Novel 4-thiazolidinone derivatives as agonists of benzodiazepine receptors: Design, synthesis and pharmacological evaluation

Article abstract of DOI:10.17179/excli2016-692

A new series of 4-chloro-N-(2-(substitutedphenyl)-4-oxothiazolidin-3-yl)-2-phenoxybenzamide derivatives were designed, synthesized and biologically evaluated as anticonvulsant agents. The designed compounds have the main essential functional groups for bi

Full text of DOI:10.17179/excli2016-692

EXCLI Journal 2017;16:52-62 – ISSN 1611-2156
Received: October 08, 2016, accepted: November 28, 2016, published: January 13, 2017
Original article:
NOVEL 4-THIAZOLIDINONE DERIVATIVES AS AGONISTS OF
BENZODIAZEPINE RECEPTORS: DESIGN, SYNTHESIS AND
PHARMACOLOGICAL EVALUATION
Mehrdad Faizia, Reza Jahania, Seyed Abbas Ebadib, Sayyed Abbas Tabatabaic,
Elham Rezaeec, Mehrnaz Lotfalieic, Mohsen Aminid, Ali Almasiradb*
a
Department of Pharmacology and Toxicology, School of Pharmacy, Shahid Beheshti
University of Medical Sciences, Tehran, Iran
Department of Medicinal Chemistry, Faculty of Pharmacy, Pharmaceutical Sciences
b
Branch, Islamic Azad University, Tehran, Iran
Department of Pharmaceutical Chemistry, School of Pharmacy, Shahid Beheshti University
c
of Medical Sciences, Tehran, Iran
Department of Medicinal Chemistry, Faculty of Pharmacy, Tehran University of Medical
d
Sciences, Tehran, Iran
Corresponding author: Tel: +98-21-22640051, Fax: +98-21-22602059,
E-mail: almasirad.a@iaups.ac.ir
http://dx.doi.org/10.17179/excli2016-692
This is an Open Access article distributed under the terms of the Creative Commons Attribution License
(http://creativecommons.org/licenses/by/4.0/).
ABSTRACT
A new series of 4-chloro-N-(2-(substitutedphenyl)-4-oxothiazolidin-3-yl)-2-phenoxybenzamide derivatives were
designed, synthesized and biologically evaluated as anticonvulsant agents. The designed compounds have the main
essential functional groups for binding to the benzodiazepine receptors and 4-thiazolidinone ring as an anticonvul-
sant pharmacophore. Some of the new synthesized compounds showed considerable anticonvulsant activity in
electroshock and pentylenetetrazole-induced lethal convulsion tests. Compound 5i, 4-chloro-N-(2-(4-methoxy-
phenyl)-4-oxothiazolidin-3-yl)-2-phenoxybenzamide, with the best activity was selected for evaluation of other
benzodiazepine pharmacological effects. This compound induced significant sedative-hypnotic activity. However,
it does not impair the learning and memory in the experimental condition. Flumazenil was able to antagonize the
sedative-hypnotic and anticonvulsant effects of compound 5i indicating that benzodiazepine receptors are highly
involved in the pharmacological properties of the novel compounds.
Keywords: benzodiazepine, sedative-hypnotic, anticonvulsant, 4-thiazolidinone derivatives, synthesis
INTRODUCTION
and epilepsy (Smith et al., 1995; Rudolph and
Knoflach, 2011). However, BZD’s usefulness
is limited by a broad range of unwanted side
effects; including sedation, amnesia, ataxia
and risk of drug dependence (Alerno et. al.,
2012). Therefore, it is important to synthesize
novel ligands of the BZD receptor with an-
tiepileptic activity and less undesirable ef-
fects.
Finding of novel anticonvulsant agents
and new ligands for the benzodiazepine
(BZD) binding site of the GABA receptor has
been an interesting field of drug design in the
recent decade. These compounds have im-
portant pharmacological effects for the treat-
ment of numerous central nervous system
(CNS) disorders such as anxiety, insomnia
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EXCLI Journal 2017;16:52-62 – ISSN 1611-2156
Received: October 08, 2016, accepted: November 28, 2016, published: January 13, 2017
Classical BZDs are considered as positive BBB permeably and physicochemical proper-
allosteric modulators by binding to a distinct ties of compounds for prediction of oral bioa-
site on the GABAA receptors (Guerrini et. al., vailability were evaluated. In order to deter-
2006), which are the greatest population of mine the anticonvulsant effects of synthesized
brain inhibitory neurotransmitter receptors compounds, the penthylentetrazole (PTZ)-in-
(Guerrini et. al., 2007). The GABAA receptors duced lethal convulsion and maximal electro-
are symmetric heteropentamers that are made shock (MES) tests were performed and the re-
of different subunits “(α1–6, β1–3, γ1–3, δ, ε, sults were compared with diazepam, a well-
θ, π and ρ1–3) with an integral channel that is known BZD agonist. The most potent com-
permeable to chloride ions” (Rudolph and pound was selected to evaluate its effect on
Knoflach, 2011). Most of the GABAA recep- learning and memory and the hypnotic activ-
tors are made of α, β and γ subunits, and are ity by passive avoidance test and potentiating
arranged around the selective chloride ion of the pentobarbital sleeping time model re-
gate with the stoichiometry of 2 α subunits, 2 spectively. Also, the neurotoxicity of this
β subunits and 1 γ subunit. The binding site of compound was evaluated by the rotarod test
BZDs is located between α and γ subunits and the involvement of BZD receptor in the
(Rudolph and Knoflach, 2011; Alerno et. al., effects of the novel compounds was con-
2012; Grunwald et al., 2006).
firmed by using flumazenil as an antagonist of
There are several pharmacophore models BZD receptor.
proposed for the binding of ligands to benzo-
diazepine receptors. Two of these models are
more commonly seen in high affinity benzo-
diazepine ligands. The first one is an aromatic
ring with a suitable distance (5Å) from a pro-
ton accepting functional group located in the
same plane and the second one is presence of
the aromatic ring located in a plane perpen-
dicular to the plane of the first aromatic ring
(Akbarzadeh et al., 2003). Furthermore, the
previously reported article revealed that thia-
zolidinone derivatives exhibit anticonvulsant
activity (Tripathi et al., 2014). Based on these
evidences and our earlier published papers on
flexible novel heterocyclic compounds (Ak-
barzadeh et al., 2003; Almasirad et al., 2004;
Zarghi et al., 2005; Almasirad et al., 2007;
Zarghi et al., 2008a; Mahdavi et al., 2010;
Faizi et al., 2012; Tabatabai et al., 2013;
Zarghi et al., 2008a, b; Faizi, et al., 2015) ,
some new thiazolidinone derivatives that
have the essential pharmacophore groups for
binding to the BZD’s receptors were devel-
oped (Figure 1). Design of the novel com-
pounds was based on hybridation between 4-
thiazolidinone and 2-phenoxyphenyl pharma-
cophores. Since both pharmacophores have
shown anticonvulsant activity, it is expected
that the final structures demonstrate compre-
hensive anticonvulsant effects. Further, the
Figure 1: The structure of the novel compounds
consist of A: an aromatic ring, B: a coplanar pro-
ton accepting group and C: a second out-of-plane
aromatic ring, D: adjunct thiazolidinone pharma-
cophore
MATERIALS AND METHODS
Chemistry
Melting points were taken on an Electro-
thermal 9300 apparatus (Ontario, Canada)
and are uncorrected. Infrared spectra and ele-
mental analysis were obtained using Shi-
madzu FT-IR 8400S spectrographs (KBr
disks) and elemental analyser (Costech, Italy)
1
respectively. H-NMR spectra was recorded
on a Bruker FT-500 MHz instrument (Bruker
Biosciences, USA) using chloroform-d as sol-
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EXCLI Journal 2017;16:52-62 – ISSN 1611-2156
Received: October 08, 2016, accepted: November 28, 2016, published: January 13, 2017
vents. Mass were obtained using a 5973 net- 4-Chloro-N-(2-(4-hydroxyphenyl)-4-oxothia-
work mass selective detector at 70 eV (Ag- zolidin-3-yl)-2-phenoxybenzamide (5b)
Yield 50 %; m. p. 126-128° C; IR (KBr) (,
ilent Technology). All chemicals and reagents
were obtained commercially from Sigma/Al-
drich or Merck Company and were used with-
out further purification. All of the intermedi-
ates were synthesized according to our previ-
ously reported articles (Akbarzadeh et al.,
2003; Almasirad et al., 2006).
cm-1): 3320, (OH, NH), 1728 (C=O), 1661
1
(C=O); H NMR (CDCl3, 500MHz): ppm
9.00 (s,1H, NH), 8.14 (d, J = 8.5 Hz, 1H, aro-
matic), 7.37 (m, 2H, aromatic), 7.30-7.16 (m,
3H, aromatic), 7.14 (dd, J = 8.5,2.0 Hz, 1H,
aromatic), 6.78 (d, J = 7.5 Hz, 2H, aromatic),
6.75 (d, J = 7.8 Hz, 2H, aromatic) 6.74 (d, J =
2.0 Hz, 1H, aromatic), 6.02 (s, 1H, Ar-CH),
3.90, 3.73 (2d, J = 16.0 Hz, 2H, SCH2), 3.71
(s, 1H, OH); MS: m/z (%) 442 (M++2, 6), 440
(M+,18), 33 (100), 231 (33), 208 (22), 168
(28), 155 (27), 139 (40), 111 (32), 97 (55), 84
(71), 69 (91), 57 (99). Anal. Calcd for
C22H17ClN2O4S: C, 59.93; H, 3.89; N, 6.35.
Found: C, 59.71; H, 3.93; N, 6.41.
General Procedures for the Synthesis of 5a-
5j
A mixture of hydrazide 4 (3.80 mmol) and
corresponding aldehyde (3.80 mmol) in abso-
lute ethanol was stirred at room temperature
for 2 to 20 hours in the presence of hydrochlo-
ric acid (2 drops) as a catalyst. After the end
of the reaction the mixture was concentrated,
neutralized by a 10 % aqueous (aq) solution
of sodium bicarbonate and the resulting hy-
drazone was washed with water, dried and
used in the next step without further purifica-
tion. To a suspension of corresponding hydra-
zone (2.8 mmol) and Thioglycolic acid (0.38
ml, 5.60 mmol) in dry toluene, catalytic
amounts of anhydrous ZnCl2 was added and
the mixture was refluxed in a round-bottomed
flask equipped with a Dean-Stark apparatus
for 20 hours. After this time, the solvent was
evaporated and NaHCO3 10 % (aq) was
added. Finally the precipitate was filtered and
recrystallized from ethyl acetate.
N-(2-(3,5-Di-tert-butyl-4-hydroxyphenyl)-4-
oxothiazolidin-3-yl)-4-chloro-2 phenoxyben-
zamide (5c)
Yield 50 %; m. p. 188-200° C; IR (KBr) (,
cm-1): 3502, (OH, bonded), 3370 (OH, non-
bonded and NH), 1732 (C=O), 1658 (C=O);
1H-NMR (CDCl3, 500MHz): ppm 8.99 (s,
1H, NH), 8.19 (d, J = 8.5 Hz, 1H, aromatic),
7.36 (t, 2H, aromatic), 7.24 (t, 1H, aromatic),
7.17 (s, 2H, aromatic), 7.13 (dd, J = 8.5, 2.0
Hz, 1H, aromatic), 6.78 (d, J = 8.0 Hz, 2H,
aromatic), 6.58 (d, J = 2.0 Hz, 1H, aromatic),
5.99 (s, 1H, Ar-CH), 5.28 (s, 1H, OH), 3.90,
3.70 (2d, J = 16.0 Hz, 2H, SCH2), 1.28 (s,
18H, CH3); MS: m/z (%) 554 (M++2,4), 552
(M+,12), 306 (100), 290 (62), 263 (24), 248
(87), 233 (77), 231 (99), 216 (16), 196 (20),
168 (28), 139 (31), 93 (27), 77 (16), 57 (64).
Anal. Calcd for C30H33ClN2O4S: C, 65.14; H,
6.01; N, 5.06. Found: C, 65.50; H, 5.96; N,
5.00.
4-Chloro-N-(4-oxo-2-phenylthiazolidin-3-
yl)-2-phenoxybenzamide (5a)
Yield 64 %; m. p. 102-104° C; FT-IR (KBr)
(, cm-1): 3311 (NH), 1726 (C=O), 1652
1
4-Chloro-N-(2-(4-(dimethylamino)phenyl)-4-
oxothiazolidin-3-yl)-2-phenoxybenzamide
(5d)
(C=O); H-NMR (CDCl3, 500MHz): ppm
9.05 (s, 1H, NH), 8.14 (d, J = 10.5 Hz, 1H,
aromatic), 7.38-7.27 (m, 4H, aromatic), 7.24-
7.21 (m, 4H, aromatic), 7.14 (dd, J = 8.5, 2.0
Hz, 1H, aromatic), 6.76 (dd, J = 9.0, 3.5 Hz,
2H, aromatic), 6.64 (d, J = 2.0 Hz, 1H, aro-
matic), 6.03 (s, 1H, Ar-CH) , 3.92, 3.75 (2d, J
= 14.5 Hz, 2H, SCH2); MS: m/z (%) 426
(M++2,11), 424 (M+,33), 247(35), 233(80),
231(100), 196(43), 168(62), 155(37),
139(67), 121(29), 93(90), 77(64). Anal. Calcd
for C22H17ClN2O3S: C, 62.19; H, 4.03; N,
6.59. Found: C, 62.40; H, 3.96; N, 6.45.
Yield 50 %; m. p. 152-153° C; IR (KBr) (,
cm-1): 3381 (NH), 1717 (C=O), 1667 (C=O);
1H-NMR (CDCl3, 500MHz): ppm 9.01 (s,
1H, NH), 8.14 (d, J = 8.5 Hz, 1H, aromatic),
7.32 (m, 2H, aromatic), 7.24-7.14 (m, 3H, ar-
omatic), 7.12 (dd, J = 8.5, 2.0 Hz, 1H, aro-
matic), 6.77 (dd, J = 6.5, 2.0 Hz, 2H, aro-
matic), 6.61(d, J = 1.5 Hz, 1H, aromatic) 6.49
(d, J = 6.5Hz, 2H, aromatic), 5.97 (s, 1H, Ar-
CH), 3.92, 3.71 (2d, J = 16.0 Hz, 2H, SCH2),
2.89 (s, 6H, CH3); MS: m/z (%) 469 (M++2,
3), 467 (M+, 9), 336 (16), 233 (100), 231 (99),
196 (32), 168 (54), 147 (79), 134 (28), 93
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EXCLI Journal 2017;16:52-62 – ISSN 1611-2156
Received: October 08, 2016, accepted: November 28, 2016, published: January 13, 2017
(38), 77 (41), 57 (34). Anal. Calcd for
C24H22ClN3O3S: C, 61.60; H, 4.74; N, 8.98.
Found: C, 61.25; H, 4.80; N, 8.93.
4-Chloro-N-(2-(4-nitrophenyl)-4-oxothiazol-
idin-3-yl)-2-phenoxybenzamide (5h)
Yield 50 %; m. p. 168-170° C; IR (KBr) (,
cm-1): 3398 (NH), 1731 (C=O), 1679 (C=O),
1528,1349 (NO2); 1H-NMR (CDCl3,
500MHz): ppm 9.04 (s,1H, NH), 8.11(d, J =
9.0 Hz , 1H, aromatic), 8.03 (d, J = 8.5 Hz,
2H, aromatic), 7.52 (d, J = 8.5 Hz, 2H, aro-
matic), 7.32-7.29 (t, 2H, aromatic), 7.22-7.17
(m, 2H, aromatic), 6.72 (d, J = 8.0 Hz, 2H,
aromatic), 6.69 (d, J = 2.0 Hz, 1H, aromatic),
6.11 (s, 1H, Ar-CH), 3.92, 3.75 (2d, J = 16.0
Hz, 2H, SCH2); MS: m/z (%) 471 (M++2, 5),
469 (M+, 15), 368 (9), 247 (18), 233 (73), 231
(100), 196 (16), 168 (31), 139 (38), 93 (87),
70 (72). Anal. Calcd for C22H16Cl1N3O5S: C,
56.23; H, 3.43; N, 8.94. Found: C, 55.98; H,
3.50; N, 8.92.
N-(2-(2-bromophenyl)-4-oxothiazolidin-3-
yl)-4-chloro-2-phenoxybenzamide (5e)
Yield 50 %; m. p. 125-127° C; IR (KBr) (,
cm-1): 3555 (NH), 1731 (C=O), 1672 (C=O);
1H-NMR (CDCl3, 500MHz): ppm 9.14 (s,
1H, NH), 8.15 (d, J = 8.5 Hz, 1H, aromatic),
7.44(d, J = 7.5 Hz, 1H, aromatic), 7.42-7.20
(m, 4H, aromatic), 7.19-7.02 (m, 3H, aro-
matic), 6.87 (d, J = 8.5 Hz, 2H, aromatic),
6.68 (d, J = 2.0 Hz, 1H, aromatic) 6.47 (s, 1H,
Ar-CH), 3.89, 3.75 (2d, J = 16.0 Hz, 2H,
SCH2); MS: m/z (%) 506 (M++4, 7),
504(M++2, 28), 502(M+, 21), 233 (38), 231
(100), 168 (19), 139 (28), 121 (16), 93 (33),
77 (21), 57 (18). Anal. Calcd for
C22H16BrClN2O3S: C, 52.45; H, 3.20; N, 5.56.
Found: C, 52.61; H, 3.16; N, 5.52.
4-Chloro-N-(2-(4-methoxyphenyl)-4-oxothia-
zolidin-3-yl)-2-phenoxybenzamide (5i)
Yield 50 %; m. p 121-123° C; IR (KBr) (,
cm-1): 3319 (NH), 1722 (C=O), 1659 (C=O);
1H-NMR (CDCl3, 500MHz): ppm 9.01 (s,
1H, NH), 8.13 (d, J = 9.0 Hz, 1H, aromatic),
7.38-7.24 (m, 6H, aromatic), 7.14 (d, J = 8.5
Hz, 1H, aromatic), 6.77 (d, J = 7.5 Hz, 2H,
aromatic), 6.72 (d, J = 8.5 Hz, 2H, aromatic),
6.64 (s, 1H, aromatic), 5.99 (s, 1H, Ar-CH),
3.91 , 3.68 (2d, J = 16.0 Hz, 2H, SCH2), 3.66
(s, 3H, CH3); MS: m/z (%) 457 (M++2,2), 455
(M+,6), 233 (51), 231 (100), 208 (96), 196
(21), 168 (33), 151 (28), 134 (60), 93 (55), 77
(78). Anal. Calcd for C23H19Cl1N2O4S: C,
60.72; H, 4.21; N, 6.16. Found: C, 60.82; H,
4.16; N, 6.09.
4-Chloro-N-(2-(4-chlorophenyl)-4-oxothia-
zolidin-3-yl)-2-phenoxybenzamide (5f)
Yield 50 %; m. p. 113-115° C; IR (KBr) (,
cm-1): 3284 (NH), 1722 (C=O), 1672 (C=O);
1H-NMR (CDCl3, 500MHz): ppm 9.03 (s,
1H, NH), 8.13 (d, J = 8.5Hz, 1H, aromatic),
7.41-7.15(m, 8H, aromatic), 6.77 (d, J = 8.0
Hz, 2H, aromatic), 6.68 (d, J = 2.0 Hz, 1H,
aromatic), 6.01 (s, 1H, Ar-CH), 3.91, 3.73
(2d, J = 16.0 Hz, 2H, SCH2); MS: m/z (%)
462 (M++4,4), 460 (M++2, 24), 458 (M+,36),
247 (44), 233 (88), 231 (100), 212 (61), 196
(24), 168 (33), 155 (30), 139 (51), 93 (99), 77
(31). Anal. Calcd for C22H16Cl2N2O3S: C,
57.52; H, 3.51; N, 6.10. Found: C, 57.61; H,
3.43; N, 5.91.
4-Chloro-N-(4-oxo-2-p-tolylthiazolidin-3-
yl)-2-phenoxybenzamide (5j)
4-Chloro-N-(2-(3-nitrophenyl)-4-oxothiazol-
Yield 50 %; m.p. 133-135° C; IR (KBr) (,
cm-1): 3326 (NH), 1731 (C=O), 1659 (C=O);
1H-NMR (CDCl3, 500MHz): ppm 10.48
(s,1H, NH), 7.43 (d, J = 8.3 Hz, 1H, aro-
matic), 7.38 (t, 2H, aromatic), 7.28-7.18 (m,
4H, aromatic), 7.03 (d, J = 7.8 Hz, 2H, aro-
matic), 6.90 (d, J = 7.7 Hz, 2H, aromatic),
6.77 (d, J = 1.7 Hz, 1H, aromatic), 5.81 (s, 1H,
Ar-CH), 3.87, 3.76 (d, J = 15.8 Hz, 2H,
SCH2), 2.20 (s, 3H, CH3); MS: m/z (%) 440
(M++2, 9), 438 (M+, 27), 247 (35), 233 (78),
231 (100), 192 (59), 168 (56), 155 (33), 139
(62), 118 (34), 93 (82), 77 (38), 57 (15). Anal.
Calcd for C23H19Cl1N2O3S: C, 62.94; H, 4.36;
N, 6.38. Found: C, 63.15; H, 4.38; N, 6.29.
idin-3-yl)-2-phenoxybenzamide (5g)
Yield 50 %; m. p. 99-100° C; IR (KBr) (,
cm-1): 3321 (NH), 1726 (C=O), 1665 (C=O),
1531, 1352 (NO2); 1H-NMR (CDCl3,
500MHz): ppm 9.12 (s, 1H, NH), 8.21 (s,
1H, aromatic), 8.08(d, J = 8.5Hz , 1H, aro-
matic), 8.01 (d, J = 8.5 Hz, 1H, aromatic),
7.72 (d, J = 8.5 Hz, 1H, aromatic), 7.40-7.21
(m, 4H, aromatic), 7.15 (d, J = 7.5 Hz, 1H,
aromatic), 6.73 (d, J = 7.5 Hz, 2H, aromatic),
6.74 (s, 1H, aromatic), 6.11 (s, 1H, Ar-CH),
3.91, 3.80 (2d, J = 16.0 Hz, 2H, SCH2). MS:
m/z (%) 471 (M++2, 5), 469 (M+, 15), 247
(15), 233 (71), 231 (100), 196 (17), 168 (38),
139 (32), 93 (87), 70 (70). Anal. Calcd for
C22H16Cl1N3O5S: C, 56.23; H, 3.43; N, 8.94.
Found: C, 57.17; H, 3.23; N, 8.85.
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EXCLI Journal 2017;16:52-62 – ISSN 1611-2156
Received: October 08, 2016, accepted: November 28, 2016, published: January 13, 2017
Pharmacology
30 minutes, MES were applied (60 Hz, 37.2
mA and 0.25 s) using ear electrodes. We rec-
orded the number of mice protected from hind
limb tonic extension (HLTE) induced by
MES for data analysis (Zarghi et al., 2005,
2008a).
Animals and drugs
For pharmacological tests, we used male
NMRI albino mice (weighing 18-22 g; 12
weeks old) obtained from Pasteur Institute
(Iran). All animals were kept in the animal
house of School of Pharmacy for 7 days in or- Potentiation of pentobarbital sleeping time
der to adapt to the new place. The mice were For the evaluation of hypnotic effects of
kept in standard cages in a typical room in the the novel compounds and diazepam, we in-
animal house with controlled conditions de- jected different doses of the novel compounds
signed for experimental animals and have un- and diazepam (1 mg/kg) and after 30 minutes,
restricted access to water and food. Before injected pentobarbital (65 mg/ kg i.p.) We
starting the experiments, mice were also recorded the duration of loss of righting re-
transferred to the testing rooms 60 minutes flex, which represents the sleeping time.
before the tests to get acclimatized to the ex- Since pentobarbital may cause hypothermia,
perimental condition. We randomly divided animals were located on the top of an electric
the mice into experimental groups and all blanket (set at 37° C) after administration of
mice were used only once in the experiments. pentobarbital (Faizi et al., 2015).
Ethical Committee of Shahid Beheshti Uni-
Passive avoidance test
versity of Medical Sciences approved all the
In order to assess the effect of the novel
protocols of the experiments before starting
compounds and midazolam on anterograde
the study. All the tests on animals were done
memory, previously described step-through
based on NIH publication #85-23. In all pro-
passive avoidance test was used. Mice were
tocols we used the least possible number of
placed into two compartment, light and dark,
animals and all ethical advises for work on ex-
apparatus. On the first day (training day) the
perimental animals were considered. In this
novel compounds or midazolam were injected
study, diazepam was used as a standard ben-
and 30 minutes later each animal was located
zodiazepine, and flumazenil was used as an-
inside the light comportment and let it to
tagonist of benzodiazepine receptors. A mix-
freely move. Thirty seconds later the door be-
ture of DMSO and water (1:10) was used as
tween two compartments was opened and
the solvent for the novel compounds, diaze-
mouse was able to easily pass the door. Im-
pam, and flumazenil were injected 5 ml/kg
mediately after entering to the dark compart-
i.p.; 30 minutes before the experiments. PTZ,
ment, an unpleasant electrical shock (0.5 mA,
pentobarbital, and midazolam were dissolved
2 seconds) was delivered to the mouse from
in distilled water and injected i.p. (volume of
bottom of the compartment. On the second
injection was 5 ml/kg).
day (testing day), animal was located in the
The anticonvulsant activity
light compartment and the delay time for each
Two experimental models including PTZ mouse to enter the dark compartment was rec-
and MES induced convulsions were used to orded (Faizi et al., 2015).
test the anticonvulsant effects of the novel
Neurotoxicity evaluation
compounds. In PTZ test, different doses of the
The neurotoxicity of the compound 5i was
novel compounds or diazepam were injected
measured in mice by the rotarod test. The
and after 30 min, lethal dose of PTZ (100
mice were trained to stay on a rotarod of di-
mg/kg) were injected. We recorded the num-
ameter 1 inch that rotates at 6 rpm and were
ber of mice protected from PTZ induced con-
given i.p. of the 5i. If in each trial mouse was
vulsion and death for data analysis. In MES
not able to stay at least 1 minute on the
model, different doses of the novel com-
pounds or diazepam were injected and after
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EXCLI Journal 2017;16:52-62 – ISSN 1611-2156
Received: October 08, 2016, accepted: November 28, 2016, published: January 13, 2017
rotarod, it was considered as neurotoxicity-in- pounds are BBB+ and the density of com-
duced deficit in coordination or balance (Sałat pounds 5a, 5d-5f, 5i-5j in the CNS could be
et al., 2013).
more than other compounds.
Data analysis
Physicochemical properties
ED50 of the anticonvulsant effects of the
The physicochemical properties and the
Lipinski’s rule of five parameters for predic-
tion of oral bioavailability were determined
by molinspiration online program (www.
molinspiration.com) and also the clogp was
achieved by Osiris Property Explorer (OPE)
(http://www.organic-chemistry.org/prog/peo/)
(Table 1) (Lipinski et al., 2012). According to
the Lipinski’s rule for oral absorption of com-
pounds, their molecular weight should be
lower than 500, the number of hydrogen bond
donor and acceptor should be ≤ 5 and ≤ 10 re-
spectively, the topological polar surface area
should be ≤ 140 and number of rotatable
bonds ≤ 10 and two violations of this rule will
result to the poor oral absorption. As shown
in Table 1, all compounds except compound
5c can be absorbed orally (Table 1).
all tested compounds were calculated by pro-
bit-regression analysis using SPSS software
(Chicago, IL; 1 version 13). To compare the
ED50s, Fisher’s exact probability test was
used. Results of PTZ and MES experiments
were reported as mean with 95 % confidence
intervals. Results of the other experiments
were reported as mean ± SEM and one-way
analysis of variance (ANOVA) with Tukey’s
HSD post hoc test were used to analyse the
results. In all tests, if P-value was less than
0.05, we concluded that a significant differ-
ence existed.
RESULTS AND DISCUSSION
Prediction of the BBB permeably of
compounds
Anticonvulsant agents similar to other
CNS active compounds should be able to pass
through blood-brain barrier (BBB). So the in
silico prediction of the BBB permeably of the
designed compounds was done by the BBB
prediction server which is a part of AlzPlat-
form (Liu et al., 2014), using support vector
machine (SVM_Molptint 2D) algorithm
(http://www.cbligand.org/BBB). The ability
or inability of a compound to penetrate blood-
brain barrier (BBB+/BBB-) depends on the
BBB score in the selected algorithm. When it
is more than zero, compound could be con-
sider as BBB+ and the best score is in the
range of 0.2-0.4. The scores of the designed
compounds were in the range of 0.139-0.681,
so it was deduced that all of the novel com-
Chemistry
The designed compounds were synthe-
sized according to the Figure 2, 4-Chloro-2-
phenoxybenzoic acid 1 was synthesized
through a nucleophilic aromatic substitution
reaction of 2,4-dichlorobenzoic acid and phe-
nol. Following esterification of 1, 4-chloro-2-
phenoxybenzohydrazide 3, was prepared in
satisfactory yield by the reaction of ester 2
with hydrazine hydrate at room temperature
(Rezaee et al., 2014a, b). Treatment of hydra-
zide 3 with corresponding benzaldehyde fol-
lowed by reaction with thioglycolic acid af-
forded final products (5a-5j) (Shingalapur et
al., 2010; Almasirad et al., 2006). The final
1
compounds were characterized through H-
NMR, FT-IR, Mass spectra and elemental
analysis.
57

EXCLI Journal 2017;16:52-62 – ISSN 1611-2156
Received: October 08, 2016, accepted: November 28, 2016, published: January 13, 2017
O
O
O
O
O
OH
O
OH
a
b
Cl
Cl
Cl
Cl
1
2
3
c
O
O
O
O
O
S
NH2
N
N
N
H
H
d
Cl
Cl
R
5a-5j
4
5a, R=H, 5b, R=4-OH, 5c, R=4-OH, 3,5=tbutyl
5d, R=4-N(CH3)2, 5e, R=2-Br, 5f, R=4-Cl
5g, R=3-NO2, 5h, R=4-NO2, 5i, R=4-OCH3
5j, R=4-CH3
Figure 2: Synthesis of the novel thiazolidinone compounds 5a-5j. (a) phenol, NaH 60 %, Cu, Dry DMF,
reflux, 20h, 61 %; (b) H2SO4, MeOH, reflux, 24h, 87 %; (c) NH2NH2.H2O, EtOH, rt, 24h, 93 %; (d) 1)
appropriate benzaldehyde, HCl 37 %, absolute EtOH, rt, 2-20h 2) thioglycolic acid, ZnCl2, dry toluene,
reflux, 20 h, 50-64 %
Table 1: Pharmacokinetic parameters important for good oral bioavailability of the synthesized com-
pounds 5a-5j
Compound
MW
425
441
553
468
504
459
470
470
455
439
Clogpa
3.33
2.99
6.15
3.23
4.05
3.94
2.41
2.41
3.26
3.68
TPSAb
58.64
78.87
78.87
61.88
58.64
58.64
104.47
104.47
67.88
58.64
HBDc
HBAd
NROTBe
LVf
0
5a
5b
5c
5d
5e
5f
1
2
2
1
1
1
1
1
1
1
5
6
6
6
5
5
8
8
6
5
5
5
7
6
5
5
6
6
6
5
0
2
0
1
0
5g
5h
5i
0
0
0
5j
0
a CLog P: logarithm of partition coefficient of compound between n-octanol and water
b Topological polar surface area.
c Number of hydrogen bond donors
d Number of hydrogen bond acceptors
e Number of rotatable bonds
f Lipinski’s Violation
58

EXCLI Journal 2017;16:52-62 – ISSN 1611-2156
Received: October 08, 2016, accepted: November 28, 2016, published: January 13, 2017
Table 2: ED50 of the novel thiazolidinone compounds and diazepam in PTZ and MES tests in mice
ED50 mg/kga
Compound
R
PTZ
MES
11.81 (7.62-19.18)b
33.22 (23/09-49.65)b
>100
5a
H
4-OH
>100
5b
>100
5c
4-OH,3,5-di-t-Bu
4-N(CH3)2
2-Br
>100
5d
>100
>100
5e
>100
>100
6.49 (4.62-9.08)b
57.61 (40.46-75.74)b
>100
20.00 (12.99-30.79)b
3.54(2.40- 5.20)b
>100
5f
4-Cl
5g
3-NO2
4-NO2
4-OCH3
4-CH3
>100
5h
>100
5i
5j
18.98 (15.41 - 23.06)b
>100
Diazepam
-
0.96 (0.66-1.20)b
1.02 (0.73-1.26)b
a n=10, 95 % confidence intervals in parenthesis.
b ED50 was significantly increased in the presence of flumazenil 10 mg/kg (p < 0.05).
Pharmacology
Potentiation of pentobarbital sleeping time
The anticonvulsant activity
Hypnotic effect of compound 5i was eval-
PTZ induced lethal convulsion test and uated by increasing pentobarbital sleeping
MES test were used for evaluation of anticon- time. According to Figure 3, the sleeping time
vulsant effects of the compounds. Results was increased by the injection of compound
shown in Table 2 indicate that, compounds 5i and the effect was dose-dependent. How-
5a, 5b, 5c, 5d, 5e, 5f, 5g, 5h and 5j were in- ever, the pharmacological effects of com-
active in PTZ model. Compound 5i was the pound 5i were blocked after injection of
only compound, which showed significant an- flumazenil, which indicates that BZD recep-
ticonvulsant effect in PTZ model. Com- tors are responsible for these effects.
pounds 5c, 5d, 5g and 5j had no significant
Passive avoidance test
anticonvulsant effect in the MES model but
The passive avoidance test was used to
other compounds, including 5i, showed sig-
evaluate the effects of compound 5i on ante-
nificant activity in this model. According to
rograde memory. As shown in Figure 4, com-
the lower ED50, it could be concluded that 5i
pound 5i in doses of 3.5 and 7 mg/kg had no
was the most potent compound between the
destructive effect on anterograde memory
novel thiazolidinone derivatives in the two
comparing to the control group, but in dose of
models. In both MES and PTZ tests, fluma-
14 mg/kg the anterograde amnesia was in-
zenil was able to significantly reduce the ef-
duced by this compound.
fect of the compounds with anticonvulsant ac-
tivity.
59

EXCLI Journal 2017;16:52-62 – ISSN 1611-2156
Received: October 08, 2016, accepted: November 28, 2016, published: January 13, 2017
In silico toxicity evaluation
The in silico toxicity risk assessment was
performed by means of OPE and except com-
pound 5e which showed high risk of tumor-
igenic effect all of them can be considered as
non-mutagenic, non-tumorigenic, non-irritant
without any reproductive effects.
Neurotoxicity evaluation
According to Figure 5, compound 5i ex-
hibited no neurotoxicity effect in doses of 3.5,
7 and 14 mg/kg. According to the BBB+
scores of compounds 5c, 5g and 5h, their in-
activity could be due to the low density in
CNS. As shown in Table 2 compound 5b and
5h are active in the MES model but their po-
tency is less than 5a, 5e and 5i. This may be
due to their lower concentrations in the CNS,
Figure 3: Effect of compound 5i on potentiation of
not less binding affinity to the receptor.
pentobarbital sleeping time: the time period of loss
of righting reflex, which is conidered as sleeping
time is presented. Results are shown as
mean±SEM. In all groups n=10. ns represents not
significant, * represents p<0.05, ** represents
p<0.01, and *** represents p<0.001 compared to
the control group. ### represents p<0.001com-
paring two indicated groups
Rotrod - 6rpm
80
60
40
20
0
Figure 5: Influence of the test compounds 5i (at
3.5, 7 and 14 mg/kg) on motor coordination of ex-
perimental animals measured in the rotarod test
revolving at 6 rpm. Results are shown as mean
time spent on the rotating rod.
CONCLUSION
Figure 4: Effect of compound 5i on memory
retrival in step-through passive avoidance
experiment: Results of the latencies to enter the
dark compartment on the second day are
presented. Results are shown as mean±SEM. In
all groups n = 10. ns represents not significant, **
represents p < 0.01, and *** represents p < 0.001
compared to the control group
In summary, the new 4-thiazolidinone de-
rivatives as novel BZD agonists were synthe-
sized and their biological effects were inves-
tigated. Some of the novel synthesized com-
pounds showed antiepileptic effects in PTZ
and MES tests. Compound 5i had the best
anticonvulsant activity among the novel 4-
60

EXCLI Journal 2017;16:52-62 – ISSN 1611-2156
Received: October 08, 2016, accepted: November 28, 2016, published: January 13, 2017
Faizi M, Sheikhha M, Ahangar N, Tabatabaei Ghomi
thiazolidinone compounds and revealed
H, Shafaghi B, Shafiee A, et al. Design, synthesis and
pharmacological evaluation of novel 2-[2-(2-
chlorophenoxy) phenyl]-1, 3, 4-oxadiazole derivatives
as benzodiazepine receptor agonists. Iran J Pharm Res.
2012;11:83-90.
considerable hypnotic effect. However, it did
not change the antrograde memory and did
not show neurotoxicity. Cosidering the fact
that the most clinically used benzodiazepins
have negative effect on memory, the novel 4-
Faizi M, Dabirian S, Tajali H, Ahmadi F, Rezaee E,
Shahhosseini S, et al. Novel agonists of benzodia-
zepine receptors: design, synthesis, binding assay and
pharmacological evaluation of 1, 2, 4-triazolo [1, 5-a]
pyrimidinone and 3-amino-1, 2, 4-triazole derivatives.
Bioorg Med Chem. 2015;23:480-7.
thiazolidinone
derivatives
(specially
compound 5i) can be used as leading
compound and related derivatives can be
designed and synthesized as novel ligands for
benzodiazepine
receptors
which
has
minimum effect on memory.
Guerrini G, Costanzo A, Ciciani G, Bruni F, Selleri S,
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Acknowledgements
This work was supported by a grant
(No.1271) from the Research Council of Sha-
hid Beheshti University of Medical Sciences.
Guerrini G, Ciciani G, Cambi G, Bruni F, Selleri S,
Besnard F, et al. Novel 3-iodo-8-ethoxypyrazolo [5, 1-
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Conflict of interest
The authors confirm that this article con-
tent has no conflict of interest.
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