Biomimetic oxidation of aromatic xenobiotics: Synthesis of the phenolic metabolites from the anti-HIV drug efavirenz

Article abstract of DOI:10.1039/c2ob25212k

We report the oxidation of the first line anti-HIV drug efavirenz (EFV), mediated by a bio-inspired nonheme Fe-complex. Depending upon the experimental conditions this system can be tuned either to yield the major EFV metabolite, 8-hydroxy-EFV, in enantiomerically pure form or to mimic cytochrome P450 (CYP) activity, yielding 8-hydroxy-EFV and 7-hydroxy-EFV, the two phenolic EFV metabolites reported to be formed in vivo. The successful oxidation of the anti-estrogen tamoxifen and the equine estrogen equilin into their CYP-mediated metabolites supports the general application of bio-inspired nonheme Fe-complexes in mirroring CYP activity.

Full text of DOI:10.1039/c2ob25212k

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PAPER
Biomimetic oxidation of aromatic xenobiotics: synthesis of the phenolic
metabolites from the anti-HIV drug efavirenz
Riccardo Wanke,* David A. Novais, Shrika G. Harjivan, M. Matilde Marques and Alexandra M. M. Antunes
*
Received 27th January 2012, Accepted 5th April 2012
DOI: 10.1039/c2ob25212k
We report the oxidation of the first line anti-HIV drug efavirenz (EFV), mediated by a bio-inspired
nonheme Fe-complex. Depending upon the experimental conditions this system can be tuned either to
yield the major EFV metabolite, 8-hydroxy-EFV, in enantiomerically pure form or to mimic cytochrome
P450 (CYP) activity, yielding 8-hydroxy-EFV and 7-hydroxy-EFV, the two phenolic EFV metabolites
reported to be formed in vivo. The successful oxidation of the anti-estrogen tamoxifen and the equine
estrogen equilin into their CYP-mediated metabolites supports the general application of bio-inspired
nonheme Fe-complexes in mirroring CYP activity.
Introduction
Efavirenz [EFV, (S)-6-chloro-4-(cyclopropylethynyl)-4-(tri-
fluoromethyl)-1,4-dihydro-2H-3,1-benzoxazin-2-one, Scheme 1]
is a non-nucleoside reverse transcriptase inhibitor (NNRTI)
administered as a first-line treatment against HIV.1 Despite its
efficacy, a major shortcoming of EFV use is its association with
neurotoxic and hepatotoxic events.2–4 Although the reasons for
the drug’s adverse effects are currently not clear, there is evi-
dence that metabolic activation to reactive electrophiles capable
of reacting with bionucleophiles leads to the formation of
covalent adducts that are involved in the initiation of species-
specific toxic outcomes.5 EFV is extensively metabolized by
Scheme 1 Structures of efavirenz (EFV) and its metabolites: 8-
cytochrome P450 (CYP), undergoing primary oxidation on the
aromatic ring to the phenolic products 7-OH-EFV (minor) and
8-OH-EFV (major), and secondary oxidation on the cyclopro-
pane ring (at C14) to 8,14-diOH-EFV (Scheme 1).6 Very
recently, an in vitro study using primary human hepatocytes
revealed that 8-OH-EFV was a more potent modulator of hepatic
cell death than the parent compound, suggesting that this
primary metabolite could potentially contribute to EFV-mediated
hepatotoxicity.7 This correlation between 8-OH-EFV and toxicity
may be very significant, due to the potentially high in vivo sys-
temic exposure to this metabolite.8 Moreover, given that EFV
increases the mRNA and protein expression of CYP2B6,9–11
chronic EFV use results in increased formation of 8-OH-EFV.
Indeed, in patients with the CYP2B6*1/1 genotype the EFV/8-
OH-EFV ratio reaches approximately 0.98 and 0.74 after 4
OH-EFV, 7-OH-EFV, and 8,14-diOH-EFV.
weeks and 16 weeks of treatment, respectively,8 and over a
period of time the concentration of the metabolite can exceed
that of the parent compound. In order to assess unequivocally the
role of metabolic EFV activation in the initiation of toxic events
elicited by chronic use of the drug, the availability of considerable
amounts of enantiomerically pure EFV metabolites is essential, so
as to enable in vitro and in vivo toxicological studies.
The syntheses of the racemic Phase I EFV metabolites have
been described;12,13 however, the low efficiency of the multistep
synthetic pathways precludes large scale preparations of the pure
enantiomers. Particularly, the racemate of the major EFV metab-
olite, 8-OH-EFV, is obtained after nine synthetic steps in 29%
yield; following chiral HPLC separation the enantiomerically
pure product is obtained in 14% overall yield.13 This limitation
motivated us to investigate new synthetic strategies in order to
simplify the procedure and maximize the yield of the enantio-
merically pure metabolite. Despite the significant challenge, we
considered the feasibility of a biomimetic direct oxidation of
Centro de Química Estrutural, Instituto Superior Técnico, Universidade
Técnica de Lisboa, 1049-001 Lisboa, Portugal. E-mail: alexandra.antu-
nes@ist.utl.pt, riccardowanke@ist.utl.pt; Fax: +351-21-8464455;
Tel: +351-21-8417627, +351-21-8419388
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EFV into its Phase I metabolites since the success of this strategy
could drastically change the common approach of research in
this field. Among the large variety of ‘bio-inspired’ catalysts that
mimic the versatile CYP mechanisms, the class of nonheme
iron-based complexes constitutes one of the most explored
groups of catalysts for the oxidation of different substrates,
including the hydroxylation of alkanes, alkenes and aromatic
compounds and the epoxidation of olefins.14–17 In particular, the
N4-tetradentate FeII family of compounds (e.g., [FeII(bpmen)
(OTf)2], bpmen: N,N′-bis-(2-pyridylmethyl)-1,2-diaminoethane)
is reported18–20 to pass through the formation of a high valence
FeVvO species, suggesting that it is capable of accessing the
formal FeV oxidation state commonly associated with the active
oxidant in CYP and other heme enzymes.21,22 Therefore, we
explored this possibility and report herein the development of a
method for direct aromatic hydroxylation of EFV and other
xenobiotics, consisting on the use of the easily accessible
[FeII(bpmen)(OTf)2]–H2O2 oxidant system in the presence of
acetic acid.
presumably due to 8-OH-EFV degradation into further oxidized
derivatives. The use of an alternative peroxide such as m-chloro-
perbenzoic acid (m-CPBA, Table 1, entry 26) provided a lower,
though still significant, yield. However, when tBuOOH was
tested, even under microwave irradiation and/or high temperature
(data not shown) no reaction occurred, plausibly due to steric
hindrance reasons. Similarly to what has been reported for other
substrates,23,24 the use of an acid, as co-catalyst (co-CAT),
improved the oxidation efficiency substantially (Table 1, entries
1 and 2 vs. 27). Since the nature of the adequate acid is highly
related with the catalytic system,25,28 different acids were
screened (e.g., nitric acid, pyrazine-2-carboxylic acid, acetic
acid; results not shown); of these, acetic acid led to cleaner reac-
tion mixtures and the best yields. The addition of ethyl acetate
(AcOEt) as co-solvent (up to 50% v/v) significantly enhanced
the yield, probably due to increased EFV solubility (Table 1,
entries 2 vs. 3 and 23 vs. 22). However, using higher percentages
of AcOEt resulted in lower reaction efficiencies (Table 1, entries
19 and 20 vs. 14 and 15 vs. 12 and 13), probably due to
decreased solubility of 1.
Whereas these types of complexes are reported to react very
fast with H2O2,29 for this system we observed an yield increment
with increasing reaction times (Table 1, entries 18 vs. 19 vs. 20,
cf. Experimental section). Likewise, better yields were obtained
when reactions were conducted at 40 °C (Table 1, entries 19 and
20 vs. 16 and 17), albeit this type of transformations are usually
conducted at room temperature.29
Results and discussion
The establishment of a procedure for the preparation of signifi-
cant amounts of 8-OH-EFV from direct oxidation of EFV was
developed considering the best yields, combined with the cheap-
est, easiest and cleanest processes, in order to extend this new
methodology to large scale preparations. To select the most
effective system, screening reactions were conducted under stan-
dard conditions,23,24 using an acetonitrile (AcCN) solution of
EFV, 10 mol% of catalyst, and hydrogen peroxide (0.45 eq.) in
the presence of acetic acid. The yields were calculated from the
HPLC peak areas, corrected for the corresponding molar absorp-
tivity at the monitored wavelength (254 nm). Among the tested
Arene oxidations by these systems tend to result in Fe(III)
bound phenolate complexes, that block catalytic activity.29 To
explore if product yields could be improved by increasing cata-
lyst loadings, parallel assays were conducted with 0.5, 1.5, 5, 10,
20, 40, 60, and 100 mol% of 1. Whereas a considerable yield
improvement was observed when the mol% of catalyst was
changed from 0.5 to 10 (Table 1, entries 25 vs. 24 vs. 23), the
use of larger amounts of Fe catalyst did not result in improved
yields (Table 1, entries 19 and 20 vs. 11 and 10 vs. 9 and 8 vs. 7
and 6). It should be outlined that, although modest, the TONs
(5.8) obtained upon use of 0.5 mol% of 1 (Table 1, entry 25)
substantiate the catalytic capacity of the system. However, nearly
stoichiometric amounts of the product were obtained with
10 mol% of 1, clearly showing a non-catalytic behaviour of the
system under these conditions, which is consistent with the yield
improvement observed with increasing reaction times. Given that
our ultimate goal was the development of a synthetic method for
the preparation of significant amounts of 8-OH-EFV, optimiz-
ation led to the selection of conditions providing the best yield.
These consisted of the use of an AcCN–AcOEt (1 : 1) solution
of EFV, 10 mol% of FeII(bpmen)(OTf)2, and hydrogen peroxide
(1.35 eq.) in the presence of acetic acid (5.0 eq.) at 40 °C for 3
days, that resulted in a 12.0% yield of 8-OH-EFV (calculated
from the HPLC peak areas). A preparative assay was performed,
starting from 100 mg of EFV under the optimized conditions,
resulting in 9.5% isolated yield of 8-OH-EFV (cf. Experimental
section). Parallel reactions were performed to test different chro-
matographic purifications of the reaction mixture and, regardless
of the technique used (e.g., semi-preparative HPLC, column
chromatography or preparative TLC) both the product and the
starting material were isolated quantitatively. A scale-up study,
starting from 1000 mg of EFV, revealed that the best results were
catalysts (Table 1) were [Fe(bpmen)(OTf)2]25,26 (1), [Fe(S,S-
23
PDP)(NCCH3)2] (SbF6)2
(2, S,S-PDP: 2-({(S)-2-[(S)-1-
(pyridin-2-ylmethyl)pyrrolidin-2-yl]pyrrolidin-1-yl}methyl)pyri-
dine), [FeCl2(Py2S2)]24 (3, Py2S2: 1,6-bis(2′-pyridyl)-2,5-dithia-
hexane) and [Cu(bpmen)(OTf)2] (4). Complexes 3 and 4
displayed limited activity (Table 1, entries 29 and 30), while
both the use of a simple FeII salt (i.e., Fe(OTf)2, Table 1, entry
31) and the absence of catalyst (Table 1, entry 32) were comple-
tely ineffective. Of all the assessed catalysts, those containing
the N4–FeII core appeared to be the most efficient. Although cat-
alysts 1 and 2 had comparable efficiencies (Table 1, entries 2
and 28), the first was chosen for subsequent reactions due to the
availability of an easy and cost-efficient method for its prep-
aration,25,26 contrasting with the high cost of the commercially
available 2. It is noteworthy that whereas 8-OH-EFV was
formed in moderate yield under Fenton’s oxidation conditions
(Table 1, entry 33), the complexity of the reaction mixture
clearly indicated non-selective oxidation and precluded a syn-
thetic scale-up. Additional studies were carried out towards
process optimization, using 1 as the selected catalyst.
Although an increment in the number of hydrogen peroxide
equivalents from 0.45 to 1.35 (Table 1, entries 2 vs. 23) led to
ca. 1.6-fold increase in the yield of 8-OH-EFV, a portionwise
addition27 of the same amount of oxidant, over an extended
period of time, resulted in a lower efficiency (Table 1, entry 5),
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Table 1 Optimization of experimental conditions for the biomimetic oxidation of EFV into 8-OH-EFV
Entry
CATa
CAT (%mol)
Time (h)
co-CAT
H2O2 (eq.)
AcCN–AcOEt
Yieldb (%)
1
2
3
4
5
6
7
8
(1)
10
10
10
10
10
100
100
60
60
20
20
10
10
10
10
10
10
10
10
10
10
10
10
1.5
0.5
10
10
10
10
10
10
None
AcOH (0.5 eq.)
AcOH (5 eq.)
0.45
0.45
0.45
0.9
100 : 0
100 : 0
75 : 25
100 : 0
100 : 0
50 : 50
50 : 50
50 : 50
50 : 50
50 : 50
50 : 50
30 : 80
30 : 80
40 : 60
40 : 60
50 : 50
50 : 50
50 : 50
50 : 50
50 : 50
50 : 50
50 : 50
100 : 0
100 : 0
100 : 0
100 : 0
100 : 0
100 : 0
100 : 0
100 : 0
100 : 0
100 : 0
2.5
4.7
6.6
6.9
5.7
2.1
1.0
1.3
1.1
2.2
1.3
0.6
0.5
0.9
72
72
72
72
72
2
24
2
24
2
24
2
2
24
24
2
5 min
2
24
48
72
72
72
72
72
72
72
72
72
72
72
1.35c
1.35
1.35
1.35
1.35
1.35
1.35
1.35
1.35
1.35
1.35
1.35
1.35
1.35
1.35
1.35
1.35
1.35
1.35
1.35
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33h
0.9
1.3d
0.6d
1.8
3.2 (2.8)e
4.4 (5.0)e
4.8 (5.7)e
12.0 (9.5)e
7.7
4.4
2.9
3.7
<1
6.2
<1
1.35
f
None
AcOH (5 eq.)
0.45
0.45
0.45
0.45
0.45
0.45
(2)
(3)
(4)
Fe(OTf)2
None
<1
n.r.g
n.r.
4.8
a (1): [FeII(bpmen)(OTf)2];29,30 (2): [Fe(S,S-PDP)(NCCH3)2] (SbF6)2;31 (3): [FeCl2(Py2S2)];32 (4): [Cu(bpmen)(OTf)2]. b Unless specified, the
reactions were conducted at 40 °C. The yields were calculated from the HPLC peak areas, corrected for the corresponding molar absorptivity at the
monitored wavelength (254 nm). The numbers in parentheses represent the yields of isolated products. c Four sequential additions of 0.33 eq. of the
oxidant at 1 hour intervals. d Reaction at room temperature. e Starting with 100 mg of EFV, the crude reaction mixture was purified by flash
chromatography on silica (CH2Cl2 to 95 : 5 CH2Cl2–MeOH). f With 0.45 eq. of m-CPBA. g n.r., no reaction. h Fenton’s oxidation conditions:
FeCl2·4H2O, 0.1 M HCl, 0.1 M H2O2.
Scheme 2 Direct hydroxylation of EFV mediated by the N4-tetradentate FeII complex [Fe(bpmen)(OTf)2].
obtained at a 50 mM substrate concentration (cf. Experimental
section).
Experimental section). This cost-efficient one-pot strategy rep-
resents a considerable improvement when compared with the
already available methods for the C8 hydroxylation of EFV,12,13
avoiding a laborious multi-step synthetic approach and the resol-
ution of racemic mixtures.
In summary, direct EFV hydroxylation, using the
[FeII(bpmen)(OTf)2]–H2O2 oxidant system in the presence of
acetic acid afforded the enantiomerically pure metabolite, 8-
OH-EFV, in 9.5% isolated yield with nearly quantitative recovery
of the unreacted EFV (ca. 98%). A three-cycle run provided a
22% total yield of the same metabolite (Scheme 2, cf.
Contrasting with previously reported arene oxidations29–36 cat-
alyzed by FeII complexes, that involve hydroxylations of a series
of simple aromatic substrates, our observation refers to an
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Fig. 1 (A) Total ion LC-ESI(−)-MS chromatogram of the reaction
mixture obtained upon oxidation of 8-OH-EFV under the optimized con-
ditions described in the text; (B) extracted ion chromatogram (m/z 344)
and corresponding mass spectrum. The elution conditions are outlined in
the Experimental section.
Fig. 2 HPLC-DAD analyses of: (A) standard of 7-OH-EFV; (B) the
reaction mixture obtained upon EFVoxidation mediated by [FeII(bpmen)
(OTf)2] using 150 mM EFV and 1.8 eq. of H2O2; (C) standard of 8-
OH-EFV. The elution conditions are outlined in the Experimental
section. The insets represent the UV spectra obtained under the con-
ditions of the HPLC run.
unprecedented complex molecule that is selectively converted
into its major phenolic metabolite. Furthermore, the examples of
most efficient catalysis mediated by nonheme FeII systems entail
the ortho-hydroxylation of benzoic acids,16,37,38 due to the
favourable formation of a cyclic iron–arene complex involving
the carbonyl group of the substrate. By contrast, the lack of an
ortho-substituent capable of assisting catalysis typically results
in a dramatic decrease of the hydroxylation efficiency. As such,
the single-cycle 9.5% isolated yield obtained for EFV hydroxy-
lation represents a very good conversion for a complex, rigid,
and hindered core lacking catalytic-assisting groups. In fact, the
EFV molecule displays an expected steric hindrance in the vicin-
ity of the ring fusion, that namely inhibited the activity of bulky
organic peroxides, such as tBuOOH, as discussed above. It
should also be noted that, similarly to what is reported for the
oxidation of other aromatic C–H bonds,39–42 any attempts to
improve the process efficiency led to partial degradation of the
phenolic product, most likely due to further oxidation to cate-
chols, and/or quinone species.28,29 Indeed, LC-ESI(−)-MS/MS
(m/z 344) analysis of the reaction mixture (Fig. 1) provided pre-
liminary evidence for the formation of a quinone derivative upon
oxidation of the isolated 8-OH-EFV under the optimized con-
ditions described above.
Scheme 3 Structures of tamoxifen and its hydroxylated metabolites, 4-
hydroxytamoxifen and α-hydroxytamoxifen.
catalysis (Fig. 2).43 The identification of the minor metabolite, 7-
OH-EFV, was performed on the basis of undistinguishable UV
profiles and identical retention times when compared with the
corresponding synthetic standard under the same elution
conditions.
To explore if this oxidation system could be successfully used
to yield phenolic metabolites from other xenobiotics of interest,
we oxidized the non-steroidal anti-estrogen tamoxifen
(Scheme 3), extensively prescribed as an adjunct
Interestingly, when more concentrated solutions (>100 mM)
were used, the formation of some byproducts of over-oxidation
became significant. Indeed, upon the use of a larger excess of
hydrogen peroxide (1.8 eq.) and a more concentrated EFV sol-
ution (150 mM), HPLC-DAD analysis provided evidence for the
formation of the two phenolic EFV metabolites, 8-OH-EFV and
7-OH-EFV, in a ratio similar to that obtained under CYP
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Scheme 4 Structures of equilin and its metabolites 4-hydroxyequilin,
4-hydroxyequilin-o-quinone and 4-hydroxyequilenin-o-quinone.
chemotherapeutic agent against breast cancer,44 and the equine
estrogen equilin (Scheme 4), one of the components of Premarin,
widely used in hormone replacement therapy.45
Tamoxifen undergoes multiple metabolic conversions, namely
involving the formation of two hydroxylated metabolites, the
phenol 4-hydroxytamoxifen and the allylic alcohol, α-hydroxyta-
moxifen (Scheme 3).44 Upon oxidation of tamoxifen with the
[FeII(bpmen)(OTf)2]–H2O2 system (cf. Experimental section),
the reaction mixture was analyzed by LC-ESI-MS/MS, which
showed an ion of m/z 388, corresponding to the protonated
mono-hydroxy-tamoxifen metabolites. Based upon comparison
of retention times and MS/MS fragmentation patterns of the
reaction mixture (Fig. 3, panel A) with those of the synthetic
standards of 4-hydroxytamoxifen (Fig. 3, panel B) and α-hydro-
xytamoxifen (Fig. 3, panel C) we obtained unequivocal evidence
for the formation of the phenolic tamoxifen metabolite.
Fig. 3 LC-ESI-MS/MS (m/z 388) chromatogram and MS/MS mass
spectra of: (A) reaction mixture obtained from oxidation of tamoxifen
catalyzed by [FeII(bpmen)(OTf)2]; (B) the synthetic standard of 4-hydro-
xytamoxifen (mixture of E and Z isomers); (C) the synthetic standard of
α-hydroxytamoxifen. The elution conditions are outlined in the Exper-
imental section.
Equilin is primarily metabolized in vivo to the catechol 4-
hydroxyequilin, which is further oxidized to 4-hydroxyequilin-o-
quinone
and,
ultimately,
4-hydroxyequilenin-o-quinone
(Scheme 4).45 LC-ESI-MS analysis of the reaction mixture
obtained upon oxidation of equilin with the system described
herein (cf. Experimental section) provided evidence for the pres-
ence of the protonated molecules of the catechol, 4-hydroxyequi-
lin (m/z 285), and the quinones, 4-hydroxyequilin-o-quinone (m/
z 283) and 4-hydroxyequilenin-o-quinone (m/z 281) (Fig. 4).
These results demonstrate that the [FeII(bpmen)(OTf)2]–H2O2
system can be used to access phenolic metabolites and their oxi-
dized derivatives in vitro, in a biomimetic fashion that mirrors
CYP activity.
metabolite, obtained in a preparative scale, is now accessible in
adequate amounts for molecular toxicology studies that are
expected to clarify its relevance to the toxic events elicited by
the parent drug. Additionally, upon tuning the experimental con-
ditions, this system successfully simulated CYP-mediated EFV
metabolism by providing the other reported phenolic EFV
metabolite, 7-OH-EFV. Moreover, the successful oxidation of
tamoxifen and equilin into their phenolic metabolites supports
the general application of bio-inspired nonheme Fe-complexes
for in vitro access to phenolic species (and their oxidized
quinone derivatives), typically formed via CYP-mediated
hydroxylation of aromatic compounds. The possibility of apply-
ing an oxidation system mirroring the CYP metabolic pathways
anticipates the usefulness of the approach described herein for
molecular toxicology studies of a large variety of toxic aromatic
xenobiotics. Indeed, the present work represents a proof of
concept regarding bioinspired oxidation technologies and opens
new opportunities for this chemistry.
Conclusions
We report herein the application of a flexible [FeII(bpmen)
(OTf)2]–H2O2 system to the oxidation of the first-line NNRTI
EFV, which represents the first model for biomimetic oxidation
of EFV. This oxidation system was successfully tuned for selec-
tively providing the enantiomerically pure major EFV metab-
olite, 8-OH-EFV, in
a single synthetic step. This EFV
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recorded on a Perkin Elmer Lambda 35 UV/VIS spectropho-
tometer. [α]D Measurements were carried out in an LDT Auto-
matic Polarimeter. 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−1. Semipreparative HPLC separations were conducted
with a Luna C18 (2) column (250 mm × 10 mm; 5 μm; Phenom-
enex) at a flow rate of 3 mL min−1. A 30-min linear gradient
from 5 to 70% acetonitrile in 0.1% aqueous formic acid, fol-
lowed by a 2-min linear gradient to 100% acetonitrile and an 8-
min isocratic elution with acetonitrile, was used in all instances.
The UV absorbance was monitored at 254 nm. Mass spectra
(MS) were recorded on a 500-MS LC ion trap mass spectrometer
(Varian, Inc., Palo Alto, CA), operated in the positive and nega-
tive electrospray ionization (ESI) modes. The spray voltage was
set at 5 kV and the capillary voltage was set at 10
V. LC-ESI-MS/MS analyses were performed with the same spec-
trometer, coupled to a Varian ProStar 410 autosampler, two 210-
LC chromatography pumps, and a ProStar 335 diode array detec-
tor. Data acquisition and processing were performed using
Varian MS Control 6.9 software. The samples were injected onto
the column via a Rheodyne injector with a 20 μL loop. Separ-
ations were conducted at 30 °C, using a Luna C18 (2) column
(150 mm × 2 mm, 3 μm; Phenomenex). The mobile phase was
delivered at a flow rate of 200 μL min−1, using a 5-min isocratic
elution with 5% acetonitrile in 0.1% aqueous formic acid, fol-
lowed by a 30 min linear gradient from 5 to 70% acetonitrile, a
2 min linear gradient to 100% acetonitrile, and an 8 min isocratic
elution with acetonitrile. The mass spectrometer was operated in
the positive and negative ESI mode; the optimized operating par-
ameters were: ion spray voltage, +5.2 kV; capillary voltage, 20
V; and RF loading, 90%. Nitrogen was used as the nebulizing
and drying gas, at pressures of 50 and 30 psi, respectively; the
drying gas temperature was 350 °C. MS/MS spectra were
obtained with an isolation window of 1.5 Da, excitation energy
values between 0.9 and 1.2 V, and an excitation time of 10 ms.
1H NMR spectra were recorded on Bruker Avance III 400 or
500 spectrometers (Bruker BioSpin GmbH, Rheinstetten,
Germany) operating at 400 and 500 MHz, respectively. 13C
NMR spectra were recorded on the same instruments, operating
at 100.62 and 125.77 MHz, respectively. Chemical shifts are
reported in ppm downfield from tetramethylsilane, and coupling
constants (J) are reported in Hz. Resonance and structural assign-
ments were based on the analysis of coupling patterns, including
the 13C–1H coupling profiles obtained in bidimensional hetero-
nuclear multiple bond correlation (HMBC) and heteronuclear
single quantum coherence (HSQC) experiments, performed with
standard pulse programs.
Fig. 4 (A) Total ion LC-ESI-MS chromatogram of the reaction mixture
obtained upon oxidation of equilin mediated by [FeII(bpmen)(OTf)2].
Extracted ion chromatograms and MS spectra of: (B) (m/z 285), corre-
sponding to the protonated molecule of 4-hydroxyequilin; (C) (m/z 283),
corresponding to the protonated molecule of 4-hydroxyequilin-o-
quinone; and (D) (m/z 281) corresponding to the protonated molecule of
4-hydroxyequilenin-o-quinone. The elution conditions are outlined in
the Experimental section.
Experimental
General remarks. Chemicals
Efavirenz was a gift from Dr F. A. Beland, National Center for
Toxicological Research, Jefferson, Arkansas, USA. 7-OH-EFV12
and the tamoxifen metabolites, α-hydroxytamoxifen and 4-
hydroxytamoxifen (mixture of E and Z isomers),44 were syn-
thesized as described. All other commercially available reagents
were acquired from Sigma-Aldrich Quimica, S.A. (Madrid,
Spain), unless specified otherwise, and were used as received.
Whenever necessary, solvents were purified by standard
methods.46
Oxidations of efavirenz
General method for optimization of experimental conditions.
Acetic acid (9 μL) was added to a solution of EFV (10 mg,
32 μmol) and catalyst (0–100% mol) in a suitable solvent
(500 μL) (Table 1). H2O2 (35 wt%, 0.45, 0.9, or 1.35 eq.) was
Instrumentation
Melting temperatures were measured in a Leica Galen III hot
stage apparatus and are uncorrected. The UV measurements were
This journal is © The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 4554–4561 | 4559

View Article Online
added at r.t. The final mixtures were stirred either at r.t. or at
40 °C. For monitorization purposes aliquots (50 μL) were col-
lected after 5 min, 2, 24, 48, and 72 h. Sodium bisulfite (100 μL,
sat. aq. solution) was added and the resulting solution was
extracted with AcOEt (50 μL) and monitored by HPLC-DAD at
254 nm.
overall yield of 8-OH-EFV was 21.8% (23 mg) after the three-
cycles run.
Scale up reaction. Acetic acid (0.9 mL, 16 mmol, 5 eq.) was
added to a solution of EFV (1 g, 3.2 mmol) and catalyst (10%
mol) in AcCN–AcOEt (64 mL, 1 : 1). The solution was stirred
and H2O2 (35 wt%, 380 μL, 4.3 mmol, 1.35 eq.) was added
dropwise at r.t. The final mixture was stirred at 40 °C for 3 days
and the reaction progress was monitored by HPLC. The mixture
was then treated with sodium bisulfite (10 mL, sat. aq. solution)
and AcOEt (20 mL) and the organic fraction was washed
sequentially with aqueous NaHCO3, water and brine, dried over
anhydrous Na2SO4, and evaporated. The crude mixture was
purified by flash chromatography on silica (CH2Cl2 to 95 : 5
CH2Cl2–MeOH), yielding 8-OH-EFV (65 mg, 6.2%).
Optimized conditions
Acetic acid (91 μL, 1.6 mmol, 5 eq.) was added to a solution of
EFV (100 mg, 320 μmol) and catalyst (10% mmol) in AcCN–
AcOEt (5 mL, 1 : 1). The solution was stirred and H2O2 (35 wt
%, 37 μL, 430 μmol, 1.35 eq.) was added at r.t. The final
mixture was stirred at 40 °C for 3 days and the reaction progress
was monitored by HPLC. The mixture was then treated with
sodium bisulfite (1 mL, sat. aq. solution) and AcOEt (2 mL) and
the organic fraction was washed sequentially with aqueous
NaHCO3, water and brine, dried over anhydrous Na2SO4, and
evaporated. The crude mixture was purified by flash chromato-
graphy on silica (CH2Cl2 to 95 : 5 CH2Cl2–MeOH), yielding 8-
OH-EFV (10 mg, 9.5%). The unreacted EFV was recovered
quantitatively (87 mg).
Oxidation of 8-OH-EFV. Acetic acid (4.3 μL, 76 μmol, 5 eq.)
was added to a solution of 8-OH-EFV (5 mg, 15 μmol) and cata-
lyst (10% mol) in AcCN–AcOEt (250 μL, 1 : 1). H2O2 (35 wt%,
1.8 μL, 20 μmol, 1.35 eq.) was added at r.t. The final mixture
was stirred at 40 °C for 24 h and the reaction progress was moni-
tored by HPLC-DAD and HPLC-ESI-MS.
Oxidation of equilin. Acetic acid (10 μL, 187 μmol, 5 eq.)
was added to a solution of equilin (10 mg, 37 μmol) and catalyst
(10% mol) in AcCN–AcOEt (500 μL, 1 : 1). Following addition
of H2O2 (35 wt%, 4.4 μL, 50 μmol, 1.35 eq.) at r.t. the final
mixture was stirred at 40 °C for 24 h. The reaction mixture was
monitored by HPLC-DAD and LC-ESI-MS after 1 and 24 h.
(S)-6-Chloro-4-(cyclopropylethynyl)-8-hydroxy-4-(trifluoro-
methyl)-1,4-dihydro-2H-3,1-benzoxazin-2-one (8-OH-EFV). Mp
171–172 °C (lit13 170–172 °C); MS [ESI(+)]: m/z: 354 [(35Cl)M
+ Na]+; 332 [(35Cl)M + H]+; MS [ESI(−)]: m/z: 330 [(35Cl)M −
H]−, 286 [(35Cl)M − (CO2)]−, 258 [M − (CO2) − (C2H4)]−;
[α]D (25 °C) = −30°, measured at 230 mg per 100 mL in metha-
1
nol (lit: −29,3°,12 and −32.0°,13); H-NMR (CD3OD): 6.93 (d,
Oxidation of tamoxifen. Acetic acid (8 μL, 135 μmol, 5 eq.)
was added to a solution of tamoxifen (10 mg, 27 μmol) and cata-
lyst (10% mol) in AcCN–AcOEt (500 μL, 1 : 1). Following
addition of H2O2 (35 wt%, 3.7 μL, 36 μmol, 1.35 eq.) at r.t. the
final mixture was stirred at 40 °C for 24 h. The reaction mixture
was monitored by HPLC-DAD and LC-ESI-MS/MS after 1 and
24 h.
1H, J = 2.15 Hz, H7), 6.92 (d, 1H, J = 2.15 Hz, H5), 1.48 (m,
1H, cyclopropyl-CH), 0.97–0.92 (m, 2H, cyclopropyl-CH2),
0.82–0.77 (m, 2H, cyclopropyl-CH2); 1H-NMR (DMSO-d6):
11.03 (br s, 1H, NH/OH), 10.3 (br s, 1H, NH/OH), 6.94 (d, 1H,
J = 2.19 Hz, H7), 6.88 (d, 1H, J = 2.19 Hz, H5), 1.58 (m, 1H,
cyclopropyl-CH), 0.87–0.84 (m, 2H, cyclopropyl-CH2),
0.79–0.74 (m, 2H, cyclopropyl-CH2); 13C-NMR (CD3OD):
149.4 (CvO), 146.4 (C8), 129.6 (C6), 129.2 (C10), 118.4 (C7),
117.8 (C5), 116.6 (C9), 96.6 (C13), 76.8 (q, C(4), J2 = 35 Hz),
67.6 (C12), 9.09 (C15/C16), 9.06 (C15/C16), −0.2 (C14);
13C-NMR (DMSO-d6): 146.7 (CvO), 145.9 (C8), 127.7 (C6),
124.2 (C10), 117.3 (C7), 116.7 (C5), 115.2 (C9), 95.8 (C13),
77.6 (q, C(4), J2 = 35 Hz), 66.5 (C12), 8.99 (C15/C16), 8.95
(C15/C16), −0.81 (C14).
Abbreviations
Bpmen
CYP
ESI
N,N′-bis-(2-pyridylmethyl)-1,2-diaminoethane
cytochrome P450
electrospray ionization
EFV
HIV
efavirenz
human immunodeficiency virus type 1
HPLC-DAD high performance liquid chromatography with
diode array detection
Three-cycles run. The EFV obtained from the reaction
described above (87 mg, 270 μmol) was dissolved in AcCN–
AcOEt (4.4 mL, 1 : 1). Following the addition of catalyst (10%
mol) acetic acid (79 μL, 1.39 mmol, 5 eq.) was added. The sol-
ution was stirred and H2O2 (35 wt%, 31 μL, 360 μmol, 1.35 eq.)
was added at r.t. The final mixture was stirred at 40 °C for 3
days. The mixture was then treated with sodium bisulfite (1 mL,
sat. aq.) and AcOEt (2 mL) and the organic fraction was washed
sequentially with aqueous NaHCO3, water and brine, dried over
anhydrous Na2SO4, and evaporated. The crude mixture was
purified by flash chromatography on silica (CH2Cl2 to 95 : 5
CH2Cl2–MeOH), yielding 8-OH-EFV (8 mg, 9%). The recov-
ered EFV (74 mg, 230 μmol), was reacted again under the
described conditions affording 8-OH-EFV (7 mg, 9%). The
MS/MS
NNRTI
S,S-PDP
tandem mass spectrometry
non-nucleoside reverse transcriptase inhibitor
2-({(S)-2-[(S)-1-(pyridin-2-ylmethyl)pyrrolidin-
2-yl]pyrrolidin-1-yl}methyl)pyridine
thin layer chromatography
TLC
Py2S2
1,6-bis(2′-pyridyl)-2,5-dithiahexane
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 grant PTDC/QUI-QUI/113910/2009
4560 | Org. Biomol. Chem., 2012, 10, 4554–4561
This journal is © The Royal Society of Chemistry 2012

View Article Online
21 B. Testa and S. D. Krämer, The Biochemistry of Drug Metabolism: Prin-
ciples, Redox Reactions, Hydrolyses. Part 2. Redox Reactions and their
Enzymes, Wiley-VCH, Weinheim, Germany, 2008.
22 J. Bernadou and B. Meunier, Chem. Commun., 1998, 2167.
23 M. S. Chen and M. C. White, Science, 2007, 318, 783.
24 R. R. Fernandes, J. Lasri, M. F. C. Guedes da Silva, J. A. L. da Silva,
J. J. R. Fraústo da Silva and A. J. L. Pombeiro, Appl. Catal., A: General,
2011, 402, 110.
25 G. J. P. Britovsek, J. England and A. J. P. White, Inorg. Chem., 2005, 44,
8125.
26 S. Poussereau, G. Blondin, M. Cesario, J. Guilhem, G. Chottard,
F. Gonnet and J.-J. Girerd, Inorg. Chem., 1998, 37, 3127.
27 N. A. Vermeulen, M. S. Chen and M. C. White, Tetrahedron, 2009, 65,
3078.
and pluriannual funds to Centro de Química Estrutural (PEst-OE/
QUI/UI0100/2011). RW and SGH also thank FCT for postdoc-
toral (SFRH/BPD/70953/2010) and doctoral (SFRH/BD/80690/
2011) fellowships, respectively.
References
1 M. A. Thompson, J. A. Aberg, P. Cahn, J. S. G. Montaner, G. Rizzardini,
A. Telenti, J. M. Gatell, H. F. Günthard, S. M. Hammer, M. S. Hirsch, D.
M. Jacobsen, P. Reiss, D. D. Richman, P. A. Volberding, P. Yeni and R.
T. Schooley, JAMA, 2010, 304, 321.
2 P. Lochet, H. Peyrière, A. Lotthé, J. M. Mauboussin, B. Delmas and
J. Reynes, HIV Med., 2003, 4, 62.
3 G. I. T. Cavalcante, V. L. M. Capistrano, F. S. D. Cavalcante,
S. M. M. Vasconcelos, D. S. Macêdo, F. C. F. Sousa, D. J. Woods and
M. M. F. Fonteles, Int. J. Neurosci., 2010, 120, 739.
28 M. Costas, M. P. Mehn, M. P. Jensen and L. Que, Jr, Chem. Rev., 2004,
104, 939.
29 O. V. Makhlynets and E. V. Rybak-Akimova, Chem.–Eur. J., 2010, 16,
13995.
4 U. Mehta and G. Maartens, Lancet Infect. Dis., 2007, 7, 733.
5 A. E. Mutlib, R. J. Gerson, P. C. Meunier, P. J. Haley, H. Chen, L.
S. Gan, M. H. Davies, B. Gemzik, D. D. Christ, D. F. Krahn, J.
A. Markwalder, S. P. Seitz, R. T. Robertson and G. T. Miwa, Toxicol.
Appl. Pharmacol., 2000, 169, 102.
30 J. P. Hage and D. T. Sawyer, J. Am. Chem. Soc., 1995, 117, 5617.
31 D. Mathieu, J. F. Bartoli, P. Battioni and D. Mansuy, Tetrahedron, 2004,
60, 3855.
32 J.-F. Bartoli, F. Lambert, I. Morgenstern-Badarau, P. Battioni and
D. Mansuy, C. R. Chim., 2002, 5, 263.
6 A. E. Mutlib, H. Chen, G. A. Nemeth, J. A. Markwalder, S. P. Seitz, L.
S. Gan and D. D. Christ, Drug Metab. Dispos., 1999, 27, 1319.
7 N. N. Bumpus, Toxicol. Appl. Pharmacol., 2011, 257, 227.
8 E. Ngaimisi, S. Mugusi, O. M. Minzi, P. Sasi, K.-D. Riedel, A. Suda,
N. Ueda, M. Janabi, F. Mugusi, W. E. Haefeli, J. Burhenne and
E. Aklillu, Clin. Pharmacol. Ther., 2010, 88, 676.
33 H. C. Tung, C. Kang and D. T. Sawyer, J. Am. Chem. Soc., 1992, 114,
3445.
34 D. Bianchi, M. Bertoli, R. Tassinari, M. Ricci and R. Vignola, J. Mol.
Catal. A: Chem., 2003, 204–205, 419.
35 T. Tezuka, N. Narita, W. Ando and S. Oae, J. Am. Chem. Soc., 1981, 103,
3045.
9 J. S. Barrett, A. S. Joshi, M. Chai, T. M. Ludden, W. D. Fiske and H.
J. Pieniaszek, Int. J. Clin. Pharmacol. Ther., 2002, 40, 507.
10 S. R. Faucette, T.-C. Zhang, R. Moore, T. Sueyoshi, C. J. Omiecinski, E.
L. LeCluyse, M. Negishi and H. Wang, J. Pharmacol. Exp. Ther., 2007,
320, 72.
11 S. Mouly, K. S. Lown, D. Kornhauser, J. L. Joseph, W. D. Fiske, I.
H. Benedek and P. B. Watkins, Clin. Pharmacol. Ther., 2002, 72, 1.
12 J. A. Markwalder, D. D. Christ, A. Mutlib, B. C. Cordova, R. M. Klabe
and S. P. Seitz, Bioorg. Med. Chem. Lett., 2001, 11, 619.
36 S. Udenfriend, C. T. Clark, J. Axelrod and B. B. Brodie, J. Biol. Chem.,
1954, 208, 731.
37 S. P. de Visser, K. Oh, A.-R. Han and W. Nam, Inorg. Chem., 2007, 46,
4632.
38 N. Y. Oh, M. S. Seo, M. H. Lim, M. B. Consugar, M. J. Park, J.-
U. Rohde, J. Han, K. M. Kim, J. Kim, L. Que, Jr and W. Nam, Chem.
Commun., 2005, 5644.
39 T. Kurata, Y. Watanabe, M. Katoh and Y. Sawaki, J. Am. Chem. Soc.,
1988, 110, 7472.
13 D. D. Christ, A. J. Cocuzza, S. S. Ko, J. A. Markwalder, A. E. Mutlib, R.
L. Parsons, Jr, M. Patel and S. P. Seitz, U.S. Pat. 5,874,430, 1999.
14 L. Que Jr, in Bioinorganic Catalysis, ed. J. Reedjik and E. Bouwman,
Marcel Dekker Inc., New York, USA, 1999, (ch.10).
15 K. Chen, M. Costas, J. Kim, A. K. Tipton and L. Que, Jr, J. Am. Chem.
Soc., 2002, 124, 3026.
16 S. Taktak, M. Flook, B. M. Foxman, L. Que, Jr and E. V. Rybak-
Akimova, Chem. Commun., 2005, 5301.
17 V. Balland, D. Mathieu, N. Pons-Y-Moll, J. F. Bartoli, F. Banse,
P. Battioni, J.-J. Girerd and D. Mansuy, J. Mol. Catal. A: Chem., 2004,
215, 81.
40 D. T. Sawyer, A. Sobkowiak and T. Matsushita, Acc. Chem. Res., 1996,
29, 409.
41 D. A. Wink, R. W. Nims, J. E. Saavedra, W. E. Utermahlen, Jr and P.
C. Ford, Proc. Natl. Acad. Sci. U. S. A., 1994, 91, 6604.
42 Iron Catalysis: Fundamentals and Applications (Topics in Organometal-
lic Chemistry), ed. B. Plietker, Springer-Verlag, Berlin, Germany, 2011,
vol. 33.
43 B. A. Ward, J. C. Gorski, D. R. Jones, S. D. Hall, D. A. Flockhart and
Z. Desta, J. Pharmacol. Exp. Ther., 2003, 306, 287.
44 G. Gamboa da Costa, L. P. McDaniel-Hamilton, R. H. Heflich, M.
M. Marques and F. A. Beland, Carcinogenesis, 2001, 22, 1307.
45 F. Zhang, Y. Chen, E. Pisha, L. Shen, Y. Xiong, R. B. van Breemen and
J. L. Bolton, Chem. Res. Toxicol., 1999, 12, 204.
18 M. Costas, K. Chen and L. Que, Jr, Coord. Chem. Rev., 2000, 200, 517.
19 K. Chen and L. Que, Jr, Chem. Commun., 1999, 1375.
20 D. Quiñonero, K. Morokuma, D. G. Musaev, R. Mas-Ballesté and
L. Que, Jr, J. Am. Chem. Soc., 2005, 127, 6548.
46 D. D. Perrin and W. L. F. Armarego, Purification of Laboratory Chemi-
cals, Pergamon Press, Oxford, UK, 3rd edn, 1988, pp. 1–391.
This journal is © The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 4554–4561 | 4561