Chem. Res. Toxicol. 1993,6, 794-799
794
Articles Identification of 4- (Methylnitrosamino)- 1-[3- (6-hydroxypyridy1)l-1-butanone as a Urinary Metabolite of 4-(Methylnitrosamino)-l-(3-pyridyl)-l-butanone in Rodents Dhimant Desai, Shashi S. Kagan, Shantu Amin, Steven G. Carmella, and Stephen S. Hecht* American Health Foundation, Valhalla, New York 10595 Received May 13, 1 9 9 3
A previously unknown urinary metabolite of 4-(methylnitrosamino)-l-(3-pyridyl)-l-butanone (NNK) was identified as 4-(methylnitrosamino)-1-[3-(6-hydroxypyridyl)]-l-butanone (6hydroxyNNK). The metabolite was initially isolated from rat urine. On the basis of its M S and NMR, it was either a 4- or 6-hydroxypyridyl derivative of NNK. Model compounds were synthesized t o distinguish between these possibilities; the results indicated t h a t the metabolite was 6-hydroxyNNK. This was confirmed by independent synthesis; the spectral and chromatographic properties of 6-hydroxyNNK were the same as those of the metabolite. F-344 rats and A/J mice treated with 100mg/kg N N K excreted approximately 1 % of urinary metabolites as 6-hydroxyNNK; i t was not detected as a sulfate or glucuronide conjugate. This is the first example of a pyridyl-hydroxylated metabolite of a tobacco-specific nitrosamine. On the basis of comparison t o published data on other pyridine derivatives, 6-hydroxyNNK may be formed by bacterial metabolism. T h e potential utility of 6-hydroxyNNK as a dosimeter of human uptake of N N K is discussed.
Introduction 4-(Methylnitrosamino)-1-(3-pyridyl)- 1-butanone (NNKY is likely to be important in the etiology of cancers of the lung, oral cavity, and pancreas in people who use tobacco products (1-3). Its role as a potential causative agent for these cancers is supported by extensive studies in laboratory animals which demonstrate its potent carcinogenicity, especially in the lung, as well as data on the exposure and uptake of NNK by smokers and users of smokeless tobacco products (4-6). Major pathways of NNK metabolism which have been elucidated previously include hydroxylation of the methylene and methyl carbons adjacent to the N-nitroso group (a-hydroxylation), reduction of the carbonyl group to produce 4-(methylnitrosamin0)-1-(3-pyridy1)-1-butanol (NNAL) followed by conjugation to its glucuronides, and N-oxidation of the pyridine ring (7-9). Some of these metabolites undergo further oxidative or reductive transformations. The a-hydroxylation pathways produce intermediates that react with DNA to form methyl and pyridyloxobutyl adducts that are believed to be involved in carcinogenesis by NNK ( 1 -3, 10, 11). The metabolites of [5-3H]NNK identified so far in rat urine account for a large proportion of the radioactivity Abstract published in Advance ACS Abstracts, October 1, 1993. 1 Abbreviations: NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-l-b~tanone; NNAL, 4-(methylnitrosamino)-1-(3-pyridyl)-l-butano1: NNALGluc, [4-(methynitrosamino)-l-(3-pyridyl) but-l-yll-~-O-~-glucosiduronic acid; CI-MS,chemical ionization mass spectrum; BSTFA, bis(trimethylsily1)trifluoroacetamide;EI-MS,electron impact mass spectrum; NNNN-oxide, N'-nitrosonornicotine 1-N-oxide;NNN, N'-nitrosonornicotine.
in urine, but unidentified peaks have been consistently observed. We are interested in the identity of these peaks because they could be used as markers of human uptake of NNK as well as providing insights on its mechanism of action. Although the unidentified peaks in rat urine are small, they may occur in significant quantities in the urine of primates or humans exposed to NNK. For example, [4-(methylnitrosamino)-1-(3-pyridyl)but- 1-yl] -8-O-D-glucosiduronic acid (NNAL-Gluc) diastereomers have been identified as urinary metabolites of NNK; one diastereomer accounting for only 2-8 % of the dose was detected in rat urine while both diastereomers were detected in patas monkey urine and comprised approximately 2030% of the urinary metabolites (8, 9). Our finding of relatively high concentrations of NNAL-Gluc in patas monkey urine led to its detection in the urine of smokers (6). In this study, we have investigated the identity of a previously unknown metabolite detected in the urine of rats treated with [5-3H]NNK. As the results will show, this metabolite, in contrast to NNAL-Gluc, will probably not be an optimal marker of NNK uptake in humans but may be of interest with respect to NNK metabolism by endogenous bacteria.
Experimental Section Apparatus. HPLC was performed with Waters Associates systems (Millipore; Waters Division, Milford, MA), equipped witha4.6" by25cmPartisphere5-pmC18column (Whatman, Clifton, NJ) and either a Model 440 UV/visible detector or a Gilson Model 116 UV detector operated at 254 nm. Radioflow analyses were carried out using a Flo-one/Beta radioactive flow detector (RadiomaticInstruments, Tampa, FL). The following
0S93-22~~/93/2~06-0~94$04.0QlQ 0 1993 American Chemical Society
6-HydroxyNNK, a Urinary Metabolite of NNK solvent programs were used (1)1mL/min of 0-35% MeOH in 20 mM sodium phosphate buffer (pH 7) in 70 min, with a 15-min hold after the first 15 min; (2) 1 mL/min of 15% MeOH in 20 mM sodium phosphate buffer (pH 7). NMR spectra were obtained in CDC18 with a Bruker Model AM 360 WB spectrometer. Chemical shifts are reported in ppm downfield from tetramethylsilane. All assignmentswere confiimedby decoupling experiments. UV spectra were determined in MeOH with a Hewlett-Packard 89530MS-DOSUV/visible spectrophotometer. MS and GUMS analyseswere carried out with a Hewlett-Packard Model 5988Ainstrument. Chemicalionization mass spectra (CIMS) were obtained with methane as reagent gas. For GC-MS, the metabolite was silylated with bis(trimethylsily1)trifluoroacetamide (BSTFA)/l%TMS from Regis Chemical Co (Morton Grove, IL). Analysis was carried out on a 30 m by 0.25 mm DB 17 column, 0.15-pm film thickness, with a temperature program of 5 OC/min to 190 OC and then 2 OC/min to 280 "C. Highresolution MS were determined with a Kratos Profile MS (Kratos Analytical, Manchester, England), courtesy of Dr. Stephen R. Wilson, New York University Chemistry Department. Chemicals. NNK and metabolite standards were synthesized (12-14). [5-3H]NNK,which has tritium at the 5-position of the pyridine ring, and [C3H3]NNK were obtained from Chemsyn ScienceLaboratories, (Lenexa,KS). Their purities (>95% ) were assessed by HPLC prior to administration to rats. BSTFA was purchased from Pierce Chemical Co. (Rockford, IL). All other chemicals used in the synthetic procedures were obtained from Aldrich Chemical Co. (Milwaukee,WI) unless noted otherwise. Syntheses. (1) 3-[2-(1,3-Dioxolanyl)]-l-[3-( 4-hydroxypyridyl)]-1-propanone (8). Under an N2 atmosphere, 4-hydroxynicotinic acid (15) (1.39 g, 10 mmol) was suspended in benzene (75 mL), and thionyl chloride (10 mL) was added. The mixture was heated under reflux for 20 h, cooled to room temperature, and evaporated to dryness to give 4-hydroxynicotinoylchloride which was suspended in dry THF (20 mL) and cooled in an ice/ H2O bath. To this mixture, a Grignard reagent [prepared by reacting Mg (0.97 g, 40 mmol) and 2-(2-bromoethyl)-l,3-dioxolane (3.52 g, 30 mmol) in dry THF (40mL)] was added dropwise.The mixture was stirred for 18 h at room temperature and poured into saturated aqueous NHlCl (100 mL). The mixture was extracted with EtOAc (4 X 100mL). The combinedorganiclayers were dried (MgSO4) and concentrated in vacuo to yield a crude product, which was purified by chromatography on silicagel with elution by EtOAc to give 8 (0.67 g, 30%) as an oil: lH NMR 6 1.8-2.0 (m, 2H, CH&H), 3.8-4.1 (m, 6H, OCH2 and COCH2), 5.0 (t,lH, CHzCH, J = 4.62 Hz), 7.42 (d, lH, pyridine H5, J = 9.21 Hz),8.58 (d, lH, pyridine H6, J = 9.34 Hz), 8.72 (d, lH, pyridine H2, J = 2.31 Hz); high-resolution MS, calcd for (M+- C2H4O) 179.0582,found 179.0564;calcd for (M+- Ca902) 122.0240,found 122.0240. (2) 34241,3-Dioxolanyl)]-l-[3-(6-hydroxypyridyl)]-l-propanone (9). In a similar manner, dioxolane 9 was prepared in 41% yield from 6-hydroxynicotinoylchloride: lH NMR 6 2.072.20 (m,2H, CH2CH),3.8-4.04 (m,4H, OCH2),4.42 (t,2H, COCH2, J = 6.51 Hz), 5.02 (t, lH, CH2CH, J = 4.61 Hz), 6.61 (d, lH, pyridine H5, J = 9.56 Hz), 8.05 (dd, lH, pyridine H4, J = 9.54, 2.44 Hz), 8.25 (d, lH, pyridine H2, J = 2.39 Hz); high-resolution MS, calcd for (M+- C2H4O) 179.0582, found 179.0587; calcd for (M+ - CsH-302) 122.0240, found 122.0240. (3) Methyl 6-(Benzyloxy)nicotinate (12) and Benzyl 6-(Benzyloxy)nicotinate (13). Under an N2 atmosphere, NaH (0.8 g of a 60 % suspension in oil, 20 mmol) was washed twice with hexane to remove the protecting oil and suspended in dry THF (20 mL). To this suspension, benzyl alcohol (2.14 g, 20 mmol) was added at room temperature and the mixture was heated at 50 "C for 2 h. After cooling to room temperature, methyl 6-chloronicotinate (1.71 g, 10 mmol) in dry THF (20 mL) was added dropwise and the mixture was heated at 50 "C for 48 h. The reaction mixture was cooled to room temperature, poured into cold 2 N HCl, and extracted with CHCls (4 X 30 mL). The combined organic layers were dried (MgS04), filtered, and evaporated. The residue was purified on a silicagel columnusing
Chem. Res. Toxicol., Vol. 6, No. 6, 1993 795 hexane/CHzClz (1:l) as eluent to give benzyl 6-(benzyloxy)nicotinate (13): 0.64 g, 20%; mp 63-65 "C; lH NMR 6 5.36 (s, 2H, COOCH2), 5.44 (s,2H, OCH2Ph), 6.82 (d, lH, pyridine H5, J = 8.63 Hz), 7.3-7.48 (m, 10H, aromatic), 8.2 (dd, lH, pyridine H4, J = 8.78,2.47 Hz), 8.9 (d, lH, pyridine H2, J = 2.31 Hz); MS m/z (relative intensity) 319 (M+,20), 274 (6), 242 (5), 213 (31), 184 (14), 122 (16), 91 (1001, 65 (30). Further elution with hexane/CHzCl2 (1:l)gave methyl 6benzyloxynicotinate (12): 0.68 g, 28%; mp 43-45 OC; lH NMR 6 3.91 (s,3H, OCHs), 5.45 (s,2H, OCHsPh), 6.83 (d, lH, pyridine H5, J = 8.89 Hz), 7.29-7.51 (m, 5H, aromatic), 8.18 (dd, lH, pyridine H4, J = 8.67, 2.23 Hz), 8.86 (d, lH, pyridine H2, J = 2.21 Hz), MS m/z (relative intensity) 243 (M+,30), 212 (15), 166 (20), 137 (40), 122 (81,91 (loo), 65 (18). (4) 3-[[3-[6-(Benzyloxy)pyridyl]]carbonyl]-l-methyl-2pyrrolidinone (14). NaH (0.17 g of a 60% suspension in oil, 4.28 mmol) was added to a 500-mL round-bottom flask. The suspension was washed twice with hexane to remove the oil, and dry toluene (50 mL) was added. These operations were carried out in an atmosphere of N2. To the stirred suspension at room temperature was added dropwise a mixture of methyl 6(benzy1oxy)nicotinate(12; 0.8 g, 3.29 mmol) and 1-methyl-2pyrrolidinone (0.33 g, 3.29 mmol) in dry toluene (30 mL). The reaction mixture was heated under reflux for 48 h, cooled to room temperature, and poured into cold 4 N HCl(100 mL). The toluene layer was separated, and the aqueous layer was extracted with CHC13 (3 X 100 mL). The combined organic layers were dried (MgS04), filtered, and concentrated. The residue was purified by chromatography on a silica gel column using EtOAc as eluent to give 14 (0.53 g, 52%) as an oil: lH NMR 6 2.17-2.3 (m, lH, pyrrolidinone H4), 2.63-2.74 (m, lH, pyrrolidinone H4), 2.86 (8, 3H, NCHs), 3.36-3.45 (m, lH, pyrrolidinone H5), 3.543.64 (m, lH, pyrrolidinone H5), 4.32-4.4 (m, lH, pyrrolidinone H3), 5.47 (8, 2H, OCHzPh), 6.86 (d, lH, pyridine H5, J = 8.81 Hz), 7.3-7.5 (m, 5H, aromatic), 8.31 (dd, lH, pyridine H4, J = 8.78, 2.51 Hz), 8.98 (d, lH, pyridine H2, J = 2.24 Hz); MS m/z (relative intensity) 310 (M+, 28), 212 (201, 184 (6), 122 (2), 98 (14), 91 (100). (5) 3-[[3-(6-Hydroxypyridyl)]carbonyl]-l-methyl-2-pyrrolidinone (15). Lactam 14 (0.5 g, 1.61 mmol) and 10% Pd/C (50 mg) in absolute EtOH (100 mL) were stirred under Hz (50 psi) for 4 h. The mixture was filtered through Celite, and the filtrate was concentrated in uacuo to afford crude hydroxylactam 15. Chromatographyonasilicagelcolumnwithelutionby EtOAc followed by EtOAc/MeOH (982) gave pure 150.27 g, 74%;mp 214-216 OC; lH NMR 6 2.13-2.25 (m, lH, pyrrolidinone H4), 2.68-2.77 (m, lH, pyrrolidinone H4), 2.85 (s,3H, NCHs), 3.353.43 (m, lH, pyrrolidinone H5), 3.53-3.62 (m, lH, pyrrolidinone H5), 4.11-4.19 (m, lH, pyrrolidinone H3), 6.58 (d, lH, pyridine H5, J = 9.69 Hz), 8.11 (dd, lH, pyridine H4, J = 9.66,2.39 Hz), 8.50 (d, lH,pyridineH2, J = 2.22 Hz);MS m/z (relative intensity) 220 (M+, 18), 164 (4), 122 (58), 98 (loo), 94 (14). (6) 4-(Methylnitrosamino)-l-[3-(6-hydroxypyridyl)]-lbutanone (5). A solution of 15 (0.25 g, 1.14 mmol) in 10 mL of 6 N HC1was heated at reflux with stirring for 96 h. The reaction mixture was cooled and concentrated to dryness on a rotary evaporator to give 4-(methylamino)-l-[3-(6-hydroxypyridyl)]1-butanone dihydrochloride which was used in the next step without purification. The dihydrochloride was dissolved in H2O (15mL) and cooled in an ice/H*Obath. The pH of the reaction mixture was adjusted to 4 with 2 N NaOH. To this mixture, a solution of NaNO2 (0.083 g, 1.21 mmol) in H2O (2 mL) was added slowly dropwise at 0 OC. The mixture was allowed to stir at room temperature for 18h. The pH was adjusted to 8 and the mixture was extracted with CHC13 (5 X 20 mL). The combined organic layers were dried (MgS04),filtered, and evaporated. The crude product was purified on a silica gel column with elution by EtOAc followed by EtOAc/MeOH (982) to give 5: 0.1 g, 40%; mp 129-131 "C; UV A, (e) 207 (1.26 X lo"), 232 (8.16 X 1@),274 (1.63 X 10'); 1HNMR61.87(m,0.66H,ZCCH2C),2.11(m,1.33H,ECCH~C), 2.81 (t, 0.66H, ZCOCH2, J = 6.91 Hz), 2.91 (t, 1.33H,E COCH2,
796 Chem. Res. Toxicol., Vol. 6, No. 6, 1993
Desai et al.
0-Gluc
b-'
2
1
!='
N=O
3
OH
N=O
Time (mln)
Figure 1. Chromatogram obtained upon HPLC analysis (solvent program 1)of 24-h urinary metabolites, excreted by an F-344 rat treated with 100 mg/kg [5-3H]NNK.The numbered peaks refer to the structures in Figure 2. J = 6.90 Hz), 3.08 (8, 2H, E NCHa), 3.68 (t, 0.66H, 2 NCH2, J = 7.05 Hz), 3.78 (8, lH, Z NCHa), 4.22 (t, 1.33H, E NCHz, J = 6.73 Hz), 6.60 (d, lH, pyridine H5, J = 9.59 Hz), 8.02 (dd, lH, pyridine H4, J = 9.62, 2.54 Hz), 8.15 (d, lH, pyridine H2, J = 2.52 Hz), 12.97 (br 8, lH, OH); MS m/z (relative intensity) 223 (M+, 4), 193 (M+-NO, 95), 164 (15), 122 (loo), 94 (32),42 (92); high-resolution MS, calcd for (M+- NO) C10H13N202,193.0977; found 193.0999. Animal Experiments. Male F-344 rats (255-290 g) were obtained from Charles River Breeding Laboratories (Kingston, NY). Four rats were each given a sc injection of [5-3H]NNK [lo0 mg (1.66 mCi)/kg] in approximately 0.7 mL of trioctanoin, and three rats were treated with [CSHaINNK of the same dose and specific activity. The 24-h urine was collected from each rat treated with [5-3H]NNKand 2-6-h urine from each rat treated with [CSHaINNK. An additional group of four rats was treated with unlabeled NNK, 100mg/kg, and the 24-h urine was collected from each rat. The radiolabeled urine was used for quantitation of metabolites and for comparison of [5JH]NNK and [CSHalNNK metabolites. The unlabeled urine was used for analysis of glucuronide or sulfate conjugates of 6-hydroxyNNK. Female A/J mice (6-8 weeks old) were obtained from The Jackson Laboratory (Bar Harbor, ME). Five mice were each given an ip injection of [EI-~HINNK[lo5 mg (5.0mCi)/kg] in approximately 0.1 mL of saline. The 24 h urine was collected and pooled for analysis. Duplicate aliquota of rat urine (250 pL) were filtered through a disposable syringe filter (Schleicher and Schuell, Keene, NH), mixed with 500 p L of HaO, filtered through Centricon 10 microconcentrator filters (Amicon Div., W. R. Grace and Co., Danvers, MA), and analyzed by HPLC solvent program 1. Peaks were quantified by radioflow counting. Duplicate aliquota of 50 pL of mouse urine were analyzed the same way. For analysis of glucuronides in rat urine, duplicate aliquota of 250 pL of urine were incubated for 60 min at 37 O C with @-glucuronidase(type X-A, Sigma),5000 units in 500 pL of H2O. Aliquota (50pL) of mouse urine were treated the same way. After incubation with @-glucuronidase,the samples were filtered through Amicon 10 filters and analyzed by HPLC as above. Similarly,duplicate samples of rat or mouse urine were incubated at 37 "C for 60 min with sulfatase (type VI, Sigma)and analyzed by HPLC.
Results A typical HPLC profile of 24-h urinary metabolites of [5-3HlNNK, administered to rats a t a dose of 100 mg/kg, is illustrated in Figure 1. Peaks 1-4,6, and 7 have been identified previously as the compounds bearing the same numbers in Figure 2 (8,13,16). The hydroxy acid 1 and keto acid 2 result from a-hydroxylation of NNAL and NNK while peak 7 is unchanged NNK. The other numbered peaks are products of carbonyl reduction (NNAL, 61, carbonyl reduction followed by glucuronidation (NNAL-Gluc, 4), and carbonyl reduction and pyridine N-oxidation (NNAL-N-oxide, 3). Peak 5 was the focus of this study. In order to determine whether all carbon atoms of NNK were still present in this metabolite, we compared
''
7,NNK
"
8
N-0 &'\CHs CHjO
10
Figure 2. Structures of compounds discussed in the text. the urinary metabolites of [5-3HlNNK and [C3H31NNK. The HPLC profile obtained from the urine of rats treated with [C3H31NNK lacked peaks 1and 2, as expected. The other peaks were all present. Therefore, the NNK skeletal structure was still present in peak 5. HPLC solvent program 2 was employed to purify peak 5. An improved separation was achieved with this program. Peak 5 appeared as a mixture of E- and 2 isomers, indicating that the N-nitroso group was present. Near-baseline resolution of the E and 2 isomers of NNAL was also achieved using this program. Two additional unknown minor metabolites eluted before and after peak 5 and NNAL. Peak 5 was collected for MS and NMR analysis. The electron impact mass spectrum (EI-MS)of peak 5 had major ions a t mlz 193 (relative intensity 98) and mlz 122 (100) and a small molecular ion a t mlz 223 (2). The CI-MS showed a molecular ion of mlz 224 (37). The CIMS of the silylated metabolite showed a molecular ion and base peak a t mlz 296. These data are consistent with a pyridyl hydroxylated derivative of NNK, molecular weight 223. The major ions in the EI-MSwould correspond to loss of NO (mlz 193) and cleavage between the carbonyl and methylene groups with charge retention in the pyridine-containing fragment (mlz 122). Analogous major ions are seen in the EI-MS of NNK (17). The 'H-NMR spectrum of the metabolite is shown in Figure 3. Although some impurities were present, all of the expected peaks for a pyridyl-hydroxylated NNK were observed. The upfield doublet a t 6.7 ppm was assigned to the pyridine proton adjacent to the hydroxyl group. Its large coupling constant of 9.8 Hz indicated that this proton was also ortho to another proton on the pyridine ring. Therefore, the hydroxyl group was located a t either the 4- or 6-position of the pyridine ring. These substitution patterns could not be unequivocally distinguished based only on the spectrum in Figure 4. Therefore, we synthesized model compounds 8 and 9 (Figure 2). The data for the pyridyl protons of these compounds are summarized in Table I. The spectrum of the unknown (Figure 4) was closer to that of the 6-hydroxy-substituted analogue 9 than to that of the 4-hydroxy-substituted 8. In the latter, all pyridyl protons were shifted downfield, perhaps resulting from hydrogen bonding between the 4-hydroxy group and the carbonyl group in 8. These data clearly indicated that
Chem. Res. Toxicol., Vol. 6, No. 6,1993 797
6-HydroxyNNK, a Urinary Metabolite of N N K
5.0
4.0
2.0
3.0
-CHCIs
pyridyl 2.H pyridyl 5-H
0.4
0.2
0.0
1.0
1.6
1.0
1.4 7.2 PPM
6.0
6.6
6.4
Figure 3. Proton NMR spectrum of peak 5 isolated from rat 229
214
hydroxy group. This allowed deprotection by the mild conditions of hydrogenolysis to yield the key intermediate 15. This intermediate could not be obtained by starting with an unprotected hydroxypyridine, and several other approaches involving deprotection of methyl ethers were not successful. Conversion of 15 to 6-hydroxyNNK (5) proceeded in good yield. The MS, NMR, and HPLC retention time of 6-hydroxyNNK confirmed the identity of the metabolite as 5. Further studies were carried out to determine whether 5 existed as the hydroxy tautomer illustrated in Figure 2 or as the corresponding pyridone. This was necessary because 2- and 4-oxygenated pyridines are known to exist predominantly as pyridones rather than hydroxypyridines (18).The UV spectra of 5 and several related compounds were useful for this analysis. The data are summarized in Figure 4 and Table 11. In agreement with literature data, Am, for the oxo tautomers were 227-228 and 29% 301 nm while for 2-methoxypyridine and 3-hydroxypyridine, , ,A were 214-218 and 271-278 nm. Comparison of the UV spectra of 5 and 10 to these values confirmed that 5 exists mainly as the hydroxy tautomer. Urine of F-344 rats and A/J mice treated with 100-105 mg/kg NNK was analyzed for 6-hydroxyNNK and other NNK metabolites before and after treatment with 8-glucuronidase or sulfatase. The results are summarized in Table 111. 6-HydroxyNNK was not detected as a conjugate and was a minor urinary metabolite in both rats and mice a t this dose. 6-HydroxyNNK was also a minor urinary metabolite in the F-344 rat after an NNK dose of 2 mg/kg and in the patas monkey after an NNK dose of 10 pg/kg. It was not detected in primary rat hepatocytes incubated with 1-1000 pM NNK (data not shown).
Discussion
I
190
I
1I
II
I
I
Wavelength (nm)
II
II
1 l
400
Figure 4. UV spectra, determined in MeOH, of 6-hydroxyNNK (-)
and NNK
(- -
- ).
Table I. NMR Data for the Pyridyl Protons of 8 and 9 OH 0
pyridyl proton H-2 H-4 H-5 H-6
chemical shift, multiplicity ( J , Hz) 8.72, d (2.3) 8.25, d (2.4) 8.05, dd (9.5, 2.4) 7.42, d (9.2) 6.61, d (9.6) 8.58, d (9.3)
the unknown was 4- (methylnitrosamino)-1-[3-(6-hydroxy)pyridyll-l-butanone (6-hydroxyNNK, 5). In order to confirm the proposed structure, we undertook the synthesis of 5. Initially, we attempted to prepare 6-methoxyNNK (10) from methyl 6-methoxynicotinate by using essentially the same chemistry that we employ for the synthesis of NNK (14). However, the preparation of 10 was achieved only in poor yield and we were not successful in converting it to 6-hydroxyNNK. Therefore, we developed an alternate synthesis which is summarized in Scheme I. The key aspect of this approach was use of the benzyl ether to protect the pyridyl
In this study, we have characterized a pyridyl-hydroxylated metabolite of NNK, 6-hydroxyNNK. This is the first complete characterization of a pyridyl-hydroxylated metabolite of a tobacco-specific nitrosamine. There do not appear to have been any previous reports of metabolic hydroxylation of the pyridine ring of nicotine or cotinine in mammals, despite extensive studies on these related compounds (19-23). In contrast, pyridine N-oxidation of tobacco-specific nitrosamines and nicotine is a commonly observed mammalian metabolic process (7, 19). Both N-oxidation and pyridine hydroxylation of pyridine have been reported in human and rat tissues (24). As discussed further below, 6-hydroxylation of the pyridine ring has been observed in the bacterial metabolism of nicotine and other pyridine derivatives (25-29). In our studies to date, 6-hydroxyNNK has been detected in the urine of mice, rats, and patas monkeys. It appears to be a minor metabolite, comprising only about 1%of the urinary metabolites of NNK in the mouse or rat, and has not been detected in the form of a glucuronide or sulfate conjugate. Ongoing studies of NNK metabolism a t doses lower than those employed here do not indicate a substantial difference in the level of this metabolite compared to the amounts reported. When this investigation was initiated, one of our goals was to identify nitrosamine-containing metabolites of NNK which would have potential utility in dosimetry studies of smokers’ urine. Some of the major urinary metabolites of NNK such as hydroxy acid 1and keto acid 2 are also formed from nicotine, potentially compromising
798 Chem. Res. Toxicol., Vol. 6, No. 6, 1993
Desai et al.
Scheme I. Synthesis of 6-HydroxyNNK (5)
A N4
t
CI
C6H5CHp0 12
11
12+ 0
0
NaH*
CH3
C&CH,O
13
B-57 N
0
14
Table 11.
CH3
UV Maxima of Selected Oxygenated Pyridine Derivatives Llal-
compound 2-pyridone N-methyl-2-pyridone 2-methoxypyridine 3-hydroxypyridine
(MeOH)
L4x-
compound
(MeOH)
227,298 6-hydroxyNNK (5) 207,232,274 228,301 6-methoxyNNK (10) 280 214,271 NNK (7) 230,229,265 218,278
Table 111. Relative Amounts of NNK Metabolites i n t h e Urine of F-344Rats a n d A / J Mice.
% of urinary metabolites' metabolite*
F-344 rat
At3 mouse
hydroxy acid (1) keto acid (2) NNAL-N-oxide (3) NNAL-Gluc (4) 6-hydroxyNNK (5) NNAL(6) NNK(7)
28 32 4.3 6.1 1.0 19 0.4
20.5 17.4 4.1 18.5 1.0 25.9 ndd
a Levels of all metabolites except NNAL-Gluc were unaffected by treatment with @-glucuronidase. Treatment of urine with @-glucuronidase resulted in disappearance of NNAL-Gluc with a corresponding increase in NNAL. Sulfatase treatment had no effect on any of the metabolites. Numbers refer to structures in Figure 2. F-344 rats were given an sc injection of [5-sHlNNK (100 mg/kg) and A/J mice were given an ip injection of [5-3HlNNK (105 mg/kg). Twenty-four-hour urine was collected and duplicate analyses were carried out as described in the Experimental Section. nd, not detected.
their use as dosimeters of NNK uptake in smokers (19). A nitrosamine-containing metabolite might be useful because of the availability of nitrosamine-selective detectors coupled to GC. NNK itself is barely detectable in rodent or primate urine (6,8,16). Thus, pyridyl hydroxylated metabolites of NNK as well as NNAL and its metabolites appeared to be the most suitable candidates. Initial studies of NNAL-Gluc as a urinary metabolite of NNK were carried out in rats and mice and did not suggest that this metabolite would be useful as a dosimeter in humans because it was not detected a t low doses approaching those experienced by smokers (8). Subsequent studies however, carried out while the present investigation was in progress, demonstrated that NNAL-Gluc diastereomers were major metabolites in patas monkey urine, thus prompting the analysis of human urine for these metabolites (9). The results of those analyses did demonstrate that NNAL-Gluc and NNAL are useful dosimeters for NNK uptake in smokers (6). While the potential utility of 6-hydroxyNNK in this regard requires further investigation, the present results and our ongoing studies in patas monkeys suggest that it may offer no advantages over NNAL and NNAL-Gluc as a dosimeter of NNK uptake in humans. Since 6-hydroxyNNK and NNK-N-oxide are both formed metabolically from NNK, it seemed possible that they may be derived from a common intermediate, possibly
H zEtOH 9 PdlC
___)
HO
& 15
1) 6N HCI, rellux 2) NaN02. pH4 - 5
CH3
an oxaziridine. Pyridine oxaziridines do not appear to have been characterized. Ongoing studies of NNK metabolism in phenobarbital treated rats indicate that 6-hydroxyNNK and NNK-N-oxide are not formed from the same intermediate since production of the latter but not the former is increased by phenobarbital pretreatment. Bacteria isolated from soil, tobacco leaves, or tobacco seeds have been shown to degrade nicotine. For example, Arthrobacter oxydans initially hydroxylates nicotine a t the 6-position of the pyridine ring. This is followed by a series of reactions resulting in oxidation and ring opening of the pyrrolidine ring (25-27). Other tobacco alkaloids such as myosmine and anabasine undergo bacterial pyridine 6-hydroxylation (28). Similarly, nicotinic acid is converted to 6-hydroxynicotinic acid by bacterial metabolism (29). On the basis of these observations, we propose that the formation of 6-hydroxyNNK from NNK is mediated by gut bacteria. Support for this hypothesis can be found in ongoing studies in which 6-hydroxyNNK is apparently not formed in rat hepatocytes or liver microsomes. Further studies are in progress to investigate the role of bacterial metabolism in the production of 6-hydroxyNNK from NNK. Only limited data are available on the tumorigenic activities of N-oxide metabolites of tobacco-specific nitrosamines. NNK-N-oxide was tested for tumorigenicity in A/J mice. I t appeared to be about one-tenth as potent as NNK for induction of lung tumors (30). N'-Nitrosonornicotine 1-N-oxide (NNN-N-oxide)was also less active than N'-nitrosonornicotine (NNN) as a lung tumorigen in A/J mice (30). In F-344 rats, NNN-N-oxide was about as potent as NNN in the induction of nasal cavity tumors, but less active in causing esophageal tumors. In Syrian golden hamsters, NNN-N-oxide was inactive compared to NNN (31). Collectively, these data suggest that 6-hydroxyNNK may be less tumorigenic than NNK. This will be examined initially in A/J mice. In conclusion, 6-hydroxyNNK has been firmly identified as a urinary metabolite of NNK. Further studies are required to determine whether it might have some utility as a dosimeter of NNK uptake or metabolism in smokers, particularly with respect to bacterial transformations of NNK.
Acknowledgment. This study was supported by Grant CA-44377 from the National Cancer Institute. This is paper 147 in "A Study of Chemical Carcinogenesis". We thank Sharon E. Murphy and Maria Nuiies for sharing unpublished data on the metabolism of NNK in phenobarbital-treated rats. Animal experiments were carried out in the American Health Foundation Research Animal Facility and spectroscopy in the Instrument Facility, supported by National Cancer Institute Cancer Center Support Grant CA-17613.
6-HydroxyNNK, a Urinary Metabolite of N N K
References (1) Hoffmann, D., and Hecht, S. S. (1985) Nicotine-derived N-nitrosamines and tobacco related cancer: current status and future directions. Cancer Res. 45, 935-944. (2) Hecht,S. S.,andHoffmann,D. (1988)Tobacco-specificnitrosamines, an important group of carcinogens in tobacco and tobacco smoke. Carcinogenesis 9, 875-884. (3) Hecht, S. S., and Hoffmann, D. (1989) The relevance of tobaccospecific nitrosamines to human cancer. Cancer Suru. 8, 273-294. (4) Carmella, S. G., Kagan, S. S., Kagan, M., Foiles, P. G., Palladino, G.,Q&,A. M.,Quart,E., andHecht,S. S. (1990)Massspectrometric analysis of tobacco-specificnitrosamine hemoglobin adducts in snuff dippers, smokers, and non-smokers. Cancer Res. 50,5438-5445. (5) Foiles, P. G., Akerkar, S. A., Carmella, S. G., Kagan, M.,Stoner, G. D., Resau, J. H., and Hecht, S. S. (1991)Mass spectrometric analysis of tobacco-specific nitrosamine-DNA adducta in smokers and nonsmokers. Chem. Res. Toxicol. 4, 364-368. (6) Carmella, S. G., Akerkar, S., and Hecht, S. S. (1993) Metabolites of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridy1)-1-butanone in smokers’ urine. Cancer Res. 53, 721-724. (7) Hecht, S. S., Castonguay, A,, Rivenson, A,, Mu, B., and Hoffmann, D. (1983) Tobacco-specific nitrosamines: carcinogenicity, metabolism, and possible role in human cancer. J.Enuiron. Sci. Health C1, 1-54. (8) Morse, M.A., Eklind, K. I., Toussaint, M., Amin, S. G., and Chung, F.-L. (1990) Characterization of a glucuronide metabolite of 44methylnitrosamino)-l-(3-pyridyl)-l-butanone(NNK) and ita dosedependent excretion in the urine of mice and rata. Carcinogenesis 11, 1819-1823. (9) Hecht, S. S., Trushin, N., Reid-Quinn, C. A., Burak, E. S., Jones, A. B., Southers, J. L., Gombar, C. T., Carmella, S. G., Anderson, L. M., and Rice, J. M. (1993)Metabolismof the tobacco-specificnitrosamine 4-(methylnitrosamino)-l-(3-pyridyl)-l-butanonein the patas monkey: pharmacokinetics and characterization of glucuronide metabolites. Carcinogenesis 14, 229-236. (10) Peterson,L.A.,andHecht,S.S. (1991)Os-Methylguanineisacritical determinant of 4-(methylnitrosamino)-l-(3-pyridyl)-l-butanone tumorigenesis in A/J mouse lung. Cancer Res. 51, 5557-5564. (11) Peterson, L. A., Liu, X.-K., and Hecht, S. S. (1993) Pyridyloxobutyl DNA adducta inhibit the repair of Os-methylguanine. Cancer Res. 53, 2780-2785. (12) Carmella, S. G., and Hecht, S. S. (1985) High-performance liquid chromatographic analysis of metabolites of the nicotine derived nitrosamines, N’-nitrosonornicotine and 4-(methylnitrosamino)-l(3-pyridyl)-l-butanoneSAnal. Biochem. 145, 239-244. (13) Castonguay, A,, Tjiilve, H., Trushin, N., and Hecht, S. S. (1984) Perinatal metabolism of the tobacco-specific carcinogen 4-(methylnitrosamino)-l-(3-pyridyl)-l-butanone in C57B1 mice. J. Natl. Cancer Znst. 72, 1117-1126. (14) Hecht,S. S.,Lin,D., and Castonguay, A. (1983)Effectaof -deuterium substitution on the mutagenicity of 4-(methylnitrosamino)-1-(3pyridy1)-1-butanone (NNK). Carcinogenesis 4, 305-310. (15) Ross,W. C. J. (1966)Thepreparationofsome4-substitutednicotinic acids and nicotinamides. J. Chem. SOC. C, 1816-1821.
Chem. Res. Toxicol., Vol. 6, No. 6, 1993 799 (16) Hecht, S. S., Young, R., and Chen, C. B. (1980) Metabolism in the F344 rat of 4-(N-methyl-N-nitrosamino)-l-(3-pyridyl)-l-butanone, a tobacco specific carcinogen. Cancer Res. 40,4144-4150. (17) Hecht, S. S., Chen, C. B., Young, R., and Hoffmann, D. (1981) Mass spectra of tobacco alkaloid-derived nitrosamines, their metabolites, and related compounds. Beitr. Tabakforsch. 11, 57-66. (18) Katritzky, A. R. (1963) Advances in Heterocyclic Chemistry, Vol. 1, pp 347-356, Academic Press, New York. (19) Gorrod, J. W., and Jenner, P. (1975) The metabolism of tobacco alkaloids. In Essays in Toxicology (Hayes, W. J., Ed.) Vol 6, pp 35-78, Academic Press, New York. (20) Kyerematen, G. A., Taylor, L. H., deBethizy, J. D., and Vesell, E. S. (1988) Pharmacokinetics of nicotine and 12 metabolites in the rat: application of a new radiometric high performance liquid chromatography assay. Drug Metab. Dispos. 16, 125-129. (21) Kyerematen, G. A., Morgan, M. L., Chattopadhyay, B., deBethizy, J. D., and Vesell, E. S. (1990) Disposition of nicotine and eight metabolites in smokers and nonsmokers: identification in smokers of two metabolites that are longer lived than cotinine. Clin. Pharmacol. Ther. 48,641451. (22) Caldwell, W. S.. Greene. J. M.. Bvrd. G. D.. Chane. K. M.. Uhrie. M. S., deBethizy, J. D., Crooks, P. A:, Bhatti, B. g; and Riggs, M. (1992) Characterization of the glucuronide conjugate of cotinine: a previously unidentified major metabolite of nicotine in smokers’ urine. Chem. Res. Toxicol. 5, 280-285. (23) Nwosu, C. G., and Crooks, P. A. (1988) Species variation and stereoselectivity in the metabolism of nicotine enantiomers. Xenobiotica 18, 1361-1372. (24) Wilke, T. J., Jondorf, W. R.,and Powis, G. (1989) Oxidative metabolism of 1%-pyridine by human and rat tissue subcellular fractions. Xenobiotica 19, 1013-1022. (25) Enzell, C. R., Wahlberg, I., and Aasen, A. J. (1977) Isoprenoids and alkaloids of tobacco. Bog. Chem. Org. Nat. Products 34, 1-79. (26) Hochstein, L. I., and Rittenberg, S. C. (1959) Bacterial oxidation of nicotine. 11. Isolation of the first oxidative product and its identification as (l)-6-hydroxynicotine. J. Biol. Chem. 234, 156-160. (27) Decker, K., Eberwein, H., Gries, F. A., and Bruhmaer, M. (1961) a e r den Abbau des Nikotins durch Bakterienenzyme. VI. L-6Hydroxynicotin ala erstes Zwischenprodukt. Biochem. 2.334,227244. (28) Wada, E. (1957) Microbial degradation of the tobacco alkaloids and some related compounds. Arch. Biochem. Biophys. 72, 145-162. (29) Tsar, L., Pastan, I., and Stadtman, E. R. (1966) Nicotinic acid metabolism 11. The isolation and characterization of intermediates in the fermentation of nicotinic acid. J.Biol. Chem. 241,1807-1813. (30) Castonguay, A., Lin, D., Stoner, G. D., Radok,P., Furuya, K., Hecht, S. S., Schut, H. A. J., and Klaunig, J. E. (1983) Comparative carcinogenicity in A/J mice and metabolism by cultured mouse peripheral lung of N’-nitrosonornicotine, 4-(methylnitrosamino)1-(3-pyridyl)-l-butanone and their analogues. Cancer Res. 43,12231229. (31) Hecht, S. S., Young, R., and Maeura, Y. (1983) Comparative carcinogenicity in F344 rata and Syrian golden hamsters of N’nitrosonornicotine and N’-nitrosonornicotine-1-N-oxide.Cancer Lett. 20, 333-340.
e:
