(Biphenyl-4-yl)methylammonium chlorides: Potent anticonvulsants that modulate Na+ currents

Article abstract of DOI:10.1021/jm4007092

We have reported that compounds containing a biaryl linked unit (Ar-X-Ar′) modulated Na⁺ currents by promoting slow inactivation and fast inactivation processes and by inducing frequency (use)-dependent inhibition of Na⁺ currents. These electrophysiological properties have been associated with the mode of action of several antiepileptic drugs. In this study, we demonstrate that the readily accessible (biphenyl-4-yl)methylammonium chlorides (compound class B) exhibited a broad range of anticonvulsant activities in animal models, and in the maximal electroshock seizure test the activity of (3′-trifluoromethoxybiphenyl-4- yl)methylammonium chloride (8) exceeded that of phenobarbital and phenytoin upon oral administration to rats. Electrophysiological studies of 8 using mouse catecholamine A-differentiated cells and rat embryonic cortical neurons confirmed that 8 promoted slow and fast inactivation in both cell types but did not affect the frequency (use)-dependent block of Na⁺ currents.

Full text of DOI:10.1021/jm4007092

Article
pubs.acs.org/jmc
(Biphenyl-4-yl)methylammonium Chlorides: Potent Anticonvulsants
That Modulate Na+ Currents
Hyosung Lee,†,¶ Ki Duk Park,†,# Xiao-Fang Yang,‡ Erik T. Dustrude,∥ Sarah M. Wilson,∥
Rajesh Khanna,‡,§,∥ and Harold Kohn*,†,⊥
⊥
†Division of Chemical Biology and Medicinal Chemistry, UNC Eshelman School of Pharmacy and Department of Chemistry,
University of North Carolina, Chapel Hill, North Carolina 27599, United States
‡Department of Pharmacology and Toxicology, Department of Biochemistry and Molecular Biology, and Program in Medical
Neuroscience, Paul and Carole Stark Neurosciences Research Institute, Indiana University School of Medicine, Indianapolis, Indiana
46202, United States
§
∥
S
* Supporting Information
ABSTRACT: We have reported that compounds containing a
biaryl linked unit (Ar-X-Ar′) modulated Na+ currents by
promoting slow inactivation and fast inactivation processes and
by inducing frequency (use)-dependent inhibition of Na+
currents. These electrophysiological properties have been
associated with the mode of action of several antiepileptic
drugs. In this study, we demonstrate that the readily accessible
(biphenyl-4-yl)methylammonium chlorides (compound class
B) exhibited a broad range of anticonvulsant activities in
animal models, and in the maximal electroshock seizure test
the activity of (3′-trifluoromethoxybiphenyl-4-yl)-
methylammonium chloride (8) exceeded that of phenobarbital
and phenytoin upon oral administration to rats. Electro-
physiological studies of 8 using mouse catecholamine A-differentiated cells and rat embryonic cortical neurons confirmed that 8
promoted slow and fast inactivation in both cell types but did not affect the frequency (use)-dependent block of Na+ currents.
acosamide1 (1) is a first-in-class antiepileptic drug (AED)
Lthat has been introduced in 34 countries, including the
U.S., as an adjunctive therapy for the treatment of partial-onset
seizures.2 Whole-cell, patch-clamp electrophysiology showed
that 1 reduced Na+ channel availability by a mechanism
consistent with its increasing the transition of Na+ channels to
the slow-inactivated state without affecting the fast inactivation
process.3−5 (For an alternative mechanism where the agent
In this study, we asked if compounds conforming to the
blocks Na+ channel fast-inactivated channels with very slow
biaryl linked unit exhibited anticonvulsant activity in rodents.
kinetics, see ref 6). We demonstrated that lacosamide analogues
Here, we focus on substituted (biphenyl-4-yl)-
in which the N-benzyl amide group was extended by an
methylammonium chlorides (B) wherein the aryl linker (X)
additional aryl unit to give compound class A exhibited
is a single bond. We report that compound class B showed a
pronounced anticonvulsant activity in proven rodent seizure
models.7 Electrophysiologic examination of A in neuronal-like
catecholamine A-differentiated (CAD) cells showed that these
compounds promoted Na+ channel slow inactivation and that
several compounds were 40−80-fold more potent than 1.8
Interestingly, we found that members of compound class A,
unlike 1, affected Na+ channel fast inactivation and exhibited
frequency (use)-dependent inhibition of Na+ channel firing. We
investigated the origin of A’s increased potency, compared to
that of 1, for Na+ channel slow inactivation and showed that
both the core “lacosamide unit” and the “biaryl linked unit”
(Figure 1) promoted slow inactivation.9
broad profile of anticonvulsant activity, and when a selected
compound of class B was given orally to rats, potent activity in
the maximal electroshock seizure10 (MES) model that was
comparable to the clinical agents phenobarbital and phenytoin
was seen.11
RESULTS AND DISCUSSION
Choice of Compounds. We selected 10 (biphenyl-4-
yl)methylammonium chlorides (B) in which the substituent
■
Received: May 12, 2013
Published: June 17, 2013
© 2013 American Chemical Society
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Figure 1. Key structural units in compound class A that affect Na+ channel slow inactivation.
of the electronic properties of the terminal aryl substituent (Y)
on anticonvulsant activity.
In mice (ip), 8 was among the most active (biphenyl-4-
yl)methylammonium chlorides. We observed ED50 values of 25
mg/kg, 43 mg/kg, and 81 mg/kg in the MES, 6 Hz, and scMet
seizure models, respectively. When 8 was tested in the MES
model in rats (po), the ED50 value was 8.7 mg/kg. Compound
8 showed no neurotoxicity in rats (po) at doses as high as 500
mg/kg, providing a protective index (PI = TD50/ED50) of >57.
The oral activity of 8 exceeded that of phenytoin,11
phenobarbital,11 and valproate11 and was approximately 2-fold
less active than 1.1 Similar activity for 8 was observed in the rat
after ip administration (MES ED50 = 16 mg/kg; TD50 = 72 mg/
kg). When tested in the iv Metrazol test18 (mice, ip) at 25 mg/
kg and 81 mg/kg there was no statistical difference from the
control group in seizure threshold.
(Y) and the site of substitution on the terminal aryl unit were
varied (Table 1, compounds 2−11). Both electron-withdrawing
and electron-donating substituents were chosen. In most
instances, the groups were placed at either the 3′ (5, 7, 8,
12, 13) or the 4′ (6, 8, 11) position since earlier structure−
activity relationship studies on 112 and compound class A7
derivatives showed substitution at these sites provided
compounds with excellent anticonvulsant activities. In the
case of the trifluoromethoxy (CF3O) substituent, we prepared
the 2′, 3′, and 4′ regioisomers (9−11). The compounds were
purified and tested as their hydrochloride salts.
The excellent activity observed for 8 led us to examine its
cellular activity by patch-clamp electrophysiology using CAD
cells. We have previously shown that CAD cells express
endogenous tetrodotoxin-sensitive Na+ currents with rapid
activation and inactivation kinetics upon membrane depolariza-
tion and are likely mediated by NaV1.7, NaV1.1, and NaV1.3
channels.5,9 Moreover, we found that the Na+ channel
properties of 1 in CAD cells5 were similar to those reported
in cultured neurons and mouse N1E-115 neuroblastoma cells.3
Accordingly, we used readily accessible CAD cells to evaluate
the effect of 8 on neuronal function, recognizing in advance
that CAD cells do not express the same complement of Na+
channels expressed in central nervous system (CNS) neurons.
We found that 8 preferentially promoted Na+ channel slow
inactivation (Figure 2A−D). The Na+ slow inactivation IC50
value at −50 mV was 2.7 μM, which was approximately 30
times more potent than 1 (IC50 = 85 μM).5 We chose the
potential of −50 mV for three reasons: (1) a large fraction of
the channels undergo steady-state inactivation, which involves
contributions from slow and fast inactivation pathways,19,20
where −50 mV is within the steep voltage-dependence range
for each; (2) it is near the resting membrane potential and
approaches the action potential firing threshold for CNS
neurons,21 where slow inactivation appears to be physiologically
relevant during sustained subthreshold depolarizations;22 and
(3) changes in the Na+ channel availability near −50 mV can
impact the overlap of Na+ current activating and inactivating
under steady-state conditions.19,23
Chemistry. The (biphenyl-4-yl)methylammonium chlorides
3−11 were prepared using Suzuki coupling13 of 4-bromobenzyl
amine (12) with the appropriate, commercially available
substituted phenylboronic acids (13−21) to give the amines,
which then were immediately converted to their hydrochloride
salts 3−11 (Scheme 1). For hydrochloride salt 2, we treated a
commercial sample of 4-(phenyl)benzylamine with HCl in
dioxane.
Pharmacological Activity. Compounds 2−11 were tested
for anticonvulsant activity at the Anticonvulsant Screening
Program (ASP) of the National Institute of Neurological
Disorders and Stroke (NINDS) at the U.S. National Institutes
of Health. Screening was performed using the procedures
described by Stables and Kupferberg.14 The anticonvulsant
activity data from the MES,10 psychomotor 6 Hz,15 and
scMetrazol16 (scMet) tests are summarized in Table 1, along
with similar results obtained for 1 and the clinical AEDs
phenytoin,11 valproate,11 and phenobarbital.11 All compounds
were administered intraperitoneally (ip) to mice and ip or
orally (po) to rats. For compounds that showed significant
activity, we report the 50% effective dose (ED50) values
obtained in quantitative screening evaluations. We also provide
the median doses for 50% neurological impairment (TD50) in
mice (rotorod test17) and in rats18 (behavioral toxicity effects).
Compounds 5 and 8 displayed activities (50−100 mg/kg) in
mice (ip) in the three seizure models (MES, 6 Hz, scMet),
whereas 2, 3, 4, 7, 9, and 10 displayed activities (30−100 mg/
kg) in two of the three assays. The observed seizure protection
of 3, 5, and 8 in the scMet model was interesting since 112 and
compounds belonging to class A7 did not display anticonvul-
sant activity in this model. The broad whole-animal
pharmacological profile for several Bs suggested that these
compounds exert their anticonvulsant activities through
multiple pathways. For the trifluoromethoxy-substituted
compounds 7−9, we found that the 3′-trifluoromethoxy
derivative 8 was the most potent in the MES test (mice, ip).
Finally, for 2−11, we did not observe a clear trend on the effect
We found that 8 did not affect Na+ channel steady-state
activation, defined as the relationship between voltage and a
shift of channels from closed to open conformations, but did
modify steady-state fast inactivation, defined as the relationship
between voltage and a shift of channel gating from open to
inactivated conformations over several hundred milliseconds
(Figure 3). Steady-state fast inactivation was assessed using a
previously described protocol designed to induce a fast-
inactivated state.5,8,9 Cells were held at −80 mV, stepped to
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a
Table 1. Anticonvulsant Activities of (Biphenyl-4-yl)methylammonium Chlorides (B)
a
b
The compounds were tested through the auspices of the NINDS ASP. The compounds were administered intraperitoneally (ip) to mice. ED50 and
TD50 values are in milligrams per kilogram. Numbers in parentheses are 95% confidence intervals. A dose−response curve was generated for all
compounds that displayed sufficient activity. The dose−effect data for these compounds was obtained at the “time of peak effect” (indicated in hours
c
d
e
in the brackets). The compounds were administered either ip or orally (po) to rats. MES = maximal electroshock seizure test. 6 Hz = 6 Hz
f
g
h
i
psychomotor seizure test (32 mA). scMet = subcutaneous pentylenetetrazol test. Tox = rotorod test. Tox = behavioral toxicity. ND = not
determined. Reference 1. Reference 11.
j
k
inactivating prepulse potentials ranging from −120 to −10 mV
(in 10-mV increments) for 500 ms, and then stepped to 0 mV
for 20 ms to measure the available current (Figure 3, top left
protocol). A 500-ms conditioning pulse was used because it
allowed all of the endogenous channels to transition to a fast-
inactivated state at all potentials examined. Steady-state, fast
inactivation curves of Na+ currents from DMSO-treated and
various concentrations of 8-treated CAD cells were well fitted
with a single Boltzmann function (R2 > 0.935 for all conditions)
and are illustrated in Figure 3 (leftmost curves). The V1/2 value
for inactivation of 0.1% DMSO-treated cells was −71.6 0.6
mV (n = 6), which was significantly different from the V1/2
values of all concentrations of 8-treated CAD cells (p > 0.05;
ANOVA with a posthoc Dunnett’s test). The 1 μM
concentration of 8 caused a significant hyperpolarizing shift
of ∼11.2 mV, while the 30 μM concentration of 8 caused a
significant hyperpolarizing shift of ∼26.5 mV with no
commensurate significant changes in slope values compared
with control cells. The slopes of fast inactivation were not
affected by 8. Steady-state activation, as measured by 15 ms
depolarizing pulses from −70 to +80 mV (in 10-mV
increments) produced equivalent V1/2 and slope values in all
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Scheme 1. Synthesis of (Biphenyl-4-yl)methylammonium Chlorides (B)
Figure 2. Effect on steady-state slow inactivation state of Na+ currents in mouse CAD cells by 8. (A) Currents were evoked by 5 s prepulses between
−120 and −20 mV, and then fast-inactivated channels were allowed to recover for 150 ms at a hyperpolarized pulse to −120 mV. The fraction of
channels available at 0 mV was analyzed. (B) Representative current traces from CAD cells in the absence (control, 0.1% DMSO) or presence of 1
μM of 8 as indicated. The blue (control) and red (8) traces represent the current at −50 mV. (C) Summary of steady-state slow activation curves for
CAD cells treated with DMSO (control) or 100 pM−30 μM 8. (D) Summary of the fraction of current available at −50 mV for CAD cells treated
with DMSO (control) or 100 pM−30 μM 8. Asterisks (*) indicate statistically significant differences in fraction of current available between control
and the indicated concentrations of 8 (p < 0. 05, Student’s t test). Data are from 4−7 cells per condition.
conditions. Finally, we found that 8 did not exhibit frequency
(use)-dependent inhibition of Na+ currents as currents
recorded from cells treated with 10 μM 8 displayed no
statistically significant differences in trend or amplitude
compared with control (Figure 4). The whole-cell, patch-
clamp electrophysiology for 8 mirrored aspects observed for 22
in CAD cells.9 For 22, the IC50 value for Na+ channel slow
inactivation was 2.1 μM, and like 8, it affected fast inactivation.
However, 22 displayed frequency (use)-dependent blockage of
Na+ currents, whereas 8 did not.
We also tested the activity of 8 in rat embryonic cortical
neurons. These neurons typically express Na+ channel isoforms
NaV1.1, NaV1.2, NaV1.3, and NaV1.6.24 The slow inactivation,
steady-state inactivation, fast inactivation, and use-dependence
of Na+ currents in cortical neurons grown for 7−10 days in
vitro (Figure 5A, D, G, and J) were examined using protocols
mostly similar to those described for CAD cells with a few
exceptions noted in the legend to Figure 5. A single
concentration (20 μM) of 8 was chosen as it represented
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was −53.9 1.2 mV (n = 4), which was significantly different
from the V1/2 value of −69.6 1.2 mV (n = 5) for 8-treated
neurons (p < 0.05; ANOVA with a posthoc Dunnett’s test).
The 20 μM concentration of 8 caused a significant hyper-
polarizing shift of ∼15.7 mV with no commensurate significant
changes in slope values compared with control cells. Finally, we
found that 8 did not exhibit frequency (use)-dependent
inhibition of Na+ currents in cortical neurons (Figure 5K, L).
Compound 8, and the other agents in this study, contained a
biphenyl unit. This motif is considered a privileged substructure
and has been shown to bind to many proteins, including G-
protein-coupled receptors.25−27 Accordingly, we determined
the binding of 2−11 at UNC’s Psychoactive Drug Screening
Program against 43 receptors. We observed appreciable binding
of most compounds at 10 μM to several serotonin (e.g., 5-
HT2A, 5-HT2B, 5-HT5A, 5-HT6, 5-HT7) and adrenergic (e.g.,
alpha 2A, 2B, 2C) receptors; the DAT, NET, and SERT
transporters; and the sigma-1 and -2 receptors (Supplementary
Table 1). The importance of these interactions on the observed
anticonvulsant activities has not been determined.
Figure 3. Effects of 8 on activation and inactivation properties of Na+
currents in mouse CAD cells. Values for V1/2, the voltage of half-
maximal activation and steady-state fast inactivation and the slope
factors (k) were derived from Boltzmann distribution fits to the
individual recordings and averaged to determine the mean ( SEM)
voltage dependence of activation and fast inactivation. The voltage
protocol used to evoke current responses for each protocol is shown
above the fits. Representative Boltzmann fits for activation and steady-
state inactivation for CAD cells treated with 0.1% DMSO (control)
and indicated concentrations of 8 are shown.
CONCLUSIONS
■
Our previous finding that the biaryl linked unit (Figure 1)
promoted Na+ channel slow inactivation in CAD cells9 led to
the discovery that compounds conforming to class B exhibited
anticonvulsant activities in proven whole animal seizure models.
Structurally, B is exceedingly simple, readily synthesized, and
soluble in water (≥300 μM). The anticonvulsant activity for 8
in the MES seizure model (rat, po) rivaled that of established
AEDs. Compound 8 appears to exert its activity, in part, by
inhibiting Na+ channel currents.
EXPERIMENTAL SECTION
■
General Methods. The general methods used in this study are
identical to those previously reported.7 The compounds were checked
1
by TLC, H NMR and 13C NMR, MS, and elemental analyses. The
analytical results, except for 6, are within 0.40% of the theoretical
value. The NMR and analytical data confirmed that the purity of the
products was ≥95%.
(Biphenyl-4-yl)methylammonium Chloride (2).28 To a sol-
ution of 4-(phenyl)benzylamine (1.00 g, 5.5 mmol) in CH2Cl2 (50
mL) was added an HCl solution in dioxane dropwise (1.5 mL, 4 N)
with stirring at room temperature (1 h). The resulting precipitate was
filtered, washed with hexanes, and dried in vacuo to give 2 (1.03 g,
86%) as a white solid: Rf = 0.00 (hexanes/EtOAc 1/1); mp 301−303
Figure 4. Lack of effect on frequency-dependent block by 8 of Na+
currents in mouse CAD cells. The frequency dependence of block was
examined by holding cells at the hyperpolarized potential of −80 mV
and evoking currents at 10 Hz by 20-ms test pulses to −10 mV (inset
middle). Representative overlaid traces are illustrated by pulses 1, 10,
20, and 30 for control (predrug) and in the presence of 8 (1 μM).
Summary of average frequency-dependent decrease in current
amplitude ( SEM) produced by control (predrug) or by the presence
of 8 (1 μM) (p > 0.05, one-way ANOVA with Dunnett’s posthoc test).
Data are from 5−7 cells per condition.
°C (lit.28 mp 308−310 °C); H NMR (CDCl3, CD3OD) δ 4.14 (s,
1
CH2N), 4.62−4.78 (br s, NH3), 7.32−7.41 (m, ArH), 7.42−7.48 (m, 2
ArH), 7.49 (d, J = 8.4 Hz, 2 ArH), 7.59 (d, J = 8.8 Hz, 2 ArH), 7.68 (d,
J = 8.4 Hz, 2 ArH); 13C NMR (CDCl3, CD3OD) δ 42.7 (CH2N),
126.5, 127.3, 127.4, 128.5, 128.9, 131.3, 139.7, 141.9 (8 ArC); LRMS
(ES+) 184.00 [M − Cl]+ (calcd for C13H14N+ 184.11). Anal. Calcd for
C13H14ClN: C, 71.07; H, 6.42; Cl, 16.14; N, 6.38. Found: C, 70.95; H,
6.44; Cl, 16.24; N, 6.34.
almost 8-fold the IC50 value for slow inactivation based on
CAD cell data. At this concentration, the extent of slow
inactivation induced by 8 was significantly greater than neurons
treated with the vehicle DMSO: 0.17 0.03 (n = 5) versus 0.41
0.05 (n = 7), respectively (p > 0.05, one-way ANOVA Figure
5B, C). Steady-state activation was also unchanged between the
two conditions with the V1/2 and k values being statistically
similar (Figure 5E, F). Steady-state, fast inactivation curves of
Na+ currents from DMSO-treated and 20 μM 8-cortical
neurons were well fitted with a single Boltzmann function
(R2 > 0.995 for both conditions) and are illustrated in Figure
5I. The V1/2 value for inactivation of 0.1% DMSO-treated cells
(3′-Fluorobiphenyl-4-yl)methylammonium Chloride (3). To a
solution of 12 (1.21 g, 6.50 mmol) in acetonitrile (65 mL) were added
3-fluorophenylboronic acid (13) (1.00 g, 7.15 mmol), tetrakis-
(triphenylphosphine)palladium(0) (0.38 g, 0.32 mmol), and aqueous
2 N K2CO3 (16.2 mL). The resulting mixture was sparged with Ar
with stirring (30 min) and then stirred again at 90 °C under Ar (16 h).
The reaction mixture was filtered and evaporated in vacuo. The
resulting residue was diluted with EtOAc (50 mL) and washed with
H2O (2 × 50 mL) and with saturated aqueous brine solution (2 × 50
mL). The organic layer was dried (Na2SO4) and concentrated in vacuo
to give the amine as a yellow oil. To a solution of the amine in ethyl
acetate was added aqueous concentrated HCl (0.8 mL) with stirring at
room temperature (1 h) to give a precipitate. To the resulting mixture
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Figure 5. Evaluation of 8 on biophysical properties of Na+ currents in rat embryonic cortical neurons. (A) Currents were evoked by 5 s prepulses
between −100 and +20 mV, and then fast-inactivated channels were allowed to recover for 1000 ms at a hyperpolarized pulse to −100 mV. The
fraction of channels available at 0 mV was analyzed. (B) Representative current traces from cortical neurons in the absence (control, 0.1% DMSO) or
presence of 20 μM 8. The black and red traces represent the current at −50 mV. (C) Summary of the fraction of current available at −50 mV for
cortical neurons treated with DMSO (control) or 20 μM 8. Values for V1/2, the voltage of half-maximal activation (D−F) and steady-state fast
inactivation (G−I) and the slope factors (k) were derived from Boltzmann distribution fits to the individual recordings and averaged to determine
the mean ( SEM) voltage dependence of activation and fast inactivation. The voltage protocol used to evoke current responses for each protocol is
shown above the fits. Representative Boltzmann fits for activation and steady-state inactivation for cortical neurons treated with 0.1% DMSO
(control) and 20 μM 8 are shown. Fast-inactivation was significantly affected by 20 μM 8. (J−L) Use-dependence was not affected by 20 μM 8.
Details are similar to those in legend to Figure 4. Five to seven cells were tested in these experiments.
was added H2O, and then the aqueous layer separated. The aqueous
layer was basified with aqueous 4 N NaOH and extracted with CH2Cl2
(2 × ), and the combined CH2Cl2 layers were dried (Na2SO4) and
evaporated in vacuo. The resulting oil was diluted in CH2Cl2, and then
4 N HCl in dioxane added. The resulting precipitate was filtered and
washed with hexanes to give 3 (1.20 g, overall yield 77%) as a white
mmol), tetrakis(triphenylphosphine)palladium(0) (1.55 g, 1.35
mmol), and aqueous 2 N K2CO3 (53.8 mL) gave the free amine29
as a yellow oil. The amine was treated with aqueous concentrated HCl
(2.5 mL) and then purified to give 4 (4.99 g, overall yield 78%) as a
1
white solid: Rf = 0.00 (EtOAc/hexanes 1/1); mp 302−303 °C; H
NMR (DMSO-d6) δ 4.06 (s, CH2N), 7.28−7.34 (m, 2 ArH), 7.61−
7.75 (m, 6 ArH), 8.70 (s, NH3); 13C NMR (DMSO-d6) δ 41.7
(CH2N), 115.8 (d, J = 21.3 Hz, C3′, C5′), 126.7 (ArC), 128.7 (d, J =
8.0 Hz, C2′, C6′), 129.6, 133.3 (2 ArC), 136.0 (d, J = 3.7 Hz, C1′),
139.1 (ArC), 162.0 (d, J = 243.2 Hz, C4′); HRMS (ESI+) 202.1022 [M
− Cl]+ (calcd for C13H13FN+ 202.1022). Anal. Calcd for C13H13ClFN:
C, 65.69; H, 5.51; Cl, 14.91; F, 7.99; N, 5.89. Found: C, 65.55; H,
5.54; Cl, 14.79; F, 7.82; N, 5.88.
1
solid: Rf = 0.00 (EtOAc/hexanes 1/1); mp 301−304 °C; H NMR
(CD3OD) δ 4.18 (s, CH2N), 7.06−7.20 (m, ArH), 7.30−7.39 (m,
ArH), 7.43−7.48 (m, 2 ArH), 7.58 (d, J = 8.2 Hz, 2 ArH), 7.71 (d, J =
8.2 Hz, 2 ArH); 13C NMR (CD3OD) δ 44.5 (CH2N), 115.1 (d, J =
20.2 Hz), 115.9 (d, J = 20.2 Hz), 124.4, 129.2, 131.2, 132.3 (d, J = 8.8
Hz), 134.6, 142.4 (d, J = 7.6 Hz), 144.4 (d, J = 7.6 Hz), 165.2 (d, J =
243.0 Hz) (10 ArC); LRMS (ES+) 201.95 [M − Cl]+ (calcd for
C13H13FN+ 202.10). Anal. Calcd for C13H13ClFN: C, 65.69; H, 5.51;
Cl, 14.91; F, 7.99; N, 5.89. Found: C, 65.68; H, 5.45; Cl, 14.77; F,
7.81; N, 5.74.
(3′-Chlorobiphenyl-4-yl)methylammonium Chloride (5). Em-
ploying the procedure for 3 and using 12 (2.16 g, 11.63 mmol),
acetonitrile (116 mL), 3-chlorophenylboronic acid (15) (4.21 g, 26.9
mmol), tetrakis(triphenylphosphine)palladium(0) (1.55 g, 1.35
mmol), and aqueous 2 N K2CO3 (53.8 mL) gave the free amine30
as a yellow oil. The amine was treated with aqueous concentrated HCl
(4′-Fluorobiphenyl-4-yl)methylammonium Chloride (4). Em-
ploying the procedure for 3 and using 12 (5.00 g, 26.9 mmol),
acetonitrile (100 mL), 4-fluorophenylboronic acid (14) (3.77 g, 26.9
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(2.5 mL) and then purified to give 5 (4.37 g, overall yield 64%) as a
white solid: Rf = 0.00 (EtOAc/hexanes 1/1); mp 264−265 °C; H
(3′-Methoxybiphenyl-4-yl)methylammonium Chloride (10).
Employing the procedure for 3 and using 12 (1.12 g, 6.0 mmol),
acetonitrile (60 mL), 3-methoxyphenylboronic acid (20) (1.00 g, 6.6
mmol), tetrakis(triphenylphosphine)palladium(0) (0.35 g, 0.3 mmol),
and aqueous 2 N K2CO3 (15.0 mL) gave the free amine as a yellow oil.
The amine30 was treated with aqueous concentrated HCl (1.6 mL)
and then purified to give 10 (0.80 g, overall yield 54%) as a white
1
NMR (DMSO-d6) δ 4.07 (s, CH2N), 7.43−7.76 (m, 8 ArH), 8.75 (s,
NH3Cl); 13C NMR (DMSO-d6) δ 40.5 (CH2N), 125.4, 126.3, 126.9,
127.4, 129.7, 130.8, 133.8, 134.0, 138.5, 141.6 (10 ArC); LRMS (ES+)
218.0 [M − Cl]+ (calcd for C13H13ClN+ 218.1). Anal. Calcd for
C13H13Cl2N·0.18 H2O: C, 60.66; H, 5.23; Cl, 27.55; N, 5.44. Found:
C, 60.30; H, 5.12; Cl, 27.16; N, 5.49.
1
solid: Rf = 0.00 (hexanes/EtOAc 1/1); mp 231−234 °C; H NMR
(DMSO-d6) δ 3.83 (OCH3), 4.05 (CH2N), 6.95 (dd, J = 2.2, 8.3 Hz,
ArH), 7.20 (s, ArH), 7.25 (d, J = 8.4 Hz, ArH), 7.39 (t, J = 7.8 Hz,
ArH), 7.50 (d, J = 8.0 Hz, 2 ArH), 7.72 (d, J = 8.0 Hz, 2 ArH), 8.40−
8.58 (br s, NH3); 13C NMR (DMSO-d6) δ 42.3 (CH2N), 55.6
(OCH3), 112.7, 113.7, 119.4, 127.3, 129.9, 130.4, 133.9, 140.5, 141.5,
160.2 (10 ArC); LRMS (ES+) 214.09 [M − Cl]+ (calcd for
C14H16NO+ 214.12). Anal. Calcd for C13H13Cl2N: C, 67.33; H,
6.46; Cl, 14.20; N, 5.61. Found: C, 67.05; H, 6.44; Cl, 13.94; N, 5.58.
(3′-Methoxycarbonylbiphenyl-4-yl)methylammonium
Chloride (11).32 Employing the procedure for 3 and using 12 (0.86 g,
4.6 mmol), acetonitrile (40 mL), 3-methoxycarbonylphenylboronic
acid (21) (1.00 g, 5.6 mmol), tetrakis(triphenylphosphine)-
palladium(0) (0.27 g, 0.2 mmol), and aqueous 2 N K2CO3 (5 mL)
gave the free amine as a yellow oil. The amine was treated with
aqueous concentrated HCl (1.2 mL) and then purified to give 11
(0.71 g, overall yield 55%) as a white solid: Rf = 0.00 (hexanes/EtOAc
(4′-Chlorobiphenyl-4-yl)methylammonium Chloride (6). Em-
ploying the procedure for 3 and using 12 (2.16 g, 11.63 mmol),
acetonitrile (116 mL), 4-chlorophenylboronic acid (16) (2.00 g, 12.79
mmol), tetrakis(triphenylphosphine)palladium(0) (0.68 g, 0.58
mmol), and aqueous 2 N K2CO3 (29.1 mL) gave the free amine as
a yellow oil. The amine was treated with aqueous concentrated HCl
(0.5 mL) and then purified to give 6 (1.00 g, overall yield 100%) as a
1
white solid: Rf = 0.00 (EtOAc/hexanes 1/1); mp 298−302 °C; H
NMR (CD3OD) δ 4.19 (s, CH2N), 7.42−7.46 (m, 2 ArH), 7.56−7.63
(m, 4 ArH), 7.67−7.71 (m, 2 ArH); 13C NMR (CD3OD) δ 40.5
(CH2N), 125.1, 126.1, 126.6, 127.3, 130.4, 131.4, 136.6, 138.4 (8
ArC); HRMS (ESI+) 218.0764 [M − Cl]+ (calcd for C13H13ClN+
218.0736).
(2′-Trifluoromethoxybiphenyl-4-yl)methylammonium
Chloride (7).31 Employing the procedure for 3 and using 12 (1.65 g,
8.9 mmol), acetonitrile (100 mL), 2-trifluoromethoxyphenylboronic
acid (17) (2.00 g, 9.8 mmol), tetrakis(triphenylphosphine)-
palladium(0) (0.51 g, 0.44 mmol), and aqueous 2 N K2CO3 (17.7
mL) gave the free amine as a yellow oil. The amine was treated with
aqueous concentrated HCl (2.3 mL) and then purified to give 7 (1.70
g, overall yield 57%) as a white solid: Rf = 0.00 (hexanes/EtOAc 1/1);
1
1/1); mp 215−218 °C; H NMR (CD3OD) δ 3.90 (OCH3), 4.08
(CH2N), 7.58−7.70 (m, 3 ArH), 7.77 (d, J = 8.0 Hz, 2 ArH), 7.98 (dd,
J = 1.0, 8.0 Hz, 2 ArH), 8.21 (s, ArH), 8.40−8.60 (br s, NH3); 13C
NMR (CD3OD) δ 41.8 (CH2N), 52.3 (OCH3), 127.0, 127.1, 128.3,
129.7, 129.8, 130.4, 131.6, 133.8, 139.1, 140.0 (10 ArC), 166.2
+
(C(O)); LRMS (ES+) 242.03 [M − Cl]+ (calcd for C15H16NO2
1
mp 175−179 °C; H NMR (CDCl3, CD3OD) δ 4.15 (s, CH2N),
242.12). Anal. Calcd for C15H16ClNO2·0.2H2O: C, 64.03; H, 5.88; Cl,
12.06; N, 4.98. Found: C, 63.65; H, 5.82; Cl, 11.84; N, 4.79.
Pharmacology. Compounds were screened under the auspices of
the National Institutes of Health’s ASP. Experiments were performed
in male rodents (albino Carworth Farms No. 1 mice (ip), albino
Sprague−Dawley rats (ip, po)). Housing, handling, and feeding were
in accordance with recommendations contained in the Guide for the
Care and Use of Laboratory Animals. Anticonvulsant activity was
established using the MES test,10 6 Hz,15 and the scMet test,16
according to previously reported methods.1
Catecholamine A-Differentiated (CAD) Cells. CAD cells were
grown at 37 °C and in 5% CO2 (Sarstedt, Newton, NC) in Ham’s
F12/EMEM (GIBCO, Grand Island, NY), supplemented with 8% fetal
bovine serum (Sigma, St. Louis, MO) and 1% penicillin/streptomycin
(100% stocks, 10,000 U/mL penicillin G sodium and 10,000 μg/mL
streptomycin sulfate).5,8 Cells were passaged every 6−7 days at a 1:25
dilution.
4.23−4.40 (br s, NH3), 7.35−7.46 (m, 5 ArH), 7.53−7.57 (br s, 3
ArH); 13C NMR (CDCl3, CD3OD) δ 43.0 (CH2N), 121.4, 127.2,
128.7, 129.1, 129.9, 131.2, 132.0, 134.3, 137.9, 146.0 (10 ArC), the
OCF3 resonance was not detected and was believed to overlap with
nearby peaks; LRMS (ES+) 268.01 [M − Cl]+ (calcd for
C14H13F3NO+ 268.09). Anal. Calcd for C14H13ClF3NO: C, 55.37; H,
4.31; Cl, 11.67; F, 18.77; N, 4.61. Found: C, 55.48; H, 4.39; Cl, 11.48;
F, 18.51; N, 4.56.
(3′-Trifluoromethoxybiphenyl-4-yl)methylammonium
Chloride (8). Employing the procedure for 3 and using 12 (1.56 g, 8.4
mmol), acetonitrile (85 mL), 3-trifluoromethoxyphenylboronic acid
(18) (2.00 g, 8.4 mmol), tetrakis(triphenylphosphine)palladium(0)
(0.38 g, 0.4 mmol), and aqueous 1 N K2CO3 (30 mL) gave the free
amine as a yellow oil. The amine was treated with aqueous
concentrated HCl (0.5 mL) and then purified to give 8 (1.00 g,
overall yield 70%) as a white solid: Rf = 0.00 (EtOAc/hexanes 1/1);
1
mp 218−219 °C; H NMR (CD3OD) δ 4.10 (s, CH2N), 7.38−7.40
(m, 1 ArH), 7.63−7.79 (m, 7 ArH), 8.81−8.85 (br s, NH3Cl); 13C
NMR (CD3OD) δ 51.4 (CH2), 128.8, 129.5 (2 ArC), 129.8 (q, J =
254.9 Hz, OCF3), 135.5, 136.6, 139.4, 140.6, 143.9, 148.0, 151.6, 158.6
(8 ArC). Anal. Calcd for C14H13ClF3NO: C, 55.37; H, 4.31; Cl, 11.67;
F, 18.77; N, 4.61. Found: C, 55.36; H, 4.31; Cl, 11.55; F, 18.58; N,
4.55.
Cortical Neurons. Rat cortical neuron cultures were prepared
from cortices dissected from embryonic day 19 brains exactly as
previously described.33,34
Electrophysiology. Whole-cell voltage clamp recordings were
performed at room temperature on CAD cells and cortical neurons
using an EPC 10 Amplifier (HEKA Electronics, Lambrecht/Pfalz
Germany). Electrodes were pulled from thin-walled borosilicate glass
capillaries (Warner Instruments, Hamden, CT) with a P-97 electrode
puller (Sutter Instrument, Novato, CA) such that final electrode
resistances were 1−2 MΩ when filled with internal solutions. The
internal solution for recording Na+ currents contained (in mM) 110
CsCl, 5 MgSO4, 10 EGTA, 4 ATP Na2-ATP, 25 HEPES (pH 7.2,
290−310 mOsm/L). The external solution contained (in mM) 100
NaCl, 10 tetraethylammonium chloride (TEA-Cl), 1 CaCl2, 1 CdCl2, 1
MgCl2, 10 D-glucose, 4 4-AP, 0.1 NiCl2, 10 HEPES (pH 7.3, 310−315
mOsm/L). Whole-cell capacitance and series resistance were
compensated with the amplifier. Series resistance error was always
compensated to be less than 3 mV. Cells were considered only when
the seal resistance was less than 3 MΩ. Linear leak currents were
digitally subtracted by P/4.
(4′-Trifluoromethoxybiphenyl-4-yl)methylammonium
Chloride (9).32 Employing the procedure for 3 and using 12 (1.65 g,
8.9 mmol), acetonitrile (100 mL), 4-trifluoromethoxyphenylboronic
acid (19) (2.00 g, 9.8 mmol), tetrakis(triphenylphosphine)-
palladium(0) (0.51 g, 0.44 mmol), and aqueous 2 N K2CO3 (17.7
mL) gave the free amine as a yellow oil. The amine was treated with
aqueous concentrated HCl (2.3 mL) and then purified to give 9 (2.18
g, overall yield 74%) as a white solid: Rf = 0.00 (hexanes/EtOAc 1/1);
mp 281−288 °C; 1H NMR (CDCl3, CD3OD) δ 4.14 (s, CH2N), 4.24
(s, NH3), 7.31 (d, J = 8.8 Hz, 2 ArH), 7.55 (d, J = 8.4 Hz, 2 ArH),
7.60−7.68 (m, 4 ArH); 13C NMR (CDCl3, CD3OD) δ 42.9 (CH2N),
121.2, 127.6, 128.4, 129.4, 131.9, 138.8, 146.0, 148.9 (8 ArC), the
OCF3 resonance was not detected and was believed to overlap with
nearby peaks; HRMS (ESI+) 290.0763 [M − HCl + Na]+ (calcd for
C14H13F3NO+ 290.0769). Anal. Calcd for C14H13ClF3NO·0.08C6H14:
C, 55.99; H, 4.58; Cl, 11.41; F, 18.35; N, 4.51. Found: C, 56.03; H,
4.22; Cl, 11.03; F, 18.52; N, 4.50.
Data Acquisition and Analysis. Signals were filtered at 10 kHz
and digitized at 10−20 kHz. Analysis was performed using Fitmaster
and origin8.1 (OriginLab Corporation, MA, U.S.). For activation
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Article
curves, conductance (G) through Na+ channels was calculated using
the equation G = I/(Vm −Vrev), where Vrev is the reversal potential, Vm
is the membrane potential at which the current was recorded, and I is
the peak current. Activation and inactivation curves were fitted to a
single-phase Boltzmann function G/Gmax = 1/{1+ exp[(V − V50)/k]},
where G is the peak conductance, Gmax is the fitted maximal G, V50 is
the half activation voltage, and k is the slope factor. Additional details
of specific pulse protocols are described in the Results and Discussion
text or figure legends.
Statistical Analyses. Differences between means were compared
by either paired or unpaired, two-tailed Student’s t-tests or an analysis
of variance (ANOVA), when comparing multiple groups (repeated
measures whenever possible). If a significant difference was
determined by ANOVA, then a Dunnett’s or Tukey’s posthoc test
was performed. Data are expressed as mean SEM, with p < 0.05
considered as the level of significance.
the official views of the National Center for Research
Resources, National Institutes of Health. H.K. has a royalty-
stake position in 1.
ABBREVIATIONS USED
■
AED, antiepileptic drug; ASP, Anticonvulsant Screening
Program; CAD, catecholamine A-differentiated; CF3O, trifluor-
omethoxy; ED50, effective dose (50%); IC50, concentration at
which half of the channels have transitioned to a slow-
inactivated state; ip, intraperitoneally; MES, maximal electro-
shock seizure; NINDS, National Institute of Neurological
Disorders and Stroke; PI, protective index; po, orally; scMet,
scMetrazol; TD50, neurological impairment (toxicity, 50%);
TEA-Cl, tetraethylammonium chloride; V1/2, voltage of half-
maximal activation
ASSOCIATED CONTENT
* Supporting Information
■
S
REFERENCES
■
Receptor binding assay profile for compounds 2−11 against 43
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AUTHOR INFORMATION
Corresponding Author
*Tel: 919-843-8112. Fax: 919-966-0204. E-mail: hkohn@email.
unc.edu.
■
Present Addresses
¶Cell Dynamics Research Center, Gwangju Institute of Science
and Technology, Gwangju, Korea.
#Center for Neuro-Medicine, Korea Institute of Science and
Technology, Seoul, Korea.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work is supported by a grant from the North Carolina
Biotechnology Center (Technology Enhancement Grant)
administered by the University of North Carolina-Chapel Hill
and awards from the Indiana Clinical and Translational
Sciences Institute funded in part by a Project Development
Team Grant Number (RR025761) from the National Institutes
of Health, National Center for Research Resources, Clinical and
Translational Sciences Award, the Indiana State Department of
Health−Spinal Cord and Brain Injury Fund (A70-9-079138 to
R.K.), a National Scientist Development Award from the
American Heart Association (SDG5280023 to R.K.), and a
Neurofibromatosis New Investigator Award from the Depart-
ment of Defense Congressionally Directed Military Medical
Research and Development Program (NF1000099 to R.K.).
We thank the NINDS and the ASP at the National Institutes of
Health with Drs. Tracy Chen and Jeffrey Jiang for kindly
performing the pharmacological studies via the ASP’s contract
site at the University of Utah with Drs. H. Wolfe, H. S. White,
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Health (NIMH) Psychoactive Drug Screening Project for
performing in vitro receptor binding studies. This work was
supported by NIMH Psychoactive Drug Screening Program,
Contract No. HHSN-271-2008-00025-C (NIMH-PDSP). The
NIMH PDSP is directed by Bryan Roth M.D., Ph.D. at the
University of North Carolina at Chapel Hill and Project Officer
Jamie Driscol at NIMH, Bethesda, MD, U.S. The content is
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