Synthesis and biological evaluation of piperazine derivatives as novel isoform selective voltage-gated sodium (Nav) 1.3 channel modulators

Article abstract of DOI:10.1007/s00044-014-1300-x

Sponges of the genus Agelas produce compounds that modulate the activity of voltage-gated sodium ion channels and contribute novel scaffolds for the development of compounds with activity against a plethora of biological targets. In particular, clathrodin and dibromosceptrin were reported to decrease the average maximum amplitude of inward sodium currents in isolated chick embryo sympathetic ganglia cells; we envisaged these compounds as a starting point to design novel Na_v channel modulators. This endeavor was part of our long-term goal of designing a comprehensive library of Agelas alkaloid analogs that would cover a broader chemical space and allow us to examine the activity of such compounds on Na_v channels. Our series of compounds was designed by maintaining the terminal structural features found in clathrodin while rigidizing the central part of the molecule and replacing the 3-aminopropene linker with a 4-methylenepiperazine moiety. Synthesised compounds were screened for inhibitory action against the human voltage-gated sodium channel isoforms Na_v 1.3, Na_v 1.4, cardiac Na_v 1.5, and Na_v 1.7 using an automated patch clamp electrophysiology technique. The results demonstrate that we have obtained a series of compounds with a modest but selective inhibitory activity against the Na_v 1.3 channel isoform. The most potent compound showed selective activity against the Na_v 1.3 channel isoform with an IC₅₀ of 19 μM and is a suitable starting point for further development of selective Na_v 1.3 channel modulators. Such compounds could prove to be beneficial as a pharmacological tool towards the development of novel therapeutically useful compounds in the treatment of pain.

Full text of DOI:10.1007/s00044-014-1300-x

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-014-1300-x
ORIGINAL RESEARCH
Synthesis and biological evaluation of piperazine derivatives
as novel isoform selective voltage-gated sodium (Nav)
1.3 channel modulators
•
•
•
Marko Jukic Rok Frlan Fiona Chan Robert W. Kirby
•
ˇ
David J. Madge Jan Tytgat Steve Peigneur
•
•
•
•
Marko Anderluh Danijel Kikelj
Received: 5 April 2014 / Accepted: 13 November 2014
Ó Springer Science+Business Media New York 2014
Abstract Sponges of the genus Agelas produce com-
pounds that modulate the activity of voltage-gated sodium
ion channels and contribute novel scaffolds for the devel-
opment of compounds with activity against a plethora of
biological targets. In particular, clathrodin and dibromo-
sceptrin were reported to decrease the average maximum
amplitude of inward sodium currents in isolated chick
embryo sympathetic ganglia cells; we envisaged these
compounds as a starting point to design novel Nav channel
modulators. This endeavor was part of our long-term goal
of designing a comprehensive library of Agelas alkaloid
analogs that would cover a broader chemical space and
allow us to examine the activity of such compounds on Nav
channels. Our series of compounds was designed by
maintaining the terminal structural features found in cla-
throdin while rigidizing the central part of the molecule
and replacing the 3-aminopropene linker with a 4-methyl-
enepiperazine moiety. Synthesised compounds were
screened for inhibitory action against the human voltage-
gated sodium channel isoforms Nav 1.3, Nav 1.4, cardiac
Nav 1.5, and Nav 1.7 using an automated patch clamp
electrophysiology technique. The results demonstrate that
we have obtained a series of compounds with a modest but
selective inhibitory activity against the Nav 1.3 channel
isoform. The most potent compound showed selective
activity against the Nav 1.3 channel isoform with an IC50 of
19 lM and is a suitable starting point for further devel-
opment of selective Nav 1.3 channel modulators. Such
compounds could prove to be beneficial as a pharmaco-
logical tool towards the development of novel therapeuti-
cally useful compounds in the treatment of pain.
Keywords Voltage-gated Á Sodium channels Á
Nav channels Á Nav channel modulators Á
Isoform selective modulators Á Piperazine derivatives
Introduction
Voltage-gated sodium channels (Nav channels) are large
transmembrane proteins capable of selective sodium ion
transmission, and they are responsible for the generation of
action potentials. Nav channels enable the spreading of
electrical impulses through nerve, muscle, and endocrine
cell systems (Catterall, 2000). Research on the mechanism
of action of antiepileptic drugs and neurotoxins such as
tetrodotoxin revealed that these molecules act as sodium
channel modulators (Termin et al., 2008). More recently,
voltage-gated sodium channel a and b subunit mutations
have been associated with pathological conditions such as
cardiac arrhythmias, myotonia, periodic paralysis, familial
hemiplegic migraine, epilepsy, congenital analgesia, and
neuropathic pain (Andavan and Lemmens-Gruber, 2011).
Moreover, research on voltage-gated sodium ion channels
represents a significant portion of academic and industrial
medicinal chemistry endeavors, and they are attractive
therapeutic targets (Clare et al., 2000). The Nav 1.3 channel
ˇ
M. Jukic Á R. Frlan Á M. Anderluh (&) Á D. Kikelj
ˇ ˇ
Faculty of Pharmacy, University of Ljubljana, Askerceva 7,
1000 Ljubljana, Slovenia
e-mail: marko.anderluh@ffa.uni-lj.si
F. Chan Á R. W. Kirby Á D. J. Madge
Xention Limited, Iconix Park, London Road, Pampisford,
Cambridge CB22 3EG, UK
J. Tytgat Á S. Peigneur
University of Leuven (KULeuven), Toxicology & Pharmacology
O&N2, PO Box 922, Herestraat 49, 3000 Louvain, Belgium
123

Med Chem Res
isoform is found in the embryonic CNS and is also
expressed in damaged nerve tissues and could be associ-
ated with various pain states. Nav channels have also been
found in heart, uterus, lung, DRG, and glia tissues, where
they contribute to pain and electrolyte imbalance condi-
ganglia cells by 27 and 40 %, respectively (Rivera Rentas
et al., 1995; Perdicaris et al., 2013). Agelas sponge alkaloids
have also been found to reduce voltage-dependent calcium
elevation in PC12 cells (Bickmeyer et al., 2002). Despite the
very limited data on the activity of Agelas sponge alkaloids,
we envisaged these compounds as a starting point to design
novel potential Nav channel modulators. This endeavor was a
part of our long-term goal of designing a comprehensive
library of Agelas alkaloid analogs that would cover a broader
chemical space and allow us to examine the potential of such
ˇ
tions (England and de Groot, 2009; Jukic et al., 2013).
Non-selective voltage-gated sodium channel modulators
are already present in materia medica and have been
¨
extensively reviewed (Bolcskei et al., 2008; Anger et al.,
2001; Taylor, 1996). Such compounds are commonly
developed without structural information on their respec-
tive targets, and they are commonly used as treatments for
conditions such as epilepsy (lamotrigine, carbamazepine),
cardiac arrhythmias (mexiletine), and numerous pain states
(lidocaine).
ˇ ´
compounds as Nav channel modulators (Tomasic et al.,
2013). The idea was also prosecuted by Hodnik et al., who
reported compound 3 (R6, R7 = Br, Fig. 1) as a Nav 1.4
channel selective state-dependent modulator with an IC50
value of 15 lM for the open-inactivated state (Hodnik et al.,
2013). This series of compounds was designed by main-
taining the terminal structural features found in clathrodin
while rigidizing the central part of the molecule with a ste-
rically restricted condensed cyclohexane ring and shortening
the 3-aminopropene linker by one methylene group (com-
pound 1, Fig. 1).
Despite their widespread use, non-selective voltage-
gated sodium channel modulators provide only modest
therapeutic potential and often exhibit unwanted side
effects. Non-selective Nav channel modulators interact with
a channel subunit S5–S6 pore lining segments; because the
pore forming region is highly conserved among Nav chan-
nel isoforms, selectivity of such compounds is low. Activity
on distinct Nav isoforms contributes to the adverse effects
observed in clinically used therapies where the modulators
presumably act on basal brain functions, skeletal muscle,
and/or the heart (England and de Groot, 2009). With
acquired and expanding knowledge on Nav channel iso-
forms and their unique role(s), selective targeting and/or
development of state-dependent modulators on individual
Nav channel isoforms could be beneficial (England and de
In contrast to reported compound 3 (R6, R7 = Br,
Fig. 1), our approach focused on the replacement of the
native 3-aminopropene linker with the less sterically
restricted 4-methylenepiperazine moiety (Teixeira et al.,
2013). Furthermore, we sought to investigate the impor-
tance of terminal structures found in the native clathrodin
compound. 2-aminoimidazole is a common structural motif
amongst Agelas alkaloids, so we postulated that the ter-
minal basic 2-aminoimidazole is a key moiety responsible
for activity against Nav channels. Interestingly, 2-amino-
imidazole, which is thought to be ionized in physiological
conditions (Storey et al., 1964), is commonly found in
marine sponge alkaloids, and is a building block used in a
ˇ
Groot, 2009; Jukic et al., 2013).
Interest in natural compounds as novel therapeutic leads
was rekindled in recent years, as such compounds cover a
significant portion of chemical space, and they often exert
favorable pharmacokinetic profiles and have activity against
numerous biological targets (Neuman, 2008; Vuorela et al.,
2004). Marine organisms represent an under-exploited source
of natural compounds; sponges and their sponge-symbiotic
microorganisms produce a variety of compounds with
selectivity against biological targets, including cytotoxins,
antibiotics, antivirals, anti-inflammatory compounds, and
antifouling agents (Laport et al., 2009). Sponges of the genus
Agelas have been shown to produce compounds that modu-
late the activity of voltage-gated sodium ion channels and
contribute novel scaffolds for the development of active
compounds against a plethora of biological targets (Rivera
Rentas et al., 1995; Cafieri et al., 1997; Richards et al., 2008;
Keifer et al., 1991). In particular, alkaloids isolated from the
marine sponge of the Agelas genus have shown modulatory
activity on voltage-gated sodium and calcium channels.
Clathrodin 1 (Fig. 1) and dibromosceptrin 2 (Fig. 1)
decreased the average maximum amplitude of inward
sodium currents in isolated chick embryo sympathetic
ˇ
multitude of small molecule drugs (Zula et al., 2013). The
pyrrole-2-carbonyl structure from clathrodin was replaced
with various heteroaromatic structures and a series of
compounds with this feature was synthesized (4). To
determine the importance of the 2-aminoimidazole moiety,
we have also replaced it with 2-aminothiazole or a 2-un-
substituted imidazole, while retaining the pyrrole-2-car-
bonyl substituent, to afford a small series of compounds
(5). We also wanted to biologically evaluate the activity of
the small focused library of synthesized piperazine analogs
(4, 5) against several Nav channel isoforms using the
voltage clamp technique (Fig. 1; Table 2).
Molecular modeling
Structural comparison of piperazine analogs to Agelas
alkaloids clathrodin and oroidin was performed using
TanimotoCombo scores calculated with the vROCS
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Med Chem Res
Fig. 1 Design of clathrodin-derived compounds. Photograph of Agelas clathrodes sponge from BioLib (Burek and Burek, 2013). Asterisk
marked atoms were selected for distance measurements
software package (vROCS version 3.1.2. OpenEye Scien-
tific Software, Santa Fe, NM. http://www.eyesopen.com,
2013; Grant et al., 1996; Hawkins et al., 2007; Tuccinardi
et al., 2009). The starting ligand geometries were opti-
mized with ChemBio 3D Ultra 13.0 (CambridgeSoft) using
the MM2 force field until a minimum 0.100 root mean
square (RMS) gradient was reached. The optimized struc-
ture was further refined with the GAMESS interface using
the semi-empirical PM3 method, the QA optimization
algorithm and Gasteiger Hu¨ckel charges for all atoms for
100 steps. 2-aminoimidazoles were kept in their ionized
state, corresponding to their ionization state in physiolog-
ical pH (that is, 7.4). Conformer libraries were prepared for
compounds 4a–g and 5a–c (Table 1; 151, 200, 74, 200,
200, 172, 136, 112, 106, and 200 conformers, respectively)
using OMEGA software from OpenEye Scientific Software
Inc. (OMEGA version 2.4.6. OpenEye Scientific Software,
Santa Fe, NM. http://www.eyesopen.com, 2013; Hawkins
et al., 2010; Hawkins and Nicholis, 2012). vROCS over-
lays the conformer library on a query structure and per-
forms ranking according to structure similarity based on
molecular shape (shape score) and types of atoms (color
score); molecules similar to the query structure obtain a
favorable ROCS_TanimotoCombo score. The distances
between two postulated key groups were also measured
using Chem3D 13.0 Ultra. Atoms selected for distance
measurements are designated in Fig. 1 and Table 1,
respectively. Structure graphic overlays were performed
using VIDA and vROCS software packages from OpenEye
Scientific Software Inc. (VIDA version 4.2.1. OpenEye
Scientific Software, Santa Fe, NM. http://www.eyesopen.
com, 2013; Fig. 2).
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Med Chem Res
Table 1 Synthesised clathrodin analogs
R4=
4a
4b
4c
5c
4d
4e
4g
5b
4f
R5=
5a
Atoms marked with an asterisk (*) were selected for distance measurements
Fig. 2 Left Overlay of compound 4d and oroidin structure (green); right overlay of compound 4d and clathrodin structure (green) (Color figure
online)
Chemistry
protected compound (Little and Webber, 1994). Due to the
instability of the triple-Boc protected compound, depro-
tection with TFA in dichloromethane was promptly per-
formed to afford the final product. Hydrochlorides of
amino compounds 4a and 4b were obtained by subjecting
the deprotected compounds to 1 M HCl in ethanol solution
(Fig. 3; Table 1), or by direct deprotection with 1 M HCl
solution.
The first two piperazine-linker analogs with an unsym-
metrical substitution on the spacer moiety (compounds 4a
and 4b) were synthesized using heteroaromatic aldehydes
that were subjected to reductive amination with mono N-
Cbz-protected piperazine and Na(OAc)3BH in dichloro-
methane or NaCNBH3 in methanol as a solvent to give
products 7a–b in good yields (Abdel-Magid et al., 1996).
Further Cbz deprotection with H2 and Pd/C (10 %) pro-
duced free-amino intermediates that were immediately
subjected to similar reductive amination conditions with
intermediate 6 to afford the unsymmetrical triple-Boc
The synthesis of other unsymmetrical piperazine-linker
clathrodin analogs was envisioned through the coupling of
heteroaromatic carboxylic acid 8 with intermediate 10
(Fig. 4, bottom) using an EDC/HOBt coupling procedure
to afford Boc-protected target compounds (Anderluh et al.,
123

Med Chem Res
Fig. 3 Synthesis of compounds 4a and 4b
Fig. 4 Synthesis of compounds 4c–g
2005). Compound 10 was obtained by reductive amination
of mono N-Cbz piperazine with intermediate 6 (Fig. 4)
using Na(OAc)3BH in dichloromethane and subsequent
Cbz deprotection under H2 and Pd/C (10 %), but 10
unfortunately proved to be unstable under storage and upon
subjection to further reaction conditions (Little and Webber
1994). Because concise convergent synthesis could thus
not be accomplished, an alternative synthesis from the
heteroaromatic starting compound was pursued.
direct and delicate Boc cleavage in trifluoroacetic acid
(TFA)/dichloromethane to yield trifluoroacetate salts of
compounds 4c–g. Hydrochlorides were prepared by sub-
jecting trifluoroacetates to 1 M HCl in ethanol solution
(Fig. 4; Table 1).
Compounds 5a–c were synthesized in a manner similar
to compounds 4c–g by initial coupling of pyrrole-2-car-
boxylic acid to mono N-protected Cbz-piperazine with
EDC/HOBt and Cbz cleavage using H2 and Pd/C (10 %) to
give common intermediate 11. 11 was subjected to
reductive amination with 1H-imidazole-5-carbaldehyde in
methanol using NaCNBH3 and a catalytic amount of acetic
acid to give compound 5a. Boc protected 5b was synthe-
sized from 11 and 2 N-Boc-amino-thiazole-5-carboxylic
acid using a coupling procedure with EDC in DMF solvent
and Et3N as a base. The deprotected trifluoroacetate salt of
compound 5b was obtained with trifluoroacetic acid in
dichloromethane. Compound 5c was obtained directly from
Heteroaromatic carboxylic acids were coupled to
monoprotected N-Cbz-piperazine using EDC/HOBt in
excellent yields to afford key intermediates 9c–g. The Cbz
group was cleaved using H2 and Pd/C (10 %) in THF/
EtOHabs. Because of the instability of the key free-amino
compounds, they were immediately subjected to reductive
amination with compound 6 using Na(OAc)3BH (Abdel-
Magid et al., 1996) in dichloromethane to afford triple-Boc
protected final compounds. Synthesis was concluded with
123

Med Chem Res
Fig. 5 Synthesis of compounds 5a–c
11 with unprotected 2-amino-4-thiazoleacetic acid using a
slightly modified coupling procedure with EDC in THF/
DMF and Et3N as a base (Fig. 5; Table 1).
were calculated from concentration–response graphs mea-
sured at 4 relevant concentrations ranging from 0.3 to
10 lM.
Key intermediate 6 was synthesized starting with 1,1-
dimethoxypropan-2-one that was subjected to a-keto bro-
mination (Fig. 6). Due to compound instability, 3-bromo-
1,1-dimethoxypropan-2-one was directly used in double
nucleophilic substitution using BOC-protected guanidine to
afford cyclized 12. The free-amino group on compound 12
was additionally protected using a classic procedure with
BOC-anhydride and DMAP catalyst in THF to afford triple
BOC protected 13. The last step was delicate dimethyl
acetal deprotection towards the final aldehyde compound.
Cells were prepared by dissociation from T175 cell
culture flasks using trypsin–EDTA (0.05 %) and were kept
in serum free media on board the QPatch HT system. Cells
were sampled, washed, and re-suspended in extracellular
recording solution by the QPatch HT before application to
well sites on the chip. 0.1 % v/v DMSO solution was
applied to the control cells (4 min total) for comparison to
cells treated with test sample concentrations (4 min incu-
bation per test concentration). Samples were prepared in
extracellular solution with serial dilutions from 10 to
0.3 lM concentration. Measurements were performed on
Nav 1.3, Nav 1.4, and Nav 1.7 cell lines using a standard
two-pulse voltage protocol. From a holding potential of
-100 mV, a 20 ms to -20 mV activating step was applied
to measure the effect of the compounds on the resting state.
The second activating pulse was applied following a 5-s
pre-pulse to achieve half inactivation potential to assess the
effect on the open-inactivated state of the channel. This
protocol was applied at an interval of 0.067 Hz. For the
Nav 1.5 isoform, 10 pulses from -20 mV to a holding
potential of -100 mV were applied at 1 Hz. This protocol
was applied at an interval of 0.016 Hz for the duration of
the experiment. Measurement of the effect of the com-
pounds on the Nav 1.3, Nav 1.4, and Nav 1.7 channel iso-
forms proceeded by determining the peak inward current
for both the closed and open-inactivated test pulses for
This reaction presumably proceeds via
a substrate
exchange mechanism using elemental iodine. Deprotection
was performed in dry acetone on an ice bath, followed by
final quenching with sodium thiosulfate to afford 6 (Sun
et al., 2004).
Biological evaluation
Biological assays were conducted at Xention Limited, as
previously described by Hodnik et al. (2013). Synthesised
compounds were screened for inhibitory action on the
human voltage-gated sodium channel isoforms Nav 1.3,
Nav 1.4, cardiac Nav 1.5, and Nav 1.7 using the automated
patch clamp electrophysiology technique on the Sophion
QPatch HT system (Sophion Bioscience A/S). IC50 values
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Med Chem Res
Fig. 6 Synthesis of key
intermediate 6
each sweep (the 10th pulse was used for the Nav 1.5
channel isoform). Data were captured using QPatch assay
software (v5.0). The percent 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 concentration–response curves were fit-
ted to the inhibition data using Xlfit (IDBS). Data are
presented as the mean standard deviation for a minimum of
three independent observations.
clathrodin structure. The IC50 of compound 4c was deter-
mined to be 25 lM on the Nav 1.3 channel isoform
(Table 2). Interestingly, the carbonyl of the pyrrole-2-car-
boxylate was proven to be necessary as compound 4b did
not show any measurable activity on the tested channel
isoforms. It should be noted, however, that during biolog-
ical testing of the compounds on the panel of Nav channel
isoforms, we selected the IC50 value of 30 lM as a ‘‘cut-
off’’ point, so all activities above this threshold were
considered to be equivalent. The substitution of indole-2-
carboxamide for the terminal pyrrole-2-carboxamide
increased potency; the IC50 of compound 4d was 19 lM on
the Nav 1.3 channel isoform. This compound could occupy
the space filled by the two additional bromines in oroidin
and possibly take advantage of the additional hydrophobic/
dispersion interactions. The measured distances between
the terminal structures of compounds 4c and 4d also nicely
Results and discussion
Previously published literature on the synthesis and activity
of the Agelas sponge alkaloids was used to design the com-
pounds aimed to selectively modulate voltage-gated sodium
channels (Rasapalli et al., 2013; Olofson et al., 1998; Little
and Webber 1994). As there are no reported crystal structures
of voltage-gated sodium channels with bound clathrodin/
oroidin or relevant analogs, we employed a simple ligand-
based design by changing the central scaffold and performing
small changes in the key structural elements of the com-
pounds. We postulated that the terminal 2-aminoimidazole
moiety of the Agelas alkaloid clathrodin separated by
3-aminopropene semi-rigid linker from the terminal pyrrole
moiety was a key structural element. We also presumed that
the two terminal moieties should be kept separate. A similar
approach with rigidization and shortening of the linker was
previously successful for the development of selective Nav
1.4 channel isoform modulators (Hodnik et al., 2013).
As seen from TanimotoCombo score calculations, we
accomplished the design of compounds similar to the
parent clathrodin and oroidin structures (Fig. 2; Table 2).
Furthermore, the piperazine linker confers a slightly
increased flexibility and increases the distance between the
key terminal heterocyclic moieties. Our direct clathrodin
˚
compared (7.7 and 7.4 A, respectively) to parent clathro-
din/oroidin structures. Compound 4e, where indole-2-car-
boxylate was replaced with indole-3-carboxylate, also
showed below-threshold activity with an IC50 of 29 lM on
the Nav 1.3 channel isoform. The observed decrease in
potency was probably due to the greater distance measured
˚
between the two key structures (9.5 A in 4e compared with
˚
7.4 A for compound 4d). The analogs 4f and 4g were
potentially too long; their measured IC50 was above the
‘‘cut-off’’ point of 30 lM. Patch-clamp measurements
confirmed the two terminal features were necessary, as the
benzyl analog 4a did not show any activity against the Nav
channel isoforms. In the final iteration, we selected our
direct analog 4c and replaced the 2-aminoimidazole
structure with bioisosteric heteroaromatic moieties
(Table 2). The measured distance between the key struc-
tures in compound 5c was evidently longer than in the
parent structure, and no activity was observed. However,
compounds 5a and 5b compared nicely to the parent cla-
throdin structure distance-wise and according to Tanimot-
oCombo score, but again, no activity was observed with
IC50 measurements hovering above the ‘‘cut-off’’ point of
˚
analog 4c had a distance of 7.7 A between designated
˚
atoms in comparison to 6.9 A measured for the parent
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Med Chem Res
Table 2 Results of biological evaluation and molecular modeling
4a-f
5a-c
TanimotoCombo scoreb IC5c0 (lM)
a
˚
Distance (A)
Compound R4
R5
Nav 1.3 Nav 1.4 Nav 1.5 Nav 1.7
1
/
/
6.9
/
/
[30
[30
[30
n.t.
[30
[30
[30
[30
4a
0.978
0.933
4b
4c
/
/
7.6
7.7
1.040
0.972
30
25
[30
[30
[30
[30
[30
[30
1.197
1.116
4d
4e
/
/
7.4
9.5
1.067
1.082
19
29
[30
[30
[30
[30
[30
[30
1.007
0.991
4f
/
/
9.3
8.6
7.5
1.057
1.009
[30
[30
[30
n.t.
n.t.
[30
[30
[30
[30
[30
[30
4g
5a
1.216
1.118
/
1.238
1.144
[30
5b
/
/
7.4
8.0
1.098
1.028
[30
[30
n.t.
[30
[30
[30
[30
5c
0.919
0.858
[30
a
Measured distance between designated atoms (marked with asterisk *)
b
TanimotoCombo score (ROCS_TanimotoCombo) calculated with OpenEye Scientific Software Inc. vROCS package. The first score is
calculated against a clathrodin reference structure and the second value is calculated against an oroidin reference structure
c
The concentration of compound that inhibited a sodium channel current by 50 %
30 lM. Based on these results, we conclude that the
2-aminoimidazole moiety is essential for activity against
the Nav channels.
Patch-clamp measurements revealed that all of our
compounds displayed a trend towards selective modulation
of the Nav 1.3 channel isoform, while the IC50 of clathrodin
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Med Chem Res
was above the selected ‘‘cut-off’’ point (IC50 of clathrodin
not shown). Nevertheless, our series of compounds dem-
onstrate that the low-potency clathrodin structure was a
solid starting point for ligand-based design, with compound
4d reaching selective activity against the Nav 1.3 channel
isoform with an IC50 of 19 lM. When compared with the
reported compounds by Hodnik et al., where selectivity
against the Nav 1.4 channels was achieved, we observed a
trend of activity towards the Nav 1.3 channel isoform by
clathrodin analogs with the less sterically restricted ami-
nopropene linker substitution and a longer 4-methylene-
piperazine moiety.
room temperature. A solution of NaCNBH3 (0.11 g,
1.68 mmol, 2.1 eq) in methanol (2 mL) was added dropwise
and the reaction mixture was stirred for 14 h under argon
atmosphere at room temperature. The reaction mixture was
filtered, the solvent evaporated under reduced pressure, and
the residue was redissolved in ethyl acetate, and washed with
a saturated solution of NaHCO3 in water (3 9 10 mL), brine
(1 9 10 mL), dried over Na2SO4, filtered, and the solvent
evaporated under reduced pressure. The crude product was
purified by flash column chromatography using ethyl ace-
tate/hexane (1:1) as an eluent to afford compound 7a. Yield,
55 %, white solid; 1H NMR (DMSO-d6, 400 MHz)
d = 2.44 (s, 4H, –N(CH2)2), 3.53–3.56 (m, 6H, –CH2,
–N(CH2)2), 5.17 (s, 2H, –O–CH2), 7.35–7.36 (m, 5H, Ar),
7.37–7.39 (m, 5H, Cbz), (Compound reported by Shrikhande
et al., 2008, structure confirmed by 1H NMR).
To conclude, our ligand-based design indicated a trend of
increasing selectivity towards Nav 1.3 inhibitory activity by
slight prolongation of the linker between the clathrodin ter-
minal heterocyclic moieties. The latter findingcan bevaluable
for further development of selective Nav 1.3 channel modu-
lators. Compounds 4c–e could also prove to be beneficial as
pharmacological tools, especially with recent literature
reports wherein the Nav 1.3 channel isoform is associated with
various pain conditions (Waxman et al., 1994).
Synthesis of benzyl 4-((1H-pyrrol-2-
yl)methyl)piperazine-1-carboxylate (compound 7b)
To a solution of N-Cbz piperazine (1.01 g, 4.58 mmol,
1.1 eq) in dry dichloromethane (20 mL), 1H-pyrrole-2-
carbaldehyde (0.40 g, 4.20 mmol, 1 eq) was added. The
reaction mixture was stirred for 20 min under argon
atmosphere at room temperature. It was then cooled to
0 °C, and Na(OAc)3BH (1.92 g, 9.62 mmol, 2.1 eq) was
added. After stirring for 1 h with gradual warming to room
temperature and additional stirring for 3 h at 40 °C, the
dichloromethane phase was washed carefully with water
(2 9 20 mL), brine (1 9 30 mL), dried over Na2SO4, fil-
tered, and the solvent evaporated under reduced pressure.
The crude product was purified by flash column chroma-
tography using ethyl acetate as an eluent to afford com-
pound 7b. Yield, 68 %, yellow-white solid; 1H NMR
(CDCl3, 400 MHz) d = 2.41–2.42 (m, 4H, N(CH2)2),
3.51–3.54 (m, 6H, –CH2, N(CH2)2), 5.15 (s, 2H, O-CH2),
6.05–6.1 (m, 1H, NCHAr), 6.13–6.16 (m, 1H, NCHAr),
6.77–6.79 (m, 1H, Ar), 7.32–7.40 (m, 5H, Cbz), 8.44–8.45
(m, 1H, NH) ppm.; 13C NMR (CDCl3, 100 MHz)
d = 43.82 (CH2N), 52.67 (CH2N), 55.42(CH2N), 60.45
(CH2O), 67.16 (CH2N), 107.92 (CHAr), 108.04 (CHAr),
117.65 (CHAr), 127.92 (CHAr), 128.07 (CHAr), 128.18
(CHAr), 128.54 (CHAr), 136.72 (CAr), 155.26 (CAr), 171.23
(CO) ppm.; IR (cm-1) m = 3333, 2899, 2864, 2809, 1685,
1428, 1285, 1238, 1118, 1093, 1024, 997, 785, 718, 696;
MS (ESI) m/z (%) = 300 (MH?); HRMS for C17H22N3O2:
calculated 300.1712, found 300.1716.
Experimental procedures
Chemistry: general
Chemicals were obtained from Sigma-Aldrich Corporation
(St. Louis, MO, USA) and Acros Organics (Geel, Belgium)
and were used without further purification. Analytical TLC
was performed on silica gel Merck 60 G F254 plates
(0.25 mm). Visualisation was performed with UV light and
ninhydrin visualisation reagent. Column chromatography was
conducted using silica gel 60(particle size 240–400 mesh). 1H
and 13C NMR spectra were recorded at 400 and 100 MHz,
respectively, on a Bruker AVANCE III 400 spectrometer
(Bruker Corporation, Billerica, MA, USA) in DMSO-d6 or
CDCl3 solutions, with TMS used as the internal standard. IR
spectra were recorded on a PerkinElmer Spectrum BX FT-IR
spectrometer (PerkionElmer, Inc., Waltham, MA, USA) or
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 spectrom-
eter (Fisons, VG Analytical, Manchester, UK).
Synthesis of benzyl 4-benzylpiperazine-1-carboxylate
(compound 7a)
To a solution of N-Cbz piperazine (0.2 g, 0.88 mmol,
1.1 eq), benzaldehyde (81.5 lL, 0.80 mmol, 1 eq) in dry
˚
methanol (8 mL), activated molecular sieves (3 A), and
Synthesis of 4-((4-benzylpiperazin-1-yl)methyl)-1H-
imidazol-2-amine (compound 4a)
glacial acetic acid (50 lL) were added, and the reaction
mixture was stirred for 20 min under argon atmosphere at
Compound 7a (0.20 g, 0.55 mmol) was dissolved in EtO-
Habs (10 mL), dry THF (10 mL), flushed with argon, and
123

Med Chem Res
degassed under reduced pressure. Ten percent Pd/C (5 %
m/m with respect to 7) was added, and the reaction mixture
was stirred under H2 atmosphere for 3 h at room temper-
ature. It was then filtered, and the solvent evaporated under
reduced pressure. The residual oil (0.07 g, 0.39 mmol,
1.1 eq) was dissolved in dry dichloromethane (15 mL).
Compound 6 (0.15 g, 0.36 mmol, 1 eq) was added, and the
reaction mixture was stirred under argon for 20 min at
room temperature. Na(OAc)3BH (0.15 g, 0.76 mmol,
2.1 eq) was slowly added, and the reaction mixture was
stirred under argon atmosphere for 40 min at room tem-
perature. The dichloromethane phase was washed carefully
with water (2 9 10 mL), brine (1 9 50 mL), dried over
Na2SO4, filtered, and the solvent evaporated under reduced
pressure. The crude product was purified by flash column
chromatography using ethyl acetate/hexane (2:1) as an
eluent to afford the Boc-protected compound. The Boc-
protected compound (50 mg, 0.09 mmol) was dissolved in
dichloromethane (2 mL), CF3COOH (200 lL) was added,
and the mixture was stirred under argon atmosphere for 2 h
at 40 °C. Solvents were evaporated under reduced pressure
and the resulting oil was redissolved in 1 M HCl in EtOH
solution (2 mL). The white crystalline product was col-
lected with filtration to afford compound 7a in excellent
yield (95 %). 1H NMR (DMSO-d6, 400 MHz) d = 2.09 (s,
4H, –N(CH2)2), 4.06–4.10 (m, 2H, CH2), 4.37 (s, 2H,
CH2), 7.03 (s, 1H, CHAr), 7.45–7.73 (m, 5H, CHAr),
12.14–12.24 (m, 2H, NH2) ppm.; 13C NMR (DMSO-d6,
100 MHz) d = 30.68 (CH2), 47.13 (CH2N), 48.16 (CH2N),
58.36 (CH2N), 128.74 (CHAr), 129.47 (CHAr), 129.56
(CHAr), 131.42 (CHAr), 131.56 (CAr), 147.31 (CAr), 206.54
(CNH2) ppm.; IR (cm-1) m = 3360, 3242, 3139, 2989,
2963, 2505, 2446, 1697, 1678, 1635, 1599, 1433, 1365,
953, 917, 747, 699; MS (ESI) m/z (%) = 342 (M-H-);
HRMS for C15H22N5Cl2: calculated 342.1252, found
342.1254.
The dichloromethane phase was washed carefully with
water (2 9 20 mL), brine (1 9 20 mL), dried over
Na2SO4, and filtered, and the solvent evaporated under
reduced pressure. The crude product was purified by flash
column chromatography using ethyl acetate/methanol (9:1)
as an eluent to afford the Boc-protected compound. The
Boc-protected compound (50 mg, 0.09 mmol) was dis-
solved in ethyl acetate (3 mL), 1 M HCl solution was
added (1.5 mL), and the reaction mixture was stirred for
50 min at room temperature. The water phase was alka-
linized to pH 13 by adding 10 M NaOH, and extracted with
ethyl acetate (2 9 5 mL). The organic phases were joined
and the solvent evaporated under reduced pressure. The
crude residue was purified by flash column chromatogra-
phy using dichloromethane/methanol (5:1) as an eluent to
1
afford compound 4b. Yield, 20 %, yellow oil; H NMR
(DMSO-d6, 400 MHz) d = 2.59 (s, 4H, –N(CH2)2), 3.08
(s, 4H, N(CH2)2), 3.44 (s, 2H, CH2), 6.81 (s, 1H, CHAr),
7.54 (s, 2H, –NH2), 8.79–8.81 (m, 2H, CHAr), 12.12 (2, 1H,
NH), 12.43–12.46 (m, 1H, NH) ppm.; 13C NMR (DMSO-
d6, 100 MHz) d = 45.31 (CH2N), 47.45 (CH2N), 48.56
(CH2N), 114.44 (CHAr), 114.98 (CHAr), 143.22 (CHAr),
147.30 (CHAr), 158.14 (CAr), 161.59 (CAr), 188.23 (CNH2)
ppm.; IR (cm-1) m = 3148, 3006, 2781, 1676, 1619, 1433,
1410, 1259, 1082, 1068, 1019, 931, 799; MS (ESI) m/
z (%) = 334 (MH?).
General procedure for synthesis of compounds 9c–g
N-Cbz piperazine (0.55 g, 2.48 mmol, 1 eq) and carboxylic
acid 8c–g (2.48 mmol, 1 eq) were dissolved in dry DMF
(10 mL), the reaction mixture flushed with argon and
cooled to 0 °C. N-methyl morpholine (NMM; 7.44 mmol,
3 eq), hydroxybenzotriazole hydrate (HOBt; 2.98 mmol,
1.2 eq) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiim-
ide hydrochloride HCl salt (EDC; 3.22 mmol, 1.3 eq) were
slowly added. The reaction mixture was stirred under argon
atmosphere for 5 h at 0 °C and an additional 15 h at room
temperature. DMF was evaporated under reduced pressure
and the residue redissolved in dichloromethane (10 mL).
The dichloromethane phase was washed with H2O
(1 9 10 mL), a 1 M HCl solution (3 9 10 mL), saturated
Synthesis of 4-((4-((1H-pyrrol-2-yl)methyl)piperazin-
1-yl)methyl)-1H-imidazol-2-amine (compound 4b)
Compound 7b (0.40 g, 1.34 mmol) was dissolved in the
mixture of EtOHabs (10 mL) and dry THF (10 mL), flushed
with argon, and degassed under reduced pressure. Ten
percent Pd/C (5 % m/m with respect to 7) was added, and
the reaction mixture was stirred under H2 atmosphere for
2 h at room temperature. The reaction mixture was filtered,
and the solvent evaporated under reduced pressure. The
residual oil (0.22 g, 1.34 mmol, 1.1 eq) was dissolved in
dry dichloromethane (20 mL). Compound 6 (0.49 g,
1.20 mmol, 1 eq) was added, and the mixture was stirred
under argon for 20 min at room temperature. Na(OAc)3BH
(0.55 g, 2.59 mmol, 2.1 eq) was added and again stirred
under argon atmosphere for 40 min at room temperature.
aqueous
NaHCO3
solution
(3 9 10 mL),
brine
(1 9 20 mL), dried over Na2SO4, filtered, and the solvent
evaporated under reduced pressure. The crude product was
purified by flash column chromatography using ethyl ace-
tate/hexane solvents as eluents to afford compounds 9c–g.
Benzyl 4-(1H-pyrrole-2-carbonyl)piperazine-1-carboxylate
(compound 9c)
Yield, 86 %, white crystalline solid; 1H NMR (CDCl3,
400 MHz) d = 3.60–3.64 (m, 4H, N(CH2)2), 3.87 (s, 4H,
123

Med Chem Res
Benzyl 4-nicotinoylpiperazine-1-carboxylate (compound
N(CH2)2), 5.19 (s, 2H, O-CH2), 6.25–6.27 (m, 1H,
N-CHAr), 6.52–6.54 (m, 1H, N-CHAr), 6.93–6.94 (m, 1H,
Ar), 7.37–7.41 (m, 5H, Cbz), 10.11–10.14 (m, 1H, NH)
ppm.; 13C NMR (CDCl3, 100 MHz) d = 43.73 (CH2N),
43.78 (CH2N), 67.50 (OCH2), 109.55 (CHAr), 112.39
(CHAr), 121.56 (CHAr), 124.14 (CHAr), 128.09 (CHAr),
128.25 (CAr), 128.61 (CHAr), 136.41 (CAr), 155.22 (CO),
162.18 (CO) ppm.; IR (cm-1) m = 3255, 3104, 2905,
2851, 1695, 1593, 1545, 1473, 1446, 1421, 1358, 1281,
1245, 1232, 1191, 1131, 1100, 1014, 961, 849, 783, 742,
697, 680; MS (ESI) m/z (%) = 312 (M-H-); HRMS for
C17H18N3O3: calculated 312.1348, found 312.1342.
9f)
1
Yield, 98 %, yellow solid; H NMR (CDCl3, 400 MHz)
d = 3.44–3.76 (m, 8H, –N(CH2)2), 5.51 (s, 2H, O-CH2),
7.32–7.39 (m, 6H, Cbz, CHAr), 7.75–7.78 (m, 1H, CHAr),
8.66–8.68 (m, 2H, CHAr) ppm.; 13C NMR (CDCl3,
100 MHz) d = 42.14 (CH2N), 43.80 (CH2N), 47.53
(CH2N), 67.59 (CH2O), 123.65 (CHAr), 128.07 (CHAr),
128.28 (CHAr), 128.60 (CHAr), 131.18 (CHAr), 135.22
(CHAr), 136.22 (CAr), 147.84 (CHAr), 151.00 (CO), 155.07
(CAr), 167.96 (CO) ppm.; IR (cm-1) m = 3386, 3027, 2919,
2835, 2857, 1697, 1627, 1472, 1426, 1410, 1278, 1225,
1159, 1115, 1071, 1003, 980, 861, 830, 754, 746, 743, 698;
MS (ESI) m/z (%) = 326 (MH?); HRMS for C18H20N3O3:
calculated 326.1505, found 326.1499.
Benzyl 4-(1H-indole-2-carbonyl)piperazine-1-carboxylate
(compound 9d)
Yield, 92 %, white solid; 1H NMR (CDCl3, 400 MHz)
d = 3.66–3.68 (m, 4H, –N(CH2)2), 3.97 (s, 4H, –N(CH2)2),
5.21 (s, 2H, O-CH2), 6.79–6.80 (m, 1H, Ar), 7.17 (ddd,
J = 8.01, 7.01, 0.98 Hz, 1H, Ar), 7.32 (ddd, J = 8.24,
7.01, 1.14, 1H, Ar), 7.36–7.42 (m, 5H, Ar), 7.46 (dd,
J = 8.29, 0.90 Hz, 1H, Ar), 7.67 (dd, J = 8.03, 0.89 Hz,
1H, Ar), 9.68 (s, 1H, NH) ppm.; 13C NMR (CDCl3,
100 MHz) d = 43.73 (CH2N), 43.76 (CH2N), 43.79
(CH2N), 43.82 (CH2N), 67.59 (OCH2), 105.49 (CHAr),
111.87 (CHAr), 120.71 (CHAr), 121.94 (CHAr), 124.65
(CHAr), 127.37 (CHAr), 128.11 (CHAr), 128.29 (CHAr),
128.62 (CAr), 128.78 (CAr), 135.84 (CAr), 136.34 (CAr),
155.20 (CO), 162.81 (CO) ppm.; IR (cm-1) m = 3252,
3058, 2878, 1700, 1595, 1530, 1460, 1443, 1408, 1346,
1221, 1120, 804, 765, 745, 727, 693, 678; MS (ESI) m/
z (%) = 362 (M-H-); HRMS for C21H20N3O3: calculated
362.1505, found 362.1498.
Benzyl 4-((tert-butoxycarbonyl)-L-prolyl)piperazine-1-
carboxylate (compound 9g)
Yield, 99 %, white solid; 1H NMR (CDCl3, 400 MHz)
d = 1.41 (s, 4H, CH), 1.47 (s, 5H, CH), 1.82–1.91 (m, 2H,
CH2), 1.97–2.21 (m, 2H, CH2), 3.45–3.65 (m, 8H,
–N(CH2)2), 4.53–4.68 (m, 1H, CH), 5.17 (d, J = 3.64 Hz,
1H, NH), 5.32 (s, 2H, O-CH2), 7.35-7.39 (m, 5H, Cbz)
ppm.; 13C NMR (DMSO-d6, 100 MHz) d = 22.96 (CH2),
28.01 (CH3), 28.14 (CH3), 29.85 (CH2), 41.11 (CH2N),
43.34 (CH2N), 43.41 (CH2N), 44.12 (CH2N), 46.29 (CH2),
56.38 (CH), 56.22 (CH2N), 66.39 (CH2O), 78.15 (CO),
127.59 (CHAr), 127.88 (CHAr), 128.41 (CHAr), 136.71
(CHAr), 153.07 (CHAr), 153.25 (CHAr), 154.40 (CO),
170.22 (CO), 170.55 (CO) ppm.; IR (cm-1) m = 2979,
2954, 2866, 1704, 1680, 1658, 1450, 1404, 1364, 1246,
1210, 1156, 1114, 1074, 1044, 947, 928, 754, 698; MS
(ESI) m/z (%) = 418 (MH?); HRMS for C22H32N3O5:
calculated 418.2342, found 418.2353.
Benzyl 4-(1H-indole-3-carbonyl)piperazine-1-carboxylate
(compound 9e)
General procedure for synthesis of compounds 4c–g
Yield, 82 %, yellow-white solid; 1H NMR (DMSO-d6,
400 MHz) d = 3.38 (s, 1H, NCH2), 3.52–3.59 (m, 5H,
N(CH2)2), 3.73 (s, 2H, NCH2), 5.19 (s, 2H, –OCH2),
7.21–7.28 (m, 2H, Ar), 7.34–7.40 (m, 8H, Ar) ppm; 13C
NMR (DMSO-d6, 100 MHz) d = 43.41 (CH2N), 43.63
(CH2N), 44.50 (CH2N), 44.95 (CH2N), 66.93 (CH2O),
112.20 (CHAr), 120.48 (CHAr), 121.19 (CHAr), 122.24
(CHAr), 128.13 (CHAr), 128.30 (CHAr), 128.39 (CHAr),
128.92 (CHAr), 134.14 (CAr), 136.02 (CAr), 137.18 (CAr),
Compound 9c–g (0.55 mmol) was dissolved in the mixture
of EtOHabs (10 mL) and dry THF (10 mL), flushed with
argon, and degassed under reduced pressure. Ten percent
Pd/C (5 % m/m with respect to compound 9) was added,
and the reaction mixture was stirred under H2 atmosphere
for 3 h at room temperature. The catalyst was then filtered
off, and the solvent evaporated under reduced pressure.
The residue (0.55 mmol, 1.1 eq) was dissolved in dry
dichloromethane (15 mL). Compound 6 (0.50 mmol, 1 eq)
was added, and the mixture was stirred under argon for
20 min at room temperature. Na(OAc)3BH (0.23 g,
1.05 mmol, 2.1 eq) was added and 40 min of stirring at
room temperature followed. Dichloromethane was washed
154.85 (CAr), 161.56 (CO), 166.69 (CO) ppm.; IR (cm-1
)
m = 3513, 3032, 2916, 2863, 1696, 1425, 1357, 1281,
1230, 1199, 1119, 1069, 1002, 732, 697; MS (ESI) m/
z (%) = 362 (M-H-); HRMS for C21H20N3O3: calculated
362.1505, found 362.1507.
123

Med Chem Res
carefully with water (2 9 15 mL), brine (1 9 15 mL),
dried over Na2SO4, and filtered, and the solvent evaporated
under reduced pressure. The crude product was purified by
flash column chromatography using ethyl acetate as an
eluent to afford the Boc-protected compound. The Boc-
protected compound (0.14 mmol) was dissolved in
dichloromethane (5 mL), CF3COOH (1 mL) was added,
and the mixture was stirred under argon atmosphere for 1 h
at 40 °C. The solvents were evaporated under reduced
pressure, and the residue was dissolved in a 1 M ethanolic
solution of HCl (2 mL). The product was obtained by
adding dry ether (500 lL) and collection by filtration.
2H, –NCH2), 4.39–4.43 (m, 2H, NCH2), 7.11–7.18 (m, 3H,
CHAr), 7.47 (d, J = 7.42 Hz, 1H, CHAr), 7.72–7.79 (m,
2H, CHAr), 11.47–11.55 (s, 1H, NH), 11.77 (s, 1H, NH),
12.04–12.05 (m, 1H, NH) ppm.; 13C NMR (DMSO-d6,
100 MHz) d = 50.23 (CH2N), 50.32 (CH2N), 67.96
(CH2N), 97.96 (CHAr), 112.05 (CHAr), 120.09 (CHAr),
120.41 (CHAr), 122.05 (CHAr), 125.89 (CHAr), 128.79
(CAr), 135.75 (CAr), 147.43 (CAr), 155.04 (CAr), 165.73
(CNH2), 178.58 (CO) ppm.; IR (cm-1) m = 3218, 3131,
2997, 2956, 2992, 2561, 1679, 1606, 1522, 1421, 1105,
947, 768, 755, MS (ESI) m/z (%) = 359 (M-HCl-).
(4-((2-Amino-1H-imidazol-4-yl)methyl)piperazin-1-
(4-((2-Amino-1H-imidazol-4-yl)methyl)piperazin-1-yl)
yl)(pyridin-3-yl)methanone (compound 4f)
(1H-pyrrol-2-yl)methanone (compound 4c)
Yield, 65 %, yellow solid; 1H NMR (DMSO-d6, 400 MHz)
d = 3.12–3.15 (m, 8H, –N(CH2)2), 4.27 (s, 2H, CH2), 7.12
(s, 1H, CHAr), 7.72 (dd, J = 7.34, 5.11 Hz, 1H, CHAr),
7.81 (s, 1H, CHAr), 8.14 (d, J = 7.65 Hz, 1H, CHAr),
8.79–8.82 (m, 2H, CHAr, NH), 12.18–12.20 (m, 2H, NH2)
ppm.; 13C NMR (DMSO-d6, 100 MHz) d = 43.79
(CH2N), 48.31 (CH2N), 49.49 (CH2N), 49.60 (CH2N),
114.79 (CHAr), 116.78 (CHAr), 125.19 (CHAr), 132.07
(CHAr), 138.85 (CHAr), 144.79 (CAr), 147.43 (CAr), 147.48
(CNH2), 165.62 (CO) ppm.; IR (cm-1) m = 3374, 3326,
3166, 3076, 3050, 2984, 2928, 2836, 2756, 2365, 1682,
1642, 1632, 1604, 1493, 1476, 1443, 1300, 1285, 953, 807,
685; MS (ESI) m/z (%) = 321 (M-HCl-); HRMS for
C14H18N6OCl: calculated 321.1231, found 321.1235.
1
Yield, 77 %, white solid; H NMR (DMSO-d6, 400 MHz)
d = 3.05–3.10 (m, 2H, –NCH2), 4.27 (s, 2H, –NCH2),
4.48–4.52 (m, 2H, –NCH2), 6.15 (s, 1H, CHAr), 6.58 (s, 1H,
CHAr), 6.94 (s, 1H, CHAr), 7.10 (s, 1H, CHAr), 7.81 (s, 2H,
–NH2), 11.58 (s, 1H, NH), 12.20–12.21 (m, 1H, NH) ppm.; 13
C
NMR (DMSO-d6, 100 MHz) d = 41.36 (CH2N), 48.38
(CH2N), 48.45 (CH2N), 50.08 (CH2N), 108.63 (CHAr), 112.57
(CHAr), 115.08 (CHAr), 116.59 (CHAr), 121.90 (CAr), 123.23
(CAr), 147.43 (CNH2), 161,47 (CO) ppm.; IR (cm-1) m = 3246,
3106, 2945, 2680, 1680, 1583, 1548, 1428, 1290, 1115, 1048,
944, 747; MS (ESI) m/z (%) = 309 (M-HCl-); HRMS for
C13H18N6OCl: calculated 309.1231, found 309.1223.
(4-((2-Amino-1H-imidazol-4-yl)methyl)piperazin-1-yl)
(1H-indol-2-yl)methanone (compound 4d)
(S)-1-((2-Amino-1H-imidazol-4-yl)methyl)-4-
prolylpiperazine (compound 4g)
Yield, 93 %, yellow solid; 1H NMR (DMSO-d6, 400 MHz)
d = 3.06–3.17 (m, 2H, –NCH2), 4.29 (s, 2H, –NCH2),
4.57–4.59 (m, 2H, –NCH2), 6.90 (s, 1H, CHAr), 7.07 (t,
J = 7.53 Hz, 1H, CHAr), 7.11 (s, 1H, CHAr), 7.21 (t,
J = 7.54 Hz, 1H, CHAr), 7.45 (d, J = 7.85 Hz, 1H, CHAr),
7.62 (d, J = 7.74 Hz, 1H, CHAr) 7.82 (s, 2H, NH2), 11.69 (s,
1H, NH), 12.21 (s, 1H, NH) ppm.; 13C NMR (DMSO-d6,
100 MHz) d = 30.68 (CH2N), 45.27 (CH2N), 48.46
(CH2N), 50.03 (CH2N), 104.80 (CH2N), 112.16 (CHAr),
115.01 (CHAr), 116.60 (CHAr), 119.88 (CHAr), 121.43
(CHAr), 123.55 (CAr), 126.68 (CAr), 128.84 (CAr), 136.10
1
Yield, 55 %, white solid; H NMR (DMSO-d6, 400 MHz)
d = 1.87–1.92 (m, 3H, CH2), 2.34–2.35 (m, 1H, CH2),
3.19–3.21 (m, 6H, –N(CH2)2), 4.22–4.29 (m, 3H, CH2),
4.62–4.63 (s, 1H, CH), 7.09–7.11 (s, 1H, CHAr), 7.75–7.81
(m, 2H, NH2), 8.51–8.52 (s, 1H, NH), 12.16 (s, 1H, NH)
ppm.; 13C NMR (DMSO-d6, 100 MHz) d = 23.63 (CH2),
28.46 (CH2), 30.70 (CH2), 41.64 (CH2N), 45.63 (CH2N),
48.29 (CH2N), 49.35 (CH2N), 49.66 (CH2N), 57.19 (CH),
115.01 (CHAr), 116.58 (CAr), 147.41 (CNH2), 166.87 (CO)
ppm.; IR (cm-1) m = 3345, 2928, 2753, 2574, 1679, 1653,
1479, 1445, 1255, 1201, 1123, 945; MS (ESI) m/
z (%) = 349 (M-H-); HRMS for C13H23N6OCl2: calcu-
lated 349.1310, found 349.1320.
(CAr), 147.45 (CNH2), 162.14 (CO) ppm.; IR (cm-1
)
m = 3272, 3093, 2932, 2662, 2571, 1677, 1604, 1423, 1249,
1199, 948, 733; MS (ESI) m/z (%) = 359 (M-HCl-); HRMS
for C17H20N6OCl: calculated 359.1387, found 359.1385.
Synthesis of (4-((1H-imidazol-4-yl)methyl)piperazin-1-yl)
(4-((2-Amino-1H-imidazol-4-yl)methyl)piperazin-1-yl)
(1H-pyrrol-2-yl)methanone (compound 5a)
(1H-indol-3-yl)methanone (compound 4e)
Compound 9c (4.30 g, 13.7 mmol, 1 eq) was dissolved in
absolute ethanol (100 mL), and glacial acetic acid (20 mL)
was added. The reaction mixture was flushed with argon
Yield, 66 %, yellow–brown solid; 1H NMR (DMSO-d6,
400 MHz) d = 3.06–3.12 (m, 2H, NCH2), 4.26–4.28 (m,
123

Med Chem Res
and degassed under reduced pressure, and 10 % Pd/C (5 %
m/m with respect to 9c) was added. The reaction mixture
was stirred under H2 atmosphere for 1 h at room temper-
ature, and the catalyst was filtered off. The solvent was
evaporated under reduced pressure, and the resulting
compound 11 was used in the next reaction step without
further purification. Compound 11 (0.70 g, 3.91 mmol,
3.5 eq) was dissolved in methanol (8 mL) and glacial
acetic acid (64 lL, 0.11 mmol, 0.1 eq) was added. 1H-
imidazole-4-carbaldehyde (0.108 g, 1.16 mmol, 1 eq) in
methanol (4 mL) was added dropwise, and the mixture was
left stirring under argon atmosphere for 30 min at room
temperature. NaCNBH3 (0.20 g, 3.35 mmol, 3 eq) dis-
solved in methanol (5 mL) was added dropwise via syringe
with further stirring under argon atmosphere at room
temperature for 1 h. The solvent was evaporated under
reduced pressure, and the residue was dissolved in water
(20 mL). 1 M aqueous NaOH was added until alkaline pH
was reached, and the water phase was extracted with ethyl
acetate (6 9 30 mL). The combined organic phases were
dried above Na2SO4 and evaporated under reduced pres-
sure, and the resulting crude product was purified by flash
column chromatography using dichloromethane/methanol
(9:1) as an eluent to afford compound 5a. Yield, 51 %,
yellow–white solid; 1H NMR (DMSO-d6, 400 MHz)
d = 2.37–2.40 (m, 4H, –N(CH2)2–], 3.43 (s, 2H, –CH2–),
3.62–3.69 (m, 4H, –N(CH2)2–), 6.07–6.10 (m, 1H, Ar),
6.43–6.47 (m, 1H, Ar), 6.84–6.91 (m, 2H, Ar, Ar-imi.),
7.52–7.56 (m, 1H, Ar-imi.), 11.40 (s, 1H, Ar–NH), 11.93 (s,
1H, Ar-imi.-NH) ppm.; 13C NMR (DMSO-d6, 100 MHz)
d = 44.41 (CNH2), 44.52 (CNH2), 52.47 (CNH2), 108.27
(CHAr), 111.65 (CHAr), 120.99 (CHAr), 124.19 (CHAr),
134.81 (CAr), 134.85 (CAr), 134.96 (CHAr), 161.32 (CO)
ppm.; IR (cm-1) m = 3122, 3072, 2955, 2868, 2820, 2778,
1597, 1459, 1436, 1293, 1266, 1141, 1090, 995, 849, 730,
633; MS (ESI) m/z (%) = 260 (MH?); HRMS for
C13H18N5O: calculated 260.1511, found 260.1514.
methanol (20:1 ? 1 % AcOH) as the eluent to afford
compound 5c. Yield, 14 %, yellow solid; 1H NMR
(DMSO-d6, 400 MHz) d = 3.50–3.56 (m, 4H, –N(CH2)2–),
3.59–3.63 (m, 2H, –CO–CH2–), 3.64–3.72 (m, 4H,
–N(CH2)2–), 6.12 (td, J = 3.49, 2.45, 2.45 Hz, 1H, Ar),
6.26 (s, 1H, –S–CH=C–), 6.51 (ddd, J = 3.67, 2.50,
1.35 Hz, 1H, Ar), 6.85 (s, 2H, –NH2), 6.89 (dt, J = 2.80,
2.70, 1.37 Hz, 1H, Ar), 11.44 (s, 1H, Ar–NH) ppm; 13C
NMR (DMSO-d6, 100 MHz) d = 21.04 (CH2), 36.69
(CNH2), 41.25 (CNH2), 45.57 (CNH2), 102.28 (CNH2),
108.42 (CHAr), 112.02 (CHAr), 121.30 (CHAr), 123.99
(CAr), 145.56 (CAr), 161.58 (CHAr), 168.03 (CNH2),
168.14 (CO), 172.01 (CO) ppm.; IR (cm-1) m = 3122,
2954, 2915, 2868, 2820, 1597, 1460, 1434, 1292, 1141,
849, 730; MS (ESI) m/z (%) = 320 (MH?); HRMS for
C14H18N5O2S: calculated 320.1181, found 320.1185.
Synthesis of (4-(1H-pyrrole-2-carbonyl)piperazin-1-yl)(2-
aminothiazol-5-yl)methanone (compound 5b)
Compound 11 (0.394 g, 1.83 mmol, 1.1 eq) was dissolved in
anhydrous DMF (20 mL), and the reaction mixture was
cooled to 0 °C. Triethylamine (0.81 mL, 5.81 mmol, 3.5 eq)
and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydro-
chloride HCl salt (EDC; 0.48 g, 2.49 mmol, 1.5 eq) were
added. The reaction mixture was removed from the ice bath
and stirred under argon for 20 h at room temperature. The
solvents were evaporated under reduced pressure, and the
crude product was purified by flash column chromatography
using hexane/ethyl acetate/methanol (5:4:1) as the eluent to
afford the Boc-protected compound. The latter (0.71 g,
1.75 mmol, 1 eq) was dissolved in dichloromethane (15 mL),
and CF3COOH was added (5 mL). The reaction mixture was
stirredunder argon for 1 hatroomtemperature. Solventswere
evaporated under reduced pressure to afford compound 5b.
1
Yield, 21 %, brown solid; H NMR (DMSO-d6, 400 MHz)
d = 3.73 (s, 4H, –N(CH2)2), 3.79 (s, 4H, –N(CH2)2), 6.14 (s,
1H, CHAr), 6.51–6.55 (m, 1H, CHAr), 6.92 (s, 1H, CHAr), 7.54
(s, 1H, CHAr), 8.09–8.12 (m, 2H, –NH2). 11.52 (s, 1H, NH)
ppm.; 13C NMR (DMSO-d6, 100 MHz) d = 44.43 (CNH2),
44.45 (CNH2), 108.49(CHAr), 112.14(CHAr), 119.59(CHAr),
121.42 (CHAr), 123.99 (CAr), 139.05 (CAr), 160.72 (CO),
161.51 (CO), 170.83 (CNH2) ppm.; IR (cm-1) m = 3254,
3076, 2734, 1663, 1585, 1425, 1180, 1126, 998, 834, 723; MS
(ESI) m/z (%) = 306 (MH?); HRMS for C13H16N5O2S: cal-
culated 306.1025, found 306.1020.
Synthesis of 1-(4-(1H-pyrrole-2-carbonyl)piperazin-1-yl)-
2-(2-aminothiazol-5-yl)ethan-1-one (compound 5c)
Compound 11 (0.30 g, 1.64 mmol, 1.1 eq) and 2-amino-4-
thiazole acetic acid (0.24 g, 1.52 mmol, 1 eq) were dis-
solved in anhydrous THF (20 mL). The reaction mixture
was flushed with argon and cooled to 0 °C. Triethylamine
(0.84 mL, 6.09 mmol, 4 eq) and 1-ethyl-3-(3-dimethyl-
aminopropyl)carbodiimide hydrochloride HCl salt (EDC;
0.38 g, 2.74 mmol, 1.8 eq) were added, and the reaction
mixture was removed from the ice bath. DMF (20 mL) was
added, and the reaction mixture was left stirring under
argon for 20 h at 60 °C. The solvents were evaporated
under reduced pressure and the crude product was purified
by flash column chromatography using dichloromethane/
Synthesis of tert-butyl 2-amino-5-(dimethoxymethyl)-1H-
imidazole-1-carboxylate (compound 12)
1,1-Dimethoxypropan-2-one (200 g, 1.7 mol) was dis-
solved in tetrachloromethane (800 mL) and cooled in an
ice bath. Bromine (87.2 mL, 1.7 mol) was added dropwise
123

Med Chem Res
over 10 min, and the reaction mixture was left stirring in
argon atmosphere for 15 h at room temperature. Sodium
bicarbonate (178 g, 2.1 mol) was then added, and the
resulting mixture was left stirring for 30 min and trans-
ferred to a separatory funnel. The organic phase was
washed carefully with water (2 9 100 mL), brine
(1 9 100 mL), dried over Na2SO4, and evaporated under
reduced pressure to afford 3-bromo-1,1-dimethoxypropan-
2-one as a clear pale-yellow oil in 89 % yield.
(CH), 97.85 (CH), 116.77 (CHAr), 136.43 (CAr), 137.54
(CO), 145.88 (CO), 148.74 (CN) ppm.; IR (cm-1
)
m = 3166, 3138, 2977, 2942, 2834, 1745, 1724, 1538,
1456, 1361, 1318, 1249, 1211, 1146, 1109, 1058, 1022,
985, 908, 872, 842, 816, 768.; MS (ESI) m/z (%) = 458.2
(MH?); HRMS for C21H35N3O8: calculated 458.2502,
found 458.2502.
Synthesis of tert-butyl 2-(di-(tert-butoxycarbonyl)amino)-
As this compound was proven to be intrinsically insta-
ble, it was used directly in a subsequent reaction step.
BOC-guanidine (5.14 g, 32 mmol) was dissolved in dry
5-formyl-1H-imidazole-1-carboxylate (compound 6)
Acetal intermediate 13 (0.2 g, 0.44 mmol, 1 eq) was dis-
solved in dry acetone (5 mL) and cooled to 0 °C. A 0.05 M
solution of iodine in dry acetone (0.9 mL) was added via
syringe and the reaction was left to stir under argon
atmosphere at 0 °C for 4 h. The reaction was quenched
with the addition of aqueous 5 % m/m solution of Na2S2O3
in water (1 mL). The aqueous mixture was then extracted
with ethyl acetate (3 9 3 mL), the organic fractions were
combined and washed with brine (1 9 5 mL), and the
solvent was evaporated under reduced pressure. The crude
product was purified using column chromatography with
ethyl acetate/hexane (1:2) as an eluent. Yield, 56 %,
white–yellow crystalline solid; 1H NMR (DMSO-d6,
400 MHz) d = 1.37 (s, 18H, N(Boc)2), 1.56 (s, 9H, NBoc),
8.52 (s, 1H, CHAr), 9.77 (s, 1H, CHO) ppm.; 13C NMR
(DMSO-d6, 100 MHz) d = 27.20 (CH3), 27.40 (CH3),
83.93 (CH), 87.76 (CH), 127.75 (CHAr), 137.66 (CAr),
139.12 (CO), 145.39 (CO), 148.62 (CN), 184.85 (CO)
ppm.; IR (cm-1) m = 3142, 2980, 2935, 1751, 1726, 1688,
1545, 1457, 1399, 1349, 1313, 1277, 1248, 1209, 1146,
1112, 1017, 991, 873, 845, 768, 734.; MS (ESI) m/
z (%) = 412.2 (MH?); HRMS for C19H29N3O7: calculated
412.2084, found 412.2076 (Peat et al., 2008, Yoke Chong
et al., 2010).
˚
THF (50 mL), activated molecular sieves (3 A) were
added, and the reaction mixture was left stirring for 10 min
under argon atmosphere at 0 °C. 3-Bromo-1,1-dimeth-
oxypropan-2-one (6.4 g, 32 mmol, 1 eq) dissolved in dry
THF (50 mL) was then added dropwise. The mixture was
left stirring under argon for 20 h at room temperature.
Na2SO4 was added and the reaction mixture filtered to
remove solids, the solvents were evaporated under reduced
pressure and the crude product was purified using column
chromatography with ethyl acetate ?1 % Et3N as an eluent
to afford an orange solid (12). Yield, 12 %, white solid; 1H
NMR (CDCl3, 400 MHz) d = 1.53 (s, 9H, Boc), 3.30 (s,
6H, OCH3), 5,22 (s, 1H, CH), 6.02 (s, 2H, NH2), 6.78 (s,
1H, CHAr) ppm.; 13C NMR (CDCl3, 100 MHz) d = 28.08
(CH3), 52.76 (OCH3), 85.18 (CH), 99.41 (CH), 109.43
(CHAr), 135.59 (CAr), 149.47 (CO), 150.74 (CNH2) ppm.;
IR (cm-1) m = 3240, 3180, 3110, 2980, 2950, 2886, 2828,
1735, 1631, 1452, 1385, 1336, 1274, 1249, 1153, 1112,
1054, 978, 907, 843, 765, 711.; MS (ESI) m/z (%) = 258.1
(MH?); HRMS for C11H19N3O4: calculated 258.1454,
found 258.1460.
Synthesis of tert-butyl 2-(di-(tert-butoxycarbonyl)amino)-
5-(dimethoxymethyl)-1H-imidazole-1-carboxylate
(compound 13)
Acknowledgments This work was supported by the European
Union FP7 Integrated Project MAREX: Exploring Marine Resources
for Bioactive Compounds: From Discovery to Sustainable Production
and Industrial Applications (Project No. FP7-KBBE-2009-3-245137).
We thank OpenEye Scientific Software, Inc. for free academic
licenses of their software.
Compound 12 (1.7 g, 6.62 mmol, 1 eq) was dissolved in
dry THF (10 mL), DMAP (242 mg, 2 mmol, 0.3 eq) was
added, and the reaction mixture was left stirring under
argon atmosphere for 20 min at 0 °C. Di-tert-butyl dicar-
bonate (4.5 g, 19.9 mmol, 3 eq) dissolved in dry THF
(5 mL) was added and stirring was continued for 20 h at
room temperature. The solvents were evaporated under
reduced pressure and the resulting crude product was
purified using column chromatography with ethyl acetate
?1 % Et3N as an eluent to afford compound 13 as bright-
yellow crystals. Yield, 78 %, yellow crystalline solid; 1H
NMR (CDCl3, 400 MHz) d = 1.34 (s, 18H, Boc), 1.53 (s,
9H, Boc), 3.20 (s, 6H, OCH3), 5.36 (s, 1H, CH), 7.42 (s,
1H, CHAr) ppm.; 13C NMR (CDCl3, 100 MHz) d = 27.24
(CH3), 27.38 (CH3), 51.69 (OCH3), 83.24 (CH), 86.29
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