Synthesis and Investigation of α-Hydroxy-N, N-didesmethyltamoxifen

cancer, tamoxifen is known to increase the risk of endometrial cancer and ... events in women; in addition, it induces liver tumors in rats and endome...
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Chem. Res. Toxicol. 2003, 16, 1090-1098

Synthesis and Investigation of r-Hydroxy-N,N-didesmethyltamoxifen as a Proximate Carcinogen in the Metabolic Activation of Tamoxifen Gonc¸ alo Gamboa da Costa,† M. Matilde Marques,*,† James P. Freeman,‡ and Frederick A. Beland*,§ Centro de Quı´mica Estrutural, Complexo I, Instituto Superior Te´ cnico, Av. Rovisco Pais, 1049-001 Lisboa, Portugal, Division of Chemistry, National Center for Toxicological Research, Jefferson, Arkansas 72079, and Division of Biochemical Toxicology, National Center for Toxicological Research, Jefferson, Arkansas 72079 Received March 3, 2003

Tamoxifen is an adjuvant chemotherapeutic agent for the treatment of breast cancer and a chemoprotective agent for breast cancer prevention. Despite being beneficial in regard to breast cancer, tamoxifen is known to increase the risk of endometrial cancer and thromboembolic events in women; in addition, it induces liver tumors in rats and endometrial tumors in rats and mice. Tamoxifen and its metabolite, N-desmethyltamoxifen, are metabolically activated to DNA binding electrophiles through R-hydroxylation, followed by O-esterification, primarily via sulfation. In the present study, we have investigated whether a second desmethylated metabolite of tamoxifen, N,N-didesmethyltamoxifen, is also involved in the metabolic activation of this antiestrogen to a genotoxic species. R-Hydroxy-N,N-didesmethyltamoxifen was synthesized, further activated by sulfation, and then reacted with DNA. After enzymatic hydrolysis to deoxynucleosides, HPLC analysis indicated the formation of one major DNA adduct, which was characterized as (E)-R-(deoxyguanosin-N2-yl)-N,N-didesmethyltamoxifen. Using 32Ppostlabeling, in combination with HPLC, the same adduct was detected in liver DNA from rats treated intraperitoneally with R-hydroxy-N,N-didesmethyltamoxifen. In contrast, only a low extent of adduct formation could be found in rats administered N,N-didesmethyltamoxifen. These data indicate that although R-hydroxy-N,N-didesmethyltamoxifen can be converted to a genotoxin in rat liver, this pathway is a minor one in the metabolic activation of tamoxifen.

Introduction Tamoxifen1 (1; Scheme 1) is an important antiestrogen for the treatment (1) and, more recently, the prevention (2-5) of breast cancer. A major concern regarding the use of tamoxifen, especially as a chemoprotective agent, is an increased incidence of endometrial cancer and thromboembolic events (1-5). Tamoxifen is also a rodent carcinogen, inducing liver tumors in rats treated as adults (6-8) and uterine tumors in rats treated neonatally or as adults (9-11). In mice, reproductive tract tumors result following transplacental or neonatal exposure to tamoxifen (12-14). * To whom correspondence should be addressed. (M.M.M.) Tel: 35121-841-9200. Fax: 351-21-846-4457. E-mail: [email protected]. (F.A.B.)Tel: (870)543-7205.Fax: (870)543-7136.E-mail: [email protected]. † Instituto Superior Te ´ cnico. ‡ Division of Chemistry, National Center for Toxicological Research. § Division of Biochemical Toxicology, National Center for Toxicological Research. 1 Abbreviations: alkO, alkoxy; Ar, aryl; Bis-Tris, bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane; t-Bu, tert-butyl; Cquat, quaternary carbon; N-desmethyltamoxifen, (Z)-1-[4-(2-methylaminoethoxy)phenyl]1,2-diphenylbut-1-ene; N,N-didesmethyltamoxifen, (Z)-1-[4-(2-aminoethoxy)phenyl]-1,2-diphenylbut-1-ene; dG, 2′-deoxyguanosine; dR, 2′deoxyribosyl; EI, electron impact; Et, ethyl; ES, electrospray ionization; FIA, flow injection analysis; R-hydroxy-N-desmethyltamoxifen, (E)-4[4-(2-methylaminoethoxy)phenyl]-3,4-diphenylbut-3-en-2-ol; R-hydroxyN,N-didesmethyltamoxifen, (E)-4-[4-(2-aminoethoxy)phenyl]-3,4-diphenylbut-3-en-2-ol; R-hydroxytamoxifen, (E)-4-[4-(2-dimethylaminoethoxy)phenyl]-3,4-diphenylbut-3-en-2-ol; MeO, methoxy; MeOtrityl, (4-methoxyphenyl)-diphenylmethyl; MS/MS, tandem mass spectrometry; Ph, phenyl; PNK, T4 polynucleotide kinase; tamoxifen, (Z)-1-[4-(2-dimethylaminoethoxy)phenyl]-1,2-diphenylbut-1-ene.

In rat liver, tamoxifen is thought to induce tumors by a genotoxic mechanism (reviewed in 15), through a pathway involving an initial hydroxylation of the allylic (R) carbon of both tamoxifen and N-desmethyltamoxifen (5; Scheme 1) followed by esterification of the hydroxy function, primarily via sulfation (16-26; Scheme 1). The major DNA adduct resulting from tamoxifen has been identified as (E)-R-(deoxyguanosin-N2-yl)tamoxifen [R-(dGN2-yl)tamoxifen, 4] (16), while the major adduct resulting from N-desmethyltamoxifen has been characterized as the structural analogue of 4, (E)-R-(deoxyguanosin-N2yl)-N-desmethyltamoxifen [R-(dG-N2-yl)-N-desmethyltamoxifen, 8] (21). An additional minor adduct detected in the livers of rats administered tamoxifen (19-22) has yet to be identified. Further metabolism of N-desmethyltamoxifen through N-demethylation gives rise to N,N-didesmethyltamoxifen (9; Scheme 1). This metabolite has been detected in incubations of tamoxifen with Hep G2 cells and human liver homogenates (27) and has also been found in the plasma and various tissues, particularly liver and lung, of rats (28) and patients treated with tamoxifen (27, 28). Incubations of N,N-didesmethyltamoxifen with primary cultures of rat hepatocytes have indicated that this metabolite can be activated to DNA binding derivatives, although to a lower extent than tamoxifen, with adduct levels estimated at 12% of those obtained with tamoxifen (19). When administered to female Sprague-Dawley rats by gavage, N,N-didesmethyltamoxifen gave very low

10.1021/tx030010o CCC: $25.00 © 2003 American Chemical Society Published on Web 08/09/2003

R-Hydroxy-N,N-didesmethyltamoxifen

Chem. Res. Toxicol., Vol. 16, No. 9, 2003 1091

Scheme 1. Metabolic Activation Pathways for Tamoxifen through r-Hydroxylationa

a The major adducts resulting from R-hydroxytamoxifen and R-hydroxy-N-desmethyltamoxifen are shown. Formation of the adduct derived from N,N-didesmethyltamoxifen has not been demonstrated in vivo. Metabolites 3, 7, and 11 are acetate (RdCOCH3) or sulfate (RdSO3-) esters.

levels of hepatic DNA binding, at approximately 1% of those detected from tamoxifen administration (22); however, substantially higher levels of adduct formation (ca. 20% of the levels obtained from tamoxifen dosing) were found in the livers of female F344 rats after treatment by intraperitoneal injection (20). Despite its lower DNA binding potential, it is apparent that N,N-didesmethyltamoxifen can form DNA adducts in rat liver. By analogy to tamoxifen and N-desmethyltamoxifen, N,N-didesmethyltamoxifen could conceivably be activated through R-hydroxylation to R-hydroxy-N,N-didesmethyltamoxifen (10; Scheme 1) and esterification, yielding an electrophilic derivative as the potential precursor of the minor adduct detected in the livers of tamoxifen-treated rats. In this work, we describe the synthesis of R-hydroxyN,N-didesmethyltamoxifen and the preparation of an R-(deoxyguanosin-N2-yl)-N,N-didesmethyltamoxifen DNA adduct standard [R-(dG-N2-yl)-N,N-didesmethyltamoxifen, 12; Scheme 1]. We have also used 32P-postlabeling analyses to compare the synthetic didesmethylated adduct standard with the DNA adduct profiles obtained in the livers of female Sprague-Dawley rats treated by intraperitoneal injection with tamoxifen, N,N-didesmethyltamoxifen, and R-hydroxy-N,N-didesmethyltamoxifen.

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 testis DNA, Bis-Tris, trioctanoin, and the enzymes used in DNA hydrolysis were purchased from Sigma Chemical Co. (St. Louis, MO). Carrierfree [γ-32P]ATP was purchased from ICN Pharmaceuticals (Costa Mesa, CA). PNK was acquired from Amersham U.S. Biochemical (Cleveland, OH). All other commercially available reagents were purchased from Aldrich Chemical Co. (Milwaukee, WI) or Sigma-Aldrich Quı´mica, S. A. (Madrid, Spain) and were used as received. N,N-Didesmethyltamoxifen hydrochloride was synthesized by the procedure described in Gamboa da Costa et al. (22), and (E,Z)-1-bromo-2-[4-(2-aminoethoxy)phenyl]-1,2-diphenylethene (13; Scheme 2) was prepared as detailed in Gamboa da Costa et al. (29). Whenever necessary, solvents were purified by standard procedures (30). Instrumentation. Melting temperatures were measured with a Leica Galen III hot stage apparatus and are uncorrected. HPLC analyses were conducted and DNA adducts were purified 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,

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Gamboa da Costa et al.

Scheme 2. Synthetic Pathway to r-Hydroxy-N,N-didesmethyltamoxifena

a Reagents and conditions: (a) 4-MeOtrityl chloride, Et N, CH Cl , room temperature. (b) (1) t-BuLi/THF, -100 °C; (2) CH CHO. (c) 3 2 2 3 (1) 25% Acetic acid in CH2Cl2, room temperature; (2) ammonium hydroxide; (3) TLC separation from the Z isomer.

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 Co., Palo Alto, CA). The UV absorbance was 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) using a Waters Associates system, as described above, equipped with a Radiomatic Flo-One model A-500 online radioactivity detector (Packard Instruments, Meriden, CT). UV spectra were recorded with either a Beckman DU-40 UV/ vis (Beckman Coulter, Fullerton, CA) or a Shimadzu 1202 UV/ vis (Shimadzu Europe, Duisburg, Germany) spectrophotometer. 1H NMR spectra were recorded on a Varian Unity 300 spectrometer (Varian Deutschland Gmbh, Darmstadt, Germany), operating at 300 MHz. The spectrum of the synthetic adduct, (E)-R-(deoxyguanosin-N2-yl)-N,N-didesmethyltamoxifen (12) was obtained using a 5 mm indirect detection probe, for increased sensitivity. 13C NMR spectra were recorded on the Varian Unity 300 instrument, operating at 75.4 MHz. Chemical shifts are reported in ppm downfield from tetramethylsilane, and coupling constants are reported in Hz. EI mass spectra were recorded on a Finnigan TSQ-700 GS/ MS system (Thermo Finnigan, San Jose, CA), with the sample being introduced via a direct exposure probe. Mass spectral analyses in the ES mode were conducted on a Finnigan TSQ7000 LC/MS system. FIA/MS/MS was conducted on the same instrument, using argon at 1 mTorr as the collision gas and a collision energy of 25 eV. The samples were loaded onto a 5 µm Prodigy ODS(3) 100A column (2.0 mm × 250 mm; Phenomenex, Torrance, CA), and the mobile phase was delivered at a flow rate of 0.2 mL/min, using a 40 min linear gradient of 5-95% acetonitrile in 0.1% aqueous formic acid. Elemental analyses were performed at the Analytical Laboratory, Instituto Superior Te´cnico, Lisboa, Portugal. Syntheses. R-Hydroxy-N,N-didesmethyltamoxifen (10) was synthesized by the sequence of steps outlined in Scheme 2. (E,Z)-1-Bromo-1,2-diphenyl-2-[4-(2-(4-methoxytritylamino)ethoxy)phenyl]ethene (14). Triethylamine (1 mL, 7.17 mmol) and p-methoxytrityl chloride (1.58 g, 5.12 mmol) were added to a solution of the bromoalkene 13 (2.03 g, 5.14 mmol) in dry CH2Cl2 (30 mL), and the mixture was stirred overnight at room temperature. After the solvent was evaporated, the oily residue was washed twice with methanol and dried under vacuum to yield a crystalline white solid containing 14 as a

mixture of the E (60%) and Z (40%) isomers (2.96 g, 86%). 1H NMR (CDCl3): δ 2.50 (2H, t, J ) 4.8, CH2N, E isomer), 2.58 (2H, t, J ) 4.8, CH2N, Z isomer), 3.78 (3H, s, CH3O, E isomer), 3.79 (3H, s, CH3O, Z isomer), 3.96 (2H, t, J ) 4.8, CH2O, E isomer), 4.11 (2H, t, J ) 4.8, CH2O, Z isomer), 6.58 (2H, d, J ) 8.7, alkOPhH, E isomer), 6.80-7.53 (26H, E isomer + 28H, Z isomer, m, ArH). 13C NMR (CDCl3): 43.05 (CH2N), 55.15 (CH3O), 68.04 (CH2O), 68.16 (CH2O), 70.18 (Ar3C), 113.12 (ArCH), 113.98 (ArCH), 120.99 (Cquat), 121.44 (Cquat), 126.22 (ArCH), 127.80 (ArCH), 128.02 (ArCH), 128.10 (ArCH), 128.53 (ArCH), 128.53 (ArCH), 129.54 (ArCH), 129.79 (ArCH), 130.29 (ArCH), 130.93 (ArCH), 131.59 (ArCH), 133.42 (Cquat), 136.18 (Cquat), 138.05 (Cquat), 141.32 (Cquat), 143.04 (Cquat), 144.04 (Cquat), 146.16 (Cquat), 157.87 (Cquat), 158.29 (Cquat). MS (EI): m/z 590 [(M - Ph)+], 588 [(M - Ph)+], 560 [(M - MeOPh)+], 558 [(M MeOPh)+], 352 {[M - (CH2CHNHMeOtrityl)+]}, 350 {[M (CH2CHNHMeOtrityl)+]}, 288 [(NHMeOtrityl)+], 274 [(MeOtritylH)+]. Anal. calcd for C42H36BrNO2‚0.25MeOH: C, 75.22%; H, 5.53%; N, 2.08%. Found: C, 75.21%; H, 5.69%; N, 1.97%. r-Hydroxy-N,N-didesmethyltamoxifen (10). The N-methoxytrityl-protected bromoalkene 14 (750 mg, 1.13 mmol) was dissolved in freshly distilled dry THF (6 mL), kept under argon, and cooled to -100 °C. A 1.5 M solution of tert-butyllithium in n-pentane (6 mL, ∼8 molar equiv) was then added, while keeping the temperature below -80 °C. After approximately 5 min, an excess of acetaldehyde was slowly added through a cannula, until a change in coloration of the reaction mixture, from dark brown to pale yellow, indicated quenching of the vinyllithium intermediate. The excess of acetaldehyde was quickly removed by evaporation, and a saturated ammonium chloride solution (150 mL) was added to the mixture. After it was extracted with methylene chloride (2 × 1 vol), the organic phase was dried and the solvent was evaporated to yield a pale yellow oily material (15). The oil was dissolved in 25% acetic acid in CH2Cl2 (25 mL), and the solution was stirred at room temperature for 30 min. TLC (silica gel, 15% MeOH in CH2Cl2) indicated complete deprotection of the amino group. The mixture was further diluted to 50 mL with CH2Cl2, washed sequentially with 30% ammonium hydroxide (2 × 1 vol) and water (1 vol), dried with anhydrous sodium sulfate, and evaporated to dryness. The product was separated from the corresponding Z isomer by preparative TLC (silica gel, 20% MeOH in CH2Cl2) to yield R-hydroxy-N,N-didesmethyltamoxifen (10) as a white solid (172 mg; 43%); mp 178-179 °C. 1H NMR (acetone-d6): δ 1.09 (3H, d, J ) 6.5, CH3CH), 3.46 (2H, t, J ) 6.2, CH2N), 4.01 (2H, t, J ) 6.2, CH2O), 4.78 (1H, q, J ) 6.5, CH3CH), 6.57 (2H, d, J ) 8.9, alkOPhH), 6.83 (2H, d, J ) 8.9, alkOPhH), 7.10-

R-Hydroxy-N,N-didesmethyltamoxifen 7.40 (10H, m, PhH). 13C NMR (acetone-d6): δ 22.29 (CH3CH), 51.12 (CH2N), 67.64 (CH2O), 68.71 (CH3CH), 114.03 (ArCH), 126.79 (ArCH), 126.96 (ArCH), 127.50 (ArCH), 127.84 (ArCH), 128.84 (ArCH), 130.39 (ArCH), 132.19 (ArCH), 132.25 (Cquat), 135.79 (Cquat), 140.14 (Cquat), 140.74 (Cquat), 143.46 (Cquat), 158.05 (Cquat). MS (EI): m/z 359 (M+), 226 (M+ - PhCCH(OH)Me + 1), 183 [(PhCHC6H4OH)+]. Anal. calcd for C24H25NO2‚H2CO3‚ 2H2O: C, 65.63%; H, 6.83%; N, 3.06%. Found: C, 65.61%; H, 6.78%; N, 3.18%. (E)-r-(Deoxyguanosin-N2-yl)-N,N-didesmethyltamoxifen (12). 1. From r-Acetoxy-N,N-didesmethyltamoxifen. Preliminary experiments to obtain 12 involved the preparation of R-acetoxy-N,N-didesmethyltamoxifen (11, R ) COCH3). Prior to reaction with DNA, the amino group of R-hydroxy-N,Ndidesmethyltamoxifen (20 mg, 56 µmol) was protected quantitatively as a p-nitrobenzyl carbamate by reaction at 0 °C with 1.2 molar equiv of p-nitrobenzyl chloroformate in a two phase system consisting of CH2Cl2 and 1 M NaOH (31). The reaction was monitored by TLC (silica gel, 15% MeOH in CH2Cl2), and upon completion, the organic phase was separated, washed with water (2 × 1 vol), and dried. The crude product was quantitatively acetylated with acetic anhydride in pyridine (16). Following evaporation of the solvent, the crude acetylated carbamate, which was pure by TLC criteria, was redissolved in dry ethyl acetate (10 mL). Total deprotection of the amino group was achieved by a 3 h catalytic hydrogenation at room temperature, using 10% Pd/C (2 mg) as the catalyst and P(H2) ) 3 atm. Following removal of the catalyst by filtration, the mixture was evaporated to dryness to afford a residue containing trace amounts of R-acetoxy-N,N-didesmethyltamoxifen. This residue was redissolved in THF (1 mL) and incubated overnight at 37 °C with a solution of salmon testis DNA (ca. 2 mg/mL) in 10 mL of 5 mM Bis-Tris and 0.1 mM EDTA (pH 7.1). Treatment of the DNA solution as described below afforded one major adduct that had chromatographic and UV characteristics identical to those of the major adduct obtained from reactions with R-sulfoxy-N,N-didesmethyltamoxifen (vide infra). 2. From r-Sulfoxy-N,N-didesmethyltamoxifen. In subsequent experiments, adduct 12 was synthesized from R-hydroxy-N,N-didesmethyltamoxifen by an adaptation of the methodology previously described for R-sulfoxytamoxifen (32) and R-sulfoxy-N-desmethyltamoxifen (21). Briefly, R-hydroxy-N,Ndidesmethyltamoxifen (20 mg, 56 µmol) and the SO3‚pyridine complex (4 molar equiv) were mixed in dry pyridine (500 µL). 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 1 mL of DMF, the insoluble materials were removed by centrifugation, and the supernatant was added to a solution of salmon testis DNA (ca. 2 mg/mL) in 20 mL of 5 mM Bis-Tris and 0.1 mM EDTA (pH 7.1). The mixture was vortexed and incubated overnight at 37 °C. Nonbonded materials were removed by extraction with n-butanol (2 × 1 vol) that had been presaturated with 5 mM Bis-Tris and 0.1 mM EDTA (pH 7.1), and the DNA was precipitated with 5 M NaCl (0.1 vol) and cold ethanol (3 vol). The DNA was pelleted by centrifugation, washed with cold 70% ethanol (2 × 20 mL), centrifuged, and redissolved in 20 mL of 5 mM Bis-Tris and 0.1 mM EDTA (pH 7.1). An aliquot of the modified DNA solution was set aside for 32P-postlabeling, and the remaining DNA was hydrolyzed enzymatically to deoxynucleosides by treatment with DNase I, followed by alkaline phosphatase and phosphodiesterase (33). The adducts were then partitioned into n-butanol (6 × 10 mL) that had been presaturated with 5 mM Bis-Tris and 0.1 mM EDTA (pH 7.1), and the n-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 n-butanol was evaporated, the residue was dissolved in methanol (1 mL) and analyzed by HPLC at a flow rate of 2 mL/min, using a linear gradient of 0-60% acetonitrile in 100 mM ammonium acetate (pH 5.7) in 17 min, followed by a linear gradient to 100% acetonitrile in 3 min, and an isocratic elution with 100% acetonitrile for 5 min. The eluate was monitored at 254 nm, and the major adduct, eluting at 13.4

Chem. Res. Toxicol., Vol. 16, No. 9, 2003 1093 min, was collected, thoroughly dried, and characterized as (E)R-deoxyguanosin-N2-yl)-N,N-didesmethyltamoxifen (12). For 1H NMR data, see Results and Discussion. MS (ES): m/z 609 (MH+). FIA/MS/MS (m/z 609): m/z 493 (MH2+ - dR), 342 (M+ - dG), 178 [(Gua + CHCH3)+]. DNA Adduct Standards. DNA samples modified with (E)R-(deoxyguanosin-N2-yl)tamoxifen (22) and (E)-R-(deoxyguanosin-N2-yl)-N-desmethyltamoxifen (21) were prepared from R-acetoxytamoxifen and R-sulfoxy-N-desmethyltamoxifen, respectively, as described in the references indicated. Treatment of Animals. Female Sprague-Dawley rats [four per group; Crl:COBS CD (SD) BR outbred; 8 weeks old; 181 ( 15 g; obtained from the breeding colony at the National Center for Toxicological Research] were treated by intraperitoneal injection with four daily doses of tamoxifen (20 mg/kg, 54 µmol/ kg, dissolved in 200 µL of trioctanoin). Four additional animals per group were treated in the same manner with equimolar doses of N,N-didesmethyltamoxifen hydrochloride (20.5 mg/kg) and R-hydroxy-N,N-didesmethyltamoxifen (19.4 mg/kg) or the solvent alone (200 µL of trioctanoin). Twenty-four hours after the last treatment, the animals were killed by exposure to carbon dioxide. The livers were quickly excised, hepatic nuclei were isolated (34), and DNA was prepared by slight modifications of the method described in Beland et al. (35). 32P-Postlabeling Analyses. 32P-Postlabeling analyses were conducted by the nuclease P1 enrichment procedure of Reddy and Randerath (36), essentially as described in Gamboa da Costa et al. (22). 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 the mixture was evaporated 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: 0-10 min, isocratic elution with 58% solvent A [1.2 M ammonium formate, 10 mM ammonium phosphate (pH 4.5)] and 42% solvent B [24% acetonitrile in 1.2 M ammonium formate, 10 mM ammonium phosphate (pH 4.5)]; 10-20 min, a linear gradient to 83% solvent B; 20-40 min, isocratic elution with 83% solvent B; 4060 min, a linear gradient to 100% solvent B. The flow rate was 1 mL/min. The adducts formed in vivo were characterized by comparison with the synthetic DNA adduct standards containing (E)-R-(deoxyguanosin-N2-yl)tamoxifen, (E)-R-(deoxyguanosinN2-yl)-N-desmethyltamoxifen, and (E)-R-(deoxyguanosin-N2-yl)N,N-didesmethyltamoxifen. The adduct levels were determined through comparison to a 32P-postlabeled DNA standard containing (E)-R-(deoxyguanosin-N2-yl)tamoxifen at a level of 5.2 adducts/106 nucleotides, assuming similar labeling efficiencies for all of the adducts. The modification level of the DNA standard was determined using HPLC-ES-MS/MS, by quantifying against a known amount of the internal standard (E)-R-(deoxyguanosinN2-yl)-N,N-bis(trideuteriomethyl)tamoxifen (29). Statistical Analyses. Statistical analyses were conducted by one way ANOVA followed by Dunnett’s test. To maintain a normal data distribution, the data were log transformed before the analyses.

Results and Discussion Synthesis of 10. The synthetic strategy toward R-hydroxy-N,N-didesmethyltamoxifen (Scheme 2) was based on the methodology originally described by Foster et al. (37) for the synthesis of R-hydroxytamoxifen, which consists of the addition of acetaldehyde to a vinyllithium precursor. The starting material was the bromoalkene 13, which was obtained as a mixture of the E and Z diastereomers (29). Prior to lithiation, it was necessary to protect the amino group in the alkyl side chain of 13 with a substituent that could provide stability under basic conditions. For this purpose, we selected the pmethoxytrityl group (38), which can be easily removed

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under mild acidic conditions (39). Thus, reaction of 13 with p-methoxytrityl chloride in the presence of triethylamine afforded the N-tritylated bromoalkene 14 (Scheme 2) as a mixture of the E and Z isomers. 1H and 13C NMR and mass spectral data (cf. Materials and Methods) confirmed the assigned structures. The mixture was used in the next synthetic step without separation of the isomers. Steric hindrance prevented the ditritylation of 13; however, one potential difficulty subsisting when generating the vinyllithium intermediate from 14 was the presence of a labile proton in the secondary amino group. A similar problem accounted for the low yield (12%) that we obtained in the synthesis of R-hydroxy-N-desmethyltamoxifen from the corresponding bromoalkene precursor (21). To minimize deprotonation of the amine, we used tert-butyllithium, which is bulkier than n-butyllithium (21, 37), as the lithiating agent and thus generated 15 by treating 14 with 8 molar equiv of tert-butyllithium at -100 °C, followed by addition of excess acetaldehyde to the organolithium intermediate (Scheme 2). Upon standard workup, the crude product was N-detritylated by solvolysis under mild acidic conditions (Scheme 2), and R-hydroxy-N,N-didesmethyltamoxifen was isolated in 43% yield after separation from the Z isomer by TLC. The improved yield, as compared to that obtained for R-hydroxy-N-desmethyltamoxifen (21), suggests that tertbutyllithium was advantageous as a lithiating agent. The product was fully characterized by spectroscopic techniques (1H and 13C NMR and MS; cf. Materials and Methods). In accord with what has been reported for other structurally similar compounds (21, 37), assignment of the E stereochemistry to 10 was based on its 1H NMR spectrum, where a pair of mutually coupled two proton aromatic doublets, ascribed to the alkoxyphenyl ring (δ 6.57 and 6.83 ppm), were shifted upfield by the combined shielding effect of the two phenyl rings (40, 41). Synthesis, Isolation, and Characterization of the Major DNA Adduct from r-Acetoxy- and r-SulfoxyN,N-didesmethyltamoxifen. The presence of the primary amino group in R-hydroxy-N,N-didesmethyltamoxifen caused some concern that esterification of the R-hydroxyl group might also lead to reaction at the amine nitrogen. Therefore, we envisioned protection of the amino group as a carbamate, followed by O-acetylation and subsequent cleavage of the carbamate by catalytic hydrogenation to yield R-acetoxy-N,N-didesmethyltamoxifen (Scheme 1; R ) COCH3), which would then be reacted with DNA. While the generation of the pnitrobenzyl carbamate of 10 by reaction with p-nitrobenzyl chloroformate (31) and the subsequent O-acetylation of the carbamate posed no difficulties, hydrogenation in the presence of Pd/C yielded a mixture of several products, with the unstable R-acetoxy-N,N-didesmethyltamoxifen being obtained in trace amounts. Although other authors did not report this problem when applying a similar strategy to the synthesis of R-acetoxy-N-desmethyltamoxifen (42), it is conceivable that competitive hydrogenolysis of the acetoxy group and/or reduction of the ethylenic double bond (43) occurred under the experimental conditions [P(H2) ) 3 atm] that we used. In addition, strong adsorption of R-acetoxy-N,N-didesmethyltamoxifen to the Pd/C catalyst may have limited the amount of isolated product. Despite the unsatisfactory yield of R-acetoxy-N,N-didesmethyltamoxifen, a preliminary reaction was conducted between the crude product

Gamboa da Costa et al.

Figure 1. HPLC of the enzymatic hydrolysate obtained from reacting in situ generated R-sulfoxy-N,N-didesmethyltamoxifen with DNA. The elution conditions are outlined in the Materials and Methods. 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.

and the salmon testis DNA. Upon hydrolysis of the DNA to deoxynucleosides, one major adduct was detected by HPLC analysis (not shown). To obtain a quantity of adduct sufficient for spectroscopic characterization, we tested an alternative activation of the R-hydroxyl group of R-hydroxy-N,N-didesmethyltamoxifen, by reaction with an excess of the SO3‚ pyridine complex, as described for the syntheses of R-sulfoxytamoxifen (32) and R-sulfoxy-N-desmethyltamoxifen (21). Although N-sulfation was expected to occur, we reasoned that subsequent N-desulfation would ensue under the hydrolytic conditions prevalent during the incubation with DNA (44). Thus, R-sulfoxy-N,N-didesmethyltamoxifen and/or its N-sulfated derivative were generated, and the crude mixture was reacted with DNA immediately after being produced. Spectroscopic characterization of the intermediate(s) was not attempted, due to their anticipated instability upon exposure to the atmosphere (21, 32). The HPLC profile of the n-butanol extract from the DNA hydrolysate (Figure 1) indicated the presence of one major (peak 1) and at least two additional minor putative DNA adducts (peaks 2 and 3) not detected in control incubations. The major peak had a retention time (13.4 min) and UV spectrum identical to the major peak observed from the reaction with R-acetoxy-N,N-didesmethyltamoxifen. This peak was isolated by HPLC and characterized on the basis of its MS and 1H NMR spectra (vide infra). The UV spectrum of the adduct (Figure 1, inset) had an absorbance pattern virtually identical to those reported for N2-deoxyguanosyl adducts through the R-carbon of tamoxifen (16, 32, 45), N-desmethyltamoxifen (21, 42), and 4-hydroxytamoxifen (46), therefore suggesting the same substitution pattern. ES mass spectrometry of the isolated adduct in the positive ion mode yielded an ion at m/z 609 (C34H37N6O5), consistent with a protonated adduct of N,N-didesmethyltamoxifen and dG. A product ion analysis of the protonated molecule was performed by FIA/MS/MS (Figure 2). Fragment ions were found at m/z 493 (MH2+ dR), 342 (M+ - dG), and 178 [(Gua + CHCH3)+]. While the m/z 493 and 342 ions stemmed from typical losses of deoxyribose and dG, respectively, the m/z 178 ion was more informative, since it provided evidence for the attachment of dG to the R-carbon of the N,N-didesmeth-

R-Hydroxy-N,N-didesmethyltamoxifen

Chem. Res. Toxicol., Vol. 16, No. 9, 2003 1095

Figure 2. MS/MS characterization of (E)-R-(deoxyguanosin-N2-yl)-N,N-didesmethyltamoxifen (12) using FIA. The major fragmentations derived from the protonated molecule are outlined.

Figure 3. Aromatic region of the 1H NMR spectrum of (E)-R(deoxyguanosin-N2-yl)-N,N-didesmethyltamoxifen recorded in methanol-d4. The chemical shift assignments are indicated in the figure.

yltamoxifen moiety. Similar cleavages of the allylic carbon from the triarylethylene segment have been found in the mass spectra of the major adducts derived from tamoxifen and N-desmethyltamoxifen (18, 21). This observation suggests that as with the tamoxifen- and N-desmethyltamoxifen-derived adducts, binding to the allylic carbon of N,N-didesmethyltamoxifen occurred through the exocyclic nitrogen of dG. Additional information was obtained from the 1H NMR spectrum, recorded in methanol-d4. The limited amount of isolated adduct prevented a full assignment, particularly in the aliphatic region, where the intensity of the residual solvent peaks obscured the adduct resonances to a significant extent. However, the signals obtained in the downfield region of the spectrum (Figure 3) provided sufficient information for structural elucidation. Thus, the H8 proton of dG was clearly present as a singlet at δ 7.97, as was the deoxyribose H1′ proton, detected as a distorted triplet at δ 6.17 (J ) 6.0 Hz). Similarly, all of the expected aromatic protons (ca.6.5-7.5 ppm) were observed, in a pattern closely resembling the one we reported previously for (E)-R-(deoxyguanosin-N2-yl)-Ndesmethyltamoxifen (21). Particularly noteworthy among the aromatic protons were two upfield, mutually coupled, doublets, each accounting for two protons, at δ 6.54 and 6.76 (J ) 9.0 Hz), which were ascribed to the protons of the alkoxyphenyl ring. The location of these signals reflected shielding by the combined effect of the two phenyl substituents (40, 41), which led to the assignment

of the E configuration to the isolated adduct. As shown in Figure 3, a broad signal, centered at δ ca. 5.34, was assigned to the proton attached to the allylic carbon. This signal appeared to be composed of two partially overlapped multiplets, presumably stemming from epimeric protons. In an attempt to assign the exchangeable protons, which would have provided definite characterization of the adduct, the 1H NMR spectrum was also recorded in DMSO-d6. We were able to detect a one proton doublet (not shown) at δ 5.93 (J ) 9 Hz), which was consistent with attachment of the N,N-didesmethyltamoxifen segment through the exocyclic nitrogen of dG. In addition, a small downfield singlet at δ 9.83 ppm (not shown) presumably stemmed from the imino proton of the dG segment, although an unambiguous assignment of this resonance would have required a greater amount of sample. Despite the limitations imposed by the small yield of the adduct, the combined spectroscopic data are consistent with characterization of the isolated adduct as (E)-R-deoxyguanosin-N2-yl)-N,N-didesmethyltamoxifen, which appears to have been obtained as a mixture of epimers at the R-carbon. Because of the limited quantities available, the minor adducts (peaks 2 and 3, Figure 1) were not isolated. By analogy with reports from reactions conducted with tamoxifen and N-desmethyltamoxifen derivatives (32, 42, 45), the minor adducts were presumably diastereomers of the major product. DNA Adduct Analyses in the Livers of Rats Treated with Tamoxifen, N,N-Didesmethyltamoxifen, or r-Hydroxy-N,N-didesmethyltamoxifen. To elucidate the potential significance of R-hydroxy-N,Ndidesmethyltamoxifen as a genotoxic metabolite of tamoxifen, female Sprague-Dawley rats were treated by intraperitoneal injection with four daily doses of tamoxifen, N,N-didesmethyltamoxifen, R-hydroxy-N,N-didesmethyltamoxifen, or the solvent alone. One day following the last treatment, the rats were killed, liver DNA was isolated, and DNA adduct levels were assessed by 32Ppostlabeling in combination with HPLC. While only background levels were detected in liver DNA from control rats (Table 1), one minor and two major DNA adducts (peaks b-d, Figure 4A) were found in liver DNA from rats administered tamoxifen. In full agreement with previous reports (16, 19-22), peak c coeluted with the (E)-R-(deoxyguanosin-N2-yl)-N-desmethyltamoxifen standard, while peak d coeluted with the (E)-R-(deoxyguanosin-N2-yl)tamoxifen standard (not shown). Simi-

1096 Chem. Res. Toxicol., Vol. 16, No. 9, 2003 Table 1. DNA Adduct Levels in Liver DNA of Female Sprague-Dawley Rats Treated by Intraperitoneal Injection with Four Daily Doses of Tamoxifen and Its N,N-Didesmethylated Derivativesa treatment

hepatic DNA adduct levels (adducts/107 nucleotides)

solvent tamoxifen N,N-didesmethyltamoxifen R-hydroxy-N,N-didesmethyltamoxifen

0(0 300 ( 220b 7(8 610 ( 350b

a Female Sprague-Dawley rats were treated by intraperitoneal injection with four daily doses of 54 µmol/kg tamoxifen. Additional animals were treated in the same manner with equimolar doses of N,N-didesmethyltamoxifen hydrochloride, R-hydroxy-N,N-didesmethyltamoxifen, or the solvent alone. The rats were killed 24 h after the last treatment. The data are expressed as the mean ( SD for four rats per group. b Significantly different (p < 0.05) from rats treated with solvent alone as determined by one way ANOVA followed by Dunnett’s test.

Gamboa da Costa et al.

supra), coeluted with the major peak (b) formed in the livers of rats administered R-hydroxy-N,N-didesmethyltamoxifen, and coelution was also observed in both samples for the minor adducts (a), presumably corresponding to diastereomer(s) of the major product (Figure 4B,C). Thus, the same pattern of adduct formation from R-hydroxy-N,N-didesmethyltamoxifen was observed in vitro and in rat liver in vivo. Table 1 summarizes the binding data for rat liver DNA. When assessed one day following the last of four daily intraperitoneal doses, the relative levels of binding were R-hydroxy-N,N-didesmethyltamoxifen ∼ tamoxifen > N,N-didesmethyltamoxifen. These data clearly indicate that R-hydroxy-N,N-didesmethyltamoxifen can be further activated in rat liver in vivo to a genotoxic derivative. Furthermore, the adduct levels obtained from R-hydroxyN,N-didesmethyltamoxifen were very similar to those that we have previously found following intraperitoneal administration of the same dose of R-hydroxytamoxifen (22), which suggests comparable activation pathways for both R-hydroxylated metabolites. The reasons for the low extent of DNA adduct formation by N,N-didesmethyltamoxifen are presently unknown. Because it is a primary alkylamine, it may become conjugated (e.g., N-glucuronidated; 47, 48) and excreted more readily than Ndesmethyltamoxifen and tamoxifen. In addition, Comoglio et al. (49) have presented evidence that N,Ndidesmethyltamoxifen inhibits cytochrome P450-catalyzed transformations; as such, it may be unable to undergo R-hydroxylation. Whatever the reason, it appears that the activation of N,N-didesmethyltamoxifen through R-hydroxylation is a relatively minor metabolic pathway in rat liver.

Conclusions

Figure 4. Representative HPLC 32P-postlabeling analyses of liver DNA from female Sprague-Dawley rats treated with (A) tamoxifen or (B) R-hydroxy-N,N-didesmethyltamoxifen and (C) DNA modified in vitro with R-sulfoxy-N,N-didesmethyltamoxifen. The 32P-postlabeling and elution conditions are outlined in the Materials and Methods.

larly to what has been found in other studies (20, 22) administration of N,N-didesmethyltamoxifen yielded very low levels of a liver DNA adduct (Table 1) that coeluted with peak b (not shown). In contrast to N,N-didesmethyltamoxifen, substantial binding occurred in the liver DNA from rats treated with R-hydroxy-N,N-didesmethyltamoxifen (Table 1), with one minor and one major adduct (peaks a and b, respectively, Figure 4B) being detected. Peak b coeluted with the minor adduct peak detected in the liver DNA from tamoxifen-treated rats. When assessed by 32P-postlabeling, DNA modified in vitro with the sulfated derivative of R-hydroxy-N,Ndidesmethyltamoxifen had one major (b) and one minor (a) adduct (Figure 4C). The major adduct in the synthetic standard, identified as the 3′,5′-bis-phosphate of (E)-R(deoxyguanosin-N2-yl)-N,N-didesmethyltamoxifen (vide

Previous reports of the DNA adduct profiles obtained in the livers of rats administered tamoxifen and Ndesmethyltamoxifen have suggested that a minor adduct arising from these treatments may originate from N,Ndidesmethyltamoxifen (19-22). However, binding to rat liver DNA by N,N-didesmethyltamoxifen was found to occur only to a low extent. In the present work, we show that R-hydroxy-N,N-didesmethyltamoxifen, a putative metabolite of N,N-didesmethyltamoxifen, binds to a substantial extent to DNA in vitro upon chemical activation by acetylation or sulfation, yielding (E)-R-(deoxyguanosin-N2-yl)-N,N-didesmethyltamoxifen as the major adduct. When administered to Sprague-Dawley rats by intraperitoneal injection, R-hydroxy-N,N-didesmethyltamoxifen afforded the same major adduct in rat liver, at a level comparable to the total extent of modification obtained from an equivalent treatment with equimolar doses of tamoxifen. These results indicate that although R-hydroxy-N,N-didesmethyltamoxifen has genotoxic activity in rat liver, a metabolic activation pathway from N,N-didesmethyltamoxifen to R-hydroxy-N,N-didesmethyltamoxifen is a minor pathway in the rat model. It remains to be established if R-hydroxy-N,N-didesmethyltamoxifen can be formed in vivo by an alternative pathway, involving N-demethylation of R-hydroxy-Ndesmethyltamoxifen, as suggested by Phillips et al. (19).

Acknowledgment. We thank Thomas Heinze for obtaining the mass spectra, Vı´tor Pereira for providing the elemental analyses, L. Patrice Hamilton for conduct-

R-Hydroxy-N,N-didesmethyltamoxifen

ing the 32P-postlabeling analyses, and Cindy Hartwick for helping 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 research grants from Programs PRAXIS XXI and POCTI, Fundac¸ a˜o para a Cieˆncia e a Tecnologia (FCT), Portugal, and by a fellowship to G.G.C. from Subprograma Cieˆncia e Tecnologia, FCT, Portugal.

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