Synthesis and anticonvulsant activity of some 2/3- benzoylaminopropionanilide derivatives

Article abstract of DOI:10.1055/s-0032-1308982

In this study, the synthesis and anticonvulsant properties of sixteen 2/3-benzoylaminopropionanilide derivatives were described. Molecular design of the compounds has been based on the modification of lacosamide which is a functionalized amino acid with a novel anticonvulsant activity. The structural confirmation of the title compounds was achieved by spectral and analytical data. The anticonvulsant activity profile of synthesized compounds was determined by maximal electroshock (MES) and subcutaneous metrazole (scMet) seizure tests, whereas their neurotoxicity was examined using rotarod test. All these tests were performed in accordance with the procedures of the Antiepileptic Drug Development (ADD) program. The majority of the compounds were effective in the MES or scMet screening tests. None of the compounds showed neurotoxicity according to the rotarod test at studied doses. Most active compounds in the series were 3, 12 and 13, which bearing 2-methyl, 2-ethyl and 2-isopropyl substituent on the N-phenyl ring, respectively. Georg Thieme Verlag KG Stuttgart - New York.

Full text of DOI:10.1055/s-0032-1308982

Original Article 295
Synthesis and Anticonvulsant Activity of Some
2/3-Benzoylaminopropionanilide Derivatives
Authors
S. Uysal1, U. Calis2, Z. Soyer1
Affiliations
1 Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Ege University, Bornova, Izmir, Turkey
2 Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Hacettepe University, Sıhhiye, Ankara, Turkey
Key words
Abstract
metrazole (scMet) seizure tests, whereas their
neurotoxicity was examined using rotarod test.
All these tests were performed in accordance
with the procedures of the Antiepileptic Drug
Development (ADD) program. The majority of
the compounds were effective in the MES or
scMet screening tests. None of the compounds
showed neurotoxicity according to the rotarod
test at studied doses. Most active compounds
in the series were 3, 12 and 13, which bearing
2-methyl, 2-ethyl and 2-isopropyl substituent on
the N-phenyl ring, respectively.
▶
propanamide
anilide
lacosamide
α/β-alanine
●
▼
▶
●
In this study, the synthesis and anticonvulsant
properties of sixteen 2/3-benzoylaminopropio-
nanilide derivatives were described. Molecular
design of the compounds has been based on the
modification of lacosamide which is a function-
alized amino acid with a novel anticonvulsant
activity. The structural confirmation of the title
compounds was achieved by spectral and ana-
lytical data. The anticonvulsant activity profile
of synthesized compounds was determined by
maximal electroshock (MES) and subcutaneous
▶
●
▶
●
▶
●
anticonvulsant activity
Introduction
acetamido-N-benzyl-3-methoxypropionamide}
developed as functionalized amino acid, was
approved in Europe and in the USA for antiepilep-
▼
Epilepsy is an important chronic neurological
disorder characterized by recurrent unprovoked
seizures. Drug therapy with current antiepileptic
drugs has important disadvantages and limita-
tions. Approximately 70–80% of epilepsy patients
are currently controlled by this drugs, the rest
are resistance to conventionally available medi-
cal therapies [1,2]. Moreover, the current drug
therapy is associated with adverse side effects
such as ataxia, gastrointestinal disturbance, gin-
gival hyperplasia, hirsutism, megaloblastic ane-
mia and hepatotoxicity. Therefore, one of the
main challenges in drug development is to dis-
cover new more effective antiepileptic molecules
with less side effe c t s [ 2 – 6 ] .
▶
tic therapy ( Fig. 1). According to the literature
●
survey, lacosamide represent a new mechanism
of action with a favorable safety profil e [ 1 0 – 2 2 ] .
Anilide is the other pharmacophore moiety
known to produce potent anticonvulsant drugs
[23–25]. The structure-activity studies on anilide
derivatives indicated the importance of substitu-
tion pattern on N-phenyl ring [26,27].
On the basis of these encouraging results, using
lacosamide and anilide as structural leads, we
aimed to synthesize some 2/3-benzoylaminopro-
pionanilide derivatives as potential anticonvul-
sant compounds and then evaluate their
anticonvulsant activities against MES and scMet
screening tests.
received 12.01.2012
accepted 02.03.2012
Bibliography
DOI http://dx.doi.org/
10.1055/s-0032-1308982
Published online:
April 2, 2012
Arzneimittelforschung 2012;
62: 295–300
© Georg Thieme Verlag KG
Stuttgart · New York
ISSN 0004-4172
The search of new antiepilept ic molecules is
mainly based on 2 different approaches. Those
are mechanism based design and structured
based design. Many of the newer antiepileptic
drugs as well as those currently in clinical devel-
opment were designed through structural modi-
fications of the pre-existing compounds [7–9].
In recent studies, functionalized amino acids
(FAA) and α-amino acids (AAA) are 2 classes of
antiepileptic molecules with pronounced anti-
convulsant activity. Lacosamide (LCM) {(R)-2-
Correspondence
Asst Prof. Dr. Z. Soyer
Department of Pharmaceutical
Chemistry
Faculty of Pharmacy
Ege University
35100 Bornova
Izmir-Turkey
Tel.: +90/232/339 99 31
Fax: +90/232/388 52 58
zeynep.soyer@ege.edu.tr
Materials and Methods
▼
Chemistry
Melting points were determined on an Electro-
thermal IA 9100 (Electrothermal, Essex, U.K.)
melting point apparatus and are uncorrected.
The IR spectra of compounds were recorded as
potassium bromide pellets on a Jasco FT/IR-400
Uysal S. Anticonvulsant Activity of Some Propanamides… Arzneimittelforschung 2012; 62: 295–300

296 Original Article
(Jasco, Tokyo, Japan) and Perkin Elmer FT-IR Spectrometer 100
(1:1) to yield N-Boc-α/β-alaninanilide. The solution of N-Boc-α/
β-alaninanilide (10.6mmol) in dichloromethane (50ml) was
cooled to 0–5°C and was saturated with hydrogen chloride gas.
The mixture was stirred for 2h, and the solvent was removed
under reduced pressure to yield an oil which solidified upon
trituration with cold diethyl ether. The crude product was crys-
tallized from ethanol.
(Perkin Elmer Inc., Massachusetts, USA). The 1H NMR spectra
were recorded on a Varian As 400 Mercury Plus NMR (Varian Inc.,
Palo Alto, CA, USA) spectrophotometer using a CDCl3 and DMSO-
d6 as solvent. Chemical shifts were reported in parts per million
(δ) j values were given in Hz. Mass spectra (API-ES) were meas-
ured on an AGILENT 1100 MSD (Agilent Technologies, Palo Alto,
CA, USA). Elemental analyses (C, H and N) were performed by Leco
CHNS-932 (Leco-932, St. Joseph, MI, USA). The analytical results
for the elements were within ±0.4% of the theoretical values.
Synthesis of 3-aminopropionanilide hydrochlorides
(15a and 16a)
The intermediates 15a and 16a were prepared according to the
method reported in the literature [30–32]. For this purpose,
appropriately substituted aniline (66mmol) was dissolved in
25ml glacial acetic acid, and 3-chloropropionyl chloride
(74mmol) was added dropwise to this solution while cooling in
ice-bath. The reaction mixture was stirred in the ice-bath for
15min and at room temperature for 45min. The mixture was
poured into saturated sodium acetate solution. The precipitate
was filtered, washed with cold water and purified by crystalliza-
tion ethanol-water (1:1). 3-Chloropropionanilide acquired by
this method (80mmol) and phthalimide potassium salt
(120mmol) were refluxed in DMF (15ml). By monitoring with
TLC, the reaction was terminated. The reaction mixture was
poured into cold water. The precipitate was filtered and washed
with water. After drying, the precipitate was purified by crystal-
lization from ethanol. The phthalimide intermediate prepared
by this method (1.2mmol) was suspended in absolute ethanol
(99%) (20ml) and hydrazine hydrate (80% aqueous) (0.2ml) was
added to this solution. The reaction mixture was refluxed for 3h.
After the mixture was cooled under 50°C, 1.2M HCl (5ml) solu-
tion was added to the reaction medium and refluxed for 1h. The
resulting crude product was filtered and crystallized from ethanol.
Synthesis of 2/3-aminopropionanilide hydrochlorides
(1a–14a)
The intermediates were prepared according to the method
reported in the literature [28,29]. For this purpose, α/β-alanine
(11.2mmol) was dissolved in a mixture dioxane (20ml) and
0.5M NaOH (20ml). After cooling with an ice-bath, di-tert-butyl
dicarbonate ((Boc)2O) (12.3mmol) was added dropwise to this
solution. The solution was stirred at room temperature over-
night and the solvent was evaporated under reduced pressure.
5% KHSO4 (150ml) solution was added to the residue and
extracted with dichloromethane (50ml) twice,, dried over anhy-
drous Na2SO4 and evaporated under reduced pressure. The resi-
due was purified by crystallization with ethyl acetate to give
N-Boc-α/β-alanine. The solution of N-Boc-α/β-alanine (5mmol)
in THF (20ml) was cooled to 0–5°C, and N-methylmorpholine
(NMM) (5mmol) was added under the nitrogen atmosphere.
After stirring for 5min, isobutyl chloroformate (IBCF) (5mmol)
was added in one portion. And then, aniline or substituted ani-
lines were added dropwise to this solution for 10min. The reac-
tion mixture was stirred at room temperature for 1h. The
precipitated N-methylmorpholine hydrochloride was filtered off
and the filtrate was evaporated under reduced pressure. The
crude product was purified by crystallization ethanol-water
Synthesis of 2/3-benzoylaminopropionanilides (1–16)
Compounds 1a–16a (2mmol) were dissolved in 50% NaOH
(15ml), and benzoyl chloride (2mmol) in chloroform (15ml)
was added to this solution. The reaction mixture was stirred at
room temperature for 4h. The organic phase was washed with
5% HCl (15ml), 5% NaHCO3 (15ml) and water (20ml) respec-
tively and then evaporated under reduced pressure. The precipi-
O
Fig. 1 Lacosamide.
H
N
N
H
O
OCH3
Table 1 Yields, melting points,
formulas, IR and API-ES data of
compounds 1–16.
Comp.
Yield
M.p.
(°C)
Formula
IR
(cm−1
API-ES
(%)
)
m/z (% intensity)
1
2
31
58
179
111
C16H16N2O2
3303, 3250, 1685, 1635, 1588, 1552
3484, 3235, 1643, 1581, 1550
176 (100), 269 (M+H, 47)
176 (100), 305 (M+2, 9),
303 (M+H, 28)
C16H15ClN2O2
3
4
5
6
7
8
9
90
75
70
86
75
13
73
131
150
170
136
178
175a
142
C17H18N2O2
C17H18N2O3
C18H20N2O2
C19H22N2O2
C18H20N2O2
C 16H16N2O2
C16H15ClN2O2
3473, 3239, 1644, 1625, 1588, 1552
3303, 3058, 1691, 1643, 1588, 1531
3288, 3250, 1656, 1631, 1581, 1538
3299, 3235, 1661, 1633, 1577, 1538
3347, 2977, 1689, 1656, 1591, 1535
3306, 3056, 1670, 1632, 1598, 1578
3353, 3284, 1664, 1633, 1579, 1531
176 (100), 283 (M+H, 84)
299 (M+H, 100)
176 (100), 297 (M+H, 32)
176 (100), 311 (M+H, 43)
176 (100), 259 (M+H, 59)
269 (M+H, 100)
176 (100), 305 (M+2, 33),
303 (M+H, 87)
10
11
12
13
14
15
16
93
80
73
92
69
93
12
172
132
166
169
220
232
249
C17H18N2O2
C17H18N2O3
C18H20N2O2
C19H22N2O2
C18H20N2O2
3353, 3237, 1668, 1635, 1538
283 (M+H, 100)
3328, 3284, 1664, 1620, 1540
299 (M+H, 100)
3335, 3239, 1671, 1631, 1570, 1542
3344, 3235, 1664, 1629, 1591, 1542
3285, 3223, 1659, 1620, 1574, 1543
297 (M+H, 100)
311 (M+H, 100)
297 (M+H, 100)
176 (100), 337 (M+, 45)
C16H14Cl2N2O2 3359, 3261, 1670, 1639, 1541, 1521
C16H15N3O4 3378, 3247, 1670, 1651, 1576, 1545
242 (100), 314 (M+H, 18)
a191℃, Ref. [34]
Uysal S. Anticonvulsant Activity of Some Propanamides… Arzneimittelforschung 2012; 62: 295–300

Original Article 297
Fig. 2 Synthesis of the compounds 1–14.
R
R'
R
R
R
R'
H
N
H2N
R''
b
+
a
Cl– H3N
n
+
Boc
n
COOH
n
H2N
N
H
COOH
O
R''
n=1, R=CH3
n=2, R=H
(1a̶14a)
R
O
R'
6
R'
H
H
N
2'
5'
N
+
n
Cl– H3N
3'
4'
n
N
H
5
c
O
6'
O
4
R''
R''
2
3
(1̶14)
(n=1, R=CH3)
(n=2, R=H)
(1) R'=H, R''=H
(8) R'=H, R''=H
(2) R'=H, R''=Cl
(9) R'=H, R''=Cl
(3) R'=H, R''=CH3
(10) R'=H, R''=CH3
(11) R'=H, R''=OCH3
(12) R'=H, R''=C2H5
(4) R'=H, R''=OCH3
(5) R'=H, R''=C H5
2
(6) R'=H, R''=CH(CH3)2 (13) R'=H, R''=CH(CH3)2
(7) R'=CH , R''=CH3
(14) R'=CH3, R''=CH3
3
(a) (Boc)2O, NaOH (b) NMM, IBCF, HCl(g), CH2Cl2 (c) Benzoyl chloride, NaOH
Fig. 3 Synthesis of the compounds 15 and 16.
O
O
R'
R'
O
a
N– K+
HN
O
+
N
Cl
N
H
R
R
O
O
O
R'
3'
6'
R'
+
2'
4'
5'
R'
b
c
H
N
Cl– H3N
H
N
H
N
N
HN
O
6
5
4
O
O
R
O
O
R
R
2
3
(15a̶16a)
(15) R=Cl, R'=Cl
(16) R=H, R'=NO2
(a) DMF (b) Hydrazine hydrate (80 % aq.), 1.2 M HCl (c) Benzoyl chloride, NaOH
tate was crystallized from ethanol-water (1:1) to furnish title
PEG 400. Metrazole was administered subcutaneously (sc) on
the back of the neck. The rotarod toxicity test was performed on
1-inch-diameter knurled wooden rod, rotating at 6rpm (the
rotarod used in phase-1 test was made by Hacettepe University
Technical Department).
▶
compounds. Yields and melting points are given in Table 1.
●
Anticonvulsant Activity Screening
The compounds were tested for their anticonvulsant activity
against maximal electroshock (MES) and subcutaneous Metra-
zole (scMet)-induced seizure treshold tests. The acute neuro-
logical toxicity was determined in the rotarod test. All these
tests were performed in male mice according to the phase-1
tests of the Antiepiletic Drug Development (ADD) program
which were developed by National Institute of Health (NIH),
National Institute of Neurological Disorders and Stroke (NINDS)
[38]. Stimulator (Grass S88, Astro-Med. Inc. Grass Instrument
Division, W. Warwick, RI, USA), constant current unit (Grass
CCU1A, Grass Medical Instrument, Ouincy, MA, USA) and corneal
electrodes were used for the evaluation of anticonvulsant activ-
ity against MES test. All synthesized compounds were suspended
in 30% aqueous of PEG 400 and administered to the mice intra-
peritoneally in a volume of 0.01mL/g at body weight. 12 Swiss
Albino male mice (20±2g) were used for each compound (mice
were obtained from The Hacettepe University Animal Farm)
according to the ADD-NINDS program. The animal were kept
under standard conditions at an ambient temperature of 25±2°C
and allowed free access to food except at the time they were
brought out of the cage. All the experimental protocols were car-
ried out with the permission of Hacettepe University, “Labora-
tory Animals Ethic Committee” decision (01.09.2010 date
2010/46-11 number). Control animals received 30% aqueous
Maximal Electroshock (MES)-induced seizure test
MES seizures were elicited with a 60-cycle alternating current of
50mA intensity (5–7 times more than that required to elicite
minimal seizures) delivered for 0.2s via corneal electrodes.
A drop of 0.9% saline was instilled into the eye prior to applica-
tion of the electrodes in order to prevent the death of the animal.
Abolition of the hind limb tonic extension component of the sei-
zure was defined as protection.
Subcutaneous Metrazole (scMet)-induced seizure test
85mg/kg of Metrazole (produces seizures in more than 95% of
mice) was administered as a 0.5% solution sc into the posterior
midline. The animal was observed for 30min to decide whether
the failure of the treshold seizure (a single episode of clonic
spasms of at least 5s duration) could be defined as protection.
Neurotoxicity
The rotarod test was used to evaluate neurotoxicity. The animal
was placed on a 1-inch-diameter knurled wooden rod rotating at
6rpm. Normal mice remain on a rod rotating at this speed indef-
initely. Neurological toxicity was defined as the failure of the
animal to remain on the rod for 1min.
Uysal S. Anticonvulsant Activity of Some Propanamides… Arzneimittelforschung 2012; 62: 295–300

298 Original Article
Table 2 1H NMR data and elemental analysis of compounds 1–16.
Compound
1
1H NMR and Elemental analysis (C, H, N, %)
1H NMR (DMSO-d6): δ 1.43 (3H, d, J=7.2Hz, CH(CH3)), 4.59–4.63 (1H, m, CH(CH3)), 7.04 (1H, td, J=1.2; 7.0Hz, H-4), 7.30 (2H, td, J=1.6;
7.4Hz, H-3 and H-5), 7.47 (2H, td, J=1.3;7.4Hz, H-3′ and H-5′), 7.52–7.56 (1H, m, H-4′), 7.60–7.62 (2H, m, H-2 and H-6), 7.90–7.93
(2H, m, H-2′ and H-6′), 8.59 (1H, d, J=7.6Hz, NH-CH(CH3)), 10.01 (1H, bs, NH-phenyl) ppm. Anal.calcd. for C16H16N2O2. 0.2H2O: C, 70.67;
H, 6.08; N,10.30. Found: C, 70.82; H, 6.11; N, 10.26.
2
3
1H NMR (DMSO-d6): δ 1.47 (3H, d, J=7.2Hz, CH(CH3)), 4.71–4.75 (1H, m, CH(CH3)), 7.17 (1H, td, J=1.6; 8.0Hz, H-4), 7.33 (1H, td, J=1.0;
8.0Hz, H-5), 7.46–7.53 (3H, m, H-3′, H-5′ and H-3), 7.54–7.57 (1H, m, H-6), 7.82 (1H, dd, J=1.6; 8.4Hz, H-4′), 7.92 (2H, dd, J=1.6; 8.8Hz,
H-2′ and H-6′), 8.73 (1H, d, J=7.2Hz, NH-CH(CH3)), 9.5 (1H, bs, NH-phenyl) ppm. Anal.calcd. for C16H15ClN2O2. 1.25H2O: C, 59.08; H, 5.42;
N, 8.61. Found: C, 59.14; H, 5.36; N, 8.74.
1H NMR (DMSO-d6): δ 1.47 (3H, d, J=7.2Hz, CH(CH3)), 2.19 (3H, s, Ar-CH3), 4.66–4.69 (1H, m, CH(CH3)), 7.07 (1H, t, J=7.2Hz, H-4),
7.14–7.21 (2H, m, H-3 and H-5), 7.40 (1H, d, J=8.0Hz, H-6), 7.47 (2H, td, J=1.6; 7.4Hz, H-3′ and H-5′), 7.52–7.56 (1H, m, H-4′), 7.92 (2H,
dd, J=1.4; 8.6Hz, H-2′ and H-6′), 8.61 (1H, d, J=6.8Hz, NH-CH(CH3)), 9.35 (1H, bs, NH-phenyl) ppm. Anal.calcd. for C17H18N2O2 .1H2O: C,
67.98; H, 6.71; N, 9.33. Found: C, 67.91; H, 7.01; N, 9.31.
4
1H NMR (DMSO-d6): δ 1.42 (3H, d, J=7.2Hz, CH(CH3)), 3.76 (3H, s, OCH3), 4.68–4.71 (1H, m, CH(CH3)), 6.89 (1H, td, J=2.0; 7.4Hz, H-3),
7.02 (2H, td, J=1.8; 8.4Hz, H-4 and H-5), 7.48 (2H, t, J=7.4Hz, H-3′ and H-5′), 7.53–7.56 (1H, m, H-4′), 7.89 (2H, dd, J=1.2; 7.2Hz, H-2′
and H-6′), 8.03 (1H, d, J=7.2Hz, H-6), 8.74 (1H, d, J=6.8Hz, NH-CH(CH3)), 9.1 (1H, bs, NH-phenyl) ppm. Anal.calcd. for C17H18N2O3: C,
68.44; H, 6.08; N, 9.39. Found: C, 68.13; H, 6.08; N, 9.15.
5
1H NMR (CDCl3): δ 1.09 (3H, t, J=7.4Hz, CH2CH3), 1.47 (3H, d, J=7.2Hz, CH(CH3)), 2.55–2.61 (2H, m, CH2CH3), 4.67–4.70 (1H, m, CH(CH3),
7.12–7.17 (2H, m, H-4 and H-5), 7.21–7.23 (1H, m, H-3), 7.39 (1H, d, J=7.6Hz, H-6), 7.47 (2H, t, J=7.4Hz, H-3 and H-5′), 7.52–7.54
(1H, m, H-4′), 7.92 (2H, dd, J=1.6; 8.0Hz, H-2′ and H-6′), 8.62 (1H, d, J=7.6Hz, NH-CHCH3), 9.32 (1H, bs, NH-phenyl) ppm. Anal.calcd. for
C18H20N2O2: C, 72.95; H, 6.80; N, 9.45. Found: C, 72.84; H, 6.60; N, 9.44.
6
1H NMR (DMSO-d6): δ 1.12 (6H, d, J=6.8Hz, CH(CH3)2), 1.47 (3H, d, J=7.2Hz, CH(CH3)), 3.13–3.19 (1H, m, CH(CH3)2), 4.68 (1H, m,
CH(CH3)), 7.13–7.20 (2H, m, H-4 and H-5), 7.29 (2H, td, J=2.4; 8, H-3 and H-6), 7.47 (2H, t, J=7.4, H-3′ and H-5′), 7.54 (1H, t, J=7.2Hz,
H-4′), 7.93 (2H, d, J=7.6Hz, H-2′ and H-6′), 8.6 (1H, d, J=7.2Hz, NH-CH(CH3)), 9.37 (1H, bs, NH-phenyl) ppm. Anal.calcd. for C19H22N2O2:
C, 73.52; H, 7.14; N, 9.03. Found: C, 73.38; H, 7.42; N, 8.90.
7
1H NMR (DMSO-d6): δ 1.48 (3H, d, J=7.2Hz, CH(CH3)), 2.12 (6H, s, 2xAr-CH3), 4.59–4.62 (1H, m, CH(CH3)), 7.03–7.04 (3H, m, H-3, H-4 and
H-5), 7.45 (2H, t, J=7.4Hz, H-3′ and H-5′), 7.50–7.51(1H, m, H-4′), 7.90 (2H, dd, J=1.6; 7.6Hz, H-2′ and H-6′), 8.54 (1H, d, J=6.4Hz,
NH-CH(CH3)), 9.29 (1H, bs, NH-phenyl) ppm. Anal.calcd. for C18H20N2O2. 1H2O: C, 68.48; H, 7.24; N, 8.93. Found: C, 68.77; H, 7.05; N,
8.91.
8
1H NMR (DMSO-d6): δ 2.62 (2H, t, J=7.2Hz, CH2-CO), 3.55 (2H, q, J=6.3Hz, NH-CH2), 7.02 (1H, t, J=7.4Hz, H-4), 7.28 (2H, t, J=6.0Hz,
H-3 and H-5), 7.45 (2H, tt, J=1.3; 7.4Hz, H-2 and H-6), 7.49–7.51 (1H, m, H-4′), 7.57 (2H, d, J=7.4Hz, H-3′ and H-5′), 7.83 (2H, td, J=1.6;
7.0Hz, H-2′ and H-6′), 8.56 (1H, t, J=8.0Hz, NH-CH2), 9.93 (1H, bs, NH-phenyl) ppm. Anal.calcd. for C16H16N2O2. 0.1H2O: C, 71.08; H, 5.99;
N, 10.36. Found: C, 71.45; H, 6.34; N, 9.91.
9
1H NMR (DMSO-d6): δ 2.63 (2H, t, J=7.0Hz, CH2-CO), 3.56 (2H, q, J=6.5Hz, NH-CH2), 7.17 (1H, td, J=1.6; 7.8Hz, H-4), 7.31 (1H, td, J=1.6;
7.6Hz, H-5), 7.43–7.48 (3H, m, H-3′, H-5′ and H-3), 7.50–7.54 (1H, m, H-4′), 7.69 (1H, d, J=7.8Hz, H-6), 7.84 (2H, dd, J=1.2; 8.4Hz, H-2′
and H-6′), 8.53 (1H, t, J=5.0Hz, NH-CH2), 9.53 (1H, bs, NH-phenyl) ppm. Anal.calcd. for C16H15ClN2O2.0.6C2H6O: C, 62.40; H, 5.63; N, 8.47.
Found: C, 62.04; H, 5.71; N, 8.87.
10
11
12
13
14
1H NMR (DMSO-d6): 2.14 (3H, s, Ar-CH3), 2.62 (2H, t, J=7.0Hz, CH2-CO), 3.54 (2H, q, J=6.5Hz, NH-CH2), 7.05 (1H, t, J=7.2Hz, H-4),
7.11–7.17 (2H, m, H-3 and H-5), 7.36 (1H, d, J=7.8Hz, H-6), 7.46–7.42 (2H, m, H-3′ and H-5′), 7.48–7.52 (1H, m, H-4′), 7.82 (2H, dd, J=
1.6; 7.8Hz, H-2′ and H-6′), 8.53 (1H, t, J=11.4Hz, NH-CH2), 9.28 (1H, bs, NH-phenyl) ppm. Anal.calcd. for C17H18N2O2: C, 72.32; H, 6.43; N,
9.92. Found: C, 72.19; H, 6.11; N, 9.89.
1H NMR (DMSO-d6): δ 2.66 (2H, t, J=6.6Hz, CH2-CO), 3.52 (2H, q, J=5.8Hz, NH-CH2), 3.75 (3H, s, Ar-OCH3), 6.87 (1H, td, J=1.2; 7.9Hz,
H-5), 6.98–7.06 (2H, m, H-3 and H-4), 7.42–7.45 (2H, m, H-3′ and H-5′), 7.48–7.52 (1H, m, H-4′), 7.82 (2H, dd, J=1.2; 8.0Hz, H-2′ and
H-6′), 7.91 (1H, d, J=8.2Hz, H-6), 8.48 (1H, t, J=14Hz, NH-CH2), 9.07 (1H, bs, NH-phenyl) ppm. Anal.calcd. for C17H18N2O3: C, 68.44; H,
6.08; N, 9.39. Found: C, 68.23; H, 6.00; N, 9.42.
1H NMR (DMSO-d6): δ 1.18 (3H, t, J=7.6Hz, CH2CH3), 2.56 (2H, q, J=7.6Hz, CH2CH3), 2.77 (2H,t, J=5.6Hz, CH2-CO), 3.84 (2H, q, J=5.8Hz,
NH-CH2), 7.15–7.26(5H, m, H-3, H-4, H-5; NH- phenyl and NH-CH2), 7.38–7.43 (2H, m, H-3′ and H-5′), 7.50–7.46 (1H, m, H-6), 7.68 (1H,
d, J=7.8Hz, H-4′), 7.77 (2H, dt, J=1.6; 6.7Hz, H-2′ and H-6′) ppm. Anal.calcd. for C18H20N2O2: C, 72.95; H, 6.80; N, 9.45. Found: C, 72.73;
H, 6.76; N, 9.42.
1H NMR (DMSO-d6): 1.06 (6H, d, J=7.0Hz, 2xCH3), 2.64 (2H, t, J=6.6Hz, CH2-CO), 3.07–3.13 (1H, m, CH(CH3)2), 3.56 (2H, q, J=6.5Hz, NH-
CH2), 7.19–7.11 (2H, m, H-4 and H-5), 7.23 (1H, d, J=7.4Hz, H-3), 7.27 (1H, d, J=7.4Hz, H-6), 7.45 (2H, t, J=7.1Hz, H-3′and H-5′), 7.52
(1H, t, J=7.4Hz, H-4′), 7.85 (2H, d, J=7.0Hz, H-2′ and H-6′), 8.54 (1H, t, J=6.0Hz, NH-CH2), 9.35 (1H, bs, NH-phenyl) ppm. Anal.calcd. for
C19H22N2O2. 0.1H2O: C, 73.03; H, 7.11; N, 8.96. Found: C, 72.81; H, 7.30; N, 8.86.
1H NMR (DMSO-d6): δ 2.10 (6H, s, 2xAr-CH3), 2.64 (2H, t, J=7.0Hz, CH2-CO), 3.57 (2H, q, J=6.5Hz, NH-CH2), 7.03–7.04 (3H, m, H-3, H-4
and H-5), 7.44 (1H, t, J=1.6Hz, H-3′or H-5′), 7.46 (1H, dd, J=1.6; 6.6Hz, H-3′ or H-5′), 7.50–7.52 (1H, m, H-4′), 7.85 (2H, td, J=1.6; 2.3Hz,
H-2′ and H-6′), 8.55 (1H, t, J=6Hz, NH-CH2), 9.26 (1H, bs, NH-phenyl) ppm. Anal.calcd. for C18H20N2O2: C, 72.95; H, 6.80; N, 9.45. Found:
C, 72.56; H, 6.39; N, 9.46.
15
16
1H NMR (DMSO-d6): 2.65 (2H, t, J=7.0Hz, CH2-CO), 3.53 (2H, q, J=6.8Hz, NH-CH2), 7.31 (1H, t, J=8.2Hz, H-4), 7.42–7.45 (3H, m,
H-3′,H-4′ and H-5′), 7.48–7.50 (2H, m, H-3 and H-5), 7.82 (2H, d, J=7.0Hz, H-2′ and H-6′), 8.51 (1H, t, J=14Hz, NH-CH2), 9.86 (1H, bs,
NH- phenyl) ppm. Anal.calcd. for C16H14Cl2N2O2: C, 56.99; H, 4.18; N, 8.31. Found: C, 56.86; H, 4.38; N, 8.42.
1H NMR (DMSO-d6): δ 2.65 (2H, t, J=7.2Hz, CH2-CO), 3.53 (2H, q, J=6.4Hz, NH-CH2), 7.31 (1H, t, J=7.9Hz, H-4), 7.41–7.45 (3H, m, H-3′,
H-4′and H-5′), 7.48–7.50 (3H, m, H-3, H-5 and H-6), 7.82 (2H, d, J=7.0Hz, H-2′ and H-6′), 8.49 (1H, t, J=9.9Hz, NH-CH2), 9.86 (1H, bs,
NH-phenyl) ppm. Anal.calcd. for C16H15N3O4: C, 61.34; H, 4.83; N, 13.41 Found: C, 57.70; H, 4.54; N, 8.47.
Uysal S. Anticonvulsant Activity of Some Propanamides… Arzneimittelforschung 2012; 62: 295–300

Original Article 299
Table 3 Anticonvulsant and neurotoxicity screening data of title compounds.
MESa
scMetb
4h (mg/kg)
Toxicityc
Compound
0.5h (mg/kg)
4h (mg/kg)
30 100 300
0.5h (mg/kg)
0.5h (mg/kg)
30 100 300
4h (mg/kg)
30
100 300
30
100 300
30
100 300
30
100
300
1
2
0/1 0/1 0/1
0/1 0/1 0/1
0/1 0/1 0/1
0/1 0/1 0/1
0/1 0/1 0/1
0/1 0/1 0/1
0/1 0/1 0/1
0/1 0/1 1/1
0/1 0/1 0/1
1/1 1/1 1/1
0/1 0/1 0/1
0/1 0/1 0/1
0/1 0/1 1/1
0/1 0/1 0/1
0/1 0/1 0/1
0/1 0/1 1/1
0/1 0/1 0/1
0/1 0/1 0/1
0/1 0/1 0/1
0/1 0/1 0/1
0/1 0/1 0/1
0/1 0/1 0/1
0/1 0/1 0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
1/1
1/1
0/1
0/1
0/1
0/1 1/1
0/1 0/1
0/1 0/1
0/1 0/1
1/1 1/1
0/1 0/1
0/1 1/1
0/1 0/1
0/1 0/1
0/1 0/1
0/1 1/1
1/1 1/1
1/1 1/1
1/1 1/1
0/1 1/1
0/1 0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/2 0/2
0/2 0/2
0/2 0/2
0/2 0/2
0/2 0/2
0/2 0/2
0/2 0/2
0/2 0/2
0/2 0/2
0/2 0/2
0/2 0/2
0/2 0/2
0/2 0/2
0/2 0/2
0/2 0/2
0/2 0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
3
4
5
6
7
8
0/1 0/1
1/1
9
0/1 0/1 0/1
0/1 0/1 0/1
0/1 0/1 0/1
0/1 0/1 0/1
0/1 0/1 0/1
0/1 0/1 0/1
0/1 0/1 0/1
0/1 0/1 0/1
10
11
12
13
14
15
16
a maximal electroshock test; b subcutaneous metrazole test; c rotarod test, 0/1: No activity at dose level; 1/1: noticeable activity at dose level; 0/4: number of animals exhibited
toxicity/number of animals tested for 05h; 0/2: number of animals exhibiting toxicity/number of animals tested for 4h
Results and Discussion
peaks were in complete agreement with the calculated molecu-
▶
lar weight for individual compounds ( Table 1) .
●
▼
Chemistry
In this study, sixteen 2/3-benzoylaminopropionanilide deriva-
tives have been synthesized to evaluate anticonvulsant activity
profile. The synthesis of the title compounds was realized in 2
steps. For this purpose, in the first step, 2/3-aminopropionani-
lide hydrochlorides were prepared according to the reported
procedures [28–32]. In the second step, those intermediates
were reacted with benzoyl chloride to furnish title compounds
▶
Anticonvulsant Activity Screening
The preclinical discovery and development of new chemical
agents for the treatment of epilepsy are based mainly on the use
of animal models. At the present time, there are 3 in-vivo screen-
ing methods used routinely, namely maximal electroshock
(MES) seizure, the subcutaneous metrazole (scMet), and the kin-
dled model. The MES and scMet screening tests are claimed to
predict the potential protection activity against generalized
tonic-clonic seizures and generalized absence seizures, respec-
tively and those tests recognized as the “gold standards” in the
early stages of anticonvulsant testing protocole according to the
Antiepileptic Drug Development (ADD) Program of National
Institute of Health (NIH). Therefore, the anticonvulsant activities
of the title compounds were evaluated against MES and scMet
tests by the standard procedures of utilized by the NINDS Anti-
convulsant Screening Program (ASP) [38].
[33]. The synthetic pathways are given in Fig. 2, 3. The struc-
●
tures of the compounds were confirmed by spectral (IR, 1H NMR,
▶
and API-ES Mass) and elemental analyses ( Table 1, 2). The
●
▶
yields and melting points are summarized in Table 1. Accord-
●
ing to the literature survey, compunds 1 and 8 are reported
derivatives [34,35] and 2–6, 9–14 are listed substances in the
literature with the CAS registry numbers 72029-13-1, 1268331-
32-3, 1266377-87-0, 1266366-95-3, 1266374-66-6, 1266376-
33-3, 108679-33-0, 909513-33-3, 909513-87-7, 909512-98-7,
178566-26-8, 909512-08-9 and 909512-89-6, respectively, but
corresponding scientific reference data are not available for
those compounds.
The title compounds were tested after intraperitoneal (i.p.)
injection to mice at doses of 30, 100 and 300mg/kg. An observa-
tion was carried out at 2 different time intervals (0.5h and 4h).
The rotarod test was used to demonstrate the possible neurotox-
The purity levels of compounds were determined by elemental
analyses (C, H, N) and results were within 0.4% of the calculated
icity. None of the compounds showed neurotoxicity at studied
▶
▶
values except for compound 16 ( Table 2) .
doses. Preliminary screening results are presented in Table 3.
●
●
In the IR spectra, the presence of C=O stretching bands were the
confirmative signals for the constructed functional groups in the
title compounds. For instance, amide C=O stretching bands of
anilide and benzamide groups were observed between 1691–
1643 and 1620–1651cm−1, respectively. N-H strecthing bands
The anticonvulsant activities of the title compounds are reported
for the first time in this study.
The results indicated that majority of compounds were effective
in MES or scMet tests. In the whole series only compound 1 was
active in both tests, whereas compounds 2, 4, 10 and 16 were
found inactive in both models.
According to the MES test results, compounds 1, 3, 6, 8 and 9
were active in the MES test indicating the ability to prevent sei-
zure spread. Compound 8 showed protection at 300mg/kg after
0.5h, whereas compounds 1, 6 and 9 were active at same dose
after 4h. Compound 3 was the most active compound of the
series against the MES test at the dose of 30mg/kg at time peri-
ods 4h. The delayed anticonvulsant activity of compounds 1, 3,
of the secondary amide structure of the title compounds were
observed between 3484 and 3056cm−1
(
Table 1), [36,37].
▶
●
1H NMR spectra of the title compounds were consistent with
expected resonance signals in term of chemical shifts and inte-
grations. The chemical shifts and splitting patterns of N-phenyl
protons in each compound differed depending on the nature of
▶
the substituents ( Table 2) .
●
The structure of the title compounds was further verified by
API-ES Mass spectra where the m/z values of molecular ion
Uysal S. Anticonvulsant Activity of Some Propanamides… Arzneimittelforschung 2012; 62: 295–300

300 Original Article
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6 and 9 at 4h could be associated with their absorbtion and dis-
tribution characteristics and long duration of action.
Compounds 1, 5, 7, 11, 12, 13, 14 and 15 were found to be active
after 0.5h in the scMet test indicating the ability to elevate sei-
zure threshold. Compounds 12 and 13 were most active com-
pounds in the scMet test. Compounds 1, 7, 11 and 15 showed
activity at a dose of 300mg/kg while compounds 5 and 14 exhib-
ited activity at doses of 100 and 300mg/kg. All of active com-
pounds in scMet screening test displayed rapid onset and shorter
duration of action.
Under the set of the compounds studied, the most active com-
pound in the both series against MES test was aroused from the
2-benzoylaminopropionanilide structure, whereas the most
active ones in scMet test were originated from the 3-ben-
13 Beyreuther BK, Freitag J, Heers C et al. Lacosamide: a review of pre-
clinical properties. CNS Drug Rev 2007; 13 (1): 21–42
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activity of a series of functionalized .alpha.-aromatic and .alpha.-
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16 Kohn H, Sawhney KN, Robertson DW et al. Anticonvulsant properties
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zoylaminopropionanilide (see compound 3 and 12, 13 respec-
▶
●
tively in
Table 3). According to the contribution of the
17 LeTiran A, Stables JP, Kohn H. Design and evaluation of affinity labels
of functionalized amino acid anticonvulsants. J Med Chem 2002; 45:
4762–4773
substituent on N-phenyl ring in both series, mono alkyl substi-
tutions seem to yield superior compounds.
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2005; 5: 69–85
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of the 3-oxy site in the anticonvulsant (R)-N-Benzyl 2-Acetamido-3-
methoxypropionamide. J Med Chem 2010; 53: 5716–5726
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and safety of lacosamide. Ther Adv Neurol Disord 2010; 3 (2): 77–83
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zures in epileptic adults. Neuropsychiatr Dis Treat 2010; 6: 465–471
22 Park KD, Salome C, Cotten SW et al. Lacosamide isothiocyanate-based
agents: novel agents to target and identify lacosamide receptors.
J Med Chem 2009; 52: 6897–6911
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4-aminobenanilides. J Med Chem 1985; 28: 1259–1262
24 Bailleux V, Valle e L, Nuyts JP et al. Comparative anticonvulsant activity
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Conclusion
▼
A series of 2/3-benzoylaminopropionanilide derivatives were
designed, synthesized and their anticonvulsant activities were
evaluated. As a result, 2 general trends may be summarized for
both series. First, majority of the compounds synthesized had
greater activity at the 0.5h than 4h and secondly, they showed
greater activity in the scMet test rather than MES test.
As observed through biological data analysis, under the set of
ortho mono substituent studied on N-phenyl ring, compounds
with methyl (3), ethyl (12) and isopropyl (13) substitutions were
found to be more potent in the MES or scMet tests. Those com-
pounds emerged as lead compounds for future investigations.
Conflict of Interest
▼
The authors report no conflicts of interest.
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