Synthesis, anticonvulsant and toxicity screening of thiazolyl-thiadiazole derivatives

Article abstract of DOI:10.1007/s00044-010-9313-6

Various thiazole-substituted thiadiazole derivatives (7a-t) were designed and synthesized using substituted acetophenones and substituted anilines as starting materials. Thiazole and thiadiazole moieties being anticonvulsants were clubbed together to get the titled compounds and their in vivo anticonvulsant screening were performed by two most adopted seizure models, maximal electroshock seizure (MES) and subcutaneous pentylenetetrazole (scPTZ). Three compounds 7i, 7l and 7n were found to be potent in both the screens with comparable ED₅₀ and better TD₅₀ values than some standard drugs. These compounds were also found to exert lesser toxic effects on liver.

Full text of DOI:10.1007/s00044-010-9313-6

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2011) 20:261–268
DOI 10.1007/s00044-010-9313-6
ORIGINAL RESEARCH
Synthesis, anticonvulsant and toxicity screening
of thiazolyl–thiadiazole derivatives
•
Nadeem Siddiqui Waquar Ahsan
Received: 8 October 2009 / Accepted: 29 January 2010 / Published online: 14 February 2010
Ó Springer Science+Business Media, LLC 2010
Abstract Various thiazole-substituted thiadiazole deriv-
atives (7a–t) were designed and synthesized using substi-
tuted acetophenones and substituted anilines as starting
materials. Thiazole and thiadiazole moieties being anti-
convulsants were clubbed together to get the titled com-
pounds and their in vivo anticonvulsant screening were
performed by two most adopted seizure models, maximal
electroshock seizure (MES) and subcutaneous pentylen-
etetrazole (scPTZ). Three compounds 7i, 7l and 7n were
found to be potent in both the screens with comparable
ED50 and better TD50 values than some standard drugs.
These compounds were also found to exert lesser toxic
effects on liver.
recent years, did not change the proportion of patients
responding to the treatment and many of those medications
cause serious side effects, which include ataxia, nausea,
mental dulling and hepatotoxicity (Locher et al., 2002;
Greenwood, 2000). Therefore, in consideration with all of
the above findings, the continued search for safer and more
effective AEDs is urgently necessary.
Earlier, we have reported (Siddiqui and Ahsan, 2009;
Siddiqui et al., 2009, 2007a, b; Rana et al., 2008) several
five membered heterocyclic compounds, which have
shown considerable anticonvulsant activities. As part of
our continuous research in the area we have designed and
synthesized several new N-[4-(4-substituted phenyl)-thia-
zol-2-yl]-N0-(substituted
phenyl)-[1,3,4]thiadiazole-2,5-
Keywords Thiazole ꢀ Thiadiazole ꢀ
diamines (7a–t) by incorporating thiadiazole moiety into
the thiazole ring.
Anticonvulsant activity ꢀ Neurotoxicity ꢀ Hepatotoxicity
Since the past few decades, the literature has been
enriched with progressive findings about the anticonvulsant
activities of various substituted thiazole derivatives (Amin
et al., 2008; DeSarro et al., 1993; Bachir et al., 1990). In
addition, many thiadiazoles have attracted much attention
due to their prominent utilization as anticonvulsants
(Archana et al., 2004; Dogan et al., 2002; Kumar et al.,
2002). The present series has been designed by clubbing
both the pharmacologically active moieties viz. thiazole
and thiadiazole, and the resulting compounds are expected
to have synergistic effect in combating epilepsy.
Introduction
Epilepsy, a common neurological disorder characterized by
recurrent spontaneous seizures arising from excessive
electrical activity in some portion of the brain, is a major,
worldwide, public health problem, which affects approxi-
mately 1% of the population (McNamara, 2001). Despite
the increasing understanding of the pathogenesis of sei-
zures, up to 30% of epilepsies are poorly treated with the
available antiepileptic drugs (AEDs) (McNamara, 2001).
Moreover, a large number of new AEDs marketed during
Materials and methods
N. Siddiqui (&) ꢀ W. Ahsan
Chemistry
Department of Pharmaceutical Chemistry, Faculty of Pharmacy,
Hamdard University, Hamdard Nagar, New Delhi 110062, India
e-mail: nadeems_03@yahoo.co.in;
The melting points were determined in open capillary tubes
in a Hicon melting point apparatus and are uncorrected.
nadeems_03@rediffmail.com
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Med Chem Res (2011) 20:261–268
The homogen elemental analyses (C, H, N) of all com-
pounds were performed on the CHNS Elimentar (Analysen
systime, GmbH) Germany Vario EL III. All the Fourier
transform infra red (FTIR) spectra were recorded in KBr
precipitate which was filtered, washed with water and
recrystallized from ethanol to get hydrazinecarbothioamides
(5a–e).
2-[(Substituted phenyl)carbamothioyl]-N-[4-(substituted
phenyl)-1,3-thiazol-2-yl]hydrazinecarboxamides
1
pellets on a Jasco FT/IR 410 spectrometer. The H NMR
(6a–t).
spectra were taken on a Bruker 400 Ultra shieldTM
(400 MHz) NMR spectrometer. Chemical shifts (d) are
expressed in parts per million relative to tetramethylsilane
(TMS) as an internal standard. The homogeneity of the
compounds was checked by thin layer chromatography
(TLC) on silica gel G (Merck) coated plates by using tol-
uene:ethylacetate:formic acid (5:4:1) as solvent system.
Iodine chamber and UV lamp were used for the visuali-
zation of TLC spots.
The solution of carbamates (3a–d, 0.01 mol) and hydra-
zinecarbothioamides (5a–e, 0.01 mol) in ethanol (25 ml)
was refluxed for 4 h. The residue was concentrated, cooled
and poured over crushed ice to the precipitate which was
filtered, washed with water, dried and recrystallized from
ethanol to get the targeted compounds (6a–t).
N-[4-(4-Substituted phenyl)-thiazol-2-yl]-N0-(substituted
phenyl)-[1,3,4]thiadiazole-2,5-diamines (7a–t). To the
cold, concentrated sulphuric acid (15 ml), was added
substituted hydrazinecarboxamides (6a–t, 0.01 mol) in
small portions and stirred for 4 h at room temperature. The
reaction mixture was poured into ice cold water and neu-
tralized with liquid ammonia. The precipitate thus obtained
was filtered, washed with water, and recrystallized from
ethanol to get the titled compounds (7a–t).
N-[4-(4-Chlorophenyl)-thiazol-2-yl]-N0-(2-methylphenyl)-
[1,3,4]thiadiazole-2,5-diamine (7a). Yield 59%, mp 251°C.
IR (KBr) Vmax cm-1: 3423 (NH str.), 3312 (NH str.), 3016
(Ar–CH str.), 1427 (C=N), 847 (C–Cl); 1H NMR (CDCl3) d
2.47 (s, 3H, CH3), 5.47 (s, 1H, ArH-thiazole), 6.87–7.79
(m, 8H, ArH), 9.37 (bs, 1H, NH, D2O exchangeable), 10.14
(bs, 1H, NH, D2O exchangeable).
N-[4-(4-Chlorophenyl)-thiazol-2-yl]-N0-(4-methylphenyl)-
[1,3,4]thiadiazole-2,5-diamine (7b). Yield 73%, mp 268°C.
IR (KBr) Vmax cm-1: 3416 (NH str.), 3325 (NH str.), 2997
(Ar–CH str.), 1406 (C=N), 816 (C–Cl); 1H NMR (CDCl3) d
2.34 (s, 3H, CH3), 5.39 (s, 1H, ArH-thiazole), 6.91–7.84 (m,
8H, ArH), 9.16 (bs, 1H, NH, D2O exchangeable), 10.06
(bs, 1H, NH, D2O exchangeable).
General procedure for the synthesis of titled compounds
(7a–t)
2-Bromo-1-(substituted phenyl)ethanones (1a–d). To the
solution of substituted acetophenones (0.1 mol) in chloro-
form (50 ml), was added bromine previously dissolved in
chloroform (0.11 mol in 50 ml) dropwise for a period of
15 min. The reaction mixture was stirred for an additional
2 h at room temperature. When the reaction was complete,
the reaction mixture was concentrated and cooled to get the
crystals of brominated acetophenones (1a–d).
4-(Substituted phenyl)-1,3-thiazol-2-amines (2a–d). A
solution of compound (1a–d, 0.1 mol) in 150 ml of acetic
acid was refluxed with thiourea (0.1 mol) for 2 h and
cooled to get thiazoles as crystals, which were filtered,
washed with water and recrystallized with ethanol.
Ethyl [4-(substituted phenyl)-1,3-thiazol-2-yl]carba-
mates (3a–d). To the substituted thiazoles (2a–d, 0.1 mol)
in benzene were added ethylchloroformate (0.11 mol) and
triethylamine (25 ml), and the reaction mixture was
refluxed for 3 h. After cooling, the reaction mixture was
poured into cold dil. HCl (50%) and the carbamate thus
formed was recrystallized from benzene.
N-[4-(4-Chlorophenyl)-thiazol-2-yl]-N0-(2-methoxyphenyl)-
[1,3,4]thiadiazole-2,5-diamine (7c). Yield 60%, mp 212°C.
IR (KBr) Vmax cm-1: 3447 (NH str.), 3356 (NH str.), 3057
1
(Ar–CH str.), 1416 (C=N), 815 (C–Cl); H NMR (CDCl3) d
1-(Substituted phenyl)thioureas (4a–e). Substituted ani-
lines (0.1 mol) were taken in water and warmed with dilute
hydrochloric acid (5 ml) until a clear solution was
obtained. To this solution was added ammonium thiocya-
nate (0.11 mol) dissolved in water (25 ml) gradually. The
reaction mixture was boiled and evaporated to less than
half of the volume. It was then cooled to get the precipitate
of phenyl thiourea which was filtered, washed with
water and recrystallized from ethanol to get the target
compounds.
3.16 (s, 3H, OCH3), 5.49 (s, 1H, ArH-thiazole), 6.75–7.79
(m, 8H, ArH), 9.16 (bs, 1H, NH, D2O exchangeable), 10.54
(bs, 1H, NH, D2O exchangeable).
N-[4-(4-Chlorophenyl)-thiazol-2-yl]-N0-(4-methyoxphe-
nyl)-[1,3,4]thiadiazole-2,5-diamine (7d). Yield 64%, mp
236°C. IR (KBr) Vmax cm-1: 3436 (NH str.), 3314 (NH
str.), 3009 (Ar–CH str.), 1421 (C=N), 845 (C–Cl); 1H
NMR (CDCl3) d 3.39 (s, 3H, OCH3), 5.67 (s, 1H, ArH-
thiazole), 6.49–7.84 (m, 8H, ArH), 9.31 (bs, 1H, NH, D2O
exchangeable), 10.47 (bs, 1H, NH, D2O exchangeable).
N-[4-(4-Chlorophenyl)-thiazol-2-yl]-N0–phenyl-[1,3,4]thia-
diazole-2,5-diamine (7e). Yield 73%, mp 187°C. IR (KBr)
Vmax cm-1: 3421 (NH str.), 3345 (NH str.), 2945 (Ar–CH
N-(Substituted phenyl)hydrazinecarbothioamides (5a–e).
To the solution of substituted phenyl thioureas (4a–e,
0.1 mol) in ethanol was added hydrazine hydrate (0.11 mol)
and the reaction mixture was refluxed for 16 h. It was then
concentrated, cooled and poured over crushed ice to get the
1
str.), 1413 (C=N), 837 (C–Cl); H NMR (CDCl3) d 5.35
(s, 1H, ArH-thiazole), 6.68–7.83 (m, 9H, ArH), 9.19
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Med Chem Res (2011) 20:261–268
263
(bs, 1H, NH, D2O exchangeable), 10.34 (bs, 1H, NH, D2O
exchangeable).
str.), 3026 (Ar–CH str.), 1464 (C=N), 1337 (NO2); 1H
NMR (CDCl3) d 3.52 (s, 3H, OCH3), 5.63 (s, 1H, ArH-
thiazole), 6.75–7.79 (m, 8H, ArH), 9.24 (bs, 1H, NH, D2O
exchangeable), 10.21 (bs, 1H, NH, D2O exchangeable).
N -(4-methoxyphenyl)-N0-[4-(4-nitrophenyl)-1,3-thiazol-
2-yl]-1,3,4-thiadiazole-2,5-diamine (7n). Yield 59%, mp
254°C. IR (KBr) Vmax cm-1: 3418 (NH str.), 3329 (NH
str.), 3036 (Ar–CH str.), 1464 (C=N), 1341 (NO2); 1H
NMR (CDCl3) d 3.57 (s, 3H, OCH3), 5.47 (s, 1H, ArH-
thiazole), 6.69–7.84 (m, 8H, ArH), 9.24 (bs, 1H, NH, D2O
exchangeable), 10.31 (bs, 1H, NH, D2O exchangeable).
N-[4-(4-Nitro-phenyl)-thiazol-2-yl]-N0-phenyl-[1,3,4]thia-
diazole-2,5-diamine (7o). Yield 73%, mp 213°C. IR (KBr)
Vmax cm-1: 3454 (NH str.), 3378 (NH str.), 3059 (Ar–CH
str.), 1458 (C=N), 1351 (NO2); 1H NMR (CDCl3) d 5.56 (s,
1H, ArH-thiazole), 6.71–7.79 (m, 9H, ArH), 9.19 (bs, 1H,
NH, D2O exchangeable), 10.21 (bs, 1H, NH, D2O
exchangeable).
N-[4-(4-Bromophenyl)-thiazol-2-yl]-N0-(2-methylphenyl)-
[1,3,4]thiadiazole-2,5-diamine (7f). Yield 76%, mp 267°C.
IR (KBr) Vmax cm-1: 3419 (NH str.), 3345 (NH str.), 3034
(Ar–CH str.), 1416 (C=N), 569 (C–Br); 1H NMR (CDCl3) d
2.38 (s, 3H, CH3), 5.42 (s, 1H, ArH-thiazole), 6.79–7.98
(m, 8H, ArH), 9.14 (bs, 1H, NH, D2O exchangeable), 10.23
(bs, 1H, NH, D2O exchangeable).
N-[4-(4-Bromophenyl)-thiazol-2-yl]-N0-(4-methylphenyl)-
[1,3,4]thiadiazole-2,5-diamine (7g). Yield 59%, mp 284°C.
IR (KBr) Vmax cm-1: 3426 (NH str.), 3324 (NH str.), 3009
(Ar–CH str.), 1435 (C=N), 557 (C–Br); 1H NMR (CDCl3) d
2.42 (s, 3H, CH3), 5.44 (s, 1H, ArH-thiazole), 6.84–7.96
(m, 8H, ArH), 9.12 (bs, 1H, NH, D2O exchangeable), 10.26
(bs, 1H, NH, D2O exchangeable).
N-[4-(4-Bromophenyl)-thiazol-2-yl]-N0-(2-methoxyphenyl)-
[1,3,4]thiadiazole-2,5-diamine (7h). Yield 53%, mp 236°C.
IR (KBr) Vmax cm-1: 3457 (NH str.), 3349 (NH str.), 2916
N-(2-Methylphenyl)-N0-4-(phenyl-1,3-thiazol-2-yl)-1,3,
4-thiadiazole-2,5-diamine (7p). Yield 55%, mp 224°C. IR
(KBr) Vmax cm-1: 3439 (NH str.), 3356 (NH str.), 3006
1
(Ar–CH str.), 1407 (C=N), 567 (C–Br); H NMR (CDCl3) d
3.59 (s, 3H, OCH3), 5.67 (s, 1H, ArH-thiazole), 6.68–7.84
(m, 8H, ArH), 9.07 (bs, 1H, NH, D2O exchangeable), 10.31
(bs, 1H, NH, D2O exchangeable).
1
(Ar–CH str.), 1418 (C=N); H NMR (CDCl3) d 2.34 (s,
3H, CH3), 5.63 (s, 1H, ArH-thiazole), 6.79–7.86 (m, 9H,
ArH), 9.26 (bs, 1H, NH, D2O exchangeable), 10.14 (bs,
1H, NH, D2O exchangeable).
N-[4-(4-Bromophenyl)-thiazol-2-yl]-N0-(4-methoxyphenyl)-
[1,3,4]thiadiazole-2,5-diamine (7i). Yield 65%, mp 251°C.
IR (KBr) Vmax cm-1: 3446 (NH str.), 3318 (NH str.), 3017
N-(4-Methylphenyl)-N0-(4-phenyl-1,3-thiazol-2-yl)-1,3,
4-thiadiazole-2,5-diamine (7q). Yield 62%, mp 235°C. IR
(KBr) Vmax cm-1: 3478 (NH str.), 3348 (NH str.), 2964
1
(Ar–CH str.), 1429 (C=N), 559 (C–Br); H NMR (CDCl3) d
3.56 (s, 3H, OCH3), 5.61 (s, 1H, ArH-thiazole), 6.71–7.81
(m, 8H, ArH), 9.14 (bs, 1H, NH, D2O exchangeable), 10.29
(bs, 1H, NH, D2O exchangeable).
1
(Ar–CH str.), 1456 (C=N); H NMR (CDCl3) d 2.42 (s,
3H, CH3), 5.66 (s, 1H, ArHthiazole), 6.84–7.76 (m, 9H,
ArH), 9.19 (bs, 1H, NH, D2O exchangeable), 10.18 (bs,
1H, NH, D2O exchangeable).
N-[4-(4-Bromo-phenyl)-thiazol-2-yl]-N0-phenyl-[1,3,4]thia-
diazole-2,5-diamine (7j). Yield 49%, mp 223°C. IR (KBr)
V
max cm-1: 3451 (NH str.), 3345 (NH str.), 3034 (Ar–CH str.),
N-(2-Methoxyphenyl)-N0-(4-phenyl-1,3-thiazol-2-yl)-
1,3,4-thiadiazole-2,5-diamine (7r). Yield 41%, mp 244°C.
IR (KBr) Vmax cm-1: 3474 (NH str.), 3351 (NH str.), 2998
(Ar–CH str.), 1447 (C=N); 1H NMR (CDCl3) d 3.54 (s, 3H,
OCH3), 5.63 (s, 1H, ArH-thiazole), 6.88–7.71 (m, 9H,
ArH), 9.23 (bs, 1H, NH, D2O exchangeable), 10.16 (bs,
1H, NH, D2O exchangeable).
1
1417 (C=N), 549 (C–Br); H NMR (CDCl3) d 5.64 (s, 1H,
ArH-thiazole), 6.55–7.78 (m, 9H, ArH), 9.23 (bs, 1H, NH,
D2O exchangeable), 10.34 (bs, 1H, NH, D2O exchangeable).
N-(2-methylphenyl)-N0-[4-(4-nitrophenyl)-1,3-thiazol-
2-yl]-1,3,4-thiadiazole-2,5-diamine (7k). Yield 75%, mp
236°C. IR (KBr) Vmax cm-1: 3429 (NH str.), 3317 (NH
str.), 3009 (Ar–CH str.), 1445 (C=N), 1354 (NO2); 1H
NMR (CDCl3) d 2.35 (s, 3H, CH3), 5.59 (s, 1H, ArH-
thiazole), 6.64–7.87 (m, 8H, ArH), 9.12 (bs, 1H, NH, D2O
exchangeable), 10.27 (bs, 1H, NH, D2O exchangeable).
N-(4-methylphenyl)-N0-[4-(4-nitrophenyl)-1,3-thiazol-
2-yl]-1,3,4-thiadiazole-2,5-diamine (7l). Yield 63%, mp
249°C. IR (KBr) Vmax cm-1: 3414 (NH str.), 3339 (NH
str.), 3034 (Ar–CH str.), 1456 (C=N), 1345 (NO2); 1H
NMR (CDCl3) d 2.39 (s, 3H, CH3), 5.61 (s, 1H, ArH-
thiazole), 6.71–7.83 (m, 8H, ArH), 9.14 (bs, 1H, NH, D2O
exchangeable), 10.32 (bs, 1H, NH, D2O exchangeable).
N-(2-methoxyphenyl)-N’-[4-(4-nitrophenyl)-1,3-thiazol-
2-yl]-1,3,4-thiadiazole-2,5-diamine (7m). Yield 42%, mp
219°C. IR (KBr) Vmax cm-1: 3418 (NH str.), 3305 (NH
N-(4-Methoxyphenyl)-N’-(4-phenyl-1,3-thiazol-2-yl)-1,3,
4-thiadiazole-2,5-diamine (7s). Yield 68%, mp 268°C. IR
(KBr) Vmax cm-1: 3435 (NH str.), 3326 (NH str.), 3018
(Ar–CH str.), 1439 (C=N); 1H NMR (CDCl3) d 3.48 (s, 3H,
OCH3), 5.66 (s, 1H, ArH-thiazole), 6.91–7.87 (m, 9H,
ArH), 9.14 (bs, 1H, NH, D2O exchangeable), 10.21 (bs, 1H,
NH, D2O exchangeable).
N-Phenyl-N0-(4-phenyl-1,3-thiazol-2-yl)-[1,3,4]thiadi-
azole-2,5-diamine (7t). Yield 64%, mp 218°C. IR (KBr)
Vmax cm-1: 3459 (NH str.), 3354 (NH str.), 3007 (Ar–CH
str.), 1457 (C=N); 1H NMR (CDCl3) d 5.54 (s, 1H,
ArH-thiazole), 6.78–7.84 (m, 10H, ArH), 9.22 (bs, 1H,
NH, D2O exchangeable), 10.19 (bs, 1H, NH, D2O
exchangeable).
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Med Chem Res (2011) 20:261–268
Pharmacology
standard error (SE) of the slope were then calculated by
means of a computer program by Litchfield and Wilco-
xon’s method (Litchfield and Wilcoxon et al., 1949).
The investigations were conducted on male albino mice
(25–30 g) that were kept under standard conditions at an
ambient temperature of 25 2°C and allowed free access
to food and water, except at the time they were brought out
of the cage. All the experimental protocols were carried out
with the permission from Institutional Animal Ethics
committee (IAEC), form no. 502. Animals were obtained
from Central Animal House Facility, Hamdard University,
New Delhi-62. Registration no. and date of registration is
173/CPCSEA, 28 Jan., 2000.
Neurotoxicity screening (NT)
The minimal motor impairment was measured in mice by
the rotorod test. The mice were trained to stay on an
O
R
C
CH3
(i)
Anticonvulsant activity
O
C
Maximal electroshock test (MES) The maximal electro-
shock seizure (MES) test was carried out according to the
standard protocol (Krall et al., 1978). Albino mice were
stimulated through corneal electrodes to 50 mA current at a
pulse of 60 Hz applied for 0.25 s. Animals were previously
administered with the test drug i.p. Abolition of hind limb
tonic extension spasm was recorded as the anticonvulsant
activity. The test compounds were dissolved in polyethyl-
ene glycol (PEG). In the preliminary screening, each
compound was administered as an i.p. injection at three
dose levels (30, 100 and 300 mg/kg body mass) and the
anticonvulsant activity assessed after 0.5 h and 4.0 h
intervals of administration.
R
CH2Br
(1a-d)
NH2
R'
(ii)
(iv)
S
R
N
N
H
C
NH2
NH2
S
R'
(2a-d)
(4a-e)
(iii)
(v)
S
R
N
N
H
C
NHNH2
NHCOOEt
S
R'
Subcutaneous pentylenetetrazole-induced seizure test
(scPTZ)
(3a-d)
(5a-e)
The subcutaneous pentylenetetrazole test was performed
according to the known protocol (Clark et al., 1984). This
method utilizes pentylenetetrazole (75 mg/kg) that pro-
duces seizures in [95% of animals as a 0.5% solution
subcutaneously in the posterior midline. The animal was
observed for 30 min; failure to observe even a threshold
seizure (a single episode of clonic spasms of at least 5 s
duration) was defined as protection.
(vi)
R
N
S
O
S
NHC
N
H
N
H
C
N
H
R'
(6a-t)
(vii)
The pharmacological parameters estimated in phase I
screening was quantified in phase II screening. Anticon-
vulsant activity was expressed in terms of the median
effective dose (ED50), and neurotoxicity was expressed as
the median toxic dose (TD50). To determine anticonvulsant
potency and toxicity, groups of 10 mice were tested with
various doses of the drug until at least three points were
established between the limits of 90% protection or toxicity
and 10% protection or toxicity. The dose of drug required
to produce the desired endpoint in 50% of animals (ED50)
in each test, the dose eliciting evidence of minimal neu-
rological toxicity in 50% of animals (TD50), the 95%
confidence interval, the slope of the regression line, and the
R'
HN
N
S
S
R
N
N
H
N
(7a-t)
R = H, Cl, Br, NO2
R' = H, 2-CH3, 4-CH3, 2-OCH3, 4-OCH3
Scheme 1 Synthetic route to the titled compounds (7a–t). Reagents
and conditions: (i) Br2, Chloroform, r.t., (ii) thiourea, glacial acetic
acid, reflux (iii) ethylchloroformate,triethylamine, benzene, reflux,
(iv) NH4SCN, HCl, (v) NH2ꢀNH2ꢀH2O, ethanol, reflux, (vi) ethanol,
reflux, (vii) conc.H2SO4
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Med Chem Res (2011) 20:261–268
265
accelerating rotorod of diameter 3.2 cm that rotates at
10 rpm. Trained animals were given i.p. injection of the
test compounds 30, 100 and 300 mg/kg. Neurotoxicity was
indicated by the inability of the animal to maintain equi-
librium on the rod for at least 1 min in each of the trials
(Yogeeswari et al., 2003).
Results and discussion
Chemistry
The synthesis of titled compounds (7a–t) was carried out
according to Scheme 1. Substituted acetophenones were
treated with bromine in chloroform to afford brominated
acetophenones (1a–d). These, on heating with thiourea in
acetic acid gave substituted thiazole amines (2a–d) which
on treatment with ethylchloroformate and triethylamine
yielded thiazolyl carbamates (3a–d). On the other hand,
substituted arylthioureas (4a–e) were prepared by the
reaction of substituted anilines with ammonium thiocya-
nate in the presence of dilute hydrochloric acid. The aryl
thioureas were made to react with hydrazine hydrate in
ethanol to get hydrazine carbothioamides (5a–e). In the
next step, the thiazolyl carbamates (3a–d) were condensed
with hydrazine carbothioamides (5a–e) in ethanol to afford
the hydrazine carboxamides (6a–t) which on treatment
with cold sulphuric acid, cyclized to form the thiadiazole
ring with the release of one molecule of water. The final
compounds thus prepared were recrystallized from ethanol
Enzyme estimation
The biochemical parameters such as serum glutamate
oxaloacetate transaminase (SGOT) and serum glutamate
pyruvate transaminase (SGPT) were estimated by Reitman
and Frankel’s method (1957) and alkaline phosphatase was
measured by using King and Armstrong method (1934).
Statistical analyses
All the statistical analyses were performed using software
GraphPad InStat 3.0 version and the method used
was ANOVA followed by dunnett’s multiple comparison
test.
Table 1 Physicochemical parameters of the titled compounds (7a–t)
Compound
R
R0
Mol. Formulaa (mol. wt.)
CLog Pb
Rcf value
Elemental analyses % found (calculated)
C
H
N
7a
7b
7c
7d
7e
7f
Cl
2-CH3
4-CH3
2-OCH3
4-OCH3
H
C18H14ClN5S2 (399.92)
C18H14ClN5S2 (399.92)
C18H14ClN5OS2 (415.92)
C18H14ClN5OS2 (415.92)
5.94 0.66
5.94 0.66
5.37 0.67
5.43 0.67
5.48 0.66
6.11 0.68
6.11 0.68
5.55 0.69
5.60 0.69
5.65 0.68
4.98 0.66
4.98 0.66
4.42 0.67
4.47 0.67
4.52 0.66
5.37 0.66
5.37 0.66
4.80 0.66
4.86 0.66
4.91 0.66
0.74
0.77
0.64
0.71
0.59
0.69
0.72
0.66
0.70
0.53
0.75
0.81
0.73
0.79
0.61
0.84
0.87
0.86
0.89
0.78
54.09 (54.06)
54.09 (54.06)
51.96 (51.98)
51.95 (51.98)
52.94 (52.91)
48.67 (48.65)
48.68 (48.65)
46.94 (46.96)
46.94 (46.96)
47.47 (47.45)
52.65 (52.67)
52.66 (52.67)
50.73 (50.70)
50.72 (50.70)
51.49 (51.51)
59.17 (59.15)
59.17 (59.15)
56.66 (56.68)
56.64 (56.68)
58.08 (58.10)
3.51 (3.53)
3.56 (3.53)
3.41 (3.39)
3.42 (3.39)
3.11 (3.13)
3.22 (3.18)
3.16 (3.18)
3.05 (3.07)
3.10 (3.07)
2.80 (2.81)
3.47 (3.44)
3.41 (3.44)
3.35 (3.31)
3.29 (3.31)
3.06 (3.05)
4.11 (4.14)
4.16 (4.14)
3.98 (3.96)
3.94 (3.96)
3.76 (3.73)
17.53 (17.51)
17.49 (17.51)
16.82 (16.84)
16.83 (16.84)
18.19 (18.15)
15.73 (15.76)
15.74 (15.76)
15.25 (15.21)
15.25 (15.21)
16.25 (16.27)
20.48 (20.47)
20.45 (20.47)
19.73 (19.71)
19.74 (19.71)
21.19 (21.20)
19.19 (19.16)
19.14 (19.16)
18.40 (18.36)
18.39 (18.36)
19.96 (19.93)
Cl
Cl
Cl
Cl
C17H12ClN5S2 (385.90)
Br
2-CH3
4-CH3
2-OCH3
4-OCH3
H
C18H14BrN5S2 (444.37)
C18H14BrN5S2 (444.37)
C18H14BrN5OS2 (460.37)
C18H14BrN5OS2 (460.37)
7g
7h
7i
Br
Br
Br
7j
Br
C17H12BrN5S2 (430.35)
7k
7l
NO2
NO2
NO2
NO2
NO2
H
2-CH3
4-CH3
2-OCH3
4-OCH3
H
C18H14N6O2S2 (410.48)
C18H14N6O2S2 (410.48)
C18H14N6O3S2 (426.47)
C18H14N6O3S2 (426.47)
C17H12N6O2S2 (396.45)
C18H15N5S2 (365.48)
C18H15N5S2 (365.48)
C18H15N5OS2 (381.48)
C18H15N5OS2 (381.48)
7m
7n
7o
7p
7q
7r
7s
7t
2-CH3
4-CH3
2-OCH3
4-OCH3
H
H
H
H
H
C17H13N5S2 (351.45)
a
Solvent of crystallization—ethanol
b
c
Log P was calculated using software ACD Labs Software 2.0
Solvent system—toluene: ethyl acetate: formic acid (5:4:1)
123

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Med Chem Res (2011) 20:261–268
Pharmacology
to get the pure titled compounds (7a–t). The physico-
chemical data are presented in Table 1.
Anticonvulsant activity
All the final compounds were characterized by different
spectral analytical techniques. The 1H NMR spectra showed
two singlets in the downfield region in the range of
9.14–10.54 ppm that corresponded for two –NH protons.
These were also D2O exchangeable. The singlet of the thi-
azole proton was observed in the range of 5.35–5.67 ppm.
The aromatic region showed multiplets which corresponded
to the number of aromatic protons present in the each com-
pound. The IR spectra of the compounds further confirmed
the structures of synthesized compounds. Sharp and intense
peaks in the region 3,305–3,478 cm-1 showed the presence
of N–H stretching. The aromatic C–H stretching was also
The anticonvulsant activity was assessed by two most
adopted animal models electroshock (MES) and chemo-
shock (scPTZ) methods. All the synthesized compounds
were administered intraperitoneally into mice using doses
of 30, 100 and 300 mg/kg and the observations were taken
at two different time intervals (0.5 and 4.0 h). Neurological
impairment was evaluated by rotorod method and the data
are presented in Table 2.
In the phase I preliminary anticonvulsant screening, all
the compounds showed protection in MES screen which
was the indicative of the good ability of these compounds
to prevent the seizure spread. Among these compounds, 7i,
7l and 7n showed protection from seizure at the lowest
dose 30 mg/kg after 0.5 h. Interestingly, 7n continued the
anticonvulsant activity after 4.0 h at the same dose. Com-
pounds 7i and 7l displayed protection after 4.0 h at
100 mg/kg dose which indicated the promising nature of
the compounds showing quick onset and long duration of
action at relatively lower doses. These compounds were
also devoid of any sign of neurotoxicity. Seven compounds
(7d, 7g, 7k, 7o, 7q, 7r and 7s) were found to be active at a
dose of 100 mg/kg after 0.5 h. Among these compounds,
7g, 7k, 7q and 7s continued to protect from seizures at the
same dose after 4.0 h. None of the compounds showed
absence of anticonvulsant activity in the MES screen.
In the scPTZ screen, compounds that were found to be
active included 7b, 7c, 7d, 7f, 7g, 7h, 7i, 7k, 7l, 7m, 7n,
7q, 7r, 7s and 7t. Among these compounds, 7d, 7i and 7r
were active at 100 mg/kg after 0.5 h. Compounds 7d and
7r were also found to be active after 4.0 h at a higher dose
300 mg/kg. Compounds which were active at 100 mg/kg
after 4.0 h included 7c, 7n and 7t indicating the delayed
onset and long duration of action of the compounds.
Compounds that were active at 300 mg/kg dose after 0.5 h
were 7g, 7l and 7q. Compound 7g was also active at the
same dose after 4.0 h.
observed in the range of 2,916–3,059 cm-1
.
Table 2 Anticonvulsant activity and minimal motor impairment data
of the synthesized compounds (7a–t)
Compound
Intraperitoneal injection in micea
MES screen scPTZ screen Neurotoxicity screen
0.5 h 4.0 h 0.5 h 4.0 h 0.5 h
4.0 h
7a
300
300
–
300
100
300
300
300
300
100
–
–
–
–
300
100
300
–
–
–
–
–
7b
7c
–
300
–
300
–
7d
100
–
100
–
7e
–
–
7f
300
100
300
30
–
300
300
300
–
100
300
300
–
–
7g
300
–
300
100
–
7h
7i
100
–
100
–
7j
300
100
30
–
300
100
–
300
300
–
7k
100
100
300
30
–
300
–
7l
300
–
7m
300
30
300
100
–
300
–
–
7n
–
–
7o
100
300
100
100
100
300
30
–
–
100
–
–
7p
–
–
–
300
–
7q
100
300
100
–
300
100
–
–
–
In the neurotoxicity screening, compounds that were
devoid of minimal motor impairment at any dose were 7a,
7b, 7d, 7e, 7i, 7l, 7n, 7q and 7r. Rest all the compounds
showed some degree of neurotoxicity, but were less neu-
rotoxic than the standard drug phenytoin as determined by
the rotorod test (Table 2).
7r
300
300
100
–
–
–
7s
300
–
–
7t
–
300
100
–
Phenytoin
Ethosuximide
30
–
100
–
–
–
100
–
300
–
Phenobarbital 100
30
100
300
In the phase II anticonvulsant screening, the most active
compounds 7i, 7l and 7n were quantitatively evaluated for
their anticonvulsant activity (ED50) and neurotoxicity
(TD50). The results are presented in Table 3. All the
compounds showed comparable anticonvulsant activity and
higher protective index than the standard drugs. Compound
7l and 7n possessed good anti-MES activity with ED50 of
a
Number of animals used = 6; solvent used—polyethylene glycol;
Dose of 30, 100 and 300 mg/kg were administered i.p. The figures in
the table indicate the minimum dose whereby bioactivity was dem-
onstrated in half or more of the mice. The animals were examined at
0.5 and 4 h after injections were administered
– indicates an absence of activity at maximum dose administered
(300 mg/kg)
123

Med Chem Res (2011) 20:261–268
267
Table 3 Phase II quantitative anticonvulsant evaluation in mice
Compound
EDa50
TDb50
PIc
MES
scPTZ
MES
scPTZ
7i
27.6 (19.2–36.4)c
19.1 (14.8–23.1)
12.6 (9.2–16.1)
9.5 (8.1–10.4)
79.4 (66.7–94.6)
232.1 (173.3–294.2)
432.6 (307.4–556.3)
[300
712.6 (531.3–904.9)
216.4 (154.2–278.3)
549.5 (351.8–741.6)
65.5 (52.5–72.9)
71.6 (45.9–135)
69 (62.8–72.9)
25.8
11.3
43.6
6.9
8.9
0.9
7l
7n
1.2
Phenytoin
Carbamazepine
Phenobarbital
Valproate
\0.2
\0.2
5.2
8.8 (5.5–14.1)
[100
8.1
21.8 (21.8–25.5)
272 (247–338)
13.2 (5.8–15.9)
149 (123–177)
3.2
426 (369–450)
1.6
2.9
Number of animals used = 10; solvent used: polyethylene glycol (0.1 ml, i.p.)
PI protective index (TD50/ED50
)
a
Dose in milligrams per kilogram body mass
b
c
Minimal toxicity which was determined by rotorod test 30 min after the test drug was administered
Data in parentheses are the 95% confidence limits
Table 4 Enzyme estimation of selected compounds
Treatment
Alkaline phosphatase (mean SEM)
SGOT level (mean SEM)
SGPT level (mean SEM)
Control
15.52 0.905
18.93 1.228
17.03 1.438
20.64 1.395*
153.20 4.866
120.15 3.549**
132.55 5.317*
152.72 4.813
32.92 2.599
34.27 3.552
34.49 3.777
37.665 3.015
7i
7l
7n
The mean levels of enzymes were calculated using ANOVA followed by Dunnett’s multiple comparison test
* P \ 0.05, ** P \ 0.01
19.1 and 12.6 mg/kg, respectively, which was comparable
to the currently used AEDs phenytoin and carbamazepine
and better than that of phenobarbital and valproate. The
much higher TD50 values exhibited by the compounds 7i,
7l and 7n indicated that these caused marked lower
neurotoxicity than the standard drugs. These compounds
showed protective indices of 25.8, 11.3, and 43.6 respec-
tively, which were significantly higher than the currently
used AEDs (PI = 1.6–8.1). In the scPTZ screen compound
7i displayed better ED50 values than most of the standard
drugs with higher PI.
followed the order NO2 \ Br \ Cl. The type of substitu-
tions at the other phenyl ring with the electron releasing
groups does not have marked effect on the activity, but the
point of attachment seemed to be important as the deriv-
atives with groups at the fourth position were compara-
tively more effective than the derivatives with groups
attached to the second position.
Enzyme estimation
Since some currently used AEDs have liver toxicity as one
of the limitation, the liver enzyme estimation of the most
active compounds 7i, 7l and 7n were also performed to
check the magnitude of liver toxicity and the data are
presented in Table 4. The concentrations of alkaline
phosphatase, SGOT and SGPT were determined in serum
and the values are represented as mean SEM. Com-
pound 7i showed moderate but significant change in the
SGOT level as compared to the control (P \ 0.01). Com-
pound 7l and 7n were also found to alter the SGOT and
alkaline phosphatase level, respectively, although it was
not much significant (P \ 0.05). All the other values were
not significant indicating the non-toxic nature of the tested
compounds.
The compounds were designed in such a way that the
phenyl ring attached to the thiazole moiety was substituted
with electron withdrawing groups and the phenyl ring
attached to the thiadiazole moiety was substituted with
different electron releasing groups at different positions.
The structure activity relationship was studied and found
that the derivatives with nitro group attached to the phenyl
ring adjacent to the thiazole moiety were the most active of
the series with most of the compounds active at the two
doses in the MES screen. The bromo-substituted deriva-
tives were comparatively more neurotoxic than the other
derivatives of the series. The size of the electron with-
drawing group played an important role in the activity as it
123

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Med Chem Res (2011) 20:261–268
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Acknowledgements One of the authors (W.A.) is thankful to
Council of Scientific and Industrial Research (CSIR), Government of
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