Dimeric carbamoylguanidine-type histamine H2 receptor ligands: A new class of potent and selective agonists

Article abstract of DOI:10.1016/j.bmc.2015.01.012

The bioisosteric replacement of the acylguanidine moieties in dimeric histamine H₂ receptor (H₂R) agonists by carbamoylguanidine groups resulted in compounds with retained potencies and intrinsic activities, but considerably improved

Full text of DOI:10.1016/j.bmc.2015.01.012

Bioorganic & Medicinal Chemistry 23 (2015) 3957–3969
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Dimeric carbamoylguanidine-type histamine H2 receptor ligands: A
new class of potent and selective agonists
Nicole Kagermeier a, Kristin Werner b, Max Keller a, Paul Baumeister a, Günther Bernhardt a,
Roland Seifert b, Armin Buschauer a,
⇑
a Institute of Pharmacy, University of Regensburg, Universitätsstraße 31, D-93053 Regensburg, Germany
b Institute of Pharmacology, Hannover Medical School, Carl-Neuberg-Str.1, D-30625 Hannover, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
The bioisosteric replacement of the acylguanidine moieties in dimeric histamine H2 receptor (H2R) ago-
nists by carbamoylguanidine groups resulted in compounds with retained potencies and intrinsic activ-
ities, but considerably improved stability against hydrolytic cleavage. These compounds achieved up to
Received 11 October 2014
Revised 21 December 2014
Accepted 7 January 2015
Available online 14 January 2015
2500 times the potency of histamine when studied in [35S]GTP
cS assays on recombinant human and gui-
nea pig H2R. Unlike 3-(imidazol-4-yl)propyl substituted carbamoylguanidines, the corresponding
2-amino-4-methylthiazoles revealed selectivity over histamine receptor subtypes H1R, H3R and H4R in
radioligand competition binding studies. H2R binding studies with three fluorescent compounds and
one tritium-labeled ligand, synthesized from a chain-branched precursor, failed due to pronounced cel-
lular accumulation and high non-specific binding. However, the dimeric H2R agonists proved to be useful
pharmacological tools for functional studies on native cells, as demonstrated for selected compounds by
cAMP accumulation and inhibition of fMLP-stimulated generation of reactive oxygen species in human
monocytes.
Keywords:
Histamine H2 receptor
Bioisosteric replacement
Bivalent ligand
Receptor subtype selectivity
Human monocytes
Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction
potency seems to result from the interaction with an additional
binding site at the same receptor molecule. However, so far the
Previously, NG-acylated hetarylpropylguanidines were identi-
fied as potent histamine H2 receptor (H2R) agonists.1–3 Surpris-
ingly, an enormous increase in H2R potency was achieved by
linking two acylguanidine moieties. The most potent of those
dimeric compounds showed up to 5000-fold potency of histamine
(depending on the type of assay and the respective H2R ortholog).3
The first generation of NG-acylated imidazolylpropylguanidines,
showed a lack of selectivity, especially towards histamine H3
receptor (H3R) and histamine H4 receptor (H4R).4 Selectivity for
the H2R was achieved by bioisosteric replacement of the imidazole
ring by an amino(methyl)thiazole moiety.2,3 Portoghese suggested
that the distance between the orthosteric binding sites of two
dimerising receptor protomers is about 22–27 Å.5 In an approach
to explore whether the bivalent H2R agonists bind to a receptor
monomer or a receptor dimer, Birnkammer et al. investigated com-
pounds with a spacer length between 6 and 27 Å.3 The highest
potency resided in compounds with an octa- or hexamethylene
chain.3 Due to insufficient spacer length for interaction with the
orthosteric binding pockets of dimeric H2R protomers, the gain in
binding mode of bivalent H2R agonists is not understood and
appropriate pharmacological tools to study the mode and stoichi-
ometry of binding are not available.
Acylguanidines turned out to undergo hydrolytic cleavage upon
long-term storage in aqueous solution. Aiming at more stable
dimeric H2R agonists, we replaced the acylguanidine moieties by
carbamoylguanidine groups according to a bioisosteric approach
(Fig. 1). Moreover, a branched linker enabled the synthesis of radi-
olabeled and fluorescent derivatives. The compounds were charac-
terized in functional and binding studies on recombinant
histamine receptors. In addition, selected agonists were investi-
gated on human monocytes.
2. Results and discussion
2.1. Chemistry
2.1.1. Synthesis of the dimeric carbamoylguanidine-type
ligands
The building blocks 3-(1-trityl-1H-imidazol-4-yl)propan-1-
amine (1) and tert-butyl [5-(3-aminopropyl)-4-methylthiazol-2-
yl]carbamate (2) (Scheme 1) were synthesized as recently
⇑
Corresponding author. Tel.: +49 941 9434827; fax: +49 941 9434820.
E-mail address: armin.buschauer@chemie.uni-regensburg.de (A. Buschauer).
http://dx.doi.org/10.1016/j.bmc.2015.01.012
0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.

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N. Kagermeier et al. / Bioorg. Med. Chem. 23 (2015) 3957–3969
converted to the diisocyanate 26. The guanidinylating reagent 27
was prepared by analogy with the procedure for the preparation
of 3–7.
The precursor (28; Scheme 3) of the labeled compounds was
synthesized from 27 according to the procedure described above
for compounds 13–17 using 2 as building block. Compound 28
was coupled to three different fluorophores: the cyanine S2197
and the small pyrylium dyes Py-1 and Py-5 (Scheme 3).11–13
S2197 was provided as succinimidyl ester and coupled to the biva-
lent precursor in the presence of triethylamine to give 29. The
pyrylium dyes reacted instantaneously in a ring transformation
(color changes from blue into red) to give the positively charged
N-substituted pyridinium compounds 30 and 31 (Scheme 3).14
Compound 28 was acylated with ‘cold’ succinimidyl propionate
(cf. 32a) to optimize the reaction conditions for the propionylation
with tritiated succinimidyl propionate (Scheme 4). This synthetic
route afforded the radioligand 32b in a yield of 90%. Purification
by HPLC gave 32b in a radiochemical purity of 99% with a specific
activity of 70 Ci/mmol.
Figure 1. Bioisosteric replacement of the acylguanidine moieties in dimeric H2R
agonists by carbamoylguanidine groups.
reported.1,2 The guanidinylating reagents (3–7) were obtained
from diamines and diisocyanates in a one-pot reaction6 (Scheme 1).
The diamines were treated with triphosgene to give the corre-
sponding diisocyanates, which were allowed to react with mono-
Boc-protected S-methylisothiourea, a well-established guanidiny-
lating reagent,7 which was prepared as described previously.2
The dimeric ligands 8–17 were prepared by treating the building
blocks 1,2 with the guanidinylating reagents 3–7 in the presence of
HgCl2 and base.8 Treating the protected carbamoylguanidine-type
intermediates with TFA gave compounds 8–17 (Scheme 1), which
were purified by preparative HPLC.
2.2. Stability of carbamoylguanidines versus acylguanidines
Acylguanidines are stable at acidic pH, but tend to decompose
under alkaline conditions. The decomposition becomes extremely
fast when an intramolecular nucleophilic attack is possible as pre-
viously demonstrated for aminoalkanoylguanidines with half-lives
between 19 s and 13 h at pH 10.4.15 Although the decomposition of
acylguanidine-type H2R agonists such as 333 (Fig. 2A) turned out to
occur at a much slower rate, hydrolytic cleavage has to be taken
into account upon long-time storage in solution. For comparison,
the stability of compounds 14 (Fig. 2B) and 33 as representative
examples of carbamoylated and acylated guanidines, respectively,
2.1.2. Toward bivalent fluorescence- and radioligands
In view of radiolabeling and introduction of fluorophores, a
branched linker comprising a primary amino group was synthe-
sized. Starting from commercially available dicyclopropylketone,
1,7-dichloroheptan-4-one was obtained as recently described.9
After nucleophilic displacement of chlorine by azides, the C@C
bond was formed by a Wittig reaction10 to yield 1-azido-4-(3-azi-
dopropyl)-8-chlorooct-4-ene (21) (Scheme 2). Via the correspond-
ing phthalimide (22), 21 was converted into the primary amine 23.
The amino group was Boc-protected (24) and the azide groups
were reduced with LiAlH4 to give the diamine 25, which was
was investigated at a concentration of 100 lM in phosphate buffer
(PBS, pH 7.4) at room temperature. After one week around 55% of
compound 33 were decomposed.
Scheme 1. Synthesis of the bivalent carbamoylguanidine-type ligands 8–17. Reagents and conditions: (a) triphosgene, DIPEA, DCM abs., 30 min, 0 °C; (b) N-tert-
butoxycarbonyl-S-methylisothiourea, 2.5 h, rt, 3: 83%, 4: 74%, 5: 75%, 6: 74%, 7: 76%; (c) 1 or 2, HgCl2, NEt3, DMF or DCM abs., 12 h, rt; (d) 30% TFA, DCM abs., 12 h, rt, 8: 7%, 9:
12%, 10: 8%, 11: 20%, 12: 27%, 13: 14%, 14: 30%, 15: 29%, 16: 9%, 17: 9%.

N. Kagermeier et al. / Bioorg. Med. Chem. 23 (2015) 3957–3969
3959
Scheme 2. Synthesis of the branched building block 27. Reagents and conditions: (a) HCl gas, rt, 100%; (b) NaN3, DMF, 12 h, rt, 78%; (c) n-BuLi, THF abs, 15 h, À72 °C ? rt,
19%; (d) phthalimide, Cs2CO3, KI (cat.), DMF, 60 °C, 12 h, 57%; (e) N2H2 x H2O, EtOH, 1 h, reflux, 88%; (f) Boc2O, NEt3, DCM, rt, 24 h, 97%; (g) LiAlH4, Et2O/abs, 2 h, reflux, 66%;
(h) triphosgene, DIPEA, DCM abs., 30 min, 0 °C; (i) N-tert-butoxycarbonyl-S-methylisothiourea, 2.5 h, rt, 44%.
2.3. Pharmacological results
the pEC50 values, in particular when [3H]UR-DE25719 was used as
radioligand. Minor differences between functional and binding
data became obvious when [3H]tiotidine was used for displace-
ment. As suggested by Kelley et al.17 [3H]tiotidine is able to address
only a subpopulation of the H2R which might explain this phenom-
enon. As expected, carbamoylguanidine-type ligands bearing two
imidazole moieties were not selective for the H2R, but also showed
high affinity for the H3R and the H4R. By contrast, the synthesized
aminothiazoles preferred the H2R (Table 2).
The synthesized compounds were investigated on membrane
preparations in the [35S]GTP
cS binding assay at the human (h)
and at the guinea pig (gp) H2R with regard to possible differences
between species orthologs (Table 1, Fig. 3). The hH2R as well as the
gpH2R were expressed as H2R-Gsas fusion proteins in Sf9 insect
cells. To test the selectivity of the compounds for the H2R com-
pared to hH1R, hH3R, and hH4R, competition binding experiments
were performed using membranes of Sf9 cells expressing the hista-
mine receptor of interest (Table 2).
2.3.3. Investigation of the fluorescence ligands 29–31 and the
radioligand 32b
2.3.1. H2R agonism in the [35S]GTP
cS binding assay
The compounds 29–31 were investigated in a flow cytometric
binding assay25 at HEK293T cells transfected with the hH2R (cf.
Supplementary data, Fig. S1). None of these compounds bound in
a saturable manner. Confocal microscopy revealed that the fluores-
cence ligands enter the cells in a receptor-independent manner (cf.
Supplementary data, Fig. S2).
Similarly, it was impossible to perform saturation binding
studies with the radioligand 32b on Sf9 membranes (cf. Supple-
mentary data, Fig. S3) and HEK293T cells because of high
unspecific binding. Interestingly, lower unspecific binding was
observed in the presence of competitors structurally similar to
32a, hinting at reversible binding to non-H2R sites at the cell
membrane. Nevertheless, in case of HEK293T cells non-H2R
mediated internalization of 32b cannot be excluded. Radioligand
32b proved to be inappropriate for H2R quantification. In spite
of that, affinities determined for various H2R ligands in competi-
tion binding experiments with 32b (membranes of Sf9 cells
expressing the hH2R), were in the range of Ki values determined
by displacement of [3H]UR-DE25719 (cf. Supplementary data,
The prepared bivalent ligands 8–17, 28 and 32a were partial or
full agonists at both the hH2R and the gpH2R. In agreement with
previous studies on guanidine- and acylguanidine-type H2R ago-
nists,2,3,16,17 higher potencies and intrinsic activities were achieved
at the gpH2R than at the hH2R. Compound 11 was 2500 times more
potent than histamine at the gpH2R. Except for 14, ligands bearing
an imidazole ring were superior to the corresponding aminothiaz-
oles at the hH2R. Dimeric ligands with a spacer length of four
methylene groups showed the lowest potency at the hH2R,
whereas the gpH2R was less sensitive to variations in chain length.
Interestingly, branching of the connecting chain hardly affected the
potency (cf. 28), though the maximal response decreased. Prop-
ionylation of the primary amino group in the side chain of 28
resulted in regaining potency and intrinsic activity at the hH2R
(32a) (Table 1, Fig. 3).
2.3.2. H2R affinities and receptor subtype selectivities
The H2R affinity of the compounds was determined in competi-
tion binding studies. The pKi values were in good agreement with

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N. Kagermeier et al. / Bioorg. Med. Chem. 23 (2015) 3957–3969
Scheme 3. Synthesis of compound 28 and preparation of the fluorescence ligands 29–31. Reagents and conditions: (a) (1) compd 2, HgCl2, NEt3, DMF, 12 h, rt, (2) 30% TFA,
DCM abs., 12 h, rt, 21%; (b) NEt3, DMF, rt, 2 h, 29: 45%, 30: 36%, 31: 38%.
Scheme 4. Synthesis of the unlabeled (‘cold’) propionamide 32a and the corresponding radiolabeled version 32b. Reagents and conditions: (a) (1) NEt3, DMF, 2 h, 81%; (2)
DIPEA, DMF, 3 h, 90%.
Table S1). However, it should be noted that, due to the low signal-
to-noise ratio, the data determined with 32b as radioligand are
less reliable.
Compounds 9 and 14 were studied at the H2R in human mono-
cytes regarding functional responses such as stimulation of cAMP
formation and inhibition of formyl peptide (fMLP)-induced pro-
duction of reactive oxygen species (ROS) (Fig. 4, Table 3). To avoid
cell lysis as observed for such cationic amphiphilic compounds
depending on the chain length of the linker (for cytotoxicity of
dimeric H2R agonists cf. Supplementary data, Fig. S5), we selected
compounds 9 and 14, which are among the most potent H2R ago-
nists and proved to be nontoxic (cf. Supplementary data, Fig. S6)
under the assay conditions.
2.3.4. Investigation of H2R agonists in human monocytes
Acute myeloid leukemia (AML) is an indication for post-consol-
idation therapy with histamine to prevent relapse.26,27 It is
assumed that histamine inhibits oxygen radical formation via
H2R mediated inhibition of NADPH oxidase, resulting in the protec-
tion of T cells and NK cells.26
Since histamine activates all four histamine receptor subtypes,
the treatment is accompanied by serious side effects like itch, ery-
thema, decrease in blood pressure and gastrointestinal prob-
lems.26,27 Patients could benefit from selective H2R stimulation
due to reduction of side effects mediated by the other histamine
receptor subtypes.
Recently, we have shown that H2R agonists increase cAMP lev-
els in a concentration-dependent manner in human monocytes.28
In accordance with these results, compounds 9 and 14 induced
cAMP accumulation (Fig. 4A). Additionally, both ligands inhibited
the fMLP-induced ROS production in a concentration-dependent
manner via H2R stimulation (Fig 4B).

N. Kagermeier et al. / Bioorg. Med. Chem. 23 (2015) 3957–3969
3961
Figure 2. Chromatograms of the H2R agonists 33 (A) and 14 (B) after different periods of incubation in PBS (pH 7.4) at rt.
Table 1
Histamine H2 receptor agonism in the [35S]GTP
c
S assaya
Compd
Human H2R
Guinea pig H2R
pEC50
Emax
Potrel
pEC50
Emax
Potrel
1.0
309.5
Histamine
8
9
5.85 0.06
7.29 0.08
7.77 0.02
8.31 0.08
8.35 0.07
8.08 0.09
7.25 0.11
8.03 0.02
7.68 0.03
7.73 0.15
7.43 0.12
7.49 0.10
7.87 0.12
1.00
1.0
27.5
83.2
288.4
316.0
169.8
25.1
151.4
67.6
75.9
38.0
43.7
104.7
6.07 0.1418,b
8.55 0.04
9.25 0.14
8.45 0.03
9.47 0.09
8.19 0.07
9.04 0.13
8.75 0.15
8.42 0.19
8.16 0.07
n.d.
1.00
0.97 0.02
0.88 0.03
1.03 0.06
1.01 0.07
0.70 0.08
0.96 0.04
0.92 0.01
0.90 0.10
0.80 0.07
0.64 0.07
0.66 0.06
0.73 0.12
0.99 0.06
0.96 0.03
0.87 0.08
1.01 0.06
0.95 0.04
1.04 0.14
1.09 0.05
0.97 0.01
0.80 0.06
n.d.
1519.9
239.7
2512.2
131.7
935.3
478.7
224.0
123.0
n.d.
10
11
12
13
14
15
16
17
28
32a
8.11 0.06
8.04 0.05
0.97 0.06
0.89 0.11
109.7
93.3
a
[
35S]GTP
c
S binding assay on membranes of Sf9 cells expressing the hH2R-Gs
as and the gpH2R-Gsas. Data were analyzed by nonlinear regression and were best fit to three-
parameter sigmoidal concentration-response curves. pEC50: ÀlogEC50; Emax: maximal response relative to histamine (Emax = 1.0); Potrel: relative potency represents the ratio
of EC50 values of test compound and histamine. Data shown are means SEM of 2-6 independent experiments, each performed in triplicate.
b
Determined in a steady-state [32P]GTPase assay on Sf9 cells expressing the gpH2R-Gsas
.

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N. Kagermeier et al. / Bioorg. Med. Chem. 23 (2015) 3957–3969
with retained potencies and intrinsic activities. Achieving up to
2500 times the potency of histamine these compounds are among
the most potent H2R agonists known so far. Unfortunately, H2R
binding studies with fluorescence and radiolabeled ligands derived
from the chain-branched precursor 28 failed due to pronounced
cellular accumulation and high non-specific binding, respectively.
In this respect, the results underline the fact that, regardless of
high affinity of the ‘cold’ form of a receptor ligand, applicability
of the corresponding labeled version as a molecular tool cannot
be taken for granted. Aminothiazoles were superior to the corre-
sponding imidazoles in terms of histamine receptor subtype selec-
tivity. Due to high potency and stability against hydrolytic
cleavage, the new H2R agonists are promising pharmacological
tools for investigations on the biological role of the H2R and are
of potential value for in vivo studies on histamine receptors in
the CNS. In human monocytes the investigated compounds
revealed high H2R agonist potency, suggesting this class of com-
pounds to point a way to selective H2R agonists as potential agents
for the treatment of AML.
4. Experimental section
4.1. General experimental section
Unless otherwise indicated, chemicals and solvents were from
commercial suppliers and were used as received. The fluorescence
dye S2197 was from FEW Chemicals GmbH (Bitterfeld–Wolfen,
Germany), the pyrylium dyes Py-1 and Py-5 were kindly provided
by Prof. Dr. O. S. Wolfbeis (Institute of Analytical Chemistry,
Chemo- and Biosensors, University of Regensburg, Germany). All
of the solvents were of analytical grade or were distilled prior to
use. THF and Et2O were distilled over sodium, DCM was stored over
CaCl2 and was afterwards distilled from P2O5. For column chroma-
tography silica gel 60 (Merck, 0.04–0.063 mm) was used. Reactions
were monitored by thin layer chromatography (TLC) on Merck sil-
ica gel 60 F254 aluminium sheets, and spots were visualized with
UV light at 254 nm, iodine vapor or ninhydrin spray. NMR spectra
Figure 3. Concentration-response curves of selected agonists at hH2R (A) and
gpH2R (B) in the [35S]GTP
cS assay. The increase in GTPcS binding is referred to the
maximal response to histamine (HIS) = 100%. Data are means SEM of 2-6
independent experiments, each performed in triplicate.
3. Summary and conclusion
The exchange of the acylguanidine moieties in bivalent H2R
agonists by carbamoylguanidine groups resulted in compounds
Table 2
Binding data of the compounds 8–17, 28, 32a on human histamine receptor subtypesa
Compd
hH1R
hH2R
hH3R
hH4R
pKi SEMb
pKi SEMc,d
pKi SEMe,f
pKi SEMg
Histamine
8
9
5.62 0.0320
6.31 0.10
5.87 0.04
6.60 0.12
6.66 0.03
6.55 0.02
6.08 0.03
6.06 0.05
5.81 0.09
5.80 0.06
5.89 0.01
5.80 0.08
6.36 0.00
6.27 0.04c,15
7.57 0.01c
7.87 0.05c
7.52 0.04d
8.47c; 8.52 0.13d
7.56 0.05d
7.76 0.08c
8.07 0.05c
7.55 0.02c
7.17 0.13d
7.21 0.10d
7.59 0.01c
7.94 0.09c
8.20 0.0421
8.56 0.02f
7.89 0.0122
7.02 0.06
7.12 0.04
7.81 0.09
8.27 0.01
7.58 0.12
5.29 0.04
5.69 0.07
5.55 0.08
6.26 0.18
5.97 0.05
6.08 0.09
5.96 0.09
8.16 0.05e; 8.52f
8.77 0.02f
10
11
12
13
14
15
16
17
28
32a
8.82 0.03f
8.21 0.03f
6.17 0.10e; 6.17f
5.94 0.16e; 6.01f
5.76 0.09f
6.00 0.10f
5.81 0.07e
6.56 0.11f
6.08 0.08f
a
Competition binding assay on membranes of Sf9 cells expressing the hH1R plus RGS4, the hH2R-Gs
a
s, co-expressing the hH3R plus G
a
i2 plus Gb1c2 or co-expressing the
hH4R plus Gai2 plus Gb1c2. Incubation period was 60 min. Data were analyzed by nonlinear regression and were best fit to 3-parameter sigmoidal concentration–response
curves. Data shown are means SEM of 2–6 independent experiments, each performed in triplicate.
b
Displacement of 5 nM [3H]mepyramine (Kd = 4.5 nM).
c
Displacement of 30 nM [3H]UR-DE257 (Kd = 31 nM).
d
Displacement of 10 nM tiotidine (Kd = 19.7 nM).
e
Displacement of 15 nM [3H]histamine (Kd = 12.6 nM).
f
a
Displacement of 3 nM [3H]N -methylhistamine (Kd = 3 nM).
g
Displacement of 10 nM histamine (Kd = 10 nM); Note: Representative compounds were investigated for functional activity at the hH1R, hH3R and hH4R. In agreement with
previous studies2,3,23 on bivalent H2R ligands, the carbamoylated imidazolylpropylguanidines behaved as agonists in the [35S]GTP
cS assay (Sf9 membranes) at the H3R and
the H4R respectively, whereas the corresponding amino(methyl)thiazolylpropylguanidines proved to be H3R and H4R antagonists and were antagonists at the hH1R (luciferase
assay, HEK293 cells24). hH1R, pKb SEM: 10: 6.48 0.14; 11: 6.29 0.21; 14: 5.71 0.06; 16: 5.69 0.13; 32a: 5.32 0.13. hH3R, pKb SEM: 14: 5.57 0.05; 16: 5.86 0.01;
32a: 5.87 0.06. hH4R: 10: pEC50 = 8.00 0.04, Emax = 0.65 0.03; 11: pEC50 = 8.04 0.09, Emax = 0.86 0.08; 14: pKb = 5.30 0.02; 16: pKb = 6.12 0.11; 32a:
pKb = 5.09 0.22.

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3963
Table 3
Potencies and efficacies in the cAMP assay and in the reactive oxygen species (ROS)
assay in human monocytesa
Compd
cAMP assay
ROS assay
pEC50
Emax
pIC50
Emax
Histamine
9
14
5.39 0.06
7.03 0.11
7.27 0.19
1.00
0.57 0.03
0.44 0.04
5.68 0.13
6.91 0.19
6.54 0.27
1.00
0.78 0.07
0.83 0.10
a
Data were analyzed by nonlinear regression and were best fit to 3-parameter
sigmoidal concentration-response curves. The maximal responses (Emax values) of 9
and 14 are referred to the efficacy of histamine defined as Emax = 1.00. Data shown
are means SEM of three to four independent experiments.
SN400 controller, a P4000 pump, an AS3000 autosampler, and a
Spectra Focus UV/Vis detector. The column was either a Euro-
spher-100 C18 (250 Â 4 mm, 5.0
l
m; Knauer, Berlin, Germany) or
a YMC C8 Column (250 Â 4.6 mm, 5.0
lm); YMC Europe GmbH,
Dinslaken, Germany), thermostated at 30 °C. As mobile phase, mix-
tures of MeCN/aqueous TFA were used. Gradient mode (unless
otherwise indicated): MeCN/TFA (0.05%) (v/v) 0 min: 5:95,
30 min: 40:60, 31–41 min: 90:10; flow rate = 0.75 mL/min,
t0 = 2.95 min (Eurospher), t0 = 2.55 min (YMC); capacity factor
k = (tR À t0)/t0. Absorbance was detected at 210 nm. Compound
purities were calculated as the percentage peak area of the ana-
lyzed compound by UV detection at 210 nm. UV Spectra were
recorded with a Cary Eclipse spectrofluorimeter (Varian, Mulgrave,
Victoria, Australia).
Figure 4. Effects of histamine (HIS), 9 and 14 on cAMP accumulation (A) and fMLP-
induced ROS production (B) in human monocytes. Samples were analyzed by HPLC–
MS/MS. A: Data were normalized to the maximal effect of HIS and represent
means SEM from three independent experiments. B: Data were normalized to the
ROS release induced by fMLP and are expressed as means SEM from four different
experiments performed in triplicate. Note: The influence of H2R agonists on ROS
production had to be studied in an indirect way, that is, after stimulation of the cells
4.2. Chemistry: Protocols and analytical data
4.2.1. N1,N4-Bis{[(tert-butoxycarbonylamino)(methylsulfanyl)
methylene]aminocarbonyl}butane-1,4-diamine (3)
with fMLP (1 l
M). In accordance with previous studies,28 we observed that the
To
a
solution of N-tert-butoxycarbonyl-S-methylisothiourea
L, 0.22 mmol) in DCM was
maximal effect of fMLP (set to 1.0 in Fig. 4B) was not fully restored in the presence
of very low concentrations of histamine or H2R agonists. So far, we have no
explanation for this phenomenon, which only minimally affects the inflections
points of the concentration-response curves. In Table 3 the maximal responses
(Emax) of 9 and 14 are given relative to the amplitude of the concentration-response
curve of histamine defined as Emax = 1.0.
(855 mg, 4.50 mmol) and NEt3 (30
l
added dropwise a solution of 1,4-diisocyanatobutane (300 mg,
2.14 mmol) in DCM. The solution was stirred for 2.5 h at room tem-
perature. The solvent was removed under reduced pressure and
the crude product was purified by column chromatography (elu-
ent: DCM/EA 15:1) (Rf = 0.19 in Hex/EA 3:2). The product was
obtained as a white solid (928 mg, 83%). mp = 144.9–146.1 °C. 1H
NMR (300 MHz, CDCl3) d (ppm) 1.47 (s, 18H), 1.55–1.65 (m, 4H),
2.29 (s, 6H), 3.22–3.26 (m, 4H), 5.62 (t, 2H, J 6.0 Hz), 12.27 (s,
2H); 13C NMR (75 MHz, CDCl3) d (ppm) 14.4, 27.3, 28.1, 39.8,
82.7, 151.2, 162.1, 167.6. HRMS (ESI) m/z (%) 521 (100) [M+H]+;
m/z [M+H]+ calculated for C20H37N6O6S+2: 521.2211, found
521.2214; C20H36N6O6S2 (520.67).
were recorded on a Bruker Avance 300 (1H: 300 MHz, 13C:
75.5 MHz) and
a
Bruker Avance 600 (1H: 600 MHz, 13C:
150.9 MHz) (Bruker, Karlsruhe, Germany) with deuterated solvents
from Deutero (Kastellaun, Germany). LRMS was performed on a
Finnigan ThermoQuest TSQ 7000 Instrument (Thermo Scientific,
Waltham, MA) using an ESI source. HRMS was performed on an
Agilent 6540 UHD Accurate-Mass Q-TOF LC/MS system (Agilent
Technologies, Santa Clara, CA) using an ESI or an APCI source. Melt-
ing points (mp) were measured on a Büchi B-540 apparatus using
an open capillary and are uncorrected. Preparative HPLC was per-
formed with a system from Knauer (Berlin, Germany) consisting
of two K-1800 pumps, a K-2001 detector and the column was
4.2.2. N1,N6-Bis{[(tert-butoxycarbonylamino)(methylsulfanyl)
methylene]aminocarbonyl}hexane-1,6-diamine (4)
To
a
solution of N-tert-butoxycarbonyl-S-methylisothiourea
L, 0.22 mmol) in DCM was
(700 mg, 3.68 mmol) and NEt3 (30
l
added dropwise a solution of 1,6-diisocyanatohexane (300 mg,
2.02 mmol) in DCM. The solution was stirred for 2.5 h at room tem-
perature. The solvent was removed under reduced pressure and
the crude product was purified by column chromatography (elu-
ent: DCM/EA 15:1) (Rf = 0.29 in Hex/EA 3:2). The product was
obtained as a white solid (820 mg, 74%). mp = 128.8–133.6 °C. 1H
NMR (300 MHz, CDCl3) d (ppm) 1.34–1.39 (m, 4H), 1.46 (s, 18H),
1.50–1.56 (m, 4H), 2.29 (s, 6H), 3.17–3.24 (m, 4H), 5.55 (t, 2H, J
5.8 Hz), 12.30 (s, 2H); 13C NMR (75 MHz, [D4]MeOH) d (ppm)
14.4, 26.7, 28.1, 29.7, 40.1, 82.7, 151.2, 162.1, 167.4. HRMS (ESI)
m/z (%) 549 (100) [M+H]+; m/z [M+H]+ calculated for C22H41N6O6S+2:
549.2524, found 549.2525; C22H40N6O6S2 (548.72).
either a Eurospher-100 C18 (250 Â 32 mm, 5
100-5 C18 (250 Â 21 mm, 5 m) (Macherey-Nagel, Düren, Ger-
many) or in case of the fluorescence ligands a Phenomenex Kinetex
(250 Â 21 mm, 5 m) (Phenomenex, Aschaffenburg, Germany). As
lm), a Nucleodur
l
l
mobile phase mixtures of acetonitrile and 0.1% aqueous TFA were
used. The temperature was 30 °C and UV detection was carried
out at 220 nm. Prior to lyophilisation (Christ alpha 2–4 LD freeze
dryer (Osterode, Germany), equipped with a RZ 6 rotary vane vac-
uum pump (Vacuubrand, Wertheim, Germany), acetonitrile was
removed under reduced pressure. Analytical HPLC was performed
on a system from Thermo Separation Products equipped with an

3964
N. Kagermeier et al. / Bioorg. Med. Chem. 23 (2015) 3957–3969
4.2.3. General procedure for the synthesis of the guanidinylating
reagents (5–7)
The bis(Boc)-bis(trityl)-(cf. imidazoles) or tetrakis(Boc)-protected
intermediate (cf. aminothiazoles) was extracted with EA (unless
indicated otherwise), purified by column chromatography (eluent:
DCM/MeOH 100:1 to 50:1), and dried in vacuo. Subsequently,
deprotection was performed by stirring with 30% TFA in DCM for
12 h. The obtained carbamoylguanidine (cf. 8–17) was purified
by preparative HPLC.
The respective diamine was dried overnight in vacuo and the
reactions were carried out in an argon purged two necked round
bottom flask. The diamines (1 equiv) were dissolved in freshly dis-
tilled DCM (8 mL) and DIPEA (5.6 equiv) was added. This mixture
was added dropwise under external cooling to a solution of tri-
phosgene (1 equiv) in anhydrous DCM over a period of 30 min.
N-tert-Butoxycarbonyl-S-methylisothiourea (4 equiv) was added
and stirring was continued for 2.5 h. The solvent was removed
under reduced pressure and the crude product was purified by col-
umn chromatography (eluent: DCM/EA 15:1).
4.2.4.1. 1-(Amino{[3-(1H-imidazol-4-yl)propyl]amino}methylene)-
3-{4-[3-(amino{[3-(1H-imidazol-4-yl)propyl]amino}methylene)
ureido]butyl}urea (8). The title compound was prepared from 3
(175 mg, 0.336 mmol), amine 1 (260 mg, 0.708), NEt3 (233 lL,
1.67 mmol) and HgCl2 (365 mg, 1.34 mmol) according to the gen-
eral procedure, yielding 8 as a white fluffy, hygroscopic solid
(21.88 mg, 7%): RP-HPLC: 99%, (tR = 11.75 min, k = 2.99); UVmax
204 nm. 1H NMR (600 MHz, [D4]MeOH) d (ppm) 1.57 (m, 4H),
2.03 (tt, 4H, 2J 7.0 Hz, 3J 7.6 Hz), 2.84 (t, 4H, J 7.7 Hz), 3.23 (m,
4H), 3.36 (t, 4H, J 6.9 Hz), 7.36 (s, 2H), 8.80 (d, 2H, J 1.2 Hz); 13C
NMR (151 MHz, [D4]MeOH, trifluoroacetate) d (ppm) 22.6, 27.7,
28.2, 40.3, 41.4, 117.1, 134.4, 135.0, 155.6, 156.0, 163.3, 163.5.
HRMS (ESI) m/z (%) 475 (29) [M+H]+, 238 (75) [M+2H]2+; m/z
[M+H]+ calculated for C20H35N12O+2: 475.3000, found 475.3003;
C20H34N12O2 Â 4 TFA (930.64).
4.2.3.1. N1,N7-Bis{[(tert-butoxycarbonylamino)(methylsulfanyl)
methylene]aminocarbonyl}heptane-1,7-diamine (5). The title
compound was prepared from heptane-1,7-diamine (262 mg,
2.01 mmol), DIPEA (1963 lL, 11.27 mmol), triphosgene (596 mg,
2.01 mmol) in anhydrous DCM and N-tert-Butoxycarbonyl-S-meth-
ylisothiourea (1530 mg, 8.08 mmol) according to the general
procedure (Rf = 0.29 in Hex/EA 3:2). The product was obtained as
colorless glassy solid (855 mg, 75%). 1H NMR (300 MHz, CDCl3) d
(ppm) 1.33 (m, 6H), 1.46 (s, 18H), 1.49 (overlap with tert-Bu, 4H),
2.29 (s, 6H), 3.16–3.23 (m, 4H), 5.55 (t, 2H, J 5.0 Hz), 12.31
(s, 2H); 13C NMR (75 MHz, CDCl3) d (ppm) 14.4, 26.9, 28.1, 29.1,
29.7, 40.2, 82.6, 151.2, 162.0, 167.3. HRMS (ESI) m/z (%) 563
(100) [M+H]+; m/z [M+H]+ calculated for C23H43N6O6S+2: 563.2680,
found 563.2690; C23H42N6O6S2 (562.75).
4.2.4.2. 1-(Amino{[3-(1H-imidazol-4-yl)propyl]amino}methylene)-
3-{6-[3-(amino{[3-(1H-imidazol-4-yl)propyl]amino}methylene)
ureido]hexyl}urea (9). The title compound was prepared from 4
(175 mg, 0.319 mmol), amine
1 (250 mg, 0.680 mmol), NEt3
4.2.3.2. N1,N8-Bis{[(tert-butoxycarbonylamino)(methylsulfanyl)
(222 L, 1.60 mmol) and HgCl2 (346 mg, 1.27 mmol) according to
l
methylene]aminocarbonyl}octane-1,8-diamine (6).
compound was prepared from octane-1,8-diamine (400 mg,
2.77 mmol), DIPEA (2701 L, 15.50 mmol), triphosgene (823 mg,
2.77 mmol) and N-tert-Butoxycarbonyl-S-methylisothiourea
(2108 mg, 11.08 mmol) according to the general procedure
(Rf = 0.34 in Hex/EA 3:2). The product was obtained as a white solid
(1183 mg, 74%). mp = 116.4–119.2 °C. 1H NMR (300 MHz, CDCl3) d
(ppm) 1.32 (m, 8H), 1.47 (s, 18H), 1.50 (m, 4H), 2.29 (s, 6H), 3.17–
3.24 (m, 4H), 5.53 (t, 2H, J 5.6 Hz), 12.32 (s, 2H); 13C NMR (75 MHz,
CDCl3) d (ppm) 14.4, 27.0, 28.2, 29.3, 29.8, 40.3, 82.7, 151.3, 162.1,
167.4. HRMS (ESI) m/z (%) 577 (100) [M+H]+; m/z [M+H]+ calcu-
lated for C24H45N6O6S2+: 577.2837, found 577.2845; C24H44N6O6S2
(576.77).
The title
the general procedure, yielding 9 as a white fluffy hygroscopic solid
(36.00 mg, 12%): RP-HPLC: 98%, (tR = 15.18 min, k = 4.15); UVmax
l
205 nm. 1H NMR (600 MHz, [D4]MeOH)
d (ppm) 1.36–1.38
(m, 4H), 1.52–1.58 (m, 4H), 2.03 (tt, 4H, 2J 7.1 Hz, 3J 7.5 Hz), 2.84
(t, 4H, J 7.7 Hz), 3.20 (t, 4H, J 7.1 Hz,), 3.37 (t, 4H, J= 6.9 Hz), 7.37
(s, 2H), 8.81 (d, 2H, J 1.3 Hz); 13C NMR (151 MHz, [D4]MeOH, tri-
fluoroacetate) d (ppm) 22.5, 27.4, 28.2, 30.3, 40.7, 41.4, 117.1,
134.3, 135.0, 155.5, 156.0. HRMS (ESI) m/z (%) 617 (4) [M+H+TFA]+,
503 (22) [M+H]+, 252 (84) [M+2H]2+; m/z [M+H]+ calculated for
C
22H39N12O+2: 503.3313, found 503.3314; C22H38N12O2 Â 4 TFA
(958.70).
4.2.4.3. 1-(Amino{[3-(1H-imidazol-4-yl)propyl]amino}methylene)-
3-{7-[3-(amino{[3-(1H-imidazol-4-yl)propyl]amino}methylene)
ureido]heptyl}urea (10). The title compound was prepared from
5 (106 mg, 0.188 mmol), amine 1 (145 mg, 0.395 mmol), NEt3
4.2.3.3. N1,N10-Bis{[(tert-butoxycarbonylamino)(methylsulfanyl)
methylene]aminocarbonyl}decane-1,10-diamine (7). The title
compound was prepared from decane-1,10-diamine (300 mg,
(128 lL, 0.92 mmol) and HgCl2 (201 mg, 0.74 mmol) according to
1.74 mmol), DIPEA (1697
l
L, 9.74 mmol), triphosgene (517 mg,
the general procedure, yielding 10 as a white fluffy hygroscopic
solid (15.30 mg, 8%): RP-HPLC: 97%, (tR = 16.65 min, k = 4.65);
UVmax 205 nm. 1H NMR (600 MHz, [D4]MeOH) d (ppm) 1.36
(m, 6H), 1.54 (m, 4H), 2.03 (tt, 4H, 2J 7.0 Hz, 3J 7.5 Hz), 2.84
(t, 4H, J 7.7 Hz), 3.19 (t, 4H, J 7.1 Hz), 3.36 (t, 4H, J 6.9 Hz), 7.36
(s, 2H), 8.80 (d, 2H, J 1.1 Hz); 13C NMR (151 MHz, [D4]MeOH, tri-
fluoroacetate) d (ppm); 22.5, 27.7, 28.2, 29.9, 30.4, 40.7, 41.4,
117.1, 134.3, 134.9, 155.5, 156.1. HRMS (ESI) m/z (%) 517 (13)
1.74 mmol) and N-tert-Butoxycarbonyl-S-methylisothiourea
(1325 mg, 6.97 mmol) according to the general procedure
(Rf = 0.35 in Hex/EA 3:2). The product was obtained as a colorless
glassy solid (798 mg, 76%). 1H NMR (300 MHz, [D4]MeOH) d
(ppm) 1.33 (m, 12H), 1.48–1.54 (m, 22H), 2.32 (s, 6H), 3.14 (t,
4H, J 7.0 Hz); 13C NMR (75 MHz, [D4]MeOH) d (ppm) 14.4, 28.0,
28.2, 30.4, 30.6, 41.0, 83.6, 152.1, 163.7, 167.2. HRMS (ESI) m/z
(%) 605 (100) [M+H]+; m/z [M+H]+ calculated for C26H49N6O6S+2:
605.3150, found 605.3154; C26H48N6O6S2 (604.83).
[M+H]+, 259 (41) [M+2H]2+
;
m/z [M+H]+ calculated for
C
23H41N12O+2: 517.3473, found 517.3473; C23H40N12O2 Â 4 TFA
(972.72).
4.2.4. General procedure for the synthesis of the bivalent
carbamoylguanidine-type ligands (8–17)
4.2.4.4. 1-(Amino{[3-(1H-imidazol-4-yl)propyl]amino}methylene)-
3-{8-[3-(amino{[3-(1H-imidazol-4-yl)propyl]amino}methylene)
ureido]octyl}urea (11). Compound 6 (175 mg, 0.303 mmol) and
amine 1 (234 mg, 0.637 mmol) were dissolved in DCM. NEt3
(210 lL, 1.51 mmol) and HgCl2 (330 mg, 1.21 mmol) were added
to the mixture. The mixture was centrifuged and the precipitate
The guanidinylating reagents (8–17) (1 equiv) and 2.1 eq of 3-
(1-trityl-1H-imidazol-4-yl)propan-1-amine (1) or tert-butyl 5-(3-
aminopropyl)-4-methylthiazol-2-ylcarbamate (2) were dissolved
in DMF (unless indicated otherwise). NEt3 (5 equiv) and HgCl2
(4 equiv) were added to the mixture and stirring was continued
for 12 h. The precipitate was centrifuged, washed with DMF
(unless otherwise indicated) and centrifuged for a second time.
was washed with DCM and centrifuged for a second time. The sol-

N. Kagermeier et al. / Bioorg. Med. Chem. 23 (2015) 3957–3969
3965
vent was removed under reduced pressure. Purification by column
chromatography (eluent: DCM/MeOH 100:1 to 50:1) yielded 11 as
a white fluffy hygroscopic solid (58.62 mg, 20%): RP-HPLC: 96%,
(tR = 18.80 min, k = 5.38); UVmax 205 nm. 1H NMR (600 MHz,
[D4]MeOH) d (ppm) 1.35 (m, 8H), 1.46–1.66 (m, 4H), 2.03 (tt, 4H,
2J 7.0 Hz, 3J 7.5 Hz), 2.86 (t, 4H, J 7.7 Hz), 3.19 (t, 4H, J 7.0 Hz),
3.39 (t, 4H, J 6.4 Hz), 7.39 (s, 2H), 8.83 (d, 2H, J 0.9 Hz); 13C NMR
J 6.9 Hz), 3.33 (t, 4H, J 6.8 Hz, overlap with solvent). 13C NMR
(75 MHz, [D4]MeOH, trifluoroacetate) (ppm) 11.4, 23.6,
27.4, 29.9, 30.3, 40.6, 41.4, 118.4, 132.6, 155.5, 156.0, 170.3.
HRMS (ESI) m/z (%) 595 (23) [M+H]+, 298 (38) [M+2H]2+; m/z
[M+H]+ calculated for C24H43N12O2S2+: 595.3068, found 595.3068;
d
C24H42N12O2S2 Â 4 TFA (1050.88).
(151 MHz, [D4]MeOH, trifluoroacetate)
d
(ppm) 22.5, 27.8,
4.2.4.8. 1-(Amino{[3-(2-amino-4-methyl-thiazol-5-yl)propyl]amino}
methylene)-3-{7-[3-(amino{[3-(2-amino-4-methyl-thiazol-5-yl)
28.2, 30.2, 30.4, 40.8, 41.5, 117.1, 134.3, 134.9, 155.3, 155.9.
HRMS (ESI) m/z (%) 531 (15) [M+H]+, 266 (47) [M+2H]2+; m/z
[M+H]+ calculated for C24H43N12O+2: 531.3626, found 531.3625;
propyl]amino}methylene)ureido]heptyl}urea (15).
compound was prepared from (175 mg, 0.311 mmol),
(177 mg, 0.652 mmol), NEt3 (215
The title
5
2
C24H42N12O2 Â 4 TFA (986.75).
lL, 1.55 mmol) and HgCl2
(338 mg, 1.24 mmol) according to the general procedure, yielding
15 as a white fluffy hygroscopic solid (95.46 mg, 29%): RP-HPLC:
99%, (tR = 19.85 min, k = 5.74); UVmax 204 nm and 264 nm. 1H
NMR (600 MHz, [D4]MeOH) d (ppm) 1.36 (s, 6H), 1.51–1.57 (m,
4H), 1.90 (tt, 4H, 2J 7.2 Hz, 3J 7.4 Hz), 2.18 (s, 6H), 2.72 (t, 4H, J
7.6 Hz), 3.19 (t, 4H, J 7.1 Hz), 3.33 (t, 4H, J 6.9 Hz); 13C NMR
(151 MHz, [D4]MeOH, trifluoroacetate) d (ppm) 11.4, 23.6, 27.7,
29.9, 30.0, 30.4, 40.8, 41.4, 118.4, 132.7, 155.5, 156.0, 170.3.
HRMS (ESI) m/z (%) 609 (23) [M+H]+, 305 (50) [M+2H]2+; m/z
[M+H]+ calculated for C25H45N12O2S2+: 609.3224, found 609.3226;
C25H44N12O2S2 Â 4 TFA (1064.91).
4.2.4.5. 1-(Amino{[3-(1H-imidazol-4-yl)propyl]amino}methylene)-
3-{10-[3-(amino{[3-(1H-imidazol-4-yl)propyl]amino}methylene)
ureido]decyl}urea (12). The title compound was prepared from 7
(150 mg, 0.248 mmol), amine
1 (191 mg, 0.520 mmol), NEt3
(172 L, 1.24 mmol) and HgCl2 (270 mg, 0.99 mmol) according to
l
the general procedure, yielding 12 as a white fluffy hygroscopic
solid (68.83 mg, 27%): RP-HPLC: 91%, (tR = 22.73 min, k = 6.71);
UVmax 205 nm. 1H NMR (600 MHz, [D4]MeOH) d (ppm) 1.32 (m,
12H), 1.49–1.57 (m, 4H), 2.02 (tt, 4H, 2J 7.0 Hz, 3J 7.6 Hz), 2.83 (t,
4H, J 7.7 Hz), 3.18 (t, 4H, J 7.0 Hz), 4H), 3.36 (t, 4H, J 6.9 Hz), 7.36
(s, 2H), 8.80 (d, 2H, J 0.7 Hz); 13C NMR (151 MHz, [D4]MeOH,
trifluoroacetate) d (ppm) 22.5, 27.8, 28.1, 30.3, 30.4, 30.5, 40.8,
41.4, 117.0, 134.3, 134.9, 155.4, 156.0. HRMS (ESI) m/z (%) 559
4.2.4.9. 1-(Amino{[3-(2-amino-4-methyl-thiazol-5-yl)propyl]amino}
methylene)-3-{8-[3-(amino{[3-(2-amino-4-methyl-thiazol-5-yl)
(10) [M+H]+, 280 (27) [M+2H]2+
C
(1014.80).
;
m/z [M+H]+ calculated for
propyl]amino}methylene)ureido]octyl}urea (16).
compound was prepared from (130 mg, 0.225 mmol),
(128 mg, 0.472 mmol), NEt3 (156
The title
26H47N12O+2: 559.3939, found 559.3935; C26H46N12O2 Â 4 TFA
6
l
2
L, 1.12 mmol) and HgCl2
(244 mg, 0.90 mmol) according to the general procedure, yielding
6 as a white fluffy hygroscopic solid (21.77 mg, 9%): RP-HPLC:
98%, (tR = 21.69 min, k = 6.36); UVmax 204 nm and 264 nm. 1H
NMR (600 MHz, [D4]MeOH) d (ppm) 1.34 (m, 8H), 1.50–1.56 (m,
4H), 1.90 (tt, 4H, 2J 7.0 Hz, 3J 7.4 Hz), 2.18 (s, 6H), 2.72 (t, 4H, J
7.6 Hz), 3.19 (t, 4H, J 7.1 Hz), 3.33 (t, 4H, J 6.9 Hz). 13C NMR
(151 MHz, [D4]MeOH, trifluoroacetate) d (ppm) 11.4, 23.6, 27.8,
29.9, 30.3, 30.4, 40.8, 41.4, 118.4, 132.6, 155.5, 156.0, 170.3.
HRMS (ESI) m/z (%) 623 (11) [M+H]+, 312 (21) [M+2H]2+; m/z
[M+H]+ calculated for C26H47N12O2S2+: 623.3381, found 623.3375;
4.2.4.6. 1-(Amino{[3-(2-amino-4-methyl-thiazol-5-yl)propyl]amino}
methylene)-3-{4-[3-(amino{[3-(2-amino-4-methyl-thiazol-
5-yl)propyl]amino}methylene)ureido]butyl}urea (13). Com-
pound 3 (200 mg, 0.384 mmol) and amine 2 (216 mg, 0.796 mmol)
were dissolved in DCM. NEt3 (265 lL, 1.80 mmol) and HgCl2
(412 mg, 1.52 mmol) were added to the mixture. The mixture
was centrifuged and the precipitate was washed with DCM and
centrifuged for a second time. The solvent was removed under
reduced pressure and the product was purified by column chroma-
tography (eluent: DCM/MeOH 100:1 to 50:1). Yielding 13 as a
white fluffy hygroscopic solid (53.34 mg, 14%): RP-HPLC: 99%,
(tR = 15.61 min, k = 4.29); UVmax 205 nm and 265 nm. 1H NMR
(600 MHz, [D4]MeOH) d (ppm) 1.57 (m, 4H), 1.90 (tt, 4H, 2J
7.0 Hz, 3J 7.3 Hz), 2.18 (s, 6H), 2.72 (t, 4H, J 7.6 Hz), 3.23 (m, 4H),
3.33 (t, 4H, J 6.9 Hz); 13C NMR (151 MHz, [D4]MeOH, trifluoroace-
tate) d (ppm) 11.4, 23.6, 27.7, 29.9, 40.34, 41.4, 118.4, 132.6,
155.6, 156.0, 170.3. HRMS (ESI) m/z (%) 567 (34) [M+H]+, 284
C26H46N12O2S2 Â 4 TFA (1078.94).
4.2.4.10. 1-(Amino{[3-(2-amino-4-methyl-thiazol-5-yl)propyl]amino}
methylene)-3-{10-[3-(amino{[3-(2-amino-4-methyl-thiazol-5-yl)
propyl]amino}methylene)ureido]decyl}urea (17).
compound was prepared from (150 mg, 0.248 mmol),
(141 mg, 0.519 mmol), NEt3 (172
The title
7
l
2
L, 1.24 mmol) and HgCl2
(269 mg, 0.99 mmol) according to the general procedure, yielding
7 as a white fluffy hygroscopic solid (24.25 mg, 9%): RP-HPLC:
99%, (tR = 25.49 min, k = 7.65); UVmax 205 nm and 263 nm. 1H
NMR (600 MHz, [D4]MeOH) d (ppm) 1.32 (m, 12H), 1.50–1.56 (m,
4H), 1.90 (tt, 4H, 2J 7.0 Hz, 3J 7.3 Hz), 2.18 (s, 6H), 2.72 (t, 4H, J
7.1 Hz), 3.19 (t, 4H, J 7.1 Hz), 3.33 (t, 4H, J 6.9 Hz). 13C NMR
(151 MHz, [D4]MeOH, trifluoroacetate) d (ppm) 11.5, 23.6, 27.8,
29.9, 30.3, 30.4, 30.6, 40.8, 41.4, 118.4, 132.7, 155.5, 156.0, 170.3.
HRMS (ESI) m/z (%) 651 (4) [M+H]+, 326 (17) [M+2H]2+, 218 (27)
(58) [M+2H]2+
;
m/z [M+H]+ calculated for C22H39N12O2S+2:
567.2755, found 567.2760; C22H38N12O2S2 Â 4 TFA (1022.83).
4.2.4.7. 1-(Amino{[3-(2-amino-4-methyl-thiazol-5-yl)propyl]amino}
methylene)-3-{6-[3-(amino{[3-(2-amino-4-methyl-thiazol-5-yl)
propyl]amino}methylene)ureido]hexyl}urea (14). Compound 4
(175 mg, 0.319 mmol) and amine 2 (177 mg, 0.652 mmol) were
[M+3H]3+
C
TFA (1106.99).
, ;
163 (42) [M+4H]4+ m/z [M+H]+ calculated for
dissolved in DCM. NEt3 (215 lL, 1.55 mmol) and HgCl2 (378 mg,
28H51N12O2S2+: 651.3694, found 651.3684; C28H50N12O2S2 Â 4
1.24 mmol) were added to the mixture. The mixture was centri-
fuged and the precipitate was washed with DCM and centrifuged
for a second time The solvent was removed under reduced pres-
sure and the product was purified by column chromatography
(eluent: DCM/MeOH 100:1 to 50:1). Yielding 14 as a white fluffy
hygroscopic solid (99.34 mg, 30%): RP-HPLC: 99%, (tR = 18.12 min,
k = 5.15); UVmax 203 nm and 265 nm. 1H NMR (600 MHz,
[D4]MeOH) d (ppm) 1.37 (m, 4H), 1.48–1.62 (m, 4H), 1.89 (tt, 4H,
2J 7.0 Hz, 3J 7.4 Hz), 2.18 (s, 6H), 2.72 (t, 4H, J 7.5 Hz), 3.20 (t, 4H,
4.2.5. 1,7-Dichloroheptan-4-one (18)9
HCl gas (10 fold excess) was passed through dicyclopropyl
ketone (15.00 g, 136.16 mmol) for 30 min. After stirring for 3 h,
HCl gas was passed through the mixture for another 30 min. The
crude product was obtained as a brown oil (24.93 g, 100%) and
was used in the next step without any further purification. 1H

3966
N. Kagermeier et al. / Bioorg. Med. Chem. 23 (2015) 3957–3969
NMR (300 MHz, CDCl3): d (ppm) 2.02 (m, 4H), 2.61 (t, 4H, J 7.0 Hz),
3.55 (t, 4H, J 6.3 Hz). 13C NMR (75 MHz, [D4]MeOH) d (ppm) 27.6,
40.3, 45.2, 210.7. HRMS (APCI; MeOH) m/z (%) 183 (100); m/z
[M+H]+ calculated for C7H13Cl2O+: 183.0337, found 183.0337. C7-
H12Cl2O (183.08).
removed by filtration. After removing the solvent in vacuo, the
product was purified by column chromatography (eluent: EA/
MeOH/NH3 9:0.8:0.2 to EA/MeOH/NH3 8:1.5:0.5), (Rf = 0.14 for
DCM/7 N NH3 in MeOH 4:1). Compound 23 was obtained as yellow
oil (580 mg, 88%). 1H NMR (300 MHz, CDCl3) d (ppm) 1.45–1.54 (m,
2H), 1.58–1.85 (m, 6H), 1.99–2.13 (m, 6H), 2.70 (t, 2H, J 7.1 Hz),
3.26 (td, 4H, 2J 6.8, 3J 2.4) 5.20 (t, 1H, J 7.1). 13C NMR (75 MHz,
CDCl3) d (ppm) 25.2, 27.3, 27.5, 33.3, 33.6, 39.5, 41.6, 51.0, 51.2,
126.6, 136.7. HRMS (ESI) m/z (%) 252 (100) [M+H]+; m/z [M+H+]+
calculated for C11H22N+7: 252.1931, found 252.1925; C11H21N7
(251.34).
4.2.6. 1,7-Diazidoheptan-4-one (19)
To
a solution of 1,7-dichloroheptan-4-one (18) (3.40 g,
18.57 mmol) in 50 ml DMF, sodium azide (3.62 g, 55.68 mmol)
was added and stirred overnight at 60 °C. After cooling and addi-
tion of water (500 mL), the mixture was extracted with EA
(3 Â 100 mL). The combined organic phases were dried over
sodium sulfate. Filtration and evaporation of the solvent yielded
a yellow oil (2.87 g, 79%). The crude product 19 was used in the
next step without any further purification. 1H NMR (300 MHz,
CDCl3) d (ppm) 1.86 (m, 4H), 2.52 (t, 4H, J 7.1 Hz), 3.31 (t, 4H, J
6.6 Hz). 13C NMR (75 MHz, CDCl3) d (ppm) 22.6, 39.0, 50.4, 208.2.
HRMS (ESI; MeOH) m/z (%) 219 (46) [M+Na+], 197 (2) [M+H]+,
169 (100) [M+HÀN2]+; m/z [M+H]+ calculated for C7H13N+6:
197.1145, found 197.1138. C7H12N6(196.21).
4.2.10. tert-Butyl [8-azido-5-(3-azidopropyl)oct-4-en-1-yl]
carbamate (24)
A solution of di-tert-butyldicarbonat (756 mg, 3.46 mmol) in
DCM was added dropwise to an external cooled solution of number
8-azido-5-(3-azidopropyl)oct-4-en-1amine
(23)
(870 mg,
3.46 mmol) and NEt3 (480 L, 3.46 mmol) in DCM over a period
l
of 1 h. The mixture was stirred at room temperature for 24 h.
The solvent was removed in vacuo. The product was purified by
column chromatography (eluent: Hex/EA 10:90 to Hex/EA 50:50),
(Rf = 0.23 for Hex/EA 3:2) and obtained as pale yellowish oil
(1.18 g, 97%). 1H NMR (300 MHz, CDCl3) d (ppm) 1.44 (s, 9H),
1.47–1.59 (m, 2H), 1.59–1.73 (m, 4H), 2.00–2.12 (m, 6H), 3.11 (t,
2H, J 6.9 Hz), 3.23–3.30 (m, 4H), 4.54 (s, 1H), 5.19 (t, 1H, J
7.1 Hz). 13C NMR (75 MHz, CDCl3) d (ppm) 25.1, 26.8, 27.3, 27.4,
28.5, 30.3, 33.5, 40.3, 51.0, 51.1, 79.1, 126.3, 137.0, 156.0. HRMS
(ESI) m/z (%) 374 (26) [M+Na+], 324 (25) [M+HÀN2]+, 268 (100)
(M+H-C4H8]+; m/z [M+Na+] calculated for C16H29N7O2Na+:
374.2275, found 374.2282; C16H29N7O2 (351.45).
4.2.7. 1-Azido-4-(3-azidopropyl)-8-chlorooct-4-ene (21)
(4-Chlorobutyl)triphenylphosphonium bromide (20) (2.50 g,
5.76 mmol) was suspended in anhydrous THF (20 mL) under an
atmosphere of argon and the mixture was cooled to À72 °C. n-
Butyllithium (1.6 M in n-hexane, 3.60 mL, 5.76 mmol) was added
to the suspension and the mixture was allowed to warm up to
À10 °C over a period of 45 min. The temperature was kept at
À10 °C for 1.5 h and then cooled again to À72 °C. 1,7-diazidohep-
tan-4-one (1.13 g, 5.76 mmol) was added and the mixture was
allowed to warm up to room temperature over a period of
60 min, stirring was continued overnight. After filtration of solid
material and evaporation of the solvent, the product was purified
by column chromatography using mixtures of Hex and EA as eluent
(Rf = 0.45 for Hex/EA 4:1), affording 21 as yellow oil (300 mg, 19%).
1H NMR (300 MHz, [D4]MeOH) d (ppm) 1.61–1.74 (m, 4H), 1.76–
1.86 (m, 2H), 2.07–2.26 (bm, 6H), 3.26–3.31 (overlap with solvent,
m, 4H), 3.56 (t, 2H, J 6.5 Hz), 5.23 (t, 1H, J 7.3 Hz). 13C NMR
(75 MHz, CDCl3) d (ppm) 25.0, 27.0, 27.4, 27.6, 32.7, 33.6, 44.6,
51.0, 51.2, 125.3, 138.0. HRMS (ESI) m/z (%) 243 (100)
[M+HÀN2]+; m/z [M+HÀN2]+ calculated for C11H20ClN+4: 243.1376,
found 243.1374; C11H19ClN6 (270.76)
4.2.11. tert-Butyl [8-amino-5-(3-aminopropyl)oct-4-en-1-yl]
carbamate (25)
A solution of 24 (200 mg, 0.57 mmol) in anhydrous diethyl
ether was dropped to a stirred suspension of LiAlH4 (100 mg,
2.63 mmol) in anhydrous diethyl ether over 1 h. The mixture was
heated to reflux for an additional hour. After cooling and quench-
ing with a solution of 2 g Seignette salt in 12 g water (pH = 9–
10), the organic phase was washed twice with 0.1 M KOH and
the solvent was evaporated in vacuo. The crude product was used
in the next step without any further purification (113 mg, 66%). 1H
NMR (300 MHz, [D4]MeOH) d (ppm) 1.42 (s, 9H), 1.47–1.57 (m,
6H), 1.63 (s, 4H), 1.98–2.06 (m, 6H), 2.66 (td, 4H, 2J 7.1, 3J 2.5),
3.05–3.12 (m, 2H), 4.67 (s, 1H), 5.11 (t, 1H J 7.0 Hz). 13C NMR
(75 MHz, CDCl3) d (ppm) 25.1, 27.4, 28.5, 30.4, 32.2, 32.4, 34.2,
40.4, 42.0, 42.3, 79.1, 124.4, 139.3, 156.1. HRMS (ESI) m/z (%) 300
(40) [M+H]+, 244 (51) [M+H-C4H8]+, 200 (100) [M+H-Boc]+; m/z
[M+H+]+ calculated for C16H34N3O+2: 300.2646, found 300.2649;
4.2.8. 2-[8-Azido-5-(3-azidopropyl)oct-4-en-1-yl]isoindoline-
1,3-dione (22)
To a solution of 21 (1.89 g, 6.98 mmol) and phthalimide (1.54 g,
10.47 mmol) in 40 mL DMF, Cs2CO3 (5.00 g, 15.34 mmol) and cata-
lytic traces of KI were added. The mixture was stirred at 60 °C over-
night. After cooling 400 mL 5% NaOH were added and the product
was extracted with EA. The combined organic phases were dried
over Na2SO4. After filtration and evaporation, the crude product
was purified by column chromatography using mixtures of Hex
and EA as eluent (Rf = 0.45 for Hex/EA 2:1) affording 22 as yellow
oil (1.51 g, 57%). 1H NMR (300 MHz, CDCl3) d (ppm) 1.56–1.58
(m, 4H), 1.69–1.78 (m, 2H), 1.96–2.15 (m, 6H), 3.24 (td, 4H, 2J
6.8, 3J 2.9), 3.65–3.73 (m, 2H), 5.21 (t, 1H, J 7.1 Hz), 7.75–7.67 (m,
2H), 7.80–7.87 (m, 2H). 13C NMR (75 MHz, CDCl3) d (ppm) 25.1,
26.9, 27.2, 27.3, 28.7, 33.4, 37.7, 50.9, 51.1, 123.2, 125.7, 132.1,
133.9, 137.3, 168.4. HRMS (ESI) m/z (%) 404 (31) [M+Na+], 354
(100) [M+HÀN2]+; m/z [M+HÀN2]+ calculated for C19H24N5O+2:
354.1925, found 354.1925; C19H23N7O2 (381.44).
C16H33N3O2 (299.45).
4.2.12. tert-Butyl ({3-[4-({3-[(tert-butoxycarbonylimino)
(methylsulfanyl)methyl]ureido}propyl)-8-(tert-
butoxycarbonylamino)oct-4-en-1-yl]ureido}
(methylsulfanyl)methylene)carbamate (27)
The title compound was prepared from 25 (122 mg, 0.41 mmol),
DIEA (393 lL, 2.3 mmol) and triphosgene (122 mg, 0.41 mmol),
giving tert-butyl [8-isocyanato-5-(3-isocyanatopropyl)oct-4-en-1-
yl]carbamate (26) as intermediate. Compound 26 was not isolated
but allowed to react with N-Boc-S-methylisothiourea (310 mg,
1.63 mmol) according to the general procedure for the synthesis
of the guanidinylating reagents (5-7). 27 was obtained as a yellow-
ish oil (Rf = 0.37 in DCM) (131 mg, 44%). 1H NMR (300 MHz,
[D4]MeOH) d (ppm) 1.43 (s, 9H), 1.48–1.54 (m, 20H), 1.59–1.66
(m, 4H), 2.03–2.07 (m, 4H), 2.08–2.12 (m, 2H), 2.33 (d, 6H, J
2.1 Hz), 3.03 (t, 2H, J 7.0 Hz), 3.14 (m, 4H), 5.22 (t, 1H, J 7.1 Hz).
4.2.9. 8-Azido-5-(3-azidopropyl)oct-4-en-1amine (23)
A mixture of 22 (1.00 g, 2.62 mmol) and hydrazine (0.50 g,
15.60 mmol) in ethanol was refluxed for 1 h. Solid material was

N. Kagermeier et al. / Bioorg. Med. Chem. 23 (2015) 3957–3969
3967
13C NMR (75 MHz, CDCl3) d (ppm) 14.4, 26.1, 28.2, 28.8, 29.1, 29.3,
31.2, 35.1, 40.8, 41.1, 83.6, 126.3, 139.8, 152.2, 163.7, 163.72, 167.2,
167.3. HRMS (ESI) m/z (%) 754 (14) [M+Na+], 732 (100) [M+H]+,
4.2.14.3.
1-{8-[3-(amino{[3-(2-amino-4-methylthiazol-5-yl)
propyl]amino}methylene)ureido]-5-{3-[3-(amino{[3-(2-amino-
4-methylthiazol-5-yl)propyl]amino}methylene)ureido]propyl}
oct-4-en-1-yl}-4-{(1E,3E)-4-[4-(dimethylamino)phenyl]buta-
266 (69) [M+2H-2Boc]2+
;
m/z [M+H+]+ calculated for
C
32H58N7O8S+2: 732.3794, found 732.3794; C32H57N7O8S2 (731.97).
1,3-dien-1-yl}-2,6-dimethylpyridin-1-ium
(31). The title compound was prepared from 28 (2.00 mg,
1.60 mol), Py-5 (294 g, 0.80 mol) and NEt3 (1.66 L, 12 mol)
according to the general procedure, yielding 31 as a red solid
(462 g, 38%): RP-HPLC: 96%, (tR = 19.68 min, k = 5.68) (eluent:
trifluoroacetate
l
l
l
l
l
4.2.13. 1-(8-Amino-4-{[3-(amino{[3-(2-amino-4-methyl-
thiazol-5-yl)propyl]amino}methylene)ureido]propyl}oct-4-
enyl)-3-[(amino{[3-(2-amino-4-methyl-thiazol-5-yl)
propyl]amino}methylene)]urea (28)
l
MeCN/TFA (0.05% aq) 0 min: 10:90, 30 min: 60:40, 31–41 min:
80:20; flow rate = 0.75 mL/min). MS (ESI) m/z (%) 939.6 (1) [M]+,
The title compound was prepared from 27 (111 mg, 0.15 mmol),
470 (90) [M+H]2+, 327 (100) [M+2H+MeCN]3+, 313 (11) [M+2H]3+
.
2
(86 mg, 0.317 mmol), NEt3 (105 lL, 0.76 mmol) and HgCl2
C50H71F3N14O4S2 Â 4 TFA (1510.41).
(165 mg, 0.60 mmol) according to the general procedure for the
synthesis of the bivalent carbamoylguanine-type ligands (8–17),
yielding 28 as a white fluffy hygroscopic solid (34.80 mg, 18%).
RP-HPLC: 96%, (tR = 17.19 min, k = 4.83); UVmax 204 nm and
263 nm. 1H NMR (600 MHz, [D4]MeOH) d (ppm) 1.59–1.67 (m,
4H), 1.70 (m, 2H), 1.90 (tt, 4H, 2J 7.0 Hz, 3J 7.3 Hz), 2.05–2.17
(bm, 6H), 2.18 (s, 6H), 2.72 (t, 4H, J 7.6 Hz), 2.90–2.94 (m, 2H),
3.19 (m, 4H), 3.34 (t, 4H, J 6.8 Hz, overlap with solvent), 5.23 (t,
1H, J 7.1 Hz). 13C NMR (151 MHz, [D4]MeOH, trifluoroacetate) d
(ppm) 11.4, 23.6, 25.6, 28.1, 28.8, 28.9, 29.1, 29.9, 34.7, 40.41,
40.44, 40.8, 41.4, 118.4, 125.3, 132.6, 140.6, 155.6, 156.0, 170.3.
HRMS (ESI) m/z (%) 678 (10) [M+H]+, 340 (23) [M+2H]2+; m/z
[M+H]+ calculated for C29H52N13O2S2+: 678.3803, found 678.3806.
4.2.15. N-{8-[3-(Amino{[3-(2-amino-4-methylthiazol-5-yl)
propyl]amino}methylene)ureido]-5-{3-[3-(amino{[3-(2-amino-
4-methylthiazol-5-yl)propyl]amino}methylene)ureido]
propyl}oct-4-en-1-yl}propionamide (32a)
The reaction was carried out in a 1.5-mL Eppendorf reaction
vessel. Compound 28 (13.62
30 L DMF and NEt3 (102.20
idyl propionate (16.36 mol, 2.80 mg) was dissolved in 20
and this solution was added to the mixture. The cup was rinsed a
second time with 10 L DMF and the reaction mixture was stirred
for 2 h at room temperature. The reaction was stopped by adding
20 L of 10% TFA. The crude product was purified by preparative
l
mol, 17.00 mg) was dissolved in
mol, 14.14 L) was added. Succinim-
L DMF
l
l
l
l
l
l
l
C29H51N13O2S2 Â 5 TFA (1248.03).
HPLC, yielding 32a as a white fluffy hygroscopic solid (13.18 mg,
81%): RP-HPLC: 94%, (tR = 21.77 min, k = 7.54) (YMC C8 column);
UVmax 204 nm and 264 nm. 1H NMR (600 MHz, [D4]MeOH) d
(ppm) 1.12 (t, 3H, J 7.6 Hz), 1.54 (tt, 2H, 2J 7.0 Hz, 3J 7.4 Hz), 1.62
(m, 4H), 1.90 (tt, 4H, 2J 7.3 Hz, 3J 7.4 Hz), 2.03–2.11 (bm, 6H),
2.16–2.21 (m, 8H), 2.72 (t, 4H, J 7.6 Hz), 3.14–3.20 (m, 6H), 3.33
(t, 4H, J 6.9 Hz), 5.23 (t, 1H, J 7.1 Hz). 13C NMR (151 MHz, [D4-
]MeOH, trifluoroacetate) d (ppm) 10.6, 11.4, 23.6, 26.1, 27.9, 28.8,
29.1, 29.2, 30.2, 30.7, 34.7, 40.1, 40.4, 40.8, 41.4, 118.4, 126.6,
132.6, 139.3, 155.5, 156.0, 170.3, 177.0. HRMS (ESI) m/z (%) 734.4
(10) [M+H]+, 367.7 (23) [M+2H]2+, 245.5 (77) [M+3H]3+; m/z
[M+H]+ calculated for C32H56N13O3S2+: 734.4065, found 734.4068.
4.2.14. General procedure for the synthesis of the bivalent
fluorescence ligands (29–31)
The reactions were carried out in a 1.5-mL Eppendorf reaction
vessel. The amine precursor 28 (2 equiv) was dissolved in 30
DMF and NEt3 (15 equiv) was added. The fluorescence dyes
(1 equiv) were dissolved in 20 L DMF and this solution was added
to the mixture, the cup was rinsed two times with DMF (20 L and
10 L). The mixture was stirred for 2 h at room temperature and
then the reaction was stopped by adding 20 L of 10% TFA. The
lL
l
l
l
l
crude products were purified by preparative HPLC.
C
32H55N13O3S2 Â 4 TFA (1190.08).
4.2.14.1. 2-{(1E,3E)-5-[(Z)-1-(5-Amino-12-{3-[3-(amino{[3-(2-
amino-4-methylthiazol-5-yl)propyl]amino}methylene)ureido]
propyl}-1-(2-amino-4-methylthiazol-5-yl)-7,18-dioxo-4,6,8,17-
tetraazatricosa-5,12-dien-23-yl)-3,3-dimethylindolin-2-ylidene]
penta-1,3-dien-1-yl}-1,3,3-trimethyl-3H-indol-1-ium trifluoro-
4.2.16. N-{8-[3-(Amino{[3-(2-amino-4-methylthiazol-5-yl)
propyl]amino}methylene)ureido]-5-{3-[3-(amino{[3-(2-amino-
4-methylthiazol-5-yl)propyl]amino}methylene)ureido]
propyl}oct-4-en-1-yl}-2,3-ditritiopropionamide (32b)
Succinimidyl [2,3-3H2]propionate was from Hartmann Analytic
(Braunschweig, Germany), provided in EA (as = 2.96 TBq/mmol,
80 Ci/mmol; av = 18.5 GBq/mL, 5 mCi/mL). The reaction was car-
ried out in a 1.5 mL Eppendorf reaction vessel. The precursor 28
acetate (29).
The title compound was prepared from 28
mol), S2197 (422 g, 0.64 mol) and NEt3
mol) according to the general procedure, yielding
29 as a dark blue solid (490 g, 45%): RP-HPLC: 99%, (tR = 24.83 min,
(1.60 mg, 1.28
l
l
l
(1.33 lL, 9.60 l
l
k = 7.42) (eluent: MeCN/TFA (0.05% aq) 0 min: 10:90, 30 min:
60:40, 31–41 min: 80:20; flow rate = 0.75 mL/min). MS (ESI) m/z
(0.375
and DIPEA (5.625
lmol, 468 lg) was dissolved in a mixture of 50 lL DMF
l
mol, 0.96 lL). This mixture was added to the
(%) 1143.8 (1) [M]+, 571.9 (34) [M+H]2+, 381.6 (100) [M+2H]3+
,
solved succinimidyl [2,3-3H2]propionate (41.46 nmol, 3.32 mCi).
The EA was removed in a vacuum concentrator for about 45 min.
296.6 (24) [M+4H+MeOH]4+. C63H88F3N15O5S2 Â 4 TFA (1713.70).
Afterwards 9
was stirred for 3 h with a magnetic stirrer at room temperature.
80 L of 2% aq TFA and 280 L Millipore water were added. The
product was isolated with a Waters HPLC system (column: YMC–
m). Eluent: mixtures of MeCN and
lL EA and 1 lL DIPEA were added, and the mixture
4.2.14.2.
1-{8-[3-(Amino{[3-(2-amino-4-methylthiazol-5-yl)
propyl]amino}methylene)ureido]-5-{3-[3-(amino{[3-(2-amino-
4-methylthiazol-5-yl)propyl]amino}methylene)ureido]propyl}
oct-4-en-1-yl}-2,6-dimethyl-4-[(E)-2-(2,3,6,7-tetrahydro-1H,5H-
pyrido[3,2,1-ij]quinolin-9-yl)vinyl]pyridin-1-ium trifluoroace-
l
l
Triart C18 (250 Â 6.0 mm, 5
l
0.04% TFA (A) and 0.05% aq TFA (B), gradient 0–20 min A/B 5:95
to 30:70. The retention time of the radioligand was 18.4 min and
the eluates were collected in a 1.5 mL reaction vessel. The com-
bined fractions were concentrated in a vacuum concentrator to a
tate (30).
(1.50 mg, 1.20
The title compound was prepared from 28
mol), Py-1 (236 g, 0.60 mol) and NEt3 (1.25 L,
l
l
l
l
9.00
lmol) according to the general procedure, yielding 30 as a
red solid (332
lg, 36%): RP-HPLC: 93%, (tR = 23.11 min, k = 6.84)
volume of around 600 lL.
(eluent: MeCN/TFA (0.05% aq) 0 min: 10:90, 30 min: 60:40,
Quantification: The concentration of the radioligand was deter-
mined by a 4 point calibration with 32a. The solutions for the cal-
ibration were prepared freshly prior to the injection. All of the
solutions were prepared separately in mobile phase from a
31–41 min: 80:20; flow rate = 0.75 mL/min). MS (ESI) m/z (%)
965.7 (1) [M]+, 483.2 (82) [M+H]2+, 336.1 (100) [M+2H+MeCN]3+
,
322.4 (15) [M+2H]3+. C52H73F3N14O4S2 Â 4 TFA (1536.45).

3968
N. Kagermeier et al. / Bioorg. Med. Chem. 23 (2015) 3957–3969
20
lM solution of 32a. Eluent: mixtures of MeCN and 0.04% TFA (A)
measured with a MicroBeta2 1450 scintillation counter (Perkin
and 0.05% aq TFA (B), gradient: 0–20 min A/B 5:95 to 26:74 (col-
Elmer, Rodgau, Germany).
umn: Phenomenex Luna C18 (150 Â 4.6 mm, 3
lm). The molarity
of the solution of 32b was calculated from the peak areas of the
4.3.3. Studies on human monocytes
standards.
The present study was approved by the Ethics Committee of the
Hannover Medical School. Monocytes were isolated from periphe-
ral blood of healthy human volunteers using density gradient cen-
trifugation and magnetic activated cell sorting. To study the H2R
mediated responses in human monocytes, increases in cAMP levels
were analyzed via reversed phase HPLC coupled to mass spectrom-
etry (HPLC–MS/MS). For this purpose, 5 Â 105 cells were treated
Determination of the specific activity: 10 aliquots of the diluted
radioligand in mobile phase were counted in 3 mL of Rotiszint eco
plus (Roth, Karlsruhe, Germany) in a LS 6500 liquid scintillation
counter (Beckmann-Coulter, München). The calculated specific
activity was 2.60 tBq/mmol (70.15 Ci/mmol). The concentration
was adjusted to 1 mCi/ml by adding 1291
pore water and EtOH (60:40). This yielded in a concentration of
14.25 M.
The radioligand was obtained in a yield of 90% (37.34 nmol,
44.41
lL of a mixture of Milli-
with the ligands (1 nM–100 lM) in presence of 100 lM IBMX at
l
37 °C for 10 min. Inhibition of fMLP-induced production of ROS
was investigated using the lucigenin chemiluminescence assay:
1 Â 105 cells loaded with the chemiluminescence dye lucigenin
l
g) with a radiochemical purity of 99%. C32H55N13O3S2 Â 4
TFA (1190.08).
were pre-incubated with the ligands (1 nM–100
l
M) at 37 °C for
5 min. After adding fMLP (1
lM), the chemiluminescence was
4.3. Pharmacological protocols
recorded at 37 °C for 60 min. These methods have been described
in detail in Werner et al.28
4.3.1. [35S]GTP
35S]GTP
S was from PerkinElmer Life Science (Boston, USA) or
Hartmann Analytic (Braunschweig, Germany). Sf9 membranes
expressing the hH2R-Gs s or the gpH2R-Gs s were thawed and sed-
cS binding assay
[
c
Acknowledgements
a
a
The authors thank Maria Beer-Krön and Dita Fritsch for excel-
lent technical assistance. This work was supported by the Graduate
Training Program (Graduiertenkolleg) GRK1910 of the Deutsche
Forschungsgemeinschaft.
imented by centrifugation at 13,000 g (4 °C) for 10 min. The mem-
branes were resuspended in cold (4 °C) binding buffer BB (12.5 mM
MgCl2, 1 mM EDTA and 75 mM Tris/HCl, pH 7.4) so that the final
concentration was 1
performed in 96-well plates (PP microplates 96 well, Greiner Bio-
One, Frickenhausen, Germany) in a total volume of 100 L, con-
taining 10 of membrane, GDP, 0.05% BSA, 20 nCi
35S]GTP
S and the investigated ligands at various concentration
lg of protein per 1 lL BB. Experiments were
Supplementary data
l
Supplementary data (Preparation of 20, 1H NMR and 13C NMR
spectra of compounds 8–17, 27 and 32a, RP-HPLC chromatograms
of compounds 8–17, 28–31, 32a,b, binding experiments with 32b
l
g
1 lM
[
c
(dissolved in water). During the incubation period of 90 min, at
room temperature, the plates were shaken at 250 rpm. Afterwards,
on membranes of Sf9 cells expressing the hH2R-Gsas fusion protein,
bound [35S]GTP S was separated from free [35S]GTP
c cS by filtration
confocal microscope images of HEK293T cells upon incubation
with 31, cytotoxicity in the crystal violet based chemosensitivity
assay, ethidium bromide/acridine orange staining of monocytes
through GF/C filters using a 96-well Brandel harvester (Brandel
Inc., Unterföhring, Germany). Filter bound radioactivity was either
quantified by liquid scintillation counting or the filters were dried
overnight and MeltiLex solid scintillator was melted onto the filter-
mats. Scintillation counting was performed with the MicroBe-
ta21450 PlateCounter (Perkin Elmer, Rodgau, Germany).
after incubation with compounds
9
and 14, concentration–
S binding assay.) asso-
response curve of histamine in the [35S]GTP
c
ciated with this article can be found, in the online version, at
http://dx.doi.org/10.1016/j.bmc.2015.01.012.
4.3.2. Competition binding experiments
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