Synthesis and anticonvulsant activity of some N-(benzoyl)glycinanilide derivatives

Article abstract of DOI:10.1007/s00044-013-0474-y

Glycine is a major inhibitory neurotransmitter and recent studies have shown that certain lipophilic glycine derivatives demonstrate anticonvulsant activity in animal epilepsy models. On the other hand, anilide is another fruitful structure for designing potential anticonvulsant agents. Ameltolide, ralitoline and some phthalimide derivatives are the examples of anilide analogs with potent anticonvulsant activity. In this study, two key structural pharmacophores were combined and a series of N-benzoylglycinanilide derivatives were designed. Their anticonvulsant activities evaluated against maximal electroshock (MES) and subcutaneous metrazole seizure tests, whereas their neurotoxicity was examined by rotarod test. The preliminary screening results indicated that majority of the compounds were effective in the MES test. None of the compounds showed neurotoxicity according to the rotarod test at studied doses. The most active compound in the series is N-(2-((4-methoxyphenyl)amino)- 2-oxoethyl)benzamide (compound 8) which bearing 4-methoxy substituent on the N-phenyl ring.

Full text of DOI:10.1007/s00044-013-0474-y

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-013-0474-y
ORIGINAL RESEARCH
Synthesis and anticonvulsant activity of some
N-(benzoyl)glycinanilide derivatives
•
•
•
Zeynep Soyer Ozlem Akgul Ayse H. Tarikogullari
Unsal Calis
Received: 11 September 2012 / Accepted: 6 January 2013
Ó Springer Science+Business Media New York 2013
Abstract Glycine is a major inhibitory neurotransmitter
and recent studies have shown that certain lipophilic
glycine derivatives demonstrate anticonvulsant activity in
animal epilepsy models. On the other hand, anilide is
another fruitful structure for designing potential anticon-
vulsant agents. Ameltolide, ralitoline and some phthali-
mide derivatives are the examples of anilide analogs with
potent anticonvulsant activity. In this study, two key
structural pharmacophores were combined and a series of
N-benzoylglycinanilide derivatives were designed. Their
anticonvulsant activities evaluated against maximal elec-
troshock (MES) and subcutaneous metrazole seizure tests,
whereas their neurotoxicity was examined by rotarod test.
The preliminary screening results indicated that majority of
the compounds were effective in the MES test. None of the
compounds showed neurotoxicity according to the rotarod
test at studied doses. The most active compound in the
series is N-(2-((4-methoxyphenyl)amino)-2-oxoethyl)benzamide
(compound 8) which bearing 4-methoxy substituent on the
N-phenyl ring.
Introduction
Epilepsy is a chronic neurologic disorder characterized by
recurrent unprovoked seizures which results from a tem-
porary electrical disturbance of the brain due to an
imbalance between excitatory and inhibitory neurotrans-
mitters. About 50 million people world wide have epilepsy.
Currently, the main treatment for epileptic disorder is the
long term and consistent administration of anticonvulsant
drugs. Although over 30 AEDs are available, approxi-
mately 30 % of epileptic patients are not seizure free. In
many cases, the clinical use of AEDs is restricted by their
side effects. Therefore, a substantial need remains to dis-
cover novel chemical entities for the development of new
¨
more effective and safer AEDs (McNamara, 2001; Loscher
and Schmidt, 2006; Lin and Kabada, 1997; Malawska,
2005).
Many of the newer antiepileptic drugs were designed
through structural modifications of available therapeutics
and were developed with a specific aim to modify neuro-
transmitter function such as enhancement of inhibitory
mediated neurotransmission or inhibition of excitatory-
mediated neurotransmission (Malawska, 2005; Czabinski
et al., 2005). It is also known that the imbalance between
inhibitory and excitatory processes leads to epileptic sei-
zures in the brain. Therefore, attenuation of excitation and
enhancement of inhibition in the brain are the prevalent
goals in designing new anticonvulsant agents. GABAergic
and glutamatergic systems and their receptor sites can be
Keywords Anilide ꢀ Glycine ꢀ Glycinamide ꢀ
Anticonvulsant activity ꢀ Amide synthesis
Z. Soyer (&) ꢀ O. Akgul ꢀ A. H. Tarikogullari
Department of Pharmaceutical Chemistry, Faculty of Pharmacy,
Ege University, Bornova, 35100 Izmir, Turkey
e-mail: zeynep.soyer@ege.edu.tr
¨
given as examples of those targets (Loscher, 1998; Porter,
1993; Fraser, 1996; Kohl and Dannhardt, 2001).
Glycine is one of the major inhibitory neurotransmitter
which plays an important role in the control of neuronal
activity in the central nervous system (CNS). Simulta-
neously, glycine acts as a co-agonist of the excitatory
U. Calis
Department of Pharmaceutical Chemistry, Faculty of Pharmacy,
Hacettepe University, Sıhhiye, 06100 Ankara, Turkey
123

Med Chem Res
neurotransmitter glutamate by interacting with the strych-
nine-insensitive glycine binding site of the N-methyl-D-
aspartate receptor complex. A deficiency in brain glycine
levels has been shown to cause convulsions or epilepsy.
Under normal physiologic conditions, glycine does not
easily cross the blood–brain barrier (BBB) due to zwitter-
ionic characteristic and the absence of an active transport
mechanism. Thus, intracerebroventricular or systemic
administrations of large doses of glycine are necessary for
significant increases in brain glycine levels. Since the entry
of glycine into the brain is negligible, several lipophilic
glycine derivatives that can cross the BBB have been
developed. These derivatives are milacemide, remacemide,
phthaloyl glycinamide, valproyl glycinamide, and N-ben-
zyloxycarbonyl glycine and its ester and amide derivatives.
Some of the derivatives mentioned above have potential to
become new antiepileptic and CNS drugs. Milacemide and
remacemide have been undergoing clinical trials in patients
with epilepsy (Fig. 1) (Sussan et al., 1999; Scriba and
Lambert, 1999; Geurts et al., 1998; Hadad and Bialer,
1995; Salach et al., 1994; Usifoh et al., 2001; Dawidowski
et al., 2011).
recorded as potassium bromide pellets on a Jasco FT/I
R-400 (Jasco, Tokyo, Japan) and Perkin Elmer FT-IR
Spectrometer 100 (Perkin Elmer Inc., Massachusetts,
USA). The NMR spectra were recorded on a Varian As 400
Mercury Plus NMR (Varian Inc., Palo Alto, CA, USA)
spectrophotometer using DMSO-d6 as solvent. Chemical
shifts were reported in parts per million (d). J was given
in Hz. Mass spectra (API-ES) were measured on an
AGILENT 1100 MSD (Agilent Technologies, Palo Alto,
CA, USA). Elemental analyses (C, H and N) were per-
formed by Leco CHNS-932 (Leco-932, St. Joseph, MI,
USA). The analytical results for the elements were within
0.4 % of the theoretic values.
General procedure for the synthesis of glycinanilide
intermediates (1a–11a)
Compounds 1a–11a were prepared according to the
method reported in the literature (Ho et al., 1998). For this
purpose, N-(tert-butoxycarbonyl)-glycine (N-Boc-glycine,
9.6 mmol) and N-methylmorpholine (NMM, 9.6 mmol)
were dissolved in DMF (10 mL). The solution was cooled to
-20 °C and isobutyl chloroformate (IBCF, 9.6 mmol) was
added. After stirring for 10 min at -20 °C, aniline or
substituted anilines (9.6 mmol) were added and the reaction
mixture was stirred at room temperature for 4 h. Then, the
precipitated NMM hydrochloride was filtered off. The sol-
vent was evaporated under reduced pressure and the residue
was partitioned between ethyl acetate (50 mL) and water
(50 mL). The ethyl acetate phase was washed with 5 %
NaHCO3 (50 mL), dried over anhydrous Na2SO4 and
evaporated under reduced pressure. Trifloroacetic acid
(TFA, 1 mL) was added to the residue and stirred for 30 min.
The solution was treated with petroleum ether–ether (1:1)
and stirred for 30 min at room temperature. The resulting
precipitate was filtered and dissolved in water (5 mL). The
solution was basified with 10 % NaOH and extracted with
dichloromethane (20 mL) twice. The dichloromethane
phase was washed with water (20 mL), dried over anhydrous
Na2SO4, filtered, and evaporated under reduced pressure.
The crude product was crystallized from ethanol.
On the other hand, anilide group has been observed as a
productive pharmacophoric structure for the potent anti-
convulsant molecules in recent studies (Clark et al., 1985;
Bailleux et al., 1995; Vamecq et al., 2000; Soyer et al.,
2003; Soyer et al., 2004).
Depending on the data described above, glycine and
anilide moieties were combined to design a series of
N-benzoylglycinanilide derivatives which can be accepted
as a open chain analogs of phthaloylglycinamides with
known anticonvulsant activity. Accordingly, the synthesis
and preliminary anticonvulsant activity evaluation of
designed N-benzoylglycinanilide derivatives are reported.
Experimental
Chemistry
Melting points were determined on an Electrothermal IA
9100 (Electrothermal, Essex, U.K.) melting point apparatus
and are uncorrected. The IR spectra of compounds were
General procedure for the synthesis of title compounds
(1–11)
NH2
H
Benzoic acid or substituted benzoic acids (1.3 mmol) were
refluxed in 5 mL thionyl chloride for 1 h and excess
thionyl chloride was removed in vacuo. The residue was
dissolved in dichloromethane (20 mL) and a solution of
appropriate amine (1a–11a) in dichloromethane (5 mL) at
0–4 °C was added dropwise. After the addition was com-
pleted, the mixture was stirred in an ice bath for 30 min, at
room temperature, for a further 2 h and washed with 10 %
N
NH
NH2
O
O
Milacemide
Remacemide
Fig. 1 Milacemide and remacemide
123

Med Chem Res
N-(2-(2-methoxyphenylamino)-2-oxoethyl)benzamide (4)
HCl (15 mL), 10 % NaOH (15 mL), and water (20 mL).
The organic phase was dried over anhydrous Na2SO4 and
then evaporated under reduced pressure. The precipitate
was crystallized from ethanol:water (1:1) to furnish title
compounds.
Yield 67 %; mp 106 °C; IR tmaks (KBr): 3330, 3056, 1681,
1648, 1538, 1486 cm-1; 1H NMR (DMSO-d6): d 3.78 (3H,
s, OCH3), 4.09 (2H, d, J = 5.5 Hz, CH2), 6.89 (1H, t,
J = 7.4 Hz, H-4), 7.01–7.05 (2H, m, H-3 and H-5),
7.47–7.55 (3H, m, H-30, H-40 and H-50), 7.90 (2H, dd,
J = 1.2; 7.6 Hz, H-20 and H-60), 8.02 (1H, d, J = 7.8 Hz,
H-6), 8.92 (1H, bs, NH–CH2), 9.14 (1H, bs, NH–phenyl);
13C NMR (DMSO-d6): d 44.3, 56.4, 111.8, 121.1, 124.9,
127.8, 128.0, 129.1, 132.2, 134.6, 150.0, 167.6, 168.5 ppm;
MS (API-ES) m/z (%):285 (M?H?, 73); Anal. calcd. for
C16H16N2O3: C, 67.59; H, 5.67; N, 9.85. Found: C, 67.37;
H, 5.61; N, 9.79.
N-(2-oxo-2-(phenylamino)ethyl)benzamide (1)
Yield 26 %; mp 216 °C (lit.24 213–215 °C); IR tmaks
(KBr): 3274, 3139, 1683, 1639, 1540, 1492 cm-1
;
1H NMR (DMSO-d6): d 4.05 (2H, d, J = 5.6 Hz, CH2),
7.03 (1H, td, J = 1.2; 7.4 Hz, H-4), 7.29 (2H, td, J = 2.0;
8.0 Hz, H-3 and H-5), 7.45–7.49 (2H, m, H-2 and H-6),
7.52–7.56 (1H, m, H-40), 7.58–7.60 (2H, m, H-30 and H-50),
7.90 (2H, dd, J = 1.6; 7.0 Hz, H-20 and H-60), 8.79 (1H,
t, J = 5.5 Hz, NH–CH2), 10.01 (1H, bs, NH–phenyl);
13C NMR (DMSO-d6): d 43.6, 119.9, 123.9, 128.0, 129.0,
129.4, 132.1, 134.7, 139.6, 167.3, 168.5 ppm; (MS
(API-ES) m/z (%): 255 (M?H?, 70); Anal. calcd. for
C15H14N2O2: C, 70.85; H, 5.55; N, 11.02. Found: C, 70.69;
H, 5.32; N, 11.05.
N-(2-(2-ethylphenylamino)-2-oxoethyl)benzamide (5)
Yield 32 %; mp 187 °C; IR tmaks (KBr): 3255, 3089, 1668,
1
1639, 1536, 1490 cm-1; H NMR (CDCl3): d 1.09 (3H, t,
J = 7.4 Hz, CH3), 2.57 (2H, q, J = 7.4; 15.0 Hz,
CH2CH3), 4.08 (2H, d, J = 5.1 Hz, CH2), 7.11–7.22 (3H,
m, H-4, H-5 and H-6), 7.39–7.53 (4H, m, H-3, H-30, H-40
and H-50), 7.90 (2H, dd, J = 1.2; 7.6 Hz, H-20 and H-60),
8.83 (1H, t, J = 5.5 Hz, NH–CH2), 9.29 (1H, bs, NH–
phenyl); 13C NMR (DMSO-d6): d 14.9, 24.4, 43.9, 126.1,
126.2, 126.6, 128.0, 129.0, 129.2, 132.1, 134.7, 136.1,
138.2, 167.4, 168.9 ppm; MS (API-ES) m/z (%):281
(M-H-, 100); Anal. calcd. for C17H18N2O2: C, 72.32; H,
6.43; N, 9.92. Found: C, 72.34; H, 6.31; N, 9.95.
N-(2-(2-chlorophenylamino)-2-oxoethyl)benzamide (2)
Yield 77 %; mp 179 °C; IR tmaks (KBr): 3274, 3110, 1679,
1643, 1538, 1488 cm-1; 1H NMR (DMSO-d6): d 4.12 (2H,
d, J = 6.2 Hz, CH2), 7.16 (1H, td, J = 1.6; 7.7 Hz, H-4),
7.32 (1H, td, J = 1.4; 7.7 Hz, H-5), 7.46–7.56 (4H, m,
H-3, H-30, H-40 and H-50), 7.82 (1H, dd, J = 1.6; 8.2 Hz,
H-6), 7.88 (2H, dd, J = 1.2; 7.0 Hz, H-20 and H-60), 8.90
(1H, t, J = 5.9 Hz, NH–CH2), 9.49 (1H, bs, NH–phenyl);
13C NMR (DMSO-d6): d 44.0, 125.7, 126.3, 126.7, 128.0,
128.2, 129.0, 130.1, 132.2, 134.5, 135.4, 167.5, 169.0 ppm;
MS (API-ES) m/z (%): 287(M-H-, 100), 289(M-H?2,
35); Anal. calcd. for C15H13ClN2O2: C, 62.40; H, 4.54; N,
9.70. Found: C, 62.25; H, 4.54; N, 9.76.
N-(2-(2-isopropylphenylamino)-2-oxoethyl)benzamide (6)
Yield 17 %; mp 120 °C; IR tmaks (KBr): 3262, 3089, 1666,
1639, 1527, 1486 cm-1; 1H NMR (DMSO-d6): d 1.11 (6H,
d, J = 6.6 Hz, 2 9 CH3), 3.13-3.17 (1H, m, CH), 4.08
(2H, d, J = 5.5 Hz, CH2), 7.14–7.17 (2H, m, H-4 and H-5),
7.28–7.30 (2H, m, H-3 and H-6), 7.45–7.49 (2H, m, H-30
and H-50), 7.51–7.55 (1H, m, H-40), 7.89 (2H, dd, J = 1.6;
7.0 Hz, H-20 and H-60), 8.82 (1H, t, J = 5.6 Hz, NH–CH2),
9.35 (1H, bs, NH–phenyl); 13C NMR (DMSO-d6): d 23.9,
27.7, 43.8, 126.2, 126.4, 126.8, 127.1, 128.0, 129.0, 132.1,
134.7, 135.3, 143.6, 167.4, 169.1 ppm; MS (API-ES)
m/z (%):295 (M-H-, 100); Anal. calcd. for C18H20N2O2:
C, 72.95; H, 6.80; N, 9.45. Found: C, 73.16; H, 6.77; N,
9.50.
N-(2-oxo-2-(o-tolylamino)ethyl)benzamide (3)
Yield 22 %; mp 195 °C; IR tmaks (KBr): 3268, 3050, 1673,
1641, 1542, 1490 cm-1; 1H NMR (DMSO-d6): d 2.19 (3H,
s, CH3), 4.02 (2H, d, J = 5.6 Hz, CH2), 7.06 (1H, t,
J = 7.4 Hz, H-4), 7.13–7.20 (2H, m, H-5 and H-6),
7.41–7.55 (4H, m, H-3, H-30, H-40 and H-50), 7.89 (2H, dd,
J = 1.2; 8.2 Hz, H-20 and H-60), 8.90 (1H, t, J = 5.9 Hz,
NH–CH2), 9.49 (1H, bs, NH–phenyl); 13C NMR (DMSO-
d6): d 18.4, 43.9, 125.4, 125.8, 126.6, 128.0, 129.0, 131.0,
132.1, 132.5, 134.7, 136.8, 167.4, 168.6 ppm; MS
(API-ES) m/z (%):267 (M-H-, 100); Anal. calcd. for
C16H16N2O2: C, 71.62; H, 6.01; N, 910.44. Found: C,
71.50; H, 5.89; N, 10.39.
N-(2-(2,6-dimethylphenylamino)-2-oxoethyl)benzamide (7)
Yield 80 %; mp 224 °C; IR tmaks (KBr): 3417, 3249, 1668,
1627, 1569, 1536 cm-1; 1H NMR (DMSO-d6): d 2.13 (6H,
s, 2 9 CH3), 4.05 (2H, d, J = 5.9 Hz, CH2), 7.03–7.04
(3H, m, H-3, H-4 and H-5), 7.45 (2H, d, J = 7.0 Hz, H-30
and H-50), 7.48–7.55 (1H, m, H-40), 7.89 (2H, dd, J = 1.6;
123

Med Chem Res
7.0 Hz, H-20 and H-60), 8.83 (1H, t, J = 5.9 Hz, CH2NH),
9.27 (1H, bs, NH–phenyl); 13C NMR (DMSO-d6): d 18.7,
43.5, 127.1, 128.1, 128.3, 128.9, 132.0, 134.8, 135.7,
136.1, 167.4, 168.4 ppm; MS (API-ES) m/z (%): 283
(M?H?, 100); Anal. calcd. for C17H18N2O2. 1H2O: C,
68.00; H, 6.67; N, 9.33. Found: C, 67.91; H, 6.99; N, 9.34.
NMR (DMSO-d6): d 4.09 (2H, d, J = 5.5 Hz, CH2), 7.03
(1H, t, J = 7.4 Hz, H-4), 7.29 (2H, t, J = 7.8 Hz, H-3 and
H-5), 7.58 (2H, d, J = 8.8 Hz, H-2 and H-6), 8.12 (2H, d,
J = 8.9 Hz, H-30and H-50), 8.33 (2H, d, J = 8.9 Hz, H-20
and H-60), 9.18 (1H, t, J = 5.9 Hz, CH2NH), 10.07 (1H,
bs, NH–phenyl); 13C NMR (DMSO-d6): d 44.1, 119.9,
124.0, 124.3, 129.4, 129.5, 139.6, 140.3, 149.8, 165.8,
168.1 ppm; MS (API-ES) m/z (%): 300 (M?H?, 100);
Anal. calcd. for C15H13N3O4: C, 60.20; H, 4.38; N, 14.04.
Found: C, 60.00; H, 4.05; N, 13.95.
N-(2-((4-methoxyphenyl)amino)-2-oxoethyl)benzamide (8)
Yield 30 %; mp 227 °C (lit.26 217–218 °C); IR tmaks
(KBr): 3269, 3057, 1675, 1639, 1538, 1515 cm-1
;
1H NMR (DMSO-d6): d 3.72 (3H, s, OCH3), 4.03 (2H, d,
J = 5.9 Hz, CH2), 6.88 (2H, d, J = 9.0 Hz, H-3 and H-5),
7.47–7.57 (5H, m, H-20–H-60), 7.89 (2H, d, J = 8.6 Hz,
H-2 and H-6), 8.80 (1H, t, J = 5.9 Hz, CH2NH), 9.89 (1H,
bs, NH–phenyl); 13C NMR (DMSO-d6): d 43.9, 55.8,
114.6, 121.4, 128.0, 129.0, 132.0, 132.8, 134.7, 155.9,
167.3, 168.0 ppm; MS (API-ES) m/z (%): 285 (M?H?,
100); Anal. calcd. for C16H16N2O3. 0.4H2O: C, 65.86; H,
5.76; N, 9.60. Found: C, 66.00; H, 5.83; N, 9.53.
Anticonvulsant activity screening
The compounds were tested for their anticonvulsant
activity against maximal electroshock (MES) and subcu-
taneous Metrazole (scMet)-induced seizure threshold tests.
The acute neurologic toxicity was determined in the rota-
rod 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
Neurologic Disorders, and Stroke (NINDS) (Krall et al.,
1978). Stimulator (Grass S88, Astro-Med. Inc. Grass
Instrument Division, W. Warwick, RI, USA), constant
current unit (Grass CCU1A, Grass Medical Instrument,
Quincy, MA, USA), and corneal electrodes were used for
the evaluation of anticonvulsant activity against MES test.
All the synthesized compounds were suspended in 30 %
aqueous of PEG 400 and administered to the mice intra-
peritoneally in a volume of 0.01 mL g-1 at body weight.
Twelve Swiss Albino male mice (20 2 g) were used for
each compound (mice were obtained from The Hacettepe
University Animal Farm) according to the ADD-NINDS
program. The animals 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 carried out
with the permission of Hacettepe University, ‘‘Laboratory
Animals Ethic Committee’’ decision (01.09.2010 date
2010/46-11 number). Control animals received 30 %
aqueous PEG 400. Metrazole was administered subcuta-
neously (sc) on the back of the neck. The rotarod toxicity
test was performed on 1-inch-diameter knurled wooden
rod, rotating at 6 rpm (the rotarod used in phase-1 test was
made by Hacettepe University Technical Department).
2-Chloro-N-(2-oxo-2-(phenylamino)ethyl)benzamide (9)
Yield 40 %; mp 182 °C; IR tmaks (KBr): 3255, 3062, 1691,
1645, 1551, 1498 cm-1; 1H NMR (DMSO-d6): d 4.03 (2H,
d, J = 5.9 Hz, CH2), 7.04 (1H, tt, J = 1.2; 7.4 Hz, H-4),
7.30–7.34 (2H, m, H-3 and H-5), 7.40–7.54 (4H, m, H-2,
H-6, H-30 and H-50), 7.59–7.61 (2H, m, H-40 and H-60),
8.70 (1H, t, J = 5.9 Hz, CH2NH), 10.01 (1H, bs, NH–
phenyl); 13C NMR (DMSO-d6): d 43.7, 119.8, 124.0,
127.7, 129.5, 130.0, 130.4, 130.7, 131.7, 137.0, 140.0,
167.4, 168.0 ppm; MS (API-ES) m/z (%): 289 (M?H?,
49), 291(M?H?2, 18); Anal. calcd. for C15H13ClN2O2: C,
62.40; H, 4.54; N, 9.70. Found: C, 62.27; H, 4.22; N, 9.84.
4-Methyl-N-(2-oxo-2-(phenylamino)ethyl)benzamide (10)
Yield 33 %; mp 222 °C; IR tmaks (KBr): 3269, 1680, 1641,
1599, 1543 cm-1; 1H NMR (DMSO-d6): 2.35 (3H, s, CH3),
4.03 (2H, d, J = 5.9 Hz, CH2), 7.02 (1H, t, J = 7.4 Hz,
H-4), 7.26–7.30 (4H, m, H-2, H-3, H-5 and H-6), 7.58 (2H,
d, J = 7.8 Hz, H-30 and H-50), 7.79 (2H, d, J = 7.8 Hz,
H-20 and H-60), 8.72 (1H, t, J = 5.9 Hz, CH2NH), 10.02
(1H, bs, NH–phenyl); 13C NMR (DMSO-d6): d 21.6, 43.9,
119.8, 123.9, 128.0, 129.4, 129.5, 131.9, 139.6, 142.0,
167.2, 168.6 ppm; MS (API-ES) m/z (%):269 (M?H?, 29);
Anal. calcd. for C16H16N2O2: C, 71.62; H, 6.01; N, 10.44.
Found: C, 71.25; H, 5.83; N, 10.33.
MES-induced seizure test
MES seizures were elicited with a 60-cycle alternating
current of 50 mA intensity (5–7 times more than that
required to elicit minimal seizures) delivered for 0.2 s via
corneal electrodes. A drop of 0.9 % saline was instilled
into the eye prior to application of the electrodes in order to
4-Nitro-N-(2-oxo-2-(phenylamino)ethyl)benzamide (11)
Yield 59 %; mp 219 °C (lit.24 213.5–215.5 °C); IR tmaks
(KBr): 3342, 3263, 1684, 1645, 1599, 1547 cm-1 1H
;
123

Med Chem Res
prevent the death of the animal. Abolition of the hind limb
tonic extension component of the seizure was defined as
protection.
Nakanishi and Solomon, 1977). For instance, amide C=O
stretching bands of anilide and benzamide groups were
observed between 1666–1691 and 1627–1648 cm-1
,
respectively. N–H stretching bands of the secondary amide
structure of the title compounds were observed between
scMet-induced seizure test
3050 and 3417 cm-1
.
85 mg kg-1 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 30 min
to decide whether the failure of the threshold seizure (a
single episode of clonic spasms of at least 5 s duration)
could be defined as protection.
1H NMR spectra of the title compounds were consistent
with expected resonance signals in term of chemical shifts
and integrations. The chemical shifts and splitting patterns
of phenyl protons in each compound differed depending on
the nature of the substituents.
13C NMR spectra, the resonance signals did not account
for the total number of carbon atoms in individual com-
pounds since as expected certain signals represent more
than one carbon atom.
Neurotoxicity
The rotarod test was used to evaluate neurotoxicity. The
animal was placed on a 1-inch-diameter knurled wooden rod
rotating at 6 rpm. Normal mice remain on a rod rotating at
this speed indefinitely. Neurologic toxicity was defined as
the failure of the animal to remain on the rod for 1 min.
The structure of the title compounds was further verified
by API-ES Mass spectra. The spectra of the compounds
were obtained by positive or negative ionization and the
m/z values of molecular ion peaks were in complete
agreement with the calculated molecular weight.
Anticonvulsant activity screening
Results and discussion
Preliminary anticonvulsant evaluation (Phase 1) of the
synthesized compounds was performed by following the
standard procedure provided by the antiepileptic drug
development program. The initial evaluation includes
qualitative assays by MES and scMet seizure tests, the
most widely employed two seizure models for early iden-
tification of candidate anticonvulsants. The MES test
generally accepted as a model which employs an electrical
stimulus to induce generalized tonic–clonic seizures and
identifies compounds preventing the spread of seizures.
The scMet test, producing myoclonic seizures induced
chemically, is capable recognizing the agents that act by
raising the seizure threshold. The rotarod test was used to
determine the possible neurotoxic effects of the compounds
under anticonvulsant screening.
Chemistry
In this study, eleven N-benzoylglycinanilide derivatives
have been synthesized to evaluate anticonvulsant activity
profile. The target compounds were prepared by a two-step
synthesis. In the first step, anilides were prepared by
reacting N-Boc-glycine with substituted anilines or aniline
and then, Boc group was removed from molecules (Ho
et al., 1998). In the second step, those intermediates were
reacted with appropriate benzoyl chlorides to furnish the
target compounds (Pabuccuoglu and Hesse, 1997). The
synthetic pathways are given in Scheme 1. The structures
of the synthesized compounds were confirmed by spectral
(IR, 1H NMR, 13C NMR, and API-ES Mass) and elemental
analyses.
Anticonvulsant activity profile of synthesized com-
pounds was established after intraperitoneally (i.p) injec-
tions into mice and evaluated in the MES, scMet at doses
of 30, 100, and 300 mg kg-1 at two different time intervals
(0.5 and 4 h). Neurotoxicity of the compounds was deter-
mined at same doses by rotarod test. The results are shown
in Table 1.
According to the literature survey, compounds 1, 3, 4,
8, and 10 are reported derivatives (Fox and Wax, 1950;
Albertson, 1951; Abernathy et al., 1958; Bird and Twibell,
1971) and 2, 5–7, and 9 are listed substances in the liter-
ature with the CAS registry numbers 941476-87-5,
1017100-89-8,
1089403-42-8,
747411-49-0,
and
903348-90-3, respectively, but corresponding scientific
reference data are not available for those compounds.
The purity levels of compounds were determined by
elemental analyses (C, H, N) and results were within 0.4 %
of the calculated values.
The initial anticonvulsant screening results indicated
that all the compounds, except compounds 2, 4, and 5, were
effective in MES or scMet tests. None of the compounds
showed toxicity in the rotarod neurotoxicity screening at
doses studied.
In the IR spectra, the presence of C=O stretching bands
were the confirmative signals for the constructed functional
groups in the title compounds (Hesse et al., 1997;
Compounds 1, 3, 6–8, and 9 exhibited activity in the
MES test. Among these compounds, 1, 3, and 6 showed
activity at doses of 300 mg kg-1 at 0.5 and they lost their
123

Med Chem Res
R
H
H2N
R
N
Boc
a
H2N
N
+
H
COO
H
O
1a-11a
O
O
O
=
H
R'
H
R
R
R'
R'
N
N
c
+
l
C
H
b
O
H2N
N
H
O
O
1a-11a
=
=
=
=
=
=
=
=
= -
(1)
R' H R H
R' H R 4
H
3
OC
,
,
,
,
,
,
,
,
(8)
(9)
(10)
(11)
= -
= -
=
(2)
(3)
(4)
(5)
(6)
(7)
R' H R 2
l
H
R' 2 l R H
C
C
OC
C
,
= -
= -
=
=
R H
R' H R 2
R' 4
= -
H
C
R' H
,
3
H
3
= -
R' H R 2
R' 4 N
O2
,
3
= -
R' H R 2 H5
C2
= -
R' H R 2 H( H )
C
C
3 2
=
-
R' H R 2 6 H
C
,
3
Scheme 1 Syntheses of the compounds 1–11. a NMM, IBCF, rt, 4 h; TFA, rt, 30 min; 10 % NaOH, CH2Cl2; b SOCl2, reflux, 1 h; c CH2Cl2,
0–4 °C, 30 min; rt 2 h
Table 1 Phase 1 anticonvulsant and neurotoxicity screening data of title compounds
Compound MESa
0.5 h (mg kg-1
scMetb
Toxicityc
)
4 h (mg kg-1
)
0.5 h (mg kg-1
)
4 h (mg kg-1
)
0.5 h (mg kg-1
)
4 h (mg kg-1
)
30
100
300
30
100 300 30
100
300
30
100 300 30
100
300
30 100 300
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
0/1
1/1
0/1
0/1
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 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
1/1
1/1
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
1/1
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
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
2
3
4
5
6
7
8
9
10
11
0/1 No activity at dose level, 1/1 noticeable activity at dose level [bold], 0/4 number of animals exhibited toxicity/number of animals tested for
05 h, 0/2 number of animals exhibiting toxicity/number of animals tested for 4 h
a
Maximal electroshock test
b
Subcutaneous metrazole test
c
Rotarod test
potencies at 4 h indicating a rapid onset of action or fast
inactivation. Compound displayed protection at
According to the results, only two compounds (10 and
11) in the series were found to be active in the scMet test.
Thus, compound 10 was active at the dose of 300 mg kg-1
only at 0.5 h. Compound 11 was active at same dose level
both at 0.5 and 4 h.
9
300 mg kg-1 dose level at 4 h period. Compound 7
showed activity at 100 and 300 mg kg-1 doses at 4 h. The
most active compound in the series was compound 8 which
demonstrated activity at 100 and 300 mg kg-1 doses at
0.5 h and 300 mg kg-1 at 4 h. These results suggested that
compound 8 has quick onset and long duration of action.
Under the set of compounds studied, it can be concluded
that, in N-benzoylglycinanilide derivatives, substitution of
N-phenyl ring enhances anti-MES activity, whereas
123

Med Chem Res
Fraser AD (1996) New drugs for the treatment of epilepsy. Clin
Biochem 29(2):97–110
Geurts M, Poupaert JH, Scriba GKE, Lambert DMJ (1998)
N-(benzyloxycarbonyl)glycine esters and amides as new anti-
convulsants. J Med Chem 41(1):24–30
Hadad S, Bialer M (1995) Pharmacokinetic analysis and antiepileptic
activity of N-valproyl derivatives of gaba and glycine. Pharm
Res 12:905–910
Hesse M, Meier H, Zeeh B (1997) Spectroscopic methods in organic
chemistry. Georg Thieme Verlag, Stuttgart, New York
Ho B, Venkatarangan PM, Cruse SF, Hinko CN, Andersen PH, Crider
AM, Adloo AA, Roane DS, Stables JP (1998) Synthesis of
2-piperidinecarboxylic acid derivatives as potential anticonvul-
sants. Eur J Med Chem 33:23–31
Kohl BH, Dannhardt G (2001) The NMDA receptor complex: a
promising target for novel antiepileptic strategies. Curr Med
Chem 8(11):1275–1289
Krall RL, Penry JP, White BG, Kupferberg JH, Swinyard EA (1978)
Antiepileptic drug development: II. Anticonvulsant drug screen-
ing. Epilepsia 19(4):409–428
substitution of phenyl ring in benzoyl residue strengthens
anti-scMet activity, depending on the substitution pattern
and the nature.
Monosubstitution at the 2 position of N-phenyl ring does
not yield superior compounds in comparison to nonsub-
stituted one. However, disubstitution at 2,6-positions
enhances and retards the activity. Surprisingly, methoxy
substitution in para position of N-phenyl ring creates the
most active compound in the series at 0.5 h in MES test.
Substitution of phenyl ring in benzoyl residue in N-ben-
zoylglycinanilide derivatives seems to determine the activity
profile. For example, 2-substituted derivative (compound 9)
was active in MES test, whereas 4-substituted derivatives
(compounds 10 and 11) were active only in scMet test.
Lin Z, Kabada PK (1997) Molecular targets for the rational design of
antiepileptic drugs and related neuroprotective agents. Med
Chem Res Rev 17(6):537–572
Conclusion
In this study, a series of N-benzoylglycinanilide derivatives
was designed, synthesized, and their anticonvulsant activ-
ities were evaluated. The preliminary anticonvulsant
screening results have demonstrated that N-benzoylglyc-
inanilide template has certain anticonvulsant activity and
can serve as a starting structure for further studies to design
new effective anticonvulsant molecules.
¨
Loscher W (1998) New visions in the pharmacology of anticonvul-
sion. Eur J Med Chem 342:1–13
Loscher W, Schmidt D (2006) New horizons in the development of
¨
antiepileptic drugs: ınnovative strategies. Epilepsy Res 69(3):
183–272
Malawska B (2005) Anticonvulsant agents. Curr Top Med Chem
5(1):69–85
McNamara OJ (2001) Drugs effective in the therapy of the epilepsies.
In: Hardman JG, Limbird LE, Gilman AG (eds) The pharma-
cological basis of therapeutics. McGraw-Hill, New York,
pp 521–548
Nakanishi K, Solomon PH (1977) Infrared absorption spectroscopy,
2nd edn. Holden-Day Inc, San Francisco
Acknowledgments This study was supported by Research Grants
from Ege University (Project Number: 05/Ecz/06).
Conflict of interest The authors report no conflicts of interest.
Pabuccuoglu V, Hesse M (1997) The total synthesis of the pavine
alkaloid thalimonine. Heterocycles 45(9):1751–1758
Porter RJ (1993) New developments in the search for improved
antiepileptic drugs. Jpn Psychiatry Neurol 47:145–162
Salach OA, Hadad S, Haj-Yehia A, Sussan S, Bialer M (1994)
Comparative pharmacokinetic and pharmacodynamic analysis of
phthaloyl glycine derivatives with potential antiepileptic activ-
ity. Pharm Res 11(10):1429–1434
References
Abernathy JL, Nakamura J, Collins BM (1958) A comparison of rates
of precipitation of substituted hippuric anilides formed by
papain-catalyzed reactions between hippuric acid and substituted
anilines at approximately pH 4.6. J Org Chem 23:586–589
Albertson NF (1951) Asymmetric enzymatic synthesis of amino acid
anilides. J Am Chem Soc 73:452–454
Scriba GKE, Lambert DMJ (1999) Synthesis and anticonvulsant
activity of N-benzyloxycarbonyl-amino acid prodrugs of phe-
nytoin. J Pharm Pharmacol 51(5):549–553
˘
Soyer Z, Kılıc¸ FS, Erol K, Pabuc¸cuoglu V (2003) Synthesis and
´
Bailleux V, Vallee L, Nuyts JP, Hamoir G, Poupaert JH, Stables JP,
anticonvulsant activity of some x-(1H-imidazol-1-yl)-N-phenylacet-
amide and propionamide derivatives. Il Farmaco 59(8):595–600
Soyer Z, Kılıc FS, Erol K, Pabuccuoglu V (2004) The synthesis and
anticonvulsant activity of some x-phthalimido-N-phenylacet-
amide and propionamide derivatives. Arch Pharm Pharm Med
Chem 337(2):105–111
Sussan S, Dagan A, Blotnik S, Bialer M (1999) The structural
requirements for the design of antiepileptic-glycine derivatives.
Epilepsy Res 34(2–3):207–220
Usifoh CO, Lambert DM, Wouters J, Scriba GKE (2001) Synthesis
and anticonvulsant activity of N,N-phthaloyl derivatives of
central nervous system inhibitory amino acids. Arch Pharm
Pharm Med Chem 334(10):323–331
Vamecq J, Bac P, Herrenknecht C, Maurois P, Delcourt P, Stables JP
(2000) Synthesis and anticonvulsant and neurotoxic properties of
substituted N-phenyl derivatives of the phthalimide pharmaco-
phore. J Med Chem 43(7):1311–1319
Vamecq J (1995) Comparative anticonvulsant activity and
neurotoxicity of 4-amino-N-(2,6-dimethylphenyl)phthalimide
and prototype antiepileptic drugs in mice and rats. Epilepsia 36(6):
559–565
Bird CW, Twibell JD (1971) Further observation on the reaar-
rangement of 3-acylaminoazetidinones. J Chem Soc Sect C Org
19:3155–3158
Clark CR, Sansom RT, Lin CM, Norris N (1985) Anticonvulsant activity
of some 4-aminobenzanilides. J Med Chem 28(9):1259–1262
Czabinski P, Blaszczyk B, Czuczwar SJ (2005) Mechanisms of action
of antiepileptic drugs. Curr Top Med Chem 5(1):3–14
Dawidowski M, Herold F, Chodkowski A, Kleps J, Szulczyk P,
Wilczek M (2011) Synthesis and anticonvulsant activity of novel
2,6-diketopiperazine derivatives. Part 1: perhydropyrrole[1,
2-a]pyrazines. Eur J Med Chem 46(10):4859–4869
Fox SW, Wax HJ (1950) Enzymatic synthesis of peptide bonds. III.
The relative effects of some amino acids and their acyl
substituents. J Am Chem Soc 72:5087–5089
123