Identification and Electrophysiological Evaluation of 2-Methylbenzamide Derivatives as Nav1.1 Modulators

Article abstract of DOI:10.1021/acschemneuro.5b00147

Voltage-gated sodium channels (Na_v) are crucial to the initiation and propagation of action potentials (APs) in electrically excitable cells, and during the past decades they have received considerable attention due to their therapeutic potential. Here, we report for the first time the synthesis and the electrophysiological evaluation of 16 ligands based on a 2-methylbenzamide scaffold that have been identified as Na_v1.1 modulators. Among these compounds, N,N′-(1,3-phenylene)bis(2-methylbenzamide) (3a) has been selected and evaluated in ex-vivo experiments in order to estimate the activation impact of such a compound profile. It appears that 3a increases the Na_v1.1 channel activity although its overall impact remains moderate. Altogether, our preliminary results provide new insights into the development of small molecule activators targeting specifically Na_v1.1 channels to design potential drugs for treating CNS diseases.

Full text of DOI:10.1021/acschemneuro.5b00147

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Letter
Identification and Electrophysiological Evaluation of 2-
Methylbenzamide Derivatives as Nav1.1 Modulators
François Crestey, Kristen Frederiksen, Henrik S. Jensen, Kim Dekermendjian, Peter H. Larsen,
Jesper F. Bastlund, Dunguo Lu, Henry Liu, Charles Yang, Morten Grunnet, and Niels Svenstrup
ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.5b00147 • Publication Date (Web): 26 Jun 2015
Downloaded from http://pubs.acs.org on July 4, 2015
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Identification and Electrophysiological Evaluation of 2-Methylbenzamide
Derivatives as Nav1.1 Modulators
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François Crestey,§ Kristen Frederiksen,§ Henrik S. Jensen,§ Kim Dekermendjian,§ Peter H. Larsen,§
Jesper F. Bastlund,§ Dunguo Lu,† Henry Liu,† Charles Yang,† Morten Grunnet *,§ and Niels Svenstrup*,§
§Neuroscience Drug Discovery, H. Lundbeck A/S, Ottiliavej 9, 2500 Valby, Denmark
†ChemPartner Co. Ltd., 998 Halei Road, Zhangjiang HiꢀTech Park, Shanghai, 201203, P. R. China
AUTHOR EMAIL ADDRESS: mgru@lundbeck.com, nisv@lundbeck.com
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RECEIVED DATE (to be automatically inserted after your manuscript is accepted if required
according to the journal that you are submitting your paper to)
TITLE RUNNING HEAD
2-Methylbenzamide derivatives as Nav1.1 modulators
CORRESPONDING AUTHOR FOOTNOTE
*
To whom correspondence should be addressed. Phone: +45 3643 2421. Eꢀmail: nisv@lundbeck.com;
Phone: +45 3643 5173. Eꢀmail: mgru@lundbeck.com
ABSTRACT. Voltageꢀgated sodium channels (Nav) are crucial to the initiation and propagation of
action potentials (APs) in electrically excitable cells and during the past decades they have received
considerable attention due to their therapeutic potential. Here, we report for the first time the synthesis
and the electrophysiological evaluation of 16 ligands based on a 2ꢀmethylbenzamide scaffold that have
been identified as Nav1.1 modulators. Among these compounds, N,N'ꢀ(1,3ꢀphenylene)bis(2ꢀ
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methylbenzamide) (3a) has been selected and evaluated in exꢀvivo experiments in order to estimate the
activation impact of such a compound profile. It appears that 3a increases the Nav1.1 channel activity
although its overall impact remains moderate. Altogether, our preliminary results provide new insights
into the development of small molecules activators targeting specifically Nav1.1 channels to design
potential drugs for treating CNS diseases.
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KEYWORDS: 2ꢀmethylbenzamide derivatives, Nav1.1 modulator, voltageꢀgated sodium channels, brain
slices, fastꢀspiking interneurons
Voltageꢀgated sodium channels1 play a pivotal role in initiating and propagating action potentials
(APs) in neurons, myocytes and endocrine cells.2 Thus, Na+ channels have been the focus of a vast body
of research including drug discovery activities.3 Indeed, various sodium channel inhibitors have
successfully been developed as human drugs and are currently approved for treating a variety of
indications such as pain, epilepsy, migraine, bipolar disorders and cardiac disrhythmia.4 By contrast,
sodium channel activators are also known but have not previously been considered relevant in
therapeutic settings mostly due to a high perceived risk of toxicity and convulsions. It has been clearly
established that sodium channels play an important role in neuronal excitability by initiation and
propagation of APs.5 Interestingly, Nav1.1 comprises the majority of the sodium current in GABAergic
fastꢀspiking inhibitory interneurons that are immunoreactive for parvalbumin. Furthermore, it plays only
a modest role in excitatory neurons due to the high redundancy of other types of sodium channels in
these cells. Moreover, mouse models of reduced Nav1.1 channel expression have revealed several
phenotypes such as autisticꢀlike behaviors, cognitive deficits, severe epilepsy and unexpected death.
Accordingly, it has been well recognized that an important number of diseases, especially Dravet
syndrome, are closely related with loss of function mutations in Nav1.1 channels.6 Jensen and coꢀ
workers recently published that selective activators of the Nav1.1 sodium channel may hold therapeutic
potential within diseases such as epilepsy, schizophrenia and Alzheimer’s disease.7 Different classes of
nonselective sodium channel activators are well known including the pyrethroid insecticides
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exemplified by allethrin and deltamethrin, the steroidꢀlike alkaloidꢀbased toxins veratridine and
batrachotoxin, as well as a number of peptideꢀtoxins isolated from diverse organisms such as spiders,
scorpions and sea anemones.8 From a drug discovery perspective nonꢀselective Na+ channel activators
are considered nonꢀviable due to general overall neuronal activation introducing a state of
hyperexcitability and thereby a number of epileptogenic related adverse effects. Some degree of
selectivity is therefore a likely prerequisite for bringing a Na+ channel activator to the market. So far, no
claims or descriptions have been made for specific Nav1.1 activators.
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In order to identify such activators, a highꢀthroughput screening (HTS) of a smallꢀmolecule library (~
55000 compounds) has been performed at a single concentration (30 ꢀM) using a voltage protocol and
veratridine and mexiletine as assay controls. Several derivatives having a 1,3ꢀdiamidobenzene core9 and
even more precisely containing a 2ꢀmethylbenzamide moiety were identified and confirmed as hits.
Herein, we wish to present the design of 16 ligands bearing a 2ꢀmethylbenzamide unit in order to study
the structureꢀactivity relationship (SAR) of such a class compound as well as their electrophysiological
evaluation on a HEK cell line with stable expression of Nav1.1 channels. Furthermore, derivative 3a was
selected for brain slice experiments in order to understand the overall impact of the compound profile.
Chemistry
The synthetic pathway to obtain 2ꢀmethylbenzamide derivatives 3a–j is outlined in Scheme 1. Benzeneꢀ
1,3ꢀdiamine 1 reacted with one equivalent of 2ꢀmethylbenzoyl chloride in the presence of triethylamine
(TEA) to furnish phenylamine 2 and bisbenzamide 3a in 45% and 35% yields, respectively. Treatment
of 2 with the appropriate acyl chlorides in a mixture of DCE–DCM provided the corresponding bisꢀ
benzamide derivatives 3b–i in 83–99% yields.10 Similarly, sulfonamide analogue 3j was obtained from
the reaction of 2 with 2ꢀmethylbenzenesulfonyl chloride in 89% yield.
The reaction sequence depicted in Scheme 2 was used as a convenient route to the compounds 3k–n.
After treatment of aminophenol 4a with 2ꢀmethylbenzoyl chloride in the presence of TEA and a
catalytic amount of DMAP ester 3k was isolated in 89% yield. Following a similar protocol as above,
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aminopyridine 4b and benzeneꢀ1,4ꢀdiamine 7 led to benzamide derivatives 3l and 3n in 66% and 94%
yields, respectively. Finally, the diamide derivative 3m was synthesized in a twoꢀstep protocol:
aminobenzoic 5 was first acylated providing carboxylic acid 6 in 82% yield. Subsequently, amidation
with oꢀtoluidine, HATU as coupling reagent and TEA in dry DMF gave 3m in 58% yield. In addition to
the series of the 14 ligands detailed above, amide derivative 3o was prepared in two steps in which one
of the usual 2ꢀmethylbenzamide moieties was replaced by a tetrazole amideꢀbioisostere. Thus, tetrazole
8 furnished after C–H activation aniline 9 in 16% yield which was subsequently acylated to lead to
tetrazole 3o in 87% yield. Moreover, constrained analogue 3p was prepared in a threeꢀsteps procedure
in order to test how the introduction of conformational restraint to a moderately flexible lead molecule
would influence the activating properties with regards to the Nav1.1 channel. Thus, nitroindoline 10 was
first acylated furnishing amide 11 which the nitro group was reduced with a slight excess of Na2S2O4 in
the presence of K2CO3. Subsequent acylation of the resulting intermediate with 2ꢀmethylbenzoyl
chloride afforded indoline 3p in 21% yield over three steps (Scheme 2).
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Results and discussion
To obtain a thorough biological understanding and reꢀconfirmation of hits identified during the
primary screen various electrophysiological approaches were performed. Since the primary screen was
performed in an automated setting (Ion works/Barracuda) that does not allow for electrophysiological
experiments in a quality that can match manual patch clamp with giga seals, the first attempt was to
confirm the hits under conditions where a true giga seal can be obtained between the cell and the
recording pipette.11 This condition allows for a very precise determination, or clamp, of the cell
membrane that can subsequently be controlled. Especially for ion channels that conduct large amount of
currents and in addition show fast activation and inactivation kinetics, like voltageꢀgated Na+ channels,
reliable continuous control of the membrane potential is a prerequisite to fully characterize compound
interactions with the channel. As an alternative to manual patch clamp experiments the automated Qꢀ
patch was applied where it is also possible to obtain giga ohm seals comparable to manual patch
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clamp.12 Nav1.1 channels ꢀ stable expressed in HEK cells ꢀ were activated by depolarizing voltage steps
from – 120 mV to + 70 mV in 10 mV increments to obtain current–voltage (I–V) curves showing the
expected voltage dependent activation revealed as an inward current deflection followed by fast
inactivation as demonstrated in Figure 1. For more detailed compound evaluation a more simple
protocol was applied, i.e. with only 6 voltage steps from – 40 mV to + 10 mV (with 10 mV increments).
Each depolarizing step was activated from – 120 mV and lasted for 20 ms and was separated with 500
ms. A holding potential (Vhold) of – 80 mV was used between each protocol with 6 voltage steps (see
Figure 2). A modulator of the Nav1.1 channels can exert its action in a number of ways and the most
relevant mode of action and associated electrophysiological profile for obtaining biological activity is at
present unknown. We chose to focus on 1) impact on peak current, 2) total area under the curve (AUC)
for the time duration of the depolarizing pulse and 3) the compound´s ability to decrease inactivation
velocity. The later parameter is addressed by determining the tau value of the inactivation current decay.
In this setting a slowing of the inactivation, and thereby more conducted current, is revealed by an
increase in the tau value. As summarized in Table 1, compounds 3a, 3c–j, 3l, 3n and 3p from this 2ꢀ
methylbenzamide family had a profile with a reduced peak current, but a compromised inactivation
revealed as an increased tau value. As an example, peak current, AUC and tau values (in %) relative to
control at – 10 mV for benzamide 3a were 52, 81 and 124, respectively (see Figure 3). Unfortunately,
no clear picture appeared with respect to the substitution pattern on the aromatic ring. However, orthoꢀ
and metaꢀsubstituted rings 3a and 3c–f, unsubstituted ring compound 3g, derivatives bearing aliphatic
moieties 3h–i, sulfonamide 3j, pyridine derivative 3l, 1,4ꢀsubstituted benzamide 3n and constrained
indoline 3p did provide novel active analogues displaying a noteworthy trend for this class of
compounds: the total AUC was significantly reduced but much less than the peak current while the tau
value was considerably increased. Tetrazole 3o, ester 3k and amide 3m were nearly inactive with no
noticeably effects on the three parameters of our designed electrophysiological protocol (see Table 1 for
more details).
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Voltageꢀgated sodium channels share high similarities of sequence, large expression in neurons
(except Nav1.4 which is expressed mainly in skeletal muscle and Nav1.5 which is found primarily in
cardiac muscle) and biophysical characteristics13 In order to quantify the selectivity profile of the
compounds the same electrophysiological protocol described above was applied using benzamide 3a on
Nav1.2, ꢀ1.4, ꢀ1.5, ꢀ1.6 and ꢀ1.7 channels ꢀ stable expressed in HEK cells. As depicted in Table 2, peak
values were significantly less reduced and tau values were significantly lower while the AUC values
remained almost constant when compared with the values obtained for Nav1.1 channel. In addition,
intrinsic hepatic clearances (Clint) of 8 synthesized compounds were also estimated for both rat and
human after incubation of the corresponding compound in rat or human liver microsomes; apart from
derivative 3p, Clint,rat and Clint,human were relatively high with values between 3 and 38 L/kg/h (rat) and
between 0.9 and 6.3 L/kg/h (human), respectively. Moreover, PgP efflux and permeability was
investigated for a selection of compounds in the MDR1ꢀMDCK cell assay. MDCK ratio14 of derivatives
3a, 3c, 3d and 3l were 0.45, 0.52, 0.31 and 0.64, respectively showing that this class of compound has a
small or modest efflux ratio (see Supporting Information for more details).
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The biological nature of a compound profile as described above is difficult to predict. To gain more
insight into whether an overall inhibitory, neutral or activation impact of these compounds was
prevailing, exꢀvivo experiments with brain slices were performed (see Supporting Information for more
details). In brief, whole cell patch clamp were performed on parvalbumin containing interneurons with
basket cell appearance in the hippocampal pyramidal cell layer. As illustrated in Figure 4A, cells were
initially challenged by ramps starting with resting membrane potentials from – 70 mV that were
gradually depolarized. The experiments were performed in the absence (control) and presence of Nav1.1
modulator 3a and as summarized data in Figure 4B application of 3a shifted the threshold for firing APs
to a more negative value. The reason for this is believed to be the activation of Nav1.1 channels that
facilitate the neuron to generate APs. To obtain a more thorough evaluation of the Nav1.1 modulators
ability to modify interneuron firing frequency depolarizing steps with different amplitude were provided
to the neuron to provoke APs (see Figure 4C). As observed, 3a was able to increase the number of
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evoked APs even though the effect was most prominent after weak depolarizations that render a larger
window available for further activity increase. Data for application at 30 and 100 µM of benzamide 3a
are depicted in Figure 4D. For inputꢀoutput and threshold experiments a number of time matched
control were performed to make certain that the observed effects were not due to sliding baseline as a
function of time (data not shown).
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To enable correct identification of fastꢀspiking parvalbumin expressing interneurons some
electrophysiological experiments were performed with biocytin in the pipette. After successful
recordings biocytin was injected into the cell and the slice was fixed for later identification. Slices were
then coꢀstained for biocytin and parvalbumin to confirm that the compound had actually exerted its
effect at a parvalbumin expressing interneuron (see Supporting Information for more details).
Conclusions
In summary, we have discovered novel 2ꢀmethylbenzamide derivatives that act as Nav1.1 modulators.
Among them, 2ꢀmethylbenzamide derivative 3a has been demonstrated in exꢀvivo experiments to act as
a functional activator of fastꢀspiking hippocampal interneurons. Even though its overall activation
impact was moderate, addition of 3a clearly resulted in an increase of activity of the Nav1.1 channel.
These preliminary yet promising results open up a novel and challenging area of investigations on
pharmacological activators of Nav1.1 channels since such compound may have important therapeutic
potential in the treatment of various CNS diseases. Further studies concerning discovery of subtype
selective small molecule activators of the Nav1.1 channel are currently in progress in our laboratories.
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Experimental
1.1 Chemistry - Material and methods
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Starting materials and reagents were obtained from commercial suppliers and used without further
purifications. Syringes which were used to transfer anhydrous solvents or reagents were purged with
nitrogen prior to use. Yields refer to isolated compounds estimated to be > 95 % pure as determined by
1H NMR and LC–MS. Thinꢀlayer chromatography (TLC) was carried out on silica gel 60 F254 plates
from Merck (Germany). Visualisation was accomplished by UV lamp (254 nm). Flash column
chromatography was performed on chromatography grade, silica 60 Å particle size 35–70 micron from
Fisher Scientific using the solvent system as stated. Melting points were determined on a Büchi
apparatus and are uncorrected. HRMS were performed on a Bruker MicroTOF instrument. 1H NMR
spectra were recorded on a Bruker 600ꢀAvanceꢀIII spectrometer equipped with a 5 mm TCI cryoprobe
operating at 600 MHz using CDCl3 or DMSOꢀd6 as solvents and TMS as internal standard. Coupling
constants (J values) are given in Hertz (Hz). Multiplicities of 1H NMR signals are reported as follows: s,
singlet; d, doublet; dd, doublet of doublets; t, triplet; dt, doublet of triplet; tt, triplet of triplets; ddd,
doublet of doublet of doublets; m, multiplet; br, broad signal. LC–MS data were obtained on a PE Sciex
API150EX instrument equipped with an ionꢀspray source and Shimadzu LC–8A/SLC–10A LC system:
column, 30 mm × 4.6 mm Waters Symmmetry C18 column with 3.5 ꢁm particle size; solvent system, A
= water–TFA (100:0.05) and B = water–acetonitrile–TFA (5:95:0.03) (TFA = trifluoroacetic acid);
method, linear gradient elution with 90% A to 100% B in 2.4 min and then 10% B in 0.4 min, with a
flow rate of 3.3 mL/min. Total time including equilibration was 2.8 min. Injection volume was 10 ꢁL
from a Gilson 215 autosampler.
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1.2 Synthesis and analytical data of representative compounds
3-Methoxy-N-(3-(2-methylbenzamido)phenyl)benzamide 3e.
To a solution of phenylamide 2 (143 mg, 0.63 mmol, 1 equiv) in a mixture DCE–DCM (5:1, 12 mL) at
room temperature was added TEA (97 ꢁL, 0.70 mmol, 1.1 equiv). The reaction mixture was cooled to 0
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°C and 3ꢀmethoxybenzoyl chloride (93 ꢁL, 0.66 mmol, 1.05 equiv) was added dropwise over 1 min.
After few min the reaction mixture was allowed to warm up to room temperature and was stirred at this
temperature for 2 h and concentrated in vacuo. The crude material was purified by column
chromatography on silica gel using a gradient elution (EtOAc–heptane, 1:1 to 3:2) to afford diamide 3e
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(222 mg, 97%) as a white solid; Rf = 0.54 (EtOAc–heptane, 1:1); mp 163.9–165.6 °C; H NMR (600
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MHz, DMSOꢀd6): δ 10.35 (br s, 1H), 10.16 (br s, 1H), 8.27–8.34 (m, 1H), 7.50–7.57 (m, 3H), 7.43–7.47
(m, 2H), 7.37–7.42 (m, 2H), 7.28–7.33 (m, 3H), 7.16 (ddd, J = 8.2 and 2.6 and 0.9, 1H), 3.84 (s, 3H),
2.39 (s, 3H); LC–MS (m/z) 361.3 (MH+); RT = 0.70; purity (UV, ELSD): 100%; 100%; HRMS
+
C26H32N3O3 [M+H2NEt2 ] calc 434.2438, found 434.2440.
N,N'-(Pyridine-2,6-diyl)bis(2-methylbenzamide) 3l.
To a solution of aminopyridine 4b (400 mg, 3.67 mmol, 1 equiv) in a mixture DCE–DCM (5:1, 48 mL)
at room temperature was added TEA (1.07 mL, 7.70 mmol, 2.1 equiv). The reaction mixture was cooled
to 0 °C and 2ꢀmethylbenzoyl chloride (0.98 mL, 7.51 mmol, 2.05 equiv) was added dropwise over 3
min. After few min the reaction mixture was allowed to warm up to room temperature and was stirred at
this temperature for 22 h and concentrated in vacuo. The crude material was purified by column
chromatography on silica gel using a gradient elution (EtOAc–heptane, 3:5 to 3:4) to afford the desired
diamide 3l (840 mg, 66%) as a colourless oil which slowly crystallized as a white solid when stored in
the fridge overnight; Rf = 0.44 (EtOAc–heptane, 3:5); 1H NMR (600 MHz, DMSOꢀd6): δ 10.51 (s, 2H),
7.79–7.93 (m, 3H), 7.41–7.48 (m, 2H), 7.38 (dt, J = 7.5 and 1.3, 2H), 7.23–7.32 (m, 4H), 2.39 (s, 6H);
LC–MS (m/z) 346.1 (MH+); RT = 0.73; purity (UV, ELSD): 96%; 100%; HRMS C25H31N4O2
+
[M+H2NEt2 ] calc 419.2442, found 419.2453.
N,N'-(1,4-phenylene)bis(2-methylbenzamide) 3n.
To a solution of benzeneꢀ1,4ꢀdiamine 7 (450 mg, 4.16 mmol, 1 equiv) in a mixture DCE–DCM (8:3, 22
mL) at room temperature was added TEA (1.27 mL, 9.15 mmol, 2.2 equiv). The reaction mixture was
cooled to 0 °C and 2ꢀmethylbenzoyl chloride (1.14 mL, 8.74 mmol, 2.1 equiv) was added dropwise over
2 min. After few min the reaction mixture was allowed to warm up to room temperature and was stirred
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at this temperature for 1.5 h then at 50 °C for 1 h. The resulting mixture was cooled to room temperature
and filtered. The resulting precipitate was washed successively with DCM, DCE and water then freezeꢀ
dried overnight to afford the desired diamide 3n (1.35 g, 94%) as a white solid; Rf = 0.52 (EtOAc–
heptane, 1:1); 1H NMR (600 MHz, DMSOꢀd6): δ 10.28 (s, 2H), 7.70 (s, 4H), 7.46 (d, J = 7.4, 2H), 7.39
(dt, J = 7.9 and 1.2, 2H), 7.26–7.33 (m, 4H), 2.38 (s, 6H); LC–MS (m/z) 345.3 (MH+); RT = 0.69;
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purity (UV, ELSD): 95%; 93%; HRMS C26H32N3O2 [M+H2NEt2 ] calc 418.2489, found 418.2485.
1.3 Electrophysiology - Methods
A HEK cell line with stable expression of Nav1.1 channels was used for inꢀvitro electrophysiological
recordings in patch clamp mode. I–V curves for both activation and inactivation was performed in order
to confirm the expected characteristic of Nav1.1 channels, both with manual patch clamp and with
automated patch clamp (Ion Works and QꢀPatch). The first HTS screening of a small library was
performed on the Ion Works platform, but the following evaluation of new compounds from medicinal
chemistry efforts was performed with a Qꢀpatch16x in single hole mode. It is possibly to obtain giga
ohm seals with the Qꢀpatch, which make higher quality recordings more comparably to manual patch.
For electrophysiological recordings HEK cells were harvested with detachin (5–10 min) and applied to
the Qꢀpatch with a cell density between 1 and 3 million cells per mL. The cell suspension could be used
up to 4ꢀ5 hours after harvesting with good performance. For evaluation of the effects of compound on
the peak current, AUC and tau values a protocol with 6 voltage steps were constructed. The voltage
steps were depolarizing steps from – 40 mV up to + 10 mV (with 10 mV increments). Each depolarizing
step was activated from –120 mV and lasted for 20 ms and was separated with 500 ms. A Vhold of – 80
mV was used between each protocol with 6 voltage steps (Figure 2). The single protocol with 6 voltage
steps was run approximately every min. For test of compounds in a single concentration the voltage
protocol was run 6 times in control (~ 6 min) followed by drug application and 6 voltage run in drug (~
6 min) and finally wash out and 6 voltage run again (~ 6 min). Effects of compounds were measured
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relative to control values before drug application for each cell. For more details concerning all the cell
lines, see Supporting Information.
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ACKNOWLEDGMENT
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The authors thank the Danish National Advanced Technology Foundation for financial support (Grant
023ꢀ2011ꢀ3) as well as Peter Brøsen for HRMS measurements and Dr. Martin Carnerup for providing
pharmacological data.
LIST OF ABBREVIATIONS
The following abbreviations are used: DCM: dichloromethane; DCE: 1,2ꢀdichloroethane; EtOAc: ethyl
acetate; TEA: triethylamine; THF: tetrahydrofuran; DMAP: 4ꢀdimethylaminopyridine; HATU: 1ꢀ
[bis(dimethylamino)methylene]ꢀ1Hꢀ1,2,3ꢀtriazolo[4,5ꢀb]pyridinium 3ꢀoxid hexafluorophosphate; DMF:
N,Nꢀdimethylformamide; NMP: Nꢀmethylꢀ2ꢀpyrrolidone; AUC: area under the curve; MDCK: Madinꢀ
Darby Canine Kidney; CNS: central nervous system; AP: action potential; current–voltage: I–V; Vhold
holding potential; Clint: intrinsic hepatic clearance.
:
SUPPORTING INFORMATION
1
Full experimental details for all synthesized compounds, H and 13C NMR spectra of selected
compounds, brain slice experiments and coꢀstaining protocols as well as table containing intrinsic
clearance and MDCK ratio. This material is available free of charge via the Internet at
http://pubs.acs.org.
COMPETING FINANCIAL INTERESTS
The authors declare no competing financial interest.
AUTHORS CONTRIBUTIONS
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H.S.J., K.D., J.F.B., M.G. and N.S. conceived and designed the project. F.C. carried out the organic
synthesis and analyzed all the compounds. K.F. carried out and analyzed the inꢀvitro experiments.
P.H.L. carried out and analyzed the coꢀstaining experiments. D.L., H.L. and C.Y. carried out and
analyzed the exꢀvivo experiments. F.C., K.F., M.G. and N.S. wrote the manuscript with the help of D.L.,
H.L. and C.Y.
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REFERENCES
(1) a) Wood, J. N.; Baker, M. (2001) Voltageꢀgated Sodium Channels. Curr. Opin. Pharmacol. 1, 17–
21; b) Yu, F. H.; Catterall, W. A. (2003) Overview of the Voltageꢀgated Sodium Channel Family.
Genome Biol. 4, 207.
(2) a) Hodgkin, A. L.; Huxley, A. F. (1952) A Quantitative Description of Membrane Current and its
Application to Conduction and Excitation in Nerve. J. Physiol. 117, 500–544; b) Amstrong, C. M.;
Hille, B. (1998) Voltageꢀgated Ion Channels and Electrical Excitability. Neuron 20, 371–380.
(3) a) Taylor, C. P.; Narasimhan, L. S. (1997) Sodium Channels and Therapy of Central Nervous
System. Adv. Pharmacol. 39, 47–98; b) Goldin, A. L. (2001) Resurgence of Sodium Channel
Research. Annu. Rev. Physiol. 63, 871–894; c) England, S.; de Groot, M. J. (2009) Subtypeꢀ
selective Targeting of Voltageꢀgated Sodium Channels. Br. J. Pharmacol. 158, 1413–1425; d)
Castle, N; Printzenhoff, D.; Zellmer, S.; Antonio, B.; Wickenden, A.; Silvia, C. (2009) Sodium
Channel Inhibitor Drug Discovery Using Automated High Throughput Electrophiysiology
Platforms. Comb. Chem. High Throughput Screening 12, 107–122.
(4) For a review on voltageꢀgated sodium channel blockers in neurological and nonꢀneurological
diseases, see: Eijkelkamp, N.; Linley, J. E.; Baker, M. D.; Minett, M. S.; Cregg, R.; Werderhausen,
R.; Rugiero, F.; Wood, J. N. (2012) Neurological Perspectives on Voltageꢀgated Sodium Channels.
Brain 135, 2585–2612.
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(5) a) Catterall, W. A.; Goldin, A. L.; Waxman, S. G. (2005) International Union of Pharmacology.
XLVII. Nomenclature and StructureꢀFunction Relationships of Voltageꢀgated Sodium Channels.
Pharmacol. Rev. 57, 397ꢀ409; b) Catterall, W. A. (2012) Voltageꢀgated Sodium Channels at 60:
Structure, Function and Pathophysiology. J. Physiol. 590, 2577–2589.
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(6) a) Yu, F. H.; Mantegazza, M.; Westenbroek, R. E.; Robbins, C. A.; Kalume, F.; Burton, K. A.;
Spain, W. J.; McKnight, G. S.; Scheuer, T.; Catterall, W. A. (2006) Reduced Sodium Current in
GABAergic Interneurons in a Mouse Model of Severe Myoclonic Epilepsy in Infancy. Nat.
Neursoci. 9, 1142–1149; b) Ogiwara, I.; Miyamoto, H.; Morita, N.; Atapour, N.; Mazaki, E.;
Inoue, I.; Takeuchi, T.; Itohara, S.; Yanagawa, Y.; Obata, K.; Furuichi, T.; Hensch, T. K.;
Yamakawa, K. (2007) Nav1.1 Localizes to Axons of ParvalbuminꢀPositive Inhibitory Interneurons:
A Circuit Basis for Epileptic Seizures in Mice Carrying an Scn1a Gene Mutation. J. Neurosci. 27,
5903–5914; c) Guerrini, R. (2012) Dravet Syndrome: the Main Issues. Eur. J. Paediatr. Neurol. 16
(Suppl. 1), S1–S4; d) Han, S.; Tai, C.; Westenbroek, R. E.; Yu, F. H.; Cheah, C. S.; Potter, G. B.;
Rubenstein, J. L.; Scheuer, T.; de la Iglesia, H. O.; Catterall, W. A. (2012) Autisticꢀlike Behaviour
in Scn1a+/- Mice and Rescue by Enhanced GABAꢀmediated Neurotransmission. Nature 489, 385–
390; e) Kalume, F.; Westenbroek, R. E.; Cheah, C. S.; Yu, F. H.; Oakley, J. C.; Scheuer, T.;
Catterall, W. A. (2013) Sudden Unexpected Death in a Mouse Model of Dravet Syndrome. J.
Clint. Invest. 123, 1798–1808.
(7) Jensen, H. S.; Grunnet, M.; Bastlund, J. F. (2014) Therapeutic Potential of Nav1.1 Activators.
Trends Pharmacol. Sci. 35, 113–118.
(8) a) Otoom, S.; Tian, L.ꢀM.; Alkadhi, K. A. (1998) Veratridineꢀtreated Brain Slices: A Cellular
Model for Epileptiform Activity. Brain Res. 789, 150–156; b) Miyawaki, T.; Tsubokawa, H.;
Yokota, H.; Oguro, K.; Konno, K.; Masuzawa, T.; Kawai, N. (2002) Differential Effects of Novel
Wasp Toxin on Rat Hippocampal Interneurons. Neurosci. Lett. 328, 25–28; c) Catterall, W. A.;
Cestèle, S.; YarovꢀYarovoy, V.; Yu, F. H.; Konoki, K.; Scheuer, T. (2007) Voltageꢀgated Ion
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Channels and Gating Modifier Toxins. Toxicon 49, 124–141; d) Soderlund, D. M. (2012)
Molecular Mechanisms of Pyrethroid Insecticide Neurotoxicity: Recent Advances. Arch. Toxicol.
86, 165–181.
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(9) A recent study showed a series of bisꢀamides as TASKꢀ1ꢀselective inhibitors. For more details,
see: Flaherty, D. P.; Simpson, D. S.; Miller, M.; Maki, B. E.; Zou, B.; Shi, J.; Wu, M.; McManus,
O. B.; Aubé, J.; Li, M.; Golden, J. E. (2014) Potent and Selective Inhibitors of the TASKꢀ1
Potassium Channel Through Chemical Optimization of a Bisꢀamide Scaffold. Bioorg. Med. Chem.
Lett. 24, 3968–3973.
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(10) AbdelꢀAziz, M.; Matsuda, K.; Otsuka, M.; Uyeda, M.; Okawara, T.; Suzuki, K. (2004)
Inhibitory Activities Against Topoisomerase I & II by Polyhydroxybenzoyl Amide Derivatives and
Their Structureꢀactivity Relationship. Bioorg. Med. Chem. Lett. 14, 1669–1672.
(11) A seal between cell and recording pipette with very high resistance (1 giga ohm or more) is
called a giga seal.
(12) This has been evaluated with Na+ channels to perform at a quality level comparable to manual
recordings.
(13) Goldin, A. L.; Barchi, R. L.; Cadwell, J. H.; Hofmann, F.; Howe, J. R.; Hunter, J. C.; Kallen, R.
G.; Mandel, G.; Meisler, M. H.; Netter, Y. B.; Noda, M.; Tamkun, M. M.; Waxman, S. G.; Wood,
J. N.; Catterall, W. A. (2000) Nomenclature of Voltageꢀgated Sodium Channels. Neuron 28, 365–
368.
(14) Membrane permeability of selected derivatives was examined and obtained in an MDCK cell
system expressing human MDR1 [ABCB1, Pꢀglycoprotein (Pꢀgp)] as previously described.15
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(15) Blom, S. M.; Rottländer, M.; Kehler, J.; Bundgaard, C.; Schmidt, N.; Jensen, H. S. (2014) From
PanꢀReactive Kv7 Channel Opener to Subtype Selective Opener/Inhibitor by Addition of Methyl
Group. PLoS One 9, e100209.
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FIGURE CAPTIONS
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Figure 1. Nav1.1 channels stable expressed in HEK cells were activated by stepping the membrane
potential from – 120 mV to + 70 mV in 10 mV steps from a holding potential of ꢀ100 mV. (A) Raw data
traces. (B) I–V curve for activation. Traces measured on the Qꢀpatch in single hole mode.
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Figure 2. Screening protocol. Nav1.1 channels stable expressed in HEK cells were activated by stepping
the membrane potential from – 120 mV in steps with 10 mV increments from – 40 mV to + 10 mV.
Screening of test compounds were performed by evaluation of the effect induced by test compounds at –
10 mV on the peak, AUC and tau values. Vhold between the step protocol was – 80 mV. Traces were
measured on the Qꢀpatch in single hole mode.
Figure 3. Effect of compound 3a at 30 ꢁM on the current trace activated to – 10 mV. 3a showed
inhibition of the peak current and the AUC, but 3a also induced a decrease in the inactivation (tau
value) (see also Table 1). Control (gray trace) and compound 3a (black trace) are showed in Figure 3A.
For comparison control trace and time matched DMSO trace is shown in Figure 3B. DMSO did not
induce any effect either on peak, AUC or tau (see also Table 1).
Figure 4. Compound 3a increases neuronal excitability in a fastꢀspiking interneuron recorded in the
stratum pyramidal in hippocampal CA1 field. (A) Top: A current ramp evoked repetitive spike firing in
the baseline preꢀdrug period. Bottom: following perfusion of 3a (100 ꢀM) for 10 mins, the same current
ramp now induced firing at a more negative membrane potential. Vhold = – 70 mV. (B) Group data from
5 interneurons tested showed that 3a was able at 100 ꢀM (but not at 30 ꢀM) to elicit firing at a more
negative membrane potential suggesting that 3a lower the threshold for firing. (C) Left top and bottom:
raw voltage traces recorded from an interneuron showed the evoked firing by a weak (200 pA)
rectangular pulse before and after perfusion of 3a. It was able to increase the excitability of the
interneuron. Right top and bottom: baseline firing evoked by a stronger depolarizing pulse was also
enhanced by 3a. (Weak depolarization is defined as the depolarizing pulses used that evoked 5~10
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spikes and strong depolarization is defined as the depolarizing pulses used that evoked 13~21 spikes).
(D) Time course of neuronal excitability change in 5 interneurons following 3a perfusion. Note that 3a
(at 30 and 100 ꢀM) can induce more firing of interneurons evoked by a weak current pulse than from a
stronger current pulse. See Supporting Information for more details.
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SCHEME TITLES
Scheme 1. Synthesis of phenylamide 2, diamides 3a–i and sulfonamide 3j. Reagents and conditions: (i)
2ꢀMePhCOCl (1 equiv), TEA (1.05 equiv), DCE, 0 °C to rt, 2: 45%, 3a: 35%; (ii) RCOCl (1.05 equiv),
TEA (1.1 equiv), DCE–DCM (5:1), 0 °C to rt, 3b: 83%, 3c: 91%, 3d: 96%, 3e: 97%, 3f: 96%, 3g: 99%,
3h: 99%, 3i: 97%; (iii) 2ꢀMePhSO2Cl (1.05 equiv), TEA (1.1 equiv), dry THF, 0 °C to rt, 89%.
Scheme 2. Synthesis of ester 3k, diamides 3l–n, tetrazole 3o and indoline 3p. Reagents and conditions:
(i) 2ꢀMePhCOCl (2.05 equiv), TEA (2.1 equiv), DMAP (0.04 equiv), DCE–DCM (5:1), 0 °C to rt, 3k:
89%; (ii) 2ꢀMePhCOCl (2.05 equiv), TEA (2.1 equiv), DCE–DCM (5:1), 0 °C to rt, 3l: 66%; (iii) 2ꢀ
MePhCOCl (1.05 equiv), TEA (1.1 equiv), CHCl3, rt, 6: 82%; 3o: 87%; (iv) 2ꢀMePhNH2 (1.5 equiv),
TEA (1.05 equiv), HATU (1 equiv), dry DMF, rt, 58%; (v) 2ꢀMePhCOCl (2.1 equiv), TEA (2.2 equiv),
DCE–DCM (8:3), 0 °C to rt then to 50 °C, 94%; (vi) 2ꢀMePhI (1.1 equiv), Cs2CO3 (1.1 equiv), CuI (1
equiv), Pd(OAc)2 (0.05 equiv), (oꢀfuryl) 3P (0.1 equiv), dry CH3CN, 65 °C then 90 °C, 16%; (vii) 2ꢀ
MePhCOCl (1.05 equiv), TEA (1.1 equiv), dry THF, 0 °C to rt, 91%; (viii) a) K2CO3 (3 equiv), Na2S2O4
(6 equiv), NMP–EtOH (5:7), 0 °C to 100 °C; b) 2ꢀMePhCOCl (1.05 equiv), TEA (1.1 equiv), CHCl3, 0
°C to rt, 23% over two steps.
TABLES TITLES
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Table 1. Peak current, AUC and tau values for compounds 3a–p tested at 30 ꢀM on a HEK cell line
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Table 1
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Response in % (± SEM) relative to control at – 10 mVa
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Compounds
DMSOc
nb
Peak (%)
AUC (%)
102 ± 2
Tau (%)
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101 ± 1
102 ± 2
10
5
52 ± 2
89 ± 1
70 ± 5
47 ± 3
54 ± 3
39 ± 2
56 ± 3
51 ± 1
75 ± 2
57 ± 2
86 ± 2
50 ± 12
93 ± 1
105 ± 1
88 ± 2
96 ± 2
81 ± 9
100 ± 5
83 ± 3
81 ± 5
86 ± 2
51 ± 2
83 ± 6
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93 ± 4
70 ± 11
92 ± 4
80 ± 13
96 ± 8
112 ± 3
86 ± 3
112 ± 10
124 ± 6
101 ± 4
115 ± 3
143 ± 7
127 ± 8
136 ± 14
128 ± 3
128 ± 4
110 ± 6
123 ± 6
104 ± 6
140 ± 11
103 ± 10
110 ± 2
95 ± 2
3a
3b
3c
3d
3e
3f
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3g
3h
3i
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3j
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3k
3l
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3m
3n
3o
3p
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a: Performed at 30 ꢀM; b: Number of individual experiments; c: DMSO control run on the same plates as
the tested compounds.
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Table 2
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Response in % (± SEM) relative to control at – 10 mVa
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Peak (%)
AUC (%)
81 ± 9
Tau (%)
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52 ± 2
124 ± 6
Nav1.1
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4
71 ± 2
94 ± 5
82 ± 4
101± 6
93 ± 6
79 ± 2
79 ± 3
84 ± 6
98 ± 5
75 ± 2
97 ± 2
85 ± 5
102 ± 5
96 ± 1
84 ± 2
Nav1.2
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Nav1.6
Nav1.7
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Scheme 1
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Scheme 2
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Figure 1
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ACS Chemical Neuroscience
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Figure 2
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ACS Chemical Neuroscience
Figure 3
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ACS Chemical Neuroscience
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Figure 4
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Baseline
A
B
-60
∗
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-40
-20
-70 mV
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Compound 3a (100uM)
20 mV
1 s
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e
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Baseline
Cpd 3a (100uM) 15min
200pA
D
AP5 50uM, DNQX 10uM, SR95531 10uM
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Compound 3a
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20mV
0.2s
Weak Depolarization (n=5)
Strong Depolarization (n=5)
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Time (min)
Weak Depolarization (5~10 APs evoked in baseline)
Strong Depolarization(13~21 APs evoked in baseline)
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ACS Chemical Neuroscience
Graphical abstract
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