We prepared 13 derivatives of N-(biphenyl-4'-yl)methyl (R)-2-acetamido-3-methoxypropionamide that differed in type and placement of a R-substituent in the terminal aryl unit. We demonstrated that the R-substituent impacted the compound's whole animal and cellular pharmacological activities. In rodents, select compounds exhibited excellent anticonvulsant activities and protective indices (PI=TD50/ED50) that compared favorably with clinical antiseizure drugs. Compounds with a polar, aprotic R-substituent potently promoted Na+ channel slow inactivation and displayed frequency (use) inhibition of Na+ currents at low micromolar concentrations. The possible advantage of affecting these two pathways to decrease neurological hyperexcitability is discussed.
We prepared 13 derivatives of N-(biphenyl-4'-yl)methyl (R)-2-acetamido-3-methoxypropionamide that differed in type and placement of a R-substituent in the terminal aryl unit. We demonstrated that the R-substituent impacted the compound's whole animal and cellular pharmacological activities. In rodents, select compounds exhibited excellent anticonvulsant activities and protective indices (PI=TD50/ED50) that compared favorably with clinical antiseizure drugs. Compounds with a polar, aprotic R-substituent potently promoted Na+ channel slow inactivation and displayed frequency (use) inhibition of Na+ currents at low micromolar concentrations. The possible advantage of affecting these two pathways to decrease neurological hyperexcitability is discussed.
(R)-N-Benzyl 2-acetamido-3-methoxypropionamide[1] (lacosamide, (R)-1) is a first-in-class antiseizure drug (ASD) that is marketed worldwide
for adjunctive treatment of partial-onset seizures.[2] Whole animal pharmacological studies showed that (R)-1 displayed potent anticonvulsant activity[1,3] in the maximal electroshock[4] (MES), 6
Hz psychomotor,[5] and hippocampal kindled[6] seizure models. Whole-cell, patch-clamp electrophysiology
studies indicated that (R)-1 reduced
neuronal hyperexcitability by a mechanism that was consistent with
its enhancing the transition of voltage-gated Na+ channels
(VGSCs) to the slow-inactivated state.[7−9] Recent structure–activity
relationship studies demonstrated that modifying either the N-benzyl[10,11] or the O-methoxy[12,13] sites in (R)-1 provided analogues
that retained excellent anticonvulsant activities. Importantly, we
found that the two N-(biphenyl-4′-yl)methyl
analogues, (R)-2[10] and (R)-3,[11] when given orally to rats in the MES model, exhibited activities
that exceeded those of (R)-1 (Figure 1). We further showed that the IC50 value
for Na+ channel slow inactivation (the concentration at
which half of the Na+ channels transitioned to the slow-inactivated
state) in catecholamine A differentiated (CAD) cells[14] was 29-fold lower (more potent) for (R)-3 than for (R)-1 (Figure 1).[15] While these findings
showed that introducing 4′-aryl substituents to (R)-1 provided pharmacologically potent compounds, they
also showed that the 4′-aryl substituent affected animal behavioral
neurotoxicity. Compound (R)-2 was significantly
more neurotoxic than (R)-1,[10] but (R)-3 was
not (Figure 1).[11]
Figure 1
Key
pharmacological properties of (R)-1,
(R)-2, and (R)-3.
Key
pharmacological properties of (R)-1,
(R)-2, and (R)-3.In this study, we report the synthesis
and pharmacological activity
of 13 substituted N-(biphenyl-4′-yl)methyl
(R)-2-acetamido-3-methoxypropionamides (compound
class (R)-A) where the R-substituent
in the terminal aryl unit was systematically varied. The (R)-A’s R-substituent significantly impacted
the compound’s whole animal and cellular pharmacological activity.
Numerous compounds with a polar, aprotic R-substituent exhibited excellent
anticonvulsant and potent Na+ channel activities while
producing minimal behavioral neurotoxicities.
Results
Compound Selection
Thirteen (R)-A derivatives, (R)-4–(R)-16, containing different R-substituents
were synthesized and evaluated in animal and cellular pharmacological
models. The compounds were divided into two groups. One set, (R)-4–(R)-12, contained polar, aprotic R-substituents (i.e., F, Cl, Br, I, CN,
CF3, OCF3), and the second set, (R)-13–(R)-16, contained
polar, protic R-substituents (i.e., N(H)C(O)CH3, CH2OH, CO2H, NH3+Cl–). We expected that the CO2H substituent in (R)-15 and the NH3+Cl– substituent in (R)-16 were likely
ionized at the physiological-like pH values used in the evaluations.
When the R-substituent was Br, I, CN, CF3, N(H)C(O)CH3, CH2OH, CO2H, or NH3+Cl–, we installed the group at the 3″-position
of the terminal aryl ring ((R)-7, (R)-8, (R)-9,
(R)-10, (R)-13, (R)-14, (R)-15, (R)-16); when the R-substituent
was F, Cl, or OCF3, we prepared both the 3″ ((R)-3, (R)-5,
(R)-11) and the 4″ ((R)-4, (R)-6,
(R)-12) regioisomers. We expected that
the R-substituents would affect the compound’s biodistribution,
anticonvulsant activities, and ability to modulate Na+ channel
function.
Chemistry
Compounds (R)-4–(R)-16 were
prepared by similar
routes (Schemes 1–6) using the mixed anhydride coupling
(MAC) method[16] and employing (R)-N-tert-butoxycarbonylserine ((R)-45), (R)-2-acetamido-3-hydroxypropionic
acid ((R)-69), or (R)-2-acetamido-3-methoxypropionic acid[10,12,17] ((R)-70) as the carboxylic
acid component. The choice of the carboxylic acid was dictated by
the ease of purification of the intermediate products and the need
to avoid competing side reactions in the latter stages of the synthesis.
For compounds (R)-14–(R)-16, we removed the protecting group in the
final step to facilitate the isolation of the desired polar products.
Scheme 1
Preparation of (Biphenyl-4-yl)methylamines 29–39
Scheme 6
Synthesis of Compound (R)-16
To prepare compounds (R)-4–(R)-13, (R)-N-tert-butoxycarbonylserine ((R)-45) was coupled with the requisite (biphenyl-4-yl)methylamine
(29–39, 43, 44) using the MAC reagents, isobutyl chloroformate (IBCF) and N-methylmorpholine (NMM), to give the amides(R)-46–(R)-55 (Scheme 3). The crude (biphenyl-4-yl)methylamines were prepared
by Suzuki coupling using tetrakis(triphenylphoshine)palladium(0).[18] For 29–39,
4-bromobenzylamine (17) was reacted with the commercially
available arylboronic acids (18–28) (Scheme 1), and for 43 and 44, p-aminomethylboronic acid (40) was coupled with the appropriate 1,3-dihalobenzene (41, 42). The serine hydroxyl group in (R)-46–(R)-55 was
methylated (CH3I, Ag2O) to provide ethers(R)-56–(R)-65, respectively. Deprotection of the tert-butoxycarbonyl
group in (R)-56–(R)-65 with acid (HCl/dioxane) followed by acetylation
(AcCl, Et3N) gave the desired products (R)-4–(R)-13, respectively.
For (R)-14 and (R)-15, we coupled (R)-69 with (biphenyl-4-yl)methylamines 37 and 38 (Scheme 5),
and for (R)-16, we combined (R)-70[10,12,17] with (3″-N-(tert-butoxycarbonyl)aminobiphenyl-4′-yl)methylamine
(39) (Scheme 6). To facilitate
the preparation of (R)-14–(R)-16, we first developed convenient synthetic
procedures for (R)-69 and (R)-70 (Scheme 4). Commercially
available d-serine benzyl ester hydrochloride ((R)-66) was selectively converted to (R)-2-N-acetamido-3-hydroxypropionate benzyl ester
((R)-67) using 0.9 equiv of AcCl at
−20 °C. Compound (R)-67 was
methylated (CH3I, Ag2O) at room temperature
to give (R)-68, without racemization.
Catalytic hydrogenation of (R)-67 and
(R)-68 using 10% Pd/C in methanol provided
the desired acids (R)-69 and (R)-70, respectively, which were used directly
in the MAC reaction without purification.
Scheme 3
Synthesis of Compounds
(R)-4–(R)-13
Scheme 5
Synthesis of Compounds (R)-14 and (R)-15
Scheme 4
Synthesis of (R)-69 and (R)-70
The enantiomeric purity of (R)-4–(R)-13 was assessed
by detecting a single acetyl
methyl peak and a single O-methyl peak in the 1H NMR spectrum (CDCl3) for each compound when a
saturated solution of (R)-(−)-mandelic acid
was added.[19] In the cases of (R)-14–(R)-16, their
poor solubility in CDCl3 prevented assessment of their
optical purity by 1H NMR. Thus, we demonstrated with 1H NMR the enantiomeric purity of their immediate precursors
(R)-73–(R)-75, respectively.In the Experimental
Section, we report
the details (synthetic procedure, characterization) of the final step
for all compounds evaluated in the anticonvulsant and cellular electrophysiology
studies. In Supporting Information, we
provide the experimental procedures and physical and spectroscopic
properties for all synthetic compounds prepared in this study.
Whole
Animal Pharmacological Activity
Compounds (R)-4–(R)-16 were
tested for anticonvulsant activity at the Anticonvulsant Screening
Program (ASP) of the National Institute of Neurological Disorders
and Stroke (NINDS), U.S. National Institutes of Health. Screening
was performed using the established protocols and procedures at the
ASP described by Stables and Kupferberg.[20] We relied principally on two rodent models to assess anticonvulsant
activity, the MES[4] and the 6 Hz psychomotor[5] seizure tests. The MES test is a model for generalized
tonic–clonic seizure and is thought to provide an indication
of a compound’s ability to prevent seizure spread when all
neuronal circuits in the brain are maximally active. Correspondingly,
the 6 Hz psychomotor test (32 mA) has been used as a model for therapy-resistant
limbic seizures. Previous studies have shown that some compounds with
minimal activity in the MES test are effective in the 6 Hz psychomotor
model.[5] The anticonvulsant data from the
MES model[4] (mice, ip; rat, po) and psychomotor
6 Hz (32 mA) seizure test for therapy-resistant limbic seizures[5] (mice, ip) are summarized in Table 1 along with similar results obtained for (R)-1,[1] (R)-2,[10] (R)-3,[11] and the ASDs phenytoin,[21] phenobarbital,[21] and
valproate.[21] For compounds that showed
significant activity, we report the 50% effective dose (ED50) values from quantitative screening evaluations. We also include
the median doses for 50% neurological impairment (TD50)
in mice, using the rotorod test,[22] and
the behavioral toxicity effects observed in rats.[23] The protective index (PI = TD50/ED50) for the test compounds, where possible, is also listed. Compounds
(R)-2–(R)-6 and (R)-11 and (R)-12 were evaluated in the subcutaneous Metrazol (scMet)
seizure model.[24] No activity was observed
below 100 mg/kg (data not shown). Similarly, we observed no activity
in the scMet model for (R)-1 and structurally
related compounds.[1,10−13]
Table 1
Structure–Activity
Relationship
for (R)-4–(R)-16a
The compounds were
tested through
the NINDS ASP.
The compounds
were administered
intraperitoneally. 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).
MES = maximal electroshock seizure
test.
6 Hz = 6 Hz psychomotor
seizure
test.
TD50 value
determined
from the rotorod test.
PI
= protective index (TD50/ED50) in the MES test.
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).
Tox = behavioral toxicity.
IC50, concentration at
which half of the Na+ channels have transitioned to a slow
inactivated state.
Reference (10).
ND = not determined.
Reference (11).
Reference (1).
Reference (3).
Reference (9).
Reference (21).
The compounds were
tested through
the NINDS ASP.The compounds
were administered
intraperitoneally. 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).MES = maximal electroshock seizure
test.6 Hz = 6 Hz psychomotorseizure
test.TD50 value
determined
from the rotorod test.PI
= protective index (TD50/ED50) in the MES test.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).Tox = behavioral toxicity.IC50, concentration at
which half of the Na+ channels have transitioned to a slow
inactivated state.Reference (10).ND = not determined.Reference (11).Reference (1).Reference (3).Reference (9).Reference (21).The R-substituent
in (R)-4–(R)-16 markedly affected the observed anticonvulsant
activity in the MES and 6 Hz seizure tests (Table 1). We identified compounds with similar potency compared with
the parent compound in this series, the unsubstituted N-(biphenyl-4′-yl)methyl (R)-2-acetamido-3-methoxypropionamide
((R)-2),[10] and the ASD(R)-1,[1] while other compounds showed little or no activity. The
most potent anticonvulsant compounds in the MES test contained a polar,
aprotic R-substituent (i.e., F ((R)-3, (R)-4); Cl ((R)-5, (R)-6); Br ((R)-7); CF3 ((R)-10); OCF3 ((R)-11, (R)-12). The MES ED50 values in mice
(ip) ranged from 4.7 to 12 mg/kg and in the rat (po) from 2.4 to <30
mg/kg. The corresponding ED50 values for (R)-2 in mice (ip) and rats (po) were 8.0 and 2.0 mg/kg,[10] and for (R)-1 they
were 4.5 and 3.9 mg/kg.[1] We observed little
differences in the anticonvulsant activities in the MES test in mice
(ip) for the 3″-substituted F, Cl, and OCF3 compounds
((R)-3, (R)-5, (R)-11) compared with their 4″-substituted
regioisomers ((R)-4, (R)-6, (R)-12) (MES ED50 (mice, ip, mg/kg): (R)-3,
12; (R)-4, 6.1; (R)-5, 9.8; (R)-6, 12; (R)-11, 4.7; (R)-12, 6.5). The 3″-CN substituted compound ((R)-9) exhibited only moderate activity in the MESseizure
model (mice, ip, mg/kg): (R)-9, 30–100).
A pronounced decrease in activity was seen for compounds (R)-13–(R)-16 containing a polar, protic R-substituent (i.e., N(H)C(O)CH3 ((R)-13); CH2OH ((R)-14); CO2H ((R)-15); NH3+Cl– ((R)-16). Most of these compounds
showed little activity in the MESseizure model in mice (ip), with
only (R)-14 exhibiting modest activity
(ED50, mice, ip, mg/kg: (R)-13, >300; (R)-14, 30–100; (R)-15, 100–300; (R)-16, ∼100).We found that the neurotoxicity of
(R)-A in the rotorod (mice, ip) and
behavioral (rat, po) tests varied
with the nature of the R-substituent and its position in the terminal
aryl ring. Many of the compounds ((R)-3, (R)-5–(R)-11, (R)-13–(R)-16) were significantly less neurotoxic in mice than
the parent, unsubstituted compound (R)-2 (TD50 = 11 mg/kg)[10] and exhibited
comparable or less neurotoxicity than the ASD(R)-1 (TD50 = 27 mg/kg).[1] Thus, the excellent activities in the MES test (mice, ip) of (R)-3, (R)-5,
(R)-6, and (R)-10 (ED50 (mg/kg), (R)-3, 12; (R)-5, 9.8; (R)-6, 12; (R)-7, 12; (R)-10, 8.3) coupled with their low neurotoxicities
(TD50 (mg/kg), (R)-3, 50;
(R)-5, 74; (R)-6, 82; (R)-7, 86; (R)-10, 50) provided compounds with PIs (4.1–7.6)
that were notably higher than that of the parent, unsubstituted compound
(R)-2 (PI = 1.4) and that were similar
to that of (R)-1 (PI = 6.0). When R
was either F or OCF3, we found that the 3″-derivative
was less neurotoxic than the 4″-substituted isomer (TD50 (mg/kg), (R)-3 (3″-F),
50; (R)-4 (4″-F), 11; (R)-11 (3″-OCF3), 23; (R)-12 (4″-OCF3), 13) and
provided higher PI values (PI, (R)-3 (3″-F), 4.1; (R)-4 (4″-F),
1.8; (R)-11 (3″-OCF3), 4.9; (R)-12 (4″-OCF3), 2.0); however, when R was Cl, there was little difference in the
neurotoxicity (TD50 (mg/kg), (R)-5 (3″-Cl), 74; (R)-6 (4″-Cl),
82) and in the PI values (PI, (R)-5 (3″-Cl),
7.6; (R)-6 (4″-Cl), 6.8) for
the regioisomers. A drop in neurotoxicity was observed for (R)-13–(R)-16, the four compounds with polar, protic R-substituents (TD50 of 100 to >300 mg/kg). These compounds showed moderate or little
protection in the seizure models. Taken together, these findings demonstrated
the importance of the R-substituent in (R)-A in eliciting the optimal responses in the animal models.
We found that specific substituents and their placement in the terminal
aryl ring afforded compounds ((R)-5 (3″-Cl),
(R)-6 (4″-Cl), (R)-7 (3″-Br), (R)-10 (3″-CF3), (R)-11 (3″-OCF3)) with excellent anticonvulsant activities
and improved PI values compared with the parent, unsubstituted compound,
(R)-2,[10] in
the mouse (ip). Similar results were observed in the rat (po) (PI:
(R)-2, 25;[10] (R)-3, >210;[11] (R)-5, >91; (R)-6, ∼78; (R)-11, 34).Three of the more active (R)-A compounds
were tested in the rat hippocampal seizure model (ip).[6] This is a test of partial complex or temporal lobe seizures,
the most common and most drug-resistant type of adult focal epilepsy.[25−27] We obtained ED50 values (mg/kg) for (R)-3, (R)-5, and (R)-11 of 13, 36, and 1.7, respectively. The
ED50 value for (R)-11 was
8-fold lower than that for (R)-1 (14
mg/kg).[3]
Whole-Cell, Patch-Clamp
Electrophysiology
Compounds
of interest were characterized for their abilities to interact with
VGSCs by whole-cell patch-clamp electrophysiological studies. In neurons,
VGSCs are responsible for action potential generation and propagation.[28−30] These channels undergo inactivation in response to activity over
two distinct time courses and mechanisms: fast inactivation occurs
over a few milliseconds, whereas slow inactivation occurs over several
hundred milliseconds. Inactivation is reversible, but for a time,
these channels are unable to produce current in response to stimulus.[31] The function of several ASDs, such as carbamazepine
and (R)-1, has been attributed to their
ability to inactivate VGSCs and reduce synaptic activity.[7−9,32] We assessed the ability of the
(R)-A compounds to alter Na+ channel kinetics of fast inactivation and slow inactivation and
also for frequency (use) dependence. These studies were carried out
in several cell models: CAD cells allow for rapid characterization
of compounds; rat cortical neurons allow for characterization in cells
related to those that generate epileptic phenotypes in humans; and
HEK293 cells allow for characterization of activity on single VGSC
isoforms.
3″- and 4″-Polar, Aprotic Substituted N-(Biphenyl-4′-yl)methyl (R)-Acetamido-3-methoxypropionamide
Derivatives in CAD Cells
We previously showed that (R)-3 inhibited several parameters of VGSC activities
in neuronal model CAD cells.[15] Here, we
tested whether (R)-4–(R)-16 would result in similar action. Initially,
we focused on (R)-A compounds with polar,
aprotic R-substituents (i.e., R = F, Cl, Br, I, CF3, OCF3) at either the 3″ or the 4″ position of the
terminal aryl ring, since these compounds exhibited the greatest anticonvulsant
activities (Table 1).CAD cells are abundant
and easy to use.[14] These cells express
endogenous tetrodotoxin-sensitive Na+ currents that display
rapid activation and inactivation kinetics upon membrane depolarization[14] and likely comprise a majority of NaV1.7 channels, with minor contributions by NaV1.1, NaV1.3, and NaV1.9 channels.[9,33,34] Importantly, we earlier demonstrated that
the Na+ channel properties of (R)-1 in CAD cells[9] were similar to
those reported in cultured neurons and in mouseN1E-115neuroblastoma
cells.[7]First, we examined (R)-3,[15] (R)-5, (R)-7,
(R)-8,
(R)-10, and (R)-11, compounds with a 3″-polar, aprotic R-substituent
(R = F, Cl, Br, I, CF3, OCF3), for their ability
to transition Na+ channels to the slow-inactivated state.
CAD cells were held at −80 mV and conditioned to potentials
ranging from −120 to +20 mV (in +10 mV increments) for 5 s.
Then fast-inactivated channels were allowed to recover for 150 ms
at a hyperpolarized pulse to −120 mV, and then the fraction
of channels available was tested by a single depolarizing pulse, to
0 mV, for 15 ms (Figure 2A, left). Brief hyperpolarization
to −120 mV allows channel recovery from fast inactivation while
limiting recovery from slow inactivation such that the property of
slow inactivation can be isolated. Representative slow inactivation
traces, at −120 and −50 mV, from CAD cells treated with
0.1% DMSO (control) or 10 μM (R)-11 are shown in Figure 2A (right). At −50
mV, a majority of the channels undergo steady-state inactivation that
involves contributions from slow- and fast-inactivating pathways.[28,35] This voltage is also near the resting membrane potential (RMP) and
approaches the action potential firing threshold for CNS neurons,[36] where slow inactivation appears to be physiologically
relevant during sustained subthreshold depolarizations.[37] Finally, changes in the Na+ channel
availability near −50 mV can affect the overlap of Na+ current activating and inactivating under steady-state conditions.[28,38] For these reasons, we utilized −50 mV conditioning pulses
to compare pharmacological effects on slow inactivation and to develop
concentration–response curves and IC50 values for
each tested compound (Figure 2B–G).
The slow inactivation IC50 values are shown in Figure 2H and summarized in Table 1. Compared with the reported IC50 calculated value of
85 μM for slow inactivation induced by (R)-1,[9] the IC50 values
for these 3″-substituted (R)-A compounds were considerably lower. For example, the IC50 values for (R)-3 and (R)-8 were 29- and 133-fold lower than (R)-1. Collectively, these data indicate that these (R)-A derivatives more effectively induced Na+ channel transition to a slow-inactivated state than did (R)-1.
Figure 2
Effects of (R)-A compounds with a
polar, aprotic substituent at the 3″-position on the steady-state,
slow inactivation state of Na currents in
CAD cells. (A) Voltage protocol for slow inactivation. Currents were
evoked by 5 s prepulses between −120 and +20 mV, and then fast-inactivated
channels were allowed to recover for 150 ms at a hyperpolarized pulse
to −120 mV. The fraction of channels available at 0 mV was
analyzed. Representative current traces from CAD cells were taken
in the absence (control, 0.1% DMSO) or presence of 10 μM (R)-11. The black and red traces represent the
currents evoked at −120 and −50 mV, respectively (also
highlighted in the voltage protocol). (B–G) Summary of steady-state,
slow activation curves for CAD cells treated with 0.1% DMSO (control)
or with various concentrations of the indicated compounds. (H) The
concentrations at which half of the channels have transitioned to
the slow-inactivated state (see text for details), the SI IC50, are indicated. Data are from four to seven cells per condition.
Effects of (R)-A compounds with a
polar, aprotic substituent at the 3″-position on the steady-state,
slow inactivation state of Na currents in
CAD cells. (A) Voltage protocol for slow inactivation. Currents were
evoked by 5 s prepulses between −120 and +20 mV, and then fast-inactivated
channels were allowed to recover for 150 ms at a hyperpolarized pulse
to −120 mV. The fraction of channels available at 0 mV was
analyzed. Representative current traces from CAD cells were taken
in the absence (control, 0.1% DMSO) or presence of 10 μM (R)-11. The black and red traces represent the
currents evoked at −120 and −50 mV, respectively (also
highlighted in the voltage protocol). (B–G) Summary of steady-state,
slow activation curves for CAD cells treated with 0.1% DMSO (control)
or with various concentrations of the indicated compounds. (H) The
concentrations at which half of the channels have transitioned to
the slow-inactivated state (see text for details), the SI IC50, are indicated. Data are from four to seven cells per condition.A similar series of experiments
were undertaken to investigate
the effects of (R)-A compounds with
polar, aprotic substituents (R = F, Cl, OCF3) at the 4″-aryl
site on Na+ current slow inactivation. We observed pronounced,
concentration-dependent slow inactivation for (R)-4, (R)-6, and (R)-12 (Supporting Information, Figure S1B–D). As for the 3″-substituted compounds,
the IC50 values of slow inactivation were calculated by
fitting the concentration responses of slow inactivation against the V1/2 of slow inactivation (Table 1; Supporting Information, Figure
S1E). The IC50 values for (R)-4, (R)-6, and (R)-12 were 101-, 708-, and 129-fold lower than for (R)-1.We next asked if the polar, aprotic(R)-A derivatives could enhance steady-state
fast inactivation
in CAD cells. For these studies we used a protocol (Figure 3A) designed to induce a fast-inactivated state,
similar to that previously described.[33] Cells were held at −80 mV, stepped to inactivating prepulse
potentials ranging from −120 to −10 mV (in 10 mV increments)
for 500 ms. Then the cells were stepped to 0 mV for 20 ms to measure
the available current. A 500 ms conditioning pulse was used because
it allowed all of the endogenous channels to transition to a fast-inactivated
state at all potentials assayed. In Figure 3, we provide the steady-state, fast inactivation curves of Na+ currents (representative family of current traces shown in
Figure 3A from control (0.1% DMSO) and Figure 3B–G for (R)-A derivatives with a polar, aprotic 3″-substituent). (R)-3-, (R)-5-,
(R)-7-, (R)-8-, (R)-10-, and (R)-11-treated CAD neurons were well fitted with a single
Boltzmann function (R2 > 0.969 for
all
conditions). The V1/2 inactivation value
for 0.1% DMSO-treated cells was −68.3 ± 3.1 mV (n = 5), which was significantly different from that of (R)-8 (10 μM) treated cells (−76.5
± 1.3 mV; n = 5; p < 0.05;
Student’s t-test; see Figure 3E). Compared with the ∼8.2 mV shift in V1/2 of fast inactivation in the hyperpolarizing direction
observed in the presence of (R)-8, the
shifts caused by the other five compounds were not significantly different
from control (Figure 3B–D,F,G) (p < 0.05 vs 0.1% DMSO (control); Student’s t-test). We also found that (R)-1 did not affect Na+ channel fast inactivation.[9]
Figure 3
Effects of (R)-A compounds
with a
polar, aprotic substituent at the 3″-position on fast inactivation
and steady-state activation state of Na currents
in CAD cells. (A) Voltage protocol for fast inactivation (left) and
activation (right). Representative families of currents in response
to these protocols are illustrated. (B–G) Representative Boltzmann
fits for steady-state fast inactivation and activation for CAD cells
treated with 0.1% DMSO (control) and various concentrations of 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 were averaged to determine the
mean (±SEM) voltage dependence of steady-state inactivation and
activation, respectively. The V1/2 and k of steady-state fast inactivation or activation other
than (R)-8 were not different among
any of the conditions tested (p > 0.05, one-way
ANOVA).
Data are from four to six cells per condition.
Effects of (R)-A compounds
with a
polar, aprotic substituent at the 3″-position on fast inactivation
and steady-state activation state of Na currents
in CAD cells. (A) Voltage protocol for fast inactivation (left) and
activation (right). Representative families of currents in response
to these protocols are illustrated. (B–G) Representative Boltzmannfits for steady-state fast inactivation and activation for CAD cells
treated with 0.1% DMSO (control) and various concentrations of 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 were averaged to determine the
mean (±SEM) voltage dependence of steady-state inactivation and
activation, respectively. The V1/2 and k of steady-state fast inactivation or activation other
than (R)-8 were not different among
any of the conditions tested (p > 0.05, one-way
ANOVA).
Data are from four to six cells per condition.The effects of (R)-A derivatives
with 4″-polar, aprotic substituents (F, Cl, OCF3) on Na+ current fast inactivation was also investigated
(Supporting Information, Figure S2). Steady-state,
fast inactivation curves of Na+ currents from 0.1% DMSO-
(control) and (R)-4-, (R)-6-, and (R)-12-treated
CAD cells were well fitted with a single Boltzmann function (R2 > 0.989 for all conditions). The V1/2 inactivation value for 0.1% DMSO-treated
cells was
not significantly different from any of the conditions (p > 0.05; Student’s t-test; Supporting Information Figure S2B–D).Because changes in current amplitudes can result from changes in
channel gating, we tested whether the nine 3″- and 4″-polar,
aprotic(R)-A derivatives ((R)-4–(R)-12) could alter voltage-dependent activation properties of Na+ currents in CAD cells. Activation changes for the (R)-A derivative treated CAD cells were measured by whole-cell
ionic conductances by comparing their midpoints (V1/2) and slope factors (k) in response
to changes in command voltage (Figure 3A, top
right). Representative traces in response to the voltage protocol
are shown in Figure 3A (bottom right). Boltzmannfits for 0.1% DMSO (control) and various concentrations of (R)-3, (R)-5,
(R)-7, (R)-8, (R)-10, and (R)-11 are shown in Figure 3B–G
and those for (R)-4, (R)-6, and (R)-12 in Supporting Information, Figure S2B–D.
An analysis of the six 3″-substituted compounds ((R)-3, (R)-5, (R)-7, (R)-8, (R)-10, (R)-11) V1/2 and k values showed that,
with the exception of two (R)-8 concentrations,
there were no changes in the steady-state activation properties of
Na+ currents between CAD cells treated with 0.1% DMSO (control)
or with any of the tested compounds (Figure 3C–G). The V1/2 steady-state activation
value for 0.1% DMSO-treated (control) cells was −12.8 ±
0.5 mV (n = 4), which was significantly different
from that of (R)-8-treated cells at
100 nM (−24.6 ± 3.9 mV, n = 4) and at
300 nM (−20.7 ± 2.4 mV, n = 5) (p < 0.05; Student’s t-test; see
Figure 3E). Similarly, we found that the V1/2 or k values for steady
state activation for 0.1% DMSO-treated (control) cells were not significantly
different from those of the 4″-substituted (R)-A compound (10 μM) treated CAD cells ((R)-4, (R)-6,
and (R)-12; p >
0.05;
Student’s t-test; Supporting
Information Figure S2B–D). These data indicate that,
except for (R)-8, polar, aprotic(R)-A derivatives did not affect a closed channel’s
transition to an open conformation.Finally, we tested whether
(R)-4–(R)-12 could elicit a frequency (use) dependent
block of Na+ currents in CAD cells. Use dependency represents
an ability to bind more efficiently to a channel undergoing repeated
transitions between closed, open, and inactivated conformations. 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 Na+ channel availability during high-frequency
(seizure-like) but not low-frequency firing.[39] Several theories have been proposed to account for the complexities
of use-dependent inhibition, including the modulated receptor hypothesis,[40] which postulates that the receptor on the Na+ channels can exist in multiple configurations and the affinity
and binding rates of drugs depend on channel’s state, which
in turn can depend on voltage. The response to voltage changes can
be viewed as reflecting voltage-sensitive shifts in equilibrium between
conducting, unblocked channels and nonconducting, blocked channels.
The modulated-receptor hypothesis postulates shifts in equilibrium
as the result of a variable-affinity receptor and modified inactivation
gate kinetics in drug-complexed channels. We tested compounds (R)-5, (R)-7,
(R)-8, (R)-10, and (R)-11 at a range of concentrations
from below to above the determined slow inactivation IC50 values. A train of 30 test pulses (20 ms to −10 mV) was delivered
from a holding potential of −80 mV at 10 Hz (Figure 4A). The available current in control cells (0.1%
DMSO) and cells in the presence of the (R)-A compounds was calculated by dividing the peak current at
any given pulse (pulse) by the peak current
in response to the initial pulse (pulse1). Representative
currents elicited by the voltage protocol are illustrated for control
and 5 μM (R)-5-treated cells (Figure 4A). Compounds (R)-5, (R)-10, and (R)-11 exhibited a statistically significant concentration-dependent
frequency (use) inhibition of Na+ currents (Figure 4B,E,F) by the last pulse, compared with control
(0.1% DMSO). The peak current was ∼50% lower in the presence
of 5 μM (R)-5, ∼18% lower
in the presence of 10 μM (R)-10, and ∼65% lower in the presence of 6 μM (R)-11. These test concentrations were 7.1–7.6
times higher than their slow inactivation IC50 values.
By comparison, as we previously reported, there was a 45% diminution
of Na+ currents in the presence of 15 μM (R)-3 (Figure 4B, purple
circles), a concentration that is 5.2 times higher than the slow inactivation
IC50 value. Similarly, the traces for (R)-7 and (R)-8 (Figure 4C,D) suggested frequency (use) inhibition of the
Na+ currents at a 10 μM concentration, but the values
did not reach statistical significance. We did not test these (R)-A compounds at higher concentrations.
Figure 4
Effect of (R)-A compounds with a
polar, aprotic substituent at the 3″-position on frequency
(use) dependent block of Na currents in CAD
cells. (A) The frequency dependence of the block was examined by holding
cells at the hyperpolarized potential of −80 mV and evoking
currents at 10 Hz by 20 ms test pulses to −10 mV (inset middle).
Representative overlaid traces are illustrated by pulses 1 and 30
for control (predrug) and in the presence of (R)-5 (5 μM). (B–F) Summary of average frequency
(use) dependent decrease in current amplitude (±SEM) produced
by control (0.1% DMSO) or by the presence of various concentrations
of the indicated compounds (p > 0.05, one-way
ANOVA
with Dunnett’s post hoc test). Data are from three to six cells
per condition. As a comparison, a use-dependent block imposed by (R)-3 is also shown.[15] Compounds (R)-3, (R)-5, (R)-10, and (R)-11 caused a significant decrease (indicated
by ∗) in current amplitude compared with 0.1% DMSO-treated
cells (control) (p < 0.05, one-way ANOVA with
Dunnett’s post hoc test). Note the rapid frequency-dependent
facilitation of the block by (R)-3 and
(R)-5, which was observed beginning
as early as pulse 4. Data are from three to six cells per condition.
Effect of (R)-A compounds with a
polar, aprotic substituent at the 3″-position on frequency
(use) dependent block of Na currents in CAD
cells. (A) The frequency dependence of the block was examined by holding
cells at the hyperpolarized potential of −80 mV and evoking
currents at 10 Hz by 20 ms test pulses to −10 mV (inset middle).
Representative overlaid traces are illustrated by pulses 1 and 30
for control (predrug) and in the presence of (R)-5 (5 μM). (B–F) Summary of average frequency
(use) dependent decrease in current amplitude (±SEM) produced
by control (0.1% DMSO) or by the presence of various concentrations
of the indicated compounds (p > 0.05, one-way
ANOVA
with Dunnett’s post hoc test). Data are from three to six cells
per condition. As a comparison, a use-dependent block imposed by (R)-3 is also shown.[15] Compounds (R)-3, (R)-5, (R)-10, and (R)-11 caused a significant decrease (indicated
by ∗) in current amplitude compared with 0.1% DMSO-treated
cells (control) (p < 0.05, one-way ANOVA with
Dunnett’s post hoc test). Note the rapid frequency-dependent
facilitation of the block by (R)-3 and
(R)-5, which was observed beginning
as early as pulse 4. Data are from three to six cells per condition.We also examined whether the three
polar, aprotic 4″-substituted
derivatives (R)-4, (R)-6, and (R)-12 affected
frequency (use) dependent block of Na+ currents using a
single concentration of 1 μM, a concentration at which near
maximal effects on slow inactivation were observed (IC50 (μM): (R)-4, 0.82; (R)-6, 0.12; (R)-12, 0.66; Supporting Information Figure
S1). Only (R)-4 and (R)-6 exhibited small but statistically significant reduction
in frequency (use) dependent bock of Na+ currents; by the
last pulse, compared with control, the peak current was ∼15%
lower in the presence of 1 μM (R)-4 and ∼16% lower in the presence of 1 μM (R)-6 (Supporting Information, Figure S3B).
Polar, Aprotic and Polar,
Protic N-(Biphenyl-4′-yl)methyl (R)-2-Acetamido-3-methoxypropionamide
Derivatives in Rat Embryonic Cortical Neurons
Next, we tested
many of the compounds with 3″-polar, aprotic R-substituents
(R = F, Cl, Br, I, CF3, OCF3) in rat embryonic
cortical neurons. These cells express channel subtypes (NaV1.1, NaV1.2, NaV1.3, NaV1.6) that
participate in seizure propagation,[41] and
we assumed contributions from all four CNS NaV isoforms
to the observed effects. Slow inactivation, fast inactivation, steady-state
activation, and frequency (use) dependence of Na+ currents
in rat cortical neurons grown for 7–10 days in vitro were examined
using protocols described earlier.[42] Because
of the reduced availability of cortical neurons and their viability
under the patch-clamp electrophysiology conditions, we used only 10
μM (R)-A compounds. Furthermore,
because the CAD cell patch-clamp electrophysiology data for the 3″-
and 4″-substituted isomers were similar, we examined only the
3″-substituted derivatives. Using cortical neurons, we also
evaluated several (R)-A compounds with
3″-polar, protic R-substituents (R = N(H)C(O)CH3, CO2H, NH3+Cl–) to try to correlate their activities on VGSCs with their diminished
anticonvulsant activities.First, we determined the ability
of the (R)-A derivatives with a 3″-polar,
aprotic R-substituent ((R)-3, (R)-5, (R)-7,
(R)-8, (R)-10, (R)-11) to modulate the VGSC transition
to a slow-inactivated state. Cortical cells were held at −70
mV and conditioned to potentials ranging from −100 to +20 mV
(in +10 mV increments) for 5 s. Then fast-inactivated channels were
allowed to recover for 1000 ms at a hyperpolarized pulse to −70
mV, and the fraction of channels available was tested by a single
depolarizing pulse, to −10 mV, for 20 ms (Figure 5A, left). The hyperpolarization pulse allows channel recovery
from fast inactivation while limiting recovery from slow inactivation.
Representative traces that illustrate the extent of slow inactivation
observed at −50 mV are compared with the prepulse at −100
mV in the absence or presence of (R)-5 in Figure 5A, with complete slow inactivation
curves (normalized peak vs prepulse potential) shown in Figure 5B. For the 0.1% DMSO (control) treated cells (−50
mV), 0.82 ± 0.03 fractional unit (n = 5) of
the Na+ current was available, suggesting a small fraction
(0.18 ± 0.03, calculated as 1 minus the normalized INa) of the channels transitioned to a nonconducting (slow-inactivated)
state (Figure 5B,C). Compared with control
(0.1% DMSO), all of the 3″-polar, aprotic substituted (R)-A compounds at 10 μM caused significant
increase in the maximal fraction of current unavailable by depolarization,
with maximal induction observed in the presence of (R)-11: 0.82 ± 0.02 (n = 5; p < 0.01, Mann–Whitney U test).
The effectiveness of these (R)-A compounds
to promote slow inactivation in cortical neurons at 10 μM approximated
the IC50 values observed for slow inactivation in CAD cells
where the order of potency was (R)-5, (R)-8 > (R)-11 > (R)-7, (R)-10 > (R)-3 (Figure 2, Table 1). When we tested
the (R)-A derivatives with a 3″-polar,
protic R-substituent ((R)-13, (R)-15, (R)-16), only (R)-15 caused an increase in
the extent of slow inactivation (Supporting Information, Figure S4A,C,E) while neither (R)-13 nor (R)-16 (Supporting
Information, Figure S4B,E) was different from 0.1% DMSO (control)
treated neurons.
Figure 5
Effects of (R)-A compounds
with a
polar, aprotic substituent at the 3″-position 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 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. Shown are representative current traces from cortical
neurons in the absence (control, 0.1% DMSO) or presence of 10 μM
(R)-5. The black and gray traces represent
the currents evoked at −100 and −50 mV, respectively
(highlighted in the voltage protocol as thick, dashed line). (B) Summary
of steady-state, slow-activation curves for neurons treated with 0.1%
DMSO (control) or 10 μM (R)-5.
Significant drug-induced slow inactivation was evident at voltages
more depolarizing than −80 mV in neurons treated with (R)-5. (C) Summary of the available current
fraction at −50 mV for neurons treated with DMSO (control)
or 10 μM (R)-3, (R)-5, (R)-7, (R)-8, (R)-10, and (R)-11. Asterisks (∗) indicate statistically
significant differences in available current fraction between control
(0.1% DMSO) and the indicated compounds (p < 0.05,
Student’s t-test). Data are from five to six
cells per condition.
Effects of (R)-A compounds
with a
polar, aprotic substituent at the 3″-position 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 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. Shown are representative current traces from cortical
neurons in the absence (control, 0.1% DMSO) or presence of 10 μM
(R)-5. The black and gray traces represent
the currents evoked at −100 and −50 mV, respectively
(highlighted in the voltage protocol as thick, dashed line). (B) Summary
of steady-state, slow-activation curves for neurons treated with 0.1%
DMSO (control) or 10 μM (R)-5.
Significant drug-induced slow inactivation was evident at voltages
more depolarizing than −80 mV in neurons treated with (R)-5. (C) Summary of the available current
fraction at −50 mV for neurons treated with DMSO (control)
or 10 μM (R)-3, (R)-5, (R)-7, (R)-8, (R)-10, and (R)-11. Asterisks (∗) indicate statistically
significant differences in available current fraction between control
(0.1% DMSO) and the indicated compounds (p < 0.05,
Student’s t-test). Data are from five to six
cells per condition.We next asked if the (R)-A derivatives
that contained a 3″-polar R-substituent that was either aprotic
((R)-3, (R)-5, (R)-6, (R)-7, (R)-8, (R)-10, (R)-11) or protic
((R)-13, (R)-15, (R)-16) could enhance steady-state
fast inactivation. To investigate, we used a protocol (Figure 6A, left) designed to induce a fast-inactivated state,
similar to that previously described.[42] Neurons were held at −70 mV, stepped to inactivating prepulse
potentials ranging from −120 to −10 mV (in 10 mV increments)
for 500 ms. Then the cells were stepped to 0 mV for 20 ms to measure
the available current. A 500 ms conditioning pulse was used because
it allowed all of the endogenous channels to transition to a fast-inactivated
state at all potentials assayed. Steady-state, fast inactivation curves
of Na+ currents (representative family of control current
traces shown in Figure 6B, left) from control
(0.1% DMSO) treated and (R)-3-, (R)-5-, (R)-6-,
(R)-7-, (R)-8-, (R)-10-, and (R)-11-treated cortical neurons were well fitted with
a single Boltzmann function (R2 > 0.983
for all conditions). The V1/2 inactivation
value for 0.1% DMSO-treated cells was −53.2 ± 1.2 mV (n = 4), which was significantly different from that of (R)-5 (−59.9 ± 3.1 mV; n = 5), (R)-7 (−58.3 ± 1.4
mV; n = 4), and (R)-11 (−60.1 ± 1.2 mV; n = 5) treated cells
(p < 0.05 vs control (0.1% DMSO); Student’s t-test; Figure 6C,D); none of the
other compounds ((R)-3, (R)-6, (R)-8, (R)-10) were different from controls (0.1% DMSO). The
slope values were unchanged between control and any of the conditions
tested. By comparison, we did not see evidence to indicate that the
(R)-A compounds with 3″-polar,
protic R-substituents, (R)-13, (R)-15, and (R)-16, affected V1/2 or k values of steady-state fast inactivation (Figure 6D).
Figure 6
Effects of (R)-A compounds with a
polar, aprotic or a polar, protic substituent at the 3″-position
on fast inactivation and steady-state activation states of Na currents in cortical embryonic neurons. (A)
Voltage protocol for fast inactivation (left) and activation (right).
(B) Representative family of currents in response to these protocols
is illustrated. (C, D) Representative Boltzmann fits for steady-state
fast inactivation and activation for cortical neurons treated with
0.1% DMSO (control) and various concentrations of 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 were averaged to determine the mean
(±SEM) voltage dependence of steady-state inactivation and activation,
respectively. Statistically significant differences between control
and fast inactivation or activation are indicated by asterisks (p < 0.05, one-way ANOVA). Data are from five to eight
cells per condition.
Effects of (R)-A compounds with a
polar, aprotic or a polar, protic substituent at the 3″-position
on fast inactivation and steady-state activation states of Na currents in cortical embryonic neurons. (A)
Voltage protocol for fast inactivation (left) and activation (right).
(B) Representative family of currents in response to these protocols
is illustrated. (C, D) Representative Boltzmannfits for steady-state
fast inactivation and activation for cortical neurons treated with
0.1% DMSO (control) and various concentrations of 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 were averaged to determine the mean
(±SEM) voltage dependence of steady-state inactivation and activation,
respectively. Statistically significant differences between control
and fast inactivation or activation are indicated by asterisks (p < 0.05, one-way ANOVA). Data are from five to eight
cells per condition.Next, we tested whether (R)-A compounds
that contained polar R-substituents, aprotic or protic, at the 3″-position
could alter voltage-dependent activation properties of Na+ currents in cortical neurons. Compound-treated cortical neuron activation
changes were measured by whole-cell ionic conductances by comparing
their midpoints (V1/2) and slope factors
(k) in response to command voltage changes (Figure 6A, right). Representative traces for the 0.1% DMSO-treated
neurons (control) in response to the voltage protocol are shown in
Figure 6B (right). Boltzmannfits for DMSO
(control) and the (R)-A compounds with
polar, aprotic and polar, protic R-substituents are shown in Figure 6C and Figure 6D, respectively.
The V1/2 value for steady-state activation
for 0.1% DMSO-treated (control) neurons was −27.3 ± 3.3
mV (n = 5), which was significantly different from
those of (R)-5 (−37.5 ±
4.9 mV; n = 5), (R)-10 (−35.4 ± 5.4 mV; n = 5), (R)-11 (−33.2 ± 1.9 mV; n = 4), (R)-13 (−39.0 ±
2.6 mV; n = 4), (R)-15 (−36.8 ± 3.0 mV; n = 5), and (R)-16 (−33.4 ± 3.2 mV; n = 5) (p < 0.05 vs control; Student’s t-test; Figure 6C,D). The hyperpolarizing
shifts induced by these compounds suggest that in the presence of
the compounds, NaV currents are likely to activate earlier
(i.e., at less-depolarized potentials).Finally, we tested whether
the (R)-A derivatives containing polar
R-substituents, aprotic or protic,
at the 3″-position could affect frequency (use) dependent block
of Na+ currents. Unlike the data obtained with CAD cells,
none of the compounds tested at a 10 μM concentration in cortical
neurons affected frequency (use) dependent block of Na+ currents, compared with controls (0.1% DMSO) (Supporting Information, Figure S5).
(R)-N-(3″-Chlorobiphenyl-4′-yl)methyl
(R)-2-Acetamido-3-methoxypropionamide ((R)-5) in HEK293 Cells
In order to assess the
relative effect of an (R)-A compound
on Na+ channel isoforms, we examined the Na+ currents in HEK293 cells that stably express CNS (hNaV1.1 and rNaV1.3), peripheral nervous system (hNaV1.7), or cardiac (hNaV1.5) isoforms. Compound (R)-5 (10 μM) was chosen for study because
it displayed excellent anticonvulsant activity in mice (ip) and rats
(po) (Table 1), and it modulated Na+ currents in CAD cells and ratembryonic cortical neurons.Properties of slow inactivation, fast inactivation, steady-state
activation, and frequency (use) dependent inhibition were examined
in the four cell lines using voltage protocols illustrated in Figure 7A,K. The results from
these experiments are summarized in Table 2. Notably, (R)-5 exhibited similar,
but not identical, effects on these biophysical properties irrespective
of Na+ channels, indicating that this compound exhibited
little isoform specificity. We observed that (R)-5 transitioned the four Na+ channel subtypes to
the slow-inactivated state and that differences were observed in the
degree to which (R)-5 affected Na+ channel fast inactivation, fast activation, and the frequency
(use) inhibition of Na+ currents.
Figure 7
Analysis of (R)-5 on electrophysiological
properties of Na1.1, Na1.3, Na1.7, and Na1.5 currents in HEK293 cells. (A, K) Voltage protocols for
examining slow inactivation, fast inactivation, activation, and frequency
(use) dependent block. NaV1.5 uses hyperpolarized protocols
because of differences in the hyperpolarized activation of this isoform.
(B, E, H, L) Summary of steady-state slow-activation curves for HEK293
cells treated with 0.1% DMSO (control) or 10 μM (R)-5. Insets illustrate representative current traces
from HEK293 cells in the absence (control, 0.1% DMSO) or presence
of 10 μM (R)-5. The black and
colored traces represent the currents evoked at −120 and −50
mV, respectively (highlighted in the voltage protocol). (C, F, I,
M) Representative Boltzmann fits for steady-state fast inactivation
and activation for HEK293 cells treated with 0.1% DMSO (control) or
10 μM (R)-5. 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
were averaged to determine the mean (±SEM) voltage dependence
of steady-state inactivation and activation, respectively. (D, G,
J, N) Summary of average frequency (use) dependent decrease in current
amplitude over time (±SEM) produced by control (0.1% DMSO) or
10 μM (R)-5. Data are from five
to six cells per condition.
Table 2
Comparative Current Densities, Cell
Capacitances, and Boltzmann Parameters of Voltage Dependence of Channel
Activation and Steady-State Fast Inactivation Curves for Control-Treated
and (R)-N-(3″-Chlorobiphenyl-4′-yl)methyl
2-Acetamido-3-methoxypropionamide ((R)-5) Treated HEK293 Cells Expressing NaV1.1, NaV1.3, NaV1.7, or NaV1.5 Channelsa
voltage dependence of activationc
voltage dependence of
fast inactivation
condition
extent of
slow inactivation (at −50 mV)b
V1/2 (mV)
slope (mV/e-fold)
V1/2 (mV)
slope (mV/e-fold)
use-dependent inhibitiond
NaV1.1
control
0.17 ± 0.04 (6)
–24.29 ± 1.52
(5)
4.92 ± 0.60 (5)
–64.29 ± 3.63
(6)
4.93 ± 1.48
(6)
1.02 ± 0.08
(5)
10 μM (R)-5
0.77 ± 0.03 (6)*
–22.43 ± 2.94 (6)
4.34 ± 1.78 (5)
–70.00 ± 4.67
(6)
6.21 ± 2.11
(6)
0.81 ± 0.07 (6)*
NaV1.3
control
0.04 ± 0.02 (5)
–29.56 ± 4.94
(5)
4.07 ± 1.96
(5)
–65.39
± 7.77
(5)
5.55 ± 1.28
(5)
1.12 ± 0.19
(5)
10 μM (R)-5
0.71 ± 0.09 (5)*
–25.44 ± 2.01
(4)
4.47 ± 1.56
(4)
–63.39
± 7.01
(5)
6.81 ± 2.67
(5)
1.03 ± 0.18
(5)
NaV1.7
control
0.07 ± 0.04 (5)
–27.15 ± 4.01
(5)
3.61 ± 1.24
(5)
–61.09
± 2.99
(5)
4.83 ± 1.12
(5)
1.03 ± 0.09
(5)
10 μM (R)-5
0.80 ± 0.03 (5)*
–27.27 ± 1.98
(5)
4.21 ± 1.43
(5)
–78.01 ± 3.69 (5)*
5.52 ± 1.98 (5)
0.92 ± 0.06 (5)
NaV1.5
control
0.18 ± 0.04 (5)
–55.67 ± 3.19
(5)
3.16 ± 1.75
(5)
–96.52
± 1.60
(5)
5.34 ± 0.87
(4)
1.05 ± 0.16
(5)
10 μM (R)-5
0.80 ± 0.03 (5)*
–57.12 ± 4.71
(5)
3.69 ± 1.69
(7)
–109.11
± 1.76
(5)
8.92 ± 0.82 (7)*
0.62 ± 0.31 (5)
N values are indicated
in parentheses. The protocols used for these parameters are illustrated
in Figure 7. Asterisks represent statistically
significant differences compared to control within each tested group
(p < 0.05, Student’s t test).
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 nonconducting (slow-inactivated) state at −50 mV.
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).
Fraction of
current remaining at
the end of the 30-pulse train, normalized to the first pulse.
Analysis of (R)-5 on electrophysiological
properties of Na1.1, Na1.3, Na1.7, and Na1.5 currents in HEK293 cells. (A, K) Voltage protocols for
examining slow inactivation, fast inactivation, activation, and frequency
(use) dependent block. NaV1.5 uses hyperpolarized protocols
because of differences in the hyperpolarized activation of this isoform.
(B, E, H, L) Summary of steady-state slow-activation curves for HEK293
cells treated with 0.1% DMSO (control) or 10 μM (R)-5. Insets illustrate representative current traces
from HEK293 cells in the absence (control, 0.1% DMSO) or presence
of 10 μM (R)-5. The black and
colored traces represent the currents evoked at −120 and −50
mV, respectively (highlighted in the voltage protocol). (C, F, I,
M) Representative Boltzmannfits for steady-state fast inactivation
and activation for HEK293 cells treated with 0.1% DMSO (control) or
10 μM (R)-5. 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
were averaged to determine the mean (±SEM) voltage dependence
of steady-state inactivation and activation, respectively. (D, G,
J, N) Summary of average frequency (use) dependent decrease in current
amplitude over time (±SEM) produced by control (0.1% DMSO) or
10 μM (R)-5. Data are from five
to six cells per condition.N values are indicated
in parentheses. The protocols used for these parameters are illustrated
in Figure 7. Asterisks represent statistically
significant differences compared to control within each tested group
(p < 0.05, Student’s t test).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 nonconducting (slow-inactivated) state at −50 mV.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).Fraction of
current remaining at
the end of the 30-pulse train, normalized to the first pulse.
Discussion
Recently,
we merged the structures of functionalized amino acids
(FAAs)[1,7−9] and α-aminoamides
(AAAs),[43] two classes of compounds that
have shown excellent anticonvulsant activities in seizure models,
to give compounds (R)-B and reported
on their anticonvulsant activities (Figure 8).[11] The substituted (R)-A compounds described herein are examples of (R)-B where R′ is a methoxymethyl moiety
(R′ = CH2OCH3) and the X-Y-Z unit is
a single bond; they are also 4′-aryl derivatives of (R)-1. In 2010, we showed that the unsubstituted N-(biphenyl-4′-yl)methyl derivative (R)-2 exhibited excellent anticonvulsant activity but
that seizure protection in mice and rats was accompanied by appreciable
neurotoxicity (Figure 1, Table 1; MES ED50 (mg/kg), 8.0 (mice, ip), 2.0 (rat, po);
TD50 (mg/kg), 11 (mice, ip), 49 (rat, po)).[10] Subsequently, we showed that the corresponding
3″-F-substituted analogue (R)-3 retained high anticonvulsant activity but that neurotoxicity was
reduced (Figure 1, Table 1; MES ED50 (mg/kg), 12 (mice, ip), 2.4 (rat, po); TD50 (mg/kg), 50 (mice, ip), rat >500 (rat, po)).[11] While the PI value in mice for (R)-3 was higher than for (R)-2, it was still below that of the ASD(R)-1 (PI (mice, ip): (R)-2, 1.4; (R)-3, 4.1; (R)-1, 6.0).[1] In this study, we explored the
effect of the terminal aryl R-substituent in (R)-A on anticonvulsant activity, neurotoxicity, and Na+ channel function.
Figure 8
Overlay of pharmacophores in FAAs and AAAs to give (R)-B.
Overlay of pharmacophores in FAAs and AAAs to give (R)-B.We found that the placement of polar, aprotic R-substituents
on
the terminal aryl ring of (R)-A provided
compounds with excellent anticonvulsant activities and that these
activities were only modestly affected by the site of aryl substitution
(3″ vs 4″). In addition, we found that several (R)-A derivatives with polar, aprotic R-substituents
had improved PI values over (R)-2 (PI
= 1.4) but that the PI values varied with the R-substituent and the
site of substitution. For the 3″-Cl and 4″-Cl derivatives
((R)-5, (R)-6), high PI values were obtained regardless of the site of substitution
(PI (mice, ip): (R)-5 (3″), 7.6;
(R)-6 (4″), 6.8) and that the
values exceeded that of (R)-2 (PI =
1.4) and even (R)-1 (PI = 6.0). Correspondingly,
for the 3″- and 4″-F derivatives ((R)-3, (R)-4) and the 3″-
and 4″-OCF3 compounds ((R)-11, (R)-12), high PI values
were observed for only the 3″-substituted isomers (PI (mice,
ip): (R)-3 (3″-F), 4.1; (R)-4 (4″-F), 1.8; (R)-11 (3″-OCF3), 4.9; (R)-12 (4″-OCF3), 2.0). Correspondingly,
when a polar, protic R-substituent was placed in the terminal aryl
ring in (R)-A to give (R)-13–(R)-16, we
observed minimal or no anticonvulsant activity. Accompanying this
loss of activity was a reduction in neurotoxicity, suggesting that
the activity loss resulted from either reduced levels of these compounds
in the CNS or a reduced interaction with the receptor(s) responsible
for the seizure protection and toxicity or both. When we tested whether
(R)-13, (R)-15, and (R)-16 modulated Na+ currents in ratembryonic cortical neurons, only (R)-15 (R = 3″-CO2H) displayed notable
activity (Supporting Information, Figure
S4). This latter finding indicated that the diminished anticonvulsant
activities for (R)-13 and (R)-16 may have resulted, in part, from their inability
to promote Na+ channel slow inactivation during the seizure
test.Both FAAs[7−9] and AAAs[44] likely exert
their anticonvulsant activities by interacting with Na+ channels. The FAA, (R)-1, has been
shown to preferentially promote the transition of Na+ channels
to the slow inactivation state without affecting fast inactivation.[7−9] We demonstrated that (R)-3 displayed
potent Na+ channel slow inactivation in CAD cells that
exceeded (R)-1 (IC50 (μM):
(R)-3, 2.9; (R)-1, 85).[15] Moreover, (R)-3 displayed notable frequency (use) dependent inhibition
of Na+ currents at concentrations (15 μM) above its
IC50 value (Figure 4B).[15] In this study, we determined the Na+ channel properties of the (R)-A compounds
in three different cell types (CAD cells, ratembryonic cortical neurons,
HEK293-transfected cells). Overall, we found it difficult to compare
the effects of individual (R)-A compounds
across the different cell systems. For several compounds, we observed
differences in the three cell types in their ability to affect Na+ channel fast inactivation and fast activation and frequency
(use) inhibition of Na+ currents. We have tentatively attributed
these differences to the different channel compositions, expression
levels, auxiliary proteins, and origins (mouse, rat, human) in the
CAD, cortical, and HEK293 cell systems and the test (R)-A concentrations used in the studies. Accordingly,
the experimental results in any given cell system were used to identify
interesting compounds, and direct comparisons were only made within
data sets from the same cell type.We found that (R)-4–(R)-8 and (R)-10–(R)-12, compounds that contained
polar, aprotic R-substituents, potently transitioned CAD cell Na+ channels to the slow-inactivated state (IC50 =
0.12–2.9 μM) and that Na+ channel fast inactivation
was not observed (Figures 2, 3; Supporting Information Figures
S1, S2). Furthermore, we observed that many compounds ((R)-4–(R)-6, (R)-10, (R)-11) showed frequency (use) dependent inhibition of Na+ currents,
provided that concentrations higher than the slow inactivation IC50 value were employed (Figure 4; Supporting Information, Figure S3). We[9] and others[7] have not
observed notable frequency (use) inhibition of Na+ currents
for (R)-1 at 100 μM. Our finding
that frequency (use) inhibition for selected (R)-A compounds was observed only at concentrations ≥5-fold
higher than their slow inactivation IC50 values in CAD
cells led us to retest (R)-1 at concentrations
above its IC50 value (85 μM). At 500 μM (R)-1, we observed noticeable frequency (use)
inhibition of Na+ currents (Figure 9) similar to that seen for the (R)-A compounds (Figure 4). Significantly, this
concentration is higher than the reported human therapeutic (R)-1 plasma concentrations (20–50 μM),[45] suggesting that frequency (use) inhibition of
Na+ currents may not be a clinically important pathway
for its drug function. In contrast, we found that several substituted
(R)-A derivatives, at low micromolar
concentrations, promoted Na+ channel slow inactivation
and frequency (use) dependent inhibition of Na+ currents.
This finding provides a potential comparative pharmacological advantage
for these compounds, since both mechanisms are proven pathways to
control neuronal hyperexcitability in the epileptic neuron.[7−9,39]
Figure 9
Effect of (R)-1 in CAD cells on frequency
(use) dependent block. Summary of average frequency (use) dependent
current amplitude decrease (±SEM) produced by control (0.1% DMSO)
or by the presence of various concentrations of (R)-1 is shown (p > 0.05, one-way
ANOVA
with Dunnett’s post hoc test). Concentrations greater than
500 μM (R)-1 caused a significant
inhibition of use-dependence starting at about pulse 8 (p < 0.05, one-way ANOVA with Dunnett’s post hoc test). Data
are from four to six cells per condition.
Effect of (R)-1 in CAD cells on frequency
(use) dependent block. Summary of average frequency (use) dependent
current amplitude decrease (±SEM) produced by control (0.1% DMSO)
or by the presence of various concentrations of (R)-1 is shown (p > 0.05, one-way
ANOVA
with Dunnett’s post hoc test). Concentrations greater than
500 μM (R)-1 caused a significant
inhibition of use-dependence starting at about pulse 8 (p < 0.05, one-way ANOVA with Dunnett’s post hoc test). Data
are from four to six cells per condition.To better approximate CNS VGSC activities, we tested many
of the
(R)-A compounds with a 3″-polar,
aprotic R-substituent in ratembryonic cortical neurons that expressed
the NaV1.1, NaV1.2, NaV1.3, and NaV1.6 channels. We observed that (R)-3, (R)-5, (R)-7, (R)-8, (R)-10, and (R)-11 all promoted
slow inactivation at 10 μM (Figure 5)
and their relative effectiveness mirrored the slow inactivation IC50 values measured in CAD cells (Figure 2). Differences were found between the cortical neurons and the CAD
cells on the ability of these compounds to affect fast inactivation
and their ability to inhibit frequency (use) dependent inhibition
of Na+ currents (Figures 3, 4, 6; Supporting Information, Figure S5). We have not explored the
factors that contributed to these differences.There are nine
Na+ channel isoforms. Despite structural
similarity, the isoforms’ distribution and biophysical properties
(e.g., kinetics of activation and inactivation, voltage, and frequency
(use) dependency) are distinct, each having specific functions.[39,41,46] Thus, we evaluated (R)-5 in HEK293 cells expressing hNaV1.1, rNaV1.3, hNaV1.5, or hNaV1.7 channels. The
patch-clamp electrophysiology protocols for the cells containing hNaV1.5 were different from those used for hNaV1.1,
rNaV1.3, and hNaV1.7 (Figure 7A,K). We found that 10 μM (R)-5 transitioned the Na+ channels to the slow-inactivated
state regardless of channel type (Figure 7B,E,H,L).
Correspondingly, differences in the fast inactivation profiles were
observed for the four isoforms. Compound (R)-5 did not affect rNaV1.3 fast inactivation compared
with 0.1% DMSO (control), while a depolarizing shift was found for
the hNaV1.1-, hNaV1.5-, and hNaV1.7-transfected
cells, with the most pronounced shift observed in hNaV1.7-expressing
cells (Figure 7C,F,I,M). Finally, we observed
notable frequency (use) inhibition of Na+ currents for
the transfected hNaV1.5 cells (Figure 7N), lesser amounts for the hNaV1.1- and hNaV1.7-expressing cells (Figure 7D,J),
and no inhibition for the rNaV1.3-expressing cells (Figure 7G). Together these findings showed that (R)-5, and perhaps the other polar, aprotic
R-substituted (R)-A derivatives, displayed
little Na+ channel isoform specificity. Rather, (R)-5 appears to act as a functionally selective
Na+ channel inhibitor[47] that
controls hyperexcitability upon excitation by transitioning a greater
fraction of Na+ channels to their slow-inactivated state
and by inhibiting Na+ currents in a frequency (use) dependent
manner.
Conclusions
The successful merger of FAAs and AAAs
(Figure 8) provided substituted N-(biphenyl-4′-yl)methyl
(R)-2-acetamido-3-methoxypropionamide derivatives
((R)-A). Select (R)-A compounds displayed low ED50 values and high
PI values that, in rodents, compared favorably with most clinical
ASDs. We found compounds that potently transitioned Na+ channels into the slow-inactivated state and displayed frequency
(use) inhibition of Na+ currents at low micromolar concentrations.
The activities of (R)-As along with
their pharmacokinetic properties are being investigated further.
Experimental Section
General Methods
Melting points were determined in open
capillary tubes using a Thomas-Hoover melting point apparatus and
are uncorrected. 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
(δ) 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 analytical thin-layer
chromatography (TLC) plates (Aldrich, catalog no. Z12272-6) and analyzed
with 254 nm UV light. The mixtures were purified by flash column chromatography
using silica gel (Dynamic Adsorbents Inc., catalog no. 02826-25).
All chemicals and solvents were reagent grade and used as obtained
from commercial sources without 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 NMR and the analytical data confirmed that the purity of the products
was ≥95%.
General Procedure for the Deprotection and
Acetylation of (R)-N-(Biphenyl-4-yl)methyl
2-N-(tert-Butoxycarbonyl)amino-3-methoxypropionamide
Derivatives (Method 1)
A CH2Cl2 solution
(0.1–0.3 M) of the (R)-N-(biphenyl-4-yl)methyl
2-N-(tert-butoxycarbonyl)amino-3-methoxypropionamide
derivative was treated with 4 M HCl in dioxane (3–4 equiv)
at room temperature (2–6 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. The resulting solution was stirred at room temperature
(2–4 h). The resulting solution was washed with an aqueous
10% citric acid solution followed 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.
Compounds
were screened under the auspices
of the National Institutes of Health’s ASP. Experiments were
performed in male rodents (albino Carworth Farms No. 1 mice (ip),
albino Sprague–Dawley rats (ip, po)). Housing, handling, and
feeding were in accordance with recommendations contained in the Guide
for the Care and Use of Laboratory Animals. Anticonvulsant activity
was established using the MES test,[4] 6
Hz,[5] and the scMet test,[23] according to previously reported methods.[1]
Catecholamine A Differentiated (CAD) Cells
CAD cells
were grown at 37 °C and in 5% CO2 (Sarstedt, Newton,
NC) in Ham’s F12/EMEM (GIBCO, Grand Island, NY), supplemented
with 8% fetal bovine serum (Sigma, St. Louis, MO) and 1% penicillin/streptomycin
(100% stocks, 10 000 U/mL penicillin G sodium and 10 000
μg/mL streptomycin sulfate).[9,14] Cells were
passaged every 6–7 days at a 1:25 dilution.
Cortical Neurons
Rat cortical neuron cultures were
prepared from cortices dissected from embryonic day 19 brains exactly
as described.[48,49]
Culturing HEK293 Cells
Expressing NaV1.1, NaV1.3, NaV1.5,
and NaV1.7
The
cDNA gene encoding NaV1.1 was codon-optimized and synthesized
using the open reading frame (accession no. NC_000002.11) and subcloned
into the vector pTarget. The cDNA genes encoding NaV1.3
from rat and NaV1.7 from human were subcloned into the
vector pcDNA3.1-mod. The cDNA encoding NaV1.5 from human[50] was introduced into pcDNA3.1(+) with the CMV
promoter. The constructs were then transfected into HEK293 cells using
the calcium phosphate precipitation technique. After 48 h, the cells
were passaged into 100 mm dishes and treated with G418 (Geneticin,
Life Technologies) at 800 μg/mL to select for neomycin resistant
cells. After 2 weeks, colonies were picked and split. The colonies
were then tested for channel expression with whole-cell patch-clamp
technique. The cell line was then maintained with 500 μg/mL
G418. NaV1.1,[34] NaV1.3,[51] NaV1.5,[52] and NaV1.7[53] stable
cells were grown under standard tissue culture conditions (5% CO2 at 37 °C) in Dulbecco’s modified Eagle medium
supplemented with 10% fetal bovine serum.
Electrophysiology
Whole-cell voltage clamp recordings
were performed at room temperature on HEK293 cells, CAD cells, and
cortical neurons using an EPC 10 amplifier (HEKA Electronics, Lambrecht/Pfalz
Germany). Electrodes were pulled from thin-walled borosilicate glass
capillaries (Warner Instruments, Hamden, CT) with a P-97 electrode
puller (Sutter Instrument, Novato, CA) such that final electrode resistances
were 1–2 MΩ when filled with internal solutions. The
internal solution for recording Na+ currents contained
the following (in mM): 110 CsCl, 5 MgSO4, 10 EGTA, 4 ATPNa2-ATP, 25 HEPES (pH 7.2, 290–310 mOsm/L). The
external solution contained the following (in mM): 100 NaCl, 10 tetraethylammonium
chloride (TEA-Cl), 1 CaCl2, 1 CdCl2, 1 MgCl2, 10 d-glucose, 4 4-AP, 0.1 NiCl2, 10
HEPES (pH 7.3, 310–315 mOsm/L). Whole-cell capacitance and
series resistance were compensated with the amplifier. Series resistance
error was always compensated to be less than ±3 mV. Cells were
considered only when the seal resistance was less than 3 MΩ.
Linear leak currents were digitally subtracted by P/4.
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 Na+ channels was calculated using the equation G = I/(Vm – Vrev), where Vrev is the reversal potential, Vm is the
membrane potential at which the current was recorded, and I is the peak current. Activation and inactivation curves
were fitted to a single-phase Boltzmann function G/Gmax = 1/{1 + exp[(V – V50)/k]}, where G is the peak conductance, Gmax is the
fitted maximal G, V50 is the half activation voltage, and k is the slope
factor. Additional details of specific pulse protocols are described
in the results text or figure legends.
Statistical Analyses
Differences between mean values
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 the mean ±
SEM, with p < 0.05 considered as the level of
significance.
Authors: Yuying Wang; Joel M Brittain; Brian W Jarecki; Ki Duk Park; Sarah M Wilson; Bo Wang; Rachel Hale; Samy O Meroueh; Theodore R Cummins; Rajesh Khanna Journal: J Biol Chem Date: 2010-06-09 Impact factor: 5.157
Authors: M E Gellens; A L George; L Q Chen; M Chahine; R Horn; R L Barchi; R G Kallen Journal: Proc Natl Acad Sci U S A Date: 1992-01-15 Impact factor: 11.205
Authors: Yuying Wang; Ki Duk Park; Christophe Salome; Sarah M Wilson; James P Stables; Rihe Liu; Rajesh Khanna; Harold Kohn Journal: ACS Chem Neurosci Date: 2011-02-16 Impact factor: 4.418
Authors: Thomas Stöhr; Harvey J Kupferberg; James P Stables; Daeock Choi; Robert H Harris; Harold Kohn; Nancy Walton; H Steve White Journal: Epilepsy Res Date: 2007-04-12 Impact factor: 3.045
Authors: Christophe Salomé; Elise Salomé-Grosjean; Ki Duk Park; Pierre Morieux; Robert Swendiman; Erica DeMarco; James P Stables; Harold Kohn Journal: J Med Chem Date: 2010-02-11 Impact factor: 7.446
Authors: Robert Torregrosa; Xiao-Fang Yang; Erik T Dustrude; Theodore R Cummins; Rajesh Khanna; Harold Kohn Journal: Bioorg Med Chem Date: 2015-04-11 Impact factor: 3.641
Authors: Ki Duk Park; Xiao-Fang Yang; Erik T Dustrude; Yuying Wang; Matthew S Ripsch; Fletcher A White; Rajesh Khanna; Harold Kohn Journal: ACS Chem Neurosci Date: 2014-12-09 Impact factor: 4.418
Figure 1
Scheme 1
Scheme 6
Scheme 3
Scheme 5
Scheme 4
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
Figure 9