Chem. Res. Toxicol. 1990, 3, 47-48
47
A Convenient Synthesis of trans -3'-H ydroxycotinine, a Major Nicotine Metabolite Found in the Urine of Tobacco Users Dhimant Desai and Shantu Amin* Division of Chemical Carcinogenesis, American Health Foundation, Valhalla, New York 10595 Received J u n e 19, 1989
trans-3'-Hydroxycotinine is a major urinary metabolite of nicotine in smokers, but no straightforward method is available for its synthesis. A simple method was developed for preparation of trans-3'-hydroxycotinine from cotinine in two steps by using NaN[ (CH3)3Si]2and dibenzyl peroxydicarbonate, followed by base-catalyzed hydrolysis.
Introduction Nicotine (1) is the principal alkaloid component of tobacco and cigarette smoke ( I ) . During smoking, nicotine is absorbed and is excreted in urine partly unchanged and partly in the form of metabolites, principally cotinine (2) and nicotine 1'-N-oxide (3) (2, 3). Cotinine undergoes further metabolism to a variety of hydroxylated and ring-opened products (2, 3). Nicotine and cotinine have been widely used as markers of exposure to tobacco smoke (4). However, recent studies have shown that trans-3'hydroxycotinine (4) is another major metabolite present in the urine of smokers ( 5 , 6 ) . In this paper, we present an improved method for synthesis of this metabolite.
Q42 H
1
\
@
G HO \
CH3
2
CH3
011
Experimental Section NMR spectra were determined in CDC13with TMS as internal standard on a Bruker AM 360 spectrometer. MS were determined with a Hewlett-Packard Model 5988A instrument. Starting materials were obtained from Aldrich Chemical Co., Milwaukee,
WI. Dibenzyl peroxydicarbonate was prepared by a modified procedure as described previously (71,taking precautions appropriate to dealing with organic peroxides. (S)-Cotinine (8) and cis-3'-hydroxycotinine(9)were synthesized by the reported literature procedures. NMR data for cis-3'-hydroxycotinine are as follows: 'H NMR (CDC13)6 1.8-2.0 (m, 1 H, Hc),2.6 (s, 3 H, N-CH,), 2.8-3.0 (m, 1 H, Hb), 4.3-4.5 (m, 2 H, Haand Hd), 7.2-7.4 (m, 1 H, pyridyl H4),7.6-7.7 (m, 1 H, pyridyl Hs), 8.5-8.6 (m, 2 H, pyridyl Hz and H6). 3,3-Bis[ [ (benzyloxy)carbonyl]oxy]-5-(3-pyridyl)-Nmethylpyrrolidinone (5) and 3-[ [ (Benzyloxy)carbonyl]oxy]-5-(3-pyridyl)-N-methylpyrrolidinone(6). A 1.0 M solution of NaN[(CH,),Si], (1.2 mL, 1.2 mmol) was added to a solution of (Sbcotinine (8) (176 mg, 1.0 mmol) in THF (30 mL) at -78 "C (see Scheme I). The solution was stirred for 30 min, and then a solution of dibenzyl peroxydicarbonate (906 mg, 3 mmol) in T H F at -5 "C was added. Stirring at -78 "C was continued for 30 min, and then the reaction mixture was allowed to warm to room temperature. Dilute HCl (5%) was added, and the resulting mixture was extracted with ethyl acetate (3 X 10 mL) and CHzClz(2 X 5 mL). The combined organic layers were dried (Na2S04),concentrated 0893-228~/90/2703-0047$02.50,I O I
,
Scheme I A
5: R,R' = OCOOCHPPh 6: R = H,j; R' = OCOOCHZPh
4
in vacuo, and chromatographed on a silica gel column using CH2C12/MeOH(99:l) as eluent to yield 142 mg (30%) of 5 as an oil: 'H NMR (CDCl,) 6 2.7 (s, 3 H, N-CH,), 2.80-3.0 (m, 2 H, Hb and Hc), 4.6-4.7 (m, 1 H, Ha), 5.2 (s, 2 H, CHZPh), 5.3 (s, 2 H, CH,Ph), 7.1-7.5 (m, 11 H, aromatic and pyridyl H4), 7.6-7.7 (m, 1 H, pyridyl H5) 8.5 (d, 1 H, pyridyl H,), 8.55-8.6 (m, 1 H, pyridyl H6). Further elution with CH2C12/MeOH(98:2) yielded 6 (26 mg, 8%)as an oil: 'H NMR (CDCl,) 6 2.3-2.4 (m, 1 H, Hb), 2.45-2.65 (m, 1 H, HJ, 2.8 (s, 3 H, N-CH,), 4.6-4.7 (m, 1 H, Hd), 5.2 ( 8 , 2 H, CH,Ph), 5.4-5.5 (m, 1 H, Ha), 7.3-7.6 (m, 7 H, aromatic and pyridyl H4and H5)8.5 (d, 1H, pyridyl HJ, 8.6-8.7 (m, 1H, pyridyl H6); MS m / e (relative intensity) 326 (M', 4), 235 (2), 191 (12), 174 (8), 91 (100). Synthesis of 6 by Inverse Addition. A 1.0 M solution of NaN[(CH3),SiI2 (3.6 mL, 3.6 mmol) was added to a solution of (S)-cotinine (528 mg, 3.0 mmol) in THF (60 mL) at -78 "C. The solution was stirred for 30 min and was added dropwise to a solution of dibenzyl peroxydicarbonate (1.8 g, 6.0 mmol) in dry THF (40 mL) at -50 OC. Stirring was continued at -50 "C for 30 min while the solution was warmed to 0 "C. The reaction was quenched with dilute HCl(5%) and extracted with EtOAc (3 X 30 mL). The combined organic layers were dried (MgS04), concentrated in vacuo, and chromatographed on a silica gel column (CH,Cl,/MeOH, 982) to yield 421 mg (43%) of 6 as an oil. The structure was confirmed by NMR (see above). trans-3'-Hydroxycotinine(4). Solid KOH (168mg, 3.0 mmol) was added to a solution of 5 (200 mg, 0.614 mmol) in 10 mL of H,O/THF (1:3). The mixture was stirred at 20 OC for 3 h and extracted with EtOAc (3 x 25 mL) and CHzClz(2 X 10 mL). The organic layers were combined, dried (MgSO,), and concentrated in vacuo. The residue was chromatographed on a silica gel column using CH2C12/MeOH (95:5) as eluent to yield 95 mg (81%) of trans-3'-hydroxycotinine (4), which was recrystallized from acetone-ether: mp 103-106 "C [lit. mp 110-111 "C (9)];'H NMR (CDCl,) b 2.2-2.3 (m, 1H, Hb),2.4-2.6 (m, 1H, Hc), 2.8 (e, 3 H, N-CH,), 4.4-4.6 (m, 2 H, Ha and HJ, 7.3-7.38 (m, 1 H, pyridyl H4),7.4-7.5 (m, 1H, pyridyl H5),8.48 (d, 1H, pyridyl H,) 8.52-8.6 (m, 1 H, pyridyl Hn);MS mle (relative intensity) 192 (M+, 60). 174 (8), id6 (100). -The material was 99.9% pure as judged by HPLC and 'H NMR. 0 1990 American Chemical Society
48
Chem. Res. Toxicol., Vol. 3, No. 1, 1990
Results and Dlscusslon Previously reported synthetic methods for 3’-hydroxycotinine resulted exclusively in the cis isomer (9). This was epimerized to trans-3‘-hydroxycotinine.One synthesis was carried out by condensing diethyl oxalacetate with pyridylidenemethylamine followed by acid-catalyzed hydrolysis to yield exclusively the cis-3’-hydroxy isomer. In another approach, a-3-pyridyl-N-methylnitrone was condensed with methyl acrylate to yield a mixture of isomeric isoxazolidines. After separation, hydrogenolysis of the 5-(alkoxycarbonyl)isoxazolidine yielded cis-3’-hydroxycotinine, which upon epimerization gave the trans isomer. Gore and Vederas (7) used dibenzyl peroxydicarbonate to form a-hydroxycarbonyl compounds via carbonate intermediates. We have used this approach for the synthesis of trans-3’-hydroxycotininefrom (S)-cotinine. We generated the enolate anion of (S)-cotinine with NaN[ (CH,),Sil2 at -78 “C, and a solution of dibenzyl peroxydicarbonate was added to trap the enolate. A major product formed was the disubstituted carbonate derivative 5 while the desired monosubstituted carbonate derivative 6 was a minor product. These two compounds were isolated, and their structures were confirmed by proton NMR. To solve the disubstitution problem, we used inverse addition. The enolate anion of (S)-cotininewas generated by treatment with NaN[(CH,),Si], at -78 “C. This was added to a solution of dibenzyl peroxydicarbonate at -50 “C. This produced only the trans-monosubstituted carbonate derivative 5 in good yield. Hydrolysis of 5 with KOH in H,O/THF (3:l) afforded trans-3’-hydroxycotinine in 81% yield as confirmed by MS and NMR. The NMR spectrum was essentially the same as those previously reported for 4 and for metabolic trans-3’-hydroxycotinine (9). In these spectra, the signal for Hb and H, was observed at 2.9 ppm. The spectrum is distinct from that of cis3‘-hydroxycotinine (see Experimental Section). The absolute configuration is 3R,5S, as in metabolic trans-3‘hydroxycotinine. This was established by comparing the CD spectra of our starting material, (S)-cotinine, with that of (5’)-cotininethat had been subjected to treatment with
Desai and Amin NaN[ (CH3),Si], under the conditions used for reaction with dibenzyl peroxydicarbonate. There was no change in the CD spectrum, indicating that racemization did not occur. The synthesis reported in this paper provides a rapid and simple method for obtaining this important metabolite.
Acknowledgment. This study was supported by Grant CA-44377 from the National Cancer Institute. We thank Dr. Bijay Misra for providing NMR data and Dr. Jyh Min Lin and Dr. Stephen Hecht for valuable discussions throughout the work. Registry No. 1, 54-11-5; 2, 486-56-6; 4, 34834-67-8; 5, 124561-73-5; 6, 124537-25-3; 9, 37096-14-3; (PhCHZOC00)2, 2144-45-8.
References (1) Schmeltz, I., and Hoffmann, D. (1977) Chemical studies on
tobacco smoke. L. Nitrogen containing compounds in tobacco and tobacco smoke. Chem. Rev. 77, 295-311. (2) Gorrod, J. W., and Jenner, P. (1975) The metabolism of tobacco alkaloids. In Essays in Toxicology (Hayes, W. J., Jr., Ed.) Vol. 6, pp 35-78, Academic Press, New York. (3) Benowitz, W. L. (1988) Pharmacokinetics and pharmacodynamics of nicotine. The Pharmacology of Nicotine (Rand, M. J., and Thurau, K., Eds.) pp 3-18, IRL Press, Oxford. (4) International Agency for Research on Cancer (1986) ZARC Monographs on the Eualuation of the Carcinogenic Risk of Chemicals to Humans 38, 127-198. ( 5 ) Neurath, G. B., Duenger, M., Orth, D., and Peen, F. G. (1987) Tran~-3’-hydroxycotinineas a main metabolite in urine of smokers. Znt. Arch. Occup. Enuiron. Health 59, 199-201. (6) Adlkofer, F., Scherer, G., Jarczyk, L., Heller, W. D., and Neurath, G. B. (1988) Pharmacokinetics of 3’-hydroxycotinine. The Pharmacology of Nicotine (Rand, M. J., and Thurau, K., Eds.) pp 25-28, IRL Press, Oxford. (7) Gore, M. P., and Vederas, J. C. (1986) Oxidation of enolates by dibenzyl peroxydicarbonate to carbonates of a-hydroxy carbonyl compounds. J. Org. Chem. 51,3700-3704. (8) Bowman, E. R., and McKennis, H., Jr. (1963) Cotinine. Biochem. Prep. 10, 36-39. (9) Dagne, E., and Castagnoli, N., Jr. (1972) Structure of hydroxycotinine, a nicotine metabolite. J. Med. Chem. 15, 356-360.
