pentadeuteriophenyl CLBZ:trideuteriomethyl CLBZ with control and phenobarbital-induced rat liver microsomes. In this way, the source of DMC derived from demethylation ([²H₅]DMC) and dedeuteriomethylation (DMC) could be determined. The values of kH/kD, determined by protio/deuterio product ratios, were calculated at 3.65 ± 0.27 (n = 3) and 2.80 ± 0.16 (n = 4) for control and induced microsomes respectively.
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