Benzimidazole-2-carboxylic acid amides and esters: A new structural class of 5-HT3 ligands

Article abstract of DOI:10.1016/S0223-5234(99)80091-9

A series of novel benzimidazole-2-carboxylic acid amides and esters with a quinuclidine or a tropane moiety were synthesized and evaluated for in vitro affinity for the 5-HT₃, 5-HT₄ and D₂ receptors. Compounds 15a, 13j and 13h exhibited affinity for the 5-HT₃ receptor (K(i) = 20.2, 18.4 and 12.7 nM, respectively) and no significant affinity for both 5-HT₄ and D₂ receptors. The amide-ester replacement did not induce significant changes in the affinity profile. The enantioselectivity for the 5-HT₃ receptor was reversed with regard to the zacopride pattern and the (R)-enantiomer 13c showed higher affinity (K(i) = 56.4 nM) than the (S)-enantiomer 13d (K(i) = 242.3 nM). An increment of the steric hindrance around the nitrogen atom at the 1-position of the benzimidazole ring led to an improvement in the affinity. The 5-HT₃ receptor antagonist activity of compounds with higher affinity was performed by evaluating the inhibition of the 5-HT induced von Bezold-Jarisch reflex. They displayed moderate 5-HT₃ antagonist activity (ED₅₀ = 10.6-29.1 μg/kg i.v.).

Full text of DOI:10.1016/S0223-5234(99)80091-9

Eur. J. Med. Chem. 34 (1999) 415−422
© Elsevier, Paris
415
Short communication
Benzimidazole-2-carboxylic acid amides and esters: a new structural class
of 5-HT3 ligands
Aurelio Orjales*, Luisa Alonso-Cires, Pedro López-Tudanca, Inés Tapia,
Ramón Mosquera, Luis Labeaga
Departamento de Investigación, FAES, S. A., Máximo Aguirre 14, 48940 Leioa (Vizcaya), Spain
(Received 8 June 1998; revised 2 November 1998; accepted 18 November 1998)
Abstract – A series of novel benzimidazole-2-carboxylic acid amides and esters with a quinuclidine or a tropane moiety were synthesized
and evaluated for in vitro affinity for the 5-HT3, 5-HT4 and D2 receptors. Compounds 15a, 13j and 13h exhibited affinity for the 5-HT3
receptor (Ki = 20.2, 18.4 and 12.7 nM, respectively) and no significant affinity for both 5-HT4 and D2 receptors. The amide-ester replacement
did not induce significant changes in the affinity profile. The enantioselectivity for the 5-HT3 receptor was reversed with regard to the
zacopride pattern and the (R)-enantiomer 13c showed higher affinity (Ki = 56.4 nM) than the (S)-enantiomer 13d (Ki = 242.3 nM). An
increment of the steric hindrance around the nitrogen atom at the 1-position of the benzimidazole ring led to an improvement in the affinity.
The 5-HT3 receptor antagonist activity of compounds with higher affinity was performed by evaluating the inhibition of the 5-HT induced von
Bezold-Jarisch reflex. They displayed moderate 5-HT3 antagonist activity (ED50 = 10.6–29.1 µg/kg i.v.). © Elsevier, Paris
benzimidazole-2-carboxamide / benzimidazole-2-carboxylate / tropane / quinuclidine / 5-HT3 ligand
1. Introduction
indicating a particularly good fit between the receptor and
these heterocycles, i.e. tropisetron 1, granisetron 2 and
zacopride 3 (figure 1).
In order to find new 5-HT3 receptor antagonists with
selectivity over 5-HT4 and D2 receptors, we prepared a
series of novel benzimidazole-2-carboxylic acid deriva-
tives including a quinuclidine or a tropane moiety.
In recent years the 5-HT3 receptor subtype has at-
tracted much attention because of the effectiveness of
5-HT3 receptor antagonists in the treatment of
chemotherapy-induced emesis [1, 2]. In addition, they are
expected to be useful for the treatment of anxiety,
schizophrenia, migraine and irritable bowel syn-
drome [3–5]. Some of these compounds, which proved to
behave as 5-HT3 receptor antagonists, also show affinity
at the 5-HT4 serotonin receptor subtype [6, 7]. Several
5-HT3 antagonist benzamides have also dopamine recep-
tor antagonist properties, which induce unfavorable ef-
fects in man such as central nervous system depression
and extrapyramidal syndrome [8].
2. Chemistry
Benzimidazole-2-carboxylic acid 6 was synthesized as
described in figure 2. 2-Hydroxymethylbenzimidazole 5
was prepared from 1,2-phenylenediamine 4 and glycolic
acid, according to the Phillips procedure [12]. Oxidation
of 5 with potassium permanganate afforded acid 6 [13].
3-Aminotropane derivatives 9 and 10 were prepared
using previously reported methods (figure 3) [14, 15].
The reaction of tropinone with hydroxylamine gave the
oxime 8 which was reduced with lithium aluminum
hydride to give the endo isomer 9; sodium reduction of 8
afforded the exo isomer 10. Other amines and alcohols
used were commercially available. Amides 11 were
Several studies have suggested that there are three
structural requirements for 5-HT3 receptor antagonism:
an aromatic ring, a linking acyl function and a basic
nitrogen group [9–11]. Many of the reported 5-HT3
receptor antagonists include an azabicycloalkane moiety,
*Correspondence and reprints
Apartado 555, 48080 Bilbao, Spain

416
Figure 1. 5-HT3 receptor antagonists.
obtained from the acid 6 following a standard procedure,
by coupling with the appropriate amine using carbonyl-
diimidazole (CDI) (figure 4). Amides 13 were synthe-
sized by reaction of the corresponding amines with
trimethylaluminium and subsequent treatment of the
resulting dimethylaluminium amides with a carboxylic
ester 12 [16]. Acid 6 was condensed with an alcohol
using CDI and 1,8-diazabicyclo[5.4.0]undec-7-ene
(DBU) to give ester derivatives 14; treatment of 14 with
sodium hydride followed by reaction with an alkyl halide
led to esters 15.
3. Pharmacology
Compounds were evaluated for their in vitro 5-HT3,
5-HT4 and D2 receptor affinities by radioligand binding
assays [17–19]. For each compound, the ability to dis-
place the specific ligand ([3H]-LY278584, [3H]-
GR113808, and [3H]-Raclopride, respectively) from re-
ceptors was determined. The antagonist activity at the
5-HT3 receptor was performed by evaluating the inhibi-
tion of the Bezold-Jarisch reflex evoked by 5-HT in
urethane-anaesthetized rats (table I).
Figure 2. Synthesis of benzimidazole-2-carboxylic acid 6. Reagents: a) HO2CCH2OH, 4 N HCl, 40 min, reflux; b) KMnO4, 10%
NaOH, 3 h, room temperature.
Figure 3. Preparation of 3-aminotropane derivati-
ves 9 and 10. Reagents: a) NH2OH, pyridine,
EtOH, 2 h, reflux; b) LiAlH4, 10% H2SO4, 2 h,
reflux; c) Na, EtOH, 2 h, reflux.

417
Figure 4. Preparation of compounds 11, 12, 13, 14 and 15.
Reagents: a) CDI, R1NH2, DMF, 20 h, room temperature; b) HNa, R2Hal, DMF, 3 h, room temperature; c) Me3Al, R1NH2, PhCH3,
3 h, reflux; d) CDI, DMF, DBU, R1OH, 24 h, 60 °C; e) HNa, R2Hal, DMF, 3 h, room temperature.
4. Results and discussion
enantiomers have higher affinity for the 5-HT3 receptor
than the (R)-enantiomers [23–25]. A similar inversion of
enantioselectivity has been reported for naphtalimides,
benzoylureas, and indolyl-2-acetamides [26–28]. The in-
crease in the potency of the (R)-enantiomer could indicate
the existence of a different and relevant bond between the
compound and the receptor site.
Most of the synthesized compounds exhibited
moderate-to-high affinity for the 5-HT3 receptor and no
significant affinity for either the 5-HT4 or the D2 recep-
tors.
The amide-ester replacement did not induce significant
changes on the affinity. Compounds 11a (Ki = 172.3 nM)
and 13j (Ki = 18.4 nM) showed a similar affinity value to
14a (Ki = 171.7 nM) and 15a (Ki = 20.2 nM), respec-
tively. Only amide 11b (Ki = 143.9 nM) displayed ca.
2-fold higher affinity than its related ester 14b (Ki = 260.1
nM). A similar equipotency of amides and esters has been
reported for several benzoic acid derivatives [20, 21].
With regard to the tropane moiety, the endo-derivative
11b showed up to 1 000-fold more potent affinity for the
5-HT3 receptor than the analogue exo-derivative 11c (Ki
> 1 000 nM). The inactivity of the axially substituted
tropane derivatives is well known and has been re-
ported [22]. On the other hand, the influence of the
chirality of the quinuclidine moiety was examined.
Whereas the (R)-enantiomer 13c had higher affinity (Ki =
56.4 nM) than the racemate 13a (Ki = 81.6 nM), the
(S)-enantiomer 13d showed weaker affinity (Ki = 242.3
nM). Consequently, the enantioselectivity for the 5-HT3
receptor was reversed with regard to that of zacopride and
related 3-quinuclidinyl amides in which the (S)-
With respect to the introduction of an alkyl substituent
at the 1-position of the benzimidazole ring, an increment
of the steric hindrance around the nitrogen atom led to an
improvement in the affinity for the 5-HT3 receptor; e. g.
compare 11b (Ki = 143.9 nM) and 13j (Ki = 18.4 nM);
11a (Ki = 172.3 nM) and 13i (Ki = 24.4 nM); 14b (Ki =
260.1 nM) and 15a (Ki = 20.2 nM). However, replace-
ment of the hydrogen in 11b by a methyl group led to 13b
(Ki = 158.5 nM), with no change in the affinity. Besides,
the affinity of the N-benzyl derivative 13j (Ki = 18.4 nM)
was somewhat lower to that of the isopropyl analogue
13h (Ki = 12.7 nM). Moreover, compounds 13a (Ki =
81.6 nM), 13e (Ki = 65.2 nM) and 13g (Ki = 70.4 nM) did
not show any remarkable difference in the affinity.
We evaluated the 5-HT3 receptor-antagonist activity of
the compounds with higher affinity (13h, 13i, 13j and
15a) in the inhibition of the 5-HT induced von Bezold-
Jarisch reflex assays in anaesthetized rats. They exhibited

418
Table I. 5-HT3, 5-HT4, and D2 receptor binding data and in vivo 5-HT3 antagonism.
Compounda
X
R1
R2
5-HT3 affinityb 5-HT4 affinityb D2 affinityb
B-J reflex inhibitionc
11a
11b
11c
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
O
A
H
H
H
Me
Me
Me
Me
Et
172.3 ± 10.2
143.9 ± 15.6
> 1000
> 1000
> 1000
> 1000
> 1000
> 1000
> 1000
> 1000
> 1000
> 1000
> 1000
> 1000
> 1000
> 1000
> 1000
> 1000
> 1000
> 1000
125.1 ± 15.8
101.2 ± 15.6
> 1000
> 1000
> 1000
> 1000
> 1000
> 1000
> 1000
> 1000
> 1000
> 1000
> 1000
> 1000
> 1000
> 1000
> 1000
> 1000
> 1000
> 1000
54.8 ± 4.8
n.t.g
endo-B
exo-B
A
endo-B
A (R)
A (S)
A
endo-B
A
endo-B
A
n.t.g
n.t.g
13ars
13b
81.6 ± 10.9
158.5 ± 17.2
56.4 ± 3.1
242.3 ± 23.6
65.2 ± 4.5
39.6 ± 5.7
70.4 ± 16.5
12.7 ± 1.9
24.4 ± 1.5
18.4 ± 1.8
171.7 ± 31.1
260.1 ± 48.9
20.2 ± 4.2
1.8 ± 0.1
n.t.g
n.t.g
13cd
13de
13e
n.t.g
n.t.g
n.t.g
13f
Et
n.t.
13gf
i-Pr
i-Pr
PhCH2
PhCH2
H
n.t.g
13hf
12.8 ± 1.5
19.1 ± 3.6
14.3 ± 2.8
n.t.g
13i
13j
14a
endo-B
A
endo-B
endo-B
14b
O
O
H
PhCH2
n.t.g
15af
10.6 ± 1.8
0.7 ± 0.2
1.7 ± 0.3
n.t.g
granisetron
tropisetron
cisapride
1.5 ± 0.1
511.8 ± 36.4
c
d
20
20
aC, H, N analyses were within ± 0.4% of the theoretical values; bKi (nM); ED50 (µg/kg i.v.); [α]D + 27° (c = 0.19, EtOH); e[α]D –27°
f
g
(c = 0.19, EtOH); fumarate; not tested.
moderate 5-HT3 antagonist activity, with ED50 values of
10.6–19.1 mg/kg i.v. (granisetron: 0.7 mg/kg i.v.).
We conclude that these compounds represent a new
structural class of 5-HT3 ligands with selectivity over
5-HT4 and D2 receptors and moderate 5-HT3 antagonist
activity when intravenously administered. Studies are in
progress in order to obtain more potent 5-HT3 receptor
antagonists in this series.
consistent with assigned structures. Specific optical rota-
tion at the sodium D line were measured in a 1 dm sample
tube on a Perkin-Elmer 341 polarimeter.
The following compounds were prepared as described
in the literature: benzimidazole-2-carboxylic acid 6 [13],
endo-3-aminotropane 9 [15], and exo-3-aminotropane
10 [14].
5. Experimental protocols
5.1.1. General procedure for compounds 11
To a solution of benzimidazole-2-carboxylic acid 6
(10 mmol) in DMF (40 mL), 1,1≠-carbonyldiimidazole
(11 mmol) was added. The reaction mixture was stirred at
room temperature for 1 h, and then an appropriate amine
(11 mmol) was added. Stirring was continued overnight
and then the solvent evaporated. The residue was poured
into water (30 mL) and extracted with CH2Cl2 (3 ×
20 mL). The organic layer was dried (Na2SO4) and the
solvent was evaporated under reduced pressure to yield
the crude product 11 which was further purified by silica
gel column chromatography (MeOH:NH4OH, 98:2).
5.1. Chemistry
Flash column chromatography was performed on silica
gel, particle size 60 Å, mesh = 23–400 (Merck). Melting
points were determined in open capillary tubes on a
Büchi SMP-20 apparatus and are uncorrected. Elemental
analyses are within ± 0.4% of the theoretical values. IR
spectra were taken on a Perkin-Elmer 1310 instrument on
KBr plates. 1H- and 13C-NMR were recorded on a Bruker
AC-200; chemical shifts (δ) are reported in parts per
million (ppm) downfield from tetramethylsilane (TMS),
which was used as an internal standard. Spectral data are

419
5.1.1.1. N-(1-azabicyclo[2.2.2]octan-3-yl)-1H-benzimi-
dazole-2-carboxamide 11a
5.1.2.1. N-(1-azabicyclo[2.2.2]octan-3-yl)-1-methyl-1H-
benzimidazole-2-carboxamide 13a
Yield: 67%; m.p.: 145–147 °C. 1H NMR (CDCl3) 1.54
(m, 1H, CH2), 1.75 (m, 2H, CH2), 1.89 (m, 1H, CH2),
2.10 (m, 1H, CH2), 2.72–3.02 (m, 5H, CH2), 3.48 (m, 1H,
CH), 4.21 (m, 1H, CH), 7.32–7.36 (m, 2H, ar), 7.51 (m,
1H, ar), 7.77 (m, 1H, ar), 7.89 (br d, 1H, CONH), 12.25
(br s, 1H, NH). 13C NMR (CDCl3) 19.97, 25.66, 25.88,
46.45, 47.14, 47.26, 55.05, 112.37, 120.19, 123.34,
124.75, 134.48, 142.65, 145.20, 158.47.
Yield: 64%; m.p.: 143–145 °C. 1H NMR (CDCl3) 1.58
(m, 1H, CH2), 1.72 (m, 2H, CH2), 1.83 (m, 1H, CH2),
2.05 (m, 1H, CH2), 2.53–2.99 (m, 5H, CH2), 3.43 (m, 1H,
CH), 4.15 (m, 1H, CH), 4.22 (s, 3H, CH3), 7.31–7.42 (m,
3H, ar), 7.45–7.79 (m, 1H, ar), 7.89 (br d, 1H, CONH).
13C NMR (CDCl3) 20.18, 25.82, 25.84, 31.96, 46.64,
46.84, 47.49, 55.79, 110.37, 120.46, 123.43, 124.51,
136.95, 140.87, 143.25, 159.69.
5.1.1.2. endo-N-(8-methyl-8-azabicyclo[3.2.1]octan-3-
yl)-1H-benzimidazole-2-carboxamide 11b
5.1.2.2. endo-N-(8-methyl-8-azabicyclo[3.2.1]octan-3-
yl)-1-methyl-1H-benzimidazole-2-carboxamide 13b
Yield: 73%; m.p.: 183–185 °C. 1H NMR (CDCl3)
1.76–2.43 (m, 8H, CH2), 2.38 (s, 3H, CH3), 3.25 (m, 2H,
CH), 4.39 (m, 1H, CH), 7.28–7.34 (m, 2H, ar), 7.54 (m,
1H, ar), 7.81 (m, 1H, ar), 8.15 (br d, 1H, CONH), 12.30
(br s, 1H, NH). 13C NMR (CDCl3) 25.70, 36.17, 40.16,
41.83, 59.83, 112.28, 120.31, 123.07, 124.56, 134. 35,
142.76, 145.22, 158.82.
1
Yield: 74%; m.p.: 143–145 °C. H NMR (DMSO-d6)
1.68–2.09 (m, 8H, CH2), 2.15 (s, 3H, CH3), 3.03 (m, 2H,
CH), 4.05 (m, 1H, CH), 4.12 (m, 3H, CH3), 7.28–7.38
(m, 2H, ar), 7.64–7.74 (m, 2H, ar), 8.32 (br d, 1H,
CONH). 13C NMR (DMSO-d6) 25.51, 31.69, 35.30,
39.07, 40.74, 59.22, 111.22, 120.04, 123.03, 124.06,
136.68, 140.48, 143.88, 158.46.
5.1.1.3. exo-N-(8-methyl-8-azabicyclo[3.2.1]octan-3-yl)-
1H-benzimidazole-2-carboxamide 11c
5.1.2.3
N-(1-azabicyclo[2.2.2]octan-3-yl)-1-ethyl-1H-
benzimidazole-2-carboxamide 13e
Yield: 71%; m.p.: 250–252 °C. 1H NMR (DMSO-
d6) 1.42–2.14 (m, 8H, CH2), 2.20 (s, 3H, CH3), 3.06 (m,
2H, CH), 4.13 (m, 1H, CH), 7.24 (m, 2H, ar), 7.58 (m,
2H, ar), 8.70 (br d, 1H, CONH), 12.25 (br s, 1H, NH).
13C NMR (DMSO-d6) 26.36, 35.46, 38.38, 39.91, 59.69,
112.94, 120.49, 123.37, 124.92, 134.84, 142.82, 145.06,
158.61.
1
Yield: 68%; m.p.: 120–122ºC. H NMR (CDCl3) 1.48
(t, 3H, CH3, J = 7.2 Hz), 1.50 (m, 1H, CH2), 1.72 (m, 2H,
CH2), 1.85 (m, 1H, CH2), 2.05 (m, 1H, CH2), 2.55–2.99
(m, 5H, CH2), 3.42 (m, 1H, CH), 4.14 (m, 1H, CH), 4.78
(q, 2H, CH2, J = 7.2 Hz), 7.27–7.48 (m, 3H, ar), 7.77 (m,
1H, ar), 7.81 (br d, 1H, CONH). 13C NMR (CDCl3)
15.51, 20.20, 25.84, 25.85, 40.40, 46.67, 46.88, 47.41,
55.72, 110.45, 120.56, 123.36, 124.46, 135.96, 141.05,
142.78, 159.47.
5.1.2. General procedure for compounds 13
To an ice-cooled solution of benzimidazole-2-
carboxylic acid 6 (30 mmol) in 60 mL of DMF, HNa
(60% suspension, 66 mmol) was added, and the mixture
was stirred for 30 min. The appropriate alkyl halide
(66 mmol) was added dropwise and the resulting suspen-
sion was stirred for 3 h at room temperature. Aqueous
sodium hydroxide (10%, 20 mL) was slowly added and
the mixture extracted with dichloromethane (3 × 20 mL).
The combined organic layers were dried (Na2SO4) and
concentrated, and compound 12 was isolated by silica gel
chromatography (Cl2CH2:MeOH, 9:1).
5.1.2.4. endo-N-(8-methyl-8-azabicyclo[3.2.1]octan-3-
yl)-1-ethyl-1H-benzimidazole-2-carboxamide 13f
1
Yield: 57%; m.p.: 197–199 °C. H NMR (DMSO-d6)
1.43 (t, 3H, CH3, J = 7.2 Hz), 2.02–2.45 (m, 8H, CH2),
2.15 (s, 3H, CH3), 3.63 (m, 2H, CH), 4.05 (m, 1H, CH),
4.81 (q, 2H, CH2, J = 7.2 Hz), 7.28–7.48 (m, 2H, ar),
7.75–7.84 (m, 2H, ar), 8.82 (br d, 1H, CONH). 13C NMR
DMSO-d6) 15.53, 23.68, 32.33, 37.56, 39.07, 40.74,
60.22, 111.19, 120.23, 123.04, 124.21, 135.51, 140.74,
143.78, 159.18.
To a solution of an appropriate amine (11 mmol) in
30 mL of toluene, a solution of trimethylaluminium in
toluene (2 M, 11 mmol) was added, and the resulting
mixture was stirred for 1 h at room temperature. Com-
pound 12 (9 mmol) was added, and the mixture was
heated to reflux for 3 h and then cooled. Water (10 mL)
was added dropwise, and the resulting precipitate was
filtered off. Evaporation of the filtrate under reduced
pressure and further purification by silica gel chromatog-
raphy (MeOH:NH4OH, 98:2) afforded 13.
5.1.2.5. N-(1-azabicyclo[2.2.2]octan-3-yl)-1-isopropyl-
1H-benzimidazole-2-carboxamide (fumarate) 13g
1
Yield: 51%; m.p.: 122 °C (dec). H NMR (DMSO-d6)
1.54 (d, 6H, CH3, J = 6.9 Hz), 1.53 (m, 1H, CH2), 1.73
(m, 2H, CH2), 1.87 (m, 1H, CH2), 2.06 (m, 1H, CH2),
2.79–3.22 (m, 5H, CH2), 3.42 (m, 1H, CH), 4.21 (m, 1H,
CH), 5.64 (m, 1H, CH), 7.21–7.38 (m, 2H, ar), 7.68–7.87
(m, 2H, ar), 9.21 (br d, 1H, CONH). 13C NMR (DMSO-
d6) 18.36, 21.01, 23.27, 25.26, 40.76, 45.16, 45.52, 48.15,

420
51.03, 110.45, 120.56, 123.36, 124.46, 135.96, 141.05,
142.78, 159.47.
5.1.3.1. 1-azabicyclo[2.2.2]octan-3-yl benzimidazole-2-
carboxylate 14a
1
Yield: 42%; m.p.: 128 °C (dec). H NMR (DMSO-d6)
1.40 (m, 1H, CH2), 1.63 (m, 2H, CH2), 2.88 (m, 1H,
CH2), 2.17 (m, 1H, CH2), 2.61–2.87 (m, 5H, CH2), 3.27
(m, 1H, CH), 5.12 (m, 1H, CH), 7.23–7.40 (m, 2H, ar),
7.60–7.79 (m, 2H, ar). 13C NMR (DMSO-d6) 19.12,
24.31, 25.21, 46.18, 46.93, 54.57, 73.68, 112.72, 114.65,
121.60, 124.51, 139.81, 142.06, 149.96, 159.92.
5.1.2.6. endo-N-(8-methyl-8-azabicyclo[3.2.1]octan-3-
yl)-1-isopropyl-1H-benzimidazole-2-carboxamide
(fu-
marate) 13h
1
Yield: 53%; m.p.: 183–185 °C. H NMR (DMSO-d6)
1.57 (d, 6H, CH3, J = 6.92 Hz), 2.04–2.40 (m, 8H, CH2),
2.15 (s, 3H, CH3), 3.61 (m, 2H, CH), 4.01 (m, 1H, CH),
5.70 (m, 1H, CH), 7.24–7.35 (m, 2H, ar), 7.74–7.86 (m,
2H, ar), 8.77 (br d, 1H, CONH). 13C NMR (DMSO-d6)
21.01, 23.84, 32.44, 37.68, 40.75,48.16, 60.19, 113.39,
120.53, 122.62, 123.75, 134.03, 141.45, 144.44, 159.90.
5.1.3.2.
endo-8-methyl-8-azabicyclo[3.2.1]octan-3-yl
benzimidazole-2-carboxylate 14b
Yield: 49%; 106 °C (dec). 1H NMR (DMSO-d6)
1.92–2.32 (m, 8H, CH2), 2.30 (s, 3H, CH3), 3.21 (m, 2H,
CH), 5.29 (m, 1H, CH), 7.27–7.40 (m, 2H, ar), 7.72 (m,
2H, ar), 12.21 (br s, 1H, NH). 13C NMR (DMSO-d6)
25.92, 36.63, 40.52, 59.82, 70.58, 115.70, 116.67, 121.85,
123.91, 138.59, 142.16, 151.01, 159.80.
5.1.2.7.
N-(1-azabicyclo[2.2.2]octan-3-yl)-1-phenyl-
methyl-1H-benzimidazole-2-carboxamide 13i
1
Yield: 64%; m.p.: 179–181ºC. H NMR (DMSO-d6)
1.24 (m, 1H, CH2), 1.59 (m, 2H, CH2), 1.75 (m, 1H,
CH2), 1.86 (m, 1H, CH2), 2.60–3.12 (m, 5H, CH2), 3.40
(m, 1H, CH), 4.07 (m, 1H, CH), 5.96 (s, 2H, CH2),
7.15–7.38 (m, 7H, ar), 7.63 (m, 1H, ar), 7.76 (m, 1H, ar),
8.87 (br d, 1H, CONH). 13C NMR (DMSO-d6) 19.88,
25.62, 25.86, 46.37, 46.62, 46.95, 47.46, 53.06, 111.68,
120.17, 123.13, 124.30, 126.98, 127.43, 128.53, 135.76,
137.31, 140.80, 143.93, 159.60.
5.1.2.8. endo-N-(8-methyl-8-azabicyclo[3.2.1]octan-3-
yl)-1-phenylmethyl-1H-benzimidazole-2-carboxamide 13j
1
Yield: 70%; m.p.: 125–127ºC. H NMR (DMSO-d6)
1.68–2.09 (m, 8H, CH2), 2.14 (s, 3H, CH3), 3.02 (m, 2H,
CH), 4.03 (m, 1H, CH), 5.95 (s, 2H, CH2), 7.17–7.36 (m,
7H, ar), 7.60 (m, 1H, ar), 7.77 (m, 1H, ar), 8.41 (br d, 1H,
CONH). 13C NMR (DMSO-d6) 25.48, 35.21, 39.91,
41.16, 47.53, 59.20, 111.72, 120.27, 123.24, 124.36,
126.95, 127.42, 128.52, 135.94, 137.23, 140.67, 143.63,
158.48.
5.1.3. General procedure for compounds 14
To a solution of benzimidazole-2-carboxylic acid 6
(10 mmol) in DMF (40 mL), 1,1≠-carbonyldiimidazole
(11 mmol) was added. After stirring the reaction mixture
for 3 h at room temperature, the corresponding alcohol
5.2. Pharmacology
5.2.1. 5-HT3 receptor binding assay [17]
Adult male Wistar rats weighing 220–280 g, were
used. Animals were killed by decapitation and the whole
brain, with the exception of the brainstem and cerebel-
lum, was quickly removed and the various areas dis-
sected, weighed and immediately frozen at –70 °C. En-
thorinal cortex used for the binding experiments was
homogenized with an Ultra-Turrax (setting 5 for 20 s) in
10 volumes of ice-cold 0.32 M sucrose buffer, centrifuged
at 1 000 g for 10 min (4 °C) and the supernatant was
(11 mmol)
and
1,8-diazabicyclo[5.4.0]undec-7-ene
(11 mmol) were added, and the mixture was heated at
60 °C for 18 h. Solvent was removed under reduced
pressure and the residue was extracted with Cl2CH2 (3 ×
20 mL). The combined organic layers were dried
(Na2SO4) and evaporated to give a residue which was
purified by silica gel chromatography (Cl2CH2: triethy-
lamine, 95:5) to yield 14.

421
recentrifuged at 17 000 g for 20 min (4 °C). The resulting
pellet was resuspended in 50 volumes of ice-cold 50 mM
Tris-HCl buffer (pH 7.4), incubated at 37 °C for 10 min
and then centrifuged three times more at 48 000 g for
10 min (4 °C). The final pellet was resuspended in 10
volumes of ice-cold 50 mM Tris-HCl buffer containing
5 mM CaCl2 and 0.1% ascorbate, and was stored at
–70 °C until use. At the time of the experiment, the
membranes were diluted in the same ice-cold buffer with
10 µM pargyline (final dilution 1:40, wt/vol). Competi-
tion assays were performed in triplicate, in a final volume
of 1 mL. To each assay tube were added the following:
0.1 mL of the displacer drug concentration (0.1 mL of
vehicle if no competing drug was added) and 0.1 mL of
[3H]-LY278584 (Amersham, 60–85 Ci/mmol) in buffer
(final concentration 2 nM). Non-specific binding was
determined using 10 µM cold 5-HT. Binding experiments
were initiated by addition of 0.8 mL of membrane sus-
pension (500–600 µg of protein). After incubation for
30 min at 25 °C the reaction was stopped for vacuum
filtration through Whatman GF/B glass pretreated filters
(1% polyethylenimine in 50 mM Tris-HCl buffer), using
a Brandel Cell Harvester, followed by two washes with
5 mL of ice-cold 50 mM Tris-HCl (pH 7.4) buffer. Filters
were placed in polyethylene scintillation vials (with 5 mL
of scintillation cocktail), equilibrated, and filter-retained
radioactivity measured in a liquid scintillation counter
(Kontron Beta V). IC50 and Ki values were calculated
using the computer program EBDA (McPherson).
membrane suspension (800–900 µg of protein). After
incubation for 30 min at 25 °C the reaction was stopped
for vacuum filtration through Whatman GF/B glass pre-
treated filters (1% polyethylenimine in 50 mM Hepes
buffer), using a Brandel Cell Harvester. Filters were
placed in polyethylene scintillation vials (with 5 mL of
scintillation cocktail), equilibrated, and filter-retained
radioactivity measured in a liquid scintillation counter
(Kontron Beta V). IC50 and Ki values were calculated
using the computer program EBDA (McPherson).
5.2.3. D2 receptor binding assay [19]
Adult male Wistar rats weighing 220–280 g were used.
Animals were killed by decapitation and the whole brain,
with the exception of the brainstem and cerebellum, was
quickly removed and the various areas dissected,
weighed and immediately frozen at –70 °C. Striatum used
for the binding experiments was homogenized with an
Ultra-Turrax (setting 5 for 20 s) in 50 volumes of ice-cold
50 mM Tris-HCl buffer (pH 7.7), centrifuged at 48 000 g
for 10 min (4 °C). The resulting pellet was resuspended in
50 volumes of ice-cold 50 mM Tris-HCl buffer (pH 7.7),
incubated at 37 °C for 10 min and then centrifuged once
more at 48 000 g for 10 min (4 °C). The resulting pellet
was resuspended in 10 volumes of ice-cold 50 mM
Tris-HCl buffer (pH 7.4) and was stored at –70 °C until
use. At the time of experiment, the membranes were
diluted in the same ice-cold buffer with 10 µM pargyline
(final dilution 1:150, wt/vol). Competition assays were
performed in triplicate, in a final volume of 1 mL. To
each assay tube were added the following: 0.1 mL of the
displacer drug concentration (0.1 mL of vehicle if no
competing drug was added) and 0.1 mL of [3H]Raclo-
pride (NEN, 60-87 Ci/mmol) in buffer (final concentra-
tion 1 nM). Non-specific binding was determined using 1
µM cold (+)-butaclamol. Binding experiments were ini-
tiated by addition of 0.8 mL of membrane suspension
(300–400 µg of protein). After incubation for 60 min at
25 °C the reaction was stopped for vacuum filtration
through Whatman GF/B glass filters, using a Brandel Cell
Harvester, followed by two washes with 5 mL of ice-cold
50 mM Tris-HCl (pH 7.7) buffer. Filters were placed in
polyethylene scintillation vials (with 5 mL of scintillation
cocktail), equilibrated, and filter-retained radioactivity
measured in a liquid scintillation counter (Kontron Beta
V). IC50 and Ki values were calculated using the com-
puter program EBDA (McPherson).
5.2.2. 5-HT4 receptor binding assay [18]
Adult male Dunkin-Hartley guinea-pigs weighing
350–400 g were used. Animals were killed by decapita-
tion and the whole brain, with the exception of the
brainstem and cerebellum, was quickly removed and the
various areas dissected, weighed and immediately frozen
at –70 °C. Striatum used for the binding experiments was
homogenized with an Ultra-Turrax (setting 5 for 20 s) in
15 volumes of ice-cold 50 mM Hepes buffer (pH 7.4) and
centrifuged at 48 000 g for 20 min (4 °C). The resulting
pellet was resuspended in 10 volumes of ice-cold 50 mM
Hepes buffer (pH 7.4) and was stored at –70 °C until use.
At the time of the experiment, the membranes were
diluted in the same ice-cold buffer (final dilution 1:80,
wt/vol). Competition assays were performed in triplicate,
in a final volume of 1 mL. To each assay tube were added
the following: 0.1 mL of the displacer drug concentration
(0.1 mL of vehicle if no competing drug was added) and
0.1 mL of [3H]GR-113808 (Amersham, 60–85 Ci/mmol)
in buffer (final concentration 0.1 nM). Non-specific
binding was determined using 10 µM cold 5-HT. Binding
experiments were initiated by addition of 0.8 mL of
5.2.4. Inhibition of the von Bezold-Jarisch reflex
Adult male Wistar rats weighing 220–300 g and fasted
for 18 h were used. Rats were anaesthetized with ure-
thane (1.25 g/kg i.p.) and placed on a heating table (Hugo

422
[5] Costall B., Naylor R.J., Pharmacol. Toxicol. 70 (1992) 157–162.
Sachs Elektronik, Freiburg, Germany) to maintain body
temperature at 37 °C. The trachea and right jugular vein
were cannulated to facilitate respiration and drug admin-
istration, respectively. The carotid artery was also cannu-
lated and connected to a Goul Statham P23Db pressure
transducer to record blood pressure. The heart rate was
measured using the blood pressure signal and a cardio-
tachometer coupler and recorded on a Graphtec Linear-
corder WR3101 (Hugo Sachs Elektronik, Freiburg, Ger-
many). The B-J reflex was evoked by rapid bolus i.v.
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Drugs were prepared daily and were dissolved in distilled
water or 0.1 M tartaric acid, with subsequent dilutions in
physiological saline. ED50 values were calculated from
3–5 doses (4–5 rats per dose) by a linear-regression
analysis.
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This work was supported in part by the Dirección
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Industry and Energy of Spain and the Department of
Industry, Agriculture and Fishery of the Basque Govern-
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