Discovery of small molecules with vasodilating characteristics and adjustable hydrolytic behavior

Article abstract of DOI:10.1016/j.bmc.2015.05.049

In this contribution the development of a new class of vasodilating compounds obtained by lead structure optimization is described. Three groups of compounds were synthesized and tested for their activity on various smooth muscle preparations of the Guinea pig. Beside the lead compound 3a, the most interesting derivative was 1H-imidazole-1-carbothioic acid O-cyclohexyl ester hydrochloride (5b) with a good selective vasodilating potential on aorta and pulmonary artery rings (EC₅₀ 14 μM and 24 μM, respectively). Due to the properties of small molecules the hydrolysis behavior of the compounds can be easily adapted hence opening a new route in terms of duration of the agent's effect. With the aid of structure-activity relationship studies, structural motifs influencing the biological activity on isolated smooth muscle cell preparations of the synthesized compounds were proposed. The presented compounds offer good tools in identifying promising molecules as emergency therapy in myocardial infarction.

Full text of DOI:10.1016/j.bmc.2015.05.049

Bioorganic & Medicinal Chemistry 23 (2015) 4710–4718
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery of small molecules with vasodilating characteristics
and adjustable hydrolytic behavior
a
a
b
a,
⇑
Gerda Brunhofer-Bolzer , Mario Gabriel , Christian R. Studenik , Thomas Erker
a
Department of Pharmaceutical Chemistry, University of Vienna, Althanstrasse 14, 1090 Vienna, Austria
Department of Pharmacology and Toxicology, University of Vienna, Althanstrasse 14, 1090 Vienna, Austria
b
a r t i c l e i n f o
a b s t r a c t
Article history:
In this contribution the development of a new class of vasodilating compounds obtained by lead structure
optimization is described. Three groups of compounds were synthesized and tested for their activity on
various smooth muscle preparations of the guinea pig. Beside the lead compound 3a, the most interesting
derivative was 1H-imidazole-1-carbothioic acid O-cyclohexyl ester hydrochloride (5b) with a good selec-
Received 13 April 2015
Accepted 28 May 2015
Available online 5 June 2015
tive vasodilating potential on aorta and pulmonary artery rings (EC50 14 lM and 24 lM, respectively).
Keywords:
Due to the properties of small molecules the hydrolysis behavior of the compounds can be easily adapted
hence opening a new route in terms of duration of the agent’s effect. With the aid of structure–activity
relationship studies, structural motifs influencing the biological activity on isolated smooth muscle cell
preparations of the synthesized compounds were proposed. The presented compounds offer good tools
in identifying promising molecules as emergency therapy in myocardial infarction.
Ó 2015 Elsevier Ltd. All rights reserved.
Dithiocarbamide derivatives
O-Thiocarbamate/carbamide derivatives
Thiocarbonic acid
Steric effect
Vasodilation
1
. Introduction
xenobiotics via degrading themselves through hydrolysis into
more polar compounds that can be excreted more easily. This
In developed countries, the leading cause of death and source of
disability are myocardial infarction and arterial thrombosis
stroke). The main determinant of clinical outcome is well
seems contradictory to the general aim of medicinal chemists
which is to prolong the duration of a compound’s activity within
the body by making it more metabolically stable. We present
6
(
established as the fast and complete restoration of blood flow to
the blocked artery1 either by thrombolysis or by acute percuta-
neous coronary interventions and reopening of the occluded vessel
compounds offering the possibility to adjust their hydrolysis prop-
erties but still keep the desired biological activity. Thinking ahead,
this is—to the best of our knowledge—a new concept, which may
open the way to the application of a specific compound/medication
depending on how long the patient needs the agent’s action.
Previous studies in which we showed that dithio- and thioben-
zanilides act as potent spasmolytic agents compared to their
2
using a stent. As a consequence anti-platelet drugs such as aspirin,
clopidogrel and its recently developed follow-up drugs are used to
preserve the open vessel.3 Additionally, vasoactive drugs like
nitrates are used to improve coronary blood flow and the hunt
for the development of new nitric oxide (NO) releasing compounds
7
,8
benzanilide derivatives and that the sulfur present in their scaf-
4
9
still continues. Whereas there have been a couple of new anti-
fold was responsible for the increased activity led us to investigate
platelet drugs promoted on the market, no new vasodilators have
the biological activity of the small molecule 3a. The base was com-
mercially available and its short synthesis (conversion to the
hydrochloride salt) as well as biological profile on smooth muscle
cell preparations made it an attractive candidate for lead structure
optimization. Conversion of the compound into its hydrochloride
salt resulted in a change of the aggregation state from fluid to solid
thus providing a better accessibility to the biological investigation
(scaling, solubility). In order to obtain compounds showing the
ability to widen selectively blood vessels we modified the structure
around the thiocarbonyl group, which was synthetically achieved
by ‘growing’ of compound 3a. Thus we gained three different
groups of compounds with partly high vasodilating activity on
aorta and pulmonary artery rings of the guinea pig as assessed
been clinically introduced.5
In this contribution, a unique collection of small sulfur-
containing compounds was synthesized and tested for vasodilating
activity, with their characteristics designed to act as emergency
drugs in the treatment of ischemia. The main idea is that the pre-
sented compounds are supposed to act immediately as vasoactive
compounds, which can for instance be applied in the status of
acute ischemia. In this case, after controlling the initial phase the
compounds should facilitate the body’s duty to get rid of the
⇑
Corresponding author. Tel.: +43 (0)1 4277 55003; fax: +43 (0)1 4277 855003.
E-mail address: thomas.erker@univie.ac.at (T. Erker).
http://dx.doi.org/10.1016/j.bmc.2015.05.049
968-0896/Ó 2015 Elsevier Ltd. All rights reserved.
0

G. Brunhofer-Bolzer et al. / Bioorg. Med. Chem. 23 (2015) 4710–4718
4711
S
S
R'''
H
N
R'
R
i or ii
R'
N
R
+
CS
2
+
X
R'''
HCl
N
N
R''
2a-f
R'' 3a-i
1
a-c
3
3
3
3
3
3
3
3
3
a
R, R’, R’’, R’’’: H
R, R’, R’’: H; R’’’: C H
6 5
R, R’, R’’: H; R’’’: (4-OCH
R, R’, R’’: H; R’’’: (3,4,5-(OCH ) )C H
R, R’, R’’: H; R’’’: CH
R, R’, R’’: H; R’’’: CH
2
2
2
2
2
2
a
X: I; R’’’: H
X: Cl; R’’’: C H
6 5
X: Cl; R’’’: (4-OCH
X: Cl; R’’’: (3,4,5-(OCH ) )C H
X: Br; R’’’: C
X: Br; R’’’: C
1
1
a
b
R,R’,R’’: H
R: CH
R’-R’’: CH=CH-CH=CH
R: CH ; R’,R’’: H
b
c
d
e
f
g
h
i
b
c
d
e
f
3
;
3 6 4
)C H
3 6 4
)C H
3 3 6 2
3 3 6 2
1
c
3
H
H
4
4
2 5
COOC H
2
2
2 5
COOC H
2
OCOCH
3
2
OCOCH
3
R: CH
R: CH
R: CH
3
3
3
; R’-R’’: CH=CH-CH=CH; R’’’=H
; R’, R’’, R’’’: H
6 5
; R’,R’’:H; R’’’: C H
3 4
Scheme 1. Conditions: (i) NaH, THF, 0 °C; diethylether, 1 M HCl; (ii) K PO , acetone, room temp; diethylether, 1 M HCl.
by isometric tension recording. Hydrolysis studies, studies
concerning their mechanisms of action as well as information of
the chemical space covered by the compounds using ChemGPS-
2
of CS was added at room temperature followed by the corre-
sponding alkyl halide. In both cases, the compounds were further
converted to their hydrochloric salt by using 1 M hydrochloric acid
in diethylether to improve water solubility, which represented an
advantage in the following biological assays in terms of repro-
ducibility. The compound 3d was not able to be converted into
the salt form and therefore was tested as its free base. The O-thio-
carbamates 5a–f as well as the thiocarbamides 5g and 5h were
1
0–12
NP
are presented.
2
2
. Results and discussion
.1. Chemistry
0
synthesized by reaction of 1,1 -thiocarbonyldiimidazole (TCDI)
and the corresponding alcohol and amine component, respectively,
in dry THF. The reaction worked equally well for both the thiocar-
bamates and -carbamides with yields above 60%. For the synthesis
of the symmetric diesters 5i and 5j the same synthetic procedure
was applied but with 2 equiv of the corresponding alcohol compo-
nent. The oxygen analogs of 5a and 5b, 6a and 6b, were obtained
Applying the approach of lead structure optimization to obtain
selectively vasodilating small molecules, we forwarded our studies
and designed three different groups of compounds: dithiocar-
bamide derivatives, O-thiocarbamate/carbamide derivatives as
well as diesters of thiocarbonic acid. The synthetic routes used to
prepare the dithiocarbamide derivatives are summarized in
Scheme 1. In general, two routes were available to obtain the
desired compounds. The first protocol includes the conversion of
imidazole or an imidazole derivative to the corresponding imida-
zolide under the influence of sodium hydride in tetrahydrofuran
0
by the same procedure but with 1,1 -carbonyldiimidazole as start-
ing material (Scheme 2). The synthesis of the compounds 8a–h
required replacing the imidazolide moiety against an alkyloxy
and -amino substituent, respectively, thus obtaining either sym-
metrical or asymmetrical diester/-amides of the thiocarbonic acid
(
THF) under argon atmosphere at 0 °C followed by adding carbonyl
(
Scheme 3). Elemental analysis was used for characterization as
well as purity confirmation of the compounds. For the compounds
a, 6a, 8c, 8d and 8g the elemental analysis provided unconvincing
disulfide (CS ). The end products were obtained by addition of the
2
13
appropriate alkyl halide at room temperature. This procedure
was applied for the preparation of compounds 3a–d and 3h. The
preparation of compounds 3e–g and 3i required the use of potas-
sium phosphate in acetone as described by Wang et al.14 An excess
5
data and therefore the bases of these compounds were used and
characterized by HRMS.
2
SAR
.2. Activities on smooth and heart muscle preparations and
A
S
HO
R
HCl
N
4
a-f
N
R
O
The vasodilating potency of the compounds was assessed
on isolated aorta and pulmonary artery rings of the guinea pig
precontracted by 90 mM potassium chloride. possible
5
a-f
H
A
N
S
S
R
R'
R
HCl
N
spasmolytic activity was studied on terminal ileum preparations
4
g-h
i
N
R'
N
N
N
R
N
N
5
g-h
HCl
HO R'
a-b
S
7
R'
R
S
S
O
O
8
a-f
2
HO
i-j
R
R
i
4
N
S
O
O
R
O
5
i-j
HCl
R
N
5
b-e
N
O
HN
B
N
8g-h
O
HCl
O
HO
R
N
4
a-b
N
N
N
R
i
O
2 3 3 2
8a R: hexyl; R’: (CH ) N(CH )
8b R: CH
R: CH
N
7
7
a
b
R’: (CH
R’: (CH
2
)
)
3
2
N(CH
N(CH
3
3
)
)
2
2
N
N
6a-b
2
CH
CH
3
; R’: (CH
; R’: (CH
2
)
)
2
N(CH
N(CH
3
3
)
)
2
2
2
8
c
2
3
2
3
a
b
c
d
e
R: CH(CH
3
)
2
f
R: cyclopentyl
R-R’: (CH
2 3 2
R, R’: CH CH(CH )
8d R: (CH
8e R: (CH
8f R: (CH
2
2
2
)
)
)
2
2
3
CH
CH
CH
3
3
3
; R’: (CH
; R’: (CH
; R’: (CH
2
2
2
)
)
)
2
3
3
N(CH
N(CH
N(CH
3
3
3
)
)
)
2
2
2
R: cyclohexyl
R: CH CH
g
h
i
2 5
)
2
3
R: (CH
R: (CH
2
)
)
2
CH
CH
3
R: (CH
R: (CH
2
)
)
2
2
N(CH
N(CH
3
3
)
)
2
3
; HCl
Cl
8g R: CH
2
CH
CH
3
+
-
2
3
3
j
2
8h R: (CH
2
)
2
3
Scheme 2. Conditions: (i) THF, reflux; diethylether, 1 M HCl.
Scheme 3. Conditions: (i) THF, reflux; diethylether, 1 M HCl.

4
712
G. Brunhofer-Bolzer et al. / Bioorg. Med. Chem. 23 (2015) 4710–4718
pre-contracted by 60 mM potassium chloride and inotropic and
chronotropic activity, respectively, was investigated on electrically
stimulated papillary muscles and isolated right atria. Except for
compound 5g and the two oxygen-analogs 6a and 6b, all the
dithiocarbamide as well as O-thiocarbamate/carbamide derivatives
caused relaxation of the pre-contracted vessels in a concentration-
dependent manner. Diesters of the thiocarbonic acid proved to be
unable to dilate KCl-pre-contracted vessel strips (Table 1). Within
the most active group of compounds, the dithiocarbamides, which
also contains the lead compound 3a, every congener showed activ-
ity on vascular smooth muscle preparations. Structural extension
of the lead scaffold through insertion of an unsubstituted phenyl
ring (3b) resulted in a potency decrease on aorta, pulmonary artery
as well as terminal ileum preparations. Concerning its inotropic
activity the compound showed about two-fold increased potential
compared to that of 3a. Considering the activity of the compounds
Elongation of the ester motif by one or two methylene groups,
respectively, resulted in compounds 5d and 5e. Compound 5d dis-
played a slight enhancement of the vasodilating activity on aortic
strips whereas 5e showed a two-fold increase in the vasodilating
potency compared to 5a and activity was also present on pul-
monary artery rings. The elongation of the alkyl chain did not show
any influence on the antispasmodic properties of the compounds.
The cyclopentyl ester 5f showed vasodilating activity similar to
that of 5a but was able to slightly improve the spasmodic potential.
Conversely, the cyclohexyl ester 5b lost the antispasmodic proper-
ties up to 100
vessel preparations of the aorta and the pulmonary artery with
EC50 values of 14 and 24 M. Because of these data it could be sug-
lM and therefore showed selective activity on the
l
gested that the number of carbons at this area of the molecule
plays a significant role in the selective vasodilating action of O-
thiocarbamates. Moreover, evaluation of the oxygen analogs 6a
2
3
c and 3d, the vasodilating as well as spasmolytic activity corre-
and 6b revealed that the sp sulfur atom (C@S) is crucial for biolog-
lates with the number of methoxy groups on the phenyl ring.
The trimethoxy benzyl derivative 3d elucidated improved vasodi-
lating properties on the one hand but on the other hand the termi-
nal ileum as well as papillary muscle preparations were also
ical activity (Table 1). Concerning the spasmolytic characteristic
the activity enhancement of this type of replacement was also pro-
ven for thio- and dithiobenzanilide derivatives previously. The sul-
fur compounds showed higher biological activity than their
corresponding amide congeners. The thiocarbamide 5g bearing
a piperidine motif did not show any biological activity up to the
8
,9
affected by this compound (15 and 27
Conversely, the para monomethoxy compound 3c had less impact
on the vasculature than 3a (13 vs 7.8 M on aorta preparations and
4 vs 6.9 M on pulmonary artery rings) but showed selectivity
l
M, respectively).
l
highest concentration tested (100 lM). Differently behaved was
the thiocarbamide derivative 5h with two iso-butyl units at the
2
l
towards vascular tissue up to more than 100
lM giving this com-
carbamide nitrogen. On the one hand, this compound exhibited
pound a highly interesting biological profile.
vasodilating activity on aorta (14
(26 M) rings and spasmolytic potential on isolated terminal ileum
preparations (17 M) and on the other hand the compound acted
as negative chronotropic (48 M) agent with a distinct negative
inotropic behavior (7 M).
Within the diesters of the thiocarbonic acid, the symmetrical
compound 5i as well as the di-ion 5j did not show any biological
activity on the tested isolated tissue preparations up to a concen-
lM) as well as pulmonary artery
Replacing the benzyl group by a propionic acid ethyl ester unit
l
(3e) slightly reduced activity on the vascular rings and terminal
l
ileum preparations compared to 3a. Compound 3f comprising an
ethylacetate motif showed a decreased activity on pulmonary
artery rings on the one hand, but on the other hand the biological
activity of the lead compound on aortic rings as well as papillary
l
l
muscles was retained (5.8 and 77 lM, respectively). In case of com-
pound 3h, insertion of a 2-methyl unit on the imidazole core area
of 3a provided similar biological activity on aorta, pulmonary
artery rings and terminal ileum preparations. But, compared to
the lead 3a, 3h did not affect the papillary muscle up to a concen-
tration of 100 lM. These data suggest—compared to the com-
pounds 8a and 8d–f—that on one side of the molecule electron-
rich substituents are contraindicated and that lipophilic aryl moi-
eties are preferred. Considering compounds 8a–f, the inotropic
activity of the diesters on isolated papillary muscles is increasing
tration of 100 lM. Conversely, insertion of a 2-methyl group
together with a phenyl motif in the imidazole unit led to decreased
activity on the vasculature and ileum, abolished inotropic potency
and suddenly to a moderately negative chronotropic activity
in the order ethyl (>100
lM) < propyl (37 lM) < cyclohexyl
(5.9 M) and butyl (4.3 M). The cyclohexyl derivative 8a was
l
l
the only representative of this set of compounds demonstrating
activity also on isolated right atria. These data suggest a fairly nar-
row chemical space for this class of compounds targeting the
chronotropic activity. Regarding the spasmolytic activity of 8a on
terminal ileum preparations it had almost similar activity as com-
pound 8f. Replacement of one ester unit by a piperidine to obtain a
thiocarbamate scaffold plus retention of the ethyl and propyl ester
function, respectively, revealed compounds 8g and 8h with biolog-
(
56 lM). This phenomenon was also observed in the case of com-
pound 3i comprising a 2-methyl group on the imidazole ring as
well as a benzyl motif linked to the dithiocarbamide unit with a
negative chronotropic potential of 15.5 lM. Furthermore, this
compound shows the highest vasodilating potential on aortic rings
within its group and slightly decreased activity was observed on
pulmonary artery and terminal ileum tissue.
In a next step we examined the effect of O-thiocarbamate/car-
bamide derivatives comprising an imidazole on the N-side of the
carbamic acid as well as a variety of aliphatic alcohols as esters
and amines to finally obtain the carbamide derivatives.
Interestingly, we observed a steric effect correlating with the
vasodilating and spasmolytic activity of the O-thiocarbamate com-
pounds. The vasodilating potential of the compounds on aortic
rings increased with the number of carbon atoms in the ester motif
whereas the antispasmodic effect of the compounds seemed to be
in inverse ratio to the size of the ester unit. Compounds 5c and 5a
representing an ethyl and isopropyl carbamic acid ester showed
ical activity higher than 100 lM. Hence, the diesters of the thiocar-
bonic acid can be regarded as the biologically preferred scaffold
concerning their potential on heart muscle preparations through-
out all groups of compounds presented herein.
2.3. Analysis of the chemical space
To substantiate the SAR studies and to get an overview of the
chemical space occupied by the compounds, they were mapped
1
0–12
using ChemGPS-NP.
Using this web-based tool, it is possible
to describe the compounds by means of structure-derived phy-
sico-chemical properties (e.g., size, shape, aromaticity, flexibility,
lipophilicity and polarity). Based on eight principle components
(PC) describing relevant descriptors compounds are able to be
mapped into the chemical space using interpolation according to
moderate activity on aorta preparations (53
respectively). 5c elucidated a high spasmolytic potential (4.8
whereas 5a showed only modest antispasmodic activity (21
l
M and 46
l
l
M,
M)
M).
l
Both compounds did not influence heart muscle preparations up
to 100 M but interestingly, in the case of 5c a vasodilating effect
on pulmonary artery preparations was observed (19 M).
1
5
l
PC analysis score prediction. In this study, the first four dimen-
sions accounting for 77% of data variance were used for
l

G. Brunhofer-Bolzer et al. / Bioorg. Med. Chem. 23 (2015) 4710–4718
4713
Table 1
EC50 values in
lM of the compounds on different smooth and heart muscle preparations of the guinea pig as well as information about the aromatic nature of the compounds
Compd
C@S present
Aromaticity present
Aorta
Pulmonary artery
Terminal ileum
Papillary muscle
Right atrium
3
3
3
3
3
3
3
3
3
5
5
5
5
5
5
5
5
5
5
6
6
8
8
8
8
8
8
8
8
a
b
c
d
e
f
g
h
i
a
b
c
d
e
f
g
h
i
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
7.8
20
13
3.0
16
5.8
23
6.3
3.4
46
14
53
30
13
36
>100
14
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
6.9
63
24
7.9
20
24
25
11
13
>100
24
19
100
23
>100
>100
26
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
5.7
40
>100
15
32
>100
26
9.5
16
21
>100
4.8
21
21
16
78
30
>100
27
>100
77
>100
>100
>100
>100
>100
>100
>100
>100
16
>100
>100
>100
>100
>100
>100
>100
48
>100
>100
>100
>100
35
>100
>100
>100
>100
>100
>100
>100
56
>100
>100
>100
>100
>100
>100
>100
>100
>100
6.5
>100
>100
>100
>100
5.9
>100
17
>100
>100
>100
>100
16
>100
>100
>100
>100
17
j
No
a
b
a
b
c
d
e
f
Yes
Yes
No
No
No
No
No
No
No
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
>100
>100
58
37
4.3
g
h
>100
>100
>100
>100
No
interpretation.12 The synthesized chemical collection contains 29
compounds in total including dithiocarbamide derivatives, O-thio-
carbamate/carbamide derivatives as well as diesters of the thiocar-
bonic acid. For the analysis of their chemical space, it was
necessary to use the basic form of the compounds as well as the
dicationic form of compound 5j since there is not more than one
structure in one SMILES allowed. As can be seen from Figure 1,
the set of compounds covers completely the range of low size
molecules as all of the compounds (spheres) are located at the neg-
ative side of the PC1-axis. PC2 describes the aromatic status of the
compounds and based on this dimension the data set presents
three volumes of chemical space and therefore allows to classify
the compounds into derivatives with high, middle and low aro-
maticity/conjugational properties. The classification correlates
with the found bioactivity of the new compounds and indicates
aromaticity as key requirement, especially concerning the biologi-
cal activity on vessel preparations (Table 1). Polarity issues of the
presented compounds are described by PC3. The compound library
shows a fairly similar amount of polar molecules, which are
located in the negative direction of PC3, and lipophilic compounds,
found in its positive direction. Mostly all of the compounds have a
moderate degree of structural flexibility as indicated by their
PC3
PC4
PC2
PC2
PC1
PC1
Figure 1. Three-dimensional representations of the chemical space occupied by the presented compounds using ChemGPS-NP.10,12 The first four dimensions of ChemGPS-NP
were used. The data are interpreted as follows: molecule size increases in the positive direction of PC1, aromaticity/conjugation-related properties increase in the positive
direction of PC2, compounds are gradually more lipophilic in the positive direction of PC3 and they show higher flexibility in the positive direction of PC4. Color code: gray:
dithiocarbamide derivatives, green: O-thiocarbamate/carbamide derivatives, red: diesters of the thiocarbonic acid.

4
714
G. Brunhofer-Bolzer et al. / Bioorg. Med. Chem. 23 (2015) 4710–4718
location on the positive site of PC4. The clusters of compounds
obtained by adding PC4 into the analysis did not show any corre-
lation with the biological activity of the compounds.
2
.4. Hydrolysis studies
Beside the evaluation of the physico-chemical properties we
went on to validate the therapeutic potential of the compounds as
emergency treatment options in ischemia and studied the hydroly-
tic behavior of three representatives in an aqueous solution.
Compounds 3a, 5b and 5c were chosen because the results were
evaluated by the compound: degradation product ratio, which
1
was clearly obtained by the H NMR spectra. The three representa-
2
tives were dissolved in D O at room temperature and measured by
1
using H NMR spectroscopy over a period of 24 hours. Time points
were measured in triplicates after 1, 3, 5, 7 and 24 h. In case of 3a,
a dithiocarbamide derivative, we did not detect any degradation
products in the reaction solution and thus we consider this com-
pound as stable over a period of 24 h. Compounds 5b and 5c, both
O-thiocarbamates, showed time-dependent hydrolysis properties
Figure 3. Activity of the compounds 5b and 5c on isolated aortic rings. The
compounds were dissolved in distilled water 24 h before the experiment and kept
at room temperature. Tested concentration was 15 and 55 lM for compound 5b
(Fig. 2) and presented a degradation behavior in releasing their alco-
and 5c, respectively; n = 3. Significance level: *P <0.05.
hol unit (cyclohexanol and ethanol). After 24 h, the compound:
degradation product ratio for compound 5b was 0.09:1 and for
derivative 5c 0.35:1 representing 8% and 26% left of the original
compound, respectively. Their half-lives (t1/2) were determined
from the equation obtained by fitting the data (Fig. S1) and were
To conclude, the latter experiments serve as a first proof of con-
cept that we developed compounds with adjustable hydrolysis
behavior. This may be of special interest in the acute treatment
of myocardial infarction as the administered compound concentra-
tion as well as the time of action open a way to be controlled more
easily.
1
2.3 and 6.5 h for 5b and 5c, respectively (Fig. 2). Another chemi-
cally conceivable degradation product might be the small molecule
COS. However, in this experimental setting, it was not possible to
prove the release of this gaseous compound.
2.5. Mode of action studies
In a further set of experiments we wanted to know whether the
compounds themselves or their degradation product, the alcohol
component, was responsible for biological activity. Therefore, we
dissolved compound 5b and 5c, respectively, in distilled water
and kept the sealed solution at room temperature. After 24 h, we
tested the dissolved compound in a concentration according to
To get a deeper insight into a possible mode of action, two com-
pounds, 3a as a representative of the dithiocarbamide class, and 5b
representing the O-thiocarbamates, were investigated. Since ATP-
dependent potassium channels (KATP-channels) are regarded as
possible key players in smooth muscle relaxation mediated by
1
6,17
its EC50 value (15
l
M for 5b and 55
l
M for 5c, respectively) on iso-
hydrogen sulfide
protective mechanism
and are suggested as an endogenous cardio-
1
8,19
lated aortic rings in the same experimental setup (pre-contracted
by 90 mM KCl) as described for isometric contraction measure-
ments. As demonstrated in Figure 3, both compounds did not show
significant vasodilating activity after 24 h dissolved in water at
room temperature. This indicates, that the primary compound acts
as active agent and that the possibly split-off alcohol unit does not
influence the contraction status of the vessel preparation.
we focused on studying the involve-
ment of these channels in the biological activity of our
compounds. To identify a possible participation of KATP-channels
we tested the effect of compound 3a and 5b in presence of 30
and 100
Compounds were tested at concentrations similar to their EC50 val-
ues, which means for compound 3a 8 M and for compound 5b
M. However, as can be seen from Figure 4A, compound 3a still
exhibited a vasodilating potential of almost 50% in 30 as well as
00 M glibenclamide-treated aortic rings indicating no influence
lM glibenclamide, a selective KATP-channel inhibitor.
l
20 l
1
l
of this type of channels in the activity of the dithiocarbamide 3a.
On the other hand, the O-thiocarbamate representative 5b was
not able to significantly dilate glibenclamide-pretreated vessel
rings anymore suggesting an involvement of the KATP-channels in
the compound’s dilating potential. The vasodilation evoked by acti-
vation of KATP-channels can also be NO-mediated in the way that
NO, produced by endothelial nitric oxide synthase (eNOS), leads
to an increase in cGMP and hence may activate KATP-channels
which results in a relaxation of vascular smooth muscles.2
Therefore, we studied the effect of the most interesting compound,
0–22
5
b, together with the eNOS inhibitor nitro-
compound did not show any significant vasodilating behavior at
M in presence of 100 M eNOS inhibitor but showed a similar
L-arginine. The test
2
0
l
l
activity profile as on KATP-channels (Fig. 4B). These data support
the hypothesis, that compound 5b acts through NO-mediated acti-
vation of KATP-channels initiated by stimulation of eNOS. Recently,
Suvorava et al. stated the importance of therapeutically used com-
pounds, which are able to improve the endothelial NO generation
Figure 2. Time-course of the hydrolysis of compound 5b and 5c after 1, 3, 5, 7 and
2
4 h incubation time in
2
D O at 20 °C. Values represent the mean of three
2
3
independent experiments ± SEM.
in cardiovascular disease.

G. Brunhofer-Bolzer et al. / Bioorg. Med. Chem. 23 (2015) 4710–4718
4715
Considering all these data, this new drug class of self-degrading
compounds might represent a safe adjunct to the current treat-
ment of for instance myocardial infarction.
4
4
. Experimental section
.1. Chemistry
All chemicals were purchased from Sigma–Aldrich Chemie
GmbH (Schnelldorf, Germany), Merck (Darmstadt, Germany) or
Apollo Scientific Ltd (Bredbury, UK) at analytical grade and used
without further purification. Dry THF was freshly distilled over
metal sodium and benzophenone. For synthesis of the hydrochlo-
ride salts dry diethylether stored over metal sodium was used as
solvent. Purification was performed using preparative separation
column chromatography (Merck silica gel 60, 230–400 mesh).
Analytical TLC (thin layer chromatography) was carried out on
pre-coated aluminum plates (Merck silica gel 60, F254) and was
used for monitoring the reactions. Melting points were determined
1
on a Kofler melting point apparatus and are uncorrected. H and
1
3
C NMR spectra were recorded on a Brucker Advance DPx200
200 and 50 MHz). Chemical shifts are reported in d values (ppm)
(
1
relative tetramethylsilane (TMS) as internal standard for H NMR
spectra and the solvent peak for C NMR. Peak splitting patterns
in the H NMR are reported as follows: s, singlet; d, doublet; t, tri-
1
3
1
plet; sept, septet; m, multiplet; br, broad. J values are reported in
Hertz. Mass spectra (MS) were obtained with a Shimadzu (GC-
1
7A; MS-QP5050A) spectrometer. Purity of the compounds was
established by combustion analysis with a Perkin–Elmer 2400
CHN elemental analyzer confirming purity P95%. HRMS was per-
formed on a Bruker micrOTOF-Q II 10240.
Figure 4. Mode of action of compound 3a (A) and 5b (B) on aortic rings pre-
contracted by 90 mM in presence of glibenclamide (Glib) and nitro- -arginine
NLA), respectively. The change in contraction force (f ) in mN is plotted on the
ordinate. Control represents the f before adding the inhibitor (glibenclamide and
nitro- -arginine, respectively) and ‘inhibitor’ characterizes the changes of f evoked
L
(
c
c
L
c
4
.1.1. General procedure of the preparation of 3a–d and 3h
To solution of imidazole or its appropriate derivative
2.5 mmol) in dry THF (2 mL), stirred at 0 °C under argon atmo-
sphere, was added suspension of sodium hydride (NaH)
3 mmol). After 20 min stirring at 0 °C, 3 mmol carbonyl sulfide
by the inhibitor without test compound. The bars symbolize the arithmetic
*
a
means ± SEM of three to four independent experiments. Significance level: P <0.05.
(
a
(
3
. Conclusion
(CS ) were added and the reaction mixture was allowed to reach
2
room temperature. After additional 60 min, the corresponding
By applying the idea of fragment-based drug discovery we were
alkyl halide (2.5 mmol) was added and stirred at room tempera-
ture till the end of the reaction (monitored by TLC). Then, the reac-
tion mixture was filtered under reduced pressure. The solvent of
the filtrate was removed in vacuo and the crude product was puri-
able to develop a new type of vasodilating agents presenting char-
acteristics of being of interest in the treatment of acute myocardial
infarction. The compounds were supposed to exhibit on the one
side a high vasodilating potential and on the other side, they were
thought to degrade within the body. The latter referring to a new
concept that is giving the compounds a certain amount of instabil-
ity in aqueous solution (t1/2 around 10 h) thus obtaining
substances which, once administered, degrade nonenzymatically
into more hydrophilic components after a definite time and thus
help the body to get rid of the xenobiotic more easily. Three
different sets of compounds were synthesized. The biological
investigations on isolated organ preparations together with the
analysis of the chemical space occupied by the compounds
1
3
fied by column chromatography. Next, the product was dissolved
in diethylether, followed by addition of 1 M hydrochloric acid in
diethylether. Finally, the so formed desired hydrochloride of the
product was filtered off. Except for compound 3d, all compounds
were transformed into their corresponding hydrochloride.
4.1.1.1. 1H-Imidazole-1-carbodithioic acid methyl ester hydro-
2
4
chloride (3a) . Yellow crystals; yield 95% (base: purchased from
1
Sigma–Aldrich 694029); mp 108–114 °C.
H
2
NMR (D O,
200 MHz) d 9.59–9.40 (m, 1H), 8.20–8.02 (m, 1H), 7.54–7.49 (m,
2
13
revealed that an sp sulfur atom as well as aromatic features pre-
1H), 2.76 (s, 3H). C NMR (D O, 50 MHz) d 197.7, 121.2, 119.9,
2
sent in the molecule have to be regarded as key requirements for
good vasodilating properties. Investigations concerning the mode
of action clearly demonstrated a difference in biological activity
between the dithiocarbamide (3a) and O-thiocarbamate (5b)
derivatives. Compound 3a did not show any involvement of
21.1.
4.1.1.2. 1H-Imidazole-1-carbodithioic acid benzylic ester hydro-
chloride (3b). Yellow crystals; yield 44%. Mp 90–110 °C. H NMR
1
(D O, 200 MHz) d 9.43 (s, 1H), 8.05 (s, 1H), 7.42 (s, 1H), 7.38–
2
1
3
KATP-channels. On the contrary, in the case of compound 5b, NO-
7.00 (m, 5H), 4.58 (s, 2H). C NMR (d -DMSO, 50 MHz) d 197.4,
6
mediated activation of KATP-channels initiated by stimulation of
eNOS might be the reason for the compound’s observed biological
activity. This fact is regarded as an advantage in the development
of compounds intended for treatment of myocardial infarction
since KATP-channels as well as endothelial-generated NO are
known as endogenous cardioprotective mechanisms.
135.8, 133.9, 129.8, 129.0, 128.3, 127.1, 119.3, 41.6.
4.1.1.3. 1H-Imidazole-1-carbodithioic acid 4-methoxybenzylic
ester hydrochloride (3c). Yellow crystals; yield 27%. Mp 84–98 °C.
1
H NMR (CDCl , 200 MHz) d 13.68 (s, 1H), 9.70 (s, 1H), 7.99 (s, 1H),
3
7.60 (s, 1H), 7.40–7.28 (m, 2H), 6.98–6.81 (m, 2H), 4.63 (s, 2H), 3.81

4
716
G. Brunhofer-Bolzer et al. / Bioorg. Med. Chem. 23 (2015) 4710–4718
(
1
s, 3H). 13C NMR (CDCl
3
, 50 MHz) d 193.6, 159.9, 132.9, 130.8 (2C),
23.3, 121.6, 118.5, 114.4 (2C), 55.3, 43.2.
the reaction mixture was purified by column chromatography.
The so-obtained product was dissolved in dry diethylether and
M HCl in diethylether was added to yield the hydrochloride salt
1
4
.1.1.4. 1H-Imidazole-1-carbodithioic acid 3,4,5-trimethoxy-
of the compound.
2
4
benzylic ester (3d) . Yellow crystals; yield 39%. Mp 110–113 °C.
1
H NMR (CDCl
3
, 200 MHz) d 8.49 (s, 1H), 7.80–7.75 (m, 1H),
4.1.3.1. 1H-Imidazole-1-carbothioic acid O-isopropyl ester
1
7
3
.12–7.09 (m, 1H), 6.61 (s, 2H), 4.56 (s, 2H), 3.87 (s, 6H), 3.85 (s,
H). C NMR (CDCl
hydrochloride (5a). White crystals; yield 65%. Mp 77–80 °C.
NMR (CDCl
(m, 1H), 7.59–7.46 (m, 1H), 5.74 (sept, J = 6.2 Hz, 1H), 1.59 (d,
H
1
3
3
, 50 MHz) d 197.3, 153.4 (2C), 137.9, 131.6
3
, 200 MHz) d 13.94 (s, br, 1H), 9.63 (s, 1H), 8.01–7.86
(
(
2C), 128.8, 117.6, 106.4 (2C), 60.8, 56.1 (2C), 42.3. MS m/z 324
2%, M ), 181 (100%), 148 (9%), 136 (5%).
+
13
J = 6.2 Hz, 6H). C NMR (CDCl
3
, 50 MHz) d 178.6, 134.0, 120.9,
1
19.2, 82.3, 20.9 (2C).
4
.1.1.5. 2-Methylimidazole-1-carbodithioic acid methyl ester
1
hydrochloride (3h). Yellow crystals; yield: 87%. Mp: 146 °C.
NMR (D
H
4.1.3.2. 1H-Imidazole-1-carbothioic acid O-cyclohexyl ester
1
2
O, 200 MHz) d 7.73 (d, J = 2.3 Hz, 1H), 7.28 (d, J = 2.3 Hz,
hydrochloride (5b). White crystals; yield 73%. Mp 93–95 °C.
NMR (CDCl , 200 MHz) d 10.26 (s, br, 1H), 9.55 (s, 1H), 8.00–7.88
(m, 1H), 7.60–7.48 (m, 1H), 5.67–5.39 (m, 1H), 2.22–1.27 (m,
H
13
1
1
H), 2.74 (s, 3H), 2.67 (s, 3H). C NMR (D
45.6, 121.9, 118.2, 21.9, 13.1.
2
O, 50 MHz) d 199.6,
3
1
3
1
0H). C NMR (CDCl
3
, 50 MHz) d 178.5, 133.8, 121.2, 119.2, 86.7,
4
.1.2. General procedure of the preparation of 3e–g and 3i
To solution of imidazole or its appropriate derivative
2.5 mmol) in acetone (5 mL), potassium phosphate (2.5 mmol)
was added. After 15 min stirring at room temperature, 3 mmol car-
bonyl sulfide (CS ) were added and the reaction mixture was stir-
red for additional 15 min. Then, the corresponding alkyl halide
2.5 mmol) was added and stirred at room temperature till the
30.4 (2C), 24.7, 23.4.
a
(
4.1.3.3. 1H-Imidazole-1-carbothioic acid O-ethyl ester
hydrochloride (5c). White crystals; yield 65%. Mp 102–105 °C.
1
2
3
H NMR (CDCl , 200 MHz) d 13.35 (s, br, 1H), 9.71 (s, 1H), 7.95
(s, 1H), 7.54 (s, 1H), 4.87 (q, J = 7.1 Hz, 2H), 1.62 (t, J = 7.1 Hz,
1
3
(
3H). C NMR (CDCl
3
, 50 MHz) d 179.6, 126.7, 121.6, 119.1, 72.6,
end of the reaction (monitored by TLC). After that, the reaction
mixture was filtered under reduced pressure. The solvent of the fil-
trate was removed in vacuo and the crude product was purified by
column chromatography. Next, the product was dissolved in over
sodium dried diethylether, followed by addition of 1 M hydrochlo-
ric acid in diethylether. Finally, the so formed desired hydrochlo-
ride of the product was filtered off.
13.5.
4.1.3.4. 1H-Imidazole-1-carbothioic acid O-propyl ester
1
4
1
hydrochloride (5d). White crystals; yield 77%. Mp 66–70 °C.
NMR (CDCl , 200 MHz) d 11.87 (s, br, 1H), 9.65 (s, 1H), 7.95 (s,
1H), 7.57 (s, 1H), 4.75 (t, J = 7.1 Hz, 2H), 2.20–1.83 (m, 3H), 1.10
H
3
1
3
(t, J = 7.1 Hz, 3H). C NMR (CDCl
3
, 50 MHz) d 179.6, 134.1, 121.4,
1
19.1, 78.0, 21.2, 10.2.
4
.1.2.1. 1H-Imidazole-1-carbodithioic acid (propionic acid ethyl
ester) ester hydrochloride (3e). Yellow crystals; yield 17%. Mp
4.1.3.5. 1H-Imidazole-1-carbothioic acid O-butyl ester
1
24
1
6
8–79 °C. H NMR (D
2
O, 200 MHz) d 9.53–9.42 (m, 1H), 8.13–
hydrochloride (5e) . White crystals; yield 70%. Mp 74–77 °C.
NMR (CDCl , 200 MHz) d 12.08, (s, br, 1H), 9.59 (s, 1H), 7.94 (s,
1H), 7.57 (s, 1H), 4.79 (t, J = 6.7 Hz, 2H), 2.13–1.80 (m, 3H), 1.62–
H
8
.05 (m, 1H), 7.51–7.41 (m, 1H), 4.04 (q, J = 7.2 Hz, 2H), 3.62 (t,
3
1
3
J = 6.8 Hz, 2H), 2.83 (t, J = 6.8 Hz, 2H), 1.10 (t, J = 7.2 Hz, 3H).
C
1
3
NMR (D
2
O, 50 MHz) d 195.9, 174.0, 134.4, 121.2, 119.9, 62.6,
3
1.40 (m, 3H), 1.02 (t, J = 7.3 Hz, 3H). C NMR (CDCl , 50 MHz) d
3
3.0, 31.8, 13.7.
179.5, 133.9, 121.3, 119.2, 76.5, 29.7, 19.0, 13.5.
4
.1.2.2. 1H-Imidazole-1-carbodithioic acid ethylacetate ester
4.1.3.6. 1H-Imidazole-1-carbothioic acid O-cyclopentyl ester
2
4
1
1
hydrochloride (3f) . Yellow crystals; yield 15%. Mp 70–87 °C.
NMR (D O, 200 MHz) d 9.62–9.50 (m, 1H), 8.23–8.14 (m, 1H),
.58–7.50 (m, 1H), 4.40 (t, J = 5.9 Hz, 2H), 3.77 (t, J = 5.9 Hz, 2H),
H
hydrochloride (5f). White crystals; yield 73%. Mp 79–82 °C.
NMR (CDCl , 200 MHz) d 12.58 (s, br, 1H), 9.60 (s, 1H), 7.92 (s,
1H), 7.53 (s, 1H), 5.98–5.76 (m, 1H), 2.22–1.53 (m, 8H). C NMR
H
2
3
13
7
2
1
1
3
.01 (s, 3H). C NMR (D
20.7, 62.0, 37.5, 21.4.
2
O, 50 MHz) d 196.7, 174.9, 135.3, 122.2,
3
(CDCl , 50 MHz) d 178.9, 133.9, 121.2, 119.2, 91.2, 32.4 (2C), 23.8
(2C).
4
.1.2.3. 2-Methylbenzimidazole-1-carbodithioic acid methyl
4.1.3.7. 1H-Imidazole-1-yl-piperidine-1-yl-methanthione hydro-
chloride (5g). White crystals; yield 95%. Mp 140–156 °C. H NMR
1
ester hydrochloride (3g). Orange crystals; yield 25%. Mp 90–
1
1
(
(
14 °C. H NMR (CDCl
m, 1H), 7.66–7.50 (m, 2H), 3.12 (s, 3H), 3.04 (s, 3H). C NMR
CDCl , 50 MHz) d 197.2, 149.1, 131.0, 129.9, 127.1, 127.0, 115.7,
12.5, 22.3, 12.8.
3
, 200 MHz) d 8.09–7.94 (m, 1H), 7.80–7.67
3
(CDCl , 200 MHz) d 9.76 (s, 1H), 7.63–7.46 (m, 2H), 4.68–3.30 (m,
13
13
4H), 2.38–1.52 (m, 6H). C NMR (CDCl
3
, 50 MHz) d 172.0, 134.9,
3
120.8, 120.1, 53.6 (2C), 23.5 (3C).
1
4
.1.3.8. N,N-Diisobutyl-1H-imidazole-carbothioamide hydro-
1
4
.1.2.4. 2-Methylimidazole-1-carbodithioic acid benzylic ester
chloride (5h). White crystals; yield 66%. Mp 110–113 °C. H NMR
(CDCl , 200 MHz) d 9.65 (s, 1H), 7.63–7.38 (m, 2H), 4.27–3.04 (m,
4H), 2.78–1.66 (m, 2H), 1.52–0.34 (m, 12H). C NMR (CDCl
1
hydrochloride (3i). Yellow crystals; yield 7%. Mp 102–110 °C.
NMR (D
H
3
1
3
2
O, 200 MHz) d 7.72–7.58 (m, 1H), 7.38–7.14 (m, 6H),
3
,
13
4
.49 (s, 2H), 2.58 (s, 3H). C NMR (D
2
O, 50 MHz) d 197.0, 145.6,
50 MHz) d 174.1, 134.3, 120.9, 120.4, 61.8 (2C), 20.0 (4C), 2C not
1
33.8, 129.8 (2C), 129.3 (2C), 128.8, 121.8, 118.3, 43.1, 13.2.
detected.
4
.1.3. General procedure of the preparation of 5a–h
To a solution of 5 mmol 1,1 -thiocarbonyldiimidazole (0.89 g) in
4.1.4. General procedure of the preparation of 5i and 5j
The compounds 5i and 5j were prepared as described for the
compounds 5a–h, but using 11 mmol of the corresponding amine
derivative as starting material. For compound 5j, it was not neces-
sary to prepare the hydrochloride.
0
dry THF, 4.5 mmol of the corresponding alcohol and amine deriva-
tive, respectively, were added under argon atmosphere and stirred
under reflux. After completion of the reaction (monitored by TLC)

G. Brunhofer-Bolzer et al. / Bioorg. Med. Chem. 23 (2015) 4710–4718
4717
4
.1.4.1. Di-[2-(dimethylamino)ethyl]-thiocarbonate hydrochlo-
4.1.6.5. Carbonothioic acid O-(dimethylamino)propyl O-propyl
2
4
1
ride (5i) . White crystals; yield 69%. Mp 135–137 °C. H NMR (d
DMSO, 200 MHz) d 11.19 (s, 2H), 5.02–4.63 (m, 4H), 3.80–3.21 (m,
H), 2.97–2.1 (m, 12H). 13C NMR (d
-DMSO, 50 MHz) d 193.3, 67.3
2C), 54.1 (2C), 42.4 (4C).
6
-
diester hydrochloride (8e). White crystals; yield 70%. Mp 102–
1
104 °C. H NMR (d
6
-DMSO, 200 MHz) d 11.10 (s, br, 1H), 4.63–
4
(
6
4.26 (m, 4H), 3.20–3.00 (m, 2H), 2.72 (s, 6H), 2.30–2.03 (m, 2H),
1
3
1.85–1.56 (m, 2H), 0.92 (t, J = 6.3 Hz, 3H). C NMR (d
6
-DMSO,
5
0 MHz) d 194.8, 74.7, 70.1, 53.3, 41.8 (2C), 23.0, 21.1, 10.1.
4
.1.4.2. Di-[2-N,N,N-trimethylammonium]ethylthiocarbonate
1
dichloride (5j). White crystals; yield 38%. Mp >350 °C. H NMR
O, 200 MHz) d 3.93–3.73 (m, 4H), 5.07–4.87 (m, 4H), 3.22 (s,
4.1.6.6. Carbonothioic acid O-butyl O-(dimethylamino)propyl
(
D
2
diester hydrochloride (8f). White crystals; yield 65%. Mp 116–
1
3
1
1
8H). C NMR (D
2
O, 50 MHz) d 193.8, 66.8 (2C), 64.5, 62.3, 54.2
118 °C. H NMR (d
6
-DMSO, 200 MHz) d 10.93 (s, br, 1H), 4.56–
(
6C).
4.31 (m, 4H), 3.20–3.00 (m, 2H), 2.72 (s, 6H), 2.26–2.03 (m, 2H),
.78–1.56 (m, 2H), 1.47–1.24 (m, 2H), 0.89 (t, J = 7.3 Hz, 3H). 13
NMR (d -DMSO, 50 MHz) d 194.9, 73.0, 70.0, 53.4, 41.9 (2C), 29.6,
23.0, 18.5, 13.5.
1
C
4
.1.5. General procedure of the preparation of 6a and 6b
The compounds 6a and 6b were prepared as described for the
6
0
compounds 5a–h, but using 1,1 -carbonyldiimidazole as starting
material.
4.1.6.7. Ethyl 4-methyl-1-piperazinethiocarboxylate hydrochlo-
1
ride (8g). White crystals; yield 81%. Mp 169–173 °C. H NMR (d
6
-
4
.1.5.1. 1-Isopropoxycarbonylimidazole hydrochloride (6a).
DMSO, 200 MHz) d 11.66 (s, br, 1H), 4.64–4.20 (m, 2H), 3.83–
1
13
White crystals; yield 72%. Mp 84–86 °C.
H
NMR (CDCl
3
,
2.91 (m, 8H), 2.72 (s, br, 3H), 1.28 (s, br, 3H). C NMR (d
50 MHz) d 186.9, 67.4, 51.1 (2C), 45.3 (2C), 41.6, 13.8.
6
-DMSO,
2
00 MHz) d 11.37 (s, br, 1H), 9.58 (s, 1H), 7.73 (s, 1H), 7.61 (s,
1
3
1
H), 5.38 (sept, J = 6.3, 1H), 1.54 (d, J = 6.3 Hz, 6H). C NMR
, 50 MHz) d 145.1, 135.9, 121.4, 118.7, 77.7, 21.5 (2C).
(
CDCl
3
4.1.6.8. Propyl 4-methyl-1-piperazinethiocarboxylate hydro-
1
chloride (8h). White crystals; yield 70%. Mp 150–162 °C. H NMR
4
.1.5.2. 1-Cyclohexylcarbonylimidazole hydrochloride (6b).
(d
6
-DMSO, 200 MHz) d 12.03 (s, br, 1H), 4.40–4.29 (m, 2H), 4.13–
1
White crystals; yield 73%. Mp 111–113 °C. H NMR (CDCl
3
,
2.19 (m, 8H), 2.74 (s, 3H), 1.82–1.55 (m, 2H), 0.92 (t, J = 7.4 Hz,
3H). C NMR (d -DMSO, 50 MHz) d 187.3, 73.1, 51.3 (2H), 45.6,
6
41.6, 41.4, 21.5, 10.3.
1
3
2
1
5
00 MHz) d 11.49 (s, br, 1H), 9.51 (s, 1H), 7.72 (s, 1H), 7.59 (s,
H), 5.27–4.29 (m, 1H), 2.28–1.04 (m, 10H). 13C NMR (CDCl
3
,
0 MHz) d 145.1, 135.8, 121.5, 118.7, 82.0, 31.0 (2C), 24.7, 23.3
(
2C).
4.2. Experiments on smooth and heart muscle preparations
In general, experiments were performed according to proce-
4
.1.6. General procedure of the preparation of 8a–h
To a solution of 1.5 mmol 5b–e in dry THF, 1.8 mmol of the cor-
2
5,7–9
dures described previously.
Guinea pigs of both sexes (340–
responding alcohol 7a–b and N-methyl piperazine, respectively,
were added under argon atmosphere and stirred under reflux for
three hours. The so-obtained crude products were then dissolved
in dry diethylether and 1 M HCl in diethylether was added to yield
the hydrochloride of the compounds.
480 g) were killed with a blow on the neck. After excision of the
heart, the aorta and the ileum were dissected. The aorta was cut
into rings of 2 mm width. The terminal portion of the ileum was
removed and the 10 cm nearest to the caecum were discarded.
The intestine was placed in a nutrient solution and cut into pieces
of 1–2 cm length. The pulmonary artery was dissected close to the
heart, cleaned and cut into rings of 2 mm width. Papillary muscles
were dissected from the right ventricle for contractility measure-
ments. Purkinje fibers were carefully removed to prevent sponta-
neous activity. Chronotropic activity was tested on guinea pig
isolated right atria. After isolation, all the preparations were stored
4
.1.6.1. Carbonothioic acid O-cyclohexyl O-(dimethylamino)
propyl diester hydrochloride (8a). White crystals; yield 86%. Mp
1
1
5
6
5
2
31–132 °C. H NMR (d
.27–5.01 (m, 1H), 4.59–4.34 (m, 2H), 3.21–3.00 (m, 2H), 2.71 (s,
H), 2.27–1.83 (m, 4H), 1.78–1.16 (m, 8H). 13C NMR (d
-DMSO,
6
-DMSO, 200 MHz) d 10.95 (s, br, 1H),
6
0 MHz) d 193.8, 81.8, 69.8, 53.4, 41.9 (2C), 30.3 (2C), 24.6, 23.1,
at room temperature in gassed (95% O
Henseleit solution with the following composition (in mM): NaCl
14.9, KCl 4.73, CaCl 3.2, MgSO 1.18, NaHCO , 24.9, KH PO
1.18, glucose 10; pH 7.2–7.4. For the experiments, the preparations
were placed in a continuously oxygenated (95% O and 5% CO
2 2
and 5% CO ) Krebs–
3.0 (2C).
1
2
4
3
2
4
4
.1.6.2. Carbonothioic acid O-(dimethylamino)ethyl O-ethyl
diester hydrochloride (8b). White crystals; yield 91%. Mp 103–
2
2
)
1
1
08 °C. H NMR (d
6
-DMSO, 200 MHz) d 11.18 (s, br, 1H), 4.92–
bath of 25 mL nutrient solution at 37 ± 1 °C. The smooth and heart
4
.65 (m, 2H), 4.62–4.32 (m, 2H), 2.74 (s, br, 6H), 1.47–1.11 (m,
muscle preparations were connected with one end to a tissue
holder and the other to a force transducer (Transbridge™,
1
3
br, 3H). C NMR (d
6
-DMSO, 50 MHz) d 193.4, 69.5, 66.4, 54.2,
4
2.5 (2C), 13.4.
4-Channel Transducer Amplifier, World Precision Instruments,
Sarasota, FL, USA). Aortic and pulmonary artery rings were precon-
tracted by 90 mM KCl and terminal ileum preparations by 60 mM
KCl. Papillary muscles were electrically driven with an Anapulse
Stimulator Model 301-T and an Isolation UnitModel 305-1 (WPI,
Hamden, CT, USA) at a frequency of 1 Hz and a pulse duration of
3 ms. Amplitude of stimulation pulse was adjusted to 10% above
threshold level. Resting tension of either 3.92 mN (papillary mus-
cles), 4.9 mN (terminal ileum), 10.37 mN (right atria) or 19.6 mN
(aortic and pulmonary artery rings) was kept constant throughout
the experiments. After a control period different concentrations of
the test compounds were added cumulatively every 45 min.
Signals were recorded with a chart recorder (BD 112 Dual
Channel, Kipp and Zonen) and evaluated. For statistical analyses
the arithmetic means and standard error of the mean (SEM) of
4
.1.6.3. Carbonothioic acid O-(dimethylamino)propyl O-ethyl
diester hydrochloride (8c). White crystals; yield 70%. Mp 103–
1
1
4
2
6
6
06 °C. H NMR (d -DMSO, 200 MHz) d 10.89 (s, br, 1H), 4.68–
.24 (m, 4H), 3.17–2.96 (m, 2H), 2.70 (s, br, 6H), 2.82–2.59 (m,
H), 1.50–1.11 (m, 3H). 13C NMR (CDCl
9.2, 53.3, 41.9 (2C), 22.9, 13.5.
, 50 MHz) d 194.6, 69.9,
3
4
.1.6.4. Carbonothioic acid O-(dimethylamino)ethyl O-propyl
diester hydrochloride (8d). White crystals; yield 73%. Mp 70–
7
1
6
8 °C. H NMR (d -DMSO, 200 MHz) d 11.19 (s, br, 1H), 4.94–4.63
(
1
1
m, 2H), 4.52–4.20 (m, 2H), 3.62–3.25 (m, 2H), 2.73 (s, 6H), 1.90–
.45 (m, 2H), 1.10–0.65 (m, 3H). 13C NMR (CDCl
94.0, 74.9, 66.9, 54.1, 42.5 (2C), 20.8, 9.9.
3
, 50 MHz) d

4
718
G. Brunhofer-Bolzer et al. / Bioorg. Med. Chem. 23 (2015) 4710–4718
3
–5 experiments were calculated. Statistical significance of the
results was evaluated by Student’s t-test for paired observations
Sigma Plot version 9.0). Stock solutions of the test compounds
preparation was performed as described in Section 4.2. After pre-
contraction of the aortic rings by 90 mM KCl, 30 M and 100
glibenclamide, respectively, or 100 nitro- -arginine were
l
L
lM
(
lM
were dissolved preferred in distilled water or, if required, in
dimethylsulfoxide (DMSO). To exclude the solvent effect experi-
ments with the solvent only were performed and the effect was
subtracted from the results of the compounds. To study the inotro-
pic and chronotropic activity, after a control period of 30 min the
different concentrations of the compounds were added to the bath-
ing solution cumulatively, until a steady state was reached. For sta-
tistical analyses the arithmetic means and standard error of the
mean (SEM) of 4–5 experiments were calculated. Statistical signif-
icance of the results was evaluated by the Student’s t-test for
paired observations (Sigma Plot version 9.0).
added to the bathing solution. After 45 min, the test compounds
were administered in a concentration similar to their EC50 values
and the change in contraction force (f ) was recorded for another
c
45 min. For statistical analyses the arithmetic means and standard
error of the mean (SEM) of 3–4 experiments were calculated.
Statistical significance of the results was evaluated by the
Student’s t-test for paired observations (Sigma Plot version 9.0).
Acknowledgement
This
study
was
supported
in
part
by
the
Hochschuljubiläumsstiftung of the City Vienna (H-2151/2010, G.B.).
4
.3. Analysis of the chemical space of the compounds
Supplementary data
Exploration of the chemical space occupied by the synthesized
and tested compounds was studied using the Principal
Component Analysis (PCA)-based chemical space navigation tool
Supplementary data (the elemental analyses and HRMS data for
the tested compounds and time-course of the hydrolysis together
with the equation used to calculate the t1/2 as well as the R values)
associated with this article can be found, in the online version, at
http://dx.doi.org/10.1016/j.bmc.2015.05.049.
1
0,12
2
ChemGPS-NP.
This analysis is generated by 2D descriptors
(
total of 35 molecular descriptors) that explain physico-chemical
properties of the compounds and are calculated from SMILES.
SMILES for all compounds were obtained using the NCI online
SMILES translator (http://cactus.nci.nih.gov/translate/). All salts
and counter-ions were excluded from the SMILES annotation.
Analysis was focused on the first four dimensions of ChemGPS-
NP (PC1–PC4). The obtained coordinates were plotted using the
software Grapher 2.1 distributed together with MacOS X.
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4
.5. Mode of action studies
The antagonist of KATP-channels, glibenclamide, and the eNOS
inhibitor nitro-L-arginine were used to elucidate a possible mode
of action in isolated aortic strips of the guinea pig. The vessel