Structure-Activity Relationship of Hetarylpropylguanidines Aiming at the Development of Selective Histamine Receptor Ligands?

Article abstract of DOI:10.1002/open.201900011

New classes of alkylated hetarylpropylguanidines with different functionality and variation in spacer length were synthesized to determine their behavior at the four histamine receptor (H₁R, H₂R, H₃R, H₄R) subtypes. Alkylated guanidines with different terminal functional groups and varied basicity, like amine, guanidine and urea were developed, based on the lead structure SK&F 91486 (2). Furthermore, heteroatomic exchange at the guanidine structure of 2 led to simple analogues of the lead compound. Radioassays at all histamine receptor subtypes were accomplished, as well as organ bath studies at the guinea pig (gp) ileum (gpH₁R) and right atrium (gpH₂R). Ligands with terminal functionalization led to, partially, highly affine and potent structures (two digit nanomolar), which showed up a bad selectivity profile within the histamine receptor family. While the benzoylurea derivative 144 demonstrated a preference towards the human (h) H₃R, S-methylisothiourea analogue 143 obtained high affinity at the hH₄R (pK_i=8.14) with moderate selectivity. The molecular basis of the latter finding was supported by computational studies.

Full text of DOI:10.1002/open.201900011

DOI: 10.1002/open.201900011
Full Papers
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Structure-Activity Relationship of Hetarylpropylguanidines
Aiming at the Development of Selective Histamine
Receptor Ligands†
Steffen Pockes,* David Wifling, Armin Buschauer, and Sigurd Elz[a]
This Paper is dedicated to the memory of Prof. Dr. Armin Buschauer (died on July 18, 2017).
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New classes of alkylated hetarylpropylguanidines with different pig (gp) ileum (gpH1R) and right atrium (gpH2R). Ligands with
functionality and variation in spacer length were synthesized to terminal functionalization led to, partially, highly affine and
determine their behavior at the four histamine receptor (H1R, potent structures (two digit nanomolar), which showed up a
H2R, H3R, H4R) subtypes. Alkylated guanidines with different bad selectivity profile within the histamine receptor family.
terminal functional groups and varied basicity, like amine, While the benzoylurea derivative 144 demonstrated a prefer-
guanidine and urea were developed, based on the lead ence towards the human (h) H3R, S-methylisothiourea analogue
structure SK&F 91486 (2). Furthermore, heteroatomic exchange 143 obtained high affinity at the hH4R (pKi =8.14) with
at the guanidine structure of 2 led to simple analogues of the moderate selectivity. The molecular basis of the latter finding
lead compound. Radioassays at all histamine receptor subtypes was supported by computational studies.
were accomplished, as well as organ bath studies at the guinea
Introduction
fully used for the treatment of allergic diseases as sedatives and
antiemetics.[9] The H2R is mainly expressed in gastric parietal
The biogenic amine histamine (1, Figure 1) is known to be the cells, in the heart, as well as in the brain and couples to a Gαs-
endogenous key modulator for histamine receptors in the protein, which activates the adenylyl cyclase (AC).[10,11] Prior to
human body.[1] There it regulates a variety of effects via the four proton-pump inhibitors, such as omeprazole and
histamine receptor (HR) subtypes H1, H2, H3 and H4, each pantoprazole,[12] overstocking the market in the 1990s, H2R
belonging to the superfamily of G-protein coupled receptors antagonists like cimetidine have been one of the first block-
(GPCRs).[2–6] The H1R is expressed in several tissues (e.g., brain, buster drugs for the treatment of gastroesophageal reflux
blood vessels, gastrointestinal tract) and couples to a Gq/11
-
disease (GERD) and peptic ulcer.[13] The H3R and H4R are both
protein.[7,8] For decades, H1-antihistamines have been success- coupled to Gαi/o-proteins, but differentiate in their localization
in the human body.[14–16] While H3Rs are widely expressed in the
central nervous system,[17] the H4R is mainly found in immune
and mast cells.[5,15,18–20] Due to its function as an auto- and
heteroreceptor in the brain, the H3R is a promising potential
target for various cognitive disorders, like Alzheimer’s disease,
Parkinson or Tourette syndrome.[21–25] Even if the biological
functions of the H4R are not completely apparent, intensive
research proved the involvement in allergic and inflammatory
processes.[26] For this reason, targeting the H4R is expected to be
crucial for the treatment of allergic rhinitis, rheumatoid arthritis
or pruritus.[27–30]
Figure 1. Structures of histamine and selected histamine receptor ligands
While the first antihistamines (H1R), like mepyramine and
(2–4).
diphenhydramine, as well as their functional behavior on
guinea-pig organs were published in the 1930s, 1940s and
[a] Dr. S. Pockes, Dr. D. Wifling, Prof. Dr. A. Buschauer, Prof. Dr. S. Elz
1950s,[31–33] a large number of highly potent H2R agonists like
Institute of Pharmacy, Faculty of Chemistry and Pharmacy
impromidine and arpromidine were released in the 1970s and
1980s.[34–36] Deriving from the lead structure SK&F 91486 (2,
Figure 1)[37], a long-known ligand addressing histamine recep-
tors, several classes of newly synthesized monomers were
characterized in this study. A couple of previous projects,
focusing on the development of potent H2R agonists, observed
an overlap of H3R- and H4R-related effects of imidazole-
containing compounds.[38] Heterocyclic replacement by amino
University of Regensburg
Universitätsstraße 31, D-93053 Regensburg, Germany
E-mail: steffen.pockes@ur.de
Supporting information for this article is available on the WWW under
https://doi.org/10.1002/open.201900011
© 2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.
This is an open access article under the terms of the Creative Commons
Attribution Non-Commercial NoDerivs License, which permits use and dis-
tribution in any medium, provided the original work is properly cited, the
use is non-commercial and no modifications or adaptations are made.
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(methyl)thiazole, following amthamine,[39] led to highly selective
dimeric H2R agonists, like URÀ Po448 (3, Figure 1)[40] and
associated molecules.[41–43] In addition, a switch away from
imidazole-bearing compounds is recommended as these struc-
tures show poor pharmacokinetic properties due to interactions
with cytochrome P450.[44] Structural modifications around the
guanidine group gave cyano-, carbamoyl-, acylguanidines and
related structures, which showed up selectivity towards the H3R
or H4R, respectively.[45,46] In this project, we aimed to attaining
insights into structure-activity relationships of novel ligands
from the hetarylpropylguanidine-type. We wanted to close the
gap between the monomeric lead structure 2, the highly affine
hH4R ligand URÀ Po194 (4, Figure 1)[40] and the dimeric ligands
described in the literature, e.g. 3.[40] Therefore, we created
alkylated guanidines with various terminal functional groups of
different basicity, like amine, guanidine, urea, including variable
spacer length. Moreover, we synthesized a class of molecules
focusing on the heteroatomic exchange at the guanidine
moiety of 2 only to attain (thio)ureas and S-methylisothioreas.
The final compounds were pharmacologically characterized
with radioligand binding assays and the GTPγS binding assay to
get binding as well as functional data. In addition, we analyzed
all compounds by organ pharmacological studies on the guinea
pig ileum and right atrium in order to receive information about
their functional behavior under physiological conditions (gpH1R
(ileum), gpH2R (right atrium)).
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Scheme 1. Synthesis of the precursors 17–27. Reagents and conditions: (a)
CH3I (1.1 equiv), MeCN, 1 h, reflux; (b) NEt3 (1 equiv), Boc2O (2 equiv),
overnight, room temperature (rt); (c) diamine (5 equiv), Boc2O (1 equiv),
°
DCM, 2 h, 0 C!rt; (d) diamine (3 equiv), 10 (1 equiv), DCM, overnight, rt.
The synthetic route for the preparation of 115–136 and
141–145 was adapted as previously described in the literature
(Scheme 2 and 3).[40,49,50,52–56] In a first step the relevant amine
11–27 attacks benzoyl isothiocyanate (28) via nucleophilic
substitution to give benzoylthioureas 29–44 (Scheme 2).[40,52]
After alkaline hydrolysis yielding the corresponding thioureas
45–60, the intermediates were treated with methyl iodide to
receive 61–76 (Scheme 2).[40,52] Prior to guanidinylation, the S-
methylisothioureas were Boc-protected obtaining 77–92
(Scheme 2).[40] Aminolysis of the guanidinylation reagents 77–92
with some of the amines 5–7 in presence of HgCl2 and
triethylamine gave 93–114 (Scheme 2).[40,57] For the synthesis of
the Boc-protected amines (93–100) and guanidines (101–109)
one equivalent of HgCl2 was used, while four equivalents of
HgCl2 were used for the preparation of the Boc-protected
carbodiimides (110–114). The carbodiimides, which were con-
verted into ureas in the next step, were unscheduled. It was
planned to create the relative dimers, which were published by
Pockes et al.[40] The original synthetic description for one-site
coupling was described with two equivalents of mercury
chloride.[41] This excess should be maintained for this two-site
coupling. Contrary to our expectations the excess of HgCl2
(4 equiv) – which facilitates the elimination of the S-methyl
group by coordination to sulfur – led to mono-Boc-carbodii-
mides, where just one aminolysis was successful. This fact could
be proven by NMR spectroscopy and mass spectrometry and a
similar issue was already reported by Kim et al. in 1993.[57]
Afterwards, the use of HgCl2 for one-site coupling was adjusted
as described in 4.2.9, as well as for two-site coupling (cf. Pockes
et al.)[40]. In a last step the precursors were Boc-deprotected
using trifluoroacetic acid (TFA) to get 115–136 as final
compounds (Scheme 2).[40]
Results and Discussion
Chemistry
Syntheses of the amines 5–7 (Figure 2), which were used for the
development of the final compounds were carried out accord-
ing to the literature.[39,41,47,48] The required precursors 17–27 for
the terminal amines and guanidines were prepared according
to previously reported procedures (Scheme 1) and
adaptions.[41,49,50] The isothiourea 10 proved to be a suitable
guanidinylation reagent for the preparation of 23–27 and was
obtained in a two-step synthesis by S-methylation of 8 and di-
Boc-protection of
9
with two equivalents of Boc2O
(Scheme 1).[41,51] Mono-Boc-protection of the respective dia-
mines 11–16 was also carried out with Boc2O to get 17–22
(Scheme 1). Due to the possibility of a di-protection a molar
ratio of at least 1:5 (Boc2O:diamine) was required in order to
achieve yields >90%.[49] The aforementioned di-Boc-protected
guanidines 23–27 were prepared by dropping the guanidinyla-
tion reagent 10 into a solution of the appropriate diamine (12–
16, 3 equiv) in DCM (Scheme 1).[50]
The synthetic strategy for the final compounds 141–145 is
depicted in Scheme 3. The same pattern was used for the
nucleophilic addition to get 137 or 139 using 28 or benzoyl
isocyanate (138) together with the amine 5 (Scheme 3).[40,52] 137
was further processed in two different ways, getting 140 by
alkaline hydrolysis and 141 by deprotection under acidic
Figure 2. Structures of the amines 5–7, which were used for the preparation
of the final compounds 115–136 and 141–145 (cf. Scheme 1–3).
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°
Scheme 2. Synthesis of the HR ligands 115–136. Reagents and conditions: (a) amine/diamine (1 equiv), 28 (1 equiv/2 equiv), DCM, 2 h/overnight, 0 C!rt; (b)
K2CO3 (2.1 equiv/4.1 equiv), MeOH/H2O (7/3, v/v), 3–5 h, rt; (c) CH3I (1.1 equiv/2.1 equiv), MeCN, 1 h, reflux; (d) NEt3 (1 equiv/2 equiv), Boc2O (1 equiv/2 equiv),
overnight, rt; (e) 5, 6 or 7 (1 equiv/2 equiv), HgCl2 (1 equiv/4 equiv), NEt3 (3 equiv/6 equiv), DCM, overnight, rt; (f) 20% TFA, DCM, overnight, reflux.
conditions (Scheme 3).[40,52] Moreover, the benzoyl isothiocya-
nate 141 was hydrolysed with potassium carbonate to give the
thiourea 142 (Scheme 3).[40,52] To complete the second route,
140 was first deprotected with hydrogen iodide (66%) and
directly handled with methyl iodide yielding 143
(Scheme 3).[53,54] To create the urea analogues, the trityl group
of 139 was first cleaved with TFA to get the final compound
144,[40] followed by alkaline hydrolysis with sodium hydroxide
solution (1 M) under reflux obtaining 145 (Scheme 3).[56] Usual
basic hydrolysis with potassium carbonate was not successful in
this case, not even after several hours of reflux. Compounds
141[53], 142[53] and 143[54,35] were already decribed in the
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°
Scheme 3. Synthesis of the SK&F 91486 analogues 141–145. Reagents and conditions: (a) 5 (1 equiv), 28 or 138 (1 equiv), DCM, overnight, 0 C!rt; (b) K2CO3
(2.1 equiv), MeOH/H2O (7/3, v/v), 3–5 h, rt; (c) 20% TFA, DCM, overnight, reflux; (d) i) 66% HI, EtOH, rt; ii) CH3I (1.1 equiv), MeOH 1 h, reflux; (e) NaOH (1 M
solution), 1 h, reflux.
literature. In this study we resynthesized these structures for
further pharmacological investigations.
ureas 132–136 with different spacer lengths. Furthermore,
heterocyclic exchange of imidazole by amino(methyl)thiazole
should give more insight in the selectivity profile of the ligands.
The compounds depicted in Scheme 3 (141–145) were mainly
altered by heteroatomic exchange at the guanidine group of 2.
The following influence on basicity should give important
information about the variability of this partial structure, with
respect to histamine receptor affinity and potency.
Pharmacology
The ligands 115–136 and 141–145 were pharmacologically
characterized using radioligand binding assays (hH1,2,3,4R), the
guinea pig ileum assay (gpH1R) as well as the guinea pig right
atrium assay (gpH2R). The most interesting compounds were
further investigated in the [35S]GTPγS binding assay (hH2,3,4R).
The radioassays were performed using membranes of Sf9 cells
expressing the respective histamine receptor described in
Table 1 and 2.
Radioligand Binding Data
A correlation was found between binding affinities of the
amines 115–122 at the hH1R and the respective lipophilicity
(Table 1). From C3- (115) to C12-spacer (120–122) there is an
upward shift of approximately 3 log units. The tendency at the
hH2R is the same, but to a lesser extent The highest affinity
Introduction of a third basic moiety was the main focus by
developing new ligands as shown in Scheme 2. Therefore, we
created terminal amines 115–122, guanidines 123–131 and
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Table 1. Binding data (pKi values) of compounds DPH, 1–4, 115–136 and 141–145 determined at human HxRs (x=1–4).[a]
1
2
3
compound
hH1R[b]
hH2R[c]
hH3R[d]
hH4R[e]
pKi
pKi
N
pKi
N
pKi
N
N
4
5
6
7
8
9
DPH
1
2
3
4
7.62�0.01
5.62�0.03
<4
<5.5
<4.5
<4
<5
<5
5.91�0.03
6.06�0.03
6.71�0.02
6.30�0.01
6.52�0.01
<4.5
4
3
3
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
n.d.
–
48
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
n.d.
–
42
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
n.d.
-
6.58�0.04
5.39�0.04[f]
7.33�0.05
5.52�0.05
6.63�0.06
6.07�0.04
6.12�0.04
6.44�0.02
6.80�0.02
7.28�0.04
6.50�0.09
6.73�0.05
6.15�0.05
6.24�0.02
6.80�0.10
6.85�0.06
7.06�0.04
6.67�0.01
7.03�0.09
6.93�0.02
6.84�0.05
5.75�0.10
6.57�0.03
6.55�0.07
6.58�0.11
6.95�0.02
4.26�0.09
<4
7.59�0.01
7.42�0.04
5.25�0.05
7.21�0.02
5.59�0.03
5.82�0.03
6.19�0.01
6.65�0.04
7.14�0.01
7.45�0.02
5.38�0.01
5.29�0.06
6.41�0.01
6.97�0.03
7.20�0.03
7.43�0.02
7.48�0.05
6.53�0.03
6.25�0.04
7.07�0.01
6.43�0.17
6.80�0.01
6.27�0.05
6.69�0.02
6.84�0.02
7.00�0.01
6.78�0.05
6.07�0.03
6.58�0.08
6.09�0.01
6.91�0.03
7.60�0.01
8.13�0.08
5.00�0.05
8.04�0.05
7.03�0.07
6.47�0.02
6.36�0.03
6.33�0.03
6.78�0.01
7.16�0.04
5.33�0.03
4.65�0.06
7.51�0.04
6.62�0.02
6.84�0.02
7.50�0.02
7.55�0.02
6.40�0.01
5.62�0.05
6.23�0.09
6.47�0.07
6.84�0.01
6.59�0.02
6.18�0.03
6.56�0.01
6.70�0.01
6.82�0.04
5.77�0.02
8.14�0.01
5.28�0.06
6.83�0.02
45
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
115
116
117
118
119
120
121
122
123
124
125
126
127
128
129
130
131
132
133
134
135
136
141
142
143
144
145
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
<5
<5.5
6.09�0.01
6.59�0.01
5.75�0.01
6.58�0.01
6.25�0.01
6.28�0.01
<4.5
<4.5
<5
<5.5
<5.5
<4
<4
<4
<4
4.98�0.09
<4
<5
6.17�0.08
[a]Data represent mean values � SEM from at least two independent experiments (N), each performed in triplicate. Radioligand competition binding
experiments performed with [b][3H]mepyramine (hH1R, Kd 4.5 nM, c=5 nM), [c][3H]tiotidine (hH2R, Kd 19.7 nM, c=10 nM), [d][3H]Nα-methylhistamine (hH3R, Kd
8.6 nM, c=3 nM) or [e][3H]histamine (hH4R, Kd 16.0 nM, c=15 nM) at membranes of Sf9 cells expressing [b]the hH1R plus RGS4, [c]the hH2R plus Gsαs, [d]the hH3R
plus Gαi2 plus Gβ1γ2, [e]the hH4R plus Gαi2 plus Gβ1γ2. [f]Displacement of [3H]UR-DE257 (hH2R, Kd 31.3 nM, c=20 nM) instead of [3H]tiotidine; n.d.=not
determined.
Table 2. Agonistic (pEC50) and antagonistic (pKB) activities of 1–3, 120, 121, 127, 130 and 136 at the hH2,3,4R determined in the [35S]GTPγS binding assay.[a]
Compound
hH2R[b]
hH3R[c]
hH4R[d]
[e]
[f]
[f]
[f]
pEC50
Emax
N
pEC50[e] (pKB)[g]
Emax
N
pEC50[e] (pKB)[g]
Emax
N
1
2
3
120
121
127
130
136
6.01�0.07
5.59�0.01[h],[46]
6.61�0.03
6.95�0.04
7.11�0.10
6.86�0.03
7.28�0.10
6.72�0.06
1.00
7
3
5
3
3
3
3
3
8.52�0.10
8.12�0.10[h],[46]
(4.53�0.05)
(6.72�0.03)
5.88�0.03
(7.18�0.04)
4.46�0.02
4.57�0.03
1.00
6
3
3
3
3
3
3
3
8.20�0.08
1.00
3
3
3
3
3
3
3
3
0.66�0.02[h],[46]
0.33�0.03
0.66�0.01
0.22�0.01
0.45�0.03
0.22�0.01
0.45�0.03
0.69�0.04[h],[46]
8.09�0.04[h],[46]
(3.83�0.03)
(3.43�0.01)
(3.75�0.01)
(3.38�0.01)
4.52�0.02
0.83�0.01[h],[46]
0
0
0
0
0
0
À 0.69�0.03
0
À 1.21�0.10
À 1.20�0.15
-0.98�0.01
4.44�0.01
-0.96�0.02
[a]Data represent mean values � SEM from at least three independent experiments (N), each performed in triplicate. Data were analyzed by nonlinear
regression and were best fitted to sigmoidal concentration-response curves (CRCs). [35S]GTPγS binding assay at membranes of Sf9 cells expressing [b]the hH2R
plus Gsαs, [c]the hH3R plus Gαi2 plus Gβ1γ2, [d]the hH4R plus Gαi2 plus Gβ1γ2; [e]pEC50: À logEC50; [f]
E
max: maximal response relative to histamine (Emax =1.00); [g]For
determination of antagonism, reaction mixtures contained histamine (1) (100 nM) and ligands were at concentrations from 10 nM and 1 mM; pKB =À logKB. [h]
Determined in a steady-state [32P]GTPase assay on Sf9 cells expressing the related receptors.
value was measured for 120 (pKi =7.28, Table 1). In comparison
with compounds bearing small alkylic side chains like
URÀ Po194 (4), showing high affinity at the hH4R,[40] introduction
of a terminal amine with similar spacer length (115, 116) led to
a remarkable loss in affinity of at least 1 log unit (Table 1).
Heterocyclic replacement by amino(methyl)thiazole resulted in
the already known affinity decrease at the hH3,4Rs. Related to
the hH2R 121 and 122 reveal moderate selectivity towards the
hH3,4Rs, but not towards the hH1R (Table 1).
Data for the guanidines 123–131 at the hH1R and the hH2R
were similar to those of the amines 115–122. Increasing spacer
length led to higher affinity values culminating in 127 (pKi
(hH1R)=6.59; pKi (hH2R)=7.06; cf. Table 1). Affinity values at the
hH3,4Rs were all in a submicromolar range and this time a switch
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from imidazole to amino(methyl)thiazole resulted in a moderate
affinity loss from maximally 1 log unit, which is noticeably low.
A slight selectivity vis-à-vis hH2R and hH1,3,4Rs is just seen for
129 (Table 1). All further compounds revealed similar but
partially high affinities, especially for the hH2,3,4Rs.
Introducing a terminal urea in the side chain (132–136)
comes along with a massive decrease in basicity, but not with a
massive loss in affinity. Overall they were in a range with the
terminal amines (115–122) and guanidines (123–131), which
means slight or no affinity at the hH1R and submicromolar
affinities at the hH2,3,4Rs (Table 1). This functionality has no
remarkable impact or change with respect to selectivity or
affinity at the histamine receptors.
Compounds 141–145 with its nonlipophilic structures
presented – as expected – no affinity for the hH1R (Table 1).
Values at the hH2R were similar, instead of 145, where the urea
analogue of 2 surprisingly demonstrated higher affinity (pKi
(145)=6.17; pKi (2)=5.39; cf. Table 1). Scoping at the hH3,4Rs
the benzoyl derivatives 141 and 144 illustrated moderate
binding values. Even if the affinities of 141 at the hH3,4Rs were
higher, 144 tends to be selective towards the hH3R (Table 1).
Compound 143 gives an even more pronounced selectivity
towards the hH4R (Figure 3). Besides the weak binding data at
the hH1,2Rs and a moderate result at the hH3R (pKi (hH3R)=
6.58), a pKi of 8.14 showed up a remarkably high tendency for
the hH4R (Table 1). In comparison with the binding data of 2,
the selectivity profile of the S-methylated analogue 143 has
improved.
agonism at the hH2R is equipotent, but less effective (Emax =
1
2
3
4
5
6
7
8
9
0.22) and functionality at the hH3R turns into a partial inverse
agonism at one digit micromolar range (Table 2). 120 and 121
are just different in its heterocyclic group (imidazole vs.
aminomethylthiazole).
The same structural decision was made for further charac-
terization of terminal guanidines. 127 and 130 both bear a C12-
chain with different hetarylic cycles. Compared to the amines,
functional data were similar and in accordance with binding
data (Table 2). 130 is a partial agonist at the hH2R (pEC50 =7.28,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
Emax =0.22) and a weak but full inverse agonist at the hH3,4Rs,
while 127 acts as a silent antagonist at these receptors (Table 2).
The switch from antagonism to inverse agonism at the hH3,4Rs
could be assigned to the aminomethylthiazole structure.
The functional experiments of the urea analogue 136 were
in line with these data. Partial agonism (pEC50 =6.72, Emax =0.45)
at the hH2R, as well as a (partial) inverse agonism at the hH3,4Rs
could be measured (Table 2). 136 was the only imidazole-
containing compound, that shows up an (partial) inverse
agonism at these receptors.
Organ Pharmacological Data
Data from organ bath studies at the guinea pig ileum (gpH1R)
and right atrium (gpH2R) provided functional values under
physiological conditions. The class of terminal amines (115–
122) showed a steady increase in their antagonistic activity at
the gpH1R by elongation of the alkyl side chain (pA2 (115–120)
=4.78–6.95; cf. Table 3). A further significant increase could be
observed by exchange of the heterocycle by aminothiazole (pA2
(122)=8.03; cf. Table 3). Agonistic data at the gpH2R gave
potencies in a submicromolar range with high intrinsic activities
culminated in 120 as a full agonist (pEC50 =6.86, Emax =1.00; cf.
Table 3). Heterocyclic exchange with amino(methyl)thiazoles
led to a slight decrease in potency and efficacy.
The raise of basicity, with respect to the terminal guanidines
(123–131), resulted in slightly higher antagonistic values at the
gpH1R compared to the respective amines (e.g. pA2 (127)=7.22;
cf. Table 3). In analogy to 122, substitution of imidazole by
aminothiazole led to a highly active antagonist at the gpH1R
(pA2 (131)=8.06; cf. Table 3). Terminal guanidines (123–131)
were developed to more potent and highly efficient agonists at
the gpH2R, in comparison to their amine and alkyl analogues
(the latter were published by Pockes et al.[40]). However, it is
striking that the alkylic spacer length seems to have no
significant influence on the agonistic potency (Table 3). The
most potent guanidine 125 (pEC50 =7.69, Emax =0.83) and the
most efficient guanidine 123 (pEC50 =7.30, Emax =1.03) bear a
C8- and a C4-spacer, respectively (Table 3).
[35S]GTPγS Binding Data
The functional data of the [35S]GTPγS assay characterize 120 as
a partial agonist at the hH2R (pEC50 =6.95) and an intrinsic
activity of 66%, relative to histamine (Table 2). At the hH3R an
antagonistic activity (pKB =6.72) of 120 could be measured, as
well as a negligible weak antagonistic effect at the hH4R
(Table 2). The values for 121 were quite similar. The partial
Organ pharmacological data for the ureas 132–136 were in
comparison with the respective amines 115–122 (Table 3).
Therefore, 135 and 136 with its lipophilic C8- and C10-spacer,
respectively, pointed up highest antagonistic activity at the
gpH1R (pA2 (135)=5.85; pA2 (136)=6.00; cf. Table 3). All
compounds, instead of 132, exhibited values in a submicromo-
Figure 3. Selectivity profile of 143 with radioligand displacement curves
from radioligand binding assays. Experiments were performed with com-
pound 143 and [3H]mepyramine (hH1R, Kd 4.5 nM, c=5 nM), [3H]tiotidine
(hH2R, Kd 19.7 nM, c=10 nM), [3H]Nα-methylhistamine (hH3R, Kd 8.6 nM,
c=3 nM) or [3H]histamine (hH4R, Kd 16.0 nM, c=15 nM) at membranes of Sf9
cells expressing the respective hHR. Data represent mean values � SEM from
at least two independent experiments, each performed in triplicate.
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Table 3. Agonistic (pEC50) and antagonistic (pA2) activities of 1–4, 115–136 and 141–145 determined by organ bath studies at the gpH1R (ileum) and the
1
2
3
4
5
6
7
8
9
gpH2R (atrium).[a]
Compound
gpH1R
gpH2R
[c],[d]
[e]
pA2[b] (pEC50)
N
pEC50
Emax
N
1
2
3
4
(6.68�0.03)
n.a.f
255
24
9
8
8
9
9
9
9
9
8
15
6
6
6
6
6
6
6
6
14
6
6
4
6
6
12
9
6.16�0.01
5.16�0.04
8.38�0.05
5.40�0.11
5.33�0.11
5.13�0.05
6.42�0.08
6.51�0.05
6.41�0.05
6.86�0.06
6.49�0.09
6.63�0.07
7.30�0.05
7.67�0.03
7.69�0.02
7.56�0.04
7.25�0.11
6.87�0.04
7.41�0.09
7.02�0.07
6.93�0.09
5.61�0.08
6.54�0.08
6.17�0.09
6.74�0.09
6.53�0.01
4.73�0.05
4.96�0.08
5.13�0.03
4.62�0.05
3.57�0.02
1.00
225
5
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
0.75�0.03
0.78�0.01
0.83�0.03
0.88�0.05
0.68�0.02
0.79�0.01
0.75�0.10
0.78�0.01
1.00�0.06
0.71�0.03
0.90�0.05
1.03�0.03
0.94�0.02
0.83�0.07
0.80�0.02
0.77�0.07
0.72�0.02
1.00�0.01
0.90�0.04
0.85�0.05
0.89�0.03
1.03�0.03
0.96�0.03
0.94�0.05
0.96�0.01
0.43�0.01
0.27�0.02
0.45�0.05
0.15�0.03
0.58�0.02
5.88�0.03
4.83�0.05
4.78�0.02
5.42�0.05
5.20�0.07
5.89�0.05
6.17�0.04
6.95�0.06
7.15�0.06
8.03�0.03
5.58�0.02
6.03�0.04
5.79�0.03
6.60�0.04
7.22�0.05
6.67�0.04
7.30�0.05
6.76�0.05
8.06�0.05
5.04�0.04
5.22�0.08
5.13�0.04
5.85�0.04
6.00�0.03
n.a.f
115
116
117
118
119
120
121
122
123
124
125
126
127
128
129
130
131
132
133
134
135
136
141
142
143
144
145
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
n.a.f
5.41�0.06
5.02�0.06
<4.5
6
6
6
[a]Data represent mean values � SEM from at least three independent experiments (N). Data were analyzed by nonlinear regression and were best fitted to
sigmoidal concentration-response curves. [b]pA2: À log c(Ant)+log (r–1); r=10ΔpEC50; ΔpEC50 was calculated from pEC50 of histamine and pEC50 of histamine in
presence of the respective antagonist; [c]pEC50: À logEC50; [d]pEC50 was calculated from the mean corrected shift ΔpEC50 of the agonist curve relative to the
histamine reference curve by equation pEC50 =6.16+ΔpEC50; [e]Emax: maximal response relative to the maximal increase in heart rate induced by histamine
(Emax =1.00). [f]n.d.=not determined.
lar range at the gpH2R with remarkable high agonistic efficacy
(Emax =0.89-1.03; cf. Table 3).
ment of the heart frequency in the guinea-pig right atrium
assay was conveyed via the H2R. The most interesting results at
the gpH2R were displayed in Figure 4, where CRCs of selected
Compounds 141–145 boasted only slight or no antagonistic
activity at the gpH1R (Table 3). Compared to 2, which reveals a
guanidine structure, the less basic thiourea (141, 142) and urea
derivatives (144, 145) presented weak partial agonism at the
guinea pig right atrium (gpH2R). Isothiourea 143 is equipotent
(pEC50 =5.13) but less effective (Emax =0.45) referred to SK&F
91486 (2). According to the literature 143 demonstrated a
potency comparable to histamine (rel. potency=0.1) at the
guinea-pig right atrium, while the maximum response was
higher (0.45 vs. 0.23).[35]
The maximum responses of the tested compounds (115–
136 and 141–145) at the right atrium were completely
antagonized after addition of the H2R antagonist cimetidine
(pA2 =6.10[58,59]) (30 μM). For compounds 120, 125 (Figure S35,
Supporting Information (SI)) and 135 full concentration-
response curves (CRCs) in the presence of cimetidine (30 μM,
30 min preincubation) were determined. The presence of an
antagonist resulted in rightward shifted curves. The calculated
values via Schild equation (Table S2, SI) were in accordance with
the experimental data. This outcome confirms that the incre-
Figure 4. Concentration-response curves of 1, 2 and 3 (black), as well as 116,
120, 124, 129, 133, 143 and 144 (colored) at the gpH2R (atrium). Histamine
(1) was used as a reference (pEC50 =6.16, Emax =1.00). Displayed curves are
calculated by endpoint determination (N=1).
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1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
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22
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24
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28
29
30
31
32
33
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37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
Figure 5. Lowest free energy (MM-GBSA) docking poses of 143 at both the hH4R (A, B) and hH3R (C, D) showing key ligand-receptor interactions in the form of
ligand interaction diagrams (A, C) or three-dimensional illustrations (B, D). Hydrogen bonds and salt bridges are colored in magenta (A, C) or yellow (B, D), and
cation-π interactions in red (A, C).
compounds of each group (colored) were depicted together
with references (black).
in the extracellular loop 2 (hH4R: E163ECL2.49, hH3R: E185ECL2.47) is
shifted by two amino acids. Therefore, the orientation of this
GLU residue seems to slightly differ between both receptor
subtypes: Whereas it seems to be still capable of properly
forming a salt bridge with the isothiourea moiety of 143 in case
of the hH4R, the interactions may be weakened in the case of
hH3R. Furthermore, this salt bridge appeared in four of five
docking poses in case of the hH4R compared to only one of five
in case of the hH3R. Consequently, these molecular differences
may, at least in parts, reflect the discrepancies in pKi values of
more than one order of magnitude between hH4R and hH3R
(hH4R: pKi =8.14, hH3R: pKi =6.58, cf. Table 1) and thus provide a
possible molecular explanation.
Computational Studies
143 was “flexibly” docked into the orthosteric binding pocket of
both the hH4R and hH3R (cf. Figure 5), two closely related
histamine receptor subtypes sharing
a
high sequence
identity.[15,60] Of the investigated protonation and/or tautomeri-
zation states of the imidazole ring (τ-H and π-H, τ-H, π-H),
docking of 143 resulted in the most reasonable binding poses
and in the lowest MM-GBSA values in case of the protonated (τ-
H and π-H) form of the imidazole ring. At first, ligand-receptor
interactions of these lowest free energy (MM-GBSA) binding
poses seemed to be highly comparable between both Conclusions
histamine receptor subtypes (cf. Figure 5): The isothiourea
moiety and the protonated imidazole ring of 143 formed salt
Novel series of alkylated hetarylpropylguanidines with function-
bridges with D943.32, E163ECL2.49 and E1825.46 (hH4R) or D1143.32
,
alized side chains or new functionality at the guanidine
structure were investigated in this project. By introduction of
three different functional groups (amine, guanidine, urea) in a
terminal position of an alkylic side chain various shades of
basicity could be displayed. The respective ligands 115—136
were obtained in a six- to nine-step synthesis in excellent yield,
E185ECL2.47 and E2065.46 (hH3R). In addition, cation-π-interactions
were detected between the isothiourea moiety of 143 and
F3447.39 (hH4R) or F3987.39 (hH3R). However, by taking a closer
look at the differences between binding of 143 to either hH4R
or hH3R, it becomes obvious that the location of a certain GLU
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atom. NMR spectra were processed with MestReNova 11.0
(Mestrelab Research, Compostela, Spain). High resolution mass
spectrometry (HRMS) was performed on an Agilent 6540 UHD
Accurate-Mass Q-TOF LC/MS system (Agilent Technologies, Santa
Clara, CA) using an ESI source. Elemental analyses (EA) were
executed on a Heraeus Elementar Vario EL III and are within �0.4%
unless otherwise noted. Melting points (mp) were detected on a
Büchi (Essen, Germany) B-545 apparatus using an open capillary
and are uncorrected. Preparative HPLC was handled with a system
from Knauer (Berlin, Germany) consisting of two K-1800 pumps and
just as for compounds 141–145 (two to three steps). Elongation
of the spacer length and, associated therewith, the increase of
lipophilicity led to higher affinities and potencies at all four
histamine receptors. The most affine and potent derivatives
(two digit nanomolar range) could be assigned to guanidines in
the terminal position (123–131), in comparison with the
appropriate amines (115–122) and ureas (132–136). None of
these classes pointed up a distinct selectivity towards any of
the four histamine receptors. Although bioisosteric replacement
of imidazole by amino(methyl)thiazole led to selectivity towards
the H2R, improvement of the selectivity profile could not be
determined, in comparison with already described H2-selective
compounds. Heteroatomic exchange at the guanidine group of
SK&F 91486 (2) led to benzoylurea derivative 144, with a
preference towards the hH3R, and isothiourea 143, with
considerable improvement of the selectivity profile towards the
hH4R. Thereby, computational studies provided molecular in-
sights into the binding modes of 143 at both hH4R and hH3R
and supported the proposal of a possible mechanism of the
enhanced selectivity profile. Furthermore, both structures,143
and 144, could be an interesting starting point for future
projects facing H3 and H4 receptor selectivity. This is of special
interest as to date there are still no drugs available for both
receptors (apart from Pitolisant[61]), although a wide field of
applications are reported for the H3R (e.g. several neuro-
degenerative diseases)[21–25] and the H4R (e.g. inflammation,
allergic diseases).[26–30]
1
2
3
4
5
6
7
8
9
a
K-2001 detector. A Eurospher-100 C18 (250×32 mm, 5 μm)
(Knauer, Berlin, Germany) or a Kinetex XBÀ C18 (250 x 21.2 mm,
5 μm) (Phenomenex, Aschaffenburg, Germany) served as stationary
phase. As mobile phase, 0.1% TFA in millipore water and
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
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28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
°
acetonitrile (MeCN) were used. The temperature was 25 C, the flow
rate 15 mL/min and UV detection was performed at 220 nm.
Analytical HPLC was implemented on an Agilent 1100 HPLC system
(Agilent Technologies, Santa Clara, CA) using a binary pump,
autosampler, and DAD detector. Stationary phase was a Kinetex
XBÀ C18 (250
x 4.6 mm, 5 μm) (Phenomenex, Aschaffenburg,
Germany). As mobile phase, mixtures of MeCN and aqueous TFA
were used (linear gradient: MeCN/TFA (0.1%) (v/v) 0 min: 5:95,
25 min: 50:50, 26–35 min: 95:5 (method A); flow rate=1.0 mL/min,
t0 =2.57 min). Capacity factors were calculated pursuant to k=
(tRÀ t0)/t0. Detection was measured at 220 nm. All compounds were
examined using method A. Filtration of the stock solutions with
PTFE filters (25 mm, 0.2 μm, Phenomenex Ltd., Aschaffenburg,
Germany) was carried out before testing. Compound purities
determined by HPLC were calculated as the peak area of the
analyzed compound in % relative to the total peak area (UV
detection at 220 nm). The HPLC purities (see analytical data and
Supporting Information) of the final compounds were�95%. For
all purity runs (see SI) the blank run was subtracted to avoid TFA-
dependent baseline drift. All the tested compounds were screened
for PAINS and aggregation by publicly available filters (http://
zinc15.docking.org/patterns/home, http://advisor.docking.org).[62,63]
None of the screened molecules have been previously reported as
PAINS or an aggregator. Since Devine et al. described 2-amino-
thiazoles as a promiscuous frequent hitting scaffold at different
enzymes,[64] full dose response curves for all experiments and
compounds – not only for the 2-aminothiazoles – were performed.
None of the curves displayed abnormalities, e.g. high Hill slopes,
what could be an indication for PAINS.[63]
Experimental Section
General Conditions
Commercially chemicals (8, 11–16, 28 and 138), reagents and
solvents were purchased from Acros Organics (Geel, Belgium), Alfa
Aesar GmbH & Co. KG (Karlsruhe, Germany), Iris Biotech GmbH
(Marktredwitz, Germany), Merck KGaA (Darmstadt, Germany),
Sigma-Aldrich Chemie GmbH (München, Germany) or TCI Europe
(Zwijndrecht, Belgium) and were used as received. Deuterated
solvents for nuclear magnetic resonance (1H NMR and 13C NMR)
spectra were purchased from Deutero GmbH (Kastellaun, Germany).
All reactions including dry solvents were carried out in dry flasks
under nitrogen or argon atmosphere. For the preparation of
buffers, HPLC eluents and stock solutions millipore water was used.
Column chromatography was accomplished using Merck silica gel
Geduran 60 (0.063–0.200 mm) or Merck silica gel 60 (0.040–
0.063 mm) (flash column chromatography). Reactions were moni-
tored by TLC on aluminium sheets with silica gel 60 F254 from
Merck. Spots were detected under UV light at 254 nm, by iodine
Chemical Synthesis and Analytical Data
General Procedure for the Preparation of the
Mono-Boc-Protected Diamines 17–22
A 0.5 M solution of Boc2O (1 equiv) in DCM was added dropwise
over a 2 h period to a 0.25 M solution of diamine 11–16 (5 equiv) in
DCM cooled with an ice-bath. The reaction mixture was stirred over
night at room temperature (rt) and filtered. The filtrate was
concentrated under vacuum and the resulting oil dissolved in
EtOAc was washed with half-saturated brine (3×150 mL), dried
(Na2SO4) and concentrated under vacuum. The crude product was
purified by column chromatography (DCM/MeOH/7 M NH3 in
MeOH 80/18/2 – 50/48/2 v/v/v).
vapor, ninhydrin or fast blue
B staining. Nuclear magnetic
resonance (1H NMR and 13C NMR) spectra were measured on a
Bruker (Karlsruhe, Germany) Avance 300 (1H: 300 MHz, 13C: 75 MHz)
or Avance 400 (1H: 400 MHz, 13C: 101 MHz) spectrometer using
perdeuterated solvents. Chemical shifts (δ) are given in parts per
million (ppm). Multiplicities were stated using the following
abbreviations: s (singlet), d (doublet), t (triplet), q (quartet), p
(pentet), m (multiplet) and bs (broad signal) and combinations
thereof. 13C NMR-Peaks were measured by DEPT 135 and DEPT 90
(distortionless enhancement by polarization transfer): “+” primary
and tertiary carbon atom (positive DEPT 135 signal), “À “ secondary
carbon atom (negative DEPT 135 signal), “quat” quaternary carbon
N-(tert-Butoxycarbonyl)-1,3-propanediamine (17)[49]
The reaction was carried out with propane-1,3-diamine (11,
3.83 mL, 45.87 mmol), Boc2O (2.0 g, 9.16 mmol) and DCM. The
product was obtained as a colorless oil (1.55 g, 97%): Rf =0.40
1
(DCM/MeOH/NH3 80:20:0.1); H NMR (300 MHz, CDCl3) δ 5.06 (t, J=
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tert-Butyl (3-thioureidopropyl)carbamate (45)[65]
5.3 Hz), 3.20 (q, J=6.2 Hz, 2H), 2.86 (bs, 2H), 2.79 (t, J=6.6 Hz, 2H),
1.65 (p, J=6.6 Hz, 2H), 1.43 (s, 9H). 13C NMR (75 MHz, CDCl3) δ
156.23, 79.15, 39.17, 38.19, 32.55, 28.42. HRMS (ESI-MS): m/z [M+
H+] calculated for C8H19N2O2+: 175.1441, found 175.1445; C8H18N2O2
(174.24).
1
2
3
4
5
6
7
8
9
45 was made out of 29 (2.90 g, 8.59 mmol) and K2CO3 (2.49 g,
18.04 mmol) in 50 ml MeOH/H2O (7/3 v/v) yielding a yellow oil
(1.90 g, 95%): Rf =0.29 (DCM/MeOH 95:5); 1H NMR (300 MHz,
CDCl3) δ 7.42 (bs, 1H), 6.39 (bs, 1H), 5.24 (bs, 1H), 3.56+3.25 (2 bs,
1.4H+0.6H (thione-thiol tautomerism)), 3.13 (q, J=6.4 Hz, 2H), 1.80
– 1.61 (m, 2H), 1.39 (s, 9H). 13C NMR (75 MHz, CDCl3) δ 183.31,
156.80, 79.72, 41.87, 37.38, 29.73, 28.43. HRMS (ESI-MS): m/z [M+
H+] calculated for C9H20N3O2S+: 234.1271, found 234.1271;
C9H19N3O2S (233.33).
General Procedure for the Preparation of the
N-Aminoalkyl-N’,N’’-di-Boc-Protected Guanidines 23–27
A solution of 10 (1 equiv) in DCM (50 mL) was added dropwise to a
solution of the respective diamine (12-16, 3 equiv) in DCM (50 mL)
at rt. The resulting mixture was stirred over night and washed with
H2O (3x25 mL) and brine (30 mL). The organic solvent was dried
over Na2SO4 and the crude product was purified with column
chromatography (DCM/MeOH/7 M NH3 in MeOH 95/3/2 – 90/8/2 v/
v/v).
The synthesis of 56–60 is described in the literature (cf. 23–27)[40]
and was carried out with the appropriate dibenzoylthiourea 40–44
(1 equiv) and K2CO3 (4.1 equiv).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
General Procedure for the Preparation of the
S-methylisothioureas 61–71
1-(4-Aminobutyl)-2,3-(di-tert-butoxycarbonyl)guanidine (23)[50]
The general procedure for the synthesis of the S-methylisothioureas
is described in the literature (cf. 4.2.10.).[40]
The synthesis was accomplished with 12 (1.37 g, 15.51 mmol) and
10 (1.50 g, 5.17 mmol) according to the general procedure. Column
chromatography gave 23 as a yellow oil (1.18 g, 69%): Rf =0.18
tert-Butyl {3-[(imino(methylthio)methyl)amino]propyl}
carbamate (61)[66]
1
(DCM/MeOH/NH3 95:5:0.1); H NMR (300 MHz, CDCl3) δ 11.47 (bs,
1H), 8.33 (bs, 1H), 3.39 (q, J=7.3 Hz, 2H), 2.71 (t, J=6.8 Hz, 2H), 1.86
(bs, 2H), 1.66 – 1.50 (m, 4H), 1.48 (s, 9H), 1.47 (s, 9H). 13C NMR
(75 MHz, CDCl3) δ 163.55, 156.16, 153.30, 83.13, 79.32, 41.60, 40.63,
30.56, 28.29, 28.07, 26.36. HRMS (ESI-MS): m/z [M+H+] calculated
for C15H31N4O4+: 331.2340, found 331.2346; C15H30N4O4 (330.43).
Compound 45 (1.80 g, 7.71 mmol) was dissolved in MeCN (30 mL)
and treated with methyl iodide (0.53 mL, 8.49 mmol) resulting a
yellow oil (61 x HI, 2.80 g, 97%): Rf =0.14 (DCM/MeOH 95:5); 1H
NMR (300 MHz, CDCl3, hydrogen iodide) δ 3.76–3.32 (m, 2H), 3.21
(q, J=6.3 Hz, 2H), 2.76 (s, 3H), 1.88 (p, J=6.6 Hz, 2H), 1.39 (s, 9H).
13C NMR (75 MHz, CDCl3, hydrogen iodide) δ 170.17, 154.28, 78.25,
40.17, 35.13, 26.66, 26.37, 13.41. HRMS (ESI-MS): m/z [M+H+]
calculated for C10H22N3O2S+: 248.1427, found 248.1429; C10H21N3O2S
x HI (375.27).
General Procedure for the Preparation of the Benzoylthioureas
29–39
To an ice-cold solution of the pertinent amine (11–27, 1 equiv) in
DCM, benzoyl isothiocyanate (28, 1 equiv) was added dropwise.
The reaction was allowed to stir at room temperature (rt) for 2 h.
The reaction mixture was washed three times with H2O and
saturated solution of NaCl (each 30 mL). The organic layer was dried
over Na2SO4 and the crude product was purified with column
chromatography (DCM/MeOH 100/0 – 98/2 v/v).
The synthesis of 72–76 is described in the literature (cf. 29–33)[40]
and was carried out with the appropriate bisthiourea 56–60
(1 equiv) and methyl iodide (2.1 equiv).
General Procedure for the Preparation of the
N’-Boc-S-methylisothioureas 77–87
tert-Butyl [3-(3-benzoylthioureido)propyl]carbamate (29)[55]
The general procedure for the synthesis of the N’-Boc-S-methyl-
isothioureas is described in the literature (cf. 4.2.11.)[40]
.
The product was developed using 17 (1.55 g, 8.90 mmol) and 28
(1.20 mL, 8.90 mmol) in DCM (30 mL) and the desired compound
1
was isolated as a yellow oil (2.91 g, 97%): Rf =0.15 (DCM); H NMR
tert-Butyl {3-[(((tert-butoxycarbonyl)imino)(methylthio)methyl)
amino]propyl}carbamate (77)
(300 MHz, CDCl3) δ 10.83 (bs, 1H), 9.12 (bs, 1H), 7.91–7.74 (m, 2H),
7.56–7.32 (m, 3H), 5.05 (bs, 1H), 3.77 (q, J=6.5 Hz, 2H), 3.21 (q, J=
7.4 Hz, 2H), 1.86 (p, J=6.6 Hz, 2H), 1.42 (s, 9H). 13C NMR (75 MHz,
CDCl3) δ 180.25, 166.91, 156.20, 133.56, 131.74, 129.10, 127.51,
79.50, 42.95, 37.06, 30.20, 28.41. HRMS (ESI-MS): m/z [M+Na+]
calculated for C16H23N3NaO3S+: 360.1352, found 360.1355;
C16H23N3O3S (337.44).
The reaction was realized with 61 (2.70 g, 7.19 mmol), NEt3
(1.00 mL, 7.19 mmol) and Boc2O (1.57 g, 7.19 mmol). After column
chromatography a colorless oil (2.30 g, 92%) was obtained: Rf =
1
0.41 (DCM/MeOH 98:2); H NMR (300 MHz, CDCl3) δ 9.52 (bs, 1H),
4.61 (bs, 1H), 3.31 (t, J=7.1 Hz, 2H), 3.12 (q, J=7.2 Hz, 2H), 2.39 (s,
3H), 1.73 (p, J=6.7 Hz), 1.43 (s, 9H), 1.37 (s, 9H). 13C NMR (75 MHz,
CDCl3) δ 173.55, 162.07, 156.11, 79.34, 79.20, 41.13, 37.75, 29.96,
28.37, 28.21, 13.65. HRMS (ESI-MS): m/z [M+H+] calculated for
C15H30N3O4S+: 348.1952, found 348.1952; C15H29N3O4S (347.47).
The synthesis of 40–44 is described in the literature (cf. 17–21)[40]
and was carried out with the appropriate diamine 11–15 (1 equiv)
and 28 (2 equiv).
The synthesis of 88–92 is described in the literature (cf. 35–39)[40]
and was carried out with the appropriate isothiourea 72–76
(1 equiv), NEt3 (2 equiv) and Boc2O (2 equiv).
General Procedure for the Preparation of the Thioureas 45–55
The general procedure for the synthesis of the thioureas is
described in the literature (cf. 4.2.9.)[40]. The NMR peak splitting due
to thione-thiol tautomerism – described in the reference – also
appears for the compounds 45–55.
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Full Papers
yielding a yellow oil (300 mg, 84%): RP-HPLC: 100%, (tR =5.94, k=
1.31). H NMR (300 MHz, CD3OD, tri-trifluoroacetate) δ 8.81 (d, J=
1.3 Hz, 1H), 7.35 (s, 1H), 3.30–3.23 (m, 4H), 3.01 (t, J=7.4 Hz, 2H),
2.81 (t, J=7.3 Hz, 2H), 1.96 (m, 4H). 13C NMR (75 MHz, CD3OD, tri-
trifluoroacetate) δ 157.66, 134.92, 134.54, 116.99, 41.68, 39.61,
38.13, 28.75, 28.02, 22.55. HRMS (ESI-MS): m/z [M+H+] calculated
for C10H21N6+: 225.1822, found 225.1822; C10H20N6 x 3 TFA. (566.38).
General Procedure for the Guanidinylation reaction of 93–109
1
2
3
4
5
1
To a suspension of the pertinent amine 5, 6, or 7 (1 equiv), the
pertinent N’-Boc-S-methylisothiourea 77–87 (1 equiv) and HgCl2
(1 equiv) in DCM, NEt3 (3 equiv) was added. The mixture was stirred
overnight at rt. A possible excess of HgCl2 was quenched with 7 N
NH3 in MeOH (3-5 mL). The resulting suspension was filtered over
Celite and the crude product was purified with column chromatog-
raphy (DCM/MeOH/7N NH3 in MeOH 98/1/1 – 95/3/2 v/v/v).
6
7
8
Synthesis of the SK&F 91486analogues 141–145
9
2-tert-Butoxycarbonyl-1-(N-tert-butoxycarbonylaminopropany-
l)-3-[3-(1-trityl-1H-imidazol-4-yl)propyl]guanidine (93)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
N-{[3-(1-Trityl-1H-imidazol-4-yl)propyl]thiocarbamoyl}
benzamide (137)
Compound 93 was prepared from 5 (500 mg, 1.36 mmol), 77
(473 mg, 1.36 mmol), HgCl2 (369 mg, 1.36 mmol) and NEt3 (0.57 mL,
4.08 mmol) in DCM (20 mL) conforming to the general procedure
yielding a yellow foamlike solid (420 mg, 46%): Rf =0.30 (DCM/
MeOH/NH3 98:2:0.1); 1H NMR (300 MHz, CDCl3) δ 9.05 (bs, 1H) 7.42–
7.27 (m, 10H), 7.15–7.07 (m, 6H), 6.56 (d, J=0.8 Hz, 1H), 5.91 (bs,
1H), 3.55–3.16 (m, 4H), 3.09 (q, J=5.6 Hz, 2H), 2.56 (t, J=6.3 Hz, 2H),
1.87 (p, J=6.7 Hz, 2H), 1.67–1.48 (m, 2H), 1.44 (s, 9H), 1.41 (s, 9H).
13C NMR (75 MHz, CDCl3) δ 164.20, 160.89, 156.55, 142.34, 140.46,
138.02, 129.72, 128.12, 128.10, 118.31, 78.74, 75.26, 75.24, 40.23,
40.20, 36.93, 33.15, 30.58, 29.16, 28.57, 28.51. HRMS (ESI-MS): m/z
[M+H+] calculated for C39H51N6O4+: 667.3966, found 667.3970;
C39H50N6O4 (666.87).
Compound 137 was prepared according to the general procedure
described in 4.2.5. using 5 (4.80 g, 13.06 mmol) and 28 (1.76 mL,
13.06 mmol) in 100 mL DCM. After column chromatography
(EtOAc/PE 1/2 – 1/1 v/v) the product was obtained as a yellow solid
(4.60 g, 66%): Rf =0.55 (EtOAc/Hex 1:1); mp 139.4 C. 1H NMR
°
(300 MHz, CDCl3) δ 10.76 (bs, 1H), 9.09 (bs, 1H), 7.85–7.74 (m, 2H),
7.63–7.54 (m, 1H), 7.52–7.42 (m, 2H), 7.37 (d, J=1.3 Hz, 1H), 7.39–
7.25 (m, 9H), 7.20–7.05 (m, 6H), 6.60 (s, 1H), 3.72 (q, J=6.9 Hz, 2H),
2.66 (t, J=7.3 Hz, 2H), 2.05 (p, J=7.3 Hz, 2H). 13C NMR (75 MHz,
CDCl3) δ 179.69, 166.76, 142.53, 140.22, 138.62, 133.46, 131.89,
129.80, 129.08, 128.05, 127.99, 127.49, 118.26, 75.14, 45.35, 27.72,
25.73. HRMS (ESI-MS): m/z [M+H+] calculated for C33H31N4OS+:
531.2213, found 531.2218; C33H30N4OS (530.69).
General Procedure for the Guanidinylation Reaction of 110–114
1-[3-(1-Trityl-1H-imidazol-4-yl)propyl]thiourea (140)
To a suspension of the amine 5 (2 equiv), the pertinent N’-Boc-S-
methylisothiourea 88–92 (1 equiv) and HgCl2 (4 equiv) in DCM, NEt3
(6 equiv) was added. The mixture was stirred overnight at rt. A
possible excess of HgCl2 was quenched with 7 N NH3 in MeOH (3–
5 mL). The resulting suspension was filtered over Celite and the
crude product was purified with column chromatography (DCM/
MeOH/7 N NH3 in MeOH 98/1/1 – 95/3/2 v/v/v).
Compound 140 was prepared according to the general procedure
described in 4.2.6. using 137 (1.50 g, 2.83 mmol) and K2CO3
(781 mg, 5.65 mmol) in 30 mL MeOH/H2O (7/3 v/v). The product
was obtained as a beige solid (920 mg, 76%): Rf =0.20 (DCM/MeOH
1
°
95:5); mp 196.1 C. H NMR (400 MHz, CD3OD) δ 7.45–7.28 (m, 10H),
7.22–7.08 (m, 6H), 6.71 (s, 1H), 3.48+3.13 (2 bs, 1.2H+0.8H,
(thione-thiol tautomerism)), 2.56 (t, J=7.3 Hz, 2H), 1.84 (p, J=
7.3 Hz, 2H). 13C NMR (101 MHz, CD3OD) δ 179.77, 143.76, 141.40,
139.35, 130.88, 129.28, 129.24, 119.92, 76.77, 45.09, 29.93, 26.03.
HRMS (ESI-MS): m/z [M+H+] calculated for C26H27N4S+: 427.1951,
found 427.1955; C26H26N4S (426.58).
2-tert-Butoxycarbonyl-1-(N’-tert-butoxycarbonylcarbodiimidopr-
opyl)-3-[3-(1-trityl-1H-imidazol-4-yl)propyl]guanidine (110)
Compound 110 was prepared from 5 (1.0 g, 2.72 mmol), 88
(572 mg, 1.36 mmol), HgCl2 (1.48 g, 5.44 mmol) and NEt3 (1.13 mL,
8.16 mmol) in DCM (20 mL) conforming to the general procedure
yielding a yellow oil (430 mg, 46%): Rf =0.44 (DCM/MeOH/NH3
N-{[3-(1H-Imidazol-4-yl)propyl]thiocarbamoyl}benzamide (141)
1
The title compound was prepared from 137 (1.0 g, 1.88 mmol), TFA
(4 mL) and DCM (16 mL) according to the general procedure (cf.
4.2.11). The crude product was purified by column chromatography
(DCM/MeOH/7 M NH3 in MeOH 95/3/2 v/v/v) yielding 141 as free
98:2:0.1); H NMR (300 MHz, CDCl3) δ 9.75 (bs, 1H), 7.34–7.21 (m,
10H), 7.14–7.03 (m, 6H), 6.51 (s, 1H), 3.52–3.08 (m, 6H), 2.68–2.46
(m, 2H), 1.99–1.71 (m, 4H), 1.41 (s, 9H), 1.39 (s, 9H). 13C NMR
(75 MHz, CDCl3) δ 164.45, 160.71, 158.01, 156.00, 142.52, 140.58,
138.41, 129.73, 128.00, 127.94, 117.91, 85.50, 78.63, 75.02, 53.52,
45.56, 43.58, 28.54, 28.26, 27.93, 25.78, 21.06. MS (LC–MS, ESI): m/z
692.39 [M+H+]; C40H49N7O4 (691.88).
base and yellow solid (350 mg, 64%): Rf =0.12 (DCM/MeOH 95:5);
1
°
mp 139.8 C. H NMR (300 MHz, CD3OD) δ 8.01–7.84 (m, 2H), 7.71–
7.62 (m, 1H), 7.61 (s, 1H), 7.57–7.46 (m, 2H), 6.88 (s, 1H), 3.72 (t, J=
7.0 Hz, 2H), 2.70 (t, J=7.5 Hz, 2H), 2.04 (p, J=7.3 Hz, 2H). 13C NMR
(75 MHz, CD3OD) δ 182.06, 169.56, 137.51, 135.97, 134.24, 133.95,
129.88, 129.19, 117.78, 45.70, 29.01, 25.07. HRMS (ESI-MS): m/z [M+
H+] calculated for C14H17N4OS+: 289.1118, found 289.1120;
C14H16N4OS (288.37); Anal. calculated for C14H16N4OS: C 58.31, H
5.59, N 19.43, found: C 58.32, H 5.66, N 19.16.
General Procedure for the Preparation of the Title Compounds
115–136
The general procedure for the synthesis of 115–136 is described in
the literature (cf. 4.2.7.).[40] All compounds were obtained as tri-
trifluoroacetates.
N-[3-(1H-Imidazol-4-yl)propyl]-S-methylisothiourea (143)
To an ice-cold suspension of 140 (500 mg, 1.17 mmol) in EtOH
(20 mL) an aqueous solution of HI (66%, 5 mL) was added dropwise.
The resulted yellow precipitate (142 x HI) was filtrated and washed
with Et2O. Subsequently, 142 x HI was dissolved in MeOH (5 mL),
treated with methyl iodide (0.08 mL, 1.29 mmol) and refluxed for
53 1-(3-Aminopropyl)-3-[3-(1H-imidazol-4-yl)propyl]guanidine
(115)
54
55
56
57
The title compound was prepared from 93 (420 mg, 0.63 mmol),
TFA (4 mL) and DCM (16 mL) according to the general procedure,
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Full Papers
1 h. After evaporation the title compound was obtained by
recrystallization in isopropanol/Et2O to give 143×2 HI as a yellow-
brown solid (300 mg, 56%): Rf =0.10 (DCM/MeOH/NH3 90:10:0.1);
form, resulting in a net charge of +1 or +2, respectively. „Flexible“
docking of 143 to both the hH4R and hH3R was performed using
the induced fit docking module (Schrödinger LLC). 143 was docked
within a box of 46×46×46 Å3 around the center of mass of the
residues D943.32, E1825.46, Q3477.42 (hH4R) or D1143.32, E2065.46 and
L4017.42 (hH3R). Redocking was performed in the extended precision
mode. Furthermore, the resulting poses were scored using MM-
GBSA (Schrödinger LLC). Among the reasonable ligand binding
poses, the pose corresponding to the lowest MM-GBSA value, was
selected as the most probable pose. Figures showing molecular
structures of the hH4R or hH3R in complex with 143 were generated
with PyMOL Molecular Graphics system, version 2.2.0 (Schrödinger
LLC), and the corresponding ligand interaction diagrams were
prepared with Maestro (Schrödinger LLC).
1
2
3
4
5
6
7
8
9
1
°
mp 104.9 C (2 HI). H NMR (300 MHz, CD3OD, di-hydrogen iodide) δ
8.85 (d, J=1.4 Hz, 1H), 7.43 (d, J=1.4 Hz, 1H), 3.46 (t, J=7.2 Hz, 2H),
2.84 (t, J=7.7 Hz, 2H), 2.66 (s, 3H), 2.02 (p, J=7.5 Hz, 2H). 13C NMR
(75 MHz, CD3OD, di-hydrogen iodide) δ 170.42, 135.07, 134.21,
117.59, 44.74, 27.96, 22.99, 15.40. HRMS (ESI-MS): m/z [M+H+]
calculated for C8H15N4S+: 199.1012, found 199.1012; C8H14N4S x 2 HI
(454.11); Anal. calculated for C7H12N4S×2 HI: C 21.16, H 3.55, N
12.34, found: C 21.23, H 3.87, N 12.08.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
1-[3-(1H-Imidazol-4-yl)propyl]urea (145)
A suspension of 144 (120 mg, 0.44 mmol) in an aqueous solution of
NaOH (1 M, 10 mL) was refluxed for 1 h. After cooling of the clear
solution (!) a colorless solid precipitated. After filtration the title
compound was washed with Et2O to give 145 (50 mg, 67%): Rf =
Acknowledgements
0.12 (DCM/MeOH/NH3 90:10); mp 128.0 C. 1H NMR (400 MHz,
°
The authors thank Christine Braun, Kerstin Röhrl and Maria Beer-
Krön for expert technical assistance, as well as the Leibnitz
Rechenzentrum (LRZ) in Munich for providing software (Schrö-
dinger Suite) and computing resources.
DMSO-d6) δ 7.61 (s, 1H), 6.77 (s, 1H), 6.00 (t, J=5.9 Hz, 1H), 5.40 (s,
2H), 2.97 (q, J=6.5 Hz, 2H), 2.47 (t, J=7.8 Hz, 2H), 1.63 (p, J=7.2 Hz,
2H). 13C NMR (101 MHz, DMSO-d6) δ 159.25, 135.91, 134.79, 117.58,
39.19, 30.38, 23.94. HRMS (ESI-MS): m/z [M+H+] calculated for
C7H13N4O+: 169.1084, found 169.1088; C7H12N4O (168.20); Anal.
calculated for C7H12N4O2 x 0.24 DMSO (sample was a recovery of a
NMR sample solved in DMSO): C 48.06, H 7.25, N 29.97, found: C
48.00, H 7.11, N 30.11.
Conflict of Interest
The authors declare no conflict of interest.
Pharmacological Methods and Materials
Keywords: histamine H1-4 receptor · ligand design · receptor
subtype selectivity · organ pharmacology · computational
chemistry
Materials
Histamine dihydrochloride was acquired from Alfa Aesar GmbH &
Co. KG (Karlsruhe, Germany). [3H]mepyramine (specific activity:
20.0 Ci/mmol), [3H]tiotidine (specific activity: 78.4 Ci/mmol), [3H]Nα-
methylhistamine (specific activity: 85.3 Ci/mmol) and [3H]histamine
(specific activity: 25.0 Ci/mmol) were purchased from Hartmann
analytic (Braunschweig, Germany). GTPγS was from Roche (Man-
nheim, Germany), and [35S]GTPγS was bought from PerkinElmer Life
Science (Boston, USA) or Hartmann Analytic (Braunschweig,
Germany). [3H]UR-DE257 was synthesized in our laboratories. All
stock solutions were dissolved in millipore water or in a mixture of
Millipore water/DMSO. In all assays, the final DMSO content
included less than 0.5%.
[1] A. Windaus, W. Vogt, Ber. Dtsch. Chem. Ges. 1907, 40, 3685–3691.
[2] A. S. F. Ash, H. O. Schild, Br. J. Pharmacol. Chemother. 1966, 27, 427–439.
[3] J. W. Black, W. A. M. Duncan, C. J. Durant, C. R. Ganellin, E. M. Parsons,
Nature 1972, 236, 385–390.
[4] J.-M. Arrang, M. Garbarg, J.-C. Schwartz, Nature 1983, 302, 832–837.
[5] T. Oda, N. Morikawa, Y. Saito, Y. Masuho, S. Matsumoto, J. Biol. Chem.
2000, 275, 36781–36786.
[6] M. C. Lagerström, H. B. Schiöth, Nat. Rev. Drug Discovery 2008, 7, 339–
357.
[7] S. J. Hill, Pharmacol. Rev. 1990, 42, 45–83.
[8] R. Leurs, M. J. Smit, H. Timmerman, Pharmacol. Ther. 1995, 66, 413–463.
[9] S. J. Hill, C. R. Ganellin, H. Timmerman, J. C. Schwartz, N. P. Shankley,
J. M. Young, W. Schunack, R. Levi, H. L. Haas, Pharmacol. Rev. 1997, 49,
253–278.
Methods
All the pharmacological methods used in this study (Membrane
Preparation of Sf9 Cells, Radioligand Binding Assay, [35S]GTPγS
Binding Assay, Histamine H1 Receptor Assay on Isolated Guinea Pig
Ileum, Histamine H2 Receptor Assay on the Isolated Guinea Pig
Right Atrium) were already described in the literature.[34]
[10] A. H. Soll, A. Wollin, Am. J. Physiol. Gastrointest. Liver Physiol. 1979, 237,
G444–G450.
[11] C. L. Johnson, H. Weinstein, J. P. Green, Mol. Pharmacol. 1979, 16, 417–
428.
[12] J. Fischer, C. R. Ganellin, A. Ganesan, J. Proudfoot, Analogue-Based Drug
Discovery, Wiley-VCH, 2010.
[13] H. Van Der Goot, H. Timmerman, Eur. J. Med. Chem. 2000, 35, 5–20.
[14] A. Rouleau, X. Ligneau, J. Tardivel-Lacombe, S. Morisset, F. Gbahou, J.-C.
Schwartz, J.-M. Arrang, Br. J. Pharmacol. 2002, 135, 383–392.
[15] R. Leurs, P. L. Chazot, F. C. Shenton, H. D. Lim, I. J. De Esch, Br. J.
Pharmacol. 2009, 157, 14–23.
[16] K. L. Morse, J. Behan, T. M. Laz, R. E. West, S. A. Greenfeder, J. C. Anthes,
S. Umland, Y. Wan, R. W. Hipkin, W. Gonsiorek, J. Pharmacol. Exp. Ther.
2001, 296, 1058–1066.
[17] T. W. Lovenberg, B. L. Roland, S. J. Wilson, X. Jiang, J. Pyati, A. Huvar,
M. R. Jackson, M. G. Erlander, Mol. Pharmacol. 1999, 55, 1101–1107.
[18] Y. Zhu, D. Michalovich, H.-L. Wu, K. B. Tan, G. M. Dytko, I. J. Mannan, R.
Boyce, J. Alston, L. A. Tierney, X. Li, Mol. Pharmacol. 2001, 59, 434–441.
[19] C. Liu, X.-J. Ma, X. Jiang, S. J. Wilson, C. L. Hofstra, J. Blevitt, J. Pyati, X. Li,
W. Chai, N. Carruthers, Mol. Pharmacol. 2001, 59, 420–426.
Computational Methods
Homology modelling of both hH4R and hH3R, based on the crystal
structure of the inactive state hH1R (PDB ID: 3RZE)[67] is described by
Pockes et al.[40] Protein and ligand preparation as well as the
assignment of protonation states (Schrödinger LLC, Portland, OR
USA) were essentially performed as described in Pegoli et al.[68]
Disulfide bonds were maintained between C873.25 and C164ECL2
(hH4R) and C1073.25 and C188ECL2 (hH3R). While the isothiourea
moiety of 143 was protonated, the imidazole ring was considered
in both deprotonated (τ-H or π-H) and protonated (τ-H and π-H)
ChemistryOpen 2019, 8, 285–297
www.chemistryopen.org
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© 2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA

Full Papers
[20] C. L. Hofstra, P. J. Desai, R. L. Thurmond, W.-P. Fung-Leung, J. Pharmacol.
Exp. Ther. 2003, 305, 1212–1221.
[21] M. J. Gemkow, A. J. Davenport, S. Harich, B. A. Ellenbroek, A. Cesura, D.
Hallett, Drug Discovery Today 2009, 14, 509–515.
[22] B. Sadek, N. Khan, F. H. Darras, S. Pockes, M. Decker, Physiol. Behav.
2016, 165, 383–391.
[23] B. Sadek, A. Saad, A. Sadeq, F. Jalal, H. Stark, Behav. Brain Res. 2016, 312,
[48] O. Mitsunobu, M. Yamada, T. Mukaiyama, Bull. Chem. Soc. Jpn. 1967, 40,
935–939.
[49] C. Dardonville, C. Fernandez-Fernandez, S.-L. Gibbons, G. J. Ryan, N.
Jagerovic, A. M. Gabilondo, J. J. Meana, L. F. Callado, Bioorg. Med. Chem.
2006, 14, 6570–6580.
[50] S. M. Hickey, T. D. Ashton, S. K. Khosa, F. M. Pfeffer, Synlett 2012, 23,
1779–1782.
[51] N. Pluym, A. Brennauer, M. Keller, R. Ziemek, N. Pop, G. Bernhardt, A.
Buschauer, ChemMedChem 2011, 6, 1727–1738.
1
2
3
4
5
6
7
8
9
415–430.
[24] M. Rapanelli, Prog. Neuro-Psychopharmacol. Biol. Psychiatry 2017, 73, 36–
40.
[25] M. Rapanelli, L. Frick, V. Pogorelov, H. Ohtsu, H. Bito, C. Pittenger, Transl.
Psychiatry 2017, 7, e1013–e1013.
[26] P. J. Dunford, N. O’Donnell, J. P. Riley, K. N. Williams, L. Karlsson, R. L.
Thurmond, J. Immunol. 2006, 176, 7062–7070.
[52] A. Buschauer, J. Med. Chem. 1989, 32, 1963–1970.
[53] J. W. Black, G. J. Durant, J. C. Emmett, C. R. Ganellin, Thioureas, 1973,
GB1305548A.
[54] J. W. Black, G. J. Durant, J. C. Emmett, C. R. Ganellin, Isothioureas, 1973,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
GB1305547A.
[27] R. L. Thurmond, E. W. Gelfand, P. J. Dunford, Nat. Rev. Drug Discovery
2008, 7, 41–53.
[28] R. Leurs, H. F. Vischer, M. Wijtmans, I. J. de Esch, Trends Pharmacol. Sci.
2011, 32, 250–257.
[29] J. A. Jablonowski, C. A. Grice, W. Chai, C. A. Dvorak, J. D. Venable, A. K.
Kwok, K. S. Ly, J. Wei, S. M. Baker, P. J. Desai, J. Med. Chem. 2003, 46,
3957–3960.
[30] E. Zampeli, E. Tiligada, Br. J. Pharmacol. 2009, 157, 24–33.
[31] A. M. Staub, D. Bovet, Compt. Rend. Soc. de biol 1937, 125, 818.
[32] B. N. Halpern, Arch. Int. Pharmacodyn. Ther. 1942, 68, 339–343.
[33] P. B. Marshall, Br. J. Pharmacol. 1955, 10, 270–278.
[34] G. J. Durant, W. A. M. Duncan, C. R. Ganellin, M. E. Parsons, R. C.
Blakemore, A. C. Rasmussen, Nature 1978, 276, 403–405.
[35] G. J. Durant, C. R. Ganellin, D. W. Hills, P. D. Miles, M. E. Parsons, E. S.
Pepper, G. R. White, J. Med. Chem. 1985, 28, 1414–1422.
[36] A. Buschauer, J. Med. Chem. 1989, 32, 1963–1930.
[37] M. E. Parsons, R. C. Blakemore, G. J. Durant, C. R. Ganellin, A. C.
Rasmussen, Inflamm. Res. 1975, 5, 464–464.
[38] H. D. Lim, R. M. van Rijn, P. Ling, R. A. Bakker, R. L. Thurmond, R. Leurs, J.
Pharmacol. Exp. Ther. 2005, 314, 1310–1321.
[39] J. C. Eriks, H. van der Goot, G. J. Sterk, H. Timmerman, J. Med. Chem.
1992, 35, 3239–3246.
[40] S. Pockes, D. Wifling, M. Keller, A. Buschauer, S. Elz, ACS Omega 2018, 3,
2865–2882.
[41] A. Kraus, P. Ghorai, T. Birnkammer, D. Schnell, S. Elz, R. Seifert, S. Dove,
G. Bernhardt, A. Buschauer, ChemMedChem 2009, 4, 232–240.
[42] T. Birnkammer, A. Spickenreither, I. Brunskole, M. Lopuch, N. Kagerme-
ier, G. Bernhardt, S. Dove, R. Seifert, S. Elz, A. Buschauer, J. Med. Chem.
2012, 55, 1147–1160.
[43] N. Kagermeier, K. Werner, M. Keller, P. Baumeister, G. Bernhardt, R.
Seifert, A. Buschauer, Bioorg. Med. Chem. 2015, 23, 3957–3969.
[44] R. Yang, J. A. Hey, R. Aslanian, C. A. Rizzo, Pharmacology 2002, 66, 128–
135.
[55] D. B. Frennesson, D. R. Langley, M. G. Saulnier, D. M. Vyas, Methods for
Preparing Macrocycles and Macrocycle Stabilized Peptides, 2012,
WO2012051405A1.
[56] W. Carling, K. Moore, Imidazolone and Oxazolone Derivatives as
Dopamine Antagonists, European Patent Office, Rijswijk, 1995, WO
95/07904.
[57] K. S. Kim, L. Qian, Int. J. Rapid Publ. Prelim. 1993, 34, 7677–7680.
[58] R. W. Brimblecombe, W. A. M. Duncan, G. J. Durant, J. C. Emmett, C. R.
Ganellin, M. E. Parsons, J. Int. Med. Res. 1975, 3, 86–92.
[59] R. W. Brimblecombe, W. A. Duncan, G. J. Durant, C. R. Ganellin, M. E.
Parsons, J. W. Black, Br. J. Pharmacol. 1975, 53, 435–436.
[60] L. B. Hough, Mol. Pharmacol. 2001, 59, 415–419.
[61] C. Robin Ganellin, J. C. Schwartz, H. Stark, Successful Drug Discovery
2018, 3, 359–381.
[62] J. B. Baell, G. A. Holloway, J. Med. Chem. 2010, 53, 2719–2740.
[63] C. Aldrich, C. Bertozzi, G. I. Georg, L. Kiessling, C. Lindsley, D. Liotta, K. M.
Merz, A. Schepartz, S. Wang, ACS Cent. Sci. 2017, 3, 143–147.
[64] S. M. Devine, M. D. Mulcair, C. O. Debono, E. W. W. Leung, J. W. M.
Nissink, S. S. Lim, I. R. Chandrashekaran, M. Vazirani, B. Mohanty, J. S.
Simpson, J. B. Baell, P. J. Scammells, R. S. Norton, M. J. Scanlon, J. Med.
Chem. 2015, 58, 1205–1214.
[65] M. Nettekoven, W. Guba, W. Neidhart, P. Mattei, P. Pflieger, O. Roche, S.
Taylor, Bioorg. Med. Chem. Lett. 2005, 15, 3446–3449.
[66] V. S. Aulakh, A. Casarez, X. Lin, M. Lindvall, G. McEnroe, H. E. Moser, F.
Reck, M. Tjandra, R. L. Simmons, A. Yifru, Preparation of Monobactam
Organic Compounds for the Treatment of Bacterial Infections, 2015, US
20150266867A1.
[67] T. Shimamura, M. Shiroishi, S. Weyand, H. Tsujimoto, G. Winter, V.
Katritch, R. Abagyan, V. Cherezov, W. Liu, G. W. Han, Nature, 2011, 475,
65–70.
[68] A. Pegoli, X. She, D. Wifling, H. Hübner, G. Bernhardt, P. Gmeiner, M.
Keller, J. Med. Chem. 2017, 60, 3314–3334.
[45] P. Igel, R. Geyer, A. Strasser, S. Dove, R. Seifert, A. Buschauer, J. Med.
Chem. 2009, 52, 6297–6313.
[46] P. Igel, E. Schneider, D. Schnell, S. Elz, R. Seifert, A. Buschauer, J. Med.
Chem. 2009, 52, 2623–2627.
[47] P. Ghorai, A. Kraus, M. Keller, C. Götte, P. Igel, E. Schneider, D. Schnell, G.
Bernhardt, S. Dove, M. Zabel, J. Med. Chem. 2008, 51, 7193–7204.
Manuscript received: January 10, 2019
Revised manuscript received: February 8, 2019
ChemistryOpen 2019, 8, 285–297
www.chemistryopen.org
297
© 2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA