Inhibition of NaV1.6 sodium channel currents by a novel series of 1,4-disubstituted-triazole derivatives obtained via copper-catalyzed click chemistry

Article abstract of DOI:10.1016/j.bmcl.2012.08.067

We have synthesized and evaluated a series of 1,4-disubstituted-triazole derivatives for inhibition of the rat Na_V1.6 sodium channel isoform, an isoform thought to play an important role in controlling neuronal firing. Starting from a series of 2,4(1H)-diarylimidazoles previously published, we decided to extend the SAR study by replacing the imidazole with a different heterocyclic scaffold and by varying the aryl substituents on the central aromatic ring. The 1,4-disubstituted 1,2,3-triazoles were prepared employing the copper-catalyzed azide-alkyne cycloaddition (CuAAC). Many of the new molecules were able to block the rNa_v1.6 currents at 10 μM by over 20%, displaying IC₅₀ values ranging in the low micromolar, thus indicating that triazole can efficiently replace the central heterocyclic core. Moreover, the introduction of a long chain at C4 of the central triazole seems beneficial for increased rNa_v1.6 current block, whereas the length of N1 substituent seems less crucial for inhibition, as long as a phenyl ring is not direcly connected to the triazole. These results provide additional information on the structural features necessary for block of the voltage-gated sodium channels. These new data will be exploited in the preparation of new compounds and could result in potentially useful AEDs.

Full text of DOI:10.1016/j.bmcl.2012.08.067

Bioorganic & Medicinal Chemistry Letters 22 (2012) 6401–6404
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Inhibition of NaV1.6 sodium channel currents by a novel series of
1,4-disubstituted-triazole derivatives obtained via copper-catalyzed click
chemistry
Mirko Rivara a, , Manoj K. Patel b, Laura Amori a, Valentina Zuliani a
⇑
a Dipartimento di Farmacia, Università degli Studi di Parma, V.le G.P. Usberti, 27/A, I-43124 Parma, Italy
b Department of Anesthesiology, University of Virginia, Charlottesville, VA 22908, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
We have synthesized and evaluated a series of 1,4-disubstituted-triazole derivatives for inhibition of the
rat NaV1.6 sodium channel isoform, an isoform thought to play an important role in controlling neuronal
firing. Starting from a series of 2,4(1H)-diarylimidazoles previously published, we decided to extend the
SAR study by replacing the imidazole with a different heterocyclic scaffold and by varying the aryl sub-
stituents on the central aromatic ring. The 1,4-disubstituted 1,2,3-triazoles were prepared employing the
copper-catalyzed azide–alkyne cycloaddition (CuAAC). Many of the new molecules were able to block the
Received 19 July 2012
Revised 15 August 2012
Accepted 16 August 2012
Available online 23 August 2012
Keywords:
rNav1.6 currents at 10 lM by over 20%, displaying IC50 values ranging in the low micromolar, thus indi-
Sodium channels
Patch clamp electrophysiology
1,4-Triazoles
rNaV1.6
Click chemistry
cating that triazole can efficiently replace the central heterocyclic core. Moreover, the introduction of a
long chain at C4 of the central triazole seems beneficial for increased rNav1.6 current block, whereas
the length of N1 substituent seems less crucial for inhibition, as long as a phenyl ring is not direcly con-
nected to the triazole. These results provide additional information on the structural features necessary
for block of the voltage-gated sodium channels. These new data will be exploited in the preparation of
new compounds and could result in potentially useful AEDs.
Ó 2012 Elsevier Ltd. All rights reserved.
Voltage gated sodium channels (VGSCs) are membrane protein
complexes that allow the passive flow of sodium ions across bio-
logical membranes.1 VGSCs open on membrane depolarization,
causing further depolarization of the membrane and the initiation
of an action potential. As such, VGSCs play a critical role in regulat-
ing cellular excitability and controlling many of the physiological
processes associated with neuronal activity.2–4 Alterations in the
behaviour of VGSCs have been associated in the pathophysiology
of epilepsy, a chronic neurological disorder characterized by recur-
rent unprovoked seizures. These seizures occur when a brief,
strong surge of electrical activity affects either one hemisphere
(partial seizure) or both hemispheres of the brain (generalized sei-
zure). In particular, mutations in VGSC genes SCN1A (encoding the
channels have a particularly low threshold for spike initiation
and are abundantly expressed along the axon initial segment
(AIS), the site of action potential initiation. They have been shown
to contribute to the spike trigger and also to regulate the repetitive
discharge properties of hippocampal CA1 pyramidal neurons. Mice
lacking NaV1.6 have lower instantaneous firing rates and maximal
firing rates in retinal ganglion cells (GCs).11,12 Impaired Nav1.6
function in SCN8A (encoding the Nav1.6
a-subunit) mutant mice
ameliorates seizure harshness in a severe myoclonic epilepsy of in-
fancy (SMEI) mouse model.13,14 In another study, the same mutant
mice displayed considerable resistance to the initiation and devel-
opment of kindling, a well-established model of abnormal plastic-
ity leading to prolonged seizures and to epilepsy.9 It was also found
that kindling is associated with higher expression of Nav1.6 sodium
channels in hippocampal CA3 neurons. All of these findings suggest
that selective pharmacological inhibition of Nav1.6 sodium chan-
nels could increase the efficacy of anti-epileptic drugs providing
them with an improved therapeutic profile.
In a recent report, a series of 2,4(1H)-diarylimidazoles were
identified as sodium channel blockers (Fig. 1).15,16 These deriva-
tives carry a central imidazole moiety with two phenyl rings at-
tached to positions 2 and 4, respectively. Different substituents
were introduced on each phenyl ring to vary the physico-chemical
properties (ionization, lipophilicity, steric hindrance) of the
Nav1.1
a-subunit), SCN2A (encoding the Nav1.2 a-subunit), SCN1B
(encoding the auxiliary b1-subunit), SCN3A (encoding the Nav1.3
a
-subunit) and SCN9A (encoding the Nav1.7 a-subunit) have been
reported in brain tissues of humans with epileptic syndromes.5–8
In animal models of epilepsy, the Nav1.6 sodium channel isoform,
which is heavily expressed with the central nervous system, has
been shown to be upregulated.9,10 The Nav1.6 isoform is thought
to play an important role in controlling neuronal firing. Nav1.6
⇑
Corresponding author.
E-mail address: mirko.rivara@unipr.it (M. Rivara).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.08.067

6402
M. Rivara et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6401–6404
Starting from this interesting series of sodium channel blockers,
R1
we decided to extend the SAR study on that class of compounds by
replacing the imidazole with a different heterocyclic scaffold and
by varying the aryl substituents on the central ring. The SAR anal-
ysis of these new series of compounds should provide us with
additional information about the structural features necessary to
inhibit voltage-gated sodium channels, providing new compounds
that may have AED activity. The new molecules were designed by
replacing the imidazole ring with a triazole, because of its interest-
ing properties from a medicinal chemistry standpoint, such as good
stability to metabolic degradation and a straightforward synthetic
route. Interestingly, several drugs contain the 1,2,3-triazole
moiety; among them, tazobactam, an inhibitor of bacterial beta-
lactamases, various cytostatic and antiproliferative agents and,
most importantly, rufinamide, an anticonvulsant medication
developed in 2004.18–21
N
R
N
H
Figure 1. 2,4(1H)-Diarylimidazoles.
resulting molecules. Some of the compounds presented in those
studies had IC50 values within the nanomolar–micromolar range
against another neuronal expressed sodium channel isoform,
NaV1.2 and showed anticonvulsant activity in an acute in vivo sei-
zure rodent model (MES: Maximal Electoshock Seizure test) with
low sedative and ataxic side effects when tested in a rat rotorod
test.17
Table 1
Electrophysiological evaluation of compounds 1–14 efficacy against rNaV1.6
R1
R
N
N
N
Compd
R
R1
Percent block of rNaV1.6 current at 10
6.2 1.6
lM (n = 4)
IC50
N.D.
(lM)
1
2
3
16.0 1.0
5.9 1.3
28.5
N.D.
O
O
4
5
10.0 2.1
11.0 1.7
125.4
N.D.
O
O
6
26.0 3.0
31.7
7
8
11.5 1.5
8.9 1.2
N.D.
N.D.
O
O
9
22.7 3.1
52.0
10
11
3.9 1.0
N.D.
N.D.
O
O
O
O
O
13.6 2.7
12
13
22.7 4.5
16.0 3.5
53.6
32.2
H
N
N
H
N
14
21.7 2.2
19.6
H3CO
N
N.D.: not determined.

M. Rivara et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6401–6404
6403
R = -CH2-O-Ph; R1 = Ph (2)
R
R = -CH2-CH2-CH2-O-Ph; R1 = Ph (3)
a
R1
+
N3
R
N
N
R1
R = Ph; R1 = -CH2-CH2-Ph (7)
N
R = -CH2-O-Ph; R1 = -CH2-CH2-Ph (8)
R = -CH2-CH2-CH2-O-Ph; R1 = -CH2-CH2-Ph (9)
2, 3, 7, 8, 9
Scheme 1. Reagents and conditions: (a) alkyne (2.0 mmol), azido-derivative (2.0 mmol), Na ascorbate (0.5 mmol), CuSO4 (0.025 mmol), tBuOH/H2O (1:1) (6 mL), 65 °C,
overnight, 70–88% yields.
R = Ph; R1 = -CH2-Ph (4)
R
R = -CH2-O-Ph; R1 = -CH2-Ph (5)
a
R1 Br
+
R
R = -CH2-CH2-CH2-O-Ph; R1 = -CH2-Ph (6)
R = Ph; R1 = -CH2-CO-Ph (10)
R = -CH2-O-Ph; R1 =-CH2-CO-Ph (11)
R = -CH2-CH2-CH2-O-Ph; R1 = -CH2-CO-Ph (12)
N
N
R1
N
4, 5, 6, 10, 11, 12
Scheme 2. Reagents and conditions: (a) alkyne (2.1 mmol), bromo-derivative (2.0 mmol), NaN3 (2.1 mmol), Na ascorbate (0.5 mmol), CuSO4 (0.1 mmol), tBuOH/H2O (1:1)
(6 mL), 65 °C, overnight, 72–96% yields.
In view of the fact that the NaV1.6 sodium channel has been
heavily implicated in epilepsy as well as other common CNS disor-
ders,14 we decided to test these new derivatives (at a concentration
phenyl-triazole derivatives 2 and 7, which demonstrated greater
inhibition of rNav1.6 currents compared to the previous com-
pounds. An interesting pattern can be observed by considering
the different substituents introduced in position 1 of the triazole.
In particular, the substituent in R (phenyl for 1, 2, 3, benzyl for 4,
5, 6; phenylethyl for 7, 8, 9 and phenylethanone for 10, 11, 12)
correlates with an increase in inhibitory activity with the length-
ening of the chain in the substituent at position 4 (R1). In fact, as
shown in Table 1, the most potent molecule for each group pos-
sesses a phenoxypropyl-chain in R1 (6, 9 and 12). Nevertheless,
considering the first group (compounds 1, 2, 3), it is possible to
observe that compound 2, having a phenoxymethyl-chain, is the
most active, behaving as an outlier. The three phenoxypropyl-
derivatives (6, 9 and 12) display comparable inhibitory activity
against rNav1.6 currents, independently of the substituent carried
on position 1 of the triazole ring.
of 10 lM) for activity against the rNav1.6 isoform stably expressed
in Human Embryonic Kidney (HEK 293) cells. We also tested two
diarylimidazoles compounds previously shown to have activity
against hNaV1.2, on rNaV1.6 for comparison purposes.
The 1,4-disubstituted 1,2,3-triazoles reported in Table 1 were
prepared according to Schemes 1 and 2, employing the copper-
catalyzed azide–alkyne cycloaddition (CuAAC), a widely used
synthetic strategy characterized by high efficiency and a simple
workup procedure.22,23 With a one-pot reaction, starting from
the appropriate alkyne and azido-derivative or bromo-derivative,
copper(II) sulfate and sodium ascorbate, used for the in situ gener-
ation of Cu(I) catalyst, after heating the reactions mixture
overnight at 65 °C in tBuOH/H2O (1:1), we were able to obtain
the desired products with yields ranging from 70% to 96% (see
Supplementary data).
The copper-catalyzed azide–alkyne cycloaddition (CuAAC) was
employed to prepare 1,4-triazoles derivatives with different aryl
substituents at the N1 and C4 positions. Although none of the new-
ly synthesized compounds resulted in more active compounds
compared to the reference compounds 13 and 14 at inhibiting
rNav1.6 sodium channels, most of them were able to inhibit
Compounds 1–14 were evaluated for their ability to inhibit
rNaV1.6 sodium channel currents at a concentration of 10
(see Supplementary data).
lM
The 1,4-triazole derivatives 1–12 were screened for activity
against the rNav1.6 sodium channel isoform at 10 M. Table 1
l
rNav1.6 currents at 10 lM by more than 20%. The newly synthsized
shows the correlation between triazole substitutions and inhibi-
tion of rNaV1.6 channel currents. For comparison purposes, we
also tested two diarylimidazoles, previously shown to have differ-
sodium channel blockers displayed IC50 values that were in the
micromolar concentration range, indicating that triazole can effi-
ciently replace the central heterocyclic core. Moreover, the intro-
duction of a long chain at C4 of the central triazole seems
beneficial for rNav1.6 current inhibition, whereas the length of
N1 substituent seems less crucial for this activity, as long as a
phenyl ring is not direcly connected to the triazole. These results
provide additional information on the structural features necessary
for the block of the voltage-gated sodium channel. These new data
could be useful on the design of future candidate therapeutics.
Activity against rNav1.6 may lead to the generation of more
effective and better tolerated anticonvulsant drugs.
ing activity against hNaV1.2 (13, 14). At a concentration of 10 lM,
compound 13 exhibited a small block of hNav1.2 (7.8%) while
compound 14 had a greater block (30.1%). Results reported in
Table 1 showed similar behaviour of both compounds 13 and
14 against hNaV1.2 and rNaV1.6 at 10 lM (16.0% block for 13
and 21.7% block for 14). The new triazole derivatives 1–12, syn-
thesized with the aim to study a new central heterocycle with
different substituents, inhibited rNaV1.6 currents between 5.8%
and 25.9% at 10 lM, thus showing comparable potencies with
the diarylimidazole blockers. Dose-response curves were gener-
ated for compounds 2, 4, 6, 9 and 12 and the calculated IC50’s
Acknowledgments
are reported in Table 1. IC50 values ranged between 28.5
lM
and 125.4 M. These findings suggest that the presence of a
l
We are grateful to the Centro Interdipartimentale Misure of the
University of Parma for providing the NMR instrumentation. Fund-
ing from the National Institutes of Health NINDS R21NS061069-02
(M.K.P.) is gratefully acknowledged.
phenyl ring direcly connected to the central triazole is less favor-
able for inhibition of rNav1.6, as demonstrated by the percent
block of compounds 1, 3, 4 and 10. Two exceptions were the

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M. Rivara et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6401–6404
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