An interactive human carbonic Anhydrase-II (hCA-II) receptor - pharmacophore molecular model & anti-convulsant activity of the designed and synthesized 5-amino-1,3,4-thiadiazole-2-thiol conjugated imine derivatives

Article abstract of DOI:10.1111/cbdd.12113

New imines, derived from aromatic aldehyde, chalcones and 5-amino-1,3,4-thiadiazole-2-thiol exhibited promising anti-convulsant activity which is explained through chemo-biological interactions at receptor site producing the inhibition of human Carbonic Anhydrase-II enzyme (hCA-II) through the proposed pharmacophore model at molecular levels as basis for pharmacological activity. The compounds 5-{1-(4-Chlorophenyl)-3-[4-(methoxy-phenyl)-prop-2-en-1-ylidene]amino}-1,3,4-thiadiazole-2-thiol (2b), 5-{[1-(4-chloro-phenyl)]-3-[4-(dimethyl-amino-phenyl)-prop-2-en-1-ylidene]amino}-1,3,4-thiadiazole-2-thiol (2c) and 5-{[1-(4-chloro-phenyl)]-3-[(4-amino-phenyl)-prop-2-en-1-ylidene]amino}-1,3,4-thiadiazole-2-thiol (2f) showed 100% activity in comparison with standard Acetazolamide, a known anti-convulsant drug. The compounds 2c, 2f also passed the Rotarod and Ethanol Potentiation tests which further confirmed them to be safe in motor coordination activity and safe from generating neurological toxicity.

Full text of DOI:10.1111/cbdd.12113

Chem Biol Drug Des 2013; 81: 666–673
Research Article
An Interactive Human Carbonic Anhydrase-II (hCA-II)
Receptor – Pharmacophore Molecular Model & Anti-
Convulsant Activity of the Designed and Synthesized
5-Amino-1,3,4-Thiadiazole-2-Thiol Conjugated Imine
Derivatives
Mohammad Yusuf1, Riaz A. Khan1, Maria Khan2
times ineffective. There is also a significant population of
patients (approximately 30%) resistant to these drugs for
and Bahar Ahmed3,*
controlling and treating the epileptic seizures. Therefore, in
1Department of Medicinal Chemistry, College of
spite of the availability of a number of prescription drugs in
Pharmacy, Qassim University, Qassim, 51452, Saudi
the market and development of other novel therapies as
Arabia
2Buraydah Colleges, Buraydah, Qassim, Saudi Arabia
well as new drugs with newer pharmacological mode of
3Department of Pharmaceutical Chemistry, Faculty of
actions, the efficiency for treatment for convulsion has not
increased significantly over decades. Today, none of the
presently approved drugs is ideal in terms of biological
activity levels, mode of action and limited or no neurological
side effects. They are best used as part of add-on therapy
and are associated with chronic and acute side effects of
adverse nature. Therefore, the search for new templates
and compounds is imperative and continued.
Pharmacy, Hamdard University, New Delhi, 110 062, India
*Corresponding author: Bahar Ahmed,
drbaharahmed@gmail.com
New imines, derived from aromatic aldehyde, chalcones
and 5-amino-1,3,4-thiadiazole-2-thiol exhibited promising
anti-convulsant activity which is explained through
chemo-biological interactions at receptor site producing
the inhibition of human Carbonic Anhydrase-II enzyme
(hCA-II) through the proposed pharmacophore model at
molecular levels as basis for pharmacological activity. The
compounds 5-{1-(4-Chlorophenyl)-3-[4-(methoxy-phenyl)-
prop-2-en-1-ylidene]amino}-1,3,4-thiadiazole-2-thiol (2b),
5-{[1-(4-chloro-phenyl)]-3-[4-(dimethyl-amino-phenyl)-
prop-2-en-1-ylidene]amino}-1,3,4-thiadiazole-2-thiol
(2c) and 5-{[1-(4-chloro-phenyl)]-3-[(4-amino-phenyl)-
prop-2-en-1-ylidene]amino}-1,3,4-thiadiazole-2-thiol (2f)
showed 100% activity in comparison with standard
Acetazolamide, a known anti-convulsant drug. The com-
pounds 2c, 2f also passed the Rotarod and Ethanol Pot-
entiation tests which further confirmed them to be safe
in motor coordination activity and safe from generating
neurological toxicity.
The interesting observation of anti-depressant activity at cer-
tain levels in some of the extended series (1) of 5-amino-
1,3,4-thiadiazole-2-thiol imines and thiobenzyl derivatives
opened the possibilities of detailed correlation between dif-
ferent CNS biological activities and set of molecular attri-
butes including molecular sizes of the compounds. The
observation that the compounds with comparatively smaller
molecular sizes and certain set of molecular & electronic
attributes exhibited negligible or no anti-depressant activity
but significant anti-convulsant activity while compounds of
larger molecular volumes and extended sizes and molecular
attributes exhibited significant anti-depressant activity, but
no anti-convulsant activity suggested a different mode and
site of pharmacological action and biological approach of
the extended and non-extended products from the 5-
amino-1,3,4-thiadiazole-2-thiol series. This also established
the versatility of 5-amino-1,3,4-thiadiazole-2-thiol synthon
as synthetically extendable entity for further structural trans-
formations to yield new bioactive compounds and provide
indications for structure activity relationships in a vast bio-
activity profiles with hereto ambiguous receptor interactions
for different biological activities, some of which are appear-
ing interesting in our ongoing experimentations.
Key words: 5-amino-1,3,4-thiadiazole-2-thiol imines, anti-
convulsant activity, human carbonic anhydrase-II, neurotoxicity
test, pharmacophore model, receptor–ligand interaction
Received 30 September 2012, revised 25 November 2012
and accepted for publication 8 January 2013
Epilepsy, the neurological disorder affecting about 1% of
worlds’ population, is among one of the hard to manage
diseases. The conventional anti-convulsants are widely pre-
scribed but induce a range of side effects and, are many
The currently employed molecular modifications affected
through different synthetic transformations yielded
comparatively (1) smaller sized, anti-convulsant active
666
ª 2013 John Wiley & Sons A/S. doi: 10.1111/cbdd.12113

Anticonvulsants 5-Amino-1,3,4-Thiadiazole-2-Thiols
compounds supporting a different receptor type interaction
for anti-convulsant biological activity and thus were chosen
to design, synthesize and analyse on the receptor-phar-
macophore molecular model.
carbon disulphide 0.25 mol (46 g). The mixture was
warmed under stirring and refluxed for 1 h and then
heated on steam bath for 4 h. Completion of the reaction
was indicated by TLC with Toluene, Ethyl Acetate and aq.
Formaldehyde (TEF) 5:4:1 as mobile phase. The solvent
was evaporated under vacuum and residue dissolved in
water (200 mL), acidified with conc. HCl to give the prod-
uct (12) (54 g), 84%, m. p. 232 °C.
The carbonic anhydrase (CA) is Zinc (II) ion, (Zn+2), based
specific enzyme biochemically catalysing the formation of
carbonic acid from water, carbon dioxide and vice-versa.
It has been suggested that anti-convulsant effect in
humans can be carried out away through inhibition of
human carbonic anhydrase-II enzyme (hCA-II) which also
causes neuro-depression by accumulation of carbon diox-
ide in brain (2). The CA complexes are reported (3–5) for
sulphamic acid, sulfamide and other drugs (6,7) including
the known (8,9) 1,3,4-thiadiazole compounds. An attempt
has been made to understand the pharmacophore func-
tioning based on these complexes where the active phar-
maceutical ingredients have been envisioned as forming
hydrogen bonds, ion-dipole and dipole–dipole interactions
at reactive sites in the enzyme substrate. Based on this
understanding, compounds were designed, synthesized
and biologically tested for their pharmacological efficacy.
Syntheses of aromatic aldehyde, imine derivatives
of 5-amino-1,3,4-thiadiazole-2-thiol (Scheme 1)
5-Amino-1,3,4-thiadiazole-2-thiol (0.02 M) was added to
benzaldehyde (0.02 M) in 25 mL methanol, and reaction
mixture refluxed till the completion of reaction (TLC, TEF
5:4:1). The mixture was concentrated in vacuo to one-
fourth volume and kept in refrigerator overnight, crystals
filtered and recrystallized from hot 95% ethanol.
5-[(2-chlorophenyl)methylene]amino-1,3,4-
thiadiazole-2-thiol (1a)
Yields 55%, m. p. 217–219 °C. 1H-NMR: d 7.09–7.29
(4H, m, Ar-Bz), 9.15 (H, s, CH=N), 2.53 (H, s, SH). IR
Methods and Materials
mmax/cm 3390–3318 (Ar C-Hstr, Bz), 2553 (S-Hstr, thiol),
1610 (Ar C=Cstr, Bz), 1540 (C=Nstr, imine), 720 (Ar C-Hdef
,
The infrared spectra were recorded as KBr pellets FTIR;
Bruker Optics, Karlsruhe, Germany; NMR spectra were
recorded on Bruker DRX-400 MHz (Karlsruhe, Germany) in
DMSO-d6 as solvent with TMS as reference, and mass
spectra were recorded on JOEL SX102/DA-6000 (Pea-
body, MA, USA) spectrometer using Argon/Xenon gas in
FAB mode with meta-nitro benzyl alcohol as matrix under
accelerating voltage of 10 KV. The elemental analysis
established 95% and above purity for all compounds.
o-disubs-Bz), 680 (Ar C-Clstr, Bz). MS m/z [M+] 256, 220,
165, 138, 112. Anal., Calcd. for C9H6ClN3S2: C, 42.27; H,
2.36; N, 16.43. Found: C, 42.24; H, 2.35; N, 16.39.
5-[(4-chlorophenyl)methylene]amino-1,3,4-
thiadiazole-2-thiol (1b)
Yields 70%, m. p. 213–214 °C. H-NMR: d 7.14–7.31 (4H,
m, Ar-Bz), 9.72 (H, s, CH=N), 2.41 (H, s, SH). IR mmax/cm
3377–3309 (Ar C-Hstr, Bz), 2546 (S-Hstr, thiol), 1612 (Ar
C=Cstr, Bz), 1541 (C=Nstr, imine), 800 (Ar C-Hdef, p-disubs-
Bz), 685 (Ar C-Clstr, Bz). MS m/z [M+] 256, 219,165, 138,
111, 92. Anal., Calcd. for C9H6ClN3S2: C, 42.27; H, 2.36;
N, 16.43. Found: C, 42.26; H, 2.34; N, 16.37.
1
Synthesis of substituted chalcones
5-Amino-1,3,4-thiadiazoles were synthesized from thiose-
micarbazide and carbon disulphide. Their aldehyde imines
and conjugated chalcone imines were synthesized accord-
ing to scheme 1 and 2 (Figure 2). Briefly, a solution of aque-
ous sodium hydroxide (2 g, 20 mL) and ethanol (12.5 mL),
both precooled at 5 °C were poured in to crushed ice con-
taining freshly distilled acetophenone (0.04 M) and benzal-
dehyde (0.04 M). The mixture was vigorously stirred and
allowed to come to RT with stirring continued for further
2 h. The mixture was kept overnight in the refrigerator at
0 °C. The completion of reaction was confirmed through
TLC with Benzene: Acetone (9:1) as mobile phase (10,11).
The chalcone product was filtered under vacuum, washed
with cold water until washings were neutral. The crude
product was recrystallized from 50 °C warm ethanol.
5-[(2-nitrophenyl)methylene]amino-1,3,4-
thiadiazole-2-thiol (1c)
Yields 40%, m. p. 231–233 °C. 1H-NMR: d 7.19–7.28
(4H, m, Ar-Bz), 9.12 (H, s, CH=N), 2.54 (H, s, SH). IR
mmax/cm 3375–3324 (Ar C-Hstr, Bz), 2556 (S-Hstr, thiol),
1607 (Ar C=Cstr, Bz), 1566 (C=Nstr, imine), 715 (Ar C-Hdef
,
o-disubs-Bz). MS m/z [M+ + 1] 268, 176, 151, 145, 92.
Anal., Calcd. for C9H6N4O2S2: C, 40.59; H, 2.27; N,
21.04. Found: C, 40.56; H, 2.24; N, 21.07.
5-[(4-nitrophenyl)methylene]amino-1,3,4-
thiadiazole-2-thiol (1d)
Synthesis of 5-amino-1,3,4-thiadiazole-2-thiol
Thiosemicarbazide 45.5 g (0.25 M) was suspended in
absolute ethanol and anhydrous Na2CO3 (24 g) with
Yields 68%, m. p. 195–197 °C. 1H-NMR: d 7.21–7.33
(4H, m, Ar-Bz), 9.74 (H, s, CH=N), 2.47 (H, s, SH). IR
mmax/cm 3379–3312 (Ar C-Hstr, Bz), 2548 (S-Hstr, thiol),
Chem Biol Drug Des 2013; 81: 666–673
667

Yusuf et al.
1619 (Ar C=Cstr, Bz), 1548 (C=Nstr, imine), 805 (Ar C-Hdef
,
(H, s, SH). IR mmax/cm 3393–3315 (Ar C-Hstr, Bz), 3029 (C-
Hstr, trans-ene), 2562 (S-Hstr, thiol), 1686 (C=Nstr), 977 (C-
Hdef, trans-ene), Anal., Calcd. for C18H14ClN3S2: C, 58.13;
H, 3.79; N, 11.30. Found: C, 58.11; H, 3.78; N, 11.11.
p-disubs-Bz). MS m/z [M+] 267, 176, 151, 145, 92. Anal.,
Calcd. for C9H6N4O2S2: C, 40.59; H, 2.27; N, 21.04.
Found: C, 40.55; H, 2.23; N, 21.04.
5-[(2-florophenyl)methylene]amino-1,3,4-
thiadiazole-2-thiol (1e)
Anti-convulsant activity
The pharmacological activity was conducted on male
albino mice (25–30 g) kept under standard conditions of
ambient temperature at 25 Æ 2 °C in the animal house.
Food and water were withdrawn prior to the experiments.
The standard drug acetazolamide and test compounds
were administered through intra-peritoneal route in doses
of 20 mg/kg in propylene glycol. The anti-convulsant activ-
ity was evaluated by maximal electroshock seizure test (3),
supra maximal electroshock of current intensity of 54 mA,
60 Hz were given to mice for 0.2 seconds. The abolition
of the hind limb tonic extensor spasm was recorded as
exhibition of increased anti-convulsant activity (Table 1).
Yields 52%, m. p. 186–190 °C. 1H-NMR: d 7.19–7.35
(4H, m, Ar-Bz), 9.23 (H, s, CH=N), 2.73 (H, s, SH). IR
mmax/cm 3389–3314 (Ar C-Hstr, Bz), 2547 (S-Hstr, thiol),
1611 (Ar C=Cstr, Bz), 1539 (C=Nstr, imine), 719 (Ar C-Hdef
,
o-disubs-Bz). MS m/z [M+ + 1] 241, 167, 145, 121, 93.
Anal., Calcd. for C9H6FN3S2: C, 45.17; H, 2.53; N, 17.56.
Found: C, 45.13; H, 2.53; N, 17.54.
5-[(4-fluorophenyl)methylene]amino-1,3,4-
thiadiazole-2-thiol (1f)
Yields 70%, m. p. 182–184 °C. 1H-NMR: d 7.23–7.31
(4H, m, Ar-Bz), 9.23 (H, s, CH=N), 2.64 (H, s, SH). IR
m
max/cm 3374–3326 (Ar C-Hstr, Bz), 2560 (S-Hstr, thiol),
Rotarod and ethanol potentiation tests for
neurotoxicity
The male albino mice were placed on a rotating rod
(24 rpm) and observed for 5 min. The skeletal muscle
1610 (Ar C=Cstr, Bz), 1574 (C=Nstr, imine), 812 (Ar C-Hdef
,
p-disubs-Bz).MS m/z [M+ + 1] 241, 166, 145, 121, 92.
Anal., Calcd. for C9H6FN3S2: C, 45.17; H, 2.53; N, 17.56.
Found: C, 45.12; H, 2.52; N, 17.56.
Table 1: Anti-convulsant activity and neurotoxicity tests
Anti-convulsant activity
(% Protection)a
Syntheses of various imines derived by different
Chalcones and 5-amino-1,3,4-thiadiazole-2-thiol
(Scheme 2, Compounds 2a–2g)
2-Amino-5-mercapto-1,3,4-thiadiazole was suspended
(0.02 mol) in 25 mL absolute ethanol, and various chal-
cones (0.02 Mol, predissolved in solvent) were added. The
reaction mixture was refluxed for 7 h, left overnight, sol-
vent evaporated under vacuum and residue crystallized
from methanol to give the products (1), 2a–2e, and prod-
ucts 2f and 2g.
Post
treatment
Control
24 h
Prior
Ethanol
After
0.5 h
After
4 h
Rotarod
testb
potentiation
c
Compound
test
1a
1b
1c
1d
1e
1f
0
0
0
0
0
0
0
0
0
0
0
0
0
0
33
66
50
66
16
66
66
100
100
83
16
50
16
16
0
50
50
16
50
16
50
66
33
50
À
À
+
À
À
À
À
À
À
À
À
+
À
À
+
+
+
+
À
À
+
+
+
5-{[1-(4-chlorophenyl)-3-(4-aminophenyl)-prop-2-
en-1-ylidene]amino}-1,3,4-thiadiazole-2-thiol (2f)
Yields 25%, m. p. 121–124 °C. 1H-NMR: d 7.69 (1H, d,
J = 15.6 Hz, trans-ene-H), 7.86 (1H, d, J = 15.6 Hz,
trans-ene-H), 6.91–7.68 (m, 8H, Ar-Bz), 5.64 (2H, s, NH2)
2.40 (H, s, SH).IR mmax/cm 3395–3320 (Ar C-Hstr, Bz),
3027 (C-Hstr, trans-ene), 2564 (S-Hstr, thiol), 1689 (C=Nstr),
976 (C-Hdef, trans-ene), 815 (C-Hdef, p-disubst. Bz), 712
(C-Hdef, monosubst. Bz), 610 (Ar C-Clstr, Bz). MS m/z [M+]
373, 256, 131. Anal., Calcd. for C17H13ClN4S2: C, 54.76;
H, 3.51; N, 15.02. Found: C, 54.71; H, 3.51; N, 15.01.
2a
2b
2c
2d
2e
2f
À
83
100
83
+
+
+
+
2g
Ref
100
aTest compounds and acetazolamide (Ref = Reference) were dis-
solved in propylene glycol and administered (20 mg/kg body
weight dose) via intra-peritoneal (i.p.) route, 30 min prior to test in
groups (n = 6). For neurotoxicity, 20 mg/kg level dose was admin-
istered (i.p.) and neurotoxicity observed after 30 min.
bThe (+) sign indicates 50%; group members or more passing the
test.
cThe mice were treated with test compounds and 1 h later with
ethanol 2.5 g/kg (i.p.); neurotoxicity was measured after 30 min.
The (+) sign indicates 50% group members or more passing the
test.
5-{[1-(4-chlorophenyl)-3-(4-methylphenyl)-prop-2-
en-1-ylidene]amino}-1,3,4-thiadiazole-2-thiol (2g)
Yields 56%, m. p. 71–73 °C. 1H-NMR: d 7.7 (1H, d,
J = 15.6 Hz, trans-ene-H), 7.84 (1H, d, J = 15.6 Hz, trans-
ene-H), 6.92–7.66 (m, 8H, Ar-Bz), 2.64 (3H, s, CH3) 2.42
668
Chem Biol Drug Des 2013; 81: 666–673

Anticonvulsants 5-Amino-1,3,4-Thiadiazole-2-Thiols
relaxation induced by test compound was evaluated by
testing the ability of mice to remain on the revolving rod (4)
which assessed the disruptive effects of compounds on
motor coordination. The dose which impaired the ability of
50% of mice to remain on the revolving rod was consid-
ered the endpoint. For ethanol potentiation test, com-
pounds and ethanol (2.5 g/kg i.p.) after 1 h were
administered to mice. This ethanol dose did not induce lat-
eral position in control animals. The number of mice that
were in the lateral position after receiving ethanol in each
group was determined (3,4) (Table 1).
constant of 15.6 Hz were characteristic of presence of
trans-protons of the -HC=CH- group. The spectral data
and elemental analyses of all the compounds were in
complete agreement with the proposed structures.
Based on previous reports (2–9) and our understanding of
the pharmacophore model (Figure 2), it was indicated that
thiadiazole ring, free thiol and imine group are part of the
main pharmacophore responsible for exhibiting the anti-
convulsant bioactivity. The two nitrogens at position 1,2 of
the thiadiazole ring forms mesoionic complex with the
hydrogen of the hydroxyl group of amino acids threonine
(Thr), positioned as Thr-200 and Thr-199 in the polypep-
tide chain of the hCA-II enzyme. The imine nitrogen inter-
acts through dipole–ion interaction with Zn+2 ion. The
hydrogen atom of the thiol group (SH) interacts (1) with the
oxygen atom of the water molecule. From the biological
evaluations, it was inferred that scheme-1 compounds
(Figure 1) forming the imine series from 5-amino-1,3,4-thi-
adiazole-2-thiol and originating from differently substituted
benzaldehyde molecules, partially occupies the pharmaco-
phore-binding site, whereas the compounds in scheme-2
completely occupies the binding site resulting in total
blockade of active site of hCA-II receptor. The compounds
in scheme-1 possessed chloro, fluoro and nitro electron-
withdrawing groups at positions 2 and 4 of the benzyl ring
and exhibited average levels of bioactivity. Notably, com-
pounds 1b and 1f were active at 50% and above potenti-
ation levels in anti-convulsant tests (Table 1). These two
Results and Discussion
The imine derivatives in both the series were synthesized
starting from 5-amino-1,3,4-thiadiazole-2-thiols (Figure 1).
A part of the synthetic details have been reported earlier
(10–12) and by us (1). The IR spectra of compounds
exhibited absorption bands in the ranges of 3390–3318/
cm due to C-H stretching, 2600–2550/cm due to SH,
1600–1550/cm due to aromatic (C=C), 1540–1500/cm
due to imine stretching and 800–720/cm due to aromatic
C-H deformations in the fingerprint region. The 1H-NMR
peaks were observed in the ranges of d 6.50–8.00 for dif-
ferent aromatic protons, while the peaks in the of d 8.00–
10.00 range were characteristic of imine protons. The d
10.00–13.1 signals were assigned to thiol-protons. The
two doublets in the range d 7.50–8.00 having coupling
Figure 1: Synthetic scheme.
Chem Biol Drug Des 2013; 81: 666–673
669

Yusuf et al.
Figure 2: Pharmacophore model with active receptor sites in vicinity of target molecule (TM). The broken black lines represent Hydrogen
bondings while solid lines represent dipole interaction. The dithiazole moiety is flanked by the amino acids (Glu at position 106, two
adjacent Thr at 199 and, 200) taking part in Hydrogen bondings while amino acids (Histidine, His at positions 94, 96 and, 119) takes part
in dipolar interactions with the Zn+2 ion, water and; Nitrogen at position 1 of the dithiazole group. The nearly squared boxes and, solid
and, broken blue lines connecting each other represent the peptidic sequence connections while the free-ending lines represent the
terminals of polypeptide chain of the hCA-II sequence. The presence of two Zn+2 metal ions means that two molecules of enzyme hCA-II
are taking part in the biochemical activity.
Figure 3: The detailed model showing compound 1b at active site of the receptor cavity. The 1,2 Nitrogens of the thiadiazole ring
participating in Hydrogen bondings with Thr-199 and, Thr-200 while the exocyclic nitrogen atom of the 5-amino-1,3,4-thiadiazole-2-thiol
moiety is in a dipole interaction with the Zn+2 ion of the His moieties at 94, 96 and, 116 positions. The benzyl ring is few A0 distances
away from the diazole ring of the His-94 moiety. The SH group of thiadiazole ring is participating through Hydrogen bonding with water
molecule which itself is in dipole interaction with Zn+2 ion. The molecular orientations of compound 1b represented as colored entity in I
and, II inside the figure represents the stereo-orientation of the thiadiazole ring. The structure I shows benzyl ring to be nearly
perpendicular to the thiadiazole ring while structure II shows the nearly perpendicular orientation of the thiadiazole ring. Both the structures
are at their minimum energy conformation status and, are energetically at same levels. The receptor cavity requirement for Hydrogen
bindings and, dipole interaction prefers the structure form I. The presence of two Zn+2 metal ions means that two molecules of enzyme
hCA-II are taking part in this biochemical action.
670
Chem Biol Drug Des 2013; 81: 666–673

Anticonvulsants 5-Amino-1,3,4-Thiadiazole-2-Thiols
compounds have 4-Cl and 4-F substitutions on the benzyl
ring which indicated that the para substitution on the ben-
zyl ring extends the length of the compound to the extent
for anti-convulsant bioactivity generation through on-site
molecular interactions with the receptor. The substituent
on the benzene ring for series-1 compounds having both
the electron-withdrawing as well as electron-donating
groups did not affect the sustained order of activity in
potentiation tests after 4 h of compounds administration to
experimental animals. However, there was some moderate
activity observed within half hour of the administration of
some of the test compounds. The active compounds 1b
and 1f pharmacophore model suggested that the benzene
ring places itself deep under the polypeptide receptor cav-
ity and is in nearly perpendicular orientation with respect
to the thiadiazole ring. It is also orienting itself in parallel
and in stacked position a few angstrom distance away
from the His-94 (Histidine amino acid’s residue at position
94) which itself is attached to the Zn+2 ion whereby the
Zn+2 ion is taking part in dipole formation with the exo-
cyclic exocyclic nitrogen atom of the 5-amino-1,3,4-thiadiaz-
ole-2-thiol moiety. Moreover, the products in series-1 finds
themselves, than products in series-2, comparatively far
away from the Zn+2 ion due to their adopted conformation
at the binding site as observed by the minimum energy
conformation of these products in the receptor cavity.
A weak ion–dipole interaction is generated between exocyclic
nitrogen atom of the thiadiazole moiety and the target
molecule (TM) from series-1 (Figure 3). The size of the
substituent atom (Cl or, F) does not seem to be effecting
the bioactivity elicitation, but the electronic nature and
position of the substituent groups are playing a part. The
substitution by an electron-withdrawing atom/group at
position 4 has favourable effects for activity generation
which further indicates that the strengthening of the dipole
interaction arising out of Zn+2 ion and slightly altered
electronic environment at exocyclic nitrogen atom of 5-
amino-1,3,4-thiadiazole-2-thiol moiety through its exocyclic
nitrogen atom lone pair of electrons has a positive role in
bioactivity generation.
The compounds in scheme-2 (Figure 1) produced anti-
convulsant activity in higher order than the series-1 com-
pounds. Compounds like 2a, 2c, 2e and 2f are notable.
Some of these products exhibited bioactivities after half an
hour of administration and retained active till 4 h after the
administration. The compound 2b also exhibited 100%
potency levels after half an hour of administration but
could not last longer beyond half an hour. The compounds
2c and 2f were among the most promising templates
exhibiting time-retained biological activity at 50% and 66%
levels after 4 h of administration. The compound 2e
followed in by exhibiting 83% and 50% potencies for anti-
convulsant activity after half an hour and 4 h of administra-
tions, respectively. The most potent products namely 2c
and 2f had a para-position substitution, while some of the
products do not have para-chloro substitution in the aro-
matic ring, and their activity is in ranges with the activity of
Figure 4: The detailed model showing compound 2f at binding site of the receptor cavity. The 1,2 Nitrogens of the thiadiazole ring
participating in Hydrogen bondings with Thr-199 and, Thr-200 while the exocyclic nitrogen atom of the 5-amino-1,3,4-thiadiazole-2-thiol
moiety is in a dipole interaction with the Zn+2 ion of the His (Histidine) moieties at 94, 96 and, 116 positions. The benzyl ring is flat-planer
to the dithiazole ring. At the other side of the thiadiazole ring, the -SH group is participating through the water molecule via dipole
interactions with the Zn+2 ion. The molecular orientations of compound 2f are represented as colored entity in I and, II which inside the
figure represents the stereo-orientation of the thiadiazole ring with all the aromatic rings of the molecule. The structure I and, II shows all
rings to be flatplaner to the thiadiazole ring. The structure II take part in receptor binding as shown in relation to the receptor model
residues and other chemical entities. Both the structures are at their minimum energy conformations status and are energetically at same
levels. The receptor cavity requirements for hydrogen bindings and dipole interaction prefer the structure form as represented in II. The
inter-atomic distances of dipole and hydrogen bondings have considerable reduced in comparison to the compounds in series-1.
Chem Biol Drug Des 2013; 81: 666–673
671

Yusuf et al.
products in series-1. This substitution pattern and the
linked aromatic ring does not makes the similar binding
coordinate as depicted for series-1 compounds but is
placed near the diazole ring of His-119 residue making
part of the Zn+2 interaction coordinates in the receptor
model as illustrated for series-2 compounds (Figure 4).
The exocyclic nitrogen atom N5 of the 5-amino-1,3,4-thi-
adiazole-2-thiol has dipole interaction with the Zn+2 placed
at right side and makes a triangular coordinate between
the N5 nitrogen atom, Zn+2 ion and carbon 1’ of the
nearest to exocyclic nitrogen atom aromatic ring substitu-
tion in products 2c and 2f.
A
B
The -N(CH3)2 and NH2 substitutions at para-positions in
C6 aromatic ring derived from chalcone part of the imine
product 2c and 2f extends beyond or at the outer limits of
the diazole ring of the His-94 residue. This conformation
has made the comparative distance of the Zn+2 and exo-
cyclic nitrogen atom a bit shorter than in series-1 binding
conformation and thus facilitates the easy dipole interac-
tion between the exocyclic N5 nitrogen atom and Zn+2 ion.
The other end of the compound 2c and 2f with thiol group
has also shortened the dipolar interaction distances
between the oxygen of the water molecule and Zn+2 ion
as well as the hydrogen bonding distances between the H
of SH group and oxygen of the water molecule. The com-
plete coordination of receptor interaction of compounds
2c and 2f has produced a compact and full extent occu-
pation of the receptor sites in hCA-II receptor (Figures 3
and 4). The nearly flat ligand occupies the receptor cavity
and compactly binds through hydrogen bondings. The
substitution groups with electron-donating and hydrogen-
binding capacities in the chalcone derived C6 aromatic ring
of the series-2 compounds seem to be preferred for better
receptor interactions which suggested a specific molecular
framework preferably having molecular volumes between
180 and 190 A3 wherein the substitutions is prime contrib-
utor in eliciting the biological activity. This observation in
the products of series-2 is substantiated by the higher
order of activity for -N(CH3)2, -NH2 and OH substitutions,
whereas the substitutions by H, OCH3 and Cl groups did
not exhibited comparative levels of activity as for other
products in the series are concerned. The R1 substitution
at para-position was preferred in comparison with substi-
tution at position 3 (meta) of the C6 aromatic ring which
did not exhibited enough potency levels after half an hour
of administration of the compound, nor the activity was
retained for other high level (100%) potent products from
the series, namely product 2c and 2f. These observations
again confirmed the para-position preference as well as
presence of electron-donating groups for the designed
product to be bioactive.
Figure 5: (A) Three-dimensional representation of the ligand-
receptor interactions (Hydrogen bondings and ion-dipole
interactions). (B) Three-dimensional representation of the ligand-
receptor interactions (space-filling model of interacting area inside
the receptors’ cavity).
ety as preferred substitutions for generating higher biologi-
cal activity for series-2 compounds (Figure 1). Herein, the
R1 substitution and the ring holding this substitution is
overlaid deep into the receptor cavity with the other two
rings namely dithiazole and benzene ring of the sub-struc-
ture H2N-C6H4-CH=CH- in a preferred flat orientation. The
inter-atomic distances for dipole interactions and hydro-
gen bondings necessary for biological activity generation
are reduced in comparison with the series-1 superiorly
active compounds (Figures 3 and 4). The biological
activity profile in series-1 compounds also preferred the
para-position substitutions or, no substitution in the lone
benzene ring. The ortho substitution seemingly interfered
with the receptor sequence residues especially with
His-94 imidazole ring (Figure 3). The series-2 compounds
did not show any interference to His-94 residue and
extended past the domain of this amino acid residue of
the hCA-II sequence at the binding site. In case of R2
substituted benzene ring as part of sub-structure H2N-
C6H4-CH=CH- in series-2 compounds, this substitution
(NH2) was well buried deep into the receptor cavity past
the imidazole ring of His-119 residue at the binding site
(Figure 4). The disposition of series-2 compounds inside
the receptor cavity provided shorter distances for molecu-
lar interactions of dipole nature with Zn+2 ions and short-
ened distances for hydrogen bondings between dithiazole
ring nitrogens with amino acids residue of Thr at 199 and
200 and free SH of the compounds in series-2 with a
The limited SAR of tested compounds suggested the
presence of electron rich and comparatively bulky group
with availability of free electrons, such as OCH3, -N(CH3)2
and -NH2 in comparison with OH, CH3 and Cl atom at
position 4 of the benzene ring away from thiadiazole moi-
672
Chem Biol Drug Des 2013; 81: 666–673

Anticonvulsants 5-Amino-1,3,4-Thiadiazole-2-Thiols
water molecule. The preferences for rings’ R1 substitution
for para-position avoided the interference of the ortho
substitutionally placed groups with amino acids residues
of the receptor chain in the series-2 compounds. The
receptor cavity is extending on both the right and left
sides of the target molecule (TM) rather than on the upper
and down sides (northern and southern parts) of the TM
as represented in receptor model (Figure 2). A closer look
is depicted in Figure 5 which represents the 3D view of
the interaction. Thus, it was concluded that scheme-1
compounds partially acquired the binding site, whereas
scheme-2 compounds perfectly acquired the binding site
resulting in complete blockade of the hCA-II receptor. The
scheme-1 compounds having electron-withdrawing group
at R position of the benzene ring showed good anti-con-
vulsant activity whereas, in scheme-2 compounds, the
electron-donating groups at R1 position and electron-with-
drawing groups at R2 position showed potent anti-convul-
sant activity.
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Conflicts of interest
12. Petrow V., Stephenson O., Thomas A.J., Wild A.M.
(1958) Preparation and, hydrolysis of some derivatives
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There are no conflicts of interest.
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