Benzimidazole derivatives. 2. Synthesis and structure-activity relationships of new azabicyclic benzimidazole-4-carboxylic acid derivatives with affinity for serotoninergic 5-HT3 receptors

Article abstract of DOI:10.1021/jm991076c

A new series of azabicyclic benzimidazole-4-carboxamides 2-21 and - carboxylates 22-30 were synthesized and evaluated for binding affinity at serotoninergic 5-HT₃ and 5-HT₄ receptors in the CNS. Most of the synthesized compounds exhibited high or very high affinity for the 5-HT₃ binding site and low to no significant affinity for the 5-HT₄ receptor. SAR observations indicated that a halogen atom at the 6-position and a nitro group at the 7-position of the benzimidazole ring is the best substitution pattern for 5-HT₃ affinity and 5-HT₃/5-HT₄ selectivity, as well as no substitution in this ring. (S)-(-)-N-(Quinuclidin-3-yl)benzimidazole-4- carboxamides 2, 8, and 14 bound at central 5-HT₃ sites with high affinity (K(i) = 2.6, 0.13, and 1.7 nM, respectively) and excellent selectivity over serotonin 5-HT₄ and 5-HT(1A) receptors (K(i) > 1000-10000 nM). Furthermore, these new 5-HT₃ receptor ligands were pharmacologically characterized as potent and selective 5-HT₃ antagonists in the isolated guinea pig ileum (pA₂ = 9.6, 9.9, and 9.1, respectively).

Full text of DOI:10.1021/jm991076c

5020
J . Med. Chem. 1999, 42, 5020-5028
Ben zim id a zole Der iva tives. 2. Syn th esis a n d Str u ctu r e-Activity Rela tion sh ip s
of New Aza bicyclic Ben zim id a zole-4-ca r boxylic Acid Der iva tives w ith Affin ity
for Ser oton in er gic 5-HT3 Recep tor s
Mar´ıa L. Lo´pez-Rodr´ıguez,*,† Bellinda Benhamu´,† M. J ose´ Morcillo,§ Ignacio D. Tejada,† Luis Orensanz,‡
M. J ose´ Alfaro, and M. Isabel Mart´ın
Departamento de Quı´mica Orga´nica I, Facultad de Ciencias Qu´ımicas, Universidad Complutense, 28040 Madrid, Spain,
Facultad de Ciencias, Universidad Nacional de Educacio´n a Distancia, 28040 Madrid, Spain, Departamento de Investigacio´n,
Hospital Ramo´n y Cajal, Carretera de Colmenar km. 9, 28034 Madrid, Spain, and Departamento de Farmacolog´ıa,
Facultad de Medicina, Universidad Complutense, 28040 Madrid, Spain
Received May 7, 1999
A new series of azabicyclic benzimidazole-4-carboxamides 2-21 and -carboxylates 22-30 were
synthesized and evaluated for binding affinity at serotoninergic 5-HT3 and 5-HT4 receptors in
the CNS. Most of the synthesized compounds exhibited high or very high affinity for the 5-HT3
binding site and low to no significant affinity for the 5-HT4 receptor. SAR observations indicated
that a halogen atom at the 6-position and a nitro group at the 7-position of the benzimidazole
ring is the best substitution pattern for 5-HT3 affinity and 5-HT3/5-HT4 selectivity, as well as
no substitution in this ring. (S)-(-)-N-(Quinuclidin-3-yl)benzimidazole-4-carboxamides 2, 8,
and 14 bound at central 5-HT3 sites with high affinity (Ki ) 2.6, 0.13, and 1.7 nM, respectively)
and excellent selectivity over serotonin 5-HT4 and 5-HT1A receptors (Ki > 1000-10000 nM).
Furthermore, these new 5-HT3 receptor ligands were pharmacologically characterized as potent
and selective 5-HT3 antagonists in the isolated guinea pig ileum (pA2 ) 9.6, 9.9, and 9.1,
respectively).
Ch a r t 1
In tr od u ction
Serotonin (5-hydroxytryptamine, 5-HT) is a neuro-
transmitter involved in a wide variety of pharmacologi-
cal effects in several peripheral as well as central
nervous tissues.1-7 Seven 5-HT receptor classes (5-
HT1-7), including 15 different subtypes (A, B, ...), have
so far been identified.8-12 Most of them belong to the
G-protein-linked receptor superfamily involving in their
action a second-messenger system such as cAMP or
diacylglycerol and inositolphosphates. The 5-HT3 sub-
type, on the other hand, has a special place because it
is a ligand-gated cation channel receptor13,14 and is
present within the central and peripheral nervous
systems.15 In recent years, there has been considerable
interest in the search for new specific 5-HT3 ligands due
to their implication in various (patho)physiological
processes16,17 and their potential applications in therapy.
Indeed, 5-HT3 receptor antagonistssondansetron, gra-
nisetron (Chart 1)sare used clinically as agents to
prevent the emesis which is a frequent, severe, and
sometimes incapacitating syndrome associated with
anticancer chemotherapy.18,19 Further possible thera-
peutic indications in the treatment of central nervous
system disorders such as anxiety, schizophrenia, drug
abuse and withdrawal, anorectic responses, and cogni-
tive disfunctions are the subject of intense investiga-
tions.16,20-23
isetron, zacopridesalso exhibit activity in the 5-HT4
subtype, significant effort has been made toward the
development of potent and selective 5-HT3 receptor
ligands. 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 serotoninergic binding sites.24 This computer-
aided conformational analysis has allowed us to confirm
the generally accepted three-component pharmacophore
for 5-HT3 receptor antagonists as well as to propose a
steric model for 5-HT4 receptor recognition. This study
led to the design of a new structural class of selective
5-HT3 and 5-HT4 receptor ligands,25-27 which are benz-
imidazole-4-carboxylic acid derivatives. Among them,
compound 1 was identified as a potent and selective
5-HT3 receptor ligand [Ki (5-HT3) ) 3.7 nM, Ki (5-HT4)
> 1000 nM].26 In this paper, we report the synthesis of
a new series of azabicyclic benzimidazole-4-carboxam-
ides 2-21 and carboxylates 22-30 related to lead
As multiple known 5-HT3 receptor antagonistsstrop-
* To whom correspondence should be addressed. Phone: 34-91-
3944239. Fax: 34-91-3944103. E-mail: mluzlr@eucmax.sim.ucm.es.
† Departamento de Qu´ımica Orga´nica I, Universidad Complutense.
§ Universidad Nacional de Educacio´n a Distancia.
‡ Hospital Ramo´n y Cajal.
Departamento de Farmacolog´ıa, Universidad Complutense.
10.1021/jm991076c CCC: $18.00 © 1999 American Chemical Society
Published on Web 11/13/1999

Synthesis of Azabicyclic Benzimidazole-4-carboxamides
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 24 5021
Sch em e 1a
Sch em e 3a
a
Regents and conditions: (a) CDI, DMF, 40 °C, 1 h; (b) Y-NH2
or Y-OH, DBU, DMF, 50 °C, 20-24 h.
Sch em e 2a
a
Reagents and conditions: (a) R1 ) Cl: H2/Ni-Ra, EtOH, rt, 2
h; (b) R1 ) Br: NH2NH2, Ni-Ra, EtOH, ∆, 15 min; (c) NH2NH2,
Pd(C), EtOH, ∆, 30 min.
tives 39 and 40, along with a minor amount (30%) of
their 5-nitro isomers 41 and 42, which were separated
by flash chromatography. The benzimidazole substitu-
tion pattern was assigned using steady state 1D-NOE
experiments through irradiation of the methyl substitu-
ent. Irradiation of the CH3 signal caused NOE enhance-
ment of the aromatic proton signal at δ 7.35 ppm for
39 and no effect of any signal for 41, indicating that
the former isomer has an aromatic proton situated at
an ortho position with respect to the methyl group, while
41 has a substituent (the nitro group) in this position.
Similar results were obtained for 40 and 42. Derivatives
39 and 40 were converted into their carboxylic acids 34
and 35, respectively, by oxidation with potassium per-
manganate. 7-Aminocarboxamides 15, 20, and 21 were
prepared from their corresponding 7-nitro derivatives
(Scheme 3). Thus, 13 was subjected to catalytic hydro-
genation over Raney nickel in ethanol to afford 15. The
treatment of 19 with hydrazine and Raney nickel
yielded 20, while its reduction with hydrazine and
palladium on carbon led to the heterolytic reduction of
the C-Br bond to give 21. As for intermediate amines
and alcohols, quinuclidine derivatives were commer-
cially available, and tropane and granatane derivatives
were synthesized according to previously described
methods.
a
Reagents and conditions: (a) HCOOH, H2O, 100 °C, 5 h; (b)
HNO3/H2SO4, 5 °C, 2 h; (c) flash chromatography; (d) KMnO4, 0.5
N NaOH, 100 °C, 5 h.
compound 1 (Chart 1) and also their affinities for 5-HT3
and 5-HT4 receptors obtained by radioligand binding
assays. In these analogues we have analyzed the influ-
ence of different substituents at the benzimidazole ring,
different azabicycloalkanes as amino moieties, and the
stereogenic center of the quinuclidine system on the
affinity for 5-HT3 receptors and selectivity over 5-HT4
sites, with respect to unsubstituted lead compound 1.
Three analogues with interesting 5-HT3 affinity and
selectivity were evaluated for 5-HT3 antagonist activity,
by analyzing their ability to inhibit the contractions
induced by 5-HT in the isolated guinea pig ileum.
All new compounds (Table 1) were characterized by
IR and NMR spectroscopy and gave satisfactory com-
bustion analyses (C, H, N). The unambiguous assign-
ment of all bicyclic proton and carbon resonances was
Ch em istr y
1
The general procedure for the preparation of target
compounds 2-14, 16-19, and 22-30 is shown in
Scheme 1. Starting benzimidazolecarboxylic acids 31-
35 were suitably activated with 1,1′-carbonyldiimidazole
(CDI), and the corresponding imidazolides were subse-
quently coupled with the appropriate 3-substituted
quinuclidine, tropane, or granatane amines and alcohols
in the presence of 1,8-diazabicyclo[5.4.0]undec-7-ene
(DBU) in N,N-dimethylformamide (DMF) solutions, to
afford the desired amides and esters. In analogy with
6-chlorobenzimidazole-4-carboxylic acid (32),27 acid 33
was obtained by condensation of 2-amino-5-bromo-3-
methylaniline (36) with formic acid and subsequent
oxidation of 6-bromo-4-methylbenzimidazole (38) with
potassium permanganate (Scheme 2). 7-Nitrobenzimi-
dazole-4-carboxylic acids 34 and 35 were prepared as
illustrated in Scheme 2. Nitration of 4-methylbenzimi-
dazoles 37 and 38 provided the desired 7-nitro deriva-
achieved by a combined use of 1D H and 13C NMR, 2D
1
NMR techniques (COSY and H-13C correlation spec-
tra), and double-resonance experiments.
A structural analysis of our 3′-substituted tropanes
and granatanes was performed by means of NMR
spectroscopy and molecular mechanics calculations. 1H
NMR spectra of esters 23, 25, and 29 show H3′ as a
triple triplet (septet-like) with coupling constant (J ) of
ca. 10.5 and 6.5 Hz, and the signal of the bridgehead
protons H1′/H5′ is a broad triplet (J ) ca. 3.5 Hz),
indicating that they are exo-isomers in a chair confor-
mation. Thus, exo-granatane derivatives adopt a chair-
chair conformation (Figure 1a). In the spectra of tropane
esters 24 and 28, the signals of H3′ and H1′/H5′ are both
triplets or broad triplets (J ) 5.1 and ca. 3 Hz,
respectively), while in the case of granatane carboxy-
lates 26 and 30 the spectra show a triple triplet (quintet-
like) with J ) 6.9 and 6.0 Hz for the H3′ signal and a

5022 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 24
Ta ble 1. In Vitro Binding Dataa
Lo´pez-Rodrı´guez et al.
24 and 28 are in a chair conformation (Figure 1b), but
in the case of endo-granatane derivatives 26 and 30 the
disubstituted ring has a boat conformation (Figure 1c).
Thus, endo-granatane derivatives adopt a boat-chair
conformation.
To obtain additional support, endo-esters 24 and 26
were subjected to computer-aided conformational analy-
sis with INSIGHT II software 97.0 using the Builder,
Discover, and Analysis programs. According to the
calculated lowest energy conformations, the piperidine
ring of endo-tropane ester 24 adopts a chair conforma-
tion, whereas endo-granatane ester 26 is in a boat-chair
conformation. On the other hand, it is known that the
vicinal coupling constants are very useful magnetic
parameters for the conformational analysis of bicyclic
systems owing to their strong dependence on the
dihedral angles. By that, the coupling parameters of H3′
were empirically estimated for the computed disubsti-
tuted ring in chair and boat conformations of 24 and
26 by using the equation proposed by Altona28 and
compared with those deduced from the analysis of the
spectra (Table 2). The experimental values are consis-
tent with those calculated for the chair conformation of
tropane 24 and the boat-chair conformation of grana-
tane 26. In the case of the carboxamides, H3′ signals
were complicated multiplets due to couplings with
amide protons. Nevertheless, H1′/H5′ signals of amides
6 and 12 showed the same pattern as those of ester 26,
indicating the same boat-chair conformation.
Ki ( SEM (nM)
5-HT3
5-HT4
compd R1
R2
X
Yb
[3H]LY 278584 [3H]GR 113808
1
2
H
H
H
NH
Q
3.7 ( 0.1
2.6 ( 0.3
38.3 ( 0.6
118 ( 1
>1000
H
H
H
H
H
H
H
H
H
H
H
NH (S)-Q
NH (R)-Q
NH exo-T
NH exo-G
NH endo-G
>1000
3
H
>10000
>10000
>10000
>1000
168 ( 6
>10000
205 ( 8
319 ( 75
>1000
4
H
5
H
230 ( 1
6
H
6.1 ( 0.8
0.29 ( 0.08
0.13 ( 0.02
0.76 ( 0.14
16.6 ( 1.6
83.0 ( 12.1
154 ( 24
5.2 ( 0.6
1.7 ( 0.2
0.24 ( 0.01
0.29 ( 0.01
0.28 ( 0.03
3.4 ( 0.1
8.8 ( 0.1
0.42 ( 0.02
2.4 (0.1
7
8
9
Cl
Cl
Cl
Cl
Cl
Cl
NH
Q
NH (S)-Q
NH (R)-Q
NH exo-T
NH exo-G
NH endo-G
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
167 ( 32
>1000
>1000
Cl NO2 NH
Cl NO2 NH (S)-Q
Cl NH2 NH
Br
Br
Br
Br NO2 NH
Br NH2 NH
H
H
H
H
H
H
Q
Q
Q
187 ( 5
168 ( 6
38.3 ( 5.0
270 ( 19
>10000
227 ( 3
779 ( 105
>10000
>10000
>10000
>10000
>10000
440 ( 87
356 ( 33
>1000
H
H
H
NH
NH (S)-Q
NH (R)-Q
Q
Q
Q
Q
NH2 NH
H
H
H
H
H
H
H
H
H
O
O
O
O
O
O
O
O
O
185 ( 3
exo-T
endo-T
exo-G
endo-G
Q
endo-T
exo-G
endo-G
>1000
>1000
678 ( 120
129 ( 1
Cl
Cl
Cl
Cl
9.3 ( 1.0
61.2 ( 12.4
88.7 ( 1.1
4.9 ( 0.8
0.77 ( 0.01
3.9 ( 0.1
P h a r m a cology
>1000
Target compounds 2-30 were assessed for in vitro
affinity at serotoninergic 5-HT3 and 5-HT4 receptors by
radioligand binding assays, using [3H]LY 278584 in rat
cerebral cortex membranes29 and [3H]GR 113808 in rat
striatum membranes,30 respectively. The inhibition
constant Ki was defined from the IC50 by the Cheng-
Prusoff equation31 (Table 1). Three highly active 5-HT3
ligands were also evaluated for in vitro affinity at
5-HT1A receptors with [3H]-8-OH-DPAT in rat cerebral
cortex membranes32 and for 5-HT3 biological activity in
the isolated guinea pig ileum (Table 3). It is well-known
that concentrations higher than 10-6 M of 5-HT induce
contractile responses in the nonstimulated guinea pig
ileum by activation of 5-HT3 receptors, whereas lower
concentrations activate 5-HT4 receptors.33-41 To test the
antagonist activity of the new compounds, their capacity
to inhibit the contractions induced by 5-HT in the
myenteric plexus-longitudinal muscle strip prepara-
tions, isolated from guinea-pig ileum, was evaluated and
the pA2 values were determined following the equation
described by Furchgott42 using one appropriate concen-
tration of the antagonists. The concentration ratios of
5-HT, in the presence or absence of antagonists, reflect
the rightward displacement of the concentration effect
measured at the point where the force of the contraction
induced by 5-HT was 50% of the maximal control value.
The 5-HT3 receptor antagonists ondansetron and gra-
nisetron were used as reference compounds. The effects
of the antagonists were also tested on the contraction
induced by low concentrations of 5-HT (<10-6 M), in
order to discard activity on 5-HT4 receptors.
ondansetron
granisetron
>1000
>1000
a
Ki values are means ( SEM of two to four assays, performed
in triplicate. Inhibition curves were analyzed by a computer-
assisted curve-fitting program (Prism GraphPad), and Ki values
b
were determined from the Cheng-Prusoff equation. Q: quinu-
clidin-3-yl (1-azabicyclo[2.2.2]oct-3-yl). T: tropan-3-yl (8-methyl-
8-azabicyclo[3.2.1]oct-3-yl). G: granatan-3-yl (9-methyl-9-azabi-
cyclo[3.3.1]non-3-yl).
F igu r e 1. (a) In exo-tropane and -granatane esters the
disubstituted ring adopts a chair conformation. (b) In endo-
tropane esters the piperidine ring adopts a chair conformation.
(c) Endo-granatane esters adopt a boat-chair conformation.
broad doublet (J ) ca. 9 Hz) for the H1′/H5′ signal.
These different patterns for H3′ and H1′/H5′ signals
suggest that the piperidine ring of endo-tropane esters

Synthesis of Azabicyclic Benzimidazole-4-carboxamides
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 24 5023
Ta ble 2. Experimental Coupling Constants and Calculated Values (in Hz) of H3′ for Compounds 24 and 26 with the Disubstituted
Ring in Chair and Boat Conformations
chaira
boata
expb
compd
J (H3′-H2′ax)
J (H3′-H2′eq)
J (H3′-H2′ax)
J (H3′-H2′eq)
J (H3′-H2′ax)
J (H3′-H2′eq)
24
26
5.04
6.10
1.93
1.50
7.80
7.55
7.85
7.98
5.1
6.0
6.9
a
Coupling constants values calculated from Altona’s equation for chair and boat conformations in the disubstituted ring obtained by
molecular mechanics calculations. Experimental coupling constants measured from 1H NMR spectra in CDCl3 solutions.
b
1000 nM). In agreement with the literature studies,40,43
compounds with endo-granatanes are more potent than
Ta ble 3. Pharmacological Activity at 5-HT3 Receptors in
Isolated Guinea Pig Ileum
b
compd
control
ondansetron
granisetron
14
8
2
5-HT ED50, Ma
pA2
Nc
their corresponding exo-analogues; e.g., 6 (Ki ) 6.1 nM)
exhibits 38-fold more affinity than 5 (Ki ) 230 nM) and
30 (Ki ) 4.9 nM) is 18-fold more potent than 29 (Ki )
88.7 nM). The only exception is compound 12 (Ki ) 154
nM), which displays slightly lower affinity than its exo-
counterpart 11 (Ki ) 83.0 nM). For all quinuclidine
derivatives S-enantiomers are more potent than the
corresponding R-isomers, but they display an affinity
for the 5-HT3 receptor similar to the racemic com-
pounds. For example, racemic bromo derivative 16 and
its S-enantiomer 17 are equipotent (Ki ) 0.29 and 0.28
nM, respectively) and both exhibit higher affinity than
the R-isomer 18 (Ki ) 3.4 nM); the same trend is
observed for unsubstituted compound 1 (Ki ) 3.7 nM),
its S-enantiomer 2 (Ki ) 2.6 nM), and the R-isomer 3
(Ki ) 38.3 nM).
Of special interest were the (S)-quinuclidin-3-yl de-
rivatives 2 (R1 ) R2 ) H, X ) NH), 8 (R1 ) Cl, R2 ) H,
X ) NH), and 14 (R1 ) Cl, R2 ) NO2, X ) NH), since
they were found to be potent 5-HT3 ligands (Ki ) 2.6,
0.13, and 1.7 nM, respectively) devoid of affinity at
5-HT4 and 5-HT1A receptors (Ki > 1000-10000 nM). For
this reason, they were selected for the evaluation of their
5-HT3 receptor activity in the guinea pig ileum. All the
tested compounds inhibited the concentrations induced
by 10-6 to 10-5 M 5-HT without modification of the effect
of low doses (5 × 10-8 to 5 × 10-7 M) of 5-HT. From
these data it can be concluded that at the tested doses
(10-9 to 10-8 M) 2, 8, and 14 selectively antagonize the
effect of 5-HT mediated through 5-HT3 receptors with-
out significant differences vs reference compounds.
Table 3 shows pA2, determined following the equation
described by Furchgott42 using the appropriate concen-
tration (5 × 10-9 M) of the antagonists, and ED50 values.
When the effect of the tested compounds on the contrac-
tions mediated through 5-HT4 receptors ([5-HT] < 10-6
M) was analyzed, no differences were found (Figure 2).
8.8 × 10-7
4.3 × 10-5
7 × 10-6
10
6
7
7
6
9.9
9.2
9.1
9.9
9.6
6.8 × 10-6
1.7 × 10-5
3.3 × 10-5
6
a
Concentration of 5-HT required to produce a 50% of maximal
contraction induced by 5-HT through 5-HT3 receptors evaluated
in the absence (control) and in the presence of tested compounds
b
(5 × 10-9 M). Single-point analysis using 5 × 10-9 M concentra-
tion of the antagonists (pA2 ) -log([B]/concentration ratio - 1);
[B] ) concentration of the antagonist). c Number of experiments.
Resu lts a n d Discu ssion
The results of the tested compounds are reported in
Tables 1 and 3. The values for 5-HT3 activation of the
compounds were compared to those of the reference
compounds: ondansetron (GR 38032F) and granisetron
(BRL 43694).
Most of the synthesized compounds exhibit high or
very high affinity for the 5-HT3 receptor and low to no
significant affinity for the 5-HT4 binding site. An
examination of the 5-HT3 binding data presented in
Table 1 shows the following:
(a) In general, the carboxamides are more potent at
5-HT3 receptors than their corresponding carboxylates;
only ester 30 (Ki ) 4.9 nM) displayed ca. 30-fold higher
affinity than its directly related amide 12 (Ki ) 154 nM).
(b) With respect to the aromatic moiety, the introduc-
tion of a chloro or bromo atom at the 6-position and/or
a nitro or amino group at the 7-position of the benzimi-
dazole ring is tolerated for binding to 5-HT3 sites. Thus,
N-(quinuclidin-3-yl)benzimidazole-4-carboxamides 1 (R1
) R2 ) H), 7 (R1 ) Cl, R2 ) H), 13 (R1 ) Cl, R2 ) NO2),
15 (R1 ) Cl, R2 ) NH2), 16 (R1 ) Br, R2 ) H), 19 (R1 )
Br, R2 ) NO2), 20 (R1 ) Br, R2 ) NH2), and 21 (R1 ) H,
R2 ) NH2) display nanomolar or subnanomolar affinity
for the 5-HT3 receptor (Ki ) 0.24-8.8 nM). Regarding
5-HT3/5-HT4 selectivity, unsubstituted compounds (e.g.,
1, 2) and derivatives with R1 ) Cl, Br, R2 ) NO2 (13,
14, and 19) show high selectivity. Most analogues with
R1 ) Cl, Br, R2 ) H (e.g., 7 and 16) and R1 ) H, Cl, Br,
R2 ) NH2 (15, 20, and 21) are moderately active at
5-HT4 receptors, and consequently they show lower
selectivity for the 5-HT3 receptor. A remarkable excep-
tion is compound 8 (R1 ) Cl, R2 ) H), which displays
the best selectivity ratio (5-HT3: Ki ) 0.13 nM, 5-HT4:
Ki > 10000 nM).
To discard effects involving other receptors present
in these tissues, the effect of 2, 8, and 14 (5 × 10-9 M)
on the contractions induced by acetylcholine (10-6-10-5
M), substance P (10-7-5 × 10-6 M), histamine (10-6-5
× 10-6 M), and bradykinin (5 × 10-7-5 × 10-6 M) or
by electrical stimulation (single square waves: 0.3 Hz,
2 ms and supramaximal voltage) was tested. None of
the compounds induced significant modifications in the
contractile responses, and did not display agonist activ-
ity in the guinea pig ileum when administered alone.
These data confirm the selectivity of the effect observed
on contractions mediated through 5-HT3 receptors.
(c) Concerning the azabicyclic amino moiety (Y),
quinuclidine derivatives are significantly more potent
than compounds with tropane or granatane systems;
only amide 6 and ester 30 (Y ) endo-granatan-3-yl)
display high 5-HT3 affinity (Ki ) 6.1 and 4.9 nM,
respectively) and selectivity over 5-HT4 receptors (Ki >
These biological results indicate that these compounds
are new potent and selective 5-HT3 receptor antagonists.
A number of pharmacophore models for antagonists
binding to the 5-HT3 receptor have been presented44-47

5024 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 24
Lo´pez-Rodrı´guez et al.
comparable to the 5-HT3 receptor antagonists on-
dansetron and granisetron. Consequently, these new
5-HT3 antagonists could be potential substrates to be
developed in different pharmacological areas, since the
5-HT3 antagonists have been reported to display a wide
range of nonantiemetic activities (control of anxiety,
psychosis, nociception, drug abuse and withdrawal,
cognitive impairments, and anorectic responses). These
therapeutic options are currently under investigation.
Exp er im en ta l Section
Ch em istr y. Melting points (uncorrected) were determined
on a Gallenkamp electrothermal apparatus. Infrared (IR)
spectra were obtained on a Perkin-Elmer 781 infrared spec-
trophotometer; the frequencies are expressed in cm-1. 1H 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. Chemical shifts (δ) are expressed in parts per
million relative to internal tetramethylsilane, coupling con-
stants (J ) are in hertz (Hz). The following abbreviations are
used to describe peak patterns when appropriate: s (singlet),
d (doublet), t (triplet), q (quartet), qt (quintet), sx (sextet), m
(multiplet), br (broad). Elemental analyses (C, H, N) were
determined at the UCM’s analysis services and were within
(0.4% of the theoretical values. Analytical thin-layer chro-
matography (TLC) was run on Merck silica gel plates (Kie-
selgel 60 F-254) with detection by UV light, iodine, acidic
vanillin solution, or 10% phosphomolybdic acid solution in
ethanol. For flash chromatography, Merck silica gel type 60
(size 230-400 mesh) was used. The enantiomeric purities of
the (R)- and (S)-quinuclidine derivatives, determined by chiral
HPLC, were confirmed to be more than 98% ee. Unless stated
otherwise, all starting materials and reagents were high-grade
commercial 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.
The following intermediates were synthesized according to
literature procedures: benzimidazole-4-carboxylic acid (31),49
6-chlorobenzimidazole-4-carboxylic acid (32),27 2-amino-5-
bromo-3-methylaniline (36),50 6-chloro-4-methylbenzimidazole
(37),27 exo-8-methyl-8-azabicyclo[3.2.1]oct-3-ylamine,51 exo-9-
methyl-9-azabicyclo[3.3.1]non-3-ylamine,51 endo-9-methyl-9-
azabicyclo[3.3.1]non-3-ylamine,52 exo-8-methyl-8-azabicyclo-
[3.2.1]octan-3-ol,53 exo-9-methyl-9-azabicyclo[3.3.1]nonan-3-
ol,54 and endo-9-methyl-9-azabicyclo[3.3.1]nonan-3-ol.55
Gen er a l P r oced u r e for th e Syn th esis of Ben zim id a -
zole-4-ca r boxa m id es a n d Ben zim id a zole-4-ca r boxyla tes
(2-14, 16-19, a n d 22-30). To a solution of acids 31-35 (5
mmol) in dry DMF (5 mL) under an argon atmosphere was
added 1,1′-carbonyldiimidazole (CDI, 811 mg, 5 mmol). The
mixture was stirred at 40 °C for 1 h, then a solution of the
appropriate amine or alcohol (6-10 mmol) and 1,8-diazabicyclo-
[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
50 °C 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% aqueous K2CO3 (20 mL).
The organic layer was dried over Na2SO4 or MgSO4 and
evaporated to afford the crude product, which was purified by
column chromatography and recrystallization from the ap-
propriate solvents.
F igu r e 2. Each point shows the mean (the SEM are omitted
for clarity) of the contraction induced by 5-HT in the second
dose-response curve, expressed as the percentage of the
maximal response reached in the first dose-response curve,
in the absence (control) and in the presence of ondansetron or
granisetron (5 × 10-9 M) as reference 5-HT3 antagonists or of
the same concentration of derivatives 2, 8, and 14 (n g 6).
The asterisk (*) indicates the difference vs control value (p <
0.05, two ways ANOVA test); it refers to the second half of
the graph.
and are in general agreement as to which pharmacoph-
oric elements are important for significant binding
affinity. In our search for new 5-HT3 receptor antago-
nists, we recently reported a computer-aided conforma-
tional analysis24 that has allowed us to confirm the
generally accepted three-component pharmacophore for
5-HT3 receptor antagonists. In this study, we also
performed a receptor mapping of the 5-HT3 binding site,
which defines the essential volume occupied by this
receptor. The synthesized benzimidazole-4-carboxylic
acid derivatives represent a new structural class of
potent and selective 5-HT3 ligands that fit the 5-HT3
receptor antagonist pharmacophore and the 5-HT3
receptor essential volume. In these compounds, the
4-substituted benzimidazole ring represents a novel
aromatic moiety where a hydrogen bond between a
benzimidazole nitrogen atom and the hydrogen of the
amide group, in the case of carboxamides, or between
the oxygen of the ester group and the hydrogen of the
benzimidazole ring, in the case of carboxylates, is likely
to exist. On the other hand, a related derivative
synthesized in our laboratory ((()-N-(1-azabicyclo[2.2.2]-
oct-3-yl)-1,3-dihydro-2H-benzimidazole-2-one-4-carboxa-
mide) has been demonstrated as inactive at the 5-HT3
receptor, despite fitting the 5-HT3 antagonist pharma-
cophore and the 5-HT3 receptor essential volume.48 In
this case, the inactivity could be due to the absence of
a hydrogen bond in the molecule. This result supports
the hypothesis that this hydrogen bond may block the
acyl group in a pseudo aromatic cycle, and this is
recognized as an important point for a better interaction
of 5-HT3 antagonists with the receptor.16
Con clu sion
(()-N-(1-Aza bicyclo[2.2.2]oct-3-yl)ben zim id a zole-4-ca r -
boxa m id e (1): yield 0.9 g (35%); chromatography CHCl3/
MeOH, from 9:1 to 7:3; mp 196-198 °C (CHCl3/MeOH); 1H
NMR (Me2SO-d6) δ 1.39-1.46 (m, 1H, H5′b or H8′b), 1.58-
1.63 (m, 2H, 2H5′ or 2H8′), 1.85-1.92 (m, 2H, H4′, H5′a or
H8′a), 2.54-2.56 (m, 1H, H2′b), 2.70 (t, J ) 7.8, 2H, 2H6′ or
2H7′), 2.75-2.83 (m, 2H, 2H6′ or 2H7′), 3.26 (dd, J ) 13.5,
9.3, 1H, H2′a), 4.03-4.05 (m, 1H, H3′), 7.33 (t, J ) 7.5, 1H,
H6), 7.74 (dd, J ) 7.8, 0.9, 1H, H7), 7.86 (dd, J ) 7.5, 0.9, 1H,
H5), 8.47 (s, 1H, H2), 10.13 (br s, 1H, CONH); 13C NMR (Me2-
SO-d6) δ 20.2, 25.4 (C5′, C8′), 25.7 (C4′), 46.1, 46.9 (C6′, C7′),
In the present paper, we have synthesized a new
series of azabicyclic benzimidazole-4-carboxamides and
-carboxylates with affinity and selectivity for serotonin-
ergic 5-HT3 receptors. The novel benzimidazoles are an
enrichment to the SAR of the 5-HT3 field. Three
compounds of the series (2, 8, and 14) displayed very
high affinity at central 5-HT3 receptors, high selectivity
over other serotonin receptors (5-HT4 and 5-HT1A), and
potent 5-HT3 antagonist activity in the guinea pig ileum,

Synthesis of Azabicyclic Benzimidazole-4-carboxamides
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 24 5025
46.3 (C3′), 56.1 (C2′), 116.0 (C7), 122.1 (C4), 122.2 (C5, C6),
134.3 (C7a), 139.6 (C3a), 143.0 (C2), 164.4 (CONH). Anal.
(C15H18N4O) C, H, N.
r oben zim id a zole-4-ca r boxa m id e (11): yield 1.3 g (39%);
chromatography CHCl3/MeOH, from 7:3 to 3:7; mp 198-200
1
°C (d) (acetone); H NMR (Me2SO-d6) δ 1.64-2.08 (m, 10H),
2.66 (s, 3H), 3.20 (br t, 2H), 4.73-4.77 (m, 1H), 7.79 (d, J )
1.5, 1H), 7.83 (d, J ) 1.5, 1H), 8.44 (s, 1H), 9.50 (br s, 1H),
12.50 (br s, 1H); 13C NMR (Me2SO-d6) δ 18.7, 25.0, 32.4, 39.1,
41.7, 53.5, 116.6, 122.2, 122.9, 126.9, 136.6, 137.9, 144.5, 163.0.
Anal. (C17H21ClN4O) C, H, N.
N-(en d o-9-Met h yl-9-a za bicyclo[3.3.1]n on -3-yl)-6-ch lo-
r oben zim id a zole-4-ca r boxa m id e (12): yield 0.9 g (27%);
chromatography AcOEt/EtOH, from 9:1 to 2:8; mp >300 °C
(CHCl3); 1H NMR (Me2SO-d6) δ 0.96 (d, J ) 12.9, 2H), 1.36 (t,
J ) 12.6, 2H), 1.47 (dm, J ) 10.9, 1H), 1.90-2.00 (m, 3H),
2.31-2.40 (m, 2H), 2.42 (s, 3H), 3.01 (br d, J ) 9.9, 2H), 4.29-
4.41 (m, 1H), 7.74 (d, J ) 0.9, 1H), 7.77 (d, J ) 0.9, 1H), 8.35
(s, 1H), 9.62 (br s, 1H); 13C NMR (Me2SO-d6) δ 14.2, 23.2, 33.0,
39.6, 40.8, 50.6, 116.6, 122.2, 122.9, 126.9, 136.6, 137.9, 144.7,
163.1. Anal. (C17H21ClN4O) C, H, N.
(S)-(-)-N-(1-Aza bicyclo[2.2.2]oct-3-yl)ben zim id a zole-4-
ca r boxa m id e (2): yield 1.1 g (80%); chromatography CHCl3/
MeOH, from 9.5:0.5 to 9:1; mp 234-236 °C (AcOEt); [R]25
-25.6 (c ) 1, MeOH).
)
D
(R)-(+)-N-(1-Aza bicyclo[2.2.2]oct-3-yl)ben zim id a zole-4-
ca r boxa m id e (3): yield 1.1 g (84%); chromatography CHCl3/
MeOH, from 9.5:0.5 to 9:1; mp 247-248 °C (water); [R]25
+26.3 (c ) 1, MeOH).
)
D
N-(exo-8-Meth yl-8-a za bicyclo[3.2.1]oct-3-yl)ben zim id a -
zole-4-ca r boxa m id e (4): yield 1.2 g (42%); chromatography
CHCl3/MeOH, 1:1; mp 233-235 °C (CHCl3/Et2O); 1H NMR
(Me2SO-d6) δ 1.57-1.64 (m, 4H, H2′ax, H4′ax, H6′b, H7′b),
1.75-1.80 (m, 2H, H2′eq, H4′eq), 1.94-1.97 (m, 2H, H6′a,
H7′a), 2.18 (s, 3H, CH3), 3.09 (br t, 2H, H1′, H5′), 4.13-4.26
(m, 1H, H3′), 7.30 (t, J ) 7.8, 1H), 7.73 (d, J ) 7.8, 1H), 7.82
(d, J ) 7.8, 1H), 8.40 (s, 1H), 9.54 (d, J ) 5.4, 1H); 13C NMR
(Me2SO-d6) δ 26.2 (C6′, C7′), 37.5 (C2′, C4′), 39.4 (CH3), 40.6
(C3′), 60.2 (C1′, C5′), 116.6, 121.9, 122.0, 122.2, 135.4, 139.0,
143.1, 164.1. Anal. (C16H20N4O) C, H, N.
N-(exo-9-Met h yl-9-a za b icyclo[3.3.1]n on -3-yl)b en zim i-
d a zole-4-ca r boxa m id e (5): yield 1.9 g (64%); chromatogra-
phy CHCl3/MeOH, from 9:1 to 1:1; mp 203-205 °C (acetone);
1H NMR (Me2SO-d6) δ 1.71-2.16 (m, 10H, 2H2′, 2H4′, 2H6′,
2H7′, 2H8′), 2.75 (s, 3H, CH3), 3.32 (br t, 2H, H1′, H5′), 4.76-
4.84 (m, 1H, H3′), 7.35 (t, J ) 7.5, 1H), 7.79 (d, J ) 7.6, 1H),
7.88 (d, J ) 7.4, 1H), 8.50 (s, 1H), 9.81 (br s, 1H), 13.18 (br s,
1H); 13C NMR (Me2SO-d6) δ 18.4 (C7′), 25.0 (C6′, C8′), 32.4
(C2′, C4′), 38.7 (C3′), 41.2 (CH3), 53.7 (C1′, C5′), 115.0, 122.1,
122.2, 134.8, 139.1, 142.9, 164.1. Anal. (C17H22N4O) C, H, N.
N-(en d o-9-Meth yl-9-a za bicyclo[3.3.1]n on -3-yl)ben zim i-
d a zole-4-ca r boxa m id e (6): yield 1.5 g (50%); chromatogra-
phy CHCl3/MeOH, 9:1; mp 240-242 °C (MeOH/AcOEt); 1H
NMR (Me2SO-d6) δ 0.95 (d, J ) 11.7, 2H, H6′eq, H8′eq), 1.37
(t, J ) 11.7, 2H, H2′b, H4′b), 1.46 (d, J ) 11.1, 1H, H7′eq),
1.89-2.03 (m, 3H, H6′ax, H7′ax, H8′ax), 2.34-2.42 (m, 5H,
CH3, H2′a, H4′a), 3.01 (br d, J ) 10.5, 2H, H1′, H5′), 4.34-
4.45 (m, 1H, H3′), 7.34 (t, J ) 7.5, 1H), 7.67 (d, J ) 7.8, 1H),
7.88 (d, J ) 7.5, 1H), 8.47 (s, 1H), 9.73 (br s, 1H), 12.80 (br s,
1H); 13C NMR (Me2SO-d6) δ 13.9 (C7′), 24.0 (C6′, C8′), 33.0
(C2′, C4′), 39.4 (CH3), 40.6 (C3′), 50.4 (C1′, C5′), 116.3, 121.6,
121.7, 121.9, 135.2, 138.8, 142.8, 163.9. Anal. (C17H22N4O) C,
H, N.
(()-N -(1-Aza b icyclo[2.2.2]oct -3-yl)-6-ch lor o-7-n it r o-
ben zim id a zole-4-ca r boxa m id e (13): yield 0.8 g (46%); chro-
1
matography CHCl3/MeOH, 9:1; mp 265-266 °C (AcOEt); H
NMR (Me2SO-d6) δ 1.69-1.74 (m, 1H), 1.79-1.83 (m, 2H),
2.04-2.06 (m, 1H), 2.13 (sx, J ) 2.7, 1H), 2.94 (dd, J ) 13.8,
4.2, 1H), 3.03 (t, J ) 7.8, 2H), 3.10-3.15 (m, 2H), 3.55 (dd, J
) 13.2, 9.0, 1H), 4.23-4.25 (m, 1H), 7.80 (s, 1H, H5), 8.29 (s,
1H, H2), 10.33 (d, J ) 6.3, 1H, CONH); 13C NMR (Me2SO-d6)
δ 18.8, 23.4, 24.9, 45.5, 45.7, 46.3, 54.0, 114.5 (C6), 119.9 (C5),
122.5 (C4), 137.0, 138.2, 142.0 (C3a, C7, C7a), 152.3 (C2), 163.6
(CONH). Anal. (C15H16ClN5O3) C, H, N.
(S)-(-)-N-(1-Aza b icyclo[2.2.2]oct -3-yl)-6-ch lor o-7-n i-
tr oben zim id a zole-4-ca r boxa m id e (14): yield 1.4 g (79%);
chromatography CHCl3/MeOH, from 9.5:0.5 to 9:1; mp 187-
189 °C (CHCl3/Et2O); [R]25 ) -15.3 (c ) 1, MeOH).
D
(()-N-(1-Aza bicyclo[2.2.2]oct-3-yl)-6-br om oben zim id a -
zole-4-ca r boxa m id e (16): yield 1.4 g (80%); chromatography
CHCl3/MeOH, from 9.5:0.5 to 9:1; mp 259-260 °C (CHCl3/
Et2O); 1H NMR (Me2SO-d6) δ 1.45-1.51 (m, 1H), 1.61-1.65
(m, 2H), 1.84-1.86 (m, 1H), 1.92 (sx, J ) 2.7, 1H), 2.53-2.58
(m, 1H), 2.72 (t, J ) 7.5, 2H), 2.77-2.84 (m, 2H), 3.27 (dd, J
) 13.8, 9.6, 1H), 4.02-4.06 (m, 1H), 7.93 (d, J ) 1.5, 1H, H5),
7.98 (d, J ) 1.8, 1H, H7), 8.49 (s, 1H, H2), 9.93 (br s, 1H,
CONH); 13C NMR (Me2SO-d6) δ 20.2, 25.7, 25.4, 46.1, 46.9,
46.6, 55.7, 114.2 (C6), 119.0 (C7), 123.0 (C4), 124.4 (C5), 136.9,
138.2 (C3a, C7a), 144.4 (C2), 163.2 (CONH). Anal. (C15H17
BrN4O) C, H, N.
-
(()-N-(1-Aza bicyclo[2.2.2]oct-3-yl)-6-ch lor oben zim id a -
zole-4-ca r boxa m id e (7): yield 1.2 g (39%); mp 264-266 °C
(water); 1H NMR (Me2SO-d6) δ 1.45-1.51 (m, 1H), 1.61-1.67
(m, 2H), 1.83-1.87 (m, 1H), 1.94 (sx, J ) 3.0, 1H), 2.58 (dd, J
) 13.8, 4.5, 1H), 2.74 (t, J ) 7.8, 2H), 2.78-2.87 (m, 2H), 3.29
(dd, J ) 13.5, 9.6, 1H), 4.06-4.10 (m, 1H), 7.84 (d, J ) 2.1,
1H, H5), 7.86 (d, J ) 2.1, 1H, H7), 8.53 (s, 1H, H2), 9.96 (d, J
) 6.3, 1H, CONH); 13C NMR (Me2SO-d6) δ 20.1, 25.3, 25.6,
46.0, 46.8, 46.5, 55.7, 116.0 (C7), 121.7 (C5), 122.7, 126.5 (C4,
C6), 136.2, 137.8 (C3a, C7a), 144.5 (C2), 163.2 (CONH). Anal.
(C15H17ClN4O) C, H, N.
(S)-(-)-N-(1-Aza b icyclo[2.2.2]oct -3-yl)-6-b r om ob en z-
im id a zole-4-ca r boxa m id e (17): yield 1.4 g (80%); chroma-
tography CHCl3/MeOH, from 9.5:0.5 to 9:1; mp 292-293 °C
(d) (MeOH); [R]25 ) -24.9 (c ) 1, MeOH).
D
(R)-(+)-N-(1-Aza b icyclo[2.2.2]oct -3-yl)-6-b r om ob en z-
im id a zole-4-ca r boxa m id e (18): yield 1.4 g (81%); chroma-
tography CHCl3/MeOH, from 9.5:0.5 to 9:1; mp 259-260 °C
(d) (CHCl3); [R]25 ) +24.2 (c ) 1, MeOH).
D
(()-N -(1-Aza b icyclo[2.2.2]oct -3-yl)-6-b r om o-7-n it r o-
ben zim id a zole-4-ca r boxa m id e (19): yield 1.6 g (82%); chro-
matography CHCl3/MeOH, from 9.5:0.5 to 9:1; mp 196-197
(S)-(-)-N-(1-Aza bicyclo[2.2.2]oct-3-yl)-6-ch lor oben zim -
id a zole-4-ca r boxa m id e (8): yield 1.2 g (80%); chromatogra-
phy CHCl3/MeOH, from 9.5:0.5 to 9:1; mp 273-274 °C (d)
1
°C (AcOEt); H NMR (Me2SO-d6) δ 1.69-1.74 (m, 1H), 1.81-
1.87 (m, 2H), 2.06-2.09 (m, 1H), 2.16 (sx, J ) 3.0, 1H), 2.96
(dd, J ) 13.5, 4.5, 1H), 3.05 (t, J ) 7.8, 2H), 3.11-3.18 (m,
2H), 3.57 (ddd, J ) 13.2, 9.6, 2.1, 1H), 4.24-4.26 (m, 1H), 7.96
(s, 1H, H5), 8.28 (s, 1H, H2), 10.29 (br s, 1H, CONH); 13C NMR
(Me2SO-d6) δ 18.8, 23.4, 24.9, 45.5, 45.6, 46.2, 54.0, 101.4 (C6),
122.5 (C4, C5), 137.5, 140.2, 142.5 (C3a, C7, C7a), 152.3 (C2),
163.5 (CONH). Anal. (C15H16BrN5O3) C, H, N.
(water); [R]25 ) -27.7 (c ) 1, MeOH).
D
(R)-(+)-N-(1-Aza b icyclo[2.2.2]oct -3-yl)-6-ch lor ob en z-
im id a zole-4-ca r boxa m id e (9): yield 1.0 g (68%); chromatog-
raphy CHCl3/MeOH, from 9.5:0.5 to 9:1; mp 272-273 °C (d)
(water); [R]25 ) +26.9 (c ) 1, MeOH).
D
N-(exo-8-Meth yl-8-azabicyclo[3.2.1]oct-3-yl)-6-ch lor oben -
zim id a zole-4-ca r boxa m id e (10): yield 1.0 g (31%); chroma-
tography CHCl3/MeOH, from 9:1 to 8:2; mp 255-257 °C
(water); 1H NMR (Me2SO-d6) δ 1.60-1.67 (m, 4H), 1.75-1.81
(m, 2H), 1.97-2.00 (m, 2H), 2.22 (s, 3H), 3.13 (br t, 2H), 4.14-
4.25 (m, 1H), 7.79 (s, 1H), 7.83 (s, 1H), 8.45 (s, 1H), 9.38 (d, J
) 5.2, 1H); 13C NMR (Me2SO-d6) δ 26.0, 37.0, 39.1, 40.7, 60.1,
116.0, 121.6, 122.2, 126.3, 136.4, 138.0, 144.5, 162.8. Anal.
(C16H19ClN4O‚H2O) C, H, N.
(()-(1-Aza bicyclo[2.2.2]oct-3-yl) ben zim id a zole-4-ca r -
boxyla te (22): yield 2.1 g (77%); mp 210-212 °C (CHCl3/
Et2O); 1H NMR (CDCl3) δ 1.48-2.08 (m, 4H, 2H5′, 2H8′), 2.21
(sx, J ) 3.3, 1H, H4′), 2.79-3.04 (m, 5H, H2′b, 2H6′, 2H7′),
3.42 (ddd, J ) 14.7, 8.4, 1.5, 1H, H2′a), 5.10-5.12 (m, 1H, H3′),
7.35 (t, J ) 7.8, 1H, H6), 7.97 (dd, J ) 7.5, 0.9, 1H, H5 or H7),
8.06 (dd, J ) 7.8, 0.9, 1H, H7 or H5), 8.18 (s, 1H, H2), 11.20
(br s, 1H, NH); 13C NMR (CDCl3) δ 19.8, 24.6 (C5′, C8′), 25.4
(C4′), 45.6, 47.4 (C6′, C7′), 55.6 (C2′), 72.3 (C3′), 114.1 (C4),
N-(exo-9-Met h yl-9-a za b icyclo[3.3.1]n on -3-yl)-6-ch lo-

5026 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 24
Lo´pez-Rodrı´guez et al.
121.7 (C6), 124.9, 125.5 (C5, C7), 133.5 (C3a), 141.8 (C2), 143.7
(C7a), 166.2 (COO). Anal. (C15H17N3O2) C, H, N.
1H NMR (Me2SO-d6) δ 1.15 (d, J ) 12.6, 2H), 1.48 (dm, J )
13.5, 1H), 1.70 (ddd, J ) 13.8, 7.5, 1.8, 2H), 1.92 (tt, J ) 13.2,
4.5, 2H), 2.23 (qt, J ) 13.2, 5.1, 1H), 2.41 (s, 3H), 2.49 (dd, J
) 14.1, 5.1, 2H), 3.00 (br d, J ) 8.7, 2H), 5.38 (tt, J ) 6.9, 6.0,
1H), 7.79 (d, J ) 2.1, 1H), 8.06 (d, J ) 1.5, 1H), 8.40 (s, 1H),
12.66 (br s, 1H); 13C NMR (Me2SO-d6) δ 14.2, 24.2, 30.8, 40.3,
50.9, 68.2, 115.3, 123.6, 123.9, 125.4, 131.5, 144.8, 145.6, 163.7.
Anal. (C17H20ClN3O2) C, H, N.
(()-N-(1-Aza b icyclo[2.2.2]oct -3-yl)-7-a m in o-6-ch lor o-
ben zim id a zole-4-ca r boxa m id e (15). A solution of 13 (1.0
g, 3 mmol) in ethanol (150 mL) was hydrogenated over Raney
nickel at 50 psi and room temperature for 2 h (TLC). The
catalyst was removed by filtration over Celite, and the solvent
was evaporated under reduced pressure. The residue was
purified by column chromatography (CHCl3/MeOH/NH3, 8:1.5:
0.5) to yield 0.25 g (26%) of 15; mp 273-274 °C (MeOH/Et2O);
1H NMR (Me2SO-d6) δ 1.41-1.51 (m, 1H), 1.61-1.70 (m, 2H),
1.82-1.88 (m, 1H), 1.91-1.93 (m, 1H), 2.71-2.93 (m, 5H), 3.41
(m, 1H), 4.05 (m, 1H), 6.11 (s, 2H, NH2), 7.76 (br s, 1H, H5),
8.41 (br s, 1H, H2), 9.90 (br s, 1H, CONH), 12.37 (br s, 1H,
NH); 13C NMR (Me2SO-d6) δ 20.1, 25.4, 25.8, 46.3, 46.2, 46.9,
55.3, 108.1 (C4, C6), 124.0 (C5), 141.6 (C2), 164.3 (CONH).
Anal. (C15H18ClN5O) C, H, N.
(()-N-(1-Aza b icyclo[2.2.2]oct -3-yl)-7-a m in o-6-b r om o-
ben zim id a zole-4-ca r boxa m id e (20). To a solution of 19 (0.6
g, 1.5 mmol) in ethanol (46 mL) warmed at 60 °C were added
Raney nickel and 80% hydrazine hydrate (0.12 mL, 2 mmol).
The reaction mixture was refluxed for 15 min (TLC) and
filtered through Celite. The solvent was removed under
reduced pressure, and the residue was purified by column
chromatography (CHCl3/MeOH/NH3, 8:2:0.2) to yield 0.3 g
(56%) of 20: mp 278-279 °C (toluene); 1H NMR (Me2SO-d6) δ
1.40-1.51 (m, 1H), 1.61-1.65 (m, 2H), 1.83-1.87 (m, 1H),
1.90-1.92 (m, 1H), 2.58-2.64 (m, 1H), 2.72-2.75 (m, 2H),
2.82-2.87 (m, 2H), 3.21-3.28 (m, 1H), 4.02 (m, 1H), 6.04 (s,
2H, NH2), 7.92 (s, 1H, H5), 8.28 (br s, 1H, H2), 9.78 (br s, 1H,
CONH); 13C NMR (Me2SO-d6) δ 20.1, 25.4, 25.8, 46.2, 46.9,
46.4, 55.4, 97.4, 110.4 (C4), 126.8 (C5, C7a), 137.2 (C3a, C7),
141.5 (C2), 164.3 (CONH). Anal. (C15H18BrN5O) C, H, N.
(exo-8-Meth yl-8-a za bicyclo[3.2.1]oct-3-yl) ben zim id a -
zole-4-ca r boxyla te (23): yield 0.7 g (28%); chromatography
CHCl3/MeOH, 1:1; mp 164-166 °C (CHCl3/Et2O); 1H NMR
(CDCl3) δ 1.65-1.70 (m, 2H), 1.90-1.96 (m, 4H), 2.01-2.06
(m, 2H), 2.29 (s, 3H), 3.22 (br t, J ) 3.3, 2H), 5.27 (tt, J )
10.5, 6.5, 1H), 7.25 (t, J ) 7.8, 1H), 7.85 (dd, J ) 7.5, 0.9, 1H),
7.95 (dd, J ) 8.1, 0.9, 1H), 8.07 (s, 1H); 13C NMR (CDCl3) δ
26.3, 36.0, 38.8, 60.2, 68.0, 114.0, 121.6, 125.1, 125.2, 132.9,
141.4, 143.4, 165.7. Anal. (C16H19N3O2) C, H, N.
(en d o-8-Meth yl-8-a za bicyclo[3.2.1]oct-3-yl) ben zim id a -
zole-4-ca r boxyla te (24): yield 1.6 g (56%); mp 205-206 °C
(CHCl3/Et2O); 1H NMR (CDCl3) δ 1.86 (d, J ) 14.1, 2H, H2′eq,
H4′eq), 2.10 (m, 4H, 2H6′, 2H7′), 2.24 (dt, J ) 14.7, 4.2, 2H,
H2′ax, H4′ax), 2.30 (s, 3H, CH3), 3.16 (br t, J ) 3.0, 2H, H1′,
H5′), 5.31 (t, J ) 5.1, 1H, H3′), 7.33 (t, J ) 8.1, 1H), 7.86 (d,
J ) 7.5, 1H), 8.03 (d, J ) 8.1, 1H), 8.15 (s, 1H), 10.90 (br s,
1H); 13C NMR (CDCl3) δ 25.9 (C6′, C7′), 37.0 (C2′, C4′), 40.7
(CH3), 59.9 (C1′, C5′), 68.6 (C3′), 114.6, 121.9, 124.6, 125.5,
133.7, 141.8, 143.8, 166.1. Anal. (C16H19N3O2) C, H, N.
(exo-9-Meth yl-9-a za bicyclo[3.3.1]n on -3-yl) ben zim id a -
zole-4-ca r boxyla te (25): yield 1.2 g (40%); chromatography
1
CHCl3/MeOH, 1:1; mp 195-197 °C (acetone/Et2O); H NMR
(CDCl3) δ 1.52 (d, J ) 13.5, 2H), 1.69-1.77 (m, 2H), 1.96-
2.10 (m, 4H), 2.26 (td, J ) 10.5, 6.0, 2H), 2.59 (s, 3H), 3.08 (br
t, J ) 4.5, 2H), 5.83 (tt, J ) 10.5, 6.6, 1H), 7.30 (t, J ) 8.1,
1H), 7.92 (dd, J ) 8.1, 0.9, 1H), 8.00 (dd, J ) 7.8, 1.2, 1H),
8.13 (s, 1H); 13C NMR (CDCl3) δ 19.4, 26.3, 31.7, 40.2, 53.5,
68.8, 114.2, 121.5, 125.0, 125.2, 133.1, 141.6, 143.5, 165.7.
Anal. (C17H21N3O2) C, H, N.
(en d o-9-Meth yl-9-a za bicyclo[3.3.1]n on -3-yl) ben zim i-
d a zole-4-ca r boxyla te (26): yield 1.2 g (40%); chromatography
CHCl3/MeOH, 9:1; mp 198-200 °C (CHCl3/Et2O); 1H NMR
(CDCl3) δ 1.25 (dd, J ) 13.5, 4.2, 2H), 1.55 (dt, J ) 13.8, 5.1,
1H), 1.66 (ddd, J ) 14.7, 6.0, 1.8, 2H), 2.01 (tt, J ) 13.5, 5.1,
2H), 2.30 (qt, J ) 13.8, 5.1, 1H), 2.52 (s, 3H), 2.59 (dd, J )
14.7, 6.6, 2H), 3.04 (br d, J ) 8.4, 2H), 5.49 (tt, J ) 6.9, 6.0,
1H), 7.33 (t, J ) 8.1, 1H), 7.94 (d, J ) 7.5, 1H), 8.04 (d, J )
8.1, 1H), 8.22 (s, 1H), 11.10 (br s, 1H); 13C NMR (CDCl3) δ
14.4, 24.7, 31.0, 40.3, 51.9, 67.7, 114.3, 121.5, 124.6, 125.2,
133.4, 141.7, 143.6, 163.7. Anal. (C17H21N3O2) C, H, N.
(()-(1-Azabicyclo[2.2.2]oct-3-yl) 6-ch lor oben zim idazole-
4-ca r boxyla te (27): yield 1.2 g (39%); chromatography
CHCl3/MeOH, 9:1; mp 201-203 °C (Et2O/hexane); 1H NMR
(Me2SO-d6) δ 1.39-1.93 (m, 4H), 2.15 (sx, J ) 3.0, 1H), 2.69-
2.97 (m, 5H), 3.26 (ddd, J ) 14.7, 8.4, 1.5, 1H), 5.03-5.07 (m,
1H), 7.83 (d, J ) 2.1, 1H, H7 or H5), 8.08 (d, J ) 2.1, 1H, H7
or H5), 8.41 (s, 1H, H2), 12.75 (br s, 1H, NH); 13C NMR (Me2-
SO-d6) δ 19.3, 24.1, 25.0, 45.9, 46.7, 54.7, 72.7, 115.2 (C4), 123.4
(C5, C7), 125.3 (C6), 132.0 (C3a), 144.2 (C7a), 145.6 (C2), 163.9
(COO). Anal. (C15H16ClN3O2) C, H, N.
(()-N-(1-Aza bicyclo[2.2.2]oct-3-yl)-7-a m in oben zim id a -
zole-4-ca r boxa m id e (21). A solution of 19 (0.6 g, 1.5 mmol)
in ethanol (46 mL) was warmed to 60 °C. Then Pd(C) (186
mg, 1 mmol) and 80% hydrazine hydrate (0.18 mL, 3 mmol)
were added, and the mixture was refluxed for 30 min (TLC).
The reaction was treated as in 15 to yield 0.35 g (81%) of 21:
1
mp 200-201 °C (MeOH/Et2O); H NMR (Me2SO-d6) δ 1.80-
2.02 (m, 3H), 2.12-2.17 (m, 1H), 2.24-2.27 (m, 1H), 3.09-
3.50 (m, 5H), 3.76 (m, 1H), 4.37 (m, 1H), 6.11 (s, 2H, NH2),
6.48 (s, 1H, H6), 7.71 (s, 1H, H5), 8.38 (br s, 1H, H2), 10.10
(br s, 1H, CONH), 12.85 (br s, 1H, NH); 13C NMR (Me2SO-d6)
δ 17.4, 21.5, 24.5, 43.9, 45.0, 45.5, 52.9, 104.5 (C6), 124.8 (C5),
140.4 (C2), 165.9 (CONH). Anal. (C15H19N5O) C, H, N.
(en d o-8-Meth yl-8-a za bicyclo[3.2.1]oct-3-yl) 6-ch lor o-
ben zim id a zole-4-ca r boxyla te (28): yield 0.8 g (25%); chro-
matography CHCl3/MeOH, 9:1; mp 248-250 °C (CHCl3/Et2O);
1H NMR (Me2SO-d6) δ 2.01 (d, J ) 15.3, 2H), 2.15 (m, 4H),
2.39 (m, 2H), 2.46 (s, 3H), 3.48 (br t, J ) 3.1, 2H), 5.24 (br t,
J ) 5.1, 1H), 7.73 (s, 1H), 8.08 (s, 1H), 8.39 (s, 1H), 12.90 (br
s, 1H); 13C NMR (Me2SO-d6) δ 24.7, 34.6, 38.7, 59.8, 67.4, 114.8,
123.0, 125.3, 132.0, 144.2, 145.6, 163.7. Anal. (C16H18ClN3O2)
C, H, N.
Ra d ioliga n d Bin d in g Assa ys. For all receptor binding
assays, male Sprague-Dawley rats (Rattus norvegicus albi-
nus), weighing 180-200 g, were killed by decapitation and the
brains rapidly removed and dissected. Tissues were stored at
-80 °C for subsequent use and homogenized on a Polytron
PT-10 homogenizer. Membrane suspensions were centrifuged
on a Beckman J 2-HS instrument.
1. 5-HT1A Recep tor . Binding assays were performed by a
modification of the procedure previously described by Clark
et al.32 The cerebral cortex was homogenized in 10 volumes of
ice-cold 50 mM Tris-HCl buffer (pH 7.7 at 25 °C) and
centrifuged at 28000g for 15 min. The membrane pellet was
washed twice by resuspension and centrifugation. After the
third wash the resuspended pellet was incubated at 37 °C for
10 min. Membranes were then collected by centrifugation, and
the final pellet was resuspended in 50 mM Tris-HCl, 5 mM
MgSO4, and 0.5 mM EDTA buffer (pH 7.4 at 25 °C). Fractions
of the final membrane suspension (about 1 mg of protein) were
incubated at 37 °C 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 µM), in a final
volume of 1.1 mL of assay buffer (50 mM Tris-HCl, 10 nM
(exo-9-Meth yl-9-a za bicyclo[3.3.1]n on -3-yl) 6-ch lor o-
ben zim id a zole-4-ca r boxyla te (29): yield 1.3 g (39%); chro-
matography CHCl3/MeOH, 9:1; mp 195-197 °C (CHCl3/
1
MeOH); H NMR (MeSO-d6) δ 1.49 (d, J ) 13.5, 2H), 1.64-
1.70 (m, 2H), 1.84-1.95 (m, 4H), 2.15 (td, J ) 11.1, 6.0, 2H),
2.47 (s, 3H), 2.95 (br t, J ) 3.5, 2H), 5.81 (tt, J ) 10.1, 6.0,
1H), 7.79 (d, J ) 1.5, 1H), 8.06 (d, J ) 1.5, 1H), 8.38 (s, 1H),
12.62 (br s, 1H); 13C NMR (MeSO-d6) δ 19.9, 26.8, 30.6, 40.2,
53.1, 70.0, 115.4, 123.5, 125.3, 131.9, 144.1, 145.5, 163.7. Anal.
(C17H20ClN3O2) C, H, N.
(en d o-9-Met h yl-9-a za b icyclo[3.3.1]n on -3-yl) 6-ch lor o-
ben zim id a zole-4-ca r boxyla te (30): yield 0.8 g (24%); chro-
matography CHCl3/MeOH, 9:1; mp 240-242 °C (acetone/Et2O);

Synthesis of Azabicyclic Benzimidazole-4-carboxamides
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 24 5027
clonidine, and 30 nM prazosin, pH 7.4 at 25 °C). Nonspecific
binding was determined with 10 µM 5-HT and represented
less than 15% of the total binding.
percentage of the maximal contraction reached during the first
curve. Then, the ED50 of 5-HT, in the presence and absence of
the antagonists, and pA2 values were calculated for the second
phase of the curve ([5-HT] g 10-6 M).
2. 5-HT3 Recep tor . Binding assays were performed accord-
ing to the procedure previously described by Wong et al.29 The
cerebral cortex was homogenized in 9 volumes of 0.32 M
sucrose and centrifuged at 1000g for 10 min. The supernatant
was centrifuged at 17000g for 20 min. The membrane pellet
was washed twice by resuspension in 60 volumes of 50 mM
Tris-HCl buffer (pH 7.4 at 25 °C) and was centrifuged at
48000g for 10 min. After the second wash the resuspended
pellet was incubated at 37 °C for 10 min and centrifuged at
48000g for 10 min. Membranes were resuspended in 2.75
volumes of assay buffer (50 mM Tris-HCl, 10 µM pargyline,
0.6 mM ascorbic acid, and 5 mM CaCl2, pH 7.4 at 25 °C).
Fractions of 100 µL of the final membrane suspension (about
2 mg/mL of protein) were incubated at 25 °C for 30 min with
0.7 nM [3H]LY 278584 (83 Ci/mmol), in the presence or absence
of six concentrations of the competing drug, in a final volume
of 2 mL of assay buffer. Nonspecific binding was determined
with 10 µM 5-HT and represented less than 25% of the total
binding.
The effect of 2, 8, and 14 (5 × 10-9 M) on the contractions
induced by acetylcholine (10-6-10-5 M), substance P (10-7-5
× 10-6 M), histamine (10-6-5 × 10-6 M), and bradykinin (5 ×
10-7-5 × 10-6 M) or by electrical stimulation (single square
waves: 0.3 Hz, 2 ms, and supramaximal voltage) was also
tested.
Ack n ow led gm en t. This work has been supported
by the DGICYT (PB97-0282) and the Universidad
Complutense (PR486/97-7483). The authors are grateful
to UNED for predoctoral grants to B.B. and I.D.T.
Ondansetron and granisetron were generous gifts from
Glaxo-Welcome and SmithKline-Beecham, respectively.
Su p p or tin g In for m a tion Ava ila ble: Synthetic proce-
dures and characterization data for new intermediate com-
pounds 33-35 and 38-40. This material is available free of
charge via the Internet at http://pubs.acs.org.
3. 5-HT4 Recep tor . Binding assays were performed accord-
ing to the procedure previously described by Grossman et al.30
The striatum was homogenized in 15 volumes of ice-cold 50
mM HEPES buffer (pH 7.4 at 4 °C) and centrifuged at 48000g
for 10 min. The pellet was resuspended in 4.5 mL of assay
buffer (50 mM HEPES, pH 7.4 at 4 °C). Fractions of 100 µL of
the final membrane suspension were incubated at 37 °C for
30 min with 0.1 nM [3H]GR 113808 (85 Ci/mmol), in the
presence or absence of six concentrations of the competing
drug, in a final volume of 1 mL of assay buffer. Nonspecific
binding was determined with 30 µM 5-HT and represented
less than 35% of the total binding.
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 (twice
with 4 mL of ice-cold 50 mM Tris-HCl, pH 7.4 at 25 °C, for
5-HT1A and 5-HT3 receptor binding assays, and once with 4
mL of ice-cold 50 mM HEPES, pH 7.4 at 4 °C, for 5-HT4
receptor binding assays) and dried. The filters were placed in
poly(ethylene) vials to which were added 4 mL of a scintillation
cocktail (Aquasol), and the radioactivity bound to the filters
was measured by liquid scintillation spectrometry. The data
were analyzed by an iterative curve-fitting procedure (program
Prism, Graph Pad), which provided IC50, Ki, and r2 values for
test compounds, Ki values being calculated from the Cheng
and Prusoff equation.31 The protein concentrations of the rat
cerebral cortex and the rat striatum were determined by the
method of Lowry,56 using bovine serum albumin as the
standard.
5-HT4 Recep tor Activity in th e Isola ted Gu in ea P ig
Ileu m . Tissues were obtained from adult guinea pigs (300-
400 g). Portions of ileum were dissected 1.5 cm proximal to
the ileocecal junction and myenteric plexus-longitudinal muscle
strips were obtained from guinea pig ileum, as previously
described by Buchheit et al.,33 and mounted in an organ bath
containing Krebs solution at 37 °C and gassed with 5% CO2/
95% O2. Methysergide was added at a concentration of 10-6
M as described by Eglen et al.,34 in order to exclude any
potential effects on 5-HT1 or 5-HT2 receptors. Ileal responses
were measured by determining changes in isometric tension
recorded with force transducers connected to an Omniscribe
polygraph.
Two 5-HT (5 × 10-8 to 10-5 M) concentration-response
curves were constructed, in a noncumulative manner, in each
tissue. Compounds 2, 8, and 14, ondansetron, granisetron, or
saline solution (control tissues) were added 30 min before the
second curves were carried out. Contractions were evoked
every 5 min by addition of the separate doses of 5-HT, and
the bathing solution was replaced before addition of the
antagonists. The effect of 5-HT has been expressed as a
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