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The use of stable isotope label — high resolution NMR technique for metabolic studies the metabolism of sparteine
1640
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pcesen~ons
I O.th.25.1 ! [
The use of stable isotope label - high resolution N M R technique for metabolic studies the metabolism of sparteine Ebner *, T., Meese *, C.O., Fischer * *, P. and Eichelbaum *, M. • Dr. Margarete Ftscher-Bosch.lnstitut fdr Klinische P ~ o l o g i e , Auerbachstr. 112, D-7000 Stuttgart 50 and * Insgtut fliw O r g ~ Chemic der Universit~ Stuttgart, Pfaffenwaldring 55, D.7000 Stuttgart 80, F.K G.
The quinolizidine alkaloid (=)-sparteine is metabolised in man by cytochrome P-450 IID6 which exhibits a genetic polymorphism. The two major metabofites of sparteine affected by this polymorphism are 2,3-didehydrosparteine (main metabofite) and 5,6-di~e~ydrosparteine. These metabolites can be formed either via enzymatic carbon hydroxylation or one electron oxidation of the nitrogen. Hitherto, the enamine structure of these two metabolites has been confirmed only by GC/MS after alkaline extraction of the urinary excreted metabofites. Carbinolamines as the hypothetical precursors of these enamines cannot be detected by this technique due to their chemical instability. In order to elucidate structure and chemical characteristics of the extremely basic enamines under physiological conditions high resolution 13C- and 1H-NMR were employed to study the synthetic conjugate acids of the sparteine metabofites, i e. the irninium salts 1,2- and 1,6-didehydrosparteinium-monopercldorate. We could demonstrate that in aqueous solution, at pH 4-9, stefic~llly3uniform (2S)-hydroxysparteine is formed from 1,2-didehydrospartcinium-monoperchlorate. A radical change in the C .~ignal pattern at pH values < 2 proves the formation of the 1,2-didehydrosparteinium ion. In contrast the 1,6-didehydrosparteinium-monoperehlorate remaines unchanged under such acidic conditions, and no signals for 6-hydroxysparteine appear in the 13C-MR spectrum. Metabolic studies were performed with deuterated [2,2-2H2]-sparteine. After oral administration of this stable-isotope-labeled analogue, deuterated [2-2I-I]-(2S)-hydroxysparteine should b¢ found in urine. 100 ml urine was collected (0-3 hr fraction), lyophilised, re-dissolved in 2 ml of deuterium-depleted water, and analysed by 2H-NMR. A resonance at 4.56 ppm was observed which was absent in blank urine. This shift value is characteristic of (2S)-hydroxysparteme. The identical carbinolamine structure is observed when undeuterated 1,2-didehydrosparteinium-monoperclflorate is dissolved in aqueous acetonitfile,and the solution monitored by 1H-NMR. When synthetic [2-2H]-l,2-didehydrosparteinium-monoperchlorate was added to the blank urine a signal identical with the metabolic carbinoIamine was observed. Similar results are obtained when urine of male Sprague Dawley rats which also excrete 2,3-didehydrosparteine as main metabolite, is examined after administration of the deuterated sparteine. The (2S)-hydroxysparteine pseudobase shows no, or only extremely slow exchange of the OH group as shown by 13C-NMR experiments with an H2180/H2160 (1 : 1) buffer and the isotope effect (upfield 0.02 ppm) on the signal of carbon-2. The unchanged carbinolAmine could, however, neither be isc,lated nor extracted from this solution without dehydratation to the corresponding enamine. Thus it is quite likely that sparteine is metabolised in man to sterically uniform (2S)-hydroxysparteine and 6-hydroxysparteine which, however, dehydratates immediately to the 1,6-iminium ion. Toe free enamine bases 2,3- and 5,6-didehydrosparteine therefore must be regarded as artefacts formed in the course of the analytical work-up for GC/MS; they cannot be detected in aqueous solution. The work presented here constitutes another example for high resolution NMR in pharmacological research which, in connection with the respective stable isotope labelled compounds, is a versatile tool for metabolic studies. Comparable to ~3C- and ~SN-NMR spectroscopy, 2H-NMR measurements of biological fluids, containing deuterated drugs or the respective metabolites, can shed a new light on metabolic reactions and reaction mechanisms. This work was supported by the Robert-Bosch Foundation Stuttgart. Excellent spectroscopic assistance of Mr. J. Rebell is gratefully acknowledged.
~l
pcesen~ons
I O.th.25.1 ! [
The use of stable isotope label - high resolution N M R technique for metabolic studies the metabolism of sparteine Ebner *, T., Meese *, C.O., Fischer * *, P. and Eichelbaum *, M. • Dr. Margarete Ftscher-Bosch.lnstitut fdr Klinische P ~ o l o g i e , Auerbachstr. 112, D-7000 Stuttgart 50 and * Insgtut fliw O r g ~ Chemic der Universit~ Stuttgart, Pfaffenwaldring 55, D.7000 Stuttgart 80, F.K G.
The quinolizidine alkaloid (=)-sparteine is metabolised in man by cytochrome P-450 IID6 which exhibits a genetic polymorphism. The two major metabofites of sparteine affected by this polymorphism are 2,3-didehydrosparteine (main metabofite) and 5,6-di~e~ydrosparteine. These metabolites can be formed either via enzymatic carbon hydroxylation or one electron oxidation of the nitrogen. Hitherto, the enamine structure of these two metabolites has been confirmed only by GC/MS after alkaline extraction of the urinary excreted metabofites. Carbinolamines as the hypothetical precursors of these enamines cannot be detected by this technique due to their chemical instability. In order to elucidate structure and chemical characteristics of the extremely basic enamines under physiological conditions high resolution 13C- and 1H-NMR were employed to study the synthetic conjugate acids of the sparteine metabofites, i e. the irninium salts 1,2- and 1,6-didehydrosparteinium-monopercldorate. We could demonstrate that in aqueous solution, at pH 4-9, stefic~llly3uniform (2S)-hydroxysparteine is formed from 1,2-didehydrospartcinium-monoperchlorate. A radical change in the C .~ignal pattern at pH values < 2 proves the formation of the 1,2-didehydrosparteinium ion. In contrast the 1,6-didehydrosparteinium-monoperehlorate remaines unchanged under such acidic conditions, and no signals for 6-hydroxysparteine appear in the 13C-MR spectrum. Metabolic studies were performed with deuterated [2,2-2H2]-sparteine. After oral administration of this stable-isotope-labeled analogue, deuterated [2-2I-I]-(2S)-hydroxysparteine should b¢ found in urine. 100 ml urine was collected (0-3 hr fraction), lyophilised, re-dissolved in 2 ml of deuterium-depleted water, and analysed by 2H-NMR. A resonance at 4.56 ppm was observed which was absent in blank urine. This shift value is characteristic of (2S)-hydroxysparteme. The identical carbinolamine structure is observed when undeuterated 1,2-didehydrosparteinium-monoperclflorate is dissolved in aqueous acetonitfile,and the solution monitored by 1H-NMR. When synthetic [2-2H]-l,2-didehydrosparteinium-monoperchlorate was added to the blank urine a signal identical with the metabolic carbinoIamine was observed. Similar results are obtained when urine of male Sprague Dawley rats which also excrete 2,3-didehydrosparteine as main metabolite, is examined after administration of the deuterated sparteine. The (2S)-hydroxysparteine pseudobase shows no, or only extremely slow exchange of the OH group as shown by 13C-NMR experiments with an H2180/H2160 (1 : 1) buffer and the isotope effect (upfield 0.02 ppm) on the signal of carbon-2. The unchanged carbinolAmine could, however, neither be isc,lated nor extracted from this solution without dehydratation to the corresponding enamine. Thus it is quite likely that sparteine is metabolised in man to sterically uniform (2S)-hydroxysparteine and 6-hydroxysparteine which, however, dehydratates immediately to the 1,6-iminium ion. Toe free enamine bases 2,3- and 5,6-didehydrosparteine therefore must be regarded as artefacts formed in the course of the analytical work-up for GC/MS; they cannot be detected in aqueous solution. The work presented here constitutes another example for high resolution NMR in pharmacological research which, in connection with the respective stable isotope labelled compounds, is a versatile tool for metabolic studies. Comparable to ~3C- and ~SN-NMR spectroscopy, 2H-NMR measurements of biological fluids, containing deuterated drugs or the respective metabolites, can shed a new light on metabolic reactions and reaction mechanisms. This work was supported by the Robert-Bosch Foundation Stuttgart. Excellent spectroscopic assistance of Mr. J. Rebell is gratefully acknowledged.