Benzyloxybenzylammonium chlorides: Simple amine salts that display anticonvulsant activity

Article abstract of DOI:10.1016/j.bmc.2013.10.031

Several antiepileptic drugs exert their activities by inhibiting Na ⁺ currents. Recent studies demonstrated that compounds containing a biaryl-linked motif (Ar-X-Ar′) modulate Na⁺ currents. We, and others, have reported that compound

Full text of DOI:10.1016/j.bmc.2013.10.031

Bioorganic & Medicinal Chemistry 21 (2013) 7655–7662
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Benzyloxybenzylammonium chlorides: Simple amine salts
that display anticonvulsant activity
Hyosung Lee a, , Alexander S. Gold a, Xiao-Fang Yang b, Rajesh Khanna b,c,d, Harold Kohn a,e,
⇑
a Division of Chemical Biology and Medicinal Chemistry, UNC Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC 27599-7568, USA
b Department of Pharmacology & Toxicology, Indiana University School of Medicine, Indianapolis, IN 46202, USA
c Department of Biochemistry & Molecular Biology, Indiana University School of Medicine, Indianapolis, IN 46202, USA
d Program in Medical Neuroscience, Paul and Carole Stark Neurosciences Research Institute, Indiana University School of Medicine, Indianapolis, IN 46202, USA
e Department of Chemistry, University of North Carolina, Chapel Hill, NC 27599-3290, USA
a r t i c l e i n f o
a b s t r a c t
Several antiepileptic drugs exert their activities by inhibiting Na+ currents. Recent studies demonstrated
that compounds containing a biaryl-linked motif (Ar-X-Ar0) modulate Na+ currents. We, and others, have
reported that compounds with an embedded benzyloxyphenyl unit (ArOCH2Ar0, OCH2 = X) exhibit potent
anticonvulsant activities. Here, we show that benzyloxybenzylammonium chlorides (+H3NCH2C6H4OCH2
Ar0 Clꢀ) displayed notable activities in animal seizure models. Electrophysiological studies of 4-(20-triflu-
oromethoxybenzyloxy)benzylammonium chloride (9) using embryonic cortical neurons demonstrated
that 9 promoted both fast and slow inactivation of Na+ channels. These findings suggest that the potent
anticonvulsant activities of the earlier compounds were due, in part, to the benzyloxyphenyl motif and
provide support for the use of the biaryl-linked pharmacophore in future drug design efforts.
Ó 2013 Elsevier Ltd. All rights reserved.
Article history:
Received 23 August 2013
Revised 14 October 2013
Accepted 22 October 2013
Available online 1 November 2013
Keywords:
Antiepileptic agents
Biaryl-linked motif
Benzyloxybenzylammonium chlorides
Na+ current inhibition
1. Introduction
currents.5–8 These three processes are proven pathways that re-
duce neuronal hyperexcitability and constitute important
Epilepsy is a group of chronic neurological disorders that arise
from dysregulations in neuronal function, including hyperexcit-
ability and hypersynchronous firing.1 They affect up to 1% of the
world’s population.1,2 Despite the many antiepileptic drugs (AEDs)à
currently in clinical use, epilepsy in nearly 30% of patients is phar-
macoresistant and does not respond to at least two of the first-line
AEDs.3 Furthermore, adverse side effects (e.g., drowsiness, dizziness,
nausea) are experienced by nearly 40% of patients with epilepsy.4
These shortcomings have prompted a search for new agents.
We have shown, using whole-cell, patch–clamp electrophysiology,
that compounds containing the biaryl-linked motif A modulated
Na+ channel slow- and fast-inactivation processes and, in some
cases, promoted frequency (use)-dependent blockage of Na+
mechanisms of action for AEDs.6,9–11 Knowing this, we demon-
8
strated that (biphenyl-4-yl)methylammonium chlorides (com-
pound class B), where a single bond linked the two aryl units in
motif A, displayed activity in the maximal electroshock seizure
(MES),12 psychomotor 6 Hz seizure (6 Hz),13 and scMetrazol
seizure (scMet)14 models.
Y
Y
X
H3N
Cl
A
Biaryl-linked Motif
B
Compound Class
Compounds 115 and 216 exhibit pronounced anticonvulsant
activities. These compounds contain an embedded benzyloxyphe-
nyl unit C, thus having a biaryl-linked motif A in which an
oxymethylene (OCH2) group separates the two aryl units. To
further document the potential of the biaryl-linked A motif to elicit
anticonvulsant activity, we show, herein, that the readily accessi-
ble and structurally simple benzyloxybenzylammonium chlorides
(compound class D) exhibited notable activity in pre-clinical
seizure models.
⇑
Corresponding author. Tel.: +1 919 843 8112; fax: +1 919 966 0204.
E-mail address: hkohn@email.unc.edu (H. Kohn).
Cell Dynamics Research Center, Gwangju Institute of Science and Technology,
Gwangju, South Korea.
à
Abbreviations: AED, antiepileptic drug; ASP, Anticonvulsant Screening Program;
ED50, effective dose (50%); ip, intraperitoneally; MES, maximal electroshock seizure;
NINDS, National Institutes of Neurological Disorders and Stroke; po, orally; scMet,
scMetrazol; TD50, neurological impairment (toxicity, 50%).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.10.031

7656
H. Lee et al. / Bioorg. Med. Chem. 21 (2013) 7655–7662
CH3
O
O
O
O
H
N
F
O
H
N
F
N
H
H2N
O
CH3
1
2
2'
3'
Y
4'
Y
O
O
H3N
Cl
D
Benzyloxyphenyl Unit C
Compound Class
(NINDS) Anticonvulsant Screening Program (ASP). Screening was
performed using the procedures described by Stables and Kupfer-
berg.17 The anticonvulsant activity data from the MES,12 6 Hz,13
and scMet14 tests are summarized in Table 1 along with similar re-
sults obtained for the AEDs lacosamide,18,19 phenytoin,20 valpro-
ate,20 and phenobarbital.20 The compounds were administered
intraperitoneally (ip) to mice and ip or orally (po) to rats. In the ini-
tial qualitative studies, the compounds were tested at the ASP at
preset dosages (e.g., MES (mice, ip): 50 mg/kg; 100 mg/kg; 30, 50,
100 mg/kg). For compounds that showed significant activity, we
report the 50% effective dose (ED50) values obtained in quantitative
screening evaluations. Also provided are the median doses for 50%
neurological impairment (TD50) in mice, using the rotorod test,21
and in rats, using the behavioral toxicity effects.22
2. Results and discussion
2.1. Selection of compounds
We prepared nine compounds from the class D (3–11), varying
the terminal aryl substituent (Y) and the site of modification. We
restricted our choices for the Y substituent to electron-withdraw-
ing groups since these moieties provided the highest anticonvul-
sant activities for compound class B. The Y substituent was
placed in the 20, 30 or 40 position of the benzyloxy aromatic ring. Fi-
nally, in D, we retained the methyleneamino (CH2N(H)) unit, which
was found in 1 and 2 in the form of the methyleneammonium
+
(CH2NH3 Clꢀ) group to increase the compounds’ water solubility.
Benzyloxybenzylammonium chlorides 3–11 showed anticon-
vulsant activities in either the MES or the 6 Hz seizure models in
mice (ip) at doses below 100 mg/kg. For compounds 4, 7–11, we
observed activity in both models. Compounds 7, 9 and 10 were
the most potent in the MES and gave MES ED50 values of 54, 20,
and 28 mg/kg, respectively. These values were similar to pheno-
barbital (MES ED50 = 22 mg/kg)20 but greater than lacosamide
(MES ED50 = 4.5 mg/kg)18 and phenytoin (MES ED50 = 9.5 mg/
kg).20 The activities of 3–11 (>30 mg/kg) in the 6 Hz model
(32 mA) were less than that of lacosamide (ED50 = 10 mg/kg).19
Compounds 7, 9, and 10 did not exhibit activity in the scMet model
at the doses tested (>80 mg/kg). In rats, 6, 7, 9, 10–12 exhibited
activity in the MES model upon ip administration at a dosage of
2.2. Synthesis
Compounds 3–11 were prepared in two steps (Scheme 1).
Treating 4-cyanophenol (12) with the appropriate substituted ben-
zyl bromide (13–21) and base gave the Williamson ether coupled
products 22–30. LiAlH4 reduction of the nitrile group in 22–30
afforded the amines, which were immediately converted to the
corresponding amine hydrochlorides 3–11. In the Experimental
(Section 4), we report the synthesis and physical and spectral data
for all new intermediates and final compounds, while in the Sup-
plementary data, we provide this information for previously re-
ported compounds. Salts 3–11 were soluble in water when tested
at concentrations of 300 lM.
30 mg/kg, with 9 (MES ED50 = 18 mg/kg) and 10 (MES ED50
=
<30 mg/kg) being the most active. Of these compounds, only 9
provided partial protection upon po administration at 30 mg/kg.
The promising results for 7, 9, and 10, led us to test these
compounds in the corneally kindled mouse seizure assay (ip).23,24
2.3. Pharmacological evaluation
Compounds 3–11 were evaluated for anticonvulsant activity at
the National Institute of Neurological Disorders and Stroke’s
4'
4'
4
Y
1'
OH
Y
1'
O
3'
O
Y
K2CO3
1) LiAlH4, THF
1
3'
2'
+
3
2'
Acetone
2) HCl
2
N
Br
13
12
N
NH3 Cl
22
3
Y = H
Y = H
Y = H
14 Y = 2-F
23 Y = 2'-F
4 Y = 2'-F
15
16
24
25
5
6
Y = 3-F
Y = 4-F
Y = 3'-F
Y = 4'-F
Y = 3'-F
Y = 4'-F
17 Y = 3-Cl
26 Y = 3'-Cl
7 Y = 3'-Cl
18
19
27
28
8
9
Y = 4-Cl
Y = 2-OCF3
Y = 4'-Cl
Y = 2'-OCF3
Y = 4'-Cl
Y = 2'-OCF3
20 Y = 3-OCF3
29 Y = 3'-OCF3
10 Y = 3'-OCF3
21
30
11
Y = 4'-OCF3
Y = 4-OCF3
Y = 4'-OCF3
Scheme 1. Synthesis of substituted benzyloxybenzylammonium chlorides (D).

Table 1
Anticonvulsant activities of substituted benzyloxybenzylammonium chlorides (D)a and (biphenyl-4-yl)methylammonium chlorides (B)a
L = –OCH2– (Compound class D)
L = single bond (Compound class B)
L
1'
2'
Compd no.
Mice (ip)b [hour] (confidence Interval)
Rat (ip)c [hour] (confidence Interval)
Rat (oral)c [hour] (confidence Interval)
Compd no.
Mice (ip)b [hour] (confidence Interval)
4'
MESd
6 Hze
scMetf
Toxg
MESd
Toxh
MESd
Toxh
MESd
6 Hze
scMetf
Toxg
3'
Y
NH3Cl
Y = H
3
>100 [0.5–2.0]
30–100 [0.5]
30–100 [0.5]
30–100 [0.5]
>50 [0.25–4.0]
NDi
NDi
NDi
100–300 [0.5–2.0]
100–300 [0.5–2.0]
>100 [0.25–4.0]
>100 [0.25–4.0]
>30 [0.25–4.0]
>30 [0.25–4.0]
>30 [0.25–4.0]
ꢁ30 [0.25]
>50 [0.25–4.0]
>50 [0.25–4.0]
>30 [0.25–4.0]
>30 [0.25–4.0]
>30 [0.25–4.0]
>30 [0.25–4.0]
>30 [0.25–4.0]
>30 [0.25–4.0]
>30 [0.25–4.0]
>30 [0.25–4.0]
>30 [0.25–4.0]
>30 [0.25–4.0]
>30 [0.25–4.0]
>30 [0.25–4.0]
>30 [0.25–4.0]
>30 [0.25–4.0]
>30 [0.25–4.0]
>30 [0.25–4.0]
>30 [0.25–4.0]
>30 [0.25–4.0]
>30 [0.25–4.0]
>500
31j
30–100 [0.25–0.5] ꢁ100 [0.5–1.0]
NDi
>100 [0.25–4.0]
Y = 20 -F
4
Not prepared
Y = 30 -F
5
<100 [0.25–0.5]
<100 [0.25]
32j
33j
34j
35j
36j
37j
38j
>50 [0.25–4.0]
ꢁ50 [4.0]
<100 [0.25, 1.0]
>50 [0.25–4.0]
ꢁ50 [1.0]
<100 [1.0–4.0]
<100 [0.5–4.0]
<100 [1.0–4.0]
ꢁ50 [2.0]
>50 [0.25–4.0]
<100 [0.5–2.0]
>50 [0.25–4.0]
>100 [0.25–4.0]
>50 [0.25–4.0]
>100 [0.25–4.0]
>50 [0.25–4.0]
72 [0.25] (59–84)
Y = 40 -F
6
>100 [0.25–4.0] NDi
Y = 30 -Cl
7
54 [0.25](49–61)
<100 [0.25]
78 [2.0] (69–91) >180 [0.25] 160 [0.25] (130–180) ꢁ30 [0.25]
<100 [2.0] >100 [0.25–4.0] >30 [0.25–4.0]
NDi
<100 [0.25–4.0] >80 [0.25] 64 [0.25] (55–77)
Y = 40 -Cl
8
Y = 20 -OCF3
Y = 30 -OCF3
Y = 40 -OCF3
Lacosamidek,l
Phenytoinm
9
20 [0.25] (17–25)
28 [0.5] (24–38)
<100 [0.5]
18 [0.5] (14–21) 81 [0.5](60–108) ꢁ30 [1.0]
<30 [0.25–0.5]
40 [0.25] (28–59) <100 [0.25–4.0] >120 [0.25]
10
11
ꢁ50 [1.0]
30–100 [2.0]
10 [0.5]
>170 [0.5] 92 [0.25] (70–100)
NDi
>30 [0.25–4.0]
>50 [0.25–4.0]
>30 [0.25–4.0]
25 [0.5] (20–34)
43 [1.0] (35–53) 81 [4.0] (60–118) 81 [6.0] (58–99)
<100 [2.0] >50 [0.25–4.0] >50 [0.25–4.0]
>100 [0.25–4.0]
ꢁ30 [0.25]
>30 [0.25–4.0]
ꢁ50 [0.5–2.0]
4.5 [0.5] (3.7–5.5)
9.5 [2.0] (8.1–10)
22 [1.0] (15–23)
270 [0.25] (250–340)
27 [0.25] (26–28)
66 [2.0] (53–72)
69 [0.5] (63–73)
430 [0.25] (370–450)
3.9 [2.0] (2.9–6.2)
30 [40] (22–39)
9.1 [5.0] (7.6–12)
Phenobarbitalm
Valproatem
61 [0.5] (44–96)
490 [0.5] (350–730) 280 [0.5] (190–350)
a
The compounds were tested through the auspices of the NINDS ASP.
The compounds were administered intraperitoneally 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
b
compounds that displayed sufficient activity. The dose–effect data for these compounds was obtained at the ‘time of peak effect’ (indicated in hours in the brackets).
c
The compounds were administered either intraperitoneally or orally to rats as shown in parentheses.
MES = maximal electroshock seizure test.
6 Hz = 6 Hz psychomotor seizure test (32 mA).
scMet = subcutaneous pentylenetetrazol (Metrazol) test.
Tox = rotorod test.
Tox = behavioral toxicity.
ND = not determined.
Ref. 8.
d
e
f
g
h
i
j
k
Ref. 18.
Ref. 19.
Ref. 20.
l
m

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H. Lee et al. / Bioorg. Med. Chem. 21 (2013) 7655–7662
Figure 1. Evaluation of 9 on biophysical properties of Na+ currents in rat embryonic cortical neurons. (A) The voltage protocol used to evoke macroscopic Na+ currents. (B)
Representative families of current responses of cortical neurons cells treated with 0.01% DMSO control (control; black traces) or 10 M 9 (red traces). (C) Peak current density
l
(pA/pF) measured at ꢀ10 mV for the two conditions are shown (n = 6 each). (D) Slow inactivation protocol: currents were evoked by 5 s prepulses between ꢀ100 mV 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 ꢀ10 mV was
analyzed. (E) Representative current traces from cortical neurons in the absence (control, 0.1% DMSO) or presence of 10
l
M 9. The black and red traces represent the current
M 9. (G) Summary of the fraction of current available
lM of 9. Steady-state activation (H) and fast inactivation (J) protocols. Values for V1/2, the voltage of half-
at ꢀ50 mV. (F) Summary of steady-state slow activation curves for cortical neurons treated with DMSO (control) or 10
l
at ꢀ50 mV for cortical neurons treated with DMSO (control) or 10
maximal activation and steady-state fast inactivation and the slope factors (k) were derived from Boltzmann distribution fits to the individual recordings (examples of fast
inactivation recordings illustrated in K) and averaged to determine the mean ( SEM) voltage dependence of activation and fast inactivation (see Table 2). Representative
Boltzmann fits for steady-state activation (I) and fast inactivation (L) for cortical neurons treated with 0.1% DMSO (control) and 20
significantly affected by 10
M of 9. (M–O) The frequency dependence of block was examined by holding cells at the hyperpolarized potential of ꢀ80 mV and evoking
M). (O)
M) (p >0.05, one-way ANOVA with
lM of 9 are shown. Fast-inactivation was
l
currents at 10 Hz by 20-ms test pulses to ꢀ10 mV. Representative overlaid traces are illustrated by pulses 1, 10, 20, and 30 for control and in the presence of 9 (10
l
Summary of average frequency-dependent decrease in current amplitude ( SEM) produced by control or by the presence of 9 (10
l
Dunnett’s post-hoc test). Data are from 5–6 cells per condition.
This model has been advanced as a sensitive screening model for
partial epilepsy in man.23 We observed that 7 (78 mg/kg), 9
(20 mg/kg), and 10 (28 mg/kg) provided protection in 3 of the 4
animals and led to a reduction of the average seizure score from
5 to 2–3.5.
We compared the anticonvulsant activities of compound class D
(i.e., 3, 5–11) with compound class B8 (i.e., 31–38) in mice (ip) and
found similar results (Table 1). Many compounds in both series
exhibited antiseizure properties at 100 mg/kg. For D, like B, the
trifluoromethoxy compounds (compound class D: 9, 10;
compound class B: 36, 37) were among the most active agents.
and induced frequency (use)-dependent inhibition of Na+ currents
at 100
M concentrations.5 Compound 40 is the corresponding free
l
amine of hydrochloride salt 5. These findings suggest that compounds
of class D, such as 5, exert their anticonvulsant activity, in part, by
affecting Na+ channel processes. Accordingly, we determined the
Na+ channel properties of 9 using rat embryonic cortical neurons.
These neurons typically express CNS Na+ channel isoforms NaV1.1,
NaV1.2, NaV1.3, and NaV1.6.25 Total macroscopic currents, slow inacti-
vation, steady-state inactivation, fast inactivation, and use-depen-
dence of Na+ currents in cortical neurons grown for 7–10 days
in vitro (Fig. 1A, D, H, J, and M) were examined using protocols de-
scribed earlier.8 Current–voltage relationships in control (0.01%
DMSO-treated) or 9-treated cells were examined by the application
of 15-ms step depolarizations ranging from ꢀ70 mV to +80 mV (in
+5 mV increments) from a holding potential of ꢀ70 mV (Fig. 1A).
The transient inward current in cortical neurons activated between
ꢀ40 and ꢀ30 mV and reached its peak at ꢀ10 mV to +0 mV. Peak
Interestingly, we found only one compound in class D (9, ED50
=
ꢁ30 mg/kg) and one in class
B
(37, ED50 = 8.7 mg/kg)8 that
exhibited activity in the rat upon po administration at 30 mg/kg
or less.
In an earlier study, we showed that 39 and 40 potently promoted
Na+ channel slow inactivation, affected the fast inactivation process,

H. Lee et al. / Bioorg. Med. Chem. 21 (2013) 7655–7662
7659
Table 2
Comparative Boltzmann parameters of voltage-dependence of channel activation and steady-state fast-inactivation curves for control (0.01% DMSO) or 9 (10
l
M) treated cortical
neurons
Condition
Voltage-dependence of activation
Voltage-dependence of fast-inactivation
V1/2 (mV)
Slope (mV/e-fold)
V1/2 (mV)
Slope (mV/e-fold)
Control
10 lM 9
ꢀ39.0 7.8 (5)
ꢀ35.3 3.2 (6)
2.8 1.3 (5)
3.3 2.0 (6)
ꢀ51.1 1.4 (5)
ꢀ66.4 0.8 (6)⁄
4.6 0.8 (5)
5.5 0.7 (6)
Values for V1/2, the voltage of half-maximal activation, and slope, were derived from Boltzmann distribution fits to the individual recordings and averaged to determine the
mean and standard error of the mean ( SEM). N Values are indicated in parentheses. Asterisks represent statistically significant differences as compared to control (i.e., DMSO
condition) (p <0.05, ANOVA with Dunnett’s post-hoc test).
inward Na+ currents were measured and expressed as peak current
density (pA/pF) to account for variations in cell size. Initial experi-
Psychoactive Drug Screening Program (PDSP) at pH 7.4.30 We ob-
served appreciable binding at 10 M, for most compounds, to sev-
l
ments were performed with a single concentration (20
l
M) of 9 so
eral serotonin (e.g., 5-HT2A, 5-HT2B) and adrenergic (e.g., alpha 2A,
2B, 2C) receptors, the DAT, NET, and SERT transporters, and the sig-
ma-1 and -2 receptors (Supplementary Table 1). A similar binding
profile was seen for class B compounds.8 The binding of class B and
class D compounds to a variety of receptors should be investigated
further (i.e., extent of binding, functional activity) as it is possible
that these additional activities may represent either an opportu-
nity to treat common co-morbidities of epilepsy such as depression
and anxiety or limit the antiepileptic potential of these agents due
to adverse effects.
as to directly compare with compound 37.8 Compound 9 is structur-
ally related to 37 differing only in the composition of the bridging unit
in the bi-aryl motif (9: oxymethylene; 37: single bond) and the regio-
nal position of the trifluoromethoxy unit (9: 20; 37: 30). However, at
20 l
M concentration, Na+ currents could not be evaluated due to
acute toxicity to the cortical cells and loss of cell membrane integrity.
Therefore, all electrophysiology experiments were performed using a
10 l
M of 9. Atthis concentration, total macroscopic Na+ currentswere
not different between control (0.01% DMSO)-treated versus 9-treated
(Fig. 1B and C). The extent of slow inactivation induced by 9 was
significantly greater than neurons treated with the vehicle 0.01%
DMSO: 0.27 0.03 (n = 6) versus 0.44 0.05 (n = 6), respectively
(p >0.05, one-way ANOVA Fig. 1F and G). Steady-state activation
wasunchanged between the two conditions with the V1/2 and k values
being statistically similar (Table 2, Fig. 1H and I). Steady-state, fast
3. Conclusion
Our findings demonstrated that appending the benzyloxyphe-
nyl motif (C) to a methyleneammonium unit yielded compounds
with notable anticonvulsant activities in several established ani-
mal models. The seizure protection is likely associated, in part,
with the ability of this pharmacophore to modulate Na+ currents.
The activities of class B and class D compounds suggest that
compounds with a biaryl motif with short bridging unit (e.g.,
oxygen, vinylic, acetylenic) may also exhibit noteworthy anticon-
vulsant activities. Finally, these results suggest that medicinal
agents containing unit C, and the more general biaryl-linked mo-
tif (A), should be tested for their Na+ channel properties31 and
that this pharmacophore may be useful in future drug design
efforts.
inactivation curves of Na+ currents from DMSO-treated and 10
lM
9-cortical neurons were well fitted with a single Boltzmann function
(R2 >0.931 for both conditions) and are illustrated in Figure 1L. The
V1/2 value for inactivation of 0.01% DMSO-treated cells was
ꢀ51.1 1.4 mV (n = 6), which was significantly different from the
V1/2 value of ꢀ66.4 0.8 mV (n = 6) for 9-treated neurons (p <0.05;
ANOVA with a post-hoc Dunnett’s test). The 10 lM concentration of
9 caused a significant hyperpolarizing shift of ꢁ15.3 mV with no
commensurate significant changes in slope values compared with
control cells. Finally, we found that
9
did not exhibit
frequency (use)-dependent inhibition of Na+ currents in cortical
neurons (Fig. 1M–O). Thus, 9, like 37,8 promoted slow and fast
inactivation, but did not affect frequency (use)-dependent block of
Na+ currents. Together, these results with previous findings,5–8
provide support that the biaryl-linked motif A can modulate Na+
channel function.
4. Experimental
4.1. General methods
The general methods used in this study are identical to those
previously reported.16 The compounds were checked by TLC, 1H
NMR, and 13C NMR, and MS, and for the final compounds by ele-
mental analyses. The analytical results are within 0.40% of the
theoretical value. The NMR and analytical data confirmed the pur-
ity of the products was P95%.
F
O
R
39
40
R = H
4.1.1. 4-(20-Trifluoromethoxybenzyloxy)benzonitrile (28)
4-Cyanophenol (12) (1.03 g, 8.6 mmol) and Na2CO3 (3.32 g,
31.3 mmol) were mixed in acetone (80 mL) and 2-trifluorometh-
oxybenzyl bromide (19) (2.00 g, 7.8 mmol) was added. The result-
ing mixture was stirred (16 h) at reflux and evaporated in vacuo.
The resulting residue was diluted in CH2Cl2 (120 mL), washed with
H2O (2 ꢂ 120 mL), dried (Na2SO4), and concentrated in vacuo to
give 28 (1.94 g, 84%) as a white solid: Rf = 0.91 (hexanes/EtOAc
7:1); mp 70–74 °C; 1H NMR (CDCl3) d 5.20 (s, OCH2), 7.02 (d,
J = 7.6 Hz, 2 ArH), 7.31–7.41 (m, 3 ArH), 7.49–7.55 (m, 3 ArH); 13C
NMR (CDCl3) d 64.8 (OCH2), 104.9, 115.7 (2 ArC), 119.2 (CN),
R = CH2NH2
The Na+ channel properties of 39 and 40 led us test these com-
pounds for anticonvulsant activity at 100 mg/kg in the MES model
in mice (ip). We observed seizure protection for 40 but not 39 (data
not shown). The factors that contribute to 39’s lack of activity have
not been determined.
Since the biaryl-linked motif (A) is found in several biologically
active agents,15,26–29 we determined the receptor binding profile
of class
D compounds 3–11 against 43 receptors at UNC’s

7660
H. Lee et al. / Bioorg. Med. Chem. 21 (2013) 7655–7662
+
+
0
120.8 (q, J = 256.4 Hz, OCF3), 120.9, 127.4, 128.7, 129.6, 130.0,
134.3, 147.0, 161.8 (8 ArC); LRMS (ESI+) 332.03 [M+H]+ (calcd for
(d, J = 241.7 Hz, C4 ); LRMS (ESI ) 232.07 [MꢀCl] (calcd for C14H15-
FNO+ 232.11); Anal. C14H15Cl2NO (C, H, Cl, F, N).
C
C
15H11F3KNOþ 332.03); HRMS (ESI+) 316.0571 [M+Na]+ (calcd for
2
15H10F3NO2Na+ 316.0561).
4.1.6. 4-(30-Chlorobenzyloxy)benzylammonium chloride (7)
Employing the procedure used for 4 and using a solution of 26
(2.10 g, 8.6 mmol) in THF (10 mL) and a suspension of LiAlH4
(0.98 g, 25.9 mmol) in THF (80 mL) gave 7 (1.70 g, 80%) as a white
solid: Rf = 0.00 (hexanes/EtOAc 1:1); mp 240–242 °C; 1H NMR
4.1.2. 4-(30-Trifluoromethoxybenzyloxy)benzonitrile (29)
Employing the procedure for 28 and using 12 (1.03 g,
8.6 mmol), Na2CO3 (3.32 g, 31.3 mmol), acetone (80 mL), and 3-tri-
fluoromethoxybenzyl bromide (20) (2.00 g, 7.8 mmol) gave 29
(1.84 g, 80%) as a colorless oil: Rf = 0.90 (hexanes/EtOAc 7:1); 1H
NMR (CDCl3) d 5.10 (s, OCH2), 6.99 (d, J = 8.8 Hz, 2 ArH), 7.16–
7.20 (m, ArH), 7.28–7.41 (m, 3 ArH), 7.55 (d, J = 8.8 Hz, 2 ArH);
13C NMR (CDCl3) d 69.5 (OCH2), 104.8, 115.8 (2 ArC), 119.2 (CN),
119.4, 120.0 (2 ArC), 120.7 (q, J = 256.3 Hz, OCF3), 120.9, 130.4,
134.3, 138.4, 149.8, 161.8 (6 ArC); LRMS (ESI+) 332.03 [M+K]+
(CDCl3)
d 3.90–3.96 (br s, NCH2), 5.16 (s, OCH2), 7.04 (d,
J = 8.0 Hz, 2 ArH), 7.38–7.51 (m, 6 ArH); 13C NMR (CDCl3) d 41.6
(NCH2), 68.2 (OCH2), 114.8, 126.1, 126.4, 127.2, 127.7, 130.4,
130.6, 133.1, 139.6, 158.0 (10 ArC); LRMS (ESI+) 248.03 [M+H]+
(calcd for C14H15ClNO+ 248.08); Anal. C14H15Cl2NO (C, H, Cl, N).
4.1.7. 4-(40-Chlorobenzyloxy)benzylammonium chloride (8)
Employing the procedure used for 4 and using a solution of 27
(2.26 g, 9.3 mmol) in THF (10 mL) and a suspension of LiAlH4
(1.06 g, 27.9 mmol) in THF (80 mL) gave 8 (1.74 g, 76%) as a white
solid: Rf = 0.00 (hexanes/EtOAc 1:1); mp 262–264 °C; 1H NMR
(DMSO-d6) d 3.92 (s, NCH2), 5.14 (s, OCH2), 7.03 (d, J = 8.4 Hz, 2
ArH), 7.42–7.50 (m, 6 ArH); 13C NMR (DMSO-d6) d 41.6 (NCH2),
68.3 (OCH2), 114.8, 126.4, 128.4, 129.4, 130.5, 132.4, 136.0, 158.1
(8 ArC); LRMS (ESI+) 248.03 [M+H]+ (calcd for C14H15ClNO+
248.08); Anal. C14H15Cl2NO (C, H, Cl, N).
(calcd for C15H11F3KNOþ 332.03); HRMS (ESI+) 316.0573 [M+Na]+
2
(calcd for C15H10F3NNaOþ 316.0561).
2
4.1.3. 4-(40-Trifluoromethoxybenzyloxy)benzonitrile (30)
Employing the procedure for 28 and using 12 (1.03 g,
8.6 mmol), Na2CO3 (3.32 g, 31.3 mmol), acetone (80 mL), and 4-tri-
fluoromethoxybenzyl bromide (21) (2.00 g, 7.8 mmol) gave 30
(2.02 g, 88%) as a white solid: Rf = 0.91 (hexanes/EtOAc 7:1); mp
59–61 °C; 1H NMR (CDCl3) d 5.11 (s, OCH2), 7.02 (d, J = 8.8 Hz, 2
ArH), 7.25 (d, J = 8.8 Hz, 2 ArH), 7.45 (d, J = 8.8 Hz, 2 ArH), 7.60 (d,
J = 8.8 Hz, 2 ArH); 13C NMR (CDCl3) d 69.6 (OCH2), 104.8, 115.7 (2
ArC), 119.2 (CN), 119.4 (q, J = 256.4 Hz, OCF3), 121.5, 129.1, 134.3,
134.6, 149.4, 161.9 (6 ArC); LRMS (ESI+) 332.03 [M+H]+ (calcd for
4.1.8. 4-(20-Trifluoromethoxybenzyloxy)benzylammonium
chloride (9)
Employing the procedure used for 4 and using a solution of 28
(2.10 g, 7.2 mmol) in THF (10 mL) and a suspension of LiAlH4
(0.82 g, 21.5 mmol) in THF (70 mL) gave 9 (2.10 g, 88%) as a white so-
lid: Rf = 0.00 (hexanes/EtOAc 1:1); mp 133–136 °C; 1H NMR (DMSO-
d6) d 3.95 (s, NCH2), 5.16 (s, OCH2), 7.06 (d, J = 8.8 Hz, 2 ArH), 7.41–
7.60 (m, 5 ArH), 7.65 (d, J = 7.2 Hz, ArH), 8.30–8.40 (br s, 3H); 13C
NMR (DMSO-d6) d 41.7 (NCH2), 64.3 (OCH2), 114.7 (ArC), 120.6 (q,
J = 230.1 Hz, OCF3), 120.7, 126.5, 127.6, 129.2, 130.3, 130.6, 130.8,
146.7, 158.2 (9 ArC); LRMS (ESI+) 298.08 [MꢀCl]+ (calcd for C15H15F3
NO2+ 298.10); Anal. C15H15ClF3NO2ꢃ0.1H2O (C, H, Cl, F, N).
C
C
15H11F3KNOþ 332.03); HRMS (ESI+) 316.0549 [M+Na]+ (calcd for
2
15H10F3NO2Na+ 316.0561).
4.1.4. 4-(20-Fluorobenzyloxy)benzylammonium chloride (4)
A solution of 23 (2.15 g, 9.5 mmol) in THF (10 mL) was added
dropwise to a suspension of LiAlH4 (1.08 g, 28.4 mmol) in THF
(85 mL) at 0 °C and stirred at room temperature (16 h). H2O
(1.2 mL) was added dropwise to the resulting mixture at 0 °C fol-
lowed by an aqueous NaOH solution (0.6 mL, 15% w/w), and then
H2O (1.2 mL). The mixture was stirred at room temperature (2 h).
The precipitate was filtered and washed with CH2Cl2. The com-
bined organic layers were concentrated in vacuo and diluted with
CH2Cl2 (100 mL). To the resulting solution a HCl solution in diox-
ane was added dropwise (3 mL, 4 N) and stirred at room tempera-
ture (30 min). The precipitate was filtered and washed with
hexanes and dried in vacuo to give 4 (1.46 g, 66%) as a white solid:
Rf = 0.00 (hexanes/EtOAc 1:1); mp 242–243 °C; 1H NMR (DMSO-d6)
d 3.94 (s, NCH2), 5.16 (s, OCH2), 7.07 (d, J = 2.2 Hz, 2 ArH), 7.23–7.25
(m, 2 ArH), 7.39–7.45 (m, 3 ArH), 7.52–7.58 (br t, ArH); 13C NMR
(DMSO-d6) d 42.4 (NCH2), 64.3 (OCH2), 115.5 (C3), 116.2 (d,
4.1.9. 4-(30-Trifluoromethoxybenzyloxy)benzylammonium
chloride (10)
Employing the procedure used for 4 and using a solution of 29
(1.80 g, 6.1 mmol) in THF (6 mL) and a suspension of LiAlH4 (0.70 g,
18.4 mmol) in THF (52 mL) gave 10 (1.06 g, 52%) as a white solid:
Rf = 0.00 (hexanes/EtOAc 1:1); mp 248–251 °C; 1H NMR (DMSO-
d6) d 3.91–3.95 (m, NCH2), 5.20 (s, OCH2), 7.05 (d, J = 8.8 Hz, 2
ArH), 7.32 (d, J = 7.6 Hz, ArH), 7.41–7.55 (m, 5 ArH); 13C NMR
(DMSO-d6) d 41.6 (NCH2), 68.2 (OCH2), 114.9, 119.4, 119.8, 120.3
(4 ArC), 120.6 (q, J = 256.4 Hz, OCF3), 123.8, 126.5, 130.6, 139.9,
148.4, 158.1 (6 ArC); LRMS (ESI+) 298.08 [MꢀCl]+ (calcd for
0
0
0
J = 20.5 Hz, C3 ), 124.5 (d, J = 14.4 Hz, C4 ), 125.3 (C5 ), 127.3 (C1),
0
131.2 (d, J = 8.3 Hz, C1 ), 131.4 (C2), 159.0 (C4), 161.2 (d,
C15H15F3NOþ 298.10); Anal. C15H15ClF3NO2 (C, H, Cl, F, N).
2
0
J = 254.2 Hz, C2 ), one aromatic resonance was not detected and is
believed to overlap with a nearby peak; LRMS (ESI+) 232.07
[M+H]+ (calcd for C14H15FNO+ 232.11); Anal. C14H15ClFNOꢃ0.5H2O:
(C, H, Cl, F, N).
4.1.10. 4-(40-Trifluoromethoxybenzyloxy)benzylammonium
chloride (11)
Employing the procedure used for 4 and using a solution of 30
(2.02 g, 6.9 mmol) in THF (7 mL) and a suspension of LiAlH4 (0.78 g,
20.7 mmol) in THF (63 mL) gave 11 (1.21 g, 55%) as a white solid:
Rf = 0.91 (hexanes/EtOAc 7:1); mp 251–254 °C; 1H NMR (DMSO-
d6) d 3.93 (s, NCH2), 5.17 (s, OCH2), 7.04 (d, J = 8.4 Hz, 2 ArH),
7.38 (d, J = 8.2 Hz, 2 ArH), 7.46 (d, J = 8.2 Hz, 2 ArH), 7.58 (d,
4.1.5. 4-(40-Fluorobenzyloxy)benzylammonium chloride (6)
Employing the procedure used for 4 and using a solution of 25
(2.05 g, 9.0 mmol) in THF (10 mL) and a suspension of LiAlH4
(1.03 g, 27.1 mmol)inTHF(90 mL)gave6(2.09 g, 100%) as awhite so-
lid: Rf = 0.00 (hexanes/EtOAc 1:1); mp 256–258 °C; 1H NMR (DMSO-
d6) d 3.90–3.95 (m, NCH2), 5.11 (s, OCH2), 7.03 (d, J = 8.0 Hz, 2 ArH),
7.20–7.25 (m, 2 ArH), 7.42–7.53 (m, 4 ArH); 13C NMR (DMSO-d6) d
J = 8.4 Hz, 2 ArH); 13C NMR (DMSO-d6)
d 41.6 (NCH2), 68.2
(OCH2), 114.8 (ArC), 120.7 (q, J = 255.6 Hz, OCF3), 121.1, 126.4,
129.5, 130.6, 136.5, 147.8, 158.1 (7 ArC); LRMS (ESI+) 298.08
41.6 (NCH2), 68.4 (OCH2), 114.8 (C3), 115.2 (d, J = 21.2 Hz, C3 ), 126.3
[MꢀCl]+ (calcd for C15H15F3NOþ 298.10); Anal. C15H15ClF3NO2 (C,
0
2
0
0
(C1), 129.9 (d, J = 8.0 Hz, C2 ), 130.6 (C2), 133.8 (C1 ), 158.2 (C4), 161.7
H, Cl, F, N).

H. Lee et al. / Bioorg. Med. Chem. 21 (2013) 7655–7662
7661
4.2. Whole animal pharmacological evaluation
from the North Carolina Biotechnology Center (Technology
Enhancement Grant) administered by the University of North Car-
olina-Chapel Hill. We thank the NINDS and the ASP with Drs. Tra-
cy Chen and Jeffrey Jiang for kindly performing the
pharmacological studies via the ASP’s contract site at the Univer-
sity of Utah with Drs. H. Wolfe, H.S. White, and K. Wilcox. We ex-
press our appreciation to Dr. Bryan L. Roth and Mr. Jon Evans at
the National Institute of Mental 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 MD, PhD, University
of North Carolina–Chapel Hill, and Project Officer Jamie Driscoll
at NIMH, Bethesda MD, USA. The authors are solely responsible
for the content of this report, and it does not represent the official
views of the National Center for Research Resources, National
Institutes of Health.
Compounds were screened under the auspices of the NINDS
ASP. Experiments were performed in male rodents [albino Car-
worth Farms No. 1 mice (ip), albino Sprague-Dawley rats (ip,
po)]. Housing, handling, and feeding were in accordance with
guidelines contained in the Guide for the Care and Use of Laboratory
Animals. Anticonvulsant activity was established using the MES,12
6 Hz,13 scMet,14 and the corneal kindled23 tests according to previ-
ously reported methods.18,23
4.3. Cortical neurons
Rat cortical neuron cultures were prepared from cortices dis-
sected from embryonic day 19 brains exactly as described.32,33
4.4. Electrophysiology
Whole-cell voltage clamp recordings were performed at room
temperature on cortical neurons using an EPC 10 Amplifier (HEKA
Electronics, Lambrecht/Pfalz Germany).8 Electrodes were pulled
from thin-walled borosilicate glass capillaries (Warner Instru-
ments, Hamden, CT) with a P-97 electrode puller (Sutter Instru-
ment, Novato, CA) such that final electrode resistances were 1–
2 MO 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 tetraethylam-
Supplementary data
Supplementary data (experimental procedures for intermedi-
ates and final products previously reported, elemental analysis
and receptor binding assay profile for compounds 3–11 against
43 receptors, and 1H and 13C NMR spectra for all new compounds
(28–30, 4, 6–11)) associated with this article can be found, in the
online version, at http://dx.doi.org/10.1016/j.bmc.2013.10.031.
References and notes
monium 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 resis-
tance was less than 3 MO. Linear leak currents were digitally sub-
tracted by P/4.
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We thank Dr. Ki Duk Park for preparing several of the interme-
diates used in this study. We thank Weina Ju and Sarah Wilson
for preparing cortical neurons. This work is supported by a grant

7662
H. Lee et al. / Bioorg. Med. Chem. 21 (2013) 7655–7662
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