Anticonvulsant Activity of Novel 1-(Substituted Benzylidene)-4-(1-(Morpholino/Piperidino Methyl)-2,3-Dioxoindolin-5-yl) Semicarbazide Derivatives in Mice and Rats Acute Seizure Models

Article abstract of DOI:10.1111/j.1747-0285.2012.01399.x

A series of novel 1-(substituted benzylidene)-4-(1-(morpholino/piperidino methyl)-2,3-dioxoindolin-5-yl) semicarbazides 6a-6t was designed and synthesized on the basis of semicarbazide-based pharmacophoric model to meet the structural requirements necessa

Full text of DOI:10.1111/j.1747-0285.2012.01399.x

ª 2012 John Wiley & Sons A/S
doi: 10.1111/j.1747-0285.2012.01399.x
Chem Biol Drug Des 2012
Research Article
Anticonvulsant Activity of Novel 1-(Substituted
Benzylidene)-4-(1-(Morpholino ⁄ Piperidino
Methyl)-2,3-Dioxoindolin-5-yl) Semicarbazide
Derivatives in Mice and Rats Acute Seizure
Models
Chinnasamy Rajaram Prakash1,*,
followed by convulsions (1). About 1% of the world populations
have epilepsy, with almost 90% of these people being in devel-
oping countries (2,3). Current treatment options include electrical
stimulation, seizure suppression, and the use of antiepileptic
Sundararajan Raja2 and Govindaraj
Saravanan3
1Department of Medicinal Chemistry, DCRM Pharmacy College,
drugs (AEDS). It has been estimated that 20% of the patients
with epilepsy are using the first-generation AEDS like phenytoin,
sodium valproate, phenobarbitone, and carbamazepine (4). Unfor-
tunately, those drugs are associated with severe side effects (5).
These limitations demand selective and lower toxicity AEDS in
the field of medicinal chemistry.
Jawaharlal Nehru Technological University, Hyderabad, Andhra
Pradesh, India
2Department of Pharmaceutical Chemistry, GITAM Institute of
Pharmacy, GITAM University, Visakhapatnam, Andhra Pradesh, India
3Department of Medicinal Chemistry, Bapatla College of Pharmacy,
Bapatla, Andhra Pradesh, India
*Corresponding author: Chinnasamy R. Prakash, crp2020@gmail.com
Isatin, a class of heterocyclic compounds, possesses a broad
spectrum of bioactivity (6–11). These properties encouraged the
efforts toward the synthesis and pharmacological screening of
many isatin derivatives. On the other hand, several studies have
identified semicarbazides as a structurally novel class of com-
pounds with particularly antiepileptic activity (12–14). It is well
documented that the essential structural features responsible for
AED is the presence of one electron donor atom (D), Hydrogen
donor ⁄ acceptor unit (HAD) and a hydrophobic domain (A) (15–17).
This common template was found in many AEDS (Figure 1).
Motivated by the above findings and considering the wide appli-
cations of the isatin molecule in medicinal chemistry, an attempt
has been made to synthesize different substituted benzylidene
semicarbazide derivatives incorporating isatin moiety as a new
antiepileptic agent.
A series of novel 1-(substituted benzylidene)-4-(1-
(morpholino ⁄ piperidino methyl)-2,3-dioxoindolin-5-
yl) semicarbazides 6a–6t was designed and
synthesized on the basis of semicarbazide-based
pharmacophoric model to meet the structural
requirements necessary for anticonvulsant activity.
The compounds were subjected to in vivo antiepi-
leptic evaluation using maximal electroshock test
and subcutaneous pentylenetetrazole seizure test
methods. The neurotoxicity was determined by ro-
torod test. In the preliminary screening, com-
pounds 6c, 6d, 6g, 6h, and 6m were found active
in maximal electroshock test model, while 6g, 6i,
6m, and 6o showed significant antiepileptic activ-
ity in subcutaneous pentylenetetrazole seizure
test model. Further, the compounds 6c, 6d, 6g, 6h,
6i, and 6m were administered orally to rats, of
which 6c and 6g showed better activity than phe-
nytoin. Among the synthesized compounds, 6g
revealed excellent protection in both models with
lower neurotoxicity.
Experimental Section
Materials and methods
The chemicals were obtained from Merck (Darmstadt, Germany) and
SD Fine (Ahmadabad, India). For Thin Layer Chromatography (TLC),
the solvent system was prepared in the following ratio: toluene ⁄ ethyl
acetate ⁄ formic acid (5:4:1). UV and iodine chamber was used as visu-
alizing methods. Melting points were determined in open glass capil-
lary and were uncorrected. 1H-NMR spectral studies were carried out
on a Bruker ultra shield NMR spectrometer using tetramethylsilane
as internal standard. Mass spectra were obtained on a JEOL-SX-102
instrument using electron impact ionization. All the IR spectra were
recorded by Jasco FT-IR 410 spectrometer. Elemental analyses were
performed on a Perkine Elmer model 240C analyzer.
Key words: convulsions, in vivo studies, Indole-2,3-dione, isatin,
neurotoxicity
Received 25 January 2012, revised 6 March 2012 and accepted for pub-
lication 9 April 2012
Epilepsy is a chronic neurological disorder, characterized by par-
oxysmal cerebral dysrhythmia, manifested as brief episodes
(seizures) of loss or disturbance of consciousness, frequently
1

Prakash et al.
Figure 1: Examples of struc-
tural similarities between known
antiepileptic drugs, compounds in
clinical and preclinical development
and the benzylidene semicarbazide
incorporated isatin heterocyclic sys-
tem (Model compound structure).
(A) Hydrophobic aryl ring system,
Hydrogen bond acceptor ⁄ donor
domain (HAD), (D) Electron donor
moiety and (B) Distal aryl ring.
Synthesis of compounds
5-Amino-1-substituted indolin-2,3-dione 3a–3b
In a round-bottomed flask containing 200 mL of absolute ethanol,
compound 2a or 2b (0.01 mol) and iron powder (0.01 mol) was
added. After the addition, the mixture was heated on an oil bath
until the temperature reaches to 80–85 ꢀC. Further, in the reaction
flask, 4 mL of hydrochloric acid (1.2 M) was added, and the content
was stirred in the same temperature for a period of 4 h. Finally,
the slurry was filtered, and the pH of the filtrate was adjusted to
7–8 with the help of sodium bicarbonate to get the precipitate of
compound 3a or 3b. Recrystallization was carried out with ethanol
to obtain the product in the pure form.
Preparation of 5-nitroisatin 1
The 5-nitroisatin 1 was prepared according to the reported litera-
ture (18).
5-Nitro-1-substituted indolin-2,3-dione 2a–2b
To the solution of 5-nitro indolin-2-one 1 (0.04 mol) in 95% abso-
lute ethanol (100 mL), aqueous formaldehyde 37% (1.0 mL) was
added. Then, secondary amino compound such as morpholine ⁄ piper-
idine (0.04 mol) was slowly added drop-wise to the above solution
under stirring. After the addition, the entire reaction mixture was
stirred at room temperature for a period of 3 h and was kept aside
for 48 h in a refrigerator to facilitate the formation of crystals.
Finally, the crystalline form of the product was separated by filtra-
tion, washed with hexane, and vacuum dried. The desired com-
pounds were finally recrystallized with ethanol to obtain the pure
product (Scheme 1).
1-(Morpholinomethyl)-5-amino indoline-2,
3-dione 3a
Yield 61%, Mp 226 ꢀC; IR (per cm): 3386, 3128, 1740, 1055; 1H
NMR (CDCl3): 2.38 (t, J = 6.0 Hz, 4H, –CH2 morpholine); 3.38 (t,
J = 5.6 Hz, 4H, –CH2 morpholine); 3.86 (s, 2H, -NH2), 4.24 (s, 2H, –
CH2); 7.42–7.76 (m, 3H, Ar-H); MS (EI) m ⁄ z: 261 [M+]; Anal. Calcd
for C13H15N3O3: C, 59.76; H, 5.79; N, 16.08. Found: C, 59.94; H,
5.81; N, 16.13.
1-(Morpholinomethyl)-5-nitro indoline-2,
3-dione 2a
Yield 68%, Mp 248 ꢀC; IR (per cm): 3018, 1734, 1565, 1352, 1070;
1H NMR (CDCl3): 2.45 (t, J = 6.0 Hz, 4H, –CH2 morpholine); 3.32 (t,
J = 5.6 Hz, 4H, –CH2 morpholine); 4.28 (s, 2H, –CH2); 7.40–7.86 (m,
3H, Ar-H); MS (EI) m ⁄ z: 291 [M+]; Anal. Calcd for C13H13N3O5: C,
53.61; H, 4.50; N, 14.43. Found: C, 53.78; H, 4.52; N, 14.38.
5-Amino-1-(piperidin-1-ylmethyl)indoline-2,
3-dione 3b
Yield 54%, Mp 234 ꢀC; IR (per cm): 3398, 3089, 1722; 1H NMR
(CDCl3): 1.24–1.57 (m, 6H, –CH2 piperidine); 2.19–2.48 (m, 4H, –CH2
piperidine); 3.74 (s, 2H, -NH2), 4.35 (s, 2H, –CH2); 7.36–7.68 (m, 3H,
Ar-H); MS (EI) m ⁄ z: 259 [M+]; Anal. Calcd for C14H17N3O2: C, 64.85;
H, 6.61; N, 16.20. Found: C, 65.05; H, 6.63; N, 16.26.
5-Nitro-1-(piperidin-1-ylmethyl)indoline-2,
3-dione 2b
Yield 56%, Mp 221 ꢀC; IR (per cm): 3024, 1710, 1544, 1332; 1H
NMR (CDCl3): 1.45–1.56 (m, 6H, –CH2 piperidine); 2.35–2.44 (m, 4H,
–CH2 piperidine); 4.40 (s, 2H, –CH2); 7.20–7.64 (m, 3H, Ar-H); MS
(EI) m ⁄ z: 289 [M+]; Anal. Calcd for C14H15N3O4: C, 58.13; H, 5.23; N,
14.53. Found: C, 58.30; H, 5.25; N, 14.58.
1-(1-Substituted 2,3-dioxoindolin-5-yl) urea
4a–4b
Compounds 3a and 3b (0.01 mol) were dissolved in 10 mL
glacial acetic acid, and the volume was diluted to 100 mL with
the same in a conical flask. A solution of NaOCN (sodium
2
Chem Biol Drug Des 2012

Mice and Rats Acute Seizure Models
Scheme 1: Synthetic protocols
of intermediates and title com-
pounds.
cyanate) (0.01 mol) and 50 mL of warm water was then added
slowly with continuous stirring. Then the reaction mixture was
allowed to stand for 30 min, cooled in crushed ice, and was
allowed to stand for further 30 min. The product thus obtained
was filtered and washed with cold water, finally dried at
100 ꢀC. The product was recrystallized at least once from etha-
nol to give the pure form.
4-((1-Substituted)-2,3-dioxoindolin-5-yl)
semicarbazide 5a–5b
The compounds 4a and 4b (0.01 mol) were dissolved in 30 mL of
absolute ethanol. To this, 2.5 mL of hydrazine hydrate was added,
and the entire mixture was refluxed for 10–12 h with stirring (19).
The contents were concentrated to half of its volume and poured
onto crushed ice. The resultant precipitate was filtered, thoroughly
washed with water, dried, and recrystallized from ethanol.
1-(1-(Morpholinomethyl)-2,3-dioxoindolin-5-
yl)urea 4a
4-(1-(Morpholinomethyl)-2,3-dioxoindolin-5-
yl)semicarbazide 5a
Yield 72%, Mp 272 ꢀC; IR (per cm): 3364, 3096, 1746, 1648, 1030;
1H NMR (CDCl3): 2.36 (t, J = 6.0 Hz, 4H, –CH2 morpholine); 3.34 (t,
J = 5.6 Hz, 4H, –CH2 morpholine); 3.76 (s, 2H, -NH2), 4.36 (s, 2H, –
CH2); 7.32–7.72 (m, 3H, Ar-H); 8.42 (s, 1H, Ar-NH); MS (EI) m ⁄ z: 304
[M+]; Anal. Calcd for C14H16N4O4: C, 55.26; H, 5.30; N, 18.41. Found:
C, 55.44; H, 5.32; N, 18.48.
Yield 71%, Mp 275 ꢀC; IR (per cm): 3387, 3058, 1720, 1640, 1042;
1H NMR (CDCl3): 2.30 (t, J = 6.0 Hz, 4H, –CH2 morpholine); 3.42 (t,
J = 5.6 Hz, 4H, –CH2 morpholine); 4.44 (s, 2H, –CH2); 5.76 (s, 2H, -
NH2), 6.84–7.62 (m, 3H, Ar-H); 8.22 (s, 1H, Ar-NH); 9.84 (s, 1H,
CONH); MS (EI) m ⁄ z: 319 [M+]; Anal. Calcd for C14H17N5O4: C,
52.66; H, 5.37; N, 21.93. Found: C, 52.82; H, 5.39; N, 22.02.
1-(2,3-Dioxo-1-(piperidin-1-ylmethyl)indolin-5-
yl)urea 4b
4-(2,3-Dioxo-1-(piperidin-1-ylmethyl)indolin-5-
yl)semicarbazide 5b
Yield 71%, Mp 246 ꢀC; IR (per cm): 3398, 3120, 1734, 1668; 1H
NMR (CDCl3): 1.28–1.53 (m, 6H, –CH2 piperidine); 2.28–2.56 (m, 4H,
–CH2 piperidine); 3.88 (s, 2H, -NH2), 4.24 (s, 2H, –CH2); 7.20–7.68
(m, 3H, Ar-H); 8.50 (s, 1H, Ar-NH); MS (EI) m ⁄ z: 302 [M+]; Anal.
Calcd for C15H18N4O3: C, 59.59; H, 6.00; N, 18.53. Found: C, 60.10;
H, 6.02; N, 18.60.
Yield 62%, Mp 284 ꢀC; IR (per cm): 3364, 3050, 1740, 1672; 1H
NMR (CDCl3): 1.32–1.62 (m, 6H, –CH2 piperidine); 2.54–2.82 (m, 4H,
–CH2 piperidine); 4.22 (s, 2H, –CH2); 5.88 (s, 2H, -NH2); 7.12–7.58
(m, 3H, Ar-H); 8.30 (s, 1H, Ar-NH); 9.88 (s, 1H, CONH); MS (EI) m ⁄ z:
317 [M+]; Anal. Calcd for C15H19N5O3: C, 56.77; H, 6.03; N, 22.07.
Found: C, 56.93; H, 6.01; N, 22.14.
Chem Biol Drug Des 2012
3

Prakash et al.
General procedure for the preparation of
1-(substituted benzylidene)-4-(1-(morpholino ⁄
piperidino methyl)-2,3-dioxoindolin-5-yl)
semicarbazide derivatives 6a–6t
Title compounds 6a–6t were synthesized by adding 5a ⁄ 5b
(0.01 mol) in fraction with the well-stirred mixture of different aro-
matic aldehydes (0.01 mol) in 50 mL ethanol and few drops of gla-
cial acetic acid. Then, this mixture was refluxed for 6–8 h and kept
aside. The separated product was filtered, dried, and recrystallized
from ethanol.
morpholine); 3.32 (t, J = 5.4 Hz, 4H, –CH2 morpholine); 4.40 (s, 2H,
–CH2); 6.94–7.90 (m, 7H, Ar-CH); 8.22 (s, 1H, Ar-NH); 8.50 (s, 1H,
CH=N); 9.94 (s, 1H, CONH); MS (EI) m ⁄ z: 444 [M + 2]; Anal. Calcd
for C21H20ClN5O4: C, 57.08; H, 4.56; N, 15.85. Found: C, 57.22; H,
4.58; N, 15.90.
1-(2-Chlorobenzylidene)-4-(2,3-dioxo-1-
(piperidin-1-ylmethyl)indolin-5-yl) semicarbazide
6f
Yield 70%, Mp 290 ꢀC; Rf 0.80; IR (per cm): 3336, 3006, 1740,
1552, 768; 1H NMR (DMSO-d6): 1.23–1.51 (m, 6H, –CH2 piperi-
dine); 2.34–2.68 (m, 4H, –CH2 piperidine); 4.23 (s, 2H, –CH2);
7.10–7.92 (m, 7H, Ar-H); 8.24 (s, 1H, Ar-NH); 8.44 (s, 1H, CH=N);
9.92 (s, 1H, CONH); MS (EI) m ⁄ z: 442 [M + 2]; Anal. Calcd for
C22H22ClN5O3: C, 60.07; H, 5.04; N, 15.92. Found: C, 60.24; H,
5.06; N, 15.95.
1-Benzylidene-4-(1-(morpholinomethyl)-2,
3-dioxoindolin-5-yl) semicarbazide 6a
Yield 66%, Mp 262 ꢀC; Rf 0.68; IR (per cm): 3368, 2998, 1720, 1546,
1
1064; H NMR (DMSO-d6): 2.40 (t, J = 5.8 Hz, 4H, –CH2 morpholine);
3.22 (t, J = 5.6 Hz, 4H, –CH2 morpholine); 4.45 (s, 2H, –CH2); 6.90–
7.45 (m, 8H, Ar-CH); 8.22 (s, 1H, Ar-NH); 8.58 (s, 1H, CH=N); 9.98 (s,
1H, CONH); MS (EI) m⁄ z: 407 [M+]; Anal. Calcd for C21H21N5O4: C,
61.91; H, 5.20; N, 17.19. Found: C, 62.08; H, 5.22; N, 17.26.
1-(4-Fluorobenzylidene)-4-(1-
(morpholinomethyl)-2,3-dioxoindolin-5-yl)
semicarbazide 6g
1-Benzylidene-4-(2,3-dioxo-1-(piperidin-1-
ylmethyl)indolin-5-yl) semicarbazide 6b
Yield 80%, Mp 228 ꢀC; Rf 0.82; IR (per cm): 3364, 3045, 1732,
1548, 1126, 1057; 1H NMR (DMSO-d6): 2.32 (t, J = 6.0 Hz, 4H, –
CH2 morpholine); 3.40 (t, J = 5.8 Hz, 4H, –CH2 morpholine); 4.34
(s, 2H, –CH2); 7.01–7.89 (m, 7H, Ar-CH); 8.14 (s, 1H, Ar-NH); 8.80
(s, 1H, CH=N); 9.96 (s, 1H, CONH); MS (EI) m ⁄ z: 425 [M+]; Anal.
Calcd for C21H20FN5O4: C, 59.29; H, 4.74; N, 16.46. Found: C,
59.14; H, 4.76; N, 16.52.
Yield 664%, Mp 238 ꢀC; Rf 0.84; IR (per cm): 3392, 3024, 1728,
1548; 1H NMR (DMSO-d6): 1.16–1.60 (m, 6H, –CH2 piperidine);
2.28–2.52 (m, 4H, –CH2 piperidine); 4.24 (s, 2H, –CH2); 7.12–7.92
(m, 8H, Ar-H); 8.32 (s, 1H, Ar-NH); 8.46 (s, 1H, CH=N); 9.88 (s, 1H,
CONH); MS (EI) m ⁄ z: 405 [M+]; Anal. Calcd for C22H23N5O3: C,
65.17; H, 5.72; N, 17.27. Found: C, 65.32; H, 5.74; N, 17.33.
1-(4-Fluorobenzylidene)-4-(2,3-dioxo-1-(piperidin-
1-ylmethyl)indolin-5-yl) semicarbazide 6h
Yield 76%, Mp 269 ꢀC; Rf 0.78; IR (per cm): 3335, 3054, 1728,
1541, 1148; 1H NMR (DMSO-d6): 1.38–1.80 (m, 6H, –CH2 piperi-
dine); 2.39–3.20 (m, 4H, –CH2 piperidine); 4.32 (s, 2H, –CH2);
6.84–7.82 (m, 7H, Ar-H); 8.22 (s, 1H, Ar-NH); 8.68 (s, 1H, CH=N);
9.82 (s, 1H, CONH); MS (EI) m ⁄ z: 423 [M+]; Anal. Calcd for
C22H22FN5O3: C, 62.40; H, 5.24; N, 16.54. Found: C, 62.62; H,
5.26; N, 16.60.
1-(4-Chlorobenzylidene)-4-(1-
(morpholinomethyl)-2,3-dioxoindolin-5-yl)
semicarbazide 6c
Yield 78%, Mp 242 ꢀC; Rf 0.92; IR (per cm): 3384, 3092, 1718, 1552,
1
1084, 748; H NMR (DMSO-d6): 2.40 (t, J = 5.8 Hz, 4H, –CH2 morphol-
ine); 3.30 (t, J = 5.6 Hz, 4H, –CH2 morpholine); 4.24 (s, 2H, –CH2);
6.90–7.92 (m, 7H, Ar-CH); 8.30 (s, 1H, Ar-NH); 8.54 (s, 1H, CH=N); 9.84
(s, 1H, CONH); MS (EI) m⁄z: 444 [M + 2]; Anal. Calcd for C21H20ClN5O4:
C, 57.08; H, 4.56; N, 15.85. Found: C, 57.18; H, 4.58; N, 15.91.
1-(4-Nitrobenzylidene)-4-(1-(morpholinomethyl)-
2,3-dioxoindolin-5-yl) semicarbazide 6i
1-(4-Chlorobenzylidene)-4-(2,3-dioxo-1-
(piperidin-1-ylmethyl)indolin-5-yl) semicarbazide
6d
Yield 60%, Mp 278 ꢀC; Rf 0.94; IR (per cm): 3336, 3051, 1733,
1567, 1548, 1356, 1038; 1H NMR (DMSO-d6): 2.32 (t, J = 6.0 Hz,
4H, –CH2 morpholine); 3.48 (t, J = 5.6 Hz, 4H, –CH2 morpholine);
4.32 (s, 2H, –CH2); 6.76–7.84 (m, 7H, Ar-CH); 8.30 (s, 1H, Ar-NH);
8.62 (s, 1H, CH=N); 9.91 (s, 1H, CONH); MS (EI) m ⁄ z: 452 [M+];
Anal. Calcd for C21H20N6O6: C, 55.75; H, 4.46; N, 18.58. Found: C,
55.94; H, 4.48; N, 18.65.
Yield 72%, Mp 287 ꢀC; Rf 0.86; IR (per cm): 3336, 2916, 1728,
1
1544, 778; H NMR (DMSO-d6): 1.20–1.62 (m, 6H, –CH2 piperidine);
2.42–2.74 (m, 4H, –CH2 piperidine); 4.32 (s, 2H, –CH2); 6.92–7.88
(m, 7H, Ar-H); 8.30 (s, 1H, Ar-NH); 8.60 (s, 1H, CH=N); 9.65 (s, 1H,
CONH); MS (EI) m ⁄ z: 442 [M + 2]; Anal. Calcd for C22H22ClN5O3: C,
60.07; H, 5.04; N, 15.92. Found: C, 60.22; H, 5.02; N, 15.98.
1-(4-Nitrobenzylidene)-4-(2,3-dioxo-1-(piperidin-
1-ylmethyl)indolin-5-yl) semicarbazide 6j
Yield 64%, Mp 258 ꢀC;Rf 0.74; IR (per cm): 3354, 3023, 1740,
1574, 1566, 1344; 1H NMR (DMSO-d6): 1.32–1.69 (m, 6H, –CH2
piperidine); 2.28–2.72 (m, 4H, –CH2 piperidine); 4.34 (s, 2H, –CH2);
1-(2-Chlorobenzylidene)-4-(1-(morpholinomethyl)-
2,3-dioxoindolin-5-yl) semicarbazide 6e
Yield 68%, Mp 218 ꢀC; Rf 0.90; IR (per cm): 3352, 3034, 1722,
1
1575, 1050, 755; H NMR (DMSO-d6): 2.44 (t, J = 5.8 Hz, 4H, –CH2
4
Chem Biol Drug Des 2012

Mice and Rats Acute Seizure Models
6.74–7.82 (m, 7H, Ar-H); 8.30 (s, 1H, Ar-NH); 8.52 (s, 1H, CH=N);
9.92 (s, 1H, CONH); MS (EI) m ⁄ z: 450 [M+]; Anal. Calcd for
C22H22N6O5: C, 58.66; H, 4.92; N, 18.66. Found: C, 58.84; H, 4.94;
N, 18.73.
1-(4-Hydroxybenzylidene)-4-(2,3-dioxo-1-(piperidin
-1-ylmethyl)indolin-5-yl) semicarbazide 6p
Yield 72%, Mp 192 ꢀC; Rf 0.81; IR (per cm): 3516, 3366, 2984,
1750, 1555; 1H NMR (DMSO-d6): 1.29–1.71 (m, 6H, –CH2 piperi-
dine); 2.52–2.94 (m, 4H, –CH2 piperidine); 4.54 (s, 2H, –CH2); 5.02
(s, 1H, OH); 7.12–7.98 (m, 7H, Ar-H); 8.36 (s, 1H, Ar-NH); 8.62 (s,
1H, CH=N); 9.88 (s, 1H, CONH); MS (EI) m ⁄ z: 421 [M+]; Anal. Calcd
for C22H23N5O4: C, 62.70; H, 5.50; N, 16.62. Found: C, 62.92; H,
5.48; N, 16.56.
1-(3-Nitrobenzylidene)-4-(1-(morpholinomethyl)-
2,3-dioxoindolin-5-yl) semicarbazide 6k
Yield 72%, Mp 287 ꢀC; Rf 0.70; IR (per cm): 3358, 3042, 1726,
1572, 1560, 1382, 1047; 1H NMR (DMSO-d6): 2.48 (t, J = 5.8 Hz,
4H, –CH2 morpholine); 3.32 (t, J = 5.6 Hz, 4H, –CH2 morpholine);
4.34 (s, 2H, –CH2); 7.12–7.87 (m, 7H, Ar-CH); 8.36 (s, 1H, Ar-NH);
8.68 (s, 1H, CH=N); 9.96 (s, 1H, CONH); MS (EI) m ⁄ z: 452 [M+];
Anal. Calcd for C21H20N6O6: C, 55.75; H, 4.46; N, 18.58. Found: C,
55.90; H, 4.44; N, 18.64.
1-(4-Methoxybenzylidene)-4-(1-(morpholinomethyl)-
2,3-dioxoindolin-5-yl) semicarbazide 6q
Yield 76%, Mp 216 ꢀC; Rf 0.79; IR (per cm): 3384, 3028, 1744,
1
1546, 1032; H NMR (DMSO-d6): 2.50 (t, J = 6.0 Hz, 4H, –CH2 mor-
pholine); 2.84 (s, 3H, OCH3); 3.42 (t, J = 5.6 Hz, 4H, –CH2 morphol-
ine); 4.16 (s, 2H, –CH2); 6.86–7.90 (m, 7H, Ar-CH); 8.29 (s, 1H,
Ar-NH); 8.52 (s, 1H, CH=N); 9.82 (s, 1H, CONH); MS (EI) m ⁄ z: 437
[M+]; Anal. Calcd for C22H23N5O5: C, 60.40; H, 5.30; N, 16.01. Found:
C, 60.24; H, 5.28; N, 16.07.
1-(3-Nitrobenzylidene)-4-(2,3-dioxo-1-(piperidin-
1-ylmethyl)indolin-5-yl) semicarbazide 6l
Yield 70%, Mp 296 ꢀC; Rf 0.88; IR (per cm): 3367, 2994, 1730, 1564,
1
1556, 1372; H NMR (DMSO-d6): 1.29–1.60 (m, 6H, –CH2 piperidine);
2.44–2.86 (m, 4H, –CH2 piperidine); 4.44 (s, 2H, –CH2); 6.85–7.86 (m,
7H, Ar-H); 8.22 (s, 1H, Ar-NH); 8.62 (s, 1H, CH=N); 9.84 (s, 1H, CONH);
MS (EI) m ⁄ z: 450 [M+]; Anal. Calcd for C22H22N6O5: C, 58.66; H, 4.92;
N, 18.66. Found: C, 58.83; H, 4.94; N, 18.70.
1-(4-Methoxybenzylidene)-4-(2,3-dioxo-1-(piperidin-
1-ylmethyl)indolin-5-yl) semicarbazide 6r
Yield 69%, Mp 235 ꢀC; Rf 0.83; IR (per cm): 3346, 2986, 1718, 1552;
1H NMR (DMSO-d6): 1.29–1.62 (m, 6H, –CH2 piperidine); 2.22–2.50
(m, 4H, –CH2 piperidine); 2.72 (s, 3H, OCH3); 4.38 (s, 2H, –CH2); 6.89–
7.76 (m, 7H, Ar-H); 8.22 (s, 1H, Ar-NH); 8.52 (s, 1H, CH=N); 9.88 (s,
1H, CONH); MS (EI) m ⁄ z: 435 [M+]; Anal. Calcd for C23H25N5O4: C,
63.44; H, 5.79; N, 16.08. Found: C, 63.62; H, 5.80; N, 16.13.
1-(4-Bromobenzylidene)-4-(1-(morpholinomethyl)
-2,3-dioxoindolin-5-yl) semicarbazide 6m
Yield 62%, Mp 222 ꢀC; Rf 0.64; IR (per cm): 3341, 3036, 1729,
1
1550, 1046, 682; H NMR (DMSO-d6): 2.44 (t, J = 6.0 Hz, 4H, –CH2
morpholine); 3.26 (t, J = 5.8 Hz, 4H, –CH2 morpholine); 4.50 (s, 2H,
–CH2); 7.04–7.76 (m, 7H, Ar-CH); 8.40 (s, 1H, Ar-NH); 8.75 (s, 1H,
CH=N); 9.90 (s, 1H, CONH); MS (EI) m ⁄ z: 488 [M + 2]; Anal. Calcd
for C21H20BrN5O4: C, 51.86; H, 4.15; N, 14.40. Found: C, 51.98; H,
4.17; N, 14.46.
1-(4-Methylbenzylidene)-4-(1-
(morpholinomethyl)-2,3-dioxoindolin-5-yl)
semicarbazide 6s
Yield 71%, Mp 198 ꢀC; Rf 0.77; IR (per cm): 3372, 3036, 1740,
1548, 1036; 1H NMR (DMSO-d6): 2.32 (s, 3H, CH3); 2.63 (t,
J = 5.8 Hz, 4H, –CH2 morpholine); 3.42 (t, J = 5.6 Hz, 4H, –CH2
morpholine); 4.20 (s, 2H, –CH2); 6.82–7.84 (m, 7H, Ar-CH); 8.18 (s,
1H, Ar-NH); 8.38 (s, 1H, CH=N); 9.98 (s, 1H, CONH); MS (EI) m ⁄ z:
421 [M+]; Anal. Calcd for C22H23N5O4: C, 62.70; H, 5.50; N, 16.62.
Found: C, 62.48; H, 5.48; N, 16.67.
1-(4-Bromobenzylidene)-4-(2,3-dioxo-1-(piperidin-1
-ylmethyl)indolin-5-yl) semicarbazide 6n
Yield 76%, Mp 237 ꢀC; Rf 0.62; IR (per cm): 3356, 3043, 1736,
1546, 658; 1H NMR (DMSO-d6): 1.20–1.58 (m, 6H, –CH2 piperi-
dine); 2.26–2.72 (m, 4H, –CH2 piperidine); 4.24 (s, 2H, –CH2);
6.88–7.74 (m, 7H, Ar-H); 8.28 (s, 1H, Ar-NH); 8.51 (s, 1H, CH=N);
9.90 (s, 1H, CONH); MS (EI) m ⁄ z: 486 [M + 2]; Anal. Calcd for
C22H22BrN5O3: C, 54.56; H, 4.58; N, 14.46. Found: C, 54.68; H,
4.60; N, 14.51.
1-(4-Methylbenzylidene)-4-(2,3-dioxo-1-(piperidin-
1-ylmethyl)indolin-5-yl) semicarbazide 6t
Yield 66%, Mp 210 ꢀC; Rf 0.71; IR (per cm): 3368, 3031, 1741,
1
1564; H NMR (DMSO-d6): 1.34–1.69 (m, 6H, –CH2 piperidine); 2.24
(s, 3H, CH3); 2.64–2.98 (m, 4H, –CH2 piperidine); 4.46 (s, 2H, –CH2);
7.08–7.94 (m, 7H, Ar-H); 8.14 (s, 1H, Ar-NH); 8.47 (s, 1H, CH=N);
9.94 (s, 1H, CONH); MS (EI) m ⁄ z: 419 [M+]; Anal. Calcd for
C23H25N5O3: C, 65.85; H, 6.01; N, 16.70. Found: C, 66.04; H, 5.99;
N, 16.77.
1-(4-Hydroxybenzylidene)-4-(1-(morpholinomethyl)
-2,3-dioxoindolin-5-yl) semicarbazide 6o
Yield 60%, Mp 208 ꢀC; Rf 0.75; IR (per cm): 3498, 3351, 3048,
1739, 1538, 1034; 1H NMR (DMSO-d6): 2.44 (t, J = 6.2 Hz, 4H, –
CH2 morpholine); 3.26 (t, J = 5.8 Hz, 4H, –CH2 morpholine); 4.38 (s,
2H, –CH2); 5.14 (s, 1H, OH); 6.87–7.88 (m, 7H, Ar-CH); 8.42 (s, 1H,
Ar-NH); 8.69 (s, 1H, CH=N); 9.92 (s, 1H, CONH); MS (EI) m ⁄ z: 423
[M+]; Anal. Calcd for C21H21N5O5: C, 59.57; H, 5.00; N, 16.54. Found:
C, 59.78; H, 5.02; N, 16.59.
Pharmacology
All the compounds' antiepileptic activity was screened by maximal
electroshock test (MES) and subcutaneous pentylenetetrazole
Chem Biol Drug Des 2012
5

Prakash et al.
seizure test (scPTZ) method. Maximal electroshock test model
employs an electrical stimulus, whereas scPTZ model utilizes chemi-
cally induced myoclonic seizures and is capable of recognizing the
agents that act by raising the seizure threshold (20). All the animal
experiments were approved by Institutional Animal Ethical Commit-
tee (IAEC ⁄ III ⁄ 22 ⁄ BCOP ⁄ 2011). Compounds are administered by i.p
injection in mice at dose levels of 30, 100, and 300 mg ⁄ kg at 0.5
and 4 h. The acute neurological toxicity (NT) was determined by ro-
torod model. To further differentiate the activity between rodent
species, the most active compounds were tested in rats.
The introduction of nitro group in compound 1 was confirmed by
the appearance of two strong bands in its IR spectrum at 1570
and 1348 per cm, which arises because of asymmetrical and sym-
metrical stretching vibrations. The formation of compounds 2a
and 2b was confirmed by the appearance of singlet between d
1
4.28 and d 4.40 respectively for two protons in its H-NMR spec-
tra, which might be accountable for CH2 group connecting the
isatin core with secondary amines through Mannich reaction. The
conversion of nitro to amino group in compounds 3a and 3b can
be acknowledged by strong absorption peak at 3386 and 3398
per cm in IR because of N-H Stretching. The IR spectrum of title
compound 6g over the 3045, 1732, and 1548 per cm, which was
assigned to Ar-H, carbonyl, and azomethine (CH=N) stretching. It
is the 1H-NMR spectrum that showed a singlet at d 8.54 ppm
because of the proton attached to the imine carbon. A group of
signals appeared between d 6.87 and 7.89 ppm corresponds to
Ar-H protons.
The maximal electroshock test
For the MES, a drop of anesthetic and electrolyte solution was
applied to the eyes of the animal before placement of the corneal
electrodes. The electrical stimulus in the MES test was 50 mA,
60 Hz, for mice and 150 mA, 60 Hz, for rats was applied for
0.2 seconds by an apparatus similar to that described by Woodbury
and Davenport (21). For the abolition of the hindleg, tonic extensor
component of the seizure was used as the endpoint. Mice were ini-
tially tested with different doses of 30, 100, and 300 mg ⁄ kg, while
rats were initially screened at a fixed dose of 30 mg ⁄ kg by oral
route.
Biological result and discussions
The maximal electroshock test
The antiepileptic activity of all the synthesized compounds has been
shown in Table 1. Compounds 6c, 6d, 6g, 6h, and 6m showed
protection at 30 mg ⁄ kg, and compounds 6e, 6f, 6j, 6k, and 6n
showed protection at 100 mg ⁄ kg, while compounds 6o, 6p, 6r,
and 6s exhibited protection at 300 mg ⁄ kg after 0.5 h. Compounds
6c, 6g, and 6i showed protection against the MES screen at
30 mg ⁄ kg, and compounds 6a, 6d, 6h, 6j, 6l, and 6m showed
protection at a dose level of 100 mg ⁄ kg, while compounds 6e, 6f,
6n–6r, and 6t showed protection at a dose level of 300 mg ⁄ kg
after 4 h. The most active compounds 6c and 6g showed activity
both at 0.5 and 4 h at dose level of 30 mg ⁄ kg indicating that they
have rapid onset, more effective and long acting. Similarly, com-
pounds 6d, 6h, and 6m were also found to be more effective but
short acting, which was evidenced by the requirement of higher
dose (100 mg ⁄ kg) at 4 h. The compound 6j showed activity both at
0.5 and 4 h period at a dose level of 100 mg ⁄ kg, indicating that
compound was effective and long acting. Compounds 6e, 6f, and
6n demonstrated protection in mice at 100 mg ⁄ kg and 300 mg ⁄ kg
after 0.5 and 4.0 h, respectively. The compounds 6o, 6p, and 6r
showed activity both at 0.5 and 4 h period at a of dose of
300 mg ⁄ kg, indicating that compounds are less potent and long act-
ing. Compound 6i showed activity at 30 mg ⁄ kg, and 6a and 6l
showed activity at 100 mg ⁄ kg only at 4 h, indicating that com-
pounds have sustained activity. Compound 6k showed activity only
at 0.5 h at a dose level of 100 mg ⁄ kg, indicating that compound
has rapid onset and shorter duration of action. Compounds 6q, 6s,
and 6t exhibited some protection in either of the tested time-
The subcutaneous pentylenetetrazole seizure
test
Subcutaneous injection of the convulsant pentylenetetrazole pro-
duces clonic seizures in laboratory animals. Animals were pretreat-
ed with various doses of the test compound by i.p. injection.
Pentylenetetrazole is injected in the midline of the neck. The ani-
mals were placed in isolation cages to minimize stress and were
observed for the next 30 min for the presence or absence of a sei-
zure. An episode of clonic spasms, approximately 3–5 seconds, of
the fore and ⁄ or hindlimbs, jaws, or vibrissae is taken as the
endpoint. Animals that do not meet this criterion are considered
protected.
Acute toxicity-minimal motor impairment
To assess a compound's undesirable side effects (toxicity), animals
were monitored for overt signs of impaired neurological or muscular
function. In mice, the rotorod (22) procedure is used to disclose
minimal muscular (MMI) or neurological impairment. When a mouse
is placed on a rod that rotates at a speed of 6 rpm, the animal can
maintain its equilibrium for long periods of time. The animal is con-
sidered toxic if it falls off this rotating rod three times during a
1-min period. In addition to MMI, animals may exhibit a circular or
zigzag gait, abnormal body posture and spread of the legs, tremors,
hyperactivity, lack of exploratory behavior, somnolence, stupor, cata-
lepsy, loss of placing response, and changes in muscle tone.
points, but
a poor onset was observed at a higher dose
(300 mg ⁄ kg). In contrast, compound 6b was not active at both the
tested dose levels.
Results and Discussion
The subcutaneous pentylenetetrazole seizure
test
Synthesis
The structures of the synthesized compounds were confirmed by
spectral (IR, 1H-NMR, and mass) and elemental analyses data.
In the chemoshock investigation, most of the compounds showed
moderate to good antiepileptic activity. The compounds 6g, 6i,
6
Chem Biol Drug Des 2012

Mice and Rats Acute Seizure Models
Table 1: Antiepileptic activity and neurotoxicity of compounds
6a–6t administered intraperitoneally to mice
Table 2: Antiepileptic activity and toxicity of compounds 6c,
6d, 6g, 6h, 6i, and 6m administered orally (30 mg ⁄ kg) to rats
MESa screening
scPTZb
NTc screening
MESa
screening
Compound
0.25 hc
0.5 hc
1 hc
2 hc
4 hc
TOXb
Compound
0.5 hd
4.0 hd
0.5 hd
4.0 hd
0.5 hd
4.0 hd
6c
6d
1 ⁄ 4
1 ⁄ 4
1 ⁄ 4
1 ⁄ 4
0 ⁄ 4
0 ⁄ 4
1 ⁄ 4
2 ⁄ 4
2 ⁄ 4
2 ⁄ 4
2 ⁄ 4
0 ⁄ 4
2 ⁄ 4
4 ⁄ 4
2 ⁄ 4
2 ⁄ 4
2 ⁄ 4
2 ⁄ 4
0 ⁄ 4
1 ⁄ 4
3 ⁄ 4
4 ⁄ 4
3 ⁄ 4
4 ⁄ 4
3 ⁄ 4
0 ⁄ 4
1 ⁄ 4
3 ⁄ 4
4 ⁄ 4
3 ⁄ 4
4 ⁄ 4
3 ⁄ 4
2 ⁄ 4
2 ⁄ 4
3 ⁄ 4
0 ⁄ 4 (–)d
0 ⁄ 4 (–)d
0 ⁄ 4 (–)d
0 ⁄ 4 (–)d
0 ⁄ 4 (–)d
0 ⁄ 4 (–)d
0 ⁄ 4 (–)d
6a
6b
–
–
100
–
–
300
–
300
–
–
–
–
–
–
30
ND
–
–
300
–
30
ND
–
–
–
6g
6h
6c
6d
30
30
100
100
30
30
100
300
300
30
6i
6e
6m
Phenytoin
6f
6g
–
–
–
100
300
100
300
–
300
300
300
300
–
–
300
–
300
–
–
–
–
–
–
–
–
300
–
300
300
–
300
300
300
ND
ND
ND
ND
ND
100
–
MES, maximal electroshock test.
6h
6i
30
–
100
100
–
100
30
100
–
100
100
300
300
300
300
300
–
aMaximal electroshock test (dose of 30 mg ⁄ kg was administrated; the data
indicate number of rats protected ⁄ number of rats tested).
bNeurotoxicity (number of rats protected ⁄ number of rats tested).
cTime after drug administration.
6j
6k
300
300
–
100
100
–
ND
ND
ND
ND
ND
100
–
6l
6m
–
d(–) No neurotoxicity at dose tested.
30
100
300
100
300
–
–
–
–
–
6n
6o
100
300
300
–
300
300
–
dose administered (300 mg ⁄ kg). The compound 6a showed neu-
rotoxicity at 30 mg ⁄ kg. Compounds 6m and 6n showed neuro-
toxicity at a dose of 100 mg ⁄ kg at 0.5 h and 300 mg ⁄ kg at
4 h, while the remaining compounds 6e, 6h, 6j, 6k, and 6o
showed neurotoxicity at a dose level of 300 mg ⁄ kg at 0.5 h
and ⁄ or 4 h period.
6p
6q
6r
6s
6t
300
30
–
–
–
300
Phenytoin
Ethosuximide
30
–
100
MES, maximal electroshock test; scPTZ, subcutaneous pentylenetetrazole
seizure test; ND, not determined.
Antiepileptic activity of selected compounds on
rats by oral administration
aMaximal electroshock test (administered intraperitoneally to mice at doses
ranging from 30 to 300 mg ⁄ kg).
As can be seen from these data in Table 2, the most active com-
pounds are 6c and 6g, which protected 100% (4 ⁄ 4) of rats at
time-points 2 and 4 h, 50% (2 ⁄ 4) at 0.5 and 1 h, and 25% (1 ⁄ 4) at
0.25 h. This molecule was more active and showed longer duration
of satisfactory action than phenytoin. The other compounds 6d and
6h were moderately effective in rat MES oral screen and protected
75% (3 ⁄ 4) at 2 and 4 h, 50% (2 ⁄ 4) at 0.5 and 1 h, while 25%
(1 ⁄ 4) at 0.25 h. Compounds 6i and 6m showed 50% (2 ⁄ 4) protec-
tion at 4 h. The compound 6m also showed 50% (2 ⁄ 4) protection
at 0.5 h and 25% (1 ⁄ 4) of protection at 1 and 2 h. All derivatives
tested were non-neurotoxic when given orally. The inhibition of
electrically induced seizures that is characteristic for phenytoin and
phenytoin-like drugs may indicate the influence of compounds on
voltage depended Na+ channels as the most reasonable mechanism
of antiepileptic action.
bSubcutaneous pentylenetetrazole test (administered intraperitoneally to
mice at doses ranging from 30 to 300 mg ⁄ kg).
cNeurotoxicity (administered intraperitoneally to mice at doses ranging from
30 to 300 mg ⁄ kg).
dTime of test after drug administration.
The sign '–' (endash) represents an absence of activity at maximum dose
administered (300 mg ⁄ kg).
6m, and 6o showed activity at a dose level of 100 mg ⁄ kg, while
compounds 6b, 6d, 6h, 6j, 6n, and 6p showed activity at dose
of 300 mg ⁄ kg at 0.5 h. Only compounds 6g, 6h, 6i, 6j, 6m, and
6o showed protection at dose of 300 mg ⁄ kg even at 4 h. Com-
pounds 6h and 6j have shown activity at dose of 300 mg ⁄ kg at
0.5 and 4 h, indicating that these compounds have moderate
activity. The compounds 6g, 6i, 6m, and 6o were found to be
most potent with rapid onset and intermediate duration of action.
None of the compounds were found active at dose level of
30 mg ⁄ kg. Compounds 6a, 6c, 6e, 6f, 6k, 6l, 6q, 6r, 6s, and
6t showed no response at both tested dose levels, while
compounds 6b, 6d, 6n, and 6p were found to be effective with
short duration of action.
Structure activity relationships analysis
With the results of the preliminary anticonvulsant screening and on
correlating the structures of the sample candidates 6a–6t with their
biological activity, it has been observed that most active were the
morpholine substituted derivatives. When morpholine ring was
replaced by piperidine, the anticonvulsant activity was altered in
both the models and has exhibited less-significant effect. The
increases in antiepileptic activity of test candidates with morpholine
derivatives may be attributed to the presence of extra one electro-
negative oxygen atom on morpholine (which is absent in piperidine)
ring that might be accountable for additional hydrogen bonding with
the binding site. Taking into consideration the influence of the sub-
stituents at the distal phenyl ring appeared to greatly influence the
Neurotoxicity screen
All the compounds evaluated for its neurotoxicity except 6b
and 6p–6t because of its poor response in antiepileptic
activity. In neurotoxic screening, compounds 6c, 6d, 6f, 6g,
6i, and 6l were not found to be neurotoxic at the maximum
Chem Biol Drug Des 2012
7

Prakash et al.
antiepileptic activity. From the results, it seems that the most favor-
able was the presence of highly electro negative chloro, fluoro, or
bromo atoms at para position of the distal phenyl ring (6c, 6d, 6g,
6h, and 6m). The ortho substituted derivatives (6e and 6f) were
less active. Presence of nitro substituent at para position (6i and 6j)
offered some protection, while meta substituents (6k and 6l) did
not greatly influence the antiepileptic activity. Three electron-releas-
ing derivatives containing p-OH, p-CH3, and p-OCH3 (6o–6t) were
also prepared. However, these compounds exhibited reduced anti-
convulsant activities in MES screening than the corresponding halo-
gen-substituted derivatives. Introduction of the –OH (6o) was found
to show better activity only in scPTZ. The other substituents like
methoxy (6q and 6r) and methyl (6s and 6t) showed very poor
activity in MES and completely devoid of activity in scPTZ.
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Conclusions and Future Directions
By choosing appropriate experimental conditions, we were able to
synthesize 1-(substituted benzylidene)-4-(1-(morpholino ⁄ piperidino
methyl)-2,3-dioxoindolin-5-yl) semicarbazide derivatives 6a–6t in
good yields, and the same was evaluated in animal models of epi-
lepsy and neurotoxicity. All the title compounds satisfied the
needed pharmacophoric structural requirement that existed in the
well-established AEDs. The most active compounds 6c, 6d, 6g,
6h, and 6m revealed protection against the MES at a dose of
30 mg ⁄ kg in 0.5 h after i.p administration, while compound 6g, 6i,
6m, and 6o showed significant antiepileptic activity in scPTZ
model. Further, compounds 6c, 6d, 6g, 6h, 6i, and 6m were
given orally to rats at a dose of 30 mg ⁄ kg. Only compounds 6c
and 6g showed better antiepileptic activity in oral dose than phe-
nytoin. Among the twenty derivatives, 6g was found to be the
most potent in the different models of epilepsy, suggesting a poten-
tial broad-spectrum anticonvulsant activity. It also showed marked
lower neurotoxicity, and therefore offered a higher protective index.
Therefore, such compounds would represent a fruitful matrix for the
development of a new class of antiepileptic agents and would
deserve further investigation and derivatization as a promising
scaffold.
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Conflict of interest
The authors report no conflict of interest. The authors alone are
responsible for the content and writing of the article.
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