524
Chem. Res. Toxicol. 1991,4 , 524-521
(4) Basu, A. K., and Marnett, L. J. (1983) Unequivocal demonstration that malondialdehyde is a mutagen. Carcinogenesis 3, 331-333. (5) Yau, T. M. (1979) Mutagenicity and cytotoxicity of malondialdehyde in mammalian cells. Mech. Ageing Deo. 11, 137-144. (6) Spalding, J. W. (1988) Toxicology and carcinogenesis studies of malondialdehyde sodium salt (3-hydroxy-2-propena11,sodium salt) in F344/N rats and B6C3F1 mice. Natl. Toxicol. Program Tech. Rep. Ser. No.331, 5-13. (7) Basu, A. K., and Marnett, L. J. (1984) Molecular requirements for the mutagenicity of malondialdehyde and related acroleins. Cancer Res. 44,2848-2854. (8) Seto, H., Okuda, T., Takesue, T., and Ikemura, T. (1983) Reaction of malondialdehyde with nucleic acid. I. Formation of fluorescent pyrimido[ 1,2-a]purin-l0(3H)-onenucleosides. Bull. Chem. SOC. Jpn. 56, 1799-1802. (9) Nair, V., Turner, G. A., and Offerman, R. J. (1984) Novel adducts from the modification of nucleic acid bases by malondi106, 3370-3371. aldehyde. J. Am. Chem. SOC. (10) Marnett, L. J., Basu, A. K., O’Hara, S. M., Weller, P. E., Rahman, A. F. M. M., and Oliver, J. P. (1986) Reaction of malondialdehyde with guanine nucleosides: formation of adducts containing oxadiazabicyclononene residues in the base-pairing region. J. Am. Chem. SOC. 108, 1348-1350. (11) Basu, A. K., O’Hara, S. M., Valladier, P., Stone, K., Mols, O., and Marnett, L. J. (1988) Identification of adducts formed by reaction of guanine nucleosides with malondialdehyde and structurally related aldehydes. Chem. Res. Toxicol. 1, 53-59. (12) Stone, K., Ksebati, M., and Marnett, L. J. (1990) Investigation
of the adducts formed by reaction of malondialdehyde with adenosine. Chem. Res. Toxicol. 3, 33-38. (13) Stone, K., Uzieblo, A., and Marnett, L. J. (1990) Studies of the reaction of malondialdehyde with cytosine nucleosides. Chem. Res. Toxicol. 3, 467-472. (14) Hadley, M., and Draper, H. H. (1990) Isolation of a guaninemalondialdehyde adduct from rat and human urine. Lipids 25, 82-85. (15) Floyd, R. A,, Watson, J. J., Wong, P. K., Altmiller, D. H., and Rickard, R. C. (1986) Hydroxyl free radical adduct of deoxyguanosine: sensitive detection and mechanisms of formation. Free Radical Res. Commun. 1, 163-172. (16) Park, J.-W., Cundy, K. C., and Ames, B. N. (1989) Detection of DNA adducts by high-performance liquid chromatography with electrochemical detection. Carcinogenesis 10, 827-832. (17) Marnett, L. J., Bienkowski, M. J., Raban, M., and Tuttle, M. A. (1979) Studies of the hydrolysis of “C-labeled tetraethoxypropane to malondialdehyde. Anal. Biochem. 99, 458-463. (18) Marinelli, E. R., Johnson, F., Iden, C. R., and Yu, P.-L. (1990) Synthesis of 1,N2-(1,3-propano)-2’-deoxyguanosine and incorporation into oligodeoxynucleotides: a model for exocyclic acrolein-DNA adducts. Chem. Res. Toxicol. 3, 49-58. (19) Ji, C., Ludeke, B. I., Kleihues, P., and Wiessler, M. (1991)DNA methylation in various rat tissues by the esophageal carcinogen N-nitrosomethyl-n-amylamineand six of its positional isomers. Chem. Res. Toxicol. 4, 77-81. (20) Golding, B. T., Patel, N., and Watson, W. P. (1989) Dimer and trimer of malondialdehyde. J. Chem. SOC.,Perkin Trans. I, 668-669.
Synthesis of a Hapten To Be Used in Development of Immunoassays for frans-3‘-Hydroxycotinine, a Major Metabolite of Cotinine Dhimant H. Desai* and Shantu Amin Division of Chemical Carcinogenesis, Naylor Dana Institute for Disease Prevention, American Health Foundation, Valhalla, New York 10595 Received January 30, 1991 4-Carboxyl-substituted analogues of truns-3’-hydroxycotininewere synthesized to be covalently was condensed linked to macromolecules for antibody production. 3-Pyridyl-N-methylnitrone with dimethyl fumarate to give two isomeric isoxazolidines. Hydrogenolysis of the major product [ 2RS- (2a,3a,3P)]-3-carbomethoxy-3-[ [ (benzyloxy)carbonyl]oxy]- 1-methyl-5-oxo-2-(3pyridiny1)pyrrolidine with Pd/C followed by hydrolysis gave [2RS-(2a,3P,4P)]-4-hydroxy-lmethyl-5-oxo-2-(3-pyidinyl)-3-pyrrolidinecarboxylic acid. The same compound was also prepared in two steps in high yield starting with dibenzyl fumarate and 3-pyridyl-N-methylnitrone. Nicotine (l),the major alkaloid present in tobacco, is extensively metabolized, primarily in liver ( 1 ) . Oxidation of the pyrrolidine ring of nicotine produces a major metabolite, cotinine (2), which accounts for the 70% of the nicotine absorbed by smokers (2). However, recent studies have also shown that tran~-3’-hydroxycotinine[ [ 2S(2a,4~)]-4-hydroxy-l-methyl-5-oxo-2-(3-pyridinyl)pyrrolidine] (3) is another major metabolite (3, 4 ) and cis-3’-hydroxycotinineis a minor metabolite (5)in the urine of smokers. (The numbering system used in structures 2 and 4 is based on pyrrolidine; trivial nomenclature for cotinine derivatives employs primes in the pyrrolidine ring and proceeds in the opposite direction, e.g., carbon 4 in 2 = carbon 3’J Radioimmunoassays for 1 and 2 permit rapid and sensitive estimation of these compounds in tissue extracts and physiological fluids (6, 7). However, a radioimmunoassay for 3 does not exist. The development of a radioimmu-
* Author to whom correspondence should be addressed.
OH
OH
1
2
3
4
noassay requires an antibody, which can be raised against a conjugate of 3 with a suitable protein. For cotinine (2), conjugation of the hapten [2RS-(2~~,3P)]-l-methyl-B-oxo2-(3-pyridinyl)-3-pyrrolidinecarboxylicacid (trans-4’carboxycotinine) with keyhole limpet hemocyanin produced a useful antigen (6). In this paper, we describe the synthesis of the analogous derivatives of 3.
Experimental Section NMR spectra were determined in CDCIBor DMSO-d6 on a Bruker AM 360 WB or Jeol FX9OQ spectrometer. Chemical shifts are expressed in ppm downfield from tetramethylsilane. MS were determined with a Hewlett-Packard Model 5988A instrument.
0893-228~/91/2704-0524$02.50/0 0 1991 American Chemical Society
Hapten for trans-3'-Hydroxycotinine Elemental analysis was carried out by Galbraith Laboratories, Knoxville, TN. Most starting materials were obtained from Aldrich Chemical Co., Milwaukee, WI.3-Pyridyl-N-methylnitrone (5) was prepared from pyridine-3-carboxaldehyde and Nmethylhydroxylamine hydrochloride (8). Dimethyl fumarate (6) and dimethyl maleate (7) were obtained from Aldrich Chemical Co., Milwaukee, WI. Dibenzyl fumarate (8) was prepared from fumaric acid and benzyl alcohol (9). [2RS-(2a,3@)]Methyl1methyl-5-oxo-2-(3-pyridinyl)-3-pyrrolidinecarboxylate(trans4'-carbomethoxycotinine) (9) was prepared according to the literature procedure (6). [ 2 R S -(2a,3a,3@)]-3-Carbomethoxy-3-[ ((benzyloxy)carbonyl)oxy]- 1-methyld-oxo-2-( 3-pyridiny1)pyrrolidine (10). A 1.0 M solution of NaN[(CH3)3Si]2(10 mL, 10 mmol) was added to a solution of [2RS-(2a,3@)]methyl l-methyl-5-0xo-2-(3pyridinyl)-3-pyrrolidinecarboxylate(9) (0.95 g, 4.06 "01) in THF' (50 mL) a t -78 "C. The solution was stirred for 30 min and was added dropwise to a solution of dibenzyl peroxydicarbonate (3.07 g, 10.2 mmol) in dry T H F (50 mL) a t -50 "C. Stirring was continued at -50 "C for 30 min, and then gradually the solution was warmed to 0 "C. The reaction was quenched with cold H 2 0 and extracted with EtOAc (3 X 30 mL). The combined organic layers were dried (MgS04),filtered, and concentrated in vacuo. Chromatography on a silica gel column with CHzClz followed by CH2C12/MeOH(91) as eluent yielded 10 (620 mg, 40%): 'H NMR (CDCl,) 6 2.78 (s, 3 H, NCH3), 2.9 (d, 1H, H4), 3.3 (s, 3 H, OCH3), 3.7 (d, 1 H, H4), 4.35 (s, 1 H, H2), 5.2 (8, 2 H, CHZPh), 7.3-7.6 (m, 7 H, aromatic and pyridyl H5 and H4),8.55 (be, 1 H, pyridyl H,), 8.6 (bs, 1 H, pyridyl He); MS m/z (relative intensity) 385 (M 1, 73), 261 (lo), 233 (54), 107 (16), 91 (100).
+
[3RS-(3a,4@,5a)]Dimethyl2-Methyl-3-(3-pyridinyl)-4,5isoxazolidinedicarboxylate (1 1). The nitrone 5 (1.36 g, 10 mmol) and dimethyl fumarate (6) (4.32 g, 30 mmol) were heated in toluene (50 mL) a t 110 "C for 4 h. The reaction mixture was cooled, poured into HzO, and basified to pH 8. The resulting mixture was extracted with ethyl acetate (3 X 75 mL), and the combined organic layers were dried (MgS04),filtered, and concentrated. Chromatography on silica gel with CHzClz followed by CH,Cl,/EtOAc (5050) as eluent yielded 11 (1.31 g, 47%): mp 60-61 "C; TLC (silica gel, EtOAc) R 0.43; 'H NMR (CDC13) 6 2.65 (s, 3 H, NCH3), 3.7 (s, 3 H, 4-06H3), 3.75 (d, 1 H, H3,J3,4 = 8.6 Hz), 3.85 (9, 3 H, 5-OCH3), 3.9 (dd, 1 H, H4), 4.92 (d, 1 H, H5, J4,5= 8.41 Hz), 7.2-7.3 (m, 1 H, pyridyl H5), 7.7-7.8 (m, 1 H, pyridyl H4), 8.6-8.7 (bs, 2 H, pyridyl H2 and Hs); MS m / z (relative intensity) 280 (M', 31), 161 (NO),135 (91), 119 (79). Anal. Calcd for C13H16N20$C, 55.71; H, 5.76; N, 9.99. Found C, 55.74; H, 5.86; N, 9.85. Further elution with CH2C12/EtOAc (5050) yielded 12 (110 mg, 4%) as an oil: TLC (silica gel, EtOAc) Rr 0.31); 'H NMR (CDCl3) 6 2.65 ( ~ , H, 3 NCHS), 3.25 (5, 3 H, 4-OCH3), 3.8 (e, 3 H, 5-OCH3), 3.85-3.95 (m, 1 H, H4), 4.05-4.15 (m, 1 H, H3), 5.15 (d, 1H, H5, J4,5= 6.9 Hz), 7.2-7.3 (m, 1H, pyridyl H5), 7.6-7.7 (m, 1H, pyridyl H4),8.4-8.55 (bs, 2 H, pyridyl H2 and H6). MS m/z (relative intensity): 280 (M', 18.1), 161 (22), 131 (100).
[3RS-(3a,4@,5/3)]Dimethyl 2-Methyl-3-(3-pyridinyl)-4,5isoxazolidinedicarboxylate (13). By use of the above procedure for the cycloaddition, the nitrone 5 (2.0 g, 14.71 mmol) was heated with dimethyl maleate (7) (4.32 g, 30 mmol) for 4 h to give 2.5 g of crude 13 and 14 as a mixture. Chromatography on silica gel with CHzClzfollowed by CH2C12/EtOAc (5050) as eluent gave the major isomer 13 (1.2 g, 43%) as an oil: TLC (silica gel, EtOAc) R, 0.33; 'H NMR (CDCl3) 6 2.7 (9, 3 H, NCHB), 3.62 (s, 3 H, 4-OCH3), 3.7 (t, 1 H, H4), 3.77 (9, 3 H, 5-OCH3), 3.98 (d, 1 H, H3, J3,4= 9.0 Hz), 4.9 (d, 1 H, H5, J4,5= 9.08 Hz), 7.25-7.3 (m, 1 H, pyridyl H5),7.7-7.78 (m, 1H, pyridyl H4),8.5-8.65 (m, 2 H, pyridyl Hz and He); MS m / z (relative intensity) 281 (M + 1, loo), 253 (24), 221 (26), 193 (loo), 177 (43), 137 (50), 119 (71), 97 (88). Further elution with CH,Cl,/EtOAc (5050) yielded the minor isomer 14 (0.14 g, 5%) as an oil: TLC (silica gel, EtOAc) R, 0.17; 'H NMR (CDCl3) 6 2.75 (9, 3 H, NCHB), 3.25 (9, 3 H, 4-OCH3), 3.75 (s, 3 H, EPOCHS), 4.1 (d, 1 H, H3; J3,4 = 7.75), 4.2 (t, 1 H, H4), 4.9 (d, 1 H, H5; J4,5= 9.42), 7.2-7.3 (m, 1 H, pyridyl H5), 7.68-7.8 (m, 1 H, pyridyl H4), 8.4-8.7 (bs, 2 H, pyridyl H, and
Abbreviations: THF, tetrahydrofuran;Pd/C,palladium on activated carbon.
Chem. Res. Toxicol., Vol. 4, No. 5, 1991 525
+
Hs); MS m / z (relative intensity) 281 (M 1,88), 257 (38), 225 (22), 209 (20), 169 (26), 129 (85), 117 (100). [2RS-(3a,4@,5a)]Dibnzyl 2-Methyl-3-(3-pyridinyl)-4,5isoxazolidinedicarboxylate(15). By use of the above procedure for the cycloaddition,the nitrone 5 (2.0 g, 14.71 mmol) was heated with dibenzyl fumarate (8) (10.88 g, 36.77 mmol) for 4 h to give 6.0 g of crude products 15 and 16 as a mixture. Chromatography on silica gel with CHzClzfollowed by CH2C12/EtOAc(5050) as eluent gave 15 (4.2 g, 66%) as an oil: 'H NMR (CDC13) 6 2.57 (9, 3 H, NCHB), 3.5 (t, 1 H, H4), 3.9 (d, 1 H, H3,53,4 = 5.0 Hz), 4.95 (d, 1H, H5, J4,5 = 9.0 Hz), 5.1 (9, 2 H, CHZPh), 5.2 (9, 2 H, CH,Ph), 6.9-7.4 (m, 11H, aromatic and ppidyl H5), 7.45-7.7 (m, 1 H, pyridyl H4), 8.3-8.6 (m, 2 H, pyridyl Hz and H6);MS m / z (relative intensity) 271 [M- (2-COOCH2Ph)], 181 (50), 91 (100). Anal. Calcd for CZ5Hz4N2O5: C, 69.43; H, 5.59; N, 6.48. Found: C, 69.19; H, 5.60; N, 6.49. Further elution with CH2C1,/EtOAc (5050) yielded product 16 (400 mg, 8%)as an oil: 'H N M R (CDCld 6 2.63 (s,3 H, NCHd, 3.8-4.2 (m, 2 H, H3 and H4), 4.64 (dd, 2 H, CH,Ph), 5.0-5.35 (m + s, 3 H, H5 and CH,Ph), 6.8-7.4 (m, 11H, aromatic and pyridyl H5), 7.4-7.7 (m, 1H, pyridyl H4), 8.2-8.6 (bs, 2 H, pyridyl Hz and He). [2 R S - (2a,3@,4@)]Methyl4-Hydroxy- 1-met hyl-5-oxo-2-(3pyridinyl)-3-pyrrolidinecarboxylate (17). Compound 11 (300 mg, 1.07 mmol) and 10% Pd/C (30 mg) in absolute EtOH (50 mL) were stirred under H, (50 psi) for 24 h. The mixture was filtered through Celite, and the filtrate was concentrated in vacuo to afford pure hydroxy ester 17 (210 mg, 78%): mp 112-114 "C; lH NMR (CDC13) 6 2.75 (s, 3 H, NCH3), 3.25 (m, 1 H, H3), 3.75 (s,3 H, OCH3),4.3(bs, 1H,OH),4.7 (d, 1H, H2,J23 = 7.62 Hz), 5.05 (d, 1 H, H4, J3,4= 5.83 Hz), 7.4-7.5 (m, 1 H, pyridyl H5), 7.5-7.6 (m, 1 H, pyridyl H4), 8.55 (s, 1 H, pyridyl H,), 8.6 (m, 1 H, pyridyl H6); MS m / z (relative intensity) 250 (M', 6.5), 232 (28), 173 (100).
[2RS-(2a,3a,4a)]Methyl4-Hydroxy-l-methyl-5-oxo-2-(3pyridinyl)-3-pyrrolidinecarboxylate(18). By use of the above procedure, compound 12 (80 mg, 0.3 mmol) in absolute EtOH (20 mL) was reduced with Hz and Pd/C (10 mg) to give 18 (66 mg, 92%): mp 122-123 "C; 'H NMR (CDC13)6 2.75 ( s , 3 H, N-CH3,, 3.4 (s, 3 H, OCH3), 3.6-3.7 (m, 1 H, H3), 4.6 (d, 1 H, H4,J3,( = 7.0 Hz), 4.8 (d, 1 H, H2,J2,3 = 7.59 Hz), 7.3-7.4 (m, 1 H, pyridyl H5), 7.6-7.8 (m, 1 H, pyridyl H4), 8.55-8.62 (d, 2 H, pyridyl Hz and He); MS m / z (relative intensity) 251 (M', loo), 233 (10).
[2RS-(2a,3@,4a)]Methyl4-Hydroxy-l-methyl-5-oxo-2-(3pyridinyl)-3-pyrrolidinecarboxylate(19). By use of the above procedure, compound 13 (760 mg, 2.7 mmol) in absolute EtOH (60 mL) was reduced with Hz and Pd/C (100 mg) to give 19 (300 mg, 44%): 'H NMR (CDC13) 6 2.62 (s, 3 H, NCH3), 3.08 (t, 1 H, H3), 3.72 (5, 3 H, OCHJ, 4.55 (d, 1 H, H2, J2.3 = 8.18 Hz), 4.6 (d, 1H, H4, J3,4= 7.70 Hz), 7.3-7.4 (m, 1 H, pyridyl H5), 7.6-7.7 (m, 1 H, pyridyl H4), 8.5-8.68 (d, 2 H, pyridyl Hz and Hs). 1-N-Methyl-3-carbomethoxy-2-(3-pyridyl)-2-pyrrolin-5-one (20). Compound 19 (200 mg, 0.8 mmol) was dissolved in dry pyridine (10 mL) a t 0 "C. After stirring for 10 min a t 0 "C, CH3SOzCl (0.19 mL, 2.4 mmol) was added slowly dropwise. Stirring was continued for 18 h a t 5 OC. The mixture was poured into ice-HpO (50 mL) and rapidly extracted with EtOAc (3 X 15 mL). Combined organic layers were dried (MgS04) and concentrated in vacuo. Chromatography on silica gel with EtOAc as eluent yielded 20 (120 mg, 65%): 'H NMR (CDC13) 6 2.9 (8, 3 H,NCH3), 3.5 (s, 2 H, CH,), 3.6 (s, 3 H, OCH3),7.4-7.5 (m, 1 H, pyridyl H5), 7.7-7.8 (m, 1 H, pyridyl H4), 8.6-8.7 (m, 1 H, pyridyl H2), 8.72-8.8 (m, 1 H, pyridyl H6); MS m / z (relative intensity) 232 (M', 88), 201 (25), 173 (loo), 145 (46), 119 (43). [2RS-(2a,3@,4a)]Methyl l-Methyl-4-(mesityloxy)-5-oxo2-(3-pyridinyl)-3-pyrrolidinecarboxylate (21). Compound 19 (510 mg, 2.04 mmol) was dissolved in CH2ClZ(20 mL) and cooled to -20 "C. After stirring a t -20 "C for 10 min, CH3SO2C1(0.19 mL, 2.48 mmol) was added slowly dropwise, followed by triethylamine (0.3 mL, 2.04 mmol). Stirring was continued for 3 h a t -20 "C. The reaction mixture was poured into ice-HzO (50 mL) and rapidly extracted with CH2Cl, (3 X 15 mL). The combined organic layers were dried (MgS04) and concentrated in vacuo. Chromatography on silica gel column with EtOAc as eluent yielded 21 (455 mg, 68%): 'H NMFt 6 2.7 (s, 3 H, NCH,), 2.8-2.95 (m, 1 H, H3), 3.28 ( s , 3 H, S0,CH3), 3.75 (8,3 H, OCH,), 4.7 (d,
Desai and Amin
526 Chem. Res. Tonicol., Vol. 4,No.5, 1991
Scheme I
(11) R 4 H 3 (15) R=CHzPh
(12) R=CH3 (16) R=CHZPh
\ 1 H,H2), 4.48 (e, 1 H,H4), 7-7.8 (m, 2 H,pyridyl H4 and H5), 8.0-8.9 (bs, 2 H,pyridyl H2 and He). [ 2RS - (2a,3&4B)]-4-Hydroxy-l-methyl-5-oxo-2-(3-
pyridinyl)-3-pyrrolidinecarboxylicAcid (4) from Isoxazolidine (17). Compound 17 (150 mg, 0.6 mmol) was stirred with 5% NaOH in 20 mL of H20/THF (1:3)at room temperature for 4 h. The solvent was evaporated to dryness to yield the crude product as a salt. It was purified on a column of ion-exchange resin (DOWEX50, cation exchange, H+ form) to yield 4 (57 mg, 40%): mp 188-189 OC; ‘H NMR (DMSO) 6 2.5 (s,3 H, NCH3), 3.68 (dd, 1 H,H3), 3.78 (d, 1 H,H2, J2,3 = 8.2 Hz), 4.7 (d, 1 H, H4, J3,4= 4.2 Hz), 7.4-7.5 (m, 1 H,pyridyl H5), 7.7-7.8 (m, 1 H, pyridyl H4),8.5-8.6 (m, 2 H,pyridyl Hzand H&, 12.93 (bs, 1 H, COOH); MS m / z (relative intensity) 237 (M + 1, 5.1), 219 (9), 191 (49), 147 (50). Anal. Calcd for CllH12N204:C, 55.93; H,5.12; N, 11.86. Found: C, 55.71; H, 5.31; N, 11.97. [ 2RS - ( 2a,3&4fl)]- 4- Hydroxy- 1-methyl-5-oxo-2-( 3 pyridinyl)-3-pyrrolidinecarboxylicAcid (4) from Isoxazolidine (15). By use of the above procedure, compound 15 (600 mg, 1.39 mmol) in absolute EtOH (50 mL) was treated with H2 and Pd/C (60 mg). The mixture was filtered through Celite and washed several times with a THF/MeOH (50:50) mixture, and the filtrate was concentrated in vacuo to afford pure 4 (267 mg, 81%);mp and NMR were identical with those of the compound that was obtained upon hydrolysis of 17.
Results and Dlscusslon We have reported the synthesis of trans-3’-hydroxycotinine (3) from cotinine by using enolate chemistry to form the a-hydroxycarbonyl compound (10).The enolate generated was trapped with dibenzyl peroxydicarbonate (11). In our initial attempt to synthesize 4, we used a similar approach starting with [2RS-(2a,3O)]methyl 1methyl-5-oxo-2- (3-pyridinyl)-3-pyrrolidinecarboxylate (trans-4’-carbomethoxycotinine)(9). The product 10, when analyzed by proton NMR,was shown to have resulted from carbonate addition at the 3-position (the carbon bearing the carbomethoxy group) instead of the 4-position. The protons at position 4 of the pyrrolidinone ring appeared as doublets at 2.9 and 3.7 ppm, and the proton at position 2 appeared as a singlet at 4.35 ppm.
9
10
Dagne and Castagnoli have reported the synthesis of 3’-hydroxycotinine, starting with 3-pyridyl-N-methylnitrone and methyl acrylate (8). This approach to the synthesis of 4 was then used (see Scheme I). We reacted
Pd/c
(17) R=CH3 (4) R=H
(18) R=CH3
/
3-pyridyl-N-methylnitrone (5) with dimethyl fumarate (6). Two isomeric isoxazolidine products were obtained after purification by silica gel chromatography. This condensation was expected to be stereoselective (e.g., retention of the trans relationship of the carbomethoxy groups), since Huisgen had reported that reaction of a nitrone with dimethyl fumarate gave exclusively the trans isoxazolidine isomer (12). Proton NMR analysis of the major product was consistent with structure 11 as the trans isomer. The signals for protons H3 and H5 of the isoxazolidine ring appear as two doublets at 3.75 (J3,4 = 8.6 Hz) and 4.92 (J4,5 = 8.41 Hz), respectively. The signal for the H4 proton appeared at 3.9 ppm. I t has been reported that, for a related fivemembered lactam system, the coupling constant for protons which are trans to each other is about 5 H and for protons which are cis to each other, about 8 Hz (13).As discussed below, coupling constants in this system are not necessarily indicative of a cis or trans configuration (13). We have observed a coupling constant of 8.41 Hz for protons 4 and 5 and 8.6 Hz for protons 3 and 4. These coupling constants are higher than would have been expected for the trans configuration of H3,4and H4,5. However, as discussed below, the corresponding carbomethoxy group displayed the anticipated difference in the shifts of the methoxycarbonyl proton signals (14). Dagne and Castagnoli have reported that, when a C02CH3group at position 4 of the isoxazolidine ring is trans to the aromatic ring in position 3, then the signal for OCH3 appears at about 3.7 ppm (8). When the C02CH3 group is cis with respect to the aromatic ring, the OCH3 signal is shifted upfield to approximately 3.2 ppm due to the shielding effect of the aromatic ring. We have observed very similar results. In the major product 11, the signal for C02CH3at position 4 occurs at 3.7 ppm, which indicates that the pyridine ring and C02CH3group are trans to each other. The signal for the methoxy group at position 5 occurs as a singlet at 3.85 ppm. NMR analysis of the minor product confirmed structure 12. The signals for the protons at positions 3 and 5 of the isoxazolidine ring appear as two doublets at 4.05-4.15 and 5.15 ppm, respectively. The signal for the proton at position 4 was a multiplet at 3.85-3.95 ppm. The coupling constant of 6.9 Hz between protons 4 and 5 is higher than would have been expected for trans configuration. However, the signal for the C02CH3groups at positions 4 and 5 appeared at 3.25 ppm and at 3.8 ppm, respectively. The upfield shift of the signal for the C02CH3at position 4 indicates that it is cis to the pyridine ring.
Hapten for trans-3’-Hydroxycotinine
Chem. Res. Toxicol., Vol. 4, No. 5, 1991 527
Scheme IIa
OCOCH,
‘a (a) CHaS02Cl/pyridine; (b) CHaSO,Cl/TEA, -20 OC; (c) AcOH/NaOAc.
These data were consistent with the assigned stereochemistry of isoxazolidine 11 and 12. Nevertheless, we wished to confirm the stereochemistry of the reaction and the configurations of the products. Therefore, nitrone 5 was reacted with dimethyl maleate (7). Two isomeric isoxazolidines, one major and one minor isomer, were obtained after purification on a column of silica gel. All four isomeric isoxazolidines obtained from the dimethyl fumarate and dimethyl maleate reactions have different Rr values on silica gel TLC. The chemical shift data for the major product 13 were consistent with the isoxazolidine ring structure. The was 9 Hz, which is larger than excoupling constant J3,4 pected for the trans configuration of these protons. The coupling constant J4,5 was 9.08 Hz, which is expected for the cis configuration of these protons (13). However, the resonance due to the OCH3protons in position 4 appeared at 3.62 ppm, indicating that the pyridine ring and C02CH, group are trans. The signal for the C02CH3group in position 5 appeared at 3.77 ppm. Proton NMR analysis of the minor product from reaction of dimethyl maleate with 5 was consistent with 14. The coupling constants J3,4and J4$were 7.75 and 9.42 Hz, respectively. These coupling constants are expected for the cis configuration of H3,4and H4 The signal for the COzCH3group at position 4 appeared at 3.25 ppm, and the signal for C02CH3at position 5 appeared at 3.75 ppm. The upfield shift of the signal for the C02CH3 at position 4 indicates that the pyridine ring and C02CH3group are cis to each other. Thus, all four isomeric isoxazolidines obtained from the dimethyl fumarate and dimethyl maleate reactions were different as confirmed by NMR and TLC analysis. Hydrogenolysis of 11 was carried out with Pd/C to yield hydroxy ester 17. Since hydrogenolysis of 11 should proceed with retention of configuration at C-3 and C-5, the stereochemistry of the resulting pyrrolidinone 17 should follow directly from the stereochemistry of 11 (8). NMR analysis of 17 was consistent with this, as was NMR analysis of 18 produced by hydrogenolysis of 12. Similarly, compound 13 on hydrogenolysis with Pd/C gave hydroxy ester 19. Proton NMR analysis was consistent with structure 19. We were interested in epimerization of 19 to 17. However, this was not successful (see Scheme 11). Epimerization at C-4 of the pyrrolidinone was attempted via the mesylate derivative (8). When pyridine and CH3S02C1 were used for the formation of the mesylate, no substitution product 21 was isolated. Instead, proton NMR analysis confirmed an elimination product 20. Protons at position 4 appeared as a singlet at 3.5 ppm. The mesylate derivative 21 was prepared by using triethylamine, CH3S02C1,and CH2C12at -20 OC. Proton NMR confirmed the
structure 21. Mesylate 21 was treated with CH,COOH/ NaOAc under a variety of reaction conditions to give the epimerized derivative 22. Instead, in all the cases we have isolated the same elimination product 20, as confirmed by proton NMR. The required hapten, 4, was produced by hydrolysis of 17. However, the yield of the hydroxy acid 4 was low. It was isolated as the sodium salt. Pure 4 was obtained after purification on an ion-exchange resin. Therefore, we developed an improved synthesis of 4. As illustrated in Scheme I, we reacted dibenzyl fumarate (8) with the nitrone 5. This gave the expected mixture of two isomeric isoxazolidine products. They were purified on a column of silica gel to yield the major product, trans isomer 15, and the minor product, cis isomer 16. Hydrogenolysis of the trans isomer 15 with Pd/C yielded pure 4 in an overall yield of 54%. The proton NMR and melting point of 4 were identical in all respeds with those for the acid obtained from hydrolysis of the methyl ester 17.
Acknowledgment. This study was supported by NCI Grant CA-44377. We thank Dr. Stephen Hecht and Dr. Jyh Min Lin for valuable discussions throughout the work, Dr. Bijay Misra for providing NMR data, and Ms. Beth Appel and Ms. Christina Lurentzatos for their editorial assistance. Registry No. 2, 486-56-6;3, 34834-67-8;4, 134938-53-7;5, 37096-15-4;6,624-49-7; 7,624-48-6; 8,538-64-7; 9,135028-97-6; 10,134938-54-8;11,134938-55-9; 12,134938-56-0;13,134938-57-1; 14,134938-58-2;15,134938-59-3; 16,134938-60-6;17,134938-61-7; 18,134938-62-8;19,134938-63-9;20,134938-64-0;21,134938-651.
References (1) Turner, D. M., Armitage, A. K., Briant, R. H., and Dolley, C. T. (1975)Metabolism of nicotine by the isolated perfused dog lung. Xenobiotica 5 (9),539-551. (2) Jacob, P., 111, Benowitz, N., and Shulgin, A. T. (1988)Recent, studies of nicotine metabolism in humans. Pharmacal. Biochem. Behav. 30,249-253. (3) Benowitz, W.L. (1988)Pharmacokinetics and Pharmacodynamics of Nicotine. In The Pharmacology of Nicotine (Rand, M. J., and Thurau, K., Eds.) pp 3-18,IRL Press, Oxford. (4) 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. Environ. Health 59, 199-201. (5) Voncken, P., Rustemeier, K., and Schepers, G. (1990)Identification of cis-3‘-hydroxycotinine as a urinary nicotine metabolite. Xenobiotica 20, 1353-1356. (6) Langone, J. J., and Van Vunakis, H. (1982)Radioimmunoassay of nicotine, cotinine, and y-(3-pyridyl)-y-oxo-N-methylbutyramide. Methods Enzymol. 84, 628-640. (7) Langone, J. J., Gjika, H. B., and Van Vunakis, H. (1973)Nicotine and its metabolites: radioimmunoassays for nicotine and cotinine. Biochemistry 12,5025-5030. (8) Dagne, E.,and Castagnoli, N., Jr. (1972)Structure of hydroxycotinine, a nicotine metabolite. J. Med. Chem. 15,356-360. (9) Bischoff, C. A.,and Heddenstrom, A. V. (1902)Uber Phenylund Benzyl-Ester der Glutar-, Fumar-, Malein- und Phtal-Saure. Ber. 35,4084-4094. (10) 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. (11) Desai, D., and Amin, S. (1990) A convenient synthesis of trans-3’-hydroxycotinine, a major nicotine metabolite found in the urine of tobacco users. Chem. Res. Toxicol. 3, 47-48. (12) Huisgen, R. (1963)Kinetics and mechanism of 1,3-dipolar cycloadditions. Angew. Chem., Znt. Ed. Engl. 2, 633-796. (13) Cushman, M., and Castagnoli, N., Jr. (1971)The condensation of succinic anhydrides with Schiff bases. Scope and mechanisms. J. Org. Chem. 36, 3404-3406. (14) Castagnoli, N., Jr. (1969)Condensation of succinic anhydride with N-benzylidene-N-methylamine, stereoselective synthesis of trans- and cis-l-methyl-4-carboxy-5-phenyl-2-pyrrolidinone. J. Org. Chem. 34, 3187-3189.
