N-1H-Benzimidazol-5-ylbenzenesulfonamide derivatives as potent hPXR agonists

Article abstract of DOI:10.1016/j.bmc.2008.02.020

The Human Pregnane X Receptor (hPXR) is a nuclear receptor that regulates the expression of phase I and phase II drug-metabolizing enzymes, as well as that of drug transporters. Because this receptor plays a critical role in protecting tissues from potentially toxic endo- and xenobiotics, highly active agonists could represent novel therapeutic tools in treating several human diseases. Using an in vitro screening reporter system that allow to characterize hPXR activators and a first step of chemical modifications of an original agonist ligand (C2BA-4, 1-(2-chlorophenyl)-N-[1-(1-phenylethyl)-1H-benzimidazol-5-yl]methanesulf onamide), we identified compounds with a N-1H-benzimidazol-5-ylbenzenesulfonamide scaffold as a potent family of hPXR agonists. Further chemical modifications allowed us to identify enhanced activators, notably N-(1-benzyl-1H-benzimidazol-5-yl)-2,3,4,5,6-pentamethylbenzenesulfonamid e (6n) with an EC₅₀ value in the subnanomolar range. Accordingly to their potent EC₅₀, these compounds induced an efficient protection of hPXR against proteolytic digestion by trypsin even at very low ligand concentrations and were able to induce the expression of the main target genes of hPXR, CYP3A4 and CYP2B6, in primary cultures of human hepatocytes.

Full text of DOI:10.1016/j.bmc.2008.02.020

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 3537–3549
N-1H-Benzimidazol-5-ylbenzenesulfonamide derivatives
as potent hPXR agonists
Cindy Benod,a Guy Subra,a Virginie Nahoum,a Aude Mallavialle,c
a
a
a
a
´
Jean-Franc¸ois Guichou, Julien Milhau, Samuel Robles, William Bourguet,
Jean-Marc Pascussi,b Patrick Balaguerc and Alain Chavanieua,*
aINSERM, U554, Montpellier, F-34090, Univ Montpellier 1 and 2, CNRS, UMR5048,
Centre de Biochimie Structurale, Montpellier F-34090, France
bINSERM, U632, Montpellier, F-34293, Univ Montpellier 1, Montpellier F-34060, France
cINSERM, U824, Montpellier, F-34298, Univ Montpellier 1, Montpellier F-34060, France
Received 10 October 2007; revised 31 January 2008; accepted 8 February 2008
Available online 13 February 2008
Abstract—The Human Pregnane X Receptor (hPXR) is a nuclear receptor that regulates the expression of phase I and phase II drug-
metabolizing enzymes, as well as that of drug transporters. Because this receptor plays a critical role in protecting tissues from poten-
tially toxic endo- and xenobiotics, highly active agonists could represent novel therapeutic tools in treating several human diseases.
Using an in vitro screening reporter system that allow to characterize hPXR activators and a first step of chemical modifications of
an original agonist ligand (C2BA-4, 1-(2-chlorophenyl)-N-[1-(1-phenylethyl)-1H-benzimidazol-5-yl]methanesulfonamide), we identi-
fied compounds with a N-1H-benzimidazol-5-ylbenzenesulfonamide scaffold as a potent family of hPXR agonists. Further chemical
modifications allowed us to identify enhanced activators, notably N-(1-benzyl-1H-benzimidazol-5-yl)-2,3,4,5,6-pentamethylbenzene-
sulfonamide (6n) with an EC50 value in the subnanomolar range. Accordingly to their potent EC50, these compounds induced an effi-
cient protection of hPXR against proteolytic digestion by trypsin even at very low ligand concentrations and were able to induce the
expression of the main target genes of hPXR, CYP3A4 and CYP2B6, in primary cultures of human hepatocytes.
ꢀ 2008 Elsevier Ltd. All rights reserved.
1. Introduction
than 60% of human drugs.7 Thus, this nuclear receptor,
which is activated by a large number of prescription
drugs, is regarded as a xenobiotic sensor but is also at
the origin of several clinically important drug–drug
interactions.6,8
The Human Pregnane X Receptor (hPXR; NR1I21),
also known as SXR2 and PAR,3 is a member of the nu-
clear receptor (NR) superfamily which includes recep-
tors for steroid and thyroid hormones as well as
retinoids, cholesterol metabolites, and vitamins. hPXR
is a ligand-dependent transcription factor which, upon
Even though hPXR was identified as a ‘xenobiotic
receptor’, it also regulates the metabolism and availabil-
ity of important endogenous compounds.9–11 Indeed,
steroid hormones and neuro-active steroids activate
hPXR,12 suggesting that this receptor plays a major role
in many biological processes in diverse tissues. Recent
studies indicated that hPXR plays a role in bilirubin
clearance, prevents hyperbilirubinemia, and hepatorenal
toxicity from cholesterol metabolites.13 Also, when cho-
lesterol metabolism is perturbed or toxic bile acids accu-
mulate, hPXR switches on feedback and feed forward
mechanisms to block bile acid production from choles-
terol and to induce bile acid and oxysterol metabolic
and transport pathways.6,14 Activation of hPXR also
inhibits the fibrosis response of the liver to chronic dam-
age and the antifibrogenic mode of action is mediated
heterodimerization with the retinoid
X
receptor
(RXR),2,4,5 interacts with a variety of DNA response
elements (direct repeats DR-3, DR-4, and DR-5, and
everted repeats ER-6 and ER-8) in the 50-flanking re-
gions of target genes. hPXR regulates the expression
of cytochrome P450s, conjugating enzymes and ABC
transporters involved in detoxification of human organ-
ism.6 In particular, hPXR is the master regulator of the
expression of CYP3A4 isoform, which metabolizes more
Keywords: Nuclear receptor; hPXR; Docking; Drug design.
*
Corresponding author. Tel.: +33 4 67 54 86 44; fax: +33 4 67 52 96
23; e-mail: alain.chavanieu@cbs.cnrs.fr
0968-0896/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.02.020

3538
C. Benod et al. / Bioorg. Med. Chem. 16 (2008) 3537–3549
through changes in the expression of genes in hepatic
stellate cells and liver macrophages.15 Finally, hPXR is
involved in lipid homeostasis by activating genes that
facilitate lipogenesis and by suppressing the b-oxidative
pathways.16 The implication of hPXR in these metabolic
pathways suggests that hPXR activators could have
therapeutic applications for the treatment of several hu-
man diseases and might represent novel therapeutic
tools.10 Several agonists of hPXR with various chemical
structures have been described including the cholesterol-
lowering drugs lovastatin, the hypocholesterolemic drug
SR12813.1,17 Nevertheless original families of agonists
with a high affinity remained to be identified.
2. Chemistry
To determine the structure–activity relationship of
C2BA-4 (1), several sulfonamide analogues were synthe-
sized. 1-Benzyl-1H-benzimidazol-5-amine moiety found
in several compounds was synthesized according to
Scheme 1. 5-Nitrobenzimidazole was alkylated with ben-
zyl bromide in the presence of potassium carbonate in
butanone19 to give a mixture of 1-benzyl-5-nitro-1H-
benzimidazole 2 and its N3 regioisomer 3. The nitro group
was then reduced by treatment with SnCl2 in dry EtOH to
afford the required compound 1-benzyl-1H-ben-
zimidazol-5-amine 4 and its N3 regioisomer 5. Com-
pounds 4 and 5 were separated by chromatography and
the desired regioisomer 4 (yield 34%) was identified
through 1H NMR experiments (NOE). The sulfonamide
analogues 6a–p and 7a–b were obtained by the condensa-
tion of compounds 4 or 5 with commercial sulfonyl chlo-
rides or 2-chlorophenylmethanesulfonyl chloride in dry
THF and in the presence of NEt3 (Schemes 2 and 3) in
good yields. To obtain the 1-benzyl-1H-indol-5-amine
moiety of compound 8, the 5-nitroindole was alkylated
with benzyl bromide,19 then the nitro group was reduced.
Compound 8 was then obtained by the condensation of
the 1-benzyl-1H-indol-5-amine moiety with 2-chloro-
phenylmethanesulfonyl chloride (Scheme 4, yield 80%).
The urea analogues, compounds 9a–c, were prepared by
the condensation of compound 4 with aryl isocyanates
(Scheme 5) in good to excellent yields. The synthesis of
amide derivatives, compounds 10a–c (Scheme 6) pro-
ceeded by the condensation of compound 4 with carbox-
ylic acids in CH2Cl2 and in the presence of TBTU,20
DIEA to afford amide derivatives 10a–c in good yields.
The 1-hexyl-1H-benzimidazol-5-amine moiety found in
compounds 14a–b and the 1-(naphthalen-2-ylmethyl)-
1H-benzimidazol-5-amine group found in compounds
15a–b were synthesized according to the Scheme 7. 5-
Nitrobenzimidazole was alkylated with 1-bromohexane
or with 2-(1-bromomethyl) naphthalene to give a mix-
ture of two regioisomer derivatives (compounds 11a–b
and 12a–b, respectively). In the case of compounds
Combining a structure-based and high-throughput vir-
tual screening method with a functional approach using
both cell- and whole animal-based assays, we recently
proposed a strategy to identify highly active hPXR
agonists.18 Thus, we reported the identification of 13
new hPXR ligands and notably identified one agonist li-
gand named C2BA-4 (1) with an EC50 of 49 15 nM
(Fig. 1) (1-(2-chlorophenyl)-N-[1-(1-phenylethyl)-1H-
benzimidazol-5-yl]methanesulfonamide).
Here, based on this initial hit, we report the chemical
optimization study of this initial hit which led to the
identification of a series of agonist ligands that exhibit
potent in vitro hPXR agonist activities and excellent effi-
cacy in regulating CYP450 expression in human primary
hepatocytes. In particular, we identified compound 6n as
a potent agonist of hPXR with active concentration in
the subnanomolar range.
H
N
O
N
N
S
C2
O
Cl
C1
Figure 1. Structure of C2BA-4 (1), a previous hit.
NO2
N
N
NO2
a
N
+
NO2
N
N
N
H
2
3
b
NH2
+
N
NH2
N
N
N
4 (yield 34%)
5 (yield 21%)
Scheme 1. Reagents and conditions: (a) BnBr, K2Co3, butanone, reflux; (b) SnCl2, EtOH, reflux.

C. Benod et al. / Bioorg. Med. Chem. 16 (2008) 3537–3549
3539
H
N
O
S
O
N
N
NH2
N
N
Ar
n
a
6a-p
4
6a : n=1, Ar=2-chlorophenyl (yield 31%)
6b : n=1, Ar=2-nitrophenyl (yield 76%)
6c : n=0, Ar=2-chlorophenyl (yield 40%)
6d : n=0, Ar=2-bromophenyl (yield 42%)
6e : n=0, Ar=2-fluorophenyl (yield 42.5%)
6f : n=0, Ar=2-nitrophenyl (yield 13%)
6g : n=0, Ar=4-nitrophenyl (yield 18%)
6h : n=0, Ar=1-naphthyl (yield 39%)
6i : n=0, Ar=2-naphthyl (yield 44%)
6j : n=0, Ar=4-biphenyl (yield 69%)
6k : n=0, Ar=2-methylphenyl (yield 18%)
6l : n=0, Ar=3,5-dimethylphenyl (yield 18%)
6m : n=0, Ar=2,4,6-trimethylphenyl (yield 46%)
6n : n=0, Ar=pentamethylphenyl (yield 66%)
6o : n=0, Ar=4-tertbutylphenyl (yield 36%)
6p : n=0, Ar=4-propylphenyl (yield 76%)
Scheme 2. Reagents and condition: (a) Ar-(CH2)n–SO2Cl, NEt3, THF, reflux.
11a–b the desired N1 regioisomer 11a (yield 54%) was
separated by chromatography on silica gel and identified
1
through H NMR experiment, before the reduction of
a
H
N
O
the nitro group. Whereas for compounds 12a–b, due to
a difficulty to separate compounds at this step, the
purification was performed after the reduction. The nitro
group was reduced to give the required compounds, 1-hex-
yl-1H-benzimidazol-5-amine (13a, yield 41%) and 1-
(naphthalen-2-ylmethyl)-1H-benzimidazol-5-amine (13b,
yield 32%). Then, compounds 14a–b and 15a–b were
obtained by the condensation with 2-chloro-
benzenesulfonyl chloride or pentamethylbenzenesulfonyl
chloride in dry THF and in the presence of NEt3 in good
yields.
NH2
Cl
N
N
N
N
S
O
n
5
7a-b
7a : n=1 (yield 46%)
7b : n=0 (yield 12%)
Scheme 3. Reagents and condition: (a) Ar-(CH2)n–SO2Cl, NEt3, THF,
reflux.
H
N
O
S
O
NO2
a, b, c
N
Cl
3. Results and discussion
N
H
8 (yield 80%)
3.1. New lead selection and synthesis of derivatives
Scheme 4. Reagents and conditions: (a) BnBr, K2CO3, butanone,
reflux; (b) SnCl2, EtOH, reflux; (c) 2-chlorophenylmethanesulfonyl
chloride, NEt3, THF, reflux.
hPXR agonist activities as well as hPXR-independent
luciferase expression were evaluated as already de-
H
N
H
N
NH2
N
N
N
Ar
a
O
N
9a-c
4
9a : Ar=2-chlorophenyl (yield 87%)
9b : Ar=2-nitrophenyl (yield 63%)
9c : Ar=1-naphthyl (yield 56%)
Scheme 5. Reagents and condition: (a) aryl isocyanate, THF, reflux.
H
N
NH2
N
N
N
N
a
Ar
O
10a-c
4
10a : Ar=2-chlorophenyl (yield 71%)
10b : Ar=1-naphthyl (yield 63%)
10c : Ar=2-naphthyl (yield 21%)
Scheme 6. Reagents: (a) Ar-CH2COOH, TBTU, DIEA, CH2Cl2.

3540
C. Benod et al. / Bioorg. Med. Chem. 16 (2008) 3537–3549
R6
N
NO2
NO2
NO2
N
N
N
a
+
N
N
H
R6
11a, 12a
11b, 12b
11a, 11b : R6=hexyl
b
12a, 12b : R6=2-naphthylmethyl
H
N
O
S
O
NH2
N
N
N
N
c
Ar
R6
R6
13a-b
14a-b, 15a-b
14a : R6= hexyl, Ar=2-chlorophenyl (yield 44%)
13a : R6= hexyl
14b : R6= hexyl, Ar=pentamethylphenyl (yield 58%)
15a : R6= 2-naphthylmethyl, Ar=2-chlorophenyl (yield 61%)
15b : R6= 2-naphthylmethyl, Ar=pentamethylphenyl (yield 23%)
13b : R6= 2-naphthylmethyl
Scheme 7. Reagents (a) R-CH2Br, K2CO3, butanone, reflux; (b) SnCl2, EtOH, reflux; (c) Ar-SO2Cl, NEt3, THF, reflux.
scribed.21,18 Briefly, to detect hPXR agonists, HGPXR
cell line22 that stably express hPXR was used and the acti-
vation values obtained with all the tested compounds
were expressed relative to the luciferase activity observed
in the presence of 1 lM of the human and rabbit PXR
activator17 SR12813 (taken as 100%). To assess the
hPXR-independent luciferase expression as well as the
toxicity, all compounds were systematically tested for
their ability to activate parent HG5LN cells.22,18
Table 1. Data of EC50 values obtained on HGPXR reporter cell lines
of compounds C2BA-4, 6a–c and 7a–b
O
N
N
N
R1
S
O
N
O
N
N
O
S
n
R2
R1
n
R2
C2BA-4, 6a, 6b, 6c
7a, 7b
Based on C2BA-4 (1), an initial attempt to identify a new
lead compound consisted in removing the CH3 group
responsible for the chirality, andthe methylene group (po-
sition C1 and C2, respectively, Fig. 1). C2BA-4 used as a
racemic solution showed an EC50 of 49 nM (Table 1 and
supporting information) and removing the CH3 group
(6a, EC50 = 176 nM) increased the EC50 value by a factor
of 3.5. Compound 6c without the CH3 group responsible
for the chirality and without the methylene group pre-
O
S
N
O
N
n
R2
R1
8
a
EC50 (nM) (n)
Compound
n
R1
R2
sented
a
twofold decrease of the EC50 value
C2BA-4 (1)
1
1
1
0
1
0
1
Me
H
Cl
Cl
49 15 (3)
176 73 (3)
428 104 (3)
23 10 (4)
(EC50 = 23 nM) which indicated that the methylene
group has a deleterious effect on the agonist activity. We
also investigated the effect of the replacement of the chlo-
ride atom on C2BA-4 by a 2-nitro moiety and found an
attenuated agonist activity (6b, EC50 = 428 nM). The syn-
thesis of 6a and 6c provided an intermediary by-product
(5), a regioisomer of compound 4 with a benzyl group at
the N3-position on the benzimidazole ring (Scheme 1).
Compound 5 led to 7a and 7b which both showed a lower
potency (EC50 of 376 nM and 340 nM, respectively) than
C2BA-4 and a high cellular toxicity at 10 lM (data not
shown). In order to investigate the influence on the ago-
nist activity of an indole moiety, compound 8 was synthe-
sized. This compound was as potent as 6a with an EC50 of
197 nM but exhibited a higher cellular toxicity at 10 lM
(data not shown).
6a
6b
6c
7a
7b
8
H
NO2
Cl
H
H
Cl
Cl
376 230 (2)
340 110 (2)
197 200 (2)
H
H
Cl
C2BA-4 is used as a racemic solution. EC50, concentration of molecule
required for 50% hPXR activation, data represent mean values SD.
a Number of independent experiments in triplicate.
potent compound (9a, EC50 = 1476 nM). This decrease
being even more marked if the chlorophenyl moiety
was replaced with a 2-nitro-phenyl (9b) or a 1-naphthyl
group (9c) (EC50 > 5000 nM). The substitution of the
sulfonamide moiety by an amide (10a) also induced a
decrease of the agonist activity (EC50 = 515 nM) and a
1-naphthyl instead of the chlorophenyl moiety slightly
restored the agonist activity (10b, EC50 = 217 nM). By
Then, to precise the importance of the sulfonamide moi-
ety, the central part of the molecule C2BA-4 was re-
placed with an urea (Table 2) which led to a much less
contrast
a
2-naphthyl was not favorable (10c,

C. Benod et al. / Bioorg. Med. Chem. 16 (2008) 3537–3549
3541
Table 2. Data of EC50 values obtained on HGPXR reporter cell lines of compounds 9a–c and 10a–c
H
N
H
N
H
N
N
N
N
N
R4
R3
O
O
Compound
R3
EC50 SDa (nM) (n)
Compound
R4
EC50 SDa (nM) (n)
Cl
Cl
9a
1456 746 (3)
10a
515 281 (3)
217 144 (3)
>5000 (2)
NO2
9b
9c
>5000 (2)
>5000 (2)
10b
10c
EC50, concentration of molecule required for 50% hPXR activation, data represent mean values SD.
a Number of independent experiments in triplicate.
EC50 > 5000 nM). Tested in competition in presence of
SR12813 (0.3 lM), compounds 9b, 9c, and 10c at
10 lM and 1 lM were not found to present an antago-
nist activity (data not shown).
1.5 nM and 0.7 nM, respectively. In contrast, bulkier sub-
stituents such as tert-butyl or propyl groups in para-posi-
tion (6o, EC50 = 42 nM, 6p, EC50 = 52 nM) led to potent
but less active compounds when compared with com-
pound 6c or the highest active compound 6n. These results
thus indicated that the phenyl group of 6c can be substi-
tuted with various—preferentially small—aliphatic or
aromatic moieties whereas bulkier lipophilic groups are
less tolerated.
Consequently, the most active compound 6c with a sul-
fonamide group, no element of chirality at position C1
and no methylene group at position C2, was selected
as the new lead compound and served as the standard
for further potency comparison.
In a next step, we investigated the influence of the benzyl
group in the activity potency of our analogues (Table 4).
Compounds 14a with a hexyl moiety in replacement of
the benzyl group presented a twofold decreased activity
(EC50 = 55 nM) when compared to compound 6c and a
2-naphthylmethyl increased the EC50 value to 385 nM
(15a). The replacement of the 2-chlorophenyl group by
a pentamethylphenyl restored the activity and led to rel-
atively potent compounds (14b, EC50 = 13 nM, 15b,
EC50 = 55 nM).
We turned our attention to the nature of the substituent
on the phenyl ring and a series of 6c derivatives were syn-
thesized (Table 3). At first, the 2-chloride atom on the phe-
nyl group was substituted by electron-withdrawing
groups. Despite the higher steric hindrance, replacement
by a bromide atom led to the same potency (6d,
EC50 = 20 nM), whereas the o-substitution of the phenyl
ring with the small electron-withdrawing fluorine atom
(6e, EC50 = 101 nM) and the larger electron-withdrawing
nitro moiety (6f, EC50 = 271 nM), resulted in a significant
loss of activity. A 15-decrease of the agonist activity was
observed with a nitro group on the para position of the
phenyl ring (6g, EC50 = 336 nM). We then investigated
the effect of the replacement of the phenyl ring by larger
aromatic moieties and found that a 2-naphthyl and pref-
erentially a 1-naphthyl led to potent compounds whereas
a biphenyl group was not favorable (6i, EC50 = 51 nM,
6h, EC50 = 20 nM, 6j, EC50 = 388 nM, respectively).
Then, 2-methyl and 3,5-dimethyl phenyl derivatives were
synthesized and led to a slight decrease of the agonist
activity (6k, EC50 = 45 nM, 6l, EC50 = 33 nM) when com-
pared to 6c whereas a sharp increase in potency was ob-
served through introduction of three (6m) and five (6n)
methyl groups on the phenyl ring as the EC50 was
3.2. Docking studies and binding mode analysis of
compound 6n
Crystal structures of the ligand-binding domain (LBD)
of hPXR have been obtained in its apo conformation,
as well as in complex with the endogenous ligand 17b-
estradiol23 or xenobiotic compounds (rifampicin,24
SR12813,25 hyperforin,26 and T090131727) and with
fragments of the human transcriptional co-activator
SRC-1. These studies revealed a large, with 20 hydro-
phobic amino acids and eight polar residues capable of
forming hydrogen bonds, and conformable binding
pocket that contributes to the ability of hPXR to re-
spond to compound of varying size and shape through

3542
C. Benod et al. / Bioorg. Med. Chem. 16 (2008) 3537–3549
Table 3. Data of EC50 values obtained on HGPXR reporter cell lines of compounds 6d–p
O
S R5
O
N
N
N
Compound
R5
EC50 SDa (nM) (n)
Compound
R5
EC50 SDa (nM) (n)
Br
F
6d
20
8
6k
45 13
6e
6f
101 33
271 98
336 217
6l
6m
6n
33 .11
1.5 0.5
0.7 0.3
NO2
6g
NO2
6h
6i
6j
20
9
6o
6p
42 10
52 12
51 16
388 105
EC50, concentration of molecule required for 50% hPXR activation, data represent mean values SD.
a Number of independent experiments in triplicate.
˚
a combination of hydrophobic and polar interactions.
Unlike other nuclear receptors, hPXR LBD displays a
60 residue insertion (sequence 175–235) which generates
an extended five-stranded antiparallel b-sheet, instead of
the canonical three-stranded antiparallel b-sheet,28 and
participates to the a2 helical region (sequence 192–209)
that change its position and its secondary structure to
accommodate different types of ligands.27,24–26,29
(3.5 A), whereas the benzyl moiety could interact with
Leu 206, Leu 240, Leu 411, Phe 420, and Met 425 (from
˚
˚
2.5 A to 4.5 A) (Fig. 2A). Intriguingly, no significant po-
lar contribution to docking scores was observed despite
the presence of several hydrophilic residues in the li-
gand-binding pocket (Ser 247, Gln 285, His 327, and
His 427) and the sulfonamide moiety of compound 6n.
Additional docking studies were performed using Sur-
flex through an implemented method that relies on a
placed molecular fragment. Interestingly, the sulfon-
amide derivative 6n shows a common N-phenyl-ben-
zenesulfonamide group with the agonist T1317
(Fig. 2B) which presented an EC50 about 9.8 nM in
our transactivation test (see supplementary data) in per-
fect agreement with the value reported by Xue et al.27
Indeed, the N-phenyl-benzenesulfonamide group of
T1317 can be used as an anchoring point during the Sur-
flex docking process. When docking was carried out un-
der this condition (see supplementary data), 6n presents
a similar orientation in the hPXR LBP as described
above (Fig. 2A–C) with the pentamethylphenyl group
directed toward the b-sheet region of hPXR and at inter-
acting distances with three hydrophobic side chain resi-
The binding mode of compound 6n into the LBD of
hPXR was analyzed by the program Surflex-2.130,31
˚
and by using the recently reported 2.8 A resolution
structure of hPXR in complex with compound
T0901317 (T1317) and a 88-amino acid fragment of hu-
man SRC-127 (code pdb: 2O9I). In this structure, T1317
interacts via Van der Waals contacts with hydrophobic
residues constantly involved in ligand binding such as
Met 243, Trp 299, and Phe 420 as well as, for polar con-
tacts, with Gln 285 and His 407 but also with never pre-
viously described residues such as Tyr 306 and His 327.
When performed in the absence of restraint, our docking
studies revealed a putative binding mode for 6n in which
the pentamethylphenyl group lies at interacting distance
of the hydrophobic side chain of Trp 299 and Y306
˚
dues Phe 288, Trp 299, and Tyr 306 (3.5 A). This

C. Benod et al. / Bioorg. Med. Chem. 16 (2008) 3537–3549
3543
Table 4. Data of EC50 values obtained on HGPXR reporter cell lines
of compounds 14a–b and 18a–b
putative mode of binding is in good agreement with
activity results obtained with compounds 6d–p, which
underlined that aliphatic or aromatic substituents on
the phenyl ring led to highly active compounds. Then,
according to fragment restraint, the N-1H-ben-
zimidazol-5-ylbenzenesulfonamide group is found close
to the place occupied by the N-phenyl-benzenesulfon-
amide group of T1317 (see supplementary data) and
could form two putative hydrogen bonds in the central
part of the cavity, one with His 307 and one with Gln
H
N
O
S
O
N
N
R7
R6
Compound R7
R7
EC50 SDa (nM) (n)
Cl
˚
285 (<3 A). In this docking condition, the benzyl group
˚
of 6n was found close to the residue Phe 420 (<3 A) from
14a
55 32 (4)
Helix-12 which is part of the AF-2 surface implied in the
recruitment of co-activators,24–26,29,27,23 as well as close
˚
to the side chain of residues Leu 240 (<3 A) and Ile
14b
15a
15b
13 5 (4)
˚
414 (<4 A). The benzyl group of 6n was also found at
˚
interacting distance (<3 A) of Leu 206, a residue of the
mobile a2 region that contributes to hPXR ligand
accommodation. Interestingly, this putative mode of
binding could explain the potent agonist activity of sev-
eral compounds bearing, instead of the benzyl group, a
bulkier hexyl (14a–b) or 2-naphthylmethyl (15a–b) moi-
eties, which then are directed toward this mobile region
upon the binding to hPXR.
Cl
385 239 (4)
55 19 (3)
This hypothesis is in agreement with a previous report of
Xue et al.,27 which demonstrated that several analogues
of T1317 bearing bulky substituent, with the goal of ste-
EC50, concentration of molecule required for 50% hPXR activation,
data represent mean values SD.
a Number of independent experiments in triplicate.
Figure 2. Putative binding mode of compound 6n within the ligand pocket of the LBD of hPXR. (A) Compound 6n docked with no restraint presents
the pentamethylphenyl group directed toward Trp 299 and Tyr 306 and the benzyl moiety close to Phe 420 and Met 425 but no polar contribution to
binding of the sulfonamide central part. Residues forming hydrophobic interactions are in green while those forming polar contacts are rendered in
blue. (B) Chemical structures of 6n and T0901317. (C) Compound 6n docked under fragment restraint using the N-phenyl-benzenesulfonamide part
of T0901317 (code pdb: 2O9I). To clarify, Leu 206 and Leu 209 were not represented. (D) Compound 6n docked with constraint within the human
PXR LBD/SRC-1 complex (monomer, 2O9I) with a-helices rendered in red except: helix 12 in orange, the mobile a2 region is purple and the SRC1
fragment in blue. b-Sheets are in yellow and the carbon atoms of compound 6n in yellow. The side chains of residues W299, Q285, and F420 are
represented in sticks.

3544
C. Benod et al. / Bioorg. Med. Chem. 16 (2008) 3537–3549
rically disrupting aAF position, led to effective agonists
rather than antagonists. Thus, crystal structures of the
hPXR LBD in complex with various ligands have re-
vealed that part of the a2 region is structurally variable,
from a helix to completely disordered, and participates
to the ligand-binding pocket accommodation to large li-
gand such as rifampicin,24 T1317 analogues.
3.4. 6c, 6m, and 6n induce CYP3A4 and CYP2B6 mRNA
expression in cultured human hepatocytes
To further characterize 6c, 6m, and 6n as potent human
PXR ligands, we studied their effects on CYP3A4 and
CYP2B6 genes transcription4,13 in primary culture of
human hepatocytes. Hepatocytes from 4 different do-
nors were treated with the solvent control (DMSO
0.1%), SR12813 (1 lM), 6c (1 lM), 6m (1 lM), and 6n
(1 lM) for 24 h. Total mRNA was isolated and real-
time PCR analysis was performed to detect the expres-
sion level of GAPDH, b-actin, CYP3A4, and CYP2B6
mRNAs. We evaluated the induction ratios (mRNA lev-
els in treated cells compared with control cells after nor-
malization with respect to GAPDH signal). b-Actin
mRNA levels were used as quality controls of RNA
preparations and RT reactions. As expected, SR12813
strongly increased CYP3A4 and CYP2B6 mRNA
expressions in these cells (Table 5 and Fig. 4), while b-
actin RNA level was unchanged whatever the com-
pound tested. Interestingly, 6c, 6m, and 6n molecules
significantly increased CYP3A4 and CYP2B6 mRNAs
levels in all culture preparations. Moreover, 6n, which
is the most active compound (EC50 = 0.7 nM) in our
HGPXR cell line, induced the expression level of
CYP3A4 and CYP2B6 mRNAs stronger than
SR12813 at the same concentration in the three culture
preparations tested (Table 5). In addition, we observed
in two different primary cultures (FT271 and FT272)
that the increases of CYP3A4 and CYP2B6 mRNAs lev-
els by 6c, 6m, and 6n were dose dependent (Fig. 4), with
6c showing the most pronounced effect at the lowest
concentration tested (0.1 lM). These results demon-
strated that, in agreement with our in vitro studies, 6c,
6m, and 6n are able to activate hPXR in human hepato-
cytes, leading to an increase of transcription of its main
target genes such as CYP3A4 and CYP2B6. Even if the
response levels were different between donors, it is
important to note that CYP3A4 and CYP2B6 are induc-
ible enzymes and their induction varies markedly (up to
40-fold) across the population due to drug-mediated
variation in CYP transcription.32,12
3.3. Limited proteolytic digestion
In a previous study,18 C2BA-4 was reported to interact
with the LBD of hPXR. Here, to confirm whether the
most highly active compounds 6c, 6m, and 6n could di-
rectly bind and induce conformational changes in
hPXR, we analyzed its resistance to limited proteolysis
(Fig. 3 and supporting information). LBD of hPXR la-
beled with [35S]methionine was pre-incubated with etha-
nol (vehicle) or increasing concentrations (0.3–10 lM)
of compounds C2BA-4, 6c, 6m, and 6n, then digested
with 250 lg/ml trypsin. Incubation of the receptor with
250 lg/ml trypsin in the absence of ligand (ETOH) led
to complete digestion of hPXR-LBD. In contrast, all
the four ligands produced a 30 kDa major protease
resistant fragment. These results indicate that com-
pounds 6c, 6m, and 6n interact directly with hPXR
LBD. In agreement with the activities of these four li-
gands on HGPXR cell line, compounds 6m and 6n,
which exhibited the best agonist activities, protected
hPXR up to very low concentrations (EC50 = 8 nM).
C2BA-4 and compound 6c were less potent on hPXR,
and the receptor protection only occurred at higher con-
centrations (EC50 = 0.81 lM and 0.16 lM, respectively).
7.5
C2BA-4
6.0
6c
6m
4.5
6n
3.0
1.5
4. Conclusion
0.0
In conclusion, hPXR has emerged as a potential thera-
peutic target in several human diseases. This nuclear
receptor presents the particularity to bind a large variety
of endogen metabolites and xenobiotics usually with a
micromolar affinity. Only few compounds with a submi-
cromolar activity have been described so far (SR12813,
T0901317, Hyperforin). Among them, we recently re-
ported an original compound C2BA-4 which was suit-
able for chemical optimizations.
10-11
10-10
10-9
10-8
10-7
10-6
10-5
10-4
[Concentration, M]
Figure 3. Binding of C2BA-4 and compounds 6c, 6m, and 6n to hPXR.
hPXR protein was synthesized and labeled with [35S]methionine in
reticulocyte lysate and incubated with the four ligands or vehicle alone
before trypsin digestion. The reaction was terminated by boiling in
SDS-containing protein sample buffer. hPXR proteolytic pattern was
analyzed by SDS–PAGE and fixed/dried gel was visualized by
PhosphorImager (see supplementary data). This figure is representative
of three experiments. Autoradiograms were analyzed using an image
analyzing software. To measure the relative levels of the 30 kDa band
protected by the four ligands on SDS–PAGE gel, the intensity of the
bandof hPXR-LBDtreatedwith ethanolwas takenas 1. The axislabeled
protection shows the ratio (level of the 30 kDa band incubated
with ligand/ level of the band treated with ethanol). C2BA-4:
EC50 = 809 150 nM, 6c: EC50 = 157 44 nM, 6m: EC50 = 8.05
0.45 nM, 6n: EC50 = 8.04 1.9 nM.
In this report, we first identified a N-1H-benzimidazol-5-
ylbenzenesulfonamide scaffold that allow the design of
potent hPXR agonist ligands. Then, as judged on a re-
porter cell line system and by limited proteolytic diges-
tion, as well as on the expression of CYP3A4 and
CYP2B6 by human hepatocytes in culture, various
chemical modifications allowed us to identify a potent li-

C. Benod et al. / Bioorg. Med. Chem. 16 (2008) 3537–3549
3545
Table 5. Effects of compounds 6c, 6m, and 6n on the expression of hPXR target genes in cultured human hepatocytes
Donor #1 (FT 268)
Donor #2 (FT 269)
Donor #3 (FT271)
Donor #3 (FT272)
CYP3A4 CYP2B6
CYP3A4
CYP2B6
CYP3A4
CYP2B6
CYP3A4
CYP2B6
SR12813
6c
4.02 0.66
7.23 0.74
ND
3.26 0.23
3.02 0.31
ND
10.90 0.79
17.04 1.93
ND
3.74 0.56
10.46 1.38
ND
11.84 1.03
9.97 0.92
15.58 1.10
14.91 3.07
2.00 0.34
3.05 0.46
1.89 0.36
4.86 1.07
24.01 0.88
36.06 2.34
31.68 2.58
32.59 0.65
3.91 0.18
11.83 0.77
7.55 0.50
11.41 1.15
6m
6n
ND
ND
29.02 4.5
14.89 2.19
Human hepatocytes from four different donors (FT268, FT269, FT271, and FT272) were treated for 24 h with solvent (DMSO 0.1%), SR12813
(1 lM), compounds 6c, 6m, and 6n (1 lM). Twenty hours later, cells were harvested in TRIzol, total RNA was extracted and cDNA was synthesized
from 1 lg of total RNA using Superscript II. CYP3A4 and CYP2B6 mRNA levels were measured via quantitative real-time PCR using the Light
Cycler apparatus. As an internal control, the b-actin mRNA level was measured similarly to normalize data. Induction ratios (mRNA in inducer-
treated cells/mRNA in untreated cells) standard errors are presented here. ND: non-determined.
5. Experimental
5.1. General methods
All reagents and solvents were purchased from commer-
cial sources and used without further purification. Melt-
ing points were determined on a Kofler block and are
uncorrected. Mass spectra were acquired under fast atom
bombardment (FAB) conditions using a Jeol JMS
DX300, and samples were introduced on a matrix. Proton
NMR spectra were collected on a Bruker 400 MHz spec-
trometer using tetramethylsilane (TMS) as internal stan-
dard, and chemical shift (d) data are reported in parts
per million (ppm) relative to internal standard TMS. Sep-
arations by chromatography on silica column were car-
ried out by using silica gel SDS 60 (mesh 35–70) with
hexane, ethyl acetate (AcOEt), cyclohexane, and metha-
nol (MeOH) as eluents with chromatographic solvents ex-
pressed in volume/volume basis. The reported chemical
yields were not optimized. Materials for cell culture,
RNA extraction TRIzol reagent, SuperScript-II First-
Strand SynthesisSystem for RT-PCR, and Lipofectamine
werefrom Invitrogen (Cergy-Pontoise, France). Luciferin
`
and G418 were purchased from Promega (Charbonnie-
res-les-Bains, France). SR12813 was purchased from
Tebu-bio (Le Perray en Yvelines, France).
5.1.1. Synthesis of 1-benzyl-5-nitro-1H-benzimidazole (2)
and 3-benzyl-5-nitro-3H-benzimidazole (3). 5-Nitrobenz-
imidazole
(30.65 mmol)
and
benzyl
bromide
(33.68 mmol) was dissolved in 155 ml of butanone. Potas-
sium carbonate was then added (33.68 mmol). The reac-
tion mixture was stirred under reflux for 3 h then
concentrated under reduced pressure and dissolved in
ethyl acetate. Formed salts were filtrated and the filtrate
was concentrated again. The solid obtained was washed
with cyclohexane (100 ml), and then dried under vacuum
to give a mixture (orange red solid, 30.63 mmol, 99%) of
the required 1-benzyl-5-nitro-1H-benzimidazole (2) and
its N3 regioisomer (3).
Figure 4. Effect of SR12813, 6c, 6m, and 6n on CYP3A4 and
CYP2B6 mRNA expression in human hepatocytes. Human hepato-
cytes (FT2171 (A) and FT272 (B)) were cultured in presence of
SR12813, 6c, 6m, and 6n molecules at 0.1 lM or 1 lM for 24 h.
CYP3A4, CYP2B6, b-actin (actin), and GAPDH mRNAs were
quantified by real-time RT-PCR analysis. Values were normalized
with respect to GAPDH mRNA level. Induction ratios (mRNA in
inducer-treated cells/mRNA in untreated cells) standard errors are
presented here.
5.1.2. Synthesis of 1-benzyl-1H-benzimidazol-5-amine (4)
and 3-benzyl-3H-benzimidazol-5-amine (5). The mixture
containing compounds 2 and 3 (15.81 mmol) was dis-
solved in 165 ml of absolute ethanol and SnCl2 dihydrate
(82.45 mmol) was then added. The reaction mixture was
stirred under reflux for 3 h then concentrated under re-
duced pressure and dissolved in 165 ml of ethyl acetate.
Sodium bicarbonate (200 ml) was then added. Formed
gand with subnanomolar EC50, which represents the
most active compound described so far for hPXR.
Therefore, N-1H-benzimidazol-5-ylbenzenesulfonamide
derivatives represent an original family of potent ago-
nists of hPXR that might serve as novel leads for further
pharmacological investigations as therapeutic agonist
agents against human metabolic or neurodegenerative
diseases.10,33

3546
C. Benod et al. / Bioorg. Med. Chem. 16 (2008) 3537–3549
salts were filtrated and washed with 50 ml of AcOEt. The
organic phase of the filtrate was washed with water
(100 ml), dried on anhydrous sodium sulfate, filtrated,
and concentrated under reduced pressure to give a mix-
ture of compound 4 and its N3 regioisomer 5. Then, this
mixture was purified by chromatography on silica gel
(AcOEt/MeOH 9:1). Two fractions were obtained, one
contained the N3 regioisomer 5 (3.45 mmol 21%,
Rf = 0.48), the other contained the required 1-benzyl-
1H-benzimidazol-5-amine 4 (5.43 mmol, 34%, Rf = 0.24).
The two fractions were pink-orange color solids.
(59.40 mmol) was then added. The reaction mixture was
stirred under reflux for 18 h then concentrated under re-
duced pressure and dissolved in 150 ml of ethyl acetate. So-
dium bicarbonate (200 ml) was then added. Formed salts
were filtrated and washed with 500 ml of AcOEt. The or-
ganic phase of the filtrate was washed with brine (1·
100 ml), dried on anhydrous sodium sulfate, filtrated,
and concentrated under reduced pressure. The solid was
purified by chromatography on silica gel (hexane/AcOEt
6:4) to afford the 1-benzyl-1H-indol-5-amine as a red solid
(10.84 mmol, 81%). The 1-benzyl-1H-indol-5-amine
(0.45 mmol), and triethylamine (0.45 mmol) were dis-
solved in 10 ml of dry THF. The mixture was placed under
agitation and the (2-chlorophenyl)-methanesulfonyl chlo-
ride (0.45 mmol, 1 equiv) was then added. The reaction
mixture was stirred under reflux for 6 h, concentrated un-
der reduced pressure, then dissolved in 20 ml of dichloro-
methane. The organic phase was washed with citric acid
(2· 10 ml) and with brine (1· 10 ml). Then, the organic
phase was dried on anhydrous sodium sulfate, filtrated
then evaporated. The crude product was purified by chro-
matography on silica gel (hexane/AcOEt 6:4) to afford the
compound 8 as a pink-red solid (0.36 mmol, 80%).
5.1.3. Synthesis of sodium 2-chlorobenzyl sulfonate. The 2-
chlorobenzyl chloride (122.99 mmol) and sodium sulfite
(124.17 mmol) were dissolved in water (100 ml). The reac-
tion mixture was heated (100 ꢁC) under reflux for 5 h then
concentrated under reduced pressure. The white solid ob-
tained was filtrated, washed with diethylic ether (100 ml)
then dried under vacuum (120.99 mmol, 97%).
5.1.4. Synthesis of 2-chlorophenylmethanesulfonyl chlo-
ride. The sodium 2-chlorobenzyl sulfonate (43.76 mmol)
was put in the presence of phosphorus oxychloride
(219.0 mmol). The reaction mixture was heated (105 ꢁC)
under reflux for 6 h then filtrated (elimination of formed
salts). Formed salts were washed with dichloromethane
(50 ml). The filtrate was successively washed with water
(1 * 20 ml), with a saturated solution of NaHCO3
(2 * 20 ml) then with 0.25 N NaOH solution (1 * 20 ml).
Then, the organic phase was dried on anhydrous sodium
sulfate, filtrated then concentrated under reduced pres-
sure to give a white solid (15.95 mmol, 36%).
5.1.7. Synthesis of urea derivatives (9a–c): general proce-
dure. Compound 4 (0.45 mmol) was dissolved in dry THF
(10 ml). The mixture was placed under agitation and the
aryl isocyanate (0.54 mmol) was then added. The reaction
mixture was stirred under reflux for 5 h then concentrated
under reduced pressure. The solid was filtrated and washed
with diethylic ether to give the urea derivatives 9a–c.
5.1.8. Synthesis of acetamide derivatives (10a–c): general
procedure. Compound 4 (0.45 mmol), carboxylic acid
(0.45 mmol), and TBTU (0.45 mmol) were dissolved in
10 ml of CH2Cl2. The reaction mixture was placed under
agitation and DIEA (1.35 mmol) was added. The mix-
ture was agitated at room temperature for 24 h. Then,
the precipitated solid was filtrated then washed with
10 ml of CH2Cl2 to give the acetamide derivatives 10a–c.
5.1.5. Synthesis of sulfonamide analogues (6a–p, 7a–b,
14a–b, and 15a–b): general procedure (except for 8). The
compound 4 or 5 or 13a or 13b (0.45 mmol) and triethyl-
amine (0.45 mmol) were dissolved in dry THF (10 ml).
The mixture was placed under agitation and sulfonyl
chloride (0.45 mmol) was then added. The mixture was
stirred under reflux for 20 h, concentrated under reduced
pressure then dissolved in dichloromethane (10 ml). The
organic phase was successively washed with a citric acid
solution (1· 15 ml), with brine (1· 5 ml), with a saturated
solution of NaHCO3 (1· 15 ml) and finally with brine (1·
5 ml). Then, the organic phase was dried on anhydrous so-
dium sulfate, filtrated then evaporated. All the sulfon-
amide derivatives 6a–p, 7a–b, 14a–b, and 15a–b were
purified by chromatography on silica gel.
5.1.9. Synthesis of 1-hexyl-5-nitro-1H-benzimidazole
(11a) and 3-hexyl-5-nitro-3H-benzimidazole (11b). 5-
Nitrobenzimidazole (30.65 mmol) and bromohexane
(33.68 mmol) was dissolved in 155 ml of butanone.
Potassium carbonate was then added (34.39 mmol).
The reaction mixture was stirred under reflux for 24 h
then concentrated under reduced pressure and dissolved
in ethyl acetate (100 ml). Formed salts were filtrated and
the filtrate was concentrated again and dried under vac-
uum to give a mixture of the required 1-hexyl-5-nitro-
1H-benzimidazole (11a) and its N3 regioisomer (11b).
Then, this mixture was purified by chromatography on
silica gel (AcOEt/hexane 7:3). Two fractions were ob-
tained, one contained the required compound 11a
(16.06 mmol, 54%, Rf = 0.60), the other contained the
N3 regioisomer 11b (6.78 mmol, 23%, Rf = 0.40).
5.1.6. Synthesis of N-(1-benzyl-1H-indol-5-yl)-1-(2-chloro-
phenyl)methanesulfonamide (8). 5-Nitroindole (30.83
mmol) and the benzyl bromide (33.91 mmol) were dis-
solved in 150 ml of butanone. Potassium carbonate
(34.03 mmol) was then added. The reaction mixture was
stirred under reflux for 23 h then concentrated under
reduced pressure and dissolved in 150 ml of ethyl acetate.
Formed salts were filtrated and the filtrate was concen-
trated under reduced pressure. The solid obtained was
washed with 100 ml of cyclohexane then filtrated and dried
under vacuum to give the 1-benzyl-5-nitro-1H-indole
5.1.10. Synthesis of 1-hexyl-1H-benzimidazol-5-amine
(13a). Compound 11a (12.67 mmol) was dissolved in
160 ml of absolute ethanol and SnCl2 dihydrate
(64.49 mmol) was then added. The reaction mixture
was stirred under reflux for 3 h then concentrated under
(29.45 mmol, 96%)
a yellow solid. This product
(11.89 mmol) was dissolved in 150 ml of absolute ethanol.
The mixture was stirred under agitation and SnCl2

C. Benod et al. / Bioorg. Med. Chem. 16 (2008) 3537–3549
3547
reduced pressure and dissolved in 165 ml of ethyl ace-
tate. Sodium bicarbonate (100 ml) was then added.
Formed salts were filtrated and washed with 50 ml of
AcOEt. The organic phase of the filtrate was washed
with water (100 ml), dried on anhydrous sodium sulfate,
filtrated, and concentrated under reduced pressure to
give an orange red solid. This product was purified by
chromatography on silica gel (AcOEt/MeOH 9:1) to af-
ford compound 13a as a pink solid (5.24 mmol, 41%).
gration of a GAL4-responsive gene (GAL4RE5-bGlob-
Luc-SV-Neo) in HeLa cells.36 The HGPXR cell line
was obtained by transfecting HG5LN cells with a plas-
mid (pSG5-GAL4(DBD)-hPXR(LBD)-puro) which en-
ables the expression of the DNA binding domain of
the yeast activator GAL4 (met 1 to ser 147) fused to
the ligand-binding domain of hPXR (met 107 to ser
434) and conferes resistance to puromycin. For the
strain culture, cells were grown in Dulbecco’s modified
Eagle’s medium (DMEM) containing phenol red and
1 g/l glucose, and supplemented with 5% fetal calf serum
(FCS) in a 5% CO2 humidified atmosphere at 37 ꢁC.
HG5LN cell medium was supplemented with 1 mg/ml
geneticin and HGPXR cell medium with 1 mg/ml genet-
icin and 0.5 lg/ml puromycin.
5.1.11. Synthesis of 1-(naphthalen-2-ylmethyl)-5-nitro-1H-
benzimidazole (12a) and 3-(naphthalen-2-ylmethyl)-5-nitro-
3H-benzimidazole (12b). 5-Nitrobenzimidazole (30.65
mmol) and bromomethylnaphthalene (33.69 mmol) was
dissolved in 155 ml of butanone. Potassium carbonate
was then added (34.39 mmol). The reaction mixture was
stirred under reflux for 3 h then concentrated under re-
duced pressure and dissolved in ethyl acetate (100 ml).
Formed salts were filtrated and the filtrate was concen-
trated again and dried under vacuum to give a mixture (or-
ange red solid, 18.03 mmol) of the required compound
(12a) and its N3 regioisomer (12b).
For tests, cells were grown in DMEM without phenol
red, supplemented with 3% dextran-coated charcoal
treated fetal calf serum (DCC-FCS).
5.3.1. Living cell luciferase assay. HGPXR and HG5LN
cells were seeded at a density of 5 · 104 cells per well, in
96-well white opaque tissue culture plates (Becton Dick-
inson) and grown in 200 ll DCC-FCS. Tested com-
pounds were added 24 h later at concentrations
between 10 nM and 10 lM and cells were incubated
for 16 h with compounds. At the end of incubation,
compound containing medium was removed and re-
placed by 0.3 mM luciferin containing culture medium.
Luciferase activity was measured in a MicroBeta Wallac
luminometer (EG&G Wallac, Turku, Finland) and
luminescence was measured in intact cells for 2 s per
well.
5.1.12. Synthesis of 1-(naphthalen-2-ylmethyl)-1H-ben-
zimidazol-5-amine (13b). The mixture containing com-
pounds 12a–b (3.13 mmol) was dissolved in 80 ml of
absolute ethanol and SnCl2 dihydrate (15.65 mmol)
was then added. The reaction mixture was stirred under
reflux for 4 h then concentrated under reduced pressure
and dissolved in 85 ml of ethyl acetate. Sodium bicar-
bonate (100 ml) was then added. Formed salts were fil-
trated and washed with 50 ml of AcOEt. The organic
phase of the filtrate was washed with water (100 ml),
dried on anhydrous sodium sulfate, filtrated, and con-
centrated under reduced pressure to give a mixture (yel-
low solid, 2.82 mmol) of compound 13b and its N3
regioisomer. Then, this mixture was purified by chroma-
tography on silica gel (cyclohexane/AcOEt/MeOH
2:7.2:0.8). Two fractions were obtained, one contained
the N3 regioisomer (0.58 mmol, 18%, Rf = 0.78), the
other contained the required compound 13b (1.06 mmol,
33%, Rf = 0.44). The two fractions were pink solids.
5.4. Limited proteolytic digestion
LBD of hPXR in pET-15b expression plasmid (2 lg)
was generated by coupled transcription/translation in
rabbit reticulocyte lysate using the TNT system accord-
ing to the manufacturer’s instructions (Promega, Madi-
son WI). [35S]Methionine was included in the
transcription/translation mix to generate [35S]PXR.
Briefly, after a 30-min incubation with ligand at room
temperature, hPXR protein was digested at 25 ꢁC with
250 lg/ml trypsin for 10 min. Proteolysis was terminated
by adding sodium dodecyl sulfate (SDS) sample buffer
and boiling for 10 min. The proteolytic fragments were
separated on a 12% SDS–polyacrylamide gel. Gels were
dried and radio-labeled digestion products were visual-
ized by autoradiography using a bio-imaging analyzer
(Fujix BAS1000 Phosphorimager). Autoradiograms
were analyzed using an image analyzing software.
Experiments were performed three times. To determine
the protection of hPXR by a test compound, the inten-
sity of the 30 kDa band of hPXR-LBD treated with li-
gand was divided by the intensity of the 30 kDa of
hPXR treated by ethanol. The protection value in pres-
ence of ethanol was thus taken as 1.
5.2. Plasmids
The pPM-LBDhPXR expression plasmid was described
previously.34 The yeast Gal4 DNA binding domain
fused to the LBD of hPXR (107–434 aa) was generated
from the pPM-LBD using PCR primers that introduced
BamHI sites and subcloned into pSG5-puro (gift from
Hinrich Gronemeyer, Strasbourg, France).
The pET15b-hPXR expression plasmid was generated by
PCR amplification of cDNA-encoding amino acids 107–
434 of hPXR using oligonucleotides 50-CGCGCGCATA
TGAAGGAGATGATCATG-30 and 50-GCGCGCGG
ATCCTCAGCTACCTGTGATGCCG-30. All plasmids
were fully sequenced.
5.3. Generation of stable reporter cell lines
5.5. Data analysis and statistics
HG5LN and HGPXR cells were described previ-
ously.35,21,36 Briefly, HG5LN cells were obtained by inte-
In the transactivation assay, each compound was tested
at various concentrations in at least three separate

3548
C. Benod et al. / Bioorg. Med. Chem. 16 (2008) 3537–3549
experiments in triplicate wells and data are presented as
means SD. HG5LN cells assessed us to evaluate
hPXR-independent luciferase expression as well as the
toxicity of the compounds. HGPXR cells were designed
to detect hPXR agonists. Individual agonist dose–re-
sponse curves on these cells were fitted using the sigmoi-
dal dose–response function of a graphics and statistics
software (Graph-Pad Prism, version 4.0, 2003, Graph-
pad Software Incorporated, San Diego, CA). EC50,
effective concentration for half-maximal luciferase activ-
ity was calculated via nonlinear regression. This equa-
tion was used to fit the data in the graphic software.
Transactivation data are presented as EC50 values for
each compound tested.
in duplicates for quantitative PCR amplification of
CYP2B6, CYP3A4, and b-actin, as internal control,
using the Light Cycler apparatus (Roche Diagnostic
Corporation, Meylan, France). The following program
was used: denaturation step at 95 ꢁC for 10 min, 45 cy-
cles PCR (denaturation at 95 ꢁC for 10 s, annealing at
65 ꢁC for 8 s, elongation at 65 ꢁC for 15 s). Sense and re-
verse primers, respectively, were as follows: b-actin: 50-t
gggcatggtgcagaaggat
and
50-tccatcacgatgccagtggt;
CYP3A4: 50-cacaaaccggaggccttttg and 50-atccatgctgta
gggccccaa; CYP2B6: 50-ggccatacgggaggcccttg and 50-a
gggccccttggatttccg and GAPDH: 50-ggtcggagtcaacg-
gatttggtcg and 50-caaagttgtcatggatgacc. The curves of
amplification were read with the Roche’s Light Cycler
Software using the comparative cycle threshold (Ct)
method. Relative quantifications of the target mRNAs
were calculated after normalization of Cts with respect
to the GAPDH levels. Values are expressed as fold
induction compared to non-treated cells (DMSO
0.1%) SD.
In the proteolysis assay, C2BA-4, 6c, 6m, and 6n were
tested at various concentrations in at least three separate
experiments in triplicate wells and data are presented as
means SD. HG5LN cells assessed us to evaluate
hPXR-independent luciferase expression as well as the
toxicity of the compounds. HGPXR cells were designed
to detect hPXR agonists. Individual agonist dose–re-
sponse curves on these cells were fitted using the sigmoi-
dal dose–response function of a graphics and statistics
software (Graph-Pad Prism, version 4.0, 2003, Graph-
pad Software Incorporated, San Diego, CA). EC50,
effective concentration for half-maximal luciferase activ-
ity was calculated via nonlinear regression. This equa-
tion was used to fit the data in the graphic software.
Transactivation data are presented as EC50 values for
each compound tested.
5.7. In silico modeling: docking of 6n to the crystal
structure using Surflex-2.1
Protein preparation for surflex docking was done in
Sybyl 7.3 (Tripos Inc., St. Louis, MO). From the Protein
Databank entry 2O9I, the chain A, Gly142-Gly433, was
chosen for a protein site preparation and water mole-
cules were deleted. Only hydrogen position energy opti-
mization was performed after addition of hydrogen
atoms, then ligand T0901317 was removed and saved
in mol2 format. The resulting protein was saved in Tri-
pos mol2 format. A ligand-based approach with
T0901317 as ligand was performed to generate the bind-
ing site (protomol) as follows: surflex-dock –adapt_pro-
to –adapt_thresh 0.4 proto. Compound 6n in mol2
format was docked into the protomol using the –pgeom
option and with or without restraints. For the restrained
docking process the N-phenyl-benzenesulfonamide moi-
ety of T0901317 was used as placed fragment. For both
strategies, scores obtained for the 20 docking poses were
from 4.8 to 6.3.
One-way analysis of variance (ANOVA) was used to
demonstrate statistical difference between the activity
percentage of control and tested compounds with the
help of GraphPad Prism software (GraphPad, San Die-
go, CA). Calculation of statistical significance (P values
<0.05) between treated and controlled groups were per-
formed using Tukey’s post hoc test.
5.6. Liver samples, hepatocyte cultures and treatment
Hepatocytes were prepared from lobectomy segments
resected from adult patients for medical purposes unre-
lated to our research program. The use of these human
hepatic specimens for scientific purposes has been ap-
proved by the French National Ethics Committee.
Acknowledgments
This study has been partly supported by the European
Union Network CASCADE (FOOD-CT-2003-506319)
and ANR JCJC-05-47810 (J.-M.P). C.B. is a recipient
of the MENRT from the French Government. V.N. is
a recipient of the French Young Researcher Program
Human hepatocytes were prepared and cultured as previ-
ously described.37 Cells were plated into collagen-coated
P12 dishes at 0.17 · 10ꢀ6 cells/cm2 in a hormonally and
chemically defined medium consisting of a mixture of
Williams’ E and Ham’s F-12 (1:1 v/v). Treatment with
10ꢀ6 M and 10ꢀ5 M effector solutions or solvent (DMSO
0.1%) started 48 h after plating and lasted 24 h.
´
(INSERM). The authors Drs. Adrien Cave and Andre
Padilla for NMR assistance. We are also grateful to
Mr. Terry Page and Dr. Julien Viaud for the critical
reading of this manuscript. This work was sponsored
by the CNRS and INSERM.
5.6.1. Total RNA purification and CYP3A4 quantitative
PCR. Total RNA was isolated with TRIzol reagent
(Invitrogen). cDNA was synthetized from 1 lg of total
RNA using the SuperScript-II First-Strand Synthesis
System (Invitrogen) for RT-PCR at 42 ꢁC for 60 min
in the presence of random hexamers (Invitrogen), and
then 10-fold diluted in water. Two microliters was used
Supplementary data
Data for the superimposition of 6n with the N-phenyl-
benzenesulfonamide fragment of T0901317. The activity
of T0901317 and other ligands on reporter cell system;

C. Benod et al. / Bioorg. Med. Chem. 16 (2008) 3537–3549
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