Characterization of the Major DNA Adduct Formed by α-Hydroxy-N

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Characterization of the Major DNA Adduct Formed by r-Hydroxy-N-desmethyltamoxifen in Vitro and in Vivo Gonç alo Gamboa da Costa,† L. Patrice Hamilton,‡ Frederick A. Beland,*,‡ and M. Matilde Marques*,† Centro de Quı´mica Estrutural, Complexo I, Instituto Superior Te´ cnico, Avenida Rovisco Pais, 1049-001 Lisboa, Portugal, and Division of Biochemical Toxicology, National Center for Toxicological Research, Jefferson, Arkansas 72079 Received November 8, 1999

Tamoxifen is hepatocarcinogenic in rats and has been associated with an increased risk of endometrial cancer in women. Recent reports suggest that it may be genotoxic in humans. N-Desmethyltamoxifen is a major tamoxifen metabolite that has been proposed to be responsible for one of the major adducts detected in liver DNA of rats treated with tamoxifen. The metabolic activation of N-desmethyltamoxifen to DNA binding products may involve oxidation to R-hydroxy-N-desmethyltamoxifen followed by esterification. In the study presented here, we report the synthesis of R-hydroxy-N-desmethyltamoxifen and the characterization of the major adduct obtained from R-sulfoxy-N-desmethyltamoxifen in vitro as (E)-R-(deoxyguanosin-N2yl)-N-desmethyltamoxifen. In addition, we use 32P-postlabeling in combination with HPLC to compare the adducts formed in the livers of female Sprague-Dawley rats treated by gavage with tamoxifen or equimolar doses of R-hydroxy-N-desmethyltamoxifen. We conclude that one of the major adducts formed in vivo and previously suggested to derive from N-desmethyltamoxifen is chromatographically identical to R-(deoxyguanosin-N2-yl)-N-desmethyltamoxifen.

Introduction Tamoxifen1 (1, Scheme 1) has been used since the early seventies as an adjuvant for the treatment of all stages of hormone-dependent breast cancer (1, 2). Recent data from a large-scale clinical trial in the United States have indicated that it may also act as a chemoprotective agent (3). Although this effect was not fully confirmed in a British study (4, 5), tamoxifen has been approved by the U.S. Food and Drug Administration for the prevention of breast cancer in healthy women considered at high risk for developing the disease. However, the prophylactic use of this anti-estrogen is controversial because tamoxifen treatment is associated with an increased risk of endometrial cancer (1-3, 6, 7). In addition, the drug is strongly hepatocarcinogenic in rats (8-10). Although hormonal effects may account, in part, for the carcinogenic properties of tamoxifen (11), the genotoxic potential of the drug has been confirmed by its ability to form DNA adducts in rat liver (9, 12-16). * To whom correspondence should be addressed. F.A.B.: HFT-110, NCTR, Jefferson, AR 72079; telephone, (870) 543-7205; fax, (870) 5437136; e-mail, [email protected]. M.M.M.: CQE, Complexo I, IST, Av. Rovisco Pais, 1049-001 Lisboa, Portugal; telephone, 351-21-8419200; fax, 351-21-846-4457; e-mail, [email protected]. † Instituto Superior Te ´ cnico. ‡ National Center for Toxicological Research. 1 Abbreviations: Bis-Tris, bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane; Bu, n-butyl; Cquat, quaternary carbon; dG, 2′-deoxyguanosine; DMSO, dimethyl sulfoxide; dR, 2′-deoxyribosyl; EI, electron impact; ESI, electrospray ionization; Gua, guanine; R-hydroxy-Ndesmethyltamoxifen, (E)-4-[4-[2-(methylamino)ethoxy]phenyl]-3,4diphenylbut-3-en-2-ol; R-hydroxytamoxifen, (E)-4-[4-[2-(dimethylamino)ethoxy]phenyl]-3,4-diphenylbut-3-en-2-ol; 4-hydroxytamoxifen, (Z)-1[4-[2-(dimethylamino)ethoxy]phenyl]-1-(4-hydroxyphenyl)-2-phenylbut1-ene; MS/MS, tandem mass spectrometry; PNK, T4 polynucleotide kinase; tamoxifen, (Z)-1-[4-[2-(dimethylamino)ethoxy]phenyl]-1,2diphenylbut-1-ene.

Tamoxifen has also been shown to induce a high frequency of mutations in liver DNA of λ/lacI transgenic rats (17). Furthermore, it has been associated with mutations in the p53 tumor suppressor gene of liver tumors in rats (18) and endometrial tumors in women (19). Recently, DNA adducts have been reported in endometrial and leucocyte samples from women undergoing tamoxifen therapy (20-22); however, it has been suggested (23) that the endometrial DNA adducts detected in one of these studies (20) could arise from endogenous compounds. In addition, other investigators (24, 25) have failed to detect tamoxifen-DNA adducts in human endometrial samples. A large body of evidence has indicated that one of the metabolic pathways of tamoxifen activation to an electrophilic derivative capable of binding to DNA involves allylic oxidation to R-hydroxytamoxifen (2, Scheme 1, pathway A), followed by esterification, most likely via sulfation (26-32). In vitro reactions with synthetic model esters of R-hydroxytamoxifen have led to the characterization of the major DNA adduct as (E)-R-(deoxyguanosinN2-yl)tamoxifen (3, Scheme 1), which exists as a mixture of epimers at the allylic carbon. Minor adducts from this reaction include the Z diastereomer of 3, and deoxyadenosine adducts, possibly at the exocyclic N6 through the allylic carbon of tamoxifen (33-35). Using the 32Ppostlabeling methodology, adduct 3 has been identified as one of the major adducts in the livers of rats treated with tamoxifen on the basis of co-chromatography with a synthetic standard (33, 36-38). Structural evidence for the formation of 3 in rat liver in vivo has been presented by Rajaniemi et al. (39) using mass spectrometry. Recent work by Shibutani and co-workers (40) has shown that all the diastereomers of R-(deoxyguanosin-N2-yl)tamoxifen are mutagenic and induce primarily G f T transversions.

10.1021/tx990187b CCC: $19.00 © 2000 American Chemical Society Published on Web 02/26/2000

Major Adduct from R-Hydroxy-N-desmethyltamoxifen

Chem. Res. Toxicol., Vol. 13, No. 3, 2000 201

Scheme 1. Metabolic Activation Pathways for Tamoxifena

a

Pathways A and C are the major activation routes in vivo. The adduct arising from pathway B has not been detected in vivo.

In addition to 3, a second major adduct and at least one additional minor adduct, which do not appear to be derived from R-hydroxytamoxifen, are found in the livers of tamoxifen-treated rats (36-38). Sequential oxidation of tamoxifen to 4-hydroxytamoxifen (4, Scheme 1, pathway B) and 4-hydroxytamoxifen quinone methide (5, Scheme 1) or R,4-dihydroxytamoxifen (41) has been proposed as a potential metabolic activation pathway of tamoxifen that may lead to DNA binding products. 4-Hydroxytamoxifen is a major phase I metabolite of tamoxifen in rat and human liver (6) and has been shown to yield the 4-hydroxylated analogue of adduct 3 (6, Scheme 1) upon oxidation and reaction with DNA in vitro (42). Similarly, R,4-dihydroxytamoxifen yields DNA adducts when reacted with DNA at acidic pH (43). However, subsequent studies in vivo (36, 44) have indicated that metabolism via 4-hydroxytamoxifen is not a major activation pathway leading to DNA adducts in the rat. The major phase I metabolite of tamoxifen in humans (45-49) and rats (50, 51) is N-desmethyltamoxifen (7, Scheme 1, pathway C), which can undergo further metabolism at the allylic carbon (48, 52) to give Rhydroxy-N-desmethyltamoxifen (8, Scheme 1). Subsequent conversion to a reactive ester may then lead to the formation of DNA adducts. Rajaniemi et al. (39, 53) presented the first evidence that one of the major tamoxifen-related adducts in the livers of rats treated with tamoxifen is N-demethylated. Comparisons between the MS fragmentation patterns of this adduct and R-(deoxyguanosin-N2-yl)tamoxifen (39) led to the suggestion that the N-demethylated adduct is R-(deoxyguanosin-N2-yl)-N-desmethyltamoxifen (9, Scheme 1). More recent data involving the administration of N-des-

methyltamoxifen have indicated that a metabolic pathway via N-desmethyltamoxifen is a significant route to DNA adducts in rat liver (37, 38), and structure 9 has been assumed to correspond to one of the adducts. However, no synthetic standards of N-demethylated tamoxifen adducts have yet been available to test this hypothesis. In this paper, we report the synthesis of R-hydroxyN-desmethyltamoxifen and the characterization of the major DNA adduct resulting from R-sulfoxy-N-desmethyltamoxifen as (E)-R-(deoxyguanosin-N2-yl)-N-desmethyltamoxifen. In addition, we compare DNA adduct formation in female Sprague-Dawley rats treated by gavage with tamoxifen and R-hydroxy-N-desmethyltamoxifen.

Materials and Methods Caution: Tamoxifen and its derivatives are potentially genotoxic and should be handled with proper care. Exposure to 32P should be kept as low as possible, by working in a confined laboratory area, with protective clothing, plexiglass shielding, Geiger counters, and body dosimeters. Waste materials must be discarded according to appropriate safety procedures. Chemicals. Tamoxifen, salmon testes DNA, bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane (Bis-Tris), and enzymes used in DNA hydrolysis were purchased from Sigma Chemical Co. (St. Louis, MO). Carrier-free [γ-32P]ATP (>7000 Ci/mmol) was purchased from ICN Pharmaceuticals (Costa Mesa, CA). T4 polynucleotide kinase (PNK) was acquired from Amersham U.S. Biochemical (Cleveland, OH). All other commercially available reagents were obtained from Aldrich Chemical Co. (Milwaukee, WI) or Sigma-Aldrich Quı´mica, S.A. (Madrid, Spain), and were used as received. Whenever necessary, solvents were purified by standard procedures (54).

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Scheme 2. Synthetic Pathway to (E)-r-(Deoxyguanosin-N2-yl)-N-desmethyltamoxifen (9)a

a

(a) (1) ClCO2CH(Cl)CH3, ClCH2CH2Cl, reflux; (2) CH3OH, reflux. (b) (1) BuLi; (2) CH3CHO. (c) (1) SO3‚pyridine, pyridine, 37 °C; (2) DNA; (3) enzymatic hydrolysis. Both 10 and 11 were used as mixtures of the E and Z diastereomers.

Instrumentation. The melting temperature of R-hydroxyN-desmethyltamoxifen was measured with an Electrothermal apparatus and is uncorrected. HPLC analyses were conducted with a µBondapak C18 column (0.39 cm × 30 cm; Waters Associates, Milford, MA), using either a Varian system consisting of a Star 9012 ternary gradient pump and a Polychrom 9065 diode array spectrophotometric detector (Varian, Inc., Palo Alto, CA), equipped with a Rheodyne model 7125 injector (Rheodyne, Cotati, CA), or a Waters Associates system consisting of two model 510 pumps and a model 660 automated gradient controller, equipped with a Rheodyne model 7125 injector and a Hewlett-Packard 1050 diode array spectrophotometric detector (Hewlett-Packard, Wilmington, DE). Peaks were monitored at 254 or 280 nm. HPLC analyses of 32P-postlabeled samples were conducted with a 5 µm Delta-Pak C18-100 column (0.39 cm × 15 cm, Waters Associates). 32P levels were monitored with a Radiomatic Flo-One model A-500 on-line radioactivity detector (Packard Instruments, Meriden, CT). UV spectra were recorded with a Beckman DU-40 UV/vis spectrophotometer. 1H NMR spectra were obtained either on a Varian Unity 300 spectrometer, operating at 300 MHz, or on a Bruker AM500 spectrometer, operating at 500 MHz. 13C NMR spectra were recorded on the same instruments, operating at 75.4 or 125.7 MHz, respectively. Chemical shifts are reported in parts per million downfield from tetramethylsilane, and coupling constants are reported in hertz. Mass spectra were recorded on either a Finnigan TSQ-700 GS/MS system, operated in the electron ionization (EI) mode, with the sample being introduced via a direct exposure probe (DEP), or a Finnigan TSQ-7000 LC/MS system, operated in the electrospray ionization (ESI) mode. For ESI spectral measurements, 50% methanol containing 0.1% ammonium formate (pH 3.5) was used at a flow rate of 0.2 mL/min. Elemental analyses were performed at the M-H-W Laboratories (Phoenix, AZ). Syntheses. (1) (E,Z)-1-Bromo-2-[4-(2-methylaminoethoxy)phenyl]-1,2-diphenylethene (11; Scheme 2). (E,Z)-1-Bromo2-[4-[2-(dimethylamino)ethoxy]phenyl]-1,2-diphenylethene (55) (10, 3 g, 7.1 mmol, 1/1 E/Z) was dissolved in 1,2-dichloroethane (40 mL), and the solution was cooled in an ice bath. A solution of 1-chloroethyl chloroformate (2 mL, 18.5 mmol) in 1,2-dichloroethane (10 mL) was added dropwise over a period of 5

Gamboa da Costa et al. min, and the mixture was subsequently allowed to reflux overnight. After the solvent was evaporated, the residue was dissolved in methanol (10 mL) and refluxed for 45 min. The solvent was then evaporated, and the mixture was redissolved in methylene chloride and washed with a saturated sodium bicarbonate solution. The organic layer was dried with anhydrous sodium sulfate, and the product was purified by flash chromatography on silica gel H (type 60, E. Merck, Darmstadt, Germany) by eluting with a stepwise gradient of 0 to 10% methanol in methylene chloride. The N-demethylated product 11 was obtained in 98% yield as a 1/1 mixture of the E and Z isomers, which were recrystallized from ethanolic hydrogen chloride as the corresponding hydrochloride salts. 1H NMR (CDCl3): δ 2.63 (3H, s, CH3NH), 2.70 (3H, s, CH3NH), 3.20 (2H, bs, CH2N), 3.29 (2H, bs, CH2N), 4.16 (2H, bs, CH2O), 4.34 (2H, bs, CH2O), 6.64 (2H, d, J ) 8.4, alkOPhH), 6.85 (2H, d, J ) 8.4, alkOPhH), 6.90-7.36 (24H, m, PhH), 9.73 (4H, bs, 2 × CH3NH2+). MS (EI): m/z 409, 407 (M+), 352, 350 (M+ CH2CH2NHCH3 + 1), 329 (M+ - Br + 1), 272 (M+ - Br - CH2CH2NHCH3 + 2), 252 (M+ - Br - C6H5 + 1), 239 (M+ - Br C7H5), 178 {[(C6H5)2C2]+}, 58 [(CH2CH2NHCH3)+]. Anal. Calcd for C23H22BrNO‚HCl: C, 62.11%; H, 5.21%; N, 3.15%. Found: C, 62.17%; H, 5.35%; N, 3.04%. (2) (E)-4-[4-(2-Methylaminoethoxy)phenyl]-3,4-diphenylbut-3-en-2-ol (r-Hydroxy-N-desmethyltamoxifen, 8). The synthetic procedure was adapted from the method of Foster et al. (55). Briefly, (E,Z)-1-bromo-2-[4-(2-methylaminoethoxy)phenyl]-1,2-diphenylethene (11, 500 mg, 1.22 mmol) was dissolved in 10 mL of freshly distilled dry THF, and the solution was cooled to -110 °C with liquid nitrogen and a 1/1 mixture of methanol and ethanol, and kept under argon. An excess of 1.6 M n-butyllithium (4 mL, 5.2 equiv) was added, while the temperature was kept below -100 °C. Acetaldehyde was then added dropwise, until the dark brown color of the organolithium intermediate disappeared and the mixture became pale yellow. The excess of acetaldehyde was quickly removed by evaporation, and the mixture was added to water (100 mL). Following extraction with methylene chloride, the product was separated from the Z isomer by TLC on silica gel (Merck) by eluting twice with a methylene chloride/methanol mixture (9/1) and recovered in 12% yield by precipitation from diethyl ether. Mp: 189-190.5 °C. UV (methanol): λmax 235 ( ) 18 700 M-1 cm-1), 267 nm ( ) 10 600 M-1 cm-1). 1H NMR (DMSO-d6): δ 0.98 (3H, d, J ) 6.6, CH3CH), 2.24 (3H, s, CH3NH), 2.69 (2H, t, J ) 5.7, CH2N), 3.81 (2H, t, J ) 5.7, CH2O), 4.57 (1H, m, J ) 6.6, J′ ) 3.0, CH3CH), 4.70 (1H, d, J ) 3.0, OH), 6.55 (2H, d, J ) 8.4, alkOPhH), 6.74 (2H, d, J ) 8.4, alkOPhH), 7.10-7.30 (8H, m, PhH), 7.37 (2H, t, J ) 7.2, PhH). 13C NMR (CDCl3): δ 22.43 (CH3CH), 35.82 (CH3N), 50.40 (CH2N), 66.21 (CH3CH), 68.04 (CH2O), 113.35 (ArCH), 126.54 (ArCH), 126.60 (ArCH), 127.67 (ArCH), 127.91 (ArCH), 129.50 (ArCH), 131.07 (ArCH), 131.41 (ArCH), 134.77 (Cquat), 138.38 (Cquat), 140.50 (Cquat), 141.68 (Cquat), 141.94 (Cquat), 156.86 (Cquat). MS (EI): m/z 373 (M+), 355 (M+ - H2O), 272 (M+ - CH2CH2NHCH3 - CH3CHOH + 2), 240 (M+ - CH2CH2NHCH3 - C6H5 + 2), 183 [(C6H5CHC6H4OH)+], 58 [(CH2CH2NHCH3)+], 44 [(CH2NHCH3)+]. Anal. Calcd for C25H27NO2‚H2CO3: C, 71.70%; H, 6.71%; N, 3.22%. Found: C, 71.83%; H, 6.55%; N, 3.40%. (3) (E)-r-(Deoxyguanosin-N2-yl)-N-desmethyltamoxifen (9). R-Hydroxy-N-desmethyltamoxifen (8, 21 mg, 56 µmol) and the SO3‚pyridine complex (41.5 mg, 4.7 equiv) were mixed in 500 µL of dry pyridine. The mixture was stirred for 1 h at 37 °C and then washed twice with 5 mL of diethyl ether. The pellet was resuspended in 2.5 mL of dry ethanol; the insoluble materials were removed by centrifugation, and the supernatant was added in two successive portions to a solution of salmon sperm DNA (2.24 mg/mL) in 20 mL of 5 mM Bis-Tris and 0.1 mM EDTA (pH 7.1). The mixture was vortexed upon each addition and then incubated overnight at 37 °C. Nonbonded materials were removed by extraction with 2 × 1 volume of n-butanol that had been presaturated with 5 mM Bis-Tris and 0.1 mM EDTA (pH 7.1). The DNA was precipitated by addition



Table 1. 1H NMR Data for (E)-r-(Deoxyguanosin-N2-yl)-N-desmethyltamoxifen δ (ppm)a N-desmethyltamoxifen fragment

deoxyribose fragment

guanine fragment

1.26b (3H, d, J ) 7.0, CH3CH) 1.85c (1H, bs, CH3NH) 2.24 (3H, s, CH3NH) 2.68 (2H, t, J ) 5.6, CH2N) 3.80 (2H, t, J ) 5.6, CH2O) 4.89 (1H, m, CH3CH) 6.53d (2H, d, J ) 8.8, alkOPhH) 6.77d (2H, d, J ) 8.8, alkOPhH) 7.03 (2H, d, J ) 6.9, PhH) 7.16 (1H, t, J ) 7.3, PhH) 7.22e (2H, t, J ) 7.3, PhH) 7.32 (1H, t, J ) 7.5, PhH) 7.41 (2H, d, J ) 6.9, PhH) 7.49f (2H, t, J ) 7.6, PhH)

2.31 (1H, m, H2′′) 2.52 (1H, m, H2′) 3.50 (1H, m, H5′/H5′′) 3.54 (1H, m, H5′/H5′′) 3.86 (1H, m, H4′) 4.37 (1H, m, H3′) 4.92c (1H, t, J ) 5.2, 5′-OH) 5.34c (1H, d, J ) 3.3, 3′-OH) 6.18 (1H, t, J ) 6.2, H1′)

5.99c (1H, d, J ) 7.4, N2H) 7.97 (1H, s, H8) 10.08c (1H, bs, N1H)

a Recorded in DMSO-d . b Abbreviations: b, broad; d, doublet; m, multiplet; s, singlet; t, triplet. c Exchangeable with D O. 6 2 from the alkoxyphenyl ring. e Coupled to the signals at 7.03 and 7.16 ppm. f Coupled to the signals at 7.32 and 7.41 ppm.

of 5 M NaCl (0.1 volume) and ethanol (3 volumes), washed with cold 70% ethanol (2 × 20 mL), and redissolved in 20 mL of 5 mM Bis-Tris and 0.1 mM EDTA (pH 7.1). The modified DNA was hydrolyzed to nucleosides by treatment with DNAse I, followed by alkaline phosphatase and phosphodiesterase I (56). The adducts were then partitioned into n-butanol (6 × 10 mL), which had been presaturated with 5 mM Bis-Tris and 0.1 mM EDTA (pH 7.1), and the butanol extracts were combined and back-extracted with 5 mM Bis-Tris and 0.1 mM EDTA (pH 7.1), presaturated with n-butanol. After the butanol was evaporated, the residue was redissolved in methanol (2.5 mL) and purified by HPLC, at a flow rate of 2 mL/min, using a 17 min linear gradient of 0 to 60% acetonitrile in 100 mM ammonium acetate (pH 5.7), followed by a 3 min linear gradient to 100% acetonitrile and a 5 min isocratic elution with acetonitrile. The major adduct, (E)-R-(deoxyguanosin-N2-yl)-N-desmethyltamoxifen (9), eluted at 14.3 min. For the 1H NMR data, see Table 1. MS (ESI): m/z 623 (MH+). MS/MS (m/z 623): m/z 507 (MH2+ - dR), 356 (M+ - dG), 330 (MH2+ - dG - CH3CH), 178 [(Gua + CHCH3)+], 58 [(CH2CH2NHCH3)+]. Treatment of Animals. Rats were treated with tamoxifen and R-hydroxy-N-desmethyltamoxifen according to the protocol of White et al. (13). Specifically, female Sprague-Dawley rats (four per group, 8 weeks old, obtained from the breeding colony at the National Center for Toxicological Research) were treated by gavage with seven daily doses of tamoxifen (20 mg/kg, 54 µmol/kg, dissolved in 200 µL of trioctanoin). Four additional rats were treated in the same manner with equimolar doses of R-hydroxy-N-desmethyltamoxifen (20.1 mg/kg, dissolved in 200 µL of trioctanoin), and four control animals were treated with 200 µL of trioctanoin alone. Twenty-four hours after the last treatment, the animals were killed by exposure to carbon dioxide. Hepatic nuclei were isolated by the method of Basler et al. (57), and DNA was prepared from the nuclei as described by Beland et al. (58). 32P-Postlabeling Analyses. 32P-Postlabeling analyses were conducted by the nuclease P1 enrichment procedure of Reddy and Randerath (59), essentially as described by Beland et al. (36). Briefly, 10 µg of DNA was hydrolyzed with micrococcal endonuclease and spleen phosphodiesterase for 3 h at 37 °C and then treated for 1 h with nuclease P1. After evaporation in a Speed-Vac concentrator, each sample was resuspended in water and labeled with 20 µCi of carrier-free [γ-32P]ATP in the presence of PNK in a total volume of 20 µL. Aliquots of each labeling mixture were analyzed by HPLC. The adducts were separated as follows: from 0 to 10 min, isocratic elution with 58% solvent A [1.2 M ammonium formate and 10 mM ammonium phosphate (pH 4.5)] and 42% solvent B [24% acetonitrile in 1.2 M ammonium formate and 10 mM ammonium phosphate (pH 4.5)]; from 10 to 20 min, a linear gradient to 83% solvent B; from 20 to 40 min, isocratic elution with 83% solvent B; and from 40 to 60 min, a linear gradient to

d

Protons

100% solvent B. The flow rate was 1 mL/min. To avoid interference from the high amounts of radioactivity associated with free 32P, unreacted [γ-32P]ATP, and 32P-postlabeled normal nucleotides, the eluent from the first 15 min was diverted from the detector.

Results and Discussion Synthesis of r-Hydroxy-N-desmethyltamoxifen (8). The bromoolefin (10), which was obtained as a mixture of the E and Z diastereomers (55), was the starting material for the synthesis of R-hydroxy-Ndesmethyltamoxifen. Quantitative N-demethylation of the tertiary amino group to 11 (Scheme 2) was easily achieved by reaction with 1-chloroethyl chloroformate, followed by methanolysis of the intermediate carbamate using the methodology of Olofson et al. (60). Although no attempt was made to separate the E and Z isomers of 11, which were obtained as the corresponding hydrochloride salts, the 1H NMR and mass spectral data (cf. Materials and Methods) confirmed the assigned structure. The synthesis of R-hydroxy-N-desmethyltamoxifen (8) was adapted from the procedure described by Foster et al. for R-hydroxytamoxifen (55). Specifically, the (E,Z)-N-demethylated bromoolefin (11) was treated with approximately 5 equiv of n-butyllithium at -110 °C, followed by reaction of the alkenyllithium intermediate with acetaldehyde (Scheme 2). This methodology had potential difficulties in this particular case, due to the presence of a labile proton in the secondary amino group of 11. We reasoned that using an excess of n-butyllithium and a temperature sufficiently low to minimize secondary reactions, it would be possible to generate 8. In fact, despite competitive deprotonation of the amino group, which was accompanied by loss of N-methylaziridine, presumably through an internal SN2 reaction, R-hydroxyN-desmethyltamoxifen was obtained in 12% yield after separation from the dealkylated products and Z isomer by TLC. Spectroscopic data (1H and 13C NMR and MS; cf. Materials and Methods) of the isolated product were fully consistent with addition of an R-hydroxyethyl fragment to the triarylethylene structure and with the presence of an intact N-methylaminoethoxy substituent. The assignment of the E configuration was based on the 1 H NMR spectrum, where an upfield shift of the AB quartet corresponding to the protons of the alkoxyphenyl ring (δ 6.55 and 6.74), reflected the combined shielding effect of the two phenyl rings (61, 62).

204


Figure 1. HPLC of the enzymatic hydrolysate obtained from reacting R-sulfoxy-N-desmethyltamoxifen with DNA. The elution conditions are outlined in Materials and Methods. The absorbance was monitored at 254 nm. The inset shows the UV absorption spectrum of the major peak (1), as detected by HPLC in 100 mM ammonium acetate (pH 5.7)/acetonitrile.

Synthesis and Isolation of the Major DNA Adduct (9). R-Hydroxy-N-desmethyltamoxifen (8) was further activated by reaction with the SO3‚pyridine complex using the methodology described by Dasaradhi and Shibutani for R-sulfoxytamoxifen (34). Since R-sulfoxyN-desmethyltamoxifen was anticipated to be unstable upon exposure to the atmosphere, it was reacted with DNA immediately after being produced and spectroscopic characterization of the intermediate was not attempted. After enzymatic hydrolysis of the modified DNA to nucleosides, the HPLC profile of the n-butanol extract (Figure 1) indicated the presence of one major (1) and at least two minor (2 and 3) peaks eluting at 12-17 min. These were presumably DNA adducts since they were not detected in control incubations. The major peak (1), with a retention time of 14.3 min, was isolated and characterized by MS and 1H NMR (vide infra). The UV spectrum of the adduct exhibited a maximum at ca. 250 nm and a shoulder at ca. 280 nm (Figure 1, inset), closely resembling those reported for N2-deoxyguanosyl adducts from R-acetoxy-, R-sulfoxy-, and 4-hydroxytamoxifen (3335, 42), which suggested the presence of essentially the same chromophore. The minor peaks (2 and 3) were not isolated, but their UV spectra (not shown) were almost identical to that of peak 1, which suggests that peaks 2 and 3 were diastereomers of the major product. Characterization of the Isolated Adduct (9). (1) Mass Spectrum. ESI mass spectrometry of the isolated product in the positive ion mode yielded an ion at m/z 623 (C35H39N6O5), which corresponded to a protonated adduct of N-desmethyltamoxifen and deoxyguanosine. A product ion analysis of the protonated molecular ion was performed in the ESI MS/MS mode (Figure 2). Prominent fragments were found at m/z 507 (52%), 356 (100%), and 330 (74%), and additional minor ions at m/z 178 (14%) and 58 (3%). The detection of strong ions at m/z 507 (MH+ - 116) and 356 (MH+ - 267) indicated that loss of deoxyribose and deoxyguanosine, respectively, were major fragmentation pathways for the adduct. The presence of the minor ion at m/z 58 [N-methylaziridinium, (CH2CH2NHCH3)+] showed that the N-methylaminoethoxy substituent was intact in the parent molecule. The most informative fragments for structural elucidation were the ions at m/z 330 [(N-desmethyltamoxifen - CH3CH + 1)+] and 178 [(guanine + CH3CH)+], which were

Gamboa da Costa et al.

Figure 2. ESI MS/MS fragmentation pattern of the MH+ ion from (E)-R-(deoxyguanosin-N2-yl)-N-desmethyltamoxifen (9). The assignments are discussed in the text.

Figure 3. Aromatic region of the 1H NMR spectrum of (E)-R(deoxyguanosin-N2-yl)-N-desmethyltamoxifen (9), recorded in DMSO-d6. Full chemical shift assignments are presented in Table 1.

fully consistent with attachment of deoxyguanosine to the allylic carbon of the N-desmethyltamoxifen segment. This fragmentation pattern is virtually identical to that of the N-demethylated adduct detected in rat liver DNA by Rajaniemi et al. (39), which gives further support to the significance of R-hydroxy-N-desmethyltamoxifen as a DNA adduct precursor in vivo. (2) 1H NMR Spectrum. Conclusive evidence for the structure of the isolated adduct was obtained by 1H NMR. The proton chemical shifts for the adduct in DMSO-d6 are summarized in Table 1. Assignments were based on comparison to the spectra of dG and R-hydroxy-Ndesmethyltamoxifen, combined with homonuclear decoupling experiments and chemical exchange of the labile protons with D2O. All the protons of the N-desmethyltamoxifen and dG moieties were detected. Elucidation of the site of attachment of dG to the N-desmethyltamoxifen segment stemmed from the presence of the imino and H8 protons of dG at δ 10.08 and 7.97, respectively, while the exocyclic amino group accounted for only one proton at δ 5.99 (Table 1). This proton was split into a doublet (Figure 3) and coupled to the methine multiplet at δ 4.89, which indicated that substitution occurred at the exocyclic nitrogen of dG through the allylic carbon of N-desmethyltamoxifen. Furthermore, two upfield, mutually coupled, aromatic doublets were present at δ 6.53 and 6.77 and assigned to the alkoxyphenyl ring protons. The position of these resonances


Figure 4. Representative HPLC profiles of liver DNA from female Sprague-Dawley rats treated with (a) tamoxifen or (b) R-hydroxy-N-desmethyltamoxifen and (c) DNA modified in vitro with R-sulfoxy-N-desmethyltamoxifen. The 32P-postlabeling and elution conditions are outlined in Materials and Methods; approximately 10 µg of DNA was postlabeled in each instance.

indicated that the protons were shielded by a combined effect of the two phenyl substituents (61, 62), which confirmed that the N-desmethyltamoxifen segment was in a trans configuration. Therefore, the major adduct from reaction of R-sulfoxy-N-desmethyltamoxifen with DNA was characterized as (E)-R-(deoxyguanosin-N2-yl)N-desmethyltamoxifen (Scheme 2). We could not establish if this adduct was obtained as a single product or a mixture of epimers at the allylic carbon. DNA Adduct Analyses in Rats Treated with Tamoxifen or r-Hydroxy-N-desmethyltamoxifen. With the aim of establishing the significance of Rhydroxy-N-desmethyltamoxifen as a DNA adduct precursor in vivo, female Sprague-Dawley rats were treated by gavage daily for 7 days with tamoxifen, R-hydroxyN-desmethyltamoxifen, or the solvent alone. The hepatic DNA adducts formed in vivo were analyzed by 32Ppostlabeling in combination with HPLC. Only background levels of adducts were detected in the liver DNA of control rats (not shown). Fully consistent with previous reports (33, 36-38), one minor and two major DNA adducts, with retention times of 27.7, 28.8, and 29.9 min (peaks 1-3, respectively, Figure 4a), were found in liver DNA from rats treated with tamoxifen. Peak 3 has been identified as R-(deoxyguanosin-N2-yl)tamoxifen on the basis of coelution with the synthetic standard (33, 36-38). Liver DNA from rats treated with R-hydroxy-N-desmethyltamoxifen had one major and two minor adducts which coeluted, respectively, with peaks 2, 1, and 3 present in liver DNA from tamoxifen-treated rats (Figure 4b). On the basis of the amount of 32P


incorporated into peaks 1-3, tamoxifen and R-hydroxyN-desmethyltamoxifen gave similar levels of hepatic DNA adducts. When assessed by 32P-postlabeling, DNA modified in vitro with R-sulfoxy-N-desmethyltamoxifen had one major (2) and three minor adducts (1, 3, and 4, Figure 4c). The major adduct present in the in vitro modified DNA standard, which was identified as the 3′,5′-bisphosphate of (E)-R-(deoxyguanosin-N2-yl)-N-desmethyltamoxifen (vide supra), had the same retention time as peak 2 detected in liver DNA from tamoxifen- and R-hydroxy-N-desmethyltamoxifen-treated rats. Upon mixed injections (not shown), peak 2 from the in vivo samples co-eluted with the major adduct (2) in the standard. In addition to the co-identity of peak 2, one of the minor adducts detected in the DNA standard had a retention time (27.8 min, Figure 4c) virtually identical to that of peak 1 (panels a and b of Figure 4), while another minor peak (retention time of 29.9 min, Figure 4c) co-eluted with peak 3. These results suggest that peaks 1 and 3 from liver DNA of rats treated with R-hydroxy-N-desmethyltamoxifen might be diastereomers of (E)-R-(deoxyguanosin-N2-yl)-N-desmethyltamoxifen. It should be noted, however, that previous studies (34, 35) have shown the reaction of R-acetoxytamoxifen with DNA in vitro yields diastereomers of (E)-R-(deoxyguanosin-N2-yl)tamoxifen as minor adducts. Since these adducts have retention times similar to those of peaks 1 and 2, it is also possible that a portion of peaks 1 and 2 in the livers of rats treated with tamoxifen may arise from R-hydroxytamoxifen.

Conclusions Recent data from rats treated with N-desmethyltamoxifen have indicated that this metabolite gives rise to one of the major adducts detected in the livers of rats treated with tamoxifen (37, 38). Our results confirm that this major N-demethylated adduct (peak 2, Figure 4) results from hydroxylation of N-desmethyltamoxifen at the allylic carbon to yield R-hydroxy-N-desmethyltamoxifen. The work by the Phillips (37) and Martin (38) groups has also suggested that peak 1 (Figure 4a) originated from N,N-didesmethyltamoxifen. As shown in Figure 4b, the liver DNA of rats treated with R-hydroxy-N-desmethyltamoxifen had a minor adduct peak with the same retention time as peak 1. In addition, one of the minor adducts present in the synthetic standard (Figure 4c) had the same retention time as peak 1. This suggests that the adduct corresponding to peak 1 in vivo may not arise from N,N-didesmethyltamoxifen but rather is a minor adduct of N-desmethyltamoxifen. Additional work is needed to determine the precise metabolic pathway leading to this minor adduct. Our data also indicate that a portion of the adducts previously attributed to R-hydroxytamoxifen (peak 3) may actually be due to R-hydroxy-N-desmethyltamoxifen.

Acknowledgment. We thank J. Pat Freeman and Jackson O. Lay, Jr., for the mass spectra, Richard Beger for some of the NMR spectra, and Cindy Hartwick for helping to prepare the manuscript. This work was supported in part by a Postgraduate Research Program administered by the Oak Ridge Institute for Science and Education, by a research grant from Program PRAXIS XXI, Fundaç aõ para a Cieˆncia e Tecnologia (FCT), Portugal, and by a fellowship to G.G.C. from Subprograma Cieˆncia e Tecnologia, FCT, Portugal.

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