Amino acid adduct formation by the nevirapine metabolite, 12-Hydroxynevirapine- A Possible Factor in Nevirapine Toxicity

Article abstract of DOI:10.1021/tx900443z

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 transmission of the virus in developing countries. However, reports of severe NVP-induced hepatotoxicity and serious adverse cutaneous effects have raised concerns about its use. NVP metabolism involves oxidation of the 4-methyl substituent to 4-hydroxymethyl-NVP (12-hydroxy-NVP) and the formation of phenolic derivatives. Further metabolism, through either oxidation to quinoid derivatives or phase II esterification, may produce electrophilic derivatives capable of reacting with bionucleophiles to yield covalent adducts. These adducts could potentially be involved in the initiation of toxic responses. To gain insight into potentially reactive sites in proteins and prepare reliable and fully characterized NVP-amino acid adduct standards for subsequent assessment as biomarkers of NVP toxicity, we have used the model electrophile, 12-mesyloxy-NVP, as a synthetic surrogate for the NVP metabolite, 12-sulfoxy-NVP. Reactions of this model ester were conducted with glutathione and the nucleophilic amino acids arginine, cysteine, histidine, and tryptophan. Moreover, because adducts through the N-terminal valine of hemoglobin are convenient biomarkers of exposure to electrophilic toxicants, we also investigated the reaction with valine. We obtained very efficient (>80%) binding through the sulfur of both glutathione and N-acetylcysteine and moderate yields (10-14%) for binding through C2 of the indole ring of tryptophan and N1 of the imidazole ring of histidine. Reaction with arginine occurred through the α-amino group, possibly due to the high basicity of the guanidino group in the side chain. Reaction at the α-amino group of valine occurred to a significant extent (33%); the resulting adduct was converted to a thiohydantoin derivative, to obtain a standard useful for prospective biomonitoring studies. All adducts were characterized by a combination of ¹H and ¹³C NMR spectroscopy and mass spectrometry techniques. The NVP conjugates with glutathione and N-acetylcysteine identified in this work were previously reported to be formed in vivo, although the corresponding structures were not fully characterized. Our results support the validity of 12-mesyloxy-NVP as a surrogate for 12-sulfoxy-NVP and suggest that NVP metabolism to 12-hydroxy-NVP, and subsequent esterification, could potentially be a factor in NVP toxicity. They further imply that multiple sites in proteins may be targets for modification by 12-hydroxy-NVP-derived electrophiles in vivo. Additionally, we obtained reliable, fully characterized standards for the assessment of protein modification by NVP in vivo, which should help clarify the potential role of metabolism in NVP-induced toxicity.

Full text of DOI:10.1021/tx900443z

888
Chem. Res. Toxicol. 2010, 23, 888–899
Amino Acid Adduct Formation by the Nevirapine Metabolite,
12-HydroxynevirapinesA Possible Factor in Nevirapine Toxicity
Alexandra M. M. Antunes,*,† Ana L. A. Godinho,† Ineˆs L. Martins,† Gonc¸alo C. Justino,†
Frederick A. Beland,‡ and M. Matilde Marques*,†
Centro de Qu´ımica 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 December 14, 2009
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 transmission of the virus in
developing countries. However, reports of severe NVP-induced hepatotoxicity and serious adverse
cutaneous effects have raised concerns about its use. NVP metabolism involves oxidation of the 4-methyl
substituent to 4-hydroxymethyl-NVP (12-hydroxy-NVP) and the formation of phenolic derivatives. Further
metabolism, through either oxidation to quinoid derivatives or phase II esterification, may produce
electrophilic derivatives capable of reacting with bionucleophiles to yield covalent adducts. These adducts
could potentially be involved in the initiation of toxic responses. To gain insight into potentially reactive
sites in proteins and prepare reliable and fully characterized NVP-amino acid adduct standards for
subsequent assessment as biomarkers of NVP toxicity, we have used the model electrophile, 12-mesyloxy-
NVP, as a synthetic surrogate for the NVP metabolite, 12-sulfoxy-NVP. Reactions of this model ester
were conducted with glutathione and the nucleophilic amino acids arginine, cysteine, histidine, and
tryptophan. Moreover, because adducts through the N-terminal valine of hemoglobin are convenient
biomarkers of exposure to electrophilic toxicants, we also investigated the reaction with valine. We obtained
very efficient (>80%) binding through the sulfur of both glutathione and N-acetylcysteine and moderate
yields (10-14%) for binding through C2 of the indole ring of tryptophan and N1 of the imidazole ring
of histidine. Reaction with arginine occurred through the R-amino group, possibly due to the high basicity
of the guanidino group in the side chain. Reaction at the R-amino group of valine occurred to a significant
extent (33%); the resulting adduct was converted to a thiohydantoin derivative, to obtain a standard
useful for prospective biomonitoring studies. All adducts were characterized by a combination of 1H and
13C NMR spectroscopy and mass spectrometry techniques. The NVP conjugates with glutathione and
N-acetylcysteine identified in this work were previously reported to be formed in vivo, although the
corresponding structures were not fully characterized. Our results support the validity of 12-mesyloxy-
NVP as a surrogate for 12-sulfoxy-NVP and suggest that NVP metabolism to 12-hydroxy-NVP, and
subsequent esterification, could potentially be a factor in NVP toxicity. They further imply that multiple
sites in proteins may be targets for modification by 12-hydroxy-NVP-derived electrophiles in vivo.
Additionally, we obtained reliable, fully characterized standards for the assessment of protein modification
by NVP in vivo, which should help clarify the potential role of metabolism in NVP-induced toxicity.
and Drug Administration for use in combination therapy of
human immunodeficiency virus (HIV)-1 infection (1). Cur-
rently, NVP is still one of the most prescribed antiretroviral
drugs, particularly in developing countries, both as a single-
dose prophylaxis to prevent mother-to-child HIV transmission
and in combination therapy (2-5). Despite its clinical
efficacy, NVP administration is associated with a variety of
toxic responses, with individual susceptibilities to the adverse
effects differing among patients (6-8). As such, the sug-
gestion (9) that extended-dose daily NVP regimens may be
adequate to decrease the risk of HIV transmission to breastfed
infants in low-resource settings has met criticism, partly on
account of toxicity considerations (10). Among the toxi-
cities associated with NVP use, the most severe is hepato-
toxicity, which can be fatal in some instances. The most
commonly reported side effect is a skin rash, which may be
life threatening and eventually leads to discontinuation of
the drug.
Introduction
The non-nucleoside reverse transcriptase inhibitor (NNRTI)1
nevirapine (11-cyclopropyl-5,11-dihydro-4-methyl-6H-dipy-
rido[3,2-b:2′,3′-e][1,4]diazepin-6-one, NVP, 1; Scheme 1)
was the first member of this class approved by the U.S. Food
* To whom correspondence should be addressed. (A.M.M.A.) Tel:
351-21-8419388. Fax: 351-21-8464457. E-mail: alexandra.antunes@
ist.utl.pt. (M.M.M.) Tel: 351-21-8419200. Fax: 351-21-8464457. E-mail:
matilde.marques@ist.utl.pt.
† Universidade Te´cnica de Lisboa.
‡ National Center for Toxicological Research.
1 Abbreviations: Boc, tert-butoxycarbonyl; t-Bu, tert-butyl; DMSO,
dimethyl sulfoxide; ESI, electrospray ionization; GSH, glutathione; HIV,
human immunodeficiency virus; HMBC, heteronuclear multiple bond
correlation; HMQC, heteronuclear multiple quantum coherence; HSQC,
heteronuclear single quantum coherence; Imid, imidazole; Ind, indole; Me,
methyl; MS/MS, tandem mass spectrometry; NNRTI, non-nucleoside reverse
transcriptase inhibitor; NOESY, two-dimensional nuclear Overhauser effect
spectroscopy; NVP, nevirapine; Ph, phenyl; TOCSY, total correlation
spectroscopy; tR, retention time.
10.1021/tx900443z  2010 American Chemical Society
Published on Web 04/14/2010

12-HydroxyneVirapine Amino Acid Adducts
Chem. Res. Toxicol., Vol. 23, No. 5, 2010 889
Scheme 1. Structures of NVP (1), NVP Metabolites (2-7),
could generate reactive electrophiles potentially involved in the
initiation of toxic events. Support for NVP sulfation in vivo
has been provided by Chen et al. (21), who detected 12-sulfoxy-
NVP (7) by LC-MS in urine and bile samples from female
Brown Norway rats administered the parent drug. This rat strain
and sex is particularly sensitive to an idiosyncratic NVP-induced
skin rash that resembles the rash occurring in humans (22, 23).
While both NVP and 5 induce the rash (21, 24), the lack of
rash induction by 12-trideuteromethyl-NVP supports the in-
volvement of 5 in the toxic response. The generation of a
quinone methide intermediate (8, Scheme 1), possibly from 7,
was proposed as a factor in this process (21), although it may
be argued that generation of a hapten adduct could stem from
direct reaction of 7 with bionucleophiles. Nonetheless, the mass
spectrometric detection of a putative 12-(glutathion-S-yl)-NVP
(10) conjugate following incubation of NVP with human liver
microsomes in the presence of glutathione (GSH) (25) suggests
that NVP activation to the same quinone methide (8, Scheme
1) may occur without phase II conjugation, either by dehydration
of 5 or by P450-induced dehydrogenation of NVP (Scheme 2).
The formation of the NVP-GSH conjugate occurred following
oxidation of NVP, primarily by P450 3A4 and to a lesser extent
by P450 2D6, 2C19, and 2A6; moreover, the oxidation of NVP
by P450 3A4 was found to cause a mechanism-based inactiva-
tion of the enzyme, which is consistent with the occurrence of
covalent binding (25). A more recent study, screening for stable
biomarkers of NVP bioactivation in rats and humans, has led
to the identification of two NVP mercapturates, the major one
through NVP-C3 and the minor one through NVP-C12, in the
urine of HIV-positive patients administered NVP as part of a
standard antiretroviral therapeutic regimen (26). The same
conjugates were detected in the bile and urine of NVP-treated
rats, as well as in incubations with rat hepatocytes and with rat
and human liver microsomes supplemented with GSH or
N-acetylcysteine (26).
Using 12-mesyloxy-NVP (9, Scheme 1) as a synthetic
surrogate for the 12-sulfoxy metabolite of NVP, we have
previously demonstrated that direct reaction of 9 with DNA in
vitro yields a variety of covalent nucleoside and depurinating
NVP-DNA adducts, consistently bound through the NVP C12
(27). Taking into consideration the potential role of covalent
binding to skin proteins in the hypersensitivity reactions
associated with NVP administration, we have further probed
the utility of the model electrophile 9 by investigating its reaction
with GSH and representative R-amino acids bearing nucleophilic
side chains (cysteine, tryptophan, histidine, and arginine), as
well as with the R-amino group of valine, whose adducted
hydantoin derivatives are frequently used in biomonitoring
studies as markers of binding to hemoglobin. We report herein
the synthesis and characterization of a series of NVP-amino
acid adduct standards and discuss their significance as potential
biomarkers of NVP toxicity. A preliminary report of this work
was presented at the 236th National Meeting of the American
Chemical Society and published as an abstract (28).
and Other NVP Derivatives (8 and 9) Mentioned in the Text
NVP is not mutagenic or clastogenic in a variety of assays,
including microbial and mammalian cell gene mutation tests
and cytogenetic assays (11). However, in mice administered 0,
50, 375, or 750 mg NVP/kg bw/day, there was an increased
incidence of hepatocellular adenomas and carcinomas at all
doses in males and at the two highest doses in females (11).
Likewise, in rats administered 0, 3.5, 17.5, or 35 mg NVP/kg
bw/day, there was an increased incidence of hepatocellular
adenomas at all doses in males and at the highest dose in females
(11). Although there have been no reports in the literature of
any correlation between NVP administration and the develop-
ment of cancer in humans, the recent suggestion (12) of a
possible association between the use of NNRTIs and the
occurrence of non-AIDS-defining cancers, predominantly
Hodgkin’s lymphoma, in HIV-positive individuals warrants
further investigation.
The reasons for the adverse effects of NVP administration
are currently not clear, although there is increasing evidence
that metabolic activation to reactive electrophiles capable of
reacting with bionucleophiles is likely to be involved in the
initiation of toxic responses. For instance, although the reactive
metabolites were not identified, Takakusa et al. (13) reported
covalent binding of [14C]NVP to rat and human liver microsomal
proteins in vitro and to liver tissue and plasma proteins from
male rats administered 20 mg NVP/kg bw as a single oral dose.
In addition to humans, NVP metabolism has been reported
in mice, rats, rabbits, dogs, cynomolgus monkeys, chimpanzees,
and baboons (14-18). In all species investigated, phase I
metabolism consistently involves oxidation to 2-, 3-, and
8-hydroxy-NVP, 4-hydroxymethyl-NVP (12-hydroxy-NVP, 5),
and 4-carboxy-NVP (2-6; Scheme 1). All of the hydroxyl
groups are prone to subsequent conjugation to the corresponding
glucuronides, which are excreted primarily in the urine. In
humans, the formation of 2-hydroxy-NVP is attributed to the
P450 3A subfamily, 3-hydroxy-NVP to P450 2B6, 8-hydroxy-
NVP to P450 3A4, 2B6, and 2D6, and 5 to P450 3A4 and
possibly 2D6 and 2C9 (16).
Materials and Methods
Caution: NVP and its deriVatiVes are potentially carcinogenic.
They should be handled with protectiVe clothing in a well-Ventilated
fume hood.
Chemicals. NVP was purchased from Cipla (Mumbai, India).
All other commercially available reagents were acquired from
Sigma-Aldrich Qu´ımica, S.A. (Madrid, Spain) or Sigma Chemical
Co. (St. Louis, MO) and used as received. (L)-R-Amino acids were
used in all instances. Compounds 5 and 9 were prepared as
Although comparatively less information is available about
other conjugation pathways, both acetylation and/or sulfonation
of the hydroxylated NVP metabolites (e.g., to 7, Scheme 1) are
plausible processes in the liver (19). Likewise, the ring-
hydroxylated metabolites may undergo oxidation to ortho-
quinone or semiquinone species (20). Any of these pathways

890 Chem. Res. Toxicol., Vol. 23, No. 5, 2010
Antunes et al.
Scheme 2. Hypothetic Pathways for in Vivo Generation of the Putative Metabolite, NVP Quinone Methide (8)a
a Compound 5 is a known NVP metabolite in all species investigated. Evidence for the formation of 7 (represented in the neutral form) in vivo has been
reported (21, 26); PAPS, 3′-phosphoadenosine-5′-phosphosulfate.
described in Antunes et al. (27). Whenever necessary, solvents were
purified by standard methods (29).
by a 90° shaped sinc pulse (with 1 cycle and size shape 1000). 13
C
resonances were 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. Reaction with GSH and N-Acetylcysteine. A
solution of 9 (8 mg, 22 µmol) in THF (200 µL) was added to a
solution of GSH or N-acetylcysteine (1.3 equiv, 29 µmol) in 1 mL
of 50 mM phosphate buffer (pH 7.4). The reaction mixtures were
incubated at room temperature for 2 h, and the products were
purified by semipreparative HPLC using a 30 min linear gradient
of 5-70% acetonitrile in 0.1% formic acid, followed by a 2 min
linear gradient to 100% acetonitrile and a 16 min isocratic elution
with acetonitrile.
Instrumentation. HPLC was conducted on an Ultimate 3000
Dionex system consisting of an LPG-3400A quaternary gradient
pump and a diode array spectrophotometric detector (Dionex Co.,
Sunnyvale, CA) and equipped with a Rheodyne model 8125 injector
(Rheodyne, Rohnert Park, 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. Semi-
preparative 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. The UV absorbance was monitored at 254 nm.
Mass spectra were recorded on either a ThermoFinnigan TSQ
Quantum Ultra LC/MS system or a Varian 500-MS LC Ion Trap
mass spectrometer, both operated in the electrospray ionization
(ESI) mode. 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 linear gradients
from 5 or 10% acetonitrile up to 95% acetonitrile in 0.1% aqueous
formic acid for durations between 15 and 40 min, depending on
the specific sample.
12-(Glutathion-S-yl)-NVP (10). Compound 10 was obtained
in 84% yield (10.6 mg); tR, 14 min. UV, λmax 292 nm. 1H NMR
[dimethyl sulfoxide (DMSO)-d6]: δ 8.62 (1H, bs, Cys-NH), 8.50
(1H, d, Jortho ) 4.4, NVP-H9), 8.14 (1H, d, Jortho ) 4.8, NVP-
H2), 8.04-8.02 (1H, m, NVP-H7), 7.89 (1H, bs, Gly-NH),
7.22-7.18 (2H, m, NVP-H8 + NVP-H3), 4.44-4.36 (1H, m,
Cys-H2), 4.21 (1H, d, Jgem ) 14.0, NVP-H12a), 4.17 (1H, d,
Jgem ) 14.0, NVP-H12a*), 3.77 (1H, d, Jgem ) 14.0, NVP-H12b),
3.75 (1H, d, Jgem ) 14.0, NVP-H12b*), 3.65-3.60 (1H, m, NVP-
H13), 3.44-3.41 (2H, m, Gly-H2), 3.31 (1H, t, J ) 6.0, Glu-
H2), 2.81-2.72 (1H, m, Cys-H3a), 2.61-2.57 (1H, m, Cys-
H3b), 2.32-2.29 (2H, m, Glu-H4), 1.92 (2H, bs, Glu-H3),
0.80-0.87 (2H, m, NVP-H14 + NVP-H15), 0.42-0.31 (2H,
m, NVP-H14 + NVP-H15). 13C NMR (DMSO-d6): δ 171.9
(Glu-CdO), 171.8 (Glu-CdO), 171.0 (Gly-CdO), 170.4 (Cys-
CdO), 170.2 (Cys-CdO*), 167.1 (NVP-C6), 159.8 (NVP-
C10a), 154.5 (NVP-C11a), 151.3 (NVP-C9), 143.7 (NVP-C2),
140.7 (NVP-C4), 140.6 (NVP-C4*), 140.0 (NVP-C7), 124.3
(NVP-C4a), 124.2 (NVP-C4a*), 121.3 (NVP-C8), 120.6 (NVP-
C6a), 119.4 (NVP-C3), 53.1 (Glu-C2), 52.4 (Cys-C2), 52.3
(Cys-C2*), 41.4 (Gly-C2), 33.3 (Cys-C3), 33.0 (Cys-C3*), 31.4
(Glu-C4), 30.7 (NVP-C12), 30.5 (NVP-C12*), 29.3 (NVP-C13),
26.8 (Glu-C3), 8.7 (NVP-C14/C15), 8.4 (NVP-C14/C15). MS
1H 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.
13C NMR spectra were recorded on the same instruments, 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.
Geminal protons are denoted with “a” and “b” subscripts, and
asterisks are used to indicate a second conformer; the proton
integrations listed below do not reflect the relative proportions of
each conformer. 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 bond correlation (HMBC) and heteronuclear multiple
quantum coherence (HMQC) or heteronuclear single quantum
coherence (HSQC)-total correlation spectroscopy (TOCSY) ex-
periments, 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, avail-
able from the Bruker library) with no decoupling during acquisition
and using gradient pulses for selection; excitation in 13C was ensured
m/z 572 [MH]+, 443 [MH - pyroglutamate]+, 287 [MH2]2+
.
12-(N-Acetylcystein-S-yl)-NVP (11). Compound 11 was ob-
tained in 82% yield (7.8 mg); tR, 16 min. UV, λmax 293 nm. 1H
NMR (acetone-d6): δ 8.49 (1H, d, Jortho ) 3.9, NVP-H9), 8.15
(1H, d, Jortho ) 3.6, NVP-H2), 8.08 (1H, d, Jortho ) 6.2, NVP-

12-HydroxyneVirapine Amino Acid Adducts
Chem. Res. Toxicol., Vol. 23, No. 5, 2010 891
(DMSO-d6): δ 10.06 (1H, s, NVP-N5H), 8.51 (1H, dd, Jortho
H7), 7.18-7.13 (2H, m, NVP-H8 + NVP-H3), 4.67 (1H, bs,
Cys-H2), 4.21-3.91 (2H, m, NVP-H12), 3.72 (1H, bs, NVP-
H13), 3.01-2.88 (2H, m, Cys-H3), 1.95 (3H, s, acetyl-CH3),
0.86 (2H, bs, NVP-H14 + NVP-H15), 0.39 (2H, m, NVP-H14
+ NVP-H15). 13C NMR (acetone-d6): δ ca. 172 (Cys-CdO),
170.4 (acetyl CdO), 167.0 (NVP-C6), 161.2 (NVP-C10a), 155.7
(NVP-C11a), 152.5 (NVP-C9), 144.9 (NVP-C2), 141.0 (NVP-
C7), 140.3 (NVP-C4), 140.1 (NVP-C4*), 125.7 (NVP-C4a),
125.6 (NVP-C4a*), 122.4 (NVP-C8), 121.5 (NVP-C6a), 119.9
(NVP-C3), 52.9 (Cys-C2), 34.5 (Cys-C3), 34.4 (Cys-C3*), 32.5
(NVP-C12), 32.3 (NVP-C12*), ca. 29.9 (NVP-C13, obscured
by the solvent resonance), 22.7 (acetyl CH3), 9.3 (NVP-C14/
C15), 9.1 (NVP-C14/C15). MS m/z 428 [MH]+, 386 [MH2 -
COCH3]+, 299 [MH2 - CH3CONHC(CH2)CO2H]+.
)
5.0, Jmeta ) 1.9, NVP-H9), 8.20 (1H, m, NVP-H2), 8.01 (1H,
d, Jortho ) 7.5, NVP-H7), 7.58 [1H, bs, imidazole (Imid)-H2],
7.56 (1H, bs, Imid-H2*), 7.32 (<1H, bs, His-NH), 7.31 (<1H,
bs, His-NH), 7.20 (1H, dd,, Jortho ) 7.5, Jortho′ ) 5.0, NVP-
H8), 7.07-7.04 (1H, m, NVP-H3), 6.84 (1H, m, Imid-H5), 6.79
(1H, m, Imid-H5*), 5.34-5.17 (2H, m, NVP-H12), 4.36-4.31
(1H, m, Hist-H2), 3.66-3.62 (1H, m, NVP-H13), 2.92-2.85
(2H, m, Hist-H3), 1.36 (9H, s, Boc-CH3), 0.92-0.85 (2H, m,
NVP-H14 + NVP-H15), 0.42-0.32 (2H, m, NVP-H14 + NVP-
H15). 13C NMR (DMSO-d6): δ 172.3 (Hist-CdO), 167.5 (NVP-
C6), 160.1 (NVP-C10a), 155.8 (Boc-CdO), 155.1 (NVP-C11a),
151.9 (NVP-C9), 144.4 (NVP-C2), 140.6 (NVP-C7), 138.9
(NVP-C4), 135.6 (Imid-C2/Imid-C4), 135.5 (Imid-C2/Imid-C4),
123.6 (NVP-C4a), 121.3 (NVP-C6a), 119.9 (NVP-C8), 118.8
(NVP-C3), 116.9 (Imid-C5), 78.9 (Boc-C-O), 62.0 (NVP-C12),
54.5 (Hist-C2), 29.4 (NVP-C13), 29.1 (Hist-C3), 28.5 (Boc-
CH3), 9.2 (NVP-C14/C15), 8.9 (NVP-C14/C15). MS m/z 520
[MH]+, 420 [MH2 - Boc]+, 302 [MH2 + 2CH3CN]2+, 281
[MH2 + CH3CN]2+, 261 [MH2]2+, 253 [MH + 2H + CH3CN
- tert-butyl (t-Bu)]2+. 12-(NR-Boc-histidin-N1′-yl)-NVP (15)
was obtained in 13% yield (5.9 mg); tR, 24 min. UV, λmax 216,
N-Deprotection of 11 (4 mg) was achieved by treatment with
trifluoroacetic acid (970 µL) at 60 °C for 5 days. The reaction
mixture was neutralized by addition of 10 M sodium hydroxide
and analyzed by HPLC-MS. The hydrolysis reaction yielded
12-(cystein-S-yl)-NVP (12). MS m/z 386 [MH]+.
Reaction with Other Amino Acids. A solution of 9 (32 mg,
89 µmol) in THF (800 µL) was added to a solution of the amino
acid (3.9 equiv, 350 µmol) in 4 mL of 50 mM phosphate buffer
(pH 7.4). The reaction mixture was incubated at room temperature
for 24 h, and the products were purified by semipreparative HPLC.
Reaction with Tryptophan. One adduct was isolated by
semipreparative HPLC, using a 14 min linear gradient of
20-30% acetonitrile in 100 mM ammonium acetate (pH 5.7),
followed by a 5 min linear gradient to 100% acetonitrile and a
10 min isocratic elution with acetonitrile. 12-(Tryptophan-2′-
yl)-NVP (13) was obtained in 14% yield (5.7 mg); tR, 13 min.
UV, λmax 220, 275, 282 nm. 1H NMR (DMSO-d6): δ 10.51 [1H,
s, indole (Ind)-N1H], 10.42 (1H, s, Ind-N1H*), 8.48 (1H, dd,
Jortho ) 4.7, Jmeta ) 1.9, NVP-H9), 8.44 (1H, dd Jortho ) 4.8,
Jmeta ) 2.0, NVP-H9*), 8.11 (1H, d, Jortho ) 4.7, NVP-H2),
8.08 (1H, d, Jortho ) 4.9, NVP-H2*), 7.63-7.58 (2H, m, Ind-
H7), 7.48 (2H, d, Jortho ) 7.3, NVP-H7 and NVP-H7*),
7.13-6.90 (10H, m, Ind-H4, H5, H6 + NVP-H3, H8; 2
isomers), 4.56-4.14 (4H, m, NVP-H12 + NVP-H12*),
3.63-3.59 (4H, m, NVP-H13 + NVP-H13* + Trp-H2 + Trp-
H2*), ca. 3.33 (Trp-H3a + Trp-H3a*, obscured by the water
resonance), 3.09 (1H, dd, Jgem ) 15.0, 3J ) 6.0, Trp-H3b), 2.86
1
297 nm. H NMR (DMSO-d6): δ 10.16 (1H, bs, His-CO2H),
8.49 (1H, d, J ) 3.3, NVP-H9), 8.29 (<1H, bs, NH), 8.08 (1H,
d, J ) 4.7, NVP-H2), 7.98 (1H, d, Jortho ) 7.2, NVP-H7), 7.59
(1H, s, Imid-H2), 7.19 (1H, dd, Jortho ) 7.4, Jortho′ ) 4.8, NVP-
H8), 6.84 (1H, bs, Imid-H5), 6.76 (1H, bs, NH), 6.32-6.29
(1H, m, NVP-H3), 5.33 (1H, bs, NVP-H12), 4.02 (1H, bs, Hist-
H2), ca. 3.40 (NVP-H13, obscured by the water resonance),
2.87-2.76 (2H, m, Hist-H3), 1.28 (9H, s, Boc-CH3), 0.86-0.85
(2H, m, NVP-H14 + NVP-H15), 0.36-0.22 (2H, m, NVP-H14
+ NVP-H15). 13C NMR (DMSO-d6): δ 174.3 (His-CdO), 167.6
(NVP-C6), 160.2 (NVP-C10a), 155.6 (Boc-CdO), 155.1 (NVP-
C11a), 151.9 (NVP-C9), 144.7 (NVP-C2), 141.2 (NVP-C4),
140.5 (NVP-C7), 138.9 (Imid-C4), 137.9 (Imid-C2), 123.5
(NVP-C4a), 121.3 (NVP-C6a), 120.0 (NVP-C8), 118.3 (NVP-
C3), 118.1 (Imid-C5), 78.2 (Boc-C-O), 54.4 (Hist-C2), 45.7
(NVP-C12), 30.6 (Hist-C3), 29.8 (NVP-C13), 28.6 (Boc-CH3),
9.2 (NVP-C14/C15), 8.9 (NVP-C14/C15). MS m/z 520 [MH]+,
281 [MH2 + CH3CN]2+, 261 [MH2]2+, 253 [MH + 2H +
CH3CN - t-Bu]2+
.
3
(1H, dd, Jgem ) 14.6, J ) 5.6, Trp-H3b*), 0.90-0.84 (4H, m,
Reaction with Histidine. One adduct was isolated by semi-
preparative HPLC, using a 30 min linear gradient of 5-30%
acetonitrile in 100 mM ammonium acetate (pH 5.7), followed by
a 5 min linear gradient to 100% acetonitrile and a 10 min isocratic
elution with acetonitrile. 12-(Histidin-N1′-yl)-NVP (16) was ob-
tained in 10% yield (3.7 mg); tR 19.5 min. UV, λmax 215, 288 nm.
1H NMR (DMSO-d6): δ 10.23 (1H, bs, NVP-N5H), 8.52 (1H, bs,
NVP-H9), 8.15 (1H, bs, NVP-H2), 8.01 (1H, m, NVP-H7), 7.68
(1H, bs, Imid-H2), ca. 7.25 (bs, His-NH2), 7.23-7.18 (1H, m, NVP-
H8), 7.00 (1H, bs, NVP-H3), 6.98 (1H, bs, NVP-H3*), 6.54 (1H,
bs, Imid-H5), 5.44-5.38 (2H, m, NVP-H12), 3.63 (1H, bs, NVP-
H13), ca. 3.40 (Hist-H2, obscured by the water resonance),
3.05-3.03 (1H, m, Hist-H3a), 2.73 (1H, bs, Hist-H3b), 0.88 (2H,
bs, NVP-H14 + NVP-H15), 0.40-0.33 (2H, m, NVP-H14 + NVP-
NVP-H14 + NVP-H15 + NVP-H14* + NVP-H15*), 0.38-0.35
(4H, m, NVP-H14 + NVP-H15 + NVP-H14* + NVP-H15*).
13C NMR (DMSO-d6): δ 172.7 (Trp-C1), 172.1 (Trp-C1*),
167.5 (NVP-C6), 167.1 (NVP-C6*), 160.3 (NVP-C10a), 160.2
(NVP-C10a*), 155.3 (NVP-C11a), 155.0 (NVP-C11a*), 151.5
(NVP-C9), 151.3 (NVP-C9*), 144.2 (NVP-C2), 143.9 (NVP-
C2*), 143.1 (NVP-C4), 142.8 (NVP-C4*), 140.9 (NVP-C7),
140.7 (NVP-C7*), 136.1 (Ind-C7a), 133.8 (Ind-C2), 133.3 (Ind-
C2*), 128.4 (Ind-C3a), 128.0 (Ind-C3a*), 124.7 (NVP-C4a),
124.5 (NVP-C4a*), 121.4, 120.9, 119.5, 119.4, 118.8, 118.6,
118.3, 118.2 (NVP-C3/C6a/C8; Ind-C4/C5/C6), 111.0 (Ind-C7),
110.9 (Ind-C7*), 109.1 (Ind-C3*), 108.2 (Ind-C3), 55.3 (Trp-
C2), 29.5 (NVP-C13), 28.2 (NVP-C12), 28.0 (NVP-C12*), 26.9
(Trp-C3), 26.5 (Trp-C3*), 9.0 (NVP-C14/C15), 8.8 (NVP-C14/
C15). MS m/z 469 [MH]+, 276 [MH2 + 2CH3CN]2+, 256 [MH2
H15). MS m/z 420 [MH]+, 231 [MH2 + CH3CN]2+, 211 [MH2]2+
.
Reaction with Arginine. One adduct was isolated by semi-
preparative HPLC, using a 14 min linear gradient of 15-70%
acetonitrile in 100 mM ammonium acetate (pH 5.7), followed by
a 5 min linear gradient to 100% acetonitrile. 12-(Arginin-NR-yl)-
NVP (17) was obtained in 6% yield (2.4 mg); tR, 8 min. UV, λmax
+ CH3CN]2+
.
Reaction with Nr-tert-Butoxycarbonyl (Boc)-histidine. Two
adducts were isolated using a 20 min linear gradient of 15-25%
acetonitrile in 0.1% formic acid, followed by a 5 min linear
gradient to 100% acetonitrile. 12-(NR-Boc-histidin-O1-yl)-NVP
(14) was obtained in 6% yield (2.5 mg) after further purification
by preparative thin-layer chromatography [dichloromethane/
1
216, 240, 302 nm. H NMR (DMSO-d6): δ 9.06 (2H, bs, NH),
8.52 (2H, bs, NVP-H9 + NVP-H9*), 8.12 (2H, bs, NVP-H2 +
NVP-H2*), 8.05-8.02 (2H, m, NVP-H7 + NVP-H7*), 7.93 (4H,
bs, NH), 7.19 (2H, bs, NVP-H8 + NVP-H8*), 7.12-7.11 (2H, m,
NVP-H3 + NVP-H3*), 3.88-3.57 (6H, m, NVP-H12 + NVPH12*
+ NVP-H13+ NVPH13*), 3.05-3.01 (4H, m, Arg-H5 + Arg-
1
methanol (10:1)]; tR, 28 min. UV, λmax 215, 295 nm. H NMR

892 Chem. Res. Toxicol., Vol. 23, No. 5, 2010
Antunes et al.
H5*), 2.91 (1H, bs, Arg-H2), 2.76 (1H, bs, Arg-H2*), 1.61-1.48
(8H, m, Arg-H3 + Arg-H3* + Arg-H4 + Arg-H4*), 0.88-0.85
(4H, bs, NVP-H14 + NVPH-14* + NVP-H15 + NVP-H15*),
0.37-0.31 (4H, m, NVP-H14 + NVP-H14*+ NVP-H15 + NVP-
H15*). 13C NMR (DMSO-d6): δ 173.3 (Arg-C1), 166.5 (NVP-C6),
166.3 (NVP-C6*), 159.9 (NVP-C10a), 159.8 (NVP-C10a*), 157.4
(guanidine-C + guanidine-C*), 153.0 (NVP-C11a + NVP-C11a*),
151.4 (NVP-C9 + NVP-C9*), 144.2 (NVP-C2), 144.0 (NVP-C2*),
142.9 (NVP-C4), 142.7 (NVP-C4*), 140.7 (NVP-C7), 140.4 (NVP-
C7*), 126.0 (NVP-C4a + NVP-C4a*), 121.9 (NVP-C3), 120.9
(NVP-C3*), 120.0 (NVP-C6a + NVP-C6a*), 118.8 (NVP-C8 +
NVP-C8*), 62.2 (Arg-C2), 61.3 (Arg-C2*), 50.3 (NVP-C12 +
NVP-C12*), 42.0 (Arg-C5 + Arg-C5*), 30.1 (Arg-C3 + Arg-C3*),
28.9 (NVP-C13 + NVP-C13*), 25.8 (Arg-C4 + Arg-C4*), 8.9
(NVP-C14/C15), 8.6 (NVP-C14*/C15*), 7.8 (NVP-C14/C15), 7.6
(NVP-C8 + NVP-C8*), 67.5 (Val-C2*), 65.9 (Val-C2), 49.7 (NVP-
C12*), 48.5 (NVP-C12), 30.8 (Val-C3/Val-C3*), 30.5 (Val-C3/
Val-C3*), 29.5 (NVP-C13 + NVP-C13*), 20.0 (Val-CH3a*), 19.6
(Val CH3a), 18.4 (Val CH3b/CH3b*), 18.2 (Val CH3b/CH3b*), 8.9
(NVP-C14/C15 + NVP-C14/C15*), 8.6 (NVP-C14/C15 + NVP-
C14/C15*). MS m/z 382 [MH]+, 336 [MH - HCO2H]+, 282 [NVP-
NH2 + H]+, 265 [MH - valine]+.
Preparation of an N-Alkyl Edman Derivative from 18. 12-[5-
Isopropyl-4-oxo-3-phenyl-2-thioxoimidazolidin-1-yl]-NVP (20). The
thiohydantoin derivative was prepared by treatment of 18 (5.0 mg,
12 µmol) with phenyl thioisocyanate (1.4 µL, 12 µmol) in dioxane
(1 mL), in the presence of 1 M KOH (10 µL). The reaction mixture
was stirred for 3 h at 55 °C, and the product was purified by
semipreparative HPLC using a 20 min linear gradient of 5-52%
acetonitrile in 0.1% formic acid, followed by a 5 min linear gradient
to 100% acetonitrile and a 2 min isocratic elution with acetonitrile.
The thiohydantoin 20 was obtained in 13% yield (0.8 mg); tR, 27.9
(NVP-C14*/C15*). MS m/z 439 [MH]+, 241 [MH2 + CH3CN]2+
,
220 [MH2]2+
.
1
Reaction with Ethyl Valinate. One adduct was isolated by
semipreparative HPLC, using a 30 min linear gradient of 5-70%
acetonitrile in 0.1% formic acid. 12-(Ethyl valinate-NR-yl)-NVP (18)
was obtained in 33% yield (12.0 mg); tR, 27.6 min. UV, λmax 239,
294 nm. 1H NMR (MeOD): δ 8.51-8.50 (2H, m, NVP-H9 + NVP-
H9*), 8.14 (4H, bs, NVP-H2 + NVP-H2* + NVP-H7 + NVP-
H7*), 7.19-7.18 (2H, m, NVP-H8 + NVP-H8*), 7.12-7.11 (2H,
m, NVP-H3 + NVP-H3*), 4.26-4.17 (4H, m, Val-OCH2CH3 +
Val-OCH2CH3*), 4.11 (1H, d, Jgem ) 13.8, NVP-H12a), 3.82 (2H,
s, NVP-H12a* + NVP-H12b*), 3.73 (2H, bs, NVP-H13 + NVP-
H13*), 3.63 (1H, d, Jgem ) 13.8, NVP-H12b), 3.32-3.00 (1H, m,
Val-H2*), 2.87-2.71 (1H, m, Val-H2), 2.07-2.02 (1H, m, Val-
H3*), 1.99-1.95 (1H, m, Val-H3), 1.32-1.27 (6H, Val-OCH2CH3
+ Val-OCH2CH3*), 1.18-1.03 (6H, m, Val-CH3a* + Val-CH3b*),
0.96-0.89 (10H, m, Val-CH3a + Val-CH3b + NVP-H14/H15 +
NVP-H14/H15*), 0.47-0.36 (4H, m, NVP-H14/H15 + NVP-H14/
H15*). 13C NMR (MeOD): δ 174.0 (Val-C1/C1*), 173.9 (Val-C1/
C1*), 167.4 (NVP-C6 + NVP-C6*), 159.9 (NVP-C10a + NVP-
C10a*), 151.6 (NVP-C9 + NVP-C9*), 143.6 (NVP-C2 + NVP-
C2*), 140.3 (NVP-C7 + NVP-C7*), 139.1 (NVP-C4 + NVP-C4*),
126.0 (NVP-C4a + NVP-C4a*), 121.5 (NVP-C3*), 120.9 (NVP-
C3), 119.1 (NVP-C8 + NVP-C8*), 67.4 (Val-OCH2CH3*), 65.4
(Val-OCH2CH3), 60.5 (Val-C2), 60.4 (Val-C2*), 50.1 (NVP-C12*),
48.8 (NVP-C12), 31.4 (Val-C3/Val-C3*), 31.1 (Val-C3/Val-C3*),
29.1 (NVP-C13 + NVP-C13*), 18.6 (Val CH3a/CH3b/CH3a*/
CH3b*), 18.4 (Val CH3a/CH3b/CH3a*/CH3b*), 17.6 (Val CH3a/
CH3b/CH3a*/CH3b*), 17.3 (Val CH3a/CH3b/CH3a*/CH3b*), 13.3
(Val-OCH2CH3/Val-OCH2CH3*), 13.2 (Val-OCH2CH3/Val-OCH2-
CH3*), 8.4 (NVP-C14/C15/C14*/C15*), 8.1 (NVP-C14/C15/C14*/
C15*). MS m/z 432 [MH + Na]+, 410 [MH]+, 265 [MH - ethyl
valinate]+.
min. UV, λmax 218, 236, 266 nm. H NMR (CDCl3): δ 9.12 (1H,
s, NVP-N5H), 8.55 (2H, bs, NVP-H9 + NVP-H9*), 8.30 (2H, bs,
NVP-H2 + NVP-H2*), 8.13 (2H, d, J ) 7.4, NVP-H7), 8.08 (1H,
d, J ) 7.3, NVP-H7*), 7.56-7.28 [10 H, m, phenyl (Ph)-H + Ph-
H*], 7.08-7.07 (2H, m, NVP-H8 + NVP-H8*), 7.01-6.94 (2H,
m, NVP-H3 + NVP-H3*), 6.27 (1H, d, Jgem ) 16.4, NVP-H12a),
5.72 (1H, d, Jgem ) 15.4, NVP-H12a*), 4.61 (1H, d, Jgem ) 15.4,
NVP-H12b*), 4.43 (1H, d, Jgem ) 16.4, NVP-H12b), 4.11 (1H, bs,
hydantoin-H5), 3.78 (2H, bs, NVP-H13 + NVP-H13*), 3.62 (1H,
bs, hydantoin-H5*), 2.55-2.49 (1H, m, isopropyl-CH), 2.32-2.27
(1H, m, isopropyl-CH*), 1.30 (3H, d, J ) 6.6, CH3a), 1.11 (3H, d,
J ) 6.4, CH3b), 1.00-0.99 (10H, m, CH3a* + CH3b* + NVP-
H14/H15 + NVP-H14/H15*), 0.63 - 0.43 (4H m, NVP-H14/H15
+
NVP-H14/H15*). MS m/z 499 [MH]+, 265 [MH
-
C12H14N2OS]+.
Computational Methods. All species were drawn and preop-
timized using a Dreiding type molecular mechanics force field (30),
implemented in the Marvin software and the Calculator plug-ins
(31). Calculations were performed with the Parametrized Model 6
(32) as implemented in MOPAC2009 (33), using a Conductor-like
Screening Model (COSMO) method to approximate the effect of
the solvent; DMSO was used for the calculations. In all instances,
geometry optimization was terminated when the gradient dropped
below 0.01. PM6DH (34), which adds correction terms for PM6
to reflect noncovalent interactions, such as hydrogen bonding, was
also used to fine-tune further the calculated geometries and energies.
All values are in kJ/mol.
Results and Discussion
We have previously demonstrated DNA adduct formation in
vitro by 9, used as a surrogate for the phase II NVP metabolite,
7. To address the potential involvement of NVP-protein binding
in the toxic events associated with NVP administration, the
availability of reliable NVP-amino acid adduct standards is
essential. To gain insight into potentially reactive sites in
proteins and prepare fully characterized NVP-amino acid
adduct standards for subsequent assessment as biomarkers of
NVP toxicity, we have investigated the reaction of 9 with the
tripeptide GSH, ethyl valinate, and selected R-amino acids
containing nucleophilic side chains.
Preliminary experiments, conducted to establish the most
suitable reaction conditions, demonstrated a correlation between
the extent of adduct formation and the anticipated nucleophilicity
of the amino acids (e.g., S > N), as expected for a bimolecular
mechanism. HPLC analyses of the reaction mixtures indicated
that the only additional product was, in all instances, the
hydrolysis product, 5, which was identified by comparison with
a synthetic standard (27).
O-Deprotection of 18 (5.0 mg, in 1 mL of methanol) was
achieved by treatment with 1 M KOH (200 µL). The solution was
stirred at 40 °C and periodically monitored by HPLC until
completion (72 h). Following neutralization with HCl, the hydroly-
sate yielded a single product, which was purified by semipreparative
HPLC. 12-(Valin-NR-yl)-NVP (19) was obtained in 86% yield (4.0
mg); tR, 12.7 min. UV, λmax 238, 302 nm. 1H NMR (DMSO-d6): δ
10.51 (1H, s, NVP-N5H), 8.52-8.51 (2H, d, NVP-H9 + NVP-
H9*), 8.14-8.13 (2H, m, NVP-H2 + NVP-H2*), 8.03-8.02 (2H,
m NVP-H7 + NVP-H7*), 7.20-7.18 (2H, m, NVP-H8 + NVP-
H8*), 7.12-7.09 (2H, m, NVP-H3 + NVP-H3*), 4.01 (1H, d, Jgem
) 14.3, NVP-H12a), 3.80 (1H, d, Jgem ) 13.8, NVP-H12a*), 3.70
(1H, d, Jgem ) 13.8, NVP-H12b*), 3.64-3.60 (2H, m, NVP-H13
+ NVP-H13*), 3.56 (1H, d, Jgem ) 14.3, NVP-H12b), 2.89 (1H,
d, J ) 5.5, Val-H2*), 2.60 (1H, d, J ) 5.4, Val-H2), 1.94-1.84
(2H, m, Val-H3 + Val-H3*), 0.98 (3H, d, J ) 6.7, Val-CH3a),
0.92 (3H, d, J ) 6.7, Val-CH3b), 0.86-0.81 (10H, m, Val-CH3a*
+ Val-CH3b* + NVP-H14/H15 + NVP-H14/H15*), 0.37-0.26
(4H, m, NVP-H14/H15 + NVP-H14/H15*). 13C NMR (DMSO-
d6): δ 171.0 (Val-C1 + Val-C1*), 164.5 (NVP-C6 + NVP-C6*),
159.7 (NVP-C10a + NVP-C10a*), 151.6 (NVP-C9 + NVP-C9*),
143.4 (NVP-C2 + NVP-C2*), 140.2 (NVP-C7 + NVP-C7*), 140.1
(NVP-C4 + NVP-C4*), 125.8 (NVP-C4a + NVP-C4a*), 119.4
The optimized reactions were typically conducted at room
temperature in 50 mM phosphate buffer (pH 7.4)/THF (5:1)
using a 3.9 molar excess of the nucleophile, with the exception

12-HydroxyneVirapine Amino Acid Adducts
Chem. Res. Toxicol., Vol. 23, No. 5, 2010 893
Scheme 3. Structures of 10 and 11, Obtained in the Reaction of 9 with GSH and N-Acetylcysteine, Respectivelya
a The arrows in structure 10 (on the left) represent the HMBC correlations of the NVP-H12 protons with specific NVP and GSH carbons. For simplicity,
arrows are drawn from only one of the geminal NVP protons, although both exhibited correlations to the same carbons. The middle structure displays a
hydrogen bond [(NVP)N5H···OdC(Cys)], present in the lowest energy conformer obtained by theoretical simulation of adduct 10 in DMSO at 298 K.
1
Figure 1. Expanded region of the H-13C HMBC spectrum of 10, displaying the connectivities between the geminal NVP-H12 protons and the
carbons of the NVP (C3, C4, and C4a) and GSH (Cys-C3) moieties. The three-bond connectivity with the Cys-3 carbon was decisive to assign the
cysteine sulfur as the binding site in GSH.
of the thionucleophiles, GSH and N-acetylcysteine, for which
a 1.3 molar excess proved to be sufficient for a significant adduct
yield. In each instance, NVP-amino acid adducts were isolated
by semipreparative reversed-phase HPLC and fully characterized
moieties. The assignment of all 1H NMR and 13C NMR
resonances was based on the correlations observed in both
HSQC-TOCSY and HMBC spectra (not shown). Evidence for
the connectivity through the NVP C12 position was initially
1
1
by H and 13C NMR spectroscopy and mass spectrometry; all
obtained from the H NMR spectrum, where the signals from
of the isolated adducts consistently involved binding through
the C12 position of NVP. The binding regiospecificity was
expressed unequivocally in the 1H NMR and 13C NMR spectra
of the isolated adducts, which showed all of the expected NVP
and amino acid signals, and by the resonances of the two
geminal H12 NVP protons (as well as the C12 carbon), which
were strongly dependent on the nature of the amino acid atom
establishing the connectivity to the NVP fragment. Further
confirmation of the binding position was achieved for each
the H12 protons appeared as duplicate (vide infra) sets of two
double duplets (at 3.75, 3.77, 4.17, and 4.21 ppm), each
accounting for one proton, with a geminal coupling constant of
14 Hz as clear evidence of magnetic anisotropy. Furthermore,
the location of the H12 (resonances indicated above) and C12
(30.5 and 30.7 ppm) NVP resonances was consistent with a
methylene bridge between the aromatic moiety and a sulfur
atom. This connectivity was unequivocally confirmed with the
HBMC correlations established by the geminal NVP H12
protons; thus, in addition to the expected two- and three-bond
interactions to carbons of the NVP moiety (C3, C4, and C4a),
a correlation was found with the C3 of the cysteine residue in
GSH at 33.0 and 33.3 ppm (Scheme 3 and Figure 1). Similarly
to the NVP H12 protons, the NVP C4, C4a, and C12 and the
cysteine C1, C2, and C3 (in the GSH fragment) had duplicate
resonances, which suggested restricted rotational mobility
between the GSH and the NVP moieties. Further confirmation
of covalent binding between the NVP and the GSH moieties
was obtained from the LC/ESI-MS spectrum of 10 (not shown),
1
adduct through observation of three-bond H-13C correlations
displayed in the HMBC spectra between the NVP H12 protons
and the relevant carbons of the amino acid moiety.
As implied above, the thionucleophiles GSH and N-acetyl-
cysteine were the most reactive. Thus, using a slight (1.3 equiv)
molar excess of the nucleophile and a short incubation time (2
h), the adducts 10 (Scheme 3) and 11 (Scheme 3) were isolated
1
in 84 and 82% yield, respectively. The H (Figure S1 of the
Supporting Information) and 13C NMR spectra of 10 showed
all of the expected resonances from both the GSH and the NVP

894 Chem. Res. Toxicol., Vol. 23, No. 5, 2010
Antunes et al.
Scheme 5. ESI-MS/MS Fragmentation Pattern for the
Scheme 4. ESI-MS/MS Fragmentation Pattern for the
Protonated Molecule (m/z 572) of 10
Protonated Molecule (m/z 386) of 12
C3 (34.4 and 34.5 ppm). The sensitivity of these specific carbons
to conformational effects is entirely consistent with binding
through the NVP C12. The observation of comparable, and
relatively shielded, resonances for the NVP C12 in adducts 10
(30.5 and 30.7 ppm) and 11 (vide supra) provides further
confirmation of the binding site. N-Deacetylation of adduct 11
was achieved by treatment with trifluoroacetic acid at 60 °C.
Following neutralization, HPLC-MS analysis of the reaction
mixture indicated the presence of adduct 12 (Scheme 5). MS/
MS of the protonated molecule (m/z 386), using a 0.9 V
excitation energy, yielded characteristic fragment ions at m/z
369 (MH+ - NH3), m/z 342 (MH+ - CO2), and m/z 265 (MH+
which displayed the protonated molecule at m/z 572, a fragment
ion resulting from loss of pyroglutamate at m/z 443, and a base
peak (m/z 287) corresponding to the diprotonated molecule.
Moreover, the ESI-tandem mass spectrometry (MS/MS) spec-
trum of MH+ showed a daughter ion at m/z 299 [(NVP + S +
H)+], stemming from cleavage of the S-C3 bond of the cysteine
residue in GSH, with the sulfur remaining attached to the NVP
moiety (Scheme 4). The same fragment ion was observed by
Wen et al. (25) for an NVP-GSH conjugate obtained in
incubations of NVP with human liver microsomes supplemented
with GSH and was assumed to involve binding through the NVP
C12, but definite proof of the site of attachment was not
provided. Likewise, although Srivastava et al. (26) reported the
detection of 10 in the bile of NVP-treated rats and in incubations
of NVP with rat hepatocytes, the structural evidence was based
upon the m/z 572 f 443 transition in the MS/MS spectrum,
and the binding position was inferred from indirect evidence
only.
Using PM6DH, which includes solvation, we calculated
enthalpies of formation for adduct 10 considering numerous
possibilities of hydrogen bond formation. The calculations were
conducted assuming a temperature of 298 K and ignoring pKa
effects, by working with nonionized molecules. The computa-
tions yielded a large number of low energy conformers, of which
the most stable (by ca. 11 kJ/mol) had an intramolecular
hydrogen bond between the NVP N5H and the carbonyl oxygen
of the cysteine residue in GSH (Scheme 3). Despite the
simplifications of the model, this result is consistent with the
detection of different conformers of 10 in DMSO solution. In
fact, the RT parameter is approximately 2.5 kJ/mol at 298 K,
which suggests that thermal motion is insufficient to allow free
interconversion at room temperature.
- cysteine); the base peak, observed at m/z 299 (MH+
-
C3H5NO2), corresponded to the already identified [(NVP + S
+ H)+] fragment (Scheme 5). The N-acetylated adduct 11 was
identified by Srivastava et al. (26) as one of the two NVP
mercapturates (designated NVP-M1) detected in the bile and
urine of NVP-treated rats, in human urine, and in incubations
of NVP with rat hepatocytes. Their structural evidence relied
1
on the 428 f 299 MS/MS fragmentation and on the H NMR
spectrum of a very diluted sample, recorded in deuterated
methanol and presented in graphic form. The authors based the
assignment of the NVP binding site on the observation of all
of the aromatic NVP signals and of an NOE cross-peak between
the H3 and the H12 protons of NVP. It is not clear whether
this NOE signal was properly identified since, judging from the
figure labels, the authors mistook a very weak signal at ca. 3.55
ppm for the signal of the NVP H12 protons, while missing two
apparent doublets of proper intensity at ca. 3.8 and 4.0 ppm,
having the characteristic pattern of nonequivalent geminal
protons. Nonetheless, despite the limitations of visual inspection
and slight variations associated with the use of different solvents,
1
the H NMR spectrum displayed by Srivastava et al. (26) for
NVP-M1 is consistent with our data for the synthetic adduct
11.
The 1H NMR (Figure S2 of the Supporting Information) and
13C NMR spectra of the N-acetylcysteine-NVP adduct (11)
showed all of the signals expected from the NVP and amino
acid moieties. Although we were unable to detect a three-bond
1H-13C correlation between the NVP H12 protons and the
cysteine C3 in the HMBC spectrum, the occurrence of nucleo-
philic attack by the thiol group of cysteine at the C-12 position
of NVP is consistent with the MS data. Thus, as observed for
10, the MS/MS spectrum of the protonated molecule (m/z 428)
showed a daughter ion at m/z 299 stemming from cleavage of
the S-C3 bond of cysteine, with the sulfur remaining attached
to the NVP moiety. In addition, similarly to the GSH-NVP
adduct, the 13C NMR spectrum of 11 showed evidence for the
presence of two conformers, as indicated by duplication of the
signals for the NVP C12 (32.3 and 32.5 ppm) and the cysteine
Reaction with tryptophan resulted in the formation of 13
(Scheme 6) in 14% yield. Both the 1H NMR and the 13C NMR
spectra were rather complex, due to the existence of duplicate
resonances. Simplification of some 1H NMR signals was
observed upon heating from 25 to 70 °C (Figure 2), which
suggests that the sample consisted of a mixture of two readily
interconvertible conformers, having comparable populations at
room temperature. The same theoretical methodology applied
for adduct 10 indicated a variety of low energy conformers for
structure 13, of which the two more stable isomers (Scheme 6)
differed by only 3.2 kJ/mol and were considerably more stable
(by at least 8 kJ/mol) than any other conformer. Again, despite
the approximations involved, these results are consistent with
1
the interconversion observed in the H NMR spectrum at a
relatively low temperature. Evidence for adduct formation

12-HydroxyneVirapine Amino Acid Adducts
Chem. Res. Toxicol., Vol. 23, No. 5, 2010 895
Scheme 6. Structure of 13, Obtained in the Reaction of 9 with Tryptophana
a The adduct is shown as two different conformers in equilibrium. Theoretical simulation of 13 in DMSO at 298 K indicated a difference of 3.24 kJ/mol
between the lowest energy conformer, having a (Trp)NRH···OdC6(NVP) hydrogen bond (left), and the next more stable conformer, having a
(Trp)C1dO···HN5(NVP) hydrogen bond (right).
1
Figure 2. Expanded downfield region of the H NMR spectrum of 13, recorded in DMSO-d6 as a function of temperature, showing the gradual
collapse of duplicate resonances upon heating. The observed changes were reversible on cooling to room temperature.
Table 1. Comparison of the 13C NMR Resonances for C12
through C2 of the Ind ring of tryptophan was first obtained from
of the NVP Fragmenta
the 1H NMR spectrum, which showed signals in the downfield
region accounting for all of the aromatic NVP protons but where
one of the aromatic Ind protons was missing. Furthermore, two
singlets at 10.42 and 10.51 ppm were unequivocally ascribed
to the exchangeable N1H protons of the Ind ring in each rotamer,
since HMBC correlations (not shown) were observed between
these protons and the Ind-C2 (133.3 and 133.8 ppm), Ind-C3
(108.2 and 109.1 ppm), and Ind-C3a (128.0 and 128.4 ppm);
this observation excluded binding of the NVP moiety through
N1 of the Ind ring. Moreover, the 13C NMR spectrum showed
that both rotamers had a relatively shielded NVP C12 (28.0
and 28.2 ppm), when compared to all other NVP adducts
characterized in this study (Table 1), which involved binding
of the NVP C12 to heteroatoms in the amino acid residue and
to the resonances observed for 5 (59.3 ppm) and 9 (66.0 ppm)
(27); this is entirely consistent with direct connectivity to a less
deshielding carbon atom. To obtain definite structural proof,
and because no correlations between the geminal NVP H12
Compound C12-O bond C12-N bond C12-S bond C12-C bond
5
9
59.3b
66.0b,c
10
11
13
14
15
17
19
30.5, 30.7d
32.3, 32.5d,e
28.0, 28.2d
62.0
45.7
50.3
48.5, 49.7d
a Unless indicated, the spectra were recorded in DMSO-d6. Chemical
shifts are in ppm, downfield from tetramethylsilane. b Data from ref 27.
c Recorded in CDCl3. d Two conformers were detected. e Recorded in
acetone-d6.
protons and any Ind carbons were detected in the standard
HMBC spectrum, a semiselective HMBC experiment was
performed to enhance the spectral resolution, using a shaped
pulse for excitation and a gradient pulse for selection. The

896 Chem. Res. Toxicol., Vol. 23, No. 5, 2010
Antunes et al.
1
Figure 3. Expanded region of the H-13C semiselective HMBC spectrum of 13, displaying the connectivities between the geminal NVP-H12
protons and the carbons of the NVP (C3, C4, and C4a) and tryptophan (Ind-C2 and Ind-C3) moieties. The connectivities to the C2 and C3 carbons
of the Ind ring of tryptophan were decisive in ascribing the Ind C2 as the binding site in tryptophan. Also shown are connectivities between the H3
protons in the aliphatic chain of tryptophan (Trp-H3) and the carbons of the amino acid, both in the aliphatic chain (the carboxylate, Trp-C1) and
the Ind ring (Ind-C2, Ind-C3 and Ind-C3a).
the two adducts presented very similar 1H NMR and 13C NMR
profiles, with all of the expected nonexchangeable resonances
from the NVP and N-Boc-histidine moieties. The corresponding
structures were discriminated on the basis of HMBC correlations
between the NVP H12 protons and specific histidine carbons
(Scheme 7). Thus, a three-bond correlation with the most
downfield histidine carbon (the carboxylate, Hist-C1, at 172.3
ppm) was observed in adduct 14, whereas adduct 15 displayed
two three-bond correlations with carbons in the Imid ring of
the histidine unit (Imid-C2, at 137.9 ppm, and Imid-C5, at 118.1
ppm). Moreover, the 13C NMR spectrum of adduct 14 (Table
1) indicated a downfield shift of approximately 16 ppm for the
NVP C12 resonance (detected at 62.0 ppm), as compared to
that of adduct 15 (detected at 45.7 ppm), which is consistent
with binding of the NVP C12 to an oxygen atom in adduct 14
(27). This observation further substantiates a direct connectivity
between the NVP C12 and one of the oxygens from the histidine
carboxylate in adduct 14.
Scheme 7. Structures of 14-16, Obtained in the Reaction of
9 with Nr-Boc-histidine (14 and 15) and Histidine (16)a
a The arrows represent the HMBC correlations of the NVP-H12 protons
with specific carbons of the amino acid moieties.
resulting spectrum (Figure 3), recorded with spectral windows
from 5.0 to 2.0 ppm (1H) and from 100.0 to 180.0 ppm (13C),
showed the expected two- and three-bond correlations between
the NVP H12 protons from both rotamers and carbons (C3, C4,
and C4a) from the NVP moiety. In addition, correlations with
two quaternary carbons of the tryptophan moiety, the Ind-C2
(at 133.3 and 133.8 ppm) and the Ind-C3 (at 108.2 and 109.1
ppm), were also observed. Taken together, these data are entirely
consistent with connectivity between the NVP and tryptophan
units through NVP-C12/Ind-C2. This regiospecificity was not
unexpected since Inds are well-known to undergo electrophilic
substitution at C2 whenever substitution blocks the reaction at
the more electron-rich C3 position (35).
Considering that adduct formation through the histidine
carboxylate will have negligible biological significance, with
the possible exception of proteins with a C-terminal histidine,
and bearing in mind that the typical pKa of the Imid side chain
is 6.5 (36), we investigated the direct reaction of 9 with free
histidine. Because histidine is well-known to be sensitive to the
microenvironment of the ionizable groups (37), we reasoned
that, under our experimental conditions (pH 7.4), a zwitterionic
form involving a protonated (and therefore non-nucleophilic)
R-amino group would predominate, while a substantial fraction
of the Imid side chain would remain unionized and thus
available for nucleophilic attack. By using the nonprotected
amino acid, we aimed to test whether predominant protonation
on the R-amino group led to increased adduct formation through
the Imid side chain. However, the reaction mixture was again
rather complex, and several putative adducts, having very close
retention times under a variety of elution conditions, were
detected. This complexity presumably results from a combina-
tion of factors, including different protonation sites and tau-
tomerism, which confer nucleophilic properties to all available
positions (i.e., the two nitrogens and the two nonsubstituted
carbons) of the Imid ring. Because of the complexity of the
reaction mixture, we could only characterize one adduct, 16
Reaction of 9 with NR-Boc-histidine gave rise to a complex
HPLC profile, due to the formation of multiple products. On
the basis of LC/ESI-MS analysis (not shown) of four fractions
separated by semipreparative HPLC, at least four adducts (m/z
520 for the protonated molecule) were present in the reaction
mixture; nonetheless, only two of these adducts could be purified
in quantities amenable to full spectral characterization. The two
adducts, formed in 6 and 13% yields, were identified as 14 and
15, respectively (Scheme 7). These species had almost identical
LC/ESI-MS spectra, displaying both the protonated (m/z 520)
and the diprotonated (m/z 261) molecules (not shown). Likewise,

12-HydroxyneVirapine Amino Acid Adducts
Chem. Res. Toxicol., Vol. 23, No. 5, 2010 897
Scheme 9. Structures of 18, Obtained in the Reaction of 9
with Ethyl Valinate, the Valine Adduct (19), and the
Thiohydantoin Derivative (20)
analysis of the three-bond correlation profile observed in the
HMBC spectrum (Scheme 8), where the arginine C2 of both
rotamers (detected at 61.3 and 62.2 ppm) had connectivities to
the NVP H12 protons (3.57-3.88 ppm), which is only possible
with binding of the NVP C12 through the R-amino nitrogen of
arginine. The lack of reaction at the terminal guanidino group
presumably stems from the high basicity of this side chain group
(pKa ) 12.0, ref 36), which suggests that arginine residues in
proteins may be unlikely targets for electrophilic NVP metabolites.
Figure 4. Expanded region of the 1H-13C semiselective HMBC
spectrum of 16, displaying the connectivities between the geminal NVP-
H12 protons and the carbons of the NVP (C3, C4, and C4a) and
histidine (Imid-C2 and Imid-C5) moieties. The connectivities to the
C2 and C5 carbons of the Imid ring of histidine were decisive in
ascribing the Imid N1 as the binding site in histidine.
Scheme 8. Structure of 17, Obtained in the Reaction of 9
with Argininea
The N-terminal valine residues in hemoglobin are primary
sites of reaction with several classes of electrophiles. The
binding products thus formed can be analyzed either as valine
adducts, upon total hydrolysis of the protein, or as hydantoins
(e.g., phenylthiohydantoins), upon an “N-alkyl Edman” proce-
dure (38). Because hemoglobin is a suitable protein for
biomonitoring studies, we also sought to obtain an NVP-valine
adduct standard. The reaction of ethyl valinate with 9 resulted
in the formation of 18 (Scheme 9) in 33% yield; subsequent
basic hydrolysis of 18 gave 19 (Scheme 9) in 86% yield. LC/
ESI-MS analysis showed the sodiated and protonated molecules
(m/z 432 and 410, respectively) for 18 and the protonated
a The arrow represents the HMBC correlation of the NVP-H12 protons
with the arginine C2, which was decisive to assign the binding site.
(Scheme 7), which was isolated in 10% yield. The LC/ESI-MS
1
molecule (m/z 382) for 19. The H and 13C NMR spectra of
spectrum of 16 showed both the protonated (m/z 420) and the
1
both species displayed all of the expected resonances, although
the spectra were rather complex, due to the existence of duplicate
resonances, again suggesting restricted rotational mobility
between the amino acid and the NVP fragments, with two
predominant conformers. Nonetheless, the NVP-C12/Val-NR
connectivity was unequivocally ascribed on the basis of three-
bond correlations in the HMBC spectra between the geminal
NVP H12 protons of each conformer (e.g., two sets of doublets
at 3.56/4.01 and 3.70/3.80 ppm in 19) and the corresponding
valine C2 (65.9 and 67.5 ppm, respectively).
The N-alkyl Edman procedure leads to the specific detachment
of adducted valine residues as phenylthiohydantoins (38). This
method was originally developed for GC-MS analysis, which
explains the common use of a fluorinated phenyl isothiocyanate,
but its application to polar, thermolabile, and high molecular
weight adducts was limited. However, recent modifications,
using LC-MS/MS for the measurement of valine adducts
derivatized with phenyl isothiocyanate, have successfully broad-
ened the scope of the method (39). Because of the anticipated
low volatility of NVP-related phenylthiohydantoins, the LC-
MS/MS methodology seems a suitable approach for the
prospective analysis of these species, following ex vivo deriva-
tization of NVP-modified hemoglobin. Thus, we also prepared
a 20 (Scheme 9) standard by reaction of 18 with phenyl
isothiocyanate. The LC-ESI-MS spectrum of the product
exhibited the expected protonated molecule (m/z 499) and a
characteristic fragment ion (m/z 265) stemming from loss of
diprotonated (m/z 211) molecules, and the H NMR spectrum
indicated the presence of all of the expected NVP protons, with
the geminal H12 protons appearing as a multiplet centered at
5.41 ppm, suggestive of C12 binding to a heteroatom. Although
it was not possible to obtain a 13C NMR spectrum of 16 with a
good signal/noise ratio, carbon resonance assignments were
performed on the basis of the correlations observed in the more
sensitive inverse bidimensional HMQC and HMBC experiments.
Moreover, a semiselective HMBC experiment allowed the
observation of three-bond correlations between the NVP-H12
protons and the Imid carbons at 119.9 (Imid-C5) and 138.0
(Imid-C2) ppm (Figure 4); these correlations were entirely
consistent with a connectivity through NVP-C12/Hist-N1′,
which corroborated the structure assignment.
Reaction of 9 with arginine resulted in the formation of 17
(Scheme 8) in 6% yield. The first evidence for the formation of
an arginine adduct was achieved by LC/ESI-MS analysis, which
indicated the presence of the protonated (m/z 439) and dipro-
tonated (m/z 220) molecules. Although displaying all of the
expected nonexchangeable signals from the NVP and arginine
moieties, both the 1H NMR and the 13C NMR spectra were again
rather complex due to the existence of duplicate resonances (not
shown). As observed for other adducts, in particular the
tryptophan derivative 13, the signal duplication suggests re-
stricted rotational mobility between the amino acid and the NVP
fragments, with two predominant conformers. Clear indication
of the binding position between the two units was achieved by

898 Chem. Res. Toxicol., Vol. 23, No. 5, 2010
Antunes et al.
1
the phenylthiohydantoin moiety. The H NMR spectrum was
spectrometry techniques. Moreover, the ready formation of
tryptophan-NVP and histidine-NVP adducts suggests that,
besides the sulfur atoms in cysteine residues, the heteroaromatic
side chains of these amino acids are plausible targets for protein
modification by 5-derived electrophiles in vivo. Likewise, the
N-terminal valine residue in hemoglobin is a good candidate
as a marker of NVP-protein binding in vivo. In conclusion,
our results support the validity of the model electrophile 9 as a
surrogate for the NVP metabolite, 7, which may be used to
provide reliable, fully characterized standards for the assessment
of protein modification by NVP in vivo. This approach should
help clarify the potential role of metabolism in NVP-induced
toxicity. We are currently using the adduct standards reported
herein for a comprehensive study of binding of 9 to model
proteins in vitro using MS-based methodology. The optimized
analytical procedures will then be applied to assess potential
correlations between NVP administration and toxic responses
in vivo.
entirely consistent with two stable conformers of 20. Moreover,
although we were unable to obtain a 13C NMR spectrum of 20
with a good signal/noise ratio, carbon resonance assignments
were performed on the basis of correlations observed in the more
sensitive inverse bidimensional HMQC and HMBC experiments.
For instance, three-bond correlations were detected in the
HMBC spectrum between each specific set of geminal NVP-
H12 protons and the thiohydantoin C5 (64.8 and 66.2 ppm) and
thiocarbonyl C2 (183.4 ppm) of the corresponding conformer,
which further confirmed the ascribed structure.
Conclusions
The role of metabolism in the toxic responses associated with
NVP administration remains largely unknown, although recent
evidence increasingly suggests the involvement of electrophilic
metabolites, particularly through functionalization at C12, either
via sulfation or generation of a quinone methide (21, 24-26).
Our first report (27) on the use of 9 as a surrogate for 7 allowed
us to synthesize and characterize a series of nucleoside and
depurinating NVP-DNA adducts which, if formed in vivo,
could be relevant to NVP-induced hepatotoxicity. Despite the
fact that the sulfate anion (SO42-) is the conjugate base of a
weak acid (pKa ∼ 2) whereas mesylate is the conjugate base of
a strong acid (pKa ∼ -2) (40), a difference that may determine
distinct reactivities and regiospecificities (i.e., C-O versus S-O
bond cleavage) toward substitution and hydrolysis reactions (41),
we were confident that our model is not unreasonable. In fact,
aliphatic nucleophilic substitution reactions on monoalkylsul-
fates, involving sulfate displacement, have been described (42)
and in some instances proceeded quite readily (43). Nonetheless,
at the time of our study, even the formation of 7 in vivo was
still speculative. However, the recent reports of the detection
of 7 in vivo (21, 26), as well as of an NVP-GSH conjugate
(25, 26) and an NVP-mercapturate (26), both inferred to
involve binding through the NVP C12, have contributed
decisively to reinforce our initial hypothesis. Although it may
be argued that trapping of 7 (or its quinone methide derivative)
by GSH can provide an efficient detoxification pathway, a
similar reaction with nucleophilic side chains in proteins could
be at the onset of toxic responses. To our knowledge,
NVP-protein binding has yet to be addressed.
Acknowledgment. We thank Thomas Heinze and Conceic¸a˜o
Oliveira for the MS analyses. Thanks are also due to the
Portuguese NMR Network (IST-UTL Center) and the Portu-
guese MS Network (IST-UTL Center) for providing access to
the facilities. This work was supported in part by a research
grant from Fundac¸a˜o para a Cieˆncia e a Tecnologia (FCT),
Portugal, and FEDER (POCI/QUI/56582/2004; PPCDT/QUI/
56582/2004), by a postdoctoral fellowship (BPD/SFRH/27563/
2006) from FCT to G.C.J., and by Interagency Agreement No.
224-93-0001 between the National Center for Toxicological
Research/Food and Drug Administration and the National
Institute for Environmental Health Sciences/National Toxicology
Program. The opinions expressed in this paper do not necessarily
represent those of the U.S. Food and Drug Administration.
Supporting Information Available: 1H NMR spectra of 10
(Figure S1) and 11 (Figure S2), recorded in DMSO-d6. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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