The bivalent ligand approach leads to highly potent and selective acylguanidine-type histamine H2 receptor agonists

Article abstract of DOI:10.1021/jm201128q

Bivalent histamine H₂ receptor (H₂R) agonists were synthesized by connecting pharmacophoric 3-(2-amino-4-methylthiazol-5-yl)-, 3-(2-aminothiazol-5-yl)-, 3-(imidazol-4-yl)-, or 3-(1,2,4-triazol-5-yl) propylguanidine moieties by N^G-acylation with alkanedioic acids of various chain lengths. The compounds were investigated for H₂R agonism in GTPase and [³⁵S]GTPγS binding assays at guinea pig (gp) and human (h) H₂R-Gsα_S fusion proteins including various H₂R mutants, at the isolated gp right atrium, and in GTPase assays for activity on recombinant H₁, H₃, and H₄ receptors. The bivalent ligands are H₂R partial or full agonists, up to 2 orders of magnitude more potent than monovalent acylguanidines and, with octanedioyl or decanedioyl spacers, up to 4000 times more potent than histamine at the gpH₂R. In contrast to their imidazole analogues, the aminothiazoles are highly selective for H₂R vs other HR subtypes. Compounds with (theoretically) sufficient spacer length (20 CH₂ groups) to simultaneously occupy two orthosteric binding sites in H₂R dimers are nearly inactive, whereas the highest potency resides in compounds with considerably shorter spacers. Thus, there is no evidence for interaction with H₂R dimers. The high agonistic potency may result from interaction with an accessory binding site at the same receptor protomer.

Full text of DOI:10.1021/jm201128q

Article
pubs.acs.org/jmc
The Bivalent Ligand Approach Leads to Highly Potent and Selective
Acylguanidine-Type Histamine H2 Receptor Agonists†
Tobias Birnkammer,‡ Anja Spickenreither,‡ Irena Brunskole,‡ Miroslaw Lopuch,‡ Nicole Kagermeier,‡
Gunther Bernhardt,‡ Stefan Dove,‡ Roland Seifert,§ Sigurd Elz,‡ and Armin Buschauer*,‡
̈
‡Department of Pharmaceutical/Medicinal Chemistry, Faculty of Chemistry and Pharmacy, University of Regensburg,
Universitatsstrasse 31, D-93053 Regensburg, Germany
̈
§Institute of Pharmacology, Medical School of Hannover, D-30625 Hannover, Germany
S
* Supporting Information
ABSTRACT: Bivalent histamine H2 receptor (H2R) agonists
were synthesized by connecting pharmacophoric 3-(2-amino-
4-methylthiazol-5-yl)-, 3-(2-aminothiazol-5-yl)-, 3-(imidazol-4-
yl)-, or 3-(1,2,4-triazol-5-yl)propylguanidine moieties by NG-
acylation with alkanedioic acids of various chain lengths. The
compounds were investigated for H2R agonism in GTPase and [35S]GTPγS binding assays at guinea pig (gp) and human (h)
H2R-GsαS fusion proteins including various H2R mutants, at the isolated gp right atrium, and in GTPase assays for activity on
recombinant H1, H3, and H4 receptors. The bivalent ligands are H2R partial or full agonists, up to 2 orders of magnitude more
potent than monovalent acylguanidines and, with octanedioyl or decanedioyl spacers, up to 4000 times more potent than
histamine at the gpH2R. In contrast to their imidazole analogues, the aminothiazoles are highly selective for H2R vs other HR
subtypes. Compounds with (theoretically) sufficient spacer length (20 CH2 groups) to simultaneously occupy two orthosteric
binding sites in H2R dimers are nearly inactive, whereas the highest potency resides in compounds with considerably shorter
spacers. Thus, there is no evidence for interaction with H2R dimers. The high agonistic potency may result from interaction with
an accessory binding site at the same receptor protomer.
INTRODUCTION
■
The histamine H2 receptor (H2R), a member of class A G-
protein-coupled receptors (GPCRs), is a well-established target
for the treatment of gastric and duodenal ulcers using
antagonists such as cimetidine or famotidine.1,2 H2R agonists
are important pharmacological tools to study the physiological
and pathophysiological role of this histamine receptor.
Although numerous compounds were described as H2R
agonists decades ago, after discovery of the histamine H3
(H3R) and H4 receptor (H4R), the H2R selectivity of
compounds such as 5-methylhistamine,1 dimaprit,3 impromi-
dine4 (3, Figure 1), or arpromidine5 and related imidazolyl-
propylguanidines6 turned out to be compromised.7−9 Most
strikingly, 5-methylhistamine is nowadays considered as
selective for the H4R.7 Recently, in search for H2R agonists
Figure 1. Histamine, selected H2R agonists, and general structure of
bivalent hetarylpropylguanidines.
derived from guanidine-type compounds such as 3,10 NG-
acylated hetarylpropylguanidines (e.g., 4) were identified in our
Over the past few decades the understanding of GPCR
laboratory as a new class of potent H2R agonists with
structure and function has been challenged by the discovery
considerably reduced basicity.10,11 These acylguanidines proved
that GPCRs are able to form homo- and hetero-oligomeric
to be highly selective for the H2R, when the imidazole ring was
complexes.14−17 The existence of homo- and heterodimers has
been demonstrated for several class A GPCRs including opioid
receptors,18−20 adrenergic receptors,21 somatostatin recep-
tors,22,23 dopaminergic receptors,24−26 muscarinergic recep-
replaced with an amino(methyl)thiazole moiety10 as in
amthamine12 (2). The structure−activity relationships revealed
that even space-filling substituents at the guanidine group were
well tolerated. This prompted us to explore the applicability of
the bivalent ligand approach, based on the working hypothesis
that such compounds should possess increased H2R agonistic
potency and could be useful to study H2R dimers.13
Received: August 23, 2011
Published: January 5, 2012
© 2012 American Chemical Society
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Scheme 1. General Procedure for the Preparation of the Boc-Protected Hetarylpropylguanidine Building Blocks 12−14 and the
a
Synthesis of the Symmetrical Bivalent Acylguanidines 15−30
a
Reagents and conditions: (i) HgCl2 (2 equiv), NEt3 (3 equiv), DCM/abs, 48 h, rt; (ii) H2, Pd/C (10%), MeOH/THF (1:1), 8 bar, 8−9 days (12,
13) or 3−4 days (14), rt; (iii) EDAC (1 equiv), HOBt (1 equiv), DIEA (1 equiv), DCM/abs, 16 h, rt; (iv) 20% TFA, DCM/abs, 3−5 h, rt.
tors,27,28 and the histamine receptor subtypes.13,29−34 The term
bivalent ligand is widely used and refers to molecules
containing two sets of pharmacophoric entities linked through
a spacer.35−37 It is assumed that duplication of the
pharmacophoric groups according to the bivalent ligand
approach leads to a supra-additive increase in potency
compared to the corresponding monovalent ligand..35,36 This
concept has been studied for various GPCRs,37 for instance, for
opioid receptors38 or aminergic GPCRs such as serotonin or
dopamine receptor subtypes,39−42 in more detail. The bivalent
ligand approach in the design of ligands targeting GPCRs has
proven to be promising to improve not only potency and
selectivity but also the pharmacokinetic profile of compounds.43
For opioid receptors, the distance between two recognition
sites of a contact dimer with a TM5/TM6 interface is about
22−27 Å, as suggested from molecular modeling.35 In an
approach to explore the structural requirements of putative
bivalent H2R agonists we synthesized and pharmacologically
investigated compounds with two pharmacophoric hetarylpro-
pylguanidine entities linked at the NG-nitrogen atoms with
dicarboxylic acids as spacers with lengths between 6 and 27 Å
(Figure 1).
zotriazole (HOBt), and N,N-diisopropylethylamine (DIEA) as
standard coupling reagents to yield the protected compounds
15a−30a. Thereby, the Boc-protected guanidine (12−14), at
its terminal position (NG), reacts similarly to a primary amine
but at lower reaction rate. Finally, removal of the protecting
groups under acidic conditions gave the symmetrical
acylguanidines 15−30 (Scheme 1), which were purified by
preparative RP-HPLC.
The unsymmetrical ligands 33 and 34 were synthesized as
depicted in Scheme 2, using the decanedioyl spacer. To reduce
the formation of byproduct, one carboxylic function of the
dicarboxylic acid was capped with a benzyl group, and the
resulting 10-benzyloxy-10-oxodecanoic acid (31) was coupled
to the 2-amino-4-methylthiazol-5-ylpropylguanidine building
block 12. After hydrogenolysis of the benzyl ester group, the
free carboxylic group of 32 was coupled to the Boc-protected
guanidine building blocks 13 and 14, respectively. In addition,
the bivalent NG-acylated 1,2,4-triazol-5-ylpropylguanidine 35
was synthesized starting from the corresponding Trt-protected
triazolylpropylguanidine building block (cf. Scheme 2).45 In
contrast to the aforementioned acylation steps, this guanidine
building block was deprotonated with NaH and coupled to
decanedioic acid, which was activated by CDI, to yield the Trt-
protected precursor 35a. Final deprotection with trifluoroacetic
acid (TFA) and purification by preparative RP-HPLC yielded
the acylguanidines 33−35.
CHEMISTRY
■
The bivalent acylguanidine-type compounds were preferentially
synthesized by analogy with the procedures developed for the
NG-acylation of monovalent imidazolyl- and aminothiazolyl-
propylguanidines.10,11 The primary amines 5−7 were treated
with the orthogonally protected isothiourea 8, yielding the
guanidines 9−11, which were subjected to hydrogenolytic
cleavage of the benzyloxycarbonyl (Cbz) group to give the tert-
butoxycarbonyl (Boc) protected 2-aminothiazolylpropylguani-
dines 1210 and 1310 as well as the NG-Boc-,NIm-Trt-protected
imidazolylpropylguanidine 1444 in good yields (Scheme 1). To
obtain the designated symmetrical bivalent ligands 15−30, the
mono Boc-protected hetarylpropylguanidines 12−14 were
coupled to alkanedioic acids of various length using 1-ethyl-3-
(3-dimethylaminopropyl)carbodiimide (EDAC), hydroxyben-
RESULTS AND DISCUSSION
■
The synthesized compounds were examined for histamine H2R
agonism on human (h) and guinea pig (gp) H2 receptors in
steady-state GTPase assays using membranes of Sf9 insect cells
expressing hH2R-GsαS and gpH2R-GsαS fusion proteins,
respectively (Table 1).46 By use of this approach, agonists of
Gs-coupled receptors can be studied directly at the level of
receptor/G-protein coupling with high sensitivity.47,48 In
addition, selected compounds were investigated on the isolated
spontaneously beating guinea pig right atrium1 as a
pharmacological standard model for the functional character-
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methylthiazolyl)propylguanidine entities (15−21), two (2-
aminothiazolyl)propylguanidine entities (22−25), or two
imidazolylpropylguanidine entities (26−30) revealed the
following results: When increasing the spacer length from 4
to 20 C-atoms, covering a distance of ∼6 to ∼27 Å between the
carbonyl groups, the highest potencies were obtained with
octanedioyl to decanedioyl spacers at both the hH2R-GsαS
Scheme 2. Synthesis of Building Block 32 and Bivalent
Acylguanidines 33−35
a
(pEC50 ≤ 8.2) and the gpH2R-GsαS fusion proteins (pEC50
≤
9.4). Further extension of the spacer length resulted in a
significant drop in potency or in a complete loss of agonistic
activity at hH2R-GsαS and switch to H2R antagonism (pKB
values: 21, 6.1; 25, 5.8; 30, 6.6).
Homobivalent 2-aminothiazoles lacking the 4-methyl sub-
stituent showed slightly decreased potencies but increased
efficacies compared to their methylated analogues (22 vs 16, 23
vs 18, and 24 vs 20) at both hH2R-GsαS and the gpH2R-GsαS.
Compounds 22 and 23 were full agonists at gpH2R-GsαS.
Compared to the corresponding 2-amino-4-methylthiazoles,
most imidazoles (27−30) were nearly equipotent at hH2R-
GsαS and slightly less potent at gpH2R-GsαS. Compound 35
bearing two 1,2,4-triazole rings showed up to 2 orders of
magnitude lower potencies compared to the corresponding
aminothiazoles 18, 23 and the imidazole 28 at hH2R-GsαS and
gpH2R-GsαS, respectively. Furthermore, the imidazoles re-
vealed the highest efficacies among the four structural classes,
resulting in full agonists at gpH2R-GsαS.
To further elaborate the contribution of the different
pharmacophores with respect to potency and efficacy, two
unsymmetrical compounds with one 2-amino-4-methylthiazole
moiety (33 and 34) were investigated (Figure 2). Interestingly,
the potencies at both receptors were always between the
potencies of the symmetrical analogues (compare 33 with 18
and 23, 34 with 18 and 28). In contrast, the efficacies of 33 and
34 were close to the high efficacies of the corresponding “bis-
imidazole” 28 and “bis-aminothiazole” 23, respectively. In
conclusion, both heterocycles of the unsymmetrical compounds
nearly additively contribute to potency, whereas efficacy seems
to be determined by the “more efficacious moiety”.
In agreement with previous studies on monovalent
acylguanidine-type H2R agonists, all bivalent compounds
exhibit higher potencies and efficacies at gpH2R-GsαS relative
to hH2R-GsαS (see Figure 3).46,55 Interestingly, compounds 15,
16, 18, and 22 are 20−76 times more potent at the gpH2R-
GsαS compared to hH2R-GsαS and exhibit the highest
selectivity toward gpH2R-GsαS among all the acylguanidines
from our laboratory. Compounds 16, 18, and 34 (EC50 values
at gpH2R-GsαS: 0.63, 0.39, and 0.51 nM, respectively) are the
most potent acylguanidine-type H2R agonists identified in the
GTPase assay.
When membrane preparations are used, G-proteins are
directly accessible to the investigated compounds; i.e., the
possibility of H2R-receptor-independent G-protein activa-
tion56−58 has to be taken into account. Therefore, selected
compounds were investigated in the presence of the H2R
antagonist famotidine in GTPase assays as shown for 33 in
Figure 4. At both, hH2R-GsαS and gpH2R-GsαS, 33-stimulated
GTP hydrolysis was inhibited in a concentration-dependent
manner by famotidine, confirming the measured GTPase
activity to be stimulated via the H2R. The calculated KB values
of famotidine (52 22 and 65 32 nM, Figure 4) determined
against 33 at hH2R-GsαS and gpH2R-GsαS, respectively, are
comparable to data obtained from GTPase assays using
a
Reagents and conditions: (i) BnOH (1 equiv), DCC (1.2 equiv),
DMAP (cat.), THF/abs, 48 h, rt; (ii) EDAC (1 equiv), HOBt (1
equiv), DIEA (1 equiv), DCM/abs, 16 h, rt; (iii) H2, Pd/C (10%),
MeOH, 1 h, rt; (iv) EDAC (1 equiv), HOBt (1 equiv), DIEA (1
equiv), DCM/abs, 16 h, rt; (v) CDI (1.2 equiv), NaH (60% dispersion
in mineral oil) (2 equiv), THF/abs, 3 h, rt; (vi) 20% TFA, DCM/abs,
3 h, rt.
ization of H2R ligands (positive chronotropic response) (Table
2) and in the GTPγS binding assay on gpH2R-GsαS fusion
proteins. The GTPγS binding assay is a valid alternative to the
GTPase assay and yields similar results.49 In addition to
occupation of the orthosteric binding site, interactions of the
bivalent ligands with individual amino acids or sequences in
other regions of the same receptor protomer, especially N-
terminus and extracellular loops, are conceivable. Different
potencies at human and guinea pig H2R orthologues might
reflect such interactions. Therefore, selected bivalent ligands
were examined on H2R mutants/chimera, in which Cys-17 and
Ala-271 in the hH2R were replaced by Tyr-17 and Asp-271 as in
the gpH2R (hH2R-C17Y-A271D-GsαS, hH2R-C17Y-GsαS),50,51
the four different amino acids in the e2 loop were reciprocally
mutated (hH2R-gpE2-GsαS, gpH2R-hE2-GsαS),52 and the N-
terminus of the hH2R was replaced by the N-terminus of the
gpH2R (hH2R-gpNT-GsαS) (Figure 8, data in Supporting
Information). Moreover, the histamine receptor subtype
selectivities of representative compounds were explored in
GTPase assays using recombinant human histamine H1, H3,
and H4 receptors (Table 3). In the following, on the basis of the
data from functional assays, the terms potency (expressed as
pEC50 of an agonist) and efficacy (intrinsic activity of an
agonist, expressed as Emax relative the maximal response
induced by histamine) and antagonistic activity (expressed as
pKB) characterize agonists and antagonists, respectively.53
Agonism at Human and Guinea Pig H2R-GsαS Fusion
Proteins. Pharmacophore duplication led to potent partial to
full agonists in the GTPase assay at hH2R-GsαS and gpH2R-
GsαS fusion proteins (Table 1). Investigations of three different
series of symmetrical compounds containing two (2-amino-4-
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Table 1. Agonist Efficacies and Potencies of Bivalent Acylguanidines and Reference Compounds at hH2R-GsαS and gpH2R-GsαS
a
Fusion Proteins Expressed in Sf9 Cell Membranes
hH2R-GsαS
gpH2R-GsαS
compd
n
Emax SEM
pEC50/(pKB) SEM
Potrel
Emax SEM
pEC50 SEM
Potrel
EC50(hH2R-GsαS)/EC50(gpH2R-GsαS)
151
251
410
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
33
34
35
1.00
5.90 0.09
6.72 0.10
7.69 0.13
7.24 0.22
7.32 0.23
7.35 0.13
8.11 0.25
7.78 0.17
7.59 0.22
1.0
1.00
5.92 0.09
6.72 0.09
8.13 0.05
8.59 0.30
9.20 0.16
8.56 0.16
9.41 0.15
8.57 0.32
7.46 0.01
6.48 0.37
8.87 0.28
8.30 0.22
7.23 0.19
6.69 0.01
7.96 0.07
8.49 0.33
8.94 0.16
7.70 0.26
7.46 0.12
8.46 0.30
9.29 0.10
7.99 0.02
1.0
1.16
0.91 0.02
0.79 0.02
0.68 0.03
0.62 0.03
0.48 0.04
0.53 0.04
0.46 0.04
0.12 0.02
6.6
1.04 0.01
0.76 0.02
0.90 0.05
0.81 0.03
0.90 0.06
0.79 0.07
0.66 0.05
0.51 0.02
0.58 0.02
1.00 0.03
0.94 0.01
0.59 0.01
0.36 0.01
1.00 0.02
1.18 0.01
0.98 0.05
0.85 0.10
0.19 0.03
0.89 0.04
1.01 0.03
0.95 0.04
6.3
1.00
61.7
21.9
26.3
28.2
162.2
75.9
49.0
162.2
467.7
1905
436.5
3090
446.7
34.7
2.75
4
22.39
75.97
16.24
19.90
6.17
6
7
8
10
14
20
6
0.74
b
6.11 0.15
3.6
0.79 0.03
0.75 0.03
0.14 0.01
7.51 0.02
7.67 0.07
7.03 0.13
40.7
58.9
13.5
891.3
239.9
20.4
22.89
4.27
1.58
8
14
20
4
b
5.77
5.9
0.68 0.04
0.77 0.12
0.81 0.02
0.29 0.08
6.67 0.34
7.25 0.16
8.21 0.07
7.61 0.18
5.9
109.7
371.5
1047
60.4
19.51
17.35
5.36
6
22.4
204.2
51.3
8
14
20
8
1.23
b
6.57 0.07
34.7
0.75 0.04
0.76 0.05
0.49 0.03
7.86 0.11
8.12 0.04
6.82 0.05
91.2
166.0
10.5
346.7
2344
117.5
3.98
8
14.88
8
14.79
a
Steady state GTPase activity in Sf9 membranes expressing hH2R-GsαS and gpH2R-GsαS was determined as described.46 Reaction mixtures
contained ligands at concentrations from 0.1 nM to 10 μM as appropriate to generate saturated concentration−response curves. Data were analyzed
by nonlinear regression and were best fit to sigmoidal concentration−response curves. Typical basal GTPase activities ranged between ∼0.5 and 2.5
pmol·mg−1·min−1, and activities stimulated by histamine (1) at a concentration of 100 μM ranged between ∼2 and 13 pmol·mg−1·min−1. The intrinsic
activity (Emax) of 1 was determined by nonlinear regression and was set to 1.0. The Emax values of other agonists were referenced to this value. For
determination of antagonism, reaction mixtures contained histamine (1) (100 nM) and ligands were at concentrations from 10 nM to 1 mM. Data
were analyzed by nonlinear regression and were best fitted to sigmoidal concentration−response curves. Typical basal GTPase activities ranged
between ∼1.5 and 2.5 pmol·mg−1·min−1, and activities stimulated by 1 (10 μM) ranged between ∼3.5 and 4.5 pmol·mg−1·min−1. Data shown are the
mean SEM of two to six independent experiments performed in duplicate. The relative potency of 1 was set to 1.0, and the potencies of other
b
agonists were referenced to this value. No agonistic activity. IC50 values were converted to pKB values using the Cheng−Prusoff equation.54
histamine as the H2R agonist (reported KB values: hH2R-GsαS,
48 10 nM; gpH2R-GsαS, 38 3 nM).59
19, 20, and 21) decreases substantially in the organ assay
compared with the GTPase assay. The combination of two
hetarylpropylguanidine moieties with octanedioyl, nonanedioyl,
or decanedioyl spacer (16, 17, 18, and 28) leads to the most
potent agonists at the guinea pig right atrium known so far,
surpassing up to 4000 times the potency of histamine in
increasing heart rate. In contrast to the GTPase assay, the
equilibration on the guinea pig atrium was extremely slow. For
the generation of cumulative concentration−response curves,
the incubation periods after addition of the H2R agonists at
concentrations below 10 nM had to be extended to 120−180
min. The positive chronotropic response was mediated by the
H2R, since it could be blocked by the H2R antagonist
cimetidine (10−300 μM, data not shown). Characteristic
concentration−response curves are shown in Figure 6 for
histamine, compound 15 alone, and 15 in the presence of
cimetidine.
For comparison, examples of acylguanidines were addition-
ally investigated in GTPγS binding assays using membrane
preparations of Sf9 cells expressing the gpH2R-GsαS fusion
protein (cf. Figure 5). The determined pEC50 values and the
intrinsic activities are in good agreement with the data from the
GTPase assay.
Histamine H2R Agonism on the Guinea Pig Right
Atrium. In addition to the studies on membrane preparations,
representative bivalent H2R agonists were investigated on the
isolated spontaneously beating guinea pig right atrium as a
more complex, well established standard model for the
characterization of H2R ligands. The obtained data (Table 2)
are largely comparable with the results from the GTPase assays
on the gpH2R-GsαS fusion protein in terms of both potencies
and intrinsic activities. The structure−activity relationships are
similar to those derived from the GTPase assay. However, the
agonist potency of the long chain members of the series (viz.
Are the Binding Sites Located at Two Protomers of an
H2R Dimer or at a Single H2R Protomer? The structure−
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Table 2. Histamine H2 Receptor Agonism at the
Spontaneously Beating Guinea Pig Right Atrium
a
b
c
compd
pEC50 SEM
Potrel
Emax SEM
1
6.00 0.02
6.21 0.09
8.59 0.07
9.61 0.03
9.08 0.05
8.93 0.12
6.56 0.05
6.26 0.14
5.10 0.13
9.22 0.06
1.0
1.6
389
1.0
260
15
16
17
18
19
20
21
28
0.95 0.02
0.88 0.03
0.64 0.03
0.71 0.05
0.62 0.03
0.46 0.04
0.53 0.11
0.62 0.07
0.91 0.04
4070
1210
847
3.66
1.82
0.13
1640
a
pEC50 was calculated from the mean corrected shift ΔpEC50 of the
agonist curve relative to the histamine reference curve by equation
pEC50 = 6.00 + ΔpEC50; data shown are the mean SEM of three to
b
c
five experiments. Potency relative to histamine (1) (=1). Intrinsic
activity: maximal response relative to the maximal increase in heart
rate induced by the reference compound histamine (=1.0).
activity relationships of bivalent H2R agonistic acylguanidines,
resulting from GTPase, GTPγS binding, and guinea pig right
atrium assays, are not compatible with the possible role of such
ligands as compounds “bridging” the recognition (orthosteric)
sites of receptor dimers (cf. Figure 7). The spacers of the highly
potent agonists 16−19, 22, 23, 27, and 28 are too short to
simultaneously occupy two H2R protomers. The presumed
optimal spacer length of ∼22−27 Å may be attained only by
compounds 21, 25, and 30 (n = 20, carbonyl−carbonyl distance
of 26.4 Å with fully extended chain). However, spacers with 14
and 20 carbon atoms result in weak agonism (gpH2R-GsαS) or
loss of agonistic activity and conversion to antagonism (hH2R-
GsαS). Thus, the remarkable increase in potency compared to
monovalent H2R agonists is presumably due to interaction with
Figure 2. Histamine H2 receptor agonism of the symmetrical bivalent
ligands 18 and 23 compared to the unsymmetrical bivalent ligand 33
in membranes expressing hH2R-GsαS (A) and gpH2R-GsαS (B) and
H2R agonism of the symmetrical bivalent ligands 18 and 28 compared
to the unsymmetrical bivalent ligand 34 at hH2R-GsαS (C) and
gpH2R-GsαS (D). Data are from two to six representative experiments
performed in duplicate, expressed as percentage change in GTPase
activity relative to the maximum effect induced by histamine (100
μM).
an accessory (allosteric?) binding site at the same receptor
molecule rather than to occupation of two protomers of a
receptor dimer (cf. Figure 7). In fact, many bivalent GPCR
ligands with drastically increased activities relative to the
Table 3. Histamine Receptor Subtype Selectivity of Selected Bivalent Ligands: Agonistic, Antagonistic or Inverse Agonistic
Effects at hH1R + RGS4, hH2R-GsαS, hH3R + Gαi2 + Gβ1γ2 + RGS4. and hH4R-RGS19 + Gαi2 + Gβ1γ2 Expressed in Sf9 Cell
a
Membranes
hH2R
hH3R
hH4R
hH1R
(pKB)
compd
pEC50 (pKB)
Emax
pEC50 (pKB)
Emax
pEC50 (pKB)
Emax
15
16
18
20
22
23
26
27
28
29
30
33
34
(<6.00)
7.24 0.22
7.32 0.23
8.11 0.25
7.59 0.22
7.51 0.02
7.67 0.07
6.67 0.34
7.25 0.16
8.21 0.07
7.61 0.18
(6.57 0.07)
7.86 0.11
8.12 0.04
0.68 0.03
0.62 0.03
0.53 0.04
0.12 0.02
0.79 0.03
0.75 0.03
0.68 0.04
0.77 0.12
0.81 0.02
0.29 0.08
(<5.00)
(<6.00)
(<6.00)
(<5.00)
(<6.00)
(6.01 0.07)
(<6.00)
(<5.00)
(<6.00)
(<6.00)
(<6.00)
(<6.00)
(6.36 0.11)
(<5.00)
(<6.00)
(<6.00)
(<6.00)
(6.13 0.22)
(6.70 0.07)
(6.32 0.16)
(<6.00)
<5.00
−0.22 0.03
0.37 0.08
0.63 0.08
−1.02 0.02
−0.77 0.02
7.10 0.12
7.38 0.02
8.07 0.19
6.47 0.04
<6.00
0.42 0.01
0.51 0.04
0.44 0.05
−0.29 0.09
−0.86 0.02
8.38 0.11
8.75 0.06
<6.00
(<6.00)
6.35 0.03
(<5.00)
(<6.00)
0.75 0.04
0.76 0.05
(<5.00)
(6.27 0.19)
8.54 0.02
0.68 0.06
8.07 0.09
0.52 0.03
a
Antagonism (pKB), agonism, and inverse agonism (pEC50, in parentheses), determined in steady state GTPase activity assays using Sf9 membranes
expressing hH1R + RGS4, hH2R-GsαS, hH3R + Gαi2 + Gβ1γ2 + RGS4, or hH4R-RGS19 + Gαi2 + Gβ1γ2 as described.46 Reaction mixtures contained
ligands at concentrations from 0.1 nM to 1 mM as appropriate to generate saturated concentration−response curves. For antagonism, reaction
mixtures contained histamine (1) (100 nM) and ligands were at concentrations from 10 nM to 1 mM. Data were analyzed by nonlinear regression
and were best fitted to sigmoidal concentration−response curves. Typical basal GTPase activities ranged between ∼1.5 and 2.5 pmol·mg−1·min−1, and
activities stimulated by 1 (10 μM) ranged between ∼3.5 and 4.5 pmol·mg−1·min−1. Data shown are mean values of one to six experiments performed
in duplicate. Efficacy (Emax) relative to the maximal response of 1 was set to 1.00. Negative Emax values refer to inverse agonistic effects.
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Figure 3. Efficacies (Emax) and agonistic potencies of compounds 15−
20, 22−24, 26−28, and 33−35 at hH2R-GsαS in comparison with
gpH2R-GsαS as determined in the steady-state GTPase assay. The
dotted lines represent the line of identity. (A) Plot of efficacies at
gpH2R-GsαS vs hH2R-GsαS. (B) Plot of pEC50 at gpH2R-GsαS vs
hH2R-GsαS.
Figure 5. Histamine H2 receptor agonism of representative bivalent
ligands 18 and 28 compared to the monovalent ligand 4 and histamine
(1) in the GTPγS binding assay using membranes expressing gpH2R-
GsαS fusion proteins. Data points are the mean of two (4, 18) or three
(1, 28) independent experiments performed in duplicate or triplicate,
analyzed by nonlinear regression for best fit to sigmoidal
concentration−response curves.
Figure 4. Concentration-dependent inhibition of GTP hydrolysis by
famotidine using 33 as the H2R agonist at 10 and 1 nM at the hH2R-
GsαS (solid line) and the gpH2R-GsαS fusion proteins (dashed line),
respectively. Data points are the mean of a representative experiment
performed in duplicate.
Figure 6. Concentration−response curves on the isolated guinea pig
■
▲
right atrium. Histamine ( , N = 4), 15 alone ( , corrected mean
leftward shift ΔpEC50 = 2.59 0.07, relative potency of 389, Emax
=
□
0.88 0.03, N = 4), and 15 in the presence of cimetidine ( , 10 μM,
pA2 = 6.09 0.08, N = 2). As expected, cimetidine (100 μM, 60 min
incubation time) also led to a fading of the maximum response
monovalent parent compounds in spite of insufficient linker
lengths for bridging of receptor protomers have been
reported.35,43,61,62 In the unsymmetrical derivatives 33 and 34
with potencies between those of their symmetrical analogues,
each of the two different pharmacophoric entities may interact
with the orthosteric and the accessory sites, respectively,
suggesting two binding modes depending on the recognition
pathway.
■
○
induced by 15 ( , 1 μM) to 46 2% ( ).
Agonistic Activities on Histamine H2R Mutants/
Chimera. Unlike small H2R agonists such as histamine (1)
and amthamine (2) (Figure 1), which are full agonists at
human and guinea pig H2 receptors, all investigated bivalent
ligands were significantly more potent and efficacious at the
gpH2R relative to the hH2R in the GTPase assay (cf. Table 1).
These differences may result from species-dependent inter-
actions with both the orthosteric and the putative accessory
binding site. The latter probably resides in the extracellular
domain, and amino acids in the e2 loop are possible candidates
for interacting with bivalent ligands. On the basis of the crystal
structure of rhodopsin,21 the participation of various residues in
the e2 loop to ligand binding was proposed63 and already
Figure 7. Bivalent ligand binding to (A) a GPCR with an accessory
binding site or to (B) a GPCR dimer (according to Portoghese et
al.35).
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experimentally demonstrated for some members of class A
GPCRs.64,65 The recently resolved crystal structures of the
turkey β1- and the human β2-adrenergic receptor indicate a
certain contribution of a phenylalanine in the e2 loop to agonist
and antagonist binding,66−68 but this residue belongs to the
orthosteric site. Since the e2 loops of the hH2R and the gpH2R
differ by only four amino acids outside the orthosteric pocket
(hH2R; G167, H169, T171, S172 vs gpH2R; D167, D169, I171,
V172), reciprocal mutation (hH2R-gpE2-GsαS, gpH2R-hE2-
GsαS) is an approach to probe whether species selectivity of
bivalent ligands depends on an accessory function of e2.
Application of this approach to N-[3-(1H-imidazol-4-yl)-
propyl]guanidines and NG-acylated analogues indicated that
the e2 loop does not contribute to species selectivity of
monovalent H2R agonists.52 Investigations of selected bivalent
acylguanidines on the reciprocal mutants led to rather
ambivalent results. All investigated compounds exhibited
similar potencies and efficacies at mutant hH2R-gpE2-GsαS
and wild-type hH2R-GsαS (for a summary of the data, see
Supporting Information). At mutant gpH2R-hE2-GsαS the
compounds are equally efficacious compared to the wild-type
gpH2R-GsαS. However, the pEC50 values are significantly
reduced by 0.5−0.9 in the case of all 2-amino-4-methylthiazolyl
compounds (16, 18, 34) except 20 and 33, whereas the
potencies of imidazolyl and 2-aminothiazolyl derivatives remain
nearly unchanged (Figure 8A). Hence, these results do not
indicate direct interactions of the mutated residues with the
bivalent ligands. However, the integrity of the e2 loop seems to
be necessary for high-affinity gpH2R binding of bivalent 2-
amino-4-methylthiazoles. It is not obvious whether the
detrimental effect of the mutations is directly based on the
modification of an accessory site in the extracellular region or
indirectly because of conformational changes of the orthosteric
site.
Furthermore, as predicted by H2R models and verified by
site-directed mutagenesis studies, the preference of the
guanidine-type agonists for the gpH2R is strongly dependent
on two amino acids, Tyr-17 and Asp-271 in TM 1 and TM 7,
respectively, which are thought to stabilize an active receptor
conformation via direct or through-water interactions.50,51 Cys-
17 and Ala-271 in the hH2R cannot fulfill this function.
Investigations of selected bivalent acylguanidines on H2R
mutants (Figure 8B), in which Cys-17 and Ala-271 of the hH2R
were replaced by the corresponding amino acids Tyr-17 and
Asp-271 of the gpH2R (hH2R-C17Y-A271D-GsαS, hH2R-
C17Y-GsαS), confirmed that both Tyr-17 in TM1 and Asp-
271 in TM7 or at least Asp-271 are key residues for highly
potent and efficacious H2R activation. The single Cys-17-Tyr
mutation has only slight (1, 2, 16, 22, 23, 28, 33, 34) or in
some cases even detrimental effects (18, 20) on hH2R potency
and efficacy.
Figure 8. Comparison of the agonistic potencies of selected bivalent
ligands at wild-type and mutant human and guinea pig H2 receptors as
determined in GTPase assays. Data shown are the mean SEM of
two to five independent experiments performed in duplicate. pEC50
values were compared with each other using one-way ANOVA,
followed by Bonferroni's multiple comparison test. (A) pEC50 values
□
of 16, 18, 20, 22, 23, 28, 33, and 34 at hH2R-GsαS ( ) vs hH2R-gpE2-
GsαS (light-gray column) vs gpH2R-hE2-GsαS (medium-gray column)
■
vs gpH2R-GsαS ( ) fusion proteins. pEC50 values are significantly
○
different for (∗) hH2R-GsαS, (+) hH2R-gpE2-GsαS, and ( ) gpH2R-
hE2-GsαS: one symbol, p < 0.05; two symbols, p < 0.01; three
symbols, p < 0.001; 95% confidence interval. (B) pEC50 values of 16,
□
18, 20, 23, and 28 at hH2R-GsαS ( ) vs hH2R-C17Y-A271D-GsαS
■
double mutant (light-gray column) vs gpH2R-GsαS ( ). The
sensitivity of the hH2R-C17Y-A271D-GsαS double mutant against
agonist stimulation is shifted from that of the gpH2R isoform. The
asterisk (∗) indicates that pEC50 is significantly different for hH2R-
GsαS: one symbol, p < 0.05; two symbols, p < 0.01; three symbols, p <
0.001; 95% confidence interval. (C) pEC50 values of 18, 22, 28, and 34
□
at hH2R-GsαS ( ) vs hH2R-gpNT-GsαS (light-gray column) vs
■
gpH2R-GsαS ( ) fusion proteins. pEC50 values are significantly
different for (∗) hH2R-GsαS, (+) hH2R-gpNT-GsαS: one symbol, p <
0.05; two symbols, p < 0.01; three symbols, p < 0.001; 95% confidence
interval.
H4 receptors for agonism and antagonism (Table 3). The
imidazolylpropylguanidine moiety proved to be a privileged
structure in terms of interactions with histamine receptors.
Such substances, initially designed as H2R agonists,11,69 were
previously identified as model compounds for the development
of H3R and H4R ligands.8,70 By contrast, recently reported
monovalent NG-acylated aminothiazolylpropylguanidine-type
H2R agonists proved to be devoid of agonistic and antagonistic
activities or to have only negligible effects on histamine
receptors other than the H2R.10 This also holds for bivalent
ligands: the investigated compounds containing two 2-amino-
thiazole moieties (15, 16, 18, 20, 22, 23, and 33) showed only
very weak antagonistic effects on H1, H3, and H4 histamine
receptors. By contrast, compounds containing at least one
imidazole ring (26−30 and 34) showed, in addition to H2R
agonism, significant agonistic, antagonistic, or inverse agonistic
activities at the other histamine receptor subtypes, depending
on the spacer length. While imidazolylpropylguanidines with
octane (27) and decanedioyl (28, 34) spacers turned out to be
highly potent hH3R and hH4R partial agonists in the low
Because of the low homology in the N-terminus of human
and guinea pig H2Rs, the potential role of this extracellular
region with respect to the species-specific pharmacological
profile of bivalent acylguanidines was also studied. Investigation
at a chimeric hH2R possessing the N-terminus of the gpH2R
(hH2R-gpNT-GsαS) revealed that the N-terminus does not
contribute to the species-selective effects (Figure 8C and
Supporting Information).
Histamine Receptor Subtype Selectivity. To determine
the histamine receptor subtype selectivity profile (human H2R
vs H1R, H3R, H4R), representative compounds were inves-
tigated in GTPase assays on recombinant human H1, H3, and
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21 mm) (Macherey & Nagel, Duren, Germany), which were attached
̈
nanomolar range, elongation of the spacer (29, n = 14; 30, n =
20) resulted in a switch to inverse agonism. Hence, by analogy
with amthamine12 as an H2R selective analogue of histamine,
the replacement of the imidazolyl with an aminothiazolyl
moiety strongly favors the selectivity for the H2R in the case of
both monovalent10 and bivalent NG-acylated hetarylpropylgua-
nidines.
to the UV detector model K-2000 (Knauer). UV detection was done
at 254 and 210 or 220 nm. The temperature was 25 °C and the flow
rate 37 mL/min (Eurosphere-100) or 20 mL/min (Nucleodur-100
C18ec). The mobile phase was 0.1% TFA in Millipore water and
MeCN. Analytical HPLC was performed on a system from Thermo
Separation Products equipped with a SN400 controller, P4000 pump,
an AS3000 autosampler, and a Spectra Focus UV/vis detector.
Stationary phase was either a Eurosphere-100 C18 (250 mm × 4.0 mm,
5 μm) column (Knauer) or a Nucleodur-C18HTec (250 mm × 4.0
mm, 5 μm) column (Macherey-Nagel) thermostated at 30 °C. For the
mobile phase, gradients of MeCN/TFA (0.05% aq) were used (flow
rate of 0.75 mL min−1). The gradient mode was as follows: 0 min,
MeCN/TFA (0.05% aq) 10:90; 20 min, 60:40; 20−23 min, 95:5; 33
min, 95:5. Absorbance was detected at 210 nm. t0(Eurosphere-100
CONCLUSION
■
The application of the bivalent ligand approach to acylguani-
dines resulted in novel hH2R and gpH2R agonists that may
serve as pharmacological tools for more detailed investigations
of the H2R. The combination of two hetarylpropylguanidine
moieties with octanedioyl or decanedioyl spacers led to the
most potent agonists at the guinea pig right atrium known so
far, surpassing up to 4000 times the potency of histamine in
increasing heart rate. However, the results of this study, in
particular the structure−activity relationships with respect to
the spacer length, do not support the hypothesis of
simultaneous occupation of the orthosteric recognition sites
of neighboring protomers. The spacer optimum suggests that
the remarkable increase in potency compared to monovalent
H2R agonists is due to interaction with an accessory binding
site at the same receptor molecule. To explore the topology of
this putative site, further investigations with H2R mutants and
novel unsymmetrical bivalent ligands are necessary. In addition,
the preparation of a structurally related bivalent radioligand
could be helpful to determine the ligand−receptor stoichiom-
etry.
C18) = 3.318 min; t0(Nucleodur-C18HTec) = 2.675 min; k′ = (tR
−
t0)/t0. Compound purities were calculated as the percentage peak area
of the analyzed compound by UV detection at 210 nm. The purities
(see Supporting Information) of the bivalent H2R agonists used for
pharmacological investigation were ≥95% except for compounds 15
(93%), 17 (92%), and 33 (94%).
Preparation of the Guanidine Building Blocks 12, 13, and
14. General Procedure for the Guanidinylation Reaction.
Compounds 5, 6, 7, and 8 were prepared according to the
literature.10,11 To a suspension of 5, 6, or 7 (1 equiv), 8 (1 equiv),
and HgCl2 (2 equiv) in DCM/abs was added NEt3 (3 equiv), and the
mixture was stirred at ambient temperature for 48 h. Subsequently,
EtOAc was added and the precipitate filtered over Celite. The crude
product was purified by flash chromatography (PE/EtOAc, 80/20 v/v)
to give the Boc- and Cbz-protected guanidines 9 and 10 and the Boc-,
Cbz-, and Trt-protected guanidine 11.
General Procedure for the Hydrogenolytic Cleavage of Cbz
Groups. To a solution of 9, 10, or 11 in a mixture of THF/MeOH
(1:1) was added Pd/C (10%), and the mixture was hydrogenated at 8
bar for 3−4 days. The catalyst was removed by filtration over Celite
and washed with MeOH. The solvent was removed in vacuo.
tert-Butyl 5-[3-(2-tert-Butoxycarbonylguanidino)propyl]-4-
methylthiazol-2-ylcarbamate (12).10 12 was prepared from 9
(5.8 g, 10.6 mmol) and 6 g of Pd/C (10%) in a mixture of 160 mL of
THF/MeOH (1:1) according to the general procedure, yielding 12 as
EXPERIMENTAL SECTION
Chemistry. General Conditions. Commercially available reagents
were purchased from Acros Organics (Geel, Belgium), Lancaster
Synthesis GmbH (Frankfurt, Germany), Sigma-Aldrich Chemie
■
GmbH (Munchen, Germany), Alfa Aesar GmbH & Co. KG
̈
(Karlsruhe, Germany), Iris Biotech GmbH (Marktredwitz, Germany),
or Merck (Darmstadt, Germany) and used as received. Where
indicated, reactions were carried out under a dry, oxygen-free argon
atmosphere. All solvents used were of analytical grade or distilled
before use. THF and Et2O were distilled over Na. CH2Cl2 was
predried over CaCl2 or distilled from P2O5 and stored under argon
atmosphere over 3 Å molecular sieves. Column chromatography was
carried out using Merck silica gel Geduran 60 (0.063−0.200) and
Merck silica gel 60 (0.040−0.063) for flash column chromatography.
Reactions were monitored by thin layer chromatography (TLC) on
Merck silica gel 60 F254 aluminum sheets and spots were visualized
with UV light at 254 nm.
1
a colorless foamlike solid (4.38 g, 100%), mp =113 °C. H NMR
3
3
(CD3OD) δ (ppm): 3.23 (t, J = 6.9 Hz, 2H, CH2-NH), 2.75 (t, J =
7.5 Hz, 2H, Thiaz-5-CH2), 2.17 (s, 3H, Thiaz-4-CH3), 1.86 (m, 2H,
Thiaz-5-CH2CH2), 1.52 (s, 9H, C(CH3)3), 1.47 (s, 9H, C(CH3)3). ES-
MS (DCM/MeOH + NH4OAc) m/z (%): 414 (MH+, 100);
C18H31N5O4S (413.53).
tert-Butyl 5-[3-(2-tert-Butoxycarbonylguanidino)propyl]-
thiazol-2-ylcarbamate (13).10 13 was prepared from 10 (5.8 g,
10.6 mmol) and 6 g of Pd/C (10%) in a mixture of 160 mL of THF/
MeOH (1:1) according to the general procedure, yielding 13 as a
1
colorless foamlike solid (3.39 g, 75%). H NMR (CDCl3) δ (ppm):
7.03 (s, 1H, Thiaz-4-H), 3.26 (t, 3J = 6.9 Hz, 2H, CH2-NH), 2.84 (t, 3J
= 7.2 Hz, 2H, Thiaz-5-CH2), 1.95 (m, 2H, Thiaz-5-CH2CH2), 1.55 (s,
9H, C(CH3)3), 1.47 (s, 9H, C(CH3)3). ES-MS (DCM/MeOH +
NH4OAc) m/z (%): 400 (MH+, 100); C17H29N5O4S (399.50).
2-(tert-Butoxycarbonyl)-1-[3-(1-trityl-1H-imidazol-4-yl)-
propyl]guanidine (14).44 The title compound was prepared from 11
(1.5 g, 2.33 mmol) and 1 g of Pd/C (10%) in a mixture of 60 mL of
THF/MeOH (1:1) according to the general procedure, yielding 14 as
Nuclear magnetic resonance (1H NMR and 13C NMR) spectra were
recorded on a Bruker Avance 300 spectrometer with perdeuterated
solvents. The chemical shift δ is given in parts per million (ppm) with
reference to the chemical shift of the residual protic solvent compared
to tetramethylsilane (δ = 0 ppm). Multiplicities were specified with the
following abbreviations: s (singlet), d (doublet), t (triplet), q
(quartet), m (multiplet), and br (broad signal) as well as combinations
thereof. The multiplicity of carbon atoms (13C NMR) was determined
by DEPT 135 and DEPT 90 (distortionless enhancement by
polarization transfer): “+” primary and tertiary carbon atom (positive
DEPT 135 signal), “−” secondary carbon atom (negative DEPT 135
signal), “quat” quaternary carbon atom. Mass spectrometry analysis
(MS) was performed on a Finnigan MAT 95, a Finnigan SSQ 710A,
and a Finnigan ThermoQuest TSQ 7000 spectrometer. Melting points
1
a colorless foamlike solid (1.05 g, 88%). H NMR (CDCl3) δ (ppm):
7.34−7.10 (m, 16H, Im-2-H, CPh3), 6.57 (s, 1H, Im-5-H), 3.41 (m,
2H, CH2NH2), 2.56 (m, 2H, Im-4-CH2), 1.86 (m, 2H, Im-4-
CH2CH2), 1.46 (s, 9H, C(CH3)3). ES-MS (DCM/MeOH +
NH4OAc) m/z (%): 510 (MH+, 100); C31H35N5O2 (509.64).
Preparation of the Boc-Protected Bivalent Acylguanidines
15a−30a. General Procedure. DIEA (1 equiv) was added to a
solution of carboxylic acid (0.5 equiv), EDAC (1 equiv), and HOBt
monohydrate (1 equiv) in DCM/abs under argon and stirred for 15
min. A solution of 12, 13, or 14 (1 equiv) in DCM/abs was added, and
the mixture was stirred overnight at room temperature. The solvent
(mp) were measured on a Buchi 530 electrically heated copper block
̈
apparatus using an open capillary and are uncorrected.
Preparative HPLC was performed with a pump model K-1800
(Knauer, Berlin, Germany). The column was either a Eurosphere-100
(250 mm × 32 mm) (Knauer) or a Nucleodur-100 C18ec (250 mm ×
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was removed under reduced pressure. EtOAc and water were added to
the residue. The organic phase was separated and the aqueous layer
extracted two times with EtOAc. After drying over MgSO4, the organic
solvent was removed in vacuo. The crude product was purified by flash
chromatography (PE/EtOAc, 70/30 to 50/50 v/v) unless otherwise
indicated.
abs and 12 (210 mg, 0.5 mmol) in 2.5 mL of DCM/abs according to
the general procedure, yielding the Bzl-protected compound as a
yellow oil, which was immediately dissolved in 10 mL of MeOH and
hydrogenated with Pd/C as catalyst for 1 h at room temperature. After
filtration over Celite, the solvent was removed under reduced pressure
1
to obtain 32 (0.21 g, 70%) as a colorless foamlike solid. H NMR
N1 ,N1 0 -Bis((tert-butoxycarbonylamino)[3-[2-(tert-
butoxycarbonyl)amino-4-methylthiazol-5-yl]propylamino]-
methylene)decanediamide (18a). The title compound was
prepared from decanedioic acid (100 mg, 0.5 mmol), EDAC (190
mg, 1 mmol), HOBt monohydrate (150 mg, 1 mmol), DIEA (0.17
mL, 1 mmol) in 5 mL of DCM/abs and 12 (410 mg, 1 mmol) in 5 mL
of DCM/abs according to the general procedure, yielding 18a (0.23 g,
46%) as a colorless foamlike solid. 1H NMR (CDCl3) δ (ppm): 12.40
(s, 2H, NH), 9.02 (t, 3J = 5.2 Hz, 2H, CH2NH), 3.45 (m, 4H,
CH2NH), 2.70 (t, 3J = 7.4 Hz, 4H, Thiaz-5-CH2), 2.35 (t, 3J = 7.5 Hz,
4H, COCH2), 2.21 (s, 6H, Thiaz-4-CH3), 1.87 (m, 4H, Thiaz-5-
CH2CH2), 1.66 (m, 4H, COCH2CH2), 1.51 (s, 18H, C(CH3)3), 1.49
(s, 18H, C(CH3)3), 1.32 (m, 8H, (CH2)4). ES-MS (DCM/MeOH +
NH4OAc) m/z (%): 993 (MH+, 100); C46H76N10O10S2 (992.5).
N1 ,N1 0 -Bis((tert-butoxycarbonylamino)[3-[2-(tert-
butoxycarbonyl)aminothiazol-5-yl]propylamino]methylene)-
decanediamide (23a). The title compound was prepared from
decanedioic acid (50 mg, 0.25 mmol), EDAC (95 mg, 0.5 mmol),
HOBt monohydrate (77 mg, 0.5 mmol), DIEA (0.08 mL, 0.5 mmol)
in 5 mL of DCM/abs and 13 (200 mg, 0.5 mmol) in 5 mL of DCM/
abs according to the general procedure, yielding 23a (0.20 g, 54%) as a
brown oil. 1H NMR (CDCl3) δ (ppm): 7.04 (s, 2H, Thiaz-4-H), 3.48
(m, 4H, CH2NH), 2.79 (m, 4H, Thiaz-5-CH2), 2.34 (m, 4H,
COCH2), 1.93 (m, 4H, Thiaz-5-CH2CH2), 1.65 (m, 4H,
COCH2CH2), 1.56 (s, 18H, C(CH3)3), 1.50 (s, 18H, C(CH3)3),
1.32 (m, 8H, (CH2)4). ES-MS (DCM/MeOH + NH4OAc) m/z (%):
965.5 (MH+, 100); C44H72N10O10S2 (964.5).
3
(CDCl3) δ (ppm): 3.47 (m, 2H, CH2NH), 2.70 (t, J = 7.1 Hz, 2H,
Thiaz-5-CH2), 2.33 (m, 4H, CH2COOH, COCH2), 2.16 (s, 3H,
Thiaz-4-CH3), 1.88 (m, 2H, Thiaz-5-CH2CH2), 1.64 (m, 4H,
COCH2CH2, CH2CH2COOH), 1.53 (s, 9H, C(CH3)3), 1.49 (s, 9H,
C(CH3)3), 1.33 (s, 8H, (CH2)4). ES-MS (DCM/MeOH + NH4OAc)
m/z (%): 598 (MH+, 100); C28H47N5O7S (597.77).
N1-((tert-Butoxycarbonylamino){3-[2-(tert-butoxycarbonyla-
mino)-4-methylthiazol-5-yl]propylamino]methylene)-N10
-
((tert-butoxycarbonylamino)[3-[2-(tert-butoxycarbonylamino)-
thiazol-5-yl]propylamino]methylene)decanediamide (33a).
The title compound was prepared from 32 (135 mg, 0.23 mmol),
EDAC (44 mg, 0.23 mmol), HOBt monohydrate (35 mg, 0.23 mmol),
DIEA (0.04 mL, 0.23 mmol) in 3 mL of DCM/abs and 13 (92 mg,
0.23 mmol) in 2 mL of DCM/abs according to the general procedure,
yielding 33a (0.12 g, 57%) as a brown oil. 1H NMR (CDCl3) δ (ppm):
7.05 (s, 1H, Thiaz-4-H), 3.47 (m, 4H, CH2NH), 2.75 (m, 4H, Thiaz-5-
CH2), 2.34 (m, 4H, COCH2), 2.21 (s, 3H, Thiaz-4-CH3), 1.91 (m,
4H, Thiaz-5-CH2CH2), 1.65 (m, 4H, COCH2CH2), 1.54 (s, 18H,
C(CH3)3), 1.50 (s, 18H, C(CH3)3), 1.32 (m, 8H, (CH2)4). ESI-MS:
m/z (rel intens, %): 979.6 (MH+, 100); C45H74N10O10S2 (978.50).
N1-{(tert-Butoxycarbonylamino)[3-(1-trityl-1H-imidazol-4-
yl)propylamino]methylene]-N10-((tert-butoxycarbonylamino)-
[3-[2-(tert-butoxycarbonylamino)-4-methylthiazol-5-yl]-
propylamino]methylene)decanediamide (34a). The title com-
pound was prepared from 32 (179 mg, 0.3 mmol), EDAC (57 mg, 0.3
mmol), HOBt monohydrate (46 mg, 0.3 mmol), DIEA (0.05 mL, 0.3
mmol) in 3 mL of DCM/abs and 14 (120 mg, 0.3 mmol) in 2 mL of
DCM/abs according to the general procedure, yielding 34a (0.07 g,
N1,N10-Bis[(tert-butoxycarbonylamino)[3-(1-trityl-1H-imida-
zol-4-yl)propylamino]methylene]decanediamide (28a). The
title compound was prepared from decanedioic acid (100 mg, 0.5
mmol), EDAC (190 mg, 1 mmol), HOBt monohydrate (150 mg, 1
mmol), DIEA (0.17 mL, 1 mmol) in 5 mL of DCM/abs and 14 (510
mg, 1 mmol) in 5 mL of DCM/abs according to the general procedure
(flash chromatography CHCl3/MeOH, 95/5 v/v), yielding 28a (0.18
1
24%) as a brown oil. H NMR (CDCl3) δ (ppm): 8.82 (s, 1H, Im-2-
H), 7.37−7.22 (m, 16H, Im-5-H, CPh3), 3.38 (m, 4H, CH2NH), 2.84
(t, 3J = 7.7 Hz, 2H, Im-4-CH2), 2.71 (t, 3J = 7.4 Hz, 2H, Thiaz-5-CH2),
2.47 (m, 4H, COCH2), 2.18 (s, 3H, Thiaz-4-CH3), 2.03 (m, 2H, Im-4-
CH2CH2), 1.90 (m, 2H, Thiaz-5-CH2CH2), 1.66 (m, 4H,
COCH2CH2), 1.52 (s, 18H, C(CH3)3), 1.35 (m, 8H, (CH2)4). ESI-
MS: m/z (rel intens, %): 989.7 (MH+, 100); C54H72N10O6S (988.54).
Preparation of the Trt-Protected Bivalent Acylguanidine
35a. N1,N10-Bis{amino[3-(1-trityl-1H-1,2,4-triazol-5-yl)-
propylamino]methylene}decanediamide (35a). To a solution
of CDI (195 mg, 1.2 mmol) in DMF (7 mL), decanedioic acid (100
mg, 0.5 mmol) was added. The mixture was stirred under argon for 1
h. In a second flask, 3-(1-trityl-1H-1,2,4-triazol-5-yl)propylguanidine45
(410 mg, 1 mmol) and NaH (60% dispersion in oil) (80 mg, 2 mmol)
in DMF (7 mL) under argon were heated to 30−35 °C for 45 min,
and the mixture was then allowed to cool to room temperature. The
two mixtures were combined and stirred for 4 h at ambient
temperature. The solvent was removed in vacuo and the crude
product was purified by flash chromatography (CHCl3/MeOH/NH3,
95/3/2 v/v/v) to obtain 35a (300 mg, 60%) as pale white foamlike
1
g, 30%) as a yellow oil. H NMR (CDCl3) δ (ppm): 7.33−7.12 (m,
32H, Im-2-H, CPh3), 6.53 (d, 4J = 1.0 Hz, 2H, Im-5-H), 3.43 (m, 4H,
CH2NH), 2.59 (t, 3J = 7.6 Hz, 4H, Im-4-CH2), 2.34 (m, 4H, COCH2),
1.90 (m, 4H, Im-4-CH2CH2), 1.65 (m, 4H, COCH2CH2), 1.49 (s,
18H, C(CH3)3), 1.27 (m, 8H, (CH2)4). ES-MS (DCM/MeOH +
NH4OAc) m/z (%): 1185 (MH+, 100); C72H84N10O6 (1184.66).
Compounds 15a−17a, 19a−22a, 24a−27a, 29a, and 30a were
prepared by analogy (see Supporting Information).
Preparation of the Boc-Protected Bivalent Acylguanidines
33a and 34a. 10-Benzyloxy-10-oxodecanoic Acid (31).71
Phenylmethanol (0.27 g, 0.25 mL, 2.47 mmol) was dropwise added
to a cooled suspension of decanedioic acid (0.5 g, 2.47 mmol) and
DMAP (cat.) in 3 mL of THF/abs. A solution of DCC (0.61 g, 2.96
mmol) in 3 mL of THF/abs was dropwise added to this mixture and
stirred for 72 h at ambient temperature. Subsequently, 1,1-
dicyclohexylurea was filtered and the solvent removed under reduced
pressure. The crude product was subjected to flash chromatography
(PE/EtOAc, 90/10 v/v) to obtain 31 (0.34 g, 47%) as a colorless
1
solid. H NMR (CD3OD) δ (ppm): 8.01 (s, 2H, Triaz-3-H), 7.37−
7.05 (m, 30H, CPh3), 3.14 (t, 3J = 7.6 Hz, 4H, CH2NH), 2.88 (m, 4H,
3
Triaz-5-CH2), 2.41 (t, J = 7.5 Hz, 4H, COCH2), 1.96 (m, 4H, Triaz-
5-CH2CH2), 1.63 (m, 4H, COCH2CH2), 1.29 (m, 8H, (CH2)4). ES-
MS (DCM/MeOH + NH4OAc) m/z (%): 987.7 (MH+, 10), 494.4
((M+2H)2+, 100); C60H66N12O2 (987.25).
1
semisolid. H NMR (CDCl3) δ (ppm): 10.88 (s, 1H, COOH), 7.34
(m, 5H, Ar-H), 5.11 (s, 2H, CH2-Ar), 2.34 (m, 4H, COCH2), 1.61 (m,
4H, COCH2CH2), 1.29 (s, 8H, (CH2)4). 13C NMR (CDCl3) δ ppm:
179.80 (quat COOH), 173.72 (quat CO), 136.12 (quat Ar-C),
128.55 (+, Ar-CH), 128.18 (+, Ar-CH), 66.11 (−, CH2-Ar), 34.30 (−,
CH2COOH), 34.04 (−, COCH2), 29.02 (−, CH2), 28.96 (−, CH2),
24.90 (−, COCH2CH2), 24.64 (−, CH2CH2COOH). EI-MS (70 eV)
m/z (%): 292 (M+•, 30); C17H24O4 (292.37).
10-((tert-Butoxycarbonylamino)[3-[2-(tert-butoxycarbonyla-
mino)-4-methylthiazol-5-yl]propylamino]methyleneamino)-
10-oxodecanoic Acid (32). Compound 32 was prepared from 31
(150 mg, 0.5 mmol), EDAC (95 mg, 0.5 mmol), HOBt monohydrate
(80 mg, 0.5 mmol), DIEA (0.09 mL, 0.5 mmol) in 2.5 mL of DCM/
Preparation of Deprotected Bivalent Acylguanidines 15−30
and 33−35. General Procedure. TFA (20%) was added to a
solution of the protected acylguanidines 15a−30a and 33a−35a in
DCM/abs, and the mixture was stirred at ambient temperature until
the protecting groups (Boc, Trt) were removed (3−5 h).
Subsequently, the solvent was evaporated in vacuo and the residue
was purified by preparative RP-HPLC and lyophilized. All compounds
were obtained as trifluoroacetic acid salts.
N1,N10-Bis[[3-(2-amino-4-methylthiazol-5-yl)propylamino]-
(amino)methylene]decanediamide Tetratrifluoroacetate (18).
The title compound was prepared from 18a (200 mg, 0.19 mmol) in 5
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mL of DCM/abs and 1 mL of TFA according to the general
procedure, yielding 18 as a colorless foamlike solid (120 mg, 57%). 1H
NMR (CD3OD) δ (ppm): 3.35 (t, 3J = 6.8 Hz, 4H, CH2NH), 2.70 (t,
3J = 7.5 Hz, 4H, Thiaz-5-CH2), 2.45 (t, 3J = 7.4 Hz, 4H, COCH2), 2.16
TFA according to the general procedure, yielding 34 as a colorless oil
1
(70 mg, 24%). H NMR (CD3OD) δ (ppm): 8.82 (s, 1H, Im-2-H),
7.37 (s, 1H, Im-5-H), 3.38 (m, 4H, CH2NH), 2.84 (t, 3J = 7.7 Hz, 2H,
3
Im-4-CH2), 2.71 (t, J = 7.41 Hz, 2H, Thiaz-5-CH2), 2.47 (m, 4H,
COCH2), 2.18 (s, 3H, Thiaz-4-CH3), 2.03 (m, 2H, Im-4-CH2CH2),
1.90 (m, 2H, Thiaz-5-CH2CH2), 1.66 (m, 4H, COCH2CH2), 1.35 (m,
8H, (CH2)4). 13C NMR (CD3OD, 400 MHz, HSQC, HMBC) δ
(ppm): 177.37 (quat CO), 155.64 (quat CNH), 134.96 (quat
Thiaz-C-4), 118.46 (quat Thiaz-C-5), 117.09 (+, Im-5-CH), 41.56 (−,
CH2NH), 37.76 (−, COCH2), 30.13 (−, CH2), 29.96 (−, Thiaz-5-
CH2-CH2), 28.10 (−, Im-4-CH2CH2), 25.41 (COCH2CH2), 23.60 (−,
Thiaz-5-CH2), 22.54 (−, Im-4-CH2), 11.41 (+, Thiaz-4-CH3).
HRLSIMS: m/z for ([C25H42N10O2S + H]+) calcd 547.3291, found
547.3299. Prep HPLC: MeCN/0.1% TFA/aq (20/80−50/50).
HPLC: k′ = 1.87 (tR = 9.51 min), purity = 94%; C25H42N10O2S·4TFA
(1002.32).
(s, 6H, Thiaz-4-CH3), 1.90 (m, 4H, Thiaz-5-CH2CH2), 1.63 (m, 4H,
COCH2CH2), 1.33 (m, 8H, (CH2)4). 13C (CD3OD) δ (ppm): 177.57
(quat CO), 170.43 (quat Thiaz-2-C), 155.41 (quat CNH), 132.59
(quat Thiaz-4-C), 118.33 (quat Thiaz-5-C), 41.56 (−, CH2NH), 37.75
(−, COCH2), 30.13 (−, Thiaz-5-CH2CH2), 29.90 (−, CH2), 29.68 (−,
CH2), 25.46 (−, COCH2CH2), 23.64 (−, Thiaz-5-CH2), 11.46 (+,
Thiaz-5-CH3). HREIMS: m/z for ([C26H44N10O2S2 + H]+) calcd
593.3163, found 593.3161. Prep HPLC: MeCN/0.1% TFA/aq (25/
75). HPLC: k′ = 2.28 (tR = 10.90 min), purity = 100%;
C26H44N10O2S2·4TFA (1048.78).
N1,N10-Bis[[3-(2-aminothiazol-5-yl)propylamino](amino)-
methylene]decanediamide Tetratrifluoroacetate (23). The title
compound was prepared from 23a (200 mg, 0.2 mmol) in 5 mL of
DCM/abs and 1 mL of TFA according to the general procedure,
yielding 23 as a yellow-brown oil (100 mg, 49%). 1H NMR (CD3OD)
δ (ppm): 7.01 (s, 2H, Thiaz-4-H), 3.37 (t, 3J = 7.1 Hz, 4H, CH2NH),
N1,N10-Bis{[3-(1H-1,2,4-triazol-5-yl)propylamino](amino)-
methylene}decanediamide Tetratrifluoroacetate (35). The title
compound was prepared from 35a (300 mg, 0.31 mmol) in 10 mL of
DCM/abs and 2 mL of TFA according to the general procedure,
1
3
3
yielding 35 (85 mg, 29%) as pale yellow oil. H NMR (CD3OD) δ
2.77 (t, J = 7.1 Hz, 4H, Thiaz-5-CH2), 2.47 (t, J = 7.4 Hz, 4H,
COCH2), 1.95 (m, 4H, Thiaz-5-CH2CH2), 1.65 (m, 4H,
COCH2CH2), 1.35 (m, 8H, (CH2)4). 13C (CD3OD) δ (ppm):
176.47 (quat CO), 172.43 (quat Thiaz-2-C), 155.92 (quat CNH),
125.55 (quat Thiaz-5-C), 123.27 (+, Thiaz-4-C), 40.66 (−, CH2NH),
36.83 (−, COCH2), 30.43 (−, CH2), 28.80 (−, CH2), 28.74 (−, Thiaz-
5-CH2-CH2), 24.41 (−, Thiaz-5-CH2), 24.08 (−, COCH2CH2).
HRLSIMS: m/z for ([C24H40N10O2S2 + H]+) calcd 565.2855, found
565.2855. Prep HPLC: MeCN/0.1% TFA/aq (10/90−50/50).
3
(ppm): 8.54 (s, 2H, Triaz-3-H), 3.42 (t, J = 7.2 Hz, 4H, CH2NH),
3
3
2.94 (t, J = 7.4 Hz, 4H, Triaz-5-CH2), 2.46 (t, J = 7.4 Hz, 4H,
COCH2), 2.11 (m, 4H, Triaz-5-CH2CH2), 1.66 (m, 4H,
COCH2CH2), 1.36 (m, 4H, (CH2)4). 13C NMR (CD3OD) δ
(ppm): 177.22 (quat CO), 163.55 (quat Triaz-5-C), 155.41 (quat
CNH), 138.37 (+, Triaz-3-C), 41.62 (−, CH2NH), 37.79 (−,
COCH2), 30.19 (−, CH2), 29.96 (−, CH2), 26.90 (−, Triaz-5-
CH2CH2), 25.41 (−, COCH2CH2), 24.11 (−, Triaz-5-CH2).
HRLSIMS: m/z for ([C22H38N12O2 + H]+) calcd 503.3319, found
503.3304. Prep HPLC: MeCN/0.1% TFA/aq (20/80−50/50).
HPLC: k′ = 1.77 (tR = 7.41 min), purity = 100%; C22H38N12O2·4TFA
(958.7).
HPLC: k′
= 2.13 (tR = 10.37 min), purity = 100%;
C24H40N10O2S2·4TFA (1020.36).
N1,N10-Bis[[3-(1H-imidazol-4-yl)propylamino](amino)-
methylene]decanediamide Tetratrifluoroacetate (28). The title
compound was prepared from 28a (160 mg, 0.13 mmol) in 10 mL of
DCM/abs and 2 mL of TFA according to the general procedure,
Compounds 15−17, 19−22, 24−27, 29, and 30 were prepared by
analogy (see Supporting Information).
1
Pharmacological Methods. Materials. Histamine dihydrochlor-
ide was purchased from Alfa Aesar GmbH & Co. KG (Karlsruhe,
Germany). Amthamine12 was from Tocris Bioscience (Avonmouth,
Bristol, U.K.). Compound 410 was synthesized in our laboratory, and
cimetidine was from Sigma-Aldrich Chemie GmbH (Munich,
Germany). [γ-32P]GTP and [γ-33P]GTP were synthesized according
to a previously described method.72 [32P]Pi (8500−9100 Ci/mmol
orthophosphoric acid) and [33P]Pi (3000 Ci/mmol orthophosphoric
acid) were purchased from Hartmann Analytic (Braunschweig,
Germany). All unlabeled nucleotides, glycerol 3-phosphate dehydro-
genase, triose phosphate isomerase, glyceraldehyde 3-phosphate
dehydrogenase, and lactate dehydrogenase were from Roche
(Mannheim, Germany). 3-Phosphoglycerate kinase and L-α-glycerol
phosphate were from Sigma-Aldrich Chemie GmbH (Munich,
Germany). Unlabeled GTPγS was from Roche (Mannheim,
Germany), and [35S]GTPγS was from Hartmann Analytics GmbH
(Braunschweig, Germany). GF/B filters were from Brandel (Gaithers-
burg, MD)
Histamine H2 Receptor Assay on Isolated Guinea Pig Right
Atrium (Spontaneously Beating).1 Guinea pigs of either sex (250−
500 g) were sacrificed by a blow on the neck and exsanguinated. The
heart was rapidly removed, and the right atrium was quickly dissected
and set up isometrically in Krebs−Henseleit's solution under a
diastolic resting force of approximately 5 mN in a jacketed 20 mL
organ bath of 32.5 °C as previously described. The bath fluid
(composition [mM]: NaCl 118.1, KCl 4.7, CaCl2 1.8, MgSO4 1.64,
KH2PO4 1.2, NaHCO3 25.0, glucose 5.0, sodium pyruvate 2.0) was
equilibrated with 95% O2 and 5% CO2 and additionally contained
(RS)-propranolol (0.3 μM) to block β-adrenergic receptors. Stock
solutions (10 mM) and all dilutions of bivalent ligands (1, 0.1, and
0.01 mM) were made in freshly prepared bath fluid instead of distilled
water in order to prevent absorption at glass surfaces. Experiments
were started after 30 min of continuous washing and an additional
equilibration period of 15 min. Two successive curves for histamine
displayed a significant desensitization of 0.13 0.02 (N = 16 control
yielding 28 as a pale yellow oil (30 mg, 23%). H NMR (CD3OD) δ
ppm: 8.81 (d, 4J = 1.3 Hz, 2H, Im-2-H), 7.37 (d, 4J = 0.9 Hz, 2H, Im-
5-H), 3.38 (t, 3J = 6.9 Hz, 4H, CH2NH), 2.84 (t, 3J = 7.6 Hz, 4H, Im-
4-CH2), 2.47 (t, 3J = 7.4 Hz, 4H, COCH2), 2.03 (m, 4H, Im-4-
CH2CH2), 1.65 (m, 4H, COCH2CH2), 1.35 (m, 8H, (CH2)4). 13C
(CD3OD) δ (ppm): 177.45 (quat CO), 155.42 (quat CNH),
134.97 (+, Im-2-C), 134.33 (quat Im-4-C), 117.14 (+, Im-5-C), 41.54
(−, CH2NH), 37.75 (−, COCH2), 30.17 (−, Im-4-CH2), 29.95 (−,
Im-4-CH2CH2), 27.98 (−, COCH2CH2CH2CH2), 25.45 (−,
COCH2CH2CH2), 22.56 (−, COCH2CH2). HREIMS: m/z for
([C24H40N10O2 + H]+) calcd 501.3408, found 501.34199. Prep
HPLC: MeCN/0.1% TFA/aq (15/85−30/70). HPLC: k′ = 1.91 (tR
= 9.66 min), purity = 96%; C24H40N10O2·4TFA (956.60).
N1-[[3-(2-Amino-4-methylthiazol-5-yl)propylamino](amino)-
methylene]-N10-[[3-(2-aminothiazol-5-yl)propylamino]-
(amino)methylene]decanediamide Tetratrifluoroacetate (33).
The title compound was prepared from 33a (110 mg, 0.11 mmol) in
10 mL of DCM/abs and 2 mL TFA according to the general
1
procedure, yielding 33 as a colorless foamlike solid (30 mg, 26%). H
NMR (CD3OD) δ (ppm): 7.01 (s, 1H, Thiaz-4-H), 3.37 (m, 4H,
3
CH2NH), 2.74 (m, 4H, Thiaz-5-CH2), 2.46 (t, J = 7.41 Hz, 4H,
COCH2), 2.18 (s, 3H, Thiaz-4-CH3), 1.93 (m, 4H, Thiaz-5-CH2CH2),
1.65 (m, 4H, COCH2CH2), 1.35 (m, 8H, (CH2)4). 13C NMR
(CD3OD) δ (ppm): 177.41 (quat CO), 171.82 (quat Thiaz-2-C),
155.29 (quat CNH), 126.36 (quat Thiaz-5-C), 123.37 (+, Thiaz-4-
C), 118.44 (quat Thiaz-5-C), 41.47 (−, CH2NH), 37.76 (−, COCH2),
30.20 (−, CH2), 29.97 (−, Thiaz-5-CH2CH2), 25.45 (−,
COCH2CH2), 24.89 (−, Thiaz-5-CH2), 11.45 (+, Thiaz-4-CH3).
HRLSIMS: m/z for ([C25H42N10O2S2 + H]+) calcd 579.3012, found
579.3006. Prep HPLC: MeCN/0.1% TFA/aq (10/90−35/65);
C25H42N10O2S2·4TFA (1034.88).
N1-[[3-(Imidazol-4-yl)propylamino](amino)methylene]-N10-
[[3-(2-amino-4-methylthiazol-5-yl)propylamino](amino)-
methylene]decanediamide Tetratrifluoroacetate (34). The title
compound was prepared from 34a in 5 mL of DCM/abs and 1 mL of
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organs). This value was used to correct each individual experiment.
For agonists, two successive concentration−frequency curves were
established: the first for histamine (0.1−30 μM) and the second for
the agonist under study in the absence or presence of cimetidine (10
μM, 30 min of incubation time). Furthermore, the sensitivity to 30,
100, or 300 μM cimetidine was routinely checked at the end of each
H2R agonist concentration−effect curve, and a significant reduction of
frequency was observed. Relative potency of the agonist under study
was calculated from the corrected pEC50 difference (ΔpEC50). pEC50
values are given relative to the long-term mean value for histamine
(pEC50 = 6.00) in our laboratory (pEC50 = 6.00 + ΔpEC50).
ATP, 100 nM GTP, 100 μM adenylyl imidodiphosphate, 5 mM
creatine phosphate, 40 μg of creatine kinase, and 0.2% (w/v) bovine
serum albumin in 50 mM Tris-HCl, pH 7.4, as well as ligands at
various concentrations. In H4R assays, NaCl (final concentration of
100 mM) was included. Reaction mixtures (80 μL) were incubated for
2 min at 25 °C before the addition of 20 μL of [γ-32P]GTP (0.1 μCi/
tube) or [γ-33P]GTP (0.05 μCi/tube). Reactions were conducted for
20 min at 25 °C and terminated by the addition of 900 μL of slurry
consisting of 5% (w/v) activated charcoal suspended in 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. An amount of 600 μL of the supernatant fluid
was removed, and 32Pi or 33Pi was determined by Cerenkov or liquid
scintillation counting, respectively. Enzyme activities were corrected
for spontaneous hydrolysis of [γ-32P]GTP or [γ-33P]GTP, determined
in tubes containing all components described above, plus a high
concentration of unlabeled GTP (1 mM) to prevent enzymatic
hydrolysis of the labeled nucleotides in the presence of Sf9
membranes. Spontaneous [γ-32P]GTP or [γ-33P]GTP hydrolysis was
<1% of the total amount of the labeled nucleotides. The experimental
conditions chosen ensured that not more than 10% of the total
amount of added [γ-32P]GTP and [γ-33P]GTP was converted to 32Pi
and 33Pi, respectively. All experimental data were analyzed by nonlinear
regression with the Prism 5 program (GraphPad Software, San Diego,
CA).
Determination of Histamine Receptor Agonism and
Antagonism in GTPase Assays. Generation of Recombinant
Baculoviruses, Cell Culture, and Membrane Preparation.
Recombinant baculoviruses encoding human H1R or a fusion protein
of the human H2R with GsαS or a fusion protein of the guinea pig H2R
with GsαS or the human H3R or a fusion protein of the human H4R
with RGS19 or fusion proteins of mutant H2Rs with GsαS (hH2R-
C17Y-A271D-GsαS or hH2R-C17Y-GsαS or hH2R-gpE2-GsαS or
gpH2R-hE2-GsαS or hH2R-gpNT-GsαS) were prepared as de-
scribed,13,50−52 using the BaculoGOLD transfection kit (BD
Pharmingen, San Diego, CA) according to the manufacturer’s
instructions. For construction of the cDNA for hH2R-gpNT-GsαS
see Supporting Information. Sf9 cells were cultured in 250 or 500 mL
disposable Erlenmeyer flasks at 28 °C under rotation at 150 rpm in
Insect-Xpress medium (Lonza, Velviers, Belgium) supplemented with
5% (v/v) fetal calf serum (Biochrom, Berlin, Germany) and 0.1 mg/
mL gentamicin (Lonza, Walkersville, MD). Cells were maintained at a
density of (0.5−6.0) × 106 cells/mL. After initial transfection, high-
titer virus stocks were generated by two sequential virus amplifications.
In the first amplification, cells were seeded at 2.0 × 106 cells/mL and
infected with a 1:100 dilution of the supernatant from the initial
transfection. Cells were cultured for 7 days, resulting in the lysis of the
entire cell population. The supernatant was harvested and stored
under light protection at 4 °C. In a second amplification, cells were
seeded at a densitiy of 3.0 × 106 cells/mL and infected with a 1:20
dilution of the supernatant from the first amplification. Cells were
cultured for 48 h, and the supernatant was harvested. After a 48 h
culture period, the majority of cells showed signs of infections (e.g.,
altered morphology, viral inclusion bodies), whereas most of the cells
were still intact. The supernatant from the second amplification was
stored under light protection at 4 °C and used as routine virus stock
for infections to obtain membrane preparations. For membrane
preparation, cells were sedimented by centrifugation (1000 rpm, 5
min, rt) and suspended in fresh medium at a density of 3.0 × 106 cells/
mL. Cells were infected with 1:100 dilutions of high-titer baculovirus
stocks encoding the various histamine receptors, histamine receptor
fusion proteins, G-protein subunits, and RGS proteins. Cells were
cultured for 48 h before membrane preparation. Sf9 membranes were
prepared as described,73 using 1 mM EDTA, 0.2 mM phenyl-
methylsulfonyl fluoride, 10 μg/mL benzamidine, and 10 μg/mL
leupeptin as protease inhibitors. Membranes were suspended in
binding buffer (12.5 mM MgCl2, 1 mM EDTA, and 75 mM Tris-HCl,
pH 7.4) and stored at −80 °C until use. Protein concentrations were
[35S]GTPγS Binding Assay. [35S]GTPγS binding assays75,76 were
performed as previously described for the H2R13,46 using Sf9 insect cell
membranes expressing the gpH2R-GsαS fusion protein. 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 (15−30 μg of protein/tube), 1
μM GDP, 0.05% (w/v) bovine serum albumin, 0.2 nM [35S]GTPγS,
and the investigated ligands at various concentrations in binding buffer
(total volume 250 μL). Incubations were conducted for 90 min at 25
°C, and shaking was at 250 rpm. Bound [35S]GTPγS was separated
from free [35S]GTPγS by filtration through GF/B 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.
ASSOCIATED CONTENT
■
S
* Supporting Information
Synthetic procedures and analytical data for compounds 15a−
17a, 19a−22a, 24a−27a, 29a, 30a, 15−17, 19−22, 24−27, 29,
and 30a, purity data and retention times (HPLC data) of all
target compounds, and construction of the cDNA for hH2R-
gpNT-GsαS. This material is available free of charge via the
Internet at http://pubs.acs.org.
determined using the DC protein assay kit (Bio-Rad, Munchen,
̈
Germany).
AUTHOR INFORMATION
Steady-State GTPase Activity Assay with Sf9 Insect Cell
Membranes Expressing Histamine H1, H2, H3, or H4 Receptors
or H2R Mutants. Membranes were thawed, sedimented, and
resuspended in 10 mM Tris-HCl, pH 7.4. In the cases of the H1R
and H2R, Sf9 membranes expressing either H1R isoforms plus RGS4
or H2R-GsαS fusion proteins were used.50,74 H3R-stimulated GTP
hydrolysis was determined with membranes coexpressing human H3R,
mammalian Gαi2, Gβ1γ2, and RGS4. Human H4R activity was
measured with membranes coexpressing an H4R-RGS19 fusion protein
with Gαi2 and Gβ1γ2. Activity on H2R mutants was measured with
hH2R-C17Y-A271D-GsαS, hH2R-C17Y-GsαS, hH2R-gpE2-GsαS,
gpH2R-hE2-GsαS, and hH2R-gpNT-GsαS fusion proteins.51,52 Assay
tubes contained Sf9 membranes (5−20 μg of protein/tube), MgCl2
(H1R, H2R, 1.0 mM; H3R, H4R, 5.0 mM), 100 μM EDTA, 100 μM
■
Corresponding Author
*Phone: +49-941 9434827. Fax: +49-941 9434820. E-mail:
armin.buschauer@chemie.uni-regensburg.de.
ACKNOWLEDGMENTS
■
The authors are grateful to Maria Beer-Kron, Christine Braun,
̈
and Kerstin Rohrl for expert technical assistance. This work was
̈
supported by the Graduate Training Program (Graduiertenkol-
leg) GRK 760, “Medicinal Chemistry: Molecular Recognition,
Ligand−Receptor Interactions”, of the Deutsche Forschungs-
gemeinschaft. The contribution of Tobias Birnkammer to this
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Journal of Medicinal Chemistry
Article
ligands at the human histamine H4 receptor: identification of 4-
methylhistamine as the first potent and selective H4 receptor agonist. J.
Pharmacol. Exp. Ther. 2005, 314, 1310−1321.
project has been awarded by the European Histamine Research
Society (“Young Investigators Award” 2011).
(8) Igel, P.; Schneider, E.; Schnell, D.; Elz, S.; Seifert, R.; Buschauer,
A. N(G)-acylated imidazolylpropylguanidines as potent histamine H4
receptor agonists: selectivity by variation of the N(G)-substituent. J.
Med. Chem. 2009, 52, 2623−2627.
DEDICATION
■
†This paper is dedicated to Prof. Dr. Dr. Dr. h.c. Walter
Schunack, in memoriam.
(9) Igel, P.; Dove, S.; Buschauer, A. Histamine H4 receptor agonists.
Bioorg. Med. Chem. Lett. 2010, 20, 7191−7199.
ABBREVIATIONS USED
■
(10) Kraus, A.; Ghorai, P.; Birnkammer, T.; Schnell, D.; Elz, S.;
Seifert, R.; Dove, S.; Bernhardt, G.; Buschauer, A. NG-Acylated
aminothiazolylpropylguanidines as potent and selective histamine H2
receptor agonists. ChemMedChem 2009, 4, 232−240.
(11) Ghorai, P.; Kraus, A.; Keller, M.; Gotte, C.; Igel, P.; Schneider,
E.; Schnell, D.; Bernhardt, G.; Dove, S.; Zabel, M.; Elz, S.; Seifert, R.;
Buschauer, A. Acylguanidines as bioisosteres of guanidines: NG-
acylated imidazolylpropylguanidines, a new class of histamine H2
receptor agonists. J. Med. Chem. 2008, 51, 7193−7204.
abs, absolute; aq, aqueous; Boc, tert-butoxycarbonyl; Cbz,
benzyloxycarbonyl; CDI, 1,1′-carbonyldiimidazole; DCM,
dichloromethane; DIEA, N,N-diisopropylethylamine; DMF,
dimethylformamide; DMSO, dimethylsulfoxide; e2, second
extracellular; EDAC, 1-ethyl-3-(3-dimethylaminopropyl)-
carbodiimide; EI-MS, electron-impact ionization mass spec-
trometry; Emax, efficacy; ES-MS, electrospray ionization mass
spectrometry; GAIP, Gα interacting protein (corresponds to
RGS19 protein); 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; Gsαs, α-subunit (short
splice variant) of the Gs protein that mediates stimulation of
adenylyl cyclase; H1R, histamine H1 receptor; H2R, histamine
H2 receptor; H3R, histamine H3 receptor; H4R, histamine H4
receptor; hH1R, human histamine H1 receptor; hH2R, human
histamine H2 receptor; H2R-gpNT, N-terminus of the guinea
pig H2 receptor; GTP, guanosine 5′-triphosphate; hH2R-GsαS,
fusion protein of the human histamine H2 receptor and the
short splice variant of Gsα; hH3R, human histamine H3
receptor; hH4R, human histamine H4 receptor; hH4R-RGS19,
fusion protein of the human histamine H4 receptor and RGS19;
HOBt, hydroxybenzotriazole; HPLC, high performance liquid
chromatography; HR-MS, high resolution mass spectrometry;
k′, capacity factor; LSI-MS, liquid-secondary-ion mass spec-
trometry; rel pot, potency relative to histamine; RGS, regulator
of G protein signaling proteins; RP-HPLC, reverse phase
HPLC; rt, room temperature; SEM, standard error of the
mean; TFA, trifluoroacetic acid; THF, tetrahydrofuran; TM,
transmembrane domain of a GPCR; tR, retention time; Trt,
trityl
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