N-propyl-2,2-diphenyl-2-hydroxyacetamide, a novel α-hydroxyamide with anticonvulsant, anxiolytic and antidepressant-like effects that inhibits voltage-gated sodium channels

Article abstract of DOI:10.1016/j.ejphar.2017.11.048

In patients with epilepsy, anxiety and depression are the most frequent psychiatric comorbidities but they often remain unrecognized and untreated. We report herein the antidepressant-like activity in two animal models, tail suspension and forced swimming tests, of six anticonvulsants α-hydroxyamides. From these, N-propyl-2,2-diphenyl-2-hydroxyacetamide (compound 5) emerged not only as the most active as anticonvulsant (ED₅₀ = 2.5 mg/kg, MES test), but it showed the most remarkable antidepressant-like effect in the tail suspension and forced swimming tests (0.3–30 mg/kg, i.p.); and, also, anxiolytic-like action in the plus maze test (3–10 mg/kg, i.p.) in mice. Studies of its mechanism of action, by means of its capacity to act via the GABA_A receptor ([³H]-flunitrazepam binding assay); the 5-HT_1A receptor ([³H]-8-OH-DPAT binding assay) and the voltage-gated sodium channels (either using the patch clamp technique in hNa_v 1.2 expressed in HEK293 cell line or using veratrine, in vivo) were attempted. The results demonstrated that its effects are not likely related to 5-HT_1A or GABA_Aergic receptors and that its anticonvulsant and antidepressant-like effect could be due to its voltage-gated sodium channel blocking properties.

Full text of DOI:10.1016/j.ejphar.2017.11.048

Author’s Accepted Manuscript
N-propyl-2,2-diphenyl-2-hydroxyacetamide,
a
novel α-hydroxyamide with anticonvulsant,
anxiolytic and antidepressant-like effects that
inhibits voltage-gated sodium channels
Valentina Pastore, Cristina Wasowski, Pedro
Martin, Andrea Enrique, Josefina Higgs, Luis E.
Bruno-Blanch, Veronica Milesi, Mariel Marder
www.elsevier.com/locate/ejphar
PII:
S0014-2999(17)30781-1
http://dx.doi.org/10.1016/j.ejphar.2017.11.048
EJP71544
DOI:
Reference:
To appear in: European Journal of Pharmacology
Received date: 11 January 2017
Revised date: 14 November 2017
Accepted date: 30 November 2017
Cite this article as: Valentina Pastore, Cristina Wasowski, Pedro Martin, Andrea
Enrique, Josefina Higgs, Luis E. Bruno-Blanch, Veronica Milesi and Marie
Marder, N-propyl-2,2-diphenyl-2-hydroxyacetamide, a novel α-hydroxyamide
with anticonvulsant, anxiolytic and antidepressant-like effects that inhibit
voltage-gated sodium channels, European Journal of Pharmacology
http://dx.doi.org/10.1016/j.ejphar.2017.11.048
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Title: N-propyl-2,2-diphenyl-2-hydroxyacetamide, a novel α-hydroxyamide with
anticonvulsant, anxiolytic and antidepressant-like effects that inhibits voltage-
gated sodium channels.
a
a
b
c
Valentina Pastore , Cristina Wasowski , Pedro Martin , Andrea Enrique , Josefina
a
c
b
a*
Higgs , Luis E. Bruno-Blanch , Veronica Milesi , Mariel Marder
a
Universidad de Buenos Aires. Consejo Nacional de lnvestigaciones Científicas y
Técnicas. Instituto de Química y Fisicoquímica Biológicas, Facultad de Farmacia y
Bioquímica, Junín 956 (C1113AAD), Buenos Aires, Argentina.
b
Universidad Nacional de La Plata. Consejo Nacional de lnvestigaciones Científicas y
Técnicas. Instituto de Estudios Inmunopatológicos y Fisiológicos, 47 street and 115
(B1900BJW), La Plata, Argentina.
c
Química Medicinal, Departamento de Ciencias Biológicas, Universidad Nacional de
La Plata, 47street and 115 (B1900BJW), La Plata, Argentina.
vpastore@qb.ffyb.uba.ar
criswasowski@qb.ffyb.uba.ar
josefinahiggs@gmail.com
pedromartinv@gmail.com
veronica@biol.unlp.edu.ar
andreaenrique@biol.unlp.edu.ar
lbb@biol.unlp.edu.ar

*
Corresponding author. Mariel Marder. Instituto de Química y Fisicoquímica
Biológicas, Facultad de Farmacia y Bioquímica, Junín 956 (C1113AAD), Buenos Aires,
Argentina. Tel.: 0054 11 4964 8289 ; fax: 0054 11 4962 5457. mmarder@qb.ffyb.uba.ar
Abstract
In patients with epilepsy, anxiety and depression are the most frequent psychiatric
comorbidities but they often remain unrecognized and untreated.
We report herein the antidepressant-like activity in two animal models, tail suspension
and forced swimming tests, of six anticonvulsants α-hydroxyamides. From these, N-
propyl-2,2-diphenyl-2-hydroxyacetamide (compound 5) emerged not only as the most
active as anticonvulsant (ED = 2.5 mg/kg, MES test), but it showed the most
5
0
remarkable antidepressant-like effect in the tail suspension and forced swimming tests
0.3-30 mg/kg, i.p.); and, also, anxiolytic-like action in the plus maze test (3-10 mg/kg,
i.p.) in mice. Studies of its mechanism of action, by means of its capacity to act via the
(
3
3
GABA receptor ([ H]-flunitrazepam binding assay); the 5-HT receptor ([ H]-8-OH-
A
1A
DPAT binding assay) and the voltage-gated sodium channels (either using the patch
clamp technique in hNa 1.2 expressed in HEK293 cell line or using veratrine, in vivo)
v
were attempted. The results demonstrated that its effects are not likely related to 5-HT1A
or GABA ergic receptors and that its anticonvulsant and antidepressant-like effect
A
could be due to its voltage-gated sodium channel blocking properties.
Keywords: anticonvulsant; antidepressant-like; anxiolytic-like; N-propyl-2,2-diphenyl-
2
-hydroxyacetamide; voltage-gated sodium channels.

1
. Introduction
Epilepsy is a common group of neurological disorders whose hallmark is unprovoked
seizures that can be distressing, harmful and even fatal. Associated comorbidities
contribute greatly to disability and impaired quality of life of people with epilepsy
(Gaitatzis et al., 2012). They present a significantly higher rate of anxiety and
depression than the general population, and the symptoms of these pathologies might
serve as a barrier to appropriate epilepsy care, as they are sometimes treated first or
instead of the major pathology that caused them. Several clinical studies have
demonstrated the “bi-directional” relationship between depression and epilepsy or the
presence of common pathogenic mechanisms that facilitate the incidence of one in the
presence of the other (Kanner A., 2003). Despite the high prevalence and significant
consequences of comorbid depression in people with epilepsy, it often remains under-
treated or untreated (Yang et al., 2014). Furthermore, these pathologies, when
recognized, are treated with psychotropic drugs which can worsen seizures (Mula and
Schmitz, 2009).
Antiepileptic drugs comprise a heterogeneous group of agents with diverse mechanisms
of action that can be grouped into four broad categories: modulation of voltage-
dependent sodium, calcium or potassium channels; increase in GABAergic inhibition
via actions on GABA receptors or on GABA synthesis, reuptake, or degradation;
A
decreased synaptic excitation via actions on ionotropic glutamate receptors and/or
modulation of neurotransmitter release via presynaptic mechanisms (Landmark, 2007;
Rogawski and Loscher, 2004). On the other hand, antidepressant drugs modify the
activity of neurons in different ways, by increasing monoamines levels and by

modulating ion channels. Voltage-gated sodium channels are involved in many
psychiatric disorders. Inhibition of voltage-gated sodium channels has emerged as a
relevant mechanism since several anticonvulsants are effective to treat depression.
Moreover, some antiepileptic drugs are also used as mood stabilizers i.e. lamotrigine,
topiramate, phenytoin, carbamazepine and valproic acid (Yatham, 2004). In addition,
voltage-gated sodium channels blockers have been proposed to be an effective non-
benzodiazepine treatment for anxiety (Bourin et al., 2009; Guy et al., 2009; Mirza et al.,
2
005; Saitoh et al., 2015; Sugiyama et al., 2012).
Continuous efforts are done by researchers to find drugs that can be active as
anticonvulsant/antidepressant/anxiolytic to minimize the side effects caused by the co-
administration of drugs to treat these pathologies. Our group has built a considerable
expertise in the synthesis of new molecular entities with anticonvulsant, antidepressant
and anxiolytic-like profiles (Pastore et al., 2013, 2014, Wasowski et al., 2012;
Wasowski and Marder, 2012).
We have previously described the synthesis and the anticonvulsant activity of novel
heterocycles N-derivative-1,2,3-oxathiazolidine-4-one-2,2-dioxides, bioisosteres of
trimethadione (oxazolidine-2,4-dione) and phenytoin, via the cyclization of a series of
α-hydroxyamides (Pastore et al., 2014). These α-hydroxyamides also showed excellent
anticonvulsant activity and were not neurotoxic (Pastore et al., 2013). The aim of this
work was to further expand the anticonvulsant profile and to evaluate the potential
antidepressant-like activities of these α-hydroxyamides. Furthermore, a first
approximation of their mechanism of action, by means of their capacity to act on the
GABA receptor, the 5HT receptor and/or the voltage-gated sodium channels was
A
1A
attempted.

2
. Materials and methods
2
.1 Drugs and chemistry
N-propyl-2-hydroxyisobutylamide (1); N-butyl-2-hydroxyisobutylamide (2); N-benzyl-
-hydroxyisobutylamide (3); N-phenetyl-2-hydroxyisobutylamide (4); N-propyl-2,2-
diphenyl-2-hydroxyacetamide (5) and N-butyl-2,2-diphenyl-2-hydroxyacetamide (6)
Figure 1) were obtained by microwave assisted free solvent synthesis as previously
2
(
described (Pastore et al., 2013). Synthesis of N-propyl-2,2-diphenyl-2-
hydroxyacetamide (5) is shown in supplementary data. Their chemical purities were
1
13
confirmed by elemental analysis (99 %). Analysis of the H NMR and C NMR spectra
showed that the compounds obtained presented analytical and spectroscopic data in full
agreement with its assigned structure. Diazepam was obtained from Roche Diagnostics,
3
3
3
[
H]-flunitrazepam and [ H]-8-hydroxy-2-(dipropylamino)tetralin ([ H]-8-OH-DPAT)
were obtained from Perkin Elmer Life and Analytical Sciences, Boston, MA, USA,
imipramine hydrochloride and veratrine from Sigma-Aldrich. All reagents for
electrophysiological recordings were of analytical grade and were purchased from local
suppliers, as well as Dulbecco's Modified Eagle's Medium and Fetal bovine serum.
Geneticin was obtained from Tecnolab S.A.
2
.2 Animals and injection procedures
Both adult male Swiss mice weighing 25–30 g used in the pharmacological tests and
adult male Wistar rats weighing 200-300 g used for binding assays were obtained from

the Central Animal House of the School of Pharmacy and Biochemistry, University of
Buenos Aires.
For behavioral assays mice were housed in groups of five in a controlled environment
(20-23 ºC), with free access to food and water and maintained on a 12h/12h day/night
cycle, light on at 07:00 AM. Housing, handling, and experimental procedures complied
with the recommendations set forth by the National Institutes of Health Guide for Care
and Use of Laboratory Animals (NIH Publication No. 8023, revised 1996) and the
Institutional Committees for the Care and Use of Laboratory Animals of the University
of La Plata, Argentina (code: 014-06-15, 2016) and the Faculty of Pharmacy and
Biochemistry, University of Buenos Aires, Argentina (code: 31682/2014). All efforts
were taken in order to minimize animal suffering. The number of animals used was the
minimum number consistent with obtaining significant data. Mice were randomly
assigned to any treatment groups and were used only once. The pharmacological tests
were evaluated by experimenters who were kept unaware of the treatment administered
and were performed between 10:00 AM and 2:00 PM.
Compounds 1-6 were dissolved by using the sequential addition of dimethylsulfoxide, a
solution of 0.25% Tween 80 and saline; up to final concentrations of 5%, 20% and 75%,
respectively. The rodents were intraperitoneally (i.p.) injected. Veratrine was dissolved
in saline at a dose of 0.125 mg/kg. In each session, a control group receiving only
vehicle was tested in parallel with those animals receiving drug treatment. Vehicle
control mice showed no significant differences in any of the tests assayed compared to
mice treated with saline (data not shown).
2
.3 Biochemical assays

2
.3.1 Tissue preparation
3
For [ H]-flunitrazepan binding assays, membranes were prepared according to literature
(Wasowski et al., 2012). Briefly the brains were rapidly dissected out on ice and the
different structures were homogenized in 10 volumes of 0.32 M sucrose and centrifuged
at 900 x g for 10 min. The resulting supernatant was centrifuged at 100,000 x g for 30
min and the pellet washed twice in 25 mM Tris–HCl buffer pH 7.4 at 100,000 x g for 30
min, and stored at -20ºC until used.
For 5HT binding assays the cortex tissues were homogenized in 30 volumes/weight of
1
A
ice-cold 50 mM Tris–HCl buffer pH 7.4 and the homogenate was centrifuged at 40,000
g for 15 min at 4°C. The resulting pellets were suspended in 30 volumes/weight of the
×
same buffer and incubated at 37°C for 20 min. The centrifugation step was repeated
twice under the same conditions as described above and the final pellets were suspended
in 30 volumes/weight of 50 mM Tris–HCl buffer pH 7.4 and stored at -80 °C until use.
Protein concentration was determined by the method of Bradford using bovine serum
albumin as standard (Bradford, 1976).
3
2
.3.2 [ H]-flunitrazepan binding assay
A radioligand binding assay was used to evaluate the putative action of the compounds
(
1–6) on the benzodiazepine binding site of the GABA receptor complex. The binding
A
3
of [ H]-flunitrazepam (81.8 Ci/mmol) to the benzodiazepine binding site was performed
in washed crude synaptosomal membranes from rat cerebral cortex prepared as
described previously (Medina et al., 1990).
The compounds were added to 0.2-0.3 mg membrane protein suspended in 1 ml of 25
3
mM Tris–HCl buffer in the presence of [ H]-flunitrazepam 0.3 nM. In the screening

assays each compound was tested at 300 µM in triplicate. Diazepam was used as
positive control. Non-specific binding was measured in the presence of flunitrazepam
1
4
0 µM and represented 5–15% of the total binding. The incubations were carried out at
ºC for 1 h. After incubation, the assays were terminated by filtration under vacuum
through Whatman GF/A glass-fiber filters followed by washing three times with 3 ml
each of incubation medium. Individual filters were incubated overnight with
scintillation cocktail (OptiPhase ‘HiSafe’ 3) before measuring radioactivity in a Wallac
Rackbeta 1214 liquid scintillation counter.
3
2
.3.3 [ H]-8-OH-DPAT binding assays
For the binding assay, membranes were thawed and suspended in 50 mM Tris–HCl pH
.4, with 1mM MnCl to a final protein concentration of 0.50-0.55 mg/ml. The
7
2
incubation was carried out at 25°C for 1 h in a final volume of 1 ml of membrane
suspension (in duplicate) in the presence of the sample assayed and with 0.55 nM of
3
[
H]-8-OH-DPAT (170.2 Ci/mmol). Nonspecific binding was determined in parallel
incubations in the presence of serotonin (10 μM). The assays were terminated by
filtration under vacuum through Whatman GF/A glass-fiber filters and three washes
with 3.5 ml each of incubation medium. Radioactivity was determined as described
above.
2
.4 Electrophysiology.
Patch-clamp experiments were performed in HEK293 cell lines stably expressing hNav
.2 α subunit (a kind gift of GlaxoSmithKline, Stevenage, UK) that have been described
1

(Burbidge et al., 2002; Mantegazza et al., 2005). Details of the experiments are
described below (Section 2.4.2)
2
.4.1 Cell culture
HEK293 cell lines were cultured in minimum essential medium, containing 10% fetal
bovine serum, and 0.5% geneticin G418 sulfate. Cells were grown in a 95% O /5% CO2
2
atmosphere at 37°C and with 95% humidity. One to two days prior to
electrophysiological recordings, the cells were plated on glass coverslips.
2
.4.2 Whole-cell voltage-clamp recordings
The cells were grown on glass coverslips and were observed with a mechanically
stabilized inverted microscope (Telaval 3, Carl Zeiss, Jena, Germany) equipped with a
4
0X objective lens. The test solutions were applied through a multi-barreled pipette, by
gravity and in continuous flux, positioned close to the target cell. After each experiment
on a single cell, the experimental chamber was replaced by another one containing a
new sample of cells. All experiments were performed at room temperature (~22 ºC).
The standard tight-seal whole-cell configurations of the patch-clamp technique (Hamill
et al., 1981) was used to record macroscopic currents. Glass pipettes were drawn from
WPI PG52165-4 glass on a two-stage vertical micropipette puller (PP-83, Narishige
Scientific Instrument Laboratories, Tokyo, Japan) and pipette resistance ranged from 2
to 3 M.
Whole-cell currents were filtered with a 4-pole lowpass Bessel filter (Axopatch 200A
amplifier) at 2 kHz and digitized (Digidata 1440, Molecular devices) at a sample
frequency of 200 kHz (5 µs). The experimental recordings were stored on a computer

hard disk for later analysis. Cells were placed in a recording chamber with 0.5 mL
extracellular solution containing (in mM): NaCl 140, KCl 5, CaCl 2, MgCl 1, HEPES
2
2
1
0 and glucose 11; pH was adjusted to 7.4 with NaOH. The patch electrodes were filled
with pipette solution containing (in mM): CsF 140, EGTA 10, HEPES 10, NaCl 5,
MgCl 2; the pH was adjusted to 7.3 with CsOH.
2
Once the whole-cell configuration was obtained, current and series resistance stability
were evaluated with a 15 ms-voltage-clamp step from a holding potential of -80 mV to a
test potential of -20 mV repeated each 10 s. The time needed for the stabilization was
variable (approximately10 min). Only cells with a stable series resistance and current
amplitude were included in the results. The series resistance was not electronically
compensated, but as it maintains constant during the experiment, the series resistance
error contamination is the same before and after drug perfusion. The same voltage-
clamp step protocol was applied in the control (vehicle) or in the presence of compound
5
, dissolved in 0.1 % dimethylsulfoxide. The voltage dependence of the steady-state
inactivation of sodium channels was evaluated using a double voltage-step protocol,
where the same depolarization to −10 mV followed different pre-conditioning steps
(from -130 to -40 mV). The available fraction of sodium channels at each membrane
potential (IVc/Imax) was calculated as the ratio of peak sodium current measured at -10
mV, at each pre-conditioning voltage test pulse (I ) and the maximum peak current
Vc
observed (Imax). The relationship between the available fraction of sodium channels and
the pre-conditioning (named h curve) was plotted and fitted with a Boltzmann equation:
IVc/Imax = 1/(1+exp((Vh-V)/k)) where the available fraction is given as IVc/Imax, Vh is the
potential of half-maximal inactivation and k is the slope parameter. Statistical

significance of the change in the Vh parameter induced by compound 5 was tested with
F method (GraphPad Prism).
2
2
.5 Behavioral assays
.5.1 Anticonvulsant effect and neurotoxicity
The anticonvulsant activity and neurotoxicity of α-hydroxyamides were performed in
acute models, using the procedures proposed by the National Institute of Health (NIH)
via the Epilepsy Therapy Screening Program (ETSP) (Rogawski and Loscher, 2004;
Stables, 1997). All procedures were previously published as well they are available for
public in NIH website at (https://www.ninds.nih.gov/Current-Research/Focus-
Research/Focus-Epilepsy/ETSP). The primary evaluation (phase 1) includes the use of
two anticonvulsant tests: Maximal Electroshock Seizure test (MES) and subcutaneous
Pentylenetetrazole Seizure test (scPTZ). Toxicity is primary detected using the
standardized Rotarod test, which is also included in this phase (Dunham and Miya,
1
957). According to this procedure the test substances were administered in 30%
polyethylene glycol 400 (PEG) and 10% water. The drugs were administered i.p. in
mice in a volume of 0.01 mL/g body weight.
2
.5.1.1 scPTZ test
The scPTZ test involves a chemical induction to generate convulsions related to
myoclonic seizures and entailed the s.c. administration of 85 mg/kg of PTZ in saline in
the posterior midline of the mice. Afterwards, the animals were placed in 15 cm × 30
cm chambers to record seizure during 30 min of observation. Protection was defined as
the failure to observe even a threshold seizure (single episode of clonic spasms of at

least 5 s duration). Compounds that are active in scPTZ may act raising seizure
threshold (Malawska et al., 2004; Rogawski and Loscher, 2004).
2
.5.1.2 MES test
The MES test is associated with the electrical induction of the seizure. The endpoint in
this test is tonic hindlimb extension, and the test is thought to be a predictive model for
generalized tonic–clonic seizures. The compounds were administrated i.p. to mice at
doses of 30 and 100 mg/kg. Quantitative biological studies (phase II) were performed
for the most promising compounds from phase I (in MES test). At this stage, the
anticonvulsant activity was expressed as median effective dose, ED , which determines
5
0
the drug concentration that is effective in the 50% of the tested animals. ED values
5
0
were calculated by treating groups of six mice with different doses at the maximal time
effect previously determined.
2
.5.1.3 Rotarod test
The Rotarod test is used exclusively in mice to assess minimal neurotoxicity. A normal
mouse can maintain its equilibrium on a rotating rod (6 rpm) for long periods of time.
Motor deficit is indicated by failure to maintain balance on a rotating rod in each of
three trials of 1 min each.
2
2
.5.2 Antidepressant-like activities
.5.2.1 Forced swimming test
The forced swimming test employed was similar to that described previously (Porsolt et
al., 1978). This test is currently the most reliable method to screen compounds for

antidepressant effects in rodents. Mice were individually dropped in glass cylinders
height: 25 cm; diameter: 10 cm; containing 10 cm of water at 24 ± 1 ºC) for 6 min. The
(
immobility time was recorded during the last 4 min of the test. Each mouse was judged
to be immobile when it ceased struggling and remained floating in the water, making
only the necessary movements to keep its head above water. The test was performed 30
min after the i.p. injection of the α-hydroxyamides, imipramine (control drug) or
vehicle.
2
.5.2.2 Tail suspension test
The tail suspension test was carried out according to the methodology proposed by
Steru and co-workers in 1985 (Steru et al., 1985). This test is frequently used in
laboratory practice to identify compounds with antidepressant-like activity. Mice were
individually suspended by the tail to a metal hook (distance from floor: 18 cm) using
adhesive tape (distance from tip of tail: 2 cm) for 6 min. Typically, mice demonstrate
several escape oriented behaviors interspersed with temporally increasing bouts of
immobility. The duration of immobility was recorded during the final 4 min interval of
the test. Mice were considered immobile only when they hung passively and completely
motionless. The test is based on the fact that animals subject to the short-term,
inescapable stress of being suspended by their tails will develop an immobile posture.
The test was performed 30 min after the i.p. injection of α-hydroxyamides, imipramine
or vehicle.
2
.5.3. Locomotor activity test

Because all behaviors measured in the tests performed depend on locomotor activity
that is probably the confounding issue; the spontaneous locomotor activity was recorded
in separate experiments. The spontaneous locomotor activity was measured in a box
made of Plexiglass, with a floor of 30 cm x 15 cm and 15 cm high walls as previously
described (Fernandez et al., 2006). The locomotor activity was expressed as total light
beam counts per 5 min.
2
2
.5.4 Anxiolytic-like effect
.5.4.1 Elevated plus-maze test
The elevated plus-maze set-up consisted of a maze of two open arms, 25 cm x 5 cm,
crossed by two closed arms of the same dimensions, with free access to all arms from
the crossing point. The closed arms had walls 15 cm high all around. The maze was
suspended 50 cm from the room floor. Mice were placed on the central part of the cross
facing an open arm. The number of entries and time spent in open arms were counted
during 5 min under red dim light. An arm entry was defined as all four paws having
crossed the dividing line between an arm and the central area. The total exploratory
activity (number of entries in both arms) was also determined (Lister, 1987). Diazepam
was used as reference compound.
2
.6 Effect of pre-treatment with veratrine on the antidepressant effect of N-propyl-2,2-
diphenyl-2-hydroxyacetamide (5) in the forced swimming test.
The possible role of voltage-gated sodium channels in the mechanism of action of N-
propyl-2,2-diphenyl-2-hydroxyacetamide (compound 5) as antidepressant was
investigated using veratrine, a selective activator of voltage-gated sodium channels.

The dose of 0.125 mg/kg of veratrine was chosen for this study as it did not induce
increase or decrease in spontaneous locomotor activity nor antidepressant like effects in
the forced swimming test, based on a previous study (Prica et al., 2008). Veratrine was
administered, in mice, in association with two active doses of compound 5. The sub
active dose of veratrine (or saline) was administered 45 min before testing in the forced
swimming test, and compound 5 (or vehicle) were administered 30 min before. The
animals were randomly allocated in six experimental groups (number of mice per
group=6-27) as follows (indicated as pretreatment-treatment): (1) saline-vehicle, (2)
saline-compound 5 (10 mg/kg), (3) saline-compound 5 (1 mg/kg), (4) veratrine-vehicle,
(5) veratrine-compound 5 (10 mg/kg), (6) veratrine-compound 5 (1 mg/kg).
2
.7 Statistical analyses
The effects of the compounds in mice were analyzed by one-way analysis of variance
ANOVA) and post-hoc comparisons between treatments and control groups were made
(
using Dunnett’s multiple comparison test. The association study with veratrine was
analyzed by two-way ANOVA (pre-treatment vs. treatment) and post-hoc comparison
was made using Bonferroni post test. As a significant interaction between pre-treatment
and treatment was observed, subsequent one-way ANOVAs and Newman-Keuls
multiple comparison post-hoc test were applied. Electrophysiological experiments were
analyzed using paired t-test. A P value <0.05 was considered statistically significant. All
data were expressed as mean ± S.E.M. and analyzed with GraphPad Prism 5.00
software.
3
. Results

3
.1 Anticonvulsant effect of α-hydroxyamides
Table 1 shows the anticonvulsant profile of compound 5. Profiles of compounds 1, 2, 3,
and 6 were previously reported (Pastore et al., 2013). The results demonstrated that
4
the administration of these compounds presented absence of tonic extension of the hind
legs after an electroshock in the MES test, even at doses lower than 100 mg/kg, being
compound 5 the most potent with an ED of 2.5 mg/kg. Compound 5 did not present
5
0
anticonvulsant activity in the scPTZ test. However, compounds 2, 3, and 4 were able to
reduce convulsions induced in this assay. Importantly, none of the mice treated with α-
hydroxyamides died during the assays or showed any failure to maintain balance on the
Rotarod assay, evidencing that they did not produce neurotoxicity or motor impairment,
at the doses tested.
3
.2 Antidepressant effects of α-hydroxyamides.
An initial antidepressant-like effect screening of all α-hydroxyamides was performed, at
a fixed dose of 10 mg/kg, using the forced swimming test, and compared to vehicle
control group and imipramine (20 mg/kg) treated group (Figure 2).
The results revealed that compounds 1, 2 and 6 did not showed an antidepressant-like
effect at this dose, as they could not significantly decrease the immobility time of mice
compared to control group. Meanwhile, compounds 3, 4 and 5 and imipramine,
significantly reduced the immobility time of mice.
Subsequently, these three α-hydroxyamides and compound 2, that although not showing
a significantly effect at 10 mg/kg revealed a tendency in decreasing the immobility time
of mice (Figure 2); were evaluated by the effect of acute administration at different
doses; compound 2 at 10, 30 and 60 mg/kg, compounds 3 and 4 at 1, 10 and 30 mg/kg

and compound 5 at 0.3, 1, 10 and 30 mg/kg (Figure 3A, 3B, 3C and 3D, respectively).
Figure 3A shows that compound 2 significantly reduced the immobility time of mice at
3
0 and 60 mg/kg compared to control mice, evidencing an antidepressant-like effect at
higher doses. Compounds 3 and 4 significantly reduced the immobility time of mice at
0 and 30 mg/kg compared to control mice. From these derivatives, compound 5
1
resulted as the most active α-hydroxyamide, showing a decrease in the immobility time
of mice even at a dose of 0.3 mg/kg (Figure 3 D). Also, all tested compounds
demonstrated a clear dose dependent effect.
Moreover, the antidepressant–like effect of compounds 2, 3, 4 and 5, at 30 mg/kg, was
evaluated using another antidepressant test of immobility, the tail suspension test
(Figure 4). The results revealed that α-hydroxyamides 2, 3 and 5 and imipramine, at 20
mg/kg, significantly reduced the immobility time of mice in this test. Compound 5 was
also tested at 10 mg/kg in the tail suspension test, but no antidepressant effect was
found at this dose (data not shown).
3
.3 Locomotor activity test
In order to discard any effect of compounds 2 to 5 on locomotion, the locomotor
activities of mice were also evaluated. The performance of α-hydroxyamides treated
mice on the locomotor activity test was unaffected at doses up to 30 mg/kg. The results
for compound 5 (at 1 mg/kg, 10 mg/kg and 30 mg/kg) are shown in Figure 5B.
3
5
.4 Anxiolytic-like effect of N-propyl-2,2-diphenyl-2-hydroxyacetamide (compound
).

As anxiety is a comorbid pathology in people with epilepsy, the potential anxiolytic-like
activity of compound 5 was also evaluated in the plus maze test. The anxiolytic-like
effect of compound 5 was compared to vehicle control group and the effect of
diazepam, a classical benzodiazepine used as a reference anxiolytic drug (Figure 5A).
Compound 5 significantly increases the percentage of open arms entries, at 3 and 10
mg/kg, and the percentage of time spent in open arms, at 10 mg/kg, when compared to
vehicle treated mice. This result revealed an anxiolytic-like effect of compound 5 at
these doses. Nevertheless, the administration of compound 5 at doses of 1 mg/kg and 30
mg/kg produce no effect in mice in this assay. Meanwhile, diazepam, at 1 mg/kg,
increased the percentage of open arm entries, the percentage of time spent in open arms,
the total arm entries (Figure 5A) and the locomotor activity of mice (Figure 5B).
3
.5 Study of the mechanism of action of compound 5.
Among all α-hydroxyamides tested, compound 5 emerged as the most active candidate,
showing significant activity as anticonvulsant in the MES test (ED of 2.5 mg/kg), but
5
0
not in the PTZ test, and even antidepressant (0.3-30 mg/kg) and anxiolytic-like (3-10
mg/kg) activities.
In order to explore possible molecular targets involve in this compound activities,
binding assays for the benzodiazepine binding site in the GABA and in the 5HT1A
A
3
3
receptors were carried out using [ H]-flunitrazepam and [ H]-8-OH-DPAT as
radioligands, respectively. These assays showed that compound 5, up to a concentration
3
3
of 300 μM, could not displace [ H]-flunitrazepam and [ H]-8-OH-DPAT from
synaptosomal rat brain membranes.

Considering that anticonvulsant drugs, which also have antidepressant-like effects, such
as lamotrigine, prevent seizures induced by MES test, and that their mechanism of
action is related to voltage-gated sodium channels inhibition, it is possible to postulate
that sodium channels could be a target for compound 5. The ability of compound 5 to
inhibit sodium current in hNa 1.2 channels stably expressed in the HEK293 cell line
v
was examined by patch-clamp technique in whole-cell configuration (Figure 6). The
current evoked by a 15 ms voltage-step from a holding potential of -80 mV to -20 mV
was significantly inhibited by compound 5 at a concentration of 100 µM. The effect of
compound 5 was washed-out as long as the seal remained stable (Figure 6 A).
Moreover, as it could be observed in Figure 6 B and C, the compound 5 is able to
produce a significant shift of h curve to more hyperpolarized membrane potentials,
suggesting that it stabilizes the inactivated state decreasing the available fraction of
sodium channels to open when membrane is more depolarized (voltage-dependent
inhibition).
The in vivo antidepressant-like effect of lamotrigine is able to be blocked by the pre-
treatment with veratrine (a voltage-gated sodium channel agonist), suggesting that
voltage activated sodium channels are involved in its in vivo action (Bourin et al.,
2
009). The study conducted in order to assess the effects of veratrine on the
antidepressant-like effect of compound 5 in the forced swimming test is shown in
Figure 7. It was already reported that veratrine tested alone in this assay, and up to a
dose of 2 mg/kg, did not modify the immobility time of mice, and that veratrine 0.125
mg/kg had no effect on the locomotor activity of mice (Prica et al., 2008). As shown in
Figure 7, veratrine (0.125 mg/kg, i.p.) did not evoke any response by itself. Compound
5
given alone induced a significant antidepressant-like effect at 1 mg/kg and 10 mg/kg.

The pre-treatment with veratrine (0.125 mg/kg) significantly reversed the
antidepressant-like effect of compound 5 at both doses. Therefore, the antidepressant-
like effect of compound 5 in the forced swimming test was able to be blocked by
veratrine, suggesting that voltage activated sodium channels are involved in the in vivo
action of compound 5.
4
. Discussion
In this work the novel synthetic compound N-propyl-2,2-diphenyl-2-hydroxyacetamide
compound 5), which induced anticonvulsant, antidepressant and anxiolytic-like effects
(
in mice, was presented. Moreover, we showed that this compound could inhibit voltage-
gated sodium channels.
Our previous and present studies demonstrated that of a series of 6 anticonvulsant α-
hydroxyamides (1 to 6), not all showed the same anticonvulsant profile. While
compounds 2, 3 and 4 are effective in both MES and PTZ test, an anticonvulsant profile
similar to valproic acid; α-hydroxyamides 1, 5 and 6 are only effective in the MES test
as phenytoin and trimethadione, probably due to their distinctive mechanisms of action
(Krall et al., 1978; Pastore et al., 2013; White et al., 2002).
Among the different tests used in animal models to evaluate antidepressant activity the
forced swimming test remains one of the most used tool for assessing pharmacological
antidepressant effect due to its relative reliability across laboratories and its ability to
detect activity in a broad spectrum of clinically effective antidepressants. The tail
suspension test is theoretically similar to the forced swimming test; it is inexpensive and
methodologically unsophisticated (Cryan and Mombereau, 2004). Although both tests
show common behavioral measure of feeling despair, the underlying pathophysiology

seems to be different (Bai et al., 2001). The synthetic compounds 1-6 were evaluated in
the forced swimming test in mice, being compounds 2 to 5 active as antidepressants in
this test. α-Hydroxyamides 2, 3, 4 and 5, at a dose of 30 mg/kg, were also evaluated in
the tail suspension test. Except for compound 4, all of them showed a decrease in the
immobility time in this assay. It is not unusual for antidepressant compounds to have
different responses in both tests due to their differential sensitivity to the immobility-
reducing effects. Some agents such as rolipram and levoprotiline reduced immobility in
the forced swimming test but have been reported inactive in the tail suspension test
(Porsolt et al., 1992). Further, the pattern of the dose response curves may be
substantially different between both procedures (Bai et al., 2001; Porsolt et al., 1978).
As psychomotor stimulants, and drugs enhancing motor activity, may give false effects
in the forced swimming and tail suspension tests, by means of a decrease in the
immobility time via stimulating locomotor activity, an additional measurement of the
possible effects of these compounds on the locomotion of mice was carried out. This
assay showed that these compounds did not produce disturbances in the locomotor
activity up to a dose of 30 mg/kg, evidencing a lack of sedative effect. Even more,
Rotarod test results indicated that they did not induce neurotoxicity at doses up to 100
mg/kg.
The elevated plus-maze stands as one of the most used in vivo animal test to screen
anxiolytic effects of drugs. It is claimed to be an ethologically valid animal model of
anxiety because it uses natural stimuli that can induce anxiety in human (Calatayud et
al., 2004). Compound 5, the α-hydroxyamide most active as antidepressant, was chosen
for further studies to evaluate its capacity to act as anxiolytic in the plus maze test at a
dose range from 1 mg/kg to 30 mg/kg. This assay showed that compound 5 increased

the percentage of entries (at doses of 3 mg/kg and 10 mg/kg) and the time spent (at a
dose of 10 mg/kg) in open arms of mice suggesting that this compound has an
anxiolytic-like action at these doses, quite similar to those related to the reference
benzodiazepine, diazepam; even though it showed no effect at doses of 1 mg/kg and 30
mg/kg in this assay. Anxiolytic drugs usually display hormetic-like biphasic dose
responses, independent of the test and animal model employed (Calabrese, 2008).
The GABA system is the main inhibitory neurotransmitter system in the brain and is the
target for many clinically used drugs to treat epilepsy and anxiety disorders. However,
3
compound 5 (at 300 µM) was not able to displace the binding of [ H]-flunitrazepam, a
GABA receptor-specific ligand, from synaptosomal rat brain membranes. This data
A
suggest that the behavioral effects induced by this compound could not involve the
benzodiazepine binding site of the GABA , at least not directly.
A
Moreover, many studies support evidence for a role of the serotonergic system in
various behavioral responses related to depression and anxiety, as well as other
psychiatric disorders (Krishnan and Nestler, 2008). The serotonergic hypothesis of
depression suggests that depressive symptoms are related to a reduced 5-HT
concentration in the brain synapse and an enhancement in the concentration of this
neurotransmitter is able to induce antidepressant action. It has been reported, also, that
5
-HT receptors were involved in the action mechanism of antidepressants in different
behavioral studies (Chenu et al., 2008; Sugimoto et al., 2010). Nevertheless, α-
hydroxyamide 5 (at 300 µM) did not show any direct effect on serotonergic 5-HT1A
receptor excluding a possible role of the serotonergic receptor in the behavioral activity
of this α-hydroxyamide. However, the possibility that compound 5 could act on 5-HT
uptake cannot be discarded.

The voltage-gated sodium channels initiate action potentials in neurons and other
excitable cells are responsible for propagation of action potentials along nerves, muscle
fibers and neuronal somato-dendritic compartment. Voltage-gated sodium channels are
the molecular targets for drugs used in the treatment of epilepsy, cardiac arrhythmias,
bipolar disorder, migraine, and in the prevention of acute and chronic pain. Many
antiepileptic drugs in use nowadays are specifically voltage-gated sodium channels
blockers i.e phenytoin, topiramate, carbamazepine, lamotrigine and their mechanisms of
action have been recently described in detail (Mantegazza et al., 2010; Qiao et al.,
2
014). Our results showed that the compound 5 (at 100 µM) induces a reversible
inhibition of hNav 1.2 sodium channels expressed in HEK293 cell line, suggesting that
this compound has a direct action on this ion channel. Moreover, the observed shift in h
curve towards more hyperpolarizes voltages indicated that the compound 5 has a
channel state dependent inhibition effect, stabilizing the inactivated state. This
mechanism is the same that was previously described for AEDs, like lamotrigine,
carbamazepine and phenytoin, which effectively block the sodium conductance and
delay recovery from inactivation, which prevents synchronized high frequency firing.
(Ragsdale and Avoli, 1998, Rogawski and Loscher, 2004).
Moreover, in vivo results demonstrated that this mechanism could be involved in the
antidepressant effect of compound 5, since veratrine, a sodium channel agonist, was
able to revert the antidepressant effect of this α-hydroxyamide.
Veratrine is an alkaloid able to increase sodium currents through voltage-sensitive
sodium channels. It was demonstrated that veratrine induced an increase in the release
of glutamate, the major CNS excitatory neurotransmitter, in cortical and cerebellum
brain slices. Inhibition of glutamate release is considered as a potentially important

mechanism for anticonvulsants (Bourin et al., 2009).Since it is known that veratrine
antagonizes the antidepressant-like effects of lamotrigine, topiramate and phenytoin
(Prica et al., 2008, Bourin et al, 2009) it was supposed that their antidepressant-like
effects were dependent on the glutamate release. On the other hand, it was also reported
that the antidepressant-like effect of paroxetine (a selective serotonin reuptake
inhibitor), desipramine and imipramine (tricyclic antidepressants) was not reversed by
veratrine, and not dependent on glutamate release, suggesting that the mechanism of
action involved in the antidepressant-like effect of anticonvulsants is different from that
of classical antidepressants. Furthermore, the antidepressant-like effect of compound 5
seems to be dependent of the glutamate release (since the antidepressant like effect of
this drug was reversed by veratrine) and not related to a serotonin reuptake inhibition.
Also, it was already demonstrated that veratrine induced an anxiogenic-like behavior of
mice in the plus maze test, and that its effect could be reversed by riluzole (a sodium
channel blocker) (Saitoh et al., 2015). So, voltage-gated sodium channels could be
involved in the mechanism of action of drugs active in the plus maze test.
Conclusion
The results of the present study provided convincing evidence that i.p. administration of
the anticonvulsant α-hydroxyamides 2 to 5, at doses that do not interfere with the motor
performance, exerted clear dose dependent antidepressant action when assessed in two
models of depression in mice. From this series of α-hydroxyamides, N-propyl-2, 2-
diphenyl-2-hydroxyacetamide (compound 5) emerged as the most promising
anticonvulsant, antidepressant and anxiolytic-like derivative. Both in vitro (on sodium
current hNa 1.2) and in vivo (using veratrine) effects could be associated with a
v

blockade of voltage-gated sodium channels. The capacity of compound 5 to stabilize the
inactivated state of the channel was also demonstrated. The combined anticonvulsant,
antidepressant and anxiolytic- like profile of compound 5 may represent a valuable tool
for the treatment of these disorders. Further studies might be necessary to determine the
effect of this compound in chronic models of epilepsy, as a kindling-test. Animals from
these experiments could also be tested looking for antidepressant and anxiolytic-like
effects. Studies of survivals, after chronic treatments with compound 5, compared to
reference drugs usually use in clinical nowadays, may be also a useful data. Moreover,
it would be necessary to determine if these compounds could exhibit enhanced
pharmacokinetic or pharmacodynamic properties with advantages as pharmacological
therapeutic agents for the treatment of various neuropsychiatric disorders.
Acknowledgments
This work has been supported by grants from Consejo Nacional de Investigaciones
Científicas y Técnicas (CONICET, PIP Numbers 112 201501 00410 and 112-201101-
0
0045), Universidad de Buenos Aires (UBA, UBACyT Number 20020100100415), and
Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT, PICT Number
011-0328), Argentina.
2
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