Synthesis and histamine H3 and H4 receptor activity of conformationally restricted cyanoguanidines related to UR-PI376

Article abstract of DOI:10.1002/ardp.201100144

Recently, we identified highly potent agonists of the human histamine H₄ receptor (hH₄R) among a series of imidazolylbutylcyanoguanidines. Aiming at improved selectivity for the hH ₄R relative to the H₃ receptor

Full text of DOI:10.1002/ardp.201100144

Arch. Pharm. Chem. Life Sci. 2011, 344, 775–785
775
Full Paper
Synthesis and Histamine H3 and H4 Receptor Activity of
Conformationally Restricted Cyanoguanidines Related to
UR-PI376*
Roland Geyer and Armin Buschauer
Department of Pharmaceutical/Medicinal Chemistry II, Faculty of Chemistry and Pharmacy, University of
Regensburg, Regensburg, Germany
Recently, we identified highly potent agonists of the human histamine H4 receptor (hH4R) among a
series of imidazolylbutylcyanoguanidines. Aiming at improved selectivity for the hH4R relative to the
H3 receptor (hH3R), the flexible tetramethylene linker connecting imidazole ring and cyanoguanidine
group was replaced by conformationally restricted carbocycles. Introduction of a para- or a meta-
phenylene spacer yielded only very weakly active compounds at both hH3R and hH4R (investigated in
[
35S]GTPgS binding assays using Sf9 insect cell membranes expressing hHxR subtypes). By contrast, the
incorporation of a more flexible cyclohexane-1,4-diyl linker resulted in EC50 or KB values ꢀ110 nM at
hH4R and hH3R. Quality of action, potency and receptor subtype selectivity of the investigated
compounds depend on the stereochemistry: Cis-configured diastereomers prefer the hH4R
and are partial agonists, whereas trans-isomers are antagonists at the hH4R. At the hH3R the
trans-diastereomers are superior to the cis-isomers by
a factor of 10. The results on
imidazolylcycloalkylcyanoguanidines suggest that variation of ring size and optimization of the
stereochemistry may be useful to increase the potency and selectivity of hH4R agonists relative to the
hH3R.
Keywords: Conformational restriction / Cyanoguanidines / Histamine / H4 receptor / UR-PI376
Received: April 14, 2011; Revised: May 9, 2011; Accepted: May 13, 2011
DOI 10.1002/ardp.201100144
Introduction
the H4R plays a crucial role in immunological and inflam-
matory processes [15–17]. Hence, the H4R is considered a
promising drug target for the treatment of inflammatory
and immunological diseases like allergic rhinitis, rheuma-
toid arthritis, bronchial asthma and pruritus [18–20].
Nevertheless, recent reports on b-arrestin-mediated
signaling [21] and partial agonistic effects of the standard
H4R antagonist JNJ7777120 [22] at certain H4R species ortho-
logs suggest that the interpretation of ligand effects in vivo in
terms of agonism or antagonism should be interpreted with
caution [23, 24]. Therefore, both selective antagonists and
agonists are required as pharmacological tools to further
explore the role of the H4R [25].
The biological effects of the biogenic amine histamine are
mediated by four histamine receptor (HR) subtypes, all
belonging to class A of G-protein coupled receptors [1, 2].
The most recently identified fourth HR subtype, the H4R,
was discovered based on its high sequence homology to
the H3R in the years 2000 and 2001, independently by several
research groups [3–9]. The H4R is mainly localized in various
cells of the immune system like eosinophils, T-lymphocytes,
dendritic cells, mast cells, and basophils [3, 6, 7, 10–14].
Furthermore, results of in-vitro and in-vivo studies suggest that
Previously, we identified hH4R agonists among NG-acylated
Correspondence: Prof. Armin Buschauer, Institut fu¨r Pharmazie,
¨
Universitat Regensburg, Universitatsstr. 31, D-93053 Regensburg,
Germany
imidazolylpropylguanidines which were initially designed
¨
E-mail: armin.buschauer@chemie.uni-regensburg.de
Fax: þ49-941 9434820
*In memoriam Prof. Dr. Dr. Dr. h. c. Walter Schunack
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R. Geyer and A. Buschauer
Arch. Pharm. Chem. Life Sci. 2011, 344, 775–785
as H2 receptor agonists [26, 27]. Structural optimization
resulted in highly potent agonists with selectivity for the
hH4R over the hH1R and the hH2R but lack of selectivity
compared to the hH3R [28–30]. Replacement of the basic
acylguanidine group with a cyanoguanidine moiety, which
imidazolylcyclohexylamines VUF5803 (3) and VUF5804 (4),
which were described as non-selective H3R and H4R agonists
((3) H4R: pKi 7.7, a 1.3; H3R: pKi 7.0; (4) H4R: pKi 6.5, a
1.1; H3R: pKi 7.4) [34].
is uncharged at physiological pH, turned out to be key in Results and discussion
terms of H4R agonism and selectivity. Highest potency
resided in imidazolylalkylcyanoguanidines with a tetra-
methylene linker, connecting the imidazole and the cyano-
guanidine moiety. 2-Cyano-1-[4-(1H-imidazol-4-yl)butyl]-3-[(2-
phenylthio)ethyl]guanidine (UR-PI376, 1) (Fig. 1) was the most
potent and selective agonist with an EC50 of 34 nM (intrinsic
activity (a) 0.93) displaying a more than 25-fold selectivity
over the hH3R and negligible activities at the hH1R
and hH2R (investigated in [32P]GTPase assays on hHxR express-
ing Sf9 cell membranes) [28]. Unlike other selective hH4R
agonists, such as 5-methylhistamine [31], VUF8430 [32] or
OUP-16 [33], UR-PI376 is devoid of agonistic activities at
hHR subtypes other than the hH4R [28].
Chemistry
The imidazolylphenylcyanoguanidines 30–44 were synthes-
ized in five steps (Schemes 1 and 2). According to ten Have
et al. [35] the cycloaddition of tosylmethyl isocyanide
(TosMIC, 5) [36] to aldimines (10–12), which are readily pre-
pared from the corresponding benzaldehydes 6–8 and N-
tosylamide (9) [37], was used. Reduction of the nitro- or the
cyano group in 13–15 gave the amines 2, 16 and 18 which
were transformed to the cyanoguanidines by analogy with a
previously described synthetic route [38]. Aminolysis of
diphenyl cyanocarbonimidate (19) [39, 40] with primary
amines 20–24 at ambient temperature gave the isourea inter-
mediates 25–29 which crystallized from diethyl ether [28].
Conversion of the isoureas to the cyanoguanidines 30–34
was performed by heating in a microwave oven with 18
in acetonitrile (Scheme 1). In case of the synthesis of 35–44
the order of the coupling steps was reverted due to the
low nucleophilicity of aniline derivatives, i.e. 19 was
treated first with building block 2 or 16 to yield 45 or 46,
respectively, and afterwards with an aliphatic amine (20–24)
(Scheme 2).
Discrimination between the closely related H3 and H4
receptors turned out to be a critical issue in the development
of selective H4R agonists. As demonstrated by Hashimoto
et al. [33] and Kitbunnadaj et al. [34], the stereochemical
requirements of selective hH3R and hH4R ligands should
be considered. Aiming at elucidating the structure-activity
and structure-selectivity relationships of cyanoguanidines
derived from UR-PI376 we explored the replacement of
the flexible tetramethylene chain by conformationally con-
strained spacers (Fig. 1). Based on a previously suggested
model of UR-PI376 binding to the hH4R four small series
of cyanoguanidines were constructed by introducing a
phenyl ring or a cyclohexyl ring, respectively, as in the
imidazolylphenylamine VUF5801 (2) which was reported
to have some hH4R affinity (pKi 5.8) [34], and the
The 1,4-disubstituted cyclohexanes 53–56 were synthesized
as outlined in Scheme 3. 4-Aminocyclohexanecarboxylic acid
(47) was Boc-protected [41] and the carboxylic acid 48 was
reduced with borane to yield the alcohol (49) [42]. Swern
oxidation [43] gave the corresponding aldehyde 50, which
was successively treated with TosMIC (5) and ammonia in
Figure 1. Replacement of the flexible tetramethylene chain in UR-PI376 with conformationally restricted substructures.
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Arch. Pharm. Chem. Life Sci. 2011, 344, 775–785
hH4R Activity of Conformationally Restricted Cyanoguanidines
777
Scheme 1. Synthesis of the cyanoguani-
dines 30–34a.
Scheme 2. Synthesis of the cyanoguani-
dines 35–44a.
methanol to introduce the imidazole ring [36]. After Boc-
protection of the imidazole nitrogen in 51, the mixture of
cis- and trans-52 was separated by flash chromatography [34].
Deprotection under acidic conditions and liberation of the
amines as free bases with the help of a basic ion exchanger
gave the cis- and trans-imidazolylcyclohexylamines 3 and 4
[28, 34]. The cyanoguanidines 53–56 were synthesized by
analogy with the above described procedure.
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R. Geyer and A. Buschauer
Arch. Pharm. Chem. Life Sci. 2011, 344, 775–785
Scheme 3. Synthesis of the cyanoguani-
dines 53–56a.
Pharmacology
The synthesized cyanoguanidines were investigated for
the biological target, and is a useful approach to explore
the bioactive conformation of flexible molecules and to
refine models of ligand-receptor interactions [44–46].
Therefore, we evaluated if the replacement of the flexible
tetramethylene chain in 1 with a phenyl ring is tolerated by
the hH4R. The substituents at the cyanoguanidine (‘‘eastern
part’’ of the molecule) were selected with respect to compari-
son of the structure-activity relationships with those of
recently published flexible compounds [28].
agonism and antagonism at hH4R and hH3R subtypes in
35S]GTPgS binding assays using membrane preparations of
[
Sf9 insect cells co-expressing the hH4R plus Gai2 plus Gb1g2 or
co-expressing the hH3R plus Gai2 plus Gb1g2. Additionally,
for reasons of comparison the amine precursors (2–4, 16, 18)
and UR-PI376 (1) were characterized.
In the following agonistic potencies are expressed as EC50
values. Intrinsic activities (a) refer to the maximal response
induced by the standard agonist histamine. Compounds
identified to be inactive as agonists (a <0.1 or negative
values, respectively, determined in the agonist mode; cf.
Tables 1 and 2) were investigated in the antagonist mode.
The corresponding KB values of neutral antagonists and
inverse agonists (Tables 1, 2) were determined from
the concentration-dependent inhibition of the histamine-
induced increase in [35S]GTPgS binding.
The amines 2, 16 and 18 were devoid of agonistic activity in
the [35S]GTPgS assay on hH3R and hH4R. VUF5801 (2) showed
inverse agonism at both, the hH4R (KB 2500 nM, a ꢁ0.64)
and the hH3R (KB 1260 nM, a ꢁ0.57); the KB values correspond
to published binding data of 2 [34]. The meta-isomer 16
is
a weak antagonist at the hH4R and the hH3R
(KB >10 000 nM), whereas the homologue 18 turned out to
prefer the hH3R: Compound 18 is a weak antagonist at
the hH4R (KB >10 000 nM) and an inverse agonist at the
hH3R (KB 430 nM, a ꢁ1.3).
Imidazolylphenylcyanoguanidines 30–44 (Table 1)
Free rotation about single bonds in a flexible linker might
result in various conformations, with the single conformers
having different affinities for the common target. The syn-
thesis of conformationally restricted analogues of a lead
compound often results in increased specific binding to
Similar to the results for the building blocks 2, 16 and 18,
none of the synthesized cyanoguanidines showed agonistic
activity, neither at the hH4R nor at the hH3R. The determined
KB values for antagonistic/inverse agonistic activity were
above 10 mM except for several compounds bearing bulkier
alkyl or phenyl(thio)alkyl substituents at the cyanoguanidine
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Arch. Pharm. Chem. Life Sci. 2011, 344, 775–785
hH4R Activity of Conformationally Restricted Cyanoguanidines
779
Table 1. Potencies and efficacies of the synthesized cyanoguanidines 30–44 and the amines 2, 16 and 18 at the hH3R and hH4R in the
35S]GTPgS assay.a
[
Compound no.
Phenyl
substitution
R
hH3R
(EC50) or KB (nM)
hH4R
(EC50) or KB (nM)
a
N
a
N
Histamine
UR-PI376 (1)
(13 ꢂ 2)
720 ꢂ 38
1260 ꢂ 50
>10 000
1
3
2
2
2
2
4
4
4
4
3
3
3
3
3
3
3
3
3
2
3
(11 ꢂ 3)
(37 ꢂ 3)
1
5
3
2
2
2
4
4
4
4
2
2
2
3
2
2
2
2
2
2
2
ꢁ0.52
ꢁ0.57
ꢁ0.09
ꢁ1.3
0.88
ꢁ0.64
ꢁ0.06
0.09
VUF5801b (2)
para
meta
meta
-NH2
-NH2
-CH2NH2
-CH3
-cPr
-CH2CH(CH3)2
-(CH2)3-Ph
-(CH2)2-S-Ph
-CH3
2500 ꢂ 126
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
5200 ꢂ 150
>10 000
>10 000
1970 ꢂ 470
2400 ꢂ 100
935 ꢂ 13
>10 000
>10 000
>10 000
>10 000
>10 000
16
18
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
430 ꢂ 10
>10 000
>10 000
ꢁ0.06
ꢁ0.01
ꢁ0.05
ꢁ0.23
ꢁ0.15
0.02
ꢁ0.16
ꢁ0.18
ꢁ0.21
ꢁ0.28
ꢁ0.19
ꢁ0.03
ꢁ0.11
ꢁ0.1
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
4100 ꢂ 200
4585 ꢂ 850
>10 000
para
para
para
para
para
meta
meta
meta
meta
meta
-cPr
ꢁ0.11
ꢁ0.05
ꢁ0.06
ꢁ0.06
0.05
ꢁ0.01
ꢁ0.09
0.01
-CH2CH(CH3)2
-(CH2)3-Ph
-(CH2)2-S-Ph
-CH3
ꢁ0.19
ꢁ0.21
0.08
-cPr
>10 000
6700 ꢂ 200
ꢁ0.1
-CH2CH(CH3)2
-(CH2)3-Ph
-(CH2)2-S-Ph
ꢁ0.13
ꢁ0.23
ꢁ0.31
>10 000
1100 ꢂ 200
ꢁ0.13
aFunctional [35S]GTPgS binding assay with membrane preparations of Sf9 cells expressing the hH3R þ Gai2 þ Gb1g2 or the
hH4R þ Gai2 þ Gb1g2 was performed as described in the Section Pharmacology. N gives the number of independent experiments
performed in duplicate each. The intrinsic activity (a) of histamine was set to 1.00 and a values of other compounds were referred to
this value. The a values of neutral antagonists and inverse agonists were determined at a concentration of 10 mM. The KB values of
neutral antagonists and inverse agonists were determined in the antagonist mode versus histamine (100 nM) as the agonist.
bPublished pKi values measured by [3H]-histamine (H4R) or [3H]-Na-methylhistamine (H3R) binding to membranes of SK-N-MC cells
expressing the human H4 or H3 receptor in presence of the ligand: pKi (hH4R) 5.8, pKi (hH3R) 6.0 [34].
moiety (KB values in the range of 1–5 mM). The activities of the
compounds at the hH4R and hH3R were not significantly
affected by the substitution pattern. Obviously, the phenyl-
ene linker is too rigid to enable optimal orientation of the
ligand in the binding pocket. Additionally, the change in
electronic properties due to the additional p-system might
contribute to the dramatic decrease in affinity for hH3R and
hH4R compared to compound 1.
subtypes. The cis-isomer prefers the hH4R (EC50 values:
hH4R 15 nM, hH3R: 115 nM) while higher potency at the
hH3R resides in the trans-isomer (EC50 values: hH4R 300 nM,
hH3R 46 nM). The potencies in the [35S]GTPgS assay corre-
spond to published binding data for 3 and 4 [34]. The
compounds 53–56 were synthesized as prototypical cyano-
guanidines reminiscent of characteristic structural features
of OUP-16 [33] and UR-PI376 (1), respectively. The investi-
gation in the [35S]GTPgS assay revealed that the phenyl-
thioethyl substituted cyanoguanidines 55 and 56 were
superior to the methyl substituted analogues. This is in
agreement with structure-activity relationships of flexible
cyanoguanidines [28], corroborating that arylalkyl residues
are suitable to increase the affinity for both receptors com-
pared to the methyl substitution. Similar to 3 and 4 the
Imidazolylcyclohexylcyanoguanidines 53–56 (Table 2)
The compounds with a cyclohexylene instead of a phenylene
spacer retain some conformational flexibility. The cis- and
trans-configured amines VUF5803 (3) and VUF5804 (4), which
were used as building blocks, proved to be partial agonists (a:
0.7–0.9) in the [35S]GTPgS assay at both histamine receptor
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Arch. Pharm. Chem. Life Sci. 2011, 344, 775–785
Table 2. Potencies and efficacies of the synthesized cyanoguanidines 53–56 and the amines 3, 4 at the hH3R and hH4R in the [35S]GTPgS
assay.a
Compound
Config.
R
hH3R
hH4R
(EC50) or KB (nM)
a
N
(EC50) or KB (nM)
a
N
Histamine
VUF5803b (3)
(13 ꢂ 2)
(115 ꢂ 11)
(46 ꢂ 7)
1
0.87
3
2
2
2
3
2
2
(11 ꢂ 3)
(15 ꢂ 1)
1
5
2
2
2
2
2
3
cis
trans
trans
cis
trans
cis
0.88
0.92
ꢁ0.01
0.55
ꢁ0.02
0.32
VUF5804b (4)
0.71
ꢁ0.38
(300 ꢂ 20)
>10 000
53
54
55
56
-CH3
-CH3
-(CH2)2-S-Ph
-(CH2)2-S-Ph
>10 000
(>10 000)
180 ꢂ 16
1900 ꢂ 300
0.41
(1840 ꢂ 40)
ꢁ0.62
ꢁ0.86
188 ꢂ 5
(110 ꢂ 7)
aFunctional [35S]GTPgS binding assay with membrane preparations of Sf9 cells expressing the hH3R þ Gai2 þ Gb1g2 or the
hH4R þ Gai2 þ Gb1g2 was performed as described in the Section Pharmacology. N gives the number of independent experiments
performed in duplicate each. The intrinsic activity (a) of histamine was set to 1.00 and a values of other compounds were referred to
this value. a values of neutral antagonists and inverse agonists were determined at 10 mM. The KB values of neutral antagonists and
inverse agonists were determined in the antagonist mode versus histamine (100 nM) as the agonist. bPublished pKi values measured by
[3H]-histamine (H4) or [3H]-Na-methylhistamine (H3) binding to membranes of SK-N-MC cells expressing the human H4 or H3 receptor in
presence of the ligand: VUF5803: pKi (hH4R) 7.7, pKi (hH3R) 7.0; VUF5804: pKi (hH4R) 6.5, pKi (hH3R) 7.4 [34].
preference for hH3R and hH4R depends on the stereo-
chemistry with higher activities at the hH4R residing in
the cis-isomers. However the cyanoguanidines are less potent
partial agonists than the corresponding amines and show
some efficacy-selectivity depending on the configuration. At
the hH4R both cis-configured compounds (54, 56) are
hH4R partial agonists (a: 0.55 and 0.32) while the trans-iso-
mers (53, 55) are neutral antagonists. At the hH3R only the
methyl substituted cis-configured compound 54 is a weak
partial agonist, whereas the other three cyanoguanidines
act as inverse agonists with intrinsic activities from ꢁ0.38
to ꢁ0.86. The trans-isomer 55 is by a factor of 10 more potent
than the corresponding cis-diastereomer 56 at the hH3R.
Conformational constraints are, in principle, tolerated in
cyanoguanidine-type hH3R and hH4R ligands. The planar
geometry of a phenylene spacer proved to be inappropriate.
By contrast, conformationally constrained analogues of
cyanoguanidine-type hH4R agonists like UR-PI376 (1), in
which the flexible tetramethylene chain was replaced by a
cis-configured 1,4-cyclohexylene spacer, turned out to be
moderately potent and selective hH4R agonists. The same
holds for the building block, cis-4-(1H-imidazol-4-yl)cyclo-
hexylamine. The situation is less clear in case of the trans-
configured analogues, but there is a tendency toward
preference for the hH3R.
balance between rigidification and flexibility, regioisomers
and stereochemical properties to explore the three-dimen-
sional requirements of high hH4R affinity and selectivity.
Experimental
Chemistry
Commercial reagents and chemicals were purchased from Acros
Organics (Geel, Belgium), IRIS Biotech GmbH (Marktredwitz,
Germany), Alfa Aesar GmbH & Co. KG (Karlsruhe, Germany),
Merck KGaA (Darmstadt, Germany), Sigma-Aldrich Chemie
GmbH (Munich, Germany), TCI Europe (Zwijndrecht, Belgium)
and used without further purification. Deuterated solvents for
NMR spectroscopy were from Deutero GmbH (Kastellaun,
Germany). All solvents were of analytical grade or distilled prior
to use. If moisture-free conditions were required, reactions were
performed in dried glassware under inert atmosphere (argon or
nitrogen). Anhydrous DMF was purchased from Sigma-Aldrich
Chemie GmbH. Flash chromatography was performed on silica
gel (Merck silica gel 60, 40–63 mM). Reactions were monitored by
TLC (DCM/MeOH 90:10 v/v) on aluminum plates coated with silica
gel (Merck silica gel 60 F254, thickness 0.2 mm). The compounds
were detected by UV light (254 nm), a 0.3% solution of ninhydrine
in n-butanol (amines) or a 1.0% solution of Fast Blue B salt (imidazole
containing compounds) in EtOH/H2O 30:70 (v/v). All melting points
are uncorrected and were measured on a Bu¨chi 530 (Bu¨chi GmbH,
Essen, Germany) apparatus. Microwave assisted reactions were per-
formed on an Initiator 2.0 synthesizer (Biotage, Uppsala, Sweden).
Nuclear magnetic resonance spectra (1H-NMR and 13C-NMR)
were recorded with Bruker Avance 300 (1H: 300.1 MHz,
13C: 75.5 MHz) NMR spectrometers (Bruker BioSpin GmbH,
In conclusion, the results suggest the optimization of
imidazolylcycloalkylcyanoguanidines with regard to ring size,
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Arch. Pharm. Chem. Life Sci. 2011, 344, 775–785
hH4R Activity of Conformationally Restricted Cyanoguanidines
781
Rheinstetten, Germany). Chemical shifts are given in d (ppm)
relative to external standards. The multiplicity of carbon atoms
(
(s, 1H, Im-H-5), 7.74 (d, 2H, 3J ¼ 8.6 Hz, Ph-H), 7.74 (s, 1H, Im-H-2).
13C-NMR (75 MHz, CD3OD): d [ppm] ¼ 20.38 (þ, 2 CH3), 29.66
(þ, CH), 50.23 (ꢁ, CH2), 116.37 (þ, Im-C-5), 119.37 (Cquat, C N),
126.48 (þ, 2 Ph-C), 126.89 (þ, 2 Ph-C), 132.79 (Cquat, Ph-C), 136.74
(Cquat, Im-C-4), 137.29 (þ, Im-C-2), 139.37 (Cquat, Ph-C), 160.46
13C-NMR) was determined by DEPT 135 (distortionless enhance-
ment by polarization transfer). Mass spectra (MS) were recorded
on a Finnigan MAT 95 (EI-MS 70 eV, HR-MS), Finnigan SSQ 710A
(CI-MS (NH3)) and on a Finnigan ThermoQuest TSQ 7000 (ES-MS)
spectrometer. The peak-intensity in % relative to the strongest
signal is indicated in parenthesis. Elemental analyses (C, H, N,
Heraeus Elementar Vario EL III) were performed by the Analytical
Department of the University Regensburg and are within ꢂ0.4%
unless otherwise noted.
(Cquat, C N). HRMS (EI-MS) calcd. for C15H18N6 [Mþ.] 282.1593;
–
–
found 282.1592. Anal. (C15H18N6 ꢃ 0.4 CH3OH) C, H, N. C15H18N6
(282.34).
2-Cyano-1-[3-(1H-imidazol-4-yl)phenyl]-3-(3-
phenylpropyl)guanidine 38
Purity of tested compounds was >95% as determined by high-
performance liquid chromatography.
Compound 38 was prepared from 45 (0.076 g, 0.25 mmol) and 3-
phenylpropan-1-amine (0.036 mL, 0.25 mmol) in MeCN (4.5 mL)
according to the general procedure. Flash chromatography
General procedure for the synthesis of compounds 35–44
[38, 47]
The respective isourea 45 or 46 (1 eq) and the pertinent primary
amine 20–24 (1 eq) in MeCN were heated by microwave irradia-
tion at 1408C for 15 min. After removal of the solvent in vacuo, the
crude product was purified by flash chromatography (DCM/
MeOH 98:2 to 80:20 v/v).
yielded
a
white solid (0.07 g, 81%); mp 888C; 1H-NMR
(300 MHz, CD3OD): d [ppm] ¼ 1.86 (m, 2H, CH2CH2Ph), 2.63
(t, 2H, 3J ¼ 7.5 Hz, CH2Ph), 3.28 (t, 2H, 3J ¼ 7.2 Hz, NCH2),
7.11–7.29 (m, 7H, Ph-H), 7.44 (s, 1H, Im-H-5), 7.73 (d, 2H,
3J ¼ 8.6 Hz, Ph-H), 7.73 (s, 1H, Im-H-2). 13C-NMR (75 MHz,
CD3OD): d [ppm] ¼ 32.29 (ꢁ, CH2), 34.12 (ꢁ, CH2), 42.67 (ꢁ, CH2),
116.39 (þ, Im-C-5), 120.18 (Cquat, C N), 126.43 (þ, 2 Ph-C), 126.89
(þ, 2 Ph-C), 127.01 (þ, Ph-C), 129.45 (þ, 2 Ph-C), 129.50 (þ, 2 Ph-C),
132.80 (Cquat, Ph-C), 136.69 (Cquat, Im-C-4), 137.29 (þ, Im-C-2), 140.41
2-Cyano-1-[4-(1H-imidazol-4-yl)phenyl]-3-
methylguanidine 35
–
(Cquat, Ph-C), 142.90 (Cquat, Ph-C), 160.32 (Cquat, C N). HRMS (EI-MS)
–
calcd. for C20H20N6 [Mþ.] 344.1749; found 344.1745. Anal.
Compound 35 was prepared from 45 (0.09 g, 0.3 mmol) and a 33%
solution of methylamine in ethanol (0.037 mL, 0.3 mmol) in MeCN
(4.5 mL) according to the general procedure. Flash chromatography
(C20H20N6 ꢃ 0.5 H2O) C, H, N. C20H20N6 (344.41).
1
yielded a white solid (0.06 g, 83%); mp 1388C; H-NMR (300 MHz,
2-Cyano-1-[3-(1H-imidazol-4-yl)phenyl]-3-[2-
(phenylthio)ethyl]guanidine 39
CD3OD): d [ppm] ¼ 2.84 (s, 3H, CH3), 7.28 (d, 2H, 3J ¼ 8.5 Hz, Ph-H),
7.44 (s, 1H, Im-H-5), 7.72 (d, 2H, 3J ¼ 8.6 Hz, Ph-H), 7.76 (s, 1H, Im-H-
2). 13C-NMR (75 MHz, CD3OD): d [ppm] ¼ 29.00 (þ, CH3), 116.42
(þ, Im-C-5), 119.36 (Cquat, C N), 126.45 (þ, 2 Ph-C), 126.78 (þ, 2 Ph-C),
132.61 (Cquat, Ph-C), 136.77 (Cquat, Im-C-4), 137.27 (þ, Im-C-2),
Compound 39 was prepared from 45 (0.076 g, 0.25 mmol) and 2-
(phenylthio)ethanamine (0.038 g, 0.25 mmol) in MeCN (4.5 mL)
according to the general procedure. Flash chromatography
yielded
a
white solid (0.08 g, 88%); mp 1408C; 1H-NMR
–
139.30 (Cquat, Ph-C), 161.01 (Cquat, C N). HRMS (EI-MS) calcd.
(300 MHz, CD3OD): d [ppm] ¼ 3.10 (t, 2H, 3J ¼ 7.3 Hz, CH2),
3.44 (t, 2H, 3J ¼ 7.3 Hz, CH2), 7.20 (m, 3H, Ph-H), 7.29 (m, 2H,
Ph-H), 7.38 (m, 2H, Ph-H), 7.45 (s, 1H, Im-H-5), 7.74 (d, 2H,
3J ¼ 8.5 Hz, Ph-H), 7.74 (s, 1H, Im-H-2). 13C-NMR (75 MHz,
CD3OD): d [ppm] ¼ 33.32 (ꢁ, SCH2), 42.29 (ꢁ, CH2), 116.45 (þ,
Im-C-5), 120.31 (Cquat, C N), 126.77 (þ, 2 Ph-C), 126.98 (þ, 2 Ph-C),
127.29 (þ, Ph-C), 130.16 (þ, 2 Ph-C), 130.31 (þ, 2 Ph-C), 133.13
(Cquat, Ph-C), 136.30 (Cquat, Im-C-4), 136.95 (Cquat, Ph-C), 137.33 (þ,
–
for C12H12N6 [Mþ.] 240.1123; found 240.1120. Anal.
(C12H12N6 ꢃ 0.4 CH3OH ꢃ 0.6 H2O) C, H, N. C12H12N6 (240.26).
2-Cyano-3-cyclopropyl-1-[4-(1H-imidazol-4-yl)-
phenyl]guanidine 36
Compound 36 was prepared from 45 (0.09 g, 0.3 mmol) and cyclo-
propylamine (0.021 mL, 0.3 mmol) in MeCN (4.5 mL) according to
the general procedure. Flash chromatography yielded a white solid
(0.05 g, 62%); mp 2158C (dec.); 1H-NMR (300 MHz, CD3OD): d
[ppm] ¼ 0.70 (m, 2H, CH2), 0.87 (m, 2H, CH2), 2.67 (m, 1H, CH),
7.36 (d, 2H, 3J ¼ 8.6 Hz, Ph-H), 7.42 (s, 1H, Im-H-5), 7.69 (d, 2H,
3J ¼ 8.6 Hz, Ph-H), 7.73 (s, 1H, Im-H-2). 13C-NMR (75 MHz, CD3OD):
d [ppm] ¼ 8.33 (ꢁ, 2 CH2), 24.47 (þ, CH), 116.45 (þ, Im-C-5), 118.94
(Cquat, C N), 125.56 (þ, 2 Ph-C), 126.30 (þ, 2 Ph-C), 131.84 (Cquat, Ph-C),
–
Im-C-2), 139.89 (Cquat, Ph-C), 160.32 (Cquat, C N). HRMS (EI-MS)
–
calcd. for C19H18N6S [Mþ.] 362.1314; found 362.1306. Anal.
(C19H18N6S ꢃ 0.1 CH3OH) C, H, N. C19H18N6S (362.45).
2-Cyano-1-[3-(1H-imidazol-4-yl)phenyl]-3-
methylguanidine 40
Compound 40 was prepared from 46 (0.09 g, 0.3 mmol) and a
33% solution of methylamine in ethanol (0.037 mL, 0.3 mmol) in
MeCN (4.5 mL) according to the general procedure. Flash
chromatography yielded a white solid (0.015 g, 21%); mp
978C; 1H-NMR (300 MHz, CD3OD): d [ppm] ¼ 2.84 (s, 3H, CH3),
7.15 (d, 1H, 3J ¼ 8.5 Hz, Ph-H), 7.40 (t, 1H, 3J ¼ 8.1 Hz, Ph-H), 7.48
(s, 1H, Im-H-5), 7.58–7.61 (m, 2H, Ph-H), 7.74 (s, 1H, Im-H-2). 13C-
NMR (75 MHz, CD3OD): d [ppm] ¼ 28.97 (þ, CH3), 116.46 (þ, Im-C-
5), 119.32 (Cquat, C N), 122.53 (þ, Ph-C), 123.89 (þ, Ph-C), 124.56
(þ, Ph-C), 124.11 (Cquat, Ph-C), 130.85 (þ, Ph-C), 135.81 (Cquat, Im-C-
137.17 (Cquat, Im-C-4), 137.27 (þ, Im-C-2), 139.25 (Cquat, Ph-C), 161.43
þ.
–
(Cquat, C N). HRMS (EI-MS) calcd. for C H N [M ] 266.1280; found
14 14
–
6
266.1280. Anal. (C14H14N6 ꢃ 0.6 CH3OH) C, H, N. C14H14N6 (266.30).
2-Cyano-1-[3-(1H-imidazol-4-yl)phenyl]-3-
isobutylguanidine 37
Compound 37 was prepared from 45 (0.09 g, 0.3 mmol) and
isobutylamine (0.03 mL, 0.3 mmol) in MeCN (4.5 mL) according
to the general procedure. Flash chromatography yielded a white
solid (0.08 g, 94%); mp 1038C; 1H-NMR (300 MHz, CD3OD): d
[ppm] ¼ 0.91 (d, 6H, 3J ¼ 6.7 Hz, 2 CH3), 1.88 (m, 1H, CH), 3.08
–
4), 137.35 (þ, Im-C-2), 138.64 (Cquat, Ph-C), 161.03 (Cquat, C N).
–
HRMS (EI-MS) calcd. for C12H12N6 [Mþ.] 240.1123; found 240.1120.
3
3
(d, 2H, J ¼ 7.1 Hz, CH2CH), 7.27 (d, 2H, J ¼ 8.6 Hz, Ph-H), 7.45
Anal. (C12H12N6 ꢃ 0.5 CH3OH ꢃ 0.3 H2O) C, H, N. C12H12N6 (240.26).
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

782
R. Geyer and A. Buschauer
Arch. Pharm. Chem. Life Sci. 2011, 344, 775–785
(300 MHz, CD3OD): d [ppm] ¼ 3.11 (t, 2H, 3J ¼ 7.3 Hz, CH2),
3.44 (t, 2H, 3J ¼ 7.3 Hz, CH2), 7.08–7.19 (m, 2H, Ph-H), 7.27
(m, 2H, Ph-H), 7.39 (m, 3H, Ph-H), 7.48 (s, 1H, Im-H-5), 7.61
(m, 2H, Ph-H), 7.75 (s, 1H, Im-H-2). 13C-NMR (75 MHz, CD3OD): d
[ppm] ¼ 33.26 (ꢁ, SCH2), 42.21 (ꢁ, CH2), 116.75 (þ, Im-C-5), 120.21
(Cquat, C N), 122.76 (þ, Ph-C), 124.18 (þ, Ph-C), 124.71 (þ, Ph-C),
127.26 (þ, Ph-C), 130.14 (þ, 2 Ph-C), 130.28 (þ, 2 Ph-C), 131.08 (þ,
Ph-C), 133.43 (Cquat, Ph-C), 136.91 (Cquat, Ph-C), 137.04 (Cquat, Im-C-
2-Cyano-3-cyclopropyl-1-[3-(1H-imidazol-4-yl)-
phenyl]guanidine 41
Compound 41 was prepared from 46 (0.09 g, 0.3 mmol) and
cyclopropylamine (0.021 mL, 0.3 mmol) in MeCN (4.5 mL)
according to the general procedure. Flash chromatography
yielded
a
white solid (0.05 g, 63%); mp 1378C; 1H-NMR
(300 MHz, CD3OD): d [ppm] ¼ 0.71 (m, 2H, CH2), 0.87 (m,
2H, CH2), 2.68 (m, 1H, CH), 7.23 (d, 1H, 3J ¼ 8.0 Hz, Ph-H), 7.35
(t, 1H, 3J ¼ 8.0 Hz, Ph-H), 7.44 (s, 1H, Im-H-5), 7.54 (d, 1H,
3J ¼ 7.8 Hz, Ph-H), 7.66 (s, 1H, Ph-H), 7.73 (s, 1H, Im-H-2). 13C-
NMR (75 MHz, CD3OD): d [ppm] ¼ 8.31 (ꢁ, 2 CH2), 24.50 (þ,
CH), 116.41 (þ, Im-C-5), 118.94 (Cquat, C N), 121.83 (þ, Ph-C),
123.24 (þ, Ph-C), 123.94 (þ, Ph-C), 130.33 (þ, Ph-C), 133.80
(Cquat, Ph-C), 137.07 (Cquat, Im-C-4), 137.29 (þ, Im-C-2), 139.03
–
4), 137.40 (þ, Im-C-2), 138.31 (Cquat, Ph-C), 160.30 (Cquat, C N).
–
HRMS (EI-MS) calcd. for C19H18N6S [Mþ.] 362.1314; found
362.1309. C19H18N6S (362.45).
General procedure for the synthesis of compounds 30–34
and 53–56 [38, 47]
–
, Ph-C), 161.58 (Cquat, C N). HRMS (LSI-MS) calcd.
–
(Cquat
Hydrochlorides of 3, 4 and 18 were converted into the bases by
passing a basic ion exchanger (Merck, ion exchanger III, mobile
phase: MeOH). The respective isourea (1 eq) and the pertinent
amine (1 eq) in MeCN were heated by microwave irradiation at
1408C for 15 min. After removal of the solvent in vacuo, the crude
product was purified by flash chromatography (DCM/MeOH 98:2
to 80:20 v/v).
for C14H15N6 [MHþ] 267.1358; found 267.1357. Anal.
(C14H14N6 ꢃ 0.9 CH3OH) C, H, N. C14H14N6 (266.30).
2-Cyano-1-[3-(1H-imidazol-4-yl)phenyl]-3-
isobutylguanidine 42
Compound 42 was prepared from 46 (0.09 g, 0.3 mmol) and
isobutylamine (0.03 mL, 0.3 mmol) in MeCN (4.5 mL) according
to the general procedure. Flash chromatography yielded a white
solid (0.06 g, 71%); mp 958C; 1H-NMR (300 MHz, CD3OD): d
[ppm] ¼ 0.91 (d, 6H, 3J ¼ 6.7 Hz, CH3), 1.88 (m, 1H, CH), 3.08
(d, 2H, 3J ¼ 7.1 Hz, CH2CH), 7.14 (d, 1H, 3J ¼ 7.8 Hz, Ph-H),
7.40 (t, 1H, 3J ¼ 7.7 Hz, Ph-H), 7.47 (s, 1H, Im-H-5), 7.60 (m, 2H,
2-Cyano-1-[3-(1H-imidazol-4-yl)benzyl]-3-
methylguanidine 30
Compound 30 was prepared from 18 (0.05 g, 0.3 mmol) and 25
(0.051 g, 0.3 mmol) in MeCN (4.5 mL) according to the general
procedure. Flash chromatography yielded a yellow solid (0.06 g,
82%); mp 778C; 1H-NMR (300 MHz, CD3OD): d [ppm] ¼ 2.82
(s, 3H, CH3), 4.44 (s, 2H, CH2), 7.18 (d, 1H, 3J ¼ 7.6 Hz, Ph-H),
7.34 (t, 1H, 3J ¼ 7.7 Hz, Ph-H), 7.43 (s, 1H, Im-H-5), 7.60 (d, 1H,
3J ¼ 7.8 Hz, Ph-H), 7.64 (s, 1H, Ph-H), 7.75 (s, 1H, Im-H-2). 13C-NMR
(75 MHz, CD3OD): d [ppm] ¼ 28.82 (þ, CH3), 46.04 (ꢁ, CH2), 116.45
(þ, Im-C-5), 121.02 (Cquat, C N), 124.76 (þ, Ph-C), 124.94 (þ, Ph-C),
126.79 (þ, Ph-C), 127.33 (Cquat, Ph-C), 130.03 (þ, Ph-C), 133.42
(Cquat, Im-C-4), 134.75 (þ, Im-C-2), 140.29 (Cquat, Ph-C), 162.13
Ph-H), 7.74 (s, 1H, Im-H-2). 13C-NMR (75 MHz, CD3OD):
d
[ppm] ¼ 20.39 (þ, 2 CH3), 29.64 (þ, CH), 50.24 (ꢁ, CH2), 116.58
(þ, Im-C-5), 119.38 (Cquat, C N), 122.53 (þ, Ph-C), 123.91 (þ, Ph-C),
124.50 (þ, Ph-C), 130.94 (þ, Ph-C), 130.28 (Cquat, Ph-C), 136.22
(Cquat, Im-C-4), 137.37 (þ, Im-C-2), 138.68 (Cquat, Ph-C), 160.47
C N). HRMS (LSI-MS) calcd. for C15H19N6 [MHþ]
–
,
–
(Cquat
283.1671; found 283.1674. Anal. (C15H18N6 ꢃ 0.75 CH3OH) C, H,
N. C15H18N6 (282.34).
(Cquat, C N). HRMS (EI-MS) calcd. for C13H14N6 [Mþ.] 254.1280;
–
–
found 254.1281. Anal. (C13H14N6 ꢃ 0.75 CH3OH) C, H, N. C13H14N6
2-Cyano-1-[3-(1H-imidazol-4-yl)phenyl]-3-(3-
phenylpropyl)guanidine 43
(254.29).
Compound 43 was prepared from 46 (0.076 g, 0.25 mmol) and 3-
phenylpropan-1-amine (0.036 mL, 0.25 mmol) in MeCN (4.5 mL)
according to the general procedure. Flash chromatography
yielded a white solid (0.07 g, 81%); mp 788C; 1H-NMR (300 MHz,
2-Cyano-3-cyclopropyl-1-[3-(1H-imidazol-4-
yl)benzyl]guanidine 31
Compound 31 was prepared from 18 (0.05 g, 0.3 mmol) and 26
(0.058 g, 0.3 mmol) in MeCN (4.5 mL) according to the general
procedure. Flash chromatography yielded a yellow solid (0.07 g,
CD3OD):
d
[ppm] ¼ 1.86 (m, 2H, CH2CH2Ph), 2.63 (t, 2H,
3J ¼ 7.9 Hz, CH2Ph), 3.29 (t, 2H, 3J ¼ 7.2 Hz, NCH2), 7.09–7.26
(m, 6H, Ph-H), 7.40 (t, 1H, 3J ¼ 8.2 Hz, Ph-H), 7.46 (s, 1H, Im-H-5),
7.59 (m, 2H, Ph-H), 7.74 (s, 1H, Im-H-2). 13C-NMR (75 MHz, CD3OD): d
[ppm] ¼ 32.27 (ꢁ, CH2), 34.11 (ꢁ, CH2), 42.68 (ꢁ, CH2), 116.43 (þ, Im-
C-5), 119.39 (Cquat, C N), 122.51 (þ, Ph-C), 123.92 (þ, Ph-C), 124.49 (þ,
Ph-C), 126.99 (þ, Ph-C), 129.44 (þ, 2 Ph-C), 129.47 (þ, 2 Ph-C), 130.96
(þ, Ph-C), 134.78 (Cquat, Im-C-4), 136.20 (Cquat, Ph-C), 137.37 (þ, Im-C-
87%); mp 628C; 1H-NMR (300 MHz, CD3OD):
d
(m, 2H, CH2), 0.82 (m, 2H, CH2), 2.52 (m, 1H, CH), 4.49
[ppm] ¼ 0.63
(s, 2H, CH2), 7.19 (d, 1H, 3J ¼ 7.6 Hz, Ph-H), 7.34 (t, 1H,
3J ¼ 7.6 Hz, Ph-H), 7.44 (s, 1H, Im-H-5), 7.59 (d, 1H, J ¼ 7.7 Hz, Ph-
3
H), 7.64 (s, 1H, Ph-H), 7.76 (s, 1H, Im-H-2). 13C-NMR (75 MHz, CD3OD): d
[ppm] ¼ 8.16 (ꢁ, 2 CH2), 23.87 (þ, CH), 46.01 (ꢁ, CH2), 116.77 (þ, Im-C-
5), 120.61 (Cquat, C N), 124.84 (þ, 2 Ph-C), 126.88 (þ, Ph-C), 126.99
(Cquat, Ph-C), 129.99 (þ, Ph-C), 134.56 (Cquat, Im-C-4), 134.62 (þ, Im-C-2),
–
–
–
2), 138.64 (Cquat, Ph-C), 142.90 (Cquat, Ph-C), 160.25 (Cquat, C N).
HRMS (EI-MS) calcd. for C20H20N6 [Mþ.] 344.1749; found
344.1740. Anal. (C20H20N6 ꢃ 0.6 CH3OH) C, H, N. C20H20N6 (344.41).
140.67 (Cquat, Ph-C), 162.64 (Cquat, C N). HRMS (EI-MS) calcd.
–
for C15H16N6 [Mþ.] 280.1436; found 280.1435. Anal.
(C15H16N6 ꢃ 1.5 CH3OH) C, H, N. C15H16N6 (280.33).
2-Cyano-1-[3-(1H-imidazol-4-yl)phenyl]-3-[2-
(phenylthio)ethyl]guanidine 44
2-Cyano-1-[3-(1H-imidazol-4-yl)benzyl]-3-
isobutylguanidine 32
Compound 32 was prepared from 18 (0.05 g, 0.3 mmol) and 27
(0.063 g, 0.3 mmol) in MeCN (4.5 mL) according to the general
Compound 44 was prepared from 46 (0.076 g, 0.25 mmol) and 2-
(phenylthio)ethanamine (0.038 g, 0.25 mmol) in MeCN (4.5 mL)
according to the general procedure. Flash chromatography
yielded
a
white solid (0.05 g, 55%); mp 518C; 1H-NMR
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2011, 344, 775–785
hH4R Activity of Conformationally Restricted Cyanoguanidines
783
procedure. Flash chromatography yielded a yellow solid (0.07 g,
82%); mp 758C; 1H-NMR (300 MHz, CD3OD): d [ppm] ¼ 0.84 (d, 6H,
3J ¼ 6.7 Hz, CH3), 1.80 (m, 1H, CH), 3.02 (d, 2H,
3J ¼ 7.1 Hz, CH2CH), 4.46 (s, 2H, PhCH2), 7.19 (d, 1H,
3J ¼ 7.6 Hz, Ph-H), 7.35 (t, 1H, 3J ¼ 7.6 Hz, Ph-H), 7.42 (s, 1H,
Im-H-5), 7.61 (d, 1H, 3J ¼ 7.9 Hz, Ph-H), 7.64 (s, 1H, Ph-H), 7.74
(s, 1H, Im-H-2). 13C-NMR (75 MHz, CD3OD): d [ppm] ¼ 20.22 (þ, 2
CH3), 29.56 (þ, CH), 46.04 (ꢁ, CH2), 50.11 (ꢁ, CH2), 115.39 (þ, Im-C-
5), 120.70 (Cquat, C N), 124.74 (þ, Ph-C), 124.98 (þ, Ph-C), 126.67
(þ, Ph-C), 127.99 (Cquat, Ph-C), 130.09 (þ, Ph-C), 134.86 (þ, Im-C-2),
32.84 (ꢁ, 2 CH2), 33.63 (ꢁ, 2 CH2), 36.68 (þ, CH-Im), 52.08 (þ,
CH-N), 115.93 (þ, Im-C-5), 120.31 (Cquat, C N), 135.68 (þ, Im-C-2),
for C12H18N6 [Mþ.
–
143.15 (Cquat, Im-C-4), 161.23 (Cquat, C N). HRMS (EI-MS) calcd.
]
–
246.1593; found 246.1592. Anal.
(C12H18N6 ꢃ 0.65 H2O) C, H, N. C12H18N6 (246.31).
2-Cyano-1-[cis-4-(1H-imidazol-4-yl)cyclohexyl]-3-
methylguanidine 54
Compound 54 was prepared from 3 (0.08 g, 0.48 mmol) and 25
(0.085 g, 0.48 mmol) in MeCN (4.5 mL) according to the general
procedure. Flash chromatography yielded a white solid (0.09 g,
76%); mp 125–1278C; 1H-NMR (300 MHz, CD3OD): d [ppm] ¼ 1.71
(m, 4H, CH2), 1.85 (m, 2H, CH2), 1.95 (m, 2H, CH2), 2.79 (s, 3H, CH3-
N), 2.84 (s, 1H, CH-Im), 3.76 (m, 1H, CH-N), 6.86 (s, 1H, Im-H-5), 7.59
(s, 1H, Im-H-2). 13C-NMR (75 MHz, CD3OD): d [ppm] ¼ 28.79
(þ, CH3), 28.84 (ꢁ, 2 CH2), 29.96 (ꢁ, 2 CH2), 34.05 (þ, CH-Im),
50.31 (þ, CH-N), 117.56 (þ, Im-C-5), 120.31 (Cquat, C N), 135.75
–
135.24 (Cquat, Im-C-4), 140.08 (Cquat, Ph-C), 161.42 (Cquat, C N).
–
HRMS (EI-MS) calcd. for C16H20N6 [Mþ.] 296.1749; found 296.1749.
Anal. (C16H20N6) C, H, N. C16H20N6 (296.37).
2-Cyano-1-[3-(1H-imidazol-4-yl)benzyl]-3-(3-
phenylpropyl)guanidine 33
Compound 33 was prepared from 18 (0.04 g, 0.23 mmol) and 28
(0.065 g, 0.23 mmol) in MeCN (4.5 mL) according to the general
procedure. Flash chromatography yielded a yellow solid (0.065 g,
79%); mp 598C; 1H-NMR (300 MHz, CD3OD): d [ppm] ¼ 1.80
(m, 2H, CH2CH2Ph), 2.52 (t, 2H, 3J ¼ 7.5 Hz, CH2Ph), 3.22
(t, 2H, 3J ¼ 7.0 Hz, NCH2), 4.44 (s, 2H, PhCH2N), 7.08 (m, 3H,
Ph-H), 7.19 (m, 3H, Ph-H), 7.35 (t, 1H, 3J ¼ 7.7 Hz, Ph-H), 7.42
(s, 1H, Im-H-5), 7.61 (d, 1H, 3J ¼ 7.7 Hz, Ph-H), 7.66 (s, 1H, Ph-H),
7.73 (s, 1H, Im-H-2). 13C-NMR (75 MHz, CD3OD): d [ppm] ¼ 32.23
(ꢁ, CH2), 33.81 (ꢁ, CH2), 42.35 (ꢁ, CH2), 46.03 (ꢁ, CH2), 115.44
(þ, Im-C-5), 120.10 (Cquat, C N), 124.71 (þ, Ph-C), 125.02 (þ, Ph-C),
126.70 (þ, Ph-C), 126.94 (þ, Ph-C), 127.44 (Cquat, Ph-C), 129.43 (þ, 4
Ph-C), 130.13 (þ, Ph-C), 134.90 (þ, Im-C-2), 136.12 (Cquat, Im-C-4),
–
(þ, Im-C-2), 141.54 (Cquat, Im-C-4), 161.20 (Cquat, C N). HRMS (EI-
–
MS) calcd. for C12H18N6 [Mþ.] 246.1593; found 246.1593. Anal.
(C12H18N6 ꢃ 0.55 H2O) C, H, N. C12H18N6 (246.31).
2-Cyano-1-[trans-4-(1H-imidazol-4-yl)cyclohexyl]-3-[2-
(phenylthio)ethyl]guanidine 55
Compound 55 was prepared from 4 (0.08 g, 0.48 mmol) and 29
(0.144 g, 0.48 mmol) in MeCN (4.5 mL) according to the general
procedure. Flash chromatography yielded a white solid (0.12 g,
68%); mp 95–968C; 1H-NMR (300 MHz, CD3OD): d [ppm] ¼ 1.45 (m,
4H, CH2), 2.03 (m, 4H, CH2), 2.54 (m, 1H, CH-N), 3.11 (t, 2H,
3J ¼ 6.9 Hz, CH2-N), 3.42 (t, 2H, 3J ¼ 6.9 Hz, CH2-S), 3.45
(m, 1H, CH-Im), 6.75 (s, 1H, Im-H-5), 7.19 (m, 1H, Ph-H-4), 7.30
(m, 2H, Ph-H), 7.40 (m, 2H, Ph-H), 7.55 (s, 1H, Im-H-2). 13C-NMR
(75 MHz, CD3OD): d [ppm] ¼ 32.68 (ꢁ, 2 CH2), 33.58 (ꢁ, 2
CH2 þ CH2-S), 36.59 (þ, CH-Im), 42.31 (ꢁ, CH2-N), 52.12 (þ, CH-
N), 115.13 (þ, Im-C-5), 119.97 (Cquat, C N), 127.34 (þ, Ph-C-4),
130.18 (þ, 2 Ph-C), 130.44 (þ, 2 Ph-C), 135.35 (Cquat, Im-C-4),
–
140.06 (Cquat, Ph-C), 142.78 (Cquat, Ph-C), 161.35 (Cquat, C N). HRMS
–
(EI-MS) calcd. for C21H22N6 [Mþ.] 358.1906; found 358.1901. Anal.
(C21H22N6 ꢃ 0.3 CH3OH) C, H, N. C21H22N6 (358.44).
2-Cyano-1-[3-(1H-imidazol-4-yl)benzyl]-3-[2-
(phenylthio)ethyl]guanidine 34
–
135.70 (þ, Im-C-2), 137.01 (Cquat, Ph-C-1), 160.28 (Cquat, C N).
–
Compound 34 was prepared from 18 (0.04 g, 0.23 mmol) and 29
(0.069 g, 0.23 mmol) in MeCN (4.5 mL) according to the general
procedure. Flash chromatography yielded a yellow solid (0.06 g,
70%); mp 648C; 1H-NMR (300 MHz, CD3OD): d [ppm] ¼ 3.05 (t, 2H,
3J ¼ 7.2 Hz, CH2), 3.40 (t, 2H, 3J ¼ 7.3 Hz, CH2), 4.40 (s, 2H,
PhCH2N), 7.12–7.18 (m, 2H, Ph-H), 7.24 (m, 2H, Ph-H), 7.31–7.37
(m, 3H, Ph-H), 7.43 (s, 1H, Im-H-5), 7.62 (m, 2H, Ph-H), 7.73 (s, 1H,
Im-H-2). 13C-NMR (75 MHz, CD3OD): d [ppm] ¼ 33.63 (ꢁ, SCH2),
HRMS (EI-MS) calcd. for C19H24N6S [Mþ.] 368.1783; found
368.1781. Anal. (C14H24N6S ꢃ 0.25 H2O) C, H, N. C14H24N6S (368.50).
2-Cyano-1-[cis-4-(1H-imidazol-4-yl)cyclohexyl]-3-[2-
(phenylthio)ethyl]guanidine 56
Compound 56 was prepared from 3 (0.08 g, 0.48 mmol) and 29
(0.144 g, 0.48 mmol) in MeCN (4.5 mL) according to the general
procedure. Flash chromatography yielded a white solid (0.13 g,
73%); mp 88–908C; 1H-NMR (300 MHz, CD3OD): d [ppm] ¼ 1.70
(m, 4H, CH2), 1.85 (m, 4H, CH2), 2.80 (m, 1H, CH-N), 3.10 (t, 2H,
3J ¼ 6.8 Hz, CH2-N), 3.43 (t, 2H, 3J ¼ 6.8 Hz, CH2-S), 3.69 (s, 1H, CH-
Im), 6.84 (s, 1H, Im-H-5), 7.17 (m, 1H, Ph-H-4), 7.28 (m, 2H, Ph-H),
7.39 (m, 2H, Ph-H), 7.58 (s, 1H, Im-H-2). 13C-NMR (75 MHz, CD3OD):
d [ppm] ¼ 28.64 (ꢁ, 2 CH2), 30.03 (ꢁ, 2 CH2), 33.70 (ꢁ, CH2-S),
34.37 (þ, CH-Im), 42.33 (ꢁ, CH2-N), 49.91 (þ, CH–N), 117.19 (þ, Im-
C-5), 120.04 (Cquat, C N), 127.35 (þ, Ph-C-4), 130.17 (þ, 2 Ph-C),
130.42 (þ, 2 Ph-C), 135.77 (þ, Im-C-2), 136.97 (Cquat, Ph-C-1), 141.92
42.16 (ꢁ, CH2), 46.17 (ꢁ, CH2), 118.36 (þ, Im-C-5), 121.36 (Cquat
,
C N), 124.73 (þ, Ph-C), 125.08 (þ, Ph-C), 126.68 (þ, Ph-C), 127.39
(þ, Ph-C), 127.39 (Cquat, Ph-C), 130.14 (þ, 3 Ph-C), 130.60 (þ, 2 Ph-C),
136.74 (Cquat, Ph-C), 137.21 (þ, Im-C-2), 137.26 (Cquat, Im-C-4),
–
139.65 (Cquat, Ph-C), 161.29 (Cquat, C N). HRMS (EI-MS) calcd.
–
for C20H20N6S [Mþ.] 376.1470; found 376.1467. Anal.
(C20H20N6S ꢃ 0.5 CH3OH ꢃ 0.8 H2O) C, H, N. C20H20N6S (376.48).
2-Cyano-1-[trans-4-(1H-imidazol-4-yl)cyclohexyl]-3-
methylguanidine 53
–
, Im-C-4), 160.32 (Cquat, C N). HRMS (EI-MS) calcd.
–
(Cquat
Compound 53 was prepared from 4 (0.08 g, 0.48 mmol) and 25
(0.085 g, 0.48 mmol) in MeCN (4.5 mL) according to the general
procedure. Flash chromatography yielded a white solid (0.11 g,
93%); mp 120–1218C; 1H-NMR (300 MHz, CD3OD): d [ppm] ¼ 1.47
(m, 4H, CH2), 2.05 (m, 4H, CH2), 2.55 (m, 1H, CH-Im), 2.80 (s,
3H, CH3-N), 3.59 (m, 1H, CH-N), 6.76 (s, 1H, Im-H-5), 7.55 (s, 1H,
Im-H-2). 13C-NMR (75 MHz, CD3OD): d [ppm] ¼ 28.78 (þ, CH3),
for C19H24N6S [Mþ.] 368.1783; found 368.1777. Anal.
(C14H24N6S ꢃ 0.25 H2O) C, H, N. C14H24N6S (368.50).
Pharmacology
Histamine dihydrochloride was purchased from Alfa Aesar
GmbH & Co. KG (Karlsruhe, Germany). Thioperamide hydrochlo-
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

784
R. Geyer and A. Buschauer
Arch. Pharm. Chem. Life Sci. 2011, 344, 775–785
ride was synthesized according to a previously described method
[48]. Guanosine diphosphate (GDP) was from Sigma-Aldrich
Chemie GmbH (Munich, Germany), unlabeled GTPgS was from
Roche (Mannheim, Germany). [35S]GTPgS was from PerkinElmer
Life Sciences (Boston, MA). GF/C filters were from Whatman
(Maidstone, UK).
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[
35S]GTPgS binding assays were performed as previously
described for the H3R [49, 50] and H4R [51]. H3R assays: Sf9
insect cell membranes coexpressing the hH3R, mammalian
Gai2 and Gb1g2 were employed, H4R assays: Sf9 insect cell mem-
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The respective membranes were thawed, sedimented by a 10-
min centrifugation at 48C and 13 000 ꢄ g. Membranes were
resuspended in binding buffer (12.5 mM MgCl2, 1 mM EDTA,
and 75 mM Tris/HCl, pH 7.4). Each assay tube contained Sf9
membranes expressing the respective HR subtype (15–30 mg
protein/tube), 1 mM GDP, 0.05% (w/v) bovine serum albumin,
0.2 nM [35S]GTPgS and the investigated ligands (dissolved in a
mixture (v/v) of 80% millipore water and 20% DMSO) at concen-
trations of 10 nM to 100 mM in binding buffer (total volume
250 mL). All H4R assays additionally contained 100 mM NaCl.
For the determination of KB values at hH3R and
hH4R (antagonist mode of the [35S]GTPgS binding assay) hista-
mine was added to the reaction mixtures (final concentration:
100 nM). IC50 values were converted to KB values using the Cheng-
Prussoff equation [52]. Incubations were conducted for 90 min at
258C and shaking at 250 rpm. Bound [35S]GTPgS was separated
from free [35S]GTPgS by filtration through GF/C filters, followed
by three washes with 2 mL of binding buffer (48C) using a
Brandel Harvester. Filter-bound radioactivity was determined
after an equilibration phase of at least 12 h by liquid scintillation
counting. The experimental conditions chosen ensured that no
more than 10% of the total amount of [35S]GTPgS added was
bound to filters. Non-specific binding was determined in the
presence of 10 mM unlabeled GTPgS.
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The authors are grateful to Mrs. Maria Beer-Kro¨n, Mrs. Gertraud Wilberg
and Mrs. Sabine Dirrigl for expert technical assistance. This work was
supported by the Graduate Training Program (Graduiertenkolleg) GRK
760, ‘‘Medicinal Chemistry: Molecular Recognition - Ligand-Receptor
Interactions’’, of the Deutsche Forschungsgemeinschaft and by ESF COST
Action BM0806, ‘Recent advances in histamine receptor H4R research’.
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The authors have declared no conflict of interest.
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