Conformational Restriction and Enantioseparation Increase Potency and Selectivity of Cyanoguanidine-Type Histamine H4 Receptor Agonists

Article abstract of DOI:10.1021/acs.jmedchem.6b00120

2-Cyano-1-[4-(1H-imidazol-4-yl)butyl]-3-[2-(phenylsulfanyl)ethyl]guanidine (UR-PI376, 1) is a potent and selective agonist of the human histamine H₄ receptor (hH₄R). To gain information on the active conformation, we synthesized analogues of 1 with a cyclopentane-1,3-diyl linker. Affinities and functional activities were determined at recombinant hH_xR (x: 1-4) subtypes on Sf9 cell membranes (radioligand binding, [³⁵S]GTPγS, or GTPase assays) and in part in luciferase assays on human or mouse H₄R (HEK-293 cells). The most potent H₄R agonists among 14 racemates were separated by chiral HPLC, yielding eight enantiomerically pure compounds. Configurations were assigned based on X-ray structures of intermediates and a stereocontrolled synthetic pathway. (+)-2-Cyano-1-{[trans-(1S,3S)-3-(1H-imidazol-4-yl)cyclopentyl]methyl}-3-[2-(phenylsulfanyl)ethyl]guanidine ((1S,3S)-UR-RG98, 39a) was the most potent H₄R agonist in this series (EC₅₀ 11 nM; H₄R vs H₃R, >100-fold selectivity; H₁R, H₂R, negligible activities), whereas the optical antipode proved to be an H₄R antagonist ([³⁵S]GTPγS assay). MD simulations confirmed differential stabilization of the active and inactive H₄R state by the enantiomers.

Full text of DOI:10.1021/acs.jmedchem.6b00120

Article
pubs.acs.org/jmc
Conformational Restriction and Enantioseparation Increase Potency
and Selectivity of Cyanoguanidine-Type Histamine H4 Receptor
Agonists
Roland Geyer, Uwe Nordemann, Andrea Strasser, Hans-Joachim Wittmann, and Armin Buschauer*
Institute of Pharmacy, Pharmaceutical/Medicinal Chemistry, Faculty of Chemistry and Pharmacy, University of Regensburg,
Universitatsstraße 31, D-93053 Regensburg, Germany
̈
S
* Supporting Information
ABSTRACT: 2-Cyano-1-[4-(1H-imidazol-4-yl)butyl]-3-[2-
(phenylsulfanyl)ethyl]guanidine (UR-PI376, 1) is a potent and
selective agonist of the human histamine H4 receptor (hH4R).
To gain information on the active conformation, we
synthesized analogues of 1 with a cyclopentane-1,3-diyl linker.
Affinities and functional activities were determined at
recombinant hHxR (x: 1−4) subtypes on Sf9 cell membranes
(radioligand binding, [35S]GTPγS, or GTPase assays) and in
part in luciferase assays on human or mouse H4R (HEK-293
cells). The most potent H4R agonists among 14 racemates
were separated by chiral HPLC, yielding eight enantiomerically
pure compounds. Configurations were assigned based on X-ray
structures of intermediates and a stereocontrolled synthetic
pathway. (+)-2-Cyano-1-{[trans-(1S,3S)-3-(1H-imidazol-4-yl)cyclopentyl]methyl}-3-[2-(phenylsulfanyl)ethyl]guanidine
((1S,3S)-UR-RG98, 39a) was the most potent H4R agonist in this series (EC50 11 nM; H4R vs H3R, >100-fold selectivity;
H1R, H2R, negligible activities), whereas the optical antipode proved to be an H4R antagonist ([35S]GTPγS assay). MD
simulations confirmed differential stabilization of the active and inactive H4R state by the enantiomers.
imifuramine (3),40,41 1-{[(2R,5R)-5-(1H-imidazol-4-yl)-
INTRODUCTION
■
tetrahydrofuran-2-yl]methyl}-2-cyano-3-methylguanidine
In 2000 and 2001, the human histamine H4 receptor (hH4R)
was identified and cloned, independently by several research
(OUP-16, 4),42 were reported as potent histamine H4R
agonists (Figure 1). Compared to the corresponding primary
amines 2 and 3, which are hH3R preferring agonists, the
introduction of the nonbasic cyanoguanidine moiety shifted the
selectivity toward the H4R. However, residual activity at the
H3R subtype is still a drawback of these and other imidazole-
type H4R ligands.18 Watanabe et al.43 identified conformation-
ally restricted histamine analogues comprising a cyclopropane
moiety (cf. Figure 1) as H3R and/or H4R antagonists with a
pharmacological profile depending on the stereochemistry. In
the case of imifuramine-type amines, all stereoisomers were
partial to full agonists with preference for the hH3R. By
contrast, both potency and intrinsic activity increased in favor
of the hH4R by introduction of a nonbasic cyanoguanidine
moiety.42 Compound 4 was reported as a full hH4R agonist
with 40-fold selectivity over the hH3R and superior to its
(2S,5S)-configured optical antipode.42 In the case of 1, bulky
substituents such as phenylsulfanylethyl at the cyanoguanidine
group were superior to smaller residues such as methyl to
increase the H4R selectivity.37
groups, due to its rather high sequence homology with the
hH3R.1−7 The H4R is expressed in various cells of the immune
system, suggesting a role in inflammatory, autoimmune, and
allergic disorders.8−16 Hence, the H4R is considered as a
promising drug target for the treatment of, e.g., allergic rhinitis,
rheumatoid arthritis, bronchial asthma, and pruri-
tus.8,10−13,17−24 Regardless of that, there are controversial
results published, for example, regarding the expression of
functional H4Rs in monocytes and in the brain16,25−27 and
species-dependent discrepancies regarding potencies, receptor
selectivities, and even opposite qualities of action of H4R
ligands are reported, in part depending on the assay used.28−36
Whereas H4R antagonists are of potential therapeutic value,
agonists are considered as molecular tools. In the case of
imidazole-type compounds, which are reminiscent of the
endogenous agonist, receptor subtype selectivity proved to be
a critical issue, especially concerning the discrimination
between the H4R and the H3R.18,21
The aim of the present study was to gain information on the
stereochemical requirements of imidazole-type H4R agonists.
Cyanoguanidines such as 2-cyano-1-[4-(1H-imidazol-4-yl)-
butyl]-3-[2-(phenylsulfanyl)ethyl]guanidine (UR-PI376 1),37
derived from imbutamine (2),38,39 and a derivative of
Received: January 25, 2016
© XXXX American Chemical Society
A
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Scheme 1. Synthesis of the Cyanoguanidines cis- and trans-
a
31−42, -46, and -47 as Racemic Mixtures
Figure 1. Selected imidazole-type histamine H4R ligands, reported
pharmacological data,37,38,42−44 and general structure of the title
compounds.
In a first attempt to optimize the structure of H4R agonists
derived from 1, we introduced carbocycles instead of a flexible
chain.44 A phenylene spacer proved to be inappropriate,
whereas a cyclohexylene linker was tolerated. This prompted
us to further explore conformational constraints by analogy
with the approach reported for compound 4. Aiming at
imidazole-type H4R agonists with improved selectivity and
taking into consideration that substances with reduced
flexibility and defined stereochemistry might help to refine
H4R−ligand interaction models, we designed a series of
compounds with a cyclopentane-1,3-diyl linker, combining
structural features of 1 and 4 (Figure 1). In the following, the
preparation of potent and selective hH4R agonists including
chiral separation, assignment of the absolute configuration
based on crystal structures of intermediates and stereoselective
synthesis is reported. Pharmacological characterization was
performed by functional assays and radioligand binding studies
on human histamine receptor subtypes. Additionally, MD
simulations of two selected entantiomers, docked into the
inactive and active state models of hH4R, were performed in
order to explain the experimental data on a molecular level.
a
Reagents and conditions: (i) RuCl3 (2.2 mol %), NaIO4 (4.1 equiv),
EtOAc/MeCN/water 2:2:3, 2 d, rt, 99%. (ii) BH3·THF (4.5 equiv),
THF, 12 h, 0 °C → rt, 80%. (iii) TBDPSCl (0.98 equiv), DIPEA,
DCM, 15 h, 0 °C → rt, 57%. (iv) DMSO (4 equiv), NEt3 (8 equiv),
(COCl)2 (2 equiv), DCM, 3 h, −78 °C → 12 h, rt, 92%. (v) (a)
TosMIC (1 equiv), NaCN (0.15 equiv), EtOH, 30 min, 0 °C; (b) 7 M
NH3 in MeOH, 18 h, 100 °C, 78%. (vi) TrtCl (1 equiv), NEt3 (2
equiv), DMF, 24 h, rt, 83%. (vii) phthalimide (1.1 equiv), PPh3 (1.1
equiv), DIAD (1.1 equiv), THF, overnight, 0 °C → rt, 75%,
diasteriomeric ratio trans-12:cis-12 = 58:42 (determined by 1H NMR).
(viii) N2H4·H2O (5 equiv), EtOH, 1.5 h at reflux →1 h at rt, 92%. (ix)
TrtCl (1.5 equiv), NEt3 (2 equiv), DCM, 24 h, rt, 95%. (x) Separation
of diastereomers by flash chromatography on silica gel, PE/EtOAc/7
M NH3 in MeOH, 100/0/0−70/27/3 v/v/v), 80%. (xi) 37% HCl,
MeOH, 3 h, reflux, ion exchanger, 88%. (xii) MeCN, microwave 150
°C, 15 min, 59−95%. (xiii) 2-propanol, 1 h, rt, 94%. (xiv) 2-propanol,
1 h, rt, 86−97%. (xv) MeCN, microwave 150 °C, 15 min, 55−69%.
RESULTS AND DISCUSSION
■
Chemistry. The cis- and trans-configured imidazolylcyclo-
pentylmethylcyanoguanidines 31−42 and 46−47 were synthe-
sized as racemic mixtures in 11 steps starting from norbornene
(5) (Scheme 1). cis-Selective oxidation of 5 yielded
norcamphoric acid (6),45 subsequent reduction with borane46
gave the diol 7,46,47 and monoprotection with a tert-
butyldiphenylsilyl group resulted in a racemic mixture of 8
and ent-8 (not depicted). Swern oxidation48 of the alcohol gave
the aldehyde 9. In this reaction epimerization occurred, giving
two pairs of enantiomers. The diasteriomeric mixture 9 was
treated with tosylmethyl isocyanide49 (TosMIC) followed by
ammonia in methanol, affording the corresponding imidazoles
10. After protection of the imidazole nitrogen with a trityl
group, the alcohol 11 was converted to the phthalimide 12
under Mitsunobu conditions50 and subsequent hydrazinolysis
gave the primary amine 13. Coupling of an additional trityl
group to the primary amino group in 13 allowed for separation
of the diastereomers 14 and 15 by flash chromatography.
Deprotection under acidic conditions and conversion to the
free bases with the help of an ion exchanger gave the racemic
cis- and trans-configured amines 16 and 17. The cyanoguani-
dines 31−42, and 46−47 were synthesized by analogy with a
previously described procedure37,51 from diphenyl cyanocarbo-
nimidate (18)52 (Scheme 1). Compound 18 is prone to
undergo 2-fold aminolysis when treated with one equivalent of
B
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an ethylenediamine derivative such as 45, resulting in the
corresponding imidazole derivative.53 Therefore, for the
synthesis of 46−47, the coupling steps were carried out in
reversed order: the amines 16 and 17 were first allowed to react
with 18 to give the isoureas 43 and 44, which were
subsequently treated with the aliphatic amine 45.
Enantioseparation of Selected Racemates. On the basis
of the pharmacological data determined for the pairs of racemic
cyanoguanidines and taking into account the reported stereo-
selectivity of tetrahydrofuran-type compounds such as 3 and
4,42 the separation of the most promising racemates by chiral
separation by HPLC was explored. Enantioseparation of the
racemates 31, 32, 39, and 40 was successful by analytical HPLC
on a Chiralcel OJ-H column, using heptane/2-propanol as
eluent and isocratic elution (Figure 2).
The optimized analytical HPLC method was applied for the
separation of the enantiomers on a semipreparative scale (10−
20 mg per injection) using a Chiralcel OJ-H column (5 μm, 20
mm × 250 mm). In case of incomplete enantioseparation after
one run, the whole procedure was repeated. The enantiomeric
excess, determined by peak integration, was >95%. The circular
dichroism (CD) of the individual enantiomers was determined
at 240 nm using a CD detector directly coupled to the HPLC
system (Figure 3; Table 1). The separated enantiomers were
numbered a and b according to the order of their elution. The
stereochemical descriptors R and S given in Table 1 are based
on the assignment of the absolute configurations as outlined
below.
Determination of the Absolute Configuration. At-
tempts to crystallize the cyanoguanidines for X-ray analysis
failed. Therefore, aiming at elucidation of the absolute
configuration of the title compounds, a stereoselective synthesis
was developed (Scheme 2) which selectively provided one cis
and one trans enantiomer of the pertinent amine building block
with known absolute configuration for conversion to the
corresponding cyanoguanidines. Comparison of the optical
rotation and HPLC data of these products with the data
collected for the chromatographically separated enantiomers
allowed unequivocal assignment of the absolute configuration
of all investigated stereoisomers.
The key step of this approach was an enzymatic cleavage
enabling the discrimination between the enantiotopic ester
groups in meso-cis-cyclopentane-1,3-dicarboxylic acid dimethyl
̂
ester (48) according to the method published by Chenevert et
al.54 Dicarboxylic acid 6 was esterified with methanol in the
presence of an acidic resin as catalyst to give 48 (Scheme 2).
According to a reported procedure,55 compound 48 was
subjected to enzymatic cleavage by cholesterol esterase (CE),
yielding the monoester (1S,3R)-49 in 89% yield with 72% ee.
The racemate (1R*,3S*)-49, obtained by dehydration of 6 with
acetic anhydride followed by reaction of the anhydride,
(1R,5S)-3-oxabicyclo[3.2.1]octane-2,4-dione56 (50), with
methanol, was used as a reference compound to assign the
Figure 2. Analytical separation of the racemates on a Chiralcel OJ-H
column (5 μm). (A) 31; (B) 32; eluent heptane/2-propanol 85/15;
(C) 39; (D) 40, eluent heptane/2-propanol 80/20; flow, 1 mL/min;
temperature, 30 °C, UV detection at 215 nm.
Figure 3. Enantioseparation by chiral HPLC using UV and CD detection. Chiralcel OJ-H column (5 μm), red, UV absorption at 240 nm; blue,
circular dichroism at 240 nm. Concentration 1 mg/mL; heptane:2-propanol 80/20; flow, 0.5 mL/min; temperature, 25 °C. (A) Racemic trans-39,
(B,C) chromatograms of purified 39a and 39b, (D) racemic cis-40, (E,F) chromatograms of purified 40a and 40b.
C
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Table 1. Stereochemical Descriptors, Chromatographic Data, Enantiomeric Purity, CD, and Optical Rotation of the Separated
Enantiomers 31a−32b and 39a−40b
f
[α]25, [deg cm3 dm−1 g−1
]
a
b
c
d
e
no.
stereochemical descriptors
RS
α
tR [min]
k′
ee [%]
CD
589 nm
546 nm
365 nm
31a
31b
32a
32b
39a
39b
40a
40b
trans-(−)-(1R,3R)
trans-(+)-(1S,3S)
cis-(−)-(1S,3R)
cis-(+)-(1R,3S)
trans-(+)-(1S,3S)
trans-(−)-(1R,3R)
cis-(−)-(1S,3R)
cis-(+)-(1R,3S)
1.23
1.23
1.23
2.67
1.33
1.27
1.27
1.72
9.85
12.16
9.11
2.26
3.03
2.01
2.59
2.86
3.23
2.92
5.72
98
96
+
−
−
+
−1.9
+1.7
−2.3
+2.1
−4.9
+6.1
>99
97
−21.0
+20.9
+0.4
−25.0
+23.9
+0.45
−1.2
−19.3
+18.8
−44.6
+39.1
+1.2
10.83
11.66
12.76
11.83
20.32
>99
96
−
+
−1.0
−16.2
+15.7
−3.8
−34.1
+33.5
>99
97
−
+
a
b
a and b were assigned according to the order of elution of the corresponding enantiomers in chiral HPLC. RS = resolution of HPLC peaks;
c
d
e
f
injection: 10 μL of a 1 mg/mL solution. α = selectivity factor (k′b/k′a). Enantiomeric excess. Circular dichroism at 240 nm. Optical rotation
measured at the following compound concentrations (g/100 mL), 31a, c 1.0, MeOH; 31b, c 0.75, MeOH; 32a, c 0.7, MeOH; 32b, c 0.66, MeOH;
39a, c 2.9, MeOH; 39b, c 2.4, MeOH; 40a, c 2.8, MeOH; 40b, c 4.0, MeOH.
Scheme 2. Stereo-Controlled Synthetic Pathway to Assign the Configuration of the Enantiomers of Cyanoguanidines 31, 32, 39,
a
and 40
a
Reagents and conditions: (i) Dowex 50x8 H+-resin, MeOH, 12 h, reflux, 88%. (ii) cholesterol esterase (4000 U), 1 M NaOH, 0.05 M phosphate
buffer pH 7.0, 1% MeCN, 37 °C, 89%, ee 72%. (iii) acetic anhydride, microwave, 150 °C, 30 min, 73%. (iv) MeOH, reflux, 4 h, 99%. (v) (a) DCMabs
,
EDC, DMAP, 0 °C, 30 min; (b) 51, rt, overnight, 92%. (vi) 32% NH3(aq), overnight, rt, 100%. (vii) BH3·THF (4.5 equiv), THF, 1 h, 0 °C → rt,
overnight, reflux, 70%. (viii) Boc2O (1.1 equiv), 1 M NaOH, dioxane/water 2/1 (v/v), overnight, 0 °C → rt, 74%. (ix) DMSO (4 equiv), NEt3 (8
equiv), (COCl)2 (2 equiv), DCM, 3 h, −78 °C → 12 h, rt, 61%. (x) (a) TosMIC (1 equiv), NaCN (0.15 equiv), EtOH, 30 min, 0 °C; (b) 7 M NH3
in MeOH, 18 h, 100 °C, 87%. (xi) 37% HCl, MeOH, overnight, rt, ion exchanger, 88%. (xii) TrtCl (6 equiv), NEt3 (10 equiv), DCM, 24 h, rt, 80%.
(xiii) (a) separation of diastereomers by flash chromatography on silica gel, PE/EtOAc/7 M NH3 in MeOH, 100/0/0−70/27/3 v/v/v), 80%,
diasteriomeric ratio trans:cis = 44:56; (b) 37% HCl, MeOH, 3 h, reflux, ion exchanger, 95−98%. (xiv) MeCN, microwave 150 °C, 15 min, 59−76%.
NMR data. The configuration of (1S,3R)-49 was assigned by
comparison of the specific optical rotation with reported
values.57 The enantiomeric purity of (1S,3R)-49 was
determined by derivatization with (S)-1-phenylethanamine
(51) and 1H NMR (600 MHz) analysis of the resulting
diastereomeric amides 52.
Compound (1S,3R)-49 was treated with aqueous ammonia
at room temperature to give the ammonium salt of the
dicarboxylic acid monoamide (1S,3R)-53.54 The absolute
configuration of 53 was confirmed by X-ray diffraction analysis
(Figure 4A). Reduction of 53 with borane46,47 yielded the
amino alcohol 54. Boc-protection of the amine and subsequent
D
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Figure 4. X-ray crystal structures. (A) cis-(1R,3S)-53·H2O; (B) cis-
(1R,3S)-17·2HCl; cf. Cambridge Crystallographic Data Center,
CCDC 965185 and CCDC 965186.
Figure 5. Structures of reference histamine receptor ligands 58−66
used for the pharmacological characterization of the title compounds
(cf. Tables 2 and 3).
Swern oxidation48 of 54 gave the corresponding aldehyde 56.
The imidazole ring was introduced according to the TosMIC
ring closure procedure.49 Under the basic conditions in this
reaction, epimerization took place at the stereogenic center
connected with the imidazole ring. By analogy with the
procedure described in Scheme 1, the Boc group in (1R,3RS)-
57 was cleaved off, the imidazole nitrogen and the amino group
were trityl-protected, and the diastereomers (1R,3R)-14 and
(1R,3S)-15 were separated by flash chromatography. Depro-
tection under acidic conditions and conversion of the salts to
the free bases with the help of a basic ion exchanger gave the
trans-(1R,3R)- and cis-(1R,3S)-configured enantiomers of the
imidazolylcyclopentylmethylamines 16 and 17, respectively.
Compound cis-(1R,3S)-17 was crystallized as hydrochloride,
and the configuration was confirmed by X-ray analysis (Figure
4B). The primary amines were converted to the corresponding
trans-(1R,3R)- and cis-(1R,3S)-configured cyanoguanidines 31a,
32b, 39b, and 40b according to the method described above.
The approach summarized in Scheme 2 gave only small
amounts of enantiomerically pure cyanoguanidines for
analytical purposes. However, the absolute configuration of all
pharmacologically characterized enantiomers (31a, 31b, 32a,
32b, 39a, 39b, 40a, 40b) could be easily assigned by comparing
optical rotation and chromatographic data (cf. Table 1) of the
subset of cyanoguanidines prepared via stereoselective synthesis
with those of the enantiomers separated in larger amounts by
chiral HPLC.
Pharmacological Investigations. The amine precursors
and the synthesized cyanoguanidines were investigated for
agonism and antagonism at the hH2R, hH3R, and hH4R
subtypes in [35S]GTPγS binding assays using membrane
preparations of Sf9 insect cells coexpressing the hH4R plus
Gαi2 plus Gβ1γ2 or the hH3R plus Gαi2 plus Gβ1γ2 or expressing
the hH2R-Gsαs fusion protein. For the characterization of the
compounds at the hH1R, steady-state GTPase assays and
radioligand binding experiments were performed using
membrane preparations of Sf9 insect cells coexpressing the
hH1R plus RGS4. Additionally, selected compounds were
investigated for agonism in luciferase reporter gene assays on
human and mouse H4R expressed in HEK-293 cells. The
histamine receptor ligands depicted in Figure 5 (58,38 59,58
60,59,60 61, 62, 63,22 64−66) were used in nonlabeled or
tritiated form as reference compounds and pharmacological
tools for functional and binding studies.
and the H2R but were moderate H4R partial agonists and H3R
antagonists (Table 2). The cis-configured amine 17 was the
preferred diastereomer at both receptor subtypes. However,
compared to the flexible analogue imbutamine38 and the
endogenous agonist histamine, efficacy and potency were
drastically reduced by the introduction of the conformationally
restricted linker.
In accordance with previous structure−activity relationship
studies on imidazole-type H4R ligands,37,42 the replacement of
an amino group by a nonbasic structural motif, the
cyanoguanidine moiety, retained the H4R affinity and, depend-
ing on substitution pattern and stereochemistry, increased the
H4R selectivity. Most of the investigated racemates revealed
partial to full agonism at the H4R with intrinsic activities (α) in
the range from 0.40 to 0.94. Only three compounds, the cis-
phenoxyethyl- and the two phenylaminoethyl-substituted
cyanoguanidines, showed antagonistic (42, 46) or inverse
agonistic (47) activity. The KB values for 42 and 46 were in the
two-digit nanomolar range at the H4R, whereas 47, the cis-
isomer of 46, was virtually inactive (KB > 10000 nM). The KB
values of these compounds at the H3R were in the range of 400
nM. All the other racemates activated the H4R with EC50 values
ranging from 14 to 500 nM. In general, more bulky substituents
compared to methyl provided higher potency at the H4R. For
instance, the isobutyl derivative 35 had an EC50 of 15 nM.
Interestingly, unlike 32, all racemic cyanoguanidines bearing
substituents other than methyl showed higher agonist activity
when trans-configured. Similar to the imidazolylbutylcyanogua-
nidine series,37 phenylsulfanylethyl substitution at the cyano-
guanidine was most favorable. Highest H4R agonistic potency
was achieved with the trans-configured derivative 39 (EC50 = 14
nM, α = 0.9). At the H3R, compounds bearing alkyl residues at
the cyanoguanidine moiety were weak partial agonists. With
EC50 values from 370 to >10000 nM, these compounds showed
some selectivity for the H4R over the H3R but no improved
activities compared to flexible imidazolylbutylcyanoguanidines
such as 1 or tetrahydrofurans such as 4. Similar to 1 and in
contrast to 4,42 compounds bearing an aromatic substituent
(37−42, 46−47) were devoid of agonistic activity at the H3R.
Compound 38 was the most potent H3R antagonist with a KB
value of 70 nM. Both phenylsulfanylethyl-substituted cyano-
guanidines, 39 and 40, were moderate H3R inverse agonists (α
= −0.4 and −0.34). Contrary to the H4R, the H3R preferred the
cis-configured isomers of most compounds. No agonistic
Functional Activities of the Racemates at the HxR
Subtypes. The racemic amine precursors 16 and 17, the carba
analogues of 3, were devoid of significant activity at the H1R
E
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Table 2. Pharmacological Data of the Racemic Amines (16, 17) and Cyanoguanidines (31−42, 46, 47) and the Reference
a
Compounds 1, 58−60, 62, and 63 at Human Histamine Receptor Subtypes, Determined in the [35S]GTPγS Assay , the GTPase
b
c,d
Assay, or in Radioligand Binding Experiments
e
hH1R
hH2R
hH3R
hH4R
compd
EC50 or (KB), nM
N
EC50 or (KB), nM
N
EC50 or (KB), nM
N
3
EC50 or (KB), nM
N
3
f
g
histamine
190
8
1410 210
13
2
11
3
α: 1.0
α: 1.0
α: 1.0
α: 1.0
37
h
h
1
(>10000)
(>10000)
720 38
2
2
2
2
3
3
2
2
2
I
I
α: 0
α: 0
α: −0.52 0.05
(103 0.5)
α: 0.88 0.08
1710 14
16
17
31
na
2
2
2
>10000
2
2
2
I
I
I
α: 0
α: 0
α: 0
α: 0.38 0.01
150 23
na
>10000
(62 10)
I
α: 0.11 0.06
α: 0.19 0.10
(>10000)
α: 0
α: 0.36 0.01
206 12
na
370
6
I
I
α: 0
α: 0
α: −0.46 0.06
Ki: 830 34
547 55
α: 0.74 0.05
Ki: 240 15
111 17
Ki: >10000
2
2
Ki: nd
2
2
4
2
32
na
(>10000)
2
I
I
α: 0
α: 0
α: 0.51 0.02
Ki: 860 48
>10000
α: 0.94 0.01
Ki: 155 25
230 62
Ki: >10000
2
2
Ki: nd
2
2
4
2
33
34
35
36
37
38
39
na
(>10000)
2
2
2
2
2
2
2
I
I
α: 0
α: 0
α: 0.36 0.03
1530 14
α: 0.74 0.07
245 78
na
2
2
2
2
2
2
(>10000)
2
2
2
2
2
2
2
2
2
2
2
3
I
I
α: 0
α: 0
α: 0.28 0.09
578 13
α: 0.64 0.02
na
(>10000)
15
3
I
I
α: 0
α: 0
α: 0.54 0.04
858 68
α: 0.84 0.07
202 66
na
(>10000)
I
I
α: 0
α: 0
α: 0.50 0.00
(325 135)
α: −0.19 0.02
(70 7)
α: 0.81 0.07
125 39
na
(>10000)
I
α: −0.19 0.17
α: 0
α: 0.70 0.05
372 63
na
(>10000)
I
I
I
α: 0
α: 0
α: 0
α: 0.40 0.11
na
(>10000)
(246 49)
14
α: 0.90 0.06
Ki: 99
3
I
I
α: 0
α: 0
α: −0.40 0.05
Ki: 1230 134
(162 3)
Ki: 6080 270
2
2
Ki: nd
2
2
8
4
4
40
na
(>10000)
2
129 10
I
I
α: 0
α: 0
α: −0.34 0.04
Ki: 1350 165
(540 50)
α: 0.72 0.09
Ki: 250 20
Ki: 3800 700
2
2
Ki: nd
2
2
4
3
41
42
46
47
58
59
60
62
63
na
(>10000)
2
2
2
2
524
8
I
I
I
α: 0
α: 0
α: 0
α: 0.63 0.10
na
2
2
2
(>10000)
(328 38)
2
2
2
(97 7)
3
3
3
I
I
I
I
α: 0
α: 0
α: 0
α: 0
na
(>10000)
(408 21)
(53 6)
I
I
I
α: 0
α: 0
α: 0
α: 0.19 0.01
(>10000)
na
(>10000)
(402 38)
I
I
I
α: 0
α: 0
α: 0
α: −0.40 0.20
70.3 44.1
h
h
j
j
16000 540
2900 12
12400 3130
α: 0.9
nd
α: 1.0
nd
α: 0.7
α: 0.9
170 11
α: 0.64 0.07
3
44
8
3
α: nd
α: nd
α: 0.81 0.03
k
k
k
k
3467
372
1.58
3.02
α: 0.3
nd
α: 0.8
nd
α: 0.4
α: 0.9
(78 9)
3
3
(73 6)
4
2
α: nd
nd
α: nd
nd
α: −0.59 0.07
(2000 400)
α: −0.56 0.02
α: −0.88 0.06
(19.1 0.3)
α: −0.54 0.10
α: nd
α: nd
F
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Journal of Medicinal Chemistry
Article
Table 2. continued
a
Functional [35S]GTPγS binding assays with membrane preparations of Sf9 cells expressing the hH3R + Gαi2 + Gβ1γ2 or the hH4R + Gαi2 + Gβ1γ2
b
c
or the hH2R-Gsαs fusion protein. Steady-state GTPase activity in Sf9 cell membranes expressing the hH1R + RGS4. Displacement of [3H]64 (5
nM), [3H]65 (10 nM), [3H]66 (3 nM), or [3H]60 (5 nM) from Sf9 insect cell membranes expressing the hH1R + RGS4, hH2R-GsαS fusion protein,
d
hH3R + Gαi2 + Gβ1γ2 or the hH4R + Gαi2 + Gβ1γ2. Reaction mixtures contained ligands at a concentration from 1 nM to 1 mM as appropriate to
generate saturated concentration−response curves. N gives the number of independent experiments performed in duplicate each. Agonistic
potencies are expressed as EC50 values. na = not applicable. EC50 values could not be determined due to lacking or extremely low agonist activity. nd
= not determined. Intrinsic activities (α) refer to the maximal response induced by the standard agonist histamine (α = 1.0). Compounds identified
to be inactive as agonists (α < 0.1 or negative values, respectively, when determined in the agonist mode) were investigated in the antagonist mode.
The corresponding KB values of neutral antagonists and inverse agonists were determined from the concentration-dependent inhibition of the
e
histamine-induced response. The α values of neutral antagonists and inverse agonists were determined at a concentration of 10 μM. Ki values were
f
determined instead of KB values: 16, 17, 31−34, 37, 41, 42, 46, 47, >10000 nM; 35, 8750 290 nM; 36, 8350 900 nM; 38, 9350 210 nM. Ref
g
h
I
61. Ref 62. Ref 37 GTPase assays on Sf9 cell membranes. α = 0 (neutral antagonism): the measured values were in the range between +0.15 and
j
k
−0.15 and not different from 0 when the SEM values were taken into consideration. Ref 39. Ref 60.
Table 3. Pharmacological Data of the Enantioseparated Cyanoguanidines 31a,b, 32a,b, 39a,b, and 40a,b at the hHxR Subtypes,
a
b
Determined in the [35S]GTPγS or the GTPase Assay (EC50 and KB Values) and in Radioligand Binding Experiments (Ki
Values)
c,d
hH1R
hH2R
hH3R
hH4R
no.
configuration
(1R,3R)
[Ki], EC50 or (KB), nM
N
[Ki], EC50 or (KB), nM
N
[Ki], EC50 or (KB), nM
N
[Ki], EC50 or (KB), nM
N
31a
[>10000]
na
2
2
[>10000]
(>10000)
2
2
[5680 316]
9200 400
α: 0.29 0.07
[958 29]
2
2
[870 57]
1100 99
4
3
e
e
α: 0
α: 0
α: 0.66 0.19
[98 11]
31b
32a
32b
39a
39b
40a
(1S,3S)
(1S,3R)
(1R,3S)
(1S,3S)
(1R,3R)
(1S,3R)
(1R,3S)
[>10000]
na
2
2
[>10000]
(>10000)
2
2
2
2
4
3
994 71
100 16
e
e
α: 0
α: 0
α: 0.59 0.04
[9500 1300]
(2812 9)
α: 0.66 0.14
[3500 75]
2675 272
α: 0.32 0.06
[120 7]
[>10000]
na
2
2
[>10000]
(>10000)
2
2
2
2
4
3
e
e
α: 0
α: 0
α: −0.14 0.07
[480 100]
212 33
[>10000]
na
2
2
[>10000]
(>10000)
2
2
2
2
4
3
119 20
e
e
α: 0
α: 0
α: 0.61 0.07
[2130 17]
(1150 112)
α: 0.86 0.02
[22 3]
[6520 570]
na
2
2
[10300 500]
(>10000)
2
2
3
3
5
3
11
2
e
e
e
f
α: 0
α: 0
α: 0
α: 0.75 0.04
[1050 120]
(7700 400)
[7910 710]
na
2
2
[9820 90]
(>10000)
2
2
[590 36]
3
2
5
2
(143 12)
e
e
e
α: 0
α: 0
α: −0.21 0.04
[745 8]
α: 0
[6780 300]
na
2
2
[7360 480]
(>10000)
2
2
3
2
[290 30]
5
3
(528 50)
549
8
e
e
α: 0
α: 0
α: −0.34 0.04
[870 22]
α: 0.54 0.09
[180 22]
293 32
40b
[3050 260]
na
2
2
[7250 140]
(>10000)
2
2
3
2
5
3
(718 12)
e
e
α: 0
α: 0
α: −0.21 0.03
α: 0.39 0.01
a
Data were analyzed for best fit to one site (monophasic) competition curves. N gives the number of independent experiments performed in
duplicate; na = not applicable, EC50 values could not be determined due to lacking or extremely low agonist activity. Functional [35S]GTPγS binding
assays with membrane preparations of Sf9 cells expressing the hH3R + Gαi2 + Gβ1γ2 or the hH4R + Gαi2 + Gβ1γ2 or the hH2R-Gsαs fusion protein.
b
c
Steady-state GTPase activity in Sf9 cell membranes expressing the hH1R + RGS4. Displacement of [3H]64 (5 nM), [3H]65 (10 nM), [3H]66 (3
nM), or [3H]60 (5 nM) from Sf9 insect cell membranes expressing the hH1R + RGS4, hH2R-GsαS fusion protein, hH3R + Gαi2 + Gβ1γ2 or the hH4R
+ Gαi2 + Gβ1γ2. Reaction mixtures contained ligands at a concentration from 1 nM to 1 mM as appropriate to generate saturated concentration−
d
response curves. N gives the number of independent experiments performed in duplicate each. Agonistic potencies are expressed as EC50 values. na =
not applicable. EC50 values could not be determined due to lacking or extremely low agonist activity. nd = not determined. Intrinsic activities (α)
refer to the maximal response induced by the standard agonist histamine (α = 1.0). Compounds identified to be inactive as agonists (α < 0.1 or
negative values, respectively, when determined in the agonist mode) were investigated in the antagonist mode. The corresponding KB values of
neutral antagonists and inverse agonists were determined from the concentration-dependent inhibition of the histamine-induced response. The α
e
values of neutral antagonists and inverse agonists were determined at a concentration of 10 μM. α = 0 (neutral antagonism): the measured values
f
were in the range between +0.15 and −0.15 and not different from 0 when the SEM values were taken into consideration. The intrinsic activities of
the eutomer 39a (α = 0.75) and the racemate 39 (α = 0.90) were not significantly different from each other (p-value: 0.2).
G
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activity was observed at the H1R and the H2R. All investigated
racemates revealed very weak H2R antagonism with KB values
>10 μM. At the H1R, the compounds were weak antagonists
(31−34 and 39−42, 46) or inverse agonists (35−38, 47),
respectively. Except for 39 (KB = 6 μM) and 40 (KB = 3.8 μM)
the KB values were in the high micromolar range.
The racemates of the phenylsulfanylethyl substituted
cyanoguanidines 39 and 40 turned out to be the most
promising candidates for more detailed investigation. There-
fore, the enantiomers were prepared as outlined above. In
addition, for reasons of comparison with the tetrahydrofuran
series, the methyl-substituted cyanoguanidines 31 and 32 were
considered as well.
Functional and Binding Data of the Separated
Enantiomers at hHxR Subtypes. The enantiomerically
pure compounds 31a,b, 32a,b, 39a,b, and 40a,b were
investigated under the same conditions as the racemates
(data cf. Table 3). The results for the enantiomers of 31 and 32
were comparable to the data reported for the tetrahydrofuran 4
and analogues. The enantiomers trans-(−)-31 (31a) and cis-
(−)-32 (32a) were both weak H4R partial agonists with EC50
values >1 μM. The eutomers, trans-(+)-31 (31b) and cis-
(+)-32 (32b), were moderately potent H4R agonists with EC50
values around 100 nM. Compared to the H4R, the potencies
and efficacies were lower at the H3R: 32a was an inverse
agonist, whereas the other three isomers were weak partial
agonists with α values in the range 0.3−0.6. Similar to the
stereodiscrimination at the H4R, 31b and 32b were by a factor
of 10 more potent than their optical antipodes at the H3R. As
for the racemates, no relevant activities were detected at the
H1R and the H2R. The most potent and selective H4R agonist
among these four stereoisomers was the carba analogue of 4,
trans-(+)-(1S,3S)-31 (31b) (4, EC50 reported, 78 nM, α =
0.99;42 31b, EC50 = 100 nM, α = 0.71). Obviously, the
exchange of the oxygen atom in 4 by a carbon atom does not
markedly affect the pharmacological activity. The correlation
between the absolute configuration of the four stereoisomers
and their pharmacological properties at the H3R and the H4R is
in agreement with the data for the structurally related
tetrahydrofuran derivatives reported by Hashimoto et al.42
The evaluation of the separated enantiomeric pairs 39 and 40
revealed 39a as a potent and selective H4R agonist: 39a
activated the H4R with an EC50 value of 11 nM and an intrinsic
activity of 0.75 in the [35S]GTPγS binding assay (Figure 6. By
contrast, at the H3R 39a was a weak antagonist (KB = 1150
nM), corresponding to a more than 100-fold selectivity for the
H4R. The activities at the other two histamine receptor
subtypes were negligible. Surprisingly, the investigation of the
optical antipode of 39a, compound 39b, revealed just the
opposite profile: 39b was a weak neutral antagonist at the H4R
(KB = 7700 nM) but almost 10 times more active as an
antagonist (inverse agonist) at the H3R (KB = 143 nM, α =
−0.21) compared to 39a. Both cis-configured enantiomers, 40a
and 40b, were moderately potent H4R partial agonists with
EC50 values in the three-digit nanomolar range and H3R inverse
agonists with KB values of 528 and 718 nM, respectively. At the
H2R and H1R, these compounds were devoid of agonistic
activity but showed weak antagonism in the micromolar range.
Comparing the whole set of eight enantioseparated
imidazolylcyclopentylcyanoguanidines, highest H4R agonistic
potency always resided in the stereoisomers with S-config-
uration in position 3. Obviously, the configuration at position 1
is less important, although (1S)-configuration is preferred, in
particular, in the case of compounds bearing substituents larger
than methyl at the cyanoguanidine moiety. Regarding the H3R,
there was no clear correlation between configuration and
potency. Nevertheless, as compound 39b, the most potent H3R
ligand in this series is trans-(1R,3R)-configured, the optimal
stereochemical requirements for H3R and H4R affinity seem to
be converse. Hence, both highest H4R agonistic potency and
H4R subtype selectivity resided in trans-(1S,3S)-configured
compounds. These results are in principle in agreement with
the spatial orientation of the substituents at the tetrahydrofuran
ring in 4. Moreover, in accordance with the imidazolylalkylcya-
noguanidine series, a phenylsulfanylethyl substituent and a
chain length of four carbon atoms between imidazole and
cyanoguanidine moiety confer high H4R potency and
selectivity.
Especially for the H4R, differences between pharmacological
data from various studies are reported,28,35 and even the quality
of action may differ,35 depending on test system and read-out.
For validation, the racemates 39 and 40 as well as the
corresponding enantiomers 39a,b and 40a,b were also
investigated in steady-state GTPase assays on hH4R and
hH3R (data not shown). The determined potencies and
efficacies were comparable to those from the [35S]GTPγS
binding assays. The [35S]GTPγS binding induced by histamine
or 39a was inhibited by standard H4R antagonists in a
concentration-dependent manner and gave comparable results,
for example: 62, KB 73 6 nM vs histamine and 100 14 nM
vs 39a; 63, KB 19 0.3 nM vs histamine and 29 2 nM vs
39a. The KB values determined for 61 (19 6 nM), 62, and 63
against 39a were in the same range as Ki values from binding
studies reported by Lim et al. (61, 16 nM; 62, 125 nM; 63, 13
nM)38 and Wifling et al. (62, 178 nM; 63, 69 nM).63 These
results confirm 39a to act as a hH4R agonist in the [35S]GTPγS
binding assay and to compete with the H4R antagonists for the
same binding site. Furthermore, 39a displaced [3H]histamine
from the hH4R, providing additional evidence of binding to the
same site.
The H4R agonist potency of 39a (Table 3) was higher than
that of 1, 58, and 59 but lower than in case of the acylguanidine
60 (Table 2). The selectivity of 39a for the H4R over the H3R
was slightly lower than that of 58. With respect to the
application of such compounds as molecular tools, in addition
to binding affinities, the quality of action has to be considered.
In contrast to the reference H4R agonists 58−60, compound
Figure 6. [35S]GTPγS binding at the hH4R. Functional [35S]GTPγS
binding assays were performed with membrane preparations of Sf9
cells expressing the hH4R + Gαi2 + Gβ1γ2. Data were analyzed by
nonlinear regression and were best fit to sigmoidal concentration−
response curves. Data points shown are the means of three
independent experiments performed in duplicate each.
H
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39a was devoid of agonist activity at H1, H2, and H3 receptors
like 1.
H4R Agonism in the Reporter Gene Assay. Selected
enantiomers were investigated in a luciferase reporter gene
assay on HEK-293 cells stably expressing the human or the
mouse H4R (Figure 7).
described for several oxime-type agonists,64 are desired with
respect to translational animal models. Therefore, the separated
enantiomers were also investigated at the mouse H4R in the
luciferase assay (Figure 7B and Supporting Information). As
becomes obvious from Figure 7, lower potency and intrinsic
activity as well as lower stereodiscrimination was characteristic
of the enantiomers at the mouse compared to the human H4R.
HxR Subtype Affinities of the Separated Enantiomers.
Binding data (Ki values) of the enantiomers of 31, 32, 39, and
40 were determined at the four hHR subtypes (Table 3, Figure
8). Competition binding studies on the hH4R were performed
Figure 7. Inhibition of forskolin stimulated luciferase activity by H4R
agonists. (A) HEK293-SF-hH4R-His6-CRE-Luc cells expressing the
hH4R; forskolin concentration, 400 nM. (B) HEK293-SF-mH4R-His6-
CRE-Luc cells expressing the mH4R; forskolin concentration, 1 μM.
Reaction mixtures contained ligands at concentrations from 1 nM to
100 μM as appropriate to generate saturated concentration−response
curves. Data represent mean values SEM of five (histamine at the
hH4R) or, in all other cases, four independent experiments performed
in triplicate. The intrinsic activity (α) of histamine was set to 1.0 and
maximal responses α of test compounds were referred to this value.
Figure 8. Representative radioligand binding experiments: (A)
displacement of [3H]60 (5 nM) from Sf9 insect cell membranes
expressing the hH4R + Gαi2 + Gβ1γ2. (B) Displacement of [3H]64 (5
nM; KD 4.49 nM), [3H]65 (10 nM; KD 32 nM), [3H]66 (3 nM; KD
8.4 nM) and [3H]60 (5 nM; KD 12.6 nM) from Sf9 insect cell
membranes expressing the hH1R + RGS4, hH2R-GsαS fusion protein,
hH3R + Gαi2 + Gβ1γ2 or the hH4R + Gαi2 + Gβ1γ2. Data were analyzed
for best fit to one site (monophasic) competition curves.
EC50 values at the hH4R: histamine, 13 4 nM, α = 1.0; 31b, 63
4
nM, α = 1.0; 39a, 25 2 nM, α = 1.0; 39b, 500 150 nM, α = 0.6.
EC50 values at the mH4R: histamine, 94 29 nM, α = 1.0; 31a, 3631
1160 nM, α = 0.96; 31b, 538 33 nM, α = 0.96; 39a, 166 20 nM,
α = 0.67; 39b, 589 162 nM, α = 0.29.
with two different radioligands, which gave comparable results:
the standard compound [3H]histamine and [3H]60,59 an
acylguanidine developed in our laboratory. All evaluated
compounds displaced [3H]64 ([3H]mepyramine) from the
hH1R, [3H]65 ([3H]tiotidine) from the hH2R, [3H]66
([3H]Nα-methylhistamine) from the hH3R, and [3H]60 or
[3H]histamine from the hH4R, giving monophasic competition
binding curves (Figure 8).
Although for most compounds the Ki values from radioligand
binding experiments were slightly higher than the EC50 values
determined in the [35S]GTPγS assay, in general, the binding
data and the rank order of the compounds were in agreement
with the potencies evaluated in the functional assays. As
expected, 39a bound with the highest affinity to the hH4R (Ki =
22 nM) and showed a remarkably lower affinity for the hH3R
(Ki = 2130 nM). At the hH2R and the hH1R, all compounds
displayed low affinity. Overall, the determined Ki values
confirmed 39a to be a highly affine and selective ligand for
the hH4R.
The data obtained for 31b and 39a at the hH4R were
comparable to the results of the [35S]GTPγS and radioligand
binding assays. Both compounds were full hH4R agonists with
EC50 values in the low nanomolar range. Interestingly, 39b,
which was a weak neutral antagonist in the [35S]GTPγS assay,
showed partial agonism with an EC50 value of 500 nM in the
luciferase assay. This apparent discrepancy, depending on either
a proximal (G-protein activation in Sf9 cell membranes) or a
distal readout (reporter gene assay), can be explained by signal
amplification downstream from G-protein activation in the
cellular assay.33 Regardless of this, the high H4R agonistic
potency of the eutomer 39a and the stereodiscrimination (cf.
39b) by the human receptor was confirmed in the cell based
luciferase reporter gene assay. Species-dependent discrepancies
regarding potencies and receptor selectivities are characteristic
of numerous pharmacological tools for the H4R,29,31,36 and
comparable potencies at the human and the murine H4R, as
Binding Mode of 39a and 39b at the hH4R. Molecular
dynamic simulations revealed a stable binding mode of 39a in
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Journal of Medicinal Chemistry
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the hH4R−Gαi2βγ complex active state. The central
cyanoguanidine moiety interacted electrostatically with the
highly conserved Asp3.32 (Figure 9A). The positively charged
hydrogen bond interactions between 39a and the inactive hH4R
was determined. In accordance with 39a, in the active state
hH4R−Gαi2βγ complex, stable hydrogen bond interactions
between the cyanoguanidine moiety and Asp3.32 and between
the positively charged imidazole moiety with Glu5.46 were
observed. Additionally, the imidazole moiety was stabilized by a
hydrogen bond with Ser6.52 (Figure 10A, left) or, alternatively,
Figure 10. Binding mode of 39a and 39b in the inactive state model of
the hH4R, obtained by molecular dynamic simulations. (A)
Orientation of 39a in the orthosteric binding pocket, forming H-
bond interactions with Asp3.32, Glu5.46, and Ser6.52 (left, top view; right,
side view). (B) Orientation of 39b in the orthosteric binding pocket,
forming H-bond interactions with Asp3.32 and Glu5.46 (left, top view;
right, side view).
with Thr6.55. Furthermore, Tyr6.51 (Figure 10A, right) interacted
with the nitrile group of the cyanoguanidine moiety. In contrast
to 39a bound to the active state hH4R−Gαi2βγ complex, an
interaction between the ligand and Gln7.42 was not observed.
The phenyl moiety was embedded in an aromatic pocket
Figure 9. Binding mode of 39a in the active state model of the hH4R−
Gαi2βγ complex, obtained by molecular dynamic simulations. (A)
Orientation of 39a in the orthosteric binding pocket, caused by the
interactions of the imidazole moiety with Glu5.46 and the
cyanoguanidine moiety with Asp3.32. (B) Contact surface between
39a and the amino acids of the orthosteric binding pocket. (C) Time
domains of direct H-bond interactions (black) and water-mediated H-
bond interactions (gray) between 39a and the hH4R.
comprising Tyr2.61, His2.64, Trp3.28, and Phe7.39
.
Molecular dynamic simulations of the neutral antagonist 39b
in the binding pocket of the inactive hH4R showed a strong
interaction of the cyanoguanidine moiety with Asp3.32 and of
the positively charged imidazole moiety with Glu5.46. Compared
to 39a, only a mean number of approximately three direct
hydrogen bond interactions between ligand and receptor was
observed during the MD simulation, which may explain the
lower hH4R affinity of 39b compared to 39a. An interaction
between the imidazole moiety and Ser6.52 or Thr6.55 was not
detected (Figure 10B, left). Thus, a second stabilizing hydrogen
bond between the imidazole moiety and the hH4R, as observed
for 39a, was not present. Furthermore, an interaction between
Tyr6.51 and the cyano group was not observed (Figure 10B,
right). In contrast to 39a, the cyano group of 39b was not
surrounded by amino acids of the hH4R but solvated by
extracellular water (Figure 10B right). Thus, compared to 39a,
there were less interactions with the receptor. Comparable to
39a at the inactive hH4R, the phenyl moiety of 39b was
imidazole moiety was embedded in a pocket between
transmembrane domain (TM) III, TM VI, and TM VII,
formed by Tyr3.33, Trp6.48, and Tyr6.51. Furthermore, the
imidazole moiety was found to interact with Glu5.46 (Figure
9B). Within the first 3 ns of the simulation, direct interactions
between the imidazole moiety and Glu5.46 and Gln7.42 were
observed (Figure 9A). After 3 ns, the imidazole moiety changed
its orientation. The interactions between the imidazole and
Glu5.46 and Gln7.42 were then mediated by two water molecules
(Figure 9C). This conformation remained stable for the
remaining 12 ns of MD simulation. Taking into account the
direct and the water-mediated hydrogen bond interactions
between 39a and the hH4R, 39a is stabilized in the binding
pocket by a total of four to five H-bonds. Additionally, the
molecular dynamic simulations revealed an aromatic interaction
of the phenyl moiety of 39a with the aromatic amino acids
surrounded by aromatic amino acids (Tyr2.61, His2.64, Trp3.28
,
Phe7.39) and showed a similar flexibility in its pocket.
The imidazole moieties of the enantiomers 39a and 39b are
embedded in slightly different orientations in the binding
pocket of hH4R, resulting in a higher number of hydrogen
bonds in case of 39a. This is in accordance with the higher
hH4R affinity of 39a compared to 39b.
Tyr2.61, His2.64, Phe7.39, and Trp7.40
.
Because 39a was a hH4R partial agonist in the [35S]GTPγS
assay (Table 3), molecular dynamic simulations of 39a at the
inactive hH4R were performed, too. A mean of ∼4.5 direct
J
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Figure 11. Comparison of the binding sites of the four human histamine receptor subtypes. Center: Section of the hH4R with bound compound 39a.
Tables: Key amino acids in the respective binding pockets of the individual receptor subtypes. Inset: Compared to the orientation of 39a in the hH4R
binding site (gray), the amino acids present at positions 6.52 and 7.42 of the hH1R, the hH2R, and the hH3R (color-coded and superimposed with
the hH4R model for visualization) disfavor the binding of 39a.
The exchange of the CH3 group in 31b by a (CH2)2SPh
moiety (39a) resulted in an increase in hH4R affinity and
agonist potency (Table 3). As suggested by the molecular
dynamic studies, the phenyl ring of 39a is embedded in an
simulation. Furthermore, compared to 39a, the imidazole
moiety of 39b was more distant from TM VI. Therefore,
presumably, there is no collision between Phe6.52 and the
imidazole ring at the hH1R and the hH2R. This may explain the
lower histamine receptor subtype selectivity of 39b compared
to its optical antipode 39a. The low homology of the binding
site of the hH4R compared to those of the hH1R and the hH2R
(Figure 11) suggests that amino acids other than those in
positions 6.52 and 7.42 also contribute to hH4R selectivity.
aromatic pocket of the hH4R (Figure 11), formed by Tyr2.61
,
His2.64, Trp3.28, and Phe7.39. These interactions are not present
in case of the methyl substituted analogue 31b.
The affinity of 39a is more than 100-fold higher to the hH4R
than to hH1R, hH2R, or hH3R (Table 3). Comparing the
binding pocket of the four human histamine receptor subtypes
(Figure 11), only Asp3.32, Trp6.48, and Tyr6.51 are conserved.
Regarding the amino acids specified in Figure 11, the hH4R
shares only 20% and 30% with the hH1R and the hH2R,
respectively. In contrast, about 63% of the respective amino
acids are identical in hH3R and hH4R. There is a difference
between the hH3R and the hH4R regarding the amino acids at
positions 6.52 and 7.42, which are in contact with the imidazole
ring and the cyanoguanidine group of 39a in the case of the
hH4R (Figure 11). Interactions of Ser6.52 and Gln7.42 via
hydrogen bonds with 39a were detected in the MD simulations.
The lower affinity of 39a to the hH3R may be attributed, at
least in part, to the presence of Leu7.42 instead of Gln7.42, which
precludes this interaction. Moreover, it may be speculated that
the methyl group of Thr6.52, which is present in the hH3R
instead of Ser6.52 in the hH4R, disfavors the orientation of the
ligand in the H3R binding pocket.
CONCLUSIONS
■
Conformationally constrained analogues of the imidazolylbu-
tylcyanoguanidine 1, a potent hH4R agonist with some
selectivity over the H3R, were obtained by incorporation of a
cyclopentane-1,3-diyl moiety and identification of the most
active enantiomer according to a step-by-step approach. At
both the H4R and the H3R, the pharmacological properties
were strongly dependent on the stereochemistry of the
cyclopentane-1,3-diyl moiety. At the H4R, a preference for
trans configured compounds was observed except for the
methyl-substituted cyanoguanidines. At the H3R, cis config-
uration was preferred in most cases. None of the investigated
compounds showed agonistic activity at the H1R and H2R.
Phenylsulfanylethyl-substituted cyanoguanidines turned out to
be superior to compounds with other substituents including
methyl as in case of the carba analogues of 4, that is the trans-
and cis-configured compounds 31 and 32, respectively. Starting
from a set of racemic compounds, the most promising H4R
agonists were selected for chiral separation by HPLC. In
addition, an enzyme assisted stereoselective synthesis enabled
the assignment of the absolute configurations. Highest receptor
subtype selectivity and H4R agonist potency, determined in a
The very low affinity of 39a at the hH1R and the hH2R
compared to the hH4R may be attributed to Phe6.52, which
prevents the formation of a hydrogen bond and causes a
collision with the imidazole ring of 39a (Figure 11, inset). In
the case of 39b, a hydrogen bond between the imidazole
moiety and Ser6.52 of the hH4R was not observed in the MD
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proximal functional assay ([35S]GTPγS binding), resided in the
trans-(1S,3S)-configured compound 39a. By contrast, the
optical antipode 39b was an H4R antagonist under the same
conditions. Obviously, trans-(1S,3S)-39a is capable of stabiliz-
ing the H4R active state, whereas trans-(1R,3R)-39b prefers the
inactive state, as supported by MD simulations. Thus, the
conformationally constrained ligand 39a may be useful as a
pharmacological tool, providing stereochemical information for
the refinement of models of the binding mode of
imidazolylalkyl-substituted cyanoguanidine-type H4R agonists.
Analytical HPLC analysis was performed on a system from Thermo
Separation Products (TSP, Egelsbach, Germany) composed of a
SN400 controller, a P4000 pump, an AS3000 autosampler, a degasser
(Degassex DG-4400, Phenomenex), a Spectra Focus UV−vis detector,
and a RP column thermostated at 30 °C ((a) Eurosphere-100 C18,
250 mm × 4.0 mm, 5 μm, Knauer, Berlin, Germany, t0 = 3.32 min; (b)
MN Nucleodur 100−5 C18 ec, 250 mm × 4.0 mm, 5 μm, Macherey
Nagel, Duren, Germany, t = 2.68 min; (c) Luna C18-2, 150 mm × 4.6
̈
0
mm, 4 μm, Phenomenex, Aschaffenburg, Germany, t0 = 2.88 min) at a
flow rate of 0.8 mL/min. UV detection was done at 220 nm. Mixtures
of acetonitrile and 0.05% aq TFA were used as mobile phase. Helium
degassing was used throughout. Compound purities were calculated as
percentage peak area of the analyzed compound by UV detection at
220 nm. HPLC conditions, retention times (tR), capacity factors (k′ =
(tR − t0)/t0), and purities of the synthesized compounds are listed in
the Supporting Information. Purity of tested compounds was >95% as
determined by high-performance liquid chromatography.
Chemistry: Experimental Protocols and Analytical Data.
Synthesis of the Cyanoguanidines 31−42, 46, and 47. General
Procedure.37,51 The isourea (1 equiv) and the pertinent amine (1
equiv) in MeCN were heated under microwave irradiation at 150 °C
for 15 min. After removal of the solvent in vacuo, the crude product
was purified by flash chromatography (DCM/MeOH/32% NH3(aq)
95/4/1 v/v/v).
( )-2-Cyano-1-{[trans-3-(1H-imidazol-4-yl)cyclopentyl]methyl}-
3-methylguanidine (31). The title compound was prepared from 16
(0.05 g, 0.3 mmol) and 25 (0.053 g, 0.3 mmol) in MeCN (4.5 mL)
according to the general procedure. Flash chromatography yielded a
yellow solid (0.07 g, 95%); mp 41 °C. 1H NMR (300 MHz, CD3OD):
δ [ppm] = 1.35 (m, 1H, CH2), 1.68 (m, 1H, CH2), 1.83 (m, 2H,
CH2), 1.94 (m, 1H, CH2), 2.08 (m, 1H, CH2), 2.39 (m, 1H, CH), 2.79
(s, 3H, CH3-N), 3.18 (m, 3H, CH-Im + CH2-N), 6.80 (s, 1H, Im-H-
5), 7.60 (s, 1H, Im-H-2). 13C NMR (75 MHz, CD3OD): δ [ppm] =
28.74 (+, CH3), 30.96 (−, CH2), 33.86 (−, CH2), 37.84 (−, CH2),
37.71 (+, CH), 39.68 (+, CH), 47.71 (−, CH2-N), 116.71 (+, Im-C-5),
120.28 (Cquat, CN), 134.98 (+, Im-C-2), 141.94 (Cquat, Im-C-4),
162.05 (Cquat, CN). HRMS (EI-MS) calcd for C12H18N6 [M+•]
246.1593; found 246.1587. IR (cm−1) = 3289 (N-H), 3142, 2940,
2864 (C-H), 2163 (CN), 1582 (CN), 1368. Anal. (C12H18N6·
0.5H2O·CH3OH) C, H, N, C12H18N6 (246.31).
EXPERIMENTAL SECTION
■
Chemistry: General Conditions. 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), and 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. Millipore water was used throughout for the
preparation of buffers and HPLC eluents. 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 and stored over 3 Å molecular sieves.
Cholesterol esterase from porcine pancreas (EC 3.1.1.13) was from
Sigma-Aldrich Chemie GmbH (Munich, Germany). Optical rotations
were measured on a PerkinElmer 141 polarimeter in the specified
solvent. Concentrations are indicated in [g/100 mL]. Flash
chromatography was performed in glass columns on silica gel
(Merck silica gel 60, 40−63 μm). Automated flash chromatography
was performed on a Varian IntelliFlash 310 using prepacked Varian
Superflash columns (Varian, Darmstadt, Germany). Reactions were
monitored by TLC 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), and a 1.0% solution of Fast Blue B salt (imidazole
containing compounds) in EtOH/H2O = 30/70 (v/v) or iodine
staining. All melting points are uncorrected and were measured on a
( )-2-Cyano-1-{[cis-3-(1H-imidazol-4-yl)cyclopentyl]methyl}-3-
methylguanidine (32). The title compound was prepared from 17
(0.05 g, 0.3 mmol) and 25 (0.053 g, 0.3 mmol) in MeCN (4.5 mL)
according to the general procedure. Flash chromatography yielded a
Buchi 530 (Buchi GmbH, Essen, Germany) apparatus. Lyophilization
̈
̈
was done with a Christ alpha 2-4 LD equipped with a vacuubrand RZ
6 rotary vane vacuum pump. Microwave assisted reactions were
performed 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),
Avance 400 (1H, 400.1 MHz; 13C, 100.6 MHz) or Bruker Avance 600
(1H, 600.1 MHz; 13C, 150.9 MHz) NMR spectrometers (Bruker
BioSpin GmbH, Rheinstetten, Germany). Chemical shifts are given in
δ (ppm) relative to external standards. Abbreviations for the
multiplicities of the signals: s (singlet), d (doublet), t (triplet), m
(multiplet), brs (broad singlet), and combinations thereof. The
multiplicity of carbon atoms (13C NMR) was determined by DEPT
135 (distortionless enhancement by polarization transfer): “+” primary
and tertiary carbon atom (positive DEPT 135 signal), “−” secondary
carbon atom (negative DEPT 135 signal), “Cquat” quaternary carbon
atom. In certain cases 2D-NMR techniques (COSY, HMQC, HSQC,
1
yellow solid (0.065 g, 82%); mp 61−63 °C. H NMR (300 MHz,
CD3OD): δ [ppm] = 1.33 (m, 1H, CH2), 1.50 (m, 1H, CH2), 1.71 (m,
1H, CH2), 1.85 (m, 1H, CH2), 2.03 (m, 1H, CH2), 2.19 (m, 1H,
CH2), 2.33 (m, 1H, CH), 2.79 (s, 3H, CH3-N), 3.09 (m, 1H, CH-Im),
3.18 (m, 2H, CH2-N), 6.79 (s, 1H, Im-H-5), 7.57 (s, 1H, Im-H-2). 13
C
NMR (75 MHz, CD3OD): δ [ppm] = 28.73 (+, CH3), 30.07 (−,
CH2), 32.93 (−, CH2), 38.78 (−, CH2), 39.01 (+, CH), 40.71 (+, CH),
47.82 (−, CH2−N), 116.75 (+, Im-C-5), 120.29 (Cquat, CN), 135.87
(+, Im-C-2), 141.94 (Cquat, Im-C-4), 162.02 (Cquat, CN). HRMS
(EI-MS) calcd for C12H18N6 [M+•] 246.1593; found 246.1593. IR
(cm−1) = 3258 (N-H), 3149, 2930, 2869 (C-H), 2160 (CN), 1575
(CN), 1448, 1364, 1026. Anal. (C12H18N6·1.2H2O·0.2CH3OH) C,
H, N, C12H18N6 (246.31).
( )-2-Cyano-3-cyclopropyl-1-{[trans-3-(1H-imidazol-4-yl)-
cyclopentyl]methyl}guanidine (33). The title compound was
prepared from 16 (0.05 g, 0.3 mmol) and 26 (0.061 g, 0.3 mmol)
in MeCN (4.5 mL) according to the general procedure. Flash
1
HMBC, NOESY) were used to assign H and 13C chemical shifts.
Infrared spectra (IR) were measured on a Bruker Tensor 27
spectrometer equipped with an ATR (attenuated total reflection)
unit from Harrick Scientific Products Inc. (Ossinning/NY, US). 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 parentheses.
Elemental analysis (C, H, N, Heraeus Elementar Vario EL III) were
performed by the Analytical Department of the University Regensburg
and are within 0.4% unless otherwise noted.
1
chromatography yielded a white solid (0.06 g, 74%); mp 43 °C. H
NMR (300 MHz, CD3OD): δ [ppm] = 0.60 (m, 2H, cPr-CH2), 0.83
(m, 2H, cPr-CH2), 1.36 (m, 1H, CH2), 1.68 (m, 1H, CH2), 1.81 (m,
2H, CH2), 1.93 (m, 1H, CH2), 2.07 (m, 1H, CH2), 2.39 (m, 1H, CH),
2.47 (m, 1H, cPr-CH), 3.17 (m, 1H, CH-Im), 3.22 (d, 2H, 3J = 7.5 Hz,
CH2-N), 6.78 (s, 1H, Im-H-5), 7.56 (s, 1H, Im-H-2). 13C NMR (75
MHz, CD3OD): δ [ppm] = 8.12 (−, 2 cPr-CH2), 23.77 (+, cPr-CH),
30.91 (−, CH2), 33.91 (−, CH2), 37.30 (−, CH2), 37.76 (+, CH),
39.89 (+, CH), 47.51 (−, CH2−N), 116.94 (+, Im-C-5), 120.11 (Cquat
,
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CN), 135.70 (+, Im-C-2), 139.67 (Cquat, Im-C-4), 162.52 (Cquat
,
CD3OD): δ [ppm] = 30.98 (−, CH2), 32.30 (−, CH2), 33.80 (−,
CH2), 33.99 (−, CH2), 37.27 (−, CH2), 37.49 (+, CH), 39.68 (+, CH),
42.38 (−, N-CH2), 47.63 (−, CH2-N), 116.57 (+, Im-C-5), 120.37
(Cquat, CN), 127.03 (+, Ph-C-4), 129.45 (+, 2 Ph-C), 129.51 (+, 2
Ph-C), 135.60 (+, Im-C-2), 139.37 (Cquat, Im-C-4), 142.86 (Cquat, Ph-
C-1), 161.27 (Cquat, CN). HRMS (EI-MS) calcd for C20H26N6
[M+•] 350.2219; found 350.2212. IR (cm−1) = 3262 (N-H), 2930,
2864 (C-H), 2158 (CN), 1574 (CN), 1452, 1426, 1362, 1104.
Anal. (C20H26N6·0.6CH3OH) C, H, N. C20H26N6 (350.46).
CN). HRMS (EI-MS) calcd for C14H20N6 [M+•] 272.1749; found
272.1743. IR (cm−1) = 3261 (N-H), 2945, 2867 (C-H), 2160 (C
N), 1570 (CN), 1428, 1344, 1106. Anal. (C14H20N6·0.4CH3OH) C,
H, N, C14H20N6 (272.35).
(
)-2-Cyano-3-cyclopropyl-1-{[cis-3-(1H-imidazol-4-yl)-
cyclopentyl]methyl}guanidine (34). The title compound was
prepared from 17 (0.05 g, 0.3 mmol) and 26 (0.061 g, 0.3 mmol)
in MeCN (4.5 mL) according to the general procedure. Flash
1
chromatography yielded a yellow solid (0.065 g, 80%); mp 46 °C. H
( )-2-Cyano-1-{[cis-3-(1H-imidazol-4-yl)cyclopentyl]methyl}-3-
(3-phenylpropyl)guanidine (38). The title compound was prepared
from 17 (0.04 g, 0.24 mmol) and 28 (0.068 g, 0.24 mmol) in MeCN
(4.5 mL) according to the general procedure. Flash chromatography
NMR (300 MHz, CD3OD): δ [ppm] = 0.60 (m, 2H, cPr-CH2), 0.82
(m, 2H, cPr-CH2), 1.32 (m, 1H, CH2), 1.50 (m, 1H, CH2), 1.71 (m,
1H, CH2), 1.83 (m, 1H, CH2), 2.03 (m, 1H, CH2), 2.18 (m, 1H,
CH2), 2.35 (m, 1H, CH), 2.47 (m, 1H, cPr-CH), 3.09 (m, 1H, CH-
Im), 3.24 (m, 2H, CH2-N), 6.78 (s, 1H, Im-H-5), 7.56 (s, 1H, Im-H-
2). 13C NMR (75 MHz, CD3OD): δ [ppm] = 8.11 (−, 2 cPr-CH2),
23.77 (+, cPr-CH), 30.01 (−, CH2), 32.94 (−, CH2), 38.79 (−, CH2),
39.06 (+, CH), 40.93 (+, CH), 47.61 (−, CH2−N), 116.78 (+, Im-C-
5), 120.33 (Cquat, CN), 136.20 (+, Im-C-2), 141.88 (Cquat, Im-C-4),
162.49 (Cquat, CN). IR (cm−1) = 3253 (N-H), 2924, 2855 (C-H),
2161 (CN), 1575 (CN), 1447, 1343, 1104. Anal. (C14H20N6·
0.35CH3OH) C, H, N, C14H20N6 (272.35).
( )-2-Cyano-1-{[trans-3-(1H-imidazol-4-yl)cyclopentyl]methyl}-
3-isobutylguanidine (35). The title compound was prepared from 16
(0.05 g, 0.3 mmol) and 27 (0.066 g, 0.3 mmol) in MeCN (4.5 mL)
according to the general procedure. Flash chromatography yielded a
yellow solid (0.07 g, 81%); mp 47 °C. 1H NMR (300 MHz, CD3OD):
δ [ppm] = 0.91 (d, 6H, 3J = 6.7 Hz, 2 CH3), 1.36 (m, 1H, CH2), 1.63−
1.87 (m, 4H, CH(CH3)2 + CH2), 1.94 (m, 1H, CH2), 2.07 (m, 1H,
CH2), 2.39 (m, 1H, CH), 3.01 (d, 2H, 3J = 7.1 Hz, N-CH2-
CH(CH3)2), 3.17 (m, 1H, CH-Im), 3.18 (d, 2H, 3J = 7.6 Hz, CH2-N),
6.78 (s, 1H, Im-H-5), 7.57 (s, 1H, Im-H-2). 13C NMR (75 MHz,
CD3OD): δ [ppm] = 20.31 (+, 2 CH3), 29.60 (+, CH(CH3)2), 30.98
(−, CH2), 33.86 (−, CH2), 37.40 (−, CH2), 37.80 (+, CH), 39.67 (+,
CH), 47.73 (−, CH2-N), 50.08 (−, N-CH2-CH(CH3)2), 116.71 (+,
Im-C-5), 120.29 (Cquat, CN), 135.09 (+, Im-C-2), 141.06 (Cquat, Im-
C-4), 161.35 (Cquat, CN). HRMS (EI-MS) calcd for C15H24N6
[M+•] 288.2062; found 288.2057. IR (cm−1) = 3266 (N-H), 2955,
2868 (C-H), 2156 (CN), 1576 (CN), 1447, 1426, 1385, 1270,
1159, 1105. Anal. (C15H24N6·0.5CH3OH) C, H, N. C15H24N6
(288.39).
1
yielded a yellow solid (0.06 g, 72%); mp 53−54 °C. H NMR (300
MHz, CD3OD): δ [ppm] = 1.31 (m, 1H, CH2), 1.49 (m, 1H, CH2),
1.70 (m, 1H, CH2), 1.85 (m, 1H, CH2), 1.86 (m, 2H, CH2-CH2-Ph),
2.03 (m, 1H, CH2), 2.18 (m, 1H, CH2), 2.31 (m, 1H, CH), 2.64 (t,
3
2H, J = 7.6 Hz, CH2-Ph), 3.09 (m 1H, CH-Im), 3.16−3.24 (m, 4H,
N-CH2 + CH2-N), 6.79 (s, 1H, Im-H-5), 7.11−7.27 (m, 5H, Ph-H),
7.58 (s, 1H, Im-H-2). 13C NMR (75 MHz, CD3OD): δ [ppm] = 30.10
(−, CH2), 32.31 (−, CH2), 32.93 (−, CH2), 33.99 (−, CH2), 38.84 (−,
CH2), 39.05 (+, CH), 40.73 (+, CH), 42.38 (−, N-CH2), 47.80 (−,
CH2-N), 118.69 (+, Im-C-5), 120.98 (Cquat, CN), 127.02 (+, Ph-C-
4), 129.45 (+, 2 Ph-C), 129.50 (+, 2 Ph-C), 135.57 (+, Im-C-2),
140.71 (Cquat, Im-C-4), 142.86 (Cquat, Ph-C-1), 161.25 (Cquat, CN).
HRMS (EI-MS) calcd for C20H26N6 [M+•] 350.2219; found 350.2212.
IR (cm−1) = 3259 (N-H), 2939, 2864 (C-H), 2158 (CN), 1575
(CN), 1451, 1426, 1362, 1104. Anal. (C20H26N6·0.55CH3OH) C,
H, N. C20H26N6 (350.46).
( )-2-Cyano-1-{[trans-3-(1H-imidazol-4-yl)cyclopentyl]methyl}-
3-[2-(phenylsulfanyl)ethyl]guanidine (39). The title compound was
prepared from 16 (0.04 g, 0.24 mmol) and 29 (0.072 g, 0.24 mmol) in
MeCN (4.5 mL) according to the general procedure. Flash
chromatography yielded a white solid (0.08 g, 90%); mp 46−47 °C.
1H NMR (300 MHz, CD3OD): δ [ppm] = 1.32 (m, 1H, CH2), 1.73
(m, 2H, CH2), 1.82 (m, 1H, CH2), 1.94 (m, 1H, CH2), 2.06 (m, 1H,
CH2), 2.35 (m, 1H, CH), 3.11 (m, 5H, CH-Im + N-CH2 + CH2-N),
3.41 (m, 2H, CH2-S), 6.77 (s, 1H, Im-H-5), 7.17 (m, 1H, Ph-H-4),
7.28 (m, 2H, Ph-H), 7.37 (m, 2H, Ph-H), 7.57 (s, 1H, Im-H-2). 13C
NMR (75 MHz, CD3OD): δ [ppm] = 31.01 (−, CH2), 33.61 (−,
CH2−S), 33.85 (−, CH2), 37.37 (−, CH2), 37.74 (+, CH), 39.47 (+,
CH), 42.27 (−, N-CH2), 47.82 (−, CH2-N), 116.69 (+, Im-C-5),
119.99 (Cquat, CN), 127.34 (+, Ph-C-4), 130.16 (+, 2 Ph-C), 130.46
( )-2-Cyano-1-{[cis-3-(1H-imidazol-4-yl)cyclopentyl]methyl}-3-
isobutylguanidine (36). The title compound was prepared from 17
(0.05 g, 0.3 mmol) and 27 (0.066 g, 0.3 mmol) in MeCN (4.5 mL)
according to the general procedure. Flash chromatography yielded a
yellow solid (0.08 g, 92%); mp 62 °C. 1H NMR (300 MHz, CD3OD):
δ [ppm] = 0.91 (d, 6H, 3J = 6.7 Hz, 2 CH3), 1.31 (m, 1H, CH2), 1.50
(m, 1H, CH2), 1.71 (m, 1H, CH2), 1.85 (m, 2H, CH + CH2), 2.03 (m,
1H, CH2), 2.19 (m, 1H, CH2), 2.33 (m, 1H, CH), 3.01 (d, 2H, 3J = 7.2
(+, 2 Ph-C), 135.92 (+, Im-C-2), 136.99 (Cquat, Ph-C-1), 141.87 (Cquat
,
Im-C-4), 161.18 (Cquat, CN). HRMS (EI-MS) calcd for C19H24N6S
[M+•] 368.1783; found 368.1778. IR (cm−1) = 3255 (N-H), 2946,
2864 (C-H), 2159 (CN), 1574 (CN), 1437, 1357, 1300, 1088.
Anal. (C19H24N6S·0.3CH3OH) C, H, N. C19H24N6S (368.50).
( )-2-Cyano-1-{[cis-3-(1H-imidazol-4-yl)cyclopentyl]methyl}-3-
[2-(phenylsulfanyl)ethyl]guanidine (40). The title compound was
prepared from 17 (0.04 g, 0.24 mmol) and 29 (0.072 g, 0.24 mmol) in
MeCN (4.5 mL) according to the general procedure. Flash
chromatography yielded a yellow solid (0.06 g, 68%); mp 48−49
3
Hz, N-CH2-CH(CH3)2), 3.09 (m, 1H, CH-Im), 3.21 (d, 2H, J = 8.3
Hz, CH2-N), 6.78 (s, 1H, Im-H-5), 7.56 (s, 1H, Im-H-2). 13C NMR
(75 MHz, CD3OD): δ [ppm] = 20.33 (+, 2 CH3), 29.61 (+,
CH(CH3)2), 30.12 (−, CH2), 32.95 (−, CH2), 38.82 (−, CH2), 39.06
(+, CH), 40.74 (+, CH), 47.84 (−, CH2-N), 50.09 (−, N-CH2-
CH(CH3)2), 116.82 (+, Im-C-5), 120.49 (Cquat, CN), 135.85 (+,
Im-C-2), 140.41 (Cquat, Im-C-4), 161.33 (Cquat, CN). HRMS (EI-
MS) calcd for C15H24N6 [M+•] 288.2062; found 288.2057. IR (cm−1)
= 3275 (N-H), 2956, 2868 (C-H), 2156 (CN), 1575 (CN),
1449, 1426, 1384, 1270, 1157, 1107. Anal. (C15H24N6·0.5CH3OH) C,
H, N. C15H24N6 (288.39).
1
°C. H NMR (300 MHz, CD3OD): δ [ppm] = 1.31 (m, 1H, CH2),
1.47 (m, 1H, CH2), 1.71 (m, 1H, CH2), 1.84 (m, 1H, CH2), 2.02 (m,
1H, CH2), 2.20 (m, 1H, CH2), 2.29 (m, 1H, CH), 3.10 (m, 5H, CH-
Im + N-CH2 + CH2-N), 3.41 (t, 2H, 3J = 7.3 Hz, CH2-S), 6.78 (s, 1H,
3
Im-H-5), 7.18 (t, 1H, J = 7.6 Hz, Ph-H-4), 7.29 (m, 2H, Ph-H-3,5),
3
7.57 (s, 1H, Im-H-2), 8.08 (d, 2H, J = 8.1 Hz, Ph-H-2,6). 13C NMR
(75 MHz, CD3OD): δ [ppm] = 30.11 (−, CH2), 32.92 (−, CH2),
33.60 (−, CH2−S), 38.79 (−, CH2), 38.98 (+, CH), 40.54 (+, CH),
42.28 (−, N-CH2), 47.90 (−, CH2−N), 116.53 (+, Im-C-5), 120.00
(Cquat, CN), 127.34 (+, Ph-C-4), 130.16 (+, 2 Ph-C), 130.44 (+, 2
Ph-C), 135.87 (+, Im-C-2), 137.01 (Cquat, Ph-C-1), 141.68 (Cquat, Im-
C-4), 161.16 (Cquat, CN). HRMS (EI-MS) calcd for C19H24N6S
[M+•] 368.1783; found 368.1774. IR (cm−1) = 3243 (N-H), 2925,
2855 (C-H), 2158 (CN), 1572 (CN), 1436, 1355, 1300, 1088.
Anal. (C19H24N6S·0.4CH3OH) C, H, N. C19H24N6S (368.50).
( )-2-Cyano-1-{[trans-3-(1H-imidazol-4-yl)cyclopentyl]methyl}-
3-(3-phenylpropyl)guanidine (37). The title compound was prepared
from 16 (0.04 g, 0.24 mmol) and 28 (0.068 g, 0.24 mmol) in MeCN
(4.5 mL) according to the general procedure. Flash chromatography
1
yielded a yellow solid (0.075 g, 89%); mp 50−51 °C. H NMR (300
MHz, CD3OD): δ [ppm] = 1.35 (m, 1H, CH2), 1.69 (m, 1H, CH2),
1.86 (m, 4H, CH2 + CH2-CH2-Ph), 1.94 (m, 1H, CH2), 2.08 (m, 1H,
3
CH2), 2.38 (m, 1H, CH), 2.64 (t, 2H, J = 7.5 Hz, CH2-Ph), 3.12−
3.25 (m, 5H, CH-Im + N-CH2 + CH2-N), 6.85 (s, 1H, Im-H-5), 7.11−
( )-2-Cyano-1-{[trans-3-(1H-imidazol-4-yl)cyclopentyl]methyl}-
3-[2-(phenoxy)ethyl]guanidine (41). The title compound was
7.27 (m, 5H, Ph-H), 7.74 (s, 1H, Im-H-2). 13C NMR (75 MHz,
M
DOI: 10.1021/acs.jmedchem.6b00120
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
prepared from 16 (0.04 g, 0.24 mmol) and 30 (0.068 g, 0.24 mmol) in
CH2), 38.80 (−, CH2), 38.92 (+, CH), 40.59 (+, CH), 42.46 (−, N-
CH2), 44.46 (−, CH2-NH-Ph), 47.90 (−, CH2-N), 113.98 (+, 2 Ph-C),
116.56 (+, Im-C-5), 118.34 (+, Ph-C-4), 120.23 (Cquat, CN), 130.21
MeCN (4.5 mL) according to the general procedure. Flash
1
chromatography yielded a white solid (0.05 g, 59%); mp 55 °C. H
NMR (300 MHz, CD3OD): δ [ppm] = 1.33 (m, 1H, CH2), 1.65 (m,
1H, CH2), 1.82 (m, 2H, CH2), 1.93 (m, 1H, CH2), 2.05 (m, 1H,
CH2), 2.38 (m, 1H, CH), 3.15 (m, 1H, CH-Im), 3.19 (d, 2H, 3J = 7.5
(+, 2 Ph-C), 135.86 (+, Im-C-2), 141.92 (Cquat, Im-C-4), 149.84 (Cquat,
Ph-C-1), 161.58 (Cquat, CN). HRMS (LSI-MS (MeOH/glycerol))
calcd for C19H26N7 [M + H]+ 352.2250; found 352.2242. IR (cm−1) =
3275 (N-H), 2971, 2901 (C-H), 2161 (CN), 1577 (CN), 1499,
1406, 1251. Anal. (C19H25N7·0.7CH3OH) C, H, N. C19H25N7
(351.45).
3
3
Hz, CH2-N), 3.61 (t, 2H, J = 5.4 Hz, N-CH2), 4.07 (t, 2H, J = 5.4
Hz, CH2-O), 6.75 (s, 1H, Im-H-5), 6.89−6.94 (m, 3H, Ph-H), 7.25
(m, 2H, Ph-H), 7.57 (s, 1H, Im-H-2). 13C NMR (75 MHz, CD3OD):
δ [ppm] = 31.03 (−, CH2), 33.87 (−, CH2), 37.33 (−, CH2), 37.69 (+,
CH), 39.64 (+, CH), 42.45 (−, N-CH2), 47.79 (−, CH2-N), 67.67 (−,
CH2-O), 115.64 (+, 2 Ph-C), 116.72 (+, Im-C-5), 120.06 (Cquat, C
N), 122.15 (+, Ph-C-4), 130.59 (+, 2 Ph-C), 135.87 (+, Im-C-2),
142.10 (Cquat, Im-C-4), 160.11 (Cquat, Ph-C-1), 161.54 (Cquat, CN).
HRMS (EI-MS) calcd for C19H24N6O [M+•] 352.2012; found
352.2007. IR (cm−1) = 3256 (N-H), 2971, 2901 (C-H), 2162 (C
N), 1578 (CN), 1406, 1241, 1071, 1049. Anal. (C19H24N6O·
0.5CH3OH) C, H, N. C19H24N6O (352.43).
Chiral Separation by HPLC. Preparative HPLC was performed at
room temperature with a system from Knauer (Berlin, Germany)
consisting of two K-1800 pumps, a K-2001 detector (UV detection at
220 nm), and a chiral NP-column (Chiralcel OJ-H, 250 mm × 20 mm,
5 μm, Daicel Chemical Industries Ltd., Fort Lee, NJ, USA) at a flow
rate of 18 mL/min. Mixtures of heptane and 2-propanol (85/15, v/v,
isocratic elution) were used as mobile phase. Solvents were removed
from the eluates under reduced pressure (final pressure: 60 mbar) at
40 °C prior to lyophilization. Chiral analytical HPLC analysis was
performed on a system from Varian (Varian 920-LC, Darmstadt,
Germany) and a NP-column thermostated at 30 °C (Chiralcel OJ-H,
250 mm × 4.6 mm, 5 μm, Daicel Chemical Industries Ltd., Fort Lee,
NJ, USA; t0 = 3.02 min) at a flow rate of 1.0 mL/min. UV-detection
was done at 215 nm. Mixtures of heptane and 2-propanol (31:32: 85/
15, v/v; 39:40: 80/20, v/v; isocratic elution) were used as mobile
phase. helium degassing was used throughout. Compound purities
were calculated as percentage peak area of the analyzed compound by
UV detection at 215 nm. Purity of tested compounds was >95% for all
compounds as determined by HPLC. Optical rotations were measured
on a PerkinElmer 141 polarimeter in the specified solvent.
Concentrations are indicated in [g/100 mL]. Circular dichroism was
determined with a Jasco CD1595 spectropolarimeter (Jasco GmbH,
Groß-Umstadt, Germany) directly coupled to analytical HPLC at 240
nm at room temperature.
Separated enantiomers (data cf. Table 1): (−)-2-Cyano-1-{[trans-
(1R,3R)-3-(1H-imidazol-4-yl)cyclopentyl]methyl}-3-methylguanidine
(31a), (+)-2-cyano-1-{[trans-(1S,3S)-3-(1H-imidazol-4-yl)-
cyclopentyl]methyl}-3-methylguanidine (31b), (−)-2-cyano-1-{[cis-
(1S,3R)-3-(1H-imidazol-4-yl)cyclopentyl]methyl}-3-methylguanidine
(32a), (+)-2-cyano-1-{[cis-(1R,3S)-3-(1H-imidazol-4-yl)cyclopentyl]-
methyl}-3-methylguanidine (32b), (+)-2-cyano-1-{[trans-(1S,3S)-3-
(1H-imidazol-4-yl)cyclopentyl]methyl}-3-[2-(phenylsulfanyl)ethyl]-
guanidine (39a), (−)-2-cyano-1-{[trans-(1R,3R)-3-(1H-imidazol-4-
yl)cyclopentyl]methyl}-3-[2-(phenylsulfanyl)ethyl]guanidine (39b),
(−)-2-cyano-1-{[cis-(1S,3R)-3-(1H-imidazol-4-yl)cyclopentyl]-
methyl}-3-[2-(phenylsulfanyl)ethyl]guanidine (40a), (+)-2-cyano-1-
{[cis-(1R,3S)-3-(1H-imidazol-4-yl)cyclopentyl]methyl}-3-[2-
(phenylsulfanyl)ethyl]guanidine (40b).
Pharmacological Methods. Materials. Histamine dihydrochlor-
ide was purchased from Alfa Aesar (Karlsruhe, Germany),
Iodophenpropit (61) from Tocris Bioscience (Ellisville, USA),
[3H]mepyramine ([3H]64), [3H]tiotidine ([3H]65), [3H]Nα-methyl-
histamine ([3H]66), and [3H]histamine were from PerkinElmer Life
Sciences (Boston, MA). The compounds 1,37 58,65 59,58 60,60 [3H]
60,59 62,66 and 6367 were synthesized according to reported protocols.
[γ-32P]GTP was synthesized according to a previously described
method.68 [32P]Pi (8500−9100 Ci/mmol orthophosphoric acid) was
from PerkinElmer Life Sciences (Boston, MA, USA). Guanosine
diphosphate (GDP) and polyethylenimine were from Sigma-Aldrich
Chemie GmbH (Munich, Germany), and unlabeled GTPγS was from
Roche (Mannheim, Germany). [35S]GTPγS was from PerkinElmer
Life Sciences (Boston, MA) or Hartmann Analytic GmbH
(Braunschweig, Germany). GF/C filters were from Whatman Ltd.
(Maidstone, UK). Glycerol-3-phosphate dehydrogenase, triose phos-
phate isomerase, glyceraldehyde-3-phosphate dehydrogenase, and
lactate dehydrogenase were from Roche (Mannheim, Germany). 3-
Phosphoglycerate kinase, L-α-glycerol phosphate, tricine, ethylene
glycol tetraacetic acid (EGTA), dithiothreitol (DTT), glycylglycine,
forskolin, DMEM, and adenosine triphosphate were from Sigma-
Aldrich Chemie GmbH (Munich, Germany). Fetal bovine serum
(FBS) and the antibiotic G418 were from Biochrom AG (Berlin,
( )-2-Cyano-1-{[cis-3-(1H-imidazol-4-yl)cyclopentyl]methyl}-3-
[2-(phenoxy)ethyl]guanidine (42). The title compound was prepared
from 17 (0.04 g, 0.24 mmol) and 30 (0.068 g, 0.24 mmol) in MeCN
(4.5 mL) according to the general procedure. Flash chromatography
yielded a yellow solid (0.05 g, 59%); mp 57 °C. 1H NMR (300 MHz,
CD3OD): δ [ppm] = 1.30 (m, 1H, CH2), 1.49 (m, 1H, CH2), 1.70 (m,
1H, CH2), 1.83 (m, 1H, CH2), 2.00 (m, 1H, CH2), 2.18 (m, 1H,
CH2), 2.31 (m, 1H, CH), 3.06 (m, 1H, CH-Im), 3.21 (d, 2H, 3J = 7.0
3
3
Hz, CH2-N), 3.61 (t, 2H, J = 5.3 Hz, N-CH2), 4.07 (t, 2H, J = 5.3
Hz, CH2-O), 6.76 (s, 1H, Im-H-5), 6.92 (m, 3H, Ph-H), 7.25 (m, 2H,
Ph-H), 7.56 (s, 1H, Im-H-2). 13C NMR (75 MHz, CD3OD): δ [ppm]
= 30.10 (−, CH2), 32.91 (−, CH2), 38.81 (−, CH2), 38.95 (+, CH),
40.68 (+, CH), 42.45 (−, N-CH2), 47.87 (−, CH2-N), 67.66 (−, CH2-
O), 115.64 (+, 2 Ph-C), 116.62 (+, Im-C-5), 120.23 (Cquat, CN),
122.14 (+, Ph-C-4), 130.59 (+, 2 Ph-C), 135.88 (+, Im-C-2), 142.04
(Cquat, Im-C-4), 160.13 (Cquat, Ph-C-1), 161.52 (Cquat, CN). HRMS
(EI-MS) calcd for C19H24N6O [M+•] 352.2012; found 352.2008. IR
(cm−1) = 3257 (N-H), 2924, 2868 (C-H), 2160 (CN), 1576 (C
N), 1453, 1239, 1080, 1050. Anal. (C19H24N6O·0.5CH3OH) C, H, N.
C19H24N6O (352.43).
( )-2-Cyano-1-{[trans-3-(1H-imidazol-4-yl)cyclopentyl]methyl}-
3-[2-(phenylamino)ethyl]guanidine (46). The title compound was
prepared from 43 (0.08 g, 0.26 mmol) and N1-phenylethane-1,2-
diamine (45) (0.034 mL, 0.26 mmol) in MeCN (4.5 mL) according to
the general procedure. Flash chromatography yielded a white solid
(0.05 g, 55%); mp 42−43 °C. 1H NMR (300 MHz, CD3OD): δ
[ppm] = 1.31 (m, 1H, CH2), 1.58−1.84 (m, 3H, CH2), 1.90 (m, 1H,
CH2), 2.05 (m, 1H, CH2), 2.32 (m, 1H, CH), 3.14 (m, 3H, CH-Im +
3
3
CH2-N), 3.27 (t, 2H, J = 6.2 Hz, N-CH2), 3.41 (t, 2H, J = 6.2 Hz,
CH2-NH-Ph), 6.63 (m, 3H, Ph-H), 6.79 (s, 1H, Im-H-5), 7.09 (m, 2H,
Ph-H), 7.66 (s, 1H, Im-H-2). 13C NMR (75 MHz, CD3OD): δ [ppm]
= 31.00 (−, CH2), 33.83 (−, CH2), 37.27 (−, CH2), 37.57 (+, CH),
39.52 (+, CH), 42.47 (−, N-CH2), 44.47 (−, CH2-NH-Ph), 47.78 (−,
CH2-N), 114.00 (+, 2 Ph-C), 116.53 (+, Im-C-5), 118.34 (+, Ph-C-4),
120.23 (Cquat, CN), 130.21 (+, 2 Ph-C), 135.80 (+, Im-C-2), 141.86
(Cquat, Im-C-4), 149.85 (Cquat, Ph-C-1), 161.61 (Cquat, CN). HRMS
(LSI-MS (MeOH/glycerol)) calcd for C19H26N7 [M + H]+ 352.2250;
found 352.2249. IR (cm−1) = 3264 (N-H), 2930, 2864 (C-H), 2159
(CN), 1575 (CN), 1499, 1426, 1323, 1257. Anal. (C19H25N7·
0.75CH3OH) C, H, N. C19H25N7 (351.45).
( )-2-Cyano-1-{[cis-3-(1H-imidazol-4-yl)cyclopentyl]methyl}-3-
[2-(phenylamino)ethyl]guanidine (47). The title compound was
prepared from 44 (0.09 g, 0.29 mmol) and 45 (0.038 mL, 0.29 mmol)
in MeCN (4.5 mL) according to the general procedure. Flash
chromatography yielded a pale-yellow solid (0.07 g, 69%); mp 55−57
1
°C. H NMR (300 MHz, CD3OD): δ [ppm] = 1.30 (m, 1H, CH2),
1.45 (m, 1H, CH2), 1.68 (m, 1H, CH2), 1.80 (m, 1H, CH2), 2.01 (m,
1H, CH2), 2.14 (m, 1H, CH2), 2.24 (m, 1H, CH), 3.05 (m, 1H, CH-
3
3
Im) 3.15 (d, 2H, J = 8.4 Hz, CH2-N), 3.27 (t, 2H, J = 5.9 Hz, N-
3
CH2), 3.41 (t, 2H, J = 5.9 Hz, CH2-NH-Ph), 6.63 (m, 3H, Ph-H),
6.76 (s, 1H, Im-H-5), 7.09 (m, 2H, Ph-H), 7.57 (s, 1H, Im-H-2). 13C
NMR (75 MHz, CD3OD): δ [ppm] = 30.10 (−, CH2), 32.90 (−,
N
DOI: 10.1021/acs.jmedchem.6b00120
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Germany). Hygromycin B was from MoBiTec GmbH (Gottingen,
μL of the supernatant were removed and 32Pi was determined by liquid
scintillation counting. Spontaneous [γ-32P]GTP degradation was
determined in tubes containing all components described above plus
a high concentration of unlabeled GTP (1 mM) that due to
competition with [γ-32P]GTP prevents [γ-32P]GTP hydrolysis by
enzymatic activities present in Sf9 membranes. Spontaneous
[γ-32P]GTP degradation was <1% of the total amount of radioactivity
added. The experimental conditions chosen ensured that not more
than 10% of the total amount of [γ-32P]GTP added was converted to
32Pi.
̈
Germany). Triton-X was from Serva Electrophoresis GmbH
(Heidelberg, Germany). D-Luciferin potassium salt was from Synchem
OHG (Felsberg, Germany). Culture flasks were from Nunc GmbH
(Wiesbaden, Germany).
Cells. Sf9 insect cells were from BD Biosciences (BD Biosciences,
Heidelberg, Germany), and human embyronal kidney cells (HEK-293
cells) were from Deutsche Sammlung fur Mikroorganismen and
̈
Zellkulturen (DSMZ, Braunschweig, Germany). The genetically
engineered cells used to perform the functional and binding studies
described in this study were generated and maintained as described (cf.
brief description of protocols and references cited below). Examination
for mycoplasma contamination was routinely performed using the
Venor GeM mycoplasma detection kit (Minerva Biolabs, Berlin,
Germany) and was negative for all cell types.
Radioligand Binding Assays..78,80 For the binding experiments the
Sf9 insect cell membranes described above were employed. The
respective membranes were thawed and sedimented by centrifugation
at 4 °C and 13000g for 10 min. Membranes were resuspended in
binding buffer (12.5 mM MgCl2, 1 mM EDTA, and 75 mM Tris/HCl,
pH 7.4). Each tube (total volume 500 μL) contained 50 μg (hH1R,
hH3R), 120 μg (hH4R), or 250 μg (hH2R) of membrane protein.
Competition binding experiments were performed in the presence of 5
nM [3H]mepyramine (hH1R), 10 nM [3H]tiotidine (hH2R), 3 nM
[3H]Nα-methylhistamine (hH3R), or 10 nM [3H]histamine or 3 nM
[3H]60 (hH4R) and increasing concentrations of unlabeled ligands.
Incubations were conducted for 60 min at 25 °C and shaking at 250
rpm. Bound radioligand was separated from free radioligand by
filtration through 0.3% polyethylenimine-pretreated (PEI) GF/C
filters, followed by three washes with 2 mL of cold binding buffer
(4 °C) using a Brandel harvester. Filter-bound radioactivity was
determined after an equilibration phase of at least 12 h by liquid
scintillation counting.
Luciferase Reporter Gene Assay. The assay was performed as
previously described.33 In brief, HEK293-SF-hH4R-His6-CRE-luc or
HEK293-SF-mH4R-His6-CRE-Luc cells were cultured in DMEM
supplemented with 10% FBS and selection antibiotics (600 μg/mL
G418 and 200 μg/mL hygromycin B) in a water-saturated atmosphere
containing 5% CO2 at 37 °C. Cells were passaged (1:10) twice a week.
For the assay, approximately 2 × 105 cells/well were seeded in 96-well
plates in DMEM + 10% FBS 17 h before the assay. After addition of
increasing concentrations of histaminergic ligands and forskolin (400
nM (hH4R) or 1 μM (mH4R)), the cells were incubated for 5 h.
Thereafter, the medium was discarded, the cells were washed with cold
PBS, and lysed in 40 μL of lysis buffer (25 mM tricine, pH 7.8, 10%
(v/v) glycerol, 2 mM EGTA, 1% (v/v) Triton X-100, 5 mM MgSO4·7
H2O, 1 mM DTT) under shaking (180 rpm) for 30 min.
Luminescence was measured with a Tecan GENios Pro microplate
reader by injection of 80 μL of D-luciferin solution (0.2 mg/mL D-
luciferin potassium salt, 25 mM glycylglycine, 15 mM MgSO4·7H2O,
15 mM KH2PO4, pH 7.8, 4 mM EGTA, pH 7.8, 2 mM ATP, and 2
mM DTT) to 20 μL of the lysate.
[35S]GTPγS Binding Assay.69,70 [35S]GTPγS binding assays were
performed as previously described for the H2R,71,72 H3R,73,74 and
H4R.75 H2R assays: Sf9 insect cell membranes expressing the hH2R-
GsαS fusion protein were employed. H3R assays: Sf9 insect cell
membranes coexpressing the hH3R, mammalian Gαi2 and Gβ1γ2 were
employed. H4R assays: Sf9 insect cell membranes coexpressing the
hH4R, mammalian Gαi2 and Gβ1γ2 were employed.
The respective membranes were thawed and sedimented by a 10
min centrifugation at 4 °C and 13000g. 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 μg protein/tube), 1 μM GDP,
0.05% (w/v) bovine serum albumin, 0.2 nM [35S]GTPγS, and the
investigated ligands (dissolved in Millipore water or in a mixture (v/v)
of 80% Millipore water and 20% DMSO) at various concentrations in
binding buffer (total volume 250 μL). All H4R assays additionally
contained 100 mM NaCl. For the determination of KB values
(antagonist mode of the [35S]GTPγS binding assay), histamine was
added to the reaction mixtures (final concentrations: H2R, 1 μM;
H3/4R, 100 nM). Incubations were conducted for 90 min at 25 °C and
shaking at 250 rpm. Bound [35S]GTPγS was separated from free
[35S]GTPγS by filtration through GF/C filters, followed by three
washes with 2 mL of binding buffer (4 °C) 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]GTPγS added was bound to filters. Nonspecific binding was
determined in the presence of 10 μM unlabeled GTPγS.
Steady-State GTPase Activity Assay. GTPase activity assays were
performed as previously described.75−79 H1R assays: Sf9 insect cell
membranes coexpressing the hH1R and RGS4 were employed. H2R
assays: Sf9 insect cell membranes expressing the hH2R-GsαS fusion
protein were used. H3R assays: Sf9 insect cell membranes coexpressing
the hH3R, and mammalian Gαi2, Gβ1γ2 and RGS4 were employed.
H4R assays: Sf9 insect cell membranes coexpressing the hH4R-GAIP
fusion protein, and mammalian Gαi2 and Gβ1γ2 were used. The
respective membranes were thawed and sedimented by centrifugation
at 4 °C and 13000g for 10 min. Membranes were resuspended in 10
mM Tris/HCl, pH 7.4. Each assay tube contained Sf9 membranes
expressing the respective HR subtype (10−20 μg protein/tube),
MgCl2 (H1,2R assays, 1.0 mM; H3,4R assays, 5.0 mM), 100 μM EDTA,
100 μM ATP, 100 nM GTP, 100 μM adenylyl imidophosphate, 5 mM
creatine phosphate, 40 μg creatine kinase, and 0.2% (w/v) bovine
serum albumin in 50 mM Tris/HCl, pH 7.4, and the investigated
ligands at various concentrations. All H4R assays additionally
contained 100 mM NaCl. For the determination of pKB values
(antagonist mode of the GTPase activity assay), histamine was added
to the reaction mixtures (final concentrations: H1R, 200 nM; H2R, 1
μM; H3,4R, 100 nM). Reaction mixtures (80 μL) were incubated for 2
min at 25 °C. After the addition of 20 μL of [γ-32P]GTP (0.1 μCi/
tube), reaction mixtures were incubated for 20 min at 25 °C. Reactions
were terminated by the addition of 900 μL of slurry consisting of 5%
(w/v) activated charcoal and 50 mM NaH2PO4, pH 2.0. Charcoal
absorbs nucleotides but not Pi. Charcoal-quenched reaction mixtures
were centrifuged for 7 min at room temperature at 13000g. Then 600
Data Analysis and Pharmacological Parameters. All data are
presented as mean of N independent experiments
SEM. Agonist
potencies were given as EC50 values (molar concentration of the
agonist causing 50% of the maximal response). Maximal responses
(intrinsic activities) were expressed as α values. The α value of
histamine was set to 1.00, and α values of other compounds were
referred to this value.
IC50 values were converted to Ki and KB values using the Cheng−
Prussoff equation.81 Ki values were analyzed by nonlinear regression
and best fit to one-site (monophasic) competition isotherms. EC50 and
KB values from the functional [35S]GTPγS, GTPase, and luciferase
gene reporter assays were analyzed by nonlinear regression and best fit
to sigmoidal dose−response curves (GraphPad Prism 5.0 software, San
Diego, CA).
Molecular Modeling. Inactive-State Model of the hH4R. The
crystal structure of hH1R 3RZE,82 with the antagonist doxepine bound,
was used as a template to construct the inactive state of hH4R by
homology modeling, as already described.83 Briefly, the lysozyme,
which was artificially cocrystallized in 3RZE, was deleted. The E2-loop
is not completely available in the hH1R crystal. Thus, for modeling of
the E2-loop, the amino acids Trp165, Asn166, and His167 of the hH1R
crystal were removed at first. Afterward, the missing amino acids
(hH4R: Val153-Lys158) were included into the models, using the “Loop-
O
DOI: 10.1021/acs.jmedchem.6b00120
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Search” module from SYBYL 7.0 (Tripos Inc.). The N-Terminus,
which is also missing in the crystal structure, was added using SYBYL
7.0 (Tripos Inc.) according to the following protocol:84 The first 15
(hH4R) amino acids of the N-terminus were added to the receptor
models with SYBYL, using a random conformation for the backbone.
Afterward, position restraints were set onto the whole inactive receptor
model except the added amino acids of the N-terminus and the amino
acids of the E2-loop, added by Loop-Search, as described above. The
structures were energetically minimized using SYBYL. Subsequently,
short gas phase MD simulations (500 ps) of the N-terminus and
inserted amino acids of the E2-loop with the same positions restraints,
as used for the minimization, were performed. The resulting model for
the inactive hH4R was used for insertion of the missing I3-loop.
Because of the lack of information concerning the conformation of the
long I3-loop of the hH4R, the amino acids Gly215 to His292 (hH4R)
were not included in the models. To close the gap between the
intracellular parts of TM V and TM VI, eight alanines were inserted
instead at hH4R. It was shown that internal water molecules play an
important role in stabilization or activation of aminergic GPCRs.85,86
Thus, these internal water molecules were included into the model of
inactive hH4R based on the 2RH1 crystal structure.82 The compounds
39a and 39b, with a positively charged histamine moiety, were docked
manually into the orthosteric binding pocket of the hH4R in an
analogous manner as described previous.37 The force field parameters
of the compounds were obtained from the PRODRG server (http://
davapc1.bioch.dundee.ac.uk/prodrg/).87 The partial charges according
acids 285−292 (Gαi2), 309−315 (Gαi2), and 318−321 (Gαi2) were
included by loops search. The loops 285−292 (Gαi2) and 309−315
(Gαi2) were chosen that were most similar to the corresponding
structures of Gαi1 (1GP2). However, it was taken care that none of
the included loops lead to collisions with the remaining part of the
model. Because the focus within the study is the binding mode of
selected compounds in the active state hH4R, the Gβγ was included, as
given in the 3SN6 crystal. The resulting complex was energetically
minimized. Afterward, the minimized receptor model was embedded
into a POPC lipid bilayer, as described previously.84 Intracellular and
extracellular water, as well as an appropriate number of sodium and
chloride ions, were added into the simulation box, and the molecular
dynamic simulations, using GROMACS 4.0.2 (http://www.gromacs.
org), were performed as already described.83 For the POPC lipid, the
force field parameters of the online source (http://moose.bio.ucalgary.
ca/index.php?page=Structures_and_Topologies) were used. Subse-
quent to a 5 ns equilibration phase for each system (force constants
(250 kJ/(mol nm2) for the first 2.5 ns and 100 kJ/(mol nm2) for the
second 2.5 ns) were put onto the backbone atoms of the TM domains
of hH4R), the 15 ns productive phases were performed.
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmed-
chem.6b00120. CCDC 965185 and CCDC 965186 contain
the supplementary crystallographic data for cis-(1R,3S)-53·H2O
and cis-(1R,3S)-17·2HCl, respectively (available free of charge
at The Cambridge Crystallographic Data Centre via www.ccdc.
cam.ac.uk/data_request/cif).
to Gasteiger and Huckel, calculated by SYBYL, were assigned to 39a
̈
and 39b. The resulting complex was energetically minimized.
Afterward, the minimized receptor model was embedded into a
POPC lipid bilayer, as described previously.83 Intracellular and
extracellular water, as well as an appropriate number of sodium and
chloride ions, were added into the simulation box and the molecular
dynamic simulations, using GROMACS 4.0.2 (http://www.gromacs.
org), were performed as already described.83 For the POPC lipid, the
force field parameters available at an appropriate online source
(http://moose.bio.ucalgary.ca/index.php?page=Structures_and_
Topologies) were used. Subsequent to a 5 ns equilibration phase for
each system (force constants (250 kJ/(mol nm2) for the first 2.5 ns
and 100 kJ/(mol nm2) for the second 2.5 ns) were put onto the
backbone atoms of the TM domains of hH4R), and the 50 ns
productive phases were performed.
Synthetic procedures and analytical data for compounds
16−17, 31−36, 43, 44, 62−65, HPLC purity data of all
target compounds, agonism of enantiomers in the
luciferase reporter gene assay at the mH4R, and
alignment of Gαs and Gαi2 (PDF)
Molecular formula strings (CSV)
AUTHOR INFORMATION
Corresponding Author
* Phone: (+49)941-9434827. Fax: (+49)941-9434820. E-mail:
armin.buschauer@ur.de.
■
Active-State Model of hH4R-Gαi2βγ. The active state model of the
hH4R−Gαiβγ-complex was modeled by homology modeling based on
the crystal structure of the hβ2R−Gαsβγ complex (3SN6).88 The
hH4R within this complex was modeled in an analogous manner to the
inactive model of the hH4R as described above and 39a was docked as
described above. The homology modeling of the Gα-subunit is based
on the amino acid alignment between Gαs and Gαi2 (cf. Supporting
Information) and was performed with SYBYL according the following
protocol: The following sequences were directly mutated (Gαs →
Gαi2): 9−59 → 2−52, 88−202 → 66−180, 205−255 → 183−233,
263−301 → 241−279, 306−308 → 282−284, 335−350 → 293−308,
354−355 → 316-317, and 361−394 → 322−355. The missing amino
acid Met1 was added. The amino acids 181−182 and 234−240 were
added by loop search because the corresponding amino acids were
missing in the 3SN6 crystal. The amino acids Met60-Glu88 of the Gαs
subunit were also missing in the 3SN6 crystal, thus the amino acids
53−65 were also added by loop search. Because the loop search of
loops >10 amino acids may lead to structural uncertainties, the loop
which was most similar to the corresponding part of the crystal
structure of Gαi1 (1GP2)89 was included into the homology model.
Because of the different numbers of amino acids, the amino acids 302−
305 of the Gαs-subunit in the 3SN6 crystal were deleted and the
amino acids Thr280-His281 of Gαi2 were included by loop search. The
alignment (cf. Supporting Information) shows that the corresponding
regions 309−334 (Gαs) → 285−292 (Gαi2), 351−353 (Gαs) →
309−315 (Gαi2), and 356−360 (Gαs) → 318−321 (Gαi2) between
Gαs and Gαi2 are of different length. Thus, instead of a direct
homology modeling, the amino acids 309−334, 351−353, and 356−
360 of the Gαs-subunit of the 3SN6 crystal were deleted. The amino
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We are grateful to Maria Beer-Kroen, Gertraud Wilberg, Dr.
Sebastian Wittmann, Helena Konkel, Dr. Manfred Zabel, and
Nikola Kastner-Pustet for expert technical assistance, to Dr.
Patrick Igel for helpful discussions and providing compounds 1
and [3H]60, and to Edith Bartole and Dr. Paul Baumeister for
resynthesis and pharmacological characterization of various
reference compounds, including 1 and [3H]60. This work was
supported by the Graduate Training Programs GRK 760 and
GRK 1910 (“Medicinal Chemistry of Selective GPCR
Ligands”) of the Deutsche Forschungsgemeinschaft.
ABBREVIATIONS USED
■
α, intrinsic activity (corresponding to the maximal response
(Emax) in the context of functional pharmacological data); aq,
aqueous; Boc, tert-butoxycarbonyl; DCM, dichloromethane;
DMF, dimethylformamide; DMSO, dimethyl sulfoxide; EDTA,
ethylenediaminetetraacetic acid; EI-MS, electron-impact ioniza-
tion mass spectrometry; ES-MS, electrospray ionization mass
spectrometry; GTP, guanosine 5′-triphosphate; GTPγS,
P
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Article
(14) Yu, F.; Bonaventure, P.; Thurmond, R. L. The future
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Med. Biol. 2010, 709, 125−140.
guanosine 5′-thiotriphosphate; GPCR, G protein-coupled
receptor; Gβ1γ2, G protein β1- and γ2-subunit; Gαi, α-subunit
of the Gi protein that mediates inhibition of adenylyl cyclase;
HMBC, heteronuclear multiple bond correlation; H1R,
histamine H1 receptor; H2R, histamine H2 receptor; H3R,
histamine H3 receptor; H4R, histamine H4 receptor; hH1R,
human H1R; hH2R, human H2R; hH2R-GsαS, fusion protein of
the hH2R and the short splice variant of Gsα; hH3R, human
H3R; hH4R, human H4R; mH4R, mouse H4R; HR-MS, high
resolution mass spectrometry; HSQC, heteronuclear single
quantum coherence; LSI-MS, liquid-secondary-ion mass
spectrometry; NOESY, nuclear Overhauser enhancement
spectroscopy; RGS, regulator of G protein signaling proteins;
SEM, standard error of the mean; TM, transmembrane domain
of a GPCR; THF, tetrahydrofuran
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