Synthesis and biological evaluation of new non-imidazole H 3-receptor antagonists of the 2-aminobenzimidazole series

Article abstract of DOI:10.1016/j.bmc.2005.09.063

A novel series of non-imidazole H₃-receptor antagonists was developed, by chemical modification of a potent lead H₃-antagonist composed by an imidazole ring connected through an alkyl spacer to a 2-aminobenzimidazole moiety (e.g., 2-

Full text of DOI:10.1016/j.bmc.2005.09.063

Bioorganic & Medicinal Chemistry 14 (2006) 1413–1424
Synthesis and biological evaluation of new non-imidazole
H3-receptor antagonists of the 2-aminobenzimidazole series
Mirko Rivara,a Valentina Zuliani,a Giuseppe Cocconcelli,a Giovanni Morini,a
Mara Comini,a Silvia Rivara,a Marco Mor,a,* Fabrizio Bordi,a Elisabetta Barocelli,b
Vigilio Ballabeni,b Simona Bertonib and Pier Vincenzo Plazzia
a
`
Dipartimento Farmaceutico, Universita degli Studi di Parma, Parco Area delle Scienze 27/A, I-43100 Parma, Italy
b
`
Dipartimento di Scienze Farmacologiche, Biologiche e Chimiche Applicate, Universita degli Studi di Parma,
Parco Area delle Scienze 27/A, I-43100 Parma, Italy
Received 26 July 2005; revised 21 September 2005; accepted 27 September 2005
Available online 2 November 2005
Abstract—A novel series of non-imidazole H3-receptor antagonists was developed, by chemical modification of a potent lead H3-
antagonist composed by an imidazole ring connected through an alkyl spacer to a 2-aminobenzimidazole moiety (e.g., 2-[[3-
[4(5)-imidazolyl]propyl]amino]benzimidazole), previously reported by our research group. We investigated whether the removal
of the imidazole ring could allow retaining high affinity for the H3-receptor, thanks to the interactions undertaken by the 2-amino-
benzimidazole moiety at the binding site. The imidazole ring of the lead was replaced by a basic piperidine or by a lipophilic p-chlo-
rophenoxy substituent, modulating the spacer length from three to eight methylene groups; moreover, the substituents were moved
to the 5(6) position of the benzimidazole nucleus. Within both the 2-alkylaminobenzimidazole series and the 5(6)-alkoxy-2-amino-
benzimidazole one, the greatest H3-receptor affinity was obtained for the piperidine-substituted compounds, while the presence of
the p-chlorophenoxy group resulted in a drop in affinity. The optimal chain length was different in the two series. Even if the new
compounds did not reach the high receptor affinity shown by the imidazole-containing lead compound, it was possible to get good
H3-antagonist potencies with 2-aminobenzimidazoles having a tertiary amino group at appropriate distance.
Ó 2005 Elsevier Ltd. All rights reserved.
1. Introduction
tion of the amine in the CNS.6 Moreover, this receptor
acts as a heteroreceptor on non-histaminergic neurons
in the brain and in periphery, inhibiting the release of
other neurotransmitters (e.g., acetylcholine, noradrena-
line, dopamine, and serotonin)7–10 and of neuropep-
tides.11 Several different histamine H3-receptor
isoforms have been characterized in rats, guinea-pigs,
and humans, and they have been shown to exhibit dis-
tinct pharmacological profiles.12,13 The high density of
H3-receptors found in the CNS14 has suggested potential
therapeutic applications of histamine H3-antagonists for
several CNS diseases, such as attention-deficit hyperac-
tivity disorders (ADHD),15 Alzheimerꢀs disease,16 obes-
ity and control of food intake,17,18 epilepsy,19–21 and
schizophrenia.22
Histamine is a biogenic amine implicated in a wide range
of physiological processes through the activation of four
G-protein coupled receptors (H1, H2, H3, and H4).1 The
postsynaptic H1- and H2-receptors are mainly involved
in the control of allergic responses and gastric acid secre-
tion, respectively, while the more recently described
H4-receptor seems to be implicated in inflammatory
processes.2,3 The third receptor subtype (H3) is mainly
located presynaptically on histaminergic neurons in the
central nervous system (CNS), where it downregulates
histamine synthesis and release acting as an autorecep-
tor.4,5 Thus, H3-receptor antagonists inhibit this
negative feedback mechanism, increasing the concentra-
Many H3-antagonists belong to the class of imidazole-
containing compounds.23,24 It is now well established
that the presence of the imidazole ring might cause phar-
macological liabilities. In fact, the coordination of this
heterocycle with the heme moiety of cytochrome P450
Keywords: Histamine; H3-receptor antagonists; 2-Aminobenzimida-
zole; Non-imidazole.
*
Corresponding author. Tel.: +39 0521 905062; fax: +39 0521
905006; e-mail: marco.mor@unipr.it
0968-0896/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.09.063

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M. Rivara et al. / Bioorg. Med. Chem. 14 (2006) 1413–1424
O
N
O
isoenzymes can lead to drug–drug interaction and meta-
bolic interferences.25
HN
N
Cl
Cl
FUB 649 (pKi=7.20)
FUB 181 (pKi=8.30)
O
O
With the aim of obtaining new H3-antagonists with im-
proved pharmacokinetic properties and enhanced access
to the CNS, several different classes of H3-antagonists,
lacking the imidazole ring, have been synthesized. Some
examples of the most recently reported non-imidazole
H3-receptor antagonists are depicted in Figure 1.26–37
These non-imidazole H3-antagonists are usually charac-
terized by the presence of an aminoalkyl moiety,
although with different chain lengths and an overall
structure. The absence of the imidazole ring does not
prevent these compounds from displaying a high poten-
cy at the H3-receptor, since many compounds are active
at nanomolar concentrations. Moreover, some of these
compounds have demonstrated high activity not only
in vitro, but also in vivo.38–41
O
N
O
HN
N
UCL 2190 (pKi=8.40)
ciproxifan (pKi=9.20)
Figure 2. Non-imidazole H3-receptor antagonists obtained by bioisos-
teric replacement of the imidazole moiety and their parent compounds.
approach, we synthesized new non-imidazole H3-recep-
tor antagonists starting from a series of imidazole-con-
taining 2-aminobenzimidazole derivatives, previously
described by us,45 which afforded a lead structure
(Fig. 3), endowed with high affinity for the H3-receptor
(pKi = 8.90, rat brain membranes and pKi = 8.31, hu-
man H3-receptor). In this series, the 2-aminobenzimi-
dazole fragment and its 5(6)-methoxy derivative
behaved as weak bases (pKa = 6.2–6.7), thus being able
Several attempts to develop new non-imidazole com-
pounds were based on the bioisosteric replacement of
the imidazole moiety of well-known and very potent
H3-receptor antagonists.42–44 This replacement has of-
ten led to a drop in H3-antagonist potency but, in some
cases, in vitro affinity values were maintained (e.g.,
FUB 649 and UCL 2190, obtained from FUB 181
and ciproxifan, respectively, Fig. 2).44 Following this
N
NH
N
HN
N
H
Figure 3. Lead structure.
O
N
CH3
N
O
N
O
N
N
O
CH3
O
NH
O
a26
b27,36
H
N
N
N
N
O
O
O
H3C
d29
c28
Cl
NH
N
O
CN
H3C
N
N
NH
NH
CH3
e30
f31,36
NH
N
N
O
N
N
g32
h33
N
O
CN
O
N
O
N
N
N
F
N
H
H3C
O
O
N
CH3
CH3
i34,36
l35
O
N
N
O
F
F
N
N
F
n36,37
m36
Figure 1. Non-imidazole H3-receptor antagonists.

M. Rivara et al. / Bioorg. Med. Chem. 14 (2006) 1413–1424
1415
to interact with the H3-receptor binding site in their
protonated or, more probably, neutral form.46 We
retained the 2-aminobenzimidazole fragment which,
according to our previous results,47 is the heterocyclic
polar fragment conferring the highest potency at the
H3-receptor, and we replaced the imidazole ring, to
investigate if it were possible to maintain high affinity
for the H3-receptor. Following the hypothesis that the
2-aminobenzimidazole moiety would constitute a criti-
cal anchor group at the H3-receptor binding site, addi-
tional interactions were supposed for a side chain with
a basic or a lipophilic ending group, introduced at dif-
ferent positions of the 2-aminobenzimidazole nucleus.
Potent non-imidazole H3-receptor antagonists, charac-
terized by the presence of a piperidine or a pheno-
xyalkylamine moiety, have been reported.36,48 Thus,
starting from the lead structure reported in Figure 3,
the imidazole ring was replaced by a basic piperidine
or by a lipophilic p-chlorophenoxy ring, and the chain
length was also modulated, preparing compounds with
three to eight methylene linkers (Table 1, 1–9). In fact,
although the replacement of the imidazol-4(5)-yl ring
by suitable moieties has been successfully applied to
different classes of imidazole-containing H3-antago-
nists, different structure–activity relationships have
been observed in the two series. The side chain was
also moved to the 5(6) position of the 2-aminobenzim-
idazole moiety (Table 2, 10–15), to test a different rel-
ative disposition of the 2-aminobenzimidazole nucleus
and the basic piperidine or the lipophilic p-chlorophen-
oxy group.
2. Chemistry
The 2-aminobenzimidazole derivatives described in this
paper were prepared following various synthetic routes,
as outlined in Schemes 1–3.
Table 1. Histamine H3-receptor affinity (pKi) and antagonist potency (pKB) of compounds 1–9
N
R
CH2 NH
n
N
H
c
Compound
n
R
Human H3 pKia
Rat H3 pKib
Guinea-pig H3 pKB
N
N
1
3
5.56 0.16
5.65 0.08
ndd
2
5
6
7
8
4
5
6
7
7.16 0.01
7.48 0.02
7.43 0.11
6.82 0.06
6.03 0.16
5.98 0.09
6.02 0.02
6.25 0.03
7.28 0.15
6.62 0.01
7.62 0.05
6.90 0.05
7.07 0.16
7.62 0.07
7.50 0.07
3
N
N
N
4
5
7.27 0.15
7.52 0.10
e
f
6
Cl
Cl
Cl
Cl
O
O
O
O
e
e
f
f
7
8
9
5.66 0.19
8.59 0.05g
6.64 0.01
9.04 0.14h
Thioperamide
a Inhibition of [3H]RAMHA binding to GPCR97-transfected SK-N-MC cells stably expressing the human histamine H3-receptor.
b Inhibition of [3H]RAMHA binding to rat brain membranes.
c Inhibition of RAMHA-induced effects on guinea-pig isolated ileum. pKB values obtained according to Furchgottꢀs method.57
d Not determined.
e Inactive until 30 lM.
f Inactive until 10 lM.
g Ref. 58.
h Ref. 59.

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M. Rivara et al. / Bioorg. Med. Chem. 14 (2006) 1413–1424
Table 2. Histamine H3-receptor affinity (pKi) and antagonist potency (pKB) of compounds 10–15
H
R
CH
2
O
n
N
NH
2
N
c
Compound
n
R
Human H3 pKia
Rat H3 pKib
Guinea-pig pKB
10
4
7.53 0.08
7.31 0.25
7.38 0.13
N
N
N
11
5
6
3
4
5
7.00 0.12
7.14 0.03
5.46 0.07
5.28 0.09
5.70 0.14
7.28 0.15
6.92 0.04
6.54 0.14
8.03 0.13
12
7.48 0.07
e
13
5.22 0.13
Cl
Cl
Cl
O
O
O
d
e
e
14
15
5.37 0.09
8.59 0.05f
Thioperamide
9.04 0.14g
a Inhibition of [3H]RAMHA binding to GPCR97-transfected SK-N-MC cells stably expressing the human histamine H3-receptor.
b Inhibition of [3H]RAMHA binding to rat brain membranes.
c Inhibition of RAMHA-induced effects on guinea-pig isolated ileum. pKB values obtained according to Furchgottꢀs method.57
d Inactive until 30 lM.
e Inactive until 10 lM.
f Ref. 58.
g Ref. 59.
N
N
a
densed with 2-chlorobenzimidazole, to give the desired
products (Scheme 2).
+
N
(CH2)n NH2
Cl
N (CH2)n NH
N
H
N
H
n = 3, 5, 6, 7, 8
1-5
The synthetic procedure followed for the 2-aminobenz-
imidazole derivatives substituted in 5(6) position (10–
15) is represented in Scheme 3. The final products were
obtained starting from the common intermediates N-[4-
(x-chloroalkoxy)-2-nitrophenyl]acetamides, which were
treated with piperidine (10–12) or 4-chlorophenol (13–
15). The 4-substituted-2-nitrophenylacetamide deriva-
tives so obtained were hydrolyzed, reduced, and treated
with BrCN to give the final products.
Scheme 1. Synthesis of compounds 1–5. Conditions: (a) isoamyl
alcohol, reflux.
The 2-[x-(piperidin-1-yl)alkylamino]-1H-benzimidazole
derivatives (1–5) were synthesized by condensation of
the corresponding x-piperidin-1-yl-alkylamine29,44,49,50
with 2-chlorobenzimidazole, according to Scheme 1.
3. Pharmacology
The 2-[x-(p-chlorophenoxy)alkylamino]-1H-benzimid-
azole derivatives (6–9) were obtained starting from
4-chlorophenol and the appropriate commercially avail-
able x-bromoalkyl cyanide. The x-(4-chlorophenoxy)
alkyl cyanide derivatives were then reduced and con-
H3-receptor affinity of the newly synthesized compounds
was measured by displacement of [3H]-(R)-a-methylhis-
tamine ([3H]RAMHA) from GPCR97-transfected
a
b
Br (CH2)n-1 CN
+
Cl
Cl
OH
O
Cl
O
c
(CH2)n-1 CN
Cl
O
(CH2)n NH2
N
N
H
N
Cl
Cl
O (CH2)n
+
(CH2)n NH2
N
H
N
H
n =4, 5, 6, 7
6-9
Scheme 2. Synthesis of compounds 6–9. Reagents and conditions: (a) K2CO3, acetone, reflux; (b) LiAlH4, THF, rt; (c) isoamyl alcohol, reflux.

M. Rivara et al. / Bioorg. Med. Chem. 14 (2006) 1413–1424
1417
NO2
+
HO
NHCOCH3
Cl (CH2)n Br
n = 3, 4, 5, 6
a
NO2
Cl (CH2)n
O
NHCOCH3
n = 3, 4, 5, 6
d,e
b,c
NO2
NH2
NO2
NH2
N
(CH2)n
O
Cl
O
(CH2)n
O
n = 3, 4, 5
n = 4, 5, 6
f,g
f,g
NH2
N
NH2
NH
N
N
(CH2)n
O
Cl
O
(CH2)n
O
NH
n = 4, 5, 6
n = 3, 4, 5
13-15
10-12
Scheme 3. Synthesis of compounds 10–15. Reagents and conditions: (a) Na2CO3, KI, DMF, 50 °C; (b) piperidine, 50 °C; (c) NaOH, reflux; (d)
4-chlorophenol, K2CO3, KI, DMF, 50 °C; (e) KOH, ethanol, rt; (f) H2, 10% Pd/C, ethanol, rt; (g) BrCN, rt.
SK-N-MC cells stably expressing the human histamine
H3-receptor51 and from rat cerebral cortex mem-
branes.52 Histamine H3-receptor antagonist potency
was evaluated on electrically stimulated guinea-pig
ileum, by inhibition of RAMHA-induced responses.53
replacement of the basic piperidine group with the lipo-
philic p-chlorophenoxy fragment resulted in a fifteen to
thirty times drop in affinity at the human H3 receptor;
this suggests a direct interaction of the basic moiety with
the H3-receptor binding site. The highest pKi value was
observed for compound 3, both at human H3 receptor
and at rat H3 receptor, and it was also the most potent
in the H3 functional assay, although with a pKB value
comparable to those of compounds 4 and 5. Within
the series of 5(6)-alkoxy-2-aminobenzimidazoles, the
highest potency was observed for the piperidine deriva-
tive having a four methylene spacer (10) and, again, an
evident decrease in affinity was observed for the lipophil-
ic p-chlorophenoxy derivatives. The piperidine deriva-
tives 10–12 showed an apparent modest selectivity for
the human H3-receptor.
4. Results and discussion
Affinity data (pKi) for human H3-receptor and for rat
brain histamine H3-receptor and H3-antagonist potency
(pKB) on guinea-pig ileum for the newly synthesized
2-alkylaminobenzimidazole derivatives having a piperi-
dine or a p-chlorophenoxy substituent (1–9) are reported
in Table 1. Table 2 reports the biological data for the
5(6)-alkoxy-2-aminobenzimidazoles, substituted with
the piperidine or the p-chlorophenoxy group in the
5(6) position (10–15). The most evident result is the dif-
ferent behavior observed for compounds having a basic
or a lipophilic group replacing the imidazole ring of the
While significantly different affinity values were
observed for thioperamide between human and rat
receptors, our compounds showed only minor differenc-
es (60.6). Higher discrepancies were observed with the
functional potency data on guinea-pig ileum (e.g., for
compound 11), which can be due to species-specificity
or to the different nature of the pharmacological tests.
lead
2-[[3-[4(5)-imidazolyl]propyl]amino]benzimidazole
(Fig. 3), for both aminobenzimidazoles substituted on
the amino-group in the 2 position and the 5(6)-alkoxy
derivatives. The piperidine derivatives acted as compet-
itive H3-antagonists on guinea-pig ileum and showed
medium to quite good affinity values for human hist-
amine H3-receptors and for rat cerebral H3-receptors.
On the contrary, the p-chlorophenoxy derivatives were
inactive or endowed with very low pKi values; moreover,
no competitive antagonism was detected for these com-
pounds, with the only exception of the weakly potent
compound 9. In particular, within the 2-alkylamino-
benzimidazole series (1–9), the highest affinity values
were observed for the hexamethylene (3) and the hep-
tamethylene (4) derivatives. As mentioned above, the
It can be concluded that the introduction of the basic
piperidine substituent led to a quite good affinity for
the H3-receptor, given an appropriate distance from
the 2-aminobenzimidazole nucleus. In fact, the optimal
length of the alkyl spacer depended on the position of
substitution: six methylene groups for the 2 position
(3) and a tetramethylene chain for the 5(6) position
(10). On the other hand, the most potent compounds
of the two series 3 and 10, although structurally differ-
ent, can be easily superposed (Fig. 4). They can place

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M. Rivara et al. / Bioorg. Med. Chem. 14 (2006) 1413–1424
Table 3. Yields and characteristic data of the final compounds
Compound
Yield (%)
Crystallization solvent
Mp (°C)
1
2
81
75
73
81
79
78
80
85
78
30
28
28
54
52
52
abs EtOH/Et2O
CCl4/CHCl3
Et2O/petroleum ether
CCl4/CHCl3
Et2O/petroleum ether
AcOEt/CCl4
AcOEt
177–178a
130–132b
153–155b
151–152b
145–147b
173–174b
127–129b
184–185b
144–145b
192–193c
220–222d
215–217e
167–169b
205–207b
156–157b
3
4
5
6
Figure 4. Superposition of compounds 3 (white carbons) and 10
(yellow carbons) obtained from energy minimized structures by a rigid
fit superposition of nitrogen atoms.
7
8
AcOEt
AcOEt
9
10
11
12
13
14
15
abs EtOH/Et2O
abs EtOH/Et2O
abs EtOH
their polar groups in a similar disposition, suggesting a
common interaction with the H3-receptor binding site;
this hypothesis is supported by the similar potencies of
the two compounds.
EtOH/H2O
EtOH/H2O
EtOH/H2O
a Dioxalate.
b Free base.
c DihydrochlorideÆ1.5H2O.
d DihydrochlorideÆ1/2C2H5OH.
e DihydrochlorideÆH2O.
The compounds described here were also evaluated for
H3-receptor selectivity with respect to the H1- and H2-
receptors on guinea-pig ileum and on guinea-pig atria,
respectively.45 They did not exhibit any remarkable
inhibition of H1 and H2 mediated responses up to
1 lM concentration.
in hertz (Hz), and number of protons; signals were char-
acterized as s (singlet), d (doublet), dd (doublet of dou-
blets), t (triplet), q (quartet), and m (multiplet) br s
(broad signal). Abbreviations are the following: Bzim,
benzimidazolyl; Ph, phenyl. Mass spectra were recorded
using a Finnigan MAT SSQ 710 instrument. Reactions
were monitored by TLC, on Kieselgel 60 F 254 (DC-
Alufolien, Merck). Final compounds and intermediates
were purified by chromatography on preparative Gilson
MPLC, using a SiO2 column (LiChroprep, Si 60, 25–
40 lm, Merck); the eluents were mixtures of CH2Cl2/
CH3OH at various volume ratios. When indicated, gas-
eous NH3 was added to the methanolic phase to obtain
a 5% w/w solution. Yields and characteristic data of the
final compounds are described in Table 3.
5. Conclusions
A novel series of non-imidazole H3-antagonists was
developed, obtained from chemical modification of a
very potent imidazole H3-receptor antagonist, charac-
terized by a 2-aminobenzimidazole heterocyclic polar
fragment (Fig. 3). When the imidazole ring was replaced
by a lipophilic p-chlorophenoxy moiety (6–9 and 13–15),
a remarkable decrease in affinity was observed, probably
due to the inability of this lipophilic fragment to under-
take a good interaction at the H3 binding site. On the
contrary, it was possible to obtain good H3-antagonist
binding affinities for 2-aminobenzimidazoles carrying a
basic piperidine substituent at appropriate distance,
although this novel structural class of non-imidazole
derivatives did not reach the high receptor affinity
shown by the lead imidazole derivative. No substantial
differences between human and rat histamine H3-recep-
tor binding data were observed for these series of com-
pounds. The results obtained suggest the series of
piperidine-substituted 2-aminobenzimidazole H3-recep-
tor antagonists as an interesting subject for further
investigations.
6.1. General method of preparation of 2-[x-(piperidin-1-
yl)alkylamino]-1H-benzimidazole derivatives (1–5)
A solution of 4.4 mmol of 2-chlorobenzimidazole and
8.8 mmol of the appropriate x-piperidin-1-yl-alkylamine
in 2.0 ml of isoamyl alcohol was heated at 130 °C for
16 h. The crude products were purified by column chro-
matography (SiO2, CH2Cl2:CH3OH(NH3) = 9:1).
6.1.1.
2-[3-(Piperidin-1-yl)propylamino]-1H-benzimid-
azole dioxalate (1Æ2C2H2O4). 1H NMR (DMSO-d6) d
1.53–1.55 (m, 2H, CH2), 1.72–1.74 (m, 4H, CH2), 2.00
(m, 2H, CH2), 3.08–3.13 (m, 6H, CH2), 3.46 (t,
J = 6.3 Hz, 2H, CH2), 7.09–7.14 (m, 2H, Bzim), 7.30–
7.34 (m, 2H, Bzim). MS (CI) 259 [M+1]+. Anal. calcd
for C15H22N4Æ2C2H2O4: C, 52.05; H, 5.98; N, 12.78.
Found: C, 52.30; H, 6.28; N, 12.57.
6. Experimental
Melting points were not corrected and were determined
with a Buchi instrument (Tottoli) and with Gallenkamp
¨
melting point apparatus. The final compounds were
analyzed on a ThermoQuest (Italia) FlashEA 1112 Ele-
mental Analyzer, for C, H, and N. The percentages we
found were within 0.4% of the theoretical values. The
1H NMR spectra were recorded on a Bruker 300 spec-
trometer (300 MHz); chemical shifts (d scale) are report-
ed in parts per million (ppm) relative to the central peak
6.1.2.
2-[5-(Piperidin-1-yl)pentylamino]-1H-benzimid-
azole (2). 1H NMR (DMSO-d6) d 1.21–1.31 (m, 2H,
CH2), 1.38–1.47 (m, 4H, CH2), 1.49–1.60 (m, 6H,
CH2), 2.20 (t, J = 7.6 Hz, 2H, CH2), 2.30–2.35 (m, 4H,
CH2), 3.36 (t, J = 7.0 Hz, 2H, CH2), 6.98–7.04 (m, 2H,
Bzim), 7.23–7.27 (m, 2H, Bzim). MS (EI) 286 [M+].
1
of the solvent. H NMR Spectra are reported in order:
multiplicity, approximate coupling constant (J value)

M. Rivara et al. / Bioorg. Med. Chem. 14 (2006) 1413–1424
1419
Anal. calcd for C17H26N4: C, 71.28; H, 9.15; N, 19.56.
Found: C, 70.91; H, 8.99; N, 19.16.
6.3. General method of preparation of x-(4-chlorophen-
oxy)alkylamine derivatives
6.1.3. 2-[6-(Piperidin-1-yl)hexylamino]-1H-benzimidazole
(3). H NMR (CDCl3) d 1.17–1.62 (m, 14H, CH2), 2.21
(t, J = 7.6 Hz, 2H, CH2), 2.32–2.37 (m, 4H, CH2), 3.33
(t, J = 6.9 Hz, 2H, CH2), 6.98–7.01 (m, 2H, Bzim),
7.23–7.26 (m, 2H, Bzim). MS (CI) 301 [M+1]+. Anal.
calcd for C18H28N4: C, 71.95; H, 9.39; N, 18.65. Found:
C, 71.70; H, 9.40; N, 18.31.
A solution of 20 mmol of the appropriate x-(4-chloro-
phenoxy)alkyl cyanide in 20.0 ml of dry THF was added
dropwise to a suspension of 100 mmol LiAlH4 in 40.0 ml
of dry THF, kept in an ice bath and under nitrogen
atmosphere. The reaction mixture was stirred at room
temperature for 30 min, then the excess of LiAlH4 was
decomposed by successive addition of 2.0 ml of a mix-
ture water:THF = 1:1 and 20.0 ml of 10% w/v NaOH.
The cake was filtered off and washed with THF, and
the combined organic filtrates were evaporated in vacuo
to dryness. The crude products were then purified by
column chromatography [SiO2, CH2Cl2:CH3OH
(NH3) = 9:1.5] and characterized by 1H NMR and mass
spectra.
1
6.1.4.
2-[7-(Piperidin-1-yl)heptylamino]-1H-benzimid-
azole (4). 1H NMR (CDCl3) d 1.12–1.32 (m, 6H,
CH2), 1.40–1.63 (m, 10H, CH2), 2.24 (t, J = 7.6 Hz,
2H, CH2), 2.34–2.40 (m, 4H, CH2), 3.37 (t, J = 6.9 Hz,
2H, CH2), 6.99–7.04 (m, 2H, Bzim), 7.23–7.28 (m, 2H,
Bzim). MS (CI) 315 [M+1]+. Anal. calcd for
C19H30N4: C, 72.56; H, 9.62; N, 17.82. Found: C,
72.53; H, 9.61; N, 17.70.
6.3.1. 4-(4-Chlorophenoxy)butylamine. 46%, 1H NMR
(CDCl3) d 1.54–1.64 (m, 2H, CH2), 1.74–1.83 (m, 2H,
CH2), 2.74 (t, J = 6.9 Hz, 2H, CH2), 3.91 (t,
J = 6.5 Hz, 2H, CH2), 6.75–6.81 (m, 2H, Ph), 7.16–
7.21 (m, 2H, Ph). MS (EI) 199 [M+].
6.1.5. 2-[8-(Piperidin-1-yl)octylamino]-1H-benzimidazole
1
(5). H NMR (CDCl3) d 1.12–1.28 (m, 6H, CH2), 1.40–
1.64 (m, 12H, CH2), 2.27 (t, J = 7.5 Hz, 2H, CH2), 2.34–
2.42 (m, 4H, CH2), 3.38 (t, J = 6.8 Hz, 2H, CH2),
6.99–7.05 (m, 2H, Bzim), 7.24–7.30 (m, 2H, Bzim). MS
(CI) 329 [M+1]+. Anal. calcd for C20H32N4: C, 73.12;
H, 9.82; N, 17.06. Found: C, 72.79; H, 9.75; N, 16.70.
1
6.3.2. 5-(4-Chlorophenoxy)pentylamine. 54%, H NMR
(CDCl3) d 1.43–1.50 (m, 2H, CH2), 1.70–1.80 (m, 4H,
CH2), 2.68 (t, J = 6.5 Hz, 2H, CH2), 3.88 (t,
J = 6.3 Hz, 2H, CH2), 6.72–6.81 (m, 2H, Ph), 7.14–
7.20 (m, 2H, Ph). MS (EI) 213 [M+].
6.2. General method of preparation of x-(4-chlorophen-
oxy)alkyl cyanide derivatives
6.3.3. 6-(4-Chlorophenoxy)hexylamine. 49%, 1H NMR
(CDCl3) d 1.28–1.48 (m, 6H, CH2), 1.68–1.77 (m, 2H,
CH2), 2.65 (t, J = 6.7 Hz, 2H, CH2), 3.86 (t,
J = 6.4 Hz, 2H, CH2), 6.72–6.78 (m, 2H, Ph), 7.14–
7.19 (m, 2H, Ph). MS (EI) 227 [M+].
A mixture of 50 mmol of 4-chlorophenol, 40 mmol of
the appropriate commercially available x-bromoalkyl
cyanide, and 40 mmol K2CO3 in 10.0 ml CH3COCH3
was refluxed under stirring for 24 h and the solid residue
was then filtered off. Removal of the solvent in vacuo
gave the crude products, which were purified by column
chromatography (SiO2, CH2Cl2:CH3OH = 99:1) and
1
6.3.4. 7-(4-Chlorophenoxy)heptylamine. 46%, H NMR
(CDCl3) d 1.30–1.48 (m, 8H, CH2), 1.68–1.78 (m, 2H,
CH2), 2.65 (t, J = 6.7 Hz, 2H, CH2), 3.87 (t,
J = 6.7 Hz, 2H, CH2), 6.75–6.80 (m, 2H, Ph), 7.15–
7.20 (m, 2H, Ph). MS (EI) 241 [M+].
1
characterized by H NMR and mass spectra.
1
6.2.1. 4-(4-Chlorophenoxy)butyronitrile. 98%, H NMR
(DMSO-d6)
d 1.97–2.06 (m, 2H, CH2), 2.64 (t,
J = 7.0 Hz, 2H, CH2), 4.02 (t, J = 6.2 Hz, 2H, CH2),
6.93–6.99 (m, 2H, Ph), 7.29–7.34 (m, 2H, Ph). MS (EI)
195 [M+].
6.4. General method of preparation of 2-[x-(p-chloro-
phenoxy)alkylamino]-1H-benzimidazole derivatives (6–9)
A solution of 5.0 mmol of 2-chlorobenzimidazole and
10.0 mmol of the appropriate x-(4-chlorophenoxy)al-
kylamine in 2.0 ml of isoamyl alcohol was heated at
130 °C for 16 h. The crude products were purified by
column chromatography (SiO2, CH2Cl2:CH3OH = 9:1).
6.2.2. 5-(4-Chlorophenoxy)pentanenitrile. 96%, 1H NMR
(DMSO-d6)
d 1.67–1.81 (m, 4H, CH2), 2.55 (t,
J = 6.8 Hz, 2H, CH2), 3.97 (t, J = 6.2 Hz, 2H, CH2),
6.91–6.96 (m, 2H, Ph), 7.28–7.33 (m, 2H, Ph). MS (EI)
209 [M+].
6.4.1. 2-[4-(p-Chlorophenoxy)butylamino]-1H-benzimid-
azole (6). 1H NMR (CDCl3) d 1.82–1.88 (m, 4H,
CH2), 3.50 (t, J = 6.6 Hz, 2H, CH2), 3.95 (t,
J = 5.9 Hz, 2H, CH2), 6.77–6.81 (m, 2H, Ph), 7.04–
7.09 (m, 2H, Bzim), 7.19–7.24 (m, 2H, Ph), 7.24–7.29
(m, 2H, Bzim). MS (EI) 315 [M+]. Anal. calcd for
C17H18N3OCl: C, 64.65; H, 5.75; N, 13.31. Found: C,
64.27; H, 5.64; N, 12.94.
1
6.2.3. 6-(4-Chlorophenoxy)hexanenitrile. 99%, H NMR
(DMSO-d6)
d 1.44–1.77 (m, 6H, CH2), 2.50 (t,
J = 7.0 Hz, 2H, CH2), 3.95 (t, J = 6.4 Hz, 2H, CH2),
6.91–6.97 (m, 2H, Ph), 7.27–7.33 (m, 2H, Ph). MS (EI)
223 [M+].
6.2.4. 7-(4-Chlorophenoxy)heptanenitrile. 98%, 1H NMR
(DMSO-d6) d 1.39–1.44 (m, 4H, CH2), 1.52–1.61 (m,
2H, CH2), 1.65–1.74 (m, 2H, CH2), 2.47 (t, J = 7.2 Hz,
2H, CH2), 3.94 (t, J = 6.4 Hz, 2H, CH2), 6.91–6.96 (m,
2H, Ph), 7.27–7.32 (m, 2H, Ph). MS (CI) 238 [M+1]+.
6.4.2. 2-[5-(p-Chlorophenoxy)pentylamino]-1H-benzimid-
1
azole (7). H NMR (CDCl3) d 1.43–1.52 (m, 2H, CH2),
1.58–1.65 (m, 2H, CH2), 1.69–1.79 (m, 2H, CH2), 3.28

1420
M. Rivara et al. / Bioorg. Med. Chem. 14 (2006) 1413–1424
(q, J = 6.4 Hz, 2H, CH2), 3.96 (t, J = 6.2 Hz, 2H, CH2),
6.52 (br s, 1H, NH), 6.81–6.87 (m, 2H, Bzim), 6.91–6.96
(m, 2H, Ph), 7.07–7.12 (m, 2H, Bzim), 7.26–7.32 (m, 2H,
Ph). MS (EI) 329 [M+]. Anal. calcd for C18H20N3OCl:
C, 65.54; H, 6.11; N, 12.74. Found: C, 65.85; H, 6.12;
N, 12.40.
6.5.4. N-[4-(6-Chlorohexyloxy)-2-nitrophenyl]acetamide.
80%, mp 89–90 °C, H NMR (DMSO-d6) d 1.48–1.60
(m, 2H, CH2), 1.70–1.89 (m, 4H, CH2), 2.00 (t, 3H,
CH3), 3.65 (t, J = 6.5 Hz, 2H, CH2), 4.04 (t,
J = 6.5 Hz, 2H, CH2), 7.27 (dd, J = 9.0 Hz and 2.4 Hz,
1H, Ph), 7.41–7.44 (m, 2H, Ph), 10.04 (s, 1H, NH).
MS (CI) 315 [M+1]+.
1
6.4.3. 2-[6-(p-Chlorophenoxy)hexylamino]-1H-benzimid-
azole (8). 1H NMR (DMSO-d6) d 1.34–1.48 (m, 4H,
CH2), 1.54–1.63 (m, 2H, CH2), 1.67–1.76 (m, 2H,
CH2), 3.26 (q, J = 7.2 Hz, 2H, CH2), 3.95 (t,
J = 6.3 Hz, 2H, CH2), 6.50 (br s, 1H, NH), 6.81–6.87
(m, 2H, Bzim), 6.90–6.95 (m, 2H, Ph), 7.07–7.12 (m,
2H, Bzim), 7.26–7.31 (m, 2H, Ph). MS (EI) 343 [M+].
Anal. calcd for C19H22N3OCl: C, 66.36; H, 6.45; N,
12.22. Found: C, 66.11; H, 6.40; N, 11.99.
6.6. General method of preparation of N-[2-nitro-4-(x-
piperidin-1-yl-alkoxy)phenyl]acetamide derivatives
A mixture of 8.0 mmol of the appropriate N-[4-(x-chlo-
roalkoxy)-2-nitrophenyl]acetamide in 80 mmol piperi-
dine was stirred at 50 °C for 5 h. The solvent was then
evaporated under reduced pressure and the crude prod-
ucts were directly used in the successive reaction without
purification.
6.4.4. 2-[7-(p-Chlorophenoxy)heptylamino]-1H-benzimid-
1
azole (9). H NMR (DMSO-d6) d 1.25–1.45 (m, 6H,
6.7. General method of preparation of N-{4-[x-(4-chlo-
rophenoxy)alkoxy]-2-nitrophenyl}acetamide derivatives
CH2), 1.52–1.62 (m, 2H, CH2), 1.65–1.74 (m, 2H,
CH2), 3.24 (q, J = 6.8 Hz, 2H, CH2), 3.94 (t,
J = 6.4 Hz, 2H, CH2), 6.46 (br s, 1H, NH), 6.82–
6.85 (m, 2H, Bzim), 6.90–6.95 (m, 2H, Ph), 7.08–
7.11 (m, 2H, Bzim), 7.26–7.31 (m, 2H, Ph). MS
(EI) 357 [M+]. Anal. calcd for C20H24N3OCl: C,
67.12; H, 6.76; N, 11.74. Found: C, 67.27; H, 6.78;
N, 11.67.
To a solution of 10 mmol of the appropriate N-[4-(x-
chloroalkoxy)-2-nitrophenyl]acetamide in 20 ml DMF
were added 20 mmol K2CO3, 10 mmol of 4-chlorophe-
nol, and a catalytic amount of KI. The reaction mixture
was stirred at 50 °C for 48 h and then the solvent was
evaporated under reduced pressure. The residue was dis-
solved in H2O, the product was extracted with CH2Cl2,
and the organic layer was dried over Na2SO4 and evap-
orated under reduced pressure. The crude products were
washed with CH3OH and crystallized from 95% EtOH.
6.5. General method of preparation of N-[4-(x-chloro-
alkoxy)-2-nitrophenyl]acetamide derivatives
A mixture of 20 mmol of N-(4-hydroxy-2-nitrophen-
yl)acetamide,54 30 mmol of the appropriate commercial-
ly available 1-bromo-x-chloroalkane, 40 mmol Na2CO3,
and a catalytic amount of KI in 40 ml DMF was stirred
at 50 °C for 24 h. The solvent was then evaporated un-
der reduced pressure, the residue was dissolved in
H2O, and the product was extracted with CH2Cl2. The
organic layer was dried over Na2SO4 and evaporated
under reduced pressure. The crude products were crys-
tallized from 50% EtOH.
6.7.1. N-{4-[3-(4-Chlorophenoxy)propoxy]-2-nitrophen-
yl}acetamide. 90%, mp 129–131 °C, H NMR (DMSO-
d6) d 2.01 (t, 3H, CH3), 2.13–2.22 (m, 2H, CH2), 4.12
(t, J = 6.2 Hz, 2H, CH2), 4.20 (t, J = 6.2 Hz, 2H,
CH2), 6.95–7.00 (m, 2H, Ph), 7.28–7.34 (m, 3H, Ph),
7.45 (d, J = 9.0 Hz, 1H, Ph), 7.46 (d, J = 2.7 Hz, 1H,
Ph), 10.01 (s, 1H, NH). MS (CI) 365 [M+1]+.
1
6.7.2.
N-{4-[4-(4-Chlorophenoxy)butoxy]-2-nitrophen-
1
yl}acetamide. 90%, mp 129–130 °C, H NMR (DMSO-
d6) d 1.85–1.87 (m, 4H, CH2), 2.01 (t, 3H, CH3), 4.03
(t, J = 5.7 Hz, 2H, CH2), 4.11 (t, J = 6.0 Hz, 2H,
CH2), 6.92–6.98 (m, 2H, Ph), 7.26–7.32 (m, 3H, Ph),
7.43 (d, J = 2.4 Hz, 1H, Ph), 7.46 (d, J = 9.3 Hz, 1H,
Ph), 9.99 (s, 1H, NH). MS (CI) 379 [M+1]+.
6.5.1. N-[4-(3-Chloropropoxy)-2-nitrophenyl]acetamide.
95%, mp 80–84 °C, 1H NMR (DMSO-d6) d 2.01 (t,
3H, CH3), 2.13–2.27 (m, 2H, CH2), 3.79 (t, J = 6.5 Hz,
2H, CH2), 4.16 (t, J = 6.0 Hz, 2H, CH2), 7.30 (dd,
J = 9.0 Hz and 2.7 Hz, 1H, Ph), 7.45–7.48 (m, 2H, Ph),
10.02 (s, 1H, NH). MS (CI) 273 [M+1]+.
6.7.3. N-{4-[5-(4-Chlorophenoxy)pentyloxy]-2-nitrophen-
1
6.5.2.
N-[4-(4-Chlorobutoxy)-2-nitrophenyl]acetamide.
yl}acetamide. 90%, mp 130–132 °C, H NMR (DMSO-
1
90%, mp 76–78 °C, H NMR (DMSO-d6) d 1.82–1.96
(m, 4H, CH2), 2.00 (t, 3H, CH3), 3.71 (t, J = 6.2 Hz,
2H, CH2), 4.08 (t, J = 6.2 Hz, 2H, CH2), 7.26 (dd,
J = 8.9 Hz and 2.8 Hz, 1H, Ph), 7.42–7.44 (m, 2H, Ph),
10.04 (s, 1H, NH). MS (CI) 287 [M+1]+.
d6) d 1.50–1.60 (m, 2H, CH2), 1.72–1.83 (m, 4H, CH2),
2.01 (t, 3H, CH3), 3.98 (t, J = 6.4 Hz, 2H, CH2), 4.06
(t, J = 6.2 Hz, 2H, CH2), 6.92–6.97 (m, 2H, Ph), 7.25–
7.33 (m, 3H, Ph), 7.43 (d, J = 3.6 Hz, 1H, Ph), 7.45 (d,
J = 9.0 Hz, 1H, Ph), 9.99 (s, 1H, NH). MS (EI) 392
[M+].
6.5.3. N-[4-(5-Chloropentyloxy)-2-nitrophenyl]acetamide.
1
80%, mp 76–79 °C, H NMR (DMSO-d6) d 1.48–1.60
6.8. General method of preparation of 2-nitro-4-(x-
piperidin-1-yl-alkoxy)phenylamine derivatives
(m, 2H, CH2), 1.70–1.89 (m, 4H, CH2), 2.00 (t, 3H,
CH3), 3.65 (t, J = 6.5 Hz, 2H, CH2), 4.04 (t,
J = 6.5 Hz, 2H, CH2), 7.27 (dd, J = 9.0 Hz and 2.4 Hz,
1H, Ph), 7.41–7.44 (m, 2H, Ph), 10.04 (s, 1H, NH).
MS (CI) 301 [M+1]+.
A mixture of 8.0 mmol of the appropriate N-[2-nitro-4-
(x-piperidin-1-yl-alkoxy)phenyl]acetamide in 29.0 ml
of 4 N NaOH was refluxed under stirring for 2 h.

M. Rivara et al. / Bioorg. Med. Chem. 14 (2006) 1413–1424
1421
The residue was then poured into a mixture of ice/water
and the resulting precipitate was collected by filtration.
The crude products were crystallized from 50% EtOH.
6.10. General method of preparation of 4-(x-piperidin-1-
yl-alkoxy)-1,2-phenylendiamine derivatives and of 4-[x-(4-
chlorophenoxy)alkoxy]-1,2-phenylendiamine derivatives
6.8.1.
2-Nitro-4-(4-piperidin-1-yl-butoxy)phenylamine.
A solution of the appropriate 2-nitrophenylamine deriv-
ative (10 mmol) in 40 ml of absolute EtOH was hydroge-
nated using a Parr apparatus with Pd/C (10%) at room
temperature, for 5 h. The products obtained after filtra-
tion of the catalyst were used in the successive reaction
without further purification.
1
82%, mp 80–81 °C, H NMR (DMSO-d6) d 1.33–1.40
(m, 2H, CH2), 1.43–1.58 (m, 6H, CH2), 1.64–1.73 (m,
2H, CH2), 2.23–2.30 (m, 6H, CH2), 3.92 (t, J = 6.3 Hz,
2H, CH2), 6.98 (t, J = 9.3 Hz, 1H, Ph), 7.15 (dd,
J = 9.1 Hz and 2.7 Hz, 1H, Ph), 7.21 (s, 2H, NH2),
7.35 (d, J = 2.7 Hz, 1H, Ph). MS (CI) 294 [M+1]+.
6.11. General method of preparation of 5(6)-[x-(piperidin-
1-yl)alkoxy]-2-aminobenzimidazole derivatives (10–12)
6.8.2. 2-Nitro-4-(5-piperidin-1-yl-pentyloxy)phenylamine.
80%, mp 81–83 °C, H NMR (DMSO-d6) d 1.29–1.50
1
(m, 10H, CH2), 1.63–1.70 (m, 2H, CH2), 2.17–2.25 (m,
6H, CH2), 3.89 (t, J = 6.4 Hz, 2H, CH2), 6.98 (t,
J = 9.2 Hz, 1H, Ph), 7.14 (dd, J = 9.2 Hz and 2.9 Hz,
1H, Ph), 7.21 (s, 2H, NH2), 7.34 (d, J = 2.8 Hz, 1H,
Ph). MS (EI) 307 [M+].
A cyanogen bromide solution (5.0 M in acetonitrile,
1.57 ml) was added to an ice-cooled solution (0 °C) of
6.56 mmol of the appropriate 4-(x-piperidin-1-yl-alk-
oxy)-1,2-phenylenediamine in 13.35 ml H2O. The reac-
tion mixture was stirred at room temperature for 12 h,
then it was cooled again and acidified with an ethanol
solution saturated with gaseous HCl. The solvent was
evaporated under reduced pressure and the residue
was extracted with abs EtOH. The crude products so ob-
tained were again changed into the free bases before
starting purification by column chromatography (SiO2,
CH2Cl2:CH3OH(NH3) = 10:1).
6.8.3. 2-Nitro-4-(6-piperidin-1-yl-hexyloxy)phenylamine.
1
85%, mp 73–74 °C, H NMR (DMSO-d6) d 1.28–1.47
(m, 12H, CH2), 1.63–1.72 (m, 2H, CH2), 2.16–2.26 (m,
6H, CH2), 3.89 (t, J = 6.3 Hz, 2H, CH2), 6.98 (t,
J = 9.0 Hz, 1H, Ph), 7.15 (dd, J = 9.6 Hz and 2.7 Hz,
1H, Ph), 7.26 (s, 2H, NH2), 7.34 (d, J = 3.0 Hz, 1H,
Ph). MS (CI) 322 [M+1]+.
6.11.1. 5(6)-[4-(Piperidin-1-yl)butoxy]-2-aminobenzimi-
dazole (10Æ2HClÆ1.5H2O). 1H NMR (DMSO-d6) d
1.48–2.08 (m, 12H, CH2), 3.01–3.16 (m, 4H, CH2),
3.98 (t, J = 5.4 Hz, 2H, CH2), 6.74 (d, J = 8.7 Hz, 1H,
Bzim), 6.89 (s, 1H, Bzim), 7.18 (d, J = 8.7 Hz, 1H,
Bzim). MS (CI) 289 [M+1]+. Anal. calcd for
C16H24N4OÆ2HClÆ1.5H2O: C, 49.48; H, 7.53; N, 14.43.
Found: C, 49.24; H, 7.21; N, 14.33.
6.9. General method of preparation of 4-[x-(4-chloro-
phenoxy)alkoxy]-2-nitrophenylamine derivatives
To a solution of 10 mmol KOH in 10 ml of absolute
EtOH was added 10 mmol of the appropriate N-
{4-[x-(4-chlorophenoxy)alkoxy]-2-nitrophenyl}acetamide.
The reaction mixture was stirred at room temperature
for 5 h, then the solvent was evaporated under reduced
pressure and the residue was extracted with ethyl ace-
tate. The organic layer was washed with water, dried
over Na2SO4, and evaporated under reduced pressure.
The crude products were used in the successive reaction
without further purification.
6.11.2. 5(6)-[5-(Piperidin-1-yl)pentyloxy]-2-aminobenzim-
1
idazole (11Æ2HClÆ1/2C2H5OH). H NMR (DMSO-d6) d
1.38–1.76 (m, 12H, CH2), 2.76–2.84 (m, 2H, CH2),
2.94–2.99 (m, 2H, CH2), 3.35–3.39 (m, 2H, CH2), 3.94
(t, J = 6.2 Hz, 2H, CH2), 6.80 (d, J = 8.5 Hz and
2.6 Hz, 1H, Bzim), 6.92 (d, J = 1.8 Hz, 1H, Bzim), 7.24
(d, J = 8.7 Hz, 1H, Bzim). MS (EI) 302 [M+]. Anal.
calcd for C17H26N4OÆ2HClÆ1/2C2H5OH: C, 54.26; H,
7.84; N, 14.06. Found: C, 54.13; H, 7.54; N, 14.44.
6.9.1.
4-[3-(4-Chlorophenoxy)propoxy]-2-nitrophenyl-
amine. 86%, mp 98–101 °C, 1H NMR (DMSO-d6) d
2.09–2.19 (m, 2H, CH2), 4.05–4.14 (m, 4H, CH2),
6.96–7.01 (m, 3H, Ph), 7.16 (dd, J = 9.1 Hz and
3.1 Hz, 1H, Ph), 7.22 (s, 2H, NH2), 7.29–7.33 (m, 2H,
Ph), 7.39 (d, J = 2.7 Hz, 1H, Ph). MS (EI) 322 [M+].
6.11.3. 5(6)-[6-(Piperidin-1-yl)hexyloxy]-2-aminobenzimi-
dazole (12Æ2HClÆH2O). 1H NMR (DMSO-d6) d 1.02–
1.49 (m, 6H, CH2), 1.65–1.76 (m, 10H, CH2), 2.75–
2.83 (m, 2H, CH2), 2.92–2.98 (m, 2H, CH2), 3.95 (t,
J = 6.3 Hz, 2H, CH2), 6.79 (dd, J = 8.7 Hz and 2.1 Hz,
1H, Bzim), 6.92 (d, J = 2.1 Hz, 1H, Bzim), 7.23 (d,
J = 8.7 Hz, 1H, Bzim). MS (CI) 317 [M+1]+. Anal. calcd
for C18H28N4OÆ2HClÆH2O: C, 53.06; H, 7.92; N, 13.75.
Found: C, 53.41; H, 7.95; N, 13.36.
6.9.2.
4-[4-(4-Chlorophenoxy)butoxy]-2-nitrophenyl-
1
amine. 90%, mp 109–111 °C, H NMR (DMSO-d6) d
1.83–1.85 (m, 4H, CH2), 3.96–4.04 (m, 4H, CH2),
6.94–7.00 (m, 3H, Ph), 7.15 (dd, J = 9.3 Hz and
2.7 Hz, 1H, Ph), 7.21 (s, 2H, NH2), 7.28–7.32 (m, 2H,
Ph), 7.37 (d, J = 3.0, 1H, Ph). MS (CI) 337 [M+1]+.
6.9.3. 4-[5-(4-Chlorophenoxy)pentyloxy]-2-nitrophenyl-
amine. 87%, mp 114–116 °C, H NMR (DMSO-d6) d
1.49–1.59 (m, 2H, CH2), 1.70–1.81 (m, 4H, CH2),
3.91–3.99 (m, 4H, CH2), 6.91–6.96 (m, 2H, Ph), 6.99
(d, J = 9.0 Hz, 1H, Ph), 7.15 (dd, J = 8.8 Hz and
2.8 Hz, 1H, Ph), 7.21 (s, 2H, NH2), 7.27–7.32 (m, 2H,
Ph), 7.36 (d, J = 2.7 Hz, 1H, Ph). MS (CI) 351 [M+1]+.
6.12. General method of preparation of 5(6)-[x-(p-chlo-
rophenoxy)alkoxy]-2-aminobenzimidazole derivatives
(13–15)
1
A cyanogen bromide solution (5.0 M in acetonitrile,
0.58 ml) was added to an ice-cooled solution (0 °C) of
2.38 mmol of the appropriate 4-[x-(4-chlorophen-

1422
M. Rivara et al. / Bioorg. Med. Chem. 14 (2006) 1413–1424
oxy)alkoxy]-1,2-phenylendiamine in 8.0 ml of absolute
EtOH. The reaction mixture was stirred at room tem-
perature for 2 h, then the solvent was evaporated under
reduced pressure and the residue was extracted with abs
EtOH. The solvent was evaporated in vacuo again and
the crude products so obtained were washed with
CH2Cl2.
10 lM histamine as competing ligand. pIC50 values were
estimated from the displacement curves of the tested
compounds (0.01 nM–100 lM) versus [3H]-(R)-a-meth-
ylhistamine (Kd in human recombinant H3 expressing
cells = 0.15 nM and Kd on rat brain = 0.40 nM)51,55
and converted to pKi values according to Cheng and
Prusoffꢀs equation.56
6.12.1. 5(6)-[3-(p-Chlorophenoxy)propoxy]-2-aminobenz-
imidazole (13). H NMR (DMSO-d6) d 2.08–2.17 (m,
6.13.2. Cell culture. GPCR97-transfected SK-N-MC
cells, a human neuroblastoma cell line stably expressing
the human histamine H3-receptor, were grown in
75 cm2 culture flasks at 37 °C in a humidified atmosphere
with 5% CO2 in Eagleꢀs minimal essential medium, sup-
plemented with 10% v/v fetal calf serum, 1% non-essen-
tial amino acids, 1% penicillin–streptomycin, 1%
L-glutamine, and 1% disodium pyruvate in the presence
of 300 lg/ml G418. Cells were used for experiments when
they reached about 70–80% confluence.
1
2H, CH2), 4.05 (t, J = 6.2 Hz, 2H, CH2), 4.13 (t,
J = 6.4 Hz, 2H, CH2), 6.45 (dd, J = 8.4 Hz and 2.1 Hz,
1H, Bzim), 6.71 (d, J = 2.1 Hz, 1H, Bzim), 6.93–7.00
(m, 3H, Bzim and Ph), 7.28–7.34 (m, 2H, Ph). MS
(EI) 317 [M+]. Anal. calcd for C16H16N3O2Cl: C,
60.47; H, 5.08; N, 13.22. Found: C, 60.72; H, 5.04; N,
12.97.
6.12.2. 5(6)-[4-(p-Chlorophenoxy)butoxy]-2-aminobenz-
1
imidazole (14). H NMR (DMSO-d6) d 1.70–1.92 (m,
6.13.3. Rat H3 binding assays. Rat (Wistar) cerebral cor-
tex membranes were incubated for 30 min with
[3H]RAMHA 0.5 nM and the compounds under study
(1 nM–30 lM), in Tris–HCl 50 mM, pH 7.4, NaCl
50 mM, and EDTA 0.5 mM, then rapidly filtered (Milli-
pore AAWP 0.8 lm) under vacuum and rinsed with ice-
cold buffer. Specific binding was defined as the binding
inhibited by thioperamide 10 lM, and the pIC50 values
were estimated from the displacement curves of the com-
pounds tested versus [3H]RAMHA bound to cerebral
membranes.52 pKi values were calculated according to
Cheng and Prusoffꢀs equation.56
4H, CH2), 3.95 (t, J = 5.7 Hz, 2H, CH2), 4.02 (t,
J = 5.7 Hz, 2H, CH2), 6.46 (dd, J = 8.7 Hz and 2.4 Hz,
1H, Bzim), 6.70 (d, J = 2.4 Hz, 1H, Bzim), 6.40–6.98
(m, 3H, Bzim and Ph), 7.27–7.33 (m, 2H, Ph). MS
(EI) 331 [M+]. Anal. calcd for C17H18N3O2Cl: C,
61.53; H, 5.47; N, 12.67. Found: C, 61.51; H, 5.48; N,
12.37.
6.12.3.
5(6)-[5-(p-Chlorophenoxy)pentyloxy]-2-amino-
1
benzimidazole (15). H NMR (DMSO-d6) d 1.50–1.60
(m, 2H, CH2), 1.70–1.81 (m, 4H, CH2), 3.91 (t,
J = 6.4 Hz, 2H, CH2), 3.97 (t, J = 6.4 Hz, 2H, CH2),
6.44 (dd, J = 8.5 Hz and 2.4 Hz, 1H, Bzim), 6.69 (d,
J = 2.4 Hz, 1H, Bzim), 6.92–6.97 (m, 3H, Bzim and
Ph), 7.27–7.33 (m, 2H, Ph). MS (EI) 345 [M+]. Anal.
calcd for C18H20N3O2Cl: C, 62.51; H, 5.83; N, 12.15.
Found: C, 62.74; H, 5.77; N, 11.87.
6.13.4. Functional assays. Portions of guinea-pig ileum
were mounted on a coaxial platinum electrode assembly
in a 10 ml water-jacketed organ bath containing Krebs–
Henseleit solution aerated with 95%O2:5%CO2 and
maintained at 37 °C. Single electrical pulses were deliv-
ered to the tissue (0.1 Hz, 1 ms, 1.5–3.0 V) from a stim-
ulator (LACE Elettronica model ES-3, Ospedaletto PI,
Italy). Cumulative concentration–response curves for
the inhibition of electrically stimulated contractions
were determined for the H3 selective agonist RAMHA
(1 nM–1 lM). The tissues were allowed to equilibrate
with the compounds under study (1 nM–10 lM) for
30 min before the generation of a second concentra-
tion–response curve to the agonist. pKB values (ꢁappar-
ent pA2ꢀ) were determined according to Furchgottꢀs
equation.57
6.13. Pharmacology
6.13.1. Human H3 binding assay. Homogenates of
GPCR97-transfected SK-N-MC cells were used for
determining affinity values of the new compounds at
the H3-receptor, in radioligand displacement studies
according to the method described by Lovenberg
et al.51 SK-N-MC cells in confluent culture plates were
harvested using a cell scraper and centrifuged for
5 min at 1100 rpm. Pellets, either fresh or stored at
À80 °C until the moment of use, were mechanically
homogenized with Potter–Elvhejem in 20 mM Tris–
HCl/0.5 mM EDTA. Supernatants from a 2000 rpm
spin (10 min) were collected and re-centrifuged at
10,000 rpm for 30 min. Pellets were rehomogenized in
50 mM Tris–HCl/5 mM EDTA (pH 7.4). Membranes
were incubated for 60 min at room temperature with
0.5 nM [3H]-(R)-a-methylhistamine (30.0 Ci/mmol,
Amersham Bioscience) in 50 mM Tris–HCl/5 mM
EDTA (pH 7.4) with or without competing ligands.
Incubation was terminated by rapid filtration over
Millipore AAWP 0.8 lm filters followed by two washes
with ice-cold buffer (50 mM Tris–HCl/5 mM EDTA).
Retained radioactivity was determined by liquid scintil-
lation counting. Non-specific binding was defined with
Antagonistic activities of compounds at H1- and H2-re-
ceptors were determined in isolated guinea-pig ileum
contracted by histamine and in isolated guinea-pig atria
stimulated by H2-agonist dimaprit, respectively.53
Acknowledgments
Financial support from Italian MIUR is gratefully
`
acknowledged. We are grateful to the Centro Interfacolta
Misure of the University of Parma for providing the
NMR and MS instrumentation. The authors thank Dr.
Massimo Coruzzi and Mr. Fabio Panni for technical
assistance. The authors thank Dr. Timothy W. Loven-

M. Rivara et al. / Bioorg. Med. Chem. 14 (2006) 1413–1424
1423
26. Shah, C.; McAtee, L.; Breitenbucher, J. G.; Rudolph, D.;
Li, X.; Lovenberg, T. W.; Mazur, C.; Wilson, S. J.;
Carruthers, N. I. Bioorg. Med. Chem. Lett. 2002, 12, 3309.
27. Cowart, M.; Faghih, R.; Curtis, M. P.; Gfesser, G. A.;
Bennani, Y. L.; Black, L. A.; Pan, L.; Marsh, K. C.;
Sullivan, J. P.; Esbenshade, T. A.; Fox, G. B.; Hancock,
A. A. J. Med. Chem. 2005, 48, 38.
berg, and Dr. Sandy J. Wilson, Johnson & Johnson
Pharmaceutical Research and Development, San Diego,
CA, for the cells stably expressing the human histamine
H3-receptor.
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