Chimeric derivatives of functionalized amino acids and α-aminoamides: Compounds with anticonvulsant activity in seizure models and inhibitory actions on central, peripheral, and cardiac isoforms of voltage-gated sodium channels

Article abstract of DOI:10.1016/j.bmc.2015.04.014

Six novel 3″-substituted (R)-N-(phenoxybenzyl) 2-N-acetamido-3-methoxypropionamides were prepared and then assessed using whole-cell, patch-clamp electrophysiology for their anticonvulsant activities in animal seizure models and for their sodium channel a

Full text of DOI:10.1016/j.bmc.2015.04.014

Bioorganic & Medicinal Chemistry xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Chimeric derivatives of functionalized amino acids and
a-aminoamides: Compounds with anticonvulsant activity in seizure
models and inhibitory actions on central, peripheral, and cardiac
isoforms of voltage-gated sodium channels
Robert Torregrosa a, Xiao-Fang Yang b, Erik T. Dustrude c, Theodore R. Cummins c,d, Rajesh Khanna b,
,
⇑
Harold Kohn a,e,f,
⇑
a NeuroGate Therapeutics, Inc., 150 Fayetteville Street, Suite 2300, Raleigh, NC 27601, United States
b Department of Pharmacology and Neuroscience Graduate Interdisciplinary Program, College of Medicine, University of Arizona, Tucson, AZ 85742, United States
c Program in Medical Neuroscience, Paul and Carole Stark Neurosciences Research Institute, Indiana University School of Medicine, Indianapolis, IN 46202, United States
d Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, IN 46202, United States
e Division of Chemical Biology and Medicinal Chemistry, UNC Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC 27599, United States
f Department of Chemistry, University of North Carolina, Chapel Hill, NC 27599, United States
a r t i c l e i n f o
a b s t r a c t
Six novel 300-substituted (R)-N-(phenoxybenzyl) 2-N-acetamido-3-methoxypropionamides were
prepared and then assessed using whole-cell, patch-clamp electrophysiology for their anticonvulsant
activities in animal seizure models and for their sodium channel activities. We found compounds with
various substituents at the terminal aromatic ring that had excellent anticonvulsant activity. Of these
compounds, (R)-N-40-((300-chloro)phenoxy)benzyl 2-N-acetamido-3-methoxypropionamide ((R)-5) and
(R)-N-40-((300-trifluoromethoxy)phenoxy)benzyl 2-N-acetamido-3-methoxypropionamide ((R)-9) exhib-
ited high protective indices (PI = TD50/ED50) comparable with many antiseizure drugs when tested in
the maximal electroshock seizure test to mice (intraperitoneally) and rats (intraperitoneally, orally).
Most compounds potently transitioned sodium channels to the slow-inactivated state when evaluated
in rat embryonic cortical neurons. Treating HEK293 recombinant cells that expressed hNaV1.1, rNaV1.3,
hNaV1.5, or hNaV1.7 with (R)-9 recapitulated the high levels of sodium channel slow inactivation.
Ó 2015 Published by Elsevier Ltd.
Article history:
Received 30 January 2015
Revised 26 March 2015
Accepted 3 April 2015
Available online xxxx
Keywords:
Chimeric agents
Functionalized amino acids
a-Aminoamides
Antiseizure agents
Na+ current inhibition
1. Introduction
ineffective for about one-third of patients, even when multiple
drugs are prescribed.3 Second, ASD use is associated with unto-
The epilepsies are serious, neurological disorders that affect up
to 1% of the world’s population.1 Although more than 40 drugs
have been used to treat these disorders,2 significant health needs
remain unmet. First, current antiseizure drugs (ASDs) are
ward side effects in more than 40% of patients, ranging from com-
mon cosmetic (e.g., gingival hyperplasia, weight gain) and quality
of life (e.g., sedation, learning, cognitive dysfunction) to rare, life-
threatening (e.g., hepatotoxicity, aplastic anemia) ones.4,5 Third,
some epilepsies, such as Lenox-Gestault and mesial temporal lobe
epilepsy, worsen over time, and for these disorders, there is a need
for a disease-modifying drug.6 Finally, most seizure medications do
not address associated comorbidities (e.g., cognitive dysfunction7).
Thus, the need still remains for ASDs that have novel mechanism(s)
of action with safe profiles.
Abbreviations: AAA, a-aminoamide; ASD, antiseizure drug; ASP, Anticonvulsant
Screening Program; CAD, catecholamine A-differentiated; CNS, central nervous
system; ED50, effective dose (50%); FAA, functionalized amino acid; IBCF, isobutyl
chloroformate; IC50, concentration at which half of the channels have transitioned
to a slow-inactivated state; ip, intraperitoneally; MAC, mixed anhydride coupling;
MES, maximal electroshock seizure; NINDS, National Institutes of Neurological
Disorders and Stroke; NMM, N-methylmorpholine; NOAEL, no observed adverse
effect level; OCF3, trifluoromethoxy; PI, protective index; po, orally; RMP, resting
We have reported on a novel series of chimeric compounds,
(R)-A,8–10 derived from the merger of key structural units
(Fig. 1B, C) present in the functionalized amino acid (FAA),
membrane potential; SAR, structure activity relationship; scMet, scMetrazol; TD50
,
lacosamide11
onamide, (R)-1) and the
((R)-N-benzyl
2-N-acetamido-3-methoxypropi-
neurological impairment (toxicity, 50%); TEA-Cl, tetraethylammonium chloride; V1/2
,
voltage of half-maximal (in)activation; VGSC, voltage-gated sodium channel.
a
-aminoamide (AAA), safinamide12,13
⇑
Corresponding authors.
http://dx.doi.org/10.1016/j.bmc.2015.04.014
0968-0896/Ó 2015 Published by Elsevier Ltd.
Please cite this article in press as: Torregrosa, R.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.04.014

2
R. Torregrosa et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
CH3
O
L
O
H
N
X
C
B
N
H
O
(R)-A
CH3
O
H
O
O
C
O
H
N
F
B
N
N
H
H2N
O
CH3
1
(S)-2 (safinamide)
(R)- (lacosamide)
Figure 1. Generation of chimeric class (R)-A from (R)-1 and (S)-2. Box B represents lacosamide ((R)-1) and Box C represents safinamide ((S)-2) derived component.
((S)-2-(40-((300-fluoro)benzyloxy)benzyl)aminopropionamide, (S)-
2). Lacosamide is a first-in-class ASD that is extensively used for
the treatment of partial-onset seizures in adults.14 Safinamide,
another anticonvulsant,15 has been advanced for the treatment of
Parkinson’s disease.16 Studies have shown that safinamide inhibits
monoamine oxidase type B,17 thus likely preventing dopamine
bioactivation in patients suffering from Parkinson’s disease. We
demonstrated that select (R)-A compounds displayed potent
anticonvulsant activities in the maximal electroshock18 (MES) and
the 6-Hz psychomotor19 seizure assays in rodents.8–10 The anticon-
vulsant activities of (R)-A have been attributed, in part, to their
actions on voltage-gated sodium channel (VGSC) properties.9,10,20
Chimeric compounds (R)-A potently transitioned VGSCs to the
slow-inactivated state and in some cases, affected fast inactivation
(R)-D derivative, exhibited pronounced anticonvulsant activity
in rodents.8 Accordingly, we restricted the site of substitution
on the terminal aromatic ring to the 300-position. The X-sub-
stituent ranged from electron-withdrawing to electron-donating
groups.
CH3
O
2"
3"
X
O
O
H
N
N
H
2
O
(R)-3; X = H
(R)-4; X = F
5
(R)-
;
X = Cl
(R)-6; X = CH3
7
(R)-
;
X = CF3
(R)-8; X = OCH3
processes and inhibited Na+ currents in
a frequency (use)-
9
;
(R)-
X = OCF3
dependent fashion. The experimental findings for slow inactivation
by (R)-A were also consistent with a mechanism in which the chi-
meric compounds block fast-inactivated channels with very slow
kinetics.21,22
2.2. Synthesis
Compounds (R)-3 and (R)-5–(R)-9 were prepared by a similar
An initial structure–activity relationship (SAR) study of (R)-A
showed excellent anticonvulsant activities for compounds in
which the two aryl units were in spatial proximity.8 Thus, we fur-
ther investigated the activities of (R)-A compounds wherein the
linker (L) was either a single bond9 or an oxymethylene (OCH2)10
group and found that the X-substituent in the terminal aromatic
ring influenced the cellular and whole animal pharmacological
activities. Here, we explore the (R)-A series that contain an oxy
(O)-linker to give (R)-D. We demonstrate in seizure models that
most X-substituents in (R)-D yielded compounds with excellent
anticonvulsant activities and minimal neurotoxicities, comparable
with many ASDs. Electrophysiology studies showed that (R)-D dis-
played sodium channel properties consistent with other members
of this general class of compounds.9,10,20
route (Scheme 1), using the mixed anhydride coupling (MAC)
method.23 Commercially available (R)-N-tert-butoxycarbonyl-
D-
serine ((R)-27) was coupled with the 4-(phenoxy)phenylmethami-
nes 21–26 using the MAC reagents isobutylchloroformate (IBCF)
and N-methylmorpholine (NMM) to give amides (R)-28–(R)-33,
respectively, without racemization of the C(2) chiral center. The
substituted 4-phenoxybenzylamines were prepared by treating
4-fluorobenzonitrile (10) with the appropriate aryl phenol
(11–15) using either K2CO3 or Cs2CO3 in DMF to give the corre-
sponding nitriles.24–27 Subsequent reduction of the nitrile group
in 16–20 with LiAlH4 afforded the amines 22–26. The correspond-
ing unsubstituted 4-phenoxybenzylamine (21) was commercially
available. Methylation (CH3I, Ag2O) of the serine hydroxyl group
in (R)-28–(R)-33 provided ethers (R)-34–(R)-39, respectively.
Deprotection of the tert-butoxycarbonyl group in (R)-34–(R)-39
with acid (HCl/dioxane) followed by acetylation (AcCl, Et3N) gave
the desired products (R)-3 and (R)-5–(R)-9, respectively. The
enantiomeric purities of (R)-3 and (R)-5–(R)-9 were assessed by
the detection of a single acetyl methyl peak and a single O-methyl
peak in the 1H NMR spectrum for each compound when a satu-
rated solution of (R)-(ꢀ)-mandelic acid was added.28
CH3
3"
O
O
X
O
H
N
N
H
O
(R)-D
We report, in the Section 5, the details (synthetic procedure,
characterization) of the final step for all compounds evaluated in
the seizure and cellular electrophysiology studies. In the
Supporting information, we provide the experimental procedures
and the physical and full spectroscopic properties for all the syn-
thetic compounds prepared in this study.
2. Results
2.1. Selection of compounds
We prepared compounds (R)-3 and (R)-5–(R)-9 where an
oxygen was the linker (L) between the two aromatic rings.
Our initial SAR study documented that (R)-4, the 300-fluorine
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R. Torregrosa et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
3
X
X
NH2
1
N
2
2
3'
3'
1
NC
3
4'
3
2'
OH
2'
4'
K2CO3 or Cs2CO3
DMF
LiAlH4
THF
4
4
+
2
O
O
X
3
F
21; X = H
22
10
11
16; X = Cl
17
;
X = Cl
12; X = CH3
13
;
X = Cl
23; X = CH3
24
;
X = CH3
18; X = CF3
19
; X = OCH3
;
X = CF3
;
X = CF3
14; X = OCH3
15; X = OCF3
25; X = OCH3
20; X = OCF3
26
;
X = OCF3
X
NH2
O
R
O
CH3
OH
OH
2"
3"
2"
21-26
O
X
1. H+
O
3"
X
O
H
O
O
O
H
N
2
O
N
H
IBCF, NMM, THF
N
2. AcCl, Et3N, DCM
2
O
N
H
2
N
O
H
O
O
27
(R)-
3
28
; R = H, X = H
(R)-
;
X = H
(R)-
(R)-5; X = Cl
(R)-29; R = H, X = Cl
(R)-30; R = H, X = CH3
31
(R)-6; X = CH3
7
(R)-
;
X = CF3
(R)-
(R)-32; R = H, X = OCH3
33
; R = H, X = CF3
(R)-8; X = OCH3
9
;
(R)-
X = OCF3
(R)-
; R = H, X = OCF3
Ag2O, CH3CN
CH3I
(R)-34; R = CH3, X = H
35
; R = CH3, X = Cl
(R)-
(R)-36; R = CH3, X = CH3
37
(R)-
(R)-38; R = CH3, X = OCH3
39
; R = CH3, X = CF3
(R)-
; R = CH3, X = OCF3
Scheme 1. Synthesis of (R)-3 and (R)-5–(R)-9.
2.3. Pharmacological evaluation
to (R)-111 and phenytoin31,32 (PI: (R)-4, 4.2; (R)-5, 5.0; (R)-6, 4.6;
(R)-8, 6.5; (R)-9, 6.2; (R)-1, 6.0; phenytoin, 6.9). In rats (po), excel-
lent activities were observed for (R)-5 and (R)-9 (ED50 (mg/kg): (R)-
5, 12; (R)-9, 8.3). The PI values for these compounds were high but
were lower than (R)-1 and phenytoin (PI: (R)-5, 18; (R)-9, 29; (R)-1,
>130; phenytoin, >36). In rats (ip), (R)-5, (R)-6, (R)-8, and (R)-9 dis-
played potent activities (ED50 values: 5.7–15 mg/kg) and low neu-
rotoxicities (TD50 values: 49–140 mg/kg) giving PI values that
ranged from 8.0 to 12, values higher than phenytoin32 (PI: (R)-5,
8.3; (R)-6, 12; (R)-8, 8.0; (R)-9, 9.5; phenytoin, 6.3). Finally, we
evaluated (R)-3–(R)-9 in the 6 Hz (32 mA) psychomotor seizure
test and found that (R)-4, (R)-5, and (R)-9 were the most active
compounds (ED50 (mg/kg): (R)-4, ꢁ10; (R)-5, 20; (R)-9, 15). By
comparison, the 6 Hz ED50 value for (R)-1 was 10 mg/kg.30
2.3.1. Whole animal pharmacological activity
Compounds (R)-3 and (R)-5–(R)-9 were tested for anticonvul-
sant activity at the Anticonvulsant Screening Program (ASP) of
the National Institute of Neurological Disorders and Stroke
(NINDS), U.S. National Institutes of Health, using the procedures
described by Stables and Kupferberg.29 The anticonvulsant data
from the MES model18 (mice, intraperitoneally (ip); rat, ip; rat,
orally (po)), and the psychomotor 6 Hz (32 mA) seizure test for
therapy-resistant limbic seizures19 (mice, ip) are summarized in
Table 1 along with similar results (where available) obtained for
(R)-1,11,30 (S)-2,15 and (R)-4,8 and the ASDs phenytoin,31,32 pheno-
barbital,31 and valproate.31 For compounds that showed significant
activity, we report the 50% effective dose (ED50) values from quan-
titative screening evaluations. We also include the median doses
for 50% neurological impairment (TD50) in mice, using the rotorod
test,33 and the behavioral toxicity effects observed in rats.34 The
protective index (PI = TD50/ED50) for the test compounds is listed,
where possible. Compounds (R)-4–(R)-6, (R)-8 and (R)-9 were eval-
uated in the subcutaneous Metrazol (scMet) seizure model,35 and
no activity was observed below 40 mg/kg (data not shown).
Similarly, we found no activity in the scMet model for (R)-111
and structurally related compounds.8–10,36
Compounds (R)-3–(R)-9 displayed excellent activities in the
MES model in mice when administered ip and in rats when admin-
istered either ip or po. For most compounds, we observed in mice
(ip) ED50 values that ranged from 5.5 to 14 mg/kg, values that were
comparable with the ASDs (R)-111 and phenytoin31,32 (ED50 (mg/
kg): (R)-3, >10, <30; (R)-4, 5.5; (R)-5, 9.4; (R)-6, 14; (R)-7, ꢁ10;
(R)-8, 13; (R)-9; 6.5; (R)-1, 4.5; phenytoin, 9.5). Furthermore, we
found that many of the compounds exhibited low neurotoxicities
in the rotorod test in mice (ip), thus affording high PI values similar
2.3.2. Whole-cell, patch-clamp electrophysiological activity
The well-described mechanisms of action by lacosamide and
safinamide, parents of (R)-D series compounds, both involve inhi-
bition of VGSCs.14,15,37,38 Promising anticonvulsant activities of
(R)-3–(R)-9 therefore prompted our examination of the VGSC activ-
ities in rat embryonic cortical neurons9,10,39 by whole-cell, patch-
clamp electrophysiology. These neurons typically express central
nervous system (CNS) sodium channel isoforms NaV1.1, NaV1.2,
NaV1.3, and NaV1.6.40 Kinetic properties of slow inactivation, fre-
quency (use)-dependence, steady-state activation, and fast inacti-
vation of Na+ currents were measured in the presence of (R)-3–
(R)-9. The cortical neurons were grown for 7–10 days in vitro and
then examined using protocols described earlier.9,10,39
Compounds (R)-3–(R)-9 were tested only at 10 lM due to con-
straints of cortical neuron viability during the course of the
patch-clamp experiments. Here, we did not separate the exact con-
tribution of the four Nav isoforms in the presence of (R)-3–(R)-9
because of the lack of subtype-specific blockers of these Nav
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R. Torregrosa et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
Table 1
Structure–activity relationship for chimeric compounds (R)-Da
CH3
O
O
X
O
H
N
N
H
O
Compd No.
X
Miceb (ip)
6 Hzd
Ratg (po)
Ratb (ip)
MESc
ED50
Toxe
TD50
PIf
MESc
ED50
Toxh
TD50
PIf
MESc
ED50
Toxh
TD50
PIf
ED50
(mg/kg)
(mg/kg)
(mg/kg)
(mg/kg)
(mg/kg)
(mg/kg)
(mg/kg)
(R)-3
(R)-4j
(R)-5
H
F
>10, <30
[0.5–2.0]
5.5 [0.25]
(3.2–6.3)
9.4 [0.5]
(7.6–11)
>10, <30
[0.5]
>30, <100
[0.5]
23 [0.25]
(18–28)
47 [0.25]
(42–51)
—
NDi
NDi
—
NDi
NDi
—
ꢁ10
4.2
5.0
<10
>10
—
NDi
NDi
—
[0.25–0.5]
20 [0.25]
(15–25)
[0.25–4.0]
12 [1.0]
(6.9–18)
[1.0–2.0]
<30
[0.5]
<30
[0.5–4.0]
ꢁ30
[0.25–4.0]
210 [0.5]
(200-250)
Cl
18
5.9 [0.5]
(3.7–7.9)
49 [0.5]
(34–61)
8.3
(R)-6
CH3
14 [0.25]
(11–17)
ꢁ10
ꢁ100
65 [0.25]
(57–73)
>100
4.6
—
>30
[0.25–4.0]
>30
[0.25–4.0]
>30
[0.25–4.0]
240 [1.0]
(160–390)
>500
—
12 [0.25]
(10–13)
NDi
140 [0.25]
(90–190)
NDi
12
—
[0.5]
(R)-7
CF3
>10, <100
[0.5]
>30, <100
[0.5]
15 [0.25]
(9.1–22)
10 [0.5]
(7.8–13)
—
[0.5]
[0.5]
(R)-8
OCH3
OCF3
13 [0.25]
(12–14)
6.5 [0.25]
(5.1–8.2)
4.5 [0.5]
(3.7–5.5)
4.1
85 [0.25]
(75–100)
40 [0.25]
(36–45)
27 [0.25]
(26–28)
6.5
6.2
6.0
—
15 [0.25]
8.0–21)
5.7 [2.0]
(3.6–7.8)
NAm
120 [1]
(86–150)
54 [2.0]
(40–66)
NAm
8.0
9.5
—
[0.25–1.0]
8.3 [2.0]
(4.9–11)
3.9 [2.0]
(2.6–6.2)
12
(R)-9
29
>130
(R)-1k,l
[0.5]
(S)-2n
NAm
NAm
—
(3.0–5.5)
9.5 [2.0]
(8.1–10)
22 [1.0]
(15–23)
270 [0.25]
(250–340)
(10–14)
28
Phenytoino,p
Phenobarbitalo
Valproateo
66 [2.0]
(53–72)
69 [0.5]
(63–73)
430 [0.25]
(370–450)
6.9
3.2
1.6
>1000
>36
6.7
0.6
2.4
15
6.3
—
(21–35)
9.1 [5.0]
(7.6–12)
490 [0.5]
(350–730)
(1.4–3.7)
(12–19)
61 [0.5]
(44–96)
280 [0.5]
(190–350)
NAm
NAm
NAm
NAm
—
a
b
c
The compounds were tested through the NINDS ASP.
The compounds were administered intraperitoneally. ED50 and TD50 values are in milligrams per kilogram.
MES = maximal electroshock seizure test. Numbers in parentheses are 95% confidence intervals. A dose–response curve was generated for all compounds that displayed
sufficient activity. The dose–effect for these compounds was obtained at the ‘time of peak effect’ (indicated in hours in the brackets).
d
6 Hz = 6 Hz (32 mA) psychomotor seizure test.
TD50 value determined from the rotorod test.
PI = protective index (TD50/ED50).
e
f
g
The compounds were administered orally. 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 for these compounds was obtained at the ‘time of peak effect’ (indicated in hours in
the brackets).
h
Tox = behavioral toxicity.
ND = not determined.
Ref. 8.
Ref. 11.
Ref. 30.
NA = data not available.
Ref. 15.
Ref. 31.
i
j
k
l
m
n
o
p
Ref. 32.
isoforms, and the possible interactions between said blockers of
various NaV channels and the (R)-D compounds themselves.
Thus, we assumed contributions from all four CNS NaV isoforms.
The excellent anticonvulsant activity observed for (R)-9 led us to
examine this compound further in both catecholamine A-differen-
tiated (CAD) cells41 that express predominantly Nav1.7 and in
recombinant HEK293 cells that express hNaV1.1, rNaV1.3,
hNaV1.5, or hNaV1.7 channels.9
to ꢀ70 mV before the extent of slow inactivation was examined by
a test pulse to ꢀ10 mV, for 20 ms (Fig. 2A, left). Representative
traces illustrating the extent of slow inactivation observed at
ꢀ50 mV compared to the prepulse at ꢀ100 mV in the absence
(control) or presence of (R)-5 and (R)-8 are shown in Figure 2A
(right), with the full slow inactivation curve (normalized peak ver-
sus prepulse potential) of (R)-5 shown in Figure 2B. At 10 lM, (R)-5
facilitated the transition of a majority of sodium channels into the
slow-inactivated state compared with control (0.1% DMSO)-treated
neurons. Slow inactivation at ꢀ50 mV was chosen as a point of
comparison between compounds due to the physiological rele-
vance of this voltage near the resting membrane potential and
action potential firing threshold of neurons. At this voltage, VGSC
activation and inactivation kinetics mediate channel availability
that determines if sustained firing, akin to that during an epileptic
2.3.2.1. Rat embryonic cortical neurons.
First, we tested the
ability of 10 M (R)-3–(R)-9 to modulate the transition of VGSCs to
l
a slow-inactivated state. Cortical cells were conditioned to poten-
tials ranging from ꢀ100 mV to +20 mV (in +10 mV increments)
for 5 s.9,39,42 Channels that underwent fast inactivation during this
conditioning pulse were then allowed to recover during a 1 s pulse
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R. Torregrosa et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
5
Figure 2. Effects of (R)-3–(R)-9 on steady-state slow inactivation state of Na+ currents in embryonic cortical neurons. (A) Voltage protocol for slow inactivation. Currents were
evoked by 5 s prepulses between ꢀ100 mV and +20 mV (in 10 mV increments), and then fast-inactivated channels were allowed to recover for 1000 ms at a hyperpolarized
pulse to ꢀ70 mV before testing for the fraction of available channels for 20 ms at ꢀ10 mV. The fraction of channels available at ꢀ10 mV was analyzed. Representative current
traces from cortical neurons in the absence (control, 0.1% DMSO) or presence of 10 lM (R)-5 or (R)-8 are illustrated. The black and dashed traces represent the currents evoked
at ꢀ100 and ꢀ50 mV, respectively (also highlighted in the voltage protocol as a dashed line). (B) Summary of steady-state slow activation curves for neurons treated with
DMSO (control) or 10
M (R)-5. Significant drug-induced slow inactivation was evident at voltages more depolarizing than ꢀ80 mV in neurons treated with (R)-5. (C)
M of the indicated compounds. Asterisks (⁄) indicate statistically
l
Summary of the fraction of current available at ꢀ50 mV for neurons treated with 0.1% DMSO (control) or 10
l
significant differences in fraction of current available between control and the indicated compounds (p <0. 05, Student’s t-test). Data are from 5 to 8 cells per condition.
event, is possible.43–47 At ꢀ50 mV, a small fraction (i.e., 0.16 0.05
n = 5; calculated as 1 minus the normalized INa) of the channels had
entered a slow-inactivated conformational state(s) in control-trea-
ted cells (Fig. 2B and C). Compared with the control, (R)-3–(R)-9
caused a significant decrease in the maximal fraction of current
that is available by depolarization, with maximal induction
observed in the presence of (R)-3 (0.82 0.03, n = 5) (p <0.01,
Mann–Whitney U test).
We next interrogated if (R)-3–(R)-9 could enhance steady-state
fast inactivation. For these studies we used a similar protocol
(Fig. 3, top left) designed to induce a fast-inactivated state.9 Cells
were subjected to inactivating prepulse potentials ranging from
ꢀ100 to ꢀ10 mV (in 10-mV increments) for 500 ms prior to a
0 mV test pulse for 20 ms to estimate the extent of fast inactiva-
tion. The 500 ms conditioning pulse allows for examination of
the linear range of fast inactivation curves for endogenous chan-
nels at assayed potentials. Steady-state, fast inactivation curves
of Na+ currents from control (0.1% DMSO)- and (R)-3–(R)-9–treated
cortical neurons were well fitted with a single Boltzmann function
(R2 >0.963 for all conditions). The value of voltage of half-maximal
inactivation (V1/2) for 0.1% DMSO-treated cells was ꢀ53.4 1.5 mV
(n = 4), which was not significantly different from that observed for
(R)-3–(R)-9 (p >0.05 vs control; Student’s t-test; Fig. 3).
Next, we tested whether (R)-3–(R)-9 could alter the voltage-de-
pendence of activation for cortical neuron VGSCs. Activation
changes for cortical neurons treated with compounds were mea-
sured by whole-cell ionic conductances in response to changes in
command voltage (Fig. 3, top right) and analyzed by comparing
Boltzmann properties of half maximal activation (V1/2) and slope
factors (k).9,39 Boltzmann fits for 0.1% DMSO (control) and (R)-3–
(R)-9 are shown in Figure 3. The V1/2 value for steady-state activa-
tion for 0.1% DMSO-treated (control) neurons was ꢀ27.1 1.3 mV
(n = 5), which was significantly different from that of (R)-7
(ꢀ35.6 4.1 mV; n = 5) and (R)-8 (ꢀ36.8 1.0 mV; n = 6) (p <0.05
vs control; Student’s t-test; Fig. 3) but not for (R)-3–(R)-6 and (R)-9.
Figure 3. Effects of (R)-3–(R)-9 on fast inactivation and steady-state activation
states of Na+ currents in embryonic cortical neurons. Voltage protocol for fast
inactivation (top left) and activation (top right). Representative Boltzmann fits for
steady-state fast inactivation and activation for cortical neurons treated with 0.1%
DMSO (control, bolded curves) and the indicated compounds are shown. Values for
V1/2, the voltage of half-maximal inactivation and activation 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 steady-state
inactivation and activation, respectively. No significant differences were observed
between control and fast inactivation or activation other than for (R)-7 and (R)-8 (*)
for any of the conditions tested (p >0.05, one-way ANOVA). Data are from 5 to 7
cells per condition.
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6
R. Torregrosa et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
Finally, we tested if (R)-3–(R)-9 could elicit frequency (use)-de-
concentration response curves for slow inactivation induction at
ꢀ50 mV as well as at +20 mV; the IC50 inactivation values are
shown in Figure 5A (boxes). Compared with our recently reported
pendent blockage of Na+ currents. The ability to block Na+ currents
in an activity- or use-dependent manner is a useful property for
ASDs since it allows for preferential decreases in sodium channel
availability during high- (i.e., seizures) but not low-frequency fir-
ing.48 Thirty, identical test pulses were applied at 10 Hz
(Fig. 4A).9,39 The difference in available current was calculated by
dividing the peak current at any given pulse (pulseN) by the peak
current in response to the initial pulse (pulse1). Representative cur-
IC50 values of 85 and 13
l
M for slow inactivation induced by (R)-
19,20 and (S)-2,10 the IC50 value for (R)-9 (0.70
lM) was ꢁ121-fold
and ꢁ19-fold lower, respectively, at ꢀ50 mV. Similar differences in
the IC50 values were measured at +20 mV. Next, we tested for the
effects of (R)-9 on fast inactivation. We used a protocol tailored
to induce a fast-inactivated state, as previously described.9,10,20
Cells were subjected to prepulse potentials ranging from ꢀ120 to
ꢀ10 mV in 10 mV increments for 500 ms. A 0 mV test pulse for
20 ms measures the available current. The 500 ms conditioning
pulse allows for examination of the linear range of fast inactivation
curves for endogenous channels at assayed potentials. As illus-
trated in Figure 5B, steady-state, fast inactivation curves of Na+
currents from control (0.1% DMSO) and CAD neurons treated with
various concentrations of (R)-9 were well fitted with a single
Boltzmann function (R2 >0.979 for all conditions). The V1/2 value
for inactivation for 0.1% DMSO (control)-treated cells was
ꢀ69.1 3.6 mV (n = 7), which was significantly different from that
rents for this protocol are shown for control and 10 lM (R)-7-trea-
ted cells (Fig. 4B). None of the tested compounds exhibited
statistically relevant frequency (use)-dependent inhibition of Na+
currents (Fig. 4C).
The anticonvulsant activity for (R)-9 led us to further explore
the electrophysiological properties of this compound in CAD and
HEK293 recombinant cells, which express a distinct population of
NaV channels.
2.3.2.2. CAD cells.
CAD cells express endogenous tetrodo-
toxin-sensitive Na+ currents that display rapid activation and inac-
tivation kinetics upon membrane depolarization39 and are likely
composed mostly of NaV1.7 channels with minor contributions
by NaV1.1, NaV1.3, and NaV1.9 channels.9,42,49 We showed that
the sodium channel properties of (R)-1 in CAD cells49 are similar
to those in cultured neurons and in mouse N1E-115 neuroblastoma
cells.50 CAD cells were easily recorded from and cultured thus
allowing us to conveniently determine (R)-9 sodium channel slow
inactivation, frequency (use)-dependence, steady-state activation,
and fast inactivation properties over a range of concentrations.
These properties were quantitatively compared with other (R)-A
compounds, (R)-1, and (S)-2. Compound (R)-9 promoted VGSC
transition to a slow-inactivated state in a concentration-dependent
manner; almost complete slow inactivation was observed at the
highest concentration (Fig. 5A). To determine the comparative
level to which (R)-9 induced slow inactivation, we calculated
of (R)-9 (10
lM)-treated cells (ꢀ84.6 1.6 mV; n = 6; p<0.05;
Student’s t-test; Fig. 5B). Because changes in Na+ current ampli-
tudes could be due to alterations in channel gating,51 we tested if
(R)-9 altered the voltage-dependent activation properties of Na+
currents. Activation changes for CAD cells treated with (R)-9 were
measured by whole-cell ionic conductances in response to changes
in command voltage and analyzed by comparing Boltzmann prop-
erties of midpoints (V1/2) and slope factors (k). We found that VGSC
steady-state activation properties were no different between 0.1%
DMSO (control; V1/2 for activation = ꢀ19.5 2.8 mV (n = 8)) and
any concentration tested for (R)-9 (Fig. 5B). Finally, we tested if
(R)-9 could confer frequency (use)-dependent Na+ current block.
Thirty, identical test pulses were applied at 10 Hz as described pre-
viously.9,10,20 The available current in control cells (0.1% DMSO)
and treated cells (various concentrations of (R)-9) was calculated
by dividing the peak current at any given pulse (pulseN) by the
peak current in response to the initial pulse (pulse1). Compound
(R)-9 exhibited frequency (use)-dependent inhibition of Na+ cur-
rents (Fig. 5C), and by the last pulse, compared with control
(0.1% DMSO), the peak current was ꢁ39% lower in the presence
of 10 lM (R)-9. When we tested 30 lM (R)-10 in a similar protocol,
we observed ꢁ44% inhibition in Na+ currents (data not shown).
2.3.2.3. HEK293 recombinant cells.
had effects on various sodium channel isoforms, we examined
the effects of 10 M (R)-9 on slow inactivation, fast inactivation,
In order to test if (R)-9
l
steady-state activation, and frequency (use)-dependence in
HEK293 cells that stably expressed CNS (hNaV1.1, rNaV1.3), periph-
eral nervous system (hNaV1.7), and cardiac (hNaV1.5) channels
using voltage protocols9 illustrated in Figure 6A and K. The results
of these experiments are summarized in Table 2. Notably, (R)-9
exhibited similar, though not identical, effects on these biophysical
properties irrespective of sodium channels, indicating that this
compound exhibited little isoform specificity. We observed that
(R)-9 robustly transitioned the four sodium channel subtypes to
the slow-inactivated state; that (R)-9 shifted the V1/2 of fast inacti-
vation for NaV1.7 by ꢁ20.2 mV in the hyperpolarizing direction
without affecting V1/2 values for any of the other channel isoforms;
and that (R)-9 had no effect on steady-state activation or on fre-
quency (use)-inhibition of Na+ currents carried by any of the
NaV1.x channels.
Figure 4. Effects of (R)-3–(R)-9 on frequency (use)-dependent block of Na+ currents
in embryonic cortical neurons. (A) The frequency (use)-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. (B) Representative overlaid traces
are illustrated by pulses 1 and 30 for control (0.1% DMSO) and in the presence of
(R)-7 (10
l
M). C. Summary of the maximal decrement in current amplitude
2.3.3. Additional studies
observed at the end of the 30-pulse train for control or 10
lM of the indicated
Among the (R)-D compounds, (R)-9 exhibited the most attrac-
tive anticonvulsant profile and potently inhibited sodium channel
function. Accordingly, we conducted additional tests on this
compounds. None of the compounds exhibited any degree of frequency (use)-
dependence (p >0.05, one-way ANOVA with Dunnett’s post-hoc test). Data are from
4 to 6 cells per condition.
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R. Torregrosa et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
7
Figure 5. Effects of (R)-9 on electrophysiological properties of Na+ currents in CAD cells. (A) Summary of steady-state slow activation curves for CAD cells treated with 0.1%
DMSO (control) or the indicated concentrations of (R)-9. Significant (R)-9-induced slow inactivation was evident; the starting voltages at which the extent of slow inactivation
was significantly different from control are indicated by asterisks (⁄p <0.05, Student’s t-test versus control). (B) Representative Boltzmann fits for steady-state fast inactivation
and activation for CAD cells treated with 0.1% DMSO (control) and various concentrations of (R)-9 are shown. Values for V1/2, the voltage of half-maximal inactivation and
activation 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 steady-state inactivation and activation, respectively. Statistically significant differences between control and fast inactivation for 10
lM (R)-9 are indicated by
the asterisk (⁄p <0.05, one-way ANOVA). (C) Summary of the maximal decrement in current amplitude observed at the end of the 30-pulse train for control or 1, 10, or 30
lM
of (R)-9. The two highest concentrations of (R)-9 induced significant frequency (use)-dependence compared to control (p >0.05, one-way ANOVA with Dunnett’s post-hoc
test). Data are from 4 to 10 cells per condition as indicated.
chimeric compound. The observed anticonvulsant activity for (R)-9
was further supported by its activities in the psychomotor 6 Hz
test (44 mA) for therapy-resistant limbic seizures19 (mouse, ip;
ED50 = 44 mg/kg at 0.5 h), the corneal kindled seizure model52
(mouse, ip; ED50 = 41 mg/kg at 0.25 h), and the rat hippocampal
kindled seizure test for partial complex seizures or temporal lobe
seizures53,54 (rat, ip; ED50 = 22 mg/kg). Significantly, the 6 Hz
(44 mA) test is a model for pharmacoresistant seizures,19,55 and
the mouse corneal kindling and the rat hippocampal tests are con-
sidered to be predicative models for partial-onset and partial com-
plex seizures.52
3. Discussion
Many of the (R)-D chimeric compounds exhibited excellent
anticonvulsant activities in the MES model (mice, ip), comparable
with the parent compounds (R)-111,30 and (S)-215 (ED50 (mg/kg):
(R)-3, >10, <30; (R)-4, 5.5; (R)-5, 9.4; (R)-6, 14; (R)-7, ꢁ10; (R)-8,
13; (R)-9, 6.5; (R)-1, 4.5; (S)-2, 4.1). Furthermore, we observed only
modest differences in seizure protection when the X-substituent in
(R)-D was varied from electron-withdrawing to electron-donating.
Earlier studies on (R)-A compounds where the linker (L) was either
a single bond9 ((R)-E) or an oxymethylene group10 ((R)-F) showed
that the electron-withdrawing, trifluoromethoxy (OCF3) group pro-
vided the greatest protection in the seizure models. For (R)-D, we
found the most active compounds to be the 300-F ((R)-4) and the
300-OCF3 ((R)-9) derivatives (MES ED50 (mice, ip, mg/kg): (R)-4,
5.5; (R)-9, 6.5; MES ED50 (rat, po, mg/kg): (R)-4, <10; (R)-9, 8.3);
however, the differences in their activities from the other com-
pounds in the series were not great.
Next, we evaluated (R)-9 (10 lM) against a panel of seven
human CYP450 enzymes and observed little or no direct inhibition
for CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and
CYP3A4/5 (data not shown). We found little or no time-dependent
inhibition of these enzymes other than for CYP2C8 by (R)-9, for
which there was an ꢁ16% inhibition increase after a 30-min prein-
cubation period. We also determined the pharmacokinetic proper-
ties for (R)-9 in Sprague-Dawley rats (iv, po). Using a single iv dose
of 5 mg/kg and an oral dose of 20 mg/kg, (R)-9 showed excellent
bioavailability (92%) and where the t1/2 (iv) value was 1.75 h, the
t1/2 (po) value was 4.63 h, and the brain/plasma ratio was 1.2:1
at 6.0 h.
X
X
CH3
O
CH3
O
O
O
H
O
H
N
N
N
H
N
H
Compound (R)-9 was evaluated at UNC’s NIMH Psychoactive
O
O
Drug Screening Program (PDSP)56 against
a
battery of 43
receptors known to adversely impact drug effectiveness. No sig-
nificant binding was observed at 10 M. Moreover, (R)-9 did not
affect hERG K+ channel activity (PatchExpress) at 30
M (data
F
(R)-
E
(R)-
For the (R)-E and (R)-F chimeric compounds, we determined their
slow inactivation IC50 values in CAD cells.9,10 Accordingly, we mea-
sured the IC50 value for (R)-9 in CAD cells to gauge the relative
potency of this series with the two earlier sets of compounds.
We found the (R)-9 slow inactivation IC50 (ꢀ50 mV) value to be
l
l
not shown). Finally, we conducted a non-GLP 7-day repeated
dose toxicity study for (R)-9 in Sprague-Dawley female rats upon
oral (gavage) administration at 20, 60, and 180 mg/kg/day dose
levels. The no observed adverse effect level (NOAEL) for (R)-9
was 60 mg/kg/day, providing a NOAEL/MES ED50 value of ꢁ10.
At higher dose levels, effects were observed in the ability to bear
weight, coordination in gait, respiratory rate, relative organ
weight (e.g., increase in liver, decrease in spleen and thymus),
clinical chemistry parameters, and gross pathology (data not
shown).
0.70 lM (Fig. 5), which is comparable to the values for the corre-
sponding (R)-E and (R)-F derivatives, (R)-40 and (R)-41, respec-
tively (IC50
M): (R)-9, 0.70; (R)-40, 0.85;9 (R)-41, 0.2410).
Similarly, we found that (R)-9 in CAD cells, like (R)-409 and (R)-
41,10 inhibited Na+ currents in
frequency (use)-dependent
manner, but unlike these two, we observed that (R)-9 affected fast
inactivation at the highest concentration (10 M) tested (Fig. 5).
(
l
a
l
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8
R. Torregrosa et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
Figure 6. Analysis of (R)-9 on electrophysiological properties of NaV1.1, NaV1.3, NaV1.7, and NaV1.5 currents in HEK293 cells. (A and K) Voltage protocols for examining slow
inactivation, fast inactivation, steady-state activation and frequency (use)-dependent block. NaV1.5 uses hyperpolarized protocols due to differences in the hyperpolarized
activation typical for this isoform. (B, E, H and L) Summary of steady-state slow activation curves for HEK293 cells treated with 0.1% DMSO (control) or 10
illustrate representative current traces from HEK293 cells in the absence (control, 0.1% DMSO) or presence of 10
M (R)-9. Traces represent the currents evoked at ꢀ120
(black or green) and ꢀ50 mV (red, purple, orange, or cyan). (C, F, I and M) Representative Boltzmann fits for steady-state fast inactivation and activation for HEK293 cells
treated with 0.1% DMSO (control) or 10 M of (R)-9. Values for V1/2, the voltage of half-maximal inactivation and activation, and the slope factors (k) were derived from
lM (R)-9. Insets
l
l
Boltzmann distribution fits to the individual recordings and were averaged to determine the mean ( SEM) voltage dependence of steady-state inactivation and activation,
respectively. (R)-9 significantly shifted the V1/2 of fast inactivation for NaV1.7 by ꢁ20.2 mV in the hyperpolarizing direction (⁄p <0.05, Student’s t-test versus control) while the
V
1/2s for NaV1.1, NaV1.3, and NaV1.5 were not affected. (D, G, J and N) Summary of average frequency (use)-dependent decrease in current amplitude over time ( SEM)
produced by control (0.1% DMSO) or 10 lM (R)-9. Data are from 5 to 6 cells per condition.
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R. Torregrosa et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
9
Table 2
Comparative current densities, cell capacitances, and Boltzmann parameters of voltage-dependence of channel activation and steady-state fast inactivation curves for control- and
(R)-N-40-((300-trifluoromethoxy)phenoxy)benzyl 2-acetamido-3-methoxypropionamide ((R)-9)-treated HEK293 cells expressing NaV1.1, NaV1.3, NaV1.7 or NaV1.5 channels
Condition
Extent of slow inactivation
Voltage-dependence of activationb
Voltage-dependence of fast inactivation
Use-dependent inhibitionc
(at ꢀ50 mV)a
V1/2 (mV)
Slope (mV/e-fold)
V1/2 (mV)
Slope (mV/e-fold)
NaV1.1
Control
10 lM (R)-9
0.19 0.04 (5)
0.87 0.04 (6)⁄
ꢀ24.69 1.52 (5)
ꢀ24.43 2.94 (6)
4.22 0.60 (5)
4.64 1.78 (5)
ꢀ64.11 3.63 (6)
ꢀ76.72 4.11 (5)
5.93 1.48 (6)
6.89 2.11 (6)
0.98 0.02 (5)
0.85 0.17 (6)
NaV1.3
Control
10 lM (R)-9
0.04 0.02 (5)
0.78 0.04 (5)⁄
ꢀ29.62 3.59 (5)
ꢀ32.48 5.48 (4)
4.17 1.97 (5)
4.47 1.46 (4)
ꢀ65.44 5.62 (5)
ꢀ72.84 4.11 (5)
5.51 1.12 (5)
7.19 2.19 (5)
1.12 0.19 (5)
1.23 0.18 (5)
NaV1.7
Control
10 lM (R)-9
0.06 0.03 (5)
0.87 0.04 (5)⁄
ꢀ27.13 4.01 (5)
ꢀ24.98 4.22 (5)
3.81 1.24 (5)
4.01 1.43 (5)
ꢀ61.11 3.02 (5)
ꢀ81.35 6.70 (5)⁄
5.13 1.22 (5)
5.52 1.68 (5)
1.03 0.09 (5)
0.82 0.22 (5)
NaV1.5
Control
10 lM (R)-9
0.07 0.03 (5)
0.81 0.04 (5)⁄
ꢀ55.71 3.81 (5)
ꢀ48.99 4.79 (5)
3.26 1.75 (5)
3.39 1.69 (7)
ꢀ96.54 1.64 (5)
ꢀ95.36 2.43 (5)
5.34 0.86 (4)
6.12 0.72 (7)
1.05 0.15 (5)
0.92 0.14 (5)
a
The extent of slow inactivation was calculated as 1 minus the normalized peak INa and denotes the fraction of channels that have transitioned to a non-conducting (slow-
inactivated) state at ꢀ50 mV.
b
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).
c
Fraction of current remaining at the end of the 30-pulse train, normalized to the first pulse. N values are indicated in parentheses. The protocols used for these parameters
are illustrated in Figure 6. Asterisks represent statistically significant differences as compared to control within each tested group (p <0.05, Student’s t-test).
These overall results suggest that the composition and size of the
different linkers in (R)-D–(R)-F did not appreciably affect the inter-
action of the chimeric compounds with the VGSCs.
MES seizure model in mice (ip) (ED50 (mg/kg): (R)-9, 6.5; (R)-1,
4.5). A similar lack of correspondence for the CAD slow inactivation
IC50 values and their in vivo seizure protection activities in the MES
test (mice, ip) were observed for the (R)-E and the (R)-F com-
pounds with (R)-1 (CAD cells slow inactivation IC50 (ꢀ50 mV,
OCF3
OCF3
CH3
O
CH3
O
lM): (R)-40, 0.85; (R)-41, 0.24; (R)-1, 85; ED50 (mg/kg): (R)-40,
O
4.7; (R)-41, 12; (R)-1, 4.5).9,10 We tentatively attribute this finding
to the multiple factors that contribute to whole animal pharmaco-
logical activity, the sodium channel composition in CAD cells ver-
sus those in the CNS, the possibility that additional, unidentified
pathways exist for seizure control, and the recognition that
patch-clamp electrophysiology studies do not recapitulate condi-
tions created in the seizure models.
O
H
O
H
N
N
N
H
N
H
O
O
41
(R)-
40
(R)-
Our finding that (R)-9 promoted sodium channel slow inactivation
in rat embryonic cortical neurons that express NaV1.1, NaV1.2,
NaV1.3, and NaV1.6 channels36 and in CAD cells that largely express
NaV1.7 channels9,42,49 suggested that this compound showed little
NaV channel subtype selectivity. We explored this by testing (R)-9
in HEK293 recombinant cells9 that expressed hNaV1.1, rNaV1.3,
hNaV1.5, or hNaV1.7 (Fig. 6). In agreement with this notion, (R)-9
4. Conclusion
Many of the chimeric (R)-D compounds displayed potent anti-
convulsant activities and high PI values, which were similar to
ASDs, in the MES test when administered to rodents. The chimeric
compounds’ anticonvulsant activities likely stemmed, at least in
part, from their ability to promote sodium channel slow inactiva-
tion. VGSCs are responsible for the initiation of and the sustained
activity of neuronal signaling during seizure events. The well
described mechanism of action for both (R)-D parent compounds,
lacosamide and safinamide, involve inhibition of these chan-
nels.14,15,37,38 Phenytoin, lamotrigine, and carbamazepine are
among other ASDs that have primary activity on VGSCs to restrict
neuronal firing during seizures.57 As evident by perturbation of
VGSC slow inactivation (Fig. 2), the class (R)-D compounds
potently shifted VGSC availability, which readily explains anticon-
vulsant activity. The type of channel block produced by these com-
pounds is also a desirable characteristic for ASDs. By selectively
affecting sodium channel slow inactivation or frequency (use)-de-
pendent block, these compounds can promote channel inhibition
in response to sustained use (i.e., epileptic activity).
(10 lM) potently transitioned the sodium channels to the slow-in-
activated state regardless of the NaV channel isoform. In the recom-
binant cells, we observed that the effect of (R)-9 was different on
sodium channel fast inactivation and frequency (use)-dependent
inhibition. A similar result was previously reported for a (R)-E com-
pound where X = Cl.9
The anticonvulsant activities for the (R)-D compounds are
attributed, in part, to their ability to modulate VGSC activities.
Using rat embryonic cortical neurons that express NaV1.1,
NaV1.2, NaV1.3, and NaV1.6 channels,40 we learned that the (R)-D
compounds uniformly promoted slow inactivation (Fig. 2). At
10 l
M, the 300-unsubstituted derivative (R)-3 was the most potent,
followed by the 300-electron-withdrawing derivatives (R)-4, (R)-5,
(R)-9, (R)-7, and then the two 300-electron-donating derivatives
(R)-6 and (R)-8 (Fig. 2C). Interestingly, the relative level of slow
inactivation for the (R)-D compounds did not parallel their corre-
sponding anticonvulsant activities. For example, (R)-3 showed
the greatest level of slow inactivation (Fig. 2C), but it was the least
potent in the MES seizure model in mice (ip) (Table 1). When we
compared the slow inactivation IC50 (ꢀ50 mV) values for (R)-9
and (R)-1 in CAD cells, we found that the IC50 value for (R)-9 was
5. Experimental
5.1. General methods
0.70
(R)-1 (85
a comparable increased protection for (R)-9 versus (R)-1 in the
l
M, which was ꢁ120-fold lower than the value reported for
l
M). Despite (R)-90s increased potency, we did not find
Melting points were determined in open capillary tubes using a
Thomas–Hoover melting point apparatus and are uncorrected.
Please cite this article in press as: Torregrosa, R.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.04.014

10
R. Torregrosa et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
Optical rotations were obtained on a Jasco P-1030 polarimeter at
the sodium D line (589 nm) using a 1 dm path length cell. NMR
spectra were obtained at 400 MHz (1H) and 100 MHz (13C) using
TMS as an internal standard. Chemical shifts (d) are reported in
parts per million (ppm) from tetramethylsilane. Low-resolution
mass spectra were obtained with a BioToF-II-Bruker Daltonics
spectrometer by Dr. S. Habibi at the University of North Carolina
Department of Chemistry. The high-resolution mass spectrum
was performed on a Bruker Apex-Q 12 Telsa FTICR spectrometer
by Dr. S. Habibi. Microanalyses were performed by Atlantic
Microlab, Inc. (Norcross, GA). Reactions were monitored by analyt-
the desired compound (R)-5 as a white solid (786 mg, 70%):
24
Rf = 0.29 (20:1 CH2Cl2/MeOH); mp 129–130 °C; [
a]
ꢀ19.9° (c
D
1.0, CHCl3); 1H NMR (CDCl3) d 2.04 (s, CH3C(O)), 3.39 (s, OCH3),
3.41–3.49 (m, CHH0OCH3), 3.75–3.86 (m, CHH0OCH3), 4.41–4.52
(m, NHCH2), 4.52–4.60 (m, CHCH2), 6.46 (d, J = 7.0 Hz, NH), 6.76–
6.85 (m, ArH), 6.85–6.91 (m, NH), 6.94–7.03 (m, 3ArH), 7.05–7.15
(m, ArH), 7.20–7.26 (m, 2ArH), 7.30–7.37 (m, ArH), addition of
excess (R)-(ꢀ)-mandelic acid to a CDCl3 solution of (R)-5 gave only
one signal for the acetyl methyl and one signal for the methoxy
protons; 13C NMR (CDCl3) d 23.1 (C(O)CH3), 42.9 (NHCH2), 52.5
(CHCH2), 59.1 (OCH3), 71.8 (CHCH2), 116.6, 118.9, 119.5, 123.3,
129.0, 130.5, 132.7, 133.6, 155.6, 158.2 (ArC), 170.0, 170.3 (C(O));
LRMS (ESI+) 377.1 [M+H]+ (calcd for C19H21ClN2O4H+ 377.1);
HRMS (ESI+) 377.1269 [M+H]+ (calcd for C19H21ClN2O4H+
377.1268); Anal. Calcd for C19H21ClN2O4: C, 60.56; H, 5.62; Cl,
9.41; N, 7.43. Found: C, 60.69; H, 5.60; Cl, 9.14; N, 7.32.
ical thin-layer chromatography (TLC) plates (Aldrich, Cat
#
Z12272-6) and analyzed with 254 nm UV light. The reactions were
purified by flash column chromatography using silica gel (Dynamic
Adsorbents Inc., Cat # 02826-25). All chemicals and solvents were
reagent grade and used as obtained from commercial sources with-
out further purification. Yields reported are for purified products
and were not optimized. Compounds were checked by TLC, 1H
and 13C NMR, MS, and elemental analyses. The analytical results
are within 0.40% of the theoretical value. The TLC, NMR and the
analytical data confirmed the purity of the products was P95%.
5.1.4. (R)-N-40-((300-Methyl)phenoxy)benzyl 2-N-acetamido-3-
methoxypropionamide ((R)-6)
Using Method 1, (R)-36 (2.10 g, 5.07 mmol), 4 M HCl (7.6 mL),
Et3N (1.54 g, 15.2 mmol), and AcCl (447 mg, 6.08 mmol) gave the
desired compound (R)-6 as a white solid (1.25 g, 69%): Rf = 0.27
24
5.1.1. General procedure for the deprotection and acetylation of
(R)-N-benzyl 2-N-(tert-butoxycarbonyl)amino-3-
methoxypropionamide derivatives (R)-34–(R)-39 to give (R)-3
and (R)-5–(R)-9 (Method 1)
(20:1 CH2Cl2/MeOH); mp 110–111 °C; [
a
]
ꢀ19.2° (c 1.0, CHCl3);
D
1H NMR (CDCl3) d 2.05 (s, CH3C(O)), 2.34 (s, CH3), 3.39 (s, OCH3),
3.42–3.48 (m, CHH0OCH3), 3.83 (dd, J = 4.3, 9.3 Hz, CHH0OCH3),
4.40–4.52 (m, NHCH2), 4.54–4.57 (m, CHCH2), 6.42 (d, J = 7.0 Hz,
NH), 6.68–6.77 (m, ArH), 6.77–6.85 (m, ArH, NH), 6.90–7.00 (m,
3ArH), 7.18–7.26 (m, 3ArH), addition of excess (R)-(ꢀ)-mandelic
acid to a CDCl3 solution of (R)-6 gave only one signal for the acetyl
methyl and one signal for the methoxy protons; 13C NMR (CDCl3) d
21.3 (ArCH3), 23.1 (C(O)CH3), 42.9 (NHCH2), 52.4 (CHCH2), 59.0
(OCH3), 71.8 (CHCH2), 115.9, 118.9, 119.5, 124.1, 128.8, 129.4,
132.5, 139.9, 156.7, 157.0 (ArC), 169.9, 170.3 (C(O)); LRMS (ESI+)
357.1 [M+H]+ (calcd for C20H24N2O4H+ 357.1); HRMS (ESI+)
357.1814 [M+H]+ (calcd for C20H24N2O5H+ 357.1814); Anal. Calcd
for C20H24N2O4: C, 67.40; H, 6.79; N, 7.86. Found: C, 67.34; H,
6.79; N, 7.85.
A CH2Cl2 solution (0.1–0.3 M) of the tert-butoxycarbonyl-com-
pound ((R)-34–(R)-39) was treated with 4 M HCl in dioxane (3–
4 equiv) at room temperature (2–12 h). The reaction mixture was
evaporated in vacuo. The resulting residue was dissolved in
CH2Cl2 (0.1–0.3 M) and then triethylamine (2–3 equiv) and acetyl
chloride (1.0–1.2 equiv) were carefully added at 0 °C and the
resulting solution was stirred at room temperature (2–16 h). The
solution was washed with an aqueous 10% citric acid solution fol-
lowed by a saturated aqueous NaHCO3 solution. The organic layer
was dried (Na2SO4) and concentrated in vacuo. The residue was
purified by column chromatography on SiO2 and/or recrystallized
with EtOAc/hexanes.
5.1.5. (R)-N-40-((300-Trifluoromethyl)phenoxy)benzyl 2-N-
Acetamido-3-methoxypropionamide ((R)-7)
Using Method 1, (R)-37 (1.65 g, 3.5 mmol), 4 M HCl (4.0 mL),
Et3N (1.07 mg, 10.6 mmol), AcCl (414 mg, 5.3 mmol) gave the
5.1.2. (R)-N-40-Phenoxybenzyl 2-N-Acetamido-3-
methoxypropionamide ((R)-3)
Using Method 1, (R)-34 (1.27 g, 3.17 mmol), 4 M HCl (5.6 mL),
Et3N (962 mg, 9.51 mmol), and AcCl (298 mg, 3.80 mmol) gave
desired compound (R)-7 as a white solid (1.23 mg, 85%): Rf = 0.32
24
the desired compound (R)-3 as a white solid (981 mg, 90%):
(20:1 CH2Cl2/MeOH); mp 115-117 °C; [
a
]
ꢀ11.1° (c 1.0, CHCl3);
D
24
Rf = 0.27 (20:1 CH2Cl2/MeOH); mp 147–148 °C; [
a]
ꢀ16.1° (c
1H NMR (CDCl3) d 2.05 (s, CH3C(O)), 3.39 (s, OCH3), 3.45 (dd,
J = 4.3, 9.1 Hz, CHH0OCH3), 3.83 (dd, J = 4.3, 9.1 Hz, CHH0OCH3),
4.41–4.52 (m, NHCH2), 4.56 (dt, J = 4.3, 6.8 Hz, CHCH2), 6.38–6.50
(m, NH), 6.73–6.85 (m, NH), 6.95–7.04 (m, 2ArH), 7.06–7.18 (m,
2ArH), 7.21–7.26 (m, ArH), 7.29 (s, ArH), 7.32–7.48 (m, 2ArH), addi-
tion of excess (R)-(ꢀ)-mandelic acid to a CDCl3 solution of (R)-7
gave only one signal for the acetyl methyl and one signal for the
D
1.0, CHCl3); 1H NMR (CDCl3) d 2.03 (s, CH3C(O)), 3.39 (s, OCH3),
3.46 (dd, J = 4.3, 9.3 Hz, CHH0OCH3), 3.81 (dd, J = 4.3, 9.3 Hz,
CHH0OCH3), 4.40–4.48 (m, NHCH2), 4.58 (dt, J = 4.3, 7.0 Hz,
CHCH2), 6.49 (d, J = 7.0 Hz, NH), 6.83–6.93 (m, 2ArH, NH), 6.93–
6.97 (m, ArH), 7.00 (d, J = 8.2 Hz, 2ArH), 7.27 (d, J = 8.2 Hz, 2ArH),
7.32 (m, 2ArH), addition of excess (R)-(ꢀ)-mandelic acid to a
CDCl3 solution of (R)-3 gave only one signal for the acetyl methyl
and one signal for the methoxy protons; 13C NMR (CDCl3) d 23.2
(C(O)CH3), 43.0 (NHCH2), 52.4 (CHCH2), 59.1 (OCH3), 71.6
(CHCH2), 118.9, 119.0, 123.3, 128.9, 132.7, 156.7 (ArC), 169.9,
170.3 (2 C(O)), the remaining two aromatic peaks were not
detected and are believed to overlap with nearby signals; LRMS
(ESI+) 365.1 [M+Na]+ (calcd for C19H22N2O4Na+ 365.1); HRMS
(ESI+) 365.1478 [M+Na]+ (calcd for C19H22N2O4Na+ 365.1478);
Anal. Calcd for C19H22N2O4: C, 66.65; H, 6.48; N, 8.18. Found: C,
66.41; H, 6.36; N, 8.14.
methoxy protons; 13C NMR (CDCl3)
d 23.2 (C(O)CH3), 42.9
(NHCH2), 52.4 (CHCH2), 59.1 (OCH3), 71.6 (CHCH2), 110.0 (ArC),
115.2 (q, J = 4.0 Hz, ArC), 119.5, 119.6 (ArC), 120.2 (q, J = 4.0 Hz,
ArC), 120.7, 121.6, 129.1 (ArC), 131.9 (q, J = 272.0 Hz, CF3), 133.6
(q, J = 32.0 Hz, ArC), 155.8 (ArC), 170.0, 170.3 (C(O)); LRMS (ESI+)
411.2 [M+H]+ (calcd for C20H21F3N2O4H+ 411.2); HRMS (ESI+)
411.1531 [M+H]+ (calcd for C20H21F3N2O4H+ 411.1527); Anal.
Calcd for C20H21F3N2O4ꢂ0.15H2O: C, 58.53; H, 5.16; F, 13.89; N,
6.83. Found: C, 58.15; H, 5.20; F, 13.80; N, 6.78.
5.1.6. (R)-N-40-((300-Methoxy)phenoxy)benzyl 2-N-Acetamido-3-
methoxypropionamide ((R)-8)
Using Method 1, (R)-38 (1.00 g, 2.32 mmol), 4 M HCl (3.5 mL),
Et3N (704 mg, 6.96 mmol), and AcCl (218 mg, 2.78 mmol) gave
the desired compound (R)-8 as a white solid (742 mg, 86%):
5.1.3. (R)-N-40-((300-Chloro)phenoxy)benzyl 2-N-Acetamido-3-
methoxypropionamide ((R)-5)
Using Method 1, (R)-35 (1.30 g, 2.99 mmol), 4 M HCl (4.5 mL),
Et3N (908 mg, 8.97 mmol), and AcCl (282 mg, 3.59 mmol) gave
Please cite this article in press as: Torregrosa, R.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.04.014

R. Torregrosa et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
11
24
Rf = 0.27 (20:1 CH2Cl2/MeOH); mp 123–124 °C; [
a]
D
ꢀ18.8° (c 1.0,
Dulbecco’s modified Eagle’s medium supplemented with 10% fetal
bovine serum and 1% penicillin/streptomycin (100% stocks,
CHCl3); 1H NMR (CDCl3) d 2.04 (s, CH3C(O)), 3.39 (s, OCH3), 3.45 (t,
J = 9.3 Hz, CHH0OCH3), 3.78 (s, OCH3), 3.82 (dd, J = 4.3, 9.3 Hz,
CHH0OCH3), 4.39–4.51 (m, NHCH2), 4.52–4.59 (m, CHCH2), 6.45
(d, J = 7.0 Hz, NH), 6.54–6.60 (m, 2ArH), 6.63–6.69 (m, ArH), 6.78
(br s, NH), 6.95–7.01 (m, 2ArH), 7.19–7.26 (m, 3ArH), addition of
excess (R)-(ꢀ)-mandelic acid to a CDCl3 solution of (R)-8 gave only
one signal for the acetyl methyl and one signal for the methoxy
protons; 13C NMR (CDCl3) d 23.2 (C(O)CH3), 43.0 (NHCH2), 52.4
(CHCH2), 55.3 (ArOCH3), 59.1 (OCH3), 71.7 (CHCH2), 104.9, 108.9,
110.9, 119.2, 128.9, 130.1, 132.8, 156.4, 158.3, 161.0 (ArC), 169.9,
170.3 (C(O)); LRMS (ESI+) 373.2 [M+H]+ (calcd for C20H24N2O5H+
373.2); HRMS (ESI+) 373.1764 [M+H]+ (calcd for C20H24N2O5H+
373.1763); Anal. Calcd for C20H24N2O5: C, 64.50; H, 6.50; N, 7.52.
Found: C, 64.51; H, 6.43; N, 7.42.
10,000 U/mL penicillin G sodium and 10,000
lg/mL streptomycin
sulfate) as described before.9
5.6. Electrophysiology
Whole-cell voltage clamp recordings were performed at room
temperature on cortical neurons, CAD cells, and HEK293 cells
expressing NaV1.x isoforms using an EPC 10 Amplifier (HEKA
Electronics, Lambrecht/Pfalz Germany) as described previ-
ously.9,10,20 Electrodes were fabricated from standard-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 M
X 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
5.1.7. (R)-N-40-((300-Trifluoromethoxy)phenoxy)benzyl 2-N-
Acetamido-3-methoxypropionamide ((R)-9)
Using Method 1, (R)-39 (1.80 g, 3.72 mmol), 4 M HCl (6.0 mL),
Et3N (1.13 g, 11.2 mmol), AcCl (350 mg, 4.46 mmol) gave the
CaCl2, 1 CDCl2, 1 MgCl2, 10 D-glucose, 4 4-AP, 0.1 NiCl2, 10 HEPES
desired compound (R)-9 as a white solid (1.03 g, 65%): Rf = 0.27
(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 for analysis only when the access resistance was less
than 3 MX. Linear leak currents were digitally subtracted by -P/4
leak subtraction.
24
(20:1 CH2Cl2/MeOH); mp 108–109 °C; [
a]
ꢀ15.1° (c 1.0, CHCl3);
D
1H NMR (CDCl3) d 2.04 (s, (CH3C(O)), 3.39 (s, OCH3), 3.45 (dd,
J = 4.3, 9.3 Hz, CHH0OCH3), 3.82 (dd, J = 4.1, 9.3 Hz, CHH0OCH3),
4.39–4.51 (m, NHCH2), 4.53–4.57 (m, CHCH2), 6.43 (d, J = 7.0 Hz,
NH), 6.70–6.80 (m, ArH), 6.98 (d, J = 8.2 Hz, 2ArH), 7.08–7.15 (m,
ArH), 7.23 (d, J = 8.2 Hz, 2ArH), 7.30–7.38 (m, 2ArH), addition of
excess (R)-(ꢀ)-mandelic acid to a CDCl3 solution of (R)-9 gave only
one signal for the acetyl methyl and one signal for the methoxy
protons; 13C NMR (CDCl3) d 23.2 (C(O)CH3), 42.9 (NHCH2), 52.5
(CHCH2), 59.1 (OCH3), 71.7 (CHCH2), 111.3, 115.2, 116.4, 119.6
(ArC), 120.3 (q, J = 257.0 Hz, CF3), 129.1, 130.5, 133.8, 150.1,
155.5, 158.5 (ArC), 170.0, 170.3 (C(O)); LRMS (ESI+) 427.1 [M+H]+
(calcd for C20H21F3N2O5H+ 427.1); HRMS (ESI+) 427.1486 [M+H]+
(calcd for C20H21F3N2O5H+ 427.1481).
5.7. 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, USA). For activation curves, conductance (G)
through sodium 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 ꢀ V1/2)/k]},
where G is the peak conductance, Gmax is the fitted maximal G,
V1/2 is the half-activation voltage, and k is the slope factor.
Additional details of specific pulse protocols are described in the
results text or figure legends.
5.2. 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 accor-
dance with recommendations contained in the Guide for the Care
and Use of Laboratory Animals. Anticonvulsant activity was estab-
lished using the MES test,18 6 Hz,19 and the scMet test,35 according
to previously reported methods.8,11
5.8. 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 post-hoc test was performed.
Data are expressed as mean SEM, with p <0.05 considered as the
level of significance.
5.3. Catecholamine A-differentiated (CAD) cells
CAD cells were grown at 37 °C and in 5% CO2 (Sarstedt, Newton,
NC) in Ham’s F12/DMEM (GIBCO, Grand Island, NY), supplemented
with 10% fetal bovine serum (Sigma, St. Louis, MO) and 1% peni-
cillin/streptomycin (100% stocks, 10,000 U/mL penicillin G sodium
Acknowledgments
and 10,000 l
g/mL streptomycin sulfate).49 Cells were passaged
every 3–5 days at a 1:5 dilution.
This work is supported by grants, in part, from the NINDS (1 R41
NS080278) and a National Scientist Development Award from the
American Heart Association (SDG5280023 to R.K.). T.R.C. is sup-
ported by NIH Grant NS053422. We thank the NINDS and the
ASP at the National Institutes of Health with Drs. Tracy Chen and
John Kehne for kindly performing the pharmacological studies
via the ASP’s contract site at the University of Utah with Drs. H.
Wolfe, H.S. White, and K. Wilcox. We express 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 sup-
ported by NIMH Psychoactive Drug Screening Program, Contract
5.4. Cortical neurons
Rat cortical neuron cultures were prepared from cortices dis-
sected from embryonic day 19 cortices exactly as described.58,59
5.5. Culturing HEK293 cells expressing NaV1.1, NaV1.3, NaV1.5,
and NaV1.7
NaV1.1, NaV1.3, NaV1.5, and NaV1.7 stable cells were grown
under standard tissue culture conditions (5% CO2 at 37 °C) in
Please cite this article in press as: Torregrosa, R.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.04.014

12
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directed by Bryan Roth MD, PhD at the University of North
Carolina at Chapel Hill and Project Officer Jamie Driscol at NIMH,
Bethesda MD, USA. The content is solely the responsibility of the
authors and does not represent the official views of the National
Center for Research Resources, National Institutes of Health.
Harold Kohn has a royalty-stake position in (R)-1 and is the foun-
der of NeuroGate Therapeutics, Inc.
Supplementary data
Synthetic procedures, experimental and spectral data for the
intermediates and final products evaluated in this study including
tables for elemental analyses and high-resolution MS data; and 1H
NMR and 13C NMR spectra for (R)-3, (R)-5–(R)-9. This material is
available free of charge via the Internet.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2015.04.014.
32. Public Access to Neuroactive
& Anticonvulsant Chemical Evaluations
(PANAChE) Database. http://panache@nins.nih.gov (accessed November 5,
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Please cite this article in press as: Torregrosa, R.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.04.014