Benzimidazole derivatives. Part 5: Design and synthesis of new benzimidazole-arylpiperazine derivatives acting as mixed 5-HT 1A/5-HT3 ligands

Article abstract of DOI:10.1016/j.bmc.2004.07.023

A series of new mixed benzimidazole-arylpiperazine derivatives were designed by incorporating in general structure III the pharmacophoric elements of 5-HT_1A and 5-HT₃ receptors. Compounds 1-11 were synthesized and evaluated for bindi

Full text of DOI:10.1016/j.bmc.2004.07.023

Bioorganic & Medicinal Chemistry 12 (2004) 5181–5191
Benzimidazole derivatives. Part 5: Design and synthesis of new
benzimidazole–arylpiperazine derivatives acting as mixed
5-HT1A/5-HT3 ligands
a,
Marıa L. Lopez-Rodrıguez, Bellinda Benhamu, M Jose Morcillo, Ignacio Tejada,
a
a
b
a
*
´
´
´
David Avila, Isabel Marco, Lucio Schiapparelli, Diana Frechilla and Joaquın Del Rıo
´
´
a
a
c
c
c
´
´
a
´
´
´
Departamento de Quımica Organica I, Facultad de Ciencias Quımicas, Universidad Complutense, E-28040 Madrid, Spain
b
´
´
´
Seccion de Quımica, Facultad de Ciencias, Universidad Nacional de Educacion a Distancia, E-28040 Madrid, Spain
c
´
Departamento de Farmacologıa, Facultad de Medicina, Universidad de Navarra, E-31008 Pamplona, Spain
Received 26 March 2004; accepted 9 July 2004
Available online 11 August 2004
´
Dedicated to Professor Jose L. Soto for a whole life devoted to Organic Chemistry
Abstract—A series of new mixed benzimidazole–arylpiperazine derivatives were designed by incorporating in general structure III
the pharmacophoric elements of 5-HT1A and 5-HT3 receptors. Compounds 1–11 were synthesized and evaluated for binding affinity
at both serotoninergic receptors, all of them exhibiting high 5-HT3R affinity (Ki = 10–62nM), and derivatives with an o-alkoxy
group in the arylpiperazine ring showing nanomolar affinity for the 5-HT1AR (Ki = 18–150nM). Additionally, all the synthesized
compounds were selective over a1-adrenergic and dopamine D2 receptors (Ki>1000–10,000nM). Compound 3 was selected for fur-
ther pharmacological characterization due to its interesting binding profile as mixed 5-HT1A/5-HT3 ligand with high affinity for both
receptors (5-HT1A: Ki = 18.0nM, 5-HT3: Ki = 27.2nM). In vitro and in vivo findings suggest that this compound acts as a partial
agonist at 5-HT1ARs and as a 5-HT3R antagonist. This novel mixed 5-HT1A/5-HT3 ligand was also effective in preventing the cog-
nitive deficits induced by muscarinic receptor blockade in a passive avoidance learning test, suggesting a potential interest in the
treatment of cognitive dysfunction.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
atric disorders8–11 such as anxiety, depression and mem-
ory loss. On the other hand, the 5-HT3 subtype is
present within the central and peripheral nervous sys-
tems, and antagonists of this receptor are of special
interest not only because of their wide clinical use as
antiemetic agents in cancer patients,12,13 but also due
to their promising therapeutic potential in the treatment
of CNS disorders14–16 such as anxiety, drug abuse and
withdrawal, and cognitive dysfunction. In light of the
potential utility in anxiety and cognitive disorders of
5-HT1A and 5-HT3 receptor ligands, it would be of inter-
est, in principle, the development of compounds with
affinity at both 5-HTR subtypes.
The discovery of ligands with affinity for the family of
serotonin receptors (5-HTRs) is an area of intense re-
search in medicinal chemistry because of the potential
to find new therapeutic drugs due to their involvement
in numerous physiological and pathophysiological proc-
esses.1–4 At present seven classes of serotonin receptors
(5-HT1–7
) including fourteen subtypes have been
found,5,6 and most of them belong to the superfamily
of G protein-coupled receptors (GPCRs); only the 5-
HT3R is a ligand-gated cation channel receptor.7
Among 5-HTRs, the 5-HT1A subtype is the best studied
and it is generally accepted that it is involved in psychi-
In the course of a wide program aimed at the discovery
of new serotoninergic agents, we have synthesized a ser-
ies of arylpiperazines17–21 I with high affinity for 5-
HT1ARs in which QSAR studies22,23 and computational
simulations23–25 have allowed us to determine the struc-
tural elements that are optimal for 5-HT1AR–ligand
Keywords: Serotonin ligands; 5-HT1A receptor; 5-HT3 receptor;
Benzimidazole derivatives; Arylpiperazines; Cognitive dysfunction.
*
Corresponding author. Tel.: +34-91-3944239; fax: +34-91-
3944103; e-mail: mluzlr@quim.ucm.es
0968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.07.023

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M. L. Lopez-Rodrıguez et al. / Bioorg. Med. Chem. 12 (2004) 5181–5191
5182
Scheme 2. Reagents and conditions: (a) HOCH2COOH, HCl; (b)
SOCl2, 80ꢁC; (c) HCl/H2O, D; (d) arylpiperazine, acetonitrile, NEt3,
60ꢁC.
Figure 1. Structure of compounds I–III.
acid with glycolic acid followed by treatment of 2-(hy-
droxymethyl)benzimidazole-4-carboxylic acid (23) with
SOCl2 and hydrolysis in acidic conditions (Scheme 2).
As for intermediate arylpiperazines, those with
Ar = phenyl, o-methoxyphenyl, m-(trifluoromethyl)phe-
nyl, and m-chlorophenyl were commercial, whereas
those with Ar = o-ethoxyphenyl, o-propoxyphenyl,
o-isopropoxyphenyl, o-butoxyphenyl, naphth-1-yl, 2,3-
dihydro-1,4-benzodioxan-5-yl, and 1-tritylbenzimida-
zol-4-yl were synthesized according to previously
described methods (see Experimental Section).
interaction, as well as a class of azabicyclic benzimid-
azole derivatives II characterized as potent and selective
5-HT3R antagonists26,27 (Fig. 1). In the present work we
have designed a series of new mixed benzimidazole–aryl-
piperazines of general structure III28 in which we have
incorporated the structural elements of 5-HT1A and 5-
HT3 pharmacophores29,30 (Fig. 1). Herein we report
the synthesis of designed compounds 1–11 and their
affinities for serotoninergic 5-HT1A and 5-HT3, adrener-
gic a1 and dopaminergic D2 receptors, obtained by radio-
ligand binding assays. Among them, analogue 3 was
selected for further pharmacological characterization:
functional activity at 5-HT1A and 5-HT3 receptors,
and behavioural effects in anxiety and cognitive dysfunc-
tion models.
1
All new compounds were characterized by IR and H
and 13C NMR spectroscopy and gave satisfactory com-
bustion analyses (C, H, N).
3. Biological results and discussion
3.1. Affinity data
2. Chemistry
The general procedure for the preparation of target
compounds III is shown in Scheme 1. Starting benzimid-
azolecarboxylic acids 12–22 were suitably activated with
1,10-carbonyldiimidazole (CDI), and subsequently
coupled with ( )-3-aminoquinuclidine in the presence
of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) in N,N-di-
methylformamide (DMF) solutions, to afford the
desired amides 1–11. 2-[(4-Arylpiperazin-1-yl)methyl]-
benzimidazole-4-carboxylic acids 12–22 were obtained
by reaction of 2-(chloromethyl)benzimidazole-4-carb-
oxylic acid (24) with the corresponding arylpiperazines
in the presence of NEt3 (Scheme 2). The intermediate
24 was prepared by condensation of 2,3-diaminobenzoic
Target compounds 1–11 were assessed for in vitro affin-
ity at serotoninergic 5-HT1A and 5-HT3 receptors by ra-
dioligand binding assays, using [3H]-8-OH-DPAT and
[3H]LY 278584, respectively, in rat cerebral cortex mem-
branes. The ligands were also evaluated for in vitro
affinity at a1-adrenergic receptors ([3H]prazosin) and
dopamine D2 receptors ([3H]raclopride), in rat cerebral
cortex and striatum membranes, respectively. In the
experimental binding assays the compounds were first
tested at fixed dose of 10ꢀ6 M, and for those that in
the screening process presented high affinity (displace-
ment of the radioligand P55%), the dose–response
curves were determined. The affinity constants obtained
for the tested compounds are listed in Table 1. All the
synthesized compounds 1–11 exhibited high 5-HT3R
affinity (Ki = 10ꢀ62nM), and derivatives with an o-alk-
oxy group in the arylpiperazine ring have shown nano-
molar affinity for the 5-HT1AR (Ki = 18ꢀ150nM).
Only analogue 11 is an exception in the series (5-
HT1A: Ki = 148nM, 5-HT3: Ki>1000nM). The weak
5-HT1AR affinity of derivative 9 was unexpected,
according to reported data for arylpiperazines bearing
a benzodioxan-5-yl group.25,31 Additionally, all the
Scheme 1. Reagents and conditions: (a) CDI, DMF, 40ꢁC; (b) ( )-3-
aminoquinuclidine, DBU, DMF, 50ꢁC.

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5183
Table 1. Binding data of compounds III (1–11)
20
10
0
(A)
-10
-9
-8
-7
-6
Log [M]
-10
Compd
Ar
Ki (nM)
5-HT3
5-HT1A
-20
40
1
Phenyl
>1000
23.1 1.5
10.3 1.1
27.2 0.9
24.6 3.2
62.1 1.3
15.1 4.8
23.9 2.9
18.3 0.4
24.0 1.0
32.5 5.3
>1000
2
o-Methoxyphenyl
o-Ethoxyphenyl
o-Propoxyphenyl
o-isoPropoxyphenyl
o-Butoxyphenyl
m-(Trifluoromethyl)phenyl
m-Chlorophenyl
Benzodioxan-5-yl
Naphth-1-yl
150 33
18.0 1.7
56.1 2.2
34.4 3.0
45.9 3.5
>10,000
>10,000
467 14
>1000
(B)
3
4
30
20
10
0
5
6
7
8
9
10
11
-10
-9
-8
-7
-6
Benzimidazol-4-yl
148
7
Log [M]
-10
-20
Values are means SEM from 2–4 separate experiments performed in
triplicate.
Figure 2. Stimulation of [35S]GTPcS binding (A) and inhibition of 8-
OH-DPAT-stimulated [35S]GTPcS binding (B) in rat hippocampal
compounds were selective over a1-adrenergic and dop-
amine D2 receptors (Ki> 1000ꢀ10,000nM). Compound
3 was selected for further pharmacological characteriza-
tion due to its interesting binding profile as mixed 5-
HT1A/5-HT3 ligand with high affinity for both receptors
(5-HT1A: Ki = 18.0nM, 5-HT3: Ki = 27.2nM).
membranes by compound 3. Values are the means
separate experiments performed in duplicate.
SEM from 5
ature in mice by approximately 1ꢁC at 60 and 120min
after administration, but only a slightly higher effect
was found when increasing the dose by 10-fold (Table
2). The hypothermic potency of 8-OH-DPAT was much
more pronounced, and the effect of this 5-HT1AR agon-
ist was not prevented by compound 3 (Table 3). Alto-
gether, in vitro and in vivo findings suggest that
compound 3 is a partial agonist at 5-HT1ARs.
3.2. Functional characterization of compound 3 at 5-HT1A
and 5-HT3 receptors
The ability of compound 3 to increase binding of
[35S]GTPcS to rat hippocampal membranes as well as
the antagonism to binding stimulated by the 5-HT1A
R
agonist 8-OH-DPAT were evaluated. The effect of
different concentrations of 3 on [35S]GTPcS binding to
hippocampal membranes is shown in Figure 2A.
Compound 3 (10ꢀ10–10ꢀ7 M) inducedminimal effects
on the basal binding of the GTP analogue. Only at a
high concentration (10ꢀ6 M) a moderate increase in
basal binding (19%) was observed, and no further
enhancement in basal binding was found after a 10-fold
higher concentration, 10ꢀ5 M (not shown). This was
in contrast to the marked stimulation observed with
the typical 5-HT1AR agonist 8-OH-DPAT (92% at
10ꢀ6 M). On the other hand, as shown in Figure 2B,
Table 2. Effect of compound 3 on rectal temperature in mice
Dose (mg/kg sc)
D Temperature (ꢁC)
30min
60min
120min
1
10
ꢀ0.09 0.1
ꢀ0.9 0.1
ꢀ0.7 0.2
ꢀ1.3 0.3
ꢀ1.1 0.2
ꢀ1.2 0.2
Values are means SEM from 8 animals.
compound
3
only inhibited the stimulation of
[35S]GTPc binding induced by 8-OH-DPAT at the low
concentrations of 10ꢀ10 and 10ꢀ9 M but this effect was
not concentration-dependent, and was much weaker
than that of the 5-HT1AR antagonist WAY-100635
(IC50 = 6nM).
Table 3. Effect of compound 3 on 8-OH-DPAT-induced hypothermia
in mice
Treatment
D Temperature (ꢁC)
60min 120min
30min
Since agonists at 5-HT1ARs consistently induce hypo-
thermia in rodents,32 the intrinsic effect of compound 3
on rectal temperature and on the hypothermia response
to the 5-HT1AR agonist 8-OH-DPAT was studied in
mice. Compound 3 (1mg/kg) also reduced body temper-
8-OH-DPAT (0.5mg/kg)
3 (1mg/kg) + 8-OH-DPAT
ꢀ1.6 0.2 ꢀ1.0 0.2 ꢀ1.4 0.4
ꢀ2.2 0.5 ꢀ1.5 0.2 ꢀ1.6 0.1
3 (10mg/kg) + 8-OH-DPAT ꢀ1.7 0.2 ꢀ1.1 0.1 ꢀ1.2 0.1
Values are means SEM from 8 animals.

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5184
Table 4. Effect of compound 3 and ondansetron on 2-Me-5-HT-
induced contraction in guinea pig LMMP
Table 6. Effect of compound 3 on scopolamine-induced impairment of
passive avoidance learning in rats
Compd
Concentration (M) Inhibition (%) IC50 (M)
Treatment
Retention latency (s)
Ondansetron 10ꢀ8
9.6
40.5
95.4
97.0
2.4· 10ꢀ7
Control
291.8 3.4
289.5 8.2
23.9 5.3a
204.5 21.6b
10ꢀ7
10ꢀ6
10ꢀ5
Compound 3 (1mg/kg)
Scopolamine (1mg/kg)
Compound 3 + Scopolamine
Scopolamine and compound 3 given 30 and 45min before the acqui-
sition trial, respectively. Retention latency measured 24h later. Values
are means SEM from 10–20 animals.
3
10ꢀ8
10ꢀ7
10ꢀ6
10ꢀ5
ꢀ31.3
39.2
90.0
1.9· 10ꢀ7
a p<0.05 versus control.
93.4
b p<0.05 versus scopolamine-treated group (ANOVA followed by
Student–Newman–Keuls test).
Values are means SEM from 4–5 separate experiments.
5-HT3R antagonists.34 These dual effects may explain
the apparent lack of anxiolytic-like effect in the present
study for compound 3, a 5-HT1AR partial agonist and
5-HT3R antagonist.
The functional profile of compound 3 at 5-HT3Rs was
characterized in the isolated longitudinal muscle-myen-
teric plexus (LMMP) preparation from guinea-pig
ileum. The contraction induced in this preparation by
the 5-HT3R agonist 2-Me-5-HT was inhibited by com-
pound 3 in the concentrations range of 10ꢀ7–10ꢀ5 M, a
full blockade of the contractions being observed at the
highest concentrations tested (Table 4). Similar effects
were found in this preparation with the 5-HT3R antag-
onist, ondansetron. The estimated IC50ꢀs for compound
3 and ondansetron were virtually identical, 0.19 and
0.24lM, respectively.
The effect of compound 3 on learning and retention was
also evaluated. In the passive avoidance learning test,
the muscarinic antagonist scopolamine, administered
30min before the acquisition session, reduced signifi-
cantly the retention latency 24h later (Table 6). Com-
pound 3, given at the dose of 1mg/kg, had no effect by
itself in the passive avoidance test. However, when this
compound was given 15min before scopolamine it sig-
nificantly prevented the retention impairment induced
by cholinergic blockade, suggesting the potential interest
of ligand 3 in the treatment of cognitive dysfunction.
3.3. Behavioural effects of compound 3
The anxiolytic-like effect of compound 3 was evaluated.
As shown in Table 5, on the second day of exposure to
the light–dark exploration test, control animals spent
less time in the white compartment and the number of
line crossings in this area also diminished. This behav-
iour was effectively prevented by administration of a
typical anxiolytic drug such as diazepam (Table 5).
However, no changes in behavioural parameters studied
were observed after administration of compound 3, indi-
cating that this compound is not endowed with an anx-
iolytic-like effect in this test, at least at the doses used.
4. Conclusion
In the present paper, we have designed and synthesized a
series of new mixed benzimidazole–arylpiperazine deriv-
atives with affinity for serotoninergic 5-HT1A and 5-HT3
receptors and selectivity over a1-adrenergic and dopam-
ine D2 receptors. Among them, compound 3 was identi-
fied as a high-affinity 5-HT1A/5-HT3 ligand, acting as a
partial agonist at 5-HT1ARs and a 5-HT3R antagonist,
and with ability to counteract the cognitive deficit in-
duced by cholinergic blockade. Consequently, this novel
mixed 5-HT1A/5-HT3 ligand could be a potential sub-
strate to be developed for treatment of cognitive dys-
function, and this therapeutic option is currently under
investigation.
5-HT1AR agonists may exert anxiolytic- or anxiogenic-
like effects by acting on presynaptic regions such as
the dorsal raphe nucleus or 5-HT terminal fields such
as the hippocampus, respectively.33 Bell-shaped dose–re-
sponse curves with enormous differences in the effective
dose–range have been reported, on the other hand, for
Table 5. Anxiolytic-like effect of compound 3 and diazepam in the
light–dark exploration test in mice
5. Experimental
5.1. Chemistry
Treatment
Dose (mg/kg) % Time in
white area
% Locomotion in
white area
Melting points were determined on a Gallenkamp elec-
trothermal apparatus. Infrared spectra (IR) were ob-
tained on a FTIR-8300 Shimadzu spectrophotometer.
1H and 13C NMR spectra were recorded on a Varian
VXR-300S or Bruker 300-AM instrument at 300 and
75MHz, respectively, or on a Bruker AC-200 spectro-
meter at 200 and 50MHz, respectively. Chemical shifts
(d) are expressed in parts per million relative to inter-
nal tetramethylsilane, coupling constants (J) are in
Saline
Diazepam
––
1
35.8 9.0
35.0 7.5
72.7 12.8a 78.5 11.6a
Saline/tween80 ––
69.3 5.6
60.2 7.3
65.8 8.3
61.1 6.7
56.3 6.8
59.1 5.7
Compound 3
0.1
1
Values are means SEM from 6–8 animals.
a p<0.05 versus the corresponding vehicle-treated controls (ANOVA
followed by Dunnettꢀs test).

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M. L. Lopez-Rodrıguez et al. / Bioorg. Med. Chem. 12 (2004) 5181–5191
5185
hertz (Hz). The following abbreviations are used to de-
scribe peak patterns when appropriate: s (singlet), d
(doublet), t (triplet), q (quartet), qt (quintet), sx (sextet),
st (septet), m (multiplet), br (broad). Elemental analyses
(C, H, N) were determined at the UCMꢀs analysis serv-
ices and were within 0.4% of the theoretical values.
Analytical thin-layer chromatography (TLC) was run
on Merck silica gel plates (Kieselgel 60 F-254) with detec-
tion by UV light, iodine, acidic vanillin solution, or 10%
phosphomolybdic acid solution in ethanol. For flash
chromato-graphy, Merck silica gel type 60 (size 230–
400 mesh) was used. Unless stated otherwise, all starting
materials and reagents were high-grade commercial
products purchased from Aldrich, Fluka or Merck.
ZINE), 3.91 (s, 2H, CH2), 6.79 (t, J = 7.2, 1H, Ph-H4),
6.94 (d, J = 8.4, 2H, Ph-H2, Ph-H6), 7.22 (t, J = 7.5,
2H, Ph-H3, Ph-H5), 7.30 (t, J = 7.8, 1H, H6), 7.81 (d,
J = 7.8, 1H, H5), 7.88 (d, J = 7.8, 1H, H7); 13C NMR
(Me2SO-d6) d 48.2 (2CH2- PIPERAZINE), 52.5 (2CH2-PIP-
ERAZINE), 54.5 (CH2), 115.4 (Ph-C2,), 118.8 (C4), 120.9
(Ph-C4, C6), 123.0 (C7), 124.1 (C5), 128.9 (Ph-C3, Ph-
C5), 134.4 (C3a), 143.5 (C7a), 151.0 (Ph-C1), 153.0 (C2),
167.1 (COOH).
5.1.4.
2-[[4-(o-Methoxyphenyl)piperazin-1-yl]methyl]-
benzimidazole-4-carboxylic acid (13). From 24 and
1-(o-methoxyphenyl)piperazine following the general
procedure was obtained 13. Yield 725mg (66%);
chromatography CH2Cl2/EtOH/NH3, from 9:1:0.05 to
8:2:0.06; mp 186–187ꢁC (d) (MeOH/Et2O); IR (KBr)
3210, 1620, 1590, 1560, 1520, 1500, 1450, 1255; 1H
NMR (Me2SO-d6) d 2.58–2.70 (m, 4H), 2.87–3.00 (m,
4H), 3.74 (s, 3H), 3.86 (s, 2H), 6.80–6.95 (m, 4H), 7.20
(t, J = 7.8, 1H), 7.69–7.79 (m, 2H); 13C NMR
(Me2SO-d6) d 50.1, 52.8, 54.8, 55.3, 111.9, 118.0, 120.7,
120.8, 121.7, 122.4, 123.8, 134.6, 141.2, 143.4, 152.0,
152.5, 168.4.
The following intermediates were synthesized according
to described procedures: 2-(hydroxymethyl)benzimidaz-
ole-4-carboxylic acid (23),35 1-(o-ethoxyphenyl)pipera-
zine,36 1-(o-propoxyphenyl)piperazine,37 1-(o-isoprop-
oxyphenyl)piperazine,36 1-(o-butoxyphenyl)piperazine,36
1-(2,3-dihydro-1,4-benzodioxan-5-yl)piperazine,38 1-(na-
phth-1-yl)piperazine,39 and 1-(1-tritylbenzimidazol-4-
yl)piperazine.40
5.1.1. Synthesis of 2-(chloromethyl)benzimidazole-4-carb-
oxylic acid (24). A solution of 2.0g (8.7mmol) of 23 in
45mL of thionyl chloride was heated at 80ꢁC for 2h.
After removing the thionyl chloride by co-distillation
with toluene under reduced pressure, the crude was
taken up in 20mL of 15% hydrochloric acid and the
solution was refluxed for 15min. The solvent was evap-
orated under vacuum to afford 2.0g (95%) of 24 as a
hydrochloride salt: mp 232–234ꢁC (MeOH/Et2O); IR
5.1.5. 2-[[4-(o-Ethoxyphenyl)piperazin-1-yl]methyl]benz-
imidazole-4-carboxylic acid (14). From 24 and 1-(o-eth-
oxyphenyl)piperazine following the general procedure
was obtained 14. Yield 753mg (66%); chromatography
CH2Cl2/EtOH/NH3, from 9:1:0.05 to 8:2:0.06; mp
145–147ꢁC (d) (MeOH/Et2O); IR (KBr) 3200, 1620,
1
1600, 1500, 1480, 1450, 1245; H NMR (Me2SO-d6) d
1.35 (t, J = 6.9, 3H), 2.62–2.76 (m, 4H), 2.95–3.08 (m,
4H), 3.89 (s, 2H), 4.02 (q, J = 6.9, 2H), 6.84–6.98 (m,
4H), 7.24 (t, J = 7.8, 1H), 7.78 (d, J = 7.7, 2H); 13C
NMR (Me2SO-d6) d 14.8, 49.9, 52.9, 54.8, 63.2, 113.1,
117.9, 118.1, 120.6, 120.8, 121.6, 122.2, 123.7, 134.5,
141.4, 143.3, 151.1, 152.5, 168.0.
(KBr) 3420, 2900, 1720, 1575, 1510, 1440, 1245cmꢀ1
;
1H NMR (Me2SO-d6) d 5.24 (s, 2H, CH2), 7.68 (t,
J = 7.5, 1H, H6), 8.11 (d, J = 7.5, 1H, H5), 8.14 (d,
J = 7.8, 1H, H7); 13C NMR (Me2SO-d6) d 34.7 (CH2),
117.0 (C4), 120.8 (C7), 124.9 (C6), 127.3 (C5), 130.8
(C3a), 135.3 (C7a), 150.8 (C2), 165.7 (COOH).
5.1.6. 2-[[4-(o-Propoxyphenyl)piperazin-1-yl]methyl]benz-
imidazole-4-carboxylic acid (15). From 24 and 1-(o-prop-
oxyphenyl)piperazine following the general procedure
was obtained 15. Yield 793mg (67%); chromatography
CH2Cl2/EtOH/NH3, from 9:1:0.05 to 8:2:0.06; mp
228–230ꢁC (d) (MeOH); IR (KBr) 3421, 1591, 1500,
1458, 1238; 1H NMR (Me2SO-d6) d 1.05 (t, J = 6.9,
3H), 1.76 (sx, J = 6.8, 2H), 2.62–2.78 (m, 4H), 2.95–
3.08 (m, 4H), 3.88 (s, 2H), 3.95 (t, J = 6.1, 2H), 6.87–
6.92 (m, 4H), 7.29 (t, J = 7.5, 1H), 7.81 (d, J = 7.5,
1H), 7.87 (d, J = 8.0, 1H); 13C NMR (Me2SO-d6) d
10.5, 22.1, 49.7, 52.7, 54.4, 68.8, 112.5, 115.1, 117.6,
120.5, 120.6, 121.9, 122.6, 123.7, 134.5, 141.0, 143.3,
151,0, 152.0, 166.8.
5.1.2. General procedure for the synthesis of 2-[(4-
arylpiperazin-1-yl)methyl]benzimidazole-4-carboxylic acids
(12–22). A mixture of 24 (740mg, 3mmol), the appropri-
ate 1-arylpiperazine (5mmol), triethylamine (1.1mL,
5mmol), and acetonitrile (6mL) was stirred at 60ꢁC
for 20–24h. After cooling, the solvent was removed
under reduced pressure, and the crude was taken up
in water and extracted with CH2Cl2 (3 · 30mL). The
organic layers were dried over Na2SO4 and evaporated
to afford the crude product, which was purified by col-
umn chromatography and recrystallization from the
appropriate solvent. Spectral data of all described com-
pounds 12–22 were consistent with the proposed struc-
tures.
5.1.7. 2-[[4-(o-Isopropoxyphenyl)piperazin-1-yl]methyl]-
benzimidazole-4-carboxylic acid (16). From 24 and 1-
(o-isopropoxyphenyl)piperazine following the general
procedure was obtained 16. Yield 828mg (70%); chro-
matography CH2Cl2/EtOH/NH3, from 9:1:0.05 to
8:2:0.06; mp 188–191ꢁC (d) (MeOH); IR (KBr) 3436,
5.1.3. 2-[(4-Phenylpiperazin-1-yl)methyl]benzimidazole-4-
carboxylic acid (12). From 24 and 1-phenylpiperazine
following the general procedure was obtained 12. Yield
504mg (50%); chromatography CH2Cl2/EtOH/NH3,
from 9:1:0.05 to 8:2:0.06; mp 272–273ꢁC (d) (MeOH/
Et2O); IR (KBr) 3200, 1630, 1595, 1500, 1450,
1
1590, 1496, 1450, 1238; H NMR (Me2SO-d6) d 1.23
(d, J = 6.1, 6H), 2.62–2.78 (m, 4H), 2.95–3.08 (m, 4H),
3.87 (s, 2H), 4.57 (st, J = 6.1, 1H), 6.87–6.92 (m, 4H),
7.20 (t, J = 7.7, 1H), 7.73 (d, J = 7.6, 1H), 7.74 (d,
1
1250cmꢀ1; H NMR (Me2SO-d6) d 2.66–2.75 (m, 4H,
2CH2-PIPERAZINE), 3.12–3.20 (m, 4H, 2CH2-PIPERA-

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5186
J = 7.8, 1H); 13C NMR (Me2SO-d6) d 22.0, 49.9, 53.0,
54.7, 69.5, 116.1, 118.2, 120.8, 121.3, 122.1, 122.7,
124.0, 134.4, 142.5, 143.3, 149.7, 153.1, 167.3.
5.1.12. 2-[[4-(2,3-Dihydro-1,4-benzodioxan-5-yl)pipera-
zin-1-yl]methyl]benzimidazole-4-carboxylic acid (21).
From 24 and 1-(2,3-dihydro-1,4-benzodioxan-5-yl)pip-
erazine following the general procedure was obtained
21. Yield 674mg (57%); chromatography CH2Cl2/
EtOH/NH3, from 9:1:0.05 to 8:2:0.06; mp 212–214ꢁC
(d) (MeOH/Et2O); IR (KBr) 3235, 1620, 1600, 1520,
5.1.8. 2-[[4-(o-Butoxyphenyl)piperazin-1-yl]methyl]benz-
imidazole-4-carboxylic acid (17). From 24 and 1-(o-but-
oxyphenyl)piperazine following the general procedure
was obtained 17. Yield 833mg (68%); chromatography
CH2Cl2/EtOH/NH3, from 9:1:0.05 to 8:2:0.06; mp
1
1485, 1470, 1455, 1255; H NMR (Me2SO-d6) d 2.57–
2.70 (m, 4H), 2.88–3.01 (m, 4H), 3.84 (s, 2H), 4.14–
4.24 (m, 4H), 6.42 (d, J = 7.8, 1H), 6.46 (d, J = 8.1,
1H), 6.67 (t, J = 8.1, 1H), 7.16 (t, J = 7.8, 1H), 7.68 (d,
J = 7.8, 2H); 13C NMR (Me2SO-d6) d 50.1, 52.6, 54.6,
63.7, 63.8, 110.2, 111.0, 117.6, 120.2, 120.7, 122.0,
123.8, 134.4, 136.2, 141.6, 143.4, 143.8, 152.5, 167.7.
1
139–141ꢁC (d) (CH2Cl2/Et2O); H NMR (Me2SO-d6) d
0.92 (t, J = 7.3, 3H), 1.44 (sx, J = 7.5, 2H), 1,70 (qt,
J = 7.9, 2H), 2.48–2.51 (m, 4H), 2.90–3.03 (m, 4H),
3.84 (s, 2H), 3.93 (t, J = 6.2, 2H), 6.33–6.89 (m, 4H),
7.19 (t, J = 7.8, 1H), 7.72 (d, J = 7.9, 2H); 13C NMR
(Me2SO-d6) d 13.8, 19.0, 31.1, 50.1, 53.0, 54.8, 67.3,
113.0, 115.0, 118.0, 120.8, 120.9, 122.3, 122.4, 124.0,
134.1, 141.5, 143.1, 151.4, 153.1, 166.8.
5.1.13. 2-[[4-(1-Tritylbenzimidazol-4-yl)piperazin-1-yl]-
methyl]benzimidazole-4-carboxylic acid (22). From 24
and 1-(1-tritylbenzimidazol-4-yl)piperazine following
the general procedure was obtained 22. Yield 1.15g
(62%); chromatography CH2Cl2/EtOH/NH3, from
9:1:0.05 to 8:2:0.01; mp 138–140ꢁC (d) (MeOH/hexane);
IR (KBr) 3651–3384, 1654, 1593, 1556, 1490, 1446,
5.1.9. 2-[[4-(m-(Trifluoromethyl)phenyl)piperazin-1-yl]-
methyl]benzimidazole-4-carboxylic acid (18). From 24
and 1-(m-(trifluoromethyl)phenyl)piperazine following
the general procedure was obtained 18. Yield 619mg
(51%); chromatography CH2Cl2/EtOH/NH3, from
9:1:0.05 to 8:2:0.06; mp 263–264ꢁC (d) (MeOH); IR
(KBr) 3225, 1625, 1595, 1495, 1450, 1250; 1H NMR
(Me2SO-d6) d 2.61–2.72 (m, 4H), 3.15–3.27 (m, 4H),
3.87 (s, 2H), 7.03 (d, J = 7.6, 1H), 7.13 (s, 1H), 7.16
(d, J = 7.8, 1H), 7.23 (t, J = 7.8, 1H), 7.38 (t, J = 7.8,
1
1269, 1234; H NMR (Me2SO-d6) d 2.62–2.65 (m, 4H),
3.20–3.30 (m, 4H), 3.78 (s, 2H), 5.84 (d, J = 8.1, 1H),
6.31 (d, J = 7.7, 1H), 6.63 (t, J = 8.1, 1H), 7.02–7.06
(m, 5H), 7.16 (t, J = 7.2, 1H), 7.23–7.26 (m, 10H), 7.69
(dd, J = 11.5, 7.1, 2H), 7.76 (s, 1H).
1H), 7.76 (d, J = 7.3, 1H), 7.80 (d, J = 7.3, 1H); 13C
5.1.14. General procedure for the synthesis of ( )-N-(1-
azabicyclo[2.2.2]oct-3-yl)-2-[(4-arylpiperazin-1-yl)methyl]-
benzimidazole-4-carboxamides (1–11). To a solution of
acids 12–22 (2.5mmol) in dry DMF (2.5mL) under an
argon atmosphere was added 1,10-carbonyldiimidazole
(CDI, 400mg, 2.5mmol). The mixture was stirred at
40ꢁC for 1h, then a solution of ( )-3-aminoquinuclidine
(2.5 g, 20mmol) and 1,8-diazabicyclo[5.4.0]undec-7-ene
(DBU, 380mg, 2.5mmol) in DMF (5mL) was added
dropwise, and the reaction mixture was stirred at 50ꢁC
for 20–24h. The solvent was removed under reduced
pressure, and the crude was taken up in CHCl3
(25mL) and washed with water (10mL) and 20% aque-
ous K2CO3 (10mL). The organic layer was dried over
Na2SO4 and evaporated to afford the crude product,
which was purified by column chromatography and re-
crystallization from the appropriate solvents. Spectral
data of all described compounds 1–11 were consistent
with the proposed structures.
3
NMR (Me2SO-d6) d 47.7, 52.4, 54.5, 110.9 (q, JC–F
3.5), 114.6 (q, JC–F = 3.8), 116.0, 118.8, 120.8,
=
3
1
122.6, 124.0, 124.5 (q, JCꢀF = 272.5), 129.9 (q,
2JC–F = 30.9), 130.0, 134.5, 143.5, 151.3, 152.8, 167.6.
5.1.10. 2-[[4-(m-Chlorophenyl)piperazin-1-yl]methyl]benz-
imidazole-4-carboxylic acid (19). From 24 and 1-(m-
chlorophenyl)piperazine following the general proce-
dure was obtained 19. Yield 623mg (56%); chromato-
graphy CH2Cl2/EtOH/NH3, from 9:1:0.05 to 8:2:0.06;
mp 247–248ꢁC (d) (MeOH/Et2O); IR (KBr) 3300,
1
1625, 1595, 1570, 1480, 1450, 1255; H NMR (Me2SO-
d6) d 2.56–2.68 (m, 4H), 3.08–3.20 (m, 4H), 3.85 (s,
2H), 6.74 (d, J = 7.9, 1H), 6.83 (d, J = 8.3, 1H), 6.88
(s, 1H), 7.16 (t, J = 8.0, 1H), 7.21 (t, J = 7.7, 1H), 7.77
(d, J = 7.7, 2H); 13C NMR (Me2SO-d6) d 47.5, 52.2,
54.4, 113.5, 114.4, 117.5, 117.9, 120.6, 121.9, 123.8,
130.2, 133.7, 134.4, 143.8, 152.1, 152.4, 167.9.
5.1.11. 2-[[4-(Naphth-1-yl)piperazin-1-yl]methyl]benzimi-
dazole-4-carboxylic acid (20). From 24 and 1-(naphth-1-
yl)piperazine following the general procedure was
obtained 20. Yield 568mg (49%); chromatography
CH2Cl2/EtOH/NH3, from 9:1:0.05 to 8:2:0.06; mp
236–238ꢁC (d) (MeOH); IR (KBr) 3225, 1620, 1590,
5.1.15. ( )-N-(1-Azabicyclo[2.2.2]oct-3-yl)-2-[(4-phenyl-
piperazin-1-yl)methyl]benzimidazole-4-carboxamide (1).
From 12 following the general procedure was obtained
1. Yield 789mg (71%); chromatography CH2Cl2/
EtOH/NH3, from 9.3:0.7:0.03 to 9:1:0.05; mp 146–
147ꢁC (CHCl3/Et2O); IR (KBr) 3430, 3255, 1645,
1
1
1575, 1520, 1510, 1490, 1455, 1255; H NMR (Me2SO-
1610, 1600, 1560, 1495, 1450 cmꢀ1; H NMR (Me2SO-
d6) d 2.77–2.90 (m, 4H), 2.99–3.10 (m, 4H), 3.95
(s, 2H), 7.10 (d, J = 7.3, 1H), 7.27 (t, J = 7.8, 1H), 7.40
(t, J = 8.0, 1H), 7.45–7.53 (m, 2H), 7.57 (d, J = 8.0,
1H), 7.80 (d, J = 7.6, 1H), 7.83–7.91 (m, 2H), 8.04–
8.12 (m, 1H); 13C NMR (Me2SO-d6) d 52.5, 52.9,
114.5, 116.2, 120.8, 122.5, 123.0, 123.2, 124.0, 125.3,
125.8, 125.9, 128.0, 128.2, 134.2, 134.5, 143.3, 149.2,
152.9, 167.6.
d6) d 1.38–1.52 (m, 1H, H5-QUINUCLIDINE or H8-QUINU-
CLIDINE), 1.54–1.68 (m, 2H, 2H5-QUINUCLIDINE or 2H8-
QUINUCLIDINE), 1.84–1.97 (m, 2H, H5-QUINUCLIDINE or
H8-QUINUCLIDINE, H4-QUINUCLIDINE), 2.54–2.59 (m,
1H, H2-QUINUCLIDINE), 2.64–2.89 (m, 8H, 2CH2-PIPERA-
ZINE, 2H6-QUINUCLIDINE, 2H7-QUINUCLIDINE), 3.12–3.23
(m, 4H, 2CH2-PIPERAZINE), 3.25–3.34 (m, 1H, H2-QUINU-
CLIDINE), 3.91 (s, 2H, CH2), 3.98–4.12 (m, 1H,

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5187
H3-QUINUCLIDINE), 6.77 (t, J = 7.3, 1H, Ph-H4), 6.92 (d,
J = 7.8, 2H, Ph-H2, Ph-H6), 7.20 (t, J = 7.3, 2H, Ph-H3,
Ph-H5), 7.29 (t, J = 7.8, 1H, H6), 7.67 (dd, J = 7.8, 1.2,
1H, H7), 7.81 (dd, J = 7.6, 1.2, 1H, H5), 10.35 (s, 1H,
CONH); 13C NMR (Me2SO-d6) d 20.3, 25.6 (C5-QUINU-
CLIDINE, C8-QUINUCLIDINE), 25.8 (C4-QUINUCLIDINE),
46.1, 47.1 (C6-QUINUCLIDINE, C7-QUINUCLIDINE), 46.3
(C3-QUINUCLIDINE), 48.3 (2CH2-PIPERAZINE), 52.6
(2CH2-PIPERAZINE), 55.4 (CH2), 56.5 (C2-QUINUCLI-
DINE), 115.0, 115.5 (Ph-C2, Ph-C6, C7), 118.9 (Ph-C4),
121.8 (C5, C6), 121.9 (C4), 129.0 (Ph-C3, Ph-C5), 135.3
(C7a), 140.2 (C3a), 151.0, 152.8 (Ph-C1, C2), 164.4
(CONH). Anal. (C26H32N6O) C, H, N.
(Me2SO-d6) d 10.8, 17.6, 21.6, 24.4, 26.4, 44.0, 45.0,
45.4, 50.0, 53.0, 57.8, 59.8, 69.0, 112.8, 115.5, 117.9,
120.8, 120.9, 122.0, 122.4, 134.5, 140.6, 141.2, 151.3,
152.9, 170.3. Anal. (C29H38N6O2) C, H, N.
5.1.19. ( )-N-(1-Azabicyclo[2.2.2]oct-3-yl)-2-[[4-(o-iso-
propoxyphenyl)piperazin-1-yl]methyl]benzimidazole-4-car-
boxamide (5). From 16 following the general procedure
was obtained 5. Yield 792 mg (63%); chromatography
CH2Cl2/EtOH/NH3, from 9.3:0.7:0.03 to 9:1:0.05; mp
125–127ꢁC (CHCl3/AcOEt); IR (KBr) 3429, 1654,
1560, 1496, 1450; 1H NMR (Me2SO-d6) d 1.27 (d,
J = 6.0, 6H), 1.49–1.60 (m, 1H), 1.61–1.75 (m, 2H),
1.90–2.00 (m, 2H), 2.28–2.36 (m, 1H), 2.65–2.80 (m,
6H), 2.82–2.92 (m, 2H), 3.00–3.12 (m, 4H), 3.18–3.22
(m, 1H), 3.93 (s, 2H), 4.55–4.65 (m, 1H), 4.61 (st,
J = 6.3, 1H), 6.87–6.96 (m, 4H), 7.35 (t, J = 7.8, 1H),
7.71 (d, J = 7.2, 1H), 7.86 (d, J = 7.2, 1H), 10.47 (d,
J = 6.6, 1H), 12.97 (br s, 1H). Anal. (C29H38N6O2) C,
H, N.
5.1.16. ( )-N-(1-Azabicyclo[2.2.2]oct-3-yl)-2-[[4-(o-meth-
oxyphenyl)piperazin-1-yl]methyl]benzimidazole-4-carbox-
amide (2). From 13 following the general procedure was
obtained 2. Yield 757mg (63%); chromatography CH2
Cl2/EtOH/NH3, from 9.3:0.7:0.03 to 9:1:0.05; mp 122–
123ꢁC (acetone); IR (KBr) 3425, 3260, 1650, 1610,
1
1575, 1560, 1500, 1450; H NMR (Me2SO-d6) d 1.41–
1.55 (m, 1H), 1.59–1.68 (m, 2H), 1.88–1.96 (m, 2H),
2.54–2.60 (m, 1H), 2.66–2.89 (m, 8H), 2.96–3.04 (m,
4H), 3.21–3.28 (m, 1H), 3.74 (s, 3H), 3.92 (s, 2H),
4.02–4.11 (m, 1H), 6.86–6.95 (m, 4H), 7.31 (t, J = 7.8,
1H), 7.69 (d, J = 7.5, 1H), 7.82 (dd, J = 7.5, 1.2, 1H),
10.45 (d, J = 7.0, 1H); 13C NMR (Me2SO-d6) d
20.1, 25.4, 25.7, 46.0, 46.9, 49.9, 52.7, 55.2, 56.4, 111.8,
114.8, 117.8, 120.7, 121.8, 121.9, 122.4, 134.3, 140.5,
141.0, 151.9, 152.4, 164.1. Anal. (C27H34N6O2)C, H, N.
5.1.20. ( )-N-(1-Azabicyclo[2.2.2]oct-3-yl)-2-[[4-(o-but-
oxyphenyl)piperazin-1-yl]methyl]benzimidazole-4-carbox-
amide (6). From 17 following the general procedure was
obtained 6. Yield 839mg (65%); chromatography
CH2Cl2/EtOH/NH3, from 9.3:0.7:0.03 to 9:1:0.05; (ace-
tone/hexane); mp 139–141ꢁC (CHCl3/AcOEt); 1H
NMR (Me2SO-d6) d 0.90 (t, J = 7.3, 3H), 1.16–1.23
(m, 1H), 1.38–1.42 (m, 3H), 1.45–1.52 (m, 5H), 1.64–
1.72 (m, 2H), 2.69–2.79 (m, 8H), 2.92–3.02 (m, 4H),
3.15–3.23 (m, 1H), 3.89–3.96 (m, 4H), 4.32–4.38 (m,
1H), 6.85–7.90 (m, 4H), 7.31 (t, J = 8.1, 1H), 7.69 (d,
J = 7.6, 1H), 7.82 (d, J = 7.6, 1H), 10.45 (d, J = 7.8,
1H,); 13C NMR (Me2SO-d6) d 13.6, 18.8, 20.2, 25.4,
25.7, 30.0, 46.0, 46.9, 49.9, 52.9, 55.3, 56.5, 67.2, 112.8,
114.8, 117.7, 120.7, 121.8, 122.2, 134.3, 140.5, 141.2,
151.2, 152.5, 164.5. Anal. (C30H40N6O2) C, H, N.
5.1.17. ( )-N-(1-Azabicyclo[2.2.2]oct-3-yl)-2-[[4-(o-eth-
oxyphenyl)piperazin-1-yl]methyl]benzimidazole-4-carbox-
amide (3). From 14 following the general procedure was
obtained 3. Yield 769mg (68%); chromatography
CH2Cl2/EtOH/NH3, from 9.3:0.7:0.03 to 9:1:0.05; mp
143–144ꢁC (CHCl3/AcOEt); IR (KBr) 3440, 3260,
1
1655, 1615, 1560, 1500, 1450; H NMR (Me2SO-d6) d
1.34 (t, J = 7.2, 3H), 1.52–1.64 (m, 1H), 1.66–1.78 (m,
2H), 1.94–2.09 (m, 2H), 2.60–2.67 (m, 1H), 2.69–2.78
(m, 4H), 2.82 (t, J = 7.5, 2H), 2.88–2.98 (m, 2H), 3.01–
3.12 (m, 4H), 3.32–3.38 (m, 1H), 3.94 (s, 2H), 4.01 (q,
J = 6.9, 2H), 4.10–4.20 (m, 1H), 6.80–7.00 (m, 4H),
7.35 (t, J = 7.8, 1H), 7.72 (d, J = 7.8, 1H), 7.86 (d,
J = 7.5, 1H), 10.50 (br d, J = 7.2, 1H); 13C NMR
(Me2SO-d6) d 14.8, 19.9, 25.0, 25.5, 45.9, 46.0, 46.8,
49.9, 52.9, 55.3, 56.2, 63.2, 113.1, 114.9, 117.9, 120.9,
121.8, 121.9, 122.3, 134.6, 140.5, 141.3, 151.1, 152.5,
164.3. Anal. (C28H36N6O2) C, H, N.
5.1.21. ( )-N-(1-Azabicyclo[2.2.2]oct-3-yl)-2-[[4-(m-(tri-
fluoromethyl)phenyl)piperazin-1-yl]methyl]benzimidazole-
4-carboxamide (7). From 18 following the general
procedure was obtained 7. Yield 1.04g (81%); chroma-
tography CH2Cl2/EtOH/NH3, from 9.3:0.7:0.03 to
9:1:0.05; mp 173–175ꢁC (d) (CHCl3); IR (KBr) 3435,
1
3260, 1655, 1610, 1565, 1495, 1450; H NMR (Me2SO-
d6) d 1.46–1.58 (m, 1H), 1.61–1.71 (m, 2H), 1.90–2.05
(m, 2H), 2.57–2.60 (m, 1H), 2.68–2.70 (m, 6H), 2.81–
2.91 (m, 2H), 3.22–3.32 (m, 4H), 3.33–3.38 (m, 1H),
3.96 (s, 2H), 4.05–4.14 (m, 1H), 7.10 (d, J = 7.8, 1H),
7.20 (s, 1H), 7.25 (d, J = 8.1, 1H), 7.34 (t, J = 7.8, 1H),
7.45 (t, J = 8.1, 1H), 7.71 (d, J = 7.5, 1H), 7.86 (dd,
J = 7.5, 0.6, 1H), 10.44 (d, J = 6.6, 1H), 12.95 (br s,
1H); 13C NMR (Me2SO-d6) d 20.2, 25.4, 25.6, 46.0,
5.1.18. ( )-N-(1-Azabicyclo[2.2.2]oct-3-yl)-2-[[4-(o-prop-
oxyphenyl)piperazin-1-yl]methyl]benzimidazole-4-carbox-
amide (4). From 15 following the general procedure was
obtained 4. Yield 817 mg (65%); chromatography
CH2Cl2/EtOH/NH3, from 9.3:0.7:0.03 to 9:1:0.05; mp
101–104ꢁC (CHCl3/AcOEt); IR (KBr) 3421, 1650,
1550, 1498, 1450; 1H NMR (Me2SO-d6) d 0.99 (t,
J = 7.3, 3H), 1.42–1.60 (m, 1H), 1.70 (sx, J = 7.1, 2H),
1.85–2.05 (m, 3H), 2.18–2.30 (m, 2H), 2.62–2.88 (m,
6H), 2.92–3.12 (m, 6H), 3.18–3.20 (m, 1H), 3.84–3.97
(m, 4H), 4.32–4.48 (m, 1H), 6.80–7.00 (m, 4H), 7.35 (t,
J = 7.6, 1H), 7.72 (d, J = 7.6, 1H), 7.84 (d, J = 7.6,
1H), 10.50 (br s, 1H), 13.10 (br s, 1H); 13C NMR
3
46.1, 46.9, 47.5, 52.3, 55.1, 56.4, 110.8 (q, JC–F = 3.9),
3
114.4 (q, JC–F = 4.2), 114.9, 118.5, 121.6, 121.8, 124.3
1
2
(q, JC–F = 272.2), 129.8 (q, JCꢀF = 30.8), 129.9,
134.3, 140.4, 151.1, 152.4, 164.1. Anal. (C27H31F3N6O)
C, H, N.
5.1.22. ( )-N-(1-Azabicyclo[2.2.2]oct-3-yl)-2-[[4-(m-chlo-
rophenyl)piperazin-1-yl]methyl]benzimidazole-4-carbox-
amide (8). From 19 following the general procedure
was obtained 8. Yield 1.03g (86%); chromatography

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5188
CH2Cl2/EtOH/NH3, from 9.3:0.7:0.03 to 9:1:0.05; mp
182–183ꢁC (acetone); IR (KBr) 3420, 3260, 1660,
CHCl3/MeOH/NH3, 8:2:0.1; (MeOH/hexane); 1H
NMR (Me2SO-d6) d 1.35–1.39 (m, 2H), 1.60–1.67 (m,
2H), 1.77–1.80 (m, 2H), 2.09–2.12 (m, 2H), 2.71–2.77
(m, 1H), 2.89–2.93 (m, 8H), 2.99–3.02 (m, 2H), 3.61–
3.64 (m, 1H), 4.08 (s, 2H), 4.21–4.23 (m, 1H), 6.66 (br
s, 1H), 7.19 (d, J = 2.9, 2H), 7.45 (t, J = 3.2, 1H), 7.82
(d, J = 3.1, 1H), 7.96 (d, J = 2.9, 1H), 8.19 (s, 1H); 13C
NMR (Me2SO-d6) d 25.2, 30.4, 45.9, 46.0, 46.9, 49.2,
52.8, 55.2, 63.4, 114.5, 115.0, 121.9, 122.8, 129.9,
134.5, 140.5, 145.7, 152.7, 164.2. Anal. (C27H32N8OÆ
HClÆH2O) C, H, N.
1
1615, 1595, 1560, 1490, 1450; H NMR (Me2SO-d6) d
1.46–1.57 (m, 1H), 1.61–1.71 (m, 2H), 1.91–2.05 (m,
2H), 2.56–2.61 (m, 1H), 2.67–2.78 (m, 6H), 2.81–2.91
(m, 2H), 3.21–3.28 (m, 4H), 3.29–3.35 (m, 1H), 3.95
(s, 2H), 4.05–4.14 (m, 1H), 6.81 (d, J = 7.8, 1H), 6.92
(d, J = 8.1, 1H), 6.97 (s, 1H), 7.23 (t, J = 8.1, 1H),
7.34 (t, J = 7.8, 1H), 7.71 (d, J = 7.8, 1H), 7.86 (d,
J = 7.8, 1H), 10.44 (br s, 1H), 12.95 (br s, 1H);
13C NMR (Me2SO-d6) d 20.3, 25.5, 25.7, 46.0, 46.1,
46.9, 47.6, 52.3, 55.2, 56.3, 113.6, 114.5, 114.8, 118.1,
121.8, 121.9, 130.4, 133.8, 134.2, 140.4, 152.2, 152.5,
164.2. Anal. (C26H31ClN6O) C, H, N.
5.2. Radioligand binding assays
For all receptor binding assays, male Sprague–Dawley
rats (Rattus norvegicus albinus), weighing 180–200g,
were killed by decapitation and the brains rapidly re-
moved and dissected. Tissues were stored at ꢀ80ꢁC
for subsequent use and homogenized on a Polytron
PT-10 homogenizer. Membrane suspensions were cen-
trifuged on a Beckman J2-HS instrument.
5.1.23. ( )-N-(1-Azabicyclo[2.2.2]oct-3-yl)-2-[[4-(naphth-
1-yl)piperazin-1-yl]methyl]benzimidazole-4-carboxamide
(9). From 20 following the general procedure was
obtained 9. Yield 878mg (71%); chromatography
CH2Cl2/EtOH/NH3, from 9.3:0.7:0.03 to 9:1:0.05;
mp 215–217ꢁC (d) (CHCl3); IR (KBr) 3425, 3265,
1660, 1615, 1590, 1575, 1560, 1510, 1490, 1450; 1H
NMR (Me2SO-d6) d 1.41–1.55 (m, 1H), 1.57–1.69
(m, 2H), 1.88–2.05 (m, 2H), 2.58–2.60 (m, 1H), 2.73 (t,
J = 7.4, 4H), 2.78–2.93 (m, 4H), 3.02–3.17 (m, 4H),
3.23–3.28 (m, 1H), 4.00 (s, 2H), 4.05–4.09 (m, 1H),
7.12 (d, J = 6.8, 1H), 7.32 (t, J = 7.8, 1H), 7.42
(t, J = 7.8, 1H), 7.46–7.51 (m, 2H), 7.61 (d, J = 8.3,
1H), 7.70 (d, J = 7.5, 1H), 7.83–7.90 (m, 2H), 8.06–
8.11 (m, 1H), 10.45 (d, J = 5.8, 1H), 12.96 (br s, 1H);
5.2.1. 5-HT1A receptor. Binding assays were performed
by a modification of the procedure previously described
by Clark et al.41 The cerebral cortex was homogenized in
10 volumes of ice-cold Tris buffer (50mM Tris–HCl,
pH7.7 at 25ꢁC) and centrifuged at 28,000g for 15min.
The membrane pellet was washed twice by resuspension
and centrifugation. After the second wash the resus-
pended pellet was incubated at 37ꢁC for 10min. Mem-
branes were then collected by centrifugation and the
final pellet was resuspended in 50mM Tris–HCl, 5mM
MgSO4, and 0.5mM EDTA buffer (pH7.4 at 37ꢁC).
Fractions of 100lL of the final membrane suspension
(about 1mg of protein) were incubated at 37ꢁC for
15min with 0.6nM [3H]-8-OH-DPAT (133Ci/mmol),
in the presence or absence of six concentrations of the
competing drug, in a final volume of 1.1mL of assay
buffer (50mM Tris–HCl, 10nM clonidine, 30nM prazo-
sin, pH7.4 at 37ꢁC). Nonspecific binding was deter-
mined with 10lM 5-HT.
13C NMR (Me2SO-d6)
d 20.1, 25.4, 25.6, 45.9,
46.0, 46.9, 52.4, 52.8, 55.1, 56.3, 114.3, 114.8, 121.7,
121.8, 122.9, 123.0, 125.2, 125.7, 125.8, 127.9, 128.1,
134.1, 140.3, 149.0, 152.4, 164.1. Anal. (C30H33N6O)
C, H, N.
5.1.24. ( )-N-(1-Azabicyclo[2.2.2]oct-3-yl)-2-[[4-(2,3-di-
hydro-1,4-benzodioxan-5-yl)piperazin-1-yl]methyl]benz-
imidazole-4-carboxamide (10). From 21 following the
general procedure was obtained 10. Yield 917mg
(73%); chromatography CH2Cl2/EtOH/NH3, from
9.3:0.7:0.03 to 9:1:0.05; mp 196–197ꢁC (d) (CHCl3/
AcOEt); IR (KBr) 3420, 3255, 1650, 1610, 1595, 1560,
5.2.2. 5-HT3 receptor. Binding assays were performed
according to the procedure previously described by
Wong et al.42 The cerebral cortex was homogenized in
9 volumes of 0.32M sucrose and centrifuged at 1000g
for 10min. The supernatant was centrifuged at 17,000g
for 20min. The membrane pellet was washed twice by
resuspension in 60 volumes of 50mM Tris–HCl buffer
(pH7.4 at 25ꢁC) and was centrifuged at 48,000g for
10min. Membranes were resuspended in 2.75 volumes
of assay buffer (50mM Tris–HCl, 10lM pargyline,
0.6mM ascorbic acid, and 5mM CaCl2, pH7.4 at
25ꢁC). Fractions of 100L of the final membrane suspen-
sion (about 2mg/mL of protein) were incubated at 25ꢁC
for 30min with 0.7nM [3H]LY 278584 (83Ci/mmol), in
the presence or absence of six concentrations of the
competing drug, in a final volume of 2mL of assay buf-
fer. Nonspecific binding was determined with 10lM 5-
HT.
1
1470, 1450; H NMR (Me2SO-d6) d 1.50–1.63 (m, 1H),
1.65–1.77 (m, 2H), 1.91–2.08 (m, 2H), 2.59–2.66 (m,
1H), 2.68–2.76 (m, 4H), 2.81 (t, J = 7.5, 2H), 2.87–2.96
(m, 2H), 2.97–3.11 (m, 4H), 3.30–3.36 (m, 1H), 3.95 (s,
2H), 4.07–4.18 (m, 1H), 4.19–4.28 (m, 4H), 6.49 (d,
J = 7.8, 1H), 6.52 (d, J = 8.1, 1H), 6.74 (t, J = 8.1,
1H), 7.35 (t, J = 7.8, 1H), 7.71 (d, J = 7.5, 1H), 7.86
(d, J = 7.8, 1H), 10.47 (d, J = 7.1, 1H), 13.00 (s,
1H); 13C NMR (Me2SO-d6) d 20.0, 25.1, 25.7, 45.9,
46.0, 46.9, 50.1, 52.7, 55.2, 56.1, 63.8, 63.9, 110.2,
111.1, 115.0, 120.4, 121.8, 121.9, 134.5, 136.2, 140.6,
141.6, 143.9, 152.6, 164.3. Anal. (C28H34N6O3) C, H, N.
5.1.25. ( )-N-(1-Azabicyclo[2.2.2]oct-3-yl)-2-[[4-(benzi-
midazol-4-yl)piperazin-1-yl]methyl]benzimidazole-4-car-
boxamide (11). The product obtained from 22 following
the general procedure was detritylated by refluxing with
a solution of THF/H2O/acetic acid, 1:1:1 for 3h to af-
ford final compound 11, which was isolated as hydro-
chloride salt: yield 606mg (45%); chromatography
5.2.3. a1 Adrenoceptor. Binding assays were performed
by a modification of the procedure previously described

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M. L. Lopez-Rodrıguez et al. / Bioorg. Med. Chem. 12 (2004) 5181–5191
5189
by Ambrosio et al.43 The cerebral cortex was homoge-
nized in 20 volumes of ice-cold buffer (50mM Tris–
HCl, 10mM MgCl2, pH7.4 at 25ꢁC) and centrifuged
at 30,000g for 15min. Pellets were washed twice by re-
suspension and centrifugation. Final pellets were resus-
pended in the same buffer. Fractions of the final
membrane suspension (about 250lg of protein) were
incubated at 25ꢁC for 30min with 0.2nM [3H] prazosin
(23Ci/mmol), in the presence or absence of six concen-
trations of the competing drug, in a final volume of
2mL of buffer. Nonspecific binding was determined with
10lM phentolamine.
GDP for 20min at 37ꢁC. The reaction was quenched
by rapid filtration through Whatman GF/B filters fol-
lowed by four washes with ice-cold 50mM Tris base
(pH7.4). Non-specific binding was determined in pres-
ence of 10M unlabeled GTPcS.
The ability of compound 3 (10ꢀ10–10ꢀ5 M) to stimulate
basal [35S]GTPcS binding was studied in comparison
with the typical 5-HT1AR agonist, 8-OH-DPAT, at the
same concentrations. This new compound was also
studied at identical concentrations for antagonism to
the stimulation of [35S]GTPcS binding induced by 8-
OH-DPAT (10ꢀ6 M), using comparatively the selective
5-HT1AR antagonist WAY-100635.
5.2.4. D2 receptor. Binding assays were performed by a
modification of the procedure previously described by
Hall et al.44 The striatum was homogenized in 50mM
Tris–HCl (pH7.7 at 25ꢁC) and centrifuged at 48,000g
for 10min. The pellet was resuspended and centrifuged
as before. The final pellet was resuspended in 50mM
Tris–HCl (pH7.7 at 25ꢁC) containing 120mM NaCl,
5mM KCl, 2mM CaCl2, 1mM MgCl2, and 0.1% ascor-
bic acid. Fractions of the final membrane suspension
(125–150lg of protein) were incubated at 25ꢁC for
60min with 0.8nM [3H]raclopride (77Ci/mmol), in the
presence or absence of six concentrations of the compet-
ing drug, in a final volume of 1.1mL of the assay buffer
(pH7.4 at 25ꢁC). Nonspecific binding was determined
with 1lM (+)-butaclamol.
5.4. Temperature measurements
Rectal temperature was measured in male Swiss mice
(24–28g) with a lubricated digital thermistor probe
(pb0331, Panlab, Barcelona) inserted to a depth of
2cm into the rectum and maintained until the tempera-
ture stabilized. Readings were taken immediately before
drug injection and 30, 60 and 120min thereafter. The
effect of compound 3 on rectal temperature was studied
at doses of 1 and 10mg/kg sc. To study the possible
antagonism to 8-OH-DPAT-induced hypothermia, this
compound was administered at the same doses
30min before 8-OH-DPAT (0.5mg/kg sc). Results
were expressed as changes in rectal temperature (n)
over basal values.
For all binding assays, competing drug, nonspecific,
total and radioligand bindings were defined in triplicate.
Incubation was terminated by rapid vacuum filtration
through Whatman GF/B filters, presoaked in 0.05%
poly(ethylenimine), using a Brandel cell harvester. The
filters were then washed with the assay buffer, dried
and placed in poly(ethylene) vials to which were added
4mL of a scintillation cocktail (Aquasol). The radioac-
tivity bound to the filters was measured by liquid scintil-
lation spectrometry. The data were analyzed by an
iterative curve-fitting procedure (program Prism, Graph
Pad), which provided IC50, Ki and r2 values for test com-
pounds, Ki values being calculated from the Cheng and
Prusoff equation.45 The protein concentrations of the rat
cerebral cortex and the rat striatum were determined by
the method of Lowry et al.,46 using bovine serum albu-
min as the standard.
5.5. Isolated longitudinal muscle-myenteric plexus
preparation (LMMP) from guinea pig ileum
The ileum from male guinea pig (300–350g) was excised
and longitudinal muscle strips with the myenteric plexus
attached (LMMP) were prepared as described.48 LMMP
strips were suspended in an organ bath containing
´
Tyrodeas solution aerated with O2/CO2, 95%/5% and
maintained at 37ꢁC. Contractile responses were
isometrically recorded with a resting tension of 0.5g.
Tissues were equilibrated for 30min and then stimulated
by the 5-HT3R agonist 2-Me-5-HT, 10ꢀ5 M. After a per-
iod of stabilization, the 5-HT3R antagonist effect of
compound 3 was determined by adding to the bath dif-
ferent concentrations 30min before 2-Me-5-HT. On-
dansetron was used as reference drug. The effect on
the contractile response induced by 2-Me-5-HT was
expressed as IC50 calculated from the corresponding
concentration–response curves.
5.3. [35S]GTPcS binding
[35S]GTPcS binding was performed in hippocampal
membranes from male Wistar rats (220–240g) as previ-
ously described47 withminor modifications. Briefly, rats
were killed and the hippocampus was dissected and
homogenized in cold Tris buffer (50mM Tris base,
pH7.4). The homogenates were centrifuged at
23,000rpm for 10min at 4ꢁC, and the remaining pellet
resuspended in the same Tris buffer and incubated at
37ꢁC for 10min in a shaking water bath. After a second
centrifugation in the same conditions, the pellet was re-
suspended in 67mM Tris base (pH7.4) containing 4mM
MgCl2, 160mM NaCl and 0.267mM EGTA. The bind-
ing assay was carried out by incubation of membranes
with [35S]GTPcS (0.1nM) in the presence of 300lM
5.6. Light–dark exploration test
A modified light–dark exploration test was used49 in
which the time spent by male Swiss mice (24–28g) in
the white compartment on the second day of exposure
was comparatively measured. Experiments were always
performed between 09.00 and 15.00h. The apparatus
consisted of an open-topped rectangular box divided
into a black small area under red light and a large white
area brightly illuminated with an opening door located
in the centre of the partition at floor level. The floor
of each compartment was marked into 9cm squares.

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M. L. Lopez-Rodrıguez et al. / Bioorg. Med. Chem. 12 (2004) 5181–5191
5190
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percentage of time spent and the number of line cross-
ings in each area. Mice were exposed to the test for
two consecutive days, the first without any treatment
and the second day after compound 3 (0.1 and 1mg/kg
sc) given 30min before the test. Diazepam (1mg/kg ip)
was used as a reference drug. Control mice received
vehicle injection.
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´
8. Schwartz, P. J.; Turner, E. H.; Garcıa-Borreguero, D.;
Male Wistar rats weighing 200–220g were used.
Experiments were always performed between 09.00
and 15.00h. The test was performed according to previ-
ously described procedures, using a two-compartment
(white/dark) passive avoidance apparatus.50 The rat
was placed in the illuminated area and 3s later the door
was raised. During 90s the animal explored the appara-
tus freely (habituation trial). After 10min, the rat was
placed again in the illuminated chamber. When the rat
entered the dark compartment, a guillotine door was
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10s, an inescapable 2mA scrambled electrical foot
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the rat in the illuminated compartment and measuring
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compound 3 was determined by administration of a
dose of 1mg/kg sc 30min before the acquisition trial.
In other experiments, learning was impaired by treat-
ment with the muscarinic antagonist scopolamine
(1mg/kg ip) 30min before acquisition. The effect of
compound 3 on retention impairment induced by
cholinergic blockade was evaluated by administration
of the compound (1mg/kg sc) 15min before scopolam-
ine. Control rats received saline or saline/tween 80
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´
´
´
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´
´
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Acknowledgements
´
´
22. Lopez-Rodrıguez, M. L.; Rosado, M. L.; Benhamu, B.;
Morcillo, M. J.; Fernandez, E.; Schaper, K.-J. J. Med.
´
This work was supported by Ministerio de Ciencia y
´
´
´
Tecnologıa (BQU2001-1459) and Comunidad Autono-
ma de Madrid (08.5/0028/2003). The authors are grate-
ful to UNED for a predoctoral grant to I. Tejada and
to CEPA SCHWARZ PHARMA for a predoctoral
grant to I. Marco, who also thanks the Spanish Society
of Therapeutic Chemistry for the award of the ꢁFAESꢀ
prize.
Chem. 1997, 40, 1648.
´
´
´
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2001, 44, 198.
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24. Lopez-Rodrıguez, M. L.; Vicente, B.; Deupi, X.; Barr-
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Ballesteros, J. A.; Salles, J.; Pardo, L. Mol. Pharmacol.
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