The phenolic metabolites of the anti-HIV drug efavirenz: Evidence for distinct reactivities upon oxidation with Fre?my's salt

Article abstract of DOI:10.1016/j.ejmech.2013.12.022

Efavirenz (EFV) is a non-nucleoside reverse transcriptase inhibitor administered as first line treatment against HIV-1. The major drawbacks of EFV therapy are neurotoxicity and hepatotoxicity, which may result from bioactivation to reactive metabolites capable of reacting with bionucleophiles. We investigated the in vitro oxidation of the phenolic EFV metabolites, 7-hydroxy-efavirenz (7-OH-EFV) and 8-hydroxy-efavirenz (8-OH-EFV), with Frémy's salt. A quinoline derivative, 6-chloro-2-cyclopropyl-4- (trifluoromethyl)quinolin-7-ol, presumably stemming from a radical rearrangement, was selectively obtained from 7-OH-EFV in 10% yield. In contrast, when subjected to the same oxidation conditions, 8-OH-EFV was considerably more prone to oxidative degradation and yielded multiple products. Among these, a quinone-imine derivative was tentatively identified upon LC-ESI-MS/MS analysis of the reaction mixture. These observations demonstrate a remarkable difference in the reactivities of the two phenolic EFV metabolites under oxidative conditions. Moreover, taking into consideration the toxicological significance of quinone-imine derivatives, these findings may explain earlier reports that 8-OH-EFV is a more potent toxicant than 7-OH-EFV in model test systems.

Full text of DOI:10.1016/j.ejmech.2013.12.022

European Journal of Medicinal Chemistry 74 (2014) 7e11
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
The phenolic metabolites of the anti-HIV drug efavirenz: Evidence
for distinct reactivities upon oxidation with Frémy’s salt
*
Shrika G. Harjivan, Riccardo Wanke, João L. Ferreira da Silva, M. Matilde Marques ,
*
Alexandra M.M. Antunes
Centro de Química Estrutural, Instituto Superior Técnico, Universidade de Lisboa, 1049-001 Lisboa, Portugal
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 28 August 2013
Received in revised form
Efavirenz (EFV) is a non-nucleoside reverse transcriptase inhibitor administered as first line treatment
against HIV-1. The major drawbacks of EFV therapy are neurotoxicity and hepatotoxicity, which may
result from bioactivation to reactive metabolites capable of reacting with bionucleophiles. We investi-
gated the in vitro oxidation of the phenolic EFV metabolites, 7-hydroxy-efavirenz (7-OH-EFV) and 8-
hydroxy-efavirenz (8-OH-EFV), with Frémy’s salt. A quinoline derivative, 6-chloro-2-cyclopropyl-4-(tri-
fluoromethyl)quinolin-7-ol, presumably stemming from a radical rearrangement, was selectively ob-
tained from 7-OH-EFV in 10% yield. In contrast, when subjected to the same oxidation conditions, 8-OH-
EFV was considerably more prone to oxidative degradation and yielded multiple products. Among these,
a quinoneeimine derivative was tentatively identified upon LCeESIeMS/MS analysis of the reaction
mixture. These observations demonstrate a remarkable difference in the reactivities of the two phenolic
EFV metabolites under oxidative conditions. Moreover, taking into consideration the toxicological sig-
nificance of quinoneeimine derivatives, these findings may explain earlier reports that 8-OH-EFV is a
more potent toxicant than 7-OH-EFV in model test systems.
2
December 2013
Accepted 19 December 2013
Available online 31 December 2013
Keywords:
Efavirenz
Phenolic metabolites
Oxidation
Quinoneeimine
Quinoline
Anti-HIV drug
Ó 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
cyclopropane ring (at C14) to 8,14-dihydroxy-efavirenz (8,14-diOH-
EFV, 4) [6].
Efavirenz (EFV, (S)-6-chloro-4-(cyclopropylethynyl)-4-(tri-
fluoromethyl)-1,4-dihydro-2H-3,1-benzoxazin-2-one, 1, Scheme 1)
Whereas the molecular mechanisms underlying EFV-induced
toxicity remain to be elucidated, an involvement of the phenolic
metabolite 8-OH-EFV (2) in the onset of the toxic events elicited by
the parent drug is consistent with results from in vitro studies. The
first evidence was provided in primary human hepatocyte cultures
[7], where 8-OH-EFV was demonstrated to be a more potent
modulator of hepatic cell death than the parent compound; this
suggests that 2 may contribute to EFV-mediated hepatotoxicity.
More recently, 8-OH-EFV was proved to be at least an order of
magnitude more toxic than EFV or 7-OH-EFV in primary rat neu-
rons, which suggests that 2 is a potent neurotoxin [8]. These ob-
servations are in accordance with our recent report demonstrating
the formation of a quinone derivative (presumably 5) upon oxida-
tion of 8-OH-EFV (2) mediated by a bio-inspired nonheme Fe-
complex catalyst [9]. Quinone metabolites are of recognized toxi-
cological significance due not only to their pro-oxidant activity and
redox cycling, which yields reactive oxygen species, but also to their
reactivity as Michael acceptors, capable of yielding covalent ad-
ducts with bionucleophiles [10,11]. Of note, covalent adducts
formed upon reaction of quinones with nucleophilic sites of pro-
teins (mainly cysteine residues) have been proposed to be at the
onset of both hepatotoxic [12] and neurotoxic [13] events. Taking
is
a non-nucleoside reverse transcriptase inhibitor (NNRTI)
administered as first-line treatment against HIV [1]. Despite its
efficacy, a major limitation of EFV use is its association with clini-
cally restrictive neurotoxic and hepatotoxic events [2e5]. EFV is
metabolized by cytochrome P450 (CYP), undergoing primary
oxidation on the aromatic ring to the phenolic products 8-hydroxy-
efavirenz (8-OH-EFV, 2, Scheme 1) (major) and 7-hydroxy-efavir-
enz (7-OH-EFV, 3) (minor) and secondary oxidation on the
Abbreviations: CYP, cytochrome P450; EFV, efavirenz; ESI, electrospray ioniza-
tion; HIV, human immunodeficiency virus type 1; HPLC-DAD, high performance
liquid chromatography with diode array detection; LCeESI(þ)eMS, high perfor-
mance liquid chromatography with mass spectrometric detection using electro-
spray ionization in the positive mode; MS/MS, tandem mass spectrometry; NNRTI,
non-nucleoside reverse transcriptase inhibitor.
*
Corresponding authors. Tel.: þ351 21 8417627, 351 21 8419200; fax: þ351 21
464455.
E-mail addresses: matilde.marques@tecnico.ulisboa.pt (M.M. Marques),
alexandra.antunes@tecnico.ulisboa.pt (A.M.M. Antunes).
8
0223-5234/$ e see front matter Ó 2014 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.12.022

8
S.G. Harjivan et al. / European Journal of Medicinal Chemistry 74 (2014) 7e11
Scheme 1. Efavirenz (1) and its phase I metabolites, 8-OH-EFV (2), 7-OH-EFV (3), and 8,14-diOH-EFV (4). Also shown are the putative oxidative bioactivation pathways to the
electrophilic quinoid species 5 and 6, conceivably capable of reacting with bionucleophiles and yield covalent adducts.
into consideration the structural features of the two phenolic EFV
metabolites, both 2 and 3 could conceivably generate the ortho-
quinone species, 7,8-EFV-quinone (5, Scheme 1), upon metabolic
oxidation. In contrast, the formation of the potentially toxic
quinoneeimine electrophile 6 is only possible from 8-OH-EFV. As
such, a better understanding of the different behaviours of these
two metabolites under oxidative conditions is essential to elucidate
if 2 and 3 are both prone to oxidative bioactivation and if different
oxidized derivatives of 2 and 3 could play distinct roles in the toxic
events associated with EFV administration. Therefore, with the
ultimate goal of preparing synthetic standards to investigate the
oxidative biotransformation of 7-OH-EFV and 8-OH-EFV in vivo, we
examined their oxidation in vitro using Frémy’s salt.
ray diffraction analysis. In contrast, no evidence for the formation of
an isomeric derivative of 7 was obtained upon oxidation of 8-OH-
EFV under similar conditions (Fig. 1); instead a more complex
mixture was obtained. Thus, the LCeESI(þ)eMS chromatograms
from the oxidation of 7-OH-EFV after 5 min and 24 h (Fig. 1B and D)
clearly show the selective formation of a product, subsequently
identified as the quinoline derivative 7, corresponding to a signal
with a 12.4 min retention time under the elution conditions used
3
5
þ
(cf. Supporting information), and with m/z 288 [( Cl)M þ H] and
37
þ
290 [( Cl)M þ H] ; the parent compound, 7-OH-EFV, was still
present in a significant amount after 24 h. In contrast, the corre-
sponding chromatograms for the oxidation of 8-OH-EFV (Fig. 1A
and C) indicate the formation of multiple products. Among these,
we were able to identify m/z values compatible with the protonated
molecule of the racemic quinoneeimine 6 (m/z 330 and 332).
Further evidence for the formation of this electrophilic metabolite
was provided upon LCeESIeMS/MS analysis of ion m/z 330. Indeed,
MS/MS of this ion yielded a fragment ion at m/z 266 (Fig. 2), cor-
responding to the loss of the ethynylcyclopropane moiety from the
protonated molecule, along with two other fragment ions at m/z
2
. Results and discussion
2.1. Oxidation of 7-OH-EFV (3) and 8-OH-EFV (2)
The synthesis of racemic 7-OH-EFV (3) was conducted by
adaptation of the multi-step procedure reported by Markwalder
et al. [14] (cf. Supporting information); 8-OH-EFV (2) was prepared
by direct oxidation of EFV mediated by a bio-inspired nonheme Fe-
complex, as described in Wanke et al. [9]. The oxidation of the two
phenolic metabolites was subsequently carried out with the radical
potassium nitrosodisulfonate (Frémy’s salt), which is frequently
employed to obtain quinones from phenolic compounds [15e17].
Additionally, this model oxidant has the advantage of mimicking
the processes involved in enzyme-mediated metabolic oxidations
3
02 and 238, corresponding to the loss of CO from the protonated
molecule and from the fragment ion at m/z 266, respectively, which
are fully consistent with the assigned structure. This oxidation
product was only detected at the beginning of the reaction, which
[18,19]. The experimental conditions used correspond to those we
optimized for the oxidation of a phenolic metabolite of the anti-HIV
drug nevirapine [20], consisting on the use of 1.5 eq. of Frémy’s salt
in a solution of CH
Following 24-h incubation, the quinoline derivative
Scheme 2) was selectively obtained from racemic 7-OH-EFV in 10%
yield and was subsequently fully characterized by NMR, MS and X-
3
CN/phosphate buffer (pH 7.4).
a
7
(
Scheme 2. Formation of the quinoline derivative 7 upon oxidation of racemic 7-OH-
EFV (3) with Frémy’s salt.

S.G. Harjivan et al. / European Journal of Medicinal Chemistry 74 (2014) 7e11
9
Fig. 1. Comparison of LCeESI(þ)eMS profiles of the reaction mixtures obtained upon oxidation of 8-OH-EFV (2) and 7-OH-EF (3) with Frémy’s salt: A. oxidation of 2 after 5 min; B.
oxidation of 3 after 5 min; C. oxidation of 2 after 24 h; D. oxidation of 3 after 24 h; and mass spectra of: E. quinoneeimine 6; and F. quinoline 7. The elution conditions are outlined in
the Supporting information.
demonstrates its transient character. It should be outlined that
although we were unable to detect m/z values compatible with the
quinone 5, its formation under the oxidative conditions used
cannot be excluded; in fact, the presumed instability of this elec-
trophilic intermediate may have prevented its detection. Multiple
products (with higher m/z values) were detected both after 5 min
and 24 h of 8-OH-EFV oxidation; the LCeESI(þ)eMS chromato-
graphic profiles were considerably different at these two time
points, demonstrating the instability of the oxidation products
initially formed. Although the relatively high m/z values corre-
sponding to these species suggest the occurrence of coupling pro-
cesses, the structural assignment of the oxidation products of 8-
OH-EFV was precluded by a number of limitations, including: i)
the scarce structural information obtained from tandem mass
spectrometry (MS/MS) analysis; ii) the large number (and small
amounts) of products, that prevented an efficient chromatographic
purification and consequently NMR analysis. One additional
distinctive feature between the behaviours of 2 and 3 is their
relative stability under the oxidative conditions used. Hence,
whereas 8-OH-EFV was only detected in trace amounts after 24 h of
incubation (Fig. 1C), approximately 90% of the initial amount of 7-
OH-EFV remained unreacted after the same time period (Fig. 1D),
in accordance with the isolated yield of the quinoline derivative 7.
Moreover, even with longer incubation times (36 and 48 h), only 7-
OH-EFV and the quinoline 7 were detected by HPLC-DAD and LCe
ESI(þ)eMS analysis of reaction mixtures from the oxidation of 7-
OH-EFV (data not shown). Taken together, these observations un-
equivocally show a remarkable difference in the reactivity of the
two phenolic EFV metabolites under oxidative conditions.
The considerable stabilities of 7-OH-EFV and its quinoline de-
rivative 7 compared to the relatively short life of 8-OH-EFV under
the oxidative conditions used in our work, and the fact that Frémy’s
salt is often used to mimic the one-electron oxidation steps of
enzyme-mediated metabolic oxidations [18,19], suggest that 8-OH-
EFV may be more prone than 7-OH-EFV to undergo oxidative bio-
activation in vivo. Moreover, the MS evidence for the transient
formation of an electrophilic quinoneeimine (6) upon oxidation of
8-OH-EFV, along with the recognized toxicological significance of
quinoneeimine derivatives [11], suggests that oxidative bio-
activation of the major EFV metabolite plays a role in the onset of
the toxic events elicited by the parent drug. Indeed, oxidation of 8-
OH-EFV to unstable quinoid electrophiles (i.e., 6 and/or 5) appears
as a plausible basis for the distinct toxicity profiles of the two
phenolic EFV metabolites in primary rat neurons [8].
On the other hand, the absence of reaction products other than
the quinoline 7, even after prolonged incubation with Frémy’s salt,
indicates that formation of the electrophilic quinone 5 from 7-OH-
EFV is improbable. Additionally, our results suggest that the quin-
oline 7 is not prone to subsequent aromatic oxidations that might
also lead to quinoid structures. Therefore, product 7 (a putative EFV
metabolite in vivo) is unlikely to have toxicological relevance.
2.2. Spectroscopic characterization of quinoline 7
The ESI(þ) mass spectral data for the compound were entirely
consistent with structure 7, with two signals at m/z 288 and 290,
3
5
corresponding to the two isotopical protonated molecules [( Cl)
þ
37
þ
1
13
M þ H] and [( Cl)M þ H] , respectively. The H- and C NMR
spectra were also compatible with the proposed structure (cf.
Supporting information); in particular, the high conjugation of
Fig. 2. Tandem mass spectrum (m/z 330) at retention time 9 min, following LCe
ESI(þ)eMS/MS analysis of reaction mixture obtained upon 5 min of the oxidation of 8-
OH-EFV (2) with Frémy’s salt.
1
19
the bicyclic aromatic ring allowed the detection of useful He
F
13
19
5
and
Ce
F
coupling constants (H5eF,
J
¼ 1:8 Hz; C3eF,
HF

10
S.G. Harjivan et al. / European Journal of Medicinal Chemistry 74 (2014) 7e11
Fig. 3. ORTEP plot representation drawn with 50% probability, showing the atomic labelling scheme for compound 7 in the two distinct asymmetric units identified in the cell.
3
¼ 5:0 Hz). However, the lack of informative He13C three-bond
1
J
oxygen (not shown). This observation supports the hypothesis that
7 was formed by a radical mechanism [22]. As such, the ring
opening mechanism is proposed to be induced by the initial for-
mation of an unstable phenoxyl radical 8, generated upon oxidation
of 7-OH-EFV (3) by Frémy’s salt radical (Scheme 3).
This phenoxyl radical is a resonance hybrid with an activated
para-position (contributor 9), which subsequently undergoes ho-
molytic bond cleavage of the carbamate moiety, assisted by the
CF
correlations in the HMBC spectrum precluded a definitive struc-
tural assignment based exclusively on NMR experiments. This
could only be possible by X-ray diffraction, which showed unam-
biguously that the product from oxidation of 7-OH-EFV was 6-
chloro-2-cyclopropyl-4-(trifluoromethyl)quinolin-7-ol (7, Fig. 3).
ꢀ
ꢀ
Bond lengths (A) and angles ( ) in the structure of 7 were in
accordance with the expected values for quinoline compounds
[
1
21]: the internal angles of the condensed rings were in the range of
15.9(4)e122.7(5) and the bicyclic cores were nearly planar (an-
2
release of CO . The resulting intermediate 10 is proposed to un-
ꢀ
dergo subsequent cyclization, followed by aromatization to yield
the quinoline core. The formation of quinoline derivatives by ana-
logues of the intermediate 9 [14,23e25] supports this hypothesis.
gles between the <C4aeC5eC6eC7eC8eC8a> and <C2eC3eC4e
C4aeC8aeN1> planes and between the <C4a eC5 eC6 eC7 eC8 e
C8a > and <C2 eC3 eC4 eC4a eC8 eN1 > planes were 3.27 and
1
N/HeO H-bonds for each asymmetric unit, namely between N1 of
one unit and the H1eO1 of the adjacent molecule that links to the
previous and the subsequent element, forming a 3D-chain (see
Fig. S1 in the Supporting information). The latter appeared to be
reinforced in the crystal packing by two
both asymmetric units, specifically between the centroid of each
0
0
0
0
0
0
0
0
0
0
0
0
0
ꢀ
ꢀ
.93 , respectively). Crystal packing revealed two intermolecular
3. Conclusions
The quinoline derivative 7 was selectively obtained upon
oxidation of the phenolic metabolite of EFV, 7-OH-EFV (3), with
Frémy’s salt. Our data indicate that this oxidative pathway of 3 is
favoured over the conceivable formation of the potentially toxic
quinone 5, that was previously identified by our group in the
oxidation of 8-OH-EFV (2) with a bioinspired non-heme Fe(II)
catalyst. One additional potentially toxic metabolite, the quinonee
imine 6, was tentatively identified upon oxidation of 8-OH-EFV
with Frémy’s salt. These contrasting behaviours under oxidative
conditions may explain the distinct toxicities of the two phenolic
EFV metabolites observed in primary rat neuron cultures [8].
Indeed, when formed metabolically, both quinones and quinonee
imines are electrophilic intermediates of unquestionable toxico-
logical significance, due not only to their pro-oxidant activity and
redox cycling ability, which contribute to generate reactive oxygen
p-stacking interactions for
0
0
0
0
0
unit (i.e., C4aeC5eC6eC7eC8eC8a and C2 eC3 eC4 eC4a eC8a e
N1 ) and the analogues of the adjacent unit (i.e., C2eC3eC4eC4ae
C8aeN1 and C4a eC5 eC6 eC7 eC8 eC8a , correspondingly).
0
0
0
0
0
0
0
2
.3. Proposed mechanism for the formation of quinoline 7
The identification of trace amounts of 7 upon HPLC-DAD anal-
3
ysis of a solution of 7-OH-EFV (3) in CH CN/50 mM phosphate
buffer (pH 7.4), in the absence of oxidant, suggests that this
phenolic derivative can (to some extent) be oxidized by molecular
Scheme 3. Proposed mechanism for the formation of the quinoline derivative 7, upon oxidation of 7-OH-EFV (3) with Frémy’s salt.

S.G. Harjivan et al. / European Journal of Medicinal Chemistry 74 (2014) 7e11
11
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[
Acknowledgements
We thank the Portuguese NMR and MS Networks (IST-UTL
Nodes) for providing access to the facilities. This work was sup-
ported in part by Fundação para a Ciência e a Tecnologia (FCT),
Portugal, through research grants PTDC/QUI-QUI/113910/2009 and
RECI/QEQ-MED/0330/2012 and strategic funds to Centro de Quí-
mica Estrutural (PEst-OE/QUI/UI0100/2013). RW and SGH also
thank FCT for postdoctoral (SFRH/BPD/70953/2010) and doctoral
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(
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[
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