In vitro bioactivation of bazedoxifene and 2-(4-hydroxyphenyl)-3-methyl-1H- indol-5-ol in human liver microsomes

Article abstract of DOI:10.1016/j.cbi.2012.03.001

Bazedoxifene is a selective estrogen receptor modulator (SERM) that has been developed for use in post-menopausal osteoporosis. However, it contains a potentially toxic 5-hydroxy-3-methylindole moiety. Previous studies on the 5-hydroxyindole and the 3-alkylindole-containing drugs indometacine, zafirlukast and MK-0524 structural analogs have shown that they are bioactivated by cytochrome P450s through a dehydrogenation process to form quinoneimine or 3-methyleneindolenine electrophilic species. In the present study, bazedoxifene was synthesized and then evaluated, together with raloxifene and 2-(4-hydroxyphenyl)-3-methyl-1H-indol-5-ol (13), a 3-methyl-5-hydroxyindole- based structural fragment of bazedoxifene, for its ability to form reactive electrophilic species when incubated with human liver microsomes (HLMs) or recombinant CYP isozymes. We showed that bazedoxifene was bioactivated only in trace amounts with recombinant CYP isozymes. In contrast, the N-dealkylated fragment of bazedoxifene (2-(4-hydroxyphenyl)-3-methyl-1H-indol-5-ol) was bioactivated in considerable amounts to an electrophilic intermediate, which was trapped with glutathione and identified by LC-MS/MS. This suggests that bazedoxifene would require initial N-dealkylation, which could subsequently lead to the formation of the reactive intermediate. However, such an N-dealkylated metabolite of bazedoxifene was not detected after the incubation of bazedoxifene in HLM or recombinant CYP isozymes.

Full text of DOI:10.1016/j.cbi.2012.03.001

Chemico-Biological Interactions 197 (2012) 8–15
Contents lists available at SciVerse ScienceDirect
Chemico-Biological Interactions
journal homepage: www.elsevier.com/locate/chembioint
In vitro bioactivation of bazedoxifene and
2-(4-hydroxyphenyl)-3-methyl-1H-indol-5-ol in human liver microsomes
⇑
´
ˇ
Tina Trdan Lušin, Tihomir Tomašic, Jurij Trontelj, Aleš Mrhar, Lucija Peterlin-Mašic
ˇ
University of Ljubljana, Faculty of Pharmacy, Aškerceva 7, 1000 Ljubljana, Slovenia
a r t i c l e i n f o
a b s t r a c t
Article history:
Bazedoxifene is a selective estrogen receptor modulator (SERM) that has been developed for use in post-
menopausal osteoporosis. However, it contains a potentially toxic 5-hydroxy-3-methylindole moiety.
Previous studies on the 5-hydroxyindole and the 3-alkylindole-containing drugs indometacine, zafirluk-
ast and MK-0524 structural analogs have shown that they are bioactivated by cytochrome P450s through
a dehydrogenation process to form quinoneimine or 3-methyleneindolenine electrophilic species. In the
present study, bazedoxifene was synthesized and then evaluated, together with raloxifene and 2-(4-
hydroxyphenyl)-3-methyl-1H-indol-5-ol (13), a 3-methyl-5-hydroxyindole-based structural fragment
of bazedoxifene, for its ability to form reactive electrophilic species when incubated with human liver
microsomes (HLMs) or recombinant CYP isozymes. We showed that bazedoxifene was bioactivated only
in trace amounts with recombinant CYP isozymes. In contrast, the N-dealkylated fragment of bazedoxif-
ene (2-(4-hydroxyphenyl)-3-methyl-1H-indol-5-ol) was bioactivated in considerable amounts to an elec-
trophilic intermediate, which was trapped with glutathione and identified by LC–MS/MS. This suggests
that bazedoxifene would require initial N-dealkylation, which could subsequently lead to the formation
of the reactive intermediate. However, such an N-dealkylated metabolite of bazedoxifene was not
detected after the incubation of bazedoxifene in HLM or recombinant CYP isozymes.
Received 21 December 2011
Received in revised form 2 March 2012
Accepted 3 March 2012
Available online 10 March 2012
Keywords:
Bazedoxifene
Raloxifene
Reactive metabolite
LC–MS/MS
Cytochrome P450
Ó 2012 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
bazedoxifene induces significantly lower uterine stimulation (low-
er promotion of endometrium cell proliferation) than raloxifene,
Raloxifene I (Fig. 1), a benzothiophene selective estrogen recep-
tor modulator (SERM), is currently the only FDA-approved SERM
available for the prevention and treatment of osteoporosis in
post-menopausal women [1]. It acts as an estrogen receptor agonist
in bone tissue, while not stimulating the breast and uterine tissues
[2,3]. Its administration is associated with a number of side-effects
of which hot flushes, leg cramps, and thromboembolism are the
most severe [5,6]. The binding of a SERM to an estrogen receptor
triggers the same sequence of molecular events as observed after
the binding of estrogen itself [4]. The agonist or antagonist activity
is tissue-dependent, so the distribution of estrogen receptors in
various tissues becomes important since modulators of estrogen
function can act not only on bone tissue but also on all other tissues
where estrogen exerts its effects. It is, therefore, desirable that SER-
Ms should act as agonists preferentially on bone tissue and in order
to circumvent negative, undesired effects in the prevention and
treatment of post-menopausal osteoporosis.
making it a promising alternative for treating post-menopausal
osteoporosis with an improved safety profile [5,6]. Glucuronida-
tion is the major pathway for the metabolism of bazedoxifene,
with bazedoxifene-5-glucuronide being the main metabolite [7].
However, no data are available on the formation of the reactive
metabolites of bazedoxifene, so we have investigated the potential
formation of such reactive intermediates through glutathione
(GSH) trapping studies.
The production of reactive metabolites following biotransforma-
tion by cytochrome P450 (CYP450) enzymes can lead to toxicity,
since they can bind covalently to proteins and/or DNA with toxic
consequences. Not only this, but when such metabolites bind cova-
lently to CYP450 they can cause its mechanism-based inactivation
(MBI) [8]. Raloxifene, for example, is a mechanism-based inhibitor
of cytochrome P450 3A4 (CYP3A4) [9]. The diquinone methide
formed after the bioactivation of raloxifene (Fig. S1) alkylates the
Cys239 residue of CYP3A4 leading to an irreversible loss of enzyme
function [10]. Raloxifene is known to undergo a CYP3A4-catalyzed
reaction with its phenolic groups in HLM in the presence of NADPH,
such that it yields a reactive diquinone methide and an o-quinone
species [11]. However, in vivo, glucuronidation of the same pheno-
lic groups in the gut and liver constitutes the principal elimination
mechanism of raloxifene in humans [12].
Bazedoxifene (II, Fig. 1) is a new, third generation, indole-based
estrogen receptor (ER)-ligand developed for use in post-
menopausal osteoporosis. Preclinical studies have revealed that
⇑
Corresponding author. Tel.: +386 (0) 1 476 96 35; fax: +386 (0) 1 425 80 31.
ˇ
E-mail address: lucija.peterlin@ffa.uni-lj.si (L. Peterlin-Mašic).
0009-2797/$ - see front matter Ó 2012 Elsevier Ireland Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.cbi.2012.03.001

T.T. Lušin et al. / Chemico-Biological Interactions 197 (2012) 8–15
9
O
O
N
N
O
H
N
N
OH
OH
OH
S
HO
HO
HO
I, raloxifene
II, bazedoxifene
13, 2-(4-hydroxyphenyl)-3-methyl-1H-
indol-5-ol
Fig. 1. Structures of raloxifene, bazedoxifene and the N-dealkylated fragment of bazedoxifene (2-(4-hydroxyphenly)-3-methyl-1H-indol-5-ol).
GSH trapping reactions can provide an insight into the mecha-
nisms of drug bioactivation and the subsequent formation of reac-
tive metabolites in the body. It is common practice for drugs under
investigation for the formation of reactive metabolites to be incu-
bated with HLM and GSH. The nucleophilic cysteine sulfhydryl
group of GSH can trap any electrophilic intermediate species in
the form of S-linked conjugates with GSH that can be analyzed
by LC–MS/MS [13]. However, interpretation of the output from
these assays is difficult as many successfully marketed drugs also
show positive results in this assay [14,15]. Although the GSH trap-
ping of electrophilic drug metabolites formed by HLM is a common
method by which potential reactive and toxic metabolites can be
identified, it can yield misleading and false positive results. For in-
stance, raloxifene has been found to form potent electrophilic
intermediates in vitro [9,12], but such intermediates have never
been observed in vivo, probably because raloxifene is conjugated
with glucuronic acid extremely quickly in vivo [16]. Bazedoxifene
glucuronidation, on the other hand, is a significantly slower pro-
cess [7,16], meaning the possibility of its electrophilic metabolites’
production by the CYP system is increased and warrants our
investigation.
Bazedoxifene possesses a 5-hydroxy-3-methyl-substituted in-
dole moiety (Fig. S2). Previous studies on the bioactivation of
5-hydroxyindole to reactive quinoneimine intermediates (Fig. S2)
have been reported for oxypertine and indomethacin, both of
which have been implicated in numerous cases of aplastic anemia,
agranulocytosis and death [17]. 5-Hydroxyindoles are bioactivated
by myeloperoxidase or by the CYP450 system to the electrophilic
quinoneimine, which can be trapped by GSH. No formalized struc-
ture–bioactivation relationship studies have been reported on
5-hydroxyindoles that address their bioactivation.
Previous studies on 3-methylindole, which is produced in animal
and human digestive systems through tryptophan degradation and
is found in cigarette smoke [18], have demonstrated that it is a po-
tent pneumotoxin that can cause severe lung injury. 3-Methylindole
is bioactivated by CYP450s to form a reactive electrophilic interme-
diate through the dehydrogenation pathway (Fig. S2) [19–22]. Their
toxicity has not been fully evaluated in humans, but the compounds
are a substrate for several human CYP450 enzymes. The toxicity of
the 3-methylindole attributed to the action of 3-methyleneindole-
nine obtained by dehydrogenation of the 3-methylindole (Fig. S2)
and its adducts with thiol nucleophiles upon incubation with micro-
somes have been observed. 3-Alkylindole-containing drugs (see
Fig. S3 for structures), such as the leukotriene receptor antagonist
zafirlukast [13,23], structural analogs of MK-0524 [24,25], and the
electron abstraction from the indole nitrogen atom, followed by a
second one-electron oxidation. The dehydrogenation atom abstrac-
tion step was believed to be the rate-limiting step of the 3-methyl-
indole dehydrogenation, but either a hydrogen atom abstraction or
a nitrogen oxidation was the potential initiating step for the dehy-
drogenation of zafirlukast and analogs of MK-0524 [23–25].
The aim of the present study was to determine whether baze-
doxifene can be dehydrogenated to an electrophilic intermediate
by CYP450 enzyme(s) in HLM. We describe the synthesis of baze-
doxifene and an N-dealkylated fragment of bazedoxifene 13
(Scheme 1), and the GSH trapping studies on bazedoxifene, the
structurally related raloxifene, and 2-(4-hydroxyphenyl)-3-
methyl-1H-indol-5-ol (13),
a 5-hydroxy-3-methylindole-based
structural fragment of bazedoxifene, in the presence of HLM or dif-
ferent recombinant CYP450 isozymes.
2. Materials and methods
2.1. Synthesis of bazedoxifene
Chemicals were obtained from Acros, Aldrich Chemical Co. and
Fluka and used without further purification. Yields refer to purified
products and were not optimized. Analytical thin-layer chromatog-
raphy (TLC) was performed using silica gel 60F254 pre-coated plates
(0.25 mm thick) with a fluorescent indicator from Merck (Ger-
many). Flash column chromatography was carried out on silica
gel 60 (particle size 0.040–0.063 mm; Merck, Germany). The com-
ponents of chromatographic eluents are given as volume-to-vol-
ume ratios (v/v). 1H NMR spectra were recorded at 300 MHz on a
Bruker AVANCE DPX300 spectrometer in CDCl3 or DMSO-d6 solution
with TMS as the internal standard. Mass spectra were obtained
using a VG-Analytical Autospec Q mass spectrometer.
The purity of the synthesized bazedoxifene (12) was deter-
mined by chromatographic separation on an Agilent 1100 system
(Agilent Technologies, Santa Clara, USA) using
a Kinetex
50 mm ꢀ 2.1 mm column coupled with an InLine filter KrudKat-
cher Ultra HPLC 0.5 mm and a guard column C18 4 mm ꢀ 2 mm
(Phenomenex, Torrance, USA) at room temperature and with UV
detection at 295 nm. The mobile phase consisted of 0.1% formic
acid in water (mobile phase A) and 100% acetonitrile (mobile phase
B). The flow rate was set at 0.5 mL/min and the separation required
gradient elution. Elution started with 25% of mobile phase B, and
continued with the following gradient: 25–63–25–25% of mobile
phase B in 0.06–6.00–6.20–12.00 min. The run time was 12 min
and the volume of injection was 5 lL.
TNF-
through
a
antagonist SPD-304 [26], are also bioactivated by P450s
similar dehydrogenation pathway to electrophilic
a
3-methyleneindolenine intermediates that are trapped with GSH.
3-Methylindole-like bioactivation has also been reported on a
3-substituted-7-azaindole derivative L-745,870, intended for the
treatment of schizophrenia [27]. It has been proposed that the for-
mation of the electrophilic 3-methyleneindolenine is a two-step
process in which dehydrogenation was initiated either by hydrogen
atom abstraction from the 3-methyl or 3-methylene carbon, or by
2.1.1. Synthesis of 1-(4-(benzyloxy)phenyl)propan-1-one (2)
Benzyl bromide (16.0 mL, 0.135 mol) was slowly added to a stir-
red solution of 1-(4-hydroxyphenyl)propan-1-one (1) (20.00 g,
0.133 mol) and potassium carbonate (18.36 g, 0.133 mol) in ace-
tone (200 mL). Having been stirred at 60 °C overnight, the reaction
mixture was cooled, the precipitate then being filtered off and the
solvent evaporated under reduced pressure. The residue was dis-

10
T.T. Lušin et al. / Chemico-Biological Interactions 197 (2012) 8–15
O
O
RO
a
c
OR
R
N
H
HO
BzO
1
5
2, R=H
3, R=Br
4, R=Bz
13, R=H
b
j
OH
OH
Cl
d
e
HO
O
O
6
7
EtO
EtO
O
O
BzO
BzO
f
OBz
g
OBz
4 + 7
N
N
O
8
9, R=OH
10
h
EtO
R
, R=Br
O
O
i
HO
BzO
j
OH
OBz
N
N
12
11
N
N
O
O
Scheme 1. Reagents and conditions: (a) benzyl bromide, K2CO3, acetone, 60 °C, overnight; (b) Br2, glacial acetic acid, r.t., 3 h; (c) (i) 4-benzyloxyaniline hydrochloride, Et3N,
DMF, 120 °C, 2 h; (ii) 4-benzyloxyaniline hydrochloride, 150 °C, 2 h; (d) ethyl bromoacetate, K2CO3, acetonitrile, 60 °C, overnight; (e) SOCl2, DMF, dichloromethane, 0 °C,
45 min; (f) NaH, DMF, 0 °C to r.t., overnight; (g) LiAlH4, THF, 0 °C, 30 min; (h) CBr4, PPh3, THF, r.t., overnight; (i) hexamethylenimine, THF, reflux, overnight; and (j) H2, Pd/C,
EtOH/THF, r.t., overnight.
solved in ethyl acetate (300 mL), then washed with water
(2 ꢀ 200 mL) and brine (200 mL). The organic phase was dried over
Na2SO4, filtered and the solvent removed in vacuo. Yield: 100.0%;
white solid; mp 98–100 °C ([28], 102–103 °C); 1H NMR (CDCl3):
d = 1.24 (t, 3H, J = 7.3 Hz, CH2CH3), 2.97 (q, 2H, J = 7.3 Hz, CH2CH3),
5.15 (s, 2H, PhCH2), 7.03 (d, 2H, J = 8.9 Hz, 2 ꢀ Ar-H), 7.31–7.48 (m,
5H, Ph), 7.97 (d, 2H, J = 8.9 Hz, 2 ꢀ Ar-H) ppm.
120 °C for 2 h. The reaction mixture was cooled and additional
4-benzyloxyaniline hydrochloride (4.100 g, 16.4 mmol) was added
before the reaction mixture was heated to 150 °C for 2 h. After the
reaction’s completion, the mixture was cooled to room tempera-
ture and poured into water (200 mL) before the product was ex-
tracted with ethyl acetate (3 ꢀ 70 mL). The combined organic
extracts were washed successively with 1 M NaOH (100 mL), water
(100 mL) and brine (100 mL), prior to being dried over Na2SO4, fil-
tered and the solvent being evaporated under reduced pressure.
The crude solid product was first washed with methanol (50 mL)
and filtered then washed with diethyl ether (50 mL) and filtered.
Yield: 84.9%; white solid; mp 146–150 °C ([29], 150–152 °C); 1H
NMR (CDCl3): d = 2.41 (s, 3H, CH3), 5.15 (d, 2H, PhCH2), 5.17 (d,
2H, PhCH2), 6.95 (dd, 1H, 3J = 2.4, 2J = 8.7 Hz, Ar-H-6), 7.10 (d, 2H,
J = 8.8 Hz, 2 ꢀ Ar-H0), 7.13 (d, 2H, 3J = 2.4 Hz, Ar-H-4), 7.26 (d, 1H,
2J = 8.7 Hz, Ar-H-7), 7.29–7.53 (m, 12H, 2 ꢀ Ph, 2 ꢀ Ar-H0), 7.84 (s,
1H, NH) ppm.
2.1.2. Synthesis of 1-(4-(benzyloxy)phenyl)-2-bromopropan-1-one (3)
Compound 2 (22.5 g, 0.094 mol) was suspended in glacial acetic
acid (70 mL) and cooled to 0 °C. A solution of bromine (4.8 mL,
0.094 mol) in glacial acetic acid (20 mL) was then added dropwise
and the reaction mixture stirred for 3 h at room temperature.
Water (200 mL) was then added to the reaction mixture and the
product extracted with diethyl ether (2 ꢀ 200 mL). The combined
organic extracts were washed successively with water (200 mL),
saturated aqueous NaHCO3 solution (200 mL) and brine (200 mL).
The organic phase was dried over Na2SO4, filtered and the solvent
evaporated under reduced pressure. The crude product was recrys-
tallized from a mixture of hexane and ethyl acetate. Yield: 32.2%;
white solid; mp 71–75 °C ([28], 75–76 °C); 1H NMR (CDCl3):
d = 1.91 (d, 3H, J = 6.6 Hz, CHCH3), 5.17 (s, 2H, PhCH2), 5.27 (q,
1H, J = 6.6 Hz, CHCH3), 7.05 (d, 2H, J = 9.0 Hz, 2 ꢀ Ar-H), 7.31–
7.50 (m, 5H, Ph), 8.03 (d, 2H, J = 9.0 Hz, 2 ꢀ Ar-H) ppm.
2.1.4. Synthesis of ethyl 2-(4-(hydroxymethyl)phenoxy)acetate (6)
A solution of 4-hydroxybenzyl alcohol (5) (15.32 g, 12.0 mmol),
potassium carbonate (17.10 g, 48.0 mmol) and ethyl bromoacetate
(15.1 mL, 12.6 mmol) in acetonitrile (150 mL) was stirred at 60 °C
overnight. Once the solvent was evaporated, the residue was dis-
solved in ethyl acetate (100 mL) and washed successively with
10% aqueous citric acid solution (2 ꢀ 50 mL), saturated aqueous
NaHCO3 solution (2 ꢀ 50 mL) and brine (50 mL). It was subse-
quently dried over Na2SO4, filtered and the solvent removed in va-
cuo. The crude product was purified with flash column
chromatography using dichloromethane/methanol (20:1) as the
2.1.3. Synthesis of 5-(benzyloxy)-2-(4-(benzyloxy)phenyl)-3-methyl-
1H-indole (4) [29]
4-Benzyloxyaniline hydrochloride (4.001 g, 16.0 mmol) was
added to a solution of compound 3 (3.964 g, 12.4 mmol) in N,N-
dimethylformamide (15 mL), following which the reaction mixture
was purged with argon for 10 min. Triethylamine (4.3 mL,
30.9 mmol) was then added and the reaction mixture heated at
eluent. Yield: 65%; brown oil; IR (KBr):
1588, 1383, 1212, 1083, 1030, 949, 789, 708 cmꢁ1
(CDCl3): d = 1.27 (t, 3H, J = 7.1 Hz, CH2CH3), 2.58 (s, 1H, OH), 4.23
m
= 2998, 1704, 1615,
.
1H NMR

T.T. Lušin et al. / Chemico-Biological Interactions 197 (2012) 8–15
11
(q, 2H, J = 7.1 Hz, CH2CH3), 4.54 (s, 2H, CH2), 4.57 (s, 2H, CH2), 6.85
(d, 2H, J = 8.7 Hz, 2 ꢀ Ar-H), 7.24 (d, 2H, J = 8.7 Hz, 2 ꢀ Ar-H) ppm.
131–134 °C); 1H NMR (DMSO-d6): d = 2.16 (s, 3H, CH3), 3.74 (t,
2H, J = 5.4 Hz, CH2CH2Br), 4.21 (t, 2H, J = 5.0 Hz, CH2CH2Br), 5.12
(s, 2H, CH2), 5.15 (s, 2H, CH2), 5.17 (s, 2H, CH2), 6.77 (s, 4H,
4 ꢀ Ar-H00), 6.82 (dd, 1H, 3J = 2.3 Hz, 2J = 8.8 Hz, Ar-H-6), 7.11–
7.14 (m, 3H, 2 ꢀ Ar-H0, Ar-H-4), 7.21 (d, 1H, 2J = 8.8 Hz, Ar-H-7),
7.31 (d, 2H, J = 8.7 Hz, 2 ꢀ Ar-H0), 7.34–7.50 (m, 10H, 2 ꢀ Ph) ppm.
2.1.5. Synthesis of ethyl 2-(4-(chloromethyl)phenoxy)acetate (7)
A solution of compound 6 (14.08 g, 67 mmol) and N,N-dimeth-
ylformamide (1 mL) in dichloromethane (200 mL) under an argon
atmosphere was cooled to 0 °C and thionyl chloride (5.4 mL,
74 mmol) dissolved in dichloromethane (80 mL) was added drop-
wise. The reaction mixture was stirred at 0 °C for 45 min and the
solvent evaporated under reduced pressure. Yield: 100%; pale yel-
2.1.9. Synthesis of 1-(4-(2-(azepan-1-yl)ethoxy)benzyl)-2-(4-
(benzoyloxy)phenyl)-3-methyl-1H-indol-5-yl benzoate (11) [29]
Hexamethylenimine (1.16 mL, 9.80 mmol) was added to a solu-
tion of compound 10 (0.650 g, 0.98 mmol) in dry tetrahydrofuran
(20 mL) and the reaction mixture refluxed overnight. The solvent
was evaporated under reduced pressure and the residue dissolved
in ethyl acetate (30 mL) and washed with saturated aqueous NaH-
CO3 solution (30 mL) and brine (30 mL). It was subsequently dried
over Na2SO4, filtered and the solvent removed in vacuo. The crude
product was purified by flash column chromatography using
dichloromethane/methanol (15:1) as eluent. Yield: 64.5%; white
solid; mp 98–100 °C ([29], 106–107 °C); 1H NMR (DMSO-d6):
d = 1.47–1.60 (m, 8H, 4 ꢀ azepane CH2), 2.16 (s, 3H, CH3), 2.59–
2.70 (m, 4H, 4 ꢀ azepane CH2), 2.79 (t, 2H, J = 5.6 Hz, CH2CH2N),
3.93 (t, 2H, J = 5.6 Hz, CH2CH2N), 5.12 (s, 2H, CH2), 5.15 (s, 2H,
CH2), 5.16 (s, 2H, CH2), 6.75 (s, 4H, 4 ꢀ Ar-H00), 6.81 (dd, 1H,
low oil; IR (KBr):
m
= 2977, 1754, 1652, 1613, 1588, 1514, 1440,
1113, 1027, 833, 733, 665 cmꢁ1
.
1H NMR (CDCl3): d 1.31 (t, 3H,
J = 7.1 Hz, CH2CH3), 4.29 (q, 2H, J = 7.1 Hz, CH2CH3), 4.57 (s, 2H,
CH2), 4.63 (s, 2H, CH2), 6.90 (d, 2H, J = 8.7 Hz, 2 ꢀ Ar-H), 7.33 (d,
2H, J = 8.7 Hz, 2 ꢀ Ar-H) ppm.
2.1.6. Synthesis of 4-(5-(benzoyloxy)-1-(4-(2-ethoxy-2-
oxoethoxy)benzyl)-3-methyl-1H-indol-2-yl)phenyl benzoate (8) [29]
A solution of compound 4 (4.213 g, 9.41 mmol) in N,N-dimeth-
ylformamide (40 mL) was cooled to 0 °C on an ice bath and sodium
hydride (0.29 g, 12.1 mmol) was added. The reaction mixture was
stirred for 20 min, followed by the addition of compound 7
(3.440 g, 15.0 mmol). After stirring at room temperature overnight,
the solvent was evaporated under reduced pressure and the resi-
due dissolved in ethyl acetate (100 mL). The organic phase was
washed with water (2 ꢀ 100 mL) and brine (100 mL), dried over
Na2SO4, and filtered. The solvent was then evaporated under re-
duced pressure. The crude product was purified by flash column
chromatography using ethyl acetate/petroleum ether (1:4) as the
eluent. Yield: 29.7%; yellow solid; mp 128–131 °C ([29], 129–
131 °C); 1H NMR (DMSO-d6): d = 1.18 (t, 3H, J = 7.1 Hz, CH2CH3),
2.16 (s, 3H, CH3), 4.13 (q, 3H, J = 7.1 Hz, CH2CH3), 4.67 (s, 2H, CO-
CH2O), 5.12 (s, 2H, CH2), 5.15 (s, 2H, CH2), 5.17 (s, 2H, CH2), 6.75
(s, 4H, 4 ꢀ Ar-H00), 6.82 (dd, 1H, 3J = 2.4 Hz, 2J = 8.7 Hz, Ar-H-6),
7.11–7.14 (m, 3H, 2 ꢀ Ar-H0, Ar-H-4), 7.21 (d, 1H, 2J = 8.7 Hz, Ar-
H-7), 7.31 (d, 2H, J = 8.5 Hz, 2 ꢀ Ar-H0), 7.34–7.50 (m, 10H,
2 ꢀ Ph) ppm.
2
3J = 2.4 Hz, J = 8.8 Hz, Ar-H-6), 7.11–7.14 (m, 3H, 2 ꢀ Ar-H0, Ar-H-
4), 7.21 (d, 1H, 2J = 8.8 Hz, Ar-H-7), 7.30 (d, 2H, J = 8.7 Hz, 2 ꢀ Ar-
H0), 7.34–7.50 (m, 10H, 2 ꢀ Ph) ppm.
2.1.10. Synthesis of 1-(4-(2-(azepan-1-yl)ethoxy)benzyl)-2-(4-
hydroxyphenyl)-3-methyl-1H-indol-5-ol (12) [29]
Compound 11 (0.420 g, 0.62 mmol) was dissolved in a mixture
of ethanol/tetrahydrofuran = 1:1 (25 mL). Following this, 10% Pd/C
(80 mg) was added and the reaction mixture stirred under a hydro-
gen atmosphere at room temperature overnight. The catalyst was
filtered off and the solvent removed under reduced pressure. The
crude product was purified by flash column chromatography using
dichloromethane/methanol (9:1) as eluent. Yield: 89.2%; light
brown solid; mp 108–113.5 °C; 1H NMR (DMSO-d6): d = 1.47–
1.63 (m, 8H, 4 ꢀ azepane CH2), 2.10 (s, 3H, CH3), 2.63–2.81 (m,
4H, 4 ꢀ azepane CH2), 2.86 (t, 2H, J = 5.6 Hz, CH2CH2N), 3.97 (t,
2H, J = 5.6 Hz, CH2CH2N), 5.10 (s, 2H, PhCH2N), 6.58 (dd, 1H,
2.1.7. Synthesis of 2-(4-((5-(benzyloxy)-2-(4-(benzyloxy)phenyl)-3-
methyl-1H-indol-1-yl)methyl)phenoxy)ethanol (9) [29]
2
A solution of compound 8 (1.700 g, 2.66 mmol) in dry tetrahy-
drofuran (20 mL) was cooled to 0 °C and 1 M solution of LiAlH4
in tetrahydrofuran (3.1 mL, 3.10 mmol) was added dropwise. After
30 min, the reaction was slowly quenched with water and tetrahy-
drofuran was evaporated under reduced pressure. The residue was
then partitioned between 1 M HCl (20 mL) and ethyl acetate
(20 mL). The organic phase was dried over Na2SO4, filtered and
the solvent evaporated under reduced pressure. The product was
used in the next step without further purification. Yield: 73.3%;
yellow solid; mp 107–111 °C; 1H NMR (DMSO-d6): d = 2.16 (s,
3H, CH3), 3.65 (t, 2H, J = 5.0 Hz, CH2CH2OH), 3.88 (t, 2H,
J = 5.0 Hz, CH2CH2OH), 5.12 (s, 2H, CH2), 5.15 (s, 2H, CH2), 5.16 (s,
2H, CH2), 6.75 (s, 4H, 4 ꢀ Ar-H00), 6.82 (dd, 1H, 3J = 2.4 Hz,
2J = 8.8 Hz, Ar-H-6), 7.11–7.14 (m, 3H, 2 ꢀ Ar-H0, Ar-H-4), 7.21 (d,
3J = 2.3 Hz, J = 8.7 Hz, Ar-H-6), 6.76 (s, 4H, 4 ꢀ Ar-H00), 6.81 (d,
3
1H, J = 2.3 Hz, Ar-H-4), 6.86 (d, 2H, J = 8.6 Hz, 2 ꢀ Ar-H0), 7.06 (d,
2
1H, J = 8.7 Hz, Ar-H-7), 7.16 (d, 2H, J = 8.7 Hz, 2 ꢀ Ar-H0), 8.67 (s,
1H, OH), 9.64 (s, 1H, OH) ppm. Purity: 97.5%. MS (ESI+): m/z (%) =
471.3 (M+H+).
2.1.11. Synthesis of 2-(4-hydroxyphenyl)-3-methyl-1H-indol-5-ol (13)
After compound 4 (0.247 g, 0.55 mmol) had been dissolved in a
mixture of ethanol/tetrahydrofuran = 1:1 (15 mL), 10% Pd/C
(50 mg) was added and the reaction mixture stirred under a hydro-
gen atmosphere at room temperature overnight. The catalyst was
filtered off and the solvent removed under reduced pressure. The
crude product was purified by flash column chromatography using
dichloromethane/methanol (9:1) as eluent. Yield: 78.3%; brown so-
lid; mp > 300 °C; 1H NMR (DMSO-d6): d = 2.27 (s, 3H, CH3), 6.57
(dd, 1H, 3J = 2.1 Hz, 2J = 8.5 Hz, Ar-H-6), 6.76 (d, 1H, 3J = 2.1 Hz,
Ar-H-4), 6.88 (d, 2H, J = 8.6 Hz, 2 ꢀ Ar-H0), 7.10 (d, 1H, 2J = 8.5 Hz,
Ar-H-7), 7.44 (d, 2H, J = 8.6 Hz, 2 ꢀ Ar-H0), 8.56 (s, 1H, OH), 9.54
(s, 1H, OH), 10.59 (s, 1H, NH) ppm. MS (ESI+): m/z (%) = 240 (100)
[M+H]+; HRMS (ESI+): m/z [M+H]+ calcd for C15H14NO2: 240.1025,
found: 240.1025.
2
1H, J = 8.8 Hz, Ar-H-7), 7.30 (d, 2H, J = 8.7 Hz, 2 ꢀ Ar-H0), 7.34–
7.50 (m, 10H, 2 ꢀ Ph) ppm.
2.1.8. Synthesis of 4-(5-(benzoyloxy)-1-(4-(2-bromoethoxy)benzyl)-3-
methyl-1H-indol-2-yl)phenyl benzoate (10) [29]
CBr4 (0.970 g, 2.92 mmol) and PPh3 (0.760 g, 2.92 mmol) were
added to a solution of compound 9 (1.107 g, 1.85 mmol) in dry tet-
rahydrofuran (30 mL). The reaction mixture was stirred at room
temperature overnight and the solvent then evaporated under
reduced pressure. The crude product was purified by flash column
chromatography using ethyl acetate/hexane (1:4) as the eluent.
Yield: 41.2%; dark yellow solid; mp 152–155 °C ([29],
2.2. Materials for the GSH trapping assay
Raloxifene, haloperidol, ketoconazole, human liver microsomes,
recombinant CYP3A4, CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2E1,

12
T.T. Lušin et al. / Chemico-Biological Interactions 197 (2012) 8–15
glutathione (GSH), NADPH, MgCl2, dimethyl sulfoxide (DMSO)
were from Sigma Aldrich (Deisenhofen, Germany).
gas flow 11 L/min, capillary entrance voltage 4000 V, nozzle volt-
age 2000 V, delta EMV 200 V. The MS detector was operated using
the negative precursor ion scan mode. The MS1 scan range was m/z
300–800 with a resolution of 0.7, while the scan time was 500 ms
and the fragmentor was set at 135 V; the collision energy was
12 eV. Instrument control, data acquisition and quantification were
performed using MassHunter Workstation software B.03.01 (Agi-
lent Technologies, Torrance, USA).
2.3. Incubations with human liver microsomes or recombinant CYP450
isozymes
All incubations (total volume, 100 lL) were conducted at 37 °C
for 60 min in a 50 mM phosphate buffer (pH 7.4) containing 1 mg/
mL of human liver microsomes or recombinant CYP450 isozymes,
5 mM MgCl2, 5 mM GSH and 1 mM NADPH. Firstly, the microsomes
were suspended in a mixture of buffer, MgCl2 and GSH. Raloxifene,
bazedoxifene or 2-(4-hydroxyphenyl)-3-methyl-1H-indol-5-ol in
3. Results
3.1. Incubation of raloxifene with HLM and with recombinant CYP3A4
DMSO was added to a final concentration of 50 lM. The final con-
centration of DMSO in the incubation media was 1%. The reaction
mixture was preincubated at 37 °C for 5 min. Twenty microliters
of 5 mM NADPH was added in order to initiate the reaction. The
While the glutathione conjugate of raloxifene was formed in the
incubation with both HLM and recombinant CYP3A4 in the pres-
ence of NADPH and GSH (Fig. 2), it was not found in the control
incubations without NADPH and/or GSH. In contrast to Chen
et al. [9] and Yu et al. [30], who suggested the formation of three
isomeric glutathione conjugates of raloxifene, only one chromato-
graphic peak corresponding to the raloxifene-glutathione adduct
was observed in the current study when searching for the precur-
sor ions of the fragment m/z 272 in negative ionization mode,
which is considered a typical fragment ion for glutathione-adducts
[19] (Fig. 2). The presence of the precursor ion at m/z 777 con-
firmed the raloxifene-glutathione adduct (with an expected mass
shift of 305). Similarly, only one glutathione conjugate of raloxif-
ene was observed in the study carried out by Dalvie et al. [12]. In
the presence of ketoconazole, the raloxifene glutathione conjugate
was not formed, a consequence of the strong inhibition of CYP by
ketoconazole; this observation was consistent with the results of
Chang et al. [31]. Moreover, so as to demonstrate the involvement
of CYP3A4 in raloxifene bioactivation, experiments on recombinant
CYP3A4 were performed and the results also showed the formation
of raloxifene glutathione conjugates. Based on successful formation
of the raloxifene glutathione conjugate, it can be concluded that
our incubation system was appropriate.
reaction was quenched by adding 25 lL of ice cold acetonitrile con-
taining haloperidol as an internal standard. The incubation mixture
was left at ꢁ20 °C for 24 h and then centrifuged at 1300g for
100 min at 4 °C. The supernatants were transferred into a 96-well
plate for subsequent LC–MS/MS analysis. Incubations that lacked
NADPH, GSH, or microsomes served as negative controls. In addi-
tion, the formation of GSH conjugates was monitored in the pres-
ence of a CYP3A4 inhibitor, ketoconazole (1 lM).
2.4. LC–MS/MS analysis of glutathione conjugates
For the purposes of metabolite profiling, the resulting superna-
tants were analyzed using an Agilent 6460 triple quadrupole mass
spectrometer equipped with a JetStream™ interface and coupled to
an Agilent 1290 Infinity UPLC (Agilent Technologies, Santa Clara,
USA). The chromatographic separation was performed on a Kinetex
100 ꢀ 3.0 mm C18 column (2.6-
4 ꢀ 2 mm C18 mm cartridge column (Phenomenex, Torrance,
USA) and kept at 50 °C. The injection volume was 1.0 L. After each
lm particles), guarded by a
l
injection, the injection needle was washed for 15 s with 80%
MeOH. The mobile phase A was 0.1% formic acid in water and
the mobile phase B was 100% acetonitrile. The flow rate was
0.5 mL/min with the following linear gradient points (time [min],
% of B): (0,10); (0.5,10); (0.8,20); (1.5,40); (1.9,60); (2.3,60);
(2.5,10); (3.5,10). The ion source parameters were set as follows:
drying gas temperature 275 °C, drying gas flow rate 5 L/min, nebu-
lizer 45 PSI (3.1 ꢀ 105 Pa), sheath gas temperature 320 °C, sheath
3.2. Incubation of bazedoxifene with HLM and with recombinant
CYP450 isozymes
In the case of bazedoxifene, no glutathione conjugates were de-
tected in the incubation with HLM (Fig. 3, m/z 272 precursor ion
scan expecting a conjugate signal at m/z 774 resulting from mass
Fig. 2. The negative precursor ion scan chromatogram of the HLM-raloxifene incubation medium (searching for precursors from fragment m/z 272) with the corresponding
spectrum extracted from the peak at 1.778 min that shows the molecular mass of the raloxifene glutathione conjugate peak (m/z 777.2).

T.T. Lušin et al. / Chemico-Biological Interactions 197 (2012) 8–15
13
Fig. 3. Total ionic chromatogram of precursor ion scan of the bazedoxifene – HLM incubation sample (searching for precursors from fragment m/z 272) where no peaks for
bazedoxifene glutathione conjugates were observed. The empty MS spectrum at the expected retention time (around 1.8 min) is presented in the upper right corner, which
shows the absence of any GSH conjugates.
Table 1
raloxifene (precursor scan m/z 272 giving a strong signal at m/z
777, Fig. 2), which validated our in vitro incubation system, the
negative results of bazedoxifene glutathione conjugate formation
can be considered reliable.
Formation of GSH conjugates by human P450 isozymes, expressed as areas of LC–MS/
MS peaks obtained from extracted precursor ion (m/z 272) scan chromatograms.
System
Bazedoxifenea
Compound 13b
Bazedoxifene was also incubated with human recombinant
cytochrome P450 isozymes, and the amounts of GSH conjugates
formed determined (Table 1). Just as in the case of the bazedoxif-
ene – HLM incubation sample, no GSH conjugates were detected
(m/z 272 precursor ion scan expecting a conjugate signal at m/z
774 or m/z 491 resulting from mass shift 305 or 321, respectively).
None of the selected human cytochrome P450 isozymes catalyzed
the formation of reactive species of bazedoxifene, while only traces
of GSH conjugates were observed in incubations with CYP2C9 and
CYP2E1 (Table 1). The N-dealkylated metabolite of bazedoxifene
(compound 13) was detected neither after the incubation of baze-
doxifene with HLM nor with recombinant P450 isozymes (molecu-
lar ion [M+H]+ m/z 240); the only exception noted was that of a
trace amount observed in CYP2C9 incubation.
Control
CYP1A2
CYP3A4
CYP2C8
CYP2C9
CYP2C19
CYP2E1
–
–
2012
3266
3306
8596c
–
1532,214
364,722
302,654
779,304
528,737
312,108
8250
a
b
c
m/z = 791 (470 + 305 + 16); tr = 1.505 min.
m/z = 543 (238 + 305); tr = 1.749 min.
In this incubation, traces of GSH conjugate of compound 13 were detected as
well (corresponding area 4641).
shift 305 from the parent as was observed with raloxifene). Based
on the positive results of glutathione conjugate formation of
Fig. 4. The negative precursor ion scan chromatogram of compound 13 in HLM incubation medium, a survey-scan for precursors of m/z 272 fragment ions characteristic for
glutathione conjugates. Upper right: extracted mass spectrum from peak at retention time 1.757 min, which shows the [MꢁH]ꢁ molecular peak of 2-(4-hydroxyphenyl)-3-
methyl-1H-indol-5-ol-glutathione conjugate (mass shift 305 from the parent [32]).

14
T.T. Lušin et al. / Chemico-Biological Interactions 197 (2012) 8–15
3.3. Incubation of 2-(4-hydroxyphenyl)-3-methyl-1H-indol-5-ol (13)
with HLM and recombinant CYP450 isozymes
HLM or recombinant CYP3A4, while bazedoxifene GSH-conjugates
were detected only in trace amounts with HLM and six selected
CYP450 isozymes (CYP1A2, CYP3A4, CYP2C8, CYP2C9, CYP2C19
and CYP2E1). The detection of raloxifene GSH-conjugates supported
previously existing evidence for the potential of raloxifene bioacti-
vation and the formation of reactive metabolites and confirmed the
suitability of the incubation conditions. The presence of ketocona-
zole as a strong CYP3A4 inhibitor [34] caused the complete disap-
pearance of the GSH-conjugate peak of raloxifene, indicating that
CYP3A4 is an important CYP450 isoform in the formation of reactive
metabolites.
One GSH adduct of 2-(4-hydroxyphenyl)-3-methyl-1H-indol-5-
ol (13), a 5-hydroxy-3-methylindole-based fragment of bazedoxif-
ene, was detected. The LC–MS/MS precursor ion scan of the
CYP3A4 incubation medium, searching for precursors of the frag-
ment ion m/z 272 in negative mode, revealed a strong peak corre-
sponding to the GSH conjugate (m/z [MꢁH]ꢁ 543: a mass shift of
305 from the parent m/z 238, Fig. 4). This strongly supports the
identification of a GSH adduct of compound 13. The GSH adduct
showed a characteristic loss of m/z 272, as evidenced by the pre-
cursor ion scan experiment. It was established that the tested baze-
doxifene fragment (compound 13) forms glutathione conjugates in
the microsomal and the human recombinant CYP450 isozyme
incubation systems (Table 1). CYP1A2 showed the highest activity,
while the activity of other selected CYPP450s decreased in the fol-
lowing order CYP2C9 > CYP2C19 > CYP3A4 ꢂ CYP2E1 ꢂ CYP2C8.
Although bazedoxifene contains a potentially toxic 5-hydroxy-
3-methylindole moiety, our studies showed that bazedoxifene
was bioactivated to electrophilic intermediates with HLM or with
six selected recombinant CYP450 isozymes only in insignificant
quantities. Based on numerous reports of the bioactivation of 5-
hydroxy- and 3-methylindole-based compounds, we tested the
N-dealkylated moiety of bazedoxifene (compound 13) in the GSH
trapping assay. The GSH adduct of compound 13 was identified with
HLM and with all six selected recombinant CYP450 isozymes with
the aid of precursor ion scan experiments (m/z 272). Among the
individual CYP450s present to a high degree in the pooled human li-
ver microsomes, CYP1A2 showed the highest activity. The activity of
other selected P450s decreases in the following rank order CY-
P2C9 > CYP2C19 > CYP3A4 ꢂ CYP2E1 ꢂ CYP2C8. Compound 13 is
bioactivated by CYP450s to the corresponding quinoneimine inter-
mediate or through a dehydrogenation pathway to an electrophilic
3-methyleneindolenine intermediate that is trapped with GSH.
Based on these results, we can conclude that bazedoxifene requires
an initial N-dealkylation reaction on the indole moiety, which can
subsequently lead to the formation of reactive intermediates.
However, an N-dealkylated metabolite 13 of bazedoxifene was not
detected after the incubation of bazedoxifene with HLM or recombi-
nant CYP450 isozymes (molecular ion [M+H]+ m/z 240), with the
exception of a trace amount observed in CYP2C9 incubation. The
in vitro data suggest that bazedoxifene is not bioactivated with re-
combinant CYP450 isozymes or HLM to form reactive species, thus
warranting further in vivo investigation of bazedoxifene bioactiva-
tion in order to confirm the in vitro results.
In conclusion, our results confirm that bazedoxifene, a new third
generation, indole-based ER-ligand, which has been developed for
use in post-menopausal osteoporosis, offers an improved safety
profile relative to SERM therapies currently available. Bazedoxifene
was not dehydrogenated with recombinant CYP450 isozymes or
HLM to form reactive electrophilic species and is therefore unlikely
to cause adverse effects by covalently binding to the nucleophilic
residues of proteins and/or DNA. The results of the studies with
bazedoxifene and its structural 5-hydroxy-3-methylindole-based
fragment, coupled with several reports on other 3- and 5-substi-
tuted indole-containing drugs, provide additional evidence that this
aromatic moiety should be used with caution in the development of
new therapeutic agents. This study provides further proof that not
all compounds possessing a potential structural fragment for bioac-
tivation (structural alert) will necessarily, under bioactivation, elicit
the formation of reactive species in vitro.
4. Discussion
In the present study, the bioactivation potential of bazedoxifene,
as a third generation SERM, was evaluated using the GSH trapping
assay with HLMs and recombinant CYP450 isozymes and compared
to the structurally related second generation SERM, raloxifene, and
to 2-(4-hydroxyphenyl)-3-methyl-1H-indol-5-ol (13), a 5-hydro-
xy-3-methylindole-based structural fragment of bazedoxifene. Our
results demonstrated that bazedoxifene forms only trace amounts
of reactive electrophilic species in incubation with HLM or recombi-
nant CYPP450isozymes (Table 1) in the presenceofNADPH and GSH.
It is, therefore, most likely that it does not cause adverse effects by
covalently binding to nucleophilic residues of proteins and/or DNA.
Bazedoxifene was synthesized, since it was not commercially
available at that time. The synthesis of bazedoxifene is outlined
in Scheme 1 [29]. Firstly, phenol 1 was protected with benzyl bro-
mide to obtain ketone 2, which was then brominated at its
a posi-
tion to obtain -bromopropiophenone 3. Indole core 4 was
a
synthesized via a Bischler-type indole synthesis from compound
3 and 4-benzyloxyaniline hydrochloride in N,N-dimethylformam-
ide using the two step – one pot procedure. The benzyl chloride
7 was prepared from 4-hydroxybenzyl alcohol (5) by alkylation
with ethyl bromoacetate (compound 6), followed by the nucleo-
philic substitution of the hydroxyl group by chlorine using thionyl
chloride and a catalytic amount of N,N-dimethylformamide. Indole
4 was N-alkylated with benzyl chloride 7, using NaH-furnished in-
dole 8. The side chain ester group of 8 was then reduced with
LiAlH4 in THF and the obtained alcohol 9 converted to bromide
10 by means of treatment with tetrabromomethane and triphenyl-
phosphine. Nucleophilic substitution of bromine in compound 10
with hexamethylenimine yielded amine 11, which, in the last step,
was deprotected using catalytic hydrogenation to obtain bazedox-
ifene 12 with a percentage purity of 97.5%, as determined by HPLC
analysis. 2-(4-Hydroxyphenyl)-3-methyl-1H-indol-5-ol (13),
a
bazedoxifene structural fragment, was synthesized by a process
of the catalytic hydrogenation of compound 4.
Previously, the most widely used LC–MS/MS method for gluta-
thione-conjugate detection was the neutral loss scan of m/z 129 in
positive ionization mode. Recently, the rather important discovery
was made that the negative precursor ion scan mode of m/z 272
yields far better detection, since the background noise in negative
ionization tends to be lower. Furthermore, any GSH bond linkage
variations that could hinder the neutral loss of m/z 129, would still
be detected by the precursor ion scan of m/z 272 [33].
Acknowledgements
This work was supported by the Slovenian Research Agency
(Grant No. P1-0208 and P1-0189). The authors thank Benedict
Dries-Jenkins for critical reading of the manuscript.
Appendix A. Supplementary data
The results obtained by HLM and recombinant CYP450 isozymes
assays were comparable. The incubations of raloxifene showed
considerable formation of the GSH-conjugate in incubations with
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.cbi.2012.03.001.

T.T. Lušin et al. / Chemico-Biological Interactions 197 (2012) 8–15
15
[19] C.M. Dieckhaus, C.L. Fernandez-Metzler, R. King, P.H. Krolikowski, T.A. Baillie,
Negative ion tandem mass spectrometry for the detection of glutathione
conjugates, Chem. Res. Toxicol. 18 (2005) 630–638.
[20] K.A. Regal, G.M. Laws, C. Yuan, G.S. Yost, G.L. Skiles, Detection and
characterization of DNA adducts of 3-methylindole, Chem. Res. Toxicol. 14
(2001) 1014–1024.
[21] G.L. Skiles, G.S. Yost, Mechanistic studies on the cytochrome P450-catalyzed
dehydrogenation of 3-methylindole, Chem. Res. Toxicol. 9 (1996) 291–297.
[22] J. Thornton-Manning, M.L. Appleton, F.J. Gonzalez, G.S. Yost, Metabolism of 3-
methylindole by vaccinia-expressed P450 enzymes: correlation of 3-
methyleneindolenine formation and protein-binding, J. Pharmacol. Exp. Ther.
276 (1996) 21–29.
[23] K. Kassahun, K. Skordos, I. McIntosh, D. Slaughter, G.A. Doss, T.A. Baillie, G.S.
Yost, Zafirlukast metabolism by cytochrome P450 3A4 produces an
electrophilic alpha, beta-unsaturated iminium species that results in the
selective mechanism-based inactivation of the enzyme, Chem. Res. Toxicol. 18
(2005) 1427–1437.
[24] B.J. Dean, S. Chang, M.V. Silva Elipe, Y.Q. Xia, M. Braun, E. Soli, Y. Zhao, R.B.
Franklin, B. Karanam, Metabolism of MK-0524, a prostaglandin D2 receptor 1
antagonist, in microsomes and hepatocytes from preclinical species and
humans, Drug Metab. Dispos. 35 (2007) 283–292.
[25] J.F. Levesque, S.H. Day, N. Chauret, C. Seto, L. Trimble, K.P. Bateman, J.M. Silva,
C. Berthelette, N. Lachance, M. Boyd, L. Li, C.F. Sturino, Z. Wang, R. Zamboni,
R.N. Young, D.A. Nicoll-Griffith, Metabolic activation of indole-containing
prostaglandin D2 receptor 1 antagonists: impacts of glutathione trapping and
glucuronide conjugation on covalent binding, Bioorg. Med. Chem. Lett. 17
(2007) 3038–3043.
References
[1] D.M. Biskobing, Update on bazedoxifene: a novel selective estrogen receptor
modulator, Clin. Interv. Aging 2 (2007) 299–303.
[2] J.R. Rey, E.V. Cervino, M.L. Rentero, E.C. Crespo, A.O. Alvaro, M. Casillas,
Raloxifene: mechanism of action, effects on bone tissue, and applicability in
clinical traumatology practice, Open Orthop. J. 3 (2009) 14–21.
[3] D. Clemett, C.M. Spencer, Raloxifene: a review of its use in postmenopausal
osteoporosis, Drugs 60 (2000) 379–411.
[4] F. Marin, M. Barbancho, Action of selective estrogen receptor modulators
(SERMs) through the classical mechanism of estrogen action, in: A. Sanchez, J.
Alsina, J. Dueñas-Díez (Eds.), Selective Estrogen Receptor Modulators,
Springer-Verlag, Heidelberg, 2006, pp. 71–77.
[5] B.S. Komm, Y.P. Kharode, P.V. Bodine, H.A. Harris, C.P. Miller, C.R. Lyttle,
Bazedoxifene acetate: a selective estrogen receptor modulator with improved
selectivity, Endocrinology 146 (2005) 3999–4008.
[6] H. Kawate, R. Takayanagi, Efficacy and safety of bazedoxifene for
postmenopausal osteoporosis, Clin. Interv. Aging 6 (2011) 151–160.
[7] A. Chandrasekaran, W.E. McKeand, P. Sullivan, W. DeMaio, R. Stoltz, J. Scatina,
Metabolic disposition of
women, Drug Metab. Dispos. 37 (2009) 1219–1225.
[
14C]bazedoxifene in healthy postmenopausal
[8] H. Yukinaga, T. Takami, S.H. Shioyama, Z. Tozuka, H. Masumoto, O. Okazaki, K.
Sudo, Identification of cytochrome P450 3A4 modification site with reactive
metabolite using linear ion trap-Fourier transform mass spectrometry, Chem.
Res. Toxicol. 20 (2007) 1373–1378.
[9] Q. Chen, J.S. Ngui, G.A. Doss, R.W. Wang, X. Cai, F.P. DiNinno, T.A. Blizzard, M.L.
Hammond, R.A. Stearns, D.C. Evans, T.A. Baillie, W. Tang, Cytochrome P450
3A4-mediated bioactivation of raloxifene: irreversible enzyme inhibition and
thiol adduct formation, Chem. Res. Toxicol. 15 (2002) 907–914.
[10] B.R. Baer, L.C. Wienkers, D.A. Rock, Time-dependent inactivation of P450 3A4
by raloxifene: identification of Cys239 as the site of apoprotein alkylation,
Chem. Res. Toxicol. 20 (2007) 954–964.
[11] T.S. Dowers, Z.H. Qin, G.R. Thatcher, J.L. Bolton, Bioactivation of selective
estrogen receptor modulators (SERMs), Chem. Res. Toxicol. 19 (2006) 1125–
1137.
[12] D. Dalvie, P. Kang, M. Zientek, C. Xiang, S. Zhou, R.S. Obach, Effect of intestinal
glucuronidation in limiting hepatic exposure and bioactivation of raloxifene in
humans and rats, Chem. Res. Toxicol. 21 (2008) 2260–2271.
[13] P. Hollenberg, U. Kent, N. Bumpus, Mechanism-based inactivation of human
cytochromes p450s: experimental characterization, reactive intermediates,
and clinical implications, Chem. Res. Toxicol. 21 (2008) 189–205.
[14] A.S. Kalgutkar, I. Gardner, R.S. Obach, C.L. Shaffer, E. Callegari, K.R. Henne, A.E.
Mutlib, D.K. Dalvie, J.S. Lee, Y. Nakai, J.P. O’Donnell, J. Boer, S.P. Harriman, A
comprehensive listing of bioactivation pathways of organic functional groups,
Curr. Drug Metab. 6 (2005) 161–225.
[15] A.S. Kalgutkar, H.T. Nguyen, Identification of an N-methyl-4-
phenylpyridinium-like metabolite of the antidiarrheal agent loperamide in
human liver microsomes: underlying reason(s) for the lack of neurotoxicity
despite the bioactivation event, Drug Metab. Dispos. 32 (2004) 943–952.
[16] D. Hochner-Celnikier, Pharmacokinetics of raloxifene and its clinical
application, Eur. J. Obstet. Gynecol. Reprod. Biol. 85 (1999) 23–29.
[26] H. Sun, G.S. Yost, Metabolic activation of
a novel 3-substituted indole-
containing TNF-alpha inhibitor: dehydrogenation and inactivation of CYP3A4,
Chem. Res. Toxicol. 21 (2008) 374–385.
[27] K.E. Zhang, P.H. Kari, M.R. Davis, G. Doss, T.A. Baillie, K.P. Vyas, Metabolism of A
dopamine D(4)-selective antagonist in rat, monkey, and humans: formation of
A novel mercapturic acid adduct, Drug Metab. Dispos. 28 (2000) 633–642.
[28] C.L. Viswanathan, M.M. Kodgule, A.S. Chaudhari, Design, synthesis and
evaluation of racemic 1-(4-hydroxyphenyl)-2-[3-(substituted phenoxy)-2-
hydroxy-1-propyl]amino-1-propanol hydrochlorides as novel uterine
relaxants, Bioorg. Med. Chem. Lett. 15 (2005) 3532–3535.
[29] C.P. Miller, M.D. Collini, B.D. Tran, H.A. Harris, Y.P. Kharode, J.T. Marzolf, R.A.
Moran, R.A. Henderson, R.H. Bender, R.J. Unwalla, L.M. Greenberger, J.P.
Yardley, M.A. Abou-Gharbia, C.R. Lyttle, B.S. Komm, Design, synthesis, and
preclinical characterization of novel, highly selective indole estrogens, J. Med.
Chem. 44 (2001) 1654–1657.
[30] L. Yu, H. Liu, W. Li, F. Zhang, C. Luckie, R.B. van Breemen, G.R. Thatcher, J.L.
Bolton, Oxidation of raloxifene to quinoids: potential toxic pathways via a
diquinone methide and o-quinones, Chem. Res. Toxicol. 17 (2004) 879–888.
[31] J.H. Chang, C.J. Kochansky, M. Shou, The role of P-glycoprotein in the
bioactivation of raloxifene, Drug Metab. Dispos. 34 (2006) 2073–2078.
[32] P. Baranczewski, A. Stanczak, A. Kautiainen, P. Sandin, P.O. Edlund, Introduction
to early in vitro identification of metabolites of new chemical entities in drug
discovery and development, Pharmacol. Rep. 58 (2006) 341–352.
[33] B. Wen, L. Ma, S.D. Nelson, M. Zhu, High-throughput screening and
characterization of reactive metabolites using polarity switching of hybrid
triple quadrupole linear ion trap mass spectrometry, Anal. Chem. 80 (2008)
1788–1799.
[17] C. Ju, J.P. Uetrecht, Oxidation of
a
metabolite of indomethacin
(desmethyldeschlorobenzoylindomethacin) to reactive intermediates by
activated neutrophils, hypochlorous acid, and the myeloperoxidase system,
Drug Metab. Dispos. 26 (1998) 676–680.
[34] M.A. Gibbs, K.E. Thummel, D.D. Shen, K.L. Kunze, Inhibition of cytochrome P-
450 3A (CYP3A) in human intestinal and liver microsomes: comparison of Ki
values and impact of CYP3A5 expression, Drug Metab. Dispos. 27 (1999) 180–
187.
[18] P.J. Formosa, T.M. Bray, S. Kubow, Metabolism of 3-methylindole by
prostaglandin H synthase in ram seminal vesicles, Can. J. Physiol. Pharmacol.
66 (1988) 1524–1530.