Benzimidazole derivatives as new serotonin 5-HT6 receptor antagonists. Molecular mechanisms of receptor inactivation

Article abstract of DOI:10.1021/jm901672k

On the basis of our previously described pharmacophore model for serotonin 5-HT₆ receptor (5-HT₆R) antagonists, we have designed, synthesized, and pharmacologically characterized a series of benzimidazole derivatives 1-20 that repres

Full text of DOI:10.1021/jm901672k

J. Med. Chem. 2010, 53, 1357–1369 1357
DOI: 10.1021/jm901672k
Benzimidazole Derivatives as New Serotonin 5-HT6 Receptor Antagonists.
Molecular Mechanisms of Receptor Inactivation
†
n-Fontecha, Jessica Sallander, Bellinda Benhamu, Mercedes Campillo, Rocıo A. Medina,
‡
‡
†
Tania de la Fuente,† Mar Martı
´
´
ꢀ †
Lucie P. Pellissier,§ Sylvie Claeysen,§ Aline Dumuis,§ Leonardo Pardo,*,‡ and Marı
´
a L. Lopez-Rodrı
´
guez*,†
ꢀ
†
´
Departamento de Quımica Organica I, Facultad de Ciencias Quımicas, Universidad Complutense de Madrid, E-28040 Madrid, Spain,
Laboratori de Medicina Computacional, Unitat de Bioestadıstica, Facultat de Medicina, Universitat Autonoma de Barcelona, E-08193
ꢀ
´
‡
´
ꢁ
§
Bellaterra, Barcelona, Spain, and Deꢀpartement de Neurobiologie, Institut de Geꢀnomique Fonctionnelle, CNRS, Universiteꢀ de Montpellier,
INSERM U661, F-34094 Montpellier, France
Received November 12, 2009
On the basis of our previously described pharmacophore model for serotonin 5-HT6 receptor (5-HT6R)
antagonists, we have designed, synthesized, and pharmacologically characterized a series of benzimi-
dazole derivatives 1-20 that represent a new family of potent antagonists at the human 5-HT6R. Site-
directed mutagenesis and a β2-adrenoceptor-based homology model of the 5-HT6R were used to predict
the mode of binding of antagonist SB-258585 and the new synthesized ligands. Substitution of W6.48,
F6.52, or N6.55 by Ala fully impedes compound 4 to block 5-HT-induced activation. Thus, we propose
that D3.32 in TM 3 anchors the protonated piperazine ring, the benzimidazole ring expands parallel
to EL 2 to hydrogen bond N6.55 in TM 6, and the aromatic ring is placed between TMs 3 and 5 in
CH2-containing compounds and between TMs 3 and 6 in CO-containing compounds. This combined
experimental and computational study has permitted to propose the molecular mechanisms by which
the new benzimidazole derivatives act as 5-HT6R antagonists.
Introduction
ciated with the control of cholinergic neurotransmission,20,21
which prompted much interest into the possible implication of
the receptor in cognitive impairment19,22,23 (memory and
learning) related to neurological diseases such as Alzheimer’s.
In the last five years, it has been shown that selective 5-HT6R
ligands are able to reduce food intake,24,25 although this effect
had been unnoticed in early studies. Thus, the 5-HT6R has
emerged as a promising target for the treatment of obesity
and related metabolic syndrome,19,22,26 a disease with an
increasing global prevalence and high unmet clinical need.
Clearly, there is much evidence that the h5-HT6R is involved in
the pathogenesis of CNS diseases4,19,22,23,26,27 related to cog-
nitive or eating disorders, so it appears to be an attractive
therapeutic target that should be exploited for drug develop-
ment. Because it is known to be expressed almost exclusively
in the CNS18,28-31 and mainly in the olfactory tubercle,
striatum, nucleus accumbens, and hippocampus, it is possible
that new therapeutic agents targeting this receptor might
have relatively few peripheral side effects. The first 5-HT6R
antagonists Ro 04-6790,32 Ro 63-0563,32 and 46 (SB-258585)33
(Chart 1), were identified in the late 1990s by high-throughput
screening at Roche and GlaxoSmithKline. Soon afterward,
Glennon et al.,34-37 pioneering in the synthesis of 5-HT6R
ligands, reported the first selective agonist EMDT35 and
antagonist MS-24536 (Chart 1), concurrently discovered by
Merck Sharp & Dohme.38 Since then, the number of
patent applications and scientific publications concerning
the 5-HT6R experienced a dizzying growth, mostly in the past
few years. At present, several research groups from both
academia and the pharmaceutical industry have contributed
to the development of structurally different 5-HT6R agonists
Serotonin (5-hydroxytryptamine, 5-HT) research is now
more than 50 years old1-3 but still represents one of the most
attractive areas in medicinal chemistry.4 Fourteen serotonin
receptor subtypes have been identified so far that are classi-
5,6
fied into seven families (5-HT1-7
)
based on amino acid
sequences, pharmacology, and intracellular mechanisms. With
the exception of the ionotropic 5-HT3 receptor, all of the 5-HT
subtypes are G protein-coupled receptors (GPCRsa).7-10 To
date many serotoninergic agents acting at 5-HT1-4 receptors
are clinically used11 for the treatment of certain central nervous
system (CNS) illnesses. One of the most recent additions to
the family of serotonin receptors is the human 5-HT6 receptor
(h5-HT6R), which was cloned in 199612,13 as a gene codifying a
polypeptide chain of 440 amino acids13,14 that is positively
coupled to the adenylate cyclase15-17 cascade via the Gs
protein. The high affinity of several antipsychotic and anti-
depressant agents13,18,19 boosted the first studies exploring
the 5-HT6R potential for the treatment of schizophrenia
and bipolar affective disorder. However, contradictory re-
sults19 were obtained about the role of the receptor in these
therapeutic indications. Later on, 5-HT6R function was asso-
*To whom correspondence should be addressed. For M.L.L.-R.: phone,
34-91-3944239; fax, 34-91-3944103; E-mail: mluzlr@quim.ucm.es. For
L.P.: phone, 34-93-5812797; fax, 34-93-5812344; E-mail: Leonardo.
Pardo@uab.es.
aAbbreviations: GPCR, G protein-coupled receptor; CNS, central
nervous system; h5-HT6R, human 5-HT6 receptor; PI, positive ionizable
atom; HBA, H-bonding acceptor group; HYD, hydrophobic; TM,
transmembrane helix; EL, extracellular loop; SEM, 2-(trimethylsilyl)-
ethoxymethyl; BOC, tert-butoxycarbonyl; WT, wild-type.
r
2010 American Chemical Society
Published on Web 01/15/2010
pubs.acs.org/jmc

1358 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3
de la Fuente et al.
and antagonists4,19,22,26,27 (such as WAY-181187, E-6801 and
SB-357134, Chart 1), which have been used as pharmacologi-
cal tools to study the receptor. To our knowledge,26 eight of
these compounds have entered clinical trials, and five of them
are still under active development in phase I or II for the
treatment of cognitive dysfunction associated with Alzhei-
mer’s disease or schizophrenia as well as for the treatment of
obesity. However, no 5-HT6R ligand has been marketed so far
and the search for new compounds acting at this receptor
remains an area of high interest for medicinal chemists, aiming
at either highly selective agents or compounds combining
5-HT6R activity with other serotonin activities thought to be
beneficial for the treatment of CNS disorders.
In this context, we are involved in a project aimed at the
development of new serotonin 5-HT6R ligands using a ra-
tional approach. As a starting point, using Catalyst soft-
ware,39 we generated a ligand-based pharmacophore model
for 5-HT6R antagonism40 from a training set of 45 structu-
rally diverse compounds previously described. On the basis of
this model, here we have designed new benzimidazole deriva-
tives containing a piperazine ring, either a carbonyl (-CO-)
or a methylene (-CH2-) group, and an aromatic ring
(benzene or naphthalene) (Figure 1). We report the synthesis
of new compounds 1-20 and their binding affinity for the
5-HT6R (Table 1). The identified ligands have been charac-
terized as antagonists in COS-7 cells transfected with the
h5-HT6R, representing a new family of 5-HT6R antagonists.
Site-directed mutagenesis and
a β2-adrenoceptor-based
homology model of the 5-HT6R were used to predict the
mode of binding of these ligands. This combined experimental
and computational study has permitted us to propose the
molecular mechanisms by which the new benzimidazole deri-
vatives act as 5-HT6R antagonists.
Results and Discussion
Pharmacophore-Based Design of New 5-HT6R Ligands.
Our previously reported 3-D pharmacophore model for 5-HT6R
antagonists showed four key pharmacophore elements: a
positive ionizable atom (PI), an aromatic ring-hydrophobic
site (AR), a hydrogen bond acceptor group (HBA), and a
hydrophobic site (HYD).40 On the basis of these structural
requirements, we have designed a series of new compounds
(Figure 1A) that contain a piperazine ring as PI, the AR
feature is a benzimidazole system, the HBA feature is a
carbonyl group (X=CO), and a benzene or naphthalene ring
as HYD. Hence, the designed derivatives could represent a
new structural class of 5-HT6R ligands, containing a benzi-
midazole scaffold in the central core. In the new compounds,
the piperazine ring is attached to the benzimidazole system
at either position 4 or position 5, providing two different
orientations of the AR, HBA, and HYD moieties relative to
the receptor-anchoring PI group. Figure 1B shows designed
compound 3: Y = 4-(piperazin-1-yl), X = CO, Ar = 1-naph-
thyl mapped onto the 3-D model. Additionally, compounds
lacking the HBA feature or the HYD region were studied
in order to assess the influence of these pharmacophore
elements on the binding to the receptor. Therefore, we also
synthesized analogues (Figure 1A) where the carbonyl group
(X = CO) was replaced by a methylene (X = CH2) with no
hydrogen bond accepting capability or where the Ar moiety
was missing (X = H).
Chart 1. Structures of 5-HT6R Agonists and Antagonists
Synthesis. The synthesis of new benzimidazole deriva-
tives 1-20 (Figure 1A, Table 1) was performed by using
two different strategies, depending on the nature of X
moiety (X = CH2 or X = CO, see Schemes 1-3). Com-
pounds 10-20 (X = CH2) were obtained by nucleophilic
Figure 1. A previously reported 3-D pharmacophore model for 5-HT6R antagonists showed four key structural elements: a positive ionizable
atom (PI, red), an aromatic ring-hydrophobic site (AR, yellow), a hydrogen bond acceptor group (HBA, green), and a hydrophobic site (HYD,
blue). (A) Pharmacophore-based designed benzimidazole derivatives 1-20. (B) Designed compound 3: Y = 4-(piperazin-1-yl), X = CO, Ar =
1-naphthyl mapped onto the pharmacophore model.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 1359
Scheme 2a
Table 1. Binding Affinity of Synthesized Compounds 1-20 at the
h5-HT6R
compd
position
R
X
Ar
Ki ( SEM (nM)a
1
4
4
4
4
4
5
5
5
5
4
4
4
4
4
5
5
5
5
5
5
H
CO
CO
phenyl
phenyl
>1000
>1000
13 ( 1
2
CH3
H
3
CO
CO
1-naphthyl
1-naphthyl
2-naphthyl
phenyl
4
CH3
CH3
CH3
CH3
CH3
H
34 ( 2
5
CO
CO
>1000
>1000
>1000
>1000
>1000
58 ( 3
66.8 ( 0.7
16 ( 2
36.22 ( 0.08
49 ( 3
>1000
>1000
>1000
>1000
>1000
>1000
6
a Reagents and conditions: (a) SnCl2, HCl, rt, 3 h, quantitative yield;
(b) HCOOH, H2O, 100 °C, 3 h, quantitative yield; (c) SEMCl, K2CO3,
DMF, 0 °C to rt, 3 h, quantitative yield; (d) 1-BOC-piperazine or
1-methylpiperazine, NaOtBu, Pd2dba3, P(tBu)3, o-xylene, 110 °C, 24 h,
43-49%; (e) ArCOOR, BuLi, THF, -78 °C to rt, overnight, 21-59%;
(f) BCl3, CH2Cl2, rt, 5 h, 50-64%.
7
CO
CO
1-naphthyl
2-naphthyl
2-naphthyl
phenyl
8
9
CO
10
11
12
13
14
15
16
17
18
19
20
H
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH3
H
phenyl
1-naphthyl
1-naphthyl
2-naphthyl
phenyl
CH3
CH3
H
Thus, the reduction of commercially available 3-chloro-
2-nitroaniline, Phillips cyclization, and SEM protection of the
obtained 4-chlorobenzimidazole (29)41,42 afforded a nonse-
parable mixture of N-protected regioisomers 30, in a 1:1 ratio
(Scheme 2). Subsequent Pd(0)-catalyzed coupling43 of 30
with 1-BOC- or 1-methylpiperazine yielded only 4-substi-
tuted intermediates 31,32. Finally, derivatives 31,32 were
acylated via butyllithium deprotonation, and SEM or BOC
groups were subsequently removed to obtain desired com-
pounds 1-5 (X = CO). Similarly, the cyclization of o-pheny-
lenediamines 27,28,44 followed by the protection of 5-substituted
benzimidazoles 38,43 and the 2-acylation of N-protected deri-
vatives 39,44, afforded final compounds 6-9 (X = CO) after
deprotection of intermediates 40-42,45 (Scheme 3).
CH3
H
phenyl
1-naphthyl
1-naphthyl
2-naphthyl
2-naphthyl
CH3
H
CH3
a Values are the mean of two to four experiments performed in
triplicate.
Scheme 1a
Binding Affinities and Structure-Affinity Relationships.
Target compounds 1-20 were assessed for in vitro affinity
at the h5-HT6R by radioligand binding assays, using
[3H]LSD in transfected HEK-293 cells (see Experimental
Section for details). The competitive inhibition assays were
first performed at a fixed dose of 10-6 M, and the complete
dose-response curve, at six different concentrations of the
ligand, was determined for those compounds that presented
a displacement of the radioligand over 50%. The inhibi-
tion constant Ki was calculated from the IC50 value using
the Cheng-Prusoff equation,45 and the values in Table 1 are
the mean of two-four experiments. Compounds binding the
5-HT6R were tested for selectivity over the 5-HT7 receptor,
using [3H]LSD in transfected HEK-293 cells (see Experi-
mental Section for details), and found to be inactive
(Ki >500 nM) at the 5-HT7R. The following structure-
affinity relationships can be drawn from the 5-HT6R
binding affinity data presented in Table 1. Most of the
4-substituted benzimidazole derivatives show high affinity
(Ki = 13-66.8 nM), whereas 5-substituted analogues are
inactive (Ki > 1000 nM). These experimental data show that
4-substituted benzimidazoles display a favorable orienta-
tion of the AR, HBA, and HYD moieties relative to the
receptor-anchoring PI group. In 4-substituted derivatives
containing a carbonyl group as HBA (1-5: X=CO), only
compounds 3 and 4, both containing 1-naphthyl ring as HYD
moiety, show 5-HT6R affinity [Ki(3) = 13 nM; Ki(4) = 34 nM].
a Reagents and conditions: (a) Cs2CO3 or K2CO3, DMF, 120 °C, 24 h,
40% to quantitative yield; (b) H2, Pd(C), MeOH, rt, 24 h, 89% to
quantitative yield; (c) ArCH2CHO, Yb(OTf)3 2H2O, CH2Cl2, rt, over-
3
night, 42-75%.
substitution of 3-chloro- or 5-chloro-2-nitroaniline with
piperazine or 1-methylpiperazine, followed by catalytic
hydrogenation of the resulting piperazinyl-2-nitroanilines
21-24 and subsequent oxidative cyclization of the obtained
o-phenylenediamines 25-28 with the appropriate readily
available arylacetaldehydes (Scheme 1). On the other hand,
those benzimidazole derivatives having a carbonyl group
(X = CO, 1-9) were synthesized by butyllithium-mediated
acylation at the 2-position of N-protected derivatives fol-
lowed by removal of 2-(trimethylsilyl)ethoxymethyl (SEM)
or tert-butoxycarbonyl (BOC) groups (Schemes 2 and 3).

1360 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3
de la Fuente et al.
Scheme 3a
a Reagents and conditions: (a) HCOOH, H2O, 100 °C, 3 h, 60-87%; (b) (i) SEMCl, NaH, DMF, 0 °C to rt, 3 h, (ii) SiO2 column chromatography
(CH2Cl2/EtOH, 9:1), 44%; (c) ArCOOR, BuLi, THF, -78 °C to rt, overnight, 33-63%; (d) BCl3, CH2Cl2, rt, 5 h, 65-77%; (e) BOC2O, DMAP, Et3N,
rt, overnight, 69%; (f) TFA, CH2Cl2, rt, 5 h, quantitative yield.
In contrast, phenyl [Ki(1)>1000 nM vs Ki(3) =13 nM, Ki(2)>
1000 nM vs Ki(4) = 34 nM)] is insufficient for high-affinity
binding, and the 2-naphthyl [Ki(5) >1000 nM vs Ki(4) =
34 nM] aromatic system is not tolerated at the HYD region.
However, when the carbonyl group is replaced by a methylene
moiety all 4-substituted compounds (10-14: X = CH2) dis-
play affinity for the 5-HT6R (Ki = 16-66.8 nM). These results
show that the hydrogen bonding capability proposed for HBA
pharmacophore element is not essential for the binding of this
new family of ligands to the 5-HT6R. Also, comparison of the
affinity data obtained for compounds 4 (X = CO, Ki =
34 nM) vs 13 (X = CH2, Ki = 36.22 nM) and 3 (X = CO,
Ki = 13 nM) vs 12 (X = CH2, Ki = 16 nM) suggest that the
aromatic ring (Ar) is accommodated at different parts of
the receptor for compounds with X = CO and X = CH2
(see Computer-Based Analysis of the Structure-Affinity Re-
lationships). The inactivity of 4-(4-methylpiperazin-1-yl)-1H-
benzimidazole (Ki >1000 nM, data not shown), lacking the
aromatic ring Ar, supports the hypothesis that the presence of
HYD region is important for the receptor binding.
Functional Activity of 5-HT6R Ligands. Compounds dis-
playing affinity for the 5-HT6R were evaluated for their
functional profile by determining the adenylate cyclase activ-
ity in COS-7 cells expressing the human receptor. Cells were
treated with the ligand for 10 min at 37 °C, in the absence
(agonist effect) or the presence (antagonist effect) of serotonin
(100 μM), and the cAMP content was then quantified by
homogeneous time-resolved fluorescence (HTRF) (see Ex-
perimental Section for details). Adenylate cyclase activity is
expressed as the percentage of the maximal effect obtained
with serotonin alone. All assayed compounds show a dose-
dependent effect in the presence of 5-HT and no effect in the
absence of 5-HT, indicating their antagonist profile. The Ki
values in Table 2 are the mean of three experiments, using
eight different concentrations of the tested compound. These
data (Ki = 5-31 nM) show that 4-substituted benzimidazoles
represent a new family of potent 5-HT6R antagonists.
Table 2. Potency of New 5-HT6R Antagonists
compd
R
X
Ar
Ki ( SEM (nM)a
3
H
CO
CO
1-naphthyl
1-naphthyl
phenyl
15 ( 2
5 ( 2
4
CH3
H
10
11
12
13
14
CH2
CH2
CH2
CH2
CH2
31 ( 4
16 ( 5
16 ( 3
27 ( 3
17 ( 4
CH3
H
phenyl
1-naphthyl
1-naphthyl
2-naphthyl
CH3
CH3
a Values are the mean of three experiments performed in triplicate.
the sequence alignment of transmembrane helices (TMs)
3, 5, 6, and 7 and extracellular loop (EL) 2 for β1- and β2-
adrenergic receptors, selected serotonin receptors, and the
5-HT6R. These domains form the ligand binding site in
the crystal structures of β1-46 and β2-47,48 adrenergic recep-
tors, as they were previously identified by site-directed
mutagenesis.49 To characterize the amino acid residues of
the 5-HT6R involved in the binding of ligand 4, mutagenesis
assays were performed with Ala substitution at C3.36, S5.43,
W6.48, F6.52, and N6.55 sites. Receptor expression levels at
the plasma membrane relative to the expression of wild-type
(WT) receptor were determined by an ELISA assay. These
mutant receptors are expressed at the 80-110% range with
the exception of W6.48A (30%). Figure 3A shows the
antagonist activity of 4 relative to 5-HT-induced activation
in the five mutant receptors and in the WT 5-HT6R. Notably,
the substitution of W6.48, F6.52, or N6.55 by Ala fully
impedes compound 4 to block 5-HT-induced activation.
The C3.36A mutation decreases the antagonist activity of
the ligand by 7-fold [Ki(WT) =5 nMvs Ki(C3.36A) = 34 nM].
Defining the Mode of Binding of Ligand 4 by Site-Directed
Mutagenesis and Computational Modeling. Figure 2 shows

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 1361
Figure 2. Sequence alignment of TMs 3, 5-7 and EL 2 of β1- and β2-adrenergic receptors, and serotonin 5-HT1A, 5-HT2A, 5-HT4, 5-HT7, and
5-HT6 receptors. Residues referenced in the manuscript are boxed. The highly conserved WxPFF motif in TM 6 is shown in dark gray.
Figure 3. (A) The antagonist activity of compound 4 was tested relative to 5-HT-induced activation in WT 5-HT6R and C3.36A, S5.43A,
W6.48A, F6.52A, and N6.55A mutant receptors. The antagonist effect is represented as the percentage of inhibition of 5-HT (100 μM)-induced
stimulation of cAMP taken as 100%. Each value represents the mean ( SEM determined from three independent experiments performed in
triplicate. The inhibition constant Ki was calculated from the IC50 value using the Cheng-Prusoff equation. IC50 was assessed as the
concentration required to inhibit 50% of 5-HT (100 μM)-induced stimulation of cAMP. EC50 values of 5-HT on WT, C3.36A, and S5.43A
receptors were 5 ( 1 nM, 0.8 ( 0.1 μM, and 3 ( 1 nM, respectively. (B,C) Computational model of the complex between ligand 4 (in white) and
the 5-HT6R. In this model, D3.32 anchors the protonated piperazine ring, the benzimidazole ring hydrogen bonds N6.55 and interacts with
F6.51, F7.35, and L182 in EL 2, and the aromatic naphthalene ring enters into a small cavity between TMs 3 and 6 to interact with V3.33,
W6.48, and F6.52. Panels B and C are rotated 90°. The color code of the helices is: TM 3 (red), 5 (green), 6 (blue), and 7 (brown).
Surprisingly, the S5.43A mutation substantially increases
the antagonist activity of 4 by 98-fold [Ki(WT)=5 nM vs
Ki(S5.43A) = 0.051 nM].
TM 6. This computational model explains the experimentally
determined importance of W6.48, F6.52, and N6.55 of the
5-HT6R in the binding of ligand 4 (Figure 3A). The finding
that the S5.43A mutation enhances the activity of 4
(Figure 3A) is attributed to the fact that S5.43 hydrogen
bonds N6.55 (Figure 3B); thus, the mutation at this site
increases the flexibility of N6.55, favoring its interaction with
4. Other significant interactions between compound 4 and the
5-HT6R, as revealed by the computational model, are: the
interaction between the highly polar methyl group attached to
the protonated piperazine ring and W3.28, the interaction of
the aromatic naphthalene ring with V3.33 in TM 3, and the
interaction between the aromatic benzimidazole ring and F6.51
in TM 6, F7.35 in TM 7, and L182 in EL 2 (Figure 3B,C).
To understand these observed effects at the molecular
level, we constructed a computer 3-D model of the complex
between compound 4 and a β2-adrenoceptor-based model of
the 5-HT6R (see Experimental Section). In this model
(Figure 3B), D3.32 anchors the protonated piperazine ring,
the benzimidazole ring expands toward TM 6, parallel to
EL 2, to hydrogen bond Oδ of the key N6.55, the coplanar
carbonyl group also hydrogen bonds the Nδ-H moiety of
N6.55, and the aromatic naphthalene ring enters deeply into a
small cavity between TMs 3 and 6 to form edge-to-face
interactions with the key W6.48 and F6.52 side chains in

1362 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3
de la Fuente et al.
S5.43A, W6.48A, F6.52A, and N6.55A mutant receptors
(Figure 5A). Clearly, with the only exception of W6.48A,
all these mutations decrease the antagonist activity of 46:
72-fold, relative to WT, in the C3.36A mutant receptor;
8-fold in S5.43A, 152-fold in F6.52A, and 36-fold in N6.55A
(Figure 5A). These mutational data are compatible with a
mode of binding of 46 to the 5-HT6R as depicted in Figure 5B:
D3.32 anchors the protonated piperazine ring, the methoxy-
phenyl system interacts with F6.52, the methoxy group hydro-
gen bonds C3.36, the sulfonamide group hydrogen bonds
both S5.43 and N6.55, and the substituted benzene interacts
with the hydrophobic side chains of V3.33 and A5.42.
Previously reported rhodopsin-based models have suggested
that arylsulfonyltryptamines bind between TMs 1, 6, and 7,50
the sulfonyl oxygens of MS-245 (Chart 1) bind S3.37 and
T5.46,51 or N6.55 in SB-357134 (Chart 1) and related sulfon-
amide derivatives.31,40
Figure 5C shows ligand 46 (white) and benzimidazole
derivatives 4 (green) and 13 (orange) bound to the 5-HT6R
for comparison purposes. With respect to the HBA feature,
the large sulfonamide moiety of 46 is capable to hydrogen
bond both S5.43 and N6.55 while the carbonyl group of
compound 4 hydrogen bonds exclusively N6.55, which also
binds the N-H of the benzimidazole ring (AR feature) of
compounds 4 and 13. Clearly, the piperazine-benzimidazole
rings of 4 and 13 are placed near the extracellular side,
parallel to EL 2, while the piperazine-methoxyphenyl rings
of 46 are located deeper in the TM bundle. Notably, the
methoxyphenyl system of 46 (AR feature) is placed at a
similar position to that of the naphthalene ring (HYD
feature) of compound 4. On the other hand, the position of
the HYD feature of 46 (iodophenyl ring) is similar to the
HYD feature of compound 13 (naphthalene ring). Thus, the
different location of the AR features in the binding site
suggests that compounds 4 and 13 elicit their antagonist
function by a different mechanism than ligand 46 (see
below).
Figure 4. Computational models of the complexes between ligand
13 (A) and 4 (B) and the 5-HT6R. An important feature of these
molecules is the linker between the benzimidazole ring and the
terminal aromatic ring. The methylene unit (-CH2-) of ligand 13
allows both rings to form an angle of approximately 90° (A),
whereas the carbonyl group (-CO-) of ligand 4 enforces both
aromatic domains to be coplanar (B). In CH2-containing com-
pounds, the aromatic ring is located between TMs 3 and 5, to
interact with V3.33 and A5.42 (A). In CO-containing compounds,
the aromatic moiety is located between TMs 3 and 6, interacting
with V3.33, W6.48, and F6.52 (B). 2-Naphthyl (in orange) is not
tolerated within the cavity between TMs 3 and 6 due to the clash
with TM 3 (B). The color code of the helices is: TM 3 (red), 5 (green),
6 (blue), and 7 (brown).
Computer-Based Analysis of the Structure-Affinity Rela-
tionships. In an attempt to get a better understanding of
the above-described structure-affinity relationships, we
have performed additional computational simulations of
the complexes between 4-substituted benzimidazole deriva-
tives and the 5-HT6R. Regarding HBA, replacement of the
sp2 carbonyl group (X = CO) by the sp3 methylene unit
(X = CH2) allows the benzimidazole ring and the aromatic
systems (Ar = phenyl, 1-naphthyl, 2-naphthyl) in the HYD
region to form an angle of approximately 90°. Thus, in
compounds with X = CH2, the HYD moiety is located
between TMs 3 and 5 to interact with the hydrophobic side
chains of V3.33 and A5.42 (Figure 4A). In contrast, the Ar
aromatic moiety in compounds containing X = CO is located
between TMs 3 and 6, interacting with V3.33, W6.48, and
F6.52 side chains (Figure 4B). The different location of the
HYD feature in the receptor explains the observed different
structure-affinity relationships at this pharmacophore ele-
ment in both types of compounds (X = CH2 or X = CO).
Clearly, all tested aromatic rings phenyl, 1-naphthyl, and
2-naphthyl are permitted in the cavity between TMs 3 and 5
(X = CH2, Figure 4A), whereas phenyl and 1-naphthyl are
permitted in the cavity between TMs 3 and 6 (X = CO,
Figure 4B). In the cavity between TMs 3 and 6, the smaller
phenyl ring, relative to 1-naphthyl, does not optimally interact
with F6.52 (Figure 4B), resulting in the corresponding loss of
affinity [Ki(1) > 1000 nM, Ki(2) > 1000 nM]. The presence of
the 2-naphthyl system is not permitted because it clashes with
TM 3 (see orange ring in Figure 4B), leading to an inactive
analogue [Ki(5) > 1000 nM].
Molecular Mechanisms of Receptor Inactivation. The un-
derstanding of the molecular mechanisms by which these
benzimidazole derivatives act as 5-HT6R antagonists, and
GPCR function in general, is a key subject in medicinal
chemistry. The recent knowledge of the molecular steps of
agonist-induced GPCR activation (see Smit et al.52 and
Nygaard et al.53 for recent reviews) facilitates this task. It
has been proposed, from engineered GPCRs with metal ion
binding sites, that TM 6 performs an inward movement of
the extracellular part toward TM 3.54 Thus, in the new family
of 5-HT6R antagonists the role of the benzimidazole ring
may be to maintain, by forming a hydrogen bond interaction
with N6.55, TM 6 away from the center of the bundle
(Figure 6A). In addition, L182 in EL 2 forms favorable
C-H π interactions55 with the benzimidazole ring, which
3 3 3
may serve to immobilize both EL 2 and TM 6 in the inactive
conformation (Figure 6A). Similar interactions are observed
between the antagonist cyanopindolol and the partial inverse
agonist carazolol with the homologous F20146 and F19348 of
the β1- and β2-adrenergic receptors, respectively. Fluores-
cence spectroscopy in the β2-adrenergic receptor provides
evidence that agonist binding induces or stabilizes relocation
of the extracellular side of TM 5 to facilitate binding of the
catechol hydroxyls of the adrenergic agonists.56 The Ar system
in CH2-containing compounds is located between TMs 3
and 5, interacting with V3.33 and A5.42, and probably block-
ing these helices in the inactive conformation (Figure 6B).
Comparison of the Binding of Ligand 46 with the Binding of
Benzimidazole Derivatives 4 and 13 to the 5-HT6R. Sulfon-
amide 4633 (Chart 1) is a commercially available potent
5-HT6R antagonist that contains the common piperazine
ring as the PI pharmacophore element, a methoxyphenyl
systemasAR, a sulfonamide groupasHBA, andaiodophenyl
ring as HYD.40 To compare the binding mode of this 5-HT6R
antagonist with the binding mode of benzimidazole deriva-
tives, we evaluated the antagonist activity of 46 in the C3.36A,

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 1363
Figure 5. (A) The antagonist activity of ligand 46 was tested relative to 5-HT-induced activation in WT 5-HT6R and C3.36A, S5.43A,
W6.48A, F6.52A, and N6.55A mutant receptors. The antagonist effect is represented as the percentage of inhibition of 5-HT (100 μM)-induced
stimulation of cAMP taken as 100%. Each value represents the mean ( SEM determined from three independent experiments performed in
triplicate. The inhibition constant Ki was calculated from the IC50 value using the Cheng-Prusoff equation. IC50 was assessed as the
concentration required to inhibit 50% of 5-HT (100 μM)-induced stimulation of cAMP. EC50 values of 5-HT were 5 ( 1 nM on WT, 0.8 (
0.1 μM on C3.36A, 3 ( 1 nM on S5.43A, 1.1 ( 0.1 nM on W6.48A, 0.18 ( 0.06 μM on F6.52A, and 45 ( 2 nM on N6.55A receptor.
(B) Computational model of the complex between ligand 46 (in white) and the 5-HT6R. In this model, D3.32 anchors the protonated piperazine
ring, the methoxyphenyl system interacts with F6.52, the methoxy group hydrogen bonds C3.36, the sulfonamide moiety hydrogen bonds both
S5.43 and N6.55, and the iodophenyl ring interacts with the hydrophobic side chains of V3.33 and A5.42. (C) Comparison of the binding modes
of ligand 46 (white) and benzimidazole derivatives 4 (green) and 13 (orange) to the 5-HT6R. The color code of the helices is: TM 3 (red),
5 (green), 6 (blue), and 7 (brown).
Figure 6. Molecular mechanisms of receptor inactivation. (A) The benzimidazole ring (in white spheres) impedes the proposed inward
movement of the extracellular part of TM 6 toward TM 3 required for receptor activation.54 The interaction of L182 in EL 2 with the
benzimidazole ring may serve to immobilize both EL 2 and TM 6 in the inactive conformation. (B) The aromatic naphthalene ring (in white
spheres) in CH2-containing compounds is located between TMs 3 and 5, interacting with V3.33 and A5.42, and probably blocking the
movement of these helices necessary for receptor activation.56 (C) The aromatic naphthalene ring (in white spheres) in CO-containing
compounds is located between TMs 3 and 6, interacting with V3.33, W6.48, and F6.52. This mode of binding blocks the proposed rotamer
toggle switch of W6.48 and the subsequent steps in the activation process.50 (D) The methoxyphenyl system of ligand 46 (in white spheres) is
located between TMs 3 and 6, interacting with V3.33, C3.36, and F6.52. The hydrogen bond between the methoxy group and C3.36 blocks the
conformational transition of C3.36 toward TM 7 during the process of receptor activation.60 The color code of the helices is: TM 3 (red),
5 (green), and 6 (blue).
In addition to these activation pathways, agonist binding
might also trigger the rotamer toggle switch of W6.48 from
pointing toward TM 7 to pointing toward the binding site
crevice,49,57,58 through the formation of specific hydrogen
bonds.59 The 1-naphthyl ring in CO-containing compounds
occupies a small cavity between TMs 3 and 6 (Figure 6C) and
might act by blocking the proposed conformational transi-
tion of W6.48 and the subsequent steps in the activation
process.52 We have also shown that the conformational
transition of W6.48 is accompanied, in a concerted manner,
by the conformational transition of the side chain at position
3.36 from pointing toward TM 6 to pointing toward TM 7.60

1364 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3
de la Fuente et al.
Thus, the methoxy group of 46 might block receptor activa-
tion by hydrogen bonding C3.36 in the inactive conforma-
tion (Figure 6D). Accordingly, the C3.36A mutation has a
significant effect in the antagonist activity of 46 (the effect for
compound 4 is less significant), while the W6.48A mutation
impairs the antagonist activity of compound 4 (the effect for
ligand 46 is minor).
run on Merck silica gel plates (Kieselgel 60 F-254) with detection
by UV light (254 nm), ninhydrin solution, or 10% phosphomo-
lybdic acid solution in ethanol. Flash chromatography was
performed on glass column using silica gel type 60 (Merck,
particle 230-400 mesh) or on a Supelco VersaFlash station
using silica gel cartridges (Supelco, particle size 20-45 μm).
Unless stated otherwise, starting materials, reagents, and sol-
vents were purchased as high-grade commercial products from
Sigma-Aldrich, Lancaster, Scharlab, or Panreac and were used
without further purification. THF was distilled from sodium
benzophenone ketyl and used immediately. Dichloromethane
was distilled from calcium hydride.
The following compounds were synthesized according to
described procedures: 23,61 24,44 28,62 and 29,42 and their spectro-
scopic data are in agreement with those previously described.
Spectroscopic data of all described compounds were consis-
tent with the proposed structures. For series 1-8, 10-20, we
include the data of compounds 5, 6, 8, 11, 12, 15, and 19. For
intermediates 25-28, 33-37, 40-42, the data of compounds
26, 35, and 42 are described.
General Procedure for the Synthesis of 2-Nitro-ω-piperazin-
1-ylanilines (21-24). To a solution of 3- or 5-chloro-2-nitroani-
line (5.12 g, 29.5 mmol) in anhydrous DMF (70 mL), piper-
azine, or 1-methylpiperazine (78 mmol), Cs2CO3, or K2CO3
(78 mmol) were added, respectively. The reaction was heated
at 120 °C for 24 h. Upon cooling, the mixture was poured
into ice-cold water and the solid was filtered, washed with
cold water, and dried under vacuum. The solid was purified
by column chromatography using the appropriate eluent, to
afford pure 21-24.
Conclusions
On the basis of our previously described pharmacophore
model for 5-HT6R antagonists,40 we have identified benzimi-
dazole derivatives as a new structural class of serotonin 5-HT6R
ligands. Computational modeling and site-directed mutagen-
esis have allowed us to characterize the amino acid residues of
5-HT6R involved in ligand binding (Figure 3). The distinctive
positively charged piperazine ring anchors the ligand to D3.32
in TM 3, whereas the benzimidazole ring is situated parallel to
EL 2, to hydrogen bond N6.55 in TM 6 (Figures 3 and 4). An
important feature of these molecules is the linker between the
benzimidazole ring and the terminal Ar system (phenyl,
1-naphthyl, 2-naphthyl). The sp2 carbonyl group (-CO-)
enforces both aromatic domains to be coplanar, whereas the
sp3 methylene unit (-CH2-) allows the rings to form an angle
of approximately 90°. Thus, in CH2-containing compounds,
the Ar system is located between TMs 3 and 5, to interact with
V3.33 and A5.42 (Figure 4A), whereas in CO-containing
compounds, the Ar aromatic moiety is located between TMs
3 and 6, interacting with V3.33, W6.48, and F6.52 (Figure 4B).
Both types of compounds display high binding affinity for
the 5-HT6R as shown for ligands 12 [Ki(12) = 16 nM] and 13
[Ki(13)=36 nM], having CH2 as a linker, and ligands 3 [Ki(3)=
13 nM] and 4 [Ki(4) = 34 nM] containing CO (Table 1). The
new compounds were pharmacologically characterized as
potent 5-HT6R antagonists (Ki = 5-31 nM).
2-Nitro-3-piperazin-1-ylaniline (21). Obtained from 3-chloro-
2-nitroaniline and piperazine, in 40% yield. Chromatography:
CH2Cl2/EtOH, 8:2; mp 212-213 °C. IR (KBr) 3308, 1514, 1443.
1H NMR (CDCl3, δ): 1.90 (br s, 1H), 3.01 (m, 8H), 4.95 (br s,
2H), 6.57 (d, J = 8.1, 2H), 7.14 (t, J = 8.1, 1H). 13C NMR
(CDCl3, δ) 46.2, 53.0, 109.3, 111.2, 122.0, 132.9, 142.9, 148.0.
MS (ESI) 223.0 (M þ H).
3-(4-Methylpiperazin-1-yl)-2-nitroaniline (22). Obtained from
3-chloro-2-nitroaniline and 1-methylpiperazine, in quantitative
yield. Chromatography: CH2Cl2/EtOH, 9:1. IR (CHCl3) 3383,
1605, 1573, 1511, 1459. 1H NMR (CDCl3) δ 2.32 (s, 3H), 2.53 (br
t, J = 4.7, 4H), 3.01 (br t, J = 4.7, 4H), 4.80 (br s, 2H), 6.38 (d,
J = 8.1, 2H), 7.11 (t, J = 8.1, 1H). 13C NMR (CDCl3) δ 46.1,
51.7, 55.1, 109.1, 111.2, 121.3, 132.9, 142.6, 147.6. MS (ESI)
258.9 (M þ Na).
This combined experimental and computational study has
permitted to understand how ligand binding blocks receptor
activation (Figure 6), which may contribute to the rational
design of new compounds acting at the serotonin 5-HT6R. As
some of the residues involved in ligand binding and receptor
inactivation are strongly conserved, these findings can likely
be extended to other members of the rhodopsin-like family of
GPCRs.
2-Nitro-5-piperazin-1-ylaniline (23). Obtained from 5-chloro-
2-nitroaniline and piperazine, in quantitative yield. Chroma-
tography: CH2Cl2/EtOH, 9:1; mp 164-165 °C (lit.61 170 °C).
5-(4-Methylpiperazin-1-yl)-2-nitroaniline (24). Obtained from
5-chloro-2-nitroaniline and 1-methylpiperazine, in 89% yield.
Chromatography: CH2Cl2/EtOH, 98:2; mp 153-154 °C (lit.44
156-157 °C).
Experimental Section
Chemistry. Melting points (uncorrected) were determined on
a Stuart Scientific electrothermal apparatus. Infrared (IR)
spectra were measured on a Shimadzu-8300 or Bruker Tensor
27 instrument equipped with a Specac ATR accessory of
5200-650 cm-1 transmission range; frequencies (ν) are ex-
pressed in cm-1. Nuclear magnetic resonance (NMR) spectra
were recorded on a Bruker Avance 500 (1H, 500 MHz; 13C, 125
MHz), Bruker Avance 300 AM. (1H, 300 MHz; 13C, 75 MHz) or
Bruker 200-AC spectrometer (1H, 200 MHz; 13C, 50 MHz) at
the UCM’s NMR facilities. Chemical shifts (δ) are expressed in
parts per million relative to internal tetramethylsilane; coupling
constants (J) are in hertz (Hz). The following abbreviations are
used to describe peak patterns when appropriate: s (singlet),
d (doublet), t (triplet), m (multiplet), br (broad), app (apparent).
Mass spectrometry (MS) was carried out on a Bruker LC-
Esquire in electrospray mode (ESI) or a HP 5989 A in electron
impact mode (EI, 70 eV). Elemental analyses (C, H, N) were
obtained on a LECO CHNS-932 apparatus at the UCM’s
analysis services and were within 0.5% of the theo-
retical values, confirming a purity of at least 95% for all tested
compounds. Analytical thin-layer chromatography (TLC) was
General Procedure for the Synthesis of ω-Piperazin-1-ylben-
zene-1,2-diamines (25-28). To a suspension of 21-24 (1.3
mmol) in absolute MeOH (10 mL), 10% Pd(C) (25 mg) was
added, and the reaction was hydrogenated at room temperature
for 24 h, with an initial hydrogen pressure of 50 psi. The mixture
was filtered over celite, and the solvent was evaporated to
dryness to give 25-28, which were used in the next step without
further purification.
3-Piperazin-1-ylbenzene-1,2-diamine (25). Obtained from 21
in quantitative yield.
3-(4-Methylpiperazin-1-yl)benzene-1,2-diamine (26). Obtai-
ned from 22 in quantitative yield. IR (CHCl3) 3325, 1600, 1478,
1455. 1H NMR (CDCl3) δ 2.31 (s, 3H), 2.60 (m, 4H), 2.94 (br t, J
= 4.7, 4H), 6.54 (dd, J = 6.9, 1.4, 1H), 6.63-6.72 (m, 2H). 13
C
NMR (CDCl3) δ 46.1, 51.3, 55.9, 111.6, 112.7, 118.9, 130.1, 134.4,
140.2. MS (ESI) 207.0 (M þ H), 228.9 (M þ Na).
4-Piperazin-1-ylbenzene-1,2-diamine (27). Obtained from 23
in quantitative yield.

Article
4-(4-Methylpiperazin-1-yl)benzene-1,2-diamine (28). Obtai-
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 1365
NaOt-Bu (250 mg, 2.7 mmol), tris(dibenzylideneacetone)dipalla-
dium(0) (190 mg, 0.19 mmol), and trit-butylphosphine (150 mg,
0.8 mmol) were added. The mixture was heated at 110 °C for 24 h.
Upon cooling to room temperature, the reaction was filtered over
celite, the solvent was removed under reduced pressure, and the
residue was purified by column chromatography using the appro-
priate eluent, to afford pure 31 or 32.
tert-Butyl 4-(1-{[2-(Trimethylsilyl)ethoxy]methyl}-1H-benzi-
midazol-4-yl)piperazine-1-carboxylate (31). Obtained from 30
and 1-BOC-piperazine in 49% yield. Chromatography: hexane/
EtOAc, 8:2. IR (CHCl3) 1697, 1603, 1543, 1477, 1450. 1H NMR
(CDCl3) δ -0.01 (s, 9H), 1.08 (app t, J = 8.6, 2H), 1.46 (s, 9H),
2.72 (br t, J = 4.8, 4H), 3.40-3.50 (m, 4H), 3.73 (app t, J = 8.3,
2H), 5.70 (s, 2H), 6.77 (dd, J = 8.0, 1.2, 1H), 7.26 (t, J = 8.0, 1H),
7.99 (dd, J = 8.4, 1.1, 1H), 8.04 (s, 1H). 13C NMR (CDCl3, δ)
-1.7, 18.1, 28.2, 51.8, 67.5, 75.3, 79.7, 114.7, 116.0, 125.0, 129.5,
138.0, 144.3, 149.9, 154.5.
ned from 24 in 89% yield; mp 101-103 °C (lit.62 101-102 °C).
General Procedure for the Synthesis of Compounds 29, 38, 43.
To a suspension of 3-chlorobenzene-1,2-diamine,41 27 or 28
(9.8 mmol) in water (14 mL), formic acid (1.2 mL, 29 mmol) was
added and the mixture was heated at 100 °C for 3 h. After the
complete disappearance of the starting material (tlc), the reac-
tion was poured on ice and 1 M KOH was added until pH 9-10.
The resulting solid was filtrated, dried, and recrystallized;
otherwise, the aqueous solution was extracted with CH2Cl2
(3 ꢀ 50 mL), the organic layers were dried (Na2SO4), and eva-
porated to dryness. The residue was purified by column chro-
matography using the appropriate eluent, to afford pure benzi-
midazoles 29, 38, 43.
4-Chloro-1H-benzimidazole (29). Obtained from 3-chloro-
benzene-1,2-diamine in quantitative yield; mp 167-169 °C
(lit.42 170-171 °C) (EtOAc).
5-(4-Methylpiperazin-1-yl)-1H-benzimidazole (38). Obtained
from 28 in 60% yield. Chromatography: CHCl3/MeOH, 95:5;
mp 81-83 °C (lit.43 82-84 °C). Spectroscopic data are in
agreement with those previously described.
5-Piperazin-1-yl-1H-benzimidazole (43). Obtained from 27 in
87% yield. Chromatography CHCl3/MeOH/NH3 6:4:0.1; mp
123-125 °C (lit.43 126-128 °C). Spectroscopic data are in
agreement with those previously described.
4-(4-Methylpiperazin-1-yl)-1-{[2-(trimethylsilyl)ethoxy]methyl}-
1H-benzimidazole (32). Obtained from 30 and 1-methylpiperazine
in 43% yield. Chromatography: CH2Cl2/EtOH, 98:2. IR
1
(CHCl3) 1499, 1453. H NMR (CDCl3) δ -0.04 (s, 9H), 0.90
(app t, J = 8.4, 2H), 2.44 (s, 3H), 2.73 (br t, J = 4.8, 4H), 3.50
(app t, J = 8.1, 2H), 3.61 (m, 4H), 5.52 (s, 2H), 6.69 (d, J = 8.3,
1H), 7.12 (d, J = 8.3, 1H), 7.24 (t, J = 8.1, 1H), 7.90 (s, 1H). 13
C
NMR (CDCl3, δ) -1.5, 17.8, 46.0, 52.8, 55.5, 65.9, 74.8, 115.3,
116.7, 122.8, 128.1, 139.9, 144.3, 145.4. MS (ESI) 347.0 (M þ H).
Synthesis of tert-Butyl 5- and 6-[4-tert-Butoxycarbonyl)-
piperazin-1-yl]-1H-benzimidazole-1-carboxylates (44). To a so-
lution of 43 (200 mg, 1 mmol) in anhydrous CH2Cl2 (6 mL), di-
tert-butyl dicarbonate (648 mg, 3 mmol), triethylamine (0.4 mL,
3 mmol), and 4-(dimethylamino)pyridine (37 mg, 0.3 mmol)
were added, and the mixture was stirred at room temperature
overnight. The reaction solution was washed with 1 M NaOH
and the organic layer was dried (Na2SO4) and evaporated at
reduced pressure. The resulting crude was purified by column
chromatography to give 44 in 69% yield, as a nonseparable
mixture of regioisomers in a 1:1 ratio. IR (KBr) 1755, 1740,
1694, 1624, 1487, 1451. 1H NMR (CDCl3) δ 1.50, 1.70 (s, 18H),
3.14-3.21, 3.62-3.66 (m, 8H), 7.05, 7.12 (dd, J = 8.9, 2.3, 1H),
7.31, 7.59 (br s, 1H), 7.66, 7.87 (d, J = 10.3, 1H), 8.30, 8.39
(s, 1H). 13C NMR (CDCl3) δ 28.3, 28.6, 50.7, 51.1, 80.0, 85.4,
102.2, 108.2, 114.7, 115.9, 117.3, 120.9, 132.8, 133.0, 138.4,
140.2, 142.4, 147.6, 149.1, 149.7, 154.7. MS (EI) m/z (%)
402 (M, 18), 346 (44), 302 (24), 290 (100), 246 (50), 216 (23), 57
(45), 41 (37).
General Procedure for the Synthesis of Compounds 33-37,
40-42, and 45. To a solution of SEM-protected or BOC-
protected benzimidazole 31, 32, 39, or 44 (1.3 mmol) in anhy-
drous THF (5.2 mL), BuLi (1.8 mmol, 1.6 M in hexane) was
added under an argon atmosphere at -78 °C. The mixture was
stirred at this temperature for 1 h, then corresponding alkyl
benzoate or naphthoate (1.4 mmol) was added slowly and the
reaction mixture was allowed to reach room temperature and
stirred overnight. The reaction was quenched with a saturated
aqueous solution of NH4Cl, then extracted with CH2Cl2, and
the organic layers were washed with water and brine, dried
(Na2SO4), and evaporated to dryness. The residue was purified
by column chromatography using the appropriate eluent, to
afford pure 33-37, 40-42, 45.
General Procedure for the Synthesis of Compounds 30 and 39.
To a suspension of K2CO3 (2.86 g, 20.7 mmol) or NaH (444 mg,
11.1 mmol, 60% mineral oil) in anhydrous DMF (10 mL), 29 or
38 (6.9 mmol) was added portionwise, respectively, under an
argon atmosphere. The mixture was stirred at room temperature
for 1 h, then 2-(trimethylsilyl)ethoxymethyl chloride (SEMCl)
(2.1 mL, 11.1 mmol) was added dropwise and the reaction was
stirred for 2 h. The reaction was quenched with water and
extracted with CH2Cl2 (30) or EtOAc (39). The organic layers
were dried (Na2SO4) and evaporated under vacuum, and the
residue was purified by column chromatography, using the
appropriate eluent, to provide 30 or 39.
4- and 7-Chloro-1-{[2-(trimethylsilyl)ethoxy]methyl}-1H-ben-
zimidazoles (30). Obtained from 29 in quantitative yield, as a
nonseparable mixture of regioisomers in a 1:1 ratio. Chroma-
tography: hexane/EtOAc, 7:3. IR (CHCl3) 1613, 1576, 1499,
1428. 1H NMR (CDCl3) δ 0.03 (s, 9H), 0.93, 0.99 (br t, J = 4.5,
2H), 3.55, 3.62 (app t, J = 8.0, 2H), 5.59, 5.88 (s, 2H), 7.20-7.36
(m, 2H), 7.46, 7.72 (dd, J = 5.4, 1.0, 1H), 7.99, 8.03 (s, 1H). 13
C
NMR (CDCl3) δ -1.4, -1.3, 17.8, 17.9, 66.3, 66.8, 74.6, 75.1,
109.2, 116.8, 119.4, 122.8, 123.4, 124.2, 125.0, 125.2, 130.1,
133.4, 143.4, 145.1, 146.3. MS (ESI) 283.0 (M þ H).
6-(4-Methylpiperazin-1-yl)-1-{[2-(trimethylsilyl)ethoxy]methyl}-
1H-benzimidazole (39). Obtained from 38 in 44% yield, after the
separation from its 5-substituted regioisomer [Spectroscopic data
of 5-(4-methylpiperazin-1-yl)-1-{[2-(trimethylsilyl)ethoxy]methyl}-
1H-benzimidazole: IR (CHCl3) 1624, 1586, 1491, 1454. 1H NMR
(CDCl3) δ-0.05(s,9H),0.90(appt,J= 8.2, 2H), 2.39 (s, 3H), 2.65
(br t, J = 4.9, 4H), 3.24 (br t, J = 4.9, 4H), 3.50 (t, J = 8.2, 2H),
5.49 (s, 2H), 7.10 (dd, J = 8.8, 2.4, 1H), 7.34 (d, J = 2.1, 1H), 7.43
(d, J= 9.0, 1H), 7.90 (s, 1H). 13CNMR(CDCl3) δ-1.6, 17.6, 46.0,
50.9, 55.2, 66.2, 74.2, 107.2, 110.2, 116.0, 128.3, 142.9, 144.9, 148.3.
MS (EI) m/z (%) 346 (M, 100), 331 (21), 229 (21), 199 (9), 73 (46),
43 (40).] (38% yield) by column chromatography (CH2Cl2/EtOH,
9:1). IR (CHCl3) 1625, 1581, 1502, 1490, 1454. 1H NMR (CDCl3)
δ -0.03 (s, 9H), 0.91 (app t, J = 8.2, 2H), 2.39 (s, 3H), 2.64 (br t,
J = 4.8, 4H), 3.26 (br t, J = 4.8, 4H), 3.51 (app t, J = 8.2, 2H), 5.49
(s, 2H), 7.00 (d, J = 2.1, 1H), 7.05 (dd, J = 9.0, 2.3, 1H), 7.69 (d,
J = 8.8, 1H), 7.84 (s, 1H). 13C NMR (CDCl3) δ -1.5, 17.6, 46.0,
50.5, 55.1, 66.2, 74.0, 96.9, 114.5, 120.4, 134.9, 137.9, 141.9, 149.5.
MS (EI) m/z (%) 346 (M, 100), 331 (21), 229 (21), 199 (9), 73 (46),
43 (40).
tert-Butyl 4-(2-Benzoyl-1-{[2-(trimethylsilyl)ethoxy]methyl}-
1H-benzimidazol-4-yl)piperazine-1-carboxylate (33). Obtained
from 31 and ethyl benzoate, in 21% yield. Chromatography:
hexane/EtOAc, 8:2.
[4-(4-Methylpiperazin-1-yl)-1-{[2-(trimethylsilyl)ethoxy]methyl}-
1H-benzimidazol-2-yl](phenyl)methanone (34). Obtained from 32
and ethyl benzoate, in 47% yield. Chromatography: CH2Cl2/
EtOH, 95:5.
General Procedure for the Synthesis of Compounds 31 and 32.
To a solution of 30 (537 mg, 1.9 mmol) in o-xylene (17 mL) under
an argon atmosphere, 1-BOC- or 1-methylpiperazine (2.7 mmol),
tert-Butyl 4-[2-(1-Naphthoyl)-1-{[2-(trimethylsilyl)ethoxy]methyl}-
1H-benzimidazol-4-yl]piperazine-1-carboxylate (35). Obtained from
31 and ethyl 1-naphthoate in 27% yield. Chromatography:

1366 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3
de la Fuente et al.
1
hexane/EtOAc, 98:2. H NMR (CDCl3) δ -0.05 (s, 9H), 0.95
[4-(4-Methylpiperazin-1-yl)-1H-benzimidazol-2-yl](2-naphthyl)-
methanone (5). Obtained from 37 in 50% yield. Chromatography
CH2Cl2/EtOH, 98:2; mp 210-211 °C. IR (KBr) 3442, 3301, 1635,
1525, 1486, 1440. 1H NMR (CDCl3) δ 2.47 (s, 3H), 2.83 (br t, J =
4.8, 4H), 3.81 (br t, J= 5.0, 4H), 6.67 (d, J=7.7, 1H), 7.11(d, J =
7.7, 1H), 7.32 (t, J = 8.0, 1H), 7.60-7.68 (m, 2H), 7.89-8.03 (m,
3H), 8.54 (dd, J = 8.7, 1.6, 1H), 9.78 (s, 1H), 10.50 (br s, 1H). 13C
NMR (CDCl3) δ 46.4, 49.9, 55.4, 104.4, 108.2, 126.1, 127.2, 127.9,
128.2, 128.4, 129.3, 130.5, 132.9, 133.1, 135.2, 135.5, 136.3, 136.6,
145.4, 145.9, 186.8. MS (ESI) 371.1 (M þ H). Anal. (C23H22N4O)
C, H, N.
(app t, J = 8.3, 2H), 1.50 (s, 9H), 3.50 (m, 8H), 3.69 (app t, J =
8.3, 2H), 6.17 (s, 1H), 6.65 (d, J = 8.0, 1H), 7.21 (d, J = 8.4, 1H),
7.36 (t, J = 8.0, 1H), 7.51-7.58 (m, 3H), 7.93-7.96 (m, 1H), 8.10
(t, J = 8.9, 2H), 8.42-8.45 (m, 1H). 13C NMR (CDCl3) δ -1.3,
18.0, 28.5, 49.7, 66.5, 74.0, 79.8, 103.9, 108.3, 124.1, 125.7, 126.3,
127.6, 128.6, 131.5, 131.8, 132.9, 133.8, 134.3, 135.0, 138.1, 144.8,
144.9, 154.9, 188.7.
[4-(4-Methylpiperazin-1-yl)-1-{[2-(trimethylsilyl)ethoxy]methyl}-
1H-benzimidazol-2-yl](1-naphthyl)methanone (36). Obtained from
32 and ethyl 1-naphthoate, in 55% yield. Chromatography:
CH2Cl2/EtOH, 98:2.
[4-(4-Methylpiperazin-1-yl)-1-{[2-(trimethylsilyl)ethoxy]methyl}-
1H-benzimidazol-2-yl)(2-naphthyl)methanone (37). Obtained from
32 and methyl 2-naphthoate, in 59% yield. Chromatography
CH2Cl2/EtOH, 98:2.
[6-(4-Methylpiperazin-1-yl)-1-{[2-(trimethylsilyl)ethoxy]methyl}-
1H-benzimidazol-2-yl](phenyl)methanone (40). Obtained from 39b
and ethyl benzoate, in 63% yield. Chromatography: CH2Cl2/
EtOH, 95:5.
[5-(4-Methylpiperazin-1-yl)-1H-benzimidazol-2-yl](phenyl)meth-
anone (6). Obtained from 40 in 75% yield. Chromatography:
CHCl3/MeOH/NH3, 9:1:0.1; mp 210-212 °C. IR (KBr) 3420,
3304, 1624, 1597, 1574, 1516, 1485, 1452. 1H NMR (CDCl3) δ 2.40
(s, 3H), 2.65(brt, J=4.9, 4H), 3.31(brt,J= 4.9, 4H), 6.91 (d, J=
2.1, 1H), 7.12 (dd, J = 9.1, 2.2, 1H), 7.53-7.58 (m, 2H), 7.62-7.67
(m, 1H), 7.81 (d, J = 9.1, 1H), 8.66 (dd, J = 7.0, 1.6, 2H), 10.60 (br
s, 1H). 13C NMR (CDCl3) δ 46.5, 50.1, 55.4, 96.8, 116.8, 122.9,
128.8, 131.5, 133.8, 135.2, 136.2, 138.9, 147.5, 150.5, 183.9. MS
(ESI) 321.0 (M þ H). Anal. (C19H20N4O) C, H, N.
[6-(4-Methylpiperazin-1-yl)-1-{[2-(trimethylsilyl)ethoxy]methyl}-
1H-benzimidazol-2-yl](1-naphthyl)methanone (41). Obtained from
39b and ethyl 1-naphthoate, in 57% yield. Chromatography:
CH2Cl2/EtOH, 95:5.
[5-(4-Methylpiperazin-1-yl)-1H-benzimidazol-2-yl](1-naphthyl)-
methanone (7). Obtained from 41 in 77% yield. Chromatography:
CH2Cl2/EtOH, 9:1; mp 142-144 °C.
[6-(4-Methylpiperazin-1-yl)-1-{[2-(trimethylsilyl)ethoxy]methyl}-
1H-benzimidazol-2-yl](2-naphthyl)methanone (42). Obtained from
39b and methyl 2-naphtoate, in 56% yield. Chromatography:
[5-(4-Methylpiperazin-1-yl)-1H-benzimidazol-2-yl](2-naphthyl)-
methanone (8). Obtained from 42 in 65% yield. Chromatography:
CH2Cl2/EtOH, 97:3; mp 231-233 °C. IR (KBr) 3444, 3299, 1633,
1616, 1570, 1517, 1493, 1446. 1H NMR (CDCl3) δ 2.39 (s, 3H),
2.63 (br t, J = 4.8, 4H), 3.31 (br t, J = 4.8, 4H), 6.93 (d, J = 2.0,
1H), 7.13 (dd, J = 9.3, 2.0, 1H), 7.50-7.68 (m, 2H), 7.84 (d, J =
9.3, 1H), 7.90-7.98 (m, 2H), 8.10 (d, J = 7.8, 1H), 8.52 (dd, J =
8.6, 1.7, 1H), 9.48 (s, 1H), 10.23 (br s, 1H). 13C NMR (CDCl3) δ
45.6, 48.8, 54.6, 96.0, 115.9, 121.6, 125.8, 126.9, 127.7, 127.9, 128.8,
129.9, 131.9, 132.9, 133.3, 135.0, 135.6, 137.6, 147.2, 150.3, 182.5.
MS (ESI) 371.0 (M þ H). Anal. (C23H22N4O) C, H, N.
1
CH2Cl2/EtOH, 97:3. H NMR (CDCl3) δ -0.11 (s, 9H), 1.02
(app t, J = 8.2, 2H), 2.52 (s, 3H), 2.80 (br t, J = 4.9, 4H), 3.48 (br t,
J= 4.9, 4H), 3.76 (app t, J=8.2,2H),6.16(s,2H),7.13(d, J=2.0,
1H), 7.26 (dd, J = 9.1, 2.2, 1H), 7.60-7.75 (m, 2H), 7.92 (d, J =
9.0, 1H), 7.98-8.13 (m, 3H), 8.38 (dd, J = 8.6, 1.7, 1H), 9.03 (s,
1H). 13C NMR (CDCl3) δ -1.6, 17.7, 45.8, 49.5, 54.8, 66.2, 73.7,
96.3, 116.3, 122.2, 125.7, 126.4, 127.6, 127.9, 128.5, 129.9, 132.3,
133.7, 134.5, 135.6, 136.2, 137.3, 145.9, 150.7, 185.6.
tert-Butyl 4-[2-(2-Naphthoyl)-1H-benzimidazol-5-yl)piperazine-
1-carboxylate (45). Obtained from 44 and ethyl 2-naphthoate, in
33% yield. Chromatography: hexane/EtOAc, 9:1. IR (CHCl3)
Synthesis of 2-Naphthyl-(5-piperazin-1-yl-1H-benzimidazol-
2-yl)methanone (9). To a solution of 45 (69 mg, 0.15 mmol) in
anhydrous CH2Cl2 (1 mL), trifluoroacetic acid(0.25 mL, 3 mmol)
was added dropwise. The reaction mixture was stirred at room
temperature for 5 h, thenevaporated under reduced pressure. The
resulting crude was resuspended in CH2Cl2 and washed with 1 M
NaOH. The organic layer was dried (Na2SO4) and evaporated,
and the obtained solid was purified by recrystallization from
Et2O, to afford pure 9 in quantitative yield; mp 155-157 °C. IR
(KBr) 3442, 1626, 1600, 1516, 1461. 1H NMR (CD3OD) δ
3.22-3.39 (m, 8H), 7.17 (br s, 1H), 7.23 (dd, J = 9.0, 2.2, 1H),
7.54-7.69 (m, 2H), 7.70 (d, J = 9.0, 1H), 7.95-8.09 (m, 3H), 8.29
(dd, J = 8.7, 1.7, 1H), 9.20 (s, 1H). 13C NMR (CD3OD) δ 44.5,
49.2, 93.4, 111.7, 118.2, 125.6, 127.1, 127.9, 128.3, 129.0, 130.0,
132.0, 132.1, 132.8, 133.6, 133.8, 136.2, 148.2, 150.3, 183.9. MS
(ESI) 357.0. Anal. (C22H20N4O) C, H, N.
General Procedure for the Synthesis of Benzimidazole Deriva-
tives 10-20 (X = CH2). To a solution of previously synthesized
25-28 (1.5 mmol) and the appropriate aldehyde (2.3 mmol) in
freshly distilled CH2Cl2 (8 mL), ytterbium triflate (93 mg, 0.2
mmol) was added and the reaction was stirred at room tem-
perature overnight. The mixture was filtered over celite, the
solvent was evaporated under reduced pressure, and the residue
was purified by column chromatography using the appropriate
eluent, to afford pure 10-20.
1
3453, 3288, 1693, 1632, 1516, 1428. H NMR (CDCl3) δ 1.52
(s, 9H), 3.24 (br t, J = 5.1, 4H), 3.64-3.67 (m, 4H), 6.95 (d, J =
2.1, 1H), 7.14 (dd, J = 9.1, 2.2, 1H), 7.56-7.68 (m, 2H), 7.87 (d,
J = 9.3, 1H), 7.88 (d, J = 9.3, 1H), 7.98 (d, J = 8.7, 1H), 8.12 (d,
J = 7.9, 1H), 8.53 (dd, J = 8.7, 1.7, 1H), 9.51 (s, 1H), 10.36 (br s,
1H). 13C NMR (CDCl3) δ 28.3, 49.9, 50.4, 50.6, 79.9, 96.8, 116.6,
122.5, 125.6, 126.4, 127.6, 128.0, 128.6, 130.1, 132.4, 132.8, 134.0,
134.4, 135.7, 138.7, 147.3, 150.9, 154.6, 182.9. MS (EI) m/z (%)
456 (M, 36), 400 (77), 356 (33), 314 (100), 155 (29), 127 (36), 56
(32), 41 (25).
General Procedure for the Synthesis of Benzimidazole Deriva-
tives 1-8 (X = CO). To a solution of 33-37, 40-42 (0.34 mmol)
in freshly distilled CH2Cl2 (3 mL), BCl3 (1.8 mmol, 1 M in
CH2Cl2) was added dropwise and under an argon atmosphere.
The reaction was stirred at room temperature for 16 h, then
quenched with saturated NaHCO3. The reaction mixture was
extracted with EtOAc, and the organic layers were dried
(Na2SO4) and evaporated at reduced pressure. The residue
was purified by column chromatography using the appropriate
eluent, to afford pure 1-8.
Phenyl(4-piperazin-1-yl-1H-benzimidazol-2-yl)methanone (1).
Obtained from 33 in 58% yield. Chromatography: CH2Cl2/
MeOH, 7:3; mp 123-125 °C.
2-Benzyl-4-piperazin-1-yl-1H-benzimidazole (10). Obtained
from 25 and phenylacetaldehyde, in 45% yield. Chromatogra-
phy: CH2Cl2/EtOH, 9:1; mp 126-128 °C.
2-Benzyl-4-(4-methylpiperazin-1-yl)-1H-benzimidazole (11).
Obtained from 26 and phenylacetaldehyde, in 42% yield. Chro-
matography: CH2Cl2/EtOH, 8:2; mp 120-123 °C. IR (KBr)
3356, 1593, 1533, 1494, 1456. 1H NMR (CDCl3) δ 2.44 (s, 3H),
2.79 (m, 4H), 3.62 (m, 4H), 4.30 (s, 2H), 6.66 (d, J = 8.0, 1H),
7.09 (m, 1H), 7.12 (t, J = 8.0, 1H), 7.30-7.38 (m, 5H). 13C NMR
(CDCl3) δ 36.3, 45.7, 49.6, 55.2, 105.6, 110.0, 123.6, 127.7, 129.4,
[4-(4-Methylpiperazin-1-yl)-1H-benzimidazol-2-yl](phenyl)meth-
anone (2). Obtained from 34 in 53% yield. Chromatography:
CH2Cl2/EtOH, 98:2; mp 151-154 °C.
1-Naphthyl(4-piperazin-1-yl-1H-benzimidazol-2-yl)methanone
(3). Obtained from 35 in 64% yield. Chromatography: CH2Cl2/
MeOH, 7:3; mp 210-213 °C.
[4-(4-Methylpiperazin-1-yl)-1H-benzimidazol-2-yl](1-naphthyl)-
methanone (4). Obtained from 36 in 58% yield. Chromatography:
CH2Cl2/EtOH, 9:1; mp 213-214 °C.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 1367
129.5, 135.1, 136.5, 137.0, 141.5, 151.3. MS (ESI) 307.0 (M þ H).
Anal. (C19H22N4) C, H, N.
drug, in a final volume of 540 μL of assay buffer. Nonspecific
binding was determined by radioligand binding in the presence
of a saturating concentration of 25 μM clozapine and repre-
sented less than 15% of total binding.
2-(1-Naphthylmethyl)-4-piperazin-1-yl-1H-benzimidazole (12).
Obtained from 25 and 1-naphthylacetaldehyde, in 45% yield.
Chromatography: CH2Cl2/EtOH, 9:1; mp 163-165 °C. IR (KBr)
3454, 1617, 1597, 1531, 1455. 1H NMR (CD3OD) δ 3.47-3.52
(m, 8H), 4.74 (s, 2H), 6.73-6.76 (m, 1H), 7.11-7.14 (m, 2H), 7.35
(d, J = 6.8, 1H), 7.42-7.50 (m, 3H), 7.83 (d, J = 8.2, 1H), 7.88-
7.91 (m, 1H), 8.10-8.14 (m, 1H). 13C NMR (CD3OD) δ 34.0,
45.3, 106.2, 108.7, 124.3, 124.7, 126.8, 127.1, 127.5, 128.3, 129.2,
129.9, 132.2, 134.2, 135.6, 136.2, 136.9, 142.2, 151.9. MS (ESI)
343.2 (M þ H). Anal. (C22H22N4) C, H, N.
For all binding assays, competing drug, nonspecific, total,
and radioligand bindings were defined in triplicate. Incubation
was terminated by rapid vacuum filtration through Wallac
Filtermat A filters, presoaked in poly(ethylenimine) (0.5% for
the 5-HT6R and 0.3% for the 5-HT7R), using a FilterMate
Unifilter 96-Harvester. The filters were then washed 5-9 times
with 500 μL of ice-cold 50 mM Tris-HCl buffer (pH 7.4 at 25 °C)
and heated at 80 °C. The radioactivity bound to the filters was
measured by scintillation spectrometry, using a Microbeta
TopCount instrument. The data were analyzed by an iterative
curve-fitting procedure using GraphPad Prism, which provided
IC50, Ki, and r2 values for test compounds, Ki values being
calculated from the Cheng-Prusoff equation.45
Plasmid Construct. HA-tagged-5-HT66receptor cDNAs in
pRK5 were generated by fusing the sequence of the HA epitope
(YPYDVPDYA) to a cleavable signal peptide (MVLLLILSV-
LLLKEDVRGSAQS) derived from the metabotropic gluta-
mate receptor 5 (mGlu5R). This sequence was inserted into the
pRK5 vector using Xba I and BsrG I restriction sites. Then, full-
length mouse 5-HT6R cDNA was subcloned in frame using
BsrG I and Hind III. HA-5-HT6-C3.36A, HA-5-HT6-S5.43A,
HA-5-HT6-W6.48A, HA-5-HT6-F6.52A, HA-5-HT6-N6.55A
receptor mutants were generated from HA-5-HT6R, cloned in
pRK5, with the Quick Change Site-Directed Mutagenesis kit
(Stratagene, Amsterdam, Netherlands).
4-(4-Methylpiperazin-1-yl)-2-(1-naphthylmethyl)-1H-benzi-
midazole (13). Obtained from 26 and 1-naphthylacetaldehyde, in
48% yield. Chromatography: CH2Cl2/EtOH, 9:1; mp 95-96 °C.
4-(4-Methylpiperazin-1-yl)-2-(2-naphthylmethyl)-1H-benzi-
midazole (14). Obtained from 26 and 2-naphthylacetaldehyde,
in 51% yield. Chromatography: CH2Cl2/EtOH, 8:2.
2-Benzyl-5-piperazin-1-yl-1H-benzimidazole (15). Obtained
from 27 and phenylacetaldehyde, in 67% yield. Chromatogra-
phy: CHCl3/MeOH/NH3, 9:1:0.1; mp 173-175 °C. IR (KBr)
3450, 1633, 1493, 1455. 1H NMR (acetone-d6) δ 2.78-2.92 (m,
8H), 2.97 (br s, 1H), 4.05 (s, 2H), 6.76 (dd, J = 8.7, 2.3, 1H), 6.84
(d, J = 2.1, 1H), 7.07-7.25 (m, 6H), 7.90 (br s, 1H). 13C NMR
(acetone-d6) δ 35.7, 46.6, 52.6, 102.0, 114.3, 114.4, 126.9, 128.8,
129.2, 133.9, 136.1, 138.4, 149.3, 152.8. MS (ESI) 293.0 (M þ H).
Anal. (C18H20N4) C, H, N.
2-Benzyl-5-(4-methylpiperazin-1-yl)-1H-benzimidazole (16).
Obtained from 28 and phenylacetaldehyde, in 53% yield. Chro-
matography: CH2Cl2/EtOH, 97:3; mp 108-109 °C.
Cell Surface ELISA. COS-7 cells were transfected with HA-
tagged-5-HT6R, WT or mutants cDNAs. Cells were grown on
96-well plates in DMEM medium with 10% dFCS and fixed as
described previously.63 Cells were then incubated with anti-HA
antibody at 0.6 μg/mL for 60 min in the same buffer and
incubated with antirabbit/HRP conjugate (Amersham Pharma-
cia Biotech) at 1 μg/mL for 60 min. Chromogenic substrate was
added (Supersignal ELISA femto-maximum sensitivity, Pierce,
2-(1-Naphthylmethyl)-5-piperazin-1-yl-1H-benzimidazole (17).
Obtained from 27 and 1-naphthylacetaldehyde, in 44% yield.
Chromatography: CH2Cl2/EtOH, 9:1; mp 112-114 °C.
5-(4-Methylpiperazin-1-yl)-2-(1-naphthylmethyl)-1H-benzi-
midazole (18). Obtained from 28 and 1-naphthylacetaldehyde, in
75% yield. Chromatography: CH2Cl2/EtOH, 9:1; mp 121-123 °C.
2-(2-Naphthylmethyl)-5-piperazin-1-yl-1H-benzimidazole (19).
Obtained from 27 and 2-naphthylacetaldehyde, in 42% yield.
Chromatography: CH2Cl2/EtOH 9:1; mp 157-159 °C. IR (KBr)
3443, 1632, 1490, 1456. 1H NMR (CD3OD) δ 3.25-3.32 (m, 8H),
4.27 (s, 2H), 6.92 (dd, J = 8.8, 2.3, 1H), 7.02 (d, J = 2.0, 1H),
7.29-7.39 (m, 4H), 7.67-7.73 (m, 4H). 13C NMR (CD3OD)
δ 36.3, 45.3, 50.1, 103.9, 116.5, 116.7, 127.0, 127.5, 128.0, 128.5,
128.8, 129.7, 134.1, 135.3, 135.5, 135.8, 140.0, 148.6, 155.3. MS
(ESI) 343.0 (M þ H). Anal. (C22H22N4) C, H, N.
ꢁ
Perbio-Brebieres, France). Chemiluminescence was detected
and quantified by a Wallac Victor2 luminescence counter.
Determination of cAMP Production in Transfected Cells.
COS-7 cells were transfected with the appropriate cDNA and
seeded into 24-well plates (100000 cells/well). Twenty-four
hours post-transfection, a 10 min-stimulation with the appro-
priate concentrations of each tested compound in absence or
presence of 100 μM 5-HT was performed as previously de-
scribed.61 Quantification of cAMP production was performed
by homogeneous time-resolved fluorescence (HTRF) using the
5-(4-Methylpiperazin-1-yl)-2-(2-naphthylmethyl)-1H-benzi-
midazole (20). Obtained from 28 and 2-naphthylacetaldehyde,
in 59% yield. Chromatography: CH2Cl2/EtOH, 95:5; mp
149-152 °C.
ꢁ
cAMP Dynamic kit (Cisbio International, Bagnols-sur-Ceze,
France), according to the manufacturer’s instructions.
Pharmacology. Radioligand Binding Assays. Membranes from
HEK-293 cells expressing human 5-HT6 or 5-HT7 receptors,
were purchased from Perkin-Elmer and conserved at -80 °C in
packaging buffer for subsequent use. Specific radioligand
[3H]LSD (79.2 Ci/mmol) was from NEN (Perkin-Elmer). Com-
petitive inhibition assays were performed according to standard
procedures, briefly detailed below.
5-HT6 Receptor. Cell paste (6.0 mg/mL) was homogenized in
200 volumes of assay buffer (50 mM Tris-HCl, 10 mM MgCl2,
0.5 mM EDTA, pH 7.4 at 25 °C). Fractions of 20 μL of the
membranes suspension were incubated at 37 °C for 60 min with
2.6 nM [3H]LSD, in the presence or absence of the competing
drug, in a final volume of 200 μL of assay buffer. Nonspecific
binding was determined by radioligand binding in the presence
of a saturating concentration of 500 μM serotonin and repre-
sented less than 10% of total binding.
Data Analysis. The dose-response curves were fitted using
GraphPad Prism and the following equation for monophasic
dose-response curves: y = (ymax - ymin)/1 þ [(x/IC50) nH] þ
ymin, whereIC50 isthe concentrationofthe antagonistnecessary to
reduce 50% of the maximal effect induced by 5-HT and nH is the
Hill coefficient. All data represented correspond to the mean (
SEM of three independent experiments performed in triplicate.
Computational Methods. Models of the Ligand-Receptor
Complexes. Modeler v9.564 was used to build a homology
model of the human 5-HT6R using the crystal structure of the
β2-adrenergic receptor (PDB code 2RH1)47,65 as template.
Internal water molecules 506, 519, 528, 529, 532, 534, 537,
543, 546, and 548 that mediate a number of interhelical inter-
actions,48 and seem conserved in the rhodopsin-like family of
GPCRs,66 were also included in the model. The Duan et al.
(2003) force field67 was used for receptors and the general
Amber force field (GAFF)68 and HF/6-31G*-derived RESP
atomic charges were used for the ligands. Molecular dynamics
simulations of the ligand-receptor complexes were performed
with the Sander module of AMBER 969 using the protocol
previously described.70
5-HT7 Receptor. Cell paste (6.8 mg/mL) was homogenized in
200 volumes of assay buffer (50 mM Tris-HCl, 10 mM MgSO4,
0.5 mM EDTA, pH 7.4 at 25 °C). Fractions of 500 μL of the
membranes suspension were incubated at 27 °C for 120 min with
3 nM [3H]LSD, in the presence or absence of the competing

1368 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3
Acknowledgment. This work has been supported by grants
de la Fuente et al.
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231.
ꢀ
from the Spanish Ministerio de Educacion (MEC, SAF-2007/
67008), Comunidad Autonoma de Madrid (CAM, S-SAL-
ꢀ
249-2006), and Instituto de Salud Carlos III (RD07/0067/
ꢀ
0008). We thank the Fundacion Ramon Areces and MEC for
predoctoral grants to T.F., J.S., and R.A.M.
ꢀ
Supporting Information Available: Spectral characterization
data of compounds 1-4, 7, 10, 13, 14, 16-18, 20, 25, 27, 29, 33,
34, 36, 37, 40, and 41; combustion analysis data. This material is
available free of charge via the Internet at http://pubs.acs.org.
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