Benzimidazole derivatives. Part 1: Synthesis and structure-activity relationships of new benzimidazole-4-carboxamides and carboxylates as potent and selective 5-HT4 receptor antagonists

Article abstract of DOI:10.1016/S0968-0896(99)00172-8

New benzimidazole-4-carboxamides 1-16 and -carboxylates 17-26 were synthesized and evaluated for binding affinity at serotonergic 5-HT₄ and 5- HT₃ receptors in the CNS. Most of the synthesized compounds exhibited moderate-to-very high affinity (in many cases subnanomolar) for the 5-HT₄ binding site and no significant affinity for the 5-HT₃ receptor. SAR observations and structural analyses (molecular modeling, INSIGHT II) indicated that the presence of a voluminous substituent in the basic nitrogen atom of the amino moiety and a distance of ca. 8.0 A from this nitrogen to the aromatic ring are of great importance for high affinity and selectivity for 5-HT₄ receptors. These results confirm our recently proposed model for recognition by the 5-HT₄ binding site. Amides 12-15 and esters 24 and 25 bound at central 5-HT₄ sites with very high affinity (K(i) = 0.11-2.9 nM) and excellent selectivity over serotonin 5-HT₃, 5-HT(2A), and 5-HT(1A) receptors (K(i) > 1000-10,000 nM). Analogues 12 (K(i)(5-HT₄) = 0.32 nM), 13 (K(i)(5-HT₄) = 0.11 nM), 14 (K(i)(5-HT₄) = 0.29 nM) and 15 (K(i)(5-HT₄) = 0.54 nM) were pharmacologically characterized as selective 5-HT₄ antagonists in the isolated guinea pig ileum (pA₂ = 7.6, 7.9, 8.2 and 7.9, respectively), with a potency comparable to the 5-HT₄ receptor antagonist RS 39604 (pA₂ = 8.2). The benzimidazole-4-carboxylic acid derivatives described in this paper represent a novel class of potent and selective 5-HT₄ receptor antagonists. In particular, compounds 12-15 could be interesting pharmacological tools for the understanding of the role of 5-HT₄ receptors. (C) 1999 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(99)00172-8

Bioorganic & Medicinal Chemistry 7 (1999) 2271±2281
Benzimidazole Derivatives. Part 1: Synthesis and Structure±Activity
Relationships of New Benzimidazole-4-carboxamides
and Carboxylates as Potent and Selective 5-HT4
Receptor Antagonists
Marõa L. Lopez-Rodrõguez, a,* Bellinda Benhamu, a Alma Viso, a
Â
M. Jose Morcillo, b Marta Murcia, a Luis Orensanz, c M. Jose Alfaro d
and M. Isabel Martõn d
a
Â
Departamento de Quõmica Organica I, Facultad de Ciencias Quõmicas, Universidad Complutense, 28040 Madrid, Spain
Â
Â
Facultad de Ciencias, Universidad Nacional de Educacion a Distancia, 28040 Madrid, Spain
b
Â
Departamento de Investigacion, Hospital Ramon y Cajal, Carretera de Colmenar km. 9, 28034 Madrid, Spain
c
Â
Â
Departamento de Farmacologõa, Facultad de Medicina, Universidad Complutense, 28040 Madrid, Spain
d
Â
Received 27 January 1999; accepted 27 March 1999
AbstractÐNew benzimidazole-4-carboxamides 1±16 and -carboxylates 17±26 were synthesized and evaluated for binding anity at
serotonergic 5-HT4 and 5-HT3 receptors in the CNS. Most of the synthesized compounds exhibited moderate-to-very high anity
(in many cases subnanomolar) for the 5-HT4 binding site and no signi®cant anity for the 5-HT3 receptor. SAR observations and
structural analyses (molecular modeling, INSIGHT II) indicated that the presence of a voluminous substituent in the basic nitrogen
atom of the amino moiety and a distance of ca. 8.0 A from this nitrogen to the aromatic ring are of great importance for high
anity and selectivity for 5-HT4 receptors. These results con®rm our recently proposed model for recognition by the 5-HT4 binding
site. Amides 12±15 and esters 24 and 25 bound at central 5-HT4 sites with very high anity (Ki=0.11±2.9 nM) and excellent
selectivity over serotonin 5-HT3, 5-HT2A, and 5-HT1A receptors (Ki >1000±10,000 nM). Analogues 12 (Ki(5-HT4)=0.32 nM), 13
(Ki(5-HT4)=0.11 nM), 14 (Ki(5-HT4)=0.29 nM) and 15 (Ki(5-HT4)=0.54 nM) were pharmacologically characterized as selective 5-
HT4 antagonists in the isolated guinea pig ileum (pA2=7.6, 7.9, 8.2 and 7.9, respectively), with a potency comparable to the 5-HT4
receptor antagonist RS 39604 (pA2=8.2). The benzimidazole-4-carboxylic acid derivatives described in this paper represent a novel
class of potent and selective 5-HT4 receptor antagonists. In particular, compounds 12±15 could be interesting pharmacological tools
for the understanding of the role of 5-HT4 receptors. # 1999 Elsevier Science Ltd. All rights reserved.
Introduction
the central nervous system (CNS).17 The presence of 5-
HT4 receptors which are supposed to facilitate release of
acetylcholine has been shown in the CNS, and thus 5-
HT4 receptor agonists may have a role in improving
cognitive function.18 To date, considerable progress has
been made in the localization of 5-HT4 receptors and
they have also been found in several animal and human
peripheral tissues, such as the gastrointestinal tract,19±21
the heart,22 and the bladder,23 where they mediate a
variety of pharmacological responses. Thus, in addition
to the clinical use of 5-HT4 receptor agonistsÐcisapride
and renzaprideÐas prokinetic agents in the treatment
of gastrointestinal motility disorders,24 a number of
potential therapeutic indications for antagonists of this
receptor are currently under investigation, including
IBS (irritable bowel syndrome),25 arrhythmias26 and
micturition disturbances.27,28 In many cases, the clinical
Since the discovery of the neurotransmitter serotonin
(5-hydroxytryptamine, 5-HT) in 1948 the knowledge
about its role in (patho)physiological processes, both
peripherally and centrally, is steadily growing.1±7 Seven
5-HT receptor classes (5-HT1±7) including 15 di€erent
subtypes have been identi®ed in recent years.8±12 There
is considerable interest in the 5-HT4 receptor13,14 which
has been recently cloned,15,16 showing that it is a mem-
ber of the G-protein-coupled receptor family and con-
®rming that it is linked positively to adenylyl cyclase in
Key words: Serotonin; 5-HT4 antagonist; benzimidazole; pharmaco-
phore.
* Corresponding author. Tel.: +34-91-394-4239; fax: +34-91-394-
4103; e-mail: mluzlr@eucmax.sim.ucm.es
0968-0896/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(99)00172-8

Â
Â
M. L. Lopez-Rodrõguez et al. / Bioorg. Med. Chem. 7 (1999) 2271±2281
2272
use of currently available drugs acting at the 5-HT4
receptor has been hampered by their lack of selectivity,
and signi®cant e€ort has been made towards the devel-
opment of potent and speci®c 5-HT4 receptor antago-
nists,29 such as SDZ 205-557, GR 113808, SB 204070,
and RS 39604.
Figure 1. Proposed pharmacophore model for 5-HT4 receptor
antagonists.
In the course of a program aimed at the discovery of
new 5-HT3 and 5-HT4 receptor agents, we have recently
reported a comparative receptor mapping of both ser-
otonergic binding sites.30 This computer-aided con-
formational analysis has allowed us to propose a steric
model for 5-HT4 receptor recognition, which o€ers
structural insights to aid the design of novel selective 5-
HT4 antagonists. This model consists of an aromatic
moiety, a coplanar carbonyl group with the oxygen
situated at ca. 3.6 A from the centroid of the aromatic
ring, a nitrogen atom situated at ca. 8.0 A from this
centroid and ca. 5.4 A from the oxygen of the carbonyl
group, and a voluminous substituent in the basic amino
framework of the molecule (Fig. 1). On the basis of
these results, in this paper we have designed a new series
of benzimidazole-4-carboxylic acid derivatives and ana-
lyzed the in¯uence of some structural variations of the
di€erent pharmacophore elements on the anity for the
5-HT4 receptor. Herein, we describe the synthesis of 1±
26, the anity for 5-HT4 receptors and the selectivity
over other serotonergic receptors (5-HT3, 5-HT2A, and
5-HT1A), obtained by radioligand binding assays. Four
analogues with interesting 5-HT4 binding properties
were also evaluated for 5-HT4 antagonist activity, by
analyzing their ability to inhibit the contractions
induced by 5-HT in the isolated guinea pig ileum.
Scheme 1. Reagents and conditions: (a) CDI, DMF, 40ꢀC, 1 h; (b) Y-
NH2 or Y-OH, DBU, DMF, 50ꢀC, 20±24 h.
alcohols in the presence of 1,8-diazabicyclo[5.4.0]undec-
7-ene (DBU) in N,N-dimethylformamide (DMF) solu-
tions. Acid 28 was obtained by condensation of 2-
amino-5-chloro-3-methylaniline (29) with formic acid
and subsequent oxidation of 6-chloro-4-methylbenzimi-
dazole (30) with potassium permanganate (Scheme 2).
The (1-alkyl-4-piperidyl)methylamines 31±34 were pre-
pared from 4-piperidylmethylamine, by reaction with
the appropriate halide (1-bromobutane, 1-bromo-2-
methylpropane, 1-chloropentane or N-(2-chloro-
ethyl)methanesulfonamide) in dry acetonitrile (Scheme
3). In all cases, small amounts of the dialkylated com-
pound and starting material were observed in the reac-
tion crudes. The rest of the amines and alcohols were
commercially available or prepared by previously
described methods.
Chemistry
Target amides and esters 1±26 were synthesized as
described in Scheme 1, by activation of benzimidazole-
4-carboxylic acid (27) or 6-chlorobenzimidazole-4-car-
boxylic acid (28) with 1,10-carbonyldiimidazole (CDI),
and subsequent coupling of the imidazolides with the
appropriate dialkylaminoalkyl or piperidine amines and
All new compounds (Table 1) were characterized by IR
1
and H and 13C NMR spectroscopy, and gave satisfac-
tory combustion analyses (C, H, N). COSY and HET-
COR experiments of several compounds were carried

Â
M. L. Lopez-Rodrõguez et al. / Bioorg. Med. Chem. 7 (1999) 2271±2281
Â
2273
Table 1. Binding anity of amides 1±16 and esters 17±26 at 5-HT3
and 5-HT4 receptorsa
Scheme 2. Reagents and conditions: (a) HCOOH, H2O, 100ꢀC, 6 h;
(b) KMnO4, NaOH 0.5 N, 100ꢀC, 5 h.
Compd
R
X
Y
Ki‹SEM (nM)
5-HT4
[3H]GR
113808
5-HT3
[3H]LY
278584
1
H NH
H NH
H NH
H NH
H NH
H NH
H NH
H NH
H NH
H NH
Cl NH
Cl NH
Cl NH
Cl NH
Cl NH
Cl NH
719‹58
>1000
>1000
290‹54
145‹10
<1000
2
3
>10,000
198‹16
Scheme 3. Reagents and conditions: (a) RX (BuBr, i-BuBr,
CH3(CH2)4Cl, Cl(CH2)2NHSO2Me), CH3CN, 0ꢀC to rt, overnight.
4
out in order to assign all the protons and carbons of
these new structures.
5
13.7‹0.9 >10,000
6
11.4‹3.0
>1000
>1000
>1000
7
Pharmacology
8
>1000
>1000
Final compounds were assessed for in vitro anity at 5-
HT4 receptors by radioligand binding assays, using
[3H]GR 113808 in rat striatum membranes31 (Table 1).
As several 5-HT4 receptor agonists and antagonists
have also been described as potent 5-HT3 receptor
antagonists, it was mandatory to evaluate the selectivity
of all active compounds over this receptor, using radio-
ligand binding assays with [3H]LY 278584 in rat cere-
bral cortex membranes32 (Table 1). Six highly active 5-
HT4 ligands were also evaluated for in vitro anity at
other serotonergic receptors by using the following spe-
9
157‹28
499‹95
290‹54
>10,000
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
54.0‹2.8 29.5‹4.4
0.32‹0.07 >1000
0.11‹0.03 >1000
0.29‹0.04 >1000
0.54‹0.10 >1000
ci®c radioligands and tissue sources: 5-HT2A
[3H]ketanserin, rat frontal cerebral cortex,33 5-HT1A
,
,
[3H]-8-OH-DPAT, rat cerebral cortex.34 The inhibition
constant Ki was de®ned from the IC50 using the Cheng±
Pruso€ equation.35 Additionally, four active and selec-
tive analogues in the 5-HT4 binding pro®le have been
evaluated for 5-HT4 biological activity in the isolated
guinea pig ileum. It is well known that concentrations
lower than 10 6 M of 5-HT induce contractile responses
in the non-stimulated guinea pig ileum by activation of
5-HT4 receptors,19,36±40 and these contractions are
antagonized by selective 5-HT4 antagonists,41 whereas
higher concentrations activate 5-HT3 receptors.19,42 In
order to test the antagonist activity of the new com-
pounds, non-cumulative concentration±response curves
9.1‹1.3
>10,000
24.6‹0.5
>1000
>1000
>1000
H
H
H
H
H
H
H
Cl
Cl
Cl
O
O
O
O
O
O
O
O
O
O
26.1‹0.3 >1000
>1000 >10,000
>10,000 >10,000
>1000
>1000
470‹35
>10,000
8
7
of 5-HT (10 to 3Â10 M) were constructed in the
6
2.9‹0.4 >10,000
2.3‹1.1 >1000
15.1‹0.3 >10,000
presence and absence of the tested compounds (10
and 10 M). Then, the ED50 of 5-HT in the presence
7
and absence of the antagonists was calculated as the
concentration of 5-HT required to produce 50% of
maximal contraction induced by 5-HT through 5-HT4
7
receptors. The e€ect of 3Â10 M 5-HT in control tis-
SDZ 205-557
RS 39604
10.7‹1.2
3.9‹0.2
155
>1000
sues was accepted as the maximal response (100%)
mediated by 5-HT4 receptors.19 The pA2 values were
determined following the equation described by Furch-
gott,43 using an appropriate concentration of the
antagonists (Table 2); the concentration-ratios of 5-HT,
a
Ki values are means‹SEM of two to four assays, performed in tri-
plicate. Inhibition curves were analyzed by a computer-assisted-curve-
®tting program (Prism GraphPad) and Ki values were determined
from the Cheng±Pruso€ equation.

Â
Â
M. L. Lopez-Rodrõguez et al. / Bioorg. Med. Chem. 7 (1999) 2271±2281
2274
Table 2. Pharmacological activity at 5-HT4 receptors in isolated
guinea pig ileum
Table 3. Energy and structural parameters of lowest energy con-
formers of analyzed compounds
5-HT ED50, Ma
pA2
Nc
Compd
Energy
(kcal mol
Ar±Oa
(A)
Ar±Nb
(A)
O±Nc
(A)
hd
(A)
b
Compd
1
)
R1
R2
4
5
9
10
97.88
135.34
150.11
128.04
3.74
3.74
3.75
3.74
7.82
8.48
7.49
8.34
4.38
5.62
4.48
5.20
0.07
3.55
1.42
2.56
Control
12
13
14
15
Ð
Ð
Ð
6
7
5
6
6
8
8
8
8
8
7
7
7
1.3Â10
2.5Â10
1.1Â10
1.7Â10
6.8Â10
2.4Â10
1.8Â10
1.4Â10
7.6
7.9
8.2
7.9
a
Distance between the centroid of the aromatic ring and the oxygen
of the carbonyl group.
8
7
RS 39604
1.5Â10
2.9Â10
8.2
6
b
Distance between the centroid of the aromatic ring and the basic
nitrogen of the amine.
a
Concentration of 5-HT required to produce a 50% of maximal
contraction induced by 5-HT to 5-HT4 receptors evaluated in the
absence (R1) and in the presence (R2) of tested compounds.
c
Distance between the oxygen of the carbonyl group and the basic
nitrogen of the amine.
d
Deviation of the basic nitrogen with respect to the aromatic ring.
b
7
Single point analysis using 10 M concentration of the antagonists
(pA2= log([B]/concentration ratio 1; [B]=concentration of the
antagonist).
c
Number of experiments.
binding sites. Furthermore, most of the compounds
displayed no signi®cant anity for the 5-HT3 receptor
(Ki>1000±10,000 nM) demonstrating their high 5-HT4/
5-HT3 selectivity. An examination of the binding data
presented in Table 1 shows the following.
in the presence and absence of the antagonists, re¯ect
the rightward displacement of the concentration-e€ect
measured at the point where the force of the contraction
induced by 5-HT was 50% of the maximal control
value. The 5-HT4 receptor antagonist RS 39604 was
used as reference compound. The e€ects of the antago-
nists were also tested on the contraction induced by
a. With respect to the aromatic moiety the introduction
of a chloro atom at the 6-position of the benzimidazole
ring induces a notable increase in potency at 5-HT4
receptors. Thus, 6-chloro substituted analogues are one
or two orders of magnitude more potent than their
directly related non-substituted derivatives (e.g. Ki
(6)=11.4 nM versus Ki (13)=0.11 nM; Ki (19)=26.1
nM versus Ki (25)=2.3 nM). The in¯uence of the 6-
chloro substitution in the benzimidazole moiety repre-
sents a structural di€erence between the 5-HT4 receptor
ligands of this class and the related conformationally
restricted 5-HT3 receptor ligands, where such a general
trend was not observed.46
6
6
10 and 3Â10 M 5-HT, in order to discard activity
on 5-HT3 receptors.
Molecular Modelling
A structural analysis of compounds 4, 5, 9, and 10 was
carried out with a Silicon Graphics INDIGO2 computer
and the multifaceted software package INSIGHT II,44
using the Builder, Discover and Analysis programs. The
molecules were built de novo in their protonated forms,
which is believed to be the bioactive form, using the
Builder module of INSIGHT II. Their geometry was
optimized by using the Optimize option of this module,
which combines three di€erent minimization algorithms
through the DISCOVER program (steepest descent,
conjugate gradients, and the BFGS algorithm). A sys-
tematic conformational search was then performed on
each compound, allowing rotatable bonds to rotate with
a 15ꢀ stepwise increment of the dihedral angles. Lowest
energy conformation(s) were identi®ed through the
analysis module of INSIGHT II, and the distances and
dihedral angles between the pharmacophoric elements
were measured (Table 3).
b. The anity for the 5-HT4 receptor is not much
a€ected by the nature of the acyl group; carboxamides
display slightly higher anity than their corresponding
carboxylates, though no signi®cant di€erence is
observed and both show the same order of magnitude
for their potency. The only exception was observed in
esters 17 and 22 which were inactive while their corre-
sponding amides 1 and 9 exhibited moderate anity.
c. In regard to the basic amino moiety of the molecule,
the presence of at least one methylene unit between the
acyl group and the 4-substituted piperidine ring is
necessary for selective binding at 5-HT4 sites over 5-
HT3 receptors. Thus, analogues 1±4, 11 and 17 (no
methylene unit) were either inactive or non-selective in
the 5-HT4 receptor. In the case of dialkylaminoalkyl
and 1-alkylpiperidine derivatives we can observe that
only 1-propylpiperidine chain leads to selective com-
pounds at 5-HT4 receptors (e.g. 16: Ki(5-HT4)=9.1 nM,
Ki(5-HT3) >1000 nM; 26: Ki(5-HT4)=15.1 nM, Ki(5-
HT3) >10,000 nM).
Results and Discussion
The results of the tested compounds are reported in
Tables 1 and 2. The values for 5-HT4 activation of the
compounds were compared to those of the reference
compounds: SDZ 205-55745 and RS 39604.38
The data from the binding assays for the synthesized
compounds presented in Table 1 highlighted their high
anity for 5-HT4 receptors since a number of them
exhibited nanomolar or subnanomolar anity for these
In order to understand the in¯uence of the methylene
chain length in 5-HT4 anity and selectivity, a struc-
tural analysis of compounds 4, 5, 9, and 10 was carried
out, and the structural parameters of lowest energy

Â
M. L. Lopez-Rodrõguez et al. / Bioorg. Med. Chem. 7 (1999) 2271±2281
Â
2275
conformers are given in Table 3. We can observe that
the distance between the aromatic ring and the basic
nitrogen atom (Ar±N) is the key structural parameter.
In non-selective analogues 4 (Ki(5-HT4)=290 nM, Ki(5-
HT3)=198 nM) and 9 (Ki(5-HT4)=157 nM, Ki(5-
HT3)=290 nM) the distance is 7.82 A and 7.49 A,
respectively, whereas in selective compounds 5 (Ki(5-
HT4)=13.7 nM, Ki(5-HT3)>10,000 nM) and 10 (Ki(5-
HT4)=499 nM, Ki(5-HT3)>10,000 nM) the nitrogen
atom is situated at a further distance from the aromatic
ring (8.48 and 8.34 A, respectively). These structural
data are in agreement with our recently proposed
hypothesis for 5-HT4 anity and 5-HT4/5-HT3 selec-
tivity,30 in which we postulated a distance of ca. 8.0 A
between the aromatic ring and the basic nitrogen atom
for the 5-HT4 antagonist pharmacophore and a distance
of ca. 7.5 A for the 5-HT3 antagonist model.
On the other hand, analogues with a substituent in the
nitrogen atom less voluminous than a butyl group are
devoid of anity at the 5-HT4 receptor, except com-
pound 11 (Ki=54.0 nM) probably due to the presence
of a chloro atom at the 6-position of the benzimidazole
ring, since non-substituted analogue 1 shows a clear
drop in binding anity (Ki=719 nM).
Figure 2. Each point shows the mean (the SEM are omitted for
clarity) of the percentage of inhibition of the contractile responses
induced by second administration of 5-HT versus the ®rst administra-
tion in the absence of drug (control), or in the presence of 0.1 mM RS
39604 as reference 5-HT4 antagonist, or in the presence of the same
concentrations (0.1 mM) of derivatives 12±15 (n=5).
These results con®rm that the presence of a voluminous
substituent in the basic nitrogen atom of the amino
moiety and the distance from this nitrogen to the aro-
matic ring are of great importance for high anity and
selectivity for 5-HT4 receptors, as we postulated in our
recently reported model for the 5-HT4 binding site.30
The crucial in¯uence of this distance has also been
recently reported by Langlois et al.47 for recognition of
esters of 4-amino-5-chloro-2-methoxybenzoic acid by 5-
HT4 receptors. When both requirements are veri®ed the
nature of the N-substituent does not seem to have
in¯uence on 5-HT4 potency, since compounds 12
(Ki=0.32 nM), 13 (Ki=0.11 nM), 14 (Ki=0.29 nM) and
15 (Ki=0.54 nM) were approximately equipotent. The
most potent 5-HT4 ligands of the series 12±15, 24, and
25 were devoid of anity at 5-HT1A and 5-HT2A sites (Ki
>1000±10,000 nM), con®rming their excellent selectivity
for the 5-HT4 receptor.
compounds (Table 2) suggesting an equipotent e€ect of
12±15 to the 5-HT4 receptor antagonist RS 39604.
A strict relationship was not observed between binding
anity (Ki, Table 1) and potency (pA2, Table 2) of the
new antagonists. This lack of close correlation between
binding anity and potency of drugs in functional in
vivo or in vitro tests is not unusual,40,42,47,48 and di€er-
ences are generally attributed to 5-HT receptor di€er-
ences in the tissues or tests used. In this study, rat
striatum membranes were used for the binding assays
and guinea pig ileum tissues were used to assess the
antagonist potency.
Conclusion
Derivatives 12±15 were evaluated for 5-HT4 receptor
activity in the guinea pig ileum. When administered
alone, they did not display agonist activity at the tested
The benzimidazole-4-carboxamides and -carboxylates
of piperidine derivatives described in this paper repre-
sent a novel class of potent and selective 5-HT4 receptor
antagonists in the CNS and in the guinea pig ileum.
Structure±activity relationships and structural analyses
have shown that the presence of a voluminous sub-
stituent in the basic nitrogen atom of the amino moiety
and a distance of ca. 8.0 A from the aromatic ring to
this nitrogen are both required for high anity and
selectivity for 5-HT4 receptors. These results con®rm
our recently proposed model for recognition by the 5-
HT4 binding site. Four compounds of the series (12±15)
displayed subnanomolar anity at central 5-HT4
receptors, high selectivity over other serotonin receptors
(5-HT3, 5-HT2A, and 5-HT1A) and potent 5-HT4
antagonist activity in the guinea pig ileum, compar-
able to the 5-HT4 receptor antagonist RS 39604. Con-
sequently, these novel 5-HT4 antagonists could be
6
7
doses (10 and 10 M). They produced a selective
inhibition of the 5-HT induced contractile response
mediated by 5-HT4 receptors without modi®cation of the
contractions mediated through 5-HT3 receptors: inter-
estingly, even when the contractions induced by low
doses of 5-HT (<10 6 M) were 100% blocked, the con-
6
tractions involving 5-HT3 receptors ([5-HT] >10 M)
were not modi®ed by none of the new tested compounds.
This di€erence in the e€ect of these compounds on con-
tractions involving 5-HT4 or 5-HT3 receptors con®rms,
doubtless, their selectivity for the 5-HT4 receptor. The
addition of derivatives 12±15 or RS 39604 induced a
parallel rightward shift of the 5-HT4-depending respon-
ses (Fig. 2), and consequently the 5-HT ED50 decreased.
The pA2 values were quite similar for the ®ve tested

Â
Â
M. L. Lopez-Rodrõguez et al. / Bioorg. Med. Chem. 7 (1999) 2271±2281
2276
interesting pharmacological tools for the understanding
of the role of 5-HT4 receptors.
2.54 (s, 3H, CH3), 7.07 (s, 1H, H5), 7.50 (s, 1H, H7),
8.29 (s, 1H, H2), 12.70 (br s, 1H, NH); 13C NMR
(Me2SO-d6) d 16.6 (CH3), 112.5 (C7), 122.0 (C5), 125.9,
126.8 (C4, C6), 136.8 (C3a, C7a), 142.9 (C2).
Experimental
Chemistry
To a solution of crude 30 (2.2 g, 13 mmol) in 0.5 N
aqueous solution of NaOH (130 mL) warmed in a steam
bath were added ®ve portions of 2.1 g (15 mmol) of
potassium permanganate, every hour. After the last
addition, the reaction mixture was re¯uxed for 2 more
hours (TLC), hot-®ltered and the solvent was evapo-
rated under reduced pressure. The solid was puri®ed by
column chromatography (CHCl3:MeOH, from 9:1 to
1:1) to yield 0.9 g (30%) of 28 which was recrystallized
Melting points (uncorrected) were determined on a
Gallenkamp electrothermal apparatus. Infrared (IR)
spectra were obtained on a Perkin±Elmer 781 infrared
spectrophotometer; the frequencies are expressed in
1
cm 1. H and 13C NMR spectra were recorded on a
Varian VXR-300S or Bruker 300-AM instrument at 300
and 75 MHz, respectively, or on a Bruker 250-AM
spectrometer at 250 and 62.5 MHz, respectively. Che-
mical shifts (d) 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), q (quartet), quint (quintet), sext
(sextet), m (multiplet), br (broad). Elemental analyses
(C, H, N) were determined at the UCM's analysis ser-
vices and were within ‹0.4% of the theoretical values.
Low resolution mass spectra were recorded by direct
injection on a HP 5989 instrument using the electronic
impact technique with an ionization energy of 70 eV.
Peaks are expressed in m/z, and molecular and base
peaks are indicated as (M) and (100), respectively.
Analytical thin-layer chromatography (TLC) was run
on Merck silica gel plates (Kieselgel 60 F-254) with
detection by UV light, iodine, acidic vanillin solution, or
10% phosphomolybdic acid solution in ethanol. For
¯ash chromatography, Merck silica gel type 60 (size
230±400 mesh) was used. Unless stated otherwise, all
starting materials and reagents were high-grade com-
mercial products purchased from Aldrich, Fluka or
Merck. All solvents were distilled prior to use. Dry
DMF was obtained by stirring with CaH2 followed by
distillation under argon.
from diluted hydrochloric acid; mp >300ꢀC. H NMR
1
(Me2SO-d6) d 7.94 (d, J=1.9, 1H, H7), 8.17 (d, J=1.9,
1H, H5), 9.46 (s, 1H, H2); 13C NMR (Me2SO-d6) d 118.6
(C4), 119.8 (C7), 126.7 (C5), 128.9, 129.0 (C3a, C6), 135.2
(C7a), 143.8 (C2), 164.5 (COOH).
General procedure for the synthesis of (1-alkyl-4-piper-
idyl)methylamines (31±34). To an ice-cold solution of 4-
piperidylmethylamine (5 g, 44 mmol) in dry CH3CN (45
mL) was added dropwise a solution of the appropriate
halide (1-bromobutane, 1-bromo-2-methylpropane, 1-
chloropentane, or N-(2-chloroethyl)methanesulfon-
amide) (35 mmol) in CH3CN (35 mL). The reaction
mixture was allowed to warm to room temperature and
stirred overnight. Then a 4% aq sol of NaOH was
added dropwise (200 mL), the mixture was extracted
with CHCl3 (3Â200 mL), and the combined organic
layers were washed with brine and dried over Na2SO4.
After evaporation of the solvent, the crude oil was pur-
i®ed by column chromatography to a€ord the corre-
sponding alkylated amine as a pure compound. In all
cases, small amounts of the dialkylated compound (5±
20%) and starting material (5±10%) were observed in
1
the H NMR spectra of the reaction crudes.
(1-Butyl-4-piperidyl)methylamine (31). Yield 3.8 g (51%);
chromatography CHCl3:MeOH:NH3, from 9:1:0.1 to
9:4:0.1; bp 45ꢀC (0.3 mm Hg); 1H NMR (CDCl3) d 0.82
(t, J=7.2, 3H, CH3), 1.08±1.27 (m, 5H, CH2CH3, H4,
H3ax, H5ax), 1.39 (quint, J=7.5, 2H, NCH2CH2), 1.62
The following intermediates were synthesized according
to literature procedures: benzimidazole- 4-carboxylic
acid,49 2-amino-5-chloro-3-methylaniline,50 4-amino-1-
methylpiperidine,51 4-amino-1-ethylpiperidine,52 4-amino-
1-propylpiperidine,52 4-amino-1-butylpiperidine,52 3-
piperidinopropylamine,53 1-methyl-4-piperidinol,54 (1-
butyl-4-piperidyl)methanol,55 N-[2-(4-hydroxymethyl-1-
piperidyl)ethyl]-methanesulfonamide,56 and 3-piperidino-
propanol.57
(d, J=9.6, 2H, H3eq, H5eq), 1.80 (t, J=11.7, 2H, H2ax
H6ax), 1.99 (br s, 2H, NH2), 2.18±2.23 (m, 2H, NCH2),
,
2.48 (d, J=6.0, 2H, CH2NH2), 2.86 (d, J=12.0, H2eq
,
H6eq); 13C NMR (CDCl3) d 14.1 (CH3), 21.0 (CH2CH3),
29.2 (NCH2CH2), 29.9 (C3, C5), 39.3 (C4), 48.0
(CH2NH2), 53.8 (C2, C6), 59.0 (NCH2).
Spectral data of all described compounds were con-
sistent with the proposed structures; for series 31±34
and 1±26, assignments are included for compounds 1,
11, 17, 24, and 31 as examples.
(1-iso-Butyl-4-piperidyl)methylamine (32). Yield 4.7 g
(63%); chromatography CHCl3:MeOH:NH3, from
9:1:0.1 to 8:2:0.1; mp 106±108ꢀC (MeOH/Et2O); 1H
NMR (CDCl3) d 0.88 (d, J=6.6, 6H), 1.15±1.30 (m,
3H), 1.63±1.80 (m, 5H), 1.83 (tm, J=10.5, 2H), 2.05 (d,
J=7.3, 2H), 2.56 (dm, J=5.6, 2H), 2.87 (dm, J=11.6,
2H); 13C NMR (CDCl3) d 20.9, 25.3, 29.7, 39.2, 47.9,
53.9, 67.2.
6-Chlorobenzimidazole-4-carboxylic acid (28). A solu-
tion of 29 (3.3 g, 21 mmol) and formic acid (2.9 g, 60
mmol) in water (20 mL) was re¯uxed for 6 h (TLC). The
reaction mixture was cooled and a cold 1 N aqueous
solution of KOH was added. 6-Chloro-4-methylbenz-
imidazole 30 (3.4 g, 98%) was recrystallized from
chloroform; mp 172±173ꢀC. 1H NMR (Me2SO-d6) d
(1-Pentyl-4-piperidyl)methylamine (33). Yield 4.4 g (54%);
chromatography CHCl3:MeOH:NH3, from 9:1:0.1 to
8:2:0.1; mp 98±100ꢀC (MeOH:Et2O); 1H NMR (CDCl3)

Â
M. L. Lopez-Rodrõguez et al. / Bioorg. Med. Chem. 7 (1999) 2271±2281
Â
2277
d 0.77 (t, J=6.7, 3H), 1.09±1.21 (m, 7H), 1.37 (m, 2H),
1.59 (dm, J=9.5, 2H), 1.76 (tm, J=11.1, 2H), 1.88 (br s,
2H), 2.16 (m, 2H), 2.45 (dm, J=4.8, 2H), 2.82 (dm,
J=11.9, 2H); 13C NMR (CDCl3) d 14.0, 22.6, 26.7,
29.9, 39.0, 47.8, 53.7, 59.2.
2.26 (t, J=7.8, 2H), 2.79 (d, J=10.2, 2H), 3.92±3.94 (m,
1H), 7.35 (t, J=8.1, 1H), 7.77 (d, J=8.1, 1H), 7.89 (d,
J=7.5, 1H), 8.49 (s, 1H), 9.97 (br s, 1H), 13.00 (br s,
1H); 13C NMR (Me2SO-d6) d 11.8, 19.6, 31.7, 45.9, 51.6,
59.8, 115.3, 121.9, 122.2, 133.5, 140.3, 142.8, 163.8.
Anal. (C16H22N4O) C, H, N.
N-[2-(4-Aminomethyl-1-piperidyl)ethyl]methanesulfon-
amide (34). Yield 5.1 g (49%); chromatography CHCl3:
MeOH:NH3, from 9:4:0.2 to 8:2:1; mp 111±113ꢀC
N-(1-Butyl-4-piperidyl)benzimidazole-4-carboxamide (4).
Yield 661 mg (44%); chromatography from AcOEt to
ꢀ
1
1
(MeOH:Et2O); H NMR (CDCl3) d 1.20±1.26 (m, 3H),
AcOEt:EtOH, 9.5:0.5; mp 94±97 C (acetone:water); H
NMR (Me2SO-d6) d 0.88 (t, J=7.2, 3H), 1.28 (sext,
J=7.2, 2H), 1.41 (quint, J=7.2, 2H), 1.56 (q, J=9.3,
2H), 1.94 (d, J=10.2, 2H), 2.11 (t, J=10.5, 2H), 2.27 (t,
J=7.2, 2H), 2.77 (d, J=11.1, 2H), 3.91±3.93 (m, 1H),
7.35 (t, J=7.8, 1H), 7.77 (d, J=7.8, 1H), 7.88 (d, J=7.5,
1H), 8.48 (s, 1H), 9.81 (br s, 1H); 13C NMR (Me2SO-d6) d
13.9, 20.1, 28.7, 31.8, 46.0, 51.7, 57.6, 116.1, 122.0, 122.1,
134.8, 139.4, 142.8, 163.9. Anal. (C17H24N4O) C, H, N.
1.72 (dm, J=10.5, 2H), 2.00 (td, J=11.7, 2.2, 2H), 2.51
(t, J=6.1, 2H), 2.57 (d, J=6.3, 2H), 2.88 (dm, J=11.5,
2H), 2.92 (s, 3H), 3.19 (t, J=5.6, 2H); 13C NMR
(CDCl3) d 29.5, 38.4, 39.5, 39.6, 47.3, 53.0, 56.9.
General procedure for the synthesis of benzimidazole-4-
carboxamides and benzimidazole-4-carboxylates (1±26).
To a solution of acid 27 or 28 (5 mmol) in dry DMF (5
mL) under an argon atmosphere was added 1,10-carbo-
nyldiimidazole (CDI, 811 mg, 5 mmol). The mixture
was stirred at 40ꢀC for 1 h, then a solution of the
appropriate amine or alcohol (6±10 mmol) and 1,8-di-
azabicyclo[5.4.0]undec-7-ene (DBU, 761 mg, 5 mmol) in
DMF (6±10 mL) was added dropwise, and the reaction
mixture was stirred at 50ꢀC for 20±24 h. The solvent
was removed under reduced pressure, and the crude was
taken up in CHCl3 (50 mL) and washed with water (20
mL) and 20% aq K2CO3 (20 mL). The organic layer
was dried over Na2SO4 or MgSO4, and evaporated to
a€ord the crude product, which was puri®ed by column
chromatography and recrystallization from the appro-
priate solvents.
N-[(1-Butyl-4-piperidyl)methyl]benzimidazole-4-carbox-
amide (5). Yield 770 mg (49%); chromatography
AcOEt:EtOH, 9:1; mp 122±125ꢀC (AcOEt); H NMR
1
(Me2SO-d6) d 0.86 (t, J=7.2, 3H), 1.24±1.38 (m, 6H),
1.55 (m, 1H), 1.70 (d, J=12.0, 2H), 1.83 (t, J=10.8,
2H), 2.23 (t, J=7.2, 2H), 2.85 (d, J=10.5, 2H), 3.34 (br
t, 2H), 7.35 (t, J=7.5, 1H), 7.77 (d, J=7.8, 1H), 7.89 (d,
J=6.9, 1H), 8.48 (s, 1H), 9.92 (br s, 1H), 12.88 (br s,
1H); 13C NMR (Me2SO-d6) d 14.0, 20.3, 28.7, 29.9, 36.3,
44.5, 53.2, 57.9, 116.0, 122.2, 122.3, 134.8, 139.6, 143.0,
164.8. Anal. (C18H26N4O) C, H, N.
N-[[1-[2-[(Methylsulfonyl)amino]ethyl]-4-piperidyl]methyl]
benzimidazole - 4 - carboxamide (6). Yield 1.1 g (59%);
chromatography from CHCl3:MeOH, 9:1 to CHCl3:
N-(1-Methyl-4-piperidyl)benzimidazole-4-carboxamide (1).
Yield 413 mg (32%); mp 122±124ꢀC (water); H NMR
MeOH:NH3, 8:2:1; mp 67±69ꢀC; H NMR (CDCl3) d
1
1
0
0
0
1.39 (qm, J=9.1, 2H), 1.71±1.81 (m, 1H, H4 ), 1.83 (d,
(Me2SO-d6) d 1.51±1.61 (m, 2H, H3 ax, H5 ax), 1.91 (d,
0
J=10.2, 2H, H3 eq, H5 eq), 2.09 (t, J=10.2, 2H, H2 ax
0
0
,
J=12.1, 2H), 2.05 (td, J=11.9, 2.4, 2H), 2.53 (t, J=5.9,
2H), 2.89 (dm, J=11.9, 2H), 2.97 (s, 3H), 3.19 (t,
J=5.5, 2H), 3.50 (t, J=5.5, 2H), 7.38 (t, J=7.9, 1H),
7.68 (d, J=7.9, 1H), 8.06 (d, J=7.5, 1H), 8.16 (s, 1H),
10.01 (br s, 1H); 13C NMR (CDCl3) d 29.8, 36.0, 39.7,
39.8, 44.9, 53.0, 58.0, 116.0, 122.0, 122.5, 122.9, 134.3,
139.7, 141.3, 166.4. Anal. (C17H25N5O3S) C, H, N.
0
H6 ax), 2.17 (s, 3H, CH3), 2.69 (d, J=10.8, 2H, H2 eq
0
,
0
0
H6 eq), 3.85±3.88 (m, 1H, H4 ), 7.33 (t, J=8.1, 1H, H6),
7.75 (d, J=8.1, 1H, H7), 7.86 (d, J=7.2, 1H, H5), 8.46
(s, 1H, H2), 9.80 (br s, 1H, CONH); 13C NMR (Me2SO-
0
0
0
0
d6) d 31.9 (C3 , C5 ), 46.0 (C4 ), 46.2 (CH3), 53.9 (C2 ,
C60), 116.5 (C7), 122.0 (C4), 122.1, 122.2 (C5, C6), 135.2
(C7a), 139.5 (C3a), 143.3 (C2), 164.3 (CONH). Anal.
(C14H18N4O) C, H, N.
N-[2-(N,N-Dimethylamino)ethyl]benzimidazole-4-carbox-
amide (7). Yield 464 mg (40%); chromatography
CH2Cl2:MeOH, from 9:1 to 7:3; mp 79±81ꢀC (CHCl3:
Et2O); 1H NMR (Me2SO-d6) d 2.24 (s, 6H), 2.51 (t,
J=6.6, 2H), 3.55 (q, J=6.0, 2H), 7.36 (t, J=7.5, 1H),
7.78 (d, J=8.1, 1H), 7.90 (d, J=7.5, 1H), 8.47 (s, 1H),
9.77 (br s, 1H); 13C NMR (Me2SO-d6) d 36.9, 45.1, 58.3,
116.2, 121.9, 122.1, 134.9, 139.1, 142.8, 164.7. Anal.
(C12H16N4O) C, H, N.
N-(1-Ethyl-4-piperidyl)benzimidazole-4-carboxamide (2).
Yield 537 mg (37%); chromatography AcOEt:EtOH,
from 9:1 to 4:1; mp 126±128ꢀC (water); 1H NMR
(Me2SO-d6) d 1.01 (t, J=7.2, 3H), 1.57 (qd, J=12.3,
3.3, 2H), 1.94±1.98 (m, 2H), 2.11 (t, J=10.2, 2H), 2.34
(q, J=7.2, 2H), 2.79 (d, J=11.4, 2H), 3.91±3.94 (m,
2H), 7.35 (t, J=7.8, 1H), 7.77 (dd, J=8.1, 1.2, 1H), 7.89
(dd, J=7.5, 1.2, 1H), 8.49 (s, 1H), 9.80 (br s, 1H); 13C
NMR (Me2SO-d6) d 12.2, 31.8, 46.0, 51.2, 51.6, 116.0,
121.9, 122.1, 134.1, 139.1, 142.8, 163.9. Anal. (C15H20
N-[2-(N,N-Diethylamino)ethyl]benzimidazole-4-carbox-
amide (8). Yield 625 mg (48%); chromatography
CH2Cl2:MeOH, from 9:1 to 4:1; 1H NMR (Me2SO-d6) d
1.00 (t, J=7.2, 6H), 2.57 (q, J=7.2, 4H), 2.65 (t, J=6.3,
2H), 3.50 (q, J=6.0, 2H), 7.34 (t, J=8.1, 1H), 7.76 (d,
J=8.1, 1H), 7.92 (d, J=7.5, 1H), 8.45 (s, 1H), 9.83 (br
s, 1H); 13C NMR (Me2SO-d6) d 11.7, 37.2, 46.6, 51.5,
115.8, 121.9, 122.0, 134.7, 138.9, 142.7, 164.7. Anal.
(C14H20N4O) C, H, N.
.
N4O H2O) C, H, N.
N-(1-Propyl-4-piperidyl)benzimidazole-4-carboxamide (3).
Yield 573 mg (40%); chromatography CHCl3:MeOH,
9:1; mp 119±121ꢀC (acetone:Et2O); H NMR (Me2SO-
1
d6) d 0.86 (t, J=7.5, 3H), 1.44 (sext, J=7.5, 2H), 1.58
(q, J=9.9, 2H), 1.95 (d, J=10.5, 2H), 2.14 (br t, 2H),

Â
Â
M. L. Lopez-Rodrõguez et al. / Bioorg. Med. Chem. 7 (1999) 2271±2281
2278
N-(2-Piperidinoethyl)benzimidazole-4-carboxamide (9).
Yield 408 mg (30%); chromatography AcOEt:EtOH,
9:1; 1H NMR (Me2SO-d6) d 1.38±1.41 (m, 2H), 1.53
(quint, J=5.4, 4H), 2.42 (br t, 4H), 2.50 (t, J=6.9, 2H),
3.53 (q, J=6.9, 2H), 7.34 (t, J=7.8, 1H), 7.75 (d,
J=7.8, 1H), 7.87 (d, J=7.8, 1H), 8.44 (s, 1H), 9.91 (br
s, 1H), 12.90 (br s, 1H); 13C NMR (Me2SO-d6) d 24.2,
25.7, 36.7, 54.1, 57.7, 116.2, 122.1, 122.2, 135.0, 139.1,
142.9, 164.8. Anal. (C15H20N4O) C, H, N.
1H NMR (CDCl3) d 0.86 (d, J=6.3, 6H), 1.42±1.58 (m,
2H), 1.73±1.87 (m, 5H), 1.98 (tm, J=13.5, 2H), 2.14 (d,
J=8.9, 2H), 2.99 (dm, J=14.4, 2H), 3.52 (t, J=7.4,
2H), 7.59 (s, 1H), 8.02 (s, 2H), 9.93 (br s, 1H); 13C
NMR (CDCl3) d 21.1, 25.4, 29.5, 36.2, 44.9, 53.8, 67.1,
115.1, 123.5, 128.7, 133.5, 139.1, 141.9, 165.1. Anal. (C18
H25ClN4O) C, H, N.
N-[(1-Pentyl-4-piperidyl)methyl]-6-chlorobenzimidazole-4-
carboxamide (15). Yield 835 mg (46%); chromato-
graphy CHCl3:MeOH, from 8:1 to 4:1; mp 176±178ꢀC
(toluene); 1H NMR (CDCl3) d 0.86 (t, J=6.3, 3H),
1.24±1.31 (m, 4H), 1.50±1.68 (m, 4H), 1.77±1.89 (m,
1H), 1.87 (d, J=12.7, 2H), 2.16 (t, J=11.9, 2H), 2.45±
2.50 (m, 2H), 3.15 (d, J=11.5, 2H), 3.57 (t, J=5.2, 2H),
7.56 (s, 1H), 8.00 (s, 2H), 9.86 (br s, 1H); 13C NMR
(CDCl3) d 13.9, 22.4, 25.8, 29.1, 29.7, 35.8, 44.4, 53.3,
58.8, 114.7, 123.6, 128.7, 134.3, 138.8, 141.9, 165.0.
Anal. (C19H27 ClN4O) C, H, N.
N-(3-Piperidinopropyl)benzimidazole-4-carboxamide (10).
Yield 601 mg (42%); chromatography AcOEt:EtOH,
ꢀ
1
9:1; mp 161±164 C (acetone); H NMR (Me2SO-d6) d
1.35±1.37 (m, 2H), 1.46±1.48 (m, 4H), 1.73 (quint,
J=6.6, 2H), 2.31±2.36 (m, 6H), 3.45 (q, J=6.3, 2H),
7.35 (t, J=7.8, 1H), 7.77 (d, J=7.8, 1H), 7.88 (d,
J=7.5, 1H), 8.45 (s, 1H), 9.74 (br s, 1H); 13C NMR
(Me2SO-d6) d 24.3, 25.7, 26.7, 37.4, 54.2, 56.3, 116.4,
122.0, 122.1, 135.1, 139.2, 142.9, 164.8. Anal. (C16H22
N4O) C, H, N.
N-(3-Piperidinopropyl)-6-chlorobenzimidazole-4-carbox-
amide (16). Yield 850 mg (53%); chromatography
CHCl3:MeOH, from 1:1 to 1:4; mp 208±210ꢀC
N-(1-Methyl-4-piperidyl)-6-chlorobenzimidazole-4-carbox-
amide (11). Yield 483 mg (33%); chromatography
CHCl3:MeOH, 9:1; mp 238±239ꢀC (acetone:AcOEt); 1H
1
(MeOH); H NMR (DMSO-d6) d 1.42±1.48 (m, 2H),
0
0
NMR (Me2SO-d6) d 1.57 (q, J=9.9, 2H, H3 ax, H5 ax),
1.54±1.80 (m, 4H), 1.85 (quint, J=6.9, 2H), 2.46±2.51
(m, 6H), 7.86 (d, J=1.9, 1H), 7.90 (d, J=2.2, 1H), 8.51
(s, 1H), 9.62 (br s, 1H); 13C NMR (DMSO-d6/
CF3CO2H) d 23.1, 24.7, 25.9, 37.9, 56.2, 116.4, 121.7,
122.5, 126.5, 137.5, 138.1, 144.3, 163.5; MS 320 (M),
230, 205, 183, 152, 108, 98 (100), 60. Anal. (C16H21
ClN4O) C, H, N.
0
1.92 (d, J=10.2, 2H, H3 eq, H5 eq), 2.09 (t, J=11.7, 2H,
0
0
H2 ax, H6 ax), 2.18 (s, 3H, CH3), 2.71 (d, J=11.1, 2H,
0
0
H2 eq, H6 eq), 3.86±3.88 (m, 1H, H4 ), 7.82 (d, J=1.5,
0
0
1H, H5), 7.84 (d, J=1.8, 1H, H7), 8.50 (s, 1H, H2), 9.58
13
0
(br s, 1H, CONH); C NMR (Me2SO-d6) d 31.5 (C3 ,
0
0
0
0
C5 ), 45.7 (C4 ), 45.9 (CH3), 53.7 (C2 , C6 ), 116.2 (C7),
121.8 (C5), 122.6, 126.5 (C4, C6), 136.2, 137.5 (C3a, C7a),
144.3 (C2), 162.7 (CONH). Anal. (C14H17ClN4O) C, H,
N.
1-Methyl-4-piperidyl benzimidazole-4-carboxylate (17).
Yield 583 mg (45%); chromatography CHCl3:MeOH,
9:1; mp 163±165ꢀC (water); 1H NMR (Me2SO-d6) d
0 0
1.86±1.96 (m, 4H, 2H3 , 2H5 ), 2.21±2.25 (m, 5H, CH3,
N-[(1-Butyl-4-piperidyl)methyl]-6-chlorobenzimidazole-4-
carboxamide (12). Yield 752 mg (43%); chromato-
graphy CHCl3:MeOH, 1:9; mp 184±186ꢀC (toluene); 1H
NMR (CDCl3) d 0.87 (t, J=7.1, 3H), 1.29 (sext, J=7.5,
2H), 1.49±1.59 (m, 3H), 1.64 (qm, J=12.7, 2H), 1.78±
1.89 (m, 1H), 1.87 (dm, J=12.3, 2H), 2.18 (t, J=11.5,
2H), 2.48±2.53 (m, 2H), 3.16 (dm, J=11.9, 2H), 3.54 (t,
J=5.2, 2H), 7.56 (s, 1H), 7.98 (s, 1H), 8.00 (s, 1H), 9.85
(br s, 1H); 13C NMR (CDCl3) d 13.8, 20.7, 28.0, 28.9,
35.7, 44.4, 53.2, 115.0, 123.1, 123.5, 128.6, 134.4, 138.9,
142.0, 165.1; MS 348 (M), 305 (100) 262, 179, 151, 138,
110, 55. Anal. (C18H25ClN4O) C, H, N.
0
H2 ax, H6 ax), 2.65±2.68 (m, 2H, H2 eq, H6 eq), 5.04 (tt,
0
0
0
0
J=8.1, 3.9, 1H, H4 ), 7.34 (t, J=7.5, 1H, H6), 7.88 (d,
J=7.5, 1H, H5/H7), 7.99 (d, J=8.1, 1H, H5/H7), 8.32 (s,
1H, H2), 12.48 (br s, 1H, NH); 13C NMR (Me2SO-d6) d
0
0
0
0
0
30.6 (C3 , C5 ), 46.0 (CH3), 52.6 (C2 , C6 ), 70.6 (C4 ),
114.2 (C4), 121.2 (C6), 124.4, 124.8 (C5, C7), 132.7 (C3a),
144.0 (C2), 144.4 (C7a), 164.8 (COO). Anal. (C14H17
N3O2) C, H, N.
(1-Butyl-4-piperidyl)methyl benzimidazole-4-carboxylate
(18). Yield 788 mg (50%); chromatography AcOEt:
EtOH, 9:1; mp 159±161ꢀC (acetone); 1H NMR (CDCl3)
d 0.87 (t, J=7.2, 3H), 1.27 (sext, J=7.4, 2H), 1.39±1.50
(m, 4H), 1.75±1.80 (m, 3H), 1.90 (t, J=11.6, 2H), 2.26-
2.32 (m, 2H), 2.95 (d, J=11.4, 2H), 4.23 (d, J=5.9, 2H),
7.29 (t, J=7.8, 1H), 7.92 (d, J=7.6, 1H), 8.00 (d,
J=8.0, 1H), 8.14 (s, 1H), 10.83 (br s, 1H); 13C NMR
(CDCl3) d 14.2, 21.0, 29.1, 29.3, 35.7, 53.5, 59.0, 69.6,
114.1, 121.8, 125.2, 125.5, 143.8, 166.6. Anal. (C18H25
N3O2) C, H, N.
N-[[1-[2-[(Methylsulfonyl)amino]ethyl]-4-piperidyl]methyl]-
6-chlorobenzimidazole-4-carboxamide (13). Yield 1.1 g
(52%); chromatography CHCl3:MeOH, from 9:1 to 3:1;
ꢀ
1
mp 85±87 C; H NMR (CDCl3) d 1.39 (qm, J=11.4,
2H), 1.71±1.82 (m, 1H), 1.80 (d, J=13.5, 2H), 2.03 (t,
J=11.5, 2H), 2.52 (t, J=5.5, 2H), 2.87 (d, J=11.1, 2H),
2.97 (s, 3H), 3.18 (t, J=5.5, 2H), 3.48 (t, J=5.9, 2H),
7.67 (s, 1H), 8.02 (s, 1H), 8.15 (s, 1H), 10.01 (br s, 1H);
13C NMR (CDCl3) d 29.9, 36.1, 39.7, 40.0, 45.1, 53.1,
56.7, 115.2, 123.7, 138.4, 141.9, 165.1. Anal. (C17H24
ClN5O3S) C, H, N.
[1 - [2 - [(Methylsulfonyl)amino]ethyl] - 4 - piperidyl]methyl
benzimidazole-4-carboxylate (19). Yield 942 mg (49%);
chromatography CHCl3:MeOH, from 10:1 to 4:1; mp
134±136ꢀC (toluene); 1H NMR (CDCl3) d 1.42 (qd,
J=11.9, 3.2, 2H), 1.68 (m, 1H), 1.84 (d, J=12.6, 2H),
2.08 (td, J=10.3, 2.4, 2H), 2.55 (t, J=5.9, 2H), 2.91
N-[(1-iso-Butyl-4-piperidyl)methyl]-6-chlorobenzimidazole-
4 - carboxamide (14). Yield 872 mg (50%); chromato-
graphy CHCl3:MeOH, 10:1; mp 184±186ꢀC (toluene);

Â
M. L. Lopez-Rodrõguez et al. / Bioorg. Med. Chem. 7 (1999) 2271±2281
Â
2279
(dm, J=11.5, 2H), 2.98 (s, 3H), 3.21 (t, J=5.5, 2H),
4.28 (d, J=5.9, 2H), 5.01 (br s, 1H), 7.36 (t, J=7.5, 1H),
7.96 (d, J=7.5, 1H), 8.06 (d, J=7.9, 1H), 8.17 (s, 1H),
10.50 (br s, 1H); 13C NMR (CDCl3) d 28.9, 35.4, 39.6,
40.0, 52.9, 56.7, 69.1, 113.7, 121.7, 124.9, 125.5, 133.3,
141.4, 143.6, 166.4. Anal. (C17H24N4O4S) C, H, N.
[1-[2-[(Methylsulfonyl)amino]ethyl]-4-piperidyl]methyl 6-
chlorobenzimidazole - 4-carboxylate (25). Yield 871 mg
(42%); chromatography CHCl3:MeOH, 10:1; mp 137±
139ꢀC (toluene); H NMR (CDCl3) d 1.40 (qd, J=11.5,
1
3.2, 2H), 1.73±1.92 (m, 3H), 2.09 (td, J=11.9, 2.0, 2H),
2.56 (t, J=5.9, 2H), 2.93 (dm, J=11.1, 2H), 2.99 (s,
3H), 3.22 (t, J=5.5, 2H), 4.28 (d, J=6.3, 2H), 5.03 (br s,
1H), 7.91 (d, J=2.0, 1H), 8.03 (d, J=2.0, 1H), 8.19 (s,
1H), 10.58 (br s, 1H); 13C NMR (CDCl3) d 29.0, 35.4,
39.9, 40.2, 53.4, 57.1, 69.7, 114.7, 125.0, 127.4, 132.2,
143.4, 144.6, 165.4. Anal. (C17H23ClN4O4S) C, H, N.
2-(N,N-Dimethylamino)ethyl benzimidazole-4-carboxylate
(20). Yield 595 mg (51%); mp 132.5±134.5ꢀC (CHCl3:
Et2O); 1H NMR (CDCl3) d 2.37 (s, 6H), 2.69 (t, J=5.5,
2H), 4.43 (t, J=5.5, 2H), 7.26 (t, J=7.8, 1H), 7.86 (dd,
J=7.6, 0.8, 1H), 7.96 (dd, J=8.0, 0.9, 1H), 8.02 (s, 1H),
12.30 (br s, 1H); 13C NMR (CDCl3) d 44.9, 57.5, 61.0,
115.0, 121.7, 125.0, 125.5, 133.9, 142.1, 144.0, 165.6.
Anal. (C12H15N3O2) C, H, N.
3-Piperidinopropyl 6-chlorobenzimidazole-4-carboxylate
(26). Yield 756 mg (47%); chromatography CHCl3:
MeOH, 9:1; mp 158±160ꢀC (acetone); 1H NMR
(CDCl3) d 1.13±1.40 (m, 4H), 1.51±1.59 (m, 4H), 1.97
(quint, J=6.3, 2H), 2.43 (m, 4H), 2.49 (t, J=6.7, 2H),
4.32 (t, J=5.9, 2H), 7.77 (s, 1H), 7.84 (s, 1H), 8.12 (s,
1H); 13C NMR (CDCl3) d 23.7, 24.9, 25.2, 54.3, 55.7,
64.1, 114.6, 124.4, 125.1, 127.1, 131.5, 143.6, 144.5,
164.8; MS 321 (M), 210, 179, 151, 124, 98 (100), 55.
Anal. (C16 H20ClN3O2) C, H, N.
2-(N,N-Diethylamino)ethyl benzimidazole-4-carboxylate
(21). Yield 747 mg (57%), chromatography CHCl3:
MeOH, 9:1; mp 139±141ꢀC (CHCl3:Et2O); 1H NMR
(CDCl3) d 1.06 (t, J=7.2, 6H), 2.67 (q, J=7.2, 4H), 2.81
(t, J=6.0, 2H), 4.43 (t, J=5.7, 2H), 7.26 (t, J=7.8, 1H),
7.87 (dd, J=7.8, 0.9, 1H), 7.96 (dd, J=8.1, 0.9, 1H),
8.01 (s, 1H), 12.34 (br s, 1H); 13C NMR (CDCl3) d 11.0,
46.0, 51.1, 60.6, 114.7, 121.5, 124.8, 125.2, 133.6, 141.6,
143.8, 165.3. Anal. (C14H19N3O2) C, H, N.
Pharmacology
Radioligand binding assays. For all receptor binding
assays, male Sprague±Dawley rats (Rattus norvegicus
albinus), weighing 180±200 g, were killed by decapita-
tion and the brains rapidly removed and dissected. Tis-
sues were stored at 80ꢀC for subsequent use and
2-Piperidinoethyl benzimidazole-4-carboxylate (22). Yield
711 mg (52%); chromatography AcOEt:EtOH, 9.5:0.5;
ꢀ
1
mp 148±150 C (AcOEt); H NMR (CDCl3) d 1.50±1.52
(m, 2H), 1.63 (quint, J=5.1, 4H), 2.51 (m, 4H), 2.68 (t,
J=5.7, 2H), 4.43 (t, J=5.7, 2H), 7.26 (td, J=8.1, 1.2,
1H), 7.86 (d, J=7.5, 1H), 7.96 (d, J=8.1, 1H), 8.05 (s,
1H), 12.40 (br s, 1H); 13C NMR (CDCl3) d 24.2, 26.2,
54.1, 57.1, 59.9, 115.1, 121.8, 125.1, 125.6, 133.8, 141.9,
144.2, 165.4. Anal. (C15H19N3O2) C, H, N.
homogenized on
a Polytron PT-10 homogenizer.
Membrane suspensions were centrifuged on a Beckman
J2-HS instrument.
5-HT1A receptor. Binding assays were performed by a
modi®cation of the procedure previously described by
Clark et al.34 The cerebral cortex was homogenized in
10 volumes of ice-cold 50 mM Tris±HCl bu€er (pH 7.7
at 25ꢀC) and centrifuged at 28,000 g for 15 min. The
membrane pellet was washed twice by resuspension and
centrifugation. After the third wash the resuspended
pellet was incubated at 37ꢀC for 10 min. Membranes
were then collected by centrifugation and the ®nal pellet
was resuspended in 50 mM Tris±HCl, 5 mM MgSO4, and
0.5 mM EDTA bu€er (pH 7.4 at 25ꢀC). Fractions of the
®nal membrane suspension (about 1 mg of protein) were
incubated at 37ꢀC for 15 min with 0.6 nM [3H]-8-OH-
DPAT (8-hydroxy-2-(dipropylamino)tetralin) (133 Ci/
mmol), in the presence or absence of the competing
drug (1 mM), in a ®nal volume of 1.1 mL of assay bu€er
(50 mM Tris±HCl, 10 nM clonidine, and 30 nM prazo-
sin, pH 7.4 at 25ꢀC). Nonspeci®c binding was deter-
mined with 10 mM 5-HT and represented less than 15%
of the total binding.
3-Piperidinopropyl benzimidazole-4-carboxylate (23). Yield
661 mg (46%); chromatography AcOEt:EtOH, 9:1; mp
ꢀ
1
153±155 C (acetone:Et2O); H NMR (CDCl3) d 1.43±
1.49 (m, 2H), 1.59 (quint, J=5.7, 4H), 1.99 (quint,
J=6.6, 2H), 2.41 (br t, 4H), 2.49 (t, J=6.6, 2H), 4.42 (t,
J=6.3, 2H), 7.31 (t, J=7.8, 1H), 7.94 (d, J=7.5, 1H),
8.01 (d, J=7.8, 1H), 8.15 (s, 1H), 11.30 (br s, 1H); 13C
NMR (CDCl3) d 24.1, 25.4, 25.7, 54.6, 56.4, 64.2, 113.9,
121.5, 125.1, 125.2, 133.0, 141.7, 143.7, 166.1. Anal.
(C16H21N3O2) C, H, N.
(1-Butyl-4-piperidyl)methyl 6-chlorobenzimidazole-4-car-
boxylate (24). Yield 1.0 g (60%); chromatography
CHCl3:MeOH, 8:1; mp 165±167ꢀC (acetone); 1H
NMR (CDCl3) d 0.90 (t, J=7.5, 3H, CH3), 1.30 (sext,
J=7.2, 2H, CH2CH3), 1.45±1.59 (m, 4H, NCH2CH2,
0
H3 ax, H5 ax), 1.79±1.88 (m, 1H, H4 ), 1.81 (d, J=11.9,
0
0
0
2H, H3 eq, H5 eq), 1.99 (t, J=11.5, 2H, H2 ax, H6 ax),
0
0
0
0
2.34±2.39 (m, 2H, NCH2), 3.03 (d, J=11.5, 2H, H2 eq
,
0
H6 eq), 4.26 (d, J=5.9, 2H, COOCH2), 7.89 (s, 1H, H5/
H7), 7.99 (s, 1H, H5/H7), 8.17 (s, 1H, H2), 10.80 (br s,
1H, NH); 13C NMR (CDCl3) d 14.0 (CH3), 20.8
5-HT2A receptor. Binding assays were performed
according to the procedure previously described by
Titeler et al.33 The frontal cortex was homogenized in 60
volumes of ice-cold 50 mM Tris±HCl, 0.5 mM
Na2EDTA, and 10 mM MgSO4 bu€er (pH 7.4 at 37ꢀC),
and centrifuged at 30,000 g for 15 min. The membrane
pellet was resuspended and incubated at 37ꢀC for 15
min. After centrifuging at 30,000 g for 15 min, the
0
0
0
(CH2CH3), 28.8 (C3 , C5 ), 29.0 (NCH2CH2), 35.4 (C4 ),
0
0
53.3 (C2 , C6 ), 58.7 (NCH2), 69.8 (COOCH2), 114.5
(C4), 125.0 (C5, C7), 127.4 (C6), 131.9 (C3a), 142.8 (C2),
144.5 (C7a), 165.4 (COO). Anal. (C18H24ClN3O2) C,
H, N.

Â
Â
M. L. Lopez-Rodrõguez et al. / Bioorg. Med. Chem. 7 (1999) 2271±2281
2280
membranes were washed twice by resuspension and
centrifugation, and the ®nal pellet was resuspended in
assay bu€er (50 mM Tris±HCl, 0.5 mM Na2EDTA, 10
mM MgSO4, 0.1% ascorbic acid, and 10 mM pargyline,
pH 7.4 at 25ꢀC). Fractions of the ®nal membrane sus-
pension (about 0.5 mg of protein) were incubated at 37ꢀC
for 15 min with 0.4 nM [3H]ketanserin (77 Ci/mmol), in
the presence or absence of the competing drug (1 mM), in
a ®nal volume of 2 mL of assay bu€er. Nonspeci®c
binding was determined with 1 mM cinanserin and
represented less than 20% of the total binding.
test compounds, Ki values being calculated from the
Cheng and Pruso€ equation.35 The protein concentra-
tions of the rat cerebral cortex and the rat striatum were
determined by the method of Lowry,58 using bovine
serum albumin as the standard.
5-HT4 receptor activity in the isolated guinea pig ileum.
Tissues were obtained from adult guinea pigs (300±400
g). According to Eglen et al.,19 portions of ileum were
dissected 1.5 cm distal to ileocecal junction and whole
segments of 2 cm long were gently ¯ushed with Krebs
solution and suspended, under 1 g tension, in an organ
bath containing 5 mL of Krebs solution at 32ꢀC and
gassed with 95% O2:5% CO2. Methysergide was added
5-HT3 receptor. Binding assays were performed accord-
ing to the procedure previously described by Wong et
al.32 The cerebral cortex was homogenized in 9 volumes
of 0.32 M sucrose and centrifuged at 1000 g for 10 min.
The supernatant was centrifuged at 17,000 g for 20 min.
The membrane pellet was washed twice by resuspension
in 60 volumes of 50 mM Tris±HCl bu€er (pH 7.4 at
25ꢀC) and centrifugation at 48,000 g for 10 min. After
the second wash the resuspended pellet was incubated at
37ꢀC for 10 min, and centrifuged at 48,000 g for 10 min.
Membranes were resuspended in 2.75 volumes of assay
bu€er (50 mM Tris±HCl, 10 mM pargyline, 0.6 mM
ascorbic acid, and 5 mM CaCl2, pH 7.4 at 25ꢀC). Frac-
tions of 100 mL of the ®nal membrane suspension (about
2 mg/mL of protein) were incubated at 25ꢀC for 30 min
with 0.7 nM [3H]LY 278584 (83 Ci/mmol), in the pre-
sence or absence of six concentrations of the competing
drug, in a ®nal volume of 2 mL of assay bu€er. Non-
speci®c binding was determined with 10 mM 5-HT and
represented less than 30% of the total binding.
6
at a concentration of 10 M, as described by Smith et
al.,59 in order to exclude any potential e€ects of 5-HT1
or 5-HT2 stimulation.
Ileal responses were measured by determining changes
in isometric tension recorded with force transducers
connected to an Omniscribe polygraph. In all experi-
ments tissues were exposed to 50 mM KCl (3 min) to
obtain an estimate of the maximal size of contraction of
the preparation. After 15 min of stabilization con-
centration±response curves to 5-HT were constructed in
8
6
a non-cumulative fashion (10 to 3 Â10 M) with an
agonist exposure period of 30 s on a 5 min dose cycle.
After the ®nal 5-HT exposure the bathing solution was
replaced and the ileum was incubated during a period of
60 min with an antagonist and, subsequently, the con-
centration±response curve was reconstructed. One dose
of one antagonist was tested in each ileal segment. Par-
allel control studies were undertaken to discard changes
in sensitivity of the tissues to 5-HT.
5-HT4 receptor. Binding assays were performed accord-
ing to the procedure previously described by Grossman
et al.31 The striatum was homogenized in 15 volumes of
ice-cold 50 mM HEPES bu€er (pH 7.4 at 4ꢀC) and
centrifuged at 48,000 g for 10 min. The pellet was
resuspended in 4.5 mL of assay bu€er (50 mM HEPES,
pH 7.4 at 4ꢀC). Fractions of 100 mL of the ®nal mem-
brane suspension were incubated at 37ꢀC for 30 min
with 0.1 nM [2H]GR 113808 (85 Ci/mmol), in the pre-
sence or absence of six concentrations of the competing
drug, in a ®nal volume of 1 mL of assay bu€er. Non-
speci®c binding was determined with 30 mM 5-HT and
represented less than 40% of the total binding.
Biphasic concentration±response curves were obtained
and ED50 for 5-HT and pA2 for tested antagonists were
6
calculated for the ®rst phase of the curve ([5-HT] <10
M) considering 100% the contraction induced by 3
Â10 7 M 5-HT in control curves.
Acknowledgements
This work has been supported by the DGICYT (PB97-
0282) and the Universidad Complutense (PR486/97-
7483). The authors are grateful to UNED for a pre-
doctoral grant to B.B. and to Universidad Complutense
for a predoctoral grant to M.M. Animals for 5-HT4
receptor activity assays were supported by Laboratorios
Esteve. M.M. thanks the Spanish Society of Thera-
peutic Chemistry for the award of the ``Ramon
Madronero'' prize.
For all binding assays, competing drug, nonspeci®c,
total and radioligand bindings were de®ned in triplicate.
Incubation was terminated by rapid vacuum ®ltration
through Whatman GF/B ®lters, presoaked in 0.05%
poly(ethylenimine), using a Brandel cell harvester. The
®lters were then washed (twice with 4 mL of ice-cold 50
mM Tris±HCl, pH 7.4 at 25ꢀC, for 5-HT1A, 5-HT2A
,
and 5-HT3 receptor binding assays, and once with 4 mL
of ice-cold 50 mM HEPES, pH 7.4 at 4ꢀC, for 5-HT4
receptor binding assays) and dried. The ®lters were
placed in poly(ethylene) vials to which were added 4 mL
of a scintillation cocktail (Aquasol), and the radio-
activity bound to the ®lters was measured by liquid
scintillation spectrometry. The data were analyzed by
an iterative curve-®tting procedure (program Prism,
Graph Pad), which provided IC50, Ki, and r2 values for
References
1. Peroutka, S. J., Ed. Serotonin Receptor Subtypes: Basic and
Clinical Aspects; John Wiley and Sons: New York, 1991.
2. Humphrey, P. P. A. In Serotonin Receptor Subtypes: Phar-
macological Signi®cance and Clinical Implications; Langer, S.
Z.; Brunello, N.; Racagni, G.; Mendlewicz, J., Eds.; Karger:
Basel, 1992, Vol. 1, pp 129±139.

Â
M. L. Lopez-Rodrõguez et al. / Bioorg. Med. Chem. 7 (1999) 2271±2281
Â
2281
3. Herndon, J. L.; Glennon, R. A. In Drug Design for Neu-
roscience; Kozikowski, A. P., Ed.; Raven: New York, 1993; pp
167±212.
4. Saudou, F.; Hen, R. Med. Chem. Res. 1994, 4, 16.
5. Boess, F. G.; Martin, I. L. Neuropharmacol. 1994, 33, 275.
6. Gerhardt, C. C.; van Heerikhuizen, H. Eur. J. Pharmacol.
1997, 334, 1.
33. Titeler, M.; Lyon, R. A.; Davis, K. H.; Glennon, R. A.
Biochem. Pharmacol. 1987, 36, 3265.
34. Clark, R. D.; Weinhardt, K. K.; Berger, J.; Fisher, L. E.;
Brown, C. M.; MacKinnon, A. C.; Kilpatrick, A. T.; Sped-
ding, M. J. Med. Chem. 1990, 33, 633.
35. Cheng, Y. C.; Pruso€, W. H. Biochem. Pharmacol. 1973,
22, 3099.
7. Baumgarten, H. G.; Gothert, M., Eds. Serotoninergic Neurons
and 5-HT Receptors in the CNS; Springer±Verlag: Berlin, 1997.
8. Hoyer, D.; Clarke, D. E.; Fozard, J. R.; Hartig, P. R.;
Martin, G. R.; Mylecharane, E. J.; Saxena, P. R.; Humphrey,
P. P. A. Pharmacol. Rev. 1994, 46, 157.
36. Buchheit, K.-H.; Engel, G.; Mutschler, E.; Richardson, B.
Naunyn-Schmiedeberg's Arch. Pharmacol. 1985, 329, 36.
37. Craig, D. A.; Clarke, D. E. J. Pharmacol. Exp. Ther. 1990,
252, 1378.
38. Hegde, S. S.; Bonhaus, D. W.; Johnson, L. G.; Leung, E.;
Clark, R. D.; Eglen, R. M. Br. J. Pharmacol. 1995, 115, 1087.
39. Gaster, L. M.; Joiner, G. F.; King, F. D.; Wyman, P. A.;
Sutton, J. M.; Bingham, S.; Ellis, E. S.; Sanger, G. J.; Wardle,
K. A. J. Med. Chem. 1995, 38, 4760.
40. Soulier, J.-L.; Yang, D.; Bremont, B.; Croci, T.; Guzzi, U.;
Langlois, M. J. Med. Chem. 1997, 40, 1755.
41. Buchheit, K.-H.; Gamse, R.; Pfannkuche, H.-J. Naunyn-
Schmiedeberg's Arch. Pharmacol. 1992, 345, 387.
42. Heidempergher, F.; Pillan, A.; Pinciroli, V.; Vaghi, F.;
Arrigoni, C.; Bolis, G.; Caccia, C.; Dho, L.; McArthur, R.;
Varasi, M. J. Med. Chem. 1997, 40, 3369.
43. Furchgott, R. F. In Handbook of Experimental Pharma-
cology; Springer: Berlin, Heidelberg, New York, 1972; Vol. 33,
pp 283±335.
9. Martin, G. R.; Humphrey, P. P. A. Neuropharmacol. 1994,
33, 261.
10. Lucas, J. J.; Hen, R. Trends Pharmacol. Sci. 1995, 16, 246.
11. Hoyer, D.; Martin, G. Neuropharmacol. 1997, 36, 419.
12. Uphouse, L. Neurosci. Behav. Rev. 1997, 21, 679.
13. Bockaert, J.; Fozard, J. R.; Dumuis, A.; Clarke, D. E.
Trends Pharmacol. Sci. 1992, 13, 141.
14. Ford, A. P. D. W.; Clarke, D. E. Med. Res. Rev. 1993, 13,
633.
15. Gerald, C.; Adham, N.; Kao, H.-T.; Olsen, M. A.; Laz, T.
M.; Schechter, L. E.; Bard, J. A.; Vaysse, P. J.-J.; Hartig, P.
R.; Branchek, T. A.; Weinshank, R. L. EMBO J. 1995, 14,
2806.
16. Blondel, O.; Vandecasteele, G.; Gastineau, M.; Leclerc, S.;
Dahmoune, Y.; Langlois, M.; Fichmeister, R. FEBS Lett.
1997, 412, 465.
44. INSIGHT II, v. 95.0, Biosym Technologies, San Diego,
CA, USA.
17. Fagni, L.; Dumuis, A.; Sebben, M.; Bockaert, J. Br. J.
Pharmacol. 1992, 105, 973.
18. Eglen, R. M.; Wong, E. H. F.; Dumuis, A.; Bockaert, J.
Trends Pharmacol. Sci. 1995, 16, 391.
19. Eglen, R. M.; Swank, S. R.; Walsh, L. K. M.; Whiting, R.
L. Br. J. Pharmacol. 1990, 101, 513.
20. McLean, P. G.; Coupar, I. M.; Molenaar, P. Br. J. Phar-
macol. 1995, 115, 47.
45. Eglen, R. M.; Alvarez, R.; Johnson, L. G.; Leung, E.;
Wong, E. H. F. Br. J. Pharmacol. 1993, 108, 376.
46. Lopez-Rodrõguez, M. L.; Morcillo, M. J.; Benhamu, B.;
Riaguas, M. D. Bioorg. Med. Chem. Lett. 1996, 6, 1195.
47. Yang, D.; Soulier, J.-L.; Sicsic, S.; Mathe-Allainmat, M.;
Bremont, B.; Croci, T.; Cardamone, R.; Aureggi, G.; Lan-
glois, M. J. Med. Chem. 1997, 40, 608.
48. Orjales, A.; Mosquera, R.; Labeaga, L.; Rodes, R. J. Med.
Chem. 1997, 40, 586.
49. Williams, A.; Salvadori, G. J. Chem. Soc., Perkin Trans. 2
1972, 7, 883.
21. Matsuyama, S.; Sakiyama, H.; Nei, K.; Tanaka, C. J.
Pharmacol. Exp. Ther. 1996, 276, 989.
22. Villalon, C. M.; den Boer, M. O.; Heiligers, J. P. C.; Sax-
ena, P. R. Br. J. Pharmacol. 1990, 100, 665.
23. Candura, S. M.; Messori, E.; Fraceschetti, G. P.; D'Agos-
tino, G.; Vicini, D.; Tagliani, M.; Tonini, M. Br. J. Pharmacol.
1996, 118, 1965.
50. Nyhammar, T.; Grivas, S. Acta Chem. Scand. B 1986, 40,
583.
51. Brookes, P.; Terry, R. J.; Walker, J. J. Chem. Soc. 1957,
3165.
24. Briejer, M. R.; Akkermans, L. M. A.; Schuurkes, J. A. J.
Pharmacol. Rev. 1995, 47, 631.
52. Harper, N. J.; Chignell, C. F. J. Med. Chem. 1964, 7, 729.
53. Ueda, T.; Ishizaki, K. Chem. Pharm. Bull. 1967, 15, 228.
54. Adlerova, E.; Seidlova, V.; Protiva, M. Cesk. Farm. 1963,
12, 122.
55. Gaster, L. M.; Jennings, A. J.; Joiner, G. F.; King, F. D.;
Mulholland, K. R.; Rahman, S. K.; Starr, S.; Wyman, P. A.;
Wardle, K. A.; Ellis, E. S.; Sanger, G. J. J. Med. Chem. 1993,
36, 4121±4123.
56. Oxford, A. W. International Patent Application WO 93/
20071.
57. Pandey, G.; Kumaraswamy, G.; Reddy, P. Y. Tetrahedron
1992, 48, 8295.
25. Hegde, S. S.; Moy, T. M.; Perry, M. R.; Loeb, M.; Eglen,
R. M. J. Pharmacol. Exp. Ther. 1994, 271, 741.
26. Kaumann, A. J. Trends Pharmacol. Sci. 1994, 15, 451.
27. Tonini, M.; Candura, S. M. Trends Pharmacol. Sci. 1996,
17, 314.
28. Ferguson, D.; Christopher, N. Trends Pharmacol. Sci.
1996, 17, 315.
29. Gaster, L. M.; King, F. D. Med. Res. Rev. 1997, 17, 163.
30. Lopez-Rodrõguez, M. L.; Morcillo, M. J.; Benhamu, B.;
Rosado, M. L. J. Comput.-Aided Mol. Des. 1997, 11, 589.
31. Grossman, C. J.; Kilpatrick, G. J.; Bunce, K. T. Br. J.
Pharmacol. 1993, 109, 618.
58. Lowry, O. H.; Rosebrough, N. J.; Farr, A. L.; Randall, R.
J. J. Biol. Chem. 1951, 193, 265.
32. Wong, D. T.; Robertson, D. W.; Reid, L. R. Eur. J.
Pharmacol. 1989, 166, 107.
59. Smith, W. L.; Sancilio, L. F.; Obwera-Atepo, J. B.; Nay-
lor, R. J.; Lambert, L. J. Pharm. Pharmacol. 1988, 40, 301.