Design and Comparative Evaluation of the Anticonvulsant Profile, Carbonic-Anhydrate Inhibition and Teratogenicity of Novel Carbamate Derivatives of Branched Aliphatic Carboxylic Acids with 4-Aminobenzensulfonamide

Article abstract of DOI:10.1007/s11064-017-2216-x

Epilepsy is one of the most common neurological diseases, with between 34 and 76 per 100,000 people developing epilepsy annually. Epilepsy therapy for the past 100⁺ years is based on the use of antiepileptic drugs (AEDs). Despite the availability of more than twenty old and new AEDs, approximately 30% of patients with epilepsy are not seizure-free with the existing medications. In addition, the clinical use of the existing AEDs is restricted by their side-effects, including the teratogenicity associated with valproic acid that restricts its use in women of child-bearing age. Thus, there is an unmet clinical need to develop new, effective AEDs. In the present study, a novel class of carbamates incorporating phenethyl or branched aliphatic chains with 6–9 carbons in their side-chain, and 4-benzenesulfonamide-carbamate moieties were synthesized and evaluated for their anticonvulsant activity, teratogenicity and carbonic anhydrase (CA) inhibition. Three of the ten newly synthesized carbamates showed anticonvulsant activity in the maximal-electroshock (MES) and 6?Hz tests in rodents. In mice, 3-methyl-2-propylpentyl(4-sulfamoylphenyl)carbamate(1), 3-methyl-pentan-2-yl-(4-sulfamoylphenyl)carbamate (9) and 3-methylpentyl, (4-sulfamoylphenyl)carbamate (10) had ED₅₀ values of 136, 31 and 14?mg/kg (MES) and 74, 53, and 80?mg/kg (6?Hz), respectively. Compound (10) had rat-MES-ED₅₀ = 13?mg/kg and ED₅₀ of 59?mg/kg at the mouse-corneal-kindling test. These potent carbamates (1,9,10) induced neural tube defects only at doses markedly exceeding their anticonvuslnat-ED₅₀ values. None of these compounds were potent inhibitors of CA IV, but inhibited CA isoforms I, II and VII. The anticonvulsant properties of these compounds and particularly compound 10 make them potential candidates for further evaluation and development as new AEDs.

Full text of DOI:10.1007/s11064-017-2216-x

Neurochem Res
DOI 10.1007/s11064-017-2216-x
ORIGINAL PAPER
Design and Comparative Evaluation of the Anticonvulsant Profile,
Carbonic-Anhydrate Inhibition and Teratogenicity of Novel
Carbamate Derivatives of Branched Aliphatic Carboxylic Acids
with 4-Aminobenzensulfonamide
David Bibi1 · Hafiz Mawasi1 · Alessio Nocentini3 · Claudiu T. Supuran3 ·
Bogdan Wlodarczyk4 · Richard H. Finnell4 · Meir Bialer1,2
Received: 12 January 2017 / Revised: 16 February 2017 / Accepted: 20 February 2017
© Springer Science+Business Media New York 2017
Abstract Epilepsy is one of the most common neurologi-
cal diseases, with between 34 and 76 per 100,000 people
developing epilepsy annually. Epilepsy therapy for the
past 100+ years is based on the use of antiepileptic drugs
(AEDs). Despite the availability of more than twenty old
and new AEDs, approximately 30% of patients with epi-
lepsy are not seizure-free with the existing medications. In
addition, the clinical use of the existing AEDs is restricted
by their side-effects, including the teratogenicity associ-
ated with valproic acid that restricts its use in women of
child-bearing age. Thus, there is an unmet clinical need to
develop new, effective AEDs. In the present study, a novel
class of carbamates incorporating phenethyl or branched
aliphatic chains with 6–9 carbons in their side-chain, and
4-benzenesulfonamide-carbamate moieties were synthe-
sized and evaluated for their anticonvulsant activity, tera-
togenicity and carbonic anhydrase (CA) inhibition. Three
of the ten newly synthesized carbamates showed anticon-
vulsant activity in the maximal-electroshock (MES) and
6 Hz tests in rodents. In mice, 3-methyl-2-propylpentyl(4-
(4-sulfamoylphenyl)carbamate (10) had ED50 values of 136,
31 and 14 mg/kg (MES) and 74, 53, and 80 mg/kg (6 Hz),
respectively. Compound (10) had rat-MES-ED50 =13 mg/
kg and ED50 of 59 mg/kg at the mouse-corneal-kindling
test. These potent carbamates (1,9,10) induced neural tube
defects only at doses markedly exceeding their anticon-
vuslnat-ED50 values. None of these compounds were potent
inhibitors of CA IV, but inhibited CA isoforms I, II and
VII. The anticonvulsant properties of these compounds and
particularly compound 10 make them potential candidates
for further evaluation and development as new AEDs.
Keywords New antiepileptic drugs · Valproic acid ·
4-Aminobenzensulfonamides · Teratogenicity · Carbonic
anhydrase inhibition
Abbreviations
CMK Corneal kindled mouse
CNS
Central nervous system
AED Antiepileptic drug
sulfamoylphenyl)carbamate(1),
3-methyl-pentan-2-yl-
SAR
Structure–activity relationship
(4-sulfamoylphenyl)carbamate (9) and 3-methylpentyl,
MES Maximal electroshock seizure
scMet Subcutaneous metrazol
PI
Protective index
* Meir Bialer
VPA
SPD
THF
Valproic acid
bialer@md.huji.ac.il
Sec-butylisopropylaceatmide
Tetrahydrofuran
1
Faculty of Medicine, School of Pharmacy, Institute of Drug
Research, The Hebrew University of Jerusalem, Jerusalem,
Israel
DCM Dichlromethane
NTD Neural tube defects
2
Affiliated with the David R. Bloom Center for Pharmacy, The
Hebrew University of Jerusalem, Jerusalem, Israel
3
NEUROFARBA Dept., Sezione di Scienze Farmaceutiche,
Introduction
Università degli Studi di Firenze, Via Ugo Schiff 6, Sesto
Fiorentino, 50019 Florence, Italy
4
Epilepsy is one of the most common neurological diseases,
with between 34 and 76 per 100,000 people developing
Department of Pediatrics, The University of Texas at Austin
Dell Medical School, Austin, TX, USA
Vol.:(0123456789)
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Neurochem Res
epilepsy annually [1]. Epilepsy is a chronic disorder of
the brain characterized by recurrent unprovoked seizures.
These seizures are signs or symptoms of abnormal neuronal
activity in the brain [2]. Epilepsy therapy for the past 100+
years is based on the use of antiepileptic drugs (AEDs),
which can be divided into two generation. First generation:
AEDs approved before 1990 that include four first-line
AEDs: carbamazepine, phenobarbital, phenytoin and valp-
roic acid (VPA). The second generation includes the AEDs
that have been introduced after 1990 [3, 4]. In addition, 12
new AEDs are currently in phase I-III clinical develop-
ments [5, 6]. Nevertheless, despite the availably of more
than 20 old and new AEDs, approximately 30% of patients
with epilepsy are not seizure-free using the existing media-
tions. Furthermore, the clinical use of many existing AEDs
is restricted by their side effects, including the teratogenic-
ity associated with the therapeutic use of VPA that restricts
its use in women of child-bearing age [7]. Consequently,
there is an unmet clinical need to discover and develop
novel chemical entities that may offer more efficacious and
safer therapeutic options over existing AEDs [3–7].
Many carbamates compounds have demonstrated poten-
tial therapeutic uses. The discovery in the 1960s that the
old dicarbamate anxiolytic drug meprobamate also pos-
sessed anticonvulsant activity prompted the design and
anticonvulsant evaluation of several anticonvulsant carba-
mates [22]. SAR studies of carbamates showed that deriva-
tives with two alkyl groups attached at C-2 possess stronger
muscle paralyzing activity, whereas the presence of a phe-
nyl group at the 2 position enhances anticonvulsant efficacy
[23]. Felbamate, 2-phenyl-1,3-propanediol dicarbamate,
lacked anxiolytic properties despite its structural similar-
ity to meprobamate yet it exhibited a broad anticonvulsant
activity profile [24]. Felbamate (FDA-approved in 1993) is
currently seldom used in clinical practice due to the fatal
aplastic anemia and hepatotoxicity associated with its ther-
apy [24]. Carisbamate is another anticonvulsant carbamate
that reached phase III clinical trials however, its regulatory
dossier (application) to the FDA and EMA was withdrawn
due to lack of consistent efficacy across a clinically-rele-
vant dose range [25, 26].
Cenobamate (YKP3089) is a new carbamate currently
undergoing Phase II/III clinical trials in epilepsy. At all
doses tested (100–400 mg) this compound was highly
effective compared to placebo in reducing the frequency
of partial-onset seizures on traditional efficacy endpoints
[5]. Cenobamate at 200 and 400 mg was highly effective
compared to placebo in achieving seizure freedom as an
adductive therapy in patients with refractory partial onset
seizures [27]. An open label safety study with a slower ini-
tial titration rate is currently ongoing.
In recent years, extensive anticonvulsant structure activ-
ity relationship (SAR) studies of numerous branched and
non-branched aliphatic carboxylic acids have been per-
formed [7–12]. Octanoic acid, like other non-branched
short fatty acids, were found to be inactive in anticonvul-
sant tests with the exception of decanoic acid which was
recently found to be active through direct AMPA receptor
inhibition [13, 14].
VPA (isoocatnoic acid) is an achiral branched monocar-
boxylic acid. The presence of eight carbons in its chemical
structure gives VPA an optimal balance between anticon-
vulsant activity and sedative/hypnotic adverse effects, since
efficacy and the sedative side effect increase in VPA homo-
logues with more than 8 carbons [15, 16].
Hen et al. designed and comparatively evaluated the
anticonvulsant activity of a series of 19 branched alkyl and
aryl carbamates, many of which were VPA derivatives [28].
Subsequently, Shekh-Ahmad et al. evaluated the enantiose-
lective pharmacodynamics and pharmacokinetics of the
most active chiral carbamates [29].
Acetazolamide is an old AED, structurally, containing a
sulfonamide group in its structure [17]. In the late 1970s
Ganz and colleagues evaluated the anticonvulsant activity
of nine derivatives of 4-aminobenzenesulfonamide (sul-
fanilamide) in electrically-(MES) and chemically-induced
(scMet) seizure tests and found that they displayed an
excellent anticonvulsant activity profile [18]. Syntheses
of various aromatic sulfonamide derivatives demonstrated
the impact of different sulfonamide moieties on their anti-
convulsant activity [18, 19]. Tasso et al. have reported that
4-(valproylamido)-benzenesulfonamide is a potent anticon-
vulsant in the mouse-MES test [20]. Hen et al., synthesized
and evaluated the anticonvulsant profile and teratogenicity
of new amide derivatives of branched aliphatic carboxylic
acids with 4-aminobenzensulfonamide. Three (sulfamoyl-
phenyl) butyramide derivatives were the most potent com-
pounds possessing in rats MES-ED50 values of 7.6, 9.9 and
9.4 mg/kg [21].
There is a critical need to discover novel chemical enti-
ties for the development of safe and potent AEDs [3, 4,
12]. Consequently, in this study we assessed the effect of
changing the length and the branching of the aliphatic side
chains in the alcohols used for the derivatization (esterifica-
tion) with 4-aminobenzenesulfonamide-carbamic acid. We
designed and comparatively analyzed the anticonvulsant
activity, neurotoxicity, teratogenicity and carbonic anhy-
drase (CA) inhibition of 10 new carbamate derivatives of
4-aminobenzenesulfonamide (depicted in Fig. 1), possess-
ing 6–9 carbons in the aliphatic side chain of the carbamate
moieties. The teratogenicity of the most potent anticonvul-
sant compounds was evaluated in mice and their CA inhibi-
tion of four human-CA isoforms was tested in vitro com-
pared to that of acetazolamide. We sought to determine the
SAR of the above mentioned carbamates in an attempt to
develop new potent and safe AEDs.
1 3

Neurochem Res
Fig. 1 Structures of the synthe-
sized and tested sulfonebenzene
carbamates
SO2NH2
designed in this study were synthesized by non-stereospe-
cific methods and thus evaluated as racemates.
O
O
a
b
Cl
R
R
OH
O
N
H
R
O
3-methyl-2-propylpentyl (4-sulfamoylphenyl)carbamate
(1)
Scheme 1 General synthesis of 4-benzenesulfonamide-carbamates
Conditions and reagents: a pyridine, triphosgene, DCM, room tem-
perature, 2 h; b 4-aminobenzenesulfonamide, THF, 12 h
White powder; mp 151–152°C. 1H NMR (300 MHz,
DMSO-d6) δ 9.94 (s, 1H), 7.76–7.65 (m, 2H), 7.65–7.54
(m, 2H), 7.19 (s, 2H), 4.15–3.92 (m, 2H), 3.36–3.23 (m,
1H), 1.56–1.04 (m, 7H), 0.92–0.77 (m, 9H). Calcd for
C16H26N2O4S: C, 56.12; H, 7.65; N, 8.18; S, 9.36.
Found: C, 56.12; H, 7.76; N, 8.0; S, 9.08.
Materials and Methods
Chemistry
Unless otherwise stated, all reagents were purchased from
commercial suppliers and used without further purification.
Solvents used in the reactions were distilled from appropri-
ate drying agents prior to use.
2-ethylhexyl (4-sulfamoylphenyl)carbamate (2)
White powder; mp 192–195°C. 1H NMR (300 MHz,
DMSO-d6) δ, 8.10 (s, 1H), 7.39 (d, J=8.6 Hz, 2H), 7.26
(s, 2H), 7.04 (d, J=8.6 Hz, 1H), 4.29 (m, 2H), 1.62–1.68
(m, 1H), 1.44–1.23 (m, 8H), 0.99–0.80 (m, 6H). Calcd
for C15H24N2O4S: C, 58.86; H, 7.37; N, 8.53; S, 9.76.
Found: C, 56.62; H, 7.09; N, 8.57; S, 9.58.
General Procedure for the Synthesis of Compounds
The general syntheses for the 4-aminobenzenesulfonamides
derivatives in Scheme 1. A solution of the alcohol (1 equiv)
in dichloromethane (DCM, 5 mL) was added dropwise to a
stirred solution of triphosgene (0.5 equiv) and pyridine (1
equiv) in DCM (10 mL). The reaction mixture was allowed
to stir for 2 h and then was evaporated. The residue was
dissolved in THF (10 mL). A solution of sulfanilamide
(500 mg) in THF (10 mL) was added and stirred over night.
The reaction was quenched with water and extracted with
EtOAc (15 mL). The organic layer was washed with water
and brine, dried over sodium sulfate, filtered, and concen-
trated in vacuo. The desired product was obtained follow-
ing flash chromatography (EtOAc/hexane). The compounds
2-propylpentyl (4-sulfamoylphenyl)carbamate (3)
White powder; mp 176–177°C. 1H NMR (300 MHz,
DMSO-d6) δ 9.92 (s, 1H), 7.76–7.65 (d, J=8.6 Hz, 2H),
7.65–7.54 (d, J=8.7 Hz, 1H), 7.17 (s, 2H), 4.00 (m, 2H),
1.66 (s, 1H), 1.42–1.18 (m, 8H), 0.97–0.81 (m, 6H). Calcd
for C15H24N2O4S: C, 54.86; H, 7.37; N, 8.53; S, 9.76.
Found: C, 55.05; H, 7.28; N, 8.43; S, 9.54.
1 3

Neurochem Res
3,3-dimethylbutyl (4-sulfamoylphenyl)carbamate (4)
for C13H20N2O4S: C, 51.98; H, 6.71; N, 9.33; S, 10.68.
Found: C, 52.03; H, 6.61; N, 9.32; S, 11.2.
White powder; mp 170–173°C. 1H NMR (300 MHz,
DMSO-d6) δ 9.99 (s, 1H), 7.67–7.74 (d, J=8.7 Hz, 2H),
7.63–7.5 (d, J=8.7 Hz, 2H), 7.21 (s, 2H) ,4.11 (m, 2H),
1.57–1.48 (m, 2H) 0.96 (s, 9H). Calcd for C13H20N2O4S:
C, 51.98; H, 6.71; N, 9.33; S, 10.68. Found: C, 52.03; H,
6.66; N, 9.2; S, 10.33.
3-methylpentyl(4-sulfamoylphenyl)carbamate (10)
White powder; mp 168–169°C. 1H NMR (300 MHz,
DMSO-d6) δ 10.00 (s, 1H), 7.70 (d, J=8.8 Hz, 2H),
7.59 (d, J=8.7 Hz, 2H), 7.20 (s, 1H), 4.18–4.07 (m, 2H),
1.73–1.56 (m, 1H), 1.11–1.21 (m, 4H), 0.85 (m, 6H).
Calcd for C13H20N2O4S: C, 51.98; H, 6.71; N, 9.33; S,
10.68. Found: C, 52.12; H, 6.54; N, 9.35; S, 10.77.
phenethyl (4-sulfamoylphenyl)carbamate (5)
White powder; mp 212–213°C. 1H NMR (300 MHz,
DMSO-d6) δ 10.02 (s, 1H), 7.56–7.63 (d, J=8.6 Hz, 2H),
7.68–7.72 (d, J=8.6 Hz, 2H), 7.29 (s, 2H), 7.31–7.19 (m,
5H) ,4.41 (t, J=6.8 Hz ,2H), 2.84 2.95 (t, J=6.8 Hz, 2H).
Calcd for C15H16N2O4S: C, 56.24; H, 5.03; N, 8.74; S,
10.01. Found: C, 55.9; H, 5.07; N, 8.61; S, 9.64.
Materials and Methods
Product formation follow-up was performed by means of
1H NMR and TLC. TLC analyses were performed on pre-
coated silica gel on aluminum sheets (Kieselgel 60 F254,
1
2-ethylbutyl (4-sulfamoylphenyl)carbamate (6)
Merck). H NMR spectra were recorded on a Varian Mer-
cury series NMR 300 spectrometer. Chemical shifts (δ
scale) are reported in parts per million (ppm) relative to
the indicated reference. Coupling constants are given in
Hz. Chemical structure and purity of the compounds newly
White powder; mp 162–165°C. 1H NMR (300 MHz,
DMSO-d6) δ 9.92 (s, 1H), 7.58–7.62 (d, J=8.9 Hz ,2H),
7.68–7.72 (d, J=8.9 Hz ,2H), 7.21 (s, 2H), 4.02 (m, 2H),
2.07 (s, 1H), 1.36 (m, 1H), 1.05–0.87 (m, 6H). Calcd for
C13H20N2O4S: C, 51.98; H, 6.71; N, 9.33; S, 10.68.
Found: C, 51.59; H, 6.65; N, 9.18; S, 10.54.
1
synthesized was assessed by TLC, H NMR and HPLC.
Melting point was determined on a 1002-230 VAC Mel-
Temp capillary melting point apparatus.
2,4,4-trimethylpentyl (4-sulfamoylphenyl)carbamate (7)
Biological Testing/Anticonvulsant Activity
White powder; mp 199–201°C. 1H NMR (300 MHz,
DMSO-d6) δ 9.96 (s, 1H), 7.76–7.65 (d, J=8.7 Hz, 1H),
7.65–7.54 (d, J=8.7 Hz, 1H), 7.18 (s, 2H), 3.97 (m, 1H),
3.79 (m, 1H), 1.83 (m, 1H), 1.92–1.76 (m, 1H), 1.29 (m,
1H), 1.00 (m, 4H), 0.89 (s, 8H). Calcd for C15H24N2O4S:
C, 54.86; H, 7.37; N, 8.53; S, 8.76. Found: C, 54.48; H,
7.29; N, 8.31; S, 8.39.
The evaluation of anticonvulsant activity in the maximal
electroshock seizure test (MES), subcutaneous metrazol
seizure test (scMet) and 6 Hz psychomotor test (6 Hz),
corneal kindled mouse (CKM) and the determination of
minimal neurotoxicity using the rotarod test and positional
sense test and others were performed at the National Insti-
tute of Health (NIH)-National Institute of neurological
disorders and stroke (NINDS) as a part of the Epilepsy
Therapy Screening Program (ETSP) according protocols
described in [30, 31].
2,4-dimethylpentan-3-yl (4-sulfamoylphenyl)carbamate
(8)
White powder; mp 193–194°C. 1H NMR (300 MHz,
DMSO-d6) δ 9.94 (s, 1H), 7.70 (d, J=8.6 Hz, 2H), 7.60
(d, J=8.7 Hz, 2H), 7.20 (s, 2H), 4.43 (t, J=6.2 Hz, 1H),
1.90 (h, J=7.0 Hz, 2H), 0.87 (d, J=6.7 Hz, 12H). Calcd
for C14H22N2O4S: C, 53.48; H, 7.05; N, 8.91; S, 10.20.
Found: C, 53.42; H, 7.25; N, 8.62; S, 9.93.
Preparation of the Compounds for Testing
The tested compounds were suspended in 0.5% methylcel-
lulose and administered intraperitoneally (i.p.) to adult male
CF no. 1 albino mice (18–25 g) in a volume of 0.01 mL/g
body weight and (b) orally to adult male Sprague–Dawley
albino rats (100–150 g) in a volume of 0.04 mL per 10 g of
body weight. The pentylenetetrazol solution at convulsing
dose was prepared by sufficient dissolution of pentylenetet-
razol in 0.9% saline to make 0.85% solution for administra-
tion to mice and 2.82% solution for administration to rats
[30, 31].
3-methylpentan-2-yl (4-sulfamoylphenyl)carbamate (9)
White powder; mp 179–180°C. 1H NMR (300 MHz,
DMSO-d6) δ 9.90 (s, 1H), 7.57–7.61 (d, J=8.7 Hz, 1H),
7.68–7.71 (d, J=8.7 Hz, 1H), 7.18 (s, 2H), 4.82–4.63 (m,
1H), 1.65–1.41 (m, 1H), 1.17 (m, 5H), 0.88 (m, 6H). Calcd
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Neurochem Res
Determination of the Median Effective Dose (ED50)
and the Median Neurotoxic Dose (TD50)
CA Inhibition
An Applied Photophysics stopped-flow instrument has been
used for assaying the CA catalysed CO2 hydration activity
[34]. Phenol red (at a concentration of 0.2 mM) has been
used as indicator, working at the absorbance maximum of
557 nM, with 20 mM Hepes (pH 7.5) as buffer, and 20 mM
Na2SO4 (for maintaining constant the ionic strength), fol-
lowing the initial rates of the CA-catalyzed CO2 hydra-
tion reaction for a period of 10–100 s. The CO2 concen-
trations ranged from 1.7 to 17 mM for the determination
of the kinetic parameters and inhibition constants. For each
inhibitor at least six traces of the initial 5–10% of the reac-
tion have been used for determining the initial velocity. The
uncatalyzed rates were determined in the same manner and
subtracted from the total observed rates. Stock solutions
of inhibitor (0.1 mM) were prepared in distilled-deionized
water and dilutions up to 0.01 nM were done thereafter
with the assay buffer. Inhibitor and enzyme solutions were
preincubated together for 15 min at room temperature prior
to assay, in order to allow for the formation of the E-I com-
plex. The inhibition constants were obtained by non-linear
least-squares methods using PRISM 3 and the Cheng-
Prusoff equation, as reported earlier [34–37] and represent
the mean from at least three different determinations. All
CA isofoms were recombinant ones obtained in-house as
reported earlier [35–38].
For the determination of the ED50 by the respective anti-
convulsant procedure, doses of the tested compounds
were varied until a minimum of 3–4 points was estab-
lished between the dose level with 0% protection and
100% protection. These data were subjected to the FOR-
TRAN probit analysis program, and the ED50 and 95%
confidence intervals were calculated. The TD50 was
determined by varying the dose of the tested compounds
until four points were established between the dose level
that induced no signs of minimal motor impairment in
any of the animals and the dose at which all of the ani-
mals were considered impaired. The TD50 and the 95%
confidence intervals were calculated by FORTRAN pro-
bit analysis. The PI values were calculated by dividing
the TD50 by the ED50 [30, 31].
Evaluation of Teratogenicity
The teratogenic properties of the newly designed com-
pounds (1–10) were evaluated in the highly inbred SWV
mice strain known for its high susceptibility to VPA-
induced neural tube defects (NTDs) [32, 33]. Two month
old nulligravid females, were mated overnight with males
and examined for the presence of vaginal plugs the fol-
lowing morning, and the onset of gestation was consid-
ered to be 10 pm. of the previous night, the midpoint of
the dark cycle. At day 8.5 of gestation, each dam received
a single i.p. injection of the test compounds in an equi-
molar dose of 0.9 or 1.8 mmol/kg and the control dams
were injected with vehicle (25% water solution of Cre-
mophore EL, Fluka Biochemica Germany). The volume
of injection was 10 µL/g of body weight. The 1.8 mmol/
kg dose was selected as this was the highest dose that did
not produce overt maternal toxicity except for deep seda-
tion and lack of ambulation lasting for approximately 2 h
post-injection. At gestation day 8.5, the dams were eutha-
nized by carbon dioxide asphyxiation followed by cervi-
cal dislocation. After the opening the uterus, the location
of all viable fetuses and resorption sites were recorded,
and the fetuses were examined for the presence of exen-
cephaly or other gross congenital abnormalities. The tera-
togenicity data (implantations, resorptions and NTDs)
were evaluated for significant differences between the
control and treated groups by analyzing the contingency
table with Fisher’s exact test. Statistical analysis was con-
ducted using GraphPad InStat (version 3.06; GraphPad
Software, San Diego, CA, USA), and the results of all
tests were considered to be statistically significant when
the p-value was less than 0.05.
Calculation of Clog P
Clog P was calculated by means of ChemDraw-Ultra Soft-
ware 8.
Results
Chemistry
The general synthesis of the carbamates designed in the
current study is presented in Scheme 1. Compounds 1–10
(Fig. 1) were obtained with isolated yields ranging between
62–85%.
Pharmacology
The anticonvulsant activity profile of compounds 1–10 was
determined using the MES test, which measures seizure
spread, scMet test, which measures seizure threshold and
the 6 Hz (32 mA) psychomotor test. These tests are mech-
anism-independent animal seizure models that enable iden-
tification of compounds preventing seizure spread [30, 31].
Compounds 1–10 were administered i.p. to mice and orally
(or i.p.) to rats at fixed doses of 30, 100 or 300 mg/kg, and
1 3

Neurochem Res
the anticonvulsant protection was observed at different
times after dosing. The method applied here allowed the
determination of the number of animals (in a group consist-
ing of 4 or 8 mice or rats) protected against electrically- or
chemically-induced seizures as well as the estimation of the
time course of anticonvulsant activity together with time of
peak effect (TPE).
(compound 1) protection at 100 mg/kg with no signs of
neurotoxicity. These compounds showed partial protection
with no neurotoxicity at doses of 30 and 100 mg/kg and full
or almost full (compound 1) protection 300 mg/kg at the
6 Hz test with no neurotoxicity.
Comparative quantitative evaluation at the mouse-MES,
scMet and 6 Hz (32 mA) (i.p.) models showed that com-
pounds 1, 9 and 10 demonstrated anticonvulsant activ-
ity in the mouse-MES and 6 Hz models with ED50 values
of 136, 31 and 14 mg/kg (MES) and 74 mg/kg, 53 mg/kg
and 80 mg/kg (6 Hz-32mA), respectively (Table 2). At the
scMet test, compound 10 had an ED50 value of 70 mg/kg
however, compounds 1 and 9 had a relatively high ED50
Table 1 presents the anticonvulsant activity and neu-
rotoxicity or minimal behavioral impairment of all tested
compounds (compounds 1–10) at the mouse-MES and
mouse-6Hz (32 mA) tests (i.p.). At the MES model, these
three most active compounds (compounds 1,9 and 10)
showed some protection at 30 mg/kg and full or almost full
Table 1 Anticonvulsant activity
and neurotoxicity (minimal
behavioral impairment)
of 4-sulfonamidobenzene
carbamates administered i.p.
to mice
Compound
1
Dose (mg/kg)
MES a
0.5 h
6Hza, b
0.5 h
Tox c
0.5 h
2.0 h
2.0 h
2.0 h
30
100
300
30
100
300
30
100
300
30
100
300
30
100
300
30
100
300
30
100
300
30
100
300
30
0/4
0/4
0/4
0/4
1/4
1/4
0/4
0/4
0/4
0/4
0/4
1/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
1/4
3/4
3/4
4/4
3/4
4/4
3/4
0/4
3/4
4/4
0/4
0/4
1/4
0/4
0/4
0/4
0/4
0/4
1/4
1/4
1/4
2/4
0/4
0/4
0/4
0/4
1/4
2/4
2/4
4/4
4/4
0/4
4/4
4/4
1/4
4/4
4/4
1/4
1/4
0/4
0/4
3/4
2/4
0/4
0/4
1/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
1/4
1/4
1/4
1/4
4/4
0/4
0/4
2/4
2/4
2/4
4/4
1/4
3/4
4/4
0/4
1/4
3/4
0/4
0/4
1/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
1/4
0/4
2/4
3/4
0/4
0/4
1/4
0/4
0/4
1/4
0/4
2/4
4/4
0/8
0/8
0/8
0/8
0/8
0/8
0/8
0/8
0/8
0/8
0/8
0/8
0/8
0/8
0/8
0/8
0/8
0/8
0/8
0/8
0/8
0/8
0/8
1/8
0/8
0/8
0/8
1/8
0/8
1/8
0/8
0/8
0/8
0/8
0/8
0/8
0/8
0/8
0/8
0/8
0/8
0/8
0/8
0/8
0/8
0/8
0/8
0/8
0/8
0/8
0/8
0/8
0/8
0/8
0/8
0/8
0/8
0/8
0/8
0/8
2
3
4
5
6
7
8
9
100
300
30
100
300
10
aData indicate number of mice protected/number of mice tested. The animals were examined at two pre-
treatment times: 0.5 and 2 h
bThe 6 Hz psychomotor test was conducted with 32 mA
cNeurotoxicity was evaluated as motor impairment or sedation (number of animals affected/number of ani-
mals tested)
1 3

Neurochem Res
Table 2 Quantitative anticonvulsant data (ED50, TD50 and PI values) in mice dosed i.p.
Compound MES-ED50
(mg/kg) [95%
CI]a
PIb
scMet- ED50
(mg/kg) [95%
CI]a
PIb
6 Hz- ED50 (mg/ PIb
CKM - ED50
(mg/kg) [95%
CI]a
PIb
Neurotoxicity-
TD50 (mg/kg)
[95% CI]
kg) [95% CI]a, c
1
136
>3.6
>300
>1.6
74
>6.8 NA
>11.3 NA
7.9 59 [36–87]
–
–
>500
>600
[89–212]
[38–125]
9
31
>19.4 369 [250–527]
50 70 [46–99]
>1.6 53 [23–94]
[20–45]
10
14
10
80d [50–127]
11.8 698 [457–1678]
[10–21]
aMedian Effective (ED50) and Toxic (TD50) values. In squared parentheses are the 95% confidence intervals (95%CI) determined by probit analy-
sis
bProtective Index (PI=TD50/ED50)
cThe 6 Hz test as done at 32 mA
dAt the 6 Hz (44 mA)-ED50 =88 mg/kg [62–135 mg/kg]
Table 3 Quantitative
anticonvulsant data (ED50,
TD50, and PI values) in rats
dosed p.o or i.p
Compound
MES-ED50 (mg/kg) PIb
[95% CI]a
scMet ED50(mg/kg) PIb
[95% CI]a
Neurotoxicity-
TD50 (mg/kg)
[95% CI]
1 (p.o.)
9 (p.o.)
36 [25–52]
23 [18–31]
NA
>21.4
NA
NA
NA
NA
>500
>250
10 (p.o.)
28 [18–35]
>17.8
>250
NA
>500
10 (i.p.)
13
>19.2
NA
NA
>250
[8–22]
aMedian Effective (ED50) and Toxic (TD50) values. In squared parentheses are the 95% confidence intervals
(95%CI) determined by probit analysis
bProtective Index (PI=TD50/ED50)
values. Compound 10 had also an ED50 value of 59 mg/kg
at the corneal kindled mouse model.
Table 4 Lipophilicity
data
Compound
Clog P
(Clog P) of the Investigated
Compounds
1
2
3
4
5
6
7
8
9
10
4.0
3.6
3.6
2.4
2.1
2.6
3.4
3.4
2.3
2.6
Following oral administration to rats compounds 1, 9 10
had MES-ED50 value of 36, 23, and 28 mg/kg, respectively
but were inactive at scMet model. Following i.p. admin-
istration to rats compound 10 had an MES-ED50 value of
13 mg/kg (95% CI=8-22 mg/kg), showing similar MES-
ED50 values after oral and i.p. administration to rats. At the
rat-MES test, compounds 9 and 10 had a wide protective
index (PI>19) (Table 3).
The optimal balance between lipophilic and hydro-
philic moieties (log P) is a very important consideration in
designing AEDs [39]. The logP values of the synthesized
carbamates are depicted in Table 4.
Clog P was calculated by utiliz-
ing the ChemDraw Ultra soft-
ware, version 12
Teratogenicity
The teratogenicity profile of the active compounds (1,
9 and 10) is presented in Table 5. These compounds
showed some indication of teratogenicity when tested
at the 1.8 mmol/kg dose but lacked a teratogenic poten-
tial at 0.9 mmol/kg. The 0.9 dosage is 4–10 times higher
than their mouse-ED50 values (except for compound 1
mouse-MES-ED50
value) and 12–20 times higher than
their rat-MES-ED50 values. The active compounds were
embryotoxic as evidenced by the high resorption rate but
again at doses significantly higher than their anticonvul-
sant ED50 values.
1 3

Neurochem Res
Table 5 Teratogenic effect in the SWV mouse model of the test compounds
Compound Dose mg/kg No. of litters No. of implants No. of resorptions No. of live fetuses No. of fetuses Fetal weight mean SD
(mmol/kg)
with NTDs
Control
1
1
9
9
10
10
25% CEL
616 (1.8)
308 (0.9)
541(1.8)
270 (0.9)
541 (1.8)
270 (0.9)
11
9
10
9
10
9
11
142
109
126
107
126
108
145
8 (5.6)
134 (94.4)
50 (45.9)
94 (74.6)
49 (45.8)
109 (86.5)
56 (51.9)
116 (80.0)
0
0.95 0.06
0.88 0.06*
0.88 0.03*
0.89 0.08
0.90 0.07
0.87 0.08*
0.88 0.03*
59 (54.1)*
32 (25.4)*
58 (54.2)*
17 (13.5)*
52 (48.2)*
29 (20.0)*
7 (14.0)*
1 (1.1)
9 (18.4)*
3 (2.8)
17 (30.4)*
9 (7.8)*
*Significantly different when compared to the control group For statistical purposes either ANOVA with Tukey post test multiple comparison
(Fetus weight) or Contingency Table Analysis with Fisher’s exact test (number of resorptions and NTDs) were performed. P value was set at
0.05
Table 6 Inhibition data of human CA isoforms hCA I, II, IV and VII
of compounds 1–10 and compared to the standard sulfonamide inhib-
itor acetazolamide (AAZ) utilizing a stopped flow CO2 hydrase assay
values of 7.6–10 mg/kg coupled with a wide PI values of
>50 [21].
In this study a novel class of 4-aminobenzenesulfon-
amide-carbamates incorporating phenethyl- or branched
alkyls with 6–8 carbon atoms in their side-chain were syn-
thesized and evaluated for anticonvulsant activity, terato-
genicity and CA inhibition.
Compound
KI (nM)
hCAI
hCA II
hCA IV
hCA VII
1
2
3
4
5
6
7
8
89.0
421.2
484.7
59.5
5.9
169.5
83.5
21.0
72.9
77.0
250
5.7
8.7
19.6
4.5
0.6
5.2
5.0
3.7
5.4
7.6
12
>10,000
>10,000
>10,000
1836.3
302.9
1690.8
2756.6
452.4
865.1
>10,000
>10,000
44.4
24.5
52.0
712.3
35.4
35.5
350.6
2.5
Three of the ten synthesized new carbamates showed
anticonvulsant activity at the MES and 6 Hz tests in mice
or rats. In mice compounds 1, 9 and 10 had ED50 values
of 136, 31 and 14 mg/kg (MES) and 74, 53 and 80 mg/
kg (6 Hz), respectively. It worth emphasizing that com-
pound 10 had similar ED50 at the 6 Hz (32 and 44 mA)
tests. Compund 10 had ED50 values of 13 and 59 mg/kg at
the rat-MES the mouse corneal kindling tests. Compound
10-PI values was 59 and above >10 mouse and rat MES
test, respectively. The aliphatic moiety of compound 1 is
an alcoholic congener of sec-butylpropylacetamdie (SPD)
a very potent antiepileptic and CNS compound [4, 5, 40].
Compounds 9 and 10 are two constitutional isomers con-
taining, 1,2-dimethybutyl and 3-methylpentyl, respectively
in their aliphatic side-chain.
9
182.6
750.0
74
10
AAZ
*Mean from 3 different assays, by a stopped flow technique (errors
were in the range of 5–10% of the reported values)
Carbonic Anhydrase (CA) Inhibition
Comparative analysis of the most active 4-aminobenze-
nesulfonamide-carbamates designed in this study with the
most active 4-aminobenzenesulfonamide amide derivatives
synthesized and evaluated in a previous study [21], yields
the followings: (a) The three most active compounds con-
tained branched butyl or pentyl moieties in their aliphatic
side-chain and had similar rat-MES-ED50 values, ranging
from 7.7 to 23 mg/kg. (b) A dimethylbutyl side-chain leads
to an active anticonvulsant compound in both the amide
and carbamate groups with rat-MES-ED50 values of 9.4
and 28 mg/kg, respectively. Compound 10-ED50 and wide
PI values were similar those of the best 4-aminobenzene-
sulfonamide amide derivatives described previously [21].
In two previous studies of unsaturated analogs of VPA
the log P values of the compounds ranged between 1.2 and
3.4 with minimal difference between the active and the
Table 6 shows the inhibition constant (Ki) of compounds
1–10 against four human carbonic anyhydrase (hCA)
isoforms. The compounds potency varied from the low
nanomolar to micromolar, depending on the substitution
pattern of the aliphatic chain of the carbamates.
Discussion
Previously, we designed and evaluated a series of branched
aliphatic amides of 4-aminobenzensulfonamide. The ana-
lyzed amides had 6–9 carbons in their carboxamide moiety.
The amides containing the aliphatic side-chain of 2-methyl-
butyramide, 2-ethyl-butyramide and 3,3-dimethyl-but-
yramde were the most potent with rat-MES(p.o.)-ED50
1 3

Neurochem Res
non-active compounds [41, 42]. The log P values of the
most active amides ranged between 1.0 and 1.5 while the
log P values of the most active carbamates ranged between
2.3 and 4, indicating that a higher (more lipophylic) log P
value does not lead directly to a more potent anticonvul-
sant activity. The active branched aliphatic carbamoyl-
sulfonamides 1, 9 and 10 had similar log P values as the
other less-active compounds. These results imply that BBB
(Blood–Brain Barrier) penetration due to lipophilicity is an
important factor in affecting the test drugs anticonvulsant
efficacy but not the only one.
role in various processes, first of all connected with pH reg-
ulation. Indeed, CA II, the physiologically dominant iso-
form, is expressed in the choroid plexus, oligodendrocytes,
myelinated tracts, astrocytes and myelin sheaths in the ver-
tebrates brain [43, 44]. The membrane-associated CA IV is
located on the luminal surface of cerebral capillaries, and
associated with the blood–brain barrier, being also con-
centrated in layers III and VI in the cortex, hippocampus
and thalamus of all investigated mammals [43, 44]. High
expression levels of CA VII were observed throughout the
cortex, hippocampus and thalamus [43–45]. Although vari-
ous functions have been assigned to the different CA iso-
forms present in the brain, such as formation of cerebro-
spinal fluid [43], regulation of its pH and ionic constituents
[43, 44], seizure activity [21, 44], regulation of GABAer-
gic signalling [45], respiratory response to carbon dioxide
[44], generation of bicarbonate for biosynthetic reactions
such as de novo glucogenesis, lipogenesis, etc [43–45]. It is
not known which isoforms contribute to the particular pro-
cesses mentioned here and whether the inhibition of one or
more isoforms is necessary to elicit anticonvulsant activity
in a CA inhibitor. The data of designed compounds point
out that CA II, the physiologically dominant and highly
effective isoform as catalyst, may be responsible for this
pharmacologic action.
These potent carbamates (1, 9 and 10) induced neural
tube defects only at doses markedly exceeding their effec-
tive dose. None of these compounds were potent inhibitors
of carbonic anhydrase IV, but did inhibit isoforms CAs I, II
and VII.
The following SAR can be drawn from the CA inhibi-
tion data depicted in Table 6.The isoform CA I was inhib-
ited with potencies ranging between 5.9 and 485 nM by the
investigated sulfonamide carbamates, with good inhibition
observed for compounds 5 and 8 which incorporate the
phenethyl moiety (5, KI of 5.9 nM) and a highly branched
aliphatic chain (8, KI of 21 nM).
The investigated carbamates inhibited CA II with KI
values comparable or better to the clinically used standard
acetazolamide, with the exception of phenethyl carbamate
(5) that was endowed with a Ki value 20 times lower than
acetazolamide, (KI of 0.6 nM). It should be mentioned that
the three compounds that showed in vivo anticonvulsant
activity were all highly effective hCA II inhibitors KI val-
ues of 5.4–7.6 nM (better than acetazolamide).
Conclusions
A novel series of carbamate derivatives containing 4-amin-
obenzenesulfonamide and branched VPA or phenethyl moi-
eties were synthesized in good yields and screened for their
anticonvulsant activities in MES and scMet and 6 Hz test.
In mice compounds 1, 9 and 10 had ED50 values of 136,
31 and 14 mg/kg (MES) and 74, 53 and 80 mg/kg (6 Hz),
respectively Compounds 1, 9 and 10 had rat (po)-MES-
ED50 values of 36, 28 and 23 mg/kg. These potent carba-
mates induced neural tube defects only at doses markedly
exceeding their anticonvuslnat-ED50 values. None of these
compounds were potent inhibitors of CA IV, but inhibited
isoforms CAs I, II and VII. All three of these compounds
exhibited better protective index than VPA suggesting that
they (and particularly compound 10) can be used as lead
compounds for future investigation to discover more effec-
tive, non-teratogenic and safer AEDs.
The transmembrane isoform CA IV was not or was
poorly inhibited by the designed carbamates. Indeed, only
compounds 5, 8 and 9, which respectively incorporate
phenethyl moiety, two highly branched aliphatic chains and
a short lowly branched chain, were medium potency inhibi-
tors, whereas the remaining ones acted in the micromo-
lar range or did not significantly inhibit the enzyme up to
10,000 nM.
Finally the hCA VII inhibition data (depicted in Table 6)
show that inhibitory properties of the investigated car-
bamates against this CA isoform is deeply related to the
degree of branching of the aliphatic chains present in the
carbamate functionality. Indeed, whereas the bulky, ramifi-
cated chain compounds 2 and 3 did not significantly inhibit
the enzyme up to 10,000 nM, the remaining ones acted as
nanomolar inhibitors of hCA VII with KIs spanning in a
wide nanomolar range (24.5–865 nM) depending on their
substitution pattern. Based on these in vitro inhibition data,
it may be inferred that isoform hCA II is probably the most
important one when considering the anticonvulsant action
of the CA inhibitors. Indeed, this isoform is highly abun-
dant in various CNS cells, where it plays probably a crucial
Supporting Information Available
Purity determination of the carbamates presented in Fig. 1
was performed by combustion analysis. The material and
description of the protocols of the animal models used for
1 3

Neurochem Res
by pentylenetetrazol and GABA content in brain of mice. Neu-
ropharmacology 22:875–879
the screening of investigational AED are available free of
charge via the internet at http://pubs.acs.
16. Morre M, Keane PE, Vernieres JC, Simiand J, Roncucci R
(1984) Valproate: recent findings and perspectives. Epilepsia
25(Suppl 1):5–9
Acknowledgements This work is abstracted from the Ph.D. thesis
of David Bibi in partial fulfillment of the Ph.D. degree requirements
for The Hebrew University of Jerusalem. The authors thank Drs. John
Keane and Shamsi Raeissi, of the NIH-NINDS Epilepsy Therapy
Screening Program (ETSP) for testing the compounds in the ETSP
and Dvora Izgelov and Bella Shusterman from the Hebrew Univer-
sity for their skillfully assistance with the HPLC and in the syntheses,
respectively.
17. Resor A SR, Resor LD, Woodbury DM, Kemp JW (1995)
Acetazolamide. In: Levy RH, Mattson RH, Meldrum BS
(eds) Antiepileptic Drugs, 4th edn, Raven Press, New York,
pp 969–985.
18. Ganz AJ, Waser PG, Pfirrmann RW (1978) Development of
new antiepileptics. V. Pharmacological activity of some deriv-
atives of sulfanilamide (author’s transl). Arzneimittelforschung
28:1331–1334
19. Masereel B, Rolin S, Abbate F, Scozzafava A, Supuran CT
(2002) Carbonic anhydrase inhibitors: anticonvulsant sulfona-
mides incorporating valproyl and other lipophilic moieties. J
Med Chem 45:312–320
References
20. Tasso SM, Moon S, Bruno-Blanch LE, Estiu GL (2004) Char-
acterization of the anticonvulsant profile of valpromide deriva-
tives. Bioorg Med Chem 12:3857–3869
1. Brodie MS, Barry SE, Bamagous GA, Norrie JD, Kwan, Pattern
P (2012) Treatment response to newly diagnosed epilepsy. Neu-
rology 78:1548–1554
21. Hen N, Bialer M, Wlodarczyk B, Finnell RH, Yagen B (2010)
Syntheses and evaluation of anticonvulsant profile and terato-
genicity of novel amide derivatives of branched aliphatic car-
boxylic acids with 4-amino-benzensulfonamide. J Med Chem
53:4177–4186
2. Fisher RS, van Emde BW, Blume W, Elger C, Genton P, Lee
P, Engel J Jr (2005) Epileptic seizures and epilepsy: definitions
proposed by the International League Against Epilepsy (ILAE)
and the International Bureau for Epilepsy (IBE). Epilepsia
46:470–472
22. Murray WJ, Kier LB (1977) Noncyclic anticonvulsants. In:
Vida JA (ed) Medicinal chemistry. Academic Press, New York,
pp 578–619
3. Perucca E, French J, Bialer M (2007) Development of new antie-
pileptic drugs: challenges, incentives, and recent advances. Lan-
cet Neurol 6:793–804
23. Ludwing BJ, Powell LS, Berger FM (1969) Carbamate deriva-
tives related to meprobamate. J Med Chem 12:462–472
24. Pellock JM, Perhach JM, Sofia RD (2002) Felbamate. In: Levy
RH, Mattson RH, Meldrum BS, m Perucca E (eds) Antiepilep-
tic drugs, 5th edn, Lippincott, Wiliams & Wilkinss, Philadel-
phia, 301–318.
4. Bialer M, White HS (2010) Key factors in the discovery and
development of new antiepileptic drugs (AEDs). Nature Rev
Drug Discov 9:68–83.
5. Bialer M, Johannessen SI, Levy RH, Perucca E, Tomson T,
White HS (2015) Progress report on new antiepileptic drugs: a
summary of the Twelfth Eilat Conference (EILAT XII). Epilepsy
Res 111:85–141
25. Bialer M, Johannessen SI, Levy RH, Perucca E, Tomson T,
White HS (2009) Progress report on new antiepileptic drugs: a
summary of the Ninth Eilat Conference (EILAT IX). Epilepsy
Res 83:1–43
6. Bialer M, Johannessen SI, Levy RH, Perucca E, Tomson T,
White HS (2017) Progress report on new antiepileptic drugs: a
summary of the Thirteenth Eilat Conference (EILAT XIII). Epi-
lepsia. doi:10.1111/epi.13634
26. Bialer M, Johannessen SI, Levy RH, Perucca E, Tomson T,
White HS (2010) Progress report on new antiepileptic drugs:
a summary of the Tenth Eilat Conference (EILAT X). Epilepsy
Res 92:89–124
7. Bialer M, Yagen B (2007) Valproic acid: second generation.
Neurother 4:130–137
8. Nau H, Loscher W (1986) Pharmacologic evaluation of various
metabolites and analogs of valproic acid: teratogenic potencies in
mice. Fundam Appl Toxicol 6:669–676
27. Karauss G, French J, Kamin J, Ilankumaran P (2016) Seizure
freedom with YKP3089 as adjunctive therapy for refractory
partial-onset seizures in double-llind placebo-controlled trials.
Neurology 86(16) P2–019
9. Bojic U, Elmazar MM, Hauck RS, Nau H (1996) Further branch-
ing of valproate-related carboxylic acids reduces the teratogenic
activity, but not the anticonvulsant effect. Chem Res Toxicol
9:866–870
28. Hen N, Bialer M, Yagen B (2012) Syntheses and evaluation of
anticonvulsant activity of novel branched alkyl carbamates. J
Med Chem 55:2835–2845
10. Liu MJ, Pollack GM (1994) Pharmacokinetics and pharmaco-
dynamics of valproate analogues in rats. IV. Anticonvulsant
action and neurotoxicity of octanoic acid, cyclohexanecarbox-
ylic acid, and 1-methyl-1-cyclohexanecarboxylic acid. Epilepsia
35:234–243
29. Shekh-Ahmad T, Mawasi H, McDonough JH, Finnell RH,
Wlodarczyk BJ, Bialer M (2014) Enantioselective pharma-
codynamic and pharmacokinetic analysis of two active chiral
CNS-active carbamate derivatives of valproic acid. Epilepsia
55:1944–1952
11. Loscher W, Nau H (1985) Pharmacological evaluation of various
metabolites and analogues of valproic acid. Anticonvulsant and
toxic potencies in mice. Neuropharmacology 24:427–435
12. Bialer M (2012) Chemical properties of antiepileptic drugs
(AEDs). Adv Drug Deliv Rev 64:887–895
30. White HS, Woodhead JH, Wilcox KS, Stables JP, Kupferberg
HJ, Wolf HH (2002) Discovery and preclinical development.
In: Levy RH, Mattson RH, Meldrum BS, Perucca E (eds),
Antiepileptic drugs, 5th edn, Lippincott Williams & Wilkins,
New York, pp 36–48.
13. Chang P, Augustin K, Boddum K, Williams S, Sun M, Terschak
JA, Hardege JD, Chen PE, Walker MC, Williams RS (2016)
Seizure control by decanoic acid through direst AMPA receptor
inhibition. Brain 139:431–443
31. Smith M, Wilcox KS, White HS (2007) Discovery of antiepi-
leptic drugs. Neurother 4:12–17
32. Finnell RH, Bennett GD, Karras SB, Mohl VK (1988) Com-
mon hierarchies of susceptibility to the induction of neural
tube defects by valproic acid and its 4-propyl-4-pentenoic acid
metabolite. Teratology 38:313–320
14. Rogawski MA (2016) A fatty acid in MCT ketogenic diet for epi-
lepsy treatment blocks AMPA receptors. Brain 139:306–309
15. Keane PE, Simiand J, Mendes E, Santucci V, Morre M (1983)
The effects of analogues of valproic acid on seizures induced
1 3

Neurochem Res
33. Wlodarczyk BC, Craig JC, Bennett GD, Calvin JA, Finnell RH
(1996) Valproic acid–induced changes in gene expression during
neurulation in a mouse model. Teratology 45:284–297
34. Khalifah RG (1971) The carbon dioxide hydration activity of
carbonic anhydrase. I. Stop-flow kinetic studies on the native
human isoenzymes B and C. J Biol Chem 246:2561–2573
35. Casini A, Antel J, Abbate F, Scozzafava A, David S, Waldeck
H, Schafer S, Supuran CT (2003) Carbonic anhydrase inhibitors:
SAR and X-ray crystallographic study for the interaction of sugar
sulfamates/sulfamides with isozymes I, II and IV. Bioorg. Med.
Chem. Lett. 13:841–845
39. Jezequel SG (1992) General nervous system penetration of
drugs: importance of phisicochemical properties, Taylor & Fran-
cis London, pp 141–178.
40. White HS, Alex AB, Pollock A, Hen N, Shekh-Ahmed T, Wil-
cox KS, McDonough JH, Stables JP, Kaufmann D, Yagen B,
Bialer M (2012) A new derivative of valproic acid amide pos-
sesses a broad-spectrum antiseizure profile and unique activity
against status epilepticus and organophosphate neuronal damage.
Epilepsia 53:134–146
41. Palaty J, Abbott FS (1995) Starcuture activity relestions of
unsaturated nalpoogs use of valrproic acid. J Med Chem
38:3398–3406
36. De Simone G, Di Fiore A, Menchise V, Pedone C, Antel J,
Casini A, Scozzafava A, Wurl M, Supuran CT (2005) Carbonic
anhydrase inhibitors. Zonisamide is an effective inhibitor of
the cytosolic isozyme II and mitochondrial isozyme V: solu-
tion and X-ray crystallographic studies. Bioorg Med Chem Lett
15:2315–2320
42. Elamzar MMA, Hauck RS, Nau H (1993) Anticonvuslnat and
neurotoxic activities of twelve anlaogues of valproic acid. J
Pharm Sci 82:1255–1258
43. Halmi P, Parkkila S, Honkaniemi J (2006) Expression of car-
bonic anhydrases II, IV, VII, VIII and XII in rat brain after kainic
acid induced status epilepticus. Neurochem Int 48:24–30
44. Supuran CT (2015) Acetazolamide for the treatment of idi-
opathic intracranial hypertension. Expert Rev Neurother
15:851–856
37. Temperini C, Innocenti A, Scozzafava A, Parkkila S, Supuran
CT (2010) The coumarin-binding site in carbonic anhydrase
accommodates structurally diverse inhibitors: the antiepileptic
lacosamide as an example. J Med Chem 53:850–854
38. Supuran CT (2008) Carbonic anhydrases: novel therapeutic
applications for inhibitors and activators. Nature Rev Drug Dis-
cov 7:168–181
45. Thiry A, Dognè JM, Masereel B, Supuran CT (2007) Carbonic
anhydrase inhibitors as anticonvulsant agents. Curr Top Med
Chem 7:855–864
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