Molecular sieves promoted, ultrasound-mediated synthesis, biological evaluation and docking study of 3-(5-substituted-1,3,4-thiadiazol-2-ylimino)indolin-2-ones as a potential anticonvulsant agents

Article abstract of DOI:10.1007/s00044-015-1458-x

In this work, the combined use of ultrasonic energy and molecular sieves was investigated for synthesis of 3-(5-substituted-1,3,4-thiadiazol-2-ylimino)indolin-2-one derivatives 5(a-j). The equimolar quantities of 5-substituted-1,3,4-thiadiazol-2-amine and

Full text of DOI:10.1007/s00044-015-1458-x

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-015-1458-x
ORIGINAL RESEARCH
Molecular sieves promoted, ultrasound-mediated synthesis,
biological evaluation and docking study of 3-(5-substituted-1,3,4-
thiadiazol-2-ylimino)indolin-2-ones as a potential anticonvulsant
agents
Anna Pratima G. Nikalje1 Sameer I. Shaikh1 Firoz A. Kalam Khan1
•
•
•
Shoaib Shaikh2 Jaiprakash N. Sangshetti1
•
Received: 26 February 2015 / Accepted: 27 August 2015
Ó Springer Science+Business Media New York 2015
Abstract In this work, the combined use of ultrasonic
energy and molecular sieves was investigated for synthesis
of 3-(5-substituted-1,3,4-thiadiazol-2-ylimino)indolin-2-
one derivatives 5(a–j). The equimolar quantities of
5-substituted-1,3,4-thiadiazol-2-amine and isatin were
Keywords Molecular sieves Á
Ultrasound-mediated synthesis Á Isatin–thiadiazole Á
Anticonvulsant activity Á Docking study
˚
sonicated in the presence of activated molecular sieves 3 A
Introduction
at a temperature of 50 °C and at a frequency 20 kHz, and
higher yields (73–92 %) and faster reaction times
(43–75 min) were obtained when ultrasonic irradiation was
used in comparison with conventional methods. The syn-
thesized compounds were evaluated for anticonvulsant
(MES and sc-PTZ model) and behavioral activity (ac-
tophotometer) in mice. The compounds 5b, 5i and 5j have
shown protection against MES model at 0.5- and 4-h period
at dose level of 100 mg/kg, while the compound 5c showed
protection against sc-PTZ model at the same dose level. To
exploit the mode of action of the synthesized compounds,
docking study against sodium channel receptor was per-
formed and the results revealed good binding interactions
with the receptor. ADME properties of synthesized com-
pounds were also analyzed and showed potential to
develop as good oral drug candidates.
Epilepsy is ubiquitous neurological disorder characterized
by recurrent attack of seizures due to continuous firing
threshold of neurons from cerebral origin, manifested as
brief episodes of loss of consciousness (McNamara, 1999;
Fisher et al., 2005). The epidemiological survey reveals
that more than 50 million population around the globe
suffer from epilepsy (Media Center, Epilepsy Fact Sheet;
WHO, 2014). Currently available first-generation anticon-
vulsant agents like phenytoin, ethosuximide, benzodi-
azepines exhibit an unwanted side effect profile and failure
to adequately control seizures (Bazil and Pedly, 1998;
McCabe, 2000). These findings demand for the develop-
ment of more effective and reliable anticonvulsant drugs
(Kramer, 2001).
Ultrasound irradiation, in recent times, has achieved an
important place in synthetic organic chemistry as a very
effective and eco-friendly method for synthetic reactions
(Mason, 1997). Ultrasound-promoted synthesis has gained
popularity as they provide potentialities like small time
cycles, cheaper reagents, increase in reaction rate and less
extreme physical conditions (Mason, 1997; Suslick, 1990;
Cella and Stefani, 2009). It can also be considered as
important tool for conservation of energy and minimization
of waste as compared to the convectional techniques (Saleh
and Abd El-Rahman, 2009).
& Anna Pratima G. Nikalje
annapratimanikalje@gmail.com
1
Department of Pharmaceutical Chemistry, Y.B. Chavan
College of Pharmacy, Dr. Rafiq Zakaria Campus, Rauza
Bagh, P.B. No. 33, Aurangabad, Maharashtra 431001, India
˚
The microporous molecular sieves 3 A are aluminosil-
2
Department of Clinical Pharmacokinetics and
Biopharmaceutics, Wockhardt Research Centre, Aurangabad,
Maharashtra 431001, India
icate minerals and have chemical formula 2/3K2OÁ1/
3Na2OÁAl2O3Á2SiO2Á9/2H2O. Since 1990s, these molecular
123

Med Chem Res
sieves have attracted considerable attention owing to their
potential use in catalysis. Avelino Corma reviewed the
synthesis of microporous to mesoporous molecular sieve
materials and their use in catalysis for various synthetic
methodologies (Corma, 1997). Molecular sieves find their
application in catalytic reactions for the synthesis of
ketimines and enamines (Westheimer and Taguchi, 1971).
In the past few years, several compounds containing
isatin molecule make up a broad class due to its wide
range of pharmacological activities (Pakravan et al.,
2013). The anticonvulsant potential of isatin-containing
compounds has been demonstrated by various research
groups (Sridhar et al., 2002; Popp, 1984; Eggadi et al.,
2013; Verma et al., 2004; Pandeya et al., 2001; Bhat-
tacharya and Chakrabarti, 1998; Pandeya et al., 2002;
Karali and Gursoy, 1994).
analysis of pharmacokinetic properties were studied
suggesting that the compounds can be exploited as good
anticonvulsant agent.
Experimental
All the starting materials and reagents were purchased from
Sigma-Aldrich, Merck and Qualigens and used without
further purification. All the solvents were either of ana-
lytical grades or dried and distilled prior to use. Phosphorus
oxychloride was used by distilling under reduced pressure.
All the reactions were performed in oven-dried glasswares.
The ultrasound sonicator (Sonics Vibra-cell, Model no.
VCX 500) equipped with solid synthetic probe, 13 mm in
tip diameter, operating at 20 kHz with a maximum power
output of 500 W, was used for the synthesis of final title
compounds. Thin-layer chromatography was performed on
silica gel G pre-coated plates (Merck), and visualization of
the developed chromatogram was performed by staining
with iodine.
As shown in Fig. 1, the structure of acetazolamide
contains thiadiazole moiety, which is carbonic anhydrase
inhibitor that has been approved by US Food and Drug
Administration (FDA) for treatment of seizures since 1953
(Foldvary-Schaefer and Falcone, 2003). Literature survey
also reveals that thiadiazole nucleus is an important hete-
rocyclic scaffold for the development of potential anti-
convulsant agents (Chapleo et al., 1988; Stillings et al.,
1986; Chapleo et al., 1986; Sharma et al., 2011). Based on
the above facts of the literature, isatin–thiadiazole coupled
moieties can be represented as privileged molecular scaf-
folds for anticonvulsant activity. So, these coupled hete-
rocycles can be effectively used in the treatment of
seizures, due to the synergistic effect of these two com-
bined moieties.
The NMR spectra of final titled compounds were
recorded on Bruker Advance II (400 MHz), and infrared
(IR) spectra were recorded for the compounds on JASCO
FTIR (PS 4000) using KBr pallet. Elemental analysis (C,
H and N) was done with a Shimadzu’s FLASHEA112
analyzer, and all analyses were consistent with theoretical
values (within
0.5 %) unless indicated. The mass
spectra were recorded on a waters Micromass ZQ 2000
spectrometer.
In continuation of our studies on the synthesis of
hybrid heterocycles as anticonvulsant agents by using
improved green methodologies (Nikalje et al., 2011,
2012, 2014), we report a new, rapid, high-yielding and
ultrasound-assisted synthesis of isatin–thiadiazole cou-
pled derivatives along with their anticonvulsant activi-
ties. The synthesized compounds were designed on the
basis of hypothesis of Unverferth et al., (1998) which
suggested a common pharmacophoric pattern that is
necessary for anticonvulsant activity, and the synthesized
compounds follow a pharmacophoric pattern required for
activity as shown in Fig. 2. The docking study and
General procedure for synthesis of 5-substituted
1,3,4-thiadiazol-2-amine 3(a–j)
5-Substituted-1,3,4-thiadiazol-2-amine derivatives were
synthesized by refluxing equimolar quantities of thiosemi-
carbazide and substituted aromatic acids in phosphorus
oxychloride, as per reported procedure (Mullick et al., 2011).
Synthesis of 3-(5-substituted-1,3,4-thiadiazol-2-
ylimino)indolin-2-one 5(a–j)
Conventional method
A mixture of substituted thiadiazole derivatives 3(a–j)
(1.0 mmol) and isatin 4 (1.0 mmol), in absolute ethanol,
was heated under reflux for 6–8 h in the presence of glacial
acetic acid (2.0 mmol) as a catalyst. Then, the reaction
mixture was poured in ice-cold water and filtered under
suction; the precipitate thus obtained was washed with
water and recrystallized from ethanol.
Fig. 1 Acetazolamide
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Med Chem Res
Fig. 2 Pharmacophoric pattern of well-known anticonvulsants and model compound structure with essential elements
Sonicated procedure
(KBr, mmax, cm-1); 3252 (NH of isatin), 3041 (C–H of Ar),
1731 (C=O),1675 (C=N), 1123 (C–S); H NMR (CDCl3,
1
A mixture of substituted thiadiazole derivatives 3(a–j)
(1.0 mmol) and isatin 4 (1.0 mmol), in absolute ethanol,
was sonicated in absolute ethanol in the presence of acti-
400 MHz): d = 7.39–8.01 (m, 9H, Ar ring), 8.26 (s, 1H,
NH of indole); 13C NMR (CDCl3, ppm): d = 117.5 (C,
C-4), 120.5 (CH, C-8), 125.8 (CH, C-6), 128.4 (CH, C-19),
131.2 (CH, C-18), 141.4 (C, C-9), 150.7 (C, C-2), 163.8
(C, C-3), 175.4 (C, C-16); MS: m/z 307.8 (M ? 1), Anal.
Calcd. For C16H10N4OS: C, 62.73; H, 3.29; N, 18.29;
Found: C, 62.75; H, 3.30; N, 18.31.
˚
vated molecular sieves 3 A (1.0 g) at a temperature of
50 °C and frequency 20 kHz for a specified time given in
Table 1. Then, the reaction mixture was poured in ice-cold
water and filtered under suction; the precipitate thus
obtained was washed with water and recrystallized from
ethanol.
3-(5-(4-Chlorophenyl)-1,3,4-thiadiazol-2-ylimino)indolin-
2-one (5b) Dark-yellowish crystals (EtOH); M.P. =
152–154 °C; IR (KBr, mmax, cm-1); 3263 (NH), 3033 (C–H
of Ar), 1725 (C=O), 1666 (C=N), 1141 (C–S), 723 (C–Cl),
3-(5-Phenyl-1,3,4-thiadiazol-2-ylimino)indolin-2-one
(5a) Yellowish crystals (EtOH); M.P. = 178–180 °C; IR
Table 1 Comparison of reaction kinetics of conventional and ultrasonic irradiation methods for synthesis of compounds 5(a–j)
Entry
Conventional
Time (h)
Ultrasonic irradiation
Time (min)
Yield (%)
Yield (%)
5a
5b
5c
5d
5e
5f
5
6
5
7
8
8
9
7
8
7
68
63
62
58
45
56
57
61
53
63
45
50
43
55
60
65
70
55
75
60
86
92
78
82
73
80
77
81
76
83
5g
5h
5i
5j
123

Med Chem Res
1H NMR (CDCl3, 400 MHz): d = 7.24–8.09 (m, 8H, Ar
ring), 8.14 (s, 1H, NH of indole); 13C NMR (CDCl3, ppm):
d = 116.6 (C, C-4), 119.4 (CH, C-8), 125.8 (CH, C-6),
128.6 (CH, C-19), 135.8 (C, C-20), 141.5 (C, C-9), 150.5
(C, C-2), 165.8 (C, C-3), 176.8(C, C-16); MS: m/z 341.5
(M ? 1), Anal. Calcd. For C16H9ClN4OS: C, 56.39; H,
2.66; N, 16.44; Found: C, 56.36; H, 2.68; N, 16.46.
C-19), 129.7 (CH, C-5), 139.5 (CH, C-17), 149.5 (C,
C-20), 151.1 (C, C-2), 163.5 (C, C-3), 175.2 (C, C-16);
MS: m/z 352.5 (M ? 1), Anal. Calcd. For C16H9N5O3S: C,
54.70; H, 2.58; N, 19.93; Found: C, 54.74; H, 2.60; N,
19.91.
3-(5-(3,5-Dinitrophenyl)-1,3,4-thiadiazol-2-ylimino)indol-
in-2-one (5 g) Greenish-yellow crystals (EtOH), M.P. =
128–130 °C, IR (KBr, mmax, cm-1); 3321 (NH), 3045 (C–H
of Ar), 1721 (C=O), 1657 (C=N), 1525 (NO2), 1171 (C–S),
1H NMR (CDCl3, 400 MHz): d = 7.09–8.54 (m, 7H, Ar
ring), 8.03 (s, 1H, NH of indole); 13C NMR (CDCl3, ppm):
d = 117.5 (C, C-4), 120.8 (CH, C-8), 125.7 (CH, C-6),
129.7 (CH, C-18), 131.3 (CH, C-5), 136.8 (CH, C-17),
141.6 (C, C-9), 149.3 (C, C-19), 151.2 (C, C-2), 164.6 (C,
C-4), 174.5 (C, C-16); MS: m/z 397.3 (M ? 1), Anal.
Calcd. For C16H8N6O5S: C, 48.49; H, 2.03; N, 21.20;
Found: C, 48.51; H, 2.05; N, 21.22.
3-(5-(2-Chlorophenyl)-1,3,4-thiadiazol-2-ylimino)indolin-
2-one (5c) Dark-yellowish crystals (EtOH); M.P. =
163–165 °C, IR (KBr, mmax, cm-1); 3302 (NH), 3078 (C–H
of Ar), 1743 (C=O), 1651 (C=N), 1173 (C–S), 746 (C–Cl),
1H NMR (CDCl3, 400 MHz): d = 7.42–8.14 (m, 8H, Ar
ring), 8.16 (s, 1H, NH of indole); 13C NMR (CDCl3, ppm):
d = 117.8 (C, C-4), 120.4 (CH, C-8), 124.8 (CH, C-6),
127.8 (CH, C-21), 128.9 (CH, C-22), 131.2 (CH, C-7),
132.8 (C, C-18), 136.8 (C, C-17), 141.2 (C, C-9), 150.5 (C,
C-2), 163.5 (C, C-3), 174.5 (C, C-16); MS: m/z 341.8
(M ? 1); Anal. Calcd. For C16H9ClN4OS: C, 56.39; H,
2.66; N, 16.44; Found: C, 56.37; H, 2.65; N, 16.47.
3-(5-(2-Hydroxyphenyl)-1,3,4-thiadiazol-2-ylimino)indolin-2-
one (5 h) Gray crystals (EtOH), M.P. = 214–216 °C, IR
(KBr, mmax, cm-1); 3554 (OH), 3315 (NH), 3035 (C–H of
Ar), 1735 (C=O), 1623 (C=N), 1142 (C–S), 1H NMR
(CDCl3, 400 MHz): d = 5.45 (s, 1H, OH attached to
phenyl), 7.42–8.16 (m, 8H, Ar ring), 8.14 (s, 1H, NH of
indole), 13C NMR (CDCl3, ppm): d = 118.7 (C, C-4),
120.5 (CH, C-8), 121.5 (CH, C-21), 123.5 (C, C-17), 128.1
(CH, C-22), 131.6 (CH, C-20), 141.2 (C, C-9), 151.7 (C,
C-2), 156.5 (C, C-18), 163.0 (C, C-3), 175.2 (C, C-16);
MS: m/z 323.5 (M ? 1), Anal. Calcd. For C16H10N4O2S:
C, 59.62; H, 3.13; N, 17.38; Found: C, 59.65; H, 3.15; N,
17.40.
3-(5-p-Tolyl-1,3,4-thiadiazol-2-ylimino)indolin-2-one
(5d) Dark-gray crystals (EtOH), M.P. = 231–232 °C, IR
(KBr, mmax, cm-1); 3312 (NH), 3022 (C–H of Ar), 1730
1
(C=O), 1659 (C=N), 1173 (C–S), 1145 (C–S), H NMR
(CDCl3, 400 MHz): d = 2.35 (s, 3H, CH3 attached to
phenyl), 7.25–8.45 (m, 8H, Ar ring), 8.21 (s, 1H, NH of
indole); 13C NMR (CDCl3, ppm): d = 21.8 (CH3, C-23),
117.5 (C, C-4), 120.8 (CH, C-8), 124.8 (CH, C-6), 127.5
(CH, C-21), 129.8 (CH, C-22), 132.7 (CH, C-20), 141.5 (C,
C-9), 150.7 (C, C-2), 163.5 (C, C-3), 175.8 (C, C-16); MS:
m/z 321.4 (M ? 1), Anal. Calcd. For C17H12N4OS: C,
63.73; H, 3.78; N, 17.49; Found: C, 63.75; H, 3.80; N,
17.52.
2-(5-(2-Oxoindolin-3-ylideneamino)-1,3,4-thiadiazol-2-yl)
phenyl acetate (5i) Gray crystals (EtOH), M.P. =
149–151 °C, IR (KBr, mmax, cm-1); 3236 (NH), 3039 (C–H
of Ar), 1718 (C=O), 1747 (C=O of ester), 1685 (C=N),
1182 (C–S), 1H NMR (CDCl3, 400 MHz): d = 2.47 (s, 3H,
CH3 of acetate), 7.14–8.47 (m, 8H, Ar ring), 8.51 (s, 1H,
NH of indole); 13C NMR (CDCl3, ppm): d = 22.7 (CH3,
C-3), 118.7 (C, C-20), 119.5 (C, C-24), 125.8 (CH, C-22),
126.6 (CH, C-8), 127.3 (CH, C-7), 129.1 (CH, C-6), 142.7
(C, C-18), 151.3 (C, C-18), 163.5 (C, C-19), 169.5 (C,
C-1), 176.8 (C, C-12); MS: m/z 365.1 (M ? 1), Anal.
Calcd. For C18H12N4O3S: C, 59.33; H, 3.32; N, 15.38;
Found: C, 59.35; H, 3.31; N, 15.40.
3-(5-(4-Hydroxyphenyl)-1,3,4-thiadiazol-2-ylimino)indolin-
2-one (5e) Gray crystals (EtOH), M.P. = 129–131 °C,
IR (KBr, mmax, cm-1); 3572 (OH), 3325 (NH), 3075 (C–H
1
of Ar), 1753 (C=O), 1643 (C=N), 1123 (C–S), H NMR
(CDCl3, 400 MHz): d = 4.23 (s, 1H, OH attached to
phenyl), 7.42–8.56 (m, 8H, Ar ring), 8.41 (s, 1H, NH of
indole); 13C NMR (CDCl3, ppm): d = 117.5 (C, C-4),
119.7 (CH, C-8), 125.8 (CH, C-6), 128.5 (CH, C-21), 132.6
(CH, C-7), 141.8 (C, C-9), 150.8 (C, C-2), 158.8 (C, C-20),
163.8 (C, C-20), 174.8 (C, C-16); MS: m/z 323.3 (M ? 1),
Anal. Calcd. For C16H10N4O2S: C, 59.62; H, 3.13; N,
17.38; Found: C, 59.63; H, 3.15; N, 17.35.
3-(5-Styryl-1,3,4-thiadiazol-2-ylimino)indolin-2-one (5j) Yel-
lowish crystals (EtOH), M.P. = 200–202 °C, IR (KBr,
3-(5-(4-Nitrophenyl)-1,3,4-thiadiazol-2-ylimino)indolin-2-
one (5f) Greenish-yellow crystals (EtOH), M.P. =
190–192 °C, IR (KBr, mmax, cm-1); 3256 (NH), 3039 (C–
H of Ar), 1737 (C=O), 1619 (C=N), 1512 (NO2), 1174 (C–
m
max, cm-1); 3236 (NH), 3039 (C–H of Ar), 1732 (C=O),
1
1653 (C=C of styryl), 1685 (C=N), 1182 (C–S), H NMR
(CDCl3, 400 MHz): d = 6.95 (d, 1H, =CH-phenyl), 6.99
(d, 1H, =CH-thiadiazole), 7.24–8.19 (m, 9H, Ar ring), 8.21
(s, 1H, NH of indole); 13C NMR (CDCl3, ppm): d = 116.2
(CH, C-17), 117.9 (C, C-4), 119.2 (CH, C-8), 124.5 (CH,
1
S), H NMR (CDCl3, 400 MHz): d = 7.12–8.36 (m, 8H,
Ar ring), 8.23 (s, 1H, NH of indole); 13C NMR (CDCl3,
ppm): d = 118.4 (C, C-4), 119.7 (CH, C-8), 125.4 (CH,
123

Med Chem Res
C-6), 128.7 (CH, C-21), 128.3 (CH, C-5), 130.3 (CH, C-7),
134.3 (CH, C-18), 138.0 (C, C-19), 142.3 (C, C-19), 151.3
(C, C-2), 159.2 (C, C-9), 164.3 (C, C-3); MS: m/z 333.4
(M ? 1), Anal. Calcd. For C18H12N4OS: C, 65.04; H, 3.64;
N, 16.86; Found: C, 65.06; H, 3.66; N, 16.89.
compounds at dose of 100 mg/kg. Neurological deficit was
indicated by the inability of the animal to maintain equi-
librium on the rod for at least 1 min in each of the three
trials. The dose at which the animal fell off the rod was
determined.
Behavioral activity
Pharmacological method
The behavioral activity of synthesized compounds was
measured by actophotometer with the i.p. administration of
drug (100 mg/kg) to mice. The mice were dropped in a
box, and after 10 min, the behavior of animal was
observed. Then, tested compounds were given to the ani-
mals, and after 0.5 and 4 h, animals were rested. The
activity score was noted, and based on these results, %
decrease in locomotor activity was calculated.
A novel series of 3-(5-substituted-1,3,4-thiadiazol-2-ylim-
ino) indolin-2-one was evaluated for anticonvulsant activ-
ity according to anticonvulsant drug development (ADD)
program protocol (Krall et al., 1978; Porter et al., 1984).
Male Swiss albino mice (CF-1 strain, 20–30 g) were used
as experimental animals. Animals were divided into twelve
groups, and each group of five animals was housed and
allowed free access to water and food. A 12-h: 12-h
light/dark cycle was maintained throughout the experi-
mental studies. All the tested compounds were adminis-
tered in suspension made up to 0.5 % Tween 80 in 0.9 %
sodium chloride solutions and are used for MES and sc-
PTZ study. Pentylenetetrazole (80 mg/kg) dissolved in
0.9 % sodium chloride solution was administered in the
posterior midline of the mice, and the onset and severity of
convulsions were noted for control group. The test group
was administered with the selected compounds 0.5 h prior
to the administration of PTZ. The statistical analyses were
performed by one-way ANOVA followed by Student’s
t test to evaluate the results using GraphPad Prism soft-
ware. All the values were expressed as mean SEM. The
approval of the Institutional Animal Ethics Committee
(CPCSEA/IAEC/Pharm. Chem.-20/2012-2013/78) of Y.B.
Chavan College of Pharmacy, Aurangabad (Maharashtra,
India), was taken prior to the start of the experiments.
Computational parameter
The docking study
The docking study of synthesized compounds and the
standard drug phenytoin was performed against crystal
structure of sodium channel receptor (PDB ID: 1BYY)
which is obtained from the Protein Data Bank (Rohl et al.,
1999). The VLife MDS 4.3 package was used for docking
study of titled compounds. The standard operating proce-
dure implemented in VLife MDS 4.3 package was fol-
lowed for GRIP batch docking of final synthesized
compounds against three-dimensional structures of sodium
channel receptors (VLife MDS 4.3, 2013).
Distance mapping
In the distance mapping study, the distance between vari-
ous groups of titled compounds and standard drugs was
calculated on 3D optimized structures by ACD/3D viewer
8.04 version program. In conformational analysis of the
clinically effective anticonvulsant drugs such as phenytoin
and lamotrigine, a molecular model was suggested on the
basis of molecular dynamics distance estimations (Wong
et al., 1986).
Anticonvulsant activity
The entire test compounds were administered i.p. injections
in a volume of 0.01 ml/g for mice at doses of 100 mg/kg.
Anticonvulsant activity was analyzed after 30 min and 4 h
of drug administration. Then, MES and sc-PTZ model were
studied for anticonvulsants evaluations using reported
procedures.
Determination of ADME properties
Neurotoxicity screening
The synthesized compounds were determined for predic-
tion of ADME properties. The various ADME properties
studied including polar surface area (TPSA), number of
rotatable bonds, molecular volume, number of hydrogen
donors, number of hydrogen acceptors and violations of
Lipinski rule were calculated by Molinspiration online
property toolkit. %ABS was calculated by using formula:
Rotarod test has been performed to detect the minimal
motor deficit in mice. Animals were divided into groups of
5 and trained to stay on an accelerating rotarod that rotates
at 10 rpm. The rod diameter was 3.2 cm. Trained animals
(able to stay on the rotarod for at least two consecutive
trials of 90 s each) were given an i.p. injection of the test
123

Med Chem Res
%ABS = 109 - (0.345 9 TPSA) (Molinspiration Che-
lower the reaction time, we performed same set of reac-
tions by ultrasound irradiation using activated molecular
moinformatics, 2014).
˚
sieves 3 A. In ultrasound-mediated synthesis of titled
compounds, the equimolar quantities of 5-substituted 1,3,4-
Results and discussion
Chemistry
thiadiazol-2-amine 3 and isatin 4 were sonicated in the
˚
presence of activated molecular sieves 3 A at a temperature
of 50 °C and frequency 20 kHz at indicated time given in
Table 1. From Table 1, it was concluded that higher yields
(73–92 %) and faster reaction time (43–75 min) were
obtained when ultrasonic irradiation was used in compar-
ison with conventional methods.
The synthetic protocol for synthesis of titled compounds
3-(5-substituted-1,3,4-thiadiazol-2-ylimino)indolin-2-one
5(a–j) is outlined in Scheme 1. The compounds 5-substi-
tuted 1,3,4-thiadiazol-2-amine 3 were synthesized by
refluxing equimolar quantities of thiosemicarbazide 1 and
substituted carboxylic acids 2 in POCl3 as per reported
procedure (Mullick et al., 2011).
The ultrasound-assisted synthesis technique works on
the principle of cavitation. As ultrasound passes through
liquid, the liquid can produce bubbles. These bubbles can
undergo a violent collapse, which generates very high
pressures and temperatures, inducing molecular fragmen-
tation, and highly reactive species are locally produced. In
addition, ultrasound is known to generate extremely fine
emulsions from mixtures of immiscible liquids to enhance
mass transfer from one phase into the other. One of the
main consequences of these emulsions is the dramatic
increase in the interfacial contact area between the reac-
tants and an increase in the region over which any reaction
between species dissolved in the different liquids can take
Further synthesis of 3-(5-substituted-1,3,4-thiadiazol-2-
ylimino) indolin-2-one 5(a–j) was achieved by using both
conventional and ultrasound irradiation methods. The
conventional synthesis of titled compounds 5(a–j) was
performed by refluxing 5-substituted 1,3,4-thiadiazol-2-
amine 3 with isatin 4 using glacial acetic acid in absolute
ethanol for the time as indicated in Table 1. The conven-
tional method has taken more time, i.e., 5–9 h, and resulted
in low yields (45–68 %). In order to increase the yields and
Scheme 1 Synthetic protocol
for titled compounds 5(a–j).
Reagents and conditions:
(a) POCl3, reflux;
(b) convectional: EtOH, AcOH,
reflux; (c) ultrasound
irradiation: EtOH, molecular
˚
sieves 3 A
123

Med Chem Res
place (Cella and Stefani, 2009). All of these parameters can
cause the reaction to take place rapidly.
and compounds 5c, 5d, 5f, 5 h and 5i showed protection at
4 h. In this study, the compounds 5b, 5e, 5i and 5j did not
show neurotoxicity in the maximum dose of 100 mg/kg
(Table 2). From the behavioral activity of synthesized
compounds as shown in Table 3 using actophotometer, the
compounds 5a, 5b, 5c, 5e, 5 g and 5j showed no behavioral
despair effect when compared to diazepam at 0.5 h. The
compounds 5b, 5c, 5e, 5 g, 5 h and 5i showed no behav-
ioral despair effect when compared to diazepam at 4 h.
Compounds 5d and 5f were found to decrease behavioral
activity of the animals when compared to diazepam
(Table 3).
Reusability of molecular sieves
We also investigated reusability and recycling of molecular
sieves. After completion of reaction mixture, molecular
sieves could be recovered by simple filtration, washed with
methanol and dried. Thus, recovery of molecular sieves
involves a simple filtration and avoids a tedious workup
procedure. Then, these recovered molecular sieves were
reused for further reactions. The recycled molecular sieves
showed no appreciable loss in activity even after the third
cycle. Listed below are the cycle number and yield of 5b
(%):1, 92; 2, 91; 3, 89, as shown in Fig. 3.
SAR analysis for synthesized compounds
The structure–activity relationship reveals some important
facts about the structure-specific variation in anticonvul-
sant activity. The significant anticonvulsant activity of
tested compounds depended on the presence of the com-
bination of Schiff’s bases of isatin and thiadiazole ring in
one molecule; thus, the Schiff’s bases of isatin and thia-
diazole were more active in comparison with Schiff’s bases
of isatin. The nature of a substituent in the aryl fragment
attached to thiadiazole ring also had an influence on the
anticonvulsant activity. Therefore, the introduction of an
electron-withdrawing group, chloro substituent showed
promising anticonvulsant activity in comparison with
electron-donor groups –OH, –CH3 and -OCH3. The sub-
stitution of 4-chloro (5b) on phenyl ring attached to thia-
diazole ring had shown protection against MES model, and
3-chloro (5c) substituent had shown protection against sc-
PTZ model. Hence, it can be proved that ortho- and para-
chloro substitution makes a large difference in MES and
sc-PTZ model. The substitution of 3-OCOCH3 of phenyl
ring (5i) gives a significant anticonvulsant activity. Alkene
linkage between thiadiazole and phenyl ring (5j) can also
be considered for a significant anticonvulsant activity
(Fig. 4).
Anticonvulsant activity
The anticonvulsant screening was performed using male
Swiss albino mice (CF-1 strain, 20–30 g). The anticon-
vulsant activity of synthesized compounds was assessed by
two models, namely maximal electroshock seizure (MES)
and subcutaneous pentylenetetrazole (sc-PTZ). Neurotoxi-
city and behavioral study was analyzed by rotarod and
actophotometer, respectively. All the synthesized com-
pounds screened for anticonvulsant activity showed pro-
tection against MES test. In the MES testing of all
synthesized compounds (Table 2), the compounds 5b, 5c,
5e, 5f, 5i and 5j showed protection at the time interval
0.5 h and 5a, 5b, 5d, 5 g, 5i and 5j showed protection at
the dose 100 mg/kg in time interval 4 h. The compounds
5b, 5i and 5j showed protection at both time intervals at the
same dose. Several compounds of these series were also
found to be active in sc-PTZ test (Table 2). The compound
5c showed protection at both time intervals. The com-
pounds 5b, 5c, 5e, 5 g and 5j showed protection at 0.5 h,
100
80
60
40
20
0
Computational parameters
The docking study
The synthesized compounds 5(a–j) and standard drug
phenytoin were docked against sodium channel receptor
(PDB ID: 1BYY) to understand the binding interactions,
docking calculation and hydrogen bond interactions by
VLife MDS 4.3 package and are shown in Table 4. The
interaction energy of the compounds 5(a–j) and their
anticonvulsant activity showed the corresponding results.
The isatin and thiadiazole rings of these compounds 5(a–
j) held in the active pocket by forming various van der
1
2
3
Runs
Fig. 3 Recycling study of molecular sieves
123

Med Chem Res
Table 2 Anticonvulsant and neurotoxicity screening of compounds
Entry
MES screen
0.5 h
sc-PTZ screen
0.5 h
Neurotoxicity screen
4 h
4 h
4 h
5a
–
100
100
–
–
–
X
5b
100
100
–
100
100
–
–
5c
100
100
–
X
5d
100
–
100
–
5e
100
100
–
100
–
5f
–
100
–
100
X
5g
100
100
100
100
100
X
100
–
5h
–
100
100
–
100
–
5i
100
100
100
X
–
5j
100
X
–
Phenytoin
Sodium valproate
X
X
100
–
X
Dose 100 mg/kg of the compound was administered, and the protection and neurotoxicity measured after 0.5 and 4 h. The figures indicate the
minimal dose required to cause protection or neurotoxicity in 50 % or more of the animals. The dash (–) indicates the absence of anticonvulsant
activity or neurotoxicity. (X) denotes not tested
Table 3 Behavioral study of compounds using actophotometer
Entry
Activity score
Posttreatmenta
0.5 h
Control (24 h before)
4 h
5a
167.76 5.757
291.38 3.829
249.59 3.970
188.41 3.894
234.82 4.821
145.36 5.046
283.01 4.037
145.41 5.163
169.40 4.697
215.17 5.122 ns
209.39 3.600
126.62 2.943
157.02 3.647
132.18 3.277
70.58 3.473
152.60 3.076
73.39 4.366 ns
111.78 3.484
57.23 3.367
66.62 3.043
124.17 5.093
87.200 3.308
110.05 4.930 ns
205.59 5.278
141.03 3.808
89.41 2.839
5b
5c
5d
5e
152.62 3.076
111.79 3.484 ns
219.08 4.658
119.60 4.771 ns
136.35 6.831
102.42 6.038 ns
116.81 3.470
5f
5g
5h
5i
5j
Diazepamb
a
Each value represents the mean SEM significantly different from the control at p \ 0.05, ns denotes not significant at p \ 0.05 (Student’s
t test), locomotor activity score was measured for 10 min
b
The compound was tested at dose level of 4 mg/kg (i. p.)
Waals interactions with the sodium channel receptor. The
various van der Waals interactions occurred between
these compounds and active site of receptor include
MET1490, LYS1495, LYS1496, TYR1498, ASN1499,
LYS1502, LEU1504 and SER1506. The active (MES
model) compounds 5b, 5i and 5j showed lowest binding
energy that is -74.02, -72.68 and -72.67 kcal/mol,
respectively. The -CH3 substituent on phenyl ring of
compounds 5d and 5i had formed hydrophobic interac-
tions with amino acid residues MET1490 and LYS1495,
and LYS1496, respectively. No other substituent of phe-
nyl ring had shown any interactions with receptor. The
various groups like O=C of isatin, nitrogen atoms of
thiadiazole, C=N of Schiff’s base and NH of isatin have
shown hydrogen bond interactions with receptor. The
amino acid residues like LYS1495, LEU1504, GLY1505
and SER1506 had taken part in hydrogen bonding.
Phenytoin had shown good binding interaction energy that
is -68.52 kcal/mol. Phenytoin had formed various
hydrophobic and van der Waals interactions with amino
123

Med Chem Res
Fig. 4 SAR analysis of
synthesized compounds 5(a–j)
Table 4 Docking statistics of synthesized compounds against sodium channel receptor
˚
H-bonds length (A)
Entry
Affinity (kcal/mol)
H-bonds
H-bonding ligand
H-binding receptor
Element
Atom No.
Residue
Element
Atom No.
5a
5b
-64.18
-74.02
01
02
O
N
N
O
N
H
N
–
22
08
09
22
09
28
09
–
SER1506
SER1506
SER1506
SER1506
SER1506
LYS1495
SER1506
–
H
H
H
H
H
O
H
–
342
342
342
342
342
138
342
–
2.565
2.452
1.600
2.580
2.372
1.978
2.452
–
5c
5d
5e
5f
-64.29
-64.07
-65.84
-65.29
-55.04
-62.00
-72.68
01
01
01
01
–
5g
5h
5i
01
03
N
H
H
O
N
–
09
31
31
22
03
–
SER1506
LEU1504
GLY1505
SER1506
SER1506
–
H
O
N
H
H
–
342
309
325
338
342
–
2.328
2.564
2.439
1.541
1.725
–
5j
-72.67
-68.52
01
–
Phenytoin
Distance mapping study
acids residues like MET1490, LYS1495 and TYR1498
and had not shown any hydrogen-bonding interaction with
sodium channel receptor.
In the distance mapping study, the distance between vari-
ous groups of titled compounds and standard drugs was
calculated. In this work, we selected well-known and
structurally different compounds with different mechanism
of action so as to propose a general pharmacophore model.
It involves the correlation of well-known compounds with
titled compounds. This correlation involves at least one
aryl unit, electron-donor atoms and NH group in the spatial
arrangement which is recommended for good pharmaco-
logical activity. The energy minimization of standard
compounds and titled compounds was performed and
analyzed. The average distance between each point was
calculated and examines whether they satisfies the
The interactions of most active compounds 5b, 5i, 5j
and the standard drug phenytoin with sodium channel
receptor are shown in Fig. 5. The amino acid SER1506
˚
(2.452 A) had formed hydrogen bonds with both nitrogen
atoms of thiadiazole ring of compound 5b. The compound
5i had formed three hydrogen bonds that is LEU1504-HN
˚
of isatin (2.464 A), GLY1505-HN of isatin (2.439 A) and
˚
˚
SER1506-O=C of isatin (1.541 A). On the basis of activity
data and docking result, it was observed that compounds
5b, 5i and 5j have potential to inhibit sodium channel
receptor and can be developed as lead for potent anticon-
vulsant agents.
123

Med Chem Res
bFig. 5 Docking study of compounds 5b, 5i, 5j and phenytoin with
sodium channel receptor (PDB ID: 1BYY). Ligands are shown in red
color. Hydrogen bonds are shown in green color (Color figure online)
condition for structural perquisites. It was found that the
synthesized compounds fulfill the essential needs for the
pharmacophores in comparison with average distance
requirement as per data presented in Table 5.
Prediction of ADME properties
We had analyzed various physical descriptors and phar-
maceutically relevant properties for ADME prediction by
using Molinspiration online property toolkit. The results
are summarized in Table 6. All the compounds 5(a–j)
showed significant values for the various parameters ana-
lyzed and are within the range of accepted values. Among
the synthesized compounds, none of the synthesized com-
pounds had violated the Lipinski’s rule of five. The value
of % absorption (ABS) and polar surface area (TPSA) for
synthesized compounds indicated good oral bioavailability
of the compounds. The parameters, like number of rotat-
able bonds and number of rigid bonds are linked with
intestinal absorption result, showed that all synthesized
compounds 5(a–j) had good absorption and making them
potentially promising agents for treatment of seizures.
Conclusion
A series of ten 3-(5-substituted-1,3,4-thiadiazol-2-ylim-
ino)indolin-2-one derivatives 5(a–j) were synthesized using
conventional and ultrasound irradiation methods. Faster
reaction time and high yields of the synthesized compounds
were obtained in ultrasonic irradiation using molecular
˚
sieves 3 A in comparison with the conventional method. The
synthesized compounds were investigated for anticonvul-
sant and behavioral activities with the hope of discovering
new structure leads serving as potential anticonvulsant
agents. The compounds 5b, 5i and 5j showed a significant
activity in MES model, and compound 5c showed protection
in sc-PTZ model at the dose of 100 mg/kg at a time interval
of 0.5 h and 4 hwhencompared withthe standard drug. Most
of the synthesized compounds exhibited lesser behavioral
activity and neurotoxicity compared to clinically effective
drugs. Further, molecular docking study strongly supports
the assumption that these compounds may be involved in the
inhibition of sodium channel receptor. Our analysis of the
distance mapping relationship showed that the titled com-
pounds fulfill the essential demand of pharmacophores when
compared with other well-known anticonvulsants. Also,
none of the synthesized compounds have violated Lipinski’s
rule of five, thus showing good drug-like properties.
123

Med Chem Res
Table 5 Distance range between the essential structure elements R, D and HBD
D
0
1 A
3
.
9
.
-
5
-
5
.
2
.
5
3
A 0
R
4.
2
0
-
8.
0
5
A
HBD
Compounds
R-HBD
R-D
D-HBD
Phenytoin
3.58
6.44
6.79
3.77
4.87
5.1
2.21
4.06
4.01
Lamotrigine
Lead compounds
Table 6 Pharmacokinetic parameter important for good oral bioavailability of compounds
Entry
Milog P
%ABS
TPSA (A2)
MW
n-ON acceptors
n-OHNH donors
Lipinski’s violations
n-ROTB
MV
Rule
5a
5b
5c
5d
5e
5f
1–5
–
–
\500
\10
4
\5
1
B1
0
3.37
4.05
4.00
3.82
2.89
3.33
3.22
3.10
2.87
3.56
88.77
88.77
88.77
88.77
81.79
72.96
57.15
81.79
79.70
88.77
58.64
58.64
58.64
58.64
78.87
104.47
150.29
78.87
84.95
58.64
305.36
2
2
2
2
2
3
4
2
4
3
257.22
270.15
270.75
273.78
265.23
280.55
303.85
265.25
301.75
284.63
339.80
339.80
319.39
321.36
350.35
395.35
321.36
363.39
331.40
4
1
0
4
1
0
4
1
0
5
2
0
7
1
0
5g
5h
5i
10
5
1
0
2
0
6
1
0
5j
4
1
0
% ABS—percentage of absorption, TPSA—topological polar surface area, n-ROTB—number of rotatable bonds, MW—molecular weight,
MV—molecular volume, n-OHNH—number of hydrogen bond donors, n-ON—number of hydrogen bond acceptors
Acknowledgments The authors are grateful to University Grant
Commission, New Delhi, for the Major Research Project Grant. The
authors are thankful to Mrs. Fatma Rafiq Zakaria Chairman, Maulana
Azad Educational Trust and the Principal, Y.B. Chavan College of
Pharmacy, Dr. Rafiq Zakaria Campus, Aurangabad-431001 (M.S),
India, for their constant support.
1,3,4-thiadiazoles with anticonvulsant activity: 1-Hydrazines.
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Screening of the anticonvulsant activity of some isatin deriva-
tives in experimental seizure models and its effect on GABA
levels in mice. Am J Pharmacol Sci 1:42–46
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