Synthesis and pharmacological evaluation of new 2-substituted-5-{2-[(2- halobenzyl)thiophenyl}-1,3,4-oxadiazoles as anticonvulsant agents

Article abstract of DOI:10.3797/scipharm.0803-10

A new series of 2-substituted-5-{2-[(2-halobenzyl)thio)phenyl}-1,3,4- xadiazoles was designed, synthesized and investigated for anticonvulsant activities. The designed compounds contain the main essential pharmacophore for binding to the benzodiazepine receptors. Conformational analysis and superimposition of energy minima conformers of designed molecules on estazolam, a known benzodiazepine receptor agonist, revealed that the main characteristics of the proposed benzodiazepine pharmacophore were well matched. Electroshock and pentylenetetrazole-induced lethal convulsion tests showed that some of the synthesized compounds had significant anticonvulsant activity. The structure-activity relationship study of these compounds indicated that the introduction of an amino group at position 2 of 1,3,4-oxadiazole ring and a fluoro substituent at the ortho position of the benzylthio moiety had the best anticonvulsant activity. Anticonvulsant effects of active compounds were antagonized by flumazenil, a benzodiazepine antagonist, which establishes the involvement of benzodiazepine receptors in these effects. Oesterreichische Apotheker-Verlagsgesellschaft m. b. H.

Full text of DOI:10.3797/scipharm.0803-10

Sci Pharm. 2008; 76: 185–201
doi:10.3797/scipharm.0803-10
185
© Österreichische Apotheker-Verlagsgesellschaft m. b. H., Vienna, Austria
Reproduction is permitted for non-commercial purposes.
Synthesis and Pharmacological Evaluation of New
2-Substituted-5-{2-[(2-halobenzyl)thio)phenyl}-
1,3,4-oxadiazoles as Anticonvulsant Agents
1
1
Afshin ZARGHI * 1, Samaneh HAMEDI , Fatemeh TOOTOONI ,
1
2
2
Behzad AMINI , Behrang SHARIFI , Mehrdad FAIZI ,
1
3
Seyed Abbas TABATABAI , Abbas SHAFIEE
1 Department of Medicinal Chemistry, Faculty of Pharmacy,
Shahid Beheshti University (M.C), Tehran, Iran
2 Department of and Pharmacology, Faculty of Pharmacy,
Shahid Beheshti University (M.C), Tehran, Iran
3 Department of Medicinal Chemistry, Faculty of Pharmacy
and Pharmaceutical Sciences Research Center,
Tehran University of Medical Sciences , Tehran, Iran
Abstract
A
new series of 2-substituted-5-{2-[(2-halobenzyl)thio)phenyl}-1,3,4-
oxadiazoles was designed, synthesized and investigated for anticonvulsant
activities. The designed compounds contain the main essential pharmacophore for
binding to the benzodiazepine receptors. Conformational analysis and
superimposition of energy minima conformers of designed molecules on estazolam,
a known benzodiazepine receptor agonist, revealed that the main characteristics of
the proposed benzodiazepine pharmacophore were well matched. Electroshock
and pentylenetetrazole-induced lethal convulsion tests showed that some of the
synthesized compounds had significant anticonvulsant activity. The structure-
activity relationship study of these compounds indicated that the introduction of an
amino group at position 2 of 1,3,4-oxadiazole ring and a fluoro substituent at the
ortho position of the benzylthio moiety had the best anticonvulsant activity.
Anticonvulsant effects of active compounds were antagonized by flumazenil, a
* Corresponding author: E-mail: azarghi@yahoo.com (A. Zarghi).

186
A. Zarghi et al.:
benzodiazepine antagonist, which establishes the involvement of benzodiazepine
receptors in these effects.
Keywords
Benzodiazepine receptor • 1,3,4-Oxadiazoles • Anticonvulsant • SAR
Introduction
Benzodiazepine (BZD) receptor ligands allosterically modulate the action of
GABA on neuronal chloride ion flux, thus eliciting a wide variety of pharmacological
actions ranging a continuum from full agonists (anxiolytic, sedative/hypnotic, and
anticonvulsant activities) to inverse agonists (proconvulsant and anxiogenic
activities), and antagonists, which do not exhibit any pharmacological effects but
can antagonize the action of both agonists and inverse agonists [1–3]. The BZD
binding sites in the brain were identified and described by radioligand receptor
binding assays and originally it was found that only 1,4-BZD derivatives bind to
these receptors. It has since been shown that many groups of compounds bind to
the BZD receptor with high affinity, e. g., triazolopyridazines, cyclopyrrolones,
quinolines and β-carbolines [4–7]. Several pharmacophore models have been
proposed for BZDs, and amongst all models suggested for binding to the BZD
receptor at least two features are common: an aromatic ring and a coplanar proton
accepting group in suitable distance (5Ao). Also, the presence of a second out-of-
plane, aromatic ring could potentiate binding to the receptor [8–12]. On this basis, a
wide variety of compounds with a chemical structure different from that of
benzodiazepines have been synthesized and tested. We recently started a wide
research program aimed to design new BZD receptor ligands characterized by a
higher degree of flexibility compared with classic BZD ligands. Accordingly, we
reported several 1,3,4-oxadiazole derivatives which showed considerable
anticonvulsant activity [13–15]. As part of our ongoing research program to design
new anticonvulsant agents, we describe herein the synthesis and biological
evaluation of a novel group of 2-substituted-5-[2-(2-halobenzylthio)phenyl]-1,3,4-

Synthesis and Pharmacological Evaluation of New …
187
oxadiazoles (Fig.1) with a flexible second out-of-plane aromatic ring, benzylthio
group which has all the essential pharmacophore groups for binding to the BZD
receptors.
N
N
N
N
N
R1
R2
O
N
Cl
S
R1 = NH2, NHPh, SH, SMe, SEt, SBz
R2 = F, Cl
Estazolam
Fig. 1.
Results and Discussion
1. Chemistry
The target 1,3,4-oxadiazole derivatives were synthesized according to
Scheme 1. Accordingly, reaction of 2-[(2-halobenzyl)thio]benzoic acid methyl ester
1 [15] with hydrazine hydrate in DMF at room temperature afforded the
corresponding 2-[(2-halobenzyl)thio]benzoic acid hydrazide 2 in high yields (87–
90%). The hydrazides were converted to 5-{2-[(2-halobenzyl)thio]phenyl}-1,3,4-
oxadiazol-2-amines 3 using cyanic bromide in methanol (70–87%). 5-{2-[(2-
halobenzyl)thio]phenyl}-1,3,4-oxadiazole-2-thiols 4 were prepared by the reaction of
hydrazide 2 with carbon disulfide under basic condition (80–90%). Sonication of
compound 4 in the presence of a suitable alkyl halide in alkaline media afforded 5-
(alkylthio)-2-{2-[(2-halobenzyl)thio]phenyl}-1,3,4-oxadiazoles 5a–f (68–92%). Treat-
ment of hydrazide 2 with phenylisothiocyanate followed by reaction with KI/I2 in
alkaline hydro-ethanol (1:1 v/v) gave 5-{2-[(2-halobenzyl)thio]phenyl}-N-phenyl-
1,3,4-oxadiazol-2-amines 7 (60–70%). The purity of all products was determined by

188
A. Zarghi et al.:
thin layer chromatography using several solvent systems of different polarity. All
1
compounds were pure and stable. The compounds were characterized by H
nuclear magnetic resonance, infrared, mass spectrometry and CHN analysis.
O
CH3
O
S
X
1a, X = F
1b, X = Cl
NH2NH2 • H2O
O
S
N
N
N
N
NH2
CS2
SH
X
NH2
X
CNBr
N
H
O
O
KOH
S
X
S
4a, X = F
4b, X = Cl
2a, X = F
2b, X = Cl
3a, X = F
3b, X = Cl
RI
N
PhNCS
O
N
H
N
H
N
SR
X
N
H
O
S
X
S
S
6a, X = F
6b, X = Cl
5a, X = F, R = Me
5b, X = Cl, R = Me
5c, X = F, R = Et
5d, X = Cl, R = Et
5e, X = F, R = Bz
5f, X = Cl, R = Bz
NaOH
KI/I2
N
N
O
NH
X
S
7a, X = F
7b, X = Cl
Sch. 1.

Synthesis and Pharmacological Evaluation of New …
189
Physicochemical data of these compounds are summarized in Table 1.
Conformational analysis of the synthesized compounds and estazolam were
preliminarily performed by MMX force field method implemented in Hyperchem 7.0
software. The conformers were optimized further by AM1 calculation using the
MOPAC 6.0 program [21]. Global energy minima conformers of the designed
compounds were superimposed on the corresponding conformer of the estazolam
molecule which was considered as a reference BZD agonist.
Tab.1. Physical data of the synthesized compounds
N
N
R1
R2
O
S
mp (oC)
Molecular
formula
C15H12N3OSF
Compound
R1
R2
Yield (%)
Molecular weight
301.33
3a
3b
4a
4b
5a
5b
5c
5d
5e
5f
NH2
NH2
F
Cl
F
146–147
191–193
175–177
170–171
68
70
75
80
90
72
92
68
70
79
75
60
70
C15H12N3OSCl
C15H11N2OS2F
C15H11N2OS2Cl
C16H13N2OS2F
C16H13N2OS2Cl
C17H15N2OS2F
C17H15N2OS2Cl
C22H17N2OS2F
C22H17N2OS2Cl
C21H16N3OSF
C21H16N3OSCl
317.79
SH
318.38
SH
Cl
F
334.83
SCH3
SCH3
SC2H5
SC2H5
SBz
332.41
Cl
F
82–83
348.86
64–65
346.43
Cl
F
87
362.89
109–110
102–104
176–178
185–187
408.50
SBz
Cl
F
424.96
7a
7b
NHPh
NHPh
377.43
Cl
393.89
Satisfactory analysis for C, H, N was obtained for all the compounds within ± 0.4% of the
theoretical values.

190
A. Zarghi et al.:
2. Pharmacology
The BZD activity of the synthesized compounds was determined through the
evaluation of the ability of the compounds to protect mice against convulsion
induced by a lethal dose of PTZ and electroshock as two routine models. Diazepam
was considered as a reference BZD agonist with anticonvulsant effect in both
models. As shown in Table 2, compound 3a with an amino group on position 2 of
the oxadiazole ring and a fluoro subtituent at ortho position of the benzylthio moiety
has the best anticonvulsant activity in both PTZ and MES models. The activity was
antagonized with flumazenil, a benzodiazepine antagonist, which establishes the
involvement of benzodiazepine receptors in this effect. Figure 2 shows the
superimposition of energy minima conformers of the compound 3a, the most potent
synthesized analogues, and estazolam. Obviously, the main BZD pharmacophores,
aromatic rings and proton accepting groups (π1 interaction), nitrogen (N-3) of the
1,3,4-oxadiazole and triazolobenzodiazepine rings, are well matched.
Fig. 2.
Replacement of the fluoro substituent with a larger electron-withdrawing group
such as Cl (3b) decreases the activity which may be explained by a steric
hindrance effect. These results are in good agreement with the classical SAR data
of BZDs [22] and our previous studies on 1,3,4-oxadiazoles [14,15]. In the series of
2-alkylthio oxadiazoles, compounds 5a and 5b possessing a small alkylthio group
at C-2 of the 1,3,4-oxadiazole ring had good anticonvulsant activity in the MES

Synthesis and Pharmacological Evaluation of New …
191
model but showed mild activity against PTZ induced convulsion. Increasing the size
of the alkyl group (5c and 5f) significantly decreases the anticonvulsant activity in
both PTZ and MES models. Accordingly, compounds 5e and 5f with a bulky
benzylthio group did not show any anticonvulsant effects. Similarly, compounds 7a
and 7b did not have any considerable anticonvulsant activity in both models.
Therefore, the size and nature of groups at C-2 position of 1,3,4-oxadiazole ring are
very important for anticonvulsant activity in both PTZ and MES models. In addition,
the size of electron withdrawing substituents at ortho position of the benzylthio
moiety is also important for their anticonvulsant effects.
Tab. 2. Pharmacological evaluation of the synthesized compounds
N
N
R1
R2
O
S
ED50 mg/kga
R1
R2
Compoud
PTZ
MES`
20.2 (11.3–32.8)b
88.9 (74.7–106.8)bc
> 100
3a
NH2
NH2
F
Cl
F
57.0(43.3–71.8)b
3b
>100
4a
SH
> 100
4b
SH
Cl
F
> 100
> 100
5a
SCH3
SCH3
SC2H5
SC2H5
SBz
75.1 (59.8–88.2)b
95.1 (80.8–110.2)bc
>100
25.1 (14.7–38.5)b
34.4 (23.5–49.4)b
65.3 (52.6–78.7)b
91.4 (75.6–110.5)bc
> 100
5b
Cl
F
5c
5d
Cl
F
> 100
5e
> 100
5f
7a
SBz
Cl
F
> 100
> 100
NHPh
NHPh
> 100
> 100
7b
Cl
> 100
> 100
Diazepam
0.7 (0.6–0.9) b
0.8 (0.5-1.1)b
a n = 10, 95% Confidence limits in parentheses, LD50 of all compounds > 300 mg/kg.
b ED50 significantly increased in the presence of flumazenil 10 mg/kg (P<0.05).
c The highest concentration of the compounds tested was 150 mg/kg.

192
A. Zarghi et al.:
Experimental
1. Chemistry
Melting points (mp) were determined using a Thomas Hoover capillary
apparatus (Philadelphia, USA). Infrared spectra were acquired on a Perkin-Elmer
1420 ratio recording spectrometer. A Bruker FT-500 MHz instrument (Bruker
1
Biosciences, USA) was used to acquire H-NMR spectra; chloroform-d, DMSO-d6
and methanol-d4 were used as solvents. Mass spectra were acquired with a
Finnigan TSQ-70 mass spectrometer. Electron-impact ionization was performed at
an ionizing energy of 70 eV; the source temperature was 250 oC. Elemental
analyses were carried out with a Perkin Elmer Model 240-C apparatus (Perkin
Elmer, Norwalk, CT, USA). The results of the elemental analyses (C,H,N) were
within ± 0.4% of the calculated amounts. All chemicals and reagents were obtained
from Aldrich (USA) or VWR (Germany) and were used without further purification.
General procedure for preparation of 2-[(2-Halobenzyl)thio]benzoic acid
hydrazides 2
To a solution of 2-[(2-halobenzyl)thio]benzoic acid methyl ester 1 (10 mmol) in
DMF (10 ml), hydrazine hydrate (50 mmol) was added and stirred at room
temperature for 10 hours. After this time, 100 ml water was added and the solid
thus separated was filtered, dried and recrystallized from ethanol [16].
2-Fluoro-2-benzylthio benzoic acid hydrazide (2a)
Yield: 87%; mp: 105-106 °C; IR (KBr): ν (cm-1) 3310, 3200 (NH2), 1680 (C=O)
Mass, m/z (%): 275.9 (M+, 20), 245 (30), 167 (50), 141.1 (20), 136.1 (30), 109.0
(100), 83.1 (60).
2-Chloro-2-benzylthio benzoic acid hydrazide (2b)
Yield: 90%; mp: 106 °C; IR (KBr): ν (cm-1) 3300, 3200 (NH2), 1680 (C=O)
Mass, m/z (%): 292.5 (M+, 30), 261.5 (40), 157.6 (55), 125.1 (100), 89.1 (70).
General procedure for preparation of 5-{2-[(2-Halobenzyl)thio]phenyl}-1,3,4-
oxadiazol-2-amines 3

Synthesis and Pharmacological Evaluation of New …
193
To a methanolic solution of 2 (5.1 mmol), cyanogens bromide (7.5 mmol) was
added. The reaction mixture was stirred at room temperature for 3 hours. The
resulting solution was neutralized with sodium bicarbonate solution. The solid thus
separated out was filtered, washed with water, dried and recrystallized from ethanol
[17].
5-{2-[(2-Fluorobenzyl)thio]phenyl}-1,3,4-oxadiazol-2-amine (3a)
Yield: 70%; mp: 146-147 °C; IR (KBr): ν (cm-1) 3380, 3290 (NH2), 1650 (C=N);
1HNMR (methanol-D4): δ (ppm) 4.23 (s, 2H, CH2), 7.01-7.31 (m, 4H, 2-fluorobenzyl
H4-H6, Phenyl H3), 7.34 (dd, 1H, Phenyl H5), 7.49 (dd, 1H, Phenyl H4), 7.56 (dd, 1H,
2-fluorophenyl H3), 7.76 (d, 1H, Phenyl H6, J= 7.8 Hz) ; Mass, m/z (%): 301.3 (M+,
20), 284.1 (20), 242.1 (25), 228.1 (50), 192.1 (30), 150.1 (50), 109 (100), 89.1 (40).
Anal. Calcd. for C15H12N3OSF: C, 59.79; H, 4.01; N, 13.94. Found: C, 59.85; H,
4.12; N, 13.85.
5-{2-[(2-Chlorobenzyl)thio]phenyl}-1,3,4-oxadiazol-2-amine (3b)
Yield: 87%; mp: 191-193 °C; IR (KBr): ν (cm-1) 3350, 3190 (NH2), 1660 (C=N);
1HNMR (methanol-D4): δ (ppm) 4.31 (s, 2H, CH2), 7.15-7.26 (m, 4H, 2-chlorobenzyl
H4-H6, Phenyl H3), 7.37 (d, 1H, 2-chlorobenzyl H3, J= 7.8 Hz), 7.41 (dd, 1H, Phenyl
H5), 7.56 (dd, 1H, Phenyl H4), 7.80 (d, 1H, Phenyl H6, J= 7.8 Hz); Mass, m/z (%):
317.1 (M+,10), 292.1 (20), 260.1 (20), 125.0 (100), 109.1 (50), 89.1 (100). Anal.
Calcd. for C15H12N3OSCl: C, 56.69; H, 3.81; N, 13.22. Found: C, 56.80; H, 3.92; N,
13.18.
General procedure for preparation of 5-{2-[(2-Halobenzyl)thio]phenyl}-1,3,4-
oxadiazole-2-thiols 4
A mixture of 2 (3.6 mmol), KOH (10 mmol) and carbon disulfide (10 mmol) in
ethanol (10 ml) was refluxed on a steam bath for 12 hours. The solution was then
concentrated, cooled and acidified with dilute HCl. The solid product that separated
was filtered, washed with cold ethanol, dried and recrystallized from ethanol [18].
5-{2-[(2-Fluorobenzyl)thio]phenyl}-1,3,4-oxadiazole-2-thiol (4a)

194
A. Zarghi et al.:
Yield: 80%; mp: 175-177 °C; IR (KBr): ν (cm-1) 2550 (SH), 1600-1490
(Aromatic); 1HNMR (DMSO-D6): δ (ppm) 4.33 (s, 2H, CH2), 7.03-7.46 (m, 6H, ArH),
7.58 (dd, 1H, 2-fluorobenzyl H3), 7.72 (d, Phenyl H6, J=7.8 Hz); Mass, m/z (%):
318.3 (M+, 20), 285.1 (30), 243.1 (20), 228.1 (30), 193.1 (10), 150.1 (60), 136 (90),
109 (100), 89.1 (45). Anal. Calcd. for C15H11N2OS2F: C, 56.58; H, 3.48; N, 8.80.
Found: C, 56.75; H, 3.55; N, 8.74.
5-{2-[(2-Chlorobenzyl)thio]phenyl}-1,3,4-oxadiazole-2-thiol (4b)
Yield: 90%; mp: 170-171 °C; IR (KBr): ν (cm-1) 2580 (SH), 1590-1480
(Aromatic);1HNMR (DMSO-D6): δ (ppm) 4.33 (s, 2H, CH2), 7.03-7.45 (m, 6H, ArH),
7.56 (d, 1H, 2-chlorobenzyl H3, J=7.9 Hz), 7.72 (d, Phenyl H6, J=7.8 Hz); Mass, m/z
(%): 335.0 (M+, 70), 301.1 (20), 247.1 (30), 209.1 (20), 151.1 (25), 125 (100), 89.1
(40). Anal. Calcd. for C15H11N2OS2Cl: C, 53.80; H, 3.31; N, 8.37. Found: C, 53.96;
H, 3.45; N, 8.24.
General procedurefor preparation of 5-(Alkylthio)-2-{2-[(2-
halobenzyl)thio]phenyl}-1,3,4-oxadiazoles 5
An ethanolic solution of appropriate alkyl iodide (3.0 mmol) was added to a
solution of compound 4 (2.4 mmol) in 10% aqueous sodium hydroxide (5 ml), and
the mixture was sonicated in a sonication bath for 30-60 minutes. The reaction
mixture was poured into cold water (50 ml) and the solid thus separated was
filtered, washed with water, dried and recrystallized from ethanol [19].
2-{2-[(2-Fluorobenzyl)thio]phenyl}-5-(methylthio)-1,3,4-oxadiazole (5a)
1
Yield: 72%; mp: 68 °C; IR (KBr): ν (cm-1) 1590-1460 (Aromatic); HNMR
(CDCl3): δ (ppm) 2.81 (s, 3H, CH3), 4.27 (s, 2H, CH2), 7.08-7.48 (m, 6H, ArH), 7.61
(dd, 1H, 2-fluorobenzyl H3), 7.88 (d, Phenyl H6, J=7.8 Hz); Mass, m/z (%): 332.4
(M+, 20), 285.2 (30), 228.1 (40), 183.1 (20), 150.1 (60), 109.1 (100), 83.1 (60).
Anal. Calcd. for C16H13N2OS2F: C, 57.81; H, 3.94; N, 8.43. Found: C, 57.96; H,
4.02; N, 8.61.
2-{2-[(2-Chlorobenzyl)thio]phenyl}-5-(methylthio)-1,3,4-oxadiazole (5b)

Synthesis and Pharmacological Evaluation of New …
195
1
Yield: 92%; mp: 82-83 °C; IR (KBr): ν (cm-1) 1580-1480 (Aromatic); HNMR
(CDCl3): δ (ppm) 2.77 (s, 3H, CH3), 4.30 (s, 2H, CH2), 7.15-7.47 (m, 6H, ArH), 7.57
(d, 1H, 2-chlorobenzyl H3, J=7.9 Hz), 7.85 (d, Phenyl H6, J=7.8 Hz); Mass, m/z (%):
348.9 (M+, 70), 301.1 (85), 244.1 (50), 223.1 (20), 150.1 (60), 125 (100), 89.1 (40).
Anal. Calcd. for C16H13N2OS2Cl: C, 55.08; H, 3.76; N, 8.03. Found: C, 55.16; H,
3.88; N, 8.01.
2-(Ethylthio)-5-{2-[(2-fluorobenzyl)thio]phenyl}-1,3,4-oxadiazole(5c)
1
Yield: 68%; mp: 64-65 °C; IR (KBr): ν (cm-1) 1570-1480 (Aromatic); HNMR
(CDCl3): δ (ppm) 1.57 (t, 3H, CH3), 3.36 (q, 2H, CH2), 4.28 (s, 2H, SCH2), 7.08-7.46
(m, 6H, ArH), 7.60 (dd, 1H, 2-fluorobenzyl H3), 7.90 (d, Phenyl H6, J=7.8 Hz); Mass,
m/z (%): 346.4 (M+, 10), 301.3 (20), 228.1 (20), 150.1 (60), 109.1 (100), 83.1 (50).
Anal. Calcd. for C17H15N2OS2F: C, 58.94; H, 4.36; N, 8.09. Found: C, 58.85; H,
4.12; N, 8.15.
2-{2-[(2-Chlorobenzyl)thio]phenyl}-5-(ethylthio)-1,3,4-oxadiazole (5d)
1
Yield: 70%; mp: 87 °C; IR (KBr): ν (cm-1) 1580-1470 (Aromatic); HNMR
(CDCl3): δ (ppm) 1.52 (t, 3H, CH3), 3.31 (q, 2H, CH2), 4.31 (s, 2H, SCH2), 7.16-7.44
(m, 8H, ArH), 7.55 (d, 1H, 2-chlorobenzyl H3, J=7.9 Hz), 7.86 (d, Phenyl H6, J=7.8) ;
Mass, m/z (%): 362.9 (M+, 20), 301.1 (100), 244.1 (50), 150.1 (60), 125 (90), 89.1
(30). Anal. Calcd. for C17H15N2OS2Cl: C, 56.26; H, 4.17; N, 7.72. Found: C, 56.35;
H, 4.22; N, 7.66.
2-(Benzylthio)-5-{2-[(2-fluorobenzyl)thio]phenyl}-1,3,4-oxadiazole (5e)
Yield: 79%; mp: 109-110 °C; IR (KBr): ν (cm-1) 1600-1480 (Aromatic); 1HNMR
(CDCl3): δ (ppm) 4.27 (s, 2H, SCH2), 4.59 (s, 2H, CH2), 7.08-7.54 (m, 11H, ArH),
7.62 (dd, 1H, 2-fluorobenzyl H3), 7.87 (d, Phenyl H6, J=7.8 Hz); Mass, m/z (%):
408.5 (M+, 20), 317.1 (20), 285.4 (20), 150.1 (30), 125 (40), 91.1 (100). Anal.
Calcd. for C22H17N2OS2F: C, 64.68; H, 4.19; N, 6.86. Found: C, 64.75; H, 4.24; N,
6.77.
2-(Benzylthio)-5-{2-[(2-chlorobenzyl)thio]phenyl}-1,3,4-oxadiazole (5f)

196
A. Zarghi et al.:
Yield: 75%; mp: 102-104 °C; IR (KBr): ν (cm-1) 1580-1490 (Aromatic); 1HNMR
(CDCl3): δ (ppm) 4.30 (s, 2H, SCH2), 4.52 (s, 2H, CH2), 7.13-7.48 (m, 11H, ArH),
7.52 (d, 1H, 2-chlorobenzyl H3, J=7.9 Hz), 7.82 (d, Phenyl H6, J=7.8) ; Mass, m/z
(%): 425 (M+, 45), 301.1 (30), 244.1 (20), 150.1 (10), 125 (40), 91.1 (100). Anal.
Calcd. for C22H17N2OS2Cl: C, 62.18; H, 4.03; N, 6.59. Found: C, 62.25; H, 4.10; N,
6.57.
General procedure for preparation of 5-{2-[(2-Halobenzyl)thio]phenyl}-N-
phenyl-1,3,4-oxadiazol-2-amines 7
A mixture of 2 (8.5 mmol), phenyl isothiocyanate (8.5 mmol) and dry THF (50
ml) was stirred at room temperature for 10 hours. It was then concentrated and
cooled. The obtained solid was filtered and dispersed in ethanol (50 ml) and to this
suspension, aqueous sodium hydroxide (5 N, 8 ml) was added with stirring to
obtain a clear solution. To this solution, iodine in potassium iodide solution (5%)
was added gradually with stirring till the colour of iodine persisted at room
temperature. The reaction mixture was refluxed for 2 hour on a steam bath. It was
then cooled and poured onto crushed ice. The solid product that separated was
filtered, dried and recrystallized from ethanol [20].
5-{2-[(2-Fluorobenzyl)thio]phenyl}-N-phenyl-1,3,4-oxadiazol-2-amine (7a)
Yield: 60%; mp: 176-178 °C; IR (KBr): ν (cm-1) 1580-1490 (Aromatic); 1HNMR
(CDCl3): δ (ppm) 4.30 (s, 2H, SCH2), 7.07 (brs, 1H, NH), 7.13-7.48 (m, 11H, ArH),
7.61 (dd, 1H, 2-fluorobenzyl H3), 7.82 (d, Phenyloxadiazole H6, J=7.8 Hz); Mass,
m/z (%): 377.4 (M+, 10), 301.4 (30), 268.1 (20), 150.1 (10), 125 (40), 91.1 (100).
Anal. Calcd. for C21H16N3OSF: C, 66.83; H, 4.27; N, 11.13. Found: C, 66.95; H,
4.34; N, 11.11.
5-{2-[(2-Chlorobenzyl)thio]phenyl}-N-phenyl-1,3,4-oxadiazol-2-amine (7b)
Yield: 70%; mp: 185-187 °C; IR (KBr): ν (cm-1) 3200 (NH), 1560-1460
1
(Aromatic); HNMR (CDCl3): δ (ppm) 4.30 (s, 2H, SCH2), 7.05 (brs, 1H, NH), 7.10-
7.45 (m, 11H, ArH), 7.58 (d, 1H, 2-chlorobenzyl H3, J= 7.8 Hz), 7.89 (d,

Synthesis and Pharmacological Evaluation of New …
197
Phenyloxadiazole H6, J=7.8 Hz); Mass, m/z (%): 393.8 (M+, 10), 317.7 (10), 246.7
(10), 230.8 (20) 150.1 (20), 89.1 (100). Anal. Calcd. for C21H16N3OSCl: C, 64.03; H,
4.09; N, 10.67. Found: C, 64.10; H, 4.14; N, 10.62.
2. Pharmacology
Anticonvulsant evaluation of the synthesized compounds was performed
following the standard procedure provided by the antiepileptic drug development
program [23, 24]. It includes qualitative assays using MES and PTZ tests. The first
assay is related to electrical induction of seizure and the second test generates
convulsion by chemical induction. The compounds were administered to adult male
albino mice (25–30 g) intraperitoneally at four or five doses (0.1,1, 10, and 100 or
150 mg/kg), and all the results are summarized in Table 2. Diazepam and all tested
compounds were dissolved in DMSO. The synthesized compounds, diazepam or
vehicle were administered 30 min before injection of PTZ 100 mg/kg or application
of electroshock (60 Hz, 37.2 mA and 0.25 s). After 30 min, the dead mice were
counted in PTZ test and occurrences of HLTE (hind limb tonic extension) were
observed in MES Model. In general, the dose-response curves were estimated by
testing three or four doses using at least 10 mice for each dose. To clarify the mode
of action of the synthesized compounds, the effects of flumazenil (10 mg/kg), a
BZD receptor antagonist, on the anticonvulsant activity of the compounds were
determined. The Institutional Animal Ethics committee approved the protocol
adopted for the experimentation of animals.
3. Statistical analysis
Statistical analysis of the anticonvulsant activity of the synthesized compounds
on animals was evaluated using a one-way analysis of variance (ANOVA). In all
cases, post-hoc comparisons of the means of individual groups were performed
using Fisher’s Exact Probability test. Differences with P< 0.05 between
experimental groups at each point were considered statistically significant. All
values were expressed as mean ± SD (standard deviations). For statistical analysis

198
A. Zarghi et al.:
we used SPSS Software 13.0 version. (SPSS Software 13.0 version, Inc. Chicago,
Ilinois, USA).
Conclusions
The results of this investigation indicate that 1,3,4-oxadiazoles having an
amino group at C-2 position with a benzylthio moiety possessing a suitable ortho
electron withdrawing substituent can show moderate benzodiazepine activity
confirming the suggested SARs for benzodiazepine agonists.
Acknowledgment
This work was partially supported by a grant from the research council of
Shahid Beheshti University of Medical sciences.
Rererences
[1]
Sieghart W.
Structure, Pharmacology, and Function of GABAA Receptor Subtypes.
Adv Pharmacol. 2006; 54: 231–263.
[doi:10.1016/S1054-3589(06)54010-4]
[2]
[3]
Gardner C R.
Interpretation of the Behavioral Effects of Benzodiazepine Receptor Ligands.
Drugs Fut. 1989; 14: 51–67.
Mohler H.
GABAA Receptors in Central Nervous System Disease: Anxiety, Epilepsy,
and Insomnia.
J Recept Signal Transduct Res. 2006; 26: 731–740.
[doi:10.1080/10799890600920035]
[4]
[5]
Yokoyama N, Ritter B, Neubert A D.
2-Arypyrazolo [4,3-c]-quinolin-3-ones: Novel Agonist, Partial Agonist, and
Antagonist of Benzodiazepines.
J Med Chem. 1982; 25: 337–339.
[doi:10.1021/jm00346a002]
Allen M S, Hagen T J, Trrudell M L, Codding P W, Skolnick P, Cook J M.
Syntheses of Novel 3-Substituted β-Carboline as Benzodiazepine Receptor
Ligands: Probing the Benzodiazepine Receptor Pharmacophore.
J Med Chem. 1988; 31: 1854–1861.
[doi:10.1021/jm00117a029]

Synthesis and Pharmacological Evaluation of New …
199
[6]
Shindo H, Takada S, Murata S, Eigyo M, Matsushita A.
Thienylpyrazoloquinolines with High Affinity to Benzodiazepine Receptors:
Continuous Shift from Inverse Agonist to Agonist Properties Depending on
the Size of the Alkyl Substituent.
J Med Chem. 1989; 32: 1213–1217.
[doi:10.1021/jm00126a012]
[7]
[8]
[9]
Trudell M L, Lifer S L, Tan Y C, Martin M J, Deng L, Skolnick P, Cook J M.
Synthesis of Substituted 7,12-Dihydropyrido [3,2-b: 5,4-b] Diindoles: Rigid
Planar Benzodiazepine Ligands with Inverse Agonist/Antagonist Properties.
J Med Chem. 1990; 33: 2412–2420.
[doi:10.1021/jm00171a015]
Ghose A K, Crippen G M.
Modeling the Benzodiazepine Receptor Binding Site by the General Three-
Dimensional Structure-Directed Quantitative Structure-Activity Relationship
Method REMOTEDISC.
Mol Pharmacol. 1990; 37: 725–734.
Codding P W, Muir A K.
Molecular Structure of Ro15-1788 and a Model for the Binding of
Benzodiazepine Receptor Ligands. Structural Identification of Common
Features in Antagonists.
Mol Pharmacol. 1985; 28: 178–184.
[10] Fryer R I, Cook C, Gilman N W, Walser A.
Conformational Shifts at the Benzodiazepine Receptor Related to the Binding
of Agonists, Antagonists, and Inverse Agonists.
Life Sci. 1986; 39: 1947–1957.
[doi:10.1016/0024-3205(86)90318-8]
[11] Diaz-Arauzo H, Evoniuk G E, Skolnick P, Cook J M.
The Agonist Pharmacophore of the Benzodiazepine Receptor. Synthesis of a
Selective Anticonvulsant/Anxiolytic.
J Med Chem. 1991; 34: 1754–1756.
[doi:10.1021/jm00109a035]
[12] Diaz-Arauzo H, Koehler K F, Hagan T J, Cook J M.
Synthetic and Computer Assisted Analysis of the Pharmacophore for
Agonists at Benzodiazepine Receptors.
Life Sci. 1991; 49: 207–216.
[doi:10.1016/0024-3205(91)90005-V]
[13] Almasirad A, Tabatabai S A, Faizi M, Kebriaeezadeh A, Mehrabi N, Dalvandi
A, Shafiee A.
Synthesis and Anticonvulsant Activity of New 2-Substituted-5- [2-(2-
fluorophenoxy)phenyl]-1,3,4-Oxadiazoles and 1,2,4-Triazoles.
Bioorg Med Chem. Lett. 2004; 14: 6057–6059.
[doi:10.1016/j.bmcl.2004.09.072]

200
A. Zarghi et al.:
[14] Zarghi A, Tabatabai S A, Faizi M,Ahadian A, Navabi P,Zanganeh V,Shafiee,
A.
Synthesis and Anticonvulsant Activity of New 2-Substituted-5-(2-
Benzyloxyphenyl)-1,3,4-Oxadiazoles.
Bioorg Med Chem Lett. 2005; 15:1863–1865.
[doi:10.1016/j.bmcl.2005.02.014]
[15] Zarghi A, Faizi M, Shafaghi B, Ahadian A, Khojastehpoor H R, Zanganeh V,
Shafiee A.
Design and Synthesis of New 2-Substituted-5-(2-Benzylthiophenyl)-1,3,4-
Oxadiazoles as Benzodiazepine Receptor Agonists.
Bioorg Med Chem Lett. 2005; 15: 3126–3129.
[doi:10.1016/j.bmcl.2005.04.018]
[16] Firoozi F, Javidnia K, Kamal M, Fooladi A, Foroumadi A, Shafiee A.
Syntheses of 1-Methyl-2-(1,3,4-Thiadiazol-2-yl)4-Nitro-Pyrroles and 2-(1,3,4-
Oxadiazole-2-yl)4-Nitropyrroles.
J Heterocycl Chem. 1995; 32: 123–128.
[17] Alekseeva V Y, Viktorovskii I V, Yakovlev S I, Vyunov K A.
Reaction of 2-Amino-5-Aryl-1,3,4-Oxadiazoles with Compounds with
Activated Double Bonds.
Chem Heterocycl Comp. 1990; 26: 689–693.
[doi:10.1007/BF00756427]
[18] Almasirad A, Sheikha M, Hosseini R, Tabatabai S A, Shafiee A.
Synthesisand Morphine Enhancement Activity of N-[5-(2-Phenoxyphenyl)-
1,3,4-Oxadiazole-2-yl]-N'-Phenylurea Derivatives.
Arch Pharm. 2004; 337: 193–200.
[doi:10.1002/ardp.200300822]
[19] Shafiee A, Naimi E, Mansoobi P, Foroumadi A, Shekari M.
Syntheses of Substituted Oxazolo –1,3,4-Thiadiazoles, 1,3,4- Oxadiazoles
and 1,2,4-Triazoles.
J Heterocycl Chem. 1995; 32: 1235–1239.
[20] Amir M, Shikha K.
Synthesis and Anti-inflammatory, Analgesic, Ulcerogenic and Lipid
Peroxidation Activities of Some New 2-[(2,6-Dichloroanilino) Phenyl]acetic
Acid Derivatives.
Eur J Med Chem. 2004; 39: 535–545.
[doi:10.1016/j.ejmech.2004.02.008]
[21] QCPE; Department of Chemistry: Indiana University, Bloomington, IN 47405,
USA.
[22] Fryer R I,
In Comprehensive Medicinal Chemistry;
Hansch, C., Ed. Pergamon: Oxford,Vol 3, p 539 (1990)

Synthesis and Pharmacological Evaluation of New …
201
[23] Krall R L, Penry J K, White B G, Kupferberg H J, Swinyard E A
Antiepileptic Drug Development: II. Anticonvulsant Drug Screening.
Epilepsia. 1978; 19: 409–428.
[doi:10.1111/j.1528-1157.1978.tb04507.x]
[24] Porter R J, Hessie B J, Cereghino J J, Gladding G D, Kupferberg H J,
Scoville B, White B G.
Advances in the Clinical Development of Antiepileptic Drugs.
Fed Proc. 1985; 44: 2645–2649.
Received March 16th, 2008
Accepted (after revision) April 14th, 2008
Available online at www.scipharm.at April 20th, 2008