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Studies on the in Vivo Biotransformation of the Tobacco Alkaloid β-Nicotyrine† Xin Liu,‡ Kay Castagnoli,‡ Cornelis J. Van der Schyf,‡,§ and Neal Castagnoli, Jr.*,‡,§ Harvey W. Peters Center, Department of Chemistry, and Department of Biomedical Sciences and Pathobiology, VA-MD Regional College of Veterinary Medicine, Virginia Tech, Blacksburg, Virginia 24061 Received July 8, 1999
This paper reports the results of studies on the in vivo metabolic fate of the tobacco alkaloid 1-methyl-2-(3-pyridinyl)pyrrole (β-nicotyrine) in New Zealand white rabbits. Two previously characterized metabolites, 5-hydroxy-1-methyl-5-(3-pyridinyl)-2-pyrrolidinone (5-hydroxycotinine) and 2-hydroxy-1-methyl-5-(3-pyridinyl)-3-pyrrolin-2-one, were present in low concentrations in the urine of the treated animals. The major urinary metabolite of β-nicotyrine was identified as cis-3′-hydroxy-1-methyl-5-(3-pyridinyl)-2-pyrrolidinone (cis-3′-hydroxycotinine), the diastereoisomer of the major urinary metabolite of (S)-nicotine. The pathway leading to cis-3′-hydroxycotinine is proposed to proceed via autoxidation of 2-hydroxy-1-methyl-5-(3pyridinyl)pyrrole, a postulated cytochrome P450-generated metabolite of β-nicotyrine, followed by reduction of the carbon-carbon double bond present in the resulting 3-hydroxy-3-pyrrolin2-one species. This proposal is supported by the in vivo biotransformation of 2-acetoxy-1-methyl5-(3-pyridinyl)pyrrole, a latent form of the putative hydroxypyrrole intermediate, to cis-3′hydroxycotinine. The in vivo conversion of 5-hydroxy-1-methyl-5-(3-pyridinyl)-3-pyrrolin-2one to 5-hydroxycotinine is offered as evidence that supports the proposed reduction step.
Introduction The use of tobacco products is known to cause serious health problems (1-3). While the pharmacology and toxicology of (S)-nicotine (1), the principal tobacco alkaloid, have been examined extensively, relatively little work has focused on the minor tobacco alkaloids. We have been interested in the metabolic fate of 1-methyl-2-(3pyridinyl)pyrrole [β-nicotyrine (2)] in part because of reports that structurally related five-membered heteroarenes may be biotransformed to reactive metabolites that contribute to their toxic properties (4-6). Results from preliminary studies on β-nicotyrine employing cytochrome P450 rich Clara cells isolated from rabbit lung suggest that this alkaloid is bioactivated in an NADPHdependent process to form pneumotoxic metabolites (7).
Previous in vivo studies have established that β-nicotyrine is biotransformed extensively in the rabbit and † A preliminary report of this work appeared in Nicotine Safety and Toxicity (Benowitz, N. L., Ed.), N. Castagnoli et al., 1998, pp 57-65, Oxford University Press, New York and Oxford. * To whom correspondence should be addressed: Department of Chemistry, Virginia Tech, Blacksburg, VA 24061-0212. Telephone: (540) 231-8200. Fax: (540) 231-8890. E-mail: ncastagnoli@ chemserver.chem.vt.edu. ‡ Harvey W. Peters Center, Department of Chemistry, Virginia Tech. § Department of Biomedical Sciences and Pathobiology, VA-MD Regional College of Veterinary Medicine, Virginia Tech.
human to unknown metabolites (8, 9). NADPH-supplemented rabbit lung and liver microsomal preparations convert β-nicotyrine to four metabolites (10). The primary metabolites (Scheme 1) were shown to be an equilibrium mixture of two unstable pyrrolinone species, 1-methyl5-(3-pyridinyl)-4-pyrrolin-2-one (3) and 1-methyl-5-(3pyridinyl)-3-pyrrolin-2-one (4). These pyrrolinones undergo autoxidation, to form 5-hydroxy-1-methyl-5-(3pyridinyl)-3-pyrrolin-2-one (5), and hydration, to form 5-hydroxycotinine (6) (11). This paper summarizes the results of our in vivo metabolic studies on β-nicotyrine in New Zealand (NZ) white rabbits.
Materials and Methods Chemistry. All reactions were carried out under a nitrogen atmosphere. Reagents, chemicals, and analytical standards not described elsewhere were purchased from Aldrich Chemical Co. (Milwaukee, WI). HPLC grade solvents, acetonitrile, triethylamine, and acetic acid were purchased from Fisher Scientific (Pittsburgh, PA). Water was obtained from a Milli-Q system (Millipore, Bedford, MA). The hydroperchlorate salt of (3R,5S)trans-3′-hydroxycotinine (7) was a gift from P. Jacob, III (12). β-Nicotyrine (13), 2-acetoxy-1-methyl-5-(3-pyridinyl)pyrrole (8)(10), and racemic cis-3′-hydroxycotinine (9) (14) were synthesized according to published methods. GC/electron ionization mass spectrometry (GC/EIMS)1 analyses were performed on a Hewlett-Packard (HP) 5970 mass selective detector interfaced with an HP 5890 gas chromatograph using an HP-1 capillary column (12.5 m × 0.2 mm × 0.33 µm). Samples were injected into a split/splitless injector port 1 Abbreviations: GC-EIMS, gas chromatography/electron ionization mass spectrometry; HP, Hewlett-Packard; DA, diode array; ICR, albino mice descended from animals from Charles River Laboratories, Wilmington, MA; NZ, New Zealand.
10.1021/tx990124t CCC: $19.00 © 2000 American Chemical Society Published on Web 04/20/2000
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Scheme 1. Metabolic Fate of β-Nicotyrine in NADPH-Supplemented Rabbit Liver Microsomal Preparations
(250 °C) operating in the splitless mode. The column head pressure was 28 psi. The temperature program used for the GC/ EIMS analyses, except as noted, was as follows. The initial temperature of 100 °C was maintained for 1 min and then was increased at a rate of 25 °C/min to 275 °C where it was maintained for 2 min before recycling (program 1). For the GC/ EIMS analysis of the chloro diastereoisomers derived from the 3′-hydroxycotinines, we used the following temperature program. The initial temperature of 70 °C was maintained for 1 min and then was increased at a rate of 20 °C/min to 270 °C where it was maintained for 2 min before recycling (program 2) (12, 15). HPLC UV-diode array (DA) analyses were performed using an HP 1040A detector, Beckman 114 pumps, a Beckman 421A gradient controller, a Rheodyne injector, and an Alltech Econosil C8 column (25 cm × 4.6 mm, 10 µm). The following gradient elution profile was employed at a flow rate of 1 mL/ min using mobile phase A [water/acetonitrile/acetic acid/triethylamine (90:10:0.5:0.1, v:v:v:v)] and mobile phase B (100% acetonitrile): from 0 to 10 min, 100% A; from 10 to 20 min, 0 to 60% B; from 20 to 30 min, 60% B; from 30 to 35 min, 60 to 0% B; from 35 to 45 min, 0% B (10). Typical retention times (detection wavelengths) were as follows: cis- and trans-3′hydroxycotinine, 7.2 min (260 nm); 5-hydroxycotinine, 8.5 min (260 nm); the 5-hydroxypyrrolinone 4, 11.7 min (260 nm); and β-nicotyrine (2), 23.0 min (280 nm). Syntheses of tert-Butyldimethylsilyl Ether Derivatives 10 and 11 of (3R,5S)-trans-3′-Hydroxycotinine (7) and Racemic cis-3′-Hydroxycotinine (9), respectively (15). Solutions of (3R,5S)-trans-3′-hydroxycotinine (7) or racemic cis3′-hydroxycotinine (9), as their free bases (2 mg), in 0.5 mL of anhydrous N,N-dimethylacetamide containing 12 mg of tertbutyldimethylsilyl chloride and 12 mg of imidazole were vortexed for 1 min in a capped incubation tube and then were placed on a heating block at 80 °C for 1 h. After the mixtures had cooled to room temperature, 0.4 mL of a toluene/butanol solution (90:10) and 1 mL of water were added. The resulting mixtures were vortexed for 1 min, centrifuged, and placed in a dry ice/acetone bath to freeze out the aqueous layer. The upper organic layers were analyzed by GC/EIMS. The same procedure as described above was followed to analyze the residue of urine samples obtained from β-nicotyrine-treated rabbits (see below for details). (3S,5S)-trans-3-Chlorocotinine (12) and Racemic cis-3Chlorocotinine (13). These compounds were synthesized starting from (3R,5S)-trans-3′-hydroxycotinine (7) and racemic cis-3′-hydroxycotinine (9) by reaction with thionyl chloride in the presence of pyridine as previously described (12, 16). The resulting diastereomeric chlorocotinines had different GC retention times (program 2, 5.30 min for the trans-isomer derived from cis-3′-hydroxycotinine and 5.45 min for the cis-isomer derived from trans-3′-hydroxycotinine) and nearly identical GC/ EIMS data: m/z (abundance), 212 (12%, M+•, 37Cl), 210 (35%, M+•, 35Cl), 175 (15%), 153 (10%), 134 (10%, 37Cl), 132 (28%,
35Cl),
118 (100%), 91 (20%), 78 (8%). This reaction also was used to convert metabolic 3′-hydroxycotinine to the corresponding 3-chloro analogue as follows. The residue obtained from the Soxhlet extraction of the lyophilized rabbit urine sample (500 µg) in 2 mL of dichloromethane was heated for 2 h at 80 °C with 200 µL of thionyl chloride and 2 drops of pyridine. After removal of the volatile components, the residue in 0.5 mL of saturated aqueous sodium carbonate was vortexed with 1 mL of ethyl acetate for 1 min. After standing, the mixture was cooled in a dry ice/acetone bath to freeze out the aqueous phase and the upper organic layer was analyzed by GC/EIMS. Metabolism. All animals were housed in the Laboratory Animal Resource at Virginia Tech. Care and use of laboratory animals followed the guidelines issued by the National Research Council. All protocols had prior approval from the Virginia Tech Animal Care Committee in accordance with NIH requirements. ICR male mice (20-30 g) were used to establish dosing regimens for subsequent in vivo β-nicotyrine metabolism studies. NZ white male rabbits (1.8-2.4 kg) were used for the in vivo studies of β-nicotyrine, (3R,5S)-trans-3′-hydroxycotinine, the 5-hydroxypyrrolinone 5, and the 5-acetoxypyrrole 8. Animals were placed individually in metabolic cages, and control urine samples were collected for 24 h. After intraperitoneal (ip) administration of the appropriate compound [50 mg/kg except for β-nicotyrine (100 mg/kg)], urine was collected over a period of 24 h (treated urine samples). All urine samples were centrifuged at 10000g for 15 min. The clear supernatants were removed and lyophilized, and the resulting residues (approximately 2 g/sample) were extracted for 2 h with 150 mL of benzene using a Soxhlet extractor. Following evaporation of the benzene, the extracts were reconstituted in 2 mL of methanol. The resulting samples were analyzed by HPLC and GC/EIMS.
Results and Discussion (S)-Nicotine is highly toxic to mammals (17, 18) due to its potent cholinergic stimulating properties that are mediated by the N-methylpyrrolidinium conjugate acid of the alkaloid (19, 20). We anticipated that the acute toxicity of β-nicotyrine should be much lower since the pyrrolyl group will not be protonated at physiological pH. At ip doses of up to 100 mg/kg, neither mice nor rabbits showed any abnormal behavior. Therefore, our in vivo metabolic studies employed an ip dose of 100 mg/kg. The high polarity of the in vitro metabolites 5 and 6 of β-nicotyrine suggested the need for an efficient extraction procedure. Consequently, urine samples were lyophilized, and the residues were subjected to continuous Soxhlet extraction with benzene for 2 h. The HPLC UV-diode array assay employed in our in vitro studies (10) was
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Figure 1. (Left) HPLC chromatogram (monitored at 260 nm) of a representative urine sample collected from a control rabbit (A) and a rabbit treated with β-nicotyrine (B). (Right) HPLC-DA spectra showing the spectral characteristics of the peaks identified as 5 (C), 6 (D), and a mixture of 9 (E) from the same sample (solid lines) and the corresponding spectral characteristics of the standard compounds (dotted lines). Note that the HPLC-DA characteristics of synthetic trans-3′-hydroxycotinine (7) were indistinguishable from those of synthetic cis-3′-hydroxycotinine (9). See the text for evidence supporting the assignment of the metabolite as the cisisomer.
utilized for these in vivo studies except that the samples, which also were to be analyzed by GC/EIMS, were reconstituted in methanol prior to analysis. Under these conditions, 100% of β-nicotyrine, more than 90% of the 5-hydroxypyrrolinone 5, and 80% of 5-hydroxycotinine (6) could be recovered from spiked control urine. The HPLC tracings of extracts obtained from control (A) and treated (B) urine samples are shown in Figure 1. One major (tR ) 8 min) and two minor (tR ) 10 and 13 min) metabolite-related peaks were present in the tracing of the extract from the treated urine samples. The background at the retention time (23 min) of β-nicotyrine was complex, but no significant increase in signal was observed in the treated urine-extracted sample. Any β-nicotyrine present was below the levels of detection in a chromatogram obtained at 280 nm, the λmax for this compound (data not shown). Assignments for the two minor peaks were made by comparisons of their HPLCDA characteristics (retention times and UV spectra) with those of synthetic 5-hydroxycotinine (6, tR ) 10 min, Figure 1D) and the 5-hydroxypyrrolinone (5, tR ) 13 min, Figure 1C). When the urine samples from treated rabbits were spiked with synthetic 5 and 6, the resulting peak intensities increased accordingly; no evidence of separation of the metabolites from the standards was evident. Both 5 and 6 were anticipated metabolites since both are formed in rabbit lung and liver microsomal incubations of β-nicotyrine (11). It should be noted that 5-hydroxycotinine also has been reported to be an in vivo metabolite of (S)-nicotine (22) and (S)-cotinine (23). The major peak eluting at 8 min had not been observed in the HPLC
chromatograms of the microsomal incubation extracts. The HPLC-DA spectrum of this metabolite (Figure 1E) was similar to the corresponding spectra of 5 and 6. The total ion chromatogram obtained by GC/EIMS analysis of the urinary extracts from the treated animals also displayed a major peak (tR ) 5.0 min) that was not present in the corresponding control tracing (data not shown and ref 21). The full mass spectrum exhibited an apparent molecular ion at m/z 192, which is consistent with a molecular formula of C10H12N2O2 corresponding to cotinine + 16 atomic mass units. The possibility that this unknown metabolite was 5-hydroxycotinine (6) could be ruled out by comparison of its LC and GC/EIMS properties with those of synthetic 6 (21). A structure isomeric with 6 is trans-3′-hydroxycotinine (7), the major metabolite detected in the urine of smokers (24) and of animals treated with (S)-cotinine (23, 25). The HPLC-DA and GC/EIMS properties of synthetic (3R,5S)trans-3′-hydroxycotinine were indistinguishable from those of the metabolite. Furthermore, the GC/EIMS characteristics of the tert-butyldimethylsilyl ether derivative(s) 10 prepared from 7 and from the metabolite also were identical (21). Unfortunately, the HPLC-DA (Figure 1E) and GC/EIMS characteristics of synthetic cis-3′hydroxycotinine (9) and its tert-butyldimethylsilyl ether derivative 11 (15) also were indistinguishable from the corresponding characteristics observed with the metabolite. Consequently, although the structure of the metabolite undoubtedly was a 3′-hydroxycotinine, it was not possible to assign the relative stereochemistry at C-3 with the available evidence since we were unable to separate
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Scheme 2. Conversion of trans-3′-Hydroxycotinine to cis-3-Chlorocotinine
Scheme 3. Proposed in Vivo Biotransformation Pathway of β-Nicotyrine (2) to cis-3′-Hydroxycotinine (9)
these diastereomers or their tert-butyldimethylsilyl ether derivatives in either system.2
We next turned our attention to the 3-chlorocotinines that may be prepared in yields of >90% by treating the corresponding 3′-hydroxycotinines with thionyl chloride (16). Treatment of highly purified synthetic (3R,5S)trans-3′-hydroxycotinine and synthetic racemic cis-3′hydroxycotinine with thionyl chloride gave the corresponding 3-chlorocotinine species with GC retention times of 5.30 and 5.45 min, respectively. This reaction, which is known to proceed by SN2 attack of Cl- on the chlorosulfonyloxy intermediate 14 as shown in Scheme 2 for (3R,5S)-trans-3′-hydroxycotinine (7), leads to inver2 Voncken et al. (26), O’Doherty et al. (27), and, more recently, Murphy et al. (28) have reported the separation of cis- and trans-3′hydroxycotinine by GC and by HPLC. Our attempts to resolve these compounds chromatographically, however, were unsuccessful.
sion of configuration at C-3 (16). Therefore, the 3-chlorocotinine produced from trans-3′-hydroxycotinine must be the cis-isomer 13, and the 3-chlorocotinine produced from cis-3′-hydroxycotinine must be the trans-isomer 12. The GC/EIMS characteristics of the product (i.e., the retention time and mass spectrum) obtained by treating the 3′-hydroxycotinine derived metabolically from β-nicotyrine with thionyl chloride were found to be identical to those of trans-3-chlorocotinine. Consequently, the 3′hydroxycotinine metabolite itself must have the cisconfiguration 9 rather than the trans-configuration of the (S)-nicotine metabolite. The amounts of the trans-isomer, if present, were below the levels of detection. The metabolic pathway leading to the formation of cis3′-hydroxycotinine from β-nicotyrine is not apparent. The possibility that trans-3′-hydroxycotinine undergoes inversion of configuration to form the cis-isomer was ruled out since we could detect only the trans-isomer in the urine obtained from a rabbit treated ip with (3R,5S)-trans-3′hydroxycotinine. A second possible pathway proceeds via the P450-generated pyrrolinones 3 and 4 (Scheme 3). The conversion of these pyrrolinones to the 5-hydroxypyrrolinone 5 has been proposed to proceed via autoxidation of 2-hydroxy-1-methyl-5-(3-pyridinyl)pyrrole (15), a tautomer of 3 and 4 (11). Proton loss and single-electron transfer from 15 to dioxygen lead to the resonance-
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stabilized radical 16. A second oxidative step coupled with the introduction of the hydroxyl group at C-5 (radical recombination with hydroxyl radical and/or with hydroperoxyl radical followed by a hydrolytic sequence) yields 5. The isomeric 3′-hydroxypyrrolinone 17 also could be formed via radical recombination at C-3 of 16. Rearrangement of 17 to 18, analogous to the known equilibrium between 3 and 4 (10), followed by reduction of the carbon-carbon double bond of 18 would result in 9. Although neither 17 nor 18 was characterized in our earlier in vitro studies on the metabolism of β-nicotyrine (10, 11), we did report the presence in the GC/EIMS tracing of a hydroxypyrrolinone with a mass fragmentation pattern almost identical to that observed for 5 (11). We also know from earlier synthetic efforts (14) that 18 is chemically unstable, behavior which could explain our inability to detect this compound as an in vitro metabolite of β-nicotyrine. These considerations led us to examine the in vivo metabolic fate of the hydroxypyrrole intermediate 15. Since 15 itself was not available synthetically, the corresponding 2-acetoxy derivative 8, which undergoes hydrolysis to yield 15 (10), was used as a precursor of 15 in these studies. HPLC-DA analysis of a urine sample obtained from a rabbit treated with 8 showed a major peak corresponding to 3′-hydroxycotinine, a minor peak corresponding to the 5-hydroxypyrrolinone 5, and a second minor peak corresponding to 5-hydroxycotinine (6). The GC/EIMS analysis confirmed the presence of the 3′-hydroxycotinine that was shown, by conversion to its chloro analogue, to have the expected cis-configuration (21). Consequently, we conclude that β-nicotyrine is biotransformed in vivo via the corresponding 2-hydroxypyrrole intermediate 15 to cis-3′-hydroxycotinine (9). The key second step proposed for the metabolic production of cis-3′-hydroxycotinine from β-nicotyrine involves carbon-carbon double bond reduction of 17 and/or 18. We have tested this hypothesis by examining the in vivo fate of the 5-hydroxy-3-pyrrolinone 5 which served as a model for such a reduction. HPLC-DA analysis of the urinary extract obtained following ip administration of 5 led to the detection of 5-hydroxycotinine as the dominant UV absorbing peak in the HPLC-DA chromatogram (21). This behavior is consistent with the proposed pathway to cis3′-hydroxycotinine and may account for the 5-hydroxycotinine (6) found in the urine of rabbits treated with β-nicotyrine. The results of these in vivo studies clearly show that cis-3′-hydroxycotinine is the principal urinary metabolite of β-nicotyrine in the rabbit. This compound, however, is not present in rabbit lung and liver mitochondrial incubation mixtures even though convincing evidence is available to suggest that the formation of cis3′-hydroxycotinine proceeds via the 5-hydroxypyrrole species 15 which, in turn, is derived from the P450generated pyrrolinones 3 and 4. The oxidation-reduction sequence outlined in Scheme 3 suggests that the reduction of 17 and/or 18 to cis-3′-hydroxycotinine is mediated by an extrahepatic pathway. Voncken et al. have reported the excretion of cis-3′-hydroxycotinine in the urine of smokers (26). The possibility that (S)-nicotine may be converted to cis-3′-hydroxycotinine via β-nicotyrine is under investigation.
Acknowledgment. We thank Dr. David Moore, Mr. David Gemmell, and the staff of the Laboratory of Animal Resources for their support and assistance and Dr.
Liu et al.
Peyton Jacob, III, for his gift of (3R,5S)-trans-3′-hydroxycotinine. This work was supported by National Institute on Drug Abuse Grant R01 DA11089, Council for Tobacco Research Grant 4728R1, and the Harvey W. Peters Center for the Study of Parkinson’s Disease and Disorders of the Central Nervous System.
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In Vivo Biotransformation of β-Nicotyrine (22) McKennis, H., Jr., Bowman, E. R., Turnbull, L. B., and Schwartz, S. L. (1962) The correct structure of a ketoamide arising from the metabolism of (-)-nicotine. J. Am. Chem. Soc. 84, 4598-4599. (23) Dagne, E., Gruenke, L., and Castagnoli, N., Jr. (1974) Deuterium isotope effects in the in vivo metabolism of cotinine. J. Med. Chem. 17, 1330. (24) Neurath, G. B., and Pein, F. G. (1987) Gas chromatography determination of trans-3′-hydroxycotinine, a major metabolite of nicotine in smokers. J. Chromatogr. 415, 400-406. (25) Cundy, K. C., and Crooks, P. A. (1987) Biotransformation of primary nicotine metabolites II. Metabolism of [3H]-S-(-)-cotinine in the guinea pig: Determination of in vivo urinary metabolites by high-performance liquid-radiochromatography. Xenobiotica 17, 785-792.
Chem. Res. Toxicol., Vol. 13, No. 5, 2000 341 (26) Voncken, P., Rustemeier, K., and Schepers, G. (1990) Identification of cis-3′-hydroxycotinine as a urinary nicotine metabolite. Xenobiotica 20, 1353-1356. (27) O’Doherty, S., Revans, A., Smith, C. L., McBride, M., and Cooke, M. (1988) Determination of cis-3′-hydroxycotinine and trans-3′hydroxycotinine by high-performance liquid-chromatography. J. High Resolut. Chromatogr. 11, 723-725. (28) Murphy, S. E., Johnson, L. M., and Pullo, D. A. (1999) Characterization of multiple products of cytochrome P450 2A6-catalyzed cotinine metabolism. Chem. Res. Toxicol. 12, 639-645.
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