2,3-dihydro-2-oxo-1H-benzimidazole-1-carboxamides with selective affinity for the 5-HT4 receptor: Synthesis and structure-affinity and structure-activity relationships of a new series of partial agonist and antagonist derivatives

Article abstract of DOI:10.1021/jm981098j

A series of 2,3-dihydro-2-oxo-1H-benzimidazole-l-carboxamide derivatives bearing a piperazine moiety was synthesized. Their in vitro 5-HT₄, 5-HT₃, and D₂ receptors affinities were evaluated by radioligand binding assay. For selected compounds functional studies at the 5-HT₄ receptor were made by using precontracted (by carbachol) preparations of rat esophageal tunica muscularis mucosae (TMM). The influence of the 3-substituent of the benzimidazole ring, the 4-substituent of the piperazine moiety, and the alkylene spacer was studied. Compounds with an ethyl or a cyclopropyl substituent in the 3-position of the benzimidazole ring showed moderate to high affinity (K(i) = 6.7-75.4 nM) for the 5-HT₄ receptor with selectivity over 5-HT₃ and D₂ receptors and moderate antagonist activity (pK(b) = 6.19- 7.73). Compounds with an isopropyl substituent in the 3-benzimidazole position exhibited moderate and selective 5-HT₄ affinity (K(i) ≥ 38.9 nM) and a partial agonist activity (5a, i.a. = 0.94) higher than that of the reference compound BIMU 8 (i.a. = 0.70). This reversal of the pharmacological activity due only to a small structural difference might confirm the existence of two binding sites on the 5-HT₄ receptor. In the alkylene spacer, a two-methylene chain is favorable to optimize the affinity and the antagonist or the partial agonist activity. In the ethyl and cyclopropyl series, 5-HT₄ antagonist activity seems to be unrelated to the size of the 4-substituent of the piperazine moiety, whereas a methyl group is optimal for high partial agonist activity in the isopropyl series; however, the presence of a butyl substituent is a favorable pattern for 5-HT₄ antagonism and even causes a reversal of the pharmacological profile in the isopropyl series (5h, pK(b) = 7.94). N-Butyl quaternization of 5a led to an improvement in affinity for the 5-HT₄ receptor and maintained the high partial agonist activity (5r, K(i) = 66.3 nM, i.a. = 0.93).

Full text of DOI:10.1021/jm981098j

2870
J . Med. Chem. 1999, 42, 2870-2880
2,3-Dih yd r o-2-oxo-1H-ben zim id a zole-1-ca r boxa m id es w ith Selective Affin ity for
th e 5-HT4 Recep tor : Syn th esis a n d Str u ctu r e-Affin ity a n d Str u ctu r e-Activity
Rela tion sh ip s of a New Ser ies of P a r tia l Agon ist a n d An ta gon ist Der iva tives
Ine´s Tapia, Luisa Alonso-Cires, Pedro Luis Lo´pez-Tudanca, Ramo´n Mosquera, Luis Labeaga,
Ana Innera´rity, and Aurelio Orjales*
Departamento de Investigacio´n, FAES, S.A., Ma´ximo Aguirre 14, 48940 Leioa, Vizcaya, Spain
Received February 22, 1999
A series of 2,3-dihydro-2-oxo-1H-benzimidazole-1-carboxamide derivatives bearing a piperazine
moiety was synthesized. Their in vitro 5-HT4, 5-HT3, and D2 receptors affinities were evaluated
by radioligand binding assay. For selected compounds functional studies at the 5-HT4 receptor
were made by using precontracted (by carbachol) preparations of rat esophageal tunica
muscularis mucosae (TMM). The influence of the 3-substituent of the benzimidazole ring, the
4-substituent of the piperazine moiety, and the alkylene spacer was studied. Compounds with
an ethyl or a cyclopropyl substituent in the 3-position of the benzimidazole ring showed
moderate to high affinity (Ki ) 6.7-75.4 nM) for the 5-HT4 receptor with selectivity over 5-HT3
and D2 receptors and moderate antagonist activity (pKb ) 6.19-7.73). Compounds with an
isopropyl substituent in the 3-benzimidazole position exhibited moderate and selective 5-HT4
affinity (Ki g 38.9 nM) and a partial agonist activity (5a , i.a. ) 0.94) higher than that of the
reference compound BIMU 8 (i.a. ) 0.70). This reversal of the pharmacological activity due
only to a small structural difference might confirm the existence of two binding sites on the
5-HT4 receptor. In the alkylene spacer, a two-methylene chain is favorable to optimize the
affinity and the antagonist or the partial agonist activity. In the ethyl and cyclopropyl series,
5-HT4 antagonist activity seems to be unrelated to the size of the 4-substituent of the piperazine
moiety, whereas a methyl group is optimal for high partial agonist activity in the isopropyl
series; however, the presence of a butyl substituent is a favorable pattern for 5-HT4 antagonism
and even causes a reversal of the pharmacological profile in the isopropyl series (5h , pKb )
7.94). N-Butyl quaternization of 5a led to an improvement in affinity for the 5-HT4 receptor
and mantained the high partial agonist activity (5r , Ki ) 66.3 nM, i.a. ) 0.93).
In tr od u ction
The 5-HT4 receptor was discovered in 1987, and
shortly later it was shown that a series of gastrointes-
tinal prokinetic benzamide derivatives (metoclopramide,
renzapride, cisapride, etc.) act as agonists at the 5-HT4
receptor.1-3 Since then, the 5-HT4 receptor has been
located in the central nervous system (CNS) (limbic
system, hippocampo-habenulo-interpeduncular path-
way, striato-nigro-tectal pathway) and peripheral
tissues (gastrointestinal tract, heart, adrenal gland,
urinary bladder).4-7 Considerable progress has been
made with the development of specific radioligands,
such as [3H]GR 113808 and [125I]SB 207710.8,9 The
5-HT4 receptor was cloned from rat brain in 1995, and
F igu r e 1. 5-HT4 receptor antagonists.
it was found that it exists in two isoforms (5-HT4S and
5-HT4L) that differ in the length and sequence of their
carboxy termini.10,11 Recently, the molecular structure
and functional characterization of four splice variants
of the human 5-HT4 receptor have been described.12
ketones, 8-naphthalimides, and benzimidazolones. Some
of the most known 5-HT4 ligands are included in Figures
1 and 2.
Recently, three-dimensional maps of the 5-HT4 ago-
nist and antagonist recognition sites have been pro-
posed.14-16 The structural features that define the 5-HT4
pharmacophore can be regarded as an aromatic moiety,
a coplanar carbonyl function, and a basic nitrogen atom;
distances between pharmacophoric elements and their
spatial orientations have been determined. For both
5-HT4 receptor agonist and antagonist recognition sites,
the existence of a hydrophobic pocket which would
To date, several 5-HT4 receptor antagonists and
agonists have been discovered.13,14 The chemical struc-
tures of the 5-HT4 antagonists belong to five different
classes including indolecarboxylates, benzoates, aryl
ketones, imidazolopyridines, and benzimidazolones. On
the other hand, there are six distinct classes of 5-HT4
agonists including indoles, benzamides, benzoates, aryl
10.1021/jm981098j CCC: $18.00 © 1999 American Chemical Society
Published on Web 07/02/1999

2,3-Dihydro-2-oxo-1H-benzimidazole-1-carboxamides
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 15 2871
F igu r e 2. 5-HT4 receptor agonists and DAU 6215 (5-HT3 receptor antagonist).
Sch em e 1a
F igu r e 3. 2,3-Dihydro-2-oxo-1H-benzimidazole-1-carboxam-
ides 1-10.
interact with the voluminous substituents of the basic
nitrogen of active ligands is accepted. On the other
hand, the existence of two binding sites at the 5-HT4
receptor has been suggested to explain the agonist and
antagonist activities of compounds with structural
similarity.17
With regard to benzimidazolone derivatives, it has
been reported that members of this class of compounds
interact with both 5-HT3 and 5-HT4 receptors with
different profiles of agonist or antagonist activity and
are devoid of affinity for the dopamine receptors.18,19
Among them, DAU 6215 (Figure 2) is a selective 5-HT3
receptor antagonist, whereas affinities of DAU 6285
(Figure 1) for 5-HT3 and 5-HT4 receptors are of the same
order of magnitude and it behaves as a competitive
5-HT4 antagonist.20 In addition, BIMU 1 and BIMU 8
a
Reagents: (a) i, 2,2-dimethoxypropane, TFA, ii, H3B-pyridine;
(Figure 2) combine 5-HT3 antagonism and 5-HT4
(b) cyclopropylamine; (c) H2, 10% Pd/C, or Zn, 1 N NaOH, EtOH;
(d) CDI, THF; (e) HNa, DMF, RI; (f) HNa, DMF, (tBOC)2O; (g)
RBr, K2CO3, DMF; (h) HNO3, Ac2O.
agonism.21-23
Although the 5-HT4 agonism or antagonism of some
benzimidazolones is well-known, very little has been
published about their 5-HT4 receptor affinity and their
structure-affinity relationships.24 It is possible that the
combination of 5-HT3 antagonism and 5-HT4 agonism
will have advantages over a selective 5-HT4 agonist ac-
tion in some areas.25 However, selective 5-HT4 receptor
ligands in this series could provide a useful tool to inves-
tigate the pharmacology of the 5-HT4 receptor and to
develop new therapies for the treatment of CNS disor-
ders (cognition, emotional and motor functional control)
and other peripherical disorders (irritable bowel syn-
drome, arrhythmia, urinary disturbance). Our aim was
to find new benzimidazolones with affinity for the 5-HT4
receptor and selectivity versus 5-HT3 and D2 receptors.
In the present paper, we report the synthesis and the
binding profile at 5-HT4, 5-HT3, and D2 receptors of a
series of 2,3-dihydro-2-oxo-1H-benzimidazole-1-carbox-
amides including a piperazine moiety, 1-10 (Figure 3).
We have studied the influence on the 5-HT4 receptor
affinity of each of these elements: the 3-substituent of
the benzimidazolone ring, the 4-substituent of the
piperazine moiety, and the alkylene spacer. In addition
the functional activity at 5-HT4 receptors has been also
evaluated. Preliminary results in structure-affinity and
structure-activity relationships are presented.
Ch em istr y
2,3-Dihydro-2-oxo-1H-benzimidazole-1-carboxam-
ides 1-10 were prepared by using 2,3-dihydro-2-oxo-
1H-benzimidazoles 13 and appropriate aminoalkylpi-
perazines 18 as key intermediates (Schemes 1-3).
According to Scheme 1 reductive amination of com-
mercially available 2-nitroanilines by using 2,2-dimeth-

2872 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 15
Tapia et al.
Sch em e 2a
Sch em e 3a
a
Reagents: (a) K2CO3, CH3CN, R′Br; (b) 48% HBr; (c) K2CO3,
CH3CN, Cl(CH2)n-1CN; (d) H4LiAl, Et2O.
oxypropane/trifluoroacetic acid and a boron-pyridine
complex yielded the isopropyl derivatives 11 (R ) i-Pr).
Cyclopropyl derivatives 11 (R ) c-Pr) were prepared
following a previously described route:26 2-chloroni-
trobenzene was heated with cyclopropylamine in a
closed vessel to afford N-cyclopropyl-2-nitroaniline 11b
(X, Y, Z ) H; R ) c-Pr). Compounds 11 (R ) i-Pr, c-Pr)
were hydrogenated over palladium/carbon to give o-
phenylenediamines 12 (R ) i-Pr, c-Pr) which, by cy-
clization with N,N′-carbonyldiimidazole, gave 1-alkyl-
2,3-dihydro-2-oxo-1H-benzimidazoles 13 (R ) i-Pr, c-Pr).
Alkylation of commercially available 2,3-dihydro-2-oxo-
1H-benzimidazole with sodium hydride and the corre-
sponding alkyl halide provided a mixture of mono- and
dialkylated derivatives; isolation of monoalkylated de-
rivatives 13 (R ) n-Pr, n-Bu, allyl, PhCH2) was ac-
complished by flash chromatography. Treatment of 2,3-
dihydro-2-oxo-1H-benzimidazole with di-tert-butyl dicar-
bonate according to reported conditions27 yielded the
alkoxycarbonyl-protected derivatives 14, which were
alkylated with an alkyl halide; the spontaneous elimi-
nation of the protecting group BOC provided derivatives
13 (R ) PhCH2CH2).
Aminoalkylpiperazines 18 were prepared following
standard procedures28 (Scheme 2). Alkylation of N-
carboxyethylpiperazine with a bromoalkane in acetoni-
trile in the presence of potassium carbonate afforded
the corresponding derivatives 15, which, after hydrolysis
with 48% HBr, yielded the alkylpiperazines 16; by
alkylation with chloroalkylnitriles in acetonitrile and
potassium carbonate, compounds 16 were converted to
17, which, after reduction with lithium aluminum
hydride in ether, provided the amines 18.
a
Reagents: (a) ClCO2CCl3, THF, charcoal; (b) 60% HNa, THF,
phosgene; (c) 18, TEA, THF; (d) Cl(CH2)nNCO, toluene; (e) K2CO3,
toluene, KI, 16, or TEA, CH3CN, KI, 16.
derivatives 20 (method C), which were converted into
the 2,3-dihydro-2-oxo-1H-benzimidazole-1-carboxamides
1-10 by reaction with substituted piperazines 16.
P h a r m a cology
The new benzimidazolone derivatives were initially
evaluated for in vitro 5-HT4 receptor affinity by radio-
ligand binding assay. For each compound the ability to
displace the specific ligand [3H]GR 113808 from 5-HT4
receptors of guinea pig striatum was determined.31
Concentrations required to inhibit 50% of radioligand
specific binding (IC50) and Ki values were calculated
from two separate competition experiments with samples
in triplicate, using 10-12 different concentrations of
displacer. Compounds with moderate to high affinity
were selected for functional studies at the 5-HT4 recep-
tor by using rat isolated thoracic esophageal tunica
muscularis mucosae (TMM) precontracted by carba-
chol.32,33
In agonism studies, responses to the cumulative
addition of 5-HT or agonist were expressed as percent-
age relaxation of carbachol-induced tone. For each
agonist, the negative logarithm of the molar concentra-
tion that relaxated 50% relative to their individual
maximun effect (pEC50) was calculated. The intrinsic
activity (i.a.) versus 5-HT was determined comparing
the maximun relaxation obtained at the concentration
of 1 or 3 µM in the same TMM preparation.
In antagonism studies, the effect of the antagonists
on 5-HT pEC50 values was registered, and the values
were compared by using the unpaired Student’s t-test.
A p value of 0.05 or less was considered statistically
significant. The apparent affinity (pKb) for antagonists
against 5-HT was determined.
For each compound, the ability to displace the specific
ligands [3H]LY 278584 and [3H]raclopride from 5-HT3
sites of rat entorhinal cortex or dopamine D2 sites of
rat striatum, respectively, was also determined.34,35 No
functional studies were carried out with the compounds
that exhibited affinity at these receptors.
2,3-Dihydro-2-oxo-1H-benzimidazole-1-carboxam-
ides 1-10 were prepared through different routes of
synthesis (Scheme 3). Chlorocarbonyl derivatives 19
were obtained according to previously reported condi-
tions by employing trichloromethyl chloroformate in the
presence of activated charcoal18 (method A) or by reac-
tion with sodium hydride and phosgene29 (method B).
Coupling with substituted piperazines 18 yielded 2,3-
dihydro-2-oxo-1H-benzimidazole-1-carboxamides 1-10.
Reaction of 2,3-dihydro-2-oxo-1H-benzimidazoles 13
with chloroalkyl isocyanates30 provided chloroalkyl

2,3-Dihydro-2-oxo-1H-benzimidazole-1-carboxamides
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 15 2873
Ta ble 1. 2,3-Dihydro-2-oxo-1H-benzimidazole-1-carboxamides
synthetic
5-HT4 binding 5-HT3 binding 5-HT4 antagonism
5-HT4
compd
R
method
mp, °C
formula
15H21N5O2
Ki (nM) ( SEa
IC50 (nM)
pKb ( SEa
agonism i.a.
1
2
H
Me
Et
A
A
A
A
A
C
A
A
C
A
188-190
87-90
C
C
=1000b
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
0.5 ( 0.1e
>1000
<6.0
0.35
0.42
<0.30
0.48
0.94
0.36
<0.30
0.63
16H23N5O2
305.8 ( 20.8
75.4 ( 19.9
252.9 ( 54.4
91.1 ( 10.8
10.2 ( 3.2
>1000b
<6.0
3a
4
5a
6a
7
194-196c C17H25N5O2‚2C4H4O4
6.79 ( 0.12
<6.0
n-Pr
205-208c C18H27N5O2
i-Pr
126-128
104-108
C
C
18H27N5O2
18H25N5O2
<6.0
c-Pr
n-Bu
allyl
PhCH2
Ph(CH2)2
7.24 ( 0.18
<6.0
202-204c C19H29N5O2‚2C4H4O4
8
9
198-201c C18H25N5O2‚2C4H4O4
>1000b
<6.0
110-113
C22H27N5O2
C23H29N5O2‚2HCl
>1000b
<6.0
<0.30
<0.30
0.70
10
BIMU 8
GR 113808
212 decd
>1000b
<6.0
63.9 ( 14.8
0.11 ( 0.01
<6.0
9.49 ( 0.06
NT
a
b
d
Standard error. IC50 (nM). c Fumarate. Hydrochloride. e Ki (nM).
F igu r e 5. Concentration-relaxation curves to 5-HT, BIMU
8, and compound 5a in rat isolated TMM. Values are mean,
with SEM, n ) 2-3.
F igu r e 4. Competition for [3H]GR 113808 binding sites in
guinea pig striatum by compounds 5a , 6a , GR 113808, and
BIMU 8.
respect to the reference compound BIMU 8. It can
therefore be concluded that in the benzimidazolone
series replacement of the rigid tropane ring (BIMU 8
and BIMU 1) by a flexible piperazine moiety signifi-
cantly reduced or abolished 5-HT3 affinity while re-
tained the 5-HT4 affinity. No compound displayed
significant affinity for the D2 receptor (IC50 > 1 µM, data
not shown).
The 5-HT4 receptor activity was also dependent on
the substituent at the 3-position of the benzimidazolone
ring. Ethyl and cyclopropyl derivatives 3a and 6a
showed moderate antagonist activity (pKb ) 6.79 and
7.24, respectively). However, isopropyl derivative 5a and
BIMU 8 acted as partial agonists, showing a classical
biphasic concentration-relaxation curve with pEC50
values of 6.54 and 6.73 and with an intrinsic activity of
0.94 and 0.70, respectively (Figure 5). At concentrations
higher than 1 µM, both compounds showed 5-HT4-
independent relaxation effects, probably due to a mus-
carinic cholinoceptor blockade.33 The 5-HT4 antagonist
GR 113808 induced a parallel, dextral shift of the
concentration-relaxation curves for 5-HT, 5a , and
BIMU 8 in the rat TMM with pKb values from 9.1 to
9.5 (data not shown). The observed change on the
pharmacological profile of benzimidazolones 1-10 sug-
Resu lts a n d Discu ssion
Pharmacological results are reported in Tables 1-4.
To study the effect of the 3-substituent of the benz-
imidazolone ring and because of the synthetic avail-
ability, we fixed the 4-methylpiperazine moiety and a
two-methylene spacer as our starting point. Examina-
tion of the results in Table 1 showed that the nature of
the substituent in the 3-position of the benzimidazolone
ring was critical for 5-HT4 receptor affinity. Only com-
pounds with ethyl (3a , Ki ) 75.4 nM), isopropyl (5a , Ki
) 91.1 nM), and cyclopropyl (6a , Ki ) 10.2 nM) substit-
uents showed moderate to high affinity for the 5-HT4
receptor, and 6a was even more potent than the refer-
ence benzimidazolone BIMU 8 (Ki ) 63.9 nM), although
it showed weaker affinity than GR 113808 (Ki ) 0.11
nM) (Figure 4). Replacement by other small groups such
as hydrogen (1, IC50 = 1 µM), methyl (2, Ki ) 305.8 nM),
or propyl (4, Ki ) 252.9 nM) led to a large decrease in
affinity. In addition, the introduction of bulkier groups,
e.g., butyl (7), benzyl (9), phenylethyl (10), severely
reduced 5-HT4 receptor affinity (IC50 > 1 µM).
All compounds showed a remarkable improvement in
selectivity versus 5-HT3 receptor (IC50 > 1 µM) with

2874 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 15
Tapia et al.
Ta ble 2. 2,3-Dihydro-2-oxo-1H-benzimidazole-1-carboxamides: 3-Ethyl and 3-Cyclopropyl Derivatives
synthetic
method
5-HT4 binding 5-HT3 binding 5-HT4 antagonism
5-HT4
agonism i.a.
compd
R
n
R′
mp, °C
formula
Ki (nM) ( SEa
IC50 (nM)
pKb ( SEa
3a
3b
3c
3d
3e
6a
6b
6c
6d
6e
6f
Et
Et
Et
Et
2
2
2
2
2
2
2
2
2
1
3
Me
Et
A
C
A
C
A
C
C
B
B
C
B
194-196b C17H27N5O2‚2C4H4O4
75.4 ( 19.9
17.9 ( 1.9
16.9 ( 2.0
25.2 ( 2.5
34.1 ( 2.0
10.2 ( 3.2
8.2 ( 1.0
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
6.79 ( 0.12
6.89 ( 0.14
7.63 ( 0.12
7.42 ( 0.10
7.73 ( 0.18
7.24 ( 0.18
7.46 ( 0.16
7.38 ( 0.15
7.51 ( 0.20
6.19 ( 0.10
6.40 ( 0.09
<0.30
<0.30
<0.30
<0.30
<0.30
0.36
0.41
<0.30
0.46
90-93
C
C
18H27N5O2
19H29N5O2
n-Pr
i-Pr
n-Bu
Me
n-Pr
i-Pr
n-Bu
Me
97-100
208-210b C19H29N5O2‚2C4H4O4
Et
203-205b C20H31N5O2‚2C4H4O4
c-Pr
c-Pr
c-Pr
c-Pr
c-Pr
c-Pr
104-108
62-64
oil
C
C
C
C
C
18H25N5O2
20H29N5O2
20H29N5O2
21H31N5O2
17H23N5O2
6.7 ( 0.4
oil
13.3 ( 0.9
49.4 ( 8.2
39.7 ( 7.4
86-88
0.53
0.46
Me
>250c
C19H27N5O2‚2HCl
a
b
Standard error. Fumarate. c Hydrochloride.
gests that the mode of binding might not be identical
through the series. A similar dramatic variation in the
5-HT4 pharmacological activity due only to a small
structural modification has been previously reported for
benzoate derivatives; as a consequence, a hypothetical
model for the 5-HT4 receptor with two sites for the
binding of agonist and antagonist molecules was pro-
posed.17
5-HT4 antagonism, e.g., compare 3b and 3e or 6c and
6d . Since those compounds were approximately equi-
potent, antagonist activity for the 5-HT4 receptor seems
to be unrelated to the size of the 4-substituent of the
piperazine moiety in the ethyl and cyclopropyl series.
The modification of the alkylene spacer was the next
step to be studied. The shortening of the two-methylene
chain of derivative 6a led to a decrease in affinity (6e,
Ki ) 49.4 nM) and also reduced the antagonist activity
(pKb ) 6.19). On the other hand, the lengthening to a
three-methylene chain caused a similar reduction in
both affinity and antagonist activity (6f, Ki ) 39.7 nM,
pKb ) 6.40). Consequently, in this series a chain length
of two methylenes seem to be optimal for high affinity
and antagonist activity at the 5-HT4 receptor site.
Similar structural modifications were also carried out
on the 3-isopropyl derivative 5a (Table 3). As a general
trend a significant reduction in 5-HT4 receptor affinity
with regard to the corresponding 3-ethyl and 3-cyclo-
propyl derivatives was observed, with a partial agonist
profile comparable to that of the reference compound
BIMU 8 (Ki ) 63.9 nM, i.a. ) 0.70). Surprisingly, butyl
derivative 5h (Ki ) 38.9 nM, pKb ) 7.94) showed the
most potent 5-HT4 antagonist activity in our series,
which confirms the key role of the butyl group in the
antagonist profile for the 5-HT4 receptor.
Replacement in derivative 5a of the methyl substitu-
ent at the 4-position of the piperazine by ethyl (5e, Ki
) 103.6 nM, i.a. ) 0.87), propyl (5f, Ki ) 87.3 nM, i.a.
) 0.68), or isopropyl (5g, Ki ) 89.8 nM, i.a. ) 0.66)
substituents retained affinity for the 5-HT4 receptor and
caused a reduction in partial agonist activity. 4-Fluo-
rophenoxypropyl, 4-fluorophenylmethyl, and piperonyl
groups have shown to be excellent substituents for
5-HT4 ligands with agonist activity (Figure 2). The
introduction of these substituents at the 4-position of
the piperazine led to 5i (Ki ) 375 nM, i.a. ) 0.78), 5j
(Ki ) 262 nM, i.a. ) 0.78), and 5k (IC50 > 1 µM, i.a. )
0.30) derivatives with reduced affinity for the 5-HT4
receptor and a decrease in the partial agonist activity.
These data suggest the existence of steric limitations
around the 4-substituent of the piperazine moiety for
5-HT4 agonism and that a methyl group seems to be
optimal in terms of both affinity and agonist activity at
the 5-HT4 receptor site.
These findings led us to evaluate a series of 3-ethyl-
and 3-cyclopropylbenzimidazolone derivatives (Table 2).
According to previously proposed 5-HT4 receptor models,
a fairly large lipophilic region is located near the basic
nitrogen recognition sites, and increasing the size and
lipophilic character of the side chain may result in
increased affinity.16 The first modification, therefore,
was to increase the size of the 4-substituent of the
piperazine moiety. Replacement of the methyl group in
compound 3a (Ki ) 75.4 nM) by ethyl or propyl groups
(3b, Ki ) 17.9 nM; 3c, Ki ) 16.9 nM, respectively) led
to an increased affinity for the 5-HT4 receptor. However,
the introduction of an isopropyl or a butyl group (3d ,
Ki ) 25.2 nM; 3e, Ki ) 34.1 nM, respectively) did not
improve the affinity of 3b. In the cyclopropyl series the
replacement of the methyl group in 6a (Ki ) 10.2 nM)
by a propyl (6b, Ki ) 8.2 nM), isopropyl (6c, Ki ) 6.7
nM), or butyl (6d , Ki ) 13.3 nM) group did not produce
a significant change on the affinity for the 5-HT4
receptor. These data also showed that a cyclopropyl
substituent in the 3-position of the benzimidazolone ring
is better than an ethyl group for 5-HT4 receptor affinity.
On the other side, these structural modifications had
divergent and slight influence on 5-HT4 antagonist
activity. Although butyl derivatives 3e and 6d displayed
moderate 5-HT4 affinity, they showed the highest 5-HT4
antagonist values (pKb ) 7.73, 7.51 respectively). This
result agrees with the hypothesized positive steric
interaction between the n-butyl group and the hydro-
phobic pocket of the 5-HT4 receptor in compounds with
5-HT4 antagonist activity.16 Besides, propyl derivatives
3c and 6b (pKb ) 7.63 and 7.46) did not show remark-
able differences on the antagonist activity with respect
to the related isopropyl derivatives 3d and 6c (pKb )
7.42 and 7.38) and methyl analogues 3a and 6a (pKb )
6.79 and 7.24). Moreover, improvement on the affinity
did not always imply a correlative improvement on the

2,3-Dihydro-2-oxo-1H-benzimidazole-1-carboxamides
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 15 2875
Ta ble 3. 2,3-Dihydro-2-oxo-1H-benzimidazole-1-carboxamides: 3-Isopropyl Derivatives
5-HT4
5-HT3
5-HT4
synthetic
binding Ki
binding
antagonism
5-HT4
compd
X
Y
Z
n
R′
method
mp, °C
formula
18H27N5O2
16H23N5O2
17H25N5O2
(nM) ( SEa IC50 (nM) pKb ( SEa
agonism i.a.
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
5q
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
NO2
NH2
H
H
H
H
H
H
H
H
H
H
H
H
Cl
F
MeO
F
F
H
2
0
1
3
2
2
2
2
2
2
2
2
2
2
2
2
2
Me
Me
Me
Me
A
A
C
A
C
C
C
A
B
A
C
C
C
B
C
f
126-128
141-143
71-73
C
C
C
91.1 ( 10.8
>1000b
>1000
<6.0
0.94
<0.30
<0.30
<0.30
0.87
0.68
0.66
<0.30
0.78
0.78
0.30
0.70
0.67
<0.30
<0.30
<0.30
<0.30
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
200.8c <6.0
309.0c <6.0
>1000b
>240d
C19H29N5O2‚2HCl
>1000b
>1000
<6.0
6.62 ( 0.13
6.89 ( 0.19
<6.0
7.94 ( 0.16
6.11 ( 0.15
<6.0
<6.0
<6.0
6.88 ( 0.11
<6.0
<6.0
<6.0
Et
190-193e C19H29N5O2‚2C4H4O4
202-205e C20H31N5O2‚2C4H4O4
210-212e C20H31N5O2‚2C4H4O4
210-212e C21H33N5O2‚2C4H4O4
103.6 ( 7.8
87.3 ( 10.3
89.8 ( 6.0
38.9 ( 3.6
>1000
>1000
>1000
>1000
n-Pr
i-Pr
n-Bu
4-FPhO(CH2)3
90-94
C26H34FN5O3
375.0 ( 23.7 >1000
4-FPhCH2
piperonyl
Me
Me
Me
Me
Me
Me
206-208e C24H30FN5O2‚2C4H4O4 262.0 ( 17.9 >1000
218 dece
134-137
130-134
109-111
198-201 C18H25FN6O4
84 dec
C25H31N5O4‚2C4H4O4
>1000b
>1000
>1000
C
C
C
18H26ClN5O2
18H26FN5O2
19H29N5O3
>1000b
108.4 ( 11.0 >1000
>1000b
>1000
>1000
>1000
>1000b
C18H27FN6O2
19H29N5O2
>1000b
B
82-84
C
216.4 ( 15.3
73.9c <6.0
a
b
d
Standard error. IC50 (nM). c Ki (nM). Hydrochloride. e Fumarate. f Reduction of 5o with Zn/1 N NaOH.
Ta ble 4. 2,3-Dihydro-2-oxo-1H-benzimidazole-1-carboxamides: Quaternary Derivatives
synthetic
method
5-HT4 binding
Ki (nM) ( SE
5-HT3 binding
IC50 (nM)
5-HT4 antagonism
5-HT4
agonism i.a.
compd
R
mp, °C
formula
pKb ( SE
5r
5s
5t
n-Bu
Me
allyl
D
D
E
170-172
60 dec
177-179
C
C
C
22H36IN5O2
19H30IN5O2
21H32IN5O2
66.3 ( 4.6
68.5 ( 26.8
56.3 ( 6.00
>1000
>1000
>1000
6.0 ( 0.23
<6.0
6.28 ( 0.17
0.93
0.75
<0.30
Modifications of the spacer in the parent compound
5a led to derivatives 5b, 5c, and 5d with a remarkable
decrease in the 5-HT4 affinity (IC50 > 1 µM) and slight
reduction in the 5-HT4 versus 5-HT3 receptor selectivity.
As observed previously for ethyl and cyclopropyl deriva-
tives, the length of the alkylene chain is an important
structural feature in recognition by the 5-HT4 receptor
binding site. A two-methylene spacer is also optimal for
high 5-HT4 agonist activity.
tives of 5a were prepared (Table 4). All of them showed
higher affinity for the 5-HT4 receptor (Ki ) 56.3-68.5
nM) than 5a , but while the butyl derivative 5r (i.a. )
0.93) retained the partial agonist activity, the methyl
derivative 5s (i.a. ) 0.75) showed a significant decrease
in the partial agonist activity and the allyl derivative
5t (i.a. < 0.30) was only weakly active as a 5-HT4
agonist.
Con clu sion
Introduction of substituents on the benzene ring had
a negative effect on 5-HT4 receptor affinity, except for
derivative 5m (Ki ) 108.4 nM) which has a fluorine in
the 6-position. The structurally related benzimidazolone
DAU 6285 has a methoxy group in the 6-position and
behaves as a 5-HT4 antagonist.20 A similar substitution
in this series led to derivative 5n (IC50 > 1 µM) with a
remarkable reduction in the affinity for the 5-HT4
receptor. It can be concluded that the 5-HT4 receptor
can only accommodate small substituents in the position
of the benzene ring in our series. Therefore, it seems
that the electronic distribution within the benzimidazole
ring has a small effect on the 5-HT4 receptor affinity,
whereas steric hindrance might play a significant role.
It has been reported that increasing the polarity by
n-butyl quaternization of renzapride increases the 5-HT4
receptor agonist potency.36 Several quaternary deriva-
1. The 2,3-dihydro-2-oxo-1H-benzimidazole-1-carbox-
amides including a piperazine moiety described in this
paper represent a novel class of ligands at the 5-HT4
receptor with selectivity over 5-HT3 and D2 receptors.
2. The 3-substituent at the benzimidazolone ring is a
determinant structural feature for both binding and
pharmacological profile. 3-Ethyl and 3-cyclopropyl de-
rivatives showed high affinity (6c, Ki ) 6.7 nM) and
moderate antagonist activity (3e, pKb ) 7.73), whereas
3-isopropyl analogues exhibited moderate affinity and
high partial agonist activity (5a , Ki ) 91.1 nM, i.a. )
0.94). The reversal of the pharmacological activity due
only to a small structural difference might confirm the
existence of two binding sites at the 5-HT4 receptor.
3. The length of the alkylene spacer is an important
structural feature in the recognition by the 5-HT4

2876 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 15
Tapia et al.
1
g, 99%). H NMR (CDCl3): δ 6.78 (1H, br d); 6.65 (1H, br s);
6.57 (1H, d); 3.52 (1H, m); 3.38 (3H, br s); 1.22 (6H, d). 13C
NMR (CDCl3): δ 135.9, 135.2, 122.9, 119.8, 116.2, 113.7, 44.3,
22.9.
receptor binding site. A chain length of two methylenes
is optimal for high affinity and also for good antagonist
or agonist activity.
4. In the ethyl and cyclopropyl series, 5-HT4 antago-
nist activity seems to be unrelated to the nature of the
4-substituent of the piperazine moiety. However, the
presence of a butyl substituent is a favorable pattern
for 5-HT4 antagonism and even in the 5-HT4 agonist
series (isopropyl derivatives) caused a reversal of the
pharmacological profile (5h , pKb ) 7.94).
5. Derivative 5a showed a partial agonist activity
greater than that of BIMU 8 and exhibited better
selectivity versus 5-HT3 and D2 receptors.
6. N-Butyl quaternization of 5a led to 5r (Ki ) 66.3,
i.a. ) 0.93) with an improvement in affinity for the
5-HT4 receptor and a similar partial agonist activity.
1,3-Dih yd r o-1-(1-m e t h yle t h yl)-2H -b e n zim id a zol-2-
on e, 13a . A solution of 12a (29.6 g, 0.19 mol) and N,N′-
carbonyldiimidazole (32.1 g, 0.19 mol) in dry THF (400 mL)
was stirred at room temperature for 20 h and then evaporat-
ed.The residue was taken up in water and extracted with CH2-
Cl2. The dried organic phase was evaporated, and the residue
was purified by flash chromatography using CH2Cl2 and
methanol (98/2) as eluent to give a product that was triturated
with hexane and filtered to yield 13a as pale-yellow solid (16.1
g, 46%). Mp: 125-127 °C. 1H NMR (CDCl3): δ 7.22-7.15 (4H,
m); 4.78 (1H, m); 1.58 (6H, d).13C NMR (CDCl3): δ 155.4, 128.9,
128.3, 121.0, 120.8, 109.9, 109.2, 44.6, 20.2.
1,3-Dih yd r o-5-flu or o-1-(1-m eth yleth yl)-6-n itr o-2H-ben -
zim id a zol-2-on e, 13f. The compound 13d (11.0 g, 56.7 mmol)
was suspended in acetic anhydride (75 mL) and cooled in an
ice bath. To this suspension was added dropwise 22.5 mL of a
mixture of nitric acid and acetic anhydride (1/2). The reaction
mixture was stirred for 3 min before being poured into ice-
water. The solid was filtered, washed with water and cold
Exp er im en ta l Section
Ch em istr y. Flash column chromatography was performed
on silica gel, particle size 60 Å, mesh ) 230-400 (Merck).
Melting points were determined in open capillary tubes on a
Bu¨chi 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 spectrom-
eter; chemical shifts δ are reported in parts per million (ppm)
downfield from tetramethylsilane (TMS), which was used as
an internal standard. Spectral data are consistent with as-
signed structures.
1
Et2O, and dried to afford 13f (11.8 g, 87%). H NMR (CDCl3):
δ 11.20 (1H, br s); 7.62 (1H, d); 6.80 (1H,d); 4.58 (1H, m); 1.41
(6H, d). 13C NMR (CDCl3): δ 154.7, 152.7 (d, J ) 245 Hz), 134.7
(d, J ) 12 Hz), 130.2 (d, J ) 7 Hz), 124.9, 104.9, 98.5 (d, J )
27 Hz), 45.0, 19.9.
1,3-Dih yd r o-1-p r opyl-2H-ben zim id a zol-2-on e, 13h . HNa
(2.0 g, 50 mmol, 60% in mineral oil) was added portionwise to
a stirred solution of 1,3-dihydro-2H-benzimidazol-2-one (6.7
g, 50 mmol) in dry DMF (50 mL). After 1 h, a solution of propyl
iodide (4.9 g, 50 mmol) in dry DMF (20 mL) was added
dropwise and the mixture stirred at room temperature for 24
h.The solvent was removed in vacuo, and the residue was
taken up into water and extracted with CH2Cl2. The organic
phase was dried over Na2SO4, the solvent was evaporated to
dryness, and the residue was chromatographed on a column
of silica gel using a mixture of dicloromethane and methanol
(98/2) as eluent to separate 13h (55%). Mp: 97-9 °C. 1H NMR
(CDCl3): δ 10.38 (1H, br s); 7.20-6.94 (4H, m); 3.90 (2H, t);
1.83 (2H, m); 1.02 (3H, t). 13C NMR (CDCl3): δ 155.8, 130.4,
128.0, 121.3, 121.1, 109.6, 107.8, 42.4, 21.7, 11.3.
2,3-Dih ydr o-2-oxo-1H-ben zim idazole-1-car boxylic Acid,
1,1-Dim eth yleth yl Ester , 14. HNa (1.6 g, 41 mmol, 60% in
mineral oil) was added portionwise to a stirred solution of 1,3-
dihydro-2H-benzimidazol-2-one (5.0 g, 37.2 mmol) in dry DMF
(100 mL) under an atmosphere of argon. After 1.5 h, a solution
of di-tert-butyl dicarbonate (8.1 g, 37.2 mmol) in dry DMF (20
mL) was added dropwise and the mixture stirred at room
temperature for 24 h. The solvent was evaporated and the
residue diluted with saturated NH4Cl solution and extracted
with EtOAc. The residue was purified by flash chromatography
using hexane and EtOAc (7/3) as eluent to give 14 as a white
solid (6.6 g, 76%). Mp: >250 °C. 1H NMR (CDCl3): δ 10.44
(1H, s); 7.69 (1H, d); 7.18-7.06 (3H, m); 1.68 (9H, s). 13C NMR
(CDCl3): δ 153.4, 148.6, 127.4, 126.8, 124.1, 122.0, 114.4, 110.0,
85.0, 28.1.
N-(1-Meth yleth yl)-2-n itr oan ilin e, 11a. 2-Nitroaniline (40.0
g, 0.29 mol), 2,2-dimethoxypropane (54.5 g, 0.52 mol), and TFA
(41.5 g, 0.29 mol) were dissolved in toluene (600 mL) and
stirred at room temperature for 1 h. A boron-pyridine complex
(26.6 g, 0.29 mol) was slowly added. The reaction mixture was
stirred for 20 h. The solvent was evaporated in vacuo, and the
residue was taken up into water and extracted with CH2Cl2.
The organic extract was dried (Na2SO4) and concentrated in
vacuo. The residue was purified by flash chromatography on
silica gel using hexane and ethyl acetate (95/5) to give 11a as
1
an orange oil (44.0 g, 84%). H NMR (CDCl3): δ 8.19 (1H, d);
7.41 (1H, t); 6.83 (1H, d); 6.49 (1H, t); 3.81 (1H, m); 1.28 (6H,
d). 13C NMR (CDCl3): δ 144.7, 136.0, 131.6, 127.0, 114.8, 114.0,
43.8, 22.6.
N-Cyclop r op yl-2-n itr oa n ilin e, 11b. A mixture of 2-chlo-
ronitrobenzene (3.1 g, 20 mmol) and cyclopropylamine (3.5 mL,
50 mmol) was placed in a high-pressure vessel and heated at
100 °C for 24 h. Then the reactor was opened, the reaction
mixture was diluted with water and extracted with CH2Cl2,
and the extract was washed with water and dried over Na2-
SO4. The solvent was evaporated in vacuo, and the residue
was purified by flash chromatography using hexane and CH2-
Cl2 (7/3) as eluent to give 11b as an orange oil (2.1 g, 60%). 1H
NMR (CDCl3): δ 8.20 (1H, d); 7.61-7.42 (2H, m); 7.30 (1H,
dd); 6.72 (1H, m); 2.60 (1H, m); 0.81-1.01 (2H, m); 0.60-0.72
(2H, m). 13C NMR (CDCl3): δ 164.0, 146.0, 135.8, 126.4, 115.8,
115.2, 24.4, 7.7.
1,3-Dih yd r o-1-(2-p h e n yle t h yl)-2H -b e n zim id a zol-2-
on e, 13l. A mixture of 14 (2.3 g, 10 mmol), 2-phenylethyl
bromide (1.8 g, 10 mmol), K2CO3 (4.1 g, 30 mmol), and DMF
(40 mL) was stirred at reflux for 24 h. The mixture was
concentrated in vacuo and the residue extracted with CH2Cl2.
The organic phase was dried over Na2SO4, and the solvent was
evaporated in vacuo. The residue was purified by flash
chromatography using a mixture of hexane and ethyl acetate
2-[(1-Meth yleth yl)a m in o]a n ilin e, 12a . A solution of 11a
(2.3 g, 12.7 mmol) in EtOH (50 mL) was hydrogenated over
10% palladium/carbon (0.5 g) at 3 atm and 20 °C for 16 h. The
catalyst was filtered off and the filtrate evaporated in vacuo
1
to give 12a (1.6 g, 83%). H NMR (CDCl3): δ 6.71-6.62 (4H,
m); 3.61 (1H, m); 3.13 (3H, br s); 1.20 (6H, d).13C NMR
(CDCl3): δ 136.7, 134.4, 120.6, 118.4, 116.7, 112.9, 44.3, 23.1.
5-Ch lor o-2-[(1-m eth yleth yl)a m in o]a n ilin e, 12c. A sus-
pension of 11c (20.0 g, 93 mmol) in EtOH (400 mL) and 1 N
NaOH (300 mL) was stirred vigorously and heated until the
solution boiled gently. The bath was removed, and zinc dust
(60.8 g, 930 mmol) was added in several portions frequently
enough to keep the solution boiling. After refluxing for 1 h,
the mixture was filtered and the filtrate was poured into water
(100 mL) and extracted with CH2Cl2. The organic phase was
dried with MgSO4 and evaporated in vacuo to give 12c (17.0
1
(7/3) as eluent to yield 13l (90%). Mp: 142-144 °C. H NMR
(DMSO-d6): δ 10.40 (1H, br s); 7.18-7.01 (4H, m); 7.01-6.81
(5H, m); 4.18 (2H, t); 3.14 (2H, t). 13C NMR (DMSO-d6): δ
155.6, 138.2,130.1, 128.8, 128.6, 128.0, 126.6, 121.4, 121.1,
109.7, 107.7, 42.4, 34.7.
Eth yl 4-(1-Meth yleth yl)p ip er a zin e-1-ca r boxyla te, 15a .
A mixture of 1-ethoxycarbonylpiperazine (7.9 g, 50 mmol),
isopropyl iodide (5 mL, 50 mmol), K2CO3 (8.3 g, 60 mmol), and

2,3-Dihydro-2-oxo-1H-benzimidazole-1-carboxamides
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 15 2877
CH3CN (100 mL) was refluxed for 24 h. The solvent was
evaporated in vacuo, and the residue was poured onto water
and extracted with dichloromethane. The organic phase was
dried over Na2SO4 and the solvent evaporated in vacuo to yield
15a as an oil (98%). 1H NMR (CDCl3): δ 4.12 (2H, q); 3.48
(4H, m); 2.70 (1H, m); 2.46 (4H, m); 1.25 (3H, t); 1.04 (6H, d).
13C NMR (CDCl3): δ 154.8, 61.1, 54.5, 48.3, 44.0, 18.3, 14.6.
1-(1-Meth yleth yl)p ip er a zin e 16a . A solution of 15a (9.8
g, 49 mmol) and 45% HBr (200 mL) was heated at reflux for
3 h. The reaction mixture was cooled, 50% NaOH solution was
added until pH > 7, and the mixture was extracted with a
solution of CH3Cl and i-PrOH (4/1). The organic phase was
dried over Na2SO4, and the solvent was evaporated in vacuo
to yield 16a (90%) as an oil. 1H NMR (CDCl3): δ 2.90 (4H, m);
2.63 (1H, m); 2.48 (4H, m); 1.21 (1H, br s); 1.04 (6H, d). 13C
NMR (CDCl3): δ 54.8, 49.9, 46.4, 18.3.
1-Bu tylp ip er a zin e (Hyd r obr om id e), 16b. A mixture of
15b (1.0 g, 4.6 mmol) and 45% HBr (30 mL) was heated at
reflux for 1 h. The solvent was evaporated and the residue
triturated with Et2O and filtered to give 16b (1.4 g, 98%).
Mp: >240 °C. 1H NMR (DMSO-d6 + D2O): δ 4.01 (5H, m);
3.41 (6H, m); 3.12 (2H, m); 1.60 (2H, m); 1.28 (2H, m); 0.86
(3H, t). 13C NMR (DMSO-d6 + D2O): δ 56.6, 48.4, 40.7, 25.6,
19.8, 14.0.
benzimidazole-1-carbonyl chloride (19a ) (5.5 g). The solid was
added to a solution of 2-(4-methylpiperazin-1-yl)ethylamine
(18a ) (2.8 g, 20.0 mmol) and triethylamine (2.8 g, 28.0 mmol)
in dry THF (60 mL) and was stirred at room temperature for
24 h. The solvent was evaporated, and the residue was taken
up into water and extracted with Cl2CH2. The organic extract
was dried over Na2SO4 and concentrated in vacuo. The residue
was purified by flash chromatography using CH2Cl2 and
MeOH (9/1 to 3/1) as eluent to give 1 (2.4 g, 41%). Mp: 188-
1
190 °C. H NMR (CDCl3): δ 8.18 (1H, m); 7.15 (3H, m); 3.48
(2H, t); 2.65-2.41 (10H, m); 2.25 (3H, s). 13C NMR (CDCl3): δ
154.1, 151.9, 127.4, 123.5, 122.2, 114.9, 109.1, 56.4, 54.5, 52.2,
45.3, 36.5.
3-Cyclop r op yl-2,3-d ih yd r o-N-[2-[4-(1-m et h ylet h yl)p i-
p er a zin -1-yl]eth yl]-2-oxo-1H-ben zim id a zole-1-ca r boxa m -
id e, 6c (Meth od B). 1-Cyclopropyl-1,3-dihydro-2H-benzimid-
azol-2-one (13b) (2.0 g, 11.5 mmol) was added to a suspension
of HNa (60% mineral oil, 0.9 g, 23.0 mmol) in dry THF (30
mL). The mixture was stirred 15 min before adding a solution
of phosgene in toluene (1.93 M) (23.8 mL, 46.0 mmol), and the
stirring was continued for 1 h. The resulting mixture was
filtered through Celite and concentrated (19f). The residue was
dissolved in dry THF (50 mL), and 2-[4-(1-methylethyl)-
piperazin-1-yl]ethylamine (18c) (2.0 g, 11.6 mmol) and tri-
ethylamine (4 mL) were added. The solution was stirred at
room temperature for 18 h. The solvent was evaporated, and
the residue was taken up into water and extracted with Cl2-
CH2. The organic extract was dried over Na2SO4 and concen-
trated in vacuo. The residue was purified by flash chromatog-
raphy using CH2Cl2 and MeOH (95/5 to 9/1) as eluent to give
1-(4-F lu or op h en ylm eth yl)p ip er a zin e, 16c. The com-
pound was prepared according to the process reported for
16b.The hydrobromide was dissolved in a solution of 10%
NaOH, extracted with CH2Cl2, and dried over Na2SO4. Evapo-
ration of solvent gave 16c as an oil.Yield: 99%. 1H NMR
(CDCl3): δ 7.30-6.98 (4H, m); 3.45 (2H, s); 2.86 (4H, m); 2.38
(4H, m); 1.80 (1H, s). 13C NMR (CDCl3): δ 161.7 (d, J ) 243
Hz), 133.6 (d, J ) 3 Hz), 130.4 (d, J ) 7 Hz), 114.7 (d, J ) 21
Hz), 62.6, 54.2, 45.9.
1
6c as an oil (1.8 g, 42%). H NMR (CDCl3): δ 8.92 (1H, br s);
8.21 (1H, m); 7.20 (3H, m); 3.59 (2H, q); 2.90 (1H, m); 2.60
(11H, m); 1.11 (10H, m). 13C NMR (CDCl3): δ 153.6, 151.7,
129.6, 126.3, 123.4, 122.7, 115.1, 108.5, 56.9, 54.4, 53.3, 48.6,
37.0, 22.4, 18.6, 6.0.
(4-Meth ylp ip er a zin -1-yl)a ceton itr ile, 17a . A suspension
of 1-methylpiperazine (9.0 g, 90 mmol), acetonitrile (60 mL),
K2CO3 (60.0 g, 430.0 mmol), and 2-chloroacetonitrile (8.2 g,
100.0 mmol) was stirred at room temperature for 20 h. Et2O
(100 mL) was added, and the suspension was filtered. The
filtrate was concentrated in vacuo to give 17a as a yellow solid
3-Cyclop r op yl-2,3-d ih yd r o-N-[2-(4-m eth ylp ip er a zin -1-
yl)eth yl]-2-oxo-1H-ben zim idazole-1-car boxam ide,6a (Meth -
od C). 1-Cyclopropyl-1,3-dihydro-2H-benzimidazol-2-one (13b)
(4.0 g, 23.0 mmol) was reflux with 2-chloroethyl isocyanate
(2.9 g, 27.5 mmol) in toluene (50 mL) for 20 h. The mixture
was cooled and the solid filtered and washed with hexane
to give N-(2-chloroethyl)-3-cyclopropyl-2,3-dihydro-2-oxo-1H-
benzimidazole-1-carboxamide (20a ) (4.7 g, 73%). Mp: 152-
153 °C. 1H NMR (CDCl3): δ 9.20 (1H, br s); 8.19 (1H, m); 7.30-
7.17 (3H, m); 3.74 (4H, m); 2.93 (1H, m); 1.28-0.95 (4H, m).
13C NMR (CDCl3): δ 153.6, 151.8, 129.6, 126.1, 123.7, 122.9,
115.1, 108.7, 43.2, 41.6, 22.5, 6.0.
A mixture of 1-methylpiperazine (0.8 g, 8.6 mmol), toluene
(50 mL), 20a (2.0 g, 7.1 mmol), K2CO3 (1.2 g, 8.6 mmol), and
a pinch of KI was stirred at reflux for 17 h. The solvent was
evaporated, and the residue was taken up into water and
extracted with Cl2CH2. The organic extract was dried over Na2-
SO4 and concentrated in vacuo. The residue was purified by
flash chromatography using CH2Cl2 and MeOH (9/1 to 3/1) as
eluent to give 6a (1.0 g, 41%). Mp: 104-108 °C. 1H NMR
(CDCl3): δ 8.91 (1H, br s); 8.19 (1H, d); 7.20 (3H, m); 3.43 (2H,
q); 2.80 (1H, m), 2.58 (10H, m); 2.22 (3H, s); 1.24-0.82 (4H,
m). 13C NMR (CDCl3): δ 153.2, 151.4, 129.3, 125.9, 123.1,
122.3, 114.7, 108.3, 56.5, 54.6, 52.5, 45.5, 36.7, 22.2, 5.8.
3-Cyclop r op yl-2,3-d ih yd r o-N-[(4-m et h ylp ip er a zin -1-
yl)m et h yl]-2-oxo-1H-b en zim id a zole-1-ca r boxa m id e, 6e.
1-Cyclopropyl-1,3-dihydro-2H-benzimidazol-2-one (13b) (3.0 g,
17.2 mmol) was heated at 60 °C with chloromethyl isocyanate
(1.9 g, 1.8 mL) in toluene (30 mL) for 24 h. The solvent was
evaporated, and the residue was triturated with Et2O to give
N-(chloromethyl)-3-cyclopropyl-2,3-dihydro-2-oxo-1H-benzimi-
dazole-1-carboxamide (20d ) (4.0 g, 87%). Mp: 202-204 °C. 1H
NMR (DMSO-d6): δ 9.23 (1H, br s); 8.16 (1H, d); 7.38-7.20
(3H, m); 4.79 (2H, d); 2.94 (1H, m); 1.21-0.82 (4H, m). 13C
NMR (DMSO-d6): δ 152.8, 151.0, 129.9, 125.8, 123.6, 122.2,
114.1, 109.1, 63.4, 22.4, 5.7.
1
(12.0 g, 96%). Mp: 50-52 °C. H NMR (CDCl3): δ 3.40 (2H,
s); 2.63 (4H, m); 2.44 (4H, m); 2.25 (3H, s). 13C NMR (CDCl3):
δ 114.5, 54.3, 51.5, 45.6, 45.5.
(4-P r op ylp ip er a zin -1-yl)a ceton itr ile, 17b. A mixture of
propylpiperazine dihydrobromide (10.0 g, 34.4 mmol), aceto-
nitrile (100 mL), K2CO3 (15.2 g, 110.2 mmol), and chloroacet-
onitrile (2.8 g, 37.7 mmol) was stirred at reflux for 5 h. The
solvent was evaporated, and the residue was taken up into
water and extracted with Cl2CH2. The organic extract was
dried over Na2SO4 and concentrated in vacuo to give 17b as
an oil (4.8 g, 83%). 1H NMR (CDCl3): δ 3.55 (2H, s); 2.66-
2.26 (10H, m); 1.51 (2H, m); 0.90 (3H, t). 13C NMR (CDCl3): δ
114.5, 60.0, 52.3, 51.5, 45.6, 19.7, 11.6.
2-(4-Meth ylp ip er a zin -1-yl)eth yla m in e, 18a . To a stirred
suspension of H4LiAl (3.3 g) in dry Et2O (100 mL), cooled at 0
°C, was slowly added a solution of 17a (11.2 g, 80.4 mmol) in
dry Et2O (150 mL) and dry THF (150 mL). The mixture was
stirred at room temperature for 24 h, cooled in an ice bath,
and hydrolyzed with a 20% NaOH solution (50 mL). After
stirring for 20 min, the mixture was filtered and the solvent
evaporated. The residue was dissolved in Et2O and dried over
Na2SO4. The solvent was evaporated to dryness to give 18a
1
as an oil (11.5 g, 99%). H NMR (CDCl3): δ 2.80 (2H, t); 2.45
(10H, m); 2.25 (3H, s); 1.61 (2H, br s). 13C NMR (CDCl3): δ
60.9, 54.9, 52.9, 45.8, 38.5.
2,3-Dih yd r o-N-[2-(4-m eth ylp ip er a zin -1-yl)eth yl]-2-oxo-
1H-ben zim id a zole-1-ca r boxa m id e, 1 (Meth od A). Trichlo-
romethyl chloroformate (5.5 g, 27.8 mmol) dissolved in dry
THF (10 mL) was added dropwise to a stirred suspension of
1,3-dihydro-1H-benzimidazol-2-one (5.0 g, 37.2 mmol), acti-
vated charcoal (0.08 g), and dry THF (100 mL). The reaction
mixture was refluxed for 8 h; then heating was removed while
stirring was continued 15 h more. The solid was removed by
filtration. Evaporation of the solvent led to a crude material
which was triturated with Et2O to give 2,3-dihydro-2-oxo-1H-
A mixture of 1-methylpiperazine (0.56 g, 5.6 mmol), aceto-
nitrile (25 mL), 20d (1.0 g, 3.7 mmol), triethylamine (0.37 g,
3.7 mmol), and a pinch of KI was stirred at room temperature

2878 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 15
Tapia et al.
for 10 min. The solvent was evaporated, and the residue was
taken up into water and extracted with Cl2CH2. The organic
extract was dried over Na2SO4 and concentrated in vacuo. The
residue was triturated with Et2O to give 6e (1.0 g, 81%). Mp:
0.1 mL of [3H]GR 113808 (Amersham, 60-85 Ci/mmol) in
buffer (final concentration 0.1 nM). Nonspecific binding was
determined using 10 µM cold 5-HT. Binding experiment was
initiated by addition of 0.8 mL of 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 pretreated filters (1% poly(ethylenimine) in 50 mM
Hepes buffer), using a Brandel cell harvester. Filters were
placed in scintillation poly(ethylene) vials (with 5 mL of
scintillation cocktail) and equilibrated, and filter-retained
radioactivity was measured in a liquid scintillation counter
(Kontron Beta V). IC50 and Ki values were calculated using
the computer program EBDA (McPherson).37
1
86-88 °C. H NMR (CDCl3): δ 9.20 (1H, br s); 8.19 (1H, m);
7.21 (3H, m); 4.37 (2H, d); 2.91 (1H, m), 2.71 (4H, m); 2.49
(4H, m); 2.28 (3H, s); 1.10 (4H, m). 13C NMR (CDCl3): δ 153.4,
152.0, 129.4, 126.0, 123.5, 122.6, 115.0, 108.5, 61.3, 54.6, 49.1,
45.6, 22.3, 5.8.
5-Am in o-2,3-d ih yd r o-6-flu or o-3-(1-m et h ylet h yl)-N-[2-
(4-m eth ylp ip er a zin -1-yl)eth yl]-2-oxo-1H-ben zim id a zole-
1-ca r boxa m id e, 5p . A suspension of 5o (1.2 g, 3 mmol) in
THF (75 mL) and 1 N NaOH (75 mL) was stirred vigorously
and heated until the solution boiled gently. The bath was
removed, and zinc dust (3.0 g, 45 mmol) was added in several
portions frequently enough to keep the solution boiling. The
mixture was refluxed for 1 h and filtered, and the filtrate was
poured into water (50 mL) and extracted with CH2Cl2. The
organic phase was dried over MgSO4 and evaporated in vacuo
2. 5-HT3 Recep tor Bin d in g Assa y.34 Adult male Wistar
rats weighing 220-280 g were used. Animals were killed by
decapitation. The whole brain with the exception of the
brainstem and cerebellum was quickly removed, and the
various areas were dissected, weighed, and immediately frozen
at -70 °C. Enthorinal cortex used for the binding experiments
was homogenized with an Ultra-Turrax homogenizer (setting
5 for 20 s) in 10 volumes of ice-cold 0.32 M sucrose buffer and
centrifuged at 1000g for 10 min (4 °C), and the supernatant
was recentrifuged at 17000g 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 48000g 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 time of experiment,
the membranes were diluted in the same ice-cold buffer with
10 µM pargyline (final dilution 1:40, 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]LY 278584 (Amersham,
60-85 Ci/mmol) in buffer (final concentration 2 nM). Nonspe-
cific binding was determined using 10 µM cold 5-HT. Binding
experiment was initiated by addition of 0.8 mL of membrane
suspension (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% poly-
(ethylenimine) 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
scintillation poly(ethylene) vials (with 5 mL of scintillation
cocktail) and equilibrated, and filter-retained radioactivity was
measured in a liquid scintillation counter (Kontron Beta V).
IC50 and Ki values were calculated using the computer program
EBDA (McPherson).37
3. D2 Recep tor Bin d in g Assa y.35 Adult male Wistar rats
weighing 220-280 g were used. Animals were killed by
decapitation. The whole brain with the exception of the
brainstem and cerebellum was quickly removed, and the
various areas were dissected, weighed, and immediately frozen
at -70 °C. Striatum used for the binding experiments was
homogenized with an Ultra-Turrax homogenizer (setting 5 for
20 s) in 50 volumes of ice-cold 50 mM Tris-HCl buffer (pH 7.7),
centrifuged at 48000g 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 48000g 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 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]-
raclopride (NEN, 60-87 Ci/mmol) in buffer (final concentration
1 nM). Nonspecific binding was determined using 1 µM cold
(+)-butaclamol. Binding experiment was initiated 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
1
to give 5p (0.95 g, 82%). Mp: 84 °C dec. H NMR (CDCl3): δ
6.78 (1H, d); 6.61 (1H, d); 5.63 (1H, br t); 4.60 (1H, m); 3.95
(2H, t); 3.61 (2H, br s); 3.45 (2H, q); 3.31 (4H, t); 2.30 (4H, t);
2.23 (3H, s); 1.41 (6H, d). 13C NMR (CDCl3): δ 157.6, 154.3,
147.5 (d, J ) 230 Hz), 128.9 (d, J ) 14 Hz), 124.0, 121.3 (d, J
) 11 Hz), 98.4 (d, J ) 3 Hz), 96.5 (d, J ) 26 Hz), 54.4, 45.8,
44.8, 43.2, 40.5, 40.3, 19.9.
1-Bu tyl-4-[2-[2,3-d ih yd r o-3-(1-m eth yleth yl)-2-oxoben z-
im id a zol-1-yloxoa m in o]eth yl]-1-m eth ylp ip er a zon iu m Io-
d id e, 5r . (Meth od D). A solution of N-[2-(4-butylpiperazin-
1-yl)ethyl]-2,3-dihydro-3-(1-methylethyl)-2-oxo-1H-benzimid-
azole-1-carboxamide (5h ) (1.2 g, 3.1 mmol) and methyl iodide
(2 mL, 32.0 mmol) in THF (30 mL) was stirred at room
temperature for 20 h. The solvent was removed, and the
residue was taken up into water and extracted with Cl2CH2.
The organic extract was dried over Na2SO4 and concentrated
in vacuo to give 5r as a solid (1.1 g, 67%). Mp: 170-172 °C.
1H NMR (CDCl3): δ 9.12 (1H, br s); 8.21 (1H, m); 7.19 (3H,
m); 4.70 (1H, m); 3.79 (4H, m); 3.61 (4H, m); 3.42 (3H, s); 3.05
(4H, m); 2.84 (2H, t); 1.84 (2H, m); 1.58 (8H, m); 1.03 (3H, t).
13C NMR (CDCl3): δ 152.5, 151.8, 127.5, 126.6, 123.3, 122.2,
115.1, 108.9, 60.6, 55.5, 47.5, 46.1, 45.3, 36.4, 23.9, 19.8, 19.5,
13.7.
1-Allyl-4-[2-[2,3-d ih yd r o-3-(1-m eth yleth yl)-2-oxoben z-
im id a zol-1-yloxoa m in o]eth yl]-1-m eth ylp ip er a zon iu m Io-
d id e, 5t (Meth od E). To a cooled solution of 2,3-dihydro-3-
(1-methylethyl)-N-[2-(4-methylpiperazin-1-yl)ethyl]-2-oxo-1H-
benzimidazole-1-carboxamide (5a ) (1.0 g, 2.9 mmol) in DMF
(12 mL) was added allyl iodide (2.4 g, 14.3 mmol). The mixture
was stirred at room temperature for 2 h. The solvent was
evaporated and the residue purified by flash chromatography
using CH2Cl2 and MeOH (9/1) as eluent to give 5t as a white
1
solid (1.0 g, 67%). Mp: 177-179 °C. H NMR (CDCl3): δ 9.08
(1H, br s); 8.25 (1H, m); 7.18 (3H, m); 4.18-5.77 (3H, m); 4.70
(1H, m); 4.51 (2H, d); 3.63 (6H, m); 3.44 (3H, s); 3.04 (4H, m);
2.83 (2H, t); 1.57 (6H, d). 13C NMR (CDCl3): δ 152.4, 151.8,
131.7, 127.2, 126.6, 123.3, 122.5, 115.4, 109.2, 65.8, 60.0, 54.8,
46.1, 45.0, 44.7, 35.1, 19.8.
P h a r m a cologica l Meth od s. 1. 5-HT4 Recep tor Bin d in g
Assa y.31 Adult male Dunkin-Hartley guinea pigs weighing
350-400 g were used. Animals were killed by decapitation.
The whole brain with the exception of the brainstem and
cerebellum was quickly removed, and the various areas were
dissected, weighed, and immediately frozen at -70 °C. Stria-
tum used for the binding experiments was homogenized with
an Ultra-Turrax homogenizer (setting 5 for 20 s) in 15 volumes
of ice-cold 50 mM Hepes buffer (pH 7.4) and centrifuged at
48000g 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 time of 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 concentra-
tion (0.1 mL of vehicle if no competing drug was added) and

2,3-Dihydro-2-oxo-1H-benzimidazole-1-carboxamides
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 15 2879
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 scintillation poly(ethylene) vials (with 5 mL of
scintillation cocktail) and equilibrated, and filter-retained
radioactivity was measured in a liquid scintillation counter
(Kontron Beta V). IC50 and Ki values were calculated using
the computer program EBDA (McPherson).37
Su p p or tin g In for m a tion Ava ila ble: Yields, melting
points, and spectral data (1H and 13C NMR) for compounds
2-20. This material is available free of charge via the Internet
at http://pubs.acs.org.
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4. Ra t Isola ted Esop h a gea l Tu n ica Mu scu la r is Mu co-
sa e (TMM).32,33 Male Wistar rats (250-300 g) were killed by
a blow on the head, and a 2-cm segment of intrathoracic
esophagus proximal to the diaphragm was removed and placed
in standard Krebs-Henseleit (K-H) solution (37 °C, pH 7.4).
The external muscularis propria containing the outer longi-
tudinal and circular muscle layers of the esophagus was
carefully removed to isolate the inner smooth muscle of TMM.
The TMM preparations were suspended longitudinally in a
20-mL tissue bath containing K-H solution at 37 °C, pH 7.4,
and continuously aerated with carbogen (95% O2/5% CO2). This
solution routinely contained 1 µM ketanserin, 10 µM zimeli-
dine, 30 µM cocaine, and 100 µM pargyline. Tissues were
placed under an initial tension of 0.5 g, equilibrated for 60
min by washing every 15 min before starting the experiment.
Responses were isometrically recorded using a F30 transducer
coupled to a two-channel chart recorder (Hugo Sachs Elek-
tronik, Freiburg, Germany). The TMM preparations were
contracted by addition of a submaximal concentration of
carbachol (3 µM).
In agonism studies, upon establishment of a stable contrac-
tion, two concentration-effect curves (relaxation) were con-
structed in the same TMM in a cumulative fashion: the first
for 5-HT itself and the second, 90 min later, for either 5-HT
(in time control experiments) or a test agonist. Responses to
the cumulative addition of agonist were expressed as percent-
age relaxation of carbachol-induced tone. For each agonist, two
or three separate TMM preparations were used and the
concentration-effect curves obtained were fitted to calculate
the negative logarithm of the molar concentration of the
agonist that relaxated 50% relative to their individual maxi-
mun effect (pEC50). The intrinsic activity (i.a.) versus 5-HT
was calculated comparing the maximun relaxation obtained
at the concentration 1 or 3 µM in the same TMM preparation.
To confirm that the agonist effect was 5-HT4 receptor-depend-
ent, another series with TMM preparations was used in which
two concentration-effect curves were constructed: the first
to agonist itself and the second, 90 min later, in the presence
of 10 nM GR 113808 added 30 min before the first concentra-
tion of agonist. Apparent affinity (pKb) for GR 113808 against
all agonists was determined from concentration ratios by the
equation: pKb ) log(X - 1) + 8, where X is the concentration
ratio and 8 is the negative logarithm of the GR 113808
concentration (10 nM). The effect of this 5-HT4 antagonist on
pEC50 value was registered and then compared by using the
unpaired Student’s t-test. A p value of 0.05 or less was
considered statistically significant.
In antagonist studies, two concentration-effect curves were
constructed in the same TMM preparation: the first for 5-HT
itself and the second, 90 min later, in the presence of a single
concentration (0.1 or 1 µM) of antagonist added 30 min before
the first concentration of 5-HT. For each antagonist, two or
three separate TMM preparations were used. Apparent affinity
(pKb) for antagonists against 5-HT were determined from
concentration ratios by the equation: pKb ) log(X - 1) - log-
[A], where X is the concentration ratio and [A] is the used
antagonist concentration (mol/L). The method assumes a
competitive interaction. The effect of the antagonists on pEC50
values was registered, and the values were compared by using
the unpaired Student’s t-test. A p value of 0.05 or less was
considered statistically significant.
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B.; Croci, T.; Cardamone, R.; Aureggi, G.; Langlois, M. New
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