New 2-piperazinylbenzimidazole derivatives as 5-HT3 antagonists. Synthesis and pharmacological evaluation

Article abstract of DOI:10.1021/jm960442e

A series of 2-piperazinylbenzimidazole derivatives were prepared and evaluated as 5-HT₃ receptor antagonists. Their 5-HT₃ receptor affinities were evaluated by radioligand binding assays, and their abilities to inhibit the 5-HT-induced Bezold-Jarisch reflex in anesthetized rats were determined. Compound 7e (lerisetron, pK(i) = 9.2) exhibited higher affinity for the 5- HT₃ receptor than did tropisetron and granisetron, while compound 7q (pK(i) = 7.5) had very low affinity for this receptor, showing that substitution on the N₁ atom of the benzimidazole ring is essential for affinity and activity. The effect of substitution on the aromatic ring of benzimidazole by several substituents in different positions is also discussed. A strong correlation between the 5-HT₃ antagonistic activity of the studied compounds and the position of substitution on the aromatic ring was established. Thus, while the 4-methoxy derivative 7m showed weak affinity for the 5-HT₃ receptor (pK(i) = 6.7), the 7-methoxy derivative 7n exhibited the highest affinity (pK(i) = 9.4). Compounds 7e and 7n are now under further investigation as drugs for the treatment of nausea and emesis evoked by cancer chemotherapy and radiation.

Full text of DOI:10.1021/jm960442e

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586
J . Med. Chem. 1997, 40, 586-593
New 2-P ip er a zin ylben zim id a zole Der iva tives a s 5-HT3 An ta gon ists. Syn th esis
a n d P h a r m a cologica l Eva lu a tion
Aurelio Orjales,* Ramo´n Mosquera, Luis Labeaga, and Rosa Rodes
Departamento de Investigacio´n, FAES, S.A., Ma´ximo Aguirre 14, 48940 Leioa (Vizcaya), Spain
Received J une 19, 1996X
A series of 2-piperazinylbenzimidazole derivatives were prepared and evaluated as 5-HT3
receptor antagonists. Their 5-HT3 receptor affinities were evaluated by radioligand binding
assays, and their abilities to inhibit the 5-HT-induced Bezold-J arisch reflex in anesthetized
rats were determined. Compound 7e (lerisetron, pKi ) 9.2) exhibited higher affinity for the
5-HT3 receptor than did tropisetron and granisetron, while compound 7q (pKi ) 7.5) had very
low affinity for this receptor, showing that substitution on the N1 atom of the benzimidazole
ring is essential for affinity and activity. The effect of substitution on the aromatic ring of
benzimidazole by several substituents in different positions is also discussed. A strong
correlation between the 5-HT3 antagonistic activity of the studied compounds and the position
of substitution on the aromatic ring was established. Thus, while the 4-methoxy derivative
7m showed weak affinity for the 5-HT3 receptor (pKi ) 6.7), the 7-methoxy derivative 7n
exhibited the highest affinity (pKi ) 9.4). Compounds 7e and 7n are now under further
investigation as drugs for the treatment of nausea and emesis evoked by cancer chemotherapy
and radiation.
Ch a r t 1
In tr od u ction
Since its discovery in the late 1940s1 5-HT has been
implicated in a wide variety of physiological roles in both
the central nervous system (CNS) and periphery. Re-
cent molecular studies and cloning strategies have
identified seven major subclasses of 5-HT receptor,
classified as 5-HT1, 5-HT2, 5-HT3, 5-HT4, 5-HT5, 5-HT6,
and 5-HT7.2-5 Over the past few years, special attention
has been paid to the 5-HT3 receptor, and intensive
efforts have been made toward the discovery of selective
5-HT3 receptor antagonists.6,7 Ondansetron 1,8 gran-
isetron 2,9 zacopride 3,10 and tropisetron 411 are repre-
sentatives of 5-HT3 antagonists (Chart 1), and they have
been shown to be highly effective in the control of
nausea and emesis evoked by citotoxic drugs such as
cisplatin.12,13 Some of them have already been mar-
keted for this therapeutic indication. Moreover, animal
behavior and binding studies on animal and human
brain tissues have suggested a role for these 5-HT3
antagonists within the CNS,14 and they may be effective
in the treatment of migraine,15 schizophrenia,16 and
anxiety.17 We started these studies with the aim of
developing potent and long-acting 5-HT3 antagonists.
On the basis of the structures of known ligands,
several studies have been conducted to define the
structural requirements for the 5-HT3 antagonistic
activity. Three structural features were shown to
contribute to 5-HT3 receptor binding:18 (1) an aromatic
ring, (2) a basic nitrogen atom, and (3) a linking acyl
functional group.
Although most of the 5-HT3 antagonists so far de-
scribed present these structural requirements, there are
several other examples of 5-HT3 radioligands not having
the acyl group which bind with high affinity for the
5-HT3 receptor,19-21 thus showing that the acyl group
is not a necessary requirement. Instead, a hydrogen-
bonding interaction with the receptor22 appears to play
an important role in the affinity. During the course of
our investigation on a series of 2-piperazinylbenzimid-
azole, the 1-(phenylmethyl)-2-piperazinyl derivative 7e
(lerisetron)23 was found to possess high-affinity binding
for the 5-HT3 receptors as well as a potent ability to
inhibit the 5-HT-evoked reflex bradycardia [von Bezold-
J arisch (B-J ) reflex] in urethane-anesthetized rats.
Afterward, the equivalent N-methyl compound of 7e has
been published.24 In this paper we report the synthesis
and initial pharmacological evaluation of compound 7e
and a series of piperazinylbenzimidazole derivatives as
a novel and potent class of 5-HT3 antagonists. These
compounds do not have any acyl group in their struc-
ture, and position 4 of the piperazine moiety is always
unsubstituted.
Ch em istr y
All new compounds are shown in Table 1, and general
synthetic procedures used for their preparation are
illustrated in Schemes 1-3. 2-(1-Piperazinyl)benzimid-
X Abstract published in Advance ACS Abstracts, December 15, 1996.
S0022-2623(96)00442-6 CCC: $14.00 © 1997 American Chemical Society

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2-Piperazinylbenzimidazoles as 5-HT3 Antagonists
Ta ble 1. 2-(1-Piperazinyl)benzimidazole Derivatives 7
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 4 587
compd
R
R1
R2
formulaa
C12H16N4
C13H18N4
C14H20N4
C14H18N4
C18H20N4
C18H19FN4
C18H19ClN4
C19H22N4
C19H22N4O
C18H20N4O
C19H22N4O
C18H20N4O BrH
C19H22N4O
C19H22N4O
C20H24N4
method
% yield
mp, °Cb
7a
7b
7c
7d
7e
7f
7g
7h
7i
CH3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
A, F
A, F
A, F
B, F
A, F
D, F
D, F
D, F
D, F
d
92
95
90
65
98
82
47
50
70
63
54
50
68
85
62
47
42
80-2
oil
175-7
56-8
CH2CH3
(CH2)2CH3
cyclo-C3H5
CH2Ph
CH2Ph
CH2Ph
CH2Ph
CH2Ph
CH2Ph
CH2Ph
CH2Ph
CH2Ph
CH2Ph
CH2Ph
CH2Ph
H
130-2
108-10
125-7
106-8
91-3
100-2
102-4
>245
125-6
101-4
191-3
160-2
>200
(5)F
(5)Cl
(5)CH3
(5)CH3O
(5)OH
(6)CH3O
(6)OH
(4)CH3O
(7)CH3O
(5)CH3
(5)Cl
7j
7k
7lc
7m
7n
7o
7p
7q
E, F
d
D, F
E, F
C, F
C, F
F
(6)CH3
(6)Cl
H
C18H18Cl2N4
C11H14N4
H
a
b
d
Analyses for C, H, N. Recrystallization solvent: hexane. c Bromohydrate. Obtained from 7i and 7k (see the Experimental Section).
Sch em e 1a
a
Reagents: (A) RX, NaH, DMF, rt, 1 h; (B) (i) cyclopropylamine, 150 °C; (ii) H2, Pd/C, EtOH; (iii) urea, ∆; (iv) POCl3/HCl, ∆; (C) (i)
urea, 3 h, 130 °C; (ii) BrCH2Ph, K2CO3, DMF; (iii) POCl3/HCl, 150 °C, 3 h; (D and E) (see Scheme 2 and 3); (F) piperazine, 150 °C.
Sch em e 2a
Unsubstituted aromatic derivatives 6a -c and 6e
were synthesized according to procedure A by N-
alkylation of the sodium salt of 2-chlorobenzimidazole
with the appropriate alkyl halide. Cyclopropyl deriva-
tive 6d was prepared following an alternative route
(procedure B) because of the known reluctance of
cyclopropyl halides to undergo nucleophilic substitution.
Thus, 2-chloronitrobenzene was heated with cyclo-
propylamine in a closed vessel to afford N-cyclopropyl-
2-nitroaniline, which was hydrogenated over palladium/
carbon, cyclized with urea, and halogenated with phos-
phorous oxychloride and hydrochloric acid under pressure
to yield 2-chloro-1-cyclopropyl-1H-benzimidazole (6d ).
Compound 7q was obtained by reaction of 2-chloro-
benzimidazole with piperazine.25
a
Reagents: (a) BrCH2Ph, K2CO3, DMF; (b) H2, Pd/C, THF; (c)
urea, ∆; (d) POCl3/HCl, ∆.
azole derivatives 7 were obtained from 2-chlorobenz-
imidazole derivatives 6 by nucleophilic substitution of
the chlorine atom by piperazine following a standard
procedure.25 2-Chlorobenzimidazole derivatives 6 were
prepared through different routes of synthesis (see
Scheme 1).

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588 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 4
Orjales et al.
Sch em e 3a
a
Reagents: (a) (i) SOCl2; (ii) NaN3; (iii) t-BuOH; (b) H2, Pd/C, EtOH; (c) BrCH2Ph, K2CO3, CH3CN; (d) K2CO3, DMF, ∆; (e) POCl3/HCl,
∆.
Ta ble 2. 5-HT3 Receptor Binding Dataa and in Vivo Receptor
Disubstituted aromatic derivatives 6o and 6p were
Antagonism
synthesized according to procedure C by cyclization of
o-phenylenediamines with urea and N-benzylation and
halogenation with phosphorous oxychloride and hydro-
chloric acid under pressure. Monosubstituted aromatic
derivatives having the substituent at the 4- or 5-position
were synthesized according to procedure D (Scheme 2):
2-nitroanilines 4 were benzylated with benzyl bromide/
potassium carbonate, hydrogenated over palladium/
carbon, cyclized with urea, and halogenated to the
corresponding 2-chlorobenzimidazole 6f-i and 6m .
Compounds having aromatic substitution at the 6- or
7-position were prepared according procedure E as
illustrated in Scheme 3; regioselectively monoprotected
1,2-diaminobenzene derivatives 9 were obtained in an
indirect way from 2-nitrobenzoic acids 5 through a
Curtius rearrangement in the presence of t-BuOH
followed by hydrogenation over palladium/carbon. These
diamines were benzylated and converted into the cor-
responding benzimidazolones 11 by intramolecular cy-
clization in the presence of K2CO3. Conventional treat-
ment of 11 with POCl3/HCl yielded the chloro derivatives
6k and 6n . Hydroxylated compounds 7j and 7l were
prepared from the corresponding methoxylated com-
pounds 7i and 7k by heating to reflux with hydrobromic
acid in acetic acid.
binding
B-J reflex inhibition
ED50c [95% + CL]d
compd
pKi ((SE)b
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
7m
7n
7o
7p
7q
7.3 ((0.1)
7.1 ((0.1)
8.0 ((0.2)
8.8 ((0.1)
9.2 ((0.3)
9.5 ((0.1)
9.5 ((0.2)
8.8((0.1)
7.2 ((0.1)
9.5 ((0.2)
8.4 ((0.1)
9.0 ((0.1)
6.7 ((0.1)
9.4 ((0.2)
8.7 ((0.1)
8.5 ((0.2)
7.5 ((0.1)
8.8 ((0.2)
8.7 ((0.1)
5.3 [4.1-7.5]
7.7 [6.2-9.0]
6.5 [5.6-7.8]
2.6 [1.7-3.8]
2.0 [1.4-2.9]
1.1 [0.7-1.6]
2.5 [1.2-3.5]
1.2 [0.8-1.8]
>100
1.3 [0.5-2.0]
26.8 [22.5-32.6]
1.0 [0.4-1.7]
>100
0.7 [0.4-1.2]
4.9 [3.8-6.0]
16.6 [13.1-20.5]
>100
4, tropisetron
2, granisetron
1.7 [1.0-2.7]
0.7 [0.5-0.9]
a
b
Displacement of [3H]-LY278584. Standard error. c ED50 from
d
dose-effect curves. Values in µg/kg iv. 95% confidence limits.
Ta ble 3. Displacement of Binding to 5-HT1A, 5-HT2A, and
5-HT4 Serotonin Receptors and to D2 Dopamine Receptors by
Compounds of Table 1 and Reference Drugs
pKi ((SE)
compd
7a -p
5-HT1A
<6a
5-HT2A
<6a
9.2 ((0.3)
5-HT4
<6a
D2
P h a r m a cologica l Resu lts a n d Discu ssion
<6a
The new 2-piperazinylbenzimidazole derivatives 7
were initially evaluated for in vitro 5-HT3 receptor
affinity by radioligand binding assay. For each com-
pound, the ability to displace the specific ligand
[3H]LY278584 from 5-HT3 sites of rat entorhinal cortex
was determined. The antagonistic activity at 5-HT3
receptors was performed by evaluating the inhibition
of the B-J reflex evoked by 5-HT in urethane-anesthe-
tized rats. None of the tested compounds per se evoked
bradycardia in anesthetized rats at doses up to 100 µg/
kg iv. A binding profile for the main 5-HT receptors
and the dopamine D2 receptor was performed by radio-
ligand binding assays using specific ligands at different
brain areas from rats or guinea pigs. 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. The
intravenous effective doses to inhibit 50% of B-J reflex
(ED50) were estimated from dose-response curves based
on measurements obtained from five rats/dose.
8-OH-DPAT 8.9 ((0.2)
ritanserin
SB-203186
haloperidol
9.0 ((0.3)
9.2 ((0.2)
a
pIC50: negative logarithm of the molar concentration required
to inhibit 50% of radioligand specific binding (IC50).
affinity, and replacement of the piperazine by other
amines as piperidine, morpholine, pyrrolidine, arylpip-
erazine, and N-methylaniline accounts for a complete
loss of activity (ED50 > 100 µg/kg iv in the inhibition of
B-J reflex). Because of that these compounds were not
selected for further studies.
From results shown in Table 2 the following conclu-
sions can be reached:
(a) N1-substituted benzimidazoles 7a -e show higher
affinities than the parent N1-unsubstituted compound
7q; moreover, compounds with more voluminous sub-
stituent (cyclopropyl and phenylmethyl) are the most
active. Therefore, the compound 1-(phenylmethyl)-2-
piperazinyl-1H-benzimidazole (7e) (pKi ) 9.2, ED50
)
2.0 µg/kg iv) shows higher affinity for the 5-HT3 receptor
than do granisetron and tropisetron (Figure 1a).
(b) The effect of substitution on the aromatic ring of
1-(phenylmethyl)-2-piperazinyl-1H-benzimidazole (7e)
was studied. Introduction of a fluorine atom or a
hydroxy group in the 5-position led to compounds 7f (pKi
) 9.5) and 7j (pKi ) 9.5) with higher affinity for the
5-HT3 receptor and greater ability to inhibit the B-J
The pKi and ED50 values for the new compounds 7
are summarized in Table 2 (data for tropisetron and
granisetron are also included in this table for compari-
son). Their affinities for other serotonin receptors (5-
HT1A, 5-HT2A, 5-HT4) and the dopamine D2 are shown
in Table 3.
The presence of the piperazine moiety in the 2-posi-
tion of benzimidazole is necessary to 5-HT3 receptor

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2-Piperazinylbenzimidazoles as 5-HT3 Antagonists
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 4 589
) 1.3 µg/kg iv) showed the highest affinity in the series.
The same trend was observed for compounds 7k (pKi )
8.4, ED50 ) 26.8 µg/kg iv) and 7l (pKi ) 9.0, ED50 ) 1.0
µg/kg iv).
In order to establish the receptor selectivity of the
compounds 7 for the 5-HT3 receptor, their affinities for
other receptors, namely 5-HT1A, 5-HT2A, 5-HT4, and D2,
were also evaluated. Results are summarized in Table
3. These 2-piperazinylbenzimidazole derivatives rep-
resent a new class of high-affinity ligands for the 5-HT3
receptor and exhibit potent activity in the B-J reflex
inhibition. 7e and 7n are now under further investiga-
tion as drugs for the control of nausea and emesis
evoked by citotoxic drugs such as cisplatin.
Exp er im en ta l Section
Ch em istr y. Each compound was characterized by elemen-
1
tal analysis and their IR, H-NMR, and 13C-NMR spectra. IR
spectra were recorded on a Perkin-Elmer 1310 instrument on
KBr plates; the frequencies are expressed in cm-1 1H-NMR
.
and 13C-NMR spectra 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
internal standard. Coupling constants are given in hertz.
Melting points were determined in open capillaries on a Bu¨chi
SMP-20 apparatus and are uncorrected. Flash column chro-
matography were performed with silica gel, particle size 60
Å, mesh ) 230-400 (Merck). Elemental analyses were within
0.4% of the theoretical values.
2-Ch lor o-1-(ph en ylm eth yl)-1H-ben zim idazole (6e). P r o-
ced u r e A. This procedure illustrates the general method of
preparation of compounds 6a -c and 6e.
Sodium hydride (60% in oil, 0.44 g, 11 mmol) was portion-
wise added to an ice-cooled solution of 2-chlorobenzimidazole
(1.52 g, 10 mmol) in 10 mL of DMF, and the mixture was
stirred at room temperature for 1 h. Benzyl bromide (1.18 mL,
10 mmol) was added, and stirring was continued for 1 h. The
reaction mixture was diluted with water, and the formed
precipitate was collected by filtration and purified by recrys-
tallization from hexane to give 2.18 g (9 mmol, 90%) of a white
F igu r e 1. Competition for [3H]LY278584 binding sites in rat
entorhinal cortex by (a) compound 7e, tropisetron, and gran-
isetron and (b) methoxy derivatives 7m , 7i, 7k , and 7n .
reflex (ED50 ) 1.1 and 1.3 µg/kg iv) than that of the
unsubstituted compound 7e. The importance of meth-
oxy substitution in 5-HT3 antagonists26 prompted us to
synthesize the 5-methoxy derivative 7i (pKi ) 7.2). This
modification unexpectedly decreased by 100-fold the
affinity for the 5-HT3 receptor over 7e, and the ED50
was higher than 100 µg/kg iv in B-J reflex inhibition.
Consequently we sought to study the effect on the
affinity and the activity of the methoxy group in the
different positions of the aromatic ring of compound 7e.
Thus, introduction of a methoxy group in the 7-position
led to compound 7n (pKi ) 9.4, ED50 ) 0.7 µg/kg iv) with
greater affinity for the 5-HT3 receptor and higher B-J
reflex inhibition than compound 7e. These pKi and
ED50 values gradually decreased upon introduction of
the same substituent in the remaining positions of the
ring according to the sequence 7 > 6 . 5 > 4 (Figure
1b). It can be concluded that 5-HT3 antagonistic activity
of the studied compounds are more directly related to
the different substitution position than to the nature
of the substituent.
(c) Disubstituted compounds 5,6-dimethyl 7o (pKi )
8.7, ED50 ) 4.9 µg/kg iv) and 5,6-dichloro 7p (pKi ) 8.5,
ED50 ) 16.6 µg/kg iv) showed good affinity, but ED50
values were higher than those for monosubstituted
compounds 7h (pKi ) 8.8, ED50 ) 1.2 µg/kg iv) and 7g
(pKi ) 9.5, ED50 ) 2.5 µg/kg iv).
(d) Hydroxy-substituted compounds exhibited higher
affinities than those of methoxy substituted ones. Thus
7i (pKi ) 7.2, ED50 > 100 µg/kg iv) had a lower affinity,
and the related hydroxy derivative 7j (pKi ) 9.5, ED50
1
solid: mp 106-108 °C; H NMR (CDCl3) δ 5.4 (s, 2H), 7.2-
7.4 (m, 8H), 7.9 (m, 1H).
2-Ch lor o-1-cyclop r op yl-1H-ben zim id a zole (6d ). P r o-
ced u r e B. (a ) N-Cyclop r op yl-2-n itr oa n ilin e. A mixture
of 2-chloronitrobenzene (3.14 g, 20 mmol) and cyclopropyl-
amine (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 Na2SO4. The solvent was evaporated in vacuo, and the
residue was purified by flash chromatography using a mixture
of hexane and CH2Cl2 (7:3) as eluent to yield after solvent
evaporation 2.15 g (12 mmol, 60%) of an orange oil: 1H NMR
(DCCl3) δ 0.6-0.7 (m, 2H), 0.8-1 (m, 2H), 2.6 (m, 1H), 6.7 (m,
1H), 7.3 (2d, 1H), 7.4-7.6 (m, 2H), 8.2 (2d, 1H); 13C NMR
(CDCl3) δ 7.74, 24.36, 115.23, 126.39, 135.83, 146, 164.
(b) 1,2-Dia m in o-N-cyclop r op ylben zen e. A solution of
N-cyclopropyl-2-nitroaniline (2.15 g, 12 mmol) in EtOH (100
mL) was hydrogenated over 10% palladium/carbon (0.5 g) at
3 atm for 4 h. The catalyst was filtered off and the filtrate
evaporated in vacuo to give 1.54 g (10.44 mmol, 87%) of the
desired product: 1H NMR (CDCl3) δ 0.5 (m, 2H), 0.7-0.8 (m,
2H), 2.4 (m, 1H), 3.4 (m, NH), 6.6-6.7 (m, 2H), 6.8 (m, 1H),
7.1 (d, 1H).
(c) 2-Ch lor o-1-cyclop r op yl-1H-ben zim id a zole (6d ). A
mixture of 1,2-diamino-N-cyclopropylbenzene (1.55 g, 10.49
mmol) and urea (0.66 g, 11 mmol) was stirred at 220 °C for 45
min. The reaction mixture was diluted with water and
extracted with CHCl3 and the extract washed with water and
dried over Na2SO4. The solvent was evaporated in vacuo and
the residue triturated with ether to yield 0.72 g (4.16 mmol,
40%) of a brown solid, which was heated in a 25 mL high-

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590 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 4
Orjales et al.
pressure vessel with POCl3 (7 mL) and HCl (2 drops) at 150
°C for 3 h. The reaction mixture was poured onto ice-water,
neutralized with 50% NaOH, and extracted with CH2Cl2. The
extract was washed with water, dried over Na2SO4, and
concentrated to yield 0.6 g (3.12 mmol, 75%) of the product as
a brown oil: 1H NMR (CDCl3) δ 1.2 (m, 4H), 3.2 (m, 1H), 7.2
(m, 2H), 7.4 (m, 1H), 7.7 (m, 1H).
2-C h lo r o -5,6-d im e t h y l-1-(p h e n y lm e t h y l)-1H -b e n z-
im id a zole (6o). P r oced u r e C. This procedure illustrates
also the preparation of compound 6p . A mixture of 4,5-
dimethyl-1,2-diaminobenzene (3.4 g, 25 mmol) and urea (1.5
g, 25 mmol) was stirred at 150 °C for 30 min. The reaction
mixture was diluted with water and 10% NaOH (10 mL), and
the precipitate was collected by filtration to give 3.5 g (21.8
mmol, 87.5%) of a brown solid. This product was heated in a
high-pressure vessel with POCl3 (30 mL) and HCl (6 drops)
at 150 °C for 3 h. The reaction mixture was poured onto ice-
water and treated with 50% NaOH, and the precipitate was
collected by filtration to give 3.53 g of 5,6-dimethyl-2-chlo-
robenzimidazole (19.6 mmol, 90%) as a brown solid.
Sodium hydride (60% in oil, 0.93 g, 21.6 mmol) was added
portionwise to an ice-cooled solution of 5,6-dimethyl-2-chlo-
robenzimidazole (3.53 g, 19.6 mmol) in DMF (30 mL), and the
mixture was stirred at room temperature for 1 h. Benzyl
bromide (2.43 mL, 20 mmol) was added and stirring continued
for 1 h. The reaction mixture was diluted with water and the
precipitate collected by filtration and purified by flash chro-
matography using a mixture of MeOH and CH2Cl2 (2:98) as
eluent to yield after evaporation of the solvent 2.97 g (10.78
mmol, 50%) of a yellow solid: mp 178-180 °C; 1H NMR
(CDCl3) δ 2.3 (2s, 6H), 5.4 (s, 2H), 7-7.5 (m, 7H).
2-C h lor o -5-flu o r o -1-(p h e n ylm e t h y l)-1H -b e n zim id -
a zole (6f). P r oced u r e D. This procedure illustrates the
general method for preparation of compounds 6f-i and 6m .
(a) 2-Am in o-4-flu or o-N-(ph en ylm eth yl)an ilin e (8f). Ben-
zyl bromide (3.16 mL, 26 mmol) was added dropwise to a
suspension of 4-fluoro-2-nitrobenzenamine (3.2 g, 20 mmol)
and K2CO3 powder (3.6 g, 26 mmol) in DMF (30 mL), and the
resulting mixture was maintained at 110-120 °C for 4 h. The
reaction mixture was diluted with water and extracted with
CH2Cl2. The extract was washed with water and dried over
Na2SO4, the solvent was evaporated in vacuo, and the residue
was dissolved in THF (70 mL) and hydrogenated at 24-25 °C
over 10% Pd/C (0.8 g) for 3 h at 3 atm. The catalyst was
removed by filtration and the filtrate evaporated. The re-
maining crude oil was purified by flash chromatography using
a mixture of AcOEt and hexane (5:1) as eluent to yield, after
solvent evaporation, 2.65 g (12.3 mmol, 75%) of the desired
product: 1H NMR (CDCl3) δ 3.4 (broad, NH), 4.2 (s, 2H), 6.4-
6.6 (cluster, 3H), 7.3-7.4 (cluster, 5 H).
was evaporated in vacuo, and the residue was dissolved in
acetone (20 mL). The solution was added dropwise to an ice-
cooled solution of NaN3 (2 g, 30 mmol) in water (20 mL), and
stirring was continued for 1 h. The reaction mixture was
diluted with water and the precipitate collected by filtration
and dried. A mixture of the crude azide and tert-butyl alcohol
(20 mL) was gradually warmed and then refluxed for 10 min.
After evaporation of the solvent in vacuo, the residue was
purified by flash chromatography using a mixture of AcOEt
and hexane (5:1) as eluent to yield, after solvent evaporation,
3.16 g (12 mmol, 60%) of tert-butyl N-(2-nitro-3-methoxy-
1
phenyl)carbamate as a pale yellow solid: mp 99-100 °C; H
NMR (CDCl3) δ 1.50 (s, 9H), 3.89 (s, 3H), 6.7 (dd, 1H, J )
0.83 and 8.43), 7.38 (t, 1H, J ) 8.43), 7.6 (broad, NH), 7.76
(dd, 1H, J ) 0.83 and 8.43); IR (KBr) 3900, 1720, 1600 cm-1
;
13C NMR (CDCl3) δ 152.67, 151.96, 132.89, 131.85, 113.16,
106.55, 81.64, 56.54, 28.07.
A solution of the above compound (3 g, 11 mmol) in EtOH
(150 mL) was hydrogenated (3 atm) over 10% palladium/
carbon (0.5 g) at room temperature for 3 h. The catalyst was
filtered off and the filtrate evaporated in vacuo to give 2.64 g
(11 mmol, 100%) of the desired product as a white solid: mp
110-112 °C; 1H NMR (CDCl3) δ 1.5 (s, 9H), 3.89 (s, 3H), 3.89
(s, NH), 6.35 (broad, NH), 6.65 (d, 1H), 6.75 (t, 1H), 6.9 (d,
1H); IR (KBr) 3350, 1680 cm-1 13C NMR (CDCl3) δ 153.66,
;
148.64, 129.50, 125.17, 118.36, 116.56, 107.14, 80.4, 57.75,
28.28.
Compound 9k was prepared by the procedure described
above.
(b) ter t-Bu tyl N-[3-Meth oxy-2-[(p h en ylm eth yl)a m in o]-
p h en yl]ca r ba m a te (10n ). To a stirred mixture of 9n (2.15
g, 9 mmol), K2CO3 (1.38 g, 10 mmol), and CH3CN (30 mL) was
added dropwise benzyl bromide (1.19 mL, 10 mmol). The
reaction mixture was stirred at room temperature for 7 h. After
evaporation of the solvent, the residue was diluted with water
and extracted with AcOEt. The extract was washed with
water and dried over Na2SO4. The solution was concentrated
in vacuo, and the residue was triturated with hexane to yield
1.55 g (4.7 mmol, 52%) of a white solid: mp 86-88 °C; 1H NMR
(CDCl3) δ 1.5 (s, 9H), 3.7 (s, 3H), 4.0 (s, 2H), 6.5 (d, 1H), 7.0 (t,
1H), 7.4 (m, 5H), 7.5 (broad, NH), 7.7 (d, 1H); 13C NMR (CDCl3)
δ 153.67, 153.06, 139.92, 134.37, 128.45, 127.25, 125.80,
124.54, 111.46, 104.94, 80.04, 55.63, 28.34.
Compound 10k was prepared following a similar procedure.
(c) 7-Met h oxy-1-(p h en ylm et h yl)-1H -b en zim id a zol-2-
on e (11n ). A mixture of 10n (1.55 g, 4.7 mmol) and K2CO3
(0.69 g, 5 mmol) in DMF (20 mL) was heated at 130 °C for 4
h. The reaction mixture was poured onto ice-water and
filtered to yield 1.42 g (4.5 mmol, 95%) of 11n as a yellowish
solid: 1H NMR (CDCl3) δ 3.8 (s, 3H), 5.3 (s, 2H), 6.6 (d, 1H),
6.7 (d, 1H), 7 (t, 1H), 7.2-7.4 (broad, 5H), 10.1 (s, NH); 13C
NMR (CDCl3) δ 155.11, 144.57, 138.41, 129.26, 127.92, 127.09,
126.68, 121.39, 118.19, 104.11, 102.85, 55.3, 45.43.
Compounds 8g, 8h , 8i, and 8m were prepared following a
similar procedure.
(b) 2-Ch lor o-5-flu or o-1-(p h en ylm eth yl)-1H-ben zim id -
a zole (6f). A mixture of 8f (2.65 g, 12.30 mmol) and urea (0.78
g, 13 mmol) was stirred at 220 °C for 0.5 h. The reaction
mixture was diluted with water and extracted with CHCl3;
the extract was washed with water and dried over Na2SO4.
The solvent was evaporated in vacuo and the residue triturated
with hexane to yield, after filtration, 2.42 g (10 mmol, 77%) of
5-fluoro-1-(phenylmethyl)-1H-benzimidazol-2-one. This prod-
uct was heated in a 25 mL high-pressure vessel with POCl3
(4 mL) and HCl (3 drops) at 150 °C for 3 h, and the reaction
mixture was poured onto ice-water, neutralized with 50%
NaOH, and extracted with CH2Cl2. The extract was washed
with water, dried over Na2SO4, and concentrated to yield a
brown solid: 1.56 g (6 mmol, 60%): 1H NMR (CDCl3) δ 5.4 (s
2H), 7-7.4 (cluster, 8H).
Compound 11k was prepared following a similar procedure.
(d ) 2-Ch lor o-7-m et h oxy-1-(p h en ylm et h yl)-1H -b en z-
im id a zole (6n ). A mixture of 11n (2.11 g, 8.3 mmol), POCl3
(30 mL), and concentrated HCl (6 drops) was heated in a high-
pressure vessel at 150 °C for 3 h. The reaction mixture was
poured onto ice-water and extracted with CH2Cl2. The
organic layer was washed with water, dried over Na2SO4, and
concentrated in vacuo to yield 1.8 g (6.6 mmol, 80%) of a brown
1
solid: mp 111-113 °C; H NMR (CDCl3) δ 3.9 (s, 3H), 5.6 (s,
2H), 6.7 (d, 1H), 7.3 (m, 7H).
Compound 6k was prepared by the procedure described
above.
1-(P h en ylm eth yl)-2-piper azin yl-1H-ben zim idazole (7e).
Gen er a l P r oced u r e for th e P r ep a r a tion of 2-(1-P ip er -
a zin yl)ben zim id a zole 7 (Ta ble 1). P r oced u r e F . A mix-
ture of 6e (2.42 g, 10 mmol) and piperazine (4.3 g, 50 mmol)
was heated at 150-160 °C for 30 min. The reaction mixture
was poured onto water, and then 10% HCl was added to acidify
the mixture, which was washed twice with CHCl3. Then a
solution of 10% NaOH was added to basify the mixture,
followed by extraction with CHCl3. The organic layer was
washed with water and dried over Na2SO4. The solvent was
evaporated under reduced pressure, and the residue was
Compounds 6g-i and 6m were prepared by the procedure
described above.
2-Ch lor o-7-m et h oxy-1-(p h en ylm et h yl)-1H -b en zim id -
a zole (6n ). P r oced u r e E. This procedure illustrates the
general method for the preparation of compounds 6n and 6k .
(a ) ter t-Bu tyl N-(2-Am in o-3-m eth oxyp h en yl)ca r ba m -
a te (9n ). A mixture of 2-nitro-3-methoxybenzoic acid (3.94 g,
20 mmol), thionyl chloride (2.2 mL, 30 mmol), and DMF (4
drops) in toluene (20 mL) was refluxed for 0.5 h. The solvent

+
+
2-Piperazinylbenzimidazoles as 5-HT3 Antagonists
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 4 591
purified by recrystallization from hexane to give the desired
protein). After incubation for 15 min at 37 °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 7.7) buffer.
Filters were placed in scintillating polyethylene vials (with 5
mL 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).28
product 7e (2.77 g, 9.5 mmol, 95%) as a white solid: mp 129-
1
130 °C; H NMR (CDCl3) δ 2.2 (broad, 1H), 2.5 (m, 4H), 2.6
(m, 4H), 4.6 (s, 2H), 7-7.4 (m, 8H), 7.7 (m, 1H).
5-H yd r oxy-1-(p h en ylm et h yl)-2-p ip er a zin yl-1H -b en z-
im id a zole (7j). A mixture of 7i (1.21 g, 4 mmol), 20 mL of
HBr (48%), and 10 mL of acetic acid was stirred at 150 °C for
3 h. After evaporation of the solvent, the residue was treated
with 10% Na2CO3 solution until pH ) 10 and extracted with
CH2Cl2. The organic layer was washed with water and dried
over Na2SO4. The solution was concentrated in vacuo to give
0.74 g (2.4 mmol, 60%) of the desired product as a yellow
solid: mp 100-102 °C; 1H NMR (DMSO-d6) δ 2.9 (m, 4H), 3.2
(m, 4H), 5.2 (s, 2H), 6.5 (m, 1H), 6.9 (m, 2H), 7.1-7.4 (m, 5H),
9.0 (broad, OH).
5-HT3 Recep tor Bin d in g Assa y.30 Adult male Wistar rats
weighing 220-280 g were used. Animals were killed by
decapitation, the whole brain with the exception of the brain
stem and cerebellum was quickly removed, and the various
areas were dissected, weighed, and immediately frozen at -70
°C. The enthorinal 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 and centrifuged
at 1000g for 10 min (4 °C), and the supernatant was recen-
trifuged 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 more times 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 the 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 vehicule if no compet-
ing 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. The
binding experiment was initiated by addition of 0.8 mL of
membrane suspension (500-600 µg of protein). After incuba-
tion 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
scintillating polyethylene vials (with 5 mL scintillation cock-
tail) 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).28
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. The striatum used for the binding experi-
ments 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 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 concentration (0.1 mL of vehicule if no compet-
ing 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 experiment was initiated by addition of 0.8 mL of
membrane suspension (800-900 µg of protein). After incuba-
tion 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 Hepes buffer), using a Brandel
cell harvester. Filters were placed in scintillating polyethylene
vials (with 5 mL of scintillation cocktail) and equilibrated, and
filter-retained radioactivity was measured in a liquid scintil-
lation counter (Kontron Beta V). IC50 and Ki values were
calculated using the computer program EBDA (McPherson).28
Compound 7l was prepared by a similar procedure.
P h a r m a cologica l Meth od s. 5-HT1A Recep tor Bin d in g
Assa y.27 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. The cerebral 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 900g for 10 min (4 °C), and the
supernatant was recentrifuged at 4800g for 25 min (4 °C). The
resulting pellet was resuspended in 10 volumes of ice-cold 50
mM Tris-HCl buffer (pH 7.5), incubated at 37 °C for 15 min,
and then centrifuged once more at 48000g for 25 min (4 °C).
The final pellet was resuspended in 4 volumes of ice-cold 50
mM Tris-HCl buffer containing 4 mM CaCl2 and 0.1% ascor-
bate 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 1:28, wt/vol).
Competition assays were performed in triplicate, in a final
volume of 1 mL. To each assay tube were added the follow-
ing: 0.1 mL of the displacer drug concentration (0.1 mL of
vehicule if no competing drug was added) and 0.1 mL of [3H]-
8-OH-DPAT (NEN, 148-163 Ci/mmol) in buffer (final concen-
tration 1.5 nM). Nonspecific binding was determined using
10 µM cold 5-HT. The binding experiment was initiated by
addition of 0.8 mL of a membrane suspension (700-800 µg of
protein). After incubation for 30 min at 37 °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 7.5) buffer.
Filters were placed in scintillating polyethylene 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
by using the computer program EBDA (McPherson).28
5-HT2A Recep tor Bin d in g Assa y.29 Adult male Wistar
rats weighing 220-280 g were used. Animals were killed by
decapitation, the whole brain with the exception of the brain
stem and cerebellum was quickly removed, and the various
areas were dissected, weighed, and immediately frozen at -70
°C. Prefrontal cortex used for the binding experiments were
homogenized with an Ultra-Turrax (setting 5 for 20 s) in 10
volumes of ice-cold 0.25 M sucrose buffer and centrifuged at
1080g for 10 min (4 °C), and the supernatant was recentrifuged
at 35000g for 10 min (4 °C). The resulting pellet was
resuspended in 40 volumes of ice-cold 50 mM Tris-HCl buffer
(pH 7.7) and then centrifuged once more at 3500g for 10 min
(4 °C). The final pellet was resuspended in 10 volumes of ice-
cold 50 mM Tris-HCl buffer 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 1:60, wt/vol).
Competition assays were performed in triplicate, in a final
volume of 1 mL. To each assay tube were added the follow-
ing: 0.1 mL of the displacer drug concentration in Tris-HCl
buffer with 10% ethanol (0.1 mL of Tris-ethanol buffer in no
competing drug was added) and 0.1 mL of [3H]ketanserin
(NEN, 60-90 Ci/mmol) in Tris-ethanol buffer (final concentra-
tion 0.8 nM). Nonspecific binding was determined using 1 µM
cold methysergide. Binding experiment was initiated by
addition of 0.8 mL of membrane suspension (400-500 µg of

+
+
592 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 4
Orjales et al.
D2 Recep tor Bin d in g Assa y.32 Adult male Wistar rats
weighing 220-280 g were used. Animals were killed by
decapitation, the whole brain with the exception of the brain
stem 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 homog-
enized with an Ultra-Turrax (setting 5 for 20 s) in 50 volumes
of ice-cold 50 mM Tris-HCl buffer (pH 7.7) and centrifuged at
48000g for 10 min (4 °C). The resulting pellet was resus-
pended 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 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 follow-
ing: 0.1 mL of the displacer drug concentration (0.1 mL of
vehicule if no competing drug was added) and 0.1 mL of
[3H]raclopride (NEN, 60-87 Ci/mmol) in buffer (final concen-
tration 1 nM). Nonspecific binding was determined using 1
µM cold (+)-butaclamol. The 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 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 scintillating polyethylene 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).28
In h ibition of th e Bezold -J a r isch Reflex. Adult male
Wistar rats weighing 220-300 g and fasted for 18 h were used.
Rats were anesthetized with urethane (1.25 g/kg ip) and placed
on a heating table (Hugo Sachs Elektronik, Freiburg, Ger-
many) to maintain body temperature at 37 °C. The trachea
and right jugular vein were cannulated to facilitate respiration
and drug administration, respectively. The carotid artery was
also cannulated 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 Linearcorder
WR3101 (Hugo Sachs Elektronik, Freiburg, Germany). The
B-J reflex was evoked by rapid bolus iv injection of 5-HT (30
µg/kg). When 5-HT-induced bradycardia (65-75% fall of heart
rate from control heart rate) returned to pretreatment levels
(within 5 min), either antagonist or saline were administered
(1 mL/kg), and 5-HT-induced bradycardia was elicited again
5 min later. Drugs were prepared daily and were dissolved
in distilled water or 0.1 M tartaric acid, with subsequent
dilutions in physiological saline. ED50 were calculated from
three to five doses (four or five rats per dose) by a linear-
regression analysis.
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Ack n ow led gm en t. This work was supported in part
by the Direccio´n General de Tecnolog´ıa Industrial of the
Ministry of Industry and Energy of Spain and the
Department of Industry and Commerce of the Basque
Government. We thank Dr. Lourdes Cortizo, Dr. Vı´ctor
Rubio, and Dr. Neftal´ı Garc´ıa for helpful discussions.
Su p p or tin g In for m a tion Ava ila ble: Yields, melting
points, and spectral data (1H NMR) for compounds 7a -d and
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