N-Alkenyl and cycloalkyl carbamates as dual acting histamine H3 and H4 receptor ligands

Article abstract of DOI:10.1016/j.bmc.2011.03.046

Previous studies have shown that several imidazole derivatives posses affinity to histamine H₃ and H₄ receptors. Continuing our study on structural requirements responsible for affinity and selectivity for H₃/H₄ receptor subtypes, two series of 3-(1H-imidazol-4-yl)propyl carbamates were prepared: a series of unsaturated alkyl derivatives (1-9) and a series possessing a cycloalkyl group different distances to the carbamate moiety (10-13). The compounds were tested for their affinities at the human histamine H₃ receptor, stably expressed in CHO-K1 cells. Compounds 1, 2, 5-7, 10-13 were investigated for their affinities at the human histamine H₄ receptor co-expressed with Gα_i2 and Gβ₁γ₂ subunits in Sf9 cells. To expand the pharmacological profile, compounds were further tested for their H₃ receptor antagonist activity on guinea pig ileum and in vivo after oral administration to mice. All tested compounds exhibited good affinity for the human histamine H₃ receptor with K_i values in the range from 14 to 194 nM. All compounds were active in vivo after peroral administration (p.o.) to Swiss mice, thus demonstrating their ability to cross the blood-brain barrier. The most potent H₃ receptor ligand of these series was compound 5, 3-(1H-imidazol-4-yl)propyl pent-4-enylcarbamate with the highest affinity (K_i = 14 nM). Additionally, compound 3 showed remarkable central nervous system (CNS) H₃R activity, increasing the N^τ-methylhistamine levels in mice with an ED ₅₀ value of 0.55 mg/kg, p.o. evidencing therefore, a twofold increase of inverse agonist/antagonist potency compared to the reference inverse agonist/antagonist thioperamide. In this study, the imidazole propyloxy carbamate moiety was kept constant. The different lipophilic moieties connected to the carbamate functionality in the eastern part of the molecule had a range of influences on the human H₄ receptor affinity (154-1326 nM).

Full text of DOI:10.1016/j.bmc.2011.03.046

Bioorganic & Medicinal Chemistry 19 (2011) 2850–2858
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
N-Alkenyl and cycloalkyl carbamates as dual acting histamine H3 and H4
receptor ligands
Małgorzata Wie˛cek a, Tim Kottke b, Xavier Ligneau c, Walter Schunack d, Roland Seifert e, Holger Stark b,
a
a,
⇑
´
Jadwiga Handzlik , Katarzyna Kiec-Kononowicz
a Jagiellonian University Medical College, Faculty of Pharmacy, Department of Technology and Biotechnology of Drugs, Medyczna 9, 30-688 Kraków, Poland
b Johann Wolfgang Goethe University, Institute of Pharmaceutical Chemistry, Biocenter, ZAFES/ICNF/CMP, Max-von-Laue-Strasse 9, 60438 Frankfurt/Main, Germany
c Bioprojet-Biotech, 4 rue du Chesnay Beauregard, BP 96205, 35762 Saint-Grégoire, France
d Institute of Pharmacy, Free University of Berlin, Königin-Luise-Strasse 2+4, 14195 Berlin, Germany
e Institute of Pharmacology, Medical School of Hannover, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Previous studies have shown that several imidazole derivatives posses affinity to histamine H3 and H4
receptors. Continuing our study on structural requirements responsible for affinity and selectivity for
H3/H4 receptor subtypes, two series of 3-(1H-imidazol-4-yl)propyl carbamates were prepared: a series
of unsaturated alkyl derivatives (1–9) and a series possessing a cycloalkyl group different distances to
the carbamate moiety (10–13). The compounds were tested for their affinities at the human histamine
H3 receptor, stably expressed in CHO-K1 cells. Compounds 1, 2, 5–7, 10–13 were investigated for their
affinities at the human histamine H4 receptor co-expressed with Gai2 and Gb1c2 subunits in Sf9 cells.
To expand the pharmacological profile, compounds were further tested for their H3 receptor antagonist
activity on guinea pig ileum and in vivo after oral administration to mice. All tested compounds exhibited
good affinity for the human histamine H3 receptor with Ki values in the range from 14 to 194 nM. All
compounds were active in vivo after peroral administration (p.o.) to Swiss mice, thus demonstrating their
ability to cross the blood-brain barrier. The most potent H3 receptor ligand of these series was compound
5, 3-(1H-imidazol-4-yl)propyl pent-4-enylcarbamate with the highest affinity (Ki = 14 nM). Additionally,
Received 10 December 2010
Revised 10 March 2011
Accepted 18 March 2011
Available online 24 March 2011
Keywords:
Histamine H3 receptor
Histamine H4 receptor
Imidazole carbamates
s
compound 3 showed remarkable central nervous system (CNS) H3R activity, increasing the N -methylhis-
tamine levels in mice with an ED50 value of 0.55 mg/kg, p.o. evidencing therefore, a twofold increase of
inverse agonist/antagonist potency compared to the reference inverse agonist/antagonist thioperamide.
In this study, the imidazole propyloxy carbamate moiety was kept constant. The different lipophilic moi-
eties connected to the carbamate functionality in the eastern part of the molecule had a range of influ-
ences on the human H4 receptor affinity (154–1326 nM).
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
synthesis and the release of histamine, as well as heteroreceptors
on non-histaminergic neurons controlling the release of many
Histamine has been found to exert tremendous influence over
variety of physiological and pathophysiological processes. It medi-
ates its effects through binding to four, so far known histamine
receptor subtypes, designated H1 to H4 (H1R–H4R). All of them be-
long to the family of G-protein-coupled receptors, and are differen-
tially expressed in various cell types. The H1R and H2R antagonists
are for many years in clinical use, in the treatment of allergic con-
ditions and gastric ulcers, respectively. The potential clinical appli-
cations for ligands of H3R and the H4R are being studied
intensively. The H3R initially described in 1983 was found to be a
constitutively active receptor mostly expressed in the brain.1,2
H3Rs are identified as presynaptic autoreceptors, regulating the
other important neurotransmitters, such as acetylcholine, norepi-
nephrine, dopamine and serotonin.3 Given their localization and
their ability to affect multiple neurotransmitter systems it is sup-
posed that H3R antagonists/inverse agonists could be useful for
the treatment of central nervous system (CNS) disorders such as
Alzheimer’s disease, schizophrenia, attention-deficit and hyperac-
tivity disorder (ADHD), narcolepsy, pain and obesity.4–6
The H4R is the latest identified receptor belonging to the hista-
mine receptor family. The human H3R and H4R have about 31% se-
quence homology (54% in the transmembrane domains) and
display similar genomic structures.7 In contrast to these structural
similarities, the expression patterns of both receptors strongly dif-
fer. Whereas the H3R is predominantly localized in the brain, the
H4R is expressed mainly in the peripheral tissues and cells of the
immune system.7 Distinct expression patterns of H4R on various
⇑
Corresponding author. Tel.: +48 12 620 55 80; fax: +48 12 620 55 96.
E-mail address: mfkonono@cyf-kr.edu.pl (K. Kiec´-Kononowicz).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.03.046

M. Wie˛cek et al. / Bioorg. Med. Chem. 19 (2011) 2850–2858
2851
S
NH
N
N
H
N
N
S
N
H
N
H
N
H
Cl
Thioperamide
Clobenpropit
hH3R: Ki = 60 12 nM
hH4R: Ki = 43 3 nM
ED50 = 1.0 0.5 mg/kg p.o.
hH3R: Ki = 2.4 0.6 nM
hH4R: Ki = 4.3 0.2 nM
ED50 = 26 7 mg/kg p.o.
O
S
NH2
N
N
O
NH
N
H
N
H
Imetit
Ciproxifan
hH3R: Ki = 0.7 0.1 nM
hH4R: Ki = 1.6 0.1 nM
ED50 = 1.0 0.3 mg/kg p.o.
hH3R: Ki = 46 4 nM
hH4R: Ki = 612 32 nM
ED50 = 0.14 0.03 mg/kg p.o.
Figure 1. Structures and pharmacological data of some reference imidazole-containing histamine H3R ligands (all Ki values are taken from Ref. 32, ED50 values are taken from
Ref. 20 for thioperamide and clobenpropit, Ref. 37 for imetit and from Ref. 47 for ciproxifan).
hematopoietic cells such as mast cells, basophils, eosinophils,
T-cells and dendritic cells suggests it has an important role in the
regulation of immune responses and inflammation.6,8
distinct ligand-receptor interactions. Tritium-labeled N1-[3-(1H-
imidazol-4-yl)propyl]-N2-propionylguanidine ([3H]UR-PI294), was
discovered as a high-affinity histamine H3 and H4 receptor radioli-
gand.12 Cyclopropane-based conformationally restricted analogs of
4-methylhistamine were designed and investigated as histamine
H3/H4 receptor ligands. Pharmacological profiles of these analogs
were shown to be different depending on the stereochemical char-
acter of the cyclopropane backbone and potent H3 and/or H4 recep-
tor antagonists with low nanomolar Ki values were identified.13,14
NG-Acylated imidazolyl-propylguanidines, originally described
several years ago as H2R agonists15–17 were shown to display great-
er activity at H3R and H4Rs.18 Recently, Lim et al.19 has described a
series of clobenpropit analogs as dual activity ligands for the hista-
mine H3R and H4Rs. These compounds showed moderate to high
affinity for both the human H3R and H4R but different intrinsic
activity at the H4R from neutral antagonism to full agonism.19 Gen-
eral structures of dual acting H3/H4R ligands are shown in Fig 2.
In our studies, we focused on the synthesis and pharmacological
evaluation of 3-(1H-imidazol-4-yl)propyl carbamates. This struc-
tural element had been intensively characterized as a potential
H3R antagonist pharmacophore in different series of imidazole
Histamine H3R inverse agonists/antagonists belong to two gen-
eral groups: imidazole containing compounds and non-imidazole
derivatives.4,9–11 Due to some structural similarities between hu-
man H3R and H4R, it is not surprising that numerous imidazole
containing H3R ligands, that is, clobenpropit, imetit and
thioperamide, have also significant affinity for the human H4R
(Fig. 1). Many dual-acting H3R and H4R ligands have been success-
fully used as reference compounds and pharmacological tools in
H3R and H4R research. It is supposed that such compounds with
hybrid structures showing dual H3R and H4R antagonistic activity
could possess therapeutic profile in the therapy of pain and
cancer.6
While the current medicinal chemistry efforts are mainly fo-
cused on selective acting GPCRs ligands, and particularly on hista-
mine H3 and H4 selective agonists/antagonists, there are also
several efforts to develop dual acting H3/H4 compounds.12–19 The
identification of novel selective H4R ligands is still of interest to en-
large the knowledge of the pharmacological profile of the hH4R and
NH2
O
N
N
3H
NH
[ ]n
N
N
H
R
3H
N
H
N
H
[3H]UR-PI294 12
Imidazolylcyclopropane compounds 13, 14
NH2
O
NH
[ ]n
N
N
N
N
R
S
N
R
H
H
N
H
N
H
Acylguanidine-type compounds 18
Clobenpropit analogs 19
Figure 2. General structures of high-affinity histamine H3 and/or H4R ligands.

2852
M. Wie˛cek et al. / Bioorg. Med. Chem. 19 (2011) 2850–2858
containing ligands.20–24 Recently, we reported about an expanded
structure affinity/efficacy relationship (SAR) of branched alkyl car-
bamates of 3-(1H-imidazol-4-yl)propanol, that showed both high
H3R antagonist activity and improved H4R affinity.25 To continue
the investigation on dual H3/H4 receptor affinity and the structural
requirements for affinity at the H3R and H4R, we introduced differ-
ent substituents in the eastern part of the molecule. Slight struc-
tural variations on sterically restricted alkenyl and cycloalkyl
residues caused subtle differences in ligand-receptor interactions
that might influence the H3R and H4R potencies.
This paper describes the synthesis and pharmacological evalua-
tions of two series of 3-(1H-imidazol-4-yl)propyl carbamates: a
series of unsaturated alkyl derivatives (1–9) and a series possess-
ing a cycloalkyl group at variable distances to the carbamate moi-
ety (10–13) (Table 1)
2. Results
2.1. Chemistry
3-(1H-imidazol-4-yl)propanol was the key intermediate for all
of the novel synthesized carbamates. It was prepared starting from
urocanic acid in its trityl-protected and deprotected form as de-
scribed by Stark et al.20 Carbamates 1, 4–6 and 9–13 were prepared
by the method described earlier20,21,23 from appropriate amines by
their reaction with excess of diphosgene, forming an intermediate
isocyanate, which subsequently reacted with 3-(1H-imidazol-
4-yl)propanol hydrochloride to furnish the desired products
(Scheme 1, route 1).
Compounds 2 and 3 were synthesized from the corresponding
(commercially available) carboxylic acids by modified Curtius
Table 1
Structures and pharmacological results of compounds 1–13
O
N
R
O
N
H
N
H
a
b
Compounds
R
ED50 (mg/kg p.o.)
IC50 (nM)
hH3R Kid (nM)
hH4R Kie (nM)
Selectivity ratio hH4R/hH3R
1
2
3.8 0.6
2.7 1.0
128
68
54
55
1053 123
378 14
20
7
CH3
3
0.55 0.16
51
27
nt
E
4
5
1.4 0.7
2.3 0.6
52
27
14
nt
Z
ntc
421 72
30
29
6
7
0.56 0.06
1.9 0.8
nt
nt
24
26
5
6
703 54
E
E
1326 76
51
8
9
1.3 0.3
7.3 1.4
29
nt
26
20
nt
nt
5
E
10
11
1.4 0.8
3.5 1.4
nt
17
25
155 24
154 15
9
6
33
12
13
4.6 2.2
4.7 1.6
43
32
86
447 41
5
194
500 105
2.5
a
b
c
s
Central H3R test in vivo: measurement of N -methylhistamine in brain after oral administration to mouse37 mean SEM.
H3R assay on guinea pig ileum35,36 (number of experiments (n): for compd 2–4 n = 6; for compd 1 n = 7, for compd 10 n = 8).
Not tested.
d
Affinity of the test compounds determined by displacement of [125I]iodoproxyfan binding to membranes of CHO-K1 cells expressing the human H3R,31 data from a single
experiment with each concentrations tested at least in triplicate, except for compounds 6, 7 and 9 with mean SEM of three independent experiments.
e
Affinity of the test compounds determined by displacement of [3H]histamine binding to membranes of Sf9 cells expressing the human H4R, co-expressed with G
ai2 and
Gb1c2 subunits,33,34 mean SD of at least three independent experiments.

M. Wie˛cek et al. / Bioorg. Med. Chem. 19 (2011) 2850–2858
2853
Route 2
NH2
Route 1
NH2
Compounds 2, 3 and 8 were prepared from commercially avail-
able intermediates (carboxylic acids for 2 and 3 and amine for 8).
Non-commercially available amines (1b–8b) were prepared by
hydrazinolysis of the corresponding N-substituted phthalimides
and isolated as hydrochlorides (Scheme 3). N-Substituted phthali-
mides (1a–8a) were prepared using the Mitsunobu reaction, from
appropriate alcohols and phthalimide in the presence of diethyl
azodicarboxylate (DEAD) and triphenylphosphine30 (Scheme 3).
The corresponding alcohols for the synthesis of these N-substi-
tuted phthalimides were commercially available.
R
R
(b)
(a)
R
N C
(c)
O
O
R
N
O
N
All final compounds 1–13 were isolated as hydrogen maleates.
Their purity was checked by TLC, and their structures were con-
firmed by standard spectral techniques (1H NMR, IR, MS) and ele-
mental analysis (Section 5.1.)
H
N
H
1, 4-13
Scheme 1. Synthesis of carbamates 1, 4–13. Reagents and conditions: (a)
ClCOOCCl3, charcoal (cat.), ethyl acetate, 4–5 h reflux; (b) di-tert-butyldicarbonate,
DMAP, acetonitrile, 10 min, rt; (c) 3-(1H-imidazol-4-yl)propanolꢀHCl, acetonitrile,
4–5 h reflux (for route 1), 24 h reflux (for route 2).
2.2. Pharmacology
Human H3R (hH3R) affinities of the described compounds were
evaluated by competition binding experiments with [125I]iodopr-
oxyfan on membranes of CHO-K1 cells stably expressing the
hH3R.31,32 Compounds (1, 2, 5–7, 10–13) were further tested
regarding their affinities at human H4R (hH4R) by a [3H]histamine
replacement assay on membranes of Sf9 cells, co-expressing the
hH4R with Gai2 and Gb1c2 subunits.33,34 In addition, these ligands
were characterized for H3R antagonist activity with a functional
in vitro test on the isolated guinea-pig ileum (compd 1–4, 8, 11–
13) and in vivo in the brain of Swiss mice. The in vitro potency
was determined by the concentration-dependent inhibition of
O
R
N
O
N
H
(a)
R-COOH
N
2, 3
H
Scheme 2. Synthesis of carbamates 2 and 3. Reagents and condiotns: (a) DPPA,
Et3N, 3-(1H-imidazol-4-yl)propanolꢀHCl, acetonitrile, 24 h reflux.
electrically evoked twitches of guinea pig ileum by (R)-a-methyl-
reaction. In this reaction, the carboxylic acid was reacted with di-
phenyl phosphorazidate (DPPA) under basic conditions (TEA), lead-
ing to an intermediate carboxylic acid azide, which is converted by
nitrogen liberation to the corresponding isocyanate.26,27 These
in situ formed isocyanates were reacted with 3-(1H-imidazol-4-
yl)propanol hydrochloride yielding the appropriate carbamates
(Scheme 2).
An alternative approach was used for the synthesis of com-
pounds 7 and 8. In these cases, the isocyanates were prepared un-
der mild conditions by DMAP-catalyzed reaction of amines with di-
tert-butyldicarbonate.28,29 The isocyanates without isolation were
added to 3-(1H-imidazol-4-yl)propanol hydrochloride and con-
verted into the appropriate carbamates (Scheme 1, route 2).
The synthesis and physicochemical data of compounds 5 and 10
were described previously.23
histamine in the presence of test compound.35,36 The in vivo tests
s
were performed by quantification of endogenous N -methylhista-
mine level in the brain of Swiss mice after oral (p.o.) treatment
with test compounds.37 Selected compounds (1, 2, 4, 8, 11–13)
were additionally investigated for their affinities at H1R and H2R
on isolated organs of the guinea-pig.38 Results of pharmacological
screening are summarized in Tables 1 and 2.
3. Pharmacological effects and discussion
All tested compounds exhibited histamine H3/H4 receptor affin-
ity. Good to moderate affinity was observed for the hH3R with Ki
values in the range from 14 to 194 nM and moderate to weak affin-
ity for the hH4R (Ki values from 154 to 1326 nM). Among carba-
O
O
(b)
(a)
+
NH
O
R OH
N R
O
R NH2
1b - 8b
1a - 8a
Compds
1a, 1b
R
Compds
R
CH3
6a, 6b
7a, 7b
2a, 2b
3a, 3b
Z
E
4a, 4b
5a, 5b
E
8a, 8b
E
Scheme 3. Synthesis of N-substituted phthalimides 1a–8a and amines 1b–8b. Reagents and conditions: (a) Ph3P, DEAD, THF, 20 h, rt; (b) H2N–NH2, EtOH, 3 h reflux; HCl
concd.

2854
M. Wie˛cek et al. / Bioorg. Med. Chem. 19 (2011) 2850–2858
Table 2
kg p.o. In the group of cyclohexyl derivatives the change of the al-
kyl linker between the carbamate group and the cyclohexyl moiety
from 1 (compound described in lit. 21) to 4 carbon atoms was
without the significant influence on the in vivo potency, but com-
pound devoid of this linker showed significantly lower activity
(ED50 > 10 mg/kg).20
Selected compounds (1, 2, 4, 8, 11–13) were additionally tested
for their H1R activity on guinea-pig ileum as well as for their H2R
potency on guinea-pig atrium38 (Table 2). All tested compounds
showed low affinity for these receptors with pA2 values below
5.0 (except of 13, pA2 <5.52 for H1R).
Activity of selected compounds at histamine H1 and H2 receptor subtypes
a
b
Compounds
H1R pA2
H2R pD2
1
2
4
4.16
<4.0
4.1
4.33
3.96
4.97
<5.52
3.75
<4.0
3.85
4.35
4.49
4.94
4.61
8
11
12
13
a
Functional H1R assay on guinea-pig ileum (SEM < 0.2).38
Functional H2R assay on guinea-pig atrium (SEM < 0.2).38
b
4. Conclusions
mates possessing an unsaturated moiety (1–9), the hH3R affinity
depended mainly on the chain length and on the position of unsat-
urated double bond. The weakest affinity is exhibited by butenyl
derivatives (1 and 2) and elongation of the chain length to five,
six and even eight carbon atoms or the introduction of bulky
cyclohexenylethyl substituent led to about 2–3-fold increase of
in vitro affinity. The highest affinity is shown by the 4-pentenyl
derivative 5 (Ki = 14 nM) and 2,6-octadienyl derivative 9 (Ki =
20 nM).
Subtle differences in lipophilic alkenyl residue in compounds 1,
2–9 and 3–5 caused distinct changes in hH4R affinity, leading to re-
duced affinities in the submicromolar concentration range. Com-
pounds 10 and 11 with an ethyl chain between cycloalkyl and
carbamate moiety showed the highest affinity for both hH3R and
hH4Rs. Elongation of the carbon chain between carbamate group
and the cyclohexyl ring resulted in a decrease of H3R and H4R affin-
ities (11?12?13).
As a continuation of the search for 3-(1H-imidazol-4-yl) propa-
nol carbamates with dual histamine H3R/H4R affinity, unsaturated
alkyl derivatives (1–9) and those possessing a cycloalkyl group in
different distances to the carbamate moiety (10–13) were synthe-
sized. Tested compounds exhibited good affinity for the hH3R and
moderate to weak affinity for the hH4R. These results indicate that
restricted alkenyl analogs may provide greater selectivity for H3R
over H4R. For example, the hex-3-enyl analog (compound 7) was
51-fold selective for the H3R over the H4R.
5. Experimental
5.1. Chemistry
Melting points were determined on a MEL-TEMP II apparatus
and are uncorrected. 1H NMR spectra were recorded on a Varian-
In addition, compounds were evaluated for their antagonist
activity for H3R in the functional in vitro test in the guinea pig
ileum (selected compounds) and in vivo in mouse brain after p.o.
administration. All compounds investigated showed moderate
H3R in vitro antagonist activities. Compounds with a cyclohexyl
moiety showed the highest potency, independently of its distance
from the carbamate moiety (11–13) or the existence of double
bond (8). Carbamates with a short unsaturated chain (1–4) exhib-
ited somewhat lower activity in this in vitro model. However, com-
pounds showed moderate to good inverse agonist/antagonist
in vivo potency with ED50 values in the range from 0.55 to
7.3 mg/kg p.o. In the group of unsaturated derivatives the activity
was dependent on the chain length and on the place of unsaturated
bond. In our series of compounds, the optimal chain length was 5–
6 carbon atoms. N-Butenyl derivatives (1 and 2) showed slightly
lower activity. Elongation of the chain to octenyl (9) moiety re-
sulted in significant decrease of the in vivo potency. Activity in this
group of compounds was determined also by the position of unsat-
urated bond. Presented data indicated that b-position was pre-
Mercury 300 MHz spectrometer or on a Bruker AC 300
(300 MHz) in DMSO-d6 or CDCl3. Chemical shifts are expressed in
ppm downfield from internal tetramethylsilane as reference. Data
are reported in the following order: multiplicity (br, broad; def, de-
formed; s, singlet; d, doublet; dq, doublet of quartets; t, triplet; m,
multiplet; qu, quintet; q, quartet; sc, sextet; Cyhexl, cyclohexyl;
Im, imidazol-4-yl; Mal, maleic acid; Pht, phthalimide; Ph, phenyl),
approximate coupling constants J in Hertz (Hz), number of protons,
⁄, exchangeable by D2O. Mass spectra (MS) were obtained on an EI-
MS Finnigan MAT CH7A and Finnigan MAT 711 (high-resolution
mass spectra). Spectrometers resolving power 12,500. Elemental
analyses were measured on Perkin–Elmer 240 B or Perkin–Elmer
240 C instruments and are within 0.4% of the theoretical values.
Preparative, centrifugally accelerated, rotatory chromatography
was performed using a Chromatotron 7924T (Harrison Research)
and glass rotors with 4 mm layers of Silica Gel 60 PF254 containing
gypsum (Merck). Column chromatography was performed using
Silica Gel 60 (0.063–0.20 mm; Merck). TLC was carried out using
Silica Gel F254 plates (Merck), and the spots were visualized with
fast blue salt BB (Aldrich) (carbamates 1–13), ninhydrin (amines
1b–8b) or by UV absorption at 254 nM (phthalimides 1a–8a).
The following abbreviations are used: DEAD, diethyl azodicarbox-
ylate; DMAP, 4-dimethylaminopyridine; DPPA, diphenyl phos-
phoryl azide; TEA, triethylamine.
ferred over d and
c position, respectively (3, 4?5; 6?7, 8).
Nevertheless, the most actives among investigated compounds
were E-isomers (3, 6) which exhibited an in vivo potency about
twofold higher than the potency of the reference inverse agonist/
antagonist thioperamide (ED50 1.0 mg/kg p.o.). Comparison of the
in vivo activity of these compounds with previously described by
our group their saturated analogs21 indicated, that straight n-alkyl
chains at the carbamate nitrogen were more beneficial to activity
than the corresponding to them rigid unsaturated ones. Only N-al-
lyl derivative (ED50 = 0.54 mg/kg p.o.)23 was about fivefold more
active than its N-n-propyl analog (ED50 = 2.7 mg/kg p.o.).21 The E-
isomers of the five- and six-membered alkenyl chains with double
bond in b position (compounds 3 and 6) showed the potency in the
same range as their saturated versions (ED50 = 0.69 mg/kg and
0.88 mg/kg, respectively21). Cyclohexylalkyl carbamates showed
moderate in vivo activity with ED50 ranging from 3.5 to 4.7 mg/
5.1.1. General synthetic procedure for the preparation of N-
substituted phthalimides (1a–8a)
The N-substituted phthalimides were prepared according to lit-
erature.30 A mixture of phthalimide (2.21 g, 15 mmol), triphenyl-
phosphine (3.93 g, 15 mmol), and appropriate alcohol (15 mmol)
in 20 mL of dry THF was cooled to 0 °C. Diethyl azodicarboxylate
(DEAD) (2.61 g, 15 mmol) in 15 mL of dry THF was slowly added
dropwise (1 h). The reaction mixture was then allowed to warm
to room temperature and stirred overnight. Solvent was evapo-
rated under reduced pressure and the residue suspended in Et2O.

M. Wie˛cek et al. / Bioorg. Med. Chem. 19 (2011) 2850–2858
2855
After the precipitate was filtered, the solvent was evaporated and
the residue purified by column chromatography [eluent CH2Cl2]
to afford appropriate N-substituted phthalimide.
5.1.2.1. (E/Z)-But-2-enamine hydrochloride (1b).
From 1a
(5 mmol, 1.0 g). Mp 110–112 °C. Yield: 98%. 1H NMR [DMSO-d6]:
d = 8.24 (br s, 3H, NH3þ), 5.87–5.76 (m, 1H, CH@CH–CH3), 5.60–
5.49 (m, 1H, CH@CH–CH3), 3.37–3.31 (m, 2H, N–CH2), 1.70–1.67
(dq, J = 6,5 Hz, J = 1,7 Hz, 3H, CH3), FAB-MS m/z (%): 72 ([M+H]+,
100), 63 (16), 56 (11), 55 (90), 39 (20), 30 (12), 29 (19), 27 (12)
5.1.1.1. N-((E/Z)-But-2-enyl)phthalimide (1a).
From (E/Z)-
but-2-enol (crotyl alcohol) (15 mmol, 1.1 g). Yield: 73%, mp 75 °C
(mp 77.5–79.5 °C, Ref. 39).
5.1.2.2. (Z)-Pent-2-enamine hydrochloride (2b).
From 2a
5.1.1.2. N-((Z)-Pent-2-enyl)phthalimide (2a).
From (Z)-pent-
(13 mmol, 2.8 g). Mp 134–135 °C. Yield: 74%. 1H NMR [DMSO-
d6]: d = 8.08 (br s, 3H, NH3þꢂ), 5.69–5.62 (m, 1H, N–CH2–CH@C),
5.44–5.38 (m, 1H, C@CH–CH2–CH3), 3.48 (d, J = 7.1 Hz, 2H, N–
CH2), 2.07 (qu, J = 7.4 Hz, 2H, CH2CH3), 0.95 (t, J = 7,5 Hz, 3H,
CH3); MS (70 eV); m/z (%) = 84 ([MꢁH]+, 4), 70 (12), 68 (40), 67
2-enol (15 mmol, 1.3 g). Colorless oil. Yield: 91%. 1H NMR [CDCl3]:
d = 7.84–7.81 (m, 2H, Pht-3,6-H), 7.72–7.68 (m, 2H, Pht-4,5-H),
5.63–5.54 (m, 1H, N–CH2–CH@CH), 5.46–5.38 (m, 1H, CH@CH–
CH2–CH3), 4.31 (d, J = 5.8 Hz, 2H, N–CH2), 2.27 (qu, J = 7.4 Hz, 2H,
CH2–CH3), 1,03 (t, J = 7.4 Hz, 3H, CH3).
(19), 56 (100); IR (KBr) (cmꢁ1) 3406, 3017
m ([N–H], 2972 m ([C–
H] CH2), 1477 d ([C–Y], N–CY2).
5.1.1.3. N-((E)-Hex-2-enyl)phthalimide (3a).
From (E)-hex-
2-enol (30 mmol, 3.0 g). Yield: 77%, mp 60–61 °C (described in
Ref. 40).
5.1.2.3. (E)-Hex-2-enamine hydrochloride (3b).
From 3a
(20 mmol, 4.6 g). Mp 180–185 °C. Yield: 48%. 1H NMR [DMSO-
d6]: d = 8.06 (br s, 3H, NH3þ), 5.83–5.73 (m, 1H, N–CH2–CH@CH),
5.52–5.43 (m, 1H, CH@CH–CH2–CH2), 3.34 (d, J = 6.6 Hz, 2H,
N–CH2), 1.98 (q, J = 6.9 Hz, 2H, CH2–CH2–CH3), 1.35 (sc, J = 7.4 Hz,
2H, CH2-CH3), 0.84 (t, J = 7.4 Hz, 3H, CH3)
5.1.1.4. N-((E)-Hex-3-enyl)phthalimide (4a).
From (E)-hex-
3-enol (30 mmol, 3.0 g). Yield: 81%, mp 49–50 °C (described in
Ref. 40).
5.1.1.5. N-((E)-3,7-Dimethyl-2,6-octadienyl)phthalimide (5a).
From (E)-3,7-dimethyl-2,6-octadien-1-ol (geraniol) (50 mmol,
7.7 g). Yield: 74%, mp 57–58 °C (described in Ref. 40).
5.1.2.4. (E)-Hex-3-enamine (4b).
From 4a (23 mmol, 5.3 g).
After concentration under reduced pressure the residue was alkal-
ized with 20 mL of 10% KOH and extracted with Et2O. The organic
solution was dried (Na2SO4), filtered and after concentration the
crude product was used. Yield: 44%.
5.1.1.6. N-[1-(2-Cyclohexyl)ethyl]phthalimide (6a).
From
2-cyclohexylethanol (15 mmol, 1.92 g). Mp 48–51 °C, Yield: 75%.
1H NMR [CDCl3]: d = 7.82 (m, 2H, Pht-3,6-H), 7,70 (m, 2H, Pht-
4,5-H), 3.70 (t, J = 7.6 Hz, 2H, N–CH2), 1.81–1.53 (m, 7H, Cyhexl-
1,3,4,5-H), 1.37–1.10 (m, 4H, Cyhexl-2,6-H), 0.96 (q, J = 11.6 Hz,
2H, CH2-Cyhexl); MS (70 eV); m/z (%) = 257 ([M+], 92), 229 (10),
162 (21), 161 (100), 160 (84), 149 (25), 148 (28), 133 (18), 130
(22), 105 (17), 104 (19), 98 (24), 81 (16), 77 (22), 76 (15), 67
5.1.2.5. (E)-3,7-Dimethyl-2,6-octadienyl amine hydrochloride
(geranylamine hydrochloride) (5b).
From 5a (33 mmol,
8.5 g). Yield: 68%, mp 155–160 °C (mp 146 °C, Ref. 42).
5.1.2.6. 2-Cyclohexylethanamine hydrochloride (6b).
From
(6a) (10 mmol, 2.57 g). Yield: 50%, mp 230 °C (mp 253–254 °C,
Ref. 43).
(15), 55 (31), 41 (30), 30 (11); IR (KBr) (cmꢁ1) 2922, 2849
([C–H] CH2), 1710 [C@O], 1396 d ([C–Y], N–CY2)
m
m
Anal. Calcd for C16H19NO2 (Mr: 257.33): C, 74.68; H, 7.44; N,
5.1.2.7.
3-Cyclohexylpropanamine
hydrochloride
(7b).
5.44. Found: C, 74.75; H, 7.56; N, 5.36.
From (7a) (10 mmol, 2.71 g). Yield: 59%, mp 215 °C (mp 224–
228 °C, Ref. 43).
5.1.1.7. N-[1-(3-Cyclohexyl)propyl]phthalimide (7a).
3-cyclohexyl-1-propanol (15 mmol, 2.13 g). Yield: 85%, mp
47–48 °C (mp 51 °C, Ref. 41).
From
5.1.2.8. 4-Cyclohexylbutanamine hydrochloride (8b).
(8a) (10 mmol, 2.85 g). Yield: 86%, mp 160–162 °C (mp 165 °C,
Ref. 41).
From
5.1.1.8. N-[1-(4-Cyclohexyl)butyl]phthalimide (8a).
From 4-
5.1.3. General procedure for the synthesis of carbamates(1, 4–6,
9–13)
cyclohexyl-1-butanol (15 mmol, 2.34 g). Mp 86–88 °C, yield: 79%.
1H NMR [CDCl3]: d = 7.89–7.82 (m, 2H, Pht-3,6-H), 7.72–7.64 (m,
2H, Pht-4,5-H), 3.67 (t, J = 7.3 Hz, 2H, N–CH2), 1.68–1.58 (m, 7H,
Cyhexl-1,3,4,5-H), 1.38–1.30 (m, 2H, N–CH2–CH2) 1.25–1.07 (m,
6H, Cyhexl-2,6-H + CH2–CH2-Cyhexl), 0.96 (q, J = 10.0 Hz, 2H, CH2-
Cyhexl); MS (70 eV); m/z (%) = 285 ([M+], 100), 202 (6), 175 (13),
174 (17), 162 (16), 161 (63), 160 (78), 148 (22), 133 (17), 130
To the solution of trichloromethyl chloroformate (0.6 g,
3 mmol) and the catalytic amount of activated charcoal in 20 mL
of dry ethyl acetate was added rapidly the corresponding amine
hydrochloride (2.5 mmol) in 10 mL of dry ethyl acetate. The reac-
tion mixture was heated to reflux for 4–5 h, the black solution
was cooled and filtered, and the solvent was evaporated carefully
under reduced pressure. The freshly prepared isocyanate was dis-
solved in 20 mL of dry acetonitrile and added to a mixture of 3-
(1H-imidazol-4-yl)propanol hydrochloride (2.5 mmol, 0.4 g) in
10 mL of dry acetonitrile. The solution was refluxed for 4–5 h
and concentrated in vacuo. The residue was purified by rotatory
chromatography [eluent: CH2Cl2/MeOH (gradient from 99:1 to
90:10), ammonia atmosphere]. Separation was monitored by TLC.
The products were obtained as colorless oils and crystallized as
hydrogen maleates in Et2O/EtOH.
(17), 105 (15), 104 (12), 83 (8); IR (KBr) (cmꢁ1) 2925, 2849
([C–H] CH2), 1704 [C@O], 1400 d ([C–Y], N–CY2)
m
m
Anal. Calcd for C18H23NO2 (Mr: 285.17): C, 75.76; H, 8.12; N,
4.91. Found: C, 75.37; H, 8.00; N, 4.77.
5.1.2. General synthetic procedure for the preparation of
amines (1b–8b)
Appropriate N-substituted phthalimide (5 mmol) and hydrazine
hydrate (5 mmol, 0.3 g) in 20 mL of EtOH were refluxed for 3 h. The
suspension was cooled, filtered, acidified with hydrochloric acid,
and once more filtered. The filtrate was concentrated under re-
duced pressure and the products were crystallized as hydrochlo-
ride salts from Et2O and used for further synthesis.
5.1.3.1. (E/Z)-3-(1H-Imidazol-4-yl)propyl but-2-enylcarbamate
hydrogen maleate (1).
From (1b) (2.5 mmol, 0.27 g). Yield
18.8%. Mp 96 °C. 1H NMR [DMSO-d6]: d = 8.85 (s, 1H, Im-2-H),

2856
M. Wie˛cek et al. / Bioorg. Med. Chem. 19 (2011) 2850–2858
7.39 (s, 1H, Im-5-H), 7.21 (s, 1H, CONH⁄), 6.04 (s, 2H, Mal), 5.54–
5.36 (m, 2H, HC@CH), 3.97 (t, J = 6.4 Hz, 2H, CH2O), 3.53 (br s, 2H,
N–CH2), 2.67 (t, J = 7.4 Hz, 2H, Im-CH2), 1.88 (qu, J = 7.2 Hz, 2H,
Im-CH2CH2), 1.62 (d, J = 6.4 Hz, 3H, CH3); FAB-MS; m/z (%) = 224
([M+H]+, 100), 185 (20), 127 (13),109 (57), 95 (6), 93 (52), 82
C
15H25N3O2 ꢃ C4H4O4 ꢃ 0.5 H2O (Mr: 404,46): C, 56.42; H, 7.48;
N, 10.39. Found: C, 56.80; H, 7.18; N, 10.36.
5.1.3.8. 3-(1H-Imidazol-4-yl)propyl 2-cyclohexylpropylcarba-
mate hydrogen maleate (12).
From (7b) (2.5 mmol, 0.44 g).
(11), 81 (10), 75 (18), 57 (14); IR (KBr) (cmꢁ1): 1701 (
m
[C@O]). Anal.
Yield 53.9%. Mp 95–96 °C. 1H NMR [DMSO-d6]: d = 8.90 (s, 1H,
Im-2-H), 7.40 (s, 1H, Im-5-H), 7.06 (t, J = 5.4 Hz, 1H, CONH⁄), 6.05
(s, 2H, Mal), 3.96 (t, J = 6.4 Hz, 2H, CH2O), 2.92 (q, J = 6.4 Hz, 2H,
N–CH2), 2.68 (t, J = 7.5 Hz, 2H, Im-CH2), 1.89 (qu, J = 7.2 Hz, 2H,
Im-CH2CH2), 1.65 (d, J = 11.6 Hz, 5H, Cyhexl-1,2,6-H), 1.37 (q,
J = 7.4 Hz, 2H, CH2–CH2-Cyhexl), 1.22–1.08 (m, 6H, Cyhexl-3,4,5-
H), 0.82 (q, J = 10.3 Hz, 2H, CH2-Cyhexl). MS (70 eV); m/z
(%) = 293 ([M]+, 9), 109 (25), 108 (100), 107 (27), 95 (60), 82 (21),
Calcd for C11H17N3O2 ꢃ C4H4O4 ꢃ 0.25 H2O (Mr: 343.85): C, 52.40;
H, 6.30; N, 12.22. Found: C, 52.62; H, 6.11; N, 12.27.
5.1.3.2. (Z)-3-(1H-Imidazol-4-yl)propyl pent-2-enylcarbamate
hydrogen maleate (4).
From (2b) (3.0 mmol, 0.36 g). Yield
11.4%. Mp 89 °C. 1H NMR [DMSO-d6]: d = 8.83 (s, 1H, Im-2-H),
7.38 (s, 1H, Im-5-H), 7.21 (s, 1H, CONH⁄), 6.04 (s, 2H, Mal), 5.44–
5.38 (m, 1H, CH@CH), 5.31–5.25 (m, 1H, CH@CH), 3.97 (t, J =
6.4 Hz, 2H, CH2O), 3.61 (t, J = 5.4 Hz, 2H, N–CH2), 2.66 (t,
J = 7.4 Hz, 2H, Im-CH2), 2.03 (qu, J = 7,4 Hz, 2H, CH3CH2), 1.88 (qu,
J = 7.2 Hz, 2H, Im-CH2CH2), 0.92 (t, J = 7.5 Hz, 3H, CH3). FAB-MS
m/z (%) = 238 ([M+H]+, 41), 127 (23),109 (100), 95 (9), 82 (17), 81
81 (36); IR (KBr) (cmꢁ1): 1699
(m[C@O]). Anal. Calcd for
C
16H27N3O2 ꢃ C4H4O4 ꢃ 0.25 H2O (Mr: 413,98): C, 58.03; H, 7.67;
N, 10.15. Found: C, 57.91; H, 7.44; N, 10.03.
5.1.3.9. 3-(1H-Imidazol-4-yl)propyl 2-cyclohexylbutylcarbamate
hydrogen maleate (13).
(22), 63 (9); IR (KBr) (cmꢁ1): 1696 (
12H19N3O2 ꢃ C4H4O4 (Mr: 353.37): C, 54.38; H, 6.56; N, 11.89.
Found: C, 54.17; H, 6.56; N, 11.88.
m
[C@O]). Anal. Calcd for
From (8b) (2.5 mmol, 0.48 g). Yield
37.7%. Mp 112–113 °C. 1H NMR [DMSO-d6]: d = 8.83 (s, 1H, Im-2-
H), 7.38 (s, 1H, Im-5-H), 7.05 (t, J = 5.3 Hz, 1H, CONH⁄), 6.04 (s,
2H, Mal), 3.96 (t, J = 6.5 Hz, 2H, CH2O), 2.94 (q, J = 6.2 Hz, 2H,
N–CH2), 2.66 (t, J = 7.4 Hz, 2H, Im-CH2), 1.88 (qu, J = 7.1 Hz, 2H,
Im-CH2CH2), 1.63 (d, J = 12.0 Hz, 5H, Cyhexl-1,2,6-H), 1.35 (q,
J = 7.2 Hz, 2H, NH–CH2–CH2–), 1.27–1.05 (m, 8H, Cyhexl-3,4,5-
H + CH2–CH2–CH2-Cyhexl), 0.83 (q, J = 10.0 Hz, 2H, CH2-Cyhexl).
MS (70 eV); m/z (%) = 307 ([M]+, 9), 109 (26), 108 (100), 107 (25),
C
5.1.3.3. Synthesis and physicochemical data of compound
5.
It was described previously.23
5.1.3.4. (E)-3-(1H-Imidazol-4-yl)propyl hex-2-enylcarbamate
hydrogen maleate (6). From (3b) (5 mmol, 0.7 g). Yield
98 (13), 95 (65), 82 (19), 81 (42); IR (KBr) (cmꢁ1): 1698 (
m[C@O]).
36.1%. Mp 82 °C. 1H NMR [DMSO-d6]: d = 8.81 (s, 1H, Im-2-H),
7.36 (s, 1H, Im-5-H), 7.20 (s, 1H, CONH), 6.01 (s, 2H, Mal), 5.54–
5.31 (m, 2H, CH@CH), 3.94 (t, J = 6.3 Hz, 2H, CH2O), 3.55 (t,
J = 5.5 Hz, 2H, N–CH2), 2.64 (t, J = 7.4 Hz, 2H, Im-CH2), 1.98–1.84
(m, 4H, CH@CH–CH2 + Im-CH2CH2), 1.30 (sc, J = 7.4 Hz, 2H, CH2–
CH3), 0.83 (t, J = 7.4 Hz, 3H, CH3). MS (70 eV); m/z (%) = 251 ([M]+,
14), 109 (37), 108 (100), 107 (37), 98 (19), 95 (64), 82 (31), 81
Anal. Calcd for C17H29N3O2 ꢃ C4H4O4 (Mr: 423,51): C, 59.56; H,
7.85; N, 9.92. Found: C, 59.41; H, 7.80; N, 9.88.
5.1.4. Synthesis of carbamates by modified Curtius reaction (2
and 3)
5.1.4.1. 3-(1H-Imidazol-4-yl)propyl but-3-enylcarbamate hydro-
gen maleate (2).
The mixture of 4-pentenoic acid (2.5 mmol,
(59), 80 (10), 72 (21), 69 (15); IR (KBr) (cmꢁ1): 1700 (
m[C@O]). Anal.
0.25 g), triethylamine (2.5 mmol, 0.25 g), diphenylphosphoryl
azide (2.5 mmol, 0.69 g), and 3-(1H-imidazol-4-yl)propanol hydro-
chloride (2.5 mmol, 0.4 g) was refluxed for 24 h in 20 mL of dry
acetonitrile. The solvent was removed under reduced pressure,
the residue was dissolved in ethyl acetate and the white precipi-
tate was filtered. The filtrate was washed twice with saturated
K2CO3 and the organic fraction after concentration was purified
by rotatory chromatography [eluent: AcOEt/i-PrOH (60:40). Sepa-
ration was monitored by TLC. Obtained colorless oil was crystal-
lized as hydrogen maleate in Et2O/EtOH. Yield 43.5%. Mp 95 °C.
1H NMR [DMSO-d6]: d = 8.83 (s, 1H, Im-2-H), 7.37 (s, 1H, Im-5-H),
7.10 (s, 1H, CONH⁄), 6.04 (s, 2H, Mal), 5.81–5.71 (m, 1H, CH@CH2),
5.07–4.99 (m, 2H, CH@CH2), 3.96 (t, J = 6.5 Hz, 2H, CH2O), 3.02 (q,
J = 6.3 Hz, 2H, N–CH2), 2.66 (t, J = 7.4 Hz, 2H, Im-CH2), 2.15 (q,
J = 7.0 Hz, 2H, CH2CH@CH2), 1.88 (qu, J = 7.2 Hz, 2H, Im-CH2CH2);
MS (70 eV); m/z (%) = 223 ([M+], 17), 182 (17), 153 (5), 109 (64),
108 (100), 107 (40), 95 (85), 82 (26); IR (KBr) (cmꢁ1): 1696
Calcd for C13H21N3O2 ꢃ C4H4O4 (Mr: 367.41): C, 55.58; H, 6.86; N,
11.44. Found: C, 55.36; H, 6.64; N, 11.32.
5.1.3.5. (E)-3-(1H-Imidazol-4-yl)propyl 3,7-dimethylocta-2,6-
dienylcarbamate hydrogen maleate (9).
From (5b) (2.5
mmol, 0.47 g). Yield 10.8%. Mp 68–70 °C. 1H NMR [DMSO-d6]:
d = 8.80 (s, 1H, Im-2-H), 7.35 (s, 1H, Im-5-H), 7.12 (s, 1H, CONH),
6.02 (s, 2H, Mal), 5.11–5.05 (m, 2H, 2 ꢃ CH@C), 3.94 (t, J = 6.6 Hz,
2H, CH2O), 3.57–3.53 (m, 2H, N–CH2), 2.65 (t, J = 7.4 Hz, 2H, Im-
CH2), 2.02–1.84 (m, 6H, Im-CH2CH2 + CH2–CH2–CH@C(CH3)2),
1.61–1.52 (m, 9H, 3 ꢃ CH3). MS (70 eV); m/z (%) = 305 ([M]+, 20),
237 (16), 183 (6), 127 (15), 109 (100), 108(52), 107 (24), 98 (31),
95 (50), 82 (34), 81 (51), 80 (13), 71 (10), 69 (36); IR
(KBr) (cmꢁ1): 1698
(
m
[C@O]). Anal. Calcd for C17H27N3O2 ꢃ
C4H4O4 ꢃ 1.25 H2O (Mr: 443.74): C, 56.84; H, 7.61; N, 9.47. Found:
C, 56.86; H, 7.74; N, 9.49.
(
m
[C@O]).
5.1.3.6. Synthesis and physicochemical data of compound
10.
Anal. Calcd for C11H17N3O2 ꢃ C4H4O4 (Mr: 339.35): C, 52.81; H,
It was described previously.23
6.24; N, 12.38. Found: C, 52.81; H, 6.23; N, 12.09.
5.1.3.7. 3-(1H-Imidazol-4-yl)propyl 2-cyclohexylethylcarbamate
hydrogen maleate (11). From (6b) (2.5 mmol, 0.40 g). Yield
5.1.4.2. (E)-3-(1H-Imidazol-4-yl)propyl pent-2-enylcarbamate
hydrogen maleate (3).
The mixture of (E)-3-hexenoic acid
24.2%. Mp 107–108 °C. 1H NMR [DMSO-d6]: d = 8.84 (s, 1H, Im-2-
H), 7.38 (s, 1H, Im-5-H), 7.03 (s, 1H, CONH⁄), 6.04 (s, 2H, Mal),
3.96 (t, J = 6.4 Hz, 2H, CH2O), 2.98 (q, J = 6.6 Hz, 2H, N–CH2), 2.66
(t, J = 7.4 Hz, 2H, Im-CH2), 1.88 (qu, J = 7.2 Hz, 2H, Im-CH2CH2),
1.66–1.59 (m, 5H, Cyhexl-1,2,6-H), 1.31–1.07 (m, 6H, Cyhexl-
3,4,5-H), 0.85 (q, J = 10.5 Hz, 2H, CH2-cyclohex). MS (70 eV); m/z
(%) = 279 ([M]+, 9), 109 (25), 108 (100), 107 (26), 95 (54), 82 (20),
(2.5 mmol, 0.29 g), triethylamine (2.5 mmol, 0.25 g), diphenyl-
phosphoryl azide (2.5 mmol, 0.69 g), and 3-(1H-imidazol-4-yl)pro-
panol hydrochloride (2.5 mmol, 0.4 g) was refluxed for 24 h in
20 mL of dry acetonitrile. The solvent was removed under reduced
pressure, and the residue was dissolved in dichloromethane and
washed successively with 5% citric acid, water and saturated solu-
tion of NaHCO3. After concentration of the combined organic frac-
tions, the residue was purified by rotatory chromatography
81 (40), 80 (8); IR (KBr) (cmꢁ1): 1704 (
m[C@O]). Anal. calcd for

M. Wie˛cek et al. / Bioorg. Med. Chem. 19 (2011) 2850–2858
2857
[eluent: CH2Cl2/MeOH (gradient from 99:1 to 90:10), ammonia
atmosphere]. Separation was monitored by TLC. The product was
obtained as colorless oil and crystallized as hydrogen maleate in
Et2O/EtOH. Yield 7.4%. Mp 94 °C. 1H NMR [DMSO-d6]: d = 8.84 (s,
1H, Im-2-H), 7.37 (s, 1H, Im-5-H), 7.20 (s, 1H, CONH⁄), 6.02 (s,
2H, Mal), 5.39–5.30 (m, 1H, CH@CH), 5.59–5.45 (m, 1H, CH@CH),
3.97 (t, J = 6.3 Hz, 2H, CH2O), 3.61 (t, J = 5.5 Hz, 2H, N–CH2), 2.66
(t, J = 7.4 Hz, 2H, Im-CH2), 1.99–1.84 (m, 4H, CH3CH2 + Im-CH2CH2),
0.91 (t, J = 7.4 Hz, 3H, CH3). FAB-MS m/z (%) = 238 ([MÅ+H]+, 100),
237 (4), 185 (15), 127 (18), 109 (77), 95 (9), 82 (17); IR (KBr)
(Na2HPO4/KH2PO4, c = 50 mM, pH 7.5). The homogenate was cen-
trifuged (23,000g, 30 min, +4 °C) and the pellet obtained was resus-
pended in the binding buffer to constitute the membrane
preparation used for the binding assay. For competition binding
experiments, membrane suspension (5–15
was incubated for 60 min at +25 °C with
l
[
g
protein/well)
125I]iodoproxyfan
(c = 25 pM) alone, or together with competing drugs dissolved in
the same buffer to give a final volume of 200 L. Incubations were
l
performed in triplicate and stopped by four additions (5 mL) of ice-
cold medium, followed by rapid filtration through glass microfibre
filters (GF/B Whatman, Clifton, NJ) presoaked in polyethyleneimine
(cmꢁ1): 1700
(
m
[C@O]). Anal. Calcd for C12H19N3O2 ꢃ -
C4H4O4 ꢃ H2O (Mr: 371.40): C, 51.74; H, 6.79; N, 11.31. Found: C,
(PEI) (
with a LKB (Rockville, MD) gamma counter (efficiency: 82%). Spe-
cific binding was defined as that inhibited by imetit (c = 1 M), a
specific histamine H3 receptor agonist.44 Assays were run in tripli-
cates at least with 9 concentrations between 0.1 nM and 1 M. The
x = 0.3%). Radioactivity trapped on the filters was measured
52.06; H, 6.37; N, 10.95.
l
5.1.5. General procedure for the synthesis of carbamates (7 and
8)
l
To a solution of di-tert-butyldicarbonate (7 mmol, 1.52 g) in
10 mL of anhydrous acetonitrile were added successively a solu-
tion of 4-dimethylaminopyridine (5 mmol, 0.62 g) in 10 mL of
anhydrous acetonitrile and a solution of appropriate amine (free
base) (5 mmol) in 5 mL of anhydrous acetonitrile. After stirring
for 10 min at room temperature 3-(1H-imidazol-4-yl)propanol
hydrochloride (5 mmol, 0.8 g) was added. The reaction mixture
was refluxed for 24 h. The solvent was removed under reduced
pressure, and the residue was alkalized by NH3/MeOH and dis-
solved in dichloromethane. The precipitate was filtered and the fil-
trate was purified first by rotatory chromatography [eluent:
CH2Cl2/MeOH (gradient from 99:1 to 90:10)], and next by column
chromatography [eluent: CH2Cl2:MeOH:NH3/MeOH (95:5:1)]. Sep-
aration was monitored by TLC. The products were obtained as col-
orless oils and crystallized as hydrogen maleates in Et2O/EtOH.
corresponding Ki values were determined from the IC50 value
according to the Cheng–Prusoff equation.45
5.2.2. In vitro [3H]histamine binding assay on hH4R
Prior to the experiments, cell membranes were sedimented by a
10-min centrifugation at +4 °C and 16,000g and resuspended in
binding buffer (12.5 mM MgCl2, 1 mM EDTA and 75 mM Tris/HCl,
pH 7.4). Competition binding experiments were carried out by
incubating membranes, 35
lg/well (prepared from Sf9 cells
expressing hH4R, co-expressed with G- protein G
a
i2 and Gb1 2 sub-
c
units) in a final volume of 200 lL containing binding buffer and
[3H]histamine (10 nM, 566.1 GBq/mmol). Assays were run in trip-
licates with seven appropriate concentrations between 0.1 nM
and 100
for 60 min at +25 °C and shaking at 250 rpm. Non-specific binding
was determined in the presence of 100 M unlabeled histamine.
lM of the test compound. Incubations were performed
l
5.1.5.1. (E)-3-(1H-Imidazol-4-yl)propyl hex-3-enylcarbamate
Bound radioligand was separated from free radioligand by filtra-
tion through GF/B filters pretreated with 0.3% (mass/vol) PEI and
washed three times with 0.3 mL of ice-cold binding buffer
(+4 °C). The amount of radioactivity collected on the filter was
determined by liquid scintillation counting. Competition binding
data were analyzed by the software GraphPad Prism™ (2000, ver-
sion 3.02, San Diego, CA, USA) using non-linear least squares fit.
Affinity values (Ki) were expressed as mean standard deviation
(SD). Ki values were calculated from the IC50 values according to
Cheng–Prusoff equation.
hydrogen maleate (7).
From (4b) (5 mmol, 0.5 g). Yield
12.8%. Mp 109–110 °C. 1H NMR [DMSO-d6]: d = 8.79 (s, 1H, Im-2-
H), 7.34 (s, 1H, Im-5-H), 7.00 (s, 1H, CONH), 6.01 (s, 2H, Mal),
5.43–5.34 (m, 2H, CH@CH), 3.94 (t, J = 6.3 Hz, 2H, CH2O), 2.95 (q,
J = 5.5 Hz, 2H, N–CH2), 2.64 (t, J = 7.4 Hz, 2H, Im-CH2), 1.98–1.84
(m, 4H, CH@CH–CH2 + Im-CH2CH2), 1.30 (m, J = 7.4 Hz, 2H, CH2–
CH3), 0.83 (t, J = 7.4 Hz, 3H, CH3). MS (70 eV); m/z (%) = 251 ([M]+,
26), 109 (85), 108 (100), 107 (29), 98 (16), 95 (55), 82 (25); IR
(KBr) (cmꢁ1): 1692 (
(Mr: 367.41): C, 55.58; H, 6.86; N, 11.44. Found: C, 55.24; H, 7.19;
m
[C@O]). Anal. Calcd for C13H21N3O2 ꢃ C4H4O4
N, 11.31
5.2.3. Histamine H3 receptor antagonist activity on guinea pig
ileum
5.1.5.2. 3-(1H-Imidazol-4-yl)propyl 2-cyclohexenylethylcarba-
For selected compounds, H3 receptor activity was measured by
concentration-dependent inhibition of electrically evoked twitches
of isolated guinea pig ileum segments induced by (R)-a-methylhis-
tamine in the presence of the antagonist according to Ligneau et
al.44 Longitudinal muscle strips were prepared from the small
intestine, 20–50 cm proximal to the ileocecal valve. The muscle
strips were mounted between two platinum electrodes (4 mm
mate hydrogen maleate (8).
From 2-(1-cyclohexenyl)ethyla-
mine (2,5 mmol, 0.31 g). Yield 10.2%. Mp 85 °C. 1H NMR [DMSO-
d6]: d = 8.90 (s, 1H, Im-2-H), 7.40 (s, 1H, Im-5-H), 7.00 (s, 1H,
CONH), 6.04 (s, 2H, Mal), 5.35 (s, 1H, CH@C), 3.94 (t, J = 6.6 Hz,
2H, CH2O), 2.99 (q, J = 6.3 Hz, 2H, N–CH2), 2.65 (t, J = 7.4 Hz, 2H,
Im-CH2), 1.99 (t, 2H, Im-CH2CH2), 1.87 (m, 6H, CH2-Cyhexl + Cy-
hexl-3,6-H), 1.57–1.44 (m, 4H, Cyhexl-3,6-H). MS (70 eV); m/z
(%) = 277 ([M]+, 23), 183 (11), 127 (16), 109 (100), 108(32), 107
apart) in 20 mL of Krebs buffer, containing 1 lM mepyramine, con-
(13) 95 (25), 82 (28); IR (KBr) (cmꢁ1): 1691 (
m[C@O]). Anal. Calcd
nected to an isometric transducer, continuously gassed with oxy-
gen containing 5% CO2 at 37 °C. After equilibration of the muscle
segments for 1 h with washing every 10 min, they were stimulated
continuously with rectangular pulses of 15 V and 0.5 ms at a fre-
quency of 0.1 Hz. After 30 min of stimulation, a cumulative con-
centration–response curve with increasing concentrations of (R)-
for C15H23N3O2 ꢃ C4H4O4 ꢃ 0.5 H2O (Mr: 402,45): C, 56.71; H,
7.01; N, 10.44. Found: C, 56.41; H, 6.68; N, 10.37.
5.2. Pharmacology
5.2.1. In vitro [125I]iodoproxyfan binding assay on hH3R
a-methylhistamine was recorded. Subsequently the preparations
were washed three times every 10 min without stimulation. Then,
Potency of the synthesized compounds was investigated in a
radioligand binding assay described by Ligneau et al.31 CHO-K1
cells stably expressing the hH3R were washed and harvested with
a PBS medium. They were centrifuged (140g, 10 min, +4 °C), and
then homogenized with a Polytron in an ice-cold binding buffer
the antagonist was incubated for 20–30 min before redetermina-
tion of the dose–response curve of (R)-a
-methylhistamine.35,36
All compounds were tested in concentrations that did not block
the ileal muscarinic M3 receptors.

2858
M. Wie˛cek et al. / Bioorg. Med. Chem. 19 (2011) 2850–2858
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In vivo testing was performed after oral administration of test
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Acknowledgment
The authors acknowledge the partial support of ESF COST
Action BM0806 ‘Recent advances in histamine receptor H4R
research’, financed by EU-FP7, Grant No. 594/N-COST/2009/0 and
that of LOEWE MFF.
32. Gbahou, F.; Vincent, L.; Humbert-Claude, M.; Tardivel-Lacombe, J.; Chabret, C.;
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.03.046.
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