Block of human NaV 1.5 sodium channels by novel α-hydroxyphenylamide analogues of phenytoin

Article abstract of DOI:10.1016/j.ejps.2004.01.004

Voltage-gated sodium (Na) channels are a critical component of electrically excitable cells. Phenytoin (diphenylhydantoin, DPH) is an established sodium channel blocker and is a useful anticonvulsant and class 1b antiarrhythmic, and has been effectively used in the treatment of neuropathic pain. In this study, we have synthesized novel α-hydroxyphenylamide analogues of diphenylhydantoin and examined their ability to inhibit human Na_v1.5 sodium channels expressed in Chinese Hamster Ovary (CHO-K1) cells. Phenyl ring substitutions were examined including para-methyl, para-fluoro, para-chloro, ortho-chloro and meta-chloro. We have found that phenyl ring substitutions with electron withdrawing properties resulted in compounds with greater activity. In comparison to diphenylhydantoin, the novel chloro-substituted α-hydroxyphenylamide compounds produced as much as a 20-fold greater tonic and frequency-dependent blockade of Na_v1.5 channels with an IC₅₀ value of 14.5μM. In addition, the chloro-substitutions have position specific state dependent blocking properties. The ortho-, meta- and para-chloro substitutions have an 8-, 13- and 3-fold increased affinity for the inactivated state, respectively. Molecular modeling suggests that these differences in affinity are due to a direct interaction with the receptor. Comparing models of diphenylhydantoin to the novel α-hydroxyphenlyamide compound suggests that the increased activity may be due to an optimized phenyl ring position and increased molecular volume. This information may be useful in the development of more potent sodium channel blockers.

Full text of DOI:10.1016/j.ejps.2004.01.004

European Journal of Pharmaceutical Sciences 21 (2004) 635–644
Block of human NaV1.5 sodium channels by novel
␣-hydroxyphenylamide analogues of phenytoin
Paul W. Lenkowskia, Seong-Hoon Kob, James D. Andersona,
Milton L. Browna, Manoj K. Patelb,∗
a
Department of Chemistry, University of Virginia, McCormick Road, Box 400319, Charlottesville, VA 22904, USA
b
Department of Anesthesiology, University of Virginia Health System, Box 800710, Charlottesville, VA 22908-0710, USA
Received 22 October 2003; received in revised form 2 January 2004; accepted 8 January 2004
Abstract
Voltage-gated sodium (Na) channels are a critical component of electrically excitable cells. Phenytoin (diphenylhydantoin, DPH) is an
established sodium channel blocker and is a useful anticonvulsant and class 1b antiarrhythmic, and has been effectively used in the treatment
of neuropathic pain. In this study, we have synthesized novel ␣-hydroxyphenylamide analogues of diphenylhydantoin and examined their
ability to inhibit human Nav1.5 sodium channels expressed in Chinese Hamster Ovary (CHO-K1) cells. Phenyl ring substitutions were
examined including para-methyl, para-fluoro, para-chloro, ortho-chloro and meta-chloro. We have found that phenyl ring substitutions with
electron withdrawing properties resulted in compounds with greater activity. In comparison to diphenylhydantoin, the novel chloro-substituted
␣-hydroxyphenylamide compounds produced as much as a 20-fold greater tonic and frequency-dependent blockade of Nav1.5 channels with
an IC50 value of 14.5 ␮M. In addition, the chloro-substitutions have position specific state dependent blocking properties. The ortho-,
meta- and para-chloro substitutions have an 8-, 13- and 3-fold increased affinity for the inactivated state, respectively. Molecular modeling
suggests that these differences in affinity are due to a direct interaction with the receptor. Comparing models of diphenylhydantoin to the
novel ␣-hydroxyphenlyamide compound suggests that the increased activity may be due to an optimized phenyl ring position and increased
molecular volume. This information may be useful in the development of more potent sodium channel blockers.
© 2004 Elsevier B.V. All rights reserved.
Keywords: Sodium channels; ␣-Hydroxyphenylamide; Phenytoin
1. Introduction
effectively used in the treatment of neuropathic pain by
virtue of its ability to block voltage-gated sodium chan-
nels (Barber et al., 1991; Willow et al., 1985; Tunnicliff,
1996; McCleane, 1999; Backonja, 2002). Sodium channel
blockers such as local anesthetics and antiarrythmics me-
diate their block of sodium channels by two mechanisms,
tonic (resting) block and use-dependent block (Hille, 1977;
Hondeghem and Katzung, 1977). Tonic block results from
low affinity binding of drug to the resting state of the chan-
nel whilst during use-dependent block drugs binds to the in-
activated state of the channel with a greater affinity resulting
in further inhibition of the current (Bean et al., 1983). Since
sodium channel isoforms differ in channel gating kinetics
these differences are thought to explain different affinities of
sodium channel blockers for the various isoforms. For ex-
ample, liodocaine has been shown to have a higher affinity
for the Nav1.5 isoform in comparison to the skeletal muscle
specific isoform Nav1.4 and neuronal sodium channel alpha
Voltage-gated sodium (Na) channels play an important
role in the generation and conduction of both cardiac and
neuronal action potentials. They are composed of a pore
forming ␣ subunit and auxiliary ␤ subunits that modulate
gating properties of the channel (Catterall, 2000). To date
nine ␣ isoforms have been cloned (Goldin, 2001) and four
␤ subunits, ␤1 (Isom et al., 1992), ␤2 (Isom et al., 1995),
␤3 (Morgan et al., 2000) and ␤4 (Yu et al., 2003).
Sodium channel blockers have proven extremely useful
targets for the treatment of many diseases. For example
Diphenylhydantoin (DPH) or Phenytoin is a clinically use-
ful anticonvulsant, class 1b antiarrhythmic and has been
∗
Corresponding author. Tel.: +1-434-924-9586;
fax: +1-434-924-2105.
E-mail address: mkp5u@virginia.edu (M.K. Patel).
0928-0987/$ – see front matter © 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.ejps.2004.01.004

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P.W. Lenkowski et al. / European Journal of Pharmaceutical Sciences 21 (2004) 635–644
subunits (Makielski et al., 1999; Pugsley and Goldin, 1998;
Nuss et al., 2000; Bean et al., 1983).
purified by flash column chromatography (10% EtOAc/90%
hexanes) to give pure substituted ketones.
Many sodium channel blocking analogues have been
synthesized in order to develop more potent and selective
sodium channel blockers. Therefore, we have synthesized
novel ␣-hydroxyphenylamide compounds designed using
the pharmacophore model developed for DPH (Brown
et al., 1999) and examined their ability to inhibit the human
sodium channel denoted Nav1.5. Our data suggest that in
comparison to DPH, the novel ␣-hydroxyphenylamide ana-
logues examined herein induce profoundly greater tonic and
frequency-dependent blockade of Nav1.5 channels. In addi-
tion, we have found that the position of chloro substitutions
on the phenyl ring in ␣-hydoxyphenylamides profoundly
affects state dependent blocking properties. We demonstrate
that DPH can be modified to have much greater blocking
potency on Nav1.5 sodium channels, providing useful data
for the design of novel antiarrhythmic, anticonvulsant and
analgesic agents.
Cyanohydrins were prepared from ketones in the follow-
ing manner. To a flame-dried, three-neck, round bottomed
flask containing a magnetic stir bar and fitted with an inlet
for maintaining an inert N2 atmosphere, the ketone (1.0 eq.)
dissolved in dry CH2Cl2 was added. Trimethylsilyl cyanide
(2.2 eq.), KCN and 18-crown-6 (10 mg for every 1.0 mmol of
ketone) were added and the reaction was monitored by loss
of the carbonyl peak (2250 cm−1) in the IR. The CH2Cl2
was evaporated in vacuo, and a minimal amount of dry
THF was added. The mixture was cooled to 0 ◦C and 15%
HCl (5 ml) was added and then stirred at room tempera-
ture for 2 h. The solution was combined with H2O and ex-
tracted with Et2O (3 × 25 ml), dried over MgSO4, filtered,
and concentrated to yield a thick dark oil. Because of sta-
bility concerns, the crude cyanohydrin was used without
purification.
␣-Hydroxyamides were prepared from cyanohydrins in
the following manner. The cyanohydrin was dissolved in
1,4-dioxane (2 ml) and added to a round-bottomed flask
while stirring. The mixture was cooled to 0 ◦C, and pre-
viously cooled (0 ◦C) concentrated HCl (0.2 ml for ev-
ery 1 mmol of cyanohydrin) was added. HCl gas was
then passed through the reaction mixture for 45 min at
0 ◦C. The mixture was allowed to stir at room temper-
ature overnight. The mixture was extracted with EtOAc
(3 × 25 ml), dried over MgSO4, filtered and concentrated
to yield the crude ␣-hydroxyphenylamides. Purification
was performed by FCC (1:1 hexanes:EtOAc), collecting all
fractions with a component of Rf = 0.28 to yield the pure
␣-hydroxyphenylamides.
2. Materials and methods
2.1. Chemistry
Compound 1 was synthesized as previously described by
Brown et al. (1999). All compounds were prepared as repre-
sented in Scheme 1. Ketones were prepared in the following
manner. To a flame-dried, round bottomed flask equipped
with a magnetic stir bar and fitted with an inlet for maintain-
ing an inert N2 atmosphere, crushed magnesium (1.2 eq.)
and one crystal I2 were added. The flask was heated gently
until I2 vapor was produced. The flask was cooled to RT and
heptyl bromide (1.1 eq.) in 10 ml ether was added dropwise.
The reaction was stirred for 30 min then the nitrile (1 eq.) in
20 ml ether and CuBr were added. The reaction was refluxed
gently under N2 for 30 min and cooled to room temperature.
Five milliliters water followed by 30 ml 15% H2SO4 were
cautiously added and the heterogeneous mixture was stirred
at RT for 24 h. The mixture was then extracted with EtOAc
(3 × 25 ml) and the organic extracts were dried (MgSO4)
and the solvent removed in vacuo. The crude ketones were
Compound 1: 2-Hydroxy-2-phenyl-nonanoic acid amide.
1H NMR: δ 0.86 (t, 3H), 1.07–1.47 (m, 10H), 2.01 (m, 1H),
2.2 (m, 1H), 3.52 (broad, 1H), 5.88 (s, 1H), 6.46 (s, 1H),
7.2–7.66 (m, 5H).
Compound 2: 2-(4-Methyl-phenyl)-2-hydroxy-nonanoic
1
acid amide. H NMR: δ 0.85 (t, 3H), 1.29 (m, 10H), 1.99
(m,1H), 2.19 (m, 1H), 2.35 (s, 3H), 3.06 (broad, 1H), 5.75
(s, 1H), 6.39 (s, 1H), 7.18–7.47 (m, 4H).
Compound 3: 2-(4-Fluoro-phenyl)-2-hydroxy-nonanoic
1
acid amide. H NMR: δ 0.85 (t, 3H), 1.28 (m, 10H), 2.01
O
O
HO
NH
2
CN
c,d
a,b
R
R
R
R = H
p-F
m-Cl
p-Cl
o-Cl
p
Scheme 1. Synthesis of the series of ␣-hydroxyphenylamides from substituted phenyl nitriles as described in detail in Section 2. The reagents used in
each step are as follows: (a) heptyl magnesium bromide, ether, CuBr, reflux, 30 min; (b) 15% H2SO4, RT 24 h; (c) TMSCN, KCN, 18-crown-6, RT 3 h;
(d) dioxane, HCl, HCl gas, 0 ◦C, 3 h.

P.W. Lenkowski et al. / European Journal of Pharmaceutical Sciences 21 (2004) 635–644
637
(m,1H), 2.22 (m, 1H), 3.09 (broad, 1H), 5.59 (s, 1H), 6.45
(s, 1H), 6.95–7.62 (m, 4H).
Data from cells with a voltage error greater than 3 mV were
excluded. Activation and steady-state inactivation data were
fitted by the equation:
Compound 4: 2-(4-Chloro-phenyl)-2-hydroxy-nonanoic
1
acid amide. H NMR: δ 0.86 (t, 3H), 1.25–1.30 (m, 10H),
1
1.99 (m,1H), 2.18 (m, 1H), 3.14 (broad, 1H), 5.64 (s, 1H),
6.46 (s, 1H), 7.30–7.54 (m, 4H).
y =
1 + exp((V − V1/2)/k)
Compound 5: 2-(2-Chloro-phenyl)-2-hydroxy-nonanoic
where y is the normalized conductance (g/gmax) or the nor-
malized current for activation and inactivation, respectively,
V1/2 the voltage of half-maximal activation or inactivation
and k the slope factor. The difference between the V1/2
value in the presence and absence of compound is shown as
ꢁV1/2 (mV). Time constants for recovery from inactivation
were obtained using a double exponential function:
1
acid amide. H NMR: δ 0.85 (t, 3H), 1.31 (m, 10H), 1.99
(m,1H), 2.25 (m, 1H), 3.2 (broad, 1H), 5.84 (s, 1H), 6.55
(s, 1H), 7.23–7.61 (m, 4H).
Compound 6: 2-(3-Chloro-phenyl)-2-Hydroxy-nonanoic
1
acid amide. H NMR: δ 0.87 (t, 3H), 1.27 (m, 10H), 1.98
(m,1H), 2.19 (m, 1H), 3.23 (broad, 1H), 5.96 (s, 1H), 6.55
(s, 1H), 7.24–7.59 (m, 4H). Log P was calculated using the
log P method described by Leo (1993).
ꢀ
ꢀ
ꢁꢁ
ꢀ
ꢀ
ꢁꢁ
t
t
y = A1 1 − exp −
+ A2 1 − exp −
τ1
τ2
2.2. Cell culture
A1 and A2 are the coefficients for the fast and slow expo-
nentials, t the time (ms) and τ1 and τ2 the fast and slow
time constants. The percentage of the current represented
by the fast time constant was calculated from the equation
100%×A1/(A1+A2), where A1 and A2 are the amplitudes of
the fast and slow gating modes, respectively. Time constants
for use-dependent block were obtained using the equation:
Chinese Hamster Ovary (CHO-K1) cells stably express-
ing Nav1.5 were grown in DMEM/F12 media (Invitrogen
Corp., CA, USA) supplemented with 10% fetal bovine
serum, penicillin (100 U/ml), streptomycin (100 ␮g/ml) and
G418 (500 ␮g/ml; Sigma, MO, USA). Cells were grown in
a humidified atmosphere of 5% CO2 and 95% air at 37 ◦C.
ꢂ
ꢃ
t
y = A exp − + C
τ
2.3. Electrophysiology
where A is the coefficient of the exponential, t the time (ms),
τ the time constant (ms) and C the fraction of unblocked
current at the end of the time course.
Sodium currents were recorded using the whole-cell con-
figuration of the patch clamp recording technique with an
Axopatch 200B amplifier (Axon Instruments, Foster City,
CA). All voltage protocols were applied using pCLAMP 8
software (Axon, USA) and a Digidata 1322A (Axon, USA).
Currents were amplified and low pass filtered (2 kHz) and
sampled at 33 kHz. Borosilicate glass pipettes were pulled
using a Brown-Flaming puller (model P87, Sutter Instru-
ments Co., Novato, CA) and heat polished to produce
electrode resistances of 0.5–1.5 Mꢀ when filled with the
following electrode solution (in mM); CsCl 130, MgCl2 1,
MgATP 5, BAPTA 10, HEPES 5 (pH adjusted to 7.4 with
CsOH). Cells were plated on glass coverslips and super-
fused with solution containing the following composition
(in mM) NaCl 130, KCl 4, CaCl2 1, MgCl2 5, HEPES 5,
and glucose 5 (pH adjusted to 7.4 with NaOH). Compounds
were prepared as 100 mM stock solutions in dimethly sul-
foxide (DMSO) and diluted to desired concentration in per-
fusion solution. The maximum DMSO concentration used
was 0.3% and had no effect on current amplitude. All ex-
periments were performed at room temperature (20–22 ◦C).
After establishing whole-cell, a minimum series resistance
compensation of 75% was applied and cells were held at
−80 mV for 5 min to account for equilibrium gating shifts.
Voltage error was calculated using the following equation:
2.4. Data analysis
All data analysis was performed using Clampfit software
(v8, Axon Instruments, CA, USA), Origin (v5, Microcal
Software, MA, USA), and Excel (Microsoft). Statistical
analyses were performed using a t-test for normally dis-
tributed data as determined by the Kolmogorov–Smirnov
test, or the Rank Sum test for non-normalized data
(Sigma Stat, Jandel). Averaged data are presented as
means standard error of the mean (S.E.M.). Values of
P < 0.05 were considered to indicate significance.
2.5. Molecular modeling
The X-ray coordinates for phenytoin (DPH) were uti-
lized in this study (Camerman and Camerman, 1971).
The ␣-hydroxyphenylamides were modified from the
X-ray structure and were energy-minimized with the Tri-
pos force field using conjugate gradient approach and
0.05 kCal/mol energy cutoff, without solvent, using default
bond distances and angles and neglecting electrostatics.
The minimization was completed by aggregating using the
SYBYL/AGGREGATE module for only the X-ray structure
atoms and allowing the modified portion to minimize. For
internal consistency, we used only the R-configuration for
all chiral compounds. For compounds 1–6, the plane angles
ꢁV = Ip(1 − a)R
where ꢁV is the voltage error, Ip the peak current, a the
series resistance compensation and R the series resistance.

638
P.W. Lenkowski et al. / European Journal of Pharmaceutical Sciences 21 (2004) 635–644
were calculated using the phenyl ring for plane 1 and the
carbonyl and nitrogen of the amide for plane 2. Plane 1 for
DPH was constructed with phenyl ring atoms and plane 2
using all hydantoin ring atoms. The plane angle was cal-
culated as the angle between planes 1 and 2. Molecular
volume was determined using the volume contour option
within SYBYL/VIEW presented with default options.
occurred with a chloro substitution on the phenyl ring (com-
pound 4). Representative current traces for the tonic block
by compound 4 are shown in the lower inset of Fig. 2A.
The order of potency determined was chloro > fluoro >
unsubstituted > methyl ∼ DPH (Table 1). We were unable
to determine IC50 values for DPH and compound 2 as their
maximum solubility’s were less than 200 ␮M. The highest
concentration tested for DPH and compound 2 was 100 ␮M
inducing 23.4 6.0% (n = 4) and 28.6 7.2% (n = 5)
block, respectively.
3. Results
The position of phenyl ring substitutions can have a
profound impact on ligand–receptor interactions (Brown
et al., 1997; Deutsch et al., 1996). In view of this, we
sought to determine the optimal phenyl ring substitution
position for our most potent inhibitor (compound 4). Com-
pounds 5 and 6 were synthesized and represent the ortho-
and meta-chloro substitutions, respectively. Fig. 2B shows
the dose response relationship for the ortho-, meta- and
para-chloro-substituted ␣-hydroxyphenylamides. Repre-
sentative currents traces for tonic block by compounds 5
and 6 are shown in the inset of Fig. 2B. Although the para-
and meta-chloro-substituted ␣-hydroxyphenylamide com-
pounds have similar IC50 values, their Hill coefficients were
significantly different and could suggest different types of
interactions. However, both of these positions were nearly
six-fold more potent then the ortho-chloro substitution. Fur-
thermore, compounds 5 and 6 had similar Hill coefficients
and thus may be interacting with the channel in a similar
3.1. Tonic block by novel α-hydroxyphenylamide analogue
In this study, we have examined ␣-hydroxyphenylamide
analogues of DPH (compounds 1–6) and focused on the re-
lationship between blockade of sodium current with substi-
tutions on the phenyl ring. Fig. 1 summarizes the compounds
examined in this study. Tonic block was assessed by a step
depolarization to +20 mV for 25 ms from a holding poten-
tial of −120 mV. We initially assessed the para position of
the phenyl ring and examined methyl (compound 2), fluoro
(compound 3) and chloro (compound 4) substitutions. These
were compared to the unsubstituted phenyl ring (compound
1) and DPH. Fig. 2A shows the dose response relationship
for these analogues. Representative currents traces for tonic
block by DPH are shown as the upper inset in Fig. 2A but
data were excluded from the dose response figure for clar-
ity. It is clear from Fig. 2A that the most efficient block
O
O
O
NH2
HO
HO
NH2
HO
NH2
H3C
F
2
1
3
O
O
O
HO
NH2
HO
NH2
HO
NH2
Cl
Cl
5
4
Cl
6
O
NH
O
HN
DPH
Fig. 1. Compound structures. DPH and novel ␣-hydroxyphenylamide substitutions examined in this study. Structures show differences in phenyl ring
substitution. Compounds 1 is unsubstituted. Compounds 2, 3 and 4 are the para-methyl, para-fluoro and para-chloro substitutions, respectively. Compounds
5 and 6 are the ortho- and meta-chloro substitutions, respectively.

P.W. Lenkowski et al. / European Journal of Pharmaceutical Sciences 21 (2004) 635–644
639
Table 1
IC50 and Hill coefficient (nH) parameters for tonic block of Nav1.5 by
novel ␣-hydroxyphenylamide compounds
Compound 1
1.0
0.8
0.6
0.4
0.2
0.0
(A)
Compound 2 (para-CH3)
Compound 3 (para-F)
Compound 4 (para-Cl)
100
30
Control
µ
M
µM
Compound
IC50 (␮M)
nH
n
Log P
1 nA
1 ms
DPH
N.D.
N.D.
1.2
N.D.
1.5
1.1
2.1
4
5
5
4
7
7
6
2.09
3.43
3.93
3.57
4.14
4.14
4.14
DPH
1
2
3
4
5
6
132.9
N.D.
32.2
14.3
81.8
14.7
31.0
0.3
7.4
2.7
7.0
1.8
0.1
0.2
0.1
0.4
100 µM
30
10
µ
M
µM
2.1
Control
1 nA
1 ms
1
10
100
IC50 and Hill coefficients were calculated using the Hill equation. Values
represent the mean S.E.M.; N.D. (not determined).
Compound 4
Concentration (µM)
were not significantly changed in the presence of any of the
compounds examined (data not shown).
Compound 4 (para -Cl)
Compound 5 (ortho-Cl)
Compound 6 (meta -Cl)
(B)
100 µM
1.0
0.8
0.6
0.4
0.2
0.0
3.3. Steady-state inactivation
30
10
µ
M
µ
M
1 nA
Control
To further investigate the mechanism of block, we ex-
amined the effects of the novel ␣-hydroxyphenylamide
compounds on steady-state inactivation. Compounds that
have a higher affinity for the inactivated state of the chan-
nel shift the steady inactivation curve in a hyperpolarized
direction. All compounds were examined at 30 ␮M with
exception of DPH, which was examined at 100 ␮M. In
addition, compound 5 was examined at 100 ␮M for com-
parison to compounds 4 and 6 in order to account for
differences in the IC50 values. Fig. 3 demonstrates the ef-
fects of compounds 4 (30 ␮M), 5 (100 ␮M) and 6 (30 ␮M)
on steady-state inactivation. The shift of the midpoint of
1 ms
Compound 5
100 µM
30
10
µM
µM
1
10
100
Control
1 nA
Concentration (µM)
1 ms
Compound 6
Fig. 2. Tonic block by novel ␣-hydroxyphenylamides. Currents were
elicited by a step depolarization to +20 mV for 25 ms from a hold-
ing potential of −120 mV. (A) Dose response relationship for the
para-substituted ␣-hydroxyphenylamide analogues. Inset shows represen-
tative current traces for the application of DPH and compound 4. (B)
Dose response relationship for ortho-, meta- and para-chloro-substituted
␣-hydroxyphenylamide analogues. Inset shows representative current
Control
1.0
Compound 4 (para-Cl) 30 µM
Compound 5 (ortho-Cl) 100 µM
Compound 6 (meta-Cl) 30 µM
traces for the application of compounds
mean S.E.M. Smooth lines represent the least squares fit when data
5 and 6. Data represent
were fitted with the Hill equation.
0.5
0.0
manner despite their different affinities for the channel.
Table 1 summarizes the IC50 values and Hill coefficients
for compounds 1 and 3–6.
3.2. Current–voltage relationship and channel activation
-100
-80
-60
-40
-20
We examined the effects of the novel ␣-hydroxyphenyl-
amides and DPH on channel activation. The current–voltage
relationship was determined with a 20 ms voltage step rang-
ing from −80 to +60 mV in steps of 5 mV from a hold-
ing potential of −120 mV at 2 s intervals. The peak of the
current–voltage relationship was +5 mV for control and was
unchanged in presence of any compound examined (data
not shown). In order to assess for effects on channel acti-
vation, the voltage dependence of channel conductance was
derived from the current–voltage relationship as described
in Section 2. The half activation and slope values for control
data were −15.8 1.7 and −7.7 0.2 mV, respectively and
Prepulse Potential (mV)
Fig. 3. Chlorinated ␣-hydroxyphenylamide compounds alter the
steady-state inactivation properties of the Nav1.5 channel. From a po-
tential of −120 mV, prepulse potentials ranging from −120 to −20 mV
were applied for 1 s followed by a depolarizing step to +20 mV to elicit
sodium current. Currents were normalized to the peak current elicited
from the −120 mV holding potential within each experiment. Data points
represent the mean S.E.M. Smooth lines correspond to the average of
the least squares fits when data were fitted by the Boltzmann equation as
described in Section 2. Compounds 4 and 6 were examined at 30 ␮M and
compound 5 was examined at 100 ␮M. The half inactivation and slope
values for control data were −60.0 2.0 and 6.0 0.3 mV, respectively.

640
P.W. Lenkowski et al. / European Journal of Pharmaceutical Sciences 21 (2004) 635–644
Table 2
1.0
The effects of novel ␣-hydroxyphenylamide compounds on Nav1.5 chan-
nel steady-state inactivation parameters
Compound
k (mV)
ꢁV1/2 (mV)
DPH (100 ␮M)
Compound 1
Compound 3
Compound 4
Compound 5
6.9
7.0
6.8
6.1
7.0
7.6
6.6
0.6
3.6
6.1
6.5
4.6
5.5
1.1
0.6
0.5
0.9
0.6
1.0a,∗∗∗
0.9a,∗∗∗
0.7
0.7
0.3
1.0
0.4
0.3
0.5
0.0
Control
Compound 4 (para-Cl) 30 µM
Compound 5 (ortho-Cl) 100 µM
Compound 6 (meta-Cl) 30 µM
Compound 5 (100 ␮M)
Compound 6
11.9
14.8
Steady-state inactivation data were fitted by the Boltzmann equation as
described in Section 2. All compounds were examined at 30 ␮M unless
otherwise noted. Values represent the mean S.E.M.
1
10
100
1000
10000
a
Denotes a lack of significance between compounds 5 and 6 (P =
Time (ms)
0.4∗0∗9∗).
P < 0.001.
Fig. 4. Novel ␣-hydroxyphenylamide compounds slow recovery from
inactivation. Compounds 4 and 6 were examined at 30 ␮M and compound
5 was examined at 100 ␮M. Recovery from inactivation was assessed
using a two-pulse protocol. A pre-pulse from −120 to +20 mV for 100 ms
was applied to inactivate all channels. Cells were then held at −90 mV for
a variable period (1–15,800 ms) to allow channels to recovery and then
the proportion of recovered channels was assessed with a voltage step to
+20 mV. Current amplitude is normalized to the peak current recorded
in the protocol. Data are plotted on a logarithmic time scale. Data points
represent the mean S.E.M. Smooth lines correspond to the average
of the least squares fits when data were fitted by a double exponential
function as described in Section 2.
the steady-state inactivation curve (ꢁV1/2) and slope (k)
values for all compounds are summarized in Table 2. The
half inactivation (V1/2) and k values for control data were
−60.0 2.0 and 6.0 0.3 mV, respectively. DPH and all of
the novel ␣-hydroxyphenylamides examined significantly
shifted the midpoint of the steady-state inactivation curve
(paired t-test). None of the compounds studied significantly
affected the k values. Compound 6 (meta-chloro) at 30 ␮M
and compound 5 (ortho-chloro) at 100 ␮M induced a signif-
icantly (P < 0.001) greater shift in the V1/2 value compared
to compound 4 (para-chloro; Fig. 3 and Table 2). These
data suggest that compounds 5 and 6 have a higher affinity
for the inactivated state of the channel than compound 4.
From the steady-state inactivation shift and the dose re-
sponse relationship, the apparent affinity for the inactivated
state (Ki) was calculated using the equation:
3.4. Recovery from inactivation
Compounds that have higher affinity for the inactivated
state of the channel may delay recovery from inactivation.
Thus, we sought to determine how the novel ␣-hydroxy-
phenlyamide compounds affect recovery from inactivation
at −90 mV. Recovery from inactivation was assessed using
a two-pulse protocol. A pre-pulse from −120 to +20 mV
for 100 ms was applied to inactivate all channels. Cells were
then held at −90 mV for a variable period (1–15,800 ms) to
allow channels to recovery and then the proportion of recov-
ered channels was assessed with a voltage step to +20 mV.
Fig. 4 demonstrates the effects of compounds 4 and 6 at
30 ␮M and compound 5 at 100 ␮M on recovery from inacti-
vation. Currents were normalized to the peak current at the
last time point and data were plotted on a logarithmic time
scale for clarity. Recovery data were fitted by a double ex-
ponential function as described in Section 2 and the effects
of DPH and the novel ␣-hydroxyphenylamide compounds
on fit parameters are summarized in Table 3.
Compounds 1–6, but not DPH significantly delayed chan-
nel recovery. However, only compounds 4–6 significantly
affected the second time constant of recovery. It is interest-
ing to note that the second time constant (τ2) for compounds
5 and 6 were significantly different from compound 4 but
not from each other suggestive that compounds 5 and 6 af-
fected the channel distinctively. The recovery data suggests
that channels blocked by 30 ␮m of compound 4 reach steady
state near 200 ms whereas only a proportion of channels are
recovered in presence of compound 5 or 6. Thus, we ex-
D
Ki =
((1 + D/Kr)/eꢁh/k) − 1
where ꢁh is the shift of the steady-state curve, k the slope
of the steady-state inactivation curve, Kr the affinity for the
resting state (IC50) taken from the dose response curve and D
the drug concentration responsible for the steady-state inac-
tivation shift (Bean et al., 1983; Gomora et al., 2001). When
the IC50 value is used as the Kr value, the Ki value represents
the IC50 for the inactivated state. The calculated affinities for
the inactivated states for compounds 4, 5 and 6 were 5.3, 10.2
and 1.1 ␮M, respectively. Examining the ratio of the affinity
for the resting state to the affinity for the inactivated state al-
lows for direct comparison of compounds with different IC50
values. The calculated ratios (Kr/Ki) were 3, 8 and 13 and
for compounds 4, 5 and 6, respectively. While compound 5
had the lowest affinity for the inactivated state (Ki), an exam-
ination of the ratio of Kr/Ki demonstrated that compounds
5 and 6 had a much increased selectivity for the inactivated
state when compared to compound 4. In order to further ex-
amine this differential affinity for the inactivated states, we
examined channel recovery from inactivation at −90 mV.

P.W. Lenkowski et al. / European Journal of Pharmaceutical Sciences 21 (2004) 635–644
641
Table 3
amined the kinetics of use-dependent block at 5 Hz (200 ms
interval).
The effects of novel ␣-hydroxyphenylamide compounds on the recovery
from inactivation
Condition
τ1 (ms)
τ2 (ms)
Fast (%)
3.5. Use-dependent block
Drug Free
6.8
18.0
14.8
13.8
22.1
35.1
100.4
35.2
1.1
8.8
1.5
2.8
2.2
5.7
17.6
4.2
43.7
57.1
67.7
62.1
97.3
357.9
2.3
5.3
10.9
6.8
9.1
76.5
67.1
57.2
68.4
71.4
56.1
88.3
84.1
4.0
12.6
2.2
4.1
0.1
12.4
7.2
4.0
DPH (100 ␮M)
Compound 1
Compound 3
Compound 4
Compound 5
Compound 5
Compound 6
Use-dependent block by DPH and the novel chlorinated
␣-hydroxyphenylamide analogues (compounds 4–6) was
assessed at 5 Hz train frequency from a holding potential of
−90 mV. Currents were normalized to the first pulse in each
experiment and the results are plotted in Fig. 5A. Example
current traces are shown in Fig. 5B. These results suggest
differences in the rate of onset and extent of use-dependent
block. Compounds 4–6 produced significantly greater
use-dependent block than DPH (P < 0.001). Compound 6
(30 ␮M) produced significantly greater use-dependent block
compared to compounds 4 (30 ␮M) and 5 (100 ␮M), block-
15.1
110.1
1.3 sa,∗∗∗
1.1 sa,∗∗∗
5.6
Recovery data were fitted by a double exponential function as described
in Section 2. Values represent the mean S.E.M.
a
Denotes a lack of significance between compounds 5 and 6 (P =
0.4∗0∗9∗).
P < 0.001.
1.0
0.5
0.0
(A)
DPH
Compound 4
Compound 5
Compound 6
0
10
20
30
40
Time (s)
(B)
t = 40 s
t = 40 s
t = 5 s
t = 5 s
t = 0 s
500 pA
100 pA
t = 0 s
1 ms
1 ms
DPH
Compound 4
t = 40 s
t = 40 s
t = 5 s
t = 5 s
t = 0 s
100 pA
1 ms
t = 0 s
100 pA
1 ms
Compound 5
Compound 6
Fig. 5. (A) Use-dependent block by DPH and novel chlorinated ␣-hydroxyphenylamides. DPH and compound 5 were examined at 100 ␮M and compounds
4 and 6 were examined at 30 ␮M. Cells were held at −90 mV and a voltage step to 20 mV was applied for 25 ms at a frequency of 5 Hz. Smooth lines
correspond to the average of the least squares fits when data were fitted by a single exponential function as described in Section 2. (B) Representative
current traces showing the first pulse, a pulse 5 s into the protocol and the last pulse.

642
P.W. Lenkowski et al. / European Journal of Pharmaceutical Sciences 21 (2004) 635–644
ing 74.1 3.3% of the current at steady state (P < 0.05).
DPH, compounds 4 and 5 blocked 19.5 2.6, 56.2 3.5
and 59.6 2.1% of sodium currents, respectively, at steady
state. There were no significant differences in the extent
of use-dependent block between compounds 4 and 6. To
assess the rate of development of use-dependent block, data
were fitted by a single exponential function as described in
Section 2. Time constants for onset of use-dependent block
were 9.5 1.5 and 10.7 1.0 s for DPH and compound
4, respectively, and were not significantly different. The
time constants for compounds 5 and 6 were 4.6 0.3 and
4.0 0.4 s, respectively, and were not significantly differ-
ent from each other, but were significantly different from
compound 4 and DPH (P < 0.001). This further confirms
that compounds 5 and 6 are acting in a distinct manner
compared to compound 4.
3.6. Molecular modeling
Compound 5 (ortho-chloro) had much lower affinity for
the closed state of the channel in comparison to compounds
4 (para-chloro) and 6 (meta-chloro). This difference could
be explained by changes in the 3D structure of compound
5 by repulsion of the phenyl ring away from the amide
carbonyl due to steric interactions from the close approxi-
mation of the ortho-substituted chloride atom. In order to
examine any potential differences in the three dimensional
structures of compounds 4–6, molecular models were com-
puted and compared as described in Section 2. The struc-
tures of compounds 4–6 were overlapped and are presented
in Fig. 6A and planes containing the phenyl ring and amide
group are delimited by purple lines. Examination of the angle
formed between the plane of the phenyl ring and the plane
of the amide revealed that the para-, ortho- and meta-chloro
␣-hydroxyphenylamides had similar angles (94.14◦, 101.16◦
and 94.11◦ for compounds 4, 5 and 6, respectively; Fig. 6A).
In order to examine potential molecular explanations for the
increased affinity of compound 6, a low energy structure of
compound 6 similar to the low energy conformer of DPH
was compared to DPH (Fig. 6B). The phenyl ring of DPH is
tilted ∼30◦ relative to the plane containing the phenyl ring of
compound 6 suggesting a significantly different phenyl ring
conformation. In order to examine the impact of these differ-
ences, overlaps of the molecular volumes of DPH (red) and
compound 6 (yellow) were created and are demonstrated in
Fig. 6C. Molecular volumes for compound 6 and DPH were
calculated to be 253.1 and 199.5 Å3, respectively. This data
suggests that the binding sites within Nav1.5 can tolerate
larger molecules resulting in increased activity.
Fig. 6. Molecular modeling. The X-ray coordinates for phenytoin (DPH)
were utilized in this study. The ␣-hydroxyphenylamides were modified
from the X-ray structure and were energy-minimized with the Tripos
force field using conjugate gradient using default bond distances and
angles and neglecting electrostatics. The minimization was completed
by aggregating using the SYBYL/AGGREGATE module for only the
X-ray structure atoms and allowing the modified portion to minimize.
For internal consistency, we used only the R-configuration for all chiral
compounds. (A) Overlap of 3D structures of compounds 4–6. The plane
containing the phenyl ring and a plane containing the amide are outlined
in purple to demonstrate the effects of chloro position on 3D structure.
(B) Overlap of compound 6 and DPH demonstrating the different relative
position of the phenyl rings. (C) Molecular volume was determined using
the volume contour option within SYBYL/VIEW presented with default
options. DPH is shown in red and compound 6 in yellow.
4. Discussion
4.1. Comparison with previous work
The development of ␣-hydroxyphenylamides as sodium
channel blockers was based on a comparative molecular

P.W. Lenkowski et al. / European Journal of Pharmaceutical Sciences 21 (2004) 635–644
643
field analysis (CoMFA) study of various hydantoins bind-
ing to neuronal sodium channels (Brown et al., 1999).
CoMFA samples the differences in steric and electrostatic
fields surrounding a set of ligands to help define important
three-dimensional properties associated with the optimum
binding of ligand to receptor. Employing the use of CoMFA,
we have previously predicted and demonstrated that an
unsubstituted ␣-hydroxyphenylamide (compound 1) pos-
sessed a four-fold lower IC50 value compared to DPH when
examined using [3H] batrachotoxinin A 20-␣-benzoate
(BTX) displacement from rat brain cerebral cortex synap-
tasomes (Brown et al., 1999). An important limitation to
the previous study was the lack of functional data on the
ability of these compounds to inhibit sodium currents. In
this study, we examined the effects of novel DPH analogues
on the human Nav1.5 sodium channel clone expressed in
a mammalian cell line. These results are relatively com-
parable to experiments with DPH in isolated ventricular
myocytes as demonstrated by Grant and coworkers (Barber
et al., 1991). Furthermore, the use of mammalian expres-
sion systems is generally accepted to accurately reflect the
channel kinetics observed in primary cells (Chen et al.,
2000).
Sodium channel gating can be described by three states;
closed, opened and inactivated. The state of the channel stud-
ied using BTX displacement is unknown. However, it has
been well established that DPH has a much higher affinity
for the inactivated state of the channel and this informa-
tion cannot be acquired using displacement studies (Kuo and
Bean, 1994). Nevertheless, consistent with BTX displace-
ment results, compound 1 blocked Nav1.5 sodium channels
with nearly three-fold greater affinity compared to DPH.
This suggests that BTX displacement data correlates with
data for functional block of sodium currents.
the para position of the aromatic ring may be attributed to
additional electrostatic and van der Waals interactions of
the larger halogen atom within the receptor site.
The most potent compound examined in this series, com-
pound 6, was able to block Nav1.5 sodium currents with
20-fold greater affinity compared to DPH. The fact that
compound 5 had a much higher IC50 value compared to
compounds 4 and 6 is most likely due to a direct steric
interaction with the receptor. Modeling of compounds 4–6
demonstrates that the position of the chloro substitution
does not significantly alter the 3D structure of the com-
pounds (Fig. 6A). Molecular modeling suggests that this
series of compounds may be more potent than DPH because
of a more optimal phenyl ring conformation and increased
bulk from the alkyl side chain (Fig. 6B and C). An inter-
esting outcome of this study was the observation that the
position of the chloro group on the phenyl ring is related to
state dependent block. Several lines of evidence support the
fact that the ortho- and meta-chloro-substituted compounds
have much greater relative affinity for the inactivated state
of the channel compared to the para position. The distinc-
tions between the ortho and the meta chloro as compared
to the para chloro compound includes: the Hill coefficient,
the steady-state inactivation shift, the relative affinity for
the inactivated state, the effects on the second phase of re-
covery from inactivation and the effects on use-dependent
block. These results may be in part due to limited access to
the receptor in the closed state of the channel for the ortho
and meta-substituted compounds. Nonetheless, this study
demonstrates for the first time that state dependent block is
dependent upon substitution position and this information
could be used in the development of compounds with state
selective blocking properties. We have developed novel
␣-hydroxyphenylamide analogues of DPH with profoundly
greater tonic and frequency-dependent blockade of Nav1.5
channels. This data may be useful in the design of novel
antiarrhythmic, anticonvulsant and analgesic agents.
4.2. Mechanism of Nav1.5 block
The dose response data suggests that the electron with-
drawing substitutions of fluoro (compound 3) and chloro
(compound 4) inhibit Nav1.5 sodium channels more ef-
fectively than a non-substituted (compound 1) or electron
donating methyl substitution (compound 2). In addition
compound 4 (para-chloro) had a greater blocking activity
than compound 3 (para-Fluoro). In agreement with previous
results, a general trend of decreasing IC50 with increas-
ing Log P was observed (Table 1) (Brown et al., 1997).
This increased block seen by compound 4 may be due
to increased stabilization of the ␲–␲ stacking interactions
between the phenyl ring of compound 4 and an aromatic
residue within the receptor site. Indeed, several studies
have implicated the role of the aromatic amino acids F1760
and Y1767 in the S6 of domain IV of the Nav1.5 sodium
channel as necessary for high affinity binding of local anes-
thetics and other compounds (Mujtaba et al., 2002; Nau
et al., 2000a; Nau et al., 2000b). Moreover, the noticeable
increase in potency by the introduction of a chloro atom at
Acknowledgements
We thank Dr. A.O. Grant (Duke University, USA) for pro-
viding the Nav1.5 stably transfected CHO cells and Dr. Carl
Lynch III for helpful comments. This work was supported
by a grant from the American Heart Association (Virginia
Affiliate; M.K.P. and S.-H.K.) and The Korean Science and
Engineering Foundation (S.-H.K.). We also thank the Med-
ical Scientist Training Program and the Cardiovascular Re-
search Center, University of Virginia (P.W.L.). We would
also like to thank the Jefferies Trust Fund (J.D.A.).
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