Synthesis and biological evaluation of a fluorescent analog of phenytoin as a potential inhibitor of neuropathic pain and imaging agent

Article abstract of DOI:10.1016/j.bmc.2012.06.042

Here we report on a novel fluorescent analog of phenytoin as a potential inhibitor of neuropathic pain with potential use as an imaging agent. Compound 2 incorporated a heptyl side chain and dansyl moiety onto the parent compound phenytoin and produced greater displacement of BTX from sodium channels and greater functional blockade with greatly reduced toxicity. Compound 2 reduced mechano-allodynia in a rat model of neuropathic pain and was visualized ex vivo in sensory neuron axons with two-photon microscopy. These results suggest a promising strategy for developing novel sodium channel inhibitors with imaging capabilities.

Full text of DOI:10.1016/j.bmc.2012.06.042

Bioorganic & Medicinal Chemistry 20 (2012) 5269–5276
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of a fluorescent analog of phenytoin
as a potential inhibitor of neuropathic pain and imaging agent
Thomas H. Walls a, Scott C. Grindrod a, Dawn Beraud b, Li Zhang a, Aparna R. Baheti c,
b
Sivanesan Dakshanamurthy a, Manoj K. Patel c, Milton L. Brown a, , Linda H. MacArthur
⇑
a Drug Discovery Program, Department of Oncology, Georgetown University Medical Center, 3970 Reservoir Rd., NW, Washington, DC 20057, USA
b Department of Neuroscience, Georgetown University Medical Center, 3970 Reservoir Rd., NW, Washington, DC 20057, USA
c 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:
Here we report on a novel fluorescent analog of phenytoin as a potential inhibitor of neuropathic pain
with potential use as an imaging agent. Compound 2 incorporated a heptyl side chain and dansyl moiety
onto the parent compound phenytoin and produced greater displacement of BTX from sodium channels
and greater functional blockade with greatly reduced toxicity. Compound 2 reduced mechano-allodynia
in a rat model of neuropathic pain and was visualized ex vivo in sensory neuron axons with two-photon
microscopy. These results suggest a promising strategy for developing novel sodium channel inhibitors
with imaging capabilities.
Received 10 April 2012
Revised 18 June 2012
Accepted 25 June 2012
Available online 3 July 2012
Keywords:
Sodium channel inhibitor
Mechano-allodynia
Dansyl
Ó 2012 Published by Elsevier Ltd.
Phenytoin
Neuropathic pain
1. Introduction
In a previous study, DPH analogs were designed based on a
QSAR model developed from [3H]-batrachotoxin-20-
a-benzoate
Human voltage-gated sodium channels (VGSCs) represent key
targets for the development of anticonvulsants, anesthetics, and
antiarrhythmic drugs.1–4 Despite the widespread use of sodium
channel inhibitors clinically, target selectivity, side effects and tox-
icity have hindered drug development.
The VGSCs have nine binding sites that cause a change in their
gating properties. Anesthetics bind to site 9 and cause a persistent
inactivation of the channel.5,6 Site 2 binds the neurotoxin batrach-
otoxin (BTX) and compounds that bind to anesthetic site 9 alloster-
ically displace BTX from site 2 providing a way to monitor site 9
binding.7
Diphenylhydantoin (DPH, phenytoin), a clinical VGSC inhibitor
used to treat epilepsy and chronic pain, binds to site 9 of VGSCs
and has served as a lead compound in the discovery of novel VGSC
inhibitors.8–10 In this model, DPH binds with higher affinity to
sodium channels in the open or inactive state than to channels in
the resting state, resulting in decreased sodium current at high
rates of stimulation.3,4,11
([3H] BTX-B) displacement in rat brain synaptoneurosomes.12
The resulting comparative molecular field analysis (COMFA)
model demonstrated that replacing one of the phenyl rings of
DPH with a heptyl side chain, reaching a hydrophobic region
of the binding site, was preferred. The addition of a meta-chloro
group on the remaining phenyl ring was optimal to binding
(Scheme 1, compound 1).2 To exploit this hydrophobically
receptive region of the drug pharmacophore, we added
a
pentyl tethered 5-(dimethylamino)-8-sulfonyl-naphthalene (Dan-
syl) moiety.13 Herein, we report the synthesis of phenytoin
analogs and their evaluation for BTX displacement, functional
blockade, toxicity, and imaging properties. A binding model of
1 and 2 is presented.
2. Results
2.1. Design and synthesis of compounds 1 and 2
The synthesis of 12 and the intermediate sulfonamide 314 have
been described previously. The sulfonamide 3 was converted to the
Weinreb amide 4 through an amide coupling in the presence of
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDCI). Follow-
ing an aryl Grignard addition to yield the ketone 5 the desired
Abbreviations: VGSC, voltage-gated sodium channel; hNav1.2, human sodium
channel 1.2; BTX, batrachotoxin; DPH, diphenylhydantoin.
⇑
Corresponding author.
E-mail address: mb544@georgetown.edu (M.L. Brown).
0968-0896/$ - see front matter Ó 2012 Published by Elsevier Ltd.
http://dx.doi.org/10.1016/j.bmc.2012.06.042

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T. H. Walls et al. / Bioorg. Med. Chem. 20 (2012) 5269–5276
Scheme 1. Design of DPH analogs based on a QSAR model using [3H] BTX-B displacement.
hydantoin scaffold 2 was prepared via the Bucherer-Berg modified
Strecker reaction (Scheme 2).
2.2. Fluorescence spectrum of compound 2
The fluorescence of the hydantoin core was confirmed by anal-
ysis of the excitation-emission spectrum (Fig. 1). The fluorescence
spectrum of 2 exhibited two excitation maxima with one exciting
at 290 nm (S0?S2) and emitting at 510 nm (S1?S0) and one excit-
ing at 400 nm (S0?S1) and emitting at 513 nm (S1?S0). This
equates to a Stokes shift of 113 nm.
2.3. VGSC affinity, functional blockade, and acute toxicity of
compounds 1 and 2
To determine if 1 and 2 bound to sodium channels like the par-
ent compound phenytoin (DPH), they were assessed for the magni-
tude of displacement of [3H] BTX-B in rat forebrain membranes,
where sodium channels are highly expressed. Both 1 and 2 showed
greater displacement of [3H] BTX-B than DPH (Table 1).
To test whether the compounds were functionally active as so-
dium channel blockers, the effects of 1 and 2 on human embryonic
kidney cells (HEK 293) stably expressing hNav1.2 sodium channels
was assessed using the whole-cell patch-clamp method. Inhibition
of sodium currents represents functional blockade of VGSCs. 1 and
2 inhibited sodium currents by 30% and 59%, respectively (Table 1).
Acute toxicity experiments were performed using the OECD Up-
and-Down Test to reduce the number of animals required. The tox-
icity of DPH and 1 were comparable. Notably, the toxicity of 2 was
reduced in mice by up to 13-fold.
Figure 1. Excitation and emission spectrum of 2.
pathic pain, they were tested in a rat model of sciatic nerve
constriction. After nerve constriction, rats developed a significant
reduction in threshold to von Frey filaments applied to the affected
hindpaw, as expected with this model (Fig. 2, Presurgery vs Base-
line-1). Both 1 and 2 increased withdrawal threshold, with a signif-
icant increase in threshold exhibited for 2. The effect of gabapentin
(an anti-seizure drug and calcium channel blocker) on mechano-
allodynia is shown in a separate cohort of rats to provide an
approximate comparison with a drug that is used clinically to treat
neuropathic pain.
2.5. Effect of compound 2 on motor control and seizure
induction
2.4. Effect of compounds 1 and 2 on mechano-allodynia
Phenytoin has been used clinically to reduce symptoms associ-
ated with neuropathic pain. To determine if 1 and 2 reduce neuro-
Because of its reduced toxicity and efficacy in a rodent model of
neuropathic pain, 2 was examined for its effects on motor control
Scheme 2. Synthetic route for 2. (a) 1 M NaHCO3, dansyl chloride, TEA, H2O/acetone, room temp, 3H, 85%. (b) EDCI, N,O-dimethylhydroxylamine HCl, DMAP, DCM, room
temp, 2.5 h, 75%. (c) 3-Chlorophenyl magnesium bromide, THF, 0 °C, room temp, 16 h, 60%. (d) (NH4)2CO3, KCN, H2O/EtOH/THF (3:2:1), 75 °C, 3H, microwave 35%.

T. H. Walls et al. / Bioorg. Med. Chem. 20 (2012) 5269–5276
5271
Table 1
in both the maximal electroshock test (MES) and subcutaneous
metrazol seizure threshold (scMET) models of epilepsy, unlike
the parent compound DPH, which increased seizure thresholds.
VGSC affinity, functional blockade, and acute toxicity15
Compound
% [3H] BTX-B
displacement
% NaV1.2
functional block
Acute toxicity
(i.p.) LD50 (mg/kg)
(40
l
M)
(10
lM)
2.6. Visualization of compound 2 ex vivo
DPH
28
11
4
15615
93
[139–178]
31
[55–175]
59
[646–2650]
Incorporation of a dansyl group into the drug pharmacophore of
2 suggested it might be useful as a fluorescent ligand. To determine
if 2 could be visualized ex vivo in neuronal tissue, sections from
dorsal root ganglia (DRG) (the nerve root and neuronal cell bodies
of the constricted sciatic nerve), were treated with 2, and visual-
ized with two-photon confocal microscopy. Differential staining
of 2 was observed in DRG tissue and showed heaviest localization
in axons and nerve fibers compared to neuronal cell bodies (Fig. 3).
Compound
1
Compound
2
70
86
5
4
2000
% BTX displacement: In vitro displacement of [3H] BTX-B by DPH, 1, and 2 in rat
forebrain membranes. Compounds were applied at 40 m. % Nav1.2 block was
measured in vitro in HEK cells expressing Nav 1.2 compounds were applied at
10 M. Compound toxicity was calculated using the OECD up-and-down test
guideline 425 (LD50). Values are statistical estimates based on the long-term fate of
7–10 mice per dose. Beneath the LD50 values are 95% confidence intervals where the
assumed sigma is 0.5 mg/kg. The DPH acute toxicity data was previously published.
l
l
2.7. SAR of novel phenytoin derivatives
The relationship between modification of the phenyl groups of
phenytoin and [3H] BTX-B displacement was assessed (Table 3).
Replacing a phenyl ring with a heptyl side chain resulted in im-
proved BTX displacement (1). Substitution of aromatic groups
was favorable (3a) although binding was diminished when the
methylene linker was replaced with an ethylene linker (3c) possi-
bly due to steric hindrance. Modification of the heptyl side chain to
increase hydrophobicity was favorable (2b, 11a) whereas activity
was lost when side chain hydrophobicity was reduced (11b). Addi-
tion of a hydrophobic dansyl group resulted in increased BTX dis-
placement (2).
2.8. Model of compound 1 and 2 at the BTX binding site of
sodium channels
The sodium channel homology model is described in a previous
report.16 Since 1 and 2 displace BTX, we hypothesized that they
bind to the open state of the sodium channel. Compounds 1 and
2 were docked into the homology model using the program Sur-
FlexDock incorporated in Sybyl X.17 The starting structure was
the unminimized constructed 3-D structure. A preliminary confor-
mation search was performed using systemic search/analysis in
Sybyl X. To be consistent with the mutation studies18–20 and previ-
ous known interactions of BTX analogs, the docked positions of 1
and 2 were remodeled using step-by-step manual docking with
restrained Molecular Dynamics simulations followed by energy
minimization. In the restrained MD simulations, the optimal H-
bond and hydrophobic distance restraints were set between the
sodium channel pore forming residues and the compounds.
A structural model of 1 and 2 docked to the sodium channel is
shown in Figure 4. Although, some uncertainly remains for several
residues, our binding model predicted residues F1283, F1579,
L1582, V1583, Y1586 in IVS6, and T1279, L1280 in IIIS6, and
L788, F791, L792, in IIS6 and F430, I433, L437 in IS6 contributed
to the tight binding interaction of 1 (Fig. 4A) and 2 (Fig. 4B). The
shape and size of 1 and 2 (shown in green) are complimentary to
the model of the BTX binding site (shown in cyan), where the imi-
dazolidine-2,4-dione fits into the sodium channel sub-cavity
formed by T1279 (IIIS6), L1280 (IIIS6), F791(II) and L792 (II).
Figure 2. Compound 2 and mechano-allodynia in rats. Withdrawal threshold to
von Frey filaments was measured 2 h after a subcutaneous injection of 1 (C1) or 2
(C2) in rats with sciatic nerve ligation and mechano-allodynia. N = 5 rats per group.
Data represent the mean and SEM; 1-way ANOVA with Tukey’s post-hoc tests. C1;
compound 1, C2; compound 2. Drug dose is in mg of drug per kg body weight of rat.
⁄p <0.05, ⁄⁄p <0.01, ⁄⁄⁄p <0.001.
and balance. A rotarod test was performed to test 2 at three differ-
ent doses. None of the mice demonstrated neurological impair-
ment at any of the drug doses tested at 0.5 and 4 h after drug
administration (Table 2).
The parent compound phenytoin is used clinically as an anti-
convulsant. To assess whether 2 reduced seizures, it was tested
in rodent models of seizure induction. Compound 2 was ineffective
Table 2
Compound 2 and the rotarod test in mice
Compound 2 (mg/kg)
Motor impairment/total tested
3. Discussion
0.5 (h)
4.0 (h)
30
100
300
0/4
0/8
0/4
0/2
0/4
0/2
Blockers of human voltage-gated sodium channels are used
clinically as anticonvulsants, antiarrhythmics, anesthetics, and
more recently, to treat neuropathic pain. While widely used, issues
of channel selectivity, off-target effects, and toxicity continue to
hinder further drug development. In this study we synthesized
compounds with increased affinity for the BTX binding site of
Mice were injected intraperitoneally with 2 and tested at 0.5 and 4.0 h on the
rotarod test. Normal controls maintained equilibrium on a rod rotating at 6 rpm for
one minute. Numbers show number of mice that did not complete each test out of
the number of mice tested.

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T. H. Walls et al. / Bioorg. Med. Chem. 20 (2012) 5269–5276
Figure 3. Two-photon confocal microscopy image of compound-2 in rat dorsal root ganglia paraffin sections. Rat DRG sections (5
lM) were deparaffinized and treated with 2
at 100 M for 5 min before washing and imaging. Samples were excited at 725 nm and monitored at 510 nm. Scale bar, 100 m B. Inset from panel A.
l
l
Table 3
Structure–activity relationships of novel phenytoin derivatives
Compound name
% BTX
28
Structure
Phenytoin (DPH)
Compound 1 (2a in Supplementary Scheme 1)
70
3a (Supplementary Scheme 1)
72
3d (Supplementary Scheme 1)
83
3c (Supplementary Scheme 1)
17

T. H. Walls et al. / Bioorg. Med. Chem. 20 (2012) 5269–5276
5273
Table 3 (continued)
Compound name
% BTX
Structure
2b (Supplementary Scheme 1)
11a (Supplementary Scheme 2)
11b (Supplementary Scheme 2)
79
87
28
86
Compound 2 (Scheme 2 and Supplementary Scheme 5)
% BTX is the percent displacement of [3H] batrachotoxin-20-
a
-benzoate (BTX) from the saturated site 2 of VGSCs in rat brain synaptoneurosomes at 40
lM of test compound.
Compounds that displaced greater than 50% of the [H] BTX-B were considered active analogs for further study.
VGSCs using deductions derived from our 3-D QSAR developed
from phenytoin (DPH).21 Incorporation of a heptyl side chain con-
ferred additional displacement of [3H] BTX-B and further func-
tional blockade of VGSCs in patch-clamp electrophysiology
experiments. The addition of a dansyl moiety (2) resulted in fur-
ther BTX displacement and functional blockade compared to DPH
or 1.
Incorporation of a dansyl group into the pharmacophore of phe-
nytoin resulted in a fluorescent analog of phenytoin. Compound 2
was visualized in the sciatic nerve and dorsal root ganglia (DRG)
ex vivo with two-photon microscopy. The drug bound primarily
to axons, consistent with structural studies on sodium channel
density which indicate higher protein levels in the axon than the
soma.22,23
Compound 2 significantly reduced mechano-allodynia associ-
ated with neuropathic pain after sciatic nerve compression compa-
rable to that observed with gabapentin, a drug used clinically to
treat chronic pain.24,25 Notably, 2 was less toxic than the parent
compound DPH suggesting it may hold improvements over other
sodium channel blockers used to treat neuropathic pain, such as
carbamazepine and lamotrigine.26–29
Structural modeling of 1 and 2 binding to the open state of
VGSCs suggests that the increased displacement of [3H] BTX-B by
2 versus 1 likely stems from the following favorable additional
interactions within hydrophobic regions of the sodium channel:
(1) the amide group of imidazolidine-2,4-dione may form hydro-
gen bonds with 1279(IIIS6); (2) hydrogen bond interactions may
occur between the dansyl sulphamide with N434(I) and
Y1586(IV); (3) the dansyl group may form favorable hydrophobic
contacts with F1283 (IIIS6), L437(I), L788(II), L1280(IIIS6) in a
hydrophobic region of the binding site.
dium channel isoform selectivity, poor brain-penetrance relative
to the parent compound, changes in selectivity for state-dependent
inhibition allowing for improved selectivity for depolarized neu-
rons, or blocking at lower frequencies of stimulation and producing
a tonic block.30
In conclusion, compound 2 may represent a novel approach for
the development of safer sodium channel inhibitors to treat neuro-
pathic pain. The enhanced hydrophobic groups in the receptive re-
gion of the anesthetic binding region improved BTX displacement
and increased functional blockade of sodium channels while
reducing compound toxicity. By using drug design to incorporate
a dansyl fluorophore into the drug pharmacophore, we synthesized
an analog of a sodium channel inhibitor with the potential to be
used as an imaging agent that may be useful in biodistribution
studies both in vivo and ex vivo.
4. Experimental
4.1. Chemistry general procedures
Commercially available reagents were purchased from Sigma-
Aldrich. 1H and 13C NMR spectra were recorded on a Varian 400-
NMR magnet. Flash chromatographic purifications were done on
Biotage SP1 chromatography system monitoring at 254 nm.
4.2. Synthesis
4.2.1. 6-(5-(Dimethylamino)naphthalene-1-sulfonamido)-N-
methoxy-N-methylhexanamide (4)
EDCI (2.93 g, 20.6 mmol), N,O-dimethylhydroxylamine HCl
(2.02 g, 20.6 mmol), and 4-dimethylaminopyridine (DMAP)
(2.54 g, 20.6 mmol) were added to a solution of acid 3 (3.02 g,
8.23 mmol) in DCM. The reaction was stirred for 2.5 h before being
quenched with brine (20 mL). The phases were separated and the
Somewhat surprisingly, 2 did not suppress seizure induction,
suggesting differences between parent drug and 2 in pharmacoki-
netics or dynamics. Possible explanations include changes in so-

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T. H. Walls et al. / Bioorg. Med. Chem. 20 (2012) 5269–5276
It was then concentrated to dryness and purified by flash chromo-
tography (1:1 EtOAc/hex) to yield a yellow oil (1.04 g, 60%). 1H
NMR (400 MHz, CDCl3):d 1.21 (m, 2H), 1.43 (m, 4H), 2.71 (t,
J = 7.2 Hz, 2H), 2.83 (s, 6H), 2.91 (dd, J = 6.8 Hz, 13.2 Hz, 2H), 5.30
(t, J = 6.2 Hz, 1H), 7.12 (d, J = 7.0 Hz, 1H), 7.34 (t, J = 7.9 Hz, 1H),
7.49 (m, 3H), 7.72 (d, J = 7.8 Hz, 1H), 7.83 (s, 1H), 8.23 (d,
J = 6.1 Hz, 1H), 8.34 (d, J = 8.7 Hz, 1H), 8.50 (d, J = 8.5 Hz, 1H). 13C
NMR NMR (100 MHz. CDCl3): d 198.6, 151.7, 138.1, 134.7, 134.6,
132.7, 130.1, 129.7, 129.6, 129.4, 129.3, 128.1, 127.8, 125.9,
123.0, 118.6, 114.9, 45.2, 45.2, 42.8, 38.0, 29.0, 25.7, 22.9.
4.2.3. N-(5-(4-(3-Chlorophenyl)-2,5-dioxoimidazolidin-4-
yl)pentyl)-5-(dimethylamino) naphthalene-1-sulfonamide (2)
The ketone 4 (300 mg, 0.65 mmol) was dissolved in THF (1 mL).
EtOH (2 mL) was added followed by KCN (213 mg, 3.3 mmol) and
ammonium carbonate (628 mg, 6.5 mmol). Water (3 mL) was
added and the vessel was sealed. The tube was placed in a CEM
Discover microwave (temperature = 75 °C, power = 150 W) for
3 h. The product was extracted with DCM (3 Â 25 mL), washed
with brine (3 Â 35 mL) and the organic layer was dried over
Na2SO4 and evaporated to dryness. It was purified by flash chromo-
tography (1:1 EtOAc/Hex) to yield a light green crystalline solid
(301 mg, 35%). 1H NMR (400 MHz, CDCl3): d 1.12 (m, 4H), 1.25
(m, 2H) 1.96 (m, 2H), 2.86 (m, 8H), 5.72 (t, J = 6.0 Hz, 1H), 7.13
(d, J = 7.5 Hz, 1H), 7.28 (m, 2H), 7.38 (m, 1H), 7.49 (dd, J = 8.6,
17.0 Hz, 3H), 7.85 (s, 1H), 8.19 (d, J = 7.3 Hz, 1H), 8.29 (d,
J = 8.6 Hz, 1H), 8.51 (d, J = 8.4 Hz, 1H), 9.21 (s, 1H). 13C NMR
(100 MHz. CDCl3): d 175.0, 157.9, 139.8, 139.4, 134.7, 134.6,
130.3, 130.0, 129.5, 129.4, 128.7, 128.4, 128.2, 125.6, 125.2,
123.6, 123.2, 115.1, 68.4, 45.3, 42.5, 38.3, 28.8, 25.7, 22.9, 14.1.
LC–MS (ESI): m/z 530 (M+H)+; HRMS (TOF): C26H29ClN4O4S. Calcd
(M+1) 529.1598. Found 529.1688.
4.3. Modeling and docking of compounds 1 and 2 to the sodium
channel
The modeling of the sodium channel is described in our previ-
ous paper.16 Docking studies between ligand and the sodium chan-
nel were carried out using the program SurFlexDock (Sybyl X.
Tripos Inc., St. Louis, USA) and AUTODOCK 4.017 with all the param-
eters set to default. Molecular dynamics simulations were carried
out using AMBER 8.031 using the default parameters. The structure
of the Nav-compound 2 complex was then refined by molecular
dynamics simulation using the Amber 9.0 program suite. The
charge and force field parameters of compounds were obtained
using the most recent Antechamber module where 2 was mini-
mized at the MP2/6-31G⁄ level using Gaussian 03. The SHAKE algo-
rithm was used to fix the bonds involving hydrogen.32 The protocol
for the molecular dynamics simulation is described below: The to-
tal charge of the system was neutralized by addition of a chloride
ion. The system was solvated in a 12 Å cubic box of water where
the TIP3P model 8 was used. 2000 steps of minimization of the sys-
tem were performed in which the sodium channel was constrained
by a force constant of 75 kcal/mol/Å2. After minimization, a 10 ps
simulation was used to gradually raise the temperature of the sys-
tem to 298 K while the complex was constrained by a force con-
stant of 15 kcal/mol/Å. Another 25 ps of equilibrium run was
used where only the backbone atoms of the complex were con-
strained by a force constant of 5 kcal/mol/Å. A final production
run of 200 ps was performed with no constraints. When applying
constraints, the initial complex structure was used as a reference
structure. All the molecular dynamics simulations were at constant
temperature and pressure. The PME method10 was used and the
non-bonded cutoff distance was set at 12 Å. The time step was
5 fs, and the neighboring pairs list was updated in every 25 steps.
Figure 4. Model showing 1 and 2 docked into the BTX binding site of the hNaV
channel. The ball and stick model shows BTX binding site (white), and the feasibility
of binding of 1 (panel A), 2 (panel B) (green) and [3H] BTX-B (cyan).
organic phase was washed with 2 M HCl (10 mL) and brine (10 mL)
before being dried over Na2SO4. The solution was concentrated and
purified by flash chromatography (1:20 MeOH/DCM) to yield a yel-
low oil (2.54 g, 75%). 1H NMR (400 MHz, CDCl3): d 1.21 (m, 2H),
1.42 (m, 4H), 2.27 (t, J = 7.3 Hz, 2H), 2.87 (m, 8H), 3.12 (s, 3H),
3.61 (s, 3H), 5.17 (t, J = 6.1 Hz, 1H), 7.16 (d, J = 7.5 Hz, 1H), 7.51
(m, 1H), 8.21 (dd, J = 1.2,7.3 Hz, 1H,), 8.31 (d, J = 8.7 Hz, 1H,), 8.52
(d, J = 8.5 Hz, 1H,). 13C NMR (100 MHz. CDCl3): d 134.9, 130.1,
129.7, 129.5, 129.3, 128.1, 123.1, 118.8, 115.0, 61.0, 45.3, 42.9,
31.3, 29.1, 25.9, 23.6.
4.2.2. N-(6-(3-Chlorophenyl)-6-oxohexyl)-5-(dimethylamino)
naphthalene-1-sulfonamide (5)
A flame dried round bottom flask was charged with amide 4
(1.56 g, 3.82 mmol) which was dissolved in THF (30 mL). The flask
was cooled to 0 °C and 3-chlorophenyl-magnesium bromide solu-
tion was added dropwise (0.50 M, 39.0 mL, 19.1 mmol). This was
warmed to room temperature and stirred overnight. The reaction
was quenched with saturated ammonium chloride solution
(20 mL). The organic phase was extracted with EtOAc
(3 Â 25 mL), washed with brine (25 mL), and dried under Na2SO4.

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5275
4.4. [3H] BTX-B displacement assay
4.6.4. Peripheral nerve injury and evaluation of tactile
sensitivity
The [3H] BTX-B binding assays were performed by Novascreen
Biosciences (Hanover, MD). Rat forebrain membranes (10 mg tis-
sue/well) were incubated with [3H] BTX-B (30–60 Ci/mmol) in
50 mM HEPES (pH 7.4) containing 130 mM choline chloride, at
37 °C for 60 min. The reaction was terminated by rapid vacuum fil-
tration of reaction contents onto glass fiber filters. Radioactivity
trapped on the filters was determined and compared to controls
to measure binding of [3H] BTX-B with the Na+ channel site 2 bind-
ing site. Reactions were pre-incubated with test compounds
Rats underwent a unilateral ligation of the sciatic nerve35 with
modifications.36 Animals that developed significant allodynia to
von Frey filaments by the up and down method37,38 were selected
for drug testing. Drug was dissolved in DMSO and diluted in
PEG400 to 200 mg/ml stock solution. Aliquots of drug stock were
diluted in vehicle (PEG400/phosphate buffered saline) prior to
injection. Rats (n = 5 per group) were injected subcutaneously at
50 or 100 mg/kg body weight. Response to von Frey filaments
was tested at 2 h after injection. Controls received vehicle without
drug. Group differences were analyzed with a one-way analysis of
variance with Tukey’s post hoc tests, with significance set at
p <0.05.
(40
(1
l
l
M) to measure percent [3H] BTX-B displacement. Aconitine
M) was used as a positive control (Sigma-Aldrich, St. Louis,
MO). Each experiment was replicated at least conce for each
compound.
Competing financial interests
4.5. Sodium hNav1.2 channel electrophysiology
A patent application has been filed by Georgetown University
on the behalf of the inventors that are listed as authors in this
article.
Sodium currents were recorded using the whole-cell configura-
tion of the patch-clamp recording technique with a Axopatch 200
amplifier (molecular devices) as described.16 Compounds were
prepared as 100 mM stock solutions in dimethyl sulfoxide (DMSO)
and diluted to desired concentration in perfusion solution. The
maximum DMSO concentration used was 0.1% and had no effect
on current amplitude. All experiments were performed at room
temperature (20–22 °C). After establishing whole-cell, a minimum
series resistance compensation of 75% was applied. Sodium cur-
rents were elicited by a depolarizing step from a holding potential
of À100 mV to +10 mV for 25 ms at 15 s intervals. Compounds
were applied after a 3 min control period and continued until stea-
dy state current amplitude was observed. All data represent per-
centage mean block standard error of the mean (SEM).
Acknowledgments
We thank Charles Bauer (Novascreen, Caliper Life Sciences,
Hanover, MD) for performing the [3H] BTX-B assays and the James
P. Stables at NINDS for help in the evaluation of compound 2 in the
Anticonvulsant Drug Development Program. Financial assistance
was provided by NIH Grants CA105435-04 and R21NS061069 in
addition to the Georgetown Drug Discovery Program and a new
investigator grant from R24HD050845 (LHM). This work was sup-
ported by the Lombardi Cancer Center shared resources.
Supplementary data
4.6. Biological evaluation
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.06.042.
4.6.1. Animals
Adult female Sprague-Dawley rats were used for peripheral
nerve injury and evaluation of tactile sensitivity (Taconic Farms,
Germantown, NY: 150 g at the start of the experiment). Rats were
housed in the Georgetown University Research Resource Facility,
maintained on a 12 h light/dark cycle and allowed free access to
food and water. For acute toxicity testing, motor impairment, and
anticonvulsant screening, male albino mice (CF-1) 18–25 g were
used. All experiments were approved by the Georgetown University
Animal Care and Use Committee and performed in accordance with
the National Institutes of Health guidelines on animal care.
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