Novel modulator of NaV1.1 and NaV1.2 Na+ channels in rat neuronal cells

Article abstract of DOI:10.1021/ml100035t

A novel modulator of sodium ion currents was synthesized in 6 steps from a protected dihydroxypyrrolidine nitrone, via 1,3-dipolar cycloaddition reaction with acrylamide. Sodium ion currents in B50 cells were evaluated in comparison to saxitoxin and tetro

Full text of DOI:10.1021/ml100035t

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Novel Modulator of NaV1.1 and NaV1.2 Naþ Channels
in Rat Neuronal Cells
Hua Mao,‡,§, Lynne A. Fieber, ,^ and Robert E. Gawley*,‡
‡Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, Arkansas 72701, and ^Division of Marine
Biology and Fisheries, University of Miami, 4600 Rickenbacker Causeway, Miami, Florida 33149
ABSTRACT A novel modulator of sodium ion currents was synthesized in 6 steps
from a protected dihydroxypyrrolidine nitrone, via 1,3-dipolar cycloaddition
reaction with acrylamide. Sodium ion currents in B50 cells were evaluated in
comparison to saxitoxin and tetrodotoxin and revealed an IC50 of 15.7 μM. The new
compound shows no evidence of binding to the C-lobe of the saxitoxin-binding
protein saxiphilin.
KEYWORDS Nitrone, 1,3-dipolar cycloaddition, saxitoxin (STX), tetrodotoxin
(TTX), sodium channel blocker, B50 cells
oltage gated sodium channels are targets for antic-
onvulsant, antiarrhythmic, and analgesic drugs.1-5
Sodium channels are responsible for excitatory trans-
saxitoxin and detected electrochemically. We therefore de-
signed the mimic, 1, and an electrochemically active deri-
vative, 2, and developed the syntheses outlined in this letter.
Although no binding to saxiphilin was detected by surface
plasmon resonance spectroscopy, sodium channel blockage
analogous to STX and TTX was found in B50 rat neuronal
cells19 that express type I and II sodium channels (NaV1.1
and NaV1.2 in the new terminology20), so we hereby disclose
our results as a novel scaffold and potential lead for further
development.
V
mission, and there are more than ten closely related iso-
forms of mammalian sodium channels known, which
primarily affect tissues of the central and peripheral nervous
systems, and skeletal muscle.6 There are a number of drug
types that target various sodium channels, and some very
potent toxins also target sodium channels. Two such toxins,
saxitoxin (STX) and tetrodotoxin (TTX), bind competitively
to the same site on voltage-gated sodium channels,7-10 and
are the only two small molecules on the US government's list
of select agents.11 Saxitoxin is a diabolically complex mole-
cule. It has more heteroatoms than carbons, two positive
charges, and an unusually low pKa of 8.24 for the five-
membered guanidinium ion (guanidinium itself has a pKa
of ∼12.5-13.2). STX and its two-dozen structural analogues
are collectively responsible for the symptom known as
paralytic shellfish poisoning.10,12 Tetrodotoxin is similarly
complex, and is the causative agent of fugu poisoning: eating
of tainted Japanese puffer fish.13,14
Common features of the structures of STX and TTX are
5- and/or 6-membered rings, multiple heteroatoms, and
guanidinium ions. Our STX mimic includes two hydroxyls,
one guanidinium, one carboxamide, and two rings. These
compounds were prepared using a convergent 1,3-dipolar
cycloaddition strategy. To maximize the possibility of observ-
ing binding (if binding is enantioselective), the synthesis was
executed using racemic tartaric acid. Regiochemical and
stereochemical features of the 1,3-dipolar cycloaddition of
pyrrolidine nitrones have been carefully studied.21-23
The key starting material, (()-bis(methoxymethoxy)pyr-
rolidine nitrone 3, was prepared from rac-tartaric acid,
according to the literature procedure.24 Scheme 1 details
For several years, we have been interested in new meth-
ods of detection of saxitoxin and its analogues,15 and one of
the avenues we have pursued is a displacement assay in a
microfluidic channel incorporating an electrochemical de-
tector16 using the c-lobe of the saxitoxin binding protein,
saxiphilin.17,18 As part of this effort, we sought a new ligand
for saxiphilin, a saxitoxin mimic, that could be displaced by
Received Date: February 17, 2010
Accepted Date: March 24, 2010
Published on Web Date: March 31, 2010
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DOI: 10.1021/ml100035t ACS Med. Chem. Lett. 2010, 1, 135–138
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Scheme 1
Scheme 2
the synthesis of the mimic (rac-1), via 1,3-dipolar cycloaddi-
tion of nitrone 3 to acrylamide 4. Cycloaddition of an
unsubstituted nitrone with methyl acrylate affords two
regioisomers, with little or no endo/exo selectivity. In the
illustrated case, the two competing transition states, 5
and 7, provided the two regioisomers 6 and 8, each as a
mixture of two stereoisomers, which were inseparable.
Hydrogenolysis gave a separable mixture of constitu-
tional isomers 9 and 10. Guanidinylation25 of 9 (mixture of
diastereomers) and chromatographic removal of the minor
epimer gave 11 in 77% yield. Cyclization was achievedeither
under Mitsunobu conditions or, more efficiently, by mesyla-
tion of the free hydroxyl group, which resulted in the
spontaneous cyclization to give MOM and Cbz protected
bicyclic compound 12. The relative configuration was estab-
lished by NMR analysis of 12. After removal of the MOM and
Cbz protecting groups, mimic 1 was isolated as its hydrochlo-
ride salt.
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Figure 1. B50 Naþ currents at -20 mV (Vh = -120 mV) in the extracellular solution (control) and upon exposure to bath-applied
tetrodotoxin (TTX; 100 nM), saxitoxin (STX, 12 μM), or 1 (24 μM).
Scheme 3
A similar 1,3-dipolar cycloaddition, employing substi-
tuted acrylamide 20, was used to prepare 2. The synthe-
sis of the substituted acrylamide was prepared as illu-
strated in Scheme 2. Bromination of trimethoxybenzene
14 and coupling26 afforded alkyne 15 in 67% overall
yield. Reduction of the alkyne to 16, mesylation of the
hydroxyl to give 17 and azide displacement gave 18 in
68% overall yield for the 3 steps. Reduction to amine 19
and carbodiimide coupling with acrylic acid gave acetamide
20 in 68% yield for the two steps (31% for the seven steps
overall).
Cycloaddition of nitrone 3 with acrylamide 20 gave pyr-
rolidinoisoxazole 21 as the major isomer, and reduction
afforded pyrrolidine 22 in 70% yield for the two steps
(Scheme 3). Guanidinylation, mesylation/cyclization, and
deprotection as before afforded the electrochemically active
mimic 2 in a total of 6 steps and 47% overall yield. Full
experimental details for the three synthetic schemes are in
the Supporting Information.
The STX mimic 1 was tested for sodium channel activity
in B50 rat neuronal cells.19 This cell line originates from the
rat CNS19 and expresses types I and II Na channel mRNAs
(NaV1.1 and NaV1.2).20 Specifically, whole cell Na currents
were recorded in B50 cells in extracellular solution (ECS)
containing (mM): 170 NaCl, 3 KCl, 2.5 CaCl2, 1.2 MgCl2, 10
HEPES-NaOH, pH 7.25. The intracellular solution con-
tained (mM) 150 KCl, 1 ethylene glycol-bis(β-aminoethyl
ether)-N,N,N0,N0-tetraacetic acid (EGTA), 1 CaCl2, 5 MgCl2, 5
Na2ATP and 40 N-[2-hydroxyethyl]piperazine-N0-[2-ethane-
sulfonic acid] (HEPES)-KOH, pH 7.0. Drugs were applied to
cells during a series of depolarizing episodes of 7 ms
separated by 4 s intervals at the holding potential (Vh) of
-120 mV. Tetrodotoxin (100 nM in ECS) was applied as a
bolus volume of 50 μL in a 1.5 mL bath. STX and our
prototype mimic were continuously bath-applied to cells
via a 1 μL pipet. Both STX and 1 mimic blocked the
same currents blocked by TTX, as shown in Figure 1. (STX
and TTX bind competitively to site 1 on voltage gated sodium
channels.)
Figure 2 shows the dose-response relationship for the
inhibition of Na current by mimic 1 in B50 neuronal cells.
The curve was fit to the equation
I=Imax ¼ Imax - ðImax=½1 þ ðexpð - ðB - IC50Þ=nÞÞꢀÞ
where I is the current amplitude, B is the concentration of
the 1, n is the Hill coefficient, and IC50 is the half-inhibitory
concentration to the electrophysiological responses to
bath-applied 1. The IC50 value derived from Figure 2 is
15.7 μM; the Hill coefficient of 2.95 implies some allo-
steric cooperativity in the binding of the mimic. In com-
parison, saxitoxin shows an IC50 value of 0.38 μM, with
a Hill coefficient of 0.99, in these same cells (data not
shown).
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CONCLUSION In summary, the synthetic STX mimic 1 and
its electroactive mimic 2 were successfully obtained via the
1,3-dipolar cycloaddition reaction. Either acrylamides 4 or
20 are suitable dipolarophiles toward the nitrone, which led
to the final STX mimic and its corresponding electrochemi-
cally active derivative. It is very encouraging that the STX
mimic exerts a similar effect on the sodium channel of
neuronal rat cells, probably by binding to the same site as
STX and TTX (site 1).
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SUPPORTING INFORMATION AVAILABLE Synthesis and
characterization of all compounds. This material is available free
of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author: *To whom correspondence should
be addressed. Tel: 479-575-6933. Fax: 479-575-5178. E-mail:
bgawley@uark.edu.
Present Addresses: § Current address: c/o NN-Laboratories, PO
Box 2168, Fayetteville, AR 72702.
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Author Contributions: All synthetic work was done by H.M.,
and the electrophysiology by L.A.F.
Funding Sources: Core facilities for this work were funded by
the Arkansas Biosciences Institute and the National Institutes of
Health (RR15569).
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DOI: 10.1021/ml100035t ACS Med. Chem. Lett. 2010, 1, 135–138
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