Clathrodin, hymenidin and oroidin, and their synthetic analogues as inhibitors of the voltage-gated potassium channels

Article abstract of DOI:10.1016/j.ejmech.2017.08.015

We have prepared three alkaloids from the Agelas sponges, clathrodin, hymenidin and oroidin, and a series of their synthetic analogues, and evaluated their inhibitory effect against six isoforms of the K_v1 subfamily of voltage-gated potassium channels, K_v1.1-K_v1.6, expressed in Chinese Hamster ovary (CHO) cells using automated patch clamp electrophysiology assay. The most potent inhibitor was the (E)-N-(3-(2-amino-1H-imidazol-4-yl)allyl)-4,5-dichloro-1H-pyrrole-2-carboxamide (6g) with IC₅₀ values between 1.4 and 6.1 μM against K_v1.3, K_v1.4, K_v1.5 and K_v1.6 channels. All compounds tested displayed selectivity against K_v1.1 and K_v1.2 channels. For confirmation of their activity and selectivity, compounds were additionally evaluated in the second independent system against K_v1.1-K_v1.6 and K_v10.1 channels expressed in Xenopus laevis oocytes under voltage clamp conditions where IC₅₀ values against K_v1.3-K_v1.6 channels for the most active analogues (e.g. 6g) were lower than 1 μM. Because of the observed low sub-micromolar IC₅₀ values and fairly low molecular weights, the prepared compounds represent good starting points for further optimisation towards more potent and selective voltage-gated potassium channel inhibitors.

Full text of DOI:10.1016/j.ejmech.2017.08.015

European Journal of Medicinal Chemistry 139 (2017) 232e241
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Clathrodin, hymenidin and oroidin, and their synthetic analogues as
inhibitors of the voltage-gated potassium channels
Nace Zidar , Ale ꢀs Zula , Tihomir Toma ꢀs i ꢀc , Marc Rogers 1, Robert W. Kirby b
a
a
a
b
,
, 1
ꢀ
,
c
c
a
a,
**
a, *
Jan Tytgat , Steve Peigneur , Danijel Kikelj , Janez Ilaꢀs
, Lucija Peterlin Ma ꢀs i ꢀc
a
University of Ljubljana, Faculty of Pharmacy, A ꢀs ker ꢀc eva cesta 7, 1000 Ljubljana, Slovenia
Xention Limited, Iconix Park, London Road, Pampisford, Cambridge CB22 3EG, UK
University of Leuven (KU Leuven), Toxicology & Pharmacology, O&N2, PO Box 922, Herestraat 49, 3000 Leuven, Belgium
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 18 January 2017
Received in revised form
We have prepared three alkaloids from the Agelas sponges, clathrodin, hymenidin and oroidin, and a
series of their synthetic analogues, and evaluated their inhibitory effect against six isoforms of the K
subfamily of voltage-gated potassium channels, K 1.1-K 1.6, expressed in Chinese Hamster ovary (CHO)
cells using automated patch clamp electrophysiology assay. The most potent inhibitor was the (E)-N-(3-
2-amino-1H-imidazol-4-yl)allyl)-4,5-dichloro-1H-pyrrole-2-carboxamide (6g) with IC50 values between
.4 and 6.1 M against K 1.3, K 1.4, K 1.5 and K 1.6 channels. All compounds tested displayed selectivity
against K 1.1 and K 1.2 channels. For confirmation of their activity and selectivity, compounds were
additionally evaluated in the second independent system against K 1.1-K 1.6 and K 10.1 channels
expressed in Xenopus laevis oocytes under voltage clamp conditions where IC50 values against K 1.3-
1.6 channels for the most active analogues (e.g. 6g) were lower than 1 M. Because of the observed
v
1
v
v
3
August 2017
Accepted 4 August 2017
Available online 7 August 2017
(
1
m
v
v
v
v
v
v
Keywords:
Voltage-gated potassium channel
K 1
v
Modulator
Inhibitor
Clathrodin
Oroidin
Hymenidin
v
v
v
v
K
v
m
low sub-micromolar IC50 values and fairly low molecular weights, the prepared compounds represent
good starting points for further optimisation towards more potent and selective voltage-gated potassium
channel inhibitors.
©
2017 Elsevier Masson SAS. All rights reserved.
1. Introduction
based on their sequence homology and function, K
v
channels can be
divided into 12 major subfamilies (K 1-12). Additionally, each of
v
Voltage-gated potassium (K
v
) channels are molecular com-
the 12 subfamilies is composed of several members that differ in
their structures, biophysical profile and expression patterns in
different tissue types which facilitate their different biological roles
plexes responsible for initiation and propagation of electrical im-
pulses in excitable cells such as neurons, myocytes and endocrine
cells, as well as for the transduction of signals in non-excitable cells
[3]. There are currently eight known pore-forming
1 subfamily (K 1.1- K 1.8), among which K 1.1 and K
are mainly expressed in the central nervous system (CNS), K
channels are typical for T and B lymphocytes and macrophages, the
rapidly inactivating K 1.4 channels can be found in CNS, skeletal
muscle, heart and pancreatic islets, K 1.5 channels are located in
cardiac myocytes, immune cells and vascular smooth muscle, while
the K 1.6 channels are expressed in spinal cord, CNS, astrocytes and
artery smooth muscle. The high expression of K 1.7 and K 1.8
channels is typical for the heart, skeletal muscle and CNS [2,4].
The loss- and gain-of-function mutations - the channelopathies
of the K 1 channel subfamily have been associated with various
pathological phenotypes which highlight the potential of K
channels as promising drug targets [3,4]. Modulators of K 1.1 and
1.2 channels have been described as potential targets for the
a
subunits of the
1.2 channels
1.3
such as immune cells. K
proteins composed of four circularly arranged
an ion conducting pore and contain voltage-sensing domains, and
one or more supplementary subunits [1,2]. K channels are a very
v
channels are homo- or heterotetrameric
K
v
v
v
v
v
a
subunits that form
v
b
v
v
large and diverse group of proteins and represent the largest
branch of the potassium channel family. In humans, there are
currently 40 known genes encoding K
v
v
channel
a
subunits and
v
v
v
*
Corresponding author.
* Corresponding author.
E-mail addresses: Janez.Ilas@ffa.uni-lj.si (J. Ila ꢀs ), Lucija.PeterlinMasic@ffa.uni-lj.si
-
v
*
v
1
(
L.P. Ma ꢀs i ꢀc ).
v
1
Current address: Metrion Biosciences Limited, Babraham Research Campus,
Cambridge, CB22 3AT, UK.
K
v
http://dx.doi.org/10.1016/j.ejmech.2017.08.015
0223-5234/© 2017 Elsevier Masson SAS. All rights reserved.

N. Zidar et al. / European Journal of Medicinal Chemistry 139 (2017) 232e241
233
treatment of epilepsy and neuropathic pain [5,6], inhibitors of K
channels exert immunosuppressive effects and have potential for
v
1.3
other species of marine sponges (acredinones and crambescins)
have recently shown inhibitory activity against K channels [24,25].
v
the treatment of various autoimmune diseases [7e9], blockers of
Because of their relatively simple structures and lead-like proper-
ties, clathrodin, hymenidin and oroidin represent interesting
starting compounds for medicinal chemistry optimisation of their
pharmacological properties.
In our study, we have prepared clathrodin, hymenidin and
oroidin, and a series of their synthetic analogues, and evaluated
K
v
1.5 channels are in development as antiarrhythmics [10e12],
while K 1.4, K 1.6, K 1.7 and K 1.8 channels have so far a less well
defined therapeutic relevance.
Among the K 1 subfamily, in recent years, the K
v
v
v
v
v
v
1.3 channel has
arguably received the most scientific attention as a drug target.
þ
Efflux of K ions through K
v
1.3 channels is necessary to repolarise
their K
1 channel subtypes expressed in Chinese hamster ovary cells
(CHO cells) using automated patch clamp electrophysiology. All of
the prepared compounds were evaluated against K 1.3 channels,
and active inhibitors also against K 1.1, K 1.2, K 1.4, K 1.5 and K 1.6
v
1 channel inhibitory activity against six different human
the membrane potential in immune cells. A negative membrane
potential is required to allow activation of calcium release-activated
channels (CRAC) channels upon a new depolarization. As such,
K
v
v
2
þ
Kv1.3 channels indirectly create a driving force for the entry of Ca
ions through CRAC channels. The resultant influx of Ca
v
v
v
v
v
2
þ
is
channels. In addition to the automated patch clamp electrophysi-
ology, compounds were also studied in the second, independent
necessary for the translocation of nuclear factors which results in T
cell activation, proliferation and inflammatory cytokine secretion.
test system under voltage clamp conditions against K
v v
1.1-K 1.6 and
v
Thus, selective K 1.3 inhibition could be regarded as a novel
v
K 10.1 channels expressed in Xenopus laevis oocytes. Based on the
approach for the treatment of autoimmune diseases such as mul-
tiple sclerosis, rheumatoid arthritis and psoriasis [3,4,13].
inhibitory activities, structure-activity relationship (SAR) of this
new structural class of voltage-gated potassium channel modula-
tors was studied.
v
There are two main binding sites for small-molecules on K 1
channels reported in the literature: the inner-pore site and a side-
pocket cavity [14]. Since the sequence of the inner pore is well
2
. Results and discussion
conserved among the K
to this site are not very selective, e.g. 4-aminopyridine (4-AP, Fig. 1)
and tetrabutylammonium (TBA, Fig. 1). Reasonable K 1.3 selectivity
has been reported for correolide (Fig. 1) [15], and for PAP-1 (Fig. 1)
16], a member of the phenoxyalkoxypsoralen family, which dis-
plays a 2 nM IC50 value against K 1.3 and a 4- to 20-fold selectivity
against K 1.1, K 1.2, K 1.4, K 1.5 and K 1.6 channels.
Despite tremendous research efforts in the K
discovery field the therapeutic potential of K channels remains
v
1 family, the majority of blockers that bind
2.1. Design
v
We have prepared clathrodin, hymenidin and oroidin (Fig. 2),
[
v v
and evaluated their inhibitory effect against K 1.1-K 1.6 channels.
With the aim of improving the activities and/or stabilities of the
natural alkaloids, we have designed two structural classes of their
analogues,
amides
v
v
v
v
v
v
v
channel drug
(E)-N-(3-(2-amino-1H-imidazol-4-yl)allyl)carbox-
and N-(3-(2-amino-1H-imidazol-4-yl)propyl)carbox-
v
I
underexploited. The main problems are (i) the lack of structural
channel data and the information on the exact binding site of
compounds, (ii) low structural diversity of leads, (iii) poor gene
family selectivity and/or (iv) unfavourable physicochemical prop-
erties of compounds, which result in non-optimal efficacy and
amides II (Fig. 2).
In the type I series we decided to retain the (E)-4-(3-aminoprop-
1-en-1-yl)-1H-imidazol-2-amide motif on the left-hand side of the
molecules and to replace the right-hand side pyrrol-2-yl (6a,
Table 1), 4-bromopyrrol-2-yl (6b, Table 1), and 4,5-dibromopyrrol-
toxicity issues. However, recent advances in crystallisation of K
v
2-yl (6c, Table 1) groups that are present in natural alkaloids, with
channels [17e19] and new in silico studies [14] are increasing our
knowledge of their structure and function. Additionally, over the
last few years development of new reliable high- and medium
throughput screening methods such as the Sophion QPatch 48
automated patch-clamp electrophysiology system have helped
accelerate the drug discovery process for ion channel modulators
other substituted pyrrol-2-yl (6d-g, Table 1), indol-2-yl (6h-k,
Table 1), indolin-2-yl (6l, Table 1), pyridin-3-yl (6m, Table 1), and
piperidin-4-yl (6n-o, Table 1) groups. In this way we examined the
influence of different size, polarity and/or acid/base properties of
the right-hand side groups on the biological activity. Additionally,
with piperidine derivatives 6n and 6o, we examined if the
[4].
aromaticity of the right-hand side groups is important for K
inhibitory activity.
v
1
Marine organisms, such as sponges, fish, cone snails, cnidarians
and sea anemones, are important sources of various bioactive
compounds that are used in their native environment to capture
pray or function as defence against predators [20]. Alkaloids iso-
lated from marine sponges Agelas sp., clathrodin, hymenidin and
oroidin (Fig. 2), and their synthetic analogues, have been shown to
In the type II series we aimed at increasing the chemical sta-
bility of compounds by eliminating the potentially reactive double
bond in the central part of the molecules and replacing it with the
saturated propylene group. In this way, we also studied the influ-
ence of increased flexibility and conformational freedom of mole-
cules on the K 1 inhibitory activity. Analogously to the type I series,
v
in addition to the analogue with naturally occurring 4,5-
dibromopyrrol-2-yl group (14a, Table 1), we have prepared
possess modulatory activities on voltage-gated sodium (Na
channels [21e23], but so far their activity on K channels has not
been reported. However, complex marine compounds isolated from
v
)
v
v v
Fig. 1. Structures of the unselective K channel inhibitors (4-AP, TBA) and K 1.3 selective inhibitors (correolide, PAP-1).

234
N. Zidar et al. / European Journal of Medicinal Chemistry 139 (2017) 232e241
Fig. 2. Agelas alkaloids clathrodin, oroidin and hymenidin, and two structural classes of the designed analogues, (E)-N-(3-(2-amino-1H-imidazol-4-yl)allyl)carboxamides I and N-(3-
2-amino-1H-imidazol-4-yl)propyl)carboxamides II.
(
analogues with bulkier indol-2-ly (14b, Table 1) and indolin-2-yl
14c, Table 1) groups on the right-hand side of the molecules.
micromolar range, with moderate isoform selectivity. The observed
potencies were generally higher for K 1.3, K 1.4, and K 1.6 chan-
nels, while none of the compounds were active against K 1.1 and
1.2 channels. By comparing the biological results for alkaloids
(
v
v
v
v
K
v
2.2. Chemistry
clathrodin (6a), hymenidin (6b), and oroidin (6c) it can be seen, that
the introduction of bromo substituents to the pyrrole ring on the
right-hand side of the molecules increases the inhibitory activity
Type I compounds were prepared according to Fig. 3. First,
pyridine (1) was acylated with benzyl chloroformate, followed by
reduction with sodium borohydride to give N-Cbz-1,2-
dihydropyridine (2). Reaction of compound 2 with N-Boc-guani-
dine in the presence of bromine afforded two regiosiomers 3a and
b, which after deprotection yielded amine 4 [26]. Final compounds
a-o were prepared by TBTU-promoted coupling of amine 4 and
against K
role-2-yl group was inactive against all K
hymenidin (6b) and oroidin (6c) displayed inhibitory activities
against K 1.3-K 1.6 isoforms. The observed differences in potencies
v
1 channels. Clathrodin (6a) with an unsubstituted pyr-
v
1 channel isoforms, while
3
6
v
v
can be rationalised either by the increase in volume and lip-
ophilicity because of the additional bromo substituent, or by the
electron-withdrawing effects of bromine which increases the acid
properties of the pyrrole NH and thus makes it a better H-bond
donor. Interestingly, hymenidin (6b) with 4-bromopyrrol-2-yl
the appropriate carboxylic acids with good regioselectivity.
Compounds 14a-c were prepared as depicted in Fig. 4. First, L-
ornithine (7) was Boc-diprotected to yield compound 8, which was
then converted to Weinreib amide 9. A second Boc protecting group
group (IC50 for K
for K M) was generally more potent than oroidin (6c)
1.6 ¼ 3.7
with 4,5-dibromopyrrol-2-yl group (IC50 for K M, IC50 for
1.3 ¼ 4.7
1.5 ¼ 2.1 M, and IC50 for K 1.6 ¼ 4.9 M). The only exception was
the K M) was more potent
1.4 channel where oroidin (IC50 ¼ 2.1
M).
v
1.3 ¼ 2.5
m
M, IC50 for K
v
1.5 ¼ 7.6
mM, and IC50
was introduced to d position of 9 to yield compound 10, followed by
m
the reduction of 10 to give aldehyde 11. Aldehyde 11 was Boc-
deprotected with gaseous HCl and then formation of a 2-
aminoimidazole ring was achieved using cyanamide to yield satu-
rated amine 13 [26]. TBTU was used for the coupling between
amine 13 and the appropriate carboxylic acids with good regiose-
lectivity to yield amides 14a-c.
v
v
m
K
v
m
v
m
v
m
than hymenidin (IC50 ¼ 5.3
m
In the type I series of clathrodin analogues, the most potent
inhibitor was the 4,5-dichloropyrro-2-yl derivative 6g, with IC50
v v v
values of 1.4 mM, 1.5 mM, and 1.6 mM against K 1.3, K 1.4, and K 1.6
2
v
.3. Inhibitory activity of compounds against K 1 channels
channels, respectively. This indicates that slightly smaller chlorine
atoms are more optimal for increasing the binding affinity than
bromine atoms. Compounds 6h-k with indole groups on the right-
hand side of the molecules were less potent than the pyrrole ana-
logues. Among the indole derivatives, the two most potent in-
Altogether 18 compounds were prepared and evaluated for their
inhibitory activities against different K 1 channel isoforms. The
channels were heterologously expressed in two different expres-
v
sion systems, mammalian CHO cells and Xenopus laevis oocytes. For
hibitors were the 5-fluoroindole 6i (IC50 for K
1.6 ¼ 7.9 M), and the 5-chloroindole 6j (IC50 for K
Compound 6j was interesting also because of its selectivity as it was
inactive against K 1.1, K 1.2 and K 1.5 channels, and only weakly
active against K M) and K M)
1.4 (IC50 ¼ 17
v
1.4 ¼ 8.8
m
M, IC50 for
the analysis of compounds against K
in CHO cells, automated patch clamp electrophysiology assay was
used. The compounds were additionally analysed on K 1.1- K 1.6
and K 10.1 channels expressed in Xenopus laevis oocytes under
voltage clamp conditions. The results are expressed as IC50 values
the concentration of compound that inhibits potassium channel
activity by 50%) or as percent inhibition at the 1 M test concen-
v v
1.1-K 1.6 isoforms expressed
K
v
m
v
1.3 ¼ 9.0
mM).
v
v
v
v
v
v
v
m
v
1.6 (IC50 ¼ 24
m
channels. Compounds with indolin-2-yl (6l), pyridin-3-yl (6m), and
piperidin-4-yl (6n-o) groups on the right-hand side of the mole-
(
m
v
cules were inactive against all of the tested K 1 isoforms. Similarly,
compounds of the type II series (14a-c) with the reduced double
bond in the central part of the molecules did not display any activity
tration and are presented in Tables 1 and 2, and Table 1S of the
Supplementary information.
Automated patch clamp electrophysiology assay was performed
on the QPatch48 HT system (Sophion, Denmark) using whole cell
against K
v
1 channels.
The results of the voltage clamp experiments on K
v
channels
v
configuration mode. The potency of compounds against the K 1
expressed in X. laevis oocytes are presented in Table 1S (Supple-
mentary information) and Table 2. First, the initial selectivity
channel subtypes was determined from concentration-response
relationships established by cumulatively applying four escalating
concentrations of test compound to a cell. Two-electrode voltage-
clamp recordings were performed using a GeneClamp 500 ampli-
fier (Molecular Devices, Sunnyvale, California, USA) controlled by a
pClamp data acquisition system (Axon Instruments, USA). For de-
tails see Experimental section.
The results of the automated patch clam electrophysiology are
presented in Table 1 and can be summarised as follows. Some of the
compounds, e.g. 6b, 6c, and 6g, exhibited promising inhibitory
profiling was performed for all 18 compounds against K
v v
1.1-K 1.6
and K 10.1 channels at 1 M concentration. The activities are pre-
v
m
sented in Table 1S as percent inhibition of potassium channel cur-
rents. Based on these results, five most potent compounds were
v v
selected for the determination of IC50 values on K 1.3, K 1.4 and
v
K 1.6 channels (Table 2). From the data presented in Tables 1S and 2
a similar trend can be observed as for the automated patch clamp
experiments. Clathrodin (6a) with no substituents on the pyrrole
ring was inactive against all of the tested K
v
isoforms. The
v
activities against different K 1 channel isoforms in the low

N. Zidar et al. / European Journal of Medicinal Chemistry 139 (2017) 232e241
235
Table 1
Inhibitory activities of (E)-N-(3-(2-amino-1H-imidazol-4-yl)allyl)carboxamides 6a-o and N-(3-(2-amino-1H-imidazol-4-yl)propyl)carboxamides 14a-c on K
v
1.1-K
v
1.6 chan-
nels expressed in CHO cells.
Compd.
R
IC50
(m
M)a
K
v
1.1
K
v
1.2
K
v
1.3
K
v
1.4
K
v
1.5
v
K 1.6
6a
n.a.b
n.a.
n.a.
n.a.
n.a.
n.a.
clathrodin
6b
n.a.
n.a.
2.5 ± 0.1
5.3 ± 0.5
7.6 ± 1.7
3.7 ± 1.0
hymenidin
6
c
n.a.
n.a.
n.a.
20 ± 14
n.a.
4.7 ± 1.3
25 ± 3
2.1 ± 0.4
12 ± 4
16 ± 10
n.a.
4.9 ± 1.6
11 ± 4
oroidin
6
d
e
6
n.a.
17 ± 5
12 ± 3
n.a.
29 ± 2
6
6
f
n.a.
n.a.
n.a.
n.a.
25 ± 8
15 ± 2
n.a.
17 ± 4
g
1.4 ± 0.1
1.5 ± 0.5
6.1 ± 1.9
1.6 ± 0.2
6
6
h
i
n.a.
n.a.
n.a.
n.a.
21 ± 3
14 ± 3
13 ± 2
n.a.
n.a.
14 ± 3
8.8 ± 2.8
7.9 ± 1.4
6
6
j
n.a.
-c
n.a.
9.0 ± 0.7
17 ± 5
n.a.
24 ± 4
k
e
n.a.
e
e
e
6
l
e
e
e
e
n.a.
n.a.
e
e
e
e
e
e
6
m
6
6
1
n
e
e
e
e
e
e
n.a.
n.a.
n.a.
e
e
e
e
e
e
e
e
e
o
4a
(
continued on next page)

236
N. Zidar et al. / European Journal of Medicinal Chemistry 139 (2017) 232e241
Table 1 (continued )
Compd.
R
IC50
(m
M)a
K
v
1.1
K
v
1.2
K
v
1.3
K
v
1.4
K
v
1.5
v
K 1.6
1
4b
4c
e
e
n.a.
e
e
e
1
e
e
n.a.
e
e
e
PAP-1d
0.25 ± 0.09
0.45 ± 0.06
0.024 ± 0.003
0.17 ± 0.03
0.088 ± 0.022
0.13 ± 0.02
a
The concentration of compound that inhibits a potassium channel current by 50%. Each IC50 value is the mean of three independent experiments.
b
c
n. a ¼ not active (IC50 value higher than 30
mM).
Not determined.
d
5-(4-Phenoxybutoxy)psoralen.
Fig. 3. Synthesis of (E)-N-(3-(2-amino-1H-imidazol-4-yl)allyl)carboxamides 6a-o. Reagents and conditions: (a) benzyl chloroformate, Et
Boc-guanidine, 45 min, 31% (3a), 18% (3b); (c) 6 M HCl/MeOH, 2 h, 33%; (d) TBTU, NMM, DMF, rt, 6 h, 22e34%.
2 4 2 3
O, then NaBH , 2 h, 54%; (b) Br , Et N, N-
Fig. 4. Synthesis of N-(3-(2-amino-1H-imidazol-4-yl)propyl)carboxamides 14a-c. Reagents and conditions: (a) Boc
2
O, NaOH, THF/H
2
O, 12 h, 99%; (b) N,O-dimethylhydroxylamine
O, pH 4.5, reflux 3 h, 30%;
ꢀ
3
hydrochloride, Et N, BOP, 2 h, 84%; (c) Boc
2
O, DMAP, MeCN, 24 h, 81%; (d) LiAlH
4
, Et
2
O, 0 C, 45 min, 67%; (e) HCl(g), Et
2
O, 30 min, 100%; (f) cyanamide, H
2
(
g) TBTU, NMM, DMF, rt, 6 h, 58e63%.
inhibitory activity increased with the introduction of bromo,
chloro, or methyl substituents to 3, 4 or 5 positions of the pyrrole
ring. Oroidin (6c) with 4,5-dibromopyrrole group showed a
experiments, the most potent compound of the series was 4,5-
dichloropyrro-2-yl derivative 6g that, besides displaying the
inhibitory activity against K
v
1.4 channels, was also very active
M IC50 value was determined.
promising 0.82
against K 1.4 channels. The most potent compounds were 6d, 6e, 6f
and 6g of the type I series, for which promising inhibitory activities
were obtained against K 1.4 channels where they displayed sub-
micromolar IC50 values, i.e. 0.38 M for 6d, 0.23 M for 6e,
.73 M for 6f and 0.60 M for 6g. Like in the patch clamp
v
mM IC50 value against K 1.3, and 5.7
m
M IC50 value
against K 1.6 channels where 0.17 m
v
v
In agreement with the results of the patch clamp experiments,
in X. laevis oocytes compounds with indole (6h-k), indolin-2-yl (6l),
pyridine-3-yl (6m), and piperidin-4-yl (6n-o) groups on the right-
hand side of the molecules were less active than the pyrrole ana-
logues. Only 5-flouroindole derivative 6i, and piperidine containing
v
m
m
0
m
m

N. Zidar et al. / European Journal of Medicinal Chemistry 139 (2017) 232e241
237
Table 2
v
and K 1.6 channels with IC50 values in the low micromolar range,
Inhibitory activities of five selected (E)-N-(3-(2-amino-1H-imidazol-4-yl)allyl)car-
boxamides (6c-g) on K 1.3, K 1.4 and K 1.6 channels expressed in Xenopus laevis
v v v
oocytes.
whereas, clathrodin was found to be inactive across the concen-
tration ranges tested. To probe the SAR, structural modifications of
hymenidin and oroidin were prepared represented by 15 ana-
logues, among which, some displayed improved inhibitory activity
against K
were used to express K
v
1.3-K
v
1.6 channels. Two different expression systems
1 channels e CHO cells and Xenopus laevis
v
oocytes. The inhibitory activities obtained in two independent test
systems were generally in good agreement. The most active natural
product analogues were compounds 6d, 6e, 6f and 6g with (E)-4-
(3-aminoprop-1-en-1-yl)-1H-imidazol-2-amide motif on the left-
hand side of the molecules and substituted pyrrol-2-yl group on
the right-hand side. 4,5-Dichloropyrro-2-yl derivative 6g displayed
Compd.
R
IC50
1.3
0.82 ± 0.21
(m
M)a
K
v
K
v
1.4
v
K 1.6
6c
5.7 ± 0.8
n.a.
IC50 values of 1.4
1.6 channels respectively in the patch clamp experiments. The
same compound displayed and IC50 of 0.60 M against K 1.4
channels and 0.17 M against K 1.6 under voltage clamp conditions.
v v
mM, 1.5 mM, and 1.6 mM against K 1.3, K 1.4, and
oroidin
K
v
m
v
m
v
6
d
e
n.a.
0.38 ± 0.09
0.23 ± 0.04
9.8 ± 1.2
Low and sub-micromolar IC50 values and relatively low molecular
weights of the prepared compounds highlight their potential for
further optimisation of their inhibitory action against voltage-gated
potassium channels.
6
n.a.
n.a.
4
4
4
. Experimental section
.1. Electrophysiology
6
6
f
n.a.
n.a.
0.73 ± 0.05
0.60 ± 0.12
n.a.
.1.1. Automated patch clamp
Chinese Hamster Ovary (CHO) cell lines stably expressing
v v v v
exogenous human a-subunits of K 1.1, K 1.2, K 1.3 and K 1.4 were
created ‘in house’ using standard techniques, hKv1.5 was purchased
from b-Sys (Switzerland). All cell lines were validated biophysically
and pharmacologically on the QPatchHTsystem. Cells were pre-
pared by dissociation from T175 cell culture flasks using trypsin-
EDTA (0.05%), cells were kept in serum free media in the cell ho-
tel on board the QPatch HT. These cells were sampled, washed and
re-suspended in extracellular recording solution by the QPatch HT
immediately before application to well site on the chip. Once in
whole-cell configuration vehicle (0.1% DMSO v/v) was applied to
the cells as two bolus additions with a 2 min recording period
between each addition needed to achieve a stable control recording
g
0.17 ± 0.03
n.a ¼ not active (IC50 value higher than 10
mM).
a
The concentration of compound that inhibits a potassium channel current by
0%. Each IC50 value is the mean of three independent experiments.
5
analogue 6o showed weak inhibitory activities against K
inhibition at 1 M) and K 1.1 (22% inhibition at 1 M) channels,
respectively. Compounds of the type II series (14a-c) were not
active against any of the tested K 1 isoforms.
In general, the activities of compounds on K
expressed in CHO cells and on K 1 channels expressed in X. laevis
oocytes were comparable (relative rank order of potencies). Small
differences can be explained with potential differences in post-
translational modifications of channels because two different
expression systems were used. Furthermore, differences in
expression of auxiliary subunits might influence kinetics of channel
v
1.3 (28%
m
v
m
(4-min total). This was followed by application of four escalating
concentrations of test sample applied as double bolus additions per
test concentration with 2 min recording time per addition. Currents
were elicited from a holding potential of ꢁ80 mV with 500 ms
activating step to þ30 mV applied at a sweep frequency of 0.067 Hz,
v
v
1 channels
v
v
except K 1.5 which was activated by 900 ms pulses to 0 mV at a
sweep frequency of 0.2 Hz. Series resistance (4e15 MU) was
compensated by 80% and leak subtraction calculated using a P/n
protocol. For each sweep of the voltage protocol charge was
calculated as the current integral over time (5e95% duration)
during the 500 ms activating test step.
gating [27]. In case of CHO cells the auxiliary
co-expressed so K channels might have somewhat different gating
b subunits were not
v
Data was captured using the QPatch assay software (v5.0). The %
inhibition was calculated from mean charge over the last three
sweeps at the end of each concentration application period relative
to that measured at the end of the control period after current
stabilisation. Concentration response curves (four parameter lo-
gistic curve) were fitted to the % inhibition data using a bio-
informatics suite developed running in Pipeline Pilot (Biovia,
USA) from which the IC50 (50% inhibitory concentration) was
determined. All data are presented as mean ± SD for a minimum of
3 independent observations.
characteristics. Moreover, the small differences observed between
both test systems can be explained based on the differences in
membrane composition together with inherent differences in os-
molarity of the recording solutions.
3
. Conclusion
In recent years, the modulators of voltage-gated potassium
channels have received increased scientific attention. Marine nat-
ural products represent a viable source of bioactive compounds that
modulate the activity of various ion channels. In this study, we have
discovered that two alkaloids produced by the Agelas sponges,
4.1.2. Two-electrode voltage clamp
For the expression of the VGPCs (rK
v v v v
1.1, rK 1.2, hK 1.3, rK 1.4,
hymenidin and oroidin, inhibit the activity of K
v
1.3, K
v
v
1.4, K 1.5,
rK
v
1.5, rK 1.6, hK 10.1) in Xenopus laevis oocytes, the linearized
v
v

238
N. Zidar et al. / European Journal of Medicinal Chemistry 139 (2017) 232e241
ꢀ
plasmids were transcribed using the T7 or SP6 mMESSAGE-
mMACHINE transcription kit (Ambion, USA). The harvesting of
stage V-VI oocytes from anaesthetized female Xenopus laevis frog
was previously described [28]. Oocytes were injected with 50 nL of
cRNA at a concentration of 1 ng/nL using a micro-injector (Drum-
mond Scientific, USA). The oocytes were incubated in a solution
dimethylformamide (1 mL) at 0 C. After 1 h, the mixture was
warmed to rt and stirred under argon for 5 h. The solvent was
evaporated under reduced pressure, and the residue was purified
by flash column chromatography using dichloromethane/methanol
3
saturated with NH (6:1) as an eluent.
2
containing (in mM): NaCl, 96; KCl, 2; CaCl2, 1.8; MgCl , 2 and
4
.3.1.1. (E)-N-(3-(2-amino-1H-imidazol-4-yl)allyl)-4-bromo-5-
methyl-1H-pyrrole-2-carboxamide (6d). Synthesized from 13
45 mg, 0.326 mmol) and 4-bromo-5-methyl-1H-pyrrole-2-
HEPES, 5 (pH 7.4), supplemented with 50 mg/L gentamicin sulfate.
Two-electrode voltage clamp recordings were performed at
(
ꢀ
room temperature (18e22 C) using a Geneclamp 500 amplifier
carboxylic acid (67 mg, 0.326 mmol) using general coupling pro-
cedure with TBTU and NMM. Yield: 35 mg (33%) as white solid; m.
p.: 124e127 C; IR (ATR):
(
Molecular Devices, USA) controlled by a pClamp data acquisition
system (Axon Instruments, USA). Whole cell currents from oocytes
were recorded 1e4 days after injection. Bath solution composition
ꢀ
n
¼ 3214, 2918, 2851, 1615, 1573, 1528,
1480, 1417, 1328, 1267, 1215, 1098, 1049, 1023, 957, 805, 717,
was ND96 (in mM): NaCl, 96; KCl, 2; CaCl
(pH 7.4). Voltage and current electrodes were filled with 3 M KCl.
Resistances of both electrodes were kept between 0.8 and 1.5 M
2
, 1.8; MgCl
2
, 2 and HEPES,
ꢁ1 1
6
29 cm ; H NMR (MeOH-d
4
, 400 MHz): 2.23 (s, 3H, -CH
-), 5.90 (td, 1H,
-), 6.30 (td, 1H, J ¼ 15.8 Hz,
-), 6.48 (s, 1H, imidazole-H), 6.75 (s, 1H,
pyrrole-H) ppm; C NMR (MeOH-d , 100 MHz): 162.59, 151.97,
31.45, 125.34, 122.49, 121.79, 117.65, 113.57, 96.67, 49.73, 42.14,
3
), 4.02
5
(
dd, 2H, J ¼ 6.1 Hz, J ¼ 1.3 Hz, -CH¼CH-CH
J ¼ 15.7 Hz, J ¼ 6.1 Hz, -CH¼CH-CH
J ¼ 1.3 Hz, -CH¼CH-CH
2
U
.
2
The elicited currents were filtered at 500 Hz and sampled at 1 kHz
using a four-pole low-pass Bessel filter. Leak subtraction was per-
formed using a -P/4 protocol. K 1 currents were evoked by 500 ms
v
2
13
4
1
depolarizations to 0 mV followed by a 500 ms pulse to ꢁ50 mV,
þ
5
11.39 ppm; HRMS (C12H14BrN O): calculated (MH ) 324.0460;
from a holding potential of ꢁ90 mV. All data represent at least three
þ
found (MH ) 324.0458.
independent experiments (n
mean ± standard error.
ꢂ
3) and are presented as
4
.3.1.2. (E)-N-(3-(2-amino-1H-imidazol-4-yl)allyl)-3,4-dibromo-5-
methyl-1H-pyrrole-2-carboxamide (6e). Synthesized from 4 (40 mg,
.289 mmol) and 3,4-dibromo-5-methyl-1H-pyrrole-2-carboxylic
4.2. Chemistryegeneral
0
acid (82 mg, 0.289 mmol) using general coupling procedure with
TBTU and NMM. Yield: 36 mg (30%) as white-yellow solid; m. p.:
Chemicals were obtained from Acros Organics (Geel, Belgium),
Sigma-Aldrich (St. Louis, MO, USA) and Apollo Scientific (Stockport,
UK) and used without further purification. Analytical TLC was
performed on silica gel Merck 60 F254 plates (0.25 mm), using
visualization with UV light and spray reagents. Column chroma-
tography was carried out on silica gel 60 (particle size 240e400
mesh). HPLC analyses were performed on an Agilent Technologies
ꢀ
1
1
4
-
23e126 C; IR (ATR):
n
¼ 3396, 3191, 2924, 1620, 1528, 1475, 1371,
ꢁ
1 1
264, 1166, 1062, 957, 760, 708 cm
;
H NMR (MeOH-d
), 4.11 (dd, 2H, J ¼ 6.1 Hz, J ¼ 1.3 Hz,
-), 5.93 (td, 1H, J ¼ 15.7 Hz, J ¼ 6.0 Hz, -CH¼CH-CH -),
.36 (td, 1H, J ¼ 15.8 Hz, J ¼ 1.3 Hz, -CH¼CH-CH -), 6.50 (s, 1H,
, 100 MHz): 161.25, 152.13,
31.94, 123.22, 123.18, 122.72, 120.99, 100.94, 100.54, 49.87, 42.33,
4
,
00 MHz): 2.28 (s, 3H, -CH
3
CH¼CH-CH
2
2
6
2
13
imidazole-H) ppm; C NMR (MeOH-d
4
1
100 instrument (Agilent Technologies, Santa Clara, CA, USA) with a
G1365B UVeVis detector, a G1316A thermostat and a G1313A
autosampler using Phenomenex Luna 5- C18 column
1
1
þ
5
2.16 ppm; HRMS (C12H13BrN O): calculated (MH ) 401.9565;
found (MH ) 401.9562.
a
mm
þ
(
4.6 ꢃ 150 mm or 4.6 ꢃ 250 mm, Phenomenex, Torrance, CA, USA)
and a flow rate of 1.0 mL/min. The eluent consisted of trifluoro-
acetic acid (0.1% in water) as solvent A and methanol or acetonitrile
as solvent B. Melting points were determined on a Reichert hot
4
.3.1.3. (E)-N-(3-(2-amino-1H-imidazol-4-yl)allyl)-4-chloro-5-
methyl-1H-pyrrole-2-carboxamide (6f). Synthesized from 4 (60 mg,
0.435 mmol) and 4-chloro-5-methyl-1H-pyrrole-2-carboxylic acid
1
13
stage microscope and are uncorrected. H and C NMR spectra
were recorded at 400 and 100 MHz, respectively, on a Bruker
AVANCE III 400 spectrometer (Bruker Corporation, Billerica, MA,
(
69 mg, 0.435 mmol) using general coupling procedure with TBTU
ꢀ
and NMM. Yield: 36 mg (30%) as a yellow solid; m. p.: 125e127 C;
IR (ATR):
n
¼ 3210, 2919, 1614, 1577, 1529, 1484, 1419, 1330, 1269,
6 4 3
USA) in DMSO-d , MeOH-d or CDCl solutions, with TMS as the
ꢁ
1 1
1
4
218, 1164, 1073, 1026, 957, 808, 753, 688 cm ; H NMR (MeOH-d ,
4
internal standard. E-configuration was determined measuring
corresponding coupling constant [30]. IR spectra were recorded on
a Thermo Nicolet Nexus 470 ESP FT-IR spectrometer (Thermo Fisher
Scientific, Waltham, MA, USA). Mass spectra were obtained using a
VG Analytical Autospec Q mass spectrometer (Fisons, VG Analytical,
Manchester, UK). HPLC was used to establish the purity of target
compounds. The purity of the tested compounds was established to
be ꢂ 95%.
00 MHz): 2.22 (s, 3H, -CH
-), 5.89 (m, 1H, -CH¼CH-CH
J ¼ 15.8 Hz, J ¼ 1.3 Hz, -CH¼CH-CH -), 6.47 (s, 1H, imidazole-H),
.68 (s, 1H, pyrrole-H) ppm; C NMR (MeOH-d , 100 MHz):
62.70, 152.07, 129.49, 124.14, 122.68, 121.57, 111.23, 111.00, 49.73,
3
), 4.02 (dd, 2H, J ¼ 6.1 Hz, J ¼ 1.3 Hz,
-
CH¼CH-CH
2
2
-), 6.30 (td, 1H,
2
13
6
1
4
þ
4
2
2.16, 10.38 ppm; HRMS (C12
80.0965; found (MH ) 280.0959.
H14ClN
5
O): calculated (MH )
þ
4.3. Synthetic procedures
4.3.1.4. (E)-N-(3-(2-amino-1H-imidazol-4-yl)allyl)-4,5-dichloro-1H-
pyrrole-2-carboxamide (6g). Synthesized from (50 mg,
4
Clathrodin (6a), hymenidin (6b), oroidin (6c), and compounds
h and 6i were synthetized as described [26,29].
0.357 mmol) and 4,5-dichloro-1H-pyrrole-2-carboxylic acid
(64 mg, 0.357 mmol) using general coupling procedure with TBTU
and NMM. Yield: 37 mg (35%) as white-yellow solid; m. p.:
6
4
0
ꢀ
.3.1. General coupling procedure with TBTU and NMM
The corresponding carboxylic acid (0.36 mmol), TBTU (137 mg,
.414 mmol) and N-methylmorpholine (0.08 mL, 0.72 mmol) were
dissolved in dry dimethylformamide (2 mL) and stirred under
argon at rt for 1 h. The prepared mixture was added dropwise to a
stirred solution of compound 4 or 13 (50 mg, 0.36 mmol) and N-
75e77 C; IR (ATR):
n
¼ 3370, 3152, 1688, 1613, 1567, 1525, 1433,
ꢁ
1 1
1329, 1246, 1017, 958, 825, 757, 627 cm
400 MHz): 4.04 (dd, 2H, J ¼ 6.0 Hz, J ¼ 1.2 Hz, -CH¼CH-CH
(td, 1H, J ¼ 15.9 Hz, J ¼ 6.0 Hz, -CH¼CH-CH -), 6.31 (td, 1H,
J ¼ 15.8 Hz, J ¼ 1.2 Hz, -CH¼CH-CH -), 6.59 (s, 1H, imidazole-H),
6.79 (s, 1H, pyrrole-H) ppm; HRMS (C11 O): calculated
;
H NMR (MeOH-d
4
,
2
-), 5.97
2
2
2 5
H11Cl N
þ
þ
methylmorpholine
(0.08
mL,
0.72
mmol)
in
dry
(MH ) 300.0419; found (MH ) 300.0420.

N. Zidar et al. / European Journal of Medicinal Chemistry 139 (2017) 232e241
239
þ
4
.3.1.5. (E)-N-(3-(2-amino-1H-imidazol-4-yl)allyl)-5-chloro-1H-
(MH ) 244.1202.
indole-2-carboxamide (6j). Synthesized from
4
(50 mg,
0
0
.357 mmol) and 5-chloro-1H-indole-2-carboxylic acid (70 mg,
4.3.1.9. tert-butyl (E)-4-((3-(2-amino-1H-imidazol-4-yl)allyl)carba-
moyl)piperidine-1-carboxylate (6n). Synthesized from 4 (55 mg,
0.393 mmol) and 1-(tert-butoxycarbonyl)piperidine-4-carboxylic
acid (91 mg, 0.393 mmol) using general coupling procedure with
.357 mmol) using general coupling procedure with TBTU and
ꢀ
NMM. Yield: 40 mg (36%) as yellow solid; m. p.: 149e152 C; IR
(
ATR):
n
¼ 3230, 2923, 1618, 1541, 1475, 1416, 1319, 1283, 1253, 1210,
ꢁ
1 1
1165,1126,1060, 957, 917, 867, 799, 732, 653 cm ; H NMR (MeOH-
TBTU and NMM. Yield: 53 mg (37%) as brown solid; m. p.:
ꢀ
d
4
, 400 MHz): 4.11 (dd, 2H, J ¼ 6.2 Hz, J ¼ 1.3 Hz, -CH¼CH-CH
.95 (td, 1H, J ¼ 15.8 Hz, J ¼ 6.1 Hz, -CH¼CH-CH -), 6.36 (td, 1H,
-), 6.50 (s, 1H, imidazole-H),
.06 (d, 1H, J ¼ 0.9 Hz, indole-H), 7.19 (dd, 1H, J ¼ 8.8 Hz,
J ¼ 2.1 Hz, indole-H), 7.42e7.45 (m, 1H, indole-H), 7.61 (dd, 1H,
2
-),
98e101 C; IR (ATR):
n
¼ 3310, 2929, 2858, 1646, 1579, 1536, 1424,
ꢁ
1 1
5
2
1365, 1322, 1221, 957 cm ; H NMR (DMSO-d5, 400 MHz): 1.42 (s,
9H, (CH ), 2.08e2.18 (m, 2H, piperidine-CH -), 2.32e2.38 (m, 2H,
piperidine-CH -), 2.73e2.79 (m,1H, piperidine-CH-), 3.49e3.53 (m,
2H, piperidine-CH -), 3.81e3.86 (m, 2H, piperidine-CH -), 3.93 (dd,
2H, J ¼ 6.0 Hz, J ¼ 1.3 Hz, -CH¼CH-CH -), 6.02 (td, 1H, J ¼ 16.0 Hz,
J ¼ 6.0 Hz, -CH¼CH-CH -), 6.31 (dd, 1H, J ¼ 16.0 Hz, J ¼ 1.5 Hz,
-CH¼CH-CH2-), 6.82 (s, 1H, imidazole-H) ppm; HRMS
J ¼ 15.8 Hz, J ¼ 1.3 Hz, -CH¼CH-CH
2
3
)
3
2
7
2
2
2
13
J ¼ 2.0 Hz, J ¼ 0.6 Hz, indole-H) ppm; C NMR (MeOH-d
4
,
2
100 MHz): 162.04, 150.59, 135.16, 132.44, 128.57, 125.31, 123.80,
2
121.45, 120.41, 119.99, 116.30, 112.99, 102.26, 48.10, 41.04 ppm.;
þ
þ
þ
þ
HRMS (C15
H14ClN
5
O): calculated (MH ) 316.0965; found (MH )
(C17
H
28
N
5
O
3
): calculated (MH ) 350.2192; found (MH ) 350.2197.
316.0961.
4
.3.1.10. (E)-N-(3-(2-amino-1H-imidazol-4-yl)allyl)piperidine-4-
4
.3.1.6. (E)-N-(3-(2-amino-1H-imidazol-4-yl)allyl)-5-methoxy-1H-
(60 mg,
.435 mmol) and 5-methoxy-1H-indole-2-carboxylic acid (83 mg,
carboxamide (6o). Synthesized from 6n (27 mg, 0.077 mmol) by
Boc deprotection in HCl/EtOH. Yield: 13 mg (66%) as brown solid;
indole-2-carboxamide (6k). Synthesized from
0
0
4
ꢀ
m. p.: 43e45 C; IR (ATR):
n
¼ 3259, 2925, 2852, 2499, 1636, 1542,
ꢁ
1 1
.435 mmol) using general coupling procedure with TBTU and
1451, 1301, 1239, 1075, 1004, 958 cm
400 MHz): 1.87e1.97 (m, 2H, piperidine-CH
piperidine-CH -), 2.59e2.65 (m, 1H, piperidine-CH-), 3.03e3.10 (m,
2H, piperidine-CH -), 3.43e3.47 (m, 2H, piperidine-CH -), 3.94 (dd,
2H, J ¼ 6.0 Hz, J ¼ 1.3 Hz, -CH¼CH-CH -), 6.01 (td, 1H, J ¼ 16.0 Hz,
J ¼ 6.0 Hz, -CH¼CH-CH -), 6.31 (ddd, 1H, J ¼ 16.0 Hz, J ¼ 1.5 Hz,
J ¼ 1.1 Hz, -CH¼CH-CH -), 6.71 (s, 1H, imidazole-H) ppm; C NMR
(MeOH-d , 100 MHz): 174.35, 148.63, 126.85, 124.27, 118.06, 112.10,
73.00, 51.89, 43.00, 40.51, 39.60, 25.38 ppm; HRMS (C12 O):
;
H NMR (MeOH-d
4
,
ꢀ
NMM. Yield: 47 mg (35%) as orange solid; m. p.: 125e128 C; IR
2
-), 2.02e2.07 (m, 2H,
(
1
(
ATR):
161, 1119, 1024, 957, 840, 800, 763, 733, 669 cm
MeOH-d , 400 MHz): 3.83 (s, 3H, -OCH
), 4.11 (dd, 2H, J ¼ 6.1 Hz,
J ¼ 1.3 Hz, -CH¼CH-CH -), 5.95 (td, 1H, J ¼ 15.9 Hz, J ¼ 6.0 Hz,
CH¼CH-CH -), 6.36 (td, 1H, J ¼ 15.8 Hz, J ¼ 1.3 Hz, -CH¼CH-CH -),
.50 (s, 1H, imidazole-H), 6.90 (dd, 1H, J ¼ 8.9 Hz, J ¼ 2.5 Hz, indole-
H), 7.03 (d, 1H, J ¼ 0.9 Hz, indole-H), 7.08 (d, 1H, J ¼ 2.1 Hz, indole-
n
¼ 3239, 2936, 1617, 1541, 1482, 1450, 1417, 1376, 1331, 1225,
2
ꢁ1
1
;
H NMR
2
2
4
3
2
2
2
13
-
6
2
2
2
4
19 5
H N
13
þ þ
calculated (MH ) 250.1668; found (MH ) 250.1667.
H), 7.33e7.36 (m, 1H, indole-H) ppm;
C
NMR (MeOH-d
4
,
100 MHz): 163.95, 155.85, 151.97, 133.68, 132.59, 129.34, 122.69,
1
21.65, 116.42, 113.89, 104.18, 103.12, 56.05, 49.85, 42.40 ppm;
4.3.1.11. N-(3-(2-amino-1H-imidazol-4-yl)propyl)-4,5-dibromo-1H-
pyrrole-2-carboxamide (14a). Synthesized from 13 (40 mg,
0.286 mmol) and 4,5-dibromo-1H-pyrrole-2-carboxylic acid
þ
þ
17 5 2
HRMS (C16H N O ): calculated (MH ) 312.1461; found (MH )
312.1465.
(
77 mg, 0.286 mmol) using general coupling procedure with TBTU
ꢀ
4
.3.1.7. (E)-N-(3-(2-amino-1H-imidazol-4-yl)allyl)-indoline-2-
and NMM. Yield: 56 mg (51%) as white crystals; m. p.: 68e70 C; IR
carboxamide (6l). Synthesized from 4 (83 mg, 0.602 mmol) and
indoline-2-carboxylic acid (115 mg, 0.602 mmol) using general
coupling procedure with TBTU and NMM. Yield: 50 mg (27%) as
(ATR):
n
¼ 3481, 3381, 3121, 2938,1689, 1644,1561, 1510, 1429, 1414,
ꢁ
1 1
1367, 1316, 1235, 1055, 1014, 973, 830, 780, 651 cm
;
H NMR
NH-), 2.51
NH-), 3.35e3.37 (m, 2H,
NH-), 6.37 (s, 1H, imidazole-H), 6.82 (s, 1H, pyrrole-H)
ppm; C NMR (MeOH-d , 100 MHz): 161.91, 149.95, 132.21, 128.97,
114.17, 111.26, 106.11, 99.87, 39.89, 30.03, 24.52 ppm; HRMS
(MeOH-d
(t, 2H,
4
, 400 MHz): 1.85 (p, 2H, J ¼ 7.2 Hz, -CH
2 2 2
CH CH
ꢀ
white solid; m. p.: 79e82 C; IR (ATR):
1
4
(
n
¼ 3328, 2915, 1638, 1578,
J
¼
7.7 Hz, -CH CH CH
2
2
2
ꢁ
1 1
526, 1483, 1465, 1245, 1097, 958, 743 cm
;
H NMR (DMSO-d
00 MHz): 2.90 (dd, 1H, J ¼ 16.0 Hz, J ¼ 8.8 Hz, -NHCHCH -), 3.36
-), 3.59 (dd, 2H,
-), 4.01 (m, 1H, -NHCHCH -), 5.71
-), 6.50
6
,
2 2 2
-CH CH CH
1
3
2
4
dd, 1H, J ¼ 16.0 Hz, J ¼ 10.5 Hz, -NHCHCH
2
þ þ
J ¼ 6.2 Hz, J ¼ 1.3 Hz, -CH¼CH-CH
2
2
(C11H13Br N O): calculated (MH ) 389.9565; found (MH )
389.9559.
2 5
(
(
m, 1H, -CH¼CH-CH
2
-), 6.09 (d, 1H, J ¼ 15.6 Hz, -CH¼CH-CH
2
s, 1H, imidazole-H), 6.61e6.65 (m, 2H, indoline-H), 6.99 (t, 1H,
J ¼ 5.51, -CONH-), 7.12e7.18 (m, 2H, indoline-H) ppm; HRMS
4.3.1.12. N-(3-(2-amino-1H-imidazol-4-yl)propyl)-1H-indole-2-
carboxamide (14b). Synthesized from 13 (53 mg, 0.378 mmol) and
indole-2-carboxylic acid (61 mg, 0.378 mmol) using general
þ
þ
(
C
15
H
18
N
5
O): calculated (MH ) 284.1511; found (MH ) 284.1510.
4
.3.1.8. (E)-N-(3-(2-amino-1H-imidazol-4-yl)allyl)nicotinamide
coupling procedure with TBTU and NMM. Yield: 63 mg (59%) as
ꢀ
(6m). Synthesized from 4 (50 mg, 0.357 mmol) and nicotinic acid
white crystals; m. p.: 76e78 C; IR (ATR):
n
¼ 3617, 3392, 2868,
(
44 mg, 0.357 mmol) using general coupling procedure with TBTU
2567, 1679, 1616, 1553, 1493, 1429, 1417, 1340, 1308, 1257, 1035,
ꢀ
ꢁ1
1
and NMM. Yield: 22 mg (24%) as a white solid; m. p.: 81e83 C; IR
745 cm
;
H
NMR (DMSO-d
NH-), 2.51 (m, 2H, -CH
CH NH-), 6.39 (s,1H, imidazole-H), 6.89 (s,1H, pyrrole-
6
,
400 MHz): 1.80 (m, 2H,
(
1
(
ATR):
027, 958, 825, 703, 627 cm ; H NMR (MeOH-d
dd, 2H, J ¼ 6.3 Hz, J ¼ 1.3 Hz, -CH¼CH-CH
J ¼ 15.8 Hz, J ¼ 6.0 Hz, -CH¼CH-CH -), 6.36 (td, 1H, J ¼ 15.8 Hz,
-), 6.51 (s, 1H, imidazole-H), 6.57 (ddd, 1H,
n
¼ 3219, 2925, 163, 1591, 1539, 1474, 1418, 1308, 1197, 1161,
2
-CH CH
2
CH
CH
2
2
CH CH NH-), 3.36e3.38 (m,
2
2
ꢁ1 1
4
, 400 MHz): 4.12
-), 5.95 (td, 1H,
2H, -CH
2
2
2
þ
2
H) ppm; HRMS (C15
(MH ) 284.1505.
H
18
N
5
O): calculated (MH ) 284.1511; found
þ
2
J ¼ 1.2 Hz, -CH¼CH-CH
2
J ¼ 8.0 Hz, J ¼ 4.9 Hz, J ¼ 0.9 Hz, pyridine-H), 8.28 (ddd, 1H,
J ¼ 8.0 Hz, J ¼ 2.3 Hz, J ¼ 1.7 Hz, pyridine-H), 8.70 (dd, 1H, J ¼ 4.9 Hz,
J ¼ 1.6 Hz, pyridine-H), 9.01 (dd, 1H, J ¼ 2.3 Hz, J ¼ 0.9 Hz, pyridine-
4.3.1.13. N-(3-(2-amino-1H-imidazol-4-yl)propyl)indoline-2-
carboxamide (14c). Synthesized from 13 (35 mg, 0.250 mmol) and
indoline-2-carboxylic acid (41 mg, 0.250 mmol) using general
H) ppm; 13C NMR (MeOH-d
, 100 MHz): 167.51, 152.63, 152.06,
coupling procedure with TBTU and NMM. Yield: 37 mg (53%) as
4
ꢀ
149.17, 137.06, 132.08, 125.18, 123.32, 120.99, 117.61, 49.73,
white crystals; m. p.: 67e69 C; IR (ATR):
n
¼ 3306, 2926, 1640,
þ
4
3.02 ppm; HRMS (C12
H
13
N
5
O): calculated (MH ) 244.1198; found
1607, 1560, 1531, 1484, 1466, 1245, 1104, 1057, 1018, 996, 883, 744,

240
N. Zidar et al. / European Journal of Medicinal Chemistry 139 (2017) 232e241
32, 602 cm 1; 1H NMR (MeOH-d
J ¼ 7.1 Hz, -CH CH CH NH-), 2.45 (t, 2H, J ¼ 7.7 Hz, -CH
, 2.97 (dd, 1H, J ¼ 16.0 Hz, J ¼ 8.9 Hz, -NHCHCH -), 3.27e3.30 (m,
H, -CH CH CH
NH), 3.47 (dd, 1H, J ¼ 16.0 Hz, J ¼ 10.5 Hz,
NHCHCH -), 6.27 (s, 1H, imidazole-H), 6.69e6.73 (m, 2H, indoline-
H), 6.99e7.06 (m, 2H, indoline-H) ppm; C NMR (MeOH-d
00 MHz): 177.19, 151.93, 150.50, 133.57, 128.78, 128.57, 125.35,
20.57, 111.70, 111.43, 62.62, 39.72, 36.49, 29.99, 25.14 ppm; HRMS
ꢁ
, 400 MHz): 1.78 (p, 2H,
CH CH NH-
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ꢀ
ꢀ