Synthesis and Characterization of DNA Adducts from the HIV Reverse

Jul 3, 2008 - Nevirapine (NVP) is a non-nucleoside reverse transcriptase inhibitor used against the human immunodeficiency virus type 1 (HIV-1), mostl...
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Chem. Res. Toxicol. 2008, 21, 1443–1456

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Synthesis and Characterization of DNA Adducts from the HIV Reverse Transcriptase Inhibitor Nevirapine Alexandra M. M. Antunes,*,† Mariana P. Duarte,‡ Pedro P. Santos,‡ Gonc¸alo Gamboa da Costa,§ Thomas M. Heinze,§ Frederick A. Beland,§ and M. Matilde Marques*,‡ REQUIMTE/CQFB, Faculdade de Cieˆncias e Tecnologia, UniVersidade NoVa de Lisboa, 2829-516 Caparica, Portugal, Centro de Quı´mica Estrutural, Instituto Superior Te´cnico, UniVersidade Te´cnica de Lisboa, 1049-001 Lisboa, Portugal, and DiVision of Biochemical Toxicology, National Center for Toxicological Research, Jefferson, Arkansas 72079 ReceiVed March 11, 2008

Nevirapine (NVP) is a non-nucleoside reverse transcriptase inhibitor used against the human immunodeficiency virus type 1 (HIV-1), mostly to prevent mother-to-child HIV transmission in developing countries. One of the limitations of nevirapine use is severe hepatotoxicity, which raises concerns about its administration, particularly in the perinatal and pediatric settings. Nevirapine metabolism involves oxidation of the 4-methyl substituent to 12-hydroxy-NVP and the formation of phenolic derivatives. Further metabolism of 12-hydroxy-NVP by phase II esterification may produce electrophilic derivatives capable of reacting with DNA to yield covalent adducts, which could potentially be involved in the initiation of mutagenic and carcinogenic events. In the present study, we have investigated the reactivity of the synthetic model electrophile, 12-mesyloxy-NVP, toward 2′-deoxynucleosides and DNA. Parallel synthetic studies were conducted with 12-bromo-NVP and 3′,5′-O-bis(tert-butyldimethylsilyl)-2′deoxynucleosides, using palladium(0) catalysis. Multiple adducts from deoxyguanosine, deoxyadenosine, and deoxycytidine were isolated in the reactions with 12-mesyloxy-NVP and characterized by NMR and MS. The adduct structures consistently involved binding through C12 of NVP and N7 or N9 of deoxyguanosine; N1, N3, or N9 of deoxyadenosine; and N3 of deoxycytidine. Reactions conducted under palladium(0) catalysis yielded adducts through O6 and N1 of deoxyguanosine, N1 of deoxyadenosine, and N3 of deoxycytidine. At least seven deoxynucleoside-NVP adducts were present in DNA reacted with 12-mesyloxy-NVP and enzymatically hydrolyzed. Four of these adducts were identified as 12(deoxyadenosin-N1-yl)nevirapine, 12-(deoxycytidin-N3-yl)nevirapine, 12-(deoxyguanosin-O6-yl)nevirapine, and 12-(deoxyadenosin-N6-yl)nevirapine. One depurinating adduct, 12-(guanin-N7-yl)nevirapine, was identified in the thermal neutral DNA hydrolysate. If formed in vivo, some of these adducts would have considerable mutagenic potential. Our results thus suggest that NVP metabolism to 12-hydroxyNVP may be a factor in NVP hepatocarcinogenicity. Introduction The use of combined antiretroviral therapeutic regimens has caused significant reductions in the morbidity and mortality associated with infection by the human immunodeficiency virus type 1 (HIV-1).1 As a result, HIV-1 infection has largely become * To whom correspondence should be addressed. (A.A.) Tel: 351-212948300 (ext. 10911). Fax: 351-21-2948550. E-mail: [email protected]. (M.M.M.) Tel: 351-21-8419200. Fax: 351-21-8464457. E-mail: [email protected]. † Universidade Nova de Lisboa. ‡ Universidade Te´cnica de Lisboa. § National Center for Toxicological Research. 1 Abbreviations: BH, adducted base; CNL, constant neutral loss; dA, 2′-deoxyadenosine; dC, 2′-deoxycytidine; dG, 2′-deoxyguanosine; DBU, 1,8-diazabicycloundec-7-ene; DMF, N,N-dimethylformamide; DMSO, dimethylsulfoxide; dR, 2′-deoxyribosyl; EI, electron ionization; ESI, electrospray ionization; HAART, highly active antiretroviral therapy; HIV, human immunodeficiency virus; HMBC, heteronuclear multiple bond correlation; HMQC, heteronuclear multiple quantum coherence; LDA, lithium diisopropylamide; MoOPH, oxodiperoxymolybdenum(pyridine)hexamethylphosphoramide; N3-NVP-Ade, 12-(adenin-N3-yl)nevirapine; N9NVP-Ade, 12-(adenin-N9-yl)nevirapine; N1-NVP-dA, 12-(deoxyadenosinN1-yl)nevirapine; N6-NVP-dA, 12-(deoxyadenosin-N6-yl)nevirapine; N3NVP-dC,12-(deoxycytidin-N3-yl)nevirapine;N1-NVP-dG,12-(deoxyguanosinN1-yl)nevirapine; N3-NVP-Gua, 12-(guanin-N3-yl)nevirapine; N7-NVPGua, 12-(guanin-N7-yl)nevirapine; N9-NVP-Gua, 12-(guanin-N9-yl)nevirapine; NNRTI, non-nucleoside reverse transcriptase inhibitor; NOESY, twodimensional nuclear Overhauser effect spectroscopy; NVP, nevirapine; O6NVP-dG, 12-(deoxyguanosin-O6-yl)nevirapine; Pd2(dba)3, tris(dibenzylideneacetone)dipalladium(0).

a chronic disease in developed countries, and long treatment periods warrant consideration of the toxicities of antiretroviral drugs (1, 2). On the other hand, pediatric HIV infection remains a major public health concern, particularly in sub-Saharan Africa, and the most efficient means to address the problem is prevention of mother-to-child transmission of the virus (3). Vertical transmission can take place in the perinatal period, either in utero or intrapartum, and also postpartum, through breast feeding (4–6). Without medical intervention, the overall risk of mother-to-child transmission has been estimated at 15-20% in Europe and the United States and 25-40% in subSaharan Africa (7–13). In developed settings, the use of highly active antiretroviral therapy (HAART) has been shown to reduce the rates of in utero and intrapartum transmission to less than 2% (14–17). Following the introduction of the World Health Organization “3 by 5 initiative” (18), HAART has become more available for administration to HIV-1-infected pregnant women in low resource countries. Although this strategy is essential to control the pediatric HIV pandemic, the long-term risks of multidrug treatment for mothers and their children remain essentially unknown. Nevirapine (11-cyclopropyl-5,11-dihydro-4-methyl-6H-dipyrido[3,2-b:2′,3′-e][1,4]diazepin-6-one, NVP, 1; Scheme 1) was the first non-nucleoside reverse transcriptase inhibitor (NNRTI) approved by the U.S. Food and Drug Administration for use in combination therapy of HIV-1 infection (19). Following pub-

10.1021/tx8000972 CCC: $40.75  2008 American Chemical Society Published on Web 07/03/2008

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Antunes et al.

Scheme 1. Structures of NVP (1), NVP Metabolites (2-6), and Other NVP Derivatives (7-9) Mentioned in the Text

lication of the results from the HIVNET 012 clinical trial in Uganda (20), which demonstrated the efficacy of a single dose during labor plus a single dose to the newborn, NVP administration became widespread in developing countries, as a singledose prophylaxis to prevent mother-to-child HIV transmission (21, 22). The low cost of the drug was also a factor in this context (23). Currently, NVP remains one of the most prescribed antiretroviral drugs in the developing world, both to prevent vertical transmission and in combination therapy (24), despite consistent reports of severe hepatotoxicity and serious adverse cutaneous events (25–28), and indications that intrapartum exposure is associated with increased virological failure during subsequent NVP treatment (29). Genetic toxicology tests, including microbial and mammalian cell gene mutation assays and cytogenetic assays, have provided no evidence that NVP is either mutagenic or clastogenic (30). However, although evidence for NVP carcinogenicity in humans has yet to be presented, a long-term administration study in mice (at doses of 0, 50, 375, or 750 mg/kg/day) showed an increased incidence of hepatocellular adenomas and carcinomas at all doses in males and at the two highest doses in females; a similar study in rats (at doses of 0, 3.5, 17.5, or 35 mg/kg/day) indicated an increased incidence of hepatocellular adenomas at all doses in males and at the high dose in females (30). The reasons for NVP carcinogenicity are currently unknown; however, it is plausible that metabolic activation to reactive electrophiles may be involved in the initiation of genotoxic responses. NVP is a known inducer of human P450 3A4 (31, 32) and is primarily metabolized by P450 3A and 2B6, through oxidation to 2-, 3-, and 8-hydroxy-NVP, 4-hydroxymethyl-NVP (12-hydroxy-NVP), and 4-carboxy-NVP (2-6; Scheme 1) (33–35). All hydroxyl groups undergo conjugation, with urinary excretion of the corresponding glucuronides as a major route of NVP elimination. Although little is known about other conjugation pathways, it is conceivable that acetylation and/or sulfation of the hydroxylated NVP metabolites (e.g., to 7; Scheme 1) may occur in the liver, similarly to what is observed for other drugs (36). Alternatively, the phenol type metabolites could undergo oxidation to ortho-quinone or semiquinone species (37). Both pathways could conceivably produce electrophiles capable of binding to DNA, yielding covalent adducts potentially involved in the genesis of mutagenic and carcinogenic events. In the present work, we have synthesized 12-mesyloxy-NVP (8; Scheme 1) as a model electrophile of the NVP metabolite 12-hydroxy-NVP (5) and investigated its reactivity toward deoxynucleosides and DNA. In parallel experiments, we have tested an alternative synthetic strategy by reacting 12-bromoNVP (9; Scheme 1) with 3′,5′-O-bis(tert-butyldimethylsilyl)2′-deoxynucleosides, under palladium(0) catalysis, using an adaptation of the Buchwald-Hartwig strategy. The characteriza-

tion of a series of DNA adducts is presented, and the results are discussed in terms of their possible relevance to NVP toxicity.

Materials and Methods Caution: NVP and its deriVatiVes are potentially carcinogenic. They should be handled with protectiVe clothing in a wellVentilated fume hood. Chemicals. NVP was purchased from Cipla (Mumbai, India). All other commercially available reagents and enzymes were acquired from Sigma-Aldrich Quı´mica, S.A. (Madrid, Spain) or Sigma Chemical Co. (St. Louis, MO) and used as received. 12-Hydroxy-NVP (5) was prepared in 49% yield by an adaptation of published methodology (38), involving generation of the C12 anion from NVP with lithium diisopropylamide (LDA) at -30 °C, followed by direct oxidation with the oxodiperoxymolybdenum(pyridine)hexamethylphosphoramide complex (MoOPH) (39). 3′,5′-O-Bis(tert-butyldimethylsilyl)2′-deoxynucleosides were quantitatively prepared by treatment of the deoxynucleoside with 10 equiv of bis(tert-butyldimethylsilyl) chloride in pyridine (40). O6-[2-(4-Nitrophenyl)ethyl]2′-deoxyguanosine was synthesized using a Mitsunobu type reaction, as described by Lee et al. (41); cleavage of the (4nitrophenyl)ethyl group was performed by treatment with 0.5 M 1,8-diazabicycloundec-7-ene (DBU) in pyridine (42). Whenever necessary, solvents were purified by standard procedures (43). Instrumentation. Melting temperatures were measured with a Leica Galen III hot stage apparatus and are uncorrected. Infrared (IR) spectra were recorded on a Perkin-Elmer 683 FTIR spectrometer; group frequencies are reported in cm-1. HPLC was conducted on 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). HPLC analyses were performed with a Luna C18 (2) column (250 mm × 4.6 mm; 5 µm; Phenomenex, Torrance, CA), at a flow rate of 1 mL/min. Semipreparative HPLC separations were conducted with a Luna C18 (2) column (250 mm × 10 mm, 5 µm; Phenomenex) at a flow rate of 3 mL/min. Unless mentioned otherwise, the elution conditions consisted of a 30 min linear gradient of 5-70% acetonitrile in 100 mM ammonium acetate (pH 5.7), followed by a 2 min linear gradient to 100% acetonitrile, and a 16 min isocratic elution with acetonitrile. The UV absorbance was monitored at 254 nm. Mass spectra were recorded on either a Finnigan TSQ-7000 GS/MS system, operated in the electron ionization (EI) mode, with the sample being introduced via a direct exposure probe, or a ThermoFinnigan TSQ Quantum Ultra LC/MS system,

12-HydroxyneVirapine DNA Adducts

operated in the positive electrospray ionization (ESI) mode. For ESI mass spectral measurements, the samples were loaded onto a 5 µm Prodigy ODS(3) 100A column (250 mm × 2.0 mm; Phenomenex) or a 3 µm Gemini C18 column (150 mm × 2 mm; Phenomenex), and the mobile phase was delivered at a flow rate of 0.2 mL/min, using 20 or 30 min linear gradients from 5 or 10% acetonitrile up to 95% acetonitrile in 0.1% aqueous formic acid. The columns were maintained at 30 °C. Fragment percent intensities and fragment assignments are indicated in parentheses and square brackets, respectively. 1 H NMR spectra were recorded on a Bruker Avance III 400, a Bruker AV 500b, or a Bruker Avance III 600 (with cryoprobe) spectrometer, operating at 400, 500, and 600 MHz, respectively. 13 C NMR spectra were recorded on a Bruker ARX 400, a Bruker AV 500b, or a Bruker Avance III 600 (with cryoprobe) spectrometer, operating at 100.62, 125.77, and 150.92 MHz, respectively. Chemical shifts are reported in ppm downfield from tetramethylsilane, and coupling constants (J) are reported in Hz; the subscripts ortho, meta, and gem refer to ortho, meta, and geminal couplings, respectively. Whenever appropriate, other long-range couplings are indicated by superscripts representing the number of bonds between the coupled protons. The presence of labile protons was confirmed by chemical exchange with D2O. Resonance and structural assignments were based on the analysis of coupling patterns, including the 13C-1H coupling profiles obtained in bidimensional heteronuclear multiple quantum coherence (HMQC) and heteronuclear multiple bond correlation (HMBC) experiments, performed with standard pulse programs. A semiselective HMBC experiment was performed on the Bruker Avance III 600 (with cryoprobe) spectrometer, using a pulse program (shmbcgpndqf, available from the Bruker library) with no decoupling during acquisition and using gradient pulses for selection. Excitation in 13C was ensured by a 90° shaped sinc pulse (with one cycle and size shape 1000), covering a 4527.536 Hz bandwidth (centered at 21879.4 Hz); the total spectral width in F2 was 4 ppm (centered at 7 ppm). 13C resonances are not discriminated whenever small sample quantities precluded the recording of one-dimensional 13C NMR spectra with good signal/noise ratios, despite having, for the most part, been detected in the inverse heteronuclear bidimensional experiments. Syntheses. 1. Preparation of NVP Derivatives. 1.1. 11-Cyclopropyl-5,11-dihydro-4-(mesyloxymethyl)6H-dipyrido-[3,2-b:2′,3′-e][1,4]diazepin-6-one (12-MesyloxyNVP, 8). Triethylamine (1.3 equiv, 290 µL) and methanesulfonyl chloride (1.3 equiv, 165 µL) were added to a solution of 5 (462 mg, 1.6 mmol) in dry THF (35 mL), under nitrogen at 0 °C, and the mixture was stirred for 30 min. Water (150 mL) was added, and the mixture was extracted with dichloromethane (3 × 50 mL). The combined organic phases were dried over anhydrous magnesium sulfate to afford a yellow solid (542 mg, 92%); mp 178-181 °C (ethyl acetate/n-hexane). IR (KBr): 1662 (CdO), 1417 (SO2). 1H NMR (CDCl3): δ 8.93 (1H, s, N5H), 8.56 (1H, dd, Jortho ) 4.7, Jmeta ) 1.6, H9), 8.29 (1H, d, J ) 4.8, H2), 8.14 (1H, dd, Jortho ) 7.6, Jmeta ) 1.6, H7), 7.15 (1H, d, J ) 4.8, H3), 7.10 (1H, dd, Jortho ) 7.6, Jortho′ ) 4.7, H8), 5.54 (2H, s, H12,12′), 3.77-3.76 (1H, m, H13), 3.23 (3H, s, CH3), 0.91-0.89 (2H, m, H14 + H15), 0.44-0.39 (2H, m, H14 + H15). 13C NMR (CDCl3): δ 168.2 (C6), 159.2 (C10a), 154.0 (C11a), 151.2 (C9), 144.4 (C2), 141.8 (C7), 136.0 (C4), 124.0 (C4a), 120.0 (C6a), 119.5 (C3), 118.4 (C8), 66.0 (C12), 38.0 (CH3), 29.9 (C13), 9.3 (C14/C15), 9.1 (C14/C15). MS (EI) m/z 360 (10) [M]+, 345 (3) [M - CH3]+, 331 (56) [M - H - CO]+,

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281 (85) [M - MeSO2]+, 265 (44) [M - MeSO3]+, 249 (100) [M - MeSO3 - CH2 (cyclopropyl) - 2H (cyclopropyl)]+. 1.2. 4-(Bromomethyl)-11-cyclopropyl-5,11-dihydro-6H-dipyrido-[3,2-b:2′,3′-e][1,4]diazepin-6-one (12-Bromo-NVP, 9). Potassium bromide (568 mg, 4.8 mmol, 2.8 equiv) was added to a solution of 8 (617 mg, 1.7 mmol) in N,N-dimethylformamide (DMF) (100 mL). The solution was stirred at room temperature overnight. The solvent was removed under reduced pressure by coevaporation with toluene, and the resulting solid was redissolved in ethyl acetate and extracted with water. The organic phase was dried over anhydrous sodium sulfate to afford a yellow solid (338 mg, 57%); mp 243-245 °C (ethyl acetate). IR (KBr): 1652 (CdO). 1H NMR [dimethylsulfoxide (DMSO)d6]: δ 10.2 (1H, s, N5H), 8.52-8.51 (1H, m, H9), 8.17 (1H, d, J ) 4.7, H2), 8.03-8.02 (1H, m, H7), 7.23 (1H, d, J ) 4.7, H3), 7.19 (1H, dd, Jortho ) 7.5, Jortho′ ) 4.9, H8), 5.08 (1H, d, Jgem ) 10.6, H12), 4.59 (1H, d, Jgem ) 10.6, H12′), 3.73 (1H, bs, H13), 0.98-0.86 (2H, m, H14 + H15), 0.41-0.30 (2H, m, H14 + H15). 13C NMR (DMSO-d6): δ 166.9 (C6), 159.6 (C10a), 154.7 (C11a), 151.5 (C9), 144.0 (C2), 139.3 (C7), 138.9 (C4), 124.3 (C4a), 121.2 (C8), 120.4 (C6a), 119.3 (C3), 29.3 (C13), 28.4 (C12), 8.8 (C14/C15), 8.5 (C14/C15). MS (EI) m/z 346 (20) [81Br-M]+, 344 (19) [79Br-M]+, 265 (100) [M - Br]+. 2. General Procedure for the Reaction of 12-MesyloxyNVP (8) with 2′-Deoxynucleosides. A solution of 8 (1.1 equiv, 50 mg, 140 µmol) in THF (1.5 mL) was added to a 108 mM solution of the deoxynucleoside (1.2 mL, 130 µmol) in DMF/ H2O (2:1). The reaction mixture was incubated at 37 °C for periods ranging from 2 to 7 days and subsequently purified by HPLC. 2.1. Reaction with 2′-Deoxyguanosine (dG). Following an incubation period of 2.5 days, two adducts were isolated by semipreparative HPLC. 2.1.1. 12-(Guanin-N7-yl)nevirapine (N7-NVP-Gua, 10). Compound 10 was obtained in 9% yield (4.9 mg); tR, 16.1 min. UV, λmax (acetonitrile/0.1% formic acid): 212, 282 nm. 1H NMR (DMSO-d6): δ 10.50 (2H, bs, NVP-N5H + Gua-N1H), 8.53 (1H, dd, Jortho ) 4.5, Jmeta ) 2.0, NVP-H9), 8.12 (1H, d, J ) 5.0, NVP-H2), 8.10 (1H, s, Gua-H8), 8.03 (1H, dd, Jortho ) 7.5, Jmeta ) 2.0, NVP-H7), 7.23 (1H, dd, Jortho ) 7.5, Jortho′ ) 5.0, NVP-H8), 6.61 (1H, d, J ) 5.0, NVP-H3), 6.17 (2H, bs, Gua-N2H2), 5.60 (1H, d, Jgem ) 16.5, NVP-H12), 5.51 (1H, d, Jgem ) 16.5, NVP-H12′), 3.66-3.61 (1H, m, NVP-H13), 0.91-0.87 (2H, m, NVP-H14 + NVP-H15), 0.44-0.32 (2H, m, NVP-H14 + NVP-H15). 13C NMR (DMSO-d6): δ 167.1 (NVP-C6), 158.8 (Gua-C4), 158.0 (NVP-C10a), 154.7 (NVPC11a + Gua-C2/C6), 152.9 (Gua-C2/C6), 151.4 (NVP-C9), 144.1 (NVP-C2), 143.8 (Gua-C8), 140.0 (NVP-C7), 139.9 (NVP-C4), 123.1 (NVP-C4a), 120.9 (NVP-C6a), 119.5 (NVPC8), 118.2 (NVP-C3), 108.0 (Gua-C5), 44.9 (NVP-C12), 29.4 (NVP-C13), 8.7 (NVP-C14/C15), 8.5 (NVP-C14/C15). LC/ESIMS m/z 416 [MH]+, 265 [MH - Gua]+, 208 [MH2]2+, 152 [GuaH]+. 2.1.2. 12-(Guanin-N9-yl)nevirapine (N9-NVP-Gua, 11). Compound 11 was obtained in 5% yield (2.5 mg); tR, 17.3 min. UV, λmax (acetonitrile/0.1% formic acid): 242, 272 nm. 1H NMR (DMSO-d6): δ 8.52 (1H, dd, Jortho ) 4.9, Jmeta ) 2.0, NVPH9), 8.11 (1H, d, J ) 5.0, NVP-H2), 8.04 (1H, dd, Jortho ) 7.7, Jmeta ) 2.0, NVP-H7), 7.74 (1H, s, Gua-H8), 7.21 (1H, dd, Jortho ) 7.6, Jortho′ ) 4.5, NVP-H8), 6.50 (1H, d, J ) 5.0, NVP-H3), 6.16 (2H, bs, Gua-N2H2), 5.40 (1H, d, Jgem ) 17.0, NVP-H12), 5.31 (1H, d, Jgem ) 17.0, NVP-H12′), 3.65-3.62 (1H, m, NVP-H13), 0.92-0.86 (2H, m, NVP-H14 + NVPH15), 0.43-0.32 (2H, m, NVP-H14 + NVP-H15). 13C NMR

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(DMSO-d6): δ 167.3 (NVP-C6), 158.7 (NVP-C10a), 157.0 (Gua-C2/C6), 154.5 (Gua-C2/C6), 154.1 (NVP-C11a), 151.6 (NVP-C9), 151.4 (Gua-C4), 144.3 (NVP-C2), 140.1 (NVP-C7), 139.2 (NVP-C4), 137.3 (Gua-C8), 123.7 (NVP-C4a), 120.9 (NVP-C6a), 119.6 (NVP-C8), 118.3 (NVP-C3), 116.5 (GuaC5), 42.0 (NVP-C12), 29.3 (NVP-C13), 8.8 (NVP-C14/C15), 8.5 (NVPC14/C15). LC/ESI-MS m/z 416 [MH]+, 265 [MH Gua]+, 208 [MH2]2+, 152 [GuaH]+. 2.2. Reaction with 2′-Deoxyadenosine (dA). After an incubation period of 1 week, the reaction mixture was purified by semipreparative HPLC using a 30 min linear gradient of 15-65% acetonitrile in 100 mM ammonium acetate (pH 5.7), followed by a 2 min linear gradient to 100% acetonitrile, and a 16 min isocratic elution with acetonitrile. Three adducts were isolated. 2.2.1. 12-(Deoxyadenosin-N1-yl)nevirapine (N1-NVP-dA, 12). Compound 12 was obtained in 3% yield (2.0 mg); tR, 12.3 min. UV, λmax (acetonitrile/0.1% formic acid): 214, 258 nm. 1H NMR (DMSO-d6): δ 8.61 (1H, s, dA-H2), 8.53-8.51 (1H, m, NVPH9), 8.21 (1H, apparent d, apparent J ) 3.0, dA-H8), 8.16 (1H, d, J ) 4.9, NVP-H2), 8.05-8.04 (1H, m, NVP-H7), 7.31 (1H, d, J ) 4.9, NVP-H3), 7.29 (1H, s, dA-N6H), 7.22 (1H, dd, Jortho ) 7.4, Jortho′ ) 4.9, NVP-H8), 6.24 (1H, t, J ) 6.7, dA-H1′), 5.31 (1H, bs, dA-5′OH/3′OH), 5.22 (1H, apparent dd, Jgem ) 14.7, apparent J′ ) 2.7, NVP-H12), 5.09 (1H, d, Jgem ) 14.6, NVP-H12′), 4.94 (1H, bs, dA-5′OH/3′OH), 4.37 (1H, bs, dAH3′), 3.85 (1H, bs, dA-H4′), 3.62-3.49 (3H, m, dA-H5′,5′′ + NVP-H13), 2.66-2.57 (1H, m, dA-H2′), 2.28-2.25 (1H, m, dA-H2′′), 0.84-0.81 (2H, m, NVP-H14 + NVP-H15), 0.38-0.27 (2H, m, NVP-H14 + NVP-H15). 13C NMR (DMSO-d6): δ 166.5 (NVP-C6), 159.4 (NVP-C10a), 154.7 (NVP-C11a), 153.8 (dA-C6), 151.3 (NVP-C9), 148.1 (dA-C2), 143.5 (NVP-C2), 141.8 (dA-C4), 139.9 (NVP-C7), 138.3 (dA-C8), 137.1 (NVPC4), 125.0 (NVP-C4a), 122.1 (dA-C5), 121.3 (NVP-C6a), 120.5 (NVP-C3), 119.5 (NVP-C8), 87.9 (dA-C4′), 83.6 (dA-C1′), 70.5 (dA-C3′), 61.5 (dA-C5′), 46.0 (NVP-C12), ca. 39.5 (dA-C2′, obscured by the solvent resonance), 29.4 (NVP-C13), 8.7 (NVPC14/C15), 8.5 (NVP-C14/C15). LC/ESI-MS m/z 538 [M + Na]+, 516 [MH]+, 400 [BH + H]+, 265 [NVP]+. 2.2.2. 12-(Adenin-N3-yl)nevirapine(N3-NVP-Ade,13).Compound 13 was obtained in 2% yield (1.0 mg); tR, 17.0 min. UV, λmax (acetonitrile/0.1% formic acid): 212, 276 nm. 1H NMR (DMSO-d6): δ 11.97 (1H, bs, NVP-N5H), 8.75 (1H, s, AdeH2), 8.53 (1H, dd, Jortho ) 4.8, Jmeta ) 2.1, NVP-H9), 8.16 (1H, d, J ) 5.1, NVP-H2), 8.06 (1H, dd, Jortho ) 7.5, Jmeta ) 2.1, NVP-H7), 7.81 (1H, s, Ade-H8), 7.26-7.23 (2H, m, NVPH3 + NVP-H8), 5.47 (2H, s, NVP-H12,H12′), 3.63-3.58 (1H, m, NVP-H13), 0.91-0.79 (2H, m, NVP-H14 + NVP-H15), 0.42-0.26 (2H, m, NVP-H14 + NVP-H15). LC/ESI-MS m/z 400 [MH]+, 265 [MH - Ade]+, 200 [MH2]2+, 136 [AdeH]+. 2.2.3. 12-(Adenin-N9-yl)nevirapine (N9-NVP-Ade, 14). Compound 14 was obtained in 1% yield (0.6 mg); tR, 17.4 min. UV, λmax (acetonitrile/0.1% formic acid): 214, 258, 295 nm. 1H NMR (DMSO-d6): δ 10.60 (1H, bs, NVP-N5H), 8.54 (1H, dd, Jortho ) 4.7, Jmeta ) 1.5, NVP-H9), 8.25 (1H, s, Ade-H8), 8.13-8.12 (2H, m, NVP-H2 + Ade-H2), 8.05 (1H, dd, Jortho ) 7.5, Jmeta ) 1.5, NVP-H7), 7.33 (2H, bs, Ade-N6H2), 7.24 (1H, dd, Jortho ) 7.5, Jortho′ ) 4.7, NVP-H8), 6.68 (1H, d, J ) 5.0, NVP-H3), 5.52 (2H, s, NVP-H12,H12′), 3.64-3.62 (1H, m, NVP-H13), 0.92-0.85 (2H, m, NVP-H14 + NVP-H15), 0.42-0.34 (2H, m, NVP-H14 + NVP-H15). LC/ESI-MS m/z 400 [MH]+, 265 [MH - Ade]+, 200 [MH2]2+, 136 [AdeH]+.

Antunes et al.

2.3. Reaction with 2′-Deoxycytidine (dC). After an incubation period of 1 week, one adduct was isolated by semipreparative HPLC. 2.3.1. 12-(Deoxycytidin-N3-yl)nevirapine (N3-NVP-dC, 15). Compound 15 was obtained in 2% yield (1.0 mg); tR, 15.6 min. UV, λmax (acetonitrile/0.1% formic acid): 216, 284 nm. 1H NMR (DMSO-d6): δ 11.90 (1H, bs, NVP-N5H), 8.51 (1H, d, Jortho ) 4.9, NVP-H9), 8.01 (1H, d, J ) 7.5, NVP-H2), 8.01 (1H, d, J ) 7.2, NVP-H7), 7.40 (1H, bs, dC-H6), 7.26-7.24 (1H, m, NVP-H3), 7.20 (1H, dd, Jortho ) 7.2, Jortho′ ) 4.9, NVP-H8), 6.22-6.19 (1H, m, dC-H1′), 5.88 (1H, d, J ) 8.0, dC-H5), 5.28 (1H, dd, Jgem ) 14.2, J′ ) 3.4, NVP-H12), 5.23 (1H, d, J ) 3.4, dC-3′-OH), 4.96 (1H, t, J ) 4.7, dC-5′OH), 4.78 (1H, d, Jgem ) 14.2, NVP-H12′), 4.21 (1H, bs, dC-H3′), 3.76 (1H, bs, dC-H4′), 3.61-3.53 (3H, m, dC-H5′,5′′ + NVP-H13), 2.31-2.02 (2H, m, dC-H2′,2′′), 0.94-0.85 (2H, m, NVP-H14 + NVPH15), 0.37-0.27 (2H, m, NVP-H14 + NVP-H15). 13C NMR (DMSO-d6): δ 167.1 (NVP-C6), 160.0 (NVP-C10a), 157.7 (dCC4), 154.9 (NVP-C11a), 151.8 (NVP-C9), 151.2 (dC-C2), 144.1 (NVP-C2), 140.4 (NVP-C7), 138.7 (NVP-C4), 133.1 (dC-C6), 125.5 (NVP-C4a), 121.7 (NVP-C3), 121.4 (NVP-C6a), 119.9 (NVP-C8), 101.1 (dC-C5), 87.8 (dC-C4′), 85.4 (dC-C1′), 70.8 (dC-C3′), 61.8 (dC-C5′), ca. 39.5 (NVP-C12 + dC-C2′, obscured by the solvent resonance), 29.9 (NVP-C13), 9.3 (NVPC14/C15), 9.1 (NVP-C14/C15). LC/ESI-MS m/z 492 [MH]+, 376 [BH + H]+, 265 [NVP]+, 188 [BH + 2H]2+, 117 [2′-deoxyribosyl (dR)]+, 112 [CytH]+. 3. General Procedure for the Palladium-Mediated Coupling of 12-Bromo-NVP (9) with 3′,5′-O-Bis(tert-butyldimethylsilyl)2′-deoxynucleosides. A suspension of 9 (20 mg, 58 µmol) in toluene (800 µL) was prepared in a screw-capped vial. Tris(dibenzylideneacetone)dipalladium(0) [Pd2(dba)3] (5 mg, 5.8 µmol) was added, and the mixture was stirred at room temperature for ca. 5 min. The 3′,5′-bis-O-(tert-butyldimethylsilyl)-2′-deoxynucleoside (87 µmol) and cesium carbonate (28 mg, 87 µmol) were then added. The resulting mixture was stirred at 95 °C until complete consumption of starting material was confirmed by HPLC (0.5-1 h). Tetrabutylammonium fluoride (78 mg, 298 µmol) was then added to cleave the silylated protection groups, and the mixture was incubated overnight at 38 °C. After centrifugation, the supernatant was decanted, and the residue was dissolved in methanol (1.5 mL) and purified by HPLC. 3.1. Reaction with 3′,5′-Bis-O-(tert-butyldimethylsilyl)-dG. Two major adducts were isolated, following reaction for 30 min at 95 °C, desilylation, and purification by semipreparative HPLC. 3.1.1. 12-(Deoxyguanosin-O6-yl)nevirapine (O6-NVP-dG, 16). Compound 16 was obtained in 44% yield (13.6 mg); tR, 19.9 min. UV, λmax (acetonitrile/0.1% formic acid): 214, 240, 288 nm. 1H NMR (DMSO-d6): δ 10.74 (1H, bs, NVP-N5H), 8.54 (1H, dd, Jortho ) 5.0, Jmeta ) 2.0, NVP-H9), 8.25 (1H, d, J ) 5.0, NVP-H2), 8.14 (1H, s, dG-H8), 8.05 (1H, dd, Jortho ) 8.0, Jmeta ) 2.0, NVP-H7), 7.34 (1H, d, J ) 5.0, NVP-H3), 7.23 (1H, dd, Jortho ) 8.0, Jortho′ ) 5.0, NVP-H8), 6.59 (2H, s, dGN2H2), 6.22 (1H, dd, J ) 7.5, J′ ) 6.5, dG-H1′), 5.67-5.59 (2H, m, NVP-H12,H12′), 5.30 (1H, bs, dG-5′OH/3′OH), 4.98 (1H, bs, dG-5′OH/3′OH), 4.37-4.35 (1H, m, dG-H3′), 3.83 (1H, dd, J ) 7.0, J′ ) 4.5, dG-H4′), 3.66-3.49 (3H, m, NVP-H13 + dG-H5′,5′′), 2.60-2.54 (1H, m, dG-H2′), 2.24-2.20 (1H, m, dG-H2′′), 0.92-0.86 (2H, m, NVP-H14 + NVP-H15), 0.42-0.33 (2H, m, NVP-H14 + NVP-H15). 13C NMR (DMSOd6): δ 167.3 (NVP-C6), 159.6 (dG-C2/C6 or NVP-C10a), 159.5 (dG-C2/C6 or NVP-C10a), 159.4 (dG-C2/C6 or NVP-C10a),

12-HydroxyneVirapine DNA Adducts

154.4 (NVP-C11a), 154.3 (dG-C4), 151.6 (NVP-C9), 144.0 (NVP-C2), 140.0 (NVP-C7), 138.4 (dG-C8), 138.2 (NVP-C4), 123.8 (NVP-C4a), 122.0 (NVP-C6a), 120.2 (NVP-C3), 118.6 (NVP-C8), 113.8 (dG-C5), 87.8 (dG-C4′), 82.9 (dG-C1′), 70.7 (dG-C3′), 62.8 (NVP-C12), 61.9 (dG-C5′), ca. 39.5 (dG-C2′, obscured by the solvent resonance), 29.4 (NVP-C13), 8.8 (NVPC14/C15), 8.6 (NVP-C14/C15). LC/ESI-MS m/z 554 [M + Na]+, 532 [MH]+, 416 [BH + H]+, 265 [NVP]+. 3.1.2. 12-(Deoxyguanosin-N1-yl)nevirapine (N1-NVP-dG, 17). Compound 17 was obtained in 2% yield (0.5 mg); tR, 16.3 min. UV, λmax (acetonitrile/0.1% formic acid): 248, 282 nm. 1H NMR (CD3OD): δ 8.52 (1H, dd, Jortho ) 4.8, Jmeta ) 1.7, NVP-H9), 8.17 (1H, dd, Jortho ) 7.6, Jmeta ) 1.7, NVP-H7), 8.14 (1H, d, J ) 5.1, NVP-H2), 8.06 (1H, s, dG-H8), 7.25 (1H, dd, Jortho ) 7.6, Jortho′ ) 4.8, NVP-H8), 6.70 (1H, d, J ) 5.0, NVP-H3), 6.33 (1H, t, J ) 7.0, dG-H1′), 5.68 (1H, m, NVP-H12), 5.10 (1H, d, Jgem ) 17.5, NVP-H12′), 4.57-4.56 (1H, m, dG-H3′), 4.03-4.02 (1H, m, dG-H4′), 3.83-3.75 (3H, m, NVP-H13 + dG-H5′,5′′), 2.41-2.39 (1H, m, dG-H2′), 2.06-2.04 (1H, m, dG-H2′′), 0.90-0.88 (2H, m, NVP-H14 + NVP-H15), 0.54-0.46 (2H, m, NVP-H14 + NVP-H15). 1H NMR (DMSO-d6): δ 8.52 (1H, dd, Jortho ) 4.8, Jmeta ) 1.7, NVP-H9), 8.08-8.04 (2H, m, NVP-H7 + NVP-H2), 8.00 (1H, s, dG-H8), 7.22 (1H, dd, Jortho ) 7.5, Jortho′ ) 5.2, NVP-H8), 7.16 (2H, bs, dG-N2H2), 6.41 (1H, bs, NVP-H3), 6.16 (1H, t, J ) 7.2, dG-H1′), 5.65 (1H, bd, Jgem ) 15.7, NVP-H12), 5.33 (1H, bs, dG-5′OH or 3′OH), 4.98 (1H, bs, NVP-H12′), 4.37-4.36 (1H, m, dG-H3′), 3.83-3.82 (1H, m, dG-H4′), 3.65-3.62 (1H, m, NVP-H13), 3.58-3.50 (2H, m, dG-H5′,5′′), 2.56-2.54 (1H, m, dG-H2′), 2.24-2.21 (1H, m, dG-H2′′), 0.90-0.87 (2H, m, NVP-H14 + NVP-H15), 0.42-0.34 (2H, m, NVP-H14 + NVP-H15). LC/ ESI-MS m/z 554 [M + Na]+, 532 [MH]+, 416 [BH + H]+, 287 [MH2 + CH3CN]2+, 267 [dG]+, 117 [dR]+. 3.2. Reaction with 3′,5′-Bis-O-(tert-butyldimethylsilyl)-dA. One major adduct was isolated, following reaction for 1 h at 95 °C, desilylation, and purification by semipreparative HPLC. 3.2.1. N1-NVP-dA (12). Compound 12 was obtained in 31% yield (9.2 mg). For spectroscopic data, see section 2.2.1. 3.3. Reaction with 3′,5′-Bis-O-(tert-butyldimethylsilyl)-dC. One major adduct was isolated, following reaction for 1 h at 95 °C, desilylation, and purification by semipreparative HPLC. 3.3.1. N3-NVP-dC (15). Compound 15 was obtained in 40% yield (11.4 mg). For spectroscopic data, see section 2.3.1. 4. Reaction with DNA. A solution of 8 (10 mg, 28 µmol) in THF (500 µL) was added to a solution of salmon testis DNA (ca. 2.5 mg/mL) in 8 mL of 5 mM Bis-Tris and 0.1 mM EDTA (pH 7.1). The mixture was incubated at 37 °C over one weekend. Following removal of the nonbonded materials by extraction with 2 × 1 vol of ethyl acetate, a second solution of 8 (10 mg, 28 µmol) in THF (500 µL) was added, and the mixture was reincubated overnight at 37 °C. The nonbonded materials were removed as indicated above, and the DNA was precipitated by addition of 5 M NaCl (0.1 vol) and ice-cold ethanol (3 vol). After centrifugation, the DNA pellet was washed with ice-cold 70% ethanol (2 × 1 vol) and redissolved in 4 mL of 5 mM Bis-Tris and 0.1 mM EDTA (pH 7.1). An aliquot of the modified DNA solution was hydrolyzed enzymatically to 2′-deoxynucleosides by treatment with DNase I, followed by alkaline phosphatase and phosphodiesterase (44). The adducts were then partitioned into n-butanol that had been presaturated with water, and the n-butanol extracts were combined and back-extracted with water presaturated with n-butanol. After the n-butanol was evaporated, the residue was redissolved in methanol for analysis by LC/MS. Another aliquot of the modified DNA solution was

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subjected to neutral thermal hydrolysis, for release of the depurinating adducts, essentially as described in Gamboa da Costa et al. (45). Briefly, the DNA solution was heated at 100 °C for 10 min; the sample was then cooled to room temperature and eluted through a prewashed Amicon Microcon 3 kDa molecular weight cutoff centrifugal size exclusion column (Millipore Co., Bedford, MA), by centrifugation at 12000g for 45 min at room temperature. The adducts were subsequently analyzed by LC/MS.

Results and Discussion Reactions of 12-Mesyloxy-NVP with 2′-Deoxynucleosides. The synthesis of 12-hydroxy-NVP (5) involved the thermodynamically controlled generation of the NVP anion at C12, followed by a simple direct oxidation with MoOPH (38). Attempts to convert this NVP metabolite into the metabolically plausible reactive electrophile 12-sulfoxy-NVP (7; Scheme 1), by reacting 12-hydroxy-NVP with the SO3·pyridine complex (46), were unsuccessful, presumably due to secondary reactions with the pyridine rings of the starting material. As an alternative, we tested the acetylation of 12-hydroxy-NVP; however, when 12-acetoxy-NVP was reacted with nucleophiles, acetyl transfer was consistently favored over nucleophilic attack on the NVP moiety. By contrast, reactions with 2′-deoxynucleosides were successfully conducted with the model electrophile 12-mesyloxyNVP (8; Scheme 1), which was used as a surrogate for 12sulfoxy-NVP (7) due to its higher stability and ease of synthesis, combined with an expected similar pattern of reactivity toward (bio)nucleophiles. The synthesis of 8 was achieved by reaction of 12-hydroxy-NVP with methanesulfonyl chloride in anhydrous THF, in the presence of triethylamine. To investigate the reactivity of 8 toward 2′-deoxynucleosides (dG, dA, and dC), reactions were conducted at 37 °C in THF/DMF/water. The overall yields followed the order dG > dA > dC, and in each instance, DNA adducts were isolated by preparative reversedphase HPLC and fully characterized by 1H and 13C NMR and mass spectrometry. All of the isolated adducts consistently involved binding through the C12 position of NVP. This was unequivocally determined from their 1H NMR spectra, which showed all of the NVP protons and where the resonance of the two H12 protons appeared either as a singlet, a multiplet, or a set of two doublets (each accounting for one proton) with a geminal coupling constant of 14-17 Hz as clear evidence of magnetic anisotropy. Adducts with dG. The reaction of 8 with dG led to the formation of three adducts, involving binding through the endocyclic base nitrogens, with subsequent loss of the sugar moiety. In all instances, evidence for the formation of depurinating adducts was achieved both by LC/ESI-MS analysis, which indicated protonated molecules at m/z 416, corresponding to adducts of NVP with Gua, and by NMR, where all proton and carbon signals from the dR moiety were absent (cf. Materials and Methods). The two major adducts, N7-NVP-Gua (10; Scheme 2) and N9-NVP-Gua (11; Scheme 2), which were isolated in relatively high yields (9 and 5%, respectively), had distinct UV spectra (Figure 1) but displayed very similar 1H NMR and 13C NMR spectral profiles. The most noticeable differences in the proton spectra were upfield shifts of the NVP H3 (0.11 ppm) and H12 (0.20 ppm) and the Gua H8 (0.36 ppm) in 11, as compared to 10 (Table 1). Likewise, as compared to 10, the carbon spectrum of 11 showed upfield shifts of the NVP C12 (2.9 ppm) and the Gua C4 (7.4 ppm) and C8 (6.5 ppm), as opposed to a downfield shift of the Gua C5 (8.5 ppm) (Table

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Scheme 2. Structures of the Depurinating NVP Adducts, N7-NVP-Gua (10) and N9-NVP-Gua (11), Characterized from the Reaction of 12-Mesyloxy-NVP with dGa

a The arrows represent the HMBC correlations between the NVP H12 protons and the specific Gua carbons that were decisive to assign the binding sites. For simplicity, arrows are drawn from only one of the geminal NVP protons, although both exhibited correlations to the same Gua carbons.

Figure 1. UV spectra of the dG-derived NVP adducts characterized from reaction of either 12-mesyloxy-NVP with dG (N7-NVP-Gua and N9NVP-Gua) or 12-bromo-NVP with 3′,5′-O-bis(tert-butyldimethylsilyl)-dG (O6-NVP-dG and N1-NVP-dG). The spectra were obtained online, by HPLC with diode array detection, in acetonitrile/0.1% aqueous formic acid.

Table 1. 1H and N7-NVP-Gua (10) N9-NVP-Gua (11)

13

C NMR Resonances for Which Significant Differences Were Observed between Adducts 10 and 11a

NVP-H12

NVP-H12′

NVP-H3

Gua-H8

NVP-C12

Gua-C4

Gua-C5

Gua-C8

5.60 5.40

5.51 5.31

6.61 6.50

8.10 7.74

44.9 42.0

158.8 151.4

108.0 116.5

143.8 137.3

a Chemical shifts are in ppm, downfield from tetramethylsilane. Carbon assignments were based upon the correlations detected in the HMQC and HMBC spectra, recorded in DMSO-d6 at room temperature.

1). Taken together, these data suggested a closer proximity of the NVP binding region to the shielding zone of the purine ring in 11 and a decreased proximity of the NVP ring system to the Gua C5 in the same adduct. Conclusive evidence for the structural assignment of 10 and 11 as regioisomers through the nitrogens of the guanine imidazole ring stemmed from the observation of heteronuclear three-bond (HMBC) correlations (Scheme 2) between both the NVP H12 geminal protons (two sets of doublets at 5.60 and 5.51 ppm in 10 and 5.40 and 5.31 ppm in 11) and the Gua C8 carbon (at 143.8 in 10 and 137.3 ppm in 11). Furthermore, the structures of the two adducts were discriminated on the basis of additional HMBC correlations (Scheme 2) between the NVP H12 protons and specific quaternary carbons in Gua. Thus, a correlation with the most upfield Gua carbon (C5 at 108.0 ppm) was observed in adduct 10, whereas adduct 11 displayed a correlation with the Gua C4 (151.4 ppm). Such interactions imply the establishment of connectivities through NVP-C12/Gua-N7 in 10 and NVP-C12/Gua-N9 in 11, which substantiated the characterization of adduct 10 as N7-NVP-Gua and adduct 11 as N9-NVPGua. While the formation of N7-substituted dG adducts from a wide variety of both small and bulky electrophiles, followed by depurination, is a well-documented event in vitro and in vivo (47), reaction at the less nucleophilic and more hindered N9 position is extremely unusual. For instance, although N7,N9-

disubstituted diastereomeric guanine adducts from the chloroprene metabolite, (1-chloroethenyl)oxirane, have been isolated from reactions in vitro with both dG and DNA (48), these adducts appear to have resulted from primary reaction at N7, followed by depurination and a second alkylation at N9 in the presence of a substantial molar excess of the electrophilic epoxide. The structure of the minor third adduct detected in the reaction of 8 with dG could not be assigned, due to the low amount of sample, combined with considerable complexity of the proton NMR spectrum. Because MS analysis (not shown) indicated that this minor product was also a guanine adduct, it is plausible that it involved substitution through the deoxyguanosine N3, followed by depurination. Reports of N3-Gua adducts are scarce, presumably because these adducts are usually formed in low abundance; for instance, the yield of 3-methyl-Gua was estimated to be