Synthesis and anticonvulsant properties of 1-(amino-N-arylmethanethio)-3- (1-substituted benzyl-2, 3-dioxoindolin-5-yl) urea derivatives

Article abstract of DOI:10.1016/j.ejmech.2011.03.004

Various 1-(amino-N-arylmethanethio)-3-(1-substituted benzyl-2, 3-dioxoindolin-5-yl) urea (5a-p) were designed keeping in view the structural requirements suggested in the pharmacophore model for anticonvulsant activity. Their in vivo anticonvulsant screenings were performed by two most adopted seizure models, maximal electroshock seizure (MES) and subcutaneous pentylenetetrazole (scPTZ). Compound 5f was found active in MES screening while compounds 5h, 5i, 5k and 5l showed significant anticonvulsant activity in both the screenings and were devoid of any neurotoxicity. Compound 5h and 5i showed marked protection at 300 mg/kg against MES and scPTZ screening. Compound 5i also showed protection against MES screening at the dose of 100 mg/kg. In 6 Hz screening these two compounds showed significant protection and emerged as lead compounds for future investigations.

Full text of DOI:10.1016/j.ejmech.2011.03.004

European Journal of Medicinal Chemistry 46 (2011) 2236e2242
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and anticonvulsant properties of 1-(amino-N-arylmethanethio)-3-
(1-substituted benzyl-2, 3-dioxoindolin-5-yl) urea derivatives
,
a
b
Nadeem Siddiqui a , M. Shamsher Alam , James P. Stables
*
a Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Hamdard University, Hamdard Nagar, New Delhi 110062, India
b Epilepsy Branch, National Institute of Neurological Disorders and Stroke, NIH, Rockville, MD 20892, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Various 1-(amino-N-arylmethanethio)-3-(1-substituted benzyl-2, 3-dioxoindolin-5-yl) urea (5aep) were
designed keeping in view the structural requirements suggested in the pharmacophore model for
anticonvulsant activity. Their in vivo anticonvulsant screenings were performed by two most adopted
seizure models, maximal electroshock seizure (MES) and subcutaneous pentylenetetrazole (scPTZ).
Compound 5f was found active in MES screening while compounds 5h, 5i, 5k and 5l showed significant
anticonvulsant activity in both the screenings and were devoid of any neurotoxicity. Compound 5h and
5i showed marked protection at 300 mg/kg against MES and scPTZ screening. Compound 5i also showed
protection against MES screening at the dose of 100 mg/kg. In 6 Hz screening these two compounds
showed significant protection and emerged as lead compounds for future investigations.
Ó 2011 Elsevier Masson SAS. All rights reserved.
Received 26 August 2010
Received in revised form
7 February 2011
Accepted 2 March 2011
Available online 10 March 2011
Keywords:
Isatin
Anticonvulsants
Neurotoxicity
1. Introduction
3CL protease inhibitor. [16] These interesting properties prompted
many efforts toward the synthesis and pharmacological screening
Epilepsy is a collective term given to a group of syndromes that
involve spontaneous, intermittent, abnormal electrical activity in the
brain, which manifest as seizures. It has been estimated that about
20% of the patients with epilepsy using the first generation of anti-
epileptic drugs (Phenobarbital, Phenytoin, Carbamazepine, Sodium
valproate and Diazepam) were not able to acquire adequate control of
seizure [1]. In fact, epilepsy is the third most frequent neurological
disorder encountered in the elderly after cerebrovascular disease and
dementia [2]. There is continuing demand for new anticonvulsant
agents as several of the currently available antiepileptic drugs (AED)
have been associated with severe side effects and fail to control
seizures in about 30% of epileptic patients [3e5]. The search for
antiepileptic agents with more selectivity and lower toxicity
continues to be an area of investigation in medicinal chemistry.
Study revealed that Isatin is a privileged lead molecule for
designing potential bioactive agents, and its derivatives have been
shown to possess a broad spectrum of bioactivity as many of which
were assessed anti-HIV, [6] antiviral, [7] anti-tumor, [8e10]
antifungal, [11,12] anti-angiogenic, [13] anticonvulsants, [14] anti-
Parkinson’s disease therapeutic, [15] and effective SARS coronavirus
of isatin derivatives.
Considering extensive applications of Isatin moiety in medicinal
chemistry and in continuation of our ongoing project biologically
active heterocycles [17e21], an attempt has been made to synthe-
size amino-N-arylmethanethio urea derivatives containing Isatin
moiety and evaluate for their in vivo screening.
2. Chemistry
The synthesis of the titled compounds (5aep) was carried out as
presented in Scheme 1.
5-Nitroindoline-2, 3-dione (1) has been prepared by the re-
fluxing of indoline-2, 3-dione with a mixture of 95e100% sulfuric
acid and 70% nitric acid in water bath at 60 ꢀC for 1 h. Compounds
(2a,b) were synthesized by stirring a mixture of compound (1),
anhydrous potassium carbonate in acetone and substituted benzyl
chloride. Compounds (2a,b) get reduced to 5-amino-1-substituted
benzylindoline-2, 3-dione (3a,b) on heating with a mixture of iron
powder and hydrochloric acid. Compounds (3a,b) on stirring with
sodium cyanate and glacial acetic acid, get converted to its urea
derivatives (4a,b). Finally titled compounds 1-(amino-N-arylme-
thanethio)-3-(1-substituted benzyl-2, 3-dioxoindolin-5-yl) urea
(5aep) were prepared on refluxing different substituted phenyl-
isothiocyanates with compounds (4a,b). The physicochemical
parameters of the synthesized compounds are presented in Table 1.
* Corresponding author. Tel.: þ911126059688x5639; fax: þ911126059688x5307.
E-mail
addresses:
nadeems_03@yahoo.co.in,
nadeems_03@rediffmail.com
(N. Siddiqui).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.03.004

N. Siddiqui et al. / European Journal of Medicinal Chemistry 46 (2011) 2236e2242
2237
O
O
O
O2N
O2N
Nitration
Benzylation
O
O
O
R
HNO3/H2SO4
N
N
N
CH2
H
CH2Cl
R
H
(1)
(2a-b)
n
Fe/HCl
O
Red
H2N
O
R
N
CH2
(3a-b)
GAA NaOCN
O
O
H2N C HN
O
N
CH2
(4a-b)
R
Ethanol
N=C=S
R1
S
O
O
HN C HN C HN
R1
O
R
N
CH2
(5a-p)
R = H, 2-Cl ; R1 = H, 2-Cl, 2-CH3, 3-CH3, 4-CH3, 2-OCH3,
3-OCH3, 4-OCH3
Scheme 1. Synthetic route to the synthesized compounds (5aep).
Table 1
Physicochemical properties of titled compounds: (5aep).
d
Compd. No.
R
R1
Mol. Formulaa
Log Pb found (calculated)
Rfc (Rm
)
Elemental analysis (%)e
C
H
N
S
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
H
H
H
H
H
H
H
H
2Cl
2Cl
2Cl
2Cl
2Cl
2Cl
2Cl
2Cl
H
2Cl
C23 H18 N4 O3
C23 H17 Cl N4 O3
S
2.48 (2.57)
3.01 (3.29)
2.98 (3.07)
2.92 (3.07)
2.96 (3.07)
2.38 (2.49)
2.40 (2.49)
2.39 (2.49)
3.18 (3.29)
3.98 (4.00)
3.70 (3.79)
3.72 (3.79)
3.73 (3.79)
3.18 (3.21)
3.19 (3.21)
3.24 (3.21)
0.94 (ꢁ1.19)
0.83 (ꢁ0.68)
0.77 (ꢁ0.52)
0.94 (ꢁ1.19)
0.77 (ꢁ0.52)
0.83 (ꢁ0.68)
0.94 (ꢁ1.19)
0.94 (ꢁ1.19)
0.78 (ꢁ0.54)
0.76 (ꢁ0.50)
0.90 (ꢁ0.95)
0.91 (ꢁ1.00)
0.94 (ꢁ1.19)
0.79 (ꢁ0.57)
0.82 (ꢁ0.65)
0.80 (ꢁ0.60)
64.17
59.42
64.85
64.85
64.85
62.60
62.60
62.60
59.42
55.32
60.18
60.18
60.18
58.24
58.24
58.24
4.21
3.69
4.54
4.54
4.54
4.38
4.38
4.38
3.69
3.23
4.00
4.00
4.00
3.87
3.87
3.87
13.01
12.05
12.60
12.60
12.60
12.17
12.17
12.17
12.05
11.22
11.70
11.70
11.70
11.32
11.32
11.32
7.45
6.90
7.21
7.21
7.21
6.96
6.96
6.96
6.90
6.42
6.70
6.70
6.70
6.48
6.48
6.48
S
S
S
S
S
S
S
S
S
2CH3
3CH3
4CH3
2OCH3
3OCH3
4OCH3
H
C24 H20 N4 O3
C24 H20 N4 O3
C24 H20 N4 O3
C24 H20 N4 O4
C24 H20 N4 O4
C24 H20 N4 O4
S
S
S
S
S
S
C23 H17 Cl N4 O3
C23 H16 Cl2 N4 O3
2Cl
2CH3
3CH3
4CH3
2OCH3
3OCH3
4OCH3
C24 H19 Cl N4 O3
C24 H19 Cl N4 O3
C24 H19 Cl N4 O3
C24 H19 Cl N4 O4
C24 H19 Cl N4 O4
C24 H19 Cl N4 O4
A logarithmic function of Rf-value was also calculated.
a
Solvent of crystallizationeEthanol.
b
Log P was determined by octanol: phosphate buffer method; CLog P was calculated using software ChemOffice 6.0.
Solvent system e Toluene: Ethyl acetate: Formic acid (5:4:1).
c
d
e
Rm ¼ log(1 ꢁ 1/Rf).
Experimental value of Elemental analysis for C, H, N and S were within ꢂ0.4% of the theoretical value.

2238
N. Siddiqui et al. / European Journal of Medicinal Chemistry 46 (2011) 2236e2242
Table 3
The structures and purity of the final compounds were confirmed
on the basis of spectral and elemental analyses and the data were
within ꢂ0.4% of the theoretical values.
Anticonvulsant screening project (ASP): Test 7 results in mice; 6 Hz assay (Dose
100 mg/kg).
Compd.
Test
Time (h)
0.25
0.5
1.0
2.0
4.0
3. Pharmacology
5h
6Hza
TOXb
1/4
0/4
1/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
The pharmacological testing of all the final compounds have
been performed by National Institute of Neurological Disorders and
Stroke (NINDS), USA under Anticonvulsant screening program
(ASP), following the protocol adopted by the Antiepileptic Drug
Development (ADD) program [22]. All the compounds prepared
herein were screened for their potential in vivo biological activities
such as anticonvulsant and minimal motor impairment test. The
Phase I pharmacological screening comprised of MES, scPTZ and
neurotoxicity. Further, some of the selected compounds were
tested for their activity at 6 Hz model.
5i
6Hza
TOXb
3/4
0/4
2/4
0/4
2/4
0/4
0/4
0/4
0/4
0/4
a
Qualitative 6 Hz assay (number of animal protected/number of animals tested).
Rotorod toxicity (number of animals exhibiting toxicity/number of animals
b
tested) endpoint: unable to maintain balance on rotorod.
screening. The most potent anticonvulsant compounds found to be
were 5f, 5h, 5i, 5k and 5l and interestingly all these compounds
were devoid of any neurotoxicity. In MES test, compound 5i was
active at dose of 100 mg//kg (1/3 animal protected) 4.0 h after
administration; interestingly compound 5i was also active at dose
of 300 mg//kg (1/1 & 1/1 animals protected) after 0.5 h & 4.0 h. In
300 mg/kg dose, compounds 5f and 5h were also found to be active
(1/1 and 1/1 animals protected) 0.5 h and (1/1 and 1/1 animals
protected) 4.0 h after administration. In 300 mg/kg dose,
compounds 5k and 5l were active (1/1 and 1/1 animals protected)
4.0 h after administration.
In scPTZ screening compounds 5h and 5i were active at dose
of 300 mg//kg (1/5 and 2/5 animals protected) 0.5 h after adminis-
tration. Compounds 5k and 5l also showed protection at dose of
300 mg//kg(1/1 and 1/1 animalsprotected) 4.0 h afteradministration.
In the neurotoxicity screening, compounds 5f, 5h, 5i, 5k and 5l
that were found to be potent were devoid of minimal motor
impairment at any doses. Rest all the compounds, although were
not potent showed lack of any neurotoxicity.
The compounds 5h and 5i that displayed a marked MES as well
as scPTZ activity in mice at the dose of 100 & 300 mg/kg i.p. were
evaluated for 6 Hz assay in mice, Test 7 results (Table 3). Compound
5h showed protection in the Minimal clonic seizure (6 Hz) test at
100 mg/kg (i.p. dose) (1/4 & 1/4 animals protected) 0.25 & 0.5 h
after administration. In 6 Hz test compound 5i showed protection
at 100 mg/kg (i.p. dose) (3/4, 2/4 & 2/4 animals protected) 0.25, 0.5
& 1.0 h after administration. Interestingly it lacks any toxicity in
given dose in any time intervals.
4. Results and discussion
4.1. Anticonvulsant activity
Phase I studies of the investigated compounds (5aep) involved
three tests: maximal electroshock seizure (MES), subcutaneous
Pentylenetetrazole (scPTZ), and rotorod test for neurological
toxicity. For the MES assay, experimental compound is adminis-
tered to sets of one, three and one animals at three respective doses
(30, 100, and 300 mg/kg) at both 0.5 h and 4.0 h time periods. The
similar standard for dosing and time is used for the scPTZ testing.
All animals were observed for potential neurological deficit at each
dose and time period.
For the identification of anticonvulsant activity in mice, test
compounds were administered intraperitoneally and challenged by
maximal electroshock and subcutaneous Pentylenetetrazole.
Compounds found to be effective in these seizure challenges are
generally regarded to be potentially useful candidates in treatment
of partial, generalized and even absence seizures. Neurotoxicity of
the test compounds was determined using the rotorod toxicity test
(TOX). The results of the in vivo tests are summarized in Table 2.
Titled compounds 1-(amino-N-arylmethanethio)-3-(1-sub-
stituted benzyl-2, 3-dioxoindolin-5-yl) urea (5aep) showed some
protection against MES and scPTZ seizures in mice in Phase 1
Table 2
Anticonvulsant screening project (ASP): Phase 1- test results in mice
Test 1 results e Intraperitoneal injection in mice
Compd.
MES screening
30 mg/kg
scPTZ screening
30 mg/kg
Minimal motor impairment screening
100 mg/kg
300 mg/kg
100 mg/kg
300 mg/kg
30 mg/kg
100 mg/kg
300 mg/kg
0.5 h
4.0 h
0.5 h
4.0 h
0.5 h
4.0 h
0.5 h
4.0 h
0.5 h
4.0 h
0.5 h
4.0 h
0.5 h
4.0 h
0.5 h
4.0 h
0.5 h
4.0 h
(N/F)
(N/F)
(N/F)
(N/F)
(N/F)
(N/F)
(N/F)
(N/F)
(N/F)
(N/F)
(N/F)
(N/F)
(N/F)
(N/F)
(N/F)
(N/F)
(N/F)
(N/F)
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
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
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
1/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/1
0/1
0/1
0/1
0/1
1/1
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
1/1
0/1
1/1
1/1
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
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
1/5
2/5
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
0/4
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
0/8
0/8
0/8
0/8
0/8
0/8
0/8
0/8
0/8
0/8
0/8
0/8
0/8
0/8
0/8
0/8
0/4
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
N/F ¼ Number of animals active or toxic over the number tested.

N. Siddiqui et al. / European Journal of Medicinal Chemistry 46 (2011) 2236e2242
2239
D
N
HBD
HBD
HBD
O
S
O
C
O
A
R
HN
A
HN
C
HN
HN
R
NH
O
D
N
CH2
O
D
A
H
O
N
H
R
HBD
Compounds (5a-p)
Phenytoin
Carbamazepine
Fig. 1. Anticonvulsant agents showing the required pharmacophoric elements.
These results suggest that thioamide and amide as two hydrogen
bonding domains (HBD), Nitrogen of Isatin as electron donar (D),
unsubstitution or 2-chloro substitution as electron withdrawing
agent in hydrophobic domain (A) and unsubstitution or methoxy/
methyl substitution aselectrondonating agentin distal hydrophobic
domain (R) made the compound potent in the MES screen at 100 &
300 mg/kg doses, scPTZ screen at 100 & 300 mg/kg (i.p. dose) in mice
as well as minimal clonic seizure (6 Hz) test at 100 mg/kg (i.p. dose)
in mice. Anticonvulsant agents having the above required pharma-
cophoric elements have been shown in Fig. 1.
solvents and reagents were of LR grade and purified before their
use. The silica gel G (160e120 mesh) used for analytical chroma-
tography (TLC) was obtained from E. Merck India Ltd. Two solvent
systems were used Benzene: Acetone (8:2) and (6:4), Toluene:
Ethyl acetate: Formic acid (5:4:1). All the melting points were
determined in open glass capillary using Kjeldahl flask containing
paraffin and are uncorrected. Both 1H NMR and 13C NMR spectra
were taken on a Bruker 400 Ultra shieldÔ (400 MHz) NMR spec-
trometer in DMSO-d6/CDCl3 using tetramethylsilane [(CH3)4Si] as
internal standard. Chemical shift (d) are expressed in ppm. All the
Fourier transform infra-red (FT-IR) spectra were recorded in KBr
pellets on a Jasco FT/IR 410 spectrometer. Elemental analyses were
realized on a PerkineElmer model 240c analyzer and were within
ꢂ0.4% of the theoretical values.
4.2. Log P determination
The dependence of biological activity in the set of congeneric
agents or lipophilic character has been shown in many types of drug
action in particular, the reports by Lien and co-workers indicate that
the anticonvulsant activity of different types of compounds were
correlated with lipophilicity [23]. However, it has been observed
that the maximum potency of the drugs that act on the central
nervous system (CNS) is obtained with congeners having an
optimum lipophilicity (log Po). In this study, we attempted to
correlate the anticonvulsant activity of synthesized compounds
with their log P value. The experimental log P values were deter-
mined using octanol-phosphate buffer method and the calculated
log P values were taken from the software ChemOffice 6.0 and the
results are shown in Table 1. Most of the synthesized compounds
met the criteria for lipophilicty and showed good anticonvulsant
activity with lesser neurotoxicity. Due to their higher lipophilicity
they are expected to have rapid onset and shorter duration of action.
6.1.1. General procedure for the synthesis of titled compounds
(5aep)
6.1.1.1. 5-Nitroindoline-2, 3-dione (1). Compound indoline-2,
3-dione (48.5 g, 0.33 mol) was added into a mixture of 50 g (35 mL,
0.5 mol) of conc. nitric acid and 74 g (40 mL, 0.75 mol) conc. sulfuric
acid with frequent shaking in 500-mL round bottomed flask,
keeping the mixture cool by immersing the flask in ice cold water.
After adding all indoline-2, 3-dione, flask was fitted with reflux
condenser and the mixture was heated on water bath maintaining
the temperature at 60 ꢀC for 1 h to obtain the desired compound
5-nitroindoline-2, 3-dione (1). Content was then poured into
a beaker containing 500 mL cold water, stirred in order to wash out
as much acid from the desired compound. When compound (1)
completely settled to the bottom, the upper acid liquor was
completely poured off from the mixture. Then the residual liquid
was transferred to the separating funnel and shaked vigorously
with about 50 mL of water. Bottom layer was collected, dried with
anhydrous calcium chloride and finally filtered to obtain the pure
compound (1). Yield 62%, mp 230 ꢀC. IR (KBr) cmꢁ1: 3306 (NHstr.),
5. Conclusions
Pharmacophore model for anticonvulsant 1-(amino-N-arylme-
thanethio)-3-(1-substituted benzyl-2, 3-dioxoindolin-5-yl) urea
has been established on the basis of results of pharmacological,
physicochemical, distance mapping and three-dimensional struc-
tural investigations. In this model, the presence of thioamide and
amide (HBD) fragment is essential for activity. Beside this, all of the
compounds contained two hydrophobic units (A & R aryl rings) as
a distal binding site and Isatin as electron donar (D). Five
compounds showed better anticonvulsant activity and among
these compounds 5h and 5i emerged as a lead compounds in the
series when they were subjected to preliminary anticonvulsant
screenings. They also showed marked lower neurotoxicity and,
therefore, a higher protective index. These can be regarded as
strong candidates for future investigations.
3015 (Ar-CHstr.), 1725 (C]O, Isatin), 1563 (NOstr.-sym.), 1325 (NOstr.
asym.), 1H NMR (CDCl3)
(ppm): 7.12e7.89 (m, 3H, Ar-H), 9.53 (s,
1H, NH-Isatin); 13C NMR (DMSO-d6)
(ppm): 107.34, 114.46, 127.24,
129.83, 144.24, 152.73, 160.26, 178.45.
-
d
d
6.1.1.2. 5-Nitro-1-substituted benzylindoline-2, 3-dione (2a,b). To
a solution of compound 1 (0.01 mol) and anhydrous potassium
carbonate (0.01 mol) in acetone (30 ml), substituted benzyl chlo-
ride (0.01 mol) was added drop wise. The mixture was stirred at
room temp for about 8 h. The mixture was then poured into water
and extracted with ethyl acetate, dried over sodium sulfate anhy-
drous and concentrated under vacuum to give the pure compound.
Desired compound 2a,b was finally recrystallized with ethanol.
6.1.1.2.1. 5-Nitro-1-benzylindoline-2, 3-dione (2a). Yield 60%, mp
260 ꢀC. IR (KBr) cmꢁ1: 3063 (Ar-CHstr.), 2825 (CHstr.), 1723 (C]O,
Isatin), 1548 (NOstr.-sym.), 1352 (NOstr.-asym.), 1H NMR (CDCl3)
6. Experimental protocols
6.1. Chemistry
d
(ppm): 4.23 (s, 2H, CH2-Ar), 7.01e7.78 (m, 8H, Ar-H), 13C NMR
(DMSO-d6) (ppm): 48.32, 120.43, 122.78, 124.26, 124.28, 125.10,
127.25, 129.23, 129.54, 130.13, 138.52, 143.27, 156.38, 158.34, 183.35.
d
The chemicals used were obtained from various chemical units
E. Merck India Ltd., CDH, and s.d. Fine Chem. and Qualigens. These

2240
N. Siddiqui et al. / European Journal of Medicinal Chemistry 46 (2011) 2236e2242
6.1.1.2.2. 1-(2-Chlorobenzyl)-5-nitroindoline-2, 3-dione (2b). Yield
(NHstr.), 2999 (NHstr.), 2955 (NHstr.), 2918 (Ar-CHstr.), 2850 (CHstr.),
62%, mp 275 ꢀC. IR (KBr) cmꢁ1: 3037 (Ar-CHstr.), 2818 (CHstr.), 1690
1758 (C]O, Isatin), 1458 (C]O, amide), 1427 (C]Sstr.); 1H NMR
(C]O, Isatin), 1540 (NOstr.-sym.), 1348 (NOstr.-asym.), 725 (CeCl);
(CDCl3) d (ppm): 3.85 (s, 2H, CH2-Ar), 7.00e7.89 (m, 10H, Ar-H),
6.97e7.96 (m, 3H, ArH-Isatin), 7.98 (s, 1H, NH, D2O exchangeable),
1H NMR (CDCl3)
d
(ppm): 4.15 (s, 2H, CH2-Ar), 7.23e7.85 (m, 7H, Ar-
H); 13C NMR (DMSO-d6)
d
(ppm): 47.45, 122.48, 123.36, 124.24,
9.08 (s, 1H, NH-Ar, D2O exchangeable), 10.72 (s, 1H, NH-Isatin, D2O
126.48, 127.12, 129.37, 129.01, 131.27, 132.37, 137.72, 142.67, 157.38,
158.92, 183.35.
exchangeable); 13C NMR (DMSO-d6)
d (ppm): 46.21, 109.51, 115.82,
119.54, 121.56, 123.62, 124.21, 124.28, 126.24, 126.47, 128.02, 128.21,
128.34, 129.71, 129.73, 134.72, 137.21, 141.32, 149.51, 152.32, 158.84,
182.36, 183.67; MS: m/z 429(M ꢁ 1).
6.1.1.3. 5-Amino-1-substituted benzylindoline-2, 3-dione (3a,b). Com-
pound 2a,b (0.01 mol) and iron powder (0.01 mol) was placed in
round bottomed flask containing 200 mL ethanol. The mixture was
heated on oil bath till temperature attained to 80e85 ꢀC. Then 4 mL
of HCl (1.2 M) was added and content was further stirred for 4 h
while maintaining the temperature. Content was then filtered at
reduced pressure and the filtrate was adjusted to pH 7e8 using
NaHCO3 to get the precipitate of compound 3a,b. It was then
recrystallized with ethanol.
6.1.1.5.2. 1-{Amino-N-(2-chlorophenyl) methanethio}-3-(1-benzyl-
2, 3-dioxoindolin-5-yl) urea (5b). Yield 59%, mp 230 ꢀC. IR (KBr)
cmꢁ1: 3525 (NHstr.), 3095 (NHstr.), 3055 (NHstr.), 2958 (Ar-CHstr.),
2950 (CHstr.),1752 (C]O, Isatin),1658 (C]O, amide),1627 (C]Sstr.),
665 (CeCl); 1H NMR (CDCl3)
d (ppm): 3.82 (s, 2H, CH2-Ar),
7.25e7.88 (m, 9H, Ar-H), 6.93e7.68 (m, 3H, ArH-Isatin), 8.96 (s, 1H,
NH, D2O exchangeable), 9.78 (s, 1H, NH-Ar, D2O exchangeable),
10.89 (s, 1H, NH-Isatin, D2O exchangeable); 13C NMR (DMSO-d6)
6.1.1.3.1. 5-Amino-1-benzylindoline-2, 3-dione (3a). Yield 71%,
d (ppm): 45.25, 108.32, 114.72, 118.57, 120.45, 122.67, 123.27, 124.51,
126.32, 126.48, 127.14, 128.16, 128.21, 129.32, 129.58, 133.74, 136.46,
140.27, 148.12, 151.37, 157.59, 181.46, 182.64.
mp 265 ꢀC. IR (KBr) cmꢁ1: 3265 (NHstr.), 3125 (Ar-CHstr.), 2848
(CHstr.), 1764 (C]O, Isatin); 1H NMR (CDCl3)
d
(ppm): 3.76 (s, 1H,
NH), 4.15 (s, 2H, CH2-Ar), 7.21e7.89 (m, 8H, Ar-H); 13C NMR (DMSO-
d6) (ppm): 46.27, 121.46, 123.59, 124.38, 124.75, 125.79, 126.28,
6.1.1.5.3. 1-(Amino-N-o-tolylmethanethio)-3-(1-benzyl-2, 3-diox-
oindolin-5-yl) urea (5c). Yield 77%, mp 120 ꢀC. IR (KBr) cmꢁ1: Yield
59%, mp 230 ꢀC. IR (KBr) cmꢁ1: 3386 (NHstr.), 3268 (NHstr.), 3071
(NHstr.), 2924 (Ar-CHstr.), 2854 (CHstr.), 1707 (C]O, Isatin), 1640 (C]
d
129.38, 129.41, 130.27, 138.56, 144.28, 157.48, 159.14, 182.65.
6.1.1.3.2. 1-(2-Chlorobenzyl)-5-aminoindoline-2,3-dione (3b). Yield
68%, mp 272 ꢀC. IR (KBr) cmꢁ1: 3356 (NHstr.), 3234 (Ar-CHstr.), 2858
O, amide), 1606 (C]Sstr.); 1H NMR (CDCl3)
d (ppm): 2.27 (s, 3H,
(CHstr.), 1726 (C]O, Isatin), 738 (CeCl); 1H NMR (CDCl3)
d
(ppm): 3.65
(s, 1H, NH), 4.02 (s, 2H, CH2-Ar), 6.84e7.85 (m, 7H, Ar-H); 13C NMR
(DMSO-d6) (ppm): 45.48, 121.26, 123.35, 124.32, 124.59, 125.24,
CH3), 3.80 (s, 2H, CH2-Ar), 7.01e7.80 (m, 9H, Ar-H), 6.98e7.93 (m,
3H, ArH-Isatin), 8.83 (s, 1H, NH, D2O exchangeable), 9.09 (s, 1H, NH-
Ar, D2O exchangeable), 9.88 (s, 1H, NH-Isatin, D2O exchangeable);
d
126.29, 129.47, 129.46, 130.28, 138.18, 144.29, 157.27, 159.17, 184.15.
13C NMR (DMSO-d6)
d (ppm): 17.68, 46.54, 107.24, 115.47, 118.32,
121.52, 123.52, 123.87, 124.42, 125.24, 125.24, 126.84, 127.01, 127.54,
128.34, 129.28, 132.34, 135.67, 141.74, 148.12, 150.23, 157.59, 181.14,
181.54.
6.1.1.5.4. 1-(Amino-N-m-tolylmethanethio)-3-(1-benzyl-2, 3-dioxo-
indolin-5-yl) urea (5d). Yield 47%, mp 110 ꢀC. IR (KBr) cmꢁ1: 3356
(NHstr.), 3224 (NHstr.), 3053 (NHstr.), 2901 (Ar-CHstr.), 2824 (CHstr.),
1704 (C]O, Isatin), 1612 (C]O, amide), 1604 (C]Sstr.); 1H NMR
6.1.1.4. 1-(1-Substituted benzyl-2, 3-dioxoindolin-5-yl) urea (4a,b). Com-
pound 3a,b (0.01 mol) was dissolved in 10 mL glacial acetic acid and
was diluted to 100 mL in a conical flask. To it a solution of NaOCN
(0.01 mol) and 50 mL warm water were added with continuous
shaking. Content was allowed to stand for 30 min, and then cooled
in ice, allowed to stand for further 30 min, filtered at the pump,
washed with water and dried at 100 ꢀC. The desired compound 4a,b
were formed and recrystallized with ethanol.
(CDCl3)
d (ppm): 2.22 (s, 3H, CH3), 3.76 (s, 2H, CH2-Ar), 7.23e7.89
(m, 9H, Ar-H), 6.35e7.98 (m, 3H, ArH-Isatin), 8.87 (s, 1H, NH, D2O
6.1.1.4.1. 1-(1-Benzyl-2, 3-dioxoindolin-5-yl) urea (4a). Yield
74%, mp 282 ꢀC. IR (KBr) cmꢁ1: 3325 (NHstr.), 3137 (Ar-CHstr.), 2839
(CHstr.), 1785 (C]O, Isatin), 1625 (C]O, Urea); 1H NMR (CDCl3)
exchangeable), 9.64 (s, 1H, NH-Ar, D2O exchangeable), 9.69 (s, 1H,
NH-Isatin, D2O exchangeable); 13C NMR (DMSO-d6)
d (ppm): 16.54,
45.64, 106.24, 114.26, 117.56, 122.24, 122.42, 123.48, 123.84, 124.34,
125.12, 125.23, 128.45, 128.63, 128.85, 129.02, 131.34, 134.37, 142.14,
147.15, 151.22, 156.57, 182.17, 183.54.
6.1.1.5.5. 1-(Amino-N-p-tolylmethanethio)-3-(1-benzyl-2, 3-diox-
oindolin-5-yl) urea (5e). Yield 63%, mp 57 ꢀC. IR (KBr) cmꢁ1: 3352
(NHstr.), 3202 (NHstr.), 3053 (NHstr.), 2942 (Ar-CHstr.), 2814 (CHstr.),
1715 (C]O, Isatin), 1614 (C]O, amide), 1602 (C]Sstr.); 1H NMR
d
(ppm): 4.26 (s, 1H, NH-Urea, D2O exchangeable), 4.85 (s, 2H, CH2-
Ar), 6.57e7.83 (m, 8H, Ar-H), 8.42 (s, 1H, NH-Isatin, D2O
exchangeable); 13C NMR (DMSO-d6)
(ppm): 49.25, 121.28, 122.38,
d
124.27, 124.36, 125.48, 127.38, 129.25, 130.42, 132.21, 137.52, 144.47,
156.43, 157.44, 162.18, 184.15.
6.1.1.4.2. 1-[1-(2-Chlorobenzyl)-2,3-dioxoindolin-5-yl]urea (4b). Yield
78%, mp 295 ꢀC. IR (KBr) cmꢁ1: 3364 (NHstr.), 3128 (Ar-CHstr.), 2848
(CHstr.), 1728 (C]O, Isatin), 1612 (C]O, Urea), 710 (CeCl); 1H NMR
(CDCl3)
d (ppm): 2.12 (s, 3H, CH3), 3.89 (s, 2H, CH2-Ar), 7.23e7.78
(m, 9H, Ar-H), 6.89e7.69 (m, 3H, ArH-Isatin), 8.89 (s, 1H, NH, D2O
(CDCl3)
2H, CH2-Ar), 6.78e7.84 (m, 7H, Ar-H), 8.37 (s, 1H, NH-Isatin, D2O
exchangeable); 13C NMR (DMSO-d6)
(ppm): 52.67, 120.46, 123.78,
124.25, 124.94, 125.28, 127.17, 129.37, 130.51, 134.61, 138.92, 146.42,
156.74, 159.47, 168.37, 186.35.
d
(ppm): 4.12 (s, 1H, NH-Urea, D2O exchangeable), 4.63 (s,
exchangeable), 9.35 (s, 1H, NH-Ar, D2O exchangeable), 9.97 (s, 1H,
NH-Isatin), D2O exchangeable; 13C NMR (DMSO-d6)
d (ppm): 16.24,
d
44.14, 105.27, 113.54, 118.03, 121.84, 122.64, 123.21, 123.87, 125.24,
125.42, 126.24, 127.26, 128.74, 128.87, 129.46, 130.41, 133.37, 143.14,
146.54, 151.84, 156.14, 183.74, 184.34.
6.1.1.5.6. 1-[Amino-N-(2-methoxyphenyl)methanethio]-3-(1-benzyl-2,
6.1.1.5. 1-(Amino-N-arylmethanethio)-3-(1-substituted benzyl-2, 3-
dioxoindolin-5-yl) urea (5aep). A mixture of compound 4a,b
(0.01 mol) and substituted phenylisothiocyanates (0.01 mol) in
20 mL of absolute ethanol was refluxed for 5e6 h. After comple-
tion of the reaction, mixture was concentrated and kept overnight
at room temperature. The needle shaped crystals thus obtained
were purified by repeated washing with petroleum ether. Desired
titled compounds 5aep were recrystallized with ethanol.
3-dioxoindolin-5-yl) urea (5f). Yield 87%, mp 132 ꢀC. IR (KBr) cmꢁ1
:
3390 (NHstr.), 2924 (NHstr.), 2853 (NHstr.), 2828 (Ar-CHstr.), 2815
(CHstr.), 1710 (C]O, Isatin), 1692 (C]O, amide), 1639 (C]Sstr.), 1115
(OCH3); 1H NMR (CDCl3)
d (ppm): 2.49 (s, 3H, OCH3), 3.36 (s, 2H,
CH2-Ar), 6.73e7.82 (m, 9H, Ar-H), 6.70e7.97 (m, 3H, ArH-Isatin),
7.98 (s, 1H, NH, D2O exchangeable), 9.01 (s, 1H, NH-Ar, D2O
exchangeable),10.59 (s,1H, NH-Isatin, D2O exchangeable); 13C NMR
(DMSO-d6)
d (ppm): 42.56, 56.42, 108.24, 114.34, 120.05, 122.74,
6.1.1.5.1. 1-(Amino-N-phenylmethanethio)-3-(1-benzyl-2, 3-diox-
122.81, 123.01, 123.86, 125.27, 125.84, 126.22, 127.24, 128.07, 128.14,
129.01, 130.37, 135.31, 145.04, 148.53, 150.54, 159.17, 182.72, 186.37.
oindolin-5-yl) urea (5a). Yield 67%, mp 300 ꢀC. IR (KBr) cmꢁ1: 3505

N. Siddiqui et al. / European Journal of Medicinal Chemistry 46 (2011) 2236e2242
2241
6.1.1.5.7. 1-[Amino-N-(3-methoxyphenyl)methanethio]-3-(1-
benzyl-2, 3-dioxoindolin-5-yl) urea (5g). Yield 68%, mp 205 ꢀC. IR
(KBr) cmꢁ1: 3385 (NHstr.), 2945 (NHstr.), 2885 (NHstr.), 2824 (Ar-
CHstr.), 2815 (CHstr.), 1701 (C]O, Isatin), 1654 (C]O, amide), 1621
121.32, 123.23, 123.54, 124.14, 125.24, 126.64, 127.24, 127.65, 128.24,
129.34, 130.57, 133.71, 143.64, 144.25, 145.74, 150.64, 185.37, 188.32.
6.1.1.5.13. 1-[1-(2-Chlorobenzyl)-2,3-dioxoindolin-5-yl]-3-(amino-
N-p-tolylmethanethio) urea (5m). Yield 57%, mp 260 ꢀC. IR (KBr)
cmꢁ1: 3453 (NHstr.), 3254 (NHstr.), 3068 (NHstr.), 2941 (Ar-CHstr.),
2935 (CHstr.),1782 (C]O, Isatin),1635 (C]O, amide),1595 (C]Sstr.),
(C]Sstr.), 1118 (OCH3); 1H NMR (CDCl3)
d (ppm): 3.12 (s, 3H, OCH3),
3.57 (s, 2H, CH2-Ar), 6.58e7.87 (m, 9H, Ar-H), 6.86e7.69 (m, 3H,
ArH-Isatin), 7.69 (s, 1H, NH, D2O exchangeable), 9.38 (s, 1H, NH-Ar,
638 (CeCl); 1H NMR (CDCl3)
d (ppm): 2.35 (s, 3H, CH3), 3.58 (s, 2H,
D2O exchangeable), 10.48 (s, 1H, NH-Isatin, D2O exchangeable); 13
C
CH2-Ar), 7.34e8.68 (m, 8H, Ar-H), 7.24e8.58 (m, 3H, ArH-Isatin),
8.79 (s, 1H, NH, D2O exchangeable), 10.48 (s, 1H, NH-Ar, D2O
exchangeable), 11.27 (s, 1H, NH-Isatin, D2O exchangeable); 13C NMR
NMR (DMSO-d6)
d (ppm): 40.25, 54.34, 106.45, 116.37, 121.95,
122.34, 122.94, 123.45, 123.83, 125.28, 125.35, 126.25, 127.54,
127.64, 128.54, 129.21, 130.34, 134.62, 144.03, 146.54, 148.24, 158.13,
181.77, 185.34.
(DMSO-d6)
d (ppm): 16.22, 55.73, 96.56, 111.15, 117.34, 121.24,
122.33,123.53, 123.64, 124.04, 125.45, 126.85, 127.32, 127.58, 128.21,
129.02, 130.37, 132.51, 144.14, 144.27, 145.64, 151.65, 186.17, 189.31.
6.1.1.5.14. 1-[1-(2-Chlorobenzyl)-2,3-dioxoindolin-5-yl]-3-[amino-
N-(2-methoxyphenyl)methanethio] urea (5n). Yield 75%, mp 123 ꢀC.
IR (KBr) cmꢁ1: 3454 (NHstr.), 3394 (NHstr.), 3080 (NHstr.), 2924 (Ar-
CHstr.), 2912 (CHstr.),1712 (C]O, Isatin),1692 (C]O, amide),1633 (C]
6.1.1.5.8. 1-[Amino-N-(4-methoxyphenyl)methanethio]-3-(1-benzyl-
2, 3-dioxoindolin-5-yl) urea (5h). Yield 55%, mp 206 ꢀC. IR (KBr)
cmꢁ1: 3384 (NHstr.), 2936 (NHstr.), 2895 (NHstr.), 2858 (Ar-CHstr.),
2814 (CHstr.), 1712 (C]O, Isatin), 1643 (C]O, amide), 1612 (C]Sstr.),
1165 (OCH3); 1H NMR (CDCl3)
d (ppm): 3.56 (s, 3H, OCH3), 3.89 (s,
2H, CH2-Ar), 6.45e7.89 (m, 9H, Ar-H), 6.58e7.76 (m, 3H, ArH-Isa-
tin), 7.57 (s, 1H, NH, D2O exchangeable), 9.54 (s, 1H, NH-Ar, D2O
exchangeable),10.37 (s, 1H, NH-Isatin, D2O exchangeable); 13C NMR
Sstr.), 1175 (OCH3), 709 (CeCl); 1H NMR (CDCl3)
d (ppm): 2.50 (s, 3H,
OCH3), 3.40 (s, 2H, CH2-Ar), 7.22e8.36 (m, 8H, Ar-H), 6.58e8.76 (m,
3H, ArH-Isatin), 8.82 (s,1H, NH, D2O exchangeable), 9.19 (s,1H, NH-Ar,
D2O exchangeable), 11.11 (s, 1H, NH-Isatin, D2O exchangeable); 13C
(DMSO-d6)
d (ppm): 38.24, 56.38, 104.46, 117.32, 121.04, 122.32,
122.74, 123.36, 123.82, 125.11, 125.34, 126.26, 127.01, 127.65, 128.24,
129.45, 130.34, 134.14, 142.01, 145.53, 146.24, 156.37, 182.74, 183.35.
6.1.1.5.9. 1-[1-(2-Chlorobenzyl)-2,3-dioxoindolin-5-yl]-3-(amino-
N-phenylmethanethio) urea (5i). Yield 68%, mp 185 ꢀC. IR (KBr)
cmꢁ1: 3451 (NHstr.), 3259 (NHstr.), 3086 (NHstr.), 2924 (Ar-CHstr.),
2950 (CHstr.), 1753 (C]O, Isatin), 1631 (C]O, amide), 1527 (C]Sstr.),
NMR (DMSO-d6) d (ppm): 41.85, 54.73, 94.31, 110.14, 116.42, 120.27,
122.34, 123.25, 123.84, 124.21, 125.34, 126.51, 127.33, 127.57, 128.64,
129.31, 130.35, 133.54, 144.24, 144.28, 145.24, 153.65, 184.13, 188.41.
6.1.1.5.15. 1-[1-(2-Chlorobenzyl)-2,3-dioxoindolin-5-yl]-3-[amino-
N-(3-methoxyphenyl)methanethio] urea (5o). Yield 52%, mp 240 ꢀC.
IR (KBr) cmꢁ1: 3456 (NHstr.), 3342 (NHstr.), 3036 (NHstr.), 2985 (Ar-
CHstr.), 2957 (CHstr.), 1732 (C]O, Isatin), 1684 (C]O, amide), 1612
646 (CeCl); 1H NMR (CDCl3)
d (ppm): 3.37 (s, 2H, CH2-Ar),
6.50e7.97 (m, 9H, Ar-H), 6.63e7.96 (m, 3H, ArH-Isatin), 9.85 (s, 1H,
NH, D2O exchangeable), 10.39 (s, 1H, NH-Ar, D2O exchangeable),
10.67 (s, 1H, NH-Isatin, D2O exchangeable); 13C NMR (DMSO-d6)
(C]Sstr.), 1123 (OCH3), 704 (CeCl); 1H NMR (CDCl3)
d (ppm): 2.46
(s, 3H, OCH3), 3.57 (s, 2H, CH2-Ar), 7.56e8.79 (m,8H, Ar-H),
6.23e8.69 (m, 3H, ArH-Isatin), 8.58 (s, 1H, NH, D2O exchange-
able), 9.48 (s, 1H, NH-Ar, D2O exchangeable), 11.35 (s, 1H, NH-
d
(ppm): 51.37, 102.64, 115.56, 120.57, 121.24, 122.54, 123.76, 123.87,
124.31, 125.45, 126.37, 127.41, 127.85, 128.31, 129.01, 130.24, 133.42,
141.24, 144.57, 145.38, 154.08, 180.77, 186.65; MS: m/z 463(M ꢁ 1).
6.1.1.5.10. 1-[1-(2-Chlorobenzyl)-2,3-dioxoindolin-5-yl]-3-[amino-
N-(2-chlorophenyl)methanethio] urea (5j). Yield 68%, mp 185 ꢀC. IR
(KBr) cmꢁ1: 3456 (NHstr.), 3245 (NHstr.), 3062 (NHstr.), 2936 (Ar-
CHstr.), 2912 (CHstr.), 1723 (C]O, Isatin), 1648 (C]O, amide), 1623
Isatin, D2O exchangeable); 13C NMR (DMSO-d6)
d (ppm): 43.32,
56.53, 96.41, 112.16, 115.52, 121.67, 122.44, 123.45, 123.49, 124.20,
125.34, 126.21, 127.51, 127.58, 128.21, 129.34, 130.75, 132.64,
143.44, 144.35, 145.28, 154.67, 189.33, 189.47.
6.1.1.5.16. 1-[1-(2-Chlorobenzyl)-2,3-dioxoindolin-5-yl]-3-[amino-
N-(4-methoxyphenyl)methanethio] urea (5p). Yield 58%, mp 197 ꢀC.
IR (KBr) cmꢁ1: 3484 (NHstr.), 3336 (NHstr.), 3085 (NHstr.), 2942 (Ar-
CHstr.), 2910 (CHstr.),1714 (C]O, Isatin),1658 (C]O, amide),1623 (C]
(C]Sstr.), 684 (CeCl); 1H NMR (CDCl3)
d (ppm): 3.59 (s, 2H, CH2-Ar),
6.54e7.59 (m, 8H, Ar-H), 6.59e7.48 (m, 3H, ArH-Isatin), 9.39 (s, 1H,
NH, D2O exchangeable), 10.48 (s, 1H, NH-Ar, D2O exchangeable),
11.67 (s, 1H, NH-Isatin, D2O exchangeable); 13C NMR (DMSO-d6)
Sstr.), 1141 (OCH3), 712 (CeCl); 1H NMR (CDCl3)
d (ppm): 2.78 (s, 3H,
OCH3), 3.58 (s, 2H, CH2-Ar), 7.45e8.69 (m, 8H, Ar-H), 6.12e8.49 (m,
3H, ArH-Isatin), 8.37 (s,1H, NH, D2O exchangeable), 9.57 (s,1H, NH-Ar,
D2O exchangeable), 11.58 (s, 1H, NH-Isatin, D2O exchangeable); 13C
d
(ppm): 52.24, 98.54, 110.25, 118.53, 120.53, 121.14, 123.06, 123.32,
124.14, 125.27, 126.19, 127.38, 127.48, 128.51, 129.34, 130.25, 133.40,
142.27, 143.25, 145.37, 151.24, 182.37, 188.39; MS: m/z 463(M ꢁ 1).
6.1.1.5.11. 1-[1-(2-Chlorobenzyl)-2,3-dioxoindolin-5-yl]-3-(amino-
N-o-tolylmethanethio) urea (5k). Yield 82%, mp 215 ꢀC. IR (KBr)
cmꢁ1: 3456 (NHstr.), 3259 (NHstr.), 3090 (NHstr.), 2925 (Ar-CHstr.),
2728 (CHstr.), 1707 (C]O, Isatin),1633 (C]O, amide),1534 (C]Sstr.),
NMR (DMSO-d6)
d (ppm): 41.72, 55.43, 99.31, 113.56, 114.58, 122.68,
123.47, 123.65, 123.75, 124.32, 125.44, 126.31, 127.57, 127.59, 128.01,
129.44, 131.74, 132.74, 143.74, 144.39, 148.18, 154.68, 188.53, 189.45.
644 (CeCl); 1H NMR (CDCl3)
d (ppm): 2.50 (s, 3H, CH3), 3.31 (s, 2H,
6.2. Pharmacology
CH2-Ar), 7.16e8.20 (m, 8H, Ar-H), 7.16e8.23 (m, 3H, ArH-Isatin),
8.76 (s, 1H, NH, D2O exchangeable), 10.42 (s, 1H, NH-Ar, D2O
exchangeable), 11.52 (s, 1H, NH-Isatin, D2O exchangeable); 13C NMR
Anticonvulsant screening at the ASP includes the maximal
electroshock test (MES), the subcutaneous Pentylenetetrazole test
(scPTZ), and evaluations of toxicity (TOX). The data of each condi-
tion is presented as N/F, where N equals to the number of animals
protected and F equals to the number of animals tested. For test of
toxicity (TOX), N equals the number of animals displaying toxic
effects and F equals the number of animals tested.
(DMSO-d6)
d (ppm): 18.54, 55.32, 96.46, 108.12, 117.34, 121.34,
122.39, 123.01, 123.34, 124.44, 125.02, 126.67, 127.23, 127.65, 128.24,
129.03, 130.54, 132.41, 143.24, 143.85, 145.24, 152.65, 183.87, 189.34.
6.1.1.5.12. 1-[1-(2-Chlorobenzyl)-2,3-dioxoindolin-5-yl]-3-(amino-
N-m-tolylmethanethio) urea (5l). Yield 58%, mp 300 ꢀC. IR (KBr)
cmꢁ1: 3452 (NHstr.), 3212 (NHstr.), 3035 (NHstr.), 2985 (Ar-CHstr.),
2941 (CHstr.), 1732 (C]O, Isatin), 1645 (C]O, amide),1627 (C]Sstr.),
6.2.1. Anticonvulsant screening
656 (CeCl); 1H NMR (CDCl3)
d
(ppm): 2.69 (s, 3H, CH3), 3.78 (s, 2H,
6.2.1.1. Maximal electroshock test (MES test). The MES test is
a model, for example, generalized toniceclonic seizures and iden-
tifies compounds that prevent seizure spread and phenytoin is
considered as the prototypical in this model. For all tests based on
MES convulsions, 60 Hz of alternating current (50 mA in mice, 150
CH2-Ar), 7.34e8.53 (m, 8H, Ar-H), 7.25e8.57 (m, 3H, ArH-Isatin),
8.69 (s, 1H, NH, D2O exchangeable), 10.38 (s, 1H, NH-Ar, D2O
exchangeable), 11.12 (s, 1H, NH-Isatin, D2O exchangeable); 13C NMR
(DMSO-d6)
d (ppm): 17.32, 54.75, 92.32, 105.14, 116.94, 120.54,

2242
N. Siddiqui et al. / European Journal of Medicinal Chemistry 46 (2011) 2236e2242
in rats) is delivered for 0.2 s by corneal electrodes which have been
primed with an electrolyte solution containing an anesthetic agent
(0.5% tetracaine HCl). For Test 1, mice are tested at various intervals
following doses of 30, 100 and 300 mg/kg of test compound given
by i.p. injection of a volume of 0.01 mL/g. In Test 2, rats are tested
after a dose of 30 mg/kg (p.o.) in a volume of 0.04 mL/10 g. In test 7,
mice are tested after a dose of 100 mg/kg in qualitative 6 Hz assay.
An animal is considered ‘protected’ from MES-induced seizures
upon abolition of the hindlimb tonic extensor component of the
seizure. [24,25].
graduated tube and 5 mg of accurately weighed drug was added.
The mixture was then shaken with the help of mechanical shaker
for 24 h at room temperature. The mixture was then transferred to
a separating funnel and allowed to equilibrate for 6 h. The aqueous
and octanol phase were separated and filtered through membrane
filter and drug content in aqueous phase was analyzed by UV
spectroscopy.
Partition coefficient was calculated by:
PC ¼ (Ct ꢁ Ca)/Ca. PC ¼ Partition coefficient, Ct ¼ Concentration
of total drug, Ca ¼ Concentration of the drug in aqueous phase.
6.2.1.2. Subcutaneous pentylenetetrazole threshold test (scPTZ). The
scPTZ test detects the ability of test compounds to raise the seizure
threshold of an animal and thus protect it from exhibiting a clonic
seizure. Animals are pretreated with various doses of the test
compound (in the similar manner to the MES test, although a dose
of 50 mg/kg (p.o.) is the standard for Test 2 scPTZ). At the previously
determined TPE of the test compound, the dose of Pentylenete-
trazole which induce convulsions in 97% of animals (CD97: 85 mg/
kg mice; 70 mg/kg rats) is injected into a loose fold of skin in the
midline of the neck. The animals are placed in isolation cages to
minimize stress [26] and observed for the next 30 min for the
presence or absence of the seizure. An episode of clonic spasms,
approximately 3e5 s, of the forte and/or hindlimbs, jaws, or
vibrissae is taken as the endpoint. Animals which do not meet this
criterion are considered protected.
Acknowledgments
One of the authors (Md. Shamsher Alam) is thankful to UGC
(Govt. of India) for financial assistance in the form of Junior
Research Fellowship (JRF). We are also thankful to the Anticon-
vulsant Screening Project (ASP), National Institute of Neurological
Disorders and Stroke, NIH, Rockville, MD 20892-9523, USA. for
anticonvulsant testing.
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6.3. Log P determination
The partition coefficient between octanol and phosphate buffer
was determined at room temperature (29 ꢀC) using a procedure
described elsewhere [29]. Ten milliliter of octanol and 10 mL
phosphate buffer (pH ¼ 7.4) were taken in a glass stoppered