Design and synthesis of 4H-3-(2-phenoxy)phenyl-1,2,4-triazole derivatives as benzodiazepine receptor agonists

Article abstract of DOI:10.1016/S0968-0896(02)00469-8

A series of new 5-substituted analogues of 4H-3-(2-phenoxy)phenyl-1,2,4-triazole and its chlorinated derivatives was designed and prepared. Conformational analysis and superimposition of energy minima conformers of the compounds on estazolam, a known benz

Full text of DOI:10.1016/S0968-0896(02)00469-8

Bioorganic & Medicinal Chemistry 11 (2003) 769–773
Design and Synthesis of 4H-3-(2-Phenoxy)phenyl-1,2,4-triazole
Derivatives as Benzodiazepine Receptor Agonists
Tahmineh Akbarzadeh,a Sayyed A. Tabatabai,b Mohammad J. Khoshnoud,c
Bijan Shafaghic and Abbas Shafieea,*
aDepartment of Medicinal Chemistry, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran
bDepartment of Medicinal Chemistry, Faculty of Pharmacy, Shaheed Beheshti University of Medical Sciences, Tehran, Iran
cDepartment of Pharmacology and Toxicology, Faculty of Pharmacy, Shaheed Beheshti University of Medical Sciences, Tehran, Iran
Received 8 July 2002; accepted 19 September 2002
Abstract—A series of new 5-substituted analogues of 4H-3-(2-phenoxy)phenyl-1,2,4-triazole and its chlorinated derivatives was
designed and prepared. Conformational analysis and superimposition of energy minima conformers of the compounds on estazo-
lam, a known benzodiazepine receptor agonist, revealed that the main proposed benzodiazepine pharmacophores were well mat-
ched. Rotarod and pentylenetetrazole-induced lethal convulsion tests showed that the introduction of an amino group in position 5
of 1,2,4-triazole ring especially in chlorinated derivatives had the best effect which was comparable with diazepam.
# 2002 Elsevier Science Ltd. All rights reserved.
Results
Introduction
Chemistry
For the past two decades, structure activity relationship
of benzodiazepine ligands, have made considerable
progress.1ꢀ6 Amongst all models suggested for binding
to the benzodiazepine receptor at least two features are
common: an aromatic ring and a coplanar proton-
accepting group in suitable distance. Also, the presence
of a second out-of-plane, aromatic ring could potentiate
binding to the receptor.7,8 On this basis, compounds 1
(Fig. 1), with a simple non-rigid structure were designed
which had all the suggested requirements for binding to
the benzodiazepine receptors. To clarify whether the
designed compounds could mimic the structure of a
benzodiazepine agonist, conformational analysis on
designed molecules as well as a known benzodiazepine
agonist, estazolam (Fig. 1) was performed followed by
superimposition of energy minima conformers. As an in
vivo model for evaluating benzodiazepine effects,
rotarod9 and pentylenetetrazole (PTZ)-induced lethal
convulsion10 tests were performed on synthesized com-
pounds, and the results were compared with diazepam,
a known benzodiazepine agonist.
The designed compounds were synthesized according to
Scheme 1. Reaction of 2-phenoxybenzoic acids 111,12
with thionyl chloride at 50–55 ꢁC gave corresponding
acid chlorides,13 the key intermediates in the prepara-
tion of 1,2,4-triazoles. Acid chlorides were converted to
3-amino-5-(2-phenoxyphenyl)-1,2,4-triazoles 2, by addi-
tion of aminoguanidine hydrogen carbonate followed
by cyclization with 5% aqueous solution of sodium
hydroxide. Reaction of acid chlorides with thiosemi-
carbazide followed by treatment with a solution of
sodium hydroxide afforded 5-(2-phenoxyphenyl)-1,2,4-
triazole-3-thiones 3 in good yield.14 Sonicating com-
pounds 3 in the presence of suitable alkyl halides in
alkaline media afforded 3-alkylthio-5-(2-phenox-
yphenyl)-1,2,4-triazoles 4 in 3–5 min. Oxidation of
alkylthio derivatives 4 with m-chloroperbenzoic acid
(MCPBA) gave corresponding 3-alkylsulfonyl-5-(2-phe-
noxyphenyl)-1,2,4-triazoles 5.14
2-Phenoxybenzoic acid hydrazides were readily prepared
via esterification of corresponding benzoic acid deriva-
tives, followed by treatment with hydrazine hydrate in
methanol.14,15 The hydrazides were converted to 2-amino-
5-(2-phenoxyphenyl)-1,3,4-oxadiazoles 6 which rear-
ranged to 3-ethoxy-5-(2-phenoxyphenyl)-1,2,4-triazoles 7
*Corresponding author. Tel.: +98-21-640-6757; fax: +98-21-646-
1178; e-mail: ashafiee@ams.ac.ir
0968-0896/03/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(02)00469-8

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T.Akbarzadeh et al./ Bioorg.Med.Chem.11 (2003) 769–773
main benzodiazepine pharmacophores, aromatic rings
and proton accepting groups, number 2 nitrogen of the
1,2,4-triazole and triazolobenzodiazepine rings, are well
matched.
Pharmacological evaluation
Benzodiazepine activity of the compounds was deter-
mined through two routine models, rotarod test and
evaluation of the ability of the synthesized compounds
to protect mice against a lethal dose of a convulsant
agent, pentylenetetrazole (PTZ). Diazepam was con-
sidered as a reference benzodiazepine agonist in both
models (Table 1). The results show that analogous with
chloro substituent on position 2 of phenoxy group and
position 4 of phenyl ring are more potent than the
corresponding unsubstituted compounds. Amongst
substituents that introduced on position 5 of the 1,2,4-
triazole ring, amino group (2b) had the best effect. The
following rank order for the contribution of sub-
stituents to the activity of the synthesized compounds
was observed:
Figure 1. The structure of designed compounds 1 and estazolam.
upon treatment with ethanolic potassium hydroxide.
Acid hydrolysis of 7 provided 5-(2-phenoxyphenyl)-
1,2,4-triazole-3-ones 8.16
Conformational analysis
Conformations of the synthesized compounds were ana-
lyzed through MMX force field followed by AM1 calcu-
lation. The procedure was also performed on a known
benzodiazepine agonist, estazolam, as a reference com-
pound. Figure 2 shows energy minima conformers of the
compound 2b, the most potent synthesized analogues,
and estazolam, which are superimposed. Obviously, the
NH2 > OEt > SMe > SO2Me > SO2Bz > SH; OH:
The activity of the compounds was significantly reduced
by flumazenil, a benzodiazepine antagonist.
Scheme 1. Reagents: (i) (1) SOCl2; (2) aminoguanidine hydrogen carbonate; (3) NaOH 5%; (ii) (1) SOCl2; (2) thiosemicarbazide; (3) NaOH 5%; (iii)
CH3I or PhCH2Br, NaOH 10%; (iv) MCPBA; (v) (1) MeOH, H2SO4; (2) NH2NH2.H2O; (3) BrCN, NaHCO3; (vi) KOH, EtOH; (vii) HCl.

T.Akbarzadeh et al./ Bioorg.Med.Chem.11 (2003) 769–773
771
Discussion
The results presented here are part of our efforts to
design simple non-rigid structures with benzodiazepine
activity based on proposed SAR.
The designed structures have the main benzodiazepine
pharmacophores: an aromatic ring and a coplanar pro-
ton-accepting group, number 2 nitrogen of 1,2,4-triazole
ring. A second out-of-plane aromatic ring, phenoxy
group, could potentiate binding to the receptor.7
In order to confirm whether the designed compounds
could mimic proper conformation for binding to the
benzodiazepine receptor, conformational analysis was
performed on synthesized compounds and a known
benzodiazepine agonists, estazolam.
Superimposition of energy minima conformers shows
that the main proposed pharmacofores are well matched.
Chloro substituted analogues on position 2 of phenoxy
group and position 4 of phenyl ring are more potent
than corresponding unsubstituted compounds. These
two positions are well matched to positions 20 and 7 of
the benzodiazepine ring; it has been established that
electron withdrawing substituent on these positions
enhance the activity.1 These confirm the results of the
superimposition studies. Amino group was the best
substituent for the 1,2,4-triazole ring, and compound 2b
was comparable with diazepam especially in rotarod
test.
Figure 2. Stereoview of the superimposition of the energy minima
conformers of estazolam (top right) and compound 2b (top left).
Table 1. Pharmacological evaluation of synthesized 1,2,4-triazoles
The fact that the activity of the compounds is sig-
nificantly reduced by flumazenil, a benzodiazepine
antagonist, confirms that this effect is mediated through
benzodiazepine receptors.
Conclusion
Compd
RX
ED
50 mg/kga
PTZ- induced lethal
convulsion test
The study indicates that some synthesized 1,2,4-triazoles
with a simple non-rigid structure in which the proposed
pharmacophores have a proper steric direction could
show benzodiazepine activity comparable with diaze-
pam confirming the suggested SARfor benzodiazepine
agonists. This could lead us to the new class of benzo-
diazepine receptor ligands.
Rotarod test
Diazepam
0.6 (0.3–1.0)b
24.0 (15.5–36.3)b
3.4 (0.1–8.3)b
>100
1.0 (0.6–1.5)b
100
12.0 (8.4–17.0)b
100
2a
2b
3a
3b
4a
4b
4c
4d
5a
5b
5c
5d
7a
7b
8a
8b
NH2
NH2
SH
H
Cl
H
Cl
H
Cl
H
Cl
H
SH
>100
100
100
SMe
SMe
SBz
SBz
SO2Me
SO2Me Cl
SO2Bz
SO2Bz Cl
OEt
OEt
OH
OH
89.0 (53.8–192.6)b
22.5 (7.5–39.8)b
NDc
61.9 (34.2–119.4)b
ND
100
100
Experimental
Chemistry
>100
89.4 (28.9–>1000)b
24.7 (4.9–49.1)b
>100
87.0 (63.5–127.6)b
100
100
H
25.7 (13.1–47.1)b
76.1 (5.3–29.6)b
10.6 (2.7–19.8)b
>100
Melting points were taken on a Kofler hot stage appa-
ratus and are uncorrected. The UV spectra were
obtained using a Perkin–Elemer Model 550 SE. The IR
spectra were obtained using a Nicolet FT-IRMagna
550 spectrographs. The 1H NMRspectra were obtained
using Bruker FT-80 or Varian 400 unity plus spectro-
meters and chemical shifts (d) are in ppm relative to
internal tetramethylsilane. Mass spectra were obtained
using a Finnigan TSQ 70 Mass spectrophotometer at
H
Cl
H
100
35.3 (25.1–50.2)b
100
Cl
>100
100
an=10, 95% confidence limits in parentheses, LD50 of all compounds
>300 mg/kg.
bED50 significantly increased in the presence of flumazenil 10 mg/kg (p
<0.05).
cNot determined.

772
T.Akbarzadeh et al./ Bioorg.Med.Chem.11 (2003) 769–773
70 ev. Elemental microanalyses were within Æ0.4% of
(750 mg, 2.13 mmol) in dichloromethane (30 mL) at 0 ꢁC
m-chloroperbenzoic acid (2.2 g, 12.78 mmol) was added.
The mixture was stirred overnight at room temperature.
Saturated sodium carbonate solution (2Â20 mL) were
added and vigorously stirred for 15 min. The organic
layer was dried (sodium sulfate) and evaporated under
reduced pressure. The residue was crystallized from
ethanol to give 600 mg (73%) of 5b, mp 178–180 ꢁC; uv
(ethanol): lmax 213 nm (log e=4.53), 256 nm (log
e=4.30), 266 nm (log e=4.22), 292 nm (log e=3.82),
302 nm (log e=3.76); ir (potassium bromide): n 3350,
3284 (N–H), 3074 cmꢀ1 (C–H aromatic), 1H NMR
(CDCl3): d 8.38 (d, 1H, H6, J5.6=8.6 Hz), 7.10–7.65 (m,
5H, aromatic), 6.70 (d, 1H, H3), 3.34 ppm (s, 3H, CH3);
ms: m/z (%) 383 (M+, 18), 350 (45), 348 (100). Anal.
calcd for C15H11Cl2N3O3S: C, 46.89; H, 2.89; N, 10.94.
Found: C, 46.81; H, 2.86; N, 10.59.
theoretical values for C, H, Cl and N.
3-Amino-5-[4-chloro-2-(2-chlorophenoxy)phenyl]-4H-
1,2,4-triazole (2b). To a stirring solution of aminogua-
nidine hydrogen carbonate (2.14 g) in dry pyridine
(26 mL), at ꢀ5 ꢁC, a solution of 4-chloro-2-(2-chloro-
phenoxy)benzoic acid chloride (5 g 16.6 mmol) in dry
benzene (26 mL) was added. The stirring was continued
for half an h at ꢀ5 ꢁC and then overnight at room tem-
perature. The solvent was evaporated. To the residue,
water (35 mL) was added. The crude white precipitate
was suspended in 5% aqueous solution of sodium
hydroxide (140 mL) and heated at reflux for 8 h. After
cooling the reaction mixture was filtered. The filtrate
was acidified with hydrochloric acid and the precipitate
was filtered, washed with ethyl acetate and crystallized
from ethanol to give 1 g (20%) of 2b, mp 265–267 ꢁC; uv
(ethanol): lmax 207 nm (log e=4.25), 264 nm (log
e=3.98), 298 nm (log e=3.72); ir (potassium bromide):
n 3371 (N–H), 3283, 3227 (NH2), 3078 (C–H aromatic),
1685 cmꢀ1 (NH2); ms: m/z (%) 320 (M+, 27), 287 (32),
285 (100). Anal. calcd for C14H10Cl2N4O: C, 52.36; H,
3.14; N, 17.45. Found: C, 52.64; H, 3.36; N, 17.30.
5-[4-Dhloro-2-(2-chlorophenoxy)phenyl]-2-amino-1,3,4-
oxadiazole (6b). Sodium bicarbonate (2.25 g, 26.8 mmol)
in water (64 mL) was added at room temperature to a
solution of 4-chloro-2-(2-chlorophenoxy)benzoic acid
hydrazide (7.9 g, 26.6 mmol) in dioxane (85 mL). After
the mixture was stirred at room temperature for 5 min,
cyanogen bromide (3.5 g, 33.03 mol) was added. The
reaction mixture was stirred for 1 h, the precipitate was
filtered and crystallized form ethyl acetate to give 8 g.
(94%) of 6b, mp 158–160 ꢁC; uv (ethanol): lmax 279 nm
(log e=4.13), 302 nm (log e=4.06); ir (potassium bro-
mide): n 3452, 3318 (NH2), 3124 (C–H aromatic),
1664cmꢀ1 (NH2); ms: m/z (%) 321 (M+, 14), 288 (65),
286 (100). Anal. calcd for C14H9Cl2N3O2: C, 52.20; H,
2.82; N, 13.04. Found: C, 52.01; H, 2.59; N, 13.12.
5-[4-Chloro-2-(2-chlorophenoxy)phenyl]-2,4-dihydro-3H-
1,2,4-triazole-3-thione (3b). To a stirring solution of
thiosemicarbazide (0.58 g, 6.4 mmol) in dry pyridine
(10 mL), at ꢀ5 ꢁC a solution of 4-chloro-2-(2-chloro-
phenoxy)benzoic acid chloride (1.92 g) in dry benzene
(10 mL) was added. The stirring was continued for half
an hour at ꢀ5 ꢁC and then overnight at room tempera-
ture. The solvent was evaporated. To the residue water
(20 mL) was added. The crude yellow precipitate was
suspended in 5% aqueous solution of sodium hydroxide
(215 mL) and heated at reflux for 8 h. After cooling the
solution was acidified with hydrochloric acid and the
precipitate was filtered and crystallized from ethanol to
give 1.8 g (84%) of 3b, mp 250–252 ꢁC; uv (ethanol):
3-[4-Chloro-2-(2-chlorophenoxy)phenyl]-5-ethoxy-4H-
1,2,4-triazole (7b). To a room temperature suspension
of 6b (2 g, 6.21 mmol) in ethanol (50 mL) potassium
hydroxide (1.37 g) was added. The solution was heated
at reflux for 3 h, and an additional potassium hydroxide
(240 mg) was added. After an additional 3 h of heating
the reaction mixture was allowed to cool at room tem-
perature and neutralized with acetic acid. The solvent
was evaporated under reduced pressure and the residue
was crystallized from ethyl acetate to give 1.5 g (69%) of
7b, mp 167–168 ꢁC; uv (ethanol): lmax 266 nm (log
e=4.08), 294 nm (log e=3.86), 305 nm (log e=3.79); 1H
NMR(CDCl 3): 8.27 (d, 1H, H6, J5,6=8.5 Hz),
7.09–7.62 (m, 5H, aromatic), 6.67 (d, 1H, H3,
J3,5=1.94), 4.39 (q, 2H, CH2), 1.45 (t, 3H, CH3); ms: m/
z (%), 349 (M+, 95), 316 (43), 314 (98), 288 (38), 286
(98). Anal. calcd for C16H13Cl2N3O2: C, 54.88; H, 3.74;
N, 12.00. Found: C, 55.09; H, 3.55; N, 11.87.
lmax, 256 nm (log e=4.26), 305 nm (log e=3.86); ir
(potassium bromide): n 3441 (N–H), 3083 (C–H aro-
matic), 1613 cmꢀ1 (N–H); ms: m/z (%) 337 (M+, 44), 304
(38), 302 (100). Anal. calcd for C14H9Cl2N3OS: C, 49.72;
H, 2.68; N,12.42. Found: C, 49.49; H, 2.46; N,14.73.
5-[4-Chloro-2-(2-chlorophenoxy)phenyl]-3-methylthio-
4H-1,2,4-thiazole (4b). Compound 3b (2 g, 5.92 mmol)
was dissolved in ethanol (3 mL) and 10% aqueous
solution of sodium hydroxide (2.4 mL) with sonication.
CH3I (0.84 g 5.92 mmol) was then added and the solu-
tion mixture was sonicated for 3 min. The precipitate
was filtered and crystallized from methanol to give 1.8 g
(87%) of 4b, mp 146–148 ꢁC; uv (ethanol): lmax 245 nm
1
(log e=4.18), 296 nm (log e=3.90); H NMR(CDCl ):
5-[4-Chloro-2-(2-chlorophenoxy)phenyl]-2,4-dihydro-3H-
1,2,4-trizole-3-one
3
d 8.32 (d, 1H, H6, J5,6=8.8 Hz), 7.13–7.60 (m, 5H, aro-
matic), 6.69 (d, 1H, H3, J3,5=1.6 Hz), 2.65 ppm (s, 3H,
SCH3); ms: m/z (%) 351 (M+, 65), 318 (100), 316 (70),
300 (15), 264 (10), 228 (5). Anal. calcd for
C15H11Cl2N3OS: C, 51.15; H, 3.15; N,11.93. Found: C,
50.86; H, 3.23; N, 12.24.
(8b).
Compound
7b
(0.4 g,
1.14 mmol) was suspended in concentrated HCl (10 mL)
and heated at reflux for 24 h. The suspension was cooled
to room temperature filtered and washed with water to
give 0.3 g (82%) of 8b, mp 286–289 ꢁC; uv (ethanol):
lmax 273 nm (log e=4.02), 300 nm (log e=3.81); ir
(potassium bromide), n 3221 (N–H), 3088 (C–H aro-
matic), 1700 cmꢀ1 (C¼O); ms: m/z (%) 321 (M+, 60),
288 (32), 286 (100). Anal. calcd for C14H9Cl2N3O2: C,
5-[4-Chloro-2-(2-chlorophenoxy)phenyl]-3-methylsulfo-
nyl-4H-1,2,4-triazole (5b). To a stirring solution of 4b

T.Akbarzadeh et al./ Bioorg.Med.Chem.11 (2003) 769–773
773
52.20; H, 2.82; N, 13.04. Found: C, 52.01; H, 3.14; N,
12.77.
test compounds as a categorical covariate and forcing
through parallel dose response. Rightward shift of the
ED50 in logarithmic scale after administration of flu-
mazenil was considered significant if both lower and
upper bonds of 95% confidence interval were greater or
less than one. All statistical calculations were performed
by SPSS for windows (Rel. 10.0.5. 1999. Chicago: SPSS
Inc.).
Compounds 2a–8a, 4c, 5c, 4d and 5d were prepared
similarly.
Calculations
Conformational analysis of the synthesized compounds
and estazolam were preliminarily performed by MMX
force field method implemented in PCMODEL 6.0
software.17 The conformers were optimized further by
AM1 calculation using MOPAC 6.0 program.18 Global
energy minima conformers of the designed compounds
were superimposed on corresponding conformer of
estazolam molecule which was considered as a reference
benzodiazepine agonist.
Acknowledgements
This work was partially supported by a grant from the
research council of Tehran University of Medical Sci-
ences, Shaheed Beheshti University of Medical Sciences
and the International Organization for Chemical Sci-
ences in Development (IOCD).
Pharmacological evaluation
References and Notes
Male NMRI mice (Pasteur Institute, Iran) weighting
20–25 g (n=10) were used in the experiments. The ani-
mals were kept in the groups of ten in cages under con-
stant temperature (24Æ1 ꢁC) and 12 h light/dark
schedule. They had free access to standard mouse diet
and tab water except during the experiment. On the day
of the experiment, animals were transferred to indivi-
dual cases randomly and allowed to acclimatize for
30 min before injection of drug or vehicle. Test com-
pounds, flumazenil (Hoffmann La Rosch) and diazepam
(Sigma) were given ip (10 mL/kg) as a freshly prepared
solution in 50% DMSO and 50% sterile normal saline.
Flumazenil were injected 5 min before administration of
vehicle, diazepam or the test compounds.
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Rotarod test
Groups of 10 mice were trained to stay for 2 min on
rotarod apparatus, 3.0 cm in diameter, at 8 rpm (Tajhiz
gostar co.). Twenty-four hours later, the animals were
injected with vehicle, diazepam or the synthesized com-
pounds and placed in the apparatus 30 min later.
Impairment of coordinated motor movement was
defined as the inability of the mice to remain on the
rotarod for the 2-min test period.9
13. Detar, F.; Hlynsky, A. J.Am.Chem.Soc. 1955, 77, 4411.
14. Shafiee, A.; Naimi, E.; Mansobi, P.; Foroumadi, A.; She-
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PTZ-induced lethal convulsion test
15. Firoozi, F.; Javidnia, K.; Kamal, M.; Fooladi, A.; For-
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The test compounds, diazepam and vehicle were admi-
nistered to groups of 10 mice 30 min before the injection
of PTZ (100 mg/kg, ip) and the dead mice were counted
30 min later.10
17. PCMODEL; Serena Software: PO Box 3070, Bloo-
mington, IN 47402, USA.
Statistical analysis
ED50 values and 95% confidence limits were determined
using probit-log (dose) model with flumazenil and the
18. QCPE; Department of Chemistry: Indiana University,
Bloomington, IN 47405, USA.