Substituted 4-phenyl-2-aminoimidazoles and 4-phenyl-4,5-dihydro-2- aminoimidazoles as voltage-gated sodium channel modulators

Article abstract of DOI:10.1016/j.ejmech.2013.12.034

Voltage-gated sodium channels play an integral part in neurotransmission and their dysfunction is frequently a cause of various neurological disorders. On the basis of the structure of marine alkaloid clathrodin, twenty eight new analogs were designed, sy

Full text of DOI:10.1016/j.ejmech.2013.12.034

European Journal of Medicinal Chemistry 74 (2014) 23e30
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European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Substituted 4-phenyl-2-aminoimidazoles and 4-phenyl-4,5-dihydro-
2-aminoimidazoles as voltage-gated sodium channel modulators
a
a
b
b
c
c
ꢀ
Nace Zidar , Ziga Jakopin , David J. Madge , Fiona Chan , Jan Tytgat , Steve Peigneur ,
a
ꢀ ꢁ a
ꢀa
ꢀ ꢀ a
Marija Sollner Dolenc , Tihomir Tomasic , Janez Ilas , Lucija Peterlin Masic ,
Danijel Kikelj a
,
*
a
ꢀ
ꢀ
University of Ljubljana, Faculty of Pharmacy, Askerceva 7, 1000 Ljubljana, Slovenia
b Xention Limited, Iconix Park, London Road, Pampisford, Cambridge CB22 3EG, UK
c University of Leuven (KU Leuven), Toxicology & Pharmacology, O&N2, PO Box 922, Herestraat 49, 3000 Leuven, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
Voltage-gated sodium channels play an integral part in neurotransmission and their dysfunction is
frequently a cause of various neurological disorders. On the basis of the structure of marine alkaloid
clathrodin, twenty eight new analogs were designed, synthesized and tested for their ability to block
human NaV1.3, NaV1.4 and NaV1.7 channels, as well as for their selectivity against human cardiac isoform
NaV1.5, using automated patch clamp electrophysiological assay. Several compounds exhibited promising
activities on different NaV channel isoforms in the medium micromolar range and some of the com-
pounds showed also moderate isoform selectivities. The most promising results were obtained for the
Received 7 November 2013
Received in revised form
16 December 2013
Accepted 22 December 2013
Available online 3 January 2014
Keywords:
NaV1.3 channel, for which four compounds were found to possess IC50 values lower than 15 mM. All of the
Voltage-gated sodium channel
NaV channel modulator
Marine alkaloid
Clathrodin
Pyrrole-2-aminoimidazole
4-Phenyl-2-aminoimidazole
active compounds bind to the open-inactivated states of the channels and therefore act as state-
dependent modulators. The obtained results validate the approach of using natural products driven
chemistry for drug discovery starting points and represent a good foundation for future design of se-
lective NaV modulators.
Ó 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction
muscle, NaV1.5 in the heart, and NaV1.7e1.9 are characteristic of the
peripheral nervous system [1e7].
Voltage-gated sodium channels (VGSC, NaV channels) are inte-
gral membrane proteins which play essential roles in the initiation
and propagation of action potentials in neurons and other electri-
The abnormally increased activity of sodium channels leads to
over-excitation of specific groups of cells which can cause different
neurodegenerative diseases, chronic pain, epilepsy, arrhythmias
and spasticity. Mutations of NaV channel genes and their encoded
proteins have been identified in heart, brain, skeletal muscle and
peripheral nerves [8e11]. Both, gain and loss of function mutations
in NaV1.1 and NaV1.2 can give rise to epilepsy [12,13] and migraine
[14]. A number of preclinical studies have implicated NaV1.3, NaV1.7,
NaV1.8 and NaV1.9 in nociceptive processing [15,16] and mutations
in their genes were linked to spontaneous chronic pain. Mutations
of NaV1.4 that result in hyperactive skeletal sodium channels have
been shown to cause myotonia or flaccid paralysis [17], whereas
mutations of NaV1.5 have profound effects on cardiac function,
leading to a range of cardiac abnormalities [18]. Owing to the
extremely broad therapeutic potential of VGSC modulators, and to
the recently disclosed first X-ray crystal structures of bacterial NaV
channels [1e3], discovery of VGSC modulators has become one of
the most attractive topics in medicinal chemistry.
cally excitable cells. They are composed of a single
forms a voltage-sensing pore and one or more auxiliary
To date, nine different -subunits (NaV1.1eNaV1.9) and four
different -subunits have been identified. The VGSC isoforms are
a
-subunit which
b
-subunits.
a
b
distributed differentially throughout the electrically excitable cells
of the body, and have different functional properties according to
their role in each tissue type. NaV1.1e1.3 and NaV1.6 are mainly
expressed in the central nervous system, NaV1.4 in the skeletal
Abbreviations: VGSC, voltage-gated sodium channel; NaV channel, voltage-gated
sodium channel; TBTU, N,N,N0,N0-tetramethyl-O-(benzotriazol-1-yl)uronium tetra-
fluoroborate; DMAP, 4-(dimethylamino)pyridine; NMM, N-methylmorpholine; Pro,
proline; THF, tetrahydrofuran; DMF, N,N-dimethylformamide; DMAP, 4-
dimethylaminopyridine; TFA, trifluoroacetic acid; ESI, electrospray ionization;
Boc2O, di-tert-butyl dicarbonate; Boc, tert-butyloxycarbonyl.
Although there are several drugs acting at NaV channels, e.g.
local anesthetics (lidocaine, procaine), antiarrhythmics (lidocaine,
* Corresponding author. Tel.: þ386 1 4769561; fax: þ386 1 4258031.
E-mail address: danijel.kikelj@ffa.uni-lj.si (D. Kikelj).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.12.034

24
N. Zidar et al. / European Journal of Medicinal Chemistry 74 (2014) 23e30
tocainide, mexiletine) and antiepileptics (phenytoin, carbamaze-
pine, lamotrigine), a more rational approach is required to exploit
the full therapeutic potential of these drug targets. Current drugs
acting on NaV channels have low potency and are relatively non-
selective, therefore there is a need for the development of
isoform-selective modulators which offers the promise of signifi-
cant advantage over current therapeutic agents [9,19,20]. Recent
studies show promise that small molecules can be made selective
for different NaV channel isoforms [8,9,21e25]. Relatively favorable
side effect profiles of some currently used drugs acting on NaV
channels are generally attributed to their state-dependent action.
They usually have a much higher affinity for inactivated channels
compared to resting channels, therefore their IC50 values are
several hundred times higher on hyperpolarized VGSCs than on
VGSCs in depolarized membranes [26,27]. This state-dependency of
action is therefore considered to be a key predictor of a high ther-
apeutic index for a VGSC blocker.
The marine ecosystem is a rich source of chemically and func-
tionally diverse toxins that interact with NaV channels and provide
inspiration for drug design [28,29]. Alkaloids from the Caribbean
sponges of the genus Agelas, e.g. monomers clathrodin, hymenidin
and oroidin, and dimers sceptrin and dibromosceptrin (Fig. 1), have
been shown to interact with muscle and nerve membrane re-
ceptors and channels, including VGSCs. Electrophysiological studies
suggested that clathrodin and dibromosceptrin act on NaV channels
by influencing channel ion conductance, and by modifying the
channel inactivation characteristics, respectively [30]. These alka-
loids belong to the pyrrole-2-aminoimidazole structural class of
marine alkaloids, with intriguing structural complexity and diverse
biological activities [31,32]. In contrast to the majority of neuro-
toxins, which have high molecular weights and many chiral centers
leading to complex 3D architectures, the structures of clathrodin-
like alkaloids are relatively simple. This makes them suitable can-
didates for optimization using established medicinal chemistry
strategies. Due to their favorable size and physicochemical prop-
erties, they have the potential to permeate further into the channel
and possibly bind to sites which are not available to large toxin
molecules [9].
Fig. 2. Design of substituted 4-phenyl-2-aminoimidazoles and 4-phenyl-4,5-dihydro-
2-aminoimidazoles as conformationally restricted clathrodin analogs.
double bond. To achieve these goals, we decided to introduce a
phenyl ring at position 5 of the 2-aminoimidazole moiety so that
the length of the molecule would remain unchanged. To explore
the correct spatial arrangement of functional groups we designed
two sets of compounds, one with 1,3- and the other with 1,4-
substitution patterns on the phenyl ring. In addition to the 2-
substituted pyrrole ring present in the clathrodin molecule, ana-
logs with pyrrol-3-yl, furan-2-yl, indol-2-yl, indol-3-yl, (R)-pyr-
rolidin-2-yl or (S)-pyrrolidin-2-yl substituents were prepared. To
assess the importance of the free amino group of the 2-
aminoimidazole ring, we designed compounds 24e32 with an
N-methyl substituent in position 2 (Schemes 4 and 5). In addition,
to examine whether aromaticity and planarity of the imidazole
ring are important for biological activity, a set of 4,5-dihydro-2-
aminoimidazoles 27e32 with a reduced imidazole C]C bond
was prepared (Scheme 5).
2. Results and discussion
2.1. Chemistry
The synthetic route towards the key intermediates 4a, 4b and
compound 6 is presented in Scheme 1. 2-Bromo-3-nitroaceto
phenone or 2-bromo-4-nitroacetophenone was reacted with 2-
aminopyrimidine and a catalytic amount of 4-dimethylamino
pyridine in acetonitrile to afford the pyrimidinium salts 1a or 1b,
respectively. A dehydrated compound 1c was formed instead of 1a,
when the reaction of 2-aminopyrimidine and 2-bromo-3-
nitroacetophenone was carried out at 85 ꢀC instead of at room
temperature. This can probably be attributed to the electron
withdrawing effect of the nitro group on the phenyl ring, which is
more prominent when the nitro group is in the para position
(compound 1b) than in the meta position (compound 1a). Hydra-
zinolysis of compounds 1aec in ethanol using microwave irradia-
tion, followed by a Dimroth-type rearrangement resulted in
formation of substituted 2-aminoimidazoles 2a and 2b. Both steps
were accomplished following a literature procedure [36] where
detailed mechanisms of the reactions are described. In a first
attempt to prepare derivatives with carboxamido substituents on
the phenyl ring, after reduction of the nitro group of 2a by catalytic
The design of the present series of compounds was based on
the structure of the alkaloid clathrodin and is schematically pre-
sented in Fig. 2. The molecule of clathrodin consists of a 2-
aminoimidazole moiety that is connected to a pyrrole ring with
a 5-atom long, partially flexible, linker. This linker gives clathrodin
a certain extent of conformational freedom which is not optimal
for the binding affinity. In addition, the linker contains in its
structure a C]C double bond conjugated to the 2-aminoimidazole
ring that can undergo various transformations, such as additions
of different nucleophiles [33] or DielseAlder cycloadditions
[34,35]. The aim of the present study was to design derivatives
with a conformationally restricted central part of the molecule
that would at the same time lack the potentially unstable alkenyl
Fig. 1. Structures of Agelas alkaloids clathrodin, hymenidin, oroidin, sceptrin and dibromosceptrin.

N. Zidar et al. / European Journal of Medicinal Chemistry 74 (2014) 23e30
25
Scheme 1. Synthesis of key intermediates 4a and 4b, and compound 6. Reagents and conditions: (a) method 1: DMAP, CH3CN, rt, 5 h (for the synthesis of 1a and 1b); method 2:
DMAP, CH3CN, 85 ꢀC, 5 h (for the synthesis of 1c); (b) hydrazine hydrate, EtOH, MW: 120 ꢀC, 40 min; (c) Boc2O, 2:1 MeOH/H2O, rt, 3 h; (d) H2/PdeC, THF, rt, 5 h; (e) 1 equiv pyrrole-
2-carboxylic acid, TBTU, NMM, DMF, rt, 15 h.
hydrogenation, the obtained amine 5 was coupled with pyrrole-2-
carboxylic acid in a TBTU-promoted reaction. We found that
coupling occurred at two possible sites, the aromatic amino group
and the imidazole N-1, to give the product 6. Interestingly, the re-
action did not occur at the 2-amino group of the imidazole ring,
probably because of its low reactivity. Therefore, the imidazole N-1
nitrogen was Boc-protected by reacting compounds 2a and 2b with
Boc-anhydride in a mixture of methanol and water. The reduction
of the nitro group of Boc-protected derivatives 3a and 3b was
accomplished through catalytic hydrogenation in tetrahydrofuran
using PdeC as a catalyst, to give the target amines 4a and 4b.
The 4-phenyl-2-aminoimidazole analogs 7e17 were prepared
according to Scheme 2. TBTU-promoted coupling of 4a or 4b and
the corresponding carboxylic acid (pyrrole-2-carboxylic acid,
Scheme 2. Synthesis of 4-phenyl-2-aminoimidazoles 7e17. Reagents and conditions: (a) corresponding carboxylic acid, TBTU, NMM, CH2Cl2, 35 ꢀC, 24 h; (b) method 1: HCl(g), THF/
EtOH, rt, 5 h (for the synthesis of 8a, 10, 13a, 15 and 17a); method 2: acetyl chloride, EtOH, 0 ꢀC, 1 h (for the synthesis of 8b, 13b and 17b).

26
N. Zidar et al. / European Journal of Medicinal Chemistry 74 (2014) 23e30
Scheme 3. Synthesis of N-methylated intermediates 22 and 23. Reagents and conditions: (a) CH3I, acetone, 75 ꢀC, 15 h; (b) NaOH, EtOH/H2O, 80 ꢀC, 0.5 h; (c) 2-bromo-3-
nitroacetophenone, DMAP, CH3CN, 50 ꢀC, 15 h; (d) hydrazine hydrate, EtOH, MW: 120 ꢀC, 40 min; (e) Boc2O, 2:1 MeOH/H2O, rt, 3 h; (f) H2/PdeC, THF, rt, 5 h; (g) H2/PdeC, THF,
rt, 24 h.
pyrrole-3-carboxylic acid, furan-2-carboxylic acid, indole-2-
carboxylic acid, indole-3-carboxylic acid, N-Boc- -proline or N-
Boc- -proline) afforded Boc-protected derivatives 7a, 7b, 9, 11, 12a,
12b, 14 and 16aec which were converted into final compounds
upon cleavage of the Boc protecting group with either gaseous
hydrochloric acid or acetyl chlorideeethanol as a convenient re-
agent for in situ HCl formation [37].
N-Methylated intermediates 22 and 23 were prepared according
to Scheme 3. 2-(N-Methylamino)-pyrimidine (18) was prepared by
heating 2-aminopyrimidine and iodomethane in acetone followed
by treatment with 10% aqueous sodium hydroxide. This was then
reacted with 2-bromo-3-nitroacetophenone in acetonitrile and a
catalytic amount of 4-dimethylaminopyridine to afford 2-hydroxy-
1-methyl-2-(3-nitrophenyl)-2,3-dihydro-1H-imidazo[1,2-a]pyr-
imidin-4-ium bromide (19). After hydrazinolysis in ethanol using
microwave irradiation and subsequent Dimroth-type rearrange-
ment, compound 19 was converted into 2-(N-methylamino)-
imidazole 20 [36]. Boc protection of the imidazole N-1 of com-
pound 20, followed by reduction of the nitro group afforded the
amine 22. A prolonged catalytic hydrogenation (24 h) enabled both,
the reduction of the nitro group and the imidazole C]C bond, to
give the 4,5-dihydro-2-(N-methylamino)-imidazole derivative 23.
4-Phenyl-2-(N-methylamino)-imidazoles 24e26, and 4-phenyl-
4,5-dihydro-2-(N-methylamino)-imidazoles 27e32 were prepared
according to Schemes 4 and 5. TBTU-promoted coupling of amines
22 or 23, and the corresponding carboxylic acid (pyrrole-2-
L
D
Scheme 4. Synthesis of 4-phenyl-2-(N-methylamino)-imidazoles 24e26. Reagents and conditions: (a) corresponding carboxylic acid, TBTU, NMM, CH2Cl2, 35 ꢀC, 24 h; (b) HCl(g), THF/
EtOH, rt, 5 h.
Scheme 5. Synthesis of 4-phenyl-4,5-dihydro-(N-methylamino)-imidazoles 27e32. Reagents and conditions: (a) corresponding carboxylic acid, TBTU, NMM, CH2Cl2, 35 ꢀC, 24 h; (b)
HCl(g), EtOH, rt, 5 h.

N. Zidar et al. / European Journal of Medicinal Chemistry 74 (2014) 23e30
27
Table 2
carboxylic acid, indole-2-carboxylic acid or furan-2-carboxylic acid)
afforded Boc-protected derivatives 24, 25, 27, 29 and 31 which were
converted into end products upon cleavage of the Boc protecting
group.
Modulatory activities of 4-phenyl-4,5-dihydro-2-(N-methylamino)-imidazoles 27e
32 on the open-inactivated state of the NaV channel isoforms.
2.2. Biological evaluation
In total, twenty eight novel compounds were synthesized and
studied for their ability to block the human sodium channel iso-
forms NaV1.3, NaV1.4 and NaV1.7, as well as their selectivity against
the human cardiac isoform NaV1.5. The screening was performed
using an automated patch clamp electrophysiology assay on the
Sophion QPatch HT system (Sophion Bioscience A/S). Compounds of
three different structural classes, (i) 4-phenyl-2-aminoimidazoles
6e17, (ii) 4-phenyl-2-(N-methylamino)-imidazoles 24e26, and
(iii) 4-phenyl-4,5-dihydro-2-(N-methylamino)-imidazoles 27e32,
were tested and their IC50 values, i.e. concentrations of compounds
that inhibit sodium channel currents by 50%, are presented in
Tables 1 and 2. The IC50 values of synthesized compounds against
each NaV isoforms were determined from concentrationeresponse
relationships established by cumulatively applying four escalating
Compd
R1
R2
IC50 (m
M)a
NaV1.3
NaV1.4
NaV1.5
NaV1.7
27
Boc
H
Boc
H
Boc
H
Pyrrol-2-yl
Pyrrol-2-yl
Indol-2-yl
Indol-2-yl
Furan-2-yl
Furan-2-yl
24
eb
eb
eb
eb
eb
eb
2
24
eb
3
23
26
18
eb
eb
27
29
28c
21
29
eb
eb
eb
22
30c
eb
eb
eb
N/Ad
31
32c
Lidocaine
10.5
a
The concentration of compound that inhibits a sodium channel current by 50%.
Each IC50 value is the mean of three independent experiments.
b
IC50 value higher than 30
HCl salt.
N/A: not available.
mM.
c
d
concentrations (0.3e10 mM) of test compound to a cell, as detailed
in the Experimental section. All of the active compounds acted as
state-dependent VGSC modulators, blocking only the open-
inactivated state of the channels (depolarization from wꢁ70 mV)
and possessing no activity on the closed state of the channels, with
not shown). IC50 values for lidocaine as reference substance were
10.5 M against NaV1.3, 2.1 M against NaV1.4 and 29 M against
NaV1.7 channel. Activity of lidocaine against NaV1.5 isoform was not
determined.
Several compounds exhibited promising activities on different
NaV channel isoforms in medium micromolar range and some of
the compounds showed also moderate isoform selectivities, e.g.,
compounds 8b, 9, 12a, 16a, 16b, 17b, 24 and 26. The most promising
results were obtained for the NaV1.3 channel where four com-
m
m
m
IC50 values higher than 30
mM (depolarization from ꢁ100 mV, data
Table 1
Modulatory activities of 4-phenyl-2-aminoimidazoles 6e17 and 4-phenyl-2-(N-
methylamino)-imidazoles 24e26 on the open-inactivated state of the NaV channel
isoforms.
pounds had IC50 values lower than 15
pound was the (R)-proline derivative 17a blocking NaV1.3 with an
IC50 value of 8 M. Compound 17a was selective towards NaV1.4
and NaV1.5, and weakly active against the NaV1.7 isoform
(IC50 ¼ 28 M). Channel-blocking activity against the NaV1.3 with
an IC50 value of 13 M was observed also for compound 17b, an (S)-
mM. The most potent com-
m
m
m
Compd R1 R2
R3
Subst IC50
NaV1.3 NaV1.4 NaV1.5 NaV1.7
(m
M)a
proline-containing analog of 17a with a 1,4-substitution pattern on
the phenyl ring. Moreover, compound 17b did not possess any ac-
tivity on other isoforms and could thus represent a starting point
for the development of selective modulators of NaV1.3. Two other
compounds of the series, pyrrol-3-yl derivative 10 and indol-3-yl
derivative 15, also possessed NaV1.3 modulatory activity, both
6
H
Pyrrol- Pyrrol-2-yl
2-oyl
1,3
25
eb
27
19
7a
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Boc
Boc
H
H
Boc
H
Boc
Boc
Boc
H
H
Boc
H
Pyrrol-2-yl
Pyrrol-2-yl
Pyrrol-2-yl
Pyrrol-2-yl
Pyrrol-3-yl
Pyrrol-3-yl
Furan-2-yl
Indol-2-yl
Indol-2-yl
Indol-2-yl
Indol-2-yl
Indol-3-yl
Indol-3-yl
(R)-Pyrrolidin-2-yl 1,3
(R)-Pyrrolidin-2-yl 1,4
(S)-Pyrrolidin-2-yl 1,4
(R)-Pyrrolidin-2-yl 1,3
(S)-Pyrrolidin-2-yl 1,4
Furan-2-yl
Indol-2-yl
Indol-2-yl
1,3
1,4
1,3
1,4
1,3
1,3
1,4
1,3
1,4
1,3
1,4
1,3
1,3
25
eb
eb
eb
23
12
27
26
22
eb
eb
27
12
eb
eb
25
8
25
eb
eb
eb
eb
15
eb
eb
28
eb
eb
26
14
eb
eb
eb
eb
eb
eb
eb
eb
2
eb
eb
eb
eb
eb
eb
eb
eb
eb
eb
eb
eb
eb
eb
20
eb
eb
eb
eb
eb
20
eb
eb
eb
27
eb
eb
22
eb
eb
eb
eb
eb
26
29
eb
19
28
eb
26
eb
eb
7b
8ac
8bc
9
with an IC50 value of 12
against the NaV1.4 isoform with IC50 values of 15
m
M. Both compounds were also active
M and 14 M,
m
m
respectively. Interestingly, isomers of compounds 10 and 15 con-
taining 2- instead of 3-substituted pyrrole or indole rings, pyrrol-2-
yl derivative 8a and indol-2-yl derivative 13a were devoid of ac-
tivity on all NaV channel isoforms, suggesting the importance of the
orientation of pyrrole or indole rings for antagonist activity.
Furthermore, based on comparison of the isomeric pyrrol-3-yl
10c
11
12a
12b
13ac
13bc
14
15c
16a
16b
16c
17ac
17bc
24
derivatives 7a (IC50 value of 25
mM against NaV1.3) and 7b (IC50
Boc
Boc
Boc
H
value >30 M against NaV1.3), the 1,3-substitution pattern on the
m
phenyl ring is preferred over 1,4-substitution pattern for the NaV1.3
modulatory activity.
The most active modulators of the skeletal muscle isoform
H
13
eb
eb
eb
10.5
CH3 Boc
CH3 Boc
CH3 H
1,3
1,3
1,3
NaV1.4 were 4-phenyl-2-aminoimidazoles 10 (IC50 ¼ 15
(IC50 ¼ 14 M) which were also active against the NaV1.3 channel
with IC50 values of 12 M. The introduction of N-methyl substituent
mM) and 15
25
m
26c
m
Lidocaine
N/Ad 29
on the primary amino group of 2-aminoimidazole ring in de-
rivatives 24e32 resulted in complete loss of activity against the
NaV1.4 isoform.
The NaV1.5 channel isoform is located in the heart. Blockade of
this channel is associated with increased risk of a cardiac side-effect
a
The concentration of compound that inhibits a sodium channel current by 50%.
Each IC50 value is the mean of three independent experiments.
b
IC50 value higher than 30
HCl salt.
N/A: not available.
mM.
c
d

28
N. Zidar et al. / European Journal of Medicinal Chemistry 74 (2014) 23e30
liability, and it is regarded as an unwanted activity in any thera-
peutic molecule. Fortunately, the majority of tested compounds
were almost inactive against the NaV1.5, with the exception of
experiment. To study NaV1.5 currents, a pulse train consisting of 10
repetitive activating test pulses to ꢁ20 mV from a holding potential
of ꢁ100 mV were applied at a 1 Hz frequency until 10 pulses were
reached, this sequence was repeated at a sweep interval of 0.016 Hz
throughout the duration of the experiment. For NaV1.3, NaV1.4 and
NaV1.7 channels the peak inward current was determined for both
the closed and open-inactivated test pulses from each sweep
applied to the cells and for NaV1.5 from the tenth pulse of each
pulse train recorded. Data was captured using QPatch assay soft-
ware (v5.0). The % inhibition of peak current was calculated as the
mean peak current value for the last three sweeps measured in
each concentration test period relative to the last three sweeps
recorded during the control vehicle period. Sigmoidal concentra-
tion response curves (four parameter logistic curves) were fitted to
the % inhibition data using Xlfit (IDBS) from which the IC50 was
determined. Fits were constrained at 0 and 100%. Data are pre-
sented as mean ꢂ SD for minimum of 3 independent observations.
compound 29 with an IC50 of 3 mM.
The NaV1.7 isoform is mainly located in the peripheral nervous
system and has been identified as a promising drug target for the
treatment of neuropathic pain [15]. The most active compound
against the NaV1.7 channel was the indol-2-yl derivative 29
(IC50 ¼ 18
m
M), but as it also displayed activity against the NaV1.5
M) it is not suitable for further development. A
isoform (IC50 ¼ 3
m
moderate activity against the NaV1.7 isoform was observed also for
4-phenyl-2-aminoimidazoles 6 and 16c, both having IC50 values of
19
mM. Compound 16c displayed also a weak NaV1.3 modulatory
activity with IC50 value of 25
mM. As both, NaV1.3 and NaV1.7
channels have been linked to nociceptive processing, finding
compounds of dual activity against both isoforms could lead to
synergistic effects and contribute to favorable therapeutic profiles
of such compounds. Interestingly, compound 16b, the R isomer of
16c, was not active neither against the NaV1.3 nor the NaV1.7
channels.
4.2. Chemistry e general
Chemicals were obtained from Acros, Aldrich Chemical Co., and
Fluka 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 ninhydrin. Column chromatography
was carried out on silica gel 60 (particle size 240e400 mesh). HPLC
analyses were performed on an Agilent Technologies 1100 instru-
ment with a G1365B UV-VIS detector, a G1316A thermostat and a
3. Conclusion
We have designed and synthesized a series of clathrodin analogs
and evaluated their effects on human NaV1.3e1.5 and NaV1.7
channels using an automated patch clamp electrophysiology assay.
Several compounds exhibited promising activities with moderate
isoform selectivities. All of the active compounds acted as state-
dependent VGSC modulators. The most promising results were
obtained for the NaV1.3 channel, against which four compounds
G1313A autosampler using a Phenomenex Luna 5 mm C18 column
(4.6 ꢃ 150 mm or 4.6 ꢃ 250 mm) and flow rate of 1.0 mL/min. The
eluent consisted of trifluoroacetic acid (0.1% in water) or ammonia
(0.1% in water) as solvent A and methanol as solvent B. Microwave-
assisted reactions were performed using a CEM Discover micro-
wave reactor (CEM Corp.). Melting points were determined on a
were found to possess IC50 values lower than 15 mM. The results
provide valuable information on structureeactivity relationships
and offer good prospect for the development of isoform selective
VGSC modulators.
Reichert hot stage microscope and are uncorrected. 1H NMR and 13
C
NMR spectra were recorded at 400 and 100 MHz, respectively, on a
Bruker AVANCE III 400 spectrometer in DMSO-d6 or MeOH-d4 so-
lution, with TMS as the internal standard. IR spectra were recorded
on a PerkineElmer Spectrum BX FT-IR spectrometer or Thermo
Nicolet Nexus 470 ESP FT-IR spectrometer. Mass spectra were ob-
tained using a VGAnalytical Autospec Q mass spectrometer. Optical
rotations were measured on a PerkineElmer 241 MC polarimeter.
The reported values for specific rotation are average values of 10
successive measurements using an integration time of 5 s. The
purity of the tested compounds was established to be ꢄ95%.
4. Experimental section
4.1. Electrophysiology
Cells were prepared by dissociation from T175 cell culture flasks
using trypsineEDTA (0.05%), cells were kept in serum free media in
the cell hotel 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 to achieve a stable control recording (4-min
total). This was followed by application of test concentrations as a
single bolus addition (4-min incubation per test concentration).
Compounds were prepared in extracellular recording solution from
4.3. Synthetic procedures
4.3.1. General procedure A. Synthesis of compounds 1aec (with 1c
as an example)
a 10 mM (100% DMSO) stock to yield a final 10
concentration from which subsequent serial dilutions in extracel-
lular solution were performed (0.3e10 M). Voltage protocols for
m
M (0.1% DMSO) test
To a solution of 2-aminopyrimidine (325 mg, 3.41 mmol) and 2-
bromo-4-nitroacetophenone (1.00 g, 4.10 mmol) in acetonitrile
(15 mL) 4-dimethylaminopyridine (4.2 mg, 0.034 mmol) was
added. After being stirred at 85 ꢀC for 5 h, the reaction mixture was
filtered, washed with acetonitrile (10 mL) and ether (2 ꢃ 10 mL),
and dried to give 1c.
m
the sodium channels being screened were designed to reflect the
high-frequency, pathophysiological state of the channels that may
be therapeutic targets (Nav1.3, Nav1.4 and Nav1.7), and the low-
frequency, physiological state of the safety target (Nav1.5). Cur-
rents were elicited from NaV1.3, NaV1.4 and NaV1.7 cell lines using a
standard two-pulse voltage protocol. From a holding potential
of ꢁ100 mV, a 20 ms activating step to ꢁ20 mV was applied to
assess the effect of compounds on resting (closed) state block. The
second activating pulse was applied following a 5-s pre-pulse to
half inactivation potential (variable depending on the sodium
channel studied, ꢁ65 to ꢁ75 mV) to assess block on the open-
inactivated state of the channel. This protocol was applied at a
sweep interval of 0.067 Hz throughout the duration of the
4.3.1.1. 2-(3-Nitrophenyl)-1H-imidazo[1,2-a]pyrimidin-4-ium bro-
mide (1c). Yield, 66% (0.726 g); orange solid; mp 251e255 ꢀC; IR
(KBr)
n
¼ 3075, 3023, 2674, 2579, 2557, 1642, 1526, 1483, 1341, 1304,
1234, 1109, 1083, 928, 915, 805, 790, 732 cmꢁ1. 1H NMR (DMSO-d6)
d
7.41 (dd, 1H, 3J1 ¼ 6.8 Hz, 3J2 ¼ 4.4 Hz, Ar-H-6), 7.86 (t, 1H,
3J ¼ 8.0 Hz, Ar-H-50), 8.29e8.32 (m, 1H, Ar-H-40/60), 8.45e8.48 (m,
1H, Ar-H-40/60), 8.83e8.85 (m, 2H, Ar-H-3, Ar-H-7), 8.86 (t, 1H,
4J ¼ 2.0 Hz, Ar-H-20), 9.20 (dd, 1H, 3J ¼ 6.8 Hz, 4J ¼ 2.0 Hz, Ar-H-5),
signal for the NH proton not seen; 13C NMR (DMSO-d6)
d 110.46,

N. Zidar et al. / European Journal of Medicinal Chemistry 74 (2014) 23e30
29
112.40, 120.53, 124.14, 130.71, 130.94, 132.20, 137.08, 137.18, 145.82,
137.69, 148.57, 148.91, 150.15; MS (ESI) m/z (%) ¼ 275 (MHþ, 100).
148.45, 155.45; MS (ESI) m/z (%) ¼ 241 ([M ꢁ Br]þ, 100). HRMS for
HRMS for C14H19N4O2: calculated 275.1508; found 275.1504.
C
12H9N4O2: calculated 241.0726; found 241.0724.
4.3.5. General procedure E. Synthesis of compounds 7a, 7b, 9, 11,
12a, 12b, 14, 16aec, 24, 25, 27, 29 and 31 (with 7a as an example)
4.3.2. General procedure B. Synthesis of compounds 2a and 2b
(with 2a as an example)
To
a suspension of pyrrole-2-carboxylic acid (122 mg,
1.09 mmol) and TBTU (380 mg, 1.18 mmol) in dichloromethane
(5 mL) N-methylmorpholine (0.501 mL, 4.56 mmol) was added, and
the mixture stirred at rt for 0.5 h upon which a clear solution
formed. Compound 4a (250 mg, 0.91 mmol) was added and the
mixture stirred at 35 ꢀC overnight. The solvent was evaporated in
vacuo, the residue dissolved in ethyl acetate (30 mL), and washed
successively with water (2 ꢃ 10 mL), saturated aqueous NaHCO3
solution (2 ꢃ 10 mL), and brine (1 ꢃ10 mL). The organic phase was
dried over Na2SO4, filtered and the solvent evaporated under
reduced pressure. The crude product was purified by flash column
chromatography using ethyl acetate/petroleum ether or dichloro-
methane/methanol as an eluent, to afford 7a.
To a suspension of 1a or 1c (0.590 mmol) in ethanol (3 mL) in a
10 mL glass vessel hydrazine hydrate (0.369 mL, 35% hydrazine in
solution, 4.13 mmol) was added, the vessel was sealed, placed in a
microwave reactor, and heated at 120 ꢀC for 40 min (maximum
power ¼ 50 W, ramp time ¼ 3 min). The mixture was cooled
to rt, the solvent evaporated and the resulting residue was purified
by flash column chromatography using dichloromethane/
methanol ¼ 10/1 with dissolved NH3(g) as an eluent, to afford 2-
aminoimidazole 2a.
4.3.2.1. 4-(3-Nitrophenyl)-1H-imidazol-2-amine (2a). Yield, 90%;
orange solid; mp 180e182 ꢀC; IR (KBr)
n
¼ 3446, 3361, 3079, 2854,
2674, 1622, 1548, 1516, 1482, 1342, 1260, 1173, 1146, 1033, 867, 799,
725 cmꢁ1 1H NMR (DMSO-d6)
. d 5.45 (s, 2H, NH2, D2O exchange-
4.3.5.1. tert-Butyl 4-(3-(1H-pyrrole-2-carboxamido)phenyl)-2-
able), 7.27 (s,1H, Ar-H-5), 7.55 (t,1H, 3J ¼ 8.0 Hz, Ar-H-50), 7.90e7.93
(m, 1H, Ar-H-40/60), 8.01e8.04 (m, 1H, Ar-H-40/60), 8.42 (s, 1H, Ar-H-
20), 10.59 (br s, 1H, NH, D2O exchangeable); 13C NMR (MeOH-d4)
amino-1H-imidazole-1-carboxylate (7a). Yield, 45% (150 mg);
white solid; mp 175e177 ꢀC; IR (KBr)
n
¼ 3404, 3354, 3142, 2976,
1731, 1644, 1555, 1517, 1431, 1361, 1311, 1255, 1199, 1157, 1122,
1089, 1038, 758, 716 cmꢁ1. 1H NMR (DMSO-d6)
1.59 (s, 9H, t-Bu),
d
d
111.54, 117.78, 119.72, 129.24,129.29, 132.87, 135.84, 148.76, 151.14;
MS (ESI) m/z (%) ¼ 205 (MHþ, 100). HRMS for C9H9N4O2: calculated
6.16e6.18 (m, 1H, Pyrr-H), 6.63 (br s, 2H, NH2, D2O exchangeable),
6.96e6.98 (m, 1H, Pyrr-H), 7.09e7.11 (m, 1H, Pyrr-H), 7.26 (s, 1H, Ar-
H-5), 7.29 (t, 1H, 3J ¼ 8.0 Hz, Ar-H-50), 7.41e7.43 (m, 1H, Ar-H-40/60),
7.67e7.69 (m, 1H, Ar-H-40/60), 8.07 (t, 1H, 4J ¼ 2.0 Hz, Ar-H-20), 9.77
(s, 1H, NH, D2O exchangeable), 11.63 (s, 1H, NH, D2O exchangeable);
205.0726; found 205.0721.
4.3.3. General procedure C. Synthesis of compounds 3a and 3b
(with 3a as an example)
13C NMR (DMSO-d6)
d 27.51 (CCH3), 84.67 (CCH3), 105.93, 108.89,
To a suspension of 2a (0.500 g, 2.45 mmol) in a mixture of
methanol (10 mL) and water (5 mL) Boc-anhydride (1.07 g,
2.90 mmol) was added and the mixture was stirred at rt for 3 h. The
precipitate was filtered off, washed with methanol (2 ꢃ 5 mL) and
dried to give 3a.
111.25, 116.28, 118.54, 119.41, 122.47, 126.06, 128.57, 133.69, 137.03,
139.47, 148.88, 150.39, 159.08; MS (ESI) m/z (%) ¼ 368 (MHþ, 20),
312 ([MH ꢁ t-Bu]þ, 100), 268 ([MH ꢁ Boc]þ, 60). HRMS for
C19H22N5O3: calculated 368.1723; found 368.1724. HPLC: Phenom-
enex Luna 5
30e80% of MeOH in TFA (0.1%) in 25 min; flow rate 1.0 mL/min;
injection volume: 20 L; retention time: 9.813 min (99.6% at
m
m C18 column (4.6 mm ꢃ 150 mm); mobile phase:
4.3.3.1. tert-Butyl 2-amino-4-(3-nitrophenyl)-1H-imidazole-1-
carboxylate (3a). Yield, 95%; yellow solid; mp 178e180 ꢀC; IR
m
254 nm, 99.3% at 280 nm).
(KBr)
n
¼ 3413, 3278, 3122, 2982, 1739, 1637, 1526, 1477, 1447, 1344,
1320, 1273, 1257, 1212, 1152, 1119, 1066, 903, 836, 773, 716 cmꢁ1. 1H
4.3.6. General procedure F. Synthesis of compounds 8a, 10, 13a, 15,
17a, 26, 28, 30 and 32 (with 8a as an example)
NMR (DMSO-d6)
d 1.60 (s, 9H, t-Bu), 6.73 (br s, 2H, NH2, D2O
exchangeable), 7.64 (t, 1H, 3J ¼ 8.0 Hz, Ar-H-50), 7.67 (s, 1H, Ar-H-5),
Solution of compound 7a (108 mg, 0.250 mmol) in a 1:1 mixture
of THF and EtOH (15 mL) was saturated with gaseous HCl and
stirred at rt for 5 h. The solvent was removed under reduced
pressure, the solid was filtered off and washed with diethyl ether
(3 ꢃ 5 mL), to afford 8a.
8.06e8.09 (m, 1H, Ar-H-40/60), 8.19e8.22 (m, 1H, Ar-H-40/60), 8.55 (t,
1H, 4J ¼ 2.0 Hz, Ar-H-20); 13C NMR (DMSO-d6)
d 28.00 (CCH3), 85.45
(CCH3), 108.88, 119.33, 121.80, 130.40, 131.31, 135.28, 135.78, 148.70,
149.25, 151.18; MS (ESI) m/z (%) ¼ 305 (MHþ, 10), 249 ([MH ꢁ t-
Bu]þ, 90), 205 ([MH ꢁ Boc]þ, 100). HRMS for C14H17N4O4: calculated
305.1250; found 305.1255.
4.3.6.1. 4-(3-(1H-Pyrrole-2-carboxamido)phenyl)-2-amino-1H-imi-
dazol-3-ium chloride (8a). Yield, 85% (76 mg); brown foam; mp
170e174 ꢀC; IR (KBr)
n
¼ 3232, 3142, 2972, 2766, 1676, 1625, 1607,
1547, 1529, 1486, 1422, 1324, 1227, 1158, 1114, 1044, 883, 742 cmꢁ1
1H NMR (DMSO-d6)
6.17e6.19 (m, 1H, Pyrr-H), 6.97e6.99 (m, 1H,
4.3.4. General procedure D. Synthesis of compounds 4a, 4b and 5
(with 4a as an example)
Compound 3a (1.00 g, 3.29 mmol) was dissolved in THF (50 mL),
Pd/C (200 mg) was added and the reaction mixture was stirred
under hydrogen atmosphere for 5 h. The catalyst was filtered off
and the solvent removed under reduced pressure to give 4a.
.
d
Pyrr-H), 7.09e7.11 (m, 1H, Pyrr-H), 7.28 (s, 1H, Ar-H-5), 7.34 (d, 1H,
3J ¼ 8.0 Hz, Ar-H-40/60), 7.41 (t, 1H, 3J ¼ 8.0 Hz, Ar-H-50), 7.48 (s, 2H,
NH2, D2O exchangeable), 7.67 (d, 1H, 3J ¼ 8.0 Hz, Ar-H-40/60), 8.03 (s,
1H, Ar-H-20), 10.04 (s, 1H, NH, D2O exchangeable), 11.80 (s, 1H, NH,
D2O exchangeable), 12.18 (br s, 1H, NH, D2O exchangeable), 12.87
4.3.4.1. tert-Butyl 2-amino-4-(3-aminophenyl)-1H-imidazole-1-
carboxylate (4a). Yield, 95%; white solid; mp 160e162 ꢀC; IR
(br s, 1H, NH, D2O exchangeable); 13C NMR (DMSO-d6)
d 108.95,
(KBr)
n
¼ 3424, 3345, 3127, 2972, 1737, 1636, 1617, 1546, 1452, 1359,
109.32, 111.86, 116.11, 119.22, 120.00, 122.65, 125.86, 126.48, 128.01,
129.20, 139.83, 147.77, 159.12; MS (ESI) m/z (%) ¼ 268 ([M ꢁ Cl]þ,
100). HRMS for C14H14N5O: calculated 268.1198; found 268.1193.
1326, 1267, 1239, 1207, 1159, 1119, 1062, 847, 769, 721 cmꢁ1. 1H NMR
(DMSO-d6)
d 1.58 (s, 9H, t-Bu), 5.02 (br s, 2H, NH2, D2O exchange-
able), 6.42e6.45 (m, 1H, Ar-H-40/60), 6.55 (br s, 2H, NH2, D2O
exchangeable), 6.86e6.88 (m,1H, Ar-H-40/60), 6.95e6.99 (m, 2H, Ar-
HPLC: Phenomenex Luna 5
mobile phase: 10e90% of MeOH in TFA (0.1%) in 20 min; flow rate
1.0 mL/min; injection volume: 20 L; retention time: 13.386 min
(95.1% at 254 nm, 95.1% at 280 nm).
mm C18 column (4.6 mm ꢃ 150 mm);
H-20, Ar-H-50), 7.12 (s, 1H, Ar-H-5); 13C NMR (DMSO-d6)
d
27.50
m
(CCH3), 84.48 (CCH3), 105.28, 110.32, 112.60, 112.80, 128.76, 133.78,

30
N. Zidar et al. / European Journal of Medicinal Chemistry 74 (2014) 23e30
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4.3.7. General procedure G. Synthesis of compounds 8b, 13b and
17b (with 8b as an example)
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To an ice cold solution of 7b (200 mg, 0.544 mmol) in ethanol
(4 mL) acetyl chloride (600 mL, 8.4 mmol) was added dropwise.
After being stirred at 0 ꢀC for 1 h the reaction mixture was allowed
to warm to rt, the solvent and the excess HCl were evaporated and
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4.3.7.1. 4-(4-(1H-Pyrrole-2-carboxamido)phenyl)-2-amino-1H-imi-
dazol-3-ium chloride (8b). Yield, 42% (69 mg); white solid; mp 114e
116 ꢀC; IR (KBr)
NMR (DMSO-d6)
n
¼ 3421, 1656, 1412, 1333, 1124, 836, 746 cmꢁ1. 1H
d 6.16e6.19 (m, 1H, Pyrr-H), 6.98 (br s, 2H, NH2,
D2O exchangeable), 7.08 (s, 1H, Pyrr-H), 7.29 (s, 1H, Pyrr-H), 7.41 (s,
3
1H, Ar-H-5), 7.61 (d, 2H, J ¼ 8.7 Hz, Ar-H-20,60/30,50), 7.84 (d, 2H,
3J ¼ 8.7 Hz, Ar-H-20,60/30,50), 9.94 (s, 1H, NH, D2O exchangeable),
11.73 (s, 1H, NH, D2O exchangeable), 12.02 (s, 1H, NH, D2O
exchangeable), 12.76 (s, 1H, NH, D2O exchangeable); 13C NMR
(DMSO-d6)
d 108.44, 108.96, 111.73, 119.88, 122.24, 122.73, 124.60,
125.89,126.45,139.20,147.46,159.08; MS (ESI) m/z ¼ 268 [M ꢁ Cl]þ.
HRMS for C14H14N5O: calculated 268.1198; found 268.1194. HPLC:
Phenomenex Luna 5
phase: 50e80% of methanol in trifluoroacetic acid (0.1%) in 30 min;
m
m C18 column (4.6 mm ꢃ 150 mm); mobile
flow rate 1.0 mL/min; injection volume: 10
mL; retention time:
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L.Y. Huang, D. Immke, B. Janosky, D. Johnson, X.W. Li, J. Ligutti, D. Liu,
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12.880 min (95.1% at 254 nm).
Acknowledgment
This work was supported by the Slovenian Research Agency
(grant no. P1-0208 and grant no. Z1-5458) and by the EU FP7 In-
tegrated Project MAREX (project no. FP7-KBBE-2009-3-245137).
We thank the Biology Department at Xention for the contributions
to this project.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2013.12.034.
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sodium channel, Mol. Pharmacol. 55 (1999) 134e141.
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